immunodeficiency presents with increased susceptibility to infection but may also manifest with conditions that reflect dysregulation of the immune response, such as allergies, autoimmunity, or lymphoproliferation

1. APPROACH TO EVALUATION
OF THE PATIENT WITH
SUSPECTED
IMMUNODEFICIENCY
DR OLA ALKHARS
GENERAL PEDIATRIC CONSULTANT

2. • immunodeficiency presents with increased susceptibility to infection but may
also manifest with conditions that reflect dysregulation of the immune response,
such as allergies, autoimmunity, or lymphoproliferation

3. • PIDs include severe combined immune deficiencies (SCIDs), complete DiGeorge
syndrome, and chronic granulomatous disease (CGD)

4. EPIDEMIOLOGY—PRIMARY
IMMUNODEFICIENCIES
• selective immunoglobulin A (IgA) deficiency, a relatively common condition,
(1/223–1/1000 people)
• in the United States SCID 1/58 000 live births

5. PRIMARY VERSUS SECONDARY
IMMUNODEFICIENCY
acquired and nonimmunological causes for recurrent infections should be first
considered in the differential diagnosis of the patient with a suspected immune
disorder

7. EVALUATING PATIENTS FOR IMMUNODEFICIENCY

8. AGE AND ENVIRONMENT
• Pediatric patients are more likely to present a PID than a secondary
immunodeficiency. Infants from birth to 3 months of age have maternal Igs
acquired through the placenta, unless they were born prematurely
• Therefore deficiencies in the immune system at this age presenting with frequent
infections most probably result from severe deficiencies in other immune
components, such as neutrophils, complement components, or T cells. Older
patients might present with increased risk of infections secondary to comorbid
conditions, such as allergic inflammation or diabetes mellitus, or to a normal
decline of immune responses, a process known as immunosenescence

9. • Infants frequently exposed to other infants with infections, such as in the setting
of a daycare facility
• By inducing an inflammatory response of the respiratory mucosa, passive
cigarette smoke inhalation also predisposes to infections, including otitis media,
pneumonia, and bronchitis

10. IMMUNIZATION AND PREVIOUS INFECTIONS
• A history of an adverse reaction to a live viral vaccine is suspicious for
immunodeficiency, as infants with T-cell defects, B-cell defects, and combined T-
and B-cell defects are susceptible to potentially fatal or severe infections from live
attenuated vaccines.
• Individuals with immunodeficiency may have infections with unusually
prolonged courses or unusual severity or that may present as unexpected
complications

11. • Recurrent infections that involve multiple sites are more suspicious for immune
deficiency than those involving a single site. Also suggestive of immune
compromise are severe and invasive infections, such as recurrent pneumonia,
meningitis, sepsis, septic arthritis, osteomyelitis, or abscess and infections with
organisms of low pathogenicity in normal individuals, such as Candida albicans or
Pneumocystis jiroveci

12. • Patients with antibody deficiency disorders tend to present with infections caused by
extracellular pyogenic organisms, such as Haemophilus spp. Pneumococcus spp., and
Streptococcus spp. In contrast, patients with defects in cell-mediated immunity are more likely
to also present with recurrent infections with viruses, fungi, protozoa, and mycobacteria.
Furthermore, infections by catalase-positive bacteria, such as Serratia marcescens, may indicate
a possible neutrophil oxidative burst defect.
• Recurrent neisserial infections may be found in individuals deficient in the terminal complement
components.
• A relatively normal incidence of infections followed by a sudden occurrence of repeated
infections in an adult or adolescent suggests a secondary immunodeficiency, including HIV
infection.

14. COMORBID CONDITIONS

15. USE OF MEDICATIONS
• Use of particular drugs might also cause immunodeficiency, which could be
predictable, such as the use of rituximab (anti-CD20 antibody) resulting in B-cell
depletion and potential antibody deficiency, or idiopathic, such as
hypogammaglobulinemia that might develop with the use of anticonvulsants.

16. FAMILY AND SOCIAL HISTORIES
• A history of early infant deaths and possible consanguinity should be sought
• A clear pattern of inheritance may be found to define an X-linked, autosomal
dominant, or autosomal recessive genetic syndrome
• Many of the most common PIDs have X-linked inheritance patterns y+ Family
members of patients with immunodeficiencies might also have a history of
autoimmune disease or of connective tissue disease. Familial cases of selective
IgA deficiency and common variable immunodeficiency (CVID) have been
reported

17. • a susceptibility trait can sometimes be traced back to many generations.
• A social history should be obtained for risk factors associated with increased risk
of acquiring HIV infection.
• Socioeconomic factors often determine malnutrition, known to be of a significant
impact on immune function

