Evaluation of Suspected Immunodeficiency

Thomas A. Fleisher

Department of Laboratory Medicine, NIH Clinical Center, National Institutes of Health, DHHS, Bethesda, MD, USA, [email protected]

Abstract. The clinical utility and capacity to evaluate immunologic function has evolved significantly over the past few decades. This chapter summarizes screening methods and more sophisticated approaches to assess the immune system when there is a suspicion of an immune deficiency.

1. Introduction

The clinical utility and capacity to evaluate immunologic function has evolved significantly over the past few decades in parallel with the marked increase in understanding the human immune system (Fleisher and Oliveira 2004). In addition, the expanding range of characterized primary immune deficiency diseases and the secondary immunodeficiency pandemic resulting from HIV have added impetus to the development of new approaches for evaluating immunologic function.

Obtaining a thorough clinical history is the appropriate starting point to direct any laboratory evaluation for immunodeficiency. For example, a history of recurrent infections with encapsulated bacteria (e.g., Haemophilus influenzae and Streptococcus pneumoniae) usually affecting the sinuses and lungs suggests an antibody deficiency (Ballow 2002). In contrast, a clinical picture of recurrent infections with opportunistic organisms (e.g., Pneumocystis jiroveci, candida species, and cytomegalovirus) should focus the initial evaluation toward a T cell abnormality (Buckley 2002). The more recent identification that persistent nontuberculous myco-bacterial (NTB) infections can be associated with defects in the IFN-y-IL-12/23 circuit has opened a new appreciation of immune deficiencies that affect the interface between the adaptive and the innate immune systems (Filipe-Santos et al. 2006). In addition, the critical role of natural killer cells in host defense has been clarified more recently based on studies of patients demonstrating increased susceptibility to herpes family viruses (e.g., EBV and HSV) and in some cases accompanied by uncontrolled inflammation (Nichols et al. 2005; Filipovich 2006; Orange 2006).

Abnormalities in Toll-like receptor (TLR) function recently has been defined in patients with a specific pattern of bacterial infections (Ku et al. 2005). These findings suggest that additional innate immune defects impacting this early response pathway are likely. Innate immune defects affecting neutrophil function typically result in cutaneous and deep-seated abscesses, pneumonia, periodontitis, and osteomyelitis (Rosenzweig and Holland 2004). These infections are often caused by characteristic bacteria (e.g., Staphylococcus aureus and Serratia marcesens) and/or fungi (e.g., Aspergillus and Nocardia species). Congenital defects in specific complement components often are associated with autoimmunity as well as recurrent bacterial infections similar to the antibody deficiencies (Walport 2001). In addition, abnormalities of the late complement components are associated with a unique increase in susceptibility to Neiserrial infections (Figueroa and Densen 1991).

Thus, the clinical history and presentation should direct the immunologic evaluation and it is necessary to consider HIV infection in the differential diagnosis with appropriate questions during the history and laboratory testing. There are now more than 120 genetically linked immune disorders, so a careful family history is also extremely important (Notarangelo et al. 2006). Finally, the physical examination may provide clues regarding specific primary immunodeficiencies (e.g., typical facies in the hyper-IgE [Job] syndrome and scars from abscess drainage sites) and secondary immune disorders (e.g., oral hairy leukoplakia or Kaposi's sarcoma in HIV infection).

2. Screening Tests of Immune Function

The standard method for screening antibody-mediated immune function involves measuring the three major immunoglobulin classes: IgG, IgA, and IgM. These results must be compared with age-matched reference ranges and interpreted with the understanding that reference ranges are usually 95% confidence intervals, meaning that 2.5% of controls are above and below the stated range. Furthermore, serum immunoglobulin levels are the net of protein production, utilization, catabolism, and loss, so decreased levels can result from increased consumption or loss as well as from decreased production.

