Department of Medical Genetics, Athens University School of Medicine, Athens, Greece.
In Vivo. 2011 May-Jun;25(3):411-7.
To design a protocol for non-invasive prenatal diagnosis of fetal Rhesus D (RhD) status.
A total of 112 single lymphocytes were used to test the efficiency of the assay. The protocol was validated using blood samples from 84 RhD-negative pregnant women at 7-24 weeks of gestation. Cell-free DNA (cfDNA) was enzymatically digested using AciI and analyzed by a polymerase chain reaction (PCR) that allowed simultaneous amplification of RHD exons 7 and 10, SRY, RASFF1A and ACTB.
On the one genome-equivalent level, the efficiency of the protocol was ≥ 94.6% for each locus amplified. Conclusive results from the first set of PCRs were obtained for 79 cases with one false-positive. In five cases the analysis was repeated and, subsequently, all cases were accurately diagnosed.
The proposed protocol is rapid, applicable in most molecular diagnostic laboratories and provides the basis for non-invasive examination of fetal RhD with 96.7% specificity and 100% sensitivity.
设计一种用于无创性产前诊断胎儿 RhD（RhD）状态的方案。
共使用 112 个单个淋巴细胞来测试该检测方法的效率。该方案使用了 84 名 RhD 阴性孕妇在妊娠 7-24 周的血液样本进行了验证。使用 AciI 对无细胞 DNA（cfDNA）进行酶切消化，通过聚合酶链反应（PCR）进行分析，该 PCR 允许同时扩增 RHD 外显子 7 和 10、SRY、RASFF1A 和 ACTB。
在一个基因组等效水平上，每个扩增位点的方案效率均≥94.6%。在第一组 PCR 中，79 例获得了明确的结果，有 1 例假阳性。在 5 例中重复了分析，随后所有病例均得到了准确诊断。
所提出的方案快速、适用于大多数分子诊断实验室，并为 96.7%特异性和 100%敏感性的胎儿 RhD 无创性检查提供了基础。
