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Article Open Access

High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections

  • Authors:
    • Ray Chun-Fai Chan
    • Richard H. Leung
    • Albert Posadas
    • Thomas S. Lorey
    • Allison J. Shaw
  • View Affiliations / Copyright

    Affiliations: Department of Flow Cytometry, Kaiser Permanente Regional Laboratory Northern California, The Permanente Medical Group, Inc., Berkeley, CA 94710, USA
    Copyright: © Chan et al. This is an open access article distributed under the terms of Creative Commons Attribution License.
  • Pages: 224-234
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    Published online on: January 24, 2018
       https://doi.org/10.3892/br.2018.1047
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Abstract

Flow cytometry is the gold standard in diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) by detecting the absence of glycol-phosphatidyl inositol (GPI)‑linked protein expression on granulocyte and monocyte surfaces. However, the current assays are not optimized and require improvement, particularly in reducing background fluorescence and optimizing sensitivity and speci­ficity. With more fluorochromes available and with advances in instrument engineering, rare populations may be identified with high sensitivity. The present study assessed an 8-color combination of comprehensive GPI-linked markers, namely fluorescein-labeled proaerolysin (FLAER), cluster of differentiation 157 (CD157), CD24 and CD14, and the lineage markers for granulocyte (CD15) and monocyte (CD64) cells to detect PNH clones. Additionally, to optimize the PNH flow assay, a ‘dump’ channel was used, comprised of CD5 and CD19, to exclude non-specific binding in order to reduce background. This method aimed to improve sensitivity and reduce the background to create an optimized PNH flow cocktail. The results demonstrated that the current 4-color PNH combination identifies a CD55- and FLAER+ population that is not PNH clones. By contrast, the 8-color panel delineated PNH clones from both monocyte and granulocytes by using granulocyte antigen (CD15) and monocyte antigen (CD64) as a gating strategy. The sensitivity was 0.01% for granulocytes and 0.05% for monocytes with an acquisition of 100,000 monocyte and granulocyte events. The background on a normal whole blood sample was 0.00076% on monocytes and 0.00277% on granulocytes. Thus, overall, the 8-color PNH assay exhibited high levels of specificity and sensitivity. The 8-color combination facilitated the improvement and enhancement of sensitivity in PNH clone identification, and may provide a useful tool for pathologists in PNH diagnosis and for monitoring patients at risk of developing classical/hemolytic PNH, to enable treatment to be delivered promptly.
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Copy and paste a formatted citation
Spandidos Publications style
Chan R, Leung RH, Posadas A, Lorey TS and Shaw AJ: High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections. Biomed Rep 8: 224-234, 2018.
APA
Chan, R., Leung, R.H., Posadas, A., Lorey, T.S., & Shaw, A.J. (2018). High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections. Biomedical Reports, 8, 224-234. https://doi.org/10.3892/br.2018.1047
MLA
Chan, R., Leung, R. H., Posadas, A., Lorey, T. S., Shaw, A. J."High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections". Biomedical Reports 8.3 (2018): 224-234.
Chicago
Chan, R., Leung, R. H., Posadas, A., Lorey, T. S., Shaw, A. J."High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections". Biomedical Reports 8, no. 3 (2018): 224-234. https://doi.org/10.3892/br.2018.1047
Copy and paste a formatted citation
x
Spandidos Publications style
Chan R, Leung RH, Posadas A, Lorey TS and Shaw AJ: High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections. Biomed Rep 8: 224-234, 2018.
APA
Chan, R., Leung, R.H., Posadas, A., Lorey, T.S., & Shaw, A.J. (2018). High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections. Biomedical Reports, 8, 224-234. https://doi.org/10.3892/br.2018.1047
MLA
Chan, R., Leung, R. H., Posadas, A., Lorey, T. S., Shaw, A. J."High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections". Biomedical Reports 8.3 (2018): 224-234.
Chicago
Chan, R., Leung, R. H., Posadas, A., Lorey, T. S., Shaw, A. J."High sensitivity 8-color flow cytometry assay for paroxysmal nocturnal hemoglobinuria granulocyte and monocyte detections". Biomedical Reports 8, no. 3 (2018): 224-234. https://doi.org/10.3892/br.2018.1047
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