Enumeration of peripheral lymphocyte subsets using 6 vs. 4 color staining: A clinical evaluation of a new flowcytometer

Technological advances in instruments allow the evaluation of many lymphocyte subsets in one step. The :aim of this study was to evaluate the new FACSCanto flowcytometer in routine conditions, using a 6 color combination, single platform, whole blood, lysis, no wash protocol. Methods: Two systems were simultaneously compared on 67 blood samples and external quality controls, :sing CD3,CD4, COB, CD19, CD16/56, and CD45 in one tube TRUCOUNT beads(TM) (BD Biosciences) or two tubes (TetraChrome(TM) and Flowcount(TM), Beckman-Counter and DakoCytomation). Results: The day-to-day instrument detection but automatic compensations were stable. Manual compensation settings were satisfactory using available facilities. Commercial and UK NEQAS quality control results were acceptable. The intra-experiment reproducibility was good (coefficient of variation (CV) <3%) but highly operator-dependant (CD4(+) T cell count CVs from 1.2 to 9.7, six operators). Storage of samples was :acceptable, but storage of stained samples altered absolute count reliability. Serial dilutions show a good count accuracy. The FACScanto T subsets and B cell data were highly correlated with our reference values (r(2) > 0.87) and absolutes values were very close (slopes > 0.89). The gating strategy, fluorochrome choice, and compensation setting are discussed. A few improvements are expected (sample loader, data management, auto-gating, acquisition parameters, sample mixing, absolute values calculation, etc). In conclusions, despite its complexity, 6 color staining is a reliable, stable, and highly informative technique for lymphocyte subset monitoring but remains to be optimized. (C) 2005 International Society for Analytical Cytology.