8:10

Laboratory & Medicine 2.1: Care For What You Wish For
Marcel Levi
AmsterdamUMC

Over the past few decades, healthcare has made remarkable advances. Across nearly all fields, diseases are better understood, and diagnostic and therapeutic options have expanded dramatically. Laboratory medicine is also developing at a rapid pace and is playing an increasingly central role in the diagnostic process, as well as in the monitoring of therapy and disease progression.For many conditions—including cardiovascular disease and cancer—patient outcomes have improved spectacularly.

However, this success has also created new challenges. Death from acute illness has increasingly been replaced by survival with chronic disease. An aging—though not necessarily healthy—population demands substantial personal, financial, and organizational support. Hyperspecialization is poorly suited to meeting these complex needs. Moreover, efficiency-driven centralization of care, encompassing both clinical services and laboratories, may be necessary but often introduces new problems that can only be addressed through innovation and technology.

Most importantly, strong professional leadership is essential. With healthcare professionals in the lead, we can ensure a resilient and sustainable healthcare system, supported by high-quality laboratory services, in which complex issues such as superspecialization, centralization, and efficiency are addressed in an optimal and integrated manner.

8:40

Hemoglobinopathies And Molecular Diagnosis
Cornelis L. Harteveld
Associate Professor at the Dept. of Clinical Genetics, Leiden University Medical Center

Presentation Topic: Laboratory diagnosis of Hemoglobinopathies and the role of Next Generation Sequencing

Cornelis L. Harteveld, PhD, Assoc. prof. Dept. of Clinical Genetics/Genome Diagnostics, Reference Lab of the Hemoglobinopathy Expert Center, Leiden University Medical Center.

Abstract

Introduction: Hemoglobinopathies are the most common monogenic disorders in human with an ever-increasing global disease burden. Thalassemia is characterized by reduced synthesis of the globin chains of hemoglobin, specifically beta-globin chains in beta-thalassemia and alpha-chains in alpha-thalassemia. Over a thousand Hb variants have been described in the HBA1/2 and HBB genes of which the majority is silent, however approximately one third is clinically relevant, the most abundant worldwide giving Sickle Cell Disease (SCD) as the major health problem in areas endemic for malaria tropica. As most hemoglobinopathies show recessive inheritance, carriers are usually clinically silent. Cases will be discussed, showing that despite the great technological advances in mutation detection, the screening of hemoglobinopathies still requires the combined use of hematologic, biochemic and molecular techniques to achieve an accurate diagnosis.

Methods: Laboratory hematology and biochemical analysis are standardized methods able to reveal carriers of sickle cell-, alpha- and beta-thalassemia. The latest technological advance in DNA variant analysis involves Next Generation Sequencing (NGS), which is progressively replacing Sanger sequencing as ‘gold standard’ in the molecular genetic lab. A great advantage is that NGS applications such as Inherited Disease Panels (IDP), Whole Exome Sequencing and Whole Genome Sequencing (WGS) allow the simultaneous analysis of multiple genes involved in hemoglobinopathies and has led to new insights in disease mechanisms.

Results: The use of WES has recently uncovered a novel disease gene SUPT5H causing a beta-thalassemia trait phenotype in individuals and families with normal HBB genes. The breakpoint sequences of multiplications and deletions of the alpha-globin gene cluster in thalassemia carriers have been elucidated using WGS. Both WES and WGS showed de novo variants explaining hemolytic anemia in individuals of families without a genetic history of anemia. During the presentation, an overview will be given of the basic requirements for adequate carrier diagnostics, the importance of genotype-phenotype correlation and how this may lead to the discovery of exceptional interactions causing a clinically more severe phenotype in carriers.

Conclusions: The discovery of disease modifiers and their influence on disease severity plays a major role in defining a better genotype-phenotype correlation and a better understanding of how variants in different genes interact. Eventually this may contribute to improvement of counselling and disease prognosis.

9:10

Laboratory Diagnosis, Monitoring And Management Of Pnh
Morag Griffin
St James Hospital, Leeds, UK

The laboratory diagnosis and monitoring of paroxysmal nocturnal haemglobinuria (PNH): Invited speaker for plenary session.

This presentation will cover pathophysiology of PNH, presentation including symptoms and laboraroty diagnostic tests, diagnosis and briefly treatment including indications based on symptoms and lab values. Monitoring of patients with PNH and how to use laboratory tests for indications to change treatment. Case studies will be included

10:15

Limits Of Haemostasis Assays
Kieron Hickey
Sheffield Haemophilia and Thrombosis Centre.

Coagulation assays remain central, alongside the clinical interpretation to diagnose haemostatic disorders, both thrombotic and bleeding. However, laboratory interpretation is often hindered by biological variability, reagent and platform related differences, alongside inadequate clinical context. This presentation provides an overview of the limitations inherent in commonly used coagulation tests and highlights how these constraints affect clinical decision making.

Despite their widespread use, routine clotting assays particularly the activated partial thromboplastin time (APTT), have significant and often under‑recognised limitations. The widespread inappropriate use of coagulation screening tests as screening tools for preoperative assessment and bleeding disorder investigations coupled with the poor correlation between laboratory abnormalities and clinical phenotype highlight key limits of the most commonly ordered coagulation investigations. Issues related to different analysers and associated reagents variable factor deficiency sensitivity, preanalytical variables such as sample quality, delays in testing, HIL , storage conditions, and transport contribute substantially to result variability and potential misinterpretation.

The session will also cover the growing complexity introduced by Xa based -direct oral anticoagulants (DOAC) in result interpretation. DOAC, heparins, direct thrombin inhibitors and emerging FXIa inhibitors all introduce unpredictable assay specific effects that can obscure true haemostatic status and limit the usefulness of traditional coagulation tests. Alongside this we have emerging strategies such as DOAC removal techniques to tackle such interference.

By establishing robust preanalytical sample policy, local validation of assay performance, laboratories can better understand the limitations of haemostasis assays that is essential to avoid misinterpretation and ensure safe, effective patient care.

10:45

Combining Functional Tests And Genetics For Inherited Thrombophilia Diagnosis
Christine Van Laer
Department of Laboratory Medicine, AZ Groeninge, Kortrijk, Belgium

Thrombotic events, including venous and arterial thrombosis, represent a major public health burden. Venous thromboembolism (VTE) affects approximately 2 per 1,000 individuals annually and arises from a multifactorial interplay between genetic and acquired risk factors. Thrombophilia encompasses inherited and/or acquired risk factors associated with an increased risk of thrombosis. Conventional thrombophilia testing focuses on deficiencies of the natural anticoagulants protein C, protein S, and antithrombin, as well as procoagulant variants such as factor V Leiden (FVL) and prothrombin G20210A.

For several years, multigene panel testing has became available for assessment of inherited thrombophilia. This genetic approach can lead to a more accurate diagnosis with a personalized approach in risk stratification, treatment, and genetic counselling. Given the complexity of VTE, stringent inclusion criteria are recommended when selecting patients for multigene panel testing.

In a cohort of up to 650 unrelated patients with suspected inherited VTE or thrombophilia, genetic testing yielded an overall diagnostic rate of 54%. As expected, the majority of pathogenic variants were identified in genes F5, PROS1, PROC, F2, and SERPINC1, including FVL and prothrombin G20210A. Notably, oligogenic inheritance was observed in 24% of patients, consistent with the multifactorial nature of thrombosis. Importantly, conventional laboratory assays for antithrombin activity, protein C activity, and free protein S antigen levels failed to detect several cases of natural anticoagulant deficiencies.

Multigene panel testing is finding its way into clinical practice, but those results emphasize the necessity for recommendations on how and when to perform genetic testing. Identifying individuals with an increased thrombotic risk can help guiding targeted thromboprophylaxis and improved prevention of VTE. Ultimately, optimal patient management requires an integrated assessment of both functional and genetic testing and patient risk factors.

11:15

Developmental Hemostasis
Ulrike Nowak-Goettl
Institute of Clinical Chemistry, University Hospital Kiel, Germany

Absolute values of reference ranges for coagulation assays in humans vary within the entire lifespan and confirm the concept of developmental hemostasis. It is known that physiologic concentrations of coagulation factors (F) gradually increase over age: they are lower in premature infants as compared to full-term babies, healthy children or adults. Additionally, the process of developental hemostasis is ongoing during adulthood and in the eldery. From adolescents, young adults to the elderly there is a further increase of F, reaching significance starting between 35 - 50 years of age compared to younger subjects. Covering the entire lifespan FVIII and von-Willebrand-factor showed the lowest values in carriers of blood group “0”. Apart from pregnancy differences related to gender, pill users, smoking habits or the presence of thrombophilic variants were reported. Laboratory test results should be compared to age-related reference intervals when hemostatic defects are suspected to avoid misclassifications as being “healthy”, prone to “bleeding” or vice versa to “thrombosis”.

10:15

Update On Mrd In Acute Myeloid Leukemia: A Consensus Document From The European Leukemianet Mrd Working Party
Jacqueline Cloos
AmsterdamUMC

10:45

Lsc Mrd In Aml
Veronika Ecker
MLL Munich Leukemia Laboratory GmbH (MLL)

LSC MRD in AML
Acute myeloid leukemia (AML) measurable residual disease (MRD) monitoring is indispensable for risk stratification, treatment guidance and as a clinical trial endpoint. The 2025 update of the ELN MRD guidelines provide enhanced recommendations for molecular and multiparameter flow cytometry (MFC) MRD. Taking into consideration AML heterogeneity, subgroup-specific guidance, low-level- and qualitative MRD response catogories have been adapted assay- and timing-specific, to facilitate the adoption of MRD monitoring into routine clinical practice.

Conventional blast‑focused MFC MRD may underestimate the persistence of therapy‑resistant disease, as relapse is thought to arise from a rare, quiescent leukemic stem cell (LSC) compartment. LSC–based MFC MRD assessment is emerging as a complementary tool to conventional MRD strategies and may refine current ELN guidelines in the future. Incorporating LSC MRD into established MFC and molecular MRD frameworks could help to better capture relapse biology and guide future therapeutic decisions. LSC measurement enables superior sensitivity than bulk MFC MRD, with detection thresholds of 0.005% when at least 1 million CD45-expressing cells are acquired, and retrospective and prospective studies at diagnosis and during remission have shown its independent prognostic value (early MRD kinetics, relapse and survival). Phenotypically aberrant LSCs can persist in morphologic or even MRD‑negative complete remission, indicating a biologically distinct layer of residual disease.

LSCs are typically defined within CD34⁺CD38⁻ compartments and can be distinguished from hematopoietic stem cells (HSC) by aberrant leukemia‑associated immunophenotypes (e.g. CD123, CD45RA, CD7, CD56, CLL-1, TIM-3). As up to ~20% of AMLs present CD34-negative, strategies for potential CD34-negative and/or monocytic LSC detection have to be established accordingly (e.g. CD117 positivity or markers of aberrancy on immature cells). Further work is needed and undertaken by the ELN LSC subgroup, to prepare LSC monitoring for clinical decision-making with robust prognostic cut-offs, particularly for specific risk groups or in the context of new therapeutic landscapes. LSC‑directed flow cytometric MRD analysis needs to be harmonized technically (marker and fluorophore selection, instrumentation, gating strategies, cut-off definitions) and biologically (prognostic relevance at diagnosis, early time points and in remission). Published protocols that enable standardized, reproducible LSC MRD detection and first retro- or prospective studies form the basis for this.

Apart from assessment of LSC kinetics, monitoring of expression levels of specific LSC therapy-targets during the course of targeted therapy provide valuable information. Therefore, we have established a flexible LSC backbone tube (dried-down) that allows us to examine different surface markers on LSCs using distinct drop-in antibodies. Validation of such a backbone panel ensures stability and reproducibility over distinct time-points and entities.

Together, these advances suggest that LSC‑inclusive MRD assessment has the potential to move the field from blast‑centered to stem cell‑oriented response definitions in AML.

11:15

Flow Cytometry Detection Of Mrd In Myeloma: Update
Bruno Paiva
Clinica Universidad de Navarra

Measurable residual disease (MRD) assessment is, from the methodological point of view, ready for the prime time in multiple myeloma (MM). Abundant evidence underscores the value of MRD status determined using highly sensitive next-generation flow cytometry and next-generation sequencing tests in evaluating response to treatment and, therefore, prognosis in patients with this disease. MRD response assessment and monitoring might present a range of opportunities for individualized patient management. Moreover, the considerable amounts of high-quality and standardized MRD data generated in clinical trials have led to the acceptance of MRD negativity as an early end point for accelerated regulatory approval of treatments for MM. The data leave no doubt that the efficacy of new regimens in inducing deeper and durable MRD-negative responses is interconnected with prolonged survival. Yet, several evidential, technical and practical challenges continue to limit the implementation of MRD-guided treatment strategies in routine practice, and the use of MRD as a surrogate end point remains controversial to some. In my presentation, I will draw on past and present research to propose opportunities for overcoming some of these challenges, and to accelerate the use of MRD assessment for improved clinical management of patients with MM.

1:15

Berend Houwen Lecture:�Laboratory Monitoring Of Unfractionated Heparin Treatments &Ndash; Unanswered Issues
Pierre Toulon
Nice University Hospital, Dept of Hematology, , France

Laboratory Monitoring of Unfractionated Heparin Treatments – Unanswered issues

Pierre Toulon

Unfractionated heparin (UFH) has long been the anticoagulant of choice. Despite the increasing use of low molecular weight heparin derivatives and more recently direct oral anticoagulants, UFH is still prescribed to prevent or treat thrombosis in specific populations at high risk of thrombosis or bleeding, including patients with severe renal impairment, critically ill patients, or those undergoing cardiac catheterization/surgery. Due to its narrow therapeutic index, with a risk of hemorrhage in case of overdosing and a risk of thrombosis in case of underdosing, monitoring its anticoagulant activity is considered standard of care even though scientific data remains limited. Despite being used for decades, some unanswered questions remain, particularly regarding the biological monitoring of its efficacy.

1. Which assay should be used? In practice, the most widely used tests are plasma-based, such as the aPTT, a clotting assay, or the anti-Xa activity, a chromogenic substrate-based assay. Whole blood tests such as the ACT are limited to specific situation such as extracorporeal circulation.

2. Which therapeutic ranges should be used? An aPTT prolongation of 1.5-2.5 x control, or an anti-Xa activity of 0.30–0.70 IU/mL, corresponding to 0.2-0.4 U/mL by protamine titration, ranges are commonly used, despite limited evidence based. Furthermore, these assays are not standardized due to some degree of heterogeneity in the sensitivity of reagents to UFH. The calculation of aPTT prolongation and the definition of the control value (mean aPTT in healthy subjects or baseline aPTT in the patient) must be adapted to the technical conditions (reagents, analyzers). Although anti-Xa activity was supposed to be more standardized, with commercially available calibrators traceable against the international standard of UFH, recent studies clearly demonstrated significant deviations from the target values with some reagents. In addition, the composition of the commercially available kits is also heterogeneous, with or without the addition of exogenous antithrombin or dextran sulphate, a chemical intended to release UFH from its form bound to e.g., PF4.

3. Which validated nomograms for dose adjustment, based on aPTT or Anti-Xa should be used?

4. Moreover, some pre-analytical questions remain, particularly regarding the collection tube to be used e.g., citrate or CTAD anticoagulant solution, full- or partial-draw (half filled) tubes, and the maximum time between collection and analysis: 1 or 4 hours.

Finally, yet importantly, the question of the actual need for monitoring of SC administered full dose UFH has been raised, as suggested by the results of the FIDO study.

2:00

Wallace H Coulter Lecture:�Investigator-Led Research To Improve The Diagnostic Assessment Of Platelet Function Disorders: Reflections On The Challenges And Rewards
Catherine Hayward
McMaster University

Investigator-led research to improve the diagnostic assessment of platelet function disorders: reflections on the challenges and rewards

Catherine P. M. Hayward, McMaster University

Introduction: Investigator-led research and quality improvement initiatives have led to important improvements in the diagnostic assessment of platelet function disorders (PFD).
Methods: Personal reflections were used to summarize our contributions to knowledge on PFD diagnostic assessment, pathogenesis and bleeding risks.

Results: Light transmittance platelet aggregometry (LTA), and whole mount electron microscopy assessment for platelet dense granule deficiency (DGD) both detect abnormalities that are highly predictive of a bleeding disorder. Observations on LTA findings that are predictive of a bleeding disorder (including those specific to certain conditions), have been incorporated into guidelines to reduce LTA interpretation errors. Efforts to improve LTA assessment of PFD with thrombocytopenia (e.g., Bernard Soulier syndrome) have led to validated, trustworthy, diagnostic procedures. Commonly encountered PFD that manifest with abnormal aggregation responses to multiple agonists, and/or DGD, are now established to have significantly increased bleeding risks, emphasizing the need for diagnosis and treatment. Unraveling of the molecular pathogenesis of some specific PFD have provided important insights and simplified diagnosis. In the case of Quebec platelet disorder (QPD), the pathogenesis is a unique, gain-of-function defect in fibrinolysis, from a mutation that repositions a megakaryocyte-specific enhancer that normally upregulates VCL expression during megakaryopoiesis, and “rewires” PLAU, increasing its expression >100-fold in megakaryocytes only. Presently, the molecular causes of many “commonly encountered” PFD awaits elucidation.

Conclusions: Research has meaningfully improved diagnostic laboratory testing for PFD. Unraveling the causes of “commonly encountered” PFD will be important to understanding their pathogenesis and increasing the yield of diagnosis by genetic investigations.

3:30

Blood Rheology Biomarkers In Sickle Cell Disease
Minke Rab
Erasmus University Medical Center Rotterdam

Sickle cell disease (SCD) is characterized by red blood cell (RBC) sickling, impaired deformability, and microvascular obstruction. Recent methodological advances—particularly oxygen‑gradient ektacytometry—have enabled precise, reproducible quantification of sickling physiology. This technique measures RBC deformability across an oxygen gradient, generating parameters such as the Point of Sickling (PoS), EImax, and EImin. These biomarkers reliably capture patient‑specific sickling thresholds and correlate strongly with physiological modulators of oxygen affinity.

Clinical studies show that oxygen‑gradient‑derived biomarkers are associated with major complications including cerebral infarction, acute chest syndrome, and vaso‑occlusive crises. They also demonstrate sensitivity to disease‑modifying therapeutics such as hydroxyurea and anti-sickling agents, reflecting improved deformability and shifts in PoS. Further work highlights their utility for individualized treatment optimization and for ex vivo evaluation of emerging therapies, including pyruvate kinase activators. Complementary microfluidic assays assessing RBC adhesion expand the biomarker repertoire by capturing adhesive and obstructive RBC phenotypes linked to hemolysis and iron overload.

Together, these integrated approaches establish deformability‑based and adhesion‑based functional assays as clinically relevant biomarkers. They offer prediction of disease severity, more precise monitoring of therapeutic response, and a foundation for advancing personalized medicine in sickle cell disease.

4:00

Laboratory Diagnosis Of Pyruvate Kinase Deficiency
Archana Agarwal
University of Utah/ARUP Laboratories

4:30

Implementation Of Ai Algorithms For Laboratory Workup Of Microcytic Anemias
Veroniki Komninaka
G.Gennimatas, General Hospital, Athens, Greece

Microcytic anemias, including iron deficiency anemia (IDA) and thalassemia, represent a significant diagnostic challenge in clinical practice due to overlapping hematological features. Traditional diagnostic approaches rely on complete blood count (CBC) parameters and confirmatory laboratory tests, which can be time-consuming, costly, and sometimes inconclusive.

The implementation of artificial intelligence (AI) algorithms has emerged as a promising solution to enhance the efficiency and accuracy of laboratory workup for microcytic anemias. Machine learning (ML) and deep learning (DL) models can analyze large volumes of hematological data, including red blood cell (RBC) indices and medical images such as peripheral blood smears, to support differential diagnosis.

Recent studies demonstrate that AI-driven approaches can improve diagnostic performance, reduce dependency on expensive confirmatory tests, and assist clinicians in decision-making.

Despite these advancements, challenges remain, including limited availability of annotated datasets, variability across populations, and the need for model interpretability. Future research should focus on developing robust, generalizable, and explainable AI systems to enable reliable integration into routine clinical workflows

3:30

Machine Learning, Anti-Cancer Immunotherapy In Hematology
Pieter Meysman
University of Antwerp

The human immune system is our first and last line of defense against the development of cancer cells. The novel generation immunotherapies recruit T-cells to specifically seek out and destroy aberrant cells. However the immune system is highly complex and highly individual.
In this talk, I will explain how we are using AI to decode the T-cell space, both its relevant clonotypes and its targets, to gain understanding in the mechanisms of action of immunotherapies, and reveal some people respond better than others.

4:00

Single-Cell Analysis, Tcr, Functional Diversity
Paul Klenerman
University of Oxford

Single-cell RNA sequencing technologies (sc-RNAseq) have had a massive impact on immunology, with the ability to resolve multiple cell types and cell states. In this talk I will give an overview of these approaches in studies of T cell subsets (both conventional and unconventional T cells)and some examples of how they can be used to define

1. Cell states ex vivo e.g. acute disease and memory

2. Cell functions after specific stimulation – including assays for antigen specific T cells using functional scRNASeq

3. Relationships between TCR usage and cell state/function.

My lab is particularly interested in abundant unconventional T cells such as MAIT cells, so these will be discussed along with antigen specific CD4+ and CD8+ T cells.

4:30

Whole Genome Analysis
James Allan
Newcastle University

Whole genome analysis of human leukaemia – a tale of two genomes

James M. Allan, Translational and Clinical Research Institute, Newcastle University, Newcastle upon Tyne.

The pathogenesis of human leukaemia is determined by complex interplay between the constitutional and somatic genomes. Interrogation of these genomes using nanoscale technologies such as high-density microarrays or next generation sequencing has significantly informed our understanding of how and why leukaemia develops. In this presentation I will discuss how whole genome analysis of the constitutional genome has led to the identification of genetic risk variants for human leukaemia which inform on the early stages of disease development. I will also discuss how whole genome analysis of the somatic leukaemia genome can inform on the latter stages of disease development and how further exploitation of whole genome technologies has the potential to identify novel therapeutic targets and improve outcomes for patients.

P100

New Approach On Testing Platelet Function In Clinical Practise
Mohammad M Algahtani
Security Forces Hospital Makkah , MAKKAH, Saudi Arabia

Background: Traditional techniques used to assess platelet function typically require large volumes of blood, making them unsuitable for settings in which sample volume is limited or when dual functional assessments are needed. To overcome this limitation, we developed a miniaturized method based on a 96-well plate format, enabling the simultaneous evaluation of platelet aggregation and platelet–leukocyte conjugate (PLC) formation using very small quantities of whole blood. Methods: Whole blood was collected from healthy volunteers and added to 96-well plates pre-coated with increasing concentrations of collagen (0.1–10 µg/mL) or vehicle control. Platelet aggregation was assessed in duplicate wells to ensure reproducibility. Samples were subsequently fixed using a stabilizing solution (AGGFix), allowing storage of prepared samples for up to nine days prior to flow cytometric analysis at a central facility. The performance of the dual assay was evaluated by examining the effects of two inhibitors: MK-0852, a GPIIb/IIIa antagonist that blocks platelet aggregation, and KPL-1, a PSGL-1 inhibitor that selectively blocks PLC formation. Results: Platelet aggregation measurements were robust and reproducible, with coefficients of variation consistently <10%. MK-0852 produced strong inhibition of aggregation at low collagen concentrations, as expected. However, at higher collagen concentrations, aggregation levels were unexpectedly reduced rather than increased. This decrease corresponded with a marked rise in PLC formation. PLCs were readily and reliably quantified from the same fixed blood samples used for aggregation assessment. Treatment with KPL-1 significantly reduced PLC formation, confirming the assay’s ability to detect inhibition of platelet–leukocyte interactions. In contrast, platelet activation in the presence of MK-0852 resulted in a substantial increase in PLC formation, demonstrating that blockade of aggregation under certain activation conditions promotes conjugate formation. Conclusion: This newly developed 96-well plate assay enables parallel assessment of platelet aggregation and PLC formation from the same low-volume whole blood sample. The method is reproducible, compatible with delayed flow cytometry analysis, and sensitive to pharmacological modulation of both platelet aggregation and platelet–leukocyte interactions. Its minimal blood volume requirements make it particularly advantageous for research involving pediatric subjects, individuals with limited sample availability, or studies requiring repeated sampling. This approach represents a practical and versatile alternative to conventional high-volume platelet function assays.

P101

Integrating Monocyte Distribution Width (Mdw) Into Routine Cbc Reporting For Early Sepsis Detection: A Systematic Review
Mohamed Al Zaabi MD, FRCPath¹, Amal Al Jabri MD, FRCPath²
¹Department of Hematology and Blood Bank, Khoula Hospital , Muscat, , Oman, ²Department of Microbiology, Khoula Hospital, Muscat , , Oman

Introduction: Timely recognition of sepsis remains challenging in emergency and acute care settings, where diagnostic delays directly worsen outcomes. Monocyte Distribution Width (MDW), a morphometric parameter automatically reported with the complete blood count (CBC), reflects early innate immune activation through shifts in monocyte size heterogeneity. Because MDW is immediately available, requires no additional testing, and is analytically stable across DxH hematology platforms, it has emerged as a practical candidate for real-time sepsis screening. Methods: A systematic search of PubMed, Embase, Scopus, and Cochrane through October 2025 identified clinical studies assessing MDW for early sepsis detection in adults, following PRISMA principles. Studies were included if MDW was measured at presentation, Sepsis-2 or Sepsis-3 criteria served as the reference standard, and extractable diagnostic performance data were available. Eighteen studies met screening criteria, and six (n = 38,956) fulfilled full eligibility. Study characteristics, analytical factors, cut-point ranges, and diagnostic accuracy were examined. Heterogeneity was evaluated qualitatively, and overall diagnostic performance was interpreted using a summary ROC framework. Results: Across eligible studies, MDW demonstrated consistent diagnostic accuracy, with sensitivity 0.79-0.84, specificity 0.63-0.78, and AUC values 0.79-0.84. Optimal thresholds clustered tightly around a 20-21 MDW-unit cut-point, indicating biologically and analytically stable performance. Variability across studies was modest and primarily clinical rather than analytical. All six studies used DxH analyzers and EDTA-based anticoagulants (K₂ or K₃), and none of these factors meaningfully affected MDW performance. Compared with C-reactive protein at initial assessment, MDW showed superior early discrimination and demonstrated accuracy comparable to procalcitonin. High sensitivity across studies suggests MDW may be particularly useful for excluding sepsis in lower-prevalence settings. Summary ROC analysis yielded pooled AUC values of 0.80-0.83 with compact clustering, consistent with reproducible diagnostic performance. Conclusions: MDW is a rapid, cost-neutral biomarker that can strengthen early sepsis detection when integrated into routine CBC reporting. Its consistent accuracy, stable cut-point range, and favorable comparison with CRP and procalcitonin support a potential role for MDW in emergency and acute care settings. These findings justify further evaluation in large prospective trials, including real-world implementation within CBC-based sepsis screening pathways.

P103

Reticulocytes Performances And Study Of Flag Of Clinical Usefulness, Sthema 601 Evaluation
N. Banh¹, B. Butt¹, C. Doinel¹, V. Nadal¹, T. Nguyen¹, D. Paulin¹, C. Ranaivoarimalala¹, S. Melin², E. Tournier², Dr. I. Habes Lenouar¹, Dr. C. Brumpt¹
¹Hematology laboratory, University Hospital Lariboisière, Paris, , France, ²Diagnostica Stago, Asnières sur Seine, , France

The Hematology Laboratory of Lariboisière University Hospital in Paris, evaluated the reticulocyte performance, CBC analytical reliability, and clinical usefulness of morphological flags on the sthemA 601 hematology analyzer. A total of 192 patient samples, including pediatric specimens, were analyzed over four weeks. Results across the analyzer’s three modes CBC, RET, and DIR (CBC + RET), were compared with those of the reference system, Sysmex XN‑3000. Reticulocytes play a crucial role in assessing anemia regeneration, particularly in normocytic and macrocytic anemia. On the sthemA 601, reticulocyte enumeration uses impedance, light extinction, and fluorescence, ensuring optimal precision. For each sample, duplicate stained smears (MGG and Cresyl Blue) were reviewed by experienced technicians. Precision, comparison, and carry‑over studies were conducted following CLSI guidelines. An ergonomic questionnaire was also completed by laboratory staff. The study confirmed that reticulocyte and CBC results from sthemA 601 are comparable to those of XN‑3000. Precision was satisfactory: 20‑day controls showed acceptable CVs for all parameters except monocytes and basophils, whose low values make CVs non‑informative. Repeatability testing on eight samples across defined ranges demonstrated consistent performance, and all within‑run precision criteria were met. Carry‑over testing using high‑ and low‑level sample pairs met specifications for WBC, RBC, hemoglobin, platelets, and reticulocytes, demonstrating no significant contamination. Accuracy was validated across CBC, RET, and DIR modes using Passing–Bablok regression, with all parameters within expected values except MPV and PDW, known to vary across technologies. Reticulocyte percentage occasionally deviated from expected values, but this was acceptable given the wide inter‑instrument variation observed in external comparisons and the clinical consistency of individual cases. Twenty‑seven pediatric samples across four FDA‑defined age groups were included to confirm accuracy in small‑volume specimens. A total of 192 samples were used to assess the clinical relevance of morphological flags. Microscopic review determined a sensitivity of 81.6%, specificity of 91.7%, and overall agreement of 86.9%. While compliant with expectations, improved sensitivity is recommended to facilitate interpretation and validation. Several users evaluated the analyzer, noting its ease of use, intuitive software, compact design, and safety features. The upcoming autoloader option was seen as a major improvement, providing 30‑tube autonomy and consistent sample mixing. Minor software enhancements were suggested, mainly regarding icon placement and QC curve display. The sthemA 601 demonstrates strong analytical performance for reticulocytes and CBC, reliable flagging capability, and excellent ergonomics, making it a robust solution for routine hematology workflows.

P104

Evaluation Of Flagging Performance For Pathological Cell Detection On The Mindray Bc-7800 Versus Siemens Advia 2120I Using Microscopy As The Reference Method
Vincenzo De Iuliis, Mario Forcella, Cristina Di Saverio, Giusy Bove, Sofia Chiatamone Ranieri
Department of Clinical Pathology, G. Mazzini Civil Hospital, Teramo, Italy

Introduction. Rapid and reliable detection of pathological cells is crucial in hematology, where timely identification of blasts, immature myeloid precursors, nucleated red blood cells (NRBCs), and atypical lymphocytes directly influences diagnostic urgency and downstream testing. Manual microscopic examination of the above-mentioned abnormal cells remains the gold standard. However,in high-volume laboratories, manual smear review is labor-intensive, making automated morphological flags crucial for effective triage. The performance of these flags varies across different analyzers [1–3]. The Mindray BC-7800 incorporates updated algorithms for abnormal cell detection, while the Siemens ADVIA 2120i is a well-established reference system. This study provides a focused, microscopy-validated comparison of their flagging accuracy, highlighting strengths, limitations, and opportunities for algorithmic improvement. Methods. Among 437 routine EDTA samples (74.6% pathological), a core set of 236 underwent full tri-modal assessment using: Mindray BC-7800, Siemens ADVIA 2120i, and manual optical microscopy by two expert pathologists (gold standard). Performance metrics (sensitivity, specificity, PPV, NPV, accuracy) were calculated for detection of blasts, immature myeloid cells, NRBCs, abnormal lymphocytes, and reactive lymphocytes. Results. Preliminary analysis on 236 samples showed heterogeneous performance across analyzers and cell categories. – Blasts: BC-7800 achieved a sensitivity of 62.5%, compared with 68.8% for ADVIA, while both instruments showed high specificity (>84%). – Immature myeloid cells: BC-7800 markedly outperformed ADVIA in sensitivity (61.0% vs 34.4%) with comparable specificity (≈95%). – NRBCs: BC-7800 achieved superior PPV than ADVIA (80.0% vs 36.8%) and excellent specificity (99.1% vs 94.4%), supporting more reliable abnormal flagging. – Abnormal lymphocytes: BC-7800 showed higher sensitivity than ADVIA (43.8% vs 25.0%) with similar specificity (86.8% vs 89.1%). – Reactive lymphocytes: available only on the BC-7800, the reactive-lymphocyte flag showed a sensitivity of 44.0% and high specificity (96.7%), consistent with the intrinsic heterogeneity of this population. Across categories, specificity remained consistently high for both analyzers, while BC-7800 generally yielded fewer false negatives for immature myeloid and atypical lymphoid populations. Conclusions. Preliminary microscopy-validated data show that automated morphological flags can effectively support abnormal cell screening, but sensitivity remains the key limiting factor. The BC-7800 demonstrates clear advantages in detecting immature myeloid and abnormal lymphoid forms, while ADVIA while ADVIA performed marginally better for blasts. These findings support targeted optimization of flagging algorithms and highlight the potential role of AI-enhanced decision tools to boost sensitivity, minimize unnecessary smears, and accelerate high-impact clinical triage in hematology.

P106

Hematologic Biomarkers To Support Procalcitonin Stewardship In Intensive Care Unit: Monocyte Distribution Width And Cbc-Derived Ratios
Laura García¹, Sandra Ramos², Jesús A. Álvarez², Antonia M. Mayol¹, Isabel Albalá¹, José C. Frías², Nicolás Suárez²
¹Department of Laboratory Medicine, Hospital Juaneda Miramar, Palma de Mallorca, Spain, ²Intensive Care Unit, Hospital Juaneda Miramar, Palma de Mallorca, Spain

INTRODUCTION Procalcitonin (PCT) is widely used in intensive care units (ICUs) to guide infection management, but overuse increases costs and leads to unnecessary testing. Hematologic biomarkers derived from the complete blood count (CBC-Diff), including monocyte distribution width (MDW) and CBC-derived ratios, are readily available and cost-effective. This study assesses the feasibility of using these markers to guide PCT testing in adult ICU patients, supporting diagnostic stewardship and optimizing resource use. METHODS A retrospective study was conducted including all adult ICU laboratory requests between 01/05/2024 and 30/04/2025 that included a CBC-Diff with MDW and PCT. K3-EDTA samples were analysed within 2 hours using a UniCel® DxH 900 (Beckman Coulter, USA). PCT was measured in serum on a DxI 800 (Beckman Coulter, USA). CBC-derived parameters and indices were assessed, including total leukocyte count (WBC), monocyte distribution width (MDW), neutrophil-to-lymphocyte ratio (NLR), systemic inflammation response index (SIRI), systemic immune-inflammation index (SII), and aggregate index of systemic inflammation (AISI) (see Table 1). Different combinations were evaluated to identify those achieving the highest negative predictive value (NPV) for infection. Selected parameters were used to develop a screening score to guide PCT testing. RESULTS 2,693 ICU laboratory requests from adult ICU patients, with or without infection, were analysed. The combination achieving the highest NPV included total leukocyte count (WBC), monocyte distribution width (MDW), and systemic inflammation response index (SIRI), using the following thresholds: WBC 4,000–12,000/µL, MDW <23.0, and SIRI <6.1×10³. A scoring rule was applied assigning 1 point for each parameter outside the normal range; samples with all parameters within range were assigned a score of 0, whereas samples with at least one abnormal parameter had a score ≥ 1. Of the 2,693 requests, 1,732 (64.31%) had a score ≥ 1, while 961 (35.69%) scored 0. Score-0 samples were classified into three subgroups: (G1) PCT <0.5 ng/mL (n = 845; 87.93%), (G2) PCT ≥ 0.5 ng/mL without infection (false-positive PCT results; n = 48; 4.99%), and (G3) PCT ≥ 0.5 ng/mL with confirmed infection (false-negative score result; n = 68; 7.08%). This later group yielded a NPV of 92.92%. CONCLUSIONS A scoring system based on WBC, MDW, and SIRI demonstrated a high NPV for infection, identifying patients at low risk in whom PCT testing may be omitted, thereby supporting diagnostic stewardship and rational use of laboratory resources in the ICU. In our cohort, it achieved a NPV of 92.92%.

P107

Use Of Monocyte Distribution Width (Mdw) For The Prediction Of Blood Culture Positivity In Emergency Department Patients
Elena Giberti¹, Irene Venturelli², Mario Sarti², Giovanni Riva¹, Vincenzo Nasillo¹, Valentina Pecoraro¹, Manuela Varani¹, Tommaso Fasano¹
¹Laboratory Medicine Unit, AUSL Modena, Modena, Italy, ²Microbiology Unit, AOU Modena, Modena, Italy

Introduction: Rapid identification of patients at risk of bloodstream infection (BSI) is essential in Emergency Department (ED) setting. Monocyte Distribution Width (MDW) is a hematological parameter automatically generated by DxH 900 haematology analyser (Beckman coulter, inc, Brea, CA, USA) from CBC-Diff analysis, reflecting the morphological heterogeneity of circulating monocytes. It has emerged as an early biomarker of sepsis. Its value for the prediction of bloodstream infections compared with commonly used biomarkers such as C-reactive protein (CRP) is still not completely understood. This study aimed to evaluate the diagnostic accuracy of MDW for blood culture positivity and analyze its behavior across different microbiological subgroups. Methods: We conducted a cross-sectional study including consecutive adults undergoing MDW plus CRP testing and blood culture (BC) sampling in a tertiary ED from March 2022 to December 2024. We compared values of MDW and CRP between culture-negative and culture-positive samplesand further stratified groups into Gram-negative, Gram-positive, anaerobic bacteria, and coagulase-negative staphylococci (CoNS). CoNS group includes patients whoresulted positive in a single set of BCs for a bacterial species that are considered as contaminating bacteria (CoNS, Corynebacterium spp, viridans streptococci, Bacillus spp, etc.). If these germs were confirmed in two or more sets of BC, the patients were included in Gram+ group. Diagnostic accuracy for MDW, CRP, and their combination was assessed using ROC curves and logistic regression models. Subgroup analyses were performed for blood culture positive for Gram-negative, Gram-positive, anaerobic, and coagulase-negative staphylococci (CoNS). Results: Among 3287 samples, 1360 (41.4%) were BC-positive, and 1927 (58.6%) were BC-negative. MDW values were significantly higher in BC-positive (mean 25.6±6.8) compared to BC-negative patients (n= 1927; mean 23.1±4.6) (p<0.001). MDW values were statistically higher in Gram-negative infections (n=471; mean 27.7±7.4), compared to Gram-positive (n= 299; mean 26.2±6.8), anaerobic infections (n=57; mean 25.2±7.8), and CoNS (n= 533; mean 23.3±5.1). When CoNS were excluded, MDW values further increased in clinically significant bacteremia(Table 1). ROC analysis demonstrated MDW had a good diagnostic accuracy (cut-off 24.5, AUROC 0.67 95%CI 0.65-0.68; SE 58%; SP 69%, NPV 79%) compared to CRP (cut-off 11.5, AUC 0.59 95%CI 0.57-0.61, SE 51%; SP 63%, NPV 75%). A combined MDW plus CRP model yielded a similar AUC to MDW alone (0.66 95%CI 0.65-0.68) (Figure1).

Conclusions: MDW is an early, widely available biomarker associated with blood culture positivity, outperforming CRP. The lack of significant improvement in blood culture discrimination from the combined model with both MDW and CRP underscores the dominant diagnostic contribution of MDW. In our cohort, MDW values in the CoNS group were comparable to those of BC-negative patients and significantly lower than those observed in confirmed Gram- and Gram+ bacteremia. This pattern strongly suggests that MDW correlates more closely with true systemic infection than with contaminant-positive cultures. Systematic use of MDW may support diagnostic stewardship and patient prioritization in ED workflows.

P108

Routine Blood Count&Ndash;Derived Markers In Patients With Mycosis Fungoides
Berrak Guven¹, Murat Can¹, Nur Cansu Erken¹, Emel Hazinedar²
¹Department of Biochemistry, Bülent Ecevit University, Zonguldak, , Turkey, ²Department of Dermatology, Bülent Ecevit University, Zonguldak, , Turkey

Introduction Mycosis fungoides (MF) is a primary cutaneous T‑cell lymphoma with an indolent but heterogeneous clinical course. While peripheral blood involvement is clinically relevant, conventional complete blood count (CBC) parameters often lack sensitivity for detecting subtle hematologic changes. Modern hematology analyzers generate fluorescence‑based research parameters reflecting cellular complexity, nucleic acid content, and activation status; however, their potential utility in MF has not been systematically evaluated. This study aimed to investigate both conventional and fluorescence‑related CBC- derived parameters in patients with MF and to compare them with healthy controls. Methods This retrospective study included 45 patients diagnosed with MF and 50 age- and sex-matched healthy controls. CBC analyses were performed using the Mindray BC-6800 Plus hematology analyzer. Evaluated parameters included conventional CBC indices, neutrophil-to-lymphocyte ratio (NLR), immature granulocyte count and percentage (IG, IG%), and fluorescence-related research parameters (neutrophil-X, -Y and -Z, lymphocyte--X, -Y and -Z, high florescence lymphocyte count [HFLC] and HFLC%). Neutrophil-and lymphocyte-X, -Y and -Z parameters reflect intracellular complexity, fluorescence intensity associated with nucleic acid content, and cell size, respectively, derived from multi-angle light scatter and fluorescence signals. HFLC represents lymphoid cells with high fluorescence intensity, corresponding to increased nucleic acid content. Group comparisons were performed using appropriate statistical tests, and receiver operating characteristic (ROC) curve analysis was used to assess the discriminative performance of selected parameters. Results Compared with healthy controls, patients with MF demonstrated significantly higher NLR, IG counts and IG%. Neutrophil-Y, neutrophil-Z, and lymphocyte-Z values differed significantly between groups, indicating altered cellular morphology and fluorescence-related characteristics. In contrast, HFLC count and HFLC% were identical in patients and healthy controls with no statistically significant difference observed. ROC analysis revealed modest discriminative performance, with the highest area under the curve (AUC) values were observed for immature granulocyte count and IG% (AUC = 0.68), followed by NLR (AUC = 0.64). Conclusions Patients with MF exhibit peripheral blood alterations predominantly reflecting inflammatory and myeloid activation rather than lymphoid fluorescence alterations. Elevated neutrophil-related parameters, immature granulocytes, and higher NLR support an inflammatory component in MF. The lack of HFLC differences is consistent with the T-cell–dominant nature of the disease and limited peripheral B-cell activation. Although the diagnostic performance of individual parameters was modest, these readily available CBC-derived indices may provide supportive information alongside clinical and histopathological evaluation. Larger prospective studies are warranted to further elucidate their role in disease characterization and monitoring.

P109

Hematologic Changes Associated With Measles Infection: A Retrospective Chart Review
Faramarz Jabbari-Zadeh¹, Michael Samuel², Benjamin Hedley², Ana Cabrera², Johan Delport², Benjamin Chin-Yee^2,3, Cyrus Hsia^2,3
¹Western University, Schulich School of Medicine & Dentistry, Department of Medicine, London Health Sciences Centre, London, ON, Canada, ²Western University, Schulich School of Medicine & Dentistry, Department of Pathology and Laboratory Medicine, London Health Sciences Centre, London, ON, Canada, ³Western University, Schulich School of Medicine & Dentistry, Division of Hematology, Department of Medicine, London Health Sciences Centre, London, ON, Canada

1. INTRODUCTION: Measles is a highly contagious viral illness caused by Morbillivirus hominis that has seen resurgence in recent years. While the classical clinical features of measles are well documented, diagnostic hematologic abnormalities associated with the infection remain poorly characterized. The current outbreak in Southwestern Ontario, Canada, one of the largest known in North America with over 900 cases reported as of April 2025, offers the opportunity to characterize laboratory abnormalities associated with measles infection, which may yield insights into key clinical features to aid in diagnosis and prognosis in this resurgent viral infection. 2. METHODS: We conducted a retrospective chart review at London Health Sciences Centre from January 1, 2025 to April 25, 2025. All patients who had measles testing performed by serology (measles IgM) and/or PCR during the study period and who had complete blood count (CBC) performed within 10 days of measles testing were eligible for inclusion. Two groups were analyzed: patients with laboratory-confirmed measles infection defined as positive serology and/or PCR test and a control group comprised of patients who tested negative for measles during the same period. Primary analysis compared CBC parameters and peripheral blood smear morphology between groups. Secondary analysis compared clinical outcomes, including hospital admission, intensive care unit (ICU) admission, length of hospital stay, length of ICU stay, and whether laboratory changes predicted clinical outcomes. Statistically significant results were defined by P <0.05. 3. RESULTS: Primary analysis showed statistically significant differences for hemoglobin and eosinophil count, with lower values in measles-positive patients (hemoglobin: t = 2.75; P = 0.0070; eosinophil count: t = 2.20; P = 0.0298). Peripheral blood smear morphology did not differ between groups. In measles-positive patients, anemia was associated with greater rates of hospital admissions (X² = 4.75; OR = 0.96; P = 0.0438), though this was not associated with differences in other clinical outcomes. There was no statistically significant relationship between eosinophil counts and any of the clinical outcome variables. 4. CONCLUSIONS: The diagnostic laboratory findings and hematologic abnormalities in measles are nonspecific. The association of anemia in measles-positive patients requiring hospitalization suggests that anemia may predict a more severe disease course. As the diagnosis of measles is based on clinical presentation and confirmed by serology, the role of other laboratory testing is limited but factors such as anemia may be helpful in predicting disease severity.

P110

Differential Responses Of Peripheral Blood Monocytes From Patients With Alzheimer&Rsquo;S Disease To Lipopolysaccharide And Amyloid-Β Stimulation
hye ryoun kim, Soonchul Hong
Chung-Ang University College of Medicine, Seoul, Korea

Introduction: Monocytes are key components of the innate immune system, responsible for the clearance of cellular debris and apoptotic cells through phagocytosis. In response to inflammatory signals, circulating monocytes are rapidly recruited to sites of inflammation, where they differentiate into macrophages and modulate immune responses. Neuroinflammation is now recognized as a central feature of Alzheimer’s disease (AD), the most common neurodegenerative disorder leading to dementia. While the role of resident microglia in AD-related neuroinflammation has been extensively studied, the contribution of systemic inflammation and peripheral monocytes remains poorly understood. In particular, how peripheral monocyte responses vary across different stages of AD and influence neuroinflammatory processes has yet to be fully elucidated. We aimed to assess whether monocyte responses to stimulation differ between individuals with and without Alzheimer’s disease. Methods: Peripheral blood monocytes were isolated from 37 patients with Alzheimer’s disease and 10 cognitively normal controls. Monocytes were cultured under unstimulated conditions or stimulated with lipopolysaccharide (LPS) or amyloid-β for 1 and 5 days. Cytokine and chemokine levels were measured in culture supernatants, and time-dependent changes were assessed by comparing responses between day 1 and day 5. Immune responses were subsequently compared between the Alzheimer’s disease and control groups. Results: Age and sex distributions were comparable between groups. Total white blood cell counts did not differ, whereas absolute monocyte counts differed significantly between groups (p <0.05). In univariate, unadjusted analyses, monocytes from patients with Alzheimer’s disease exhibited significantly attenuated IL-8 and TNF-α responses to amyloid-β stimulation, along with increased IL-4 responses to LPS, suggesting dysregulated innate immune reactivity. Conclusion: This study suggest that Peripheral monocytes from patients with Alzheimer’s disease show attenuated IL-8 and TNF-α responses to inflammatory and disease-relevant stimuli, indicating dysregulated innate immune reactivity. Rather than reflecting hyperinflammation, these findings suggest immune exhaustion or maladaptive tolerance in chronic neuroinflammatory states. Such impairment may limit effective immune cell recruitment and amyloid clearance, thereby contributing to persistent low-grade neuroinflammation and Alzheimer’s disease pathophysiology.

