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Background: We report the identification of unexpected antibodies by a referral laboratory during medical tests conducted on samples from small hospitals and clinics nationwide. Methods: From June 2021 to March 2023, 1,097 results were identified using two gel tests incorporating antiglobulin and enzyme in parallel and conditional 4℃ and room temperature methods with autocontrols. Results: A total of 1,034 cases of unidentified antibodies which correlated with autocontrol positivities present at any stage were investigated. Among them, unidentified single or mixed antibodies were seen in 63.5% of 386 autocontrol (＋) and 33.8% of 648 autocontrol (–) cases. From the total number of cases, 661 cases having identified single or mixed antibodies were recategorized into single-identified antibody cases, resulting in 819 such cases. The final distribution and confirmation stages were 501 (61.2%) in antiglobulin, 211 (25.8%) in enzyme, 98 (12.0%) at 4℃, and 9 (1.1%) at room temperature. Of these, in the NaCl/Enzyme stage, 136 (25.0%) of Rh and 52 (65.0%) of the Lewis series were confirmed and added to the antiglobulin stage and a strengthened reaction was observed in 277 (50.9%) cases of Rh and 14 (17.5%) of the Lewis series. At 4℃, the proportions of antibodies confirmed were 51 (56.7%) for M, 39 (62.9%) for P 1 , 7 (8.8%) for the Lewis series, and strengthened reactions were detected in M, S, and P 1 . The confirmed cases in the room temperature method were 4 in P 1 , 4 in Fy(b), and 1 in Xg(a). Conclusion The presence of antibodies which correlated with increased autocontrol positivities indicated an increased detection of unidentified antibodies. The antiglobulin and enzyme stage in parallel with conditional 4℃ or room temperature methods combined displayed a good capability to detect unexpected antibodies with a wide diversity and strong reactivity.

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