Purpose: The Intracellular cytokine staining (ICS) assay is a flow cytometric assay designed to identify cytokine-producing antigen-specific T cells in a mixed and highly heterogeneous sample. The assay relies on the ex vivo stimulation of the sample in the presence of protein transport inhibitors, leading to the retention of produced cytokines within the cell, enabling their detection by flow cytometry. Antigen-specific T cells in a sample can be identified through cytokine expression/production.
“Fit-for-purpose” validation of a method involves experimental testing to establish that assay performance characteristics meet intended use of the data and the associated regulatory requirements. The validation enables the establishment of prospective acceptance criteria that will be applied on clinical sample analysis.
Methods: We performed a “fit-for-purpose” analytical method validation following Good Laboratory Practices (GLP) regulations of an ICS assay intended for use as a secondary endpoint in a clinical trial. The assay validation was performed on CMV pp65 peptide pool-stimulated PBMC from two prescreened CMV seropositive healthy donors. Data from one healthy donor are presented herein. Reportable results of the ICS assay that underwent assay validation included three cytokines (IFNγ, IL2 and TNFα), expressed either alone or in combination and analyzed using the Boolean approach. Multiple assay parameters were validated, namely specificity, precision, linearity, lower limit of quantification (LLOQ), limit of detection (LOD), and stained sample stability.
Results: Four CD4+ T cell response reportable results (single or triple cytokine producers) were used to assess assay performance. Cytokine-producing antigen-specific CD4+ T cell frequencies exhibited acceptable inter-instrument, inter-operator, intra- and inter-assay precision (%CV<30%).
Conclusion: The LLOQ, defined as the last consecutive dilution that can be measured with acceptable bias and CV, was signature-specific and ranged between 0.01-0.05% in cytokine-producing CD4+ T cells. The LOD was determined using the isotype panel and determined to be lower than the LLOQ. Expression of cytokines of antigen-specific CD4+ T cells was detectable 24hr following staining procedure.
Genevieve Levesque– Lead Research Assistant, CAPRION BIOSCIENCES INC., Quebec
Chanel Cadieux-Dion– Research Assistant, CAPRION BIOSCIENCES INC., Quebec
Clémence Meunier– Research Assistant, CAPRION BIOSCIENCES INC., Quebec
Jo-Annie Fontaine– Research Assistant, CAPRION BIOSCIENCES INC., Quebec
Gloria Giono Chiang– Research Assistant, CAPRION BIOSCIENCES INC., Quebec
Bader Yassin Diab– Lead Scientist, CAPRION BIOSCIENCES INC., Quebec
Sandrina Da Fonseca– Manager, Data Analysis Unit, CAPRION BIOSCIENCES INC., Quebec
Jean-François Poulin– Associate Director, CAPPION BIOSCIENCES INC., Quebec
Michael Schirm– Associate Director, R&D Proteomics, Caprion Biosciences Inc., Montreal, Quebec