Pediatric Common Variable Immunodeficiency Workup

A diagnosis of common variable immunodeficiency (CVID) is based on defective functional antibody formation, usually accompanied by decreased (not absent) serum immunoglobulin (Ig)G and IgA levels, generally (not invariably) decreased serum IgM, and exclusion of other known causes of antibody deficiency.

Serum Ig levels are consistently depressed but are generally higher in patients with common variable immunodeficiency than in patients with X-linked agammaglobulinemia. Interpretation of results must consider the marked variations in normal Ig levels that accompany aging; always use age-related normal values for comparison. Normal values should be provided by the laboratory. Serum immunoglobulins are commonly measured by radial immunodiffusion or immunoturbidimetric methods. Electrophoresis and immunoelectrophoresis are not satisfactory techniques for the quantification of immunoglobulins.

IgG subclass determination is of limited value in assessing patients with clinical immunodeficiency because functional antibody deficiency may be present despite normal IgG subclass levels. Conversely, deficient levels of a single subclass of IgG may be found in individuals who have effective specific antibody production and are clinically normal.

Diminished immunoglobulin levels may be caused by loss and decreased synthesis. An indirect indication of loss may be obtained by measuring serum albumin or alpha-1-antitrypsin, which is usually lost concomitantly through GI or renal tracts.

Assessment of the ability to produce functional antibodies can be obtained by measuring antibody responses to natural antigens or those antigens to which the population commonly is exposed. This assessment can also be done by measuring antibody responses following active immunization with protein or polysaccharide antigens.

Isohemagglutinins are IgM antibodies to ABO blood group antigens that are polysaccharide. All individuals, except the 3% of the population who have blood group AB, develop isohemagglutinin titers during the first year of life; therefore, normal infants aged up to 8-10 months and people with the AB blood group lack significant isohemagglutinins.

In children who have completed immunizations with diphtheria, pertussis, and tetanus (DPT) or Hib-conjugated vaccines, the antibody response to protein antigens can be tested in adults and older children by measuring IgG antibodies to tetanus or diphtheria toxoid and H influenzae type b (Hib) polysaccharide antigen. Approximately 80% of children have detectable antibodies to these antigens after 3 immunizations during their first year of life. If the child's antibody level is low, administer 1 booster injection, then measure for antibodies 4 weeks later.

For nonimmunized children, recommended doses of diphtheria-tetanus (DT) or Hib-conjugate vaccines may be administered. Obtain blood 4 weeks after the last immunization and determine IgG antibodies. Alternatively, administer 3 doses of killed poliomyelitis vaccine (1 mL IM at 2-wk intervals); take a blood sample 4 weeks after the last injection and determine antibody level (usually by virus neutralization).

Antibody responses to polysaccharide antigens depend less on T cells; they are poor immunogens in children younger than 2 years. Assessment of responses to polysaccharide antigens is important in patients older than 18-24 months because these responses may be deficient in some patients who can respond normally to protein antigens. Pneumococcal (unconjugated) or meningococcal vaccines are commercially available polysaccharide antigens. Protein conjugated pneumococcal vaccine elicit antibody responses that are T-cell dependent. Therefore, antibody responses should be measured to polysaccharide antigens that are not present in the protein-conjugated pneumococcal vaccine. Alternatively antibody response to typhoid-Vi antigen can be measured following typhoid vaccine administration.

Hepatitis B is not a reliable antigen for testing immune competence because of the high frequency of nonresponders in the population, particularly in persons older than 40 years. Bacteriophage PhiX174 (a bacterial virus noninfective in humans) has been shown to be a potent, safe, and useful antigen; it allows measurement of antigen clearance and primary and secondary immune responses. Bacteriophage fX174 is not commercially available, and only a few research laboratories have used it for human in vivo testing.

In vivo T-cell function can be measured by skin testing for delayed cutaneous hypersensitivity (DCH). The prototype is the tuberculin skin test.

DCH is a localized immunological skin response and depends on functional thymus-derived T lymphocytes. Frequently used antigens include mumps (1 mg/mL), trichophyton (1:30), purified protein derivative (PPD) (2-10 IU), Candida (1:100 dilution, or 1:10 if negative with 1:100), or tetanus fluid toxoid (1:100). Several antigens must be used for DCH testing. Perform the tests by intradermal injection of 0.1 mL of antigen. Read results in 48-72 hours for the maximal diameter of induration. This test must be carried out by intradermal injection of antigens.

Erythema is not an indication of DCH. A positive DCH finding is informative, whereas negative DCH test findings may be difficult to interpret. This is because DCH is influenced by age, steroid therapy, severe illness, previous exposure to testing antigens, concurrent stress, and recent immunization.

Enumerate circulating T and B lymphocytes.

This is achieved by immunofluorescent staining of lymphocytes with monoclonal antibodies. Commonly used antibodies are CD19 and CD20 (for B cells), CD3 (for T cells), CD4 (for subsets of helper T cells), and CD8 (for T-suppressor lymphocytes). However, CD3 and CD8 are also expressed on natural killer (NK) cells. Monoclonal antibodies against CD16, CD56, and CD57, although not lineage-specific, may be useful for enumeration of NK cells to distinguish these cells from T cells.

