Processing of blood samples influences PBMC viability and outcome of cell-mediated immune responses in antiretroviral therapy-naïve HIV-1-infected patients.
Author(s): Bourguignon P, Clément F, Renaud F, Le Bras V, Koutsoukos M, Burny W, Moris P, Lorin C, Collard A, Leroux-Roels G, Roman F, Janssens M, Vandekerckhove L
Publication: J Immunol Methods, 2014, Vol. 414, Page 1-10
PubMed ID: 25224748 PubMed Review Paper? No
Purpose of Paper
This paper investigated how the duration of a delay to processing, pre-stimulation resting period, and stimulation period and method may affect lymphocyte recovery, viability, and response to HIV (human immunodeficiency virus) peptide pools in peripheral blood mononuclear cells (PBMCs) and whole blood from HIV-positive patients.
Conclusion of Paper
Optimal lymphocyte recovery was observed when a processing delay was limited to 2 h and a post-thaw resting period was avoided, while optimal viability was observed when a processing delay was limited to 2 h followed by a 6.5 h post-thaw resting period. Time to processing and post-thaw time to stimulation had no effect on the magnitude of the response from CD8+ T-cells, but the antigen-specific response was higher when stimulation was overnight rather than 6 h; the magnitude antigen-specific response also depended on the peptide used for stimulation. The HIV-specific CD8+ T-cell response was comparable after stimulation for 6 h or overnight, but differed between cytokines analyzed after a processing delay of 7 h. The author’s state HIV-specific responses of CD40L+ and CD4+ T cells expressing one or more cytokines were too low for an observable effect of processing delay or post-thaw resting period. There were no significant correlations between inflammatory markers and viability/T-cell mediated immune responses. Strong correlations for CD8+ T-cell responses against HIV antigens were observed between whole blood and PBMCs following stimulation with Nef, reverse transcriptase (RT), and P24.
Studies
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Study Purpose
This study investigated how the duration of a delay to processing, pre-stimulation resting period, and stimulation period and method may affect lymphocyte recovery, viability, and response to HIV peptide pools in PBMCs and whole blood from HIV-positive patients. Blood from 22 HIV patients who were not eligible for antiretroviral therapywas collected in lithium heparin tubes. Blood was stored and transported at room temperature for 2, 7, or 24 h before isolation of PBMCs by Lymphoprep™ gradient. PBMCs were counted by flow cytometry and frozen in liquid nitrogen. Specimens were stored in liquid nitrogen for 9-23 weeks and then rapidly thawed and counted again. PBMCs were allowed to rest for 0, 2, 6, or 18 h at room temperature before stimulation with HIV peptide pools (reverse transcriptase, Nef, p24, or p17) or medium-only controls for 2 h at 37˚C. After stimulation, brefeldin A was added and PBMCs were incubated at 37˚C for 4 h or overnight before intracellular cytokine staining with antibodies to CD3, CD40L, IL-2, IFN-γ, and TNF-α. Fresh whole blood was stimulated 2 or 4 h after collection using the same method as for PBMCs.
Summary of Findings:
Storage of blood before PBMC isolation had little effect on the percentage of PBMCs recovered, but storage of thawed PBMCs for >2 h before stimulation resulted in <50% recovery. The authors calculated that recovery was optimal (81.5%) when there the delay to processing was limited to 2 h, and when specimens were stimulated immediately after thawing. Lymphocyte recovery increased slightly following a 24 h delay to processing when there was an 18 h delay that occurred after thawing but before stimulation. Interestingly, these increases were not accompanied by changes in the proportion of granulocytes or large mononuclear cells. Optimal cell viability (87.5%) was achieved when the processing delay was limited to 2 h and the delay to stimulation was 6.5 h, although effects were modest with 82.9% viability observed after a 7 h processing delay and no resting time.
Delays before processing and post-thaw but before stimulation had no effect on the magnitude of response from CD8+ T-cells expressing interleukin (IL)-2, interferon (IFN)-γ, or tumor necrosis factor (TNF)-α to reverse transcriptase (RT), but the antigen-specific response was higher when stimulation was overnight rather than 6 h. The authors report similar findings after stimulation with Nef, p24, or p17 peptides, despite a modestly higher response to response to Nef in comparison to that obtained in response to p24 and p17. The HIV-specific CD8+ T-cell response was comparable after a 6 h or overnight stimulation, but differed between cytokines assayed after a processing delay of 7 h. The authors state HIV-specific responses of CD40L+ and CD4+ T cells expressing one or more cytokines were low compared to that of CD8+ T-cells and that there were no significant correlations between inflammatory markers and viability or T-cell mediated immune responses.
Similar to PBMCs, when whole blood was used there was no effect of a 2 or 4 h delay to stimulation on HIV-specific CD8+ T-cell response for each of the antigens, and the responses of CD40L+ and CD4+ T cells were too low for an effect to be observed. Finally, strong correlations among CD8+ T-cell responses against HIV antigens were observed between whole blood and PBMCs following stimulation with NEF, RT, and P24 but the response in PBMCs was lower than observed with whole blood for P17.
Biospecimens
Preservative Types
- Frozen
- None (Fresh)
Diagnoses:
- AIDS/HIV-related
Platform:
Analyte Technology Platform Cell count/volume Flow cytometry Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Biospecimen Aliquots and Components Centrifugation Centrifugation delays investigated
Biospecimen Aliquots and Components Blood and blood products Peripheral blood mononuclear cells
Whole blood
Storage Time at room temperature 2 h
7 h
24 h
0 h
6 h
18 h