Detailed Immunologic Laboratory Evaluation

Although frank hypogammaglobulinemia, neutropenia, and complete deficiency of a component of the classic complement pathway can be detected by the screening laboratory tests described previously, more detailed testing is necessary to detect more subtle immune deficiencies. This level of testing is also frequently necessary to characterize severe defects more completely.

Because of the possibility that clinically significant antibody deficiency may be present even when the total serum concentrations of the major immunoglobulin classes and IgG subclasses are normal, specific antibody production should be assessed in all cases in which the clinical presentation suggests recurrent bacterial infections, particularly of the respiratory tract, unless the major immunoglobulin classes themselves are absent or severely depressed. Specific antibody titers should be measured against polysaccharide as well as protein antigens (51,52). Although measurement of isohemagglutinins may be used to screen for the ability to produce antibodies against polysaccharides (the A, B, or both blood group substances in patients of other blood groups), the availability of measurement of antibodies against specific bacterial antigens (see later) has decreased dependence on those assays.

In cases in which pathogens have been isolated and identified (e.g., from effusions at the time of insertion of tympanostomy tubes, endoscopic drainage of paranasal sinuses, or expectorated or induced sputum samples), antibodies against those specific organisms should also be measured. In addition, antibodies against common immunizing agents should be measured. We usually request measurement of antibodies against tetanus and diphtheria toxins and several pneumococcal polysaccharides as well as H. influenzae type B polysaccharide (42,51). Testing for these and additional antibody titers are available in many commercial laboratories and are sometimes referred to as a humoral immunity panel.

An advantage of using these particular antigens is that they are contained in readily available, well-tested vaccines, which often have already been given to or will be clinically indicated for the patients in question, so that exposure to the antigen is definite. Obtaining titers before, as well as 4 to 8 weeks after, immunization allows comparison of the response to each antigen. The absence of a threefold rise in titer after immunization or failure to achieve protective levels indicates that the patient is unable to mount specific antibody responses. This may be seen either with protein or polysaccharide antigens and may indicate a failure to process properly or recognize an entire class of antigens, such as in what has been termed specific polysaccharide antibody deficiency, or certain particular antigens in what may be considered a "lacunar" defect.

In some rare cases, patients already receiving immunoglobulin infusions may require assessment of their own specific antibody production, which may be difficult because antibodies against many common antigens will have been acquired passively. In most cases, the immunoglobulin therapy can be stopped for a few months so that the patients can be immunized and their own antibody production measured while they are being reassessed clinically. If this is not possible, special test antigens, such as keyhole limpet hemocyanin and the bacteriophage fX174, can be obtained from specialized centers ( 53). Because most individuals and plasma donors have not been commonly exposed to these antigens, commercial immunoglobulin preparations do not contain antibodies against them, and they can be used to assess de novo specific antibody formation.

Specific T-cell function is most commonly tested by measuring the incorporation of 3H-thymidine into the newly formed DNA of rapidly proliferating lymphocytes after cultures of peripheral blood mononuclear cells are stimulated in vitro (54). Lectins, proteins generally derived from plants that bind specific polysaccharides, commonly present in surface glycoproteins on human cells and are frequently used as the stimuli in such assays. Because these proteins stimulate most human lymphocytes, regardless of prior antigen sensitization, they are called mitogens, and tests using them should be referred to as lymphocyte mitogen proliferation assays. Plant lectins often used as stimuli for mitogen proliferation assays include concanavalin A, phytohemagglutinin, and pokeweed mitogen. Incorporation of 3H-thymidine, a low-molecular-weight precursor, into high-molecular-weight cellular DNA in newly proliferating lymphocytes serves as the basis for the measurements, and the results may be expressed as the amount incorporated (in counts per minute) or as the ratio of incorporation in parallel cultures of mitogen-stimulated versus unstimulated lymphocytes, also referred to as the stimulation index. Mitogen stimulation tests are useful even in newborns who have not received any immunizations and may be particularly informative about lymphocyte function and immune competence in babies with partial T-cell deficiency, such as those with DiGeorge syndrome (55). Disadvantages of these tests include the requirements for several milliliters of blood, which may be prohibitive for small newborns; time constraints that may be imposed by the laboratory to facilitate isolation of the mononuclear cells during normal working hours; and the fact that the cells must be cultured for several days (usually 48 to 72 hours) before they are "pulsed" with 3H-thymidine to assess its incorporation.

To surmount these difficulties, many laboratories are now using flow cytometry assays based on the appearance on the lymphocyte plasma membrane of early activation markers such as CD69 (56). Mixed lymphocyte cultures, in which a patient's (or potential donor's) T cells are stimulated by a relative's lymphocytes that have been irradiated to prevent them from proliferating, are also used to test T-cell competence and to determine histocompatability in cases in which bone marrow transplantation is contemplated. Staphylococcal enterotoxins are also often employed as stimuli in proliferation assays because they function as "superantigens," which stimulate broad families of T cells by binding to parts of their T-cell receptors other than the antigen-binding site. The response to these superantigens is thus also independent of prior antigen sensitization.

The Cowen strain of Staphylococcus aureus may be used as a T-cell-independent stimulus for B-cell proliferation. T-cell proliferative responses to recall antigens may also be assessed using similar techniques, although because a smaller number of T cells will respond to any given antigen than to the more broadly reacting mitogens discussed previously, these tests commonly involve 4- to 5-day incubation periods before the 3H-thymidine is added and its incorporation determined.

Obviously, antigen responses can only be expected if it is documented that the patient has been exposed to the antigen in question. Thus, antigen stimulation tests are usually not useful in early infancy. However, if an older child is known to have received his or her scheduled immunizations, or if candidal infection has been obvious, the response to soluble candidal preparations and vaccine antigens such as tetanus toxoid may be useful. Thus, patients with normal responses to mitogens who fail to respond to candidal preparations may be considered to have chronic mucocutaneous candidiasis rather than a more pervasive T-cell defect, as might be seen in DiGeorge syndrome or HIV infection. In patients with opportunistic infections suggestive of AIDS or positive screening tests for HIV, confirmatory tests, such as Western blot, and quantitation of p24 antigen or viral load should be performed, and absolute CD4 number as well as T-cell function should be assessed as part of the detailed evaluation.

Detailed laboratory analysis in patients suspected of phagocyte disorders should include assessment of neutrophil chemotaxis and the oxidative respiratory burst that accompanies phagocytosis (37,57,58). Chemotaxis is assessed by measuring the migration of polymorphonuclear leukocytes through agar gels or across filters in specially designed Plexiglas (Boyden) chambers. The oxidative burst can be assessed by the nitroblue tetrazolium test, in which a soluble yellow dye is reduced to an easily visible insoluble blue intracellular precipitate ( 59). This is available in most hematology laboratories. Flow cytometric assays in which oxidized products are detected by fluorescence may also be employed (58). If the CH50 was abnormal on screening, the actual deficient component can be identified in reference laboratories that stock commercially available purified complement components and test systems. These laboratories can also screen for abnormalities of the alternative pathway, which may be indicated in patients who have recurrent bacterial infections or bacteremia and sepsis but in whom antibodies and the classic pathway have been found to be normal.

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