title: Enumeration of peripheral lymphocyte subsets using 6 vs. 4 color staining: a clinical evaluation of a new flowcytometer.
abstract: 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.
authors.affiliation: Immunology Laboratory, University Hospital of St Etienne, France. Claude.lambert@univ-st-etienne.fr
authors.forename: Claude
authors.initials: C
authors.lastname: Lambert
authors.forename: Iobagiu
authors.initials: I
authors.lastname: Cristina
authors.forename: Genin
authors.initials: G
authors.lastname: Christian
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D003116:Color; D005260:Female; D005434:Flow Cytometry / Q000295:instrumentation / Q000379:methods*; D006801:Humans; D018655:Lymphocyte Count; D008214:Lymphocytes / Q000145:classification* / Q000166:cytology*; D008297:Male; D008875:Middle Aged; D011786:Quality Control; D015203:Reproducibility of Results; D013194:Staining and Labeling; D016176:T-Lymphocyte Subsets / Q000166:cytology