Optimization of a whole blood phenotyping assay for enumeration of peripheral blood leukocyte populations in multicenter clinical trials

Abstract

Vaccination with viral vectors or adjuvants can induce early changes in circulating peripheral blood leukocytes that are predictive of a protective immune response. In this study, we define an 11- color whole blood antibody staining Trucount Panel (TP1) to enumerate and phenotype the major leukocyte populations in a human vaccine experimental medicine trial setting. TP1 can be prepared up to 8 weeks prior to use, enabling bulk preparation at a central laboratory and distribution to clinical sites. Cells in whole blood must be stained within 4 hours of draw to detect the major cell populations. Staining of cells with TP1 followed by storage and shipping at −80°C to a central laboratory has little to no effe ct on the cell concentrations observed. We also present data from an HIV vaccine multicenter clinical trial obtained using the optimized TP1 assay protocol and show that the data produced accurately correlates with complete blood count (CBC) data. Taken together, these data indicate the optimized TP1 panel assay can be used in a multicenter clinical trial setting to increase our understanding of systemic responses to vaccination or disease.

Cryopreservation-related loss of antigen-specific IFNγ producing CD4+ T-cells can skew immunogenicity data in vaccine trials: Lessons from a malaria vaccine trial substudy

“…Focus on the development of whole blood assays could contribute to reducing the influence of sample processing on immunological data from clinical trials however the reliance on established functional techniques and inherent issues with standardising cell counts in whole blood assays mean that efforts are largely restricted, certainly in flow cytometry, to phenotyping assays [33]. Efforts to reduce sample processing in the context of clinical trials can strengthen commitment to develop the capacity of trial sites where local immunology laboratories are a fundamental component, this is especially relevant in disease endemic countries where the vaccine target populations are those participating in trials.…”

Impact of granulocyte contamination on PBMC integrity of shipped blood samples: Implications for multi-center studies monitoring regulatory T cells

“…PBMCs were stained with a cocktail of 12 antibodies, shown in Table 1 to identify cell subsets in samples that were deemed to pass or fail quality control. Supplementary Figure 1 shows gating strategy to identify cell subsets, which was based on a publication by Hensley-McBain et al (Hensley-McBain et al, 2014). …”

Implementation of Highly Sophisticated Flow Cytometry Assays in Multicenter Clinical Studies: Considerations and Guidance

“…Using this freezing procedure it is possible to measure complex immunophenotyping panels including panels for B cell subsets, T cell subsets, comprehensive leukocyte subsets and rare cell population in multicenter studies. A similar approach is also described by Hensley et al [39]. This group compared the stability of samples at 4 versus -80°C and demonstrated that freezing provides a better stability than 4°C.…”

“…This group compared the stability of samples at 4 versus -80°C and demonstrated that freezing provides a better stability than 4°C. They also show that samples kept at -80°C maintain their integrity for up to 120 days for surface markers but that activation markers showed deterioration after only 13 days [39].…”