1C, gate E) by the number of total leukocytes (Fig. 1A, gate F). This gating strategy was tested on a series of 20 samples by three operators all experienced in the analysis of list mode data files. There was no significant difference in the results obtained for all data files examined P = 0.95, the proportion of CLL cells ranged from 0.01 to 0.09%. Using the single tube 10-color assay, levels of residual disease in the 129 samples analyzed varied from 0.003 to Erlotinib manufacturer
22%. Twenty-five samples (19%) showed residual disease >1%, 57 samples (44%) showed residual disease between 1.0 and 0.01%, and 47 samples (37%) showed residual disease below 0.01% (Fig. 2). In seven samples from which a minimum of 1.8 �� 106 events were acquired, it was possible to quantify residual disease levels between 0.003 and 0.01% (Fig. 2). Paired peripheral blood and bone marrow samples were obtained from 12 patients following fludarabine, cyclophosphamide, and Rituximab (FCR) therapy. In nine patients, MRD was detected in both peripheral blood and bone marrow, whereas three patients showed residual disease in bone marrow only. Bone marrow samples (n = 4) and peripheral blood (n = 7) from Selleckchem GSK126
six patients at various time points post allogeneic stem cell transplant (SCT) were also examined for residual disease. Eight samples showed levels above 0.01% and three (all from peripheral blood) samples were <0.01%. Only one of the latter patients had a corresponding bone marrow sample taken and the MRD level was 0.3%. Two patients were monitored for MRD at regular time intervals over a period of 18 months to assess the robustness of the assay for clinical use. Patient 1 (Fig. 3A) was a 57-year-old man who received a second course of six cycles of FCR between April and September 2009. He remained in clinical CR and low but stable levels of MRD were detected from January to September 2011. Subsequently, there was a <a href="http://en.wikipedia.org/wiki/Vasopressin_receptor
">Vasopressin Receptor progressive rise in MRD levels in blood in association with reappearance of splenomegaly. Patient 2 (Fig. 3B) was a 69-year-old man previously treated with oral chlorambucil and in 2009 with three cycles of FCR. From January to October 2011 increasing levels of disease were detected in blood in the presence of a stable lymphocytosis. From November 2011 to March 2012 he received five cycles of FCR because of soft tissue infiltration by CLL cells in the conjunctivae and sinuses. To validate the single tube 10color assay, we directly compared results from a cohort of 85 samples analyzed using the ISA method performed according to the standard operating procedure described by Rawstron et al. (14) with results obtained using the single tube 10-color method. Of the 85 samples, five (5.8%) could not be evaluated by the ISA methodology and were excluded from the validation study: four samples had insufficient events to obtain a sensitivity of 0.01% and in one sample the level of contamination due to the co-expression of CD19 and CD3 exceeded the residual CLL population.