Deka Roopam, Pati Hara P, Chandra Dinesh, Manivannan Prabhu, Chauhan Richa, Tyagi Seema, Saxena Renu
Oncopathology/Pathology/Hematopathology, Dr Bhubaneswar Borooah Cancer Institute, Guwahati, IND.
Hematology/Pathology, All India Institute of Medical Sciences, New Delhi, IND.
Cureus. 2022 Apr 8;14(4):e23965. doi: 10.7759/cureus.23965. eCollection 2022 Apr.
Introduction As per current guidelines, detection of paroxysmal nocturnal hematuria (PNH) clones on leucocytes requires the demonstration of the loss of at least two glycosyl-phosphatidyl-inositol (GPI)-linked molecules on both neutrophils and monocytes by flow cytometry. CD24 and CD14 are GPI-linked molecules expressed on neutrophils and monocytes respectively, whereas another GPI-linked molecule, CD157, is expressed on both neutrophils and monocytes. This prospective study evaluated the ability of CD157 to replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes. Materials and methods PNH clones were newly detected in 52 patients by an existing "standard" single-tube six-color flow-cytometric method, which was routinely performed in our laboratory at the time of undertaking this study. Six antibodies (CD45/CD15/CD64/CD24/CD14/FLAER) were used in this "standard" technique. Subjects were divided into two groups: (i) PNH disease (n=10), and (ii) aplastic anemia/myelodysplastic syndrome (AA/MDS) (n=42). Diagnosis of PNH disease and AA/MDS were made as per standard literature and guidelines. Results were compared with a single-tube five-color "test" assay using the antibodies CD45/CD15/CD64/CD157/FLAER by flow cytometry. Samples from 20 healthy control subjects were used to calculate cut-off values for the "test" assay. Results By the "test" method, cut-off values for detecting PNH clones obtained from receiver operating-characteristic curve analysis were >0.4% for neutrophils (sensitivity=96.15%, specificity=95%), and >0.9% for monocytes (sensitivity=98.08%, specificity=95%). There was significant correlation between PNH clone sizes measured by both the "standard" and "test" assays in neutrophils (PNH disease: r=0.976, p<0.001; AA/MDS: r=0.980, p<0.001) as well as monocytes (PNH disease: r=0.806, p=0.005; AA/MDS: r=0.915, p<0.001). Bland-Altman analysis showed agreement between both assays in all the 52 patients and in individuals with AA/MDS. The cost of the test to the patients was about 15% less in the "test" method than the "standard" technique, with improved technical efficiency. Conclusion CD157 can replace both CD24 and CD14 in a single-tube flow-cytometric assay to detect PNH clones on both neutrophils and monocytes, with reduced cost to the patients and improved technical efficiency.
引言 根据当前指南，检测白细胞上的阵发性夜间血红蛋白尿（PNH）克隆需要通过流式细胞术证明中性粒细胞和单核细胞上至少两种糖基磷脂酰肌醇（GPI）连接分子的缺失。CD24和CD14分别是在中性粒细胞和单核细胞上表达的GPI连接分子，而另一种GPI连接分子CD157在中性粒细胞和单核细胞上均有表达。这项前瞻性研究评估了在单管流式细胞术检测中，CD157替代CD24和CD14两者以检测中性粒细胞和单核细胞上PNH克隆的能力。
材料与方法 通过现有的“标准”单管六色流式细胞术方法在52例患者中首次检测到PNH克隆，在开展本研究时该方法在我们实验室常规使用。此“标准”技术使用六种抗体（CD45/CD15/CD64/CD24/CD14/FLAER）。受试者分为两组：（i）PNH疾病组（n = 10），和（ii）再生障碍性贫血/骨髓增生异常综合征（AA/MDS）组（n = 42）。PNH疾病和AA/MDS的诊断依据标准文献和指南。通过流式细胞术使用抗体CD45/CD15/CD64/CD157/FLAER的单管五色“试验”检测对结果进行比较。使用20名健康对照受试者的样本计算“试验”检测的临界值。
结果 通过“试验”方法，从受试者工作特征曲线分析获得的检测PNH克隆的临界值，中性粒细胞>0.4%（敏感性=96.15%，特异性=95%），单核细胞>0.9%（敏感性=98.08%，特异性=95%）。“标准”和“试验”检测所测的中性粒细胞（PNH疾病：r = 0.976，p<0.001；AA/MDS：r = 0.980，p<0.001）以及单核细胞（PNH疾病：r = 0.806，p = 0.005；AA/MDS：r = 0.915，p<0.001）中PNH克隆大小之间存在显著相关性。Bland-Altman分析显示在所有52例患者以及AA/MDS个体中两种检测方法结果一致。“试验”方法对患者的检测成本比“标准”技术低约15%，且技术效率提高。
结论 在单管流式细胞术检测中，CD157可以替代CD24和CD14两者以检测中性粒细胞和单核细胞上的PNH克隆，同时降低了患者成本并提高了技术效率。
