XXXIst International Symposium on Technological Innovations in Laboratory Hematology

Printable Program

May 9-11, 2019

Vancouver, Canada

Wednesday, May 8, 2019

8:00 - 4:00 PMEast Ballroom A
Educational Workshop: An Interactive Review of New and Recurrent Topics in Laboratory Hematology

Chair(s): Joyce Rogers, Anna Merino
Coagulopathy Of Liver Dysfunction In The Critically Ill Patient: Clinical And Laboratory Implications
Ted Warkentin
McMaster University, Canada

Coagulopathy of Liver Dysfunction in the Critically Ill Patient: Clinical and Laboratory Implications

Sometimes, ischemic limb injury occurs in critically ill patients with hypotension and lactic acidemia (“shock”). The ischemic limb injury can be strikingly symmetrical, affecting toes/feet, and sometimes also fingers/hands, a syndrome known as “symmetrical peripheral gangrene (SPG).” The pathogenesis is microvascular thrombosis, which explains preservation of arterial pulses in most instances. Ischemic limb injury with pulses is usually accompanied by thrombocytopenia and/or coagulopathy, triggering frequent referral to a hematologist. Fundamentally, SPG in critically ill patients involves a profound disturbance in procoagulant/anticoagulant balance. “Procoagulant” aspects of SPG feature prominent disseminated intravascular coagulation (DIC), usually secondary to septic or cardiogenic shock. “Anticoagulant” failure in SPG most often is explained by prominent liver dysfunction, either acute or chronic, which plays a crucial role in explaining failure of the natural anticoagulants, protein C and antithrombin, to protect against thrombosis in the microcirculation. Approximately 90% of patients with SPG have preceding “shock liver” (acute ischemic hepatitis), a risk factor for severe depletion of (hepatically-synthesized) protein C and antithrombin. In a minority of SPG patients, there is preexisting chronic liver disease. Also key to understanding SPG is recognizing the role of circulatory shock in explaining localization of tissue injury, as impaired peripheral blood flow in severely hypotensive patients (with or without use of vasopressors) predisposes to microvascular thrombosis in those vascular beds. While physicians sometimes blame vasopressors for causing SPG, the typical occurrence of ischemic limb injury beginning 2 to 5 days (median, 3 days) after onset of hypotension (and initiation of vasopressors) points rather to critical time-dependent factors in explaining microthrombosis, namely the progressive decline in the hepatically-synthesized natural anticoagulants. Illustrative cases will demonstrate the role of acute or chronic liver dysfunction in explaining SPG occurrence. While preventive and treatment approaches for SPG remain to be established, better understanding of the pathobiological mechanisms that underlie SPG could help in formulating rational prevention and treatment approaches.

KEY WORDS = antithrombin; disseminated intravascular coagulation (DIC), protein C, shock liver (acute ischemic hepatitis); symmetrical peripheral gangrene (SPG)

Hiding In Plain Sight: Identifying Myeloid Neoplasms With Germline Predisposition
David Czuchlewski
University of New Mexico, USA

Prognostic value of anti-FVIII immunoglobulin isotypes and IgG

subclasses in acquired haemophilia A subjects with long term follow-up

Arnaud Bonnefoy1, Clémence Merlen1, Hadrien Claus-Desbonnet1, Molly Warner2, Stéphanie Cloutier3, Christine Demers3, Jean-François Castilloux4, Jean St-Louis1 and Georges-Etienne Rivard1*.

1 CHU Sainte-Justine, 2CHU McGill,3CHU de Québec, 4CHU de Sherbrooke.

*Presenting author


Introduction. The diagnosis of acquired hemophilia A (AHA) is based on the measure of factor VIII (FVIII) activity level and inhibitor titers. The lack of reliable correlation between FVIII activity and the inhibitor titer can make the diagnosis of AHA challenging. Also, there is little information about the predictive value of inhibitor titers, residual FVIII activity, immunoglobulin isotypes and IgG subclasses on clinical outcomes. We measured titers of FVIII-binding antibody immunoglobulin isotypes and IgG subclasses and correlated these with clinical outcomes in subjects with AHA.

Methods. Baseline demographic characteristics and outcomes were collected from 118 consecutive AHA subjects diagnosed at the Quebec Inhibitor Reference Centre with a median follow up of 29.9 months (range 0.4-142.3). Factor VIII inhibitors were measured with the Nijmegen modification of the Bethesda assay. Titers of FVIII-binding antibodies of different immunoglobulin isotypes and of IgG subclasses were measured by ELISA on baseline plasma available for 75/118 subjects. All assays were centralized at the Quebec Inhibitor Reference Centre. Frequencies of clinical and/or biological events were compared using Fisher’s exact test or χ2 test.

Results. FVIII-binding immunoglobulins were detected in all subjects’plasma with the following distribution: IgG (99%), IgA (35%), IgM (12%) and IgE (7%). The most prevalent subclasses of FVIII-binding IgG were IgG4 (92%), IgG2 (88%), IgG1 (74%), IgG3 (62%) with IgG1 and IgG4 having the highest antibody titers (median 1:1600 and 1:800; range: 1:400-1:3200 and 1:200-1:3200). No correlation was found between titers of FVIII-binding immunoglobulins and FVIII activity. However, Bethesda unit titers significantly correlated with titers of FVIII-binding immunoglobulins: IgG1 (r=0.492), IgG2 (r=0.373), IgG4 (r=0.472) and IgM (r=0.863), all with p<0.01. The presence of FVIII-binding IgM was significantly associated with death related to acute bleeding (p<0.003), whereas FVIII-binding IgA and IgG4 were associated with auto-immune (p<0.008) or underlying malignant (p<0.001) disorders.

Conclusion. Testing for FVIII-binding immunoglobulin isotypes and IgG subclasses in baseline plasma of subjects with AHA revealed distinctive immunological profiles some of which statistically correlate with clinical and/or biological parameters liable to guide precision patient management and aid in the prediction of outcomes for subjects with AHA.


Coffee Break

Morphology Cases With Difficult Clonal Cells
Gina Zini
Fondazione Policlinico Universitario A. Gemelli IRCCS -Universita' Cattolica sacro Cuore, Rome Italy

Morphology Cases with Difficult Clonal Cells

Gina Zini, Hematology Professor

Fondazione Policlinico Universitario A. Gemelli IRCCS - Roma

Università Cattolica del Sacro Cuore

In the era of rapid technological advances, the role of microscopy for cell identification and evaluation in the diagnostic workup of hematological disorders is periodically debated:  the landscape of diagnostics in malignant hematology is moving toward greater specialization, producing relevant results for prognostication and the development of targeted therapies.

The WHO 2016 classification of hematology neoplasms confirms the role of morphology as a diagnostic tool which should be integrated with flow cytometry, cytogenetics/molecular genetics, histology, and clinical characteristics: quantitative and qualitative recommendations are clearly mentioned in these guidelines for the diagnostic algorithm. Morphology represents the first diagnostic screening in the majority of hematological disorders, with a high predictivity in both myeloid‐ and lymphoid‐ neoplasms involving peripheral blood and bone marrow.

Moreover a prompt morphologic diagnosis in hematologic emergencies, such as in APL, Burkitt lymphoma, thrombotic thrombocytopenic purpura (TTP), or sickle cell crisis, is critical to save patient's lives.

Based on these considerations, it appears obvious that the microscopy skills of hematology cytomorphologists should be maintained. In the era of ICT image digitalization allows an easy cross‐analysis between morphologists as well the possibility to be trained via on-line courses.

In this scenario some criticisms derive from the correct identification of some cells: wheb missed, the diagnosis is delayed inexorably.

The evaluation of “similar cells” having a different diagnostic impact does represent another clue.

Among these, the correct differentiation of promonocytes from immature monocytes still represents a challenge: promonocytes, as blasts-equivalent, should be included within the blast count in the appropriate diagnostic setting, while immature monocytes should be included within the count of the maturating monocytic series.

Some morphologic variants of abnormal promyelocytes can be also misdiagnosed with a possible impact on the patient’s survival. 

For an appropriate morphologic identification of difficult cells, it is mandatory to look at peculiar different cell details as well as at the company they keep.  

Hemoglobinopathy Update
Cornelis Harteveld
Leiden University, Netherlands

The hemoglobinopathies constitute the most common recessive monogenic disorders worldwide [1, 2]. Over 1700 different mutations have been reported which either affect the synthesis of globin chains (causing thalassemia) or alter the structure and properties of the hemoglobin tetramer (hemoglobin variants or abnormal hemoglobins) [3]. Hemoglobinopathies are mostly autosomal recessive disorders and heterozygotes are symptom-free but present various hematological characteristics which are used for their identification in carrier screening programs. The homozygous states and compound-heterozygous states result in four main groups of clinically significant conditions, each with a variable degree of phenotypic severity: the b-thalassemias, a-thalassemias, sickle cell syndromes, and Hb E syndromes [4]. The heterogeneity of the hemoglobinopathies is caused by the numerous types of thalassemia and abnormal hemoglobin genotypes which can interact when co-inherited, creating a complex range of hematological phenotypes that often cause difficulties in interpretation. Moreover, some phenotypes can arise from several different genotypes, such as heterozygous alpha zero thalassemia and homozygous alpha plus thalassemia, and the genotypes cannot be distinguished by simple hematological parameters.  Finally, the electrophoresis or chromatography techniques used to screen for abnormal hemoglobins only provide a presumed diagnosis for the variant, and further tests are required for a definitive diagnosis. Thus in many cases of carrier screening, an accurate diagnosis requires expertise in the interpretation of the hematological results and confirmation of the genotypes by DNA analysis [1].

The presentation will highlight some unexpected molecular mechanisms showing interesting genotype-phenotype correlations, either ameliorating or deteriorating the clinical presentation of hemoglobinopathy.

[1] John Old , Cornelis Harteveld , Carrier Screening for the Haemoglobinopathies: Past, Present and Future,

[2] Modell B, Darlison M. Global epidemiology of haemoglobin disorders and derived service indicators. Bulletin of the World Health Organization. 2008;86(6):480-7.


[3] Giardine B, Borg J, Viennas E, Pavlidis C, Moradkhani K, Joly P, et al. Updates of the HbVar database of human hemoglobin variants and thalassemia mutations. Nucleic acids research. 2014;42(Database issue):D1063-9.


[4] Weatherall DJ, Clegg JB. The thalassaemia syndromes: John Wiley & Sons; 2008.

Lunch On Your Own

Infection-Related Hematology Laboratory Abnormalities
Ruth Padmore
University of Ottawa, Canada

Flow Cytometry Illustrative Cases
Marie Christine Bene
Centre Hospitalier Universitaire de Nantes, France

This educative session, as indicated, will guide the participants through a series of immunophenotypic strategies based on real-life cases.

Hematology Test Validation
Kandice Marchant
Cleveland Clinic, USA

Test Validation in Hematology

Kandice Kottke-Marchant, MD, PhD; Cleveland Clinic, Cleveland, OH, USA

This talk will discuss the appropriate validation of laboratory tests for clinical use, and indicate specific examples for hematology testing. From a regulatory perspective under ISO 15189, CLIA (Clinical Laboratory Improvement Amendments in the USA) or SCC (Standardization Council of Canada), all clinical analytical methods must be verified or validated prior to testing patient specimens. There are different requirements for verification (in-lab testing of approved laboratory tests to show that performance characteristics fulfil specified requirements) and for validation (for laboratory-developed or modified tests to establish that specified requirements are adequate for intended use). The standards for test validation and verification between ISO15189 and CLIA will be compared and contrasted. Approved laboratory tests being brought into a hospital laboratory must be verified for accuracy, precision, reportable range and the manufacturer’s reference range. For laboratory-developed tests, validation must include, in addition, analytical sensitivity and analytical specificity, and to meet ISO 15189 requirements, validation should include measurement uncertainty and diagnostic specificity and sensitivity. Hematology-specific validation parameters, such as mixing studies, factor sensitivity, heparin therapeutic ranges, and pathologist agreement will be discussed. Examples of validation plans and a validation compliance program will be presented.

Thursday, May 9, 2019

8:20 - 10:00 AMEast Ballroom BC
Opening Plenary

Chair(s): Tracy George, Ruth Padmore, Parul Bhargava
Catherine Hayward
McMaster University, Canada

Rna-Seq: Clinical, Diagnostic, And Novel Findings Arising From These Studies
Aly Karsan
University of British Columbia, Canada

Clinical genetic testing in the myeloid malignancies is undergoing a rapid transition from the era of cytogenetics and single-gene testing to an era dominated by next-generation sequencing (NGS). This transition promises to better reveal the genetic alterations underlying disease, but there are distinct risks and benefits associated with different NGS testing platforms. NGS offers the potential benefit of being able to survey alterations across a broader set of genes, but analytic and clinical challenges associated with incidental findings, germline variation, turnaround time, and limits of detection must be addressed. Additionally, transcriptome-based testing may offer several distinct benefits beyond traditional DNA-based methods. This presentation will examine the potential for genome-scale testing in the clinic with a particular focus on RNA-Seq as a diagnostic, and its potential role in revealing mechanisms of therapy resistance. 

Heparin-Induced Thrombocytopenia
Theodore (Ted) Warkentin
McMaster University, Canada

Heparin-induced thrombocytopenia (HIT) is a clinical-pathological disorder; thus, laboratory testing for the pathogenic platelet-activating anti-platelet factor 4 (PF4)/heparin antibodies is central for diagnosis. The “iceberg” model summarizes the interrelationship between platelet activation assays and PF4-dependent immunoassays, with platelet-activating antibodies comprising a subset of anti-PF4/heparin antibodies. The platelet serotonin-release assay (SRA), performed by reference laboratories, has high sensitivity and specificity for HIT (~95% each), and is especially suited for detecting highly-pathogenic HIT sera containing both heparin-dependent and heparin-independent platelet-activating antibodies; this latter subgroup of antibodies explains “autoimmune HIT” disorders (delayed-onset, persistent, spontaneous, heparin “flush”, fondaparinux-associated). Recently, SRA-negative HIT has become recognized, in which serum from some HIT patients contains subthreshold levels of platelet-activating antibodies (by SRA) that become detectable using a PF4-enhanced platelet activation assay. Unusual immunologic features of HIT include early antibody detectability (at onset of platelet count fall) and antibody transience (seroreversion). Widely-available PF4-dependent enzyme-immunoassays (EIAs) have high sensitivity but poor specificity for HIT, although specificity is enhanced with IgG-specific EIAs and strong-positive results; unfortunately, EIA results are usually not available in real time. Automated rapid immunoassays, such as the chemiluminescence immunoassay (CLIA) and latex immunoturbidimetric assay (LIA), facilitate real-time laboratory diagnosis. Recently available likelihood ratio (LR) data for positive (LR+) and negative (LR–) test results allow clinicians to adjust their pretest probabilities for HIT, using Bayesian analysis, into real-time posttest probabilities that are dramatically increased (test-positive) or decreased (test-negative). Moreover, (semi)quantitative CLIA and LIA positive results (weak-, moderate-, strong-positive) can further refine the posttest probability of HIT.


(autoimmune) heparin-induced thrombocytopenia, Bayesian analysis, chemiluminescence immunoassay, latex immunoturbidimetric assay, rapid immunoassays, serotonin-release assay

Erythrocytes And Vascular Function: Oxygen And Nitric Oxide
Mark Gladwin
University of Pittsburgh

Nitric oxide (NO) is a critical regulator of vascular homeostasis, increasing basal and flow-mediated vasodilation. Red blood cells can regulate NO bioavailability through an intrinsic nitrite (NO2-) reductase mechanism that generates NO during physiological and pathological hypoxia. In contrast, red blood cell hemolysis releases hemoglobin into plasma, where can react with and scavenge NO. In this context, hemoglobin functions as an oxido-reductase: A nitrite-reductase and NO synthase when it deoxygenates and an NO oxidase when it is oxygenated, particularly in the setting of hemolysis. The mechanistic linkage between hemolytic anemia and vasculopathy has been the subject of extensive study in pre-clinical animal models, in vascular studies in patients, and in large human cohort studies.  Intravascular hemolysis releases cell-free hemoglobin into the plasma, which can scavenge NO and generate reactive oxygen species, impairing redox balance and leading to proliferative systemic and pulmonary vasculopathy. It has been recently appreciated that products released from the red cell during hemolysis, including heme released from hemoglobin, can be considered danger associated molecular pattern molecules or erythrocyte “DAMPs” (eDAMPs). These studies support a more general pathological role for intravascular hemolysis and cell-free hemoglobin in various human diseases and in transfusion medicine.

10:00 - 10:30 AMEast Exhibit Hall B
Coffee Break

10:30 - 12:00 PMEast Ballroom BC
CONCURRENT 1: Hemostasis/Platelets/Thrombosis

Chair(s): Kristi Smock, Martha Eva Viveros
Acquired Hemophilia: Laboratory Perspectives On Inhibitors
Georges E Rivard
Centre Hospitalier Universitaire Sainte Justine, Canada


 Prognostic value of anti-FVIII immunoglobulin isotypes and IgG subclasses in acquired haemophilia A subjects with long term follow-up

Bonnefoy A1, Merlen C1, Claus-Desbonnet H1, Warner M2, Cloutier S3, Demers C3, Castilloux JF4, St-Louis J1 and Rivard GE1.

1Division of Hematology-Oncology, CHU Sainte-Justine, Montreal, Canada, 2Division of Hematology, McGill University Health Centre, Montreal, Canada,3Hôpital de l’Enfant Jésus, Centre Hospitalier Universitaire de Québec, Quebec, Canada, 4Division of Hematology-Oncology, Centre Hospitalier Universitaire de Sherbrooke, Université de Sherbrooke, Canada.


Introduction. The diagnosis of acquired hemophilia A (AHA) is based on the measure of factor VIII (FVIII) activity level and inhibitor titers. The lack of reliable correlation between FVIII activity and the inhibitor titer can make the diagnosis of AHA challenging. Also, there is  little information about the predictive value of inhibitor titers, residual FVIII activity, immunoglobulin isotypes and  IgG subclasses on clinical outcomes . We measured titers of FVIII-binding  antibody immunoglobulin isotypes and IgG subclasses and correlated these with clinical outcomes in subjects with AHA.  Methods. Baseline demographic characteristics and outcomes were collected from 118  consecutive AHA subjects diagnosed at the Quebec Inhibitor Reference Centre  with a median follow up of 29.9 months (range 0.4-142.3). Factor VIII inhibitors were measured with the Nijmegen modification of the Bethesda assay. Titers of FVIII-binding antibodies of different immunoglobulin isotypes and of IgG subclasses were measured by ELISA on baseline plasma available for 75/118 subjects. All assays were done at the Quebec Inhibitor Reference Centre.  Frequencies of  clinical and/or biological events were compared using Fisher’s exact test or χ2 test. Results. FVIII-binding immunoglobulins were detected in all subjects’plasma with  the following distribution: IgG (99%), IgA (35%), IgM (12%) and IgE (7%). The most prevalent subclasses of FVIII-binding IgG were  IgG4 (92%), IgG2 (88%), IgG1 (74%), IgG3 (62%) with IgG1 and IgG4 having the highest antibody titers (median 1:1600 and 1:800; range: 1:400-1:3200 and 1:200-1:3200). No correlation was found between titers of FVIII-binding immunoglobulins and FVIII activity. However, Bethesda unit titers significantly correlated with titers of FVIII-binding immunoglobulins: IgG1 (r=0.492), IgG2 (r=0.373), IgG4 (r=0.472) and IgM  (r=0.863), all with p<0.01. The presence of  FVIII-binding IgM  was significantly associated with death related to acute bleeding (p<0.003), whereas FVIII-binding IgA and IgG4 were associated with auto-immune (p<0.008) or underlying malignant (p<0.001) disorders.

Conclusion. Testing for FVIII-binding immunoglobulin isotypes and IgG subclasses in baseline plasma of subjects with AHA revealed distinctive immunological profiles some of which statistically correlate with clinical and/or biological parameters liable to guide precision patient management and aid in the prediction of outcomes for subjects with AHA.

Update On Testing For Platelet Function Disorders: What Is Useful?
Catherine Hayward
McMaster University, Canada

Update on Diagnostic Testing for Platelet Function Disorders: What is Practical and Useful?

Catherine P.M. Hayward, Karen A. Moffat, Justin Brunet, Stephen A. Carlino, Elizabeth Plumhoff, Piet Meijer, James L. Zehnder


Introduction: Platelet function disorders (PFD) are an important group of bleeding disorders that require validated and practical laboratory strategies for diagnosis.

Methods: This review summarizes the authors’ experiences, current literature and an international survey to evaluate the practices of diagnostic laboratories that offer tests for PFD.

Results: Blood counts, blood film review and aggregation tests are the most commonly performed investigations for PFD and help determine if there is thrombocytopenia and/or defective platelet function due to a variety of causes. The performance characteristics of tests for PFD, and the level of evidence that these test detect bleeding problems, are important issues to determine where tests are useful for diagnostic or correlative purposes, or research only uses. Platelet aggregation assays, and quantitative analysis of platelet dense granule numbers, are tests with good performance characteristics that detect abnormalities associated with increased bleeding in a significant proportion of individuals referred for PFD investigations. Lumiaggregometry estimates of platelet adenosine triphosphate release shows greater variability which limits the diagnostic usefulness. Diagnostic laboratories report that fiscal and other constraints, including a lack of high quality evidence, limit their ability to offer an expanded test menu for PFD.

Conclusion: PFD are clinically important bleeding disorders that remain challenging for diagnostic laboratories to investigate. While some PFD tests are well validated for diagnostic purposes, gaps in scientific evidence and resource limitations influence diagnostic laboratory decisions on which PFD tests to offer.

What We Have Learned From Coagulatory Laboratory Participation In External Quality Programs?
Kristi Smock
University of Utah, USA

Coagulation laboratory external quality assurance (EQA) programs provide information that satisfies regulatory requirements and improves laboratory quality, patient care, and safety.  In addition to the value to individual laboratories, data from EQA programs benefits the laboratory community in multiple ways by providing a snapshot of laboratory practice and summarizing the performance of various methods in identifying normal and abnormal specimens and the effects of therapies or interfering substances.  This presentation aims to summarize and provide examples of some of the important lessons learned from coagulation EQA data. 

10:30 - 12:00 PMEast Ballroom A
CONCURRENT 2: Cellular Analysis and Flow Cytometry

Chair(s): Sindhu Cherian, Bruce Davis
Informatics In Flow Cytometry: Automated Flow Data Analysis
Ryan Brinkman
University of British Columbia, Canada

Manual analysis of flow cytometry data is subjective and time consuming. Automated algorithms have reached a level of maturity that enables them to match and, in many cases, exceed the results produced by human experts. The current state-of-the-art will be reviewed though example applications of these algorithms in automating the entire data analysis pipeline. Examples will include automated quality checking at the event and sample level, rapid, robust and reproducible gating and biomarker discovery algorithms.The utility of these algorithms will be shown through their application on patient data for basic research, clinical trials and drug discovery.

Immunotherapy Interference In Flow Analysis
Sindhu Cherian
University of Washington, USA

Flow cytometry is a cornerstone of diagnosis and characterization of hematopoietic neoplasms, in particular leukemia and lymphoma.  Measurable or minimal residual disease (MRD) that persists post therapy is a reproducible and powerful predictive factor in hematopoietic neoplasms and is often measured by flow cytometry.  Many current flow cytometric assays developed for residual disease detection were developed in the setting of conventional cytotoxic chemotherapies.  Such strategies may have limitations and hence require re configuring in the current therapeutic environment with increasing use of targeted and immunologically mediated therapy.  During this session, we will review the impact of targeted immunotherapy interference on flow cytometric analysis using the example of B lymphoblastic leukemia.

Immunotherapy Using Car-T Cells (Monitoring)
Hao-Wei Wang

The recent successes with chimeric antigen receptor (CAR) T-cell therapy in hematologic malignancies have revolutionized anticancer therapy. However, a substantial number of patients do not achieve durable remissions. Understanding the mechanisms of incomplete tumor elimination is critical to harnessing the full potential of this novel treatment. Growing experience with these agents has revealed that resistance to CAR T-cell therapy can arise from tumor antigen loss or modulation, poor CAR T cell persistence and/or T-cell exhaustion. Monitoring these parameters has become an important part to successful CAR T-cell therapy. During this session, we will review the roles of flow cytometry in CAR T-cell therapy, with a focus on monitoring of the target cells and effector CAR T cells.

12:00 - 1:30 PMEast Meeting Room 2
Abbott Luncheon Workshop

12:00 - 1:30 PMEast Meeting Room 8
Instrumentation Laboratory Luncheon Workshop

12:00 - 1:30 PMEast Meeting Room 11
CellaVision Luncheon Workshop

3:30 - 5:00 PMEast Ballroom BC
CONCURRENT 3: Red Cell Disorders

Chair(s): John Frater, Cornelis Harteveld
Sickle Cell Disease: A Global Perspective
Isaac Odame
The Hospital for Sick Children, Toronro & University of Toronto, Canada

Sickle cell disease (SCD)  is one of the commonest genetic disorders and was the first molecular disease described in humans. Strong evidence supports selection pressure from endemic malaria as the reason for the geographical distribution of the sickle gene. Areas of high gene frequencies include sub-Saharan Africa (SSA), the Indian sub-continent, Middle East and Brazil. In high-income countries with newborn screening (NBS) programs consisting of early diagnosis and interventions (penicillin prophylaxis, pneumococcal vaccinations and comprehensive care) over 90% of children born with SCD survive to adulthood. By contrast, in SSA where these programs are nonexistent, 50-90% of children born with SCD die before the age 5 years. The emergence of innovative point-of-care (POC) tests have potential to bridge the NBS implementation gaps if ways can be found to deploy them within health systems in low-income countries (LICs). Recently, clinical trials have provided evidence for the safety and efficacy of hydroxyurea (HU) therapy in LICs. New approaches to develop POC tests for monitoring HU efficacy and toxicity will boost widespread implementation of HU therapy in these regions, where it is most needed. Public-private partnerships are needed to develop sustainable programs for SCD diagnosis and treatment globally, particularly in LICs that shoulder the heaviest disease burden.

What Is The Gold Standard In Laboratory Testing For Inherited Hemolytic Anemia?
Madeleine Verhovsek
McMaster University, Canada

In an undifferentiated case of hemolytic anemia, the initial list of potential causes can be diverse.  However, in a subset of patients the history, symptoms, and signs will be suggestive of an inherited cause.  Following clinical assessment, a carefully selected set of laboratory screening investigations, including morphology, is essential in honing in on the likely diagnosis, which can then be followed up with specialized testing. With modern advances in laboratory diagnostics for inherited hemolytic anemias (e.g. eosin-5-malemide testing for band 3 abnormalities and molecular testing, including next generation sequencing panels), we are increasingly able to provide precise diagnoses to patients and their families.

Beta Thalassemia- Challenges In Diagnosis And Prevention Especially In Developing Countries
Saleem Ahmed Khan
Armed Forces Institute of Pathology and National University of Medical Sciences

Beta Thalassemia: Challenges in Diagnosis and Prevention especially in developing countries

Beta Thalassemia is among the world’s most prevalent inherited disorder affecting people from the Mediterranean Region, Africa, Middle East, Indian Subcontinent and South East Asia. Since the disease requires long-term care and management with significant social and economic burden, therefore its prevention is a major target worldwide. The presentation emphasizes upon the main challenges in diagnosis of Beta Thalassemia and ways to prevent it. The laboratory diagnosis consist of typical red cell morphology, red cell indices, serum ferritin levels, one tube osmotic fragility test, hemoglobin electrophoresis, family studies, quantitative assessment of hemoglobin fractions and molecular genetic analysis. Most of the cases can be diagnosed accurately by cellulose acetate electrophoresis or HPLC. Molecular analysis and DNA sequencing are needed in patients presenting with recent transfusions and in challenging patients such as those having Silent Beta Thalassemia Trait, Coexisting Beta Thalassemia Trait and iron deficiency. Similarly Beta Thalassemia with hemoglobin variants, Coexisting beta and alpha Thalassemia, unusually severe beta thalassemia trait having Co-inheritance of mutations in globin or unrelated gene such as triplicated alpha genes, dominantly inherited thalassemia and mutations in the genes controlling globin gene expression require extensive workup.  Beta Thalassemia is a preventable disease. It requires government will, appropriate legislation, public awareness, education and carrier counselling. The preventive strategies employed in various countries include population-based carrier screening programs, premarital screening, antenatal screening, prenatal diagnosis and special methods such as Preconception or Preimplantation diagnosis. The discussion highlights the major preventive programs mainly drawing from the experiences in countries who overcame this problem such as Italy, Sardinia, Cyprus and Greece. However, thalassemia prevention programs had not been successful in many developing countries, thus maintaining the high burden of thalassemia homozygotes. The reasons of this failure have multiple reasons but there is a need to act jointly to prevent Beta Thalassemia in these countries.

5:00 - 6:30 PMEast Exhibit Hall B

Study On The Detection Of Remaining Platelets In Plasma Samples By Clot Waveform Analysis Of Prothrombin Time -The Evaluation Using Clinical Samples-
Yugo Shimada1, Kaneo Satoh1, Fuminori Kazama1, Takeshi Suzuki2, Yutaka Komiyama3, Tsukasa Suetake2, Katsue Suzuki-Inoue1
1University of Yamanashi, Chuo, Japan, 2Sysmex Corporation, Kobe, Japan, 3Hokuriku University, Kanazawa, Japan

Introduction: Coagulation test is affected by remaining platelets in plasma; however exact count of the number of remaining platelets in plasma is difficult. As one of us (T. Suzuki) presented in the different poster, clot waveform analysis (CWA) of prothrombin time (PT) are different between plasma with and without remaining platelets. In the present study, we investigated the clinical application of the CWA of PT to detect remaining platelets in plasma. 
Methods: PT and platelet counts were measured by CS-5000i with Thromborel S and XN-9000 (Sysmex), respectively. 1. Sodium citrate-added plasma was obtained from healthy subjects. To mimic the plasma with remaining platelet, patient plasma was diluted with platelet-rich plasma (0 to 30 × 109 /L) from the same donor, and then PT was measured. In the CWA of PT, the increase of slope (A/DS) between 1 and 90 seconds was analyzed. 2. Patient samples (n=87) from 30 to 120 minutes after blood collection were centrifuged at room temperature, at 1700 ×g for 10 minutes, and then PT and the remaining platelet counts were measured.
Results: 1. A/DS for 5 seconds from the end point was the most strongly correlated with remaining platelet counts in plasma from healthy donors. 2. Correlation between A/DS and remaining platelet counts for 5 seconds from the end point of coagulation reaction in plasma form patients was y = 0.0017 x - 0.0643, R = 0.88.
Conclusions: In plasma from both healthy donors and patients, the best correlation was observed between platelet counts and A/DS for 5 seconds from the end point of coagulation reaction, suggesting a remarkable relationship between the coagulation response curve and the number of remaining platelets. As shown in the expert consensus on standardization of sample preparation for clotting time assay by Working Group in Japanese society of laboratory hematology, the remaining platelet count in plasma should be less than 10 × 109 /L; however several clinical laboratories do not recognize this consensus in Japan. The proposed detection method by CWA of PT would help technicians recognize remaining platelets and adequately carry out centrifugation.

Improvement Of Chromogenic Anti-Xa Assay To Measure Betrixaban Concentration In Plasma
Romain Siriez1, Jonathan Evrard1, Jean-Michel Dogn1, Lionel Pochet1, Franois Mullier2, Jonathan Douxfils1,2
1University of Namur, Department of Pharmacy, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for LIfe Sciences (NARILIS), Namur, Belgium, 2Universit catholique de Louvain, CHU UCL Namur, Hematology Laboratory, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for Life Sciences (NARILIS), Yvoir, Belgium, 3Qualiblood s.a., Namur, Belgium

Introduction: Chromogenic anti-Xa assays are the most appropriate tests to estimate the amount of betrixaban in plasma but the sensitivity of available tests is limited and improvements are needed to encompass the on-therapy range.

Methods: Betrixaban was spiked at concentrations ranging from 0 to 500ng/mL in plasma from healthy donors. Three commercial tests were used (Biophen®DiXaI®, STA®Liquid Anti-Xa, HemosIL®Liquid-Anti-Xa) and adaptation of their sample dilution scheme were performed. These new methodologies were also tested on plasma spiked with amounts of unfractionated heparin (UFH), low molecular weight heparins (LMWH) or fondaparinux covering the on-therapy ranges to evaluate their sensitivity to indirect factor Xa inhibitors. 
Results: Results showed concentration-dependent decreases in OD/min inversely proportional to the dilutions. While modifications improve the sensitivity of these tests to betrixaban (e.g. ½*OD/min of 502ng/mL (95% CI: 495ng/mL-508ng/mL) for Biophen®DiXaI®(1:50) is reduced to 51ng/mL (95% CI: 50ng/mL-52ng/mL) for improved Biophen®DiXaI®(1:5)), results also showed an increased sensitivity to indirect factor Xa inhibitors, except for Biophen®DiXaI®which remains insensitive to UFH and LMWH.
Conclusions: Results showed that the improvement of current chromogenic anti-Xa methodologies enhances the sensitivity of these assays to betrixaban but also to indirect factor Xa inhibitors. This lack of specificity may lead to overestimation of betrixaban concentrationsin patients bridged with heparins. To avoid this cross-interference, the use of the Biophen®DiXaI®may be a solution except for fondaparinux which remains active even in presence of the Biophen®DiXaI®’s specific buffer. For the other chromogenic assays,the conception and validation of specific buffer is required.

Coexistent Acquired Hemophilia A And Lupus Anticoagulant? A Case Identified By Silica Clotting Time Assay
Dennis Sosnovske1, Kelly Townsend2, Richard A. Marlar1, Marian A. Rollins-Raval1
1University of New Mexico, Albuquerque, NM, United States, 2TriCore Reference Laboratories, Albuquerque, NM, United States

Introduction: In patients without congenital hemophilia, autologous antibodies to Factor VIII can result in acquired hemophilia A (AHA), a condition usually presenting with bleeding into the skin and muscles. Several reports of AHA associated with a positive lupus anticoagulant (LA) (which may result in thrombosis)  by Kaolin clotting time or the STA-Clot LA assays have been described, but we could not find any reports measured by silica clotting time (SCT) LA assay.
Methods: Demographic information, clinical history, and coagulation testing results were obtained from the treating physician and electronic medical record.
Results: The patient is a 72 year-old female with a 30 year history of rheumatoid arthritis, treated with prednisone, hydroxychloroquine and leflunomide. As she presented with a few months of easy skin bruising, a spontaneous black eye, large bruises around her upper neck, and a hematoma around her right knee, her PCP ordered initial and subsequent (two weeks later) laboratory studies(Table 1). She was then referred to a hematologist for additional work-up(Table 2). Given the patient’s clinical presentation, the significance of AHA with apparent coexisting LA was unclear. She was treated with a short course of high dose steroids followed by a taper. Over the next two months, her factor VIII activity increased into the normal range with no additional aPTT, LA or antiphospholipid antibody testing ordered. Several months after initial diagnosis, she was admitted to another hospital for a C1 burst fracture and found to have a mildly prolonged aPTT (no further evaluation requested). Approximately two days after admission, she was unresponsive and attempts at resuscitation were unsuccessful; no autopsy was performed.
Conclusions: The laboratory studies and clinical features support the diagnosis of AHA in this case. However, it is unclear from the studies ordered if the LA identified by SCT represents an in vitro effect due to the aPTT overlap between the two laboratory phenomenon (FVIII and LA) or a true in vivo condition. While this diagnostic dilemma has been previously described using other aPTT based LA assays, SCT has not been well evaluated. Further studies, such as immunologic-based antiphospholipid antibody studies as well as chromogenic FVIII, may be helpful for discrimination in similar situations.

Study On The Detection Of Remaining Platelets In Plasma Samples By Clot Waveform Analysis Of Prothrombin Time -The Basic Analysis Using Artificial Samples-
Takeshi Suzuki1, Yutaka Komiyama2, Tsukasa Suetake1, Yugo Shimada3, Kaneo Satoh3, Fuminori Kazama3, Sho Shinohara1, Hiroshi Kurono1, Katsue Suzuki-Inoue3
1Sysmex, Kobe-city, Japan, 2Hokuriku University, Kanazawa-city, Japan, 3University of Yamanashi, Chuo-city, Japan

Introduction: Validating the quality of plasma samples are important for coagulation tests. In Japan, centrifugal conditions (centrifugal force, time and temperature) are presented in expert consensus on standardization of sample preparation for clotting time assay by the working group in Japanese society of laboratory hematology (Ieko et al.,2016), making it possible to reduce the remaining platelets in plasma to less than 10x10e9 /L. But there are several laboratories that do not comply with this condition. Actually, because there are also several cases where measurement becomes impossible due to the large number of residual platelets, we investigated the relationship between the influence on the measured values when changing the number of platelets in the plasma and the coagulation waveform.
Methods: Citrated plasmas of healthy volunteers were obtained, and to mimic the plasma with remaining platelet, plasma sample was diluted with its platelet-rich plasma (0 to 30x10e9 /L). Then PT and others were measured with CS-5100, and the measured values and coagulation waveforms by the number of platelets were confirmed. For confirmation of coagulation waveform, coagulation waveform analysis (CWA) was performed from Time Course (Time vs Transmitted light (A / D value)) in each item.
Results: When the number of platelets reached 20x10e9 /L, PT showed the error message, whereas, other APTT and Fibrinogen measurements were possible. When the coagulation waveform was confirmed, a change in A / D value was confirmed in PT and Fibrinogen depending on the number of platelets after the end of coagulation. In order to develop the quantification method, the inclination of a certain section (1, 3, 5, 10 . 90 sec.) after that based on the place where CS-5100 determined to be the coagulation end point was evaluated, it was found that a fixed section, 5 sec, was judged most effective.
Conclusions: We could estimate remaining platelets in plasma sample by CWA using with A / D value after coagulation reaction. Since there are non-negligible effect by remaining platelets on APTT mixing test, lupus anticoagulant and FXIII, this method is considered to be useful as an index in conducting these measurements.

Performance Evaluation Of Siemens Innovance Free Ps Ag Reagent On The Sysmex Cs-5100 Analyzer
A. Anil Timur, Laila Vengal, Haiyan Lu, Heesun J. Rogers, Kandice Kottke-Marchant
Cleveland Clinic, Department of Laboratory Medicine, Cleveland, OH, United States

Introduction: Protein S is a vitamin K-dependent plasma glycoprotein which circulates as free or bound to C4b-binding protein. Free protein S functions as a cofactor of anticoagulant protein C. Decreased free protein S levels associate with increased risk of venous thrombosis. INNOVANCE® Free PS Ag launched by Siemens Healthcare Diagnostics Products is an immunoturbidimetric assay for measuring free protein S antigen in human citrated plasma. Herein, we evaluated INNOVANCE® Free PS Ag assay on the Sysmex® CS-5100 coagulation analyzer.
Methods: Precision studies were performed using three sample pools having low, medium, and high free protein S levels and two controls with normal and low protein S levels for 20 days with 2 runs per day and 2 replicates per run (20x2x2). Method comparison studies were carried out between the INNOVANCE® Free PS Ag assay on the Sysmex® CS-5100 and the Stago STA®-Liatest® Free Protein S assay on the STA-R Evolution® using fresh or frozen patient citrated plasma samples.
Results: INNOVANCE® Free PS Ag assay revealed a good within-run and between-run reproducibility. Data from a total of 116 patients were analyzed for the method comparison. Regression analysis showed a very strong correlation between the INNOVANCE® Free PS Ag and Stago STA®-Liatest® Free Protein S assays (R=0.965, p-value < 0.001).
Conclusions: The INNOVANCE® Free PS Ag assay is reproducible and reveals reliable results. It can be used in laboratory settings to measure free protein S antigen in human citrated plasma.

The Diagnostic Ability Of Monocyte Distribution Width (Mdw) Is Not Affected In Patients With Hematological Malignancy Or Neutropenia*
Iris Castro1, Diana Careaga1, Robert Magari1, Elliott Crouser2, Joseph Parrillo3, Octavia Palmer4, Liliana Tejidor1
1Beckman Coulter , Miami, FL, United States, 22The Ohio State University Medical Center, Columbus, OH, United States, 33Hackensack University Medical Center, Hackensack, NJ, United States, 44University of Pittsburgh Medical Center, Pittsburgh, PA, United States

Introduction: Sepsis is a systemic inflammatory response to infection and a rapidly progressing, life-threatening condition that can lead to organ failure and shock if not treated immediately. The likelihood of developing sepsis increases in patients with hematological malignancies, as systemic chemotherapy results in immune suppression. When neutropenic, the patient is vulnerable to invasive infection which can potentially cause overwhelming sepsis and death. Consequently, early diagnosis of sepsis in these high risk population of patients is very important for reducing mortality and alleviating healthcare costs. MDW is a novel parameter that has been shown to identify sepsis in the emergency department (ED) with good diagnostic accuracy (Crouser et. al, CHEST 2017; 152(3):518-526).
Methods: Patient Enrollment:The study, registered with (NCT03145428) was a blinded, prospective cohort study enrolling patients presenting to three academic centers.  The study enrolled adults, age 18 to 89 years, whose evaluation included a complete blood count (CBC) with differential upon presentation to the ED. White Cell Volume Determination:  All blood samples were analyzed on a UniCel® DxH 800 analyzer (Beckman Coulter, Inc.) with version 3.0 software within 2 hours of collection.  Statistical Approach: Diagnostic ability was evaluated in terms of the area under the curve (AUC) sensitivity, specificity, and analysis of area receiver operating characteristic (ROC) curves.
Results: AUCs and ROC curves of malignant and non-malignant groups were not statistically different. Indicating that diagnostic ability of MDW is the same in both groups. Furthermore, analyses in a subset of patients with neutropenia (ANC< 1500) demonstrated aperformance trend that is similar to that of all ED patients.
Conclusions: In a multi-center prospective clinical trial (2,158 adults), we showed that MDWcould be useful for early detection of adults having or developing sepsis in the emergency department (Crouser et. al. 2019 manuscript submitted for publication). In addition, we observed that the diagnostic ability of MDW does not seem to be affected in patients with hematological malignancy or neutropenia. Additional studies are needed to further establish performance in these critical populations. CE Marked. Pending clearance by the US FDA; not yet available for in vitro diagnostic use in the US.

Whole Blood Is Not Equivalent To Plasma Using The Triage D-Dimer Assay
Elona Turley1,2, Bobbi-Lynn Goudreau2, Susan Nahirniak1,2, Artur Szkotak1,2, Melanie Bodnar1,2
1University of Alberta, Edmonton, AB, Canada, 2Alberta Public Laboratories, Edmonton, AB, Canada

Introduction: Venous thromboembolism (VTE) imparts high morbidity and mortality; definitive radiographic diagnosis is costly and may not be readily accessible. In outpatients, a negative D-Dimer result is used to help exclude VTE without imaging, thus false negative results are of significant clinical concern. The Quidel Triage D-Dimer is a quantitative, point-of-care assay for whole blood (WB) or plasma EDTA samples, which is used in smaller urgent/emergent care centers throughout our region. During a recent instrument evaluation, we noted a high rate of discordantly negative Triage D-Dimer results performed on whole blood compared to the STA-Liatest D-Dimer (Stago) method.
Methods: Patients with clinician-ordered D-Dimer testing were collected in sodium citrate and tested by STA-Liatest on Stago Compact instruments (routine process/reference method). K2 EDTA WB and/or plasma was obtained from concurrently ordered CBC specimens, and tested using Triage D-Dimer kits on Triage Meter Pro instruments. Manufacturer reference materials defined a negative result as < 500ng/mL for both the STA-Liatest and Triage D-Dimer kit.
Results: Initial results comparing 23 WB Triage D-Dimers to the STA-Liatest revealed seven (30%) discordantly negative Triage results (Table 1). Subsequent testing of 20 different EDTA plasma Triage D-Dimers showed fewer discordant results (3/20 discordant results; one negatively discordant).  Fourteen specimens were tested by all three methods: 4/14 cases were negative by WB Triage but positive by both Plasma Triage and STA-Liatest.    
Conclusions: Compared to EDTA plasma, Triage D-Dimer results performed on whole blood showed inferior agreement for negative results with the STA-Liatest assay (threshold < 500ng/mL). The low number of Liatest-negative results in the EDTA-plasma Triage D-Dimer patient cohort limits our analysis. As the STA-Liatest was conducted first, impact of specimen age on Triage Meter results cannot be excluded (all testing was performed within the manufacturer-specified 24 hour stability limit). Our study did not assess diagnostic accuracy for VTE exclusion with clinical outcomes. Nevertheless, due to the clinical importance of negative D-Dimer results, we have modified local procedures to cease whole blood testing and accept only EDTA plasma for Triage D-Dimer testing.

Hemostasis System Study After Early Cancellation Of Fondaparinux In Uzbekistan Patients Hospitalized With Acute Coronary Syndrome Without Stable Rises Of The St Segment.
Azamatjon Umarov
Tashkent Medical Academy, Tashkent, Uzbekistan

Introduction: Anticoagulants are one of the components of the early treatment of acute coronary syndrome without persistent ST segment (ACSwpST) elevations on an ECG and after their premature cancellation, reactivation of thrombosis processes is described. The aim of the study is to assess the state of the hemostatic system with early cancellation of fondaparinux in this category of patients.
Methods: The prospective non-comparative study included 53 patients urgently hospitalized in the first 48 (median 2.3) hours after anginal attack. In all cases, the sum of points on the GRACE scale was ≤108, cardiac troponin T was not elevated (≤0.03 ng / ml) and there was no ST segment misalignment ≥0.1 mV. All patients in the hospital received acetylsalicylic acid and beta-blockers; Clopidogrel was used in 35 cases. After a single subcutaneous injection of fondaparinux, no anticoagulants were used. At the peak of the action of fondaparinux and during a period of significant weakening of its effect (median time of blood sampling 18.0 and 42.5 hours after injection), the concentration of the complexes thrombin-antithrombin (TAT), plasmin-antiplasmin (PAP) and D-dimer (DD) were determined in the blood plasma. At the same time, Cholter-monitoring ECG (CM) was monitored in 12 leads for at least 36 hours (median start after injection of fondaparinux 21.2 h, median duration 38.7 h). 
Results: After cessation of treatment with fondaparinux, the concentrations of the TAT, PAP and DD complexes increased: medians 3.1 and 3.3 ng / l (p = 0.002), 471 and 498 ng / l (p = 0.052), 359 and 486 ng / l (p = 0.002), respectively. Ischemic displacement of the ST segment in CM was observed in 13 patients (24.5%). The levels of TAT, PAP, DD and their changes were not associated with the presence of myocardial ischemia detected in CM. There were no cases of myocardial infarction and deaths during the hospitalization period; 3 patients had angina attacks.
Conclusions: In a small “pilot” study with early cancellation of fondaparinux in patients hospitalized with ACSwpST and having a low risk of adverse outcome, during the period of significant reduction of the effect of anticoagulant, there were laboratory signs of thrombosis reactivation that were not associated with the onset of myocardial ischemia.

Validation Of Activated Clotting Time Target Levels For Interventional Cardiac Catheter Ablation
Anne Vincent1, Shari Neal1, Benedict Glover1,2, Damian Redfearn1,2, Graeme Quest1,3, David Good1,3
1Kingston General Hospital, Kingston, ON, Canada, 2Division of Cardiology,Department of Medicine, Queen's University, Kingston, ON, Canada, 3Department of Pathology and Molecular Medicine, Queen's University, Kingston, ON, Canada

Introduction: The activated clotting time (ACT) is used to monitor anticoagulation with high dose unfractionated heparin (UFH) during cardiac procedures, as it is amenable to point-of-care testing (POCT).  Target ACT levels in cardiology guidelines are largely generic, not considering methodology or reagents.  Newer POCT devices (Abbott i-STAT) measure a different endpoint than other devices, placing the validity and reliability of published target ACT levels in question, possibly leading to bleeding or clotting risks for patients.  In this study, we attempted to correlate ACT levels with anti-Xa levels to determine an appropriate ACT target level for cardiac catheter ablation (recommended target ACT of >300s).
Methods: Twelve patients undergoing catheter ablation in the electrophysiology lab at Kingston Health Sciences Centre (KHSC) had blood drawn for anti-Xa testing, aiming for three time points with concurrent ACTs: baseline before heparin; 20 minutes after heparin bolus and before protamine reversal. Anti-Xa testing was performed on the Sysmex CS2100i instrument (Siemens, Marburg, Germany) using Siemens Innovance Anti-Xa reagent.  ACT testing was performed on the Abbott i-STAT POCT using ACT-Kaolin cartridge.  Comparison between anti-Xa and ACT values was done using linear regression analysis.
Results: Twenty eight anti-Xa results with corresponding ACT values were obtained, with strong correlation between the two methods (r2=0.915).  The mean baseline ACT prior to heparin administration (anti-Xa levels < 0.10 IU/mL) was 131 seconds.  After UFH administration, the mean ACT was 242 seconds with a mean anti-Xa of 2.73 IU/mL.  None of the patient samples reached the target of >300 seconds despite recommended doses of UFH.  Although literature varies on acceptable anti-Xa targets, a level of 2.0 IU/mL is considered safe for cardiac surgery.  Based on our data, this corresponds to an ACT target of 225 seconds, much lower than accepted value of >300 seconds.
Conclusions: Contrary to published literature, we found a strong correlation between our anti-Xa and ACT results, albeit in a small sample size.  Nonetheless, this data demonstrates the value of correlating the ACT to a gold standard measurement, due to the potential impact on heparin dosing and resultant bleeding or clotting complications for patients.  Further studies including those requiring higher and lower ACT targets are needed to validate this process.

Comparative Study Between Thromboelastography And Conventional Coagulation Tests In Ischemic Cerebral Vascular Disease
Bin Yan, Xin Li
The Affiliated Nanyang City Center Hospital of Zhengzhou University, Nanyang, China

Introduction: Coagulation disorder is the vital pathogenesis of ischemic cerebral vascular disease (ICVD). Thromboelastography (TEG) is a novel platform for evaluating coagulation status, and could monitor the whole process from coagulation to thrombosis then to thrombolysis. The aim was to investigate the consistency between TEG andconventional coagulation tests (CCTs) in ICVD.
Methods: Transient ischemic attack (n=54) and acute ischemic stroke patients (n=54) were involved as ICVD patients. The correlation and consistency analysis between TEG and CCTs were assessed by the Spearman correlation and Kappa value. Receiver operating characteristic (ROC) curve was selected to analyze the predictive value of TEG parameters for abnormal results of CCTs.
Results: (1) PLT was positively correlated with MA value (r=0.309, P=0.001) and G value (r=0.312, P=0.001); PT was positively correlated with K value (r=0.210, P=0.029); TT was positively correlated with R value (r=0.199, P=0.038) and K value(r=0.232,P=0.016); FIB was positively correlated with Angle value (r=0.242, P=0.012), MA value (r=0.353, P< 0.0001) and G value (r=0.352, P< 0.0001). TT was negatively correlated with Angle value (r=-0.232, P=0.016) and CI value (r=-0.228, P=0.017); FIB was negatively correlated with K value (r=-0.246, P=0.010); (2) PT and MA values, FIB and MA values were accordant in valuing the hypocoagulable state of ICVD patients, respectively; (3) PLT and Angle values, PLT and MA values were accordant in assessing hypercoagulable status of ICVD patients, respectively; FIB and Angle values, FIB and MA values were consistent in evaluating the hypercoagulable state of ICVD patients, respectively; (4) For detecting TT>20s, the area under the curve (AUC) of K value and Angle value were 0.648, 0.651, respectively; For detecting FIB>4g/L, The AUC of Angle value and MA value were 0.717 and 0.747, respectively; For detecting PLT>300, the AUC of the MA value was 0.808 (all P< 0.05).
Conclusions: There was weak correlation and consistency between TEG and CCTs parameter in ICVD patient. The TEG parameters have good predictive value in evaluating the abnormal results of CCTs. Combing the advantages of two methods will better reflect the coagulation status of ICVD patients.

Comparative Study Of Platelet Function As A Bio Marker In Childhood Burkitt Lymphoma And Plasmodium Falciparum Malaria
Renate Asare1,2, Clement Opoku-Okrah1,2, Kwabena Owusu-Danquah2, Otchere Addai-Mensah2, Daniel Gyamfi2, Nana Opare-Sem1, Michael Frimpong3, Edward Yaw Afriyie2, Francis Agyei-Amponsah2, Richard Vikpebah Dunneh2
1Komfo Anokye Teaching Hospital, Kumasi, Ghana, 2Kwame Nkrumah university of science and Technology, Kumasi, Ghana, 3Kumasi Center for Collaborative Research in Tropical Medicine, Kumasi, Ghana

Introduction: Children living in endemic areas of Plasmodium (P) falciparum malaria are highly susceptible to Burkitt lymphoma (BL), but the underlying mechanisms remain unclear. The association between P. falciparum malaria and Burkitt lymphoma still remains debatable. Unfortunately, no studies have assessed and compare platelet function in children with BL and P. falciparum malaria. Thus, this study sought to investigate the significance of the difference in platelet function in BL and P.falciparum malaria to establish a possible association  between the two disease condition.The objectives included; to compare platelet function in Burkitt lymphoma and P. falciparum malaria, to determine the expressions of platelet membrane glycoproteins and parameters in  Burkitt lymphoma or P. falciparum malaria and to establish the common bleeding episodesusing the bleeding score assessment.  
Methods: An unmatched case control study design was used in selection of 50 (16 BL, 19 Malaria and 15 Control groups) study participants. Platelet surface membrane glycoproteins (GPIIb/IIIa [PAC-1], P-selectin [CD62p] and GPIV [CD36]) expression and platelet indices (Platelet count (PLT), Plateletcrit (PCT), Mean Platelet Volume (MPV), Platelet Distribution Width (PDW) and Platelet- Large Cell Ratio (P-LCR) were determined in children with P.falciparum, Burkitt lymphoma and controls by using flow cytometry and automated blood cell analysis techniques. In addition, bleeding score assessment tool was used in the investigation of bleeding episodes.
Results: The study observed a significant (p< 0.004) low median PLT and PCT in children with P. falciparum malaria than in BL when compared with the controls. However, the other platelet indices were significantly (p< 0.05) lower in patients with BL than P. falciparum malaria. Platelet activation was common in both BL and P.falciparum malaria cases. In addition, platelets showed normal function inP.falciparum malaria while platelet functionality in children with BL was abnormal after the addition of the agonist (ADP). CD62p was probably associated with an increased (OR 4.12, RR 1.78, 95% CI 0.96-17.64; p=0.09) risk of Burkitt lymphoma in children with P. falciparum malaria. Oral bleeding was the commonest bleeding episodes found in the malaria and Burkitt lymphoma patients and was associated with decreased risk of Burkitt lymphoma and P. falciparum malaria (odd ratio [OR] 0.76, 95%CI 0.11-5.23; p=0.58).
Conclusions: This study has indicated the significance of platelet membrane glycoproteins and platelet parameters in children with Burkitt lymphoma and P. falciparum malaria. It has been observed that, the pathogenesis of BL in children with P. falciparum malaria might probably be linked to platelet membrane glycoproteins.  

Platelet Reactivity Testing Over Time In Vascular Patients.
Aarent Brand1,2, Suzanne korporaal2, Rolf Urbanus2, Gerard Pasterkamp2, Gert Jan de Borst1
1UMC Utrecht Vascular Surgery, Utrecht, Netherlands, 2UMC Utrecht Laboratory of Clinical Chemistry and Haematolgy, Utrecht, Netherlands

Introduction: Many patients who have atherosclerosis still experience thromboembolic complications and cardiovascular events (CVE) despite antiplatelet medicines. High-on-clopidogrel platelet reactivity (HCPR) occurs in 40.4% of cases and bears an increased risk of CVE (RR 2.80, 95%CI: 2.40-3.27). HCPR can be detected using platelet reactivity tests. Platelet reactivity testing to guide therapy or aid in prognostic assessment has not been proven effective, nor ineffective yet. A wide variation in study design, heterogeneous populations and the use of a variety of platelet reactivity tests all contribute to this problem. Previous trials have assessed platelet reactivity at varying time points. Also, intra-individual platelet response to APT over time, the type of test that is best suited to measure platelet reactivity and the appropriate time point for testing are still unknown.
Methods: Between January 2017 – January 2019, we conducted a prospective, single-centre, observational study aiming to uncover intra-individual fluctuation or stability in platelet reactivity by investigating the variability of platelet reactivity measurements at three perioperative time-points in 50 patients undergoing carotid endarterectomy. The tests used were the VerifyNow, VASP and PACT assay (a flow cytometry-based platelet reactivity assay measuring platelet reactivity after stimulation with multiple agonists). In addition, we assessed the impact of cytochrome P450 2C19 (CYP2C19) gene polymorphisms on the test results.
Results: We have concluded our inclusion period and are conducting statistical analyses. 
Conclusions: The results of the PLOT trial will provide a basis for uniformity in applying platelet reactivity testing, which will propel the search for the ideal platelet reactivity test to guide tailored APT forward. We look forward to the opportunity of presenting our findings at the ISLH 2019 conference.

Eosin-5-Maleimide (Ema)- Binding Test Is Not Only For Scanning Of Herediter Spherocy Tosis (Hs) But Also For Herediter Stomatocytoses (Hst)
mesude Falay1, Guchan Alanoglu2, Ebru Keskin2, M.Seda Aydın1, İmdat Dilek3, Gulsum Ozet1
1numune education /trainning hospital , Ankara, Turkey, 2Suleyman Demirel University, Isparta, Turkey, 3Ataturk Eğitim ve Araştırma Hastanesi, Ankara, Turkey

Introduction: Eritrocyte membrane deficiencies are either caused by structural protein loss in membrane proteins (HS, herediter eliptocytosis etc) or definciencies in the membrane transport (Hst).
Methods: We, as Ankara Numune Education and Research Hospital Flow Cytometry Lab, evaluated retrospectively all clinical and lab data such as blood count indexes and reticulosyte indexes  for a total of 103 cases, which were  EMA binding tested for eritrocyte membrane deficiency scanning,
Results:  We performed the EMA binding test as described by King et al with few very minor modification. We then evaluated the results according to reference intervals obtained from healthy persons based on their age groups as defined by CLSI. We found 36 cases coherent with HS ( with EMA binding ratio: 0.08-0.82) which was further supported by the patient’s clinical and lab findings. 5 cases had EMA binding test ratio over 1.2 (1.22-1.28) and all of them, upon reviewing their clinical and lab findings, were found out to have high MCV values (98 fL up to). We further evaluated these 5 cases according to megaloblastic anemia etiology and found that 1 case had lack of  pruvate kinase , 1 case had deficiency of Vit B12  and 3 cases had ongoing haemolytic anemia which were previously recieved splenectomy, and 2 cases out of this 3 splenectomy cases were having a history of reccürent tromboembolism.  We were able to perform Family screening only for 2 cases, and found out that EMA binding test ratio has over 1.2 in the father of the first case and in the mother of the second case. 
Conclusions: Our study is the first study which evaulates and express  that the EMA binding test can be utilized as a screening test for rarely observed HSt as well as it is used for HS screening. HS and HSt may have some similar and overlapping clinical findings. While splenectomy is generally used in the treatment of HS, the same has no benefit in treatment of HSt. Therefore, EMA binding test can also be used to distinguish the diagnosis of this two disease.

Early T-Cell Precursor Acute Lymphoblastic Leukemia/Lymphoma (Etp-All/Lbl) In Chinese Adolescent And Adult Patients: The Differential Clinical Characteristics, Risk And Prognosis Factors
Hongyan Liao, Zhuoyi Sun, Yongmei Jin, Nenggang Jiang
West China Hospital, Sichuan University, Chengdu, China

Introduction: Recently, an aggressive and infiltrative subtype of T-ALL derived from thymic cells at the early T-cell precursor (ETP) differentiation stage has been recognized as ETP-ALL/LBL. Sporadic studies have reported that the subtype of ETP-ALL is associated with deleterious outcome. Therefore, we sought to determine whether this subtype has different characteristics of great clinical significance in adolescent and adult patients of Asian population. 
Methods: We retrospectively analyzed 126 cases of T-ALL/LBL patients to identify the clinical characteristics and prognosis factors of adult patients with ETP-ALL/LBL, and to evaluate the clinical significance of the risk stratification of ETP-ALL/LBL subtypes representing the Asian population.
Results: In 126 patients involved in this study, the male to female ratio is 2.5:1, with a median age of 25 years old. T-ALL patients exhibited a higher ratio of complete remission after first induction chemotherapy (CR1) than T-LBL patients (64% vs 31%,P = 0.03). The patient group with WBC count > 50×109/L had a higher CR1 than those with WBC count ≤ 50×109/L (78% vs 51%,P= 0.0095). Unexpectedly, ETP-ALL/LBL accounted for 47.6% of all T-ALL/LBL patients. ETP group had a lower CR1 than non-ETP group (37% vs 85%,P< 0.0001). Furthermore, ETP group had a marginally shorter overall survival duration than non-ETP group (P = 0.0733). Patients with CD1a+, CD8+ or CD4+ had a higher CR1 than their negative counterparts (P = 0.0016, P = 0.0003 and P = 0.001, respectively), while myeloid lineage antigen CD33- and CD56-positive group showed a lower CR1 than their negative counterparts (P = 0.035, P = 0.035, respectively).
Conclusions: Immunophenotyping and the detection of specific antigens of the leukemic cells based on flow cytometry are of great clinical significance in the diagnosis, subtyping and risk and prognosis evaluation of T-ALL/LBL. In Chinese adolescent and adult patients with T-ALL, the percentage of ETP-ALL/LBL is 47.6%, which is unexpectedly higher than the reported 7.4% in adult T-ALL/LBL from previous studies based on different populations. Meanwhile, ETP-ALL/LBL is a high risk subtype that needs more precise diagnosis and updated treatment strategies to achieve a favorable prognosis.

Interest Of Horiba Medical Yumizen H1500/H2500 Technology In Patients With Wbc-Diff Abnormalities Due To Malignant Blood Disease Or Chemotherapy Treatment.
Pascal CORREIA1, Julie BLANCHI1, Franck SEGUY2, Francoise DURRIEU1

Introduction: The HORIBA Medical Yumizen® H1500/H2500 is a new automated hematology analyzer, generating complete blood counts with WBC (White Blood Cell) differential. In this study we evaluated the flagging performance in the WBC differential of the Yumizen®H1500/H2500 in comparison with our routine analyzer HORIBA Medical Pentra Nexus in patients with pathology or treatment affecting hematopoiesis (under chemotherapy affecting hematology parameters or with oncohematologic disorder).
Methods: Samples with WBC-Diff Flag on the Pentra Nexus were systematically rerun within 4 hours on the Yumizen®H1500/H2500. Standard rules of flagging were applied on both analyzers (ISLH and GFHC* rules). Differential counts were performed by optical microscopy as reference methods, FCM was performed to confirm differential and identify abnormal cell population (abnormal lymphocytes, blast, immature granulocytes. *French Group for Cell Hematology
Results: The WBC-diff identifies  groups of cells (lymphocytes, neutrophils, monocytes, eosinophils) by definition of a lower discriminator and upper discriminator. Flags are generated when the distinction between groups of determined cells is difficult or impossible.  On the Pentra Nexus discriminators are previously set on the analysis software, as on the Yumizen®H1500/H2500 discriminators are variable and determined by algorithm for each sample. On our group of patients, with respect to the different flags, the Pentra Nexus analyzer flagged more frequently than the Yumizen®H1500/H2500 (Table 1). Table 1 : frequency of WBC-Diff flag on the 2 analyzers
  Pentra Nexus Yumizen H1500/H2500
Atypical Lymphocytes 13% 5%
Lympho/neutrophils alone 22% 15%
Lympho/neutrophils with any other flag 20% 17%
Abnormal Neutrophils 36% 36%
Immature granulocytes 8% 8%
Blast 10% 2,5%
Abnormal monocytes 8% 8%
False positive (i.e. flag triggering slide review with no change in WBC-diff) was significantly decreased  from 70.5% to 38.6% with the Yumizen®H1500/H2500 as compared to the Pentra Nexus and Positive predictive value for slide reviewing (i.e. flag triggering slide review with change in WBC-diff and/or abnormal cell detection) was improved from 29.5% to 61.4%. With the Pentra Nexus as the reference, no false negative was observed with the Yumizen®H1500/H2500.
Conclusions: Our study focused on WBC-flag triggering slide review by microscopy. Overall, the Yumizen®H1500/H2500 analyzer demonstrated improvement of WBC-diff analysis as compared to our routine analyzer Pentra Nexus and a significant decrease for unnecessary morphology reviewing by microscopy.

Evaluation Of Automated Differential Count For Immature Granulocytes On Sysmex Xn-9000 Analyser
Yu Ting Ng, Frederick M.llagan, Erica Chiang, Siti Thuraiya A.Latiff, Ping Ying Heng, Vishnu Prasad, Moh Sim Wong
Khoo Teck Puat Hospital, Singapore, Singapore

Introduction: Khoo Teck Puat Hospital (KTPH) is a 761-bed general and acute-care hospital, serving the northern sector of Singapore. The Department of Laboratory Medicine receives an average of 6000 full blood count (FBC) requests monthly. Approximately 20% of FBC requests with detected immature granulocytes (IG) require manual slide reviews. We evaluated the automated IG count function of XN-9000 analyser (Sysmex Asia Pacific Pte Ltd) which utilises fluorescence flow cytometry in white blood cells (WBC) differentiation. This study aims to evaluate the performance of XN-9000 in measuring IG absolute count and percentage estimation.
Methods: Trueness study was performed using 100 anonymised patient samples. Manual differential count was performed by two medical technologists, and the mean of both manual IG counts were evaluated against the automated differential count generated by XN-9000. Reproducibility and repeatability were assessed using XN-Check QCs consecutively for a month and patient samples (n=15) with low and high IG count respectively. Carryover studies were also performed using anonymised patient samples.
Results: The slope and the intercept from the trueness study were 1.00 and 1.59 respectively with R value of 0.95. For repeatability, the CVs for IG percentage, were 8.74%, 8.90% and 9.19% for XN-9000-1, 2 and 3 analysers respectively while the CVs for IG absolute, were 8.82%, 9.03% and 8.23% for XN-9000-1, 2 and 3 analysers respectively. The Sysmex-recommended acceptable limit for repeatability is less than 25%. For reproducibility, the CVs for IG percentage, were 3.37%, 2.87% and 3.23% for median QCs of 10.4, 11.0 and 11.8 respectively while the CVs for IG absolute, were 3.67%, 3.17% and 3.40% for median QCs of 0.32, 0.80 and 1.93 respectively. There is no significant carryover interference between samples with low and high IG percentage. Discordance between automated and manual IG count was observed when IG exceeded 5% of total WBC.
Conclusions: The study demonstrated good correlation between automated differential count by XN-9000 analyser and manual differential count. Furthermore, the use of automated count reduces the need for manual cell count by a medical technologist. It is estimated that by reporting automated IG count, 200 man-hours can be saved each month. The proposed cut off to reflex to additional manual IG count is set at 5%.

Bone Marrow Aspirate Examination Using The Automated Blood Cell Analyzer Sysmex Xn-2000
Koji Tsuchiya1,2, Konobu Kimura3,4, Takamasa Yamamoto2, Kaori Nagasaka2, Hiroyuki Takemura2, Kaori Saito3, Shigeki Misawa2, Takashi Horii2, Kinya Uchihashi4, Yoko Tabe3, Akimichi Ohsaka1,2,3
1 Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University Graduate School of Medicine, Tokyo, Japan, 2Department of Clinical Laboratory, Juntendo University Hospital, Tokyo, Japan, 3Department of Next Generation Hematology Laboratory Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan, 4Sysmex Corporation, Kyoto, Japan

Introduction: The bone marrow (BM) samples are conventionally tested by the microscopic examination which is labor intensive and time consuming. The Sysmex XN-2000® is the latest model of automated blood cell analyzer for peripheral blood and body fluid samples. The aim of this study was to apply the Sysmex XN-2000® to assess the BM samples, including the number of total nucleated cells (TNC), erythroblasts (NRBC) and white blood cells (WBC) and M/E ratio.
Methods: The study was conducted at Juntendo University Hospital (Tokyo, Japan), using a total of 41 BM aspirations collected in K2-EDTA. The number of TNC, NRBC and WBC was measured by the XN-2000® employing WNR and WDF channels. WBC differential included NEUT: band and segmented neutrophils, LYMP: lymphocytes, MONO: monocytes, EO: eosinophils, BASO: basophils, IG: immature granulocytes. The M/E ratio was calculated from the number of myeloid cells (NEUT+EO+BASO+IG) and NRBC. As a reference, the manual microscopic examination by board-certified laboratory technologist was performed. Lipid content was calculated by the gating algorithm using the XN-2000® and compared with BM cellular density obtained by the BM biopsy.
Results: The correlation between TNC count determined by XN-2000® and the microscopic method was y=0.92x+12.6(r=0.993). The classification results were as follows: NRBC: y=0.79x+0.12(r=0.966), NEUT: y=0.66x+11.3(r=0.840), LYMP: y=0.93x+6.76(r=0.919), MONO: y=0.04x+7.75(r=0.013), EO: y=1.18x+0.50(r=0.911), BASO: y=0.77x+1.53(r=0.383), IG: y=0.95x+3.36(r=0.690), and M/E: y=0.94x+0.41(r=0.776).The sensitivity and specificity of the lipid content detection by XN-2000® for BM cellular density were 100% and 88% for hypoplasia and 89% and 86% for hyperplasia, respectively.
Conclusions: The fully automated XN-2000® quickly provided quantitative BM nuclear cell counts and M/E ratio along with the information of BM cellular density, which are comparable to the microscopic method. These results suggest that the measurement of BM aspirates using the automated XN-2000® might be useful for the clinical screening before performance of microscopic examination.

Clinical Characteristics Of Patients With Positive Paroxysmal Nocturnal Hemoglobinuria (Pnh) Testing
Weiwei Wang1,2, Haibo Li2, Ying Wang2, Guang Fan2, Lisong Shen1
1 Clinical laboratory, Xinhua hospital, Shanghai Jiaotong University of Medicine School, Shanghai, China, 2Department of Pathology, Oregon Health and Science University, Portland, OR, United States

Introduction: PNH testing is suitable for a wide range of diseases. However, it’s not appropriate to screening every patient. So, we reviewed the positive PNH patients’ clinical history to figure out what’re common disorders and associated laboratory results.
Methods: High sensitivity PNH flow cytometry (0.01% limit of detection) was performed with FLAER-FITC, CD64-PE, CD14-ECD, CD15-PC5, CD24-AA7500, CD45-KO for neutrophils & monocytes; CD59-PE and CD235a-AA750 for RBCs.   Retrospective analysis was done in the positive PNH cases from 2013-2019 at OHSU. Total 173 cases for 57 patients; including 27 females and 30 males, 52 adults and 5 pediatrics, with a mean age of 45 (range 9-78). We also reviewed results of serums LDH, bone marrow biopsy and molecular/cytogenetics of these patients.
Results: Among 57 patients, there are 30 aplastic anemia (AA) patients (53.63%), 7 AA patients progressing to PNH (AA&PNH, 12.28%), 5 myelodysplastic syndromes (MDS, 8.77%), 12 PNH patients (21.05%), 1 pancytopenia, 1 autoimmune disease, 1 thrombosis&diabetes. The diagnosis of AA and MDS were confirmed by bone marrow biopsy and molecular/cytogentics.   The mean of PNH clone for AA patients are RBC-type-II 0.06% (range 0-0.5), RBC-type-III 0.38% (0-9.17), neutrophils 3.01% (0.01-15.43), and monocytes 2.80% (0.01-14.50). For AA&PNH group, mean of PNH clone are RBC-type-II 1.601% (0.01-33.22), RBC-type-III 9.21% (0.41-50.15), neutrophils 28.06 (0.92-98.94), and monocytes 27.03% (1.5-99.07). In MDS patients, they are RBC-type=II 0.040% (0.01-0.10), RBC-type-III 0.22% (0-0.82), neutrophils 4.79% (0.04-17.09), monocytes 3.83% (0-10.80). PNH patients have the highest PNH clone, RBC-type-II 3.70% (0.01-21.99), RBC-type-III 32.63% (0.75-100), neutrophils 84.22% (16.36-99.05), monocytes 86.08% (4.40-99.89).   Significantly higher levels of all PNH clones were observed in PNH and AA & PNH groups, compared to AA (all P < 0.001) and MDS (all P< 0.05).   LDH was higher in PNH and AA&PNH than AA and MDS groups (P< 0.001). PNH clone sizes in RBC-type-III, neutrophils and monocytes have positive correlation with LDH (all P< 0.0001, R= 0.4447, 0.5469, 0.5711, respectively).
Conclusions: Positive PNH was most frequently seen in AA patients. AA has lower PNH clones and LDH than those of PNH patients or AA &PNH patients. For all patients, PNH population showed positive correlation with LDH.

Re-Expression Of Shp-1 And Prg2 By Azacytidine Could Enhances Sensitivity Response To Tyrosine Kinase Inhibitors In Myeloid Leukemic Cell Lines
Hamid Al-Jamal1, Wan Rohani Wan Taib 1, Farid Johan2
1Universiti Sultan Zainal Abidin, Kuala Terengganu, Malaysia, 2University Sains Malaysia, Kuban Kerian, Kelantan, Malaysia

Introduction: Epigenetic silencing of tumor suppressor genes (TSG) has been reported to play important roles in leukemogenesis. Tyrosine kinase inhibitors (TKI) such as imatinib specifically targets BCR/ABL protein and induces hematological remission in patients with chronic myeloid leukemia (CML). However, majority of CML patients treated with imatinib develop resistance under prolonged therapy. CEP-701 and PKC-412 are also TKI that potentially inhibit FLT3-ITD in patients with acute myeloid leukemia (AML) in clinical trial. However, such response is only temporarily. 
Methods: Three resistant cell lines were developed by overexposure of K562 and MV4-11 cells TKI. The treatment was started with low doses with stepwise increments for 12 months. Theresistance was confirmed by cytotoxicity and apoptosis assays. The resistant cells were treated with 5-Azacytidine (5-Aza) and cytotoxicity of TKI and apoptosis were determined by MTS and Annexin V-FITC, respectively. Gene expression of SHP-1, SOCS-1, SOCS-3, STAT5a, JAK2, PRG2 and MAP2K6 was validated by RQ-PCR.  
Results: DNA methylation status of those genes showed re-expression after treatment with 5-AZA (SHP-1 and PRG2)was determined using pyrosequencing analysis. The phosphorylation status of STAT1, STAT2 and STAT3 were examined by Western blot. All resistant cells demonstrated higher vitality and significant lower apoptosis (p< 0.001) in response to TKI compared to their parental cells. DNA methyltransferases (DNMTs) showed a significant down regulation (p< 0.001) in 5-Aza treated cells correlated with re-expression of SHP-1 and PRG2 genes. Interestingly, PRG2 and SHP-1 genes were significantly up-regulated in all 5-Aza treated cells. The re-expression of SHP-1 and PRG2 was associated with inhibition of activated STAT3 and high sensitivity response to TKI.
Conclusions: This study addressed PRG2  and SHP-1 function as TSG in AML and CML cells and enhanced expression of both genesis associated with higher sensitivity response to TKI. The findings support the hypothesis that, transcriptional silencing TSG  genes could lose their suppressor functions to inhibit the activated STAT3 resulting resistance to TKI and re-expression of SHP-1 and PRG2 by 5-Aza could enhance the effects of TKI in the treatment of AML and CML patients. 

Novel Copy Number Variants In Cytogenetically Normal Acute Myeloid Leukaemia (Aml-Cn) With Poor Survival
Angeli Ambayya, Jameela Sathar
Haematology Department, Hospital Ampang, Selangor, Malaysia

Introduction: Genomic characterization is crucial in unravelling prognostically heterogeneous cytogenetically normal acute myeloid leukaemia (AML-CN). In this study, we elucidated genomic aberrations by systematic profiling of AML-CN patients that could predict their survival status by utilizing customized CGH+SNP microarray.
Methods: Paired tumour and germline DNA were studied using customized CGH+SNP microarray in 53 AML cases (including 20 cases were AML-CN with the absence of FLT3-ITD and NPM1 mutations). Somatic aberrations were ascertained by comparing the remission DNA of each patient and by referring to personalized track of copy number variants (CNVs) seen Malaysians established by our laboratory. We then delineated specific recurrent aberrations seen in AML-CN versus other subtypes of AML. Following that, we compared these recurrent aberrations with their outcome after intensive chemotherapy and their overall survival (OS) between 2013 and 2018.
Results: A total of 979 somatic aberrations were discovered in this study of which 319 were detected in AML-CN patients(n < 1Mb = 278, n 1-5 Mb = 29, n 5-10 Mb = 5 and n >10 Mb = 7).  We identified 255 regions with gains, 63 regions with losses and 1 region of CN-LOH in AML-CN patients.  Further analysis enabled exclusion of 105 CNVs that are commonly seen in Malaysian based on the personalized track integrated in the analysis. Subsequent analysis revealed five novel recurrent regions of gains involving chromosomes 17p13.1 (7/20 cases), 4p16.3 (6/20 cases), 7q11.23 (6/20 cases), 15q11.1 - q11.2 (5/20 cases), and 2p23.3 (5/20 cases) exclusively seen only in AML-CN patients with poor OS(P < 0.01 by Fisher’s exact test).
Conclusions: With the use of our customized DNA microarray with paired germline DNA analysis and personalized CNVs seen in Malaysian track, we have successfully elucidated exclusive cryptic and novel somatic genomic aberrations that could be related to the pathogenesis of AML-CN patients in Malaysia. Although the number of cases in this study is small, these aberrations could serve as an independent predictor of OS in AML-CN as these regions were exclusive and recurrent.

Observations From The First 1,000 Suspected Ahus Patients Sequenced By A Targeted Genetic (Ngs) Panel
James Anderson-Furgeson, Gayatri Raghuraman, Sujatha Venkataramani, James Kain, Jooeun Lim, Connie Ng, Brad Lewis, Michael Ero
Machaon Diagnostics, Oakland, CA, United States

Introduction: Due to the severity of aHUS and the expense of treatment, rapid diagnosis of the disease is desirable. We compared the frequencies of mutations found in various genes in our panel in positive samples to those found by other groups sequencing aHUS patients (Bu et al. 2018 and Norris et al. 2010). Results for our panel reflect a different patient population (test ordered STAT for patients with suspected aHUS) than the population reported on by other groups (patients with a confirmed aHUS diagnosis).
Methods: We reviewed de-identified aggregate results for a subset of patients tested using this panel (416 patients). We tabulated the frequency of clear-cut causative genetic variants in each of the genes among the patients tested positive for aHUS. Furthermore, we report the frequency of positive, equivocal and negative results, and frequency of novel variants in the genes sequenced.
Results: The most common variant among the positives tabulated is the large deletion in CFHR1, at 23%. 23% of positives have a variant in MCP, 15% in CFH, 10% in THBD, 9% in CFI, 6% in C3, 6% in PLG, 5% in CFB, 3% in CFHR5, and 1% in DGKE. 20.5% of test results were positive, 28.8% were negative, 49.2% were equivocal, and 1.4% were uncategorized. 111 novel variants were found. 32.4% were in CFH, 1.8% in CFHR1, 5.4% in CFHR3, 4.5% in CFHR4, 6.3% in CFHR5, 20.7% in C3, 9% in PLG, 6.3% in DGKE, 8.1% in CFB, and 5.4% in THBD.
Conclusions: Frequencies of causative genetic variants in several genes, notably MCP, CFH, C3, and THBD differed notably from those reported by Bu et al. (2018). Frequencies of causative genetic mutations in MCP, CFH, CFB, and C3 differed from those reported by Norris et al. (2010). These differences possibly reflect different patient populations tested by the two panels. A plurality of novel variants were found in CFH, with the second highest number of novel variants found in C3.

Genetic Mutations Outside Bcr-Abl1 Determining Tyrosine Kinase Inhibitor Resistance In Chronic Myeloid Leukemia
.Salah Aref1, Nada Abdalla1, Martin Muller2, Solofa El Sharaway1, Sherin Abdel Aziz1, Sussane Sauselle2
1Mansoura University, Mansoura, Egypt, 2Heidelberg University Germany, Mannheim, Germany

Introduction: The aim of the current study was to identify novel genetic mutations outside BCR-ABL1 that may have a role in the development of resistance to tyrosine kinase inhibitor therapy    in CML patients
Methods: This study included 59 CML patients resistant to TKI. They divided into 2 cohorts; the first cohort included 16 patients in secondary resistance; this group served as screening cohort for detection of somatic mutations that may appear at time of disease relapse by WES of mutated paired samples at 2 points (diagnosis and relapse). The second cohort used as a validation cohort on 43 CML resistant patients (primary or secondary resistance) by deep sequencing with a panel of 18 genes.
Results: The mutations detected in both cohorts included 16 genes in the total number of 30 patients. Mutations in ASXL and RUNX1 genes were the most frequent to be detected; each 13.5% of all mutations followed by ABL1 mutation(11,8%); DNMT3A, IDH1 and WT1 mutations(6,7% each); TP53(5%); BCOR and TET2(3.3%) ; CBL; EZH2;IKFZI; IKFZ3,NRAS,KRAS and SETBP each represented(1.6%) of all detected mutations in both cohorts. The detected mutations either single or combined with other mutations in the same patients. The overall survival was significantly shorter and the blastic crisis was significantly higher in CML patients developed somatic mutations as compared to those with no mutations (P =0.0016; P=0.0001). The Blastic crisis was significantly higher in The mutations development was statistically correlated with higher WBCs count; lower Haemoglobin level; higher basophilic and eosinophilic count; intermediate or high Sokal and Hasford score failure to achieve complete cytogenetic response at 12 months or MMR at 24 months. Inconclusion: BCR-ABL1 independent somatic mutations play a role in the development of TKI resistance and disease progression and had deleterious impact on the CML patient’s outcome. The most frequently detected mutations are ASXL1 and RUNX1 gene mutations. 
Conclusions: BCR-ABL1 independent somatic mutations play a role in the development of TKI resistance and disease progression and had deleterious impact on the CML patient’s outcome. The most frequently detected mutations are ASXL1 and RUNX1 gene mutations.  

Rapid Verification Of Variant D Phenotype By Genotyping In A Regional Laboratory
Philip Berardi1,2, Melanie Tokessy1, Marc Pilon1, Liane Cousineau3, Doris Neurath1, Antonio Giulivi1
1The Ottawa Hospital, Ottawa, ON, Canada, 2Canadian Blood Services, Ottawa, ON, Canada, 3Montfort Hospital, Ottawa, ON, Canada

Introduction: Red cell genotyping can complement phenotyping to minimize the limitations of using serology alone. This is particularly relevant to expectant mothers who harbour a variant RHD allele that confers a different alloimmunization risk should their fetus harbour a conventional RHD allele. In this setting, genotyping offers a high-throughput method to resolve these challenging cases and to identify expectant mothers who can safely forego unnecessary Rh(D) immune globulin (RhIG) prophylaxis. The aim of this project is to operationalize a regional prenatal testing algorithm that would allow for rapid verification of variant D phenotype to help guide clinical decision-making around RhIG prophylaxis.
Methods: The transfusion medicine laboratory at our center is a regional reference site for 16 hospitals in eastern Ontario. For 5 years we have been using red cell genotyping to help resolve complex serological results, which include cases with variable D reactivity using traditional phenotyping methods. Our laboratory also offers high risk prenatal testing for expectant mothers within our region. This is typically done following their first prenatal visit between 8-12 weeks gestation. At this time a blood group and antibody screen is requested by the health care professional most responsible for their care through the pregnancy.
Results: To date we have genotyped 51 samples from expectant mothers and females of child bearing potential (< 45 years old) with variable D typing. Weak D type 1, 2 or 3 was identified in 33 (65%) of cases. The majority of samples were analyzed in 2018 and of 33 tests performed, 19 (57.6%) were identified as weak D type 1, 2 or 3. For these females, if pregnant, we would not recommend the use of RhIG at 28 weeks and post-partum, saving 38 doses. 
Conclusions: Our goal is to operationalize a rapid-turnaround method of resolving variable RhD phenotyping results using an approved genotyping platform within a regional TM laboratory. This will allow many expectant mothers with weak D type 1, 2 or 3 to safely forego RhIG prophylaxis. This will result in more efficient and appropriate utilization of RhIG, may help decrease anxiety among expectant mothers who have a low risk of alloimmunization and reduce the unnecessary use of RhD negative units.

Expression Of B7-H6 In Chronic Myeloid Leukemia And Its Clinical Significance
Yanglin Cao1, Li Huo1, Jianfeng Yang2, Zhen Weng2, Jiannong Cen1, Yang He1
1Jiangsu Institute of Hematology, the First Affiliated Hospital of Soochow University, Suzhou, China, 2Cyrus Tang Hematology Center, Collaborative Innovation Center of Hematology,Soochow University, Suzhou, China

Introduction: Chronic myeloid leukemia (CML) is a malignant myeloproliferative disease,characterized by clonal expansion and accumulation of bone marrow (BM) cells. The prognosis of CML is still extremely poor due to the rapid malignant property and adverse reactions associated with long-term tyrosine kinase inhibitor (TKI) treatment. In this work, we aimed to examine the expression level of B7-H6 in peripheral blood mononuclear cells (PBMCs) and bone marrow mononuclear cells (BMMCs) of CML patients and to explore its clinical significance in order to seek effective disease biomarker to monitor disease progression and prognosis.
Methods: 105 PBMCs and 97 BMMCs samples of  CML patients were collected forB7-H6 mRNA expression analyses by quantitative real time polymerase chain reaction (RT-PCR).
Results: The expression of B7-H6 mRNA was successfully detected in all samples. According to the clinical characteristics of CML patients, no difference was found on B7-H6 level of PBMCs. However, B7-H6 level in BM samples with BCR-ABL1/ABL > 0.1% (10 copies of BCR-ABL1/10000 copies of ABL) decreased significantly compared to ≤0.1% (P = 0.0001), and negative correlation was found between the expression level of B7-H6 in BMMCs and the number of BCR-ABL1/ABL transcripts (r = -0.27, P = 0.0064). Besides, a significant difference in PFS was observed (c2=15.19, P< 0.0001) and patients with high B7-H6 level had longer PFS than those with low level (Hazard Ratio: 0.07 vs  14.31, 95% CI: 0.02-0.27 vs 3.75-54.55).
Conclusions: Our study demonstrated for the first time the clinical significance of B7-H6 expression in CML. We found that the decreased level of B7-H6 is an unfavorable indicator and is inversely related to the copy number of the fusion gene BCR-ABL1 transcript, Compare with detecting BCR-ABL transcripts, the detection of B7-H6 mRNA in BMMCs by RT-PCR was not restricted by low-copy number transcripts or control gene and there was little difference in each laboratory, suggesting that the detection of B7-H6 expression level in BMMCs maybe further employed to assist CML progression monitoring and prognosis prediction in clinical practice. 

Association Of Integrin Alpha 2 C807T And L-Selectin P213S Alleles Polymorphism With Clinical Severity Of Sickle Cell Disease Among Sudanese Patients
Alaaeddin Elzubeir1, Shima Elsheikh 2, Nazik Husain3, Abu Elgasim Awad Elkareem4, Hind Mirghani 5
1Sudan International University, Medical Laboratory Science, Hematology Department, Khartoum, Sudan, 2University of Khartoum, Faculty of Medical Laboratory Science, Hematology Department, Khartoum, Sudan, 3Omdurman Islamic University, Faculty of Medicine, Pathology department, Khartoum, Sudan, 4Sudan University of Science and Technology, College of Medical Laboratory Science, Khartoum, Sudan, 5Gaafar Ibn-Auf Paediatric Tertiary Hospital, Hematology clinic, Khartoum, Sudan

Introduction: Sickle cell disease (SCD) accompanied by hemolytic and vaso-occlusive crisis are still responsible for high morbidity and early mortality. Integrins, and selectins are a family of cell surface receptors, interact with vascular cell adhesion molecules and leading to vaso-occlusion.
Methods: Venous blood samples were collected from SCD patients (n=133) admitted to the referral hematology clinic at Gaafar Ibn-Auf Paediatric Tertiary Hospital in Khartoum from June 2015 to June 2017.Samples from healthy Sudanese individuals were collected (n=112) as controls. Clinical data were collected by using constructed questionnaire, and from records, by authorized clinician. A modified scoring system was used to assess disease severity. It includes number of transfusions, hospitalizations and lifetime cumulative incidence of specific complications of SCD. Blood was genotyped by polymerase chain reaction-restriction fragment length polymorphism. Complete blood counts were measured by an automated hematology analyzer, and reticulocytes were manually counted.
Results: The genotype and alleles frequencies of ITGA2 C807T and L-Selectin SELL P213S were found to be significantly different between patients and controls groups (P=0.002 and 0.000), respectively. The ITGA2relative risk analysis of alleles frequency showed that patients with the T allele were 5.4 times more likely to suffer from hemolytic crisis, vaso-occlusive and ischemic stroke rather than patients with the C allele. There was a significant association between these gene polymorphisms and high expression of L-selectin by leukocytes and the development of complications in SCD (P= 0.000).Hb and RBCs counts were found to be significantly higher in controls than in patients (P = 0.000, P= 0.045, respectively) while retics significantly lower in controls (P= 0.034) and also when compared between severity groups for SCD showed statistically significant results (P=0.000).
Conclusions: These results indicated that the (P213S) polymorphism of SELL gene and (C807T) polymorphism of ITGA2 are associated with more complications and crisis, withITGA2T allele at C807T that appears to deliberate increased susceptibility to ischemic stroke and vasoocclusive and hemolytic crisis in Sudanese patients.

