CHAPTER 36 PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
CHAPTER 36 PAROXYSMAL NOCTURNAL HEMOGLOBINURIA
Definition and History
Etiology and Pathogenesis
Therapy, Course, and Prognosis
Paroxysmal nocturnal hemoglobinuria is an acquired hematopoietic stem cell disease characterized by chronic hemolytic anemia, thrombotic episodes, and often pancytopenia. It is a clonal disorder, caused by a somatic mutation of the X-linked gene PIG-A, which is required for formation of the phosphatidylinositol anchor. As a result, many membrane proteins, including some inhibitors of the complement cascade, are missing from the cell surface, and the erythrocytes are usually sensitive to the hemolytic effect of complement. The disease is diagnosed provisionally with the sucrose hemolysis test and definitively by the Ham acid hemolysis test, which is being replaced by flow cytometric demonstration of a deficiency in CD55 and CD59. Treatment with glucocorticoids and/or androgenic steroids is sometimes helpful. The median survival is approximately 10 years. Stem cell transplantation is curative.
Acronyms and abbreviations that appear in this chapter include: DAF, decay accelerating factor; FACS, fluorescent-activated cell sorting; G-CSF, granulocyte colony–stimulating factor; GPI, glycosylphosphatidylinositol; HEMPAS, Hereditary Erythroblastic Multinuclearity with a Positive Acidified Serum lysis test; HRF, homologous restriction factor; LFA-3, lymphocyte function–associated antigen-3; MIRL, membrane inhibitor of reactive lysis; PIG-A, phosphatidylinositol glycan class A; PNH, paroxysmal nocturnal hemoglobinuria.
DEFINITION AND HISTORY
Although commonly regarded as a type of hemolytic anemia, paroxysmal nocturnal hemoglobinuria (PNH) is in reality a hematopoietic stem cell disorder characterized by the formation of defective platelets, granulocytes, and possibly lymphocytes as well as abnormal erythrocytes. The abnormality of the red cells predisposes them to intravascular complement-mediated lysis, which waxes and wanes in severity. The name suggests that cyclic variation in hemoglobinuria is an important feature of this disease. However, in many patients hemoglobinuria is quite irregular or occult. The classical diagnostic feature of PNH is the increased sensitivity of the red blood cells to the hemolytic action of complement.
In a scholarly historical review of PNH, Crosby1 attributed the first definitive account of this disease to Strübing,2 who in 1882 described a patient with hemoglobinuria after sleep. The patient’s plasma was red, and Strübing suggested that the erythrocytes were being destroyed within the blood stream. He also detected in the urine a fine-grained yellowish-brown material, which must have been hemosiderin. Hijmans van den Bergh3 demonstrated that erythrocytes from a similar patient were lysed in normal serum as well as in the patient’s serum if the mixture was acidified with carbon dioxide. Marchiafava and Micheli also were early students of the disease, and for a time it was designated the Marchiafava-Micheli syndrome, an appellation that has fallen into disuse.
Because many different proteins that had in common their attachment to a phosphatidylinositol anchor are missing from the surface of blood cells in PNH, it was recognized that the underlying defect in PNH would be likely to affect this structure. The detection of the defect in an X-linked gene designated PIG-A (for phosphatidylinositol glycan class A)4 has been the culmination of over 100 years of research into this once mysterious disorder.
