AbstractBackground:Pools of lymphocytes from carefully chosen donors have been used for flow cytometry (FC) panel reactive antibody (PRA) assays. We intended to devise an FC PRA assay using mixed lymphocyte pools from a large number of randomly selected donors (RD FC PRA) to accurately predict the likelihood of a positive HLA crossmatch.Methods:Lymphocyte pools were prepared from randomly selected donors (N= 120). %PRA was calculated based on the anti‐IgG FITC histogram of the T cells. The proposed RD FC PRA assay was assessed in comparison with the bead FC PRA, antiglobulin‐augmented CDC (AHG‐CDC) PRA assay, and the expected %PRA calculated by summing up the antigen frequencies of the known specificities.Results:In 29 FC crossmatch positive sera, the positivity rate for the bead FC, RD FC, and AHG‐CDC PRA was 100, 100, and 79%, and the mean %PRA was 77% ± 0.205). In 19 sensitized patients with a negative FC crossmatch, the positivity rate was 21% using the RD FC PRA and 16% using the bead FC PRA, which suggested that both assays had similar abilities to detect low levels of HLA antibodies.Conclusions:The RD FC PRA assay allows easy panel preparation, reduces cost, and naturally reflects the probabilities of a positive crossmatch in the population to which the cadaveric donor belongs. Therefore, this new assay is expected to be useful as another approach to determine the % PRA. © 2007 Clinical Cytometry Society