The prognostic impact of monocyte fluorescence, immunosuppressive monocytes and peripheral blood immune cell numbers in HIV-associated Diffuse Large B-cell Lymphoma.

INTRODUCTION: Diffuse large B-cell lymphoma (DLBCL) is a high grade non-Hodgkin lymphoma which is common among immunodeficient people. Derangements of peripheral blood immune cells have been described to have a prognostic impact in DLBCL in high income countries, including a monocytosis, the ratios of lymphocytes to both monocytes (L:M) and neutrophils (N:L), as well as the numbers of regulatory T-cells (Tregs) and immunosuppressive monocytes (HLA-DRlow monos). To date, the impact of these variables has not been assessed in the setting of HIV-associated DLBCL (HIV-DLBCL), which is among the most common malignancies seen in people living with HIV. In this study, we assessed these factors in a cohort of South African patients with DLBCL and a high HIV-seropositivity-rate. In addition, we evaluated the prognostic value of monocyte activation (as reflected by monocyte fluorescence (MO-Y) on a Sysmex haematology analyser). This parameter has to date not been assessed in the setting of DLBCL. METHODS: A full blood count and differential count as well as flow cytometry for HLA-DRlow monocyte and Treg enumeration were performed in patients with incident DLBCL referred to the Chris Hani Baragwanath Academic Hospital in Johannesburg, South Africa between November 2019 and May 2022. Additional clinical and laboratory data were recorded from the patient charts and laboratory information system. RESULTS: Seventy-six patients were included, of whom 81.3% were people living with HIV with a median CD4 count of 148 cells/ul. Most patients had advanced stage disease (74.8%) and were predominantly treated with cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP)-based chemotherapy (without Rituximab). At a median follow-up period of 19 months, the median survival time was 3.5 months, with a 12-month survival rate of 27.0%. All of the immune-cell-related variables (with the exception of the CD4 count) were similar between the people living with HIV and the HIV-negative individuals. In contrast to previous studies, a high monocyte count, the L:M and increased numbers of HLA-DRlow monocytes were not significantly associated with survival in HIV-DLBCL, while a neutrophilia (>8 x 109/L), the N:L (>6:1), high numbers of Tregs (≥5.17% of CD4s) and lymphopenia (<1.3 x 109/L) were. In addition, increased monocyte fluorescence (MO-Y >115.5) was associated with superior outcomes, which we speculate to reflect a more robust antitumour immune response among individuals with high levels of monocyte activation. On Cox Proportional hazard analysis, immune-cell factors independently associated with survival included a CD4 count <150 cells/ul and a neutrophilia. CONCLUSION: The monocyte count, L:M and the number of HLA-DRlow monos are not strong prognostic indicators in HIV-DLBCL, while a low CD4 count and neutrophilia are. Elevation of the MO-Y shows some promise as a potential biomarker of antitumour immunity; further study in this regard would be of interest.

Commercial DURAClone panels for extending the repertoire of multicolour immunophenotypic panels in an academic flow cytometry laboratory in South Africa.

