Simsek S, Faas B H, Bleeker P M, Overbeeke M A, Cuijpers H T, van der Schoot C E, von dem Borne A E
Central Laboratory, The Netherlands Red Cross Blood Transfusion Service, Amsterdam.
Blood. 1995 May 15;85(10):2975-80.
Rh (rhesus) D is the dominant antigen of the Rh blood group system. Recent advances in characterization of the nucleotide sequence of the cDNA(s) encoding the Rh D polypeptide allow the determination of the Rh D genotype at the DNA level. This can be of help in cases in which red blood cells are not available for phenotyping, eg, when in concerns a fetus. We have tested three independent DNA typing methods based on the polymerase chain reaction (PCR) for their suitability to determine the Rh D genotype. DNA derived from peripheral blood mononuclear cells from 234 Rh-phenotyped healthy donors (178 Rh D positive and 56 Rh D negative) was used in the PCR. The Rh D genotypes, as determined with a method based on the allele-specific amplification of the 3' noncoding region of the Rh D gene described by Bennett et al (N Engl J Med 329:607, 1993), were not concordant with the serologically established phenotypes in all cases. We have encountered 5 discrepant results, ie, 3 false-positive and 2 false-negative (a father and child). Rh D genotyping with the second method was performed by PCR amplification of exon 7 of the D gene with allele-specific primers. In all donors phenotyped as D positive tested so far (n = 178), the results of molecular genotyping with this method were concordant with the serologic results, whereas a false-positive result was obtained in one of the D-negative donors (also false-positive in the first method). Complete agreement was found between genotypes determined in the third method, based on a 600-bp deletion in intron 4 of the Rh D gene described by Arce et al (Blood 82:651, 1993), and serologically determined phenotypes. The Rh blood group system is complex, and unknown polymorphisms at the DNA level are expected to exist. Therefore, although genotypes determined by the method of Arce et al were in agreement with serotypes, it cannot yet be regarded as the golden standard. More experience with this or other methods is still needed.
Rh（恒河猴）D是Rh血型系统的主要抗原。编码Rh D多肽的cDNA核苷酸序列特征分析方面的最新进展，使得在DNA水平上确定Rh D基因型成为可能。这在无法获得红细胞进行表型分析的情况下，例如涉及胎儿时，可能会有所帮助。我们测试了三种基于聚合酶链反应（PCR）的独立DNA分型方法，以确定它们用于确定Rh D基因型的适用性。PCR中使用了来自234名经Rh表型分析的健康供者（178名Rh D阳性和56名Rh D阴性）外周血单个核细胞的DNA。采用Bennett等人（《新英格兰医学杂志》329:607, 1993）描述的基于Rh D基因3'非编码区等位基因特异性扩增的方法所确定的Rh D基因型，在所有情况下与血清学确定的表型并不一致。我们遇到了5个不一致的结果，即3例假阳性和2例假阴性（一对父子）。第二种方法通过使用等位基因特异性引物对D基因的第7外显子进行PCR扩增来进行Rh D基因分型。在所有迄今为止经表型分析为D阳性的供者（n = 178）中，该方法的分子基因分型结果与血清学结果一致，而在一名D阴性供者中获得了一个假阳性结果（在第一种方法中也是假阳性）。在基于Arce等人（《血液》82:651, 1993）描述的Rh D基因第4内含子中600 bp缺失的第三种方法所确定的基因型与血清学确定的表型之间，发现完全一致。Rh血型系统很复杂，预计在DNA水平上存在未知的多态性。因此，尽管Arce等人的方法所确定的基因型与血清型一致，但它尚未能被视为金标准。仍需要对这种或其他方法积累更多经验。
