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Updated: Aug 14, 2026
Isolation of CD4+ T-cells and Analysis of Circulating T-follicular Helper (cTfh) Cell Subsets from Peripheral Blood Using 6-color Flow Cytometry
Published on: January 7, 2019
Enumeration of peripheral lymphocyte subsets using 6 vs. 4 color staining: a clinical evaluation of a new
Claude Lambert1, Iobagiu Cristina, Genin Christian
1Immunology Laboratory, University Hospital of St Etienne, France. Claude.lambert@univ-st-etienne.fr
Unlabelled:
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, 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).
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-dependent (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.
