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