FY*X real-time polymerase chain reaction with melting curve analysis associated with a complete one-step real-time FY genotyping

BACKGROUND AND OBJECTIVES: The Duffy (FY) blood group system is controlled by four major alleles: FY*A and FY*B, the Caucasian common alleles, encoding Fy(a) and Fy(b) antigens; FY*X allele responsible for a poorly expressed Fy(b) antigen, and FY*Fy a silent predominant allele among Black population. Despite the recent development of a real-time fluorescent polymerase chain reaction (PCR) method for FY genotyping FY*X genotyping has not been described by this method. This study focused on the real-time FY*X genotyping development associated with a complete, one-step real-time FY genotyping, based on fluorescence resonance energy transfer (FRET) technology. MATERIALS AND METHODS: Seventy-two blood samples from Fy(a+b-) Caucasian blood donors were studied by real-time PCR only. Forty-seven Caucasian and Black individual blood samples, referred to our laboratory, were studied by PCR-RFLP and real-time PCR. For each individual, the result of the genotype was compared to the known phenotype. RESULTS: The FY*X allele frequency calculated in an Fy(a+b-) Caucasian blood donors population was 0.014. With the Caucasian and Black patient samples we found a complete correlation between PCR-RFLP and the real-time PCR method whatever the alleles combination tested. When the known phenotype was not correlated to FY*X genotype, the presence of the Fy(b) antigen was always confirmed by adsorption-elution. CONCLUSION: The real-time technology method is rapid and accurate for FY genotyping. From now, we are able to detect the FY*X allele in all the alleles combinations studied. Regarding its significant frequency, the detection of the FY*X allele is useful for the correct typing of blood donors and recipients considering the therapeutic use of blood units and the preparation of test red blood cells for antibody screening.     

Large-scale blood group genotyping – clinical implications

The molecular background of blood group antigen expression of the major clinically significant blood group antigens has been largely accomplished. Despite this large body of work, blood group phenotype prediction by genotyping has a marginal supporting role in the routine blood bank. It has however had a major impact in the prenatal determination of fetal blood group status in the management of haemolytic disease of the fetus and newborn. In the past few years several high throughput systems have been in development that have the potential capacity to perform genotyping on a mass scale. Such systems have been designed for use on donor- and patient-derived DNA and provide much more comprehensive information regarding an individuals blood group than is possible by using serological methods alone. DNA-based typing methodology is easier to standardize than serology and has the potential to replace it as a front line diagnostic in blood banks. This review overviews the current situation in this area and attempts to predict how blood group genotyping will evolve in the future.     

Sequence-specific primers for MNS blood group genotyping

Background

Various techniques of genotyping the MNSs blood group have been described, but none of them enables the complete detection of all MNS antigens.

Materials and Methods.

Blood samples were obtained from blood donors. Primers were created using the published DNA sequences for glycophorins A and B. Genotyping was performed using polymerase chain reaction sequence-specific primers (PCR-SSP).

Results

A total of seven primers were found to specifically amplify the most common MNS antigens. The use of these primers has enabled us to correctly genotype all blood samples tested so far (n=116).

Discussion.

Specifically created primers enable genotyping of the MNS antigens in a single PCR-SSP run. The method is reliable, easy to perform, and can be used in routine practice.     

Blood group genotyping: from patient to high-throughput donor screening

Blood group antigens, present on the cell membrane of red blood cells and platelets, can be defined either serologically or predicted based on the genotypes of genes encoding for blood group antigens. At present, the molecular basis of many antigens of the 30 blood group systems and 17 human platelet antigens is known. In many laboratories, blood group genotyping assays are routinely used for diagnostics in cases where patient red cells cannot be used for serological typing due to the presence of auto-antibodies or after recent transfusions. In addition, DNA genotyping is used to support (un)-expected serological findings. Fetal genotyping is routinely performed when there is a risk of alloimmune-mediated red cell or platelet destruction. In case of patient blood group antigen typing, it is important that a genotyping result is quickly available to support the selection of donor blood, and high-throughput of the genotyping method is not a prerequisite. In addition, genotyping of blood donors will be extremely useful to obtain donor blood with rare phenotypes, for example lacking a high-frequency antigen, and to obtain a fully typed donor database to be used for a better matching between recipient and donor to prevent adverse transfusion reactions. Serological typing of large cohorts of donors is a labour-intensive and expensive exercise and hampered by the lack of sufficient amounts of approved typing reagents for all blood group systems of interest. Currently, high-throughput genotyping based on DNA micro-arrays is a very feasible method to obtain a large pool of well-typed blood donors. Several systems for high-throughput blood group genotyping are developed and will be discussed in this review.     

