Laboratory Medicine Program, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada.
Cytometry B Clin Cytom. 2014 Jan;86(1):44-55. doi: 10.1002/cyto.b.21111. Epub 2013 Aug 6.
Recent Flow Cytometric guidelines to detect Paroxysmal Nocturnal Hemoglobinuria (PNH) in white blood cells recommend using FLAER-based assays to detect granulocytes and monocytes lacking expression of GPI-linked structures. However national proficiency testing results continue to suggest a need for improved testing algorithms, including the need to optimize diagnostic analytes in PNH.
CD157 is another GPI-linked structure expressed on both granulocytes and monocytes and here we assess its ability to replace CD24 and CD14 in predicate 4-color granulocyte and monocyte assays respectively. We also assess a single tube, 5-color combination of FLAER, CD157, CD64, CD15, and CD45 to simultaneously detect PNH clones in granulocyte and monocyte lineages.
Delineation of PNH from normal phenotypes with 4- or 5-color CD157-based assays compared favorably with 4-color predicate methods and PNH clone size data were similar and highly correlated (R(2) >0.99) with predicate values over a range (0.06%-99.8%) of samples. Both CD157-based assays exhibited similar high levels of sensitivity and low background levels in normal samples.
While CD157-based 4- and 5-color assays generated closely similar results to the predicate assays on a range of PNH and normal samples, the 5-color assay has significant advantages. Only a single 5-color WBC reagent cocktail is required to detect both PNH granulocytes and monocytes. Additionally, sample preparation and analysis time is reduced yielding significant efficiencies in technical resources and reagent costs. All 4- and 5-color reagent sets stained stabilized whole blood PNH preparations, used in external quality assurance programs.
最近的流式细胞术检测阵发性夜间血红蛋白尿症（PNH）的指南建议使用基于 FLAER 的检测方法来检测缺乏糖基磷脂酰肌醇（GPI）连接结构表达的粒细胞和单核细胞。然而，国家能力验证结果继续表明需要改进检测算法，包括优化 PNH 中的诊断分析物。
CD157 是另一种在粒细胞和单核细胞上表达的 GPI 连接结构，在此我们评估其在分别替代预测性 4 色粒细胞和单核细胞检测中的 CD24 和 CD14 的能力。我们还评估了一种单管、5 色 FLAER、CD157、CD64、CD15 和 CD45 的组合，以同时检测粒细胞和单核细胞谱系中的 PNH 克隆。
与 4 色预测性方法相比，基于 CD157 的 4 或 5 色检测法可清晰地区分 PNH 与正常表型，并且 PNH 克隆大小数据与预测值相似且高度相关（R(2)＞0.99），样本范围为（0.06%-99.8%）。基于 CD157 的两种检测方法在正常样本中均表现出相似的高灵敏度和低背景水平。
虽然基于 CD157 的 4 色和 5 色检测法在一系列 PNH 和正常样本上产生了与预测性检测法非常相似的结果，但 5 色检测法具有显著的优势。仅需一种 5 色白细胞试剂鸡尾酒即可同时检测 PNH 粒细胞和单核细胞。此外，样本制备和分析时间减少，从而在技术资源和试剂成本方面具有显著的效率。所有 4 色和 5 色试剂均可对稳定的全血 PNH 制剂进行染色，用于外部质量保证计划。