ETIOLOGY AND PATHOGENESIS
In contrast to all the other intrinsic abnormalities of the erythrocyte, PNH is an acquired disorder; in a number of cases only one of a pair of identical twins was affected.5,6 and 7 Before the basic lesion had been discovered, the expression of glucose-6-phosphate dehydrogenase alleles,8 methylation of polymorphic restriction sites,9 and cytogenetic studies10 had all demonstrated the clonal nature of PNH. Thus, PNH arises, like a neoplasm, from the transformation of a single cell. The underlying defect in PNH is one or several of many different mutations in the PIG-A gene, an X-linked gene that plays a major role in the formation of the phosphatidylinositol anchor. Many different mutations have been documented in PNH patients,11 most of them nonsense mutations or insertions or deletions producing frameshifts. It is no accident that the gene that is mutated is X-linked. In this way a single somatic mutation is enough to affect formation of the anchor. If an autosomal gene were to cause the disease, damage to both copies of the gene would be required, a statistically unlikely event. The abnormal clone appears to arise most commonly in a damaged marrow; many patients with PNH have a prior history of aplastic anemia,12 either idiopathic or drug-induced. Cells with PIG-A mutations do not appear to have a proliferative advantage in vitro or in hybrid animal models made with PIG-A knockouts,13 but they are resistant to apoptosis,14,15 which may account for their survival advantage and the evolution of PNH into leukemia. Somewhat surprisingly, some patients have multiple clones, each with a distinct mutation.16,17 This implies that mutations of the PIG-A gene may not be altogether rare, and when conditions are such that a PNH clone has a selective advantage, several independent mutational events may come to light. The PIG-A gene plays a vital role in a very early step in the conversion of N-acetylglucosamine and glucosamine-phosphoinositol into mature mannolipids.18 Transformed lymphoblasts are unable to incorporate labeled mannose into glycosylphosphatidylinositol (GPI) anchor precursors and, when provided with uridine diphospho-N- acetyl[3H] glucosamine, there is a marked reduction in the production of anchor phospholipids. Transfection with the PIG-A cDNA corrects the defect in GPI anchor synthesis.4
A large number of membrane protein deficiencies have been observed in PNH. These include deficiencies of acetylcholinesterase,19 leukocyte alkaline phosphatase,20 the “decay accelerating factor” (DAF, CD55),21 CD59 antigen (membrane inhibitor of reactive lysis—MIRL), homologous restriction factor (HRF, C8-binding protein),22 CD58 (lymphocyte function–associated antigen-3—LFA-3),23 5′-ectonucleotidase,24 CD16 (the low-affinity Fc receptor of granulocytes),25 urokinase-type plasminogen activator,26 and CD14 antigen.27 The common denominator appears to be that all of these proteins are attached to the GPI anchor (see Chap. 27).
The classic abnormality of PNH erythrocytes is their increased sensitivity to complement-mediated lysis, whether the complement is activated by the classic or the alternative pathway (see Chap. 6). Activation of complement may be achieved by a variety of means28: lowering of pH, as in the acid hemolysis test; reducing ionic strength, as in the sucrose hemolysis test; treating the plasma with cobra venom; increasing the magnesium concentration; or coating the cells with antibodies such as anti-A. Using graded amounts of complement, it is possible to identify several populations of cells which have been designated PNH I, PNH II, and PNH IIIa and IIIb, manifesting progressively increasing sensitivity to complement lysis and deposition of increasing amounts of C3 on the PNH membranes.12 The existence of multiple populations, once difficult to explain in a clonal disorder, is presumably due to the coexistence of several different clones with different mutations.16,17
Although several of the abnormalities involve proteins that modulate complement function, a variety of findings suggest that the absence of the CD59 antigen (MIRL) plays the most critical role in the complement sensitivity of PNH erythrocytes. Inherited deficiency of DAF is not associated with clinical hemolysis, although a weakly positive acid hemolysis test may be present,29,30 while a hereditary deficiency of CD59 is associated with PNH.31 Restoration in vitro of CD59 corrects the complement sensitivity of red cells more completely than restoration of DAF,32 and PNH may occur in the absence of DAF deficiency.33 The trimodal distribution of complement sensitivity of the red cells of some patients with PNH19 appears to be related to the degree of deficiency of CD59 and DAF among the erythrocytes of such patients.34,35
Granulocytes and platelets, like red cells, show increased sensitivity to complement-mediated lysis36 and are deficient in DAF.37 Chemotactic responses of PNH granulocytes are also impaired.38 Even blood lymphocytes39 and lymphoblastoid cell lines from some patients with PNH show abnormalities in CD59 and DAF.40
The nocturnal hemoglobinuria from which PNH derives its name, that is, the passage of red or brownish urine in the morning on rising, occurs in only a small proportion of the patients. When it does follow the classic cyclic pattern, the hemoglobinuria occurs during sleep regardless of the time of day.41 It was originally believed that nocturnal hemoglobinuria was a function of a lowered blood pH during sleep, but this is not the case.42 Hemoglobinuria may also be the result of the production of increased numbers of abnormal cells rather than of an increase in hemolytic processes.
