Single-Tube Multiplex PCR-SSP for the Detection of RHD Variant Alleles Commonly Found in Serologically D− Phenotype Individuals in a Thai Population

RhD (RH001) is the most highly antigenic minor blood group antigen in transfusion medicine. Anti-D alloimmunization occurs when RhD-negative (D−) individuals are exposed to RhD-positive (D+) red blood cells (RBCs) during pregnancy or after a blood transfusion, which can cause severe hemolytic transfusion reactions and hemolytic disease of the fetus and newborn. The D− phenotype is common among Caucasians, at around 15–17%, although Africans and Asians only reach 5% and 3%, respectively [1]. The majority molecular basis of the D− phenotype in Caucasians is due to total deletion of the RHD gene. In Africans, the D− phenotype is frequently caused by the RHD pseudogene (RHDψ) [2, 3]. In East Asia, around 10–30% of D− individuals exhibit the DEL phenotype, a genetic variation of the RHD gene causing extremely weak D antigen expression [4]. DEL alleles derive from several mechanisms, and roughly 50 alleles have been recognized by the International Society of Blood Transfusion (ISBT). Among these is the Asian-type DEL allele, also known as RHD*01EL.01, RHD*DEL1, or RHD c.1227G>A (K409K), which is the predominant DEL allele in the East Asian population [5].

In Thailand, the occurrence rate of the D− phenotype is 0.3% [6]. The molecular basis of RHD alleles in serologically D− phenotype Thai blood donors was revealed. Approximately 98% of the allele frequencies were identified as RHD*01N.01, RHD*01EL.01, RHD*01N.03, and RHD-CE(3–10), which represent the four most common variant RHD alleles. It was reported that 14–18% of serologically D− phenotype cases carry the RHD*01EL.01 allele [7‒9]. Importantly, RBCs from Asian-type DEL individuals express the complete repertoire of epitopes such as D+ RBCs, contributing to the expression of the RhD antigen. Consequently, patients with Asian-type DEL may safely receive D+ RBCs transfusion, including discontinuation Rh immunoglobulin prophylaxis in pregnant women with Asian-type DEL [10‒12]. From the perspective of blood donors, RBCs from Asian-type DEL individuals can trigger alloanti-D when transfused to recipients who are truly D− phenotype. Recently, anti-D alloimmunization was reported in D− Thai patients who received Asian-type DEL RBC units in transfusions [13, 14]. It is, however, difficult to differentiate the common variant alleles, especially RHD*01N.01 and RHD*01EL.01, in the routine blood bank laboratory. DEL RBCs cannot be detected by conventional serological testing, and are mistyped as D− phenotype. Although DEL can be identified using the adsorption-elution technique, it is a laborious process and not routinely implemented.

In this study, single-tube multiplex PCR-sequence specific primer (multiplex PCR-SSP) was developed to detect RHD*01N.01, RHD*01EL.01, and RHD-CE-D hybrid as RHD variant alleles commonly found in serologically D− phenotype individuals in a Thai population. This single-tube multiplex PCR-SSP is a simple and reliable molecular method for analyzing RHD variant alleles in both small- and large-scale studies. Additionally, the single-tube multiplex PCR-SSP in this study is capable of distinguishing between Asian-type DEL and true D− individuals. Implementation of this method will improve blood transfusion safety.

A total of 205 blood samples with a serological D− phenotype, including 161 D− blood donors and 44 D− patients, were collected from the Blood Transfusion Center, Faculty of Medicine, Khon Kaen University. The RhD phenotyping was analyzed by blended monoclonal IgM/IgG anti-D clones LDM2/LHM77, National Blood Centre, Thai Red Cross Society, Thailand. Genomic DNA was extracted from 6 mL of EDTA blood or buffy coat using the guanidine-hydrochloride method and an automated DNA extractor (Maxwell 16, Promega, USA), while the concentration was estimated using a NanoDrop spectrophotometer (Dynamica Scientific, Dietikon, Switzerland). This study was approved by the Khon Kaen University Ethics Committee for Human Research (HE661043).

