Contributed by S. Chiosea, MD and F. Bontempo, MDFINAL DIAGNOSIS:
DISCUSSION:
NATURAL COURSE - The average age of onset of PNH is 25-45 years. About 50% of patients will survive more than 15 years. Fatalities result from thrombotic and infectious complications. 10-25% of PNH patients will develop a secondary anaplastic anemia and about 1% of PNH patients will develop acute non-lymphoblastic leukemia (ANLL)(Meletis and Terpos 2003).
PATHOGENESIS OF PNH - PNH results from a somatic mutation in the phosphatidylinositol glycan - A (PIG-A) gene mapped to X-chromosome. PIG-A is required for the construction of the glycosylphosphatidylinositol (GPI) anchor protein needed for membrane attachment of proteins lacking hydrophobic domains able to span the cellular membrane. More than 30 GPI-anchored proteins expressed in hematopoietic lineages are identified (Meletis and Terpos 2003). CD16 and CD14 are GPI-anchored receptors present on monocytes, neutrophiles and T-lymphocytes. The lack of CD14 and CD16 detected by flow cytometry was used to diagnose PNH in our patient. The absence on the cellular membrane of two other GPI-anchored proteins, CD55 (Decay Accelerating Factor) and CD59 (protectin, or membrane inhibitor of reactive lysis) results in complement mediated intravascular hemolysis. In the absence of CD59, C8 stimulates C9 polymerization, a process required for the complement system to bore a hole in cell membrane (Meyers and Parker 2003).
In the absence of CD59, polymeric C9 complexes are removed from PNH platelets by exocytosis. These vesicles are very thrombogenic and serve as a platform for the prothrombinase complex. In addition, the abnormal monocytes in PNH lack the receptor for plasminogen activator and are less effective in fibrinolysis (Rosse and Nishimura 2003).
A proliferative and survival advantage of an affected stem cell with a mutation of the PIG-A gene allows the expansion of PNH clone. Current experimental data indicate that PIG-A mutant cells are relatively resistant to cytotoxic attack by natural killer cells and cytotoxic T-lymphocytes (Nakakuma and Kawaguchi 2003).
CLINICAL MANIFESTATIONS OF PNH - In PNH the intravascular hemolysis results is hemoglobinemia. When the binding capacity of haptoglobin is surpassed, free hemoglobin circulates in the plasma and is filtered by the glomeruli of the kidney. As hemolysis rate increases, the level of hemoglobin in the glomerular filtrate will go beyond the resorption capacity of the proximal tubules. This may result in temporary Fanconi syndrome. In dehydrated patients, high concentration of hemoglobin in the tubular filtrate leads to acute renal failure. The progressive renal failure is the cause of death in approximately 8% of PNH patients.
Free hemoglobin in the plasma binds nitric oxide (NO) and causes local NO deficiency leading to smooth muscle contraction (i.e., esophagus, manifested as retrosternal tightness). Penile erectile dysfunction (i.e., priapism) among men with PNH is explained my NO deficiency and is controlled by sildenafil.
The leading cause of death among patients with PNH is venous thrombosis. The thrombosis incidence is about 40% of all patients in Europe and in the United States. The incidence among Asian and Mexican patients is 8 fold lower. Hepatic and portal veins are commonly involved. In fact, venous thrombosis in an unusual location strongly suggests the diagnosis of PNH. Other sites commonly involved by thrombosis are cerebral veins, sinuses, and splanchnic veins. Venous thrombosis is a serious complication of pregnancy, with mortality rate of 20% and the fetal death rate of 8%.
Typical PNH patients presents with pancytopenia. The current hypothesis is that GPI-deficient hematopoietic stem cells exist in the bone marrow of many healthy persons. Pancytopenia is caused by the expansion of an affected stem cell with a mutation of the PIG-A gene (vide supra).
LABORATORY DIAGNOSIS - Screening laboratory tests relevant to PNH include a complete blood count (CBC), stained blood smear, and bone marrow examination. Leukocyte alkaline phosphatase (LAP) is usually decreased in PNH.
The classic and highly specific way to identify the RBC PNH clone is Ham's test. Only one other disease is associated with a positive Ham's test: hereditary erythroid multinuclearity with positive acidified serum (HEMPAS), distinguished from PNH by a negative sucrose hemolysis test. Hence, Ham's test is highly specific for PNH. The Ham's test is based on the precept that complement will attach to RBCs at low pH and that PNH RBCs are more sensitive to complement. Whole defibrinated blood collected in heparin is utilized. Cells from the patient and controls are tested for hemolysis with (1) unmodified serum, (2) serum, pH 6.8, (3) heat-inactivated serum (55°C for 3 min.), and (4) heated serum with guinea pig complement. Positivity should be encountered with: (1) PNH cells only and (2) acidified serum; (3) destroying complement (heating) prevents the hemolysis, and (4) not with guinea pig complement (Krauss 2003).
The sucrose lysis test is another standard screening test for PNH. Low ionic strength isotonic sucrose induces serum globulin to bind complement on the RBC membrane.
Flow cytometry (FCM) recently became the definitive test for PNH. Usually, CD55 and CD59 are both measured by FCM. Depressed levels of both of these glycoproteins are consistent with PNH. FCM identification of small PNH clones (<5.0%) shows the greater sensitivity of FCM, relative to Ham's test. Clone size is more accurately evaluated with white blood cells (WBCs), because the RBC half-life is shortened by hemolysis. For FCM PNH granulocyte studies, antibodies to two GPI-linked antigens (CD55/CD16, CD59/CD16) and another antibody to non-GPI-linked antigens (CD15/CD33) are recommended (Krauss 2003).
A GPI-binding aerolysin toxin can be used to differentiate PNH cells. Inactivated aerolysin (fluorescent proaerolysin variant, FLAER) binds to the GPI anchor more specifically and sensitively than anti-CD59. Furthermore, FLAER GPI-anchor specificity precludes the need for a second marker (i.e., both CD55 and CD59)(Brodsky, Mukhina et al. 1999).
TREATMENT - The only radical treatment is bone marrow transplant and is indicated after the aplastic crisis. Antithymocyte or antilymphocyte globulin induces remission in about 40% to 60% of PNH patients with aplastic anemia.
Long-term anticoagulation with coumadin are suggested for patients of European descent who are not thrombocytopenic; alternatively, daily aspirin therapy may be considered.
Heparin is most useful in acute thrombosis. In acute Budd-Chiari syndrome, the timely use of tissue plasminogen activator can dramatically reduce the size of the liver and improve patient's survival.
Prophylaxis with LMWH or heparin is used during extended immobilization or during the prolonged use of an intravenous catheter. Prophylaxis with heparin or LMWH should be started in the first trimester of pregnancy and continued until 6 weeks postpartum.
An acute hemolytic crisis (especially when refractory to corticosteroids) is an indication for red blood cell transfusion. In order to avoid increase in complement levels, washed cell have been recommended.
Several complement inhibitors are in phase II trials in humans. Recently, Hillmen and colleagues reported the results of a study using a humanized monoclonal antibody against complement C5. This reagent is effective in controlling the PNH hemolysis without significant adverse (Hillmen, Hall et al. 2004).
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Contributed by S. Chiosea, MD and F. Bontempo, MD
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