Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic disorder caused by a genetic mutation in hematopoietic stem cells, leading to defects in red blood cell membranes. It is considered a benign clonal disease. The primary clinical manifestation involves intravascular hemolytic anemia, which may be accompanied by thrombosis and bone marrow failure. Classic presentations include characteristic intermittent nocturnal hemoglobinuria after sleep. The peak onset age is 20 to 40 years, with a higher prevalence in males compared to females.
Etiology and Pathogenesis
The disease results from the mutation of the phosphatidylinositol glycan class A (PIGA) gene on the X chromosome in one or more hematopoietic stem cells. The PIGA protein product is a glycosyltransferase required for the synthesis of glycosylphosphatidylinositol (GPI) anchors. Mutated hematopoietic stem cells and their progeny (including red blood cells, granulocytes, monocytes, lymphocytes, and platelets) exhibit impaired GPI anchor synthesis, which results in the absence of various functional proteins (known as GPI-anchored proteins) on the cell surface.
Complement regulatory proteins CD55 (decay-accelerating factor, DAF) and CD59 (membrane inhibitor of reactive lysis, MIRL) are GPI-anchored proteins. CD55 inhibits the formation of complement C3 convertase, while CD59 prevents the transformation of complement C9 into the membrane attack complex. The absence of CD55 and CD59 on mature red blood cell membranes underlies the intravascular hemolysis characteristic of PNH.
The blood in PNH patients is a "mosaic" comprising both normal and abnormal cells, with the size of the PIGA-mutated clone varying significantly between individuals. The nature of the GPI anchor protein deficiency is determined by the phenotypic mosaicism of the PIGA gene. In type III PNH cells, GPI-anchored proteins are completely absent, while in type II cells, they are partially deficient, and type I cells express these proteins normally. The proportions of different cell types correlate with the severity of hemolysis.
A prothrombotic tendency is observed in PNH, although its mechanism remains unclear. Potential factors include complement activation-induced platelet activation, increased procoagulants resulting from hemolysis, and abnormalities in fibrin generation or fibrinolytic activity.
Clinical Manifestations
Anemia
Varying degrees of anemia are observed. In addition to anemia due to intravascular hemolysis, a subset of patients may transition to an aplastic anemia-PNH (AA-PNH) syndrome, where bone marrow failure leads to anemia. Frequent hemolytic episodes with persistent hemosiderinuria can cause iron deficiency, exacerbating anemia.
Hemoglobinuria
Morning hemoglobinuria is a characteristic feature of this disease, with approximately 1/4 of patients presenting with this as their initial symptom. In severe cases, urine may appear dark brown or red, resembling "soy sauce" or "red wine," and symptoms may include fatigue, retrosternal and lumbar/abdominal pain, as well as fever. In mild cases, only urinalysis reveals positive occult blood. The mechanism for increased nocturnal hemolysis remains unclear, but it may be associated with blood acidification during sleep. Triggers for hemoglobinuria may include infections, blood transfusions, physical exertion, or iron therapy.
Manifestations of Cytopenias
As a marrow failure syndrome, PNH frequently leads to not only anemia but also reductions in neutrophils and platelets. Neutropenia and functional defects increase susceptibility to infections such as bronchitis, pneumonia, and urinary tract infections. Thrombocytopenia increases the risk of bleeding, and severe hemorrhage is among the leading causes of death in PNH.
Thrombosis
Thromboembolic events are common in PNH, occurring in approximately 1/3 of patients. Venous thrombosis frequently occurs in atypical locations, with hepatic veins being the most common site, leading to Budd-Chiari syndrome, the most common cause of death in PNH. Other common sites include the mesenteric veins, cerebral veins, and deep veins of the lower extremities, each with corresponding clinical presentations. Arterial embolism is rare.
Smooth Muscle Dysfunction
Symptoms such as abdominal pain, esophageal spasms, dysphagia, and erectile dysfunction are common and may be linked to hemolysis-induced release of large amounts of free hemoglobin, which depletes nitric oxide (NO) and impairs smooth muscle function.
Laboratory Tests
Peripheral Blood Findings
Anemia often presents as normocytic or macrocytic, though microcytic hypochromic anemia may also occur. Reticulocyte counts are elevated, but the increase is less marked compared to other types of hemolytic anemia (HA). Granulocyte counts are typically reduced, and platelet counts are moderately to severely decreased. Approximately half of patients exhibit pancytopenia. Peripheral blood smears may show nucleated red blood cells and fragmented erythrocytes.
Bone Marrow Examination
Bone marrow activity ranges from hypercellular to markedly hypercellular, particularly in the erythroid lineage. In some cases, bone marrow may be hypoplastic. Chronically excessive urinary iron loss may result in reduced iron stores in bone marrow as indicated by iron staining.
Intravascular Hemolysis Testing
Details are provided in the related sections.
Diagnostic Tests
Specific tests focus on the complement sensitivity of PNH red blood cells and the absence of GPI-anchor proteins on blood cell membranes.
Flow Cytometry for CD55 and CD59
Reductions in CD55 and CD59 expression on the membranes of granulocytes, monocytes, and red blood cells can be detected by flow cytometry.
