Laboratory Studies
Levels of serum immunoglobulin
The evaluation of patients with suspected hypogammaglobulinemia should include quantitative measurement of serum immunoglobulins. If these levels are normal and a humoral immunodeficiency still is suggested, antibody response to specific antigens (polysaccharide and protein antigens) should be determined. [13, 8, 7, 5] The impaired antibody responses to various pathogens in hypogammaglobulinemic states may make serological diagnosis of certain infections (eg, HIV, Epstein-Barr virus [EBV]) difficult. In these patients, nucleic acid detection methods (ie, PCR or reverse PCR) may be the best diagnostic tests for certain viral infections.
Perform serum protein electrophoresis for presumptive diagnosis of hypogammaglobulinemia or monoclonal protein. Quantitative methods using immunodiffusion or nephelometry are used for the precise measurements of each isotype of Ig. Enzyme-linked immunosorbent assay is used for IgE quantitation.
Values must be compared with age-standardized reference ranges.
Common variable immunodeficiency (CVID) is defined by IgG levels less than 2 standard deviations below the mean, with equally low levels of IgA, IgM, or both. [13, 10]
Serum IgA is less than 5 mg/dL, with normal IgG and IgM levels, in selective IgA deficiency. levels of IgG2 and IgG4 also may be decreased, especially in patients with sinopulmonary infections.
In hyper-IgM syndromes, IgM may be markedly increased to levels frequently higher than 1000 mg/dL. However, the level of IgM often gradually increases with time and may be normal in children. levels of IgG, IgA, IgE, and the lymphocytes bearing these antibodies are decreased. IgM response to antigens is possible, but IgG and IgA responses are absent or diminished.
Antibody response after immunization
Vaccination-associated antibodies to diphtheria, tetanus toxoid, and HIB are normally demonstrable in patients who have received these vaccines, reflecting memory B-cell responses. Neoantigen responses may better reflect a patient’s current ability to mount antibody responses.
Typically, immunization with unconjugated pneumococcal vaccine is used to assess the response to polysaccharides by comparison of pre- and post-immunization titers (generally, a 4-fold rise is considered adequate). [7] Vaccine-induced antibodies should be determined 4-8 weeks after pneumococcal immunization. Pneumococcal immunization should be repeated if the response is inadequate after the first immunization, and remaining titers should be determined 8-12 months later, if impaired immunologic memory is suspected.
Isohemagglutinins
IgM antibodies to A and/or B blood group antigens should be checked if the other tests results are normal and the patient is unable to mount a response to specific antigens. Antibodies to blood group antigens A or B would not be expected to be present if the patient's blood group is A or B respectively, or AB. These antibodies normally develop in the first year of life in response to ingestion of cross-reacting animal antigens in food.
The production of these antibodies is normal in protein-losing states, in contrast to extremely low levels in XLA.
Peripheral blood lymphocyte immunophenotyping
Peripheral B cell levels are variable.
Their number is normal in 75% of patients with CVID, but their surface phenotype may be immature.
T-lymphocyte number and function are intact in most cases of pure B-cell disorders.
Reversal of the ratio of helper (CD4) to suppressor (CD8) T cells has been reported in CVID, leading to nonreactive delayed-type hypersensitivity (DTH) test results. In combined T-cell and B-cell disorders, peripheral T cells are absent or decreased, with negative DTH test results.
Evaluation of cellular immunity
Cutaneous delayed-type hypersensitivity
Delayed-type hypersensitivity testing helps evaluate the memory response of cellular immunity to a previously encountered antigen. This test is not reliable in children younger than 1 year, and the response frequently is suppressed following viral and bacterial infections and during or after glucocorticoid therapy.
The test is read by measuring the induration 48-72 hours following administration of mumps skin test antigen or candidal antigen (at 1:100 wt/vol dilution; if no reaction, use 1:10 dilution), tuberculin (0.1 mL containing 2-10 IU of purified protein derivative), and trichophytin (1:30 wt/vol dilution). The test result is considered positive if the induration is greater than 5 mm (or >2 mm in children). Aqueous tetanus toxoid is no longer available for anergy panel testing.
Complete blood count (CBC)
The CBC may indicate lymphopenia or lymphocytosis, which may be seen with secondary causes of hypogammaglobulinemia (intestinal lymphangiectasia and chronic lymphocytic leukemia [CLL], respectively). The absolute lymphocyte count must be compared to age-specific norms because infants normally have higher counts than older children and adults. Immunophenotypic lymphocyte studies are useful in determining the most likely defect in infants with severe combined immunodeficiency (SCID) and may be required to diagnose CLL.
Renal studies
Renal disease in which protein loss causes hypogammaglobulinemia is easily diagnosed by quantitation of the total 24-hour urinary protein excretion.
Gastrointestinal studies
Protein-losing enteropathy that causes hypogammaglobulinemia may be more difficult to diagnose. Increased alpha1-antitrypsin (which is not present in normal diet) loss in the stool can be quantified in a 24-hour clearance procedure. Alternatively, a nuclear scan using technetium 99m dextran can be used to diagnose and localize protein-losing enteropathy.
