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 TACI and BAFF-R in patients presenting

with clinical symptoms and other laboratory features consistent

with CVID

T- and B-Cell Quantitation by Flow Cytometry:

See Individual Unit Code

 

Immune Assessment B Cell Subsets, B:

The adaptive immune response includes both cell-mediated

(mediated by T cells and 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 1:25,000 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

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: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 leveles 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(2)

 - TACI - transmembrane activator and CAML (calcium modulator

    and cyclophilin ligand) interactor(3)

 - CD19(4)

 - BAFF-R-B cell activating factor belonging to the tumor necrosis

    factor (TNF) receptor family(5)

 

Of these, the TACI mutations probably account for about 10% of

all CVID cases.(3) 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 #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.

T- AND B-CELL QUANTITATION BY FLOW CYTOMETRY

T & B SURFACE MARKER

      % T-cells (CD3)

                0-2 months: 53-84%*

                3-5 months: 51-77%*

                6-11 months: 49-76%*

                12-23 months: 53-75%*

                2-5 years: 56-75%*

                6-11 years: 60-76%*

                12-17 years: 56-84%*

                > or =18 years: 52-84%

      % B-cells (CD19)

                0-2 months: 6-32%*

                3-5 months: 11-41%*

                6-11 months: 14-37%*

                12-23 months: 16-35%*

                2-5 years: 14-33%*

                6-11 years: 13-27%*

                12-17 years: 6-23%*

                > or =18 years: 5-25%

      % Natural killer (CD16 CD56)

                0-2 months: 4-18%*

                3-5 months: 3-14%*

                6-23 months: 3-15%*

                2-11 years: 4-17%*

                12-17 years: 3-22%*

                > or =18 years: 5-30%

      % Helper cells (CD4)

                0-2 months: 35-64%*

                3-5 months: 35-56%*

                6-11 months: 31-56%*

                12-23 months: 32-51%*

                2-5 years: 28-47%*

                6-11 years: 31-47%*

                12-17 years: 31-52%*

                > or =18 years: 30-61%

      % Suppressor cells (CD8)

                0-2 months: 12-28%*

                3-5 months: 12-23%*

                6-11 months: 12-24%*

                12-23 months: 14-30%*

                2-5 years: 16-30%*

                6-17 years: 18-35%*

                > or =18 years: 12-42%

ABSOLUTE COUNTS

      CD45 lymph count, flow:  0.66-4.60 thou/uL

      T-cells (CD3)

                0-2 months: 2,500-5,500 cells/uL*

                3-5 months: 2,500-5,600 cells/uL*

                6-11 months: 1,900-5,900 cells/uL*

                12-23 months: 2,100-6,200 cells/uL*

                2-5 years: 1,400-3,700 cells/uL*

                6-11 years: 1,200-2,600 cells/uL*

                12-17 years: 1,000-2,200 cells/uL*

                > or =18 years: 582-1,992 cells/uL

      B-cells (CD19)

                0-2 months: 300-2,000 cells/uL*

                3-5 months: 430-3,000 cells/uL*

                6-11 months: 610-2,600 cells/uL*

                12-23 months: 720-2,600 cells/uL*

                2-5 years: 390-1,400 cells/uL*

                6-11 years: 270-860 cells/uL*

                12-17 years: 110-570 cells/uL*

                > or =18 years: 71-567 cells/uL

      Natural killer (CD16 CD56)

                0-2 months: 170-1,100 cells/uL*

                3-5 months: 170-830 cells/uL*

                6-11 months: 160-950 cells/uL*

                12-23 months: 180-920 cells/uL*

                2-5 years: 130-720 cells/uL*

                6-11 years: 100-480 cells/uL*

                12-17 years: 70-480 cells/uL*

                > or =18 years: 80-597 cells/uL

      Helper cells (CD4)  

                0-2 months: 1,600-4,000 cells/uL*

                3-5 months: 1,800-4,000 cells/uL*

                6-11 months: 1,400-4,300 cells/uL*

                12-23 months: 1,300-3,400 cells/uL*

                2-5 years: 700-2,200 cells/uL*

                6-11 years: 650-1,500 cells/uL*

                12-17 years: 530-1,300 cells/uL*

                > or =18 years: 401-1,532 cells/uL

      Suppressor cells (CD8)

