Laboratory evaluation of primary immunodeficiencies

Abstract

Primary immunodeficiencies are congenital disorders caused by defects in different elements of the immune system. Affected patients usually present clinically with recurrent infections, severe infections, or both, as well as autoimmune phenomena that are associated with many of these disorders. Early diagnosis is essential for referral to specialized care centers and the prompt initiation of appropriate therapy. In this article the authors describe a general approach for the investigation of the most common primary immunodeficiencies, outlining the typical clinical symptoms and most appropriate laboratory investigations.

The clinical spectrum of characterized primary immunodeficiencies (PID) has expanded significantly over the past 2 decades, and the underlying genetic basis of the majority of primary immunodeficiencies (PIDs) also has been identified. The accurate diagnosis of patients with PIDs is critical for appropriate therapy and also affords the opportunity to provide appropriate genetic counseling to the patient and his or her family. In virtually all cases the clinical symptoms involve increased susceptibility to infection, and early diagnosis and therapy provides the greatest opportunity to prevent significant disease-associated morbidity. In this setting the laboratory serves as the primary source of diagnostic information used to define the immunologic defect. The optimal use of the laboratory for the diagnosis and characterization of PIDs is the focus of this chapter.

Evaluating Suspected Antibody Deficiency Disorders

When to suspect

The majority of patients with primary antibody deficiencies present with recurrent bacterial infections of the sinopulmonary tract, including recurrent otitis media, sinusitis, and pneumonia (Table I).^1,2 The most commonly isolated organism is Streptococcus pneumoniae, but Haemophilus influenzae (often untypeable), Staphylococcus and Pseudomonas species are also seen. Diarrhea affects up to 25% of these patients, often associated with Giardia lamblia infection. However, infections with rotavirus, enterovirus, Campylobacter, Salmonella, and Shigella species might also be found.¹ In addition, autoimmune manifestations are seen in up to 25% of these patients, with autoimmune hemolytic anemia and autoimmune thrombocytopenia being most commonly observed. Finally, granulomatous disease involving various organs with particular predilection for the lung might also be present, and in some patients this process can result in significant morbidity.¹

Table I. Common pathogens and infection sites according to the underlying immune defect.

Affected immunity arm	Typical site of infection	Common pathogens
B cells	Sinopulmonary tract, GI tract, joints, CNS	Pyogenic bacteria: streptococci, staphylococci, Haemophilus influenzae
		Enteroviruses: ECHO, polio
		Mycoplasma species
T cells	Sepsis, lung, GI tract, skin	Viruses: CMV,
		adenovirus, measles, molluscum
		Fungi: Candida and Aspergillus species, Pneumocystis jiroveci
		Pyogenic bacteria
		Protozoa: Cryptosporidium species
Phagocytes	Skin infections, lymphadenitis, liver, lung, bone, GI tract, gingivitis/periodontitis	Bacteria: staphylococci, Serratia marcescens, Burkholderia cepacia, Klebsiella species, Escherichia coli, Salmonella species, Proteus species
		Fungi: Candida, Aspergillus, and Nocardia species
Complement	Systemic infections, meningitis	Pyogenic bacteria: streptococci, Haemophilus influenzae, Neisseria species

GI, gastrointestinal; CNS, central nervous system; ECHO, echovirus; CMV, cytomegalovirus.

PIDs that commonly manifest some degree of hypogammaglobulinemia include selective IgA deficiency, common variable immunodeficiency, and congenital agammaglobulinemias (both X-linked and autosomal recessive inheritance, Table II). Less common causes include agammaglobulinemia with thymoma (Good syndrome) and X-linked lymphoproliferative syndrome (XLP).¹ X-linked agammaglobulinemia should be suspected in all male patients with recurrent otitis and even a single episode of pneumonia, even if the family history is negative. This condition also might present with neutropenia and sepsis by Pseudomonas or Staphylococcus.³ Occasionally, the ataxia-telangiectasia syndrome manifests with recurrent infections and upper respiratory tract symptoms associated with IgA deficiency before the onset of overt neurologic signs.⁴ Concomitant bacterial sinopulmonary and opportunistic infections, including low pathogenic mycobacteria, should raise suspicion of a cellular defect that also affects antibody production, such as nuclear factor κB essential modulator (NEMO; also called IKK-γ) or CD40 ligand (CD154) deficiencies.^5,6 Selected complement deficiency and phagocytic defects might also have a clinical presentation similar to that of antibody deficiency and could be considered for investigation (Table II).

Table II. Differential diagnosis of antibody deficiencies and associated laboratory findings.

