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Definition

Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired chronic disorder characterized by persistent intravascular haemolysis, subject to recurrent exacerbations, often associated with pancytopenia, and with a distinct tendency to venous thrombosis. The triad of haemolytic anaemia, pancytopenia, and thrombosis makes PNH a truly unique clinical condition: however, even in the absence of one or more of these manifestations a conclusive diagnosis can be made by appropriate laboratory investigations (see below).

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Clinical features

The patient may seek medical attention because, one morning, he or she has ‘passed blood instead of urine’. This distressing or frightening event—the direct evidence of haemoglobinuria—may be regarded as the classical presentation; however, not infrequently the patient presents as a problem in the differential diagnosis of anaemia, whether symptomatic or discovered incidentally. The anaemia may be associated with jaundice (suggesting haemolytic anaemia), and/or with neutropenia, thrombocytopenia, or both. Venous thrombosis may be the first clinical manifestation in other patients. Although any vein may be affected, the most common localization is intra-abdominal: indeed, recurrent attacks of severe abdominal pain defying a specific diagnosis, and sometimes eventually found to be related to thrombosis, have given to PNH the attribute of being a great impostor. On the other hand, when thrombosis affects the hepatic veins it may produce acute hepatomegaly and ascites—i.e. a fully fledged Budd–Chiari syndrome.

The natural history of PNH can extend over decades. Without treatment the median survival is estimated to be about 8 to 10 years (see Fig. 22.3.12.1) but now several forms of treatment are possible in the past the most common causes of death have been thrombosis, or infection associated with severe neutropenia, or haemorrhage associated with severe thrombocytopenia. PNH may evolve to bone marrow aplasia undistinguishable from AAA; and more frequently PNH may manifest itself in patients who previously had AAA. Rarely (estimated 1–2% of all cases), PNH may terminate in acute myeloid leukaemia. On the other hand, full spontaneous recovery from PNH has been also well documented.

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Fig. 22.3.12.1

PNH is a chronic disorder, the time course of which is often measured in decades. From a series of 80 patients who received only minimal supportive treatment we estimate a median survival of about 10 years. Allogeneic bone marrow reansplanation has still been associated with significant mortality, and therefore may have reduced the survival of some patients; but more encouraging results have been reported on a small series from a single centre.

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Laboratory investigations and diagnosis

The most consistent blood finding is anaemia, which may range from mild to moderate to very severe. The anaemia is usuallynormomacrocytic; a high mean cell volume (MCV) is usually largely accounted for by reticulocytosis, which may be quite marked—up to 20%. The anaemia may become microcytic if the patient is allowed to become iron-deficient as a result of chronic urinary blood loss through haemoglobinuria. The red cell morphology is otherwise usually normal. There may be neutropenia and/or thrombocytopenia. Unconjugated bilirubin is mildly or moderately elevated; lactate dehydrogenase (LDH) is typically markedly elevated; haptoglobin is usually undetectable. All these findings make the diagnosis of haemolytic anaemia compelling. Haemoglobinuria may be overt in a random urine sample: if it is not, it may be helpful to obtain serial urine samples, since haemoglobinuria can vary dramatically from day to day, and even from hour to hour (it is more common, but not always, in the early morning: hence the adjective ‘nocturnal’). Obviously, haemoglobinuria must be distinguished from haematuria. Surprisingly, even today a patient may undergo extensive urological investigations before it is realized that the patient has PNH. There may be free haemoglobin in the serum, and sometimes this is so high as to interfere with clinical chemistry. These findings clearly indicate intravascular haemolysis, thus increasing, by an order of magnitude, the likelihood that the haemolytic anaemia is in fact PNH (see Table 22.3.12.1). The bone marrow is usually cellular, with marked to massive erythroid hyperplasia, often with mild to moderate dyserythropoietic features. However, at some stage of the disease the marrow may become hypocellular or even frankly aplastic (see below).

The definitive diagnosis of PNH must be based on the demonstration that a substantial proportion of the patient’s red cells have an increased susceptibility to complement, due to the deficiency on their surface of proteins that normally protect the red cells from activated complement. For decades this has been done reliably b using the acidified serum (Ham–Dacie) test. Nowadays the gold standard is the demonstration of a discrete population of ‘PNH red blood cells’ by flow cytometry, using anti-CD59 or anti-CD48. This analysis is quantitative, and it has a higher sensitivity when applied to granulocytes (see Fig. 22.3.12.2).

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Fig. 22.3.12.2

Flow cytometry analysis of blood cells in a patient with PNH. On the left, red cells and granulocytes from a normal person display a unimodal distribution of surface expression of the GPI-linked protein CD59, which protects red cells against complement-mediated lysis. On the right, a similar analysis reveals, in a patient with PNH, a clearly bimodal distribution: from this analysis the size of the PNH cell population can be quantified.

