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Use of Real Time PCR for Rapid Detection of D_el Phenotype in Taiwan

Address correspondence to Dr. Chien-Feng Sun, Department of Clinical Pathology, Linkou Medical Center, Chang Gung Memorial Hospital, 5 Fushin Street, Kueishan, Taoyuan, 333, Taiwan; tel 886 3 328 1200 ext. 2554; fax 886 3 397 1827; e-mail: suncgj{at}adm.cgmh.org.tw.

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.

Keywords: D_el blood group, real-time PCR, melting curve analysis, transfusion medicine