Measurement of a functional antibody response is particularly useful when the total immunoglobulin levels are modestly depressed or normal in the face of a strong history of recurrent bacterial infection. This can include evaluating "spontaneous" specific antibodies (e.g., antiblood group antibodies [isohemagglutinins] and antibodies to past immunizations). However, the definitive method is assessing pre- and postimmunization antibody levels using protein antigens (e.g., tetanus toxoid) or polysaccharide antigen (e.g., Pneumovac) vaccines (Go and Ballas 1996). Guidelines for interpreting the results are usually provided by the testing laboratory and typically consist of at least a fourfold increase in antibody and/or generation of protective antibody levels following immunization. Additional approaches to evaluating specific antibody production revolve around using a neoantigen such as a bacterio-phage phi X 174 (Pyun et al. 1989).

An additional and readily available antibody screening test is quantitation of IgG subclass levels; however, in most settings detection of a laboratory finding of IgG subclass deficiency would still require demonstrating an abnormality in functional antibody production in order to establish a clinically meaningful diagnosis.

Screening of the T cell compartment has far fewer test options and generally includes obtaining an absolute lymphocyte count (i.e., white blood cell count with differential) and possibly testing the cutaneous delayed-type hypersensitivity (DTH) response to recall antigens. 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 absolute lymphocyte count. This comparison must be made using age-matched reference intervals as the absolute lymphocyte count is substantially higher in infants and young children than in adults. The DTH response provides an in vivo window of T cell function in response to previously encountered antigens (recall antigens such as tetanus toxoid, candida antigen, and mumps antigen) (Blatt et al. 1993). However, failure to respond may reflect T cell dysfunction (T cell anergy), but it also could indicate, that the host has not been exposed (sensitized) to the antigen(s) being used. Consequently, it is prudent to use more than one recall antigen for this test and the reliability is dependent on the antigen preparations, test application, and interpretation (evaluation) of the response.

It is important to consider HIV infection as part of the screening process in an immune evaluation and this may require viral load testing in addition to serologic testing for antibodies to HIV.

Screening options of the innate immune system are rather limited consisting primarily of evaluating for the presence of neutrophils including reviewing their morphology and obtaining a total hemolytic complement assay (CH50).

Upon completion of medical history-directed screening tests, it may be necessary to utilize more sophisticated testing to develop or confirm the final diagnosis. This typically would involve quantitating and characterizing cells of the immune system, testing in vitro immune function, and potentially performing mutation analysis. These three general categories of testing will be considered in more detail.

3. Immune Cell Quantitation and Characterization

The capacity to study the various cells of the immune system depends on flow cy-tometry and is based on the surface expression of specific proteins. These can be detected using fluorochrome-labeled monoclonal antibodies with more than 300 specificities currently available. In the setting of primary immunodeficiency disorders, the application of this technology enables accurate determination of absent cell populations, cell subpopulations, or specific cell surface proteins as well as the biological effects of the primary immune deficiency. These studies are enhanced by the use of "multicolor" flow cytometry, and the future may see even greater possibilities with a recent report of 17-color analytical flow cytometry using a commercially available instrument (Perfetto et al. 2004). The utility of flow cytometry has been further extended by the introduction of intracellular flow cytometry to detect proteins found within the cell but not expressed on the cell surface. This approach can also be applied to cell function testing, a method that will be considered in the following section.

Evaluation of lymphocyte populations is accomplished by assessing the percentage and number of T, B, and NK cells. This is particularly useful in identifying and categorizing severe combined immunodeficiency (SCID) when evaluating an infant with failure to thrive and/or opportunistic infections usually in the setting of lymphopenia (Buckley 2002). From these studies, it is possible to generate four major groupings: T- B- NK- SCID (typical of ADA deficiency), T- B- NK+ SCID (characteristic of RAG 1/2 deficiency), T- B+ NK- SCID (found in common y-chain and JAK3 deficiency), and T- B+ NK+SCID (seen with IL-7 receptor alpha-chain deficiency). Low T cell percentage and number is also found in severe DiGeorge syndrome. Low CD8 T cell number in the setting of recurrent infection is compatible with ZAP70-deficient combined immune deficiency. In the setting of recurrent sinopulmonary infections, the absence of B cells (<1%) is strongly suggestive of either X-linked agammaglobulinemia (XLA) or autosomal recessive agammaglobulinemia (Figure 1) (Ballow 2002). However, it is not diagnostic as some patients with common variable immunodeficiency have very low to absent B cells.

