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Paroxysmal nocturnal hemoglobinuria (PNH)

Rare, acquired chronic haemoglobinuria characterized by persistent intravascular hemolysis subject to recurrent exacerbations.

  • Only hemolytic anemia caused by an acquired (rather than inherited) intrinsic defect in the cell membrane (deficiency of glycophosphatidylinositol leading to the absence of protective proteins on the membrane).

History:

While the term paroxysmal nocturnal hemoglobinuria (PNH) was introduced by Enneking in 1925, case reports dating back to the 1880s can be found. One of the earliest was that of Strubing, who documented the case of a young adult man with fatigue, abdominal pain, and intermittent hemoglobinuria. Strubing also noted that the patient’s plasma was red following the most severe episodes, and he deduced that intravascular hemolysis was the cause. Later in 1937, Ham was able to discover that erythrocytes of individuals with PNH hemolyzed when incubated with normal acidified urine. This resulted in the first diagnostic test for PNH, known as the Ham test (acidified serum test). While complement activation was suspected as the etiology for hemolysis, the theory was not formally proven until 1954. Over the following years, the nature of protein deficiencies affecting PNH erythrocytes was identified, and this paved the way for the identification of the responsible genetic mutation.

Etiopathogenesis

PNH is characterized by destruction of RBCs by the complement system (innate immune system)

X-linked gene phosphatidylinositol glycan class A (PIGA) mutation
↓
Glycosylphosphatidylinositol (GPI) protein deficiency: CD55, CD59
(responsible for anchoring other protein moieties to erythrocyte surface)
↓
Complement-mediated lysis of RBCs

This chronic state of hemolysis can be exacerbated if the complement system is activated by stress due to surgery, trauma, or other triggers for inflammation.

Intravascular and extravascular haemolysis in PNH: Complement decay-accelerating factor (also known as CD55) and CD59 glycoprotein (CD59) are glycosylphosphatidylinositol (GPI)-anchored self-protective complement regulatory factors. CD55 is a widely expressed membrane protein that accelerates the decay of C3 convertases (C3 con) bound to the cell surface, thereby limiting the formation of C5 convertases. CD59 is also widely expressed: it blocks the generation of the membrane attack complex (MAC) and is, therefore, the major inhibitor of terminal complement | a) Normal RBCs are protected from activated complement. (b) By contrast, the CD55-defective and CD59-defective PNH RBCs are highly sensitive to complement activation, which causes intravascular haemolysis. (c) Upon eculizumab treatment, CD59 deficiency is compensated for and intravascular haemolysis is prevented owing to inhibition of C5 activation and subsequent MAC formation. However, CD55 deficiency remains on unlysed PNH RBCs, which causes inefficient downregulation of C3 convertases and in turn could lead to an accumulation of C3b and its processed forms iC3b and C3dg. iC3b and C3dg are ligands of integrin Ξ±M, Ξ²2 (CR3), a receptor expressed on macrophages in the spleen and liver; thus, these macrophages can recognize PNH RBCs, leading to extravascular haemolysis. | Hill, A., DeZern, A. E., Kinoshita, T., & Brodsky, R. A. (2017). Paroxysmal nocturnal haemoglobinuria. Nature reviews. Disease primers, 3, 17028. https://doi.org/10.1038/nrdp.2017.28

Presentation

Traditionally PNH was characterized by recurrent episodes of intravascular hemolysis, venous thrombosis, and cytopenias associated with bone marrow failure.

Most patients present with nonspecific and variable signs that do not fit any specific syndrome.

  • Constitutional symptoms (anemia): Fatigue, generalized malaise, dyspnea
  • Dark urine (due to marked hemoglobinuria)
  • Renal insufficiency (hemosiderin deposition leading to tubulointerstitial inflammation)
  • Smooth muscle dystonia:
    • Dysphagia or esophageal spasms
    • Abdominal pain, back pain
    • Erectile dysfunction

Diagnosis

Because signs and symptoms are so variable, it is often difficult to diagnose this condition, and hence, diagnosis is often delayed.

