Controlling laboratory variables to improve precision and accuracy of CD4+ T-cell enumeration across flow cytometry methods

Abstract

This study assessed the effect that certain logistical and methodological factors in the laboratory could have on influencing precision and accuracy of enumeration of CD4+ cells. The efficacy of a new blood stabiliser to extend the window of CD4 testing, was also evaluated. CD4+ counts were derived using the 2-colour Pan-leucogating, 4-colour TetraONE and MultiTEST/TruCount protocols on the EPICS-XL, FC-500 or FACSCalibur flow cytometers. Statistical analyses included the paired-t-test, Spearman’s correlation and Bland Altman comparisons. The results showed that the reliability of CD4+ count results was heavily dependent on how blood samples were handled prior to and after receipt into the laboratory and on how samples were processed and analysed. The factors, motion, operator pipetting and analysis skills, storage temperature, use of different protocols, different gating strategies and the use of different flow cytometers, were found to influence accurate and precise enumeration of CD4+ counts.