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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.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
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

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
The RhD antigen is highly immunogenic and is clinically involved in immune hemolytic anemia in transfusion medicine [1]. The frequency of RhD-negative phenotype varies among different ethnic groups, with a frequency of approximately 15% in Caucasians and <0.5% in persons from the Far East 1,2]. Among these phenotypically RhD-negative individuals, the D_el phenotype is present with a frequency of 1 in 1,000 in Europeans and 1 in 3 among Asians [3–7].

For years, physicians and blood bankers have not been concerned about the weak D phenotypes [8]. However, some weak D’s, such as weak D types 1, 2, and 26, have been reported to cause anti-D immunization [3,9,10]. A few reports have recently demonstrated the ability of the D_el units of RBCs to induce an anti-D immunologic reaction in RhD-negative recipients [11,12]. RBCs with <30 D sites have now been shown to be immunogenic [8]. Although current routine serological methods are satisfactory in detecting most weak D’s [13], the D_el RBCs are detectable only by adsorption and elution tests. Such methods are time-consuming and not feasible as a routine screening test. Thus, the D_el blood units are not usually identified in routine serologic procedures and are often labeled as RhD-negative and subsequently transfused to RhD-negative patients. A rapid and easy method for detection of the D_el donor units is thus of value in preventing potential sensitization of the recipient by transfusion of D_el units.

The RHD-deleted haplotypes are observed in approximately 99.7% of European RHD-negative individuals [3,7]. The D_el type was noted in only 0.1% of RhD-negative European blood donors and the RHD gene is present in about 0.2% [3,7]. Wagner et al [7] have demonstrated 14 rare RhD-negative alleles other than the classical RHD- deleted haplotype with a cumulative population frequency of 1:1,500 among Europeans. Genotyping for RHD-negative and D_el Caucasians is rather complicated [3,14]. Contrarily, the allele distribution of RhD-negative for Asians appears to be much simpler [15–17]. Approximately one-third of the apparent RhD-negative individuals in Asia have the D_el phenotype [4–6]. The RHD(K407K) with RHD1227G>A change is present in almost all the D_el individuals of Korean, Japanese, and Chinese descent [15–17]. The RHD1227G>A change is an important marker for D_el phenotype in east Asians, including the Taiwanese [16].

Real-time PCR methods for genotyping single nucleotide polymorphisms have proven to be useful in biallelic discrimination of genomic sequence [18]. The LightCycler (Roche Corp., Mannheim, Germany) is a commercially available instrument designed to perform rapid real-time fluorescence-based detection of the PCR product in a closed system. Using a method based on the principle of fluorescence resonance energy transfer (FRET) with two adjacent fluorophores, a ‘detection probe’ spanning the polymorphic site and an ‘anchor probe’ recognizing an adjacent sequence, the polymorphic alleles can be distinguished by the melting temperature (Tm) of the ‘detection probe’ [19]. Continuous fluorescence monitoring of the reaction as the temperature is raised from annealing to denaturation results in a sharp decrease in fluorescence at the temperature at which the ‘detection probe’ dissociates from the template. The single base change caused by the D_el polymorphism results in a decrease of the Tm of the ‘detection probe’ that can easily be distinguished using the LightCycler instrument.

Since D_el RBCs are potentially immunogenic [8] and have been shown to induce anti-D in RhD-negative recipients [11,12], a rapid and efficient way to detect D_el red cell units is of value in Asian countries. In this study, we describe the development of a diagnostic strategy using a real-time PCR-melting curve technique to bypass the tedious and labor-intensive adsorption-elution test for efficient detection of D_el donor units in Taiwan. The use of this method will facilitate identification of blood units with the D_el blood type and thus prevent potential sensitization of recipients.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Study design.  A set of 1000 leftover blood samples was collected from the Department of Clinical Pathology, Chang Gung Memorial Hospital (CGMH). Real-time PCR analyses for the 1227 pattern were then performed. The results of real-time PCR were validated on all samples with the 1227 G+A pattern [1227A(+)/1227G(+)] and 50 randomly selected samples with the 1227G pattern [1227A(–)/1227G(+)] by direct DNA sequencing analysis of RHD exon 9. The RHDEL allele was then calculated using RHD1227A as a marker for RHDEL. The efficiency study of real-time PCR 1227A analysis for detection of the D_el phenotype was analyzed on the samples of the apparently RhD-negative Taiwanese donors from blood donors during a 2-mo interval at the Hsin-Chu Blood Donation Center (HCBDC). Our program was approved by the Institutional Review Board of the CGMH.

