Paroxysmal Nocturnal hemoglobinuria a case report and review of the
literature.
By Luis Almonte,MD
Introduction
Paroxysmal nocturnal hemoglobinuria was first described clinically by Gull in 1866 and as a distinct clinical syndrome by Paul Strubing in 1882 (1). Now we understand that paroxysmal nocturnal hemoglobinuria (PNH) is an acquired stem cell disorder of a clonal nature with absence of glycosylphosphatidylinositol anchored proteins resulting from a somatic mutation of the pig-a gene located in chromosome X. The clinical manifestations are mainly due to lack of the glycosylphosphatidylinositol (GPI) anchored proteins. Hemolysis is the major clinical feature and is used for the diagnosis of PNH. Hemoglobinuria and hemosidenuria indicates that PNH hemolysis is intravascular. The hemolysis is chronic, mild with nocturnal exacerbations and precipitated by infections, transfusion, and surgery (2). The incidence has been estimated at about 1 in 100,000 patients and is thought to be higher in Taiwan, Thailand, and China but specific data is scant and there is no clear explanation for the difference (3)
Case report
A 32 y.o hispanic female presented with complaints of dark urine and progressive weakness of 3 days duration prior to admission. Her urine is especially dark in the morning, subsides during the day and again becomes darker at night. She is 15 weeks pregnant and has not received prenatal care. There is no complaint of abdominal pain, change in color of stools, no fever / chills/recent transfusions. No dysuria but increase frequency for the last week. She has a history of paroxysmal nocturnal hemoglobinuria diagnosed 5 years ago and her only medication is ferrous sulfate 325 mg by mouth three times a day. No history of kidney stones or liver disease. Family history is unremarkable and she does not drink or smoke. On admission, well nourished well developed female who appears pale and weak in no acute distress. Blood pressure, 113/58; pulse rate, 68; respiration, 16; temperature, 98.3; and oxygen saturation, 100% on room air. Eyes appeared icteric with pale conjunctiva, abdomen with intrauterine pregnancy consistent with 15 weeks of gestation, heart/lungs/mouth/neuro and extremities were unremarkable.
The urine was amber colored and without gross hematuria.Urinalysis revealed large blood, 10-14 WBCs, moderate leukocyte esterase, positive nitrites, moderate bacteria and 500 proteins. Reticulocyte count was 32.8, LDH was 3172, beta Hcg was 83,239, hgb 6.7, hct 20.3, wbc 4.8 and platelets 92. MCV 116, rdw 15, bun 7, creatinine .6, total bili 1.8, direct bili .6, indirect bili 1.2, AST 172, ALT 17, alkaline phosphatase 53 and electrolytes within normal limits. Pelvic sonogram was consistent with intrauterine pregnancy of 15 weeks gestation.Coomb's negative. Ham's test was positive. Exacerbation of paroxysmal nocturnal hemoglobinuria secondary to urinary tract infection was diagnosed and the patient underwent treatment with amoxicillin for urinary tract infection, transfusion with washed PRBCs, hydration with normal saline, dvt prophylaxis with heparin and prednisone at 60 mg po qd. Other diagnostic evaluation s were undertaken such as hepatitis profile, iron , ferritin , B12, folate, haptoglobin, VDRL, and TIBC. Hepatitis profile was only significant for Hep A IgG, folate 14.5, iron 230, B12 269, TIBC 230, ferritin 90, VDRL negative and haptoglobin < 6.0. Abdominal sonogram demonstrated sludge in the gallbladder and no evidence of hepatic vein or portal vein thrombosis.The patient responded well to treatment and symptoms of dark urine resolved by the third day after admission. A repeat urinalysis still showed large blood with 7 RBCs but no bacteria, no leukocyte esterase and no nitrites.The total bilirubin decreased to 1.1, direct bili decreased to 0.2, ALT decreased to 40, hemoglobin after 3 units of blood increased to 10.5, hematocrit to 31.5 and platelets to 85. She was discharged on a prednisone taper, subcutaneous heparin to be continued during her pregnancy and was given amoxicillin for a total of 10 days. On follow up with the outpatient hematology oncology clinic the patient was stable. Approximately 5 months later she presented again with dark urine, cough, and headaches. On admission her hemoglobin was 8.0, hct 24.3, plalelets 73 and wbc 8.4. Late decelerations of fetal heart tracing and membrane rupture prompted a cesarean section 1 day after admission. She had an uneventful cesarean section and required 2 units of PRBCs after surgery for a hemoglobin of 6.9 and hct of 20.3. Sputum culture grew Haemophilus influenza and she was treated with zithromax with excellent clinical response. Her hemoglobin and hematocrit remained stable at about 8/25 and she was discharged on a prednisone taper.