Intestinal lymphangiectasia, which is sometimes considered a subset of protein-losing enteropathies, manifests not only with protein loss but also with lymphopenia. This occurs because of intestinal lymphatic blockade with resulting leakage of lymphatic fluid and cellular components into the lumen. Imaging and endoscopy are useful in diagnosing intestinal lymphangiectasia. However, this is often a "patchy lesion," and the diagnosis may be difficult.
Imaging Studies
Chest radiograph
In many patients with CVID and primary hypogammaglobulinemia, recurrent or chronic infections lead to abnormal findings on chest radiograph, such as interstitial infiltrates, bronchiectasis, emphysema or bullae, and scarring. Chest radiograph findings may be normal despite the presence of structural abnormalities. CVID patients often have hilar adenopathy and/or granulomata. [14]
Although chest radiograph is an appropriate follow-up test for these patients, some argue for the use of high-resolution computed tomography (HRCT) as the criterion standard.
The absence of a thymic shadow is a common finding in patients with SCID. Thymomas may be identified on chest radiograph in patients with Good syndrome.
Cupping and flaring of the costochondral junctions is typical for adenosine deaminase (ADA) deficiency.
High-resolution computed tomography (HRCT) and nuclear scanning
HRCT scans may uncover important lung abnormalities in patients with CVID and primary hypogammaglobulinemia. [15] These include, but are not limited to, pulmonary fibrosis, bronchiectasis, parenchymal scarring, pleural thickening, and, less commonly, emphysema or parenchymal nodules.
HRCT scans are more sensitive than chest radiograph for detecting asymptomatic structural changes of airways and lung parenchyma that sometimes occur despite appropriate intravenous immunoglobulin (IVIG) therapy. [15]
Imaging studies of the abdomen may show organomegaly. Splenomegaly may be observed in CVID in the absence of lymphoma or lymphoproliferative disease. Pathologic-appearing para-aortic and other abnormal abdominal lymph nodes may be stable findings in CVID; they should be monitored carefully and may require studies using other modalities (fluorodeoxyglucose positron emission tomography [FDG-PET] and/or biopsy) to rule out malignancy.
Other Tests
ADA levels should be measured in patients with SCID. The diagnosis of ADA deficiency is made by finding ADA levels less than 1% of the reference range. Cost-benefit analysis dictates that enzyme assays should be checked before genetic analysis. Also in the differential are mutations in purine nucleoside phosphorylase; this should be evaluated along with ADA levels. Tests can be done prenatally on amniotic fluid.
Absent or decreased Wiskott-Aldrich syndrome protein (WASP) can be determined by flow cytometry or western blotting. For Wiskott-Aldrich syndrome (WAS), sequence analysis determines 99% of mutations known to cause the disease entity.
Prenatal diagnosis of X-linked agammaglobulinemia (XLA), X-linked hyper-IgM syndrome (XHM), WAS, and ADA deficiency can be accomplished by restriction fragment length polymorphism (RFLP) using fetal blood, amniotic cells, or chorionic villus tissue.
The most consistent feature of individuals with XLA is the absence or extreme decrease in the number of B cells (CD19+ cells). The BTK gene contains the mutation.
Umbilical cord blood can be used in the prenatal diagnosis of some of these disorders. B cells are absent in XLA. T cells are absent in X-linked SCID. "Bald" lymphocytes found on scanning electron microscopy is diagnostic of WAS. Red blood cell ADA is decreased in fetuses with ADA deficiency.
Commercial laboratories are available for many of these tests. More information can be found at www.genetests.org.
Histologic Findings
Lymph node biopsy is not a necessary diagnostic test in these disorders and can be complicated by poor healing and infection. However, it should be considered for rapidly enlarging lymph nodes to rule out infection or malignancy.
Rectal biopsy in CVID and IgA deficiency may show plasma cell and lymphoid cell infiltrate in rectal tissue. The presence of G lamblia or cryptosporidia can be documented via intestinal biopsy, which may show findings similar to sprue.
Thymus biopsy is indicated only in the presence of thymoma.
In XLA, lymph node biopsy reveals underdeveloped or rudimentary germinal centers. The same finding also can be documented in the tonsils, Peyer patches, and appendix.
In CVID, lymphoid follicles in lymph nodes, spleen, and gut are characterized by hyperplastic B-cell areas.
The thymus in patients with X-linked SCID resembles fetal thymus and is characterized by lobules of undifferentiated epithelial cells and depleted T-cell areas and, occasionally, both T-cell and B-cell areas.
In ADA deficiency, remnants of Hassall bodies can be seen in the thymus.
Tables
IVIG |
SCIG |
Advantages |
|
Dosing — large dose Intermittent and infrequent intervals High levels of IgG |
No venous access Self infusion Small volume Steady state levels of IgG — less "wear-off’ effects Compatible with working lifestyle Home treatment — convenient Less expensive Rare IgA reactions |
Disadvantages |
|
Needs venous access Trained personnel and healthcare facilities for administration Large fluctuations — "wear-off" effects More adverse effects May not suit a busy working lifestyle |
More frequent dosing and injections Smaller volume/dose and multiple infusion sites Needs education Nonadherence issues likely |
