Hematology

Diagnosis and treatment of paroxysmal nocturnal hemoglobinuria

Diagnosis and treatment of paroxysmal nocturnal hemoglobinuria:

INTRODUCTION — Paroxysmal nocturnal hemoglobinuria (PNH) is a disorder characterized by a defect in the glycosylphosphatidylinositol (GPI) anchor, due to an acquired abnormality in the PIG-A gene. This leads to partial or complete absence of all GPI-linked proteins, particularly CD59 (membrane inhibitor of reactive lysis) and CD55 (decay accelerating factor), resulting in an increased sensitivity of the affected cells to the action of complement [1]. (See “Pathogenesis of paroxysmal nocturnal hemoglobinuria: Absence of the GPI anchor” and “Pathogenesis of paroxysmal nocturnal hemoglobinuria: Missing cell proteins”.)

The clinical manifestations of PNH are primarily related to abnormalities in the hematopoietic system, including hemolytic anemia, a hypercoagulable state, and diminished hematopoiesis. Progression to aplastic anemia, myelodysplasia, and acute leukemia can also occur. In the past, the treatment of PNH had been largely empirical. As with most medical disorders, therapy is largely given to ameliorate the symptoms or complications rather than the cause of the disease. However, with improved understanding of the underlying pathogenesis, more rational therapies are emerging, such as the use of eculizumab. (See ‘Eculizumab’ below.)

The diagnosis and treatment of PNH will be reviewed here [1-4]. The clinical manifestations of this disorder are discussed separately. (See “Clinical manifestations of paroxysmal nocturnal hemoglobinuria”.)

DIAGNOSIS

Clinical presentation of the classic disease — The diagnosis of PNH should be considered in a patient with any of the following manifestations:

  • Evidence of acquired hemolysis, specifically with a negative direct antiglobulin (Coombs) test of the red cells
  • Evidence of intravascular hemolysis (eg, hemoglobinemia, hemoglobinuria, hemosiderinuria, elevation of plasma lactate dehydrogenase levels, reduction in plasma haptoglobin levels)
  • Granulocytopenia and/or thrombocytopenia in the presence of an elevation in the reticulocyte count
  • Venous thrombosis, particularly of the abdominal or cerebral veins (eg, Budd-Chiari syndrome, mesenteric or portal vein thrombosis, thrombosis of cerebral or dermal veins). (See “Overview of the causes of venous thrombosis”, section on ‘Myeloproliferative neoplasms and PNH’.)
  • Aplastic anemia. (See “Aplastic anemia: Pathogenesis; clinical manifestations; and diagnosis”, section on ‘Clonal disorder’.)
  • Myelodysplastic syndrome, refractory anemia variant. (See “Treatment of intermediate-1 or low risk myelodysplastic syndromes”, section on ‘PNH-positive cells’.)
  • Episodic dysphagia or abdominal pain with evidence for intravascular hemolysis. (See “Clinical manifestations of paroxysmal nocturnal hemoglobinuria”.)

In the past, PNH was diagnosed indirectly based upon the sensitivity of PNH red cells to lysis by complement. The sucrose lysis test was used as a screening test and the diagnosis was confirmed by the Ham acid hemolysis test [5-7].

  • In the sucrose lysis test, the patient’s red cells are incubated with fresh serum diluted in an isotonic sucrose solution; complement is activated under these circumstances and hemolysis of the red cells indicates a positive test.
  • In the Ham test, complement is activated by reduction of the pH of fresh serum to 6.4; hemolysis of red cells indicates a positive test.

However, the recognition of a deficiency of GPI-linked proteins in PNH has resulted in the development of flow cytometric methods for diagnosis, which have largely replaced the sucrose lysis and Ham tests [8]. (See ‘Expression of GPI anchored proteins’ below.)

Expression of GPI anchored proteins — At least 20 GPI (glycosyl-phosphatidyl-inosityl)-linked proteins are missing from the surface of PNH hematopoietic cells (see above); the absence of any of them can be used as evidence of PNH. Red cells are most frequently analyzed, using monoclonal antibodies to the GPI-anchored proteins CD55 and CD59 followed by flow cytometry [9-11]. Deficient populations that comprise more than 1 percent of the red cells can be identified by this technique. Using special techniques, ever smaller numbers of cells can be identified, especially in patients with aplastic anemia and myelodysplastic syndromes [12,13]. Such patients are considered to have subclinical PNH [2]. (See “Aplastic anemia: Pathogenesis; clinical manifestations; and diagnosis”, section on ‘Clonal disorder’ and “Treatment of intermediate-1 or low risk myelodysplastic syndromes”, section on ‘PNH-positive cells’.)