Presumptive diagnosis:

Recurrent episodes of intravascular hemolysis and cytopenias, and hemoglobinuria or hemosiderosis can be seen in PNH. Thus markedly increased LDH, low haptoglobin, and unconjugated bilirubinemia would be seen due to intravascular hemolysis.
  • Complete blood count with differential (CBC w/ diff):
    • Increased reticulocyte count
    • Anemia, leukopenia, and thrombocytopenia
  • Basic metabolic panel (BMP):
    • Renal dysfunction: Increase in serum creatinine and blood urea nitrogen (BUN)
    • Electrolyte abnormalities: Seen in chronic kidney disease (CKD)
  • Urinalysis (UA):
    • Intravascular hemolysis: Hemoglobinuria and hemosiderosis

Clinical blood tests:

  • Direct antiglobulin test (DAT, or direct Coombs’ test): Negative (non-immune hemolysis)
  • Now obsolete:
    • Sucrose lysis test
    • Ham’s acid hemolysis test

Flow cytometry (GOLD STANDARD)

It utilizes various monoclonal antibodies, and special reagent called fluorescent aerolysin reagent (FLAER) that binds directly to glycosylphosphatidylinositol (GPI) anchored protein, specifically their glycan portion.
  • GPI-anchored proteins:
    • Decay-accelerating factor (DAF/CD55)
    • Membrane inhibitor of reactive lysis (MIRL/CD59)

Differential diagnosis:

Differential diagnosis includes other hemolytic anemias, other causes of atypical thrombosis, and conditions that cause bone marrow failure.
Common causes of hemolysis classified by type. | G6PD: glucose-6-phosphate dehydrogenase; CLL: chronic lymphocytic leukemia; NHL: non-Hodgkin lymphoma; SLE: systemic lupus erythematous; TIPS: transjugular intrahepatic portosystemic shunt; TTP: thrombotic thrombocytopenic purpura; HUS: hemolytic uremic syndrome; HELLP: hemolysis, elevated liver enzyme levels, and low platelet levels; DIC: disseminated intravascular coagulation; EV: extravascular: IV: intravascular. | Ruiz, E. F., & Cervantes, M. A. (2015). Diagnostic approach to hemolytic anemias in the adult. Revista brasileira de hematologia e hemoterapia, 37(6), 423–425. https://doi.org/10.1016/j.bjhh.2015.08.008

Management

In PNH, complement-mediated hemolysis and chronic dysregulation of the alternative complement pathway are the main culprits. Commonly there is a loss of anchoring proteins such as CD55 and CD59, which causes cells to hemolyze and lead to complications like thrombosis, which causes morbidity and mortality.

Anti-C5 monoclonal antibodies therapies

Drugs to block alternative complement pathways are the mainstay of current therapy as in PNH, complement-mediated hemolysis and chronic dysregulation of the alternative complement pathway are the main culprits. These drugs pevent C5 conversion into C5a and C5b factors; thus, effectively inhibit membrane attack complex (MAC) formation and complement-mediated lysis.
  • Eculizumab
  • Ravulizumab (longer action, more cost-effective and fewer breakthrough hemolysis episodes)
  • Daily oral antibiotic prophylaxis: Due to associated risk of encapsulated bacterial infections
Complement cascade inhibition: The lectin, classical and alternative pathways converge at the step of complement component 3 (C3) activation. Haemolysis in PNH is usually chronic because the alternative pathway is always in a state of low-level activation through a process known as tickover. Terminal complement is initiated by C5 convertases, leading to cleavage of C5 to C5a and C5b. C5b oligomerizes with C6, C7, C8 and multiple C9 molecules to form the membrane attack complex (MAC). The complement decay-accelerating factor (CD55) inhibits proximal complement activation by accelerating the decay of C3 convertases; CD59 glycoprotein (CD59) inhibits terminal complement activation by preventing the incorporation of C9 into the MAC. There is a potent amplification loop within the alternative pathway. The absence of CD55 and CD59 on PNH cells leads to haemolysis, inflammation, platelet activation and thrombosis. Eculizumab prevents C5 convertases from cleaving C5 into C5a and C5b. C5 activation promotes coagulation via various mechanisms, including activating thrombin82. Thrombin cleaves C3 and also generates C5a in the absence of C3. The fibrinolytic factors plasmin and kallikrein also directly cleave C3. | MASP, mannose-binding lectin-associated serine protease. | Hill, A., DeZern, A. E., Kinoshita, T., & Brodsky, R. A. (2017). Paroxysmal nocturnal haemoglobinuria. Nature reviews. Disease primers, 3, 17028. https://doi.org/10.1038/nrdp.2017.28

Allogeneic hematopoietic stem cell transplantation (allo-HSCT):

Only curative therapy for PNH.

Summary:

Paroxysmal nocturnal haemoglobinuria
Somatic PIGA mutations in haematopoietic stem cells generate erythrocytes that are deficient in glycosylphosphatidylinositol-anchored proteins. These erythrocytes are susceptible to haemolysis by complement activation, which manifests as paroxysmal nocturnal haemoglobinuria. | Paroxysmal nocturnal haemoglobinuria. Nat Rev Dis Primers 3, 17029 (2017). https://doi.org/10.1038/nrdp.2017.29

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