B-Cell Phenotyping Profile for Immunodeficiency and Immune Competence Assessment, Blood
Method Description Describes how the test is performed and provides a method-specific reference
T- and B-Cell Quantitation by Flow Cytometry:
The T- 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-2:1-29)
Immune Assessment B Cell Subsets, Blood:
Peripheral blood mononuclear cells (PBMC) 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 BD FACSCanto 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 the BD Multitest TBNK Panel (BD BioSciences) kit, which contains monoclonal antibodies for CD45, CD3, CD4, CD8, CD19, and CD16+CD56+. The TBNK Panel utilizes a 6-color, lyse-no wash procedure and the absolute counts are calculated for each of the above subsets using the FACSCanto clinical software using the internal bead standards. The absolute lymphocyte count per microliter is used to calculate the absolute counts o the various B-cell subsets in this assay using the following formula:
Absolute CD19+ and CD20+ B cells/mcL (Formula 1)
=% CD19+ or % CD20+B cells (as % lymphocytes from the B-cell subset assay-PBMC) x absolute lymphocyte count (from TBNK panel)/100
Absolute count of other B-cell subsets/uL (Formula 2)
=% B-cell subset (as % B cells from the IABC-IABCS/88800 B-Cell Phenotyping Profile for Immunodeficiency and Immune Competence Assessment, Blood x absolute CD19+ B cells (from Formula 1)/100(Unpublished Mayo information)
Common Variable Immunodeficiency Confirmation Flow Panel:
Peripheral blood mononuclear cells are isolated and stained with CD19, TACI, and BAFF-R, each conjugated to a fluorochrome. After the staining with specific antibody, the cells are washed, fixed with paraformaldehyde, and then analyzed by flow cytometry on a BD FACS Canto instrument. The cell-surface expression is denoted as the percent of CD19+ B cells expressing TACI and BAFF-R.(Unpublished Mayo information)
Day(s) and Time(s) Test Performed Outlines the days and times the test is performed. This field reflects the day and time the sample must be in the testing laboratory to begin the testing process and includes any specimen preparation and processing time required before the test is performed. Some tests are listed as continuously performed, which means assays are performed several times during the day.
Monday through Friday
Do not send specimen after Thursday. Specimen must be received by 10 a.m. on Friday.