Paroxysmal Nocturnal Hemoglobinuria (PNH)


Paroxysmal nocturnal hemoglobinuria (PNH) is a rare, acquired, fatal blood disease characterized by red blood cell destruction, blood clot formation, and impaired bone marrow function.1 PNH is caused by acquired mutations in the PIGA gene, which codes for phosphatidylinositol N-acetylglucosaminyltransferase subunit A, which is required for the production of certain membrane-associated complement regulatory proteins.2,3

The blood cells of individuals with PNH are deficient in 3 glycosylphosphatidylinositol (GPI)-linked proteins that control complement activity: CD59 (membrane inhibitor of reactive lysis), CD55 (decay-accelerating factor), and C8-binding protein. The most important of these is CD59, a strong C3 convertase inhibitor involved in preventing spontaneous activation of the alternative complement pathway.3 Red cells that lack GPI-linked components are particularly vulnerable to complement-induced lysis or damage. The C5b-C9 membrane attack complex causes intravascular hemolysis, which is the main manifestation of PNH. However, only 25% of episodes of hemolysis are paroxysmal and nocturnal. Chronic hemolysis without severe hemoglobinuria is more common.3

A diagnosis of PNH is considered in patients in whom evidence of intravascular hemolysis, such as hemoglobinuria and markedly high serum levels of lactate dehydrogenase (LDH), is found with no identifiable etiology. A comprehensive clinical assessment, full patient history, and range of specialized tests may be used to diagnose PNH. Flow cytometry offers the clearest diagnostic path for suspected PNH, and other testing, including laboratory testing and imaging, can provide support for this diagnosis.4 

Read more about PNH diagnosis

Laboratory testing for PNH

Flow Cytometry

Diagnostic flow cytometry is regarded as the gold standard method for diagnosing PNH. It uses several monoclonal antibodies and a reagent called fluorescent aerolysin reagent (FLAER), which specifically binds to the glycan region of GPI-anchored proteins. This test, which is highly sensitive and specific, can assess a number of GPI-anchored proteins, particularly CD55 and CD59.5 PNH can be identified by a lack or diminished expression of both CD59 and CD55 on red cells.6

It is recommended that 2 GPI-linked proteins be studied to ensure that no false-negative results have occurred. These may be caused by rare congenital deficiencies of individual antigens like CD55 or CD59 or by polymorphisms in individual antigens such as CD16, which make it impossible for some monoclonal antibodies to recognize them. All GPI-negative red blood cells, monocytes, and granulocytes are detected. The test classifies PNH red blood cells as type 1, 2, or 3. In type 1 cells, expression of GPI anchor proteins is normal. In type 2 cells, expression is partially deficient, and in type 3 cells, expression of GPI anchor proteins is completely absent. The sensitivity of flow cytometry tests can be high or low. A high-sensitivity test is more effective at detecting PNH in the presence of another bone marrow disease, although low-sensitivity flow cytometry tests may be sufficient for a diagnosis of PNH.5

Hematology and Biochemistry Laboratory Tests

A basic hematology test, such as a complete blood cell count, should always be done to evaluate for low hemoglobin values and low blood cell counts. A reticulocyte count to determine the number of immature red blood cells in the blood is also important because the reticulocyte count may be increased in PNH. LDH levels are also measured; increased levels indicate hemolysis or tissue damage. Monitoring of the LDH levels is important in patients with PNH. High bilirubin levels also indicate red blood cell destruction.7 The urine is examined to identify hemoglobinuria (hemoglobin in the urine) and hemosiderosis (excess iron deposits in organs and tissues). Low levels of haptoglobin also indicate damage to red blood cells.8 

Ancillary Tests

Ham Test

The Ham test, devised by Thomas Ham, is based on the theory that nocturnal hemolysis in patients with PNH is caused by a drop in blood pH during sleep. In this assay, washed red blood cells are incubated with acidified serum, and the free hemoglobin released during red blood cell lysis is measured with spectrophotometry.9 

