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Laboratory evaluation of the immune system

Laboratory evaluation of the immune system
Manish J Butte, MD, PhD
Section Editor:
Luigi D Notarangelo, MD
Deputy Editor:
Anna M Feldweg, MD
Literature review current through: Nov 2022. | This topic last updated: Feb 08, 2022.

INTRODUCTION — Inborn errors of immunity (IEI), which is the preferred terminology for primary immunodeficiency disorders, most often come to clinical attention because of an increase in the incidence or severity of infectious illness beyond what is considered "normal." Patients with IEI diseases may also have immune dysregulation that includes unusual or severe autoimmunity, lymphoproliferation, and fevers and severe inflammation that are unprovoked (autoinflammation). Some forms of IEI are also characterized by an increased risk of malignancies. This topic review will provide a general approach to the laboratory evaluation of the immune system, beginning with screening tests and progressing through the indications for more advanced immunologic testing. Indications for referral to a specialist are discussed, and links to more detailed topics about the different groups of disorders are provided here and throughout the topic.

(See "Primary humoral immunodeficiencies: An overview".)

(See "Combined immunodeficiencies".)

(See "Primary disorders of phagocyte number and/or function: An overview".)

(See "Laboratory evaluation of neutrophil disorders".)

(See "Overview and clinical assessment of the complement system".)

(See "Autoimmunity in patients with inborn errors of immunity/primary immunodeficiency".)

INITIAL APPROACH TO THE PATIENT — Inborn errors of immunity (IEIs) should be considered once the more common causes of recurrent infection, autoimmunity, and inflammation have been excluded. The initial approach to a child or adult with recurrent infections is described separately. (See "Approach to the child with recurrent infections" and "Approach to the adult with recurrent infections".)

Immune dysregulation can result in disorders other than recurrent infections including:

Autoimmune disorders, such as autoimmune hemolytic anemia

Inflammatory disorders, such as inflammatory bowel disease or inflammatory arthritis

Nonmalignant lymphoproliferation (lymphadenopathy, splenomegaly, lymphoid aggregates in target organs)

Malignancies, such as lymphoma

Allergic disease, such as atopic dermatitis, food allergy, allergic rhinosinusitis, and asthma

Before initiating immunologic testing, the clinician should perform a thorough clinical history and physical examination. In infants and children, height and weight records should be reviewed, as failure to thrive and poor growth are consistent with immunodeficiency. In patients with possible IEIs, important historical elements include:

The nature of the infections – This should include the frequency, chronicity, severity, and response to therapy. Other considerations include what organ system(s) is involved and what type of organism has been identified in the past (ie, viral, bacterial, fungal, opportunistic). Patterns of infections can suggest specific immune defects.

Age of onset of illness, since different immune problems present in infancy, childhood, and adulthood.

The patient's sex, because X-linked defects are mostly or exclusively seen in boys.

Family history of infections, abnormal inflammatory responses, and autoimmune disorders, as well as any childhood deaths.

Family history of lymphoproliferative illnesses, including occurrence of splenectomy.

Any associated nonimmunologic symptoms and signs, as revealed by a complete review of systems.

Physical examination — Physical examination findings that suggest inborn errors of immunity in children and adults are reviewed separately. (See "Approach to the child with recurrent infections", section on 'Physical examination' and "Approach to the adult with recurrent infections", section on 'Physical examination'.)

Initial screening laboratory tests — In patients of any age, the laboratory evaluation of the immune system begins with general studies including:

Complete blood count (CBC) with differential – Lymphopenia is characteristic of a variety of combined immunodeficiencies (ie, cellular and antibody deficiency). Lymphopenia is defined as an absolute lymphocyte count <1500 cells/microL in adults or <2500 cells/microL in infants. Neutropenia can be found in primary phagocyte disorders, as well as in neutrophil disorders that lead to secondary immunodeficiency [1]. Leukocytosis is sometimes noted and suggests chronic infection. Monocytopenia is seen in GATA2 deficiency. Eosinophilia can be seen in primary atopic disorders and in several IEIs.

Chemistry panels – Chemistry panels should be evaluated to assess for metabolic disorders (diabetes mellitus, renal disease) that might cause secondary immunodeficiency. Hypoalbuminemia or low serum proteins suggest malnutrition or protein loss. Markedly elevated globulin levels may be seen in gammopathies or chronic infections.

Urinalysis – Urinalysis should be performed to detect proteinuria, casts, or cells, which suggest nephritis.

Tests to evaluate for specific infections, if indicated by the presentation – Tests for specific infections include appropriate cultures, serologies, and sinus imaging. Note that antibody testing for acute or historical evidence of infection may not accurately reflect past infections in patients with IEIs that involve impaired antibody function. Sinus imaging may uncover chronic rhinosinusitis in patients with immunodeficiency. Children or adolescents with nasal polyposis should be evaluated for cystic fibrosis, which is a cause of frequent sinopulmonary infections.

Chest radiograph – In infants, a chest radiograph showing the absence of a thymic shadow is a potentially critical finding (image 1). An ultrasound of the anterior chest should be performed next to confirm the lack of thymic tissue, which is a feature of severe combined immunodeficiency (SCID) and should prompt an urgent evaluation for severe forms of IEI. However, there are other causes of thymic involution as the thymus responds to stress by shrinking in infants, and the presence of a thymic shadow does not exclude SCIT. (See "Severe combined immunodeficiency (SCID): An overview", section on 'Clinical manifestations'.)

In older children and adults, chest radiographs may show scarring from past infections, interstitial lung disease, or bronchiectasis. Hyperinflated lung fields suggest chronic obstructive lung disease or chronic asthma.

Erythrocyte sedimentation rate and/or C-reactive protein – Nonspecific elevations in acute-phase reactants can be seen with infectious and inflammatory disorders and suggest the need for further evaluation.

Referral — More advanced immunologic tests require expertise to perform and interpret, may not be widely available, and are often costly. In addition, knowledge about the possible diagnoses in question is invaluable in deciding the type of testing to pursue first [2]. Thus, immunologic testing is best performed in a graded fashion, and referral to an immunologist should be sought early in the process, when possible.

Ultimately, definitive diagnosis may require specialized flow cytometric, genetic, and other advanced tests available in reference laboratories [3,4] or some children's hospitals. The website of the Immune Deficiency Foundation offers the ability for clinicians to request consultations with expert immunologists.

