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Unit Code 88800:
B-Cell Phenotyping Profile for Immunodeficiency and Immune Competence Assessment, Blood

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Method Description

T- and B-Cell Quantitation by Flow Cytometry:

See Individual Unit Code

 

Immune Assessment B Cell Subsets, B:

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/uL (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 - #88800,

                "B-Cell Phenotyping Profile for Immunodeficiency and Immune

                Competence Assessment, Blood) x absolute CD19

                B cells (from Formula 1)/100

 

(Unpublished Mayo information)

 

CVID 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 FACSCanto instrument. The cell-surface

expression is denoted as the percent of CD19 B cells

expressing TACI and BAFF-R. (Unpublished Mayo Information)

Performing Laboratory Location

Rochester

Key