X-linked severe combined immunodeficiency

Context.-To review the applications of flow cytometry in the diagnosis and management of primary immunodeficiency disease.

Data Sources.-Articles describing the use of flow cytometry in the diagnosis of several primary immunodeficiency diseases were obtained through the National Library of Medicine database.

Study Selection.-Publications that described novel and known applications of flow cytometry in primary immunodeficiency disease were selected. Review articles were included. Articles describing the different immunodeficiency diseases and methods of diagnosis were also selected.

Data Extraction.-Approximately 100 data sources were analyzed, and those with the most relevant information were selected.

Data Synthesis.-The diagnosis of many primary immunodeficiency diseases requires the use of several laboratory tests. Flow cytometry has become an important part of the workup of individuals suspected to have such a disorder. Knowledge of the pathogenesis of many of these diseases continues to increase, hence we acquire a better understanding of the laboratory tests that may be helpful in diagnosis.

Conclusions.-Flow cytometry is applicable in the initial workup and subsequent management of several primary immunodeficiency diseases. As our understanding of the pathogenesis and management of these diseases increases, the use of many of these assays may become routine in hospitals.

The primary immunodeficiency diseases (PIDs) are rare disorders that reflect abnormalities in the development, maturation, or performance of cells required for immune function. Affected individuals are susceptible to repeated infections, allergies, autoimmune diseases, and malignancies.1 The incidence of these diseases varies and ranges from 1:400 to 1:500 000 live births in the United States. More than 95 inherited immunodeficiency disorders are currently recognized, and the number continues to increase as more genetic defects are identified.2 The initial classification of these diseases was based mainly on the clinical features of the disease or syndrome. The use of flow cytometry and understanding of the molecular defects involved has been helpful in regrouping some of these disorders. The PIDs are currently classified into 5 groups: antibody/humoral deficiencies, combined immunodeficiency diseases, other well-defined immunodeficiency syndromes, complement deficiencies, and defects in phagocytosis. Table 1 presents a summary of the more common PIDs. During the past few years, our understanding of the pathogenesis and molecular and genetic bases of many of these diseases has expanded, thus improving the use of laboratory diagnostic studies and the development of new assays.

The initial evaluation of patients suspected to have an immunodeficiency disorder include enumeration of crucial cell populations and assessment of immunological competence.1,3 Flow cytometry using monoclonal antibodies to analyze and identify cells involved in immune function is useful in the diagnosis and management of many primary immunodeficiency disorders. It is a rapid and sensitive assay that has the advantage of evaluating several characteristics of a cell type. It has basically replaced previous tests, such as E-rosette formation for T-cell detection and identification of surface immunoglobulins for B-cell detection.4 Flow cytometry can be used to enumerate a particular cell type, evaluate function, or detect a particular gene product. Initial results, together with other available clinical data, help in making a decision on further testing, especially genetic testing.

This article reviews the applications of flow cytometry in some of the more common PIDs. Tables, figures, and an example of a functional assay are included. Figure 1 illustrates B- and T-lymphocyte development and the different stages at which the defects leading to some of the primary immunodeficiency disorders occur. The different cells involved in immune function and their normal counts are summarized in Table 2.

All cells involved in immune and phagocytic functions express a number of surface and cytoplasmic proteins unique to the cell lineage or stage of development. Some of these proteins are referred to by the initials "CD" (cluster of differentiation), followed by an identifying number. The common CD markers used in clinical practice are summarized in Table 3.