P111

A Substantial Proportion Of Body Fluid Samples Fall Below The Limit Of Quantification: Implications For Automated Hematology Analyzer Use By Body Fluid Type
Young Kyung Lee^1,2, Ju Yeon Shim², Youngmin Kim²
¹Department of Laboratory Medicine, Hallym University College of Medicine , Chuncheon, South Korea, ²Department of Laboratory Medicine, Hallym University Sacred Heart Hospital, Anyang, South Korea

Introduction. Automated hematology analyzers (AHAs) are increasingly used for body fluid (BF) cell counting due to improved efficiency and standardization. However, their clinical applicability is limited when white blood cell (WBC) or red blood cell (RBC) counts fall below the limit of quantification (LOQ), necessitating manual counting. The frequency of such low-count samples and its variation by BF type remain insufficiently characterized. This study evaluated the analytical performance of the Sysmex XN-350 (Sysmex Co., Kobe, Japan) BF mode and assessed the proportion of BF samples below LOQ according to BF type to inform practical laboratory strategies. Methods. WBC-BF and RBC-BF parameters of the Sysmex XN-350 were evaluated. Limit of blank (LOB), limit of detection (LOD), and LOQ were determined in accordance with CLSI guidelines. A total of 3,299 BF samples submitted for WBC and RBC counts between January and December 2025 were included. Specimen types included ascitic fluid (n=1,076), pleural fluid (n=934), cerebrospinal fluid (CSF; n=561), Peritosol (n=319), bile juice (n=173), joint fluid (n=62), pericardial fluid (n=34), pus (n=47), and others (n=93). The distribution of WBC and RBC counts and the proportion of samples below LOQ were analyzed overall and by BF type. Results. The LOBs for both WBC-BF and RBC-BF were 0.0/µL. LODs were 1.0/µL for WBC and 1,000/µL for RBC, while LOQs were 2.0/µL and 2,000/µL, respectively. The analytical measurement range was 2–10,000/µL for WBC and 2–5,000×10³/µL for RBC. Overall, 299 samples (8.7%) had WBC counts below LOQ and 598 samples (18.1%) had RBC counts below LOQ. WBC counts below LOQ were most frequent in CSF (42.4%), followed by bile juice (7.5%) and Peritosol (6.9%), whereas such results were rare in pleural and ascitic fluids (<1%). Similarly, RBC counts below LOQ were highly prevalent in Peritosol (57.1%) and CSF (49.6%), but uncommon in pleural fluid (3.0%) and absent in pus samples. The proportion of samples below LOQ differed significantly according to BF type for both WBC and RBC (p<0.05). Conclusions. A substantial proportion of BF samples, particularly CSF and Peritosol, yield WBC or RBC counts below the LOQ of the XN-350. These marked differences by BF type highlight the need for specimen-specific strategies when applying automated BF cell counting. Incorporating LOQ- and BF type–based decision rules may optimize the appropriate use of AHAs in routine clinical laboratories.

P112

Scatterplot Analysis As A Potential Guide For Peripheral Blood Smear Review In Samples With Low Schistocyte Counts
Maria del Mar Meijon¹, Beatriz Astibia¹, Sandra Lopez¹, Fernando Martin¹, Miguel Piris¹, Juan Marquet¹, Javier Lopez¹, Jesus Villarrubia², Gemma Moreno¹
¹Department of Hematology, Hospital Universitario Ramon y Cajal-IRYCIS, Madrid, Spain, ²Laboratorio Central de Madrid, UR Salud UTE, Madrid, Spain

Introduction: Schistocytes on the peripheral blood smear are a key morphological finding for the suspicion and monitoring of thrombotic microangiopathies (TMA). According to recommendations from the International Society for Laboratory Hematology, a schistocyte percentage ≥1% is highly suggestive of TMA; however, lower values may be observed in certain clinical situations. In this context, hematology analyzer scatterplots could constitute a resource to support peripheral blood smear performance, as certain scatterplot patterns suggestive of red cell fragmentation may be detectable when schistocyte percentages remain below this threshold. The aim of this study was to evaluate the utility of scatterplot interpretation in samples with schistocyte percentages <1%. Methods: A retrospective observational study was conducted including samples processed during 2025 at Hospital Universitario Ramón y Cajal with schistocytes identified on peripheral blood smear. Hematologic analysis was performed using Alinity hq analyzers (Abbott). Samples were classified according to schistocyte percentage as <1% or ≥1%. Hematimetric parameters were described as median and interquartile range (IQR). IAS1×IAS3 scatterplots were independently and blindly evaluated by two observers and classified using a semi-quantitative grading system (grades 1 and 2). Interobserver agreement was subsequently assessed using Cohen’s kappa coefficient. Results: Thirty-five samples were included; 31/35 (88.6%) corresponded to secondary TMA and 4/35 (11.4%) to acquired thrombotic thrombocytopenic purpura. Based on schistocyte percentage, 16/35 samples (45.7%) showed values <1% and 19/35 (54.3%) ≥1%. The ≥1% group was included for comparative purposes. The median schistocyte percentage was 0.8% (IQR 0.8–0.8) in the <1% group and 1.6% (IQR 1.2–2.4) in the ≥1% group. In the <1% group (n = 16), median (IQR) values were: hemoglobin 8.56 g/dL (7.84–9.66), hematocrit 25.1% (23.5–28.7), RDW 18.2% (16.1–20.7), and platelets 66.4 ×10³/µL (17.0–163.5). In the ≥1% group (n = 19), median (IQR) values were: hemoglobin 8.69 g/dL (8.00–9.91), hematocrit 24.4% (22.7–29.0), RDW 16.6% (16.2–19.3), and platelets 26.7 ×10³/µL (17.7–62.0). Interobserver agreement for scatterplot interpretation was κ = 0.86 in the <1% group and κ = 0.73 in the ≥1% group. Interobserver discrepancies were observed in 3/35 samples (8.6%), at schistocyte percentages ranging from 0.8% to 1.6% (median 1.2%), with heterogeneous hematimetric findings. Conclusions: The results of this study suggest that, in samples with schistocyte percentages <1%, scatterplot interpretation may provide complementary information to support peripheral blood smear performance. However, studies with larger sample sizes are required to confirm these findings.

P113

&Ldquo;Infected Rbc Flag&Rdquo; On Mindray Cal8000 Leading To Development Of A Novel Screening Algorithm For Prevention Of Donor-Derived Malaria In Live-Donor Liver Transplantation (Ldlt) Patients.
ADITI MITTAL¹, ANIL HANDOO¹, TINA DADU¹, PRATYUSH MISHRA¹, ABHIDEEP CHAUDHARY²
¹Dept of Hematology, BLK-Max Super Speciality Hospital, NEW DELHI, India, ²Dept of HPB Surgery & Liver Transplantation, BLK-Max Super Speciality Hospital, NEW DELHI, India

Introduction:India is one of the large hubs for live donor liver transplantation, with many patients travelling from other countries, especially middle-east and Africa for this procedure. While there are many-a-factor for successful transplantation, a thorough work up of the donor prior to transplantation is imperative. Amongst many pre-procedural investigations, screening for malaria is done using rapid diagnostic test (RDT) based on the identification of plasmodial antigens using immunochromatography, with our centre being no exception. The RDT kit used has specificity for Plasmodium falciparum histidine-rich protein (PfHRP) and Plasmodium vivax-specific pLDH, however, it does not assess the other species of Plasmodium. The challenge though is the sensitivity of RDTs to pick up Plasmodium infection. We herein report 5 cases of non-vivax, non-falciparum Plasmodium infection with negative/non-reactive RDT and positive “Infected RBC flag” (In) on Mindray CAL 8000 and subsequent development of PCR based screening algorithm of malaria screening in Live Donors for organ transplantation, as a screening tool for picking up cases of asymptomatic parasitemia. Methods: 5 cases of asymptomatic malaria were picked up on CBC analysis using Mindray CAL 8000 and MC80 digital morphology analyzer as apart of the pre-operative screening of donors for Liver Transplantation. Subsequent to RDT screening, PCR based confirmation and identification stands adopted as an algorithm. Results: Positive “In” flag on Mindray CAL8000 raised the suspicion of smear evaluation, subsequent identification of parasite by digital morphology and thick smear, eventually confirmed by PCR. An algorithm to include PCR as a modality for screening of malaria as a part of the Tropical Infection Screening panel stands introduced since then. ~1.5% cases were detected to have asymptomatic malaria based on this algorithm, subsequent to its adoption in our practice. Conclusions: Our case series demonstrates the pressing need for improved screening strategy for malaria in solid organ transplant patients from only RDT to RDT and PCR.

P114

Nt-Probnp As A Biomarker In Monoclonal Gammopathy Of Undetermined Significance And Multiple Myeloma: A Retrospective Analysis
Eun-Hee Nah¹, Yong Jun Choi², Jooheon Park², Ha Jin Lim³, Yong Jun Kwon³, Myung Geun Shin²
¹MEDIcheck LAB, Korea Association of Health Promotion, Seoul, , South Korea, ²Department of Laboratory Medicine, Chonnam National University Hwasun Hospital, Hwasun, , South Korea, ³Department of Laboratory Medicine, Chonnam National University Hospital, Gwangju, , South Korea

Introduction: N-terminal pro–B-type natriuretic peptide (NT-proBNP) is a well-established biomarker of cardiac stress and has recently been implicated in hematologic malignancies. However, research on how NT-proBNP changes from monoclonal gammopathy of undetermined significance (MGUS) to multiple myeloma (MM), and its association with disease severity and progression, remains limited. This study evaluated whether NT-proBNP levels are associated with disease severity and progression in patients with MGUS and MM. Methods: This retrospective cross-sectional study included 121 patients with MGUS and 472 patients with MM. MGUS risk was stratified based on the presence of three major risk factors, while MM was staged according to the ISS, R-ISS, and R2-ISS systems. Associations between NT-proBNP and clinical or laboratory parameters were evaluated using univariate and multivariate regression. Results: NT-proBNP levels did not significantly differ between the MGUS and MM groups. In MGUS, NT-proBNP levels were positively associated with β2-microglobulin (P = 0.018) and creatinine (P <0.001). In MM, NT-proBNP levels increased with advancing disease stage in all staging systems (P <0.001), but these associations were no longer significant in multivariate models. Instead, β2-microglobulin, LDH, creatinine, and albumin remained independently associated with NT-proBNP levels. Conclusions: NT-proBNP levels were not associated with MGUS risk factors and showed limited value for risk stratification in MGUS. In MM, NT-proBNP may reflect disease burden but lacks independent value as a marker of disease stage. NT-proBNP may serve as an indicator of overall disease burden, but has limited value as an independent biomarker for disease severity in MM.

P115

Comparison Of Neutrophil-Lymphocyte Ratio, Neutrophil-Monocyte Ratio, Pan-Immune-Inflammatory Value, C-Reactive Protein, And Procalcitonin In Determining Prognosis Of Neonatal Sepsis
Asvin Nurulita^1,2, Mansyur Arif^1,2, Arifin Seweng², Sakina Usman²
¹dr. Wahidin Sudirohusodo Hospital, Makassar, , Indonesia, ²Department of Clinical Pathology, Faculty of Medicine, Hasanuddin University, Makassar, , Indonesia

Background: Neonatal sepsis remains a leading cause of global morbidity and mortality,contributing to approximately 13% of all neonatal deaths.The immaturity of the neonatal immune system results in heightened vulnerability to infections and severe systemic immune dysregulation. Imbalances in peripheral blood cell activity—including neutrophils, monocytes, platelets, and lymphocytes—have highlighted the potential of inflammatory biomarkers and derived hematologic indices such as the Neutrophil-Lymphocyte Ratio (NLR), Neutrophil-Monocyte Ratio(NMR),and Pan-Immune-Inflammatory Value(PIV). Although promising,the diagnostic performance of these newer indices compared with established biomarkers such as procalcitonin and C-reactive protein (CRP) requires more comprehensive evaluation for clinical validation. Objective: To evaluate and compare the diagnostic accuracy of NLR, NMR, PIV, CRP, and procalcitonin as predictors of mortality in neonates with sepsis. Methods:This retrospective observational study involved 244 neonates diagnosed with sepsis at dr. Wahidin Sudirohusodo General Hospital, Makassar, during the 2023–2024 period. Subjects were categorized into non-survivor (n=135) and survivor (n=109) groups. Demographic and laboratory data were analyzed using Kolmogorov-Smirnovnormalitytests, Mann Whitney U tests, Independent T-tests, and Pearson correlation tests. Receiver Operating Characteristic (ROC) analysis was performed to determine the Area Under the Curve (AUC) and optimal cutoff values, with p<0.05 considered statistically significant. Results: Birth weight and gestational age differed significantly between groups (p<0.05). Procalcitonin demonstrated the strongest prognostic performance with AUC 0.927(sensitivity 89.6%, specificity 88.1%) with cutoff ≥1.61 ng/mL. NLR showed high accuracy (AUC 0.813) with cut off ≥2.50. Other biomarkers showed moderate accuracy: CRP (AUC 0.734; cut off ≥23.3 mg/dL), NMR (AUC 0.707; cut off ≥4.32), and PIV(AUC0.649; cut off<344.31). Significant positive correlations were observed between procalcitonin and CRP (r=0.418) and between procalcitonin and NLR (r=0.219), indicating a strong association with systemic inflammatory response. Conclusion: Procalcitonin proved to be the superior mortality predictor compared to NLR, NMR, PIV and CRP. The use of Procalcitonin ≥1.61 ng/mL and NLR ≥2.50 is highly recommended as effective early risk stratification instruments in clinical practice to predict poor outcomes and guide more aggressive interventions in neonatal sepsis patients.

P116

Autoverification For Optimizing Cbc Workflow And Laboratory Efficiency
Karan Paisooksantivatana
Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

Introduction Accurate and reliable reporting of laboratory results is essential for medical laboratories, especially for high-volume tests such as the complete blood count (CBC) (1). The high workload poses challenges in maintaining analytical quality while improving efficiency (2). Advances in hematology analyzers, middleware, and laboratory information systems have enabled robust autoverification systems that support faster and more consistent reporting (3,4). Reflex testing using optical or fluorescent analytical modes provides added diagnostic value and improves error detection within automated workflows (5). This study aims to develop and validate an autoverification system for CBC testing in our laboratory. Methods A verification workflow was developed to detect potential errors in RBC, WBC, and PLT parameters across the pre-examination, examination, and post-examination phases. The workflow integrated algorithms consisting of verification criteria and corresponding troubleshooting actions (Table 1). Verification criteria included delta checks, individuality indices, cross-channel comparisons, critical thresholds, and analyzer flags/messages. Troubleshooting actions comprise reflex testing using optical or fluorescent analysis, manual specimen manipulation, slide review, and specimen rejection when necessary. Specimens that satisfied all verification criteria were auto-authorized. The rules were evaluated using routine samples processed between 1 and 31 October 2025 to assess their efficiency. Results Using the established workflow results in autoverification of 19,559 of total 23,062 specimens (84.81%). Of the 3,503 CBC results that were blocked from autoverification (15.19%), only 3,378 cases (14.69%) ultimately required slide review. The most frequent conditions being blocked from autoverification included PLT abnormal distribution with PLT-F = Null (6.8%) and the presence of blasts or abnormal lymphocytes when Q > 80 (5.8%). Hemoglobin levels <10.5 occurring for the first time in three months, together with either MCV <80, MCV > 100, RDW-CV > 15%, or a fragments flag, accounted for 4.8% of triggers. Platelet-related parameters were also notable, with PLT-I <100,000 accompanied by PLT-F = Null (3.3%) and PLT-F <100,000 alone (3.3%). Other less frequent triggers included atypical lymphocytes when Q > 140 (1.2%), RBC abnormal distribution with %RET = Null (1.0%), and lymphocyte counts exceeding 5,000/µL when occurring for the first time within 30 days (1.0%). Abnormal WBC scattergrams (0.9%) and IG > 10% (0.8%) contributed to a smaller proportion of cases. Conclusion
The established autoverification algorithm shows strong potential to improve efficiency by reducing manual interventions and shortening turnaround time. These are preliminary findings, and further evaluation of its accuracy, reliability, and clinical impact is ongoing.

P117

Comparison Of Blood Film Review Rates Between Atellica Hema And Advia 2120I Hematology Analyzers
Sharon Radford¹, Apostolia Barmpoudi¹, Andrew Smith¹, Graham Gibbs²
¹Leeds Teaching Hospitals NHS Trust, Leeds, United Kingdom, ²Siemens Healthcare Laboratory Diagnostics, Camberley, United Kingdom

Introduction: Atellica HEMA 570 and Atellica HEMA 580 analyzers were recently launched by Siemens Healthcare Diagnostics Inc. to replace their legacy ADVIA 2120i Hematology Systems. Low blood film review rates are a welcome and, increasingly, an expected feature of hematology analyzers in modern high-throughput laboratories. Low blood film review rates improve workflow efficiency while allowing technologists or clinicians time to focus upon cases that add clinical value to the overall patient pathway. During 2025 we replaced our seven ADVIA 2120i systems with three Atellica HEMA 570 and five Atellica HEMA 580 across two sites. We moved from ADVIA 2120i at St James’s University Hospital (SJUH) to Atellica HEMA in our new-build Centre for Laboratory Medicine (CFLM) mid-June 2025. We transferred our general practitioner (GP) work from our Leeds General Infirmary (LGI) site to CFLM at the end of June. We then moved from ADVIA 2120i at LGI to Atellica HEMA in our new Acute Hospital Laboratory (AHL) mid-August 2025. The purpose of this study was to compare blood film review rates between the new Atellica HEMA and our legacy ADVIA 2120i automated hematology analyzers. Methods: Blood count (n=882,555) and blood film review data (n = 25,478) were extracted from our Laboratory Information Management System (LIMS) from January to October 2025 to reflect pre- and post- conversion to Atellica HEMA. Percentage blood film review rates were calculated for each month. Results: Blood film review rates were 2.80% (January), 2.74% (February), 2.82% (March), 2.87% (April), 3.04% (May), 2.86% (June), 3.08% (July), 2.90% (August), 2.79% (September), and 2.95% (October). The average blood film review rate was 2.89%. The highest blood film review rate was recorded in July and was 0.19% above the average. Conclusions: ADVIA 2120i Hematology Systems are typically associated with low blood film review rates and our data supported that. We observed a small increase in July 2025 of 0.19% above average, coinciding with the largest element of our conversion to Atellica HEMA in mid to late June. Staff rapidly familiarised themselves with the new technology and the blood film review rates settled down to those seen previously with our ADVIA 2120i systems, as evidenced by the film review rates recorded from August to October. We are satisfied that there have been no significant changes and blood film review rates remain low with Atellica HEMA hematology analyzers.

P118

Evaluation Of Analytical Precision Of Monocyte Distribution Width (Mdw) In Hospitalized And Emergency Department Patients.
Jennifer Rodriguez-Dominguez, Alba Leis-Sestayo, Isabel Aparicio-Calvente, Laura Jimenez-Añon, Cristian Morales-Indiano, Alicia Martinez-Iribarren
Laboratory department of medicine. Germans Trias i Pujol University Hospital , Badalona, Spain

INTRODUCTION Sepsis is a major global health problem because of its high prevalence and mortality, and because early diagnosis and timely treatment are essential to improve outcomes. For this reason, sepsis biomarkers are widely used in clinical practice. Monocyte distribution width (MDW) is a recently introduced biomarker for sepsis, obtained routinely as part of the complete blood count with differential. Reported cut-off values include 21.5 for emergency department patients (sensitivity 75%, specificity 73%) and 24 for intensive care unit patients (sensitivity 80.6%, specificity 84.5%), both measured in EDTA K3 whole blood. Its negative predictive value makes it a highly useful biomarker for the exclusion of sepsis. However, like all laboratory parameters, MDW measurement is subject to analytical variability. Therefore, this study aimed to evaluate the impact of intra‑assay imprecision on clinically relevant decision thresholds through a repeatability study. METHODS A total of 38 EDTA K3 whole blood samples from hospitalized and emergency department patients were analyzed. Patients were categorized into four groups based on MDW results: group 1 (n=10) MDW 16–21.5; group 2 (n=10) MDW 21.5–24; group 3 (n=10) MDW 24–30; and group 4 (n=8) MDW >30. Groups 1 and 2 include the clinical decision cut-off values. Repeatability was assessed by performing 10 consecutive measurements per sample using a DxH900 analyzer (Beckman Coulter). The mean, standard deviation (SD) and coefficient of variation (CV%) were calculated for each sample. Analytical precision was evaluated against the manufacturer’s specification of a maximum CV of 10% for MDW in patient samples. RESULTS The repeatability assessment demonstrated that the CV% for MDW remained below the manufacturer-specified limit across all groups (Table 1). Three samples showed CV% values slightly above 10% (10.75%, 10.23%, and 10.32%), one in group 3 and two in group 4, respectively). Nevertheless, none of these groups included clinical decision cut-off values. Although overall imprecision remained within acceptable analytical limits, the observed variability may have clinical relevance for samples with MDW values close to established decision thresholds. CONCLUSIONS The coefficient of variation remained within acceptable limits in all patient groups, fulfilling the analytical quality specifications for imprecision. However, despite satisfactory repeatability, analytical imprecision may impact the clinical interpretation of MDW results in patients with values near decision thresholds. Therefore, MDW results should be interpreted alongside the patient’s clinical context and other laboratory findings, and exhaustive follow-up evaluation may be advisable when values are close to established cut-off points.

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First Comparative Analysis Of The Behavior Of Hematimetric Biomarkers Associated With Sepsis (Neu-X, Neu-Y, And Mon-X) In Adult Ambulatory Population Versus Intensive Care Patients In Montevideo, Uruguay (2024-2026).
Lucila I Silva¹, Cecilia Canessa¹, Marco Alpuin², Gabriela Chocho¹, Macarena Acosta¹, Miguel Lujan¹, Ana K. Ambrosetti¹, Jimena Viera¹, Gabriela Geymonat¹, Rafael Bertullo¹, Mariela Luppi²
¹Medica Uruguaya (MUCAM), Montevideo, , Uruguay, ²Biodiagnostico, Montevideo, , Uruguay

Introduction The Mindray CAL 8000-211 platform, through the BC-6800 Plus analyzers and SF Cube technology (flow cytometry with forward scatter, side scatter, and fluorescence signals), provides high-resolution three-dimensional information on leukocyte morphology, including cell volume, cytoplasmic/nuclear complexity, and nucleic acid content. Among the derived parameters, Neu-X, Neu-Y, and Mon-X have been proposed as reportable biomarkers for the early detection of sepsis, as they reflect changes in leukocyte activation and maturation. Sepsis is a life-threatening condition and one of the leading causes of mortality; describing the behavior of these parameters in contrasting clinical settings (ambulatory vs Intensive Care Unit (ICU) patients), provides local evidence and supports their clinical implementation. This study represents the first experience in Uruguay with these parameters, with our institution being the pioneer in reporting them in routine local practice. Objective:To compare the values of Neu-X, Neu-Y, and Mon-X between adult ambulatory patients and ICU patients at Médica Uruguaya, as an initial step in evaluating our experience implementing these new parameters. Methods: A retrospective observational study was conducted. • Ambulatory population: anonymized data from 98,891 hemograms processed between December 2024 and October 2025, in patients aged 18–99 years attending general outpatient clinics at MUCAM (specialty clinics were excluded). • ICU population: anonymized data from 3,737 hemograms of patients admitted to the Intensive Care Unit at the central hospital of MUCAM, between June 2025 and January 2026. For each parameter, mean, median, mode, and standard deviation were calculated. Comparisons between populations were performed using Welch’s t-test, Mann–Whitney U test, and calculation of effect size (Cohen’s d). Results: All parameters showed statistically significant differences (p <0.0001). • Neu-Y was the most discriminant parameter, with a large effect size. • Neu-X showed medium-sized differences, clinically relevant. • Mon-X presented a small to medium effect, although statistically significant. Conclusion: The parameters Neu-X, Neu-Y, and Mon-X demonstrated significant differences between ambulatory and critically ill patients. Neu-Y stands out as the biomarker with the greatest discriminative power, followed by Neu-X, while Mon-X contributes more modest differences. These initial results provide local evidence on their behavior in critical populations and support future evaluations with clinical stratification and correlation with outcomes. This work represents the first national evidence on Neu-X, Neu-Y, and Mon-X, positioning our institution as the initial reference in their implementation and reporting.

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Performance Evaluation Of Mindray Bc-6800 Plus Versus Sysmex Xn 1000 And Dxh 800 Hematology Analyzers
Sonya F. Soliman¹, Mohamed Kamal², Roxan Shafik¹, Mohamed Zaki¹, Amr Hussein ¹, Mai Abdelwahab¹, Mohamed Ahmed¹
¹Clinical Pathology Department, Children Cancer Hospital of Egypt , 57357, Cairo, , Egypt, ²Clinical Research Department, Children Cancer Hospital of Egypt, 57357, Cairo, , Egypt

Introduction: Automated hematology analyzers are indispensable for modern laboratory medicine. They provide rapid, reliable, and standardized results essential for clinical decisionmaking. The Mindray BC6800 Plus represents a new generation of hematology analyzers. It offers advanced technologies for improved accuracy and efficiency. Key advantages include high throughput and robust differential analysis. However, adoption of new analyzers requires rigorous validation. Benchmark instruments such as DxH800 and Sysmex XN1000 are widely trusted worldwide. They serve as reference standards for hematology testing. Therefore, systematic comparison with DxH800 and Sysmex XN1000 is essential. This study addresses that need by evaluating performance across key hematology parameters. Methods: A total of 3952 EDTA-anticoagulated blood samples were analyzed. 1999 complete blood counts (CBCs) were processed on the DXH800 and Mindray BC-6800 Plus, while another 1953 were run on the Sysmex XN1000 and Mindray BC-6800 Plus. Comparative performance was evaluated against Clinical Laboratory Improvement Amendments (CLIA) 2025 total allowable error limits for red blood cell ( RBC), white blood cell (WBC), hemoglobin (HGB), and platelets (PLT) parameters, ensuring analytical accuracy and clinical acceptability. Results: Comparing Sysmex XN 1000 with Mindray BC-6800 Plus, excellent correlation was observed for all CBC parameters (r >0.995). WBC showed minimal negative bias (-1.68%) with 96.7% of results within CLIA limits. RBC demonstrated the lowest agreement, with 93.2% within CLIA criteria. HGB showed the best performance, with negligible bias (-0.61%), perfect proportionality (slope=1.000), and 99.1% of results within CLIA limits. PLT exhibited the greatest positive bias (+7.34%) but maintained 95.8% of results within acceptable limits. As regards results of DXH 800 versus Mindray BC-6800 Plus, WBC counts showed the lowest compliance with CLIA criteria, with 89.2% of results meeting acceptance limits. PLT measurements exhibited the highest systematic bias (+7.09%) but remained largely within CLIA limits (96.8%). HGB showed the best performance, with minimal bias (-1.39%), the highest CLIA compliance (99.3%). RBC parameters also showed good agreement, with (97.0%) of results meeting CLIA acceptance criteria. Conclusion: Mindray BC-6800 Plus analyzer demonstrated excellent analytical comparability with both the DXH800 and Sysmex XN 1000 reference systems. All CBC parameters showed strong correlation (r >0.995) and high clinical agreement, with HGB consistency showing the highest compliance (>99%). While minor systematic biases were observed in PLT and WBC counts, the vast majority of results remained within acceptable CLIA limits. These findings support the clinical suitability and reliability of the Mindray BC-6800 Plus for routine hematological analysis.

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Evaluation Of Complete Blood Count-Derived Ratios In Ischemic Stroke. A Preliminary Report
Konstantina Tsioni¹, Eftychia Nikolakopoulou², Angeliki Karyoti¹, Aristeidis Chalvantzis¹, Marianthi Issa¹, Aikaterini Karantani¹, Dimitrios Komilis³, Eleni Vagdatli¹
¹Biopathology Laboratory, Hippokration General Hospital,, Thessaloniki, Greece, ²Biopathology Laboratory, Xanthi General Hospital, Xanthi,, Xanthi, Greece, ³Department of Environmental Engineering, Democritus University of Thrace, Xanthi, Greece

Introduction: Ischemic stroke (IS) is a leading cause of morbidity and mortality worldwide. Inflammation and immune dysregulation plays a critical role in atherogenesis, atherosclerosis, IS pathophysiology and clinical outcome. A variety of biomarkers like hsCRP, D-Dimers, neuron-specific enolase(NSE), glial fibrillary acidic protein (GFAP) have been studied in IS to investigate and establish a relationship with inflammation, neuronal damage, endothelial dysfunction and thrombosis. Several had associations with IS severity, outcome, infarct size and hemorrhagic transformation. Nevertheless, hsCRP and D-Dimers lack specificity and the novel biomarkers are not widely available due to cost, lack of technical installations and standardized methods. Ratios derived from Complete Blood Count (CBC) parameters such as Neutrophil-to Lymphocyte ratio (NLR), Neutrophil-to-Monocyte ratio (NMR), Lymphocyte-to-Monocyte ratio (LMR) and Platelet-to-Lymphocyte ratio (PLR) are ratios of interest for a lot of scientists because they are easy to access, cost-effective and readily available. Neutrophils induce innate cellular immune response, Lymphocytes induce adaptive cellular immune response, activated macrophages and T cells infiltrate atherosclerotic plaque. Novel studies demonstrated that several diseases with inflammatory pathophysiology, such as cardiovascular diseases, malignancies, infectious and inflammatory disorders benefit from these ratios. Likewise, in IS these ratios may contribute to management and prognosis. CBC was determined, CBC-derived ratios were calculated in patients with IS and were compared with a control group, investigating their relevance with IS. Methods: 97 individuals admitted to a tertiary care hospital were studied. 56 suffered an IS, had a median age of 61(33-76)years and constituted Group A. 46 healthy individuals constituted the control Group (Group B) with median age 47(19-75)years. Patients with hematological malignancies, solid tumors and women in pregnancy were excluded. CBC was determined on UniCell DXH800 (Beckman Coulter). To compare medians between the two groups, the SPSS^® v. 29 and the non-parametric Mann-Whitney U test were used. The discriminatory ability of CBC-derived biomarkers to differentiate between the two groups was evaluated using a ROC curve. Level of statistical significance, p<0.05. Results: NLR was statistically significant higher, p<0.05 and LMR was statistically significant lower, p<0,001 in Group-A. No statistically significant difference was found between the two groups regarding NMR and PLR, p=0.950 and p=0.762, respectively. NLR showed moderate but statistically significant discriminatory ability between groups and better discriminatory ability compared to the other ratios(AUC=0.677, p<0.05). Conclusion: CBC-derived ratios, NLR, NMR, LMR and PLR may not be statistically superior to novel biomarkers but they are practically superior. They are readily available, low-cost and worldwide available because CBC is determined upon admission everywhere. They reflect systemic inflammation, immune response and thromboembolic burden which are crucial in IS. NLR and LMR were significantly higher and lower, respectively in patients with IS reflecting the inflammatory response and the changes in neutrophils, lymphocytes and monocytes. Neutrophils move to Central Nervous System within a few hours after IS. Lymphocytes produce various cellular mediators that modulate the inflammatory response. Monocytes infiltrate atherosclerotic plaque. NLR and LMR may facilitate early risk stratification and may be useful for patients management and monitoring because they are easy to repeat and detect dynamic changes of patients clinical condition. It is crucial to continue the study with a larger patient sample size and correlate NLR and LMR with outcomes.

P123

Dynamics Of Hematological Parameters During Iron Therapy In Patients With Iron Deficiency Anemia And Latent Iron Deficiency
Irina V Tyutlina¹, Yuliana Y Agametova², Elena A Sukhacheva³
¹LLC "Center for Laboratory Diagnostics", Novosibirsk, Russia, ²Beckman Coulter Ltd., Moscow, Russia, ³Beckman Coulter Eurocenter, Nyon, Switzerland

Introduction. Iron deficiency anemia (IDA) is a common condition, particularly among women. Latent iron deficiency (LID) occurs when hemoglobin remains normal despite depleted iron stores, which, if untreated, can progress to IDA. Both conditions are managed with iron supplementation. This study aimed to evaluate changes in hematological and reticulocyte-related parameters during iron therapy, including research-use-only (RUO) parameters—Red Blood Cell Size Factor (@RSF), Microcytic Anemia Factor (@MAF), and Low Hemoglobin Density (@LHD)—available on the UniCel DxH 800 and UniCel DxH 900 hematology analyzers. Methods. Fifteen female patients (aged 21–48 years) presenting with symptoms such as fatigue, weakness, and hair loss were enrolled at LLC "Center for Laboratory Diagnostics", Novosibirsk (Russia). Initial evaluation included complete blood count with differential and reticulocyte analysis (CBC-Diff-Ret) on UniCel DxH 800 hematology analyzer (Beckman Coulter Inc., USA), iron metabolism tests (iron, UIBC, TIBC, transferrin, ferritin, transferrin saturation, high-sensitivity CRP) on DxC 700 AU analyzer (Beckman Coulter Inc., USA), and vitamins B9 (folate) and B12 on UniCel DxI 800 analyzer (Beckman Coulter Inc., USA). Patients diagnosed with IDA or LID received oral or intravenous iron therapy. Follow-up assessments (CBC-Diff-Ret, ferritin, CRP) were performed on days 10, 30, and 90 after treatment started. Results. Of the 15 patients, 6 had LID, 3 had IDA (one with inflammation defined by elevated CRP), 3 had combined LID with B12 and/or folate deficiency, and 3 had IDA with B12 and/or folate deficiency. IDA patients without inflammation showed low hemoglobin, MCV, MCH, ferritin, @RSF, and mean reticulocyte volume (MRV), with normal-high @LHD. LID patients had low ferritin, normal hemoglobin and MCV, low-normal MRV and @RSF, and normal @LHD. During treatment, MRV, @RSF, and @MAF increased, while @LHD decreased in both groups, as presented on the examples for IDA and LID patients on figure 1. Notably, in IDA patients, increases in @RSF and MRV occurred earlier than significant rises in hemoglobin and MCV (figure 1). In LID patients, iron therapy improved clinical symptoms and ferritin levels, while hemoglobin and MCV remained within normal ranges. Conclusions. Reticulocyte-related and RUO parameters provide valuable insights for monitoring iron therapy in IDA and LID patients, enabling earlier detection of treatment response and supporting clinical decision-making. For LID patients, early treatment is critical as it may eliminate symptoms and reduce the risk of progression from LID to overt anemia. @For research use only. Not for use in diagnostic procedures.

P124

Predictive Model Based On Cell Population Data For 30 Days -Mortality In Community-Acquired Pneumonia
Eloisa Urrechaga, Monica Fernandez, Ane Uranga, Urko Aguirre
Hospital Universitario Galdakao Usansolo, Galdakao, Spain

Background: Community-acquired pneumonia (CAP) is a major health system burden due to its high death and morbidity rates. Biomarkers of inflammation, like C-Reactive Protein and Procalcitonin and prognostic scores, ie CURB-6 , have been developed to support the assessment of severity in patients with CAP, but the predictive power is limited or expensive. Cell Population Data (CPD) quantify the granularity, volume, and nucleic acid content of leukocytes and their morphological variations and changes of functionality in response to infectious stimuli. We aimed to evaluate the predictive power of CPDs in the prognosis of patients with CAP in terms of mortality in 30 days. Methods: A prospective study was conducted including hospitalized patients (age≥ 18 years) with a confirmed CAP diagnosis by chest radiography and compatible symptoms; patients with immunosuppression or recent hospitalization were excluded. Demographic, clinical, and analytical data were recorded at admission. Complete Blood counts were analyzed with Sysmex XN20 counter. CPD were recorded at admission (arbitrary optical units, au). Non-parametric Wilcoxon test was used to compare CPDs according to vital status. A logistic regression model was developed in order to determine those CPDs relevant to the prognosis. A score based in analytical and clinical variables was defined. The area under the ROC curve (AUC) was calculated to verify the robustness of this model. Statistical significance was set p<0.05. Results: One hundred and eighty eight patients were studied. NE-WY≥ 617 au, MO-Y≥ 130 au, MO-WY≤ 667 au, male gender and age ≥ 65 years were identified as predictors of 30-day mortality. The score was composed with NE-WY≥ 617 weight of 2 points, MO-Y≥ 617, MO-WY≤ 667 and male gender scored with 3 points respectively and age ≥ 65 years scored with 8 points; the predictive accuracy was AUC 0.82 , 95%, confidence interval 0.77-0.86). The score categorized into risk groups showed the highest risk among patients with ≥16 points. Conclusions: CPDs could be useful in the evaluation of patients at admission to predict the severity of CAP. Those related to the innate immune response were found to be relevant to predict mortality of patients admitted for CAP.

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A Comparative Evaluation Of Domain-Specific Versus General-Purpose Large Language Models For Complete Blood Count Report Interpretation
Di Wang¹, Donglan Yao², Jianping Xiao², Xiaosen Bian², Xiaomei Zhang², Haoqing Jiang¹
¹Department of Laboratory Medicine, Huashan Hospital, Shanghai Medical College, Fudan University, Shanghai, , China, ²Hematology Product Development Department, Shenzhen Mindray Bio-Medical Electronics Co. Ltd, Shenzhen, , China

Background: Large language models (LLMs) have demonstrated significant potential in laboratory medicine, particularly for interpreting complex diagnostic data. However, systematic evaluations remain insufficient regarding whether general-purpose LLMs can match the performance of domain-specific models in interpreting laboratory reports. This study aims to compare the performance of domain-specific LLMs versus general-purpose LLMs for interpreting complete blood count (CBC) reports. Methods: We developed a domain-specific model for CBC report interpretation (CBC-LLM), which uses clinical context, CBC parameters, historical results, and peripheral blood morphology data as inputs. CBC-LLM was built on the Qwen3-14B backbone and trained using a two-stage domain-adaptive fine-tuning strategy: construction of a structured knowledge base covering nearly 10,000 diseases to support standardized retrieval; and optimization through expert preference alignment. We compared CBC-LLM with three general-purpose models (GPT-5.2, Gemini-3 Pro, and the base Qwen3-14B model). The evaluation comprised 100 CBC analysis cases involving suspected hematologic disorders, infectious diseases, anemia, coagulation abnormalities, and critically ill patients. All models generated interpretation reports using a unified prompt. The outputs were independently reviewed by an expert committee comprising two laboratory medicine specialists and one hematologist, with reviewers blinded to model identify. Reports were assessed using a 5-point Likert scale across four dimensions: completeness of abnormality descriptions, accuracy of disease analysis, usefulness of clinical recommendations, and clinical safety. Group differences were analyzed using ANOVA. Results: Among the 100 included cases, CBC-LLM achieved performance comparable to GPT-5.2. For completeness of abnormality description, Gemini-3 Pro scored significantly higher than CBC-LLM and GPT-5.2 (4.73 vs 4.47; 4.73 vs 4.36; both p<0.05), while CBC-LLM outperformed its base model Qwen3-14B (4.47 vs 4.09; p<0.05). For accuracy of disease analysis, CBC-LLM showed no statistically significant differences compared with Gemini-3 Pro or GPT-5.2 (4.80 vs 4.91; 4.80 vs 4.75; p>0.05). For usefulness of clinical recommendations, CBC-LLM, Gemini-3 Pro, and GPT all scored significantly higher than Qwen3-14B (4.75, 4.73, 4.60 vs 3.85; all p<0.05). For clinical safety, all models achieved scored ≥4.90, with no statistically significant between-group differences. Conclusions: This evaluation of CBC report interpretation indicates that, through domain-adaptive pretraining and fine-tuning, CBC-LLM achieved performance comparable to commercial paid models with fewer parameters, suggesting substantial effectiveness advantages and application potential for domain-specific models in specialized medical settings

P126

Leukopenia, Thrombocytopenia, And Beyond: Hematological Signatures Of Dengue Infection In A Malaysian Cohort
Shafini M. Yusoff^1,2, Marini Ramli^1.2, Rosnah Bahar^1,2, Noor Haslina M Noor^1,2, Mohd Nazri Hassan^1,2, Zefarina Zulkafli^1,2
¹Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia, ²Hospital Pakar Universiti Sains Malaysia, Kubang Kerian, Kelantan, Malaysia

Introduction: Recently the incidence and mortality of dengue fever have shown a consistent rise in tropical regions, including Malaysia. The clinical manifestations of dengue infection frequently resemble those of other febrile illnesses, complicates early and accurate diagnosis. Although the serology diagnostic test offers high specificity, their application is limited by cost, availability and time constraints. In contrast, routine hematological parameters provide a rapid, affordable, accessible and informative alternative that reflects disease progression. However, real-world data characterizing comprehensive hematological signatures, including derived inflammatory indices in dengue-suspected patients remain limited in Malaysian cohorts. This study, therefore, aims to investigate the distribution of dengue-positive cases and evaluate hematological variations between dengue-positive and dengue-negative patients. Methods: This was a cross-sectional single center study from a tertiary Hospital in Northeast of Malaysia. All patients who were presented with acute fever suspected to have dengue from 2023 till 2025 were included.Diagnosis was confirmed using NS1 antigen and IgM antibody tests. Those with serology negative were the control group. A total of 200 patients were analyzed, comprising 126 dengue-positive and 74 dengue-negative cases. Hematological parameters were measured using automated hematology analyzers Mindray BC-6000 series, independent t-test was performed to compare mean values between dengue-positive and dengue-negative groups. A p-value of less than 0.05 was considered statistically significant. Results:Among the 126 dengue-positive patients, 82.5% were positive for NS1 antigen, 10.3% for IgM antibody, and 7.1% for both markers. Females accounted for 61.1% of positive cases, while males represented 38.9%. The most affected age group was between 16 and 45 years (53.2%). Hematological findings revealed leukopenia in 63.5% and thrombocytopenia in 64.3% of dengue-positive patients. The mean age of dengue-positive individuals (36.4 years) was significantly lower than dengue-negative patients (52.5 years). Significant decreases were observed in WBC, hemoglobin, hematocrit, and MCHC levels (p <0.001). Conversely, MCV, platelet count, PDW, MPV, and PCT showed no significant differences. Neutrophil counts and absolute neutrophil levels were markedly reduced (p <0.001), while absolute lymphocyte, monocyte, and eosinophil count also showed significant reductions (p <0.05). Moreover, inflammatory indices derived including NLR (p= 0.013) demonstrated statistical significance, suggesting their diagnostic relevance. Conclusions:Dengue infection was more prevalent among females and younger adults. Leukopenia, thrombocytopenia, and significant reductions in key hematological parameters were characteristic findings among dengue-positive patients. Routine hematological testing thus serves as a rapid, cost-effective diagnostic tool to support early detection and management of dengue infection.

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Rapid Detection Parameter For Sepsis (Rdps): A Multiparameter Hematological Biomarker For The Early Diagnosis Of Sepsis In Emergency Departments
Xiaohe Zheng¹, Weidong Chen², Yating Zhao³, Shiyao Pan⁴, Liang Zhou⁴, Yan Liu⁴, Shihong Zhang¹
¹Department of Laboratory Medicine, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, , China, ²Emergency Department, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, , China, ³Department of Laboratory Medicine, Nansha, The First Affiliated Hospital, Sun Yat-Sen University, Guangzhou, , China, ⁴Department of Clinical Research & Medical Affairs, Shenzhen Mindray Bio-Medical Electronic Co., Ltd., Shenzhen, , China

Background: Sepsis is a life-threatening condition characterized by a dysregulated host response to infection, leading to organ dysfunction and high mortality. Early recognition and intervention are critical for improving clinical outcomes, yet timely diagnosis in the emergency department (ED) remains challenging. Despite the existence of multiple hematological parameters and biomarkers for sepsis diagnosis, most suffer from insufficient sensitivity or specificity. They often fail to distinguish sepsis from other inflammatory conditions due to overlapping clinical manifestations, resulting in delayed diagnosis, inappropriate treatment, and poor prognosis. Procalcitonin (PCT) and monocyte distribution width (MDW), as commonly used biomarkers, still have limitations in clinical application, which highlights the urgent need to develop more reliable and efficient diagnostic tools to address unmet clinical needs. Methods: A retrospective study was conducted using data from the Mindray BC-6800 plus hematology analyzer, including training and validation cohorts. The training cohort involved screening 6520 emergency department (ED) patients aged ≥18 years (June–August 2024), of whom 827 met inclusion criteria (complete clinical data, C-reactive protein (CRP)/procalcitonin(PCT)/routine blood test results, and 30 random patients monthly). From these 827 patients, 195 (146 non-sepsis, 49 sepsis) were randomized for monocyte distribution width (MDW) testing. The validation cohort included 2297 ED Observation Room patients (November 2023–October 2024); 356 were eligible (matching training cohort inclusion criteria, excluding overlap with the training cohort), comprising 302 non-sepsis and 54 sepsis patients. Diagnostic performance was evaluated via receiver operating characteristic (ROC) curve analysis, with area under the curve (AUC) as the primary metric: the training cohort identified promising parameters, and the independent validation cohort confirmed their clinical utility. Results: In the training cohort, the parameter N_WBC_SFL_W showed an AUC of 0.81 (95% CI: 0.74-0.88). A novel combination parameter (N_WBC_SFL_W and D_Mon_SSC_W), named the Rapid Detection Parameter for Sepsis (RDPS), demonstrated the best performance with an AUC of 0.85 (95% CI: 0.78-0.92), sensitivity of 79.6%, and specificity of 80.8%. This outperformed PCT (AUC: 0.80; 95% CI: 0.72-0.86) and MDW (AUC: 0.76; 95% CI: 0.69-0.84). In the validation cohort, the RDPS maintained a specificity of 80.8% with a sensitivity of 66.7%. Conclusion: The novel hematological parameter RDPS shows promising diagnostic efficacy for sepsis compared to PCT and MDW. Its implementation in the ED could facilitate earlier, more accurate identification of septic patients, supporting timely intervention and potentially improving outcomes. Keywords: sepsis, emergency department, diagnosis, hematological parameter

P128

Machine Learning-Based Early Screening Of Myelodysplastic Syndromes Using Complete Blood Count
Guoqing Zhu¹, Juan Zhang²
¹Institute of Hematology & Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College, Tianjin, China, ²Sichuan Academy of Medical Sciences & Sichuan Provincial People’s Hospital, Chengdu, China

Background: Myelodysplastic syndromes (MDS) are hematopoietic stem cell-originated disorders with high progression risk to acute myeloid leukemia. Current diagnosis relies on invasive bone marrow aspiration and cytogenetic testing, which are time-consuming, costly, and susceptible to interference from cytopenic caused by inflammation or immune disorders. There is an urgent need for a cost-effective, rapid laboratory screening tool to differentiate MDS from other cytopenias. This study aims to develop an MDS risk prediction model using routine blood test parameters to reduce unnecessary invasive procedures and improve early screening. Method: This retrospective cohort study included 854 samples: 80 confirmed MDS patients, 412 cytopenia patients without MDS morphological evidence, and 362 age-matched healthy controls. All diagnoses followed international standards. Complete blood count parameters were analyzed using the Dymind DH-800 serial hematology analyzer. Multiple machine learning algorithms (decision trees, random forests, XGBoost, SVM) were trained with SMOTE for class imbalance. Model performance was evaluated by AUC, with SHAP analysis for feature importance and interpretability. Results: Feature selection analysis using the Gain algorithm identified eight key parameters, including two conventional hematological parameters (hemoglobin, HGB; platelet count, PLT) and six leukocyte scatter plot-derived parameters (DIFF_NEU_Y, WNR_WBC_X, DIFF_LYM_Y, DIFF_WBC_Y, DIFF_MON_Y, DIFF_MON_Z). These parameters are ranked in descending order of their Gain values, reflecting their respective contributions to MDS risk prediction. The support vector machine (SVM) demonstrated superior diagnostic performance among tested algorithms. In an independent validation set (27 MDS, 120 cytopenia, 110 healthy controls), the SVM model achieved 81.48% sensitivity, 99.57% specificity, and an AUC of 0.98 for MDS warning. This performance significantly surpassed the Horiba-MDS scoring system (65% sensitivity, 90.6% specificity, AUC 0.841), showing clear advantages in differentiating MDS from non-malignant cytopenias. Conclusion: We successfully developed an MDS screening model based on routine parameters from the Dymind DH-800 serial hematology analyzer. The high specificity (99.57%) and good sensitivity (81.48%) of the model support its feasibility as a clinical warning tool. This digital solution enables efficient differentiation of MDS from other cytopenias, promoting a shift from invasive to non-invasive screening paradigms.

