Evaluation of suspected immunodeficiency. (Cover Story).

The need to evaluate immune function has moved from the concern of specialists caring for the rare patient with primary immune disorders to an issue for nonspecialized clinicians, resulting at least in part from the emergence of HIV infection. This discussion reviews the general clinical situations that suggest immune dysfunction and links these to the approaches used to assess immune function.

The clinical scenario that suggests immune deficiency is a history of increased susceptibility to infection. The characteristic features of the recurrent infections provide critical insights into the most likely site of immune dysfunction, serving as an important guide to direct the laboratory evaluation of immune function. Thus, the medical history is critically important in determining which patient should be evaluated and what part of the immune system is most likely affected. In this context, the immune evaluation typically focuses on the adaptive immune response. A defense system of higher organisms that provides specifically tailored responses consisting of two "arms," the B lyrnphocyte (cell) or humoral system that produces antibodies and the cellular or T lymphocyte (cell) system that responds to a variety of intracellular microbes. (1,2) In addition, the phylogenetically older innate immune system is also crucial for normal host defense and components that can be readily evaluated, include phagocytic cells (e.g. neutrophils) and the complement system. (1) Initially, the common clinical findings associated with defects in each of the major categories noted above will be considered, and then a brief review of approaches to evaluate each component will be presented. It is important to recognize that the components noted above work cooperatively in the normal host defense but--for the sake of evaluation--are considered separately.

Defects in host defense that impact on specific antibody (immunoglobulin) production (humoral immunity) frequently result in recurrent infections that typically involve the lungs and sinuses caused by encapsulated bacteria such as Haemophilus influenzae or Streptococcus pneumoniae. (3,4) The normal immune response involves the production of antibodies directed against the capsular carbohydrates of these organisms, and this facilitates the elimination of the pathogen. Without this antibody response, the host has a diminished capacity to eliminate the organisms, resulting in decreased control of infection and recurrent pneumonias and/or sinusitis.

The typical clinical finding associated with defective T cell (cellular) immunity is recurrent and/or persistent infections with opportunistic organisms including Pneumocystis carinii, Mycobacterium avium intracellulare (MAJ), cytomegalovirus and Epstein Barr virus. (4,5) These infectious are uncommon in a normal host, but may be seen as a consequence of immunosuppressive therapy used in autoimmunity, cancer and transplantation. In the absence of this type of therapy, an opportunistic infection is strongly suggestive of immune deficiency that most often involves defective T cell function. These clinical observations establish that T cells are necessary to respond to and clear a variety of intracellular organisms.

Abnormalities in phagocytic cell (neutrophil) function, as well as significantly decreased numbers of these cells, permit recurrent and/or chronic cutaneous and deep-seated bacterial and fungal infections. (6) These typically include abscesses, pneumonias, periodontitis, osteomyelitis and occasionally, life-threatening sepsis. The clinical link between these infections and defective neutrophil function point to the critical role of mobile phagocytic cells in the normal host defense. Congenital defects in specific complement components can also be associated with recurrent infections, although in many cases these are also linked to the development of autoimmune disease. Deficiency of the complement component, C3, leads to repeated sinopulmonary infections similar to agammaglobulinemia, while deficiencies in the terminal complement components (C6, C7, C8, C9) result in recurrent systemic infections or meningitis with neisserial organisms. (7)

Assessment of immune function is appropriate when the medical history includes recurrent infection. Careful attention must be paid to the frequency and sites of infections, the types of organisms involved and the therapy required, as these provide clues that direct the laboratory evaluation. The family history may also prove important, since many defined immune deficiencies are genetically linked, although the incidence of these congenital (primary) immune disorders is quite rare. Any patient with a history of increased susceptibility to infection must be carefully questioned about risk factors for HIV infection, as this is the most common cause of nontherapy-related immune deficiency in the United States. It also is important to follow an orderly approach to evaluating the immune system based on the clinical history and findings.

The next sections outline the standard tests that facilitate evaluating the acquired and innate immune systems.

B cell function

As previously noted, the clinical findings suggestive of an abnormality in antibody production are recurrent or chronic bacterial infections, typically involving the lungs and/or sinuses. (3, 4) Additionally, there may be chronic conjunctivitis and rhinitis, as well as associated gastrointestinal, hematologic and autoimmune disorders.

