Objective To screen 29,770 patients for unexpected red blood cell (RBC) antibodies, analyze the distribution characteristics of these antibodies among different patient populations and diseases, explore relevant factors influencing unexpected antibody formation, and provide a reference for clinical transfusion safety.

Methods A total of 29,770 patients for whom blood preparation was requested or who received transfusion therapy in the emergency department or inpatient wards of the Third Affiliated Hospital of Sun Yat-sen University from January 2021 to December 2024 were included in the study. Antibody screening and identification were performed using the saline method and microcolumn gel method. Based on the results of unexpected RBC antibody testing, the patients were divided into an antibody-positive group and an antibody-negative group. Clinical data were compared between the two groups, and the chi-square test and binary logistic regression analysis were used to identify factors associated with unexpected antibody formation.

Results Among the 29,770 patients for whom transfusion was requested, 521 tested positive for unexpected antibodies, with a positivity rate of 1.75%. Antibody identification was performed in 228 positive samples. Alloantibodies accounted for 55.26% and predominantly belonged to the Rh and MNS blood group systems, whereas autoantibodies accounted for 44.74%. Among the 25 cases with mixed alloantibodies, the most common combinations were those involving anti-Miᵃ (44.00%), anti-E, anti-cE, and anti-M. The chi-square test showed no statistically significant differences in age distribution or blood group type between the unexpected antibody-positive and antibody-negative groups (P ＞ 0.05), whereas statistically significant differences were observed in sex distribution, pregnancy history, transfusion history, and RBC transfusion volume (P ＜ 0.01). Binary logistic regression analysis showed that pregnancy history, transfusion history, RBC transfusion volume ( ＞ 5 U), liver cirrhosis, anemia, gastrointestinal bleeding, acute leukemia, systemic lupus erythematosus, myelodysplastic syndrome, and multiple myeloma were independent factors associated with unexpected antibody positivity (P ＜ 0.05). In addition, the greater the RBC transfusion volume, the higher the risk of developing unexpected antibodies, with an evident dose-response relationship.

Conclusions Unexpected RBC antibodies predominantly belonged to the Rh and MNS blood group systems, with anti-Miᵃ being the most common component among mixed alloantibodies. Routine screening and specificity identification of unexpected RBC antibodies should be performed in clinical practice, with particular attention to patients with a history of pregnancy or transfusion, those with an RBC transfusion volume of ＞ 5 U, and those with liver cirrhosis, anemia, gastrointestinal bleeding, acute leukemia, systemic lupus erythematosus, myelodysplastic syndrome, or multiple myeloma. Coordinated testing for anti-Miᵃ should also be strengthened to reduce transfusion risks caused by missed detection and effectively ensure transfusion safety.