Paroxysmal Nocturnal Hemoglobinuria

Article written and reviewed by Dr Luca Moretti

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare acquired hematologic disorder characterized by chronic intravascular hemolysis, episodes of hemoglobinuria and a marked predisposition to thrombotic events. In the vast majority of cases, the disease results from a somatic mutation in the PIGA gene in hematopoietic stem cells, causing a defect in synthesis of the glycosylphosphatidylinositol (GPI) anchor required for expression of numerous surface proteins, including the complement-cascade regulators CD55 (DAF) and CD59 (MIRL); rare PIGT-related forms have been described, in which transfer of the preassembled GPI anchor to proteins is defective. Deficiency of these factors makes red blood cells vulnerable to complement-mediated lysis, resulting in chronic hemolysis and multiorgan complications.

PNH may occur as an isolated disorder or in association with other bone marrow diseases, particularly aplastic anemia or myelodysplastic syndromes. Onset may be insidious or acute, and prognosis depends on the severity of hemolysis, thrombotic risk and the presence of bone marrow failure.

Although rare, PNH is a critical diagnosis in hematology because of the high morbidity and mortality associated with its complications, particularly thrombosis, and because innovative treatments have radically changed the natural history of the disease in recent years.

Etiology and Genetics

Paroxysmal nocturnal hemoglobinuria is an acquired clonal disorder caused, in the vast majority of cases, by a somatic mutation in the PIGA gene (phosphatidylinositol glycan class A), located on the X chromosome, in hematopoietic stem cells; rare PIGT-related forms have been described in which a predisposing germline variant is associated with a second somatic event. This mutation causes a defect in GPI-anchor biosynthesis, a structure required to anchor specific surface proteins to cell membranes.

The GPI defect prevents expression of numerous proteins on blood cells, including:

• CD55 (DAF, decay accelerating factor): regulates complement inactivation on cell membranes
• CD59 (MIRL, membrane inhibitor of reactive lysis): inhibits formation of the complement membrane attack complex (MAC)

The absence or marked reduction of these proteins exposes mainly erythrocytes to complement-mediated intravascular lysis; in platelets, complement predominantly promotes activation, whereas in GPI-deficient leukocytes it does not cause generalized peripheral lysis.

PNH develops as an expanded hematopoietic clone derived from the mutated stem cell, which coexists with the normal cell population. The disease may arise de novo or, more often, in association with hematopoietic disorders, particularly aplastic anemia, which provides a selective advantage to PNH cells.

PNH is sporadic and acquired in the vast majority of cases; however, rare PIGT-related forms have been described in which a germline variant predisposes to the disease and a second somatic event inactivates the other allele.

Pathogenesis and Pathophysiology

Understanding the pathophysiology of PNH requires focusing on the role of complement and GPI-anchored proteins in protecting red blood cells and other blood cells. Under normal conditions, CD55 accelerates decay of C3/C5 convertases and limits upstream complement activation, whereas CD59 directly inhibits formation of the membrane attack complex (MAC) by preventing incorporation of C9; together they help protect erythrocytes from complement-mediated lysis.

In PNH, deficiency of these proteins causes:

• Susceptibility of erythrocytes to intravascular lysis: activation of the complement cascade leads to MAC formation and disruption of the erythrocyte membrane, with release of hemoglobin into plasma and urine (hemoglobinuria).
• Effects on platelets and leukocytes: complement activation contributes to platelet activation and thrombophilia; leukocyte abnormalities do not amount to generalized peripheral lysis.

Chronic hemolysis releases free hemoglobin into the circulation, which may bind and consume nitric oxide, resulting in endothelial dysfunction, vasoconstriction and increased thrombotic risk.

The term “paroxysmal nocturnal” is historical and derives from the observation of dark urine in the morning. Complement-mediated hemolysis is continuous or intermittent and is not attributed to physiological acidosis during sleep.

In addition to hemolysis, another fundamental mechanism is the thrombotic tendency: platelet activation and NO depletion promote venous thrombosis, particularly at atypical sites such as hepatic, splanchnic and cerebral veins, which is the leading cause of disease-related mortality.

The cytopenia that accompanies PNH is mainly due to concomitant bone marrow failure, particularly in disease associated with aplastic anemia or myelodysplasia; it should not be attributed generically to peripheral destruction of all GPI-deficient cells.

