Survey and characterization of nonfunctional alleles of FUT2 in a database.

The expression of ABO antigens in human saliva is regulated by the FUT2 gene, which encodes a secretor type α(1,2)fucosyltransferase. Secretors express ABO substrates in saliva and non-secretors do not. Secretor status is an object of concern, especially for susceptibility to various infectious diseases. A multitude of single nucleotide polymorphisms (SNPs) and copy number variations (CNVs) have been reported, and they show unique distributions among different populations. In this study, we selected 18 uncharacterized FUT2 alleles listed in the Erythrogene database and obtained genomic DNA having these alleles. We experimentally confirmed the haplotypes, but 10 of 18 alleles disagreed with those in the database, which may be attributed to their low frequency. We then examined the activity of the encoded α(1,2)fucosyltransferase for 13 alleles by flow cytometry of H antigen expression. The impact of each nonsynonymous SNP on the enzyme was also estimated by software. We finally identified two non-secretor alleles (se610and se357,856,863) and one weak secretor allele (se262,357), while in silico analysis predicted that many alleles impair the function. The present results suggest that correct haplotyping and functional assays are desirable for analysis of the FUT2 gene.

Maintaining adequate donations and a sustainable blood supply: Lessons learned.

BACKGROUND: The availability of a safe blood supply is a key component of transfusion medicine. A decade of decreased blood use, decreased payment for products, and a dwindling donor base have placed the sustainability of the US blood supply at risk. STUDY DESIGN AND METHODS: A literature review was performed for blood center (BC) and hospital disaster management, chronically transfusion-dependent diseases, and appropriate use of group O-negative red blood cells (RBCs), and the Choosing Wisely campaign. The aim was to identify current practice and to make recommendations for BC and hospital actions. RESULTS: While BCs are better prepared to handle disasters than after the 9/11 attacks, messaging to the public remains difficult, as donors often do not realize that blood transfused during a disaster was likely collected before the event. BCs and transfusion services should participate in drafting disaster response plans. Hospitals should maintain inventories adequate for patients in the event supply is disrupted. Providing specialty products for transfusion-dependent patients can strain collections, lead to increased use of group O RBCs, and create logistical inventory challenges for hospitals. The AABB Choosing Wisely initiative addresses overuse of blood components to optimally use this precious resource. Group O-negative RBCs should be transfused only to patients who truly need them. CONCLUSIONS: Collecting and maintaining a blood supply robust enough to handle disasters and transfusion-dependent patients in need of specialty products is challenging. Collaboration of all parties should help to optimize resources, ensure appropriate collections, improve patient care, and ultimately result in a robust, sustainable blood supply.

Automated typing of red blood cell and platelet antigens: a whole-genome sequencing study.

BACKGROUND: There are more than 300 known red blood cell (RBC) antigens and 33 platelet antigens that differ between individuals. Sensitisation to antigens is a serious complication that can occur in prenatal medicine and after blood transfusion, particularly for patients who require multiple transfusions. Although pre-transfusion compatibility testing largely relies on serological methods, reagents are not available for many antigens. Methods based on single-nucleotide polymorphism (SNP) arrays have been used, but typing for ABO and Rh-the most important blood groups-cannot be done with SNP typing alone. We aimed to develop a novel method based on whole-genome sequencing to identify RBC and platelet antigens. METHODS: This whole-genome sequencing study is a subanalysis of data from patients in the whole-genome sequencing arm of the MedSeq Project randomised controlled trial (NCT01736566) with no measured patient outcomes. We created a database of molecular changes in RBC and platelet antigens and developed an automated antigen-typing algorithm based on whole-genome sequencing (bloodTyper). This algorithm was iteratively improved to address cis-trans haplotype ambiguities and homologous gene alignments. Whole-genome sequencing data from 110 MedSeq participants (30 × depth) were used to initially validate bloodTyper through comparison with conventional serology and SNP methods for typing of 38 RBC antigens in 12 blood-group systems and 22 human platelet antigens. bloodTyper was further validated with whole-genome sequencing data from 200 INTERVAL trial participants (15 × depth) with serological comparisons. FINDINGS: We iteratively improved bloodTyper by comparing its typing results with conventional serological and SNP typing in three rounds of testing. The initial whole-genome sequencing typing algorithm was 99·5% concordant across the first 20 MedSeq genomes. Addressing discordances led to development of an improved algorithm that was 99·8% concordant for the remaining 90 MedSeq genomes. Additional modifications led to the final algorithm, which was 99·2% concordant across 200 INTERVAL genomes (or 99·9% after adjustment for the lower depth of coverage). INTERPRETATION: By enabling more precise antigen-matching of patients with blood donors, antigen typing based on whole-genome sequencing provides a novel approach to improve transfusion outcomes with the potential to transform the practice of transfusion medicine. FUNDING: National Human Genome Research Institute, Doris Duke Charitable Foundation, National Health Service Blood and Transplant, National Institute for Health Research, and Wellcome Trust.