Evaluation Of An Automated Bone Marrow Slide Scanning Unit (Part 2)
Kathleen Deiteren, Marie-Berthe Maes, Jan Van den Bossche, Ronald Malfait
University Hospital Antwerp, Edegem, Belgium

Introduction: Counting bone marrow (BM) cells is very time consuming and requires highly trained individuals. We evaluated an automated BM slide scanning system, the Vision Hema Bone Marrow 4Pro (Vision Hema). Our main goal was to save time but maintain the same high standard as with manual microscopy. 
Methods: The Vision Hema (West Medica) first scans the BM slide (10x). Regions of interest (ROI’s) are automatically selected but can be adapted manually. In a second scanning step (100x) cells are counted and pre-classified automatically with the digital microscope preset to count 550 cells. The pre-classification was reviewed by a clinical pathologist. The aim of the study was to evaluate for both normal (n=108) and pathological (n=158) BM samples 1) the pre-classification agreement (this is the % of cells remaining in the same cell category after reviewing pre-classification results), 2) the sensitivity and 3) specificity of the cells of the myelogram and artifacts after reviewing pre-classification results.
Results: 1) Pre-classification agreement was higher for normal (n=108) than for pathological (n=158) BM samples except for blasts and plasma cells. Pre-classification agreement for normal/pathological BM samples respectively was 56,1%/79,2% for blasts, 85,8-93,2%/78,6-91,8% for myeloid cells (excluding basophils), 20,0%/18,3% for basophils, 61,8%/44,5% for monocytes, 68,2%/61,0% for lymphocytes and 58,8%/83,9% for plasma cells, 67,2-87,5%/49,2-79,7% for erythroid cells, and 99,6%/96,6% for artifacts. 2) In general, sensitivity was higher for normal (n = 108) compared to pathological (n = 155) BM samples. Sensitivity for normal BM cells was 75,1%/62,1% for blasts, 85,2-98,7%/77,6-97,0% for myeloid cells (excluding basophils), 28,6%/16,0% for basophils, 87,3%/88,5% for monocytes, 93,1%/88,2% for lymphocytes, 67,6%/64,2% for plasma cells, 78,9-92,8%/77,0-95,4% for erythroid cells and 66,6%/67,5% for artifacts. 3) Specificity was ≥97,6%/≥97,7% for all cell types except for lymphocytes (95,1%/90,8%).
Conclusions: The Vision Hema has a moderate to very high pre-classification agreement and sensitivity for all BM cells. Specificity was ≥95,1% except for pathological lymphocytes. Moreover, the pictures of the BM cells remain available for consultation which is an advantage over manual microscopy for education or when discussing BM results with colleagues.

Distribution Chemotherapy Response Modulating Genetic Polymorphisms In Armenian Population
Yervand Hakobyan
Hematology Center of Armenia, Yerevan, Armenia

Introduction:   Primary chemotherapy drug resistance is a one of the major issues of medical hematology-oncology affecting about 20% of all patients. In Armenia, clinical data suggest that the rate of primary resistance depends on the disease and treatment type and varies in the range of 10-30%. One of the mechanisms of resistance is a drug inactivation mediated by metabolizing enzymes and it has been known that the variability of those genes contributes to the response to drugs and treatment efficacy. In Armenia a number of drugs are used for treatment acute and chronic hematology-oncological diseases, such as Doxorubicin and Vincristine (lymphoma), Rituximab (lymphoma), Cladribine (hairy cell leukemia), interferon alfa-2a (chronic myeloid leukemia) and asparaginase (acute lymphoblastic leukemia). However, the distribution of genetic polymorphisms that modulate response to  the above mentioned drugs in unknown in Armenians. In this study we were aimed at performing pilot characterization of distribution of single nucleotide polymorphisms affecting chemotherapy treatment response in Armenian population. 
Methods: We used genome wide genotyping data of 168 healthy Armenians from three freely available datasets used in population genetics studies. 112 single nucleotide polymorphisms (SNPs) interfering the mentioned treatment agents were obtained from Pharmgkb database.  The distribution of allele frequencies in Armenians were compared to the allelic rates in European population available in 1000 Genomes and HapMap project (retrieved using LDLink software).
Results: Minor allele frequency (MAF) associated with altered response to drugs (table 1) was varying in the range of M±SD ( min-max): 0.25±0.14 (0.00-0.49). The minor allele frequency of 18 SNPs was significantly higher in Armenians compared with European population. Another 18 SNPs were significantly underrepresented in Armenians. Six SNPs (MAF min-max: 0.22-0.48) were associated with the altered response to 5 and more drugs. 
Drug/Treatment N, SNPs MAF min-max
Doxorubicin and Vincristine 28 0.05-0.49
Rituximab 14 0.05-0.44
Cladribine 2 0.10-0.28
Interferon alfa-2a 13 0.28-0.46
Asparaginase 2 0.24-0.46

Conclusions: Our results demonstrate the overall enrichment of genetic factors connected to the risk of failure and toxicity of chemotherapy in Armenian population and necessitate the further research on cumulative impact on the  on the chemotherapy response in patients with blood cancers.

Microrna-661 Upregulation In Myelodysplastic Syndromes Induces Apoptosis Through P53 Activation And Associates With Decreased Overall Survival.
Seong-Ho Kang1, Ji-Seon Choi2
1Chosun University, Gwangju, Korea, 2Catholic Kwandong University, Incheon, Korea

Introduction: MicroRNA (miRNA) dysregulation contributes to myelodysplastic syndromes (MDS), and one pathogenic feature of MDS is apoptosis. The present study aimed to investigate the dysregulation of miRNA and its biological significance in MDS.
Methods: The present study compared the expression profiles of miRNAs (miR-598-5p, miR-599, and miR-661)encoded by chromosome 8 between 65 MDS patients and 11 controls and analyzed in vitro effect of upregulated miRNA by transfection of the miRNA into OCI-AML3 cells.
Results: We found thatthe expression of miR-661 was upregulated in patients with MDS by 5.28-fold relative to the levels in controls (P=0.021).The miR-661 expression data were used to generate a receiver operating characteristic (ROC) curve for miR-661, for which the area under the curve (AUC)was 0.752 (95% confidence interval, 0.532–0.972; P = 0.021). Patients with high miR-661 expression showed a significantly decreased overall survival (P=0.026). Furthermore, an in vitro study demonstrated that transfection with a miR-661 mimic inhibited cell growth compared with transfection of negative control (P=0.001) and induced apoptosis through activation of p53. Besides, TP53 mutations are known to be associated with poor prognosis in MDS and increased expression of p53.
Conclusions: These findings suggest that high miR-661 expression can be associated with decreased overall survival for MDS and recapitulate apoptosis, the characteristic feature of MDS.

Interleukin-10 (1082G/A) Polymorphism Is Associated With Susceptibility Of Acute Myeloid Leukemia Patients In Sudanese Population
ibrahim khidir ibrahim1, omnia almak2
1Al Neelain University, khartoum, Sudan, 2Sudan University of Sciences and Technology , Khartoum, Sudan

Introduction:   Interleukin-10 (IL-10) is a multifunctional cytokine with both immunosuppressive and anti-angiogenic functions and may have both tumor-promoting and -inhibiting properties. We do this study to determine the association of IL-10 promoter polymorphisms with increased risk of Acute myeloid leukaemia and its association with patients haematological and demographic data.      
Methods:  A total of 30 patients with acute myeloid leukaemia and 30 control subjects were enrolled in this study. Blood samples were collected from all patients in EDTA containing tubes. Genomic DNA was extracted from all blood samples using salting out method. The genotypic variants of IL-10(-1082G/A) polymorphism were detected by allele specific-PCR. 
Results: We found that (36.7%) of cases have homogenous IL-10 (1082G/A)genotype, (43.3%) have heterogeneous (IL-10 -1082G/A)genotype, (20.0%) have normal IL-10(-1082G/A)genotype. The association between IL-10 (-1082G/A) polymorphism and AML is statistically significant, there is no association between IL-10(-1082G/A) polymorphism and AML sub-type, gender, age group, mean of the haematological parameter (TWBs, Blast%, Hb and platelet count).
Conclusions: In summary, we conclude that Dominant (GA+GG) genotype of IL-10 -1082G/A (rs1800896) polymorphism is a risk factor for AML.

Igh-Fr1Analysis Using Next-Generation Sequencing For The Detection Of Clonality In High-Risk Korean Multiple Myeloma Patients
Miyoung Kim1, Young Kyung Lee1, Hyo Jung Kim2, Jee-Soo Lee1, Han-Sung Kim1, Hee Jung Kang1
1Department of Laboratory Medicine, Hallym University Sacred Heart Hospital, Anyang, Korea, 2Department of Internal Medicine, Hallym University Sacred Heart Hospital, Anyang, Korea

Introduction: Next-generation sequencing (NGS)-based immunoglobulin gene rearrangement assays may be useful for assessing clonality in multiple myeloma (MM), although their applicability has not been convincingly demonstrated. Hence, we evaluated the primer set most widely used with the only commercially available NGS-based clonality assay in high-risk MM patients for the first time. 
Methods: Thirty bone marrow aspirate samples from 29 patients with MM were evaluated for IGH rearrangements using the LymphoTrack® IGH-FR1 assay (InVivoScribe, San Diego, CA) on a MiSeqDx instrument (Illumina, San Diego, CA). The results were interpreted according to the manufacturer’s instructions and analyzed while considering disease-related factors that might influence the outcomes.
Results: IGH clonality was observed in 21 of the 30 samples (70.0%); the median percentage of clonality (in relation to the total lymphoid cells) was 68.12% (range: 4.85–86.60%). While 2 samples showed biclonality, the remaining 19 showed monoclonality (including the relapse of 1 of the 2 aforementioned samples with biclonality). V3-J4 was the most common type of rearrangement (28.6%) followed by V3-J6 (19.0%), V3-J5 (19.0%), and V4-J6 (14.3%). Neither the existence of IGH clonality nor its quantity was associated with the percentage of plasma cells in the specimens, the median percentage of plasma cells in the 30 samples was 41.4% (range, 10.4–81.7%); 21 samples (70.0%) had more than 30.0% of plasma cells. The median percentage of plasma cells among the 21 samples with IGH clonality was 41.0% (range, 10.4–80.3%) and that among the 9 that were negative for IGH clonality was 42.4% (range, 12.7–81.7%). International Staging System (ISS) stage and karyotype were not associated with the IGH clonality; 17/24 samples with ISS-3 (70.8%), 3/4 with ISS-2 (75.0%), and 1/2 with ISS-1 (50.0%) showed IGH clonality. Moreover, 13/20 samples with normal karyotype (65.0%), 5/5 with hyperdiploid karyotype (100.0%), and 2/3 with non-hyperdiploid karyotype (66.7%) showed IGH clonality.
Conclusions: The IGH-FR1 assay detected clonality in approximately 70% of high-risk Korean MM patients. Clonality was not influenced by the percentage of bone marrow plasma cells, ISS stage, or karyotype. To validate this assay, the negative clonality of the remaining samples ought to be confirmed using a different set of primers. 

Mutational Spectrum Of Setbp1 In Myeloid Neoplasms
Daniel Larson, David Viswanatha, Dong Chen, Rong He
Mayo Clinica, Rochester, MN, United States

Introduction: SETBP1encodes the poorly characterized SET binding protein 1 located predominantly in nuclei. SETBP1 has shown to bind and stabilize SET, a negative regulator of the tumor suppressor protein phosphatase 2A. The mutational spectrum of SETBP1 has not been thoroughly characterized. This study aimed to explore the spectrum and mutation pattern of SETBP1  in myeloid neoplasms (MN).
Methods: Following Institutional Review Board approval, we retrospectively reviewed the in-house patient results of a 35-gene OncoHeme NGS panel performed at our CLIA-certified Molecular Hematopathology Laboratory.  DNA was extracted from bone marrow/peripheral blood samples from patients with known/suspected hematologic neoplasms. NGS was performed using 200ng sheared DNA with a custom hybridization-capture reagent (SureSelectXT, Agilent, Santa Clara, CA) and sequenced on MiSeq or HiSeq (Illumina, San Diego, CA). Data were processed through a proprietary bioinformatics analysis pipeline (Mayo NGS Workbench).  Patient medical records were reviewed for pathology diagnoses.
Results: Among 1,014 patients tested from 6/2015-11/2016, 24 harbored pathogenic/likely pathogenic mutations in SETBP1. The disease distribution in descending order was  myelodysplastic/myeloproliferative neoplasm (MDS/MPN) (n=7,  5 chronic myelomonocytic leukemia, 2 MDS/MPN-unclassifiable); MDS (n=6, 2 MDS-multilineage dysplasia, 1 MDS-excess blasts-1, 1 MDS- excess blasts-2,1 MDS-unclassifiable), acute myeloid leukemia (n=4),  MPN (n=4, 3 primary myelofibrosis and  1 post-ET myelofibrosis). The remaining four cases showed clonal cytopenia of undetermined significance without distinct morphologic features of myeloid neoplasm. SETBP1mutations identified include p.Asp868Asn (n=15), p.Gly870Ser (n=7), p.Asp868Tyr (n=1), p.Gly870Asp (n=1), p.Ile871Thr (n=1), involving the highly conserved region of the SKI homologous domain. Their variant allele frequencies (VAF) were 43.3% (mean, range 34.0-48.0%), 21.4% (mean, range 5.6-41.0%), 30.5%, 14.1%, and 47.3% respectively. One patient co-harbored two mutations (p.Asp868Asn and p.Gly870Ser). Concurrent mutations of other genes were seen in 22 (91.7%) cases, including ASXL1 (20), SRSF2 (13), CBL (7), RUNX1 (4), JAK2 (4), TET2 (3), U2AF1 (3), FLT3 (2), EZH2 (2), NRAS (2), TP53 (2), KRAS (1), ETV6 (1), DNMT3A (1), WT1 (1), ZRSR2 (1), and SF3B1 (1). The mean number of concurrent mutations was 2.9 (0-6).
Conclusions: SETBP1mutations are rare in myeloid neoplasms and enriches in the highly conserved region of the SKI homologous domain. They occur in a spectrum of myeloid neoplasms and frequently co-occur with other mutations. Further study is warranteed to improve our understanding of the intricate interplay of comutations and ultimately improve patient outcome.

The Mitochondrial Damage, Represented By The Expression Level Of Prohibitin 2, Could Predict The Prognosis Of Hematological Malignancies
JunHyung Lee1, Ha Jin Lim1, Hye Ran Kim2, Yonggwan Won3, Jong Hee Shin1, Myung Geun Shin1
1Department of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hwasun Hospital, Hwasun, South Korea, 2College of Korean Medicine, Dongshin University, Naju, South Korea, 3Department of Electronics and Computer Engineering, College of Engineering, Chonnam National University, Gwangju, South Korea

Introduction: Prohibitins (PHBs) are a highly conserved class of proteins first discovered as inhibitors of cellular proliferation. Recently, prohibitin 2 (PHB2) has been known to be involved in mitochondrial damage as an inner mitochondrial membrane receptor. In this study, we investigated how the severity of mitochondrial membrane damage, represented by the level of PHB2 expression, correlates with the prognosis of hematological malignancy.
Methods: To measure the expression level of PHBs, messenger RNA sequencing (mRNA-Seq) was performed in 11 patients, diagnosed as hematological malignancies with recurrent genetic abnormalities (4 AMLs, 2 APLs, 2 B-ALLs, and 3 CMLs). Libraries were prepared with 1 ug of total RNA from bone marrow aspirates at initial diagnosis. Indexed libraries were then sequenced in the HiSeq2500 platform (Illumina). The sequencing data was mapped to human reference transcripts (GRCh37 assembly in Ensembl) using HiSAT2 aligner (v2.1.0). Gene expression levels was measured as TPM unit using StringTie (v1.3.4) and normalized to 5 housekeeping genes.
Results: In expression analysis, PHB2 showed the possibility as a prognostic predictor of hematologic malignancies. In AML, when listed in descending order of PHB2 expression level, it coincided with AML prognosis exactly: 319.8 as normalized TPM in AML with t(6;9) (Poor-risk); 183.3 in AML with t(9;11) (Intermediate risk); 138.9 in AML with t(8;21), 134.8 in in AML with inv(16), 125.0 and 90.1 in AML with t(15;17) (favorable risk) (Figure 1). Also, in ALL, the expression level of PHB2 represented disease risk: 157.2 in B-ALL with t(9;22) (Poor risk); 118.6 in B-ALL with t(12;21) (Good risk). In 3 CML cases, the expression levels were relatively suppressed as 136.2, 95.5, and 68.1 than those of acute leukemia.
Conclusions: In this study, the severity of mitochondrial membrane damage, represented by the level of PHB2 expression, could exactly stratify the disease prognosis in 11 hematological malignancy cases. We expect that PHB2 will provide important information for risk stratification of various hematological malignancies.

Methodological Evaluation Of Otsuka Wt1 Real-Time Fluorescence Commercial Kit For The Quantitative Detection Of Wt1 Expression
Xiaoli Ma1,2, Fang Wang1,2, Qisheng Wu1,2, Na Wang1,2, Lin Zhou1,2, Ying Zhang1,2, Chunye Cao1,2, Hongxing Liu1,2,3
1Beijing Lu Daopei Institute of Hematology, Beijing, China, 2HebeiYanda Lu Daopei Hospital, Langfang, China, 3Beijing Lu Daopei Hospital, Beijing, China

Introduction: Wilms tumor type I (WT1) gene is overexpressed in most AML. WT1 expression has been evaluated as a marker for risk stratification and MRD detection in AML. The using RT- PCR to detect WT1 expression has become a standard method in clinical laboratories and has been widely used. The laboratory uses the Otsuka Pharmaceutical of Japan WT1 Quantitative Test Kit (Otsuka®) to compare the in-house assay that European Multicenter study recommended method to verify the performance of the commercial kit.
Methods: Between March and October 2018,a total of 104 specimens(100 cases of  BM, 4 cases of PB)were collected from 47 patients, and 26 patients were submitted multiple times. The expression of WT1 was detected simultaneously by Otsuka®and the in-house assay. The results of the two methods were statistically analyzed by linear regression analysis to investigate the correlation between the two methods. Eight of the 26 patients who were sent for multiple examinations were selected for more than three times and the samples were taken at different times (mean interval 29.68 days, 7-48 days). The Otsuka ® kit for detection of WT1 Expression was compared to determine the extent of compliance with MRD, and to assess its clinical usefulness.
Results:  104 cases of clinical samples were simultaneously quantified WT1 using the in-house assay and Otsuka® kit.  Linear regression analysis showed that the correlation between the two methods was better (Y=0.8735x+0.1621,R2=0.9304)(Fig.1). The quantitative results of Otsuka ® kits in 8 patients were consistent with the results of MRD(Fig. 2). WT1 expression changes with the development of the disease and the MRD, suggesting that it can be monitor the therapeutic effect.
Conclusions: The commercial Otsuka® WT1 PCR quantification kit can monitor the expression of WT1 mRNA to aid diagnosis and as a monitoring indicator for disease progression.

High Frequency Of I Allele Of Angiotensin-Converting Enzyme Gene In Sudanese Patients With Chronic Lymphocytic Leukaemia
ibrahim Osman
Al neelain university, khartoum, , Sudan

Introduction: Renin angiotensin system (RAS) comprising Angiotensin converting enzyme (ACE), Angiotensin II (Ang II) and its receptor Angiotensin II receptor type I (AGTR1), plays a critical role in several diseases including cancer. An insertion/deletion (I/D) polymorphism present in intron 16 of ACE gene has been associated with many diseases, but their association with chronic lymphocytic leukaemia (CLL) is still debatable. Here, we for the first time investigated the association of this polymorphism in Sudan.  The aim of this study was to study the association between ACE (I/D) polymorphism and CLL in Sudanese patients.
Methods: A total of 40 patients with chronic lymphocytic leukemia and 40 control subjects were enrolled in this study. Blood samples were collected from all patients in EDTA. Genomic DNA was extracted from all blood samples using salting out method.ACE I/D polymorphism as determined using polymerase chain reaction (PCR) method. 
Results: We present a case control study that includes 40 Sudanese patients with chronic lymphocytic leukaemia(CLL). Among them, 21 (52.5%) were male and 19 (47.5%) were female. In addition, 40 subjects (19 (47.5%) male and 21 (52.5%) female), who were healthy and without a medical history of CLL, were included as a control group. Molecular analysis showed that the most frequent genotype in CLL patients was I/I, followed by genotypes D/I and D/D. In the control group, D/D was the most frequent genotype, followed by I/D. The I/I genotype was absent (Table 1). Patients carrying I allele were found had 3.5 folds increased the risk of CLL (OR: 6.5, 95%, C.I: 1.98-20.0, P= 0.001). The frequencies of the D allele were 0.61 and 0.90 in patients with CLL and the control group, respectively, whereas those of the I allele were 0.39 and 0.10 in patients with CLL and the control group, respectively. A significant deviation from the Hardy–Weinberg equilibrium was observed in patients with CLL (χ2 = 1.7, df = 2, P = 0.001).
Group ACE Genotype
I/I            I/D DD
Patient 17 (42.5 %) 15 (37.5 %) 8(20%)
Control 0 (0%) 8 (20%) 32 (80%)

Conclusions: In conclusion, a significant association was found between ACE I/D polymorphism and CLL among Sudanese patients. 

Newly Developed Monosomy 7 Confirmed By Digital Droplet Pcr In Philadelphia Negative Cells Of A Chronic Myeloid Leukemia Patient After Tyrosine Kinase Inhibitor Treatment
Sholhui Park1, Yeung Chul mUN2, Chu-Myong Seong2, Jungwon Huh1
1Deparment of Laboratory Medicine, Ewha Womans University, College of Medicine, Seoul, Korea, 2Department of Internal Medicine, Ewha Womans University, College of Medicine, Seoul, Korea

Introduction: Two to 17% of chronic myeloid leukemia (CML) patients treated by tyrosine kinase inhibitor (TKI) were reported to develop clonal chromosomal abnormalities (CCA) in Philadelphia negative cells (Ph-).  Among frequently reported CCA/Ph-, -7/del(7q) abnormality is considered as a warning according to the 2013 European Leukemia Net recommendations. It is still controversial that CCA/Ph- appeared whether before or after TKI treatment. To evaluate the presence of CCA at different responding status, we conducted digital droplet PCR (ddPCR) targeted at chromosome 7 (PrimePCR ddPCR Copy Number Assay: MET, human, Bio-RAD,Hercules, CA) in a CML patient who developed myelodysplastic syndrome (MDS) with monosomy 7, after 206 months of tyrosine kinase inhibitor (TKI) treatment.
Methods: A 60-year-old female patient with 19-years follow-up from CML diagnosis developed MDS under TKI treatment 17 years after diagnosis. Cytogenetic, hematologic, and molecular response was assessed from the bone marrow study at least once a year (Table 1). ddPCR was performed using 6 bone marrow samples including Ph+, normal karyotype, del(20q), and monosomy 7 and 1 peripheral blood sample with monosomy 7.
Results: The patient received imatinib since the initial diagnosis of CML. Complete cytogenetic response was achieved in 6 months of treatment. Philadelphia clones transiently reappeared in 10, 36, and 51 months of imatinib treatment. Therapeutic agent was replaced with 2nd generation TKI, nilotinib, 7 years after diagnosis. In 150 months of treatment for CML, del(20q) appeared in cytogenetics and persisted for one year. She returned to normal karyotype next year. In 206 months from CML diagnosis, new CCA/Ph- of monosomy 7 appeared as a sole cytogenetic abnormality. And one year later, her bone marrow showed dysplastic features in erythroid and megakaryocytic lineages enbling to make a diagnosis of MDS. ddPCR for monosomy 7 showed negative results from 3 to 15 years since CML diagnosis. At the same time as monosomy 7 appeared in cytogenetics, ddPCR revealed reduced copy number of chromosome 7.
Conclusions: The ddPCR revealed that monosomy 7 in Ph negative cells in a CML patient newly developed during TKI treatment, not at the time of diagnosis before TKI treatment. 

The Detection Of P2Ry8-Crlf2 Fusion In Acute Lymphoblastic Leukemia
Dollawat Sae-chua1, Teerapong Siriboonpiputtana1, Nittaya Limsuwanachot1, Budsaba Rerkamnuaychoke1, Samart Pakakasama2
1Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, 2Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

Introduction: Acute lymphoblastic leukemia (ALL) is recognised as the most common cancers in childhood and a major cause of sickness and death in adults. Recently, CRLF2 rearrangements were frequently identified in Ph-like acute lymphoblastic leukemia with the aggressive disease phenotype. Aim: In this report, we performed a conventional RT-PCR method to detect P2RY8-CRLF2 fusion which known as the most common CRLF2 rearrangements in Ph-like ALL in childhood.
Methods: Conventional RT-PCR was performed to analyseP2RY8-CRLF2 fusion in RNA isolated from bone marrow samples of 87 newly diagnosed childhood ALL patients. The P2RY8-CRLF2 fusion sequence was subsequently confirmed by using Sanger sequencing technique.
Results: We found that 2 out of 87 patients (2.3%) are positive for P2RY8-CRLF2 fusion. Additionally, one patient with P2RY8-CRLF2 fusion was affected with Down syndrome (trisomy 21). Sequencing analysis on P2RY8-CRLF2 fusion sequences in both two positive samples revealed that the fusion breakpoint is located at 111 nucleotides upper the start codon of P2RY8 sequence (XM_011545631.2)and 2 nucleotides before the start codon of CRLF2 sequence(NM_022148.3).
Conclusions: We could detect a P2RY8-CRLF2 fusion by using a simple RT-PCR technique. Furthermore,P2RY8-CRLF2 fusion sequence was conserved in our tested samples. In recent future, we are going to deeply investigate the function of P2RY8-CRLF2 fusion in the initiation of Ph-like ALL in both in vitro and in mouse model.

Achieving The Third 90: Keeping Adolescents Living With Hiv Virally Suppressed In Rural Nigeria In The Era Of Test And Treat Using Continuous Quality Improvement (Cqi) Model Of Peer Counseling & Support Group
Saheed Usman1,2, Prosper Okonkwo2, Oluwatoyin Jolayemi2, Jay Osi-Samuels2, Patrick Akande2, Babatunde Ladi-Akinyemi2, Oluremi Olaitan2, Femi Owolagba2, Matthias Alagi2, Eke Ofuche2, Chiedozi Akueshi2, Babatunde Akinbinu2
1Nnamdi Azikiwe University, Awka, Nigeria, 2APIN Public Health Initiatives, Abuja, Nigeria

Introduction: In 2016, Nigeria transitioned to “Test & Treat”, a policy where all people living with HIV (PLHIV) are treated with lifelong antiretroviral therapy (ART). There are unique challenges achieving viral suppression in ALHIV mainly due to increased stigma & lack of social support. Hypothesis tested was ART adherence effect on viral load outcome. We examined viral suppression among adolescents living with HIV in Western Nigeria.
Methods: This study was an observational prospective cohort study of adolescents living with HIV (ALHIV) already initiated on antiretroviral therapy for at least six months, enrolled in health facilities across supported facilities in Western Nigeria, during a 12-month observation period starting October 2016 till September 2017. Quantitative viral load analysis was done using Polymerase Chain Reaction, Roche Cobas Taqman 96 Analyzer.
Results: A total of 126 (64 males & 62 females) subjects were recruited. The mean age of 13.58 ± 4.26 years. 83 (65.9%) & 71 (56.3%) had viral suppression of < 1000 & < 50 RNA copies per ml respectively. The 43 subjects went through peer counseling by trained ALHIV and enhanced adherence counseling (EAC) for three months and viral load test repeated three further months after, which made 113 (89.7%) & 101 (80.1%) of the subjects have < 1000 RNA & < 50 RNA copies per ml respectively during the observation. The ALHIVs joined the institutionalized social-media driven support group & adolescent decentralized care model ensuring they achieve the third 90 at an undetectable level. ART adherence has significant effect on viral load outcome (χ² = 6.42, df = 1, P = 0.001).
Conclusions: ART adherence counseling is key to the achieving viral suppression and determine infection prognosis, thus, developing robust continuous quality improvement (CQI) plans to address issues across the cascade ultimately helping in the monitoring of HIV/AIDS disease progression and decrease treatment failure tendencies.

Spectrum Of Genetic Alterations In Npm1-Positive Acute Myeloid Leukemia (Aml) Observed Using A Targeted Next Generation Sequencing (Ngs) Panel.
David Viswanatha, Surendra Dasari, Dan Devine, James Hoyer, Jennifer Oliveira, Rong He
Mayo Clinic, Rochester, MN, United States

Introduction: An important consideration in AML biology concerns the identification of new molecular subtypes and effects of co-occurring mutations. AML with NPM1 mutation has been recognized as a distinct subtype per the WHO classification and until recently, only the common association with mutated FLT3 was recognized. Recent large AML studies have suggested that NPM1+ AML may be more heterogeneous and that concurrent alterations in other genes may be relevant, particularly in the absence of FLT3 mutation. However, the findings are not uniform, suggesting additional characterization of NPM1 positive patients is yet of value. We reviewed our experience with ~10,000 NGS cases analyzed using a 35 gene NGS panel for myeloid neoplasms and describe our findings for NPM1+ cases.
Methods: Peripheral blood or bone marrow specimens from patients with known or suspected myeloid malignancies were received in our Molecular Hematopathology laboratory for evaluation using a 35 gene targeted clinical NGS panel.  DNA was extracted and library preparation was performed using hybrid capture probes (Sure Select XT, Agilent) with sequencing on MiSeq or HiSeq platforms (Illumina). Following initial data processing, quality assessment and read alignment to the human genome reference (HG19), called variants were analyzed using the Mayo Clinic NGS Workbench. Variants were classified as benign/likely benign, uncertain significance or pathogenic/likely pathogenic.  This study was conducted with IRB approval. 
Results: A total of 457 NPM1 mutated myeloid neoplasms were identified. The most commonly co-mutated gene was FLT3 (49%, mainly internal duplications).  Frequent concurrent alterations were also seen in DNMT3A, TET2, IDH1, NRAS, WT1. Interestingly, a significant minority of NPM1+ cases showed alterations involving SRSF2 or CEBPA.  Mutations in these genes were evident in both FLT3 positive and negative cases; alterations in other kinase genes (KIT, JAK2) were not significantly present. The overall data summary is shown in Figure 1.
Conclusions: In addition to confirmation of previously described co-mutation patterns seen in NPM1+ AML, our retrospective study identifies other common recurrent associations. The data suggests that NPM1+ AML is a more diverse subtype than previously assumed. Detailed knowledge of cooperating gene mutations and pathways is critical for improving patient outcomes. 

Identification Of A Novel Npm1-Rarg-Npm1 Chimeric Fusion In Acute Myeloid Leukemia Resembling Acute Promyelocytic Leukemia
Wei Wang1, Lili Yuan2, Fang Wang2, Yang Zhang2, Wen Teng2, Panxiang Cao2, Xiaoli Ma2, Mingyue Liu2, Yaoyao Tian1, Tong Wang2, Daijing Nie2, Jing Zhang2, Hongxing Liu2,3,4, Xue Chen2
1The 2nd Affiliated Hospital of Harbin Medical University, Harbin, China, 2Hebei Yanda Lu Daopei Hospital, Langfang, China, 3Beijing Lu Daopei Institute of Hematology, Beijing, China, 4Beijing Lu Daopei Hospital, Beijing, China

Introduction: The RARG gene is a member of the nuclear hormone receptor superfamily and shares high homology with RARA and RARBRARA is involved in translocation with PML in acute promyelocytic leukemia (APL). Little is known about RARB or RARG rearrangement. RARG fusions were reported in only five APL patients and the partner genes were NUP98PML, and CPSF6. We report a novel NPM1-RARG-NPM1 chimeric fusion in a APL patient.
Methods: Multiplex-nested RT-PCR showed abnormal positive bands in one reaction which was designed to amplify NPM1-RARA. Sanger sequencing of the PCR products demonstrated fusions between NPM1 and RARG. Whole genome sequencing (WGS) was performed to clarify the genomic breakpoints.
Results: Laboratory, morphology and immunophenotypic analysis of the patient suggested the diagnosis of APL. Conventional cytogenetics and RT-PCR for PML-RARA and common APL variant translocations were negative. However, multiplex-nested RT-PCR showed 3 abnormal positive bands in one reaction which was designed to amplify NPM1-RARA. Sequencing of the PCR products revealed NPM1-RARG transcripts derived from fusion of NPM1 exon 4 to RARG exon 1, exon 2, or exon 4, respectively. The extensive homology between RARA and RARG made it possible to amplify the NPM1-RARG fusion using the reverse primer designed to amplify RARA. WGS revealed breakpoints in intron 4 of NPM1 and 5’ UTR of RARG. Interestingly, there was another breakpoint in each of the two genes, which located in intron 10 of NPM1 and intron 9 of RARG, respectively. Both NPM1 intron 4-RARG 5’-UTR and RARG intron 9-NPM1 intron 10 genomic fusions were confirmed by Sanger sequencing. Thence the genomic alterations of this patient were a deletion of NPM1 intron 4 to intron 10 accompanied by an insertion of RARG 5’ UTR to intron 9. Moreover, RT-PCR and Sanger sequencing verified the presence of NPM1-RARG-NPM1 chimeric fusion with 3 fusion transcripts. All the three fusion transcripts generated a NPM1 mutation-like domain.The missing of NPM1 exon 5-9 and the mutation-like C-terminus of the NPM1-RARG-NPM1 transcripts may result in ectopia of nucleophosmin in cytoplasm and contribute to impaired differentiation and leukogenesis. Besides, all the three transcripts caused deletions of 25 amino acids of the ligand-binding domains of RARG, which may lead to ATRA insensitivity. Actually, the patient was treated with arsenic trioxide and then switched to ATRA and both showed no response.
Conclusions: We first report NPM1 as a partner gene of RARG in APL and identify a unique NPM1-RARG-NPM1 chimeric fusion. 

Plasma Von Willrebrand Factor Antigen And Activity Levels Are Associtaed With The Severity Of Coronary Stenosis
Yiming Zhao, Bin Yan, Qi Wang, Changgeng Ruan
The First Affiliated Hospital of Soochow University, Suzhou, China

Introduction: Coronary artery disease (CAD) is the leading cause of mortality and morbidity worldwide, and the rupture of plaque in coronary arteries would lead to artery stenosis and even blockage, then occurrence of acute myocardial infarction (AMI). Von Willebrand factor (VWF) is a plasma multimeric glycoprotein, which is acted as a bridge of platelets and subendothelial matrix following vessel damaged. The aim of this study is to investigate the value of VWF in appraising the severity of coronary artery quantified by the Gensini score in AMI.
Methods: Plasma VWF antigen (VWF:Ag) and VWF-collagen-binding assays (VWF:CB) in normal controls (n=123) and patients with AMI (n=205) were tested by ELISA. Patients with AMI underwent coronary angiography were divided into three groups (G1, G2 and G3) in accordance with Gensini score.
Results: The levels of VWF:Ag and VWF:CB (219.87 ± 1.32 % and 257.58 ± 2.18 %) in AMI were significantly higher than those in control group (78.90 ± 0.38 % and 68.40 ± 0.48 %) (p< 0.001). Patients with AMI with a history of stroke had higher level of VWF:Ag and VWF:CB than those without stroke history (p< 0.05). Plasma levels of VWF:Ag and VWF:CB were positively correlated with Gensini score in patients with AMI (r = 0.257, p < 0.0001;r = 0.228, p = 0.001, respectively). VWF:Ag and VWF:CB were positively correlated with Gensini core as well as implicated vessels.ROC analysis was undertaken for VWF:Ag and VWF:CB to assess the ability of VWF to predict AMI. The sensitivity and specificity of VWF:Ag in predicting AMI were 80.5% and 73.2% when the cutoff value was 105.10%; while VWF:CB in predicting AMI were 79.5% and 79.7% when the cutoff value was 90.91%. Hence, plasma VWF:Ag and VWF:CB were valuable in predicting AMI.    
Conclusions: VWF:Ag and VWF:CB in patients with AMI were elevated, and were positive correlation with the severity of coronary artery. This study enriched the value of VWF in pathogenesis of AMI. Thus, VWF detection could be a relative sample, non-invasive test to evaluate the severity of coronary stenosis before angiography.

Circulating Mir-451A As A Potential Biomarker To Predict Prognosis In Patients With Multiple Myeloma
ling zhong1, Jiao Chen2, tao jiang3
1Department of Clinical laboratory, Sichuan Academy of Medical Sciences and Sichuan Provincial Peoples Hospital, School of medicine, University of Electronic Science and Technology of China, chengdu , China, 2Department of Hematology, Sichuan Academy of Medical Sciences and Sichuan Provincial Peoples Hospital, School of medicine, University of Electronic Science and Technology of China, chengdu, China, 3Department of Hematology, Sichuan Academy of Medical Sciences and Sichuan Provincial Peoples Hospital, School of medicine, University of Electronic Science and Technology of China, chengdu, China

Introduction: The current R-ISS stratification criteria for multiple myeloma (MM) patients cannot reflect the dynamic changes of tumor load nor monitor Minimal Residual Disease(MRD). The usually- focally-located myeloma brings false negative results. Targeting IL-6R and sIL-6R, miR-451a could suppress proliferation and accelerate apoptosis in MM.
Methods: This study aims to find a new marker for dynamic monitoring, and to stratify for earlier prediction on MM patients.   The clinical sample comprised 10 subjects from donor of transplantation as control and 52 below-65-years-old MM patients(15 in R-ISS I, 17 in R-ISSⅡand 20 in R-ISS Ⅲ). We evaluated miR-451a expression levels from BM (bone marrow) and circulation by qRT-PCR, measured IL-6 and sIL-6R by ELISA, assessed MRD by multi-parameter flow cytometry (MFC) in follow-up, and confirmed miR-451a to target IL-6R by dual-luciferase reporter assay.
Results: The miR-451a expression in MM patients was 0.43±0.11 times of the control group (P=0.001). Circulating miR-451a related inversely with sIL-6R and IL-6 (r=-0.92, -0.98, P< 0.05), but positively with miR-451a in BM (r=0.97, P< 0.05). Circulating miR-451a expressed differently at different stages (0.73±0.05 times in R-ISS I, 0.41±0.04 times in Ⅱ, 0.21±0.02 times in Ⅲ). In R-ISSⅠand Ⅱ, the lower-miR-451a-level patients ---below the median group value --- showed a worse survival (X2=5.49, P=0.019; X2=15.96; P=6.45E-5). In R-ISS Ⅲgroup, the median of miR-451a level was 0.21; below 0.21, the patients displayed a worse survival, without statistical significance (X2=0.021, P=0.88). 11 post-chemotherapy patients achieved complete remission (CR). Their follow-up showed constantly negative MRD for 7 patients with high miR-451a levels, and sudden decline of miR-451a in the other 4 patients. Recurrence of miR-451a was observed 4–8 weeks earlier in circulation than in BM relapse. Dual-luciferase reporter assay verified the target effect between miR-451a and IL-6R.
Conclusions: Circulating miR-451a can be a new marker to sub-stratified MM patients for earlier relapse prediction.

Bcl2Overexpression In Patients With Acute Myeloid Leukemia Identified Specific Subtypes But Did Not Benefit From Hematopoietic Stem Cell Transplantation
Jing-dong Zhou
Zhenjiang First People's Hospital, Zhenjiang, China

Introduction: BCL2 protein inhibitor ABT-199/venetoclax has been granted breakthrough designation by Food and Drug Administration for relapsed or refractory chronic lymphoid leukemia with 17p deletion. Although ABT-199 also causes on-target cell death in acute myeloid leukemia (AML), whether it can be applied to clinical treatment needs further studies. Herein, we reported BCL2 expression pattern and its clinical implication in AML, and may provide theoretical basis for targeted therapy using venetoclax.
Methods: BCL2 expressionwas analyzed in a cohort of 200 AML patients from The Cancer Genome Atlas (TCGA) datasets and confirmed by another independent cohort from our own data.
Results: BCL2 expression was significantly up-regulated in AML patients from TCGA datasets and confirmed by our own data. High expression of BCL2 was significantly correlated with FAB-M0/M1, whereas low expression of BCL2 was associated with FAB-M5. No significant differences were observed in overall survival (OS) and leukemia-free survival (LFS) between BCL2low and BCL2high group. In the BCL2low group, patients undergoing hematopoietic stem cell transplantation (HSCT) had significantly better OS and LFS compared with patients treated with chemotherapy. In the BCL2high group, no significant differences in OS and LFS were observed between chemotherapy and HSCT groups. 
Conclusions: Collectively, BCL2 overexpression identified specific FAB subtypes of AML, but not affected prognosis. Patients with BCL2 overexpression did not benefit from HSCT among both whole-cohort AML and CN-AML. These results may provide theoretical basis for those AML patients with BCL2 overexpression using BCL2 inhibitor venetoclax rather than HSCT.

A Deep Learning Approach To Automatically Classify Pathological Cell Images In Peripheral Blood
Santiago Alferez1, Anna Merino2, Laura Bold2, Andrea Acevedo1, Angel Molina2, Jos Rodellar1
1Technical University of Barcelona, Barcelona, Spain, 2Hospital Clinic. Biochemistry and Molecular Genetic Department. CDB., Barcelona, Spain

Introduction: Discrimination among reactive lymphocytes (RL) or malignant cells, such as abnormal lymphoid cells (ALC) and blast cells (BL) by cell morphology requires extensive experience and skill. Previously, we published the recognition of these cell types using a traditional computer vision pipeline, involving segmentation, feature extraction/selection and classification steps. In this work, we propose a deep learning approach to automatically classify reactive and malignant cells in a straight step.
Methods: A total of 46469 digital cell images from PB were obtained from 1560 blood smears (785 patients), acquired by the CellaVision DM96 and stained with May-Grünwald-Giemsa. These images corresponded to lymphocytes (N), and the remaining were: 1) ALC corresponding to chronic lymphocytic leukemia (CLL), splenic marginal zone lymphoma (SMZL), mantle cell lymphoma (MCL), hairy cell leukemia (HCL), follicular lymphoma (FL), Sézary's syndrome (SS); 2) blasts corresponding to promyelocytic leukemia (APL) and other myeloid or lymphoid leukemias (BL); 3) plasma cells (PC) and; 4) reactive lymphocytes (RL). We used a deep learning approach by fine tune a Resnet-101 convolutional deep net, producing a model trained with a part of the above cell images.
Results: To evaluate independently the performance of the classification model, we smear-divided the total images set in 80% for training and a 20% for test. Thus, the test set contains blood smears not present in the training set, avoiding a smear-biased evaluation. The test classification accuracy of this procedure was 82.23%. Figure 1 shows the confusion matrix corresponding to the whole process. Rows represent the true cell types and columns the predicted cell types (in percentage per true cell type). The diagonal gives the true positive rates for each type.
Conclusions: This work developed a deep learning approach, able to automatically recognize between 12 different groups of cells including normal, reactive, abnormal lymphocytes and blast cells. This methodology processes the cell images in only one step (calculate the weights of the deep net), being faster to develop and deploy a model than the traditional approach. This methodology has been tested using a independent set, as it would be in a clinical trial. The final model could help to the pathologists in the recognition of pathological cell images, being a practical diagnosis support tool.

Neoplastic Blasts Or Non-Neoplastic Immature Mononuclear Cells? Towards Rule Based Identification Using Electronic Health Records Data Mining
Nicholas J. Bevins, H. Elizabeth Broome
UCSD, La Jolla, CA, United States

Introduction: Distinguishing neoplastic blasts from non-neoplastic immature mononuclear cells (IMC) on blood smears is important since neoplastic blasts are often a presenting finding in hematologic neoplasms.  However, neoplastic blasts and IMC can have identical morphology, requiring additional testing (e.g. flow cytometry) for definitive identification. At the University of California at San Diego (UCSD) clinical hematology laboratory, historical findings are used to determine whether to report neoplastic blasts versus IMC, reducing the need for the additional testing in many cases. Our hypothesis is that a data mining approach may help semi-automate decisions about reporting neoplastic blasts versus IMC. 
Methods: Technologists at UCSD report immature cells as “Others” for a hematopathologist to categorize as neoplastic blasts or IMC in a text comment. In 2018, of the 2.76 million patients receiving care in at UCSD 1,565 patients had “Others” called with 452 identified as having neoplastic blasts and 1,113 identified as IMC. Patient data from the EHR was extracted only for the month of the “Others” finding and is selectively summarized in Table 1. Additional variables (22) were analyzed but not shown. 
Results: Table 1. Comparisons between groups
 Patient Data within Month Neoplastic Blasts (%) IMC (%) P Value
Received stem cell mobilizing drug 20.9 33.2 2.7E-4**
Received corticosteroids 36.2 45.6 9.8E-4*
Received anti-neoplastic therapy 74.1 56.5 8.3E-6**
RBC flagged abnormal 97.2 88.9 1.7E-6**
PLT flagged abnormal 90.1 79.9 1.4E-6**
Myelocytes, promyelocytes, or metamyelocytes resulted 88.6 80.9 1.3E-4**
LDH resulted 81.7 54.3 3.2E-10**
If resulted -> LDH flagged abnormal 97.0 93.2 2.0E-3*
Blood culture resulted 32.0 42.6 1.5E-3*
If resulted -> Organism identified 17.8 19.2 0.7
Diagnosis of CML/AML/MDS 36.0 18.7 3.1E-9**
Bone marrow biopsy performed 13.6 5.9 1.5E-3*
Two-tailed t-test of significance * Significant at p=0.01 level ** Significant at 2.9E-4 level (p=0.01 with Bonferroni correction applied for 34 comparisons)
Conclusions: Our data indicate that “Others” classified by hematopathologists into neoplastic blasts versus IMC categories show significant differences with multiple variables encoded in the UCSD EHR. These findings will inform the development of a multi-variate rules-based mechanism to aid the distinction of blasts from IMC in proper clinical context.

Use OfDigital Morphology In Hematology Training: An Added Value?
Anna Maria Cenci, Fabrizio Papa, Barbara Casolari, Maria Golato, Marco Moretti, Valentino Miconi, Maria Diquattro, Luciano Pasini, Bruno Biasioli
Italian Society of Pathology and Laboratory Medicine - Hematology Study Group (SIPMeL-GdSE), Castelfranco Veneto (TV), Italy

Introduction: Morphology still plays a basic role in hematological diagnostics, overtime enriched by the contribution of instrumental data for Blood Cell Count (CBC). Nowadays we see an important, increasing and successful diffusion of digital morphology, employed in facilitating the CBC pathways, and improved by easy and quick use, link to automated systems/hematologic chains, while doubts related to the ability to appreciate the complete blood contest remain.
Methods: SIPMeL GdS-E inserted in 2018 a new one-day course, with 3 theoretical-practical modules in its teaching activity. The 2 practical parts discuss clinical cases and review cell images in plenary session. 4 courses on technological innovations, new CBC parameters and morphology contribution have been performed in central and southern Italian regions.  259 users were involved and examined 15-30 smears/course, implemented as follows: 1 used images from traditional paths only (morphology taken by operator on to routine or dedicated with digital cameras); 2 “blended mode”, traditional and automated digital material; 1 Digital morphology alone realized by automated instrument. Even in the latter case, however, two images compared traditional and new method results on purpose.The answer were evaluated immediately, in 2 cases by automatic responders or by Smartphone apps. The agreement was reached via questionnaire.
Results: - 259 Laboratory Professionals tested had the same degree of correct performance both with traditional and digital morphology (60-92%). Correct response seemed to be tied more to the difficulty of the proposed image (Worse performance: dysplastic morphologies; asynchronies; blood parasites). - No special negative reactions nor declared difficulties in understanding the submitted digital images were observed, even for operators, mostly without experience in using digital morphology.
Conclusions: This experience results support SIPMeL GdSE in using this training facility in the future, with the current instrumental ongoing improvements. Useful in discussing single item cytology, however, the lack of the scenario vision constitutes the most important limit in studying the relationships between blood cells. In contrast, the good quality needed in the preanalytical phase for this technique (optimized smears) can constitute a first step to promote the  standardization of language not yet present in the cell description and in cell reports. The experience confirms digital morphology as an excellent tool for team training.

Screening Of Chronic Lymphocytic Leukemia Using Wpc Scatterplots Of Sysmex Xn Analyzers
Eunhee Han, Kyungja Han
Department of Laboratory Medicine, The Catholic University of Korea, College of Medicine, Seoul, Korea

Introduction: The chronic lymphocytic leukemia (CLL) is the most common leukemia of the mature lymphocyte with no characteristic signs and symptoms. It shows marked lymphocytosis on CBC. Many acute lymphoblastic leukemia (ALL) samples show lymphocytosis on CBC analyzer too. Therefore, if the automated hematology analyzer can detect CLL during CBC, it would be valuable. The leukemic cells in most CLL cases are B lymphocytes with expression of CD 19 and CD 5. The CLL cells are mainly small lymphocytes with a few prolymphocytes. Sysmex XN analyzer has 3 different reagents channels for WBC and shows 4 scatterplots per each channel. Each scatterplot shows various morphologic characteristics of each cell population in each reagent bath. The lymphocytes are divided into 2 populations in WPC channel, low FSC and high FSC populations, but shows only 1 lymphocyte population in WDF and WNR channels. We reviewed all of these WBC scatterplots in samples from patients with various diseases to find out characteristic findings of CLL.
Methods: From May 2017 to December 2018, we analyzed XN WBC scatterplots of 1,055 samples including 119 normal samples, and samples from 87 CLL, 2 T-PLL, 2 leukemic phase of mantle cell lymphoma, 81 ALL, 76 AML, 39 chronic phase of CML patients and 649 samples from patients with other diseases or after treatment of leukemia using XN-20 (Sysmex, Kobe).
Results: All 87 CLL samples showed characteristic scatterplots in WPC channel. The low FSC cell populations are markedly increased in number and they showed widely dispersed SSC and SFL and increased FSCW. The scatterplots of WDF and WNR channel were not different from other samples with lymphocytosis. Two samples of leukemic phase of mantle cell lymphoma showed similar findings to CLL. These characteristic findings are not found in any other samples from patients with other diseases including ALL. The 2 samples of T-PLL patients showed very interesting findings with increased high FSC lymphocyte population and not low FSC population.
Conclusions: The XN scatterplots are very valuable for screening of CLL. If the samples are from B-cell lymphoma, similar scatterplots could mean leukemic transformation of the lymphomas. It could help differential diagnosis of CLL from other diseases with lymphocytosis on CBC including ALL also.

Clinico-Hematological Profile And Morphological Features On Bone Marrow Aspirate In Visceral Leishmaniasis From Non-Endemic Region
Harish Chandra, Vandna Bharti, Arvind Gupta, Neha Singh, Sanjeev Kishore
All India Institute of Medical Sciences, Rishikesh, India

Introduction: Visceral leishmaniasis is vector borne parasitic disease caused by Leishmania donovani involving macrophages in reticuloendothelial system. The higher altitudes negatively affect distribution of vector and therefore leishmaniasis is considered to be almost absent in highlands. However, cases demonstrating Leishmania donovani bodies on bone marrow aspirate have been observed in our center which caters to non-endemic hilly regions. The present study was conducted to evaluate clinico hematological profile and morphological features on bone marrow examination in leishmaniasis from non-endemic region along with categorization of morphological features into common, uncommon and atypical features.
Methods: The study included all the cases of leishmaniasis which were diagnosed on bone marrow aspirate cytology by demonstration of LD bodies on Giemsa stained smears over period of one and half year. Relevant clinical details, residential address, laboratory investigations and morphological features on bone marrow aspirate were recorded for every case. The average parasite density, hemophagocytosis and morphological findings observed on bone marrow aspirate cytology were also noted and categorized into common, uncommon and atypical according to frequency of presentation.
Results: The study included total 10 cases of visceral leishmaniasis which were diagnosed on bone marrow aspirate cytology over period of one and half years. Out of these, 8 cases were observed in males and 2 cases in females and all the cases were resident of non-endemic region. Out of these 6 cases (60%) were residents of places which are at the height of 800 m or above sea level. Fever (90%) and hepatosplenomegaly (80%) was the most common clinical presentation with pancytopenia (90%) and anemia (80%) being the most common hematological finding. 80% of cases showed low parasitic density score of 1-2+. On bone aspirate plasmacytosis (90%), hem phagocytosis (80%) were the common findings, plasma cell with abnormal inclusions (30%), dysmelopoiesis (20%) were uncommon findings and Donovoni body with kinetoplast only (10%), giant cells (10%) were atypical features.
Conclusions: Leishmaniasia may be associated with common, uncommon and atypical hematological features which may be helpful in clinching an early and correct diagnosis especially in non-endemic areas where clinical suspicion is low. They may also guide the pathologist for vigilant search of leishmania donovani bodies in marrow aspirate for definite diagnosis and prevent unnecessary workups.

Significance Of Persistent T(15;17) By Fluorescence In Situ Hybridization In Acute Promyelocytic Leukemia Patients With Morphologic Response To Induction Chemotherapy
Jonathan Galeotti1, Stephanie Mathews2, Marian Rollins-Raval3
1UNC Healthcare, Chapel Hill, NC, United States, 2University of North Carolina, Chapel HIll, NC, United States, 3University of New Mexico, Albuquerque, NM, United States

Introduction: While the gold standard of response to induction therapy in acute promyelocytic leukemia (APL) is < 5% blasts/promyelocytes (B/P) by aspirate marrow differential (AMD), cytogenetic FISH and/or molecular studies are also evaluated. In most cases, morphologic and cytogenetic/molecular findings are consistent, but in a subset of cases the findings are discrepant. When B/P are ≥5% by AMD, but FISH studies demonstrate < 5% involvement, regeneration of the marrow is presumed. As significance of persistent t(15;17) by FISH in patients with < 5% B/P by AMD is unclear, we sought to evaluate this discrepancy in relation to risk of relapse and disease-related mortality.  
Methods: Retrospective review of medical records was performed from 4/2012-6/2017 to identify all consecutive patients newly diagnosed with APL treated at a university hospital. We identified 36 patients, 5 of whom were excluded for lack of follow-up, including death prior to or insufficient information from the first post-induction marrow. In the 31 remaining patients with post-induction marrow evaluations,  morphologic findings, ancillary studies, and clinical information were documented. Categorical variables were analyzed by Fisher’s exact test, and statistical significance was defined as p< 0.05.
Results: Five of 31(16%) patients demonstrated < 5% B/P by AMD but ≥5% cells with t(15;17) by FISH. These 5 patients had persistent PML-RARα transcripts. Compared to patients with no morphologic or cytogenetic evidence of persistent disease, those with < 5% B/P by AMD but ≥5% cells with t(15;17) by FISH actually had no increased risk of relapse or death (p=0.7 and 1.0, respectively).  Furthermore, this discrepant group trended towards lower incidences of DIC and differentiation syndrome (p=0.1 and 0.1, respectively).
Conclusions: t(15;17) status by FISH in patients with morphologic response to therapy at time of post-induction marrow evaluation does not appear to impact risk of relapse or mortality. Patients with persistently positive FISH results, but < 5% B/P by AMD, showed a lower rate of DIC and differentiation syndrome compared to all other groups. The broader significance of these findings is unclear, especially given the small number of patients, and larger studies are needed to confirm these preliminary results.

Clinical Features And Diagnosis Of Loa Loa InA Bone Marrow Aspirate
Ping Guo
Shanghai Jiaotong University School of Medicine Ruijin Hospital , Shanghai, China

Introduction: Filariasis has been a disabling parasitic disease worldwide particularly in tropical and subtropical countries of the world. Apart from the blood and the lymph node aspirates, microfilariae have been demonstrated in the fine needle aspiration cytology smears from various sites like the thyroid, breast and subcutaneous nodules. These cases were most caused by Wuchereria bancrofti and Brugia malayi. But there was rarely cases of filariasis infiltrating the bone marrow leading to eosinophilia by Loa Loa.
Methods: Patient’s clinical history and symptoms were collected. Peripheral blood specimen was collected at 11:00 a.m. and 9:00 p.m. Routine blood test was performed after aspiration and blood smears were made immediately. The remaining specimen (about 8ml) were centrifuged (378×g,5minitues) and hemolyzed with distilled water. Discard the supernatant and take the sediment to make the smears. Peripheral blood smears, centrifuged sediment smears and bone marrow aspirates were stained with Wright’s staining for parasite examination. The internal transcribed spacer 1 (ITS-1) region of the parasite was sequenced after PCR amplification and compared with BLAST program of GenBank.
Results: On examination, the patient showed swellings and darkening of the skin in distal left upper extremity. He worked in DR Congo three years ago for six months and returned to China in January 2016. Routine blood test in local hospital showed increased eosinophil count, and magnetic resonance imaging showed inflammatory lesions of partial muscle groups in the left forearm and tendons in the wrist joint, which lead to a diagnosis of "eosinophilic fasciitis". In our laboratory, eosinophils count was 8.2×109/L. Microfilariae were found in blood smears and bone marrow aspirates (Fig1A, Fig1B). More Microfilariae were found in centrifuged sediments (Fig 1C). PCR amplification resulted in a band of ~457 bp (Fig 2),sequence of which had 100% homology to that of Loa loa (GenBank Accession No:DQ995497). The microfilariae were identified as Loa loa microfilariae by morphological observation and gene sequencing. The patient was diagnosed as loiasis and oral ivermectin at 150μg/(kg·d) was performed. After 2 weeks of treatment,The eosinophil count was 6.39×109/L and no microfilaria was found in the peripheral blood,but 12 microfilariae were found in concentrated blood smears. 
Conclusions: Loa loa in bone marrow can be diagnosed by parasitological examination and molecular detection.

Burkitt- Lymphoma And Burkitt-Like Lymphoma/B-Cell Lymphoma Unclassifiable With Features Intermediate Between Diffuse Large B-Cell Lymphoma And Burkitt Lymphoma: Clinico-Pathological Features And Survival From An Academic Hospital In Cape Town
Ania Koller1,2, Katherine Antel1, Nicolas Novitzky1,2, Jessica Opie1,2
1University of Cape Town, Cape Town, South Africa, 2National Health Laboratory Services, Cape Town, South Africa

Introduction: South Africa has the highest global burden of Human Immunodeficiency Virus (HIV) infection. The HIV seropositive (+ve) population is at markedly increased risk of developing non-Hodgkin lymphoma, particularly high grade aggressive subtypes such as Burkitt- and Burkitt-like lymphoma (BLL), previously also called B-cell lymphoma unclassifiable with features intermediate between diffuse large B-cell lymphoma and Burkitt lymphoma (BCL-U).
Methods: Retrospective review of clinico-pathological features and survival of adults newly diagnosed with Burkitt lymphoma (BL) and BLL/BCL-U between 2005 – 2014, at Groote Schuur Hospital, a tertiary academic hospital in Cape Town, South Africa. 
Results: 149 patients were included for analysis; 60% female (n=90) and 85% HIV positive (n=125). Burkitt lymphoma (n=109) was more frequent than BLL/BCL-U (n=40).  There were no significant differences between the BL and BLL/BCL-U groups in HIV prevalence, median CD4 count; bone marrow involvement and leukaemic dissemination (Table 1). The most frequent site of diagnosis in both groups was nodal (30%), with 23% of all BL cases diagnosed on bone marrow biopsy.  Patients with BL were more likely to be younger (p=0.03), and have a higher LDH level (p< 0.01) (Table 1). Overall survival (OS) at 2 years was 39% (95% CI 31-47%) and at 5-yr 36% (95% CI 28-44%). There was no significant difference in mortality between BL and BLL/BCL-U (HR 0.96, 95% CI 0.62-1.5). The HIV positive group had a non-statistically significant increase in mortality (HR 1.6 95% CI 0.87-2.95). Leukaemic presentation was associated with a 4-fold increase risk in mortality (HR 3.6 (95% CI 2.3-5.5).
Conclusions: This is the largest cohort of BL and BLL/BCL-U described in South Africa. There was a high HIV prevalence. Leukaemic presentation was frequent and associated with less favourable survival. The majority achieved OS  comparable to similar local and international middle income settings.

Performance Of The Sysmex Di-60 With Special Focus On Neutrophil Granulocytes
Johan Mathillas, Emma Strandgren
Klinisk Kemi, Ume, Sweden

Introduction: The Sysmex Di-60 is an automated digital cell morphology analyzer. The performance of this
instrument was explored in comparison with manual differential count and the Sysmex XN cell counter.
Methods: 148 samples were taken from clinical laboratory routine. These samples had either been flagged for abnormalities by the Sysmex XN and converted to manual microscopy by the standard operating procedure or directly requested as manual microscopies by the clinician. They were reviewed by experienced morphologists. After completion of manual microscopy, the samples were analyzed on the Di-60. The pre-classification made by the Di-60 was then compared to the manual microscopy. Subsequently the same morphologist who
originally reviewed the slide in manual microscopy performed a differential on the Di-60. This result
was then compared to the original microscopy. Correlations were explored with weighted Deming
regression on the Method Validator software (Philippe Marquis, 1999). In addition, 20 of the 148
samples with reported neutrophil granulocyte counts < 1*10^9/L were analyzed by 3 different
morphologists. The Rumke binomial distribution 95% confidence limits were applied to their median
neutrophil count, and the Di-60 and Sysmex Xn neutrophil values were compared to these limits.
Furthermore, ten peripheral blood slides from the Swedish external quality assurance program
EQUALIS were analysed on the di-60. Correlation was again explored with weighted Deming
Results: Correlation for neutrophil granulocytes when comparing microscopy to Di-60 pre-classification was
0,966, and after verification by morphologist 0,991. Correlation for lymphocytes when comparing
microscopy to Di-60 pre-classification was 0,969, and after verification by morphologist 0,979.
Correlation for monocytes when comparing microscopy to Di-60 pre-classification was 0,963, and
after verification by morphologist 0,991. Correlation for eosinophils when comparing microscopy to
Di-60 pre-classification was 0,138, and after verification by morphologist 0,392. Correlation for
basophils when comparing microscopy to Di-60 pre-classification was 0,597, and after verification by
morphologist 0,797. Correlation for blasts when comparing microscopy to Di-60 pre-classification
was 0,988, and after verification by morphologist 0,996. When applying the Rumke 95% confidence
limits to the 20 samples with reported neutrophil granulocyte counts < 1*10^9/L, the Di-60 neutrophil
preclassification was within limits in 17/20 cases. When counting neutrophil granulocytes in the 10
EQUALIS samples, pre-classification correlation with supplied expert group reviews was 0,764.
Conclusions: The Di-60 has acceptable performance regarding leukocyte classification after slide verification.

Multicenter Analysis Of Smear Review And Laboratory Parameters Contributing To The Detection Of Unexpected Malaria
Anna Merino1,9, Rosa Fernndez1, Laura Bigorra2,9, Angel Molina1,9, Cristian Morales-Indiano3,9, Javier Nieto4,9, Maite Serrando4,9, Xavier Tejedor3,9, Elosa Urrechaga5,9, Teresa Villalba6,9, M Jos Alcaide7,9, Eduardo Arellano1,9, M Helena Redn8,9, Mara Sanz7,9
1Hospital Clinic., Barcelona, Spain, 2Synlab Global Diagnostics, Barcelona, Spain, 3Germans Trias i Pujol University Hospital, Badalona, Spain, 4Hospital Dr. Josep Trueta, Girona, Spain, 5Hospital Galdakao Usansoslo, Bilbao, Spain, 6Catlab, Terrassa, Spain, 72La Paz University Hospital, Madrid, Spain, 8Donostia University Hospital, San Sebastin, Spain, 9SEQC_ML Hematological Biology Committee, , Spain

Introduction: Malaria is a parasitic infection, caused by Plasmodium species. Prompt and accurate diagnosis is critical to the effective management. The peripheral blood (PB) film revision remains the gold standard in diagnosing malaria and allows identifying species and recognizing parasite forms. We present laboratory parameters and morphology findings obtained in patients diagnosed of unexpected malaria. Patients were from hospitals related to the members of the Committee on Hematological Biology of the SEQC_ML.
Methods: A total of 9 patients, in which an unexpected malaria was diagnosed during the last six-month period in 2018 were included. Analyzers used for the CBC were Advia 2120i (Siemens), Coulter DxH (Beckman Coulter) and Sysmex XN (Sysmex). PB samples were stained with MGG and PB review were done using the microscope or in the CellaVision DM96.
Results: Fever was the reason for consulting in 7/9 patients in the emergency department (6). In one patient, fever was presented after 13 days of a heart transplant. The remaining 2 were visited for medical work review (1) or at the family medicine department (1). We summarize the abnormal parameters found in the CBC and other biochemical data (Figure 1). With respect to PB film review, cell digital images showed the parasites in 6/9 cases and the microscope was used in 3 laboratories. Figure 2 shows the most representative images. Parasitemia was 14% in patient recipient of a heart transplant, < 1% in 4 patients and unknown in the remaining. Species of plasmodium in these unexpected infections were: falciparum (4), vivax (4) and ovale (1).
Conclusions: The results presented in this work demonstrated the great value of the PB film review (microscope or digital images) to detect unexpected different Plasmodium species in clinical laboratories. Hematological parameters, such as high leukocyte count and low hemoglobin or low platelet values were the most common trigger hematological abnormalities. In this sense, the hematology laboratory has a relevant role in the detection of malaria.

Detection Of Malaria Infection By A Novel All-In-One Point-Of-Care Device Using Deep-Learning Algorithm
Youngmin Shin1, Mijin Kim1, Younghoon Song1, Yoontaik Hong1, Jiyeon Lee1, Chae Yun Bae1, Douglas Lungu2, Seongsoo Jang3, Hans-Peter Baek4, Dongyoung Lee1
1NOUL, Inc, Yongin, South Korea, 2Daeyang Luke Hospital, Lilongwe, Malawi, 3Dept. of Laboratory Medicine, Asan Medical Center and University of Ulsan College of Medicine, Seoul, South Korea, 4Swiss Tropical and Public Health Institute, University of Basel, Basel, Switzerland

Introduction: Malaria remain a major health risk around the world, especially in low-to-middle income countries. Despite of progress in rapid diagnostic test (RDT) and molecular diagnostics, manual microscopic examination of blood smears remain as the gold standard in malaria diagnosis. Manual microscopic examination suffers from its own shortcomings such as stechnician error and inconsistencies inherent in manual blood smear. In order to address these issues, an all-in-one, point-of-care miLab® (noul, Korea) platform was developed for automated blood smear, liquid-free stain and deep-learning based malaria detection and diagnosis.
Methods: 5ul of whole blood was loaded into miLab®, which automatically smeared, stained and scanned 250,000 red blood cell (RBC). The deep-learning based diagnostic algorithm was trained with 987 P. falciparum positive RBCs collected from 9 Malawian patients. The device performance was assessed based on blood samples from 1,869 (male 864, female 1,004) Malawian patients (Daeyang Luke Hospital & Likuni community hospital, Malawi) and was compared to the results from thick smear microscopy examination by Swiss TPH professionals. The result was presented as the ratio of the number of infected RBCs to the total number of detected RBCs.
Results: miLab® consistently smeared, fixed and stained samples in 15 minutes with coefficient of variation around 3% for RBC count. The device consistently produced evenly stained samples with more than 90% of RBCs smeared as monolayers (Figure 1. a-b). The sensitivity of 97.22% with specificity of 77.78% was achieved when the threshold was set to high sensitivity. Specificity of 100% corresponded to sensitivity of 88.9%. In the balance point where sensitivity is equal to specificity (94.44%), the ROC curve intercepts with the 45 degree line (Figure 1c). The specificity of miLab® (94.44%, n=52) was 9.44%p higher than that of RDT (85%, n=101) when both were compared to manual microscopy.
Conclusions: Results show that malaria infection could be screened with miLab®, an all-in-one point-of-care device, with the benefits of quick turnaround, minimal training and ease of use. Despite being trained with relatively limited training set (n=987), miLab® malaria detection algorithm showed sensitivity and specificity of 94.4%. The performance is expected to improve with a larger training set.

Abnormal Neutrophil Nucleus Morphology Detected Due To Abnormal Differential White Blood Cells Analysis
Maria Stamou1, Agapi Kotanidou1, Athanasios Tsiligiris2, Paraskevi Aslanoglou1, Athanasia Agorasti1
1Haematology Laboratory, General Hospital of Xanthi, Xanthi, Greece, 2Microbiology Laboratory, General Hospital of Xanthi, Xanthi, Greece

Introduction: On hematology analyzer Sysmex®XE-5000TM in the differential (DIFF) scattergram, side scattered light (SSC), providing information on internal cell structure (x-axis), and side fluorescence (SFL), providing information on RNA/DNA content (y-axis), are employed. NEUT-X and NEUT-Y are the mean values for both side scatter diffraction and fluorescence measurement for the neutrophil population. NEUT-X is representative of the inner complexity and the granularity of neutrophils, while NEUT-Y reveals the neutrophil nucleic acid / protein content and is related to production or release of proteins and reactive oxygen intermediates. Granularity Index (GI) is defined based on NEUT-X value. Red blood cells (RBC) are lysed and a ghost population is formed.
Methods: A 75-years old woman presented to the Emergency Department with the following symptoms and signs: side (flank) pain, burning sensation when urinating, nausea, vomiting and fever. Full blood count, urine analysis, urine culture, and blood culture were ordered. Full blood count was analyzed on Sysmex®XE-5000TM. Standard urine analysis and standardurine culture quantitativemethod were performed. Blood bacterial growth was monitored by BACT/ALERT®3D (BIOMÉRIEUX). Bacterialidentification and antibiotic susceptibility testingwere performed on VITEK®2 (BIOMÉRIEUX).    
Results: The patient has normocytic-normochromic anemia (Hb = 8.4g/dL) and leukocytosis (white blood cells = 35.15 x103/μL). The DIFF scattergram was abnormal, without differential white blood cells. Neutrophils & basophils population and RBC ghosts were gray (instead of turquoise and blue respectively). There was a discrepancy between NEUT-X and NEUT-Y values. NEUT-X was low (122.8 ch, reference interval 135.6-138.8 ch, indicative of hypogranularity with GI < -3 and thus dysfunction), while, on the opposite, NEUT-Y was high (40.7 ch, reference interval 36.4-39.3 ch, indicative of enhanced activation, as seen in bacteremia). The blood smear analysis on CellaVisionTM DM1200 revealed abnormal neutrophil morphology; in particular, abnormal nucleus segmentation, presence of cytoplasmic toxic granulation, vacuoles and Döhle bodies (Figures 1, 2). Escherichia coliwas isolated in both urine and blood culture.   
Conclusions: The abnormal DIFF scattergram was due to neutrophil abnormal inner structure. The low NEUT-X value was due to abnormal nucleus segmentation and not to hypogranularity and the high value of NEUT-Y did reflect the reactive neutrophilia.  

Vaccine Development & Participation In Sub Saharan Africa: How Willing Are Young People In Western Nigeria
Saheed Usman1, Ibiwumi Usman2
1Nnamdi Azikiwe University, Awka, Nigeria, 2Ladoke Akintola University of Technology, Ogbomoso, Nigeria

Introduction: An estimated 36.7 million people live with HIV/AIDS in 2015, with more than 3 million people living with the virus in Nigeria. Because adults are mostly affected by this epidemic, their inclusion in HIV vaccine trials is of utmost importance in obtaining an effective and acceptable vaccine. This study is thus aimed at evaluating the factors determining adults (young persons) willingness-to-participate (WTP) as well as their entire knowledge and perception about HIV vaccine trials.
Methods: Data was obtained from 3500 young persons (18-49 years) recruited by a multi-stage sample technique. The cross-sectional study was carried out using a face-to-face interview. An informed consent was obtained through a pre-tested structured questionnaire, with questions addressing socio-demographics, HIV vaccine studies knowledge and perception, sexual behaviour and possible stigma from HIV vaccine trial participation.
Results: The mean age ± SD was 27.53 ± 3.46 years. 1094 (31.3%) expressed their willingness to definitely participate in the vaccine studies while 999 (28.5%) reported that they may participate especially if a very tangible incentive will be given. Unwillingness to participate was associated with safety concerns (12.0), side effects (5.0%), fear of HIV infection from vaccine (4.1%), time required for study (1.9%) and partner’s sexual intercourse refusal (1.2%). 983 (28.3%) reported people in good health, HIV negative individuals and at low risk of HIV infection, are eligible for HIV vaccine trial. There was a significant association between willingness to participate in HIV vaccine trials and age as well as gender.
Conclusions: Participation in an HIV vaccine trial in a Nigerian context is likely to be influenced by comprehensive education about the vaccine trial concept, addressing issues relating to concerns and possible risks pertaining to participation and incentives, as the WTP in the vaccine trial is quite low probably due to the participants’ perception and inadequate knowledge.

Deep Learning-Based Nuclear Lobe Count Method For Differential Count Of Neutrophils
Mayu Yabuta1, Kana Nanato1, Kazunori Okada2, Sanae Kaga2, Nobuo Masauzi2
1Graduate School of Health Sciences, Hokkaido University, Sapporo, , Japan, 2Faculty of Health Sciences, Hokkaido University, Sapporo, , Japan

Introduction: The differential count of neutrophils by counting nuclear lobes for the diagnosis of various diseases such as sepsis, megaloblastic anemia or myelodysplastic syndrome requires an intensive labor. Therefore, we developed an automated method for this using deep learning (DL).
Methods: Peripheral blood (PB) films were prepared using venous PB obtained from 4 healthy volunteers and stained with May-Grünwald Giemsa stain. Six hundred nuetrophil images with 5 different types of nuclear lobes, i.e., band-formed (band), two-segmented (2-seg), three-segmented (3-seg), four-segmented (4-seg), and five-segmented (5-seg) lobes, were automatically captured using CellaVision® DM96. Of these, 500 images were used to make a training image set (ETR). The remaining 100 images made up a test image set (ETE). Several neural networks (NNs) containing multi-convolution layers were developed using the Neural Network Console (Sony Network Communications Inc.). Each image both in training and test image set was labeled according to the nuclear lobe count. The number of optimization iterations for machine learning (ML) was up to 300 cycles.
Results: All developed NNs were converged with ETR within 300 cycles of ML. However, the maximum accuracies in 5-times distinction among the band and 2-seg to 5-seg for each type of image in ETE were ranged from 0.834 to 0.198. These were too small, especially so between 4-seg and 5-seg. Therefore, we made those types into a single group (4&5-seg) of 500 and 100 images for a training image set (ATR) and a test image set (ATE), respectively. Then we created three new extended training image sets (BTR, CTR, and DTR), comprising 2,000, 2,000, and 3,500 images, respectively. Those were the inversion, rotation, and distortion of each copied image in ATR. Next, 300 cycles of ML were performed 5 times using ATR, BTR, CTR, and DTR. Using a NN with the best accuracy for each training set, the average accuracy values (maximum-minimum) for the ATE in 5-times trials evaluated each for 4 training sets. These were 0.584 (0.700-0.403), 0.877 (0.983-0.728), 0.829 (0.978-0.720), and 0.889 (0.990-0.740), respectively.
Conclusions: We developed a DL system that facilitates the differential count of neutrophils by 4 different types of nuclear lobes  —band, 2-seg, 3-seg and 4&5-seg— with up to 99% accuracy.  

Evaluation Of The Body Fluid Cycle Of The YumizenH2500 Analyzer (Horiba Medical Sas).
Anais Inquel1, Martin Broly1, Myriam Chevalier1, Khadija Viau1, Marie C Bene1, Elise Tomini2, Camille Debord1, Elise Darrambide2, Marion Eveillard1
1Service d'Hematologie Biologique, CHU de Nantes, Nantes, France, 2Societe Horiba Medical SAS, Montpellier, France

Introduction: Automated analysis of biological fluids is now required to ensure the robustness and reliability of results reporting. In this study, we evaluated the performance of the body fluid cycle of the Yumizen®H2500.
Methods: Ninety-eight samples including cerebro-spinal (6), pleural (18), ascitic (5), synovial (54), pericardic (2) and bronchoalveolar liquid (BAL, 13) were collected over 10 weeks in a prospective study at Nantes University Hospital. The samples were analyzed in duplicate by the Yumizen®H2500 to determine the number of leukocytes and erythrocytes, the partial formula (% of mononuclear cells, %MN and % of polynuclear cells, %PN) and to detect abnormal cells. Results were compared to a conventional microscopic count and to the body fluid mode of the XN10 (Sysmex®) reference analyzer.  Both instruments were calibrated prior to the samples analysis using external calibrators. Passing-Bablok regression was used to analyze results.
Results: Seventy-four out of 98 samples were used to compare leukocyte and erythrocyte count. Eleven samples were excluded because they were outside the range of linearity of the analyzers and the BAL were excluded due to viscosity related flags. The correlation coefficient between the 2 analyzers was 0.991 for leukocyte and 0.955 between Yumizen®H2500 and the conventional chamber counting method. Discrepant results were observed in lipemic samples and samples containing crystals. For erythrocyte counts, the correlation coefficient was 0.996 between both analyzers. For the differential count, seventy-one samples were retained. Comparison of the analyzers yielded a slope of 0.931, intercept of +0.18 and a correlation coefficient of 0.933. The coefficient was 0.914 between the manual mode and Yumizen®H2500. The presence of abnormal plasma cells in the sample caused discrepant results between both analyzers and between the Yumizen®H2500 and manual count. The %MN correlation coefficient between the two analyzers, and between the Yumizen®H2500 and the manual mode was respectively 0.933 and 0.917.
Conclusions: This study confirmed the good analytical performance of the Yumizen®H2500 in comparison with conventional microscopic count as well as with the XN10 analyzer. In addition, data from this study allowed the scattergram thresholds used for the leukocyte count and the differential to be optimized.

Does Platelet Count Impact On Measurements Of Platelet Adhesion Under Static Condition Using Collagen Coated 96 Well Plates?
Mohammad Algahtani
Comperhensive Specialised Clinics -Security Forces Ministry Of Interior, JEDDAH, Saudi Arabia

Introduction: Platelets play an important role in preventing bleeding whenever vessel injury takes place. Adhesion is the first step toward thrombus formation in which the platelets interact through receptors with collagen in the subendothelium. Assays of platelet adhesion under static conditions have been widely used to measure the initial stage of this process. We have developed a Static Platelet Adhesion assay and have examined the influence of platelet count on the results obtained.
Methods: Blood was taken from healthy volunteers who denied taking antiplatelet drugs; which was anti-coagulated with hirudin. Platelet rich plasma (PRP) was prepared by centrifugation and then treated with the GPIIb/IIIa antagonist,MK-852 to prevent platelet aggregation or withsaline as control. Black 96 well plates with a clear bottom coated with collagen type I from rat tail were obtained commercially.  PRP of differentcounts was prepared by dilution with platelet poor plasma (PPP) and added to the plates. The plates were then incubated at 370C in a CO2 incubator for 45 minutes; following this platelets were fixed for 30 minutes at room temperature.  Immunohistochemistry is carried out using FITC-labelled CD42a antibody and detected using an IRdye800 conjugated secondary antibody. Imaging and analysis was carried out using the LICOR Odyssey 3.0.29 software which measures the fluorescent intensity. This reflects the amount of platelet adherence to the collagen.
Results: The results demonstrated in the table show that the fluorescent intensity, expressed as the mean percentage of maximum response (n=7),are directly proportional to the platelet count. However, results were lower in the presence of MK-852 except for the lowest platelet count (50X109/L). 
Platelet number (109/L) 50 100 200 400
Mean count of PRP after dilution with PPP 43 90 191 391
(-) MK-852 17.7 40.2 80.9 138.1
(+) MK-852 18.2 10.6 23.6 43.2

Conclusions: This study shows clearly the direct relationship between the platelet number and adhesion. MK-852 blocked any platelet aggregation thus facilitating clear studies of platelet adhesion. It is necessary to be aware of any aggregation, particularly in PRP with higher platelet counts which may interfere with adhesion measurement. Future studies of PRP with low platelet counts may provide an alternative platelet function test for thrombocytopenic patients.

Prevalence Of Pseudothrombocytopenia In A Major Teaching Hospital In Nigeria
Abiola Bolarinwa, Ademola Adewoyin, Ann Ogbenna, Adejoke Olatinwo
Lagos University Teaching Hospital, Lagos, Nigeria

Introduction: Pseudo-thrombocytopenia (PT) is a relatively common occurrence in our day to day laboratory practice. Usually due to platelet clumping, some identified causes of platelet clumping includes; improper sample collection method, EDTA induce Clumping , Type 2 VWD, May-Heglin anomaly and other pre-analytical and post analytical factors.  Findings of Platelet clumps, large and giant platelet on peripheral blood film suggests that the true platelet count might be higher than reported on the haemogram. This condition leads to improper counting of platelet and report of low platelet count. This has a huge implication on patient care, leading to delay in surgeries, chemotherapy and other intervention, spending of precious, insufficient fund on investigation of thrombocytopenia, unnecessary usage of blood component leading to wastage. Our laboratory is the main haematology laboratory in a major tertiary hospital located in the southwest region of Nigeria. We are not excluded in the challenges of pseudo-thrombocytopenia. This study will aim at determining the prevalence of thrombocytopenia and pseudo-thrombocytopenia.
Methods: We retrospectively reviewed the Full blood count data of 2000 patients received over a period of 3 months. Peripheral blood film report of all cases of thrombocytopenia was accessed and in few cases fixed smears were stained and reviewed. For the sake of this study Thrombocytopenia was described as platelet count < 100 and Pseudo-thrombocytopenia was described as Platelet count < 100 with peripheral blood film findings of adequate platelet with platelet clump and giant platelet. The patients clinical summary, red cell indices and total WBC were also reviewed and analysed
Results: The prevalence of Peudo-thrombocytopenia amidst our study population was found to be 0.6%,(4.8% of all cases of Thrombocytopenia). No cases of malignant disease have pseudo thrombocytopenia, in the PT group, the mean corpuscular volume is higher. We found no statistically significant correlate with occurrence of PT and age, degree of anaemia, MCH, RDW, and total WBC.
Conclusions: This study showed a low prevalence of pseudo-thrombocytopenia in the routine Full Blood Count sample, thrombocytopenia in patient being managed for malignant disease is not likely to be a pseudo thrombocytopenia. Pseudo-thrombocytopenia is associated with higher MCV than other cases of thrombocytopenia. This study is a pilot to an ongoing prospective study to further describe this condition and its associations.

Diagnostic Utility Of Platelet Parameters In Thrombocytopenic And Thrombocythemic Disorders
Ji Seon Choi, Myung Jin Cho, Song Hee Oh, Dong Yon Park, Jayoung Kim
Department of Laboratory Medicine, International St. Marys Hospital , Catholic Kwandong University College of Medicine, Incheon, South Korea

Introduction: Platelets are cell fragments that function in the clotting system. Platelet disorders include an abnormal increase of decrease in platelets and platelet dysfunction. Automated blood cell counters provide additional information on the circulating platelets. Among several platelet indices, platelet distribution width (PDW), mean platelet volume (MPV), platelet larger cell ratio (P-LCR), and plateletcrit (PCT) are a group of platelet parameters determined together in automatic CBC profiles. In this study, we hypothesized that these platelet parameters reflect the pathophysiologic profiles of different hematologic diseases, i.e., hyperdestructive thrombocytopenia with compensatory thrombopoiesis, hypoproductive thrombocytopenia due to bone marrow failure and hyperproductive thrombosis with clonal thrombopoiesis. We analyzed the platelet parameters in thrombocytopenic and thrombocythemic disorders and compared them to those of healthy individuals.    
Methods: Complete blood count (CBC) results including platelet counts, PDW, MPV, P-LCR, and PCT were measured using XN-9000 hematology analyzer (Sysmex, Kobe, Japan) in 40 healthy individuals, 57 essential thrombocythemia (ET), 76 idiopathic thrombocytopenic purpura (ITP) and 78 aplastic anemia (AA) patients; none of the patients in either group had previously received treatment.
Results: MPV was significantly increased in ITP patients compared to healthy individuals (p< 0.0001) and decreased in ET patients compared to both ITP and healthy individuals (p< 0.0001). ET patients showed lower PDW and P-LCR values than healthy individuals (p< 0.0001) and ITP patients showed higher PDW and P-LCR (p< 0.0001). AA patients showed no difference in MPV, PDW and P-LCR values except platelet counts compared to healthy individuals.
Conclusions: MPV, PDW and P-LCR were higher in ITP, suggesting upregulation in the production of young, large platelets in conjunction with the indiscriminate destruction of circulating platelets. MPV, PDW and P-LCR were lower in ET patients, suggesting that the proportion of small platelets was higher than normal even though platelet production is increased. MPV, PDW, and P-LCR may help in predicting thrombocytopenic patients as having ITP or hypoproductive thrombocytopenia, and provide the guidelines in delaying/avoiding unnecessary bone marrow study in ITP patients or supplement a request for bone marrow study in hypoproductive thrombocytopenic patients. A rapid and inexpensive automated measurement of platelet indices can be integrated as a standard parameter to evaluate the thrombopoietic state.

Electron Microscopy Examination Of Platelet Dense Granules: Evaluation OfInter-Reader Variability And ImplicationsFor AssessmentOf Platelet Function Disorders In DailyPractice
Pieter De Kesel1, Lisa Florin1, Marleen Praet2, Katrien Devreese1
1Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium, 2Department of Pathology, Ghent University Hospital, Ghent, Belgium

Introduction: Electron microscopy (EM) is useful in diagnosing δ-storage pool disease, a platelet function disorder (PFD) characterized by dense (δ-)granule (DG) deficiency. We aimed to evaluate inter-reader variability in platelet DG counts in healthy volunteers and to correlate EM with relevant diagnostic tests and bleeding severity in patients for whom platelet EM was requested.
Methods: To assess inter-reader variability, DG and platelets in fixed platelet preparations from healthy volunteers (n=9) were counted (10 fields, magnification 7000x) by five investigators. Electron-dense, dark, round, clearly delineated structures were counted as DG. Due to high variability in quantitative DG counts in these normal samples, DG in patient samples were semi-quantitatively judged (present, absent or scarce) by three investigators. Patient data were reviewed for platelet counts, light transmission aggregometry (LTA), ATP-release and bleeding score (ISTH-BAT). 
Results: Mean DG count/platelet in healthy volunteers was 0.13 (range 0.05-0.41), with inter-reader coefficient of variation ranging from 14.9 to 40.0%. In patient samples (n=76), DG were clearly present in 55 patients, scarce in 9 and absent in 6 cases. The latter two subgroups were considered as DG deficiencies (DGD). Six samples were excluded (EM inconclusive). Platelets counts in platelet-rich plasma for LTA were not significantly different in DGD (n=15) and non-DGD (n=55) samples. The DGD group showed lower ATP-release (p=0.0012), with all results below the in-house cut-off versus 70% of values below cut-off in non-DGD samples. Impaired LTA (≥2 agonists) was seen in 53% and normal LTA in 20% of DGD samples, highlighting the importance of ATP-release. In the non-DGD group, impaired LTA was found in 34% and normal LTA in 55% of samples. In both groups, LTA was most frequently impaired with epinephrine, ADP and U46619. A tendency towards more severe bleeding was observed in the DGD group, with abnormal bleeding scores (children ≥3, men ≥4, women ≥6,) for 39% of patients versus 26% in the non-DGD group, although not significantly different.
Conclusions: We found high inter-individual variability in interpretation of platelet EM in healthy volunteers. In patients with suspected PFD, DGD was associated with decreased ATP-release and more frequently impaired LTA. No clear association between bleeding severity and DG number was found.

