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Immunophenotyping of Paroxysmal Nocturnal Hemoglobinuria (PNH)

  • Andrea J. IllingworthEmail author
  • Iuri Marinov
  • D. Robert Sutherland
Protocol
  • 1.5k Downloads
Part of the Methods in Molecular Biology book series (MIMB, volume 2032)

Abstract

Paroxysmal nocturnal hemoglobinuria (PNH) is a rare but often debilitating disease which may lead to death in up to 35% of patients within 5 years if unrecognized and untreated. Detection of PNH and assessment of PNH clone size in RBC and WBC lineages by flow cytometric analysis has increased in importance due to the availability of novel therapies. These therapies typically block the hemolysis of red blood cells and thus significantly lower the morbidities and mortality associated with this disease. This chapter describes validated, state-of-the-art, high-sensitivity flow cytometric methodologies based on latest published testing guidelines for PNH.

Key words

Paroxysmal nocturnal hemoglobinuria (PNH) Glycophosphatidylinositol (GPI)-anchored protein Aplastic anemia (AA) Myelodysplastic syndrome (MDS) CD59 FLAER 

References

  1. 1.
    Parker CJ (2007) The pathophysiology of paroxysmal nocturnal hemoglobinuria. Exp Hematol 35:523–533CrossRefGoogle Scholar
  2. 2.
    Oni SB, Osunkoya BO, Luzzatto L (1970) Paroxysmal nocturnal hemoglobinuria: evidence for monoclonal origin of abnormal red cells. Blood 36:145–152PubMedGoogle Scholar
  3. 3.
    Young NS, Calado RT, Scheinberg P (2006) Current concepts in the pathophysiology and treatment of aplastic anaemia. Blood 108:509–519CrossRefGoogle Scholar
  4. 4.
    Kordasti S, Marsh J, Al-Khan S et al (2012) Functional characterization of CD4+ T cells in aplastic anemia. Blood 119:2033–2043CrossRefGoogle Scholar
  5. 5.
    Solomou EE, Rezvani K, Mielke S et al (2007) Deficient CD4+CD25+FOXP3+ T regulatory cells in acquired aplastic anemia. Blood 110:1603–1606CrossRefGoogle Scholar
  6. 6.
    Kordasti SY, Afzali B, Lim Z et al (2009) IL-17-producing CD4 (1) T-cells, proinflammatory cytokines and apoptosis are increased in low-risk myelodysplastic syndrome. Br J Haematol 145:64–72CrossRefGoogle Scholar
  7. 7.
    Mufti GJ, Marsh J (2018) Somatic mutations in aplastic anemia. Hematol Oncol Clin N Am 32:595–607CrossRefGoogle Scholar
  8. 8.
    DeZern AE, Symons HJ, Resar LS et al (2014) Detection of paroxysmal nocturnal hemoglobinuria clones to exclude inherited bone marrow failure syndromes. Eur J Haematol 92:467–470CrossRefGoogle Scholar
  9. 9.
    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 147:102–112CrossRefGoogle Scholar
  10. 10.
    Raza A, Ravandi F, Rastogi A et al (2014) A prospective multicenter study of paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow failure. Cytometry B Clin Cytom 86:175–182CrossRefGoogle Scholar
  11. 11.
    Morado M, Freire Sandes A, Colado E et al (2017) Diagnostic screening of paroxysmal nocturnal hemoglobinuria: Prospective multicentric evaluation of the current medical indications. Cytometry B Clin Cytom 92B:361–370CrossRefGoogle Scholar
  12. 12.
    Dezern AE, Borowitz MJ (2018) Consensus guidelines for the flow cytometry testing of patients with suspected paroxysmal hemoglobinuria (PNH) and related disorders part 1—clinical utility. Cytometry B Clin Cytom 94:16–22CrossRefGoogle Scholar
  13. 13.
    Hill A, Rother RP, Wang X et al (2010) Effect of eculizumab on haemolysis-associated nitric oxide depletion, dyspnoea, and measures of pulmonary hypertension in patients with paroxysmal nocturnal haemoglobinuria. Br J Haematol 149:414–425CrossRefGoogle Scholar