19. PHYSICAL EXAMINATION FINDINGS
• The physical examination might provide findings that indirectly address the immune system;
• for example, scarred bilateral tympani suggest recurrent ear infections.
• More commonly, patients with immunodeficiency might otherwise look like normal
individuals, unless severe infections had produced an organ damage or had delayed growth
and development.
• However, attention to details in the physical examination may supply important clues that
suggest immune dysfunction.
• In a normal child, a paucity of lymphoid tissue, such as tonsils and lymph nodes, might reflect
impaired development resulting from immune deficiency. This is especially seen in patients
with X-linked agammaglobulinemia. Certain physical findings are suggestive of syndromic
immunodeficiencies, such as with telangiectasia over the bulbar conjunctivae and face with or
without ataxia in ataxia–telangiectasia (AT)

20. PHYSICAL EXAMINATION FINDINGS
• chronic eczema and delayed shedding of primary teeth in hyper-IgE syndrome
(HIES)
• severe eczema in immunodeficiency, polyendocrinopathy and enteropathy, X-linked
(IPEX) syndrome, and WAS
• chronic periodontitis in defects of the neutrophils; or silvery hair, pale skin, and
photophobia in Chediak-Higashi syndrome.
• Investigation for Shwachman-Bodian-Diamond syndrome (SBDS) should be
considered in patients with neutropenia, especially if they also present with skeletal
dysplasia.
• Patients with DiGeorge syndrome and nuclear factor-κ B (NF-κ B) essential
modulator (NEMO) deficiency present with characteristic facies.

21. • Children with severe immune defects are small for their age, with growth delay
secondary to recurrent infections.
• Hepatosplenomegaly and diffuse lymphadenopathy might suggest HIV infection or a
disorder of immune dysregulation.
• Children with leukocyte adhesion defect (LAD) can present with severe
gingivostomatitis and dental erosion as a consequence of abnormal leukocyte
function .
• Multiple scars from skin abscesses might suggest neutrophil defects, and scarred
tympani with reduced hearing might indicate a history of recurrent otitis media, which
can be associated with antibody deficiency.

22. LABORATORY TESTING FOR IMMUNE FUNCTION
• Immunology Testing
Serum Immunoglobulin Levels

23. UV87VV362

24. IMMUNOLOGY TESTING
Serum Immunoglobulin Levels:
• The IgA level is especially helpful in that it is low in all permanent types of agammaglobulinemia and in selective IgA
deficiency.
• IgE level measurement is of significance for the diagnosis of HIES.
• Serum IgG subclasses levels can be determined. However, rather than using IgG subclass levels to screen for
immunodeficiency, they are best utilized when patients have clinical conditions associated with specific antibody
deficiencies but normal total IgG levels.
• In some of these patients, IgG subclass deficiency, particularly IgG2 and IgG3 deficiencies, might be present.
• IgG2 subclass deficiency has been linked with selective IgA deficiency and deficiency of antipolysaccharide
antibodies.
• IgA subclass low levels, IgA1, IgA2, have not been associated with a specific immune defect, and there is no validity
for measuring these.
• The variation of normal ranges of human serum Igs with age is an important consideration in children, since IgA and
IgG subclass levels may not reach normal adult reference ranges until 6 years of age.

25. B-CELL FUNCTION: SPECIFIC ANTIBODY PRODUCTION
• To properly assess B-cell function, specific antibody production must be measured. Patients with
normal Ig and Ig subclass levels might exhibit deficient antigen-dependent antibody responses.
• An initial screen of antibody production may involve the quantification of isohemagglutinins.
Isohemagglutinins occur in all individuals except those with blood type AB; isohemagglutinins are
natural IgM antibodies to polysaccharide blood group antigens A and/or B, which are not expressed in
the red blood cells (RBCs) of the patient tested.
• Individuals form isohemagglutinins as a result of environmental exposure to ubiquitous antigens that
share epitopes with blood antigens.
• Children less than 1 year of age do not reliably have measurable serum isohemagglutinins because of
the limited exposure to the environment.
• A patient with blood type A should have anti-B IgM; patients with blood type B should have anti-A
IgM; and patients with blood type O should have both anti-A and anti-B IgM. These antibodies
are normally present in titers greater than 1 : 10; individuals with poor antibody production may have
low or absent titers.