P129

Evaluation Of Different Methods Of Reporting And Interpretation Of Drvvt Testing
Zahra Al-Ghammari¹, Muntadhar AL-Moosawi ²
¹Oman Medical Specialty Board, Muscat, Oman, Oman, ²Cleveland Clinic, Abu Dhabi, UAE. , United Arab Emirates

Evaluation of Different Methods of Reporting and Interpretation of DRVVT Testing Dr Zahra Al-Ghammari1, Dr Muntadhar AL-Moosawi 2 1 Hematopathology Residency Training Program, Oman Medical Specialty Board, Muscat, Oman. 2 Pathology And Laboratory Medicine, National Referance Laboratory, Cleveland Clinic Abudhabi,UAE. Abstract:
Background: Antiphospholipid syndrome (APS) is an autoimmune thrombophilia characterized by recurrent thrombosis and pregnancy morbidity in the presence of persistent antiphospholipid antibodies (aPL). Diagnosis requires both clinical manifestations and persistently positive aPL, including lupus anticoagulant (LA). The Dilute Russell’s Viper Venom Time (DRVVT) assay is a central tool in LA detection; however, variability in reporting and interpreting DRVVT results across laboratories introduces inconsistency in diagnosis and clinical decision-making. Aim: To compare three DRVVT reporting methods and determine the most reliable interpretive approach to support standardization and improve diagnostic accuracy. Methods: We conducted a cross sectional study, including the results of all samples with positive DRVVT screen tests performed in the period from September 2022 to September 2023 at a referral coagulation laboratory in Oman. Three different reporting methods were compared. These methods are (1) index-based method (IB): [DRVVT screen - DRVVT confirm / DRVVT screen], (2) ratio-based method (RB): [DRVVT screen / DRVVT confirm], and (3) normalized ratio method (NR): [DRVVT screen ratio / DRVVT confirm ratio] which involves obtaining ratios at each step by comparing the patients result with normalized pooled plasma (NPP) By evaluating different approaches, the study seeks to identify optimal practices at a referral coagulation laboratory in Oman,to enhance diagnostic accuracy and facilitate standardized reporting in diverse clinical settings. The Agreement between methods was assessed using Kappa Coefficient. Results: A total of 101 results were analyzed. Concordance among all three methods was observed in 64% of cases (65/101). The strongest agreement was found between the NR and IB methods (κ= 0.742; 95% CI: 0.598–0.885). Between these two methods, 5 cases were positive by NR but negative by IB, while 6 cases were positive by IB but negative by NR. Agreement was substantially lower between NR and RB (κ= 0.301; 95% CI: 0.014–0.589) and between IB and RB (κ= 0.300; 95% CI: 0.120–0.479). Conclusion: The study highlights the need for standardized DRVVT reporting to reduce inconsistencies in LA interpretation. Among the evaluated methods, the normalized ratio approach demonstrated the highest reliability and strongest agreement, supporting its use as the preferred reporting method in clinical laboratories.

P131

Haematological Biomarkers: Von Willebrand Factor, Adamts13 And Fvw/Adamts13 Ratio In Atrial Fibrillation Patients With Or Without Ischemic Stroke.
Carlos Arean-Martinez¹, Alan F Cano-Mendez², Nallely Garcia-Larragoiti², Larissa Herrera-Rodríguez¹, Martha Eva Viveros-Sandoval²
¹Servicio de Cardiología. Hospital General Miguel Silva, SSM, Morelia , Mexico, ²Laboratorio de Hemostasia y Biología Vascular. "Ignacio Chavez" School of Medicine, UMSNH., Morelia, Mexico

Background: Atrial fibrillation (AF) is the most common arrhythmia worldwide and is directly associated with thrombotic events. Clinical scales such as CHA₂DS₂-Vasc can identify thrombotic risk; however, they may have limitations. The measurement of haematological biomarkers such as von Willebrand factor (vWF), the restriction enzyme ADAMTS13, and the FvW/ADAMTS13 ratio, as well as imaging biomarkers such as arterial calcium, has been shown to impact the identification of thrombotic risk in patients with AF. Aim: To assess FvW, ADAMTS13, FvW/ADAMTS13, and coronary calcium scores in patients with AF with and without a history of ischemic stroke (IS) who were receiving anticoagulant therapy or not. Patients and Methods: 88 patients in 5 groups: patients with AF without IS receiving oral anticoagulant therapy (DOAC), patients with AF with IS receiving DOAC therapy, patients with AF without IS receiving no treatment, patients with AF with IS receiving no treatment, and a group of patients without AF matched for age and comorbidities. The concentration of FvW and ADAMTS13 was determined by ELISA, and the FvW/ADAMTS13 index was calculated. Calcium scores were determined by cardiac tomography. Results: 40% female patients, average age 71.55 years. Higher FvW concentrations were found in AF patients not receiving anticoagulant therapy (391.82 ± 208.46 and 394.43 ± 153.55, p=<0.01). A positive correlation was found between FvW and the CHA₂DS₂-Vasc scale, with higher concentrations in patients at greater risk of embolism. A negative correlation was found between ADAMTS13 and the CHA₂DS₂-Vasc scale. No correlation was found between the coronary calcium score and biomarkers; however, a calcium score >100 AU was observed in 48.8% of patients. Conclusions: FvW is a haematological biomarker of endothelial dysfunction, useful for assessing the risk of cardiovascular complications, which is elevated in patients with AF who are not receiving DOAC therapy. ADAMTS13 concentrations were lower in patients with a history of IS. Assessment of coronary calcium score may be useful for identifying patients at higher risk of embolic complications and for the etiological diagnosis of AF.

P132

Validation Of The Revohem&Trade; Fib Lrt Reagent On The Cn-3000 Analyzer: Analytical Performance And Method Comparison
Ivana Buttice, Anne-sophie Vandoorne, Samuel Vandooren, Jonathan Brauner, Phuong Ngyuen
Clinical laboratory CH EpiCURA , 7800 Ath, , Belgium

Introduction Fibrinogen is a plasma glycoprotein involved in platelet aggregation and fibrin formation during secondary hemostasis. Congenital or acquired fibrinogen disorders lead to quantitative or qualitative abnormalities affecting clot stability and are routinely assessed using the Clauss method, the reference technique based on thrombin-induced clotting time in diluted plasma. The aim of this study was to evaluate the analytical performance of the Revohem™ FIB LRT reagent on the Sysmex CN-3000 analyzer and to assess its commutability with established fibrinogen reagents. Materials and Methods Analytical performance was assessed for repeatability, within-laboratory precision, accuracy, linearity, limit of quantification (LOQ), stability, carryover, and hemolysis interference. Measurements were performed using Revohem™ FIB LRT, Dade Thrombin, and QFA Thrombin reagents with standardized calibration and daily internal quality controls. Repeatability and within-laboratory studies were conducted using pooled plasma samples and commercial controls, while accuracy was evaluated using Sciensano external quality assessment samples. Method comparison was performed on forty patient samples using Deming regression and Bland–Altman analyses on the Sysmex CN-3000 and ACL TOP CTS 300 analyzers. Hemolysis interference was assessed by artificially inducing hemolysis in vitro through freezing plasma with red blood cells, with measurements performed at hemoglobin concentrations up to 400 mg/dL. Linearity and LOQ were determined by serial dilutions, stability was evaluated over 48 hours, and carryover was assessed by alternating high- and low-fibrinogen samples. Results Revohem™ FIB LRT demonstrated excellent repeatability and within-laboratory precision, with coefficients of variation below 5.4%. Accuracy was confirmed by Z-scores below 3. Revohem™ FIB LRT demonstrated linearity from 0.84 to 7.18 g/L, LOQ at 0.84 g/L, stability deviation below 5% after 48 hours, and a carryover rate of 1.12%. Hemolysis interference testing revealed reagent-dependent effects. Revohem™ FIB LRT and Dade Thrombin maintained analytical stability with negligible bias even at high hemoglobin concentrations, while QFA Thrombin showed fibrinogen overestimation and increased variability under moderate to severe hemolysis (≥ 200 mg/dL hemoglobin). Method comparison showed no systematic or proportional bias when compared with Dade Thrombin, whereas both types of bias were observed with QFA Thrombin. Conclusions The Revohem™ FIB LRT reagent on the Sysmex CN-3000 analyzer showed robust analytical performance and good commutability with reference methods. Unlike QFA Thrombin, it preserved analytical accuracy in the presence of hemolysis. These results support its suitability for reliable fibrinogen quantification in routine clinical hemostasis laboratories.

P133

Characterizing Coagulation Dynamics In Hormonal Contraception Users Using Thromboelastography
Stefania Coroneos¹, Regan Bucciol¹, Yousra Tera^1,2, Maica Yunon³, Caroline Mwubaha^3,4, Maha Othman^1,2,3
¹Queen's University, Kingston, ON, Canada, ²Mansoura University, Mansoura, Egypt, ³St. Lawrence College, Kingston, ON, Canada, ⁴Infectious Disease Research Collaboration, Kampala, Uganda

Introduction: Hormonal contraception (HC) is widely used for both fertility regulation and management of hormonally mediated conditions, yet it is associated with an increased thrombotic risk. This risk is largely attributed to estrogen-mediated shifts toward a procoagulant state, though the magnitude and clinical relevance of these changes vary by formulation, route of administration, and individual susceptibility. Conventional coagulation assays are limited in their ability to detect hypercoagulability or predict the risk. This study interrogates coagulation dynamics among HC users and non-users using thromboelastography (TEG) to characterize differences in clot initiation, propagation, and thrombus generation. Methods: In this cross-sectional study, 83 healthy women aged 18-48 years were recruited, including 44 HC users and 39 non-users. Blood samples were collected during the first 14 days of the menstrual cycle to minimize hormonal variability. TEG analysis was performed using the TEG® 5000 system, assessing standard parameters (R, K, α-angle, MA, CI, LY30) and thrombin generation-derived measures obtained from first-derivative velocity curves (TG, maximum rate of thrombus generation [MRTG], and time to maximum rate of thrombus generation [TMRTG]). Group comparisons were conducted using Wilcoxon rank-sum tests due to non-normal distributions; MRTG was additionally evaluated using a parametric t-test as a sensitivity analysis. Effect sizes were calculated, and principal component analysis (PCA) was used to assess multivariate coagulation patterns. Results: No TEG parameters differed significantly between HC users and non-users at the conventional α = 0.05 threshold using non-parametric testing. At a more permissive α = 0.10, HC users demonstrated shorter K times, higher α-angles, and increased MRTG, suggesting modest acceleration of early clot formation and thrombus generation. MRTG was significantly higher in HC users on parametric testing (p = 0.025; Cohen’s d = 0.50), though this difference was not preserved using rank-based methods, indicating heterogeneity within the HC group. Measures of clot strength and fibrinolysis (MA, CI, LY30, TG) were preserved. PCA revealed substantial overlap between groups, with no distinct multivariate coagulation phenotype associated with HC use. Conclusion: HC use was not associated with overt global hypercoagulability detectable by TEG in this low-risk cohort. However, modest, heterogeneous shifts toward accelerated clot propagation and thrombus generation were observed in a subset of HC users. These findings support TEG as a sensitive exploratory tool for detecting subtle coagulation changes and highlight its potential role in individualized thrombotic risk stratification rather than population-level screening.

P134

Comparison Of Different Interpretative Methods Of Activated Partial Thromboplastin Time Immediate Mixing Test In Paediatric Patients
Yee Zhe Heng, Qiu Yan Wong, Soh Hong Ang, Rebecca A. Barton, Joyce C. M. Lam
KK Women's and Children's Hospital, Singapore, Singapore

Introduction Coagulation mixing tests are performed to assist in determining the cause of an unexpected prolongation of activated partial thromboplastin time (APTT), i.e. factor deficiencies versus inhibitors. Different methods exist to determine whether a correction in APTT has occurred. This study aims to compare three common methods of interpreting the immediate APTT mixing test in the paediatric population. Materials and Methods APTT immediate mixing tests were performed on 225 specimens from paediatric patients (<16-years-old) between September 2023 and September 2025. The results were interpreted using three methods: correction to within reference range (RR method), the Rosner Index with correction threshold value of 15, and the percent correction with threshold value of 70 as recommended by the International Council for Standardization in Haematology (ICSH). Factors VIII and IX levels testing were performed in-house, whereas testing of factors XI and XII, von Willebrand antigen levels and lupus anticoagulant were performed in an external reference laboratory. Results Concordance was excellent between the RR method and Rosner Index (91.1%), but poorer between the RR and the percent correction method (50.2%). 104 specimens were tested for factor deficiencies; the sensitivity of RR, Rosner Index and percentage correction method was 91.7%, 88.9% and 33.3% respectively, while their respective specificity was 16.2%, 26.4% and 73.5%. 49 specimens were tested for lupus anticoagulant; the sensitivity of RR, Rosner Index and percentage correction method was 70.0%, 80.0% and 90.0% respectively, while their respective specificity was 76.9%, 71.8% and 17.9%. Conclusions There is no single interpretation method that completely distinguishes between factor deficiencies and the presence of inhibitors. For factor deficiencies, the RR method and Rosner Index show good agreement with regards to sensitivity for paediatric patients, whereas a lower correction threshold than currently recommended may be required for the percent correction method. All three methods show comparable sensitivity for lupus anticoagulant, however limited sample size precludes recommendation of the superior method. Incubated mixing tests were not performed therefore time and temperature-dependent inhibitors could not be detected in this study.

P135

Evaluation Of The Sysmex Automated Blood Coagulation Analyser Cn^Tm-700: Precision Assessment And Comparative Analysis With The Automated Blood Coagulation Analyser Sysmex Cn^Tm-6000.
Kevin Horner ¹, Kerensa Leeson ¹, Irfan Patel ², Seigo Munehisa ³, Sakayori Tasuku ³, Matsuno Eriko ³, Kieron Hickey ¹, Steve Kitchen ¹
¹Haemostasis Department, South Yorkshire and Bassetlaw Pathology Royal Hallamshire Hospital, Sheffield, United Kingdom, ²Sysmex UK Ltd, Milton Keynes, United Kingdom, ³Sysmex Corporation, Kobe, Japan

Introduction
Automated Blood Coagulation Analyzer CN-700 (Sysmex Corporation, Japan) is a novel compact addition to the CN family of coagulation analysers, designed as an accessible alternative with advanced features comparable to higher-end models. This study assessed the performance of the newly developed CN-700 coagulation analyser capable of performing routine clotting, chromogenic and immuno-turbidimetric assays. Methods The analyser's reproducibility was evaluated through duplicate measurements of commercial controls and pathological sample pools, conducted twice daily over 20 days. Method comparison with Automated Blood Coagulation Analyzer CN-6000 the Sysmex CN-6000 (Sysmex Corporation, Japan) involved at least 120 samples for each assay type, utilising plasma from both anonymised patients and healthy donors to assess correlation across a broad measurement range. The following tests, parameters, and reagents were assessed: Prothrombin Time (PT), measured in seconds, and PT-derived fibrinogen using Innovin and Thromborel S; PT percentage and INR were calculated using two standards (SHP and PT Multicalibrator); Activated Partial Thromboplastin Time (APTT), measured in seconds and APTT ratio using Actin FS, Actin FSL, and Pathromtin SL; Thrombin Time (TT), assessed using Test Thrombin; Clauss Fibrinogen, quantified with Dade Thrombin; Antithrombin activity, measured with Berichrom Antithrombin III and INNOVANCE Antithrombin; and D-Dimer, assessed using INNOVANCE D-Dimer. All reagents were supplied by Sysmex (Hamburg, Germany). Results The CN-700 exhibits a high level of precision across all reagent and assay parameters, as shown in Table 1. All control and pathological sample pools had reproducibility CVs of less than 6%, which are well within the acceptance limits that were used for the FDA 510K submission for the CN/CS series instruments. The only exception was antithrombin measured with Berichrom ATIII reagent against Control P, which had a moderately raised CV of 10.2%. However, this value is still within the acceptance limit of 12.5%. A strong correlation was observed between the CN-700 and CN-6000, with slopes ranging from 0.927 to 1.073 and correlation coefficients between 0.995 and 0.999 for all assays. Importantly, no misclassifications occurred between the two systems, and data points were closely aligned along the line of identity. Conclusions The results indicate that the CN-700, despite being the smallest low-end analyser in the CN family, provides user-friendly operation, high functionality, and exceptional reliability. It facilitates routine coagulation and specialised assays with acceptable imprecision levels and demonstrates excellent comparability with the CN-6000.

P136

Identification Of Underlying Genetic Variants In Sporadic Factor Viii Deficiency Through Precision Genomics
Tehmina Khan¹, Shahla Sohail², Masood Fareed Malik³, Arshi Naz⁴
¹IMU University, Kuala Lumpur, Malaysia, ²Shahla Sohail, Lahore, Pakistan, ³Masood Fareed malik, Lahore, Pakistan, ⁴Liaquat University of Medical and Health Sciences, Jamshoro, Pakistan

Introduction: Haemophilia A is an X-linked genetic disorder, characterized by a deficiency in the production or function of the blood clotting factor VIII. Factor VIII or Antihemophilic factor, plays a vital role in human blood clotting system. It is involved in the intricate series of events resulting in the development of an insoluble fibrin clot. Deficiency can lead to haemorrhagic manifestations. We aim to report a case of Haemophilia A in a child who is first born son with a negative family history of F8 deficiency through clinical presentation and genetic analysis. Methods: Coagulation studies including a prolonged activated partial thromboplastin time (aPTT) and factor VIII activity was done on proband ,expectant mother (P₁G₂) and father. Chorionic villus sampling was performed to evaluate gene variant in feotus . Later, mutational analysis was performed using whole exome sequencing (WES) to identify variants in the F8 gene.Pathogenecity scoring on identified genetic variant was done using different computational software tools to evaluate the disease causing mutation from neutral polymorphism in genetic sequence. Results: The results revealed a novel mutation in the F8 gene in a proband, however no variant identified in the foetus and their mother, confirming the sporadic mutation in proband. The pathogenecity tools predicted this mutation to be deleterious and disease causing .
This case underscores the usefulness of genetic testing, specifically exome sequencing, in diagnosing the cases as new gene flaw and managing this condition across generations. Early detection and tailored treatment strategies are crucial in improving outcomes and quality of life for affected individuals.

P137

Modern Hemostasis Testing: Comparison Of The Sysmex Cs-2500 And Cn-3000 Analyzers&Rsquo; Impact On Laboratory Efficiency
XJ Koa, HM Zulkifli, N Ruzlan, NA Kamaludin , TC Aw
Department of Laboratory Medicine, Changi General Hospital, Singapore, Singapore, Singapore

Introduction:
We evaluated the Sysmex CN-3000 coagulation analyzer in 2025 as a possible replacement for the Sysmex CS-2500 that was in use since 2016. Equipped with new technology, the compact CN-3000 is attractive as it allows STAT sampling, easier reagent access, and requires lower sample volume (50uL). With our rising workload we were concerned with throughput and result turnaround times (TAT). Methods: We verified the analytical performance of the CN-3000. The reference ranges derived from healthy subjects (n=40) for PT, aPTT, fibrinogen and D-dimer were compared to the CS2500. We also compared the TAT (from receipt in the laboratory till transmission of results to the hospital medical records) for PT alone and PT+aPTT assays on the 2 platforms over similar 2-month periods. Results: The CN3000 performance is as follows. Intra-assay precision: normal control samples - 0.4% for both Prothrombin Time (PT) & Activated Partial Thromboplastin Time (aPTT); Abnormal controls - 0.5% for PT & 0.8% for aPTT. Inter-assay precision: Normal control - 1.1% (PT) and 0.4%(aPTT); Abnormal control - 2.1% (PT) and 0.8% (aPTT). Correlation and regression analyses showed close agreement between the CN3000 (y) and CS2500 (x) analytical platforms. For PT: y = 1.0033x – 0.0656 and r² = 0.9972 . For APTT y = 1.0158x – 0.4365 and r² = 0.9988. For Fibrinogen y = 1.0344x – 0.007 and r² = 0.9966. For D-dimer y = 1.0363x – 0.0467 and r² = 0.9960. Reference ranges (n=40) from healthy adult samples for PT, aPTT, fibrinogen, and D-dimer were very similar on both Sysmex platforms. The CN-3000 achieved shorter overall TATs. For PT, CN3000 (n=7263) Median TAT & Inter-quartile range (IQR) was 16min (13min, 18min) versus CS-2500 (n=7669) Median TAT & IQR was 17min (15min, 21min). For PT+aPTT, CN-3000 (n=5835) Median TAT & IQR was 16min (14min, 19min) versus CS-2500 (n=5789) Median TAT & IQR was 18min (15min, 21min). Like the CS2500, the performance of the CN3000 on the College of American Pathologists (CAP) external quality assurance program for coagulation assays was satisfactory. Conclusion: The performance of CN3000 compares favorably with the CS2500. Assay TAT is much better with the CN3000. Its ergonomics and smaller footprint is another attractive feature. We adopted the CN-3000 analyzer into service since July 2025.

P138

Comparative Assessment Of The Sysmex Automated Blood Coagulation Analyser Cn^Tm-700 With The Automated Blood Coagulation Analyser Sysmex Ca^Tm-660
Kerensa Leeson ¹, Kevin Horner ¹, Irfan Patel ², Seigo Munehisa ³, Sakayori Tasuku ³, Matsuno Eriko ³, Kieron Hickey ¹, Steve Kitchen ¹
¹Haemostasis Department, South Yorkshire and Bassetlaw Pathology, Royal Hallamshire Hospital, Sheffield, United Kingdom, ²Sysmex UK Ltd, Milton Keynes, United Kingdom, ³Sysmex Corporation, Kobe, Japan

Introduction Automated Blood Coagulation Analyzer CN-700 (Sysmex Corporation, Japan) is the latest model of compact haemostasis analyser, that will replace Automated Blood Coagulation Analyzer CA-600 series (Sysmex Corporation, Japan). There are significant hardware and software differences between these two devices that could influence the results they produce. A method comparison was performed between the CA-600 and CN-700 analysers. Methods A performance evaluation was carried out between the two analysers for: Prothrombin Time (PT), Activated Partial Thromboplastin Time (APTT), Clauss Fibrinogen, Thrombin Time (TT), chromogenic Antithrombin, and immuno-turbidimetric D-Dimer assays. The following reagents were used: Innovin for PT, Actin FS for APTT, Dade Thrombin for Clauss Fibrinogen, Test Thrombin for TT, Berichrom Antithrombin III for Antithrombin, and Innovance D-Dimer for the D-Dimer assay. All reagents were sourced from Sysmex (Hamburg, Germany). A minimum of 120 samples per assay were analysed, consisting of plasma from anonymised patients and healthy donors, to assess correlation across a range of measurements. Statistical analysis was conducted using Passing-Bablok regression and Spearman’s Rank Correlation Coefficient (rs). Results A strong correlation was observed between the CN-700 and Automated Blood Coagulation Analyzer CA-600 series CA-660 across most assays, as summarised in Table 1. The slopes for these comparisons varied from 0.911 to 1.093, with the exceptions of Clauss fibrinogen and Thrombin Time, which were marginally outside the acceptance threshold of 1.000 ± 0.100, recording values of 1.132 and 1.131, respectively. The correlation coefficients for most assays ranged between r = 0.964 and r = 0.988, except for thrombin time (r = 0.740), which fell below the acceptance limit of 0.950. Regarding Thrombin Time, prolonged results were noted on the CA-660, while the CN-700 yielded results compatible with those of normal individuals. Notably, 11 samples exhibited an analysis time over flag on the CA-660 yet produced normal results on the CN-700. Conclusion The findings of this study show a strong agreement between the CN-700 and CA-660 devices. However, some inconsistencies in Thrombin Time were observed, likely due to differences in the coagulation endpoint detection algorithm. Although the Thrombin Time showed poor correlation, the CN-700's ability to correct previously flagged analysis errors represents a significant advantage. Additionally, the CN-700 is more user-friendly, offers improved functionality, and demonstrates exceptional reliability, making it a suitable option as a low throughout analyser.

P139

Procoagulant Shift Induced By Oral Contraceptives In The St Genesia&Reg; Thrombin Generation Assay: A Distinct Subgroup In An Ongoing Local Reference Range Study In Portugal
Tiago Luís¹, Dalila Marques¹, Joana Rita¹, Ana Campos¹, Paula Leal², Goncalo Orfao¹, Nelya Syamro¹, Olena Lavrukhina¹, Marco Matias¹, Jorge Tomaz¹
¹Congenital Coagulopathies Reference Centre-Ramón Salvado, Thrombosis and Haemostasis Unit, Blood Bank and Transfusion Medicine, Coimbra, Portugal, ²Resident Pharmacist in Clinical Pathology, Coimbra, Portugal

Introduction
The ST Genesia (STG) is a fully automated analyser for thrombin generation (TG), increasingly recognized for its value in assessing bleeding and thrombotic risk and in monitoring both inherited and acquired haemostatic disorders. Its clinical utility depends on robust, population-specific reference intervals (RIs) and on strong analytical standardisation to ensure accurate and reproducible measurement of global haemostatic function.
This study aimed to establish local TG reference values in a healthy Portuguese population using three STG panels, emphasizing the differences between women with and without oral contraceptives (W+OS and W-OCs, respectively), and men, including their sensitivity to thrombomodulin. Methods
TG was measured in platelet-poor plasma obtained by double centrifugation (2500g, 15 min) from 270 healthy donors (43.8 ± 12.2 years): 162 men (46 ± 11.3y), 101 W−OCs (51 ± 11.7y) and 68 W+OCs (32.5 ± 9.2y). Three STG reagent panels, STG-BleedScreen (BS, n=113), ThromboScreen (TS, n=114); and STG-DrugScreen (DS, n=104) were used. In 44 samples, two or three assays were performed in parallel (331 tests in total). Statistical analyses were performed with IBM®SPSSv30.0. Differences were considered significant at p<0.05. RIs (95% CI) were calculated as 2.5–97.5th percentiles for absolute and normalised TG parameters in men and W-OCs, while descriptive statistics and ranges were generated separately for W+OCs.
Results
Men and W-OCs showed similar TG profiles across all reagent panels, whereas W+OCs had significantly higher TG values, particularly in peak height, endogenous thrombin potential (ETP) and velocity index (VI), alongside a marked reduction in natural anticoagulant response to thrombomodulin (ETP reduction 24.9%; p<0.001 vs. other groups). Local RIs for BS, DS and TS were defined for the population without OCs, while W+OCs displayed right-shifted ranges for peak height, ETP and VI across assays. Inter-individual variability was highest for VI (55.5% BS, 22.2% DS, 52.1% TS) and peak height in BS and TS, and lowest for Start Tail. Variability in peak height decreased with higher tissue factor concentrations (TS and DS).
Conclusions
These data provides preliminary STG TG reference values for a Portuguese population and confirms the clear procoagulant shift and attenuated thrombomodulin sensitivity in W+OCs, alined with recent literature. Excluding W+OCs from global RIs seems to enhance assay sensitivity and may improve clinical interpretation, while defining separate TG ranges for W+OCs supports a most shaped approach to risk assessment. The high inter-individual variability observed highlights the need for assay- and population-specific RIs and for longitudinal studies to further elucidate the effects of OCs on coagulation dynamics.

P140

Impact Of Cartridge Lot-To-Lot Variability On Extrinsic Thromboelastometry Clotting Time: Risk Of Misinterpretation Across Clinical Decision Thresholds
Hoyoung Moon¹, Taekyun Kim¹, Mijeong Kim¹, Daehyun Chu^1,2, Miyoung Kim^1,2, Young-Uk Cho^1,2, Seongsoo Jang^1,2
¹Department of Laboratory Medicine, Asan Medical Center, Seoul, Korea, ²Department of Laboratory Medicine, University of Ulsan College of Medicine, Seoul, Republic of Korea, Seoul, Korea

Rotational thromboelastometry (ROTEM) assesses the entire blood clotting process and supports transfusion decision-making. ROTEM-guided bleeding risk management algorithms use the thromboelastometry with extrinsic activation clotting time (EXTEM CT) threshold of 80 sto guide coagulation factor therapy. During verification for our institutional transition from the ROTEM Delta to the ROTEM Sigma system, we observed shifts in EXTEM CT. In this study, we evaluated cartridge lot-to-lot variability and its impact on reference interval (RI) establishment. Residual citrated whole blood samples from 95 healthy adults (48 males, 47 females; 20–50 years) collected after routine health screening between December 2024 and November 2025 were analyzed. All subjects had prothrombin time, activated partial thromboplastin time, and platelet count within the reference range, with no history of anticoagulant use. EXTEM CT was measured using 10 cartridge lots (A to J) under routine laboratory conditions. RIs were calculated using the non-parametric percentile method (2.5th–97.5th) in accordance with the Clinical and Laboratory Standards Institute (CLSI) guideline EP28-A3c. The manufacturer-provided RI for EXTEM CT (51–73 s) was narrower than the RI calculated in our institution (53–112 s). EXTEM CT differed across the 10 cartridge lots (Kruskal–Wallis test, p=0.008). Lot H showed significantly higher EXTEM CT values than the other nine lots combined (median 76.50 s [interquartile range, 62.00–97.25 s] vs. 62.00 s [interquartile range, 59.00–66.00 s]; Mann–Whitney U test, p=0.008) (Figure). Lot H exceeded the manufacturer’s upper RI limit of 73 s more frequently than all other lots combined (6/10 [60.0%] vs 10/85 [11.8%]; Fisher’s exact test, p=0.001), corresponding to an odds ratio of 11.25 (95% confidence interval, 2.70–46.86). After excluding 12 statistical outliers identified by the robust regression and outlier removal (ROUT) method (Q=1%), the upper RI limit remained 78 s. Significant lot-to-lot variability in EXTEM CT on the ROTEM Sigma system can lead to substantial shifts in calculated RIs and individual results. Importantly, such shifts may cause results to cross the clinical decision threshold of 80 s, with potential implications for transfusion or therapeutic decision-making. Therefore, rigorous lot-specific verification and parallel testing before implementation of new cartridge lots are essential to ensure diagnostic consistency and patient safety.

P141

Machine Learning-Based Detection Of In Vitro Activated Coagulation Using Aptt Clotting Curves
Keisuke Nishi¹, Akiyo Kawamoto¹, Eri Adachi¹, Takesh Suzuki¹, Nobuo Arai¹, Hiroshi Kurono¹, Kaho Kurashima^2,3, Itsuko Sato³, Tomoya Fukuoka^3,4, Ruri Kono^2,3, Kohei Morimoto², Yoshinori Nakamura², Yoshiharu Miyata^2,5, Takamitsu Imanishi³, Yoshihiko Yano³, Hiroshi Matsuoka²
¹Sysmex Corporation, Kobe, Hyogo, Japan, ²Kobe University Hospital Bioresource Center, Kobe, Hyogo, Japan, ³Department of Clinical Laboratory, Kobe University Hospital, Kobe, Hyogo, Japan, ⁴Department of Clinical Laboratory Science, Tenri University, Tenri, Nara, Japan, ⁵Department of Artificial Intelligence and Digital Health Science, Kobe University Graduate School of Medicine, Kobe, Hyogo, Japan

Introduction
Coagulation testing is essential for assessing hemostatic function, identifying bleeding or thrombotic tendencies, and monitoring treatment efficacy. However, pre-analytical errors, such as in vitro activated coagulation, can occur and may significantly affect test results, even in the absence of visible clot formation. Automated detection integrated into analyzers may help mitigate undetected cases and streamline laboratory workflows.

Methods
Samples suspected of in vitro activated coagulation were defined by one or more of the following criteria: (1) clot detection during visual inspection at sample receipt,(2) suspicion based on complete blood count (CBC) findings from simultaneously collected samples, (3) abnormal test results, including deviation from previous values of APTT and PT, shortened APTT (<25 seconds), or instrument measurement errors (e.g., Early Reaction Error),and (4) clot observed in the buffy coat post-analysis. For samples meeting these criteria, both the initial and re-collected samples were obtained as pairs. Pairs with minimal change in thrombin–antithrombin complex were excluded, as these were considered unlikely to represent in vitro activated coagulation, leaving 150 pairs (300 samples) for analysis. Measurements were performed using Automated Blood Coagulation Analyzer CN-6500 (Sysmex) with Actin FS and Actin FSL reagents (Siemens Healthineers). Features were extracted from APTT clotting curves and used to develop machine learning models for each reagent.

Results
The model using Actin FS achieved a sensitivity of 90.7%, specificity of 97.3%, and an AUC of 0.97. The model using Actin FSL achieved a sensitivity of 93.9%, specificity of 96.6%, and an AUC of 0.98.

Conclusions
This machine learning-based approach demonstrated favorable performance in detecting in vitro activated coagulation with both Actin FS and Actin FSL reagents. Implementing this technique may help reduce pre-analytical errors and improve laboratory workflow in routine coagulation testing.

P142

Method Comparison Of The Revohem Anti-Xa Lrt Assay Compared To Other Commercial Reagents &Ndash; Monitoring Of Unfractionated And Low Molecular Weight Heparin
Thomas Pitchford¹, Kieron Hickey¹, Steve Kitchen¹, Tasuka Sakayori², Sho Shinohara³, Miyu Kitagami²
¹Haemostasis Department, South Yorkshire and Bassetlaw Pathology, Royal Hallamshire Hospital, Sheffield, United Kingdom, ²Sysmex Corporation, Kobe, Japan, ³Hyphen BioMed, Neuville-sur-Oise, France

Introduction Accurate measurement of heparin levels is important in the management of anticoagulated patients. Unfractionated heparin (UFH) requires dose monitoring and adjustment and the use of anti-Xa assays over the traditional APTT ratio may be beneficial.Low molecular weight heparin (LMWH) doesn’t require monitoring if used according to licence but there are many settings where accurate drug concentrations are beneficial.^1,2Revohem Anti-Xa LRT is a ready to use liquid reagent, which can be used for the measurement of heparin as well as DOAC’s (Rivaroxaban, Apixaban and Edoxaban). The assay uses a single hybrid calibration curve for the measurement of both UFH and LMWH. BSH guidelines recommend that hybrid calibration curves should be locally validated against separate curves if adopted. ³ Method Plasma from anticoagulated patients (frozen aliquots from Sodium citrate samples (0.109M) stored at -70^OC) was tested with the Revohem Anti-Xa LRT (Sysmex, UK) on a Sysmex CN-6000 (Sysmex, UK) results were compared to HemosIL Liquid Anti-Xa (Werfen,UK) tested on an ACL Top 700 (Werfen, UK), STA Liquid Anti-Xa (Stago, UK ) tested on STA-R Max (Stago, UK ) and Hyphen Heparin LRT (Sysmex, UK) and Siemens INNOVANCE Anti-Xa both tested on a CN-6000 (Sysmex, UK). 120 samples were tested for both UFH and LMWH to assess comparability. The STA Liquid Anit-Xa and Hyphen Heparin LRT methods use unique calibration and separate calibrators for both UFH and LMWH measurement while the HemosIL Liquid Anti-Xa, INNOVANCE Anti-Xa and Revohem Anti-Xa LRT use hybrid calibration. Results See figure Conclusion Revohem Anti-Xa LRT assay demonstrated high levels of comparability with the Hyphen Heparin LRT reagent. These results support the use of the Revohem hybrid curve if switching from Hyphen to Revohem Revohem Anti-Xa LRT assay demonstrated good levels of comparability with the HemosIL Liquid Anti-Xa and the Siemens INNOVANCE Anti-Xa with an overall pattern of clinically irrelevant positive bias Revohem Anti-Xa LRT assay demonstrated a notable positive bias and poorer correlation when compared against the Stago-STA Liquid Anti-Xa during the measurement of UFH. Stago-STA Liquid Anti-Xa reagent does not contain dextran sulphate while all other reagents in the study do. Results match a pattern of higher results seen in dextran sulphate reagents previously reported ^4,5,6,7 Use of the Revohem hybrid curve if switching from Stago-STA Liquid Anti-Xa would require local clinical review

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Introduction Of A New Paired Reagent Combination For Lupus Anticoagulant Assays By Activated Partial Thromboplastin Time
Jude Platton^1,2, Minal Dave^1,2, Anne Horton^1,2, Katarzyna Kniola^1,2, Nada Yartey^1,2, Sean Platton³
¹Haemostasis Laboratory, Royal London Hospital, London, United Kingdom, ²National Health Service East and South East London Pathology Partnership, London, United Kingdom, ³The Royal London Hospital Haemophilia Centre, London, United Kingdom

Introduction International and national guidelines recommend paired reagents or phospholipid addition for Lupus Anticoagulant [LA] testing by dilute Russell’s Viper venom time [DRVVT] and activated partial thromboplastin time [APTT], but paired reagents for APTT have not been widely available. For Siemens and Sysmex users, Siemens Actin FSL has the same activator as Actin FS but different phospholipids; Pathromtin SL has different activator and phospholipids; none are paired reagents, and neither Actin FSL nor Pathromtin SL is sufficiently sensitive to LA for routine use. Hyphen Biomed Cephen LS and Cephen both use a silica activator; only phospholipid concentration differs. A recent study of these paired reagents found an overall sensitivity of 48.6%, and specificity of 89.0% for DRVVT-positive LA. We used the installation of Sysmex CN-6500 analysers as an opportunity to evaluate them. Methods Residual double-spun citrated plasmas from patients previously investigated for the presence of a lupus anticoagulant were selected at random. All had previously been assayed on a Sysmex CN-6000 analyser for DRVVT using Siemens LA1 and Siemens LA2, and for APTT using Stago PTT-LA and Siemens Dade Actin FS. Lyophilised Platelet Poor Plasma [PPP] (Technoclone) was used for mixing studies. Samples were re-assayed on a Sysmex CN-6500 analyser for DRVVT using LA1 and LA2, and for APTT using Cephen LS and Cephen with PPP for mixing studies. Reference ranges were derived from >120 patients whose clinical details gave no indication of disorders associated with antiphospholipid syndrome (APLS), and whose samples were LA negative by DRVVT and APTT. All were from non-pregnant, non-anticoagulated adults from general practice or outpatients. 99th-percentiles were used in line with guideline recommendations. A further 134 samples from patients being investigated for disorders associated with APLS were tested, and results correlated and compared with previous results. Results Refer to Figure 1A-C. Conclusions Hyphen Biomed Cephen LS and Cephen offer a paired-reagent solution for APTT testing for LA that is more sensitive to DRVVT-positive LA (42%) than Siemens Actin FSL (22%) and Siemens Pathromtin SL (13%). These reagents are now in routine use at our laboratory.

P144

Performance Evaluation Of Revohem^Tm Vwf Ag Reagent Featuring An Extended Measuring Range For High-Level Von Willebrand Factor Antigen
Hayato Saito¹, Takahiko Bando¹, Tasuku Sakayori¹, Sho Shinohara², Sophie Molton², Claire Dunois², Kaori Otake¹
¹Sysmex Corporation, Kobe, , Japan, ²Hyphen BioMed, Neuville-sur-Oise, , France

Introduction: Von Willebrand Factor (vWF) is a large multimeric protein that plays a crucial role in hemostasis, the process that stops bleeding at the site of injury. Blood vWF antigen levels, in combination with vWF activity levels, are essential for the diagnosis and classification of von Willebrand disease (vWD). Several reports suggest the potential clinical utility of high vWF antigen levels in various patient conditions, such as the prognosis of chronic liver failure. The ability to accurately measure high levels of vWF antigen is becoming increasingly important. Revohem^TM vWF Ag (Hyphen BioMed, France) is an in vitro diagnostic reagent utilizing the immunoturbidimetric method to measure vWF antigen levels. The reagent is in liquid form and ready-to-use with automated coagulation analyzers. In this study, we evaluated the performance of this reagent on an automated coagulation analyzer.
Methods: Analytical performance was evaluated including measurement range (linearity and limit of quantitation), precision, after opening stability, and on-board stability. Method comparison with two reference reagents was performed across measurement range.
Results: The linearity and limit of quantitation studies demonstrated a measuring range from 2% to 1600% with redilution/extrapolation. Within-laboratory precision studies showed coefficients of variation (CV) of 1.5% for normal range, 2.6% for abnormal range, respectively. Reagent was stable after opening up to 90 days at 2 to 8 degrees. The reagent was also stable at on-board condition up to 14 days. Method comparison studies showed excellent correlation with the existing liquid reagents: a correlation coefficient of 0.995 (y = 1.032x – 2.258) with VWF Ag (Siemens, Germany), and 0.989 (y = 0.895x – 1.514) with von Willebrand Factor Antigen (Werfen, Spain).
Conclusion: Revohem^TM vWF Ag demonstrated excellent performance on an automated coagulation analyzer. This reagent is useful for measuring high levels of von Willebrand Factor antigen.

P145

Discrepant Results In Fxiii Assays Due To Fxiii Replacement Therapy?
Sanne Vanthourenhout, Katrien M.J. Devreese
Ghent university hospital, Ghent, Belgium

Introduction A 12-year-old girl with a congenital factor XIII (FXIII) deficiency attends the day clinic monthly for administration of Cluvot® (purified human plasma-derived FXIII concentrate) and blood sampling to monitor the FXIII levels. Dosing is individualized based on body weight, laboratory values, and clinical condition, with administration every 28 days to maintain trough FXIII activity of approximately 5% to 20%. FXIII is routinely measured using the Hexamate Factor FXIII assay on the STAR MAX3 analyzer (Stago), a latex turbidimetric assay employing polyclonal anti-FXIII antibodies directed against the FXIII A subunit which has shown good performance and concordance with the clot solubility (CST) in previous in-house evaluation studies. The CST is a qualitative screening assay detecting severe FXIII deficiency (<3%) by clotting the plasma with thrombin and using urea to evaluate stability of the clot. During follow-up of the patient, discrepant results were occasionally observed, with a dissolved clot in the CST after 24 hours despite FXIII antigen levels of 8–10%. We wondered whether Cluvot® behaved differently compared to native FXIII. Methods A method comparison was performed between the Hexamate Factor FXIII assay (STAR MAX3) and the TECHNOFLUOR FXIII Activity assay on the Ceveron S100 analyzer (Technoclone), a fluorescence-based assay using a quenched peptide substrate cleaved by activated FXIII (FXIIIa). Analytical imprecision, limit of detection (LOD), and reference ranges were determined for both methods. Additionally, a dilution series was prepared by spiking FXIII-deficient plasma with Cluvot® and normal pooled plasma. FXIII was measured using both quantitative assays and CST in parallel. Results Both assays met predefined analytical imprecision criteria. The limit of detection was 3.2% for the Hexamate Factor FXIII assay and 2.2% for the TECHNOFLUOR FXIII Activity assay. In the dilution series, concordant results were observed between the quantitative FXIII assays and CST (see Table 1). Conclusion Although discrepant results between quantitative FXIII measurements and the CST were observed during clinical follow-up, these discrepancies could not be reproduced in a controlled dilution series. This suggests that analytical assay performance alone does not explain inconsistencies in FXIII monitoring at low activity levels, underscoring the importance of cautious interpretation of FXIII results near clinical decision thresholds.

