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Diagnosis of Paroxysmal Nocturnal Hemoglobinuria: Recent Advances

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Indian Journal of Hematology and Blood Transfusion Aims and scope Submit manuscript

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematopoietic stem cell disorder with its protean clinical manifestations. This is due to partial or complete absence of ‘glycophosphatidyl-inositol-anchor proteins’ (GPI-AP). The main aim of this review is to highlight various diagnostic modalities available, basic principle of each test and recent advances in the diagnosis of PNH. Recently among various tests available, the flow cytometry has become ‘the gold standard’ for PNH testing. In order to overcome the difficulties encountered by the testing and research laboratories throughout the world, International Clinical Cytometry Society has come up with guidelines regarding the indications for testing, protocol for sample collection, processing, panel of antibodies as well as gating strategies to be used, how to interpret the test and reporting format to be used. It is essential to test at least two GPI-linked markers on at least two different lineages particularly on red cells and granulocytes/monocytes. The fluorescent aerolysin combined with other monoclonal antibodies in multicolour flow cytometry offered an improved assay not only for diagnosis but also for monitoring of PNH clones. It is equally important to diagnose this rare entity with high index of suspicion.

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References

  1. Parker C, Omine M, Richards S et al (2005) Diagnosis and management of paroxysmal nocturnal hemoglobinuria. Blood 106:3699–3709

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Pu JJ, Brodsky RA (2011) Paroxysmal nocturnal hemoglobinuria from bench to bedside. Clin Transl Sci 4:219–224

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  3. Richards SJ, Hill A, Hillmen P (2007) Recent advances in the diagnosis, monitoring and management of patients with paroxysmal nocturnal haemoglobinuria. Cytom B Clin Cytom 72:291–298

    Article  Google Scholar 

  4. Hill A, Richards S, Hillmen P (2007) Recent developments in the understanding and management of paroxysmal nocturnal hemoglobinuria. Br J Haematol 137:181–192

    Article  CAS  PubMed  Google Scholar 

  5. Brodsky RA (2008) Advances in the diagnosis and therapy of paroxysmal nocturnal hemoglobinuria. Blood Rev 22:65–74

    Article  CAS  PubMed  Google Scholar 

  6. Hu R, Mukhina GL, Piantadosi S et al (2005) PIG-A mutations in normal hematopoiesis. Blood 105:3848–3854

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Prince GM, Nguyen M, Lazarus HM et al (1995) Peripheral blood harvest of unaffected CD34+ CD38− hematopoietic precursors in paroxysmal nocturnal hemoglobinuria. Blood 86:3381–3386

    CAS  PubMed  Google Scholar 

  8. Zacks MA, Garg N (2006) Recent developments in the molecular, biochemical and functional characterization of GPI8 and the GPI-anchoring mechanisms. Mol Membr Biol 23:209–225

    Article  PubMed  Google Scholar 

  9. Bain BJ, Win N (2006) Acquired haemolytic anaemias. In: Lewis SM, Bain BJ and Bates I (eds) Dacie and Lewis Practical Haematology. Philadelphia; 10th ed, pp 239–266

  10. Zupanska B, Bogdanik B, Pyl H (2002) A gel microtyping system for diagnosis of paroxysmal nocturnal hemoglobinuria. Immunohematology 18:9–12

    CAS  PubMed  Google Scholar 

  11. Hall SE, Rosse WF (1996) The use of monoclonal antibodies and flow cytometry in the diagnosis of paroxysmal nocturnal hemoglobinuria. Blood 87:5332–5340

    CAS  PubMed  Google Scholar 

  12. Madkaikar M, Gupta M, Jijina F, Ghosh K (2009) Paroxysmal nocturnal haemoglobinuria: diagnostic tests, advantages, & limitations. Eur J Hematol 83:503–511

    Article  Google Scholar 

  13. Sutherland DR, Kuek N, Davidson J et al (2007) Diagnosing PNH with FLAER and multiparameter flow cytometry. Cytom B Clin Cytom 72:167–177

    Article  Google Scholar 

  14. Sutherland R, Keeney M, Illingworth A (2012) Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytom B Clin Cytom 82:195–208

    Article  Google Scholar 

  15. Brodsky RA, Mukhina G, Li S et al (2000) Improved detection and characterization of paroxysmal nocturnal hemoglobinuria using florescent aerolysin. Am J Clin Pathol 114:459–466

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Peghini PE, Fehr J (2005) Clinical evaluation of an aerolysin-based screening test for paroxysmal nocturnal hemoglobinuria. Cytom B Clin Cytom 67:13–18

    Article  Google Scholar 

  17. Movalia MK, Weitz IC, Lim SH, Illingworth A (2011) Incidence of PNH clones by diagnostic code utilizing high sensitivity flow cytometry. Blood (Abstracts from the 2011 American Society of Hematology Annual Meeting) 118:Abstract 1033

  18. Latour RP, Mary JY, Salanoubat C et al (2008) Paroxysmal nocturnal hemoglobinuria: natural history of disease subcategories. Blood 112:3099–3106

