The ratio of TH1:TH2 cytokines in the cytoplasm of CD3+CD4+ lymphocytes can be determined by flow cytometry. Lymphocytes (mononuclear cells) are stimulated with phorbol myristate acetate (PMA) and ionomycin in the presence of GolgiPlug (brefeldin A), a Golgi transport inhibitor, and IVIg (a preparation of immunoglobulin derived from human plasma) for 18 hours. The cells are then harvested, permeabilized, and stained with phycoerythrin-conjugated antibodies against TNF-α, IFN-γ, or IL-10. Four-color flow cytometry is used to measure the percentages of intracellular cytokines in CD3+CD4+ lymphocytes.
Stimulation down-regulates CD4 expression on the cell surface, preventing direct identification. Therefore, CD3+CD4+ cells are identified by negative gating, using ECD-anti-CD3 and FITC-anti-CD8. Lymphocytes that are CD3+CD8− represent the CD3+CD4+ population. The percentages of cytokine-positive CD3+CD8− cells are determined for each of the three cytokines, and ratios are calculated for TNF-α:IL-10 and IFN-γ:IL-10.
Results of this test are compared with those from the TH1:TH2 Intracellular Cytokine Ratio assay performed without IVIG to assess the ability of IVIG to inhibit CD3+CD4+ lymphocyte activation.
Send at room temperature. Do not refrigerate. Deliver to the laboratory within 24 hours.
If a specimen is older than 48 hours, lymphocytes will be isolated and viability assessed. If viability is >80%, the assay will be performed; if <80%, the specimen will be rejected.
The TH1:TH2 Intracellular Cytokine Ratio with IVIG may only be performed in conjunction with the TH1:TH2 Intracellular Cytokine Ratio test.
Flow Cytometry