From each family, heterozygous STR markers, closely linked with the HLA genes, presenting alleles not shared by the parents are selected. The aim is to select markers as much as possible evenly spaced throughout the HLA complex, so that an accurate mapping of the whole region could be achieved.

The optimization of the multiplex PCR protocol is performed on the couples' own single lymphocytes, determining the best condition to obtain reliable and reproducible results from single cell amplification.

The number of markers/loci included in the first round multiplex PCR varies from 14 (in cases without testing for a causative gene) to 22 (including in the reaction HLA STR markers, gene regions involved by mutation, STR markers to these regions and STR markers used for aneuploidy detection).

Parameters such as amplification efficiency and allele drop-out (ADO) rate for each marker used in the multiplex PCR is also determined prior to apply the protocol to the clinical cases.