Clinical presentation
The characteristic clinical features of paroxysmal nocturnal hemoglobinuria include intravascular hemolysis, venous thrombosis, bone marrow hypoplasia, frequent episodes of infection, and rarely development into leukemia. This condition is most frequently seen in adults 30 to 50 years of age but it may occur at any age (1). Hemolysis is one of the major features of PNH and this characteristic is exploited to diagnose it. The hemolysis of erythrocytes from PNH patients requires far less complement that that required for the hemolysis of normal erythrocytes (2). Red blood cells in PNH are classified into 3 groups:
I- normal or almost normal sensitivity to complement
II- moderately sensitive cells
III- markedly sensitive cells. These may require as little as 1/25 the concentration of human serum for lysis compared to normal erythrocytes. (2). This is especially true of reticulocytes which are almost always type III (4).
Hemolysis is clinically characterized as chronic mild hemolysis, nocturnal exacerbations, and hemolytic precipitation induced by infection, transfusion, and operation (5). The extent of hemolysis varies from patient to patient (1). All patients with PNH have some intravascular hemolysis, which can range from barely detectable to massive, requiring repeated transfusions. Reticulocyte is usually elevated but may be lower than expected for the degree of anemia. Red cells are usually normal in appearance (5).
The amount of hemolysis depends on a number of factors
1- Size of the abnormal clone(s):proportion of complement sensitive red blood cells in the circulation.
2- Abnormality of the red cells. Depending on the amount of complement defense proteins cells may be more prone to lysis. The more cell membrane proteins they lack the more likely they are to lyse.
3- Degree of complement activation: complement activation even on other cells as it occurs in infections triggers lysis by complement of the abnormal cells in PNH. Nocturnal hemolysis may be due to activation of complement by the absorption of endotoxin (e.g., lipopolysaccharide) from the gut, leading to C5b/7 complexes that initiate attacks through the reactive pathway (5,3).
Hemolysis is most significant when complement is activated by viral (particularly GI viruses ) or bacterial infections (3). A greater amount of C3 is fixed to PNH erythrocyte membrane than to the normal erythrocyte membrane and this has been associated to DAF deficiency (2,6). In humans about 30 GPI anchored membrane proteins have been detected and about 15 of them are missing from the PNH cells. This membrane defect is detectable by flow cytometry and is used for diagnosis (2). For hemolysis to occur, intravascular complement activation is indispensable (2). The sensitivity to hemolysis is due to the absence, partial or complete, of CD59 (membrane inhibitor of reactive lysis) and CD55 ( decay accelerating factor). Absence of CD59 has been shown to be the most important in promoting hemolysis (3).
Differential diagnosis
Hemoglobinuria is easily confused with myoglobinuria and hematuria.
In myoglobinuria the serum is without red pigment. Hemoglobin is more easily precipitated by ammonium sulfate or specific antibodies against myoglobin and hemoglobin. With Hematuria red cells are present in the freshly collected urine and the supernatant does not contain heme reactive pigments after centrifugation (5).
Other conditions that may cause hemoglobinuria may include severe autoimmune hemolytic anemia ( paroxysmal cold hemoglobinuria ), severe traumatic hemolytic anemia from prosthetic cardiac devices or from microangiopathic causes. Rarely, toxic hemolytic anemia may result in hemoglobinemia.Others conditions that may test positive for Ham's are congenital dyserythropoietin anemia type II and inherited CD59 deficiency (5,7).
