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Corrado Cilio

Professor

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

Author

  • Georgia Afonso
  • Matthieu Scotto
  • Amedee Renand
  • Jeanette Arvastsson
  • Dominique Vassilieff
  • Corrado Cilio
  • Roberto Mallone

Summary, in English

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.

Department/s

  • Diabetes - Immunovirology
  • EXODIAB: Excellence in Diabetes Research in Sweden

Publishing year

2010

Language

English

Pages

28-36

Publication/Series

Journal of Immunological Methods

Volume

359

Issue

1-2

Document type

Journal article

Publisher

Elsevier

Topic

  • Immunology in the medical area

Keywords

  • ELISpot
  • Peripheral blood mononuclear cells
  • T cell
  • Tetramer

Status

Published

Research group

  • Diabetes - Immunovirology

ISBN/ISSN/Other

  • ISSN: 1872-7905