Initial Workup for Suspected Paroxysmal Nocturnal Hemoglobinuria (PNH)
The initial workup for suspected PNH should include flow cytometry of peripheral blood to detect CD55 and CD59 deficiency on red blood cells and granulocytes, which is the gold standard diagnostic test for PNH. 1
Understanding PNH
PNH is a rare acquired hematopoietic stem cell disorder characterized by:
- Complement-mediated intravascular hemolysis
- Bone marrow failure
- Thrombosis (particularly in unusual sites)
The condition results from somatic mutations in the PIG-A gene, leading to deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins, particularly the complement regulatory proteins CD55 and CD59.
Diagnostic Algorithm
Step 1: Initial Laboratory Tests
- Complete blood count (CBC) with differential
- Reticulocyte count
- Comprehensive metabolic panel
- Lactate dehydrogenase (LDH) - typically elevated in PNH
- Haptoglobin - typically decreased in PNH
- Urinalysis (for hemoglobinuria/hemosiderinuria)
- Serum ferritin, transferrin, and transferrin saturation
- Vitamin B12 and folate levels (to rule out other causes of macrocytic anemia)
Step 2: Definitive Diagnostic Testing
- Flow cytometry of peripheral blood - This is the most accurate technique for PNH diagnosis 1
- Test for CD55 and CD59 deficiency on red blood cells
- Test for CD55, CD59, and CD16 deficiency on granulocytes
- Flow cytometry is more specific, quantitative, and sensitive than older complement lysis tests
Step 3: Bone Marrow Evaluation
- Bone marrow aspiration and biopsy with cytogenetics
- Helps evaluate for concurrent bone marrow failure syndromes
- Prussian blue stain for iron evaluation
- Assess for dysplastic features and cellularity
Step 4: Additional Testing Based on Clinical Presentation
If thrombosis is present (especially in unusual sites like splanchnic veins):
- Imaging studies (Doppler ultrasound, CT angiography, or MR venography)
- Thrombophilia workup
If bone marrow failure features are prominent:
- HLA-DR15 typing (may predict response to immunosuppressive therapy) 2
- Testing for other bone marrow failure syndromes
Important Considerations
Clinical Scenarios Warranting PNH Testing
- Unexplained hemolytic anemia (especially with negative direct Coombs test)
- Cytopenias with evidence of hemolysis
- Unexplained thrombosis, particularly in unusual sites (splanchnic veins, cerebral veins)
- Aplastic anemia or myelodysplastic syndrome (small PNH clones can accompany these conditions) 2
- Unexplained iron deficiency anemia (due to hemoglobinuria)
- Episodic dark urine (hemoglobinuria)
Interpretation of Flow Cytometry Results
- PNH clone size is reported as percentage of cells with absent or reduced GPI-anchored proteins
- PNH III cells: completely deficient cells
- PNH II cells: partially deficient cells
- The proportion of abnormal granulocytes is usually greater than abnormal red cells 1
- Anti-CD59 best demonstrates PNH II red cells 1
Pitfalls to Avoid
- Relying solely on older tests like Ham test or sucrose lysis test, which are less sensitive and specific than flow cytometry 1
- Missing the diagnosis in patients with small PNH clones (flow cytometry can detect clones as small as 0.01%)
- Failing to consider PNH in patients with unexplained thrombosis, especially in unusual sites
- Not testing for PNH in patients with aplastic anemia or myelodysplastic syndrome
Follow-up After Diagnosis
- Assess for complications: thrombosis, renal insufficiency, pulmonary hypertension
- Evaluate for treatment with complement inhibitors (eculizumab, ravulizumab, or crovalimab) 3
- Monitor clone size and hemolysis parameters periodically
- Consider HLA typing if stem cell transplantation might be an option
By following this comprehensive diagnostic approach, clinicians can accurately diagnose PNH and initiate appropriate management to reduce morbidity and mortality associated with this rare but serious condition.