PNH

Overview of Paroxysmal Nocturnal Hemoglobinuria (PNH)

Definition: A rare, acquired, life-threatening hematopoietic stem cell disorder characterized by a deficiency of glycosylphosphatidylinositol (GPI)-anchored proteins on the surface of blood cells
Pathophysiology:
- Acquired Mutation: PNH arises from an acquired mutation in the PIGA gene (phosphatidylinositol glycan anchor biosynthesis class A), located on the X chromosome
- Defective GPI Anchor Synthesis: The PIGA gene is essential for the synthesis of the glycosylphosphatidylinositol (GPI) anchor, which attaches certain proteins to the cell surface
- Deficiency of GPI-Anchored Proteins: The PIGA mutation leads to a deficiency of all GPI-anchored proteins on the surface of affected blood cells, including:
  - CD55 (Decay-Accelerating Factor): Regulates complement activation on cell surfaces
  - CD59 (Membrane Inhibitor of Reactive Lysis): Inhibits the formation of the membrane attack complex (MAC), the terminal component of the complement cascade
  - Other GPI-linked Proteins: CD16 (FcγRIII), CD48, CD14, and many others
- Unregulated Complement Activation: The deficiency of CD55 and CD59 results in uncontrolled complement activation on the surface of RBCs, WBCs, and platelets
- Intravascular Hemolysis: Complement activation on RBCs leads to formation of the MAC, causing RBC lysis within the blood vessels (intravascular hemolysis)
Clinical Features:
- Hemolytic Anemia:
  - Fatigue
  - Weakness
  - Jaundice (yellowing of the skin and eyes)
  - Dark urine (hemoglobinuria), especially in the morning (paroxysmal)
- Thrombosis:
  - Increased risk of blood clots due to platelet activation and complement-mediated endothelial damage
  - Venous thrombosis (DVT, PE) is more common than arterial thrombosis
  - Unusual sites of thrombosis (e.g., hepatic vein thrombosis)
- Bone Marrow Failure:
  - Cytopenias (anemia, thrombocytopenia, leukopenia) may occur due to bone marrow dysfunction
  - Some patients with PNH may also have underlying aplastic anemia or myelodysplastic syndromes (MDS)
- Other Manifestations:
  - Smooth muscle dystonia (abdominal pain, dysphagia, erectile dysfunction)
  - Pulmonary hypertension
  - Chronic kidney disease

Flow Cytometry Immunophenotyping for PNH

Principle:
- Flow cytometry is the gold standard for diagnosing PNH
- It detects the absence or reduced expression of GPI-linked proteins on blood cells (RBCs, granulocytes, and monocytes)
- Fluorescently labeled antibodies are used to bind to specific GPI-linked proteins, and the fluorescence intensity is measured by flow cytometry
Cell Populations Analyzed:
- Granulocytes:
  - CD15, CD45: Used to identify and gate on the granulocyte population
  - GPI-linked proteins: CD16, CD24
- Monocytes:
  - CD14, CD45: Used to identify and gate on the monocyte population
  - GPI-linked proteins: CD14, CD48
- Red Blood Cells (RBCs):
  - Anti-glycophorin A or anti-CD71: Used to identify and gate on the RBC population
  - FLAER (Fluorescently Labeled Aerolysin): A bacterial toxin that binds to the GPI anchor itself. It stains all GPI-anchored cells, including RBCs.
Antibodies Used:
- CD55 (Decay-Accelerating Factor, DAF): A GPI-linked protein that regulates complement activation
- CD59 (Membrane Inhibitor of Reactive Lysis, MIRL): A GPI-linked protein that inhibits the formation of the membrane attack complex (MAC)
- Other GPI-Linked Proteins: CD16 (FcγRIII), CD24, CD14, CD48, CD66b
- Fluorescently Labeled Aerolysin (FLAER): A bacterial toxin that binds to the GPI anchor itself
Gating Strategy:
1. Forward Scatter (FSC) and Side Scatter (SSC): Used to identify and gate on the granulocyte and monocyte populations
2. CD45 Expression: Used to further refine the gating and exclude debris and non-leukocyte events
3. CD15 or CD14 Expression: Used to specifically identify granulocytes (CD15+) and monocytes (CD14+)
Analysis and Interpretation:
- Normal Cells: Express normal levels of all GPI-linked proteins (CD55, CD59, CD16, CD24, CD14, CD48)
- PNH Cells: Show decreased or absent expression of one or more GPI-linked proteins
- Types of PNH Cell Populations:
  - Type I Cells: Normal cells with normal expression of GPI-linked proteins
  - Type II Cells: Cells with partial deficiency of GPI-linked proteins (intermediate expression)
  - Type III Cells: Cells with complete deficiency of GPI-linked proteins (absent expression)
- Quantification: The percentage of PNH cells (Type II and Type III cells) is calculated for each cell population (granulocytes, monocytes, and RBCs)
- Clinical Correlation: The percentage of PNH cells correlates with the severity of the disease and the risk of complications

Reporting Results

Report the percentage of PNH cells (Type II and Type III cells) in each cell population (granulocytes, monocytes, and RBCs)
Include histograms or dot plots showing the expression of CD55 and CD59 (or other GPI-linked proteins)
Provide an interpretation of the results, noting the presence or absence of PNH and the size of the PNH clone
Correlate the laboratory findings with the patient’s clinical information

Key Terms

Paroxysmal Nocturnal Hemoglobinuria (PNH): An acquired stem cell disorder characterized by deficiency of GPI-anchored proteins
GPI Anchor: Glycosylphosphatidylinositol, a glycolipid that anchors certain proteins to the cell surface
CD55 (DAF): Decay-accelerating factor, a GPI-linked protein that regulates complement activation
CD59 (MIRL): Membrane inhibitor of reactive lysis, a GPI-linked protein that inhibits the formation of the membrane attack complex (MAC)
FLAER: Fluorescently Labeled Aerolysin, a bacterial toxin that binds to the GPI anchor
Flow Cytometry: A technique for identifying and quantifying cells based on their surface markers
Immunophenotyping: Using flow cytometry to identify cell surface markers
Clone: A population of cells derived from a single progenitor cell

Lymphocyte Subsets

Molecular/Cytogenetic