Single-tube multiplex PCR-SSP was performed to analyze the RHD gene for the detection of RHD*01N.01, RHD*01EL.01, and RHD-CE-D hybrid. Four RHD exons (exons 1, 4, 7, 10) and c.1227G>A in RHD exon 9 were selected for amplification in one reaction mixture (Fig. 1a, b). RHD exon 8 was used as an internal control due to the similarity to RHD and some RHCE alleles. The specific primer sequences used in this study were comparable to those previously described [15‒17]. The PCR reaction mixture consists of 8 µL of 2× PCR buffer (67 mm Tris-HCL pH 8.8, 17 mm ammonium sulfate, 0.1% Tween 20), 2 mm dNTPs (Vivantis, Kuala Lumpur, Malaysia), 8 mm MgCl₂, 5 U of Taq DNA polymerase (Invitrogen, Madison, WI, USA), 100–200 ng of genomic DNA, and 7 µL of five-specific primer pairs. The concentrations of each primer are shown in online supplementary Table 1 (for all online suppl. material, see https://doi.org/10.1159/000544107). The PCR reaction was amplified in a final volume of 15 µL using a thermocycler (Applied Biosystems Veriti™ Thermal Cycler, Life Technologies, Foster City, CA, USA). The cycling conditions for the PCR program were as follows: 1 cycle of initial denaturation at 94°C for 2 min, followed by 30 cycles of denaturation at 94°C for 30 s, annealing and extension at 68°C for 4 min, and 1 cycle of final extension at 72°C for 10 min. The PCR products were analyzed on 1.5% agarose gels using 0.5x Tris borate ethylenediaminetetraacetate (TBE) buffer stained with ethidium bromide (AppliChem GmbH, Darmstadt, Germany). The PCR bands were visualized under UV light using the gel documentation system (Syngene, MD, USA) (Fig. 2a).

The hybrid Rhesus box was identified as the RHD gene deletion in all samples by PCR-SSP. The sequence of specific primers used for the hybrid Rhesus box was similar to the previous report [15, 18]. The PCR reaction mixture is composed of 8 µL of 2× PCR buffer (67 mm Tris-HCL pH 8.8, 17 mm ammonium sulfate, 0.1% Tween 20), 1.4 mm dNTPs (Vivantis, Kuala Lumpur, Malaysia), 6 mm MgCl₂, 5 U of Taq DNA polymerase (Invitrogen, Madison, WI, USA), 100–200 ng of genomic DNA, and 5 µL of 0.5 µm hybrid Rhesus box of forward and reverse primer. Internal control was performed using the human growth hormone (HGH) gene at a concentration of 0.02 µm. The PCR amplification and analysis of PCR products were performed under the conditions described in single-tube multiplex PCR-SSP (Fig. 2b).

All samples were determined for the RHCE*C, RHCE*c, RHCE*E, and RHCE*e genotypes using PCR-SSP. The sequences of specific primers used were similar to the previous report [19]. All PCR mixtures comprised 8 µL of 2× PCR buffer (67 mm Tris-HCL pH 8.8, 17 mm ammonium sulfate, 0.1% Tween 20), 1.4 mm dNTPs (Vivantis, Kuala Lumpur, Malaysia), 6 mm MgCl₂, 5 U of Taq DNA polymerase (Invitrogen, Madison, WI, USA), 100–200 ng of genomic DNA, 5 µL of 0.5 µm of forward and reverse specific primers, and 0.1 µm of HGH as an internal control. Four PCR mixtures were tested in the same PCR conditions. The cycling parameters for the PCR procedure were as follows: 1 cycle of initial denaturation at 96°C for 2 min, followed by 5 cycles at 96°C for 30 s, 68°C for 60 s, and 72°C for 40 s; then 21 cycles of 96°C for 30 s, 60°C for 60 s, and 72°C for 40 s; then 4 cycles of 96°C for 30 s, 55°C for 1.15 min, and 72°C for 2 min, and followed by 1 cycle of final extension at 72°C for 10 min. The analysis of PCR products was performed under the conditions described in single-tube multiplex PCR-SSP (Fig. 2c).