The standard method for screening antibody-mediated immune function is accomplished by measuring the three major immunoglobulin classes, IgG, IgA and IgM. (8) Currently, the most common laboratory method for assessing immunoglobulin levels is done by nephelometry. It is critical to compare patient results with age-matched reference ranges, since there are significant differences between infants, children and adults. It is also important to recognize that reference ranges are most commonly expressed as 95 percent confidence intervals meaning that, 2.5 percent of controls (normals) are above and below the stated range. In addition, serum immunoglobulin levels are the net of protein production, utilization, catabolism and loss. This means that decreased levels can result from increased consumption or loss, as well as from decreased production.

There are no rigid standards regarding the diagnosis of immunoglobulin deficiency (hypogammaglobulinemia), although an IgG value below 3 g per liter (300 mg per deciliter), other than in early childhood, is strongly suggestive of antibody deficiency and--at the very least -- requires careful clinical monitoring. When any degree of hypogammaglobulinemia is associated with significant recurrent bacterial infection, it suggests the need for replacement therapy with intravenous immunoglobulin, and the patient should be referred to a specialist for evaluation and follow-up.

Measurement of a functional antibody response is particularly useful when the total immunoglobulin levels are normal or only slightly depressed, particularly when linked to a history of recurrent infection. The simplest method is evaluation for spontaneous antibodies (e.g., anti-blood group antibodies [isohemagglutinins], antibodies to prior immunizations). The definitive method is immunizing with specific antigens and assessing preimmunization vs. three-to four-week post-immunization antibody levels. This is typically done using both protein antigens (e.g., tetanus toxoid) and polysaccharide antigens (e.g., Pneumovax fl, Aventis Pasteur MSD Ltd). Guidelines for normal responses are usually provided by the testing laboratory and typically consist of at least a fourfold increase in antibody to a protein antigen and at least a twofold increase and/or protective levels of antibody following exposure to a polysaccharide antigen. There are caveats to these general rules, in that a protective titer prior to immuniz ation may not result in a significant rise in antibody level postimmunization, particularly following immunization with carbohydrate antigens. (9) The interpretation of this data is best left to specialists who see patients with immune disorders.

Despite the preponderance of recurrent opportunistic infections associated with HIV infection, appropriate testing to rule this out should be considered, even in the face of primarily recurrent bacterial infection. This type of clinical presentation is more common among young children infected with HIV. Special testing, such as a virus-specific nucleic acid test (reverse transcribed polymerase chain reaction [RT PCR] or a branched chain DNA testing) may be needed to rule out HIV infection in the face of absent or diminished antibody production, since the screening tests evaluate anti-HIV antibodies (ELISA and Western blot assays), or to circumvent possible contamination with maternal anti-NW antibodies in infants under 18 months of age.

An additional and readily available laboratory test is quantitation of IgG subclass levels. In most settings, however, detection of an IgG subclass deficiency still requires the demonstration of an abnormality in functional antibody production before replacement therapy (intravenous immunoglobulin) is indicated. Due to the expense of this test and the limited utility of the results, it has very little practical use in evaluating most cases of possible immune deficiency. A possible exception is in the evaluation of an IgA-deficient patient with significant recurrent infections, although utility in this setting remains to be fully substantiated.

Secondary testing of humoral immunity is performed in centers specializing in immunologic testing and typically includes evaluation of B cell number and testing of in vitro B cell function. The former assesses the number of B cells/B cell subsets and is performed with a panel of monoclonal antibodies and flow cytometry. (10) The latter involves studies that evaluate in vitro immunoglobulin biosynthesis induced by specific stimuli that may include selected cytokines to induce immunoglobulin class switching. The interpretation of these tests should be done by a clinical immunologist and must be considered in the context of the clinical story and the standard testing used to assess humoral immunity.

T cell function

A clinical history of recurrent opportunistic infections suggests an abnormality in T cell function. Immune deficiency involving T cells most commonly accompanies HIV infection, and initial screening assays should always include testing for NW infection. (4,5) In addition, the absolute lymphocyte count (i.e., white blood cell count with differential) and cutaneous delayed-type hypersensitivity (DTH) response to recall antigens can prove to be useful. (8) The significance of the former relates to the fact that T cells constitute approximately three-fourths of the circulating lymphocytes, thus a substantial decrease in circulating T cells typically results in a decreased lymphocyte count. In this setting, it is important to recognize that the absolute lymphocyte count differs significantly between infants, children and adults; thus, comparison with age-matched reference intervals is critical for the correct interpretation of patient results.

