What are the diagnostic steps for Paroxysmal Nocturnal Hemoglobinuria (PNH)?

Diagnostic Approach to Paroxysmal Nocturnal Hemoglobinuria (PNH)

Flow cytometry testing of peripheral blood is the gold standard for diagnosing PNH and should be performed in all suspected cases to detect GPI-anchored protein deficiency on multiple cell lineages. 1

Clinical Presentations That Warrant PNH Testing

• Unexplained intravascular hemolysis with evidence of hemolysis on peripheral smear 2
• Venous thrombosis in unusual sites, particularly splanchnic vein thrombosis (hepatic, portal, or mesenteric veins) 2
• Cytopenias associated with bone marrow failure syndromes 2
• Unexplained hemolytic anemia with negative direct antiglobulin test (Coombs test) 2
• Young patients with normal cytogenetics and hypoplastic myelodysplastic syndrome (MDS) 2
• Budd-Chiari syndrome (9-19% of patients have underlying PNH) 2

Diagnostic Algorithm

Step 1: Initial Laboratory Evaluation

• Complete blood count (CBC) with differential and reticulocyte count 2
• Peripheral blood smear examination for evidence of hemolysis 2
• Hemolysis markers: LDH, haptoglobin, bilirubin (direct and indirect), free hemoglobin 2
• Renal function tests 2
• Coagulation profile 2

Step 2: Flow Cytometry Testing (Gold Standard)

• Test at least two GPI-linked markers on at least two different cell lineages 1
• Primary cell populations to analyze:
  • Red blood cells (RBCs): Test for CD59 and/or CD55 deficiency 3
  • Granulocytes: Test for CD24, CD16, CD66b deficiency 3
  • Monocytes: Test for CD14, CD48 deficiency 3

Step 3: Additional Testing Based on Clinical Presentation

• For patients with thrombosis:
  • JAK2V617F mutation testing to rule out myeloproliferative neoplasms 2
  • Thrombophilia screening (protein C, protein S, antithrombin, Factor V Leiden, prothrombin gene mutation) 2
• For patients with cytopenias:
  • Bone marrow aspiration and biopsy 2
  • Cytogenetic analysis 2
  • HLA-DR15 typing (for potential immunosuppressive therapy response) 2

Interpretation of Flow Cytometry Results

• PNH clone size classification 3:

  • Small clone: <10% GPI-deficient granulocytes
  • Medium clone: 10-50% GPI-deficient granulocytes
  • Large clone: >50% GPI-deficient granulocytes

• Clinical correlation:

  • Patients with >60% PNH granulocytes have higher thrombosis risk 2
  • Clone size correlates with hemolysis severity and thrombosis risk 4

Important Considerations and Pitfalls

• Recent transfusions may affect flow cytometry results by diluting the PNH clone 5
• High-sensitivity flow cytometry can detect PNH clones as small as 0.01% 3
• Fluorescent aerolysin (FLAER) combined with monoclonal antibodies improves detection sensitivity 1
• PNH can occur in association with other bone marrow failure syndromes (aplastic anemia, MDS) 2
• Patients with splanchnic vein thrombosis should be routinely tested for PNH, especially those with Budd-Chiari syndrome 2
• Flow cytometry should be performed before initiating complement inhibitor therapy (eculizumab or ravulizumab) to establish baseline clone size 4

Follow-up Testing

• Monitor PNH clone size every 6-12 months in patients with established diagnosis 3
• More frequent monitoring may be needed in patients on complement inhibitor therapy 4
• Repeat testing is warranted with any significant clinical change or disease progression 1