CATEGORIES AND PREVALENCE OF INBORN ERRORS OF IMMUNITY — Inborn errors of immunity (IEIs) may be grouped into categories based upon the aspects of the immune system that are predominantly affected and their approximate frequency [5]:

Predominantly antibody deficiencies (55 percent)

Immunodeficiencies affecting cellular and humoral immunity (15 percent)

Congenital defects of phagocyte number or function (10 percent)

Diseases of immune dysregulation (5 percent)

Autoinflammatory disorders (5 percent)

Complement deficiencies (4 percent)

Combined immunodeficiencies with associated or syndromic features (3 percent)

Defects in intrinsic and innate immunity (3 percent)

Bone marrow failure syndromes

Each of these categories of disorders has characteristic clinical manifestations, although there are overlaps among the clinical presentations of the groups. In addition, several conditions have been described that phenocopy inborn errors of immunity. These include disorders due to somatic mutations in immune-related genes, as well as diseases associated with autoantibodies against cytokines, growth factors, and other immune molecules [5].


Antibody deficiency and defects — Antibody deficiency most frequently results in recurrent and severe sinopulmonary infections with encapsulated bacterial strains (eg, Streptococcus pneumoniae, Haemophilus influenzae). Children commonly present with recurrent otitis media, rhinosinusitis, and pneumonia. Adults present similarly, although otitis media is less common. Viral infections of the respiratory tract also occur with greater frequency and severity in these patients. Clinical presentation is discussed in more detail separately [6]. (See "Primary humoral immunodeficiencies: An overview".)

The age of the patient can help narrow the differential diagnosis:

The most common antibody defects that present in infancy are physiologic hypogammaglobulinemia (to age six months), transient hypogammaglobulinemia of infancy (after six months, often to the age of six years), selective antibody deficiency (after the age of two years), and selective immunoglobulin A (IgA) deficiency.

The most common antibody deficiencies in young children are specific antibody deficiency and selective IgA deficiency.

The most common antibody disorders that present in adulthood include the common variable immunodeficiency phenotype, selective IgA deficiency, and selective antibody deficiency.

The disorders mentioned above are reviewed in detail separately.

(See "Transient hypogammaglobulinemia of infancy".)

(See "Specific antibody deficiency".)

(See "Selective IgA deficiency: Clinical manifestations, pathophysiology, and diagnosis".)

(See "IgG subclass deficiency".)

(See "Common variable immunodeficiency in children".)

(See "Clinical manifestations, epidemiology, and diagnosis of common variable immunodeficiency in adults".)

Measurement of antibody levels — Measurement of serum IgG, IgA, IgM, and IgE is useful in all cases of suspected antibody deficiency. There are several methods for determining serum immunoglobulin levels, and laboratories use different systems. Therefore, it is critical that age-adjusted normal reference ranges are used when making comparisons (table 1).

Hypogammaglobulinemia is defined as an IgG less than two standard deviations below normal, and agammaglobulinemia is usually considered when IgG is <100 mg/dL.

Panhypogammaglobulinemia is defined as low levels of IgA, IgG, and IgM and is a hallmark of B cell deficiencies, most forms of severe combined immunodeficiency, and can rarely be seen in certain genetic variants of the common variable immunodeficiency phenotype. In combined immunodeficiencies, as well as in several predominantly humoral immunodeficiencies, there are characteristic alterations in the profile of immunoglobulin isotypes that may aid in diagnosis (eg, selective IgA deficiency, selective IgM deficiency, and hyper-IgM immunodeficiencies).

An isolated low level of IgA is relatively common and does not necessarily signify an IEI. (See "Selective IgA deficiency: Clinical manifestations, pathophysiology, and diagnosis", section on 'Epidemiology of IgA deficiency'.)

Measurement of IgG subclasses is usually not helpful and it is useless in young children because the expression of IgG subclasses is quite variable in these ages. However, IgG subclasses are sometimes assessed later in the evaluation of antibody defects. (See "IgG subclass deficiency".)

IgE levels can be useful if elevated or low. Elevated IgE levels are helpful in the identification of several monogenic causes of antibody deficiency (eg, STAT3, IL21R, IL6R, and IL6ST deficiencies, among others). Measurement of IgE is helpful in patients with recurrent sinopulmonary infections or scaly or eczematoid skin disorders, as an elevation is consistent with underlying allergic disease (eg, >100 international units/mL). A very elevated IgE (eg, >2000 international units/mL) in a patient with recurrent bacterial or fungal infections and dermatitis would raise suspicion for a hyper-IgE syndrome and several other IEIs (table 2) (see "Autosomal dominant hyperimmunoglobulin E syndrome"). Very low or undetectable serum IgE <2 ng/mL is often seen in the common variable immunodeficiency (CVID) phenotype that can be used to distinguish it from other causes of hypogammaglobulinemia [7].

Serum IgD is not used in the diagnosis of any disorder, though elevated levels of IgD are often seen in one of the rare autoinflammatory disorders (MVK deficiency). This and related disorders are described in detail separately. (See "Hyperimmunoglobulin D syndrome: Clinical manifestations and diagnosis".)

Measurement of antibody function — Clinically significant impairment in antibody function can be present even when serum antibody levels are normal. Antibody function can be assessed by measuring antibody titers (usually IgG isotype) to specific antigens (also known as specific antibody) in response to intentional immunization or natural infection. There are two major goals to measuring vaccine responses: first, to assess if naïve B cells can respond to a new antigen; second, to assess if memory B cells respond appropriately to an antigen seen in the past. Choosing the right vaccine to test with is critical for properly assessing B cell function.

Antibody function is assessed by examining the patient's response to the two general types of antigens: protein antigens and polysaccharide antigens. Routine vaccinations provide examples of both types:

Vaccines that assess responses to protein antigens - Measurement of antibody titers to tetanus, diphtheria, H. influenzae B, and protein-conjugated pneumococcal vaccines (eg, Prevnar) are used to assess response to protein antigens. Antibody titers to other vaccines (eg, hepatitis A and hepatitis B, measles, others) can also be used.

Vaccines that assess responses to polysaccharide antigens - Measurement of antibody titers to multiple serotypes in pneumococcal polysaccharide vaccines (eg, Pneumovax 23) are used to assess response to polysaccharide antigens. This evaluation is useful in adults and children older than two years. This response is particularly important in making a diagnosis of specific antibody deficiency. (See "Specific antibody deficiency".)

If a patient has received immune globulin in the prior six months, measuring vaccine response is difficult because antibodies to most vaccine-associated antigens are abundant in immunoglobulin preparations. Testing can be achieved if the patient can be vaccinated with a vaccine antigen that is not routinely used such as rabies vaccine (representing a protein antigen) or Salmonella Typhim M , Typhim Vi (a polysaccharide antigen) [8]. (See "Assessing antibody function as part of an immunologic evaluation", section on 'Other polysaccharide vaccines'.)

Principles of interpretation:

The combination of low levels of IgG accompanied by low IgA or IgM or both, and poor vaccine responses is a feature of many IEIs, including both antibody defects and combined (antibody and cellular) defects.

Normal responses to immunization (ie, normal antibody function) can occur with subnormal levels of total IgG or of IgG subclasses. This pattern is typically seen with secondary causes of hypogammaglobulinemia, such as that caused by medications, protein loss, or severe malnutrition. (See "Secondary immunodeficiency induced by biologic therapies".)

