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Test ID: IABCS    
B-Cell Phenotyping Profile for Immunodeficiency and Immune Competence Assessment, Blood

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Useful For Suggests clinical disorders or settings where the test may be helpful

Screening for common variable immunodeficiency (CVID) and hyper-IgM syndromes

 

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

 

Assessing response to B-cell-depleting immunotherapy

 

Identifying defects in transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI) and B-cell-activating factor receptor (BAFF-R) in patients presenting with clinical symptoms and other laboratory features consistent with CVID

Testing Algorithm Delineates situation(s) when tests are added to the initial order. This includes reflex and additional tests.

If immune assessment B-cell subsets test is abnormal, then confirmation will be performed at an additional charge.

 

When multiple specimen types are required to perform a panel of tests, the laboratory will perform the tests for which the appropriate specimen type was received and the laboratory will cancel those for which the appropriate specimen was not received. Please be advised that this may change the degree of interpretation received with the report. If only the refrigerate EDTA sample is received, this test will be canceled and converted to RBCS / Relative B-Cell Subset Analysis Percentage which provides the relative B-cell subset values without quantitation.

Clinical Information Discusses physiology, pathophysiology, and general clinical aspects, as they relate to a laboratory test

T- and B-Cell Quantitation by Flow Cytometry:

Normal immunity requires a balance between the activities of various lymphocyte subpopulations with different effector and regulatory functions.

 

Different immune cells can be characterized by unique surface membrane antigens described by a cluster of differentiation nomenclature (eg, CD3 is an antigen found on the surface of T lymphocytes). Abnormalities in the number and percent of T (CD3), T-helper (CD4), T-suppressor (CD8), B (CD19), and natural killer (CD16+CD56) lymphocytes have been described in a number of different diseases. In patients who are infected with HIV, the CD4 count is measured for AIDS diagnosis and for initiation of antiviral therapy. The progressive loss of CD4 T lymphocytes in patients infected with HIV is associated with increased infections and complications.

 

The United States Public Health Service has recommended that all HIV-positive patients be tested every 3 to 6 months for the level of CD4 T lymphocytes.

 

The absolute counts of lymphocyte subsets are known to be influenced by a variety of biological factors, including hormones, the environment, and temperature. The studies on diurnal (circadian) variation in lymphocyte counts have demonstrated progressive increase in CD4 T-cell count throughout the day, while CD8 T cells and CD19+ B cells increase between 8:30 a.m. and noon, with no change between noon and afternoon. Natural killer (NK) cell counts, on the other hand, are constant throughout the day.(1) Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration.(2-4) In fact, cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus effector CD4 and CD8 T cells.(2) It is generally accepted that lower CD4 T-cell counts are seen in the morning compared with the evening,(5) and during summer compared to winter.(6) These data, therefore, indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.

 

Immune Assessment B Cell Subsets, Blood:

The adaptive immune response includes both cell-mediated (mediated by T cells and natural killer [NK] 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 3 subsets: marginal zone B cells (MZ or nonswitched 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 to 25,000 to 1 to 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 of age, or occasionally even later. Four different genetic defects have been associated with CVID including mutations in the ICOS, CD19, BAFF-R, and TACI genes. The first 3 genetic defects account for approximately 1% to 2%, and TACI mutations account for 8% to 15% of CVID cases.

 

CVID is characterized by hypogammaglobulinemia usually involving most or all of the Ig 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 CVID patients (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 to CD8 ratio (<1). Studies have shown the clinical relevance of classifying CVID patients by assessing B-cell subsets, since changes in different B-cell subsets are associated with particular 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--mutations in the CD40L, CD40, AID (activation-induced cytidine deaminase), UNG (uracil DNA glycosylase), and NEMO (NF-kappa B essential modulator) genes.(5) Mutations in CD40L and NEMO are inherited in an X-linked fashion, while mutations 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 increased in nonswitched 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-depicting therapies, such as Rituxan (Rituximab) and Zevalin (Ibritumomab tiuxetan).

 

CVID Confirmation Flow Panel:

The etiology of CVID is heterogeneous, but recently 4 genetic defects were described that are associated with the CVID phenotype. Specific mutations, all of which are expressed on B cells, have been implicated in the pathogenesis of CVID.

 

These mutations encode for:

-ICOS-inducible costimulator expressed on activated T cells(1)

-TACI-transmembrane activator and CAML (calcium modulator and cyclophilin ligand) interactor(2)

-CD19(3)

-BAFF-R-B cell activating factor belonging to the tumor necrosis factor (TNF) receptor family(4)

 

Of these, the TACI mutations probably account for about 10% of all CVID cases.(2) Patients with mutations in the TACI gene are particularly prone to developing autoimmune disease, including cytopenias as well as lymphoproliferative disease. The other mutations each have been reported in only a handful of patients. The etiopathogenesis is still undefined in more than 50% of CVID patients.