Primary B-cell disorders

Common variable immunodeficiency: low IgG and IgA levels, variable IgM levels, usually normal B-cell numbers

Selective IgA deficiency: low IgA levels, normal IgG and IgM levels, normal B-cell numbers

Congenital agammaglobulinemia: low IgG, IgA, and IgM levels; undetectable or very low B-cell numbers (<2%)

Specific antibody deficiency: normal IgG, IgA, and IgM levels; normal B-cell numbers; defective antibody response to vaccination

Agammaglobulinemia with thymoma (Good syndrome): low IgG and IgA levels, variable IgM levels, low B-cell numbers

Combined cellular and humoral disorders

Hyper-IgM syndromes: low IgG and IgA levels, normal, low or high IgM levels, normal B-cell numbers

Ectodermal dysplasia with immunodeficiency syndrome (NEMO/IκBα deficiency): variable immunoglobulin levels, normal B-cell numbers

XLP: low IgG and IgA levels, variable IgM levels, typically normal B-cell numbers

Ataxia-telangiectasia syndrome: low IgA levels

Other causes to consider

Drug-induced hypogammaglobulinemia; sickle cell disease with secondary hyposplenism; primary asplenia; immunodeficiency, centromeric instability, facial anomalies syndrome; cystic fibrosis; complement component deficiency; myelodysplasia; chronic lymphocytic leukemia; multiple myeloma; dysmotile cilia syndrome; warts, hypogammaglobulinemia, immunodeficiency and myelokathexis (WHIM) syndrome

Laboratory evaluation

The initial clinical laboratory screening of antibody-mediated immune function can be accomplished by measuring the levels of the major immunoglobulin classes IgG, IgA, IgM, and IgE (Table III). The results must be compared with age-matched reference intervals (normal ranges) that are typically provided as 95% CIs. There are no rigid standards regarding the diagnosis of immunoglobulin deficiency, although an IgG value of less than 3 g/L (300 mg/dL) in an adolescent or adult, as well as values clearly below the age-appropriate reference (95% confidence interval) in a child should trigger further evaluation. An additional and readily available test is quantitation of IgG subclass levels. This test is most useful in evaluating an IgA-deficient patient with significant recurrent bacterial infections. However, in most settings, detection of an IgG subclass deficiency still requires documentation of an abnormality in specific antibody production before initiating therapy, making this test of more limited utility. Measurement of specific antibody responses is useful in confirming defective antibody production and is essential when the total immunoglobulin levels are only modestly decreased (or even normal) in the setting of recurrent bacterial infection. The simplest method is evaluation for spontaneous specific antibodies (eg, anti–blood group antibodies [isohemagglutinins]) and antibodies to previous immunizations or infections. The definitive method to evaluate in vivo antibody production involves immunizing a patient with protein antigens (eg, tetanus toxoid) and polysaccharide antigens (eg, Pneumovax, Merck & Co, Inc, Whitehouse Station, NJ) and assessing preimmunization and 3- to 4-week postimmunization antibody levels. Guidelines for normal responses, which are usually provided by the testing laboratory, typically consist of finding at least a 4-fold increase in antibody levels and/or protective antibody levels after immunization. An alternative method to access the humoral immune response that is specifically useful in patients already receiving immunoglobulin replacement therapy involves vaccination with a neoantigen, such as the bacteriophage Phi X174; however, this is only available in some specialized centers.⁷

Table III. Evaluation of suspected antibody deficiency.

Screening tests

Quantitative immunoglobulins

Specific antibody levels

Circulating specific antibody levels to prior vaccines and blood group antigens (isohemagglutinins)

Pre/postimmunization antibody levels

Protein antigens

Carbohydrate antigens

IgG subclasses

Secondary tests

B-cell immunophenotyping

In vitro functional studies

Tests to exclude rare and secondary causes

Thoracic computed tomography to exclude thymoma (particularly useful if patient is >50 years old with low B-cell numbers)

Intracellular flow cytometry or genetic evaluation for BTK (XLA) or SAP/XIAP (XLP)

Genetic evaluation of NEMO to rule out anhydrotic ectodermal dysplasia with immune deficiency

Fecal α1-antitrypsin, urinary protein, serum albumin, absolute lymphocyte count to exclude gastrointestinal or urinary protein loss or lymphatic loss

HIV testing to exclude AIDS

Complement function (CH50, AP50) to exclude complement deficiency

Karyotype to exclude immunodeficiency, centromeric instability, facial anomalies syndrome

Sweat chloride or genetic evaluation to exclude cystic fibrosis

BTK, Bruton tyrosine kinase; XLA, X-linked agammaglobulinemia; SAP/XIAP, SLAM-associated protein/X-linked inhibitor of apoptosis.

Additional testing focuses on determining the presence or absence of B cells by using flow cytometry. This is particularly useful as a marker for congenital forms of agammaglobulinemia because this group of disorders typically is characterized by absent or extremely decreased circulating B-cell numbers based on the underlying defects that block B-cell development.² More recently, characterization of B-cell subsets, particularly directed at memory and immature B cells, has been put forward as a means of further characterizing patients with common variable immunodeficiency.⁸ Studies that test in vitro B-cell signaling and immunoglobulin biosynthesis are generally performed only in research centers.