Some patients with common variable immunodeficiency have low numbers or undetectable peripheral B cells. These patients, especially males, may have atypical X-linked agammaglobulinemia and should be studied for mutations of the Btk gene. Female patients with agammaglobulinemia and undetectable peripheral B cells may have phenotypic X-linked agammaglobulinemia. Some female patients with agammaglobulinemia were found to have mutations in the µ heavy-chain gene or l5 gene.

Enumeration of the B-cell subsets in peripheral blood may be useful in classifying of common variable immunodeficiency. These subsets include class-switched memory B cells (CD27+IgM-IgD-), non-switched memory B cells (CD19+CD27+IgM+IgD+), IgM-only memory B cells (CD19+CD27+IgM+IgD^dim), transitional B cells (CD19+CD38+++IgM+++), plasmablasts (CD19+CD38+++IgM-), mature B cells (CD19+CD21+), and CD21^lo B cells (CD19+CD21^lo).

To test functional integrity of lymphocytes in a patient, lymphocytes can be isolated and stimulated with various agents in vitro.

Lymphocytes can be activated in vitro by the following:

• Mitogens, such as phytohemagglutinin (PHA) or concanavalin A (Con A), stimulate T-cell proliferation. Pokeweed mitogen (PWM) stimulates T-cell and B-cell proliferation.

• Antigens, such as PPD, Candida, streptokinase, and tetanus toxoid, can activate lymphocytes if the patient has had a prior encounter with the antigen. Superantigens, such as toxic shock syndrome toxin (TSST), can activate lymphocytes without prior encounter.

• Allogeneic cells stimulate T-cell proliferation in mixed lymphocyte culture.

• Lymphocytes can be activated in vitro by antibodies against T-cell surface molecules involved in signal transduction, such as to CD3, CD2, CD28, and CD43.

T-lymphocyte activation can be directly assessed by the following:

• Assess the expression of activation markers on T cells, such as CD69, IL-2 receptor a (CD25), transferrin receptors (CD71), and major histocompatibility complex (MHC) class II molecules (HLA-DR). Detection of activation markers by immunofluorescent or immunohistochemical staining can provide rapid results.

• Measure blastogenesis and/or proliferation of cells. The blastogenic response to soluble PHA or Con A requires presence of monocytes for stimulation of T cells. PWM stimulates both T cells and B cells, although T cells must be present for the B cells to be stimulated. The mixed lymphocyte reaction (MLR) results from T-cell reactivity to MHC antigens displayed on B cells and monocytes. Note that when normal irradiated or mitomycin C-treated lymphocytes are the stimulators of an MLR, the normal T cells in the culture may secrete factors that induce blastogenesis in the patient's lymphocytes. Therefore, using B-cell lines or T-cell depleted normal cells as the stimulators is preferable. Following in vitro stimulation of B cells with B-cell activators, culture supernatant can be tested for secreted Ig.)

• Measure the release of cytokines or mediators, such as interleukin (IL)-2, IL-4, IL-5, IL-6, interferon-gamma, and tumor necrosis factor (TNF), in the culture supernatant.

• Measure the secretion of immunoglobulin in the culture supernatant.

Anemia may be secondary to autoantibodies. Severe lymphopenia may raise suspicions that a patient has severe combined immunodeficiency or other primary T-cell defects. Small platelets in an infant boy suggest Wiskott-Aldrich syndrome. Neutropenia or thrombocytopenia may occur secondary to autoantibodies. Various organ-specific autoantibody production has been reported in patients with common variable immunodeficiency. Tests for autoantibody production should correlate to the patient's presenting symptoms and organ involvement.

Protein expression for BAFF-R, TACI, and CD19 on B cells and ICOS on activated T cells by flow cytometry are available in only a few specialized reference laboratories.

Genetic testing for mutation of TNFRSF13B, the gene encoding TACI (transmembrane activator and CAML interactor), can be performed using a small amount of blood sample through a polymerase chain reaction (PCR). Genetic testing can help to distinguish common variable immunodeficiency and other antibody deficiency syndromes. (Reference laboratory for genetic testing: Correlagen Diagnostics (617) 577-0152; Mayo Medical Laboratories (800) 533-1710, (507) 266-5700)

High-resolution CT scanning of the chest may be more sensitive in monitoring of pulmonary abnormalities in these patients than chest radiography or pulmonary function testing.

Studies for infectious agents: Make every effort to diagnose infections and identify infectious agents. The diagnosis of infection is complex and beyond the scope of this chapter.

Pulmonary function test: All patients who are able to perform forced expiratory maneuvers, usually those older than 6 years, should have periodic monitoring of pulmonary function.

Bronchoscopy or endoscopy may be necessary for diagnosis of specific lesions or infectious process.

Noninfectious pulmonary disease should be aggressively pursued with diagnostic lung biopsy because the pathology may indicate the prognosis. ^[9]

Lymph node biopsy

Typical histologic findings of lymph nodes from patients with common variable immunodeficiency are reactive follicular hyperplasia, atypical hyperplasia, or granulomatous inflammation.

Intestinal biopsy

Histology of intestine may demonstrate villous atrophy or infection with cryptosporidia or G lamblia. Presence or absence of plasma cells in the submucosal tissue can be examined by hematoxylin and eosin (H and E) stain, and immunohistologic stain may be informative. Lymphoid cells are found in intestinal submucosa of normal infants older than 15-20 days.

Malignancies complicating common variable immunodeficiency are staged by conventional guidelines for immunocompetent patients.