 

A BAFF-R defect should be suspected in patients with low to very low class switched and nonswitched memory B cells and very high numbers of transitional B cells (see IABC/87994 B-Cell Phenotyping Screen for Immunodeficiency and Immune Competence Assessment, Blood). Class switching is the process that allows B cells, which possess IgD and IgM on their cell surface as a part of the antigen-binding complex, to produce IgA, IgE, or IgG antibodies. A TACI defect is suspected in patients with low IgM with normal to low switched B cells, with autoimmune and/or lymphoproliferative manifestations, and normal B cell responses to mitogens.

 

The absolute counts of lymphocyte subsets are known to be influenced by a variety of biological factors, including hormones, the environment, and temperature. The studies on diurnal (circadian) variation in lymphocyte counts have demonstrated progressive increase in CD4 T-cell count throughout the day, while CD8 T cells and CD19+ B cells increase between 8:30 a.m. and noon, with no change between noon and afternoon. Natural killer (NK) cell counts, on the other hand, are constant throughout the day.(5) Circadian variations in circulating T-cell counts have been shown to be negatively correlated with plasma cortisol concentration.(6-8) In fact, cortisol and catecholamine concentrations control distribution and, therefore, numbers of naive versus effector CD4 and CD8 T cells.(6) It is generally accepted that lower CD4 T-cell counts are seen in the morning compared with the evening,(9) and during summer compared to winter.(10) These data, therefore, indicate that timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets.

Reference Values Describes reference intervals and additional information for interpretation of test results. May include intervals based on age and sex when appropriate. Intervals are Mayo-derived, unless otherwise designated. If an interpretive report is provided, the reference value field will state this.

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

Interpretation Provides information to assist in interpretation of the test results

T- and B-Cell Quantitation by Flow Cytometry:

When the CD4 count falls below 500 cells/mcL, HIV-positive patients can be diagnosed with AIDS and can receive antiretroviral therapy.

 

When the CD4 count falls below 200 cells/mcL, prophylaxis against Pneumocystis jiroveci pneumonia is recommended.

 

Immune Assessment B Cell Subsets, Blood:

The assay provides quantitative information on the various B-cell subsets (percentage and absolute counts in cells/microliter). 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 nonswitched 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 low ("immature") B cells

-CD19+ CD21+=mature B cells

-CD19+ CD20+=B cells co-expressing both CD19 and CD20 as a percent of total lymphocytes

 

For isotype class-switching and memory B-cell analyses, the data will be reported as being consistent or not consistent with a defect in memory and/or class switching. If a defect is present in any of these B-cell subpopulations, further correlation with clinical presentation and additional functional, immunological, and genetic laboratory studies will be suggested.

 

Since each of the 11 B-cell subsets listed above contributes to the diagnosis of common variable immunodeficiency (CVID) and hyper-IgM syndromes and provides further information on the likely specific genetic defect, all the B-cell subsets are carefully evaluated to determine if further testing is needed for confirmation, including functional assays and genotyping, which is then suggested as follow-up testing in the interpretive report as detailed below.

 

If abnormalities are found in the B-cell phenotyping panel, the specimen will be reflexed to the CVID confirmation panel for assessment of defects in surface expression of B-cell-activating factor receptor (BAFF-R) and transmembrane activator and calcium modulator and cyclophilin ligand (CAML) interactor (TACI) (2 genes/proteins associated with CVID). To conclusively determine if TACI mutations are present, the TACI mutation analysis test by gene sequencing can be ordered (TACIF / Transmembrane Activator and CAML Interactor [TACI] Gene, Full Gene Analysis).      

 

CVID Confirmation Flow Panel:

BAFF-R is normally expressed on over 95% of B cells, while TACI is expressed on a smaller subset of B cells and a proportion of activated T cells.

 

The lack of TACI or BAFF-R surface expression on the appropriate B-cell population is consistent with a CVID defect.

 

Results will be interpreted in the context of the B-cell phenotyping results and correlation to clinical presentation will be recommended.

Cautions Discusses conditions that may cause diagnostic confusion, including improper specimen collection and handling, inappropriate test selection, and interfering substances

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, results may not be identical to those performed on whole blood (eg, TBBS / T- and B-Cell Quantitation by Flow Cytometry).

