What is the approach to ordering and interpreting immunology blood tests, and what are the treatment options for related conditions?

Approach to Ordering and Interpreting Immunology Blood Tests

Begin with a complete blood count (CBC) with differential as the foundational screening test, followed by targeted immunoglobulin levels and lymphocyte subset analysis based on clinical presentation. 1

Initial Screening Tests

Complete Blood Count with Differential

• Order a CBC with differential to identify lymphopenia, leukopenia, or cytopenias that suggest immunodeficiency or autoimmune disease. 1
• Lymphopenia (particularly age-adjusted) is a critical finding that warrants immediate further investigation for combined immunodeficiency or SCID. 1
• Examine the peripheral blood smear for neutrophil morphology and staining patterns to identify phagocytic cell disorders. 1
• Assess for anemia and thrombocytopenia which may indicate autoimmune cytopenias or bone marrow involvement. 2, 3

Inflammatory Markers

• Measure erythrocyte sedimentation rate (ESR) and C-reactive protein (CRP) to assess disease activity in suspected autoimmune conditions. 2
• These markers help distinguish inflammatory from non-inflammatory processes but lack specificity for particular diagnoses. 2

Humoral Immunity Assessment

Immunoglobulin Levels

• Measure serum IgG, IgA, and IgM levels as the primary screening for antibody deficiencies. 1
• In SCID, IgA and IgM are typically low or absent, while IgG may appear normal due to transplacental maternal transfer. 1
• Low immunoglobulin levels require functional antibody testing to confirm clinical significance. 1

Functional Antibody Testing

• Assess specific antibody titers to vaccine antigens (tetanus, diphtheria, pneumococcus) to evaluate antibody production capacity. 1
• If baseline titers are low, administer booster immunization and recheck titers in 4-6 weeks to assess response. 1
• Failure to mount adequate antibody responses despite normal immunoglobulin levels indicates functional antibody deficiency. 1

Cellular Immunity Evaluation

Lymphocyte Subset Enumeration by Flow Cytometry

• Enumerate CD3+ T cells, CD4+ helper T cells, CD8+ cytotoxic T cells, CD19+ B cells, and CD16/56+ NK cells as essential screening. 1
• Measure naive T cells (CD45RA+) and memory T cells (CD45RO+) to assess thymic function and immune maturity. 1
• Very low or absent naive T-cell counts with profoundly reduced proliferation to mitogens indicate SCID. 1

Advanced T-Cell Testing (When Indicated)

• Perform T-cell proliferation assays with mitogens (PHA, ConA) and antigens to assess functional capacity. 1
• Evaluate T-cell activation markers (CD25, CD69, HLA-DR) and immune checkpoint expression (PD-1) in specific clinical contexts. 4
• Assess regulatory T cells (CD4+CD25+Foxp3+) when immune dysregulation is suspected. 4

NK Cell Function

• Measure spontaneous NK cytotoxicity when recurrent viral infections or hemophagocytic syndromes are present. 1

Autoantibody Testing

ANA Screening

• Perform indirect immunofluorescence assay (IIFA) on HEp-2 cells as the reference method for ANA screening at a dilution of 1:160. 1
• Report both titer and pattern (homogeneous, speckled, nucleolar, centromere) as both provide diagnostic information. 1
• A positive ANA (≥1:160) in appropriate clinical context warrants testing for specific extractable nuclear antigens. 1

Disease-Specific Autoantibodies

• For suspected systemic lupus erythematosus: test anti-dsDNA and anti-Smith antibodies. 1
• For suspected autoimmune hepatitis: test anti-smooth muscle antibodies (SMA), anti-liver kidney microsomal-1 (LKM1), anti-liver cytosol-1 (LC1), and anti-soluble liver antigen/liver pancreas (SLA/LP). 1
• Anti-SLA/LP is the only disease-specific autoantibody for autoimmune hepatitis and has high diagnostic value. 1
• For rheumatoid arthritis: test rheumatoid factor (RF) and anti-cyclic citrullinated peptide (anti-CCP) antibodies. 2

Important Caveats for Autoantibody Interpretation

• Never rely solely on autoantibody testing without clinical correlation, as positive results occur in healthy individuals and various non-autoimmune conditions. 1, 2
• ANA positivity occurs in up to 5% of healthy controls and increases with age. 1
• The pre-test probability significantly affects the predictive value of autoantibody results. 1