The impact of blood type O on mortality of severe trauma patients: a retrospective observational study.

BACKGROUND: Recent studies have implicated the differences in the ABO blood system as a potential risk for various diseases, including hemostatic disorders and hemorrhage. In this study, we evaluated the impact of the difference in the ABO blood type on mortality in patients with severe trauma. METHODS: A retrospective observational study was conducted in two tertiary emergency critical care medical centers in Japan. Patients with trauma with an Injury Severity Score (ISS) > 15 were included. The association between the different blood types (type O versus other blood types) and the outcomes of all-cause mortality, cause-specific mortalities (exsanguination, traumatic brain injury, and others), ventilator-free days (VFD), and total transfusion volume were evaluated using univariate and multivariate competing-risk regression models. Moreover, the impact of blood type O on the outcomes was assessed using regression coefficients in the multivariate analysis adjusted for age, ISS, and the Revised Trauma Score (RTS). RESULTS: A total of 901 patients were included in this study. The study population was divided based on the ABO blood type: type O, 284 (32%); type A, 285 (32%); type B, 209 (23%); and type AB, 123 (13%). Blood type O was associated with high mortality (28% in patients with blood type O versus 11% in patients with other blood types; p <  0.001). Moreover, this association was observed in a multivariate model (adjusted odds ratio = 2.86, 95% confidence interval 1.84-4.46; p <  0.001). The impact of blood type O on all-cause in-hospital mortality was comparable to 12 increases in the ISS, 1.5 decreases in the RTS, and 26 increases in age. Furthermore, blood type O was significantly associated with higher cause-specific mortalities and shorter VFD compared with the other blood types; however, a significant difference was not observed in the transfusion volume between the two groups. CONCLUSIONS: Blood type O was significantly associated with high mortality in severe trauma patients and might have a great impact on outcomes. Further studies elucidating the mechanism underlying this association are warranted to develop the appropriate intervention.

Rapid ABO genotyping by high-speed droplet allele-specific PCR using crude samples.

BACKGROUND: ABO genotyping has common tools for personal identification of forensic and transplantation field. We developed a new method based on a droplet allele-specific PCR (droplet-AS-PCR) that enabled rapid PCR amplification. We attempted rapid ABO genotyping using crude DNA isolated from dried blood and buccal cells. METHODS: We designed allele-specific primers for three SNPs (at nucleotides 261, 526, and 803) in exons 6 and 7 of the ABO gene. We pretreated dried blood and buccal cells with proteinase K, and obtained crude DNAs without DNA purification. RESULTS: Droplet-AS-PCR allowed specific amplification of the SNPs at the three loci using crude DNA, with results similar to those for DNA extracted from fresh peripheral blood. The sensitivity of the methods was 5%-10%. The genotyping of extracted DNA and crude DNA were completed within 8 and 9 minutes, respectively. The genotypes determined by the droplet-AS-PCR method were always consistent with those obtained by direct sequencing. CONCLUSION: The droplet-AS-PCR method enabled rapid and specific amplification of three SNPs of the ABO gene from crude DNA treated with proteinase K. ABO genotyping by the droplet-AS-PCR has the potential to be applied to various fields including a forensic medicine and transplantation medical care.

