What laboratory tests should be run to diagnose Autoimmune Lymphoproliferative Syndrome (ALPS) in a young patient?

Laboratory Testing for ALPS Diagnosis

For diagnosing ALPS, you must run flow cytometry to measure TCRαβ+ CD3+ CD4-CD8- double-negative T cells (DNTs), which must be ≥1.5% of total lymphocytes or ≥2.5% of CD3+ T cells in the setting of normal or elevated lymphocyte counts. 1

Required Laboratory Tests

Flow Cytometry Panel (Essential)

• TCRαβ+ CD3+ CD4-CD8- double-negative T cells (DNTs): This is the hallmark laboratory finding and a required criterion for ALPS diagnosis 1, 2

  • Must be ≥1.5% of total lymphocytes OR ≥2.5% of T lymphocytes 1
  • Requires normal or elevated lymphocyte counts; lymphopenia invalidates this criterion 1
  • DNT levels >3% of total lymphocytes (or >5% of T lymphocytes) are essentially pathognomonic for ALPS 1
  • Must distinguish from TCRγδ+ DNTs by costaining with TCR-directed antibodies 1

• Complete blood count with differential: Essential to document lymphocyte counts and assess for cytopenias 3

Primary Accessory Tests (For Definitive Diagnosis)

Lymphocyte apoptosis functional assay (if available): 1, 2

• Performed on activated primary T cells using FAS activation with cross-linked agonistic antibodies, recombinant FAS ligand, or TCR restimulation 1
• Abnormal if patient cells show ≤50% of cell death compared to healthy controls run in parallel 1
• Requires repeat testing for confirmation due to high interlaboratory variability 1
• Important caveat: This test may be normal in patients with somatic FAS mutations or germline FASLG mutations 1

Genetic testing: 1, 2

• Sequencing of FAS, FASLG, and CASP10 genes for germline or somatic mutations 1, 2
• Next-generation sequencing is the preferred method 3

Secondary Accessory Tests (For Probable Diagnosis)

Biomarkers: 2, 3

• Soluble FAS ligand (sFASL): Elevated levels, particularly when combined with abnormal apoptosis function, are highly predictive of ALPS-FAS 3
• Interleukin-10 (IL-10): Elevated levels serve as an additional indicator 3
• Interleukin-18 (IL-18): Elevated levels serve as an additional indicator 3
• Vitamin B12: Often elevated and can help distinguish ALPS from other lymphoproliferative conditions 4

Autoimmune markers: 5, 6

• Direct Coombs test (often positive) 5, 6
• Antinuclear antibody (ANA) 5
• Anti-double-stranded DNA 5
• Rheumatoid factor 5
• Immunoglobulin levels (hypergammaglobulinemia is common) 5, 7

Additional hematologic studies: 6, 4

• Reticulocyte count (to assess for hemolytic anemia) 6
• Platelet count (to assess for immune thrombocytopenia) 6
• Serum ferritin (to help exclude hemophagocytic lymphohistiocytosis) 4

Diagnostic Algorithm

1. Screen with DNT percentage by flow cytometry in any patient with chronic (>6 months) lymphadenopathy and/or splenomegaly plus autoimmune features 1, 2

2. If DNTs ≥1.5% of total lymphocytes: Proceed to primary accessory testing (apoptosis assay and/or genetic testing) for definitive diagnosis 1, 2

3. If DNTs are borderline or primary tests unavailable: Measure secondary biomarkers (sFASL, IL-10, IL-18, vitamin B12) and autoimmune markers for probable diagnosis 2, 3

4. Confirm diagnosis: Requires both required criteria (chronic lymphoproliferation + elevated DNTs) plus one primary accessory criterion (abnormal apoptosis OR pathogenic mutation) for definitive ALPS 1, 2

Critical Pitfalls to Avoid

• Do not screen for ALPS based solely on DNT levels between 1.0-1.5%, as these can occur in healthy individuals or reactive conditions like systemic lupus erythematosus 1

• Do not rely on apoptosis testing alone for patients with suspected somatic FAS or FASLG mutations, as these may show normal results 1

• Ensure lymphocyte counts are normal or elevated when interpreting DNT percentages; lymphopenia invalidates the DNT criterion 1

• Request both percentage and absolute numbers of DNTs, as absolute counts vary by age 1

• Exclude infectious and malignant causes before pursuing ALPS workup, as these can mimic the presentation 1, 4