HUMORAL/ANTIBODY IMMUNODEFICIENCIES

X-Linked Hypogammaglobulinemia

X-linked hypogammaglobulinemia is inherited as an X-linked recessive trait. There is a mutation at the Xq21.3-Xq22 region, which leads to a defect in the Bruton tyrosine kinase (BTK) gene. The defect leads to a deficiency of Btk, an enzyme required for B-lymphocyte maturation.5 Evaluation of the peripheral blood lymphocyte population by flow cytometry will reveal a decrease (1%-2%) or absence of B cells.5,6 T cells are usually normal in number. The CD4-CD8 ratio is normal to low. CD45RA, which is found on naive T cells, predominates on the CD4^sup +^ T cells.4 This distribution suggests a defect in T-cell differentiation. In the bone marrow, a few CDW19^sup +^CD10^sup ±^ B-cell progenitors are present. CD38 and cytoplasmic [mu] may be expressed. Mature B cells expressing surface immunoglobulin are absent. These findings support a maturational block in the B-cell lineage. Futani et al7 described a flow cytometry assay using a monoclonal antibody to Btk. Their study revealed the lack of Btk expression on the monocytes of individuals with this disorder.7,8 By this method, the authors were able to detect Btk deficiency in a group of patients who had been registered in the immunodeficiency registry as having common variable immunodeficiency.9

The same group also demonstrated the lack of Btk expression on the platelets of most patients (37 of 45) with this disorder. Platelets from carrier females had both normal and abnormal platelet populations.10

Common Variable Immunodeficiency

Common variable immunodeficiency constitutes a group of undifferentiated syndromes characterized by defects in B-cell maturation and antibody formation. No well-defined mode of inheritance has been identified, and it typically manifests around the second to third decade of life.1,3 B-cell maturation does occur, but immunoglobulin (Ig) levels (IgG, IgA, and IgM) are decreased. Multiple studies suggest several etiologies. T-cell and B-cell defects have been revealed. No definite flow cytometry profile has been described for this syndrome.3 Farrant et al11 reported the common immunophenotypic findings in 71 common variable immunodeficiency patients. They showed a decrease in CD4^sup +^ T cells, especially the CD45RA subset, and decreased B cells with little or no expression of surface IgM and IgG.11 Recent reports have noted diminished numbers or absence of the mature class switched CD27^sup +^ IgD^sup -^ IgM^sup -^ memory B cells in subgroups of patients with this disease. The findings have lead to a proposal for a classification of common variable immunodeficiency.12

Selective IgA Deficiency

Selective IgA deficiency occurs more often than other PIDs. Flow cytometry may not be very helpful in the diagnosis, unless it coexists with common variable immunodeficiency. Individuals with IgA deficiency alone will have a normal number of B and T cells. Conley et al13 showed that more than 80% of IgA-positive cells in some patients with IgA deficiency coexpress IgM and IgD, suggesting a maturational arrest.

COMBINED IMMUNODEFICIENCY DISEASES

Severe Combined Immunodeficiency

Severe combined immunodeficiency (SCID) comprises a group of inherited disorders that characteristically show abnormalities in B-, T-, and natural killer (NK)-cell function. More than 10 types have been described, based on the genetic defects involved (Table 1). Recently, many advances in our understanding of the molecular basis of this disease have been made; thus, we have a better understanding of the immunophenotypic findings.6,14,15 Almost all forms of this disease show a decrease or absence of T cells. Further classification can be made based on the presence or absence of B and NK cells (Table 4). After evaluating the immunophenotypic characteristics of the lymphocytes, further diagnostic tests can be selected, in order to arrive at a definite diagnosis. The more common forms are reviewed here.

X-Linked SCID.-X-linked SCID is the most common form of SCID, and it is characterized by a defect in the common y chain (yc). The yc (CD132) is an essential component of the interleukin (IL)-4, IL-7, IL-9, and IL-15 cytokine receptor complex, which is necessary for normal B-, T-, and NK-cell development.14,16,17 In addition to the absence or decrease in T cells, NK cells are absent. B cells are present, but they do not exhibit normal function. Most of these cells are of the naive B-cell phenotype, expressing surface IgM. This is due to a defect in isotype switching following the loss of IL-4 function.16 In addition, several authors have demonstrated the absence of [gamma]c expression by flow cytometry on mononuclear cells of children with X-linked SCID.6,14,15 A similar phenotype (T^sup -^ B^sup +^NK ^sup -^) is observed in JAK 3 SCID, which is inherited in an autosomal recessive fashion.15 The JAK 3 molecule interacts with the [gamma] chain in the [gamma]c/JAK 3 pathway.14,16

