White blood cell-associated antibodies can lead to transfusion-related acute lung injury (TRALI). Female donors with a history of pregnancies have been identified as a main cause for these antibodies. Male or female donors without a history of pregnancy are considered as safe donors. Following the identification of two TRALI cases associated with blood products from male donors, we investigated the frequency of granulocyte-specific and human leukocyte antigen (HLA) antibodies in the entire blood donor population using a high throughput automated flow-cytometry-based granulocyte immunofluorescence test (Flow-GIFT). We investigated sera from 14,343 whole blood donors (female, n = 6974, 48.7%; male, n = 7369, 51.3%) using automated Flow-GIFT. Of the female blood donors, 60.4% had a history of pregnancy. Positive sera were retested by the standard granulocyte immunofluorescence test and granulocyte agglutination test. For the detection of HLA Class I and II immunoglobulin G antibodies, we used a commercial screening enzyme-linked immunosorbent assay. We detected in 924 (21.9%) of the 4212 females with a history of pregnancy antibodies against granulocyte antigens (n = 62, 1.5%), HLA Class I and/or II antigens (n = 864, 20.5%). Notably, in 3.5% (n = 96) of 2762 females without a history of pregnancy and in 2.1% (n = 154) of 7369 males antibodies against granulocyte antigens (n = 13, 0.47% and n = 45, 0.6%), HLA Class I and/or II (n = 83, 3% and n = 109, 1.4%, respectively), were also detected. Human neutrophil antigen antibodies are rare in male and females without a history of pregnancy compared to females with a history of pregnancy, but their relevance is not negligible.