Fetal cells in maternal circulation : fetal cell separation and FISH analysis (2005)

This work focuses on the isolation of fetal cells from the blood of pregnant women, with the&#13;\naim of developing safe, efficacious, non-invasive alternatives for prenatal diagnosis. Although&#13;\nthe fetal cells were first detected in maternal blood in the 1893, an effective protocol for noninvasive&#13;\nanalysis is still not firmly established. This is due, on the one hand, to the scarcity of&#13;\nfetal cells in maternal blood, which is of the order of 1 fetal cell to 106 - 107 maternal&#13;\nnucleated cells and on the other hand, to the fact that fetal cells have no specific cell markers.&#13;\nEfforts were made to improve development and evaluation of new fetal cell enrichment&#13;\nprocedures. One of the tasks of this study was to evaluate galactose specific enrichment via&#13;\nsoybean agglutinin, a galactose-specific lectin for isolation of erythroblast from maternal&#13;\nblood, and to compare this new technique with the conventional CD71 enrichment technique.&#13;\nAnother technical obstacle which had to be overcome was how to analyze the chromosomal&#13;\ncontent of few fetal cells enriched from the maternal circulation. Since these fetal&#13;\nerythroblasts were not actively dividing it was impossible to use standard cytogenetic&#13;\nmethods. To address this issue multicolor fluorescence in situ hybridisation (FISH), or single&#13;\ncell polymerase chain reaction (PCR) procedures for analysis of fetal cells were developed&#13;\nand optimized. In the largest series of articles published to date, the efficacy of detecting fetal&#13;\ncells by the use of FISH for X and Y chromosomes was below what was needed. We tried to&#13;\noptimize the FISH procedure by applying different treatments to the nucleus and using&#13;\ndifferent kinds of fluorescent probes firstly on cord blood erythroblasts as a model system.&#13;\nThen, after optimization, the best FISH protocols were applied to electronically marked&#13;\nerythroblasts from maternal blood. The analysis of FISH signals in maternal blood revealed&#13;\nthat about half of erythroblasts did not hybridize. Additionally, we checked whether the&#13;\nability to successfully perform FISH depended on chromosome choice. FISH analysis for&#13;\nchromosome 18 gave the same result. Thereafter we searched for possible reasons of FISH&#13;\nsignal absence. The morphometric analysis of erythroblasts indicated that erythroblasts which&#13;\nhad hybridized efficiently were of larger nuclear size than those which had been impervious&#13;\nto the FISH procedure; that is, the efficiency of FISH procedure is connected with nuclear&#13;\nsize. We then compared the erythroblasts from maternal blood with those from cord blood.&#13;\nThe morphometric analysis indicated a significant difference in size between erythroblasts&#13;\ncirculating in maternal blood and cord blood.&#13;\nAdditionally, we wanted to determine whether the presence of fragmented DNA hindered the&#13;\nFISH analysis. The results of Terminal deoxynucleotidyl Transferase Biotin-dUTP Nick End&#13;\nLabeling (TUNEL) analysis suggested that effective FISH analysis had been hindered by the&#13;\n&#13;\npresence of dense nuclei rather than nuclei containing fragmented DNA. A point of interest&#13;\nwas for us to look for possible reasons for nucleus size reduction of erythroblasts in the&#13;\nmaternal circulation. We hypothesized that the changes in the nuclear size of erythroblasts&#13;\ncould be attributed to the different oxygen tensions in the fetal and maternal circulatory&#13;\nsystems. We checked and confirmed this hypothesis on model systems such as culture at low&#13;\nand normal oxygen conditions.&#13;\nAnother interesting issue for us was to look in detail at the chromatin and cytoplasm&#13;\norganization of erythroblasts on a spectral level, using spectral imaging analysis, and then to&#13;\ncompare the results for erythroblasts from maternal and cord blood. This analysis also&#13;\nconfirmed differences between erythroblasts from maternal and cord blood.&#13;\nWhich fetal target cell is best suited for analysis remains an open question. Our task was to&#13;\nevaluate the ability to perform FISH analysis of fetal cells in whole blood without any&#13;\nenrichment. One further task was to improve fetal cell recovery by applying XYY FISH as&#13;\nalternative to conventional XY FISH&#13;\nAs reported by many researchers, single cell PCR analysis of fetal erythroblasts is more&#13;\neffective than FISH analysis. We checked the fetal status of small dense erythroblasts which&#13;\nappeared to be refractory to FISH analysis by Taqman PCR after laser microdissection&#13;\ncapture (pool). Furthermore, we explored the opportunity of single cell Taqman analysis of&#13;\nerythroblasts enriched by soybean lectin-based (SBA) method and microdissected by laser&#13;\nmicrodissection and pressure catapulting (LMPC) technology from membrane covered slides.&#13;\nAll the above–listed questions comprised the subject matter for our investigation. In what&#13;\nfollows, we give a detailed description of the studies performed.