Method Description

CD8 T-Cell Immune Competence, Global:

Peripheral blood mononuclear cells (PBMC), which contain CD8 T cells, are stimulated with a mixture of phorbol myristate acetate and ionomycin, and with stimulatory signals derived using antibodies against the costimulatory molecules CD28/CD49d. The cells are simultaneously treated with a mixture of brefeldin A and monensin, which blocks extracellular secretion of interferon-gamma (IFN-gamma), enabling intracellular retention and detection of the protein. PBMC that have not been stimulated are used as a control to determine the background levels of IFN-gamma and CD107a and CD107b. The cells are analyzed on the Becton Dickinson FACS Canto flow cytometer, and analysis involves gating (defining) of the CD8 T cells using an antihuman CD8 antibody. Specific IFN-gamma, CD107a, and CD107b signals are determined within the "gated" CD8 T-cell population. Global CD8 T-cell immune competence is measured by the amount of IFN-gamma produced (CD8 T-cell functional activity) and surface expression of CD107a and CD107b (cytotoxicity assessment) relative to the unstimulated control and is interpreted on the basis of the reference range determined from healthy adult donors.(Unpublished Mayo method)

Quantitative Lymphocyte Subsets: T, B, and Natural Killer:

The T, B, and natural killer-cell surface marker assay uses monoclonal antibodies to identify the various membrane antigens, and flow cytometry to enumerate the number of cells expressing these differentiation antigens. CD14 is used to exclude monocytes, thereby improving accuracy and enhancing the purity of the lymphocyte population. The results are reported as the percent of lymphocytes that are total T cells (CD3+), CD3+CD4+ T cells, CD3+CD8+ T cells, natural killer (CD16+56+, CD3-), and B-lymphocytes (CD19+), and the absolute number of each cell type per mL of blood. The assay is a 7-color no-wash procedure and the absolute counts are calculated from internal bead standards. In addition, the total lymphocyte count and the CD4:CD8 ratio are reported.(Hoffman RA, Kung PC, Hansen WP, Goedstien G. Simple and rapid measurement of human T lymphocytes and their subclasses in peripheral blood. Proc Natl Acad Sci USA. 1980:77(8):4914-4917; Mandy FF, Nicholson JK, McDougal JS; CDC. Guidelines for performing single-platform absolute CD4+ T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. Centers for Disease Control and Prevention. MMWR Recomm Rep. 2003;52(RR-2):1-13)