Interleukin-7 Receptor [alpha] Deficiency.-The IL-7 receptor is composed of an IL-7 [alpha] and [gamma] chain. Mutations impairing the normal expression of the [alpha] chain lead to a defect in T-cell maturation. Individuals with a defect of this receptor show a T^sup -^B^sup +^NK^sup +^ phenotype, as well as absence or abnormal expression of the IL-7[alpha] receptor (CD127) by flow cytometry.17

Adenosine Deaminase Deficiency.-Adenosine deaminase (ADA) deficiency accounts for 20% to 50% of all cases of SCID. The lack of this enzyme results in the accumulation of deoxyadenosine, 5-adenosylhomocysteine, and deoxyadenosine triphosphate (dATP). These toxic metabolites inhibit DNA synthesis. Lymphocytes, which exhibit the highest amount of ADA expression in lymphoid tissue, are especially sensitive to these metabolites and their maturation is therefore limited.14 B- and T-cell numbers may be normal at birth, and then eventually drop and become low or absent. The number of NK cells is decreased in most cases.6,14,15,17 A definite diagnosis can be made by the analysis of dATP concentration and ADA activity in washed red cells. A flow cytometry assay to estimate levels of intracellular ADA has been described recently by Otsu et al.18

Purine Nucleoside Phosphorylase Deficiency.-Purine nucleoside phosphorylase deficiency is a rare cause of SCID. T-cell function is severely affected, revealing a T^sup -^BN^sup +^K^sup -^ phenotype.6,14

Recombination Activating Genes 1 and 2 Deficiency.-Mutations in recombination activating genes 1 or 2 (RAGl or RAG2) lead to a defect in antigen receptor gene recombination activity and the inability to assemble the B- and T-cell receptor complexes. Mature B and T lymphocytes are absent.6,14,17,19 A phenotype similar to ADA SCID, but with a normal NK-cell population (T^sup -^B^sup -^NK^sup +^) is observed in this form of SCID.

Omenn Syndrome.-Omenn syndrome is a clinical disorder marked by the normal infective complications of SCID plus an erythrodermic rash, lymphadenopathy, hepatosplenomegaly, eosinophilia, and elevated IgE levels. RAG gene mutations have been found to be responsible in this syndrome. The mutation leads to partial RAG activity, therefore residual V(D)J recombination. Immunophenotyping reveals low or complete absence of B cells. T cells are present but usually display an activated phenotype. The literature contains reports of an increased amount of [gamma][delta]^sup +^ cells in these individuals.15

Major Histocompatibility Complex Class I Deficiency.-This deficiency is a milder and more limited disease compared to major histocompatibility complex (MHC) class II deficiency. Only a few cases have been described to date. Patients with this condition have chronic repeated bacterial infections, skin ulcerations, and vasculitis.5 Normally, the class I molecules are expressed on all cells. They participate in antigen presentation to CD8^sup +^ T cells and activate their cytotoxic function. Class I molecules are also necessary for intrathymic maturation of CDS^sup +^ T cells. Affected individuals have normal class I proteins, but these proteins fail to reach the cell surface. This failure is due to the deficiency of TAP-1, a transporter antigen required for the expression of the HLA class I molecules on the cell surface. Instead, these molecules are retained in the endoplasmic reticulum.1,20 The absence of the class I molecule leads to a lack of CDS^sup +^/[alpha][beta]^sup +^ T cells. CDS^sup +^/[gamma][delta]^sup +^ Tcells are present.20 Flow cytometry using monoclonal antibodies against pan-HLA class I molecules or anti-ß^sub 2^-microglobulin will show an absence of expression.6,20

Major Histocompatibility Complex Class II Deficiency.-Originally called the bare lymphocyte syndrome, MHC class II deficiency is a rare form of SCID inherited in an autosomal recessive fashion. Normally MHC class II molecules are expressed on antigen-presenting cells, such as B lymphocytes, activated T cells, dendritic cells, the monocyte/macrophage cell lineage, and thymic epithelial cell. Major histocompatibility complex class II binds to CD4^sup +^ T cells, leading to activation and humoral/cell-mediated immunity. In addition, the MHC class II molecule also plays a role in the maturation of CD4^sup +^ T cells, and the life span of these cells is dependent on their interaction with this molecule.1,21

