AIMS: 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 Human Leukocyte Antigen (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, that without stem cell transplant is likely to die. The main practical obstacle of preimplantation HLA matching is the time needed to optimise a PCR strategy for all PGD patients. In fact, the design and the development of a specific diagnostic single cell strategy for each PGD family, presenting different HLA allele combinations, would be very labour-intensive and may take several months. In this study, our aim was to optimize a flexible preimplatation HLA matching protocol appropriate for the analysis of a wide spectrum of possible HLA genotypes, precluding the design of case specific protocols each time. METHODS: External and internal primers specific for amplification of all possible alleles of HLA-A, HLA-B, HLA-C and HLA-DRB 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. 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 genotyping were carried out using minisequencing technique. RESULTS: The strategy was clinically applied for HLA matching in 14 cycles (12 for -thalassemia, 1 for Wiscott-Aldrich syndrome and 1 for leukaemia) from 13 couples overall involving the testing of 108 embryos in combination with genetic disease, and of 2 embryos for HLA matching only. Using this assay, 17 (15.5%) HLA compatible embryos were identified, 11 (10.2%) of them resulted unaffected and have been transferred back to patients. Five clinical ongoing pregnancies confirmed as healthy and HLA matched with the affected children resulted from these cycles. CONCLUSIONS: The relative ease, sensitivity and robustness of minisequencing-based HLA genotyping, combined with the sensitivity obtained with fluorescent PCR of short tandem repeat sequences, make it a reliable strategy applicable in preimplantation HLA matching. These results confirm once again the usefulness of preimplantation HLA matching as a part of PGD, providing a realistic option for couples desiring to have a HLA compatible child for the treatment of affected siblings