Impaired vaccine responses in a patient with normal levels of IgG, IgA, and IgM may be evidence of specific antibody deficiency, although medications, malignancies, and some infections can also cause impaired vaccine responses. (See "Specific antibody deficiency".)

Assessing and interpreting vaccine responses are reviewed in greater detail separately. (See "Assessing antibody function as part of an immunologic evaluation".)

Isohemagglutinins are antibodies mostly of the IgM isotype generated in response to polysaccharides of gut flora that cross-react with A or B blood group erythrocyte antigens. They generally appear in the blood by six months of age in individuals who have blood types other than AB. Very low titers in a child suggests poor antibody function. Similarly, antibodies to streptococcal antigens (eg, Streptolysin O and Anti-DNAase) are normally present in all subjects after two years of age; very low titers also hint at antibody deficiency. However, vaccine responsiveness is generally considered to be a more reliable indicator of intact humoral immune function. Isohemagglutinin testing is reviewed in more detail separately. (See "Assessing antibody function as part of an immunologic evaluation", section on 'Isohemagglutinin testing'.)

Measurement of immunoglobulin loss — Low levels of immunoglobulins are occasionally due to loss of immune globulin into the gastrointestinal tract, urine, lung, skin, pleural space, bronchi, or peritoneal fluid during dialysis in patients with certain chronic diseases. However, there is usually a loss of other serum proteins, including albumin and alpha-1 antitrypsin, and often accompanying lymphopenia in these disorders. A method of evaluating the rate of protein loss is reviewed separately. (See "Protein-losing gastroenteropathy", section on 'Diagnosis'.)

Defects in cellular immunity — Specific cellular immunity is mediated by T cells, and defects affecting these T cells underlie some severe IEIs. However, because much of the specific antibody production by B cells requires intact T cell function, most T cell defects lead to combined (cellular and humoral) immunodeficiency.

Thus, evaluation of T cell numbers and function is critical in patients with "predominantly" antibody deficiencies, especially in those with certain infections or significant autoimmunity. An evaluation of cellular immunity is appropriate for patients with severe viral and/or bacterial illnesses or opportunistic infections. The disorders that can impair cellular immunity are different in various age groups:

In children younger than one year of age, IEIs are the most common cause of impaired cellular immunity, although perinatal cytomegalovirus (CMV) and other herpes virus infections can cause transient or persistent cellular immunodeficiency [9]. Maternal exposure to immunosuppressive medications (eg, fingolimod, azathioprine) can cause transient cellular immunodeficiency [10].

In older children and adults, the major causes are human immunodeficiency virus (HIV) infection and iatrogenic immune suppression due to therapy for autoimmune disease, malignancy, or transplantation. Mild forms of primary combined immunodeficiency or DiGeorge syndrome can escape diagnosis until adolescence or adulthood.

The most profound combined immunodeficiencies are classified under the heading "severe combined immunodeficiency" or SCID. SCID disorders usually present in infancy, while less severe combined immunodeficiencies present in children and occasionally in adolescents or adults. (See "Severe combined immunodeficiency (SCID): An overview" and "Combined immunodeficiencies".)

Complete blood count with differential and blood smear — The complete blood count (CBC) with differential and blood smear provides valuable information about the cellular immune system. Lymphocyte counts in infants are normally much higher than in older children and adults [11,12]. In many primary immunodeficiency disorders, cell populations decline over time. Thus, normal results in the past cannot be relied upon as a reflection of the patient’s current state.

In patients suspected of having a defect in cellular immunity, the CBC evaluates for lymphopenia and any associated gross hematologic abnormalities, some of which may greatly assist in diagnosis. For example, patients with Wiskott-Aldrich syndrome have low numbers of small platelets.

Evaluation of lymphopenia — The definition of lymphopenia depends considerably on the age of the subject, and normal values for adults may be critically low for infants. Thus, it is especially important to consider the age and appropriate reference values, and not the general "reference range" that is often provided alongside laboratory results. The ranges of normal lymphocyte counts in premature infants, low birth weight infants, and through early infancy have been published [13]. In young children through adolescence, normal values are quite variable and can be seen in this reference [12]. Normal lymphocyte counts through adulthood and into elderly years are more stable [14]. Lymphopenia can be caused by an array of disorders (table 3). A single finding of lymphopenia should be interpreted with caution, since transient lymphopenia is frequently found in a variety of common infectious illnesses [15]. However, significant lymphopenia that does not rapidly correct should not be ignored, since lymphopenia may be the first indication of cellular immunodeficiency or another serious disease (eg, lymphoma) [16]. (See "Approach to the child with lymphocytosis or lymphocytopenia", section on 'Lymphocytopenia' and "Approach to the adult with lymphocytosis or lymphocytopenia", section on 'Lymphocytopenia'.)

Flow cytometry to evaluate lymphocyte populations should be performed in all patients suspected of cellular immunodeficiency with any significant infection in a patient where the absolute lymphocyte count is consistently low. In the absence of other indicators of immune dysfunction, there should (at the very least) be subsequent measurements of the lymphocyte count to document normalization. Persistent lymphopenia requires further investigation of immune function and to ascertain which type of lymphocytes are low. In rare situations, a normal total lymphocyte count can be seen in the presence of a severe immunodeficiency (eg, normal lymphocyte counts due to expanded B cell counts in infants with Jak3 deficiency SCID). If the clinical presentation is suggestive of an underlying disorder (eg, a patient with Pneumocystis pneumonia or invasive Candida), then lymphocyte subsets should be evaluated with flow cytometry, even if the total lymphocyte count is normal. (See 'Flow cytometry for cell populations' below.)

Cutaneous delayed-type hypersensitivity — Cutaneous delayed-type hypersensitivity (DTH) is the classic in vivo test of cellular immunity. This test measures the recall response to an intradermal injection of an antigen to which an individual has already been exposed over a period of time [17]. For that reason, skin testing is usually not of much value under the age of one year. DTH testing has fallen out of common use in most centers but is reviewed briefly here.

A positive response to intracutaneous antigen injection requires uptake and processing of antigen by antigen-presenting cells, their interaction with CD4+ helper T cells, cytokine production by T cells, and subsequent recruitment and activation of monocytes and macrophages. Thus, skin testing is a sensitive indicator of intact cellular immunity, but negative results must be interpreted with caution, because an impairment in any step of the response pathway will lead to a negative response. In addition, the DTH response is unreliable in children under one year of age (even with prior antigen exposure) [18] and is often suppressed during viral and bacterial infection (even with live-attenuated vaccines as in the combined measles, mumps, and rubella vaccine). DTH skin reactivity is also suppressed by anti-inflammatory drugs (glucocorticoids) and other immunosuppressants (cyclosporine, tacrolimus, mycophenolic acid). (See "Glucocorticoid effects on the immune system" and "Secondary immunodeficiency induced by biologic therapies".)

