Sanquin Research and Landsteiner Laboratory, Academic Medical Centre, University of Amsterdam, and MRC-Holland, Amsterdam, the Netherlands.
Transfusion. 2013 Jul;53(7):1559-74. doi: 10.1111/j.1537-2995.2012.03919.x. Epub 2012 Oct 9.
The presence of a D variant may hamper correct serologic D typing, which may result in D immunization. D variants can be determined via RHD genotyping. However, a convenient single assay to identify D variants is still lacking. We developed and evaluated a multiplex ligation-dependent probe amplification (MLPA) assay to determine clinically relevant RHD and RHCE variant alleles and RHD zygosity.
We analyzed 236 cases (73 normal and 163 selected samples) with the RH-MLPA assay, which is able to determine 79 RHD and 17 RHCE variant alleles and RHD zygosity. To confirm the results, mutations were verified by RHD and/or RHCE exon-specific sequencing and RHD zygosity was verified by quantitative real-time polymerase chain reaction (PCR) for 18 cases.
In 99% of the cases, the RH-MLPA assay correctly determined whether a person carried only wild-type RHD and RHCE alleles (n = 69) or (a) variant RHD allele(s) and/or (a) variant RHCE allele(s) (n = 164). In only three cases, including two new RHD variant alleles, the variant allele was not identified, due to lack of detecting probes. These were RHDDCS2, a new partial RHD allele, RHD525T (Phe175Leu), and a new D- null allele, RHD*443G (Thr148Arg). All RHD (n = 175) and RHCE variant alleles (n = 79) indicated by the RH-MLPA assay were confirmed by sequencing. RHD zygosity was confirmed by quantitative PCR. Two hematopoietic chimeras were recognized.
The RH-MLPA genotyping assay is a fast, easy, and reliable method to determine almost all clinically relevant RHD and RHCE variant alleles, RHD zygosity, and RHD+/RHD- chimeras in blood donors, blood recipients, and pregnant women.
D 变异体的存在可能会干扰正确的血清学 D 型定型，从而导致 D 免疫。D 变异体可通过 RHd 基因分型确定。然而，仍然缺乏一种方便的单一检测方法来鉴定 D 变异体。我们开发并评估了一种多重连接依赖性探针扩增（MLPA）检测方法，以确定临床相关的 RHd 和 RHCE 变异等位基因和 RHd 基因型。
我们分析了 236 例（73 例正常和 163 例选定样本）的 RH-MLPA 检测结果，该检测能够确定 79 种 RHd 和 17 种 RHCE 变异等位基因和 RHd 基因型。为了确认结果，我们通过 RHd 和/或 RHCE 外显子特异性测序验证了突变，并通过定量实时聚合酶链反应（PCR）验证了 18 例的 RHd 基因型。
在 99%的情况下，RH-MLPA 检测正确地确定了一个人是仅携带野生型 RHd 和 RHCE 等位基因（n=69）还是携带（一个或多个）变异型 RHd 等位基因和/或（一个或多个）变异型 RHCE 等位基因（n=164）。仅在三种情况下，包括两个新的 RHd 变异等位基因，由于缺乏检测探针，未识别出变异等位基因。这两个新的 RHd 变异等位基因是 RHDDCS2，一个新的部分 RHd 等位基因，和一个新的 D- 无效等位基因，RHD443G（Thr148Arg）。RH-MLPA 检测所指示的所有 RHd（n=175）和 RHCE 变异等位基因（n=79）均通过测序得到证实。RHd 基因型通过定量 PCR 得到确认。两个造血嵌合体被识别出来。
RH-MLPA 基因分型检测是一种快速、简便、可靠的方法，可用于确定献血者、受血者和孕妇中几乎所有临床相关的 RHd 和 RHCE 变异等位基因、RHd 基因型以及 RHd+/RHd-嵌合体。
