Both intact fetal cells as well as cell-free fetal DNA are present in the maternal circulation and can be recovered for non-invasive prenatal genetic diagnosis. Although methods for enrichment and isolation of rare intact fetal cells have been challenging, diagnosis of fetal chromosomal aneuploidy including trisomy 21 in first- and second-trimester pregnancies has been achieved with a 50-75% detection rate. Similarly, cell-free fetal DNA can be reliably recovered from maternal plasma and assessed by quantitative PCR to detect fetal trisomy 21 and paternally derived single gene mutations. Real-time PCR assays are robust in detecting low-level fetal DNA concentrations, with sensitivity of approximately 95-100% and specificity near 100%. Comparing intact fetal cell versus cell-free fetal DNA methods for non-invasive prenatal screening for fetal chromosomal aneuploidy reveals that the latter is at least four times more sensitive. These preliminary results do not support a relationship between frequency of intact fetal cells and concentration of cell-free fetal DNA. The above results imply that the concentration of fetal DNA in maternal plasma may not be dependent on circulating intact fetal cells but rather be a product of growth and cellular turnover during embryonic or fetal development.

Microparticles (MPs) that circulate in blood may be a source of DNA for molecular analyses, including prenatal genetic diagnoses. Because MPs are heterogeneous in nature, however, further characterization is important before use in clinical settings. One key question is whether DNA is either bound to aggregates of blood proteins and lipid micelles or intrinsically associated with MPs from dying cells. To test the latter hypothesis, we asked whether MPs derived in vitro from dying cells were similar to those in maternal plasma. JEG-3 cells model extravillous trophoblasts, which predominate during the first trimester of pregnancy when prenatal diagnosis is most relevant. MPs were derived from apoptosis and increased over 48 hours. Compared with necrotic MPs, DNA in apoptotic MPs was more fragmented and resistant to plasma DNases. Membrane-specific dyes indicated that apoptotic MPs had more membranous material, which protects nucleic acids, including RNA. Flow cytometry showed that MPs derived from dying cells displayed light scatter and DNA staining similar to MPs found in maternal plasma. Quantification of maternal MPs using characteristics defined by MPs generated in vitro revealed a significant increase of DNA ؉ MPs in the plasma of women with preeclampsia compared with plasma from women with normal pregnancies. Apoptotic MPs are therefore a likely source of stable DNA that could be enriched for both early genetic diagnosis and monitoring of pathological pregnancies. (Am J

Isolation and analysis of intact fetal cells in maternal blood is an attractive method of non-invasive prenatal diagnosis; however, detection levels are not optimal. The poor sensitivity and inconsistent recovery of fetal cells is compounded by small numbers of circulating fetal cells and loss of fetal cells during enrichment procedures. Optimizing selection criteria by utilizing less complicated methods for target cell enrichment is essential. We report here salutary results using a simple density-based depletion method that requires neither MACS (magnetic-activated cell sorting) nor flow cytometric separation for enrichment of progenitor cells. Maternal blood samples (n = 81) were obtained from women prior to invasive prenatal genetic diagnostic procedures and processed randomly within 24 h using one of two density-based enrichment methods. For progenitor cell enrichment, samples (n = 49) were labeled with a RosetteSep progenitor antibody cocktail to remove unwanted mature T-cells, B-cells, granulocytes, natural killer cells, neutrophils and myelomonocytic cells. For CD45-negative cell enrichment, samples (n = 14) were labeled with RosetteSep CD45 antibody to remove unwanted maternal white cells. The desired cellular fraction was collected and analyzed by either fluorescent in situ hybridization (FISH) or real-time PCR for the presence of intact fetal cells and to quantify Y-chromosome-specific DYS1 sequences, respectively. Overall, FISH and real-time PCR correct detection rates for the progenitor cell enrichment approach were 53% and 89% with 3% (1 out of 30 cases) and 0% false-positive detection, respectively. Fetal sequences were detected in the range from 0.067 to 1.167 genome equivalents per milliliter of blood. No fetal cells were detected using the CD45-negative enrichment method. Flow cytometric analysis of cord blood showed that a unique myeloid population of cells was recovered using RosetteSep trade mark progenitor enrichment compared with the CD45-negative enrichment method. Sensitivity of the RosetteSep progenitor enrichment approach for detection of fetal cells in this pilot study shows great promise with recovery of cells that are suitable for FISH and automated microscope scanning. This simple and rapid method may also allow expansion in culture and characterization of the fetal cell type(s) that circulate in maternal blood, hence, greatly improving reliability of non-invasive prenatal diagnosis.

Fetal DNA is found in the plasma of pregnant women that appears to be stable for PCR amplification. Although the underlying mechanism giving rise to this DNA in plasma remains unclear, the source of these fragments may be from apoptotic bodies (Apo-Bodies) created from dying cells. Trophoblast apoptosis is essential for normal placental development, given the enormous amount of proliferation, differentiation, and migration during pregnancy. Through flow cytometric analysis coupled with real-time PCR, our lab has shown that aggregates of acridine orange (AO)-stained material (apoptotic particles) are resistant to DNase treatment, disrupted by sodium dodecyl sulfate (SDS), and contain fetal DNA. Because the placenta continuously re-models in an hypoxic environment, our hypothesis is that fetal DNA in maternal plasma comes from hypoxia-induced dying trophoblasts and that this DNA circulates predominantly in the form of Apo-Bodies. We have developed a model culture system for analysis of Apo-Bodies derived from JEG-3 cells, an extravillous trophoblastic cell line, undergoing various methods of cell death: hypoxia-induced, etoposide-induced, and heat stress (necrosis like)-induced cell death. Under conditions of similar propidium iodide (PI) uptake, suggesting comparable levels of death, both hypoxia- and etoposide-induced Apo-Bodies increase in concentration over time, whereas heat-induced levels of particles remain fairly constant, indicating that production of DNA-associated Apo-Bodies is a continuous process. Hypoxia, which is likely to be responsible for trophoblast cell death in vivo, produced membrane-bound Apo-Bodies containing DNA. Our results are consistent with the characteristics of membrane-bound particles containing fetal DNA found in maternal plasma.

Both intact fetal cells and cell-free fetal DNA are present in the maternal circulation and have been used for non-invasive prenatal genetic diagnosis. However, broad clinical application awaits development of robust methods for collecting, transporting and enriching maternal blood samples to recover rare fetal cells. To circumvent this impediment, we have devised a reliable method of fetal DNA detection using dried maternal blood spots and real-time polymerase chain reaction. Fetal Y-specific (DYS1) sequences were detected in all 19 (100%) maternal blood specimens from women carrying male fetuses, in genome equivalents of 4.20-24.68 per ml of blood; the ubiquitous glyceraldehyde 3-phosphate dehydrogenase (GAPDH) gene, reflecting both maternal and fetal DNA, concurrently showed 43,684 to 680,357 genome equivalents per ml of blood. The results demonstrate that fetal DNA detection using dried maternal blood spots is highly feasible and easily adaptable for population screening.