Phenotypic and functional stability of leukocytes from human peripheral blood samples: considerations for the design of immunological studies.
Author(s): Navas A, Giraldo-Parra L, Prieto MD, Cabrera J, Gómez MA
Publication: BMC Immunol, 2019, Vol. 20, Page 5
PubMed ID: 30658588 PubMed Review Paper? No
Purpose of Paper
This paper evaluated potential effects of a room temperature delay that occurred immediately after collection of blood into EDTA tubes on the frequency of granulocytes and peripheral blood mononuclear cell (PBMC) subpopulations, PBMC viability, mRNA expression of immune-related genes, and cytokine secretion.
Conclusion of Paper
The relative frequency of monocytes, NK cells, B-cells, and granulocytes in EDTA blood was stable among specimens that experienced a delay immediately after venipuncture of 7 to 24 h at room temperature with gentle agitation compared to case-matched controls that were analyzed within 2 h of collection. However, the frequency of CD3+ T cells declined significantly from 65.06% after a 2 h delay to 53.46% after a 24 h delay at room temperature that occurred in concert with an increase in CD3- T cells, suggesting a delay-induced change rather than an effect on cell viability. A room temperature delay after collection of up to 24 h at room temperature did not alter absolute counts of viable cells or isolated PBMCs, or the percentage of viable leukocytes. However, significant and rapid changes in mRNA levels of immune-related genes in isolated PBMCs were observed during delays immediately after venipuncture. TNFα mRNA levels in PBMCs were significantly higher (>3-fold) when EDTA blood experienced a 24 h delay at room temperature after collection compared to case-matched controls that experienced a delay of 2 h, while mRNA levels of CCL8, CCR2, and CXCL10 were all significantly albeit modestly lower after a delay of ≥7 h compared to 2 h controls (p<0.001 for all). No differences in RNA integrity were observed via gel electrophoresis among delay durations. A room temperature delay after collection of EDTA blood for up 24 h did not significantly affect the concentration of cytokines secreted during subsequent PBMC culture. The authors conclude that a room temperature delay of 12 h or longer after collection of EDTA blood warrants cautious analysis of cell viability and/or function.
Studies
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Study Purpose
This study evaluated the potential effects of a room temperature delay with gentle agitation that occurred immediately after collection of blood into EDTA tubes on PBMC viability, the frequency of granulocytes and PBMC subpopulations, mRNA expression of immune-related genes, as well as the concentration of cytokines secreted during PBMC culture. Blood (100 ml) was collected from eight healthy volunteers (18-60 y) into tubes containing EDTA. Four aliquots (25 ml each) from each volunteer received gentle agitation on a rocker at 24°C for 2 h, 7 h, 12 h, or 24 h after phlebotomy. One ml of EDTA blood was then analyzed immediately by flow cytometry to determine the relative frequency of T lymphocytes (CD3+/-), B lymphocytes (CD19+), NK cells (CD56+), and monocytes (CD14+) cell subpopulations using mean fluorescence intensity and an antibody-based gating strategy. Forward and side scatter features were also used to estimate the percentage of granulocytes in each sample. The remaining portion of each aliquot was centrifuged using a Ficoll-Hypaque gradient to isolate PBMCs. PBMC viability was assessed by trypan blue staining. Total RNA was extracted from 10 million PBMCs that were centrifuged at 450 x g and resuspended and stored in TRIzol at -80°C for one week prior to reverse transcription and real-time PCR amplification with Taqman probes for immune-related genes. Gene expression was quantified using the ∆∆Ct method and expressed as a fold change relative to GAPDH and the 2 h control. Isolated PBMCs used to assess cellular function were stored in fetal bovine serum (FBS) with 10% DMSO at -80°C for 1 week. Prior to culture, cryopreserved PBMCs were thawed in a 37°C water bath and washed with RPMI with FBS. Cytokine secretion was quantified in PBMC cultured by ELISA following lipopolysaccharide stimulation.
Summary of Findings:
The relative frequency of monocytes, NK cells, B cells, and granulocytes in EDTA blood was stable among specimens that experienced a delay after collection of 7 to 24 h at room temperature with gentle agitation compared to case-matched controls that were analyzed within 2 h of collection. However, the frequency of T cells (CD3+) declined significantly from 65.06% after 2 h to 53.46% after 24 h (p<0.05). This decrease in the CD3+ T population occurred in concert with an increase in CD3- T cells suggesting a delay-induced change rather than an effect on cell viability. While a slight decline in the mean percentage of viable PBMCs was observed among EDTA blood that experienced a delay after collection of 24 h compared to 2 h controls (85% vs. ~97%), differences were not significant. In fact, PBMC viability was maintained at or above 85% for all PBMC samples except for a single sample. Further, no significant differences were observed in the percentage of viable cells among total leukocyte samples isolated from EDTA blood following a delay after venipuncture of 7-24 h compared to 2 h controls.
TNFα mRNA levels in PBMCs were significantly higher (>3-fold) when EDTA blood experienced a 24 h delay at room temperature after venipuncture compared to case-matched controls that experienced a delay of 2 h (p<0.001). Conversely, mRNA levels of CCL8, CCR2, and CXCL10 were significantly albeit modestly lower after a 7, 12, or 24 h delay after blood collection compared to 2 h controls (p<0.001 for all). A post-collection delay of up to 24 h did not induce any observable differences in RNA integrity by gel electrophoresis.
A room temperature delay post-venipuncture of up to 24 h did not significantly affect the concentration of cytokines secreted during subsequent PBMC culture. The authors note that CXCL10 secretion by PBMCs during culture displayed nonsignificant differences among EDTA blood that experienced different delays immediately after venipuncture; CXCL10 secretion was somewhat higher among specimens that experienced a 7 or 12 h delay after blood collection compared to the 2 h control, but secretion by specimens that experienced a 12 h post-collection delay was equivalent to 2 h controls.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Light scattering Cell count/volume Flow cytometry RNA Electrophoresis Cell count/volume Light microscopy RNA Real-time qRT-PCR Protein ELISA RNA Low density array Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Storage Time at room temperature 2 h
7 h
12 h
24 h
Real-time qRT-PCR Specific Targeted nucleic acid CCL8
CCL2
CXCL3
CXCL10
CCR2
CCR7
ELISA Specific Targeted peptide/protein TNFα
IL-1β
CXCL10