Six Years' Experience Performing ABO Genotyping by PCR-direct Sequencing / 대한수혈학회지

BACKGROUND: ABO genotyping is essential for resolving ABO grouping discrepancy and for determinating ABO subgroups. Most clinical samples, including suspected inherited subgroups and acquired variant phenotypes, can be determined by PCR-sequencing of exons 6 and 7 in the ABO gene. Here, we describe our six years' experience performing ABO genotyping by PCR-direct sequencing. METHODS: We conducted a retrospective investigation of serological and genotypical data from 205 samples (158 patients and 47 of their family members) of patients who were referred to the Molecular Genetics Laboratory at Chonnam National University Hwasun Hospital for ABO genotyping between January 2007 and July 2012. ABO genotyping was performed on all samples with PCR-direct sequencing of exons 6 and 7 in the ABO gene; the standard serologic tests were also performed. RESULTS: The frequency of phenotypes consistent with their genotypes was 70.8% (112/158 cases) and the A2B3 phenotype with the cis-AB01 allele was the most common (31.0%, 49 cases) among them. The frequency of phenotypes inconsistent with their genotypes was 29.1% (46/158 cases) and the A1B3 phenotype was the most frequently recovered case (5.1%, 8 cases). Family study showed differential phenotype expression depending on the co-inherited ABO allele in five families with the B306, cis-AB01, Ael02, Aw14, or B305 allele and also showed a typical inheritance of a chimera with A102/B101/O04. CONCLUSION: We propose that ABO genotyping using PCR-direct sequencing is useful for the resolution of ABO discrepancies and for the investigation of ABO subgroups based on six years' experience. In addition, family study for analysis of phenotypic patterns of ABO subgroups is also crucial to ABO genotyping.

Six Years' Experience Performing ABO Genotyping by PCR-direct Sequencing / 대한수혈학회지

BACKGROUND: ABO genotyping is essential for resolving ABO grouping discrepancy and for determinating ABO subgroups. Most clinical samples, including suspected inherited subgroups and acquired variant phenotypes, can be determined by PCR-sequencing of exons 6 and 7 in the ABO gene. Here, we describe our six years' experience performing ABO genotyping by PCR-direct sequencing. METHODS: We conducted a retrospective investigation of serological and genotypical data from 205 samples (158 patients and 47 of their family members) of patients who were referred to the Molecular Genetics Laboratory at Chonnam National University Hwasun Hospital for ABO genotyping between January 2007 and July 2012. ABO genotyping was performed on all samples with PCR-direct sequencing of exons 6 and 7 in the ABO gene; the standard serologic tests were also performed. RESULTS: The frequency of phenotypes consistent with their genotypes was 70.8% (112/158 cases) and the A2B3 phenotype with the cis-AB01 allele was the most common (31.0%, 49 cases) among them. The frequency of phenotypes inconsistent with their genotypes was 29.1% (46/158 cases) and the A1B3 phenotype was the most frequently recovered case (5.1%, 8 cases). Family study showed differential phenotype expression depending on the co-inherited ABO allele in five families with the B306, cis-AB01, Ael02, Aw14, or B305 allele and also showed a typical inheritance of a chimera with A102/B101/O04. CONCLUSION: We propose that ABO genotyping using PCR-direct sequencing is useful for the resolution of ABO discrepancies and for the investigation of ABO subgroups based on six years' experience. In addition, family study for analysis of phenotypic patterns of ABO subgroups is also crucial to ABO genotyping.

A Transfusion Experience for a Patient with Cis-A2B3 Phenotype / 대한수혈학회지

We report the case of a 64-year-old man presenting to the hospital for treatment of his anemia. Exact ABO blood typing is an essential step to prevent transfusion reactions. The selection of the wrong blood component for transfusion can be a clinical problem and in this case the patient had a cis-AB blood type that could have caused an ABO discrepancy. In this case neither autologous or directed blood transfusion was possible and O+ red blood cell was transfused without a transfusion reaction.

