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Background CD4+ T-lymphocyte enumeration is vital for monitoring disease progression in individuals positive for the human immunodeficiency virus (HIV), and as a result, there is a need to develop cost-effective protocols that provide accuracy, precision, and affordability. Recently, PanLeucoGating has been shown to fulfill these requirements; however, although comparable to state-of-the-art single-platform protocols (SP), there is still a requirement for an accurate total white cell count. To overcome this limitation, we recently developed a flow-rate based calibration method that enables the PanLeucoGating protocol to be used as a SP approach, and in this study show that this approach can be used for CD4+ T-lymphocyte enumeration. Methods A total of 113 HIV samples were analyzed using three protocols: (a) state-of-the art SP bead-based method (MultiSet; predicate protocol), (b) PanLeucoGating protocol used as a dual-platform (DP) approach, and (c) the newly developed flow rate–based SP approach. We demonstrate that flow rate calibration can be achieved easily and that the method is highly comparable to the state-of-the-art SP method. Results A high correlation was observed between the predicate protocol and the SP PanLeucoGating approach over the whole range of CD4 counts tested (r2 = 0.9928; bias 8 cells/μl), including the clinically relevant range (e.g., 0–200 CD4 cells/μl; bias 0 cells/μl). For batched samples, the cost of providing a CD4+ T-lymphocyte count was reduced to approximately US $1. Conclusions The SP PanLeucoGating is a cost-effective approach to CD4+ T-lymphocyte enumeration that maintains accuracy and precision. Cytometry Part B (Clin. Cytometry) 55B:8–13, 2003. © 2003 Wiley-Liss, Inc.

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/l). 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. © 2003 Wiley-Liss, Inc. Key terms: flow cytometry; single platform; absolute counts; quality control; CD4 T lymphocytes

The human immunodeficiency virus (HIV) global epidemic has necessitated the routine enumeration of T-lymphocyte subsets, which has created a need for external quality assurance (EQA). The United Kingdom National External Quality Assessment Scheme (UK NEQAS) for Immune Monitoring provides EQA for 296 laboratories in 40 countries. In 1993, UK NEQAS developed and incorporated into its program stabilized whole blood that enables the accurate monitoring of laboratory performance. Overall, the mean interlaboratory coefficient of variation (CV) for percentage CD4+ T-lymphocyte subset enumeration has fallen from 15% to less than 5%, as a direct result of the increased use of CD45/ side scatter (SSC) gating. Laboratories using alternative gating strategies (i.e., CD45/CD14 or forward scatter [FSC]/SSC) were about 7.4 times more likely to fail an EQA exercise. Furthermore, the adoption of single-platform technology resulted in a reduction of the overall mean interlaboratory CV for absolute CD4+ T lymphocytes from 56% (prior to the widespread use of single-platform technology) to 9.7%. Individual laboratory deficiencies were also identified using a performance monitoring system and, through re-education by collaboration with the coordinating center, satisfactorily resolved. In conclusion, during the last 9 years, the UK NEQAS for Immune Monitoring program has highlighted the significant technological advances made by laboratories worldwide that undertake lymphocyte subset enumeration. Cytometry (Clin. Cytometry) 50:102–110, 2002. © 2002 Wiley-Liss, Inc.