Evaluation of serological assays for the diagnosis of HIV infection in adults

Serological tests based on the enzyme immunoassay (EIA) are the primary tool for the diagnosis of human immunodeficiency virus (HIV) in adults and have rapidly evolved to quicker, affordable and more accurate test formats to detect early HIV infection. Secondand third-generation HIV rapid tests detect the immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to the HIV and are used at the point of care and in HIV self-testing. The tests are affordable and accessible in state and private diagnostic laboratories. The presentday fourth- and fifth-generation EIAs can detect both p24 antigen and IgG and IgM HIV antibodies and thereby diagnose early HIV infection at approximately 2 weeks. The fourthand fifth-generation EIAs also report sensitivity and specificity of more than 99%. The correct interpretation of HIV diagnosis of false-positive and false-negative EIA test results requires collaborative scrutiny of patient factors and laboratory test methodologies.https://safpj.co.za/index.php/safpjChemical PathologyMedical Virolog

Flow cytometric analysis of apoptotic biomarkers in actinomycin D-treated SiHa cervical cancer cells

Apoptosis biomarkers were investigated in actinomycin D-treated SiHa cervical cancer cells using a benchtop flow cytometer. Early biomarkers (Annexin V and mitochondrial membrane potential) and late biomarkers (caspases 3 and 7, and DNA damage) of apoptosis were measured in experimental and control cultures. Cultures were incubated for 24 hours in a humidified incubator at 37 °C with 5% CO2. The cells were then detached using trypsin and enumerated using a flow cytometric cell count assay. Cells were further analyzed for apoptosis using an Annexin V assay, a mitochondrial electrochemical transmembrane potential assay, a caspase 3/7 assay, and a DNA damage assay. This article provides an overview of apoptosis and traditional flow cytometry, and elaborates flow cytometric protocols for processing and analyzing SiHa cells. The results describe positive, negative, and sub-optimal experimental data. Also discussed are interpretation and caveats in performing flow cytometric analysis of apoptosis using this analytical platform. Flow cytometric analysis provides an accurate measurement of early and late biomarkers for apoptosis.The National Research Foundation (NRF) and the South African Medical Research Council (SAMRC).https://www.jove.com/journalam2022Chemical Patholog

Potential anticancer actions of cholecalciferol on a cervical squamous carcinoma cell line

Cervical cancer is the fourth most common female malignancy worldwide and is substantively higher in low-income and middle-income countries. In South Africa, cervical cancer is a leading cause of mortality amongst women. The anti-cancer actions of the vitamin D and its numerous metabolites are an active field of research. The family of vitamin D metabolites regulate numerous cellular pathways which are implicated in tumorigenesis. Pre-clinical studies and clinical studies have yielded promising, although conflicting results in various cancers. Some healthy and cancerous tissue express an autocrine vitamin D metabolising system (VDMS) which is capable of tightly regulating intracellular metabolism and growth. The VDMS expresses activating and inactivating enzymes and a vitamin D receptor (VDR). At the cellular level, the VDMS can activate and inactivate vitamin D precursors and transduce signals to the nucleus to regulate various cell health genes, including cell growth, metabolism and survival. Healthy and cancerous cervical tissue express a VDMS. The anti-cancer actions of cholecalciferol, an early precursor of activated vitamin D, is poorly studied in cervical cancer. This study aimed to characterise cholecalciferol’s action on cell growth, cell death and the VDMS in a high-grade cervical cancer cell line, SiHa. SiHa cell cultures were treated with a range of cholecalciferol doses (26 nM, 104 nM, 260 nM and 2600 nM) for 72 hours. Cell count and viability were assessed by crystal violet and trypan blue assays, respectively. Cell proliferation was enumerated by Ki67 nuclear antigen and the cell cycle profile analysed by flow cytometry. Apoptotic cell death was investigated by measuring mitochondrial membrane potential (∆Ψm), phosphatidylserine (PS) externalisation, effector caspase activation and evaluation of DNA damage markers by flow cytometric analysis. The biochemical markers microtubule-associated proteins 1A/1B light chain 3B-II (LC3-II) and lactate dehydrogenase (LDH) were also measured by flow cytometry and spectrophotometric analysis to identify autophagic cell death and necrosis, respectively. In addition, brightfield microscopy and transmission electron microscopy (TEM) were respectively used to characterise morphological and ultrastructural features of apoptosis, autophagic cell death and necrosis. The VDMS in SiHa control and experimental cultures were characterised by the investigation of intracellular gene and protein expression of the cholecalciferol activating (CYP2R1 and CYP27A1) and inactivating (CYP24A1) enzymes, and the VDR. Qualitative microscopical analysis evaluated classical characteristics of cell death and semi-quantitative analysis of apoptosis was performed. Data were analysed using a one-way ANOVA and Bonferroni post-hoc test. p < 0.05 was considered statistically significant. A significant decrease in cell count and cell viability was identified in SiHa cell cultures treated with 2600 nM cholecalciferol. Furthermore, significant increase in biochemical markers of apoptosis were identified including, decreased ∆Ψm; PS exposure; terminal caspase activation; and nuclear damage at 2600 nM cholecalciferol treatment of SiHa cell cultures. Moreover, the biochemical findings were supported by brightfield microscopy and TEM, which observed classical apoptotic features viz. membrane blebbing, apoptotic bodies and nuclear fragmentation. Also, a significantly increased number of apoptotic cells were enumerated. There was no evidence of autophagic cell death and necrosis. Additionally, a significant increase in 25-hydroxylase (CYP2R1) gene and protein expression was identified in SiHa cells treated with 2600 nM cholecalciferol. Conversely, a significant decrease in 1α-hydroxylase (CYP27B1) gene and protein expression was identified in SiHa cells treated with 2600 nM cholecalciferol. Furthermore, significant increase in both 24-hydroxylase (CYP24A1) and VDR expression at gene and protein levels were observed in 2600 nM experimental SiHa cultures. In conclusion, cholecalciferol exerts growth inhibition and apoptosis in SiHa cells at 2600 nM. This is accompanied by CYP2R1 and VDR upregulation which suggests autocrine activation to calcidiol and intracellular nuclear signalling, respectively. It is therefore hypothesised that calcidiol synthesised de novo binds to VDR and induces apoptosis in SiHa cell line.Dissertation (MSc (Chemical Pathology))--University of Pretoria, 2020.Chemical PathologyMSc (Chemical Pathology)Restricte

