How to cite this article: Sartor M. M., Gottlieb D. J. A single tube 10-color flow cytometry assay optimizes detection of minimal residual disease in chronic lymphocytic leukemia. Cytometry Part B 2013; 84B: 96–103.
A single tube 10-color flow cytometry assay optimizes detection of minimal residual disease in chronic lymphocytic leukemia†
Article first published online: 2 JAN 2013
Copyright © 2012 International Clinical Cytometry Society
Cytometry Part B: Clinical Cytometry
Volume 84B, Issue 2, pages 96–103, March 2013
How to Cite
Sartor, M. M. and Gottlieb, D. J. (2013), A single tube 10-color flow cytometry assay optimizes detection of minimal residual disease in chronic lymphocytic leukemia. Cytometry, 84B: 96–103. doi: 10.1002/cyto.b.21067
- Issue published online: 20 FEB 2013
- Article first published online: 2 JAN 2013
- Manuscript Accepted: 30 NOV 2012
- Manuscript Revised: 29 NOV 2012
- Manuscript Received: 24 SEP 2012
- chronic lymphocytic leukemia;
- minimal residual disease;
- multiparameter flow cytometry
Levels of residual disease (RD) are an independent predictor of progression-free survival (PFS) and overall survival (OS) in patients treated for chronic lymphocytic leukemia (CLL). We modified the international standardized approach (ISA) to RD detection using flow cytometry by developing a single tube 10 color antibody assay.
A single tube incorporated the following monoclonal antibodies: CD81FITC, CD22PE, CD3ECD, CD5PercP5.5, CD20PECY7, CD79bAPC, CD38A700, CD43APC Alexa750, CD19eFluor 450, and CD45KO. A modified ISA gating strategy was developed that removed contaminating events. Sensitivity assays were performed using dilution with normal peripheral blood and bone marrow. Clinical samples were compared using the ISA and the single tube assay.
Dilution studies showed that sensitivity of 0.001% was achievable when a minimum of 1.8 × 106 total events were acquired. One hundred twenty-nine samples were analyzed and showed RD levels from 0.003 to 22%. In 80 samples analyzed with both assays, there was an excellent correlation between the two methods (slope = 1.0, intercept = 0.07 and R2 = 0.992) and results from Bland–Altman analysis showed a bias of 0.04 ± 0.38 with 95% confidence interval of −0.71 to 0.79. Removal of contaminating events in the single tube assay led to a significant reduction in RD values (P = 0.0014).
The single tube 10-color assay for the detection of RD in CLL provides equivalent results to the ISA but requires fewer cells, uses fewer reagents, and allows for simpler analysis. By directly removing contaminating events, it improves the accuracy of CLL RD detection and may reclassify the status of some patients following chemotherapy. © 2013 International Clinical Cytometry Society