P146

Trbc1 Flow Cytometry In The Assessment Of T-Cell Clonality: A Single-Center Experience
Tanja Antunovic¹, Ivana Milasevic¹, Enisa Zaric²
¹Centre for Clinical Laboratory Diagnostic, Clinical Centre of Montenegro, Podgorica, , ²Centre for Hematology, Clinic for Internal Medicine, Clinical Centre of Montenegro, Podgorica,

Introduction Flow cytometric diagnosis of chronic T-cell lymphoproliferative neoplasms (T-CLPD) is challenging because of overlapping reactive and malignant immunophenotypes. In recent years, the monoclonal antibody clone JOVI-1 has been introduced as a practical tool for assessing T-cell clonality through TRBC1 expression, mirroring the kappa/lambda paradigm in B-cells. Methods We retrospectively analyzed 58 samples (40 peripheral blood, 12 bone marrow, 1 ascites, and 5 cerebrospinal fluid) examined between 2024 and 2025 at the Centre for Clinical Laboratory Diagnostics, Clinical Centre of Montenegro. T-cell clonality was assessed using PE-conjugated TRBC1 (JOVI-1, BD Pharmingen). Cut-off values of <15% or >85% TRBC1 expression in CD4 or CD8 T-cell subsets were considered indicative of monoclonality. The median patient age was 62.5 years (51.0–67.0); 69% were male and 31% female. Forty-eight patients were referred for initial diagnostic workup mainly because of pancytopenia, lymphadenopathy, or splenomegaly, while 10 patients were referred for follow-up of peripheral T-cell lymphoma or Mycosis fungoides. In the first group of forty-eight patients T-cell clonality was assessed due to suspected expansion of T-cell subsets. Results In the peripheral blood group, the median leukocyte count was 7.61 × 10⁹/L (5.91–9.74), with median lymphocyte and T-lymphocyte counts of 2220 cells/µL (1502–5237) and 1602 cells/µL (1066–4128), respectively. The mean T-lymphocyte percentage was 77.5 ± 14.9%. Monoclonal CD4 or CD8 T-cell populations were detected in 6 of 40 peripheral blood samples (15%; 4 CD4 and 2 CD8). In 5 of these 6 cases (83%), clonality was confirmed by molecular / histopathological findings, while one case was reported as reactive. TRBC1 expression in polyclonal peripheral blood samples was 46.8 ± 5.0% in CD4 T cells and 44.3 ± 11.0% in CD8 T cells. In bone marrow samples, monoclonal T-cell populations were identified in 4 of 12 cases (33%; 1 CD4 and 3 CD8). Three of these patients were subsequently diagnosed with chronic T-cell neoplasms, while one patient died before completion of the diagnostic workup. TRBC1 expression in polyclonal bone marrow samples was 41.5 ± 7.0% in CD4 T cells and 38.3 ± 8.9% in CD8 T cells. Among five cerebrospinal fluid samples, monoclonal TRBC1 expression was detected in two cases, both involving CD8 T cells. Conclusions TRBC1 assessment using the JOVI-1 clone provides rapid and reproducible flow cytometric method for evaluating T-cell clonality in variety of biological samples including peripheral blood, bone marrow, cerebrospinal fluid, serous fluids, and significantly improves routine diagnostic workup of suspected T-cell lymphoproliferative neoplasms.

P147

Optimization Of Immunophenotypic Evaluation Of Myelodysplastic Neoplasms By Flow Cytometry: A 2-Tube, 13-Color PanelOptimization Of Immunophenotypic Evaluation Of Myelodysplastic Neoplasms By Flow Cytometry: A 2-Tube, 13-Color Panel
Laiz C Bento, Flavia A Sousa, Priscila C Myamoto, Barbara F Bueno, Elizabeth X Souto, Nydia S Bacal
Einstein Hospital Israelita, São Paulo, Brazil

Introduction Multiparametric flow cytometry (FC) enables the detection of subtle immunophenotypic abnormalities associated with bone marrow dysplasia, particularly in cases with inconclusive morphology. The use of compact and well-designed panels improves standardization, reproducibility, and applicability in routine diagnostic settings for myelodysplastic neoplasms (MDS). Objective To compare two FC panels organized in 2 tubes and 13 colors in order to optimize the immunophenotypic evaluation of MDS. Methods Twenty bone marrow samples were analyzed. The routine diagnostic MDS panel consisted of three tubes. Tube 1 (DuraClone Dry reagent+liquid reagent in violet) included CD4+Kappa/FITC, CD8+Lambda/PE, CD3+CD14/ECD, CD5+CD33/PC5.5, CD20+CD56/PC7, CD34/APC, CD19/A700, CD10/A750, CD38/PB, CD45/KO, CD11b/BV610, CD16/V660, and CD13/V780.
Erythroid evaluation was performed using Tube 2 (CD36/FITC, CD105/PE, CD64/ECD, CD33/PC5.5, CD34/PC7, CD71/A700, HLA-DR, CD45/KO), and monocytic evaluation using Tube 3 (CD64/FITC, CD16/PE, CD14/ECD, CD33/PC5.5, CD117/PC7, IREM/APC, CD11b/A750, HLA-DR/PB, CD45/KO). The comparative panel maintained Tube 1 unchanged and reorganized Tubes 2 and 3 into a single tube containing CD36/FITC, CD34/PE, CD14/ECD, CD33/PC5.5, CD117/PC7, IREM/APC, CD71/A700, CD4/A750, HLA-DR/PB, CD45/KO, CD105/BV605, CD64/BV650, and CD2/BV785. Data acquisition was performed on a DxFlex (Beckman Coulter) flow cytometer with gaintration and compensation adjustments. Analyses were conducted using a semi-automated protocol in Kaluza software (Beckman Coulter). Results Both panels enabled adequate identification of the main bone marrow cellular populations. (R²: 0,98). However, the comparative panel demonstrated improved integration of myeloid, monocytic, and erythroid lineage analysis within a compact two-tube design, while preserving maturational resolution and increasing sensitivity for detecting asynchrony, aberrant antigen expression, and fluorescence intensity abnormalities consistent with MDS. Marker reorganization enhanced routine diagnostic assessment, including Ogata Score evaluation, granulocytic maturation analysis, detection of aberrant antigen expression in blasts, granulocytes, and monocytes, classification of monocytic subpopulations, and estimation of eosinophil populations. The panel also allowed plasma cell identification as a screening tool and detection of abnormal CD56, CD19, and CD45 expression. The integration of erythroid (CD36, CD71, CD105), progenitor (CD34, CD117, HLA-DR), and monocytic (CD14, CD64, IREM) markers improved lineage characterization and enhanced assessment of maturation stages, facilitating dysplasia identification, while CD2 supported detection of cross-lineage antigen expression. In addition, semi-automated Kaluza analysis provided objective parameters, including Ogata Score calculation, lineage-specific alerts, CD4/CD8 and Kappa/Lambda ratio deviations, hemodilution indicators, and standardization. Conclusion Rational marker reorganization into a compact 2-tube, 13-color panel preserves analytical robustness, reduces operational complexity, and enhances standardization when combined with semi-automated analysis, in line with international recommendations for FC in MDS evaluation.

P148

Real-World Data Of T-Cell Clones Of Uncertain Significance: An Observational Study Of Prevalence And Immunophenotypic Characteristics In A Large Community Laboratory
Song Chen¹, Sophie Suke¹, Brydon Panozzo^1,2, Ben Hawkins¹, Sophie Sanguine¹
¹Australian Clinical Labs, Melbourne, Australia, ²Alfred Health, Melbourne, Australia

Introduction The recent introduction of monoclonal antibody targeting the T-cell receptor β chain constant region1 (TRBC1) has enabled rapid and cost-effective assessment of T-cell clonality. However, the interpretation of clonal T-cell populations remains challenging, particularly in distinguishing benign or reactive clones from early T-cell malignancies. Our one-year observational study aimed to evaluate the prevalence and immunophenotypic characteristics of T-CUS in a large community-based cohort. Methods We retrospectively reviewed peripheral blood flow cytometry results performed between 2024 and 2025. The screening panel for lymphocyte surface markers included CD45/SSC, FSC/SSC, CD3, CD4, CD5, CD8, CD10, CD16, CD38, TRBC1, CD19, CD20, CD200, surface Kappa and Lambda immunoglobulin. TRBC1 expression against pan-T cell markers (CD3 and CD5) and FSC/SSC was gated prior to evaluation to avoid missing CD3 negative T-cells or large abnormal T-cells. Cases demonstrating monotypic T-cell clones were identified based on TRBC1 expression (defined by<15% or >85%). Lymphosum (sum of %CD3+, %CD19+, and %CD3-/CD19-/CD16+) of >95% was considered valid analysis. Abnormal populations were reported as a percentage of total lymphocytes. The incidence and immunophenotypic characteristics of monotypic T-cell clones were summarized. Clinical and laboratory data, including full blood examination and relevant clinical history where available, were also reviewed in conjunction with flow cytometry results. Results A total of 259 results from 252 patients (102 male and 150 female patients, 6 patients having multiple T-CUS clones ) with T-CUS were reviewed. Monotypic T-cell populations were identified in approximately 2-10% of samples processed on a day-to-day basis. The most common phenotype of monotypic T-cell population was CD8+/TRBC1-, followed by CD4+/ CD8+/TRBC1-, CD4+/CD8+/TRBC1+, CD8+/TRBC1+, CD4-/CD8-/TRBC1-, CD4+/TRBC1-, and CD4+/TRBC1+ in descending frequency. Aberrant antigen expression, particularly CD5+dim, CD5-, CD8+dim,CD16+dim-mod and surface CD3+dim or bright, was frequently observed in most cases. 42% of cases had mild lymphocytosis, while the remainder had low lymphocyte count with no cytopenia(s). The requests for lymphocyte surface marker testing were mainly from general practitioners for routine health checks. Most patients had normal full blood examination results, and only a minority demonstrated cytopenias. Persistent and unchanged T-CUS clones were observed in 16 patients and 6 patients exhibited multiple T-CUS clones. Conclusion The detection of monotypic T-cell clones by multicolor flow cytometry is not uncommon and results should not be overinterpreted as evidence of T-cell malignancy in isolation. The study findings highlight the importance of cautious interpretation of T-CUS and support haematohistopathologists to establish confidence in recognizing the immunophenotypes of T-CUS populations.

P149

Simple Flow Cytometry Assay Uncovers Ar-Cgd Missed By Whole Exome Sequencing: A Multi-Specialty Diagnostic Odyssey
Gabrielle Dammers¹, Priyanka Pandey¹, Leslie Koutoufaris¹, Jeri Dorsey¹, Michele Paessler^1,2, Soma Jyonouchi^1,3
¹Clinical Immunology Lab, Abramson Research Center, Children’s Hospital of Philadelphia, Philadelphia, PA, United States, ²Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA, United States, ³Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA, United States

Introduction: Autosomal recessive (AR) Chronic Granulomatous Disease (CGD) is a genetic disorder characterized by defective NADPH oxidase production in neutrophils, resulting in impaired microbial killing and increased susceptibility to infections. This is the story of a child from Saudi Arabia who was referred to Children’s Hospital of Philadelphia (CHOP) in 2014 and 2024 for a second opinion. Since the age of 3 years (born 2007), patient had severe chronic lip swelling and ulcers in the mouth without specific triggers. Antibiotic treatment showed limited effectiveness. The patient was evaluated by GI, Immunology, Dermatology, and Rheumatology specialties at CHOP. Initial workup in 2014 suspected patient of Hereditary Angioedema. Later in 2024, the patient was suspected of Orofacial Granulomatosis, rare manifestation of inflammatory bowel disease (IBD). Since, almost 25% of patients with CGD have IBD, a dihydrorhodamine (DHR) assay was requested. Methods:The DHR assay measures neutrophil oxidative burst through flow cytometry, serving as a reliable method to evaluate neutrophil functional activity. The assay was conducted by incubating EDTA-anticoagulated whole blood from patients with the non-fluorescent, cell-permeable dye DHR 123, in the presence or absence of the free radical stimulator PMA, at 37°C for 15 minutes. The oxidation-induced change in DHR 123 fluorescence was then analyzed by flow cytometry. Results: In 2014, Hereditary Angioedema workup was negative, C1 esterase functional levels were reported to be normal. GI endoscopy did not reveal any significant inflammation. Clinicians from multiple specialties at CHOP could not come to a unifying diagnosis. A few years later, whole exome sequencing (WES) performed in Saudi Arabia resulted in no conclusive findings. The patient continued to have severe lip swelling and oral ulcers. He also started to have chronic purulent eye discharge and crusting. Eye discharge and lip swelling sometimes seemed to get better with antibiotic therapy. In 2024, the DHR assay results strongly indicated autosomal recessive CGD. AR-CGD is more prevalent in the Middle East, with NCF1 gene mutations being the most common cause. Conclusions: AR CGD caused by NCF1 gene mutations can be missed by WES due to presence of a non-coding pseudogene. NCF1 gene deletion was confirmed by research whole genome sequencing. After a month of treatment, patient had marked improvement in symptoms. Clinicians should take the patient’s ethnic background into account when evaluating challenging cases. Autosomal recessive CGD is more prevalent in regions where consanguineous marriages are frequent, whereas X-linked CGD is more commonly observed in Western populations.

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Flow Cytometric Backbone Panels For Flexible Antigen Quantification On Lymphoid Or Myeloid Populations
Sarah Klapproth¹, Angelika J. Fischbeck¹, Manja Jargosch¹, Vasco Dos Reis Goncalves², Sabrina Mueller², Chrysanthi Tsamadou¹, Wolfgang Kern¹, Veronika Ecker¹
¹MLL Munich Leukemia Laboratory, München, , Germany, ²T-CRUX GmbH, Würzburg, , Germany

Introduction: In addition to determining antigen positivity/negativity, quantification of antigen expression provides valuable information, especially in the context of targeted and immunotherapies. Assessment of antigen density (molecules per cell, mpc) or absolute numbers of CAR T-cells can predict likelihood of response/relapse after therapy and is frequently applied in clinical trials. Aim: Establishment of a lymphoid and a myeloid backbone panel with unoccupied PE channel for flow cytometry analysis of human peripheral blood (PB) and bone marrow (BM) samples to quantify the expression of antigen of interest on lymphoid or myeloid cells. Methods: Backbone panels were prepared as dry pre-formulated tubes (DURAclone, Beckman Coulter) and PE-labeled antibodies against various target antigens were added as liquid drop-in. Both panels cover CD45 and CD33 with addition of CD3, CD4, CD8, CD5 and CD19 (lymphoid) and CD34, CD117, CD13 and CD64 (myeloid), respectively. QuantiBRITE PE-beads (BD technologies) were applied for quantification. Results: PB CLL samples (n=11) were analyzed by lymphoid backbone panel. MFI (geometric mean) of the target antigen on CLL cells revealed a median of 2,501 compared to fluorescence minus one (FMO) control (473), T-cell internal negative control (283) and B-cell internal positive control (10,945). Absolute quantification by parallel measurement of PE-beads revealed 121 mpc (versus 22, 14 and 436 mpc, respectively). Moreover, the lymphoid backbone panel with drop-in of an anti-CAR (PE) antibody was applied to quantify CAR T-cells (% of CD3-positive T-cells, cells/µl). Dilution experiments (n=4) of CAR T-cells into PB samples revealed, considering the limit of blank, a sensitivity of 1-10 detectable CAR T-cells/µl and confirmed linearity up to 1,000/µl. Myeloid backbone panel was tested for 3 distinct antigens which were evaluated on myeloid and/or monocytic blast populations in a total of n=17/53 PB/BM AML samples that resulted in very low/negative (30 mpc), low (170 mpc) and intermediate (895 mpc, range [1.4-9,638 mpc]) expression levels, respectively, as compared to lot-specific QuantiBRITE PE-range. In addition to specificity also sensitivity, precision, sample stability and comparison of instruments and operators were validated for both backbone panels. Conclusions: Generated backbone panels provide a stable immunophenotyping framework for main lymphoid and myeloid compartments. While maintaining the flexibility to insert different markers without having to redesign the entire assay each time, different antigens can be quantified in a standardized, comparable manner across experiments, studies, and instruments.

P151

Minimal Expression Of Cd300E (Irem-2) In Paroxysmal Nocturnal Hemoglobinuria (Pnh) Monocyte Clones &Ndash; A Novel Gpi-Anchored Or Gpi-Linked Receptor In Monocytes?
Angeliki Kotsiafti¹, Christina Karela¹, Evangelia Kouvata², Georgios Oudatzis¹, Maroula Milaiou¹, Aikaterini Mandaraka¹, Anna Pigaditou³, Styliani Kokoris⁴, Nikolaos Giannakoulas², Eleni Gavriilaki⁵, Paraskevi Vasileiou⁶, Elisavet Grouzi⁷, Georgios Paterakis¹
¹Department of Immunology, Athens General Hospital “G. Gennimatas”, Athens, Greece, ²Department of Hematology, University Hospital of Larisa, Larisa, Greece, ³A’ Hematology Department & Bone Marrow Transplant Unit Athens Medical Center, Athens, Greece, ⁴Laboratory of Hematology and Blood Bank Unit, , Athens, Greece, ⁵2nd Propedeutic Department of Internal Medicine AUTH, Thessaloniki, Greece, ⁶«Flowdiagnosis» Diagnostic Center, Athens, Greece, ⁷Transfusion Service and Clinical Haemostasis, "St Savvas" Oncology Hospital, Athens, Greece

Introduction:CD300e (also known as IREM-2) is a cell surface immune activating receptor of the Ig-superfamily that is selectively expressed by monocytes and myeloid dendritic cells. In normal individuals it has been proved to be compatible with monocyte differentiation. The important role of CD300e in MDS progression was also revealed, which indicated that high expression of CD300e might be a favorable prognostic marker for MDS. So far, there has been no correlation between IREM2 and PNH. Aim of this study was to assess the significance of CD300e expression in PNH monocytes. Methods: The expression of CD300e in monocytes of patients with PNH was studied, comparing the intensity of CD300e expression between normal monocytes and pathological monocytes belonging to the PNH-clone.In 9patients in whom the presence of a PNH clone was initially documented through CD55, CD59 and FLAER, immunophenotypic separation of normal from pathological monocytes was followed and the difference in expression of CD300e in the different monocyte populations was examined.The protocol used was CD11c(FITC) CD300e(PE) CD14(ECD) CD33(PC5.5), CD34(PC7), CD117(APC) CD123(A700) CD38(A700) CD36(PB) CD45(KO), Total monocytes were defined by CD36/CD33 gating. Results: In 9patients with documented presence of PNH clone, a difference in CD300e expression was observed between normal monocytes and pathological monocytes belonging to the PNH clone.In normal monocytes, an increased expression intensity of CD300e was found with an average percentage of 84.05% as well as an increased mean fluorescence intensity (MFI) with a median value of 4.1. On the contrary, in the pathological monocytes of the PNH-clone there is an absence or minimal expression of CD300e with a median percentage of 0% as well as a significantly reduced MFI with a median value of 0.3.The expression of Glycosylphosphatidylinositol-anchored proteins (GPI-AP) like CD14 and CD16 were absent in monocytes associated with the PNH as expected.The repeated finding of CD300e absence in all patients with PNH monocyte clones substantiates the identification of CD300e as a novel GPI-AP/ GPI-linkedrelated molecule. Conclusions: To our knowledge this is the first study that CD300e (IREM-2) is identified with negative expression in PNH monocyte clones in cases of diagnosed PNH, contrary to the clearly positive CD300e (IREM-2) expression in the normal monocytes of the same patients. Thus, clear evidence is provided that CD300e (IREM2) may be a Glycosylphosphatidylinositol-anchored or GPI-linked protein and should be included in the diagnostic assessment of monocytes in PNH.

P152

Prognostic Impact Of Hematogones And Cd34+ Myeloblasts In Bone Marrow Of B-Cell Acute Lymphoblastic Leukemia Patients Without Measurable Residual Disease Post-Induction: A Single-Center Cohort Study
Prabhu Manivannan¹, Gifta Catharine¹, Smita Kayal²
¹Department of Pathology, JIPMER, Puducherry, India, ²Department of Medical Oncology, JIPMER, Puducherry, India

Introduction: Measurable residual disease (MRD) negativity at the end of induction (EOI) is a critical prognostic marker in B-cell acute lymphoblastic leukemia (B-ALL). However, a considerable proportion of MRD-negative patients still relapse, indicating the need for additional biomarkers. Hematogones (HG) and CD34⁺ myeloblasts (MB) are routinely observed in post-chemotherapy marrow and may provide prognostic insights. This study evaluates the significance of HG and CD34⁺ MB levels in predicting relapse and survival among MRD-negative B-ALL patients. Methods: This retrospective cohort study included all B-ALL patients diagnosed at JIPMER, India between January 2020 and June 2024 who were MRD-negative at EOI by flow cytometry, with follow-up until June 2025. Clinical, hematological, and cytogenetic data were collected. A total of 125 patients were analyzed: 58% children (0–14 years), 38% adolescents and young adults (AYA; 15–39 years), and 4% adults (>40 years). Treatment regimens included ICiCLe (67%), BFM-95 (30%) and others (3%). HG and CD34⁺ MB percentages were quantified in EOI MRD bone marrow samples. Median HG and CD34⁺ MB levels were 0.04% (range: 0–16.46%) and 0.22% (range: 0–3.35%), respectively. ROC curve analysis determined optimal relapse-predictive cut-offs, based on which patients were stratified. Survival analyses were performed using Kaplan–Meier and Cox regression methods. Results: Despite achieving MRD negativity, 31% (n=39) of patients relapsed during follow-up. Overall, HG levels showed no significant association with relapse-free survival (RFS) or overall survival. However, among children, higher HG levels (≥0.12%) correlated with improved hematological recovery, including higher hemoglobin and platelet counts after chemotherapy. Conversely, lower CD34⁺ MB levels (<0.06%) in children were associated with longer RFS, although statistical significance was borderline and requires validation in larger cohorts. These associations were less consistent in AYA patients. Several clinical and biological variables were significantly associated with inferior RFS, including high total leukocyte count at diagnosis, abnormal karyotype, high-risk cytogenetics, CD123 negativity, and CD9 negativity. Of these, abnormal karyotype emerged as a strong independent predictor of poor outcome on multivariate analysis. Conclusion: In MRD-negative B-ALL patients, HG and CD34⁺ MB levels provide insights into marrow recovery but do not independently predict survival. The high relapse rate within this MRD-negative cohort underscores that MRD alone is insufficient for risk stratification. Incorporating diagnostic tumor burden and cytogenetic features offers a more comprehensive assessment of relapse risk. Larger multicenter studies are warranted to validate the potential prognostic value of HG and CD34⁺ MB, particularly in pediatric patients.

P153

Flow-Cytometric Profiling Of Leukocyte Markers And Their Association With Sofa-Based Severity And Mortality In Sepsis
Shruti Mishra¹, Rohit Daga², Kailash Kumar², Nilesh Kumar²
¹Bone Marrow Transplantation and Stem Cell Research Centre, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India, ²Department of General Medicine, Institute of Medical Sciences, Banaras Hindu University, Varanasi, India

Introduction: Sepsis is characterised by heterogeneous immune dysregulation that is not fully captured by clinical severity scores such as the Sequential Organ Failure Assessment (SOFA). Flow cytometric profiling of leukocyte surface markers offers a practical way to assess immune status at the bedside, but its added prognostic value for mortality prediction remains uncertain. We evaluated whether toll-like receptor (TLR) and checkpoint markers, measured on routine whole-blood cytometry, refine SOFA-based risk assessment in adult sepsis. Methods: We conducted a prospective observational study in an adult intensive care unit. Consecutive patients meeting sepsis criteria underwent whole-blood flow cytometry within 24 hours of enrolment (baseline) and at an early follow-up “landmark” timepoint. Monocyte subsets, monocyte and neutrophil TLR2, TLR4 and TLR9, monocyte PD-L1, neutrophil CD64 and CD25⁺ lymphocytes were quantified. A Sepsis Index integrated SOFA with selected immune parameters. Associations with SOFA and 28-day mortality were assessed using Spearman correlations and logistic regression. Parsimonious prediction models combining SOFA with individual markers and composite Immune Scores were evaluated by area under the receiver operating characteristic curve (AUC) and decision-curve analysis. Results: One hundred thirty-three samples from 111 sepsis episodes were analysed. Higher SOFA and Sepsis Index values were associated with increased neutrophil and monocyte TLR4/TLR9 expression, a shift from classical to non-classical monocytes, and lower CD25⁺ lymphocyte proportions, consistent with mixed immune activation and exhaustion. Baseline SOFA (SOFA1) showed good discrimination for 28-day mortality (AUC ≈ 0.76). Among single markers, monocytic TLR9 provided the strongest signal (AUC > 0.62, inverse association), and a SOFA1 + mTLR9 model modestly improved discrimination (AUC ≈ 0.78) by increasing sensitivity. Composite Immune Scores derived from mTLR9, neutrophil activation, PD-L1 and the Sepsis Index achieved AUCs ~0.68–0.70 alone and ~0.76 when combined with SOFA, but added little over an optimised SOFA model. Dynamic modelling combining SOFA1 with change in neutrophil activation (ΔNAI) improved discrimination (AUC ≈ 0.69), and landmark mTLR2 increased net clinical benefit at intermediate risk thresholds. Conclusions: A focused TLR- and checkpoint-based flow cytometry panel captures sepsis-related immune dysregulation and modestly refines SOFA-based mortality prediction. Dynamic measures, particularly changes in neutrophil activation and monocytic TLR expression, appear more informative than single baseline measurements, supporting the role of serial clinical cytometry as an adjunctive immunomonitoring tool in sepsis.

P155

Development And Preliminary Validation Of A 10-Color Multiparameter Flow Cytometry Panel For Acute Myeloid Leukemia
Najwan Salih Nima¹, Renjie Liang², Libing Xu², Xin Guo³, Xin Chen³
¹Medical City, Baghdad, Iraq, ²Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, China, ³Henan Caprico Biotechnology Co., Ltd., Henan, China

Introduction Flow cytometric immunophenotyping plays a critical role in the diagnosis of acute myeloid leukemia (AML). The establishment of a standardized, robust diagnostic panel is essential for accurate identification of leukemic blasts and characterization of immunophenotypic heterogeneity. This study aimed to develop and preliminarily validate an AML diagnostic flow cytometry panel based on a Mindray flow cytometry platform using antibody (Caprico Biotechnologies) . Methods An AML diagnostic flow cytometry panel was designed to support leukemic blast identification and immunophenotypic characterization. The panel included markers for leukocyte population orientation and blast localization (CD45), myeloid lineage association (CD13, CD33, CD117), stem and progenitor cell–associated markers (CD34), immune-related and characterization markers (HLA-DR), as well as markers commonly used to identify aberrant antigen expression patterns in AML (CD7, CD56, CD9, CD25). Lineage exclusion markers were included to aid differential diagnosis. Flow cytometric analysis was performed on a Mindray flow cytometer using Capri antibody reagents. Leukemic blasts were identified based on CD45 dim expression in combination with low side scatter, together with antigen expression patterns. The panel was evaluated in six patients with newly diagnosed AML, and immunophenotypic data were analyzed descriptively. Results All six AML cases demonstrated a distinct cell population characterized by dim CD45 expression and low side scatter, consistent with leukemic blasts. CD117 was expressed in all cases, supporting myeloid lineage association. HLA-DR expression was observed across all samples, with variable intensity. Myeloid-associated markers CD13 and/or CD33 were detected in most cases, while CD34 expression was present in four of six patients, reflecting heterogeneity in stem and progenitor cell–associated antigen expression. Aberrant expression of markers including CD7, CD56, CD9, CD25, and CD11b was identified with heterogeneous patterns among individual cases. The distribution and frequency of aberrantly expressed markers detected in each AML sample are summarized in Figure 1 (A bar height of 1.0 indicates positive expression, and a height of 0.5 indicates dim expression. Negative markers were are not displayed.), highlighting inter-patient immunophenotypic variability. Lineage exclusion markers, including TdT, monocytic, and megakaryocytic markers, were consistently negative. Conclusion The AML diagnostic panel developed using the Mindray flow cytometry platform and Antibody (Caprico Biotechnologies) demonstrated reliable performance in a preliminary validation cohort of six AML cases. The panel effectively identified leukemic blasts, confirmed myeloid lineage, and characterized immunophenotypic heterogeneity and aberrant antigen expression. These findings support the feasibility of this panel for routine AML diagnosis, with further validation in larger cohorts warranted.

P156

Expanding Diagnostic Insights: Ema Binding Assay And Ngs In Red Cell Membrane Pathologies
Priyanka Pandey¹, Marilyn Li^1,2,3, Michele Paessler^1,3
¹Department of Pathology and Laboratory Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA, United States, ²Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, United States, ³Department of Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania , Philadelphia, PA, United States

Introduction: Hereditary spherocytosis (HS) is a common inherited disorder characterized by abnormal red blood cell morphology and hemolytic anemia. Mutations in genes encoding cytoskeletal proteins disrupt membrane stability, leading to spherocyte formation. Flow cytometry using Eosin-5′-Maleimide (EMA), a fluorescent dye that binds to transmembrane protein Band-3, provides an effective screening method. Reduced EMA fluorescence indicates destabilization of Band-3 and associated membrane proteins due to genetic defects. In this retrospective analysis, we assessed the incorporation of EMA-based screening into routine clinical workflows to enable not only early detection of red cell membrane disorders but also to identify additional abnormalities affecting erythrocyte biology. Methods: The HS assay was conducted by incubating EDTA-anticoagulated whole blood with EMA dye for one hour. Fluorescence intensity was standardized against a normal control sample in each run. The HS Index was calculated by dividing the patient’s mean channel fluorescence (MCF) by the healthy control MCF. In our laboratory, the established normal EMA Binding Index range is 0.87–1.16; an HS Index below 0.87 is considered indicative of disease. The Inherited Red Blood Cell Disorder Panel is an in-house NGS-based test designed to identify genetic causes of red cell disorders, including anemias and membrane defects, by analyzing genes involved in cell structure, enzyme function, and ribosomal proteins. Results: Between January 12, 2023, and January 12, 2026, our laboratory performed 137 EMA binding assays. Next-generation sequencing (NGS) was conducted for 11 patients. Of these, seven patients had an HS Index <0.87, while three patients with HS Index values >0.87 (0.94, 0.95, and 0.91) exhibited inconclusive genetic findings. Notably, patients with borderline HS Index (0.91 and 0.95) harbored sequence variants in the CDAN1 and AK1, genes associated with erythropoiesis and energy homeostasis, respectively. Among patients with HS Index <0.87, causal pathogenic variants were identified in the following red cell cytoskeletal genes: ANK1 (n = 2), SLC4A1 (n = 1), SPTB (n = 3), and SPTA1 (n = 1). The three cases with HS Index >0.87 did not reveal causal pathogenic variants in cytoskeletal genes. Conclusions: EMA binding assay is a reliable screening tool for red cell cytoskeletal disorders. However, HS Index values near the lower end of the reference range should be interpreted with caution. While NGS panels for inherited red cell disorders primarily target cytoskeletal genes, comprehensive genetic analysis can uncover variants in other genes critical for erythrocyte physiology, such as those involved in energy metabolism, thereby expanding diagnostic insights.

P157

Security Guards Of Immune System: A Flow Cytometric Study Of Regulatory T Cells In Paediatric Idiopathic Nephrotic Syndrome
SWATI SHARMA¹, P LALITA JYOTSNA¹, SHAILAJA SHUKLA¹, ABHIJEET SAHA²
¹Department of Pathology, Lady Hardinge Medical College, New Delhi, India, ²Department of Paediatrics, Lady Hardinge Medical College, New Delhi, India

Introduction: Idiopathic nephrotic syndrome (INS) in children is characterized by proteinuria, oedema and hypoalbuminemia. Traditionally hypothesized to be a T-cell–mediated disorder, increasing evidence suggests a broader immune dysregulation involving multiple lymphocyte subsets. Flow cytometric immunophenotyping provides a robust platform to evaluate these immune alterations in peripheral blood. This study aimed to analyse peripheral blood lymphocyte subsets in children with INS and compare them with healthy controls, to better understand immune mechanisms contributing to disease pathogenesis. Methods: This observational case–control study was conducted at a tertiary care centre. Peripheral blood samples were obtained from 30 children diagnosed with the first episode of nephrotic syndrome and age-matched healthy controls. The children were followed up at 6 weeks and 12 weeks of starting standard steroid therapy. Multiparametric flow cytometry was performed to assess lymphocyte subsets, including total T cells (CD3⁺), helper T cells (CD4⁺), cytotoxic T cells (CD8⁺), B cells (CD19⁺), natural killer cells (CD16⁺/CD56⁺), and regulatory T cells identified by CD4⁺CD25⁺CD127dimFoxP3 expression. Absolute counts and relative proportions of lymphocyte subsets were analysed and compared between study groups using appropriate statistical methods. Results: Children with idiopathic nephrotic syndrome demonstrated significant alterations in peripheral blood lymphocyte subsets compared to controls. A key finding was a reduction in the percentage and absolute count of regulatory T cells in children with INS at disease onset as compared to controls (p=0.007, p=0.025 respectively). At the 6 weeks and 12 weeks follow up, it was observed that the percentage and absolute counts of regulatory T cells increased with steroid therapy and was comparable to healthy controls (p=0.004, p=0.007 respectively). Receiver operator curves were plotted and a cut-off of 85/µL and 3% was identified for the absolute and percentage of regulatory t cells at disease onset as a predictor of relapse during the disease course. The relative deficiency of regulatory T cells suggests impaired immune regulation, potentially contributing to disease activity and relapse tendency in INS. Conclusions: This flow cytometric study demonstrates that idiopathic nephrotic syndrome in children is associated with significant immune dysregulation, prominently involving regulatory T cells. The reduction in peripheral blood regulatory T cells supports their role in the pathogenesis of INS and reinforces the concept of defective immune tolerance in this condition. Flow cytometric immunophenotyping provides valuable insights into immune alterations in INS and may aid in identifying immunological markers relevant for disease monitoring and future targeted therapies.

P158

Autoimmune Lympho-Proliferative Syndrome Unmasked By Detection Of A Rare Fas Splice Acceptor Variant On An Expanded Next-Generation Sequencing Hematologic Malignancy Panel
Daniel C. Akuma, Emily Ling-Lin Pai, Olga O. Pozdnyakova, Guang Yang
Department of Pathology and Laboratory Medicine, Hospital of the University of Pennsylvania, Philadelphia, PA, United States

Introduction: Autoimmune lymphoproliferative syndrome (ALPS) is a rare primary immune disorder characterized by defective FAS-mediated apoptosis of lymphocytes, leading to chronic non-malignant lymphoproliferation, autoimmune cytopenias, and increased risk of lymphoma. The 2009 NIH International Workshop designates pathogenic variants in FAS, FASLG, or CASP10 as a primary diagnostic criterion, underscoring the critical role of next-generation sequencing (NGS), in disease definition and subclassification. The absence of a standardized ALPS-enriched sequencing panel across academic and commercial laboratories hinders consistent FAS detection and early diagnosis of ALPS. Here, we report a unique case of a 69-year-old man with longstanding intractable multifocal lymphadenopathy and cytopenias, whose ALPS diagnosis was clinched by an expanded NGS panel. Initial bone marrow and lymph node (LN) biopsies revealed non-specific findings ranging from polytypic plasmacytosis to benign lymphoid proliferation with follicular hyperplasia and sinus histiocytosis, most consistent with autoimmune lymphadenopathy. In contrast, a porta hepatis LN biopsy revealed an atypical lymphoid proliferation with expansion of γδ T cells and a clonal T-cell receptor (TCR) rearrangement, raising concern for a T cell neoplasm. Molecular analyses by NGS were unrevealing except for a persistent TET2 c.2941delinsGT (NM_001127208.2) variant (Table 1). Given equivocal findings, a diagnosis of clonal cytopenia of undetermined significance (CCUS) was favored, and the patient was treated with 6 cycles of CHOEP chemotherapy and later pralatrexate for presumed peripheral T-cell lymphoma, without substantial clinical benefit. After three years of diagnostic uncertainty, a follow-up LN biopsy revealed interfollicular expansion of TCRαβ-positive CD4⁻/CD8⁻ T cells, raising strong suspicion for ALPS and prompting FAS mutational analysis by NGS. Methods: Genomic DNA from left inguinal LN was analyzed using a hybrid-capture NGS panel, which was redesigned to provide full coverage of 202 hematologic malignancy-associated genes, including FAS. Results: NGS of inguinal LN specimen identified a somatic FAS splice-acceptor variant (c.652–1G>C, NM_000043.6; VAF 11%). Retrospective analysis of sequencing data from prior LN biopsies (from 2022 and 2023) also revealed the same FAS variant. In the context of the patient’s longstanding lymphadenopathy, splenomegaly, cytopenias, and characteristic double-negative T-cell expansion, a revised diagnosis of ALPS was established. Clinical management has since shifted from cytotoxic chemotherapy to ALPS-directed immunomodulatory therapy. Conclusions:This is the first report of the FAS splice acceptor variant c.652-1G>C, expanding the molecular spectrum of ALPS. As exemplified by retrospective sequencing analysis, this report underscores the importance of FAS-enriched NGS panels, as earlier diagnosis of ALPS might prevent misdiagnosis and unnecessary cytotoxic therapy.

P159

Detection Of Flt3 Mutations In Acute Myeloid Leukemia Using Next-Generation Sequencing, Fragment Analysis And Direct Sequencing
Soon Hee Chang, Sang Mook Kim, Soong Ki Roh, Eun Jin Kim, Yu Kung Kim, Dong Il Won, Jang Soo Suh
Kyungpook National University, Daegu, South Korea

Introduction:FLT3 mutations are the most common genetic alterations in AML, found in about one third of newly diagnosed patients. There are two distinct types of FLT3 mutations: internal tandem duplication (ITD) in the juxtamembrane domain occur in about 20-25% of adult AML patients, and point mutations in tyrosine kinase domain (TKD), primarily affecting D835, occur in approximately 5-10% of patients. FLT3-ITD mutation is an independent prognostic factor associated with increased relapse and poorer overall survival in AML. As extensive next-generation sequencing (NGS) panels have become increasingly available, additional FLT3 mutations outside of the ITD and D835 regions have been described. This study aimed to investigate FLT3 mutations detected by targeted NGS in AML patients and compare the results with fragment analysis or direct sequencing. Methods: This study enrolled 185 patients including 176 newly diagnosed and 9 relapsed AML, and compared FLT3 mutations detected by targeted NGS with the results from fragment analysis or direct sequencing. Targeted NGS for FLT3 mutation was performed using the Oncomine Myeloid Research Assay (OMA, Thermo Fisher Scientific, Waltham, MA, USA). Fragment analysis for FLT3-ITD was performed on a 3500 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) for exon 14 and 15. For detection of FLT3-TKD mutation, exon 20 of FLT3 was PCR amplified and then direct sequencing was performed. Results: The overall positivity rate for FLT3 mutations in newly diagnosed AML patients was 28.4% (50/176), including 21.6% (38/176) for FLT3-ITD, 8.5% (15/176) for FLT3-TKD, and 3.4% (6/176) for non-ITD/TKD FLT3. Most (36/38) of FLT3-ITD were detected by both NGS and fragment analysis showing high concordance. Of the four FLT3-ITDs detected by NGS in relapsed AML patients, two with low AF (0.1 and 1.0) were not detected in fragment analysis. Among 15 FLT3-TKD positive patients detected by NGS, 5 cases with low AF (3.85-12.91) were not detected by direct sequencing. Additionally, multiple FLT3-ITD, multiple FLT3-TKD, and FLT3-ITD-TKD dual mutations occurred in 4.0% (7/176), 1.1% (2/176), and 1.1% (2/176) of newly diagnosed AML patients. Conclusions: This study demonstrates reliable results with better sensitivity of NGS compared to conventional fragment analysis or direct sequencing in detecting FLT3 mutations. This study revealed additional comprehensive information from NGS, such as multiple FLT3-ITD or FLT3-TKD mutations, FLT3-ITD-TKD dual mutations, and especially NC FLT3 mutations, which showed quite heterogenous patterns among patients.

P160

Ultra-Sensitive Superrca Assay Enables Detection Of Kit D816V In Systemic Mastocytosis Patients With Low Vaf
Andrew S. Dixon¹, Natalia Sigal¹, Erik Kish-Trier¹, Mark Rosenzweig², Guang Yang², Rentian Wu², Rachel L. Erlich², Lei Chen³, Sara Bodbin³, Tracy I. George¹, Leo Lin¹, Peng Li¹
¹ARUP Laboratories, Salt Lake City, UT, United States, ²Blueprint Medicines, Cambridge, MA, United States, ³Rarity Bioscience, Uppsala, Sweden

Introduction
Detection and quantification of the KIT D816V mutation is critical for diagnosing systemic mastocytosis (SM). Conventional methods such as ddPCR typically achieve a LoD near 0.03% VAF in validated commercial assays, which we (George et al) and others (Navarro-Navarro et al) previously demonstrated fails to detect KIT D816V mutation in peripheral blood in up to 24% of patients with SM. To overcome this limitation, we modified a superRCA assay to deliver ultra-high sensitivity and precision for KIT D816V detection, which was validated to 0.001% VAF (LoQ). This level of sensitivity may enable earlier detection of disease in SM patients with low KIT D816V VAF due to low circulating mast cell burden and improve the overall patient diagnostic journey. Methods
The new ultra-sensitive superRCA KIT D816V assay includes an increased genomic DNA input (2,640 ng), double reaction volume, optimized amplification enzymes and optimized flow cytometry conditions versus the original superRCA assay. Validation was performed assessing accuracy, precision, linearity, sensitivity, specificity, stability, and carryover. Testing included healthy donor and patient samples spanning VAFs from 50% to below 0.03% (not detectable by ddPCR). Results
The ultra-sensitive superRCA KIT D816V assay demonstrated robust performance across all evaluated parameters. Accuracy studies showed 100% qualitative concordance with ddPCR and excellent quantitative correlation (R² = 1.00). Sensitivity exceeded expectations, with the analytical LoD calculated at 0.0006% , leading to detection of KIT D816V in 10/10 ISM patient samples that were previously undetectable by ddPCR. The assay maintained precision even at VAFs <0.03%, with %CVs ranging from 3 – 32%. Linearity was confirmed from 0.0003% to 10% VAF (R² = 1.00). Specificity testing in healthy donor cohorts demonstrated minimal background, ranging from 0 – 0.0004% VAF. Stability studies confirmed DNA integrity up to 39 months when stored frozen at -20°C and minimal impact from freeze–thaw cycles. Carryover analysis indicated one wash well was sufficient to remove residual events from samples with VAFs up to 10%. The detected/not detected calling threshold of the new, clinically validated ultra-sensitive superRCA KIT D816V assay was set at the LoQ – 0.001%, over 30-fold more sensitive than the calling threshold of ddPCR. Conclusions
The ultra-sensitive superRCA KIT D816V assay enables detection of KIT D816V at levels previously undetectable by ddPCR, supporting its use in clinical practice and research, and highlighting potential for future diagnostic applications. The increased sensitivity demonstrated here may prove beneficial by improving KIT D816V detection and enabling earlier diagnosis of SM.

P161

An Aggressive Disease: Pediatric Bcr::Abl1 Positive T-Lymphoblastic Leukemia
Asma Al Jahwari¹, Nawal Al Mashaykhi², Fatma Al Bulushi¹, Suha Al lawati¹, Mohammed Al Rawahi³, Yasser Wali⁴, Amr Dahdouh², Abdulhakim Al-rawas², Halima Al-shehhi⁵
¹Department of Hematology, Sultan Qaboos University Hospital, University Medical City, Muscat, Oman, ²Department of Child Health, Sultan Qaboos University Hospital , University Medical City, Muscat, Oman, ³Department of Hematology, College of Medicine, Sultan Qaboos University, Muscat, Oman, ⁴Department of Child Health, College of Medicine, Sultan Qaboos University, Muscat, Oman, ⁵Department of Genetics, Sultan Qaboos University Hospital, University Medical City, Muscat, Oman

Introduction T lymphoblastic leukemia (T-ALL) with BCR::ABL1 fusion is an exceedingly rare entity in both adults and pediatrics. A recent literature review reported 16 pediatric cases¹. Available literature indicates that this subtype is associated with an aggressive clinical behavior, limited therapeutic responses to conventional regimens and poor overall prognosis^1,2.Herein,we report our first encounter of a pediatric case with BCR::ABL1 positive T-ALL Research Methodology This is a retrospective case report. Clinical and laboratory data were obtained from the hospital information system after informed consent. Results A 7-year-old boy presented with fever, abdominal pain, hepatosplenomegaly and lymphadenopathy. Initial investigations showed marked leukocytosis, anemia, thrombocytopenia and acquired hypofibrinogenemia likely due to disseminated intravascular coagulation (DIC). Blood film revealed a large population of circulating blasts. Bone Marrow Examination (BME) and immunophenotyping were consistent of T-ALL of non-early T cell precursor phenotype. G-banded chromosomal analysis and Fluorescence In Situ Hybridization (FISH) showed TCRA/D rearrangement as a result of translocation t(4;14)(q?25;q11) and BCR::ABL1 fusion resulting from t(9;22)(q34;q11). DNA and RNA targetednext generation sequencing (NGS) showed BCR::ABL1 with B4A4 isoform. Furthermore, it showed an oncogenic variant in PTEN with a variant allele frequency of 50%. The patient was initially started on UKALL 2019 regimen B protocol which was switched to ALL 0622 Ph positive protocol including tyrosine kinase inhibitor (TKI), Dasatinib after the conformation of BCR::ABL1 positivity. Post induction bone marrow assessment showed refractory disease with 20% blasts on BME. Within one week after post induction assessment, the patient presented with fever and high WBC count of 200x10⁹/L. BME confirmed infiltration with >95% blasts similar to the diagnostic phenotype. Repeat molecular panel confirmed the presence of both BCR::ABL1 and TCRA/D rearrangement with an additional oncogenic variant in RUNX1. The patient was salvaged with NECTAR protocol and Ponatinib. On assessment post Cycle 1 NECTAR, BME showed residual disease at 5-7%. Furthermore, CNS involvement was confirmed on CSF sample with similar immunophenotype. Treatmentwas escalated to FLA-ID, Ponatinib, and IT chemotherapy, with allogenic stem cell transplant planned on first remission. Conclusion T-ALL with BCR::ABL1 fusion is a rare disease associated with unfavorable prognosis based on the limited number of reported cases so far. The clinical course of our case highlights the aggressiveness and chemoresistance that could be associated with this disease. Reporting such cases is essential to improve the understanding of this entity and refine the therapeutic guidelines that target its underlying biology and improved patient outcomes.