Evaluating Suspected T-Cell or Combined T- and B-Cell Immunodeficiency Disorders

When to suspect

Patients affected by severe combined immunodeficiency (SCID) or other primary conditions with markedly abnormal T-cell function usually manifest failure to thrive and recurrent infections with opportunistic pathogens, such as Candida albicans (thrush), Pneumocystis jiroveci, or cytomegalovirus very early in life (Table I).⁹ Other common findings are chronic diarrhea, recurrent bacterial infections affecting multiple sites, and persistent infections despite adequate conventional treatment. SCID is a pediatric emergency because early diagnosis can dramatically improve the clinical outcome. Skin rashes are common, particularly with specific T-cell disorders, including Omenn and Wiskott-Aldrich syndromes.¹⁰ Other severe cellular or combined defects present with varied clinical symptoms, as listed briefly in Table IV.

Table IV. Most common T-cell and combined immunodeficiencies and distinctive features.

SCID: failure to thrive, chronic diarrhea, oral thrush, recurrent or severe bacterial, viral and/or fungal infections

CD40 and CD40 ligand deficiency: recurrent sinopulmonary and opportunistic infections with low IgG and IgA levels and variable IgM levels

Wiskott-Aldrich syndrome: easy bruising, eczema, recurrent otitis media, diarrhea, thrombocytopenia with small platelets

DiGeorge syndrome: hypoparathyroidism, cardiac malformations, dysmorphic features, variable T- and B-cell defects

Anhydrotic/hypohidrotic ectodermal dysplasia with immunodeficiency (NEMO or IκBα deficiency): recurrent mycobacterial or pyogenic infections, with or without skin, hair, and nail abnormalities; poor fever responses

XLP: hypogammaglobulinemia, persistent or fatal EBV infection

Chronic mucocutaneous candidiasis: recurrent oroesophageal and skin Candida species infection

Laboratory evaluation

Careful analysis of the white blood cell count and differential is of utmost importance when evaluating patients suspected of cellular immunodeficiency disorders. The absolute lymphocyte count must be compared with age-matched control ranges for proper interpretation. Severe lymphopenia in an infant (<3,000/mm³) is a critical finding that should prompt immediate immunologic evaluation if confirmed on a repeat test. The caveat in using low T-cell number during infancy as the screen to detect defects in T-cell development is that this would not identify patients with Omenn syndrome. In this disorder normal or increased T-cell numbers are typically found in the face of profound cellular immunodeficiency caused by an oligoclonal expansion of T cells.¹⁰ In addition, circulating T cells might also be seen in the face of a severe cellular immune defect as a result of maternal T-cell engraftment. The maternal T cells will consist of primarily memory CD45RO⁺ cells (compared with naive CD45RA⁺T cells found in a healthy infant) that do not provide host protection.¹¹ Finally, transfusion of nonirradiated blood products in the setting of a severe cellular immune defect will result in circulating donor T cells that can produce graft-versus-host disease, a potentially fatal process. This scenario emphasizes the need to irradiate any blood product used in an infant with a suspected T-cell deficiency.

HIV infection has to be ruled out in all patients with symptoms of cellular immunodeficiency, and this typically requires testing for the presence of virus (ie, HIV viral load assay) rather than serologic testing for anti-HIV antibody (Table V).

Table V. Evaluation of suspected T-cell and combined immunodeficiency.

Screening tests

HIV testing

Lymphocyte immunophenotyping

Delayed-type hypersensitivity skin testing

Secondary tests

T-cell proliferation (mitogens, alloantigens, recall antigens)

T-cell cytokine production

Flow cytometric evaluation of surface or intracellular proteins, such as CD40 ligand (CD154 on activated T cells), IL-2 receptor γ chain (CD132), MHC class I and II, IL-7 receptor α chain (CD127), CD3 chains, WASP

Enzyme assays: adenosine deaminase, PNP

FISH for 22q11 deletion

TREC numbers

TCR repertoire analysis

Mutation analysis

WASP, Wiskott-Aldrich syndrome protein; PNP, purine nucleoside phosphorylase; FISH, Fluorescence in situ hybridization; TREC, T-cell receptor excision circle.

After T-cell screening tests, the next step would be a directed assessment of cellular immunity (Table V). This includes immunophenotyping of T cells by means of flow cytometry together with in vitro functional testing (eg, proliferation and cytokine production assays).¹² The immunophenotyping for a patient suspected of having SCID not only helps to establish the diagnosis, but it can also point to the potential underlying genetic defect (Table VI).¹² It is important to carefully review the percentage and absolute numbers for all lymphocyte subsets, comparing them with age-appropriate reference ranges. Typically, defects in cytokine signaling molecules result in a T⁻B⁺NK⁻ phenotype, whereas mutations in DNA-editing proteins required for T- and B-cell receptor expression are associated with a T^-B^-NK⁺ phenotype; severe metabolic defects usually are toxic for all lymphocyte types, resulting in a T^-B^-NK^- phenotype (Table VI).

