Skip to main content
SpringerLink
Log in

Establishment of a flow cytometry assay for detecting paroxysmal nocturnal hemoglobinuria-type cells specific to patients with bone marrow failure

  • Original Article
  • Published:
Annals of Hematology Aims and scope Submit manuscript

Abstract

Minor populations of glycosylphosphatidylinositol-anchored protein-deficient (GPI[−]) cells in the peripheral blood may have a prognostic value in bone marrow failure (BMF). Our objective is to establish the optimal flow cytometry (FCM) assay that can discriminate GPI(−) populations specific to BMF from those of healthy individuals. To identify a cut-off that discriminates GPI(−) rare cells from GPI(+) cells, we determined a position of the borderline that separates the GPI(−) from GPI(+) cells on a scattergram by testing more than 30 healthy individuals, such that no GPI(−) dot fell into the upper left quadrant where fluorescein-labeled aerolysin (FLAER)−CD11b+ granulocytes and CD55−CD59− glycophorin A+ erythrocytes were positioned. This method allowed us to define ≥ 0.003% CD11b+FLAER− granulocytes and ≥ 0.005% glycophorin A+CD55−CD59− erythrocytes to be specific to BMF patients. Longitudinal cross-validation studies showed minimal (< 0.02%) inter-laboratory differences in the GPI(−) cell percentage. An analysis of 1210 patients with BMF revealed a GPI(−) cell population in 56.3% of patients with aplastic anemia and 18.5% of patients with myelodysplastic syndrome. The GPI(−) granulocyte percentages was 0.003–0.01% in 3.7% of patients. This FCM assay effectively identified an increase in the percentage of GPI(−) rare cells that are specific to BMF patients and allowed different laboratories to accurately detect 0.003–0.01% of pathological GPI(−) cells.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  1. Dunn TE, Tanawattanacharoen P, Boccuni P, Nagakura S, Green SW, Kirby MR, Kumar MS, Rosenfeld S, Young NS (1999) Paroxysmal nocturnal hemoglobinuria cells in patients with bone marrow failure syndromes. Ann Intern Med 131:401–408

    Article  CAS  PubMed  Google Scholar 

  2. Maciejewski JP, Rivera C, Kook H, Dunn D, Young NS (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 

  3. Sugimori C, Mochizuki K, Qi Z, Sugimori N, Ishiyama K, Kondo Y, Nakao S (2009) Origin and fate of blood cells deficient in glycosylphosphatidylinositol-anchored protein among patients with bone marrow failure. Br J Haematol 147(1):102–112

    Article  CAS  PubMed  Google Scholar 

  4. Hosokawa K, Sugimori N, Katagiri T, Sasaki Y, Saito C, Seiki Y, Mochizuki K, Yamazaki H, Takami A, Nakao S (2015) Increased glycosylphosphatidylinositol-anchored protein-deficient granulocytes define a benign subset of bone marrow failures in patients with trisomy 8. Eur J Haematol 95:230–238

    Article  CAS  PubMed  Google Scholar 

  5. Kulagin A, Lisukov I, Ivanova M, Golubovskaya I, Kruchkova I, Bondarenko S, Vavilov V, Stancheva N, Babenko E, Sipol A, Pronkina N, Kozlov V, Afanasyev B (2014) Prognostic value of paroxysmal nocturnal haemoglobinuria clone presence in aplastic anaemia patients treated with combined immunosuppression: results of two-centre prospective study. Br J Haematol 164:546–554

    Article  CAS  PubMed  Google Scholar 

  6. Maciejewski JP, Follmann D, Nakamura R, Saunthararajah Y, Rivera CE, Simonis T, Brown KE, Barrett JA, Young NS (2001) Increased frequency of HLA-DR2 in patients with paroxysmal nocturnal hemoglobinuria and the PNH/aplastic anemia syndrome. Blood 98:3513–3519

