Open AccessCD45‐assisted Pan Leuco gating for accurate, cost‐effective dual‐platform CD4+ T‐cell enumeration
Author(s) -
Glencross Deborah,
Scott Lesley E.,
Jani Ilesh V.,
Barnett David,
Janossy George
Publication year - 2002
Publication title -
cytometry
Language(s) - English
Resource type - Journals
eISSN - 1097-0320
pISSN - 0196-4763
DOI - 10.1002/cyto.10068
Subject(s) - gating , enumeration , flow cytometry , cytometry , nuclear medicine , hematology analyzer , accuracy and precision , lymphocyte , peripheral blood , diagnostic accuracy , analytical chemistry (journal) , mathematics , chemistry , microbiology and biotechnology , medicine , biomedical engineering , statistics , chromatography , immunology , biology , pathology , biophysics , combinatorics
BACKGROUND North American and European guidelines for dual‐platform (DP) flow cytometry recommend absolute CD4 T‐cell counts to be calculated from two parameters: the absolute lymphocyte counts obtained on a hematology analyzer and the percentages of CD4+ cells among lymphocytes (CD4%/ lympho ) obtained by flow cytometry. Nevertheless, the identification of lymphocytes is error‐prone: a poor match between these common denominators in the two systems is the main source of inaccuracy. In contrast, total leucocyte counts (white cell counts [WCC]) and CD4% among the gated CD45+ leucocytes (CD4%/ leuco ) can be determined with greater accuracy. Methods: We introduced “Pan Leuco gating,” i.e., we used total leucocytes as the common denominator for improving the precision of DP absolute CD4 counting. Correlations and Bland‐Altman tests were used for statistical analysis. Results: First, 22 stabilized blood product samples were provided by U.K. National External Quality Assessment Scheme (NEQAS) and a higher accuracy and precision of CD4 counts were documented using Pan Leuco gating compared with lymphocyte gating. Next, 183 fresh and 112 fixed (TransFix) whole blood samples were used to compare DP methods and single‐platform (SP) methodology, including both volumetric and bead‐based techniques. A particularly high correlation and comparable precision of absolute CD4 counts were observed between the SP volumetric method and DP Pan Leuco gating (R 2 = 0.990; bias 6 ± SD 17%). The SP volumetric method showed lower levels of agreement with the DP lymphocyte gating (R 2 = 0.758; bias 14 ± SD 51%) and with the SP bead‐based method (R 2 = 0.923; bias 4 ±SD 31%). Conclusions: These observations show that DP leucocyte counts (WCC) should replace lymphocyte counts as the “common denominator” although CD4%/ lympho values can, as an extra step, be also provided readily if requested. When coupled with quality control for WCC on hematology analyzers, the DP method with CD45 Pan Leuco gating represents a robust CD4 T‐cell assay that is as accurate as the SP volumetric technique. This DP method uses only two, CD45 and CD4, antibody reagents and can be run on any pair of hematological analyzer plus flow cytometer. Cytometry (Clin. Cytometry) 50:69–77, 2002. © 2002 Wiley‐Liss, Inc.