The abnormal red cells in PNH are either completely lacking the GPI-linked proteins (PNH III cells) or, less commonly, partially lacking (bearing about 10 percent of normal amounts of the protein, PNH II cells). Some patients have both types of abnormal PNH cells. Granulocytes can also be analyzed for the missing proteins; in addition to antibodies against CD55 and CD59, antibodies against a number of other GPI-linked antigens (CD14, CD16, CD24, CD48, CD52, and C66) may be used. A bacterial channel-forming toxin (proaerolysin) which binds specifically to the GPI anchor may be used with a fluorescence label (called FLAER) [8,13]. This reagent may be more sensitive and more specific than the available monoclonal antibodies mentioned above. A consensus document on the optimal methods of flow cytometry for the diagnosis of PNH has been published [14].

In general, the population of PNH granulocytes detected is larger than the population of PNH red cells [15], and more closely measures the degree to which the abnormal clone has taken over in the bone marrow, since the abnormal red cells are lysed in the circulation while the abnormal granulocytes are not.

To be certain of the diagnosis of PNH, if FLAER is not used, antibodies against two GPI-linked antigens and at least two cell lines should be used to be certain that rare genetic abnormalities of these antigens are not present.

PROGNOSIS — PNH is a chronic disease with significant morbidity and mortality. However, a substantial number of patients live for extended periods, and spontaneous recovery may occur. Three reviews have examined the natural history of patients with this disorder. (See “Clinical manifestations of paroxysmal nocturnal hemoglobinuria”, section on ‘Disease overview’.)

  • In one report of 80 patients, the median survival after the onset of the disease was approximately 10 years, with 28 percent of patients surviving 25 years or more [16]. Twelve of 35 patients who survived for longer than 10 years experienced a spontaneous recovery. Approximately 60 percent of deaths were due to venous thrombosis or bleeding; one or more episodes of venous thrombosis occurred in almost 40 percent of the patients. There were no cases of acute leukemia.
  • A large retrospective study of 220 patients found that the median survival was 14.6 years with Kaplan-Meier survival estimates of 78, 65, and 48 percent at 5, 10, and 15 years after diagnosis, respectively [17]. The eight-year rates of the major complications of PNH (pancytopenia, thrombosis, and myelodysplastic syndrome) were 15, 28, and 5 percent, respectively. Adverse prognostic factors included thrombosis, evolution to pancytopenia, myelodysplastic syndrome, or acute leukemia, and age >55 years at onset of disease. Evidence of deficient hematopoiesis at disease onset, such as aplastic anemia or thrombocytopenia, was a less powerful predictor.
  • A third large retrospective study of American and Japanese patients showed major differences between these two populations [18]. The onset of PNH among Japanese patients was more commonly in the setting of an aplastic or hypoplastic marrow than for the American patients. More strikingly, the prevalence of thrombosis was much greater among the American patients than among Japanese patients (38 versus 6 percent). Among the American patients, a large population of abnormal platelets was a likely indicator of thrombosis. The mean survival time for the Japanese patients was 32.1 years compared with 19.4 years for the American population, although the Kaplan-Meier survival curves were not different for the two groups.

TREATMENT OF ANEMIA

Overview — The treatment of PNH has been markedly changed by the addition of eculizumab to the therapeutic armamentarium. This monoclonal antibody stops the hemolysis characteristic of the disorder, resulting in marked improvement in the anemia in most cases. (See ‘Eculizumab’ below.)

Otherwise, the treatment of anemia among patients with PNH consists of supportive therapy, including the replacement of iron and folic acid, and the administration of red blood cell transfusions if clinically required. In addition, suppression of ongoing red cell destruction and/or the stimulation of hematopoiesis may result from the administration of prednisone and/or androgens.

Immunosuppression with antithymocyte serum may be effective, particularly when signs of hematopoietic deficiency are present (eg, granulocytopenia, thrombocytopenia, reticulocytopenia). Inhibition of the lytic effect of complement by an antibody to C5 has been very successful in the treatment of the symptoms of PNH (eg, fatigue, esophageal spasm, abdominal pain, erectile dysfunction, pulmonary hypertension, renal dysfunction). (See ‘Eculizumab’ below.)