Sucrose Hemolysis Test 

The sucrose hemolysis test, or Hartmann and Jenkins “sugar water” test, can also be used to diagnose PNH. In this test, complement activation and red blood cell hemolysis occur when blood from a patient with PNH is incubated in a hypotonic sucrose solution.9 This test is less specific but more sensitive than the Ham test for diagnosing PNH.6 

Complement Lysis Sensitivity Test

The Rosse and Dacie complement lysis sensitivity test is an efficient technique for diagnosing PNH. Red cells and small volumes of normal serum, which is a source of complement, undergo hemolysis after they have been sensitized with a strongly lytic anti-I antigen. The test identifies 3 groups of red blood cells in patients with PNH: PNH I cells, which exhibit normal sensitivity to complement; PNH II cells, which are moderately more sensitive to complement than PNH I cells; and PNH III cells, which are markedly sensitive to complement (only one-fifteenth to one-twentieth of the quantity of complement required to lyse normal cells is required for the same level of lysis in these cells). The number of PNH III cells is elevated in relatively severe cases of PNH, and their mean life span is 10 to 15 days.6

Bone Marrow Examination

Examination of the bone marrow is not required but can be done to rule out other illnesses.10 The 2 basic purposes of bone marrow testing are to support the diagnosis of PNH and to evaluate effective or ineffective functioning of the bone marrow.7 The appearance of the bone marrow can vary from erythroid hyperplasia to marked hypoplasia.11

Imaging Studies for PNH

Imaging studies have a key role in diagnosis of PNH. Echocardiography can be used to evaluate pulmonary hypertension. Doppler abdominal ultrasonography can be useful for identifying thrombosis or evaluating hepatic blood flow. Computed tomography pulmonary angiography (CTPA) is helpful if a silent pulmonary embolism is suspected. Abdominal CT can be useful when Budd-Chiari syndrome is suspected. For patients with central nervous system thrombosis, magnetic resonance imaging (MRI) of the head may be beneficial.5 

Read more about PNH complications

Genetic Studies 

Mutation analysis of the PIGA gene is still limited to research laboratories. Additionally, despite being highly specific, mutation analysis is not a diagnostic test for PNH.6 

Read more about PNH clinical studies

References

  1. Paroxysmal nocturnal hemoglobinuria (PNH). John Hopkins Medicine. Accessed November 8, 2022.
  2. Paroxysmal nocturnal hemoglobinuria. MedlinePlus. Accessed November 8, 2022.
  3. Chapter 14: Red blood cell and bleeding disorders. In: Kumar V, Abbas AK, Aster JC. Robbins & Cotran Pathologic Basis of Disease, 10th ed. New York, NY: Elsevier; 2020:648.
  4. Paroxysmal nocturnal hemoglobinuria. NORD. Accessed November 8, 2022.
  5. Shah N, Bhatt H. Paroxysmal nocturnal hemoglobinuria. StatPearls [Internet]. Updated August 1, 2022. Accessed November 8, 2022.
  6. Besa EC. Paroxysmal nocturnal hemoglobinuria workup. Medscape. Updated May 20, 2021. Accessed November 8, 2022.
  7. Paroxysmal nocturnal hemoglobinuria, Diagnosis. AA • MDS International Foundation. Accessed November 8, 2022.
  8. Paroxysmal nocturnal hemoglobinuria. Cleveland Clinic. Accessed November 8, 2022.
  9. Lima M. Laboratory studies for paroxysmal nocturnal hemoglobinuria, with emphasis on flow cytometry. Pract Lab Med. 2020;20:e00158. doi:10.1016/j.plabm.2020.e00158
  10. Braunstein EM. Paroxysmal nocturnal hemoglobinuria (PNH). MSD Manual, Professional Version. Reviewed/revised June 2022. Modified September 2022. 
  11. Erem AS, Asakrah S. Paroxysmal nocturnal hemoglobinuria (PNH). PathologyOutlines.com. Updated August 5, 2022. Accessed November 8, 2022.

Reviewed by Hasan Avcu, MD, on 11/16/2022.