Pediatric Severe Combined Immunodeficiency Workup

Lymphopenia is the classic hallmark of severe combined immunodeficiency (SCID); however, normal or even elevated lymphocyte counts can be seen in a significant proportion of patients. Failure to make the diagnosis because the child is not frankly lymphopenic may present a problem, particularly in patients with Omenn syndrome, bare lymphocyte syndrome, and interleukin (IL)–2 deficiency. Obtaining lymphocyte markers and test results of antibody and lymphocyte proliferation can help physicians to avoid this pitfall.

Other laboratory studies can be performed on the basis of clinical judgment, depending on the nature of the infection and the organ system involved. Specifically, assays that measure the ability of lymphocytes to respond to activating agents, such as pokeweed mitogen and phytohemagglutinin, are valuable. Imaging studies are not useful for diagnosis of the primary condition; however, obtaining a chest radiograph may be necessary to evaluate pneumonia secondary to SCID.

Conduct a complete blood count (CBC) with differential to help detect lymphopenia. Children with SCID have a lymphocyte count lower than 3000/µL; however, a normal number of lymphocytes does not rule out SCID, because the lymphocytes may be nonfunctional. An absolute lymphocyte count lower than 2500/µL in an infant definitely warrants further workup, but any infant with severe infection or opportunistic infection should have the full initial workup.

Obtain total serum immunoglobulin (Ig) levels, including IgG, IgA, IgM, and IgE. Immunoglobulin levels, especially IgM levels, can be low. However, soon after birth, IgG levels may be falsely elevated because of maternal IgG.

Draw lymphocyte markers at the same time as the CBC to obtain percentages and absolute counts of CD3⁺ T cells, CD4⁺ T cells, CD8⁺ T cells, CD19⁺ B cells, and natural killer (NK) cell markers (CD16 and CD56).

Lymphocyte function should be assessed by measuring responses to phytohemagglutinin, a nonspecific stimulant of T-cell proliferation, concanavalin A directed at T-cell proliferation, and pokeweed mitogen directed at T-cell and B-cell proliferation.

A complete absence of T-cell function by mitogen tests can occur in association with a normal lymphocyte count for age in some forms of SCID, including X-linked SCID (XL-SCID), in which all the lymphocytes are B cells. DiGeorge syndrome is another example in which lymphocyte counts may be higher than 2000/µL with no T-cell function, or, conversely, normal T-cell function may be observed in spite of lymphopenia.

Specific antigens, such as tetanus and Candida, stimulate lymphocyte proliferation and represent a later step in lymphocyte function than responses to the nonspecific mitogens. Healthy young infants may not respond well to these specific antigens due to lack of exposure and/or immature T-cell functions.

Another T cell function used for screening is their ability to proliferate in response to allogeneic cells; this response aids in defining the type of SCID but also is relevant to determining the need for immunosuppressive therapy in preparation for stem cell reconstitution. Additional activators of lymphocyte proliferation are phorbol myristate acetate (PMA) with ionomycin or anti-CD3 and anti-CD28.

Cellular hallmarks that help differentiate between various forms of SCID, as well as other combined immune deficiencies that are sometimes severe enough to be classified as SCID, are as follows:

