Paroxysmal nocturnal hemoglobinuria

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Editor(s)-in-Chief: C. Michael Gibson, M.S.,M.D. [mailto:mgibson@perfuse.org] Phone:617-632-7753; Robert Killeen, M.D. [mailto:aak324@gmail.com]

Overview
Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired, potentially life-threatening disease of the blood characterised by hemolytic anemia, thrombosis and red urine due to breakdown of red blood cells. PNH is the only hemolytic anemia caused by an acquired intrinsic defect in the cell membrane.

History
The first description of paroxysmal hemoglobinuria was by the German physician Paul Strübing (1852). A more detailed description was made by Dr Ettore Marchiafava and Dr Alessio Nazari in 1911, with further elaborations by Marchiafava in 1928 and Dr Ferdinando Micheli in 1931.

Classification
PNH is classified:
 * Classic PNH. Evidence of PNH in the absence of another bone marrow disorder.
 * PNH in the setting of another specified bone marrow disorder.
 * Subclinical PNH. PNH abnormalities on flow cytometry without signs of hemolysis.

Pathophysiology
All cells have proteins attached to their membranes that are responsible for performing a vast array of functions. There are several ways for proteins to be attached to a cell membrane. PNH occurs as a result of a defect in one of these mechanisms.

It is thought to be an acquired disease with the clonal expansion of pluripotent stem cells containing the somatic mutation of an X-linked (short arm of X-chromosome) PIG-A (for phosphatidylinositol glycan class A) gene. The gene that codes for PIG-A is inherited in an X-linked fashion. This gene is involved in the first step of the synthesis of the glucosylphosphatidyl-inositol anchor of GPI membrane proteins such as CD55, CD59, CD14 and others (CD is an acronym for 'cluster of differentiation'). Mutations in the PIG-A gene cause a deficiency of the glucosylphophatidylinositol-anchored proteins in PNH hematopoietic cells (all 3 cell lines can be affected). Two of these proteins, CD55 and CD59, are complement regulatory proteins; the absence of these proteins is fundamental to the pathophysiology of this disease. The complement system is the part of the immune system that helps to destroy invading microorganisms. The presence of CD55 and CD59 confers resistance to the body's blood cells from lysis by complement. CD55 inhibits C3 convertase and CD59 blocks the formation of the membrane attack complex (MAC) by inhibiting the incorporation of C9 into the MAC. The loss of these complement regulatory proteins renders PNH erythrocytes susceptible to both intravascular and extravascular hemolysis but it is the intravascular hemolysis that contributes to much of the morbidity of this disease.

The increased destruction of red blood cells results in anemia. The increased rate of thrombosis is due to dysfunction of platelets. They are also made by the bone marrow stem cells and will have the same GPI anchor defect as the red blood cells. The proteins which use this anchor are needed for platelets to clot properly, and their absence leads to a hypercoagulable state.

Signs and symptoms
Quite paradoxically, the destruction of red blood cells (hemolysis) is neither paroxysmal nor nocturnal the majority of the time (this constellation of symptoms is seen in only 25% of patients). Patients with PNH manifest the clinical and laboratory signs of chronic hemolytic anemia. Weakness, dyspnea and pallor are common. Splenomegaly may be present.

A common finding in PNH is the presence of breakdown products of RBCs, hemoglobin (26% pf patients) and hemosiderin, in the urine. Hemosiderinuria is a more constant feature of this disease and typically doesn't occur in other forms of anemia unless there is considerable intravascular erythrocyte destruction. Hemolysis is increased in the evening however, the (classic) passage of dark, hemoglobin-containing urine upon rising in the morning is seen in only a minority of cases. Haptoglobin is decreased and LDH can be increased to as much as 2-10 times normal. Neutropenia as well as thrombocytopenia may be evident. The leukocyte alkaline phosphatase (LAP) score is decreased.

An inconsistent, but potentially life-threatening, complication of PNH is the development of venous thrombosis (~40% of patients). These thrombi are often found in the hepatic (causing Budd-Chiari syndrome; the most comon cause of mortality), portal (causing portal vein thrombosis), and cerebral veins (causing cerebral venous thrombosis). The risk of thrombosis has been directly linked to the size of the PNH clone. The thrombotic risk increases with pregnancy.

PNH can present with or as other disease entities such as aplastic anemia or myelodysplasia (MDS). Patients who present with pancytopenia or thrombosis compounding anemia should be suspected of having PNH. Many patients with bone marrow failure (aplastic anemia) develop PNH (10-33%). Aplastic anemia can be caused by an attack by the immune system against the bone marrow. For this reason, drugs that suppress the immune system are being researched as a therapy for PNH. A small percentage of PNH patients can have dysplasia that leads to Acute Myelocytic Leukemia. Interestingly, the blasts of the PNH-derived AML also lack leukocyte alkaline phosphatase (LAP) and decay accelerating factor (CD59;DAF).