 

This test is a screening test and further analyses will be required to complete a diagnostic workup for common variable immunodeficiency (CVID) (eg, CVID / Common Variable Immunodeficiency Confirmation Flow Panel; TACIF / Transmembrane Activator and CAML Interactor [TACI] Gene, Full Gene Analysis) and hyper-IgM (XHIM / X-Linked Hyper IgM Syndrome, Blood and CD40 / B-Cell CD40 Expression by Flow Cytometry, Blood for CD40 ligand and CD40 expression, respectively).

 

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

 

Timing and consistency in timing of blood collection is critical when serially monitoring patients for lymphocyte subsets. See data under Clinical Information.

Clinical Reference Provides recommendations for further in-depth reading of a clinical nature

T- and B-Cell Quantitation by Flow Cytometry:

1. Carmichael KF, Abayomi A: Analysis of diurnal variation of lymphocyte subsets in healthy subjects and its implication in HIV monitoring and treatment. 15th Intl Conference on AIDS, Bangkok, Thailand, 2004, Abstract B11052

2. Dimitrov S, Benedict C, Heutling D, et al: Cortisol and epinephrine control opposing circadian rhythms in T-cell subsets. Blood 2009;113(21):5134-5143

3. Dimitrov S, Lange T, Nohroudi K, Born J: Number and function of circulating antigen presenting cells regulated by sleep. Sleep 2007;30:401-411

4. Kronfol Z, Nair M, Zhang Q, et al: Circadian immune measures in healthy volunteers: relationship to hypothalamic-pituitary-adrenal axis hormones and sympathetic neurotransmitters. Psychosom Med 1997;59:42-50

5. Malone JL, Simms TE, Gray GC, et al: Sources of variability in repeated T-helper lymphocyte counts from HIV 1-infected patients: total lymphocyte count fluctuations and diurnal cycle are important. J AIDS 1990;3:144-151

6. Paglieroni TG, Holland PV: Circannual variation in lymphocyte subsets, revisited. Transfusion 1994;34:512-516

7. 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;52:1-13

8. Centers for Disease Control: 1997 Revised guidelines for performing CD4+ T-cell determinations in persons infected with human immunodeficiency virus (HIV). MMWR Morb Mortal Wkly Rep 46 no. RR-2: 1997, pp 1-29

9. U.S. Department of Health and Human Services: Recommendations for prophylaxis against Pneumocystis carinii pneumonia for adults and adolescents infected with human immunodeficiency virus. MMWR Morb Mortal Wkly Rep 43 no. RR-3: 1994, pp 1-21

 

Immune Assessment B Cell Subsets, Blood:

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;99:1544-1551

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

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

4. Alachkar H, Taubenheim N, Haeney MR, et al: 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;120:310-318

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

 

CVID Confirmation Flow Panel:

1. Grimbacher B, Hutloff A, Schlesier M, et al: Homozygous loss of ICOS is associated with adult-onset common variable immunodeficiency. Nat Immunol 2003;4(3):261-268

2. Salzer U, Chapel HM, Webster ADB, et al: Mutations in TNFRSF13B encoding TACI are associated with common variable immunodeficiency in humans. Nat Genet 2005;37(8):820-828

3. van Zelm M, Reisli I, van der Burg M, et al: An antibody-deficiency syndrome due to mutations in the CD19 gene. New Engl J Med 2006;354:1901-1912

4. Warnatz K, Salzer U, Gutenberger S, et al: Finally found: human BAFF-R deficiency causes hypogammaglobulinemia. Clin Immunol 2005;115(Suppl 1):820

5. Carmichael KF, Abayomi A: Analysis of diurnal variation of lymphocyte subsets in healthy subjects and its implication in HIV monitoring and treatment. 15th Intl Conference on AIDS, Bangkok, Thailand, 2004, Abstract B11052

6. Dimitrov S, Benedict C, Heutling D, et al: Cortisol and epinephrine control opposing circadian rhythms in T-cell subsets. Blood 2009 (prepublished online March 17, 2009)

7. Dimitrov S, Lange T, Nohroudi K, Born J: Number and function of circulating antigen presenting cells regulated by sleep. Sleep 2007;30:401-411

8. Kronfol Z, Nair M, Zhang Q, et al: Circadian immune measures in healthy volunteers: relationship to hypothalamic-pituitary-adrenal axis hormones and sympathetic neurotransmitters. Pyschosom Med 1997;59:42-50

9. Malone JL, Simms TE, Gray GC, et al: Sources of variability in repeated T-helper lymphocyte counts from HIV 1-infected patients: total lymphocyte count fluctuations and diurnal cycle are important. J AIDS 1990;3:144-151

10. Paglieroni TG, Holland PV: Circannual variation in lymphocyte subsets, revisited. Transfusion 1994;34:512-516

11. 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;99:1544-1551