Severe hematopoietic dysfunction leading to marked cytopenias can be successfully treated with hematopoietic cell transplantation (see ‘Hematopoietic cell transplantation’ below), or immunosuppression by cyclosporine and/or antithymocyte globulin.

For transfusion-dependent patients with severe hemolytic anemia and disabling symptoms (eg, fatigue, thromboses, frequent paroxysms of pain, end-organ damage), treatment with eculizumab has been successfully employed. (See ‘Eculizumab’ below.)

Asymptomatic patients, especially those with a PNH clone <10 percent, or those with only mild symptoms should not be treated. PNH clone size should be determined every 6 to 12 months in order to monitor for disease progression prior to the onset of symptoms.

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Iron — Even when gross hemoglobinuria is not observed, the urinary loss of iron in PNH is significant because of hemosiderinuria from ongoing intravascular hemolysis [19]. In this setting, urinary losses may be as much as 10 to 20 times normal, leading to the development of iron deficiency anemia. Even larger amounts of iron are lost by individuals with hemoglobinuria. (See “Approach to the diagnosis of hemolytic anemia in the adult”, section on ‘Intravascular hemolysis’.)

Thus, patients with PNH usually require supplemental iron unless they are receiving frequent red blood cell transfusions. (See “Treatment of anemia due to iron deficiency”.)

The administration of iron occasionally precipitates a hemoglobinuric episode, because of the outpouring of reticulocytes from the marrow. This event can be suppressed with the administration of prednisone or a red blood cell transfusion (see ‘Red blood cell transfusions’ below) [20].

Folic acid — Supplemental folic acid is usually recommended for all patients with chronic hemolytic anemia (eg, sickle cell anemia) because of the expected increased need of the hyperplastic marrow for this cofactor.

This vitamin is almost routinely prescribed, although there are no reports of patients with PNH who have become folic acid deficient if folic acid supplementation had not been prescribed. The reutilization of red blood cell folate that is released into the plasma with hemolysis may result in a lower requirement for folic acid supplementation.

Red blood cell transfusions — As with any anemic patient, those with PNH may be transfused if clinically necessary. However, transfusions may result in transient hemoglobinuria due to hemolysis of the patient’s complement sensitive red cells [21]. The reaction is usually fairly mild, but may be sufficiently severe to cause acute renal failure [22,23].

Transfusion-associated hemolysis in PNH occurs most frequently when whole blood is transfused, is seen less often when packed red cells are transfused, and is seen rarely when washed red cells are employed [24-26]. Removal of white blood cells from the transfused product also lessens the occurrence of this event.

It is thought that this reaction results from minor immunologic incompatibilities that activate complement on the abnormal PNH cells. Such reactions are now sufficiently rare as to permit the use of the usual preparation of packed red cells without washing in most cases, particularly if the cells have been leukocyte depleted.

Glucocorticoids and ACTH — The use of ACTH has been abandoned because of toxicity resulting from the relatively high doses required to provide any possible benefit [27]. In addition, the overall beneficial effect of prednisone is a subject of disagreement among experts [2,28,29]. Anecdotally, prednisone has been most effective in patients with anemia due to hemolysis without alterations in hematopoiesis. Prednisone may act by diminishing complement activation, thereby halting hemolysis.

The effective dose of prednisone is generally higher than can be easily tolerated if given on a daily basis. As a result, moderate doses (15 to 30 mg) are usually administered on alternate days. During marked hemolytic episodes, however, higher doses given daily may help allay the paroxysm. The effect of prednisone can be rapid. As an example, in patients with nightly hemolysis, a dose of prednisone will suppress the hemolysis for that night, particularly if given in the evening.

Androgenic hormones — Androgenic hormones are effective in diminishing the anemia of PNH in some cases [30]. Derivatized androgens (such as danocrine or danazol) with less masculinizing effect are also effective. The mechanism by which these agents work is not entirely clear. They may increase hematopoiesis or downregulate the activation of complement.Eculizumab — Eculizumab is a humanized monoclonal antibody that binds to the C5 component of complement and inhibits terminal complement activation [31]. This agent reduced hemolysis and transfusion requirements in an open-label pilot study of 11 transfusion-dependent patients with PNH [32,33]. Following this pilot study, eculizumab was compared with placebo in a double-blind, randomized, multicenter phase III trial (the TRIUMPH study) in 87 patients with PNH who had received at least 4 red cell transfusions during the previous 12 months [34]. Eculizumab (600 mg) or placebo was given by infusion every week for four weeks, followed one week later by 900 mg given every two weeks through week 26.