Method — Delayed hypersensitivity skin responses can be assessed using an intradermal injection of Candida antigen (Candin) or Trichophyton. These are the only commercially available reagents intended for use in DTH testing. However, mumps skin test antigen and purified protein derivative (PPD) of tuberculosis are also available. Tetanus toxoid is no longer available for use as a skin test reagent. A 0.1 mL dose of undiluted Candin or Trichophyton is injected intradermally in the volar surface of the forearm. The response is measured 48 to 72 hours after injection. Induration of more than 5 mm diameter is considered indicative of appropriate cellular immunity in a patient of any age. In children, induration of more than 2 mm is sometimes accepted as an appropriate response [19]. Erythema alone without induration is not an appropriate response.

Historically, a panel of three reagents has been used for DTH testing:

Candida antigen (Candin, as above)

Tuberculin skin test (TST) (5 units or 0.1 mL)

Trichophyton (1:500 weight to volume [w/v] ratio)

The main advantages of DTH testing are its ease and economy. The main disadvantage is the difficulty in obtaining the reagents. The second disadvantage is that low T cell counts will result in poor DTH results even if the T cells are functional. However, it is a useful screening test in many instances of suspected cellular immunodeficiency. A DTH test that results in no induration when one is expected should be followed by measurement of lymphocyte populations by flow cytometry, combined with in vitro assays of T cell function. In vitro assays (particularly mitogen stimulation) are less sensitive to interference during intercurrent illnesses or by drugs. (See 'T cell function proliferation assays' below.)

Flow cytometry for cell populations — Flow cytometry uses monoclonal antibodies to identify and quantitate cells of the hematopoietic system that have specific antigens termed "cluster designations" or "cluster of differentiation" (CDs). The tables list the fundamental set of markers commonly used and the lymphocyte populations that they define (table 4 and table 5). A typical panel of markers used to identify the major subsets of lymphocytes includes CD3, CD4, CD8, CD19 or CD20, and CD16 and CD56. Analysis of the expression of markers of naïve (CD45RA in combination with CD62L or CCR7) and memory (CD45RO) T cells is important in the diagnostic approach to patients with combined immunodeficiency (CID). The nature and derivation of the nomenclature of the markers are discussed elsewhere. (See "Normal B and T lymphocyte development".)

A CBC with differential should be performed on a blood specimen obtained at the time of the flow cytometric analysis, or the cytometer itself should be used to determine the lymphocyte number. This analysis permits the calculation of the absolute numbers of each lymphocyte subset. It is possible for the percentage of a particular subset to be abnormal while the total number of cells is within the normal range and vice versa. For the major subsets of lymphocytes, an absolute deficiency, rather than a relative (percentage) deficiency, is of much greater clinical significance.

Flow cytometry is invaluable in the assessment of lymphocyte subpopulations in patients with opportunistic infections or severe or persistent lymphopenia [20,21]. Standard flow cytometry analysis will be abnormal in almost all cases of SCID, and in many instances of other combined immunodeficiencies (table 6).

Note that while total B cell numbers are often normal in CVID, subpopulations of B cells are usually abnormal. In patients with CVID and autoimmunity, CD21 low B cells are elevated in numbers; these B cells show defective central and peripheral tolerance to self-antigens. In CVID patients with poor antibody responses, memory B cells (CD27+) are low, and especially those that have switched their immunoglobulin isotype (IgM-negative, IgD-negative). (See "Clinical manifestations, epidemiology, and diagnosis of common variable immunodeficiency in adults".)

T cell numbers are broadly normal in the CVID phenotype, but when T cell counts are low or patients have opportunistic infections in what otherwise looks like a predominantly antibody deficiency, one should consider a diagnosis of CID instead. The analysis of lymphocyte subsets may be diagnostic for various forms of lymphoma as well. The use of flow cytometry in the diagnosis of specific immunodeficiencies is reviewed in more detail separately. (See "Flow cytometry for the diagnosis of primary immunodeficiencies".)

Abnormalities in immunodeficiency — The table summarizes anticipated alterations in the representations of various lymphocyte populations in several immunodeficiency diseases (table 6). Specific disorders are reviewed separately. (See "Severe combined immunodeficiency (SCID): An overview".)

While certain immune defects are associated with characteristic patterns of lymphocyte subsets, lymphocyte populations may appear to be entirely normal even with clinical evidence of significant immune dysfunction. Conversely, as with total lymphocyte numbers, lymphocyte subsets may be profoundly altered by common infectious illnesses and other factors [12,15]. Thus, for the purpose of diagnosis, flow cytometry data must be considered in conjunction with functional tests of the immune system.

Decreased numbers or absent natural killer CD16/56 cells (≤100 cells/microL), particularly in the presence of recurrent infection with herpes viruses, should warrant further studies for natural killer cell deficiencies, including cytotoxicity studies. (See "NK cell deficiency syndromes: Clinical manifestations and diagnosis".)

Advanced tests — Many advanced tests are available to study specific cellular immune defects. These tests are best ordered and interpreted by an immunology expert. Advanced tests include:

Advanced flow cytometry – Advanced flow cytometry using monoclonal antibodies specific for activated cells, regulatory T cells, naïve or memory T and B cells, or various stages of B cell development are of value in further characterizing many antibody deficiencies or cellular immunodeficiencies (eg, immune dysregulation, polyendocrinopathy, enteropathy, X-linked or IPEX syndrome). Flow cytometry can be used in the definitive diagnosis of several genetic immunodeficiencies by assessing the absence or abnormal expression of a specific protein (eg, X-linked agammaglobulinemia, Wiskott-Aldrich syndrome). Two possible caveats limiting the accuracy of this kind of testing include the existence of missense variants that result in normal expression of the protein but abnormal function, and the existence of missense variants that alter the epitope recognized by the antibody during flow cytometry. (See "Flow cytometry for the diagnosis of primary immunodeficiencies".)

TREC analysis – Measurement of T cell receptor excision circles (TRECs) by quantitative polymerase chain reaction (PCR) analysis of peripheral blood provides an assessment of thymic function by measuring recent thymic emigrant cells. It is primarily used as a screening test for newborn T cell lymphopenia due to SCID on newborn blood samples. The reconstitution of T cells following hematopoietic stem cell transplantation should be accompanied by a steady increase in TRECs. (See "Severe combined immunodeficiency (SCID): An overview", section on 'Newborn screening'.)

KREC analysis – Measurement of kappa-light chain recombination circles (KRECs) by quantitative PCR analysis of peripheral blood for the assessment of the number of B cells is often used in conjunction with TRECs in newborn screening procedures. Both TRECs and KRECs can be done on dried blood spots. At present, analysis of KRECs is not commercially available in most of the world.