P163

Detection Of Measurable Residual Disease By Measurement Of Wt1 Expression In Acute Myeloid Leukemia Patients
Perapon Junturat¹, Adcharee Kongruang¹, Dollawat Sae-chua¹, Kwannapa Aiyarapong¹, Takol Chareonsirisuthigul¹, Budsaba Rerkamnuaychoke¹, Sasivimol Rattanasiri², Chanatpon Aonnuam², Orathai Munggaranonchai², Teerapong Siriboonpiputtana¹
¹Department of Pathology, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand, ²Department of Clinical Epidemiology and Biostatistics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok, Thailand

Introduction: Measurable residual disease (MRD) assessment during morphological remission is critical for evaluating treatment response and prognosis in acute myeloid leukemia (AML). Wilms' tumor gene 1 (WT1) has been proposed as an alternative MRD biomarker due to its frequent overexpression in AML, although previous studies have reported conflicting prognostic implications. This study aimed to characterize WT1 expression patterns and evaluate its potential as an MRD marker in a single patient cohort. Methods: Quantitative real-time reverse transcription PCR (qRT-PCR) was performed on RNA samples from the bone marrow of 22 AML patients, collected at multiple treatment time points. Patients were divided into high- and low-WT1 expression groups using a threshold of 2.5%. The outcome of interest in these two groups was the occurrence of relapse, with the median follow-up duration of 50.4 months (range: 0.5–81.4 months). Results: WT1 overexpression was detected in 13 of 14 (93%) diagnostic baseline samples. WT1 expression levels decreased consistently in all 15 patients following therapy, mirroring treatment response. Median WT1 levels were 0.36% (range: 0.08–1.29%) post-induction, 0.15% (0.03–0.98%) post-consolidation, and 0.24% (0.08–0.76%) post-maintenance. Longitudinal monitoring revealed no significant fluctuations in WT1 expression throughout the follow-up period. Relapse was observed in both patients with high and low WT1 expression at diagnosis, as well as in a case with sustained WT1 suppression after therapy. No significant association was observed between WT1 expression levels at diagnosis or at subsequent time points and the occurrence of relapse. Conclusion: WT1 overexpression was common at the time of AML diagnosis and declined after therapy, reflecting the treatment response. However, WT1 expression did not demonstrate prognostic utility as an MRD biomarker in this cohort, underscoring the need for complementary MRD assays to improve prognostic accuracy. These findings should be interpreted as preliminary due to the limited sample size and warrant validation in larger, prospective studies. Keywords: Acute myeloid leukemia, measurable residual disease, WT1, biomarker, qRT-PCR

P164

Ngs-Based Comparative Analysis Of Multiple Myeloma According To 1Q21Gain
Jung Ah Kwon¹, Jong Kwon Lee², Seong Gyu Yun², Dongjin Shin²
¹Korea University guro hospital, seoul, Korea, ²Korea university anam hospital, seoul, Korea

Introduction Chromosome 1q gain (1q+) is one of the most common secondary cytogenetic abnormalities in multiple myeloma (MM) and is recognized as an adverse prognostic factor. Genes located on chromosome 1q (e.g., CKS1B, MCL1, ADAR1) play important roles in cell proliferation, treatment resistance, and clonal evolution. Although conventional fluorescence in situ hybridization (FISH) can detect these abnormalities, next-generation sequencing (NGS) provides a more comprehensive genomic perspective,including small variant, copy number variation (CNV), and structural variants. This study aimed to determine whether MM patients with 1q gain and concomitant IGH rearrangements exhibit distinct genomic patterns. Methods A total of 124 patients diagnosed with multiple myeloma between January 2022 and September 2025 at Korea University Medical Center were included in this study. For cytogenetic evaluation, fluorescence in situ hybridization (FISH) analyses targeting IGH::FGFR3, IGH::MAF, IGH::CCND1, 1q21 gain/1p32 deletion, del(13q), and del(TP53) were performed on CD138⁺plasma cell–enriched samples. Targeted next-generation sequencing was performed on unenriched bone marrow aspirate samples using targeted myeloma gene panels. Somatic variants were interpreted according to 2022 ClinGen/CGC/VICC guidelines, and only Tier I, II variants were analyzed. Result Among 124 MM patients, 1q gain was observed in 61 cases (49.2%). Concomitant IGH rearrangements were present in 33 of these cases, most commonly IGH::FGFR3 (n=15), followed by IGH::CCND1 (n=13) and IGH::MAF (n=5) in FISH analysis. Among patients with 1q gain, the most frequent concomitant IGH rearrangement was IGH::FGFR3, observed in 15 of 61 cases (24.6%). In contrast, IGH::CCND1 was the most common rearrangement in patients without 1q gain, occurring in 24 of 63 cases (38.1%). In the 1q gain without IGH arrangement group, oncogenic variants were identified in KRAS, RB1, NRAS, BRAF, and TP53, in that order. In the 1q gain with IGH rearrangement group, oncogenic variants were identified in KRAS, TP53, NRAS, ASXL1, and IRF4, in that order. In contrast, in the group without 1q gain, oncogenic variants were observed in NRAS,KRAS,BRAF,TET2, and TP53, in that order. Conclusion Oncogenic variant analysis demonstrated that multiple myeloma with 1q gain exhibits a distinct genomic profile characterized by the accumulation of activating oncogenic signaling variants together with recurrent oncogenic alterations affecting tumor suppressor genes. This pattern was observed in both 1q gain subgroups with and without IGH rearrangements, whereas cases without 1q gain were predominantly associated with isolated oncogenic signaling and epigenetic modifier mutations.

P165

Development Of A Highly Sensitive Real-Time Qpcr Reagent For The Screening Of Vexas Syndrome
Miwa Matsubara, Mayu Ikeda, Shota Nakada, Yuki Namba, Hikaru Hattori, Yuji Izumisawa
Sysmex Corporation, Kobe, Japan

Introduction VEXAS syndrome, initially reported in 2020, is an adult-onset autoinflammatory disease characterized by progressive bone marrow failure. A diagnosis necessitates the identification of somatic mutations in the UBA1 gene within myeloid cells. However, there is an absence of IVD reagents at present, and testing is conducted in research settings or as LDTs. “The American College of Rheumatology Guidance Statement for Diagnosis and Management of VEXAS Developed by the International VEXAS Working Group Expert Panel (2025)”, underscores the significance of genetic testing for UBA1 mutations in the diagnosis of VEXAS syndrome. This consensus recommends initial screening for mutations in exon 3 and splice sites using Sanger sequencing or NGS. However, Sanger sequencing has been observed to lack sensitivity in detecting low variant allele frequency (VAF) somatic mutations. Furthermore, highly sensitive methods such as NGS or digital PCR are costly for routine screening. Consequently, there is an urgent need for a low-cost, simple, and highly sensitive assay for UBA1 mutations is urgently needed in clinical practice. We aimed to develop a real-time qPCR clamp assay technology for the sensitive detection of four hotspot mutations in exon3 and splice sites (c.122T>C, c.121A>G, c.121A>C, c.118-1G>C), ensuring both analytical performance and clinical applicability. Methods Contrived samples with mutation rates ranging from 0.3% to 1% were prepared by combining synthetic DNA and genomic DNA extracted from healthy male whole blood. Utilizing these samples, an evaluation was conducted of the analytical performance of the developed reagent for the four target mutations through real-time PCR. Results A real-time qPCR-based assay was established to determine the presence of UBA1 variants based on Ct values. The LoD was achieved at a mutation rate of 0.75% for all four target mutations. Conclusions Our reagent has been demonstrated to exhibit a high degree of reliability in the detection of four clinically relevant UBA1 mutations, thereby ensuring high sensitivity. This assay combines the simplicity of Sanger sequencing with sensitivity comparable to NGS or digital PCR and can provide clinical testing aligned with the diagnostic workflow for VEXAS syndrome based on international consensus.

P166

Refining B‑All Diagnostics: Unlocking Rna‑Seq&Rsquo;S Potential Against The Fish Gold Standard
Manja Meggendorfer, Torsten Haferlach, Wencke Walter
MLL Munich Leukemia Laboratory, Munich, Germany

Introduction The current WHO 5th edition and International Consensus Classification classify B‑ALL primarily through genetic alterations that inform prognosis and targeted therapies. RNA‑sequencing plays a key role by detecting fusion transcripts, gene‑expression patterns, and driver lesions, enabling precise assignment to established and emerging molecular B‑ALL entities. The aim of this study was to evaluate the potential of RNA‑Seq in a real‑life adult B‑ALL cohort compared with current gold standard diagnostics. Methods RNA‑Seq libraries were prepared using the NEBNext Ultra Directional RNA Library Prep Kit and sequenced on a NovaSeq 6000/X (~50 million cluster/sample). Fusions were identified with Arriba, STAR‑Fusion, and Manta and retained only if called by at least two algorithms and absent from controls; expression data were normalized and ALLCatchR was applied for gene expression‑based subtype stratification. Results A cohort of 110 adult patients (median age 53 years [18–86]) diagnosed by immunophenotyping and complementary cytogenetics (chromosome banding analysis, FISH) was investigated by RNA‑Seq. ALLCatchR stratified 109/110 cases with variable confidence into BCR::ABL1 (n=40), BCR::ABL1‑like (n=17), hypodiploid (n=13), KMT2Ar (n=13), DUX4 (n=6), ZNF384r (n=5), TCF3r(n=4), hyperdiploid (n=3), PAX5‑altered (n=3), PAX5 P80R (n=2), MEF2Dr (n=2), CDX2/UBTF (n=1), and one case not classifiable by RNA‑Seq with other genetic lesions. Blast counts ranged from 10-99% without relevant impact on RNA‑Seq-based subclassification confidence. In total, 77 patients were assigned “high‑confidence” scores, 27 “candidate”, and 5 “low confidence”; the “low‑confidence” group comprised BCR::ABL1 (3/5), BCR::ABL1‑like (1/5), and hypodiploid (1/5) cases, 4/5 showing concordant fusion transcripts or CRLF2 overexpression in RNA‑Seq analyses. RNA‑Seq-only detectable entities were supported by parallel readouts, like high CRLF2 expression for BCR::ABL1‑like cases or fusion calls for DUX4r cases. In addition, RNA-Seq significantly enhanced diagnostic accuracy in phenotypically complex cases. For instance, it identified a patient as belonging to the B-ALL PAX5 P80R subgroup despite immunophenotyping suggesting minimally differentiated AML. The PAX5 mutation was later verified by single amplicon sequencing. In another case, where phenotypic assessments were inconclusive, RNA-Seq revealed HOX gene activation and an expression profile consistent with AML-NPM1, subsequently confirmed by routine panel sequencing. Additionally, for a patient initially suspected to be a common B-ALL without available cytogenetics due to non-proliferating cells, RNA-Seq uncovered a CBFB::MYH11 fusion transcript, later validated by FISH. Conclusion RNA‑Seq was compared to established cytogenetic methods and enabled comprehensive detection of defining genetic lesions, recognition of emerging molecular entities, and timely therapeutic stratification, supporting its implementation in modern B‑ALL diagnostic algorithms and clinical trials.

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Routine Tp53 Testing And Clinical Impact In Dlbcl: An Open Question
Veronika Navrkalova^1,2,3, Andrea Mareckova¹, Vaclav Kubes⁴, Lenka Radova^2,3, Michael Doubek^1,2,3, Sarka Pospisilova^1,2,3, Jana Kotaskova^1,3
¹Department of Internal Medicine, Hematology and Oncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Brno, Czech Republic, ²Central European Institute of Technology (CEITEC), Center of Molecular Medicine, Masaryk University, Brno, Czech Republic, ³Department of Medical Genetics and Genomics, Faculty of Medicine, Masaryk University and University Hospital Brno, Brno, Czech Republic, ⁴Department of Pathology, University Hospital Brno, Brno, Czech Republic

Introduction Diffuse large B-cell lymphoma (DLBCL) is the most common non-Hodgkin lymphoma with marked genetic heterogeneity and variable clinical outcomes. While approximately 60% of patients achieve durable remission following standard first-line immunochemotherapy (R-CHOP), up to 40% develop relapse or refractory disease. TP53 defects occur in 20-30% of patients and are associated with poor prognosis and refractoriness to R-CHOP. However, routine TP53 testing is not settled and usually limited to immunohistochemistry (IHC), while sequencing and cytogenetics are rarely applied. Consequently, the allelic composition and clinical impact of TP53 defects is under-recognized in DLBCL diagnostics. Methods Diagnostic tissue samples from 69 DLBCL patients were retrospectively analyzed using the established NGS panel LYNX for comprehensive profiling in lymphoid malignancies (PMID: 34082072, 40346678).TP53 mutations (mut), deletions (del) and copy-neutral loss of heterozygosity (cnLOH) were identified and correlated with progression-free survival (PFS) and overall survival (OS). TP53 status determined by NGS was compared with IHC results in 46 cases. Results TP53 defects were detected in 48% (33/69) of patients, including 21 mutations identified in 20 patients (predominantly missense, n=18) and 33 cytogenetic alterations in 31 patients (28 deletions, 5 cnLOHs; in two cases concurrent). Based on allelic status, patients were classified as having single-hit TP53 defect (13 del, 2 mut) or double-hit defects (15 mut/del, 2 mut/cnLOH, 1 mut/mut). The presence of TP53 alterations was not significantly associated with aggressive disease, defined as survival shorter than the cohort median (PFS 0.9 years, OS 1.8 years). However, double-hit alterations were more frequent among patients with short PFS (<0.9 year; p=0.05). Survival analysis showed that patients with double-hit TP53 defects tend to have the shortest PFS and OS, but the difference did not reach statistical significance likely due to low numbers of cases. Concordance between TP53 status assessed by NGS and IHC was 78% (36/46). Ten discordant cases were misclassified as TP53 wild-type by IHC despite the presence of TP53 defects by NGS (7 single-hit and 3 double-hit patients). Conclusion While we observed high frequency of TP53 defects affecting nearly half of our DLBCL cohort, the overall clinical impact remains debatable. Evaluation of TP53 allelic status suggests that double-hit alterations are associated with more aggressive disease and early progression. Integrative NGS-based TP53 testing outperformed IHC and should be considered for improved risk stratification in routine diagnostics and patient enrollment in clinical trials investigating novel therapies. Supported by MH CR (FNBr, 65269705), MEYS CR (CZ.02.01.01/00/23_020/0008555) and MUNI/A/1733/2025.

P170

Β-Globin Gene Mutations In Β-Thalassaemia Patients Across Cambodia And Malaysia
zefarina zulkafli^1,4, Razan Hayati Zulkeflee^1,4, Rosnah Bahar^1,4, Rosline Hassan^1,4, Zilfalil Alwi^1,4, Muhammad Faris Danish Syukri³, Srey Viso², Chean Sophal²
¹Department of Haematology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Malaysia, ²National Pediatric Hospital, Phnom Penh, Cambodia, ³School of Health Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian, Malaysia, ⁴Hospital Pakar Universiti Sains Malaysia, Kubang Kerian, Malaysia

Introduction: β-thalassaemia is a genetically inherited haemoglobin disorder and represents a significant global public health concern. It is particularly prevalent in Southeast Asia, including Cambodia and Malaysia. Understanding the molecular spectrum of β-globin gene mutations in affected populations across Malaysia and Cambodiais crucial for accurate diagnosis, effective screening, and the development of targeted prevention and control strategies. Methods: A retrospective cross-sectional study was conducted upon biobanked samples (n=62); 31 samples obtained from Haematology Unit of Hospital Pakar Universiti Sains Malaysia (HPUSM) and 31 samples from Cambodia. The samples were analysed to study the mutation allele prevalences specific to each population including allelic zygosity, and the effectiveness of multiplex ARMS (MARMS) assay when compared to sequencing method. Results: β-globin mutations detected were to be IVS1-5 (G>C), CD41/42 (−TTCT), CD26 (G>A), IVS1-1 (G>T), CD8/9 (+G), CD71/72 (+A), CD19 (A>G), and IVS2-654 (C>T) in Malaysian cohort, while CD41/42 (−TTCT), CD26 (G>A), Poly-A (A>G), and IVS2-654 (C>T) were detected in Cambodian cohort using the MARMS panel. However, we also detected β⁰Filipino deletion, CD41(−C), and IVS1-2 (T>G) in four Malaysian patients using next generation sequencing these mutations were not detected by MARMS. 43.5% of the total patients were detected for compound heterozygous HbE/β-thalassaemia with 19.4% and 67.7% within Malaysian and Cambodian cohorts respectively. Only one patient in Cambodian group was found to have homozygous IVS2-654 (C>T) mutation. The Fisher’s exact test (α = 0.05) was applied to assess the association between different β-globin gene mutations and coinheritance with CD26 (G>A) (HbE). In the overall study population, CD41/42 (−TTCT) and IVS2-654 (C>T) showed a statistically significant association with coinheritance with HbE (p <0.05). However, in the Cambodian cohort, only CD41/42 (−TTCT) demonstrated a significant association (p <0.05). In contrast, the Malaysian cohort revealed that CD8/9 (+G) and the β⁰ Filipino deletion were significantly associated with coinheritance with CD26 (G>A) (HbE) (p <0.05). Conclusions: The identification of population-specific mutation patterns can enhance early detection of β-thalassaemia while optimising the efficiency of national screening programmes. Keywords: β-thalassaemia; mutations; Malaysia, Cambodia

P171

Early Detection Of Acute Megakaryoblastic Leukemia Secondary To A Chronic Myeloproliferative Neoplasm
Miriam Albéniz¹, Ángel Molina², Maite Serrando², José Luis Bedini², Anna Merino²
¹Biochemistry and Molecular Genetics Department, Biomedical Diagnostic Centre (CDB), Hospital Clínic, Barcelona, Spain, ²Core Laboratory, Biochemistry and Molecular Genetics Department, Biomedical Diagnostic Centre (CDB), Hospital Clínic, Barcelona, Spain

Introduction Transformation to acute myeloid leukemia in chronic myeloproliferative neoplasms is an uncommon complication associated with a poor prognosis, especially when it evolves into acute megakaryoblastic leukemia (AMkL). Early recognition is challenging, particularly when initial laboratory findings include anemia, thrombocytopenia, and elevated lactate dehydrogenase (LDH), requiring consideration and exclusion of hemolytic anemia in the differential diagnosis. In this context, the clinical laboratory plays a critical role in identifying early signs of leukemic transformation. Methods A 66-year-old woman with a 5-year history of Polycythemia Vera treated with hydroxyurea, was admitted to the Emergency Department of our Hospital presenting small bruises on the upper extremities and generalized musculoskeletal pain. Complete blood count and leukocyte scattergram analysis were performed using a Sysmex XR analyzer, and peripheral blood (PB) smears were examined using the CellaVision DM9600 digital image analyzer. Due to a non-evaluable bone marrow aspirate, flow cytometry immunophenotyping was performed on PB. Cytogenetic and molecular analyses included conventional karyotyping, FISH and next generation sequencing. Results Laboratory testing revealed low values of hemoglobin (112 g/L; reference range (RR): 120-150) and reticulocyte counts (9.7x10⁹/L; RR: 25-90), severe thrombocytopenia (42x10⁹/L; RR: 130-400), and markedly elevated LDH levels (3,027 U/L; RR: <234), with negativity for direct antiglobulin test. The initial clinical suspicion based on laboratory results was hemolytic anemia. Although total leukocyte count remained within reference range, leukocyte scattergram analysis demonstrated an abnormal population overlapping the monocytic region, with increased fluorescence intensity and altered internal complexity (Figure 1A). PB smear examination showed preserved red blood cell morphology, making hemolysis unlikely, and a total of 23% of blasts with the following morphological characteristics: medium to large size, high nuclear-cytoplasmic ratio, predominantly regular nucleus, with immature chromatin and occasional nucleoli. The cytoplasm was scant, slightly basophilic and agranular, with occasional cytoplasmic protrusions (Figure 1B). Flow cytometry confirmed a blastic population expressing megakaryocytic markers (CD41, CD61, CD42b), myeloid-associated markers (CD33), and immaturity markers (CD34), with absence of myeloperoxidase expression, consistent with AMkL. Cytogenetic studies showed a complex karyotype with TP53 deletion, and molecular analysis identified mutations in JAK2, ASXL1 and TP53. Despite initiation of treatment, the patient experienced rapid clinical deterioration and died within weeks of diagnosis. Conclusions This case emphasizes the critical role of the clinical laboratory, integrating PB morphology and complementary tests, in the early detection of the unusual transformation of a myeloproliferative neoplasm (Polycythemia Vera) to an acute myeloid leukemia of megakaryocytic lineage.

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Automatic Identification Of Acute Myeloid Leukemia With Nucleophosmin Gene Mutation Using A Vision Transformer-Based Artificial Intelligence Model
Kevin Barrera², Anna Merino¹, José Rodellar³
¹CORE Laboratory. Biochemistry and Molecular Genetics Department. Biomedical Diagnostic Center. Hospital Clinic, Barcelona, Spain, ²Control, Data and Artificial Intelligence (CoDAlab), Department of Mechanical Engineering, Escola d’Enginyeria de Barcelona Est (EEBE), Universitat Politècnica de Catalunya, Barcelona, Spain, ³Control, Data and Artificial Intelligence (CoDAlab), Department of Mathematics, Escola d’Enginyeria de Barcelona Est (EEBE), Universitat Politècnica de Catalunya, Barcelona, Spain

Introduction: Acute myeloid leukemia (AML) with mutation in nucleophosmin gene (NPM+) is associated with a good response to treatment and is characterized by blasts showing a fingerprint-like nuclear indentation. However, distinguishing these blast cells from atypical promyelocytes (AP) of acute promyelocytic leukemia remains a morphological challenge. To contribute to the differential diagnosis of APL and AML NPM+, this study proposes a ViT-DINO-tSNE model for the automatic classification of these cellular subtypes.

Methods: A set of 5,909 images of blast cells was used, obtained from peripheral blood (PB) smears stained with May Grünwald-Giemsa using the Sysmex SP-50 and analyzed using the CellaVision DM96 and DM9600 morphological analyzers. The dataset included 2,892images of blast cells without NPM mutation (NPM-), 2,943 images of atypical promyelocytes, and 74 blast cells NPM+. After evaluating different architectures based on convolutional neural networks and Vision Transformers (ViT), a ViT trained using the self-supervised, DINO approach was selected (see Figure 1A). In this framework, a ViT Giant (teacher) guides the learning of a ViT Tiny (student) to extract 768 features per image. This design allows the tiny model to learn meaningful representations similar to those of a larger model while maintaining a lower computational load. The resulting feature vectors were then projected into a two-dimensional space using t-SNE to analyze the separability among the three classes. Finally, a simple neural classifier was trained to assign each image to a class based on these features, resulting in a lightweight automatic identification system. .

Results: Model validation was performed using an independent test set composed of 723 images of NPM- blasts, 736 of AP and 75 of NPM+ blasts.The system showed perfect performance in detecting blastsNPM+, achieving 100% sensitivity and 100% specificity (Figure 1B). For the remaining categories, sensitivity and specificity were 96.4% and 97.7% for NPM- blasts, and 97.4% and 96.7% for AP, respectively, reaching an overall accuracy of 97.1%. The t-SNE projection revealed a clearly separated visual cluster for NPM+ blasts, as well as a clear separation between NPM- blasts and atypical promyelocytes, delineating the three classes, as shown in Fig. 1C.

Conclusion: The ViT-DINO-tSNE model shows high performance in distinguishing NPM+ blasts from other myeloid blasts and atypical promyelocytes. By combining high accuracy with computational efficiency, this model can serve as a support tool in the initial diagnostic of patients with AML with NPM mutation and its morphological differentiation from APL.

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Pediatric Erythroblastic Sarcoma: A Unique Hematopoietic Neoplasm With Cns Tropism And Nfia Fusions.
Mira/G Basuino¹, Anton Rets¹, Jeffrey Jacobsen¹, Peng Li¹, Maria Aguiar², William Owen³, Linda Pegram³, Madhu Menon¹
¹University of Utah and ARUP Laboratories, Salt Lake City, UT, United States, ²Children's Hospital of the King's Daughters Department of Pathology, Norfolk, VA, United States, ³Children's Hospital of the King's Daughters Department of Hematology and Oncology, Norfolk, VA, United States

Introduction: Erythroblastic sarcoma (ES), a rare hematologic neoplasm, is the mass-forming presentation of acute erythroid leukemia (AEL). Given its rarity and the limited number of published cases, diagnosis is challenging especially in a pediatric setting. We describe a case of ES in a male child 22 months of age who presented with abnormal neuroimaging and CSF analysis. Methods: Outside consultation was performed at ARUP Laboratories in Salt Lake City, Utah. Results: The patient presented in March 2025 with a history of seizures. Cytologic examination of cerebrospinal fluid (CSF) revealed increased numbers of medium to large mononuclear cells with cytoplasmic blebs, moderate amounts of dense blue cytoplasm, and nuclei with moderate chromatin clumping and visible nucleoli. By flow cytometry, the neoplastic cells were intermediate to large by scatter properties and expressed CD117, CD36, CD71 (bright), CD235a and CD45 (dim/negative) while lacking expression of CD34, CD38, CD33, CD56, and CD105. T-cell gene rearrangement by PCR was negative. Bone marrow biopsy showed a normocellular marrow with trilineage hematopoiesis and no flow cytometric evidence of myeloid neoplasia or lymphoproliferative disorder. A brain biopsy showed clusters and sheets of atypical cells with a high nuclear/cytoplasmic ratio and fine chromatin. By immunohistochemistry, those cells were positive for CD117, E-cadherin, CD71, GLUT-1, and EMA and negative for MPO, CD68, CD33, CD34, S100, SOX10, CD45, Glycophorin A, SMA, TdT, CD3, AE1/AE3, and synaptophysin. P53 immunostain demonstrated a wild-type pattern. FoundationOne testing revealed NFIA::RUNX1T1 fusion. Despite treatment with systemic and intrathecal chemotherapy, craniospinal radiation therapy, and haploidentical stem cell transplant, the patient died in November 2025. Conclusions: The overall findings were diagnostic of ES. Our report contributes to the sparse literature on this entity and supports previous findings of increased soft tissue and CNS involvement by AEL/ES in pediatric patients as compared to adults. Divergent molecular pathways have been shown in adult versus pediatric cases; In adults, AEL/ES are primarily TP53 mutation driven while in pediatric patients, AEL/ES appear to be enriched for gene fusions particularly involving the NFIA gene (similar to our case). In addition, pediatric cases seem to demonstrate CNS tropism. In conclusion, pediatric ES is a unique CNS tropic hematopoietic neoplasm, which is a potential pitfall for neuropathologists and requires careful morphologic, immunophenotypic and cytogenetics/molecular evaluation before arriving at a diagnosis.

P174

Integrated Longitudinal Laboratory And Morphologic Assessment For Risk Stratification Of Disease Progression In Chronic Lymphocytic Leukemia
Yi-Yun Chen¹, Chuan-Po Lee¹, Hsiang-Ling Ho^{1, 2}
¹Department of Pathology and Laboratory Medicine, Taipei Veterans General Hospital, Taipei, Taiwan, ²Department of Biotechnology and Laboratory Science in Medicine, National Yang Ming Chiao Tung University, Taipei, Taiwan

Introduction Chronic lymphocytic leukemia (CLL) demonstrates marked heterogeneity in disease progression. While molecular and cytogenetic analyses provide prognostic information, these tests are not routinely performed in all clinical settings. Therefore, there is a practical need for laboratory-based approaches that utilize routinely available hematologic parameters to provide early warning signals of disease acceleration. This study aimed to evaluate whether longitudinal changes in absolute lymphocyte count (ALC), lymphocyte doubling time (LDT), hemoglobin (Hb), platelet count (PLT), and peripheral blood morphology could be integrated to stratify disease progression risk in CLL. Methods We retrospectively analyzed 36 patients with CLL who had long-term serial complete blood count data available prior to treatment. LDT was calculated using log-linear regression based on the most prominent pre-treatment exponential increase phase of ALC for each patient. Longitudinal decline rates of Hb and PLT were calculated using linear regression. Patients were stratified into four progression tiers according to the concordance or discordance between LDT shortening and cytopenia progression: Tier 1, highly progressive disease; Tier 2, biologically rapid progression with preserved marrow function; Tier 3, slow lymphocyte kinetics with worsening cytopenia; and Tier 4, concordantly indolent disease. Peripheral blood morphology was independently reviewed and categorized based on the presence of intermediate-sized cells, large CLL cells with prominent nucleoli, irregular nuclear contours, and extra-high nuclear-to-cytoplasmic (N/C) ratio. Morphologic features were semi-quantitatively assessed and correlated with progression tiers. Results Patients classified as Tier 1 demonstrated the shortest LDT and the most rapid deterioration of hematologic parameters. The median Hb decline rate in Tier 1 was 1.8 g/dL/year (interquartile range [IQR], 1.8–3.9), with all cases exceeding a decline of 1 g/dL/year, whereas Tier 4 patients showed minimal Hb decline (median 0.3 g/dL/year). Similarly, PLT decline was most pronounced in Tier 1 (median 62,284/µL/year; IQR, 57,179–146,013), compared with a median decline of 14,027/µL/year in Tier 4. A semi-quantitative morphologic scoring system was applied to peripheral blood smears, integrating four predefined morphologic features associated with cellular atypia. Rapidly progressive cases exhibited a higher proportion of intermediate-sized cells and large CLL cells with prominent nucleoli, whereas indolent cases showed more uniform small mature lymphocytes. Conclusions An integrated longitudinal assessment combining LDT, Hb and PLT kinetics, and peripheral blood morphology provides a practical, laboratory-based framework for stratifying disease progression risk in CLL. This approach may serve as an accessible early warning system in routine laboratory practice, particularly when advanced molecular testing is unavailable.

P175

From Optical To Digital: Performance Evaluation Of The Scopio X100 In Bone Marrow Morphology.
Nathan Debunne, Nick Verhavert, Annemie De Craemer, Carolien Bonroy, Barbara Denys, Stijn Lambrecht, Mattias Hofmans
Department of Laboratory Medicine, Ghent University Hospital, Ghent, Belgium

Background:Digital microscopy solutions aim to improve efficiency, reproducibility, and standardization of hematological morphology while reducing the limitations of conventional manual microscopy. The Scopio X100 is a full-field digital microscopy system that generates high-resolution images of large areas of hematological slides (whole slide imaging), enabling digital review, classification, archiving, and remote consultation. Its bone marrow module allows automated or manual selection of scan regions and analysis areas. The systems also proposes a differential count, but expert revision of the digital images are mandatory. This study evaluated the performance of the Scopio X100 bone marrow module to assess its suitability for routine clinical implementation in a large tertiary university hospital. Methods: Workflow optimization was first assessed by comparing four acquisition strategies (combining automatic or manual full-field scanning with automatic or manual selection of analysis areas) using five bone marrow samples. Image quality, number of unclassified cells, and analysis time were evaluated. Analytical performance was assessed through within-run, between-run, inter-operator, and intra-patient variability analyses, using binomial statistics (Rumke table) as acceptance criteria. Quantitative method comparison was performed on normal and pathological bone marrow samples (n=30) analyzed by both conventional light microscopy and Scopio X100. Finally, a qualitative clinical validation was conducted in which 15 diagnostic bone marrow samples were assessed using the Scopio system by two independent laboratory specialists and compared to the routine diagnosis to evaluate diagnostic feasibility. Results: Workflow evaluation demonstrated that strategies involving manual selection of analysis areas, regardless of full-field selection mode, provided the best performance, yielding higher image quality, fewer unclassified events, and shorter analysis times. All imprecision analyses showed acceptable reproducibility within expected statistical limits. Method comparison with manual microscopy demonstrated good overall agreement, with minor discrepancies attributable to scan area selection, In the qualitative clinical validation, in one-third of diagnostic samples interpretation was inconclusive and additional conventional microscopic review was suggested. However, diagnostic classification remained concordant for the remaining samples. The cases that needed additional conventional microscopy primarily involved hypocellular specimens, samples with increased plasmacells, or slides of suboptimal quality where confidence in the system had not yet been established, highlighting the importance of high-quality smear preparation for reliable digital evaluation. Conclusion: The Scopio X100 bone marrow module demonstrates robust analytical and diagnostic performance and is suitable for routine implementation. However, for diagnostic bone marrow samples, morphological review by conventional microscopy remains advisable, particularly for cell-poor samples, and slides of limited technical quality.

P176

Evaluation Of Chemotherapy Induced Erythrocyte Injury Beyond Routine Blood Counts Using Peripheral Blood Smears And Mizar&Reg; Syllectometry
Caroline Ding¹, Yousif Al-Aboosi¹, Lucas Chronis¹, Caroline Mwubaha³, Regan Bucciol¹, Yousra Tera^1,2, Maha Othman^1,2,3
¹Department of Biomedical and Molecular Sciences, Queen’s University, Kingston, ON, Canada, ²Clinical Pathology Department, Faculty of Medicine, Mansoura, Egypt, ³School of Baccalaureate Nursing, St. Lawrence College, Kingston, ON, Canada

Introduction Chemotherapy exerts substantial cytotoxic effects that impair quality of life in patients with cancer. Prior cancer studies documented chemotherapy-associated erythrocyte (RBC) membrane toxicity and abnormal morphology. Recent work from our group using MIZAR® Syllectometry demonstrated that a single chemotherapy cycle significantly impairs RBC biomechanics, including elasticity and deformability. Peripheral blood smears are a low-cost, widely available underutilized tool that can reveal RBC structural abnormalities and potentially inform patient care. We aimed to characterize chemotherapy-associated RBC morphological abnormalities and examine their associations with conventional hematological indices and functional RBC biomechanics. Methods Peripheral blood smears were obtained from women diagnosed with cancer before and after first chemotherapy cycle (15 patients; 30 smears). Smears were stained using the Giemsa technique and examined by light microscopy at 100× oil immersion. Standardized monolayer images were captured using a predefined protocol. Per smear, 140–180 erythrocytes from randomly selected fields were evaluated and manually graded into predefined abnormal morphology categories. Inter-rater reliability was assessed by independent re-evaluation of 27% of smears. Morphological changes were assessed in relation to complete blood count (CBC) parameters, and RBC elasticity and deformability measured by MIZAR® Syllectometry pre- and post-chemotherapy. Results The median percentage of abnormal erythrocytes increased from 19.4% (IQR 14.0–22.0) pre-chemotherapy to 23.9% (IQR 17.3–26.7) following cycle one, with a median paired increase of +4.7% (IQR 0.8–10.5). This increase was statistically significant (Wilcoxon signed-rank V=102, p=0.018), with an estimated paired shift of +5.5% (95% CI 1.3–19.0). Changes did not differ by cancer type (all p>0.4), and no individual morphology category reached significance. Inter-rater agreement was excellent for total cell counts (ICC=0.995) and abnormal cell counts (ICC=0.993). HB, MCV and RDW showed no significant changes after chemotherapy (all p>0.10). In contrast, RBC biomechanics shifted significantly: the distribution integral decreased (1878±537 vs. 1567±475; adjusted p=0.032), as did base point low (184±29 vs. 161±25; adjusted p=0.032), while redistribution peak remained unchanged (p=0.108). No significant correlations were observed between percentage morphology changes and biomechanical parameters (all p>0.08). Conclusions Peripheral blood smears detect significant RBC morphological abnormalities early during chemotherapy despite stable CBC indices. Concurrent biomechanical impairment by MIZAR® Syllectometry supports the presence of early functional and structural RBC injury. These findings suggest chemotherapy-associated RBC damage precedes routine hematologic abnormalities and supports smear morphology as a feasible, reproducible early detection tool for detecting early RBC injury, warranting validation in larger cohorts.

P177

Evaluation Of The New Cellavision Bone Marrow Aspirate Application For Morphological Examination And Classification Of Bone Marrow Cells
JOSE RAMON FURUNDARENA¹, ALASNE URANGA¹, SARA URRUTIA ¹, LOURDES AGUIRREZABALA ¹, AMAIA ARAMBARRI ¹, NEREA URESANDI ¹, NAGORE ARGOITIA ¹, LEIRE URRITZA ¹, CARLOS BARBARIN ¹, CLARA DIEZ ¹, SHEILA CARRASCO ¹, JESSICA IRANZO ¹, JULIA SANTA CATALINA ¹, CRISTINA SARASQUETA², CARLOS MANUEL PANIZO¹
¹Hematology Laboratory. Donostia University Hospital., DONOSTIA, Spain, ²Clinical epidemiology. Donostia University Hospital., DONOSTIA, Spain

Introduction For many years, our laboratory has used the CellaVision Peripheral Blood Application for the morphological examination of peripheral blood cells based on automated microscopy, image analysis, and artificial intelligence (AI). Methods CellaVision has recently introduced the CellaVision Bone Marrow Aspirate (BMA) Application, which runs on the CellaVision DC-1 instrument using a 100× objective. The system consists of the CellaVision BMA Analyzer Software, which automatically locates and preclassifies bone marrow aspirate cells using advanced AI-driven technology, and the CellaVision BMA Review Software, which allows users to review cell images and verify preclassification results. We evaluated CellaVision BMA Review Software version 1.0 by comparing its performance with conventional morphological examination of 500 cells using optical microscopy. Between September and December 2025, a total of 158 bone marrow aspirate samples were analyzed. Method comparison was performed using Passing–Bablok regression analysis, and agreement between methods was assessed using Bland–Altman analysis. Results The study included 95 men (60.1%) and 63 women (39.9%), with a mean age of 59 years (range: 1–89). Diagnoses comprised 12 myeloproliferative neoplasms (MPN), 10 myelodysplastic/myeloproliferative neoplasms (MDS/MPN), 21 myelodysplastic syndromes (MDS), 38 acute myeloid leukemias (AML), 21 acute lymphoblastic leukemias (ALL), 2 acute leukemias of ambiguous lineage, 12 lymphomas, 27 multiple myelomas or monoclonal gammopathies, and 15 non-neoplastic conditions. Regarding disease status, 56 samples (35.4%) were obtained at diagnosis, 95 (60.1%) during follow-up, and 7 (4.4%) during disease progression or relapse. Passing–Bablok regression analysis demonstrated comparability between the two methods for erythroblasts, granulocytic cells, blasts, eosinophils, basophils, monocytes, lymphocytes, and plasma cells (Table 1). A slight systematic difference was observed for blasts, while a slight proportional difference was noted for lymphocytes. Linearity between methods was observed for all cell types except basophils and plasma cells. Bland–Altman analysis showed good agreement between the CellaVision BMA Application and optical microscopy, with no relevant systematic bias or random error (Table 2). Conclusions The CellaVision BMA Application provides a reliable tool for automating and simplifying the morphological examination of bone marrow aspirate cells. Our results demonstrate that its performance is comparable to the current gold standard of conventional optical microscopy.

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Real-World Application Of Horiba D20 Digital Morphology Analyzer For Remote Access Peripheral Blood Film Reporting And Its Impact Patient Management: An Experience From A Tertiary Care Hospital Laboratory In India
Anil Handoo¹, Mudita Bhargava², Ankur Lath³, Richa Sharma², Parul Babbar³, Paramjit Kaur³
¹BLK-Max Super Speciality Hospital, Pusa Road, New Delhi, India , New Delhi, India, ²Max Super Speciality Hospital, Dwarka, New Delhi, India , New Delhi, India, ³Horiba India Pvt Ltd, New Delhi, India

Introduction:Manual peripheral blood film review for assessment for abnormalities in analyzer-flagged samples, an expert-dependent manual job, is a challenging “last-mile connect” in the journey of a CBC sample, due to non-availability of a trained expert 24 x 7. Artificial intelligence based digital morphology cell analysis for blood cell classification and assessment reduces manual effort, and provides consistent results. Most of digital morphology analyzers (DMA), mandate use of propriety slide makers, slides stains/ staining protocols, increasing costs as well as adding a different dimension to the routine work-flow. This study, shares a real-world experience of using Horiba^TM D20, an economical, slide/stain agnostic DMA with “cloud-based” remote data access for peripheral blood film review with impact on patient management. Methods: 1769 patient slides were scanned and reviewed on the Horiba D20 DMA from November 2024 till Dec 2025. 47 slides (2.73%) did not meet scan quality specifications. Rest of the 1722 off hour slides (97.34%) were scanned for expert. Remote access expert review with manual re-assessment by the same person was done. 3 expert hemato-pathologists with median experience of 15 years, reviewed the smears and relative concordance was recorded. Digital Morphology was scored from 1 to 5 (1=Poor, 2=fair, 3=good, 4=very good and 5=excellent) for image quality, background cleanliness, correctness of AI based identification of cells and presumptive diagnosis versus expert manual morphology review. Additionally, the impact of remote access and quick reporting on subsequent patient management was also assessed into 3 groups: Major, Minor and no impact. Results:Of the 1722 scanned cases,312 cases (18.12%) scored 2 while 1410 cases (81.88%) had a score of 3 or 4 for image quality, background cleanliness. No case scored 5 for these parameters. For AI based identification of cells and presumptive diagnosis versus expert manual morphology review, scores of 2, 3, 4 and 5 were observed in 309 (17.94%), 1201 (69.74%), 111 (6.45%) and 101 (5.87%), respectively, indicating good to excellent correlation in approximately 82.1% cases. Major impact on patient management was seen in 464 (26.95%) cases, while 207 (12.02%) cases had a minor impact, with overall some impact on 38.97% cases. Conclusions: Horiba^TMD20 DMA with for remote access peripheral blood film reporting is a cost-effective slide and stain agnostic digital morphology analyzer with good image quality and excellent AI driven cell analysis. Off-hour reporting of peripheral smears using Horiba^TMD20 DMA has a significant impact on patient management.

P179

Case Report: Hematological Findings In Case Of Intravascular Hemolysis Caused By Clostridium Perfringens Infection
Urška Kramberger, Elizabeta Božnar Alič
University Medical Centre Ljubljana, Institute of Clinical Chemistry and Biochemistry, Ljubljana, Slovenia

Introduction:Clostridium perfringens is a strictly anaerobic, Gram-positive bacillus. Rare but mostly lethal complications of sepsis due to C. perfringens are gas gangrene and massive intravascular hemolysis. In our case, a 73-year-old male patient was brought to the emergency department and was diagnosed with septic shock, there was no record of recent trauma or surgery. Methods:EDTA blood samples were analyzed with Sysmex XN1000 analyzers (Sysmex Corporation, Kobe, Japan). Manual differential leukocyte count of 100 cells was performed in May-Grünwald-Giemsa stained blood smears. Results:Samples sent to the laboratory were visibly, grossly hemolyzed and all further samples were hemolyzed too. Basic blood count showed leukocytosis (32,1 x 10⁹/L), low red blood cell (RBC) count (2,53 x 10⁹/L), low hemoglobin concentration (87 g/L) and high MCHC value (412 g/L). When a result of MCHC exceeding 356 g/L is obtained, sample is automatically retested in the reticulocyte channel and optical method provides a calculated result of hemoglobin actually present in RBCs (CBC-O algorithm). Photometric methods for the determination of hemoglobin can give falsely elevated results in case of intravascular hemolysis. In our case calculated hemoglobin was 47 g/L; the difference in both methodologies was significant, raising suspicion for the possibility of in vivo hemolysis. We also observed some distinctive morphological features in the blood smear: left shift, vacuolization of neutrophil cytoplasm, spherocytes and anisocytosis were seen. The most characteristic feature we observed was so-called ghost cells, microcytic erythrocytes, which are very pale due to the loss of hemoglobin. α-toxin, the main virulent factor of C. perfringens, degrades the membrane of erythrocytes, resulting in cell lysis. Spherocytes were misidentified as mature reticulocytes in the scattergram (in severe hemolytic anemia an abundant population of immature reticulocytes is expected), furthermore ghost cells were misidentified as platelets in the optical platelet channel. Therefore, scattergrams encouraged us in recognizing those features. Conclusions:Blood cultures remain the gold standard for identifying the causative pathogen of sepsis but are time-consuming. There are some routinely laboratory markers that can alert us that massive intravascular hemolysis is due to a C. perfringens infection, which are available much earlier than results of blood cultures can be obtained. Massive hemolysis, distinct spherocytosis, and the presence of ghost cells in the peripheral blood smear, accompanied by other test results indicating severe infection, can serve as indirect indicators, which could aid clinicians in early recognition and adequate treatment.

P180

Evaluation Of The Hematology Digital Imaging System Scopio X100Ht In A University Hospital Setting
Alina Kuepper, Sladana Nikolic, Michael Spannagl, Michael Weigand, Daniel Teupser, Mathias Bruegel
Institute of Laboratory Medicine, Ludwig-Maximilians-University, Munich, Germany

Introduction: In the field of peripheral blood count testing, hematology analyzers are used for automated classification and quantification of peripheral cells. Pathological results are verified by manual microscopy as the gold standard in cell morphology. To overcome diverse limitations of manual microscopy, digital imaging platforms have been introduced many years ago; however, routine application of current instruments is limited based to the limited diagnostic quality in detecting abnormal cells. The Scopio X100HT instrument is a newly developed digital imaging platform, introducing full-field computational imaging. The aim was to evaluate this new technology in a university hospital setting. Methods: In the adult study, 400 adult samples - 300 randomly selected routine samples and 100 with predefined pathology criteria - were analyzed with the Scopio X100HT. In the pediatric study, 85 samples from one work week were included, which got a manual differential in routine. Preclassified and user-verified results were compared to those obtained from the Sysmex XN hematology analyzer and manual microscopy as the gold standard. Additionally, a workflow study including 20 routine samples per day over one week compared turnaround times between digital imaging analysis and manual microscopy.Evaluations were performed in accordance with CLSI standards. Results: Method comparison evaluations showed strong correlations for normal peripheral blood cells, ranging from 0.64 to 0.99. For blasts, considered as the standard abnormal cell type, correlations ranged from 0.76 to 0.99. In the pediatric study, correlations for normal peripheral blood cells were also good, ranging from 0.41 to 0.99. Regarding blasts in this context, there were 3 true positives, 8 false positives, 73 true negatives, and 3 false negatives. Scopio X100HT platelet counts were less correlated with Sysmex XN at higher concentration ranges. The workflow study revealed an average reduction of hands-on time of 26% when comparing user-verified digital imaging analysis with manual microscopy. Conclusion: The results of our validation study demonstrate a good performance of the Scopio X100HT digital imaging analyzer and indicate potential for routine application in a university hospital setting. The results of our workflow study, revealing reduced hands-on times, support the idea that the Scopio X100HT is suitable for increasing efficiency in peripheral blood count testing.