The following significant benefits were seen in eculizumab-treated patients [34]:

  • A higher rate of stabilization of hemoglobin levels in the absence of transfusion (49 versus zero percent with placebo).
  • Fewer packed red cell transfusion (median of zero versus 10 units with placebo).
  • Less intravascular hemolysis, as shown by an 86 percent decrease in the median area under the curve for serum lactate dehydrogenase concentrations plotted against time.
  • An improvement in the quality of life, including improvement in fatigue and the symptoms of nitric oxide (NO) depletion (eg, esophageal spasm, abdominal pain, erectile dysfunction, renal dysfunction, and pulmonary hypertension) [35].

There were no treatment-related serious adverse events. A follow-up one-year phase III study (the SHEPHERD study) in 97 patients receiving long-term treatment with eculizumab showed similar significant reductions in hemolysis (87 percent), decreases in the degree of anemia and transfusion requirement (52 percent), increased transfusion independence (51 percent), and improvement in fatigue and quality of life [36,37]. In all PNH studies using eculizumab to date, there has been a marked reduction in thromboembolic event rates while taking this medication, as compared with rates prior to treatment [38,39]. Comparable reductions were seen in patients who were or were not taking antiplatelet agents or anticoagulants.

The reduction in intravascular hemolysis in eculizumab-treated subjects resulted in a significant increase in PNH type III red cells (ie, red cells with a complete absence of GPI-linked proteins) from a mean of 28 percent at baseline to 57 percent at week 26 and week 52, compared with 34 to 36 percent at these time points with placebo. These cells completely lack GPI-linked proteins and might be prone to hemolysis if eculizumab were discontinued. However, 19 patients reported to date have discontinued treatment with this agent, which was not associated with serious hemolysis [36]. (See “Pathogenesis of paroxysmal nocturnal hemoglobinuria: Missing cell proteins”, section on ‘Erythrocytes’.)

By contrast, the median proportions of PNH granulocytes did not change during the course of the study (96 percent at baseline and 97 percent at 52 weeks) [36].

In a preliminary report, eculizumab was found to be equally effective in patients with PNH and a prior history of myelodysplastic syndrome or aplastic anemia [40]. Although experience is severely limited, eculizumab appears to be safe when used during pregnancy [41,42].

The United States FDA has approved the use of eculizumab (Soliris™, Alexion Pharmaceuticals, Inc.) for the reduction of hemolysis in patients with PNH. The cost of treatment with this medication has been estimated to be approximately 400,000 US dollars per year [31]. Since this agent has no effect on the underlying cellular abnormality in PNH, treatment, once started, may require prolonged administration.

Other drugs that control complement and its activation are under study [43].

Extravascular hemolysis after eculizumab — While dramatic decreases in intravascular hemolysis have been noted following treatment with eculizumab, many patients still have persistent anemia, reticulocytosis, positive direct antiglobulin tests (ie, positivity for C3d), and biochemical evidence of hemolysis [43,44]. That this persistence of hemolysis might be due to immune-mediated extravascular hemolysis was shown in one study in 41 eculizumab-treated subjects, in whom a substantial fraction of red cells had C3 bound on their surface [45]. C3 binding was entirely restricted to red cells with the PNH phenotype (ie, CD59 negative) and correlated directly with the reticulocyte count and indirectly with the hematologic response to eculizumab. In three of these patients, Cr-51 labeling of red cells demonstrated reduced in vivo survival, with excess tracer uptake in spleen and liver, consistent with extravascular hemolysis. This accumulation of C3 on the affected red cells is due to the fact that these cells lack CD55, a cell-bound inhibitor of C3 activation. The prolongation of circulating life span by eculizumab allows the accumulation to occur.

In most cases, this phenomenon has relatively little clinical relevance and requires no additional therapy [46]. However, in single case reports in which hemolysis worsened in conjunction with a positive direct antiglobulin (Coombs) test, or in which transfusions were still required, addition of glucocorticoids (initial dose 75 mg/day) [47], or performance of splenectomy [48], reduced the degree of hemolysis. The use of a monoclonal antibody specific for the C3/C5 convertase may ultimately be of value in patients in whom this phenomenon becomes clinically important [43].

