Perfect count:: A novel approach for the single platform enumeration of absolute CD4+ T-lymphocytes

Background: The derivation of reliable CD4(+) T lymphocyte counts is vital for the monitoring of disease progression and therapeutic effectiveness in HIV+ individuals. Flow cytometry has emerged as the method of choice for CD4(+) T lymphocyte enumeration, with single-platform technology, coupled with reference counting beads, fast becoming the "gold standard." However, although single-platform, bead-based, sample acquisition requires the ratio of beads to cells to remain unchanged, there is no available method, until recently, to monitor this. Methods: Perfect Count beads have been developed to address this issue and to incorporate two bead populations, with different densities, to allow the detection of inadequate mixing. Comparison of the relative proportions of both beads with the manufacture's defined limits enables an internal QC check during sample acquisition. In this study, we have compared CD4(+) T lymphocyte counts, obtained from 104 HIV+ patients, using TruCount beads with MultiSet software (defined as the predicated method) and the new Perfect Count beads, incorporating an in house sequential gating strategy. Results: We have demonstrated an excellent degree of correlation between the predicate method and the Perfect Count system (r(2) = 0.9955; Bland Altman bias +27 CD4(+) T lymphocytes/mul). Conclusions: The Perfect Count system is a robust method for performing single platform absolute counts and has the added advantage of having internal QC checks. Such an approach enables the operator to identify potential problems during sample preparation, acquisition and analysis. (C) 2003 Wiley-Liss, Inc.