Cytotoxicity assays – Cytotoxicity assays measure the functional capacity of cytotoxic T cells or natural killer cells and are critical for assessing effector functions that are often defective in the familial hemophagocytic lymphohistiocytosis (fHLH) set of monogenic autoinflammatory disorders. One such method utilizes antigenic peptides attached to specific major histocompatibility complex (MHC) molecules labeled with a fluorochrome as targets. Binding of the cytotoxic cell to the peptide is measured by flow cytometry [22].

Natural killer cell cytotoxicity is of value in the diagnosis of natural killer cell disorders, hemophagocytic lymphohistiocytosis, and Chediak-Higashi syndrome. (See "Clinical features and diagnosis of hemophagocytic lymphohistiocytosis", section on 'Immunologic profile' and "Chediak-Higashi syndrome", section on 'Laboratory and imaging findings' and "NK cell deficiency syndromes: Clinical manifestations and diagnosis", section on 'Cytotoxicity testing'.)

T cell cytotoxicity is measured in the diagnosis of functional T cell deficiencies, where the number of T cells is normal or near normal. (See "Severe combined immunodeficiency (SCID): An overview", section on 'Diagnosis'.)

Defects in T cell cytotoxicity are seen in some patients with herpes virus infections (eg, CMV, Epstein-Barr virus [EBV]) or HIV, when T cells are challenged with the patient's own virally-infected cells and found to have deficient function.

Cytokine assays

Plasma inflammatory cytokines, such as interleukin (IL)-1, TNF-alpha, IL-6, and IL-18, are elevated in autoinflammatory disorders and in cytokine storms associated with severe infections, graft-versus-host reactions, and acute flares of primary or secondary hemophagocytic lymphohistiocytosis (HLH). Serial assays may be of value in tracking the therapeutic response. Measurement of these cytokines at baseline and during autoinflammatory "flares" can help identify potential therapies (eg, treatment with anti-IL1 agents).

Cytokine autoantibodies have been associated with immune disorders including anti-IFN-alpha (associated with SARS-CoV-2 susceptibility), anti-IFN-gamma (mycobacterial infection), anti-IL-6 (staphylococcal infections), anti-IL-17A, -17F, and -22 (mucocutaneous candidiasis), and anti-GM-CSF (pulmonary alveolar proteinosis) [23].

Cytokine production assays, measured in the supernatants of a patient's cells after culture with various antigens, may be of value in identifying defects in selected patients with severe infections or noninfectious inflammatory disorders [24], or for monogenic disorders where skewing of T cell cytokine production is affected.

Single cytokine assays are of limited value in the diagnosis of immunodeficiencies and are mainly used in research.

Genetic assays – Genetic testing to identify genetic variants are available for many primary immunodeficiency diseases [6,25-27]. (See "Genetic testing in patients with a suspected primary immunodeficiency or autoinflammatory syndrome" and "Combined immunodeficiencies" and "Severe combined immunodeficiency (SCID): An overview".)

Other advanced tests

Chromosome microarray is of value in the diagnosis of DiGeorge syndrome. (See "DiGeorge (22q11.2 deletion) syndrome: Clinical features and diagnosis", section on 'Genetic analysis'.)

Short tandem repeats (STR) analysis of sorted leukocyte subsets is used to identify chimerism in newborns with SCID.

HLA typing is used to identify an appropriate stem cell donor. (See "Hematopoietic cell transplantation for non-SCID inborn errors of immunity" and "Hematopoietic cell transplantation for severe combined immunodeficiencies".)

CD3/T cell receptor analysis and function. (See "CD3/T cell receptor complex disorders causing immunodeficiency".)

X-inactivation tests can analyze the distribution of X-inactivated cells to determine the influence of X-chromosome variants on hematopoiesis or survival of downstream cell [28].

T cell function proliferation assays — In vitro studies of T cell function measure peripheral blood T cell proliferation in response to several different types of stimuli [29]:

Mitogens (such as the plant lectins phytohemagglutinin, concanavalin A or pokeweed mitogen, or anti-CD3).

Specific antigens (such as tetanus toxoid).

Allogeneic lymphocytes (ie, mixed lymphocyte culture).

These studies may not be possible in patients with profound lymphopenia. In parallel with (or prior to) studies of T cell function, flow cytometric measurement of peripheral blood lymphocyte subpopulations should be performed. (See 'Flow cytometry for cell populations' above.)

In T cell function studies, the patient's purified peripheral blood mononuclear cells (lymphocytes and monocytes) are incubated with the stimulant or cells for three to six days. A control tube with cells and media alone is also incubated. In the most common procedure, tritiated thymidine is added to the cells during the last 24 hours of culture. Dividing lymphocytes incorporate the thymidine into their DNA. The extent of proliferation is determined by measuring the radioactivity taken up by cells. Many laboratories report results as a stimulation index (SI), the ratio of radioactivity (as counts per minute [CPM]) with stimulation over the CPM in the control tube without stimulation (background). This method is dependent on the number of lymphocytes in the sample, and when during the cell cycle the thymidine is added, so as to identify maximal cell proliferation. Tritiated thymidine incorporation should be interpreted with caution when there are very low numbers of T cells in the blood.

Suppression of T cell responses to mitogens and antigens may be seen with significant nutritional deficiencies, moderate-to-severe concurrent illnesses, or with administration of immunosuppressive drugs. Thus, these tests should be performed when the patient is relatively well and not receiving glucocorticoids or other immunosuppressive medications, whenever possible. T cell responses appear to normalize rapidly (ie, within one day or two) after glucocorticoids are discontinued [30].

Newer assays to assess the proliferative responses of lymphocytes utilize flow cytometry, eliminating the need for radioactive thymidine. These tests are less susceptible to the effects of very low numbers of T cells and should exclusively be used when evaluating functional responses of T cells in SCID or severe lymphopenia. The assays can be performed using mitogens or specific antigens (eg, tetanus antigen) as stimuli and usually measure the uptake of a thymidine analogue linked to a fluorescent dye (5-ethynyl-2'deoxyuridine [EdU]). Alternatively, the permeable fluorescent dye carboxyfluorescein succinimidyl ester (CFSE) can be added to the cells prior to stimulation and its diminution with each cell division can be measured [31,32]. The "CFSE dilution" test is often reported as percent of the initial cells that have undergone proliferation.

However the test is performed, it is interpreted as showing low proliferation, partial proliferation, or normal proliferation by comparing patient results with healthy control lymphocytes assayed simultaneously, and by comparison with the normal ranges for controls in the laboratory [29].

Response to mitogens — Most mitogens require functional antigen-presenting cells (ie, monocytes and B cells) for T cell stimulation, although the mechanisms of antigen processing and presentation are bypassed. Mitogens are powerful stimulants for T cells, and responses may be assessed in patients of any age, even newborns, regardless of immunization status.

