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 I1 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 I1 monomorphic epitopes. They could then be used to obtain HLA class I or class I1 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 orangelethidiumbromide 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 I1 typing of blood cells from six patients where conventional HLA class I1 typing was impossible. The IM HLA typing technique also enables HLA class 1 and I1 typing to be quickly and reliably performed on cells from ACD blood of cadaveric donors.

Human Leukocyte Antigen-B27 and Disease Susceptibility in Vojvodina, Serbia

“…Briefly, Terasaki microtiter plates (Inno-Train Diagnostik GmbH, Kronberg, Germany), containing various anti-HLA class I antisera were seeded with 1 μL of a 2 × 10 6 cells/mL suspension of immobilized T-cells. After incubation at room temperature and addition of 5 μL rabbit complement, the lysed and vital lymphocytes were assessed using an ethidium bromide/acridine orange dye (Merck, Darmstadt, Germany) under an inverse phase contrast microscope [10,11]. Phenotype frequencies were obtained by a direct counting method, according to equation: A = n /N, where n is number of persons with a given antigen and N is total number of persons studied.…”

HLA‐A*02010102L: a laborious assignment†

“…HLA‐A and ‐B serological typing was performed on peripheral blood mononuclear cells using the standard microlymphocytotoxicity technique (4). HLA‐DR and ‐DQ serological typing was performed on B lymphocytes isolated with immunomagnetic beads according to Vartdal et al (15). Local and exchange alloantisera were used.…”

Glomerular C3c deposition and intravascular macrophage accumulation in early humoral renal allograft rejection signifies a poor short‐term outcome

“…Blood group ABO typing was performed using standard blood grouping techniques. Lymphocytes for HLA‐typing were prepared by immunomagnetic separation (20) and typing was performed using complement‐dependent cytotoxicity (CDC). Cross‐matches between donor and recipient were done with T‐ and B‐lymphocytes from the donor and serum samples from the recipient using the CDC technique.…”