title: Multisite comparison of CD4 and CD8 T-lymphocyte counting by single- versus multiple-platform methodologies: evaluation of Beckman Coulter flow-count fluorospheres and the tetraONE system. The NIAID DAIDS New Technologies Evaluation Group.
abstract: New analytic methods that permit absolute CD4 and CD8 T-cell determinations to be performed entirely on the flow cytometer have the potential for improving assay precision and accuracy. In a multisite trial, we compared two different single-platform assay methods with a predicate two-color assay in which the absolute lymphocyte count was derived by conventional hematology. A two-color method employing lymphocyte light scatter gating and Beckman Coulter Flow-Count fluorospheres for absolute counting produced within-laboratory precision equivalent to that of the two-color predicate method, as measured by coefficient of variation of replicate measurements. The fully automated Beckman Coulter tetraONE System four-color assay employing CD45 lymphocyte gating, automated analysis, and absolute counting by fluorospheres resulted in a small but significant improvement in the within-laboratory precision of CD4 and CD8 cell counts and percentages suggesting that the CD45 lymphocyte gating and automated analysis might have contributed to the improved performance. Both the two-color method employing Flow-Count fluorospheres and the four-color tetraONE System provided significant and substantial improvements in between-laboratory precision of absolute counts. In some laboratories, absolute counts obtained by the single-platform methods showed small but consistent differences relative to the predicate method. Comparison of each laboratory's absolute counts with the five-laboratory median value suggested that these differences resulted from a bias in the absolute lymphocyte count obtained from the hematology instrument in some laboratories. These results demonstrate the potential for single-platform assay methods to improve within-laboratory and between-laboratory precision of CD4 and CD8 T-cell determinations compared with conventional assay methods.
authors.affiliation: Division of Viral Pathogenesis, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA 02215, USA. kreimann@caregroup.harvard.edu
authors.forename: K A
authors.initials: KA
authors.lastname: Reimann
authors.forename: M R
authors.initials: MR
authors.lastname: O'Gorman
authors.forename: J
authors.initials: J
authors.lastname: Spritzler
authors.forename: C L
authors.initials: CL
authors.lastname: Wilkening
authors.forename: D E
authors.initials: DE
authors.lastname: Sabath
authors.forename: K
authors.initials: K
authors.lastname: Helm
authors.forename: D E
authors.initials: DE
authors.lastname: Campbell
mesh_terms: D016516:CD4-CD8 Ratio / Q000295:instrumentation* / Q000379:methods*; D005434:Flow Cytometry / Q000295:instrumentation* / Q000379:methods* / Q000592:standards; D006801:Humans; D016130:Immunophenotyping / Q000295:instrumentation / Q000379:methods / Q000592:standards; D007753:Laboratories / Q000592:standards; D017493:Leukocyte Common Antigens / Q000032:analysis; D015203:Reproducibility of Results; D013048:Specimen Handling
chemical_list: D017493:Leukocyte Common Antigens