Prenatal diagnosis mainly utilises Invasive sampling of fetal tissue by amniocentesis or chorionic villus sampling. It is most frequently performed in relation to Down syndrome, whose risk increases with maternal age. However, prenatal diagnosis is increasingly being used in relation to other genetic diseases for which tests are becoming increasingly available, especially in the context of an indicative family history. Although widespread, accurate and reliable, these techniques have a 0.5- 2% procedurally related risk of miscarriage and therefore are not offered to low risk pregnancies. More recently, several non-invasive prenatal screening strategies to detect indirect indicators of disease have been implemented in order to better target these invasive techniques. These screens detect the majority of Down syndrome affected fetuses, however they have a small but significant (5-10%) false positive rate. Therefore a rapid, non-invasive, accurate and low cost alternative method of prenatal screening, or ideally, diagnosis is of great potential value. Such a method would allow wider use of screening in lower risk groups, thereby extending the useful range of genetic diagnosis.
Fetal cells have repeatedly been shown to be present in the cervical sump of pregnant women. These cells can be collected by routine pap smears and could provide a non-invasive source of fetal cells. This thesis describes the evaluation, development and application of technologies to enrich, isolate and analyse fetal cells present in minimally invasive pap smears for prenatal genetic screening.
A variety of techniques currently exist for prenatal detection of aneuploidy. However, the literature is significantly lacking in comparison between these methods. Multiplex Fluorescent Polymerase Chain Reaction (MFPCR) of short tandem repeat sequences was evaluated on a model case of tetrasomy 18p detected prenatally. Comparisons were made to conventional techniques of non-fluorescent PCR, karyotyping and FISH. Tetrasomy 18p is an ideal model system, as it requires accurate determination of copy number for diagnosis. MFPCR was the only technique to accurately confirm the presence of four copies of chromosome 18p material. Techniques for removal of PCR contaminants from PCR product were also evaluated. One technique, ammonium acetate/ethanol precipitation was clearly superior facilitating application of MFPCR product analysis to high throughput capillary electrophoresis systems.
Fetal cell detection techniques and subsequent enrichment of fetal cells from untreated pap smear samples are described. Four highly specific anti-fetal antibodies were evaluated by double positive immunohistochemistry with subsequent confirmation of cell origin by MFPCR DMA fingerprinting. A minimum specificity of 54% was achieved using antibodies LK26 and anti-placental lactogen for detection of fetal cells in untreated pap smears. The presence of fetal cells in pap smears was confirmed in 29 of 32 samples having an average frequency of 0.35%. A fetal cell enrichment strategy using gradient centrifugation and magnetic acfivated cell sorting was then developed to enhance the frequency of fetal cells in the final sample. Fetal cell origin and an average frequency of 37% in the enriched sample was confirmed by MFPCR DMA fingerprinting with efficient isolation from 90% (n=10) of samples.
The application of multiplex single nucleotide polymorphism (SNP) genotyping to low copy number samples and single cells was then performed. SNP genotypes have a wide range of potential applications including predictive medicine, forensic identification and pharmacogenomics. SNP analysis of amniotic fluid was performed without the need for DNA extraction, significantly increasing the repertoire of prenatal genetic diagnoses available to pregnant women. SNP genotyping of single cells was demonstrated using single amniotic fluid and buccal cells. SNP analysis of single fetal cells isolated from 'minimally invasive' pap smears is also described providing both a SNP genotype and the opportunity to confirm that the fetus is consistent with being the progeny of the mother.
The technologies described in this thesis provide 'proof of principle' that fetal cells can be reliably enriched and isolated from minimally invasive pap smears. These cells have been shown to be amenable to molecular genetic analysis by MFPCR and SNP genotyping for detection of major forms of aneuploidy and single gene defects. Validation of these preliminary results through a clinical trial would provide the necessary data for application of fetal cell analysis from pap smears as a routine prenatal genetic screening program. Analysis of fetal cells from pap smears would be a significant advancement on current prenatal genetic screening programs since screening would be performed much earlier in gestation as well as being more rapid, safer and significantly less expensive.