BACKGROUND The diagnosis of paroxysmal nocturnal hemoglobinuria (PNH) relies on flow cytometric demonstration of loss of glycosyl-phosphatidyl inositol (GPI)-anchored proteins from red blood cells (RBC) and white blood cells (WBC). High-sensitivity multiparameter assays have been developed to detect loss of GPI-linked structures on PNH neutrophils and monocytes. High-sensitivity assays to detect PNH phenotypes in RBCs have also been developed that rely on the loss of GPI-linked CD59 on CD235a-gated mature RBCs. The latter is used to delineate PNH Type III (total loss of CD59) and PNH Type II RBCs (partial loss of CD59) from normal (Type I) RBCs. However, it is often very difficult to delineate these subsets, especially in patients with large PNH clones who continue to receive RBC transfusions, even while on eculizumab therapy. METHODS We have added allophycocyanin (APC)-conjugated CD71 to the existing CD235aFITC/CD59PE RBC assay allowing simultaneous delineation and quantification of PNH Type III and Type II immature RBCs (iRBCs). RESULTS We analyzed 24 medium to large-clone PNH samples (>10% PNH WBC clone size) for PNH Neutrophil, PNH Monocyte, Type III and Type II PNH iRBCs, and where possible, Type III and Type II PNH RBCs. The ability to delineate PNH Type III, Type II, and Type I iRBCs was more objective compared to that in mature RBCs. Additionally, total PNH iRBC clone sizes were very similar to PNH WBC clone sizes. CONCLUSIONS Addition of CD71 significantly improves the ability to analyze PNH clone sizes in the RBC lineage, regardless of patient hemolytic and/or transfusion status.

中文翻译：

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CD71改善阵发性夜间血红蛋白尿患者的PNH III型，PNH II型和正常未成熟RBCS的轮廓。

背景技术阵发性夜间血红蛋白尿（PNH）的诊断依赖于流式细胞术证明红细胞（RBC）和白细胞（WBC）中糖基磷脂酰肌醇（GPI）锚定蛋白的丢失。已经开发出高灵敏度的多参数测定法来检测PNH中性粒细胞和单核细胞上GPI连接的结构的缺失。还已经开发了检测RBC中PNH表型的高灵敏度检测方法，该检测方法依赖于CD235a门控的成熟RBC上GPI连接的CD59的丢失。后者用于从正常（I型）RBC划定PNH型III（CD59的总损失）和PNH II型RBC（CD59的部分损失）。但是，通常很难区分出这些亚型，尤其是在具有大量PNH克隆的患者中，即使在接受依库丽单抗治疗的情况下，这些患者仍继续接受RBC输血。方法我们在现有的CD235aFITC / CD59PE RBC分析中添加了与藻蓝蛋白（APC）偶联的CD71，可以同时描绘和定量PNH III型和II型不成熟RBC（iRBC）。结果我们分析了24个中到大克隆PNH样品（> 10％PNH WBC克隆大小）的PNH中性粒细胞，PNH单核细胞，III型和II型PNH iRBC，并在可能的情况下分析了III型和II型PNH RBC。与成熟RBC相比，描绘PNH III型，II型和I型iRBC的能力更为客观。此外，PNH iRBC总克隆大小与PNH WBC克隆大小非常相似。结论无论患者溶血和/或输血状态如何，添加CD71均可显着提高分析RBC谱系中PNH克隆大小的能力。