Failure of the normal T-cell activation process leads to the clinical presentation of severe infections due to the inability to respond appropriately to foreign antigens. When the peripheral blood of affected individuals is evaluated by flow cytometry, there are normal numbers of B and T lymphocytes (T^sup +^/B^sup +^ SCID).1,4,6,14,21 The number of CD4^sup +^ T cells is reduced, and the severity of this reduction varies among different individuals with this disease. The response of the residual CD4 T ^sup +^ cells to antigen presentation has not been explored completely. Most of these CD4+ lymphocytes express CD45RA, supporting the fact that they are not activated.4 The B cells express high levels of IgM and IgD, and fail to express the class II proteins (HLA-DR, HLA-DP, HLA-DQ, and HLA-DM).5,14 The B- cell immunophenotype supports the finding of hypogammaglobulinemia in patients with this disease. Residual MHC class I molecule expression is seen, and T-cell receptor (TCR) expression is normal.1,21

CDS Deficiency.-The phenotype of the CD3 deficiency is variable even among family members with the disease, owing to variable expression of CD3 on the T cell.22,23 This condition commonly affects the [gamma] or [epsilon] chains of the CD3 molecule. There are mutations in the CD3 subunit, and abnormal assembly of the TCR-CD3 complex. Flow cytometry reveals a decrease or complete absence of CD3 expression. T-cell receptor expression is also decreased.22-24 Other T-cell markers are expressed normally. The overall percentage and number of T cells is usually decreased. B lymphocytes are present in normal amounts.6 It should be noted that most CD3 monoclonal antibody preparations are targeted against CDSe.22

ZAP-70 Protein Deficiency.-The ZAP-70 protein normally binds to the [delta] chain of the CD3 complex.14,22 It is thought that this molecule is essential for positive selection of CDS^sup +^ T cells during thymus maturation.14 Individuals with mutations of the ZAP-70 protein show a lymphocytosis with CDS lymphopenia. There is a relative increase in CD4^sup +^ lymphocytes.6,14,22 B cells are normal in number, but defects in B-cell function have been reported.22

Reticular Dysgenesis.-Reticular dysgenesis is a very rare form of SCID characterized by defective lymphoid and myeloid differentiation at the stem cell level. Individuals with this disorder are pancytopenic with a decrease in all cell lines.6,17

Hyper IgM Syndrome

Hyper IgM syndrome is a rare disorder characterized by normal to high levels of polyclonal IgM and IgD, but decreased or absent IgG, IgE, or IgA. Affected individuals present with repeated infections, including Pneuniocystis carinii infections. They also have an increased incidence of autoimmune disease and malignancy.23 In the X-linked form, there is a mutation in the gene that encodes for the CD40 ligand (CD154). CD154 is normally expressed primarily on activated CD4 ^sup +^T cells and a small number of CD8+ T cells, and its absence interferes with the ability of the B cells to undergo isotype switching. Evaluation for the CD40 ligand requires initial lymphocyte activation. The full procedure in whole blood has been described by O'Gorman et al.26 Carriers can also be identified by this method. Platelets have also been shown to be capable of expressing CD40 ligand when activated. Flow cytometric detection of this protein on platelets has been described.27 The absence of the CD40 ligand has also been reported in individuals with common variable immunodeficiency and in children infected with human immunodeficiency syndrome.26