• X-linked SCID - Lymphopenia occurs primarily from the absence or near absence of T cells (CD3⁺) and NK cells; variable levels of B cells occur, which do not make functional antibodies
• JAK3 deficiency - Lymphopenia occurs primarily from the absence or near absence of T cells (CD3⁺) and NK cells; normal or high levels of B cells occur, which do not make functional antibodies
• Adenosine deaminase (ADA) deficiency - Lymphopenia occurs from the death of T and B cells secondary to the accumulation of toxic metabolites in the purine salvage pathway; functional antibodies are decreased or absent
• ZAP-70 deficiency - Lymphopenia occurs because of the absence of CD8⁺ T cells; as in all types of SCID, no antibody formation is present
• Reticular dysgenesis - Lymphopenia occurs from the absence of myeloid cells in the bone marrow; red blood cells and platelets are present and functioning
• Omenn syndrome - Normal or elevated T-cell numbers are present, but these are of maternal, not fetal, origin; B cells are usually undetectable, NK cells are present, and the total Ig level is markedly low with poor antibody production; eosinophils are elevated, as is total IgE
• Purine nucleoside phosphorylase (PNP) deficiency - Lymphopenia occurs from the death of T cells secondary to the accumulation of toxic metabolites in the purine salvage pathway; this deficiency differs from ADA deficiency because circulating B cells are normal in number, but B-cell function is poor, as evidenced by the lack of antibody formation; PNP deficiency can be severe enough to be classified as SCID
• Bare lymphocyte syndrome - The lymphocyte count is normal or mildly reduced, CD4⁺ T cells are decreased, and CD8⁺ T cells are normal or mildly increased; B-cells are normal or mildly decreased, but the ability to make antibodies is decreased; bare lymphocyte syndrome is sometimes classified as SCID
• IL-2 deficiency - Normal, or near normal, numbers of T cells exist (both CD4⁺ and CD8⁺), but they fail to proliferate in vitro when stimulated with mitogens unless IL-2 is added to the culture medium; production of functional antibody is decreased; IL-2 deficiency may be severe enough to be classified as SCID

Determine the ADA and PNP levels in lymphocytes, erythrocytes, or fibroblasts. Measurement of leukocyte ADA enzyme activity is both sensitive and specific for the detection of ADA-deficient SCID.

Consider X-inactivation studies to determine whether the SCID is X-linked. Approximately 50% of patients have sporadic mutations with no history of affected family members.

Perform molecular studies to identify any specific known genetic defects or to identify new defects. These tests are now commercially available. If identifying a laboratory to perform these tests is difficult, consult a referral center for primary immune deficiency to assist in this matter.

Even when SCID is not suspected until the infant’s death, lymphocyte markers, mitogen responses, and DNA studies can still be carried out. Anticoagulated blood should be saved because lymphocytes are viable for at least 48 hours after death. An autopsy to assess the thymus and peripheral lymphoid tissues, including the spleen, gut, and tonsils, is needed.

Compromise of other hematopoietic cell lines is observed in reticular dysgenesis, in which myeloid cells are decreased, and platelets and erythrocytes may be deficient. Autoimmune hemolytic anemia can complicate forms of SCID in which autoimmune phenomena are present. Hypoplastic anemia occurs in cartilage-hair hypoplasia.

Patients with SCID are anergic. However, the reliability of delayed hypersensitivity skin testing depends on adequate exposure to the antigen. Candida and tetanus are the most useful antigens, but exposure requires 4-6 weeks, and more than 1 immunization is required in the case of tetanus. Mumps and Trichophyton antigens are of minimal use in infants.

T-cell defects can be difficult to define. The clinical manifestations of T-cell–associated opportunistic infections, such as mycobacteria, cytomegalovirus (CMV) and associated viruses, and P jiroveci, are usually interpreted by immunologists as defining a T-cell defect, even in the presence of apparently adequate mitogen responses (eg, IKK-γ deficiency for which impaired T-cell receptor [TCR]–mediated signaling is present despite normal mitogen responses).

Somech and Roifman suggest mutation analysis in patients with apparently normal immunologic tests to diagnose atypical cases of γC deficiency.^[28]

When a T-cell disorder is suspected, the Immune Deficiency Foundation has a consultative service for physicians. Laboratories in Seattle (the University of Washington), Boston (Children’s Hospital), and New York City are funded to provide molecular analysis (Jeffrey Modell Foundation) or they can assist in contacting other research facilities.

To exclude HIV infection, perform HIV-DNA testing using polymerase chain reaction (PCR) testing; because of maternal antibody, anti-HIV tests are of no value in this setting. To help exclude congenital infection, perform serum testing of IgM against any suspected infection.