Iron deficiency often occurs with PNH patients because of urinary loss and should be treated. The administration of oral iron is usually sufficient. Although there may be an increase in hemoglobinuria with iron therapy, due to the increased production of PNH cells by the marrow, the net positive effect in red blood cell production may lessen the requirements for blood transfusion. Folate should be given concurrently with the iron to help augment hematopoiesis.

Nitric Oxide depletion / thrombosis; During episodes of acute hemolysis free plasma hemoglobin that is released as a consequence of erythrocyte lysis may overpower haptoglobin, a hemoglobin-scavenging protein. Excess free hemoglobin depletes plasma nitric oxide, which can play an important role in the maintenance of normal platelet function. It has been postulated that nitric oxide down-regulates platelet aggregation, adhesion and regulating molecules in the coagulation cascade. Nitric oxide depletion may therefore lead to platelet activation and aggregation. With this in mind the chronic consumption of nitric oxide by intravascular hemoglobin can play a role in the thrombotic events that occur in patients with PNH. Depletion of nitric oxide at the tissue level contributes to numerous PNH manifestations including smooth muscle dystonia (eg esophageal spasm, abdominal pain, male erectile dysfunction), pulmonary hypertension, severe fatigue and renal insufficiency as well as thrombosis.

Diagnosis
A sugar or sucrose lysis test, in which a patient's red blood cells are placed in low ionic strength solution and observed for hemolysis, is used for screening. A more specific test for PNH, called Ham's acid hemolysis test, is performed if the sugar test is positive for hemolysis. In a positive sucrose lysis test ionic strength facilitates the complement binding whereas in a positive Ham acid hemolysis test acidic strength facilitates the complement binding. The differential diagnosis of a positive sugar lysis test includes some autoimmune hemolytic anemias; even leukemias can give a false positive result. The differential diagnosis for a positive Ham test includes congenital dyserythropoietic anemia; note that a negative Ham test doesn't rule out PNH. These assays do not reliably quantitate the percentage of PNH cells and can be falsely negative in patients who have received red blood cell transfusions. Occasionally the characteristic complement-sensitive erythrocytes cannot be demonstrated in patients with well-established PNH. This probably occurs when the production of PNH cells is relatively low and most of the PNH cells that have been made have already been destroyed either in the marrow or in the circulation. Therefore a single normal sucrose hemolysis test cannot be considered absolute evidence that a patient does not have PNH.

Modern methods include flow cytometry for CD55, CD16, CD59 and other GPI anchored proteins on white and red blood cells. Laboratories favor flow cytometry to evaluate PNH due to its high sensitivity and specificity. Flow cytometry of the peripheral blood, not the bone marrow aspirate, is required to evaluate the presence or absence of GPI linked proteins. The bone marrow biopsy in PNH shows erythroid hyperplasia. In addition, because of the short life of granulocytes, the peripheral blood samples need to reach the lab in an expedited manner. The most commonly used antibodies are CD59 (expressed on all hematocellular lineages), and CD55 but other GPI anchored antigens (CD14, CD16, CD24) can also be studied on leukocytes. Dependent on the predominance of these molecules on the red blood cell surface, they are classified as type I, II or III PNH cells.

PNH type II & III cell populations; definitions. Some patients may have erythrocytes with low but detectable GPI anchored proteins; these cells are designated PNH type II. By contrast, cells that are completely devoid of GPI anchored proteins are referred to as PNH type III. Patients with large populations of PNH type II erythrocytes may have less hemolysis than those with comparable populations of PNH III cells but these patients are still at risk for both hemolysis and thrombosis.

MRI

 * Renal cortical signal intensity loss (hemosiderin accumulates in the renal cortex when intravascular hemolysis results in the direct release of hemoglobin into the plasma).
 * Venous thrombosis.
 * Liver and spleen are usually of normal signal intensity in paroxysmal nocturnal hemoglobinuria, unless repeated transfusions have resulted in hepatic and splenic signal intensity loss owing to transfusional siderosis.

(Images shown below are courtesy of RadsWiki)

Treatment
There is no widely accepted evidence-based indication for the treatment of PNH. In classic PNH it is recommended to treat patients with disabling fatigue, thromboses, transfusion dependence, frequent painful paroxysms, renal insufficiency or other end-organ complications from this disease. Watchful waiting is appropriate for the asymptomatic patient or the patient with mild symptoms.

Long-term
PNH is a chronic condition. In patients who have only a small clone and few problems, monitoring of the flow cytometry every six months gives information on the severity and risk of potential complications. Given the high risk of thrombosis in PNH, preventative treatment with warfarin decreases the risk of thrombosis in those with a large clone (50% of white blood cells type III). Episodes of thrombosis are treated as they would in other patients, but given that PNH is a persisting underlying cause it is likely that treatment with warfarin or similar drugs needs to be continued long-term after an episode of thrombosis.