OTHER WELL-DEFINED IMMUNODEFICIENCY SYNDROMES

DiGeorge Syndrome

One of the features of this syndrome is thymic hypoplasia or aplasia. Because of the thymic aplasia, normal Tcell maturation does not occur. Flow cytometry of peripheral blood will reveal a decrease in CD3^sup +^, CD4^sup +^, or CD8^sup +^ T cells. Of the T cells remaining, TCR[alpha]ß^sup +^ cells are decreased, whereas the number of TCR[gamma][delta]^sup +^ cells is normal.6 The total number of CDS^sup +^ cells may initially be decreased due to a decrease in suppressor cell activity.4 The CD4- CDS ratio is therefore increased. The suppressor activity will increase as these individuals get older, and the number of CDS^sup +^ cells will increase. In some individuals, the T-cell population has increased and eventually become normal. This improvement with age may be due to the presence of residual or ectopic thymic tissue, enough to allow normal T-cell development and maturation.4

Wiskott-Aldrich Syndrome

Wiskott-Aldrich syndrome (WAS) is an X-linked disorder characterized by the triad of thrombopathia, eczema, and combined B- and T-cell deficiency. This disease is caused by a defect in the WAS protein gene on the X chromosome. The WAS protein (WASP) normally has multiple functional domains that contribute to actin polymerization, cell motility, intracellular signaling, and apoptosis.28 A milder form of this condition (X-linked thrombocytopenia) has been described.

Early in the initial stages of this disease, lymphocyte subset numbers are normal, but with increasing age, the numbers of T and B cells decrease. The CD4-CD8 ratio varies; it is elevated in some individuals and low to normal in others.4,6 Lymphocytes and platelets are smaller than normal. Expression of cell surface glycoproteins, including CD43 (normally expressed on all lymphocytes, neutrophils, macrophages, and platelets), is decreased.29 These defects are thought to interfere with the trafficking of leukocytes to the sites of inflammation. Identification of both affected individuals and carriers can be performed by flow cytometry evaluating for the expression of WASP on lymphocytes or monocytes. Using a monoclonal antibody against WASP, affected individuals will reveal WASP-dim lymphocytes or monocytes. Carriers will show 2 populations of cells (WASP bright and WASP dim).30-31

Ataxia Telangiectasia

This rare condition presents as progressive cerebellar ataxia and telangiectasias involving the conjunctiva or the arms. Most patients with this disorder also show a marked decrease in serum immunoglobulins and poor cell-mediated responses.25 No diagnostic flow cytometry pattern is observed in these patients. Carbonari et al12 studied 10 patients with ataxia telangiectasia and found that most of them had a relative increase in T cells expressing the [gamma]/[delta] receptor. Selective deficiency of the CD4^sup +^/CD45RA (naive) T lymphocytes has also been observed.33

Autoimmune Lymphoproliferative Syndrome

Autoimmune lymphoproliferative syndrome is characterized by diffuse nonmalignant lymphadenopathy, hepatosplenomegaly, autoimmune hemolytic anemia, thrombocytopenia, neutropenia, and other autoimmune diseases.34 Basically, inherited defects are found in mechanisms that induce lymphocytes to undergo apoptosis. Mutations in the genes that encode FAS and its ligand FasL, caspase 10, and other proteins involved in the apoptosis pathway are responsible for this condition.3,35 One of the criteria required for the diagnosis of this disorder is the presence of an increased amount of CD3^sup +^CD4^sup -^CD8^sup -^ [alpha]/[beta]^sup +^ cells ([alpha]/ [beta]^sup +^ double-negative [DNT] cells).3,34,35 Other findings in these persons include low numbers of CD3^sup +^CD25^sup +^ T cells due to a reduction of the CD4^sup +^CD25^sup +^ T cells, expansion of [gamma]/[delta] DNT cells, CD3^sup +^HLA-DR^sup +^ T cells, CD8^sup +^ T cells, CD8^sup +^CD57^sup +^ T cells, total B cells, and CD5^sup +^ B cells.35