Dihydrorhodamine Test

ptftt stimulated pm«.- Stimulated

Figure 1. Panel A: Histograms from a dihydrorhodamine 123 (DHR) test showing normal oxidative burst (green histogram) on the left side and markedly decreased oxidative burst from a patient with autosomal recessive CGD (right side). Panel B: Histograms from a DHR test from an X-linked CGD patient (left side) and the maternal carrier (right side).

ptftt stimulated pm«.- Stimulated

Figure 1. Panel A: Histograms from a dihydrorhodamine 123 (DHR) test showing normal oxidative burst (green histogram) on the left side and markedly decreased oxidative burst from a patient with autosomal recessive CGD (right side). Panel B: Histograms from a DHR test from an X-linked CGD patient (left side) and the maternal carrier (right side).

Focusing on the lack of expression of specific cell surface proteins can be an important approach to establishing the diagnosis of specific immunodeficiencies. As a group, these disorders usually involve genetic defects that do impact cell differentiation and release but also alter cell function. Examples include absence of CD40 on circulating B cells and monocytes or CD40 ligand (CD 154) on activated CD4 T cells, findings characteristic of two specific forms of the hyper-IgM syndrome (Notarangelo et al. 2006).

A more recently described group of disorders characterized by recurrent non-tuberculous mycobacterial (NTM) infection should direct the evaluation of such a patient to the function of IFN-y and IL-12/23 circuit (Filipe-Santos et al. 2006). At the cell characterization level, this would involve assessment of specific cytokine receptor expression, but due to limited reagent availability, only specific receptor components can currently be evaluated by flow cytometry (e.g., IFNyRl).

Flow studies in a patient with recurrent bacterial infections involving the skin, oral cavity, and other internal organs in the face of neutrophilia and inability to form pus suggest a problem with p-2 integrin expression (Rosenzweig and Holland 2004). Decreased or absent CD18 expression is found in the leukocyte adhesion deficiency type 1 (LAD1) and results in diminished CD11a, b, and c on neutrophils and lymphocytes.

The application of intracellular flow cytometry has found its way into the evaluation of a number of primary immune deficiencies. This approach requires fixation and permeabilization of the cell membrane to allow intracellular entry of the antibody reagent. The primary application in this setting is linked to those disorders in which there is a defect in a specific cytoplasmic or nuclear protein. It is important to recognize that this approach has the same limitation as any protein detection method (e.g., immunoassay) in detecting disease—the absence of the protein is essentially diagnostic but the presence of the protein generally does not distinguish between the presence of a functional and that of a nonfunctional protein. An example of intracellular flow cytometry for the evaluation of an immunodeficiency is detecting the presence of Bruton's tyrosine kinase (BTK) expression in monocytes or platelets (Lopez-Granados et al. 2005). This is a useful screening test for patients suspected of having XLA or in potential carriers of XLA where one would expect a mixture of normal (BTK expressing) and abnormal (BTK nonexpressing) monocytes or platelets. This type of analysis has also been applied in the evaluation of patients suspected of having Wiskott-Aldrich syndrome-testing for WASp; X-linked lym-phoproliferative syndrome (XLP)-evaluating for SH2D1A; hemophagocytic lymphohistiocytosis (HLH)-assaying for perforin; immune deregulation, polyendo-crinopathy, and enteropathy X-linked (IPEX)-testing for FOXP3; and chronic granulomatous disease (CGD)-evaluating for gp91 phox and p47 phox.

Another approach to cell characterization focuses on evaluating the distribution of the T cell receptor (TCR) repertoire using reagents that distinguish between each of the VP families. This method is complementary to TcR spectratyping, a PCR-based method that evaluates the diversity of each VP family and has proven useful in characterizing patient T cells in disorders such as the DiGeorge syndrome (Davis et al.