Table VI. Immunophenotypic findings and associated genetic defects in patients with SCID.

Phenotype	Pathway affected and genetic defect(s)
T−B+NK−	Cytokine signaling: IL-2 receptor γ, JAK3
T−B−NK+	DNA editing: RAG1/2, Artemis, ligase 4, Cernunnos
T−B−NK−	Metabolic defects: adenosine deaminase, AK2
T−B+NK+	Cytokine signaling: IL-7 receptor α chain
CD8+CD4−B+NK+	Positive selection/signaling: MHC class II, p56lck
CD4+CD8−B+NK+	Signaling: ZAP70

JAK3, Janus kinase 3; RAG, recombination-activating gene; AK2, adenylate kinase 2; ZAP70, zeta-chain associated protein kinase, 70 kD.

Other useful tests in special circumstances include fluorescence in situ hybridization for the 22q11 microdeletion found in the majority of patients with DiGeorge syndrome and specific enzyme assays to evaluate for adenosine deaminase and purine nucleoside phosphorylase (PNP) deficiencies.¹³ Evaluation for intracellular Wiskott-Aldrich syndrome protein expression by means of flow cytometry can be performed in selected centers to screen for possible Wiskott-Aldrich syndrome.¹⁴ Direct evaluation of T-cell function, as assessed by the proliferative response to mitogens, recall antigens, and/or alloantigens, is an important part of evaluating cellular immunity. The same sort of culture conditions can also be used to evaluate for cytokine production using the culture supernatant (alternatively, one can evaluate cytoplasmic cytokine expression using flow cytometry).¹⁵

Quantification of T-cell receptor excision circles (TRECs) and evaluation of the T-cell repertoire can be used for additional evaluation of immune status. TRECs are formed during the normal editing of the T-cell receptor (TCR) genes during T-cell differentiation and maturation within the thymus and persist within the cell as extragenomic circular pieces of DNA. TREC copies are diluted over time as the T cells proliferate after antigen encounter. Therefore naive T cells that have recently emigrated from the thymus will present relatively high TREC levels compared with those of aged, antigen-experienced T cells.¹⁶ TREC evaluation (also CD4⁺CD45RA⁺CD31⁺ T cells by flow cytometry) can be used as a diagnostic confirmation of low thymic output that would be found in DiGeorge syndrome or to monitor immune reconstitution after bone marrow transplantation. More recently, the quantification of TRECs on blood derived from the Guthrie card obtained from infants after delivery has been initiated as a neonatal screening tool for SCID (and other significant T-cell defects) in both Wisconsin and Massachusetts.¹⁷ The finding of low TREC levels in neonates should prompt immediate follow-up with immunophenotyping by means of flow cytometry. A recent report from Wisconsin suggests that this test has a very low rate of false-positive or inconclusive results (approximately 0.00009% and 0.0017%, respectively).¹⁸

Analysis of the T-cell repertoire can be useful in specific clinical situations. The T-cell repertoire in circulating T cells from healthy subjects includes expression of the majority of the 24 TCR Vβ chain families, which can be promptly assessed by flow cytometry.¹⁹ Alternatively, evaluation of TCR Vβ CDR3 region diversity can be performed by PCR and is commonly referred to as spectratyping. The PCR-amplified product from each of these Vβ families normally demonstrates a Gaussian distribution of variously sized PCR products, each differing by 3 nucleotides. In settings in which there is an oligoclonal T-cell population, such as is found in patients with Omenn and atypical DiGeorge syndromes, a very limited number of Vβ families will be represented, with each demonstrating a very distorted (non-Gaussian) distribution.¹⁹

Evaluating Suspected Phagocyte Dysfunction Syndromes

When to suspect

The clinical features of neutrophil dysfunction (including neutropenia) usually include recurrent bacterial and fungal infections of the skin, lymph nodes, lung, liver, bone, and, in some cases, the periodontal tissue (Table I).²⁰ The clinical pattern of infection often can help to discriminate the underlying problem. Common phagocyte defects and accompanying laboratory findings are presented in Table VII. Patients with neutropenia and those with leukocyte adhesion deficiency (LAD) tend to have recurrent cellulitis, periodontal disease, otitis media, pneumonia, and rectal or gastrointestinal infections with diminished inflammation and lack of pus formation.²⁰ Although LAD is accompanied by a persistent granulocytosis, there is effectively a tissue neutropenia caused by the underlying adhesion defect that prevents the directed movement of these phagocytic cells to sites of infection. Delayed umbilical cord separation is commonly seen in patients with LAD; however, LAD is very rare, and most infants whose cords persist for up to 1 month are actually healthy. In patients with cyclic neutropenia, there are short periods of fever, mouth ulcers, and infections recurring at intervals of 18 to 21 days in concert with the decreased neutrophil count. Other more common instances of neutropenia include drug-induced and immune-mediated neutropenia.

Table VII. Differential diagnosis of phagocyte defects and associated laboratory findings.