Background: Commercial multicolour fixed immunophenotyping panels can improve flow cytometric diagnostic immunophenotyping repertoire. Objective: This study validated the commercially available, standardised Beckman Coulter lyophilised DURAClone RE panels to discriminate specific haematolymphoid subtypes. Methods: We compared the diagnostic capability of the DURAClone acute leukaemia B (ALB), chronic leukaemia B (CLB), and plasma cells (PC) panels to the predicate second-line panels in Charlotte Maxeke Johannesburg Academic Hospital, Johannesburg, South Africa, from April to August 2020. Clinical diagnostic concordance between the in-house second-line immunophenotyping (the predicate method) and DURAClone was established. The ALB panels tested for precursor B-cell acute lymphoblastic leukaemia (n = 11) or normal bone marrow haematogones (n = 9); CLB panels established haematolymphoid subtypes of mature B-cell lymphoproliferative disorders (B-LPD) (n = 20), while PC panels detected plasma cell dyscrasias (PCD) (n = 17). Flow cytometer setup and data interpretation to discriminate normal and aberrant immunophenotypes were per manufacturer's instructions. Results: There was 100% clinical diagnostic concordance between the predicate and the test panels for second-line diagnostic investigation of B-ALL (with additional CD56), mature B-LPD (with additional discernment of CD81, ROR-1, CD79b and CD43) and PCD. Conclusion: The DURAClone CLB exceeded the predicate second-line performance, offering extended second-line diagnostic discernment of mature B-LPD subtypes and discernment of CD5+ B-LPD from other non-CD5+ (or CD5-) B-LPD; likewise, the PC panels enabled discovery of PCD. While ALB testing offered no additional diagnostic advantage over existing predicate investigation, CD58 did offer additional information to discern haematogones from B-ALL.

Evaluation of fixed-panel, multicolour ClearLLab 10C at an academic flow cytometry laboratory in Johannesburg, South Africa.

Background: Flow cytometric immunophenotyping is well established for the diagnosis of haematological neoplasms. New commercially available systems offer fixed, pre-aliquoted multi-parameter analysis to simplify sample preparation and standardise data analysis. Objective: The Beckman Coulter (BC) ClearLLab™ 10C (4-tube) system was evaluated against an existing laboratory developed test (LDT). Methods: Peripheral blood and bone marrow aspirates (n = 101), tested between August 2019 and November 2019 at an academic pathology laboratory in Johannesburg, South Africa, were analysed. Following daily instrument quality control, samples were prepared for LDT (using > 20 2-4-colour in-house panels and an extensive liquid monoclonal reagent repertoire) or ClearLLab 10C, and respectively analysed using in-house protocols on a Becton Dickinson FACSCalibur, or manufacturer-directed protocols on a BC Navios. Becton Dickinson Paint-a-Gate or BC Kaluza C software facilitated data interpretation. Diagnostic accuracy (concordance) was established by calculating sensitivity and specificity outcomes. Results: Excellent agreement (clinical diagnostic concordance) with 100% specificity and sensitivity was established between LDT and ClearLLab 10C in 67 patients with a haematological neoplasm and 34 participants with no haematological disease. Similar acceptable diagnostic concordance (97%) was noted when comparing ClearLLab 10C to clinicopathological outcomes. Additionally, the ClearLLab 10C panels, analysed with Kaluza C software, enabled simultaneous discrimination of disease and concurrent background myeloid and lymphoid haematological populations, including assessing stages of maturation or sub-populations. Conclusion: ClearLLab 10C panels provide excellent agreement to existing LDTs and may reliably be used for immunophenotyping of haematological neoplasms, simplifying and standardising sample preparation and data acquisition.

The utility of extended differential parameters as a biomarker of bacteremia at a tertiary academic hospital in persons with and without HIV infection in South Africa.