Biologic Variation In Platelet Counts Of Inpatients With Thrombocytopenia When Monitored By The Sysmex Xn 9000
Hanan Gerges1, Eric Xu2, Janice Park3, Bruce Davis4, Gwen Clarke5, George Cembrowski6,7
1University of Alberta Hospital, Edmonton, AB, Canada, 2University of Manitoba, Winnipeg, MB, Canada, 3Alberta Health Services, Edmonton, AB, Canada, 4Bruce H Davis, MD, Inc., Bangor, ME, United States, 5Canadian Blood Services, Edmonton, AB, Canada, 6University of Alberta, Edmonton, AB, Canada, 7CC Quality Control Consulting, Edmonton, AB, Canada

Introduction: Biologic variation (BV)  has become the primary criterion for specifying maximum allowable analytic error.  While BV is usually assessed in healthy subjects, (generally with strict observation of preanalytical conditions), it is not clear whether similarly derived BVs are applicable in subjects with abnormal concentrations of the analyte in question.  We use a unique methodology to derive the total variation from consecutive patient data [a mixture of preanalytic variation (PA) and biologic  variation (BV) and analytical variation (AV)].  We demonstrate adequacy of the Sysmex XN for thrombocytopenic patients and increased short term relative BV of platelets of thrombocytopenic patients. 
Methods: A data repository provided low platelet results measured over a 2 year period at the University of Alberta and Royal Alexandra Hospitals in Edmonton, Canada. These measurements were made on Sysmex XN 9000 analyzers.  If platelet values were associated with abnormal hemoglobin levels or neutrophils counts, they were eliminated.  We tabulated the pairs of intra-patient platelets that were separated by 0-4, 4-8, 8-12,… up to 48 hours. The standard deviation of duplicates (SDD) of the paired platelet determinations was calculated for each time interval. The graphs of SDD vs. time interval were approximately linear; the y intercept provided by the linear regression equation represents the sum of short term BV and AV: y02=(AV2 + BV2) ½.  AV was determined by extrapolating the QC imprecisions to those of the median platelet counts. BV was determined for multiple patient platelet ranges.  
Results: The Table summarizes the short term platelet biologic variations for various patient platelet ranges. There were too few platelet observations to study the < 20 x109/L range.
Conclusions: The relative short term platelet variation for patients with thrombocytopenia is about twice that of normal individuals (3.2% from Buoro et al, Clin Chim Acta 470 (2017), 125-132). 

Superior Levels Of Soluble P-Selectin In 5 Day-Stored Platelets With Higher Expression Of Intra-Platelet Reactive Oxygen Species (Ros)
Ehteramolsadat Hosseini, Mehran Ghasemzadeh
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

Introduction: So far, several lines of evidence showed the importance of intra-platelet ROS generation in the modulation of platelet activation and thrombus formation. This study aimed to evaluate the correlation between the levels of soluble P-selectin and ROS generation during platelet storage  
Methods: To analyze ROS production, platelets (107 /mL) were incubated with 1 μM DHR 123. Cells were then subjected to flow cytometer (CyFlow®Space, Partec GmbH, Germany) where a total of 20000 PLT events were acquired. To evaluate soluble P-selectin, microparticle-free PPP samples obtained from 5 day-stored PCs with the same platelet count were also subjected to ELISA assay.
Results: We evaluated ROS generation in the stored platelet concentrates where we showed increasing pattern of ROS generation in platelet population from day 1 to day7 of storage. According to the results obtained from intra-platelet ROS examination, 5 day-stored platelets were divided into two groups of samples including those with intra-platelet ROS expression more or less than 40%. In this study we demonstrated that the levels of soluble P-selectin were significantly higher in the samples with ROS expression more than 40% (p< 0.05).
Conclusions: These results were in line with our previously published data in which we showed the higher expression of P-selectin in platelets with superior levels of ROS generation during storage. Taken together while we have introduced intra platelet ROS as a marker of platelet storage lesion, our result also suggest its important role in P-selectin ectodomain shedding during platelet storage. Keywords:Platelet; P-selectin; Reactive oxygen species; Shedding; Storage

Comparison Of Manual Peripheral Smear Platelet Count And Platelet Count By Unicel Dxh800 With International Reference Method By Flow Cytometry In Thrombocytopenic Patients
Sindhura Lakshmi Koulmane Laxminaraya1, Ranjitha Nair1, Sushma Belurkar1, Michael Keeney2
1Pathology, Kasturba Medical College,Manipal, MAHE, Udupi, India, 2Flow cytometry, London Health Sciences Centre, London, ON, Canada

Introduction: Platelet count at critically low levels must be accurate as they are transfusion triggers. Automated analysers provide accurate platelet counts, however, manual peripheral smear platelet counts (MPC) are still widely used in resource poor settings. Few studies have compared automated platelet counts with MPC. In contrast to automated counts, MPC have not been validated against the international reference method by flow cytometry (IRM). In this study, we compared MPC and platelet count by Unicel DxH800 with IRM in thrombocytopenic patients.
Methods: Cases included Unicel DxH 800 platelet counts < 50000/µL with no flag related to platelet parameters. Peripheral smears containing platelet clumps on manual review were excluded. Flow cytometry analysis with CD41/CD61 FITC was performed according to ICSH guidelines. Peripheral smears were reviewed by two blinded independent users. Ten oil immersion fields were screened in the zone of morphology and number of counted platelets were multiplied by 1500 (user1_15k and user2_15k) and 2000 (user1_20k and user2_20k) separately. 
Results: Study included109 cases. Mean age was 47 years and male:female ratio was 1:1.3. DxH800 platelet count and MPC were correlated with IRM by Spearman’s correlation coefficient. All methods showed strong correlation with IRM (>0.7). Intraclass correlation coefficient showed excellent reliability with DxH 800 counts and good reliability with all methods of MPC. The details are given in table 1 and 2.
  Mean /µL Standard Deviation   Minimum /µL Maximum /µL
IRM 20500 11360 2446 48934
DXH800 25853 12813 5000 49000
USER1_15K 26257 16303 1500 79500
USER1_20K 35009 21737 2000 106000
USER2_15K 28142 17242 1500 82500
USER2_20K 37523 22989 2000 110000
Table 1:Descriptive statistics 
In comparison with IRM Spearman’s correlation coefficient   Intraclass correlation coefficient
DXH800 0.909 0.946
USER1_15K 0.803 0.765
USER1_20K 0.803 0.829
USER2_15K 0.749 0.779
USER2_20K 0.749 0.771
Table 2: Correlation coefficients
Conclusions: MPC has excellent correlation and good reliability when compared to IRM. However, MPC (particularly the 20K method) significantly overestimated the counts in many instances. These differences may affect the decision to transfuse at critical threshold counts. Results of this study may be used to train manual reviewers to improve agreement using this data.

Prevalence Of Edta-Anticoagulation Associated Platelet Agglutination In A Large Out-Patient Laboratory: Specificity And Sensitivity Of Sysmex Xe 5000 Flagging For Platelet Aggregates
Joe Mansour1,2, Steffen Mannu1, Angela Gropp1, Klaus Scharfenberg2, Peter Schuff-Werner1,3
1MDI Labor Limbach Berlin, Berlin, Germany, 2University of Applied Sciences Emden-Leer, Emden, Germany, 3Institute of Clinical Chemistry and Laboratory Medicine (ILAB), Rostock University Medical center, Rostock, Germany

Introduction: Time- and temperature-dependent in vitro platelet agglutination is rarely observed in
EDTA-anticoagulated blood samples, leading to low platelet counts (“pseudo-thrombocytopenia”).
Anticoagulation with EDTA is a necessary pre-requisite for automated blood cell counting but is accompanied in 0.01 to 0.2 % by time- and temperature-dependent in vitro aggregation of platelets, with false low platelet counts. To avoid wrong clinical conclusions, spontaneous anticoagulant-induced platelet agglutination should therefore be recognized already during analysis.
Methods: Using our routine hematology analyzer (Sysmex XE 5000), CBCs of thousand EDTA-anticoagulated blood samples from unselected patients were measured in the order of their arrival in the laboratory. In parallel blood smears were produced and microscopically analyzed for platelet aggregates.
CBC flags, abnormalities of either the IMI channel and / or the platelet distribution curve indicating platelet aggregates were compared with the result of the microscopic analysis of the blood smears and calculated for diagnostic sensitivity, specificity and efficiency.
Results: Pseudo-thrombocytopenia was found with a prevalence of 0.61 % (6 out of 977 patients). Only two of the 6 PTCP were flagged by the respective aggregate warning. Other warnings of the device that are reported to be associated with platelet aggregates were also found unsuitable to safely identify PTCP, as shown by the calculation of diagnostic specificity, sensitivity and efficiency.
The prevalence of EDTA-induced platelet agglutination in a cohort of unselected out-patients was 0.61 % and therefore in accordance with the literature. All 6 cases were identified by microscopic examination of the blood smears (correct positive cases). Only two cases were flagged by the hematology analyzer XE 5000 (sensitivity 33%; specificity 97%). The analysis of the IMI channel resulted in the identification of 5 (sensitivity 83%, specificity 63%) blood samples with platelet aggregates. Characteristic changes of the platelet distribution curve (“sawtooth similar”) were also found in 5 out of the 6 positive samples (sensitivity 83%, specificity 43%). 
Conclusions: Our study revealed a 0.61 % prevalence of pseudo-thrombocytopenia in EDTA-anticoagulated blood samples from out-patients. XE 5000 analyzer flags and separate analysis of IMI channel and /or the platelet distribution curve are not very helpful for detecting platelet aggregates in EDTA-anticoagulated blood.

Performance Evaluation Of Anysis-200 Platelet Function Analyzers In Cardiology Patients
Seongjun Park1, Jun Kim1, Jinwoo Jang1, Chaeseung Lim1, Hongseog Seo2, Sehyun Shin3
1Korea university medical center, Department of Laboratory Medicine, department of Laboratory medicine, seoul, Korea, 2Korea university medical center, Division of cardiology, seoul, Korea, 3School of mechanical engineering, korea university, seoul, Korea

Introduction: Platelets play an important role in maintaining normal hemo­stasis via various mechanisms. Malfunction of these mecha­nisms can result in the formation of pathologic thrombi and cause vascular occlusion, leading to cardiovascular disease and ischemic stroke. The analysis of platelet function is a crucial component in the assessment of the hemostasis status in the perioperative setting; primarily in patients receiving anti-platelet medication. Anysis-200 analyzer (RheoSCAN Ltd., Seoul, Republic of Korea) is a lab on-a-chip based new easy automated platelet activation assay that re­quires a short time to obtain the test results. The purpose of this study to evaluate a new platelet function analyzing system, Anysis-200 which based on migration distance with Platelet Function Analyzer-200 (PFA: Siemens Canada, Mississauga, Ontario, Canada)  widely used in hospitals to estimate bleeding time in cardiology patients.
Methods: Citrated bloods were collected from 100 patients (170 tests) who visited department of cardiology of Korea University Guro Hospital between December 2017 and May 2018. We performed a PFA-200 collagen and epinephrine (C/Epi)assay both by the Anysis-200 and PFA-200 analyzers simultaneously. The percent agreement is directly interpreted as the percent of data that showed no difference between the values which were assigned either within the reference range or not (categorical agreement). P values < 0.05 were considered statistically significant.
Results: Normal range of closuretime(CT) in PFA-200 Epi/Col assay was from 61 to 110 sec and this value put into the linear correlation function and normal range of Anysis-200 assay was 34.1 to 72.7 mm of sample migration. Pairwise agreement between the two platelet function assays was assessed using Cohen’s kappa coefficient and percent agreement within the reference limit. The percent of agreement between the two assays was 52.9% and Pearson’s correlation value between the two assays were 0.525. Linear regression analysis of the PFA(y) vs. the migration distance of Anysis-200 yielded the following equation:y = 0.9459x + 74.041,R² = 0.3064 . Kappa values were moderate (0.5).
Conclusions: Anysis-200 analyzer has shown high agreement with PFA-200, and can be used as a reliable assay for screening of platelet function in cardiology patients.

Developing An Image Analyzing Software Discriminating Mature Neutrophil Subsets
Hajimu Kawakami, Hirokazu Kurata
Sysmex Corporation, Kobe, Japan

Introduction:  Blood image analysis is a critical component of hematology testing. Recent advances in artificial intelligence (AI) have been enabling the high-quality automated digital image analysis (DIA), which replaces the subjective and laborious optical microscopic inspection.
 In this study, to investigate the critical parameters of automated DIA, we constructed an algorithm discriminating accurately mature peripheral neutrophils (Band- and Segmented-form). 
Methods:  Using several thousands of images of neutrophils from normal volunteers in DI-60 (Sysmex), we developed the discriminating algorithm using the following methods: (1) constructing a cell-image measuring system ('Metrics') by strictly following the criteria of ISLH and the Japanese joint committee (Association of Medical Technologists (JAMT) and Society for laboratory Hematology (JSLH)), (2) examining whether the known and novel parameters are essential for the discrimination, (3) comparing 'Metrics' with other mechanical learning methods and expert judgment (as the gold standard), and (4) examining the reasons of discordance among the above judgments. 
Results: (a) We established several cell-image measuring techniques (nuclear widths and their ratio, nuclear separation and overlapping, etc.) and confirmed their feasibility and reproducibility. (b) Combining them, we developed an algorithm ('Metrics') with the accuracy of more than 98% in discriminating Band and Seg. (c) Compared with other methods, including Deep Learning, 'Metrics' provided higher accuracy of discrimination. (d) By analyzing the discordant results, we recognized the limitations of the present mechanical judgment as well as the obscurity of optical microscopic judgment. 
Conclusions:  In this study, we investigated the algorithm discriminating mature neutrophils - Band and Seg, as a proof-of-concept subject of AI-mediated next-generation DIA system. Through strictly applying the academic criteria into the algorithm and combining with novel parameters, we attained the high-accuracy of judgment. Moreover, we revealed the discrepancies between human (optical inspection) and mechanical judgments. By utilizing this 'Metrics', we can accumulate significant numbers of supervised image data with high accuracy, and develop the high-quality mechanical learning.

Thrombocytosis And Probability Of Calreticulin Mutation
Munazza Rashid, Rifat Ahmed, Uzma Zaidi, Tahir Shamsi
NIBD, Karachi, Pakistan

Introduction: Essential thrombocythemia (ET) is one of the three BCR-ABL1-negative myeloproliferative neoplasms (MPN) that primarily involves the megakaryocytic lineage, and is characterized by increased numbers of large, mature megakaryocytes in bone marrow as well as sustained thrombocytosis. When evaluating thrombocytosis, the detection of Janus Kinase 2 (JAK2), Calreticulin (CALR), or Myeloproliferative Leukemia Virus Oncogene (MPL) mutations confirms the presence of an underlying MPN but their absence does not rule out the possibility. Somatic insertions/deletions in the CALR gene have recently been discovered to be causative alterations in clonal ET.
Methods: Our study population consisted of 42 patients with clinically suspected as ET, 22 males, 20 females; median age: 42.5 years, (range: 12– 80 years) at National Institute of Blood Diseases & Bone Marrow Transplantation, Karachi, Pakistan. Traditional polymerase chain reaction was performed on DNA previously extracted from peripheral blood. Amplified products were purified and bidirectionally sequenced.
Results: CALRmutations was found 21.4% by sanger sequencing and JAK2 V617F was 29.7% by ARMS-PCR. One patient had both CALR and JAK2V617F mutations. CALR mutant thrombocytosis is associated with younger age (median 45 years), higher platelet counts (median= 1067 X109/L), lower leukocyte counts (median= 11.0 X109/L) and also lower haemoglobin (median =11.7 g/dL) in comparison with JAK2 V617F-positive ET.  Patients with JAK2 (V617F) were older (median 66 years), had lower platelet count (median= 938X 109/L), high white blood cell count (median= 14.5 X 109/L), and higher hemoglobin level (median= 13.07 g/dL) than those with CALR mutation. More splenomegaly was observed in CALR mutated thrombocytosis.
Conclusions: CALRmutations as a new molecular marker in diagnosing and predicting the prognosis of patients with previously difficult-to-characterize.

Platelet Indices As Markers For Korean Patients With Coal Workers&Rsquo; Pneumoconiosis
HeeJin So, Beom tak Park
Korea worker's compensation and welfare service Ansan Hospital , Ansan, South Korea

Introduction: Coal workers’ pneumoconiosis (CWP) is caused by inhalation and deposition of coal dust in the lungs. Coal dust provides a stimulus for the macrophage to release various enzymes, cytokines, oxygen radicals. A reactive oxygen species (ROS) released from macrophages and leucocytes play an important role in the inflammation and fibrosis of CWP. Several studies have investigated the relationship between ROS and platelet function. We evaluate platelet indices in Korean patients with CWP.
Methods: A total of 37 patients with CWP and 27 healthy subjects were enrolled in this study. Complete blood count were retrospectively analysed.
Results: Haemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC) were all significantly lower in patients with CWP compared to those in healthy subjects (p< 0.05). Only mean platelet volume (MPV) was significantly higher in patients with CWP compared to that in healthy subjects (p = 0.04). There were no significant differences in red blood cells (RBC), white blood cells (WBC), red blood cell distribution width (RDW), platelet count, platelet distribution width (PDW), plateletcrit (PCT) between the patients with CWP and healthy subjects. The characteristics and laboratory values of the study population are presented in Table 1.      
Conclusions: Our findings indicate that only MPV among platelet indices has significant difference between Korean patients with CWP and healthy subjects. Further investigation is needed to clarify the usefulness of these markers for pneumoconiosis.

Diagnostic Performance Of The Zymutest Immunoassay For Heparin-Induced Thrombocytopenia - A French Prospective Multicentre Study
Brigitte Tardy-Poncet1, Thomas Lecompte2, Bernard Tardy1
1CHU, Saint-Etienne, France, 2HUG, Geneva, Switzerland

Introduction: Diagnosis of heparin-induced thrombocytopenia (HIT) is challenging, and on clinical grounds and changes in platelet counts the diagnostic probability is often intermediate. Thus laboratory testing is required. A negative immunoassay for HIT antibodies can reasonably rule out a substantial fraction of patients, for whom a change of anticoagulant therapy would not be necessary, and a platelet activation assay improves specificity of laboratory diagnosis since it detects those antibodies able to activate platelets, which are involved in thrombogenesis.
Methods: Among available immunoassays for the detection of HIT IgG antibodies,Zymutest HIA®(Hyphen Biomed, Neuville sur Oise, France) is unique since a platelet extract interacts with immobilized heparin. Thus besides PF4, IL-8 and NAP-2, the two chemokines that can also be the target of HIT antibodies, are present. Among 1266 patients with an intermediate diagnostic probability, 82 (7%) were classified as HIT by two independent experts on the basis of parameters prospectively collected in a CRF (HIT score study – Groupe Français sur l’Hémostase et la Thrombose - GFHT), and SRA performed in four French reference laboratories.
Results: The AUROC was excellent: 0.980; 95% CI 0.971-0.987; z statistic: 128. Sensitivity of 100% (the 95% CI including 100%) was obtained up to 0.634 OD with a 90 % specificity. Specificity further increased with increasing the OD, advocating for taking into account the intensity of the signal. A cut-off value can even be proposed for which the cost-effectiveness of a platelet activation assay it is debatable. The AUROC when IgA and IgM were detected together with IgG was slightly lower, suggesting that there were few patients with isolated IgM in the cohort.
Conclusions: Our results strongly support the use of this ELISA as a laboratory diagnostic tool for HIT, when the diagnostic probability is intermediate.

Assessment Of The Predictive Value Of Von Willebrand Factor Antigen And Activity Combined Detection In Patients With Acute Myocardial Infarction
Bin Yan1, Yiming Zhao2, Changgeng Ruan2
1The Affiliated Nanyang City Center Hospital of Zhengzhou University, Nanyang, China, 2The First Affiliated Hospital of Soochow University, Suzhou, China

Introduction: Von Willebrand factor (VWF) acts as a bridge between platelet and the subendothelial matrix, and plays a vital role during thrombosis. Acute myocardial infarction (AMI) is an arterial thrombotic disease. Plasma VWF levels in AMI patients are not only elevatedquantity, but also have qualitative change. The aim was to analyze the levels of VWF antigen (VWF:Ag) and activity (VWF:GPIbR and VWF:CB) in AMI patients, then investigate the predictive value of combined detection for AMI.
Methods: Plasma levels of VWF:Ag, VWF:GPIbR and VWF:CB were measured in AMI patients (n=105) and healthy controls (n=101) by ELISA. Spearman correlation was chosen to analyze the correlation between VWF and AMI tests. The Receiver operating characteristic curve was used to evaluate the predictive value of VWF single detection and combined detection for AMI.
Results: (1) Plasma VWF:Ag, VWF:GPIbR and VWF:CB levels in AMI patients were significantly higher than those in control group (P< 0.0001). (2) VWF:GPIbR was positively correlated with platelet level in AMI patients (r=0.241, P=0.013), while VWF:Ag and VWF:CB were negatively correlated with APTT (r=-0.308, -0.297 and -0.289, respectively, P< 0.05); Only plasma VWF:GPIbR level was positively correlated with creatine kinase in AMI patients(r=0.212, P=0.030). (3) For single detection, the diagnostic efficacy of VWF:GPIbR (AUC=0.967) was significantly higher than VWF:Ag (AUC=0.880) and VWF:CB (AUC=0.828, all P< 0.0001); For combined detection, VWF:Ag+VWF:CB (AUC=0.881) has significantly lower diagnostic efficacy for AMI than VWF:Ag+VWF:GPIbR (AUC=0.967) or VWF:GPIbR+VWF:CB (AUC=0.967, all P< 0.0001); The three combined detection of VWF (AUC=0.967) was also significantly higher than VWF:Ag+VWF:CB (P< 0.0001), and there was no difference between three combined detection and VWF:Ag+VWF:GPIbR or VWF:GPIbR+VWF:CB (P>0.05).
Conclusions: Plasma VWF: Ag, VWF:GPIbR and VWF:CB in AMI patients were higher than those in controls. VWF:GPIbR had better predictive value for AMI than VWF:Ag and VWF:CB. Hence, the inclusion of VWF:GPIbR contributed to obtain higher predictive power, and the three combined tests cannot significantly improve the diagnostic value of AMI.

Iron Status And Some Dependent Factors Among Pregnant Women In Sub-Urban Setting In South-West, Ogun State, Nigeria
Opeyemi.O ADESINA1, Mathias. Abiodun Emokpae2
1Babcock University, Ogun, Nigeria, 2University of BenIn, Benin, , Nigeria

Introduction: Anaemia in pregnancy is a global public health problem. There is paucity of data on anaemia among pregnant women in Ogun State Southwest, Nigeria. This prospective cross-sectional case-control study was carried out to investigate the association of anaemia in pregnancy with iron status and dependent factors.   
Methods: 1200 pregnant women were recruited in the study with 400 each from the 3 health centre: Olabisi Onabanjo Teaching Hospital (oouth), General Hospital Ijebu-Ode (GHI) and Abeokuta (GHA). Age ranged 18 - 40 yrs with a mean age of 20.8 ± 0.6 yrs. 774 multiparous and 426 primiparous; 162 ,560 and 478 in Ist,2nd and 3rd trimester.  Socio-demographic and other information after an informed consent was obtained with  structured questionnaire. Serum iron, ferritin, total iron binding capacity (TIBC), transferrin, percentage transferrin saturation, full blood Count (FBC), BMI, Folate, vitamin A, Malaria parasite and urinalysis were analyzed using standard methods. Data analyzed using the Chi-square, t-test, Anova, and Odds ratio . Anaemia in pregnancy was classified based on WHO classification.  
Results: Pregnant women with anaemia was 566(47.2%) . The incidence in highest at GHA 232(58%). The mean levels of Hb, iron, transferrin, ferritin were significantly lower (p< 0.001) while TIBC and BMI (p< 0.05) was significantly higher than non-anaemic pregnant  subjects.   The prevalence of  iron deficiency, iron depletion and iron deficiency anaemia were significantly higher (p< 0.05) among pregnant women in GHI and GHA compare to OOUTH while Folate and vit.A levels significantly lower (p< 0.05) . The prevalence of malaria, mean parasite density and HIV was higher P>0.05 in anaemic pregnant women while mean levels of Fe concentration were highest (P< 0.001) in first trimester and lowest in third trimesters and among multigravidas. 
Conclusions: The prevalence of anaemia among our cohort of pregnant women was high.These differences in iron status results from the three centers may likely be attributed to level of antenatal care ,nutritional deficiencies, parasitic infection and low socio-economic factors identified. We recommend that free supplements should be routinely prescribed for pregnant women in the area as well as intermittent preventive antimalaria.    Key words: Iron Status, Anaemia, Iron deficiency, dependent, Ogun  

Analysis Of Alpha-Globin Gene (Hba1 And Hba2) For Deletion Type Mutations In Alpha-Thalassemia Patients Using Mlpa
Zeeshan Ahmed, Asghar Nasir , Kahkashan Imam, Tariq Moatter
Aga Khan University Hospital, karachi, Pakistan

Introduction: Alpha thalassemia disorders are a group of hereditary anemias caused by absent or decreased synthesis of the α chain of hemoglobin. They are one of the most common single gene disorders, affecting 5% of the world’s population. The severity of the α- thalassemia disorders is quite variable. At one extreme is a completely asymptomatic condition resulting from one of the four α genes(HBA1/2)  being mutated. Molecular genetic testing of HBA1 and HBA2 detects deletions in about 90% and point mutations in about 10% of affected individuals. Identificationof at-risk couples by population screening is strongly recommended but largely not successfully implemented
Methods: Genomic DNA was isolated and HBA1 and HBA2 gene were amplified by Multiplex Ligation-dependent Probe Amplification (MLPA), detected by Capillary electrophoresis and analyzed by Coffalyser software. This method is relatively simple and robust multiplex PCR method for detecting chromosomal DNA copy number changes in multiple targets.(MRC Holland)
Results: In this study, a total number of 36 samples were analyzed from May 2017 until Sep 2018. Out of which, 28 cases were males and 08 were females Mean age 8 years and male female ratio 20: 16. All patients were diagnosed as α-thalassemia based on their family history, clinical and laboratory findings. Among all 08 patients were found mutation of Alpha gene (HBA1/2).  3.7 Deletion, 01 as Alpha thalassemia trait and 01 as compound mutation detected as HS40 deletion, based on their family history, hematological parameters and other clinical and laboratory findings. Among a total of 36 patients, 09 had a history of blood transfusion.
Conclusions: The molecular testing of α-thalassemia has been challenging due to this complex molecular basis. As MLPA is a convenient alternative method for detecting deletions, it has recently been a compelling choice for mutation analysis of deletion alpha-thalassemia in non-Beta thalassemia.

Association Of Some Blood Group Phenotypes And Risk Of Myeloid Leukaemias In Kano, Nigeria

Introduction: Myeloid leukaemias are heterogeneous clonal disorders characterized by the production of immature and non-functional cells of myeloid origin known as blasts or production of abnormal blood cellsand subsequently result in bone marrow failure and organs infiltrations.Blood group antigens have been linked with infections and diseases etiology. However, not much has been studied on the association between the rare blood types and myeloid leukaemias in this part of the world. This study therefore investigated the association of some rare blood group phenotypes with myeloid leukaemias among subjects in North-Western Nigeria.
Methods: A total of 63 myeloid leukaemic subjects (age: 33.1±18.4 years, male/female ratio: 35:28) were studied with 63 healthy blood donors (controls) with mean ages of 29.3±5.8 years and male/female ratio: 54:9. Out 63 myeloid leukaemic subjects, 38 were chronic myeloid leukaemia (CML) while 25 were acute myeloid leukaemia (AML). Myeloid leukaemias were diagnosed using the clinical and cytological criteria in Aminu Kano Teaching Hospital (AKTH). The blood groups were determined using Tube method for ABO, Rh-D, Lewis, Duffy and MNS while Rh C, c, E, e and Kell by Gel card method. Data was presented as tables and figures and analysed using SPSS (version 25.0). Association of some blood groups and myeloid leukaemias were determined using Chi-square test whileOdds Ratio (OR) and Relative Risk (RR) were used to ascertain their risk values.
Results: All myeloid leukaemias (CML and AML) had male gender predominance. The results showed that B, C, e, Lea, K and M antigens were significantly associated with CML (OR: 0.18, 3.38, 0.09, 4.80, 33.30 and 5.34 respectively) while Lea, Leb, K and M antigens were significantly associated with AML (OR: 4.75, 7.11, 11.29 and 7.67). 
Conclusions: We conclude that individuals with Rhesus antigen C, Lewis antigens (Lea and Leb), Duffy antigen (Fyb), Kell antigen (K), M antigen may have increased risk of developing leukaemia especially CML while individuals with ABO antigen B, Rhesus e antigen and antigen N may have lesser risks of developing leukaemias

Prenatal Diagnosis And Selective Abortion In Thalassaemia Prevention: Experience Of Azerbaijan
Gunay Aliyeva1, Chingiz Asadov1, Tahira Mammadova1, Eldar Abdulalimov1, Ruslan Shamilov2, Shahla Rahimova2, Mirvari Aliyeva2, Surmaya Gafarova1, Yegana Guliyeva1
1Institute of Hematology and Blood Transfusion, Baku, Azerbaijan, 2Republican Perinatal Center, Baku, Azerbaijan

Introduction: Azerbaijan is known for the high prevalence of hemoglobinopathies, with 4% carrier frequency of β-thalassemia and less prevalent α-thalassemia, δ/β-thalassemia and clinically significant hemoglobin variants. Therefore, “State Program for Thalassemia Prevention”, initiated in 2015, stipulated the compulsory countrywide premarital screening for thalassemia. All identified carrier couples as well as the parents of sick children were consulted to receive prenatal diagnosis in their following pregnancies. According to the local legislation, first trimester abortion is legal on request, whereas pregnancies where fetus is identified with predicted phenotype of transfusion-dependent thalassemia or sickle cell disease can be terminated in the second trimester. The aimof the program was to consult at-risk couples for the termination of affected pregnancies.
Methods: At-risk couples, where both partners were identified as carriers of clinically significant hemoglobinopathies, were referred to genetic counseling at the Institute of Hematology and Blood Transfusion from across the country. Although the program prioritized the couples where both partners were prone to terminate affected pregnancies, prenatal diagnosis was applied even to those who were against abortion due to religious or ethical beliefs. Couples were referred to obstetric examination following the molecular testing and conformation of the carrier state. Transabdominal amniocentesis was performed between 16th-18th weeks of gestation following fulfilled prerequisites and received consent. Amniotic fluid was examined for the presence of globin gene mutations via reverse dot-blot hybridization or sequencing techniques.
Results: The total of 100 fetuses have been examined during three-year period (2015-2018). 24 of them were revealed as homozygous or compound-heterozygous affected fetuses, 22 were healthy and 54 were heterozygous carriers. Prenatal diagnosis was performed in 3 consecutive  pregnancies in 1 couple, and 2 times in 7 couples; it included 2 twin and 1 triple pregnancies. 24.5% of the couples were in consanguineous marriages (third-degree relatives). The most common mutations among parents were Codon 8 [-AA] – 37.9%, IVS-II-1 [G>A] – 15% and IVS-I-110 [G>A] – 8.6%. Termination of the affected pregnancies was performed solely on the decision of parents.  
Conclusions: The initiation of the program increased public awareness and decreased incidence rate of thalassemia in Azerbaijan.

Validation Of A Red Blood Cell Pitting Assay For Monitoring Splenic Function
Chanda Cheng, Susan Shibutani, Michele Paessler, Amrom Obstfeld
Children's Hospital of Philadelphia, Philadelphia, PA, United States

Introduction: The spleen is responsible for the removal of senescent red blood cells (RBCs), abnormal RBCs, or RBC inclusions. Loss of splenic function leads to increases in circulating RBCs with vacuoles and inclusions and importantly, increased risk of infection. The RBC pit count test estimates the number of RBC with small granular depressions, or “pits”, on their surface. An inverse relationship between the pit count and the degree of splenic dysfunction has been well established. We set out to technically validate the RBC pit count assay to assess splenic function in pediatric patients.
Methods: Peripheral whole blood cell samples collected in potassium EDTA containing tubes were fixed with a buffered glutaraldehyde solution. Samples were visualized using differential interference contrast microscopy (with Nomarski optics). The number of “pitted” cells seen in 1,000 RBCs counted by two technologists is expressed as the pit count percentage. Pit counts were evaluated in patients known to have elevated counts. Precision was assessed by repeated measurement of high and intermediate samples. A reference interval was established by measuring the pit counts in individuals with no known splenic dysfunction.
Results: Amongst fifty individuals with no known splenic dysfunction, the average pitted RBC levels 0.35% (standard deviation [sd] = 0.27%). In order to establish the expected values for patients without splenic function pit counts were assessed in 65 patients who had undergone splenectomy. Amongst these patients, pit counts ranged from 19.7% to 63.9% (mean=39.4%, sd=11%). Newborns have abnormal splenic function within the first month of birth. Twelve such patients were assessed and found to have elevated pit counts ranging from 1.2% to 32.1% (mean=12%, sd=9.2%). Precision was assessed by measurement of samples with high and intermediate levels over five days. The high sample had an average pit count of 44.3% and a coefficient of variation of 4.3%. The intermediate sample, which had an average pit count of 13.3%, the coefficient of variation was 13.1%.
Conclusions: The RBC pit count is a robust assay that easily distinguishes between individuals with normal and absent splenic function. Additionally it identifies those that are expected to have subnormal but not complete dysfunction of the spleen. Future work will establish its utility in guiding therapy for those at increased risk for infection due to splenic dysfunction.

Bone Marrow Findings Of Pediatric Patients With Hemophagocytic Lymphohistiocytosis
Min-sun Kim1, Chan-Jeoung Park1, Seongsoo Jang1, Young-Uk Cho1, Kyung-Nam Koh2, Ho-Joon Im2, Jong-Jin Seo2
1Laboratory Medicine, Seoul, South Korea, 2Pediatrics, Seoul, South Korea

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is uncommon, a life-threating hyperinflammatory syndrome caused by severe hypercytokinemia and fatal immunodysregulatory disorder caused primarily by the uncontrolled activation and proliferation of T cells and macrophages. To diagnose HLH, bone marrow (BM) study is necessary to confirm the presence of hemophagocytic histiocytes. We reviewed and analyzed BM findings of 43 pediatric HLH patients.
Methods: From July, 2009 to June, 2016, we reviewed BM aspirates, clot sections and biopsies obtained from 43 children who were diagnosed with HLH according to HLH 2004 criteria. We estimate the median value about each finding. The median age of patients was 58.7 months (12~74). The male and female patients were 25 (58%) and 18 (42%), respectively. We analyzed the cellularity, presence of hemophagocytosis with an average count of hemophagocytic histiocytes, histiocytic aggregates, proportions of histiocytes, monocytes and lymphocytes, megakaryocyte count, and cellularity of BM biopsy.
Results: The median values of CBC (WBC-hemoglobin-platelet count (1st quartile~3rd quartile)) were 3,700 /ul (2,600~7,800) -9.0g/dl (8.4~11.0)- 49k (36~82). In BM aspirates, among 43 cases, 39 (91%) cases showed hemophagocytosis with median 0.5 hemophagocytic histiocytes/HPF (0.2-0.6). Twenty-two cases (51%) showed histiocytic aggregates (≧ 5histiocytes). The median values of proportions of histiocytes, monocytes, and lymphocytes were 3.4% (1.7~4.9), 1.6% (0.1~3.2) and 22.3% (15.4~29.6), respectively. In BM biopsies, the median cellularity was 70% (40~80), and the median megakaryocyte count was 3.3/HPF (1.8~4.9). Increased micromegakaryocytes in 9 cases were observed. The serum levels [the median value, (range)] of triglyceride, fibrinogen and ferritin were 221 mg/dL (71-608), 115 mg/dL (40-309) and 4,801 ng/mL (78.4-175,363.6). NK cell activity was decreased in 39 patients (97.5%) among 40 patients in whom the flow cytometric NK cell activity test was performed, and the median value was 15.8% (0.36-31.24). The median quantitative EBV DNA copy number by a real-time polymerase chain reaction of blood and bone marrow specimen was 6.36 log/ml in 16 cases and 6.82 log/ml in forth cases, respectively. 3 cases relapsed, and 5 cases died after 7 days~12 months from the diagnosis of HLH. 
Conclusions: Most pediatric HLH patients showed normocellular marrow for age with hemophagocytic histiocytosis, histiocytes, lymphocytosis, and megakaryocytes hyperplasia. 

Learning From Eqa: The Delta Dilemma
Duljeet Chohan, Nikki Emodi, Barbara De la Salle
UK NEQAS Haematology, Watford, United Kingdom

Introduction: Haemoglobin A2 (HbA2)is a minor Hb fraction in adults which is increased in beta thalassaemia carriers. The National Screening Progamme in England uses a cut-off HbA2 of 3.5% for the identification of a beta thalassaemia carrier in antenatal screening. Hb A2 consists of 2 alpha and 2 delta chains (α2δ2). Carriers of a delta chain variant display a Hb A2 variant peak (Hb A2 Prime (A2’)) in addition to the main HbA2 peak. With any probable Hb A2 variant, the total Hb A2 % is calculated by adding the Hb A2 and the A2’ values. Although delta chain variants are clinically insignificant, they are common and failure to detect one would result in the possible underestimation of the total Hb A2 concentration and a potential missed diagnosis if the patient is also a beta thalassaemia carrier.   
Methods: UKNEQAS specimen 1806AH3 simulated a specimen from a 33 years old Northern European female undergoing antenatal screening. Her full blood count was normal and haemoglobinopathy screening showed a Hb A2 variant peak (Hb A2 Prime (A2’)) resulting from the presence of a delta globin chain variant. The distribution was circulated to 332 participants for analysis.
Results: A total 177/293 (60%) participants who returned a haemoglobin variant identification for this ‘patient’ suggested the presence of a HbA2 variant. HbA2% results returned showed a bimodal distribution of HbA2%, demonstrating that some participants reported the quantitation of the HbA2 peak only and others the sum of the HbA2 and HbA2 variant peaks. Some variation by instrument type was also noted. This specimen was withdrawn from performance assessment due to lack of consensus in the HbA2% but reported for its significant educational content.
Conclusions: This case shows a variation between participating laboratories in the interpretation and HbA2% quantitation in the presence of a HbA2 variant and raises concern about the possibility of misdiagnosis of a beta thalassaemia carrier, especially in antenatal patients. The case highlights the importance of sharing best practice through EQA to improve the standardisation of diagnostic testing.

Cold Agglutinins : A Diagnostic Challenge In Laboratory

Introduction:  •Autoimmune haemolytic anaemia (AIHA) is a complex process characterized by an immune reaction against red blood cell self-antigens. AIHA is classified into warm and cold reactive antibody types ,Cold agglutinin is an autoantibody that causes autoimmune haemolytic anaemia by binding to  (I/i) antigens on the surface of Red blood cells. Cold agglutinins have been one of the banes of blood group serologists since pretransfusion testing protocols were first implemented. Cold auto-agglutinins interfere with ABO/Rh typing tests, yield unwanted positive tests for unexpected antibodies, and may mask the presence of concomitant, clinically significant alloantibodies. •The cold agglutinins are vicious preanalytical and analytical factors that leads to  interference in  the laboratory results.
Methods: Retrospective study of 15 cases with flaggings for "cold agglutinins" during routine screening for CBC s on  HORIBA Analyser  ( PENTRA 120 DX Nexus) and monitored accordingly and 8 cases of cold agglutinin  in six  months causing erroneous results in blood grouping done by column agglutination technique (ORTHO CLINICAL AUTOVEU) ,hence proves to be a diagnostic challenge in the laboratory practice,
Results: •Erroneous results for RBC INDICES, decrease in RBC count ,Falsely elevated  (MCHC,MCH)  •Haemogobin and HCT Incompatibility ,though Hb values were not affected. •Mostly bimodal population  of RBCs observed in histograms •After Pre warming the EDTA tubes, results became valid •Discrepancy in reverse grouping causing false positive reaction occurred due to additional antibody that was  resolved by prewarming the samples.
Conclusions: Cold Agglutinins can cause interferance in laboratory test. Pre-Analytical check for ensuring proper temperature conditions during transportation of samples will lead to accurate results as well as decrease workload and laboratory cost. If ABO discrepency is found , it has to be diligently approached through thorough serological workup

The Value Of Performing Both Enzymatic And Molecular Testing In Cases Of Pyruvate Kinase (Pk) Deficiency
James Hoyer, Aruna Rangan, Nikita Mehta, David Viswanatha, Jennifer Oliveira
Mayo Clinic, Rochester, MN, United States

Introduction: Hemolytic anemia (HA) due to PK deficiency is the second most common red blood cell enzyme disorder. Diagnosis of PK deficiency can be established by either enzymatic or molecular methods. We present three cases which demonstrate the complementary value of performing both assays.
Methods: PK enzymatic testing was by Beutler method modification. PKLR gene mutation testing was performed by Sanger sequencing combined with a PCR-based assay to detect large PKLR deletions.
Results: Patient 1 was a 19 year-old (yo) Caucasian female with a lifelong history of HA. PK enzyme activity was 5.0 U/gHb  (48% of mean normal PK range (MNPK)). DNA sequencing of the PKLR gene detected a homozygous  c.1529 G>A, p.R510Q mutation, a known pathogenic PKLR variant. No PKLR deletions were detected. Patient 2 was a 23 yo Hispanic female with a lifelong history of HA. PKLR gene sequencing detected two missense mutations. The first (c.1456 C>T,  p.R486W) is a known pathogenic PKLR variant. The second (c.1378 G>A, V460M), was classified as a Variant of Uncertain Significance (VUS) but was suspicious for pathogenicity. PK enzyme activity was 2.5 U/gHb (24% MNPK), which supported pathogenicity and indicated these two variants were likely in a trans configuration. Patient 3 was a 10 yo Arab male with a lifelong history of HA. PKLR gene sequencing detected two missense mutations. The first (c.1456 C>T,  p.R486W) is a known pathogenic PKLR variant. The second (c.1070 T>A, p.I357N) has not been previously reported but was classified as Likely Pathogenic per ACMG criteria. PK enzyme activity was 2.8 U/gHb (27% MNPK), which  supported pathogenicity and indicated these two variants were likely in a trans configuration.
Conclusions: Performance of both enzymatic and molecular testing can be beneficial in cases of suspected PK deficiency. Molecular testing can establish the diagnosis in cases where the PK enzyme values are in the intermediate range (usually due to reticulocytosis). Conversely, PK enzyme values can help to support the pathogenicity of novel mutations, and provide support to determine variant phasing. The level of PK enzyme activity is of particular importance with the development of the small molecule activator AG-348 (Agios Phamaceuticals), which is currently in clinical trials as a treatment for PK deficiency.

Antenatal Screening For Prevention Of Severe Haemoglobinopathies: Experience From Tertiary Care Teaching Hospital In Western India
Anjali Kelkar, Anuja Patil, Anu Christopher, Amit Nisal, Ravindra Nimbargi
Bharati Vidyapeeth Univ Med College Pune, Pune, India

Introduction: Diagnosis of haemoglobinopathy in the first trimester in antenatal cases is a crucial step in prevention of births of children with transfusion dependent anaemias. Severe haemoglobinopathies pose significant economic, social and mental burden on the families and society; β thalassemia major being the most prevalent form. Timely diagnosis of the heterozygous conditions in the parents combined with effective counseling help in the prevention of severe disorders. 
Methods: Haemoglobinopathy screening is part of the routine antenatal profile in our hospital - a Tertiary care Teaching hospital of University medical college. We use the Variant II Beta Thalassemia Short Program (Bio-Rad) utilizing principles of ion-exchange liquid chromatography (HPLC). 876 antenatal cases were studied from April 2018 to October 2018. The HPLC results were interpreted along with the RBC indices, serum iron studies.  
Results:               64 antenatal cases (7.3% of total) were positive for presnece of some form of haemoglobinopathy.                                                                                                     
  Number % out of cases positive for haemoglobinopathy  % out of toatal cases studied
Heterozygous for β thalassemia 51 79 5.8
Heterozygous for Hb-Sickle 05 7.8 0.5
Heterozygous for Hb-Sickle with associated alpha thalassemia 03 4.7 0.34
Heterozygous for dβ thalassemia 02 3.1 0.22
Heterozygous for Hb D (Punjab) 01 1.5 0.14
Hb Hofou - β globin chain variant 01 1.5 0.14
Heterozygous for Hb E 01 1.5 0.14

Conclusions: In the present study, Heterozygous β thalassemia was the commonest abnormality, followed by Heterozygous Hb Sickle. All the positive patients were further counseled for the testing of spouse. 15 spouses' samples were studied; all were found to be normal. Major obstacles still faced in the haemoglobinopathy screening program are reluctance for testing asymptomatic population, lack of awareness, lack of facilities and trained staff.  At our center, the systematic efforts by the laboratory and clinical faculty resulted in incorporation of the test of Hb-HPLC in the routine ANC profile benefiting the patient care. 

Cold Agglutinin Sample Results On The Sysmex Xn-1000 And Cobas M 511 Integrated Hematology Analyzer
Tania Khartabil, Yolanda de Rijke, Henk Russcher
Department of Clinical Chemistry, Erasmus MC, University Medical Center, Rotterdam, Netherlands

Introduction: Cold Agglutinin Disease (CAD) is a type of autoimmune hemolytic anemia in which antibodies attach to the red blood cells (RBCs) in cold temperatures causing them to form clumps, known as agglutination. These particular types of samples often cause discrepancies and abnormal results in the laboratory, particularly for the RBC parameters in a complete blood count (CBC). Currently, the process to obtaining accurate results for these samples requires warming the sample, manual methods and repeat testing. Most hematology technology for CBC parameters uses a flow cytometry-based method, such as in the Sysmex XN-1000, but more recent technological advances have introduced digital analysis of cellular imaging as in the cobas m 511 integrated hematology system. With this new technology lies the potential to produce more accurate results for CAD samples with less time and difficulty.
Methods: Eight samples with varying degrees of confirmed CAD were processed on both the Sysmex XN-1000 and cobas m 511 system and the CBC results were compared. Samples were processed in both open and closed modes on each analyzer both warmed (37°C) and at room temperature. If the sample did not process on an analyzer in its first attempt due to extreme agglutination, a second attempt was made. If the analyzer still failed to complete the analysis of the sample then that run was excluded from the data. The results of each analyzer were compared to the results reported, either from an analyzer or a manual method.
Results: Overall, results on the cobas m 511 system were more consistent compared to the XN-1000, in particular for RBC parameters, which showed overall more RBC recovery compared to the reported result compared to the Sysmex XN-1000 for all samples both warmed and at room temperature. In addition, the room temperature the cobas m 511 system results were generally much closer to the reported results, especially for hematocrit (HCT).
Conclusions: The cobas m 511 system can provide more accurate RBC results for samples with cold agglutination at both room temperature and warmed as compared to the Sysmex XN-1000. This largely eliminates the need for sample warming or manual methods and reduces the turn-around-time in order to produce accurate results for CAD samples.

Using Reticulocyte Haemoglobin Equivalent And Delta-Haemoglobin To Differentiate Thalassaemia And Iron Deficiency Anaemia.
Pei Yu Lee, Noor Hafizah Noordin, Christina Yip, Shir Ying Lee
National University Hospital (Singapore), Singapore, Singapore

Introduction: Thalassaemia and iron deficiency anaemia (IDA) are different conditions exhibiting similar parameters like microcytosis and hypochromia, causing frequent misdiagnosis. Reticulocyte haemoglobin equivalent (Ret-He) measures the mean haemoglobin content in reticulocytes, while delta-haemoglobin (Delta-He) calculates the haemoglobin content difference between reticulocytes and erythrocytes. These novel parameters reflecting the haemoglobinization of newly formed red blood cells could potentially differentiate thalassaemia and IDA. Our study aimed to evaluate the effectiveness of using Ret-He and Delta-He to differentiate between four conditions:alpha-thalassaemia, beta-thalassaemia, absolute IDA and functional IDA.
Methods: We surveyed patient samples sent to our laboratory for full blood count and iron panel and/or thalassaemia screen over a year’s period. Full blood count was performed using Sysmex XN-10 (Sysmex, Kobe, Japan), which provides haemoglobin, Ret-He and Delta-He values. Ferritin levels and iron saturation were obtained from Beckman Coulter AU (Beckman Coulter, Miami, FL). Thalassaemia screening was performed using Sebia Capillary electrophoresis (Sebia, Norcross, GA) to give HbA2 levels, and stained with Brilliant Cresyl Blue to screen for Hb H inclusion bodies.
Results: In our preliminary results, 702 samples were analysed. The median Ret-He of normal control, alpha-thalassaemia and beta-thalassaemia are 32.9pg, 23.7pg and 21.5pg respectively. ROC analysis of Ret-He yield a cut-off value of 25.3pg (sensitivity 86.5%, specificity 91.9%) to screen for both alpha and beta-thalassaemia.  The median Ret-He for absolute IDA and functional IDA are 25.1pg and 29.2pg respectively. Ret-He between these two groups are shown to be significantly different with a p-value of < 0.0001. An overall significant correlation was found between Ret-He and Delta-He (Spearman r = 0.7371; p < 0.001) in all the patient cohorts. Our preliminary finding showed that beta-thalassaemia patients has significantly lower Delta-He when compared to alpha-thalassaemia patients. Absolute IDA results showed lower Delta-He than those of functional IDA although the difference is not significant.
Conclusions: Ret-He and Delta-He are potentially useful in differentiating various type of anaemia. Since these parameters are readily available in modern blood counter, these routine indices could value-add as part of initial anaemia laboratory screening test. 

Homocysteine, Imt And Fmt Correlates To Iron Homeostasis Changes In Obstructive Sleep Apnea Patients
Victor Manolov1, Ognyan Georgiev1, Vasil Vasilev1, Zlatina Gramatikova2, Ventcislava Pencheva-Genova1, Radoslava Grozdanova3, Iulia Petrova1, Kamen Tzatchev1, Savina Hadjidekova1, Silvyia Voleva3, Georgi Angov1, Todor Kunchev1, Maria Karadjova1, Latchezar Traykov1
1Medical University Sofia, Sofia, Bulgaria, 2R.E.D. Laboratories N.V./S.A., Zellik, Belgium, 3NCIPD, Sofia, Bulgaria

Introduction: Obstructive sleep apnea syndrome (OSA) is defined as a combination of symptoms as a result of intermittent, recurrent constraint and/or complete airway overflow (sleep disturbance). OSA is associated with the development of insuline resistance, arterial hypertension, metabolic syndrome, systemic atherosclerosis and increased cardiovascular risk.
Methods: 49 patients with OSA were included; age 44.1 +- 7.7. Their results were compared to sex and age matched healthy controls. CBC, serum iron, ferritin, hsCRP, hepcidin, homocysteine and vitamin B12 were measured in the included groups. IMT and FMT were used for atherosclerotic changes evaluation.
Results: We found increased serum hepcidin levels in OSA patients with IMT and FMD changes (129.9 +- 20.4 ug/L) compared to control group (20.9 +- 1.7 ug/L); P< 0.001. A positive correlation was found in OSA patients with atherosclerotic changes between IMT and FMD to serum hepcidin levels (r=0.811, r=0.829, resp.; P< 0.005). Serum hepcidin correlates positively to homocysteine and vitamin B12 in OSA patients (r=0.809, r=0.855, resp., P< 0.01).  
Conclusions: Brain-vascular disease risk factors are connected to obstructive sleep apnea syndrome. Disregulation of iron homeostasis is one of the main risk atherogenesis factors. Early hepcidin quantification might predict an atherosclerosis occurence in OSA patients. Acknowledgements: This project is sponsored by MU-Sofia, as part of Grant Д-52/2018.  

Relationship Between Morphological Rbc Pattern And The Graves' Disease
Junko Matsumoto, Aya Sasaki, Ayumi Takehira, Risah Hayashida, Keisuke Morisaki, Hiroshi Nishiyama, Kenta Fujiwara
Kure Kyosai Hospital, Hiroshima, Japan

Introduction: In the RBC cytogram of ADVIA 2120 (Siemens healthcare diagnostics), we encountered 2 cases with the characteristic pattern of a portion extending from a normal region to a high concentrated hemoglobin region (% Hyper) and a large variation of hemoglobin distribution width(HDW).After the investigation, the common background was Graves' disease. Therefore, we found significance of screening for Graves' disease in RBC cytogram of ADVIA 2120.
Methods: For 18 cases with the characteristics pattern, the following items were examined. 1. Commonality of cases 2. Changes in cytogram with treatment course of Graves' disease. 3. Investigation of RBC cytogram utility concerning screening for Graves' disease.
Results:  1. Commonality was high in ALP and 14 out of 18 cases had Graves' disease. 2. Thyroid hormone were normalized by treatment, whereas the cytogram changed to a normal pattern about 2 to 3 months later. 3. High correlation was found in the order of HDW>% Hyper> RBC among the RBC parameters (p < 0.0001) between thyroid function and RBC cytogram parameters, and the HDW cutoff value was 2.56 g / dL (sensitivity 80.0%, specificity 81.2%).
Conclusions: The RBC cytogram of ADVIA 2120 was shown to be useful for screening for Graves' disease. "Part of the normal range extends to the %Hyper area and HDW ≧2.56 g / dL" is recognized, and if ALP is high, it may be necessary to consider the existence of Graves' disease. Regarding the relationship between Graves' disease and erythrocytes, Yoshida and colleagues reported that thyroid hormone inhibits the synthesis of carbonic anhydrase I isozyme (CA1) in erythrocyte maturation process, and the concentration of zinc in erythrocytes, which are constituents there of, is decreasing. And since the lifetime of erythrocytes is 120 days, it reflects the averaged thyroid hormone concentration about two months ago like HbA1c. Changes in the RBC cytogram seen in this ADVIA 2120 may also be related to them. The screening for Graves' disease by CBC measurement might be very useful for clinical use, and it suggested a new value of ADVIA 2120.

Haemoglobin H Disease And Pitfalls
Yona Mimanda1, Diana Aulia2
1Syarif Hidayatullah State Islamic Uni, Jakarta, Indonesia, 2University of Indonesia, Jakarta, Indonesia

Introduction: Thalassemia α is an inherited disorder of α globin chain synthesis. Mutation of the α-globin gene lead to a decrease or absence of α-globin chain synthesis that affects the severity of the disease such as haemoglobin H disease. The incidence is quite a lot in our country which is in thalassemia belt and proper screening test for early diagnosis is essential. Technical and interpretive problem can lead to misdiagnosis.
Methods: Peripheral blood test in our laboratory found hypochromic microcytic anemia, leukopenia and thrombocytopenia. We Confirmed with peripheral blood smear there are schistocytes (fragmented erythrocytes), target cell and pencil cell. We used hematology analyzer with PLT-F method for platelet count. And then we performed hemoglobin H inclusions test due low of HbA2 level, the result of HbH inclusion was 86/100 erythrocytes. Hb analyzed with capillary electrophoresis (CE) method obtained 25.3% HbH fraction and 1% Hb Bart’s fraction. We diagnosed HbH disease.

Results: Pitfalls in this case are false thrombocytosis, no HbH inclusion body test and misinterpreted graphical output of HPLC. The impedance method calculates schistocytes and microcytic erythrocyte as platelets. Very high thrombocytosis need to be confirmed by peripheral blood smear or optical platelet count or the newest PLT-F method. Haemoglobin H inclusion test should performed if low HbA2 fraction present. And the chromatogram (HPLC) must always be inspected by an experiences person since the software algorithms of the instrument may lead to peak being mislabeled, Hemoglobin Bart’s and Hemoglobin H are not identified or quantitated. Fig 1. Peripheral blood smear  Fig 2. Hemoglobin H inclusion bodies Fig 3. Capillary Hb and HPLC    
Conclusions: Screening tests of HbH disease can widely performe like HbH inclusion body and HPLC and definitive diagnosis requiring either DNA analysis or protein analysis.  Technical and interpretative problems can lead to misdiagnosis. Some pitfalls are known and should be recognized and must be avoided whenever possible.

A Method For Assessing A Platelet Population Ability To Respond To Adenosine Diphosphate
Alena Litvinenko1, Vyacheslav Nekrasov1,3, Dmitry Strokotov1,3,4, Valeri Maltsev1,3,4
1Voevodsky Institute of Chemical Kinetics, Novosibirsk, Russia, 2Optics and Dynamics of Biological Systems Laboratory, Novosibirsk state university, Novosibirsk, Russia, 3Physics Department, Novosibirsk State University, Novosibirsk, Russia, 4Chair of Normal Physiology, Novosibirsk State Medical University, Novosibirsk, Russia

Introduction: Platelets are complex particles that are involved in various physiological processes, such as inflammation, thrombosis, and healing. Modern methods of platelet research without reaction with coagulation proteins are laborious and inapplicable for routine analysis. In addition, pre-analytical sample preparation has a strong influence on the platelet state, and this fact need to be taken into account when interpreting the results. All the above indicates the need to create a method for assessing the reaction of platelets in the absence of coagulation factors and taking into account the initial state of platelets.
Methods: For obtain optical information related to the morphological state of platelets we used the Scanning Flow Cytometer. This devise is measured the angle-resolved light-scattering patterns of individual cells. After measure, we solved an inverse light-scattering problem for obtaining the size and the shape index of each platelet. In addition, we have developed a method for assessing the ability of the platelet population response to adenosine diphosphate influence, taking into account the native state.
Results: We modified sample preparation protocol to reduce the influence of the pre-analytical stage. We also examined the platelet state in five healthy donors using the new method and the sample preparation. In addition, for each sample, the degree of response to adenosine diphosphate was determined.
Conclusions: The presented method showed the effectiveness of determining the degree of platelet reaction to adenosine diphosphate. We hope that this technology has promising possibilities for diagnosing diseases in the early stages and poorly pronounced pathology of platelets.

New Laboratory Approach To Iron Deficiency Anemia In Hospitalized Children
Rungrote Natesirinilkul, Jetniphat Khamyuang, Kulnipa Kittisakmontri, Suphara Manowong
Faculty of Medicine, Chiang Mai Universi, Chiang Mai, Thailand

Introduction: Although iron deficiency anemia (IDA) has been reported as the most common cause of anemia in children, the current available investigations for IDA still have some limitations for diagnosing IDA in hospitalized children who may have inflammation. Reticulocyte hemoglobin concentration (CHr) is recommended by American Academy of Pediatrics (APP) for diagnosing IDA in children and not affected by inflammation. The objective of this study is to see the effectiveness of adding of CHr with the routine investigations for diagnosing IDA in hospitalized children.
Methods: One hundred and seventy two hospitalized children, aged 0.5-15 years, without known hematologic disorders or evidences of hemolysis were enrolled. Completed blood count (CBC), red blood cell (RBC) indices, reticulocyte count (RC), CHr, serum iron (SI), total iron binding capacity (TIBC) and serum ferritin (SF) were tested in all subjects. All anemic subjects defined by World Health Organization (WHO) criteria. IDA was defined by transferrin saturation (TSAT) < 16% or SF < 30 ng/dL combined with diagnosis of anemia for age.
Results: The mean age (±standard deviation) of the subjects was 4.6 (±4.4) years and 61% were male. One hundred and five children (61%) were diagnosed as IDA [95% confident interval (CI) 53-85%]. Among children with IDA, only 58 subjects (55%) had low MCV (< 70 fL) and high RDW (≥ 16%) which are the characteristic features of IDA with the positive predictive value (PPV) of 79% (95% CI 71-86%) and negative predictive value (NPV) of 53% (95% CI 46-59%), respectively. When combining RBC indices with the reticulocyte production index (RPI) < 2 and CHr < 27 pg, the PPV decreased to 69% (95% CI 64-72%) while the NPV increased to 73% (95% CI 57-84%) regardless of thalassemia carrier status. The accuracy of adding RPI and CHr to RBC indices in diagnosis of IDA increase from 63 to 70%.
Conclusions: Adding of RPI and CHr as a part of screening for the cause of anemia in hospitalized children seems to be effective to diagnose IDA in the hospitals where SI, TIBC and SF are not available in regardless of thalassemia carrier status.    

Genetics Of Iranian Patients With Hbh Disease: A Comprehensive Original Study
Mostafa Paridar1, Abbas Khosravi2
1Ministry of Health and Medical Education, Tehran, Iran, 2Transfusion Research center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

Introduction: Despite prevention programs, thalassemia is still one of the most common genetic disorders in Iran. Loss of function of the three alpha-globin genes is called Hemoglobin H disease(HbH), a severe form of α-thalassemia. People with HBH have a variable phenotype, from mild to severe symptoms, depending on their genetics. Hence, the most important step in improving preventive programs is to determine the genetic variation of the disease. In the current study, we have aimed to evaluate the genetics of patients with HbH disease.
Methods: The total of 80 patients suspected of having alpha thalassemia ( mild to moderate anemia, microcytosis, and normal iron levels) referred to hemoglobinopathy and thalassemia center of Ahvaz University of Medical Science were enrolled in the study. Genomic DNA was extracted and analyzed using ARMS PCR, Multiplex Gap, and direct sequencing
Results: Twelve different genotypes were observed in the study population. The most prevalent genotype was -α3.7/-MED (45%) followed by Poly A2 Homozygote (17.5%). Moreover, A total of ten different α-globin mutations have been found in the patients. The most common α-globin mutation was  --MED I which accounts for 26.27% of all mutated alleles, followed by -α3.7 (24.37%) and poly A2 (18.12%). Hematological parameters including Hb, MCV, MCH, and HbH level showed significant differences among patients with different genotypes(adjusted for age and sex).
Conclusions: The findings of this study provide useful information for the design of national thalassemia prevention program. The results can also be used for pre-marriage counseling and prenatal diagnosis.

Effective Screening Of Thalassemia In Hiv Infected Patients
Nattaphol Prakobkaew1, Sitthichai Panyasai2, Surachat Buddhisa2
1Faculty of Allied Health Sciences, Burapha University, Chonburi, Thailand, 2Faculty of Allied Health Sciences, University of Payao, Payao, Thailand

Introduction: Human immunodeficiency virus (HIV) is a cause of acquired immunodeficiency syndrome.  Hematological alterations are common manifestations in HIV infected patients and maybe effect on the screening for thalassemia. In this study, we aimed to investigate the efficacy of thalassemia screening using red cell indices, osmotic fragility (OF) test and Dichlorophenol indophenol precipitation (DCIP) test in HIV positive subjects. 
Methods: Two hundred subjects of HIV infected patients and heathy individuals were enrolled to the study. Complete blood counts, OF test, DCIP test were performed in all samples.Confirmation of common thalassemia genes in Thailand was carried out using polymerase chain reaction. The data were analyzed according to thalassemia genes and screening results. 
Results: Normocytic red cell were observed in a-thalassemia and Hemoglobin (Hb) E carriers. All of HIV infected patients who were carrier with a-thalassemia 1 trait had OF test positive, whereas mean corpuscular volume (MCV) shown the false negative results.Among HIV infected with Hb E trait, false positive rate of Hb E screening using DCIP test was similar to healthy, while a false negative wasn’t detected. 
Conclusions: The results indicate that OF test and DCIP test still has efficiency for screening of thalassemia in HIV infected patients.Therefore, in order to provide accurate screening of thalassemia, the red cell indices, OF test and DCIP test should be carefully evaluated

Symptomatic Southeast Asia Ovalocytosis In A Pregnant Adult Pacient &Ndash; A Case Report And Ongoing Family Study
Samuel Rodrigues1, Carla Nobre2, Celeste Bento3, Maria Joo Fermisson 1, Janet Pereira3, Lus Relvas3, Bela Nogueira1
1Patologia Clnica, CHBM, Barreiro, Portugal, 2Medicina Interna, CHBM, Barreiro, Portugal, 3Hematologia Molecular, CHUC, Coimbra, Portugal

Introduction: Southeast Asia Ovalocytosis (SAO) is a rare autosomal dominant inherited defect of the erythrocyte membrane band3 caused by a 27bp deletion in the SLC4A1 gene, relatively frequent in the Indic-Pacific countries. It confers a characteristic RBC morphology: ridged ovalocytes and stomato-ovalocytes. Hemolytic anemia is usual in newborns and infants. However, symptomatic adults are very uncommon. Worldwide, this mutation is extremely rare with a global prevalence of < 1/1000000. 
Methods: We report the case of a 28-year-old portuguese woman from a Mozambique family, in late-term pregnancy, whose laboratory work-up was remarkable for hemolysis. She had a history of non-immune chronic compensated hemolytic anemia since childhood, seldom hemolytic crisis episodes upon infection and previous pregnancies, 2 spontaneous abortions and cholecystectomy at the age of 15 due to symptomatic gallstones. Family history unveiled chronic anemia, scleral icterus, gallstones on the maternal side and a 7-year-old son with neonatal jaundice. No specific diagnosis was ever established, nor genetic counseling offered. 
Results: Laboratory results showed persistently low RBC count, hemoglobin < 110g/L, borderline/high MCH (35pg), MCHC (35,4g/dL) and RDW (19,1%); sustained reticulocytosis; blood smears documenting anisopoikilocytosis with marked polychromasia, ridged ovalocytes, stomatocytes and elliptocytes. Indirect signs of chronic hemolysis were also present. Hemoglobinopathies were ruled-out. G6PD was within normal range, but the maintained reticulocytosis rendered this result uninformative motivating erythrocyte membrane protein electrophoresis which was also, apparently, normal. A 28 genes NGS panel for hereditary hemolytic anemia was then performed. Two variants in the gene SLC4A1 (band3) were identified in heterozygosity: c.1199_1225del (SAO mutation) and c.166A>G (band3 Memphis polymorphism). An unexpected G6PD Betica variant (p.Leu323Pro/p.Asn126Asp) was also identified.
Conclusions: These results when found in heterozygosity should associate with a no-disease phenotype. This prompted the need for mandatory ongoing family genetic study, further functional tests and segregation analysis to better understand the presented phenotype and its relation to these genetic abnormalities. SAO mutation is independently rare on its own and the presence of these other polymorphisms in the same symptomatic patient is even more so. Studies of such cases greatly enrich the clinical practice, providing the clinicians with important genotype/phenotype data that might help manage these rare patients.

Screening Of Antenatal Women For Beta Thalassemia Trait

Introduction: Thalassemias are autosomal recessive disorders with morbidity, mortality and the economic burden. It is essential to identify carriers in order to assess the risk of couple giving birth to a  child with homozygous state of thalassemia. This study was carried out to detect β- thalassemia carrier state in antenatal women and to develop a model program. Also to create a validated flag in the hematology analyser by evaluating the utility of RBC screening indices.
Methods: This  was a cross sectional study done in two years. At 95% confidence limits and 2.5% absolute precision, the sample size was 183.  The participants were antenatal women(22-40 years of age and between 6 to 12 weeks of pregnancy)with microcytic hypochromic anemia (MCV< 80fL,MCH< 27pg,Hb< 11g/dL).Their blood samples were processed by high performance liquid chromatography for haemoglobinopathies. The sensitivity and specificity of screening indices calculated. Spouses of positive women were counselled for testing and those consented were tested. 
Results: β- thalassemia trait was detected in 43 women(23.5%). 24(13.11%) had other hemoglobinopathies. 116(63.33%) were normal.In β-thalassemia trait, the haemoglobin values ranged from 7.8 to 12.2 g/dl. (Mean 10.2 g/dl). Mean RBC count, MCV and MCH were 5.21million/µl, 65.12fl and 20.86pg respectively.   Mentzer index < 14, Green &King < 84.9,Sirdah < 33.74, Ehasini < 20.1, England&Fraser < 10.98, Sehgal index < 971.55 had sensitivity of > 97% and specificity of > 85%. Except Mentzer and Sehgal indices, the cut offs differed from the original values designed. Flag to indicate positive Mentzer or Sehgal index is included in the hematology analyser. HPLC was done for 24 (55.81%) of the spouses of carriers for β-  thalassemia.
Conclusions: Antenatal women who are microcytic hypochromic with Mentzer or Sehgal indices positive(flag alert) are to be subjected for HPLC. Spouses of women who are heterozygous need to be counselled and tested. If both are positive, options of prenatal testing to be provided. This model if adopted in all health sectors will definitely bring down the burden of thalassemia.

Reticulocyte Hemoglobin (Chr) Reported By Mindray Bc8600 + In The Study Of Anemia

Introduction: Reticulocyte hemoglobin content (CHr) is an estimation of the functional availability of iron into the erythron, and it  has been extensively used in diagnosing different erythropoiesis conditions. Mindray BC 8600+ counter reports  CHr, we  study its value in anemia of different ethiologies and the reliability  for the diagnosis of iron deficient erythropoiesis. 
Methods: Four hundred and sixteen  samples were run : 80 healthy subjects, 202 microcytic anemia  (thalassemia carriers and iron deficiency anemia, IDA), 91 normocytic  anemia (hematology malignancies and anemia of chronic disease, ACD) , 37 macrocytic anemia (lack of vitamin B12  or MDS). C reactive protein, Serum iron, transferrin saturation, ferritin and soluble transferrin receptor (sTfR) were assayed. Kolmogorov-Smirnoff  was used  to verify normal  distribution of data. Differences among groups were assessed using analysis of variance, considering P < 0.05 to be significant. Correlation coefficient between erythrocyte indices and CHr was calculated using the method of Pearson. Receiver operating characteristic analysis was used to assess  the diagnostic performance of CHr   for detecting iron deficient erythropoiesis. Gold standard for iron deficiency  was sTfR >52 nmol/L.
Results: Gaussian distribution was proven. Whole range  CHr 15.1-47.2 pg, Mean and range in healthy subjects CHr 33.2 pg, 28.9-37.5 pg.  The groups had CHr values significantly different P=0.0001, except the healthy group and ACD patients, P=0.0754. In the microcytic group, the values in patients with IDA (CHr mean 23.8 pg, SD 1.7pg) and thalassemia carries (CHr mean 23.1 pg, SD 3.0 pg) were not significantly different P=0.0756. So patients with restricted erythropoiesis, due to lack of iron  or globin, had similar low values. Values over the reference range in the macrocytic group is not related to iron status, reflects the megaloblastosis. Correlation MCH/CHr R2=0.9232, P< 0.0001 (95%CI 0.9069-0.9367). Using a cut off  CHr < 29.0 pg, iron deficient erythropoiesis  could be diagnosed sensitivity 82.9%, specificity 98.6%.  Area under the curve was 0.934 (95% CI 0.892–0.963).
Conclusions: Disturbances in erythropoiesis and iron metabolism may occur in many patients, the challenge is to identify these patients as early as possible. CHr provides a sensitive method for quantifying the hemoglobinization of reticulocytes. It is a reliable marker to identify iron deficient erythropoiesis, CHr may allow the complete scope of disorders of iron metabolism to be identified quickly and managed.

Differential Diagnosis Of Microcytic Anemia

Introduction: The Mindray 6800+  analyzer  reports red cells (RBC)  extended parameters , which represent the subsets of erythrocytes : % Hypo, the percentage of hypochromic RBC, Hb < 280g/L, % Hyper, the percentage of hyperchromic RBC  Hb >410g/L % Micro, the percentage of microcytic RBCs  volume< 60 fL;  % Macro, the percentage of macrocytic RBCs  volume > 120 fL. We aimed to  evaluate the reliability of an algorithm combining  the values of RBC extended parameters in the differential diagnosis of microcytic anemia.
Methods: Only adults were included in the study The learning set comprised samples from 250 patients with microcytic anemia,  mean cell volume (MCV < 80 fL), which were measured on a Mindray 6800+   analyzer. Subjects With Mild IDA: 150 patients with Hemoglobin (Hb) < 120g/L.  Patients with Hb < 90 g/L were excluded because these cases of severe anemia are not confused with beta thalassemia carriers in our daily practice. 100 patients had a previous diagnosis of beta thalassemia trait The values of the RBC extended parameter were recorded; microcytic/hypochromic ratio (MH ratio ) was calculated; the optimal cut off for detecting thalassemia carriers was established using Receiver operating characteristic (ROC). This value was  used in the validation set of  95 patients with microcytic anemia (30 carriers , 65 IDA)
Results: Area under curve  0.918 (95% CI 0.871-0.966) for a cut off >20 , sensitivity 90.3 % specificity 81.1% In the validation set using MHratio>20,   28/30 patients were correctly classified as carriers; these two misclassified had % Hyper >5%. In the IDA group 10  patients had MH ratio>20 and thus considered carrieres, but 8 of them Hyper < 10 %. The combination of MHratio>20 and % Hyper>10 % correctly classified 100 % of carriers and 96.9 %  of IDA patients .  
Conclusions:   RBC subsets give insights of physiology underlying microcytic anemia. Thalassemia is characterized by an increase in of RBCs,  and high rate of microcytosis. Thalassemic microcytes are more microcytic, Hb concentration,  seems to be more preserved, despite low Hb content, so the percentages of hyperchromic cells tend to be higher than those obtained for IDA patients with a similar Hb values. An algorithm  derived from RBC extended parameters provided by the Mindray 6800+  analyzer  could be a useful tool in the differential diagnosis of microcytic anemia. Samples can be chosen for further analysis to confirm the diagnosis of thalassemia.  

The Diversity Of Myeloproliferative Leukaemia Virus (Mpl) W515L, Jak2 V617Fmutations And Erythropoietin Level In Patients With Polycythemia Vera
Nasr Eldeen Ali Mohammed , Muzna Mohammed Ali Sulima , Ibrahim khider Ibrahim
1Department of Haematology, Faculty of Medical Laboratory Sciences, Alneelain University,, , Sudan

Introduction: Myeloproliferative leukemia virus Oncogene MPL (W515L) mutation was described before in patients with myeloproliferativedisorders including polycythemia vera (PV). Its found in chromosome 1p34, resulting from substitution of tryptophane to leucine amino acids at codon 515. Mutation in (MPL W515) leading to receptor activation in the absence of thrombopoietin binding.
Methods: This is a case control study, in which a total of 37 patients were known as cases with polycythemia vera according to World Health Organization (WHO) diagnostic criteria 2008 were participant in this study;their mean age was 48.5 years. DNA was extracted from peripheral blood leukocytes using salting out methods and the presence of MPL (W515L) mutations were analyzed by using allele specific polymerase chain reaction.
Results: The study observed that 10 (27.0%) of patients (out of 37) were positive for MPL (W515L) mutation,based on the gener all mutated patients were males.The coexistence (JAK2 V617F and MPLW515L) mutations were present in 90% patients.MPL W515L-positive and JAK2 V617F-positive polycythemia vera cases had more sever than other patients .The haemoglobin concentration, red blood cells count, white blood cells count and platelets count were increased among mutated MPL W515L compared non mutated. As for package cell volume (PCV) and erythropoietin level (EPO) there is significant differences mutated MPLW515L compared to nonmutated with (P.value 0.042, 0.00) respectively.
Conclusions: This study concluds that the MPL W515L mutation was found in 27% of Sudanese patients with polycythemia Vera and the coexistence (JAK2 V617F and MPLW515L) mutations were present in 90% patients.

Automated Haematology Qc Survey Indicates That The Actionable Tests (Wbc, Neutrophils, Platelets, Lymphocytes And Haemoglobin) Are Minimally 3 Sigma And That Haemoglobin Should Be Reported Instead Of (Low Sigma) Haematocrit
George Cembrowski1,2, Barbara Delasalle3, Gwen Clarke4, Ismail Surtina-Azarov5, Richard Mccafferty6, Samuel Machin7
1University of Alberta Hospital, Edmonton, AB, Canada, 2CC Quality Control Consulting, Edmonton, AB, Canada, 3UK NEQAS Haematology, Watford, United Kingdom, 4Canadian Blood Services, Edmonton, AB, Canada, 5Northern Alberta Institute of Technology, Edmonton, AB, Canada, 6St. James's Hospital, Dublin, Ireland, 7University College London Hospitals NHS Foundation Trust, London, United Kingdom

Introduction: Under the auspices of the International Council for Standardization in Hematology (ICSH) we conducted a survey of QC practices in automated haematology.  The ultimate purpose of this survey is to provide guidance for selecting optimal QC  rules for each of the full blood count  (FBC) components.  Laboratories provided estimates of their long term normal level QC averages and standard deviations for each FBC component.  Sigma, a measure of analytical goodness was derived from the ratio of Biologic Variation (BV) to Analytical Variation (AV) and helps select optimal QC rules.  Based on the BV and AV, we  simulated patient results to demonstrate the effect of sigma on specific components.
Methods: Survey participants used Survey Monkey to record instrument model, laboratory environment, QC frequency, usual QC range and mean and standard deviation of the normal level QC at 60 days.  Sigma was derived for each laboratory and FBC constituent from published BV estimates and survey-determined QC AV. We simulated the analysis of both low and high sigma analytes.  As clinicians primarily classify normal and abnormal based on institutional reference intervals, we simulated serial patient data close to their reference limits. 
Results: 55 survey results represented 3 manufacturers:  Coulter:(1 DXH and 7  DXH 800); Siemens:(14 Advia 2120); and Sysmex:(9 XE2100 and 24 XN).  Figure 1 shows median QC defined sigmas with the manufacturer anonymized.  Figure 2 compares simulated Hgb (sigma=3.3) and Hct (sigma=1.4) measurements of a nonHispanic black woman close to her upper and lower reference intervals.  There is negligible scatter of Hgb in comparison to Hct.
Conclusions: Use of tests with low sigma (< 2) will result in analytical error affecting the accuracy of the measurement.  Reporting of Hgb rather than Hct will always provide a more useful, less variable measure.  Luckily, the actionable tests, those which are associated with specific clinical protocols, generally have sigmas exceeding 3, and thus produce highly reproducible results.  

&Ldquo;Standardization Of Workflow Management Along With Developing Quality & Screening Algorithm For Wbc Pathologies, In A Busy Hematology Laboratory By Utilizing Middle Link Software&Rdquo; Combining Islh Rules & Technical Instrument Flagging&Rsquo;S.

Introduction: Effective utilization of HORIBA Software “MIDDLE LINK “applications along with ISLH rules & instrument technical flags for; 1)Standardization & Harmonization of two 5 part Automated Haematology analyzers        2)Applying ISLH rules along with MIDDLE LINK rules for report standardizing 3)To study effective utilization of technical “Flags & Alarms” system of instrument 4)Creating universal guidelines for slide review & reflex testing. · 5)Improvement and analysis on quality control 6)Development of algorithm for abnormal results for better clinical outcome 7)Effective control & monitoring of TAT 
Methods: The study was conducted at SUBURBAN DIAGNOSTICS, a multidiagnostic center with a core lab, performing approximately 2,50,000 complete blood count analysis per year. Two HORIBA ABX 120, 5 part haematology instrument were installed in 2013 & 2014, harmonized both by using MIDDLE LINK Software validation with defined pathological rules in the form of technical flags of instruments combining with ISLH Rules (27 applied) for automated 5 part haematology analyzer following ICSH guidelines. 
Results: A total of 75000 complete blood count with technical flag, reflex ISLH rules were analyzed for obtaining statistical data observations of 1) Sample integrity issues (interfering substances) & 2)Standardizing slide reiew criteria & 3)Creating a screening algorithm for WBC pathology categorizing, for improving validation, better reporting of results and clinical outcomes.  
Conclusions: After using ML & ISLH rules together, we found to have better harmonization between the instruments. The result interpretation has been precise as the quality control is on real time basis with technical flags & reflex rule application on sample integrity. It is being observed that combining both ISLH & ML rules together provide better understanding of abnormal WBC pathologies along with instrument Flagging & Alarming system, improving result validation, also developing newer rules for common pathologies using RUO parameters like LIC, IMG, IMM % & # with technical flag & ISLH rules, so as to help more in workflow management in a busy lab & managing turnaround time (TAT) ultimately improving productivity with quality.

Usefulness Of CellMorphologyParameters ForDetection And Diagnosis Of Acute Leukemias.
Alicia Martnez-Iribarren1, Mara Lpez-Molina 1, Xavier Tejedor-Gandux 1, M ngels Sala-Sanjaume 1, Agueda Ancochea2, Luca Galiano1, Alicia Castillo1, Carla Fernndez-Prendes1, M Antonia Llopis-Daz1, Cristian Morales-Indiano1
1Laboratori Clnic Metropolitana Nord. Hospital Universitari Germans Trias i Pujol, Badalona, Spain, 2Institut Catal dOncologia. Hospital Universitari Germans Trias i Pujol, Badalona, Spain

Introduction: Acute leukemia (AL) starts in the marrow bone and is characterized by an uncontrolled, fast and, if not treated, fatal process, in which immature cells invade blood. It’s necessary to have an integrated laboratory diagnosis, includingcell blood count (CBC) with differential leukocyte, morphology, cytogenetics, immunophenotype and molecular biology. The CBC and differential leukocyte is the first step in AL detection. In recent years, technology has improved and new morphological parameters that could help in AL approach have been developed. The aim of this study was to assess the usefulness of cell morphology parameters (CMP) obtained from combining volume (V), conductivity (C) and laser dispersion (Sn) of different cells in AL diagnosis.
Methods: We evaluated 92 samples (43 new AL diagnoses and 49 healthy controls). The patients presenting AL were classified into two groups: 35 patients with acute myeloid leukemia (AML) and 9 with acute lymphocytic leukemia (ALL). Patients of AML group were divided according to morphological FAB classification in 29 AML (M0+M1), 3 promyelocytic AL (PAL;M3) and 3 monocyte AL (LAMM;M4+M5). All samples were run on the DxH-800 Analyzer (Beckman Coulter; Miami, FL, USA). We analyzed CMP in all groups. ANOVA test and ROC curve were performed to assess differences between groups. Significant p values were those below 0.05.
Results: There were no differences in quantitative results of CBC between AML and ALL. Concerning CMP values, blasts in AML presented higher significant V and Sn in neutrophils distribution zone compared to ALL (V-Ne-AML:159.8±24.3vs V-Ne-ALL:146.3±9.0; p< 0.013. Sn-Ne-AML:16.8±5.1 vs Sn-Ne-ALL:12.4±2.0; p< 0.001). ALL blasts showed higher significant Sn in monocytes distribution zone (Sn-Mo-AML:13.9±2.7 vs Sn-Mo-ALL:18.1±3.3; p=0.001). PAL showed higher significant monocytes’ conductivity compared with the rest of AL (p< 0.001). CMP with better diagnostic accuracy for distinguishing between AML and ALL were Sn-Ne (AUC: 0.788 (IC95%: 0.631-0.945; p=0.012)) and SU-Mo (AUC:0.852 (IC95%:0.715-0.990; p=0.012)).
Conclusions: The CBC along with the CMP, especially those related with laser dispersion, could be useful in early diagnosis and could help to discriminate between AML and ALL. In this preliminary study, monocytes’ conductivity allowed the PAL identification, but future research should be done.

Malaria Parasite Proficiency Testing- 11 Year Review
Fernando Estepa1, John Giannoutsos2, Emanuel Dales1
1RCPAQAP, St Leonards, Australia, 2NSWH Pathology, Nepean, Australia

Introduction: The definitive diagnosis of malaria requires the identification of the parasite on a stained blood film. There are currently 5 species of the malaria parasite and each species has unique morphological features that distinguished the specie. RCPAQAP provides an external quality assurance (EQA) program for malaria parasite identification. This paper reviews the performance of the programs in its 11 years of providing proficiency testing in malaria parasite morphology.
Methods: The EQA program consists of 2 surveys per year and 2 case studies per survey. Each case study consists of a thick and thin blood film, stained at pH 7.2. A small expert committee, using a similar format as the participants, conducted selections of case studies. In each survey, participants receive the two case studies, brief clinical details and limited full blood count results.  Using a predetermined code list, participants report the morphological feature(s) and a diagnosis. Using a predefined scoring system, an expert committee assigns scores to the submitted descriptions and diagnoses. A graphical report including results, peer comparisons and educational commentary is then provided to the participants. The results of  44 case studies over 11 years from 2008 to 2018 have been reviewed.
Results: Case studies submitted for assessment include: P. falciparum (15), P. vivax (13), P. Malariae (6), P. ovale (3), mixed infections (6) and a normal blood film. Of the 15 cases of P. falciparum, 2 of these cases returned < 50% with acceptable response. In contrast the other three species returned acceptable response ranges from 52% to 97%. The 2 case studies with P. falciparum +P. malariae returned >70% acceptable responses. Whereas, the 2 case studies with P falciparum +P ovale returned acceptable responses of < 50%. There were differences of acceptable responses for P falciparum +P vivax infection, one returned 21%, the other the 96%. The normal blood film returned a 76% acceptable response. Obtaining an acceptable diagnosis did not correlate consistently with accuracy in reporting the morphological feature.
Conclusions: The identification and differentiation of the 5 species of malaria parasite through microscopy remains clinically relevant. The review highlights the need for continued professional development, and participation in an EQA program facilitate and improved capabilities necessary for best practice in the identification and differentiation of the morphological feature of each malarial species.

Advantages Of Bd Microtainer Map Microtube - A Comparison With Bd Microtainer Edta Microtube
Hani Hadrianto, Kah Jing Lee, Lai Ngah Leong, Soh Hong Ang, Ching Mei Lam
KK Women's and Children's Hospital, Singapore, Singapore

Introduction: The current blood collection tube used for paediatric sample collection for Full Blood Count (FBC) analysis in our institution is the BD Microtainer EDTA Microtube. The disadvantage of this tube is the need for manual (open mode) sampling, requiring cap removal prior to analysis and potential aerosol exposure risk. The BD Microtainer MAP Microtube is an alternative which comes with a membrane cap allowing sample analysis without cap removal, reducing aerosol exposure risk. The tubes are also compatible with autoloader (closed mode) sampling, enabling walkaway analysis. We compared the performance of the EDTA Microtube and MAP Microtube using paired paediatric samples. Comparison studies were performed on the following parameters - White Blood Cell Count (WBC), Red Blood Cell Count (RBC), Haemoglobin (HGB) and Platelet Count (PLT). We also assessed the throughput of analysis and test volume based on autoloader sampling.
Methods: Whole blood samples were obtained from paediatric patients into EDTA Microtubes and MAP Microtubes and analysed on the Sysmex XN analyser. The R, slope and intercept values were obtained to determine the correlation strength of the parameters stated above. Determination of the minimum volume required for MAP Microtube using autoloader sampling was performed by analysing whole blood samples in adult EDTA tubes against 3 sample volumes (150, 200 and 250 µL) in MAP Microtube using autoloader sampling. Throughput of analysis was determined by analysing samples continuously and measuring the time taken between result generation.
Results: There was excellent correlation in the parameters assessed when comparing between the EDTA Microtube and MAP Microtube (Table 1):  
Parameter R Slope Intercept n
WBC 1.00 0.98 0.05 42
RBC 0.99 1.03 -0.13 42
HGB 0.99 1.02 -0.29 42
PLT 1.00 0.99 -1.59 41
According to Sysmex XN user manual, minimum sample volume is 250 µL. However sample volumes of 200 and 250 µL using the MAP Microtube produced good correlative results. Throughput of analysis for the MAP Microtube was comparable to the EDTA Microtube at 63 samples per hour.
Conclusions: MAP Microtube produces comparable results and throughput to EDTA Microtube with small sample volume requirements. Therefore it is a suitable substitute for EDTA Microtube, with the advantages of time savings and improved staff safety.

Evaluation And Analysis Of Non-Standardized Hematology Reagents And Original Hematology Reagents On Patient Results
Neeraj Jain1, Arvinder Singh2, Shubham Rastogi3, Ajay Sajwan4
1Jain Diagnostics, New Delhi, India, 2Arth Diagnostics , Udaipur, India, 3HORIBA Medical, Montpellier France, France, 4HORIBA Medical , Haridwar , India

Introduction: Original equipment manufacturers produce technologically adaptable chemical reagents for their respective instruments. Nowadays, the in vitro diagnostics industry has expanded with many manufacturers producing non-standardized chemical reagents. In this study, we evaluated on the ABX Micros 60 from HORIBA Medical the impact of widely available reagents in the market on patient’s results and also the impact on quality of the analyzer.
Methods: Hematology reagents of HORIBA medical and non-standardized company were striated for fungal culture on SDA (Sabouraud Dextrose Agar Media) and for bacterial culture on R2A Media on petri dishes. One ABX Micros 60 (instrument 1) was installed with three original reagents (ABX Minidil LMG, ABX Cleaner, ABX Lysebio) from HORIBA Medical while another ABX Micros 60 (instrument 2) was installed with all three reagents of non-standardized reagents. Patient samples were analyzed simultaneously on both instruments. 80 random patient’s samples were processed in the laboratory. These samples were processed after having been duly calibrated and subjected to quality control in accordance with the original reagent manufacturer's prescription. We found that the quality controls and calibrators were not available from the manufacturer of non-standard reagents.
Results: The analysis of this study is divided into two sections. The first has microbiological tests and second has the correlation of parameters, flags and alarms between both the analyzer’s results. Microbiology tests were very important to understand bacterial and fungal contamination of reagents. This was useful because it can affect the results of the patient’s samples. These tests show the growth of bacterial and fungal with non-standardized reagents. We also compared 10 parameters (WBC, RBC, HGB, HCT, PLT, MCV, LYM%, MON %, GRA % and RDW) on both instruments. WBC results correlation was acceptable. RBC, HGB, HCT were in “To verify” category, parameters like LYM%, MON%, GRA% and RDW were rejected. In addition, flags and alarms are numerous when patient samples were run on non-standardized reagents in comparison to samples processed on standardized reagents.
Conclusions: Microbial culture shows a contamination during the manufacturing of non-standardized reagents and therefore their use may affect the results. Patient’s results are more important than lower cost. For this reason, we recommend that you use only original reagents.

