Introduction:

• It is an acquired hemolytic anemia characterized by non-malignant clonal expansion of one or more hematopoietic stem cells that have undergone somatic mutation of the PIG-A gene located on X chromosome.
• Hence blood in PNH patients is a mosaic of normal and abnormal cells
• PNH is characterized by
  • Chronic intravascular hemolytic anemia from uncontrolled complement activation
  • Propensity for thrombosis
  • Bone marrow failure in some cases
• PNH clone is present in considerable proportion of general population without symptoms.
• It is also called as Marchiafava- Micheli syndrome

Epidemiology:

• Peak incidence in 3rd-4th decade of life
• As it is acquired mutation, females are also equally affected (As 1 chromosome is inactivated during embryogenesis)
• Prevalence- 15.9/1million

Etio-pathogenesis:

Stem cell somatic mutations in Phosphotidyl inositolglycan class A (PIG-A) gene which is located chromosome Xp, which codes for enzyme acetyl glucosaminyl transferase, which in needed for the synthesis of Glycosyl PhosphatidylInositol (GPI) anchor

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Proteins that require GPI anchor (nearly 150 proteins) are unable to become membrane bound as they are degraded in endoplasmic reticulum

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As some of these GPI linked proteins normally inactivate complements (CD55 and CD59), in their absence complements remain on RBC surface.

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Mild respiratory acidosis of sleep leads to enhanced complement activity

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Intravascular hemolysis through alternate complement pathway

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Release of free hemoglobin in circulation

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Scavenging of nitric oxide and depletion of nitric oxide at tissue level

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Fatigue

Esophageal spasm

Erectile dysfunction

Renal insufficiency

• GPI– linked proteins that are deficient in PNH blood cells
  • CD 55 (Decay accelerating factor)- Accelerates rate of destruction of membrane bound C3 convertase
  • CD 59- Interacts with membrane attack complex and prevents lytic pore formation by blocking aggregation of C9
  • Acetyl cholinesterase (function is unknown)
  • CD 58 (LFA 3; cell adhesion molecule of immunoglobulin super family; acts as ligand for CD 2 on T – lymphocytes): function in erythrocyte unknown
  • C 8 binding protein (HRF 60, MIP)
  • Dombrock protein
  • JMH Protein.
• Other GPI – Linked Proteins deficient in Blood Cells in PNH
  • Alkaline phosphatase (leukocytes)
  • Urokinase receptor
  • 5¹- Ectonucleotidase (lymphocytes)
  • Folate receptor
  • Endotoxin binding protein receptor (CD 14)
  • CD 48 (lymphocytes)
  • CD w52 (lymphocytes, some monocytes)
  • CD 24
  • CD 66
  • CD 67
  • P-50-80 (granulocytes)
• Because defect affects stem cells, along with anemia there is granulocytopenia& thrombocytopenia.
• Due to unknown reasons, these cells have proliferative advantage, so these patients are prone to develop.
  • Acute myeloid leukemia
  • Chronic lymphocytic leukemia
  • Myeloproliferative disorders.
  • MDS
• Thrombosis is also often seen in PNH, as
  • Urokinase-plasminogen activator receptor is a GPI linked protein.
  • Tissue pathway inhibitor is also GPI linked protein
  • Complement activation in platelets causes hypercoagulability
  • Depletion of nitric oxide increased platelet aggregation and adhesion.
• Definite relationship of PNH and aplastic anemia has been noted.
  • It occurs essentially due to organ specific autoimmune disease that is mediated by “activated” cytotoxic T lymphocytes.
  • So this is treated with intensive immunosuppressive therapy which includes ATG and cyclosporine A

Classification:

• Classic: 
  • Associated with florid hemolysis
  • BM is cellular with erythroid hyperplasia
  • >50% population of GPI deficient granulocytes
  • Eculizumab is useful
• PNH in setting of another marrow failure syndrome:
  • Mild to moderate hemolysis
  • Hypocellular BM
  • Population of GPI deficient granulocytes is generally <30%
  • Benefit of Eculizumab depends on the size of PNH clone
• Subclinical
  • No evidence of hemolysis
  • <1% population of GPI deficient granulocytes 
  • No use of Eculizumab

Clinical Features:

• Intravascular hemolysis and hemoglobinuria in first morning urine (Coca-Cola urine)
  • It is precipitated by stress, infection, exercise, pregnancy, vaccination, menstruation, blood transfusion, surgery etc.
• Multiple venous thrombosis
  • Involves especially portal and cerebral veins.
  • It is the most common cause of death in PNH patients.
  • Occurs in 40% of patients with PNH 
  • Arterial thrombosis is also observed in some cases
• Repeated infections due to granulocytopenia.
• Anemia
  • Due to hemolysis and iron deficiency anemia which occurs due chronic blood loss in the form of hemoglobinuria and hemosiderinuria
  • It can also be due to bone marrow failure
• Fever
• Smooth muscle dystonia- Esophageal spasms, erectile dysfunction (Occurs due to scavenging of nitric oxide by free plasma hemoglobin)