Finally, chronic intravascular hemolysis and urinary hemoglobin loss may cause iron deficiency and contribute to renal hemosiderosis and tubular injury; renal function may deteriorate in a proportion of patients.

Clinical Presentation

The clinical presentation of paroxysmal nocturnal hemoglobinuria is extremely heterogeneous and depends on the extent of the mutated clone, the severity of hemolysis, and the presence of thrombosis and bone marrow failure.

Chronic hemolysis and anemic symptoms

The most characteristic finding is intermittent hemoglobinuria, often noticed in the morning, which gives the urine a reddish-brown (“port-wine”) color. Anemia varies in severity and is frequently accompanied by severe fatigue, dyspnea, tachycardia and mild or overt jaundice due to increased indirect bilirubin. Reticulocytosis is frequent but may be attenuated or absent in the presence of bone marrow failure or iron deficiency.

Hemolytic crises may be triggered by infections, stress, surgery or medications and are often associated with abdominal pain, headache and fever.

Thrombotic events

The most feared complication of PNH is venous thrombosis, which characteristically may affect the hepatic veins (Budd-Chiari syndrome), splanchnic, renal and cerebral veins and, less commonly, arterial territories. These events are the leading cause of death in untreated patients and may occur even when anemia is mild.

Cytopenias and bone marrow failure

A substantial proportion of patients have leukopenia, thrombocytopenia and bone marrow failure. The risk of myelodysplastic syndrome or acute leukemia is related to the marrow disorder and clonal evolution, not to small PNH clone size in itself.

Common manifestations and clinical complications:

Regardless of phenotype, several manifestations are common:

• Chronic jaundice and pigmenturia
• Erectile dysfunction and symptoms related to nitric oxide deficiency
• Abdominal pain and nonspecific abdominal symptoms
• Pulmonary hypertension in advanced disease
• Chronic kidney disease due to tubular injury

The natural history of PNH varies considerably: some patients remain stable for years, whereas others rapidly develop fatal complications. Thrombotic risk persists even when anemia is modest.

Investigations and Diagnosis

The diagnosis of paroxysmal nocturnal hemoglobinuria is based on an integrated pathway that begins with clinical suspicion, continues with identification of deficient GPI-anchored proteins, and is completed by characterization of the clone and assessment of complications.

Suspicion arises in the presence of intravascular hemolytic anemia with hemoglobinuria, markedly elevated LDH, low or undetectable haptoglobin, reticulocytosis, normocytic or macrocytic anemia, and a history of atypical thrombotic events. The presence of leukopenia or thrombocytopenia further strengthens the suspicion.

Laboratory tests:

• Complete blood count: anemia, reticulocytosis, and possible leukopenia/thrombocytopenia
• Peripheral blood smear: nonspecific findings, with possible polychromasia when a reticulocyte response is present
• Elevated LDH, increased indirect bilirubin, low or undetectable haptoglobin
• Hemoglobinuria and urinary hemosiderin
• Negative direct antiglobulin test

Confirmatory diagnosis: Diagnosis is based on identification of deficient GPI-anchored proteins by flow cytometry on peripheral blood:

• Detection of the PNH clone with FLAER and GPI-anchored markers on granulocytes and monocytes, which may be complemented by CD55 and CD59 assessment on erythrocytes
• Quantification of the PNH clone (ratio of GPI-negative to normal cells)

This technique allows diagnosis even in the absence of hemoglobinuria, in patients with cytopenias or in subclinical disease.

Additional investigations: Selected according to the clinical picture to assess bone marrow function, associated syndromes and complications:

• Bone marrow aspiration and biopsy in the presence of significant cytopenias or suspected bone marrow failure or myelodysplasia
• Selective assessment for inherited thrombophilia only when the personal or family history suggests a specific defect and the result could alter management; routine thrombophilia screening is not indicated solely on the basis of a family history of thromboembolic events.
• Renal function tests and urinalysis for proteinuria and hemosiderinuria
• Abdominal imaging (ultrasonography, CT, MRI) to detect splanchnic thrombosis

Differential diagnosis: PNH must be distinguished from other causes of hemolytic anemia (autoimmune hemolytic anemia, microangiopathies, enzyme deficiencies) and from myelodysplastic or aplastic syndromes.

Specialist assessment: Diagnosis and management require multidisciplinary collaboration among hematologists, nephrologists and internal medicine specialists.

