## Paroxysmal Nocturnal Hemoglobinuria: Flow Cytometry Diagnosis **Key Point:** PNH is caused by a somatic mutation in the PIGA gene, leading to deficiency of glycosylphosphatidylinositol (GPI)-anchored complement regulatory proteins CD55 (DAF) and CD59 (MAC-inhibitory protein). Flow cytometry demonstrating absence of these proteins is the gold standard diagnostic test. ### Pathophysiology 1. **PIGA gene mutation:** Occurs in hematopoietic stem cells (somatic, acquired mutation) 2. **Loss of GPI anchor:** Cells cannot display GPI-anchored proteins 3. **Missing complement regulators:** - **CD55 (DAF):** Decay-accelerating factor — inhibits C3/C5 convertase - **CD59 (MAC-IP):** Membrane inhibitor of reactive lysis — blocks C5b-9 complex formation 4. **Result:** Uncontrolled complement activation → intravascular hemolysis, thrombosis, and cytopenias ### Diagnostic Approach | Test | Finding in PNH | Specificity | |------|---|---| | **Flow cytometry (CD55/CD59)** | Absent on RBCs, WBCs, platelets | **Gold standard — most specific** | | **Ham test (acidified serum)** | Hemolysis in acidified serum | Positive but less sensitive; rarely used now | | **Sucrose lysis test** | Osmotic lysis in sucrose | Positive but less specific | | **Hemoglobinuria** | Dark urine (especially morning) | Supportive but not diagnostic | | **Reticulocytosis** | Elevated | Nonspecific | | **LDH, bilirubin** | Elevated | Nonspecific | **High-Yield:** Flow cytometry for CD55 and CD59 is the **modern diagnostic gold standard** for PNH. It is more sensitive and specific than the older Ham test or sucrose lysis test. **Clinical Pearl:** PNH presents with a triad: 1. **Hemolysis** (intravascular, episodic) 2. **Thrombosis** (venous, especially hepatic vein — Budd-Chiari syndrome) 3. **Cytopenias** (due to bone marrow involvement) **Mnemonic:** **PNH = GPI loss → CD55/CD59 absent → Complement uncontrolled → Hemolysis + Thrombosis + Cytopenias**
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