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.


Two systems were simultaneously compared on 67 blood samples and external quality controls, using CD3,CD4, CD8, CD19, CD16/56, and CD45 in one tube TRUCOUNT beads™ (BD Biosciences) or two tubes (TetraChrome™ and Flowcount™, Beckman-Coulter and DakoCytomation).


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 (r2 > 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. © 2005 Wiley-Liss, Inc.