    Article  CAS  PubMed  Google Scholar 

  7. Narita A, Muramatsu H, Sekiya Y, Okuno Y, Sakaguchi H, Nishio N, Yoshida N, Wang X, Xu Y, Kawashima N, Doisaki S, Hama A, Takahashi Y, Kudo K, Moritake H, Kobayashi M, Kobayashi R, Ito E, Yabe H, Ohga S, Ohara A, Kojima S, Japan Childhood Aplastic Anemia Study Group (2015) Paroxysmal nocturnal hemoglobinuria and telomere length predicts response to immunosuppressive therapy in pediatric aplastic anemia. Haematologica 100:1546–1552

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Sugimori C, Chuhjo T, Feng X, Yamazaki H, Takami A, Teramura M, Mizoguchi H, Omine M, Nakao S (2006) Minor population of CD55-CD59- blood cells predicts response to immunosuppressive therapy and prognosis in patients with aplastic anemia. Blood 107:1308–1314

    Article  CAS  PubMed  Google Scholar 

  9. Wang H, Chuhjo T, Yasue S, Omine M, Nakao S (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 

  10. Araten DJ, Nafa K, Pakdeesuwan K, Luzzatto L (1999) Clonal populations of hematopoietic cells with paroxysmal nocturnal hemoglobinuria genotype and phenotype are present in normal individuals. Proc Natl Acad Sci U S A 96:5209–5214

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Hu R, Mukhina GL, Piantadosi S, Barber JP, Jones RJ, Brodsky RA (2005) PIG-A mutations in normal hematopoiesis. Blood 105:3848–3854

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Borowitz MJ, Craig FE, Digiuseppe JA, Illingworth AJ, Rosse W, Sutherland DR, Wittwer CT, Richards SJ (2010) Clinical Cytometry Society. Guidelines for the diagnosis and monitoring of paroxysmal nocturnal hemoglobinuria and related disorders by flow cytometry. Cytometry B Clin Cytom 78:211–230

    PubMed  Google Scholar 

  13. Dalal BI, Khare NS (2013) Flow cytometric testing for paroxysmal nocturnal hemoglobinuria: CD64 is better for gating monocytes than CD33. Cytometry B Clin Cytom 84:33–36

    Article  PubMed  Google Scholar 

  14. Tutelman PR, Aubert G, Milner RA, Dalal BI, Schultz KR, Deyell RJ (2014) Paroxysmal nocturnal haemoglobinuria phenotype cells and leucocyte subset telomere length in childhood acquired aplastic anaemia. Br J Haematol 164:717–721

    Article  CAS  PubMed  Google Scholar 

  15. Perfetto SP, Chattopadhyay PK, Roederer M (2004) Seventeen-colour flow cytometry: unravelling the immune system. Nat Rev Immunol 4:648–655

    Article  CAS  PubMed  Google Scholar 

  16. Katagiri T, Kawamoto H, Nakakuki T, Ishiyama K, Okada-Hatakeyama M, Ohtake S, Seiki Y, Hosokawa K, Nakao S (2013) Individual hematopoietic stem cells in human bone marrow of patients with aplastic anemia or myelodysplastic syndrome stably give rise to limited cell lineages. Stem Cells 31:536–546

    Article  CAS  PubMed  Google Scholar 

  17. 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 82:195–2008

    Article  PubMed  Google Scholar 

  18. Sutherland DR, Kuek N, Davidson J, Barth D, Chang H, Yeo E, Bamford S, Chin-Yee I, Keeney M (2007) Diagnosing PNH with FLAER and multiparameter flow cytometry. Cytometry B Clin Cytom 72:167–177

    Article  PubMed  Google Scholar 

  19. Wang SA, Pozdnyakova O, Jorgensen JL, Medeiros LJ, Stachurski D, Anderson M, Raza A, Woda BA (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(1):29–37

    Article  PubMed  Google Scholar 

Download references

Acknowledgments

We thank all participating physicians and registered patients who took part in this study.

Funding

Research fund from Alexion.

