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.

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.

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.

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.