Effects of storage time and temperature on highly multiparametric flow analysis of peripheral blood samples; implications for clinical trial samples

Abstract

We sought to determine the effect of time and temperature of blood sample storage before preparation of human PBMCs by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4oC before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.

Instructions for Flow Cytometric Detection of ASC Specks as a Readout of Inflammasome Activation in Human Blood

“…This also seems more reasonable in the context of multicenter studies. In contrast, when blood samples were stored at 4 • C, we observed cell clumping in the PBMC interface as previously described by Jerram et al [47] which might be caused by spontaneous platelet activation at 4 • C [48,49].…”

“…On the other hand, in many multicenter studies, the blood sample has to be sent to another location. A recent publication recommends storing whole blood rather at RT than cooling it at 4 °C for a longer period of time to minimize the negative effect on PBMCs [ 47 ]. We were able to confirm this observation, as PBMCs of sufficiently good quality could only be isolated from blood stored at RT ( Figure S2A ).…”

Peripheral B‐cell dysregulation is associated with relapse after long‐term quiescence in patients with multiple sclerosis

“…PBMCs were isolated from blood within 0–8 h of collection in EDTA vacuette tubes (Greiner Bio‐One International, Kremsmünster, Austria) using a Ficoll‐Paque PLUS (GE Healthcare, Chicago, IL) density separation gradient. When blood was not processed immediately, samples were stored at room temperature to minimize cell loss 40 . Although leaving cells at room temperature can alter receptor expression (particularly chemokine receptors), 40 variance of receptor expression between patients was comparable to internal controls, suggesting that experiment/instrument variability played a larger role than blood processing time (Supplementary figure 5).…”

“…When blood was not processed immediately, samples were stored at room temperature to minimize cell loss 40 . Although leaving cells at room temperature can alter receptor expression (particularly chemokine receptors), 40 variance of receptor expression between patients was comparable to internal controls, suggesting that experiment/instrument variability played a larger role than blood processing time (Supplementary figure 5). Cells were counted and blood volume was recorded, such that the concentration of cells in blood could be calculated.…”

Investigation of the effect of N-acetylcysteine on aluminum phosphide toxicity in rats

“…Plasma of the blood samples was obtained by the standard method using a centrifuge at 2500 rpm for 20 minute. 10 Plasma was used for the activity of antioxidant enzymes glutathione S-transferase (GST), Superoxide Dismutase (SOD), Catalase (CAT). The rats' livers and kidneys were washed with cold saline.…”