  14. 14.
    Socie G, Mary JY, de Gramont A et al (1996) Paroxysmal nocturnal haemoglobinuria: long-term follow-up and prognostic factors. French Society of Haematology. Lancet 348:573–577CrossRefGoogle Scholar
  15. 15.
    Sugimori C, Chuhjo T, Feng X et al (2006) Minor population of CD55-CD59- blood cells predicts response to immunosuppressive therapy and prognosis in patients with aplastic anemia. Blood 107:1308–1314CrossRefGoogle Scholar
  16. 16.
    Borowitz MJ, Craig FE, DiGiuseppe JA et al (2010) Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom 78B:211–230Google Scholar
  17. 17.
    Killick SB, Bown N, Cavenagh J et al (2016) Guidelines for the diagnosis and management of adult aplastic anaemia. Br J Haematol 172:187–207CrossRefGoogle Scholar
  18. 18.
    Sutherland DR, Keeney M, Illingworth A (2012) Practical guidelines for the high-sensitivity detection and monitoring of paroxysmal nocturnal hemoglobinuria clones by flow cytometry. Cytometry B Clin Cytom 82:195–208. and Supplementary DataCrossRefGoogle Scholar
  19. 19.
    Illingworth A, Marinov I, Sutherland DR et al (2018) ICCS/ESCCA consensus guidelines to detect GPI-deficient cells in PNH and related disorders part 3—data analysis, reporting and case studies. Cytometry B Clin Cytom 94:49–66. and Supplementary DataCrossRefGoogle Scholar
  20. 20.
    Oldaker T, Whitby L, Saber M et al (2018) ICCS/ESCCA Consensus Guidelines for the Flow Cytometry Testing of Patients with suspected Paroxysmal Hemoglobinuria (PNH) and related Disorders Part 4 - Assay Validation and quality assurance. Cytometry B Clin Cytom 94:67–81CrossRefGoogle Scholar
  21. 21.
    Illingworth A, Oldaker T, Sutherland DR et al.: Verification of PNH assay sensitivity through spiking experiment. (2018). https://www.cytometry.org/web/modules/Module10.pdf
  22. 22.
    Davis BH, Keeney M, Brown R et al (2014) CLSI H52-A2 red blood cell diagnostic testing using flow cytometry; approved guideline, 2nd edn. Clinical and Laboratory Standards Institute, Wayne, PA. ISBN Number: 1-56238-957-2Google Scholar
  23. 23.
    Sutherland DR, Richards SJ, Keeney M et al (2015) High-sensitivity detection of PNH red blood cells, red cell precursors and white blood cells. Curr Protoc Cytom 72(Unit 6.37):1–29Google Scholar
  24. 24.
    Sutherland DR, Illingworth A, Marinov I et al (2018) ICCS/ESCCA Consensus Guidelines to detect GPI-deficient cells in Paroxysmal Nocturnal Hemoglobinuria (PNH) and related disorders part 2—reagent selection and assay optimization for high-sensitivity testing. Cytometry B Clin Cytom 94:23–48CrossRefGoogle Scholar
  25. 25.
    Sutherland DR, Kuek N, Azcona-Olivera J et al (2009) Use of FLAER-based white blood cell assay in the primary screening of PNH clones. Am J Clin Pathol 132:564–572CrossRefGoogle Scholar
  26. 26.
    Dalal BI, Khare NS (2013) Flow cytometric testing for paroxysmal nocturnal hemoglobinuria: CD64 is better for gating monocytes than CD33. Cytometry B Clin Cytom 84:33–36CrossRefGoogle Scholar
  27. 27.
    Sutherland DR, Ortiz F, Quest G et al (2018) High-sensitivity 5-, 6-, and 7-color PNH WBC Assays for both Canto II and Navios Platforms. Cytometry B Clin Cytom 94:637–651CrossRefGoogle Scholar
  28. 28.
    Keeney M, Illingworth A, Sutherland DR (2017) Paroxysmal Nocturnal Hemoglobinuria assessment by flow cytometric analysis. Clin Lab Med 37:855–867CrossRefGoogle Scholar
  29. 29.
    Sutherland DR, Richards SJ, Ortiz, F et al. Addition of CD71 to high-sensitivity CD235a/CD59 PNH RBC assay allows the additional accurate delineation of PNH Type III, PNH Type II and normal (Type I) nucleated RBCs: Cytometry B 2019, in pressGoogle Scholar
