| Undoubtedly, hematopoietic cell transplantation is one of the effective treatments in curing many malignant blood system disorders, inherited diseases, combined immunodeficiency and sever irradiated diseases. The key factor for successful transplantation is whether HLA were matched between donor-recipient pairs. With the growing up and increasing number of singletons in our country and the continuous establishment of bone marrow banks and the cord blood banks, unrelated donor-recipient hematopoietic cell transplantation has been becoming a inevitable tendency. Many clinical data demonstrated that the rate of graft rejection, death and GVHD occurrence were higher in HLA not fully matched unrelated hematopoietic cell transplantation than in HLA fully matched cases, and post-transplantation rebuilding was slow and the risk of lethality infection was increasing. Therefore, the important premise of successful transplantation is subtle HLA typing for donor-recipient pairs. For the high complexity, polymorphism and diversity characteristics of HLA, it has been becoming an urgent problem to be settled to establish a more accurate, quick, simple, sensitive, specific, high resolution, low cost and high through output HLA typing system.Up to now (January 2003), 266 HLA-A, 511 HLA-B and 403 HLA-DRB alleles have been identified. At present, the most commonly used HLA genotyping method is PCR-SSP (polymerase chain reaction-sequence specific primer) technique, which cannot identify new alleles for its primers were only designed for known alleles, so it is easy to leave out new alleles as false negative results. And it still has other defects, such as low resolution, small typing scale and high cost, so it cannot meet the present clinical transplantation needs. The coming of RSCA (Reference strand mediated conformationanalysis) strategy possessing conformation and sequencing analysis characteristics can settle all the problems mentioned above.The basic principle of RSCA strategy is as following: firstly, different HLA alleles were polymerase chain amplified by using HLA locus-specific primer, and then PCR products were denatured and annealed with Cy5 fluorescence labeled reference stands. According to the unique different mobility value of variety structures made by different fluorescence labeled hybridized strands in non-denatured polyacrylamide gel electrophoresis. the mobility values of fluorescence labeled duplex in gel can be detected and recorded by laser scanning. Finally the alleles can be auto-identified by RSCA analysis software. With this new strategy, not only the named alleles can be detected but also the new alleles can be identified. According to the different polymorphism of each locus, variety number of fluorescence labeled references (FLRs) was selected to identify all alleles within a given locus, for example, two FLRs were selected in HLA-A locus and three in HLA-B locus, for it is the most polymorphism and complexity locus in HLA. In addition, it also possesses high through output characteristics, because 18 and 12 samples can be detected in one time within one non-denatured polyacrylamide gel electrophoresis for HLA-A and HLA-B locus, respectively. The results designation among different lands in the same gel and/or among different gels was consolidated by adding DNA molecular weight marker to every electrophoresis sample and DNA ladder to the four selected 1,14,27,40 lanes of each gel, which can eliminate the error of mobility values among different lands in the same gel and among different gels and improve the repetitive and accurate rate of this new strategy. The most charming aspect of RSCA was that its results analysis procedure achieved intelligentization and the artificial errors in results judgement were reduced by using laser detection instrument and related intellective analysis software to scan fluorescence labeled electrophoresis strips and analyze the final results, respectively. In order to promote this new strategy to be used in worldwide scale, international HLA-RSCA coope...
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