26. B-CELL FUNCTION: SPECIFIC ANTIBODY PRODUCTION
• Specific IgG antibody production can be measured following immunization with
protein antigens, such as toxoids derived from Tetanus and Diphtheria organisms,
and polysaccharide antigens, such as those produced by pneumococci and
Haemophilus influenzae.
• For pneumococcal immunization, there are two vaccines that need to be
differentiated. The conjugated vaccine containing 13 pneumococcal serotypes
(PREVNAR13, Wyeth) is currently included in the universal schedule of immunizations
for infants and toddlers and induces a robust, T cell–dependent immune response.
• The unconjugated 23-valent pneumococcal polysaccharide vaccine (Pneumovax,
Merck) is available for immunization to adults and children aged 2 years and older.
The immune response for this vaccine is considered less dependent on T cells and
also less lasting than the conjugated vaccine.

27. B-CELL FUNCTION: SPECIFIC ANTIBODY PRODUCTION
• The pneumococcal antigen challenge using the unconjugated vaccine is not recommended for children under 2 years of age
because healthy children are not thought to reliably respond to the unconjugated pneumococcal antigen at this age. However,
this view has been challenged by data showing that 1-year-old children produce normal antibody responses to this
unconjugated vaccine.
• Normal antibody responses are usually demonstrated with an over twofold rise in specific antibody levels within 2–3 weeks
for protein antigens and within 4–6 weeks for polysaccharide antigens.
• Patients with agammaglobulinemia are expected not to produce antibody responses, whereas others, such as those with IgG2
subclass deficiency and normal levels of total IgG, may only have difficulty with antibody production following immunization
with polysaccharide antigens.
• Patients with selective IgA deficiency, alone or with transient hypogammaglobulinemia of infancy, have normal specific IgG
antibody production, by definition. The pneumococcal serotypes included in the current conjugated antipneumococcal
vaccine, serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F, were estimated to be responsible for approximately 90%
of invasive pneumococcal disease in children less than 5 years of age worldwide.
• Previous immunization with the conjugate vaccine does not preclude use of the unconjugated pneumococcal polysaccharide
vaccine. The 23-valent polysaccharide vaccine provides the potential for stimulation and measurement of a protective immune
response to additional 11 serotypes (2, 8, 9N, 10A, 11A, 12F, 15B, 17F, 20, 22F, 33F) not included in the conjugated vaccine.
Testing for antibodies against serotypes not included in the two vaccines and comparing the antibody titers in the pre- and
postimmunization blood samples helps in the assessment of specific increase of particular antiserotype antibody titers as a
response to the vaccine administration.

28. EVALUATION OF CELLULAR IMMUNITY
• Lymphocyte subset enumeration.
• B-cell panels and NK-cell panels.
• Lymphocyte functional analysis.

29. PHAGOCYTES
• The laboratory evaluation of a patient with a suspected phagocyte deficiency should always begin
with a CBC.
• Neutropenia is the most frequently encountered disorder of the phagocyte system.
• Neutrophilia, at values exceeding those associated with acute infection, is a common finding in LAD
type 1 (LAD-1).
• Abnormalities of WBC function involve difficulty with adherence, locomotion, deformability,
recognition, attachment, engulfment, phagosome formation, phagocytosis, degranulation, microbial
killing, and elimination of engulfed material.
• Clinical assays to evaluate neutrophil function are limited in number.
• CGD is diagnosed by demonstrating absent or markedly reduced oxidase activity in neutrophils in
response to stimulation.

30. • Oxidase activity can be detected by a flow cytometry assay measuring the oxidation of
dihydrorhodamine (DHR) 123 in phagocytes, resulting in fluorescent rhodamine-123.
• The nitroblue tetrazolium (NBT) test measures oxidative burst activity as well, but it is a more
subjective test and can miss the diagnosis of CGD. For patients with suspected LAD-1 deficiency,
neutrophils are labeled with mAb directed against the adhesion molecule CD11/CD18 heterodimer.
• Absence of fluorescence intensity indicates lack of expression of the adhesion molecule.
• In addition, an increase of fluorescence intensity after stimulation can be documented in normal
individuals, indicating the normal upregulation of this molecule after cell activation.
• Other laboratory techniques used to identify phagocytic defects include assays for chemotaxis and
bactericidal activity.
• A major pitfall for neutrophil studies is the spontaneous cell activation that might occur in vitro when
cells are not tested within a few hours of when the sample was drawn, resulting in artifactual values
that might falsely suggest poor function.