P182

Detection Of Monoblasts In Urine In A Patient With Acute Myeloid Leukemia: Integration Of Cytology And Next-Generation Sequencing For Diagnosis
Carlos Martín Alonso, Maite Serrando Querol, Anna Merino, Rosa Fernández Bonifacio, Ángel Molina Borras, Mari Carmen Salgado
CORE Laboratory, Biochemistry and Molecular Genetics Department, Biomedical Diagnostic Center, Hospital Clínic, Barcelona, Spain

Introduction: The presence of blast cells in urine is a rare finding. It has been described in cases of renal myeloid sarcoma and leukemic renal infiltration in patients with acute myeloid leukemia (AML) with monocytic differentiation. Several mechanisms of kidney injury have been described in acute monocytic myeloid leukemia, including direct leukemic infiltration, leukostasis with microvascular obstruction, lysozyme-induced tubular damage, and tumor lysis syndrome. Methods and results (Case Report): We report the case of an 87-year-old woman who presented with a one-month history of progressive dyspnea and constitutional symptoms. Her medical history included a family history of AML. At admission, laboratory tests revealed severe leukocytosis (79.2×10⁹/L), anemia (hemoglobin 71 g/L), and thrombocytopenia (31×10⁹/L). Peripheral blood (PB) smear showed a total of 72% neoplastic cells including monoblasts, promonocytes, and monocytes. The patient also presented acute kidney injury (creatinine 3.4 mg/dL) and hyperuricemia (15 mg/dL), without other signs of tumor lysis syndrome. Bone marrow immunophenotyping demonstrated expansion of immature monocytic cells (CD34⁻, CD45⁺, CD64⁺⁺, CD33⁺⁺, among others), composed of 16% monoblasts, 22% promonocytes, and 34% monocytes. Next-generation sequencing evidenced mutations in ASXL1, NF1, RUNX1, STAG2 and TET2. This, along with the lack of alterations in the karyotype, confirmed the diagnosis of AML, myelodysplasia-related. Urinary sediment examination revealed the presence of monoblasts morphologically similar to those in PB, in the absence of significant hematuria. Urinary tract infection by Gram-negative bacilli was also detected. The urine flow cytometry study revealed an 88% of monocytes which lacked CD34 expression, supporting their origin from the leukemic clone. The patient died three days after admission due to acute pulmonary edema. Conclusions: In this patient with AML, the rare observation of monoblasts in urine has been demonstrated. It is, to our knowledge, the first case of these characteristics which lacked concomitant hematuria. Renal impairment was likely multifactorial, involving leukostasis and direct renal infiltration by circulating blast cells. Lysozyme-induced tubular damage may also have contributed, although lysozyme levels were not assessed. This case highlights the potential diagnostic value of urine cytology in acute leukemia patients and underscores the importance of integrating different areas of laboratory medicine to achieve a comprehensive understanding of the disease.

P183

Early Detection Of Myelodysplastic Syndromes And Chronic Myelomonocytic Leukemia With A New Model Based On Transformers And Sysmex-Xr Cell Population Data
Anna Merino¹, Kevin Barrera², Alex Barragán¹, Angel Molina¹, Jose Rodellar²
¹CORE Laboratory. Biochemistry and Molecular Genetics Department. Biomedical Diagnostic Center. Hospital Clinic of Barcelona-IDIBAPS, Barcelolna, Spain, ²Technical University of Catalonia, Barcelona, Spain

INTRODUCTION This study aims to develop and evaluate a transformers-based deep learning model for the automatic detection of Myelodysplastic Syndromes (MDS) and Chronic Myelomonocytic Leukemia (CMML) using numerical variables obtained from peripheral blood (PB) samples with the Sysmex-XR hematological analyzer. METHODS A total of 377 patients were included in this study, 201 with MDS, 48 with CMML and 128 with anemia not due to MDS or CMML (other). The diagnosis of the 249 patients with MDS or CMML was made after bone marrow examination and other tests. In all patients, the Sysmex-XR analyzer was used to obtain cell counts and Cell Population Data (CPD) from peripheral blood (PB). These variables were used to develop a transformer encoder adapted for tabular data, trained to automatically extract quantitative features from the input data paying attention to the most relevant. The final features are passed through a classical neural network that predicts the output in one of two classes: 1) MDS/CMML, which includes those patients with either MDS or CMML; 2) the other class. Data from 213 patients were used for training the model. The final model was validated using data from 164 patients, 86 diagnosed of MDS, 23 CMML and 55 others. This model was compared in 82 of the patients (50 MDS/CMML and 32 others) with the MDS-score (with 0.165 threshold) previously published. RESULTS The best results for the automatic discrimination of patients with MDS/CMML were obtained using 20 variables. Among the most relevant variables we found blood cell counts, hemoglobin and MCV, and CPDs related to neutrophil morphology (NE-WY, NE-WX, NE-WZ). The confusion matrices of automatic classification are shown in Figure 1(A). Among 109 MDS/CMML patients, our model correctly identified 102, meaning a sensitivity of 93.6%. With respect to the 55 non-MDS patients, only five were wrongly classified in the MDS/CMML group (90.9% specificity). The comparison of our model with the MDS-score previously published showed higher values of sensitivity, specificity, precision and F1-Score, Figure 1(B). CONCLUSIONS In this work, a model based on transformers was designed using hematological variables from PB, which showed high values of sensitivity, specificity and precision for the automatic detection of patients with MDS/CMML.

P184

Large Immature Cell (Lic) Parameter Accurately Detects Blast Cells On The Horiba Yumizen H2500
Tri Ratnaningsih¹, Lusia Nasrani²
¹2Department of Clinical Pathology and Laboratory Medicine, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, , Indonesia, ²Specialist Medical Education Program in Clinical Pathology, Faculty of Medicine, Public Health and Nursing, Universitas Gadjah Mada, Yogyakarta, , Indonesia

Introduction Hematologic malignancies remain a significant health concern in Indonesia, with increasing healthcare costs each year. Early detection of blast cells in peripheral blood is essential for screening, diagnosis, and disease monitoring. While manual microscopic examination remains the gold standard, it has limitations in terms of subjectivity, the need for specialized expertise, and turnaround times. The Large Immature Cell (LIC) parameter provided by hematology analyzers offers a simple, rapid method for detecting blast cells; however, research evaluating its efficacy remains limited. This study aimed to assess the performance of the extended Large Immature Cell (LIC) parameter on the Horiba Yumizen H2500 hematology analyzer for blast cell detection. Methods This cross-sectional diagnostic study included 251 patients at Dr. Sardjito General Hospital (May–November 2024), grouped according to the presence or absence of blast cells on microscopic examination. Peripheral blood samples were analyzed using the Horiba Yumizen H2500 and confirmed by microscopic examination of a smear. A comparative analysis was performed between groups, and correlations between hematologic parameters and blast cell percentage were assessed. Diagnostic performance was evaluated using receiver operating characteristic (ROC) curves to determine the optimal LIC% cutoff. Results LIC% values were significantly higher in the blast-positive group (median 17.8%, p<0.001). LIC% showed the strongest correlation with blast cell percentage (r=0.694, p<0.001) among the other parameters. ROC analysis yielded an AUC of 0.978 (95% CI: 0.963–0.993, p<0.001). An optimal cut-off of 12% yielded a sensitivity of 91.23%, specificity of 96.39%, PPV of 88.14%, NPV of 97.40%, LR+ of 25.28, and LR– of 0.09. Using the analyzer’s default cut-off of 3% increased sensitivity to 94.74% but reduced specificity to 84.54%. Conclusion LIC% on the Horiba Yumizen H2500 demonstrates excellent diagnostic accuracy for blast cell detection. A 12% cutoff is recommended for confirmation, while 3% may be suitable for initial screening. Implementing this parameter may expedite the detection of hematologic malignancies, particularly in resource-limited settings. Keywords: Large Immature Cell, Horiba Yumizen H2500, blast cells, hematologic malignancy, ROC curve

P185

Performance Evaluation Of An Ai-Powered Point-Of-Care Hematology Analyzer In A Tertiary Care Setting
Tushar Sehgal¹, Ashutosh Patra², Praveen Kumar², Megha Sharma²
¹Department of Laboratory Medicine, AIIMS, New Delhi, India, ²Neuranics Lab, School of International Biodesign, AlIMS, New Delhi, India, ³Neuranics Lab, School of International Biodesign, AlIMS, New Delhi, India, ⁴Neuranics Lab, School of International Biodesign, AlIMS, New Delhi, India

Introduction The demand for decentralized hematology diagnostics is increasing due to the need for rapid clinical decision-making in rural, emergency, and resource-limited settings. Conventional complete blood count (CBC) testing relies on centralized laboratory infrastructure, often causing diagnostic delays. Point-of-care (POC) hematology solutions integrating speed, analytical accuracy, and morphological assessment can improve frontline care. MorphX, an artificial intelligence (AI)–powered POC CBC analyzer developed by Neuranics Lab, provides quantitative CBC parameters with real-time peripheral smear analysis within seven minutes using a single disposable cartridge using venous blood. By combining quantitative counts with AI-based morphological interpretation, MorphX bridges the gap between central laboratories and bedside testing. This study evaluates the analytical concordance of MorphX with a standard laboratory hematology analyzer in venous blood samples under real-world conditions.MethodsThis prospective, single-center, blinded clinical study was conducted at the Department of Laboratory Medicine, All India Institute of Medical Sciences (AIIMS), New Delhi. A total of 375 leftover blood samples obtained from routine laboratory testing were included for analysis. Comparative evaluation was performed between MorphX using venous blood samples and a reference Sysmex hematology analyzer using corresponding venous samples. The evaluated parameters included total leukocyte count (WBC), red blood cell count (RBC), hemoglobin (Hb), hematocrit (HCT), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW), platelet count (PLT), and differential leukocyte count (DLC). Statistical analysis comprised correlation analysis expressed as coefficient of determination (R²), along with sensitivity and specificity, to determine analytical agreement and diagnostic performance. ResultsMorphX demonstrated strong analytical concordance with the reference analyzer across all evaluated CBC parameters (Fig. 1). The AI-driven image analysis algorithm successfully identified key morphological abnormalities, including microcytosis, thrombocytopenia, and anisocytosis, with an accuracy exceeding 90%. Sensitivity and specificity for anemia detection were both greater than 90%. Real-time peripheral smear examination using MorphX produced clinically satisfactory results for detection of malaria parasite, nucleated red blood cells, myeloid left shift and blast identification in leukemias. Notably, platelet counts generated by MorphX showed superior correlation with gold-standard manual microscopy compared to the reference analyzer. ConclusionMorphX demonstrates robust analytical equivalence to standard laboratory hematology analyzers and reliable performance in venous blood samples. By integrating AI-augmented CBC analysis with real-time peripheral smear evaluation at the point of care, MorphX supports scalable hematology screening and aligns with national public health initiatives such as India’s anemia control programs.

P186

Detection Of Histoplasma Sp. During Complete Blood Count Analysis In A Newly Diagnosed Hiv Patient: A Case Report Supported By Artificial Intelligence
Thulio F. Theodoro^1,2, Camila N. M. Araujo^1,2, Pamela R. V. Figueredo¹, Lucas J. C. Ferreira¹, Sally C. M. Monteiro², Rossy-Eric P. Soares¹
¹Laboratorio Cedro, Sao Luis, Brazil, ²Universidade Federal do Maranhao, Sao Luis, Brazil

Introduction Histoplasmosis is a systemic mycosis caused by Histoplasma capsulatum, and represents a major opportunistic infection in immunosuppressed patients, particularly patients infected with human immunodeficiency virus (HIV). Although morphological findings suggestive of histoplasmosis in peripheral blood smears are rare, they play a decisive role in early laboratory-driven diagnosis. In parallel, artificial intelligence (AI)-assisted digital morphology integrated into automated hematology analyzers has emerged as a valuable screening tool for detecting cellular abnormalities associated with infectious diseases, such as fungal infections. This study reports a rare case of peripheral blood morphological findings suggestive of Histoplasma sp. in a newly diagnosed HIV patient, highlighting the contributory tole of AI-based hematology systems in laboratory screening. Case Description A 31-year-old male patient was admitted to a public hospital for diagnostic investigation. Biochemical, immunological, and hematological analyses were performed, and a reactive HIV result was identified using a chemiluminescence assay. A complete blood count was analyzed using the Mindray CAL 6000 hematology analyzer equipped with the MC-80 digital morphology module. Laboratory Findings and Diagnosis The automated leukocyte differential analysis generated an “excessive artifacts” flag. Subsequently, digital microscopy analysis revealed intracellular structures with yeast-like morphology compatible with Histoplasma sp., observed within neutrophils and monocytes. These findings were confirmed through manual microscopic examination of the peripheral blood smear. Immunodiffusion testing for anti-Histoplasma antibodies was negative. However, considering the characteristic morphological findings, the patient’s clinical presentation, and the epidemiological context, the suspicion of histoplasmosis was strongly supported and communicated to the multidisciplinary clinical team. Conclusion The negative serological result for Histoplasma sp. was likely attriutable to the patient’s severe immunosuppression, which might impair the humoral immune response, as the patient presented with severe anemia with erythroblastosis, leukocytosis with left shift, lymphopenia, and monocytosis. This case illustrates the complementary roles of AI-assisted digital morphology and expert human review in hematology laboratories. Although the analyzer did not directly identify the fungal organism, the AI-generated flag prompted manual smear evaluation, enabling early recognition of an opportunistic infection. Despite increasing automation in laboratory diagnostics, morphological expertise remains indispensable while AI adds value as a screening tool for atypical and clinically significant findings.

P187

Functional Hyposplnism Post Hematopoietic Stem Cell Transplantation: A Retrospective Analysis
Kaori Uchino^1,2, Yuya Nakagami³, Megumi Enomoto³, Yukie Sugita^1,2, Yuto Isaji^1,2, Sakura Saigusa^1,2, Yusuke Iida^1,2, Saki Shinohara^1,2, Tomohiro Horio^1,2, Satsuki Murakami^1,2, Shohei Mizuno^1,2, Kazuhiro Ikegame^1,2, Ichiro Hanamura^1,2, Akiyoshi Takami^1,2
¹Division of Hematology, Department of Internal Medicine, Aichi Medical University School of Medicine, Nagakute, Japan, ²Hematopoietic Cell Transplantation Center, Aichi Medical University Hospital, Nagakute, Japan, ³Department of Clinical Laboratory, Aichi Medical University Hospital, Nagakute, Japan

Introduction: Functional hyposplenism is an underrecognized complication following hematopoietic stem cell transplantation (HSCT). Its incidence, etiologies, and optimal diagnostic approaches are not well established, making accurate diagnosis challenging. We previously reported that Howell–Jolly bodies (HJBs) can serve as an indicator of functional hyposplenism and that patients with HJBs tend to have smaller splenic volumes than their predicted ideal volumes. To further clarify the characteristics and risk factors of functional hyposplenism after HSCT, we conducted a retrospective study using combined evaluation of HJBs and splenic volume. Methods: We retrospectively analyzed 77 consecutive adult patients who underwent allogeneic HSCT at our hospital between January 2010 and December 2023. Patients in whom HJBs were newly detected in peripheral blood smears after transplantation were identified. The presence of HJBs was defined as at least one erythrocyte containing a HJB in a single microscopic field (×400). Splenic volume was calculated from CT images using ZIOSTATION software (Ziosoft Inc., Tokyo, Japan). Splenic atrophy was defined as a post-HSCT splenic volume <10 mL or a relative reduction >90% compared with the predicted ideal volume. Results: HJBs newly detected after transplantation were identified in 6 patients (7.8%), and splenic atrophy newly detected after transplantation was observed in 3 patients (3.9%). In these three patients, post-HSCT splenic volumes were <10 mL, whereas pre-HSCT volumes had exceeded 70 mL. Among them, two patients (2/77, 2.6%) received 12 Gy total body irradiation (TBI) as part of the conditioning regimen. Chronic GVHD occurred in all three patients and was classified as moderate to extensive. At the last follow-up, one patient had died of sepsis and two were alive.In contrast, three patients developed HJBs without evident splenic atrophy; however, the cause could not be determined because all had died before detailed functional evaluation could be performed. Conclusions: These findings suggest that high-dose TBI and chronic GVHD are associated with reduced splenic volume and functional hyposplenism after HSCT. The combined evaluation of HJBs and splenic volume may facilitate early detection of post-HSCT splenic dysfunction. Early diagnosis of functional hyposplenism could enable implementation of infection-prevention measures, potentially improving transplant outcomes. Further case accumulation and prospective studies are warranted to validate this approach.

P188

Neu-X And Neu-Y Obtained With Mindray Bc-6200 As Laboratory Markers Of Inflammation In Emergency Department Patients
Agnieszka Wiśniewska^{1, 2}, Małgorzata Wituska², Olga Ciepiela^{1, 2}
¹Department of Laboratory Medicine, Medical University of Warsaw, Warsaw, Poland, ²Central Laboratory, University Clinical Center of Medical University of Warsaw, Warsaw, Poland

Introduction:
Early identification of patients at risk of severe inflammation remains a major diagnostic challenge in clinical practice. In recent years, increasing attention has been focused on novel laboratory markers that may complement classical inflammatory parameters such as C-reactive protein (CRP). Among these, neutrophil-derived parameters NEU-X and NEU-Y have emerged as potential indicators that may facilitate faster clinical decision-making and earlier initiation of appropriate treatment. Objective:
The aim of this study was to evaluate the relationship between NEU-X, NEU-Y values and CRP concentrations in patients admitted to the Emergency Department, and to assess their potential utility in identifying individuals with suspected inflammation and/or sepsis. Materials and Methods:
A total of 357 Emergency Department patients (180 women and 177 men) presenting with diverse clinical symptoms were selected between April and December 2025. Routinely performed laboratory tests were analyzed, including complete blood count parameters (NEU-X and NEU-Y) and serum CRP levels. Hematological analyses were conducted using the MINDRAY BC-6200 analyzer (Mindray, China) on K₃-EDTA-whole blood samples. Serum CRP concentrations were measured using an immunoturbidimetric method on the Allinity analyzer (Abbott, USA). Statistical analyses were performed on GraphPad Prism 10 to assess associations between the evaluated parameters. Results:
Median value of CRP in admitted patients was 11.1 mg/l (Q1=2.3; Q3=69.7). Mean NEU-X was 391±45.6 and mean NEU-Y 516±66.7. Both hematological parameters positively correlated with CRP (r=0.44 for NEU-X and r=0.46 for NEU-Y, both p<0.0001). ROC curve analysis revealed that both NEU-X and NEU-Y well differentiate between patients with normal (<10 mg/l) and elevated (>10 mg/l) CRP concentrations. AUC for NEU-X was 0.73, and AUC for NEU-Y was 0.74, p<0.001. NEU-X at a cut-off value of 400.3 had sensitivity of 70.5 and specificity of 65.52, at the likelihood ratio of 2.04. At the same likelihood ratio values of sensitivity and specificity were 64.48 and 67.82 for NEU-Y of 510.8. Conclusion Since NEU-X and NEU-Y had good differentiation value for patients with high and normal CRP concentrations, their analysis could be included as screening test for ER patients in which only complete blood count was referred. High values of both NEU-X and NEU-Y could be additional reason to test patient for inflammation and infection.

P189

Thal-Rbcnet: A Lightweight Deep Learning System For Red Blood Cell Morphology Analysis In Thalassemia
Vasita Wongsirikul¹, Bin Zhao², Chalisa Parnsamut¹, Patrawadee Pitakpolrat¹, Hathaiphon Aphaiwiwat¹, Parinda Limprasert¹, Chutitorn Ketloy¹, Phandee Watanaboonyongcharoen¹
¹Department of Laboratory Medicine, Faculty of Medicine, Chulalongkorn University, Bangkok, , Thailand, ²Shenzhen Mindray Bio-Medical Electronics Co., Ltd., Shenzhen, , China

Introduction:
Red cell morphology is one of the key elements in thalassemia diagnosis, yet manual smear review remains time-consuming and operator-dependent. To address this, we developed Thal-RBCNet, a lightweight deep-learning system designed to classify red blood cell (RBC) morphology from digital images in thalassemic patients. Methods:
High-resolution blood smear images were collected from 370 thalassemia patients, yielding 46,574 individual RBCs that were manually annotated and divided into training, validation, and test sets in a 7:2:1 ratio.
For the shape classification task, the training/validation/test sets comprised schistocytes (4,285/1,250 / 579), target cells (2,798/806/399), teardrop cells (3,474/1,000/548), and others (22,044/6,259/3,132).
For inclusions, there were basophilic stippling (312/105/53), Howell–Jolly bodies (96/27/18), Pappenheimer bodies (571/166/75), and others (31,622/9,017/4,512).
For hemoglobin content, hypochromic (8,773/2,441/1,231) and non-hypochromic (23,823/6,874/3,427) cells were labeled.
The pipeline consisted of a YOLO11 detector for RBC cropping and a MobileNetV3-based classifier that simultaneously predicted cell shape, inclusion type, and hemoglobin content. The model was tuned on the validation set and evaluated on the held-out test set. Results:
Generalizability was assessed on an external temporal test set of 156 thalassemia samples (≈ 400,000 RBCs) collected in 2025, including 8,250 schistocytes, 22,889 target cells, 2,475 teardrop cells, and 391,588 others for shape; 1,027 basophilic stippling, 128 Howell–Jolly bodies, 4,375 Pappenheimer bodies, and 419,672 others for inclusions; and 25,007 hypochromic vs 400,195 others for hemoglobin content.
Against expert-validated labels, Thal-RBCNet achieved accuracies from 0.71 to 0.96. Sensitivity (Se) and specificity (Sp) by class were: Hypochromic: Se 0.97, Sp 0.76, F1 0.69, Accuracy (Acc) 0.81 Schistocytosis: Se 1.00, Sp 0.69, F1 0.37, Acc 0.71 Target cells: Se 0.39, Sp 0.99, F1 0.56, Acc 0.85 Teardrop cells: Se 0.71, Sp 0.77, F1 0.78, Acc 0.73 Basophilic stippling: Se 0.96, Sp 0.80, F1 0.83, Acc 0.86 Howell–Jolly bodies: Se 1.00, Sp 0.95, F1 0.63, Acc 0.96 Pappenheimer bodies: Se 0.72, Sp 0.88, F1 0.75, Acc 0.82
Overall, F1-scores ranged 0.37–0.83, with strong negative predictive value (0.84–1.00) and negative predictive value up to 0.98 in most categories. Conclusions:
Thal-RBCNet is a compact AI system capable of analyzing multiple RBC morphological features simultaneously, maintaining high accuracy across tasks, and demonstrating stable performance on a large external cohort. Its application may enable standardized, high-throughput morphology screening and reduce reporting variability in routine hematology practice.

P190

Establishment Of Reference Intervals For Platelet Parameters And Immature Platelet Fraction (Ipf) In The Omani Population
Musleh Al Musalhi , Abbas Dastani, Shital Parag, Zayad Al Maskari , Yahya Al Rashdi, Ahmed Al Aamri
Ibra Hospital , Ibra , Oman

Background: Platelet parameters are critical biomarkers for the evaluation of thrombocytopenia and thrombotic disorders. While standard parameters like Platelet Count (PLT) and Mean Platelet Volume (MPV) are routinely used, the Immature Platelet Fraction (IPF) has emerged as a novel marker for assessing real-time thrombopoietic activity. Reference intervals (RIs) for these parameters are known to vary significantly based on ethnicity, age, geography, and the analytical platform used. To date, there is a paucity of data establishing specific reference ranges for parameters, particularly IPF, within the Omani population. This study aims to establish reference intervals for platelet parameters in healthy Omani individuals. Methods: A cross-sectional retrospective study was conducted on a cohort of healthy Omani individuals. Peripheral blood samples collected in EDTA tubes were analyzed using the Mindray BC-6200 Hematology Analyzer. The study established RI for platelet indices, including MPV, Platelet Distribution Width (PDW), Plateletcrit (PCT), Absolute IPF, percentage IPF (IPF%) and High Fluorescence IPF (H-IPF). Reference intervals were calculated in accordance with Clinical and Laboratory Standards Institute (CLSI) C28-A3 guideline. Results: A total of 129 healthy Omani individuals ranging in age from 13 to 95 years were included in the final analysis to establish reference intervals (RIs). The IPF% showed a median of 4.10% with a calculated reference interval of 1.1% – 13.0% (90% CI: lower limit 0.4–1.4, upper limit 11.5–14.6). The absolute IPF count had a mean of 11.86 x 10⁹/L and an RI of 2.0 – 29.8 x 10⁹/L. H-IPF ranged from 0.4% to 6.3% with a median of 1.7%. For standard platelet indices, the MPV showed a mean of 10.95 fL, SD 1.3 with an RI of 8.3 – 13.6 fL, PDW had a mean of 16.00, SD 0.6 with an RI of 14.9 – 17.1. PCT values ranged from 1.5% to 4.5% with a median of 2.6%. A significant positive correlation was observed between H-IPF and IPF% (Spearman’s coefficient rs = 0.964, P<0.0001). Conclusion: This study establishes the first comprehensive reference intervals for platelet parameters, including the IPF marker, specifically for the Omani population using the Mindray BC-6200 platform. The derived reference interval for IPF (1.1% – 13.0%) provides essential baseline data for local clinical practice. The strong correlation between H-IPF and IPF confirms the analytical reliability of the high-fluorescence fraction in this population. Implementation of these local reference intervals will enhance diagnostic accuracy in Omani clinical laboratories.

P191

Utility Of Platelet Indexes For Parvovirus B19 Diagnosis .
Tiffany I. V. Boaventura, Liliana M. S. Otsuka, Alexandre E. Kayano, Newton F. Centurião, Erivaldo S. Junior, Andreza O. Santos, Nikolas R. Constantino, Andrea A. R. Villarinho, João C. C. Guerra
Hospital Israelita Albert Einstein, São Paulo, Brazil

Introduction: Parvovirus B19 (PvB19) infection is a common viral disease worldwide and has the potential to cause serious hematologic complications, including transient aplastic crisis and pure red cell aplasia, particularly in patients with chronic hemolytic anemia and in immunocompromised individuals. A well-established feature of acute PvB19 infection is a marked decrease in reticulocyte count. Platelet indices have been increasingly investigated for clinical applications, including their role as unfavorable prognostic markers in sepsis. However, data regarding their usefulness in the diagnostic prediction of viral infections remain scarce. This study aimed to evaluate the association between platelet indices and PvB19 infection and to explore their potential role as supportive diagnostic markers. Methods: A cross-sectional study was conducted including patients with suspected PvB19 infection between January 2023 and December 2025. Peripheral blood samples were collected for complete blood count analysis and qualitative PvB19 polymerase chain reaction (PCR) testing. Hematological analyses were performed using the Sysmex® XN‑10 analyzer, and PCR testing was carried out using the RealStar® Parvovirus B19 PCR Kit 1.0. Continuous variables were expressed as medians and interquartile ranges (IQR), according to the Shapiro–Wilk test for normality. The qualitative PvB19 PCR result was considered the primary outcome variable. Age, platelet count, mean platelet volume (MPV), and immature platelet fraction (IPF) were included as explanatory variables. Group comparisons were performed using the Mann–Whitney U test. A p-value <0.05 was considered statistically significant. Statistical analyses were conducted using R software version 4.2.1. Results: A total of 200 samples were analyzed, including 25 PvB19 PCR–positive and 175 PCR–negative cases. Patients with positive PvB19 PCR results were younger than PCR‑negative patients (27.44; IQR 20.99 and 44.05; IQR 24.40, p = 0.005). Among platelet indices, IPF was significantly higher in PvB19 PCR–positive patients compared with PCR‑negative individuals (6.89; IQR 4.40 and 13.67; IQR 7.04 , p = 0.002). No significant differences were observed in platelet count (192.71 IQR 140.46 and 211.40 IQR 104.17, p = 0.258) or MPV (10.27 IQR 1.01 and 10.59 IQR 1.05, p = 0.157). Conclusion: An increased immature platelet fraction was associated with positive PvB19 PCR results, suggesting that IPF may serve as a supportive laboratory marker for suspected PvB19 infection. Limitations of this study include the absence of clinical correlation and serological testing. Further prospective studies are warranted to validate the clinical applicability of platelet indices in the diagnostic evaluation of PvB19 infection.

P193

Investigating The Utility Of Whole Blood Flow Cytometry For The Assessment Of Platelet Function In Patients With Normal Or Low Platelet Counts
Joanne E Joseph^1,2,3, David E Connor^2,3, David Rabbolini⁴, Bronwyn Thorp^1,3
¹St Vincent's Hospital , Sydney , Australia, ²St Vincent's Centre for Applied Medical Research , Sydney , Australia, ³St Vincent's Clinical School, University of NSW, Sydney , Australia, ⁴Kolling Institute of Medical Research, Sydney NSW, Australia

Introduction Light transmission aggregometry is the gold standard test for assessing platelet function, however testing is generally limited to patients with platelet counts >100x10⁹/L. Flow cytometric analysis of platelet function can be performed in patients with any platelet count using whole blood, allowing for minimal manipulation of samples. This study evaluated the utility of flow cytometry for the assessment of platelet function in patients with acquired and inherited thrombocytopenia, as well as patients with suspected platelet function disorders and normal platelet counts. Methods Whole blood was obtained from 56 individuals with suspected inherited thrombocytopenia, 22 with acquired thrombocytopenia (14 with immune thrombocytopenic purpura and 8 with myelodysplasia), as well as 69 with suspected inherited platelet function disorders and normal platelet counts. 38 healthy controls were also assessed. In addition to evaluation of glycoprotein expression, platelet reactivity was assessed by measuring expression of activation makers P-Selectin (CD62p), LAMP-1 (CD63) and activated glycoprotein IIb/IIIa (PAC-1) in whole blood stimulated with either thrombin receptor activating peptide (TRAP), collagen-related peptide (CRP) or adenosine diphosphate (ADP). Bleeding history was assessed using the ISTH bleeding assessment tool. Results In agonist stimulated samples overall, the expression of platelet activation markers was significantly decreased in both inherited and acquired thrombocytopenia when compared to healthy controls following TRAP or CRP stimulation. Patients with platelet function disorders demonstrated decreased PAC-1 binding for all agonists, decreased CD62p expression following CRP stimulation, but otherwise did not have decreased platelet activation. When comparing to healthy controls, the highest degree of platelet dysfunction was found in patients with myelodysplasia, with all patients demonstrating decreased reactivity to at least one platelet activation marker and agonist. Approximately 50% of patients with inherited thrombocytopenia demonstrated normal platelet reactivity. Notably, two patients had platelet counts lower than 10x10⁹/L and evaluation of platelet reactivity was possible. There was no significant difference between average bleeding scores for patients with inherited and acquired thrombocytopenia (p=0.3 for both), however bleeding scores were significantly higher for patients with suspected inherited platelet function disorders (p<0.001). There was a significant correlation between bleeding scores and platelet reactivity to TRAP (all markers), CRP (CD62p and PAC-1) and ADP (PAC-1) in inherited thrombocytopenia, but not in acquired thrombocytopenia. Conclusions Platelet function can be assessed in patients with normal and low platelet counts using flow cytometry, even in patients with profound thrombocytopenia who would not have been otherwise able to have functional testing performed.

P194

The End Of The Citrate Tube In The Hematology Field?
Elise KRATTINGER¹, Léo VOISIN¹, Nabil CHIOUKH ¹, Pascale DUSSERT¹, Enrico ZULIANI², Christophe VALLET², Terence LIANG³, Emilie GEREMIA⁴
¹Hôpital Nord Franche-Comté, BELFORT, France, ²Menarini Diagnostics France, RUNGIS, France, ³Shenzhen Mindray Bio-Medical Electronics Co., Ltd. , SHENZHEN, China, ⁴Mindray France, CRETEIL, France

Introduction: Hematology laboratories are commonly confronted with EDTA-dependent false thrombocytopenia and need to use alternative anticoagulants such as citrate or Thromboexact tube (Sarstedt®). At the Nord Franche-Comté Hospital (HNFC), the Mindray CAL 8000 line installed in November 2024, integrates two BC-6800Plus hematology analyzers, a SC-120 slide-stainer, and a MC-80 automatic digital microscope, all managed by the LabXpert expert software (version 1.55).For platelet count, three automated methods are available: impedance (PLT-I), hybrid (PLT-H) and optical measurement in the ERP channel triggered in case of aggregates (PLT-O), always a priority.Is the relevance of the citrated tube still relevant? Material and methods: Between January and June 2025, 67000 CBC were passed on the 2 BC-6800Plus analyzers. A retrospective study was performed on 207 thrombocytopenic patient profiles: PLT-I <150 G/L with platelet clumps flag. These results were compared to those of the same period in 2024, which required a sampling on a citrate tube. Results: The results of the PLT-I and PLT-O methods were compared on samples with the platelet aggregate flag, by threshold (Student's test, box plot with 95% CI). Our results confirm that the value of the PLT-O is always higher than the PLT-I count, especially in the low values (p<0.001). 31 typical cases of platelet depolymerization were identified (PLT-I<100 G/L), 13 improved (Average improvement ~200%) (<100 G/L) and 18 normalized after passage through the ERP channel. 14 had an H-IPF (hyperfluorescent platelet fraction) <10%, which leads to the conclusion that all the aggregates were depolymerized. The remaining cases had H-IPF between 10.3 and 30.8% and normal MPVs (<12 fL), suggesting the persistence of some aggregates resistant to depolymerization. Some moderate thrombocytopenia (>100 G/L impedance, n=23), are also improved in the optic channel with 6 H-IPF>10% including 3 with VPM>12 fL (12.1-14) in favor of highly fluorescent macro platelets in the optic channel. The actual thrombocytopenia is comparable between the 3 methods and has been verified under an automatic digital microscope. All the results meet the clinical acceptance criteria for their clinical background. For the same period in 2024, prior to the acquisition of Mindray analyzers, we had observed 50 cases of aggregated platelets, requiring citrate tube sampling. Conclusion: In 2025, the management of platelet aggregates has been simplified. Indeed, all thrombocytopenia with platelet aggregates were corrected in the ERP channel (PLT-O) after depolymerization, which improves the delivery time and allows a rapid clinical decision (transfusion, epidural, surgery,.).

P195

Comparison Of Automated Platelet Counting Methodologies Against A Cd61-Based Immunological Reference Method And Their Potential Impact On Clinical Decision-Making
Maria del Mar Meijon¹, Beatriz Astibia¹, Sandra Lopez¹, Fernando Martin¹, Miguel Piris¹, Juan Marquet¹, Javier Lopez¹, Jesus Villarrubia², Gemma Moreno¹
¹ Department of Hematology, Hospital Universitario Ramón y Cajal-IRYCIS, Madrid, Spain, ²Laboratorio Central de Madrid/UR Salud UTE, Madrid, Spain

Introduction: Automated hematology analyzers use different analytical principles for platelet counting, including optical methods, electrical impedance, and fluorescence-based channels. Some systems incorporate an immunological platelet count based on monoclonal antibodies against CD61, which is commonly used as a comparative reference method. Differences among these technologies may result in analytical variability at low platelet counts, potentially affecting clinical decisions. The aim of this study was to compare the performance of different automated platelet counting methodologies against a CD61-based immunological reference method and to evaluate their impact on clinical decision-making. Methods: A retrospective method comparison study was conducted using consecutive routine samples analyzed at Hospital Universitario Ramón y Cajal. In all cases, the time from phlebotomy to analysis was less than 6 hours. Samples with leukocyte counts between 4.0 and 15 ×10⁹/L and an MCV >80fL were included to minimize analytical interferences. Platelet counts were obtained using Alinity HQ (optical method with light scatter and fluorescence) and CELL-DYN Sapphire, which provides platelet counts through optical, electrical impedance, and a CD61-based immunological channel. The CD61 platelet count was used as the comparative reference method. Method comparison was performed using absolute Bland–Altman analysis, Lin’s concordance correlation coefficient (CCC), and Passing–Bablok regression. Additional analyses focused on discrepancies around the 50 and 20 ×10⁹/L thresholds, evaluating reclassification. Results: A total of 114 samples were included. The median platelet count by the CD61 method was 118.38 ×10⁹/L (interquartile range: 43.11–395.03; range: 0.00–768.28), with 46.5% of samples below 100 ×10⁹/L. Global Bland–Altman analysis showed similar negative biases for all automated methodologies compared with the CD61 method (−19.68 ×10⁹/L for Alinity HQ, −18.24 ×10⁹/L for the Sapphire optical channel, and −18.27 ×10⁹/L for impedance). Lin’s CCC values were high (>0.98) for all methodologies and Passing–Bablok regression slopes were close to unity (Figure 1). In the 20–50 ×10⁹/L range (n=26), absolute biases were close to zero for all methodologies. In the <20 ×10⁹/L range (n=8), positive biases and reclassification above the 20 ×10⁹/L threshold were observed in a limited number of samples; however, the reduced sample size in this interval limited robust assessment of interchangeability. Conclusions: Automated platelet counting methodologies showed high overall concordance with the CD61-based immunological reference method. Platelet counts were comparable in the 20–50 ×10⁹/L range, whereas increased variability and limited interchangeability were observed below 20 ×10⁹/L, although interpretation in this range is limited by sample size.

P196

Distinction Of Real Thrombocytopenia From False Low Platelet Counts Using Differently Anticoagulated Blood
Anna-Louisa Paul¹, Annika Stueven¹, Alexandra Hapke¹, Peter Kohlschein¹, Peter Schuff-Werner^1,2
¹Medical Laboratory Osnabrueck, D-49124 Georgsmarienhuette, , Germany, ²University Medical Center, Institute for Clinical Chemistry and Laboratory Medicine, D-18057 Rostock, , Germany

Introduction
Before planned invasive medical procedures and, in general, for the assessment of bleeding risks, it is obligatory to report thrombocytopenia (<150*10⁹/L). Possible causes of low in vivo platelet counts are e.g. cytotoxic medications, acute and chronic infectious diseases, bone marrow infiltration by tumor cells or inborn reasons. In vitro low platelet counts are predominately associated with pre-analytical errors, such as incorrect filling of the collecting tube or insufficient mixing with the anticoagulant with consecutive clot formation. The in vitro phenomenon of anticoagulant dependent formation of platelet aggregates, leading to falsely low platelet counts (“pseudo-thrombocytopenia”) is underestimated and difficult to recognize. Methods
EDTA-anticoagulated blood samples sent-in for routine blood cell count were measured by the XN 9000 analyzer. Thrombocytopenic samples (<150 * 10⁹ platelets /l), and samples presumably with “normal” platelet counts but flagged for “clumps” or “aggregates” were identified. “Unflagged” samples served as controls.
Finally, we asked the ordering physicians to send us with the patient's consent a new EDTA and a magnesium sulfate anticoagulated sample drawn in parallel.
Pseudo-thrombocytopenia (PTCP) was defined either by positive analyzer flagging, conspicuous platelet distribution curves or by morphological evidence of clumps and / or aggregates.

Results
Before starting our study, we compared blood cell counts measured in parallel in either EDTA- or MgSO4-anticoagulated blood samples from 222patients showing platelet counts within the reference range and from 38 real thrombocytopenic patients. By this approach we wanted to experience the comparability of platelet counts in differently anticoagulated blood samples. Platelet counts from real thrombocytopenic patients, ranged from 32 to 127*10⁹/L in EDTA- and from 32 to 98*10⁹/L in MgSO4- anti-coagulated blood.
Inconspicuous platelet counts measured in parallel ranged from 149 to 396 *10⁹/L (mean 229.55*10⁹/L ; SD 61.73*10⁹/) when measured in EDTA-anticoagulated blood and from 140 to 398 *10⁹(mean 229.55*10⁹/L ; SD ^61.73*10⁹/) when anticoagulated with MgSO₄(mean 226.90*10⁹/L , SD 62.48*10⁹/L). Platelet counts of 134 individuals with pseudo-thrombocytopenia measured in EDTA-anticoagulated blood ranged from 10to 150 *10⁹/L (mean 106*10⁹/L; SD 27.50*10⁹/L). Platelet counts measured in parallel in MgSO₄-anticoagulated samples ranged from 117 to 467*10⁹/L(mean 216.99*10⁹/L; SD 58.72*10⁹/L). Conclusions
With the here presented data we intend to show that platelet counts of suspected individuals with PTCP, when measured in parallel in EDTA and in magnesium sulfate anticoagulated samples allow to distinguish anticoagulant-dependent false low platelet counts from true thrombocytopenia.

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Primary Analytical Performance Evaluation Of The Mindray Bc‑720 Immature Platelet Fraction Parameter (Ipf‑D)
Kunal Sehgal, Vasudha Kaul, Pratidnya Parab, Jyotsna Rathod
Sehgal Path Lab, Mumbai, India

Background:
Immature platelet fraction (IPF) is an emerging biomarker for assessing thrombopoietic activity and predicting platelet recovery in conditions such as dengue-associated thrombocytopenia. Mindray has implemented an innovative algorithm enabling IPF measurement using data from the DIFF channel on the BC‑720 hematology analyzer (IPF‑D). However, the analytical performance of IPF‑D has not yet been systematically evaluated. Methods:
Analytical repeatability was assessed using 10 consecutive measurements on 10 whole‑blood samples. Precision was evaluated following CLSI EP05‑A3 using three control levels (High/Normal/Low, n = 80 replicates each). Method comparison against Sysmex XN‑Series was conducted across 115 clinical samples using Bland–Altman and stratified acceptance criteria (absolute difference ≤2 units for IPF <7%; relative difference ≤30% for IPF ≥7%). Results:
All samples met the predefined repeatability specification (CV ≤25% for PLT ≥50×10⁹/L and IPF ≥3%). Observed CVs ranged from 6.10% to 12.76%, demonstrating strong concentration‑dependent precision.
Precision studies showed within‑laboratory CVs of 18.5% (High), 20.6% (Normal), and 16.5% (Low), with no detectable between‑run variability, confirming robust day‑to‑day stability.
Method comparison revealed minimal systemic bias (−0.12 IPF units), with excellent agreement at lower IPF levels: 100% of samples with IPF <7% met absolute-difference criteria. In high IPF samples (≥7%), 94.8% met the relative‑bias criterion (≤30%). Variability increased at higher concentrations, consistent with biological and analytical expectations. Conclusions:
The BC‑720 IPF‑D parameter exhibits strong analytical performance, including high repeatability, stable multi‑day precision, and clinically acceptable agreement with the Sysmex XN‑Series. These findings support the suitability of IPF‑D for routine hematology testing and for monitoring thrombopoietic dynamics in clinical practice. Further validation in disease‑specific cohorts (e.g., dengue) is warranted.

P198

Assessment Of Beliefs, Education And Habits In The Prevention Of Sickle Cell Disease Among College Students In Osun State: The Need For Genetic Counselling
Florence I. Aboderin¹, Busayo G. Ologun², Joshua S. Olajide², Emmanuel Adepoju¹
¹University of Ilesha, Ilesha, Nigeria, ²Obafemi Awolowo University, Ile-Ife, Nigeria

INTRODUCTION Sickle cell disease (SCD) is a major hereditary blood disorder and public health challenge, especially among people of African descent. Over 500,000 infants are born with SCD each year globally, with more than 75% in sub-Saharan Africa. In Nigeria, 20–30% of the population carry the sickle cell trait, while 2–3% have SCD, contributing significantly to childhood morbidity and mortality, as 50–80% of affected children may die before age five without proper care. This study examines how cultural and religious beliefs and habits influence SCD prevalence, emphasizing the need for genetic counselling among young people to reduce its burden. METHODS An online questionnaire was used to collect data from students. It included socio-demographics, SCD knowledge, prevention-related habits and practices, genetic counselling, and prevention barriers. Data were analysed using IBM SPSS version 27 for inferential statistics overall. RESULTS A total of 269 respondents aged 18–25 participated in the survey. Male and female respondents were 52.4% and 46.8%, respectively. About 98.9% had heard of SCD through school lectures, health workers, media platforms, or peers. Overall, 77.7% knew their haemoglobin genotype, while 22.3% did not. Most respondents correctly identified SCD as a genetic condition and agreed it can be prevented through premarital genotype screening. Socially, there was general acceptance of SCD patients, including in marriage. Approximately 73.6% had undergone voluntary genotype testing, and the majority (88.5%) insisted on genotype testing before marriage or avoiding marriage if results were incompatible. Barriers to SCD prevention included poor awareness, testing costs, fear of results, and limited access to facilities. Cultural and religious beliefs and peer pressure were also contributing factors. A significant association existed between sex and knowledge of personal genotype (χ² = 9.293, p = 0.010), but not between genotype knowledge and SCD knowledge. Binary logistic regression showed sex as a significant independent predictor, with males nearly twice as likely to know their genotype compared to females (AOR = 1.87, 95% CI: 1.16–3.01, p = 0.010). CONCLUSION This study highlights a positive preventive attitude toward reducing the burden of SCD. Sex emerged as a significant determinant of genotype awareness, underscoring the need for targeted interventions, particularly among young female students.

P199

Haemoglobin Jeddah, A Rare Α-Globin Variant Highlighting Diagnostic Challenges In Hemoglobinopathy Screening: A Case Series From Oman.
Thekra I. Al-Obaidani¹, Maha S. Al-Yahyai², Ghalia Al-Harthi², Afrah Al-Fulaiti²
¹Oman Medical Specialty Board (OMSB), hematopathology residency program, Muscat, Oman, ²The Royal Hospital, Muscat, Oman

Introduction: Hemoglobin Jeddah is an extremely rare α-globin gene variant resulting from a point mutation at codon 68 in exon 2 of the α1 gene. This causes a substitution from asparagine to histidine (Asn→His) due to a nucleotide change from AAC to CAC. To date, hemoglobin Jeddah has been described in only a single case series involving three middle eastern families, representing its initial report in the literature. No cases have been reported from Oman. This case series enhances the understanding of hemoglobin Jeddah’s phenotype and highlights characteristic High-Performance Liquid Chromatography (HPLC) findings that may lead to misclassification as a β-globin variant, impacting hemoglobinopathy screening and premarital counselling in regions with high prevalence of hemoglobinopathies and consanguineous marriages. Methods: We conducted a retrospective review on three asymptomatic siblings who were identified during routine hemoglobinopathy screening (2017-2024) at a tertiary hospital in Oman. Complete Blood Counts (CBC), HPLC, and confirmatory molecular testing by bi-directional Sanger Sequencing of α‑, β‑ and δ‑globin genes were evaluated. Result: The siblings included in this case series were two males aged 77 and 69 years and one 36-year-old pregnant female. Their CBC results showed hemoglobin levels ranging from 11.0 - 14.8 g/dL with normocytic and normochromic red cell indices (range mean corpuscular volume (MCV) 88.0-96.3 fL and mean concentration hemoglobin (MCH) 30.0-32.8 g/dL). HPLC demonstrated an abnormal peak eluting in the HbA2 window with proportions ranging from 12.9% to 17.8%, initially suggesting a possible β-globin variant. A small additional peak in the D‑window (0.5%) was consistently observed in all three cases, which was not described in previous reports. Although analytical artefact cannot be fully excluded, the results reproducibility warranted further testing. Molecular analysis in all three cases confirmed heterozygosity for α-globin variant hemoglobin Jeddah: Cd68 AAC>CAC (HBA2:c.205 A>C). No mutations have been detected in the α2‑, β‑ and δ‑globin genes. The siblings were clinically asymptomatic with no evidence of hemolysis supporting a benign phenotype. However, long-term clinical significance remains unclear due to limited data. Conclusions: This case series represents the first reported cases of hemoglobin Jeddah in Oman. It is an addition to the limited existing literature by describing other familial cases and laboratory characteristics of hemoglobin Jeddah. This can help enhance laboratory experiences and improve diagnostic accuracy. Awareness of Hemoglobin Jeddah and its HPLC profile is important to avoid misclassification as a β‑variant and to support accurate interpretation of hemoglobinopathy screening, particularly in consanguineous populations.