INTRODUCTION: Extended differential parameters (EDPs) are generated with the automated differential count by Sysmex XN-series automated hematology analysers, and include the immature granulocyte count (IG%), the neutrophil fluorescent light intensity (NE-SFL) and the neutrophil fluorescent light distribution width (NE-WY). These have been proposed as early biomarkers of bacteremia. This study aimed to evaluate the NE-SFL, NE-WY and IG% in comparison to neutrophil CD64 (nCD64) expression (as a high quality sepsis biomarker) among patients with suspected bacterial sepsis at the Chris Hani Baragwanath Academic Hospital in Johannesburg, South Africa. METHODS: A daily search of the laboratory information system identified samples submitted for a blood culture (BC) and a concurrent full blood count (FBC). Automated differential counts using a Sysmex XN-9000 haematology analyser and neutrophil CD64 expression by flow cytometry were assessed on the residual FBC samples. RESULTS: A total of 151 samples were collected, of which 83 were excluded due to equivocal results with regards to the presence of bacterial infection. The remaining 68 samples included 23 with bacteremia, 28 with evidence of non-bacteremic bacterial infection, 13 with no evidence of bacterial infection and 4 with Tuberculosis. HIV status was documented in 90 of the patients, with a seropositivity rate of 57.8%. The EDPs were all significantly higher among patients with bacteremia as compared to those without bacterial infection, but on ROC curve analyses, only the NE-SFL showed good performance (AUC>0.8) for discriminating cases with bacteremia from those without bacterial infection at a cut-off value of 49.75. In comparison to the nCD64, the NE-SFL showed moderate agreement (kappa = 0.5). On stratification of the ROC analysis by HIV status, the NE-SFL showed superior performance among persons with HIV infection (AUC = 1), while the automated IG% showed better performance among the patients without HIV infection (AUC = 0.9). CONCLUSION: In this study, EDPs showed differential performance as biomarkers for bacteremia according to HIV-status in the South African setting, with the most promising results seen with the NE-SFL and IG% parameters among people with and without HIV infection, respectively. Further assessment of these parameters without pre-selection of patients likely to have infection is required to further determine their clinical utility, particularly among patients with underlying inflammatory conditions or malignancy.

Comparison of Lymphocyte Subset Populations in Children From South Africa, US and Europe.

Background: Typically, African healthcare providers use immunological reference intervals adopted from Europe and the United States (US). This may be inappropriate in a setting with many differences including exposure to different environmental stimuli and pathogens. We compared immunological reference intervals for children from Europe and the US with South African children to explore whether healthy children living in settings with high rates of infectious diseases have different baseline immunological parameters. Methodology: Blood was taken from 381 HIV-uninfected children aged between 2 weeks and 13 years of age from a Child Wellness Clinic in an informal settlement in Cape Town to establish local hematological and lymphocyte reference intervals for South African children. Flow-cytometry quantified percentage and absolute counts of the B-cells, NK-cells, and T-cells including activated, naïve, and memory subsets. These parameters were compared to three separate studies of healthy children in Europe and the US. Results: Increased activated T-cells, and natural killer cells were seen in the younger age-groups. The main finding across all age-groups was that the ratio of naïve/memory CD4 and CD8 T-cells reached a 1:1 ratio around the first decade of life in healthy South African children, far earlier than in resource-rich countries, where it occurs around the fourth decade of life. Conclusions: This is the largest data set to date describing healthy children from an African environment. These data have been used to create local reference intervals for South African children. The dramatic decline in the naïve/memory ratio of both CD4 and CD8 T-cells alongside increased activation markers may indicate that South African children are exposed to a wider range of environmental pathogens in early life than in resource-rich countries. These marked differences illustrate that reference intervals should be relevant to the population they serve. The implications for the developing pediatric immune system requires further investigation.

The influence of haemodialysis on CD4+ T-cell counts in people living with human immunodeficiency virus with end-stage kidney disease.