Flow Cytometry Using Fluorescent Aerolysin (FLAER)
The precursor of aerolysin, produced by Aeromonas hydrophila, specifically binds to GPI-anchor proteins. Flow cytometry can identify fluorescently-labeled aerolysin variants on peripheral granulocytes and monocytes, distinguishing GPI-positive and GPI-negative cells. FLAER is typically used to test nucleated cells, as it cannot assess PNH clones in mature red blood cells. This method represents a more sensitive and specific diagnostic approach, particularly for detecting small PNH clones. It is not affected by recent transfusions or ongoing hemolysis.
Specific Serological Assays
Acidified serum hemolysis test (Ham test), sugar water hemolysis test, snake venom factor hemolysis test, and micro-complement sensitivity test are examples of these assays, though their sensitivity and specificity are relatively low.
Diagnosis and Differential Diagnosis
PNH is diagnosed when the clinical presentation aligns with the disease and one or both of the following laboratory criteria are met. The two diagnostic criteria may complement each other.
In acidified serum hemolysis (Ham test), sugar water hemolysis test, snake venom factor hemolysis test, and urinary hemosiderin/hemoglobin tests, specific conditions for a definitive diagnosis include:
- Two or more positive results.
- One positive result with additional supportive evidence, such as:
- At least two instances of positive results.
- Direct or indirect evidence of hemolysis, including confirmed episodes of hemoglobinuria.
- Exclusion of other hemolytic disorders.
Flow cytometry detects >10% of peripheral neutrophils or red blood cells with CD55 or CD59 deficiency (5–10% deficiency is considered borderline) or >1% of cells with FLAER deficiency.
PNH requires differentiation from autoimmune hemolytic anemia (especially paroxysmal cold hemoglobinuria or cold agglutinin syndrome), myelodysplastic neoplasms, and aplastic anemia (AA).
PNH Classification (International PNH Working Group)
Classical PNH
Patients exhibit typical hemolysis and/or thrombosis, significantly elevated LDH levels, and the absence of other bone marrow failure (BMF) syndromes. Flow cytometry typically shows >50% GPI-deficient neutrophils.
PNH with Associated Bone Marrow Failure Syndromes
This group includes cases with conditions such as aplastic anemia (AA), myelodysplastic syndrome (MDS), or myelofibrosis (MF). These patients often have subtle biochemical evidence of hemolysis, and flow cytometry reveals <50% GPI-deficient neutrophils.
Subclinical PNH
Patients exhibit small PNH clones (<10% GPI-deficient neutrophils) detected using highly sensitive flow cytometry techniques. However, there is no laboratory or clinical evidence of hemolysis or thrombosis.
Treatment
Supportive and Symptomatic Treatment
Blood Transfusion
Red blood cell transfusion may be administered when necessary, preferably using leukocyte-depleted red blood cells.
Androgens
Androgens such as testosterone undecanoate, danazol, or stanozolol may be used to stimulate erythropoiesis.
Immunosuppressants
For patients with PNH-AA, immunosuppressive therapy, such as cyclosporine, can be considered.
Iron Therapy
For patients with evidence of iron deficiency, low-dose iron supplementation (1/10 to 1/3 of the standard dose) may be provided, with discontinuation in the presence of active hemolysis.
Control of Hemolytic Episodes
Anti-Complement Monoclonal Antibodies
Complement pathway inhibitors, such as eculizumab, represent first-line therapy for classical PNH. Eculizumab is a humanized monoclonal antibody targeting complement protein C5, preventing the formation of membrane attack complexes. It effectively reduces intravascular hemolysis, lowers thrombotic risks, and prolongs survival. The recommended dosing is 600 mg intravenously per week for 4 weeks, followed by 900 mg starting from the fifth week and subsequently every two weeks thereafter. Patients should receive meningococcal vaccination prior to treatment, with revaccination every 2.5 to 3 years. Although eculizumab controls hemolytic symptoms, it does not completely cure PNH and carries a risk of breakthrough hemolysis.
Glucocorticoids
Glucocorticoids are effective in some patients. Prednisone at a dose of 0.25–1 mg/kg/day may be used but should be administered for short cycles to avoid side effects associated with long-term use.
Sodium Bicarbonate
Administration of 5% sodium bicarbonate solution, either orally or intravenously, alkalinizes blood and urine.
Antioxidants
Agents such as high-dose vitamin E, which may protect cell membranes, have uncertain efficacy.
Prevention and Management of Thrombosis
Patients with thrombosis should receive anticoagulant therapy. There is no consensus regarding the use of prophylactic anticoagulation.
Allogeneic Hematopoietic Stem Cell Transplantation
Allogeneic hematopoietic stem cell transplantation remains the only potentially curative approach for PNH. However, since PNH is not a malignant disorder and transplantation carries inherent risks, patient selection must be cautious and based on strict indications.
Prognosis
In the pre-complement-inhibitor era, the median survival time for PNH patients was 10–15 years. The use of eculizumab has significantly improved survival, with cumulative survival rates comparable to those of healthy individuals of the same age and sex. Some long-term patients experience gradual disease alleviation with varying degrees of spontaneous remission. Primary causes of death include infections, thrombotic events, and bleeding. PNH may progress to aplastic anemia, and in rare cases, to myelodysplastic syndrome (MDS) or acute leukemia, which are associated with poor prognoses.