Policing cancer : vitamin D arrests the cell cycle

Vitamin D is a steroid hormone crucial for bone mineral metabolism. In addition, vitamin D has pleiotropic actions in the body, including anti-cancer actions. These anti-cancer properties observed within in vitro studies frequently report the reduction of cell proliferation by interruption of the cell cycle by the direct alteration of cell cycle regulators which induce cell cycle arrest. The most recurrent reported mode of cell cycle arrest by vitamin D is at the G1/G0 phase of the cell cycle. This arrest is mediated by p21 and p27 upregulation, which results in suppression of cyclin D and E activity which leads to G1/G0 arrest. In addition, vitamin D treatments within in vitro cell lines have observed a reduced C-MYC expression and increased retinoblastoma protein levels that also result in G1/G0 arrest. In contrast, G2/M arrest is reported rarely within in vitro studies, and the mechanisms of this arrest are poorly described. Although the relationship of epigenetics on vitamin D metabolism is acknowledged, studies exploring a direct relationship to cell cycle perturbation is limited. In this review, we examine in vitro evidence of vitamin D and vitamin D metabolites directly influencing cell cycle regulators and inducing cell cycle arrest in cancer cell lines.South African Medical Research Council (SAMRC)http://www.mdpi.com/journal/ijmspm2021Chemical Patholog

Variation in the measurement of anti-Mullerian hormone – what are the laboratory issues?

Anti-Müllerian Hormone (AMH) is a 140 kDa homodimeric glycoprotein consisting of two identical subunits linked by disulphide bonds and is synthesised by the testes and ovaries. Its clinical applications are prediction of ovarian response and gonadotropin dose selection upon in vitro fertilization. In males, AMH is used to investigate sexual developmental disorders and gonadal function. AMH is commonly assayed by enzymelinked immunosorbent assay or automated immunoassay formats that show variation between methods. This review applies fundamental chemical pathology concepts to explain the observed analytical variation of AMH measurement. We examine the lack of standardisation between AMH assays, the impact of antibody design on variable measurements, consider the analytical detection of AMH isoforms, review analytical interference in AMH measurement, and briefly assess systematic bias between AMH assays. The improved attempt at standardising AMH measurement by the recent approval of a WHO Reference Reagent offers promise for harmonising immunoassay results and establishing consensus medical cut-off points for AMH in disease. Standardisation, however, will need to redress the issue of poor commutability of standard reference material and further assign a standard reference procedure to quantify AMH standard reference material. The improvement of the analytical phase of AMH testing will support harmonised method development and patient care.http://www.frontiersin.org/Endocrinologyam2022Chemical Patholog

Policing Cancer: Vitamin D Arrests the Cell Cycle

Vitamin D is a steroid hormone crucial for bone mineral metabolism. In addition, vitamin D has pleiotropic actions in the body, including anti-cancer actions. These anti-cancer properties observed within in vitro studies frequently report the reduction of cell proliferation by interruption of the cell cycle by the direct alteration of cell cycle regulators which induce cell cycle arrest. The most recurrent reported mode of cell cycle arrest by vitamin D is at the G1/G0 phase of the cell cycle. This arrest is mediated by p21 and p27 upregulation, which results in suppression of cyclin D and E activity which leads to G1/G0 arrest. In addition, vitamin D treatments within in vitro cell lines have observed a reduced C-MYC expression and increased retinoblastoma protein levels that also result in G1/G0 arrest. In contrast, G2/M arrest is reported rarely within in vitro studies, and the mechanisms of this arrest are poorly described. Although the relationship of epigenetics on vitamin D metabolism is acknowledged, studies exploring a direct relationship to cell cycle perturbation is limited. In this review, we examine in vitro evidence of vitamin D and vitamin D metabolites directly influencing cell cycle regulators and inducing cell cycle arrest in cancer cell lines