Effect Of Autoverification Of Results On Hematology Laboratory Workflow
1Kanuni Sultan Suleyman Training&Research Hospital, Istanbul, Turkey, 2Sisli Hamidiye Etfal Training&Research Hospital, Istanbul, Turkey

Introduction: Our aim was to evaluate the effect of autoverification process on our test turnaround times, sample rejection rates, and sample repeatability rates on our newly established hematology analyzers.
Methods: We used the middleware of Sysmex XN9000 series (Sysmex Cooperation, Kobe, Japan) for the autoverification process. We compared the hematology laboratory test turnaround times (TAT), test rejection rates and test repeatability rates of the preceding 3 months with those of the following three months during which we started to use the autoverification process of Sysmex XN Hematology Analyzers. Only outpatients were enrolled; emergency department patients and inpatients were excluded from our study. Body fluid analysis and cell counts were excluded. Our target turnaround time for complete blood count was 120 minutes for outpatients.
Results: In the first three months with no autoverification process, total tests analyzed were 57234; whereas in the following three months with autoverification process it was 67287. Out of 67287 tests analyzed, 48155 results were verified with the new autoverification software which comprised 71.6% of the results analyzed totally. Turnaround times in the same periods were 69.4 and 51.5 minutes, respectively. Reaching the target TAT were 91.4% and 92.8%, respectively. Sample rejection rates  decreased from 3.85% to 2.85%, and also test repeatability rates also decreased from 1.28% to 0.38%, respectively.
Conclusions: This is the first time we used autoverification in our hematology laboratory. We found that as well as being effective in accelerating the hematology lab’s workflow, it also helps to decrease the sample rejection and repeatability rates which have a negative effect on the extra costs paid for analyzed tests with no results, the tubes wasted, the time spent, and most importantly the patients’ blood drawn to no purpose. We conclude that using autoverificationshould also be of concern in healthcare management.

Comparison Of Differential Leukocyte Counts Using Microscopic Analysis As Reference Procedure And Immunoflowcytometric Analysis
Hiroshi Kondo1,6, Tomohiro Takeda1,6, Etsuko Ogawa2,6, Kunihiro Takano3,6, Yutaka Nagai1,4,6, Kaoru Tohyama5,6
1Knasai University of Health Sciences, Osaka, Japan, 2BD Bioscience, Tokyo, Japan, 3Beckman Coulter, Osaka, Japan, 4Nihon Kohden, Saitama, Japan, 5Kawasaki Medical School, Okayama, Japan, 6Japanese Society for Laboratory Hematology, Tokyo, Japan

Introduction: The Japanese Society for Laboratory Hematology (JSLH) has continued to employ EQA as a differential leukocyte count using anticoagulated fresh blood samples for manufacturer's reference analyzers since 2001.  In order to verify the immunoflowcytometric method for determining the five-part differential leukocyte counts developed by JSLH (JSLH-Diff) as a reference method to determine the EQA target value, three methods for differential leukocyte counts were compared.  The differential leukocyte counts were determined by microscopic analysis and immunoflowcytometric analysis (JSLH-Diff and ICSH-Diff) and compared with values obtained using automated hematology analyzers for EQAs in 2010, 2013, and 2016.
Methods: The microscopic reference method defined by the Clinical and Laboratory Standards Institute (CLSI) guideline H20-A2 was used for every EQA.  The International Council for Standardization in Hematology (ICSH)-Diff (French method) as the immunoflowcytometric reference method was used for EQAs in 2010 and 2013, and   JSLH-Diff was used for every EQA.  Fresh whole blood anticoagulated with K2EDTA was collected from healthy volunteers.  The survey samples were measured within 4 hours after drawing using the representative reference hematology analyzers at six manufacturers’ laboratories.  The manufactures were: Beckman-Coulter, Sysmex, Siemens, Abbott, Horiba, and Nihon Kohden.
Results: Monocyte counts determined using the microscopic method were lower than on using immunoflowcytometry and automated hematology analysis for every EQA.  Monocyte counts using ICSH-diff and JSLH-Diff were 39 and 18% higher than the microscopic method, respectively.  Lymphocyte, neutrophil, monocyte, and basophil counts using automated hematology analyzers were closer using immunoflowcytometric analysis than using microscopic analysis.
Conclusions: Monocyte counts determined using the microscopic method were lower than on using immunoflowcytometry and automated hematology analysis for every EQA.  JSLH-Diff is recommended as a reference method to determine the target value of the differential leukocyte count for EQA.

Evaluation Of One-Stage Aptt-Based Factor Ix Assays For Monitoring N9-Gp (Rebinyn), A Recombinant Pegylated Fix Concentrate.
Karen Moffat1,2, Stephen Carlino1, Michael Keeney3, Michelle Sholzberg4, David Good5, Catherine Hayward1,2,6
1Hamilton Regional Laboratory Medicine Program, 2Department of Medicine, McMaster University, 3London Health Sciences, 4Departments of Medicine and Laboratory Medicine and Pathobiology, University of Toronto, 5Department of Pathology and Molecular Medicine, Queen's University, 6Department of Pathology and Molecular Medicine, McMaster University

Introduction: The APTT-based factor IX (FIX) assay is robust and aids clinicians in diagnosis and monitoring of therapy for hemophilia B patients. Recently, Canadian Blood Services introduced N9-GP (Rebinyn), a recombinant PEGylated FIX concentrate, as a treatment option. Chromogenic FIX assays are reported to be accurate for monitoring N9-GP, however low volume tests add laboratory costs in environments of significant fiscal constraints. To meet clinical need and minimize costs, we evaluated one-stage FIX assays using the two APTT reagents recommended for monitoring N9-GP: Instrument Laboratories SynthAFax and Diagnostica Stago’s Cephascreen.
Methods: FIX one-stage APTT-based assays were run on Diagnostica Stago’s STAr/Evolution with APTT reagent being the only test variable between assays. Standard Human Plasma (WHO referenced) with quality controls (QC) were used (Siemens, Marburg, Germany). SynthAFax and Cephascreen FIX assays were run over 3 days on four calibration curves. Twenty individual normal plasma samples and three commercially prepared N9-GP samples of known concentrations were assayed (Precision BioLogic, Dartmouth, NS). Samples were also run using routine FIX assay (Siemens Actin FS APTT reagent). N9-GP interlaboratory data were compared with three laboratories. Statistical analysis included: mean, standard deviation, %CV, t-test and linear regression.
Results: Individual normal plasma samples FIX results were similar between SynthAFax:Actin FS: (y=0.81x+0.19;R2=0.70); Cephascreen:Actin FS (y=1.15x+0.25;R2=0.72); SynthAFax:Cephascreen (y=1.30x-0.38;R2=0.86). Although limited QC values (n=4) for SynthAFax and Cephascreen, values were within established Actin FS QC ranges. N9-GP samples demonstrated excellent recovery with SynthAFax (y=1.13x+0.06;R2=0.999), and compared well with two laboratories (Lab A:y=1.03x-0.01;R2=0.999; Lab B:y=1.12x-0.01;R2=0.997). Cephascreen and Actin FS demonstrated significant underestimation of N9-GP assigned values (p< 0.05).
Conclusions: There was acceptable performance with all FIX assays for individual normal plasma samples and QC. Inhouse and interlaboratory results for N9-GP samples demonstrated good FIX recovery using SynthAFax. Underestimation of N9-GP FIX activity with Actin FS had been reported; however, underestimation using Cephascreen was not expected. A limitation was the small number of prepared N9-GP samples. Our study confirms N9-GP can be reliably monitored using a one-stage FIX assay with SynthAFax APTT reagent.

Stability Of Complete Blood Cell Count (Cbc) Parameters At Room Temperature And Refrigerated Using Xn Sysmex Hematological Analyser.
Rosana Penteado, Erivaldo Junior, Rodrigo Barroso, Liliana Suganuma, Andrea Villarinho, Cludio Mendes, Camila Monteiro, Heloise Costa, Elvira Velloso, Joo Guerra
HIAE, So Paulo, Brazil

Introduction: Numerous studies show that 70% of laboratory errors are attributed to failures in the preanalytical phase. Inappropriate transport and storage conditions of the samples directly affect the quality of the test. Numerous articles published on the stability of the blood samples, but demonstrated  inconsistency and variability interequipment. This study aims to investigate the stability of the CBC parameters, using Sysmex XN equipment.
Methods: 60 samples of CBC within normal parameters of healthy patients were collected in tubes containing EDTA and processed in less than 1 hour. These samples were divided into two groups, room temperature and refrigerated at 4-8°C, and were repeated every 4 hours up to maximum of 44 hours. The differences between the results compared to the initial time (< 1 hous) were performed using ANOVA with repeated measures followed by multiple Bonferroni comparisions. The analyzes were performed with a significance level of 5%.
Results: The results of this study demonstrate that the Hematocrit (HT), Mean Corpusculat Volume (MCV), Mean Corpusculat Hemoglobin Concentration (CHCM) and Red Cell Distribution Width (RDW) showed significant differences in room temperature and refrigeration. Inlike ok leukocyte indices, that did not show statistical diference time and temperature dependent. Erythroblastos counts showed lower stability when refrigerated, and, conversely, improved the satbility of the Reticulocyte hemoglobin equivalent  (Ret-HE), parameter from 4hs to 8 hours. Dispersin results were observed in platelet indexes, with improved stability of the Mean Platelet Volume (MPV) and Immature Platelet Fraction (IPF) parameter when the sample was refrigerated, but with loss of stability of te platelet counts by fluorescent and impedance methodology after 24 hours.
Conclusions: These results suggest that the sample for CBC should preferably be abalysed before 4 hours after collection, but in cases of ler transport times we suggest that this sample be conditioned in a refrigerated environment, preferably up to 24 hours, with a loss of reliability of the VPM and Ret-He results. Studies to evaluate the qualitative flags and percentage of microscopic revisions should be performed.

Regional Quality Improvement Initiatives For Bone Marrow Specimen Processing In British Columbia
Audi Setiadi1,2, Monika Hudoba1,2, Kanwal Deol1, Geoffrey Chan1, Kathy Stockford1, Loretta Rothstein1, Smith Tyler1,2, Edin Kojic1, David Pi1,2
1Vancouver General Hospital, Vancouver, BC, Canada, 2University of British Columbia, Vancouver, BC, Canada

Introduction: The examination ofgood quality bone marrow (BM) specimens is essential to the diagnosis of hematological disorders. Methods of processing of BM specimens can vary considerably across different laboratories, which may lead to diagnostic inconsistencies. We conducted a regional, inter-laboratory BM specimen quality improvement initiative in British Columbia (BC) to identify specific challenges that technologists encounter during BM procurement/processing, and to address these through workshops and reach out programs.
Methods: In 2016, we sent a survey and unstained BM aspirate slides (1 normal, 1 myelodysplastic syndrome and 1 multiple myeloma) to 12 hospitals across BC to process and stain according to their protocols. Six blinded scorers at Vancouver General Hospital evaluated the stained slides and assigned a rating from 1 to 4 (1=unacceptable, 2=poor, 3=fair, 4=excellent) for each criteria evaluated (nuclear detail, cytoplasmic and granular staining, acidity/alkalinity, paleness/darkness, stain debris and fixation artifacts). We conducted two educational BM workshops at the BC Society of Laboratory Science Congress in 2016 and 2017, which included best practices in BM procurement, processing, staining, and differential count. We shared resources for staining trouble-shooting, protocols, and Standard Operating Procedures. Feedback and opportunities for on-site training for BM technologists was provided at a central laboratory. Two years later, a follow up study was conducted by re-sending unstained slides, documenting any changes in their protocols since the previous interventions. The stained BM slides were scored utilizing the same criteria as previous.
Results: Ten hospitals participated in the pre-intervention evaluation of BM slide staining in 2016, and 8/10 sites participated in the post-intervention study in 2018. There is an improvement in the mean scores across laboratories pre- vs post-intervention with regards to nuclear details (65.1% vs 78.6%, P=0.01), cytoplasmic staining (65.1% vs 74.7%, P=0.08), staining of granules (65.1% vs 78.1%, P=0.01), acidity/alkalinity of stain (73.4% vs 84.9%, P=0.03) and fixation artifacts (74.5% vs 89.1%, P=0.01). Overall, there is a significant improvement in stain quality across all centres over the two-year period (mean 74.2% vs 80.7%, P=0.045).
Conclusions: Our results highlighted that quality improvement initiatives involving reach-out programs and educational workshops had a positive impact in the quality of BM slide processing and staining across laboratories in BC.

Procedure To Minimize Interference Of Hypertriglyceridemia In Haemogram Parameters Of Lipemic Samples In Inadequate Fasting
Xavier Tejedor Gandux, Alicia Martnez Iribarren, Cristian Morales Indiano, Angels Sala Sanjaume, Merc Espinosa Nieto, Selena Rodrguez Castro, Silvia Torres Romero, Xantal Abadia Ruiz, Joana Minchinela Gir, MAntnia Llopis Daz
Department of Clinical Analysis and Biochemistry. Laboratori Clnic Metropolitana Nord (LCMN). Hospital Universitari Germans Trias i Pujol. , Badalona, , Spain

Introduction: Lipemia interferes with the accurate determination of hemoglobin by spectroscopy on most hematology analyzers and those derived calculated parameters from it. There are several case reports in literature concerning association between inadequate fasting and (hypertriglyceridemia) HTG, but few intend to evaluate aspects of laboratory diagnosis, methodological limitations imposed by lipemia and management for resolution. This study is an approach to laboratory interference caused by HTG in plasma from patients with inadequate fasting with the objective to present actions aimed at achieving reliable and useful laboratory tests in clinical decisions.
Methods: 48 samples of patients in inadequate fasting with triglyceride values between 329 mg/dL and 11171 mg/dL were used. Complet haemograms were determined in an automated hemocytometer. The lipemia correction was carried out by replacing the plasma volume by an equal volume of saline solution (NaCl 0,9%) after centrifugation of the whole blood (with EDTA K3). For each sample, it was calculated the variation between analyzes (before and after the correction) for the following parameters: leukocytes (WBC), erythrocytes (RBC), haemoglobin (Hb), haematocrit (HCT), mean corpuscular volum (MCV), mean corpuscular haemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), platelets (PLT). For each parameter the reference change value (RCV) was calculated. The concentration of triglycerides required to cause a clinically significant change in most parameters was determined by ROC curve.
Results: The ROC curve presented an Area Under Curve (AUC) of 0,9683 (95% CI: 0.85746 - 0.99491) for the concentration of TG as a predictor of interference. The optimal cutoff point was 1271 mg/dL (Se(%)=100; Sp(%)=88,89). Above this concentration the variation between analyzes (before and after the correction) was greater than 6.08% (RCV) for Hb, which directly impacts on RBC parameters calculated (MCH, MCHC).
Conclusions: Thus, since the TG levels found are greater than the interference limit for Hb and RBC calculated parameters, the replacement of lipemic plasma for saline solution was needed to minimize interference clinically significant. These data show the relevant TG interference in blood count, and how may impact in medical decision, and the importance of correction.

Assessment Of Quality Control System By Six Sigma Metrics: An Applicable Tool For Laboratory Tests Of Hemostasis?
Rana Turkal1, Tlay evlik1, Ozan nl1,2, nder Şiriki1,2, Goncagl Haklar1,2
1Marmara University Pendik E&R Hospital, Istanbul, Turkey, 2Marmara University, School of Medicine, Department of Biochemistry , Istanbul, Turkey

Introduction: Six Sigma (6σ) is a widely-accepted quality management system, quantifying the performance of processes as a rate of ‘Defects-per-Million Opportunities’. The quantitative goal of 6σ is to create a process that minimizes variation until six standart deviations can fit within the tolerance limit. The purpose of this study was to assess performance of our tests of hemostasis and to compare CLIA and biological variation (BV) allowed total allowable error (TEA) requirements on sigma scale.
Methods: The 6σ values were calculated as follows: Sigma = (TEA−Bias)/CV, with all values expressed as percent (%). CV% from the internal quality control (IQC) at normal and abnormal levels were evaluated for imprecision. IQC data were taken for a period of one year from January 2018 to December 2018 for the parameters of prothrombin time(PT), activated partial thromboplastin time (aPTT), fibrinogen, factor VII, VIII, von Willebrand antigen (vW:Ag), protein C (PC), free protein S (PS) and antitrombin (AT) which were run on a hemostasis analyzer STA-R (Diagnostica Stago, France), in the setting of Marmara University Pendik E&R Hospital Biochemistry Laboratory. Bias% was calculated based on the comparison between laboratory result and the peer group mean, available from 2018 external quality control scheme ECAT. Mean bias was obtained from calculated bias of all surveys. The performance of all parameters was successful in all of these surveys.
Results: Routine coagulation markers; PT, aPTT and fibrinogen had acceptable but mostly poor performance of ≥3-< 4 sigma score at level 1 and level 2 control when CLIA was used as TEA. When BV was used as TEA, all parameters except free PS at level 1 with a sigma value of 4.94, had unacceptable sigma (< 3).
Conclusions: Calculating sigma values offers the advantage of setting a quality base line and evaluating a procedure by combining the results of internal and external assessments. However, sigma metrics, widely used for chemical determinations, was disappointing for most of the tests in our study, especially when BV was used as TEA. The result was compatible with the literature. Since sigma is an industrial standart we conclude that 6σ might not be an applicable quality tool for the tests of hemostasis.    

Clinical Significance Of Increased Hflc And Its Use In Mononucleosis Infection
Anna Marull, Javier Nieto-Moragas, Orlando Jimenez, Mariona Hernandez, Patricia Tejerina, Maite Serrando
Laboratory Trueta Hospital, Girona, Spain

Introduction: Atypical lymphocytes can be observed in  peripheral blood in a large number of clinical situations, including immune reactions, malignant disease, drug reactions, and infections. Lymphocytes play an important role in the adaptative  immune response. High-fluorescence lymphocytes count (HFLC) is a hematological research marker which indicates a cellular activity that may correspond to an active immune response.  According to Sysmex, this parameter reflects the presence of highly fluorescent lymphocytes, which represent antibody-secreting B lymphocytes/plasma cells. The aim of this study is to determine the clinical significance of increased HFLC and its potential use as marker mononucleosis infections (MI) promoted by cytomegalovirus and Epstein-Barr virus
Methods: From October to December 2018, 188  patients were classified in 5 groups according to their principal diagnosis. 38 patients presents MI, 26 sepsis, 19 autoimmune disorders, 42  lymphoproliferatives disorders (LPD) and 38 healthy subjects. MI was detected using PCR or serological test. Sepsis was diagnosed by positive blood culture isolation and clinical parameters of sepsis contemplated in The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). Hematological disorders were diagnosed by flow cytometry. CBC and HFLC results were compared between the groups with Kruskal-Wallis test. Receiver operating characteristic (ROC) curve analysis was improved to determine the usefulness of HFLC for the different groups.
Results: Lymphocyte absolute count and HFLC absolute and relative values for the MI group show statistical differences compared to the other (p< 0.001). Lymphocyte absolute count was higher for MI compared to LPD (p< 0.001), but HFLC presented great values for MI. ROC curve analysis was improved to compare between SLP and MI groups and it was obtained an Area Under the Curve (AUC) of 0.937 and 0.902 for %HFLC and absolute HFLC, respectively. Using a cutoff of 0.35 for HPLC% a sensitivity of 87,% and a specificity of 84,8% were obtained
Conclusions: HFLC has been proposed to evaluate an active immune response including sepsis or virus infections among others. HFLC % were not significantly different among patients with immunological or lymphoproliferative disorders respect to the healthy group. In our study, as in other recent studies, HFLC counts are not effective as a marker for early sepsis. However, it is a good marker to differentiate CMV-EB infections from other active immune response disorders.

A Multi-Site Inter-Regional Utilization Management And Quality Initiative For Autoimmune Testing
Michelle Wong1, Richard Cleve1, Sonja Turnbull1, Michael Nimmo2, Sophia Wong2, Carmen Phoon2, Lorraine Romanin3, Gerald James3
1Royal Columbian Hospital, New Westminster, BC, Canada, 2Royal Columbian Hospital, New Westminster, BC, Canada, 3Royal Columbian Hospital, New Westminster, BC, Canada, 4Vancouver General Hospital, Vancouver, BC, Canada, 5Vancouver General Hospital, Vancouver, BC, Canada, 6Vancouver General Hospital, Vancouver, BC, Canada, 7Royal Inland Hospital , Kamloops, BC, Canada, 8Royal Inland Hospital, Kamloops, BC, Canada

Introduction: Autoimmune tests, including anti-nuclear antibody (ANA), are frequently ordered.  Unnecessary testing imposes excess cost on our health care system.  The British Columbia Guidelines for ANA Testing recommend that ANA need only be ordered once.  Three large health authorities consisting of a total of 58 sites (13 in Fraser Health, 15 in Vancouver Coastal Health and surrounding region, and 30 in Interior Health) participated in a utilization management and quality initiative.  These sites limited autoimmune testing to only once within a 6 - 12 month timeframe.  In addition, there was an attempt to standardize protocols and testing where possible.  Both Interior Health and Fraser Health also replaced their machines resulting in alignment in the testing platforms with both sites changing from immunofluorescence assay (IFA) to enzyme-linked immunoassay (ELiA, Phadia 250).  Vancouver General Hospital (Vancouver Coastal Health) had already incorporated the Phadia 250 into their testing algorithm.
Methods: Retrospective data analysis for the 6 months pre- and post-implementation periods was performed.
Results: The number of ANA tests performed has decreased from 2532 to 2185 per month (P < 0.01).  All three health authorities saw a decrease.  Positivity rates differed significantly between the three health authorities (range 13-48%) due to different methodologies for testing (IFA versus ELiA).  Subsequent standardization at a later timeframe resulted in much more consistent positivity rate (range 15-21%).  There was no statistically significant change in the positivity rate.
Conclusions: The decrease in ANA testing was consistent with expectations.  Utilization management initiatives such as limiting repeat testing for ANA is a sustainable, cost effective method, consistent with provincial guidelines based on clinical recommendations.  Quality initiatives such as standardization of testing can also provide alignment between hospital sites and improve clinical care.

The Role Of Stem Cells In A Model Of Hepatic Regeneration
Laila Montaser1, Dina El-Azab2, Gehan Tawfeek 3, Sara Saied4
1Menoufia University, Faculty of Medicine, Shebin El-Kom, Egypt, 2Menoufia University, National Liver Institute, Shebin El-Kom, , Egypt, 3Menoufia University, Faculty of Medicine, Shebin El-Kom, , Egypt, 4Menoufia University, Faculty of Medicine, Shebin El-Kom, , Egypt

Introduction: Acute liver failure  is a multisystemic disorder with a high mortality rate. Adult bone marrow contains progenitors capable of generating hepatocytes. 
Methods: In bone marrow mononuclear cells (BMMCs) treated group , BMMCs were injected into the rats 24 h after liver damage with CCL4. In hepatocyte lineage treated group , the animals were infused with BMMCs derived hepatocyte lineage 24 hours after CCL4 injection.Transaminases and survival rate were measured after injection of cells. BMMCs in rats were detected by flow cytometry after 4 weeks.                   
A liver failure model is introduced by CCL4 to assess whether bone marrow-derived progeny contribute to liver regeneration after acute hepatotoxic liver failure. Homing of BMMCs was detected in the treated rats by flow cytometric analysis with monoclonal antibodies specific for CD 45, CD34 and CD14. Liver histopathology revealed both significant decreases in the degree of necrosis, steatosis and inflammation with significant increases in the mitotic index of treated groups than non-treated. liver immunohistochemical stained section from treated groups showed significant positivity for both proliferating cell nuclear antigen (PCNA) and anti-human albumin indicating the presence of functional human cells in rat livers and proliferation of liver cells. PCNA results of the studied groups are presented at Table 1.  
Group Control N = 6 Group 1 (MNCs) N = 6 Group2 (Differ.) N = 6 U test P value
After   2w Range X ± SD     5 – 20 12.5±5.24     30 – 40 33.3±4.08     45 – 75 59.17±12.42     2.92 2.90 2.91   0.0041 0.0042 0.0043
After   4w Range X ± SD     10 – 35 20.83±10.68     80 – 90 83.3±4.08     80 – 100 89.17±8.01     2.91 2.90 1.34   0.0041 0.0042 0.183
Conclusions: Experimenting on rats as CCL4–induced acute liver failure model, injected  with BMMCs or hepatocyte lineage have potential as a new therapeutic option for acute liver disease and suggest that these cells may contribute to hepatic recovery.

Reduction Of Ordered Peripheral Blood Smear Reviews
Megan Nakashima, Eric Hsi, Gary Procop
Cleveland Clinic , Cleveland , OH, United States

Introduction: Peripheral blood smear review (PBSR) is a traditional method to detect hematologic abnormalities. PBSR requires a significant amount of time and skilled operators compared to complete blood count and differential (CBC/DIF) by automated analyzers. Clinicians may be unaware of how well modern multi-channel flow cytometry-based analyzers are able to screen for abnormalities including abnormal lymphocytes, immature granulocytes, blasts, and schistocytes. Thus, clinicians may over-utilize PBSR. At our institution, PBSR by pathologist has traditionally been orderable as a “Staff Review” (STFREV). STFREV are performed Monday-Saturday, with as many a 40/day, requiring a significant amount of time for both pathologist (to perform) and technologists (to result). We sought a rational strategy to reduce non-indicated STFREV.
Methods: CBC/DIF were performed on hematology analyzers and PBS prepared by automated slide-maker/stainer (XN-9000, Sysmex, Kobe, JP). If there were abnormalities which triggered PBSR by technologists per laboratory procedure (e.g. lymphocytosis, blasts), PBSR was performed, then sent for STFREV. If technologist review was not indicated, but the analyzer flagged a qualitative abnormality, PBS was sent for STFREV. If there were no flags the STFREV was cancelled, slides were stored, and the CBC/DIF result had a comment added: “The Staff Review on this sample was cancelled because the hematology analyzer did not flag any parameters as requiring manual review. If there is a specific clinical concern for which you would like a staff pathologist to review the blood smear, please call Lab Client Services within 28 days.” Clinicians were able to call to override cancellation. Hematologists/oncologists at remote sites were also able to bypass the system upfront.
Results: Between February and December 2018 the number of staff reviews ordered per month ranged from 762-904. After the intervention STFREV performed ranged from 493-584, a net reduction of 253-338 (33.2-40%) per month. We estimate a savings of over an hour per day each for both pathologists and technologists. The medical director received less than five complaints regarding this process, and there were no reports of patient harm related to cancellations.
Conclusions: We were able to markedly reduce the number of STFREV by relying on instrument flagging. This resulted in significant time saving for both our technologists and pathologists. This was accomplished with minimal clinician dissatisfaction, and to our knowledge no patient harm has resulted due to this policy.

Comparison Of White Blood Cells Automated Differential Count Using Sysmex Xn-550 And Advia2120I
Halimatun Radziah Othman1, Qian Wei Choong2, Madyhah Abdul Monir1
1Universiti Teknologi Mara, Sungai Buloh, Malaysia, 2Sysmex (Malaysia) Sdn Bhd, Petaling Jaya, Malaysia

Introduction: Sysmex XN-550 is a newer automated haematology analyser compared to Advia 2120i. The Advia 2120i incorporates white blood cells (WBC) lysis and a gold standard myeloperoxidase stain for the determination of WBC differential. The XN-550 also undergoes nearly similar process but it uses different dye entities. Both analysers utilise fluorescent flow cytometry and light scatter technology for classification and measurement of WBC differential. Now that the XN-550 is newly available in our laboratory therefore we aimed to compare the XN-550 to the Advia 2120i for the measurement of WBC automated differential.
Methods: We measured WBC and differential count (absolute count and percentage) for Neutrophils (NEUT), Lymphocytes (LYMPH), Monocytes (MONO), Eosinophils (EOS) and Basophils (BAS) in 62 and 59 EDTA-anticoagulated samples respectively. The analysis were performed on the XN-550 (Sysmex, Japan) as an analyser being evaluated and the Advia 2120i (Siemens, USA) as a reference analyser. Results were assessed using Pearson correlation (r2), Passing-Bablok (PB) regression and Westgard medical decision level method.
Results: Generally, the XN-550 demonstrated an excellent correlation with Advia 2120i for WBC, NEUT# and LYMPH# with r2=1.00 and NEUT%, LYMPH% and EOS% with r2 values of 0.96. 0.96 and 0.98 respectively. Others; MONO#, EOS#, BASO#, MONO% and BASO% yielded a wide range of values (r2 = 0.85, 0.00, 0.18, 0.83 and 0.16). These parameters are physiologically low in count hence the r2 is not suitable for assessing these parameters. PB was then used to assess the parameters however they still did not meet the criteria for slope (CI Slope) and/or y-intercept (CI intercept) (Figure 1). These may be contributed by the difference in measurement principle, calibration stability between analysers and statistical limitation due to low count. Nevertheless, the Westgard medical decision level guideline (Figure 2) was used to verify concordance of the data and the outcome showed that all parameters either fell within the acceptable limit or there was no significant difference in the absolute bias value.
Conclusions: A few guidelines are needed when evaluating method comparison between two different methods as often the requirement is not met. Overall, the comparison of WBC automated differential between XN-550 and Advia 2120i is acceptable. However, the acceptance for diagnostic practice shall be evaluated with performance of other blood count parameters.

Cell Population Data Of Automated Hematology Analyzer Sysmex Xn For Sepsis
Mikyoung Park, Hanah Kim, Mina Hur, Hee-Jung Chung, Hee-Won Moon, Yeo-Min Yun
Department of Laboratory Medicine, Konkuk University School of Medicine, Seoul, South Korea

Introduction: Cell population data (CPD) using multi-parametric algorithms are available in modern automated hematology analyzers. We investigated clinical utility of CPD obtained on Sysmex XN-9000 (Sysmex XN, Sysmex, Kobe, Japan) for risk stratification and mortality prediction in critically ill septic patients.
Methods: We enrolled 169 patients (sepsis, n = 85 and septic shock, n = 84) diagnosed by Sepsis-3 criteria. CPD was as follows; immature granulocytes (IG, number [#] and percentage [%]), neutrophil granularity intensity (NEUT-GI), neutrophil reactivity intensity (NEUT-RI), antibody synthesizing lymphocytes (AS-LYMP, # and %), reactive lymphocytes (RE-LYMP, # and %), and highly fluorescent lymphocytes (HFLC, # and %), reticulocyte hemoglobin equivalent (RET-He), RBC hemoglobin equivalent (RBC-He), difference of RET-He-RBC-He (Delta-He), reticulocytes, and immature reticulocytes fraction (IRF, # and %), and immature platelets fraction (IPF, # and %) for platelets. The CPD was analyzed according to the sepsis severity and 30-day mortality using Mann-Whitney U test and the receiver operating characteristic (ROC) curve analysis.
Results: The NEUT-RI, IRF% and IPF% values were significantly higher in septic shock patients than septic patients, respectively (median: 53.6 FI vs. 60.5 FI, 10.0% vs. 12.4%, and 3.1% vs. 4.4%, all P < 0.05, M-W test). These three parameters significantly predicted septic shock and 30-day mortality, respectively (AUC: 0.588-0.723 and 0.614-0.696, all P < 0.05, ROC curve analysis).
Conclusions: Cell population data such as the NEUT-RI, IRF%, and IPF% may provide useful information for risk stratification and mortality prediction of critically ill septic patients.

Using Smudge Cells Percentage On Routine Blood Smear In Chronic Lymphoytic Leukemia As Prognostic Factor: Senegalese Experience
Abibatou Sall1, Moussa Seck2, Blaise Faye3, Fatimata Bintou Sall4, Saliou Diop5, Awa Oumar Toure6
1Hematology, Dakar University, Dakar , Senegal, 2Hematology, Dakar University, Dakar, Senegal, 3Hematology, Dakar University, Dakar, Senegal, 4Hematology, Dakar University, Dakar, Senegal, 5Hematology, Dakar University, Dakar, Senegal, 6Hematology, Dakar University, Dakar, Senegal

Introduction: The heterogeneity of chronic lymphocytic leukemia (CLL) has important consequences which will impact on clinical approaches or treatment strategies. Prognostic markers as CD38 expression, cytogenetic abnormalities or IgVH gene mutation have all been very useful. However, these tests are expensive and require considerable technical expertise and equipment and thus are not available in many developing countries. Therefore less expensive prognostic markers are needed. In this study we evaluated the prognostic significance of smudge cells percentage on a blood smear in CLL patients.   
Methods: 42 untreated CLL patients have participated after signing a consent form. The diagnosed was based on an absolute lymphocyte count greater than 5 G/L and flow cytometry using Matutes scoring. Staging was done according to the Binet staging system. CD38 was determined in all patients. The cytogenetic abnormalities were performed by FISH and available for 25 patients. The smudge cell (SC) (figure 1) percentage is estimated by using this formule SC= number of SC/200x(lymphocytes+SC) x 100. Each slide was evaluated by 2 hematologists and the blood smears were prepared using a manual wedge method. 
Results: The mean age was 63 years (48 to 85) and the sex ratio was 2.81. At the time of diagnosis 81% of the patients were classified as having advanced Binet stages B or C. The median lymphocyte count was 189.9 109/L (5.2 - 887 G/L). The prognostic marker CD38 was positive in 28 patients, while 11 patients had 13q del, 7 patients Trisomy 12, 3 del 11d and 3 del 17p. The median smudge cell percentage was 20.1% (range, 4 - 80%) and 30 patients had a percentage less than 30. We found no correlation of proportion of smudge cells with age (p=0.19) and sex (p=0.76). However there was a significant correlation with the Binet stage (p=0.0002), the CD38 expression (p=0.04) and a negative correlation with the lymphocyte count (p=0,01) (r= -0,38).   
Conclusions: Simple, accessible and inexpensive, the percentage of smudge cells on a routine blood smear could be a prognostic test available to all patients with CLL especially those in countries with limited resources.   

Exploration Of Machine Learning In PeripheralBlood Blast Intelligent Detection
WANG Shuang, CHENG Si, FU Yang, MAO Zhigang, JIANG Hong
1,2,3,4,5, Chengtu, China

Introduction: To establish a blast intelligent detection model by computer learning software for blast cell screening, based on machine learning. The model is a foundation for the intelligent examination of blood routine reports and the intelligent diagnosis of laboratory test results.
Methods: A total of 6173 cases of outpatient and inpatient laboratory data were collected through Laboratory Information System (LIS) and Hospital Information System (HIS) of West China Hospital Sichuan University .We enrolled 64 blood cell indexes and the P value of blast was obtained by statistical analysis of computer software, the sensitivity and the specificity were used to evaluate the detection performance of the model.
Results: 20 indicators were invovled in the analysis including HGB、HCT、LYMP、NRBC%、LYMPH、LY-WZ、IG、NE-WZ、WBC-D、WBC、WBC-N、TNC-D、TNC、TNC-N、NE-WX、MO-WY、NE-WY、MO-WX、NRBC、MONO. The P value of blast was 96.4%。In this model, 989 cases of outpatient were verified by blind method, the sensitivity was 100%, and the specificity was 53.2%; 493 cases of inpatient were verified by blind method, the sensitivity was 68.1%, and the specificity was 52.4%.
Conclusions: The model of peripheral blood blast intelligent detection based on machine learning has certain value for blood cell analysis, which provides experience for the application of artificial intelligence in intelligent diagnosis in the future.

Performance Evaluation Of The New Beckman Coulter Dxh-900 Hematology Analyzer.
Xavier Tejedor Gandux, Alicia Martinez-Iribarren, M Angels Sala Sanjaume, Mara Lpez-Molina, Merc Espinosa, Ester Viets, Silvia Torres, Chantal Abadia, M Antonia Llopis Daz, Cristian Morales-Indiano
Laboratori Clnic Metropolitana Nord. Hospital Universitari Germans Trias i Pujol, Badalona, Spain

Introduction: DxH900 is a new fully automated analyzer that provides cell blood count, white blood cell (WBC) differential, reticulocyte count (RET) and NRBC enumeration. The aim of this study was to validate DxH900 and compare it with its predecessorDxH800.
Methods: 321 samples (EDTA-K3 blood specimen) were analyzed over a 1-month period. Following the guidelines of the Clinical and Laboratory Standards Institute (CLSI,H26-A2) we evaluated background, carryover, reproducibility, intrinsic interference (I), linearity, limit of quantification (LQ), abnormality messages (flags), differential count (DC) and correlation with DxH800.

Results: The results for background, carryover, reproducibilityand interferences met manufacturer’s specifications.Excellent linearity was obtained for red blood cells (RBC) (y=1.005x–0.026;R=1.00), WBC (Y=0.984x–4.867;R=.99), haemoglobin (Hg) (Y=1.011x–0.106;R=1.00) and platelet count (PLT) (Y=1.007x+58.726;R=1).The samples used to assess LQ presented WBC values between 0.236 and 0.311x109/L with coefficient of variation (CV) < 15%, while the samples used to quantify PLT (values between 4.1 and 8.35x109/L) yielded a CV< 10%.Passing Bablok method showed no differences between both analyzers for WBC (y=0.05+1.00x;R=.99), Hg (y=0.24+0.98x;R=.99) and RET (y=0.06+1.04x;R=.99). PLT showed a good correlation (y=1.23+0.92x;R=.99) but were higher in comparison to the DxH-800 with a 7% bias (3.1 to 16.5%). Comparison of 50 leukocyte DC results analyzed in parallel with blood smear observation (reference method) revealed a good correlation for neutrophils (R=.93), lymphocyte (R=.95), monocyte (R=.82)and eosinophils (R=.79),while basophils showed a poor correlation (R=.41). Sensitivity and specificity for the detection of flags are described in the table below:
Conclusions: DxH900 provides reliable results and fully comparable to DxH800.  One of the highlights is sensitivity and NPV of flags, which has been improved. DxH900 is an accurate, highly precise analyzer with good analytical performances to be used effectively in high-volume laboratories.

Performance Evaluation Of Abbott&Rsquo;S Alinity Hq Hematology Analyzer
Bastien Tossens, Anne Kornreich, Pascale Van Daele
Grand Hpital de Charleroi, Charleroi, Belgium

Introduction: The Abbott Alinity hq is a new hematology analyzer that performs Complete Blood Count (CBC) with a 6-part White Blood Cell (WBC) differential and several parameters using Advanced MAPSS (Multi Angle Polarized Scatter Separation) technology. We evaluated its performance for the following parameters: red blood cells (RBC), white blood cells (WBC), platelets (Plt), hemoglobin (Hb) and reticulocytes (Retic). 
Methods: The blood specimens used for the evaluation were residual material from patient samples sent to the clinical laboratory for routine testing, collected in K2 EDTA Vacutainer (BD®), maintained at room temperature and tested within 8 hours of collection. Within-run precision was evaluated by performing 10 consecutive RBC, WBC, Plt and Retic numerations and 10 consecutive Hb measurements on 3 levels of Abbott controls CELL-DYN 29 PLUS and 3 levels of patient samples (low, normal range and high). Between-day precision was evaluated by processing the 3 levels of Abbott controls CELL-DYN 29 PLUS for the same parameters twice a day for 15 days. We then calculated the coefficients of variations (CV) for the precisions obtained and compared them to Ricos’ table for biological variation to determine acceptability (table 1)1. Carry-over was assessed for RBC, WBC, Plt and Hb using the International Council for Standardization in Haematology procedure (table 2)2. Linearity was evaluated in duplicate for Plt (range 700 to 7x103/µL) and WBC (range 20 to 0.4x103/µL) using serial dilutions in NaCl 9‰ of patient blood samples (table 2). 
Results: See Table 1: Within-run precision and between-day precision. See Table 2: Carry-over and Linearity
Conclusions: Precision surpassed the manufacturer’s specifications for all parameters and were well within the more stringent limits of Ricos. The reduced precision for plasmas with very low WBC, Retic and Plt counts is considered acceptable since it entails no clinical impact. The carry-over was minimal to nil and the linearity was very good including for low WBC and Plt values. The Alinity hq hematology analyzer is a user-friendly instrument that showed high performances for the parameters studied.  1.                                        2. Int. Jnl. Lab. Hem. 2014, 36, 613-627 

The Role Of Mindray Bc 6800+ AnalyzerS Neut-X For The Detecting Dysplasia In Neutrophils

Introduction: The new counter BC 6800+ (Mindray Diagnostics) reports the cell population data of leukocytes, which reflect the morphology of cells including granularity, size and nucleic acid content. NEUT-X is the parameter related to the cytoplasm structure of the neutrophils and strongly correlates with their granularity. As hypogranularity is the most consistent feature of myelodysplasia we evaluate the reliability of NEUT-X in the discrimination of Myelodysplastic Syndromes (MDS).
Methods: Two hundred and eighty samples were selected from the routine workload. 100 samples from healthy individuals were used established the reference  interval  of NEUT-X for our Laboratory. 50 patients diagnosed of MDS: 50 patients with macrocytosis, due to lack of vitamin B12 or folate, 80 samples of patients with leukopenia.Complete blood counts were run on BC6800+ analyzer (Mindray Corporation, Shenzhen, China);  blood smears were revised with a CellaVision DM96 CellVision, Lund, Sweden)  to verify the morphology cells to detect dysplasia and hypogranularity. Kolmogorov-Smirnoff  was used  to verify normal distributions. Differences among groups were assessed using analysis of variance, considering P < 0.05 to be significant. For post hoc comparisons of outcomes between each pair of groups Scheffé correction was applied. Receiver Operating Curves analysis (ROC) was used to established the performance of NEUT-X In the detection of  morphologic feature of MDS.
  Healthy MDS Leukopenia Macrocytosis
 Mean 332.7 266.7 371.9 368.4
standard deviation 23.9 33.8 59.1 43.2
 P  < 0.0001 -- < 0.0001 < 0.0001
P significance between MDS and the other groups. Morphology of the leukocytes of patients with leukopenia and macrocytic anemia was normal, no hypogranularity was evident. 2 patients with MDS and concomitant infection  had high NEUT-X values, >390, no hypogranulation  was evident , whereas 48 presented marked dysplasia, and reduced NEUT-X values. ROC results for NEUT-X in the detection of dysplasia in neutrophils: Area under curve was (AUC) 0.935 (95% CI 0.875–0.972), considering a cut off < 318.3 sensitivity 85.7 %, specificity 88.5 %.  Positive predictive value 88.1 %, negative predictive value 90.0%
Conclusions: NEUT-X is a reliable tool to screen the presence of hypogranular neutrophils and thus could aid in the detection of dysplastic neutrophils and MDS, with high diagnostic performance. A value < 318.3 ch can be used to select samples for further confirmatory tests.

Preliminary Evaluation Of The Abbott Alinity Hq Wbc Morphological Flagging
Martijn W.H.J. Demmers1, Christiaan L. Slim2, Brigitte A. Wevers3, Henk J. Adriaansen1, Huib Storm2, Jurgen A. Kooren3, Johannes J.M.L. Hoffmann4, Gabriellla Lakos5
1Department of Clinical Chemistry, Gelre Hospital Apeldoorn, Apeldoorn, Netherlands, 2Department of Clinical Chemistry, Certe, Groningen, Netherlands, 3Department of Clinical Chemistry, Atalmedial Medical Diagnostic Centers, Hoofddorp, Netherlands, 4H3L Consult, Nuenen, Netherlands, 5Abbott Diagnostics Division , Santa Clara, CA, United States

Introduction: The Alinity hq high throughput hematology analyzer(Abbott Diagnostics, Santa Clara, CA) reports a six-part WBC differential and displays morphological flags that suggest the presence of blasts cells (BLAST), reactive or malignant lymphocytes (VAR LYM) and immature granulocytes (band neutrophils with or without more immature forms) (Left Shift).
Methods: The performance of the BLAST, VAR LYM and Left Shift flags was evaluated on 1461 samples in three clinical laboratories in The Netherlands, before the commercial launch of the analyzer, using a Performance Evaluation Only software version. Flagging was compared to manual WBC differential results. The study included patients with a diverse range of diseases, including large number of hematological malignancies.
Results: A BLAST flag was triggered for 54.4% of samples with ≥ 1% blasts in the manual differential and for 70.8% of samples with ≥ 5% blasts. Specificity was 96.6%. Out of the 7 samples that were not flagged at the 5% blast level, three had a WBC count of < 1.0x109/L. In another two samples, with the diagnosis of myelodysplastic syndrome and > 8% immature granulocytes (IG) in the manual differential, Alinity hq reported a Left Shift flag, and > 10% IG, increasing the sensitivity of blast detection to 89.5%. The sensitivity of the VAR LYM flag was 41.9% and 88.9% at the thresholds of ≥ 5% and ≥ 20%, with a specificity of 94.9% and 94.3%, respectively. When left shift was defined as ≥ 5% band neutrophils in the manual differential, the Left Shift flag was 52.6% sensitive and 91.2% specific. When left shift was defined as ≥ 5% band neutrophils and/or ≥ 1% IG in the manual differential, it was detected with 64.3% sensitivity and 91.1% specificity by the combination of the Left Shift flag and/or ≥ 1% IG in the automated differential. 
Conclusions: Detection of WBC abnormalities, including blast cells, reactive and abnormal lymphocytes and left shift was shown to be efficient with Alinity hq and in line with published performance of other hematology analyzers.

Diagnostic Power Of Fibrin Monomer In Disseminated Intravascular Coagulation In Sepsis Patients: Preliminary Results
Tlay evlik1, Fethi Gl2, Ahmet F. Tekin1,3, Rana Turkal1, Gneş Eskidemir4, İsmail Cinel2, nder Şiriki1,3, Goncagl Haklar1,3
1Marmara University Pendik E&R Hospital, Biochemistry Laboratory, Istanbul, Turkey, 2Marmara University, School of Medicine, Department of Anesthesiology and Reanimation, Istanbul, Turkey, 3Marmara University, School of Medicine, Department of Biochemistry, Istanbul, Turkey, 4Marmara University, School of Medicine, Department of Pulmonology, Istanbul, Turkey

Introduction: Disseminated intravascular coagulation (DIC) is always secondary to an underlying disorder such as sepsis, solid organ or hematological malignancies. The soluble fibrin monomer (sFM) is considered to be a useful marker for the diagnosis of DIC in early stages. We aimed to evaluate the diagnostic value of fibrin monomer (FM) and D-dimer measurements in the diagnosis of DIC in the sepsis patients. 
Methods: This prospective study was conducted in Ministry of Health-Marmara University Pendik E&R Hospital. Eighteen (8 female, 10 male) adult intensive care unit (ICU) patients with sepsis were recruited upon admission. The patients were categorized into overt and non-overt DIC groups, based on the International Society of Thrombosis and Hemostasis (ISTH) scoring system including platelet count, prothrombin time (PT), D-dimer, fibrinogen. Non-overt DIC score additionally included Antitrombin III, protein C activity, factor V and VII activity. During the study period, 130 plasma samples of patients were analyzed. All coagulation tests were measured by STA-R (Diagnostica Stago, France) coagulation analyzer and platelets were counted by Unicel DxH800 Coulter Cellular Analyzer (Beckman Coulter, USA). The data was analyzed using SPSS Statistics 17 software. Results were expressed as the mean ± standard deviation (SD) and compared by Mann Whitney U test and the correlation between DIC score and the sFMs was also determined.
Results: Six patients (33.3%) were categorized as overt DIC and twelve (66.7%) as non-overt DIC calculated from overt DIC score. The PT, platelet count, fibrinogen, D-dimer, sFM values were significantly different in overt DIC than in non-overt DIC and the differences were statistically significant (P< 0.001). Median D-dimer and sFM values in patients with overt DIC were 11.1±6 mg/L, 81.9± 153 μg/mL when compared to median values of 5.7±5.5 mg/L 25± 42 μg/mL in the non-overt DIC respectively. D-dimer and sFM were also significantly correlated in overt DIC and non-overt DIC (r=0.650, P =0.002 r=0.350 P < 0.001) respectively.
Conclusions: Further studies may indicate that sFM is a better indicator than D-dimer in the early diagnosis of overt DIC.

Haemostasis And Inflammation Cross-Talk In Tuberculosis Disease
Patience Akpan, Josephine Akpotuzor, Eme Osim
University of Calabar, Calabar, Nigeria

Introduction: Tuberculosis is an infectious disease which induces a state of chronic granulomatous inflammation and inflammation and haemostasis is said to exhibit an interdependent relationship as part of host defences against infection.This study is therefore aimed at assessing the relationship between haemostasis and inflammation in tuberculosis disease.
Methods: Cross sectional study design was employed. One hundred and twenty TB patients and 120 healthy mantoux-negative subjects (control) aged 15-60 years were enrolled. Diagnosis of TB was by microscopy, radiography or clinical assessment. Platelet count (PLT) was by haemocytometry, prothrombin time (PT) and activated partial thromboplastin time (APTT) by Quick’s one stage technique, thrombin clotting time (TCT) and fibrinogen (FIB) by Clauss technique while platelet factor 4 (PF4), fibrin degradation product (FDP), interferon gamma (IFN-γ) and interleukin 10 (IL-10) were determined by enzyme linked immunosorbent assay. Data were analysed on statistical package for social sciences (version 20) software. Pearson’s correlation was used to express relationship with significance set at P ≤ 0.05.
Results: Platelet factor 4, PT, APTT, FIB, FDP, IFN-γ and IL10 levels of TB patients were significantly higher (P< 0.05) while the thrombin clotting time was significantly lower (P< 0.05) versus control. Significant positive correlations (p< 0.05) were observed between PT and IFN-γ (r=0.247), PT and IL 10 (r=0.189), APTT versus IL 10 (r=0.208), FIB and IL 10 (r=0.218) while a significant negative correlation was seen between TCT and IL 10 (r-0.222).
Conclusions: A mutually dependent relationship exists between the haemostatic and inflammatory systems in tuberculosis disease which significantly enhances its pathogenesis and course progression.

Strategies For Assessing The D-Dimer Reference Values In Healthy Population Over 50 Years: Conventional Versus Aged Adjusted.
Tailiny AF Alonso1,2, Karen Y Kimoto1,2, Dayane M Jacinto1,2, Christiane P Gouvea1,2
1University of So Paulo, So Paulo, Brazil, 2Fleury Medicina e Sade, So Paulo, Brazil

Introduction: Venous thromboembolism (VTE) comprises deep vein thrombosis and pulmonary embolism and its diagnosis is based on imaging and D-dimers (DD) quantification. In patients with low / moderate clinical VTE probability, DD is a useful tool with a high negative predictive value in exclusion of the thrombosis. On the other hand, elevated DD may be due to several pathological and physiological conditions, such as gestation and age. Studies evaluating DD levels in healthy elderly (≥ 70 years) demonstrated that 50% had concentrations above the reference value (500 ng / mL FEU) and the current guidelines recommend the adjustment of DD levels through of the multiplication of the patient's age by 10 from 51 years, associated with the clinical risk score rather than considering the standard cut-off value.
Methods: A retrospective observational study of the 8,424 DD performed by the laboratory of the Hospital das Clínicas of the São Paulo University, from January 2016 to 2018, was carried out to verify among ambulatory patients with increased DD and age over 50 years, a reduction in the percentage of positive results when cut-off values were adjusted for age.106 (1.25%) patients were selected because did not present comorbidities that justified the elevation, VTE and disseminated coagulopathy (DIC).  
Results: 75% of patients were females with a mean age of 71 years. When calculating the DD adjusted for age, only 13% patients remained with high values. Patients were classified into age groups every 10 years: 51 to 60:13, 61 to 70:39, 71 to 80:40, 81 to 90:11, 91 to 100:3 to evaluate the DD behavior in each group. Between 61-70 and 71-80 years there was a greater number of individuals with high results, even with the adjustment of the reference value for age (13% and 20% respectively), but in contrast, in the groups between 81-90 and 90-100 years none of the results remained positive after adjustment.
Conclusions: The adjusting normal values of DD according to age seems to be an adequate strategy, since it would avoid performing imaging tests in 87% of the patients in this study and this DD values were not associated with thrombosis or other comorbidities. 

Laboratory Profile Of Haemophilia Patients In Nigeria.
Abiola Bolarinwa, Fatai Bello, Titilope Adeyemo
Lagos University Teaching Hospital, Lagos, Nigeria

Introduction: Haemophilias are the most common inherited bleeding disorders. The transmission is by X- linked recessive fashion affecting the males, and females are the carriers of the disease. Haemophilia has a worldwide distribution and heterogeneous presentation, depending upon its severity. Knowledge of spectrum of presentation of haemophilia helps in early diagnosis and planning of management.
Methods: A cross section study was carried out by the Department of Haematology and Blood transfusion, Lagos University Teaching Hospital, Lagos. Biodata and some relevant clinical history were obtained via  a structured interviewer- administered questionnaire and samples were collected from patients attending Haemophilia clinics in 4 different sites across the country. The clotting factor deficiencies were determined following routine coagulation screening and the severity of such deficiencies were determined by the respective factor assays. The patients were also screened for transfusion transmittable infections.
Results: A total of 88 patients were enrolled in the study, comprising of 87 males and 1female. The mean age was 13 years with a range of 2 to 42 years. 78 (88.6%) were diagnosed as Haemophilia A and 5(5.7%) as Haemophilia B. 5(5.7%) had neither factors VIII nor IX deficiencies. Among the Haemophilia A, 59 (75.7%) severe deficiency, 18(23.1%) has moderate deficiency and 1(1.3%) had mild deficiency.   All the Haemophilia B had moderate deficiency. We found correlation between the clinical presentation (number and pattern of bleed) and the laboratory profile.41% of the Haemophilia A had positive time dependent inhibitors screening. 75% of the Haemophilia A with positive inhibitor screening has severe deficiency. 
Conclusions: Risk factors for inhibitor development include severity of the deficiency even with low factor exposure. The level of diagnosis of non-severe haemophilia is very low. This may be due to poor level of presentation of mild haemophilia to the treatment centers in the absence of clinical symptoms.

Cryopreservation Of Peripheral Blood Stem Cell Using Mechanical Freezers : Initial Experience From An Indian Tertiary Care Hospital
nitin agarwal, prashant pandey
Jaypee Hospital, Noida, India

Introduction: Cell therapies based on hematopoietic stem cells (HSCs) have become the standard of care for a large number of clinical indications, and the number of patients and disorders being treated using HSCs continue to grow. Here, we report an analysis of first 10 cryopreservation of peripheral-blood stem-cell (PBSC) at our center in terms of CD 34+ viability and cell engraftments in those patients.
Methods: Peripheral blood stem cells (PBSCs) were collected from auotologus donors using P1YA kits on Com.tec (Fresenius kabi). A solution made up of 10% DMSO, 20% human serum albumin (HSA) with 6% HES was used for cryopreservation of cells. Whole procedure was done under fully aseptic technique using laminar air flow. A mechanical freezer (Thermo Scientific) was used to store stem cells at -80 degree Celsius.
Results: All 10 patients were posted for re-transplant (2nd /3rd) and heavily pre treated with chemotherapy. Average time duration of storage for cryopreserved stem cells was 27 days (range 10 to 56 days). The initial yield of the products before cryopreservation ranged from 3 X 106 to 10.2 X 106 per Kg of the patient (mean dose 7.85 X 106 per Kg) which decrease to a mean of 5.85 X 106 per Kg post thawing of frozen stem cells. Stem cells viability after thawing ranged from 77 percent to 90 percent of total CD 34+ cells.
All the patients had neutrophil and platelet engraftment before they were discharged. Days taken for engraftment of neutrophil ranged from 10 to 15 days (mean 11.5 days) whereas for platelets, it ranged from 14 to 43 days (mean 23.5 days)
Conclusions: Peripheral blood stem cells can be cryopreserved in mechanical freezers at -80 degree celsius using a solution of 10% DMSO, 20% albumin with 6% HES up till 8 weeks without significant decrease in number and viability of CD34+ cells.

Sensitivity And Specificity Of Jaam And Isth Dessiminated Intravascular Coagulation Scors At Hemobiology Laboratory In Chu Oran-Algeria
souaad Bouali, Driss Belaledj, Fatima Seghier
Centre D'Hmobiologie Transfusion Sanguine, Oran, Algeria

Introduction: Our objectives were the  determination of the sensitivity and specificity of the JAAM scores (Japanese Association for Acute Medicine) and ISTH (The International Society of Thrombosis and Hemostasis) used for the diagnosis of disseminated intravascular coagulation (DIC) in order to adapt the most Sensitive and specific one to the department of Hemobiology Pr SEGHIER and CHU-Oran as the tool of diagnosis of the disease.
Methods: All patients admitted to the clinical services of CHU d'Oran from 01 January 2011 to 31 October 2011 with suspicion of DIC - whose samples are sent to the service of Hemobiology Pr SEGHIER - were part of the study. A questionnaire was filled by the clinicians to accompany the blood samples. Biological examinations carried out at our level: Hemoglobin and blood counting parameters (COULTER BECKMAN HmX), PT, PTT, Fibrinogen level (STA-STAGO), serum fibrin degradation products (FDP) and soluble complexes. The statistical analysis was done in collaboration with the Epidemiology Department CHU Oran using the software EPI604dFr.exe
Results: 50 patients were analyzed by the two diagnostic scores: ISTH and JAAM, of which 32% were hospitalized in maternity and 26% in medical intensive care. The average age of the population is 39.7 years and 48.4% are male. DiIC was diagnosed in 58% of patients according to the JAAM score; and 48% according to the ISTH score. The diagnostic concordance between JAAM score and ISTH is 86%. ISTH score has a sensitivity of 84.61% and a specificity of 95.83%. JAAM score has a sensitivity of 100% and a specificity of 74.07%
Conclusions: DIC is a serious disease that is life-threatening for the patient requiring a very sensitive score that is that of JAAM according to our study. The great sensitivity of the JAAM score is due to the consideration of the variation level of the platelets, whereas the ISTH score only takes into account the platelet count at the day of calculation of the score and the absence of a parameter which is the fibrinogen level in the JAAM score while it is present in the ISTH score.

Effect Of Factor Activities On Pt And Aptt-Based Clot Waveform Analysis
Yvonne Chu, Wan Hui Wong, Sim Leng Tien, Heng Joo Ng
Department of Haematology, Singapore General Hospital, Singapore, Singapore

Introduction: Clot Waveform Analysis (CWA) is the extended study of the wave generated by an optical detection during routine coagulation assays. Prothrombin time (PT) and activated partial thromboplastin time (APTT) are routinely used to assess coagulopathy and are sensitive to factor levels. This study assesses how individual factor activities affect PT and APTT-based CWA in vitro.
Methods: Samples of factor activity 0% to 100% were prepared for factors II, V, VII, VIII, IX, X, Xl and XII by dilution of normal standard human plasma (Siemens Healthcare, Marburg, Germany) with deficient plasmas. PT and APTT were analysed on Sysmex CS2100i coagulometer (Sysmex Corporation, Kobe, Japan) using Dade Innovin and Actin FSL reagents and CWA parameters min1, min2 and max2 were obtained. Statistical analysis for paired t-test and correlations were performed using IBM SPSS Statistics.
Results: Single factor deficiency predictably prolonged PT and APTT. PT was prolonged beyond the reference interval (RI) for samples with less than 40% FII, 60% FV, 100% FVII, 80% FX and 20% FXI with no influence by FVIII, IX or XII. APTT in samples with less than 40% FII, FV, or FVIII, or 20% FIX, FX, FXI or FXII were prolonged. In contrast, the CWA parameters were more tolerant to low factor activities. There was strong correlation between FII activity and PT Max2 (r=0.929) as well as the APTT CWA parameters. FV was strongly correlated to PT min1 and inversely to APTT min1. The APTT CWA parameters were also positively correlated to FVIII, FX and FXII.  PT CWA parameters were dependent on FVII and normal in samples with more than 10% FII, 20% FV or 2% FX. FVIII, IX, XI and XII activities did not affect PT CWA. APTT CWA was normal for samples with more than 40% FII, 20% FV, 20% FVIII, 4% FIX, 4% FX, 6% FXI and 4% FXII. FVII did not affect APTT CWA. However, most CWA parameters were statistically different in samples of altered factor activities to that with normal activities.
Conclusions: Strong correlation of CWA parameters to factor activities suggests the potential utility of CWA to discern other coagulopathy from single factor deficiency.  The effects of single factor activities on CWA parameters will help to further our understanding of clot kinetics.

Autoverification Of Routine Coagulation Tests Gerald M Davis Mt(Ascp), Mph University Of Michigan Health Center; Ann Arbor, Mi Usa
UNIVERSITY OF MICHIGAN, Ann Arbor, MI, United States

Introduction:     An autoverification algorithm was validated via analysis of more than five thousand specimens during a seven-day period.  The autoverification rate was greater than eighty percent of all daily routine coagulation specimens.  Thrnaround times for routine coagulation testing were decreased by greater than fifty percent.  Tangible benefits the new workflow process included increased efficiency and improving workflow and lowering cost in the clinical laboratory.
Methods: The autoverification algorithm was designed to emulate the exact algorithm used for manual verification.  Analysis was performed on four Dade-Behring BCS-XP coagulation analyzers.  All ordered tests for the device was set to evaluate in block at the LIS (Softcomputer) interface level with the LIS Rules-Based-System deployed to suppress autoverification when set criteria were not met.  The algorithm evaluated all coagulation tests on the order and autoverification was limited to only routine coagulation testing only, whick included PI, international normalized ratio (INR), PTT, Anti IIa & Xa unfractionated Heparin, Dimer and plasma fibrinogen (FIB).
Results: The overall autoverification rate of routine coagulation testing exceeded eithty percent of the patient test sample population. TEST                n               Percent Autoverified              Percent Manual PT                   2,023                       82.25                                  17.75 INR                 2,023                       82.25                                   17.74 PTT                    709                      78.90                                    21.10 ANTI IIa              477                      89.12                                    11.88 ANTI Xa              203                      88.00                                    12.00 DIMER                172                      83.89                                    16.11 FIBROGEN         101                      86.62                                    13.38 Turnaround times ere decreased by greater than fifty percent for each test.
Conclusions: Evaluation of all the coagulation tests on the order performed perfectly at the LIS interfaced device level.  This is important so that results for one or more tests are not released until all tests meet criteria.  This ensures that results are never retracted due to a clotted specimen.  This deployment supported high level autoverification and reduced turnaround times of routine coagulation tests in the clinical laboratory.

Effect Of Automated Pre-Analytical Sample Transport By Accelerator A3600 On Platelets, Platelet Function Test And Coagulation Assays
Pieter De Kesel, Katrien Devreese
Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium

Introduction: Blood samples that are transported within the laboratory by automated pre-analytical transport systems, as part of total laboratory automation, might be prone to various influences (e.g. continuous vibration, acceleration, deceleration). Data on the effect of this type of sample transport on platelet and coagulation assays are sparse. Therefore, we aimed to evaluate the effect of sample transport by an Inpeco Accelerator a3600 track system on platelets, a platelet function test and several functional coagulation assays.
Methods: Citrated blood was collected from 20 healthy volunteers without known history of coagulation disorders. For each volunteer, half of the collected tubes were transported by the Accelerator a3600 track along a trajectory (±30 meters) similar to what is applied for routine samples. The other half of the samples was not transported. The following analyses were performed on both sample groups: platelet count (Sysmex hematology analyzer), platelet activation test via flow cytometric detection of p-selectin (CD62p) expression on platelets (FACSCanto™ II, BD), INNOVANCE® PFA-200 with collagen/EPI and collagen/ADP cartridges (Siemens), PT, aPTT, PTT-LA and dRVVT screen (STA-R Evolution, Stago). 
Results: Blood sample transport by the Accelerator a3600 track leaded to significantly increased platelet activation (38.0 ± 15.1% CD62p expression vs. 28.9 ± 11.7% in samples that were not transported, p=0.03). Platelet counts, PFA-200 results, PT, aPTT and PTT-LA clotting times were not significantly affected by transport on the Accelerator a3600 track. dRVVT screen clotting times were mildly reduced (mean difference 1.0 s, p=0.001) in samples that were transported on the Accelerator a3600.
Conclusions: In a population of healthy volunteers, blood sample transport by an Accelerator a3600 pre-analytical transportation system induced platelet activation and leaded to limitedly shortened dRVVT screen clotting times. Further evaluation is needed in samples from patients with platelet function disorders and/or prolonged clotting times to verify whether this may cause clinically relevant effects. PFA, PT, aPTT and PTT-LA results were unaffected.

A Rapid And Inexpensive Point Of Care Method To Verify Aliquot Specimens As Citrate Plasma, Edta Plasma, Or Serum
Jeffrey Dlott, Laura Worfolk, Edward Wong
Quest Diagnostics, Chantilly, VA, United States

Introduction: Common laboratory practice involves the separation of plasma or serum from whole blood collection tubes. The remaining cellular component is usually discarded, and the aliquots labeled with patient identifiers, sample type, and date of collection. A potential pitfall of this process is that specimen types can be mislabeled. For coagulation tets, specimen types other than citrated plasma can greatly skew some clot-based assays. For example, Factor VIII activity assays that are otherwise normal in citrated plasma will yield hemophilia range levels in EDTA and serum. Here we describe a rapid and inexpensive microsampling method for point of care quality control (QC)  to verify citrated plasma, EDTA plasma, and serum sample types.
Methods: This method exploited the different eletrolyte properties that result from the addition of chelating agents to collection tubes or absence of such agents, as is the case with serum. Focusing on Calcium (Ca++) as a discriminator, it was eventually determined that water hardness test strips effectively measure Ca++ concentration. Three different manufacturer kits were ordered and tested against known Citrate, EDTA,  and serum samples. Testing involved pipetting a few microliters of specimen directly onto the test strip, allowing 15-20 seconds for development, then interpreting the colorimetric change from the manufacturer's provided chart. The 1 suitable water hardness strip was then validated as a sample type discriminator for  coagulation clot-based assays.
Results: Only the Sofchek Total Hardness kit by Hach Company produced distinct colorimetric differences without blancing or runoff. The colorimetric changes were: citrate (brown),  EDTA (green), and serum (orange). The cost per test   is a few cents and nominally more than a Ca++ performed on a chemistry analyzer. The main advantage is the convenience of point of care testing. Still, the chemistry analyzer was useful for the rare ambiguous case (e.g. significant hemolysis). Validation of this method identified select coagulation tests to QC prior to result reporting.
Conclusions: This water hardness test strip method is a rapid and inexpensive way to mitigate pre-analytic specimen error type. Awareness of the potential hazard of mislabeled specimen type and the recognition of which coagulation assays are most afected can guide laboratories to implement specimen processing changes and QC checks.

Depressed Neutrophil&Rsquo;S Functions In Nigerian Myeloid Leukaemia Subjects

Introduction: Diverse leucocyte functions have been described in different forms of leukaemia while specific neutrophil’s dysfunctions have been highlighted in chronic myeloid leukaemia. The exact mechanism of these non-specific abnormalities is however poorly understood. The analysis of the neutrophil’s functional parameters could be used in prophylactic assessment of patients with the disease and other forms of myeloid leukaemia. However, studies of this nature have not been considered in Nigerian subjects with myeloid leukaemia.  This study therefore highlighted some functionalities of leucocytes in acute and chronic myeloid leukaemia (Naïve and the treated groups) in order to document specific mechanism for neutrophil functional failures despite its numerous advantage.
Methods: A total of twenty six (26) leukemic subjects comprising of nine AML and seventeen CML subjects as well as twenty apparently healthy controls were studied. They were diagnosed with standard clinical and cytological criteria and were age and sex -matched with the controls. Also, they verbal consented to the study and ethical approval were obtained from appropriate bodies.  They were studied for various neutrophil’s functions such as viability, Nitrozolium blue tests, Chemotaxis and Phargocytic indices. These tests were performed at pre and post-treatment (with or without remission) stages of the diseases using standard methods of assay.
Results: There was a significant increase (p < 0.05, respectively) in total white blood cells in newly diagnosed acute myeloid leukaemia patients and those on treatment (with or without remission) compared with controls.  There were no significant differences (p > 0.05, respectively) recorded in percentage viability, oxidative burst activity and chemotaxis index in AML and CML on remission compared with controls, although significantly decreased in untreated subjects compared to controls and during remission (P< 0.05, respectively. Phagocytic index was statistically decreased (p < 0.05) in newly diagnosed AML and on remission compared with that of controls.
Conclusions: We inferred that depressed phagocytosis function coupled with altered chemotaxis and oxidative burst activity in treatment naive  myeloid leukaemia may influence susceptibility to infections especially in AML. We suggest that these parameters could serve clinical advantages during treatment monitoring.

Identifying Factor Deficiency With The Coagpia Aptt Fs Reagent And The Automated Mixing Study Application Of The Cp3000 Coagulation System
Sarah Harper1, Peter Baker1, Larry Smith2, Gabriella Lakos2
1Oxford Haemophilia and Thrombosis Centre, Oxford, United Kingdom, 2Abbott Diagnostic Division - Hematology, Santa Clara, CA, United States

Introduction: Factor VIII and Factor IX deficiency are the most important severe bleeding disorders. Identifying these deficiencies relies on screening tests with APTT, factor level determination and mixing studies to differentiate inhibitors from true factor deficiencies.
Methods: The ability of the Sekisui Coagpia® APTT FS reagent to identify FVIII and FIX deficiency with the CP3000 Coagulation System(Sekisui Diagnostics, Lexington, MA, distributed by Abbott Laboratories), was assessed in the laboratory of the Oxford Haemophilia and Thrombosis Centre, in comparison with Dade® Actin® FS Activated PTT reagent on the Sysmex® CS-5100 Hemostasis System. In addition, the performance of the CP3000 automated mixing study feature was evaluated with FVIII and FIX deficient samples.
Results: The Coagpia APTT FS reagent has demonstrated a within-run and within-laboratory %CV of 2.3-4.7% and 9.0-12.1%, respectively, for samples with FVIII level of 3.0% to 82.9%. When results were compared to those obtained with the CS-5100 on 150 clinical samples (including samples with FVIII and FIX deficiency), a strong correlation (r=0.94) and agreement (slope=1.13, intercept= 1.3) was demonstrated, with a qualitative total agreement of 86 % for prolonged APTT. The sensitivity limits of the APTT FS reagent for detecting FVIII and FIX deficiencies were established at 42.6% and 48.4%. Mixing study was performed with 11 and 9 samples with known FVIII and FIX deficiency on CS-5100 and by using the automated mixing study application on CP3000, utilizing a 5-level mixing setting. Rosner index values obtained by CP3000 were lower compared to those obtained by CS5100 (P< 0.0001) but showed good correlation (r=0.64) and were below 12% in all 20 cases, correctly identifying factor deficiency. The visual interpretative feature of CP3000 mixing application displayed a concave mixing curve for each factor deficient sample, which remained practically unchanged after incubation for 2 hours.   
Conclusions: The Coagpia APTT FS reagent has optimal sensitivity for detecting FVIII and FIX deficiencies. The CP3000 Coagulation System correctly identified FVIII and FIX deficient samples by using the APTT FS reagent and the automated mixing study feature. The visual display of the mixing study results facilitates the interpretation of results.

Assessment Of Normative Range And Deriving Cut-Off Values For Lupus Anticoagulant Testing: An Experience From A Tertiary Care Centre In Southern India
Rakhee Kar, Arikrishnan Ramaraj, Vir Singh Negi
Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India

Introduction: Lupus anticoagulant (LA) testing is a demanding laboratory test. The most common among recommended tests are dilute Russell viper venom time (dRVVT) and LA sensitive activated partial thromboplastin time (LA-aPTT). Final interpretation depends on normalized ratio (NR) or percentage correction of ratio. Although integrated tests come with manufacturer-assigned cut-off, but according to guidelines cut-off should be locally-derived. This study over one-year derived normative range of LA in south Indian population and used locally-derived cut-off to interpret patient samples. 
Methods: Initially, 100 healthy controls (54 females,46 males; mean age 29 years) were tested for LA-aPTT, aPTT and dRVVT screen and confirm. Aliquot of normal pooled plasma (NPP) was used with every batch. All ratios were calculated in two ways, using NPP control values and taking reference interval mean (RI mean) as denominator. 152 patients (143 females,9 males; mean age 28.8 years; clinical profile- SLE 46%, pregnancy complications-32.9%, venous thrombosis-5%, arterial thrombosis-3%, other miscellaneous-13.2%) were enrolled. Patients ion anticoagulants were excluded. The sensitivity, specificity, positive predictive value (PPV) and negative predictive value (NPV) of screening tests was calculated.
Results: Cut-off (mean+2.3 SD)for aPTT, LA-aPTT, dRVVT screen and dRVVT confirm assays were 37.5, 45.7, 45.5 and 41.3 seconds. Cut-off for NR and percentage correction of ratio taking as denominator NPP were 1.16 and 15.8% and RI mean were 1.13 and 13.2% respectively. There were 2 false-positive controls. As RI mean cut-off was much lower than manufacturer cut-off of 1.2, NPP cut-off was used and 25(16.4%) patients were positive (14 NR, 7 LA-aPTT, 4 both). Four additional patients could be identified using local cut-off. The specificities and NPV of all the screening tests were high. Sensitivity of aPTT, LA-aPTT and dRVVT taking as gold standard NR was 11.1% and 22.2% and 94% and LA positivity (NR and/or LA-aPTT) was 16%, 44% and 76% respectively.
Conclusions: All LAtesting laboratories should derive normative range and locally-derived cut-off and result should be interpreted correlating with clinical history. Sensitivity of only dRVVT screen was high making it an ideal screening test.  However, combining an additional test like LA-aPTT done in this study helped in picking up cases negative by dRVVT.              

Analysis Of Teg Performance And Comparison To Hemostasis Screening Tests In Cirrhotic Patients
Karen Y Kimoto1,2, Dayane M Jacinto1,2, Tailiny A F Alonso1,2, Christiane P Gouvea1,2
1University of So Paulo, So Paulo, Brazil, 2Fleury Medicina e Sade, So Paulo, Brazil

Introduction: Hepatic cirrhosis is one of the leading death causes. In advanced stages is characterized by hemostasis disorders, whose nature is complex and multifactorial. Despite the widespread use of coagulation tests in cirrhotic patients, these assays are ineffective predictors of hemorrhagic probability. In this scenario, thromboelastography (TEG®) becomes an auxiliary tool in this evaluation.
Methods: To evaluate the correlation between hemostasis tests (INR, APTT, Fibrinogen, Platelets and Factor V) and TEG® parameters, 63 patients with liver cirrhosis confirmed through imaging tests or hepatic biopsy were included consecutively from May to June 2017. The TEG® was performed as soon as the sample was delivered and analyzed from kinetic (R: time to initial fibrine’s formation and K: clot formation speed) and structural (alpha angle: clot formation rate and MA: clot strength) coagulability. Correlations were calculated using the Spearman test, considering correlation degree: weak (r< 0,3), moderate (0,3>r>0,7) and strog (>0,7).   
Results: The patient´s mean age was 51 years. The most common cause of cirrhosis was autoimmune hepatitis (21%) and the MELD score median was 13 (7-30). There was a strong correlation between  platelets and K (r = -0.79), fibrinogen and K (r = -0.73) and factor V and K (r =-0.72). Equally significant and moderate correlation between platelets and MA (r = 0.63), fibrinogen and MA (r = 0.64),INR and MA (r = -0.61), APTT and MA (r = -0.69), factor V and R (r = -0.40), INR and R (r = 0.49), APTT and R (r = 0.61), platelets and alpha angle (r = 0.70) and fibrinogen and alpha angle (r = 0.64). The correlation between MELD score and  MA (r = -0.26) was not observed. 27% presented reduction in kinetic and structural coagulability simultaneously and all showed INR’s prolongation, 53% APTT prolongation and 47% had platelets lower than 50,000 / mm³. Factor V activity mean was 45%. Of the 63 patients, 44.5% had hypocoagulability in at least one of TEG’s® phases and only two had no alterations in the basic parameters of hemostasis, suggesting a high association between hemostasis parameters and bleeding probability.  
Conclusions: This study reveals the usefulness of thromboelastography in the cirrhotic advanced stages patients evaluation and follow-up and shows a high correlation with hemostasis basic parameters.

Correlation Between Inr And Factor V And Vii Activities In Pediatric Acute Liver Failure
Vadim Kostousov, Yebin Chae, Karen Bruzdoski, Lisa Hensch, Shiu-Ki Hui, Tamir Miloh, Jun Teruya
Texas Children's Hospital, BCM, Houston, TX, United States

Introduction: Factor V (FV) and factor VII (FVII) activities are often ordered to assess synthetic liver function when considering liver transplant based on the belief that FV and FVII as levels and trends might be earlier markers of liver function recovery or failure when compared to international normalized ratio (INR). However, the utility of FV and FVII in addition to INR, calibrated against patients on stable warfarin therapy, in pediatric acute liver failure (ALF) is unknown. The study aims to determine a correlation between INR values and FV and FVII activities, creating a conversion equation that predicts factor level using INR.
Methods: The retrospective study (April 2015-July 2018) was conducted at a tertiary care pediatric hospital. Simultaneously measured INR and FV or/and FVII tests were extracted from database of patients with confirmed ALF (acute liver disease with INR>2 and encephalopathy with INR>1.5). INR values were measured using STA-Neoplastine-plus reagent (ISI=1.3) on STA-Revolution analyzer (Diagnostica Stago). FV and FVII values were measured by one-stage clotting method. Statistical analysis was performed using MS-Excel 2010; data is presented as mean±SD (range) with significance at p< 0.05.
Results: Two hundred twelve data points were found from 34 children (F:M=3:1, 6.5±6.4 years old). INR value was 2.3±1.0 (0.9-5.4), FVII and FV activities were 25±22% (2-128) and 47±27% (10-176), respectively. After logarithmic transformation, INR values had a stronger linear negative correlation with FVII compared to FV.     
  Slope Pearson's correlation, p value
Log INR vs Log FVII -1.89 0.91, < 0.0001
Log INR vs Log FV -0.98 0.73, < 0.0001
In the critical (INR=3.0-5.4) range (n=49), the calculated FVII activity was lower in 27% and FV was lower in 30% compared to the measured levels. For the majority of the discrepant results (16 out of 28), higher actual FVII activity than the calculated could be explained by concomitant lower FV level in the specimen, and vice versa. The remaining 12% of discrepant data could be explained by other factor deficiencies.
Conclusions: The study overall demonstrated that FV and FVII were strongly negatively correlated with INR values. The formulated conversion accurately predicts majority (≥70%) of FV and FVII activities from known INR values, ultimately helpful for prompt clinical decisions.

The Clinicopathological Impact Of Notch 1 Mutations Among Acute Myeloid Leukemia Patients
.Salah Aref1, Mohamed Sabry1, Rasha Rizk1, Wafaa Fakhry1, Maha Elzafrany2
1Hematology Unit Mansoura University, Mansoura, Egypt, 2Medical Oncology Unit Internal Medicine Department Mansoura University, Mansoura, Egypt

Introduction: This study aimed to determine the prevalence and the clinical  impact of  NOTCH1  mutations  in acute myeloid leukemia patients.
Methods: Mutations in the NOTCH1 gene were investigated in 50 primary acute myeloid leukemia (AML) cell samples by  Sanger  method using ABI  sequencer. The NOTCH 1 areas  for sequencing were the heterodimerization (HD) domain (exon 26), exon 34 (tad domain) and exon 34 (pest domain) .  
Results: NOTCH 1 mutation was detected in 6 cases out of 50 AML cases (12%). Two mutant cases were detected in the heterodimerization (HD) domain (exon 26) and 4 mutant cases were found in the proline, glutamic acid, serine, threonine-rich (PEST) domain (exon 34) of the NOTCH1 receptor. All mutant NOTCH1 cases died during the study period, their mean OS was 1.2 months, whereas, mean OS for wild NOTCH cases was 21.2 months. Mutant NOTCH cases showed significantly shorter OS when compared to wild NOTCH cases (p< 0.001). 
Conclusions: NOTCH1 mutations displayed bad clinical outcome in AML patients. Targeting  NOTCH1 mutations might have benificcial therapeutic value. 

Effects Of Polyphosphate On Clotting Reaction Triggered By Activated Partial Thromboplastin Time Assay Reagents
Yuko Kuroda1, Masatoshi Wakui2, Yuta Fujimori3, Shoko Nakamura1, Yoshino Kondo1, Shusaku Oka1, Terumichi Nakagawa1, Hisako Katagiri1, Mitsuru Murata2
1Clinical Laboratory, Keio University Hospital, Tokyo, Japan, 2Department of Laboratory Medicine, Keio University School of Medicine, Tokyo, Japan, 3Office of Clinical Laboratory Technology, Keio University Hospital, Tokyo, Japan

Introduction: Polyphosphate (polyP) is a linear polymer formed by polymerization of inorganic phosphoric acid, which is polymorphic in length. Very long chain polyP is present in bacteria while short polyP is present in platelets and cancer cells. The relevance of polyP to physiological and pathological conditions is of considerable interest. Since polyP leads to activation of the contact phase in the blood coagulation pathway, it is considered to be one of the major intrinsic coagulation activators in vivo to give rise to hypercoagulability. This study aims to evaluate effects of polyP on clotting reaction triggered by activated partial thromboplastin time (APTT) reagents. 
Methods:  An APTT reagent (Dade Actin, Siemens) or its 10% dilution was added to CRYO Check Pooled Normal Plasma (Precision BioLogic) and spiked with polyP with different chain lengths (Bioenex).  APTT measurement and clot waveform analyses were performed using the apparatus CS-5100 (Sysmex). With respect to serial dilutions (2.5 to 100%) of six types of APTT reagent, similar experiments were also performed using extralong-chain polyP, which was estimated to have the strongest influence. 
Results:  When 100% APTT reagents were used, prolongation of APTT was observed dependently on the chain length and concentration of polyP. The maximum coagulation velocity as well as the maximum coagulation acceleration were decreased. Addition to 10% diluted APTT reagent shortened clotting time. There were no obvious differences in the shape of the clot waveform. When APTT reagents were diluted to 2.5 to 10%, polyP caused a decrease in clotting time and an increase in each waveform parameter value. This was obvious especially when diluted to 2.5%. There were remarkable differences in the effects of polyP between APTT reagents.
Conclusions: The effects of polyP were observed especially when APTT reagents were diluted to low concentrations. It seems that laboratory tests using diluted APTT reagents, such as lupus anticoagulant detection assays, may be affected in the case of diseases under which polyP is increased in the blood. The differences in the polyP effects among APTT reagents might be related to the quality and quantity of activators or phospholipids contained.

Effects Of Citrate Tube Fill Volume On Pt- And Aptt-Based Clot Waveform Analysis (Cwa) Parameters
Shan Shan Lim, Wan Hui Wong, Sim Leng Tien, Heng Joo Ng
Department of Haematology, Singapore General Hospital, Singapore, Singapore

Introduction: Accurate routine coagulation (prothrombin time, PT and activated partial thromboplastin time, aPTT) results are dependent on optimal blood: sodium citrate ratio (v/v) of 9:1 which is affected by the fill volume during sample collection. The kinetics of the coagulation process can be expressed by CWA but the influence of fill volume is little known. This study investigates the effects of this preanalytical factor on PT- and aPTT-based CWA parameters.
Methods: Pooled platelet poor plasma (PPP) from 20 healthy volunteers were serially diluted with sodium citrate and a haematocrit level of 0.4 was deemed to reflect blood: anticoagulant (v/v) ratios of 8:1, 7:1, 13:2, 5:1, 4:1, 3:1 and 2:1, corresponding to fill volumes of 90%, 80%, 75%, 60%, 50%, 40% and 30%. PT and aPTT were then analysed by the Sysmex CS2100i coagulometer (Sysmex Corporation, Kobe, Japan) using Dade Innovin and Actin FSL reagents (Siemens Healthcare, Marburg, Germany) respectively. The corresponding CWA parameters min1 (maximum velocity), min2 (maximum acceleration) and max2 (maximum deceleration) were obtained from the built-in algorithm of the analyser. Regression analysis and paired t-test was performed using Microsoft Excel.
Results: All measured CWA parameters showed a numerical decrease with lower blood: anticoagulant ratios while PT and aPTT showed a corresponding increase. The sharpest decline occurred between fill volumes of 40% and 30% in both PT and aPTT-based CWA. PT CWA parameters for samples with less than 80% fill were significantly different (p< 0.05) compared to samples filled to 100%. aPTT CWA parameters in all sub-optimally filled samples were significantly different from the 100% filled. All PT CWA parameters were within the lab established reference interval (RI) if samples were filled to 40% or more. The aPTT min1 was within RI once 50% filled and 60% filled for aPTT min2 and max2. In contrast, only samples more than 75% filled were within the reference intervals for PT and aPTT.
Conclusions: PT and aPTT CWA parameters are affected by the under filling of citrated tubes at sample collection, with aPTT CWA parameters being more affected than PT CWA parameters. Samples of 60% fill volume or less should be rejected while other underfilled sample results should be interpreted with caution due to the statistical difference. 

The Impacts Of Sample Storage Conditions And Mechanical Agitation As Well As Their Interaction On Coagulation Tests
Masato Matsuda1, Yutaka Komiyama2, Yutaka Takino2, Masahiro Ieko3, Yoshiki Hoshiyama1, Tohru Minamino1, Yasuo Saijo4, Masato Moriyama4
1Medical Laboratory Division, Niigata University Medical and Dental Hospital, Niigata, , Japan, 2Faculty of Health and Medical Sciences, Hokuriku University, Kanazawa, , Japan, 3Department of Internal Medicine, Health Sciences University of Hokkaido, Sapporo, , Japan, 4Department of Medical Oncology, Niigata University Graduate School of Medical and Dental Sciences, Niigata, , Japan

Introduction: Pre-analytical variables (e.g. sample storage and transportation) can alter the results of coagulation tests, which is crucial when the medical institutions outsource the tests. Previous our questionnaire conducted in Japan revealed that 96% commercial laboratories have received whole blood samples, transported by car for 3 or more hours, stored at ambient or refrigeration temperature, indicating the difficulties of following the CLSI guidelines. To establish a guideline considering the circumstances, this study investigated the impacts of the three pre-analytical variables and their interaction on hemostasis tests: (1) storage time; and (2) temperature; (3) mechanical agitation mimicked for transportation.
Methods: The aliquoted whole blood samples, obtained from nine healthy volunteers, were stored for 0, 4, 8, 12, 24 hours at room temperature or 2-8℃, with or without mechanical agitation (50 or 200 rpm with a shaker). After the centrifugation of the samples (1500 g for 15 min), seven hemostasis tests were performed on the samples: prothrombin time (PT); activated partial thromboplastin time (APTT); fibrinogen (Fbg); normotest (NT); dilute Russell viper venom time (dRVVT); factor VIII activity (FVIII:C); and soluble fibrin (SF). The effects of the variables were considered as moderate when < 25% of the samples showed change under ±10%, as large when >25% samples showed over ±10% changes.
Results: The large effects of APTT prolonging were found after storage for 24-h at 4℃ with 50 rpm or without mechanical agitation. The large effects of FVIII:C decreasing were identified after 4-h at every condition, and the FVIII:C continued decreasing. Interestingly, FVIII:C was higher in agitated samples . The NT showed large effects, over time, depending on the combination. The PT, Fbg, dRVVT and SF showed moderate effects at every condition.
Conclusions: The impacts of the variables were quite different depending on their combination, but the effects were crucial in the limited tests, indicating requirement of reconsideration of the current guidelines.

