流式检测PNH克隆的方法学探讨及临床筛检和意义

目的:探讨流式细胞仪高敏感方法检测PNH克隆的必要性和血细胞减少患者PNH克隆的发生率及临床意义。方法:采用CD59检测红细胞及FLAER联合CD24或CD14检测粒细胞和单核细胞的方法检测了20例健康志愿者和1 095例血细胞减少患者的PNH克隆比例,同时对31例患者采用传统的CD59方法检测粒细胞和单核细胞中CD59~-细胞比例。结果:根据健康志愿者正常背景和患者获取的细胞数,确定粒细胞、单核细胞和红细胞PNH克隆的最低检测限(LOD)分别为0.04%、0.10%和0.05%;827例患者FLAER~-CD24~-粒细胞、FLAER~-CD14~-单核细胞和CD59~-红细胞的中位比例分别为0.02%、0.02%和0.03%;318例(38.45%)患者粒细胞PNH克隆高于LOD,180例FLAER~-CD24~-粒细胞1.00%,粒红和粒单细胞的一致性只有43%～45%。138例FLAER~-CD24~-粒细胞≥1.00%,粒红、粒单和粒单红细胞的一致率分别为91.30%、97.83%和89.86%;比较CD59~-和FLAER~-CD24~-粒细胞、CD59~-和FLAER~-CD14~-单核细胞比例,CD59~-细胞比例明显更低(P0.000 1和P=0.000 9);26.85%和24.54%患者因出现FLAER~-CD24~+粒细胞与FLAER~-CD14~+单核细胞,导致FLAER单阴比例高于FLAER和锚连蛋白双阴比例,对PNH克隆比例低于0.10%的患者影响更大;36例PNH患者和50例MDS或AA患者PNH克隆比例分别为90.30%(44.49%～99.05%)和1.30%(0.10%～96.07%)(P0.000 1);PNH克隆比例等于41.81%时,诊断PNH的敏感度和特异度分别为100.0%和96.0%。结论:在本实验条件下根据正常背景值,确定粒细胞、红细胞的LOD分别为0.04%和0.05%。粒细胞PNH克隆比例≥1.00%,与单核和红细胞诊断PNH克隆阳性结果更一致,PNH克隆比例高于41.81%时PNH疾病可能性更大。

Methodological Study on Flow Detection of PNH Clone and Its Clinical Screening Significance

Objective: To explore the importance and clinical indications of high-sensitivity assay detecting PNH clones by flow cytometry and the incidence and clinical significance of PNH clones in patients with hematopenia.Methods: FLAER combined with CD24 or CD14 was used to detect the percentage of PNH clones in granulocytes and monocytes, and CD59 was used to detect the percentage of PNH clones in erythrocytes of 20 healthy volunteers and 1 095 patients with hematopenia. Meanwhile, CD59 negative percentage in granulocytes and monocytes of 31 patients was detected by traditional CD59 method.Results: According to background of volunteers and numbers of cells, limited of detection (LOD) of granulocytes, monocytes and erythrocytes were 0.04%, 0.10% and 0.05%, respectively. The median percentages of FLAER ^-CD24 ^- granulocytes, FLAER ^-CD14 ^- monocytes and CD59 ^- erythrocytes were 0.02%, 0.02% and 0.03% in 827 patients, separately. The proportions of PNH clones in granulocytes were above LOD in 318 patients. Among of 180 patients with PNH clones below 1.00%, the consistent rates of granulocytes with monocytes and erythrocytes were from 43% to 45%. When the percentages of PNH clones in granulocytes were more than 1.00%, the consistency rates of granulocytes with monocytes and erythrocytes were around 90.00%. Comparing the percentage of CD59 negative granulocytes and monocytes with that of FLAER and anchor protein double negative, the former was significantly lower than the latter (P< 0.000 1 and P=0.000 9). The percentages of FLAER single negative granulocytes were higher than that of FLAER and anchor protein double negative cells in 26.85% patients. The similar results occured in monocytes of 24.54% patients. The patients with PNH clones below 0.10% were affected more easily. PNH clones were 90.30% (44.49%~99.05%) in 36 patients with PNH and 1.30% (0.10%~96.07%) in 50 patients with MDS or AA, separately (P<0.000 1). When PNH clone was 41.81%, the sensitivity and specificity of PNH diagnosis were 100.0% and 96.0%, respectively. Conclusion: In this study, the LOD of granulocyte and erythrocyte was 0.04% and 0.05% respectively. When the proportion of PNH clones in granulocytes is more than 1.00%, the positive results of monocytes and erythocytes are more consistent. PNH disease is more likely when the proportion of PNH clones is higher than 41.81%.