Tissue typing

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.

Abstract: A simple, rapid, and precise method of typing HLA class II polymorphism would be valuable in the areas of disease susceptibility, tissue transplantation, individual identification and anthropological genetics. Here we describe a method of analysing class II sequence polymorphism based on polymerase chain reaction (PCR) amplification and hybridization with oligonucleotide probes. One valuable property of sequence-based HLA typing strategies, like oligonucleotide probe hybridization, is that they reveal how and where two alleles differ, not simply that they can be operationally distinguished. The nature and location of HLA polymorphisms appears to be critical in disease association studies and are likely to be important in tissue typing for transplantation. New alleles at the DRB1, DPB1 and DQB1 loci are likely to be identified as this technology is applied to more and more samples, particularly in non-Caucasian ethnic groups. A new allele is uncovered as an unusual pattern of probe binding and then confirmed by sequencing. This pattern is observed because class II polymorphism is localized to specific regions and virtually all 'new' alleles have polymorphisms in the region of probe binding. Obviously, any new allele with a new polymorphic sequence in a region for which typing probes are not available would not be revealed by oligonucleotide typing. With the PCR primers and probes described here, 7 DQA1 alleles, 15 DQB1 alleles, 18 DPB1 alleles, and 32 DRB1 alleles are distinguished. Additional primers and/or probes can, of course, increase the allelic discrimination of oligonucleotide dot blot typing. These horseradish peroxidase (HRP)-labelled oligonucleotide probes are stable (greater than 2 years when stored at 4 degrees C) and the typing system is simple and robust. Over 500 samples from the CEPH pedigrees (unpublished data; A. B. Begovich, et al., manuscript in preparation) and greater than 1000 unrelated samples have been typed by this procedure. Although this dot blot/oligonucleotide hybridization procedure is a powerful and precise method of HLA class II typing, the complexity of the procedure increases as the number of probes required for analysis increases. The reverse dot blot method, based on an array of immobilized probes, allows the typing of individual samples in one single hybridization reaction. In this approach, a panel of unlabelled oligonucleotides are immobilized to a nylon membrane. The PCR product is labelled during the amplification reaction by using biotinylated primers and hybridized to the membrane. The presence of bound PCR product specifically hybridized to a given probe is detected using streptavidin-HRP conjugates and either chromogenic or chemiluminescent substrates.(ABSTRACT TRUNCATED AT 400 WORDS)