1997) and Omenn syndrome as well as the response to hematopoietic stem cell reconstitution therapy. An additional means of evaluating T cells using flow cytome-try has emerged to characterize the frequency of antigen-specific CD4 and CD8 T cells based on the binding of defined MHC-antigenic peptide tetramers (multimers) that are fluorescently labeled (Mallone and Nepom 2004).

Taken together, flow cytometry has become an effective laboratory adjunct in the evaluation and diagnosis of primary immune deficiencies. The addition of intracellu-lar tests to evaluate for the presence of protein defects associated with a number of these disorders has expanded the utility of flow cytometry as an important tool in the diagnosis of immunologic disorders.

4. Testing of Immune Function

The standard method for ex vivo evaluation of immune function has historically consisted of evaluating lymphocyte proliferation in response to mitogens (e.g., PHA and ConA) and recall antigens (e.g., tetanus toxoid). This usually involves evaluating cell division poststimulation by measuring tritiated thymidine incorporation. In addition, one can assess early cell activation by evaluating specific receptor expression and later events such as cytokine production either intracellularly by flow cy-tometry or secreted by ELISPOT at the cell level or by immunoassay to evaluate cell free supernatants (Letsch and Scheibenbogen 2003). Testing of cytokine expression and secretion have applications beyond immunodeficiency evaluations and are being applied in the evaluation of immunomodulatory therapy and the characterization of inflammatory diseases. Assessment of cytokine production forms the mainstay of the approach to evaluating patients with recurrent NTM infections. This also resulted in a new application of flow cytometry, evaluation of protein phosphorylation associated with cytokine stimulation. In the setting of patients with persistent NTM infections, quantitating STAT1 phosphorylation following IFN-y stimulation of monocytes has proven to be a very useful test in evaluating the IFN-y-IL12/23 circuit (Fleisher et al. 1999). Evaluation of cell activation at the phosphorylation of intracel-lular messengers has been expanded to include other cytokines such as IL-2 and IL-12 and their specific signaling molecules (STAT5 and STAT3, respectively) as a method to assess for other possible immune defects. The list of intracellular targets using these methods continues to expand and has the distinct advantage of providing high-sensitivity results in real time.

Cytolytic function can be evaluated in vitro and is directed at cytotoxic T cells, NK cells, and myelomonocytic cells. T cell-mediated cytotoxicity is MHC and antigenic peptide restricted that requires prior exposure to develop the cyto-lytic function. In contrast, NK cell-mediated cytolysis and antibody-dependent cellular cytotoxicity do not require prior sensitization and are dependent either on naturally occurring receptors (NK cells) or on antibody bound to the target (NK cells, monocytes, and macrophages) in a process referred to as antibody-dependent cellular cytotoxicity (ADCC).

The function of the thymus in generating new T cells can be evaluated using a PCR-based method to assess the frequency of T cells displaying T cell receptor excision circles (TRECs) (McFarland et al. 2000). These small pieces of extra chromosomal DNA are generated in T cells during the thymic selection process. Because this DNA segment is episomal, it does not increase in frequency during post-thymic cell division. Hence, identifying the frequency of TREC-positive T cells is a reflection of the number of recent thymic immigrants and likewise a measure of thymic output. As one would expect, TREC analysis is useful in the setting of evaluating the DiGeorge syndrome as well as assessing thymic dependent immune reconstitution following hematopoietic stem cell and thymic transplantation.

The third arm of the lymphoid system consists of circulating cells distinct from B and T cells, the natural killer (NK) cells. Deficiency in NK cell function has been described in a limited number of patients with recurrent herpes infections (Orange 2006). In addition, experimental models point to a role for the NK cell in allograft and tumor rejection. Another category of NK cell defects found in disorders with an uncontrolled inflammatory response (initiated by an infection) that can lead to multiple organ damage (HLH and XLP) (Filipovich 2006; Nichols et al. 2005). Testing of NK cell function includes immunophenotyping NK cells by flow cytometry and assaying cytotoxic activity using standard in vitro assays.