Prompt diagnosis and assessment of complications are essential to establish appropriate treatment and prevent the most severe outcomes.

Treatment and Prognosis

treatment of paroxysmal nocturnal hemoglobinuria is based on an individualized strategy that takes into account the severity of hemolysis, thrombotic risk and bone marrow function. The goals are to prevent hemolysis and its complications, reduce thrombotic risk and improve both quality and length of life.

Complement-targeted therapy: The major therapeutic advance in recent years has been the introduction of complement inhibitors:

• Eculizumab: an anti-C5 monoclonal antibody that blocks MAC formation and prevents hemolysis, drastically reduces thrombotic risk and improves survival
• Ravulizumab: a long-acting analogue that allows less frequent infusions
• Crovalimab: a subcutaneously administered anti-C5 monoclonal antibody authorized in the European Union for adult and pediatric patients aged at least 12 years and weighing at least 40 kg with PNH, according to regulatory criteria relating to hemolytic activity or stability during previous C5-inhibitor therapy.
• Pegcetacoplan (C3 inhibitor) and iptacopan (factor B inhibitor) are monotherapy options authorized in the European Union for adults with PNH and hemolytic anemia; danicopan (factor D inhibitor) is authorized as add-on therapy to ravulizumab or eculizumab in adults with residual hemolytic anemia.

Complement-inhibitor therapy is indicated in the presence of clinically significant hemolysis, thrombosis or relevant symptoms, according to the drug and patient profile, after the required vaccination and meningococcal prophylaxis.

Treatment of thrombotic complications: Anticoagulation is indicated for documented thrombosis and, in high-risk patients, long-term prophylaxis may be considered. Clinical and laboratory monitoring is essential to prevent recurrence.

Transfusion therapy: Reserved for cases of severe symptomatic anemia; units should be compatible and leukoreduced to reduce the risk of transfusion reactions.

Supportive therapies: These include:

• Iron supplementation in iron deficiency anemia
• Management of renal, cardiovascular and nutritional complications
• Monitoring of bone marrow function and surveillance for neoplastic progression

Hematopoietic stem cell transplantation: It is the only option that may potentially eradicate the clone, but it is reserved mainly for patients with severe bone marrow failure or clonal evolution and for exceptional selected cases. Transplantation carries substantial risks and must be assessed case by case.

Prognosis: The introduction of complement inhibitors has radically improved survival and quality of life in patients with PNH, reducing mortality from thrombosis and organ failure. In treated patients, 5-year survival exceeds 80–90%. However, in patients with associated bone marrow failure, risk related to the marrow disorder and clonal evolution persists; this risk does not depend on small PNH clone size in itself.

prevention of complications relies on early diagnosis, careful management of hemolytic crises and targeted therapy in patients at risk.

Complications

complications of paroxysmal nocturnal hemoglobinuria are the main cause of morbidity and mortality, especially in the absence of targeted treatment. The risk of complications is related to the severity of hemolysis, thrombotic burden and associated bone marrow failure.

Thrombotic complications: They are the leading cause of death and disability in untreated patients:

• Deep venous thrombosis and thrombosis at atypical sites (hepatic, splanchnic and cerebral veins)
• Pulmonary embolism
• Budd-Chiari syndrome

Early treatment with complement inhibitors has drastically reduced these events.

Renal complications: Chronic intravascular hemolysis may cause renal hemosiderosis and contribute to acute or chronic kidney injury; proteinuria and hypertension may be present, while acute tubular injury may occur during severe hemolytic crises.

Hematologic complications: Concomitant bone marrow failure may be associated with aplastic anemia and, in a minority of patients, myelodysplastic or leukemic evolution. This risk depends on the marrow disorder and clonal evolution, not on small PNH clone size in itself.

Infectious complications: Immunosuppressive therapy and transplantation, when indicated for concomitant bone marrow failure, increase the risk of severe and opportunistic infections.

Metabolic and nutritional complications: Chronic urinary hemoglobin loss may cause iron deficiency. Malnutrition is not a characteristic complication of PNH and requires a separate etiologic assessment.

Psychosocial complications: Dependence on infusion therapies, the risk of residual or breakthrough hemolysis, thrombotic complications and bone marrow failure may significantly affect quality of life.

Overall, management of PNH complications requires a multidisciplinary approach, close monitoring and timely intervention to reduce mortality and preserve good quality of life.

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