Diagnosis
The diagnosis of paroxysmal nocturnal hemoglobinuria rest on three tests, the ham test, the sugar water test and flow cytometry. The Ham test is based on the knowledge that complement in PNH is activated by acidification of the serum to a pH of 6.2 leading to hemolysis in PNH patients blood but not in normal red blood cells (5). The sensitivity of the test is dependent on the concentration of magnesium in serum. Many false positive and false negative reactions occur during routine lab practice (5). The sugar water test (more specific) is based on the fact that complement is activated when the serum is mixed with a medium of low ionic strength. This test is quite sensitive but is less specific because cells in other conditions ( autoimmune hemolytic anemia, leukemia ) may also be lysed (5).
The best mean for diagnosing PNH is demonstrating that the GPI linked proteins are deficient on the PNH blood cells (5,8). For this, monoclonal antibodies to CD59 or CD55 (DAF) are used with flow cytometric analysis. In patients with aplastic anemia tested for GPI anchoring defects, granulocytes are the first line of cells reported to be affected (8). Studies of patients with aplastic anemia have identified affected cells in the bone marrow without evidence in peripheral blood and this may be useful someday to predict which patients with aplastic anemia and pancytopenia will develop PNH (7).
Pathogenesis
Membrane defects of PNH cells
1- Susceptibility to autologous complement
Hemolysis of erythrocytes in PNH occurs when complement is activated by Ab or exposure to low pH ( Ham’s acidified serum test ) or low ionic strength ( sugar water test ). Inactivation of complement abolishes the hemolysis but in PNH the amount of complement required for hemolysis is much less. The proportion of red blood cells that are sensitive to complement varies among patients with PNH and this may explain the variety of severity that patients may experience.
2- Disappearance of complement regulatory membrane proteins
Decay accelerating factor (CD55) is absent in the membranes of affected erythrocytes, leukocytes, and platelets. It functions to inhibit the formation of or accelerating the decay of complement complexes with C3 / C5 convertase activity in both the classical and the alternative pathway. Its absence causes the loss of complement regulatory activity, decreasing the rate at which convertase complexes are dissociated or decayed which ultimately lead to a increase in the amount of C3 and increasing the number of membrane attack complexes that are formed (5). Deficiency of decay accelerating factor (DAF) results in greater activity of the convertases; hence much more C3 is deposited on the membrane when either the classic or alternative pathway is activated (5). DAF is only absent in RBCs. Membrane inhibitor of reactive lysis (CD59) is more important than DAF in the cellular regulation of complement action. In PNH the lack of CD59 appears more critical pathophysiologically than the lack of DAF. Absence of CD59 is primarily responsible for the increased susceptibility to complement lysis. The abnormal platelets in PNH appear to be specifically sensitive to the aggregating activity of thrombin. The combination of these two reactions increased production of thrombin and increase sensitivity to thrombin aggregating activity probably accounts in large part for the markedly increased incidence of thrombosis in the patients. The absence of the primary IgG receptor present on granulocytes on PNH granulocytes may contribute to the propensity of these patients to infections, especially blood-borne infections (5).
3- Lack of glycosylphosphatidylinositol ( GPI ) anchored proteins
The inability of PNH patients to synthesize the GPI anchor is related to a somatic mutation of the PIG-A gene located on chromosome X at Xp.22 (5,9). The biochemical defect resulting from this mutation has been localized to an early step in the glycosyl phosphatidylinositol (GPI) anchor biosynthetic pathway, transfer of acetylglucosamine to the phosphoinositol molecule (12,8,10). About 15 membrane proteins have been found to be lacking on the abnormal cells of patients with PNH. A single mutagenic locus gives rise to the PNH phenotype due to a single carriage in males and lyonization ( inactivation of one x ) in females (11). At least 12 or more genes are involved in the biosynthesis of the GPI anchor and Mutation of any one of them could result in GPI anchor deficiency. The large majority of reported PIG-A mutations are single base substitutions, deletions, or insertions (74%) and in about 82% of cases only 1 or 2 bases are involved in mutations (1). These mutations lead to a frameshift of the coding sequence in the majority of cases (57%). The clinical manifestations of PNH are inherently related to the dominance of hematopoiesis by PIG-A mutant clone. Whether the mutations of the PIG-A gene are spontaneous or induced is unknown but their presence has profound effects on the function of the abnormal cells (6,5).
