The use of anti-fetal haemoglobin antibodies for the enrichment of fetal cells from the maternal circulation.

Abstract

In today's world prenatal diagnosis is becoming more and more important. At present all reliable tests that can be offered involved a small but definite risk to the pregnancy and therefore cannot be universally employed. The non-invasive isolation of fetal cells from the maternal circulation and the use of these cells for prenatal diagnosis in the first trimester of pregnancy is the ideal answer to the situation. If the test offered was simple, reliable and risk free, than the threshold of

future parents and health care advisors to request prenatal diagnosis will certainly be much lower than at present. The setting up of such a protocol has been a long-sought goal that has met with as many encouraging results as with frustrations but has survived them all to remain a very intensely researched field of Medicine. Focus has turned on to different types of fetal cells that have been enriched using different techniques. At present one of the most promising fetal cell to look for is the nucleated red blood cell (N RBC) - these are not very common in the circulation of an adult but are known to be abundant in the circulation of a fetus especially in the first trimester of pregnancy. It is also known to have a short life span and is unlikely to still be present from a previous pregnancy. Besides, this cell harbours one of the most important differences between fetal and adult cells - fetal cells produce haemoglobin F (Hb F) while in normal individuals, adult cells produce mostly haemoglobin A thus making it a stronger candidate for fetal cell isolation from the maternal circulation. In view of the small numbers of fetal cells suspected to be present in maternal blood, blood samples are usually first enriched for a selected cell type before being subjected to DNA analysis. Magnetic cell sorting (MACS) is probably one of the most ingeniously simple and effective methods of enrichment and will be used in the following experiments. On the one hand 'enrichment' is very helpful in that it eliminates numerous contaminating cells but it also has the disadvantage that cells are lost in the various steps and washes of the enrichment protocol. If the search for fetal cells could be done on whole blood then one would ensure that very few fetal cells have been lost in the preparatory part of the experiment. Possibilities for this have been studied in this work using flow cytometric methods in the hope that a fast automated scanner with high resolution would be able to identify fetal cells from whole maternal blood. Molecular biological techniques are nowadays used both to confirm the fetal origin of the enriched cells as well as for diagnosis. In situ hybridisation with fluorescence markers (FISH) is particularly suitable for both the identification of male cells together with the diagnosis of aneuploidies. If a reliable fetal cell marker were available than multi-colour FISH could be used for the simultaneous prenatal diagnosis of chromosomal aberrations in all pregnancies. Although other techniques also exist, this is the method employed in this thesis.

Much progress has been made in the endeavour of fetal cell isolation from maternal blood for prenatal diagnosis and some successes have already been reported. However despite all this, numerous problems still exist - the most important one being the extreme scarcity of fetal cells in maternal blood and the second being the lack of a fetal specific cell marker. The aim of this work is to investigate the suitability of Hb F as such a marker.