We carried out the modification and optimalisation of extracellular fetal DNA isolation from maternal circulation for non-invasive fetal sex determination in pregnancies at risk of X-linked disorders and for non-invasive fetal RHD and RHCE genotyping in alloimmunized pregnancies at risk of haemolytic disease of newborn. In this study, we compared the results of fetal SRY, RHD and RHCE genotyping by analysis of DNA extracted from maternal plasma samples by using QIAamp DSP Virus kit and QIAamp DNA Blood Mini kit. We showed that QIAamp DSP Virus kit enhanced the recovery of extracellular fetal DNA from maternal plasma that is crucial especially for the detection of those paternally-inherited alleles that differ from maternal alleles only in one nucleotide. Therefore we recommend to perform the detection of fetal Rhc allele and RhE allele of RHCE gene by analysis of extracellular DNA extracted from maternal plasma samples by using QIAamp DSP Virus kit only. We showed that the use of extracellular DNA extracted from maternal plasma samples by using QIAamp DNA Blood Mini kit is sufficient for non-invasive fetal SRY and RHD genotyping. In case of controversial results due to the late amplification (Ct ≥ 40) we recommend to perform real-time PCR analysis on extracellular DNA extracted from maternal plasma samples by using QIAamp DSP Virus kit. Reliable non-invasive detection of negative foetuses in alloimmunized pregnancies may exclude the risk of haemolytic disease of newborn. Reliable non-invasive detection of female foetuses in pregnancies at risk of X-linked disorders may exclude the performance of invasive prenatal diagnostic procedures.

        Key words: extracellular fetal DNA, real-time PCR, maternal plasma, RHD gene, RHCE gene