  30. 30.
    Hernández-Campo PM, Almeida J, Sánchez 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. Cytometry B Clin Cytom 70:71–81CrossRefGoogle Scholar
  31. 31.
    Sutherland DR, Acton E, Keeney M et al (2014) Use of CD157 in FLAER-based assays for high-sensitivity PNH neutrophil and PNH monocyte detection. Cytometry B Clin Cytom 86:44–55CrossRefGoogle Scholar
  32. 32.
    Owens MA, Vall HG, Hurley AA, Wormsley SB (2000) Validation and quality control of immunophenotyping in clinical flow cytometry. J Immunol Methods 243:33–50CrossRefGoogle Scholar
  33. 33.
    International Organization for Standardization (ISO) Statistics-Vocabulary and Symbols. ISO 3534-1. Geneva; 1993Google Scholar
  34. 34.
  35. 35.
    Libeer J-C (2001) Role of external quality assurance schemes in assessing and improving quality in medical laboratories. Clin Chim Acta 309:173–177CrossRefGoogle Scholar
  36. 36.
    CLSI EP12-A2 (2008) User Protocol for Evaluation of Qualitative Test Performance- Second Edition – Approved Guideline. National Committee for Clinical Laboratory Standards, Wayne, PAGoogle Scholar
  37. 37.
    Clinical and Laboratory Standards Institute (2004) Protocols for Determination of Limits of Detection and Limits of Quantitation Approved Guideline. CLSI document EP17. CLSI, Wayne, PAGoogle Scholar
  38. 38.
    New York State Department of Health (2011) Clinical Laboratory Evaluation Program, Assay Approval in Cellular Immunology. New York State Department of Health, Albany, NYGoogle Scholar
  39. 39.
    Marinov I, Kohoutova M, Tkacova V et al (2013) Intra- and inter-laboratory variability of paroxysmal nocturnal hemoglobinuria testing by flow cytometry following the 2012 Practical Guidelines for high-sensitivity paroxysmal nocturnal hemoglobinuria testing. Cytometry B Clin Cytom 84:229–236CrossRefGoogle Scholar
  40. 40.
    Fletcher M, Sutherland DR, Whitby L et al (2014) Standardizing leucocyte PNH clone detection: An international study. Cytometry B Clin Cytom 86B:311–318CrossRefGoogle Scholar
  41. 41.
    Illingworth A, Keeney SDR (2016) High-sensitivity detection of PNH Red and White Blood Cells by Multiparameter Flow Cytometry. In: Detrick B, Schmitz JL, Hamilton RG (eds) Manual of Clinical Laboratory Immunology, 8th edn. ASM Press, Washington DC, pp 168–181. Chapter 18Google Scholar
  42. 42.
    Marinov I, Kohoutová M, Tkáčová V et al (2015) Clinical relevance of CD157 for rapid and cost-effective simultaneous evaluation of PNH granulocytes and monocytes by flow cytometry. Int J Lab Hematol 37:231–237CrossRefGoogle Scholar
  43. 43.
    Blaha J, Schwarz K, Fischer C et al (2018) The Monoclonal Anti-CD157 Antibody Clone SY11B5, used for High Sensitivity detection of PNH Clones on WBCs, fails to Detect a Common Polymorphic Variant Encoded by BST-1 published in line. Cytometry B Clin Cytom 94:652CrossRefGoogle Scholar
  44. 44.
    Sutherland DR, Musani R (2019) Re Blaha J et al.: Re: Blaha J et al.: The monoclonal anti-CD157 antibody clone SY11B5, used for high sensitivity detection of PNH clones on WBCs, fails to detect a common polymorphic variant encoded by BST-1 Cytometry B Clin Cytom 96(1):16-18CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Andrea J. Illingworth
    • 1
    Email author
  • Iuri Marinov
    • 2
  • D. Robert Sutherland
    • 3
  1. 1.Dahl-Chase Diagnostic ServicesBangorUSA
  2. 2.Institute of Hematology and Blood TransfusionPrague 2Czech Republic
  3. 3.University of TorontoTorontoCanada

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