RESUMEN

Peripheral blood mononuclear cells (PBMCs) are commonly isolated from whole blood samples in clinical trials. Isolated PBMCs can be cryopreserved for use in downstream assays such as flow cytometry, single-cell RNA sequencing (scRNA-seq) and enzyme-linked immunosorbent spot (ELISpot) assays to aid understanding of disease biology and treatment effects, and biomarker identification. However, due to logistical practicalities, delays from blood collection to PBMC processing may exceed 24 h, which can potentially affect PBMC function and, ultimately, downstream assay results. Whole blood samples from 20 healthy adults were collected and incubated at 20-25 °C for 2-48 h before PBMC processing. PBMC viability was measured, and flow cytometry immunophenotyping, scRNA-seq and ELISpot were performed following increasing PBMC processing delays. The RosetteSep™ granulocyte depletion kit was used to evaluate the impact of granulocyte contamination following processing delay. Processed scRNA-seq reads were used to identify cell clusters based on marker genes. scRNA-seq data was further used to determine gene expression correlation and pathway activity score in major PBMC cell types (T cells, B cells, natural killer cells, monocytes and dendritic cells) between PBMC preparations subjected to shorter (2-4 h) and longer (8-48 h) processing delays. ELISpot assays evaluated the impact of processing delays on the number of interferon-γ (IFN-γ) secreting cells from ex vivo stimulated PBMCs. PBMC viability was reduced after a 48-h processing delay. Flow cytometry showed that granulocyte contamination of PBMCs increased after 24 h. Cluster analysis of scRNA-seq data identified 23 immune cell type gene expression clusters that were not significantly changed upon granulocyte depletion. Gene expression correlations across the major PBMC cell types were < 0.8 after 24 h of delay compared with 2 or 4 h of delay. Inflammatory, proliferation and signaling pathway activities increased, whereas IFN-γ and metabolic pathway activities decreased with increasing PBMC processing delays. The number of IFN-γ secreting cells trended towards a reduction as PBMC processing delays increased. PBMC processing delays should be minimised when designing clinical trials to reduce outcome variability in downstream assays. Ideally clinical trial sites should have on-site PBMC processing capabilities or be located close to such facilities.

Asunto(s)

RESUMEN

BACKGROUND: Standardisation of procedures for performing cellular functional assays across laboratories participating in multicentre clinical trials is key for generating comparable and reliable data. OBJECTIVE: This article describes the performance of accredited laboratories in Africa and Europe on testing done in support of clinical trials. METHODS: For enzyme-linked immunospot assay (ELISpot) proficiency, characterised peripheral blood mononuclear cells (PBMCs) obtained from 48 HIV-negative blood donors in Johannesburg, South Africa, were sent to participating laboratories between February 2010 and February 2014. The PBMCs were tested for responses against cytomegalovirus, Epstein Barr and influenza peptide pools in a total of 1751 assays. In a separate study, a total of 1297 PBMC samples isolated from healthy HIV-negative participants in clinical trials of two prophylactic HIV vaccine candidates in Kenya, Uganda, Rwanda and Zambia were analysed for cell viability, cell yield and cell recovery from frozen PBMCs. RESULTS: Most (99%) of the 1751 ELISpot proficiency assays had data within acceptable ranges with low responses to mock stimuli. No significant statistical difference were observed in ELISpot responses at the five laboratories actively conducting immunological analyses. Of the 1297 clinical trial PBMCs processed, 94% had cell viability above 90% and 96% had cell yield above 0.7 million per mL of blood in freshly isolated cells. All parameters were within the predefined acceptance criteria. CONCLUSION: We demonstrate that multiple laboratories can generate reliable, accurate and comparable data by using standardised procedures, having regular training, having regular equipment maintenance and using centrally sourced reagents.