Laboratory considerations for reporting cycle threshold value in COVID-19

The Coronavirus Disease 2019 (COVID-19) pandemic is caused by the SARS-CoV-2 RNA virus. Nucleic acid amplification testing (NAAT) is the mainstay to confirm infection. A large number of reverse transcriptase polymerase chain reaction (RT-PCR) assays are currently available for qualitatively assessing SARS-CoV-2 infection. Although these assays show variation in cycle threshold values (Ct), advocacy for reporting Ct values (in addition to the qualitative result) is tabled to guide patient clinical management decisions. This article provides critical commentary on qualitative RT-PCR laboratory and clinical considerations for Ct value reporting. Factors contributing to Ct variation are discussed by considering relevant viral life-cycle factors, patient factors and the laboratory total testing processes that contribute to the Ct variation and mitigate against the reporting of Ct values by qualitative NAAT.https://ifcc.org/ifcc-communications-publications-division-cpd/ifcc-publications/ejifcc-journalhj2023Chemical PathologyMedical VirologySDG-03:Good heatlh and well-bein

A case study comparing the flipped hybrid classroom and traditional classroom in a post-graduate chemical pathology module

[EN] This case study compared student performance and experiences in a flipped hybrid classroom (FHC) and a traditional classroom (TC) in a post-graduate chemical pathology programme. Nine students participated in the study. The final summative grades based on clinical case vignettes assessing high cognitive domains were 55.83% (±26.94) and 60.61% (±36.02) for the FHC and TC, respectively. Students obtained higher scores in the synthesis domain in the FHC compared to the TC. In contrast, higher scores were obtained in evaluating pathophysiology and biochemical test results in the TC. The thematic analysis of the open-ended questions identified three themes: (1) flipping is fun and informative; (2) TC is better with a bit of flip; and (3) we know what we like.This study showed that the TC showed slightly better summative assessment performance, and that students are positive about flipped approaches but have their own preferences.Punchoo, R.; Bhoora, S.; Wolvaardt, L. (2022). A case study comparing the flipped hybrid classroom and traditional classroom in a post-graduate chemical pathology module. En 8th International Conference on Higher Education Advances (HEAd'22). Editorial Universitat Politècnica de València. 39-46. https://doi.org/10.4995/HEAd22.2022.14521394

Evaluation of serological assays for the diagnosis of HIV infection in adults

Serological tests based on the enzyme immunoassay (EIA) are the primary tool for the diagnosis of human immunodeficiency virus (HIV) in adults and have rapidly evolved to quicker, affordable and more accurate test formats to detect early HIV infection. Second- and third-generation HIV rapid tests detect the immunoglobulin G (IgG) and immunoglobulin M (IgM) antibodies to the HIV and are used at the point of care and in HIV self-testing. The tests are affordable and accessible in state and private diagnostic laboratories. The present-day fourth- and fifth-generation EIAs can detect both p24 antigen and IgG and IgM HIV antibodies and thereby diagnose early HIV infection at approximately 2 weeks. The fourth- and fifth-generation EIAs also report sensitivity and specificity of more than 99%. The correct interpretation of HIV diagnosis of false-positive and false-negative EIA test results requires collaborative scrutiny of patient factors and laboratory test methodologies

Cholecalciferol inhibits cell growth and induces apoptosis in the CaSki cell line

Vitamin D has displayed anti-cancer actions in numerous in vitro studies. Here, we investigated the anti-cancer actions of cholecalciferol, a vitamin D precursor, on a metastatic cervical cancer cell line, namely, CaSki. Experimental cultures were incubated for 72 h and treated with cholecalciferol (10–1000 ng/mL). In the present study, cell count, viability, proliferation and cell cycle were analyzed by a crystal violet assay, trypan blue assay, Ki67 proliferation, and a cell cycle assay, respectively. Biomarkers of apoptosis, necrosis, and autophagic cell death were measured by the Caspase 3/7 and Annexin V/7-AAD Muse™ assays, a LC3-II assay, and a lactate dehydrogenase release assay, respectively. The ultrastructural features of cell death were assessed by transmission electron microscopy. A statistical analysis was performed using a one-way ANOVA and Bonferroni’s post-hoc analysis test, and p < 0.05 is considered statistically significant here. The results identify statistical decreases in cell count and viability at high-dose treatments (100 and 1000 ng/mL). In addition, significant increases in apoptotic biochemical markers and apoptotic ultrastructure are shown to be present at high-dose treatments. In conclusion, high-dose cholecalciferol treatments inhibit cell count and viability, which are both mediated by apoptotic induction in the CaSki cell lineNational Research Foundation (NRF); Research Committee (School of Medicine) of the University of Pretoria (RESCOM); Research Development Program from the University of Pretoria and South African Medical Research Council.http://www.mdpi.com/journal/medscihj2021Chemical PathologyPhysiolog