DEFECTS IN PHAGOCYTOSIS

Chronic Cranulomatous Disease

Chronic granulomatous disease is characterized by a defect in phagocytic killing. The neutrophils lack one of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits required for a normal oxidative burst, which normally occurs after phagocytosis. Patients with this disease present with recurrent bacterial and fungal infections. The nitroblue tetrazolium test was once the diagnostic test for this disease. Flow cytometry is now replacing the nitroblue tetrazolium test and is easy to perform and more accurate. The flow assay measures the neutrophils' ability to generate an oxidative burst by indirectly measuring the increase in fluorescence generated by the oxidation of a laser-sensitive dye (dihydrorhodamine 123).36 The neutrophils are incubated with the dye and stimulated with phorbol myristate acetate to generate an oxidative burst. In the presence of reactive intermediates generated during the burst, the dye is oxidized and becomes highly fluorescent. The level of fluorescence is proportional to the amount of dye oxidized. In patients with chronic granulomatous disease, the oxidative burst does not occur and there is no increase in fluorescence. Female carriers in the X-linked form will show 2 populations of neutrophils, one with and the other without increased fluorescence (Figure 2).

Leukocyte Adhesion Deficiencies

Leukocyte Adhesion Deficiency-1.-In leukocyte adhesion deficiency (LAD)-I, there is a mutation in the gene encoding CD18, the common [beta] chain of the leukocyte adhesion proteins. This mutation leads to a decreased expression of the CD11/CD18 ([beta]2 integrins) family of proteins, a group of proteins essential for leukocyte adhesion. They include leukocyte function-associated protein (LFA- 1 or CDlla/CD18), Mac-1 (CDllb/CD18), and pl50/95 (CDllc/CDlS).25 Patients with this condition present with recurrent bacterial infections with no pus formation, and infants have delayed umbilical cord separation.

Leukocyte Adhesion Deficiency-2.-Leukocyte adhesion deficiency-2 is similar to LAD-1, but is not due to integrin defects. Rather, a defect in fucose metabolism results in the absence of sialyl Lewis X (CD15s), a carbohydrate ligand on the surface of neutrophils. This ligand is required for the binding of E selectin and P selectin to activated endothelium. Individuals affected with this disorder present with recurrent infections.

Flow cytometry is useful in both LAD disorders. Gating on the granulocytes and the expression of CD11 and CD18 will be decreased or absent in LAD-1. In LAD-2, there is a decrease or absence of expression of CDlSs.37

Chediak-Higashi Syndrome

In Chediak-Higashi syndrome, patients have giant granules in their phagocytes, melanocytes, and other granuleforming cells. They suffer repeated infections, peripheral neuropathy, partial oculocutaneous albinism, and lymphoproliferative disease with diffuse organ involvement. There is a mutation of the LYST gene, which is responsible for cellular signal response coupling. No definite changes are evident on flow cytometry, even though the presence of large granules would suggest otherwise. Cario et al38 showed that the right angle scatter was normal in these individuals despite the large inclusions. This finding suggests that the total cell density in the granulocytes of individuals with this disease is not increased. Lymphocyte subsets of 6 patients with this disease were evaluated and revealed an increase in the CDS^sup +^ population of T cells and a decrease in CD4^sup +^ T cells. Interestingly, the mothers of these patients also showed a similar immunophenotype.39

Complement Deficiency

Flow cytometry is of limited value in the evaluation of a patient with complement deficiency. B- and T-cell subsets are usually normal.

COMMENT

Immunophenotyping of leukocytes has become a very important part in the initial workup and management of patients with PIDs. Many of the flow cytometric studies can be performed in hospital laboratories, while extensive or unusual cases may have to be performed in specialized laboratories or research settings. Initial results can help the physician determine what other investigative procedures are needed to arrive at a definite diagnosis. This procedure will also be helpful in prenatal screening for some of the immunodeficiency diseases. Not all cases of PID will yield the expected changes by flow cytometry. Molecular and genetic studies are useful in such cases. As we gain a better understanding of the pathogenesis of these diseases, and with the identification of new antigens and antibodies, the number of diagnostic flow cytometric assays will certainly increase in the near future.

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Accepted for publication August 14, 2003.

From the Department of Pathology, University of Virginia Health System, Charlottesville.

Reprints: Orieji Illoh, MD, Department of Pathology, Box 800214, University of Virginia Health System, Charlottesville, VA 22908 (e-mail: oci8t@virginia.edu).

Copyright College of American Pathologists Jan 2004
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