Dilution studies demonstrating increased sensitivity of MRD Flow compared with conventional flow cytometry.

Peripheral blood leukocytes from 3 patients were serially diluted (4-fold dilutions from 1:1 to 1:16 348) into normal peripheral blood leukocytes pooled from 3 healthy individuals. Figure 3 is a representative plot from one experiment, showing the percentage of CLL cells identified by conventional 4-color flow cytometry (CD19 + 5⁺kappa⁺ or CD19 + 5⁺lambda⁺) in dotted lines, and percentage of CLL cells identified by MRD Flow (CD19/CD5/CD20/CD79b) in solid lines, against actual CLL cell percentage. At the top of the graph, results are shown as positive (+) or negative (−) for the presence of CLL cells at each dilution for the different methods of assessing residual disease. Criteria for detection of CLL cells were as follows: (1) IgH-PCR: there is at least 2-fold greater PCR product with the same size as the CLL cell rearrangement product than would be expected for a normal distribution23,31; (2) conventional 2-color flow: over 25% of the B cells coexpress CD517-19; (3) conventional 4-color flow: light chain restriction within the CD5⁺ B-cell compartment; and (4) MRD Flow: the number of gated B cells with an aberrant phenotype is at least 2-fold greater than background (ie, the number generated by the same gating strategy applied to a sample containing normal leukocytes only). R²values from linear regression analysis of MRD Flow results for observed versus actual percentages of CLL cells are shown at the bottom right of the graph, demonstrating linearity over the series.