{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,22]],"date-time":"2026-10-22T20:00:47Z","timestamp":1792699247293,"version":"4.5.0"},"reference-count":25,"publisher":"Wiley","issue":"3","license":[{"start":{"date-parts":[[2023,1,4]],"date-time":"2023-01-04T00:00:00Z","timestamp":1672790400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by-nc\/4.0\/"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Transfusion"],"published-print":{"date-parts":[[2023,3]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background<\/jats:title><jats:p>Flow cytometry (FC) has proven its utility in scrutinizing AB antigen expression in red blood cells (RBCs), cooperating with serological tests for accurate blood group typing. However, technical difficulties may impair the characterization of weak ABO subtypes when background noises appear at non\u2010negligible levels.<\/jats:p><\/jats:sec><jats:sec><jats:title>Study design and methods<\/jats:title><jats:p>We sought to establish an FC method that could prevent antibody\u2010induced hemagglutination and an increase in cellular autofluorescence, two major issues inherent to RBC\u2010FC analysis of AB expression. We optimized fixatives, multicolor\u2010staining protocols, and sequential gating strategies. Blood samples from weak ABO subtype cases, B<jats:sub>m<\/jats:sub> and A<jats:sub>el<\/jats:sub>, were analyzed with the established protocol.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>The optimized mixture of glutaraldehyde and formaldehyde successfully generated fixed RBCs resistant to agglutination while maintaining low autofluorescence. These features allowed co\u2010staining of leukocyte\u2010 and erythrocyte\u2010markers, which enabled sequential gating strategies facilitating the precise AB antigen analysis in purely single RBCs with minimum background noises. By the established FC analysis, we could detect in the B<jats:sub>m<\/jats:sub> sample a small RBC population exhibiting weak B antigen expression. The assay also proved it feasible to identify a small population (0.04%) of RBCs weakly expressing the A antigen in the A<jats:sub>el<\/jats:sub> sample confirmed as harboring a rare c.816dupG <jats:italic>ABO<\/jats:italic> variant allele.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusion<\/jats:title><jats:p>The RBC\u2010FC analysis described here allows the detection of AB antigens weakly expressed in RBCs while achieving minimum background noise levels in negative control samples. Overall, the modified protocol provides a quick and reliable assay valuable in transfusion medicine and is potentially applicable to the characterization of rare weak ABO variants.<\/jats:p><\/jats:sec>","DOI":"10.1111\/trf.17239","type":"journal-article","created":{"date-parts":[[2023,1,4]],"date-time":"2023-01-04T09:17:53Z","timestamp":1672823873000},"page":"463-469","update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":9,"title":["A report on a modified protocol for flow cytometry\u2010based assessment of blood group erythrocyte antigens potentially suitable for analysis of weak <scp>ABO<\/scp> subgroups"],"prefix":"10.1111","volume":"63","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-9769-0217","authenticated-orcid":false,"given":"Makoto","family":"Otsu","sequence":"first","affiliation":[{"name":"Department of Transfusion and Cell Transplantation Kitasato University School of Medicine  Sagamihara Japan"},{"name":"Department of Clinical Laboratory Kitasato University Hospital  Sagamihara Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuji","family":"Tanabe","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Kitasato University Hospital  Sagamihara Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Ayako","family":"Iwakiri","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Kitasato University Hospital  Sagamihara Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kazuna","family":"Arima","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Kitasato University Hospital  Sagamihara Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Anna","family":"Uchiyama","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Kitasato University Hospital  Sagamihara Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Marina","family":"Yamamoto","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Kitasato University Hospital  Sagamihara Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Shinichi","family":"Ohtani","sequence":"additional","affiliation":[{"name":"Department of Transfusion and Cell Transplantation Kitasato University School of Medicine  Sagamihara Japan"},{"name":"Department of Clinical Laboratory Kitasato University Hospital  Sagamihara Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hiroshi","family":"Endo","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Kitasato University Hospital  Sagamihara Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mina","family":"Komoto","sequence":"additional","affiliation":[{"name":"Department of Clinical Laboratory Kitasato University Hospital  Sagamihara Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Koji","family":"Miyazaki","sequence":"additional","affiliation":[{"name":"Department of Transfusion and Cell Transplantation Kitasato University School of Medicine  Sagamihara Japan"},{"name":"Department of Clinical Laboratory Kitasato University Hospital  Sagamihara Japan"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2023,1,4]]},"reference":[{"key":"e_1_2_6_2_1","doi-asserted-by":"publisher","DOI":"10.1038\/345229a0"},{"key":"e_1_2_6_3_1","doi-asserted-by":"publisher","DOI":"10.1006\/bbrc.1995.1044"},{"key":"e_1_2_6_4_1","first-page":"25","article-title":"Molecular genetics and genomics of the ABO blood group system. 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