|Values are valid only on day of printing.|
Peripheral blood mononuclear cells (PBMCs), 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. PBMCs 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 BDFACS Canto flow cytometer and analysis involves gating (defining) of the CD8 T cells using an antihuman CD8 antibody. Specific IFN-gamma and 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 (cytoxicity assessment) relative to the unstimulated control and is interpreted on the basis of the reference range determined from healthy adult donors.(Unpublished Mayo method)
The T-cell and B-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. The results are reported as the percent of lymphocytes that are T-helper (CD3+, CD4+), T-suppressor (CD3+, CD8+), 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 4-color no-wash procedure and the absolute counts are calculated from internal bead standards. In addition, the total lymphocyte count and the helper-suppressor ratio (T[H]/T[S]) is 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, 1980:77:4914-4917; US Department of Health and Human Services: Guidelines for performance of CD4+ T-cell determinations in persons with human immunodeficiency virus infection. MMWR Morb Mortal Wkly Rep 1997;46 no. RR-2 pp 1-29)
Monday through Friday
Specimens are required to be received in the lab weekdays and by 4 p.m. on Friday. No weekend processing.