In most patients with PNH, hemoglobinuria occurs irregularly. Bouts of hemolysis may be initiated by infections, surgery, and possibly even strenuous exercise.43 The injection of contrast dyes, as in intravenous pyelography or myelography, may precipitate hemolysis by activating complement.44
Patients with PNH manifest all the clinical and laboratory signs of chronic hemolytic anemia. Weakness, dyspnea, and pallor are common, particularly when the anemia is quite severe. Splenomegaly is present in some patients, but the enlargement of the spleen is usually quite modest.
Iron deficiency is often a manifestation of PNH because of iron loss in the urine, in the form of both hemosiderin and hemoglobin. The administration of iron to patients with iron deficiency sometimes results in overt signs of hemolysis, manifested by the appearance of frank hemoglobinuria. Although this effect of iron has sometimes been attributed to its peroxidatic effect, increasing damage to the red cell membrane,45 it seems more likely that it is due to increased production of both normal and abnormal red cells by the marrow, with the newly formed abnormal cells undergoing hemolysis.46
Thrombocytopenia varies greatly in severity. It may be very mild and persist for years, or it may be very severe. In the latter instance extensive hemorrhagic complications may be a prominent part of the clinical presentation of patients with PNH.
Although thrombotic complications occur in other forms of hemolytic anemia as well, they are particularly prominent and severe in PNH. The reason for this is not entirely clear, but it may be related to activation of platelets by complement,12 the procoagulant activity of red cell membranes, or the intravascular release of adenosine diphosphate (ADP) from red cells, leading to platelet aggregation. The prevalence of factor V Leiden is not increased in PNH.47 Venous thromboses represent one of the most frequent clinical manifestations of PNH. The Budd-Chiari syndrome, resulting from hepatic vein thrombosis, has been observed repeatedly. In one study48 of 40 patients with Budd-Chiari syndrome, 5 were found to have PNH. Thus PNH should be a serious consideration in any patient with hepatic vein thrombosis. Budd-Chiari syndrome has an ominous prognosis when fully developed.49,50 It may also occur in a milder, subclinical form detectable by ultrasonography,51 and early therapy has been recommended.48 Pain in the abdomen or in the lower part of the back also appears to be more common in patients with PNH than in those with other types of hemolytic anemia. The abdominal pain is often colicky in nature and the abdomen is tender on palpitation. Frank intestinal infarction or bleeding into the intestinal wall has sometimes been found.52,53 Esophageal spasm has been observed in patients who are undergoing hemolysis; it has been likened to the symptoms that have occurred in patients who are receiving hemoglobin solutions as a blood substitute, symptoms that are probably related to removal of ambient nitric oxide.12 Pulmonary hypertension has occurred and has been attributed to widespread thromboses in the pulmonary microvasculature.54 Arterial as well as venous thrombosis has been documented.55
Pregnancy in PNH patients has been associated with abortion and venous thromboembolism, but the outcome is sometimes normal.56,57 and 58 In a study of 38 pregnant patients with PNH, pregnancy was uncomplicated in one-third of the cases and life-threatening complications in mothers were uncommon.57
A variety of abnormalities of renal function are observed. Included are hyposthenuria, abnormal tubular function, and declining creatinine clearance. Hypertension was observed in 8 of a series of 21 patients who had been followed for a long period. Radiologic findings included enlarged kidneys and cortical infarcts, cortical thinning, and papillary necrosis. Most patients have some episodes of hematuria and proteinuria distinct from hemoglobinuria.44 Acute and chronic renal failure may occur.44,59,60 and 61
Severe headaches or pains in the eyes occur in patients with PNH without any objectively demonstrable neurologic abnormalities. These complications may be due to small venous occlusions. Frank cerebral venous thrombosis is a grave and fortunately uncommon complication of PNH.62
Anemia may be very severe, with hemoglobin concentrations below 5 g/dl, but in some cases the hemoglobin level is normal. A mild to moderate reticulocytosis is usually present; the reticulocyte count tends to be lower than in other patients with chronic hemolysis who manifest the same degree of anemia. A modest degree of macrocytosis commensurate with the increased reticulocyte count is usually present. However, if the patient has developed iron deficiency, the red cells may be microcytic and hypochromic. In this case, the plasma iron and ferritin levels are usually low and the iron-binding capacity elevated.