Phagocytic Cell Assessment

Neutrophil Function Tests

• Perform dihydrorhodamine (DHR) reduction test or nitroblue tetrazolium test to screen for chronic granulomatous disease. 1
• Evaluate expression of adhesion molecules (CD11b, CD18) by flow cytometry when leukocyte adhesion deficiency is suspected. 1
• Advanced testing includes chemotaxis assays, phagocytosis assays, and bacterial/fungal killing assays. 1

Complement System Evaluation

• Measure CH50 (total hemolytic complement activity) to screen for classical pathway defects. 1
• Measure AH50 (alternative pathway hemolytic activity) to screen for alternative pathway defects. 1
• If screening tests are abnormal, measure individual complement components (C3, C4, C1q, etc.) to identify specific deficiencies. 1

Specialized Testing for Primary Immunodeficiency

TREC Screening

• T-cell receptor excision circles (TRECs) in dried blood spots identify SCID and other T-cell lymphopenias through newborn screening. 1
• Very low or absent TRECs indicate SCID and require urgent evaluation. 1
• Premature infants have lower TREC numbers that typically increase over time. 1

Maternal Engraftment Assessment

• When T cells are present in suspected SCID, determine if T cells are of maternal origin through chimerism studies. 1
• Maternal T cells indicate SCID despite presence of T cells and require urgent intervention. 1

Algorithmic Approach Based on Clinical Presentation

For Recurrent Infections in Infants

1. Obtain CBC with differential and lymphocyte subset enumeration immediately. 1
2. If severe lymphopenia (especially T-cell lymphopenia) is present, expedite evaluation for SCID. 1
3. Measure immunoglobulin levels, but note IgG may be falsely normal from maternal transfer. 1
4. Assess T-cell proliferation to mitogens—profound reduction confirms SCID. 1
5. Do not delay treatment while awaiting complete diagnostic workup in severe presentations. 2

For Suspected Autoimmune Disease

1. Obtain CBC, comprehensive metabolic panel, ESR, and CRP. 2
2. Screen with ANA by IIFA at 1:160 dilution. 1
3. If ANA positive with appropriate clinical features, test disease-specific autoantibodies. 2
4. For suspected autoimmune hepatitis, measure serum immunoglobulins and liver-specific autoantibodies (ANA, SMA, anti-LKM1, anti-LC1, anti-SLA/LP). 1
5. Consider liver biopsy for definitive diagnosis of autoimmune hepatitis. 1, 2

For Recurrent Bacterial Infections

1. Measure serum immunoglobulin levels (IgG, IgA, IgM). 1
2. Assess specific antibody titers to vaccine antigens. 1
3. If titers are low, administer booster and recheck in 4-6 weeks. 1
4. Enumerate B-cell subsets including naive and switched memory B cells. 1
5. Perform DHR test to exclude chronic granulomatous disease. 1

Quality Considerations and Standardization

• Use central laboratories with standardized assays for immune monitoring in clinical trials or complex cases to minimize variation. 1
• Flow cytometry for lymphocyte subsets requires proper gating strategies and quality controls. 4
• ELISPOT assays for antigen-specific T cells are highly standardized but require serum adalimumab concentrations <2 mcg/mL for antibody detection. 1, 5

Common Pitfalls to Avoid

• Do not order isolated ANA testing without clinical suspicion of autoimmune disease, as positive results are common in healthy individuals. 1
• Do not interpret a single positive band on Western blot as evidence of Lyme disease—specific criteria must be met. 1
• Do not delay immunosuppressive treatment in severe autoimmune presentations while awaiting complete serologic workup. 2
• Always screen for infectious diseases (HIV, hepatitis B and C, tuberculosis) before initiating immunosuppressive therapy. 2
• Do not rely on IgM Western blot for Lyme disease beyond 30 days of symptoms, as this increases false-positive results. 1
• Avoid interpreting fewer than required bands on autoantibody blots as positive, as cross-reactivity is common. 1

When to Refer to Immunology/Rheumatology

• Refer immediately when SCID is suspected based on severe lymphopenia and recurrent infections in infants. 1
• Refer when screening tests suggest primary immunodeficiency but specific diagnosis is unclear. 1
• Refer early for suspected autoimmune disease to prevent organ damage from delayed treatment. 2
• Consult when uncertainty exists regarding evaluation or management of abnormal immunology results. 1