The twenty-one PCR products that were positive for c.1227G>A in RHD exon 9 were chosen at random as a cohort to verify c.1227G>A, a substitution in the RHD codon 409, by Sanger sequencing (Bio Basic Inc., Canada) using fluorescent dye-terminator sequencing on an analyzer (ABI Prism™ 3730x/DNA sequencers) using forward and reverse primers as described above. Sequencing data were analyzed using SnackVar V2.4.3 software.

Single-tube multiplex PCR-SSP was used to investigate RHD exons (exons 1, 4, 7, 10) and c.1227G>A in RHD exon 9 in a total of 205 samples with a serological D− phenotype. The results revealed three patterns of amplified RHD exons: total deletion of the RHD gene, Asian-type DEL, and RHD-CE-D hybrid (Fig. 2a). Of the samples investigated, 141 (68.8%) exhibited total deletion of the RHD gene, while Asian-type DEL was observed in 50 samples (24.4%). Eight samples were assumed to contain the RHD-CE-D hybrid allele, characterized by a substitution of RHD with RHCE that resulted in the absence of RHD exons. Among these, six samples exhibited exons 1 and 10 of the RHD gene, probably indicating the RHD*01N.03 allele. The other two samples displayed only exon 1, likely suggesting the RHD-CE(3–10) allele. The remaining six samples were unclassified RHD variants as they contained RHD exons 1, 4, 7, and 10. The allele frequency in 205 samples with a serological D− phenotype is shown in Figure 3. As expected, the homozygous deletion of the RHD gene was found to be the most common genotype (RHD*01N.01/RHD*01N.01, 68.8%), followed by the hemizygous state of Asian-type DEL (RHD*01EL.01/RHD*01N.01, 22.9%). The RHD genotypes that this approach was able to identify are exhibited in Table 1.

The detection of the hybrid Rhesus box, an unequal crossing of the upstream and downstream Rhesus boxes, has determined the RHD zygosity (Fig. 2b). From the overall samples, 68.8% (141/205) were presumably homozygous for RHD deletion (RHD−/RHD− homozygotes), identified by samples testing positive only for hybrid Rhesus box and the absence of RHD exons in the total deletion of RHD. A further 29.3% (60/205) of the samples were RHD+/RHD− hemizygotes, which had detectable RHD exons, while another RHD allele had a hybrid Rhesus box. Finally, 1.9% (4/205) lacked the hybrid Rhesus box that determined RHD+/RHD+ homozygotes (Table 1).

The frequencies of predicted RhCE phenotypes based on RHCE genotyping are summarized in Table 1. In the homozygous deletion of the RHD gene subset (141 samples), the RHCE genotyping revealed 44.7% RHCE*Ce/RHCE*ce (63/141), followed by 44.0% RHCE*ce/RHCE*ce (62/141). Interestingly, all of the Asian-type DEL samples present the RHCE*C/E allele (predicted RhCE phenotype: C/E+). RHCE*Ce/RHCE*ce was detected in 63.3% (31/50), representing the highest occurrence. Among the 205 samples, the predicted RhCE phenotypes from RHCE genotyping were Ccee (102, 49.8%), followed by ccee (62, 30.3%), CCee (30, 14.6%), CcEe (6, 2.9%), and ccEe (5, 2.4%).

To confirm the genotyping results of RHD*01EL.01 (Asian-type DEL), the twenty-one PCR products that were positive for c.1227G>A in RHD exon 9 were chosen at random as a cohort to verify c.1227G>A, a substitution in the RHD codon 409, by Sanger sequencing (Bio Basic Inc., Canada). The results of the twenty-one samples by Sanger sequencing were all consistent with single-tube multiplex PCR-SSP (Fig. 4).

In East Asia and Thailand, individuals with the D− phenotype are rare, accounting for 0.1–0.5% [4, 6]. Among the serologically D− phenotype in Thai blood donors, the four most common RHD variant alleles were RHD*01N.01, RHD*01EL.01, RHD*01N.03, and RHD-CE(3–10), accounting for 98% of the total RHD alleles [8, 9]. Importantly, routine blood bank serological methods are inadequate for detecting certain variations, like the RHD*01EL.01 allele. Therefore, simple and accurate molecular methods to discriminate these alleles are needed but present challenges since the clinical outcomes and management of Asian-type DEL RBCs are different in these groups.

