Defining blood processing parameters for optimal detection of cryopreserved antigen-specific responses for HIV vaccine trials.
Author(s): Bull M, Lee D, Stucky J, Chiu YL, Rubin A, Horton H, McElrath MJ
Publication: J Immunol Methods, 2007, Vol. 322, Page 57-69
PubMed ID: 17382342 PubMed Review Paper? No
Purpose of Paper
Conclusion of Paper
Studies
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Study Purpose
The purpose of this study was to determine the effects of anticoagulant, blood storage prior to PBMC isolation, and freezing media on PBMC recovery and viability. PBMC were either isolated within 8 h or after overnight shipping of blood. Following isolation and cryopreservation at -70 degrees C, all PBMC were stored in liquid nitrogen for 3-21 days.
Summary of Findings:
Specimens processed within 8 h of blood collection had greater than 94% viability, but in specimens shipped overnight recovery was 85.7-91.8%. The only exception to the reduced recovery in specimens shipped overnight was observed in specimens collected in heparin tubes and isolated with the ficoll method. The trend toward decreased recovery with overnight shipping was not significant across all methods and anticoagulants. Within each timepoint no effect of anticoagulant or isolation method was observed. In conclusion, PBMC should be isolated within 8 h of blood collection.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- AIDS/HIV-related
Platform:
Analyte Technology Platform Cell count/volume Flow cytometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Acid-citrate-dextrose
EDTA
Heparin
Biospecimen Aliquots and Components Blood and blood products Peripheral blood mononuclear cells
Analyte Extraction and Purification Analyte isolation method Ficoll method
Accuspin
Cell separation tubes
Biospecimen Preservation Fixative additive/buffer RPMI buffer with DMSO and human serum albumin
Fetal bovine serum with DMSO
Storage Between site transportation method Mailed
Not transported
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Study Purpose
The purpose of this study was to determine the effects of anticoagulant, blood storage (on a cold pack) prior to PBMC isolation, and peptide stimulation on IFN-gamma secreting cells by ELISpot and flow cytometry.
Summary of Findings:
Delay in PBMC isolation resulted in loss of IFN-gamma secreting cells by ELISpot and flow cytometry. The decrease in IFN-gamma spot forming cells (SFC) was particularly evident in low responders (<200 SFC per 1x106 cells) but also occurred in intermediate responders (200-1000 SFC per 1x106 cells) regardless of anticoagulant used. The effect of the delay in isolation was greatest when EDTA was used as an anticoagulant and was not always significant when heparin was used. No effect of isolation method was identified by ELISpot, and with flow cytometry the reduction was only significant for PBMC isolated with Accuspin tubes. The authors also state that CD4+ T cell population was lost when shipped overnight in 6 of 7 specimens. In conclusion, PBMC should be isolated within 8 h to prevent loss of IFN-gamma secreting cells particularly among low responders, regardless of anticoagulant and isolation method used.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- AIDS/HIV-related
Platform:
Analyte Technology Platform Cell count/volume Flow cytometry Protein ELISpot Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Analyte Extraction and Purification Analyte isolation method Accuspin
Cell preparation tubes
Ficoll
Storage Time at room temperature Less than 8 h
Approximately 24 h
Preaquisition Prognostic factor Strong response (more than 1000 sfc per 10^6 cells)
Intermediate response (200-1000 sfc per 10^6 cells)
Low response (less than 200 sfc per 10^6 cells)
Biospecimen Acquisition Anticoagulant Acid-citrate-dextrose
EDTA
Heparin
Biospecimen Aliquots and Components Blood and blood products Peripheral blood mononuclear cells
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Study Purpose
The purpose of this study was to determine the effects of shipping PBMC overnight in liquid nitrogen 24 h after isolation or on dry ice 24 h or 3 weeks after isolation on PBMC recovery and IFN-gamma producing cells. Different anticoagulants (ACD, sodium heparin) and isolation methods (Accuspin, Ficoll) were also investigated. All PBMC were cryopreserved at -70 degrees C for 24 h prior to shipping.
Summary of Findings:
The recovery of PBMC, but not the number of IFN gamma secreting cells was dependent on the shipping method and isolation protocol utilized. While significant, the changes observed were small and the authors believe they may not be biologically relevant. The use of liquid nitrogen resulted in decreased recovery in PBMC isolated with accuspin or ficoll methods if the PBMC were shipped after 24 h, but only in those isolated with ficoll if the cells were shipped after 3 weeks. No significant differences were observed between PBMC shipped on dry ice 24 h or 3 weeks after isolation. No effects of anticoagulant were noted. In conclusion, the method by which PBMC are shipped after isolation does not appear to significantly alter the recovery or the number of IFN-gamma secreting cells.
Biospecimens
Preservative Types
- Frozen
Diagnoses:
- AIDS/HIV-related
Platform:
Analyte Technology Platform Cell count/volume Flow cytometry Protein ELISpot Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Acquisition Anticoagulant Acid-citrate-dextrose
Heparin
Biospecimen Aliquots and Components Blood and blood products Peripheral blood mononuclear cells
Analyte Extraction and Purification Analyte isolation method Ficoll method
Accuspin
Storage Storage temperature -196 degrees C
-78 degrees C
Storage Storage duration 24 h
3 weeks