(Courtesy of Dr David Araten.)

Pathophysiology

Haemolysis

Haemolysis in PNH is due to an intrinsic abnormality of the red cell, which makes it exquisitely sensitive to activated complement, whether it is activated through the alternative pathway or through an antigen–antibody reaction. The former mechanism is probably the reason why there is chronic intravascular haemolysis in PNH. The latter mechanism explains why the haemolysis can be dramatically exacerbated in the course of a viral or bacterial infection. Hypersusceptibility to complement is due to the deficiency of several protective membrane proteins, of which CD59 is the most important, because it hinders the insertion into the membrane of C9 polymers.

The molecular basis for the deficiency of these proteins has been pinpointed not to a defect in any of the respective genes, but rather to the shortage of a unique glycolipid molecule, glycosyl phosphatidyl inositol (GPI), which, through a peptide bond, anchors these proteins to the surface membrane of cells. The shortage of GPI is due in turn to a mutation in an X-linked gene, called PIGA, required for an early step in GPI biosynthesis. In virtually each patient the PIGA mutation is different. This is not surprising, since these mutations are not inherited: rather, each one takes place de novo in a haemopoietic stem cell (in other words, they are somatic mutations). As a result, the patient’s bone marrow is a mosaic of mutant and nonmutant cells, and the peripheral blood always contains both GPI-negative PNH cells and GPI-positive cells (see Fig. 22.3.12.2).

Thrombosis

This is one of the most immediately life-threatening complications of PNH, and yet one of the least understood pathogenetically. It could be due to impaired fibrinolysis, because the urokinase plasminogen Activator Receptor (uPAR) is a GPI-linked protein; more likely, complement activation could cause hypercoagulability, or hyperactivity of platelets, or both.

Bone marrow failure and the relationship between PNH and AAA

PNH has an intimate link with AAA, which manifests in several ways. (1) As stated above, sometimes a patient with PNH becomes ‘less haemolytic’ and ‘more pancytopenic’ and ultimately evolves to frank AAA. (2) In terms of pathogenesis, AAA is regarded as an organ-specific autoimmune disease mediated by ‘activated’ cytotoxic (CD8+) T lymphocytes, which are able to inhibit haemopoietic stem cells. Recently, skewing of the T-cell repertoire, indicating the presence of abnormally expanded T-cell clones, has also been observed in patients with PNH. (3) Most important, intensive immunosuppressive treatment is the standard of care in those with AAA, and a beneficial response to the same treatment can be seen also in patients with PNH (see below).

Thus, it seems that an element of bone marrow failure in PNH is the rule rather than the exception: an extreme view is that PNH is a form of AAA, in which bone marrow failure is masked by the enormous expansion of the PNH clone that populates the patient’s bone marrow. In other words, it appears that two different mechanisms co-operate in producing PNH (see Fig. 22.3.12.3): autoimmune damage to stem cells, and a somatic mutation in the PIGA gene. This notion is supported by two further lines of evidence. (1) By targeted inactivation of the Piga gene in mouse embryonic stem cells one can produce mice with a PNH cell population. However, this population does not grow further, as it does in patients with PNH. (2) By using refined flow cytometry technology, PNH cells harbouring PIGA mutations can be demonstrated in normal people at a frequency in the order of 10 per million. Both these findings indicate that some other factor is required, in addition to a somatic mutation in the PIGA gene, in order to cause PNH. Most likely, the same cytotoxic damage to stem cells that would otherwise cause AAA spares the PNH stem cells, thus allowing the PNH clone to grow to the size when it gives clinical PNH. The mechanism whereby the PNH cells escape damage is not yet known.

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Fig. 22.3.12.3

The role of somatic mutation and bone marrow failure in causing PNH. This diagram aims to emphasize that two separate factors are required to bring about PNH as a clinical disease. On the one hand, a PIGA mutation on its own will produce a PNH clone, but there will be no basis for it to expand; on the other hand, damage to haemopoietic stem cells (HSC) can cause aplastic anaemia without PNH. When both factors co-operate, and if the damage to HSC is GPI-mediated, then there will be selective expansion of the PNH clone.

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Complications

The most important complication is thrombosis, which is nearly always venous, and can be life-threatening especially if it affects either the abdominal veins (see Fig. 22.3.12.4) or the intracranial veins. The Budd–Chiari syndrome has already been mentioned: because of its characteristic clinical picture it is usually easy to recognize. However, in PNH it is sometimes associated with portal vein thrombosis, and this may limit the extent of liver enlargement. Thrombosis of the splenic vein should be suspected whenever a patient with PNH has, or develops, splenomegaly. Thrombosis of one of the mesenteric veins is much more difficult to diagnose clinically. Appropriate investigations include Doppler ultrasonography, contrast-enhanced CT, and MRI: in our experience, the most sensitive methodology is magnetic resonance (MR) venography. Recognizing venous thrombosis is of great practical importance, because thrombolytic therapy with tissue plasminogen activator (Fig. 22.3.12.4) has been carried out successfully even after 6 weeks from the onset of signs and symptoms.