P200

Assesment Of B-Thalassemia Trait Occurrence In An Outpatient Sample From Southern Chile: A Retrospective Study.
Mario Balcazar, Luis Carrasco, Angélica Mancilla, Sandra Navia
Tecno-Medic, Puerto Montt, Chile

Introduction: β-thalassemia trait (BTT) is a common cause of microcytic anemia worldwide, with highest prevalence in the Mediterranean, Middle East, and Southeast Asia; however, migration has contributed to its growing presence in non-endemic regions, including Latin America. In this region, BTT is a recognized cause of microcytosis, yet published data remain scarce and markedly limited in certain countries such as Chile, where nationwide estimates are lacking. Generating local evidence is essential to improve diagnostic accuracy for microcytosis and to inform public health strategies. This retrospective study sought to determine the frequency of BTT among referred outpatients in Puerto Montt, southern Chile, addressing a significant gap in national data.

Methods: A retrospective search was performed in the Laboratory Information System (LIS) of 24,634 unique outpatient Complete Blood Counts (CBCs) performed between January 2021 and November 2024. Cases were confirmed as BTT using a Hemoglobin A2 (HbA2) fraction >3.5% by capillary electrophoresis (Capillarys 2, Sebia). Hematological parameters were collected (Mindray BC-5380), and morphological characteristics from May-Grünwald-Giemsa-stained blood smears were evaluated. Iron Deficiency Anemia (IDA) was ruled out in all confirmed BTT patients (iron studies, Mindray BS-480, Maglumi-800). Results: A total of 60 cases were confirmed as BTT (HbA2> 3.5) out of 24,634 CBCs. This yielded a frequency of 0.24% (95% CI: 0.18-0.31%) in the studied cohort. The mean age of affected individuals was 44.0 years (SD: 23.2 years) 1515, with a predominance of females (68.3%, 41/60)16. The hematological profile showed pronounced microcytosis and hypochromia, with a mean MCV of 64.9 fL (SD: 3.13) and mean MCH of 20.5 pg (SD: 1.06). Anemia (WHO adjusted) was present in 58.3% (35/60) of BTT patients. Crucially, basophilic stippling was observed in 88.3% (53/60) of BTT patients on peripheral blood smears. Conclusions: This study identified a low, but not negligible, frequency of BTT (0.24%) in southern Chile , positioning the country with frequencies similar to Uruguay and lower than most neighboring countries. The high presence of basophilic stippling (88.3%) provides a useful diagnostic clue for the BTT phenotype in this population. These findings represent one of the first BTT prevalence estimates in Chile , contributing new evidence for the region, and highlighting the relevance of including BTT in both clinical and public health agendas for microcytic anemia differential diagnosis and genetic counseling.

P201

Fast Hemoglobin: A Sign Of Alpha Thalassemia Or A Red Herring?
Melissa Delio¹, Christopher Tormey², Alexa Siddon²
¹University of Colorado Anschutz School of Medicine, Aurora, CO, United States, ²Yale School of Medicine , New Haven, CT, United States

Introduction Alpha thalassemia is caused by mutations in the alpha globin gene resulting in decreased or absent production of alpha globin chains. Complications of the disease are dependent on the number of alpha globin genes mutated, ranging from no symptoms in adult carriers with one mutated gene to death in fetuses with 4 mutated genes. Given the potentially morbid outcome for the fetus, screening for alpha thalassemia in newborns and parents who could potentially pass it on to their children is a common practice. Hemoglobin (Hb) analysis is a common method used for screening, as a characteristic finding is varying amounts of fast hemoglobin including HbH and Hb Barts. Unfortunately, screening for alpha thalassemia has a high false positive rate as normal patients or patients with other hemoglobinopathies are often found to have fast Hb. This may result in unnecessary clinical work-up including expensive molecular tests to exclude alpha thalassemia. This study aims to find clinical and laboratory parameters to identify patients who truly have alpha thalassemia that would benefit from confirmatory molecular testing. Methods A search was conducted for patients with fast hemoglobin detected by capillary electrophoresis using the Sebia Capillarys 2 Flex-Piercing instrument between 2012 and 2024 at Yale New Haven Hospital. Cases were reviewed retrospectively and the following data points were collected: age, total Hb, red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular Hb concentration (MCHC), red cell distribution width (RDW), percentage of Hb (H, Barts, A, A2, S, C, E and F), history of hemoglobinopathies and results of next generation sequencing (NGS) studies. Patients were divided into the following groups based on their hemoglobin study results: normal, sickle cell disease, sickle cell trait, hemoglobin SC disease, alpha thalassemia (hemoglobin H disease and alpha thalassemia major), alpha thalassemia carrier/trait and beta thalassemia. The mean and standard deviation were calculated for all continuous parameters and the means between groups were compared by an ANOVA using prism software with a p value of <0.05 indicating statistical significance. Results 34 patients were identified with fast hemoglobin. 3/34 (8.82%) patients were placed in the alpha thalassemia group. There was no statistically significant difference between any of the groups in terms of age, total hemoglobin, RBC, MCV or MCHC. The percentage of HbH was borderline significantly higher in the alpha thalassemia group than in normal patients (17.53% ± 12.72% versus 0.54% ± 0.34%, p=0.056). Conclusions Patients with a % HbH that is 17.53% ± 12.72% may benefit from confirmatory NGS studies as these patients are borderline statistically more likely to have clinically significant alpha thalassemia. CBC parameters such as total hemoglobin, MCV, MCHC, RBC and RDW may not be helpful in distinguishing a patient with alpha thalassemia versus a patient with normal hemoglobin versus a patient with another hemoglobinopathy.

P202

Sample Stability Of A Photometric Rheology-Based Esr Method After Transient Heat Exposure And Return To Room Temperature
Steve Giarrusso, Megan McCutcheon
ALCOR Scientific LLC, Smithfield, RI, United States

Introduction: Erythrocyte sedimentation rate (ESR) is a non-specific marker of inflammation and one of the most common clinical laboratory tests worldwide. Samples must be at room temperature before testing, and elevated temperatures during testing are known to falsely increase ESR values. However, there is limited evidence on the effect of transient heat exposure prior to returning samples to room temperature for analysis. With specimen collection sites often located far from testing laboratories, temperature control during transport is frequently suboptimal, and samples may be exposed to temperatures well beyond room temperature in warm climates. The iSED ESR analyzer family uses photometric rheology to assess RBC aggregation – the first step of erythrocyte sedimentation - at a controlled 37°C and may be less sensitive to temperature fluctuations than Westergren-based ESR methods. Method: Residual whole blood samples (n = 131) <24 hours from venipuncture were analyzed for ESR at room temperature (18-25°C) using iSED ELITE. Samples were then incubated at 95°F for 4 hours to simulate extreme transport or storage conditions, brought back down to room temperature, and re-analyzed for ESR. Agreement between initial room temperature and post-heating values was assessed using intraclass correlation coefficient (ICC) with 95% confidence intervals. Results: ESR values at room temperature had a mean of 21.4 ± 16.3 mm/hr (median 15, IQR 8–31). After heating, the mean was 22.9 ± 16.2 mm/hr (median 17, IQR 10–32). The mean difference between conditions was 1.5 mm/hr (95% CI: 1.1–1.8). The ICC for absolute agreement was 0.987 (95% CI: 0.987–0.993), indicating excellent reliability between the two conditions. The observed difference of 1.5 mm/hr falls within acceptable clinical variability for ESR testing. Conclusion: Exposure to elevated temperature (95°F for 4 hours) prior to returning to room temperature does not produce clinically significant changes in iSED ELITE ESR results. The excellent ICC demonstrates that heated samples yield values interchangeable with room temperature measurements for clinical interpretation. These findings suggest photometric rheology-based ESR testing remains reliable despite short-term exposure to elevated temperatures that may occur during specimen transport.

P203

A Digital Transformation Of The Kleihauer-Betke Removes The High Coefficient Of Variation During The Screening Of Fetomaternal Haemorrhage; A Pilot Study
Phoebe King¹, Riya Raghuram², Kenneth Salisbury³, Joanna Ward¹, Aaron Niblock^4,5
¹School of Engineering, Ulster University, Belfast, United Kingdom, ²Department of Engineering, University of Cambridge, Cambridge, United Kingdom, ³KAIoptix Ltd., Antrim, United Kingdom, ⁴School of Medicine, Ulster University, Derry, United Kingdom, ⁵Antrim Area Hospital, Antrim, United Kingdom

Introduction: Fetomaternal haemorrhage (FMH) is the passage of fetal red blood cells into the maternal bloodstream. It is a clinically important issue, as inadequate recognition can lead to maternal alloimmunisation and haemolytic disease of the newborn. The Kleihauer–Betke (KB) acid-elution test is widely used for screening FMH, but is limited by a high coefficient of variation between users and laboratories, due to poor cell visibility and preparation issues (see Figure). While flow cytometry, the current gold-standard, gives more accuracy, it is unsuitable for routine screening due to significant cost considerations and need for specialised staff. This pilot study evaluated a deep-learning and image-analysis-based digital method for FMH quantification from KB-stained blood films. Methods: 10 UK National External Quality Assessment Service (NEQAS) slides were imaged using an adapted form of optical microscopy for enhanced cell delineation (see Figure), and a MATLAB-based workflow incorporating deep-learning cell segmentation and colour- and texture-based classification was used to automatically identify foetal and adult red blood cells to estimate FMH volume. Results were compared with inter-laboratory manual KB counts and NEQAS flow cytometry reference values using Bland–Altman analysis, correlation and paired t-tests. Results: Across the samples, the digital automated model produced FMH volume estimates that were closer to flow cytometry reference values than manual KB counting. The manual method, in general, overestimated bleed volume, with a mean bias of +2.4 mL compared with flow cytometry, whereas the digital model showed a negligible mean bias of –0.26 ml. Paired t-testing demonstrated a significant difference between manual KB and flow cytometry (p = 0.0001), with substantially less significant difference between the digital model and flow cytometry (p = 0.67). Both methods correlated strongly with flow cytometry (r² ≥ 0.96). The digital model produced fully repeatable results on re-analysis and showed no operator-dependent variability, in contrast to wide inter-laboratory variation in manual KB results (0.0–52.5 mL). The model also reliably excluded a range of common artefacts, although two thin, sub-optimally prepared slides led to underestimation in volume, highlighting an area for future work. Conclusions: Digital transformation of the KB test provides more accurate, consistent, and reproducible FMH volume estimation than manual KB counting, with performance more comparable to flow cytometry on standard-quality slides. These data support its potential role as a reliable FMH screening tool.

P204

Association Of Hematimetric Indices With Ema Binding And Osmotic Fragility In Hereditary Spherocytosis
Isabella C. Lima, Liliana M. S. Otsuka, Alexandre E. Kayano, Newton F. Centurião, Erivaldo S. Junior, Andreza O. Santos, Cristina M. Ito, Andrea A. R. Villarinho, João C. C. Guerra
Hospital Israelita Albert Einstein, São Paulo, Brazil

Introduction: Hereditary spherocytosis (HS) is the most common inherited hemolytic anemia, resulting from defects in erythrocyte membrane proteins that cause premature extravascular hemolysis. Diagnosis may be challenging, particularly in mild or atypical cases, requiring integration of clinical features, peripheral blood morphology and laboratory investigations. Hematimetric indices obtained from the complete blood count (CBC), together with specialized tests such as eosin-5-maleimide (EMA) binding by flow cytometry and the osmotic fragility test (OFT), are frequently used in the diagnostic workup. This study aimed to evaluate the association between CBC parameters and EMA and OFT results in an institutional cohort of patients investigated for suspected HS. Methods: This cross-sectional study included 94 patients evaluated at a tertiary care hospital between 2020 and 2024. CBC parameters analyzed were hemoglobin (Hb), hematocrit (Hct), red blood cell count (RBC), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), red cell distribution width (RDW) and reticulocyte count. Patients were grouped according to EMA results (reduced or normal) and OFT results (right shift, left shift, or normal). The Shapiro–Wilk test assessed data distribution. Student’s t-test and ANOVA were applied to normally distributed variables (MCV, MCH, MCHC, RBC), while the Mann–Whitney and Kruskal–Wallis tests were used for non-normally distributed variables (reticulocytes, Hb, Hct, and RDW). Statistical significance was defined as p <0.05. Analyses were performed using R software version 4.2.1. Results: Among the 94 patients, 55% were female and the median age was 30 years. EMA testing was performed in 25 patients, of whom 7 (28%) showed reduced fluorescence. OFT was performed in 72 patients, with 11 (15%) demonstrating a right shift. Only five patients underwent both tests, with one (20%) yielding concordant positive results. MCHC values were significantly higher in EMA-reduced patients (p = 0.01), whereas no significant association was observed with OFT alterations. RDW was increased exclusively in the EMA-positive group (p = 0.037). Reticulocyte counts were significantly higher in EMA-positive patients (p = 0.01) and in those with OFT right shift (p = 0.003). No significant associations were found for Hb, Hct, RBC count, or MCV. Conclusion: Despite the superior sensitivity and specificity of the EMA test compared with OFT, its limited use in routine practice may reflect higher costs and restricted reimbursement. These findings reinforce the value of CBC-derived parameters as initial screening tools for HS. Elevated MCHC, increased RDW, and reticulocytosis should heighten clinical suspicion and guide the selection of confirmatory tests, underscoring the importance of integrated laboratory evaluation in the diagnosis of HS.

P205

“Electroglobins” �" Haemoglobin Capillary Electrophoresis: A One Year Experience
Ana Catarina B. Marques , Joana Sevilha, Carla Ferreira , Helena Ferreira da Silva
Clinical Pathology Department, ULS Médio Ave, Vila Nova de Famalicão, Portugal

Introduction: Hemoglobinopathies (HBP), including structural and quantitative disorders, are the most common monogenic autosomal recessive diseases worldwide. The increasing population mobility has led to a rising prevalence of these conditions in non-endemic countries. While severe forms such as sickle cell disease and α/β-thalassemia major cause significant morbidity, carriers are usually asymptomatic. Nevertheless, carrier couples have a 25% risk of having an affected child in each pregnancy, underscoring the importance of effective preconception and antenatal screening strategies. Haemoglobin Capillary Electrophoresis (CE) has emerged as a valuable tool for the detection and characterization of haemoglobin variants. Our aim is to determine the prevalence and spectrum of hemoglobinopathies in individuals from our healthcare area during the first year of implementation of CE. Methods: A retrospective observational study was conducted, including CE results obtained in 2025 using the Capillarys 3 Octa (Sebia). Demographic data and hemogram parameters were also collected. Statistical analysis was performed using Excel® software. Results: Seventy-one CE results were reviewed from 71 patients (51 females and 20 males, with a mean age of 43 years). Most tests were directly requested, while 24 were added following the detection of abnormal hemoglobin patterns during routine HbA1c testing. Hemoglobinopathies were identified in 38 samples (53%), comprising eight different variants. The most frequent findings were Hb S trait (34%) and hereditary persistence of foetal haemoglobin (HPFH) (29%), with similar distributions in both sexes. Almost half of the studied population were pregnant women (n=32), and hemoglobinopathies were identified in half of these cases, most frequently Hb S trait and β-thalassemia minor. Similar findings were observed in the paediatric population (n=8), with hemoglobinopathies detected in four cases. Among patients with microcytic anaemia (n=18), hemoglobinopathies were detected in 10 cases, predominantly HPFH and β-thalassemia minor. Portuguese patients accounted for 45 results, 25 with HBP, mainly HPFH and β-thalassemia trait. In the non-Portuguese population (n=26), mostly Brazilian, hemoglobinopathies were detected in half of the cases, with Hb S trait being the most prevalent. Conclusions: Capillary electrophoresis proved to be an effective method for detecting a wide range of haemoglobin variants, including incidental findings during HbA1c testing. This first-year experience highlights the impact of migration on the local prevalence of hemoglobinopathies and supports the need for structured diagnostic pathways and targeted screening, particularly in prenatal care. A specialized medical consultation has been implemented to optimize diagnosis and follow-up. There are no conflicts of interest.

P206

Exploring The Utility Of Advanced Red Blood Cell Parameters In Microcytic Hypochromic Anemia
Tandry Meriyanti^1,2, Fransisca Probo Setyoningrum²
¹Siloam Hospitals Lippo Village, Banten, Indonesia, ²Pelita Harapan University, Banten, Indonesia

Introduction. Classification of anemia requires morphological classification via measurement of red blood cell (RBC) indices. Modern automated hematology analyzer offers advanced RBC parameters to deliver detailed data on erythrocyte morphology and hemoglobin status in erythrocyte, such as percentage of microcytic (MicroR) and hypochromic RBC (HYPO-He), immature reticulocyte fraction (IRF) and reticulocyte hemoglobin content (RET-He), which allow fast and explicit description of erythrocyte maturation and erythropoiesis without additional tests. This study aimed to explore the utility of advanced red blood cell parameters in microcytic hypochromic anemia, particularly in differentiating hemoglobinopathy, iron deficiency anemia, and anemia of inflammation as the common causes of microcytic hypochromic anemia. Methods. This study was a cross-sectional analytic study with total of 163 samples, consisting of hemoglobinopathy (50 samples), iron deficiency anemia (53 samples), anemia of inflammation (30 samples), and normal (30 samples). This study was conducted between January – July 2025 at Siloam Hospitals Lippo Village, Banten, Indonesia. Advanced red blood cell parameters (IRF, MicroR, HYPO-He, Ret-He) were included in RET channel, performed by Sysmex XN2000. Statistical data analysis was done using SPSS version 26. Results. A Kruskal Wallis test demonstrated significant difference in IRF, MicroR, HYPO-He, Ret-He among iron deficiency anemia, hemoglobinopathy, anemia of inflammation, and normal group (p<0.001 for all parameters). Pairwise comparison using Dunn’s test with Bonferonni correction indicated IRF, MicroR, HYPO-He, and RET-He were significantly different in anemia of inflammation compared to iron deficiency anemia and hemoglobinopathy. Meanwhile, MicroR and the ratio of MicroR/HypoHe were significantly different between iron deficiency anemia and hemoglobinopathy (p<0.001, p=0.004, respectively). Analysis on ROC curve, Ret-He had AUC of 0.912 (95% CI: 0.851 – 0.974) in distinguishing anemia of inflammation from iron deficiency anemia and hemoglobinopathy. MicroR had AUC of 0.792 (95% CI: 0.698 – 0.887) and MicroR/HYPO-He had AUC 0.741 (95% CI: 0.645 – 0.836) to differentiate hemoglobinopathy from iron deficiency anemia. Conclusions. Advanced RBC parameters and its combination represented a valuable tool to improve screening of differential diagnosis of microcytic hypochromic anemia.

P207

Prevalence And Clinical Correlates Of Anaemia Among Hospitalized Geriatric Patients In Malaysia: A Cross-Sectional Study
noor haslina mohd noor¹, esba ahamed², maryam azlan², nurul izzah abdul razak¹, asiah nabihah¹
¹haematology department, universiti sains malaysia, KUBANG KERIAN, , Malaysia, ²biomedicine department, universiti sains malaysia, KUBANG KERIAN, , Malaysia

Introduction:
Anaemia is common in older adults and is associated with adverse outcomes including functional decline and increased mortality. Hospitalised geriatric patients are particularly vulnerable due to multimorbidity and acute illness, yet data from Southeast Asia remain limited. This study aimed to determine the prevalence, severity, morphological patterns, haematological characteristics, and common aetiologies of anaemia among geriatric inpatients in a Malaysian tertiary teaching hospital. Methods:
A retrospective laboratory-based cross-sectional study was conducted at Hospital Pakar Universiti Sains Malaysia. All inpatients aged >65 years who had a full blood count performed between January and June 2023 were included. Anaemia was defined using World Health Organization criteria and classified by severity and morphology based on haemoglobin levels and red cell indices. Haematological parameters were compared by gender. Available clinical records were reviewed to identify documented causes of anaemia. Results:
Among 3,610 geriatric inpatients, anaemia was identified in 1,914 patients, giving a prevalence of 53.0%. Mild anaemia predominated (59.9%), followed by moderate (29.2%) and severe anaemia (10.9%). Normocytic normochromic anaemia was the most common morphological pattern (75.1%), while microcytic hypochromic and macrocytic anaemia accounted for 22.6% and 2.3%, respectively. Female patients had significantly lower haemoglobin levels and a higher proportion of moderate-to-severe anaemia compared with males. Among patients with available clinical data (n=104), chronic illnesses—particularly cardiovascular disease, diabetes mellitus, and hypertension—were the most frequently documented causes, followed by infection, chronic kidney disease, and malignancy. Conclusion:
Anaemia is highly prevalent among hospitalised older adults and, although often mild, frequently reaches clinically significant severity. These findings highlight the importance of routine anaemia screening and systematic evaluation in geriatric inpatients to enable timely intervention and potentially improve functional and clinical outcomes.

P208

Evaluation Of The New Mindray H120 Instrument For Determining Hba1C By Hplc: Comparison Of Two Analysis Modes Offered By The H-120 With Capillary Electrophoresis In Common Situations And In The Presence Of Haemoglobin Variants.
Stephane Moutereau¹, Soraya Fellahi¹, Emilie Geremia², Terence Liang³, Jean-Philippe Bastard¹
¹Département de Biochimie-Pharmacologie, Hôpital universitaire Henri Mondor, AP-HP INSERM U955, Université Paris-Est-Créteil, Creteil, France, ²Hematology IVD Product - Mindray France, Creteil, France, ³International Scientific Strategy & Partnerships Mindray IVD Division -Shenzhen Mindray Bio-Medical Electronics Co., Ltd. , Shenzen, China

Background: Assessing average blood glucose levels by measuring glycated haemoglobin is a valuable tool for the long-term monitoring of diabetic patients. The aim of this study was to evaluate the Mindray H-120 analyser for measuring HbA1c in routine settings. Methods: Two analysis modes were studied: ‘standard mode’ (30-second elution) and ‘variant mode’ (60-second elution). Repeatability and reproducibility were studied at several levels using both supplier controls and patient samples. A comparative study was conducted between the Mindray H120 and the Capi3Tera (Sebia) using Passing-Bablok regression and Bland-Altman plots. The interference of Hb variants was studied using samples containing HbS, HbC, HbD, HbE, Hb Hope, HbH and a few other rare haemoglobins. Results: For each of the ‘standard’ and ‘variant’ modes, the intra-series repeatability CVs are less than 0.67% and 0.34%, respectively. The inter-series CVs are less than 2.88% and 2.36%, respectively. Apart from a few rare haemoglobin variants, no non-linearity was observed: the correlations between the different modes and with capillary electrophoresis were very satisfactory for samples without haemoglobin variants (slopes of 0.95 to 1.026; y-intercepts of 0.0263 to 0.1585) or containing common haemoglobin variants (HbS, HbC: slopes of 1.022 to 1.069; y-intercepts of -0.409 to -0.070). Only a few rare variants (Hb Hope, Hb Athens Georgia, etc.) revealed discrepancies between the different types of analysis: all elution abnormalities, including the presence of haemoglobin variants, were correctly reported by the automated system. Conclusion: The Mindray H120 analyser proved to be extremely easy to use and highly reliable, with good reproducibility, good concordance and excellent linearity across the wide range of HbA1c levels commonly observed in diabetic patients, without being significantly influenced by common haemoglobin variants.

P209

The Rising Prevalence Of Elevated Haematocrit In A Large Uk Population: A Five Year Retrospective Analysis
Toby Nicholson, Ana B. Martins, Andzelika Aydin
Mersey and West Lancashire Teaching Hospitals NHS Trust, Prescot, United Kingdom

Introduction: An elevated haematocrit is a common laboratory abnormality found both in acute care and community settlings. Causes are varied, be it due to dehydration, secondary erythrocytosis or myeloproliferative diseases. Whilst changes in population demographics, health, societal behaviour and prescribing patterns could theoretically influence haematological pararmeters over time, there is little published evidence describing temporal trends in the prevalence of raised haematocrit in routine full blood count (FBC) testing. Methods: We conducted a retrospective analysis of all FBCs processed at a large district general hospital serving a population of approximately 600,000 people. The proportion of samples with haematocrit values above the established reference range was calculated from 2021 to 2025 and stratified by sex. No changes in analysers, calibration procedures, or reference intervals occurred during this period. Results: A progressive increase in the proportion of elevated haematocrit results was observed in both sexes. In men, the percentage of FBCs with haematocrit above the reference range rose from a mean of 0.27% in 2021 to 0.34% in 2025. In women, the corresponding increase was from 0.35% to 0.41%. This represents a relative rise of approximately 17-25% over five years. There appears to be seasonal variation with higher proportions in the winter months but this did not impact the upward trend. Conclusion: This study identifies a steady and clinically relevant increase in the proportion of elevated haematocrit results in a large UK population over a five‑year period. It does not reveal the cause of the progressive rise but we speculate it might be secondary to increasing prescription of SGLT2 inhibitors which have gained new indications over that period; testosterone use or obesity are other potential causes, whilst alcohol and smoking are declining and unlikely to be contributing. The findings highlight the need for broader epidemiological investigation into emerging drivers of erythrocytosis and underscore the value of laboratory data as an early signal of population‑level health trends.

P210

Pediatric Polycythemia: Frequent Transient Finding With Few Underlying Diagnosis
Sotirios Papadeas^1,2, Yves D Pastore^1,2,3,4, Thomas Pincez^1,2,3,4
¹CHU Sainte-Justine Azrieli Research Center, Montreal, QC, Canada, ²Faculty of Medicine, Université de Montréal, Montreal, QC, Canada, ³Division of Pediatric Hematology-Oncology, CHU Sainte-Justine, Montreal, QC, Canada, ⁴RED, Réseau Érythrocytes et Drépanocytose, Montreal, QC, Canada

Introduction Polycythemia can be driven by hematopoiesis-intrinsic or hypoxia-alterations, defining primary and secondary polycythemia, respectively. Primary polycythemia due to myeloproliferative neoplasms is the leading cause in adults but these diseases are uncommon in children. However, there are limited data on the underlying causes identified in children and adolescents with polycythemia. We aimed to describe the characteristics, underlying diagnosis, and outcomes of children referred for polycythemia. Methods Patients ≤21 referred for polycythemia in the hematology department of the CHU Ste-Justine pediatric hospital in Montreal, Canada from 1992 to 2025 were retrospectively reviewed. Polycythemia was defined using age-specific cutoff. Results After exclusion of 12 patients for insufficient data, we included 92 patients in this study. Most patients (81%) were boys. Age of consultation was considerably lower for girls than boys (mean ± standard deviation: 6.2 ± 4.9 years vs 13.3 ± 5.2 years, p <0.0001). Male patients frequently had an elevated body mass index (28% > z-score=2 and 7% > z-score=3) (figure 1). Hemoglobin values were <190 g/L at the exception of one girl who was diagnosed with idiopathic polycythemia and reached hemoglobin levels of 230 g/L. Four patients (4%) had low EPO levels and were initially suspected of primary polycythemia. Eight patients (9%) presented high EPO levels, and 76 (83%) had normal EPO levels (the remaining 4 patients (4%) did not have an EPO dosage). A somatic variant of myeloproliferative neoplasm was researched in 16 patients (17%) and identified in only one (1%) (JAK2 gene). Eleven patients (12%) underwent gene- or panel-based germline sequencing. Two had pathogenic variants in EPAS1 (n=1), and EPOR (n=1). Three other patients had genetic variations that may have contributed to polycythemia: HFE H63D variant (n=1), variants of unknown significance in RYR1 (n=1), and in TSC2, INVS, and BBS1 (n=1). These were not retained as primary causes and 89 patients (97%) were considered to have secondary polycythemia without genetic anomalies. Of those, 30 (33%) had one potential contributing factor: lung disease (n=18), kidney disease (n=12), cardiopathy (n=2), hepatopathy (n=2), or medication (n=1). Conclusions Overall, our data highlight the specificities of pediatric polycythemia. Most cases were observed in male adolescents who were frequently overweighted, and hemoglobin level was modest in most cases. Polycythemia was more often secondary without monogenic cause and mandate search of potential contributing factors as first investigations. Genetic work-up including inherited or acquired genetic variations should be considered in the absence of such contributing factors.

P211

When To Suspect Co-Inherited Alpha-Thalassemia In Common Structural Hemoglobin Variants
Armin P. Piehler¹, Lea Dewi Schlieben¹, Eva C. Langsjøen², Gregor Hoermann¹, Torsten Haferlach¹, Wolfgang Kern¹, Manja Meggendorfer¹
¹MLL Munich Leukemia Laboratory, Munich, Germany, ²Fürst Medical Labortory, Oslo, Norway

Background Alpha‑thalassemia and common beta‑globin variants such as HbS, HbE, HbD, and HbC are inherited independently and may therefore co‑occur in the same individual. Hemoglobin separation methods, including capillary electrophoresis (CE) and high‑performance liquid chromatography (HPLC), reliably identify these variants but generally fail to detect the alpha‑thalassemia trait. Indirect markers, such as the relative size of the hemoglobin variant fraction, can indicate concomitant alpha‑thalassemia. This study aimed to characterize the impact of co‑inherited alpha‑thalassemia trait on hemoglobin variant fraction size and to provide guidance for interpreting hemoglobin patterns in patients with common structural hemoglobin variants and suspected alpha‑thalassemia. Methods The study includes data from 21,500 individuals undergoing hemoglobinopathy work‑up at two large European laboratories (Fürst Medical Laboratory, Norway, and MLL Munich Leukemia Laboratory, Germany). Hemoglobin differentiation was performed by CE, and structural variants were confirmed by HPLC or beta‑globin gene sequencing. Alpha‑thalassemia was identified by gap‑PCR or multiplex ligation-dependent probe amplification (MLPA), and serum ferritin was measured by nephelometry or immunoassay. Statistical significance was assessed by Wilcoxon signed‑rank test, with adjusted p <0.01 considered significant. The study was approved by the institutional review boards and the regional ethics committee (REK). Results In total, the structural hemoglobin variant traits HbS, HbE, HbD-Punjab and HbC were identified in 697 (3.2%), 558 (2.6%), 229 (1.1%) and 133 (0.6%) of 21,500 cases, respectively. The number of concomitant inherited defective alpha-globin genes exhibited a clear gene-dosage effect on the proportion of the hemoglobin variant relative to total hemoglobin (Table 1). Across all traits, concomitant alpha-thalassemia was not observed in HbS, HbE, HbD-Punjab and HbC cases with variant fractions exceeding 39.6%, 25.0%, 41.8% and 33.2%, respectively. Similar effects were observed in patients with iron deficiency (serum ferritin <15µg/L). Conclusion The amount of structural hemoglobin variants is significantly influenced by concomitant alpha‑thalassemia, exhibiting a clear gene‑dosage effect corresponding to the number of co‑inherited defective alpha‑globin alleles. Because accurate detection of alpha-thalassemia in hemoglobin variant carriers is essential to prevent misdiagnosis and to support appropriate genetic counseling, these findings provide useful guidance for interpreting hemoglobin separation profiles and for identifying cases that warrant further testing for alpha-thalassemia.

P212

Quantitative Reticulocyte Changes Precede Hemoglobinization After Short-Term Oral Iron Supplementation In Regular Blood Donors
Tri Ratnaningsih¹, Noor Mukantari², Fuad Anshori¹, Umi S Intansari¹, Usi Sukorini¹
¹Department of Clinical Pathology and Laboratory Medicine, Faculty of Medicine, Public Health and Nursing, Gadjah Mada University, Yogyakarta, , Indonesia, ²Medical Specialist Education Program in Clinical Pathology, Faculty of Medicine, Public Health, and Nursing, Gadjah Mada University, Yogyakarta, , Indonesia

Introduction: Iron deficiency without anemia is common among regular blood donors and can develop into iron deficiency anemia if not detected early. Standard donor screening relies mainly on hemoglobin tests, which often miss initial iron depletion. Reticulocyte parameters, especially advanced indices from modern hematology analyzers, can signal early erythropoietic activity and may help monitor iron repletion. However, there is limited data on the effects of short-term oral iron supplementation on these parameters in regular donors. This study examines how reticulocyte count and extended reticulocyte parameters change after short-term oral iron therapy in this population. Methods: This was a single-arm pre–post clinical study involving thirty regular blood donors. Participants received 65 mg of elemental iron by mouth daily for 10 days. Blood samples were collected before and after supplementation. The parameters measured included reticulocyte percentage, absolute reticulocyte count, fluorescence-based reticulocyte fractions (RET-L, RET-M, RET-H), immature reticulocyte fraction (IRF), mean reticulocyte volume (MRV), and reticulocyte hemoglobin content (RHCC), all analyzed with the Horiba Yumizen 2500 hematology analyzer. Data were analyzed using paired statistical tests; p-values <0.05 were considered statistically significant. The study was approved by the Ethics Committee of Universitas Gadjah Mada (KE/FK/0330/EC) and the Indonesian Red Cross Blood Center in Yogyakarta, with all participants providing written informed consent. Results: Short-term oral iron supplementation led to significant rises in the absolute reticulocyte count (0.06 ± 0.02 to 0.08 ± 0.02 ×10⁹/L, p<0.001) and reticulocyte percentage (1.24 ± 0.34 to 1.72 ± 0.52, p<0.001). The reticulocyte maturation profile changed notably, with RET-L decreasing (86.69 ± 6.74 to 82.64 ± 6.25, p=0.005) and RET-M increasing (10.73 ± 4.76 to 14.39 ± 4.53, p=0.002); RET-H remained unchanged (p=0.150). The immature reticulocyte fraction increased significantly (0.12 ± 0.05 to 0.15 ± 0.05, p=0.011), as did MRV (106.87 ± 8.13 to 109.50 ± 7.93 fL, p=0.002). RHCC showed no significant change (30.99 ± 3.49 to 31.16 ± 3.26 pg; p=0.525). The overall changes, illustrated in the figure, indicate that short-term oral iron increases reticulocyte count and immature reticulocytes, while mature reticulocytes decrease. Changes in MRV and RHCC were minor, suggesting early erythropoietic activation prior to hemoglobinization. Conclusions: In regular blood donors, short-term oral iron therapy triggers an early erythropoietic response. This response is more easily identified by reticulocyte count parameters than by reticulocyte hemoglobin content. Extended reticulocyte parameters serve as useful early indicators of iron-dependent erythropoiesis and could enhance existing blood donor screening techniques.

P213

Untangling Iron Deficiency From Inherited Microcytosis: The Utility Of Ferritin Measurement In Hemoglobinopathy Investigations
Nicholas Sandercock¹, Jessica Graham¹, Barry Eng¹, John Waye^1,2, Sarah Patterson^1,3
¹1. Hamilton Regional Laboratory Medicine Program, Hamilton Health Sciences, Hamilton, ON, Canada, ²2. Department of Pathology and Molecular Medicine, McMaster University, Hamilton, ON, Canada, ³3. Division of Hematology and Thromboembolism, Department of Medicine, McMaster University, Hamilton, ON, Canada

Introduction:
Hemoglobin electrophoresis is central to the evaluation of hemoglobinopathies; however, α-thalassemia deletions are not detectable by electrophoresis and require DNA analysis. Thalassemia trait causes microcytosis that may mimic iron deficiency. Incorporating ferritin measurement into hemoglobinopathy investigations may both guide reflex genetic testing and identify patients at risk for iron deficiency anemia. Methods:
We performed a retrospective single-centre study in Canada of hemoglobinopathy investigations conducted between September 1, 2023, and September 1, 2025. Testing included hemoglobin electrophoresis confirmed by high-performance liquid chromatography and ferritin measurement. Low ferritin was defined as <30 µg/L. DNA testing was reflexed for abnormal hemoglobin variants and/or microcytosis (MCV <82 fL). Genetic testing included gap Polymerase Chain Reaction (PCR) and Sanger sequencing for common β-thalassemia mutations and gap PCR for seven α-thalassemia deletions (SEA, FIL, THAI, MED, 20.5 kb, 4.2 kb, and 3.7 kb). Data were collected anonymously from the electronic medical record. Results:
A total of 4,994 hemoglobinopathy investigations were completed; 1,522 (30.5%) demonstrated low ferritin. When broken down by sex, 1278 samples were female (83.97% of low ferritin samples, and 25.59% of all samples) and 241 were male (15.83% of low ferritin samples, and 4.83% of all samples). Among samples with low ferritin, 342 (22.5%) underwent genetic testing due to microcytosis. Low ferritin was present in 24.2% of all 1416 samples sent for DNA analysis. Among 1002 samples assessed for α-thalassemia, only 26.5% had concomitant low ferritin. Conclusions:
Iron deficiency was common in this population and frequently occurred without microcytosis, identifying individuals at risk for future anemia. Iron deficiency was markedly more prevalent in females, likely due to menstrual blood loss. The higher frequency of iron deficiency observed compared with other Canadian cohorts likely reflects selection for microcytosis and family planning investigations. Routine ferritin assessment adds value to hemoglobinopathy testing by helping distinguish iron deficiency from inherited causes of microcytosis and identifying clinically actionable deficiencies.

P214

Null Α-Spectrin Mutations In Trans With Alpha-Lely Leading To Mild Hemolytic Anemia; Presentation Of Four Cases.
Ryan C Shean^1,2, Archana Agarwal^1,2
¹University of Utah Department of Pathology, Salt Lake City, UT, United States, ²ARUP Laboratories, Salt Lake City, UT, United States

Introduction Mild hereditary hemolytic anemia represents a heterogeneous group of patients with diverse genotypes and phenotypes. Laboratory findings often vary significantly and may overlap with normal individuals. Hereditary spherocytosis (HS) is the most common form of inherited hemolytic anemia due to mutations impacting ankyrin, band 3, protein 4.2, and alpha (α) or beta (β) spectrin. HS is genetically and phenotypically heterogenous, with severity ranging from mild or asymptomatic to transfusion-dependent anemia. Most cases of HS are autosomal dominant. Autosomal recessive HS usually results from homozygous or compound heterozygous variants in α-spectrin gene (SPTA1), which markedly reduce the level of α-spectrin protein. In normal individuals, α-spectrin is overproduced by roughly fourfold. α^LELY, a common polymorphism in α-spectrin gene, reduces the amount of spectrin to approximately 50%. It was previously considered clinically insignificant when combined with HS-related spectrin mutations. However, Bianchi et al recently reported five patients with lifelong hemolysis and mild anemia with this combination. Herein, we present four cases of null alpha spectrin mutations with α^LELY leading to mild hemolytic anemia. Methods We retrospectively reviewed data from our hereditary hemolytic anemia cascade panel performed at University of Utah health/ARUP laboratories from 2021 to 2025 and identified four cases of mild hereditary hemolytic anemia with α^LELYand a null alpha spectrin mutation. All cases had peripheral smear evaluation, osmotic fragility, EMA band 3 testing, and targeted NGS performed. Results Median age of patients was 40 years old (range 35-80+), all were female. All presented with at least mild anemia (Median hgb 11.6 g/dL; range 8.1-13.1). EMA testing was normal in all cases. Osmotic fragility was slightly to moderately increased in all cases. Peripheral smear findings in all cases showed at least mildly increased spherocytes, with certain cases showing other abnormal erythrocyte forms. Null SPTA1 and α^LELY mutations were seen in all cases (Table 1). Conclusions Our case series reinforces the hypothesis that null SPTA1 alleles in compound heterozygosity with low-expression polymorphisms may result in spectrin deficiency not detectable by EMA binding test but causes lifelong mild hemolytic anemia. It is possible that the frequency of these cases may be underrecognized due to the diagnostic overlap with normal individuals. These findings underscore the need for molecular analysis in the workup of all potential cases of hereditary hemolytic anemia.

P215

Validation Of Assay For Glucose-6-Phosphate Dehydrogenase Concentration Using An Automated Haemostasis Analyser
Karen Thompson^1,2, Jude Platton^1,2, George Tarpley^1,2, Sophia Katiri^1,2, Andrea Alkins³, Juswal Dadhra^1,2
¹Red Cell Laboratory, Royal London Hospital, London, United Kingdom, ²National Health Service East and South East London Pathology Partnership, London, United Kingdom, ³Sysmex UK, Milton Keynes, United Kingdom

Introduction Glucose-6-phosphate dehydrogenase (G6PD) deficiency is a red-cell enzyme deficiency that shows sex-linked inheritance; it is associated with haemolysis and neonatal jaundice. G6PD catalyses the conversion of glucose-6-phosphate to 6-phosphogluconate, where nicotinamide-adenine-dinucleotide-phosphate (NADP) is reduced to NADPH. The rate of formation of NADPH measured at 340 nm is proportional to G6PD. We describe validation of a G6PD assay on Sysmex CN-6500 haemostasis analysers (CN-6500). Methods Pointe G6PD Reagent Set (Pointe) and quality control (QC) materials (Horiba Scientific) and residual patient samples were assayed on the CN-6500. Reagents R1 and R2 are combined 1:2 to create working reagent, and placed on the CN-6500. 25 µL of patient sample or QC is pre-lysed at room temperature for five minutes using 975 µL of Lyse Reagent. The CN-6500 adds 200 µL working reagent to 26 µL haemolysate; dOD is measured for five minutes at 340nm at 37°C. For comparison, quantitative assays were performed on a Lambda 365 spectrophometer, using the manual method as described by the manufacturer. Results The limit of the blank on the CN-6500 was 0.0001 dOD (coefficient of variation (CV) 29%), with limit of detection and quantitation 0.0002 and 0.0004 dOD respectively. The assays is linear between 0.3 and 10.9 IU gHb^-1 (r²=0.9841). CV was 9.5% (deficient QC) and 7.3% (normal QC). Measurement Uncertainty for low, intermediate, and normal QC was 0.17, 0.42, and 0.68 IU gHb^-1 respectively. Manual quantitative assays results agree with CN-6500 results (r²=0.8194): median difference between methods was 0.4 IU gHb^-1. There was no carryover into FXIII chromogenic assays at 340 nm, FVIII chromogenic assays at 405nm, or clotting assays. Conclusions Pointe can be used on a variety of automated platforms, for which Application Notes have been produced. These typically use very small volumes of haemolysate and working reagent, which was not possible on the CN-6500, where the minimum sample volume is 20 µL. By combining R1 and R2 reagents into a working reagent, and by decreasing the concentration of the initial haemolysate to compensate for a larger sample volume than in the manual assay, good comparison was observed between all assays. There was a positive bias at normal G6PD concentrations (>8 IU gHb^-1), which does not affect the clinical interpretation of the result. Overall, our analysis shows that using the Pointe G6PD Reagent Set on the Sysmex CN-6500 analyser is an acceptable method for quantitative measurement of G6PD.

P216

Reticulocyte Volume (Mrv) Reported By Mindray Bc8600 + In The Study Of Iron Deficient Erythropoiesis
Eloisa Urrechaga, Eugenia Axpe, Andrea Nieto, Julen Madrazo
Hospital Universitario Galdakao Usansolo, Galdakao, Spain

Background: The Mindray BC 8600+ counter reports MRV, we study its values in anemia of different etiologies and its reliability for the detection of iron deficient erythropoiesis. Methods: 416 samples were analyzed. The scope of the pathology included a variety of diseases representative of the daily workload: 90 healthy subjects, 280 anemia (102 microcytic, 121 normocytic, 57 macrocytic) . Kolmogorov-Smirnoff was used to verify normal distribution of data. Differences among groups were assessed ANOVA, considering P <0.05 to be significant, post hoc comparisons of groups with Scheffé method. Pearson correlation was applied to compare erythrocyte indices and MRV. Receiver operating characteristic analysis was used to assess the diagnostic performance of MRV for detecting iron deficient erythropoiesis, gold standard sTfR >2.2 mg/L. Results: Gaussian distribution was proven. Whole MRV range 51.8-151.3 fL Mean and range in healthy subjects 107.8 fL, 94.6-121.2 fL Microcytic anemia mean 78.5 fL standard deviation (SD) 13.5 fL Normocytic anemia Mean 105.1 fL, SD 16.9 fL Macrocytic anemia Mean 122.5 fL, SD 18.6 fL In the microcytic group, the values in patients with IDA (MRV mean 78.8 fL, SD 14.3 fL) and thalassemia carries (MRV mean 76.6 fL, SD 10.2 fL) 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/MRV R2=0.86, (95%CI 0.8469-0.9067). Using a cut off MRV 94.6 fL, iron deficient erythropoiesis could be diagnosed sensitivity 81.5 %, specificity 95.6 %. Area under the curve was 0.922 (95% CI 0.888–0.943). Conclusions: MRV provides a sensitive method for identifying iron deficient erythropoiesis, MRV may allow the complete scope of disorders of iron metabolism to be identified quickly and managed.

P217

Comparison Of Esr Measurement Results Obtained Using The Modified Westergren Method And An Alternative Method Integrated Into A Hematology Analyzer
Malkov Vladimir, Kolupaev Vsevolod
Scientific and Practical Center for Laboratory Research of the Moscow City Health Department, Moscow, , Russia

Introduction: In the context of high analytical workloads, modern laboratories require optimized workflows. Integrating erythrocyte sedimentation rate (ESR) measurement into a hematology analyzer alongside a complete blood count (CBC) presents a promising strategy for improving efficiency, standardization, and turnaround time. Mindray has developed an easy ESR, integrated into its hematology analyzer. The objective of this study was to evaluate the analytical agreement between the Ves-Matic Cube 200 (modified Westergren method) and the Mindray BC-6800 Plus (integrated method) for ESR measurement, ensuring result consistency during a technology transition.
Methods: A method comparison study was performed using 414 clinical samples with ESR values distributed across the entire analytical range. Agreement was assessed using Passing-Bablok regression and Bland-Altman plot analysis. Precision was evaluated based on 20 internal quality control (IQC) runs for each system.
Results: A high correlation was observed between the methods (r = 0.93), with a Passing-Bablok regression equation of y = 3.387 + 0.695x. The alternative method on the BC-6800 Plus demonstrated superior precision, with a coefficient of variation (CV) of 0.78–2.18%, compared to 5.26–5.76% for the modified Westergren method. Bland-Altman analysis revealed a consistent relative bias of 5.55%, with 94.5% of data points within the limits of agreement, indicating good clinical comparability. Systematic differences between the methods were statistically insignificant (p > 0.05).
Conclusions: The integrated ESR module of the Mindray BC-6800 Plus analyzer provides accurate results with excellent reproducibility, ensuring consistency when transitioning from a standalone Westergren-based system. This integration offers significant advantages for high-volume laboratories: it improves efficiency by enabling simultaneous CBC and ESR testing from a single tube, enhances standardization, reduces operational costs, and significantly shortens the time to result. The solution represents a fast, reliable, and workflow-optimized method for ESR determination.