The DTH response provides an in vivo window of cell function in response to previously encountered antigens (recall antigens such as tetanus toxoid, candida antigen, mumps antigen). Failure to respond may reflect T cell dysfunction (T cell anergy) or it may indicate that the host has not been exposed (sensitized) to the antigen being used. Consequently, it is prudent to use more than one recall antigen for this test. Reliability of DTH testing is dependent on the antigen preparations, test application and interpretation (evaluation) of the response. This requires careful training and consistency among those performing DTH testing. Cutaneous response to poison ivy and other contact hypersensitivity reactions are equivalent reactions to the DTH skin test that may be noted during a clinical history.

Screening tests for T cell function are often followed by additional testing to complete the assessment of cellular immunity, and these typically are only available through large centers with immunology laboratories. The approach parallels that for evaluating humoral immunity with quantitation of T cells/T cell subsets using monoclonal antibodies and flow cytometry together with in vitro functional testing (e.g., proliferation assays, cytokine production, cellular cytotoxcity assays). (10,11) Recent data suggest that abnormalities of protein components of specific cytokine receptors may be associated with recurrent infections to a more limited range of opportunistic organisms such as Mycobacterium avium complex. This new information suggests that there are forms of immune deficiency that may be manifested as recurrent or chronic infections with a more limited range of microbes, or even one organism. The common feature in these patients is the failure of the infection to respond to conventional antimicrobial t herapy. (12) This represents an area of active investigation that may define a number of alternative immune disorders. Just as is the case for the B cell compartment, the interpretation of these additional tests should be provided by a clinical immunologist in the context of the clinical findings and the results of the screening tests.

Neutrophil function

The clinical features of neutrophil dysfunction usually include recurrent bacterial and fungal infections of the skin, periodontal tissue, lymph node, lung, liver and bone. (6) This clinical presentation is most commonly observed with neutropenia from decreased production and/or altered localization resulting from suppressive drug therapy. In addition, immune-based destruction of neutrophils, as well as certain primary and secondary abnormalities of neutrophil function, also can demonstrate patterns of increased susceptibility to infections.

The clinical pattern of infection often can help to discriminate the underlying problem. Patients with neutropenia and those with the leukocyte adhesion deficiency type 1 (LAD-l) tend to have recurrent cellulitis, periodontal disease, otitis media, pneumonia and rectal or gastrointestinal abscesses and poor wound healing. Although LAD-i is accompanied by a persistent granulocytosis, there is, in effect, a tissue neutropenia due to the underlying adhesion defect that interferes with neutrophil migration to sites of infection. In contrast, patients with chronic granulomatous disease (CGD) have a defective intracellular killing mechanism that results in significant problems with liver and bone abscesses, as well as pneumonias. This group of patients tends to have less difficulty with cellulitis or otitis media and lower frequency of beta-step and Escherichia coli infections than patients with neutropenia.

Screening studies directed at the evaluation of neutrophil function should start with the leukocyte count, differential and morphology." It us tinportant to obtain multiple absolute neutrophil counts because cyclic neutropenia can result in recurrent infections, and this could be missed if only a limited number of neutrophil counts are obtained. If neutropenia and morphologic abnormalities are ruled out, the evaluation should be directed at assays that provide functional information about neutrophils. Included are the flow cytometric assessment of neutrophil adhesion molecules to assess for the expression of [beta]-intergrins CD 11 and CD18 surface antigens since these are absent or depressed in patients with LAD-1. (13) The neutrophil oxidative burst pathway can be screened using either the nitroblue tetrazolium (NBT) test or a flow cytometric assay with dihydrorhodamine 123, both of which are abnormal in patients with CGD. (13) Finally, evaluation of neutrophil-directed movement (chemotaxis) can be performe d in vivo using the Rebuck skin window technique, as well as in vitro with a Boyden chamber or a soft agar system. The clinical utility of the chemotaxis assays remains to be defined, and they are primarily used in a research setting.

