Paroxysmal Nocturnal Hemoglobinuria

Clinical Laboratory Science, Summer 2004 by Smith, Larry J

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal stem cell disorder resulting from a somatic mutation in the hematopoietic stem cell. It is characterized by intravascular hemolysis, cytopenias, frequent infections, bone marrow hypoplasia, and a high incidence of life-threatening venous thrombosis. An absent glycosylphosphatidylinositol (GPI)-anchored receptor prevents several proteins from binding to the erythrocyte membrane. These include the complement-regulatory proteins, CD55 and CD59, whose absence results in enhanced complement-mediated lysis. Patients present with anemia and hemoglobinuria. Laboratory diagnosis includes the sucrose hemolysis test, Ham acid hemolysis test, and fluorescent-activated cell analysis. There is considerable overlap between PNH, aplastic anemia, and myelodysplastic syndrome and some cases evolve into acute leukemia. Treatment is mainly supportive consisting of transfusion therapy, anticoagulation, and antibiotic therapy. Hematopoietic stem cell transplantation may be curative.

ABBREVIATIONS: DAF = decay accelerating factor; GPI = glycosylphosphatidylinositol; PNH = paroxysmal nocturnal hemoglobinuria; MIRL = membrane inhibitor of reactive lysis.

INDEX TERMS: paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria is an acquired stem cell disorder characterized by intravascular hemolysis, hemoglobinuria, and life-threatening thrombotic episodes.1 PNH occurs with an incidence of two to six per million persons. In addition, it may be considered a chronic hemolytic anemia caused by a defect intrinsic to the erythrocyte. PNH is unique, in that it is the only hemolytic anemia due to an intrinsic defect that is acquired and not inherited. The disorder was described in 1882 by Strübing as a case of intermittent hemoglobinuria, wherein a patient presented with hemoglobinuria upon awakening.2 Strübing went on to suggest that the hemoglobinuria was due to erythrocyte destruction within the blood vessels. Strubing's main contribution was to distinguish his patient from a common condition known as paroxysmal cold hemoglobinuria which was associated with syphilis. PNH is mainly a disease of adults; however, children and adolescents may be affected. Approximately 50% of PNH patients present with nocturnal hemoglobinuria, the hallmark of PNH. Darkening of the urine is most noticeable in the morning, either because the urine is more concentrated or there is increased hemolysis at night. Additional findings include hemosiderinuria, thrombocytopenia, decreased hematopoietic activity, and thrombosis. Thrombotic events often occur in unusual locations.

PATHOPHYSIOLOGY

PNH is an acquired stem cell disorder. Defective stem cells arise from the clonal expansion of a totipotent hematopoietic stem cell containing a somatic mutation in the phosphatidylinositol glycan complementation group A gene (PIG-A gene).3 The PIG-A gene, located on the X chromosome, is responsible for the synthesis of the glycosylphosphatidylinositol anchor that serves to attach a number of proteins to the cell membrane surface (Figure 1). As a result of this mutation, defective erythrocytes, leukocytes, and platelets are produced that are deficient in various surface membrane phosphatidylinositol-linked proteins such as CD14, CD16, CD24, CD48, CD55, CD58, CD59, CD66, CD67, and CD73.4 The combined protein deficiencies consist of the following: 1) complement regulatory proteins such as CD55 and CD59, 2) membrane enzymes such as acetylcholinesterase and alkaline phosphatase, and 3) a group of miscellaneous membrane-associated proteins: CD14, CD16, CD55, and CD59 on granulocytes; CD48 and CD59 on lymphocytes; CD48 and CD95 on monocytes; and CD55 and CD59 on erythrocytes. Two very important proteins that are members of the complement regulatory protein family and are missing from the erythrocyte cell surface in PNH are the decay accelerating factor (DAF, CD55), and the membrane inhibitor of reactive lysis (MIRL, CD59).5,6,7 These contribute to the normal red cell's ability to resist complement-mediated lysis. The importance of these two proteins has been demonstrated in experiments using antibodies to either DAF and/ or MIRL.8 Inhibition of MIRL results in greater hemolysis and is similar to the hemolysis seen in patients with a congenital defect in the MIRL molecule. In cells experimentally challenged with antibody to MIRL alone and with normal DAP expression, there is an increased susceptibility to complement-mediated lysis while in cells with antibody to DAF alone there is only a small increase in complement-mediated inhibition of erythrocytes.8,9

A better understanding of the pathologic basis of PNH can be seen by looking at the interaction of DAF, MIRL, GPI, and complement on the erythrocyte surface. In 1993, Miyata and colleagues cloned cDNA for the PIG-A gene and demonstrated its ability to restore the expression of GPI-linked proteins that were missing from lymphoblastoid cell lines derived from patients with PNH.10 The actual gene product of the PIG-A gene is a glycosyltransferase that transfers N-acetylglucosamine from UDP-N-acetylglucosamine to phosphatidylinositol at the rough endoplasmic reticulum.11 GPI anchors are complex glycolipid structures that when synthesized attach various proteins to the membrane.