Test Id : RBCS

This test should be ordered only when percentages are needed for the reportable B-cell subsets. If both percentages and absolute counts are needed for the reportable B-cell subsets, order IABCS / B-Cell Phenotyping Profile for Immunodeficiency and Immune Competence Assessment, Blood.

Testing performed Monday through-Friday. Specimens not received by 4 p.m. Central time on Fridays may be canceled.

Specimens arriving on the weekend and observed holidays may be canceled.

Collect and package specimens as close to shipping time as possible. Ship specimens overnight.

It is recommended that specimens arrive within 24 hours of collection.

The ordering healthcare professional's name and phone number are required.

Container/Tube: Lavender top (EDTA)

Specimen Volume:

< or =14 years: 4 mL

>14 years: 10 mL

Collection Instructions:

1. Send whole blood specimen in original tube. Do not aliquot.

2. Label specimen as blood for RBCS.

Additional Information: For serial monitoring, it is recommended that specimens are collected at the same time of day.

< or =14 years: 3 mL; >14 years: 5 mL

Screening for humoral or combined immunodeficiencies, including common variable immunodeficiency, hyper IgM syndrome, among others, where B-cell subset distribution information is desired

Assessing B-cell subset reconstitution after hematopoietic cell or bone marrow transplant

Assessing B-cell subset reconstitution following recovery of B cells after B-cell-depleting immunotherapy

This test is not indicated for the evaluation of lymphoproliferative disorders (eg, leukemia, lymphoma, multiple myeloma).

This test should not be used to monitor B-cell counts to assess B-cell depletion in patients on B-cell-depleting therapies.

The adaptive immune response includes both cell-mediated (mediated by T cells and natural killer cells) and humoral immunity (mediated by B cells). After antigen recognition and maturation in secondary lymphoid organs, some antigen-specific B cells terminally differentiate into antibody-secreting plasma cells or become memory B cells. Memory B cells are of 3 subsets: marginal zone B cells (MZ or non-switched memory), class-switched memory B cells, and IgM-only memory B cells. Decreased B-cell numbers, B-cell function, or both, result in immune deficiency states and increased susceptibility to infections. These decreases may be either primary (genetic) or secondary. Secondary causes include medications, malignancies, infections, and autoimmune disorders.

Common variable immunodeficiency (CVID), a disorder of B-cell function, is the most prevalent primary immunodeficiency with a prevalence of 1:25,000 to 1:50,000.(1) CVID has a bimodal presentation with a subset of patients presenting in early childhood and a second set presenting between 15 and 40 years, or occasionally even later. Various genetic defects have been associated with CVID, including variants in the ICOS, CD19, BAFF-R, and TACI genes.; TACI variants account for 8% to 15% of CVID cases.

CVID is characterized by hypogammaglobulinemia usually involving most or all immunoglobulin classes (IgG, IgA, IgM, and IgE), impaired functional antibody responses, and recurrent sinopulmonary infections.(1,2) B-cell numbers may be normal or decreased. A minority of patients with CVID (5%-10%) have very low B-cell counts (<1% of peripheral blood leukocytes), while another subset (5%-10%) exhibit noncaseating, sarcoid-like granulomas in different organs and also tend to develop a progressive T-cell deficiency.(1) Of all patients with CVID, 25% to 30% have increased numbers of CD8 T cells and a reduced CD4:CD8 ratio (<1). Studies have shown the clinical relevance of classifying patients with CVID by assessing B-cell subsets, since changes in different B-cell subsets are associated with specific clinical phenotypes or presentations.(3,4)

The B-cell phenotyping assay can be used in the diagnosis of hyper-IgM syndromes, which are characterized by increased or normal levels of IgM with low IgG and/or IgA.(5) Patients with hyper-IgM syndromes can have 1 of 5 known genetic defects in the CD40L, CD40, AID (activation-induced cytidine deaminase), UNG (uracil DNA glycosylase), and NEMO (NF-kappa B essential modulator) genes.(5) Variants in CD40L and NEMO are inherited in an X-linked fashion, while variants in the other 3 genes are inherited in an autosomal recessive fashion. Patients with hyper-IgM syndromes have a defect in isotype class-switching, which leads to a decrease in class-switched memory B cells, with or without an increase in non-switched memory B cells and IgM-only memory B cells.