Evaluation of kodecytes using function-spacer-lipid constructs as a survey material for external proficiency testing for ABO subgrouping.

BACKGROUND: It is not easy to find natural red blood cells (RBCs) with weak A (Aw ) or weak B phenotype (Bw ) for use as quality controls in ABO subgroup testing (subgrouping). The aim of this study was to prepare RBC kodecytes with synthetic blood group A and/or B function-spacer-lipid (FSL) constructs and to evaluate the possibility of using such kodecytes as a survey material for an external proficiency test (PT) to improve the quality of subgroup analysis. METHODS: Three types of survey samples, including O phenotype RBCs and A kodecytes with Aw (0.02 mg/mL FSL-A solution) and B kodecytes with Bw (0.15 mg/mL FSL-B solution) were sent to 53 laboratories for an educational trial of PT for subgrouping. Cell typing was done using the manual tube technique. RESULTS: Forty-three laboratories responded, and the re-activities of the survey samples varied from 0 to 4+ against anti-A and anti-B monoclonal reagents(MoAbs). Twenty-nine laboratories (67%) correctly grouped the Bw kodecytes as Bw . Fifteen (35%), 21 (48%), and 6 (13%) laboratories grouped the Aw kodecytes as Aw , A2 , and O phenotypes, respectively. The anti-A MoAb clone affects the results of cell typing for Aw kodecytes. The stability of kodecytes was similar to that of natural O RBCs during storage. CONCLUSION: Our kodecytes were useful as a survey material, and the survey results showed the necessity of materials for PT for subgrouping to improve the quality of laboratory analysis regardless of the different reactions according to the MoAb.

Distribution and clinal trends of the ABO and Rh genes in select Middle Eastern countries.

An understanding of the ABO and Rh blood group systems is important for blood transfusions and is also pertinent due to their potential association with certain morbidities and susceptibilities to infections. To investigate the diversity and differentiation of the ABO and Rh loci in Middle Eastern populations, data from twelve representative Middle Eastern populations were analyzed. Six populations were in conformity with Hardy-Weinberg equilibrium at the ABO locus. The pooled heterozygosity at both loci was calculated to be highest in the sample from Jordan and lowest in Bahrain. Heterogeneity was pronounced in the Northern compared to the Southern Middle Eastern populations. Overall, the absolute gene diversity was 0.0046 and gene differentiation was calculated to be 0.0100. Genetic diversity of the studied loci across all populations (HT) was estimated to be 0.4594, while the diversity within the populations (HS) was 0.4548. Nei's genetic distance analyses revealed highest affinities between the populations of Kuwait and Qatar, Oman and Yemen, and between Qatar and the United Arab Emirates. These results were displayed through a UGPMA dendrogram and principal component analyses, which established clustering of certain populations. Clinal trends of the allelic systems were observed by generating contour maps that allow a detailed appreciation of the distributions of alleles across the geography of the Arabian Peninsula and the Middle East. Taken together, these analyses are helpful in understanding the differentiation of blood group loci and for designing prospective studies for establishing the associations of these loci with health variables in the populations studied.

The association baseline NIH Stroke Scale score with ABO blood-subtypes in young patients with acute ischemic stroke.

OBJECTIVES: The presence of the A and B blood group antigens has been associated with risk of arterial thrombosis. The aim of the current study was to design a new simpler form of National Institutes of Health Stroke Scale (NIHSS) for use on admission, and assess the association of blood groups with NIHSS score in young stroke patients. METHODS: We conducted this study in 1311 young Chinese adults with acute ischemic cerebral stroke. The outcome measures included a composite favorable outcome (defined as a modified Rankin Scale (mRS) of 0 or 2) and poor outcome (defined as a modified Rankin Scale score of 3 or 6) at discharge; a minor strokes (NIHSS scores 0-5) and severe strokes (NIHSS scores ≥6). Logistic regression analyses were used to determine the association between ABO blood groups and stroke severity. RESULTS: Regression analysis confirmed in relative to patients with AB subtype, Oxfordshire community stroke project classification (OCSP) subtype and serum white blood cell (WBC) were the major predictors for stroke severity. Meanwhile, diabetes, serum triglyceride and uric acid levels were determined as independent indicators of stroke severity in A, B and O blood subtype respectively. The optimal cutoff score of the baseline NIHSS was ≤5 for patients with non-O subtype, the optimal cutoff score of the baseline NIHSS was ≤7 for patients with blood O subtype. CONCLUSIONS: Our analysis provide compelling information regarding the ABO blood groups differences in predictors of stroke severity and the different validity of NIHSS scores in predicting prognosis at discharge between O subtype and non-O subtype.