Investigations:

• Hemogram
  • Hemoglobin content- Reduced to 8-10gm/dl.
  • Macro-ovulocytosis
  • Microcytic hypochromic RBCs if there is iron deficiency due to chronic urinary iron loss
  • Polychromatophilic RBCs and nucleated RBCs are seen.
  • Erythrophagocytosis.
  • Thrombocytopenia.
  • Granulocytopenia.
• Reticulocyte count- Raised to 5-10%
• Urine examination- Both hemoglobin and hemosiderin are present.
• Serum LDH-Increased
• Bilirubin-Raised
• Haptoglobin-Decreased
• Leukocyte alkaline phosphatase score- Decreased
• Bone marrow examination
  • Normal/ normoblastic hyperplasia in most of the cases
  • Required to rule out MDS/ aplastic anemia which can develop in patient with PNH
• Demonstration of RBC lysis by complement activation 
  • Ham’s test (Acidified serum lysis test). 
    • PNH cells undergo hemolysis in compatible acidified serum at 37^oC
    • Acidification activates alternate complement pathway
    • In PNH 10-50% cells are lysed.
    • It is false positive in congenital dyserythropoietic anemia type II
  • Sucrose lysis test
    • Reduction in ionic strength (by using sucrose) promotes binding of complements to erythrocytes.
    • More than 10% hemolysis indicates positive test.
• Flow cytometry.
  • For analysis of GPI linked proteins 
  • Antibodies against CD 55, CD59, CD14, CD16 and CD67 are used.
  • Most sensitive and specific test
  • Analysis must be done both on RBCs and WBCs (As GPI deficient RBCs may be selectively destroyed)
  • 3 types of cells
    • PNH I cells- Normal complement resistant red blood cells
    • PNH II cells- Mildly complement sensitive cells
    • PNH III cells- Markedly abnormal and complement sensitive cells
  • Rarely any one of these antigens may be congenitally absent. Hence absence of any 2 antigens should be demonstrated for diagnosis of PNH.
  • Fluoresceinlabelledproaerolysin (FLAER)- is more sensitive method of analysis, as it specifically binds with high affinity to GPI anchor of most of cell lineages.
• Coagulation studies- Show a hypercoagulable state.
• Osmotic fragility test- Normal.
• Autohemolysis test- Increased after 48 hours and increased more when glucose is added.
• Coomb’s test- Negative

Criteria for Diagnosis:2X2 Rule

• At least 2 different GPI protein deficiencies within 2 different cell lines from granulocyte, monocyte or erythrocyte

When PNH should be suspected?

• Hemoglobinuria due to intravascular hemolysis
• Hemolytic anemia with low platelet count and/or low neutrophil count
• Hemolytic anemia and recurrent abdominal pain with or without blood in stool
• Hepatic vein thrombosis
• Any venous thrombosis anywhere in young adult with low platelet and/or neutrophil counts/ hemolytic anemia
• Idiopathic pancytopenia

Prognosis:

• Median survival without treatment is 5-10years
• 35% patients die within 5 years of diagnosis
• Usually has a waxing and waning course
• Course and prognosis is enormously variable. In some cases, after many years, full spontaneous recovery is seen

Indications for treatment:

• Disabling fatigue
• Thrombosis
• Transfusion dependency
• Frequent pain paroxysms
• Renal insufficiency/other end organ damage
• Presence of high disease activity, which is defined as LDH more than 1.5 times the normal with any one of following features
  • Hemoglobinuria
  • Abdominal pain
  • Dyspnea
  • Anemia (Hb<10gm/dL)
  • Major thrombosis
  • Dysphagia
  • Erectile dysfunction

Treatment:

• Eculizumab
  • It is a humanized monoclonal antibody against complement protein C5 and it prevents conversion of C5 to C5a. Hence there is no terminal activation of complement pathway.
  • Decreases hemolysis and also thrombosis
  • Dose: 600mg- IV- over 30min- weekly for 4 weeks, then once in 2 weeks- continue indefinitely
  • Long term use increases the survival up to the level of general population
  • If there is breakthrough hemolysis, decrease the interval between 2 injections to 12 days. 
  • Side effects: Infection with encapsulated (Especially Neisseria) organisms. Hence 15 days prior to starting Eculizumab, give meningococcal vaccine and then repeat it every year.
• Blood  transfusion- 
  • Ameliorate hemolysis by suppressing erythropoiesis. 
  • Iron overload is unlikely.
• Iron, folic acid and Vitamin B12 supplementation (Iron deficiency can exacerbate hemolysis)
• For thrombosis
  • Thrombolysis with rtPA if indicated
  • Regular anticoagulant therapy- Preferably life long
• Prevent precipitating factors for hemolysis
• Prednisolone
  • Dose- 15 -30 mg only during episodes for short time(Long term use is contraindicated)
  • If no effect after 8 weeks, taper and stop
  • Steroids must be better combined with androgens (Oxymetholone – 10-15 mg OD/ Danazol- 400mg BD)
• Bone marrow transplantation     
  • Only method of cure but has very high risk of morbidity and mortality. 
  • 2 year survival- 56%.
  • Outcome is extremely poor if there is associated thrombosis
  • Indications
    • Associated marrow aplasia
    • Associated myelodysplastic syndrome
    • Resistance to Eculizumab
    • Non-affordable patient for Eculizumab
  • Conditioning- Same as aplastic anemia (Cy-ATG)
• Role of splenectomy is unclear
• Hypoplastic PNH- Treat similar toaplastic anemia