Normal reference ranges are derived from studies on the cells of healthy adults. Newborns generally have higher mitogen responses compared with adults [33]. When using a tritiated thymidine incorporation assay, depending upon the mitogens and the laboratory, the range of CPM reported for normal controls is approximately 50,000 to 300,000, with a SI between 10 and 200. Phytohemagglutinin (PHA) is the mitogen most commonly used. Results for patients in comparison with controls are roughly interpreted as normal (>50 percent of control), low (25 to 50 percent), very low (10 to 25 percent), or absent (<10 percent). Results expressed as SI vary widely between laboratories. In general, a SI <10 may be considered as no response.

Some laboratories use the monoclonal antibody anti-CD3 (clone OKT3) as a stimulant of T cell proliferation. OKT3 cross-links the epsilon chain of T cell CD3 complexes and stimulates T cells (in the presence of accessory or antigen-presenting cells) in a manner analogous to T cell mitogens [34]. These tests are superior to mitogens in directly assessing the ability of the T cell receptor (TCR) itself to initiate activation. Proliferative responses to mitogen or OKT3 are generally similar, although they may sometimes give different patterns in the context of distinct immune defects [35]. There are no published comparisons in a large group of normal individuals or immune-deficient patients. Most clinical reference laboratories use one or more mitogens and one or more antigens, but do not routinely use OKT3.

Diminished or absent proliferation indicates a serious derangement of T cell function. Mitogen responses will be severely depressed (usually well below the control fifth percentile) in the vast majority of patients with profound T cell lymphopenia (eg, complete DiGeorge syndrome), SCID [36], or advanced acquired immunodeficiency syndrome (AIDS). By comparison, the response may be partial or normal in milder syndromes of combined deficiency (eg, Wiskott-Aldrich syndrome), in mainly humoral deficiency, or in those with infections (especially CMV and other herpes viruses) [37].

Response to specific antigens — Specific antigen tests are more sensitive than mitogen assays as indicators of defects in T cell function since the mechanisms of antigen-specific activation go through the TCR and are more complex than those for mitogens. These tests require antigen processing and presentation by antigen-presenting cells and thus are affected by states where antigen presenting cells are low (eg, B cell depletion following treatment with rituximab or with monogenic deficiencies of circulating dendritic cells). As with DTH testing, in vitro-specific antigen proliferation tests are not useful in infants or other patients who have not been vaccinated.

Tetanus and diphtheria toxoids and monilia (Candida) are antigens frequently used for this assay. Since only a small number of cells respond to the specific antigens offered in these assays, the cultures must be maintained longer, and proliferation is often lower than for mitogen stimulation. Proliferation as assessed by CFSE or EdU incorporation dilution may also be lower than is seen for mitogen stimulation. Results expressed as CPM are often reported from 5000 to 50,000; results as SI generally range from 4 to 50. In general, a SI for specific antigen >4 is usually considered adequate, although an isolated SI <4 is not a reliable indicator of impaired cellular immunity.

In many forms of combined immunodeficiency (eg, in those that diminish the signaling downstream of the TCR), responses to specific antigens are diminished or even absent while mitogen responses remain intact. In secondary immunodeficiency, specific antigen responses will also generally be suppressed more easily than mitogen responses. However, these generalizations are not always true [38], and interpretation of these tests requires expertise. As with DTH testing, booster immunization with tetanus followed by a repeat in vitro proliferation assay may detect a significant response.

Another way to assess function of T cells is to measure cytokine release, rather than proliferation, following antigen exposure. An example of this type of test that has become widely used in the diagnosis of latent tuberculosis infection is the interferon gamma release assay, which can be used instead of tuberculin testing. Cytokine production upon stimulation with mitogens or tetanus antigen is typical. When low or absent, cytokine production defects indicate a failure of T cell activation that often parallels proliferative defects.

Chromosomal instability assays for radiation-sensitive patients — Tests for chromosomal breakage, as are present in Fanconi anemia and Nijmegen breakage syndrome, are commercially available. (See "Clinical manifestations and diagnosis of Fanconi anemia" and "Nijmegen breakage syndrome".)

Tests for radiation sensitivity are not widely commercially available. These require radiation of lymphocyte or fibroblast cell lines followed by analysis of chromosomal breaks and fragility [39]. A newer generation of radiation sensitivity testing is performed by flow cytometry and relies on the activation of specific pathways that recognize DNA damage such as gamma-H2AX and SMC1 [40].

Tests for phagocytic abnormalities — Neutrophil defects result in a range of illness from mild recurrent skin infections to overwhelming, fatal systemic infection. Affected patients are more susceptible to bacterial and fungal infections, but have a normal resistance to viral infections. Most are diagnosed in infancy due to the severity of the infection or the unusual presentation of the organism, but some escape diagnosis until adulthood.

Primary phagocytic deficiencies characteristically lead to recurrent and severe fungal (eg, Candida and Aspergillus) and bacterial (eg, Staphylococcus aureus, Pseudomonas aeruginosa, Nocardia asteroides, Salmonella typhi) infections. Response to nontuberculous mycobacteria may also be abnormal, particularly in patients with chronic granulomatous disease. The most common sites of infection are the respiratory tract and skin. Tissue and organ abscesses also occur. Other frequent manifestations include abnormal wound healing, dermatitis/eczema, stomatitis, and delayed umbilical cord detachment. Many patients have growth failure. Many patients have super-abundant inflammation in sites of injury or in the colon. (See "Primary disorders of phagocyte number and/or function: An overview".)

Phagocytic disorders can be caused by extrinsic or intrinsic defects. Extrinsic defects include opsonic abnormalities secondary to deficiencies of antibody and complement factors, suppression of granulocyte production, and leukocyte autoantibodies or isoantibodies that decrease the number of circulating neutrophils. Intrinsic disorders of granulocytes may be divided into defects of sensing infections, defects in granulocyte killing ability and defects in chemotaxis (cell movement).

The initial test for evaluation of phagocyte disorders is the CBC with differential (see "Laboratory evaluation of neutrophil disorders"):

Leukocytosis raises the possibility of a leukocyte adhesion defect (ie, a defect in chemotaxis or extravasation), and flow cytometry is indicated next to assess for the presence of specific adhesion molecules. (See "Leukocyte-adhesion deficiency".)

Severe neutropenia can be seen with congenital agranulocytosis or cyclic neutropenia. (See "Congenital neutropenia" and "Cyclic neutropenia".)

Pancytopenia (combined deficiency of all blood cells) can be seen with bone marrow failure syndromes, such as aplastic anemia and dyskeratosis congenita. (See "Treatment of acquired aplastic anemia in children and adolescents" and "Aplastic anemia: Pathogenesis, clinical manifestations, and diagnosis" and "Dyskeratosis congenita and other telomere biology disorders", section on 'Clinical features'.)