[ABO blood group genotyping based on amplification refractory mutation system].

OBJECTIVE: To develop a rapid, cost-effective and reliable method for ABO genotyping. METHODS: Six pairs of primers were designed for the amplification refractory mutation system (ARMS). Thirty peripheral blood DNA samples, derived from Chinese Han unrelated individuals, were genotyped by ARMS. The results were compared with the corresponding data obtained from serological methods and DNA sequencing. RESULTS: The ABO genotyping results using ARMS were perfectly in line with the serological methods and DNA sequencing. CONCLUSION: The method developed and optimized in this study is able to accurately and rapidly detect 28 kinds of ABO genotypes, indicative of a new approach applicable to clinical practice.

Evaluation of an automated microplate technique in the Galileo system for ABO and Rh(D) blood grouping.

BACKGROUND: A number of automated devices for pretransfusion testing have recently become available. This study evaluated the Immucor Galileo System, a fully automated device based on the microplate hemagglutination technique for ABO/Rh (D) determinations. METHODS: Routine ABO/Rh typing tests were performed on 13,045 samples using the Immucor automated instruments. Manual tube method was used to resolve ABO forward and reverse grouping discrepancies. D-negative test results were investigated and confirmed manually by the indirect antiglobulin test (IAT). RESULTS: The system rejected 70 tests for sample inadequacy. 87 samples were read as "No-type-determined" due to forward and reverse grouping discrepancies. 25 tests gave these results because of sample hemolysis. After further tests, we found 34 tests were caused by weakened RBC antibodies, 5 tests were attributable to weak A and/or B antigens, 4 tests were due to mixed-field reactions, and 8 tests had high titer cold agglutinin with blood qualifications which react only at temperatures below 34 degrees C. In the remaining 11 cases, irregular RBC antibodies were identified in 9 samples (seven anti-M and two anti-P) and two subgroups were identified in 2 samples (one A1 and one A2) by a reference laboratory. As for D typing, 2 weak D+ samples missed by automated systems gave negative results, but weak-positive reactions were observed in the IAT. CONCLUSIONS: The Immucor Galileo System is reliable and suited for ABO and D blood groups, some reasons may cause a discrepancy in ABO/D typing using a fully automated system. It is suggested that standardization of sample collection may improve the performance of the fully automated system.

[Molecular basis and clinical transfusion of a family with Bw subtype of ABO blood group system].

OBJECTIVE: To study a family with Bw subtype of ABO blood group system, and to review safety issues in relation with clinical transfusion. METHODS: The molecular basis for the blood type was studied with serological assay, polymerase chain reaction-sequence specific primer (PCR-SSP) and DNA sequencing, TA clone and haplotype analysis in one blood donor whose ABO blood group were difficulty typed and her family. The bioinformatics analysis was carried out by biological analysis software to investigate the change of structure and function of enzymes influenced by the change amino acid. A retrospective survey was carried out to investigate what is the actual position that the donor blood was used in the clinical transfusion. RESULTS: Three members from the family were found to have a Bw subtype. A substitution of nucleotide C by T at position 721 in exon 7 was discovered, which resulted in replacement of amino acid Arg to Trp. Review of clinical record suggested that there has been no significant abnormality association with past three blood transfusions. CONCLUSION: A 721C>T mutation of the ABO gene probably underlies the Bw subtype. Further research is needed for understanding the clinical significance of this subtype in the blood transfusion.