An area of intense current investigation involves the identification of disorders potentially associated with defective signaling by TLRs (von Bernuth et al. 2006). This is a family of at least 10 receptors that represent a phylogenetically more primitive arm of the immune system dependent on pattern recognition of bacterial, fungal, and viral products. An example of such a process is the activation of monocytes and macrophages by bacterial lipopolysaacharide (LPS). This pathway of activating the immune system appears to be one of the first lines in host defense, as it does not require prior exposure to the pathogenic organism. Recently, two different clinical phenotypes have been identified with genetic defects specifically involving TLR signaling. In one, there is a genetic susceptibility to serious bacterial infection that presents in childhood and generally improves during adolescence and is associated with an autosomal recessive defect in the intracellular protein IRAK4 (Ku et al. 2005). The most recently described defect is associated with the development of herpes simplex encephalitis and is linked to an autosomal recessive defect in an intracellular protein (UNC 93B). This function is part of the TLR signaling pathway although its exact function has not yet been definitively elucidated (Casrouge et al. 2006).

Evaluation of neutrophil function after neutropenia (including cyclic neutropenia) has been ruled out is generally focused on NADPH oxidase activity in patients with recurrent bacterial and fungal infections that is consistent with CGD. The primary means of testing currently involves a flow cytometry test with leukocyte loading of a fluorochrome precursor (dihydrorhodamine 123, DHR) that fluoresces following normal activation of NADPH oxidase-dependent electron transfer using an agonist like PMA (Vowells et al. 1995). The DHR assay is extremely reliable in diagnosing CGD and X-linked CGD carriers (Figure 1). Other methods have also been used to assess NADPH oxidase activity including the nitro blue tetrazolium (NBT) test and luminol-enhanced chemiluminescence. Although the clinical utility remains somewhat controversial, neutrophil-directed movement (chemotaxis) can be tested either in vivo using the Rebuck skin window or collection chamber technique or in vitro with a Boyden chamber or a soft agar system. Abnormalities of chemotaxis have been observed secondary to certain pharmacologic agents as well as the leukocyte adhesion deficiency, Chediak Higashi syndrome, Pelger-Huet anomaly, and juvenile periodontitis; however, the test is not specific for any diagnosis. A hallmark clinical feature of significantly abnormal chemotaxis is diminished neutrophil infiltration and decreased inflammation.

Evaluation of the complement pathway begins by screening the classical pathway using a CH50 assay and/or the alternate pathway using an AP50 test (Walport 2001). Interpretation of any result demonstrating decreased complement activity assumes correct handling of the serum sample (complement components are very heat labile) and should be repeated to confirm the abnormality. The next step would be to assess specific components using specific immunoassays recognizing the limitation of this approach relative to a component being produced that is dysfunctional. Ruling this possibility out requires component functional testing that is available only in very specialized complement laboratories.

5. Mutation Analysis

Identifying a specific gene mutation associated with a primary immunodeficiency provides the most definitive means of diagnosis. There is a growing list of specific genetic defects associated with primary immunodeficiencies (Notarangelo et al. 2006). Previously, screening methods (e.g., single-strand conformational polymorphism [SSCP] and dideoxy finger printing) were often applied in patient evaluations. However, with the ready availability of fluorescence-based sequencing and the advent of automated capillary sequencers, it is now practical to directly screen for mutations for virtually all genes associated with primary immunodeficiencies (Niemela et al. 2000). This is obviously applied only if there is strong evidence for a particular type of immunodeficiency based on a family history or a clinical history with definitive abnormalities on screening tests. The initial laboratory often serves to direct the mutation analysis by identifying specific characteristics associated with a diagnosis (e.g., absent or very low B cells in XLA and absent T and NK cells with B cells present in X-linked SCID). The information generated by mutation analysis has significant implications not only in establishing a definitive diagnosis but also for carrier assessment for prenatal diagnosis, and for offering genetic counseling.