BACKGROUND: In South Africa it is estimated that 7.9 million people are living with human immunodeficiency virus (HIV). HIV is associated with an increased risk of kidney disease. For people living with HIV (PLWH) who develop end-stage kidney disease (ESKD), access to renal replacement therapy can be difficult. Kidney transplantation is a cost-effective option, with improved overall survival and better quality of life. In Johannesburg, the eligibility criteria for kidney transplantation include a sustained CD4+ T-cell count of > 200 cells/µL and suppressed HIV replication. OBJECTIVE: To investigate the influence of haemodialysis on the lymphocyte subsets in PLWH with ESKD. In addition, all available %CD4+ T-cell counts, absolute CD4+ T-cell counts and viral load measurements were collected to assess the longitudinal trends of these measurements in PLWH with ESKD. METHODS: This was a cross-sectional study comparing two groups. The HIV-infected study participants (n = 17) and HIV-uninfected controls (n = 17) were recruited from renal dialysis centres in Johannesburg from 2017 to 2018. Demographic data and social data were collected from all the study participants (n = 17). Blood samples were collected from all the study participants (before and after a haemodialysis session), and the lymphocyte subsets were then measured. The available longitudinal data for the serial CD4+ T-cell counts and HIV viral loads were collected (n = 14). RESULTS: Our cohort showed a statistically significant increase in the post-dialysis percentage of CD4+ T cells (5%, p < 0.001) and the absolute CD4+ T-cell counts (21 cells/µL, p < 0.03). The longitudinal trend analysis for the percentage of CD4+ T cells revealed a significant increase in five participants (36%), and a single patient (7%) had a significant decrease in the longitudinal trend analysis for the absolute CD4+ T-cell counts. The longitudinal trend analysis for HIV viral load revealed the majority of our participants were not virologically suppressed. CONCLUSION: This study showed that haemodialysis does not have an immediate negative impact on CD4+ T-cell count, suggesting that immunologic recovery is not impeded by treatment of the underlying ESKD.

Assessment of the AQUIOS flow cytometer - An automated sample preparation system for CD4 lymphocyte PanLeucogating enumeration.

BACKGROUND: Flow cytometry has been the approach of choice for enumerating and documenting CD4-cell decline in HIV monitoring. Beckman Coulter has developed a single platform test for CD4+ T-cell lymphocyte count and percentage using PanLeucogating (PLG) technology on the automated AQUIOS flow cytometer (AQUIOS PLG). OBJECTIVES: This study compared the performance of AQUIOS PLG with the Flowcare PLG method and performed a reference interval for comparison with those previously published. METHODS: The study was conducted between November 2014 and March 2015 at 5 different centres located in Canada; Paris, France; Lyon, France; the United States; and South Africa. Two-hundred and forty samples from HIV-positive adult and paediatric patients were used to compare the performances of AQUIOS PLG and Flowcare PLG on a FC500 flow cytometer (Flowcare PLG) in determining CD4+ absolute count and percentage. A reference interval was determined using 155 samples from healthy, non-HIV adults. Workflow was investigated testing 440 samples over 5 days. RESULTS: Mean absolute and relative count bias between AQUIOS PLG and Flowcare PLG was -41 cells/µL and -7.8%. Upward and downward misclassification at various CD4 thresholds was ≤ 2.4% and ≤ 11.1%. The 95% reference interval (2.5th - 97.5th) for the CD4+ count was 453-1534 cells/µL and the percentage was 30.5% - 63.4%. The workflow showed an average number of HIV samples tested as 17.5 per hour or 122.5 per 8-hour shift for one technician, including passing quality controls. CONCLUSION: The AQUIOS PLG merges desirable aspects from conventional flow cytometer systems (high throughput, precision and accuracy, external quality assessment compatibility) with low technical operating skill requirements for automated, single platform systems.

The iron status of South African blood donors: balancing donor safety and blood demand.

BACKGROUND: Several studies in developed countries have demonstrated high levels of iron deficiency (ID) among blood donors. There is a paucity of data for developing countries where blood shortages remain a major concern. STUDY DESIGN AND METHODS: A total of 4412 donors were enrolled in the study. Specimens were collected for full blood count, iron, transferrin saturation, and ferritin assessment. Donor demographics were recorded. ID was indicated by a ferritin level of less than 20 ng/mL for men and less than 12 ng/mL for women. Anemia was defined as hemoglobin levels less than 12.5 g/dL. Regression models for predictors of ID were developed. RESULTS: A total of 17.5% of all donors had ID, with 16.3% prevalence in women and 18.6% in men. Low hemoglobin had the highest association with ID (adjusted odds ratio [AOR], 11.078; 95% confidence interval [CI], 7.915-15.505); male donors had twice the odds of ID compared to female donors (AOR, 2.501; 95% CI, 1.964-3.185), while increasing age was associated with lower odds (AOD, 0.965; 95% CI, 0.956-0.975). Among male donors, an interdonation interval of less than 3 months (AOR, 2.679; 95% CI, 1.929-3.720) was associated with ID. Compared to other females combined, colored female donors (AOR, 2.335; 95% CI, 1.310-4.160) had higher odds and black female donors (AOR, 0.559; 95% CI, 0.369-0.845) lower odds of ID. CONCLUSION: ID is common among South African donors; low hemoglobin, gender, ethnicity, and past donation history is independently associated with ID. Recommendations aimed at protecting donor health may increase blood shortages in South Africa.