Advanced assays of lymphocytes, if present, include measurements of the proliferative response of B cells and T cells to mitogens and lymphocyte subset analysis with flow cytometry. Analysis of specific genes associated with immunodeficiency may be helpful.

Once lymphocyte populations are enumerated by flow cytometry, mutational analysis usually can be initiated based on the distribution of cell surface markers and clinical findings, including the sex of the infant.

Chest radiographs in classic SCID show a small or absent thymus. However, infants who are immunologically normal may have no visible thymus if they have an overwhelming infection, such as sepsis or meningitis. Other T-cell defects, especially DiGeorge syndrome, also lack thymic tissue. Presence of thymic tissue does not exclude SCID. Patients with SCID who have mutations in ZAP70 or CD3 typically have normal-sized thymuses.

Chest radiographs are essential for early recognition of pneumonitis caused by viral pathogens and P jiroveci.

Patients with ADA deficiency and cartilage-hair hypoplasia may have bony abnormalities observed in the ribs and vertebrae on chest radiography. In ADA deficiency, chest radiographs show typical cupping and flaring of the costochondral junction.

Prenatal diagnosis may be attempted when the family history is positive for SCID. Available DNA tests allow for the identification of mutations involving ADA, RAG1/RAG2, JAK3, γC, IL-7 receptor, and Artemis, as well as many other gene mutations associated with the SCID phenotype.

Prenatal diagnosis is possible by chorionic villus sampling at 10 weeks’ gestation (or later) by amniocentesis, using DNA methodology in families for whom the exact mutations have been established.

Fetal blood sampling for fluorocytometric testing, mitogen responses, and enzyme levels can establish the diagnosis when DNA analysis is not available. Percutaneous umbilical blood sampling is performed to examine fetal blood for T-cell deficiency, as well as ADA enzyme levels.

The techniques for mutational analysis include screening by single-strand conformation polymorphism (SSCP), which detects about 85% of mutations, and dideoxy fingerprinting (ddF), a more sensitive test. The criterion standard to detect the exact DNA change is determination of genomic DNA; direct DNA sequencing must be carried out for some molecular defects, such as those at the 3’ and 5’ ends of exons and where the full exon-intron structure of the gene has not been delineated.

When the exact mutation cannot be found, linkage analysis and restriction fragment length polymorphism (RFLP) studies may be performed within families. With the advent of specific mutation analysis, these options are needed less frequently.

Polymorphisms in the androgen receptor are used to define nonrandom inactivation of the X chromosome in the mother and other female relatives in families in which an infant boy has SCID but no extended family pedigree is informative.

The newborn screening test for T-cell receptor excision circles (TRECs) has been used to identify infants with T-cell lymphopenia. No TRECs were detected in newborns with SCID.^[29]

Bronchoscopy frequently is indicated to identify the etiologic agent for pulmonary infection. Endoscopy and biopsies are important in delineating the extent and identifying the cause of diarrhea and other GI symptoms.

In classic SCID, the thymus is small with few thymocytes, and it lacks corticomedullary distinction and Hassall corpuscles (see the image below). The epithelium is normal.

The skin and gut may show infiltration with histiocytes, eosinophils, or activated dysfunctional T cells. The epidermis can have foci of hyperkeratosis with parakeratosis or irregular acanthosis with spongiosis and exocytosis. The papular dermis has edema and a diffuse perivascular infiltrate with some eosinophils.

The spleen and peripheral lymph nodes are characteristically atrophic, but, in maternal and transfusion-mediated graft-versus-host disease (GVHD) or in Omenn syndrome, they may be hyperplastic, with histiocytes and eosinophils. The spleen is depleted of lymphocytes. Although a lymph node biopsy is not necessary for diagnosis, findings may indicate a paucity of T and B cells and a lack of germinal centers. The tonsils, adenoids, and Peyer patches are underdeveloped or absent.

Hemophagocytic lymphohistiocytosis is reported in XL-SCID and cartilage-hair hypoplasia.