Mackay Memorial Hospital Transfusion Medicine Laboratory, Tamsui, Taiwan.
Transfusion. 2013 May;53(5):962-71. doi: 10.1111/j.1537-2995.2012.03861.x. Epub 2012 Aug 23.
GP.Mur (Mi.III) is a glycophorin B-A-B hybrid sialoglycoprotein expressing several potent immunogens, including Mi(a), Mur, and Hil. GP.Mur is considered one of the most important red blood cell (RBC) phenotypes in blood banking in Southeast Asia. However, there are no antibodies commercially available for the screening of GP.Mur RBCs.
To develop a direct blood polymerase chain reaction (PCR) approach for the screening of GP.Mur cells, we first confirmed the genomic sequence differences among four GP.Mur and three Mi(a-) samples by sequencing their GYP.Mur and GYPB genes. With these data, we designed PCR primers that best discriminate GYPB and GYP.Mur. Our primer design also allows the detection of other Hil+ glycophorin variants. We also constructed two plasmids--pGBi2i3 and pMiIIIi2i3--which serve as the negative and positive control DNA, respectively, for the PCR procedure. Additionally, we designed a control PCR to be run side by side with the typing PCR.
Because of the high specificity of our primers, we found it unnecessary to extract DNA from blood samples for PCR. We have tested this PCR method on 379 fresh and frozen blood samples. The results were further validated by serology and DNA sequencing and were shown to be completely accurate in our hand. We also found that the rapid genotyping method--high-resolution melting--can be a timesaving alternative for DNA sequencing.
This direct blood PCR approach for determination of GP.Mur and related Hil+ phenotypes is reliable and economical and is expected to be useful for blood banking in Southeast Asia.
GP.Mur（Mi.III）是一种糖蛋白 B-A-B 杂合唾液糖蛋白，表达多种有效的免疫原，包括 Mi(a)、Mur 和 Hil。GP.Mur 被认为是东南亚血液银行中最重要的红细胞（RBC）表型之一。然而，目前没有商业上可用于筛选 GP.Mur RBC 的抗体。
为了开发用于筛选 GP.Mur 细胞的直接血液聚合酶链反应（PCR）方法，我们首先通过测序其 GYP.Mur 和 GYPB 基因来确认四个 GP.Mur 和三个 Mi(a-)样本之间的基因组序列差异。利用这些数据，我们设计了最佳区分 GYPB 和 GYP.Mur 的 PCR 引物。我们的引物设计还允许检测其他 Hil+糖蛋白变体。我们还构建了两个质粒-pGBi2i3 和 pMiIIIi2i3-它们分别作为 PCR 程序的阴性和阳性对照 DNA。此外，我们设计了一个对照 PCR，与分型 PCR 一起平行运行。
由于我们的引物具有很高的特异性，我们发现没有必要从血液样本中提取 DNA 进行 PCR。我们已经在 379 个新鲜和冷冻的血液样本上测试了这种 PCR 方法。通过血清学和 DNA 测序进一步验证了结果，证明在我们手中完全准确。我们还发现，快速基因分型方法-高分辨率熔解曲线-可以替代 DNA 测序，节省时间。
这种用于确定 GP.Mur 和相关 Hil+表型的直接血液 PCR 方法可靠且经济，预计将对东南亚的血液银行有用。