Observed full blood count and lymphocyte subset values in a cohort of clinically healthy South African children from a semi-informal settlement in Cape Town.

BACKGROUND: The paediatric full blood count and lymphocyte subset reference intervals used by the National Health Laboratory Service (NHLS), South Africa (SA), are taken from two international reference interval publications. Differences in reference intervals suggest that international data sets may not be appropriate for use in SA. OBJECTIVE: To study immunohaematological values of a group of clinically healthy children from an informal settlement in Cape Town, SA, to assess whether international paediatric reference intervals used by the NHLS are appropriate. METHODS: A cross-sectional study of 207 female and 174 male HIV-uninfected children living in an informal settlement in Cape Town was performed. Full blood counts, automated differential counts and lymphocyte subset analysis were done using internationally accepted technologies. Data were categorised by age and reference intervals compiled using medians and 95% confidence intervals (CIs). Gender comparisons were calculated by non-parametric tests. RESULTS: Although median and 95% CI values differed slightly, physiological trends for red cell, platelet, white blood cell differential and lymphocyte subsets were similar to international reference intervals currently in use at the NHLS. Benign ethnic neutropenia was not a significant finding, and gender-specific intervals were not necessary until 12 years of age. Lower overall median values for haemoglobin and haematocrit, and higher median values for mean cell volume and red cell distribution width, were noted. Assessment of haemoglobin, red cell distribution width and calculated Mentzer ratios suggested underlying iron deficiency in 14.2% of participants. CONCLUSION: Paediatric immunohaematological reference intervals observed in this study are similar to, and support continued use of, international paediatric reference intervals. Underlying iron and related nutritional deficiencies may be contributing to lower haemoglobin levels noted in local children. A larger nationwide study, including all ethnic groups, is recommended.

Natural killer cell activation distinguishes Mycobacterium tuberculosis-mediated immune reconstitution syndrome from chronic HIV and HIV/MTB coinfection.

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 tuberculosis-related immune reconstitution inflammatory syndrome (uTB-IRIS) after ART initiation; control groups were subjects with HIV and HIV/tuberculosis-coinfected subjects with comparable ART treatment. Testing was conducted with whole blood-based 4-color flow cytometry and plasma-based Luminex cytokine assessment. RESULTS: Natural killer cell activation, C-reactive protein, and interleukin 8 serum concentration were significantly higher in uTB-IRIS subjects compared with both control groups. In addition, all MTB-coinfected subjects, independent of clinical presentation, had higher neutrophils and T-cell activation, together with lower lymphocytes, CD4⁺ T-cell, and myeloid dendritic cell counts. Using conditional inference tree analysis, we show that elevated natural killer cell activation in combination with lymphocyte count characterizes the immunological profile of uTB-IRIS. CONCLUSION: 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.

Metabolic and anthropometric parameters contribute to ART-mediated CD4+ T cell recovery in HIV-1-infected individuals: an observational study.