The leukocyte count is characteristically low, principally because of a diminution of the number of granulocytes. The leukocyte alkaline phosphatase activity20 and surface urokinase receptors63 are diminished. The platelet count is often low, but it may be 150,000/µL or more in about 20 to 50 percent of the patients.64 Platelet survival is usually normal.36
Erythroid hyperplasia is usually present in the marrow of patients with PNH. However, the marrow cellularity is generally not greatly increased, and the marrow may even be aplastic. Stainable iron is often absent.
Hemoglobin is sometimes but by no means always present in the urine. Hemoglobin casts may be present. Hemosiderinuria is one of the most constant features of the disease and is of considerable diagnostic importance.
The diagnosis of PNH should be entertained in any patient with pancytopenia of unknown origin, particularly when accompanied by reticulocytosis. Isolated defects in a single lineage, such as thrombocytopenia, may also be the presenting finding. PNH arises within the context of marrow failure states such as aplastic anemia. When such patients manifest moderate numbers of reticulocytes in the blood, tests for PNH may demonstrate that a complement-sensitive clone has appeared. A search for PNH occasionally proves rewarding in the case of patients who have repeated unexplained thrombotic episodes.
The most convenient screening tests for PNH are the sucrose hemolysis test65 and the examination of urine for hemosiderin. Occasionally the characteristically complement-sensitive red cells 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. Thus a single normal sucrose hemolysis test cannot be considered strong evidence that a patient does not have PNH. Hemosiderinuria is a more constant feature of the disease and is helpful in identifying patients who may have PNH with a transiently normal sucrose hemolysis test. If the sucrose hemolysis test is positive, the diagnosis should be confirmed with fluorescent-activated cell sorting (FACS) analysis or with the complete Ham acid hemolysis test.66 The latter test establishes that (1) hemolysis is a property of the patient’s erythrocytes, (2) hemolysis requires the presence of serum, (3) the hemolytic effect of the serum is increased by acidification, (4) the hemolytic properties of serum are destroyed by heating, and (5) the hemolytic properties of heated serum are not restored by guinea pig serum. Although historically the Ham test has been the “gold standard” for the diagnosis of PNH, it is a cumbersome and time-consuming procedure with a considerable number of potential technical pitfalls. Therefore flow cytometry of erythrocytes using anti-CD59 or of granulocytes using either anti-CD55 or anti-CD59 antibodies is rapidly replacing the older techniques once used for diagnosis67,68,69,70 and 71 and can be regarded as a sensitive and specific diagnostic measure.
Thrombocytopenia and leukopenia are features of PNH that help to differentiate this disorder from other types of hemolytic anemia. Hemosiderinuria, a constant feature of PNH, does not usually occur in other forms of hemolytic anemia, except for those in which there is considerable intravascular destruction of erythrocytes, such as in the hemolytic anemia associated with prosthetic cardiac valves. Although HEMPAS (Hereditary Erythroblastic Multinuclearity with a Positive Acidified Serum lysis test) is characterized, as its name indicates, by a positive acidified lysis test, there should be no difficulty in distinguishing this disorder from PNH (see Chap. 35). Lysis of HEMPAS cells occurs because of the presence in normal serum of antibodies to unusual antigens on the surface of HEMPAS cells. The serum of these patients lacks the required alloantibody, and HEMPAS cells will not lyse in their own serum. Moreover, HEMPAS is a hereditary disorder and is not associated with leukopenia or thrombocytopenia.
THERAPY, COURSE, AND PROGNOSIS
Treatment of PNH consists chiefly of supportive measures such as transfusion, antibiotics, and anticoagulants as may be required. Suitable patients may be cured by marrow transplantation.
Transfusions with red cells are often necessary in the management of patients with severe PNH. Although washed red cells are usually recommended in order to avoid transfusing the complement contained in plasma, analysis of a large number of transfusions given to PNH patients suggests that packed red cells are equally safe.72,73
The iron deficiency that often occurs in patients with PNH because of the urinary loss of iron should be treated. The oral administration of iron is usually entirely satisfactory (see Chap. 38). Although an increase in hemoglobinuria may occur during iron therapy because of increased production of PNH cells by the marrow, the net positive effect of the administration of iron may lessen the requirements for blood transfusion.