Complications
1- Relative and absolute bone marrow failure
All patients with PNH have some degree of diminished hematopoiesis and aplastic anemia is the most severe manifestation of bone marrow failure in PNH. The cause of the bone marrow failure is not clear but may be related to the missing GPI-anchored protein on the abnormal stem cell or on the monocytes and lymphocytes that may control hematopoiesis. Another possibility is that the abnormal cells can not grow in a normal bone marrow and PNH develops when the bone marrow is suppressed perhaps by an immunologic process that affects the normal cells leading to an aplastic process (5). Others argue that PNH clones have a growth and survival advantage relative to normal hematopoiesis and that normal hematopoiesis is depressed in PNH patients (12). PNH has been found to be associated with aplastic anemia (3,8). Some studies have shown that 15 - 30% of patients undergoing treatment with anti-thymocyte for aplastic anemia have PNH. PNH developing in the clinical spectrum of aplastic anemia may be transient (3). The clinical course of these patients have been noted to be more indolent than the patient who develop PNH de novo (13). In patients with aplastic anemia the development of PNH usually occurs late in the disease process. About 2/3 of patients with PNH develop granulocytopenia or thrombocytopenia at some point during the course of their disease. Paroxysmal nocturnal hemoglobinuria most commonly becomes acute myelogenous leukemia in those patients that develop leukemia. This usually occurs 5 years after the first symptoms of PNH. Hemolysis associated with blood transfusion is least common when washed red blood cells are transfused (3). The mechanism leading to the appearance of PNH spontaneously in aplastic anemia during the recovery phase treated aplastic anemia is unknown (13). Patients with aplastic anemia who develop PNH tend to have a worse clinical response to immunosuppressive therapy (8).
2- Thrombotic events
Several theories exist to explain the thrombotic tendencies in PNH. One proposes that the loss of CD59 on platelets results in an increase formation of C9 complexes / vesicles in response to the activation of a given amount of complement, with a resultant increased generation of thrombin (11). Another theory explains that the lack of the receptor of the urokinase type plasminogen activator (u-par) on blood monocytes and granulocytes lead to greater clot stability thus promoting the vascular thrombotic events described in PNH patients (11).
The risk of thrombosis on PNH patients is estimated at about 20% and is the major cause of death. The incidence of thrombotic events is less in Asian and Caucasian populations for reasons that are not known (1). Hepatic vein thrombosis often developes during a hemolytic crisis and patients presents with acute severe right upper quadrant pain, jaundice, hepatomegaly, and ascites. This process, once begun, tends to persist, with periodic exacerbations and remissions and is usually ultimately fatal. Since this is associated with hemolytic crisis it is believed that complement activation is responsible for this complication (5,8 ). Thrombosis of other intra-abdominal veins such as inferior vena cava, portal vein, and splenic vein is also common (3). Abdominal vein thrombosis of the small and large veins may result in severe abdominal pain which usually lasts up to 5 days. The thrombosed areas are difficult to diagnose radiologically and may progress to intestinal infarction requiring resection. Microvascular thrombosis is another presentation with severe abdominal pain and increase d-dimer and crossed linked fibrinogen during these attacks. In pregnant patients it tend to occur in the last stages. Diagnosis of thrombosis of larger veins of the abdomen is by CT scan, MRI or ultrasound with doppler flow measurement (3). Cerebral vein thrombosis of the sagittal sinus and veins covering the parietal lobes are particularly prone to thrombosis in patients with PNH. Magnetic resonance imaging or doppler flow ultrasound may be required to demonstrate the presence of the thrombosed veins. Thrombosis of cerebral veins tend to be chronic and is negative prognostic factor. Dermal vein thrombosis may present as painful, swollen, discolored lesions, usually measuring 5 to 10 cm, over various parts of the body. Thrombosis of the veins of the lower extremity occurs with greater frequency than in the general population, but death by pulmonary embolism is rare (5).
3- Infections
The rate of infection in PNH patients is disproportionately high and may occur secondary to leukocyte dysfunction (11). There is little evidence of immunologic deficiency. Abnormalities of the granulocytes (e.g., absence of IgG receptor ) and monocytes may impose some predisposition to infection (3).