BACKGROUND: The degree of immune reconstitution achieved in response to suppressive ART is associated with baseline individual characteristics, such as pre-treatment CD4 count, levels of viral replication, cellular activation, choice of treatment regimen and gender. However, the combined effect of these variables on long-term CD4 recovery remains elusive, and no single variable predicts treatment response. We sought to determine if adiposity and molecules associated with lipid metabolism may affect the response to ART and the degree of subsequent immune reconstitution, and to assess their ability to predict CD4 recovery. METHODS: We studied a cohort of 69 (48 females and 21 males) HIV-infected, treatment-naïve South African subjects initiating antiretroviral treatment (d4T, 3Tc and lopinavir/ritonavir). We collected information at baseline and six months after viral suppression, assessing anthropometric parameters, dual energy X-ray absorptiometry and magnetic resonance imaging scans, serum-based clinical laboratory tests and whole blood-based flow cytometry, and determined their role in predicting the increase in CD4 count in response to ART. RESULTS: We present evidence that baseline CD4+ T cell count, viral load, CD8+ T cell activation (CD95 expression) and metabolic and anthropometric parameters linked to adiposity (LDL/HDL cholesterol ratio and waist/hip ratio) significantly contribute to variability in the extent of CD4 reconstitution (ΔCD4) after six months of continuous ART. CONCLUSIONS: Our final model accounts for 44% of the variability in CD4+ T cell recovery in virally suppressed individuals, representing a workable predictive model of immune reconstitution.

Comprehensive & cost effective laboratory monitoring of HIV/AIDS: an African role model.

We present the video about assisting anti-retroviral therapy (ART) by an apt laboratory service - representing a South-African role model for economical large scale diagnostic testing. In the low-income countries inexpensive ART has transformed the prospects for the survival of HIV seropositive patients but there are doubts whether there is a need for the laboratory monitoring of ART and at what costs - in situations when the overall quality of pathology services can still be very low. The appropriate answer is to establish economically sound services with better coordination and stricter internal quality assessment than seen in western countries. This video, photographed at location in the National Health Laboratory Services (NHLS-SA) at the Witwatersrand University, Johannesburg, South Africa, provides such a coordinated scheme expanding the original 2-color CD4-CD45 PanLeucoGating strategy (PLG). Thus the six modules of the video presentation reveal the simplicity of a 4-color flow cytometric assay to combine haematological, immunological and virology-related tests in a single tube. These video modules are: (i) the set-up of instruments; (ii) sample preparations; (iii) testing absolute counts and monitoring quality for each sample by bead-count-rate; (iv) the heamatological CD45 test for white cell counts and differentials; (v) the CD4 counts, and (vi) the activation of CD8+ T cells measured by CD38 display, a viral load related parameter. The potential cost-savings are remarkable. This arrangement is a prime example for the feasibility of performing > 800-1000 tests per day with a stricter quality control than that applied in western laboratories, and also with a transfer of technology to other laboratories within a NHLS-SA network. Expert advisors, laboratory managers and policy makers who carry the duty of making decisions about introducing modern medical technology are frequently not in a position to see the latest technical details as carried out in the large regional laboratories with huge burdens of workload. Hence this video shows details of these new developments.

A model for continuous quality control incorporating sample-to-sample assessment of optical alignment, fluorescence sensitivity, and volumetric operation of flow cytometers.

BACKGROUND: Bead count rate (BCR) monitoring successfully identifies pipetting error during single platform CD4 enumeration. Despite rigorous prescribed quality control performed, preliminary data suggested that BCR outliers could also be attributed to occasional failure of flow cytometric volumetric operation. The aim of this report was to use counting beads in a model of continuous quality control (CQC) to monitor overall flow cytometric performance (laser alignment, fluorescence stability and volumetric operation). METHODS: The proposed CQC model used FlowCheck and IMMUNOTROL blood controls daily. Extended monitoring of fluidics (FPV; beads and sheath only) and sample preparation (SPV; blood, IMMUNOPREP and beads) was done daily on five flow cytometers over five consecutive days prior to testing patient samples. Sample-to-sample CQC included monitoring BCR, selected time/fluorescence histograms (Time vs. Count; Time vs. Fluorescence and Forward Scatter vs. Fluorescence) and full peak coefficient of variation (FPCV) for 2000 samples tested. RESULTS: Prescribed quality controls showed Half Peak CV values of <2% (FlowCheck) with Immunotrol within 0.5SD of the target means. Laser stability was confirmed (FPCV values <2%). However, fluidics (volumetric operation) fluctuated as indicated by a 3.2% BCR outlier rate of 2,000 samples tested (minus pipetting error) despite optimal fluidics performance verified at start-up (FPV CV < 3%). CONCLUSIONS: Sustained laser stability was confirmed with Time vs. Fluorescence histograms, but Time vs. Count histograms were insufficient to detect intermittent volumetric failure. The proposed CQC model, incorporating BCR monitoring with time/fluorescence histograms and FPCV monitoring can identify all volumetric inconsistencies in real-time.

