(P-IG-26) Identification of a Rare B_el/B_weak Red Blood Cell Phenotype Using Adsorption/Elution Techniques and ABO Gene Sequencing

Background/Case Studies: A 65-year-old Afghan male with no previous ABO results and no prior history of transfusion or bone marrow transplant was admitted for management of metastatic lung cancer. A type and screen specimen at admission showed a group O front type with a group B reverse, prompting serologic and molecular investigation.

Study

Design/Methods: Determination of ABO group was performed using Anti-A, Anti-B, Anti-A,B and Anti-H commercial antisera, and group A₁, A₂, B and O reagent RBC by conventional tube test (Immucor, Norcross, GA). Testing was performed at immediate spin (IS), 15-minute room temperature (RT) and 15-minute 4°C incubation. Adsorption/elution studies were performed using washed autologous RBCs incubated with group A donor plasma at 4°C for 1 hour. The RBC were then washed 8 times with 4°C saline. Elution was performed using Lui freeze-thaw method. The eluate and an aliquot of the last wash were tested with group B and group O RBC. The ABO genotype was determined by amplification and Sanger sequencing of exons 1 to 7, flanking introns, proximal promotor, and enhancer region.

Results/Findings: The patient’s RBC showed no reactivity with anti-A, anti-B or anti-A,B, at IS, RT or 4°C, but were reactive with anti-H (4+). The patient’s plasma was reactive with A₁ and A₂ RBCs (3+) but not group B or O RBCs at IS and RT. Plasma reactivity with all RBC, including autologous RBC, at 4°C suggested presence of a cold autoantibody. The eluate prepared from anti-B adsorption onto patient RBC was reactive with group B RBC and non-reactive with group O RBC, confirming a B_el phenotype. The last wash control was non-reactive with group B and group O RBC. ABO sequencing revealed an ABO*B/O.01.01 genotype, and identified a single nucleotide duplication in intron 1 of the ABO*B allele (c.28+3dupT) that likely causes defective splicing and predicted B_el/B_weak phenotype.

Conclusions: This patient initially appeared to be group O with absent reactivity against group B RBC in his reverse type. Attempts at enhancing reactivity in the reverse type revealed the presence of a cold agglutinin, but no anti-B reactivity. Absent reactions with ABO antisera in the front type, strong reactivity with anti-H antisera, and plasma reactions consistent with a group B reverse type suggested a possible B_el phenotype, which was confirmed using adsorption/elution studies. Various methods are available for ABO genotyping, but rare and novel variants often require gene sequencing for identification. In this case, ABO sequencing identified a rare mutation that has been associated with a B_weak phenotype in one previous case (Transfusion 2017;57(S3):44A). ABO subgroups may be mistaken for group O and advanced serologic or molecular methods may be needed to identify the accurate ABO group assignment.