Use of real time PCR for rapid detection of Del phenotype in Taiwan

During routine serologic procedures, Rh D(el) blood is often not identified and subsequently labeled as RhD-negative. Recently, several reports have shown the ability of D(el) blood to induce anti-D in RhD-negative recipients. Among Korean, Japanese, and Chinese, almost all individuals with D(el) blood have a nucleotide change in the RHD gene of 1227G>A (RHDK407K). Thus, nucleotide 1227A at RHD exon 9 can be used as a marker for the D(el) phenotype in Asians. Real-time PCR for single nucleotide polymorphisms has been useful in biallelic discrimination of genomic sequence. Use of this methodology to identify 1227A will facilitate identification of blood units with the D(el) blood type and thus prevent potential sensitization of the RhD-negative recipients. In this study, real-time PCR-melting curve analysis at nucleotide 1227 of RHD exon 9 was performed on 990 leftover blood samples. PCR analysis identified 22 samples with the 1227G+A pattern, 965 samples with the 1227G pattern, and 3 with negative real-time results. The RHDEL allele frequency is 0.0116 (22/1980) among Taiwanese. These real-time PCR patterns were validated through DNA sequencing analyses of RHD exon 9 on 22 samples with the 1227G+A pattern and on 50 randomly selected samples from 1227G individuals. The real-time PCR test was then analyzed in 118 apparently RhD-negative Taiwanese donors, including 38 D(el) and 80 true RhD-negative donors, for efficiency studies. All of the D(el) samples (38, 100%) were found to have the 1227A pattern. Among the 80 serologic true RhD-negative samples, 77 were negative for real-time PCR results [1227A(-) /1227G(-)], 2 had the 1227G pattern [1227A(-)/1227G(+)], and one had the 1227A pattern [1227A(+)/ 1227G(-)]. Results of the melting curve analysis of RHD 1227A for the detection of D(el) among apparent RhD-negative individuals in Taiwan had the following characteristics: 100% sensitivity; 98.75% specificity, positive predictive value of 97.44%; negative predictive value of 100%; and an efficiency of 99.15%. Melting curve analysis using RHD 1227A for detection of D(el) phenotype can be efficiently applied in eastern Asian countries, since almost all the D(el) type in eastern Asians have the characteristic 1227A mutation.