Manivannan Prabhu, Tyagi Seema, Pati Hara Prasad, Saxena Renu
Department of Pathology, JIPMER, Puducherry, India.
Department of Hematology, All India Institute of Medical Sciences (AIIMS), Ansari Nagar, New Delhi, 110068 India.
Indian J Hematol Blood Transfus. 2020 Jul;36(3):526-534. doi: 10.1007/s12288-019-01220-8. Epub 2019 Nov 5.
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 < 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 '' 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.
流式细胞术已成为检测阵发性夜间血红蛋白尿（PNH）、再生障碍性贫血（AA）和骨髓增生异常综合征（MDS）中异常克隆的“金标准”。这项初步研究于2015年进行，主要目的是评估基于单管荧光气溶素（FLAER）检测的效用及其与基于两管非FLAER检测（CD55、CD59、CD24和CD66b）在检测这些新诊断病例中异常PNH克隆方面的比较。次要目的是试图根据临床、实验室特征以及诊断时的克隆大小，将PNH与伴有PNH克隆的AA/MDS病例区分开来。在本研究中，对所有PNH病例（n = 12）、健康受试者（n = 18）和伴有PNH克隆的AA/MDS病例（n = 9），使用基于单管FLAER的检测和基于两管非FLAER的检测来检测异常PNH克隆，并与诊断时的临床和实验室特征进行比较。受试者操作特征曲线（ROC）分析确定了粒细胞（> 0.7%）和单核细胞（> 0.9%）中FLAER的最佳临界值。在这些细胞中，基于FLAER的检测与基于非FLAER的检测之间存在显著正相关（r > 0.3且< 0.05）。基于FLAER的检测帮助我们发现了4例患者中被后一种技术遗漏的较小克隆，从而提高了其敏感性，并且在技术上也证明具有成本效益（1800卢比对2100卢比）。即使在PNH患者中，与基于非FLAER的抗体组合相比，使用FLAER时克隆大小也略高。在PNH和AA/MDS组中，单核细胞的克隆大小始终高于粒细胞。诊断时骨髓细胞密度以及粒细胞和单核细胞克隆的平均大小在这些组之间显示出惊人的统计学显著“值”< 0.0001。在这项初步研究中，基于单管FLAER的PNH检测在所有PNH病例、伴有PNH克隆的AA/MDS病例中提高了克隆检测率。大多数PNH病例中的克隆大小> 30%，而在AA/MDS中，诊断时通常< 10%。因此，这项新技术不仅提高了PNH克隆检测的敏感性，而且还证明具有成本效益。
