Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence

Sofie Bruun Hartmann, Jenny Emnéus, Anders Wolff, Gregers Jungersen

Research output: Contribution to journalJournal articleResearchpeer-review

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 × 106 cells/ml PBMC and 5 × 106 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.
Original languageEnglish
JournalJournal of Immunological Methods
Volume434
Pages (from-to)24-31
ISSN0022-1759
DOIs
Publication statusPublished - 2016

Keywords

  • Immunology
  • Immunology and Allergy
  • Culture conditions
  • Interferon-gamma release assay
  • PBMC
  • Whole blood

Cite this

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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 × 106 cells/ml PBMC and 5 × 106 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{\circledR} (cat# 3595) (plate d) compared to two different flat-bottom plates from Corning{\circledR} (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.",
keywords = "Immunology, Immunology and Allergy, Culture conditions, Interferon-gamma release assay, PBMC, Whole blood",
author = "Hartmann, {Sofie Bruun} and Jenny Emn{\'e}us and Anders Wolff and Gregers Jungersen",
year = "2016",
doi = "10.1016/j.jim.2016.04.003",
language = "English",
volume = "434",
pages = "24--31",
journal = "Journal of Immunological Methods",
issn = "0022-1759",
publisher = "Elsevier",

}

Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence. / Hartmann, Sofie Bruun; Emnéus, Jenny; Wolff, Anders; Jungersen, Gregers.

In: Journal of Immunological Methods, Vol. 434, 2016, p. 24-31.

Research output: Contribution to journalJournal articleResearchpeer-review

TY - JOUR

T1 - Revisiting the IFN-γ release assay: Whole blood or PBMC cultures? - And other factors of influence

AU - Hartmann, Sofie Bruun

AU - Emnéus, Jenny

AU - Wolff, Anders

AU - Jungersen, Gregers

PY - 2016

Y1 - 2016

N2 - 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 × 106 cells/ml PBMC and 5 × 106 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.

AB - 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 × 106 cells/ml PBMC and 5 × 106 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.

KW - Immunology

KW - Immunology and Allergy

KW - Culture conditions

KW - Interferon-gamma release assay

KW - PBMC

KW - Whole blood

U2 - 10.1016/j.jim.2016.04.003

DO - 10.1016/j.jim.2016.04.003

M3 - Journal article

VL - 434

SP - 24

EP - 31

JO - Journal of Immunological Methods

JF - Journal of Immunological Methods

SN - 0022-1759

ER -