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FLAER Based Assay According to Newer Guidelines Increases Sensitivity of PNH Clone Detection

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

Abstract

Flow cytometry has become ‘gold standard’ for detecting abnormal clones in paroxysmal nocturnal hemoglobinuria (PNH), aplastic anemia (AA) and myelodysplastic syndrome (MDS). This pilot study was conducted in 2015 with a primary aim to evaluate the utility of single tube fluorescent aerolysin (FLAER) based testing and its comparison with two tubes non-FLAER based testing (CD55, CD59, CD24 and CD66b) in detecting abnormal PNH clones in these newly diagnosed cases. The secondary aim was an attempt to distinguish PNH from AA/MDS cases associated with PNH clones based on clinical, laboratory features and clone size at diagnosis. In this study, the abnormal PNH clones were detected using a single tube FLAER based testing and two tubes non-FLAER based testing in all cases of PNH (n = 12), healthy subjects (n = 18) and AA/MDS with PNH clone (n = 9) and compared with clinical and laboratory features at diagnosis. The receiver operator curve (ROC) analysis defined the optimal cut-offs for FLAER in granulocytes (> 0.7%) and monocytes (> 0.9%). There was significant positive correlation between FLAER and non-FLAER based testing in these cells (r > 0.3 and p < 0.05). FLAER based testing helped us in picking up smaller clones which were missed by latter technique in four patients thereby increasing its sensitivity and also technically proved to be cost-effective (Rs. 1800 vs. Rs. 2100). Even in PNH patients, the clone size was slightly higher by using FLAER when compared to non-FLAER based antibodies panel. The clone size of monocytes was always higher than granulocytes in both PNH and AA/MDS groups. Bone marrow cellularity and mean size of granulocytes and monocytes clone at diagnosis showed a striking statistically significant ‘p’ value of < 0.0001 between these groups. In this pilot study, a single tube FLAER based PNH testing had improved clone detection in all cases of PNH, AA/MDS with PNH clones. The clone size was > 30% in majority of PNH cases whereas in AA/MDS, it was usually < 10% at diagnosis. Hence this newer technique not only increased the sensitivity of PNH clone detection but also proved to be cost-effective.

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Acknowledgements

We thank “Indian Society of Hematology and Blood Transfusion” for funding Rs. 50,000/– Grant for this study.

Funding

Funding was provided by HERS and Indian Society of Hematology and Blood Transfusion (Grant No. IESC/T-161/02.05.2014).

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Authors and Affiliations

  1. Department of Pathology, JIPMER, Puducherry, India

    Prabhu Manivannan

  2. Department of Hematology, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, 110068, India

    Seema Tyagi, Hara Prasad Pati & Renu Saxena

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  1. Prabhu Manivannan
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  2. Seema Tyagi
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Correspondence to Seema Tyagi.

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Manivannan, P., Tyagi, S., Pati, H.P. et al. FLAER Based Assay According to Newer Guidelines Increases Sensitivity of PNH Clone Detection. Indian J Hematol Blood Transfus 36, 526–534 (2020). https://doi.org/10.1007/s12288-019-01220-8

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  • DOI: https://doi.org/10.1007/s12288-019-01220-8

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