Evaluation Of Activated Partial Thromboplastin Time Coagulation Waveforms Generated By Acl Top Series Coagulation Analyzers Can Help Identify Patients With Acquired Factor Viii Inhibitors.
Eric McGinnis1, Steven K.W. Wong2, Tyler W. Smith1,3
1University of British Columbia, Vancouver, BC, Canada, 2Saint Paul's Hospital, Vancouver, BC, Canada, 3Vancouver General Hospital, Vancouver, BC, Canada

Introduction: ACL TOP instruments measure activated partial thromboplastin time (aPTT) turbidimetrically and generate coagulation waveforms (CWs), which graphically represent change in light absorbance over time. Isolated prolonged aPTT results pose challenges for clinical laboratories because they are frequently caused by a lupus anticoagulant (LA), which is generally not associated with bleeding but often requires laboratory investigation to exclude uncommon disorders that are associated with bleeding, including acquired inhibitors of coagulation factors (particularly FVIII). Early identification of factor inhibitors is of critical importance, as delayed recognition can result in significant bleeding-related morbidity and mortality. We sought to identify features in the CW to differentiate LAs from factor inhibitors and facilitate rapid initial testing to identify patients at risk of severe bleeding.
Methods: We retrospectively assessed CWs collected at two tertiary care centers from ACL TOP 700 CTS instruments using SynthASil aPTT reagent, including normal controls and patients with LA (or probable LA; PLA), acquired FVIII inhibitors, congenital intrinsic pathway factor deficiencies, and medical anticoagulation. Initial examination of CWs identified the presence of a double peak in the first derivative curve (DP1D) as potentially useful to differentiate factor inhibitors from LAs (Figure 1). CWs were inspected for DP1D and numeric parameters of CW first and second derivative (2D) curves were used to compute receiver operator characteristic (ROC) curves.
Results: 182 CWs were assessed, including 60 LA/PLA (6 with DP1D) and 21 FVIII inhibitors (17 with DP1D). In cases with prolonged aPTT (>38 seconds, n=143), DP1D had 81.0% sensitivity and 89.3% specificity for FVIII inhibitors. Among other CW parameters, 2D minimum demonstrated the highest area under the ROC curve for identification of FVIII inhibitors (AUC=0.892; sensitivity 88.9%, specificity 82%).
Conclusions: CW assessment for DP1D and derivative curve parameters readily available on ACL TOP instruments appears to be of value in differentiating acquired factor inhibitors from other causes of prolonged aPTT. Incorporating CW analysis in routine laboratory practice may prove useful in directing initial investigations of unexplained prolonged aPTT and facilitate early diagnosis of acquired hemophilia.

Evaluation Of Anti-Factor Xa Assays For Monitoring Indirect Xa Inhibitors Using External Quality Data
Piet Meijer, Martine Hollestelle
ECAT Foundation, Voorschoten, Netherlands

Introduction: Chromogenic anti-factor Xa (FXa) assays are currently the “gold standard” for monitoring the indirect anticoagulants; like unfractionated heparin (UFH), low-molecular-weight-heparin (LMWH), fondaparinux and danaparoid sodium. However, the anti-FXa has been shown to vary with the choice of reagents. In the present study, interlaboratory variation in indirect anti-FXa measurements is evaluated to get more insight in the clinical applications of the various methods.
Methods: Laboratory tests were evaluated for UFH, LMWH, fondaparinux and danaparoid sodium using spiked plasma samples. External quality assessment data of 2013 till 2017 is used of more than 100 laboratories, derived from the ECAT foundation.
Results: The results of danaparoid and fondaparinux did not show significant differences in absolute measured values between the three most frequently used methods by the participants of the ECAT. On the other hand, large differences between the measured values using various methods were observed for LMWH (till maximum 50% difference) and even more for UFH (till maximum 100% difference). These difference may be caused due to differences in method composition, like for example the addition of dextran sulphate. Substantial interlaboratory variation in anti-FXa monitoring was observed for all parameters (4% - 42%, Table 1), in particularly at low concentrations. Our results show that especially below 0.35 IU/mL the coefficient of variation for UFH and LMWH increases almost 2 times compared to higher concentrations. Table 1. Overall interlaboratory variation in the anti-FXa measurements
Anti-FXa concentrationa CV - UFHb CV - LMWHb CV - Danaparoidb CV - Fondaparinuxb
< 0.35 20 [7-42] 17 [12-33] 12 [7-17] 14 [9-22]
0.35-0.70 10 [6-21] 10 [8-13] 8 [5-12] 9 [6-14]
>0.70 7 [5-10] 8 [4-13] 7 [4-11] 9 [5-12]
aConcentration in IU/mL for UFH, LMWH and danaparoid and in µg/mL for fondaparinux. bMean CV and [range] both in %.
Conclusions: Our study demonstrates that the choice of anti-FXa method is particularly important for UFH and LMWH measurement. Therapeutic ranges should be adapted to the used combination of methodology. Furthermore, results below 0.35 IU/mL for especially UFH and LMWH should be used with caution.

Influence Of Direct Oral Anticoagulants On Lupus Anticoagulant Measurement
Sumiyoshi Naito1,3, Masahiro Ieko2, Takeshi Suzuki2,5, Mika Yoshida1, Osamu Kumano2,5, Kazumasa Ohmura2, Nobuhiko Takahashi2, Mitsuru Moriya1,4, Satoshi Fujii3
1Department of Clinical Laboratory, Health Sciences University of Hokkaido Hospital, Sapporo, Japan, 2Department of Internal Medicine, Health Sciences University of Hokkaido, Ishikari-Tobetsu, Japan, 3Department of Clinical and Laboratory Medicine, Asahikawa Medical University, Asahikawa, Japan, 4Department of Internal Medicine, Health Sciences University of Hokkaido Hospital, Sapporo, Japan, 5Sysmex Corporation, Kobe, Japan

Introduction: Direct oral anticoagulants (DOACs), developed for prophylaxis and treatment of patients affected by thromboembolic disorders, are known to effect the results of clotting time assays. We investigated the influence of DOACs on lupus anticoagulant (LA) testing.
Methods: Three DOACs were examined; dabigatran, rivaroxaban, and apixaban. Each was separately added to 4 different normal plasma samples (2 from commercially available sources, pooled normal plasma from 6 healthy subjects, plasma from healthy donor) at concentrations ranging from 0-400 ng/ml, considered to be adequate for anticoagulant therapy. We then examined those samples using activated partial thromboplastin time (APTT), dilute Russell’s viper venom time (dRVVT), confirmation testing based on APTT, and APTT cross-mixing test (CMT) assays.
Results: APTT and clotting time in the confirmation tests were prolonged by the addition of dabigatran, rivaroxaban, and apixaban in a concentration-dependent manner. The ratio of dRVVT was also increased by the addition of each DOAC. That ratio increase with rivaroxaban was in proportion to its concentration, while it was not proportional with the concentration of dabigatran or apixaban. Results of CMT with the DOACs showed a pattern similar to those for LA.
Conclusions: The present findings confirmed that DOAC administration influences the results of LA testing. In particular, samples containing rivaroxaban may cause a false-positive result. In addition, CMT results with samples containing a DOAC showed a similar pattern to the LA results, thus sufficient attention is necessary to distinguish between those.

Performance Evaluation Of A New Hematology Analyzer: Unicel Dxh 900 Coulter Cellular Analysis System
Gnel Bahramzada1,2, Rana Turkal2, Tlay evlik2, nder Şiriki1,2, Goncagl Haklar1,2
1Department of Biochemistry, School of Medicine, Marmara University, Istanbul, Turkey, 2Biochemistry Laboratory, Marmara University Pendik E&R Hospital, Istanbul, Turkey

Introduction: A certain level of verification of any new hematology analyzer has to be done according to the ISO 15189 in accordance to a professional standard. The aim of this study was to evaluate the analytical performance of DxH 900 (Beckman Coulter, FL, USA) for its accuracy and imprecision, in which a new hematology parameter, monocyte volume width distribution (MDW), intended for use as an early sepsis indicator and offered as part of a routine cell blood count (CBC) with differential test, was included.
Methods: Evaluation was performed in Marmara University Pendik E&R Hospital Laboratory according to the recommendations of the H26-A2 of the CLSI in one-month period. Venous blood samples were collected into EDTA-tubes (Vacutainer-BD, NJ, USA). All samples were chosen randomly among the residual samples of the patients whose CBC was ordered and were analyzed within 4 hours of collection. Within-run precision coefficient of variations (CV%) for all parameters of CBC were assessed on DxH 900 by performing 10 consecutive measurements of the low, normal, and high level samples. Between-run precision were calculated by processing the manufacturer’s control material with low, normal, and high twice daily over a period of 20 days. All parameters analyzed on DxH 900 were compared to DxH 800 (n=550).
Results: Within-run precision was satisfactory according to the manufacturer’s specifications. CV for leukocyte count of 0–2 ×10³/μL was 1.8%, and for the platelets 0-50x10³/μL it was 3.49%. Hb value 6-10g/dL also presented satisfactory results, with CV 0.67%. CV for within-run and between-run precision of MDW were < 6% and < 2, respectively. All the CVs observed in between-run precision were lower than the specification suggesting that the maximum intra-analyzer imprecision for hematological parameters must be lower than half of the intraindividual biological variability (CV (analytical) or imprecision < 0.5×CV (intra)). DxH 900 displayed very good correlation (r>0.97) with the DxH 800 for CBC and cell differential parameters except for monocyte% and basophil% (respectively r=0.93 and r = 0.71).
Conclusions: We conclude that DxH 900 HA ensures satisfactory interchangeability, providing reliable hematological data.  

Dilute Prothrombin Time Is Associated With The Plasma Drug Concentrations, Contributing To Be Comparable With Anticoagulant Effects As A Measuring Test For Anti-Xa Doacs Therapy: Results From Multicenter Study.
Kazumasa Ohmura1, Masahiro Ieko1, Sumiyoshi Naito2, Mika Yoshida2, Takeshi Suzuki3, Nobuhiko Takahashi1, Shoko Ono4, Kozue Ikeda5, Ichiro Sakuma6
1Health Sciences University of Hokkaido, Ishikari-Tobetsu, Japan, 2Health Sciences University of Hokkaido, Department of Clinical Laboratory, Sapporo, Japan, 3Sysmex Corporation, Kobe, Japan, 4Division of Endoscopy, Hokkaido University Hospital, Sapporo, Japan, 5Shinoda General Hospital, Cardiovascular Medicine, Yamagata, Japan, 6Caress Sapporo Hokko Memorial Clinic, Cardiovascular Medicine, Sapporo, Japan

Introduction: Direct oral anticoagulants targeted to factor Xa (Anti-Xa DOACs) have been administered for prevention against several venous thrombotic diseases and the routine monitoring is not required. However, assessment of anticoagulant effect is needed to prevent severe events including major bleeding and/or refractory thrombosis.
The aim of this study is to explore correlation between the Ratio of Inhibited Thrombin Generation (RITG) based on dilute prothrombin time (dPT) and some coagulant markers or laboratory tests for drug effects.
Methods: Citrated plasma samples were obtained from Japanese patients with receiving Rivaroxaban (n=882), Apixaban (n=1214) and Edoxaban (n=820) at four sites. Laboratory tests such as PT, activated partial thromboplastin time (APTT), fibrin monomer complex (FMC) and D-dimer were determined by Sysmex CS series. The plasma concentrations of anti-Xa DOACs were measured by anti-Xa assay, specially calibrated for each drug (Hypen Biomed, Technoclone and Daiichi-Sankyo). The drug concentrations were divided into peak and trough groups within 5 hours of administration of drugs or longer.
Results: RITG was positively correlated with PT, APTT and drug concentrations, and negatively with D-dimer in each DOACs group. RITG was negatively correlated with FMC in Edoxaban group. Both PT and APTT were significantly different between peak and trough groups in Rivaroxaban and Edoxaban, while neither were different between two groups in Apixaban. In contrast, RITG was significantly different between peak and trough groups in Apixaban as well as Rivaroxaban and Edoxaban.
Conclusions: This study indicated that RITG was positively correlated with plasma concentrations of anti-Xa DOACs and negatively with coagulant markers. PT and APTT have not been shown to be comparable with DOACs anticoagulant activity, however RITG measuring might be useful to evaluate bleeding and thrombotic tendency.

Assessment Of Thrombotic Disorders As A Risk Factor To Maternal Mortality In Southwestern Nigeria.
Bamisaye Oluwaseyi1, Akanni Emmanuel2, Okungbowa Michael3, Akinbo David1, Feyisola Tomide1
1Department of Medical Laboratory Science, Afe Babalola University, Ado Ekiti, Nigeria, 2Department of Medical Laboratory Science, LAUTECH, Osogbo, Nigeria, 3Department of Medical Laboratory Science, University of Benin, Benin, Nigeria

Introduction: The incidence of maternal mortality is still unacceptably high around the world with about 830 women being lost to pregnancy or childbirth related complications. However, almost all of these deaths have been observed to occur in low-resource settings and most of them could have been prevented. There is therefore need to determine how these deaths could be controlled through the monitoring and assessment of their risk factors.
Methods: Exactly 270 pregnant women  across the three trimesters were recruited for this study. The parameters estimated include packed cell volume (PCV) and platelet count (Mind ray autoanalyser); prothrombin time (PT) and activated partial thromboplastin time (APTT) (Diagen reagents); D-dimer (Tina Quant D-dimer Roche Cobas CII) and activated protein C resistant (APC) (Chromogenix Coatest APC Resistance V) assays.
Results: The mean±SD of the PCV, platelet count, PT, APTT, D-dimer and APC-V ratio assessed in the subjects are 33.94±4.53, 173.33±46.91, 11.15±1.71, 31.90±5.95, 0.85±0.99 and 2.72±0.54 respectively. Six (2%) and thirty-two (11.9%) subjects who had reduced and normal PT/APTT had increased D-dimer level respectively with two (0.74%)  from both group (Table 1) being positive to the APCr assay and possess abnormally increased D-dimer values. Table 1:  Distribution of the coagulation and thrombotic Markers among pregnant subjects.
APTT(seconds) PT(seconds)  D-Dimer(< 0.5 ug FEU/ml)  D-Dimer(>0.5 ug FEU/ml)
< 25 < 11 12(4.4%) 6(2.2%)
11-15 12(4.4) 6(2.2%)
25-35 < 11 52(19.3%) 10(3.7%)
11-15 72(26.7%) 32(11.9%)
>35 < 11 8(3.0%) 6(2.2%)
11-15 32(11.9%) 22(8.1%)

Conclusions: Two subjects displayed results and symptoms with tendencies of developing deep venous thrombosis which could eventually lead to post-partum disseminated intravascular coagulation as is observed in some maternal mortality in this region. Also this study has shown the presence of APC resistance in this region which is a risk factor to thrombotic disorders and this calls for serious attention in monitoring and managing the condition in the populace especially pregnant women in the various antenatal clinics in Nigeria with the introduction of D-dimer assay in routine screening and APCr functional assay in suspected patients presenting with related symptoms in order to reduce the maternal mortality rate in this region.

Factor Viii - Binding Assay In Diagnostic Workflow OfType 2 N Von Willebrand Disease
Dalila Patricio, Joana Rita Pinheiro, Tiago Luis, Rogrio Barreira, Gonalo Orfo, Ramn Salvado, Jorge Tomaz
Blood Bank and Transfusion Medicine, Coimbra University Hospital, Coimbra, Portugal

Introduction: Type 2N von Willebrand Disease(2NvWD), a qualititative variant, is caused by a defect in the ability of von Willebrand Factor(vWF) to bind FactorVIII(FVIII), which reduces FVIII’s half-life and plasma levels, while vWF usually remains normal. It´s phenotypic profile mimics mild haemophiliaA(MHA) and have been reported patients misdiagnosed with type 1 vWD(1vWD)either. 2NvWD final diagnosis, as well as differential diagnosis between MHA, is accomplished by molecular studies. However, measurement of vWF capacity to bind FVIII using a vWF:FVIII Binding(B) assay is considered a taken point in diagnostic-oriented workflow, specially the vWF:FVIIIB/vWFAntigen(Ag)ratio(R) - 2NvWD suspicion if < 0.3. This assay is only available in reference laboratories with expertise for vWD diagnosis. The aim of this study is to evaluate the first vWF:FVIIIB results from previously diagnosed 2NvWD  patients and its role in diagnosis workflow such as predicting disease severity.            
Methods: vWF:FVIIIB assay was performed with a commercial available ELISA kit(Asserachrom® VWF:FVIIIB; Stago)  in a total of 14 patients(13 unrelated families):6 with 2NvWD previously diagnosed by molecular studies(5F;1M;47.1[2-66]years); 4 patients  with MHA(57[31-72]years) and  4 with type 1vWD(3F;1M;34.3[6-52]years). There were no 2NvWD carriers included. This vWF:FVIIIB method cut-off for 2NvWDdiagnosis is< 20%; carriers/heterozygous might present30-60%, that doesn’t exclude MHA.
Results: From 2NvWD (FVIII20±11%,vWF:Ag62±10%), 5 showed markledy descreased vWF:FVIIIB results (9.4±8.2%;2 nil values) and 1 borderline 20,6%. MHA showed normal values (one with 64.4%); one 1vWD obtained 28.3%. vVWF:FVIIIB Ratio was always < 0.3 (0-0.27) for all 2NvWD(Table1) while MHA(0.6±0.2; with one R0.36) and 1vWD (4±2.6) were normal. The lowest vVWF:FVIIIB R was found in 2NvWD with Arg186Trp mutation(Table2).
Conclusions: Disability to bind FVIII were detected with no doubt by vWF:FVIIIB in 5 patients, in comparison with HA normal values; one patient with borderline values has been under treatment. One MHA  wouldn’t be differentiated from a possible 2NvWD carrier.  The assay requirement of vWF:Ag>10% was a limitation that might difficult 2NvWD defectdetection when it’s combined with a quantitative vWF variant. Nevertheless, vWF:FVIIIB assay is still the only functional test for differential diagnosis between 2NvWD and MHA and thereby the first step to lead molecular diagnosis strategy

Retrospective Evaluation Of Hit Pre-Test Probability And Association With Hit Assays
Majed Refaai1, Ferhat Kara1, Hannah McRae1, Khaled Refaai1, Ryan Whitmarsh2, Michael Spigarelli2
1University of Rochester, Rochester, NY, United States, 2Immucor, Inc, Norcross, GA, United States

Introduction: Heparin-induced thrombocytopenia (HIT) is a critical complication of heparin therapy characterized by a decrease in platelet count connected to heparin exposure.  Immune-mediated HIT occurs in approximately 3-5% of patients receiving unfractionated or low-molecular weight heparin within 5-14 days of exposure and increases with repeated administration.  Undiagnosed HIT poses a serious risk for thrombotic events, known as heparin-induced thrombocytopenic thrombosis (HITT). HIT develops when heparin forms complexes with platelet factor-4 (PF4) which their immunogenicity has led to the development of commercial test products.  Serotonin Release Assay (SRA) is currently the gold standard for HIT diagnosis; however, results are not consistent as platelet responses to HIT antibodies vary significantly between donors when exposed to the same HIT plasma. 
Methods: Retrospective investigation utilizing clinical samples obtained from 299 patients suspected of having HIT (Average Age 60.8 years, 53.8% male, and 54% ICU patients) sent for testing utilizing LIFECODES PF4 assay (Immucor GTI Diagnostics, Inc., Waukesha WI), remaining samples were aliquoted into 3 vials, stored at -80°C until further testing.  For the purposes of the trial, the 4Ts scoring (Lo GK et al) was blindly and independently scored by two laboratory attendings.  Samples were tested on each LIFECODES PF4; STAGO Asserachrom HPIA-IgG (Diagnostica Stago, Inc., Parsippany NJ); and HemosIL HIT-Ab on the ACL TOP 500 CTS instrument (Instrumentation Laboratories, Bedford MA) assays according to manufacturer’s package insert instructions by the same trained laboratory technician.  All SRA Assays were performed in a single laboratory.
Results: Each commercial kit outperformed the SRA test with a positive cut off of 4T > 4 (Table-1). Analyzing results by 4T Score (Table-2) reveal that two assays LIFECODES and STAGO performing above 88% concordant with scores of 7 or 8, while HemosIL HIT-Ab and SRA were below 24% concordant with these highly clinically scored samples.
Conclusions: The Serotonin Release Assay (SRA) is considered the gold standard for HIT diagnosis; however, responses to HIT antibodies vary significantly between donors.  This retrospective study compared SRA to commercially available tests as well as the 4T pretest probability which demonstrate less than ideal performance.  In addition, this investigation compares the 4T pretest probability to the results of commercially available assay methodologies demonstrating the clinical performance of the various assays with some outperforming others.      

Evaluating The Use Of Age-Adjusted D-Dimer Cutoffs In A Healthy Donor Population
Audrey Sato1, Kelly Townsend2, Richard A. Marlar1, Marian A. Rollins-Raval1
1Dept. of Pathology, University of New Mexico , Albuquerque, NM, United States, 2TriCore Reference Laboratories, Albuquerque, NM, United States

Introduction: Recently, the American Society for Hematology (ASH) published guidelines for exclusion of pulmonary embolism (PE) using an age-adjusted D-dimer cutoff (AADC) in outpatients >or=50 years with low/intermediate clinical pretest probability (LPTP). Currently, per FDA-approved manufacturer package insert, we use the cutoff < 500 ng/mL FEU (fibrinogen equivalent unit), with LPTP, to exclude venous thromboembolism (VTE). We evaluated the use of AADC from healthy donor population D-dimer reference range validation values.
Methods: Our core laboratory performed HemosIL D-dimer HS 500 assay using ACL TOP coagulation analyzers on normal healthy donor citrated plasma samples (N=132; age and gender available) for reference range validation. Ten additional sites within our system using ACL TOP instruments analyzed >or=20 samples each to verify the established reference range.
Results: Our study included 334 healthy donors (18-80 years, 1:1 female:male). Females had higher mean D-Dimer levels (ng/mL FEU) than males (276 vs. 256, respectively; p=0.002). D-Dimer levels increased slightly with age (Figure 1)(Spearman r=0.24;p=< 0.0001). Mean D-Dimer level was only slightly higher overall in donors >or=50 (n=84;mean 270;range 41-1057) than in those < 50(n=250;mean 265;range 8-4481)(p=0.01). By Kruskal-Wallis analysis between decades, only 4th (p=0.007) and 6th (p=0.018) had significantly higher D-Dimer compared to 2nd.  Twenty-one donors had D-Dimer levels >or=500, (15/250(6%) < 50 vs. 6/84(7%) >or=50 years old(p=0.8); only one of the latter was below AADC (Figure 2, starred data point). The number of older donors with elevated D-Dimer in whom additional testing would have been performed to rule out PE decreased from 6 donors (if evaluated by FDA approved cutoff) to 5 (using AADC)(p=1.0).
Conclusions: There is a weak correlation between increasing age and D-dimer levels, but a subset of normal donors will be above the FDA-approved VTE exclusion cutoff regardless of age. An AADC for Hemosil D-Dimer HS 500 assay in our normal population would not have significantly precluded additional testing for PE exclusion. This study is limited by the paucity of normal donors >or=70 years and demonstrates the challenge of finding an adequate number of healthy older donors for evaluation of AADC. Further studies are indicated to evaluate the utility of AADC in our population with this assay.

Awareness For Diagnosis By Molecular Testing As An Option For Thalassemia Free Pakistan
Tanzeel Imran, C.L Hearteveld
jamila sultana foundation for thalassemi, rawalpindi, Pakistan

Introduction: Thalassemia is the most prevalent inherited blood disorder in Pakistan with more than 5000 new cases every year. The purpose of this study was to assess the frequency of affected individuals by molecular testing after normal HPLC reports in Jamila Sultana Foundation Pakistan. 
Methods: Six patients were selected over 10 months after the designed questionnaire including demographics, normal serum ferritin  and  HPLC results. Two CVS samples were analysed by ARMS while four samples of peripheral blood were analyzed by Vieena  Thalassemia Strip Assay by reverse Hybridization. All samples were out sourced as a pilot study.
Results: We diagnosed thalassemia major in both our CVS patients (100%) and were offered termination of pregnancy .While to our surprise we found 2 patients with alpha thalassemia one homozygous for 3.7 mutation  while other  alpha 2  cd 59 heterozygous. Beta thalassemia  IVS 2.745 (C>G) Heterozygous was identified in one patient while last patients had normal molecular study.
Conclusions: Awareness regarding diagnosis by different molecular  modalities  of selected high risk patients should be mandatory at national level in developing country like Pakistan. As we can miss thalassemia diagnosis by route available tests i.e CBC morphology, serum ferritin and HPLC To minimize risk of new cases national health policy must be updated for budgeting as prevention is better than cure. 

Reference Intervals For Classic And New Parameters Of The Hemogram In Healthy Adults Living At Two Different Altitudes In Colombia
Silvia Sanchez
Clinica Colsanitas S.A., Bogot, Columbia

Introduction: The Clínica Colsanitas clinical laboratory in Colombia has many facilities at different altitudes around the country. Currently, the laboratory uses the same hematological reference intervals (RI) for classical parameters in low and high altitudes. The laboratory also lacks RI for novel parameters. To suggest hematological reference intervals of novel and classical parameters for Colombian adults at two different altitudes (2600 and 18 MAMSL).
Methods: In this preliminary approach samples were obtained from 143 volunteer blood donors ages 18 to 64. The analysis was performed on a Sysmex XN-10 instrument. 95% reference intervals were calculated in excel 2016 for RetHe, IRF, #Re, %Re, IPF, IG%, IG#., following the CLSI guidelines EP28 – A3c. 
Results: The RI obtained in this preliminary report were: RetHe: 31,4–39,1pg; IRF: 1,8–12,7%; Re#: 0,048–0,146x106/ml; Re%: 1–2,45%; IPF: 1,1–8,9%; IG%:0,2–0,8%; IG#:0,01–0,08x103/ml. For most of the parameters, the 2.5th and the 97.5th percentiles calculated in this study are similar to those suggested by one or two studies reviewed. However, in the case of Ret# and Re% they differ from those suggested by the mentioned authors.
Conclusions: In general, the new RI obtained in this preliminary approach are similar to those currently used by the laboratory. However, some differences in parameters such as IG%, RetHe, and IRF were found as well as significant differences between the RI for reticulocytes proposed by our team and those from the studies reviewed. The final report would provide RI for 23 parameters and would show the differences between altitudes, ages, and genders, which would help guide better diagnosis and treatment decisions.

New Kid On The Block: An Approach To Implementing A Modernization To Operating Coagulation Laboratories.
Dashini Pillay
National health laboratory Services, Durban, South Africa

Introduction: The aim of this project was to develop and implement an approach to overseeing 30 provincial coagulation laboratory institutions, including identifying the challenges starting new assays and implement changes as required. Part of the new challenge was to implement and verify the anti-factor Xa assay (AFXa) testing for the monitoring of low molecular weight heparin (LMWH) due to the demand from clinicians for on-site testing of this analyte. 
Methods: This was a prospective descriptive study of the implementation of 29 coagulation laboratories at small outlying hospitals and one central laboratory. Baseline data which included scope of current coagulation testing and final optimum testing, internal quality control procedures and external quality assurance (EQA) performance were collected. After the review of baseline EQA results, a root cause analysis was performed in all laboratories with poor EQA performance. The AFXa assay was verified on the Sysmex CS2500i at the central laboratory, using commercial controls for precision studies and 28 plasma samples from patients on LMWH were tested in the validation studies.
Results: The central laboratory and 7/29 (24%) peripheral laboratories had automated coagulation analysers. Whereas 22/30 (73%) laboratories were performing manual PT and APTT. The common causes for the poor EQA performance, their root cause analysis and corrective action are illustrated in Table 1.   Table 1: Reasons for poor EQA performance
Reason for poor EQA performance Root cause analysis Corrective action
Incorrect method registration All laboratories registration information was reviewed. All laboratories were registered with the proficiency testing scheme (PTS) for correct method.
Non-returns There was a delay in shipping of EQA sample from central site. A timeline was compiled for all laboratories to ensure receipt of EQA samples in time to submit to the PTS deadline.
Manual PT and APTT showed intermittent failure, depending on technical staff analysing sample Difficulty reconstituting samples and performing manual PT and APTT Staff members were retrained and competency records completed.
Positive bias observed on trend analysis for PT and APTT internal quality controls. Inconsistent application of Westgard rules on Levi Jennings (LVJ) charts Monthly meetings were held with senior technologist to review LVJ charts and troubleshoot causes and solutions for trends/bias
  The EQA performance over the next 6 months improved and was within the acceptable performance range for the EQA program.
Conclusions: Ongoing training and competency of technical staff is essential to maintain a quality management system at small provincial hospital coagulation laboratories. A team effort is required to ensure continuity of laboratory processes in the background of a high turnover of staff. 

The Prognosis Predictive Value Of Fms-Like Tyrosine Kinase 3- Internal Tandem Duplications Mutant Allelic Ratio (Flt3-Itd Mr) In Patients With Acute Myeloid Leukemia Detected By Genescan
Qin Zheng1, Mengyuan Lyu1, Hongyan Liao1, Xiao Shuai2, Yongmei Jin1, Jun Su1
1West China Hospital of Sichuan Universit, Chengdu, China, 2Department of Hematology, West China Hospital of Sichuan University, Chengdu, China

Introduction: FMS-like tyrosine kinase 3-internal tandem duplications (FLT3-ITD) confers poor prognosis for patients with acute myeloid leukemia (AML). This study aimed to explore the value of FLT3-ITD mutant allelic ratio (MR) in prognostic evaluation of AML patients (AML-M3 patients and non-M3 patients) and further identify the cut-off values of FLT3-ITD MR.

Methods: A total of 249 Chinese AML patients were enrolled in this study. Genomic polymerase chain reaction was carried out to amplify exons 14 and 15 of the FLT3 gene. The FLT3-ITD MR was detected by GeneScan, and mutation types were further determined by single nucleotidesequencing.AML patients were grouped into high- and low-ratio FLT3-ITD AML (FLT3-ITD MRhighand FLT3-ITD MRlow) applying the median of FLT3-ITD MR as the cut-off ratio, and patients without FLT3-ITD were grouped as FLT3-ITD-wild type (FLT3-ITD-wt). Duration of complete remission (CR), relapse rate (RR) and overall survival (OS) were examined.
Results: FLT3-ITD was detected in 58 of all AML patients. The FLT3-ITD MR ranged from 0.04 to 0.83 (median for all patients, 0.31; median for M3 patients, 0.36; median for non-M3 patients, 0.29). The CR rate and death risk of FLT3-ITD MRhighgroup was 0.194 times (95% CI: 0.093-0.404; p < 0.001) and 2.675 times (95% CI: 1.726-4.146; p < 0.001) higher than that of FLT3-wt group, 0.102 times (95% CI: 0.027-0.378; p < 0.001) and 1.879 times (95% CI: 1.061-3.328; p = 0.031) higher than that of FLT3-ITD MRlowgroup, respectively. For non-M3 patients, the OS of both FLT3-ITD MRlow and MRhighgroups (median, 9.5 months; range, 1.00-74.00 months for the former and median, 10.00 months; range, 1.00-38.00 months for the latter) were significantly shorter than FLT3-wt group (median, 35.00 months; range, 2.00-92.00 months). Furthermore, FLT3-ITD MRhighgroup had higher death risk than FLT3-wt group (hazard ratio: 3.301, 95% CI: 1.423-7.661; p = 0.005). However, comparable OS, RR and OS were identified among the groups of M3 patients. 
Conclusions: FLT3-ITD MR was an independent prognostic factor for CR and OS in AML patients (cut-off value: 0.31), and for OS in non-M3 patients (cut-off value: 0.29). Classifying risk grades based on FLT3-ITD MR is more appropriate from the perspectives of individualized treatment and prognosisevaluation.

Friday, May 10, 2019

7:00 - 8:00 AMEast Meeting Room 2
NASCOLA Breakfast workshop: Interesting cases in hemostasis and thrombosis

Chair(s): Dot Adcock
7:00 - 8:00 AMEast Meeting Room 8
How to get your Work Published

Chair(s): Tracy George, Szu-Hee Lee, Giuseppe dOnofrio
8:00 - 9:00 AMEast Ballroom BC
SPECIAL PLENARY: Making Evidence Based Decisions in Healthcare/Choosing Wisely

Chair(s): Ruth Padmore
Reducing Daily Blood Work
Ian Chin- Yee
University of Western Ontario, Canada

Ash Choosing Wisely For Hematology Laboratory Testing
Lisa Hicks
St. Michael's Hospital, Canada

Choosing Wisely is an international campaign that challenges health care workers to identify and stop doing tests, treatments or procedures that are unnecessary.  Choosing Wisely was originally spearheaded by the American Board of Internal Medicine Foundation in the United States and has subsequently spread to countries around the world. The American Society of Hematology (ASH) joined the campaign in 2013 with the release of its first list of five hematologic tests and treatments to question. I will review the method and content of the ASH Choosing Wisely Campaign and highlight recommendations of special interest to lab hematology. Efforts by ASH and other groups to encourage implementation strategies targeting overuse will be discussed. Overutilization of laboratory tests is an ideal target for implementation efforts because overutilization of lab tests is common, because there is evidence that some current testing patterns are unhelpful, because changing practice in this realm is feasible, and most importantly because lab medicine has high awareness of overutilization and an interest in tackling it.  Examples of successful projects addressing overutilization of hematology lab tests will be discussed.      

9:00 - 10:30 AMEast Ballroom BC
CONCURRENT 6: Update on Hemostasis Testing including the Evaluation of Individuals on Therapy

Chair(s): Stefan Tiefenbacher, Kandice Marchant
Doacs And The Laboratory: Measurement And Interference
Robert Gosselin
University of California Davis, Hemophilia Treatment Center, USA


The first direct oral anticoagulant (DOAC) to be approved for clinical use was dabigatran,


a direct thrombin inhibitor, in 2010. Since that time, four additional DOACs,


all direct anti‐Xa inhibitors, have been approved, including rivaroxaban, apixaban,


edoxaban and betrixaban. Our knowledge about the effect of DOACs on laboratory


testing, as well as the use of the laboratory for measuring DOACs has been an evolving


process. These drugs are not routinely monitored in the same fashion as coumadin,


but there is an increasing demand on the laboratory to have the capacity to


adequately assess DOAC anticoagulant effect (pharmacodynamics) or levels (pharmacokinetics)


in either emergent or the routine situations. This presentation provides


an update on laboratory guidance and progress of methods for measuring DOACs.


Oral Anticoagulant Monitoring: Are We On The Right Track?
Pall Onundarson
National University Hospital of Iceland

Vitamin K antagonists (VKAs) cannot be administered without regular monitoring in order to assure their efficacy and safety. Indeed, if well managed, the VKAs appear to be no less efficacious or safe than the newer direct oral anticoagulants (DOACs). Although it is claimed that no regular monitoring of the DOACs is needed, their levels are increasingly being measured under a variety of circumstances, e.g. prior to surgery, in suspected overdose, to confirm effective reversal, in patients with malabsorption and to assess patient compliance. Although no therapeutic range has been identified for the DOACs, it has been demonstrated for dabigatran and edoxaban that their antithrombotic effect increases gradually with increasing concentrations and that the risk of major bleeding also gradually increases. Furthermore, it has been determined that almost all dabigatran related thrombotic events occur in patients with the lowest quartile concentration of the drug. This suggests that to assure an ideal effect of DOACs in all patients taking them, some form of regular monitoring and dose tailoring should be performed. For the vitamin K antagonists, the best outcome is obtained using formal algorithms and centralized management. Furthermore, data suggests that replacing the standard prothrombin time as a monitoring test may increase the stability of VKA anticoagulation with consequent reduction in thromboembolism without an increase in bleeding. Thus, it is likely that the outcome of all current oral anticoagulants can be improved in the coming years by improving monitoring and tailoring their effect.

Laboratory Testing For New Hemophilia Treatments
Stefan Tiefenbacher
LabCorp, USA

This presentation will provide an overview of the common coagulation assays used in clinical laboratories to monitor novel hemophilia A and B therapies, including those currently in development or recently approved.  The challenges facing clinical laboratories when trying to measure the efficacy of these treatments using existing coagulation assays will be discussed.  Examples of observed assay differences for particular therapies and associated challenges that may be encountered by the clinical laboratory for individual therapies will be presented.  The novel hemophilia treatments reviewed will include extended half-life (EHL) factor VIII and factor IX replacement therapies, hemophilia A (factor VIII) and hemophilia B (factor IX) gene therapies, as well as some of the novel target therapies (e.g. bispecific antibody mimicking activated FVIII).  Approaches to achieve accurate monitoring for some of the currently approved hemophilia therapies will be presented.

9:00 - 10:30 AMEast Ballroom A
CONCURRENT 5: Joint Session with ICCS

Chair(s): Mike Keeney, John Frater
Minimal Residual Disease Analysis In Acute Myeloid Leukemia
Shaoying Li
MD Anderson Cancer Center, USA

Approximately 80-90% of patients with acute myeloid leukemia (AML) can achieve a morphological complete remission (CR) (defined by morphologic bone marrow blast count <5%, absolute neutrophil count >1.0 x 109/L) after current standard chemotherapy. However, many patients who originally achieve a morphological CR will eventually relapse. It is well-known that morphological assessment is neither sensitive nor specific in the detection of residual leukemic cells after chemotherapy. The presence of submicroscopic leukemic blasts in the setting of morphological CR is generally called “minimal residual disease” (MRD) which contributes to ultimate leukemia relapse. Several lab techniques are useful for the detection of minimal residual disease in AML, mainly including multi-color flow cytometry (MFC) analysis and molecular techniques (PCR, next generation sequencing). A lot of studies in the literature have shown that a positive MRD detected by MFC study after induction/consolidation or prior to allogeneic hematopoietic stem cell transplantation is correlated with an increased risk of relapse and a poor survival. MRD detected by MFC is an independent prognostic factor in patients with AML. Here I am going to share our institution’s experience in AML-MRD detection by using MFC, including antibody panel design, data analysis and interpretation, and some diagnostic pitfalls.  The aim is to provide some practical information for pathologists or hematologists who are interested in AML-MRD detection by MFC.

Acute Myeloid Leukemia Flow And Clinical Perspectives
Roland Walter
Clinical Research Division, Fred Hutchinson Cancer Research Center

Role Of Flow Cytometry In Immunodeficiency
Vijaya Knight
Children's Hospital, Colorado, USA


Primary immunodeficiencies (PID) or in-born errors of immunity affect phenotype and/or function of one or more components of the immune system. The exponential growth of genetic analysis has enabled identification of known and novel mutations. However, genetic analysis continues to be expensive and is not easily accessible. Flow cytometry, on the other hand, has been well established for many years in clinical laboratories and it’s use for the analysis of immunodeficiencies is expanding. Surface, intracellular and intranuclear proteins can easily be evaluated by flow cytometry, enabling both phenotypic and functional identification of specific cell populations and therefore facilitating the identification of a variety of PIDs. While genetic analysis provides a definitive diagnosis for PIDs, flow cytometry is necessary to confirm or establish the immune phenotype of a gene mutation. Furthermore, flow cytometry provides a rapid means to identify an immunological defect at a relatively low cost.

10:30 - 11:00 AMEast Exhibit Hall B
Coffee Break

11:00 - 12:00 PMEast Ballroom BC

Chair(s): Albert Huisman, Richard Marlar, Cathy Ross
A New Image-Based Machine-Learning System (Cellsimatic) For The Automatic Recognition Of Hematologic Neoplasia Versus Infections In Peripheral Blood
Anna Merino1, Santiago Alfrez2, Laura Bold1, Angel Molina1, Laura Puigv2, Andrea Acevedo2, Jose Rodellar2
1Hospital Clinic., Barcelona, Spain, 2Technical University of Catalonia, , Spain

Introduction: The objective is to design and evaluate a machine-learning software (CellsiMatic) to operate in real-time as follows: 1) the input is a set of pathological images of an individual peripheral blood smear (PBS); 2) the outcome is the prediction of one of the following diagnosis: lymphoma, acute leukemia (AL) or infection.
Methods:   In a first stage, we designed and tested a full computational procedure implementing automatic image segmentation and classification using 667 descriptors. In a second stage, we tested this procedure using PBS as a diagnostic unit. Tests consisted in classifying PBS images in: abnormal lymphocytes, blasts, reactive lymphocytes or unknown. After several experiments, a threshold value was established such that the diagnosis is predicted from the cell class with the percentage of images classified above this value. Once the software was developed, we designed a web application for real-time on-site or remote operation. Images are uploaded from any computer or mobile device to a server hosting the software and to receive back the outcome in a timely way. All images in the previous tests and in the further system evaluation were stained with MGG and acquired with CellaVision®DM96.
Results: In the system evaluation, 544 PBS were analysed from 230 patients: 39 with infections, 97 with lymphoma and 94 with AL. These smears contained a total of 21,108 individual cell images. The time for the classification of individual images was 4 seconds. The confusion matrix (Figure 1) summarizes the predicted diagnosis compared to the true diagnosis. All the 43 smears containing reactive lymphocytes were predicted as belonging to patients with infection (100% accuracy). For the smears containing abnormal lymphoid cells, 95% were predicted as patients with lymphoid neoplasm. Finally, 92% of the smears with blasts were predicted as patients with AL. The overall classification accuracy was 95.3%.
Conclusions: CellsiMaticis an efficient software system for diagnosis prediction in patients with acute leukaemia, lymphoma or infection using digital images. On-site or remote implementation is feasible in terms of operation and calculation time, and may be a straightforward tool helping the pathologist for computer assisted diagnosis in the morphological evaluation of PB cells.

Big Flow Cytometry Data Analysis Methods And Applications
Albina Rahim1, 4, Lauryl Nutter2, Ryan R. Brinkman1, 3
1Terry Fox Lab, BC Cancer Agency, Vancouver, BC, Canada, 2The Centre for Phenogenomics, Toronto, ON, Canada, 3Department of Medical Genetics, University of British Columbia, Vancouver, BC, Canada, 4Department of Bioinformatics, University of British Columbia, Vancouver, BC, Canada

Introduction: Even though we have the full sequence of many mammalian genomes, the function of most genes remains unknown. The International Mouse Phenotyping Consortium (IMPC) is creating 20,000 knockout mouse strains, characterizing each strain through a standardized phenotyping protocol including immunophenotyping of spleen and other organs by flow cytometry, generating approximately 77,000 FCS files. We have developed and applied an automated analysis pipeline that (1) Detects batch effects and outliers at both sample and dataset level; (2) Enumerates cell populations using supervised and semi-supervised computational analysis to help understand gene function; (3) Identifies cell populations significantly different between sample groups (knockout lines and wild type controls).
Methods: The pipeline can be divided into pre-processing, quality checking, cell population identification, biomarker identification, and visualization.  Like manual analysis, automated analysis is performed on a per tissue and per panel basis. Here we focus on data from IMPC’s The Centre for Phenogenomics (TCP) group. The dataset is based on a 16 and 20 dimensional panels and applied to 2532 knockout FCS files from 215 knockout mouse lines and 279 wild type FCS files.
Results: This automated pipeline we developed is designed to identify cell populations similar to those found by manual gating and thus high correlations were observed between the two. Figure 1 illustrates the comparison of the automated versus manual result proportions of the Natural Killer (NK) cells of 700 FCS files, collected from 2014 to 2016. The black line in the plot represents the linear regression fit between the automated versus manual.
Conclusions: The automated pipeline can also identify novel biomarkers. Automated gating can offer considerable time savings.The time spent on initial setup of the pipeline is approximately the same for manual and automated analysis. Once this setup is completed, manual gating can take between 30-60 minutes per sample [1]. For automated analysis, computer run time is 5 seconds per sample resulting in the same gates. This represents a time save of 59–117 days for analyzing all panels of TCP.

1. Ivison, Sabine, et al. "A standardized immune phenotyping and automated data analysis platform for multicenter biomarker studies." JCI insight 3.23 (2018)

Detection Of Anti-Domain I Antibodies Enables The Identification Of High-Risk Antiphospholipid Syndrome Patients: Results Of A Multicenter Study
Dongmei Yin1,2, Walid Chayou1,2, Bas de Laat1,2, Philip de Groot1, Gary Moore3, Jean-Christophe Gris4, Stphane Zuily5, Jacek Musial6, Katrien Devreese7, Hilde Kelchtermans1,2
1Synapse Research Institute, Maastricht, Netherlands, 2Cardiovascular Research Institute Maastricht, Maastricht, Netherlands, 3Guy's and St Thomas' Hospitals, London, United Kingdom, 4Centre Hospitalier Universitaire, Nmes, France, 5Centre Hospitalier Regional Universitaire de Nancy, Nancy, France, 6Jagiellonian University Medical College, Krakow, Poland, 7Ghent University Hospital, Ghent, Belgium

Introduction: Classification of the antiphospholipid syndrome (APS) relies predominantly on measuring antiphospholipid antibodies (aPLs), including lupus anticoagulant (LAC), immunoglobulin (Ig)G/IgM anti-cardiolipin (anti-CL) and anti-β2glycoprotein I (anti-β2GPI) antibodies. Especially aPLs against a domain I (DI) epitope of β2GPI are pathogenic in APS. We aimed to investigate the clinical value of detecting anti-DI IgG in a multicenter study.
Methods: From eight European centers 1005 patients were enrolled: 379 APS patients, 237 non-APS thrombotic/obstetric controls, 196 patients with autoimmune diseases and 193 normal controls. LAC was determined by the local center; anti-CL, anti-β2GPI (HemosIL AcuStar™) and anti-DI IgG (QUANTA Flash β2GPI Domain I) by a single technician. To avoid a classification bias, data were analyzed with outcome variable ‘clinically affected versus not-affected’.
Results: Odds ratios (ORs) of anti-DI IgG for clinical manifestations were higher than those of the conventional assays. Upon restriction to patients positive for anti-β2GPI IgG/IgM or anti-β2GPI IgG, anti-DI IgG positivity still resulted in significant ORs for clinical complications (Table 1-2).  When anti-DI IgG was added to the criteria or used as substitute for anti-β2GPI IgG and anti-CL IgG, ORs hardly improved (OR(95%CI) of (2.9(2.2-3.8)/2.7(2.0-3.6) versus 2.8(2.1-3.8) for thrombosis; 5.4(3.6-8.2)/5.0(3.3-7.5) versus 5.2(3.5-7.9) for pregnancy morbidity). Upon removing anti-DI IgG positive patients, LAC remained significantly correlated with clinical complications (OR2.5(1.7-3.5) and 3.9(2.4-6.4) for thrombosis and pregnancy morbidity), in contrast with anti-CL and anti-β2GPI IgG. Most of the anti-DI IgG reactive patients were triple-positive (83%/70% for the thrombotic/pregnancy morbidity subpopulation) and triple-positive patients were characterized by higher levels of anti-DI compared to single/double-positive patients.
Conclusions: Detection of anti-DI resulted in the highest ORs for clinical manifestations in comparison with the current criteria. Although addition of anti-DI to the criteria hardly improved the association with clinical symptoms, detection of anti-DI can be used as a risk stratification tool, identifying high-risk patients.

Myelodysplastic Syndrome With Germline Gata2 Mutation Shows Significant Differences In Clinical, Immunophenotypic And Genetic Alterations In Comparison ToMyelodysplastic SyndromeWith Wild Type Gata2
Alina Dulau-Florea, Weixin Wang, Stephenie Droll, Vollter Anastas, Amy Hsu, Bhavisha Patel, Danielle Townsley, Lisa McReynolds, Christopher Hourigan, Dennis Hickstein, Raul Braylan, Neal Young, Steven Holland, Katherine Calvo
National Institutes of Health, Bethesda, MD, United States

Introduction: Germline heterozygous mutations in GATA2 result in GATA2 deficiency, and confer increased risk of MDS/AML. The distinction of GATA2-deficiency-associated-MDS (GATA2-MDS) from MDS with wild-type GATA2 (GATA2-wt-MDS) is important for therapy and donor selection for hematopoietic cell transplant. In this study we compared clinical, morphologic, immunophenotypic, and molecular genetic features of GATA2-MDS vs. GATA2-wt-MDS.
Methods: 42 bone marrow (BM) samples from patients with GATA2-wt-MDS and 53 BM samples from individuals with germline GATA2 mutations including 38 MDS (GATA2-MDS), 7 with BM and immunodeficiency disorder not meeting criteria for MDS, and 8 healthy relatives, were assessed by morphology, flow cytometry (FC), cytogenetics and molecular analysis using targeted next generation sequencing for somatic mutations in over 50 genes associated with myeloid neoplasms.
Results: GATA2-wt-MDS were predominantly males (27M; 15F) and older (median age 64 years) in comparison to GATA2-MDS (13M; 25F; median age 28.5 years). MDS with multilineage dysplasia was the predominant MDS subtype in both groups. Hypocellular MDS was more common in GATA2-MDS (63.2% vs 33.3%). FC analysis revealed significantly different feaures in GATA2-MDS compared to GATA2-wt-MDS, with nearly absent monocytes (0.45% vs 4.9%; p< 0.0001) and dendritic cells (0.0 vs 0.18%, p< 0.0001). Among lymphocytes were decreased mature B cells (2.27 vs 5.70, p< 0.0001), precursor B cells (0.0% vs 0.43%, p< 0.0001) and NK cells (1.02% vs 10.93%, p< 0.0001). CD34-positive cells were decreased in GATA2-MDS vs GATA2-wt-MDS (0.41% vs 1.19%), particularly CD34-positive B cell precursors (0.01% vs 0.06%). Cytogenetic abnormalities were found in similar proportions in the 2 cohorts (69.2% of GATA2-wt-MDS and 58.3% of GATA2-MDS). The profile of somatic mutations was different: GATA2-MDS had very few splicing factor mutations (2.6% vs 31.25%) and a high proportion of mutations in cohesin complex genes (23.7% vs 3.1%) in comparison to GATA2-wt-MDS. Of note, all cohesin mutations in GATA2-MDS were in STAG2.
Conclusions: FC findings of decreased-to-absent BM monocytes, dendritic cells, NK and B-lymphocytes including B cell precursors; younger age at presentation; paucity of splicing factor mutations and increased proportion of STAG2 mutations, are features seen in MDS with germline GATA2 mutation.

12:00 - 1:30 PMEast Meeting Room 2
Beckman Coulter Luncheon Workshop

12:00 - 1:30 PMEast Meeting Room 8
Sebia Luncheon Workshop

12:00 - 1:30 PMEast Meeting Room 11
Sysmex Luncheon Workshop

2:00 - 3:00 PMEast Ballroom A
Oral Abstract Session: Flow Cytometry

Chair(s): Catherine Ross, Marie Christine Bene
Cbc Parameters Based Derived Predictive Factor For Acute Promyelocyte Leukemia
Rana Zeeshan Haider1, Sabrina Buoro2, Ramon Simon-Lopez3, Erich De Paula4, Tahir S. Shamsi1
1NIBD & BMT, Karachi, Pakistan, 2Referente Qualit Dipartimento di Medicina di Laboratorio Referente Emocitometria UOC SMeL 2 Analisi Chimico Cliniche Azienda Socio Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, , Italy, 3Sysmex Europe, Norderstedt, , Germany, 4Department of Internal Medicine - Hematology, University of Campinas, Campinas, , Brazil

Introduction: Acute promyelocytic leukemia (APML) due to coagulopathies drives hematological distress in clinical practices. A targeted and timely offered treatment can be a benefitting tool for patients. Current round of study made use of potential research parameters (cell scattering intensities) generated as a result of routine hematology analyzers during CBC analysis. This helped in successfully offering pre-microscopic screening of APML patients.
Methods: Blood samples of patients suffering from APML and other hematological neoplasms were collected and analyzed by using hematology analyzer (Sysmex XN-1000) in comparison with control group. For morphological diagnosis peripheral blood films was examined. For detailed CBC parametric analysis, and also intensity values of side, forward, and side florescence light scatter of white blood cells (WBC) was recorded utilizing analyzer software. Statistical and machine learning (unsupervised) tools including principle component analysis (PCA) and heat map pilots (figure-1) helped in the evaluation of predictive capacity of routine and research CBC parameters. This led to development of novel APML factor, which was derived from WBC and platelet count along mean value of neutrophil side fluorescence light (NE-SFL) using logistic regression approach. The proposed cutoff value for this factor is -2.011, at which the prediction of APML was challenged among present study cases.
Results: An exceptionally discriminative value of white blood cell (5.65), platelet (29.5), and NE-SFL (54.4) in APML samples was recorded in comparison to non-APML subjects 7.96, 219, and 47.00 respectively. In combination, it was found that WBC and platelet count led to an increase in the predictive power of NE-SFL when utilized for the derivation of our proposed “APML factor”: -4.5070 + (0.049213 x NE-SFL) + (-0.017431 x PLT) + (-0.0058366 x WBC). At the defined cutoff value (-2.011), this factor can have robust impact in the differentiation on APML with high values for sensitivity (0.935) and negative predictive power (0.985).
Conclusions: This study proposes an APML factor that can be potentially utilized in clinical practice by lab staffs and physicians) to increase the rate of APML diagnosis on time.

Fabrication Of A Microfluidic Impedance Cytometer (Mic) For Cell Counting(Cbc)
Prakhar Jain1, Usama Abbasi1, Sasikala Subramaniam1, Subham Banerjee1, Prasanta Chowdhury2
1MicroX Labs, Society for Innovation Development, Indian Institute of Science, Bangalore, India, 2National Aerospace Laboratories-CSIR, Bangalore, India

Introduction: Microfluidic Impedance Cytometery(MIC) enables probing for electrical cell signatures at multiple high-frequencies when the cells transits across an electric field. We present a novel fabrication technique for such sensors having 3 pairs of parallel facing electrodes across a microchannel resulting in uniform electric field .We further demonstrate enumeration of platelets, RBCs, 3 part differential WBCs in human blood samples without use of sheath fluids and without requirement for position correction algorithms.
Methods: Fabrication of sensor can be divided into three parts (i)Patterning of 3 pair of platinum electrodes (30µm wide at sensing region,30µm inter-separation) on substrate A (glass-wafer) and on substrate B(silicondioxide) (ii) Etching on substrateA to make access holes for fluid flow; patterning of a microfluidic channel (30 µm(h)x 45 µm(w) at sensing region) using lithography on substrateB (iii)Bonding and alignment of both substrates and finally dicing to get the devices. Detection electronics: Custom FPGA lock-in amplifier ,output signal was sampled at 16bit 250 ksps, 50kHz bandwidth Data processing: MATLAB script. Sample preparation: (a)RBC and PLT -dilution of blood in 1:10,000ratio (b)WBC- blood to lysing to quench 1:12:5.65ratio . For sensitivity analysis a mixture of 3µm, 4µm and 5µm PS beads with known concentration was flown and CV was computed. Blood samples were procured from healthy donors
Results: Sensor(figure 1A) characterization was done for 50 hours by continuous flow of 1x PBS. Sensors can withstand 1.5 bar pressure without delaminating;withstand 24Vpp without damaging electrodes. Part B:(Fig. 1B) .Sensor is able to clearly separate mix of 3µm, 4µm , 5µm beads.Part C:(Fig. 2A): Sensor is able to enumerate RBC and PLT at applied frequency of 500 kHz at 20Vpp.Part D:(Fig. 2B): Enumeration for 3 part WBCs with applied frequency a mix of 500 kHz and 2 MHz @10Vpp. Process parameters: All flow rates at 40 µL/min with a +ve displacement pump
Table 1:Summary  from our MIC sensor vs  from Sysmex 2100
Cell type Number of samples Correlation-R2
RBC 30 0.961
PLT 30 0.970
LYMP 30 0.989
MON 30 0.981
GRN 30 0.984

Conclusions: MIC sensors enable a full CBC panel ,if coupled with automated sample preparation on disposable cartridges can enable solution for CBC at Point of care.

Performance Of 8-Color Dry Reagent Tube For Mrd Detection In Chronic Lymphocytic Leukemia By Flow Cytometry
Rodolfo Patussi Correia, Laiz Cameiro Bento, Rodrigo de Souza Barroso, Nydia Strachman Bacal
Hospital Israelita Albert Einstein, So Paulo, Brazil

Introduction: The detection of minimal residual disease (MRD) in chronic lymphocytic leukemia (CLL) has presented great improvements in methods of detection and in clinical/therapeutic approaches. Regarding flow cytometry (FC) method, the use of pipetting-free antibody staining procedures, such as dry antibodies cocktail (DuraClone), is an example of this advances. In this study, we demonstrated the performance of the DuraClone tube in MRD detection of CLL by FC in comparison with the traditional and standardized method that uses liquid monoclonal antibodies.
Methods: The commercial 8-color DuraClone tube, named RE CLB Tube from Beckman Coulter, is composed by CD81-FITC, ROR1-PE, CD79b-PC5.5, CD19-PC7, CD5-APC, CD43 APC-AlexaFluor750, CD20-PB and CD45-KrO. For standardized 9-color liquid reagent tube we used CD43-FITC, ROR1-PE, CD3-ECD, CD5-PC5.5, CD20-PC7, CD79b-APC, CD19 APC-AlexaFluor750, CD81 APC-H7 and CD45-KrO. The liquid and dry tubes were applied in 18 MRD positive CLL samples: 10 peripheral blood, 6 bone marrow and 2 external quality control from UKNEQAS program. The total volume of sample required for MRD evaluation, which ranged from 300µL to 2000µL, was lysed using VersaLyse for DuraClone and ammonium chloride for liquid reagent, concentrated in 300µL, stained with monoclonal antibodies, lysed again if necessary, washed and resuspended. Samples were analyzed in Navios Flow Cytometer and Kaluza software. The CLL MRD frequency was used for comparison between both methods.
Results: The statistical correlation between liquid and dry reagents was good and acceptable, R2=0.953. The average of total number of acquired events in DuraClone was 750,645 (363,632 to 2,290,387) that allowed good sensitivity for FC assay. It was possible to detect MRD in all CLL samples evaluated by both methods. The lowest MRD frequency detected by DuraClone was 0.01% corresponding a cluster with 106 events in a total of 737,030. In addition, the use of DuraClone demonstrated satisfactory performance in external quality control samples. Furthermore, the Kaluza Radar tool allowed the easily discrimination of the CLL cells from the normal population.
Conclusions: It is well established that the use of DuraClone in clinical flow cytometry laboratories increases quality, productivity, avoid pipetting error, reduces cost and allows for standardization. Together, these improvements in workflow could promote rapid, sensitive and specific evaluation of MRD in CLLmonitoring patients.

Flow Cytometry Based Study Of Crlf2 Expression In B-Cell Acute Lymphoblastic Leukemia
Gautam Bhagwat1, Subhajit Brahma1, Sambhunath Banerjee1, Arun SR2, Sundar Shewale1, Rishu Vidhatri1, Tanvi Gupta 2, Mayur Parihar2, Neeraj Arora3, Deepak Mishra3
1Department of Laboratory Hematology, Tata Medical Centre, Kolkata, India, 2Department of Laboratory Hematology and Cytogenetics, Tata Medical Centre, Kolkata, India, 3Department of Laboratory Hematology and Molecular Genetics, Tata Medical Centre, Kolkata, India

Introduction: Cytokine receptor–like factor 2 (CRLF2) gene is located in the pseudo autosomal region 1 (PAR1) at both sex chromosomes, X (Xp22.3) and Y (Yp11.3). CRLF2 genomic rearrangements are found in approximately 50% of Ph-like B-cell acute lymphoblastic leukaemia (B-ALL). CRLF2 rearrangements are often associated with its overexpression and can be detected by flowcytometry using the antibody against TSLPR - thymic stromal lymphopoietin receptor (TSLPR) and by real time PCR.  We share our experience of evaluation of CRLF2 expression using the flowcytometry based assay.
Methods: A prospective CRLF2 expression analysis using BV510 mouse antihuman TSLP receptor (BD horizonTM) was done in 96 consecutive cases of B-ALL (82 diagnostic, 14 relapsed) by flow cytometry from February 2018 to December 2018. Karyotyping and Interphase FISH analysis using CRLF2 gene break-apart probe was carried out in cases which had CRLF2 overexpression (cut-off >10%).
Results: Of the total 96 cases evaluated (age range 1 year to 75 years, Male: Female ratio 1.7); 14.6% cases (n=14) overexpressed CRLF2. Majority of the cases (11/14) had CRLF2 expression >50%. These 14 cases were diploid (n=9) and hyperdiploid (n=4) by flow ploidy. Eleven cases were diagnostic (13.4%, 11/82) whereas three cases presented as relapsed B-ALL (21.4%, 3/14). Overall In adults (>15 yrs) 23% (7/31) cases had CRLF2 overexpression (Diagnostic 20.8%; 5/24) while in paediatrics CRLF2 overexpression was seen in 11% (7/65) B ALL cases (Diagnostic 10.3%; 6/58). Cytogenetic analysis performed in 13/14 cases revealed CRLF2 rearrangement in three cases. The rest ten CRLF2 FISH negative cases included hyperdiploid (n=3),c-myc rearrangement(n=2), t(1;19)(n=1), t(9;22)(n=1) and KMT2A gene rearrangement(n=1). Normal karyotype was found in remaining two cases. Post induction (day +28-35) MRD status was available for nine newly diagnosed cases and 55.5% (5/9) had a positive MRD. Two adult cases died within 1 month of the diagnosis.
Conclusions: Flowcytometry based analysis of CRLF2 over expression is rapid, inexpensive and simple technique in triaging the cases of B-ALLs to further investigate for CRLF2 rearrangements by other techniques. CRLF2 by flowcytometry can be included in diagnostic algorithms of B-ALL as a screening tool for further investigation.

2:00 - 3:00 PMEast Meeting Room 2
Oral Abstract Session: Cellular & Morphology

Chair(s): John Frater, Albert Huisman
Evaluation Of An Automated Bone Marrow Slide Scanning Unit (Part 1)
Kathleen Deiteren, Marie-Berthe Maes, Jan Van den Bossche, Ronald Malfait
University Hospital Antwerp, Edegem, Belgium

Introduction: Counting bone marrow (BM) cells is very time consuming and requires highly trained individuals. We evaluated an automated BM slide scanning system, the Vision Hema Bone Marrow 4Pro (Vision Hema). Our main goal was to save time but maintain the same high standard as with manual microscopy.
Methods: The Vision Hema (West Medica) first scans the BM slide (10x). Regions of interest (ROI’s) are automatically selected but can be adapted manually. In a second scanning step (100x) cells are counted and pre-classified automatically with the digital microscope preset to count 550 cells. The pre-classification was reviewed by a clinical pathologist. The aim of the study was to 1) investigate whether there is a difference between the ROI’s selected by the software or manually (n=94) by comparing the 1a) total number cells counted, 1b) % artifacts 1c) myelogram (after reviewing) with manual microscopy (correlation coefficient, Passing-Bablok regression analysis, Wilcoxon test), 2) evaluate the speed of the system.
Results: 1a) The total number of counted cells is less for BM samples with ROI’s selected automatically compared to manually (n = 94)) (p = 0,018). In 81,5% or 76,0% of BM samples ≥300 cells are reached after reviewing the manual or automatic ROI selection respectively. 1b) Also, the % artifacts is higher for samples with ROI’s selected automatically (n = 94) (p=0,0006). Samples with < 200 counted cells (n = 15) are excluded from further analysis. 1c) We found no difference in correlation coefficient (p=0,28) or slopes (regression analysis) (p = 1) between BM samples with ROI’s selected by the software/manually (n = 79) compared to microscopy. Slopes are 0,66 - 1,39 (0,99 for blasts). Manual selection remains superior when the BM is focally infiltrated by malignant cells (e.g. plasma cell neoplasm) or when the BM is very fatty with small ROI’s. 2) Median scan time (10x) is 2 min 28 s (n = 84). 94,5% of the BM samples are counted and pre-classified (100x) automatically within 15 min (n = 237).
Conclusions: The Vision Hema is a fast instrument to count BM cells. The ROI’s selected by the software are satisfactory in most cases. However less cells and more artifacts are counted when accepting ROI’s selected by the software. To reach ≥500 counted BM cells the number of selected ROI’s (automatically or manually) will have to be increased.

Distinction Between Myelodysplastic Syndrome And Nonclonal Cytopenias By Cellular Morphometric Analysis.
1Core-Hematology Department. Clinical Laboratory Metropolitana Nord (LCMN). Hospital Universitari Germans Trias i Pujol, BADALONA, Spain, 2Hematology Department. Institut Catal dOncologia. Hospital Germans Trias i Pujol., BADALONA, Spain

Introduction: Myelodysplastic syndromes (MDS) are clonal diseases of the hematopoietic stem cells that are characterized by cytopenia and dysplasia in peripheral blood. The diagnosis of morphological dysplasia is complex, subjective, presenting a high interobserver variability. Besides MDS, different pathologies can present cytopenias and differentiating between a benign and a malign pathology is necessary. The complete blood count(CBC) and differential leukocyte analysis allow the detection of cytopenias reliably. In recent years, hematology analyzers provide information about cell morphology through new population’s parameters. These morphology parameters (CMPs) could be useful in early MDS diagnosis. The aim was to assess diagnosis efficiency of CMPs in MDS diagnosis and compared it with benign cytopenias.
Methods: 102 patients were included in the study and divided in 3-groups:23-MDS, 48 patients with hepatopathy(HP) and 31 with chronic renal disease(CRD). The inclusion criteria were: total leucocytes< 4,0x109/L and cytopenia (hemoglobin< 10g/dL, neutrophils< 1,8x109/L and/or platelets< 100x109/L). All samples were run on the DxH-800 Analyzer (Beckman Coulter,  Miami, FL,USA). We obtained the CMPs of leucocytes: volume(V), conductivity(C) and different angle laser dispersion (SM, SU, SL, SA and AL2). The means differences of CMPs were compared by ANOVA-test. Significant p-values were those< 0.05. The diagnostic capacity to identify correctly the MDS with cytopenia was assessed with ROC curve analysis.
Results: We found statistical difference in neutrophil SU (MDS:132.5±8.0 vs HP:140.0±4.8 vs CRD:137.7±6.3;p< 0.001) and SL (MDS:127.6±7.4 vs HP:137,0±7,9 vs CRD:132.4±11.7; p< 0.001) but not statistical in V and C (table-1). Neutrophil SU and SL angle laser dispersion showed an AUC of 0.782 (IC:0.661-0.903;p< 0.001) and 0.775 (0.671-0.879;p< 0.001), respectively. The MDS index [(SU-NEU + SL-NEU) x NEU%/100] showed the best efficiency in distinguishing MDS-cytopenia from benign cytopenia with AUC:0.911 (IC:0.848-0.975;p< 0.001)(table-2).  
Conclusions: Many pathologies present cytopenia and detecting MDS is essential. The combination of neutrophils count with SU and SL dispersion angle could be useful in the identification of MDS. The MDS index showed the highest diagnostic efficiency differentiating between malignant and benign cytopenias, especially when white cell count is lower.

Machine Learning Algorithms For The Detection Of Spurious White Blood Cell Differentials Due To Erythrocyte Lysis Resistance
Laura Bigorra1,2, Iciar Larriba1, Jordi Huguet1, Ricardo Gutirrez-Gallego2
1Synlab Global Diagnostics, Barcelona, Spain, 2Pompeu Fabra University, Barcelona, Spain

Introduction: Red blood cell (RBC) lysis resistance results in overlapping regions in the impedance histogram between RBC and white blood cells (WBC) for the smallest volumes (lymphocyte subpopulation), interfering with WBC count and the differential, phenomenon not clearly adverted by the Beckman Coulter DXH 800 instrument.
Methods: The WBC count, addressed by impedance and optical measures, in the DXH 800, the WBC differentials and the Cell Population Data (CPD) obtained through the Volume-Conductivity-Scatter technology from 100 healthy controls (HC) were compared to 232 patients with RBC lysis resistance (anemia or liver disease). Corrective actions to the RBC lysis resistance included sample dilution with saline solution. WBC and CPD were compared from pre and post-diluted samples, and the differentials with the obtained using the CellaVision DM 1200 and ANOVA. Then, a Machine Learning (ML) model was built for identification and classification using lymphoid CPD.
Results: We identified 1) That impedance WBC are not affected by the RBC lysis resistance interference, 2) the abnormal scattergram pattern for both pathological categories with dual or dispersed lymphoid populations, 3) that the differentials obtained by the DXH 800 overestimated lymphoid subpopulations when RBC lysis resistance occurred (~ 20%) and it is corrected when sample dilution was performed, 4) that all 14 CPD displayed significant differences (p ≤0.05) among the three categories compared when using ANOVA, being such differences suppressed for the volume, the mean upper median and the mean low light scatter angles when the corrective action was carried out. The ML, support vector machine (SVM) algorithm allowed the classification of the three groups with validation accuracies corresponding to 97.98%, 100% and 88.78% for the global, anemia and liver disease groups, respectively. ML classification of diluted samples indicate that these are indistinguishable from HC in 72.64% of the pathological cases, as part of the performed corrective action.
Conclusions: The SVM-ML algorithm has an impressive discriminatory potential and its application would be a valuable support system to detect this phenomenon. The impedance WBC counts are not affected by the RBC lysis resistance and the differentials and CPD from RBC lysis resistant samples can be easily corrected by sample dilution, being also indistinguishable from HC in terms of classification in the 72.64% of the cases.

Peripheral Blood Films: Adding More Value To An Important Value-Added Test
Karen Dallas
Providence Health Care, Vancouver, BC, Canada

Introduction: The Peripheral Blood Films (PBF) remains an important tool in the diagnosis, management & monitoring of a host of medical conditions. PBF reviews can be ordered by clinical physicians or, more commonly, can be reflexively generated by the Laboratory after the Hematology analyzer encounters an abnormality in the CBC data. 
Unfortunately, many clinical physicians do not know that the Laboratory reflexively generates PBFs and thus, they are not expecting or looking for a Hematopathologist report and valuable clinical information may be missed. This is not only a very real patient safety risk, but it also decreases the clinical value of the important work we do in the Laboratory every day and it most likely results in unnecessary re-work.
Methods: We employed a stepwise approach (PDSA) to this Quality Improvement endeavor, starting with an informational e-survey of Emergency Medicine, Internal Medicine and Critical Care physicians. We then used small tests of change to implement automatic rules notifying clinical teams about when to expect a PBF review and Pathologist report.  The main Outcome metric was number of physician-ordered PBFs generated each month (which is really a surrogate marker for clinical awareness) Process metrics include what percentage of the time the rules are applied. And finally, Balancing metrics included complaints from physicians ordering PBFs and any increase in Clinical Hematology consultations based upon PBF reviews.
Results: Not surprisingly, 47% of clinical physicians surveyed did not know that the Laboratory auto-generated PBFs. But importantly, all but one respondent felt that they system could work better together to improve patient care.  During the course of this project we decreased the number of physician-ordered PBFs from 136/month to 22/month which has been sustained. This decrease translates to an annual cost-avoidance of over $10,600 (not including Pathologist time).
Conclusions: Oftentimes in Healthcare we work in silos, unaware of important work that other clinical areas are doing, and convinced that improving care will require extra resources/money. This PBF project is a great example of how we can work better together for the benefit of patient care and not costing the system extra money.  Additionally, it provides a simple framework for how another institution might go about introducing and implementing a similar PBF standard notification process. This would prove beneficial especially as many Laboratories are searching for cost avoidance strategies. 

2:00 - 3:00 PMEast Meeting Room 8
Oral Abstract Session: Molecular, Red Cell & Standards

Chair(s): Imran Mirza, Giuseppe d'Onofrio
Optimization Of Malaria Diagnostic Algorithm Using Rapid Lamp Screening AssayWithin A Regional Health Care Setting
1Vancouver General Hospital, Vancouver, BC, Canada, 2University of British Columbia, Vancouver, BC, Canada

Introduction: Malaria can be a rapidly fatal disease and testing must be performed on a STAT basis for effective disease management. WHO gold standard microscopic assessment is time consuming, expertise-dependent, and lacks sensitivity. Rapid screening tests are available but their sensitivity and specificity can be limited. Introducing rapid Illumigene LAMP malaria assay with high sensitivity to all malaria species as a screening test precludes performance of rapid diagnostic test (RDT) for P. falciparum, microscopic slide review and repeated testing in cases with negative results.  It can improve malaria detection rate with low parasitemia, laboratory workflow and workload.
Methods: We implementedIllumigene LAMP malaria assay as a screening step in the diagnostic algorithm. Negative tests did not require microscopic assessment of thick and thin blood films by technologist or pathologist. Repeated orders in cases with negative screening tests resulted in cancellation of the orders. Positive tests were assessed in the standard way performing RDT for P. falciparum, microscopic assessment of thin and thick films, parasitemia calculation, pathologist review and referral to Centre for Disease Control for confirmation of Malaria species.  We compared two 3-month periods (pre and post-implementation of the new diagnostic algorithm) for total number of ordered tests, cancellation of repeated tests, malaria positivity rate, turn-around-time (TAT), technologist and pathologist workload.
Results: Three-month post-implementation period (October 24, 2018 – January 24, 2019) was compared to the same periods a year ago (October 24, 2017-January 24, 2018). Total number of orders in this period was 96 and 106 respectively.  Malaria positivity rate remained similar in both periods 3% and 2% respectively. Average TAT from collection to resulting RDT P. falciparum and microscopic thin film in pre-implementation period was 1.5 ± 1.8 2SD and 2.4 ± 2.5 2SD hours versus 2.4 ± 1.6 2SD hours for IllumigeneLAMP malaria assay. Repeated orders for negative malaria tests in 2017/18 (13/106; 12%) were canceled in the new algorithm (13/96; 14%) resulting in total net technical and pathologist workload saving of 61.3 hours (45 min/test) and 27 hours (20 min/test) respectively.
Conclusions: Rapid LAMP screening assay for malaria was easy to implement. It resulted in reduction of workload and improvement of patient care without compromising the turn-around-time.

Prognostic Impact Of Copy-Number Alterations In Multiple Myeloma: Integration Of Cytogenetic Abnormalities With Gene Expression Profiles
Jung Yoon1, Jae Won Yun1, Jaebon Lee2, Jaesun Kim2, Hee-Jin Kim1, Sun-Hee Kim1
1Department of Laboratory Medicine & Genetics, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, Korea, 22Sungkyunkwan University School of Medicine, Seoul, Korea

Introduction: Almost all multiple myeloma (MM) patients harbor chromosomal abnormalities, yet the established prognostic significance is limited to only a few. DNA damage response (DDR) pathway is important in cancer, and the defect in many of DDR related genes are causally connected to tumor initiation. In MM, deletion and/or mutation of TP53 is regarded as genetic indicators of high-risk myeloma, yet the other DDR pathway is largely unknown. We hypothesized that the recurrent chromosomal deletions may be one of mechanism resulting in aberrant downregulation of DDR related genes in MM. The aim of this study was to identify putative tumor suppressive gene in MM by integration of cytogenetic abnormalities with gene expression profiles. 
Methods: Conventional cytogenetics of 106 newly diagnosed patients with MM was analyzed along with fluorescent in situ hybridization study covering all panels according to IMWG consensus recommendations. Recurrent chromosomal deletions, except for previously well-known chromosomal regions such as 1p, 13q and 17p deletion, were identified and correlations between the international staging system (R-ISS) were assessed. The expression and prognostic value of the gene expression pattern within the deleted lesion was analyzed using three independent array data sets from previous studies.
Results: Among the MM patients, 70.8% (75/106) harbored deletional CNAs. The frequent (>5%) chromosomal lesions with deletion were 6q23, 8p21, 12p12-13 and 16q23 found in 7.5% (8/106), 13.2% (14/106), 7.5% (8/106) and 14.1% (15/106) of MM patients, respectively. Of these four chromosomal lesions, deletions of 8p21 and 12p12-13 was significantly associated with higher R-ISS (p=0.02 and p=0.03). We then investigated the expression of genes within 8p21 and 12p12-13 to find the under-expressed genes associated with significant prognostic value. Eleven candidate genes located on 8p21 and 16 candidate genes, including GABARAPL1, one of autophagic flux regulators, located on 12p12-13 were significantly associated with the prognosis.
Conclusions: Integration of expression of genes exist in recurrent  copy number aberrations (CNAs) may further extend the understanding of the molecular mechanism of each CNAs driving poor prognosis in MM. Identification of putative tumor suppressor genes would help to predict the outcome in MM.

Automatic Classification Of Red Blood Cells Infected With Malaria By Means Of Digital Image Analysis
Angel Molina1, Santiago Alfrez2, Laura Bold1, Andrea Acevedo2, Jose Rodellar2, Anna Merino1
1Hospital Clinic of Barcelona, Barcelona, Spain, 2Polytechnic University of Catalonia, Barcelona, Spain

Introduction: Malaria is a life-threatening disease caused by Plasmodium parasites that infect red blood cells (RBC). It is an acute febrile illness that can have a great impact on patients’ health if urgent therapeutic measures are not taken. Peripheral blood smear (PBS) examination under a microscope remains the gold standard for the detection of the parasite. However, microscopic analysis is time-consuming, requires high experience and suffers from certain degree of subjectivity. The objective of this work is to design a method for the automatic classification of malaria by means of digital image processing techniques. This methodology should be able to recognize malaria-infected RBC among other RBC inclusions such as Howell-Jolly bodies (HJ), Pappenheimer bodies (PP), basophilic stippling (BS) and platelets (PLT) allocated over RBC.
Methods: A total of 69 PBS stained with May Grünwald-Giemsa were collected from different patients with malaria, HJ, PP, BS or PLT. From all PBS 6,870 RBC images were acquired using an Olympus BX43 microscope and an Olympus DP73 digital camera at 1,000 magnifications. The preprocessing, segmentation and extraction of the images’ characteristics were done through the python module scikit-image. 92 color and texture descriptors of 34 different color components were analyzed. A classifier based on a Linear Discriminant Analysis (LDA)model was trained using the machine learning library scikit-learn library. PBS were divided into 75 % for training (with 5-fold cross-validation) and the remaining 25 % for testing
Results: By means of the maximum relevance, minimum redundancy algorithm, 700 descriptors with the greatest impact in the classification were selected. Table 1 shows the 10 most important features and the color component obtained. Using these descriptors in a LDA model, an overall accuracy of 97 % was obtained in the recognition of malaria and the different erythrocyte inclusions (see Table 2).
Conclusions: The proposed methodology allows the automatic recognition of malaria-infected RBC from PBS images. This methodology is highly specific, being able to discriminate the Plasmodium parasites not only from normal RBC, but also from other similar RBC inclusions. This approach supposes an improvement with respect to the works already published, leading to a better standardization in the report of these alterations.

Uk Neqas Haematology Eqate: A New Digital Platform In Haematology Eqa
Barbara De La Salle, Jon Sims, Yvonne John
UK NEQAS Haematology, Watford, United Kingdom

Introduction: UKNEQAS Haematology has operated a web-based, continuing professional development (CPD) programme for haematology morphology since 2008, using high quality, virtual slides delivered using Digital Slidebox™ software. From approximately 1500 scientists and clinicians in 2008, the number of registrants rose to more than 3000 in over 16 countries by 2018. The cases issued have included a conditions such as: AML, CLL with AIHA, G6PD deficiency, Hb SC disease, Liver disease with hyposplenism, Pelger-Huet Anomaly, T-ALL and TTP. Recent feedback reported 99% of participants satisfied with the image quality and 100% satisfied with the cases provided (reported as satisfactory or better).The Digital Slidebox™ software reached its ‘end-of-life’ in 2018 and a new solution has been developed with the assistance of participants.
Methods: The UKNEQAS EQATE (External Quality Assessment, Training and Education) system launched in 2018, initially for digital haematology morphology. Slides continue to be imaged using optical light transmission microscopy on a motorised automated microscope equipped with a video camera operated by Zeiss Axiovision software and each participant submits their individual interpretation of the case using a secure log-in. UKNEQAS Haematology has undertaken a workshop with expert participants to identify the next phases of development for the EQATE service, which offers greater flexibility and opportunities for education than its predecessor.
Results: The workshop scored the provision of high quality education, competency and training as the best use of the technology, advising that the results returned are related to the skill level of the participant and that cases are extended to assess interpretation as well as morphology skills. The priorities identified for the next modules for development include programmes for competency assessment of medical as well as scientific staff and access to the considerable gallery of images and self-assessment tools available within UKNEQAS Haematology. The EQATE digital platform is seen as an acceptable means of delivering EQA and interpretive cases to complement the more traditional UKNEQAS services in blood and bone marrow morphology, the haemoglobinopathies and automated cell counting.
Conclusions: Ten years’ UKNEQAS experience in the provision of web-based CPD has demonstrated the application of digital services in haematology education. Although participants still prefer the use of ‘traditional’ specimens, digital cases in EQA are now seen as an acceptable alternative for rare conditions.

2:00 - 3:00 PMEast Meeting Room 11
Oral Abstract Session: Coagulation & Platelets

Chair(s): Richard Marlar, Jun Teruya
Innovative Rapid Pcr Technique Without Dna Extraction For The Combined Diagnosis Of Factor V Leiden And Prothrombin G20210A Mutations
Alexandre Desjonqueres1, Liselot Detemmerman 2, Audrey Mnard1, Catherine Ternisien1, Marc Fouassier1, Benjamin Gillet1, Marie C Bn1, Yannick Le Bris1
1Hematology Biology, Nantes, France, 2LaCAR MDX, Lige, Belgium

Introduction: Factor V Leiden and prothrombin G20210A mutations (F2G20210A ) are genetic factors predisposing to thrombosis often retrieved in populations of thrombotic patients. It is recommended to investigate for these mutations in the exploration of thrombophilia. Loop-Mediated Isothermal Amplification (LAMP PCR), a recently developed method which can be used for the detection of these mutations was validated here for the simultaneous detection of F5 and F2G20210A  mutationson whole blood.
Methods: The LC-FII&FVL-LP-24® (LaCAR MDX Technologies) kit was used, with 1µL of whole blood or DNA. Five steps are performed in this technique, respectively lysis, isothermal amplification, denaturation, hybridization and fusion with fluorescence detection. The polymerase chain reaction (PCR) uses 6 different primers for each target, allowing for the concomitant amplification of F2 et F5. Fusion profiles are then analyzed in two distinct fluorescence channels with specific probes (mutation for F2 and wild site for F5) and a quencher. All amplifications were directly carried out on an LC96® (Roche) and analyzes used an automated online software (Mobiolink).
Results: DNA from 23 patients (stored at +4°C) and a prospective series of 17 whole blood samples were tested. Usual methods of the laboratory were used as reference, respectively an allele-specific PCR for F2 wt, F2 G20210Aand F9 with SYBR®Green detection, and a FRET PCR on RotorGene® for F5. All samples were selected based on the initial results obtained onsite. They were 19 mutated FII and 21 mutated FV, homozygous (10 FII and 7 FV) or heterozygous (11 FII and 12 FV). In all cases, the status identified initially was confirmed, and reproducibility tests were 100% concordant.
Conclusions: Overall, this innovative LAMP-PCR method allows to obtain results within 40 minutes for both mutations, using classical molecular biology instruments. A small aliquot of whole blood is directly mixed with the reagents of amplification and detection in a single step. Data are thus obtained faster than with conventional techniques. The method is versatile, allowing for individual detection or batch tests in microwell plates.

The Added Value Of Anti-Cardiolipin And Anti-Β2Glycoprotein I Igm Antibodies In The Antiphospholipid Syndrome, A European Multicenter Study
Walid Chayoua1,2, Hilde Kelchtermans1,2, Gary W. Moore3, Jean-Christophe Gris4, Jacek Musiał5, Denis Wahl6, Bas de Laat1,2, Katrien M.J. Devreese7
1Cardiovascular Research Institute Maastricht, Synapse Research Institute, Maastricht, Netherlands, 2Cardiovascular Research Institute Maastricht, Synapse Research Institute, Maastricht, Netherlands, 3Viapath Analytics, Guy's and St Thomas' Hospitals, London, United Kingdom, 4Centre Hospitalier Universitaire, Nimes, France, 5Inserm, Centre Hospitalier Regional Universitaire de Nancy, Universit de Lorraine, Nancy, France, 6Jagiellonian University Medical College, Krakw, Poland, 7Cardiovascular Research Institute Maastricht, Synapse Research Institute, Maastricht, Netherlands, 8Coagulation Laboratory, Ghent University Hospital, Ghent, Belgium

Introduction: The antiphospholipid syndrome (APS) is characterized by thrombosis and/or pregnancy morbidity with the persistent presence of antiphospholipid antibodies (aPL). Diagnosis of APS predominantly relies on laboratory assays detecting lupus anticoagulant (LAC), anti-cardiolipin (aCL) and anti-β2glycoprotein I (aβ2GPI) IgG/IgM antibodies. However, the role of IgM in APS is debated. Moreover, comparison of clinical studies is difficult as multiple study designs are used with a wide variety of aPL assays, characterized with a large inter-assay and inter-laboratory variation. Here, we aimed to investigate the association of aCL and aβ2GPI IgM and thrombosis/pregnancy in APS.
Methods: From eight European medical centers 1008 patients were enrolled: 259 thrombotic APS patients, 122 obstetric APS patients, 204 non-APS thrombosis patients, 33 non-APS obstetric patients, 196 patients with autoimmune diseases and 194 normal controls. aPL were detected with Bioplex 2200®, ImmunoCap®EliA,ACL AcuStar®and QUANTA Lite ELISA® by a single technician, using manufacturer’s recommended cut-off values. LAC was determined by the local center.
Results: Positivity for IgM aPL was detected in 25-36% (depending on the assay) APS patients and in 6.4-16% patients with an autoimmune disease and controls. Isolated IgM positivity was rare in APS patients (3.6-6.6%) and the control population (1.8-10%). In a multivariate logistic regression analysis of aPL, no significant association was found between IgM antibodies and thrombosis (Table 1). However, we did find a significant association of IgM antibodies with pregnancy morbidity (Table 2). From the current aPL panel, positivity for IgG reached the highest odds ratio (OR) for thrombosis. Positivity for LAC reached the highest OR for pregnancy morbidity.
Conclusions: Within the current aPL-panel, aCL and aβ2GPI IgM antibodies are of added value in obstetric APS, but not in thrombotic APS. Our data suggest that testing for aCL and aβ2GPI IgM positivity should only be performed in women suspected of obstetric APS.  

Light Transmission Aggregometry On Automated Coagulation Analyzer Systems Compared To A Manual Aggregometer (Atellica Coag 360 &Ndash; Cs 2500I &Ndash; Chronolog 700)
Florian Prller1, Jasmin Rabensteiner1, Carina Koller1, Dirk von Lewinski2, Elisabeth Mahla3, Markus Herrmann1
1Clinical Institute of Medical and Chemical Laboratory Diagnostics, University Hospital Graz, Medical University of Graz, Graz, Austria, 2Department of Cardiology, Medical University of Graz, Graz, Austria, 3Department of Anesthesiology and Intensive Care Medicine, Medical University of Graz, Graz, Austria

Introduction: Light transmission aggregometry (LTA) is still considered as the gold standard for platelet function testing e.g. to monitor dual anti-platelet therapy (DAPT), to uncover platelet dysfunction. The method described by Gustav Born has to be still manually performed, needs substantial blood volumes, and is technically challenging and time-consuming. Aim: To evaluate LTA performed on routine coagulation analyzers in comparison with a manual optical aggregometer.
Methods: Samples from 47 patients on DAPT or with bleeding disorders were tested. Routine LTA was performed on the Chronolog 700 aggregometer (Chrono-log Corporation, Havertown, PA, USA) and compared with commercially available coagulation analyzers (i.e. CS-2500i and Atellica COAG 360 with a pre-release software, Siemens Healthineers, Marburg, Germany). LTA was performed using standard platelet activating reagents in platelet rich plasma (PRP) with platelet counts ranging from 150 to 500 G/L. PRP was tested within 2 hours on all three devices.
Results: The tested PRP samples showed no significant difference in aggregation results [presented as medians and interquartile ranges (IQR), see Table] and aggregation curves led to concordant diagnoses of platelet inhibitors used or platelet dysfunction.
Conclusions: Automated platelet function testing on routine coagulation analyzers shows good correlation with the gold standard LTA manual method and will allow for broad-based access in future patient care overcoming the limitations of the present manual method: 24/7 availability, standardizing testing procedures, and minimizing sample volumes according to patient blood management strategies. 

