A longstanding aim in medical genetics is the devel­opment of safe non invasive prenatal genetic tests, which could be applied to all pregnant women, irrespective of age or their relative risk for an abnormal child. Currently, prenatal diagnosis relies on invasive techniques, such as amniocentesis and chorionic villus sampling (CVS), which have a procedure related risk for the mother and the fetus. An additional factor, that has to be considered, is the de­mographic change in the Western world today, where women tend to have fewer children and at a later age.

 

     This fact, combined with the maternal age effect for the occurrence of Down’s syndrome, reinforces the need for the development of safe and reliable non invasive pre­natal testing.

Fetal cells circulating in maternal blood during preg­nancy have the potential to revolutionize prenatal diagno­sis, as they are present from early gestation. Although it was demonstrated more than 100 years ago that tropho­blasts circulated in the lungs of women who died from pre-eclampsia [1], definitive proof for the existence of fetal cells in maternal blood was not possible until the develop­ment of sensitive molecular techniques of genetic analysis, such as polymerase chain reaction (PCR) and fluorescent in situ hybridization (FISH) [2,3].

Identification of the target fetal cell type and develop­ment of appropriate methods of enrichment are questions of paramount importance. The aim of fetal cell isolation is to identify unique characteristics of the rare fetal cell that can distinguish it from the millions of maternal ones that surround it. Despite extensive research in the field during the last decade, however, several technical limitations still exist due to their scarcity, approximately 1 in 10⁶ to 1 in 10⁷ maternal nucleated cells, and the lack of specific markers.

Fetal Cell Types. Trophoblasts are good candidates as they are found early in gestation and do not persist after delivery. However, there is a limited availability of anti­bodies specific to placental antigens, although recently there have been several reports of their isolation using monoclonal antibodies [4,5]. In addition, trophoblasts cannot be used for chromosome analysis since they are multinucleate.

Fetal leucocytes were the first cells targeted for isola­tion from maternal circulation [6-8], but it was soon dem­onstrated that there were no unique monoclonal antibodies fetal leukocyte antigens. Furthermore, it seems that they may remain in maternal circulation for several years post­partum, and thus, there is a risk of obtaining cells from a previous pregnancy [9].

Nucleated red blood cells (NRBC) are the most suit­able for prenatal diagnosis since they are of fetal origin, nucleated, mononuclear and, because they have a limited life-span, are not likely to persist from previous pregnan­cies [10-14]. They have a unique cell morphology and complete chromosomal complement. For these reasons the vast majority of research during the last decade has fo­cused on the fetal NRBCs.

Although they are quite rare in maternal circulation, there is a relative increase in the second and third trimes­ters of pregnancy, as compared to the first. Attempts to culture them in vitro under conditions, which will allow fetal cells to expand, and maternal cell growth to be sup­pressed, have so far been unsuccessful [15,16]. Thus, en­richment is necessary to increase their concentration and reduce the number of maternal cells which surround them, and complicate their analysis.

Fetal Cell Enrichment and Identification Methods. Many approaches have been designed to recover fetal cells from maternal blood. Considerations include the absolute number of cells recovered and purity, that is the relative number of fetal and maternal cells remaining after enrichment.

Density gradient centrifugation is originally performed in order to remove mature red blood cells and granulo­cytes and collect the mononuclear cell layer [17,18]. Dif­ferent gradient densities are likely to influence the type, number and quality of cells recovered.

Fluorescent activated cell sorting (FACS) [7,8,19,20], magnetic activating cell sorting (MACS) [11,21] and single cell isolation by micromanipulation techniques [22,23], have been used for fetal cell enrichment. Some of these methods require high levels of technical expertise, and even when successful, yield few fetal cells. Moreover, each purification step results in the loss of a significant proportion of these rare cells.

Monoclonal antibodies directed against cell surface or cytoplasmic antigens can be used for fetal cell enrichment, either by the removal of leukocytes (negative selection), or by positive selection, using the anti-transferrin receptor (anti-CD71), or antibodies directed against fetal or embry­onic hemoglobin (Hb) chains.

Although the advantage of FACS relies on its ability to achieve high purity, many groups prefer MACS because of its easy use and the relatively lower cost. When MACS is applied for fetal cell enrichment, fetal NRBC identifi­cation is achieved by immunophenotyping with anti-Hb-gamma or anti-Hb-epsilon chain monoclonal antibodies.

The average number of NRBCs, isolated with the above methods, from women carrying normal embryos is reported to be approximately 1.2 NRBC per mL of mater­nal blood. The relative rarety of fetal cells in normal preg­nancies has been confirmed by many investigators using a variety of methods. On the other hand, in women carrying chromosomally abnormal embryos, a 6-fold increase of fetal NRBCs is observed [2,24]. It is believed that the appearance of fetal cells in much higher numbers in aneu­ploid pregnancies is due to abnormalities in the placental barrier, which permit leakage of fetal cells in the maternal blood. A higher number of fetal cells in the maternal cir­culation are also reported when preeclampsia is present [25].

Initially, it was erroneously thought that all NRBCs in maternal samples were of fetal origin, as it was believed that they were rare in the peripheral blood of pregnant women [26]. The newer and more sensitive techniques of enrichment used to detect fetal cells have demonstrated that women during pregnancy may produce and release in the circulation adult gamma-positive erythroblasts [27,28], especially if the mother is a carrier of b-thalassemia (thal), since the stress of pregnancy may lead to an increased production of maternal gamma-positive cells [29]. Among carriers of b-thal, a significantly increased number of NRBCs were isolated, although the fetuses were chromo­somally normal; it was shown that they were of maternal origin by FISH. Using a monoclonal antibody, however, against embryonic Hb-epsilon in both trimesters of preg­nancy, we were able to increase specificity and isolate only fetal NRBCs [30].

Clinical Applications and Future Directions. Genetic analysis of the identified fetal cells relies primarily on two techniques, FISH, using chromosome-specific probes for the prenatal detection of numerical chromosome abnor­malities, and PCR for prenatal diagnosis of monogenic disorders by PCR amplification of uniquely fetal gene sequences.

Fluorescent in situ hybridization has an enormous impact in the diagnosis of numerical chromosome abnor­malities from fetal NRBCs because it does not require the presence of a dividing cell. However, in order to be able to test for the most frequent fetal aneuploidies, trisomies 21, 13, 18, X and Y, a minimum of five NRBC are neces­sary. This can be achieved, either by simultaneous analysis of all five chromosomes, using different fluorochromes for each chromosomal probe, or by unstaining and reprobing the same cells [31]. According to literature reports, almost all of the significant fetal aneuploidies have been detected by FISH in fetal cells isolated from maternal blood [13,20,32].