Chronic granulomatous disease: defective oxidative burst by means of DHR assay or NBT

Leukocyte adhesion defects

LAD1: low/absent CD18 and CD11 expression by means of flow cytometry; persistent leukocytosis

LAD2: Bombay phenotype; absent CD15 (Sialyl-Lewis X) expression

LAD3: mutation analysis only

Chediak-Higashi syndrome: giant lysosomal inclusion bodies observed on morphologic review of granulocytes (with partial albinism)

Griscelli syndrome type 2: neutropenia without inclusion bodies (with partial albinism)

Severe congenital neutropenia: persistent neutropenia; maturation arrest on bone marrow studies

Cyclic neutropenia: intermittent neutropenia requiring serial measurements

X-linked neutropenia: altered WASP expression by means of flow or mutation analysis

G6PD and MPO deficiency: abnormal functional enzymatic assay

Hyper-IgE syndrome: IgE level >2,000 IU/mL; low TH17 cell numbers

Other disorders to be considered

Drug-induced neutropenia; autoimmune/alloimmune neutropenia; hypersplenism; chronic mucocutaneous candidiasis; TCII deficiency; hyper-IgM syndrome, XLA; Schwachman-Bodian-Diamond syndrome; warts, hypogammaglobulinemia, immunodeficiency and myelokathexis (WHIM) syndrome

NBT, Nitroblue tetrazolium; WASP, Wiskott-Aldrich syndrome protein; G6PD, glucose-6-phosphate dehydrogenase; MPO, myeloperoxidase; XLA, X-linked agammaglobulinemia.

In contrast, patients with chronic granulomatous disease have significant problems with liver and bone abscesses, as well as pneumonias with selected organisms, including Staphylococcus aureus, Serratia marcescens, Burkholderia cepacia, and Nocardia and Aspergillus species.²¹ Furthermore, they tend to have a lower frequency of Escherichia coli and streptococcal species infections compared with patients with neutropenia or LAD.

Finally, patients with hyper-IgE syndrome present with recurrent skin abscesses and cavitary pneumonias caused by S aureus and other pyogenic bacteria and demonstrate chronic mucocutaneous candidiasis.²² In addition, they typically demonstrate specific nonimmunologic findings, such as coarse facial features, scoliosis, hyperextensible joints, increased risk for bone fractures, and delayed or failed shedding of primary dentition.²³

Laboratory evaluation

Screening studies directed at the evaluation of neutrophil function should start with a leukocyte count, differential, and morphologic review (Table VIII). The diagnosis of cyclic neutropenia requires multiple absolute neutrophil counts 2 to 3 times a week for at least 4 to 6 weeks.²⁴ A diagnosis of severe congenital neutropenia (Kostmann syndrome) is made with neutrophil counts of less than 0.5 × 10⁹/L on several occasions.²⁴ Bone marrow analysis is useful to exclude insufficient production because of neoplasia or other causes and to document other abnormalities, such as the maturation arrest typical of Kostmann syndrome.

Table VIII. Evaluation of suspected phagocyte defects.

Absolute neutrophil count and morphologic analysis: congenital neutropenia syndromes and Chediak-Higashi syndrome

Oxidative burst by means of DHR or NBT assays: chronic granulomatous disease; rarely complete G6PD or MPO deficiency

CD18 (also CD11a, CD11b, and CD11c) expression by means of flow cytometry: LAD1

CD15 expression by means of flow cytometry: LAD2

Bombay phenotype: LAD2

Anti-neutrophil antibodies: autoimmune neutropenia

Bone marrow biopsy: exclude defective myeloid production in neutropenia syndromes

Chemotaxis/phagocytosis assays: limited utility

NBT, Nitroblue tetrazolium; G6PD, glucose-6-phosphate dehydrogenase; MPO, myeloperoxidase.

If neutropenia and morphologic abnormalities are ruled out, the evaluation should be directed at assays that provide functional information about neutrophils. LAD workup involves flow cytometric assessment of the neutrophil adhesion molecules CD11 and CD18, the expression of which is absent or decreased on neutrophils (and other leukocytes) from patients with LAD1.²⁵ CD15 (Sialyl-Lewis X) expression is absent on neutrophils from patients with LAD2.²⁶

The neutrophil oxidative burst pathway can be screened with either the nitroblue tetrazolium tests or a flow cytometric assay (dihydrorhodamine 123 [DHR]), the results of both of which are abnormal in patients with chronic granulomatous disease, but the latter is a more sensitive test.²⁷

The diagnosis of autosomal dominant and sporadic hyper-IgE syndrome has been associated with heterozygous pathogenic mutations in the gene encoding signal transducer and activator of transcription (STAT) 3.^28,29 A consistent feature in this disorder is a very increased IgElevel(>2,000IU/mL),andmore recently, low to absent IL-17–producing T cells (T_H17) have been demonstrated.³⁰

Finally, evaluation of neutrophil-directed movement (chemotaxis) can be performed in vivo by using the Rebuck skin window technique, as well as in vitro with a Boyden chamber or a soft agar system. Abnormalities of chemotaxis have been observed after use of certain pharmacologic agents, as well as in patients with LAD, Chediak-Higashi syndrome, Pelger-Huet anomaly, and juvenile periodontitis. However, chemotactic tests are difficult to perform, very hard to standardize, and available in only a limited number of laboratories.