Course and Prognosis
The clinical outcome in PNH is extremely variable. Estimates of mean life span from the time of diagnosis have varied from 8 to 10 years to 15 years or more (5,1,3,14). The most common causes of death are the consequences of thrombosis or the effects of hypoproliferation of the bone marrow, infections, and bleeding (5). Adverse prognostic factors may include thrombosis (since there is a tendency for recurrence), transformation to pancytopenia or myelodysplastic syndrome or acute leukemia. Age greater than 55 is another negative prognostic factor (3).
Three to five percent of patients progress to leukemia usually of myeloid origin. It usually presents 5 years after onset of symptoms of PNH. As the number of leukemic cells increase the abnormal red cells disappear but will reappear if the leukemia is successfully treated (5).The prognosis of paroxysmal nocturnal hemoglobinuria that developes in the setting of acute leukemia, myelodysplasia and myeloproliferative diseases is that of the underlying condition and not PNH (6). In PNH 10-20% of patients may undergo spontaneous remission after a protracted course (14,15).
Unusual presentations
Dysphagia during episodes of hemolysis have been described. It is theorized that this results from hemoglobin induced reduction in nitric oxide leading to excessive contraction of the esophageal musculature. Esophageal manometry during these complaints has shown peristaltic waves of great intensity (3). Impotence in men can become permanent even after the episode of hemoglobinuria resolves (1). The reason is not completely understood but may be related to lack of nitric oxide (3). Fatigue is another complaint that may not be directly related to the amount of hemoglobin. Low back pain of unclear etiology has been theorized to be caused by undetectable thrombosis in the muscles (2).
Treatment
The hemolytic anemia is often treated with steroids and by transfusion with washed normal erythrocytes. Only bone marrow transplantation may replace the affected clone with a normal clone and consequently abolish the manifestations (2,14,16). Bone marrow transplant (BMT) is recommended for PNH patients with severe hypoplasia of bone marrow. In a study involving 48 patients who underwent HLA identical sibling transplant the 2 year survival was reported as 56% (14).
Treatment modalities depends on the symptoms. For correction of anemia one should consider interrupting complement activation, replace missing nutrients, transfusion, prevent and treat thrombosis, modify the bone marrow by transplantation or stimulation. Correction of anemia- Glucocorticoids at present are the only medications that prevent activation of complement thus preventing hemolysis and anemia. The dose is relatively large 0.3 to 0.5 mg/kg/day and may be increased to 1 mg/kg/day during an acute episode of hemoglobinuria. Sixty percent of patients with hemolysis respond to this treatment (5).
Most patients require iron supplementation because they lose it as hemosiderin and hemoglobin in the urine (5). Iron loss through the urine is significant even when hemoglobinuria is not obvious. Patients not undergoing blood transfusion may need iron supplementation but precaution should be taken initially since iron supplementation can lead to hemoglobinuria due to increase production of reticulocyte (3). Blood transfusion may result in the lysis of a small number of the patient’s own cells and result in hemoglobinuria. This can be prevented by washing donor cells before transfusion. Thrombosis should be treated with thrombolytic agents such as T-pa, streptokinase, urokinase and they should be administered immediately unless otherwise contraindicated. It may be used even days later. Once safe start heparin in the usual fashion
as in any other thrombotic event and then anticoagulate with warfarin for six or more months (5). Caution is warranted in patients with cerebral vein thrombosis as it can evolve into a hemorrhagic event. Long term anticoagulant, unfortunately, does not guarantee recurrence of thrombosis (3). Modification of hematopoiesis is yet another method that can be employed to treat PNH. Paroxysmal nocturnal hemoglobinuria can be cured with replacement of the abnormal cells of the bone marrow with normal bone marrow. This may be considered in children in whom the prognosis of the disease is known to be relatively poor (5,3).The relative infrequency of this condition and experience with bone marrow transplantation has made it difficult to make specific recommendations as to when to proceed with bone marrow transplantation. Factors to consider in making the decision should include the recipient's age, performance status, type of donor available and coexisting infections. Prospective trials should be done to better define when to proceed with this treatment modality (14). In a study involving 16 patients who underwent BMT, the 5 year survival rate for the 16 patients was 58 +/- 13%. An absolute neutrophil count > 1.0 x 10 to the ninth and hemoglobin greater than 9 at transplant were found to be statistically significant for a better outcome (16). Syngeneic transplantation is easier to perform than allogeneic and has been noted to be more successful (5). The first successful application of BMT for Treatment of PNH was in 1973. Out of the 17 initially treated patients 5 relapsed. It was noted that relapse had been due to new PNH clones rather than from persistence of the original PNH clone (10). The most common cause of treatment failure are graft failure and infections (14,16). In one study BMT restored normal bone marrow function in approximately 50% of PNH patients (14).