Sample Collection and Serologic Tests.  Blood samples (n = 990) with sufficient specimen for DNA extraction and serological Rh D_el typing were collected from 1000 leftover blood specimens from the Department of Clinical Pathology, CGMH. Apparently RhD-negative blood samples (n = 118) were collected from blood donors during a 2-mo interval from the HCBDC. RhD phenotyping was performed using a semiautomated microplate method. The Organo Technika (Boxtel, Netherlands) anti-D (human monoclonal IgM+IgG) reagent was used for RhD phenotyping at the HCBDC. The blood samples were preserved in EDTA and sent to CGMH for analysis. No Du tests were performed at the HCBDC. There were no irregular antibodies present in any of the 118 samples. These apparently RhD-negative blood samples were then retyped at CGMH after receipt of the specimens. Novaclone Anti-D (IgM+IgG Monoclonal Blend, Dominion Biologicals, Dartmouth, Nova Scotia, Canada) was used. Adsorption/elution tests were also performed on all 118 samples. The eluate was prepared by the chloroform elution method, as described in the AABB Technical Manual. All assessed subjects belonged to the Taiwanese population.

DNA preparation.  Genomic DNA was prepared from peripheral blood cells using the QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany).

SSP-PCR of RHD exon 9.  Approximately 200 ng of DNA was used for SSP-PCR (total reaction volume, 20 µl) with 20 pmol RHD-9 forward primer (5-tactgtcgttttgacacacaatatttc-3') and 20 pmol RHD-9 reverse primer (5'-tgcaaacttcgcttcccagg-3'), and 0.2 U HotStar Taq DNA polymerase (Qiagen, Valencia, CA, USA), 200 µM dNTP, 1 X reaction buffer, and 1.5 µM Mg²⁺. The PCR reaction was performed in the GeneAmp PCR system 9700 (Applied Biosystems) with the following cycle conditions: 1 cycle of 95° for 10 min, 35 cycles of 95° for 30 sec, 60° for 30 sec, 72° for 1 min, and 1 cycle of 72° for 10 min. The final elongation step was 10 min at 72°.

After finishing SSP PCR of RHD exon 9, 9 µl of PCR product was mixed with 100 nM 3'-FL-labeled detection probe TTCTGGAAGataagatttttcacctat, and 100 nM 5'-LC labeled anchor probe, aacgtgatagattttgagtgcatgaagt. The reaction mixtures were loaded into glass capillary cuvettes (Roche, Mannheim, Germany) and centrifuged to place the sample at the capillary tip before capping. The temperature transition rates were all programmed at 20°/sec from denaturation to annealing, annealing to extension, and extension to denaturation. Fluorescence was measured at the end of the annealing period of only one cycle to monitor the concentration of amplicon. After one amplification cycle was complete, a final melting curve was recorded by heating to 95° <1 sec and then cooling to 40° with 20°/sec, followed by a 60-sec hold before heating slowly at intervals of 0.1°/sec until a temperature of 80° was attained. Fluorescence was measured continuously during the slow temperature rise to monitor the dissociation of the LightCycler Red 640-labeled detection probe. The fluorescence signal (F) was plotted in time against the temperature (T) to produce melting curves for each sample (F versus T). Melting curves were then converted to melting peaks by plotting the negative derivative of F with respect to T against T (-dF/dT versus T). The entire process required approximately 10 min.

Genotyping by direct sequencing of PCR product of exon 9/ intron 9.  For confirmation of genotypes, the DNA fragment encompassing exon 9 through intron 9 of the RHD gene was sequenced directly. The PCR product of the RHD exon 9 was used as the templates for DNA sequencing using the BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA) and the RE-83 primer (5'-gagattaaaaatcctgtgctcca-3') that is located 93 bp upstream to exon 9.

	Results

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Validation of melting curve analysis.  The 1227A pattern with a Tm of 50.91°C (SD ± 0.20, range 50.53–51.44) would detect the RHDEL allele. The 1227G pattern with a higher melting temperature (Tm) of 53.99°C (SD ± 0.16, range 53.58–54.52) would detect the RHD allele. The 1227G+A pattern with two melting peaks, corresponding to those observed in the 1227 A (Tm 50.91°) and 1227G (Tm 53.99°) would detect the heterozygous RHD/ RHDEL alleles (Fig. 1). Since noPCR RHD product was obtained from the RHD-deletion allele of true RhD-negative individuals, negative melting curve reaction would be noted in those true RhD-negative individuals.

View larger version (19K): [in this window] [in a new window]   Fig. 1. Three melting patterns, ie, 1227G, 1227G+A and 1227A, are identified. The 1227G pattern (dotted line) with a higher melting temperature (Tm) of 53.99° (SD ± 0.16, range 53.58–54.52) detects the RHD allele. The 1227A pattern (dashed line) with Tm of 50.91° (SD ± 0.20, range 50.53–51.44) detects the RHDEL allele. The 1227G/A pattern (solid line) having 2 melting peaks detects the heterozygous RHD/RHDEL alleles.