6. Conclusions

The clinical pattern of recurrent infections provides the critical starting point in pursing a diagnosis of immunodeficiency and directing the best laboratory approaches for patient evaluation. Infections that are recurrent and difficult to treat or those that involve unusual organisms should raise suspicion of an underlying immunodeficiency. HIV infection has become the most likely cause of immunodeficiency, and early appropriate diagnostic testing for HIV is critical. The prudent use of laboratory tests requires that they be used in an orderly fashion, starting with the simpler screening tests chosen based on the patient history. The results generated are usually easy to interpret when they are either clearly normal or abnormal. The difficulty arises in determining the actual degree of immune dysfunction when the results fall in a gray zone, a circumstance that can be clarified by applying additional tests that can clarify the status of immune function or dysfunction. The increasing capacity to identify gene mutations linked to specific immunodeficiencies provides the most definitive evidence for a diagnosis and affords the opportunity to perform family studies and provide appropriate genetic counseling. The laboratory has emerged as the most critical source of diagnostic information in the characterization of primary immunodeficiency disorders.


Ballow, M. (2002) Primary immunodeficiency disorders: antibody deficiency. J. Allergy

Clin. Immunol. 109, 581-591. Blatt, S.P., Hendrix, C.W., Butzin, C.A., Freeman, T.M., Ward, W.W., Hensley, R.E., Melcher, G.P., Donovan, D.J. and Boswell, R.N. (1993) Delayed-type hypersensitivity skin testing predicts progression to AIDS in HIV-infected patients. Ann. Intern. Med. 119, 177-184.

Buckley, R.H. (2002) Primary cellular immunodeficiencies. J. Allergy Clin. Immunol. 109, 747-757.

Casrouge, A., Zhang, S.Y., Eidenschenk, C., Jouanguy, E., Puel, A., Yang, K., Alcais, A., Picard, C., Mahfoufi, N., Nicolas, N., Lorenzo, L., Plancoulaine, S., Senechal, B., Geissmann, F., Tabeta, K., Hoebe, K., Du, X., Miller, R.L., Heron, B., Mignot, C., de Villemeur, T.B., Lebon, P., Dulac, O., Rozenberg, F., Beutler, B., Tardieu, M., Abel, L. and Casanova, J.L. (2006) Herpes simplex virus encephalitis in human UNC-93B deficiency. Science 314, 308-312. Davis, C.M., McLaughlin, T.M., Watson, T.J., Buckley, R.H., Schiff, S.E., Hale, L.P., Haynes, B.F. and Markert, M.L. (1997) Normalization of the peripheral blood T cell receptor V beta repertoire after cultured postnatal human thymic transplantation in Di-George syndrome. J. Clin. Immunol. 17, 167-175. Figueroa, J.E. and Densen, P. (1991) Infectious diseases associated with complement deficiencies. Clin. Microbiol. Rev. 4, 359-395. Filipe-Santos, O., Bustamante, J., Chapgier, A., Vogt, G., de Beaucoudrey, L., Feinberg, J., Jouanguy, E., Boisson-Dupuis, S., Fieschi, C., Picard, C. and Casanova, J.L. (2006) Inborn errors of IL-12/23- and IFN-gamma-mediated immunity: molecular, cellular, and clinical features. Semin. Immunol. 18, 347-361. Filipovich, A.H. (2006) Hemophagocytic lymphohistiocytosis and related disorders. Curr.

Opin. Allergy Clin. Immunol. 6, 410-415. Fleisher, T.A., Dorman, S.E., Anderson, J.A., Vail, M., Brown, M.R. and Holland, S.M. (1999) Detection of intracellular phosphorylated STAT-1 by flow cytometry. Clin. Immunol. 90, 425-430.

