Luminex-based virtual crossmatching for renal transplantation in South Africa

Background. Current practice in the Johannesburg renal transplantation programme is to perform a transplant when the patient’s complement-dependent cytotoxicity and flow cytometric crossmatches are negative. However, even in patients with negative crossmatches early graft rejections have occurred. We retrospectively evaluated the use of Luminex anti-human leukocyte antigen (HLA) antibody detection technology, often termed ‘virtual crossmatching’, compared with the flow cytometric crossmatch, for predicting graft outcome in renal transplant patients. Methods. Sixty-four recipients were crossmatched against multiple donors during their routine work-up for transplant (111 crossmatches); 17 of these patients received transplants during the study period. Anti-HLA antibody detection was performed using Luminex technology and the results were compared with the flow cytometric crossmatch results and with short-term graft success. Results. Compared with flow cytometric crossmatch results, the sensitivity and specificity of Luminex virtual crossmatching was 85.7% and 90.7% for the T-cell crossmatch and 100% and 87.2% for the B-cell crossmatch. Both the sensitivity and specificity of Luminex for predicting short-term graft success were 100%. Conclusions. Strong evidence is provided that single-antigen assays provide improved sensitivity to detect clinically relevant anti-HLA antibodies and can reliably be used to predict shortterm graft success. We recommend incorporation of single-antigen Luminex methodology into the routine work-up algorithm of renal transplant recipients in South Africa.S Afr Med J, 2012;102:40-4

Inducing apoptosis using chemical treatment and acidic pH, and detecting it using the Annexin V flow cytometric assay

Cell death is important in physiology, and can happen as a result of structural damage, or as a sequence of programmed cellular processes known as apoptosis. Pathogenic alterations in apoptosis occur in a number of diseases, including cancer, viral infections, autoimmune diseases, immunodeficiencies, and degenerative conditions. Developing accurate and reproducible laboratory methods for inducing and detecting apoptosis is vital for research into these conditions. A number of methods are employed to detect cell death, including DNA fragmentation, the TUNEL assay, and electron microscopy although each has its limitations. Flow cytometry allows for the distinction between live, early apoptotic, late apoptotic and necrotic cells. In this protocol we successfully induce apoptosis using chemical treatment and treatment with low pH in solid tumour cell lines, and have optimized detection using the Annexin V/PI apoptosis assay.The National Research Foundation (NRF) Thuthuka program.http://www.plosone.orgdm2022Immunolog

The acidic tumour microenvironment : manipulating the immune response to elicit escape

The success of cancer treatment relies on the composition of the tumour microenvironment which is comprised of tumour cells, blood vessels, stromal cells, immune cells, and extracellular matrix components. Barriers to effective cancer treatment need to be overcome, and the acidic microenvironment of the tumour provides a key target for treatment. This review intends to provide an overview of the effects that low extracellular pH has on components of the tumour microenvironment and how they contribute to immune escape. Further, potential therapeutic targets will be discussed.The National Research Foundation (NRF) Thuthuka program.http://www.elsevier.com/locate/humimmhj2023Immunolog

FOXP3 Expression Is Upregulated in CD4+T Cells in Progressive HIV-1 Infection and Is a Marker of Disease Severity

Understanding the role of different classes of T cells during HIV infection is critical to determining which responses correlate with protective immunity. To date, it is unclear whether alterations in regulatory T cell (Treg) function are contributory to progression of HIV infection.FOXP3 expression was measured by both qRT-PCR and by flow cytometry in HIV-infected individuals and uninfected controls together with expression of CD25, GITR and CTLA-4. Cultured peripheral blood mononuclear cells were stimulated with anti-CD3 and cell proliferation was assessed by CFSE dilution.HIV infected individuals had significantly higher frequencies of CD4(+)FOXP3(+) T cells (median of 8.11%; range 1.33%-26.27%) than healthy controls (median 3.72%; range 1.3-7.5%; P = 0.002), despite having lower absolute counts of CD4(+)FOXP3(+) T cells. There was a significant positive correlation between the frequency of CD4(+)FOXP3(+) T cells and viral load (rho = 0.593 P = 0.003) and a significant negative correlation with CD4 count (rho = -0.423 P = 0.044). 48% of our patients had CD4 counts below 200 cells/microl and these patients showed a marked elevation of FOXP3 percentage (median 10% range 4.07%-26.27%). Assessing the mechanism of increased FOXP3 frequency, we found that the high FOXP3 levels noted in HIV infected individuals dropped rapidly in unstimulated culture conditions but could be restimulated by T cell receptor stimulation. This suggests that the high FOXP3 expression in HIV infected patients is likely due to FOXP3 upregulation by individual CD4(+) T cells following antigenic or other stimulation.FOXP3 expression in the CD4(+) T cell population is a marker of severity of HIV infection and a potential prognostic marker of disease progression

Age, Disease Severity and Ethnicity Influence Humoral Responses in a Multi-Ethnic COVID-19 Cohort