If neutrophil numbers are normal and the patient's history is consistent with a neutrophil disorder, then an evaluation of neutrophil function is appropriate. (See "Laboratory evaluation of neutrophil disorders".)

A general approach to the patient with neutropenia, including identification of the cause and determination of the infectious risk, is presented separately. (See "Overview of neutropenia in children and adolescents" and "Approach to the adult with unexplained neutropenia".)

Tests for complement defects — Complement disorders may be inherited or acquired. Broadly, monogenic complement deficiencies can either incur susceptibility to infection or susceptibility to autoimmunity, with some overlaps. Screening for a classical complement pathway defect is indicated in patients with any of the following:

Recurrent, unexplained pyogenic infections in whom the white blood count and immunoglobulin levels and specific antibody responses are normal

Recurrent Neisserial infections at any age

Multiple family members who have experienced Neisserial infections

In addition, it is reasonable to evaluate the complement system in any patient with systemic lupus erythematosus (SLE) or a familial tendency of SLE. However, this is particularly relevant in those with familial lupus or subacute cutaneous lupus, in whom C1q or C2 deficiency should be excluded.

The initial screening test for complement defects is a total hemolytic complement assay (CH50), which assesses classical pathway function and is widely available. If the CH50 is significantly reduced or zero, then the levels of individual complement components are measured. Low levels of multiple components, particularly low C3 and C4, suggest complement consumption, rather than a primary complement deficiency. If the CH50 is normal and a complement defect is still suspected, the AH50, a screening test for alternative pathway defects, may be obtained. This test is largely performed in specialized laboratories [41]. Alternative complement deficiencies include properdin and factor D deficiencies, both of which are rare. Each specific complement factor and complement inhibitor can be assessed for its abundance and function in specialized laboratories. Inherited and acquired defects in complement components are reviewed in more detail elsewhere. (See "Inherited disorders of the complement system" and "Acquired disorders of the complement system".)

Deficiencies or defects of C1 esterase inhibitor are associated with hereditary angioedema, although these disorders are not associated with increased susceptibility to infection. (See "Hereditary angioedema: Epidemiology, clinical manifestations, exacerbating factors, and prognosis".)

Tests for innate immune defects — Defects in innate immune mechanisms should be suspected in patients with chronic mucocutaneous candidiasis, invasive fungal infections, invasive bacterials infections, and severe mycobacterial disease, when other immunodeficiencies have been excluded. Many innate immune defects arise in early life (infancy and preschool years), and should be suspected, for example, in a newborn with mycobacterial or fungal sepsis. Many are associated with specific infections (eg, herpes or EBV infections in natural killer cell defects, atypical mycobacterial infection in interleukin 12/23 (IL-12/23)-interferon-gamma (IFN-gamma) pathway defects, herpes simplex encephalitis in the toll-like receptor 3 (TLR3) signaling pathway and other severe infections, often associated with poor inflammatory response).

Natural killer cell defects — Natural killer cell defect can be primary or secondary. Primary natural killer deficiency should be suspected in patients with low natural killer cells and/or recurrent severe herpes virus infections. Natural killer defects are also noted in patients with X-linked lymphoproliferative disorders, hemophagocytic lymphohistiocytosis, and Chediak-Higashi syndrome. (See "NK cell deficiency syndromes: Clinical manifestations and diagnosis", section on 'Introduction'.)

Defects of IL-12/23-IFN-gamma pathways — Patients with invasive infections caused by low virulence Mycobacterial and Salmonella species may have defects of the genetic components of the IL-12/23-IFN-gamma pathways, including the following genes in order of incidence: the IL-12 receptor beta-1 gene (IL12RB1), IFN-gamma receptor 1 gene (IFNGR1), the IL12B gene (IL-12p40), the IFN-gamma receptor 2 gene (IFNGR2), signal transducer and activator of transcription 1 (STAT1) gene, NEMO, and the ubiquitin-like protein ISG15 gene. Special tests involving western blotting or flow cytometry can pinpoint the exact molecular defects and must be performed in a research laboratory at present [26]. (See "Mendelian susceptibility to mycobacterial diseases: Specific defects".)

Defects of IL-17 pathways — Patients with chronic mucocutaneous candidiasis can have defects in a number of specific pathways, including the IL-17 receptor chains IL-17RA and Il-17RC, in the IL-17F cytokine, in the STAT1 gene that lead to gain-of-function defects, and in the protein ACT1 (gene name TRAF3IP2) that allows for responses to the IL-17 cytokines. Defects in making T helper 17 cells due to deficiencies of the IL-23 receptor and in the master transcription factor RORC lead to susceptibility to candidiasis as well. Finally, autoantibodies that block IL-17 or its receptor have been described as a secondary cause of chronic mucocutaneous candidiasis in AIRE deficiency (APECED or APS1). (See "Chronic mucocutaneous candidiasis".)

Toll-like receptor pathway defects — Pattern recognition receptors (PRRs) are receptors specific for molecular components of micro-organisms widely expressed on phagocytic cells (neutrophils, macrophages, and monocytes and several other cells). Defects in PRRs can cause increased susceptibility to infections that is most severe in infancy, before the adaptive (ie, humoral and cellular) immune system has developed. The frequency and severity of infections generally lessen as the patient matures, in contrast to most other types of immunodeficiency [42]. (See "An overview of the innate immune system".)

The toll-like receptors (TLRs) are one group of PRRs that, after ligation with specific micro-organisms, initiate a series of steps that eventually activate the nucleus to initiate cytokine synthesis. Several genetic disorders of the signaling pathways have been identified, including an IL-1 receptor-associated kinase 4 defect (IRAK4), a myeloid differentiation primary response gene 88 defect (MYD88), a nuclear factor (NF)-kappa-B essential modulator defect (NEMO), a TLR3 receptor defect, and a UNC93B defect. Screening tests to evaluate these defects are available, including a flow cytometric assay [6,26,27]. (See "Toll-like receptors: Roles in disease and therapy", section on 'TLR signaling defects in primary immunodeficiency' and "Flow cytometry for the diagnosis of primary immunodeficiencies".)

Advanced genomic studies for all forms of IEIs — There are over 500 monogenic inborn errors of immunity (IEIs), which are named after the genes that undergo either loss- or gain-of-function due to pathogenic variants in the gene. Thus, a central step in making the diagnosis of IEI includes genetic testing. Next-generation sequencing (NGS) tools include whole exome sequencing (WES), whole genome sequencing (WGS), and target enrichment NGS ("gene panels"). Genetic testing techniques had previously been performed at research centers, but availability has expanded through a number of academic and commercial laboratories. Due to the decrease in cost and increased availability, every patient with suspected primary immunodeficiency should have a genetic diagnosis as part of their workup [43-53]. Increasingly, genetic testing is performed earlier in the workup, and results of genetic tests can inform the need for other laboratory tests. Older techniques such as linkage analysis and homozygosity mapping worked well for identifying monogenic traits, particularly in patients with syndromic features, although a large pedigree or several smaller pedigrees were needed. Microarray methods of comparative genomic hybridization can detect relatively large areas of gain or loss of copy number and may identify clinically relevant change. (See "Next-generation DNA sequencing (NGS): Principles and clinical applications".)

