Laboratoire d'Immunologie Cellulaire, AP-HP, Hôpital Pitié-Salpêtrière, INSERM UMR S 543, Université Pierre et Marie Curie—Paris 6, Paris, France,¹ INSERM SC10, Villejuif, FranceLaboratoire d’Immunité Antivirale, AP-HP, Hôpital Kremlin-Bicêtre,,² Faculté de Médicine Paris-Sud, Université Paris XI, Kremlin-Bicêtre, France,³ INSERM U567, Hôpital Cochin, Paris, France,⁴ Unité d'Immunologie Biologique Hôpital AP-HP Européen Georges Pompidou, UMR S 255, Université Paris V René Descartes, Paris, France⁵

Received 15 November 2005/ Returned for modification 30 January 2006/ Accepted 27 March 2006

The gamma interferon (IFN-) enzyme-linked immunospot (ELISPOT) assay is a reference method for the ex vivo monitoring of antigen-specific T cells and a primary tool for assessing clinical trials of human immunodeficiency virus (HIV) or cancer vaccines. Four experienced laboratories in Paris compared their results with this method by exchanging frozen blood samples from eight HIV-seronegative and eight HIV-seropositive subjects. Each laboratory measured the IFN--producing cells specific for HIV, Epstein-Barr virus, cytomegalovirus, and influenza using the same set of peptides and the same ELISPOT reader but its own ELISPOT technique. The cutoff values for positive responses (50 or 100 spot-forming cells/10⁶ peripheral blood mononuclear cells over background) were consistent with the binomial statistic criterion. The global qualitative concordance, as assessed by the kappa index, ranged from 0.38 to 0.92, that is, moderate to excellent, and was better for non-HIV 9-mer peptide pools than for HIV 15-mer peptide pools. The interlaboratory coefficient of variation for the frequency of virus-specific T cells was 18.7% (data are expressed on a log scale). Clustering analysis of HIV-positive subjects showed qualitative agreement for ELISPOT results from all four laboratories. Overall, the good interlaboratory qualitative concordance of IFN- ELISPOT assays with only the peptide source and ELISPOT reader in common suggests that a qualitative comparison of interlaboratory findings is feasible. Nonetheless, a single set of standard operating procedures should be used in multicenter trials to improve standardization.

A.S. and C.D. contributed equally to this work.

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