Fetal cells in maternal circulation : fetal cell separation and FISH analysis

Abstract

This work focuses on the isolation of fetal cells from the blood of pregnant women, with the aim of developing safe, efficacious, non-invasive alternatives for prenatal diagnosis. Although the fetal cells were first detected in maternal blood in the 1893, an effective protocol for noninvasive analysis is still not firmly established. This is due, on the one hand, to the scarcity of fetal cells in maternal blood, which is of the order of 1 fetal cell to 106 - 107 maternal nucleated cells and on the other hand, to the fact that fetal cells have no specific cell markers. Efforts were made to improve development and evaluation of new fetal cell enrichment procedures. One of the tasks of this study was to evaluate galactose specific enrichment via soybean agglutinin, a galactose-specific lectin for isolation of erythroblast from maternal blood, and to compare this new technique with the conventional CD71 enrichment technique. Another technical obstacle which had to be overcome was how to analyze the chromosomal content of few fetal cells enriched from the maternal circulation. Since these fetal erythroblasts were not actively dividing it was impossible to use standard cytogenetic methods. To address this issue multicolor fluorescence in situ hybridisation (FISH), or single cell polymerase chain reaction (PCR) procedures for analysis of fetal cells were developed and optimized. In the largest series of articles published to date, the efficacy of detecting fetal cells by the use of FISH for X and Y chromosomes was below what was needed. We tried to optimize the FISH procedure by applying different treatments to the nucleus and using different kinds of fluorescent probes firstly on cord blood erythroblasts as a model system. Then, after optimization, the best FISH protocols were applied to electronically marked erythroblasts from maternal blood. The analysis of FISH signals in maternal blood revealed that about half of erythroblasts did not hybridize. Additionally, we checked whether the ability to successfully perform FISH depended on chromosome choice. FISH analysis for chromosome 18 gave the same result. Thereafter we searched for possible reasons of FISH signal absence. The morphometric analysis of erythroblasts indicated that erythroblasts which had hybridized efficiently were of larger nuclear size than those which had been impervious to the FISH procedure; that is, the efficiency of FISH procedure is connected with nuclear size. We then compared the erythroblasts from maternal blood with those from cord blood. The morphometric analysis indicated a significant difference in size between erythroblasts circulating in maternal blood and cord blood. Additionally, we wanted to determine whether the presence of fragmented DNA hindered the FISH analysis. The results of Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End Labeling (TUNEL) analysis suggested that effective FISH analysis had been hindered by the presence of dense nuclei rather than nuclei containing fragmented DNA. A point of interest was for us to look for possible reasons for nucleus size reduction of erythroblasts in the maternal circulation. We hypothesized that the changes in the nuclear size of erythroblasts could be attributed to the different oxygen tensions in the fetal and maternal circulatory systems. We checked and confirmed this hypothesis on model systems such as culture at low and normal oxygen conditions. Another interesting issue for us was to look in detail at the chromatin and cytoplasm organization of erythroblasts on a spectral level, using spectral imaging analysis, and then to compare the results for erythroblasts from maternal and cord blood. This analysis also confirmed differences between erythroblasts from maternal and cord blood. Which fetal target cell is best suited for analysis remains an open question. Our task was to evaluate the ability to perform FISH analysis of fetal cells in whole blood without any enrichment. One further task was to improve fetal cell recovery by applying XYY FISH as alternative to conventional XY FISH As reported by many researchers, single cell PCR analysis of fetal erythroblasts is more effective than FISH analysis. We checked the fetal status of small dense erythroblasts which appeared to be refractory to FISH analysis by Taqman PCR after laser microdissection capture (pool). Furthermore, we explored the opportunity of single cell Taqman analysis of erythroblasts enriched by soybean lectin-based (SBA) method and microdissected by laser microdissection and pressure catapulting (LMPC) technology from membrane covered slides. All the above–listed questions comprised the subject matter for our investigation. In what follows, we give a detailed description of the studies performed.