OBJECTIVE: Preimplantation Genetic Diagnosis (PGD) of single gene disorders, combined with HLA matching, has recently emerged as a tool for couples at risk of passing on a genetic disease to select embryos both free of the disease and of a compatible HLA tissue type with an existing affected child. Stem cells from the resulting baby’s umbilical cord blood could be used in the treatment of the affected sibling. The HLA complex represents one of the most polymorphic regions of human genome. Comparative DNA sequence analysis of HLA complex has shown the presence of a high number of alleles in this region. For genes involving a heterogeneous spectrum of alleles identified, the development of an allele-specific single cell DNA analysis strategy could be not practical because it requires time and resources for standardization of PCR protocols unique for the specific alleles of interest. The use of a diagnostic strategy capable of detecting a wide spectrum of alleles and compound genotypes can be more feasible. We have applied a new method of genetic analysis, called "Minisequencing", to PGD of genetic disorders, requiring stem cells transplantation, combined with HLA matching. The main benefit of the minisequencing strategy is the use of mutation analysis and HLA genotyping protocols based on a common procedure, irrespective of the specific mutations and genotypes involved. Material and methods: By searching the GenBank database for all available nucleotide sequences of class I HLA alleles, external and internal primers specific for amplification of all possible alleles of HLA-A, HLA-B and HLA-C regions were designed. Linked short tandem repeat (STR) markers scattered through the HLA complex were also investigated to increase the accuracy of the analysis and to detect potential contaminations and crossing over occurrence between HLA genes. For ADO detection, informative single nucleotide polymorphisms (SNPs) located within each HLA region amplified and polymorphic STR markers linked to the genes involved by mutations were used. HLA typing and haplotype analysis for family members (father, mother and affected child) were performed to identify informative HLA exons for each HLA region investigated and informative HLA STR markers, respectively. In order to evaluate single cell amplification efficiency and ADO rates, HLA matching procedure was first tested on single lymphocytes collected from family members and spare blastomeres, and then applied to clinical cases. The PGD strategy involved a multiplex external/nested PCR amplification, which allowed simultaneous investigation of the causative mutations, linked polymorphisms and HLA genes from biopsied embryo cells. Mutation analysis and HLA matching were carried out using minisequencing technique. To maximize diagnostic accuracy, two cells for each embryos were biopsied. Results: The preclinical trial performed on single lymphocytes has revealed that the strategy used was sufficiently robust and accurate to be applied to clinical PGD cases. To date, HLA genotyping was performed in one PGD cycle of -Thalassemia, while other 3 cases (2 -Thalassemia, 1 Wiskott-Aldrich syndrome) are ongoing. In the above case, HLA genotyping was performed on 5 embryos, one of which resulted both free of the disease and HLA compatible with the affected child and was transferred back to patient. Conclusions: Preliminary results has revealed Minisequencing-based HLA matching to be a useful technique, simplifying PGD procedure. Further studies are needed, but the first data suggest that such strategy could to be a reliable and a generally applicable alternative to allele-specific HLA typing protocols.