    Article  PubMed  Google Scholar 

  19. Schrezenmeier H, Muus P, Socie G et al (2014) Baseline characteristics and disease burden in patients in the International Paroxysmal Nocturnal Hemoglobinuria Registry. Haematologica 99:922–929

    Article  PubMed  PubMed Central  Google Scholar 

  20. Mahapatra M, Singh PK, Agarwal M et al (2015) Epidemiology, clinico-haematological profile and management of aplastic anemia: AIIMS experience. J Assoc Physicians India 63:30–35

    CAS  PubMed  Google Scholar 

  21. Wang SA, Pozdnyakova O, Jorgensen JL et al (2009) Detection of paroxysmal nocturnal hemoglobinuria clones in patients with myelodysplastic syndromes and related bone marrow diseases, with emphasis on diagnostic pitfalls and caveats. Haematologica 94:29–37

    Article  PubMed  Google Scholar 

  22. Mukhina GL, Buckley JT, Barber JP et al (2001) Multilineage glycosylphosphatidylinositol anchor deficient hematopoiesis in untreated aplastic anaemia. Br J Haematol 115:476–482

    Article  CAS  PubMed  Google Scholar 

  23. Sachdeva MU, Varma N, Chandra D et al (2015) Multiparameter FLAER-based flow cytometry for screening of paroxysmal nocturnal hemoglobinuria enhances detection rates in patients with aplastic anemia. Ann Hematol 94:721–728

    Article  CAS  PubMed  Google Scholar 

  24. Wang H, Chuhjo T, Yamazaki H et al (2001) Relative increase of granulocytes with a paroxysmal nocturnal hemoglobinuria phenotype in aplastic anemia patients: the high prevalence at diagnosis. Eur J Haematol 66:200–205

    Article  CAS  PubMed  Google Scholar 

  25. Wang H, Chuhjo T, Yasue S et al (2002) Clinical significance of a minor population of paroxysmal nocturnal hemoglobinuria-type cells in bone marrow failure syndrome. Blood 100:3897–3902

    Article  CAS  PubMed  Google Scholar 

  26. Agarwal R, Chapple P, Brown M et al (2015) Analysis of abnormal clones by the fluorescent aerolysin method in paroxysmal nocturnal haemoglobinuria and other marrow disorders. Int J Lab Hematol 37:14–21

    Article  CAS  PubMed  Google Scholar 

  27. Maciejewski JP, Rivera C, Kook H et al (2001) Relationship between bone marrow failure syndromes and the presence of glycophosphatidyl inositol anchored protein deficient clones. Br J Haematol 115:1015–1022

    Article  CAS  PubMed  Google Scholar 

  28. Young NS, Abkowitz JL, Luzzattoet L (2000) New insights into the pathophysiology of acquired cytopenias. Hematology Am Soc Hematol Educ Program, pp 18–38

  29. Brodsky RA (2006) New insights into paroxysmal nocturnal hemoglobinuria. Hematology Am Soc Hematol Educ Program, pp 24–28

  30. Bessler M, Hiken J (2008) The pathophysiology of disease in patients with paroxysmal nocturnal hemoglobinuria. Hematology Am Soc Hematol Educ Program, pp 104–108

  31. Borowitz MJ, Craig FE, DiGuiseppe JA, On behalf of the Clinical Cytometry Society et al (2010) Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytom B Clin Cytom 78:211–230

    Google Scholar 

  32. Sharma VR (2013) Paroxysmal nocturnal hemoglobinuria: pathogenesis, testing and diagnosis. Clin Adv Hematol and Oncol. 11:2–8

    Google Scholar 

  33. Gupta R, Pandey P, Choudhry R et al (2007) A prospective comparison of four techniques for diagnosis of paroxysmal nocturnal hemoglobinuria. Int J Lab Hematol 29:119–126

    Article  CAS  PubMed  Google Scholar 

  34. Oelschlaegel U, Besson I, Arnoulet C et al (2001) A standardized flow cytometric method for screening paroxysmal nocturnal haemoglobinuria (PNH) measuring CD55 and CD59 expression on erythrocytes and granulocytes. Clin Lab Haematol 23:81–90

    Article  CAS  PubMed  Google Scholar 

  35. Piedras J, López-Karpovitch X (2000) Flow cytometric analysis of glycosylphosphatidylinositol–anchored proteins to assess paroxysmal nocturnal hemoglobinuria clone size. Cytometry 42:234–238

    Article  CAS  PubMed  Google Scholar 

  36. Hernandez-Campo PM, Almeida J, Sanchez ML et al (2006) Normal patterns of expression of glycosylphosphatidylinositol-anchored proteins on different subsets of peripheral blood cells: a frame of reference for the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytom B Clin Cytom 70:71–81

    Article  Google Scholar 

  37. Richards SJ, Whitby L, Cullen MJ et al (2009) Development and evaluation of a stabilized whole-blood preparation as a process control material for screening of paroxysmal nocturnal hemoglobinuria by flowcytometry. Cytom B Clin Cytom 76:47–55

