title: Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence.
abstract: The interferon-γ release assay (IGRA) is a widely used test for the presence of a cell-mediated immune (CMI) response in vitro. This measure is used to test for infection with intracellular pathogens or for validating vaccine efficacy, and it is a widely used test for both human as well as cattle. However, there is no consensus whether to use whole blood cultures or purified PBMCs for the assay, and both cell populations are being used and results compared. Therefore the aim of this study was to compare different culture settings using immune cells from previously vaccinated calves, and to shed light on external factors that could influence the read out in terms of IFN-γ levels. It was found that optimal culture conditions varied between individual animals; when polyclonal activated, cells from whole blood cultures were most responsive, but when activated specifically, the optimal cell concentration/population varied with whole blood, 10×10(6)cells/ml PBMC and 5×10(6)cells/ml PBMC being the highest performing conditions. A further investigation of the distribution of cell populations in PBMCs compared to whole blood was conducted, and a significant (p<0.001) decrease in the percentage of CD3(+) T lymphocytes within the PBMCs was found. More specifically, this reduction was due to a significant (p<0.01) decrease in the percentage of γδ(+) T lymphocytes. Thus measuring immune responses on purified PBMCs might not give a physiologically relevant output. Additionally, it was tested if the choice of incubation plate would interfere with the level of secreted IFN-γ in whole blood cultures from five calves. Six plates (a-f) were tested and no significant difference in absolute levels of IFN-γ was detected in the six plates when cells were polyclonal and specifically activated. However, we observed a significant (p<0.05) higher background level in a flat-bottom plate from Corning® (cat# 3595) (plate d) compared to two different flat-bottom plates from Corning® (cat# 3596) (plate b) and Nunc™ (cat# 167008) (plate a). Furthermore 4 out of 5 calves had maximum specific IFN-γ expression on plate b, and the relative-to-maximum level on this plate was significant (p<0.05) compared to plate a. Altogether these findings highlight the potential weaknesses of the IFN-γ release assay in terms of the many variables that can influence the results, including the cell culture population, the concentration of cells being cultured, and the plastic ware used for the in vitro culture. These findings stress the importance of documenting the precise assay conditions when publishing results of in vitro IFN-γ release assays.
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark.
authors.forename: Sofie Bruun
authors.initials: SB
authors.lastname: Hartmann
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Jenny
authors.initials: J
authors.lastname: Emnéus
authors.affiliation: Department of Micro- and Nanotechnology, Technical University of Denmark, Kgs. Lyngby, Denmark.
authors.forename: Anders
authors.initials: A
authors.lastname: Wolff
authors.affiliation: National Veterinary Institute, Technical University of Denmark, Frederiksberg C, Denmark. Electronic address: grju@vet.dtu.dk.
authors.forename: Gregers
authors.initials: G
authors.lastname: Jungersen
mesh_terms: D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D007111:Immunity, Cellular; D007371:Interferon-gamma / Q000032:analysis*; D059425:Interferon-gamma Release Tests / Q000379:methods*; D007963:Leukocytes, Mononuclear / Q000276:immunology*; D016014:Linear Models; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D007371:Interferon-gamma
keywords: Culture conditions; Interferon-gamma release assay; PBMC; Whole blood