**9. Diagnosis**

Initial magnetic resonance imaging (MRI) or computed tomography (CT) scan reveal no abnormality; however, cortical or subcortical atrophy, ventricular dilatation, periventricular changes, and hydrocephaly may be observed later in life. There are no typical histopathology findings at brain biopsy. Illness is progressive and mortality is very high. As a result of disseminated enteroviral infection, dermatomyositis-like syndrome may develop with erythematous rash and peripheral edema. Hepatitis with increased alanine aminotransferase

Gastrointestinal disorders are common problems of XLA patients. Patients may have infec‐ tions, autoimmunity, or rarely malignancy. *Campylobacter jejuni* is the most frequent pathogen causing gastrointestinal tract infection. Typically, affected patients suffer from fever, skin rash, and persistent diarrhea [64–66]. Parasitic infection especially *Giardia lamblia* infection may cause abdominal pain, diarrhea, poor growth, or loss of serum proteins. *Giardia* can be isolated

Inflammatory disorders in XLA patients unexpectedly occur. A web-based patient survey was conducted in patients with XLA. Based on 128 patient responses, the majority of respondents (69%) reported having at least one inflammatory symptom. Just 28% of them had been diagnosed with an inflammatory condition. Arthritis had been diagnosed only in 7% XLA patients, despite 20% reported painful joints and 11% reported swelling of the joints. Similarly, 21% reported symptoms of chronic diarrhea and 17% reported abdominal pain. However, only 4% had been diagnosed with Crohn's disease. Data from the United States Immune Deficiency Network (USIDNET) Registry on 149 patients with XLA revealed that 12% had pain, swelling, or arthralgia, while 18% had been diagnosed with arthritis [67]. Kawasaki disease is also reported in an XLA patient, providing opportunity to understand the relationship between

Noninfectious or infectious arthritis may occur in patients with X-linked agammaglobuline‐ mia. Juvenile rheumatoid is relatively common in patients with XLA. The mechanisms are not clear of noninfectious arthritis. *H. influenzae*, *S. pneumoniae*, and mycoplasma are pathogens causing septic arthritis. Pain and swelling of joints are presenting symptoms. Erythrocyte sedimentation rate increase, rheumatoid factor (RF), and antinuclear antibody (ANA) tests are negative. Arthritis may be the first presenting symptom of X-linked agammaglobulinemia.

Although the relationship between the Bruton kinase mutation and the development of malignant tumors is unknown, XLA patients seem to be at risk for colorectal cancer and lymphoid malignancies. B-cell precursor acute lymphoblastic leukemia (BCP-ALL) has been reported in a case with XLA. It has been reported that somatic mutation found in MLL2 suggests that the alterations of BTK and MLL2 synergistically function as leukemogenesis [72,

Cases with vaccine-associated paralytic poliovirus infection as a consequence of attenuated oral polio (Sabin) have been reported in XLA patients [74]. The incubation period of the infection is more than 30 days, and chronic encephalomyelitis develops eventually [75].

That is the reason why physicians must be aware of immunodeficiencies [69, 70]. Growth hormone deficiency associated with XLA patient has been reported [71].

(ALT) may accompany [60–63].

226 Immunopathology and Immunomodulation

autoimmunity and XLA [68].

73].

from stool samples of patients and too hard to eradicate.

Significant progress in the field has been achieved in the light of collaborative studies. Guidelines for screening primary immunodeficiencies have been published by experts, nonimmunologists and immunologists [51, 76–78]. The classification of primary immunodeficien‐ cy diseases had been updated by the *ad hoc* Expert Committee of the International Union of Immunological Societies [79]. The hallmark of clinical features and laboratory evidences were provided for each immunodeficiency, which help physicians to recognize and diagnose patients with immunodeficiency timely.

#### **9.1. Clinical clues**

X-linked agammaglobulinemia (XLA) should always be considered in children with recurrent, persistent, unusual sinopulmonary infections or a life-threatening severe bacterial infection below 5 years of age. Small tonsils and lymph nodes on physical examination are warning signs of disorder. Patients with strong family history of immunodeficiency relevant with Xlinked inheritance should be investigated further.