                0-2 months: 560-1,700 cells/uL*

                3-5 months: 590-1,600 cells/uL*

                6-11 months: 500-1,700 cells/uL*

                12-23 months: 620-2,000 cells/uL*

                2-5 years: 490-1,300 cells/uL*

                6-11 years: 370-1,100 cells/uL*

                12-17 years: 330-920 cells/uL*

                > or =18 years: 152-838 cells/uL

LYMPHOCYTE RATIO

      H/S ratio:  > or = 1.0

*Shearer WT, Rosenblatt HM, Gelman RS, et al:  Lymphocyte

subsets in healthy children from birth through 18 years of age: The

Pediatric AIDS Clinical Trials Group P1009 study. J Allergy Clin

Immunol 2003;112(5):973-980

 

B-CELL PHENOTYPING SCREEN FOR IMMUNODEFICIENCY

AND IMMUNE COMPETENCE ASSESSMENT

The 95% reference values were established for healthy adult

(19-70 years) and pediatric donors (0-18 years).

 

B-Cell Subset	Pediatric Reference Values (n=98)
Results Expressed as a Percentage of Total Lymphocytes	Percentage	Absolute Count (Cells/uL)
CD19	4.3-23.2	92.0-792.0
CD19 CD20	4.2-24.1	85.0-767.9
Results Expressed as a Percentage of CD19 B-cells	Percentage	Absolute Count (Cells/uL)
CD19 CD27	4.6-49.1	9.4-136.0
CD19 CD27 IgM IgD	0.2-12.0	0.8-42.7
CD19 CD27 IgM- IgD-	1.9-30.4	5.2-74.2
CD19 CD27 IgM IgD-	0.3-13.1	0.8-37.8
CD19 IgM	32.8-82.6	46.0-596.0
CD19 CD38 IgM-	2.9-51.8	8.2-275.1
CD19 CD38 IgM	7.6-48.6	14.2-229.6
CD19 CD21	94.5-99.8	89.4-780.1
CD19 CD21-	0.2-5.5	0.9-25.5

 

B-Cell Subset	Adult Reference Values (n=96)
Results Expressed as a Percentage of Total Lymphocytes	Percentage	Absolute Count (Cells/uL)
CD19	2.8-17.4	90.0-539.0
CD19 CD20	3.2-16.8	95.0-580.8
Results Expressed as a Percentage of CD19 B cells	Percentage	Absolute Count (Cells/uL)
CD19 CD27	6.3-52.8	18.0-145.0
CD19 CD27 IgM IgD	1.7-29.3	4.0-85.0
CD19 CD27 IgM- IgD-	2.3-26.5	7.0-61.0
CD19 CD27 IgM IgD-	0-5.3	0-12.0
CD19 IgM	26.0-78.0	37.0-327.0
CD19 CD38 IgM-	4.1-42.2	7.0-153.0
CD19 CD38 IgM	1.2-50.7	2.0-139.4
CD19 CD21	92.1-99.6	85.0-533.0
CD19 CD21-	0.2-8.6	0.3-22.0

 

 

An interpretive report will be provided.

T- and B-Cell Quantitation by Flow Cytometry:

See Individual Unit Code

 

Immune Assessment B Cell Subsets, B:

The assay provides quantitative information on the various B-cell

subsets (percentage and absolute counts in cells/microliter).

Each sample 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 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

sample will be reflexed to the CVID confirmation panel for

assessment of defects in surface expression of BAFF-R and

TACI (2 genes/proteins associated with CVID). To evaluate

defects in ICOS, a flow cytometry test for assessing ICOS protein

on activated T cells can be ordered (#89010 "Inducible

Costimulator [ICOS] Protein, Blood). To conclusively determine if

TACI mutations are present, the TACI mutation analysis test by

gene sequencing can be ordered (#84388 "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.

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. #9336 "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 CVID (eg. #87993, "CVID

Screen"; #89010, "ICOS Protein"; #84388, "TACI Full Gene

Sequencing"), and hyper-IgM (#82964 "X-Linked Hyper IgM

Syndrome Panel, Blood" and #89009 "Be-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).

T- and B-Cell Quantitation by Flow Cytometry:

See Individual Unit Code

 

Immune Assessment B Cell Subsets, B:

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;120:310318

 

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.     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.   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