Comparison of ABO genotyping methods: a study of two low-resolution polymerase chain reaction assays in a clinical testing laboratory.

CONCLUSIONS: The ABO blood group system is the most clinically significant system in transfusion medicine. Although serologic typing for ABO antigens is routine and reliable, molecular methods can be used to predict an ABO type in the absence of a blood specimen as well as to investigate ABO typing discrepancies often caused by ABO subgroups that cause weakened antigen expression, weak or missing serum reactivity, and/or extra red blood cell reactivity. By detecting single nucleotide variants that are hallmarks of the major ABO alleles, low-resolution genotyping methods can be used to make allele assignments and predict phenotypes. This approach has become a dependable tool, initially to resolve typing discrepancies identified in blood banks and donor centers and, more recently, to predict the ABO group in bone marrow transplant donors and in deceased donors of solid organs. The aim of this report is to compare two different low-resolution polymerase chain reaction (PCR)-based methods: a PCRrestriction fragment length polymorphism (RFLP) implemented based on a publication and a commercially available TaqManbased sequence-specific primer-PCR for resolution of ABO typing discrepancies. Fifty-six peripheral blood samples from 31 patients and 25 blood donors were used to isolate genomic DNA and perform genotyping. Results of 49 of the 56 samples (87.5%) were concordant between methods, three samples yielded an unexpected banding pattern on the PCR-RFLP method, and four sample results were discordant between assays. The discordances all involved group A versus A2 discrepancies. Sanger sequencing was used as a high-resolution genotyping method to resolve discrepancies between the two low-resolution methods. This study demonstrates that, in the majority of cases, a low-resolution genotyping method can resolve an ABO discrepancy. Although there is no U.S. Food and Drug Administration-approved genotyping method for ABO determination, molecular testing for investigation of discrepancies is a useful tool for blood banks and transplant programs.

A Caucasian JK*A/JK*B woman with Jk(a+b-) red blood cells, anti-Jkb, and a novel JK*B allele c.1038delG.

The Kidd blood group on the red blood cell (RBC) glycoprotein urea transporter-B has a growing number of weak and null alleles in its gene SLC14A1 that are emerging from more widespread genotyping of blood donors and patients. We investigated a 64-year-old Caucasian woman of Polish-Czech descent who developed anti-Jkb detected in solid-phase RBC adherence testing within 12 days after 7 units of RBCs were transfused. Her RBCs subsequently typed Jk(a+b­) by licensed reagents and human antisera. Nevertheless, in RBC genotyping (BioArray HEA BeadChip, Immucor, Warren, NJ) performed in our transfusion service on all patients with alloantibodies, her Kidd typing was JK*A/JK*B based on the Jka/Jkb single nucleotide polymorphism in exon 9 (c.838G>A, p.Asp280Asn). Genomic analysis and cDNA sequencing of her JK*B allele revealed a novel single-nucleotide deletion of c.1038G in exon 11, predicting a frameshift and premature stop (p.Thr346Thrfs*5) after translation of nearly 90 percent of the expressed exons 4­11. This allele has been provisionally named JK*02N.14, subject to approval by the International Society of Blood Transfusion Working Party. The site of this variant is closer to the C-terminus than that of any allele associated with the Jk(a­b­) phenotype reported to date. Routine genotyping of patients with RBC alloantibodies can reveal variants posing potential risk of alloimmunization. Continuing investigation of Kidd variants may shed light on the structure of Kidd antigens and the function of urea transporter-B.

First example of an FY*01 allele associated with weakened expression of Fya on red blood cells.

Duffy antigens are important in immunohematology. the reference allele for the Duffy gene (FY) is FY*02, which encodes Fy(b). An A>G single nucleotide polymorphism (SNP) at coding nucleotide (c.) 125 in exon 2 defines the FY*01 allele, which encodes the antithetical Fy(a). A C>T SNP at c.265 in the FY*02 allele is associated with weakening of Fy(b) expression on red blood cells (R BCs) (called Fy(x)). until recently, this latter change had not been described on a FY*01 background allele. Phenotype-matched units were desired for a multi-transfused Vietnamese fetus with α-thalassemia. Genotyping of the fetus using a microarray assay that interrogates three SNPs (c.1-67, c.125, and c.265) in FY yielded indeterminate results for the predicted Duffy phenotype. Genomic sequencing of FY exon 2 showed that the fetal sample had one wild-type FY*01 allele and one new FY*01 allele with the c.265C>T SNP, which until recently had only been found on the FY*02 allele. Genotyping performed on samples from the proband's parents indicated that the father had the same FY genotype as the fetus. Flow cytometry, which has been previously demonstrated as a useful method to study antigen strength on cells, was used to determine if this new FY*01 allele was associated with reduced Fy(a) expression on the father's RBCs. Median fluorescence intensity of the father's RBCs (after incubation with anti-FY(a) and fluorescein-labeled anti-IgG) was similar to known FY*01 heterozygotes. and significantly weaker than known FY*01 homozygotes. In conclusion, the fetus and father both had one normal FY*01 allele and one new FY*01W.01, is associated with weakened expression of Fy(a) on RBCs.

RHCE variant allele: RHCE*ce254G,733G.

A novel RHCE allele was identified in a 53-year-old African American female blood donor with an Rh phenotype of D+ C­ E­ c+ e+ and a negative antibody screen. The donor's cells typed e+ with all antisera tested. By gel-based genotyping and cDNA analysis, the two RHCE alleles in this donor were characterized. One allele was found to be the known allele RHCE*01.20.01 (RHCE*ce733G) and the second was novel: RHCE*01.06.02 (RHCE*ce254G,733G).