In this study, we established a single-tube multiplex PCR-SSP in order to detect RHD variant alleles commonly found in serologically D− phenotype individual in a Thai population. This approach is designed to use four RHD exons (exons 1, 4, 7, 10) and c.1227G>A in RHD exon 9. Additionally, the hybrid Rhesus box was included to detect the RHD deletion allele and has the potential to predict RHD zygosity. Accordingly, our molecular platform is helpful for distinguishing Asian-type DEL from true D− phenotype individuals.

In our cohort, the homozygous deletion of the RHD gene (RHD*01N.01/RHD*01N.01) was found to be the most common RHD genotype. Similar findings were reported in other studies from East Asian populations [6, 20‒22]. Interestingly, in this subset, half of the samples carry RHCE*C, E, or both alleles. This observation is concordant with the previous study that revealed ∼42% of the 290 samples from the northeastern part of Thailand with RHD*01N.01/RHD*01N.01 were C/E+ phenotypes (regional prevalence range: 24.9%–42.4%) and even more specifically, C+ [8]. Based on these results, the C+ phenotype was widely involved in RHD variant alleles in a Thai population. This observation indicates important results to help design and implement a potential molecular diagnostic strategy for RHD genotyping in Thailand.

Interestingly, six samples showed only exons 1 and 10 of the RHD gene and were assumed to be the RHD-CE-D hybrid allele. Based on our results, we believe that these samples may carry RHD*01N.03, which is the most common type of hybrid in East Asia and Thailand [23]. Since this hybrid comprises RHD exons 1 and 10, with the RHCE sequence spanning exons 2 through 9, RHD exons 4 and 7 were thereby not detected. Similarly, the other two samples exhibited only exon 1, leading to the assumption of the RHD-CE(3–10). RHD*01N.03 and RHD-CE(3–10) are known to lack production of the D antigen. Consequently, RBCs from blood donors who carry these hybrids may safely be omitted because their RBC units are actually true D− phenotype. Thus, specific detection of large hybrid genes, such as the RHD*01N.03, may lack a known clinical benefit but still be valuable for research aiming to discriminate between the mutational mechanisms caused by the large hybrid RHD-CE-D and the total deletion of the RHD gene. Additionally, most of the samples in this group carried the Ccee phenotype. This hybrid allele and RHCE combinations are consistent with data from a previous study, indicating that typically there is a strong positive bias toward C/E+ individuals [21, 23, 24].

About 24.4% of the serologically D− phenotype cases observed in this study carried the Asian-type DEL allele, a frequency comparable to that found in a previous investigation of the Asian-type DEL allele in the northeastern Thai population [8, 17], but higher than the reported frequency in other regions and Bangkok [9]. Furthermore, all Asian-type DEL individuals in our cohort carried the C/E+ phenotype, without any samples carrying the ccee phenotype. The result aligns with a prior study that found that the Ccee phenotype is the predominant RhCE phenotype among ethnic Asians [25]. Since the DEL phenotype is associated with the RhC+ phenotype (CDe or cDE haplotypes), RhCE phenotyping is recommended as a potential screening approach for DEL variants in serologically D− phenotype individuals and to select those who are C/E+ phenotype for future analysis of the DEL variant alleles [7, 26, 27]. This approach can be implemented in East and Southeast Asian countries, which together include ∼ 2.0 million individuals with Asian-type DEL [10].

Asian-type DEL RBCs transfusion in D− patients has been observed to result in both primary and secondary anti-D alloimmunization [28, 29]. Recent studies have provided multiple lines of evidence that individuals with Asian-type DEL may safely receive D+ RBCs transfusions without risk of alloanti-D immunization, including discontinuation Rh immunoglobulin prophylaxis in pregnant women with Asian-type DEL [10‒12]. Indeed, Asian-type DEL individuals are affirmed to be classified as RhD-positive. Thus, molecular identification of Asian-type DEL carried in patients and blood donors with the serologically D− phenotype is necessary and also challenging. It will safely and consistently increase patient protection against anti-D alloimmunization by employing blood transfusions. Besides, the management of supplying D− RBC units for D− recipients is critical for blood transfusion services, especially involving those from East Asian backgrounds where D− RBC units are scarce.

