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Critical parameters in blood processing for T-cell assays: Validation on ELISpot and tetramer platforms

Afonso, Georgia; Scotto, Matthieu; Renand, Amedee; Arvastsson, Jeanette LU ; Vassilieff, Dominique; Cilio, Corrado LU and Mallone, Roberto (2010) In Journal of Immunological Methods 359(1-2). p.28-36
Abstract
Assays detecting antigen (Ag)-specific T-cell responses in immune-mediated processes are increasingly employed to understand disease pathogenesis and "immune staging". The quantity and quality of starting peripheral blood mononuclear cell (PBMC) preparations are important factors in the performance of such assays. We therefore compared final PBMC yield and function by modifying parameters at the blood drawing, storage and processing steps. While drawing blood in vacuum-driven tubes or syringes and separating PBMCs on density gradients using standard or membrane (Leucosep (R)) tubes made no difference, storing tubes for 18 h without any agitation led to PBMC preparations contaminated with granulocytes and decreased interferon (IFN)-gamma... (More)
Assays detecting antigen (Ag)-specific T-cell responses in immune-mediated processes are increasingly employed to understand disease pathogenesis and "immune staging". The quantity and quality of starting peripheral blood mononuclear cell (PBMC) preparations are important factors in the performance of such assays. We therefore compared final PBMC yield and function by modifying parameters at the blood drawing, storage and processing steps. While drawing blood in vacuum-driven tubes or syringes and separating PBMCs on density gradients using standard or membrane (Leucosep (R)) tubes made no difference, storing tubes for 18 h without any agitation led to PBMC preparations contaminated with granulocytes and decreased interferon (IFN)-gamma enzyme-linked immunospot (ELISpot) responses. Even agitated blood showed a trend towards reduced ELISpot responses and increased human leukocyte Ag (HLA) multimer readouts when stored for 18 h compared to 3 h. These changes were reduced by diluting blood prior to storage. Washing PBMCs with media containing 10% human serum increased PBMC yields by 40.5%, without affecting ELISpot responses and multimer counts. However, washes with >10% human serum decreased multimer counts, with no additional improvement in PBMC yields. These findings may be relevant for optimizing and harmonizing PBMC processing procedures for T-cell assays. (C) 2010 Elsevier B.V. All rights reserved. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
ELISpot, Peripheral blood mononuclear cells, T cell, Tetramer
in
Journal of Immunological Methods
volume
359
issue
1-2
pages
28 - 36
publisher
Elsevier
external identifiers
  • wos:000280619200004
  • scopus:77954384687
ISSN
1872-7905
DOI
10.1016/j.jim.2010.05.005
language
English
LU publication?
yes
id
5736d713-7d8e-44c7-9dc3-84aab9cd6fad (old id 1678253)
date added to LUP
2010-09-21 09:49:37
date last changed
2018-05-29 11:24:22
@article{5736d713-7d8e-44c7-9dc3-84aab9cd6fad,
  abstract     = {Assays detecting antigen (Ag)-specific T-cell responses in immune-mediated processes are increasingly employed to understand disease pathogenesis and "immune staging". The quantity and quality of starting peripheral blood mononuclear cell (PBMC) preparations are important factors in the performance of such assays. We therefore compared final PBMC yield and function by modifying parameters at the blood drawing, storage and processing steps. While drawing blood in vacuum-driven tubes or syringes and separating PBMCs on density gradients using standard or membrane (Leucosep (R)) tubes made no difference, storing tubes for 18 h without any agitation led to PBMC preparations contaminated with granulocytes and decreased interferon (IFN)-gamma enzyme-linked immunospot (ELISpot) responses. Even agitated blood showed a trend towards reduced ELISpot responses and increased human leukocyte Ag (HLA) multimer readouts when stored for 18 h compared to 3 h. These changes were reduced by diluting blood prior to storage. Washing PBMCs with media containing 10% human serum increased PBMC yields by 40.5%, without affecting ELISpot responses and multimer counts. However, washes with >10% human serum decreased multimer counts, with no additional improvement in PBMC yields. These findings may be relevant for optimizing and harmonizing PBMC processing procedures for T-cell assays. (C) 2010 Elsevier B.V. All rights reserved.},
  author       = {Afonso, Georgia and Scotto, Matthieu and Renand, Amedee and Arvastsson, Jeanette and Vassilieff, Dominique and Cilio, Corrado and Mallone, Roberto},
  issn         = {1872-7905},
  keyword      = {ELISpot,Peripheral blood mononuclear cells,T cell,Tetramer},
  language     = {eng},
  number       = {1-2},
  pages        = {28--36},
  publisher    = {Elsevier},
  series       = {Journal of Immunological Methods},
  title        = {Critical parameters in blood processing for T-cell assays: Validation on ELISpot and tetramer platforms},
  url          = {http://dx.doi.org/10.1016/j.jim.2010.05.005},
  volume       = {359},
  year         = {2010},
}