Local reference ranges for full blood count and CD4 lymphocyte count testing.

OBJECTIVE: Recent advances in full blood count and CD4 technology, coupled with the changing population demographics of the Gauteng region, have necessitated reevaluation of the reference ranges currently in use. METHODS: A cross-sectional study of 631 female and 88 male HIV-negative participants from the Gauteng region was performed. Full blood count, automated differential and CD4 count analyses were done using the latest internationally accepted technology. Reference ranges were compiled from the 2.5th and 97.5th percentiles for both male and female participant groups, and gender and ethnic comparisons calculated by non-parametric tests. RESULTS: Results of 41 females were removed from the statistical analysis because their results were suggestive of possible anaemia. Full blood count reference interval comparison confirmed gender-specific differences in red blood cell and platelet parameters. Ethnic-specific differences were found for some red blood cell parameters in the black female cohort. In addition, black males and females both generally had lower neutrophil and higher lymphocyte counts than a combined Asian/Caucasian/coloured ethnic group. CONCLUSION: Comparison of the currently calculated reference ranges with published data and reference values in use indicated that a separate ethnic-specific reference range should be introduced for the percentage/absolute neutrophil count and percentage lymphocytes. In addition, locally derived reference ranges for red cell distribution width (RDW) and CD4 percentage of lymphocytes should be implemented for routine diagnostic testing.

From research tool to routine test: CD38 monitoring in HIV patients.

BACKGROUND: CD38 expression on CD8+ 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 HIV+ patient monitoring. METHODS: A single-platform (SP) CD38/CD8 protocol was "piggy-backed" onto the standardized "panleucogating" CD45/CD4+ protocol. A weekly QC was established to monitor instrument stability (FlowSET) and absolute cell count accuracy and reproducibility (stabilized blood product, Immuno-Trol). The Mean Fluorescence Intensity (MFI) of CD38 expression on CD8(+)-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 HIV+ 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 (+/-100%) and precision for absolute CD4 and CD8 counts (CVs < 5%). Fluorosphere MFI- (FlowSet, FlowCount) and color compensation values were exceptionally stable over time. CD38 MFI values established on monocytes as biological control was 4.0 and <2.0 for HIV-control lymphocytes. CONCLUSIONS: Monitoring FCM with fluorosphere MFI values, color compensation, and biological controls, can ensure that CD38 analyses are technologically stable. Flow cytometry is thus the preferred method to monitor fluctuations in CD38 MFI (CD38 molecules/cell) associated with HIV-disease progression and/or response to ART and has potential for application across instruments and centers.

Large-scale affordable PanLeucogated CD4+ testing with proactive internal and external quality assessment: in support of the South African national comprehensive care, treatment and management programme for HIV and AIDS.