However, the multiplex PCR assay targeting different RHD exons has successfully been developed to detect RHD variant alleles for D− East Asians. Kim and colleagues proposed a multiplex PCR for eight RHD exons and modular PCR-SSP and used PCR-restriction fragment length polymorphism for hybrid Rhesus box and for Asian-type DEL by sequencing [30]. Suksard and colleagues, Luettringhaus and colleagues, and Kim and colleagues proposed a single or double set of multiplex PCR for RHD genotyping, modular PCR-SSP for RHD*01EL.01, and sequencing, respectively [13, 21, 31]. The aforementioned multiplex PCR setup still required two or more sets of PCR and included different conditions for RHD genotyping strategies. In contrast, single-tube multiplex PCR-SSP in this study allows for the analysis of total RHD gene deletion, Asian-type DEL, and RHD-CE-D hybrid in a single tube. This RHD genotyping approach is very simple to use and ought to be applied more often in routine blood bank laboratories.

Unexpectedly, six samples in this study remained unclassified. This could be attributed to the limitations of the technique, which led to the misidentification of certain variations. For instance, variant alleles have an identical pattern to the RHD-CE-D hybrid allele, RHD*D-CE(4–9)-D (termed RHD*01EL.44 by the ISBT), the second most frequent DEL allele in Thais, which has an allele frequency of 0.15% [8, 9]. In addition, various mechanisms play a role in RHD mutations. For example, RHD*01N.83, a frameshift mutation with an allele frequency of 0.12% [9], was not detected using this method. However, these alleles occur far less frequently. Interesting data from the RHD genotype of D− phenotype Thai blood donors revealed the presence of a wild-type RHD allele (RHD*01) [8, 9]. Hence, sequencing the complete RHD gene, including the promoter, introns, and all exons, allows further exploration of the feasible functional variations altering the production of the D antigen. For this reason, quantitative methods such as quantitative multiplex polymerase chain reaction of short fluorescent fragments and next-generation sequencing are crucial in characterizing novel alleles and expanding the comprehensive molecular basis of RHD gene polymorphism.

Our results indicated that the single-tube multiplex PCR-SSP targeted RHD exons (exons 1, 4, 7, 10) and c.1227G>A in RHD exon 9 in combination with hybrid Rhesus box for analyses of RHD*01N.01, RHD*01EL.01, and RHD-CE-D hybrid as a simple and accurate method. The method was developed in this study and is useful to determine RHD variant alleles commonly found in the serologically D− phenotype in a Thai population. Thus, this assay may be used as an alternative technique for the routine clinical differential diagnosis of RHD*01EL.01 and RHD*01N.01 and may be especially useful for blood transfusion in the prevention of anti-D alloimmunization in regions with a high prevalence of Asian-type DEL.

The authors thank the staff at the Blood Transfusion Center, Faculty of Medicine, Khon Kaen University, for providing the blood samples and for their excellent cooperation.

The project was approved by the Khon Kaen University Ethics Committee for Human Research (HE661043). All participants provided their informed consent.

The authors have declared no conflicting interests.

This research was funded by the research fund for supporting lecturers to admit high potential students to study and research within his expert program, year 2021, Graduate School, Khon Kaen University, Thailand (Grant No. 641JH106). The funder had no role in the design, data collection, data analysis, and reporting of this study.

O.P. was involved in the experiments, acquisition of data, analysis and interpretation of data, and writing the original draft. C.L. and A.V.R. were involved in critical revision of the article. P.S. was involved in conceptualization, analysis and interpretation of data, and writing and editing and funding acquisition. All authors have read and approved the final version of the article.

All data generated or analyzed during in this study are included in this article and its supplementary material file.