#### **9.2. Laboratory approach**

Based on studies, detailed family and medical history combined with careful physical examination further guide specific blood tests of the immune system, which will be performed step by step to confirm the diagnosis of XLA. Severe defects of immune systems should be ruled out at the first steps of diagnostic protocol for suspected immunodeficiency. Infection with human immunodeficiency virus (HIV) has to be excluded rapidly. Tests have to be performed and interpreted by a specialist immunologist. The use of age-matched reference values for lymphocyte subsets and immunoglobulin levels are highly recommended to avoid misinterpretation.

Baseline blood tests give useful information. The complete blood test (CBC) is a crucial test to reveal anemia, neutropenia, lymphopenia, thrombocytopenia, or eosinophilia, which may give a clue to which type of PID is present. Neutropenia is associated with a small subpopulation of patients with XLA (11%), which can be misdiagnosis as congenital neutropenia [8, 80, 81]. Severe neutropenia is usually in association with pseudomonas or staphylococcal sepsis. A CBC and a manual leukocyte differential can aid in the identification of striking lymphopenia, which is a very important clue for severe combined immunodeficiencies (SCID) accepted as medical emergency.

Screening tests for antibody deficiencies as recommended by experts are presented as follows [8, 50, 76–78, 82]:


Serum immunoglobulin concentration should be measured by quantitative techniques, and IgG, IgA, and IgM are routinely measured in serum. Values of determined immunoglobulin levels have to be compared with normal-for-age values. The majority of patients with XLA have less than 200 mg/dL serum IgG level. However, serum IgG concentration is more than 200 mg/dL in 10% of children with XLA. Serum IgM and IgA are classically less than 20 mg/dL. Low serum IgG concentration may also be determined in patients who have proteinlosing enteropathy and nephrosis. The concomitant serum protein levels of these patients are low; hence, they produce antibodies normally. Under certain conditions, the determination of IgE and IgD levels may be required.

Isohemagglutinins are natural IgM-class antibodies against A and B blood group antigens. Therefore, they are not found in patients who have type AB blood group. In addition, the measurement of isohemagglutinin titers in the serum is not reliable below 6 months of age. Normal values for anti-A titer is 1:16 or higher and anti-B titer is 1:8 or higher. Isohemagglu‐ tinins titer is low in XLA patients due to poor IgM synthesis.

Individuals with XLA fail to make antibodies against vaccine antigens or pathogens such as tetanus, *H. influenzae*, or *S. pneumoniae*. Since children are vaccinated with diphtheria–tetanus —acellular pertussis (DTaP), conjugated *H. influenzae* type b, and conjugated pneumococcal vaccine (PVC), the measurement of antibodies against these antigens is informative. The measurement of specific antibodies against diphtheria and tetanus before and 2 weeks after DTaP or booster DT immunization reveals a patient's ability to produce IgG antibodies against protein antigens. Pneumococcal polysaccharide vaccine or conjugated pneumococcal vaccine (PVC) is used to evaluate a patient's ability to respond to polysaccharide antigen (IIb C). The measurement of pneumococcal antibody titers before and 4 to 8 weeks after vaccination should be done in patients more than 2 years of age (Ib A). The normal response to each pneumococcal serotype is defined as a titer equal to or greater than 1.3 mg/mL antibody (IIb C) [83]. The ability of antibody production against antigens and the response to vaccination are severely impaired in XLA patients.

Patients who have agammaglobulinema need lymphocyte phenotyping. The number of B lymphocytes in the peripheral blood can be enumerated by flow cytometry using dyeconjugated monoclonal antibodies (CD 19 and CD 20), which are specific B-cell markers. B cells constitute 4–10% of the peripheral lymphocyte. The reduced number of CD19+ B cells in the peripheral blood would be indicative of defective B-cell differentiation and would suggest XLA if not combined with depleted T-cell numbers. B cell is markedly reduced below <1% in patients with XLA [76–79, 83].

Mutations in *BTK* gene can be scanned by sequence analysis, which can detect approximately 90% of mutations in *BTK*.