Neisserial infections — All patients enrolled in the TRIUMPH study discussed above were vaccinated against N. meningitidis, due to the observation that persons with a genetic deficiency of terminal complement proteins have an increased risk of neisserial infections, including N. meningitidis and N. gonorrhoeae. In all clinical studies of eculizumab, meningococcal sepsis developed in 2 of 196 patients (1.0 percent) receiving the drug. Both had previously received meningococcal vaccination.

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Accordingly, patients should be told that there is a 0.5 percent/year risk of neisserial sepsis even after vaccination; they should be re-vaccinated every three to five years after starting treatment with eculizumab, and should seek immediate medical attention if they develop signs and symptoms of such infection [49]. (See “Inherited disorders of the complement system” and “Epidemiology of Neisseria meningitidis infection”, section on ‘Complement deficiency and meningococcemia’.)

Prescribing information for this agent includes a boxed warning describing the risk for serious meningococcal infection and the need for meningococcal vaccination at last two weeks prior to treatment and revaccinations according to current medical guidelines [49,50].

Other agents — Some patients with diminished erythropoiesis derive benefit from high doses of erythropoietin (40,000 units/week) or darbepoetin, particularly if renal impairment is present [51]. Recombinant granulocyte stimulating factor (G-CSF) has been used to increase the granulocyte count in granulocytopenic patients [52].

Recombinant modified CD59 (Prodaptin) capable of binding to the red cell membrane has been shown to reduce complement-mediated red cell lysis in vivo in a mouse model [53]. This approach for the potential treatment of PNH has not yet been tested in human subjects.

TREATMENT OF THROMBOSIS — Acute thrombosis in PNH is treated similarly to venous thrombosis occurring in other settings [54]. However, prednisone is also used, since complement activation probably initiates thrombosis in patients with PNH. The role of eculizumab in the treatment of acute thrombosis is under consideration. (See “Treatment of acute pulmonary embolism” and “Treatment of lower extremity deep vein thrombosis”.)

Thrombolysis — Thrombolysis should be instituted in the patient with PNH and large vein or life-threatening thrombosis if the clot is less than three to four days old and in whom there are no contraindications [4,55,56]. In patients who develop Budd-Chiari syndrome, such therapy may decrease the probability of long-term complications. However, fibrinolysis may be dangerous in those with cerebral vein thrombosis, since it may convert a thrombotic stroke into a hemorrhagic one. (See “Clinical manifestations, diagnosis, and treatment of the Budd-Chiari syndrome”.)

Anticoagulation — Anticoagulation for an acute episode of thrombosis should be instituted with heparin. Heparin appears to be effective when given in full therapeutic doses [54]. (See “Therapeutic use of heparin and low molecular weight heparin”.)

Any patient with a significant thrombosis, particularly intra-abdominal or intracerebral, should be treated with eculizumab. Most patients who have had significant thrombosis also receive continuing anticoagulation following the acute event, using agents such as warfarin or heparin/low molecular weight (LMW) heparin. For the first episode, they are generally managed as other patients with a similar thrombotic event. (See “Treatment of lower extremity deep vein thrombosis”, section on ‘Summary and recommendations’.)

Patients with recurrent episodes are managed as other patients with a clinically manifested hypercoagulable state, using LMW heparin or warfarin for an indefinite period. In the absence of concomitant treatment with eculizumab, antithrombotic therapy alone had not been completely effective in preventing further episodes of thrombosis. In a preliminary report of three patients with prior episodes of recurrent thrombosis, treatment with eculizumab was effective in eliminating subsequent episodes of thrombosis even after treatment with anticoagulants had been discontinued [57]. (See ‘Eculizumab’ above.)

Prophylactic anticoagulation — Thrombosis in PNH appears to be related to the size of the abnormal platelet clone; those patients with large numbers of abnormal platelets are at greater risk. In one study, the 10-year risk of thrombosis was 44 percent in those with large PNH clones (ie, PNH granulocytes >50 percent of the total), and was 5.8 percent in those with small clones [58]. Accordingly, in one medical center, patients with large PNH clones and no contraindication to anticoagulation were offered warfarin prophylaxis (target INR 2.0 to 3.0). At a median follow-up of six years, the following observations were made [58]:

  • There were no thrombotic episodes in the 39 patients who received primary prophylaxis.
  • The 56 patients not taking warfarin had a 10-year thrombosis rate of 36.5 percent.
  • There were only two serious bleeding episodes in more than 100 patient-years of warfarin treatment.

