Method Description

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

The T, B, and natural killer (NK)-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, NK (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, Goldstien 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. Guidelines for performing single-platform absolute CD4+T-cell determinations with CD45 gating for persons infected with human immunodeficiency virus. Center for Disease Control and Prevention. MMWR Morb Mortal Wkly Rep. 2003;52[RR-2]:1-13)

Immune Assessment B Cell Subsets:

Peripheral blood mononuclear cells are isolated from whole blood using a Ficoll gradient and used in the staining protocol. The assay involves a multicolor 5-tube panel for the following antibodies: CD45, CD19, CD20, CD27, IgD, IgM, CD38, and CD21. After the staining with specific antibody, the cells are washed and fixed with paraformaldehyde and then analyzed by flow cytometry on a Becton Dickinson FACS Canto instrument. The cell-surface expression is denoted as the percent of CD19+ B cells expressing each of the specific markers. CD19+ and CD20+ B cells are expressed as a percent of the total lymphocytes (CD45+). The absolute counts for the B-cell subsets are derived from flow cytometry analysis of whole blood using monoclonal antibodies to identify CD45, CD3, CD4, CD8, CD19, and CD16+CD56+. CD14 is used to exclude monocytes, thereby improving accuracy and enhancing the purity of the lymphocyte population. The assay is a 7-color, lyse-no wash procedure and the absolute counts are calculated from internal bead standards. The absolute lymphocyte count per microliter is used to calculate the absolute counts of the various B-cell subsets in this assay.(Unpublished Mayo method)