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

No abstract

Background With increased access to antiretroviral treatment (ART), Immune Reconstitution Inflammatory Syndrome (IRIS) in Mycobacterium tuberculosis (MTB)-infected populations remains a clinical challenge. We studied a cross-sectional cohort of HIV-infected subjects in Johannesburg (South Africa) to help define the immune correlates that best distinguish IRIS from ongoing MTB cases. Methods We studied HIV+ subjects developing MTB-related unmasking IRIS (u-TB-IRIS) after ART initiation; control groups were HIV subjects and HIV-TB co-infected subjects with comparable ART treatment. Testing was conducted with whole blood-based 4-color flow cytometry and plasma-based Luminex cytokine assessment. Results NK cell activation, C-reactive protein and IL-8 serum concentration were significantly higher in u-TB-IRIS subjects as compared to both control groups. In addition, all MTB co-infected subjects, independent of clinical presentation, had higher neutrophils and T cell activation, together with lower lymphocytes, CD4+ T cell and myeloid DC counts. Using conditional inference tree analysis we show that elevated NK cell activation in combination with lymphocyte count characterizes the immunological profile of u-TB-IRIS. Conclusions Our results support a role for innate immune effectors in the immunopathogenesis of unmasking MTB-related IRIS, and identify new immune parameters defining this pathology.

Background: CD38 expression on CD81 T lymphocytes in HIV-infected patients is monitored by flow cytometry (FCM).There is however no consensus re CD38 protocols, analyses or result reporting within/ between laboratories. Internal quality control measures (QC) were established for a standardized CD38 protocol and a system proposed for reporting CD38 fluctuation in longitudinal HIV1 patient monitoring.Methods: A single-platform (SP) CD38/CD8 protocol was ''piggy-backed'' onto the standardized ''panleucogating'' CD45/CD41 protocol. A weekly QC was established to monitor instrument stability (Flow-SET TM ) and absolute cell count accuracy and reproducibility (stabilized blood product, Immuno-Trol TM ). The Mean Fluorescence Intensity (MFI) of CD38 expression on CD81 -lymphocytes was monitored on both stabilized blood and HIV-control samples. Linearized MFI values were determined from biological controls, i.e. healthy donor monocytes and granulocytes, and tested as a method of reporting CD38 expression on selected HIV1 patients on ART.Results: The CD45/CD4/CD8/CD3 method for lymphocyte enumeration compared well with the CD38 protocol (CD45/CD4/CD8/CD38) with excellent similarity (6100%) and precision for absolute CD4 and CD8 counts (CVs < 5%).