Approach to diagnosis of primary immunodeficiency, with emphasis on SCID

Definition

1. Clinical suspicion

• Recurrent, severe, persistent, or poorly responsive infections
• Infection with unusual or opportunistic organisms
• Repeated sinopulmonary bacterial infections
• Chronic diarrhea, persistent oral candidiasis, or failure to thrive
• Deep abscesses, recurrent skin infections, or poor wound healing
• Complications after live vaccines, especially BCG, rotavirus, oral polio, or varicella vaccines
• Family history of early infant deaths, known immunodeficiency, or consanguinity
• Autoimmune disease, lymphoid malignancy, or absent lymphoid tissue in an appropriate setting

Clues to the immune arm involved

2. First exclude secondary immunodeficiency

• HIV infection
• Malnutrition
• Immunosuppressive therapy, chemotherapy, corticosteroids, biologics
• Hematologic malignancy
• Protein loss: nephrotic syndrome, protein-losing enteropathy
• Severe systemic disease, diabetes, renal or liver failure

3. Initial screening investigations

1. Complete blood count with differential
   • Absolute lymphocyte count is particularly important in infants.
   • Persistent infantile lymphopenia suggests a T-cell or combined immunodeficiency.
   • Neutropenia suggests a phagocyte disorder.
2. Peripheral blood smear
   • Assess leukocyte morphology and platelet size.
   • Small platelets with thrombocytopenia suggest Wiskott-Aldrich syndrome.
3. Quantitative serum immunoglobulins
   • IgG, IgA, IgM, and sometimes IgE.
   • Interpret by age, particularly in infants because maternal IgG is present.
4. Specific antibody function
   • Measure antibodies to previous vaccines, for example tetanus, diphtheria, pneumococcal antigens.
   • Poor vaccine response indicates defective humoral immunity.
5. HIV testing
   • Mandatory in suspected cellular immunodeficiency.
6. Complement screening
   • CH50 for classical pathway.
   • AH50 for alternative pathway.
   • C3 and C4 levels where indicated.

4. Second-line immune evaluation

A. Lymphocyte immunophenotyping by flow cytometry

• T cells: CD3+
• Helper T cells: CD3+CD4+
• Cytotoxic T cells: CD3+CD8+
• B cells: CD19+ or CD20+
• NK cells: CD3-CD16+/CD56+

B. Functional lymphocyte studies

• T-cell proliferative response to mitogens, especially phytohemagglutinin (PHA)
• Responses to antigens
• Delayed hypersensitivity testing, where appropriate
• Neutrophil oxidative burst by DHR flow cytometry for chronic granulomatous disease
• Neutrophil adhesion-marker studies for leukocyte adhesion deficiency

C. Genetic confirmation

• Targeted testing for a suspected familial mutation
• Primary immunodeficiency gene panel
• Whole-exome or whole-genome sequencing when phenotype is unclear

Diagnosis of SCID

1. When to suspect SCID

• Onset in the first few months of life
• Persistent thrush
• Chronic diarrhea
• Failure to thrive
• Severe or recurrent pneumonia, especially Pneumocystis jirovecii
• Persistent viral infection, such as CMV, adenovirus, or RSV
• Disseminated infection after BCG or another live vaccine
• Severe eczema or erythroderma, especially in Omenn syndrome
• Family history of male infant deaths or established SCID

2. Screening for SCID

A. Newborn screening

• Low or absent TRECs indicate reduced thymic output of naïve T cells.
• It is a screening test, not a final diagnosis.
• Abnormal TREC must be followed urgently by CBC, lymphocyte subsets, and functional testing.

B. CBC

3. Confirmatory diagnostic tests for SCID

1. Flow-cytometric lymphocyte subset analysis
   • Markedly decreased or absent CD3+ T cells.
   • Defines T-B-NK phenotype.
2. Naïve versus memory T-cell markers
   • Evaluate CD45RA+ naïve and CD45RO+ memory T cells.
   • A predominance of memory phenotype cells in a newborn may suggest maternal engraftment or oligoclonal expansion.
3. T-cell proliferation to PHA
   • Profoundly reduced or absent proliferative response supports severe functional T-cell deficiency.
4. Maternal T-cell engraftment studies
   • Chimerism testing identifies maternal lymphocytes.
   • Maternal cells may give a misleading impression of detectable T cells.
5. Molecular genetic testing
   • Identifies the molecular subtype, including defects in IL2RG, JAK3, ADA, RAG1/2, DCLRE1C (Artemis), IL7R, or CD3/TCR-signaling genes.
6. Additional tests
   • Serum immunoglobulins and antibody responses
   • ADA enzyme/metabolite assay when ADA deficiency is suspected
   • HLA typing of child and family once SCID is strongly suspected

4. Immediate measures while confirming SCID

• Urgent referral to a pediatric immunologist and transplant center
• Protective infection-control measures
• Avoid all live vaccines
• Use CMV-negative, irradiated blood products if transfusion is necessary
• Begin antimicrobial prophylaxis as advised by the treating team
• Consider immunoglobulin replacement
• Assess for active infection before hematopoietic stem-cell transplantation

Robbins pathology correlation

• Defective cytokine receptor signaling, especially common gamma-chain pathway defects
• Failure of V(D)J recombination, such as RAG defects
• Toxic metabolite accumulation, such as ADA deficiency
• Defective T-cell receptor or CD3 signaling

Clinical suspicion -> exclude secondary causes -> CBC and immunoglobulins -> lymphocyte subsets -> T-cell functional assay -> molecular diagnosis and maternal-engraftment studies -> urgent transplant evaluation.