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 "PanLeucogating," 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 PanLeucogating compared with lymphocyte gating. Next, 183 fresh and 112 fixed (TransFix) whole blood samples were used to compare DP methods and singleplatform (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 PanLeucogating (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 PanLeucogating 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.

Background: In order to expand the treatment of human immunodeficiency virus-1 (HIV) infected patients in Africa, millions will require cost-effective CD4 counts. Supporting laboratories therefore, need to move away from crisis management and haphazard practices to organized pathology services. The authors reviewed the performance of the simplified single platform (SP) PanLeucogated (PLG) CD4 methodology, introduced into 52 laboratories across the South African National Health Laboratory Service (SA-NHLS), with a proactive approach to training, internal quality control (IQC), and external quality assessment (EQA).Methods: Two-color flow cytometry for SP PLG (CD4/CD45) was combined with the sample-by-sample bead-count-rate (BCR) IQC for bead pipetting. PLG þ BCR was validated versus conventional predicate SP and dual-platform (DP) 4-color flow cytometric methods used in the first world-on 1181 samples from 250 HIVþ patients followed longitudinally on anti-retroviral therapy (ART). EQA (accuracy) was performed through the United Kingdom National External Quality Assessment Scheme (UK-NEQAS). Further EQA was performed across the 52 SA-NHLS SP-PLG laboratories participating on the CD4 African Regional External Quality Assessment Scheme (AFREQAS), to assess both accuracy and/precision between NHLS PLG laboratories.Results: There was virtually no bias noted between SP PLG and SP predicate methods. On DP, bias and variability increased but the errors introduced were minor without affecting CD4-related clinical decisions. The simpler 2-color PLG was less expensive with additional advantages: CD4þ T-cells were discriminated from monocytes without a need for CD3-staining, and the training was faster and easier for

BackgroundThe South African National Health Laboratory Service (NHLS) responded to HIV treatment initiatives with two-tiered CD4 laboratory services in 2004. Increasing programmatic burden, as more patients access anti-retroviral therapy (ART), has demanded extending CD4 services to meet increasing clinical needs. The aim of this study was to review existing services and develop a service-model that integrated laboratory-based and point-of-care testing (POCT), to extend national coverage, improve local turn-around/(TAT) and contain programmatic costs.MethodsNHLS Corporate Data Warehouse CD4 data, from 60–70 laboratories and 4756 referring health facilities was reviewed for referral laboratory workload, respective referring facility volumes and related TAT, from 2009–2012.ResultsAn integrated tiered service delivery model (ITSDM) is proposed. Tier-1/POCT delivers CD4 testing at single health-clinics providing ART in hard-to-reach areas (<5 samples/day). Laboratory-based testing is extended with Tier-2/POC-Hubs (processing ≤30–40 CD4 samples/day), consolidating POCT across 8–10 health-clinics with other HIV-related testing and Tier-3/‘community’ laboratories, serving ≤40 health-clinics, processing ≤150 samples/day. Existing Tier-4/‘regional’ laboratories serve ≤100 facilities and process <350 samples/day; Tier-5 are high-volume ‘metro’/centralized laboratories (>350–1500 tests/day, serving ≥200 health-clinics). Tier-6 provides national support for standardisation, harmonization and quality across the organization.ConclusionThe ITSDM offers improved local TAT by extending CD4 services into rural/remote areas with new Tier-3 or Tier-2/POC-Hub services installed in existing community laboratories, most with developed infrastructure. The advantage of lower laboratory CD4 costs and use of existing infrastructure enables subsidization of delivery of more expensive POC services, into hard-to-reach districts without reasonable access to a local CD4 laboratory. Full ITSDM implementation across 5 service tiers (as opposed to widespread implementation of POC testing to extend service) can facilitate sustainable ‘full service coverage’ across South Africa, and save>than R125 million in HIV/AIDS programmatic costs. ITSDM hierarchical parental-support also assures laboratory/POC management, equipment maintenance, quality control and on-going training between tiers.