Determination Of Sigma Score Based On Biological Variation For Haemostasis Assays: Fit-For-Purpose For Daily Practice?
Piet Meijer1, Martine Hollestelle1,2, Janneke Ruinemans-Koerts3, Ren Idema4, Moniek de Maat2
1ECAT Foundation, Voorschoten, Netherlands, 2Department of Hematology, Erasmus Medical Center, Rotterdam, Netherlands, 3Department of Clinical Chemistry and Hematology, Rijnstate Hospital, Arnhem, Netherlands, 4Laboratory for Clinical Chemistry and Hematology, Amphia Hospital, Breda, Netherlands

Introduction: Internal quality control (QC) rules for laboratory tests can be derived from analytical performance specifications (APS) using the six-sigma method. We tested the applicability ofthis paradigm to routine haemostasis measurements.
Methods: Three laboratories using different instruments and reagents calculated sigma scores for their prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen and antithrombin (AT) measurements. Sigma scores were calculated using biological variation (BV) data from the literature in combination with internal and external QC data. Internal data were derived from one quality sample in the normal range collected in three consecutive months and external data were based on 6 external quality surveys with 2-3 data points per survey.
Results: Wide ranges in sigma scores for the PT (0.1-6.8), APTT (0.0-4.3), fibrinogen (1.5-8.3) and AT (0.1-2.4) were observed when QC data was combined with the minimum, median and maximum value of BV data, due in particular to a large variation in within-subject and between-subjects coefficients of variation. When the median BV values were applied, most sigma scores were below 3.0, for internal QC data; 75% and for external QC data; 92%.
Conclusions: Our findings demonstrate that: 1) The sigma scores for common haemostasis parameters are relatively low and 2) The application of the six-sigma method to BV-derived APS is hampered by the large variation in published BV data. An updated database is needed, in which only BV studies are included which fulfil standardised criteria. Since the six-sigma concept is based on requirements for monitoring, and many haemostasis tests are only designed for diagnostic purposes, a fit-for-purpose APS is needed to achieve clinically relevant quality goals.

3:00 - 4:00 PMEast Exhibit Hall B

Possibility To Detect Samples With Increased Band Count On The Beckman Coulter Unicel Dxh 800 And Improvements In Laboratory Workflow*.
Victoria Burova1, Dmitry Sukhachev2, Diana Kovchenko1, Eleonora Khalyuzeva1, Dmitry Butlitski3, Victoria Gerasimenko3, Elena Sukhacheva4
1LLC GlobusMed, St. Petersburg, Russia, 2LabTech Ltd, St. Petersburg, Russia, 3BC LLC, Moscow, Russia, 4Beckman Coulter Eurocenter, Nyon, Switzerland

Introduction: For high-throughput laboratories, it is difficult to identify patients with increased band count without performing blood film review, which is time-consuming and requires manual steps. UniCel DxH 800 provides @Cellular Morphometric Parameters (@CMP) for leukocytes differentiation, which potentially may help detect the presence of abnormal cells. Our goal was to investigate if @CMP, together with other parameters can be used to estimate the probability of increased band count in blood specimens.
Methods: Data from 2,384 patients were used as training set: 1,350 women, 1,034 men (1,533 adults, 851 children 1- 15 years old). 1,700 venous blood samples were collected in K2 EDTA BD Vacutainer tubes, 684 capillary blood samples - in K2 EDTA BD Microtainer tubes. Microscopic review and band count was performed for all samples. Statistical analysis was performed with MedCalc statistical software (ver.17.2, Ostend, Belgium). Samples from 10,192 patients were used to verify results.
Results: In 36 patients from the training set, increased band count was detected (range 7-21). We developed regression model to discriminate samples with more than 6% of bands from samples with normal band count. This model included 2 @CMP (@MN-V-MO–Mean Monocytes Volume; @SD-V-MO–SD of Mean Monocytes Volume) and 2 IVD parameters NEUT# and WBC. Following results were obtained for training set: 117 true positive (TP), 4 false negative (FN), 1,138 false positive (FP), 1,125 true negative (TN). Sensitivity and specificity were 96.69% and 49.71%, respectively. To verify prospectively the performance of the model, the equation was included in the LIS and applied to 10,192 patients. From this cohort, 327 samples with bands >6% were detected (TP-306, FP-3,295, FN-21 and TN-6,570). Among FP, there were samples with atypical mononuclear cells, metamyelocytes and myelocytes according to microscopy; among FN – samples with Pelger cells, atypical mononuclear cells and myelocytes. Specificity and sensitivity were 66.6% and 93.6% respectively. 
Conclusions: Our approach to detect samples with increased band count may be useful in routine practice to reduce the percentage of unnecessary blood film review and improve the efficiency of the laboratory*. *Requires validation through a controlled clinical trial @For research use only. Not for use in diagnostic procedures

Performance Of The Thrombodynamics-4D Videomicroscope For Real-Time Visualization Of Clot-Growth
Marianne Schoorl, Tess Jansen, Johannes van Pelt
Northwest Clinics, Alkmaar, Netherlands

Introduction: The actual coagulation status can only be reliably measured using highly sensitve global functional tests. The Thrombodynamics-4D videomicroscope allows real-time visualization of in vitro clot growth using immobilized tissue factor. The biochemical reactions of the coagulation cascade and clot formation process are characterized by measurement of clot size (CS) and calculation of the spatial clot growth rate (initial rate Vi and average rate Vs) along with clot density (CD) and lag-time of clot formation (Tlag). The Thrombodynamics assay would be sensitive to hypo- and hypercoaguable states, because spontaneously formed fibrin clots can also be detected. In this study, the analytical performance as well as sample stability and reference values of healthy individuals were established. Furthermore, a correlation study was performed with patient samples with normal to extended APTT and PT values.  
Methods: The Thrombodynamics-4D videomicroscope and Thrombodynamics TDX kit (HemoCore LLC, Moscow, Russia) were used to perform measurements of spatiotemporal dynamics of fibrin clot formation. Within 2 hours after venipunture (BD Vacutainer Citrate 3.2%), blood samples were centrifuged at 3300g for 10 minutes. In order to obtain platelet poor plasma without microparticles, the plasma was centrifuged for another 5 minutes at 12500g. Reproducibility was performed in normal and abnormal plasma samples (n=10). For sample stability, normal and abnormal plasma samples were four times freezed (-20°C) and thawed (5 minutes at 37°C). Before measurement, plasma was centrifuged for 5 minutes at 12500g. Reference ranges were established in healthy volunteers without any medication that could affect coagulation (n=20). For correlation studies, samples with normal and extended PT (n=20) and APTT (n=20) were compared with Thrombodynamics clot formation parameters.
Results: Reproducibility of CS, Tlag, Vi, Vs and CD were respectively 4.1%, 17.5%, 4.0%, 6.0% and 7.1%. Regarding the analytical performance of the Thrombodynamics parameters, a four times freezing and thawing process did not affect the results. Reference ranges were established as CS1000-1600 µm, Tlag 0.5-1.5 min, Vi 45-70 µm/min, Vs 20-45 µm/min and CD 9000-20000 AU. Linear regression resulted only for Tlag in a positive correlation with PT (r=0.95, p=0.000) and APTT (r=0,73, p=0.026).
Conclusions: Analytical performance of the Thrombodynamics-4D videomicroscope is satisfactory. Additional studies with samples that reveal hypo- and hypercoaguable states are needed to prove its use in clinical practice.

Preperation Of Highly Active Concentrate Of Blood Coagulation Factor Viii By The Use Dye-Silica Sorbents
Natalia Shurko1, Taras Danysh1, Vasyl Novak1
1 State Institution Institute of Blood Pathology and Transfusion Medicine NAMS of Ukraine, Lviv, , Ukrenia

Introduction: The ion-exchange chromatography is main method of obtaining of plasma concentrate of factor VIII (FVIII). The active dyes are importantalternatives to natural ligands for specific affinity chromatography, offer clear advantages over biological ligands, in terms of economy (inexpensive), ease of immobilization, safety, stability and adsorbent capacity.
Methods: Precipitation of proteins, ion-exchange chromatography and dye-ligands affinity chromatography.
Results: The scheme for obtaining a highly purified concentrate of the FVIII to create. The research was conducted on combinations of the pre-fractionation method with the method of negative affinity sorption. FVIII is not absorbed by any of the synthesized sorbents,however, its specific activity increases in the supernatant. This is due to its sorption from the investigated solution of additional proteins. The data are confirmed statistically by a two-sample t-test with different dispersions in determining the significance of the difference between the groups (P≤0.05).  We were used in these work 16 different sorbents with immobilized active dyes. Selected 5 sorbent for cleaning the FVIII:  Diasorb-Procion Gelb M4R,  Diasorb-Procion Blue HB, Diasorb-Procion Blue MXR, Diasorb-Active Bright Blue КНand Diasorb-Active purple 4GT.  It has also been demonstrated that the use of ion-exchange chromatography on DEAE-Sepharose FAST FLOW and affinity chromatography on selected sorbents allows obtaining FVIII from Cryoprecipitate with a degree of purification of 129 to 242 times and preservation of up to 73.0–76.0 % of the initial activity. The main losses from the initial activity of FVIII and the vWF (up to 27.0 %) occurred at the stage of ion-exchange chromatography. The phenomenon of negative affinity sorption provides almost 100.0 % (96.34 %) yield of the product. It has been found that the combination of pre-fractionation, ion-exchange and affinity chromatography stages provides a purification rate of 239.0–700.0 times, depending on the type of selected sorbents.
Conclusions: The new method to obtain purified concentrate of the blood coagulation FVIII was created. The best result (the highest degree of purification FVIII) was achieved when using Diasorb-Active purple 4GT: the specific activity 50.58±1.68 IU/mg protein, vWF:Rcof – 3.07±0.18 IU/mL, FVIII/vWF:Rcof – 1.73±0.2.

Comparison Of The Impact Of Doacs On Hemostasis Diagnostic Tests
Romain Siriez1, Jonathan Evrard1, Franois Mullier2, Jean-Michel Dogn1, Jonathan Douxfils1, 3
1University of Namur, Department of Pharmacy, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for LIfe Sciences (NARILIS), Namur, Belgium, 2Universit catholique de Louvain, CHU UCL Namur, Hematology Laboratory, Namur Thrombosis and Hemostasis Center (NTHC), Namur Research Institute for Life Sciences (NARILIS), Yvoir, Belgium, 3Qualiblood s.a., Namur, Belgium

Introduction: There is a laboratory and clinical need to know the impact of anticoagulants on diagnostic tests to avoid misinterpretation of results. Although the labeling documents of these medicines (i.e. EMA summary of products characteristics, FDA prescribing information) provide some information about the influences of each DOAC (factors Xa antagonists including apixaban, edoxaban, rivaroxaban and betrixaban and factors IIa antagonist such as dabigatran) on diagnostic tests, these are usually limited to some of the most common tests, with few information about the reagents used, and above all, not head to head comparison. This study aims to compare the impact of DOACs on hemostasis diagnostic tests and provide practical recommendations for clinicians.
Methods: The impact of increasing concentrations of DOACs of a normal pooled plasma has been assessed on several thrombophilia testing, including antiphospholipid assays, the assessment of protein C, protein S and antithrombin activity, measurements of clotting factors levels and the detection of activated protein-C resistance with a large panel of reagents. The results are compared and discussed with data obtained from a cumulative review of literature.
Results: Overall, factors Xa and factors IIa antagonists significantly affect clot-based hemostasis diagnostic tests resulting in false-positive results.  Impacts on thrombin-based hemostasis diagnostic tests are observed with dabigatran but not with anti-Xa and conversely for FXa-based hemostasis diagnostic tests. No impact was observed with antigenic or chromogenic methods for the assessment of protein S and C activity.
Conclusions: Interpretation of hemostasisdiagnostic tests results should be done with caution in patients on DOACs since many of them may be impacted. The use of a device/chemical compound able to remove or antagonize the effect of DOACs or the development of new diagnostic tests insensitive to DOACs should be considered to further minimize the risk of false results.

Anti-Iia Heparin Assay For Patients On Direct Oral Anticoagulants
Morgan Stuart, Linda Johnson, Sarah Hanigan, Steven W. Pipe, Shih-Hon Li
Michigan Medicine, University of Michigan, Ann Arbor, MI, United States

Introduction: Direct Oral Anticoagulants (DOACs) (e.g. rivaroxaban, apixaban) are increasingly prescribed for atrial fibrillation and venous thromboembolic disease. Such patients transitioning to unfractionated heparin (UNFH) during a hospital admission were monitored via an Anti-Xa assay. However, due to DOAC interference, patients exhibited critically high (CH) UNFH results with discordant aPTT, confounding heparin dosing. Since thrombin is insensitive to DOACs, an Anti-IIa assay was evaluated.
Methods: The BIOPHEN Anti-IIa (Aniara Diagnostica) and INNOVANCE Anti-Xa heparin assays (Siemens Medical Solutions USA, Inc.) were compared on the BCS XP system (Siemens). Forty consecutive specimens from Emergency Department (ED) patients on DOAC therapy transitioning to UNFH were tested with/out DOAC-REMOVE (Aniara). Linearity/recovery, accuracy, and precision were evaluated using BIOPHEN Heparin Calibrators and Controls (Aniara) diluted into CRYOcheck Pooled Normal Plasma (Precision Biologics, Inc.). DOAC interference was tested in heparinized plasma spiked with 192 ng/mL apixaban or 158 ng/mL rivaroxaban. CH results were retrospectively analyzed 4 months post-implementation. Data were analyzed using EP Evaluator (Data Innovations, LLC) and Prism 7.0 (GraphPad Software). Significance was evaluated using the Mann-Whitney U test.
Results: Anti-IIa UNFHs better approximated DOAC-REMOVE-treated versus untreated Anti-Xa UNFHs in ED specimens, indicating interference in the untreated assay. Anti-IIa UNFH accuracy studies using 0.21 and 0.51 IU/mL UNFH yielded 0.20±0.03 and 0.49±0.05 IU/mL (mean±SD, n=11), respectively. Within-run and between-run coefficients of variation were 3.5% and 15.2% for the low-control, and 2.1% and 9.6% for the high-control. The method comparison slope was 0.97 (R-square 0.96). The linear analytical range was 0.1-1.3 IU/mL UNFH (mean recovery 111%, 95% CI 103.8-118.1%). The Anti-IIa assay measured UNFH linearly across the expected range in the presence of spiked apixaban or rivaroxaban; however, all Anti-Xa UNFH results from prepared DOAC-containing samples exceeded the assay's reportable range. The monthly percentage of CH UNFH results for DOAC-prescribed patients was 6.0% (4.5-8.8) from the Anti-Xa assay and 1.3% (0.3-2.9) from the Anti-IIa assay in the same high-use inpatient units (median, 25th-75th percentile; n=4, P=0.0286). Per test, Anti-IIa and Anti-Xa UNFH cost $3.83 and $2.66, respectively.
Conclusions: The Anti-IIa UNFH assay is more expensive than the Anti-Xa assay, but yields proportionately fewer CH results. UNFH measurement without DOAC interference may help optimize heparin management. A prospective clinical outcomes study is underway.

A Direct Comparison Of Aptt-Based Lupus Anticoagulant Confirmatory Assays
Julie Tange, Jansen Seheult, Nahla Heikal, Dong Chen, Rajiv Pruthi
Mayo Clinic Rochester, Rochester, MN, United States

Introduction: Laboratory diagnosis of lupus anticoagulant (LAC) is challenging. In this study, we compared Silica Clot Time (SCT) and Staclot-LA on the ACL TOP 700 (Instrumentation Laboratory) (STLA-TOP), and STLA on the Evolution (Diagnostica Stago) (STLA-EVOL) to determine if these assays could be used interchangeably in samples negative for the dilute Russel viper venom time (DRVVT).
Methods: Commercially available SCT and STLA assays were validated on the TOP instrument. The SCT assay was validated per manufacturer's instructions. The STLA-TOP assay (FDA approved on the Evolution) was validated per CLSI guidelines as a laboratory developed test. The STLA-EVOL has been previously validated and is currently utilized within the lab. Normal donors (n=120) were performed to verify (SCT Screen/Confirm Normalized Ratio and STLA-EVOL) and establish (SCT Screen Ratio and STLA-TOP) normal cutoffs. Archived patient samples (n=50) negative or equivocal using the DRVVT methodology were tested using a moderately-sensitive APTT, SCT, STLA-TOP, and STLA-EVOL.
Results: Thirty samples (60.0%) had positive screening APTT and SCT, while two samples each (6.3%) had positive screening APTT or SCT. Seventeen (53%) of the APTT screen-positive samples demonstrated inhibition on mixing studies compared with 14 (43.8%) of the SCT screen-positive samples. For the APTT screen-positive samples, 17 (53%) and 14 (44%) showed evidence of phospholipid-dependent inhibition using the STLA-TOP and STLA-EVOL, respectively, compared with 9 (50%) and 11 (61%) of the APTT screen-negative samples respectively. For the SCT screen-positive samples, 15 (47%) showed evidence of phopholipid-dependent inhibition based on the SCT Screen/Confirm Normalized Ratio compared with only one (5.6%) of the SCT screen-negative samples. There was no significant difference in the rate of thrombosis among samples that were positive using the STLA-TOP, STLA-EVOL or SCT methodologies (9/17 vs 8/18 vs 7/15, p=0.84). Comparing results for only the confirmatory assays, there was moderate agreement among all three assays (κ=0.54). For samples with positive screening and confirmatory assays, there was moderate agreement using the three methodologies (κ=0.69)
Conclusions: The SCT and STLA assays have modest agreement suggesting they may detect different antiphospholipid antibodies. The higher frequency of positive results by the STLA methodology indicates higher sensitivity but likely poorer specificity. Clinic performance of both assays will be determined using a larger prospecitve study.

Lack Of Standardization Of The Anti-Factor (F)Xa Activity Reagents Used In Monitoring Treatments With Unfractionated Heparin
Pierre Toulon1, Motalib Smahi2, Martine van Essen-Hollestelle3, Neila De Pooter4
1Hematology, Pasteur University Hospital, Nice , France, 2Hematology, Simone Veil Hospital, Eaubonne, France, 3ECAT foundation, Leiden, Netherlands, 4Hematology, CH, Grasse, France

Introduction: UFH treatments are usually monitored by using either the anti-FXa activity, with the therapeutic range between 0.30 and 0.70 IU/mL, or the anti-FXa-correlated aPTT. The aim of that study was comparing the inter-reagent agreement of anti-FXa activity in patients on UFH.
Methods: 125 inpatients on full-dose UFH were sampled at one center. Tubes containing 3.2% tri-Na citrate were centrifuged twice within 2h after collection. Plasma was stored frozen in aliquots at -70°C before being shipped in dry ice to 3 accredited coagulation laboratories to be locally evaluated for anti-FXa activity using their routine chromogenic assay(s): Biophen Heparin LRT (Hyphen BioMed), HemosIL Liquid Anti-Xa (Werfen), Innovance Heparin (Siemens), and STA-Liquid Anti-Xa (Stago). Participating centers used combinations of reagents and analyzers from the same manufacturer i.e. ACL TOP 700 (Werfen), CS-5100 (Siemens) and STAR (Stago). Pool of normal plasmas spiked  with dilutions of the 6th NIBSC Standard to achieve anti-FXa activities up to 1.0 IU/mL was evaluated using the same techniques.
Results: Anti-FXa results evaluated in patients’ plasmas using different reagents were found to be significantly different, even though test results were well correlated (r>0.91 in all cases). Median activity ranged from 0.57 IU/mL with one reagent to 0.37 IU/mL with another, two reagents giving intermediate results (0.44 IU/mL). Comparisons of test results performed according to Bland-Altman demonstrated unacceptable bias for some reagents. If considering 0.30 to 0.70 IU/mL as the therapeutic range, such discrepancy in test results led to a lack of agreement as to whether a sample was subtherapeutic, therapeutic or supratherapeutic in more than 25% of the patients. The same trend was demonstrated when plasma spiked with dilutions of the 6th NIBSC Standard for levels within the therapeutic range were evaluated using these different reagents, differences being lower for activity around 1.0 IU/mL. Such a discrepancy was also revealed by analyzing results from 2017 exercises of the ECAT EQAP.
Conclusions: The reported discrepancy between test results obtained using commercially available anti-FXa assays clearly suggests a lack of standardization of that assay with potential significant impact on anticoagulation of patients monitored using anti-FXa activity, and on calculation of the anti-FXa-correlated aPTT therapeutic range.

Utility Of Lupus Anticoagulant Assays (Aptt-La, Kct, Dpt And Drvvt) In Detection Of Antiphospholipid Syndrome (Aps) In High Risk Pregnancy Cases"
Seema Tyagi1, Ankur Ahuja2
1All India Institute of Medical Sciences, New Delhi, India, 2Department of Lab Sciences and Molecular Medicine, Army Hospital (Research and Referral), New Delhi, India

Introduction: Antiphospholipid antibody syndrome (APS) is an autoimmune disorder  leading  to multisystem manifestations from recurrent thrombosis to pregnancy loss and other obstetric morbidities. The diagnosis is based on  anticardiolipin (acl) antibody, Anti β2 glycoprotein (aB2GP) and  lupus anticoagulants (LAC). While aB2GP and acl are being detected  by ELISA, LAC is detected by clot based test. Four LAC tests such as  Diluted Russel Viper Venom test (dRVVT), Activated Partial Prothrombin Time -Lupus Anticoagulant (APTT-LA), Kaolin clotting time (KCT) and  Dilute Prothrombin Time (dPT)  are being recommended in various studies. International society of Thrombosis and Hemostasis (ISTH) has recommended dRVVT and aPTT-LA for diagnosis of APS and discouraged KCT and dPT due to poor diagnostic performance.  However, there is still a huge confusion over the test of choice .The present study was done to evaluate the utility and sensitivity of four LACS test  to identify the best combination for diagnosis of  APS in high risk pregnancy.
Methods:   A total of  526 patients with high risk pregnancy and  50 Sex and age matched   healthy controls. LAC was done  by four methods, 12 weeks apart with a minimum gap of 3 months and maximum of 5 years  of last abortion .
Results: Sixty five  cases were positive for LAC, of which 25 became negative after 12 weeks,  therefore were not considered as per 2006  Sydney guidelines for diagnosis of APS . Overall dRVVT could able to diagnose 36 cases followed by APTT-LA (28 cases), KCT (23 cases) and dPT could diagnose 14 cases.  12 cases showed LAC positivity with all assays shown in table 1.
Conclusions: Diagnosis of APS in high risk  pregnancy requires LAC testing by  more than one assay . The combination of dRVVT with either APTT-LA or KCT is superior especially with history of still births and thrombosis   as compared to recurrent abortions.

Extremely Low Activated Partial Thromboplastin Time (Aptt) In An Individual: A Case Report

Introduction: Low Activated Partial Thromboplastin Time (APTT), which are always accompanied by a decrease in Prothrombin Time (PT), are commonly attributed to pre-analytical errors, inflammation, and hypercoagulability. The aim of this study is (i) to report the extremely low APTT levels documented in an individual, and (ii) to explain the aforementioned paradox measurement.
Methods: The routine laboratory testing of a patient suffering from myelofibrosis revealed a significant low APTT value (1.53sec/APTTcontrol:27sec). This measurement was performed by means of a BCSXP analyzer. To investigate the reasons why such a low APTT value was retrieved, a)new samples were considered to account for potential pre-analytical error issues, b)a mixing test with a mixture of normal plasmas was executed, c)fibrinogen and CRP were measured to exclude the presence of inflammation, d) intrinsic pathway factors activity was measured, and e) a manual APTT measurement was undertaken.
Results: In the subsequent two days, the patient’s APTT values were still very low (i.e., 12 and 14 secs), hence excluding the presence of a pre-analytical error. The APTT waveform(Figure 1) was biphasic since an increase in absorption (turbidity?) occurred after the addition of CaCl2. During the mixing test, although an increase in the amount of light scattered was observed at the baseline, this particular rise was not included in the measurement time, thus correcting the APTT to 26.45 sec(Figure 2). PT was slightly elevated (14.1sec/C:11.1sec), while the fibrinogen, CRP, FXII, FXI, FIX and FVIII were within the normal limits. During the manual APTT measurement, turbidity was observed after the addition of CaCl2, rendering fibrin detection a difficult task to be accomplished. The measured APTT was 37sec/ APTTcontrol:33sec and in concordance with the slightly elevated PT. Concerning the remainder of the laboratory tests, protein immunoprecipitation revealed a large amount of monoclonal immunoglobulin IgMκwhich was possibly attributed to Ca+2-dependent precipitation.
Conclusions: Technical errors during the testing process are implicated in the documentation of very low coagulation times, which are not consistent with the findings from other coagulation tests. It is also highlighted that the manual measurement of clotting time is of paramount importance because direct observation is implemented. In cases with extremely low APTT measurements, the possibility of monoclonal immunoglobulin IgMκexistence interfering with APTT measurement should be born in mind. 

Towards A Better Understanding Of Factor Viii Inhibitors With The Thrombin Generation Assay.
Marie-Astrid van Dievoet, Sandrine Desmet, Catherine Lambert, An Van Damme, Cedric Hermans, Stphane Eeckhoudt
Cliniques universitaires Saint-Luc, Brussels, Belgium

Introduction: Most coagulation tests routinely used in clinical practice and hemophilia follow-up only assess the first part of the coagulation process. Global tests, such as the thrombin generation assay (TGA), better approach in vivo coagulation taking into account both procoagulants and anticoagulants. We studied the effect of factor VIII inhibitors on normal pool plasma (NPP), after heat inactivation of patient plasma, thereby excluding interference of residual coagulation factors.
Methods: 185 platelet poor plasma (PPP) samples from 84 congenital and 2 acquired hemophilia A patients with inhibitor (n=36; ≥0,6  BU/mL in the Nijmegen-Bethesda assay) and without inhibitor (n=149) were heat inactivated (30 minutes at 58°C), mixed 1:1 with buffered NPP (pH: 7,4; CRYOcheck, Precision Biologic, Dartmouth, Canada) and incubated 2 hours at 37°C. TGA was then performed with the Thrombinoscope (Diagnostica Stago, Asnieres sur Seine, France). The parameters lag time (LT), peak hight (peak), endogenous thrombin potential (ETP) and velocity index (VI) were normalized to the reference plasma. Statistical analysis was performed using MedCalc for Windows, version 14.8.1 (MedCalc Software, Ostend, Belgium).
Results: The following medians with 95% confidence intervals (CI) were found for samples with (+) and without (-) inhibitor: LT+ =  80% [95%CI: 77-85%], LT- = 83% [95%CI: 81-85%]; peak+ = 43,5% [95%CI: 26,7-69,7%], peak- = 116% [95%CI: 110,0-123,0%]; ETP+ = 54% [95%CI: 33.4-79.4%], ETP- = 108% [95%CI: 103,0-111,0%]; VI+ = 21,5% [9,4-118,8%], VI- = 116,5% [95%CI: 110,8-126,0%]. A significant difference in median between samples with and without inhibitors was seen for peak, ETP and VI (p< 0,001) but not for LT (p=0,4). In the lower range (0, 6-1.1 BU/mL) medians for LT, peak, ETP and VI were 86,0; 93,0; 93,5 and 82,0% respectively. In the higher range (BU>1.9 BU/mL) medians for LT, peak, ETP and VI were 79,5; 26,5; 33,0 and 13,0% respectively.  
Conclusions: Lag time is of no value in the detection of factor VIII inhibitors by TGA. A significant difference in median between samples with and without inhibitors was seen for peak, ETP and VI. In the lower Bethesda unit range TGA seems to be less affected.

A Novel Whole Blood Thrombin Generation Model To Study The Involvement Of Blood Cells In Coagulation
Jun Wan1,2, Joke Konings1,2, Hilde Kelchtermans1,2, Bas de Laat1,2, Mark Roest1,2
1Synapse Research Institute, Maastricht, Netherlands, 2Cardiovascular Research Institute, Maastricht University, Maastricht, Netherlands

Introduction: Plasma thrombin generation (TG) is the most commonly used model of global coagulation phenotype. Whole blood (WB)-TG is one step closer to physiology because it involves the intrinsic blood cells instead of synthetic phospholipids. However, red blood cells (RBCs) seriously disturb WB-TG by causing variable quenching of the fluorescence signal. We have solved this issue by continuously mixing the sample during the whole measurement course.
Methods: The WB-TG model was validated by evaluating the reproducibility and comparing with a previously developed filter paper based WB-TG model. Reconstitution experiments were performed to study the effects of RBCs and platelet numbers on WB-TG. WB samples from 119 healthy donors uwere tested with this novel model and multivariate linear regression analysis was done to analyze the effect of cell counts, age, sex and oral contraceptive (OC) use on WB-TG.
Results: Under optimized assay conditions, a reproducible light transmission was established in both non-clotting and clotting WB samples during TG. The intra- and inter-assay variations of the TG parameters were all below 7%. This novel model showed good correlation with the previous model when WB samples from 10 donors were tested in both models (Pearson correlation coefficients were 0.82, 0.79 and 0.75 for ETP, lag-time and time-to-peak, respectively, P < 0.05 for all).  Importantly, the lag-time from the old model was significantly shorter than in the new model (P < 0.0001). Reconstitution experiments showed that: (1) thrombin-peak increased with ascending numbers of RBCs or platelets, and RBCs addition augmented thrombin-peak even in the presence of high platelet numbers (Fig 1A & C); (2) the addition of platelets into PPP markedly shortened the lag-time, while RBCs did not show the same effect (Fig 1B & D). The inter-individual variations of the WB-TG parameters were 14.8 - 21.1%. Multivariate linear regression analysis showed that thrombin-peak was significantly affected by the RBC count, as well as age and OC use (P < 0.05 for all).    
Conclusions: We have developed and validated a novel WB-TG model with good reproducibility. Our novel model was less influenced by contact activation than the previous model. In addition, our model showed that RBCs (and platelets) significantly augment the thrombin-peak, while platelets are essential for a fast TG initiation.

In Chronic Myeloid Leukemia, Granulocytic Myeloid-Derived Suppressor Cells (Mdscs) In Complete Hematologic Response Might Reflect Non-Leukemic Granulopoiesis, But Monocytic Mdscs Are Associated With Poor Prognosis
Ari Ahn1, Min-Sun Kim1, Chan-Jeoung Park1, Young-Uk Cho1, Seongsoo Jang1, Mi-Hyun Bae2, Jung-Hee Lee3, Je-Hwan Lee3, Kyung-Nam Koh4, Ho-Joon Im4
1Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea, 2Department of Laboratory Medicine, Hanyang University Guri Hospital, Hanyang University College of Medicine, Guri, Korea, 3Department of Internal Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea, 4Department of Pediatrics, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea

Introduction: Myeloid-derived suppressor cells (MDSCs) represent phenotypically heterogeneous populations of myeloid cells that can suppress tumor-specific T cell responses. MDSCs are produced from normal granulocytic precursors in emergent states and are increased in several hematologic malignancies. We measured MDSCs and evaluated significance and disease correlation of MDSCs in patients with chronic myeloid leukemia (CML).
Methods: Peripheral blood (PB)(n = 77) and bone marrow (BM) (n = 76) aspirates were obtained from patients with CML. The status of CML included chronic (n = 30), accelerated (n = 3), and blastic (n = 4) phases and complete hematologic response (CHR) (n = 40). Age-matched and sex-matched individuals were recruited as healthy controls (HCs)(n = 40), and patients with non-Hodgkin lymphoma but without BM involvement were recruited as negative controls (NCs) (n = 38). The percentage and number of granulocytic MDSCs (gMDSCs) and monocytic MDSCs (mMDSCs) were quantified by 5 color-flow cytometry with panel (HLA-DR/CD11b/CD15/CD33/CD14) (Figure 1).
Results: The gMDSC% and mMDSC% were well correlated between BM and PB samples (P< 0.001). The median BM-gMDSC% and PB-gMDSC% at diagnosis were lower than those of the CHR, BM NC, and HC (P< 0.001, respectively). Conversely, PB-mMDSC number at diagnosis was higher (P< 0.001). BM-gMDSC%, PB-gMDSC%, and PB-gMDSC number were negatively correlated with BCR-ABL1 level, WBC count, platelet count, basophil%, and blast%, and positively correlated with the hemoglobin level, but PB-mMDSC number showed opposite results. The survival rate was higher with high PB-gMDSC number (P=0.021) (Figure 2A) and lower with high PB-mMDSC% (P=0.182) (Figure 2B).
Conclusions: gMDSCs in CHR of CML could reflect non-leukemic granulopoiesis, i.e., the presence or recovery of normal granulocytic precursors, and a high PB-gMDSC number suggests better prognosis. But mMDSCs might be associated with malignant conditions and poor prognosis.

Cd49D And Cd43 Expression Is Useful To Detect Erythroid Dysplasia
Rui Bartolo1, M. Oliveira1, P. Laranjeira2, M. Fortuna1, A. Ribeiro3, M. Santos4, E. Corteso3, G. Marques5, A. Sarmento-Ribeiro2,3, H. Vitria4, L. Ribeiro3, A. Paiva1,2
1Unidade de Gesto Operacional de Citometria, Servio de Patologia Clnica, Centro Hospitalar e Universitrio de Coimbra, Coimbra, Portugal, 2Coimbra Institute for Clinical and Biomedical Research (iCBR), Coimbra, Portugal, 3Servio de Hematologia Clnica, Centro Hospitalar e Universitrio de Coimbra, Coimbra, Portugal, 4Servio de Hematologia, Centro Hospitalar Viseu-Tondela, Viseu, Portugal, 5. Servio de Patologia Clnica, Centro Hospitalar e Universitrio de Coimbra, Coimbra, Portugal

Introduction: Despite bone marrow (BM) immunophenotyping by flow cytometry has progressively been recognized as an important tool for the diagnosis of myelodysplastic syndromes (MDS), the sparse knowledge about the normal erythroid maturation and the lack of markers for erythroid characterization is a major shortcoming.  
Methods: Thus, the objective of this work was to analyze the expression pattern of CD43 and CD49d, two markers included in the diagnostic panel for B cell chronic lymphoproliferative disorders, in the CD34 compartment of normal BM, as well as along the normal and dysplastic erythroid maturation process. For this, 13 normal BM aspirates and 18 BM aspirates from MDS patients (2 MDS with single lineage dysplasia, 6 MDS with multilineage dysplasia, 3 MDS with excess blasts-1 and 7 MDS with excess blasts2), were studied by flow cytometry.
Results: In normal BM, we found a higher expression of CD43 and CD49d among CD34 erythroid precursors, compared to the CD34 cells committed to the remaining hematopoietic cell lineages. CD43 expression progressively decreased along the normal erythroid maturation. In turn, CD49d levels increased in earlier stages and decreased in the last stage of maturation. In MDS, the expression of CD43 and CD49d followed the same pattern, however, a statistically significant decrease in the mean fluorescence intensity values was observed for all maturation stages of erythroid lineage.
Conclusions: Our results point to the usefulness of CD43 and CD49d markers in the identification of dysplastic phenotypic features in the erythroid lineage, allowing the selection of patients who would benefit from a more extensive BM immunophenotypic study to evaluate the presence of MDS.

Standardization Of A Two-Tube 13-Color (15-Antibodies) Flow Cytometric Assay For The Evaluation Of Detailed Immune Cell Profiling (Sixty-Two Immune-Cell Subsets) In Human Peripheral Blood
Dilshad Dhaliwal, Twinkle Khanka, Avinash Gupta, Neha Jodhawat, Pratyusha Gudapati, Sitaram Ghogale, Yajmanan Badrinath, Nilesh Deshpande, Ashok Kumar, PG Subramanian, Nikhil Patkar, Sumeet Gujral, Prashant Tembhare
Tata Memorial Hospital, Navi Mumbai, Navi Mumbai, India

Introduction:  The human immune system consists of many white blood cell subsets that coordinate actions to maintain body’s immune environment in health and disease. The balance between these cell populations is critical to protect humans from various types of infections and autoimmune disorders. Immune cell profiling is becoming important to study outcomes in allogenic hematopoietic stem cell transplant. Detailed analysis of the immune cell profile has a tremendous potential to distinguish patients at risk for specific infections or immune-mediated diseases and inform vaccination strategies. Hence, there is need to standardize immune cell profiling assay for routine clinical practice. In this study, we standardized a two-tube 13-color flow cytometric assay which allows an evaluation of 62 immune-cell subsets in human peripheral blood.    
Methods:  A two-tube 13-color panel antibodies was designed (table 1) and standardized for the human peripheral blood sample(PBS). Samples were processed using bulk-lysewash technique and data was acquired using a 3-laser 13-color flow cytometer i.e. CytoFlex (Beckman Coulter). Fluorescence-minus-one assay was studied to evaluate inter-antibody interactions and to study the background fluorescence due to the reagents. This panel was studied for the quantitation of variety of T, B, NK cell, monocytes, basophils, dendritic cells subsets in PBS from 20 healthy individuals in Tata Memorial Centre. 
Results: We standardized a two-tube 13-color assay that allows studying 62 immune-cell subsets simultaneously. Intra assay variation was studied by processing the single sample 10 times and CV/SD of each subset was determined. The panel was studied in PBS from 20 healthy individuals. The results (mean, median and range) of 62 immune-cell subsets from study 20 healthy adults are given in Table 2. 
Conclusions:  We first time standardized a two-tube 13-color (15-antibodies/tube) flow cytometric assay that allows quantitation of 62 immune-cell subsets in human peripheral blood. This tube is useful in the evaluation of immune-reconstitution and immune response in many physiological and pathological conditions. It reduces intra-assay variations caused due to multiple tube assay and highly useful in the samples with limited volume. 

Acute Leukemia Diagnostic Detecting Blasts In Peripheral Blood With Cytodiff

Introduction: Hematology analyzers are not effective in recognizing and classifying blasts although they may provide flag messages when such cells are present. Microscopic evaluation is a laborious, time-consuming and requires expertise. Moreover, it may show variable reproducibility. Blast count is important for the diagnosis of hematologic diseases and prognosis of patients,
especially in cases such as acute leukemia and Myelodisplasic syndrome. Using a combination of monoclonal antibodies for an extended white blood cell differential (WBC-Diff) by flow cytometry introduced by Beckman Coulter we improve the specificity of several hematologic pathologies diagnosis. Our study focuses on comparing manual and CytoDiff® blast count and screening blast origin using CytoDiff®.
Methods: 41 patients were analyzed by both Sysmex XN (Kobbe, Japan) and Cytodiff®. The last one is a combination of 6 monoclonal antibodies/5 colors which performs a rapid WBC-Diff. 10 patients were diagnosed of acute lymphocytic leukemia (ALL) and 31 were diagnosed of acute myeloid leukemia (AML). Manual WBC differential count was performed using Cellavision DM96 analyzer. Manual and CytoDiff® blast count were compared using Passing Bablock regression and Pearson correlation test.
Results: 41 samples were analyzed showing in 33 cases “Blast” flag.  Passing Bablock regression showed neither proportional nor systematic differences with 95% confidence interval containing the value 1 and 0 when comparing manual and CytoDiff® blast count.Pearson correlation testshowed good correlation for blast count (r=0.95).In 3 cases (7.3%) blast origin was identified wrongly: 1 T-ALL blast (91% manual count) were found in Xn gate (86.85%), 1 AML blasts (26% manual count and 69% Xb and 2.2% Xt) were included in Xb and 1 B-ALL blast (1.3% manual count and 1,81% CytoDiff® count) were located in Xn gate.
Conclusions: CytoDiff®blast count show good correlation with manual count and did not depend on experience. It provides more specificity than WBCdiff in blast detection although its classification is not possible due to the lack of some specific antigens. CytoDiff®can be considered a middle step between PB morphology and specific flow cytometry.

Trem-Like Transcript 1: A More Sensitive Marker Of Platelet Activation Than P-Selectin And Lysosomal Associated Membrane Protein 3 In Patients With Acute Coronary Syndromes
Shundong Ji1,2,3,4, Linyan He1,2, Mingqin Zhu1,2,3, Changgeng Ruan1,2,3,4
1The First Affiliated Hospital of Soochow University, Suzhou, China, 2Jiangsu Institute of Hematology, Suzhou, China, 3MOH Key Laboratory of Thrombosis and Hemostasis, Suzhou, China, 4Collaborative Innovation Center of Hematology, Suzhou, China

Introduction: Platelets have a central role in cardiovascular thrombosis.P-selectin is a type 1 transmembrane protein present in platelet and megakaryocyte a-granules. P-selectin is commonly used as a gold standard marker of a-granule secretion and irreversible platelet activation. TREM (triggering receptor expressed on myeloid cells) – like transcript 1 (TLT-1) is another membrane receptor found in platelet and megakaryocyte a-granules and is rapidly translocated to the surface upon activation.Lysosomal associated membrane protein 3 (CD63) is a cell surface glycoprotein that is known to complex with integrin. It may function as platelet activation marker. In this study, we show that TLT-1 is a more sensitive marker of platelet activation than P-selectin and CD63in patients with acute coronary syndromes.
Methods: Adult patients who developed acute coronary syndromes over the past six months in our hospital were enrolled. After provided written informed consent, citrated blood was obtained from patients for assay. Surface protein expression of TLT-1, P-selectin and CD63 was analyzed in resting and stimulated platelets (Beckman Coulter Epicx XL-1 flow cytometer) following fixation and staining with FITC- or Alexa Fluor 488 conjugated antibodies. Platelets directly stained with FITC- or Alexa Fluor 488- anti-mouse IgG antibodies were set up as controls. Data was analyzed using Flow Jo 10 (Tree Star, Ashland, OR). Mean fluorescence intensity (MFI) of cells in each sample was used directly for comparison.
Results: Of this 107 patients studied, 72 were males and 35 were females. TLT-1 and P-selectin was only detectable in activated platelets after ADP addition. The MFI of TLT-1 and P-selectin on resting platelets were 1.01±0.41 and 0.45±0.27, while activated were 5.01±1.60 and 18.11±3.52, respectively. CD63 was detected in both resting and stimulated platelets, the MFI of CD63 on platelets without or with ADP was 18.01±1.58 and 23.49±2.71, respectively. TLT-1 was also more rapidly upregulated to the surface following thrombin and CRP stimulation compared with P-selectin and CD63. 
Conclusions: In summary, we show that TLT-1 is more rapidly and abundantly upregulated on the surface of activated platelets than CD63 and P-selectin. 

Assessment Of Viability Of Haemopoietic Stem Cells In Cord Blood Through Cd34 Marker By Immunophenotyping Technique
1EDO UNIVERSITY, IYAMHO, AUCHI, Nigeria, 2Ladoke Akintola University of Technology Teaching Hospital, Osogbo Dept of Haematology,, Osogbo, Nigeria, 3Ladoke Akintola University of Technology, Department of Obstetrics and Gynaecology, Osogbo, Nigeria

Introduction: Cord blood is a potential source of primitive haemopoietic stem cell (HSC) and is available in advanced countries of the world, for clinical application to reconstitute the hematopoietic system in affected individuals requiring treatment. Cord blood can be used as an alternative source for bone marrow transplantation and its use is developing into a new field of treatment for patients presenting with hematological disorders, immunological defects and specific genetic diseases; including haemoglobinopathies. This work was carried out to assess the viability of cord blood obtained from Osogbo and stored frozen as a source of haemopoietic stem cell which may be suitable for transplantation.
Methods: Blood specimens were obtained from umbilical cords of 30 consenting mothers and dispensed into 5 cryovials with glycerin for freezing at -200C; while quantitative assay was carried out on a fresh citrated sample by immunophenotyping using CD34 as marker of haemopoietic stem cell. Partec Cyflow cube 6 was used to measure viable cells after labeling the cells with specific fluorochrome/antibody obtained from sysmex. A repeat quantitation was carried out at one month interval for 5 consecutive months and results generated were analysed using T- independent test.
Results: The mean ± SEM for the 6 consecutive counts were 20,798±2750, 19849±2691, 19223±2637, 18363±2582, 17052±2583 and 16184±2423. The p values obtained when the cryoprotected samples were compared to the baseline were 0.806, 0.681, 0.521, 0.325 and 0.213 reflecting that all values, though descending, were insignificantly different from the baseline count.
Conclusions: Thus, it is a safe alternative in resource-poor setting to store stem cells in a cryoprotective agent like glycerin and freeze at -200C for up to 6 months, without significant depreciation in viability. It is recommended that this alternative be explored, while more researches are advocated to be conducted on larger number of participants with possibility of extending the number of months and using liquid nitrogen storage as gold standard.

Comparison Of Cell Counting Methods: Standard Hematology Analyzer Versus Volumetric Cell Counting Flow Cytometry.
Fabio Sgnotto, Maral Silva, Rodrigo Barroso, Guilherme Lopes, Felipe Maciel, Cecilia Konecsni, Maria Mirtes Sales
Hospital das Clnicas FMUSP, Sao Paulo, Brazil

Introduction: Cell counting is crucial for the hematology laboratory and many methods are available nowadays. Major neutrophils, lymphocytes and its subsets enumeration is very important for diagnosis and monitoring of a variety of conditions affecting the immune and hematopoietic system. Hematology analyzers use impedance and flow cytometry for cell number and percentage determination, as SYSMEX XE 2100 lines.  New flow cytometry technologies also use beadles volumetric counting for single platform enumeration showing precision and reproducibility.  In this study we demonstrated the comparison of volumetric cell counting and the standardized hematology analyzer one in peripheral blood (PB) and bone marrow (BM) samples.  
Methods: 14 BM and 17 PB samples were processed at the SYSMEX XE 2100 analyzer (one time) and at Beckman Coulter CytoFLEX flow cytometer (three times). The samples were antibodies free, and the results were compared using Paired t-test and Pearson's correlation.
Results: 14 BM samples had media count of 32571 (2450-108930) / uL cells in the SYSMEX XE 2100 and 32146 (2596-107008) / uL cells in the CytoFLEX (X2 = 1 p< 0,001 / paired t-Test p = 0,128) . The 17 PB samples had media count of 8545 (3860-23230) / uL cells in the SYSMEX XE 2100 and 8713 (3697-23509) / uL cells in the CytoFLEX (X2 = 0,996 p< 0,001 / paired t-Test p = 0,172). PB neutrophil absolute counting:  media count of 4178 (900-13250) / uL cells in the SYSMEX XE 2100 and 4125 (950-13150) / uL cells in the CytoFLEX (X2 = 0,994 p< 0,001 / paired t-Test p = 0,217) . PB Lymphocyte counting : media count of 3357 (540-6100) / uL cells in the SYSMEX XE 2100 and 2536 (529-7399) / uL cells in the CytoFLEX (X2 = 0,979 p< 0,001 / paired t-Test p = 0,454). PB monocyte counting : media count of 672 (113-3706) / uL cells in the SYSMEX XE 2100 and 798 (80-1620) / uL cells in the CytoFLEX (X2 = 0,961 p< 0,001 / paired t-Test p = 0,04).
Conclusions: These results show a good correlation between the two methods allowing the use of beadless volumetric cell counting for hematopoietic cell determination. Based on this we are intending to perform lymphocyte subset (CD4, CD8, CD19, CD20, CD16, CD56) enumeration and compare this new methodology with the standardized beads based one, looking forward to have less costly and time saving workflow.

Flow Cytometric Analysis Of Patients With Hereditary Spherocytosis &Ndash; An Indian Scenario.
Seema Tyagi1, Prabhu Manivanan2
1All India Institute of Medical Science, New Delhi, India, 2JIPMER, Pondichery, India

Introduction:   Tests such as red cell osmotic fragility test (OFT), acidified glycerol lysis time test, osmotic gradient ektacytometry, hypertonic cryohemolysis test, eosin-5-maleimide binding test and flow cytometric osmotic fragility test (FC-OFT) are used for diagnosis of hereditary spherocytosis(HS). Expensive and cumbersome molecular tests, like  sodium dodecyl sulfate polyacrylamide gel electrophoresis are available to diagnose the protein defects.  Still, incubated  red cell OFT is considered as the gold standard due to  its easy  availability  and simpler technique.  FC-OFT  is  a recently introduced screening test for HS. It is a simple, quantitative, sensitive and cost-effective technique for effective screening of  HS patients. This study was conducted to evaluate the utility of FC-OFT in all newly diagnosed cases of HS patients, to compare its diagnostic value with conventional  incubated OFT and to correlate with disease severity.
Methods: A total of 100 blood  samples  including  HS (n = 40), healthy subjects (n = 40) and beta-thalassemia traits (BTT) (n = 20).The percentage of residual red cells (%RRC) was measured using flow cytometer with red cell suspension. Subsequently, this was spiked with deionized water for FC-OFT. The percentage of residual red cells (%RRC value) was calculated from region R7 and R8 events.  Increased osmotic fragility is indicated by low %RRC value and  high % RRC ratio. The degree of hemolysis was expressed as “%RRC value” and was calculated by  formula given in the figure 1. Thus, a low %RRC value and a high %RRC ratio indicated increased FC-OFT.
Results: The receiver operator curve analysis defined the optimal cut-offs for FC-OFT-derived indices, such as %RRC value (≤16.29%) and % RRC ratio (>1.72), for HS cases vs  healthy subjects and BTT (p < 0.05). The FC-OFT (96%) achieved higher test efficiency than the conventional OF test (68.9%). A significant positive and a negative correlation were found between number of spherocytes/hpf and %RRC ratio (p = 0.001) and %RRC values (p = 0.0486). No significant correlation was observed between %RRC value (p = 0.8934), %RRC ratio (p = 0.6348) and HS disease severity score. 
Conclusions: FC-OFT could be a better screening test for HS cases in developing countries where molecular testing is not cost effective.

The Possible Significance Of Cd19 Positive Expression In Acute Promyelocytic Leukemia
Yael Zimra1, Zohar Rotem1, Doris Parnas1, Judy Chezar1, Ofir Wolach2, Pia Raanani2, Esther Rabizadeh1
1Hematology Laboratory, Beilinson Hospital, Petach tikva, Israel, 2Hematology Dep. Davidov center, Beilinson Hospital, Petach tikva, Israel

Introduction: Acute promyelocytic leukemia (APL) defined by t(15;17) or (PML-RARa) has a relatively favorable prognosis. Despite this, up to 25% of patients relapse after attaining complete remission.  Identification of prognostic markers and novel targets in APL may be useful in this setting. Expression of atypical immunophenotypic markers as, CD2, CD56, CD34, has been reported in APL. Some data suggested a correlation of these markers to microgranular morphology, bcr3 PML-RARa isoforms, poor outcome and early death. Expression of the B lymphoid antigen CD19 was reported in up to 15% of APL cases. To our knowledge, the significance of CD19 expression in APL has not been determined so far.
Methods: Between 1/1/2011 and 10/2018, 214 AML cases were diagnosed in our institute, including 17 APL cases. Different antibody combinations and fluorochromes were used to confirm CD19 positivity (>30%). Blood count, coagulation parameters, immunephenotypingandPML-RARa isoform types were collected. 
Results: Similar to the literature, we detected CD19 expression in 3.8% of none-APL-AML and in 50% of Mixed phenotype leukemia patients. However, the expression of CD19 in APL patients in our institute, was 47% compared to the reported  15%. Within the APL cases, no significant differences were found in bcr3 PML-RARa isoform, microgranular morphology, mean age of diagnosis (Table 1), white blood cell count and coagulating parameters (data not shown) between the CD19+ and CD19- APL. Only CD19+ APL expressed TdT and CD79a. The CD19+ group included more females (table1), higher aberrant antigen expression and multiple aberrations per patient (5 vs 1) compared to the CD19- group.
Conclusions: The significance of CD19+ blasts in APL is unclear. It may be important for understanding of the pathogenesis  and therapeutic implications.  Potential applications of CD19CART-cell therapy for CD19+AML cells has been recently suggested. This treatment may also be beneficial in cases of relapsed CD19+ APL patients. Further studies need to be conducted due to the small number of cases and the short follow-up times.

Identification Of Novel Chimeric Rnas In Cytogenetically Normal Acute Myeloid Leukaemia Using Deep Transcriptomic Sequencing
Angeli Ambayya1,3, Chong Siew Lian1, Yap Yee Yee1, Putri Intan Hafizah Megat Mohd Azlan1, Syed Carlo Edmund1, Leong Wai Mun2, Wee Kai Li2, Jameela Sathar1, Rosline Hassan3
1Hospital Ampang, Selangor, Malaysia, 2Neoscience, Selangor, Malaysia, 3Universiti Sains Malaysia, Kelantan, Malaysia

Introduction: Cytogenetically normal acute myeloid leukaemia (AML-CN) displays a heterogeneous response to therapy and overall outcome with underlying unknown genomic alterations undetectable by conventional analysis yet crucial in understanding the pathogenesis and dynamics of AML-CN.  In this study we performed deep transcriptome sequencing of nine AML-CN patients to comprehensively detect the presence of chimeric fusion RNAs.
Methods: Transcriptome sequencing was performed on Illumina NovaSeq 6000 with 150bp paired-end reads with an average coverage of 200 x106reads per sample on nine adult AML-CN patients who were treated with intensive chemotherapy. Raw sequence reads were cleaned and trimmed using Trimmomatic and only paired reads were selected for further analysis. TopHat-Fusion package (v2.1.0) was used to identify candidate chimeric fusion RNAs along with Bowtie aligner to align short reads to the genome. Customized algorithms were established to select putative chimeric transcripts for further validation.
Results: We identified 55 chimeric transcripts, of which 40 were formed between the adjacent genes in the same chromosome and distributed at different frequencies between the samples. Interestingly two novel chimeric transcripts, CTD-2583A14.10-ZNF552 (n=6) and ZNF746-ZNF767P (n=3) were unveiled in this study. The ZNF746-ZNF767P chimeric RNAs were exclusively distinguished in patients with poor overall survival (OS) whereas CTD-2583A14.10-ZNF552 was discerned in patients with poor OS as well as OS> 5 years.
Conclusions: Our findings suggest that the recurrent novel chimeric transcripts formed between adjacent genes as observed in this study may have possible roles in pathogenesis of AML-CN, paving a way in identification of new genetic markers. However, further studies in a larger cohort of AML spectrum is crucial to interrogate the clinical relevance of these chimeric transcripts exclusively in AML-CN.

Identifying Cml Stem Cells And Subpopulations As A Potential Solution For Prediction To Treatment Response And Cure
Jitakshi De1
1DeePath Medical Diagnostics, Costa Mesa, CA, United States, 2KromePath, Costa Mesa, CA, United States

Introduction: A major hurdle to cure of stem cell neoplasms such as chronic myeloid leukemia (CML) is treatment-resistant residual stem cell population(s) with relapse-conferring capability. Although BCR-ABL1-targeted tyrosine kinase inhibitors (TKIs) have demonstrated success in treatment of CML through selective kinase inhibition, the residual stem cell compartment poses risk of resistance and relapse. There is significant variation in BCR-ABL1 level (at diagnosis) and the rate of elimination, with the classic esponse curve a rapid initial (alpha) with a much slower (beta) elimination of BCR-ABL1.   About 40 – 60% of CML patients who achieve an optimal response on TKIs and sustain a deep molecular response experience relapse upon treatment discontinuation. Persistent stem cells are a source of BCR-ABL1 but is not a reliable predictor of outcome.The characteristic features for distinguishing CML progenitor cells are largely unknown, and primary CML cell identification based on activation of proliferative and/or apoptotic pathways has not previously been explored. Identification and characterization of CML cell types can shed light on features differentiating relapse-causing cells which could serve as a potential cell-based predictive marker in CML.
Results: In a n=1 case study by mass cytometry analysis of peripheral blood from a relapsed CML patient revealed unusual subpopulations of cells in the freshly fixed sample, based on markedly elevated p-STAT5 and p-p38MAPK activation levels. Features differentiating these subpopulations were lymphoid and myeloid lineage-specific markers, indicative of variable lineage commitment. A greater than 10-fold higher frequency of p-STAT5hi lymphoid cells compared to p-STAT5hi/CD34+ myeloid cells was found. Co-expression of an early development marker, IL-7 receptor, on the two CD19 and/or CD3-expressing lymphoid subsets of p-STAT5hi cells, which in toto comprised about 1% of the total leukocytes analyzed, raised the interesting possibility that these lymphoid cell types were co-activated with the much rarer (0.05%) CD34+ myeloid blast subset at relapse.
Conclusions: Further efforts to derive pure p-STAT5hi cell subpopulations from CML patients would illuminate the characteristics of these cell types, and explore whether STAT5 and p38 MAPK expression and activation correlate with that of BCR-ABL1. The significance of lymphoid progenitor cells in CML including possible role in immunity requires further study to identify novel cell-based biomarkers by routine cytometry.

Elucidation Of Prognostically Useful Gene-Expression Profiles In Cytogenetically Normal Acute Myeloid Leukaemia
Angeli Ambayya1, Leong Wai Mun2, Wee Kai Li2, Rosline Hassan1
1Universiti Sains Malaysia, Kelantan, Malaysia, 2Neoscience, Selangor, Malaysia

Introduction: Although about 50% of the acute myeloid leukaemia are cytogenetically normal (AML-CN), current classification system does not fully reflect the heterogeneity of the disease and variable responses have been observed in intensive chemotherapy. Although discoveries of molecular markers have begun to subdivide AML-CN, more accurate risk stratification is required to predict clinical outcome. In this study, we examined the gene expression patterns in AML-CN (with wild type NPM1 and FLT3-ITD) patients based on their response to intensive chemotherapy to identify molecular markers that could aid in prognostic stratification.   
Methods: Eight AML-CN patients were dichotomized based on the following criteria: NPM1 and FLT3-ITD wildtype and response to intensive chemotherapy (attainment of remission after intensive chemotherapy). Transcriptome sequencing was performed on Illumina NovaSeq 6000 with 150bp paired-end reads with an average coverage of 200 x106 reads per sample. Raw sequence data was processed using FASTQC v0.11.7 and low-quality data was removed using Trimmomatic v0.38. Hisat2 was used for data alignment (hg38) and identification of individual gene expressions using Cufflinks. Next, featureCounts v1.6.0 was used to count aligned reads by genomic features and followed by differentially expressed genes were studied using R/Bioconductor package DESeq2. Differentially expressed genes (DEGs) were determined by p-adjusted (padj) value and the log2 fold change (log2FC) after read count normalization using regularized logarithm (rlog) method provided in DESeq2.
Results: Rigorous analysis of the sequencing data revealed that 16 genes were differentially expressed between the AML-CN patients who attained their remission after induction and patients who failed to attain remission following intensive chemotherapy, including genes that are undiscussed in the context of AML pathogenesis. The most overexpressed genes in the patients who attained remission after intensive chemotherapy were TPTE2P1, RP1-35C21.1, GPC6, ROS1 and RP11-287D1.4 whereas the most under-expressed genes were IL1R2, PITX1, MMP7, SP7, ALOX15B, RP11-356M20.1, DDIT4, TNFRSF18, AC147651.3, FKBP5 and GAS2L1. We discovered that under-expression of IL1R2 gene was observed in AML-CN patients who remained longer under complete remission following intensive chemotherapy.
Conclusions: Our study demonstrates that high throughput deep transcriptomes sequencing leads to discovery of discriminatory genes that distinguishes AML-CN patient’s outcome following intensive chemotherapy.

Next Generation Sequencing In Predicting Outcome In Mds- Dawn Of New Era In Pakistan
Nida Anwar, Aisha Arshad, Saba Shahid, Shariq Ahmed, Naveena Fatima, Tahir Shamsi
National Institute of blood diseases BMT, Karachi, Pakistan

Introduction: Myelodysplastic syndromes (MDS) are a heterogenous group of hematologic malignancies characterized by clonal expansion of bone marrow myeloid cells with impaired differentiation. Studies utilizing next-generation sequencing (NGS) have identified a core set of recurrently mutated genes in the majority of patients with AML and MDS. The identification of recurrent mutations in MDS has led to incipient insights into the pathophysiology of this disorder including those with mundane cytogenetics. DNA-level mutations in several of these genes including ASXL1, ETV6, EZH2, RUNX1 and TP53 in MDS contribute to disease pathogenesis, outcome and currently is an active area of research. Our institution is first in the country to perform next generation sequencing analysis and this study was done to assess mutation analysis in MDS and correlate the data with the clinical outcome of the patients.
Methods: A total of 20 MDS diagnosed patients were included. Informed consent and detailed history was taken from all patients. The next generation sequencing analysis for a panel of 54 genes including tumor suppressor and oncogenic hotspots associated with myeloid malignancies (AML, MDS, MPN, CMML and JMML) was performed using DNA samples.
Results: A total of 20 MDS patients meeting the criteria of WHO classification were included. The mean age of patients was 42 years. Most common presenting complain was weakness in 13 (65%) patients followed by fever and lethargy in 6 (30%). The mean IPSS was 01. Cytogenetic analysis revealed 13(65%) patients with abnormal karyotype. Using next generation sequence analysis, 10 (50%) patients had mutations including 2 novel mutations {RUNX1 p.lle428Thr(het) and GATA2 p.Thr358Pro} and 8 reported mutations including p.Pro384Leu (het) RunX1,p. Pro75Leu CDKN2A,Tet-2 c5162 T>G mutation,p.Gln1039Ter (het) ASXL1,p.Gly12Ser , NRAS and DNMT3A NPM. Mutations in MDS are associated with changes in patient outcomes and it was found statistically significant. Numbers of expiry in patients identified with mutations were comparably higher than those who did not carry any mutation.
Conclusions: Multiple mutations are required for MDS initiation and progression to acute myeloid leukemia. These gene mutations can be used to monitor patient disease burden through the use of ultrasensitive detection techniques like NGS. Additional studies will be required to understand the prognostic implications of these mutations.

Application Of Whole-Exome Sequencing Technology In Diagnosis Of Inherited Platelet Disorders
Xia Bai, Hongjie Shen, Jundan Xie, Miao Jiang, Changgeng Ruan
The First Affiliated Hospital of Soochow University, suzhou, China

Introduction: Inherited platelet disorders (IPDs) are a group of hereditary hematologic diseases that affect platelet number and function. The incidence of most types is very low and often misdiagnosed as immune thrombocytopenia (ITP).  Clinically, it is urgently needed for a convenient and rapid diagnosis. We introduced the whole-exome sequencing (WES) into the routine diagnosis of IPDs patients and analyzed the results of gene sequencing.
Methods: We screened 75 patients with hereditary features from 302 patients with platelet disorder. The blood samples of these patients were performed pathogenic gene detection using WES technology. The pathogenic variations detected were verified by Sanger sequencing.
Results: Of the 75 patients, 89 variations in 41 genes were detected.  Combined with clinical features and other findings, based on the ACMG guideline, 23 patients (30.7%) were diagnosed with IPDs.  Of these 23 patients, a total of 29 pathogenic variations that cause IPDs were detected, and 8 of them are newly discovered.
Conclusions: Combined with traditional morphological and functional tests, WES could play an important role in the molecular pathology diagnosis of inherited platelet disorders.

Role Of Hoct1 Gene Expression, Sequence Polymorphism & Promoter Hypermethylation On Response To Imatinib In Cml
Namrata Bhutani1, alpana saxena2
1vardhman mahavir medical college, New Delhi, India, 2maulana azad medical college, new delhi, India

Introduction: hOCT1 (Human Organic Cation Transporter 1)mediates influx of imatinib into cells.Variation in clinical response to Imatinib has been observed with pretherapeutic expression & nonsynonymous SNPs ,namely M420del and M408V in some populations.Role of promoter hypermethylation of hOCT1 gene in CML has not been studied much.
Methods: 30 newly diagnosed BCR-ABL positive CP-CML patients &30 healthy controls were recruited. hOCT1 gene expression  was quantified by SYBR Green based qRT-PCR. M420del &M408V SNPs were examined by allele specific PCR.of hOCT1 gene hypermethylation was studied by Methylation Specific PCR.After initiation of imatinib therapy ,hematological response monitored at regular intervals&molecular response (BCR-ABL1/ABL1 ratio) assessed after 6 or 12 months.  
Results: Cases were divided into two groups, high expression(n=15) & low expression (n=15), based on median fold change in hOCT1 gene expression.(median =5.6).11 (73.33%)  low expression patients achieved CHR by the end of 3 months.all patients with high hOCT1 gene expression achieved CHR by the end of 3 months.(p=0.10).also mean time to CHR in low expression group was higher.(p=0.046).While all 15 patients with high expression had an optimal response,only 13.33 %  patients with low expression had it. 40% low expression patients had treatment failure(n=6)while remaining 23.33 % were categorized as warning.(p=0.000) Minor allele frequencies for M420del were 0.18&0.1 in CML patients and controls ; for M408V 0.4 and 0.27 respectively.No significant  association between different genotypes of M420del  and M408V observed with achieving response to imatinib.To analyze the combined effect of  two SNPs,cases  divided into 4 groups.Patients with mutant M420del & wild type homozygous M408V ,failed to achieve an optimal molecular response to imatinib, unlike those with mutant  genotypes for both SNPs (p=0.02). Methylation was seen in 83.33 % of CML cases whereas in only 23.33 % of controls.(p=0.001)Methylation was observed in all 15 patients(100%) with low expression whereas 10  high expression patients(66.66%)were methylation positive.(p=0.042) No significant association was seen between methylation status and achievement of CHR or optimal molecular response to imatinib.
Conclusions:  high expression of hOCT1 gene leads to early achievement of CHR & significantly associated with achievement of optimal response to imatinib.Mutant M420del allele may be linked to poor outcome of imatinib treatment in CML,however simultaneous presence of mutant M408V allele appears to circumvent this effect.Promoter hypermethylation of hOCT1 gene leads to silencing of gene expression.

Panel Genotyping Of Pharmacogenomic And Application In A Case Of Acute Lymphoblastic Leukemia With Hematopoietic Stem Cell Transplantation
Jiaqi Chen1,2, Yang Zhang1,2, Fang Wang1,2, Yu Li3, Yuanli Xu2, Hongxing Liu1,2,3
1Beijing Lu Daopei Institute of Hematology, Beijing, China, 2HebeiYanda Lu Daopei Hospital, Langfang/Hebei, China, 3Beijing Lu Daopei Hospital, Beijing, China

Introduction: To assist clinicians in optimizing the individualized medication for a patient with acute lymphoblastic leukemia who had hematopoietic stem cell transplantation by using pharmacogenomic test.
Methods: A B-ALL patient was enrolled and given a pharmacogenomic panel test. We customized the panel based on the Ion torrent PGM platform, consulted PharmGKB database and related guideline and finally selected 17 genes associated with seven commonly used drugs of hematological diseases (eg.methotrexate, tacrolimus, cyclosporine A) .
Results: The patient (male, 31 years old, 50 kg.) was diagnosed as BCR-ABL1-positive B-ALL on December 29, 2016, and was given prednisone-induced chemotherapy combined with dasatinib. On February 8, 2017, the patient was in complete remission by bone marrow morphology examination. However, the patient relapsed at the 5th month after the initial diagnosis with 1.09% of BCR-ABL1 fusion gene.On June 14, he was admitted to our hospital for further treatment and tested with the pharmacogenomic panel. The results showed that MTHFR (c.677C>T) was TC heterozygous; ABCB1 (c.3435T>C ) was TC heterozygous and CYP3A5*3 (c.6986G>A) was GA heterozygous,the metabolic capacity of CYP3A5 expresser increased (Table 1).Evaluate patients with a higher risk of drug toxicity when using methotrexate, which is recommended to be adjusted to 2/3 of the conventional dose, he has no adverse reactions such as hematemesis and headacheand, he successfully underwent allo-HSCT at the 9th month after initial diagnosis. It is recommended that the initial dose of tacrolimus be adjusted to 1.5 times the conventional dose (initial dose is 1.1 mg qd). So during the treatment, the dosage of tacrolimus given to him was higher than the conventional one (Fig. 1). After transplantation, the patient experienced GVHD involving multiple organs which was significantly improved after treatment, and discharged after 61 days of transplantation. As of September 12, 2018, he was reviewed for 1 year, the signs were stable with micro-residues were negative, BCR-ABL1 fusion gene quantitatively negative and complete donor chimerism status.      
Conclusions: Panel genotyping of pharmacogenomics can help clinicians optimize the individualized drug administration in hematological patients with complicated medications, further improve the efficacy of drugs, minimize adverse reactions and reduce the cost of treatment.

The Clinical And Prognostic Significance OfFis1, Spi1, Pdcd7AndAng2Expression Levels In Acute Myeloid Leukemia
Marwa Gamaleldin1, Reham Aboelwafa1, Omar Ghallab1
1Alexandria Faculty of Medicine, Alexandria, Egypt, 2Alexandria Faculty of Medicine, Alexandria, Egypt, 3Alexandria Faculty of Medicine, Alexandria, Egypt

Introduction: The marked heterogeneity of acute myeloid leukemia (AML) renders precisely predicting patient prognosis extremely difficult. Genetic alterations, fusions and mutations, may result in misexpression of key genes in AML. We aimed to investigate the expression patterns of 4 novel genes; FIS1, SPI1, PDCD7 and Ang2 to determine their potential prognostic role in AML patients.
Methods: Bone marrow mononuclear cells were analyzed for of FIS1, SPI1, PDCD7 and Ang2 expression levels by real-time quantitative PCR as well as of FLT3/ITD and NPM1 mutations in 100 newly diagnosed cytogenetically normal (CN-AML) patients, and 100 non-malignant controls.
Results: FIS1, SPI1, PDCD7 and Ang2 were significantly overexpressed in CN-AML patients (p< 0.001). Their high expression levels were significantly associated with lower complete remission (CR) rate, shorter relapse-free survival (RFS) and overall survival (OS). On multivariate analysis, high FIS1 expression showed a significant impact on CR response after induction therapy (OR=88.777, 95% CI 2.85–2765.78, p=0.011) while high PDCD7 appeared to be independent risk factor for RFS (HR=5.107, 95% CI 1.731–15.066, p=0.003) and OS (HR=7.353, 95% CI 1.859–29.079, p=0.004) in CN-AML patients.
Conclusions: FIS1 and PDCD7 expression are considered independent risk factors and should be integrated into the current AML stratification system.

&Ldquo;Antioxidrogram&Rdquo; As A New Useful Molecular Biomarker For The Diagnosis And Follow-Up Of Myelodysplastic Syndromes
Olivier Herault1,2, Christine Vignon2, Frederic Picou1,2, Sbastien Lachot1, Amlie Foucault1,2, Nomie Ravalet1,2, Marie-Hlne Estienne1, Olivier Kosmider3, Michaela Fontenay3, Emmanuel Gyan4
1CHRU de Tours, Service dhmatologie biologique, Tours, France, 2CNRS ERL7001 LNOx, Tours, France, 3APHP, Institut Cochin, Service d'Hmatologie Biologique, Paris, France, 4CHRU de Tours, Service dhmatologie et therapie cellulaire, Tours, France

Introduction: Myelodysplastic syndromes (MDS) are heterogeneous group of clonal stem cell disorders with an inherent tendency for leukemic transformation in secondary acute myeloid leukemia (sAML). This study focused on the redox metabolism of bone marrow (BM) cells from 97 patients compared to 16 healthy controls. By comparing these samples, we found an increase in intracellular reactive oxygen species (ROS) level in CD34posCD38low cells and we determined specific antioxidant signatures of clinical interest in low-grade MDS, high-grade MDS and sAML.
Methods: The level of reactive oxygen species (ROS) was quantified in the subpopulations of BM cells by flow cytometry (BD FACSCanto II) after CM-H2DCFDA staining. The expression level of 28 transcripts was determined by qRT-PCR using Universal Probe Library technology on Roche LightCycler 480 encoding for major enzymes implicated in the antioxidant cellular response (PRDXGLRX and GPX families and CAT). All statistical analyses were conducted using R v3.2.2 software.
Results: Our results highlight increased ROS level in BM non-lymphoid cells and especially in primitive CD34posCD38low progenitor cells. Moreover, we identified a specific antioxidant signature, named antioxidogram, for the different MDS entities or sAML. More particularly, the progression of the disease is characterized by a 3-step response at the molecular level: (i) overexpression of enzymes reducing disulfides bonds in protein (low-grade MDS - < 5% blasts - GLRX family) then (ii) increased expression of enzymes detoxifying H2O2 (high-grade MDS - 5% blasts - PRDX and GPX families), and finally (iii) decreased expression of these enzymes in sAML. The antioxidant score (AO-Score) defined by logistic regression from the expression levels of transcripts made it possible to score the stage of disease progression, and interestingly, AO-Score was independent of the IPSS-R.
Conclusions: Altogether, this study demonstrates that MDS and sAML present an important disturbance of redox metabolism, especially in BM stem and progenitor cells and that the specific molecular antioxidant response (antioxidogram, AO-Score) could be considered as biomarkers useful for diagnosis and follow-up of the disease. The antioxidogram is patented (publication number: WO2012085188 A1).

Association With Tp53 Codon 72 Polymorphism And The Risk Of Chronic Lymphocytic Leukemia In Sudanese Population
ibrahim khidir , Ameen Abdelaziz
Al neelain university, khartoum, Sudan

Introduction: The P53 tumor suppressor is a transcription factor that plays a central role in preventing tumorgenesis by inducing apoptosis, senescence, and/or cell-cycle arrest in response to various cellular stresses.In addition to this, it also has a critical role in regulating tumor growth and survival in CLL demonstrated by the fact that mutational inactivation of this tumor suppressor is associated with aggressive disease and poor prognosis. This present study was conducted to examine the association between the P53 Arg72Pro polymorphism and Risk of Chronic Lymphocytic Leukemia.
Methods: A total of 110 patients newly diagnosed with chronic lymphocytic leukemia seen over a year ( April 2017-April 2018) at Flowcytometry Laboratory Center Khartoum- Sudan and 80 control subjects were enrolled in this study. Analysis of the P53 exon 4 codon 72 (rs1042522) genotype was performed by Allele-Specific PCR (AS-PCR Data was analyzed by SPSS Ver 23 to compare means according to age and sex with variance of means and correlations.
Results: The Arg/Pro was the most frequent genotype in patients with CLL (50%), followed by Arg/Arg (25.5%) and Pro/Pro (24.5%) whereas in healthy control group Arg/Pro was the most frequent (47.5%), followed by Arg/Arg (45%) and Pro/Pro (7.5%) . These frequencies were consistent with the Hardy-Weinberg equilibrium (HWE). In our patient's samples, the allelic frequencies of Arg and Pro were 0.50 while in control samples were 0.69 and 0.31, respectively. The Pro/Pro genotype of P53 SNP showed higher risk of B-CLL compared with Arg/Arg; which was statistically significant (P value=0.002198, OR4.01, 95% CI=1.57-10.26). Not surprising, the risk was lower when adding Arg/Pro + Pro/Pro in patients compared to controls (P value=0.004887, OR 2.4, 95% CI=1.3-4.43), . Not only P53 codon 72 genotypes but also P53 allele’s frequencies were studied. Arg and Pro allele’s frequencies of P53 codon 72 SNP in CLL patients were 110 (50%). Compared to these alleles in the control group, 110.4 (69%) and 49.6 (31%), respectively. The Pro allele showed higher risk compared with Arg allele (P value=0.000211, OR 2.23, 95% CI=1.45-3.41
Conclusions: Our result suggested that P53 Pro72Arg (rs1042522)  polymorphism contribute to increasing B-Chronic Lymphocytic Leukemia risk in our population.

Mutational Status Of Nucleophosmin Gene In Cytogenetically Normal Acute Myeloid Leukaemia: Its Associations With Specific Clinical Profiles And Concomitant Molecular Abnormalities
Si Jie Khoo, Gee Fung How, Charles Chuah
Department of Haematology, Singapore General Hospital, Singapore, Singapore

Introduction: Genomic mutations in Acute Myeloid Leukaemia (AML) are associated with disease outcome. Frameshift insertions of Nucleophosmin gene (NPM1) are important markers for AML and are already included as a provisional entity of AML in the WHO classification in 2008. Several different NPM1 insertions have been identified, with the Type A variant being the most common, constituting approximately 70% in adult AML. Some studies found that patients with different variant types had different outcomes whereas others did not. The influence of coexisting mutations was also unclear.
Methods: In this study, we evaluated the mutation status of NPM1 in 124 cytogenetically normal AML patients. Genetic alterations in FLT3, DNMT3A, IDH1 and IDH2 were also determined. Correlation of NPM1 mutational status with other genetic mutations and biological parameters was investigated.
Results: A total of 75(60%) patients were identified with NPM1 exon 12 mutations. Type A mutation was found most frequently (n=57), constituting 76% of all variants detected. Of the remaining 18 variants, type D was the most common (n=7). Other mutation types detected were B (n=1), Km (n=2), Pm (n=2) and Qm (n=1) while there were also 4 novel variants. Correlation of other gene mutations and clinical parameters with NPM1 mutational status was determined by Fisher exact test (categorical variables) and Mann Whitney U test (continuous variables). Concomitant occurrences of DNMT3A and FLT3/ITD mutations with NPM1 mutations were statistically significant (n=21, p=0.0023 and n=39, p=0.0014 respectively).Platelet counts were significantly higher in NPM1-positive patients than in NPM1-negative patients (p=0.0034).NPM1 mutation status also appeared to be associated with FAB subtype (p=0.0182), with incidence in M2 subtype being significantly lower (p=0.0190). Other biological parameters such as age, gender, white blood cell count and blast count showed no difference between NPM1-wild type and NPM1-mutated groups. There was also no correlation of IDH1 and IDH2 mutational status with NPM1 mutations.
Conclusions: Although this study cohort is not large, the significant association of FLT3/ITD and DNMT3A mutations with NPM1 mutations corroborates the evaluation of DNMT3A mutation status together with FLT3 mutations in NPM1-mutated patients. Further work to investigate the relationships of coexisting mutations including event-free and overall survival analysis in a larger patient cohort needs to be conducted.

A Case Of Chronic Neutrophilia Leukemia With ConcurrentCsf3R, Setbp1, AndCalrMutation
Sang-Gyung Kim1, A-Jin Lee1, Cheon Gang Park1, Soon-Ho Mun1, Sung Hwa Bae2
1Department of Laboratory Medicine, Daegu Catholic University School of Medicine, Daegu, Korea, 2Department of Internal Medicine, Daegu Catholic University School of Medicine, Daegu, Korea

Introduction: Chronic neutrophilic leukemia (CNL) is a rare myeloproliferative neoplasm characterized by sustained neutrophilia, splenomegaly, hypercellular bone marrow without BCR-ABL1fusion transcript. Colony-stimulating factor 3 receptor gene (CSF3R) gene mutation has been associated with CNL. 

Methods: Here, we report a CSF3R T618I-mutated CNL patient with concurrent mutation. 
Results: A 69-yr-old man presented in 2016 with systemic erythematous papules, pustules, crusts and marked neutrophilia. Peripheral blood parameters were as follows: white cell count 91.2 x 109/L with a differential of 80% neutrophils, 12% band neutrophils, 2% metamyelocytes, 1% myelocytes, 2% promyelocytes, and 3% lymphocytes, Hb 8.8 g/dl, platelets 110 x 109/L. A physical examination and computed tomography of the abdomen demonstrated splenomegaly. Bone marrow aspirate showed hypercellularity with granulocytic hyperplasia. Chromosomal study showed 46,XY[20]. There was no evidence of JAK2V617F and BCR-ABL1rearrangement. The patient was treated with hydroxyurea. However, his neutrophilia persistently progressed. About 6 months later, he developed left upper quadrant pain. Leukoerythroblastic feature was also shown at the peripheral blood smear. The CSF3RT618I mutation was detected using direct sequencing. He was treated with additional interferon-alpha, however, he expired due to pneumonia. Targeted next-generation sequencing (NGS) was retrospectively performed. In addition to CSF3Rmutation, missense mutations in SRSF2, SETBP1, CALRgenes were detected. In ASXL1gene, nonsense mutation (c.1773C>G, p.Tyr591Ter) was detected. 
Conclusions: At diagnosis, targeted NGS panel test can help risk stratification and guide therapeutic strategy.  

Evaluation Of Dengue And Malaria Speciation Suspect Flags On Compact 5 Part Differential Horiba Medical Blood Cell Counter
Parag Dharap1, Sebastien Raimbault2
1Dr. Dharap's Diagnostic Centre, Mumbai, India, 2Horiba Medical, Montpellier, France

Introduction: Though spread by different subspecies of vector insects, Malaria and Dengue are frequently found to be coexistent in economically challenged endemic area of developing countries. Common symptomatology poses challenges to health care givers.  Encouraged by the performance of a previous malaria flagging algorithm applications developed through contemporary computer machine-learning techniques on blood cell counter, Horiba decided to develop similar tools to screen for Dengue fever, as well as sub speciation of Malarial parasites, as a part of continual improvement endeavor. We evaluated in this study the performance of these flags on the HORIBA Yumizen YH 550 analyzer.
Methods: Residual blood specimens were serially analyzed on Horiba Yumizen YH-550 analyzer. Besides the primary patient selection criteria of fever, diagnostic microscopic blood smear examination for malarial parasite and confirmation by antigenic testing for Malaria and Dengue NS1 antigen were performed for all cases. Malaria cases were classified for speciation, dominant life cycle forms and parasite density. Persons for routine health checkup without history of specific ailments were considered the control group (N = 60). Samples were analyzed (N=1424, Vivax Positive N=186, Falciparum Positive N=21, Dengue fever N=217, other Pyrexia N=941) with prototype integrated instrument software comparing flagging results obtained with confirmatory testing. Results were statistically analyzed for sensitivity, specificity, predictive values, likelihood ratio and diagnostic accuracy with ROC curves in comparison with routine laboratory confirmatory methods, except for Falciparum cases due to insufficiency of cases.
Disease Specific Flag ROC AUC Sensitivity Specificity PPV NPV Youden PLR NLR
P. Vivax - default (0.50) 0.900 0.663 0.989 0.901 0.953 0.652 62.3 0.034
P. Vivax -optimized (0.31) 0.900 0.730 0.980 0.840 0.960 0.713 35.7 0.272
Dengue Fever 0.810 0.789 0.715 0.308 0.951 0.504 2.770 0.030
Performance of malaria flags is shown at both default and optimized cut-offs. The performance for vivax malaria flag was linked to the observed microscopic level of parasitemia. Due to
Conclusions: Many diseases affect different blood cell parameters in direct or indirect way resulting in detectable or undetectable changes in blood cell counts. With the help of computer assisted machine learning techniques it is possible to develop an effective, economical and helpful tool (suspect flags) to predict, screen and diagnose specific ‘signatures’ of the disease, as malaria and dengue.

Performance Evaluation Of Oncomine Myeloid Research Assay For Acute Myeloid Leukemia
Jae Hee Lee1, Jong Ho Lee1, Chae Hoon Lee1
1Yeungnam University Medical Center, Daegu, South Korea, 2Yeungnam University Medical Center, Daegu, South Korea, 3Yeungnam University Medical Center, Daegu, South Korea

Introduction: Recently, many next-generation sequencing (NGS) panels about acute myeloid leukemia (AML) are accessible to screen for pathogenic mutations of AML related genes. But the process of planning and familiarization of customized NGS panel can be difficult for a small clinical laboratory. In this study, we evaluated the analytical performance of Oncomine Myeloid Research Assay, a commercial NGS panel contained automated library preperation, including accuracy, precision, limit of detection (LOD) and total hands-on time for estimating labor intensity.
Methods: Total 32 reactions through 4 batches (each batch can analyze 8 samples) were performed by Ion Torrent S5 and Ion Chef system using 15 clinical specimen, 2 commercial reference materials and on specimen of external quality assessement service. The criteria of quality assurance in sequencing were total bases > 8x10^8, total reads > 4x10^6, and usable reads % x total reads > 4x10^6. Bioinformatics pipeline was Oncomine Myeloid Research 530 w2.3.1. The criteria of quality assurance in sequencing data were mean depth >x300, on target % >80%, and uniformity > 80%. Genetic variantions validated were single nucleotide polymorphism (SNP), small insertion and deletion such as FLT-ITD. The specimen of LOD evaluation were prepared by mixing reference material and clinical samples which were identified variant allele frequency (VAF) by previous NGS analysis. Validated LOD ranged 10%, 5%, 2.5% of VAF.  
Results: All reactions passed the criterial of quality assurance of sequencing and bioinformatics process. The average of total bases, total reads and usable reads were 16.5x10^8, 15.9x10^6, and 9x10^6 respectively. The average of mean depth, on target rate, and uniformity were 1,697, 97.72 and 98.13. The accuracy of clinical samples and reference material was 100%. The precision assessment also showed consistent results in inter-run and intra-run process with similar VAF. A proper LOD was measured at 5% through this evaluation. But the results of FLT-ITD plug-in, a seperated program provided by the manufacturer, showed higher VAF for FLT-ITD variants than that of main results in Ion Reporter. The average of total hands-on time was 83 mininutes.
Conclusions: The Oncomine Myeloid Research Assay can provided reliable results of detection of diverse variation in AML and relatively short hands-on time.

Detection Of Fusion Genes Using Targeted Rna Sequencing (Rnaseq) In Leukemia Patients
Ha Jin Lim1, Jun Hyung Lee1, Seung Yeob Lee1, Sejong Chun1, Seung-Jung Kee1, Soo-Hyun Kim1, Jong-Hee Shin1, Seo-Yeon Ahn2, Hyeoung-Joon Kim2, Myung-Geun Shin1,3
1Department of Laboratory Medicine, Chonnam National University Medical School and Chonnam National University Hwasun Hospital, Hwasun, South Korea, 2Department of Hematology-Oncology, Chonnam National University Medical School and Chonnam National University Hwasun Hospital, Hwasun, South Korea, 3Brain Korea 21 Project, Center for Biomedical Human Resources, Chonnam National University, Gwangju, South Korea

Introduction: Gene fusions caused by recurrent genetic abnormalities play key roles in the pathophysiology of leukemia. Detection of such fusions plays important roles in diagnosis, risk stratification, targeted therapy and follow-up of disease. Conventional methods used to detect fusion genes are sensitive but detect only specified breakpoints. RNA sequencing (RNAseq) of Next-generation sequencing (NGS) overcomes the limitation of conventional methods, but adequate filtering strategies are essential. We established such a strategy and compared our results to those of multiplex RT-PCR.
Methods: Nine bone marrow specimens from patients with acute lymphoblastic leukemia/lymphoma (four cases), acute myeloid leukemia (one case), and chronic myeloid leukemia (four cases), diagnosed in Chonnam National University Hwasun Hospital from September to December 2017, were tested. We subjected extracted RNA from specimens to multiplex RT-PCR (HemaVision kit; DNA Technology, Aarhus, Denmark). Targeted RNAseq was performed using the HEMEaccuTest Library Prep kit (NGeneBio, Seoul, Korea) that targeting 53 genes associated with hematologic malignancies (5 housekeeping genes served as controls). The RNAseq data were analyzed using STAR and STAR-fusion software (figure 1).
Results: The RNAseq data of nine specimens yielded 133 fusion candidates detected within an average of 64 min. We employed six filtering strategies (Figure 1); we chose fusion genes for which the read counts and qualities were good, and that encoded functional oncogenic proteins; this strategy identified 12 final candidates. We detected all fusion genes (BCR-ABL1, ETV6-RUNX1, KMT2A-AFF1, and RUNX1-RUNX1T1) previously identified by multiplex RT-PCR (Table 1). We found two additional fusion breakpoints in ETV6-RUNX1 and KMT2A-AFF1; these had not been previously detected.
Conclusions:   RNAseq used to detect fusion genes associated with leukemia was more sensitive than conventional multiplex RT-PCR in that RNAseq detected additional breakpoint of fusion genes. Thus, detection of fused genes via targeted RNAseq will facilitate the implementation of personalized medicine; and provide more accurate molecular targeting during diagnosis, treatment, and follow-up of patients with leukemia.

Gstm1 And Gstt1 Polymorphisms And Susceptibility To Acute Myeloid Leukemia: A Case-Control Study Of The Sudanese Population
ibrahim Osman, ebtihal ahmed, nour mahmoud, saadia osman
Al neelain university, khartoum, SC, Sudan

Introduction: Glutathione S-transferase (GST) enzyme levels are associated with risk of many cancers, including hematological tumors. We here aimed to investigate the relationships between GSTM1 and GSTT1 polymorphisms and the risk of AML. Conflicts in the published results and the absence of similar in depth studies in Sudan prompted us to perform the present case-control study of GSTM1 and GSTT1 polymorphisms and their possible association with AML in a Sudanese population. 
Methods: A total of 40 patients with AML and 40 control subjects were enrolled in this study. Blood samples were collected from all patients in EDTA. Genomic DNA was extracted from all blood samples using salting out method.Genotyping for detection of GSTM1, and GSTT1 polymorphisms was performed for both patients and controls using a multiplex PCR 
Results: We reported that there is an association between the GSTM1 null genotype and AML risk (OR= 2.7, 95% CI= 1.2-6.04; P = 0.012), the GSTT1 null genotype appeared also to have an influence in the development of AML (OR= 4.93, 95% CI= 1.6-15.07; P = 0.005). 
Conclusions: These findings indicate that genetic variants of GST s (GSTM1 and GSTT1) genes may increase individual susceptibility to AML.