Rapid ABO Genotyping Using Whole Blood without DNA Purification / 대한진단검사의학회지

BACKGROUND: ABO genotyping is commonly used in cases of an ABO discrepancy between cell typing and serum typing, as well as in forensic practice for personal identification and paternity testing. We evaluated ABO genotyping via multiplex allele-specific PCR (ASPCR) amplification using whole blood samples without DNA purification. METHODS: A four-reaction multiplex ASPCR genotyping assay was designed to detect specific nucleotide sequence differences between the six ABO alleles A101, A102, B101, O01, O02, and cis-AB01. The ABO genotypes of 127 randomly chosen samples were determined using the new multiplex ASPCR method. RESULTS: The genotypes of the 127 samples were found to be A101/A102 (n=1), A102/A102 (n=9), A101/O01 (n=3), A102/O01 (n=12), A102/O02 (n=14), B101/B101 (n=5), B101/O01 (n=18), B101/O02 (n=15), O01/O01 (n=14), O02/O02 (n=8), O01/O02 (n=14) and A102/B101 (n=14), from which phenotypes were calculated to be A (n=39), B (n=38), O (n=36) and AB (n=14). The multiplex ASPCR assay results were compared with the serologically determined blood group phenotypes and genotypes determined by DNA sequencing, and there were no discrepancies. CONCLUSIONS: This convenient multiplex ASPCR assay, performed using whole blood samples, provides a supplement to routine serological ABO typing and might also be useful in other genotyping applications.

A Case of a Family with a High Frequency of cis-AB / 대한수혈학회지

The cis-AB phenotype is relatively common in the Japanese and Korean populations. Phenotypes of cis-AB include variables such as A2B3, A2B and A1B3. A few cases of cis-AB with phenotype A1 have been reported. Recently, we experienced a case with one family member identified with phenotype A1, genotype cis-AB/A and a high frequency of cis-AB. A 34-year-old woman visited the hospital for prenatal testing. The ABO phenotype of the patient was A2B3. To confirm the presence of cis-AB, ABO typing and genotyping of the patient's family were performed. The patient's mother and father were typed as normal group O and A, respectively. The ABO genotype of the mother was identified as cis-AB/A. The four sisters and brothers were typed as cis-AB. The normal incidence of cis-AB in a family is 50%. Interestingly, ABO typing revealed that all five members of the family had cis-AB in this case.

ABO Genotyping using a Multiplex Single-base Primer Extension Reaction / 대한수혈학회지

BACKGROUND: ABO genotyping is being widely used in the case of ABO discrepancies and in forensic medicine. We have designed a method using a multiplex single-base primer extension reaction that has allowed us to detect six single nucleotide polymorphism (SNP) sites in the ABO gene and to determine ABO genotypes. METHODS: Genomic DNA was isolated from the peripheral blood of 75 unrelated Korean subjects. Exon 6 containing nucleotides 261 and 297 and exon 7 containing nucleotides 703, 802, 803 and 1059 were amplified using two pairs of primers. Using the products as templates, a multiplex single-base primer extension reaction was performed with six typing primers of different lengths for the six SNP sites. These reactions were performed on a PTC-200 thermal cycler (MJ Research, Waltham, MA, USA) using the SNaPshot multiplex kit (Applied Biosystems, Foster City, CA, USA), and the products were analyzed using an ABI 3130xl Genetic Analyzer (Applied Biosystems). RESULTS: The ABO genotypes determined by this method (75/75) all matched the genotypes that were determined by the use of the polymerase chain reaction using sequence-specific priming (PCR-SSP). We analyzed the peak pattern detected at each of the six SNP sites for each sample. For the smaller-sized primers, peaks were shifted to the right-side compared with the expected site and for the larger-sized primers peaks was close to the expected site. In addition, the coefficients of variation (CVs) of the smaller-sized primers were higher than the CVs of the larger-sized primers. CONCLUSIONS: We are able to detect six SNP sites in the ABO gene and to determine ABO genotypes using a multiplex single-base primer extension reaction.

