Delayed processing of blood increases the frequency of activated CD11b+ CD15+ granulocytes which inhibit T cell function.
Author(s): McKenna KC, Beatty KM, Vicetti Miguel R, Bilonick RA
Publication: J Immunol Methods, 2009, Vol. 341, Page 68-75
PubMed ID: 19041316 PubMed Review Paper? No
Suggested by: ISBER
Purpose of Paper
The purpose of this paper was to determine the effects of room temperature and refrigerated storage of blood on contamination of peripheral blood mononuclear cells (PBMC) with granulocytes.
Conclusion of Paper
Storage of blood specimens at room temperature or 8 degrees C for 22-26 hours prior to PBMC isolation caused significant increases in granulocytes. Incubation of fresh blood specimens with N-Formyl-Met-Leu-Phe (FMLP) to induce granulocyte activation also resulted in an increase in granulocytes in subsequently isolated PBMC. Contamination of PBMC with granulocytes led to inhibition of T cell function and decreased T cell proliferation which were mitigated by removal of contaminating granulocytes by flow cytometry or dilution of blood prior to storage.
Studies
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Study Purpose
The purpose of this study was to determine the effects of room temperature and refrigerated storage of blood on contamination of PBMC with granulocytes. Sodium heparin was used as an anticoagulant.
Summary of Findings:
Storage of blood specimens at room temperature for 22-26 hours prior to PBMC isolation caused a significant, rapid, and progressive increase in granulocytes ranging from 1.2 to 679 times the percentage of granulocytes observed in PBMC isolated from fresh blood (<3 hours at room temperature). Refrigeration of blood specimens at 8 degrees C for a comparable amount of time resulted in an 84-fold increase in contamination with granulocytes. Incubation of fresh blood specimens with FMLP to induce granulocyte activation also resulted in an increase in contamination with granulocytes of subsequently isolated PBMC. CD3zeta chain expression was reduced by 68% and 24% in PBMC from blood stored at room temperature and FMLP incubated blood, respectively, when compared to PBMC from fresh blood, indicating inhibition of T cell function. Further, T cell proliferation was significantly reduced in both treatment groups. Removal of contaminating granulocytes by flow cytometry restored some T cell proliferation. Dilution of blood in RPMI 1640 medium or PBS, prior to storage at room temperature, reduced granulocytes and increased T cell proliferation.
Biospecimens
Preservative Types
- Other Preservative
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Flow cytometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature Room temperature
8 degrees C
Storage Storage duration <3 h
3 h
6-8 h
12-14 h
22-26 h
Storage Storage conditions Diluted ½ in RPMI-1640 medium
Diluted ½ in PBS
Undiluted
Biospecimen Preservation Type of fixation/preservation Paraformaldehyde
Cytofix/Cytoperm solution