Functional testing of neutrophils has its greatest yield when evaluating patients with recurrent infections associated with a genetic neutrophil abnormality. Many patients with histories of recurrent cutaneous abscesses fail to demonstrate abnormalities in the above tests. This likely is related to the very specific measurement provided by certain tests and the relative insensitivity of the other tests in discerning mild abnormalities.

Complement testing

The best screening test for the classical complement pathway is the total hemolytic complement activity (CH50) assay. (7) Assuming correct handling of the serum sample, a markedly depressed CH50 result strongly suggests a complement component deficiency. This can be the result of C3 deficiency associated with recurrent sinopulmonary infections or late component (C6, C7, C8, C9) deficiency that is associated with recurrent neisserial infection. If the clinical history supports a possible complement component deficiency, and the CH5O is abnormal, the next step is to assess selected component levels using immunoassays that are available in larger laboratories. The final step in this type of evaluation is complement component functional testing, since it is possible to have a protein that is immunologically detectable using an immunoassay, but is defective when evaluated using a functional assay. These latter assays are only available, however, in very specialized complement laboratories.

Natural killer cell function

The third arm of the lymphoid system consists of circulating lymphocytes distinct from B and T cells, the natural killer (NK) cells. These cells function as "armed-and-ready" killer lymphocytes, as well as partners with T cells, in responding to a number of intracellular pathogens. Deficiency in NK cell function has been described in a very limited number of patients who most commonly have recurrent herpes infections. In addition, experimental models point to a role for the NK cell in allograft and tumor rejection. Testing of NK cells includes quantitating the cells by flow cytometry and assaying killing activity with standard in vitro assays. (10,11) The clinical utility of NK cell assessment has yet to be clearly established, despite their role in a variety of immunologic processes.

Recommendations

The clinical pattern of recurrent infections remains the single most useful clue in determining the likelihood of immune deficiency and identifying the best approach for laboratory evaluation. HIV infection has become the most likely cause of immune deficiency and appropriate diagnostic testing for HIV is critical, particularly in the setting of recurrent opportunistic infection. When the history identifies repeated bacterial infections involving the sinopulmonary tract, abnormalities in antibody production and C3 deficiency (extremely rare) should be considered. Infections with intracellular opportunistic organisms suggest T cell dysfunction, while bacterial and fungal infections of the skin, lungs and bone strongly suggest defective neutrophil function. All of these suggestions must be tempered by the fact that the range of infections between individuals can vary significantly and the line distinguishing normal from abnormal is not always clear. More limited immune dysfunctions (e.g., recurrent MAI) are now recognized clinically, but the immunologic basis for only a small proportion of these patients has been clearly defined.

Laboratory studies are essential for evaluating the status of immune function. The prudent use of these tests requires, however, that they not only be used in an orderly fashion, starting with the simpler screening tests, but that they also be selected according to the clinical clues provided by the history and physical exam. Furthermore, the results of these tests are relatively easy to interpret when they are either clearly normal or totally abnormal. The difficulty arises in determining the actual degree of immune dysfunction when the results fall in a gray zone. To address this, a combination of laboratory tests often helps to clarify the status of immune function, and interpretation should be left to a specialist who sees patients with immune disorders. It is very likely that, in the future, new testing approaches will evolve that can clearly identify and categorize more subtle changes in immune function that have clinical consequences at the level of diagnostics and therapeutics.

Table 2

EVALUATION OF ACQUIRED IMMUNITY

Screening tests

B cell function

* Quantitative immunoglobulins

* Specific antibody
   - Circulating specific antibodies
   - Postimmunization antibodies (protein and carbohydrate antigens)
   - IgG subclasses (+/-utflity)
   - HIV testing

T cell function

* HIV testing

* Lymphocyte count

* Delayed-type hypersensitivity skin tests

Secondary tests

B Cell function

* B cell enumeration (flow cytometry)

* In vitro B cell function tests

T cell function

* T cell enumeration (flow cytometry)

* T cell proliferation (mitogen, antigen)

* T cell cytokine production

* T cell cytotoxicity

Cells destined to become immune cells, like all blood cells, arise in
the bone marrow from so-called stem cells. Some develop into myeloid
cells, a group typified by the large, cell- and particle-devouring
white blood cells known as phagocytes; phagocytes include monocytes,
macrophages and neutrophils. Other myeloid descendants become granule-
containing inflammatory cells such as eosinophils and basophils.
Lymphoid precursors develop into the small white blood cells called
lymphocytes. The two major classes of lymphocytes are B cells and T
cells.