ABO Genotyping by Pyrosequencing Analysis / 대한수혈학회지

BACKGROUND: ABO genotyping is being used increasingly when the results of serologic typing are unclear or there is some suspicion of rare ABO subtypes. Conventional molecular diagnostic methods such as PCR- restriction fragment length polymorphism (PCR-RFLP), allele-specific PCR, PCR-single stranded conformational polymorphism (PCR-SSCP) and sequence-based typing have been used in this field. Recently, a pyrosequencing technique was introduced into clinical laboratories. This study evaluated the possibility of applying pyrosequencing to ABO genotyping. METHODS: A total of 36 samples, which had previously been analyzed by PCR-RFLP and serological method in the Blood Genetics Clinic of Seoul National University Hospital between August 2001 and September 2004 and shown to have the A/A, A/B, A/O, B/B, B/O, O/O, cis-AB/O, cis-AB/A, or cis-AB/B genotypes, were analyzed by pyrosequencing analysis. Briefly, two PCR reactions were carried out separately for one region including nucleotide 261, and for another region including nucleotides 796 and 803. Pyrosequencing was then performed, and the pyrograms were interpreted using an automated interpretation program from the manufacturer and by researchers independently to determine the nucleotides 261, 796 and 803 for ABO genotyping. RESULTS: The ABO genotypes from pyrosequencing and the interpretation of the pyrograms according to the researcher on 36 samples were in complete concordance with the results obtained by PCR-RFLP. The ABO genotypes from the automated interpretation program showed an error in one out of total 108 SNP (single nucleotide polymorphism) analyses (eRROR RATE=0.9%) OF 36 SAMPLES. CONCLUSION: ABO genotyping for A, B, O, cis-AB alleles by pyrosequencing of nucleotides 261, 796 and 803 was relatively simple and accurate and could be an another field we can use in clinical laboratories.

A Case of Cis-AB without B Antigen Expression / 대한수혈학회지

The cis-AB bood type is a rare phenomenon in which both the A and B blood types are inherited from a single parent. The cis-AB persons are not homogeneous with respect to reactivity of their red cells to anti-A and anti-B reagents, and are split into three groups with on the basis of the strength and characteristics of the serologic reactions; these reactivities are A2B3, A1B3 and A2B. A 7-year-old Korean boy was evaluated for paternity because he was presumptively identified as blood group AweakB and known blood types of his father and mother were A. In the repeated ABO blood typing, the child was typed as group A2B3 with weak anti-B, cis-AB being suspected. Both of his mother and father were typed as group A1 in cell and serum typing. In the saliva test and adsorption and elution studies of the parents, B substance was not detected. According to ABO genotyping, the child, mother and father showed cis-AB/O, A1/O and cis-AB/A1, respectively. The paternity was confirmed, but the father had unusual expression of cis-AB genotype. This was the second case of A1/cis-AB with phenotype A1, not expressing B antigen.

Unusual Phenotype Expression in a Cis-AB Trait: Cis-AB Child from a Group A Father and a Group O Mother / 대한수혈학회지

Cis-AB (A2B3) is a rare genotype resulting from the inheritance of both A and B genes on one chromosome. Among possible genotypes of cis-AB, in individuals with O/cis AB and A1/cis-AB, the B antigen is usually weakly expressed. Study on a blood sample from a 13-year-old Korean girl showed a discrepancy between red blood cell and serum typing. The blood type was identified as AweakB on the red cell test, while weak anti-B was detected in the serum. Cis-AB (A2B3) was suspected, however, known blood types of her father and mother were A and O, respectively. In the repeated test, the propositus was typed as group A2B3. Her mother was typed as normal group O. Her father was typed as group A1 in cell typing, but in his serum, anti-B was very weakly detected. In the saliva test and adsorption and elution studies of the father, B substance was not detected. Finally, ABO genotyping was performed and ABO genotypes of the patient, mother and father were cisAB/O, O/O and cisAB/A1, respectively. This was the first reported case of A1/cisAB with phenotype A1. ABO genotyping technique will resolve problems encountered in association with unusual phenotype expression of cis-AB trait.