Molecular genetic analysis of ABO blood group variations reveals 29 novel ABO subgroup alleles.

BACKGROUND: Identifying genetic variants of the ABO gene may reveal new biologic mechanisms underlying variant phenotypes of the ABO blood group. We report the molecular genetic analysis of 322 apparently unrelated ABO subgroup individuals in an estimated 2.1 million donors. STUDY DESIGN AND METHODS: We performed phenotype investigations by serology studies, analyzed the DNA sequence of the ABO gene by direct sequencing or sequencing after cloning, and evaluated promoter activity by reporter assays. RESULTS: In 62 rare ABO alleles, we identified 29 novel ABO subgroup alleles in 43 apparently unrelated subgroup individuals and their four available pedigrees. Of these alleles, one was a deletion-mutation allele, four were hybrid alleles, and 24 were point-mutation alleles. Most of the point mutations were detected in Exons 6 to 7, while several others were also detected in Exons 1 to 5 or splicing regions. One ABO promoter mutation, -35 to -18 del, was found and verified to reduce promoter activity, as determined by dual luciferase assays. Two mutations, 7G>T and 52C>T, carrying the premature terminal codons E3X and R18X in the 5'-region, were found to be associated with the very weak ABO subgroups "Ael" and "Bel." CONCLUSION: Twenty-nine ABO subgroup alleles were newly linked to different kinds of ABO variations. We provide the first evidence that promoter abnormality is involved in the formation of weak ABO phenotypes. We also described the first naturally occurring ABO alleles with premature terminal codons in the 5'-region that led to Ael and Bel phenotypes.

Synthesis and NMR studies on the ABO histo-blood group antigens: synthesis of type III and IV structures and NMR characterization of type I-VI antigens.

The ABO histo-blood group antigens are best known for their important roles in solid organ and bone marrow transplantation as well as transfusion medicine. Here we report the synthesis of the ABO type III and IV antigens with a 7-octen-1-yl aglycone. Also described is an NMR study of the ABO type I to VI antigens, which were carried out to probe differences in overall conformation of the molecules. These NMR investigations showed very little difference in the (1)H chemical shifts, as well as (1)H-(1)H coupling constants, across all compounds, suggesting that these ABO subtypes adopt nearly identical conformations in solution.

Subgrouping of A and AB blood groups in Indian blood centres: is it required?

Anti A1 antibody in the serum of A2 and A2B individuals is rare but when present can have laboratory and clinical significance. Routine subgrouping of all A and AB blood groups in blood centres in India is difficult due to economic constraints and has always been a point of debate. This study thus brings out the prevalence of anti A1 antibody and the clinical significance related to its presence. The results of the study showed a low prevalence of anti A1 antibody and when present, it had a low thermal amplitude and titre. Further, no blood group discrepancy or problems during compatibility testing were encountered with these (A1 antibody positive) blood units. Thus, it may be concluded that in India and other developing countries where resources are scarce, routine subgrouping of A and AB may not be really worthwhile unless there is a group discrepancy, problem during compatibility testing or history of a transfusion reaction.

Enhanced agglutination reaction of ABO subgroups by gold nanoparticle solution: implication for identification of ABO subgroups.

Although the ABO blood group is the most significant in blood group system in human, other subgroups system is also important to be concerned in blood banking laboratory. ABO subgroups have weak antigen potency on red blood cell. In some cases, they could not been detected by cell grouping and serum grouping methods. This may lead to misinterpretation of ABO typing which will cause serious problems for transfusion and transplantation. Gold nanoparticle solution can increase the agglutination reaction of ABO typing. Thus far, the investigation of ABO blood group system has been performed using gold nanoparticle solution. Samples were tested comparing between with and without gold nanoparticle solution. After reading the agglutination reaction, supernatants were collected and measured at the optical density at 760 nm by spectrophotometer. The optical density of 2-5% cell suspension and monoclonal antibody was higher than in the tube of 2-5% cell suspension, monoclonal antibody and gold nanoparticle solution. By adding the gold nanoparticle solution, the agglutination reaction was increased ranging from 7.0-37.7% (median 15.0%) for ABO grouping system whereas 12.1-50.9% (median 23.4%) was observed in ABO subgroups. It could decrease the chance of misinterpretation by 33.3%. By using gold nanoparticle solution might be the alternative way for investigation of weak antigen potency on red blood cell.

