Stabilization of Human Whole Blood Samples for Multicenter and Retrospective Immunophenotyping Studies

Abstract

Whole blood is often collected for large‐scale immune monitoring studies to track changes in cell frequencies and responses using flow (FC) or mass cytometry (MC). In order to preserve sample composition and phenotype, blood samples should be analyzed within 24 h after bleeding, restricting the recruitment, analysis protocols, as well as biobanking. Herein, we have evaluated two whole blood preservation protocols that allow rapid sample processing and long‐term stability. Two fixation buffers were used, Phosphoflow Fix and Lyse (BD) and Proteomic Stabilizer (PROT) to fix and freeze whole blood samples for up to 6 months. After analysis by an 8‐plex panel by FC and a 26‐plex panel by MC, manual gating of circulating leukocyte populations and cytokines was performed. Additionally, we tested the stability of a single sample over a 13‐months period using 45 consecutive aliquots and a 34‐plex panel by MC. We observed high correlation and low bias toward any cell population when comparing fresh and 6 months frozen blood with FC and MC. This correlation was confirmed by hierarchical clustering. Low coefficients of variation (CV) across studied time points indicate good sample preservation for up to 6 months. Cytokine detection stability was confirmed by low CVs, with some differences between fresh and fixed conditions. Thirteen months regular follow‐up of PROT samples showed remarkable sample stability. Whole blood can be preserved for phenotyping and cytokine‐response studies provided the careful selection of a compatible antibody panel. However, possible changes in cell morphology, differences in antibody affinity, and changes in cytokine‐positive cell frequencies when compared to fresh blood should be considered. Our setting constitutes a valuable tool for multicentric and retrospective studies. © 2020 International Society for Advancement of Cytometry

Protocol for large scale whole blood immune monitoring by mass cytometry and Cyto Quality Pipeline

“…Confirm that the targeted antigens are resistant to the fixation: the pattern of the expression is preserved, as shown in Figure 4 and in ( Rybakowska et al., 2021a ).…”

“…The stimulation agents were the toll-like receptor agonists LPS, ODN2006 (CpG), R848 (RSQ), R837 (IMQ), and medium alone (UNS). These were prepared at 2× concentration in RPMI, and the final blood stimulation was performed at 1× concentration in a final volume of 1 mL (500 μL of stimuli + 500 μL of blood) in sterile conditions as in ( Rybakowska et al., 2021a ). Prepare stimuli.…”

“…We recommend to prepare at least one extra aliquot of antibody cocktail in case needed, thus in this example antibody cocktail is calculated for a total of 4 experiments. Two separate cocktails for cell surface and intracellular staining were prepared, as in ( Rybakowska et al., 2021a ). Calculate the amount of antibody needed.…”

“…We then detail the use of barcoding and reference samples to facilitate multicenter and multi-batch experiments. For complete details on the use and execution of this protocol, please refer to Rybakowska et al. (2021a ) and ( 2021b ).…”

“…For complete details on the use and execution of this protocol, please refer to Rybakowska et al. (2021a ) and ( 2021b ).…”