P218

Superior Performance Of A Novel Thalassemia Screening Model Over Traditional Indices Using Automated Hematology Analyzers
Ping Yin, Jialin Tan, Honghai Hong
Department of Clinical Laboratory, Third Affiliated Hospital of Guangzhou Medical University, Guangzhou, China

Introduction: Differentiating thalassemia carriers from individuals with other anemias — particularly microcytic types like iron deficiency — is a critical yet challenging task in laboratory hematology. Conventional screening indices, such as MCV, MCH, and the Mentzer index, are limited by suboptimal sensitivity and specificity. This study aimed to develop and validate an advanced analytical model using automated hematology analyzer data to overcome these limitations and provide a more accurate tool for thalassemia screening. Methods: A retrospective cohort of 968 participants was categorized into three groups: 1) Thalassemia Group (n=442): Genetically confirmed, including α-thalassemia trait (n=160), α-thalassemia intermedia (n=7), β-thalassemia (n=103), and unspecified genotypes (n=172). 2) Non-thalassemia Anemia Group (n=378): Included iron deficiency anemia (n=13), anemia of chronic disease (n=324), and hemolytic anemia (n=41). 3) Healthy Controls (n=148). Complete blood count and extended research-use parameters from a DH-800CS automated hematology analyzer (Dymind Biotechnology) were used. An advanced classification algorithm was developed and validated using a 70%/30% stratified split and five-fold cross-validation. SHapley Additive exPlanations (SHAP) analysis was employed to interpret the predictive model. Results: Seven machine-learning classifiers — including Logistic Regression (LR), Decision Tree (DT), Random Forest (RF), Gradient Boosting (GB), eXtreme Gradient Boosting (XGBoost), Support Vector Machine (SVM), and Multi-Layer Perceptron (MLP) — were trained on a set of 12 selected variables. The best-performing model (Gradient Boosting) demonstrated high accuracy on an independent test set (AUC: 0.955, Sensitivity: 97.7%, Specificity: 88.6%). Its performance markedly exceeded that of the Mentzer index (Sensitivity: 42.7%, Specificity: 73.1%) and the conventional MCV/MCH rule (Sensitivity: 73.8%, Specificity: 37.6%). SHAP analysis identified the most influential predictive features as mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), microcyte percentage (Micro%), and red blood cell count (RBC). Conclusions: This high-performance screening model provides a substantial advancement for the clinical laboratory, enabling significantly more accurate identification of thalassemia carriers and their differentiation from other anemias directly from routine CBC data. Its implementation can streamline the analytical workflow, enhance the specificity of screening reports, and improve overall efficiency in the hematology laboratory.

P219

Hypo-He And Micror As Early Indicators Of Iron Deficiency In First-Trimester Pregnant Women: Beyond Ferritin And Transferrin Saturation
Razan Hayati Zulkeflee¹, Rosline Hassan¹, Zefarina Zulkafli¹, Shafini Mohamed Yusoff¹, Wan Nor Fazila Hafizan Wan Nik², Nur Hidayah Mohd Fauzi³, Anees Abdul Hamid⁴, Munira Mahmud⁴, Siti Nur Iman Sofiah Zulkifli⁵
¹Department of Hematology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian , Kelantan, Malaysia, ²Department of Chemical Pathology, School of Medical Sciences, Health Campus, Universiti Sains Malaysia, Kubang Kerian , Kelantan, Malaysia, ³Department of Obstetrics & Gynaecology, School of Medical Sciences, Universiti Sains Malaysia, Health Campus, Kubang Kerian, Kelantan, Malaysia, ⁴Primary Care Unit, Kelantan State Health Department, Tingkat 5, Wisma Persekutuan, Jalan Bayam, 15590 Kota Bharu, Kelantan, Malaysia, ⁵Faculty of Biosciences and Medical Engineering, Universiti Teknologi Malaysia, Skudai, Johor Bharu, Malaysia

Introduction: Early recognition of iron deficiency enables timely management before anaemia develops. Conventional biochemical markers such as serum ferritin, serum iron, and transferrin saturation (TSAT) remain standard tools but are limited by low sensitivity and longer turnaround times. Extended RBC parameters, including MicroR, HypoHe, Delta He, and Ret-He, may provide rapid and potentially superior diagnostic insights. This study assessed the diagnostic performance of these parameters compared with conventional biochemical markers across progressive stages of iron deficiency. Methods: A prospective case–control study was conducted at selected primary health clinics on the East Coast of Malaysia between February and July 2025. A total of 178 participants were classified into four groups: Normal (n=71), Pre-Latent Iron Deficiency (Pre-LID, n=47), Latent Iron Deficiency (LID, n=45), and Iron Deficiency Anaemia (IDA, n=15). Haematological indices (Hb, MCV, Ret, MicroR, HypoHe, Delta-He, Ret-He, RPI, IRF) were measured using the Sysmex XN-1500, while biochemical markers (serum iron, ferritin, and TSAT) were analysed using standard laboratory methods. Correlations between haematological and biochemical parameters were assessed using Pearson’s correlation. Diagnostic performance was evaluated using ROC analysis, and optimal cut-off values were identified using Youden’s Index.Results: HypoHe and MicroR showed the best diagnostic accuracy. In detecting LID, HypoHe ≤ 30% and MicroR ≥ 3.4% yielded AUCs of 0.774 and 0.775, respectively. Both improved further in IDA. Delta He showed moderate accuracy, while Hb, MCV, and Ret-He performed poorly in detecting Pre-LID and LID. Conclusions: HypoHe and MicroR, demonstrated superior diagnostic accuracy to traditional iron studies, particularly for detecting latent and pre-latent iron deficiency where haemoglobin levels remain normal.

P220

Benchmarking Large Language Models Against Interpretive External Quality Assessment In Haematological Oncology
Ashley Cartwright, Samuel Nti, Stuart Scott
UK NEQAS for Leucocyte Immunophenotyping, Sheffield Teaching Hospitals NHS Foundation Trust, Sheffield, United Kingdom

Introduction: Haematological malignancies account for 6.6% of all cancer diagnoses and 7.2% of cancer-related deaths. Diagnosis requires a multidisciplinary approach involving evaluation of results from multiple pathology disciplines and provision of a conclusion based on World Health Organisation (WHO) Classification of Tumours of Haematopoietic and Lymphoid Tissues framework(s). However, misdiagnosis rates range from 17-33%, highlighting challenges in review of clinical information for accurate diagnosis. Use of large language models (LLMs) in healthcare is rapidly evolving. A recent study showed that LLMs can outperform clinicians in clinical reasoning and differential diagnosis identification, outlining the potential for LLMs to be utilised in an augmented capacity to improve misdiagnosis. This study evaluated the ability of LLMs to provide accurate diagnostic conclusions from interpretive external quality assessment (iEQA) cases. Methods: Thirty iEQA cases from the UK NEQAS for Leucocyte Immunophenotyping Leukaemia Diagnostic Interpretation programme (June 2021-April 2025) were included in benchmarking. Cases were assigned complexity based on agreement amongst participating laboratories (>90%, low; 60-90% medium; <60% high). Fifteen LLMs from five developers (OpenAI, Anthropic, Google, Perplexity and xAI) were tested, in a zero-shot setting. Models were provided with standardised instruction prompts, requiring review of multidisciplinary results, provision of a WHO Revised 4th Edition (WHO-HAEM4R) diagnostic conclusion and the International Classification of Diseases for Oncology (ICD-O) code. LLMs were evaluated on four criteria: correct disease entity (broad disease category); WHO-HAEM4R diagnostic conclusion (semantically correct, fully specified disease); ICD-O coding and internal consistency (WHO-HAEM4R and ICD-O concordance). Accuracy was assessed by criterion and case complexity, with overall accuracy also determined. Results: Across all cases, LLM evaluations and criteria, accuracy was 77.0%. When considering individual criteria, accuracy was 88.7% for disease entity recognition (range 83.3-96.7%); 80.2% for WHO-HAEM4R conclusion (74.2-92.5%); 65.3% for ICD-O coding (46.7-86.7%) and 73.8% for internal consistency (50.0-96.7%). When evaluating accuracy by case complexity, accuracy decreased with increasing complexity; however, the highest performing models were consistent across complexity tiers. For low complexity cases (n=21), accuracy was 84.0% (range 71.1-94.3%); medium complexity case (n=5) accuracy was 66.4% (38.8-88.8%). High complexity case (n=4) accuracy was 53.6% (31.3-81.3%). Conclusions: LLMs achieved an overall diagnostic accuracy of 77.0% when evaluating iEQA cases, highlighting the innate abilities of non-specialist, general access LLMs in diagnostic haematological oncology; however, accuracy decreased with increasingly complex cases. The design of iEQA schemes needs to evolve to address the potential diagnostic use of LLMs, safeguarding the clinical benefit, utility and integrity of EQA.

P221

An Italian Project Aimed At Harmonizing Hematology Reporting: Navigating Through Innovation, Demands And Requirements Of A Changing World
Anna Maria Cenci, Marco Moretti, Sofia Chiatamone Ranieri, Barbara Casolari, Fabrizio Papa, Elisa Piva, Maria Lorubbio
On behalf of Italian Society of Pathology and Laboratory Medicine Hematology Working Group (SIPMeL GdS-E), Castelfranco Veneto (TV), Italy

Introduction. SIPMeL-GdS-E promoted an update of the widespread 2002 SIMeL Guidelines for the Peripheral CBC Report, providing recommendations (RECs) based on new scientific findings, technological developments, current healthcare scenario, perceived needs and requests of innovation in a changed laboratory world, standardization of language and behavior. Methods. In December 2024, GdS-E-Coordination-Group (80 members) introduced the promoted methodology, with a “core-work” assigning RECs for CBC and differential parameters through intermediate sharing steps by all GdS-E consensus, with an assessment of the evidence strength and quality from available literature (GRADE approach). As specific high impact documents on these topics are often unavailable, (i.e. for lack of meta-analyses), several works were identified as basic sources considered reliable and characterized by a consolidated practical use among national and international consensus documents. As for taxonomic language, the main current classifications from literature were followed. Results. After the description of the “rationale” and methodological aspects, RECs concern numerical data, measure units, data frames (reference ranges, decision limits, critical values), detection of morphological counts or anomalies and morphological revision, interpretive reports, management of non-standardized/technology-dependent parameters. 2002 Guidelines RECs were "confirmed" or "modified" based on new evidence. In case of divergent levels of evidence, "challenges" were carried out for definitive “GdS-E consensus”. The main challenges concerned instrumental indices use/reporting; SI units diffusion; reference ranges for age, gender, ethnicity, specific conditions (pregnancy). Interpretive reports were carefully studied as for language, content, presentation, timing, patient types, diagnostic suspect supplying, follow-up suggestions, In October 2025, representative RECs were brought forward at the SIPMeL National Congress; final release of the revised document is expected in the first half of 2026, following approval by GdS-E survey. To disseminate and debate the work all over the country, three courses are scheduled, starting in February 2026. Conclusions. Key points to be highlighted: The chosen approach has fostered a meaningful "consensus process," hopefully useful in the dissemination, feedback and evaluation phases, the main goals of GdS-E and SIPMeL. RECs represent a useful tool for the standardization in daily laboratory practice, enhancing the efficiency of clinical/analytical reporting and providing the user with clear, reliable and consistent data. An innovative perspective consists in RECs developed by laboratorians, aware of the technical value of the produced data, their use in clinical practice, their check by a solid evidence system, in collaborative connection with healthcare professionals. From here, increasingly clear and precise words and numbers can result in the report.

P222

Verification Of Results Comparison Between Several Hematology Analyzers, Through The Ep31-A-Ir Clsi Guideline
Xavier Tejedor Ganduxé¹, Fernando Marques Garcia², Cristina Martínez Bravo¹, Jennifer Rodríguez Domínguez¹, Alba Leis Sestayo¹, Isabel Aparicio Calvente¹, Alicia Martinez Iribarren¹
¹Laboratory Medicine Department. Germans Trias i Pujol Hospital, Badalona, , Spain, ²Laboratory Medicine Department. Donostia Universitity Hospital, Donostia, , Spain

Introduction: Results from laboratories using multiple instruments may present at multiple locations within a health care system. For this reason, it is necessary that the comparability of test results produced by different measurement systems must be verified periodically. Objectives: To evaluate the applicability of a comparability methodology based on the EP31-A-IR guideline published by the Clinical and Laboratory Standards Institute (CLSI) to ensure regular comparability of patient results on various hematology analyzers. Methods: Comparability of nine hematology analyzers (Beckman CoulterDXH-900) was evaluated for seventeen hematological parametersincluding complete blood count variables, leukocyte differentials, monocyte distribution width (MDW), and reticulocytes. Following the EP31-A-IR framework, approximate target concentrations were estimated from internal quality control data to guide the selection of native patient samples. Acceptance criteria were established specifically for each concentration level and quality specification was selected, considering the analytical performance of the available technology. The maximum allowable difference (MAD) was then calculated for the comparability test and also the number of runs and replicates that would be executed. Finally, after performing the comparison, maximum differences between analysers were calculated. Results were considered comparable when the maximum difference was less than or equal to the MAD. Results: For all seventeen parameters evaluated, including reticulocytes assessed on a subset of analyzers reflecting routine workflow, the maximum observed differences between instruments were within the predefined acceptance limits at all concentration levels. Conclusions: In our setting, the applicability of the EP31-A-IR protocol was successfully evaluated across nine hematology analyzers, under conditions close to the maximum of ten instruments defined by the guideline. This experience demonstrates that the EP31-A-IR framework can be effectively implemented to provide objective and reliable verification of patient result comparability within a health care system, thereby supporting consistent interpretation of patient results over time and accreditation requirements.

P223

A Retrospective Evaluation Of A Web-Based Myelodysplastic Syndrome Predictive Calculator In A Tertiary Hospital In Singapore
Frederick M. Ilagan, Kieren Lenin Cheng, Vishnu Prasad
Khoo Teck Puat Hospital, Singapore, Singapore

Introduction Myelodysplastic syndrome is a heterogenous group of haematopoietic disorders frequently encountered in tertiary haematology practice. Definitive diagnosis requires BMA, an invasive procedure that may be unnecessary in a proportion of patients with low clinical suspicion. Laboratory-based predictive algorithms have been developed to support early risk stratifications using routine haematology and chemistry parameters. This study evaluates the performance of a web-based MDS predictive calculator in assisting the exclusion or identification of MDS cases, using bone marrow findings as the diagnostic reference standard in Khoo Teck Puat Hospital in Singapore. Methods A retrospective review of bone marrow cases performed between January 2024 and December 2025 was conducted. A total of 88 unique patient cases were included. Demographic data (age and sex) and laboratory parameters were collected, including haemoglobin concentration, WBC, MCV, absolute neutrophil count, monocyte count, serum creatinine, and glucose levels. Final bone marrow diagnoses were categorized as MDS or non-MDS. Each patient’s laboratory data was entered into the University of York web-based MDS predictive modelling calculator, generating outcomes categorized as probable MDS, indeterminate, or probably not MDS. Results were compared with final marrow diagnoses. Results Of the 88 cases analysed, 20 (22.7%) were confirmed as MDS and 68 (77.3%) as non-MDS on bone marrow examination. Among confirmed MDS cases, the predictive algorithm classified 9 cases as probable MDS, corresponding to sensitivity of 45.0%. Six MDS cases (30.0%) were classified as probably not MDS, representing false-negative results, while 5 cases (25.0%) were categorised as indeterminate. When indeterminate outcomes were considered non-concordant, the overall false-negative rate was 55.0% (11/20). In the non-MDS cohort, 49 cases were correctly classified as probably not MDS, yielding a specificity of 72.1 %. Five non-MDS cases (7.4%) were classified as probable MDS and represented false-positive results, while 14 cases (20.6%) were categorized as indeterminate. Overall, concordance was higher for exclusion of MDS than confirmation. Conclusions The laboratory-based MDS predictive algorithm demonstrated utility as a supportive screening tool, particularly in ruling out MDS in patients with low-risk laboratory profiles. While sensitivity for confirmed MDS was moderate, the tool showed good discriminatory performance in non-MDS cases, potentially aiding clinical decision-making and prioritization for bone marrow examination. Indeterminate outcomes highlight the limitations of laboratory parameters alone and reinforce the necessity of bone marrow evaluation in equivocal cases. Integration of such predictive tools into laboratory practice may support risk stratification and resource optimisation but should complement, not replace, comprehensive haematological assessment.

P224

Impact Of Moderated Calibration Factor Application On Quality Control Behavior In A High-Throughput Hematology Analyzer
Hyun Lee^1,2, Yoonhwan Chang¹
¹Seoul National University Hospital, Seoul, , South Korea, ²Dankook University College of Health Sciences, Chungnam, , South Korea

Introduction: Calibration is essential for maintaining analytical accuracy in automated hematology analyzers. However, abrupt application of newly derived calibration factors may induce transient analytical shifts and destabilize quality control (QC) performance. In routine laboratory practice, moderated or partial application of calibration factor adjustments is sometimes adopted to buffer sudden analytical changes, although its impact on QC behavior has not been systematically evaluated. This study aimed to assess the effect of calibration factor scaling on relative analytical changes and analyte-specific QC behavior in automated hematology testing. Methods: This retrospective study analyzed calibration factor and QC data generated from a high-throughput hematology analyzer (Sysmex XN-9100) between January 2022 and July 2025, during which calibration was performed semiannually. Calibration-induced changes were evaluated under the routine scaled application and a counterfactually modeled full-application condition. Factor-based absolute shifts were quantified, and QC instability was assessed using Westgard multirule criteria (1-3s, 2-2s, and R-4s) with standardization based on post-calibration target means and standard deviations. Results: A total of 288 analyte-specific calibration events were included. Routine scaled application reduced factor-based absolute calibration-induced shift compared with the modeled full-application condition (0.69% ± 0.99% vs 1.41% ± 1.34%). In the baseline-referenced run-level analysis, multirule violation rates were 20.61% under routine scaled application and 29.31% under the modeled full-application condition. In the primary paired comparison, routine scaled application showed a lower run-level multirule violation rate, corresponding to an absolute reduction of 8.70 percentage points and a relative reduction of 29.68% (P <0.001, McNemar’s test). Conclusions: Moderated application of calibration factors functioned as an effective buffering strategy that mitigated abrupt analytical shifts while preserving QC stability in this high-throughput hematology setting.

P225

Improving Peripheral Blood Slide Review With Cellavision Digital Hematology Microscopy
Zahra Madani, Gabe Fellows, Lauren Namovic, Jan DiGiacinto, Tina Ishii, Jiehao Zhou
Mayo Clinic Arizona, Phoenix, AZ, United States

Introduction: Peripheral blood (PB) slide review plays a critical role in hematologic diagnostics, allowing the identification of abnormal cells that guide clinical decisions, particularly in the diagnosis of hematologic malignancies. The accuracy and timeliness of these results are crucial, as they directly influence patient management and treatment strategies. Our laboratory recently introduced the CellaVision (CV) digital hematology system, an automated digital workflow designed to support PB morphological evaluation. Digital microscopy consistently provides standard high-resolution images that reduce variability compared to traditional manual light microscopy, leading to improved diagnostic accuracy, reproducibility, and enhanced standardization across reviewers. Methods: Our study specifically aimed to evaluate the correlation of manual light microscopic review with the CV digital slide review across a variety of hematologic cases, including normal and abnormal peripheral blood samples. Workflow analysis, validation data, and staff survey (20 technologists and 5 pathologists) were utilized to evaluate the impact of digital slide review. The turnaround time (TAT) data from the first quarter of 2024 were reviewed and compared to the fourth quarter of 2024, before and after implementing CV. Data from slides sent for pathologist review were retroactively reviewed to assess improved standardization. Results: Statistical analysis showed that CellaVision reduced the median slide review turnaround time by 29% by eliminating redundant manual steps and enhancing workflow efficiency. The survey results indicated that technologists experienced improved standardization in routine slide reviews and faster TATs. Pathologists reported greater efficiency in PB case reviews and a reduced need for travel to the hospital during off-hours. Validation data demonstrated an agreement rate of 90-100% between manual light microscopy and CV identifying various types of white blood cells (WBC), including mature granulocytes, immature granulocytes, plasma cells, and lymphocytes. In the majority of cases for pathologist review, the WBC differential and morphology interpretation generated from CV were approved by the hematopathology pathologists. Conclusions: The integration of the CV digital hematology system into our laboratory’s diagnostic workflow has proven to be an effective tool in enhancing both operational efficiency and diagnostic reliability. This study supports the adoption of digital hematology systems as a solution for modernizing hematopathology practices and improving patient care outcomes.

P226

Evaluation Of Criteria Used To Define Allowable Intervals In External Quality Assessment Programs For Hematology Measurands
Yutaka Nagai^1,2, Tomohiro Takeda², Sayaka Mori³, Kazuto Tsuruda³, Hiromitsu Matsushita¹
¹Keio University School of Medicine, Tokyo, Japan, ²Kansai University of Health Sciences, Osaka, Japan, ³Nagasaki University Hospital, Nagasaki, Japan

Introduction:
The Japanese Society for Laboratory Hematology (JSLH) has promoted the standardization of automated blood cell analysis. As part of this initiative, external quality surveys using fresh blood and manufacturers’ standard analyzers compare with reference values have been conducted regularly (JSLH survey). In line with international practice, allowable ranges have been defined using bias criteria from the European Federation of Clinical Chemistry and Laboratory Medicine Biological Variation Database (EFLM-BV), with medians set by the highest calibration hierarchy, i.e., the international harmonization protocol (IHP) or IHP-compliant measurement procedure (IHP-compliant MP). With the dissemination of ISO 17511:2020, manufacturers are now required to present maximum allowable expanded measurement uncertainty (MAU) and its rationale. Consequently, MAU has been incorporated into the analytical performance specifications (APS) derived from EFLM‑BV, in addition to Bias and total allowable error (TEa). We examined the setting of allowable intervals (AI) based on these APS.

Methods:
Data were obtained from the 2024 JSLH survey in this study. The selected measurands were red blood cell count (RBC), hemoglobin concentration (Hgb), hematocrit (Hct), mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), and white blood cell differential (WBC-Diff: %lymphocytes, %monocytes, %neutrophils, %eosinophils and %basophils). In IHPs, Hct was defined as packed cell volume (PCV), hemoglobin concentration was measured by the cyanmethemoglobin method, IHP‑compliant MP are used for WBC-Diff, and RBC was based on the mean value obtained from the standard analyzers verified calibration in six manufacturers' reference laboratory. The APS was calculated using biological within- and between-subject variation (CV_I/CV_G) to derive MAU, Bias, and TEa. Formulas applied were: MAU <2×0.5×CV_I; Bias＜0.25 × (CV_I² + CV_G²)^1/2, TEa<1.65×(0.5×CV_I) + 0.25×(CV_I² + CV_G²)^1/2).

Results:
For RBC‑related measurands with small CV_G, high agreement was observed for Hgb and Hct, whereas substantial differences were noted for MCV, MCH, and MCHC due to differences in measurement principles. In contrast, for measurands with large CV_G, applying TEa or bias resulted in excessively wide allowable intervals (AI), indicating that MAU‑based AI was more appropriate. The largest discrepancy between bias and MAU was observed for neutrophil percentage, with bias/MAU ratios ranging 61.8–64.1 (52.9–73.0/60.7–65.4), 48.5–51.5 (42.0–58.0/48.2–51.9), and 35.0–39.5 (29.3–40.4/33.5–36.2).

Conclusions:
Regarding the setting of AIs based on APS, Bias and TEa are suitable for integrated evaluation of multiple specimens by single measurement, whereas MAU is more appropriate for assessing analytical performance in detecting intra-individual variation.

P227

Leveraging Process Mining To Compare Platelet Usage Practices And Wastage Within A Health Care Network
Andreea/M Palage¹, Calvino Cheng², Neal Callaghan³, Jason Quinn², Natalie Chisholm², Robert Liwski²
¹Faculty of Medicine, Dalhousie University, Halifax, NS, Canada, ²Department of Pathology and Laboratory Medicine, Division of Hematopathology, Halifax, NS, Canada, ³Department of Medicine, Division of Internal Medicine, Halifax, NS, Canada

Introduction Platelet products are critical resources in transfusion medicine that must be used as efficiently as possible. Previous work has utilized the process mining tool Disco to uncover actionable processes for the optimization of the disposition of red blood cell products, but not for platelets. Given their escalating cost, short shelf life, and clinical consequences associated with suboptimal utilization, this study aimed to leverage process mining to compare platelet usage and wastage within a Canadian healthcare system. Methods Process mining encompasses a suite of analytical methods used to visualize and evaluate processes through their operational pathways. In this study, the process-mining platform Disco was employed to characterize the movement of platelet products across the healthcare network. Laboratory data from 2022 to 2025 was extracted and analyzed using Disco and Microsoft Excel. Within Disco, predominant product pathways were identified, and transfer times were quantified. Excel pivot tables were used to determine transfusion rates, discard rates, and the residual shelf life of platelet units at the time of receipt. Analyses were stratified by platelet type, with non–psoralen-treated units from 2022–2023 and psoralen-treated units from 2024–2025 evaluated independently and compared. Results Products in 2022-2023 had rates of transfusion between 93.3% and 96.6% while products in 2024-2025 had rates between 96-97%. Rates of disposal for psoralen-treated platelets in 2024-2025 were half of those for non-psoralen products (3.4-3.8% compared to 5.4-6.9%). Rates of disposal were higher for HLA matched platelet products in both time frames. For ABO grouping, rates of disposal were highest for AB negative and B negative products in both time frames. Median duration between hospital transfers was 8.3 hours in 2022-2023 and 8.5 hours in 2024-2025 while the maximum time between transfers was 3.4 and 4.6 days respectively. Average lifetime days remaining after institutional product receipt was lower for non-psoralen treated platelets (1.7 - 3.7 days) compared to non-psoralen treated platelets (3.5-5 days). Conclusions Platelet products in 2024-2025 were handled by the same unchanged methods in the healthcare network but had overall lower rates of disposal. This can be attributed to the increased lifespan of the psoralen-treated platelets on arrival. Additionally, this study is the first to examine and compare transfer times, a crucial step given the products’ short shelf-life. We demonstrate the utility of process mining as a tool to interrogate data sets over years and confirm effective internal standard operating procedures for product management.

P228

Applying Indirect Approaches To Define Hematology And Cell Population Data Reference Intervals In A Spanish Population
Alvaro Piedra-Aguilera, Alba Leis-Sestayo, Jennifer Rodriguez-Dominguez, Isabel Aparicio-Calvente, Cristian Morales-Indiano, Alicia Martinez-Iribarren
Laboratory Medicine Department. Germans Trias i Pujol University Hospital, Badalona, Spain

INTRODUCTION: Accurate estimation of reference intervals (RI) is essential for clinical decision-making. However, hematological RI currently used in most laboratories are derived from published literature and may not be representative of the corresponding population. Therefore, current guidelines recommend that laboratories establish their own RI. To this end, indirect methods based on routinely generated laboratory data represent a suitable alternative to classical approaches. Our aim was to establish more accurate RI for Cell Blood Count and Differential (CBC-DIFF) parameters in our population, using indirect methods. Additionally, we established RI for other white blood cell parameters, known as Cell Population Data (CPD), which are currently considered research-use only but may be highly useful for disease screening. METHODS: CBC-DIFF results, as well as CPD, were collected from over 300,000 and 150,000 adult primary care patients, respectively, over 2024. Samples were analysed in Beckman Coulter DxH900. Repeated measurements from the same patient were excluded. RI were estimated using refineR algorithm, an indirect method that applies Box-Cox transformation, separates non-pathological from pathological distributions and identifies the model that best fits the non-pathological data. Harris and Boyd method was used to determine whether partitioning the population by sex and/or age was necessary. The newly generated and classical bibliographic RI were compared, and all of them were verified following CLSI EP28-A guidelines. RESULTS: We identified significant age-related differences for red blood cell count (RBC), hemoglobin and hematocrit, which were not previously considered. Additionally, sex-related differences were observed in monocytes and platelets, which are not accounted for in classical RI. The newly established intervals for CBC-DIFF and CPD were successfully validated according to CLSI guidelines. In contrast, the classical interval for RBC in women aged 18–79 and men aged ≥80 did not pass validation. For SD-V-MO, from which Monocyte Distribution Width (MDW) is derived, the reference interval was 16.46–22.03, in agreement with the published cutoff value of 21.5 proposed for ruling out sepsis (see Fig.1). The 95% confidence intervals for the upper and lower limits of most RI did not exceed 25% of the total interval, indicating a reliable estimation. CONCLUSIONS: Our results for CBC-DIFF provide more accurate RI for our population, with previously unrecognized sex and age-related differences identified. RI derived from CPD offer new information on parameters that have previously shown utility in the diagnosis of hematological and non-hematological diseases. These new RI may encourage laboratories and manufacturers to implement their use in routine laboratory practice.

P229

The Artificial Intelligence Challenge: Assessing Diagnostic Quality Of Large Language Models
Christian Pohlkamp, Christopher Maier, Benjamin Braun, Sven Maschek, Torsten Haferlach, Vivian Würf
MLL Munich Leukemia Laboratory, Munich, Germany

Introduction: Evaluating Artificial Intelligence (AI)-generated diagnostic reports across thousands of cases is challenging. Human review is relevant to ensure clinical validity of Large Language Models (LLMs) in diagnostics, but enormously time-consuming at large scale. We implemented a separate judge LLM as an additional evaluation framework. It assesses the performance of diagnostic LLMs generating reports for cytomorphology in bone marrow and peripheral blood. Methods: We established Claude Sonnet 4 as a separate judge LLM to evaluate LLM-generated diagnostic reports (using Llama 3.1) against corresponding human expert reports from our repository. The judgment process incorporated 24 specific criteria categorized into critical errors (factual inaccuracy, hallucinations), major errors (clinically significant findings omissions), and minor errors (less critical morphological detail omissions). The evaluation considered patient laboratory values, short diagnoses and tabular cytomorphological raw data as well as the AI-generated or human diagnostic reports based on these parameters. The judge LLM provided step-by-step reasoning with error counts by category, detailed explanations with criterion references and traceable paths to source data. It calculated scores starting with a baseline of 10, subtracting 5/3/1 point(s) for each critical/major/minor error to a bottom line of zero. This process was systematically applied to a balanced test dataset of 250 cases containing the 20 most common hematological diagnoses. For each case, reports of two diagnostic LLM versions were assessed in parallel and compared with the corresponding human experts’ reports as a benchmark. The judge model's assessments were validated by human reviewers across 50 out of the 250 test cases (corresponding to 2x50 AI-generated and 1x50 human reports). Results: Transparent reasoning allowed efficient human validation of judge decisions. Agreement between the judge's error counts and categorizations and human expert review reached 67.4%. In 32.5%, the judge missed or wrongly declared errors, and human review led to modified scorings. Interestingly, the judge identified higher alignment between underlying diagnostic raw data and final report narratives in AI–generated reports compared with human-authored reports. This is represented by the two AI-generated report versions achieving an average judge score of 8.45 and 8.65 versus 7.11 for the human-authored reports on the test set. Conclusions: Diagnostic LLMs transition from research prototypes to clinical decision-support tools. The LLM-as-a-judge framework establishes automated and transparent, criterion-based evaluation paradigms for diagnostic AI in laboratory hematology. It enables systematic quality monitoring, scalable beyond human capabilities, and may serve as a prototype for future regulatory evaluation frameworks and post-market surveillance of diagnostic LLMs.

P230

Implementation And Evaluation Of A Peripheral Blood Morphology External Quality Assessment Program In South Africa
Elise Schapkaitz^{1, 2}, Sarvashni Moodliar¹, Bongiwe Mofokeng¹, Mandisa Zikhali¹, Thendo Ramaliba ¹
¹Department of Quality Assurance, National Health Laboratory Service, Johannesburg, South Africa, ²Department of Hematology, University of Witwatersrand Medical School, Johannesburg, South Africa

Introduction The National Health Laboratory Service (NHLS) haematology external quality assessment (EQA) scheme functions to evaluate the quality of diagnostic sample assessment by clinical laboratories in South Africa. Recently, a peripheral blood morphology EQA scheme was introduced using EQAlite (KPMD IT Solutions Ltd, Sheffield, United Kingdom), a web-based EQA management system. This scheme was implemented in accordance with guidelines established by the International Committee for Standardization in Haematology (ICSH). Methods During the period of 2023 to 2025, replicates of 25 glass peripheral blood slides were distributed monthly to 147 EQA participants for cell morphology evaluation, interpretation, and manual differential (DIFF) counting. Each case was accompanied by a clinical history and full blood count (FBC) results. Participants were required to use a provided coding list to select up to eight FBC descriptors, eight DIFF descriptors, and five significant morphological features involving red blood cells, white blood cells, and platelets, as well as up to four differential diagnoses and a single most likely diagnostic interpretation. For each survey, individual laboratory results were compared with morphological reference results established by an expert committee. Descriptive statistics were used to summarise overall performance and stratified by laboratory classification. one-way Anova test was performed to evaluate performance before and after training interventions. The study was approved by the Human Research Ethics Committee of the University of the Witwatersrand (M250563). Results Participants comprised 10 (6.8%) academic, 14 (9.5 %) tertiary, 39 (26.5%) regional, and 84 (57.1 %) district laboratories; 110 (74.8 %) were ISO 15189 accredited. During the reporting period, the mean ±standard deviation (SD) overall performance was 79.9 ±7.9%. In 2023, the mean performance was 76.0 ±7.3%. In response to performance below 80 %, 10 targeted morphology “look-back” educational training sessions were implemented. Post-intervention mean performance increased to 79.5 ±7.3% in 2024 and 84.3 ± 6.9% in 2025 (p<0.001). Conclusion Training in morphological assessment and familiarity with EQA scheme requirements are essential for the successful implementation of a peripheral blood morphology EQA program across a laboratory network with varying levels of technical expertise.

P231

Impact Of Heat Block Drying On Morphology Of Peripheral Blood Smears
Ryan C Shean^1,2, Nicolas Mavromatis^1,2, Gary Isom², Angie Turnbow², Lauren Pearson^1,2
¹University of Utah Department of Pathology, Salt Lake City, UT, United States, ²ARUP Laboratories, Salt Lake City, UT, United States

Introduction Morphologic evaluation of peripheral blood is essential in diagnosing many hematologic disorders. Smear findings can rapidly suggest life-threatening conditions like acute promyelocytic leukemia and thrombotic microangiopathies and also guide diagnosis and treatment of less acute diseases. ICSH guidelines stress standardized nomenclature and grading to improve reproducibility and communication. Equally important is consistent smear preparation to minimize artifacts and ensure accurate morphology. Known sources of artifact include thermal injury, with in-vitro studies showing significant changes in blood parameters and morphology with excessive heating. While CAP and CLSI emphasize preanalytical quality control, they lack specific guidance on smear drying temperatures, allowing practice variation. Our literature review found no systematic studies on heat block use during smear preparation. This study aims to assess how varying heat block temperatures effects smear quality for manual pathologist review. Methods Residual clinical samples representing various hematologic conditions were selected and prepared in triplicate using three drying conditions: ambient air and heat blocks set to 37 °C and 45 °C. Smears were randomized and blinded for evaluation. Two trained pathology residents assessed WBC differentials, RBC morphology (graded 0–4+), and overall smear quality (scored 0–5). Subjective comments and discrepancies were reconciled post-unblinding. Ethics approval was obtained under a quality improvement protocol. Results Thirteen unique patient samples were analyzed. No statistically significant differences were found in overall subjective smear quality score or normal WBC differential counts across drying conditions. However, RBC morphology showed notable differences: in 5 cases spherocytes were identified only at 45 °C and in another 5 cases, schistocytes were identified only at 45 °C. Two cases showed blast cells exclusively in the 45 °C condition despite clinical negativity. Slides dried at 45 °C exhibited subjectively distorted leukocyte morphology, including irregular borders and increased vacuolation. Conclusions Heat block drying at 37 °C yields smear morphology comparable to ambient air drying. In contrast, drying at 45 °C introduces clinically significant artifacts, including RBC fragmentation and distorted WBC morphology, which may lead to misdiagnosis. These findings support limiting drying temperatures to ≤37 °C to preserve smear integrity. Standardization of smear preparation methods should be incorporated into ICSH, CAP, and CLSI guidelines to ensure diagnostic accuracy and reproducibility.

P232

Enhancing Clinical Excellence: A Comparative Analysis Of National And International External Quality Assessment Programs.
Marco Stillavato¹, Anna Maria Massobrio¹, Emiliano Carmelo Sartania², Marco Zompa², Huijing Hu², Vincenzo David Russo³, Tiziana Francisci²
¹Clinical Biochemistry-Città della salute e della Scienza PO OIRM, Torino, , Italy, ²Blood Bank -Città della salute e della Scienza, Torino, , Italy, ³Analytical Development-SITLab, Torino, , Italy

INTRODUCTION: External Quality Assessment (EQA) is a system that controls laboratories’ performance and it’s necessary to guarantee the accuracy and reliability of measurements. The diagnostic testing centers analyze samples and release results to the institution that evaluate individual performances and the overall trends of the group. This system is designed to impartially check the accuracy of the results. In consideration of the evolution of the quality analytical parameters, the focus is shifted towards the quality controls performances of clinical laboratories and enhancement programs of the Total Testing Process (TTP). This study is intended to analyze and compare national and international EQS programs, to evaluate advantages and criticalities of each approach and the opportunity of following a specific one.
METHODS: The study is centered on a comparative discussion between two different EQSs in two Italian clinical laboratories: a national centric from the Centro di Ricerca Biomedica di Padova (CRB), and a second of international relevance from UK NEQAS with several locations in Great Britain. The two programs are the most representative and used. The data have been collected by analyzing official documents, evaluation reports and semi-structural interviews from involved experts and operators. In addition, quality controls of the evaluation results are performed to highlight differences of efficacy and reliability.
RESULTS: The CRB had EQDs with favorable elements such as report’s readability with performances from great to non-acceptable, and well-structured histograms. Also, fast and simplify communication with the Italian team and the fast submission of data. A negative side is the absence of a test for the presence of Reticulocyte for pediatric subjects. In further observations, the clinical laboratory exhibits test submission only for Dasit but not for Simens systems, the latter used in pediatric subjects for leukemia diagnosis. For the UK NEQAS, the core issue observed is communication despite being an international institution.
CONCLUSION: This methodology supported a complete and in-depth overview of the characteristics and performances of the two EQSs during evaluation and supported strategic improvements. The study suggests implementation to the EQSs of specific analytes, that could support clinical laboratories to use proper tests for the requests, and to better evaluate the outputs of external quality control results.

P233

Development Of A Cell Culture Model For Studying Monocytopoiesis: Impact Of Tet2 Gene Mutations On Monocyte Maturation
Lucy LOCHER^1,2, Bérénice SCHELL^1,2, Jules CRETIN², Sihem TARFI^1,2, Lydia ROY^2,3, Valeria BISIO⁴, Nicolas DULPHY^4,5, Ivan SLOMA^1,2, Orianne WAGNER-BALLON^1,2
¹Department of Biological Haematology and Immunology, Henri Mondor Hospital APHP, Creteil, , France, ²INSERM Unit 955, Université Paris Est Créteil, Creteil, , France, ³Department of Clinical Haematology, Henri Mondor Hospital APHP, Creteil, , France, ⁴Université Paris Cité, Institut de Recherche Saint Louis, INSERM UMR1342, Paris, , France, ⁵Laboratory of Immunology and Histocompatibility, Saint-Louis Hospital APHP, Paris, , France

Introduction Chronic myelomonocytic leukaemia (CMML) is a clonal hematopoietic stem cell disorder characterized by sustained monocytosis and a relative accumulation of the circulating classical monocyte subset CD14⁺⁺CD16^- (cMo). The majority of CMML patients exhibit TET2 mutations, most frequently leading to biallelic inactivation. Currently, no in vitro cell culture model faithfully recapitulates monocytic differentiation and maturation into the three main monocyte subpopulations described in peripheral blood. Here, we developed a three-dimensional in vitro model of monocytopoiesis to investigate the role of TET2 gene inactivation in monocyte maturation in CMML. Methods Ten to 20x10³ CD34⁺ cells were cultured in a three-dimensional spheroid model, known as artificial marrow organoid (AMO*), combined with 8x10⁴ murine fibroblasts. The AMO* medium was supplemented with a cytokine cocktail (TPO, FLT3-L, IL-3, SCF and GM-CSF). Monocyte subpopulations were analysed by flow cytometry (DxFLEX, Beckman Coulter®) using a validated antibody panel. Gene editing of TET2 was performed in mobilized CD34⁺ cells from healthy donors using the CRISPR/Cas9 ribonucleoprotein complex (crRNA, tracrRNA, and Cas9), and the 4D-Nucleofector system (Lonza®). Guide RNA were designed to target either the exon 7 of the TET2 gene or the neutral genomic region AAVS1 as a control. Editing rate in CD34⁺ cells was quantified by drop-off digital PCR. Results Culture of CD34⁺ cells from CMML patients in the AMO* model reproduced two CMML hallmark features after 10 days: increased monocyte proliferation (31,770 ±9,596 monocytesin CMML patients vs 24,078 ±5,645 monocytesin healthy donors (n=3)) and a trend toward a relative accumulation of cMo ≥94% (94.8% ±1.8in CMML patients vs 87.4% ±1.3in healthy donors (n=3)) after 10 days of culture. Editing of the TET2 gene in CD34⁺ cells from healthy donors cultured in the AMO* model after 10 days of culture: (i) similarly reproduced a relative accumulation of cMo exceeding 94% (94.6% in TET2-edited cells (editing rate: 31.8%) vs 90.4% in AAVS1-edited cells (editing rate: 21.4%)); and (ii) revealed a significant positive correlation between TET2 editing rate and the relative proportion of cMo (Spearman correlation, r_s=0.886 ; p=0.03 ; n=6). Conclusions We developed a three-dimensional co-culture model that recapitulates both physiological and pathological monocytopoiesis. Our findings suggest that CMML-associated accumulation of cMo is driven by intrinsic alterations of CD34⁺ cells and provide functional evidence that TET2 inactivation directly contributes to defective monocyte maturation in CMML.

8:00

Anti-Pf4 Induced Thrombotic Disorders �
Edelgard Lindhoff-Last
CardioAngiology Center Bethanienhospital (CCB), Head of the CCB Coagulations Center and CCB Coagulation Research Center, Frankfurt, Germany

8:30

Acquired Von Willebrand Disease
Jeroen Eikenboom
Leiden University Medical Center

Title

Acquired Von Willebrand Disease

Author

Jeroen Eikenboom

Affiliation

Department of Internal Medicine, division of Thrombosis and Hemostasis, Leiden University Medical Center, the Netherlands

Abstract

Von Willebrand factor (VWF) is essential for normal platelet adhesion and aggregation, and it is the carrier protein for factor VIII. Quantitative and qualitative defects in VWF lead to the inherited bleeding disorder Von Willebrand disease (VWD), which is usually caused by genetic defects in the VWF gene.

In rare cases, VWD is not inherited, but acquired during life. The laboratory findings are very similar to the inherited form of VWD, and also the clinical picture may be similar. However, in acquired VWD the patient will have no previous, personal bleeding history and a family history of bleeding is also lacking. Making the distinction between inherited and acquired VWD is very important as the therapeutic management is very different. Treatment is not only focused on management of bleeding episodes, but also targets the cause of acquired VWD. Underlying causes are various, like autoimmune diseases, monoclonal gammopathy of undetermined significance, plasma cell disorders, lympho- and myeloproliferative disorders, hypothyroidism, and cardiovascular conditions like aortic stenosis, left ventricular assist devices, and extracorporeal membrane oxygenation.

In acquired VWD several pathophysiological mechanisms play a role. Clearance of VWF from the circulation may be faster due to absorbance of VWF to malignant cells or by formation of immune complexes. Enhanced proteolysis of VWF in cardiovascular conditions of high shear stress may lead to loss of high molecular weight VWF multimers. Neutralizing antibodies, as usually seen in acquired hemophilia, are extremely uncommon in acquired VWD.

The diagnostic approach in acquired VWD will consist of measuring factor VIII activity, VWF antigen (VWF:Ag), and the VWF platelet-binding activity (VWF:Act). Although neutralizing antibodies are very rare, these can be detected by regular mixing studies. More often, auto-antibodies binding VWF will lead to enhanced clearance of VWF and those antibodies cannot be detected by mixing studies. ELISA based assays will be required, but no standardized assays are available. Finally, the VWF propeptide (VWFpp) assay may indicate accelerated clearance when the VWFpp/VWF:Ag ratio is increased, however this does not distinguish acquired from inherited VWD as congenital VWD may also be characterized by increased VWF clearance. It is important to realize that acquired VWD in cardiovascular conditions is mainly characterized by reduced VWF:Act/VWF:Ag ratio, whereas the absolute levels of antigen and activity may be within normal range. Sometimes, a test infusion of VWF concentrate and assessment of its clearance from the circulation may be required for a final diagnosis.

9:00

Platelet Response To Dengue Virus Infection
Martha Viveros
Universidad Michoacana de San Nicolás de Hidalgo

Professor Martha Eva Viveros Sandoval. Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Mexico.

Session Date/Time: Saturday, April 18th from 8:00 AM - 9:30 AM
Session Name: CONCURRENT 5: Acquired Haemostasis Disorders
Presentation Title: Platelet response to Dengue virus infection.

Dengue virus (DENV) infection is a re-emerging disease responsible of 50-100 million clinical cases and 10,000 deaths every year worldwide. It is characterized by a wide spectrum of clinical manifestations ranging from mild febrile illness to severe dengue with hemorrhage and shock. Platelets play a central role in dengue pathogenesis, as thrombocytopenia is one of the most consistent laboratory findings and a key determinant of disease severity. Increasing evidence demonstrates that platelets are not merely passive targets but play an important role in the interface of immunity and haemostasis during viral infections. DENV can directly interact with and infect platelets, leading to platelet activation, mitochondrial dysfunction and enhanced clearance from circulation. Activated platelets contribute to immune modulation by releasing inflammatory mediators, interacting with leukocytes promoting NETosis and causing endothelial dysfunction, thereby amplifying vascular permeability and bleeding risk.

Different mechanisms have been hypothesized to explain platelet response to this infection, one of them is the participation of the non-structural protein 1 (DENV-NS1) which can be secreted into circulation during DENV infection promoting a more efficient infection. It has been demonstrated that full-length DENV NS1 protein and its domains (β-ladder, wing and NS1 mutant) induce P-selectin and glycoprotein αIIbβ3 (gpIIb/IIIa, integrin) complex expression on platelets surface.

Laboratory Hematology is essential for understanding these mechanisms and for the clinical management of dengue patients. Quantitative and qualitative platelet analyses including platelet count, mean platelet volume, and leucocyte and lymphocyte/platelet ratio, activation markers and functional assays provide critical insights into disease progression and prognosis. Serial hematological monitoring allows early detection of severe dengue, guides clinical decision-making and support risk stratification.