Sequencing of the RHD exon 9 was performed on 50 of the 965 1227G individuals and on all 22 1227G+A samples. The RhD-positive 1227G samples were shown to carry a G nucleotide at the 1227 position of the RHD exon 9. All 22 individuals with the 1227G+A melting pattern were shown to be heterozygous for 1227G+A at the 1227 nucleotide of exon 9 by sequence analysis of RHD exon 9. All 3 RhD-negative samples were negative after PCR, implying an absence of RHD exon 9.

Application of real-time PCR-melting curve analysis for detection of D_el  Among the 118 apparently RhD-negative individuals, there were 38 D_el samples and 80 true RhD-negative samples using the adsorption/elution results. All 38 D_el samples showed the 1227A melting pattern and were confirmed to possess an A nucleotide at 1227 position of the RHD exon 9. Among the 80 true RhD-negative samples by adsoption/elution results, 77 had a negative real-time PCR result [1227A(–)/1227G(–)], 2 had a 1227G pattern [1227A(–)/1227G(+)], and 1 had a 1227A pattern. [1227A(+)/ 1227G(–)](Table 1). Three of these individuals, 2 with 1227G [1227A(–)/1227G(+)] and 1 with 1227A [1227A(+)/1227G(–)], were found to possess the RHD exon 9. The sequencing results of the RHD exon 9 showed a RHD-deletion/RHD for the 2 1227G individuals and a RHD-deletion/RHDEL for the 1227A individual.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
In our series, 96.25% (77/80) of the true RhD-negative samples showed no real time PCR results for either 1227A or 1227G (Table 1). This implies that the most common genotype for RhD-negative in Taiwan is deletion of the RHD genome. The RHD exon 9 was detected with either 1227A or 1227 G in about 3.75% (3/80) of the true RhD-negative individuals (Table 1). Our observation is compatible with a report by Shao et al [15], where the RHD exon 9 was detected in 3.9% (3/76) of RhD-negative Chinese.

The D_el units of RBCs with a very small number of D sites are capable of inducing an anti-D reaction in RhD-negative recipients [11,12]. It may be beneficial to avoid transfusion of these D_el units to RhD-negative recipients. Currently, the gold standard for detection of the D_el phenotype is the absorption-elution test. However, this test is difficult and time-consuming to perform, and is not a feasible test for screening a large number of donors for the D_el phenotype. In this study, we propose the use of novel real-time PCR-melting curve analysis of 1227A for the detection of D_el units among apparently RhD-negative donors. This method is more practical, easier to perform, and economically justifiable in Taiwan, and offers several advantages over serological methods. The real-time PCR genotyping technique is precise, in contrast to the serological blood typing method, which depends on observation of RBC agglutination and may occasionally have a non-objective interpretation. The PCR genotyping test has better sensitivity and specificity, with shorter hands-on time than the traditional serological method. The cost for PCR genotyping is <$4.00 (USA) per test, and may be cheaper when done in larger batches. Although the method presented and used here involves use of an instrument and materials from a particular manufacturer, the Roche LightCycler and the use of FRET probes, this approach could be applicable to other platforms that use melting curve analysis, such as the TaqMan probe.

The Ce phenotype is highly associated with D_el in Chinese populations [15,17]. Since an overwhelming (88.1%–91.4%) proportion of the D_el alleles are associated with the RhC+ phenotype, C phenotyping and RHD1227A analysis by SSP-PCR in the RhD-negative population has been proposed as a screening measure for the detection of D_el in the clinical laboratory in order to avoid the tedious absorption test [20]. However, the protocol of selecting C+ among the RhD-negative population and then performing SSP-PCR RHD1227A typing is not a simple task for routine blood unit processing. Most important, approximately 9% of the D_el phenotype is associated with the ce phenotype [15], which would be missed using this approach [15]. However, since virtually all with the D_el phenotype have the RHD1227G>A allele in Taiwan [15–17], we have shown here that RHD1227A could used as an efficient marker for the detection of D_el in RhD-negative blood donors without testing of C.

In this study, our strategy of performing real-time PCR-melting curve analysis for polymorphic 1227A detection among the RhD-negative blood units is very efficient (99.15%). It has high sensitivity (100%), high specificity (98.75%), high positive predictive value (97.44%), and high negative predictive value (100%). We believe this strategy could also be applicable in eastern Asians, since almost all D_el subjects among eastern Asians have the characteristic 1227A mutation [15–17].

	Acknowledgements

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 
Technical assistance of TIB MOLBIOL Company (Berlin, Germany) is gratefully acknowledged. This work was supported by Grant NMRPD131233 from Chang Gung Memorial Hospital and Grant NSC95-2320-B-182-025-MY3 from the National Science Council of Taiwan.

	References

Top Abstract Introduction Materials and Methods Results Discussion Acknowledgements References

 

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