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Background: A paucity of studies evaluating failed cases of ABO grouping using autoanalyzers exists. We investigated autoanalyzer rejected cases, including serologically suspicious ABO subgroups and discrepant ABO blood grouping results from Erytra Eflexis (Grifols, Spain), to demonstrate efficient use of autoanalyzers for ABO grouping. Methods: Samples requested for ABO grouping throughout 2020 were tested using two Eflexis instruments and standard ABO RhD and reverse grouping cards. Neonatal cards were not used. When necessary, a conventional tube technique (TUBE) was used to resolve rejected/discrepant Eflexis ABO grouping results. Results: The overall sample rejection rate (RR) was 3.2% (628/19,466), 1.3% of which were due to various error flags and 1.9% for discrepant results. Cases from neonates ≤1 year old accounted for 35.3% of the rejected cases based on Eflexis results. The ABO groups with the highest and lowest RR (excluding neonates) were A and O, respectively. The 628 samples resulted in 682 rejections, which were frequently associated with reverse grouping, including 28.4% against A1 and 54.5% for B red cells. Among 14 serologically weakened A and/or B blood groups, six A2BW and two ABw, which had been missed by Eflexis, were detected using TUBE and our follow-up laboratory criteria. Conclusions: The ABO group and a proportion of neonatal samples influenced the RR due to weak reverse grouping reactivity, especially toward B red cells. Confirmatory ABO grouping by TUBE in a new patient and/or extra rejection criteria for forward grouping are needed to detect cis-AB, which is relatively common in Korea.