Effect of storage time on peripheral blood mononuclear cell isolation from blood collected in vacutainer CPT™ tubes.
Author(s): Linggi B, Cremer J, Wang Z, Van Viegen T, Vermeire S, Lefevre P, Shackelton LM, Jairath V, Teft W, Vande Casteele N, Verstockt B
Publication: J Immunol Methods, 2023, Vol. 519, Page 113504
PubMed ID: 37257687 PubMed Review Paper? No
Purpose of Paper
This paper compared peripheral blood mononuclear cell (PBMC) viability and T-cell composition among PBMCs isolated from blood that was centrifuged and then stored in Cell Preparation Tubes (CPT) for 24 to 96 h at room temperature prior to PBMC isolation and analysis.
Conclusion of Paper
Cell number and viability was highly variable among the specimens examined, but viability was >75% in all specimens when PBMCs were isolated immediately after blood specimen centrifugation. Cell viability was significantly lower in PBMCs from blood stored at room temperature for ≥ 24 h after centrifugation than in PBMCs that were isolated immediately after centrifugation. High coefficients of variance (CV) (>25%) only occurred for T-cell types that made up less than 5% of the total T-cell population. No overall trends were observed in terms of a change in cell type abundance with delayed isolation, but larger changes were often noted with longer delays. Normalization of cell type counts to the other T-cell types counts reduced the observed variability with delayed PBMC isolation.
Studies
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Study Purpose
This study compared PBMC viability and T-cell composition among PBMC isolated from blood that was centrifuged and then stored in CPT for 24 to 96 h at room temperature prior to analysis. Blood from three patients diagnosed with ulcerative colitis was collected into CPT. Blood specimens were then centrifuged (duration and speed not specified) within 2 h of venipuncture and matched tubes were stored at room temperature for 0, 24, 48, 72 and 96 h before PBMC isolation following the tube manufacturer’s protocol. PBMCs were resuspended in cryopreservation media (90% fetal bovine serum and 10% DMSO) and frozen in a Corning CoolCell at -80°C for 24 h before storage in liquid nitrogen. Cells were thawed in a 37°C water bath and resuspended in Hank’s balanced salt solution (HBSS) with 0.5% bovine serum albumin, pelleted by centrifugation at 300 g for 10 min and resuspended in HBSS with DNase. Red blood cells were lysed by incubation with ACK red blood cell lysing solution at room temperature for 10 min, and PBMCs were pelleted and resuspended in phosphate buffered saline. Cell viability was assessed by trypan blue dye exclusion. PBMCs were stained with antibodies against and enumerated using a BD LSR Fortessa SORP cell analyzer with FACSDiva v8.0 software.
Summary of Findings:
The number and viability of PBMCs were highly variable among the blood specimens and storage durations evaluated, but viability was >75% in all specimens when PBMCs were isolated immediately after centrifugation. PBMC viability was significantly lower when PBMCs were isolated from blood that was centrifuged and then stored at room temperature for ≥ 24 h than when PBMCs were isolated immediately after centrifugation (P<0.01, all timepoints). The CV was >25% for counts of 10 of the 26 cell types evaluated in replicate specimens from one patient, but only two cell types (T helper 17 and T helper 1 like T follicular helper cells) had CVs >25% in blood specimens from the remaining two patients. The cell types with the lowest technical variability were CD4+ (CV = 1.91%), effector memory (TEM) CD8+ (CV = 2.13%), effector CD8+ (CV = 4.22%), naive CD4+ (CV = 4.54%), CD8+ (CV = 4.82%), central memory (TCM) CD8 + (CV = 4.98) and naive effector CD8+ (CV = 4.95) T-cells. Importantly, high variability (CV >25%) was only observed for T-cell types that accounted for less than 5% of the total T-cells quantified. No overall trends were observed in the change of cell-type abundance with delayed isolation, but larger changes in individual cell types were often noted with longer delays. Importantly, when isolation was delayed by ≥72 h, counts of some cell types fell below 20, which the authors state is approaching the limit of detection. Counts of CD4+, CD8+, naïve effector CD8+, and TCM CD4 + T cells changed by <50% at all timepoints examined in specimens from all three patients. Normalization of cell type counts to the other T-cell types counts reduced the observed variability with delayed PBMC isolation.
Biospecimens
Preservative Types
- None (Fresh)
Diagnoses:
- Ulcerative Colitis
Platform:
Analyte Technology Platform Cell count/volume Light microscopy Cell count/volume Flow cytometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Time at room temperature 0 h
24 h
48 h
72 h
96 h