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Fig. 22.3.12.4

Abdominal vein thrombosis in PNH can resolve with thrombolytic therapy. (a) Extensive thrombus in the inferior vena cava in a patient with known PNH who had developed Budd–Chiari syndrome a few days earlier: it is not infrequent in PNH for thrombosis to involve multiple veins in the abdomen all at once. (b) A thrombus-free vena cava 2 days after an intravenous infusion of tissue plasminogen activator.

(Courtesy of Dr Raymond Thertulien.)

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Treatment

Unlike other acquired haemolytic anaemias, PNH may be lifelong, and this is important in our approach to management. Until recently, there were essentially two options. On one hand, allogeneic bone marrow transplantation is the only form of treatment that can provide a cure for PNH: therefore, it should be offered for consideration to any young patient with PNH for whom a human leucocyte antigen (HLA)-identical sibling is available. Results similar to those for AAA can be expected, with long-term disease-free survival ranging from 60 to 100% in the few series that have been published (see Fig. 22.3.12.1: by contrast, the past record of bone marrow transplantation from unrelated donors in PNH is poor). On the other hand, supportive management supervised by somebody who has previous experience of PNH can help the patient to ‘live with PNH’ for years, sometimes for decades, and sometimes with a good quality of life. The mainstay of support is the transfusion of filtered red cells whenever necessary. Folic acid supplements (≥3 mg/day) are mandatory; the serum iron concentration should be checked periodically and iron supplements added as indicated. There is no evidence that prednisone (which used to be administered at a dose of 15–30 mg on alternate days) decreases the rate of haemolysis, and long-term administration of prednisone, even at a low dosage, is contraindicated, in view of its well known serious potential side effects (a short course of prednisone may sometimes appear helpful in dealing with an episode of massive haemoglobinuria associated with intercurrent infection). Any patient who has had a deep vein thrombosis should be given anticoagulant prophylaxis.

A major advance in the management of PNH has been the introduction of complement blockade by the use of a humanized monoclonal antibody, eculizumab, specific for the C5 component of complement. In an international double-blind placebo-controlled trial carried out on patients with severe haemolytic PNH who were dependent on periodic red cell transfusions, eculizumab has proven effective in controlling intravascular haemolysis: so much so, that haemoglobinuria disappears, about one-half of the patients are no longer transfusion-dependent (see Fig. 22.3.12.5), and in the other half the number of transfusions required is generally decreased. Thus, the quality of life is markedly improved. Why not all patients improve as dramatically as others is currently under investigation. It is already known that in treated patients a proportion of PNH red cells become coated with C3 fragments and these may be susceptible to extravascular haemolysis. Given its mechanism of action, eculizumab is clearly not a curative treatment: its benefits will last as long as the agent is administered, through an intravenous infusion, at fortnightly intervals. Because the distal complement pathway is blocked in patients on eculizumab, they are at an increased risk for infection by meningococcus: thus, immunization against this organism is mandatory before starting eculizumab. In most patients this treatment has been remarkably free of serious side effects: however, there have been a few instances of severe infection which have responded to antibiotics.

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Fig. 22.3.12.5

Eculizumab can abrogate the need for blood transfusion. Patients with severely haemolytic PNH (n = 87) all of whom had needed at least 4 units of blood during the previous year, were randomized to receive either placebo or eculizumab. Within 14 weeks all patients on placebo required blood transfusion; of those receiving eculizumab, 49% remained transfusion-free at 6 months: of these, all those who continued treatment are still transfusion-free some 3 years later.

Eculizumab will have clearly no effect on the bone marrow failure component of PNH. When the manifestations of bone marrow failure predominate, the approach to treating PNH becomes similar to that indicated for AAA: accordingly, a logical option is intensive immunosuppressive treatment with antilymphocyte globulin (ALG) and ciclosporin. Although no formal trial has ever been conducted, this approach has particularly helped to relieve severe thrombocytopenia and/or neutropenia in patients in whom these were the main problem(s): by contrast, there is often little beneficial effect on the haemolysis itself. Thus, the therapeutic effects of ALG and eculizumab are in a sense complementary.

From recent data, it appears that administration of eculizumab, in addition to abrogating intravascular haemolysis, also decreases the risk of thrombosis: this is especially important, since patients with PNH are not fully protected from thrombosis even by painstaking anticoagulant treatment. Thus, it is clear that the availability of eculizumab, though very expensive, will influence significantly therapeutic choices, including bone marrow transplantation.