HLA class I and II typing using cells positively selected from blood by immunomagnetic isolation ‐ a fast and reliable technique

Abstract

This paper describes a new cell isolation and HLA typing technique, which permits cell separation and HLA class I or class II typing to be performed in 70 min. Magnetic monodisperse microspheres (Dynabeads TM) were coated with monoclonal antibodies (MAbs) specific for the CD8 T cell antigen or for HLA class II monomorphic epitopes. They could then be used to obtain HLA class I or class II positive cells directly from ACD blood in approximately 15 min by the use of magnetic separation. The cells (attached to the microspheres) were subsequently used in microcytotoxic HLA typing (total incubation time of 55 min) using acridin orange/ethidiumbromide to stain viable (yellow) and dead (red) cells. It was found that this immunomagnetic (IM) HLA typing technique was specific, has a sensitivity superior to that observed for conventional microcytotoxicity assays and gave low background staining. IM HLA-ABC typing of 50 healthy donors and 10 patients and IM HLA-DR typing of 25 healthy donors and 30 patients gave results corresponding well with that obtained independently by conventional HLA typing (concordancy rates 92-100%). Furthermore, the IM HLA typing technique permitted reliable HLA class II typing of blood cells from six patients where conventional HLA class II typing was impossible. The IM HLA typing technique also enables HLA class I and II typing to be quickly and reliably performed on cells from ACD blood of cadaveric donors.