High Level Of Gdf-15 In Ineffective Erythropoiesis: Cause, Effect, Or Biomarker?
Reza Ranjbaran1, Elahe Rahimian1, Mojdeh Abbasi1,2, Niloofar Amirian1, Negin Shokrgozar1, Gholamreza Rafiei Dehbidi1, Noorossadat Seyyedi1, Farahnaz Zare1, Abbas Behzad-behbahani1
1Diagnostic Laboratory Sciences and Technology Research Center, School of Paramedical Sciences, Shiraz University of Medical Sciences, Shiraz, Iran, 2Faculty of Medicine and Health Sciences, Macquarie University, Sydney, NSW, Australia

Introduction: Growth differentiation factor-15 (GDF-15), a member of TGF-β superfamily, is mainly produced by erythroid series and has been recently introduced as a novel biomarker of ineffective erythropoiesis (IE). However, whether IE results in enhanced expression of GDF-15 or is prompted by, has remained elusive. According to the identified role of GDF-15 in cell maturation arrest and apoptosis activation, this study aimed to explore the possible mechanisms by which high levels of GDF-15 contributes to ineffective erythropoiesis.
Methods: GDF-15 mRNA level was assessed using qRT-PCR during erythroid maturation and in peripheral blood of patients with IE, where association of its expression with erythropoiesis severity biomarkers was evaluated. GDF-15-treated hematopoietic cell lines of different origin were subjected to CFSE proliferation and apoptosis assays by flow cytometry. Colony forming cell assay was performed to evaluate any changes in erythroid colony forming capacity of GDF-15-exposed hematopoietic stem cells (HSCs). The effects of GDF-15 high levels on maturation of differentiating erythroid progenitor cells were analyzed by expression analysis of erythroid-associated transcription factors (TF) and flow cytometry assessment of CD235a expression.
Results: GDF-15 mRNA significantly increased during erythroid differentiation as well as in β-thalassemia and MDS patients which was directly correlated with erythropoiesis severity. Moreover, GDF-15 high levels induced apoptosis by nearly 15% in erythroleukemia cell line and CFSE assay confirmed dose-dependent inhibitory effect of GDF-15 on erythroid cell proliferation. Erythroid colony formation of HSCs and the number of CD235a positive cells were also significantly reduced when treated with GDF-15 (%15±4 and 30±2% respectively) and β-thalassemia serum with high GDF-15 levels (%13±2 and 31±6 respectively). Being treated with GDF-15, erythroid-specific TFs were significantly down-regulated in early erythroid stages whereas no significant variation was observed in late-stages.
Conclusions: These findings suggest that GDF-15 high levels and IE could be reciprocal positive regulators of each other. The more severe IE the more compensatory erythropoietic activity and the resultant higher GDF-15-producing cells which will ultimately raise GDF-15-mediated erythroid death and maturation impairment. Conclusively, high level GDF-15 is a regulatory response in modulating excessive erythropoiesis.

Microrna-126 As APlasmaBiomarkerFor TheDiagnosisOfDvt
Fei Shen, Liqian Xie, Yunxiao Zhao, Jeney Ma
The First Affiliated Hospital of Soochow University, Suzhou, China

Introduction: DVT is the formation of thrombi in the deep veins, most commonly the large veins of the legs or pelvis. Numerous efforts have been made to identify reliable and predictive biomarkers to detect the early signs of deep vein thrombosis. Circulating microRNAs (miRNAs) are stably present in the plasma. Their diagnostic function has been investigated in many diseases. The aims of present study were to assess the diagnostic ability of plasma miRNA-126 in DVT.
Methods: 47 DVT patients, 36 healthy controls were enrolled in this study. The TaqMan miRNA microarray was used to identify dysregulated miRNAs in the plasma of APE patients. The TaqMan-based miRNA quantitative real-time reverse transcription polymerase chain reactions were used to validate the dysregulated miRNAs. The receiver-operator characteristic (ROC) curve analysis was conducted to evaluate the diagnostic accuracy of the miRNA-126 identified as the candidate biomarker.
Results: Plasma miRNA-126 level was significantly higher in the DVT patients than in the normal controls. The ROC curve showed that plasma miR-126 was a specific diagnostic predictor of DVT with an area under the curve of 0.782 (95% confidence interval, P < 0.01).
Conclusions: Our findings indicated that plasma miR-126 is associated with DVT and it could be an important biomarker for the diagnosis of DVT.

Factors Associated With Mother-To-Child Hiv Transmission In Western Nigeria: The Importance Of 90-90-90 Goals In The Elimination Strategy
Saheed Usman1, Prosper Okonkwo2, Oluwatoyin Jolayemi2, Jay Osi-Samuels2, Patrick Akande2, Babatunde Ladi-Akinyemi2, Oluremi Olaitan2, Femi Owolagba2, Matthias Alagi2, Eke Ofuche2, Chiedozi Akueshi2, Babatunde Akinbinu2, Adetosoye Adebanjo2, Jennifer Ale2, Chisom Udechukwu2
1Nnamdi Azikiwe University, Awka, Nigeria, 2APIN Public Health Initiatives, Abuja, Nigeria

Introduction: HIV pandemic has continued to be a huge challenge in Nigeria, with the problem of stigmatization reducing the chances of early determination of the HIV status of pregnant women. Hypotheses tested were the influence of maternal antiretroviral therapy (ART) use and infant’s feeding option on baby’s final early infant diagnosis (EID) outcome. The study was aimed at evaluating the variables associated with mother-to-child transmission of HIV & the factors associated with the transmission of the infection from the mothers.
Methods: This study was a prospective cohort study of HIV-exposed infants conducted in Western Nigeria, between January 2015 and September 2017. Dried Blood Spots (DBS) were analysed using polymerase chain reaction technique. All data collected using Epidata and were statistically analysed, using statistical package for the social sciences (SPSS).
Results: A total of 197 pregnancies resulting in 200 live births, 91 (45.5%) female and 109 (54.5%) male exposed to HIV were recruited. The overall MTCT rate was found to be 1.5% after cessation of all exposures, with two of these babies given mixed-feeding and their mothers not taking anti-retroviral therapy during pregnancy. The factors associated with MTCT in the univariate analysis include HIV diagnosis late during pregnancy, virological suppression & late ART commencement.Maternal antiretroviral therapy (ART) use & infant feeding option were found to have significant effect on baby early infant diagnosis (EID) outcome(χ² = 15.40, df = 2, P = 0.001; χ² = 12.67, df = 2, P = 0.001).
Conclusions: The diagnosis of HIV and antiretroviral therapy coverage prior to pregnancy are the main factors in the prevention and elimination of mother-to-child transmission of HIV, thus, the achievement of the 90-90-90 goals in HIV-infected women would make it possible to achieve the goal of eliminating perinatal HIV transmission in Nigeria.

Few Epigenetic Changes In Acute Myeloid Leukemia: Experience From A Tertiary Care Centre In North India
Neelam Varma, Raj Kumari Banashree, Shano Naseem, Pankaj Malhotra, Subhash Varma, Jogeshwar Binota
PGIMER, Chandigarh, India

Introduction: Novel biomarkers like epigenetic changes are likely to be useful in prognosis and therapy of AML. DNMT3A mutations, IDH1 mutations and IDH2 mutations are reported in >30%, 6-16% and 8-19% of cytogenetically normal AML (CN-AML) patients, respectively. Significant association of DNMT3A mutated cases with AML-M1 and AML-M4/5 FAB  subtypes has been reported in few studies.
Methods: This prospective study was conducted between January 2016-June 2017, enrolling 103 newly diagnosed de-novo AML cases. Restriction fragment length polymorphism (RFLP)analysis was performed for detection of DNMT3A (R882H G>A) mutation. High resolution melt (HRM) curve analysis was performed for IDH1 (R132) mutation and amplification-refractory mutation system (ARMS) method for IDH1 (R132) mutation. Appropriate positive control samples were used.
Results: Among 103 AML cases, DNMT3A R882H mutation was detected in 9 (8.7%), IDH1 R132 in 9 (8.7%) and IDH2 R140Q mutation in 13 (12.6%) patients. Two out of 9 (22.2%) cases with IDH1 R132 mutation showed PML-RARA, and 1 (11.1%) case had associated DNMT3A R882H mutation. Out of 13 AML patients harbouring IDH2 R140Q mutation, coexistent DNMT3A R882H mutation was noted in 2 (15.4%) patients; 3 (23.1%) had t(8;21)(q22;q22);RUNX1-RUNX1T1 and 1 (7.7%) had inv(16)(p13.1q22);CBFB-MYH11. None of the IDH1 R132 mutated AML cases had concomitant IDH2 R140Q gene mutation, depicting mutual exclusivity. DNMT3A R882H mutated AML were significantly associated with AML-M1 FAB subtype. AML patients harbouring IDH1 (R132) mutation and IDH1 (R132) mutation showed AML-M2 FAB subtype more frequently, though the p value was not significant.
Conclusions: Baseline frequencies of few epigenetic changes like DNMT3A(R882H G>A), IDH1(R132) and IDH1 (R132) mutations have been ascertained in our AML patients. This information is likely to be useful in patient management in future.

The Application Of Dna Fragment Analysis Of Bone Marrow Smear In Next Generation Sequencing
Fang Wang1,2, Yincheng Tan2, Yuanli Xu2, Yongxin Guo2, Chunye Cao2, Ying Zhang2, Xiaoli Ma2, Yang Zhang1,2, Xue Chen1,2, Daijing Nie1,2, Jiaqi Chen1,2, Lili Yuan1,2, Hongxing Liu1,2,3
1Beijing Lu Daopei Institute of Hematology, Beijing, China, 2Pathology & Laboratory Medicine Division, Hebei Yanda Lu Daopei Hospital, Langfang/Hebei, China, 3Pathology & Laboratory Medicine Division, Beijing Lu Daopei Hospital, Beijing, China

Introduction: Genetic mutation testing of hematology patients usually use fresh bone marrow or peripheral blood specimens. But many patients who have received remission after treatment cannot obtain freshly diagnosed specimens for this testing. It has been reported that bone marrow smears can be used for it, but the fragmentation of bone marrow smears DNA may add difficulty to next generation sequencing (NGS). To investigate the application of DNA fragment analysis of bone marrow smear in NGS, we analyzed the DNA fragment analysis results and the sequencing success rate of bone marrow smear DNA.
Methods: We retrospectively analyzed 135 DNA fragment analysis results of bone marrow smears and 160 sequencing results of NGS panel (328 amplicons, length range 125-275bp) on Ion Torrent PGM platform which from 135 hematology patients. The sequencing success criteria according to the sequencing parameters is On Target>95 and Uniformity>93 and Mean Depth>700.
Results:  In 135 patients, the first sequencing success rate of bone marrow smears is 71.9%, the resequencing success rate is 80%, and the final sequencing success rate is 86.7% (Table 1). In sequencing success specimens,mean value of Mapped Reads is 382572.5(309051.5-461347.5), of On Target is 98.6(98.1-98.9), of  Uniformity is 95.5(94.9-95.9), and of  Mean Depth is 1156.5(926.9-1385.8). But in sequencing failure specimens, mean value of Mapped Reads is 223357(161106-349375), of On Target is 98.1(96.9-98.6), of  Uniformity is 93.8(83.3-95.0), and of Mean Depth is 616.9(460.8-789.7)(Table 2).We divided bone marrow smears DNA into 5 types according to the fragment analysis graphs: type a (79 patients), type b (8 patients), type c (9 patients), type d (27 patients), and type e (12 patients) (Figure 1). The first sequencing success rate and resequencing success rate of type a-e are respectively: type a: 86.1%、90.0%,type b:62.5%、100%,type c:33.3%、0,type d: 63.0%、100%,and type e:33.3%、100%. The final sequencing success rate of type a-e are 97.5%、100%、33.3%、88.89%、41.7%, respectively (Table 1).
Conclusions: We can assess the specimen quality based on the different DNA fragment analysis results of bone marrow smears, and thereby improve the one success rate of NGS.

Next-Generation Sequencing For Plasma Cell Myeloma In A Real Clinical Setting
Shinae Yu, Sae Am Song, Kyung Ran Jun, Jeong Nyeo Lee
Haeundae Paik Hospital, Inje University, Busan, South Korea

Introduction: Plasma cell myeloma (PCM) is a bone marrow based plasma cell malignancy characterized by wide clinical presentation and heterogeneous genetic background. The evaluation of genomic profiles for PCM patients using next generation sequencing (NGS) were established in several studies. We performed gene panel test for PCM patients using NGS in a real clinical setting from sample collection to result report. 
Methods: We constructed a 88 gene panel for targeted hematologic malignancy including PCM and investigated ten newly diagnosed PCM patients. NGS was performed using the MiSeq system (Illumina, USA) and analyzed using bioinformatics pipeline of NGB-DNA-somatic-v1.4 (NGeneBio, Korea). The results were interpreted and reported as tier I, II and III based on the Association for Molecular Pathology (AMP) guideline. The target turnaround time (TAT) was 21days.
Results: We detected 37 variants in 25 genes (28.4%) and all patients had two or more variants. Three tier I variants were detected in TP53, eight tier II variants were detected in KRAS, NRAS, andPTPN11. NRASwas the most frequently mutated gene (40%), followed by KRAS(30%), RELN(30%), and TP53 (30%). The mean TAT was 18.75 days (13-25).
Conclusions: The clonal heterogeneity observed in this study in spite of small size of patients.  Interpretation of NGS result according to AMP guideline was valuable to predict therapeutic or prognostic impacts on PCM patients. TAT is always concerned to match clinician’s needs in a real clinical setting.

Gender And Age-Dependency Of Advanced Reticulocyte Parameters On The Abbott Alinity Hq Hematology Analyzer
Manoj Gandhi1, Hitomi Hoshino1, Raj Chandran2, Kai Qu2, Gabriella Lakos1, Zainab Mukhtar1, Mona Patel1
1Abbott Laboratories, Santa Clara, CA, United States, 2Abbott Laboratories, Lake Forest, IL, United States

Introduction: Cell by cell volume and hemoglobin (HGB) analysis, along with fluorescence technology for reticulocyte staining enables the reporting of extended Red Blood Cell (RBC) parameters on the Alinity hq hematology analyzer (Abbott Diagnostics, Santa Clara, CA). These parameters include the HGB content of reticulocytes (MCHr), immature reticulocyte fraction (IRF) and reticulated platelets (%rP), which have proven to be useful in a number of clinical scenarios. Examples include anemia diagnosis and therapy monitoring, assessing bone marrow recovery after stem cell transplantation or chemotherapy and thrombocytopenia. Previous publications have shown that reference intervals for reticulocytes and related parameters are technology-dependent. Therefore, instrument specific reference ranges are recommended.
Methods: Reference ranges for the above-mentioned parameters were established on Alinity hq using samples from apparently healthy adult subjects. Gender and age-dependency of the values were assessed.
Results: Results obtained on samples from 213 adults (46.6 ± 12.0 years, 19 to 91 years) were analyzed. Data distributions were Gaussian for IRF and MCHr, while %R and %rP results followed a non-normal distribution. The central 95th percentile ranges were calculated according to the CLSI EP28-A3c guideline using a parametric method for normally distributed measurand and a non-parametric method for non-normally distributed data. A small difference was observed between genders for %rP values only (p=0.012), with slightly higher values in males. Age dependency was noted for IRF and %rP only in males (with higher values with increasing age) and for %R values only in females (peaking between the age of 30 to 50 years). A statistically significant positive correlation was found between IRF and %R (r=0.208, p=0.0023) and a negative correlation between IRF and MCHr (r=-0.381 (p< 0.0001). The cumulative reference ranges for %R, IRF and MCHr respectively, were  0.86 – 2.82%, 0.17 – 0.44, and 25.3 – 35.5 pg. The reference range for %rP was 1.14 – 6.87%. 
Conclusions: Reference ranges for %R, IRF, MCHr and %rP were established on Alinity hq. These ranges can serve as guide for Alinity hq users and as basis for comparison to other analyzers.

Identification And Validation Of Sry-Box Containing Gene Family Member Sox30 Methylation As A Prognostic And Predictive Biomarker In Myeloid Malignancies
Jing-dong Zhou
Zhenjiang First People's Hospital, Zhenjiang, China

Introduction: Methylation-associated SOX family genes have been proved to be involved in multiple essential processes during carcinogenesis, and acts as potential biomarkers for cancer diagnosis, staging, prediction of prognosis, and monitoring of response to therapy. Herein, we revealed SOX30 methylation and its clinical implication in acute myeloid leukemia (AML) and myelodysplastic syndromes (MDS). 
Methods: SOX30 expression was assessed by real-time quantitative PCR, whereas SOX30 methylation was detected by methylation-specific PCR and bisulfite sequencing PCR.
Results: In the discovery stage, we identifiedthat SOX30 methylation,a frequent event in AML, was negatively associated with SOX30 expression, and correlated with overall survival (OS) and leukemia-free survival (LFS) in cytogenetically normal AML among SOX family members from The Cancer Genome Atlas (TCGA) datasets. In the validation stage, we verified that SOX30 methylation level was significantly higher in AML even in MDS-derived AML compared to controls, whereas SOX30 hypermethylation was not a frequent event in MDS. SOX30 methylation was inversely correlated with SOX30 expression in AML patients. Survival analysis showed that SOX30 hypermethylation was negatively associated with complete remission (CR), OS, and LFS in AML, where it only affected LFS in MDS. Notably, among MDS/AML paired patients, SOX30 methylation level was significantly increased in AML stage than in MDS stage. In addition, SOX30 methylation was found to be significantly decreased in AML achieved CR when compared to diagnosis time and markedly increased in relapsed AML when compared to the CR population. 
Conclusions: Our findings revealed that SOX30 methylation was associated with disease progression in MDS, and acted as an independent prognostic and predictive biomarker in AML.

Automatic Detection Of Dysplastic Cells Using Deep Learning
Andrea Acevedo1, Anna Merino2, Santiago Alfrez1, Laura Bold2, Angel Molina2, Jose Rodellar1
1Technical University of Catalonia, Barcelona, Spain, 2Hospital Clinic of Barcelona, Barcelona, Spain

Introduction: Morphologic abnormalities in blood cells are associated to myelodysplastic syndromes (MDS). Lower o absence of granulation in the cytoplasm of neutrophils circulating in peripheral blood (PB) is a morphologic abnormality characteristic of MDS. The objective of this work is to evaluate the use of a convolutional neural network model to automatically detect dysplastic neutrophils.

Methods: We collected 5,935 digital images, corresponding to : 1) normal neutrophils (N=4,054) and 2) dysplastic neutrophils (N=1,881). Each group was organized in three subsets: training, validation and testing. Regarding the group of normal neutrophils, 3,054 images were selected for training, 500 for validation and 500 for testing. In the same way, from the group of dysplastic neutrophils, 881 images were selected for training, 500 for validation and 500 for testing. To balance the training set, we augmented up to 3,200 the number of training images of each group, implementing techniques of image processing. To perform a final test, new images of neutrophils corresponding to 41 smears from control individuals and 57 smears from 40 dysplastic patients were selected. A convolutional neural network model named VGG-16 was selected to perform the automatic recognition of normal and dysplastic neutrophils. The final model was tested in two ways: 1) by cell and 2) by smear.
Results: Confusion matrices of the automatic classification by cell and by smear are shown in Figure 1. The diagonal of each table represents the true positive rates (TPR). Considering the classification by cell, we obtained TPR of 94.2% and 97% for normal and dysplastic neutrophils respectively, being 96% the global accuracy of the system. For the smears containing dysplastic neutrophils, 96.5% were predicted as belonging to patients with dysplasia. Moreover,the 95.2% belonging to control individuals, were classified in the correct class, being the global accuracy of 96%. Sensitivity, specificity, precision and accuracy values for each test are shown in Figure 1, and the ROC curve obtained in the classification by smear in Figure 2.
Conclusions: The results obtained in this work indicate that a deep learning strategy, such as the classification with convolutional neural networks, can be implemented to automatically recognize dysplastic neutrophil images obtaining very high accuracy values.

Evaluation Of A Novel All-In-One, Point-Of-Care Device With Deep Learning-Based Blood Cell Processing Algorithm
Chae Yun Bae1, Raeeun Chung1, Oleksandr Bailo1, Minkyo Lee1, Jooyeon Park1, Seongsoo Jang2, Jongha Yoo3, Jaewoo Song4, Dongyoung Lee1
1NOUL, Inc., Yongin, South Korea, 2Department of Laboratory Medicine, Asan Medical Center and University of Ulsan College of Medicine, Seoul, South Korea, 3Department of Laboratory Medicine, National Health Insurance Service Ilsan Hospital, Goyang, South Korea, 4Department of Laboratory Medicine, Yonsei University College of Medicine, Seoul, South Korea

Introduction: Access to laboratory-scale diagnosis is limited to large clinics in developing countries. These devices are expensive and require skilled technician to prepare samples and often perform additional manual microscopy examination. Thus, the demand for all-in-one, point-of-care hematology device is high. miLab®  (noul, Korea) automates the preparation of blood smear images (smearing, fixation, staining, and acquisition) and analyzes them using deep learning-based techniques. We have evaluated the performance of miLab®’s automated process in identifying and differentiating various blood cell types.
Methods: 5ul of whole blood was loaded into miLab®, which smeared, stained and acquired 400x images within fifteen minutes. A set of 22,000 images were collected and manually annotated for red blood cell (RBC), five types of white blood cells (WBC) and platelet (PLT). The sample preparation performance was evaluated as the coefficient of variation calculated by comparing RBC counts of 10 repeated smears from a single patient. In addition, blood samples from 12 patient (Yonsei University, Korea) were smeared by both miLab® and XN-20 (SYSMEX, Japan). For both smears, the RBCs were counted by miLab® cell detector and the coefficient of determination was calculated by correlating the two data set. The accuracy of deep learning algorithm was assessed as the average precision (AP) for the detection of each cell types and percent accuracy for the classification of WBCs types.
Results: miLab® consistently smeared, fixed and stained samples all within 15 minutes. The coefficient of variation from the 10 times repeatability experiment was around 3% for RBC cell count (Figure 1). The RBC count from blood smear made by miLab® showed high correlation against that from slides made by Sysmex with the coefficient of determination around 96.2% (n=12). The deep-learning algorithm from  miLab®  device was able to detect RBCs, WBCs and PLTs with the AP score of 0.985, 0.975 and 0.960, respectively, and was able to differentiate five WBC types at 98.1% percent accuracy.
Conclusions: The result shows the all-in-one, point-of-care device, miLab®, is able to consistently generate highly correlated, auto-stained blood smear and acquire images. Deep learning based algorithm also analyze the blood cell components including RBCs, PLTs and WBCs.

Automatic Recognition Of Different Types Of Acute Leukaemias In Peripheral Blood By Image Analysis
Laura Bold1, Santiago Alfrez2, Angel Molina1, Andrea Acevedo2, Jos Rodellar2, Anna Merino1
1Hospital Clinic of Barcelona, 2Technical University of Catalonia

Introduction: Morphological differentiation among different blast cells circulating in peripheral blood (PB) is a difficult task and there are not automated analysers which can morphologically recognise these abnormal cells. The goal of this work is the development of an extended machine-learning approach for the automatic recognition of different types of acute leukaemia.
Methods: We analysed 442 PB smears from 206 patients: 86 healthy individuals, 53 with infections and 67 with acute leukaemia from the following origins: myeloid (AML:18), lymphoid (ALL-B:23), monocytic (AMONL:17) and promyelocytic (APL:9). These smears contained a total of 7,468 cell images. The training set was arranged with 75% of these smears, while the remaining 25% was the testing set. PB films were stained with MGG and images were acquired in CellaVision®DM96. Automatic segmentation procedure was done using MATLAB®, which allowed the extraction of 2,867 geometric and colour-texture features. Feature selection was implemented within the tuning of parameters of the five classifiers studied to obtain the most relevant features from the best classification technique. To evaluate the efficiency of each classifier, a 5-fold cross validation was performed over the training set. The automatic recognition system was assessed with the testing set of new PB smears. It was given to the classifier, performing a blind automatic recognition whose outcomes were the different cell types under study.
Results: The highest classification accuracy was achieved with the selection of 700 features with linear discriminant analysis (Figure 1). Figure 2 shows confusion matrices which summarize the results of the classification performed for each cell type (I) and for individual smears (II). Regarding cell classification, accuracies for normal mononuclear cells and reactive lymphocytes were above 90%, 81% for myeloblasts, 73% for abnormal promyelocytes and 79% for B-lymphoblasts. With respect to individual smear classification, accuracies superior to 95% were obtained for smears containing infections and normal mononuclear cells. 88% of smears with AML, 85% with ALL-B and 80% with APL were correctly classified.
Conclusions: The key contribution of this work is a system able not only to detect blast cells, but also to recognize the lineage origin of blast cells. From a diagnosis perspective, it is relevant since it automatically allows to distinguish among different types of acute leukaemia.

Hairy Cell Leukemia: The Point On Laboratory Diagnostics Of Hcl In Italy
Anna Maria Cenci, Barbara Casolari, Maria Golato, Fabrizio Papa, Elisa Piva, Marco Moretti, Maria Diquattro, Mariacaterina Maconi, Paolo Doretto, Luciano Pasini, Mauro Buttarello, Piero Cappelletti, Bruno Biasioli
Italian Society of Pathology and Laboratory Medicine - Hematology Study Group (SIPMeL-GdSE), Castelfranco Veneto (TV), Italy

Introduction: GdSE- SIPMeL evaluated the laboratory performance in managing Hairy Cell Leukemia (HCL) in Italy by reviewing the diagnostic pathways of the disease, (prevalence: 2% leukemias and 8%/linfoproliferative disorders) and distributing a questionnaire, intended to verify HCL incidence; laboratory contribution to disease  management (morphological recognition/instrumental alarms); Guidelines actually followed in diagnosis and follow-up; instrumental and professional performance.
Methods: In 2018, 16 cases (14 HCL and 2 HCLv) were collected. CBC samples were tested in 12 institutions (stand-alone or network Laboratories) and performed by different counters. Specialists of GdSE-SIPMeL managed results and reviews. Final studied data came from 11 scenarios fully representing the Italian background.
Results: -HCL incidence: similar to literature (1.9 new cases/year).  -Diagnostic Flow Charts (immunophenotypic/diagnostic follow-up; second opinion): 9 present, 2 absent; 4 departmental; 2 second opinion/extra-laboratory consulting.  -Diagnostic pathway priming: 3 from outpatients clinical suggestions; 13 triggered by CBC results.  In these 13 cases, identified in laboratory, we observed: 12 CBC parameters atypiae; 10 lymphocytes/blasts single flag, 2 double flag; 11 atypical lymphocytic data; 7 positive cell-population parameters; 1 negative for lymphocytic items; 12 others blood-cell features (anaemia, thrombocytopenia, leucopenia, monocytopenia, pseudomonocytosis). Difference in presence/strengh/lack of CBC warning data reflected the characteristics of Hairy Cell type (HC) involved, so as presumably the presence of instrumental monocytosis. -HC report: 7 HC number included in differential; 3 HC vs lymphocytes number provided; 7 semiquantitative reports (present/absent; rare…) performed; 1 HC presence suggested. -Digital morphologyused in 5: resulted particularly useful in presence of severe leukopenia and/or instrumentals monocytosis. -Comments: widespread heterogeneity about cell description/whole hematologic picture. Each Laboratory proposed local/standard notes, similar phrases present in 11 cases; only 4 laboratories reported the same comments.
Conclusions: The review highlights: microscopic review mostly triggered by aspecific flag for atypical lymphocytes; microscopic counts entered in about 50% reviewed differential; 50% reported semiquantitative evaluation/single cell description; predefined dedicated test lists are not currently structured, while relevant task in HCL diagnostic pathways is commonly acknowledged to the hematology laboratory. All laboratory Professionals involved in the review are claiming a specialist role. HCL related reports still need standardization and performance improvement. 

Evaluation Of An Automated Image Analyzer For Microvessel Density Measurement In Bone Marrow Biopsy
Yousun Chung1, Hyoeun Shim2, Seungwon Shin3, Kwang Gi Kim4, Ji Yeon Sohn5, Hyeon-Seok Eom6, Sun-Young Kong2
1Department of Laboratory Medicine, Kangdong Sacred Heart Hospital, Seoul, South Korea, 2Department of Laboratory Medicine, National Cancer Center, Goyang, South Korea, 3Optical Research Team, Z-tec Co., Ltd., Seoul, South Korea, 4Department of Medical Engineering, Gachon University, Incheon, South Korea, 5Department of Laboratory Medicine, Eone Laboratories, Incheon, South Korea, 6Center for Hematologic Malignancies, National Cancer Center, Goyang, South Korea

Introduction: Angiogenesis is important for the proliferation and survival of multiple myeloma (MM) cells. Bone marrow (BM) microvessel density (MVD) has been reported a useful marker for evaluation of angiogenesis, therefore, it has been associated with poor prognosis of MM patients. Here we evaluated an automated image analyzer which had been developed for the assessment of MVD in bone marrow biopsy of MM patients. 
Methods: For development an algorithm, we used image of BM sections stained with anti-CD34 antibodies using two color models; RGB (red, green, and blue) model and HSV (hue, saturation, value) model. MVD was calculated on the merged image of red channel and hue channel after elimination of non-microvessels using regression equation (Figure 1). For comparison, MVD was assessed by two hematopathologists in a blinded manner using BM samples of MM patients and it compared with the counts by automatic analyzer. 
Results: A total of 84 BM samples from MM patientswere used for the evaluation. The mean and standard deviation (SD) of the results from manual count were 19.8/HPF and 11.7/HPF. And from developed analyzer, the mean and SD were 19.5/HPF and 11.1/HPF. The correlation between manual results and automatic results were shown as Pearson’s r value of 0.905 (p < 0.001).
Conclusions: The MVD results from developed automatic analyzer showed very good correlation with manual count. Considering that manual count is labor-intensive and time-consuming, this developed analyzer for BM MVD will provide more objective results which can provide additional information for the prognosis of MM patients.

Copper Deficiency: Don&Rsquo;T You Forget About Me.
Rebecca Haack, Shreyashee Mallik, Nagendraprasad Sungala, Adam Bryant, Lye Lin Ho, Penelope Motum
Haematology Department, Liverpool Hospital, Liverpool, Australia

Introduction: Copper deficiency (hypocupremia) is a rare nutritional deficiency. Like a myelodysplastic syndrome (MDS), it may present with cytopenias and macrocytosis, or may mimic B12/folate deficiency with neurological symptoms. Bone marrow features of vacuolated erythroid and myeloid precursors should raise the suspicion of copper deficiency. Patients at risk for hypocupremia include those with dietary deficiencies and/or malabsorption, including a subgroup who have undergone bariatric surgery.
Methods: Two cases of hypocupremia were reviewed. Peripheral blood and bone marrow films stained with May-Grunwald -Giemsa were examined in conjunction with clinical notes.
Results: Case 1: 35/F (G1P0 38+3) with gastric bypass (Roux-en-Y, 2004) presented with lower limb swelling, macrocytic anaemia (Hb:75g/L. MCV:110fL), a leucoerythroblastic blood film and neurological symptoms.  Although mildly hypertensive, a fulminant HELLP syndrome was unlikely as no erythroid fragmentation was seen. A low copper level was noted at 6.8umol/L (Ref: 12-22umol/L), with normal B12/folate levels. This was consistent with nutritional deficiency exacerbated by pregnancy demands.  IV copper was administered with recovery of the anaemia, resolution of the macrocytosis and symptomatic improvement. Case 2: 23/M with autism presented with extreme fatigue in conjunction with a pancytopenia (Hb:72g/L, platelets:32x109/L, neutrophils:0.5x x109/L), low serum folate (4.1nmol/L) but normal serum B12. Progressive bloods showed occasional blasts triggering bone marrow evaluation. Megaloblastic erythroid changes were noted consistent with folate deficiency.  However, additional changes not typically associated with megaloblastosis were noted including atypical megakaryocytes with frequent nuclear separation and some small hypolobulated forms; hypogranulated giant metamyelocytes and granulocytes. The cytopenias persisted despite cessation of valproate. Nutritional deficiency was suspected as the patient had an extremely selective diet.  Copper levels were found to be low 10umol/L (Ref: 12-22umol/L) with normal zinc levels. The full blood count normalised only after the commencement of oral supplements and reestablishment of normal copper levels.
Conclusions: Hypocupremia is a rare dietary deficiency with haematological ramifications; including macrocytosis, anaemia and/or cytopenias; and is a described mimicker of other serious disorders. Although vacuolation of bone marrow precursors are noted, this unusual feature is not entirely unique to copper deficiency alone. Thus the diagnosis of hypocupraemia should be considered in patients nutritional deficiency or malabsorption, particularly those with a history of gastric surgery. Cop per replacement therapy is a simple and effective measure to completely reverse these features

Influence Of K2-Edta And K3-Edta Tubes For Monocyte Distrubution Width Measurement
Maria Lopez-Molina, Xavier Tejedor Gandux, Alicia Martinez Iribarren, Merce Espinosa, Silvia Torres, MAngels Sala, Carla Fernandez, Chantal Abadia, MAntonia Llopis, Cristian Morales-Indiano
Hematology-CoreLab Department. Clinical Laboratory Metropolitana Nord (LCMN). Hospital Universitari Germans Trias i Pujol., Badalona(Barcelona), Spain

Introduction: Ethylenediaminetetraacetic acid(EDTA) is the anticoagulant of choice for hematology testing. Mainly, EDTA comes in blood tubes as dipotassium (K2) and tripotassium (K3) salts. It has been recognized that potassium’s concentration salt may affect the accuracy of cell blood counting, cell sizing and probably its stability. EDTA-salt may cause shrinkage of erythrocytes, affect mean corpuscular volume and mean platelet volume. These effects are more significant in K3-EDTA than in K2-EDTA. Monocyte Distribution Width(MDW) is a new morphology parameter available on the new DxH-900 hematology analyzer of Beckman Coulter, which could potentially be useful for early sepsis detection. The aim was to evaluate the differences in MDW using 2 different EDTA-tubes(K2 and K3 salts) and to study its stability over time in both anticoagulants.
Methods: A 25 healthy volunteers(80% women) were studied. For each one we recollected two different EDTA-tubes(K2 and K3, BD Vacutainer®). Samples were tested at different times(0hour (h), 1h, 2h, 4h, 6h, 8h, 24h and 48h) and stored at room temperature during the study. All samples were run on the DxH-900 Analyzer (Beckman Coulter; Miami, FL, USA). The differences between K2 and K3 at time 0h were assessed by paired Student’s t-test. Significant P values were values below 0.05. Toassess stability, the percentage change [(the mean of result at time X – mean of result at 0h)/mean of result at 0h]*100 was calculated. We established a significant difference for MDW when the change percentage(CP%) was >10%
Results: A significant 2 points difference was found between K2 and K3 for MDW at 0h (K2: 16.12±1.57 vs K3: 18.33±1.75;p< 0.001). This difference was maintained for most of the analyzed times. The value of the MDW remained stable until 8h in both EDTA-tubes (CP%-K2: 1h=5.2%; 2h=2.6%; 4h=5.0%; 6h=6.4%; 8h=8.4%; 24h=19.4% and CP%-K3: 1h=4.2%; 2h=2.6%; 4h=5.3%; 6h=5.9%; 8h=8.6%; 24h=19.8%).
Conclusions: In our preliminary study, we found significant differences in the MDW over 2 points between K2 and K3 EDTA-tubes in all the analyzed times. MDW showed CP< 10% in the first 8 hours of the venipuncture. New morphological parameters such as MDW should be evaluated according to the EDTA tube used.

LeA Antigen Predisposes Nigerians To LymphoidLeukaemias

Introduction: Lymphoid leukaemias are characterized by progressive accumulation of immature (blasts) or mature-appearing, functionally incompetent cells of lymphoid origin in peripheral blood, bone marrow, lymph nodes and sometimes other organs in the body.Blood group antigens have been linked with infections and diseases etiology. However, there is paucity of information on the association of blood groups especially the rare types and lymphoid leukaemias in this part of the world.
Methods: This study investigated the association of some rare blood group phenotypes with lymphoid leukaemias among subjects in North-Western Nigeria. A total of 37 lymphoid leukaemic subjects (age: 28.2±25.1 years, male/female ratio: 18:19) were studied with 37 healthy blood donors (controls) with mean ages of 29.9±6.4 years and male/female ratio: 34:3. Out 37 lymphoid leukaemic subjects, 16 were chronic lymphoid leukaemia (CLL) while 21 were acute lymphoblastic leukaemia (ALL). Lymphoid leukaemias were diagnosed using the clinical and cytological criteria in Aminu Kano Teaching Hospital (AKTH). The blood groups were determined using Tube method for ABO, Rh-D, Lewis, Duffy and MNS while Rh C, c, E, e and Kell by Gel card method. Data was presented as tables and figures and analysed using SPSS (version 25.0).
Results: Association of some blood groups and lymphoid leukaemias were determined usingChi-square test whileOdds Ratio (OR) and Relative Risk (RR) were used to ascertain their risk values. The age distributions of lymphoid leukaemias (CLL and ALL) vary significantly (P< 0.05). Chronic lymphocytic leukemia (CLL) had female gender predominance while ALL had male gender predominance. The results showed that only Lea antigen was expressed significantly in both CLL and ALL (OR: 13.0 and 8.50 respectively) while other blood groups considered were normally distributed with no significant association except the Lea
Conclusions: We conclude that individuals with Lea antigen inheritance may have increased risk of developing leukaemia especially CLL or ALL while individuals with other blood groups may have lesser risks of developing lymphoid leukaemias.

Preliminary Evaluation Of The Body Fluid Mode Of The Abbott Alinity Hq Hematology Analyzer
Kolja Hegel1, Stephen Young2, Martin Krockenberger3, Queency Lee3, Zainab Mukhtar3, Gabriella Lakos3
1Labor Berlin Charit Vivantes Services , Berlin, Germany, 2TriCore Reference Laboratories, Albuquerque, NM, United States, 3Abbott Diagnostics, Santa Clara, CA, United States

Introduction: Laboratories have increasingly been performing body fluid (BF) cellular analysis on hematology analyzers. Current methodological limits of precisely and accurately assessing low WBC and RBC counts are well-known barriers to accepting automated cell counting, particularly for cerebrospinal fluid (CSF) samples.
Methods: We have investigated the analytical performance of the prototype BF mode on the Abbott Alinity hq hematology analyzer, which employs advanced MAPPS™ technology and fluorescence flow cytometry as operating principles. The BF mode requires < 90 uL sample, diluted 1:20 with the routine reticulocyte reagent. Altogether 160 CSF, pleural, peritoneal and synovial fluid samples were tested with the Alinity hq, equipped with a Research Use Only software version, and with the Sysmex XN-9000 BF mode, in two clinical laboratories. The comparison was performed between the two instruments, and with results obtained with the manual reference method.
Results: There was good correlation between Alinity hq and Sysmex XN-9000 total nucleated cell count (TNCC), as well as between %Mononuclear (%MN) and %Polymorphonuclear (%PMN) results (r=0.90 for all). Ideal correlation (r=1.00) was obtained between manual and Alinity hq as well as Sysmex TNCC methods, with a slight positive bias by Passing Bablok regression (slope=1.24 and 1.11 with Alinity hq and Sysmex XN-9000, respectively). Alinity hq and Sysmex XN-9000 RBC counts demonstrated robust agreement with the manual RBC results on 48 samples with a manual RBC concentration of 1,000-70,000/uL (r=0.98 and 0.97; slope=1.19 and 1.05, respectively). Of the 71 samples with manual RBC count of < 1,000/uL, however, Sysmex XN-9000 was unable to enumerate RBCs in 47 cases. Alinity hq accurately determined the RBC concentration in these samples (r=0.93, slope=1.29). In addition, Alinity hq reported < 10 RBC/uL in 19 samples out of the 20 with manual RBC count of < 10/uL. When evaluated on CSF specimens alone,  Alinity hq demonstrated a high degree of accuracy (r= 1.00 and slope=1.19) in comparison to the manual RBC result.
Conclusions: The Alinity hq BF mode has demonstrated robust correlation and agreement with manual WBC and RBC counts on BF samples with low cell concentrations, and is a promising alternative to manual cell counting.

Comparison Of Morphological Flagging Generated By The New Abbott Alinity-Hq VersusBeckman Coulter Dxh800, And Siemens Advia2120I, In A Clinical Laboratory
Guilhem MAYORAL1, Quentin CHEVRIER2, Corinne BOUCHAHDA3, Zainab MUKHTAR4, Gabriella LAKOS5
1Medilab66 (INOVIE Group), St Laurent de la Salanque, France, 2Medilab66 (INOVIE Group), St Laurent de la Salanque, France, 3Biople66 (INOVIE Group), Cabestany, France, 4Abbott Diagnostic Division, Santa Clara, CA, United States, 5Abbott Diagnostic Division, Santa Clara, CA, United States

Introduction: Alinity-hq Hematology Analyzer (HA) is a fully automated recently launched system equipped with dynamic machine learning algorithm. However, when it comes to clinical performance, it is well-known that flagging capabilities of HA for morphological abnormalities is an important criterion for clinical pathologist because it helps in detection of pathological cells and improve laboratory workflow. The aim of this study was to evaluate the sensitivity and specificity of blast, malignant lymphocytes, immature granulocytes and NRBC flags for three HA compared to the PBS review reference method
Methods: Random samples were selected from the routine CBC testing workflow as per laboratoy guidelines.PBS were made for each sample. Two trained hematologists,  performed each a 100-cell hand count. The analytical and/or morphological alerts were generated respecting the algorithms of the different suppliers. During our evaluation, Alinity-hq had its on market AlgoSoftware, which is under further advancements (use of two relevant data invalidation flags of Alinity-hq had been added to blast flag ). Our laboratory is also evaluating Flagging efficiency results with the upcoming software (RUO) not included in this poster.  
Results: Microscopic review results were compared to flagging alerts of all three HA. Defining a > 0% cut-off for presence of blasts in blood smear, ≥ 2% cutt-off for presence of variant lymphocytes, cut-off ≥ 2% for promyelo/myelo/metamyelo-cytes for presence of IG, cut-off ≥ 1% for presence of NRBC.Out of total 498 samples, microscopy identified 17 samples with blasts, 10 with suspected malignant lymphocytes, 41 with IGs and 11 with NRBC. Sensitivity and Specificity of each morphological flag with the cutoff values are shown in tables 1 and 2  
Conclusions: This is one of the first comparison of the performances of the new Alinity-hq with existing high volume HA to detect abnormal blood cells.This study is a first step and it highlights the importance to combine efficient methodologies with advanced algorithms to post process raw results of a hematology analyzer, to achieve the best pathological blood cells detection performances for a high throughput laboratory

Evaluation Of Bone Marrow Study In Patients With Ewing Sarcoma/Primitive Neuroectodermal Tumors At A Tertiary-Care Hospital
Kuenyoul Park1, Min-Sun Kim1, Sihwan Kim1, Hyeri Kim2, Kyung-Nam Koh2, Young-Uk Cho1, Seongsoo Jang1, Chan-Jeoung Park1
1Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea, 2Department of Pediatrics, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea

Introduction: It is well-known that metastasis is the most important prognostic factor in Ewing sarcoma family tumor (ESFT). Due to the importance of bone marrow (BM) metastasis as a risk factor, BM examination still plays a big role in staging of ESFT.  This study aimed to investigate the BM findings and other prognostic factors in ESFT at a tertiary-care hospital since January 2000.
Methods: All patients who were newly diagnosed with ESFT and underwent BM study were enrolled during 19 years from January 2000 to September 2018.  The first BM study from enrolled patients were reviewed, and BM diagnosis was made from a combination of morphological findings of BM specimens including BM aspirates and biopsies, and immunohistochemistry (IHC) results. Electronic medical records of these patients were reviewed for previous history of ESFT, pathology results of primary lesion and primary tumor site, tumor size and metastasis.
Results: For 19 years, a total of 55 patients who were diagnosed with ESFT and underwent BM examination were identified. Unilateral BM study was performed for 10 patients, the other 45 patients received bilateral BM examination. Primary tumor sites were 9 extremity area, 31 chest/spine/head and neck area, 15 abdomen-pelvis area. Bone metastasis was observed in 10 patients: single in 3 patients, 2 to 5 in 3 patients, more than 5 in 4 patients. Only 2 cases showed lung metastasis. Only one of 53 IHC staining for CD99 of primary tumor specimen showed negativity unusually. BM metastasis was revealed in 7 patients. Three cases were diagnosed as BM metastasis only with morphological findings. Other 4 cases were assisted with IHC staining: 2 with CD56, one with CD99, one with vimentin. Bone scan didn’t show distant bone metastasis in 2 cases with BM metastasis, of which, primary sites were sacrum and lumbar spine.
Conclusions: Not only morphological findings, but also IHC staining was helpful for diagnosing BM metastasis of ESFT. This study concludes that BM study in Ewing sarcoma is important for detecting metastasis although the utility of imaging modalities is rapidly improving.

The Neutrophil Structural Parameter Neut-X In Iron Deficiency
Maria Stamou, Athanasia Agorasti
General Hospital of Xanthi, Xanthi, Greece

Introduction: Sysmex®XE-5000TM haematology analyzer provides during full blood count analysis the parameter NEUT-X, which is the mean value of the side scatter measurement of the neutrophils population. NEUT-X is representative of the inner structure of the neutrophils, is not correlated to the neutrophil count and is age independent. High values of NEUT-X in vitamin B12 deficiency reflect the presence of hypersegmented giant neutrophils. The aim of this study is to investigate the NEUT-X in iron deficiency as well as in combined iron and vitamin B12 deficiency.
Methods: The patients enrolled in this study were recruited from a larger cohort of patients who were referred to our Laboratory for investigation. Full blood count was performed with Sysmex®XE-5000TM analyzer, ferritin, vitamin B12 and folate levels were determined by Roche cobas®e-411 analyzer. 140 patients (54 males) with iron deficiency, defined as ferritin levels below the relevant threshold for gender and age (Group A), 99 patients (43 males) with combined iron and vitamin B12 deficiency (Group B), matched in haemoglobin levels with group A (9,7±2,7 vs 10,1±2,7, respectively; P=0,18),were enrolled in the study. 144 healthy individuals (54 males) were included in the study, as control group (Group C). Statistical analysis: Values are reported as mean±SD. One-way ANOVA, Tukey HSD and Mann-Whitney U tests were applied. Values of P< 0,05 were considered to indicate statistical significance.
Results: There was a statistically significant difference in MCV (mean corpuscular volume) and NEUT-X values between groups, as determined by one-way ANOVA (F=71,5; P=0,0001 and F=16,7; P=0,0001, respectively) (Table 1). The Tukey HSD revealed that group A presents statistically significant lower MCV and NEUT-X values compared to groups B and C (all P=0,0001), whereas there was no statistically significant difference between groups B and C in NEUT-X values (P=0,973). Group B presents statistically significant higher values of both MCV and NEUT-X compared to group A.  Table 1.
Group MCV, fL NEUT-X, ch
A: iron deficiency 77,8±8,6 135,2±3,7
B: iron & vit. B12 deficiency 82,6±10,2 137,2±3,2
C: healthy individuals 89,0±4,6 137,3±3,2

Conclusions: Iron deficiency apart from the well known morphological effect on red blood cell volume has an impact on the neutrophil structure as documented by the low NEUT-X value. The concomitant presence of vitamin B12 deficiency affects both erythrocyte and neutrophil morphology as documented by increasing the values of both MCV and NEUT-X. 

Hemolytic Disease Of The Newborn In A Group A Caucasian-European Infant Delivered By A Group O Mother
Xavier Tejedor Gandux, Alicia Martnez Iribarren, Cristian Morales Indiano, Carla Fernndez Prendes, Merc Espinosa Nieto, Silvia Torres Romero, Ana Dueas Mrquez, Trinidad Lema Pual, MJos Cnovas Bueno, Cristina Juan Lizana, Komal QyYum, MAntnia Llopis Daz
CoreLab-Hematology. Department of Clinical Analysis and Biochemistry. Laboratori Clnic Metropolitana Nord (LCMN). Hospital Universitari Germans Trias i Pujol, Badalona, Spain

Introduction: Maternal-fetal ABO incompatibility is a common hematological problem affecting the newborn. In general, hemolysis is minimal and the clinical course is relatively benign, rarely causing significant anemia commonly associated with Rh hemolytic disease of the newborn (HDN). We describe a case of a A/Rh positive term newborn born to an O/Rh positive Caucasian-European mother demonstrating hemolysis and a robust response of the bone marrow.
Methods: It was carried out at first total and conjugated bilirubin hemogram, reticulocytes, blood smear. Immunohematology tests: ABO / Rh group, Direct Coombs, Indirect Coombs, and antibody titration test. After the first 24 hours, hemogram, reticulocytes and total and direct bilirubin were repeated daily during 6 days.
Results: The neonate’s red cells typed A, D positive, while the mother’s red cells typed O, D positive. The mother’s anti-A antibody titer was 256. The direct antiglobulin test (DAT) was positive with the newborn red cells with an IgG type specificity, and anti-A, but not anti-B, antibody was detected in the neonatal red cell eluate. The infant’s blood hemoglobin and serum total bilirubin (TBIL) concentrations were 12.4 g/dl and 10.81 mg/dl, respectively  with the presence of reticulocytosis.   The value of reticulocytes (Ret) reached its maximum value with a value of 12.9% on the second day (Fig1). The peripheral blood smear demonstrated numerous nucleated RBCs, schistocytes, prominent spherocytes, polychromasia. The infant’s TB peaked at 12.9 mg/dl on day two (Fig. 2), which prompted 2 sessions of phototherapy. Simple RBC transfusion or exchange transfusion was not required. The infant was discharged on day 6 with a TBIL of 6.8 mg/dl.
Conclusions: This case demonstrate that the etiology of ABO hemolytic disease of the newborn (ABO-HDN) is complex because anti-A and anti-B antibodies are composed mainly of IgM. Since only IgG antibodies cross the placenta, those pregnant women with high levels of IgG anti-A,B, anti-A, or anti-B with an ABO incompatible fetus will be the ones to give birth to an infant with ABO-HDN.

Telemicroscopy: An Innovative Solution To An Age-Old Problem
Craig Vilk, Dan Lee, Jacky Chow, Kyle Congo
MultiCare Health System, Tacoma, WA, United States

Introduction: Background: Telemicroscopy is the digitizing of images from a microscope and transmitting those images to another location via the internet. MultiCare Health System has adopted this technology in hematology and microbiology to provide real-time support for satellite hospitals and remote clinics, especially in the areas of interpreting challenging cells on a differential and identifying organisms on a Gram stain. This telemicroscopy model allows the Core Lab to see exactly what the outlying locations are seeing in real-time and provide feedback in making an accurate decision on differentials and Gram stains, thereby improving patient care.
Methods: Methods: When a technologist at an off-site location is looking at a slide and needs a second-opinion consultation from an experienced technologist, they call the Core Lab. A ProScope 5MP Microscope Camera (Bodelin, Wilsonville, OR), under $500, is inserted into an ocular of a Nikon or Leica microscope at the off-site location and the Micro Capture Software (Bodelin, Wilsonville, OR) is opened. When the Core Lab receives the consult request, a web conference using GoToMeeting (Logmein,Boston, MA) is initiated. The Core Lab technologist will then “Share Your Screen” from the GoToMeeting, and “Change Presenter to” the off-site location. This allows the Core Lab to dynamically look at the subject under the off-site microscope. The protected health information (PHI) are discussed over the phone, and not documented by both software. With the real-time video streaming and discussion of the clinical case, the Core Lab can give an accurate second-opinion consultation.
Results: Results: Below are the images the technologist from the Core Lab would see from the ProScope 5MP Camera through the GoToMeeting software:
Conclusions: Conclusion: This technology offers a relatively inexpensive option for locations that do not have the clinical expertise that the Core Lab has in analyzing slides. Moreover, telemicroscopy offers a valuable educational opportunity with real-time feedback on difficult cells or organisms.

Performance Comparison Of Malaria Scanner With Microscopy And Flow Cytometric Method For Malaria Detection And Parasitemia Determination
Jung Yoon, Jung Ah Kwon, Soo Young Yoon, Chae Seung Lim
Department of Laboratory Medicine, Korea University College of Medicine, Seoul, Korea

Introduction: The rapid diagnosis and parasite density monitoring is crucial in timely management of malaria. Microscopic examination of Giemsa stained peripheral blood smear has been considered a reference method for diagnosis of malaria, despite its limitations. Recently, microscopic image based automatic malaria detection systems were developed. Here, we evaluated the analytical performance of a new automated image-based malaria detection system for malaria detection and parasitemia determination.
Methods: The Malaria scanner (BioZentech, Seoul, South Korea) is an automated image based microscopic counter system composed of digital microscopy, plastic microchannel chip, malaria staining methods using fluorescent (SYBR green I), and image analysis program for differentiation of RBCs and malaria. With diluted samples of cultured P. falciparum (strain 3D7), and EDTA collected 9 P. vivax positive samples collected at Korea University Guro Hospital from June 2017 to September 2017, presence of malaria and parasitemia levels were analyzed by Malaria scanner, conventional microscopy and flow cytometric method, simultaneously. The linearity, precision and LOD of Malaria scanner was evaluated and compared to the results of conventional microscopic examination and flow cytometry.
Results: When evaluated using malaria culture with infected RBC % range of 0% to 8.03%, the automated microscopic malaria scanner showed high degree of linearity (R2=0.958, P=0.005) and the CV% range was 12.57% to 34.67%. In case of malaria infected samples with the infected RBC% range of 0.03% to 0.22%, the automated microscopic malaria scanner also shoed high degree of linearity (R2=0.931, P=0.008) and the CV% range of 28.11% to 53.22%. Malaria scanner was more precise than microscopic examination of PB below the parasitemia range of 4.92%. The limit of detection for malaria scanner was 0.0017863% and high correlation was observed between all three methods.
Conclusions: Malaria scanner could offer many advantages over other methods for parasitemia determination. Malaria scanner achieved more precise parasitemia results when compared with microscopy and is technically easier and faster method when compared with the flow cytometric method.

The Surface Markers And Survival Rate Of Platelets During Storage At 4C; The Influence Of Sodium Octanoate
vahid baghdadi, Fatemeh Yari, Mohammad Hessam Rafiee
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, TEHRAN, Iran

Introduction: Storage of platelet (PLT) concentrate (PC) at room temperature (20-24°C) limits its storage time to 5 days due to both the destructive effects of PLT storage lesion (PSL) and bacterial contamination.Although, prolonged storage of PLTs at 4°C reduces bacterial contamination and PSL levels but it is accompanied by an increase in the clearance rate and changes in the surface markers of PLTs. The goal of this study was to evaluate the effect of sodium octanoate(SO) as a stabilizer on PLTs during storage at 4°C.
Methods: PCs were divided into three portions and stored for 5 days in three different conditions; 20-24°C with agitation, 4°C temperature and 4°C in presence of sodium octanoate.PLTs enumeration was performed using automated hematology analyzer. To measure the metabolic activity and survival rate of PLTs, the WST-1 assay was performed. The activity of lactate dehydrogenase enzyme (LDH) was measured by a biochemical analyzer. Additionally, the levels of GPIbα and CD62P were measured on PLTs by flow cytometry technique.
Results: The trend for platelet count was as follows:  SO (4°C) PLTs > 4°C PLTs > 22°C PLTs.The viability was higher in the SO-treated PLTs than other groups.LDH amount was lower in the SO-treated PLTs than other groups.  GPIbα expression was higher in SO-treated PLTs than other groups at 4°C. On the other hand, the expression of CD62P was lower at 4°C in PLTs in the presence of SO. 
Conclusions: SO could modulate the effects of cold temperatures on PLTs. It can also keep PLT survival better at 4°C .

The Use Of Platelet Activation Measurements For Predicting The Occurrence Of Peroperative Solid Micro-Emboli During Carotid Endarterectomy.
Aarent Brand1,2, Tesse Leunissen1, Suzanne Korporaal2, Rolf Urbanus2, Gerard Pasterkamp2, Gert Jan de Borst1
1UMC Utrecht Vascular surgery, Utrecht, Netherlands, 2UMC Utrecht Laboratory of Clinical Chemistry and Haematology, Utrecht, Netherlands

Introduction: Post-operative white-matter brain lesions on diffusion-weighted magnetic resonance imaging (DWI-MRI) after carotid endarterectomy (CEA) are associated with an unfavourable outcome. Up to 17% of patients undergoing CEA show DWI lesions post-intervention. These lesions are regarded as a sign of acute ischemia and thought to be caused by both peroperative hemodynamic changes and, more importantly, by arterial micro-emboli. The main component of the micro-embolic signals (MES) that are detected with transcranial Doppler imaging (TCD) are platelet aggregates. Peroperative MES detected with TCD increase the risk for these lesions. To date, there is no known predictor for the occurrence of MES. High on-treatment platelet reactivity (HTPR) is associated with thrombo-embolic events after CEA and can be detected using platelet reactivity tests. Therefore, our study aimed to investigate the predictive value of platelet reactivity (PR) measurements on the incidence of MES.
Methods: Peroperative MES were recorded with TCD in 197 patients undergoing CEA. Venous trisodium citrate anticoagulated blood samples were analysed for platelet reactivity with the VerifyNow P2Y12-assay and with a flow cytometry-based (FC) assay, measuring ADP and thrombin receptor activating peptide (TRAP) initiated platelet response. Secondary outcome was major adverse cardiovascular events (MACE).
Results: 49/197 patients had peroperative MES. PR measured with FC after ADP stimulation correlated with MES [P=.032] and high PR towards ADP was associated with MACE [OR 2.346 (95%CI 1.126 - 4.890), P=0.023]. In contrast, PR measured with the VerifyNow P2Y12-assay did not. The correlation between PR measured with FC and MES dissolved after correcting for clopidogrel use.
Conclusions: PR measured with FC after ADP stimulation correlates with the occurrence of MES whereas the VerifyNow P2Y12-assay does not. The use of clopidogrel mitigates this effect, suggesting that this FC assay can detect clopidogrel treatment. The expediency of FC PR measurement for the prediction of adverse outcome is encouraging and compels further investigation.

Immature Platelet Fraction Normal Reference Range During Pregnancy
Kara Bureau , Rajan Dewar , Gerald Davis
University of Michigan Health Center , Ann Arbor , MI, United States

Introduction: Thrombocytopenia is a common finding in healthy woman during pregnancy. The immature platelet fraction (IPF) reference intervals are about 1% to 7% of the total platelet count in a normal population. The IPF reference range has not yet been established for women during pregnancy. Bone marrow thrombopoiesis is estimated by counting the immature reticulated platelets in the peripheral blood. By finding the normal IPF reference range for healthy pregnant women, these values can be used to help with diagnosis of thrombocytopenia in women during pregnancy.
Methods: Blood samples in EDTA tubes were recieved from labor and delivery floors of Michigan Medicine and analyzed by the Sysmex XN-9000. Out of 189 smaples collected, 51 pregnant women with normal thrombocyte values (150-400 K/uL) and no gestational or preexisting health conditions were chosen. Women included in this study were between the 20th and 40th week of gestation. Immature platelet fraction values of the 51 women were then quantified by the hematology analyzer. The normal population IPF ranges were collected on a broad spectrum of outpatients with normal white cell, hemoglobin, and platelet counts.
Table 1. XN-9000 Immature Platelet Fraction Reference Intervals
%IPF Healthy Pregnant Women Normal Population
Mean 5.5 3.4
SD 3.9 1.6
N 51 58
Range 0.9-20.5 0.0-7.7
Only 51 of the 189 samples were used due to current and preexisting complications including pre-eclampsia, gestational diabetes, gestational hypertension, cholestasis during pregnancy, traumatic injury, hypothyroidism, and samples acquired post-delivery.  
Conclusions: The normal immature platelet fraction reference range for pregnant women is considerably larger than that of the normal population (non-pregnant). The importance of establishing normal IPF reference values for pregnant women will be helpful in identifying normal IPF values in pregant women with thrombocytopenia.

Two Cases Of Autosomal Dominant Macrothrombocytopenia Caused By A Rare Gpibb Variant
Pieter De Kesel1, Anna Vantilborgh2, Jan Dierick3, Ariane Luyckx3, Sarah Debussche4, Kathleen Freson5
1Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium, 2Department of Hematology, Ghent University Hospital, Ghent, Belgium, 3Department of Laboratory Medicine, AZ Maria Middelares, Ghent, Belgium, 4Department of Hematology, AZ Maria Middelares, Ghent, Belgium, 5Department of Cardiovascular Sciences, Center for Molecular and Vascular Biology, University of Leuven, Leuven, Belgium

Introduction: The platelet transmembrane glycoprotein (GP) Ib-IX-V receptor plays an essential role during the initial steps of primary hemostasis by adhering platelets to damaged subendothelium via VWF. A variety of mutations in genes encoding GPIb-IX-V cause Bernard-Soulier syndrome, an autosomal recessive disorder characterised by macrothrombocytopenia and often severe bleeding. Recently, several monoallelic variants of GP1BB, encoding the GPIb β-chain, were found to be associated with autosomal dominant macrothrombocytopenia1-2. We present two cases of unrelated patients referred to a hematologist for persisting macrothrombocytopenia. The first patient, a 32-year old woman, presented with frequent ecchymoses, gingival bleeding, metrorrhagia and bright red blood loss per rectum. The second patient, a 22-year old man, showed no bleeding symptoms. 
Methods: The following analyses were performed: complete blood count; light transmission aggregometry (LTA) using ristocetin, ADP, epinephrine, arachidonic acid, collagen and U46619 as agonists; lumiaggregometry-based ATP-release assay; electron microscopy (EM) of platelets; flow cytometric detection of CD42b (GPIbα)expression on platelets and next generation sequencing (NGS) for inherited platelet disorders (ThromboGenomics platform).
Results: Both patients had moderate thrombocytopenia (patient 1: 95-141 x109/L; patient 2: 88-103 x 109/L), increased platelet volume (patient 1: MPV 12.9-14.0 fL; patient 2: multiple large platelets on blood smear), elevated immature platelet fraction (patient 1: 15.4-17.0%; patient 2: 23.4-29.5%), normal LTA with all agonists (including ristocetin), but reduced ATP-release. EM showed normal platelet dense granule content for patient 1 and an inconclusive result for patient 2 (low platelet count). Slightly reduced GPIbα-expression on platelet surface was found for patient 2 only, with 6.3% of platelets lacking CD42b-expression. NGS revealed the presence of a heterozygous c.47T>C variant in GP1BB encoding Leu16Pro. This missense mutation has been associated with autosomal dominant macrothrombocytopenia and variable bleeding diathesis1-2.
Conclusions: The presented cases highlight the value of mutation analysis when congenital macrothrombocytopenia is suspected, as platelet aggregation and flow cytometry might not be sufficient to detect monoallelic subtypes. 1Sivapalaratnam et al. Blood. 2017;129(4):520-524. 2Babuty et al. Br J Haematol. 2019;doi: 10.1111/bjh.15739.

Performance Evaluation Of The Nucleated Red Cell Count In Neonates Using The Abbott Alinity Hq Hematology Analyzer
In-Hwa Jeong, Gyu-Dae An, Hyeon-Ho Lim, Kwang-Sook Woo, Jin-Yeong Han
Dong-a University College Of Medicine, Busan, South Korea

Introduction: Nucleated red cells (NRBC) are physiologically present in the peripheral blood of newborns and fetus. The detection of NRBC in the blood can be diagnostic and prognostic markers in many medical conditions and therefore, an accurate quantitation of NRBC is an important procedure. In this study, we evaluated the performance of Abbott Alinity hq NRBC measurement.
Methods: We evaluated reproducibility (within-run precision) and between-batch precision using abnormal high quality control (QC) materials. For comparison of the Alinity hq with a manual count of NRBC, 149 neonatal blood samples were analyzed. The NRBC count results from the Sysmex XN-9000 were also compared with those of the Abbott Alinity hq in 40 neonates.
Results: The reproducibility and between-batch precision were excellent with abnormal high QC materials and the coefficient of variation (%CV) was 2.0 and 2.8, respectively within precision limits provided by the manufacturer. The NRBC counts obtained with the Abbott Alinity hq were strongly correlated with the Sysmex XN-9000 with correlation coefficients of 0.992. For comparison with a 400-cell manual count, the Alinity hq NRBC measurement also showed a high degree of correlations (g= 0.960).
Conclusions: To the best of our knowledge, for the first time we describe here the NRBC measurement of a new Abbott Alinity hq hematology analyzer. NRBC are counted using fluorescence and light scatter characteristics and are expressed as both concentration and percentages. The Alinity hq automated hematology analyzer provides a precise NRBC count and is a safe and efficent system for the measurement of NRBC in newborns.

In Vitro Platelet Activation Response Induced By Dengue Non Structural Protein 1
Nallely Garca-Larragoiti1, Young Kim2, Cesar Lpez-Camacho2, Soledad Vzquez-Garcidueas1, Arturo Reyes-Sandoval1, Martha Eva Viveros-Sandoval1
1Postgraduate Division, School of Medicine, UMSNH, Morelia, Mexico, 2Jenner Institute, NDM, Oxford University, Oxford, United Kingdom

Introduction: In recent years, besides their important role in hemostasis and thrombosis, platelets have been recognized as mediators in immune response and host defense against  several pathogens including viruses. Dengue virus (DENV) infection is an increasing health problem in tropical and subtropical areas, its clinical presentation goes from mild disease to severe and haemorragic fever. NS1 is a highly conserved DENV glycoprotein that can be detected in serum during the febrile phase infection. Recent results suggest a critical role for NS1 in the vascular leak and thrombocytopenia found in severe DENV infection, however, the mechanisms involved in DENV-induced platelet activation are not completely understood.  
Methods: NS1 DENV2 was cloned into pHLSec plasmid. Competent E. coli cells were transformed and plasmid DNA was extracted to verify the presence of NS1 by agarose electrophoresis. HEK293 cells were transfected with plasmid DNA, the secreted protein was analyzed by silver staining and western blot while a liquid chromatography system was used to purify it. Platelet-rich plasma (PRP) was obtained from healthy donors by centrifugation of citrated blood at 800 rpm for 10 minutes; PRP was incubated with NS1 protein. After protein stimulation, platelets were stained with specific antibodies and cells fixed with paraformaldehyde solution. Platelets were detected by flow cytometry using anti-CD41-PC7 antibody. Expression of platelet activation markers P-selectin and GpIIb-IIIa was assessed using anti-CD62P-PE and PAC1-FITC antibodies, respectively.
Results: Flow cytometry analysis showed that DENV NS1 protein is able to induce expression of P-selectin and GpIIb-IIIa complex on platelet surface. Platelets activated with DENV NS1 have an activation response similar to that obtained when stimulated with collagen.
Conclusions: Our results suggest that DENV NS1 induces the expression of activation markers on platelet surface. These results may help to explain thrombocytopenia in dengue and emphasize the important role of DV NS1 in the pathogenesis. Further studies are needed to characterize platelets immune response against viruses.

Reverse Correlation Of Platelet Microparticle Formation And The Expression Of The Adhesive Receptors In Stored Platelets
Mehran Ghasemzadeh, Ehteramolsadat Hosseini
Blood Transfusion Research Center, High Institute for Research and Education in Transfusion Medicine, Tehran, Iran

Introduction: Platelet activation was shown to be associated with microparticle release. However, during platelet storage, microvesiculationmay also occur by the ignition of the apoptotic pathways especially in long-stored platelets.Decreasing levels of GPIbα and GPVI expression during the storage ofplatelet concentrates (PCs) were reported by several studies. Receptor ectodomain shedding is considered as the main mechanism of this phenomenon. However, whether the down-regulation of these receptors could be also relevant to plateletmicrovesiculation during storage is not clear yet.
Methods: PRP-PCs were subjected to flowcytometry analysis to examine the expression of platelet adhesive receptors GPIbα and GPVI on day 1 and 5 post-storage.The levels of microparticle formation were also evaluated by flowcytometric analysis of CD61 positive particles which detected around polysciences FITC-Microbeads (1.00 μm in diameter).
Results: Our data showed increasing levels of platelet microparticle formation during storagewhereas expression of platelet adhesion receptors GPVI and GPIbαwere significantly reduced in 5 day-stored platelets
Conclusions: We found significant reverse correlation between storage-dependent platelet microparticulation and decreasing levels of adhesion receptors. This may suggest the platelet membrane loss due to microparticulation as another important mechanism for the down-regulation of platelet adhesion receptors during storage.    Keyword:GPIbα; GPVI; microparticle; Platelet; Shedding; Storage  

Role Of Mean Platelet Volume (Mpv), Platlet Distribution Width (Pdw) And Platelet Large Cell Ratio (Plcr) In Diagnosis Of Hyperdestructive Thrombocytopenia.
hamzullah khan1, dr adnan masood2, dr fazli bari 3
1Nowshera Medical college, nowshera, PA, Pakistan, 2nowshera medical college, nowshera, AK, Pakistan, 3nowshera medical collge, nowshera, AK, Pakistan

Introduction:   Thrombocytopenia has been shown to have significant mortality if ignored. Platelet indices have been reported to be useful prognostic indicators. The objectives of this study was to determine the diagnostic importance of the platelet indices in diagnosis of hyperdestructive thrombocytopenia i.e ITP.
Methods: This was a cross sectional observational study was conducted in the department of Pathology  (MTI) Qazi Hussain Ahmed Medical Comeplex Nowshera Medical College , from  Aug 2017 to Jan 2018. These blood samples were analyzed in clinical Pathology laboratory of QHAMC. Required information’s were recorded on predesigned proforrma as per objectives of the study.
Results: The peripheral smears of 139 cases were reported in the study. Detailed  history and Thorough clinical examination was conducted. Mean age of the study population of the patients with standard deviation was 30.90(±6.4) years. Mean platelet count was 27. 37(±12.8) x109/. Mean platelet volume MPV was 11.4(±1.4) fl. Mean platelet distribution width (PDW) was 15.4(±3.3) fl. Mean platelet large cell ratio (PLCR) was 39.6(±8.9) %. Eight cases with MPV lower than 11fl and cases with PDW more than 15fl that were also having pancytopenia or bycytopenic picture were advised bone marrow aspiration for further diagnosis if clinically indicated. Six cases out of eight to whom bone marrow was advised were sent for bone marrow examination by the clinicians and we found that three of them were idiopathic thrombocytopenia and one Megaloblastic anemia,one case with pancytopenia due to hyperspleenism and one with acute leukemia with eosinophilia.
Conclusions: From the above we concluded that all cases with MPV>11fl and PDW>14fl are sensitive and specific indicators for ITP and These indices help to distinguish hyper-destructive thrombocytopenia and hypo-productive thrombocytopenia very easily and it is also cost effective on a very simple test that is special smear. We must look for platelet indices very keenly while reporting a case with bi-cytopenia and pancytopenia.

Gene Expression Profiling Of Platelet Receptors In A Mouse Model Of Perivascular Cuff Induced Stenosis
Fangfang Lu1, Zhen Weng2, Yang He1,2
1MOH Key Lab of Thrombosis and Hemostasis, Jiangsu Institute of Hematology, The First Affiliated Hospital of Soochow University, Suzhou, China, 2Cyrus Tang Hematology Center, MOE Engineering Center of Hematological Disease, Soochow University, Suzhou, China

Introduction: Platelets are considered to play a critical role during the pathogenesis of atherosclerosis and the vessel response to vascular injury. However, systemic evaluation of the expression of platelet receptors in the setting of stenosis was rarely reported. To investigate the gene expression change of major platelets receptors in a mouse model of stenosis.
Methods: Stenosis was established in the unilateral femoral artery of10-weekwildtype male C57BL/6mice by perivascular cuff placement and 4 weeks’ high fat chow breeding, while mice without no cuff placement was designated as control. Blood was then drawn from the abdominal aorta of above mice for platelets isolation, followed by RNA extraction from these platelets with Trizol method and real time reverse-transcription quantitative PCR to examine the expression levels of platelet receptors.
Results: Successfully induction of the stenosis was confirmed by H&E staining of cross section of femoral artery tissues. Significantly increased levels of GPIb (2.85 times), αIIb (8.15 times), β3 (6.14 times), GPV (2.30 times), GPIX (2.84 times), P2Y1(3.56 times) and P2Y12 (3.15 times) were observed in mice with stenosis compared to controls , whereas no difference was found on GPVI, PAR3, PAR4, TP and CLEC-2.
Conclusions: Altered expression of several platelet receptors during the stenosis, suggesting the possible involvement of these platelet receptors in the stenosis. Further function studies about these platelets receptors should be performed to elucidate the exact roles during the stenosis, make it an important entry point for the diagnosis and treatment of clinical vascular restenosis.

Platelet Count And Platelet Indices Of Symptomatic And Asymptomatic Children Infected By Malaria In Calabar, Nigeria

Introduction: Malaria infection has been reported to be a major problem affecting developing countries. The aim was to study the effect of malaria parasite infection on platelet count and platelet indices [mean platelet volume (MPV), platelets distribution width (PDW), plateletcrit (PCT)] among symptomatic subjects.
Methods: A total of one hundred and twelve (112) subjects (males and females) comprising of fifty-eight (58) symptomatic and fifty-four (54) asymptomatic children infected by malaria who were within the age range of 0-15 years (mean age of 8.44 and 9.21) were enrolled in this study. Full automatic blood cell counter was used to determine the platelet count and platelet indices while thick and thin film method was used for to ascertain malaria parasite density. Student t-test, ANOVA and Pearson correlation was used (SPSS 20.0 version) in the analysis of the data.
Results: The mean values of symptomatic malaria subjects were found to be significantly reduced in PCT, PDW and PC (0.16±0.04, 15.84±0.89 and 181.45±43.62) and significant high in MPV and MPD (9.36±1.05 and (418.80±554.40) against that of asymptomatic malaria subject (p˂0.05). Mean MPV was significantly increased in 6 -10 and 11–15 years (9.35±0.90 and 9.81±1.16) when compared with 0-5 years (8.60±0.62) age range symptomatic malaria subject groups while mean PC was significantly decrease in 6-10 and 11–15 years (172.14±35.52 and 181.55±48.49) when compared to 0-5 years (204.36±36.92) age range groups. There was a moderate positive significant correlation between PCT versus MPV (r=0.327) while PC versus PCT shows a strong (r=0.895) positive significant correlation (p˂0.05). ParametersSymptomaticn(58)Asymptomaticn(54)TP-value PCT (%)m MPV (fl) PDW (fl) PC (×109/L) MPD (p/µl)
Table 1: Comparison between the symptomatic and the asymptomatic group
0.16±0.04 0.22±0.03 -6.997 0.000*
9.36±1.05 8.59±0.73 4.409 0.000*
15.84±0.89 16.21±0.58 -2.573 0.002*
181.45±43.62 246.06±41.20 -8.047 0.000*
418.97±591.87 120.80±80.00 3.599 0.001*

Conclusions: There is significant increase MPD and MPV, decrease PC, PCT and PDW in the symptomatic group compared to the asymptomatic. It is important that platelet indices and platelet count should be included as a routine screening investigation to detect malaria parasite infection and treated accordingly.

Correlation Between Fibrinogen-Like Protein 2 Activity And Pet/Ct In Follow Up Of Aggressive Lymphomas
Esther Rabizadeh, Ronit Gurion, Yael Zimra, Pinhas Hasin, Anat Gafter , Ofir Wolah, Izhack Cherny
Rabin Medical Center, Petah Tikva, Israel

Introduction: Fibrinogen-Like Protein 2 (FGL2/fibroleukin) is a key player in the coagulation-immune system axis. It has two forms. The membrane-bound possesses a serine protease activity capable of directly cleaving prothrombin into thrombin (i.e., prothrombinase) independent of the canonical coagulation route, while the secreted form possesses significant immunosuppressive properties. Accumulating evidence underlines the critical role of FGL2 in cancer development and metastasis. Essentially, FGL2 has been shown to be overexpressed by all solid tumor tissues tested thus far. Increased FGL2 expression or activity has been observed in the peripheral blood of various malignancies including aggressive lymphomas. Lymphoma, the most common blood cancer, is characterized by a fast-growing disease of B/T cells.   Most patients with aggressive lymphoma will enter remission due to effective treatments available today. However, approximately 25% of the patients will experience relapse within 24 months. Nevertheless, there is a lack of a simple, non-invasive biomarker to monitor response of lymphoma to therapy and predict relapse. The diagnosis is largely based on the pathologic workup of patients with suspicious clinical presentation and the response to therapy is based mainly on clinical assessment tools such as PET/CT. AIM:  To develop a simple and reliable liquid biopsy diagnostic test, based on the FGL2 activity in peripheral blood, to monitor remission status and to predict relapse of aggressive lymphoma.
Methods: Over 100 peripheral blood samples were collected from healthy individuals and lymphoma patients every 2-3 months. FGL2 prothrombinase activity, mRNA and protein levels and PET/CT were determined.
Results: We found a strong correlation between FGL2 prothrombinase activity and PET/CTresults of patients in follow up process. On average, FGL2 activity is 30% higher in active disease compared to healthy or remission. In 90% of cases, remission accompanied an average decrease in FGL2 activity and was correlated with PET/CT results.  All cases of relapse were accompanied by a significant increase in FGL2 activity.
Conclusions:  A simple, minimally-invasive, rapid, semi-automated and low cost test was developed which allows monitoring of aggressive lymphoma patients. In view of our findings, the measurement activity of FGL2 has a potential to be used as a biomarker for follow up of aggressive lymphoma patients. 

Glanzmann Thrombasthenia From Selected Regions Of Pakistan. A Health Related Quality Of Life Survey.
Muhammad Yunus Jamal Siddiqi1, Arshi Naz1, Akbar Najmuddin2, Ayisha Imran3, Tahir Sultan Shamsi1
1National Institute of blood diseases , Karachi, Pakistan, 2Fatimid Foundation, Karachi, Pakistan, 3CHughtai Lab, Lahore, Pakistan

Introduction: Glanzmann thrombasthenia (GT) is one of the well-known inherited platelet functional defect. It is a familial disorder being autosomal recessive in character. The defect is caused by mutations in the genes encoding ITGA2B or ITGB3 located on chromosome 17, resulting in qualitative or quantitative abnormalities of the expression of αIIb-β3 integrin, a fibrinogen receptor positioned on platelets. It is characterized by a bleeding diathesis which is variable. Incidence is increased in those territories where consanguineous marriages are practiced. Quality of life assessment in these patients is seldom evaluated which is very important for their well-being and existence.
Methods: 20 patients with GT were selected for the study after obtaining informed and written consent. History was recorded in a structured data sheet and bleeding score was assessed in a questionnaire based on MCMDM-1VWD Bleeding Questionnaire. HRQoL assessment questionnaire (Likert scale type) was based on different aspects of their well-being and functioning, their social status, as well as their interpretations on medical care. Flowcytometry was performed on BD FACSCALIBUR (using CD 41, 61, 42a, 42b antibodies BD Biosciences) to classify the GT patients. 
Results: GT type I had the highest average bleeding score and secured the highest points on the severity assessment for poor healthcare, social deprivation, lack of education and self-esteem followed by GT type II. GT type III scored least points.
Conclusions: Patient with GT type I were the worst affected needing revision in medical care and improving social support and right to education.

A Clinical Evaluation Of Various Predictive Markers Of Platelet Recovery In Dengue Patients

Introduction: Dengue related thrombocytopenia, poses a critical challenge in patient management, especially in predicting Platelet recovery. Since many parameters are available for predicting platelet recovery, it was required to evaluate all of them, and find the most efficacious one. We evaluated different parameters and their trends to predict platelet recovery within a defined period. Availability of these parameters in predicting platelet recovery, can help clinicians, in decision making.
Methods: Dengue positive patients (n=62) confirmed using  NS1 Ag or Dengue IgM Elisa, with platelet count < 150000, exhibiting rising and falling trend for platelets and predictive markers were selected for this study. The criteria for significant Platelet recovery was an increase in platelet count by 10,000 or 10%, whichever is lower; over preceding value. The testing was done on Mindray BC 6200 Hematology Analyzer. The predictive markers were classified as peaks, troughs, jumps and falls. The parameter where the Platelet recovery was seen in reference to “peaks” were Immature Platelet Fraction(IPF), High Fluorescence IPF(H-IPF), High Fluorescence Cells(HFC), Mean Platelet Volume(MPV). Whereas the platelet recovery was seen vs. troughs of parameters such as Neutrophil Lymphocyte Ratio(NLR), Platelet Lymphocyte Ratio(PLR) & Platelet HFC Ratio(PHR). We classified ‘HFC Jump’ with base values >0.5% and increase of at least 3 times over preceding value. ‘Falls’ were classified as decrease in 50% from preceding values, like observed in NLR, PLR & PHR.
Results: All these parameters were observed in terms of predictability of platelet recovery, from the day of Peak/trough/rise/fall of parameter. Cumulative percentage of recovery of population tested, was tabulated for each parameter, wherein recovery between 24 to 96 hours was established by Delta Value. The results, as in table no 1, exhibited that, the PHR Trough & PHR Fall were the best predictor of Platelet recovery amongst PEAK/Trough and Rise/fall parameters respectively. PHR trough had a delta value of 52% and PHR Fall had a delta value of 71%. Thus, PHR trend analysis both in terms of trough and Fall had the best predictive value, amongst all the parameters evaluated.
Conclusions: We could establish the clinical utility of all the above said markers for platelet recovery, in Dengue patients. PHR Fall was the most efficacious marker amongst, the various parameters analyzed.

Establishment OfReferenceIntervals For Platelet Count And Indices In Healthy Greek Term-Pregnant Women

Introduction: Pregnancy is characterized by a chronic disseminated intravascular coagulation status. Given the fact that the Mean Platelet Volume (MPV) and Platelet Distribution Width (PDW) are markers of platelet activity, those simple parameters can indirectly assess thrombotic complication after labor. The rationale of this study was(i) to determine the platelet count (PLT), MPV, and PDW reference values in healthy Greek term-pregnant women, and (ii) to evaluate those markers before and after the delivery process with the aim to prevent a possible thrombotic episode in postpartum period.
Methods: Blood samples from 673 healthy pregnant women before labor were collected in tubes with EDTA as an anticoagulant. The hematologicalparameters of PLT, MPV, and PDW were considered in all blood samples and measured by COULTER LH780 analyzer pre-labor and one or two days postpartum. The samples with the indication “platelet clumps” and those that had an MCV< 75fl were excluded, in order to avoid the interference of small red blood cells with platelet count measurement. After subtracting 5% of the extreme values from PLT, MPV and PDW the Median, Minimum and Maximum values were calculated using the SPSS 21 statistical software. In terms of statistically comparing pre- and post- delivery PLT, MPV, and PDW values in women with a “normal birth“, the Wilcoxon signed-rank test was employed. Of note, women who underwent caesarian section were excluded due to the possibility of blood transfusion.
Results: The Median, Minimum, and Maximum PLT, MPV and PDW values are shown in Table 1. With regard to comparisons between pre- and post-natal values in 256 women giving a “normal birth“, there was a statistically significant reduction in platelet count, MPV, and PDW (p< 0.001).
Conclusions: At the end of the pregnancy, MPV and PDW are elevated as a physiological response to postpartum bleeding and a decrease of those parameters is noticed in the subsequent days. MPV and PDW are simple and inexpensive parameters which can be easily used for the prediction of a thrombotic complication after labor.

Platelet Counting In Thrombocytopenic Samples Using Mindray Bc-6800 Plus
Sumi Yoon, Hanah Kim, Mina Hur, Hee-Jung Chung, Hee-Won Moon, Yeo-Min Yun
Department of Laboratory Medicine, Konkuk University School of Medicine, Seoul, Korea

Introduction: The performance of low PLT counting is crucial for clinical practice especially in transfusion management. We evaluated low PLT counting performance of Mindray BC-6800 Plus (BC-6800P, Mindray, Shenzhen, China) and compared it with that of Sysmex XN-9000 (XN, Sysmex, Kobe, Japan).
Methods: We observed precision of both PLT-I channels (PLT-I) in BC-6800P and XN in 11 consecutive samples (10.7 - 385.0 x 10^9/L) and 10 thrombocytopenic samples (6.7 - 20 x 10^9/L). We also observed precision of PLT-O channel with CR mode in BC-6800P (PLT-O/CR) and PLT-F channel in XN (PLT-F) in 10 thrombocytopenic samples. We analyzed correlation of PLT-Is in both analyzers in 2,339 clinical samples. We also analyzed correlation of PLT-O/CR and PLT-F in reflex tested (n = 398) and thrombocytopenic (=< 50 x 10^9/L, n = 145) samples.
Results: In 11 consecutive samples, precision of PLT-Is were all < 10% CV in both analyzers. In 10 thrombocytopenic samples, precision of PLT-Is ranged 8.0 - 26.7% in BC-6800P and 5.5 - 28.7% in XN; however, precision of PLT-O/CR and PLT-F ranged 2.1 - 7.2% in BC-6800P and 2.6 - 17.8% in XN. Correlation was very strong (r < 0.900, all) in all samples as well as reflex tested, and thrombocytopenic samples.
Conclusions: BC-6800P would be a promising and reliable option in clinical hematology laboratories and transfusion management with good analytical performance in PLT counting especially in thrombocytopenic samples.

Bone Marrow Findings Of Pediatric Patients With Hemophagocytic Lymphohistiocytosis
Min-Sun Kim1, Chan-Jeoung Park1, Seongsoo Jang1, Young-Uk Cho1, Hyery Kim2, Kyung-Nam Koh2, Ho-Joon Im2, Jong-Jin Seo2
1Departments of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea, 2Pediatrics, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea

Introduction: Hemophagocytic lymphohistiocytosis (HLH) is uncommon, a life-threating hyperinflammatory syndrome caused by severe hypercytokinemia and fatal immunodysregulatory disorder caused primarily by the uncontrolled activation and proliferation of T cells and macrophages. To diagnose HLH, bone marrow (BM) study is necessary to confirm the presence of hemophagocytic histiocytes. We reviewed and analyzed BM findings of 43 pediatric HLH patients.
Methods: From July, 2009 to June, 2016, we reviewed BM aspirates, clot sections and biopsies obtained from 43 children who were diagnosed with HLH according to HLH 2004 criteria. We estimate the median value about each finding. The median age of patients was 58.7 months (12~74). The male and female patients were 25 (58%) and 18 (42%), respectively. We analyzed the cellularity, presence of hemophagocytosis with an average count of hemophagocytic histiocytes, histiocytic aggregates, proportions of histiocytes, monocytes and lymphocytes, megakaryocyte count, and cellularity of BM biopsy.
Results: The median values of CBC (WBC-hemoglobin-platelet count (1st quartile~3rd quartile)) were 3,700(2,600~7,800)/ul -9.0(8.4~11.0) g/dl - 49(36~82)k. In BM aspirates, among 43 cases, 39 (91%) cases showed hemophagocytosis with median 0.5 hemophagocytic histiocytes/HPF (0.2-0.6). Twenty-two cases (51%) showed histiocytic aggregates (≧ 5histiocytes). The median values of proportions of histiocytes, monocytes, and lymphocytes were 3.4(1.7~4.9) %, 1.6(0.1~3.2)%, and 22.3 (15.4~29.6) %, respectively. In BM biopsies, the median cellularity was 70% (40~80), and the median megakaryocyte count was 3.3/HPF (1.8~4.9). Increased micromegakaryocytes in 9 cases were observed. The serum levels [the median value, (range)] of triglyceride, fibrinogen and ferritin were 221 (71-608) mg/dL, 115 (40-309) mg/dL and 4,801(78.4-175,363.6) ng/mL. NK cell activity was decreased in 39 patients (97.5%) among 40 patients in whom the flow cytometric NK cell activity test was performed, and the median value was 15.8 (0.36-31.24) %. The median EBV DNA copy number was 6.36 log/ml in 16 blood samples, and 6.82 log/ml in 14 bone marrow samples, respectively. Three cases relapsed, and 5 cases died after 7 days~12 months from the diagnosis of HLH. 
Conclusions: Most pediatric HLH patients showed normocellular marrow for age with hemophagocytic histiocytosis, histiocytes, lymphocytosis, and megakaryocytes hyperplasia. 