Real-time multiplex allele-specific polymerase chain reaction for genotyping of the Duffy antigen, the Plasmodium vivax invasion receptor

BACKGROUND AND OBJECTIVES: Duffy blood group is of major interest in clinical medicine as it is not only involved in blood-transfusion risks and occasionally in neonatal haemolytic disease, but it is also the receptor for the human malaria parasite Plasmodium vivax in the erythrocyte invasion. The aim of this study was to develop a rapid and inexpensive approach for high-throughput Duffy genotyping. MATERIALS AND METHODS: This paper reported the development of a Duffy genotyping assay based on multiplex real-time polymerase chain reaction (PCR) using SYBR Green I fluorescent dye. RESULTS: By using this approach for Duffy genotyping we obtained the same results as that for the conventional allele-specific PCR, however, in a high-throughput assay. The Duffy genotyping of field samples demonstrated that P. vivax-infected individuals showed a significantly higher prevalence of two functional alleles than Plasmodium falciparum-infected and non-infected individuals. This finding corroborates the hypothesis that the presence of two functional alleles increases the risk of P. vivax infection. CONCLUSION: This methodology may be suitable for epidemiological studies, particularly for exploring the relationship between Duffy alleles and malaria susceptibility, and also for identification of transfusional incompatibility in blood banks.     

Prenatal genotyping of RHD and SRY using maternal blood

BACKGROUND AND OBJECTIVES: The aim of the study was to perform fetal RHD genotyping in maternal plasma using a fluorescent polymerase chain reaction (PCR) technique. Duplex PCR, amplifying RHD and SRY in the same tube, was undertaken. The effect of varying storage temperatures on the concentration of fetal DNA was investigated in a separate study involving 10 RhD-negative pregnant women. MATERIALS AND METHODS: Primers and probes for the RHD gene's exon 7 and the sex-determining region, Y, were designed, and monoplex and duplex PCR were performed. Blood samples from 10 RhD-negative women were split into four and treated in four different ways before measuring the concentration of fetal DNA by quantitative PCR. RESULTS: DNA extracted from the plasma of 114 RhD-negative pregnant women was tested for the presence of fetal RHD. The discrepancy between genotyping and serological RhD typing of the babies postpartum was 8% when counting one positive replicate as a positive result. Duplex PCR, amplifying RHD and SRY in the same tube, showed a reduced sensitivity for amplification of the SRY gene segment. There was a statistically significant reduction of fetal DNA in blood samples stored at room temperature for 48 h compared with the same sample stored at a temperature of <10 degrees C for the same length of time. CONCLUSIONS: This method is not suitable for routine analysis because of the lack of a positive control for RHD-negative female fetuses and a decrease in PCR sensitivity when performing duplex PCR. Fetal DNA in maternal plasma is better preserved when the blood sample is kept cool.     

澳门地区鸡源产气荚膜梭菌的污染情况及基因分型

目的 调查澳门地区鸡源性产气荚膜梭菌的污染情况及基因分型。方法 利用双管法分离产气荚膜梭菌,再作生化测试以鉴定;利用多重PCR测定分离菌株的基因分型。结果 从120个样本中检出率为37.5%,所分离到的产气荚膜梭菌均为 A 型产气荚膜梭菌。结论 初步研究指出本澳供应的鸡肠、新鲜鸡和冰鲜鸡中存在A 型产气荚膜梭菌污染,为产气荚膜梭菌所致的公共卫生问题提供了参考数据。     

Distribution of Diego blood group alleles and identification of four novel mutations on exon 19 of SLC4A1 gene in the Chinese Han population by polymerase chain reaction sequence–based typing

Background The Diego blood group system plays an important role in transfusion medicine. Genotyping of DI1 and DI2 alleles is helpful for the investigation into haemolytic disease of the newborn (HDN) and for the development of rare blood group databases. Here, we set up a polymerase chain reaction sequence–based typing (PCR‐SBT) method for genotyping of Diego blood group alleles. Study Design and Methods Specific primers for exon 19 of the solute carrier family 4, anion exchanger, member1 (SLC4A1) gene were designed, and our PCR‐SBT method was established and optimized for Diego genotyping. A total of 1053 samples from the Chinese Han population and the family members of a rare proband with DI1/DI1 genotype were investigated by the PCR‐SBT method. An allele‐specific primer PCR (PCR‐ASP) was used to verify the reliability of the PCR‐SBT method. Results The frequencies of DI1 and DI2 alleles in the Chinese Han population were 0·0247 and 0·9753, respectively. Six new single nucleotide polymorphisms (SNPs) were found in the sequenced regions of the SLC4A1 gene, and four novel SNPs located in the exon 19, in which one SNP could cause an amino acid alteration of Ala858Ser on erythrocyte anion exchanger protein 1. The genotypes for Diego blood group were identical among 41 selected samples with PCR‐ASP and PCR‐SBT. Conclusion The PCR‐SBT method can be used in Diego genotyping as a substitute of serological technique when the antisera is lacking and was suitable for screening large numbers of donors in rare blood group databases.     