Although these retrospective observations need to be confirmed, they suggest that warfarin prophylaxis is effective in patients with PNH if the granulocyte clone size is >50 percent, the platelet count is >100,000/microL, and there are no contraindications to anticoagulation [59]. (See “Therapeutic use of warfarin”.)

Prophylaxis with heparin (7500 to 10,000 units twice a day) or LMW heparin should be instituted in any perioperative period, during immobilization, or when an indwelling intravenous catheter is used. Such prophylaxis should also be started in the first trimester of pregnancy and continued until four to six weeks post-partum. (See “Anticoagulation during pregnancy”, section on ‘Management of specific conditions requiring anticoagulation’.) The role of eculizumab in these settings is under consideration.

Prednisone — Since activation of complement may be involved in thrombotic episodes, moderate to high doses of prednisone (0.5 to 1 mg/kg per day) can be given, although the effectiveness of this agent has been questioned. (See ‘Glucocorticoids and ACTH’ above.)

HEMATOPOIETIC CELL TRANSPLANTATION — Identical twin (syngeneic) and HLA identical sibling hematopoietic cell transplantation (HCT) have been used successfully in selected patients with PNH [60-64]. Marrow ablative conditioning regimens are commonly administered to eliminate the PNH clone [60,62,63]. In one syngeneic transplant, the absence of conditioning was associated with gradual loss of the transplanted cells and symptom recurrence at 17 months, suggesting a survival advantage for the PNH clone [65]. Matched unrelated HCT has also been successfully used in some patients. Complications with this procedure appear to be no more frequent in PNH than in other disorders [61-63].

The largest reported series, from the International Bone Marrow Transplant Registry, described the outcome of HCT in 57 patients with PNH [63]. Forty-eight of the donors were HLA-identical siblings, two were identical twins, one was a phenotypically HLA-identical parent, and six were unrelated HLA-matched donors. The median age at transplant was 28 years (range 10 to 47 years) and the median interval from the time of diagnosis to transplant was 26 months (range 2 to 240 months). Severe aplastic anemia was present pretransplant in 32 percent. The following results were noted [63]:

  • Sustained engraftment occurred in 77 percent of the evaluable patients; acute (≥ grade II) and chronic graft versus host disease were present in 34 and 33 percent, respectively.
  • Overall survival following HLA-matched sibling transplantation was 56 percent at a median follow-up of 44 months. Of the seven patients receiving HCT from a parent or a matched unrelated donor, only one was alive at five years.
  • Two patients received identical twin transplants, one with prior conditioning, resulting in engraftment and complete recovery at more than eight years. The one who received no prior conditioning had graft failure; a second transplant preceded by cyclophosphamide and total body irradiation resulted in complete recovery and survival at more than 12 years.

The observation of graft failure or relapse after syngeneic transplants has been observed in other patients [62,65]. Although HCT might be expected to cure PNH, one review of 17 patients found that relapse of PNH occurred in all five recipients of syngeneic transplants without conditioning [66]. In one patient, polymerase chain reaction analysis of the PIG-A gene showed that relapse was due to the emergence of a new clone, rather than persistence of the original clone [66]. This finding supports the hypothesis that the bone marrow environment may create selective conditions favoring the emergence of PNH clones.

Indications — Candidates for HCT generally include those with life-threatening disease:

  • Since there is considerable experience with the successful use of HCT for aplastic anemia, HCT is indicated in the patient with PNH, significant degrees of neutropenia and thrombocytopenia, and severe bone marrow hypoplasia, as demonstrated on bone marrow aspiration and biopsy. (See “Aplastic anemia: Prognosis and treatment”.)
  • Severe thrombotic events, particularly involving the hepatic veins, carry such a serious prognosis that consideration for transplantation is warranted in patients eligible for this procedure (eg, having an HLA matched sibling donor, age less than 45 to 50 years). By reversing the thrombotic tendency, HCT can lead to reversal of the Budd-Chiari syndrome [60]. The use of eculizumab may effectively prevent further thrombosis, thus eliminating thrombosis as a reason for undergoing HCT; further follow-up on this question is needed.
  • Transplantation early in the course of the disease has been suggested for children, since transplantation is better tolerated in children than in adults and the long-term prognosis of children with PNH is somewhat limited [67,68]. In one report of 26 PNH patients ≤21 years of age, only 4 (15 percent) survived more than 15 years after diagnosis [68]. As noted above, the use of eculizumab may alter the need for such HCT.
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In many patients, however, clear indications for HCT are not present. Although many of these patients do well over a long period of time, PNH is associated with appreciable morbidity and mortality. The potential benefits of HCT in this setting must be balanced against the untoward consequences of this procedure, such as its 15 percent mortality rate. (See ‘Prognosis’ above.)