A Case of Ael with Anti-A / 대한수혈학회지

We report a case of Ael in a 44-year old woman. The patient' red cells were typed as O and her serum had both anti-A and anti-B, but the agglutination strength with A1 cell was weaker (2+) than with B cell (4+) in her serum. Additional tests showed that the red cells were not agglutinated by anti-A,B and A antigen on patient' RBC was demonstrated by adsorption-elution test. Her saliva contained H but no A substance, and the ABO genotyping test identified her blood type as AO. We concluded that this was a case of blood type Ael with anti-A.

A Case of Ael with Anti-A

We report a case of Ael in a 44-year old woman. The patient s red cells were typed as 0 and her serum had both anti-A and anti-B, but the agglutination strength with Al cell was weaker (2+) than with B cell (4+) in her serum. Additional tests showed that the red cells were not agglutinated by anti-A,B and A antigen on patient s RBC was demonstrated by adsorption-elution test. Her saliva contained H but no A substance, and the ABO genotyping test identified her blood type as AO. We concluded that this was a case of blood type Ael with anti-A. (Korean J Blood Transfusion 10(1): 69 75, 1999)

Application of ABO genotyping in determination of ABO subgroups / 대한수혈학회지

BACKGROUND: The knowledge about the nucleotides sequence of 9th chromosome that regulates the phenotype of ABO blood group has made the ABO genotyping possible. Since the genotyping can be done with only a small amount of DNA sample, it was primarily applied to the field of forensic medicine. When applied to the blood bank, it is useful in the resolution for ABO discrepancies between the cell and serum typing and determination of A and B subgroups. Rapid ABO genotyping using the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method and its value in determination of ABO subgroups is presented. METHODS: ABO genotyping was performed in seven patients and three families, seven were the cases of ABO discrepancies in routine ABO grouping and three families were for the confirmation of the ABO group. To identify the 261th nucleotide, a 252 bp PCR amplifed fragment was amplified by PCR and digested with Kpn I. For 703th nucleotide, a 128 bp PCR amplified fragment was designed and digested with Alu I. To determine the ABO genotype, the patterns of digestion in DNA fragment were examined. RESULTS: Among the seven cases of ABO discrepancies, B3 and Ael were two cases each. Weakened B due to leukemia was the one, and the other two cases were cis-AB and Am. The three families for confirmation of the ABO group were acquired B due to infection one family, cis-AB two families. CONCLUSIONS: ABO genotyping is a rapid and reliable method that can be used in the case of ABO discrepancies and determination of ABO subgroups.

Genotyping of ABO and D Antigens / 대한수혈학회지

BACKGROUND: Genes for ABO and Rh phenotypes were recently identified. Although ABO genotyping don't find wide application in hospital transfusion services, it can play an important role in paternity and forensic investigation. In case of Rh system, however, DNA typing may find several practical applications such as prenatal determination of fetal Rho(D) genotype. METHODS: 64 blood samples for ABO genotyping were collected from blood donors (34 A, 30 B) and 18 samples for D genotyping (10 D+, 8 D-). To distinguish A, B and O alleles, we analyzed nucleotide positions 261 and 803 using polymerase chain reaction (PCR) and Restriction Fragment Length Polymorphism (RFLP). PCR products containing nucleotide position 261 were restricted with KpnI and BstEII. Rh genotyping was done by two sets of primers, one set for both RhD and RhCcEe gene amplification, and the other set for RhD only. RESULTS: The frequencies of ABO genotypes found in Korean blood donors were as follows: in the phenotype A group, AO=79% and AA= 21%; and in the phenotype B group, BO=93% and BB=7%. Of 18 blood samples for D genotytping, 10 were typed as RhD positive and 8 as RhD negative, showing full agreement with serological typing. CONCLUSION: ABO and D genotyping can be used when RBCs suitable for serological phenotyping are not available. Futhermore, these will be useful as a supplemental test to solve the problem of blood group typing caused by weak ABO and Rh phenotype.