Fleisher, T.A. and Oliveira, J.B. (2004) Functional and molecular evaluation of lymphocytes. J. Allergy Clin. Immunol. 114, 227-234; quiz 235. Go, E.S. and Ballas, Z.K. (1996) Anti-pneumococcal antibody response in normal subjects:

a meta-analysis. J. Allergy Clin. Immunol. 98, 205-215. Ku, C.L., Yang, K., Bustamante, J., Puel, A., von Bernuth, H., Santos, O.F., Lawrence, T., Chang, H.H., Al-Mousa, H., Picard, C. and Casanova, J.L. (2005) Inherited disorders of human Toll-like receptor signaling: immunological implications. Immunol. Rev. 203, 10-20.

Letsch, A. and Scheibenbogen, C. (2003) Quantification and characterization of specific T cells by antigen-specific cytokine production using ELISPOT assay or intracellular cytokine staining. Methods 31, 143-149.

Lopez-Granados, E., Perez de Diego, R., Ferreira Cerdan, A., Fontan Casariego, G. and Garcia Rodriguez, M.C. (2005) A genotype-phenotype correlation study in a group of 54 patients with X-linked agammaglobulinemia. J. Allergy Clin. Immunol. 116, 690-697.

Mallone, R. and Nepom, G.T. (2004) MHC Class II tetramers and the pursuit of antigen-specific T cells: define, deviate, delete. Clin. Immunol. 110, 232-242.

McFarland, R.D., Douek, D.C., Koup, R.A. and Picker, L.J. (2000) Identification of a human recent thymic emigrant phenotype. Proc. Natl. Acad. Sci. USA 97, 4215-4220.

Nichols, K.E., Ma, C.S., Cannons, J.L., Schwartzberg, P.L. and Tangye, S.G. (2005) Molecular and cellular pathogenesis of X-linked lymphoproliferative disease. Immunol. Rev. 203, 180-199.

Niemela, J.E., Puck, J.M., Fischer, R.E., Fleisher, T.A. and Hsu, A.P. (2000) Efficient detection of thirty-seven new IL2RG mutations in human X-linked severe combined immunodeficiency. Clin. Immunol. 95, 33-38.

Notarangelo, L., Casanova, J.L., Conley, M.E., Chapel, H., Fischer, A., Puck, J., Roifman, C., Seger, R. and Geha, R.S. (2006) Primary immunodeficiency diseases: an update from the International Union of Immunological Societies Primary Immunodeficiency Diseases Classification Committee Meeting in Budapest, 2005. J. Allergy Clin. Immunol. 117, 883-896.

Orange, J.S. (2006) Human natural killer cell deficiencies. Curr. Opin. Allergy Clin. Immunol. 6, 399-409.

Perfetto, S.P., Chattopadhyay, P.K. and Roederer, M. (2004) Seventeen-colour flow cytometry: unravelling the immune system. Nat. Rev. Immunol. 4, 648-655.

Pyun, K.H., Ochs, H.D., Wedgwood, R.J., Yang, X.Q., Heller, S.R. and Reimer, C.B. (1989) Human antibody responses to bacteriophage phi X 174: sequential induction of IgM and IgG subclass antibody. Clin. Immunol. Immunopathol. 51, 252-263.

Rosenzweig, S.D. and Holland, S.M. (2004) Phagocyte immunodeficiencies and their infections. J. Allergy. Clin. Immunol. 113, 620-626.

von Bernuth, H., Ku, C.L., Rodriguez-Gallego, C., Zhang, S., Garty, B.Z., Marodi, L., Chapel, H., Chrabieh, M., Miller, R.L., Picard, C., Puel, A. and Casanova, J.L. (2006) A fast procedure for the detection of defects in Toll-like receptor signaling. Pediatrics 118, 2498-2503.

Vowells, S.J., Sekhsaria, S., Malech, H.L., Shalit, M. and Fleisher, T.A. (1995) Flow cy-tometric analysis of the granulocyte respiratory burst: a comparison study of fluorescent probes. J. Immunol. Methods 178, 89-97.

Walport, M.J. (2001) Complement. First of two parts. N. Engl. J. Med. 344, 1058-1066.

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