Androgenic hormones are effective in diminishing the anemia of PNH. Danacrine or danazol have been shown to be effective. Their mechanism of action is not clear but may be related to down regulation of activation of complement or perhaps increase in hematopoiesis (3).
Paroxysmal nocturnal hemoglobinuria is a rare acquired genetic disorder that presents with hemolysis precipitated by infections, surgery and transfusions. We presented a young patient with multiple bouts of exacerbation of this condition all related to infectious processes. Despite her gravid status she responded well to conventional treatment and delivered a heathy premature baby. Unfortunately, treatment of this condition is to relieve the consequences rather than treating the root of the problem.
References
1. Nishimura J, Murakami Y, Kinoshita T. Paroxysmal nocturnal hemoglobinuria: An
acquired genetic disease. Am J Hematol 1999;62:175-182.
2. Nakakuma H. Mechanism of intravascular hemolysis in paroxysmal nocturnal
hemoglobinuria. Am J Hematol 1996;53:22-29.
3. Rosse WF. Paroxysmal nocturnal hemoglobinuria as a molecular disease. Medicine1997;76:63-93.
4. Ware RE, Rosse WF, Hall SE. Immunophenotypic analysis of reticulocytes in
paroxysmal nocturnal hemoglobinuria. Blood 1995;86:1586-1589.
5. Rosse WF. Paroxysmal nocturnal hemoglobinuria.
6. Pramoonjago P, Wanachiwanawin W, Chinprasertsak S, et al. Somatic mutations of PIG-A in Thai patients with paroxysmal nocturnal hemoglobinuria. Blood
1995;86:1736-39.
7. Nakakuma H, Nagakura S, Iwamoto N, et al. Paroxysmal nocturnal hemoglobinuria
clone in bone marrow of patients with pancytopenia. Blood 1995;86:1371-76.
8. Schubert J, Vogt H, Zielinska M, et al. Development of the
glycosylphosphatitylinositol-anchoring defect characteristic for paroxysmal nocturnal hemoglobinuria in patients with aplastic anemia. Blood 1994;83:2323-28.
9. Norris J, Hall S, Ware RE, et al. Glycosyl-phosphatidylinositol anchor synthesis in paroxysmal nocturnal hemoglobinuria: Partial or complete defect in an early step. Blood 1994;83:816-821.
10. Nafa K, Bessler M, Deeg HJ, Luzzatto L. New somatic mutation in the PIG-A gene emerges at relapse of paroxysmal nocturnal hemoglobinuria. Blood 1998;92:3422-27.
11. Doukas MA, Fleming D, Jennings D. Identical twin marrow transplantation for
venous thrombosis in paroxysmal nocturnal hemoglobinuria; long term complete remission as assessed by flow cytometry. Bone marrow transplant 1998;22:717-21.
12. Bessler M, Mason P, Hillmen P, Luzzatto L. Somatic mutations and cellular selection in paroxysmal nocturnal haemoglobinuria. The Lancet 1994;343:951-53.
13. Nagarajan S, Brodsky R, Young N, Medof ME. Genetic defects underlying
paroxysmal nocturnal hemoglobinuria that arises out of aplastic anemia. Blood 1995;86:4656-61.
14. Saso R, Marsh J, Cevreska L, et al. Bone marrow transplants for paroxysmal
nocturnal haemoglobinuria. Br J Haematol 1999;104:392-96.
15. Wallach J:Interpretation of diagnostic tests, p367. New York:Little, Brown and Company, 1996.
16. Bemba M, Guardiola P, Garderet L, et al. Bone marrow transplantation for
paroxysmal nocturnal haemoglobinuria. Br J Haematol 1999;105:366-68.