Higher frequency of secretor phenotype in O blood group - its benefits in prevention and/or treatment of some diseases.

ABO blood groups and secretor status are important in clinical and forensic medicine and in relation to some diseases. There are geographic and racial differences in their frequencies, but the frequency of secretor status in different ABO blood group systems has not been determined yet. Therefore, the aim of this study was mainly to determine this point. Blood and saliva from 762 randomly selected apparently healthy adult individuals (480 men and 282 women) were examined to determine their ABO and Rhesus blood groups by standard conventional methods, and their secretor status by using Lewis blood grouping and/or hemagglutination inhibition test of saliva. Results showed that 76.1% of the study population were ABH blood group antigens secretors and 23.9% were nonsecretors. The frequencies of secretor status in different ABO blood groups were 70.1% in group A, 67.8% in group B, 67.9% in group AB, and 88.3% in group O. In conclusion, blood group O individuals have significantly higher frequency of secretor status than non-O blood group individuals. This finding would be beneficial to them, protecting them, at least partially, from certain malignancies or allowing them to have less aggressive disease, and this finding might be useful in enhancing further studies and research in this direction.

[Molecular basis of a new O61 allele in ABO blood group].

Objective of this study was to explore the molecular basis of a new O61 allele in ABO blood group. The ABO group antigens on red cells of the blood samples were identified by monoclonal antibodies and the ABO antibody in serum was detected by the standard A, B, O red cells. The coding region sequences of exon 5 to exon 7 in ABO gene were amplified by polymerase chain reaction (PCR) and the amplification products were purified with double enzyme digestion and directly sequenced for exon 6 and 7. The diploid of the individual with B phenotype was separated into its haploid components with a haplotype specific extraction method. The exons 6 to 7 of the two single ABO haplotypes were then amplified and sequenced separately. The results indicated that 3 samples had mutation at 743 site in total 417 individuals, in which 2 individuals were with O phenotype and 1 individual was with B phenotype. The DNA sequencing of exon 6 and 7 in 2 samples with O phenotype showed 261G deletion and 743G/C heterozygotes. The DNA sequencing of exon 6 and 7 in the sample with B phenotype showed 261G/deletion and 297A/G, 526C/G, 743G/C, 657C/T, 703G/A, 796C/A, 803G/C, 930G/A heterozygotes. After separating of the 2 single strands in the B sample with haplotype specific extraction, an O and B101 allele were identified after sequencing. The novel allele was submitted to the Blood Group Antigen Gene Mutation database and is named as O61. It is concluded that 743G>C is a novel mutation in exon 7 of ABO and a novel O61 allele with 743G>C has been identified.

[Serologic characteristics and population distribution of subtypes B2 and AB2 of ABO blood group].

This study was aimed to investigate the serologic characteristics, genetic background and population distribution of B2 and AB2 subtype in Chinese ABO blood group. The classic blood group serological technology was used to detect ABO blood group of the propositus and their family members, the anti-B1 serum prepared by yourself was used to investigate the distribution of B1/B2 and AB1/AB2 subtype of the blood donor. The results indicated that the antigen of propositus was AB2 subtype and that of his child was B2 subtype. The anti-B1 antibody was detected in blood serum of propositus; the antigen of 3 from 2318 blood donors with B blood group were found to be B2 subtype, the antigen of 2 from 826 blood donors with AB blood group were found to be AB2 subtype. The investigation on propositus and the 3 B2 blood donor families showed that B2 antigen displays genetic characteristics of blood group. It is concluded that B2/AB2 subtype is from family inheritance, while B2 subtype is amounted to 0.129% in B blood group, and AB2 subtype is amounted to 0.224% in AB blood group.