In addition to its utility in the diagnosis of the above-described primary immunodeficiencies, B-cell phenotyping may be used to assess reconstitution of B-cell subsets after hematopoietic stem cell or bone marrow transplant. This test is also used to monitor B-cell-depleting therapies, such as Rituxan (rituximab) and Zevalin (ibritumomab tiuxetan).

The appropriate age-related reference values will be provided on the report.

The assay provides semiquantitative information on the various B-cell subsets. Each specimen is evaluated for B-cell subsets with respect to the total number of CD19+ B cells present in the peripheral blood mononuclear cell population, compared to the reference range. In order to verify that there are no CD19-related defects, CD20 is used as an additional pan-B-cell marker (expressed as percentage of CD45+ lymphocytes).

The B-cell panel assesses the following B-cell subsets:

CD19+=B cells expressing CD19 as a percent of total lymphocytes

CD19+ CD27+=total memory B cells

CD19+ CD27+ IgD+ IgM+=marginal zone or non-switched memory B cells

CD19+ CD27+ IgD- IgM+=IgM-only memory B cells

CD19+ CD27+ IgD- IgM-=class-switched memory B cells

CD19+ IgM+=IgM B cells

CD19+ CD38+ IgM+=transitional B cells

CD19+ CD38+ IgM-=plasmablasts

CD19+ CD21-=CD21-negative B cells

CD19+ CD21+=CD21-positive B cells

CD19+ CD20+=B cells coexpressing both CD19 and CD20 as a percent of total lymphocytes

This assay and the reference range reported are based on analysis of B cells derived from the mononuclear cell fraction of peripheral whole blood and, therefore, total CD19+ B cell quantitation may not be identical to those performed on whole blood (eg, TBBS / Quantitative Lymphocyte Subsets: T, B, and Natural Killer (NK) Cells, Blood).

This test should not be used to monitor B-cell counts to assess B-cell depletion in patients on B-cell-depleting therapies; order CD20B / CD20 on B Cells, Blood for that purpose; this test is meant to be used specifically for assessing the relative distribution of B-cell subsets within the total B-cell pool.

Timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.

1. Warnatz K, Denz A, Drager R, et al. Severe deficiency of switched memory B cells (CD27+ IgM- IgD-) in subgroups of patients with common variable immunodeficiency: a new approach to classify a heterogeneous disease. Blood. 2002;99(5):1544-1551

2. Brouet JC, Chedeville A, Fermand JP, Royer B. Study of the B cell memory compartment in common variable immunodeficiency. Eur J Immunol. 2000;30(9):2516-2520

3. Wehr C, Kivioja T, Schmitt C, et al. The EUROclass trial: defining subgroups in common variable immunodeficiency. Blood. 2008;111(1):77-85

4. Alachkar H, Taubenheim N, Haeney MR, Durandy A, Arkwright PD. Memory switched B-cell percentage and not serum immunoglobulin concentration is associated with clinical complications in children and adults with specific antibody deficiency and common variable immunodeficiency. Clin Immunol. 2006;120(3):310-318

5. Lee WI, Torgerson TR, Schumacher MJ, Yel L, Zhu Q, Ochs HD. Molecular analysis of a large cohort of patients with hyper immunoglobulin M (hyper IgM) syndrome. Blood. 2005;105(5):1881-1890

6. Ramirez NJ, Posadas-Cantera S, Caballero-Oteyza A, Camacho-Ordonez N, Grimbacher B. There is no gene for CVID - novel monogenetic causes for primary antibody deficiency. Curr Opin Immunol. 202172:176-185. doi:10.1016/j.coi.2021.05.010

7. Kumanovics A, Sadighi Akha AA. Flow cytometry for B-cell subset analysis in immunodeficiencies. J Immunol Methods. 2022;509:113327. doi:10.1016/j.jim.2022.113327

8. Sadighi Akha AA, Csomos K, Ujhazi B, Walter JE, Kumanovics A. Evolving approach to clinical cytometry for immunodeficiencies and other immune disorders. Clin Lab Med. 2023;43(3):467-483. doi:10.1016/j.cll.2023.05.002

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 BD FACSCanto II 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+).(Unpublished Mayo method)

Monday through Friday

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This test was developed using an analyte specific reagent. Its performance characteristics were determined by Mayo Clinic in a manner consistent with CLIA requirements. This test has not been cleared or approved by the US Food and Drug Administration.

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