Special Situations:

• MDS in association of PNH
  • Less pronounced morphological abnormalities of blood cells
  • More severe thrombocytopenia
  • Lower rates of karyotypic abnormalities
  • High incidence of HLA-DR-15
  • Lower rate of progression to AML
  • Higher probability of response to Cyclosporine
• PNH with pregnancy
  • Associated with high risk of morbidity and mortality
  • Main stay of treatment includes transfusions, folic acid and iron supplements
  • Anticoagulation prophylaxis with heparin is recommended
  • Eculizumab is not recommended, but may be considered as Category C option.

Recent advances:

New medications for treatment of PNH:

• Ravulizumab: Complement 5 inhibitor
• Crovalimab:  Complement 5 inhibitor
• Pozelimab: Complement 5 inhibitor
• Tesidolumab: Anti C5 monoclonal antibody
• Zilucoplan: 
• Nomacopan
• Aptamer

Pegcetacoplan in PNH 

Pegcetacoplan is apegylated peptide that binds to and inhibits Complement protein C3. It was approved for use in 2011 for PNH. In arecent study it was found that Pegcetacoplan was superior to eculizumab with respect to improvement in haemoglobin levels. No cases of meningococcal meningitis were observed in both groups.  

https://doi.org/10.1056/NEJMoa2029073  

Iptacopanin  Paroxysmal Nocturnal Hemoglobinuria 

Iptacopan is an orally administered proximal complement inhibitor of Factor B. Up to 60% of PNH patients have clinically meaningful residual anemia with standard of care therapy which is secondary to C3-mediated extravascular hemolysis. In the present study Iptacopanshowed superiority vs. SoC with respect to correction of anemia. Iptacopan monotherapy also showed superiority in transfusion avoidance. There were no deaths and no serious encapsulated bacteria infections. 

https://doi.org/10.1182/blood-2022-171469

Addition of danicopan to ravulizumab or eculizumab in patients with paroxysmal nocturnal haemoglobinuria

The ALPHA trial, a phase 3, double-blind, placebo-controlled study, assessed the efficacy and safety of danicopan (ALXN2040), an oral complement factor D inhibitor, as add-on therapy to ravulizumab or eculizumab in patients with paroxysmal nocturnal haemoglobinuria (PNH) and clinically significant extravascular haemolysis. The interim analysis revealed that danicopan significantly increased hemoglobin concentrations compared to placebo at week 12, suggesting an improved benefit–risk profile. Adverse events were generally manageable, with no new safety concerns reported.

https://doi.org/10.1016/S2352-3026(23)00315-0

IptacopanMonotherapy in Paroxysmal Nocturnal Hemoglobinuria

Iptacopan, an oral factor B inhibitor, was assessed in two phase 3 trials for patients with paroxysmal nocturnal hemoglobinuria (PNH) and hemoglobin levels below 10 g/dL. In the first trial, patients previously treated with anti-C5 therapy who switched to iptacopan experienced significant increases in hemoglobin levels without transfusion, while those continuing anti-C5 therapy did not meet the endpoint. In the second trial, iptacopan effectively raised hemoglobin levels in PNH patients who had not received complement inhibitors. Overall, iptacopan demonstrated promising efficacy as a monotherapy for PNH, offering a potential alternative to anti-C5 therapy and benefitting patients regardless of prior treatment with complement inhibitors.

https://doi.org/10.1056/NEJMoa2308695

Crovalimab versus eculizumab in patients with paroxysmal nocturnal hemoglobinuria naive to complement inhibition

The phase 3 COMMODORE 2 trial evaluated crovalimab, a novel C5 complement inhibitor, against eculizumab in C5 inhibitor-naive patients with paroxysmal nocturnal hemoglobinuria. Crovalimab, administered subcutaneously every 4 weeks, demonstrated non-inferiority to eculizumab in hemolysis control (79.3% vs. 79.0%) and transfusion avoidance (65.7% vs. 68.1%) over a 24-week period. Secondary endpoints, including breakthrough hemolysis and hemoglobin stabilization, were comparable between the groups. Both treatments improved fatigue, and crovalimab maintained complete terminal complement inhibition. Safety profiles were similar, with no meningococcal infections reported. Patients switching to crovalimab favored its convenience, supporting its positive benefit–risk profile.

https://doi.org/10.1002/ajh.27412