四川地区鸡源产气荚膜梭菌的分离鉴定与基因分型

目的检测四川地区健康鸡群产气荚膜梭菌的分布状况。方法从四川地区规模化鸡场采集新鲜粪便样品,按产气荚膜梭菌α、β、ε、ι毒素基因cpa、cpb、etx及iA序列,设计针对4种毒素基因的4对特异引物,应用多重PCR方法对产气荚膜梭菌进行基因分型。最后利用重复序列PCR进一步做亚型分析。结果从150份样品中分离到8株(5.3%)产气荚膜梭菌,多重PCR都只扩增出α条带,毒素基因分型结果均为A型。ERIC-PCR、REP-PCR图谱显示两者均适用于产气荚膜梭菌亚型分析,ERIC-PCR是一种更简便、快速和有效的分子流行病学调查方法。结论四川地区健康鸡群中存在的产气荚膜梭菌主要是A型。     

Report of the fourth International Workshop on molecular blood group genotyping

The fourth International Society of Blood Transfusion (ISBT) workshop on molecular blood group genotyping was held in 2010, with a feedback meeting at the ISBT Congress in Berlin, Germany. Fifty laboratories participated, 17 more than in 2008. Six samples were distributed. Samples 1-3 were DNA samples for all red cell blood group tests available to the participants. Of the 46 laboratories that tested these samples, 37 obtained completely correct results, although the extent of testing varied considerably. Sample 4, also a DNA sample, was an Rh problem in which RHDΨ and RHCE*ceCF were present, but the participants were only informed that the donor's red cells typed as positive with some monoclonal anti-D. Of the 42 laboratories that participated in this exercise, seven performed the sequencing necessary to obtain the correct result. Samples 5 and 6 were plasma samples from RhD-negative pregnant women, for foetal RhD testing. These were sent to 25 laboratories, and two incorrect results were reported. Overall, the level of accuracy was about equal to that of the previous workshop. The main conclusion for the last two workshops can be reiterated: with greater care and attention to detail, very high standards could be set for molecular blood group genotyping.     

Report of the First International Workshop on molecular blood group genotyping

The use of molecular genetic technology for blood group typing is becoming routine procedure in many reference laboratories worldwide. A First International Workshop was organized on behalf of the International Society of Blood Transfusion (ISBT) and the International Council for Standardization in Haematology (ICSH). Thirty laboratories that provide a molecular diagnostic service participated in the workshop. Six samples were distributed: two represented DNA from transfusion-dependent patients for testing for multiple polymorphisms; two represented fetal DNA prepared from amniotic fluid for RhD, Rhc and K-testing; and two represented plasma from RhD-negative pregnant women for fetal RhD testing. Error rates varied from 0 to 11% for different polymorphisms. A consensus arising from discussion on the workshop results between participants at a feedback meeting and by e-mail has resulted in seven recommendations for molecular blood group genotyping. Further international workshops will take place every 2 years, with a more limited exercise being organized in the intervening years.     

International reference reagents to standardise blood group genotyping: evaluation of candidate preparations in an international collaborative study

Background and Objectives The aim of the study was to evaluate, in an international collaboration, four lyophilised genomic DNA preparations, selected from genotyped and phenotyped donors by the study organisers, for their suitability to standardise and control blood group genotyping procedures for common ancestral Caucasian and Black African alleles. Materials and Methods Twenty‐nine laboratories performed ‘blind’ testing of replicated ampoules of the candidate reference reagents, RBC1 (10/232), RBC4 (10/236), RBC5 (10/238) and RBC12 (10/234), using a range of genotyping procedures, most commonly classical PCR using allele or sequence specific primers. Results The majority of laboratories reported blood group genotypes in accordance with those determined by the study organisers and the serological phenotypes. Despite an overall high level of accuracy in genotyping, the identified errors and inconsistencies, and the limited genotyping capabilities of many laboratories, confirmed the need for validated reference materials to control test procedures. Conclusions The establishment of RBC1, RBC4, RBC5 and RBC12 as World Health Organization Reference Reagents will facilitate international standardisation of blood group genotyping and ensure that such tests are sufficiently sensitive and specific.     

Report of the Second International Workshop on molecular blood group genotyping

The second International Society of Blood Transfusion and International Council for Standardization in Haematology workshop on molecular blood group genotyping was held in 2006. Forty-one laboratories participated. Six samples were distributed: two representing DNA from transfusion-dependent patients for testing for all clinically important polymorphisms; two representing DNA from amniotic fluid for RhD, Rhc, and K testing; and two plasma samples from RhD-negative pregnant women for fetal RhD testing (only tested by 20 laboratories). Overall, a high level of accuracy was achieved by most of the laboratories, although the error rate caused by RHDPsi was not acceptable and needs to be addressed. With greater care and attention to detail, very high standards could be set for molecular blood group genotyping.     