Evaluating Suspected Natural Killer and Cytotoxic T-Cell Defects

When to suspect

Deficiency in natural killer (NK) cell function has been described in a limited number of patients with recurrent herpes virus family infections. Another category of NK and cytotoxic T-lymphocyte defects results in an uncontrolled inflammatory response initiated in association with certain specific infections that produces multiple organ damage (hemophagocytic lymphohistiocytosis [HLH]). One of these disorders is XLP, which is usually asymptomatic until the patient has an EBV infection and then leads to an uncontrolled lymphoproliferative disorder that is often fatal without aggressive treatment.³¹ Importantly, approximately 30% of patients with XLP present with hypogammaglobulinemia without other symptoms. Bone marrow transplantation is the only long-term cure for XLP.³¹

The clinical manifestations of familial HLH are rather nonspecific, requiring a high suspicion index for early diagnosis.³² They include persistent fever, hepatosplenomegaly, neurological symptoms (ataxia and seizures), lymphadenopathy, and skin rashes. Diagnosis mandates an immediate therapeutic response and prompt referral for bone marrow transplantation because this is currently the only curative approach. Disorders caused by defective intracellular vesicle trafficking, such as Chediak-Higashi syndrome and Griscelli syndrome type 2, also commonly manifest with a secondary lymphohistiocytic syndrome.³²

Laboratory evaluation

Testing of NK cell function includes immunophenotyping NK cells by means of flow cytometry and assaying cytotoxicity with standard in vitro assays. Patients with XLP1 will demonstrate absent invariant-chain NK T cells in peripheral blood, as measured by CD3⁺Vα24⁺Vβ11⁺ staining.³¹ Additionally, intracellular flow cytometry can be used to evaluate for expression of SAP (SLAM-associated protein) and XIAP (X-linked inhibitor of apoptosis), the proteins defective in XLP1 and XLP2, respectively.^33,34 Absent protein would indicate disease, whereas normal expression could be the result of an abnormal protein that is not distinguished from the normal protein by means of antibody staining. Therefore this screening test would require further investigation directed at cell function when the protein is detected in a patient suspected of having XLP. HLH is commonly associated with cytopenias, including anemia and thrombocytopenia; increased liver function test results; hypofibrinogenemia; and hypertriglyceridemia.³² High ferritin and circulating soluble CD25 levels are also typical and represent laboratory findings used to establish the diagnosis of HLH.³² Low intracellular perforin expression, as determined by flow cytometry, can be used to diagnose HLH2, and decreased surface expression of CD107a (LAMP1, lysosomal-associated membrane protein 1) on NK cells after activation can predict the presence of mutations in MUNC13-4 and syntaxin 11.^35,36

Evaluating Suspected Defects Involving The Adaptive-Innate Immunity Interface

IL-12/23–IFN-γ pathways

An emerging concept in the field of PIDs is that monogenic disorders can cause recurrent severe infections involving 1 or a very restricted range of pathogens.³⁷ Recently, patients with severe invasive infections caused by low virulence or environmental Mycobacteria and Salmonella species have been found to harbor defects in genes encoding different components of the IL-12/23–IFN-γ pathway: the IFN-γ receptor 1 gene (IFNGR1), the IFN-γ receptor 2 gene (IFNGR2), the IL-12 receptor β1 gene (IL12RB1), IL12B, and STAT1.³⁸ The 2 most prevalent genetic defects among this group involve IL12RB1 and IFNGR1, typically resulting in absent cell-surface protein expression.³⁹ This can be readily assessed by using flow cytometry with monoclonal reagents specific for these 2 proteins.²⁵ In addition, there is an autosomal dominant defect affecting IFNGR1 that results in overexpression of the protein, and this also can be detected with flow cytometry.⁴⁰ Screening for other defects in IFN-γ signaling (abnormalities in IFNGR2 or STAT1) can be done by evaluating monocyte STAT1 phosphorylation (by means of flow cytometry or Western blotting) ex vivo in response to IFN-γ.⁴¹ Defects in IL-12 production can be tested by evaluating IL-12 production in response to ex vivo stimulation of mononuclear cells with LPS and IFN-γ.