Targeted NGS is a cost-effective method to screen for defects in known primary immunodeficiency-associated genes. It can identify patients with atypical presentations of known genetic defects and can also be used to identify the defect in patients with a primary immunodeficiency that has multiple associated candidate genes. Most panels allow for identification of copy number variation because they use high numbers of NGS reads. If targeted NGS screening is uninformative, WES is usually performed next. As the cost of WGS decreases, the role of WES and targeted NGS may become less clear.

WES assesses about 2 percent of the genome that encompasses most of the coding genes (the exons) and the few nucleotides that surround each exon (regions of splicing). In contrast, WGS includes the entire genome including the introns. Thus, WES is less laborious and expensive, and easier to analyze. Variants found by WGS in the deep intronic regions or in the promoter regions of genes are difficult to interpret by themselves. Therefore, WGS is frequently performed in the research setting and is often coupled with RNA sequencing to allow for interpretations of alternative splicing and transcriptional levels. Both WES and WGS are used for identifying rare genetic defects, particularly in patients with early-onset severe disease. Validating a new variant either in a newly-described or a known gene is difficult, as the underlying biology needs to be understood, and the putatively pathogenic variants often have to be recreated in the lab to understand their impact on signaling or other aspects of the immune response.

These techniques also work well if several genes are involved (polygenic traits), although analysis can be challenging and laborious in that circumstance. Variants identified by NGS methods still require further study to determine the biochemical, transcriptional, and immunology impacts of the variant identified.

SOCIETY GUIDELINE LINKS — Links to society and government-sponsored guidelines from selected countries and regions around the world are provided separately. (See "Society guideline links: Inborn errors of immunity (previously called primary immunodeficiencies)".)


Inborns errors of immunity (IEIs), previously called primary immunodeficiencies, should be considered once the more common causes of recurrent infection have been excluded. (See "Approach to the child with recurrent infections" and "Approach to the adult with recurrent infections".)

Before initiating immunologic testing, the clinician should perform a thorough clinical history and physical examination. In infants and children, height and weight records should be reviewed to detect failure to thrive and poor growth. Routine laboratories can provide clues about other causes of infections and help focus further immunologic evaluation. Screening laboratories include complete blood count (CBC) with differential, chemistry panels, urinalysis, test to diagnose specific organ involvement (eg, sinus computed tomography [CT] or chest radiograph), and C-reactive protein or erythrocyte sedimentation rate to look for elevations that may accompany chronic infections. (See 'Initial approach to the patient' above.)

Every patient with a clinical phenotype compatible with an IEI should be referred to an immunology specialist early in the evaluation, when possible. Immunologic testing is best performed in a graded fashion because many tests require varying degrees of expertise to perform and interpret, may not be widely available, and are often costly. (See 'Referral' above.)

Defects in humoral immunity, which account for the majority of primary immunodeficiencies, usually result in recurrent and severe sinopulmonary infections. Immunoglobulin G (IgG), immunoglobulin A (IgA), and immunoglobulin M (IgM) should be measured initially. The next step in evaluation is the measurement of serum-specific antibody titers (IgG) in response to intentional immunization (vaccine response) or natural infection. (See 'Antibody deficiency and defects' above and "Assessing antibody function as part of an immunologic evaluation".)

Defects in cellular immune function (T cell disorders) are often accompanied by antibody defects, yielding combined immunodeficiencies. These disorders should be considered in any child presenting with recurrent or severe viral and/or bacterial illnesses or opportunistic infections in the first year of life. Presentation in adulthood is uncommon, although it does occur. (See 'Defects in cellular immunity' above.)

Lymphopenia is an early indicator of severe combined immunodeficiency (SCID) in infants and of combined immunodeficiencies in older children and occasionally adults. Lymphopenic newborns are usually identified by T cell receptor excision circles (TRECs) analysis of heel stick blood. The presence of lymphopenia should be followed by flow cytometry to determine which lymphocyte subsets are deficient (table 4 and table 5). In most cases, the evaluation should be repeated at several points in time, as viral infections and other factors can influence lymphocyte counts. (See 'Complete blood count with differential and blood smear' above and 'Evaluation of lymphopenia' above.)

An overview of the anticipated alterations in various lymphocyte populations observed in several immunodeficiency diseases is provided (table 6). (See 'Abnormalities in immunodeficiency' above and "Flow cytometry for the diagnosis of primary immunodeficiencies".)

In vitro studies of T cell function measure peripheral blood T cell proliferation in response to mitogens or antigens. Mitogen responses may be assessed in patients of any age, regardless of immunization status. Specific antigen tests are only useful in patients who have been previously immunized to that antigen, but may be more sensitive than mitogen assays as indicators of defects in T cell function. (See 'T cell function proliferation assays' above.)

Advanced tests for T cell deficiencies may include cytokine levels, cytokine release assays, autoimmune anti-cytokine antibodies, radiosensitivity assays, and cytotoxic assays using human leukocyte antigen (HLA)-matched target cells.

Primary phagocytic cell deficiencies characteristically lead to recurrent and severe fungal (eg, Candida and Aspergillus) and bacterial infections. The most common sites of infection are the respiratory tract and skin, but deep tissue and organ abscesses are also seen. Initial testing for these disorders is the CBC with differential, followed by various specific tests of phagocyte function, depending upon the disorder suspected. (See 'Tests for phagocytic abnormalities' above.)

Complement defects should be considered in patients with recurrent pyogenic infections with normal white blood cell counts and immunoglobulins and in patients with a personal or family history of recurrent neisserial infections. A total hemolytic complement (CH50) is the initial screening test. (See 'Tests for complement defects' above.)

Defects in innate immune mechanisms are rare disorders that should be suspected in patients with recurrent infections, often of a specific type (eg, herpes viruses), in which other immunodeficiencies have been excluded. (See 'Tests for innate immune defects' above.)

IEIs are monogenetic diseases, and with the advent of low cost next-generation sequencing, all patients with IEI should be offered a genetic diagnosis. Whole exome sequencing (WES) or next-generation sequencing (NGS) by "gene panels" are well-established approaches to identify pathogenic variants underlying IEIs. Whole genome sequencing (WGS) and RNA sequencing offer more advanced approaches but are typically performed in the research setting due to the difficulty in analysis. (See 'Advanced genomic studies for all forms of IEIs' above.)

ACKNOWLEDGMENT — The UpToDate editorial staff acknowledges Francisco A Bonilla, MD, PhD, and E Richard Stiehm, MD, who contributed to an earlier version of this topic review.

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