31. COMPLEMENT
• Laboratory tests for complement components include tests for functional activity of the classical pathway with a
CH50 assay and the alternative pathway with an AH50 assay, as well as immunochemical methods to measure
complement component levels.
• The CH50 evaluation tests the ability of fresh serum from the patient to lyse antibody-coated sheep erythrocytes.
• This reflects the activity of all numbered components of the classical complement pathway, C1–C9, and terminal
components of the alternative complement pathway.
• A total deficiency of one of the classical complement pathway components will result in a CH50 assay approaching
zero
• Patients with complement deficiency are rare, and complement test abnormalities are often transient because of
increased consumption or activation.
• It is usually recommended that that in case of an abnormal result, the complement test be repeated if the sample
was taken when the patient had an acute illness.
• Quantitative tests for components C3 and C4 are utilized in testing for complement deficiencies and for evaluation of
complement activation

32. INNATE IMMUNITY: INTERFERON-Γ LEVELS, TOLL-LIKE
RECEPTOR ASSAY
• The importance of the many components of innate immunity are increasingly
recognized, as single gene defects in this immune compartment have been found to
cause susceptibility to specific infections.
• For example, patients with defects in the proteins that are part of the interferon-
γ (IFN-γ ) receptor may have elevated serum IFN-γ levels, even when there is no
infection to explain these levels.
• The IFN-induced response associated kinase 4 (IRAK4) defect, observed with
susceptibility to pneumococcal infection, might be accompanied with abnormal Toll-
like receptor (TLR) assay responses.
• It should be noted that the clinical value of most of these innate immunity tests as
screening or diagnostic tools for immune defects has not been clearly established.

33. • The testing of lymphocyte apoptosis in a patient who may have ALPS and the
evaluation of NK-cell function for suspected familial hemophagocytic
lymphohistiocytosis are examples of specific functional assays that suggest
immunodeficiency syndromes.
• Many patients with increased frequency of infections may not have abnormal
results in clinically available immunological testing, which may not give clear
evidence of a secondary etiology in the medical evaluation.
• In these difficult cases, referral to tertiary care and research centers for
investigation of rare diseases is recommended.

34. MOLECULAR TESTING FOR PRIMARY
IMMUNE DEFECTS
• Molecular testing for specific PIDs is available through commercial and research
laboratories. Biochemical and genetic testing should be considered. If autosomal recessive SCID
is suspected, the adenosine deaminase (ADA) and purine nucleoside phosphorylase (PNP)
enzyme activities in the RBCs should be determined.
• White blood cells must be used to measure the activity of these enzymes in recently transfused
individuals, since donor RBCs will elevate the enzyme activity in deficient patients.
• AT has the consistent laboratory finding of elevated alpha-fetoprotein (AFP) levels along with
variable abnormalities in B- and T-cell function.
• Nearly 300 defective genes and gene products have been identified to result in congenital
immunodeficiency syndromes.
• In these cases, the diagnosis can be confirmed with molecular genetic analysis . For example,
gene mutations in BTK leading to the absence of Bruton tyrosine kinase (BTK) results in arrest of
B-cell development at the pre–B cell stage in congenital X-linked agammaglobulinemia.

35. • Similarly, abnormal T-cell development leading to SCID results from mutations in at least 15 genes,
including IL2RG and JAK3.
• Patients with gene mutations that may result in disease but have not been investigated need to be
carefully evaluated to demonstrate the pathogenic nature of the genetic change.
• Some genetic changes do not have clinical significance and are known as single nucleotide
polymorphisms (SNPs).
• Standard protocols using Southern, Northern, and Western blot analyses, PCR analysis, and DNA
sequence analysis are helpful to identify affected patients, affected fetuses prenatally, and carriers of
genetic mutations .
• Most recently, the use of whole exome sequencing for immunodeficiency syndromes has facilitated the
identification of new genes causing immunodeficiencies by examining all known gene exons without
bias, and simultaneously.
• This methodology for diagnosis is particularly helpful when the clinical presentation does not match
any of the already described immunodeficiency syndromes.

36. CONCLUSIONS
• The approach to the patient with suspected immune deficiency requires knowledge of developmental pathways
and function of the different compartments of the immune system, as well as the clinical presentation of these
disorders.
• The medical history, particularly the frequency, severity, and etiology of infections, is most helpful to orient the
diagnostic workup. Commonly ordered tests in primary care, such as a CBC and serum Ig levels, are helpful to
support possible diagnosis and referral to the clinical immunologist.
• Immunological testing according to clues obtained from the medical history helps narrow the differential
diagnoses to specific immunodeficiencies, which are confirmed by molecular methods.
• Description of new T-cell subsets (e.g., Th17 and regulatory T cells [Tregs]) has helped explain the
immunopathogenesis of certain clinical manifestations, such as the occurrence of autoimmunity in patients with
combined immunodeficiency, and “cold abscesses” in the autosomal dominant HIES.
• Testing for these lymphocyte phenotypes is being integrated in the clinical evaluation. Identification of genetic
defects can now be accomplished by increased availability of whole exome sequencing, as an alternative to
genetic analysis of candidate genes. Technological advances are making molecular diagnosis available for most
patients with immunodeficiency conditions.