Toll-like receptor and NF-κB signaling defects

Recently, recurrent infections involving S pneumoniae and Staphylococcus species have been associated with defects involving molecules of the Toll-like receptor (TLR) pathway, including IL-1 receptor–associated kinase 4 (IRAK4), MYD88 (myeloid differentiation primary response gene 88), and NEMO.^42-44 One of the distinctive features of patients with IRAK4 and MYD88 mutations is the markedly diminished inflammatory response to infection with little or no fever and acute-phase reactants observed.⁴⁵ NEMO deficiency is a more complex X-linked recessive disorder with a wide-ranging clinical phenotype and varied degree of immunologic abnormalities.⁵ Finally, susceptibility to herpes simplex encephalitis has been linked to mutations in the genes encoding the receptor, TLR3, and an accessory protein of the TLR pathway, unc-93 homolog (UNC-93B).^46,47 Additional defects in TLR function associated with specific clinical phenotypes are likely to be identified and represent an evolving field in clinical immunology. Currently, the evaluation of TLR function is confined to a limited number of centers that usually screen response by stimulating mononuclear cells with various TLR-specific ligands and measuring cytokine production. This can then be followed by direct sequencing of the suspected mutant gene or genes involved in the specific TLR signaling process. Recently, von Bernuth et al⁴⁸ described a simplified assay for the screening of TLR function that is reported to detect functional defects in the signaling process by using whole blood samples. This assay involves stimulation of leukocytes with a series of specific TLR ligands and then evaluating for CD62L shedding from granulocytes by using flow cytometry. In cells with intact TLR signaling pathways, CD62L is promptly shed from the cell surface in contrast to the failure of CD62L shedding in cells from patients with IRAK4 or UNC-93B deficiency. One caveat is that the sample has to be analyzed shortly after obtaining the blood sample to prevent interpretation problems resulting from spontaneous CD62L shedding.

The identification of this new class of defects has also opened up potentially new therapeutic approaches, including the use of IFN-γ to augment antibiotics in selected patients with recurrent mycobacterial disease. In the case of herpes simplex encephalitis, the findings that patients with UNC-93B and TLR-3 defects have diminished virally induced type 1 interferon production suggests that supplementation of conventional antiviral therapy with IFN-α could be beneficial in terms of decreasing morbidity, but this study has yet to be undertaken.⁴⁹

Evaluating Suspected Complement Disorders

When to suspect

The clinical setting in which complement defects should be suspected depends on the site of the defect. Abnormalities in the early components of the classical complement pathway (C1, C4, and C2) typically manifest as systemic lupus erythematosus–like autoimmunity, but recurrent sinopulmonary infections are also seen, especially in C2 deficiency.⁵⁰ Defects in C3 produce a clinical phenotype that is indistinguishable from an antibody defect, although this complement deficiency is markedly less frequent than humoral immunodeficiencies.⁵¹ Defects in the late components of complement producing defects in the generation of the membrane attack complex (C5-C9) present with increased susceptibility to infections with Neisseria species that might not manifest until adolescence or young adulthood.⁵¹ Clinically, these patients manifest neisserial meningitis, sepsis, or gonococcal arthritis. Alternative complement pathway defects, including properdin, factor B and factor D deficiencies also present with severe neisserial and other bacterial infections. Factor H deficiency is associated with atypical (not associated with diarrhea) hemolytic uremic syndrome or glomerulonephritis and also with secondary C3 deficiency that can result in recurrent pyogenic infections.⁵¹ Finally, C1 esterase inhibitor deficiency causes hereditary angioedema, whereas DAF (decay-accelerating factor) and CD59 defects are seen in patients with paroxysmal nocturnal hemoglobinuria.⁵¹

Laboratory evaluation

The best screening test for defects in the classical complement pathway is the total hemolytic complement activity (CH50) assay, whereas the AH50 assay screens for defects in the alternative complement pathway. Assuming correct handling of the serum sample (complement components are very labile), a classical complement component deficiency will result in virtual absence of hemolysis on CH50 testing in contrast to the markedly decreased but not absent results seen in diseases like systemic lupus erythematosus. A decreased AH50 test result suggests a deficiency in factor B, factor D, or properdin. A decrease in both CH50 and AH50 test results suggests deficiency in a shared complement component (from C3 to C9).

Selected component immunoassays are available in larger laboratories, whereas specific component functional testing is typically only available in a very limited number of specialized complement laboratories.

Evaluating Suspected Immune Dysregulation Disorders

When to suspect

Under this category are included monogenic autoimmune disorders, such as the autoimmune lymphoproliferative syndrome (ALPS); the immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX); and the autoimmune, polyendocrinopathy, candidiasis, ectodermal dystrophy syndrome (APECED; Table IX). Patients with ALPS present early in life with persistent nonmalignant lymphadenopathy and splenomegaly commonly accompanied by immune thrombocytopenia, hemolytic anemia, or both.⁵² Organ-specific or vasculitic-type autoimmunity is rarely seen in patients with ALPS. IPEX is an immunologic emergency and typically presents in the neonatal period with severe watery or bloody diarrhea, skin eczema, and type 1 diabetes.⁵³ An immediate diagnosis is mandatory because these children require aggressive immunosuppression to control the acute symptoms, and bone marrow transplantation is currently the only curative therapy that should be undertaken before islet cells are destroyed, if at all possible. Finally, APECED is characterized by endocrine organ–directed autoimmunity (adrenal insufficiency and hypothyroidism) and chronic mucocutaneous candidiasis.⁵⁴ Patients might also have type 1 diabetes, gonadal failure, pernicious anemia, autoimmune hepatitis, and cutaneous manifestations. This is usually not a life-threatening condition, and immunosuppression is usually not required, with specific therapy directed at the endocrine abnormalities.