Table 3

EVALUATION OF INNATE IMMUNITY

Neutrophil function

* Absolute neutrophil count (multiple times)

* Neutrophil morphology

* Evaluation of [beta]-integrins (CD11, CD18)

* Evaluation of neutrophil oxidative burst

Complement testing

* Total hemolytic complement activity (CH50)

* Specific component testing

   - Immunoassay for specific components
   - Functional testing for specific components

Natural killer (NK) cell testing

* NK cell enumeration (flow cytometry)

* In vitro functional cytotoxicity assay

To attack, cytotoxic T cells need to recognize a specific antigen,
whereas natural killer or NK cells rely on an alternative recognition
system. Both types contain granules filled with potent chemicals, and
both types kill on contact. The killer binds to its target, aims its
weapons and delivers a burst of lethal chemicals.

The complement system consists of a series of proteins that work to
"complement" the work of antibodies in destroying bacteria.
Complement proteins circulate in the blood in an inactive form. The
so-called "complement cascade" is initiated via three different
initiation pathways. The classical pathway, diagrammed above, is
inactivated when the first complement molecule, Cl, encounters antibody
bound to antigen in an antigen-antibody complex. Each of the complement
proteins performs its specialized job in turn, acting on the molecule
next in line. The end product of all of these pathways is a cylinder
that punctures the cell membrane and, by allowing fluids and molecules
to flow in and out, dooms the target cell.

References

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(2.) Fleisher TA and Blessing JJH. Immune function. 2000. Peds Clin NA. 47:1197-1210

(3.) Bellow M. Primary immunodeficiency disorders: antibody deficiency. 2002. J Al-lergy Clin Immunol. 109:581-591

(4.) Buckley RH. Primary immunodeficiency diseases due to defects in lymphocytes. 2000. N Engl J Mod. 343: 1313-1324

(5.) Buckley RH. Primary cellular immunodeficiencies. 2002. J Allergy Clin Immunol. 109:747-757

(6.) Lekstrom-Himes JA and Gallin JI. Primary immunodeficiency diseases due to defects in phagocytes. 2000. N Engl J Mod. 343:1703-1714

(7.) Walport MJ. Advances in immunology: complement. 2001. N Engl J Med. 344: 1058-1066 and 1140-1144

(8.) Fleisher TA. Methods. In Sampter's Immunologic Diseases 6th edition. Eds Austen KF, Frank MA, Atkinson JP, Cantor H. 2001. Lipincott Williams and Wilkins. Philadelphia, 1229-1240

(9.) Go E and Ballas Z. Anti-pneumococcal antibody response in normal subjects. 1996. J Allergy Cue Immunol. 98:205-215.

(10.) Fleisher TA, Bleesing JJH, Marl G. Flow cytometry. In Clinical Immunology: Principles and Practice 2nd edition. Eds Rich RR, Fleisher TA, Shearer WT, Kotzin BL, Schroeder HW. 2001. Mosby. London. pp 121.1-121.12.

(11.) Nowak-Wegrzyn A and Lederman HM. Assessment of lymphocyte end monocyte function. In Clinical Immunology: Principles and Practice 2nd edition. Eds Rich RR, Finisher TA, Shearer WT Kotzin BL, Schroeder HW. 2001. Mosby. London. 1222.1-122.12

(12.) Uzel G and Holland SM. Th 1 T Cell and menocyte defects. 2000. Peds Clin NA. 100:270-276

(13.) Kuhns DB. Assessment of neutrophil function. In Clinical Immunology: Principles and Practice 2nd edition. Eds Rich RR, Fleisher TA, Shearer WT, Kotzin BL, Schroeder HW. 2001. Mosby. London. 123.1-123.10

Thomas A. Finisher, M.D., is chief of the Department of Laboratory Medicine at the Warren G. Magnuson Clinical Center of the National Institutes of Health, Department of Health and Human Services, in Bethesda, MD.