Author information

Authors and Affiliations

  1. Department of Hematology and Respirology, Kanazawa University, 13-1 Takaramachi, Kanazawa, Ishikawa, 920-8641, Japan

    Kohei Hosokawa, Ken Ishiyama, Hiroyuki Takamatsu & Shinji Nakao

  2. Japan PNH Study Group, Tokyo, Japan

    Kohei Hosokawa, Chiharu Sugimori, Ken Ishiyama, Hiroyuki Takamatsu, Hideyoshi Noji, Tsutomu Shichishima, Naoshi Obara, Shigeru Chiba, Haruhiko Ninomiya, Yukari Shirasugi, Yoshihiko Nakamura, Kiyoshi Ando, Yasutaka Ueda, Yuji Yonemura, Tatsuya Kawaguchi, Jun-ichi Nishimura, Yuzuru Kanakura & Shinji Nakao

  3. Department of Hematology, Ishikawa Prefectural Central Hospital, Ishikawa, Japan

    Chiharu Sugimori

  4. Department of Hematology, Fukushima Medical University, Fukushima, Japan

    Hideyoshi Noji & Tsutomu Shichishima

  5. Department of Hematology, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

    Naoshi Obara & Shigeru Chiba

  6. Department of Medical Sciences, Faculty of Medicine, University of Tsukuba, Tsukuba, Japan

    Haruhiko Ninomiya

  7. Department of Hematology and Oncology, Tokai University, Tokai, Japan

    Yukari Shirasugi, Yoshihiko Nakamura & Kiyoshi Ando

  8. Department of Hematology and Oncology, Osaka University, Osaka, Japan

    Yasutaka Ueda, Jun-ichi Nishimura & Yuzuru Kanakura

  9. Department of Hematology and Infectious Diseases, Kumamoto University, Kumamoto, Japan

    Yuji Yonemura & Tatsuya Kawaguchi

Authors
  1. Kohei Hosokawa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Chiharu Sugimori
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Ken Ishiyama
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hiroyuki Takamatsu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hideyoshi Noji
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Tsutomu Shichishima
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Naoshi Obara
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Shigeru Chiba
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Haruhiko Ninomiya
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Yukari Shirasugi
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Yoshihiko Nakamura
    View author publications

    You can also search for this author in PubMed Google Scholar

  12. Kiyoshi Ando
    View author publications

    You can also search for this author in PubMed Google Scholar

  13. Yasutaka Ueda
    View author publications

    You can also search for this author in PubMed Google Scholar

  14. Yuji Yonemura
    View author publications

    You can also search for this author in PubMed Google Scholar

  15. Tatsuya Kawaguchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  16. Jun-ichi Nishimura
    View author publications

    You can also search for this author in PubMed Google Scholar

  17. Yuzuru Kanakura
    View author publications

    You can also search for this author in PubMed Google Scholar

  18. Shinji Nakao
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

KH, CS, HN, TS, NO, YS, YN, YU, YY, and TK performed the research; TS, SC, HN, KA, YY, and TK collected patients’ samples. KI and HT collected samples from healthy volunteer. CS, JN, YK and SN designed the study; KH, CS, and SN wrote the paper.

Language check, by an independent medical writer, Caroline Dunstall, was provided by Alexion.

Corresponding author

Correspondence to Shinji Nakao.

Ethics declarations

All human subjects were collected after obtaining written informed consent for their participation in accordance with the Declaration of Helsinki through clinical protocols approved by the Institutional Review Board.

Conflicts of interest

Dr. Ninomiya, Dr. Shichishima, Dr. Yonemura, Dr. Kawaguchi, and Dr. Nakao report personal fees from Alexion Pharma G.K., outside the submitted work; Dr. Ninomiya, Dr. Chiba, Dr. Ueda, Dr. Yonemura, and Dr. Nakao report grants from Alexion Pharma G.K., outside the submitted work; Dr. Kanakura and Dr. Nishimura report grant and personal fees from Alexion Pharma G.K. during the conduct of the study; Dr. Shirasugi report honoraria from Novartis; Dr. Hosokawa, Dr. Sugimori, Dr. Ishiyama, Dr. Takamatsu, Dr. Noji, Dr. Obara, Dr. Nakamura, and Dr. Ando have nothing to declare.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Hosokawa, K., Sugimori, C., Ishiyama, K. et al. Establishment of a flow cytometry assay for detecting paroxysmal nocturnal hemoglobinuria-type cells specific to patients with bone marrow failure. Ann Hematol 97, 2289–2297 (2018). https://doi.org/10.1007/s00277-018-3443-1

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00277-018-3443-1

Keywords

Search

Navigation