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

Diagnostic Approach to Paroxysmal Nocturnal Hemoglobinuria (PNH)

Flow cytometry performed on peripheral blood is the gold standard test for diagnosing PNH and should be ordered when clinical suspicion exists. 1

Clinical Presentations That Warrant PNH Testing

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

Initial Laboratory Evaluation

• Complete blood count with differential and reticulocyte count 2
• Peripheral blood smear examination for evidence of hemolysis 2
• Hemolysis markers: LDH, haptoglobin, bilirubin, and free hemoglobin 2
• Renal function tests and coagulation profile 2
• JAK2V617F mutation testing and thrombophilia screening for patients presenting with thrombosis 2

Diagnostic Testing Algorithm

1. First-line testing: Flow cytometry of peripheral blood

   • Test at least two GPI-linked markers on at least two different cell lineages, particularly red cells and granulocytes/monocytes 3
   • High-sensitivity flow cytometry can detect PNH clones at a sensitivity level of around 0.01% in red cells and neutrophils 4

2. Bone marrow evaluation

   • Bone marrow aspiration and biopsy with cytogenetic analysis for patients with cytopenias 2
   • Helps identify associated conditions like aplastic anemia or MDS 2

Interpretation of Flow Cytometry Results

• PNH clone size correlates with clinical manifestations:

  • Patients with >60% PNH granulocytes have a higher thrombosis risk 2
  • Clone size helps determine treatment approach and monitoring frequency 4

• Classification based on flow cytometry findings:

  • Classical PNH: Large PNH clones with clinical hemolysis
  • PNH in the setting of another bone marrow failure syndrome
  • Subclinical PNH: Small PNH clones without clinical evidence of hemolysis 5

Important Considerations and Pitfalls

• Recent red blood cell transfusions may affect flow cytometry results by diluting the proportion of PNH cells 4
• Testing should be performed before initiating complement inhibitor therapy to establish baseline clone size 6
• False negative results can occur if testing is limited to red cells only, as hemolysis may reduce the detectable PNH red cell population 5, 4
• Follow-up testing is essential to monitor disease progression and treatment response, especially in patients receiving complement inhibitors like eculizumab or ravulizumab 6

When to Initiate Treatment

• Treatment decisions should be based on:

  • Presence of hemolysis
  • Thrombotic events
  • Transfusion dependence
  • Clone size
  • Associated bone marrow failure 1, 6

• Complement inhibitors are indicated for patients with significant hemolysis or thrombosis 6

• Proximal complement inhibitors (targeting C3) may be beneficial for patients with extravascular hemolysis not fully controlled by terminal complement inhibitors 6