    Article  Google Scholar 

  38. Richards SJ, Rawston AC, Hillmen P (2000) Application of flow cytometry to the diagnosis of paroxysmal nocturnal hemoglobinuria. Cytometry 42:223–233

    Article  CAS  PubMed  Google Scholar 

  39. Richards SJ, Hillmen P (2002) Immunophenotypic analysis of PNH cells. Curr Protoc Cytom. doi:10.1002/0471142956.cy0611s20

    PubMed  Google Scholar 

  40. Navenot JM, Muller JY, Blanchard D (1998) Investigation of the survival of paroxysmal nocturnal hemoglobinuria red cells through the immunophenotyping of reticulocytes. Transfusion 38:337–342

    Article  CAS  PubMed  Google Scholar 

  41. Ware RE, Rosse WF, Hall SE (1995) Immunophenotypic analysis of reticulocytes in paroxysmal nocturnal hemoglobinuria. Blood 86:1586–1589

    CAS  PubMed  Google Scholar 

  42. Diep DB, Nelson KL, Raja SM et al (1998) Glycosylphosphatidylinositol anchors of membrane glycoproteins are binding determinants for the channel-forming toxin aerolysin. J Biol Chem 273:2355–2360

    Article  CAS  PubMed  Google Scholar 

  43. Brodsky RA, Mukhina GL, Nelson KL et al (1999) Resistance of paroxysmal nocturnal hemoglobinuria cells to the glycosylphosphatidylinositol-binding toxin aerolysin. Blood 93:1749–1756

    CAS  PubMed  Google Scholar 

  44. Sreedharanunni S, Sachdeva MU, Bose P et al (2016) Frequency of Paroxysmal nocturnal hemoglobinuria clones by multiparametric flow cytometry in pediatric aplastic anemia patients of Indian Ethnic origin. Pediatr Blood Cancer 63:93–97

    Article  CAS  PubMed  Google Scholar 

  45. Parker CJ (2016) Update on the diagnosis and management of paroxysmal nocturnal hemoglobinuria. Hematology Am Soc Hematol Educ Program, pp 208–216

  46. Sutherland DR, Acton E, Keeney M et al (2014) Use of CD157 in FLAER-based assays for high-sensitivity PNH granulocyte and PNH monocyte detection. Cytom B Clin Cytom 86:44–55

    Article  CAS  Google Scholar 

  47. Weitz IC, Razavi P, Rochanda L et al (2012) Eculizumab therapy results in rapid and sustained decreases in markers of thrombin generation and inflammation in patients with PNH independent of its effects on hemolysis and microparticle formation. Thromb Res 130:361–368

    Article  CAS  PubMed  Google Scholar 

  48. Wanachiwanawin W, Siripanyaphinyo U et al (2006) A Cohort study of the nature of paroxysmal nocturnal hemoglobinuria clones and PIG-A mutations in patients with aplastic anemia. Eur J Haematol 76:502–509

    Article  CAS  PubMed  Google Scholar 

  49. Sugimori C, Mochizuki K, Qi Z et al (2009) Origin and fate of blood cells deficient in glycosylphosphatidylinositol-anchored protein among patients with bone marrow failure. Br J Haematol 147:102–112

    Article  CAS  PubMed  Google Scholar 

  50. Raza A, Ravandi F, Rastogi A et al (2014) A prospective multicentre study of paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow failure. Cytom B Clin Cyto 86:175–182

    Article  Google Scholar 

  51. Mayo VM, Mukhina GL, Garrett ES et al (2004) Natural history of paroxysmal nocturnal hemoglobinuria using modern diagnostic assays. Br J Haematol 126:133–138

    Article  Google Scholar 

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Acknowledgements

Currently Dr. Prabhu Manivannan is working as Assistant Professor in the department of Pathology, JIPMER, Puducherry would like to extend his acknowledgement to all laboratory technicians and Dr. Roopam Deka (Senior Resident), All India Institute of Medical Sciences (AIIMS), New Delhi for their dedicated work and for contributing CD157 plot for this manuscript. He would also like to acknowledge his wife Mrs. Manjari for final proof-reading of this manuscript. Dr. (Lt. Col) Ankur Ahuja is currently working as Assistant Professor in the department of Laboratory services, Army hospital Research and Referral, New Delhi. He would like to acknowledge his department as well as the hospital for letting him to contribute to this manuscript.

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Authors and Affiliations

  1. Department of Pathology, JIPMER, Puducherry, India

    Prabhu Manivannan

  2. Department of Laboratory Services, Army Hospital for Research and Referral, New Delhi, India

    Ankur Ahuja

  3. Department of Hematology, All India Institute of Medical Sciences (AIIMS), New Delhi, India

    Hara Prasad Pati

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  1. Prabhu Manivannan
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Correspondence to Hara Prasad Pati.

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Manivannan, P., Ahuja, A. & Pati, H.P. Diagnosis of Paroxysmal Nocturnal Hemoglobinuria: Recent Advances. Indian J Hematol Blood Transfus 33, 453–462 (2017). https://doi.org/10.1007/s12288-017-0868-y

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