Since peripheral blood progenitor cells can be harvested by leukocytapheresis, some investigators have suggested that the “normal” GPI-positive stem cell population can be separated and reinfused. If this approach were to be successful, it would probably be necessary to “condition” the marrow by chemotherapy and/or radiation, since GPI-positive cells do not grow well in the marrow environment. This approach has not yet been attempted.

However, autologous transplantation is unlikely to be successful because of the difficulty in obtaining sufficient numbers of normal stem cells. In one study, the majority of the most primitive peripheral blood stem cells were of normal phenotype in patients with PNH; however, the cells that were mobilized into the peripheral blood with G-CSF were phenotypically similar to mature neutrophils, ie, mainly PNH cells [69]. (See “Sources of hematopoietic stem cells”.)

TREATMENT OF APLASIA OTHER THAN TRANSPLANTATION

Cyclosporine — In a small number of patients, cyclosporine has been tried as an alternative to HCT for aplastic anemia [70,71]. The rationale for the use of cyclosporine is the hypothesis that immune-mediated bone marrow damage in PNH is primarily directed against the normal GPI-positive cells, producing a growth advantage for the GPI-negative PNH cells. One report of three such patients noted a complete response in two after 6 and 24 months, and a partial response in the third after 12 months [70]. The PNH clone and hemolysis persisted, suggesting that cyclosporine acted via immune mechanisms and not on the PNH cells. The disease relapsed in two patients in whom cyclosporine was withdrawn.

The role of cyclosporine for marrow aplasia in PNH remains to be determined in a larger number of patients. Cyclosporine does not appear to be beneficial in patients without aplasia in whom the hematologic and PNH characteristics are not changed [71].

Antithymocyte globulin — In one series, seven patients with PNH and aplastic anemia received antithymocyte globulin (ATG, 20 mg/kg per day) for eight days along with prednisone to prevent or control serum sickness [72]. Three patients experienced a sustained improvement in at least one peripheral blood cytopenia, including one patient who had a complete trilineage response. These patients had hypoproliferative features and low-grade hemolysis which persisted at the same rate after therapy, whereas the degree of hemolysis was more pronounced in the nonresponders.

In one series of 100 patients with aplastic anemia, the response to treatment with ATG in patients with normal and GPI-anchored protein-deficient granulocytes was 71 percent and 82 percent, respectively [73]. No statistically significant evidence of expansion in the size of the PNH clone was seen during long-term follow-up of these patients.

These preliminary observations suggest that impaired hematopoiesis in PNH may respond to cyclosporine or ATG treatment. However, the hemolytic component of the disease, and therefore the PNH clone, is not affected.

FUTURE DIRECTIONS — The defective PIG-A gene in PNH has been cloned. It is therefore theoretically possible to correct the defect in the abnormal stem cells using gene therapy [74,75]. (See “Pathogenesis of paroxysmal nocturnal hemoglobinuria: Absence of the GPI anchor”.)

At present, the mechanism that enables the PNH clone to expand in the bone marrow is not understood since PIG-A inactivation alone does not confer a proliferative advantage to the hematopoietic stem cell. In view of the relationship between PNH and aplastic anemia, it may be that the cause of the failure of normal hematopoiesis in aplastic anemia enables the PNH clone to proliferate. Correcting the PNH cell defect may allow exposure to the insult causing bone marrow failure [76]. For these reasons, a better understanding of the problems of hematopoiesis will be required prior to such an attempt.

Protein transfer — Cell-to-cell transfer of GPI proteins has been demonstrated in a variety of systems. Transfer of GPI-linked proteins has been shown to be feasible using high density lipoproteins or washed RBC microvesicles [77], or an alternative artificial glycolipid anchor (Prodaptin) [53]. The PNH red cells exposed to these soluble preparations manifested both increased cell-associated CD55 and CD59 levels and decreased in vitro complement-mediated hemolysis. The advantage of these preparations over red blood cell transfusions is that they can be sterilized and do not result in iron overload.