In patients with aplastic anemia / PNH treatment should be directed toward the underlying bone marrow failure with careful monitoring of the PNH clone using flow cytometry. Patients who meet the criteria of severe aplastic anemia should be managed with either an allogeneic bone marrow transplant or immunosuppressive treatment dependent on the age of the patient and the availability of a bone marrow donor. Treatment of bone marrow failure in PNH is similar to that for aplasia. Immunosuppressives can be administered such as antithymocyte globulin or cyclosporine. Supportive measures in terms of GCSF or erythropoeitin (EPO) can also be given. An allogeneic bone marrow transplant is the only curative treatment and is an option for younger patients. A bone marrow transplant is curative but is associated with significant morbidity and mortality. The 2 year survival with this modality is 56%; the majority of deaths occur within the first year of transplant (International Bone Marrow Registry).

Acute attacks
There is debate as to whether steroids (such as prednisolone) can be useful in decreasing the severity of hemolytic crises. Steroids can decrease complement activation which, subsequently, decreases hemolysis however high doses are usually necessary. Transfusion therapy may be needed; in addition to correcting significant anemia this suppresses the production of PNH cells by the bone marrow, and indirectly the severity of the hemolysis. Some sources advocate the use of washed red cell transfusions. Leucocyte-depleted transfusions are also recommended for those requiring chronic transfusion therapy.

Iron deficiency develops with time, due to losses in urine, and may have to be treated if present. Iron therapy can result in more hemolysis as more PNH cells are produced. It may be necessary to give folate too in order to augment hematopoiesis. Erythropoeitin (EPO) can be given (10-20,000 u tiw) to help. As with steroid therapy tranfusions are given when the iron treatments do not suffice.

Eculizumab (AKA Soliris) is a monoclonal antibody against the complement protein C5, halting terminal complement-mediated intravascular hemolysis. It binds to a subunit of the C5 convertase enzyme. It prevents C5 convertase from hydrolyzing C5 to C5a and C5b, the latter combining with C9 to form the terminal complement complex.

Selection of patients to be treated with Eculizumab should be guided by the degree of hemolysis and the risk of thrombosis. Although most of the patients with PNH have some degree of ongoing hemolysis not all are transfusion dependent nor even anemic.

Patients who take Eculizumab are at increase risk of life-threatening meningococcal infection. Patients must receive the meningococcal vaccine at least 2 weeks before Eculizumab is given. If the patient had already received the vaccine, they may need a booster. Patients have a 0.5% yearly risk of acquiring neisserial sepsis even after vaccination. Patients should be revaccinated against Neisseria meningitidis every 3-5 years after starting the treatment and they should seek medical care if they develop any signs or symptoms suggestive of neisserial infection. These include headache, nausea, vomiting, fever, stiff back or neck, rash, confusion, visual sensitization to light and myalgias with flu-like manifestations. Note that the most common toxicity of Eculizumab is headache which occurs in about 50% of patients given the first dose or two but, typically, this rarely recurs afterwards. Patients still need to be monitored for meningitis for at least 8 weeks after discontinuing Eculizumab.

Long term terminal complement inhibition by Eculizumab doesn't increase the incidence of myeloproliferative disease, myelodysplasia, acute leukemias or aplasia / pancytopenias in PNH patients. Eculizumab administration decreases hemolysis leading to stabilization of the hemoglobin concentration and reticulocyte count. This is manifest clinically with a decrease in the need for transfusions.

Breakthrough intravascular hemolysis and a return of PNH symptoms occurs in < 2% of PNH patients treated with Eculizumab. This typically occurs a day or two before the next scheduled dose and is accompanied by a spike in the LDH. The LDH usually returns to normal or near normal within days to weeks after Eculizumab. Since the (episodic) hemolysis of PNH is partly intravascular, the finding of urine hemosiderin is consistent with continued erythrocyte destruction. The reticulocyte count often remains elevated because most PNH patients on Eculizumab continue to have some extravascular hemolysis. If this occurs on a regular basis then the dosing interval can be shortened or the dose increased in order to compensate. It is also important to remember that increased complement activation accompanies infection (eg. flu or viral gastroenteritis) or trauma which can result in transient breakthrough hemolysis. It is not recommended to change the dosing with regard to a single episode of breakthrough hemolysis.

Anticoagulation is only partly effective in preventing thrombosis in PNH. Some sources state that thrombosis is an absolute indication for initiating treating with Eculizumab. Prophyllactic anticoagulation has never been proven to prevent thrombosis in all PNH patients and can be dangerous given the thrombocytopenia seen in this malady. Some sources state that patients who do not meet criteria for Eculizumab therapy should not receive anticoagulation. Possible exceptions to this rule might include patients with persistently elevated D-dimer levels, pregnant PNH patients and patients in the perioperative period.

Pregnant patients with PNH have an even greater need for folate and iron supplementation because of the hemolysis. Pregnancy, as with oral contraceptive use, increases the risk of thrombosis in PNH. Anticoagulation with a LMWH is recommended as long as there are no contraindications for full anticoagulation. Give 1 mg/kg subcutaneously every 12 hours when the pregnancy is confirmed in a PNH patient with a large PNH clone. Some sources state that it is often necessary to switch to unfractionated heparin around the time of delivery if a C-section is planned.