Abstract
Abstract 1460
The eradication of minimal residual disease (MRD) in CLL predicts for improved outcome. Approaches to CLL MRD detection vary in sensitivity and cost. Historically, CD5/CD19 co-expression with demonstration of clonality has been the principal method of MRD evaluation in CLL. However this approach is limited by the identification of normal hemopoietic cells and the inability to demonstrate light chain restriction with very low B cell numbers. More recently, an international standardised approach (ISA) to the assessment of MRD in CLL that utilises 10 monoclonal antibodies in a 4 tube test system and permits a sensitivity of 0.01% (Rawstron et al, Leukemia 2007) was published. Here we compare the ISA to a 10 colour flow cytometric assay incorporating all the mononclonal antibodies utilised in the ISA in a single tube.
Method:
Peripheral blood (n=21) and bone marrow (n=7) was collected from patients at various time points post treatment for CLL (including bone marrow from 2 patients day 30 post-allogeneic stem cell transplant). Immunophenotyping was performed using a Gallios flow cytometer (Beckman Coulter). Monoclonal antibodies were used either according to the ISA (antibody/fluorochrome combination as per Rawstron et al) or in a single tube incorporating all of the following monoclonal antibodies: CD3 ECD, CD5 PercP5.5, CD19 eFluor, CD20 PE CY7, CD81 FITC, CD22 PE, CD43 APC CY7, CD79b APC, CD38 A700, CD45 CO. A whole blood or bone marrow lyse method was used and analysis was performed according to the published ISA methodology or for the single tube using templates based on the ISA approach. Results from the two methods were compared.
Results:
Levels of residual disease in the 28 samples analysed varied from <0.01 to 22%. Sixteen samples showed residual disease of <1%. Analysing all samples showed an excellent correlation between the two methods slope = 0.989, intercept =0.1 and R(2)=0.992. There was also excellent correlation for disease levels below 1.0%, (median 0.03 range <0.01–0.63%, n=16) slope = 1.15, intercept =0.007 and R(2)=0.994. Bland Altman analysis showed a mean of 0.008 +/− 0.058 (2SD) for values below 1%.
Conclusion:
The single tube ten colour flow cytometric assay for detection of MRD in CLL gives equivalent results to the ISA. There is a potential for improved sensitivity resulting from increased event detection since there is no need to divide the sample into multiple tubes, particularly post-treatment when cell numbers are frequently limiting. The single tube assay is also simple, rapid and cost effective.
Disclosures:
No relevant conflicts of interest to declare.
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