Next‐generation sequencing is a credible strategy for blood group genotyping

Although several medium/high‐throughput tools have been engineered for molecular analysis of blood group genes, they usually rely on the targeting of single nucleotide polymorphisms, while other variants remain unidentified. To circumvent this limitation a strategy for genotyping blood group genes by next‐generation sequencing (NGS) was set up. Libraries consisting of exons, flanking introns and untranslated regions of 18 genes involved in 15 blood systems were generated by the Ion AmpliSeq^? Library Kit 2.0 and by fragmenting polymerase chain reaction products, normalized by two different approaches, mixed and sequenced by the Ion Torrent Personal Genome Machine (PGM^?) Sequencer. In our conditions, defined to limit both intra‐ and inter‐sample variability, sequences from mixed libraries were read in a single run for a total coverage of 86·03% of the coding DNA sequences, including all loci defining the most clinically relevant antigens in all genes, except ABO. Importantly, the challenging attempt to generate gene‐specific data for the homologous genes was successful. This work, which combines two complementary approaches to generate libraries, defines technical conditions for genotyping blood group genes, illustrates that NGS is suitable for such an application and suggests that, after automation, this novel tool could be used for molecular typing at the laboratory level.     

Specificity and sensitivity of RHD genotyping methods by PCR-based DNA amplification

We have compared the sensitivity and specificity of four PCR methods of RHD gene detection using different sets of primers located in the regions of highest divergence between the RHD and RHCE genes, notably exon 10 (method I), exon 7 (method II), exon 4 (method III) and intron 4 (method IV). Methods I–III were the most sensitive and gave a detectable signal with D-pos/D-neg mixtures containing only 0.001% D-positive cells. Moreover, method II could detect the equivalent DNA amount present in only three nucleated cells in the assay without hybridization of PCR products, whereas the sensitivity of the other methods was 10–50 times less. Investigation of D variants indicated that false-negative results were obtained with method II (D^IVb variant), method III (D^VI and DFR variants) and method IV (D^VI variants), but not method I. Weak D (D^u) was correctly detected as D-positive by all methods, but most cases of Rh_null appeared as false-positives, as they carry normal RH genes that are not phenotypically expressed. Some false-positive results were obtained with method I in a few Caucasian DNA samples serotyped as RhD-neg but carrying a C - or E -allele, whereas a high incidence of false-positives was found among non-Caucasian Rh-negative samples by all methods. In the Caucasian population, however, we found a full correlation between the predicted genotype and observed phenotype at birth of 92 infants. Although we routinely use the four methods for RHD genotyping, a PCR strategy based on at least two methods is recommended.     

A1,2BO1,2 genotyping by multiplexed allele-specific PCR

The ABO blood group is the most clinically important human alloantigen system in transfusion medicine. The system involves three antigens A, B and H. H antigen is converted to either A or B by the activity of α1 → 3- n -acetyl-galactosaminyl transferase (A transferase) or α1 → 3 galactosyl transferase (B transferase). The O phenotype is the result of an inactive glycosyltransferase, which is unable to glycosylate the H antigen.
The immunological properties of the ABO system were identified at the turn of the century; however, the genetic basis of the ABO system has only recently been characterized. This has enabled the development of a number of molecular ABO typing methods. Described here is a two-reaction multiplex allele-specific PCR (ASPCR) genotyping assay for the A¹, A², B, O¹ and O² subtypes. 11 different allele-specific oligonucleotide primers were selected to detect the presence or absence of the O¹ associated G →  (−) deletion at base 261, the O² associated G → A substitution at base 802, the B associated G → A substitution at base 803, and finally the A² associated C → (−) deletion at base 1059.
A total of 122 peripheral blood samples were genotyped and serologically forward and reverse typed. A concordance rate of 98.4% (120/122 samples) was observed between the actual genotype and the serologically-based predicted genotype. These results indicate that this assay provides a rapid, accurate, and simple method for A^1,2BO^1,2 genotyping that serves as a useful supplement to standard serological ABO typing.     

Rapid genotyping of human platelet antigen 5 with fluorophore-labelled hybridization probes on the LightCycler

Genotyping of human platelet antigens (HPAs) can be useful for the diagnosis and therapy of alloimmune thrombocytopenic syndromes such as neonatal alloimmune thrombocytopenic purpura, post-transfusion purpura and refractoriness to platelet transfusion therapy. We developed a single-tube method for HPA-5 genotyping on the LightCycler that combines rapid-cycle polymerase chain reaction with allele-specific fluorescent probe melting for mutation detection. Our method is fast, robust and suitable for routine HPA-5 typing. This work extends recent studies on HPA-1 typing using the LightCycler.