The COVID-19 pandemic has affected all individuals across the globe in some way. Despite large numbers of reported seroprevalence studies, there remains a limited understanding of how the magnitude and epitope utilization of the humoral immune response to SARS-CoV-2 viral anti-gens varies within populations following natural infection. Here, we designed a quantitative, multi-epitope protein microarray comprising various nucleocapsid protein structural motifs, including two structural domains and three intrinsically disordered regions. Quantitative data from the microarray provided complete differentiation between cases and pre-pandemic controls (100% sensitivity and specificity) in a case-control cohort (n = 100). We then assessed the influence of disease severity, age, and ethnicity on the strength and breadth of the humoral response in a multi-ethnic cohort (n = 138). As expected, patients with severe disease showed significantly higher antibody titers and interestingly also had significantly broader epitope coverage. A significant increase in antibody titer and epitope coverage was observed with increasing age, in both mild and severe disease, which is promising for vaccine efficacy in older individuals. Additionally, we observed significant differences in the breadth and strength of the humoral immune response in relation to ethnicity, which may reflect differences in genetic and lifestyle factors. Furthermore, our data enabled localization of the immuno-dominant epitope to the C-terminal structural domain of the viral nucleocapsid protein in two independent cohorts. Overall, we have designed, validated, and tested an advanced serological assay that enables accurate quantitation of the humoral response post natural infection and that has revealed unexpected differences in the magnitude and epitope utilization within a population

Luminex-based virtual crossmatching for renal transplantation in South Africa

Background. Current practice in the Johannesburg renal transplantation programme is to perform a transplant when the patient’s complement-dependent cytotoxicity and flow cytometric crossmatches are negative. However, even in patients with negative crossmatches early graft rejections have occurred. We retrospectively evaluated the use of Luminex anti-human leukocyte antigen (HLA) antibody detection technology, often termed ‘virtual crossmatching’, compared with the flow cytometric crossmatch, for predicting graft outcome in renal transplant patients. Methods. Sixty-four recipients were crossmatched against multiple donors during their routine work-up for transplant (111 crossmatches); 17 of these patients received transplants during the study period. Anti-HLA antibody detection was performed using Luminex technology and the results were compared with the flow cytometric crossmatch results and with short-term graft success. Results. Compared with flow cytometric crossmatch results, the sensitivity and specificity of Luminex virtual crossmatching was 85.7% and 90.7% for the T-cell crossmatch and 100% and 87.2% for the B-cell crossmatch. Both the sensitivity and specificity of Luminex for predicting short-term graft success were 100%. Conclusions. Strong evidence is provided that single-antigen assays provide improved sensitivity to detect clinically relevant anti-HLA antibodies and can reliably be used to predict short-term graft success. We recommend incorporation of single-antigen Luminex methodology into the routine work-up algorithm of renal transplant recipients in South Africa

Pathogenic factors associated with development of disseminated intravascular coagulopathy (DIC) in a tertiary academic hospital in South Africa.

Disseminated intravascular coagulopathy (DIC) is a thrombotic microangiopathy arising from consumption of both coagulation factors and platelets. DIC is triggered by a number of clinical conditions including severe infection, trauma and obstetric complications. Early diagnosis and treatment of the underlying condition is paramount. A high clinical index of suspicion is needed to ensure that patients at risk of developing DIC are appropriately investigated.In order to establish the clinical conditions most frequently associated with DIC, we reviewed all DIC screens received at a tertiary hospital in Johannesburg, South Africa over a 1 year period.The commonest clinical condition associated with DIC in our population was infection with 84% of patients infected with an identified pathogen. The most frequently diagnosed pathogen was HIV followed by Mycobacterium tuberculosis and other bacterial infections. In the majority of cases, bacteria were isolated from blood cultures. In 47 patients, HIV was the only pathogen which could be isolated. A relative risk ratio of 2.73 and an odds ratio of 29.97 was attributed to HIV for development of a DIC. A malignancy was present in 51 of the patients of which approximately 60% had co-existing infection. No cause could be attributed in 30 patients.Infection was identified in the majority of the patients diagnosed with DIC in this study. HIV showed the highest relative risk ratio of all pathogens although previous studies have not suggested that HIV was strongly associated with DIC. In almost half of the HIV infected patients, there was no other pathogen isolated despite extensive investigation. This suggests that HIV has a strong association with the development of DIC, warranting further research into the relationship between HIV and disseminated microvascular thrombosis

The effect of freezing and thawing of samples for anti-factor Xa testing for the determination of enoxaparin activity

The quality and validity of laboratory results, especially for coagulation analyses, are influenced by pre-analytical variables including the collection technique, transportation, pre-analytical processing and storage of samples. Laboratory networks often rely on centralization of specialized testing, which necessitates on-site preparation of samples and transportation to referral centres. Low molecular weight heparin (LMWH) offers improved clinical predictability of anticoagulation and often does not require routine monitoring.http://www.wileyonlinelibrary.com/journal/ijlhhj2021Haematolog

The effect of freezing and thawing of samples for anti‐factor Xa testing for the determination of enoxaparin activity.

The quality and validity of laboratory results, especially for coagulation analyses, are influenced by pre-analytical variables including the collection technique, transportation, pre-analytical processing and storage of samples. Laboratory networks often rely on centralization of specialized testing, which necessitates on-site preparation of samples and transportation to referral centres. Low molecular weight heparin (LMWH) offers improved clinical predictability of anticoagulation and often does not require routine monitoring.http://www.wileyonlinelibrary.com/journal/ijlhhj2021Haematolog