Both androgens and glucocorticoids have been used in the treatment of PNH. Fluoxymesterone (Halotestin) in doses of 20 to 30 mg per day usually produces some increase in the hemoglobin concentration of the blood.74 The administration of danazol failed to produce a therapeutic effect in two patients.75
The administration of glucocorticoids has also been reported to be useful in the treatment of both hemolysis and thrombotic episodes.12 Doses ranging from 20 to 60 mg of prednisone on alternate days may be tried. In view of the potential side effects, particularly when these drugs are administered chronically, steroid therapy should be limited to those patients whose transfusion requirement is significantly decreased at well-tolerated doses.
Use of prophylactic anticoagulants in PNH has been advocated,76 but there is no clear-cut evidence of a beneficial effect. The principal role of anticoagulants in the management of PNH is in the treatment of thrombotic complications such as the Budd-Chiari syndrome.77 Thrombolytic therapy with streptokinase and urokinase was considered to be safe and effective in two patients with PNH.78 Sometimes a trial of anticoagulation therapy is used in patients who have repeated episodes of abdominal and back pain, but the usefulness of this approach remains to be established. The administration of drugs associated with an increased incidence of thrombosis, particularly oral contraceptives, should be avoided in patients with PNH.
Generally, splenectomy is not indicated, although favorable responses have been reported in some patients. Because of the considerable risk of thromboembolic complications in patients with PNH, elective surgery of any type, including splenectomy, is best avoided. When surgery is necessary, the administration of low-dose or low-molecular-weight heparin postoperatively may be prudent because of the high risk of thrombosis.
STEM CELL TRANSPLANTATION
As in other stem cell disorders, stem cell transplantation is an effective albeit high-risk method for the treatment of PNH79,80,81 and 82 (see Chap. 18). As might be expected, the abnormalities in the phosphoinositol-anchored proteins are corrected by this procedure.83
Erythropoietin therapy seemed to have a beneficial effect in some patients but not in others.84,85 Hemolysis may be diminished temporarily by the infusion of a dextran solution,86,87 but this measure does not seem to have a role in the clinical management of PNH. Some selective suppression of PNH cells was documented with 6-mercaptopurine treatment, but this did not prove to be of any clinical value.88 Cyclosporine, sometimes in combination with granulocyte colony–stimulating factor (G-CSF), has seemed helpful in some patients.89,90
The clinical course of PNH is enormously variable. In rare instances, the patient may succumb to this disease within a few months of the first onset of symptoms. Other patients experience a chronic course in which the severity of the disease may wax and wane as the normal cells and the PNH clone alternately appear to gain ascendancy. Sometimes the abnormal clone disappears altogether and the patient appears to be cured. It has been suggested that the course is more severe in children and adolescents with PNH.91
The development of acute leukemia is rare but well documented.92,93 Myelodysplastic syndrome also occurs in patients with PNH.94,95 and 96 Not surprisingly, the defect seems to arise in the PNH clone.92
As with so many other diseases, initial reports on PNH tended to emphasize the more severely affected patients, so the prognosis was generally deemed to be very grave. As physicians developed a higher index of suspicion concerning this disorder, and as simplified methods for diagnosis became available, milder cases were diagnosed, and these tend to have a better long-term outlook. Nonetheless, even today the disease must be considered a very serious one, and most patients eventually succumb to its complications. The most commonly lethal complication probably is thrombotic episodes such as the Budd-Chiari syndrome, but the various complications of pancytopenia also may lead to death, and in a few patients the terminal episode has been the development of acute leukemia.97 In a study of 220 patients with PNH with follow-up as long as 46 years, the Kaplan-Meier survival estimate was 65 percent at 10 years and 48 percent at 15 years after diagnosis.98 In another study of 80 consecutive patients, the outlook was similar: the median survival after diagnosis was 10 years, and 28 percent of the patients survived for 25 years.99 Eight-year cumulative incidence rates of the main complications (pancytopenia, thrombosis, and myelodysplastic syndrome) were 15 percent, 28 percent, and 5 percent, respectively. Poor survival was associated with age over 55 years at the time of diagnosis, the occurrence of thrombosis as a complication, evolution to pancytopenia, a myelodysplastic syndrome or acute leukemia, and thrombocytopenia at diagnosis. The prognosis for patients in whom aplastic anemia antedated PNH was better than that for those in whom it did not.98
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Ernest Beutler, Marshall A. Lichtman, Barry S. Coller, Thomas J. Kipps, and Uri Seligsohn