SUMMARY AND RECOMMENDATIONS

Suspecting the diagnosis — The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) should be suspected in patients with one or more of the following presentations. (See ‘Clinical presentation of the classic disease’ above.)

  • Coombs negative acquired hemolytic anemia
  • Intravascular hemolysis with or without hemoglobinuria
  • Granulocytopenia and/or thrombocytopenia in the presence of an elevated reticulocyte count
  • Aplastic anemia
  • Myelodysplastic syndrome (refractory anemia and hypoplastic variants)
  • Episodic dysphagia or abdominal pain
  • Venous thrombosis along with evidence of hemolysis, particularly thrombosis of the abdominal or cerebral veins (eg, Budd-Chiari syndrome, mesenteric or portal vein thrombosis, thrombosis of cerebral or dermal veins)

Laboratory testing — The following laboratory tests are helpful in assessing the patient with a presumptive diagnosis of PNH:

  • Complete blood count with white blood cell differential, platelet count, red cell indices (ie, MCV, MCH, MCHC), and reticulocyte count
  • For anemic patients — direct antiglobulin (Coombs) test, serum lactate dehydrogenase (LDH), direct and indirect bilirubin, haptoglobin, urinalysis for the presence of hemoglobin and hemosiderin, serum iron, total iron binding capacity, ferritin
  • Bone marrow aspiration and biopsy for subjects with pancytopenia, granulocytopenia, thrombocytopenia, or reticulocytopenia, in order to assess for the presence of bone marrow hypoplasia or aplasia. (See ‘Hematopoietic cell transplantation’ above and ‘Treatment of aplasia other than transplantation’ above.)

Making the diagnosis — To be certain of the diagnosis of PNH, the absence or near-absence of two glycosyl-phosphatidyl-inosityl (GPI)-linked antigens (eg, CD55, CD59) in at least two cell lines (eg, red cells, granulocytes), or the absence of the GPI anchor by the FLAER technique, should be demonstrated. This is performed using flow cytometric methods. (See ‘Expression of GPI anchored proteins’ above.)

Treatment — The only potentially curative treatment for PNH is allogeneic hematopoietic cell transplantation. All other treatments are supportive and are directed against the major manifestations of the disease.

  • Asymptomatic patients, especially those with a PNH clone <10 percent, or those with only mild symptoms should not be treated. PNH clone size should be determined every 6 to 12 months in order to monitor for disease progression.
  • Because hemoglobinuria is a prominent complication of PNH, all patients with PNH should be evaluated for the presence of iron deficiency. If found, iron deficiency should be treated with an oral iron preparation. (See ‘Iron’ above and “Treatment of anemia due to iron deficiency”, section on ‘Oral iron therapy’.)
  • As a general precaution, oral folic acid supplementation (1 mg/day) should be given prophylactically to those with hemolytic anemia in order to avoid the development of folic acid deficiency. (See ‘Folic acid’ above.)
  • For patients with symptomatic anemia due to PNH, supportive treatment with periodic blood transfusions or the use of erythropoiesis-stimulating agents, androgens, and/or glucocorticoids have all been helpful in alleviating the signs and symptoms of anemia. There are no randomized studies that give guidance on the selection of an appropriate agent for this complication of PNH. (See ‘Treatment of anemia’ above.)
  • For selected transfusion-dependent patients or patients with disabling symptoms (eg, fatigue, thromboses, frequent paroxysms of pain, end-organ damage), we suggest treatment with eculizumab (Grade 2A). Any patient with a history of thrombosis of abdominal or cerebral veins should be treated with eculizumab; the necessity of additional anticoagulation therapy has not been established. Enthusiasm for this agent is reduced because of its expense, potential need for indefinite treatment, and increased risk for Neisserial infection. (See ‘Eculizumab’ above.)
  • The prophylaxis and treatment of thrombosis in PNH is not well established. Although both warfarin and heparin are useful, particularly acutely, their ability to prevent further thrombosis is not complete. Eculizumab is likely to provide such prophylaxis, but this needs to be established by further data.
  • For patients with severe aplastic anemia due to PNH, available choices include allogeneic hematopoietic cell transplantation or immunosuppressive regimens, depending upon patient age and the availability of an HLA-matched donor. These important issues are discussed in depth separately. (See ‘Hematopoietic cell transplantation’ above and ‘Treatment of aplasia other than transplantation’ above and “Hematopoietic cell transplantation in aplastic anemia” and “Aplastic anemia: Prognosis and treatment”.