## **10. BTK protein testing**

Screening tests for antibody deficiencies as recommended by experts are presented as follows

Serum immunoglobulin concentration should be measured by quantitative techniques, and IgG, IgA, and IgM are routinely measured in serum. Values of determined immunoglobulin levels have to be compared with normal-for-age values. The majority of patients with XLA have less than 200 mg/dL serum IgG level. However, serum IgG concentration is more than 200 mg/dL in 10% of children with XLA. Serum IgM and IgA are classically less than 20 mg/dL. Low serum IgG concentration may also be determined in patients who have proteinlosing enteropathy and nephrosis. The concomitant serum protein levels of these patients are low; hence, they produce antibodies normally. Under certain conditions, the determination of

Isohemagglutinins are natural IgM-class antibodies against A and B blood group antigens. Therefore, they are not found in patients who have type AB blood group. In addition, the measurement of isohemagglutinin titers in the serum is not reliable below 6 months of age. Normal values for anti-A titer is 1:16 or higher and anti-B titer is 1:8 or higher. Isohemagglu‐

Individuals with XLA fail to make antibodies against vaccine antigens or pathogens such as tetanus, *H. influenzae*, or *S. pneumoniae*. Since children are vaccinated with diphtheria–tetanus —acellular pertussis (DTaP), conjugated *H. influenzae* type b, and conjugated pneumococcal vaccine (PVC), the measurement of antibodies against these antigens is informative. The measurement of specific antibodies against diphtheria and tetanus before and 2 weeks after DTaP or booster DT immunization reveals a patient's ability to produce IgG antibodies against protein antigens. Pneumococcal polysaccharide vaccine or conjugated pneumococcal vaccine (PVC) is used to evaluate a patient's ability to respond to polysaccharide antigen (IIb C). The

[8, 50, 76–78, 82]:

**•** Serum total protein level

228 Immunopathology and Immunomodulation

**•** Specific antibody responses

**•** Tetanus, diphtheria (IgG1)

**•** Lymphocyte proliferation tests

**•** Genetic determinations of defect

IgE and IgD levels may be required.

**•** Serum immunoglobulin assay: IgG, IgA, IgM

**•** Viral respiratory pathogens (IgG1 and IgG3)

**•** B-cell maturation analysis in bone marrow

**•** Isohemagglutinins (IgM antibodies to A and B blood group antigens)

**•** Other vaccines: hepatitis B, influenza, MMR, and polio (killed vaccine)

**•** Lymphocyte subpopulation by flow cytometry (B-cell quantitation)

**•** Pneumococcal and meningococcal polysaccharides (IgG2)

tinins titer is low in XLA patients due to poor IgM synthesis.

*BTK* mutations occur in the absence of the BTK protein in monocytes. The detection of BTK protein in monocytes by immunofluorescence or western blot [84, 85] can confirm the diagnosis of XLA.

Female carriers may be determined by mutation analysis or Btk protein expression on blood cells by flow cytometry. They have normal immune functions, but they have a 50% chance of transmitting the disease to each of her sons.

The following diagnostic criteria for X-linked agammaglobulinemia were published by Conley et al. in 1999 [86]:

*Definitive diagnosis*—males with less than 2% CD19+ B cells and at least one of the following:


*Probable diagnosis*—males with less than 2% CD19+ B cells and the following:


*Possible diagnosis*—males with less than 2% CD19+ B cells in whom other causes of hypogam‐ maglobulinemia have been excluded and who has at least one of the following:


*Chest X-ray or CT scan* of patients with XLA reveals bronchiectasis most commonly distributed in the middle or lower lobes, atelectasis, and bronchial wall thickening. CT scan of sinuses may suggest the presence of chronic sinusitis.

*Prenatal diagnosis—*this may be achieved by using mutation analysis in amniotic fluid cells and Btk protein expression on cord blood cells by flow cytometry.

#### **11. Newborn screening**

Kappa-deleting recombination excision circles (KRECs) are chosen as markers for B lympho‐ penia at birth, indicative of X-linked agammaglobulinemia. The measurement of KRECs in newborn would help the early diagnosis of XLA patients [87].