Advanced Serological Evaluation Of A Case Of Anti D Autoantibody Induced Aiha And Immune Thrombocytopenia (Evans Syndrome) In A D Positive Patient: An Experience Of A Tertiary Healthcare Center
nitin agarwal, prashant pandey
Jaypee Hospital, Noida, India

Introduction: Evans syndrome (ES) is an autoimmune disorder characterized by the simultaneous or sequential development of autoimmune hemolytic anemia (AIHA) and immune thrombocytopenia (ITP) in the absence of any underlying cause. In 30% of cases of Evans syndrome, thrombocytopenia can precede AIHA, and in 10% AIHA can precede thrombocytopenia. Here we present a case of suspected Evan’s Syndrome where we found thrombocytopenia and immune hemolytic anemia due to an autoantibody against D antigen in a Rh (D) positive patient.
Methods: ABO Blood grouping, Rh phenotyping, antibody screening (Capture ready screen) and identification (Capture-R Ready-ID) were performed by SPRCA on NEO (Immucor, USA). DAT and Elution of DAT positive cells with Gamma Elukit-II (Immucor, USA) were performed using conventional tube technique (CTT). All the procedures were performed according to manufacturer’s instruction and departmental standard operating procedures (SOP).
Results: A previously healthy 56 yrs old woman was admitted in ICU for breathlessness and malena for 2 days. Two units of Packed Red Cells (RC) were requested as her hemoglobin was gradually decreasing (from 8.6 to 6.7 gm/dl in 2 days) with increasing bilirubin (0.8 to 2.1mg/dl) and LDH (160 to 510 IU/L) and thrombocytopenia (platelet count 16000). Patient blood group was B Rh (D) Positive but on cross matching with multiple Rh(D) Positive Packed Red Cell, it showed 2+ incompatibility with all. Only Rh (D) negative of her corresponding ABO group’s, packed red cells were found compatible with her serum. Peripheral blood smear showed few spherocytes and few large platelets indicating in vivo hemolysis and some platelet abnormality. Bone marrow biopsy showed increased number of megakaryocytes, suggesting ITP. Table 1 summarizes the results of the serologic studies performed on patient’s serum. Patient was treated further considering it a case of Evans syndrome (AIHA + ITP) but showed refractoriness to conventional therapy (steroids etc.). She responded well with a second line therapeutic agent and was discharged in stable condition with raised platelet counts and hemoglobin.
Conclusions: A blood bank having an immunohematology laboratory with all advanced techniques and proceduresshould not only able to diagnose these cases early, but also to provide blood to the patient in earliest possible time.

Screening Of Β-Globin Gene (Hbb) For Rare Mutations In Β-Thalassemia Patients Using Sanger Sequencing
Zeeshan Ahmed, Asghar Nasir , Tariq Moatter
Aga Khan University Hospital, karachi, Pakistan

Introduction: β -thalassemia is an autosomal recessive disorder which results in the formation of abnormal hemoglobin due to a variety of different mutations found in the HBB gene. These mutations   render patients incapable of producing correct form of hemoglobin. The aim of this study was to identify HBB gene mutations in β-thalassemic patients, not included in the common-mutation panel of ARMS PCR, by sequencing HBB coding, intronic and promoter.
Methods: A total of 27 samples previously tested for HBB gene mutations by ARMS PCR common-panel (i.e. IVS 1-1, IVS 1-5, Codon 8/9, Codon 41/42 and 619bp deletion) were analyzed by Sanger sequencing. Two healthy subjects were included as negative controls. Genomic DNA was isolated and HBB gene was amplified. Column purified amplified products were utilized for bidirectional cycle sequencing (Big Dye Terminator, ABI, USA).
Results: In the present study, a total of 27 samples were analyzed. Fifteen were males and twelve  females. The Mean age of the patients was three years. All patients were diagnosed as β-thalassemia major based on their family history, clinical and laboratory findings.  On average, patients were receiving transfusions every 2nd week. Seven rare mutations in HBB gene were detected including point mutations. The mutations spanned in the promoter  region HBB:c.138C>A (-88 C>A), exon1 HBB:c.17_18 delCT (Codon5 –CT), HBB:c.47G>A (Codon15 G>A), HBB:c.92G>C (Codon30 G>C), HBB:c.50A>C (CAP+1 A>C), exon2 HBB:c.118C>T (Codon39), and intron2 HBB:c.315+1G>A (IVS II-I G>A). All control subjects showed normal HBB gene sequence. In addition, a polymorphism T>C in codon3 at position HBB:c.59 was detected in majority of the patients and controls.
Conclusions: Although ARMS PCR is a fast and convenient method for detection of common mutations in the HBB gene, a small subset of patients may be missed because of rare mutations, which would require other means for diagnosis.  Sanger sequencing is an accurate and robust technique to manage such patients.

Large-Scale Genotyping Of Hemoglobinopathies In Azerbaijan
Gunay Aliyeva, Chingiz Asadov, Tahira Mammadova, Eldar Abdulalimov, Surmaya Gafarova, Yegana Guliyeva
Institute of Hematology and Blood Transfusion, Baku, Azerbaijan

Introduction: Azerbaijan is located in Transcaucasia which is a high prevalent region of hemoglobinopathies. Besides β-thalassemia, the local population is also known for the incidence of α, δ/β-thalassemia and some hemoglobin variants. Although previous studies reported the mutation spectrum of β-thalassemia in the local population, these were mainly conducted in small study groups limited on β-globin genotyping, which necessitated a large-scale genotyping to reveal mutational background of all common hemoglobinopathies in Azerbaijan.
Methods: The study included the samples of patients referred to Hemoglobinopathy Reference Laboratory at the Institute of Hematology and Blood Transfusion. The samples of the patients which were confirmed by routine methods as carriers of clinically significant hemoglobinopathies (α-thalassemia, β-thalassemia and variants) were further proceeded for genotyping which was performed by reverse dot-blot hybridization or sequencing techniques.
Results: 1331 samples were genotyped for β-globin mutations and 80 samples for α-globin mutations from June 2016 to December 2018. As a result, we revealed that mutation spectrum of  β-thalassemia is highly heterogenous including 33 mutations with the majority of severe β0 mutations. Genotyping of α-globin genes, on the other hand, revealed 8 mutations with the prevalence of deletions leading to milder clinical manifestations.  
Conclusions: The first large-scale genotyping study including hemoglobinopathies other than β-thalassemia revealed that the profile of hemoglobinopathies and mutation spectrum is highly heterogenous in Azerbaijan.

Reticulocyte Hemoglobin Equivalent: A Study On Comparison And Effectiveness In Assessment Of Iron Deficiency
Wardah Aslam1, Maryam Habib2, Humera Mahmood1, Afrose Liaquat2, Saeeda Aziz1, Shazia Fatima1, Madiha Habib3
1Nuclear Medicine Oncology & Radiotherapy Institute, Islamabad, Pakistan, 2Shifa College Of Medine (STMU), Islamabad, , Pakistan, 3University Of Malaya, Kualalumpur, , Malaysia

Introduction: Anemia is a global public health problem affecting 24-30% of population worldwide.Iron deficiency is the most common cause of anemia. It is a major health problem in many developing and under developed  countries of the world including Pakistan. However prompt diagnosis and treatment can cause significant reduction in mortality and morbidity.This study is done to prove usefullness of Reticulocyte Hemoglobin Equivalent (RET-HE) as a reliable and effective tool in dignosis of iron deficiency in different states.
Methods: This prospective study was approved by ethical comitee of Nuclear Medicine Oncology and Radiotherapy Institute Islamabad.154 blood samples were obtained and analyzed on Sysmex XN-350.Serum ferritin & biochemical data was measured using an automated chemistry analyzer.Patients were divided into 4 groups Iron Deficiency Anemia (IDA), Latent Iron Deficiency (ID), Anemia of Chronic Disorder combined with Iron Deficiency(ACDC) and Controls .Statistical analysis was done by SPSS 23
Results: There were (66) Controls (28 males, 38 females) with age range (28-68). (66) IDA patients ,(22 males and 44 females),with age range (14-71) .(10) ACDC patients (4 males and 6 females) with age range  (43-69). (12) ID patients , (7 males and 5 females) with age range (43-70). The median RET-HE levels were 22.1 (12.3-35.6).After ROC analysis area under curve (AUC) of RET-HE for detecting IDA was 0.95 at cut off 29.9 with maximum specificity of 91% and sensitivitry of 92%.AUC of  RET-HE  in ACDC IS 0.94. AUC of RET-HE in diagnosing ID is0.93.Ptients in IDA and ID groups had significantly lower values of RET-HE than controls (p< 0.001).RET-HE showed moderate  positive spearman correlation with Hb,RDW CV and MCV in IDA group and negative correlation with RDW SD.However it showed positive spearman correlation with ferritin in  IDA and ID groups , but showed negative correlaion with ferritin in ACDC group. 
Conclusions: Our study suggests the measurement of RET-HE as a usefull, rapid and costeffective method for diagnosing iron deficiency in IDA,ID and ACDC.

Impact Of Iron Deficiency On Diagnosis Of Beta Thalassemia Trait
Asma Cheema1, Tariq Saleem1
1University Medical & Dental College, The University of Faisalabad, Faisalabad, Pakistan, 2Madinah Teaching Hospital, Faisalabad, Pakistan

Introduction: Among the genetic disarrays, hemoglobinopathy is the only disorder in which carrier state can be determined by hematological parameters instead of DNA testing. Hemoglobin A2 (HbA2) value plays central role in the diagnosis of Beta Thalassemia trait. Iron deficiency (ID) might affect the HbA2 value, hence masking the presence of Beta thalassemia trait. The objective was to evaluate the effect of ID on HbA2 levels.
Methods: The study was approved by Ethical Committee of Madinah Teaching Hospital and 137 subjects were initially enrolled after written informed consent for thalassemia trait screening. Blood samples were collected in plain tubes for estimating serum ferritin and in EDTA plasma tubes for determining red cell indices and Hemoglobin variants. Serum Ferritin was estimated on Cobas 6000 e611 and red cell indices were measured on Sysmex XN1000. Hb variants were determined on BioRad D10. Since ferritin is an acute-phase reactant protein, 25 subjects with high white blood cell count were omitted from study analysis to avoid any potential bias. Subjects with serum ferritin < 10µg/L were considered iron deficient. The statistical analyses were completed using SPPS 23.00.
Results: We selected 112/137 subjects with clinical suspicion of thalassemia trait to determine Hb variants. Of 112, 64 subjects were found with high HbA2 (>3.5%) levels. Majority subjects (40/64) were females. Mean Hb value was 9.0±0.5g/dL in females and 10.5±0.10g/dL in males. We found 26 subjects with Iron deficiency (Group A) and 38 without Iron deficiency (Group B). Mean value of HbA2 in group A was 4.56±0.04 and in group B it was 5.80±1.06 with statistical significant difference ofP< 0.001. Interestingly Mean values of other Red cell indices were also significantly different between two groups except for RBC count (Table 1). Table 1: Comparison of red cell indices in Group A* & Group B^
Red Cell indices Group A (Mean±SD) Group B(Mean±SD) P-value
RBC count (1012/L) 6.50±0.38 5.93±0.57 0.497
MCV (fL) 50.43±6.08 58.36±2.71 0.010
MCH (pg) 14.90±2.93 19.02±1.69 < 0.001
MCHC (g/dL) 29.46±2.10 31.70±0.38 0.004
*Subjects with Iron deficiency ^Subjects without Iron deficiency
Conclusions: Our results show that ID may decrease HbA2 levels. However, it does not preclude the diagnosis of Beta thalassemia trait. Iron deficiency should be corrected before measuring HbA2 levels.

Diagnostic Performance Of Reticulocyte Haemoglobin Equivalent In Assessing The Iron Status
Pawadee Chinudomwong, Aleeyas Binyasing, Rangsiri Trongsakul, Karan Paisooksantivatana
Department of Pathology, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

Introduction: Bone marrow iron staining, the gold standard method for diagnosing iron deficiency,is invasive and subjective. Conventional iron biomarkers are also affected by acute-phase responses. Due to these diagnostic difficulties, an alternative marker is sought. This study evaluated reticulocyte haemoglobin equivalent (Ret-He) for its diagnostic performance in assessing the iron status.
Methods: This is a hospital-based retrospective analytical study. One thousand seven hundred and eight (304 healthy volunteers and 1,404 patients) clinical data and laboratory results – complete blood count, reticulocyte count, Ret-He, and serum ferritin – collected from April 2017 to May 2018 were reviewed. Patients on iron therapy were excluded. Iron status was defined by serum ferritin as the reference method in this study. Diagnosis of iron deficiency anaemia (IDA) was performed by using a combination of serum ferritin, haemoglobin, red cell indices, and medical history (iron therapeutic trial). Receiver operating characteristic (ROC) analysis was employed to evaluate the diagnostic performance of Ret-He and determine its optimal cut-off value.
Results: Ret-He values were significantly different between healthy controls (n = 156) and IDA patients (n = 133) (median 20.6 vs. 33.0; p< 0.0001). ROC curve analysis revealed area under the curve of 0.993 (95% CI 0.975-0.999; p< 0.0001) at cut-off 30.3 pg/cell, by which the two groups were discriminated with 100% sensitivity, 96.2% specificity and positive likelihood ratio of 26 (95% CI 11.9-57). In addition, from the medical records, a significant fraction of cases (n = 106, 12.2%) diagnosed with IDA demonstrated low Ret-He value (< =30.3 pg/cell) despite normal-to-high ferritin levels. According to their clinical data, these cases are afflicted by medical conditions that are associated with inflammation, namely, autoimmune diseases, malignancies, and infections. 
Conclusions: Ret-He may be a substitute for ferritin in the diagnosis of IDA because it is unaffected by such factors as inflammation in addition to its excellent diagnostic sensitivity, specificity, convenience, and cost-effectiveness.    

Establishing Locally Derived Reference Intervals For Full Blood Count Parameters And White Cell Differential Counts In The Western Cape Region Of South Africa
Annemarie De Koker1, Arthur Bird2, Caroline Hilton2, Jessica Opie1
1Division of Haematology, University of Cape Town & National Health Laboratory Service, Cape Town, South Africa, 2Western Province Blood Transfusion Services, Cape Town, South Africa

Introduction: The recognised variation observed in normal haematological parameters in different populations and geographic locations, emphasizes the need to establish locally derived reference intervals (RI) with appropriate representation of the various ethnic groups. Accurate RI are essential to distinguish between health and disease. We aimed to establish locally derived RI for full blood count (FBC) and white blood cell  (WBC) differential count parameters in healthy adults in the Western Cape region of South Africa.
Methods: A prospective, descriptive study was performed, utilizing blood samples of healthy first-time blood donors, presenting voluntarily for blood donation to the Western Province Blood Transfusion Service (WPBTS) between November 2016 and October 2017. African, Coloured and Caucasian participants aged between 18 and 60 years of age were included based on convenience sampling. All samples were tested for human immune deficiency virus, hepatitis B, hepatitis C, syphilis and iron deficiency (using serum ferritin), and excluded if positive. Donors with an elevated serum ferritin were also excluded. Reference intervals were derived using non-parametric statistical methods and expressed to include the central 95% of the sample population (2.5th to 97.5th percentiles). Outliers for individual parameters were identified and excluded from the analysis.
Results: A total of 376 females and 244 males were included for analysis; 31.61% were African, 39.68% Coloured and 28.71% Caucasian. For all race groups combined, gender-based differences were found in most FBC parameters, including the haemoglobin (Hb), WBC count,  neutrophil count and platelet count. When comparing RI for males and females in the three ethnic groups, statistically significant differences were found for parameters including the Hb, WBC count and other red cell indices. The ranges differed for a number of FBC variables compared to the National Health Laboratory Service coastal reference ranges in current use.
Conclusions: Locally established and population-specific RI reference ranges are essential for accurate interpretation of blood counts. Implementation of separate RI for the main ethnic groups in the Western Cape should be considered and would have implications for the diagnosis of anaemia and other blood count abnormalities. 

Evaluation Of Fragmented Red Cells Counting For Detection Of Schistocytes Using Sysmex Xn-3000
Motoki Hisasue1,3, Yoko Tabe2, Konobu Kimura2, Akimichi Ohsaka1,2,3
1Department of Transfusion Medicine and Stem Cell Regulation, Juntendo University Graduate School of Medicine, Tokyo, Japan, 2Department of Next Generation Hematology Laboratory Medicine, Juntendo University Graduate School of Medicine, Tokyo, Japan, 3Department of clinical Laboratory, Juntendo Hospital, Tokyo, Japan

Introduction: Schistocytes are fragments of red blood cells (RBCs) produced by extrinsic mechanical damage in the blood flow. Because the presence of schistocytes on the peripheral blood smear is a characteristic feature of microangiopathic hemolytic anemia. The morphological identification and diagnostic value of schistocytes has been recommended by the International Council for Standardization in Hematology (ICSH).  However, the morphologic identification of schistocytes remains subjective and time consuming. The purpose of this study is to evaluate the utility and to determine Fragmented Red blood cells (FRC)  range for assessing schistocytes utilizing the latest model of automated blood cell counter XN-3000 (Sysmex, Kobe, Japan).
Methods: Morphological identification of schistocytes and detection of FRC by XN-3000 were conducted using 1026 peripheral blood samples (513 of XN-flag ”fragments?” positive and 513 of negative) collected for hematological tests at Juntendo University Hospital. Morphological schistocytes counting (>1,000RBCs /slide) was performed by a board-certified hematology laboratory technician using the digital images of May Grunwald-Giemsa-stained blood smear, captured by the automated image analyzer DI-60 (Sysmex). Based on the ICSH guideline, the sample with 1% or more schistocytes was defined as positive. The Mann-Whitney U test and receiver operation characteristic (ROC) analysis were applied.
Results: We observed that 184 samples (36% of the XN-flag positive) were schistocytes positive. Moreover, none of the XN-flag negative samples showed < 1% morphological schistocytes. Seventy-three percent of the schistocytes positive samples demonstrated < 12g/dL hemoglobin and 43% showed <15,000/L platelets.With regard to the XN RBC parameters, Micro-R (microcytic RBCs) and %Hypo-He (hypo-hemoglobinised Red cells) and FRC% (p< 0.001) significantly correlated to the positive. The area under curve (AUC) of the FRC% was 0.85 (95%CI: 0.82-0.88) for a cutoff value of 2.4%, Micro-R was 0.75 (95%CI: 0.72-0.78) for a cutoff of 3.3%, and  Hypo-He was 0.77 (95%CI: 0.74-0.80) for a cutoff of 3.0%    
Conclusions: These results demonstrate the most effective combination of XN flag “fragments?” with FRC% parameter of the XN-series for the screening of schistocytes positive cases.

Evaluation Of Red Cell Concentrate Haematocrit As ImprovedIndicator For Post Transfusion Increment In Thalassaemia Patients
tanzeel imran, Humera Altaf
jamila sultana foundation for thalassemi, rawalpindi, Pakistan

Introduction: The rationale of this study was to evaluate post transfusion increment as a representative of improved  quality  red blood cell concentrates chosen for transfusion of thalassaemic patients.
Methods: A total of 1098 red cell concentrates were evaluated during 6 months of study, of which 697 were transfused as buffy coat reduced   while  401 were washed and transfused with in 24 hrs. Post transfusion increment was noted at 1 hour post transfusion and was significantly high in buffy coat removed than in washed RCC. The washing procedure was manual .The haematocrit was  evaluated by a blood cell counter (Sysmix 2000i).
Results: Sixty seven percent (697/1098) of the red cell concentrates that were buffy coat removed as pre-storage had hematocrit of 80% while washed RCC,36% (401/1098) red cell concentrates  had haematocrit of 71% .Post transfusion increment was.5% higher in pre storage buffy coat removed RCC indicating good post transfusion increment

Conclusions: It was concluded that increment in hemoglobin of washed RCC  was lower than leucodepleted  RCC. Reveling that washing loses some RCC. So it is recommended that for avoiding transfusion reactions pre storage reduced RCC are better option than washed RCC that certainly increase transfusion interval. Keywords: Red cell Concentrate, post-storage increment

Sysmex Xn White Precursor Channel Evaluation
Agus Kosasih, Lyana Setiawan, Hubertus Hayuanta
Dharmais Cancer Hospital - National Cancer Center, Jakarta, Indonesia

Introduction: Acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) have different treatment. Hyperleukocytosis in such leukemias needs prompt management, so it is important to classify early. However, immunophenotyping is not always available and takes longer time. Therefore, simpler method is needed to do the task. Sysmex XN White Precursor Channel (WPC) has potential to fulfill the role. This study aims to evaluate Sysmex XN WPC in differentiating AML and ALL compared to immunophenotyping as gold standard.
Methods: Subjects were patients that requested immunophenotyping of peripheral blood in Dharmais Cancer Hospital - National Cancer Center from June-October 2018, with peripheral blast cells ≥20%. White Precursor Channel results were evaluated by three clinical pathologists. The end result was the result picked by the greatest number of clinical pathologists (≥2). The end results were compared with their immunophenotyping counterparts.
Results: As many as 127 subjects were included. The subjects consisted of 64 (50.39%) males, and 63 (49.61%) females, with a median age of 18 (0-66) years old, and a median blast count of 65 (21-94)%. As many as 63 (49.61%) subjects had myeloid lineage (15 with monocytic markers, 48 without; 16 with aberrant markers, 47 without), and 64 (50.39%) had lymphoid lineage (56 with B markers, 8 with T markers; 15 with aberrant markers, 49 without). To diagnose AML, sensitivity was 95.24%, and specificity was 93.75%. To diagnose ALL, sensitivity was 93.75%, and specificity was 95.24%. There were 7 incompatible results, 3 had myeloid lineage (all without monocytic markers; 2 with aberrant markers, 1 without) and 4 had lymphoid lineage (3 with B markers, 1 with T markers; all without aberrant markers). Among the incompatible results, there were no false positives. Results were incompatible with immunophenotyping because in those results, lineage could not be determined from WPC (inconclusive).
Conclusions: Sysmex XN WPC channel has good sensitivity and specificity in differentiating acute leukemia into AML or ALL, allowing for early clinical intervention.

Serum And Red Cell Folate Levels Among Low Birth Weight Neonates In Wad Madani Obstetrical And Gynaecological Teaching Hospital,Gezira State,Sudan (2016)
Alfatih Khadir1, Sulafa Eltayeb2, Awad elseed Mustafa3
1university of Gezira, medani, Sudan, 2university of Gezira, medani, Sudan, 3university of Gezira, medani, Sudan

Introduction: A central feature of embryonic and fetal development is widespread cell division; folate is central because of its role in nucleic acid synthesis. During gestation folate deficiency can impair cellular growth and replication in the fetus or placenta. Low birth weight is closely associated with inhibited growth and cognitive development, and chronic diseases later in life.Many researches found a relationship between umbilical cord folate status and intrauterine growth restriction. In sudan no similar study was found. It was a case-control study aimed to measure folate levels among groups of normal and low birth weight neonate to evaluate the correlation between umbilical cord folate levels and birth weight.
Methods: Haematological parameters were measured using automated cell counter,microscopic examination of peripheral blood smears and reticulocytes preparations. Serum and red cell folate were measured by electrochemiluminscence technology (cobas e 411 analyzer) .Statistical analysis was done using SPSS program version 20.
Results: From the study it was found that umbilical cord RBC folate status to be an important predictor of neoborn birth weight, with increasing cord RBC folate being associated with increasing neoborn birth weight. Statistically significant association was found between red blood cell folate levels and birth weight (Pvalue of 0.047 in case group). Thirteen out of 43 cases had low red cell folatelevels . This indicates the presence of relationship between folate levels in fetus and birth weight. No statistically significant association was found between levels of serum folate and birth weight in the case group (P Value 0.59 in case group).This may be due to fact that serum folate is a marker of recent dietary intake and it is subjected to prandial variation.
Conclusions: -Red blood cell folate is considered the most reliable biomarker of folate status,as it reflects tissue folate stores. - A single measurement of serum/plasma folate reflects only the time of blood collection and cannot differentiate between occasional low dietary intake of the vitamin and folate deficiency. - Our study revealed that there is clear association between folate deficiency and increase incidence of low birth weight and preterm delivery.

Experience Of Diagnosing Inherited Hemolytic Disease In Resource Limited Setting In Nepal
Surendra Koju, Ramita Shankhadev
Dhulikhel Hospital-Kathmandu University Hospital, Dhulikhel, Nepal

Introduction: Inherited hemolytic anemia is genetic disorder presenting anemia due to increase red cell destruction. Confirmatory diagnosis of such disease is vital for care and treatment of disease. Although significant advances have been made around the world in diagnosis of inherited hemolytic anemia, many centers in Nepal are still not able to practice cost effective method for diagnosis of uncommon hematological disorder. Hence due to lack of proper diagnostic technique, patient is being mis-diagnosed and getting wrong treatment. Due to lack of special diagnostic service in health care centers of Nepal, patients are being obliged to refer to neighboring country for diagnosis of disease. Few centers performing diagnostic service of such disease is using limited available resource and operational experience.
Methods: We analyzed patients visited in different outpatient department of our institute with mild to severe anemia. Patients diagnosed with normocytic anemia were further evaluated for peripheral blood smear examination and reticulocyte count. Ten cases were suspected for hemolytic anemia based reticulocytosis and peripheral blood smear showing normocytic red cell with few cases showing spherocytes and few cases with schistocytes. We excluded autoimmune hemolytic anemia as Direct Coomb’s test was negative for all cases. Hence, Based on history and initial investigation, inherited hemolytic anemia was suspected. Osmotic fragility test was performed in 6 cases for diagnosis of hereditary spherocytosis in which spherocytes was seen in peripheral blood smear and for remaining cases methemoglobin reduction test was performed for diagnosis of G6PD deficiency
Results: Four cases form 6 patient, where osmotic fragility tests was performed shows increased osmotic fragility test while remaining 2 was normal for red cell fragility. Likewise, four cases in which methemoglobin reduction test was performed for diagnosis of G6PD deficiency, found to be deficiency in three cases while remaining one cases was normal for G6PD. Although missed cases were also highly susceptible for inherited hemolytic anemia, based on less sensitive test in our resource limited setting, it was unable to reach the final diagnosis of disease.
Conclusions: This study aim to diagnose inherited hemolytic disorder for the confirmation of disease by utilizing limited cost effective resources. Many diseases are under diagnosed in developing countries like Nepal, but utilization of simple training and cost effective reagent will be helpful in diagnosing such disease.

Validation Of An Assay For The Rapid Quantification Of Hemoglobin S. One Centre'S Experience.
Kelly Lightfoot1, Karen Moffat1,2, Joseph Macri1,3, Madeleine Verhovsek1,2,3
1Hamilton Regional Laboratory Medicine Program, 2Department of Medicine, 3Department of Pathology and Molecular Medicine, McMaster University

Introduction: Sickle cell disease (SCD) is due to an inherited variant hemoglobin. In a deoxygenated state, Hemoglobin S (HbS) polymerizes resulting in the sickle cell red blood cell shape and cause vaso-occlusion and hemolytic anemia. Our laboratory provides routine measurement of HbA1c for clinical management of diabetes. We hypothesized that using the electropherograms from the HbA1c assay would allow quantification of HbS and provide a rapid result to our clinicians on confirmed HbS patients, to monitor effects of therapy. Our laboratory validated the HbA1c kit on the Capillarys2-Flex Piercing analyzer (Sebia, France) for rapid HbS quantitation for confirmed HbS patients receiving therapy.
Methods: Capillary electrophoresis separates and quantitates proteins in a capillary based on mobility in electrically charged fluid. Under high voltage, the HbA1c kit separates hemoglobin fractions based on their electrophoretic mobility and electroosmotic flow. The quantity of each fraction, including variant Hemoglobin F,A,S,X are identified by direct measurement at 415 nm.  Accuracy and precision studies were performed on the HbA1c kit. HbS linearity (3.2% - 100%) and carryover studies across all eight capillaries using a HbSC sample directly after a high HbA1c sample, were also assessed. Commercial Streck normal and abnormal quality control (QC) samples for HbA1c (Nebraska, USA), and an in-house HbSS patient control were run. Results from 13 previously confirmed SCD patient samples were compared to HbS results obtained using the HbE kit (Sebia, France). Statistical analysis included: mean, standard deviation, coefficient of variation, regression analysis and %-difference calculations.
Results: Precision and accuracy for the HbA1c kit was acceptable. The HbS linearity challenge was acceptable (y=0.93x-0.99;R2=1.00). There was no evidence of carryover across channels. Commercial and in-house QC were within acceptable parameters. The HbA1c kit was noted to underestimate HbS by 5% on patient samples as compared with the HbE kit.  This difference was considered clinically significant.
Conclusions: Our laboratory validated the HbA1c kit for rapid quantification of HbS in confirmed SCD patients. With the identified underestimation of HbS up to 5%, a standardized comment was added to all results to inform clinicians. As our laboratory runs HbA1c daily, we provide a faster turnaround time and improved patient care for confirmed patients with HbSS, HbSC or HbSD receiving therapy.

Epidemiologic Distribution Of Haemoglobinopathies And &Prop;-Thalassemia Detection In A Mediterranean Area Between 2016-2018
Anna Marull, Javier Nieto-Moragas, Orlando Jimenez, Javi Hernando, Diana Pedrola, Maite Serrando
LaboratoryTrueta Hospital, Girona, Spain

Introduction: ∝-thalassemia has always been more difficult to be diagnosed than others hemoglobinopahies because it is not always possible to be detected when HPLC (high performance liquid chromatography) is performed.  ∝-thalassemia can be only detected by molecular DNA. Techniques such as GAP PCR can detect the most common ∝-thalassemia deletions.  However, in most countries these determinations are only available in specific laboratories. We previously studied the epidemiologic distribution of hemoglobinopahties in our area during 2013-2015. In that study we were aware of the low detection for this hemoglobinopathy according to other epidemiologic studies carried out in other Mediterranean regions. For this study, we adopted a new protocol to improve the sensitivity of the hemoglobinopathy diagnosis and have applied several changes in our detection flowchart (iron deficiency parameters, Ret-He, and matching of the hemoglobinopathy with the features described in www.globin.bx) Our goal was to determine whether the detection of ∝-thalassemia can be improved as well as we can assemble a more accurate distribution of hemoglobinopathies in our area.
Methods: From 2016 to 2018, 5731 samples were processed by HPLC (Variant, BioRad). This test was ordered according to medical or laboratory criteria with regard to age and full blood count and after iron deficiency parameters were discarded. In 2013-2015, 835 samples were positive for a hemoglobinopathy. The samples were processed using the same criteria as above mentioned.
Results: 1114 out of 5731 (19,5%) samples tested presented a haemoglobinopathy of some type. β-thalassemia minor (434, 39%) was the most common followed by ∝-thalassemia (244, 22%) and hemoglobin S (220, 20%). When we compared our results to the previous study, we observed an increase in ∝-thalassemia detection as well as compound variant detection, mainly Hb∝S.
Conclusions: The detection of ∝-thalassemia has improved due to molecular diagnosis methods. However, these procedures are still only accessible in specialty diagnostic centers, of which are few. This is one of the reasons that ∝-thalassemia could be under diagnosed. In this study we have demonstrated the diagnosis could be improved by applying more stringent and sensitive diagnosis parameters. The aim of screening is to identify carriers of hemoglobin disorders to provide information to couples at risk.

Reticulocyte Count Increases On Hematopoietic Stress In Sickle Cell Disease
SATYABRATA MEHER1,2, Pradeep Kumar Mohanty1, Siris Patel1, Kishalaya Das1, Bisnu Prasad Dash2
1Veer Surendra Sai Institute of Medical Science and Research, Sambalpur, India, 2Bioscience and Biotechnology, Fakir Mohan University, Balasore, India

Introduction: Reticulocytes are associated with hematopoietic stress. In this communication we correlate the Reticulocyte count (RC) with various hematological, biochemical parameters in Sickle Cell Disease (SCD) suggesting possible role of count as a marker of stressed hematopoeisis.
Methods:  Five hundred cases were subjected to sickling slide test, alkaline agarose gel electrophoresis (pH- 8.6), CE-HPLC was used to detect SCD and quantify various Hb fractions. Reticulocytes count was done by flowcytometry using Thiazole Orange reagent. CBC, Biochemical parameters were done. Principal Component Analysis (PCA) was done.
Results: Among 500 cases, number of HbSS, HbSβ, HbAS and HbAA cases were 350, 20, 70, 60 respectively.  Percentage of RC of HbSS, HbSβ, HbAS and HbAA cases were 4.29±2.9, 4.28±3.3, 1.7±0.8, 1.6±0.9 respectively. Significant higher RC were observed in cases of HbSS and HbSβ compared to HbAS and HbAA (p< 0.001). Similarly, RBC count, hemoglobin, hematocrit and platelet count were significantly lower in case of HbSS and HbSβ when compared to HbAS and HbAA (p< 0.001). PCA suggests RC as a hematopoietic stress marker. 
Conclusions:  Higher RC in HbSS and HbSβ indicate higher rate of hematopoietic stress. Lower estimates of RBC, hemoglobin, hematocrit and increased LDH in patients with HbSS and HbSβ correlated with RC. Splenomegaly, hepatomegaly, anemia and VOC may be considered as additional factors for elevated RC causing hematopoietic stress. As per our results, reticulocytes increases during hematopoietic stress condition. Hence, it considered as a good hematopoietic stress marker in patients with SCD.

Subclinical Iron Deficiency In Health Checkups
Eun-Hee Nah1, Seon Cho1, Suyoung Kim1, Han-Ik Cho2
1Department of Laboratory Medicine, Korea Association of Health Promotion, Seoul, South Korea, 2MEDIcheck LAB, Cheongju, South Korea

Introduction: Apparently healthy individuals who are within the normal range for the hemoglobin (Hb) may have undetected subclinical iron deficiency (SID). The aims of this study were to determine the prevalence of SID and identify the associated factors for SID. In addition, the screening performance of RBC indices for SID in health checkups was assessed .
Methods: A cross-sectional study was conducted with16,485 non-anemic health examinees (3,567 males and 12,918 females) who underwent tests for iron variables (serum iron, TIBC, ferritin, and iron saturattion) at 16 health-promotion centers in 13 cities in Korea between 2017 and 2018. SID was defined as a decreased ferritin level (< 24 mg/L in males and < 15 mg/L in females) and either a decreased serum iron level or an iron saturation.
Results: The prevalence of SID were 0.6% and 3.3% in males and females, respectively. In terms of age and sex, SID was most prevalent in males aged≥70 yrs (7.8%) and females aged 15–49 yrs (7.6%). The factors associated with SID in males and females are presented in Table 1. The AUC for the MCH (0.877, 95% CI=0.793–0.960 in male; 0.872, 95%CI=0.853–0.890 in female) indicates that the MCH at cutoffs of 29.2 pg and 29.3 pg are the best discriminators of SID in males and females, respectively (P< 0.001).   
Table 1. Factors contributing to subclinical iron deficiency
              Males OR (95% CI) Females OR (95% CI) Total OR (95% CI) P value
Age (yrs) 1.07 (1.025, 1.117) 0.955 (0.946, 0.965) 0.962 (0.953, 0.971) < 0.001
Sex - - 1.588 (0.881, 2.861) 0.124
BMI, kg/m2 0.927 (0.781, 1.101) 0.909 (0.872, 0.947) 0.906 (0.871, 0.943) < 0.001
Hb, g/dL 2.937 (0.249, 34.63) 0.264 (0.198, 0.351) 0.295 (0.223, 0.39) < 0.001
RBC, 106m/L 0.008 (< 0.001, 10.485) 2.352 (1.179, 4.693) 1.794 (0.908, 3.541) 0.092
MCV, fL 1.06 (0.924, 1.216) 0.988 (0.933, 1.048) 0.999 (0.945, 1.055) 0.960
MCH, pg 0.209 (0.058, 0.755) 0.65 (0.555, 0.762) 0.623 (0.533, 0.727) < 0.001
RDW, % 2.04 (1.067, 3.898) 1.779 (1.564, 2.024) 1.822 (1.604, 2.07) < 0.001
Conclusions: Reproductive-age females with a lower BMI and elderly males are high-risk groups for SID. MCH is a reliable RBC index for the screening of SID. 

Haematological Parameters As Alternative Risk Of Stroke Predictor In Children With Sickle Cell Disease In Poor-Resource Setting
olusola olowoselu, oluwakemi otokiti, oyedeji o.a, ogunmola j.a, ogunlade abosede
lagos University Teaching Hospital, lagos, Nigeria

Introduction: The role of trans-cranial Doppler (TCD) in the identification and prediction of the risk of stroke in children with sickle cell disease(SCD) is crucial. However, in developing countries where TCD is uncommon, very expensive and not readily accessible; finding cheaper and readily available alternative as predictors is needful. Hence, this study described the association between haematological parameters and TCD findings in the prediction of stroke among SCD patients attending a clinic in Nigeria.
Methods: This cross-sectional study included Nigerian children with SCD (n=62) between age 2-15 years old. On confirmation of their haemoglobin phenotype, 3 mL of blood was collected into K-EDTA bottles and used for complete blood count (CBC) analysis. TCD was done on the participants’ middle cerebral artery to determine the cerebral blood velocity. A time averaged mean velocity (TAMV)< 170mHz was taken as limit for normal TCD. p< 0.05 was considered statistically significant. 
Results: Mean age of the participants was 7.79+3.422, male to female ratio of 1:1.3. The participants(33.87%) with TAMV of < 170cm/s were classified as standard risk; those(32.26%) with TAMV of ≥170< 200cm/s were classified as conditional risk; and 33.87% of the participants with TAMV of ≥200cm/s were classified as high risk. There was a statistically significant difference and association in the mean and p-values between the standard and high risk participants in PCV (mean-23.243+3.03 & 21.24+2.96)(p-value-0.0363*), HB(mean-7.86+0.91 & 6.762+1.09)(p-value-0.0011*), total WBC(mean-11.19+2.75 & 16.47+3.11)(p-value-0.000*) and platelets(mean-504.57+211.83 & 613.17+111.98)(p-value-0.0443*). Higher WBC, Platelets and lower  PCV were reported in the conditional and high risk group compared to standard risk. There was no statistically significant difference in the MCH, MCV and MCHC across groups                  
Conclusions: Conclusion Though the TCD remains the gold standard for predicting risk of stroke in SCD, a cheaper and readily available complete blood count, especially PCV, Hb, total WBC and plateletscounts, can help predict the risk of stroke in a poor resource setting. Keywords: Trans-cranial Doppler, Sickle cell disease, Stroke, Complete blood count, Time averaged mean velocity

Assessment Of CellavisionTm Dm Advanced Rbc Application For Detecting Red Cell Abnormalities
Seongjun Park, Jeong Ah Kwon, Soo Young Yoon
Korea university medical center, seoul, Korea

Introduction: Examination of peripheral blood is the primary procedure to provide important information that supports in the diagnosis of hematologic diseases. The CellaVision DM96 system (CellaVision AB, Lund, Sweden) is an automated image analysis system capable of morphological classification and determination of differential counts of WBCs and RBCs in peripheral blood  smears. This Advanced RBC Application is a new software that automatically perform a pre-characterization and sorting of RBCs into 21 morphological categories based on size color, shape, inclusions. We evaluated the performance of this automated system to characterize RBC poikilocytes and its potential for substitute for labor-intensive manual microscopic examination.
Methods: 68 ‘normal’ blood samples that it’s blood counts were within reference range and 65 ‘pathological’ samples with abnormal RBC morphology were selected during daily routine slide examination of hematology laboratory.Each two medical pathologists manually examined all normal and abnormal peripheral blood smear slides for RBC morphology differentiation. Differential results obtained by direct microscopy were compared with the final results from the CellaVision DM96. Poikliocytes were grouped and graded according to 2015 ICSH guidelines and we also applied it to the CellaVisionTMsoftware.(Table 1,2) Results per morphological category were analyzed both semi-quantitatively grades and quantitatively as percent of the RBCs. Microsoft Excel 2010 (Microsoft Corp., Redmond, WA, USA) and MedCalc statistical software version (MedCalc Software BVBA, Mariakerke, Belgium) were used for the statistical data analysis.
Results: Observed grading agreement were generally high (>50%) in normal group except elliptocytes and shistocytes. Otherwise, Only target cells and tear drop cells were satisfactory in abnormal group. When it comes to Weighted Kappa coefficients and Intraclass Correlation Coefficient, elliptocytes (ICC 0,43), tear drop cells (0.28), acanthocytes (0.49) and echinocytes(0.35) were fair in normal group while target cells (ICC 0.82) and elliptocytes (ICC 0.72) were moderate in abnormal group.
Conclusions: The general accordance and the number of cell type that show high coefficient value were lower in the abnormal group. That is because the digital analyzer tends to count artifacts as poikilocytosis in abnormal group as well as due to the lack of samples with “many”(Grade 2&3) abnormalities. The CellavisionTMsystem would be a reproducible and labor-saving device alternative to manual microscopy for RBC abnormality detection although further studies may be needed to confirm these findings

The Oxygenscan: A Rapid And Reproducible Characterization Of Sickling During Automated Deoxygenation In Sickle Cell Disease Patients.
Minke Rab1,2, Brigitte van Oirschot1, Jennifer Bos1, Annet van Wesel1, Maite Houwing3, Marjon Cnossen3, Roger Schutgens2, Gerard Pasterkamp1, Marije Bartels2, Eduard van Beers2, Richard van Wijk1
1Laboratory of Clinical Chemistry and Haematology, University Medical Center Utrecht, Utrecht, Netherlands, 2Van Creveldkliniek, University Medical Center Utrecht, Utrecht, Netherlands, 3Department of Pediatric Hematology, Erasmus University Medical Center, Rotterdam, Netherlands

Introduction: In sickle cell disease (SCD) hemoglobin S (HbS) polymerizes upon deoxygenation, resulting in sickling of red blood cells (RBCs). These sickled RBCs have strongly reduced deformability, leading to vaso-occlusive crises and chronic hemolytic anemia. To date, there are no reliable laboratory parameters or assays capable of predicting disease severity or monitoring treatment effects.
Methods: The oxygenscan is a newly developed method to measure RBC deformability (expressed as Elongation Index – EI) as a function of pO2 (Figure 1A). Upon a standardized, 22minute, automated cycle of deoxygenation (pO2 nadir 16mmHg, standard error of the mean 0.17) and reoxygenation, a number of clinically relevant parameters are produced in a highly reproducible manner (coefficients of variation < 5%).
Results: Physiological modulators of oxygen affinity, i.e. pH and 2,3-diphosphoglycerate showed a significant correlation (respectively R=-0.993 and R=0.980) with Point of Sickling (PoS5%), which is defined as the pO2 where a 5% decrease in EI is observed during deoxygenation. Furthermore, in vitro treatment with anti-sickling agents, including GBT440, which alter the oxygen affinity of hemoglobin, caused a reproducible left-shift of the PoS, indicating improved deformability at lower oxygen tensions. When RBCs from 21 SCD patients were analyzed we observed a significantly higher PoS in untreated homozygous SCD patients compared to treated patients and other genotypes (Figure 1B).
Conclusions: The oxygenscan is a state-of-the-art technique that allows for rapid analysis of sickling behavior in SCD patients. The method is promising for personalized treatment, development of new treatment strategies and could have potential in prediction of complications.

Evaluation Of The Added Value Of New Hematological Parameters For Anemia Screening During Pregnancy
Marianne Schoorl, Johanna Rodriquez, Johannes van Pelt, Margreet Schoorl
Northwest Clinics, Alkmaar, Netherlands

Introduction: Worldwide, a high prevalence of anemia during pregnancy has been reported, ranging from 2 to 30% in the developed countries. Although a mild degree of anemia is common in the third trimester of pregnancy, it remains a challenge to establish whether a decrease in hemoglobin (Hb) concentration is physiological (due to hemodilution) or pathological (due to insufficient availability of essential nutrients, such as iron, folic acid or vitamin B12). Several European investigators recommended Hb cut-off values between 6.3-6.8 mMol/L to discriminate anemia and providing an indication for subsequent iron supplementation. The aim of this study was to establish the added value of the percentages microcytic red blood cells (%MicroR) and hypochromic red blood cells (%HypoHe), on parameters indicating hemoglobin content of reticulocytes and erythrocytes (RET-He, RBC-He, delta-He) in case of suspected iron deficient erythropiesis (IDE) in the third trimester of pregnancy.
Methods: Blood samples (K2EDTA) were collected during the third trimester of pregnancy. A total of n=168 cases were classified based on Hb concentration, MCV, ZPP, RET-He and delta-He into a group with symptoms of IDE (n=55) or without IDE (n=113). A subgroup of pregnant women with IDE (n=43) was treated with iron supplementation (200 mg ferrofumarate/day, according to local practice). After four weeks, effects of treatment were evaluated. Hemocytometry parameters were measured on a Sysmex XN-9000 Hematology Analyzer (Sysmex Corporation). Zinc protoporphyrin (ZPP) concentrations were measured on a hematofluorometer (AVIV Biomedical Inc.).
Results: In the group of pregnant women with IDE results of %MicroR and %Hypo-He were significantly increased (p=0.000), compared to the group of pregnant women without symptoms of IDE, whereas RET-He, RBC-He and delta-He were decreased (p=0.000). A significant positive correlation to increased values of %MicroR (r=0.75, p=0.000) and %Hypo-He (r=0.79, p=0.000) with ZPP was established. However, in the ZPP range 75-100 µMol/Mol Hb a slight overlap was demonstrated between subjects with and without symptoms of IDE. After iron supplementation, %Hypo-He decreased significantly (p=0.031) while %MicroR remained stable. RET-He, delta-He and RDW-SD increased significantly (p=0.000)
Conclusions: Although the individual predictive value of %MicroR and %Hypo-He is poor, combined interpretation of %MicroR and %Hypo-He with parameters indicating hemoglobinization of mature and immature red blood cells (RET-He, delta-He) has added value in the discrimination of IDE during pregnancy.

Blast Detection On The Advia 2120IAnalyser
Laura Macas, Laura Bold, Angel Molina, Francisca Bascn, JL Bedini, Anna Merino
Hospital Clnic, Barcelona, Spain

Introduction: Analysers use flags to identify samples that require manual review by clinical pathologists. Blast flag (BF) is related to the percentage of blast cells circulating in blood. Large Unstained Cells (LUC) in the Advia 2120i® analyser (Siemens) is associated with the presence of abnormal, reactive lymphocytes or blast cells on the blood sample. The objectives of this study are 1) to evaluate the rate of blood samples containing blasts on peripheral blood smear (PBS) with negative BF in the ADVIA2120i® and the haematological diseases related to this finding and 2) to compare the BF with LUC percentage in blood samples containing blasts.
Methods: From May to November 2018, CBC from a total of 182,626 samples was performed on the ADVIA2120i. According to our laboratory criteria, a total of 17,442 of these blood samples were selected for PBS analysis. Peripheral blood films were automatically stained with MGG and manual leukocyte differentials were performed on Cellavision®DM96. Then, we evaluated the LUC percentage and the BF in the entire PBS positive for blast cells.
Results: Considering samples included in this study, blasts were observed in digital images provided by CellaVision in 793 smears (4.5%). From these smears, the BF was triggered in 764 samples (96.35%). However, the BF was absent in 29 (3.6%). Figure 1 show the number of samples in which blasts was observed in the smear, the positive or negative BF and LUC values.In 2/29 samples in which BF was negative, abnormal LUC values (> 5) were obtained. With this in mind, the sensitivity in blast detection increased only slightly (0.3%). The diagnosis of the different haematological diseases corresponding to these patients is shown in Figure 2. In most of the negative samples for BF, the diagnosis of the patients was a myelodysplastic syndrome (MDS) and the most common findings were: low blast percentages (median: 2%), low haemoglobin values (median: 98.5 g/L) and low leukocyte and platelet counts (median: 2.99x109/L and 71x109/L, respectively).
Conclusions: The sensitivity of the Advia2120i in blast cells detection through the BF reached very high values. It is interesting to note that most of samples containing blasts with negative flags belonged to patients with MDS, low haemoglobin values and low leukocyte and platelet counts. The review of these cases should be considered by the clinical pathologist.

Correlation Between Glycated Hemoglobin And Red Blood Cell Distribution Width (Rdw)
Georgia Tsakiroglou 1, Eleni Vagdatli2, Vasiliki Konstantinidou1, Katerina Karantani2, Eugenia Limberaki1, Konstantina Tsioni2, Theodoros Lialiaris 1
1Department of Medical Laboratories Studies, Alexander Technological Educational Institute , Thessaloniki, Greece, 2Hippokratio General Hospital, Thessaloniki, Greece

Introduction: Hyperglycemia has various effects on red blood cells, such as the glycation of hemoglobin and the reduction of red blood cells lifespan, as a result of their impaired deformability. Therefore, elevated blood glucose concentration affect red blood cells volume. Red blood cell volume variation is associated with Red Blood Cell Distribution Width (RDW). The aim of the present study is to investigate the correlation between Glycated Hemoglobin (HbA1c) and RDW.
Methods: In this retrospective study HbA1c levels and RDW-SD of complete blood count were investigated in 1848 individuals for the period September 2017 to August 2018. Both measurements were performed the same day. Samples with platelet clumps flag and interference on platelets or red blood cells measurement, because of small erythrocytes and giant platelets respectively, were excluded. The studied samples were divided in three groups (American Diabetes Association. Diagnosis and classification of diabetes mellitus. Diabetes Care 2011;34): Group A with normal glucose homeostasis (HbA1c < 5.7 %), group B with pre-diabetes (HbA1c: 5.7– 6.4%) and group C with diabetes (HbA1c >= 6.5%). SPSS Statistics Version 21 and Pearson's correlation coefficient were used for statistical analysis and comparison among groups, in order to find the association between HbA1c and RDW.  The p-value< 0.05 was considered statistically significant.
Results: Ninety two samples were excluded from the study due to  interference. In the rest 1756 samples, no statistically significant correlation was observed between RDW-CV and HbA1c (r=-0.024, p=0.313). According to Mann-Whitney test, a statistically significant increase of RDW-CV was observed among the groups A , B and  C (A-B and A-C: p< 0.001, B-C: p=0.005)  (figure 1)
Conclusions: The progressive increase of RDW-SD from normal glucose homeostasis group towards  pre-diabetes and diabetes groups demonstrates the significant impact of hyperglycemia on red blood cells. Since RDW is a simple and inexpensive parameter, it may be regarded as a potential, innovative biomarker for improving risk assessment of developing diabetes.

Microapp: A Useful Tool For TheScreening Of Thalassemia Carriers
ELOISA URRECHAGA1, Urko Aguirre2, Johannes Hoffmann3
1HOSPITAL GALDAKAO USANSOLO, GALDAKAO VIZCAYA, Spain, 2Research Unit, REDISSEC, Health Services Research on Chronic Patients Network, GALDAKAO VIZCAYA, , Spain, 3H3L Consult , Nuenen, , Netherlands

Introduction: Iron deficiency anemia (IDA) and thalassemia are the most common causes of microcytic anemia. Identification of thalassemia is important for reducing morbidity and mortality, whereas IDA can simply be treated with iron administration. The differential diagnosis of IDA and thalassemia should start with efficient and inexpensive screening, followed by further analysis for confirming the disease. Computer simulation has become a useful tool of mathematical modelling in medicine. Usually it is developed by data scientists/statisticians to assess the performance of the statistical model in relation the final diagnosis. We present MicroApp, a statistical tool  for evaluation and interpretation of  hemogram results, optimized for detecting thalassemia carriers.
Methods: MicroApp is a user-friendly, free web-based application. The application is internally programmed in RStudio and stored on a server.  Input for the tool are basic numerical hemogram data: red blood cell count (RBC), Hemoglobin (Hb), mean cell volume (MCV), and red cell distribution width (RDW). Through interactive user queries the dynamically generated web page eventually results in a proposed classification: thalassemia or IDA. We used 1858 datasets for developing the model and 768 for verifying its performance. In the set of 1858 patients with microcytic anemia (Hb < 120 g/L, MCV < 80 fL) 1304 suffered from IDA, 534 were thalassemia carriers (346 beta, 208 alpha). In the validation group 315 IDA, 391 carriers (263 beta, 107 alfa 83 with concomitant IDA).
Results: Cut offs of the selected variables RBC > 5.0*1012/L, Hb>112 g/L, MCV< 72.8 fL, RDW < 17.3 %, rendered odds ratios 9.4, 2.4, 22.8 and 10.9 respectively to discriminate carriers from IDA. The verification group demonstrated that MicroApp provided an accurate classification of 96.8 % thalassemia cases  and 92.0% of IDA patients. Overall, the accuracy was 94.9%. Incorrect classifications were mainly encountered in alpha thalassemia carriers with single alpha globin deletion and very mild hemogram abnormalities.
Conclusions: The screening of hemoglobinopathies must rely on inexpensive methods, adding in the selection of highly suspicious samples for further analysis, more sophisticated and expensiveOur sophisticated MicroApp predicts thalassemia trait accurately in 94.9%, making it a highly useful tool for thalassemia screening.

Differential Pharmaco-Proteomic Analysis Of Hydroxyurea Response In Β-Thalassemia
Muhammad Zohiab1, Saqib Ansari2, Tahir Shamsi2, Roman Zubarev3, Shamshad Zarina1
1 University of Karachi, Karachi, Pakistan, 2National Institute of Blood Diseases and Bone Marrow Transplantation, Karachi, Pakistan, 3Division of Physiological Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden

Introduction: Β-thalassemia is the most common genetic disorder caused by mutations in β-globin gene results in absence and reduced production of Adult Hemoglobin (HbA). Regular blood transfusions and effective iron chelation is necessary for survival of β-thalassemia patients. An emerging therapeutic approach to handle β thalassaemia is production of fetal hemoglobin (HbF). Hydroxyurea (HU), a potent HbF inducing agent, has shown to be an effective drug for β-thalassemia management with variable responses from transfusion independence to null outcome. Diversified response of β-thalassemia patients to HU therapy remains ambiguous for hematologist. Clinical proteomics has revolutionized the study of differential protein expression associated with disease and offers a unique technique to monitor the response to treatment .
Methods: In current study, we focused on comparative analysis of plasma proteome in pre- and post- HU-treated β-thalassemia patients, as well as responders and non-responders to HU treatment. Plasma was collected from β-thalassemia patients before and after 6 months of HU treatment, and the treated group were sub-categorized on the basis of response to HU. Label-free LC-MS/MS analysis was performed on nano-HPLC coupled Q-Exactive Orbitrap Plus mass spectrometer. Quantitative data and statistical analysis was carried out using MaxQuant and Perseus respectively.
Results: Proteomics analysis revealed identification of 400 proteins in all groups, among them the abundances of twenty eight proteins were significantly different in pre- vs post- HU treated groups, with, twenty six proteins being differentially expressed in responder vs non-responder groups. Transferrin receptor protein 1 (TfR) was the most down-regulated and hemopexin and haptoglobin were the most up-regulated proteins in plasma after HU treatment whereas proteins with most significant difference between responder and non-responder was carbonic anhydrase 1, hemoglobin subunit gamma-1 and peroxiredoxin-2.
Conclusions: A number of plasma proteins show significantly different expression in response to HU treatment in β thalassemia.HU therapy in β thalassemia patients changed protein profile towards normal pattern, in addition with reduction in transfusion requirements. A noteworthy difference in plasma protein profile of HU responder and non-responder β thalassemia patients was also demonstrated. Differentially expressed proteins in HU responder may be used for validation in large cohort to establish these proteins as potential predictive biomarker for HU therapy.

Sigma Metrics And Quality Goal Index Ratio As A Tool For Evaluating The Performance In A Hematology Laboratory
Tlay evlik1, Rana Turkal1, Ozan nl1,2, nder Şiriki1,2, Goncagl Haklar1,2
1Marmara University Pendik E&R Hospital, Istanbul, Turkey, 2Marmara University, School of Medicine, Department of Biochemistry , Istanbul, Turkey

Introduction: Sigma is a quality management tool that helps in continuous monitoring and improvement of the performance of the analytes in the clinical laboratory. The Six Sigma (6σ) methods are usually applied when the outcome of the process can be measured. Quality goal index (QGI) ratio represents the relative extent to which both bias and precision meet their respective quality goals. The aim of the present study was to calculate sigma metrics and QGI for several tests of the hematology laboratory, using European Society for External Quality Assessment (ESFEQA) total allowable error (TEA) requirements.
Methods: This retrospective study was conducted in Marmara University Pendik E&R Hospital Hematology Laboratory. 6σ values were calculated (Sigma = (TEA − Bias)/CV) by using Bias %, CV%, and TEA of the tests. To calculate precision (CV) and bias, internal quality control (IQC) (Coulter 6 C Cell Control) and external quality control (EQC) (RIQAS) data were used respectively, which were analyzed at Unicel DxH800 Coulter Cellular Analyzer (Beckman Coulter, USA) between June 2018 and December 2018. The mean values obtained in the last six EQC surveys in which our laboratory participated at the same time were used for bias calculation. QGI was calculated using the following formula: QGI = Bias/1.5 CV. QGI score of < 0.8 indicates imprecision, QGI > 1.2 indicates inaccuracy, and QGI score 0.8-1.2 indicates both imprecision and inaccuracy.
Results: The performance of each analyte was evaluated as sigma metrics and quality goal index ratio. WBC (all level IQC), RBC (level 3), hemoglobin (level 2), hematocrit (level 2 and level 3), MCV (level 2 and level 3) showed an ideal performance of >6 sigma level. RBC (level 1), hemoglobin (level 1) showed poor performance of ≥3-< 4 sigma level. For all analytes, showing < 6 sigma metric level, quality goal index ratio was < 0.8 indicating the area of imprecision requiring improvement except PLT with a QGI ratio of > 1.2 indicating inaccuracy.
Conclusions: The study shows that sigma metrics and QGI ratio is a good quality tool to evaluate the analytical performance of a hematology laboratory. However, we might need to design a very tight internal quality control protocol for analytes showing poor assay performance.    

Pyruvate Kinase Activity: The Need For Eqa
Duljeet Chohan, Zeno Abid, Alexandra Makin, Barbara De la Salle
UK NEQAS Haematology, Watford, United Kingdom

Introduction: Pyruvate Kinase (PK) catalyses the final step in the Embden-Meyerhof pathway, generating adenosine triphosphate (ATP) essential for cellular integrity and energy metabolism. PK deficiencyis a major glycolytic enzymopathy, causing hereditary, chronic non-spherocytic haemolytic anaemia.The clinical severity varies widely, ranging from a mildly compensated anemia to severe anemia of childhood. Pyruvate kinase enzyme activity is the gold standard for initial testing for PK deficiency. Although low enzyme activity is consistent with the diagnosis, falsely normal levels are sometimes encountered due to transfusion, incomplete leucocyte removal or a reticulocytosis compensating for haemolysis. Good laboratory practice is essential for its accurate quantitation.Due to the rarity of the disease, there are relatively few laboratories undertaking this assay in any one country and no established external quality assessment programme.
Methods: UKNEQAS Haematology distributed a first pilot PK assay exercise in December 2018. Eight expert laboratories in the UK, the Netherlands, Italy and Spain were recruited for the pilot study, each receiving two samples for analysis. A questionnaire accompanied the samples requesting methodology, the temperature at which the assay is performed and the laboratories' reference ranges. 
Results: All eight participants returned results and all perform the assay based on Beutler’s method. All participants returned the correct clinical diagnosis/outcome for both samples. Sample 1 had a mean enzyme activity of 10.4u/g Hb and a standard deviation of 1.8. Sample 2 had a mean of 11.0u/g Hb and a standard deviation of 2.0. The results of the pilot exercise, together with internal stability studies showed the survey material to be viable for the duration of the EQA exercise.
Conclusions: The good inter-laboratory agreement of results is expected from a cohort of expert laboratories. A second exercise will be conducted on a larger scale and incorporate PK deficient blood. Although molecular analysis is important in the diagnosis of PK deficiency, this does not necessarily reflect the patient’s phenotype. AG-348, an allosteric activator of the red cell isoform of PK (PKR), has been trialled in humans as a treatment for PK deficiency and monitoring of AG-348 therapy requires PK quantitation. Accurate measurement of PK enzyme activity remains an essential component of diagnosis and monitoring of this disorder.

D-Dimer Testing: A 6 Year Review Of Proficiency Testing Data From The Institute For Quality Management In Healthcare (Iqmh).
Carolyne Elbaz1, Sumedha Kulkarni2, Anna Johnston2, Karen Moffat3, Michelle Sholzberg1,2, Rita Selby1
1Department of Laboratory Medicine & Medicine, University of Toronto, Toronto , ON, Canada, 2Institute for Quality Management in Healthcare, Toronto, ON, Canada, 3Hamilton Regional Laboratory Medicine Program, McMaster University, Hamilton, ON, Canada

Introduction: D-dimer testing is used extensively for ruling out venous thromboembolism in symptomatic outpatients. However, D-dimer assays are not standardized or harmonized, and use different types and magnitude of units leading to significant inter-assay variability in performance characteristics. There is ongoing concern about insensitive, non-validated D-dimer methods being used for VTE exclusion. The Institute for Quality Management in Healthcare (IQMH) Centre for Proficiency Testing (PT) offers quality management services through ISO 17043-accredited programs, providing an opportunity for laboratories to assess their results compared to their peers.    
Methods: IQMH D-dimer PT challenges are conducted biannually. Participants test the sample like a patient sample and submit results online with assay method, units and instrument. Although numeric results are provided by participating laboratories, since 2011 only qualitative interpretation of the sample (positive or negative) is assessed based on the assay’s reference range or VTE exclusion threshold. D-dimer results for surveys from 2013 to 2018 were reviewed.
Results: Between 2013 and 2018, 137 to 143 laboratories participated in D-dimer PT challenges. The majority (86%) used quantitative D-dimer assays and this has steadily increased over time from 70.7% in 2013 to 97.9% in 2018. Inversely, the use of qualitative assays is decreasing from 29% in 2013 to 2.1% in 2018. Inter-assay coefficient of variation (%CV) of numeric results is high ranging from 14.1% to 79.4% on D-dimer positive samples, and 13.2% to 38.3% on D-dimer negative samples, staying consistent over the last six years. When assessing inter-assay performance on qualitative interpretation of results based on assay-specific thresholds, laboratories achieved 98.9% agreement.
Conclusions: The majority of laboratories use appropriate, high sensitivity, quantitative D-dimer assays suitable for VTE exclusion. Moreover, over six years, we found an increasing proportion of laboratories adopting quantitative methodologies. Despite significant inter-assay variability and poor agreement on numeric results, the qualitative agreement on interpretation of the D-dimer (positive/negative) provided by laboratories was almost 100% which indicates sufficient test accuracy to allow for appropriate clinical decision making, as long as interpreting clinicians adhere to assay specific thresholds. 

A Clinical Validation Of Delta Check Model In Hematology Test For A Quality Improvement
Yang Fu1, Guoju Luo1, Qiang Li2, Zhuoyun Tang1, Mengyu Li1, Si Chen1, Hong Jiang1
1West China Hospital, Sichuan University, chengdu, China, 2Scientific & Application Div. Sysmex Shanghai Ltd, shanghai, China

Introduction: Delta Check has been applied in hematology test for quality control and report verification for quite a long time. But there have been problems in routine application for most laboratories, mainly including parameter selection, limit setting and software support. This study aims to provide a simplified and practical process in hematology test for a clinical quality improvement.
Methods: A total of 1260 patients with sequential complete blood cell counting results in one week admitted to West China Hospital of Sichuan University from October 2017to March2018 were enrolled in this studyand all specimens were subjected to microscopic examinations.Parameters of complete blood cell analysis for delta check were chosen by ROC. Limitation lines of the delta check equations were simulated by Sigma Plot 13.0 software. Delta check processes were established by clinical rules and equations and validations were performed by samples of patients from the hematology, oncology and other departments. 
Results: MCV, Hb, WBC and PLT were selected for delta check by ROC analysis. Based on the delta check graph reported by Berend Houwen, the delta value(%) was calculated for fitted curve as a ordinate and the higher value of the two results as a abscissa. Equations of delta check limits for each item were simulated and optimum parameters in each formula were obtained. And the re-inspection rate was reduced by 7.01%, 4.66%, 7.86% in oncology, hematology and other patients in clinical validation.
Conclusions: Delta check limitation equations have solved difficulties of parameter selection and formula simulation in delta check application. With the application of delta check, the clinical process can be simplified in practice, which will further improve the clinical quality.

Improving Peripheral Blood Smear Turn-Around Time In A Distributed Laboratory Network With The Cellavision Dc-1 Digital Morphology Analyzer
Diane Higa1, Cheri Mayes1, Tracey Gwilliam1, Tracey McFarland1, Maureen Cyfra1, Meer-Taher Shabani-Rad1,2, Etienne Mahe1,2
1Alberta Public Laboratories, Calgary, AB, Canada, 2University of Calgary, Calgary, AB, Canada

Introduction: Digital pathology is becoming increasingly valuable in the delivery of timely, high-quality clinical services; CellaVision® is the leader in the field of hematology. While most systems have been designed with high-volume/high-throughput in mind, CellaVision® has recently developed a lower-volume/throughput and affordable instrument, the CellaVision® DC-1. The potential utility of the CellaVision® DC-1 was tested in a distributed laboratory system (one with smaller laboratories situated at significant distance from a central referral laboratory), with a focus on turn-around times (TATs).
Methods: Research Ethics Board approval was obtained. We selected the Calgary Lab Services (CLS) peripheral High River Laboratory (HRL) to test the CellaVision® DC-1. HRL performs on-site complete blood counts and peripheral blood smears (PBSs) but more complex PBSs are referred for review to a central CLS laboratory. Baseline TAT data was collected for 30 cases by retrospective assessment of PBS time-stamps logged by HRL from March to June, 2017. Subsequently, 21 cases (collected from June to Nov, 2018) flagged for PBS were prospectively analyzed using the CellaVision® DC-1 followed by remote review by HRL staff in consultation with central CLS staff. The same 21 cases were also analyzed by the standard manual workflow with physical slide referral. Time-stamp data were collected for both workflow.  
Results: Significant improvement was found in the distribution of TATs using the CellaVision® DC-1 relative to both the retrospective HRL TAT data (baseline median TAT 1794 minutes vs DC-1 workflow median TAT 82 minutes; Mann-Whitney U=26, p< 0.0001) and the parallel manual PBS review with physical slide referral (manual PBS review median TAT 1447 minutes vs CellaVision® DC-1 PBS review median TAT 82 minutes; Wilcoxon W=190, p< 0.0001). Exclusive of scanning time, the CellaVision® DC-1 also permitted a significant reduction in case-assessment times (mean time for CellaVision® DC-1 case evaluation 1.92 minutes vs 4.05 minutes by manual assessment; Wilcoxon W=105, p=0.0001). No significant diagnostic discrepancies were identified during the CellaVision® DC-1 testing timeframe.
Conclusions: We describe a real-world assessment of the CellaVision® DC-1 digital morphology analyzer in a distributed laboratory network, linking low-volume laboratories to high-throughput sites. Our evaluation highlights significant improvements in case TATs with a CellaVision® DC-1 assisted digital pathology workflow.

Bone Marrow Sample Preparation And Processing: An Iqmh Survey On Laboratory Practices
Anna Johnston1, Andrew McFarlane1, Hong Chang2, Elaine Leung2, Brian Olsen2, David J Good2, Miranda Wozniak2, Ruth Padmore2
1IQMH, Toronto, ON, Canada, 2Hematology Scientific Committee, IQMH, Toronto, ON, Canada

Introduction: Examination of the bone marrow, including aspirate smear, clot section, imprint morphology and trephine biopsy are essential for correct diagnosis and management of hematological disorders. The Institute for Quality Management in Healthcare (IQMH) offers a bone marrow proficiency testing (PT) survey where glass slides of bone marrow aspirate or biopsy, peripheral blood smear and relevant clinical data are provided. A questionnaire was developed by the IQMH Hematology Scientific committee to gather information regarding practices for preparation and processing of bone marrows.  
Methods: A questionnaire consisting of 17 questions was distributed to 59 participants from July to September 2015 with the rotational slide survey to determine laboratory practices for bone marrow sample preparation and staining.
Results: Fifty-one (87%) participants submitted results; summarized below: 100% prepare direct bone marrow aspirate smears, 67% prepare particle crush smears, 63% clot sections and 51% biopsy touch imprints. 82% place bone marrow biopsy tissue sections in fixative immediately at the bedside. The most commonly reported fixative used for clot sections and trephine biopsies was buffered formalin (61% and 67%, respectively). There was great variability in the fixation times for clot sections and for trephine biopsies, with the greatest number reporting a minimum of 24 hours (35% and 41%, respectively). Decalcification time varied, ranging from < 1 hour (19%) up to 24 hours (4%). 98% performed paraffin embedding for trephine biopsy sections post decalcification. One participant uses surface decalcification after formalin fixation and paraffin embedding. The most commonly used stain for bone marrow aspirate slides was Wright-Giemsa stain (49%). 96% routinely perform an iron stain on the marrow aspirate; Perls’ Prussian Blue being the most commonly used method (88%). 98% routinely stain bone marrow biopsies with H&E.
Conclusions: The results from this questionnaire demonstrate wide variability and lack of standardization amongst laboratories in preparation and processing of bone marrow specimens. This may lead to inconsistencies in the interpretation of the marrow findings and ultimately in the classification of disease. These findings present an opportunity to review published guidelines to standardize current laboratory practice.

Establishment Of Gender-Specific Reference Intervals For Research Parameters Of An Automated Hematology Analyzer In Healthy Korean Adults: A Large Population Study
Miyoung Kim, Kibum Jeon, Jee-Soo Lee, Han-Sung Kim, Hee Jung Kang, Young Kyung Lee
Department of Laboratory Medicine, Hallym University Sacred Heart Hospital, Anyang, Korea

Introduction: The selection and recruitment of large numbers of healthy individuals representing relevant demographic groups are critical for establishing representative reference intervals. Automated hematology analyzers for complete blood cell counts provide not only basic parameters routinely reported to clinicians, but also various research parameters some of which have shown significant clinical implications. We aimed at establishing gender-specific reference intervals for research parameters in healthy Korean adults along with investigating the necessity of separating the reference intervals for men and women. As a preliminary study, we performed the analysis on 17 research parameters. 
Methods: Among 9,474 individuals (5,449 men and 4,298 women) aged ≥20 years and < 60 years who underwent medical checkups, 1,483 healthy subjects (15.7%, 919 men and 563 women) were enrolled based on stringent criteria including laboratory, imaging and endoscopy results; previous medical history; and medication history based on the CLSI EP28A-3C. CBC research parameters, as well as basic parameters, were measured using an Advia 2120i (Siemens, Munich, Germany) instrument. The reference intervals for measured CBC parameters were established according to a nonparametric method based on the CLSI EP28A-3C. The Harris and Boyd method was used to determine the necessity of separating the reference intervals for each gender group. 
Results: Gender-specific reference intervals for 9 RBC (%High CH, %Low CH, CHDW, %Hyper, %Hypo, %Macro, %Micro, RBC Covar and R count) and 8 platelet parameters (Large Plt, MPC, MPM, P count, PCDW, PMDW, RBC Fragments and RBC Ghosts) were established. Among those 17 parameters, the median value was significantly different in all 9 RBC parameters and 3 platelet parameters (MPC, P count and PCDW) between men and women. However, only 3 RBC parameters (%Hyper, %Hypo and %Macro) required separate reference intervals for each gender group. None of the 8 platelet parameters required separate reference intervals for each gender group.
Conclusions: We established representative reference intervals for research parameters of CBC in healthy Korean adults using a large cohort with stringent selection criteria for the first time and detailed the need for separate reference intervals between the two genders.

Role Of Ne-SflAnd Ne-Ssc Parameters In Early Detection Of Sepsis
Anna Marull, Javier Nieto-Moragas, Orlando Jimenez, Javier Hernando, Patricia Tejerina, Maite Serrando
Laboratory Trueta Hospital, Girona, Spain

Introduction: Sepsis is a leading cause of mortality in critically ill patients.  A fast and accurate diagnosis followed by a rapid treatment is essential. However, differentiating sepsis from non-infectious can sometimes be very difficult. Several markers have been proposed as a sepsis biomarker (procalcitonin, reactive C protein.), but none of them are specificfor sepsis.  When blood infection occurs, neutrophils are activated by microorganisms and inflammatory cytokines. SysmexXN hematology analyzer provides information about these changes measuring sideward scatter light (NE-SSC) and the cellular nucleic acid content by sideward fluorescence light (NE-SFL). The aim of this study is to know whether NE-SSC/NE-SFL can be a favorable parameter in differential diagnosis of sepsis.
Methods:   From October to December 2018, a total of 65 adults’ patients were analizedand distributed into sepsis group (30 patients)  and reference group (38 subjects). The CBC and NE-SSC, NE-SFL parameters were obtained from Sysmex-XN. Diagnosis of sepsis required positive blood culture isolation and clinical parameters of sepsis following The Third International Consensus Definitions for Sepsis and Septic Shock (Sepsis-3). Results between both groups were compared with Mann-Whitney U test. Receiver Operating Curves (ROC) were analysed for the best parameter.
Results: Both NE-SSC and NE-SFL presents higher values in sepsis group compared to the control group (median and interquartile range in scatter intensity were: NE-SSC 157.1 (153.8-160.0) vs 153.1 (151.1-157.2), p< 0.01; NE-SFL 53.0 (50.1-58,55) vs 46.4 (45.18-47.95), p< 0.001) . The best NE-SFL cutoff observed was 49.9 SI (sensitivity 80% and specificity 89.5%). ROC analysis showed Area Under the Curve (AUC) for NE-SFL of 0.877. It was stablished a cutoff of 49.9 and NE-SFL was 80% sensitive and 89.5% specific.
Conclusions: Neutrophils are activated when bacterial infection occurs, especially in cases of sepsis. Sysmex XN provides information of the activity of these neutrophils through NE-SSC and NE-SFL analysis.  According to recent studies these parameters can be used in the differential diagnosis of sepsis. In this study, we suggest NE-SFL as a reliable marker for sepsis. A cutoff value of 49.9 SI is appropriate to distinguish septic patients from the control group.

Long-Term Biological Variation Estimates Of 18 Hematological Parameters In Healthy Subjects
Chenbin Li1, Mingting Peng1, Ji Wu2, Zhongli Du1, Wenbin Zhou1
1National Center for Clinical Laboratories, Beijing Hospital, National Center of Gerontology, Beijing, China, 2Guangdong Provincial People's Hospital, Guangzhou, China

Introduction: Complete blood cell count(CBC) is a basic test ordered by physicians routinely as a part of initial diagnostic workup on their patients. To ensure safe and effective clinical application of the CBC, reliable biological variation (BV) data are required to establish analytical performance specification. The aim of this study was to define BVs of CBC parameters employing a protocol with all essential elements of Biological Variation Data Critical Appraisal Checklist (BIVAC) recently published by the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM) Working Group on BV (WG-BV).
Methods: Blood samples, drawn from 41 apparently healthy subjects (22 females, 19 males,aged 23-59 years old) once monthly for six consecutive months by an expert phlebotomist, were analyzed using a ABX Pentra 80 instrument. Its performance had been verificated, and it was effectively calibrated and regularly controlled. All samples were analyzed in duplicate for 18 CBC parameters. Cochran test and Reeds criteria were used to identify outliers. Shapiro-Wilk test was used to check the normality of between- and within-subjects data. The differences between females and males CVI were determined by Mann-Whitney test. Analytical precision (CVA) was calculated from duplicate measurements. CVI and CVG were calculated by nested ANOVA.
Results: The numbers of remaining data for each subject were 458-484 after removal outliers. Only for WBC, significant differences were found between female/male CVI estimates. The ranges of CVA, CVI and CVG for 18 parameters were 0.6%(Hb)-18.3%(Bas), 0.5%(MCHC)-20.6%(Bas)and 1.1%(MCHC)-53.9%(Eos). Except RBC, Hct, Hb and Lym, BV of other parameters were lower than those on Westgard website. Except Bas with higher BV data, the BV data of other parameters was close to those of the European Biological Variation Study (EuBIVAS).
Conclusions: The BV data of this study is close to those of EuBIVAS and different from those on Westgard website. The reason is the protocol of this study and EuBIVAS met the BV evaluation criteria issued by EFLM. It is recommended that the BV data, from the studies meeting the BV evaluation criteria, should be used. When several studies met the evaluation criteria, we suggest the more stringent targets should be employed after meeting the current practice.

Establishment Of Third Korea National Standard Of Blood Coagulation Factor Viii For Potency
Sangmi Park, Hyunjeong Kim, Kikyung Jung, Jaeok Kim
Blood Products Division, National Institute of Food & Drug Safety Evaluation , Cheongju, South Korea

Introduction: In 2009, the National Institute of Food and Drug Safety Evaluation of Korea has established the 2nd Korean national biological  reference standard for FVIII:C concentrate(09/035). However remaining stocks will be exhausted, therefore in this study we prepared a 3rd Korean national biological reference standard for blood coagulation FVIII:C concentrate for replace the 2nd standard and established the potency of the candidate material.
Methods: The candidate material for the 3rd Korean national standard for blood coagulation was manufactured to comply with the 'Green-Mono Injection' protocol for the highly pure plasma-derived standard established by GC Pharma Corp. The potency of FVIII was measured by one-stage clotting assay and chromogenic assay.
Results: The candidate material of 3rd national standard FVIII was produced from final product of factor VIII:C Monoclonal Antibody-purified, Freeze-dried Human Blood Coagulation Factor VIII:C, the potency of candidate material was about 9.51 IU/vial and total vials were 10,285 vials. The long-term stability is ongoing, according the accelerated degradation stability study, the potency decrement is represented in the case of storing in 25 ℃ and 40 ℃. The collaborative study involving 4 laboratories of 3 manufacturers and 1 national control laboratory has been undertaken to evaluate the 3rd national standard for Factor VIII.              
Conclusions: The quality of the candidate material which manufactured in manufacturer was validated to meet acceptance of QC test. The candidate of the 3rd national standard FVIII is verifying the potency and long-term stability test. After verifying quality, it's avaliability for the candidate as the 3rd natioanl standard FVIII shall be verified with further collaborative study with 4 participated institutions to assign the potency. Finally the national standard can be used for quality control of blood coagulation facotor VIII.  

Myeloperoxidase Deficiency Manifested As Pseudoneutropenia With Abnormally High Monocyte And Luc Counts
Soongki Roh, Jang Soo Suh
Department of Clinical Pathology, School of Medicine, Kyungpook National University, Daegu, Korea

Introduction: Myeloperoxidase (MPO), which is abundant in the azurophilic granules of neutrophils and lysosomes of monocytes, plays a key role in amplifying the toxicity of hydrogen peroxide generated by the respiratory burst. MPO deficiency, one of the most common inherited phagocyte defects, is rarely associated with clinical symptoms, and may exist as a partial or transient phenomenon in combination with clinical conditions such as diabetes, severe infection, or pregnancy. MPO deficiency is easily detected at present because of the widespread use of automated flow cytochemical analysis in clinical hematology laboratories for enumerating peripheral blood neutrophils with peroxidase activity. Hematological analyzer ADVIA 2120i is used to routinely identify the different types of leukocytes [neutrophils, monocytes, eosinophils, lymphocytes, and large unstained cells (LUCs)] based on their size and staining properties, and by mean peroxidase index (MPXI). When MPO deficiency is present, neutrophils may be incorrectly counted as monocytes with lower MPXI values, and LUCs, which are reported on the hemogram when large peroxidase-negative cells in the peripheral blood cannot be characterized further. We encountered a few cases of MPO deficiency with abnormally high monocyte and LUC counts resulting in extremely low neutrophil counts (pseudoneutropenia). These abnormal counts could lead to a mistaken diagnosis of severe neutropenia, which could result in unnecessary therapy such as the infusion of G-CSF in the patients. Manual differential count and automated differential count using a distinct cell analyzer, which utilized an alternative differential technology, exhibited the normal differential count in every case, while no cases of neutropenia were observed. Although the reference range for these MPXI analyses was from -10 to 10, every case yielded a markedly low MPXI value below -20. Cytochemical staining of peroxidase in the neutrophils was dramatically decreased in the patients as compared to healthy controls. In conclusion, we suggest that MPO deficiency must be considered in patients with incompatibility of neutropenia between smear and automated cell analyzer, especially when abnormally high monocyte or LUC counts combined with low MPXI values are observed.

Indirect Estimation Of Blood Cells Reference Intervals From Clinical Data In The Commercial Reference Laboratory.
Yong-Hak Sohn1, Min Kyung Kim 1, Choong-Hwan Cha 2
1Seegene Medical Foundation , Seoul, South Korea, 2Department of Laboratory Medicine, Gangneung Asan Hospital, University of Ulsan College of Medicine, Gangneung, South Korea

Introduction: The recommend method of determining a reference interval (RI) based on Clinical and Laboratory Standards Institute (CLSI) guideline EP28-A3C, require statistically sufficient healthy reference subjects (more than 120). It is difficult to define "healthy" person and occasionally enroll relevant subjects. Recently, Internal federation of Clinical Chemistry reviewed the indirect methods for RIs and encouraged their use for establishing and verifying reference material. Thus, we calculated RIs of hematological assays using the indirect method from clinical data tested in the commercial reference laboratory.
Methods: A total of 329026 laboratory data of WBC count, Platelet count and hemoglobin concentration assays for 3 months (from September to December 2018) were collected from laboratory information system in Seegene reference laboratory, Korea. All hematological assays are tested by using Sysmex XN-9000 (Sysmex, Japan). Estimated RIs are calculated by Bhattacharya method using open spreadsheet ( according to age (18~59 and 60~99 years) and sex. The locations of central bin were set as median values of raw data, and the size and number of bins were adjusted for determining the line of best fit (r2 >0.99).
Results: The summary of estimated RI are presented at Table 1. The upper and lower limits of estimated RIs were lower than them of current RIs, except hemoglobin concentration assay in male adult group.
Table 1. Indirect reference intervals of hematological tests estimated from patient data
Paramter Current RI Age (year) N Estimated RI
Hb_Male (g/L) 13.0 ~ 17.5 18~59 95199 13.3~17.7
  60~99 55472 12.2~17.0
Hb_Female (g/L) 12.0 ~ 16.0 18~59 106894 11.3~15.3
  60~99 71461 11.2~15.2
WBC (109/L) 4.0 ~ 10.0 18~59 141500 3.31~8.39
  60~99 102045 3.38~8.14
Platelet (109/L) 150 ~ 450 18~59 136724 138~366
  60~99 97353 111~347
* RI, referece interval: Hb, hemoglobin; WBC, white blood cell  

Conclusions: Our analysis shows the estimated RIs acquired by indirect methods are slightly different with currently used RIs. Indirect estimation of RIs from clinical data may be useful, but more delicate techniques excluding pathological data are needed. 

Evaluation Of An Automated Alloantibody Titration System
Xavier Tejedor Gandux1, Alicia Martnez Iribarren1, Cristian Morales Indiano1, Eva Alonso Nogus2, Joan Ramon Grfols Ronda2, Ana Dueas Marcos1, Trinidad Lema Pual1, MJos Cnovas Bueno1, MAntnia Llopis Daz1
1CoreLab-Hematology. Department of Clinical Analysis and Biochemistry. Laboratori Clnic Metropolitana Nord (LCMN). Hospital Universitari Germans Trias i Pujol, Badalona, Spain, 2Banc de Sang i Teixits. Hospital Universitari Germans Trias i Pujol, Badalona, , Spain

Introduction: The titration of an alloantibody to a red cell antigen is a useful semi-quantitative screening tool that, when compared to a previous titer performed by the same technique, can detect an increased production of maternal antibody during pregnancy. This study evaluates a new automated alloantibody titration system, IH-500 System (BioRad) by comparison with the manual standard technique in column gel card, assessing the accuracy and concordance with this established manual reference method
Methods: Prospective study in which a total of 43 samples of patients, from the Blood and Tissue Bank, were processed, which contained some irregular antibody for which the prior identification and manual titration were carried out. They were then processed by afully automated random access system for  ABO blood grouping, reverse testing, phenotype, Rh-subgroups, single antigen testing, direct AHG testing (DAT), antibody screening, antibody identification and antibody titration. Each sample was tested a total of 3 times, on different days, by each method for precision as an indicator of accuracy. Samples were stored frozen between test days. For the statistical analysis, the assessment of reproducibility for variables with more than two categories was used with the weighted kappa index.
Results: The precision of repeat titer results performed in the automated system was slightly more consistent (p = 0.0095) when compared to manual standard titration. There was no difference in titer in 26 of 43 samples (60,5%) when testing was performed by automated and manual method, while 17 of 43 samples (39,5%) showed a difference in titer of 1 dilution when repeat testing was performed using manual standard titration. The assessment of agreement for variables with more than two categories that uses the weighted kappa index, showed values of 0.9304 (0.8979 to 0.9629) (p=0.0000)
Conclusions: Although the titers in the automated system could be slightly higher than manual standard method, performing antibody titration using this automated technology presents acceptable values in terms of precision and agreement with the manual reference method.

A Web-Based Tool For Generating Bone Marrow Synoptic Reports
Brian Wong, Elona Turley
University of Alberta, Edmonton, AB, Canada

Introduction: Synoptic reporting is increasingly prevalent in pathology. However, there is a paucity of tools available for composing synoptic bone marrow reports. Our objective was to develop an easily accessible and efficient tool for generating synoptic-style bone marrow reports as part of a quality improvement project in our division. We used the PDSA (Plan, Do, Study, Act) methodology to systematically incorporate user feedback in each iteration of the tool.
Methods: Using standard Web technologies and libraries (HTML5, CSS, JavaScript/jQuery and Bootstrap), we created an interactive tool named the Edmonton Zone Standardized Marrow Reporting Tool (EZ-SMaRT) which is optimized for Google Chrome. Initial planning included consultation with representatives from laboratory leadership, potential hematopathologist users, and information technology. Typical reporting workflow patterns were examined.EZ-SMaRT incorporates our local bone marrow synoptic-style template and features numerous input types, including dropdown lists, radio buttons, checkboxes and free text (Figure 1), increasing efficiency and flexibility for pathologists. User input is autosaved and encrypted. The tool prevents entry of uniquely identifying patient information with warnings and data pattern recognition. Access is restricted to authorized users on a secure network drive. As part of the quality improvement process, we distribute feedback surveys to hematopathologist users following each EZ-SMaRT release, and make improvements accordingly.
Results: EZ-SMaRT is routinely used for bone marrow cases by most hematopathologists in our division, with feedback cycles resulting in numerous improvements (Table 1). In feedback surveys, frequent users note that it is faster than dictation, more stable and robust than other programs, and generates satisfactory synoptic output. The non-technical barrier most frequently mentioned was preference for an in-person demo prior to using EZ-SMaRT, which led to the creation of instructional videos in a subsequent release. A small subset of users commented that installing Google Chrome was a technical barrier; detailed installation instructions were consequently added.
Conclusions: We created a robust, practical and secure Web-based synoptic reporting tool for bone marrow specimens. Using the PDSA methodology, we distributed feedback surveys following each release, incorporated user feedback, and documented user responses to changes. This approach has contributed to the widespread adoption of EZ-SMaRT within the division, and is applicable to other pathology informatics projects. A future version of the tool integrates ancillary study results to facilitate acute leukemia classification and report amendment.

Flow Cytometry- And Sysmex Hematology Analyzer-Based Determinations Of Cell Counts In Bone Marrow- And Apheresis-Harvested Hematopoietic Stem Cell Transplant Products Demonstrate Inter-Platform Correlation.
Eric McGinnis1, Alastair K. Williams1, Suzanne M. Vercauteren1,2, Kirk R. Schultz1,2, Kate M. Chipperfield1,2
1University of British Columbia, Vancouver, BC, Canada, 2British Columbia Children's Hospital, Vancouver, BC, Canada

Introduction: Hematopoietic stem cell transplantation (HSCT) is a potentially life-saving therapy for malignant and non-malignant diseases. Cellular donor products for HSCT are derived from umbilical cord blood, bone marrow, or mobilized peripheral blood apheresis. HSCT outcomes have been shown to depend on cellular content of infused products, particularly the per-kilogram dose of CD34-positive (CD34+) and total nucleated (TNC) cells. Enumeration of CD34+ and TNC is readily accomplished by flow cytometry (FC), but this method requires specialized technologist training, is relatively expensive and time-consuming, and may not be available outside routine laboratory hours. Automated hematology analyzers provide rapid TNC measurement, have lower operating costs, and have less need for specialized technologist training. We sought to determine the correlation between cell counts determined by FC and those measured by Sysmex XE (SXE) or XN (SXN) series hematology analyzers.
Methods: PB- and BM-source HSCT products processed at our pediatric institution for first HSCT during a five-year period were retrospectively reviewed, documenting TNC determined using SXE or SXN instruments (STNC) and FC measurements (using commercial Stem-Kit reagents) of TNC (FTNC), mononuclear cells (FMNC), and CD34+ cells (FCD34). Pearson product-moment correlation coefficients were computed to assess rel