By Carrie Chenault, M.D. and Kathleen M. Murphy, Ph.D.

 

Background

Paroxysmal nocturnal hemoglobinuria (PNH) is a hematopoietic stem cell disease characterized by complement-induced intravascular hemolysis, thrombosis, and bone marrow failure. PNH is caused by an acquired (somatic) mutation in the X-linked phosphatidyl-inositol glycan (PIG-A) gene which results in reduced/absent synthesis of glycophosphatidylinositol (GPI) anchor proteins. Defici­ency of GPI anchor proteins leads to an absence of GPI-linked proteins on the cell membrane of all hematopoietic cell lineages. Depending on the mutation, PNH cells have either complete (Type III cells) or partial (Type II cells) absence of an entire class of GPI-linked proteins.

 

GPI-linked proteins serve many functions, but many of the PNH symptoms are related to their function in regulating comple­ment mediated cell destruction. CD55 (decay accelerating factor) and CD59 (membrane inhibitor of reactive lysis) are GPI-linked proteins normally expressed on red blood cells. CD55 is responsible for preventing the formation of and destabilizing C3 convertase and CD59 inhibits assembly of the membrane attack complex. In PNH these proteins are decreased or absent resulting in increased complement-mediated lysis and recurrent episodes of intravascular hemolysis.

 

PNH may develop on its own (primary PNH) or in the context of other bone marrow disorders such as aplastic anemia or myelodysplasia (secondary PNH), and subclinical forms have also been described. PNH was historically diagnosed using comple­ment based testing, but this has been replaced by flow cytometry which is now the gold standard and allows for the detection of small PNH clones even in patients with a history of recent transfusion. Consensus guidelines for immunophenotyping PNH cells are also available and have allowed for interlaboratory comparison and a more unified approach to the diagnosis. High sensitivity testing which can detect PNH clones smaller than 1% is also available.

Who should be tested?

• Patients with unexplained hemoglobinuria

• Patients with Coomb’s-negative hemolytic anemia

• Patients with intravascular hemolysis and iron deficiency

• Patients with hemolysis and thrombosis

• Patients with thrombosis in uncommon sites

• Patients with cytopenias and hemolysis or thrombosis

• Patients with hypoplastic or aplastic anemia

• Patients with myelodysplasia (especially those with refractory cytopenia with unilineage dysplasia RCUD)

• Patients with known PNH should have clone size testing regularly

 

Treatment 

Soliris (eculizumab) is a monoclonal antibody directed against the complement protein C5, which is the blood protein responsible for destroying red blood cells in patients with PNH. Eculizumab inhibits the C5 terminal complement functions involved in complement-mediated hemolysis while the early complement functions such as immune complex clearance, microbial opsonization, and weak anaphylatoxin function remain intact. Eculizumab has been shown to be effective at reducing intravascular hemolysis, reducing the need for blood transfusions, and reducing the risk of thrombosis.

PNH Flow Cytometry Assay 

At ProPath, we use the fluorescent aerolysin protein (FLAER) in addition to several antibodies directed toward GPI-linked proteins (including CD14, CD24, and CD59) to detect GPI-deficient blood cells. RBC, monocytes, and granulocytes are analyzed and reported separately using an ultra-sensitive 7-color flow cytometry assay. Testing is available Monday-Saturday and requires 2-3 ml fresh (<48 hours old) peripheral blood collected in an EDTA or heparin tube (testing cannot be performed on bone marrow).

References:

1. Parker C, Omine M, Richards et al. Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood 2005; 106(12):3699-3709

2. Borowitz MJ, Craig FE, Digiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ; Clinical Cytometry Society. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom. 2010 Jul;78(4):211-30.