Table IX. Main clinical and laboratory findings of immune dysregulation syndromes and causative genes.

Disorder	Distinctive clinical findings	Key laboratory findings	Gene(s) involved
ALPS	Lymphadenopathy, splenomegaly, autoimmune hemolytic anemia and/or thrombocytopenia, high risk for lymphomas	↑ CD3+αβ-TCR-αβ+ CD4−CD8− cells, hypergammaglobulinemia, Coomb positive, ↑ plasma IL-10 levels, ↑ serum vitamin B12 levels, ↑ soluble Fas ligand levels	FAS, FASL, CASP8, CASP10, NRAS
IPEX	Early-life enteritis, dermatitis, autoimmune endocrinopathy (usually type 1 diabetes)	↑ IgE levels, diminished FoxP3+ CD4 T-cell subpopulation	FOXP3
APECED	Adrenal insufficiency, hypothyroidism, chronic mucocutaneous candidiasis	Organ-specific autoantibodies	AIRE

FASL, Fas ligand; CASP8, caspase 8; CASP10, caspase-10, NRAS, neuroblastoma RAS viral oncogene homolog; FOXP3, forkhead box protein 3; AIRE, autoimmune regulator.

Laboratory evaluation

The diagnosis of ALPS currently requires the presence of compatible clinical symptoms and the presence of a characteristic T-cell population on immunophenotyping that expresses CD3 and αβ-TCR but does not express CD4 or CD8 markers, which are referred to as double-negative T cells (Table IX). Determination of this T-cell subpopulation requires the use of antibodies to αβ-TCR because most double-negative T cells in normal samples are γδ-TCR⁺ and are not relevant for establishing a diagnosis of ALPS. Normal ranges for αβ double-negative T cells should be established for each laboratory. At the National Institutes of Health, more than 1% of the total lymphocyte population is considered abnormal in adults. Other supporting features include hypergammaglobulinemia and increased plasma IL-10, vitamin B12, and soluble Fas ligand levels (J.B.O. and T.A.F., unpublished observations).⁵⁵ In addition, for a diagnosis of certainty, one must demonstrate defective lymphocyte apoptosis in vitro or the presence of a mutation on FAS, FASL (FAS ligand), CASP8 (caspase-8), CASP10 (caspase-10), or NRAS (neuroblastoma RAS viral oncogene homolog).^56-61

Screening for IPEX is based on demonstrating absent or diminished population of forkhead box protein 3 (Foxp3)– expressing CD4 T cells in the peripheral blood, as assessed by intracellular flow cytometry. Another common laboratory finding is a marked increase in IgE levels. The gold standard for diagnosis is the demonstration of mutations on the FOXP3 gene. However, in approximately 50% of patients with clinical findings compatible with IPEX, no mutation is demonstrated (Troy Torgerson, personal communication). Diagnosis of APECED in the setting of a clinically consistent phenotype currently requires sequencing of the AIRE (autoimmune regulator) gene.

Conclusion

Laboratory testing serves as the critical approach necessary for evaluating immune function in the setting of a patient with a history of recurrent infections, unusual infections, or both. The appropriate and directed use of immune function testing provides not only critical diagnostic information but also directs decisions regarding the most appropriate therapy. The latter is crucial to limit disease-associated morbidity. The use of the laboratory in evaluating the immune system should not follow a shotgun approach but rather should be a focused evaluation using specific testing in an orderly process based on the clinical history.

Abbreviations used

ALPS

Autoimmune lymphoproliferative syndrome

APECED

Autoimmune polyendocrinopathy, candidiasis, ectodermal dystrophy syndrome

DHR

Dihydrorhodamine 123

FOXP3

Forkhead box protein 3

HLH

Hemophagocytic lymphohistiocytosis

IFNGR1

IFN-γ receptor 1 gene

IFNGR2

IFN-γ receptor 2 gene

IL12RB1

IL-12 receptor β1 gene

IPEX

Immunodysregulation, polyendocrinopathy, enteropathy, X-linked syndrome

IRAK4

IL-1 receptor–associated kinase 4

LAD

Leukocyte adhesion deficiency

NEMO

Nuclear factor κB essential modulator, also called IKK-γ

NK

Natural killer

PID

Primary immunodeficiency

SCID

Severe combined immunodeficiency

STAT

Signal transducer and activator of transcription

TCR

T-cell receptor

TLR

Toll-like receptor

TREC

T-cell receptor excision circle

XLP

X-linked lymphoproliferative syndrome