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 + 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 HIV+ 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: CD4+ T-cells were discriminated from monocytes without a need for CD3-staining, and the training was faster and easier for the trainees and trainers alike. The accuracy of SP-PLG was satisfactory: all PLG results submitted to the UK NEQAS were within +/-1 Trimmed Standard Deviation (SD) of the UK NEQAS CD4 Pool Trimmed Mean. Further, on the CD4 AFREQAS, the SA-NHLS laboratories using SP-PLG + BCR showed better precision (mean %CV = 7.2%) than the CD4 methods employed in other laboratories in Africa (mean %CV = 10.7%) or on other continents (mean %CV = 12.9%). PLG + BCR accommodated high workloads, exceeding 3,000 tests/laboratory/month, with capacity for further growth around 10% per month across the SA-NHLS. CONCLUSIONS: The superior performance of PLG + BCR over other methods has been demonstrated. In resource-conscious countries, where large-scale ART is being introduced, flow-cytometry using PLG + BCR can make a significant impact-due to simpler operation, easier training, stricter quality assurance, and better cost-efficiency. These cost-effective flow methods can legitimately replace the more cumbersome predicate technology of the first world for ART monitoring whilst accommodating an ever-expanding national ART program and consequent extremely high workloads reached country-wide.

CD8/CD38 activation yields important clinical information of effective antiretroviral therapy: findings from the first year of the CIPRA-SA cohort.

BACKGROUND: The affordable, reliable, and simplified flow cytometric method on single platform with 2-color (CD45+, CD4+) Panleucogating (PLG-CD4) is used to monitor HIV+ patients on antiretroviral therapy (ART) in South Africa (SA). Viral load (VL) assays are also used to monitor response to ART but are labor-intensive with costs that, in the long run, may remain unsustainable. Cheaper and quicker alternatives to VL such as CD38 tests on CD8+ T cells may therefore play a role in securing the continuation of ART programs in high-volume resource-restricted settings. METHODS: The single-tube PLG assay (CD45/CD4) was modified to use the full capacity of flow cytometers for 4-color immunofluorescence by including two more parameters: the CD38-activation marker on CD8 T-cells (CD8/CD38). This was introduced at baseline prior to the start of ART and then the changes of CD38+ mean fluorescent intensity (MFI) on CD8+ T-cells were then regularly assessed during follow-up-in comparison with the VL. RESULTS: A total of 103 patients received ART. By the end of their 4-, 8-, 12-, and 24-week observation periods, 29 (32.2%), 58 (64.4%), 74 (82.2%), and 83 (92.2%) had undetectable VL. This was also reflected by the gradual decrease of CD38 MFI expression on CD8+ T cells, irrespective of whether patients were "high," "moderate," or "low" CD38 responders at baseline. As expected, the CD4+ T cell counts substantially increased during the first 4 weeks from baseline 186 +/- 8.3 (SEM) to 267 +/- 12.3 cells/microl blood. But the recovery was slower over the rest of the 1-year follow-up to reach 334 +/- 18.2 cells/microl at week 48. As these CD4 increments were meager, the longitudinal follow-up of the continuously decreasing CD38 MFI values has become a particularly useful laboratory parameter to ascertain that the patients had indeed been responding well to ART. This monitoring protocol, in uneventful cases, may assist in reducing the frequency of VL testing. Conversely, a rise of CD38 MFI, if significantly higher than that seen at the previous visit even without fully reverting back to high baseline CD38-MFI values, provides an immediate indication for VL testing to judge whether or not the rebound of immune-activation is due to HIV VL-related irregularities. Therefore the CD8/CD38 test can provide the early, albeit not fully HIV-specific, warning signs about nonadherence to ART and/or developing drug resistance. CONCLUSION: This single-tube 4-color PLG-CD4 + CD8/38 activation assay (CD4/CD45/CD8/CD38), can replace the conventional 4-color CD4 protocols by substituting the redundant CD3 reagent with the informative CD38 antibody. This modification carries virtually no extra costs, while adding extra value to CD4 monitoring and enabling real-time, practical management of patients on ART. As a result, the numbers of VL tests required in patients on ART can be reduced-to save costs across our national treatment program which is already equipped with the necessary flow-cytometric screening capacity.