Britta Christensen

Characterization of fetal cells from the maternal circulation by microarray gene expression analysis--could the extravillous trophoblasts be a target for future cell-based non-invasive prenatal diagnosis?

Introduction: Circulating fetal cells in maternal blood provide a tool for risk-free, non-invasive prenatal diagnosis. However, fetal cells in the maternal circulation are scarce, and to effectively isolate enough of them for reliable diagnostics, it is crucial to know which fetal cell type(s) should be targeted. Materials and Methods: Fetal cells were enriched from maternal blood by magnetic-activated cell sorting using the endothelial cell marker CD105 and identified by XY fluorescence in situ hybridization. Expression pattern was compared between fetal cells and maternal blood cells using stem cell microarray analysis. Results: 39 genes were identified as candidates for unique fetal cell markers. More than half of these are genes known to be expressed in the placenta, especially in extravillous trophoblasts (EVTs). Immunohistochemical staining of placental tissue confirmed CD105 staining in EVTs and 76% of fetal cells enriched by CD105 were found to be cytokeratin-positive. Discussion: The unique combination of mesodermal (CD105) and ectodermal (cytokeratin) markers in EVTs could be a potential marker set for cell enrichment of this cell type in maternal blood and could be the basis for future cell-based non-invasive prenatal diagnosis.

Studies on the Isolation and Identification of Fetal Nucleated Red Blood Cells in the Circulation of Pregnant Women before and after Chorion Villus Sampling

Objective: To investigate the feasibility of various molecular forms of hemoglobin as markers for fetal nucleated red blood cells (NRBCs). Methods: The presence of epsilon and gamma globin positive NRBCs was investigated in pure fetal blood and in blood from pregnant women before and after chorion biopsy. Maternal samples were enriched for NRBCs by various conventional methods, including limited enrichment by only positive CD71 selection or single density gradient. We searched for fetal cells on slides by automated scanning. Fetal cells were defined by (1) the presence of epsilon or gamma globin and (2) simultaneously by the presence of a Y chromosome signal. Results: 18 of 25 gamma globin positive cells identified in blood samples after chorion biopsy were chromosome Y signal positive, and 1 cell had two X chromosome signals. 263 of 339 epsilon globin positive cells identified in blood samples after chorion biopsy were hybridized with X and Y chromosome probes. None had two X signals, and 249 were Y positive. In blood samples before chorion biopsy, only 1 epsilon globin positive fetal NRBC and no epsilon globin positive maternal NRBCs were found. Conclusions: Epsilon globin may be specific for fetal NRBCs. Only 1 epsilon globin positive fetal cell was identified in 1 of 12 blood samples before chorion biopsy, representing a total of 182 ml of maternal blood. This suggests that most fetal cells found in maternal blood by fluorescence in situ hybridization methods may not be NRBCs.

Rapid prenatal diagnosis of chromosome aneuploidies by interphase fluorescence in situ hybridization: a one‐year clinical experience with high‐risk and urgent fetal and postnatal samples

Objectives. To evaluate the clinical utility of rapid prenatal and postnatal detection of common chromosome aneuploidies by interphase fluorescence in situ hybridization (FISH) analysis with DNA probes.

A new marker set that identifies fetal cells in maternal circulation with high specificity.

Fetal cells from the maternal circulation (FCMBs) have the potential to replace cells from amniotic fluid or chorionic villi in a diagnosis of common chromosomal aneuploidies. Good markers for enrichment and identification are lacking.

Identification of circulating fetal cell markers by microarray analysis

Different fetal cell types have been found in the maternal blood during pregnancy in the past, but fetal cells are scarce, and the proportions of the different cell types are unclear. The objective of the present study was to identify specific fetal cell markers from fetal cells found in the maternal blood circulation at the end of the first trimester.

Fetal Cells in Maternal Blood: A Comparison of Methods for Cell Isolation and Identification

Objective: A variety of methods have been used to select and identify fetal cells from maternal blood. In this study, a commonly used 3-step selection method is compared with selection directly from whole blood. Identification of fetal origin by XY FISH of male cells was also evaluated. Methods: Maternal blood was drawn either before invasive chorion villus sampling (pre-CVS) or after (post-CVS) from women carrying a male fetus. Fetal cells were isolated either by density gradient centrifugation succeeded by CD45/CD14 depletion and CD71-positive selection from CD45/CD14-negative cells, or by CD71-positive selection directly from whole blood. The true origin of fetal cells recovered by the two methods was established by two rounds of XY chromosome FISH in reverse colors, in some instances combined with anti-zeta (ζ) or anti-ζ/anti-gamma (γ) antibody staining. Results: In blood samples taken post-CVS and enriched by CD71 selection directly from whole blood, fetal cells were identified with a frequency that was almost four orders of magnitude higher than in post-CVS samples enriched by the 3-step method. In blood samples taken pre-CVS and enriched by the 3-step procedure, no fetal cells were identified by reverse color FISH in 371 ml of blood. In similar samples enriched by CD71 selection on whole blood, two fetal cells were identified in 27 ml of blood. Rehybridization with X and Y chromosome probes with reverse colors was necessary to exclude false Y chromosome signals. Not all fetal cells identified by the presence of a true Y chromosome signal stained with anti-ζ antibody. Conclusions: Selection of fetal NRBCs from maternal blood by CD71-positive selection directly from whole blood is superior to density gradient centrifugation succeeded by CD45/CD14 depletion and CD71 selection of CD45/CD14-negative cells. Combining two markers for fetal origin is recommended for unambiguously identifying a cell as fetal.

Microselection – affinity selecting antibodies against a single rare cell in a heterogeneous population

Rare cells not normally present in the peripheral bloodstream, such as circulating tumour cells, have potential applications for development of non‐invasive methods for diagnostics or follow up. Obtaining these cells however require some means of discrimination, achievable by cell type specific antibodies. Here we have generated a microselection method allowing antibody selection, by phage display, targeting a single cell in a heterogeneous population. One K562 cell (female origin) was positioned on glass slide among millions of lymphocytes from male donor, identifying the K562 cell by FISH (XX). Several single cell selections were performed on such individual slides. The phage particles bound to the target cell is protected by a minute disc, while inactivating all remaining phage by UV‐irradiation; leaving only the phage bound to the target cell viable. We hereby retrieved up to eight antibodies per single cell selection, including three highly K562 cell type specific.

The Fetal Erythroblast Is Not the Optimal Target for Non-invasive Prenatal Diagnosis: Preliminary Results

Fetal cells, present in the blood of pregnant women, are potential targets for non-invasive prenatal diagnosis. The fetal erythroblast has been the favorite target cell type. We investigated four methods of enrichment for fetal erythroblasts, identifying only three fetal erythroblasts in 573 ml of maternal blood. This is much less than the expected two to six fetal cells per ml of maternal blood. Hamada and Krabchi used a cell type-independent marker, i.e., the Y chromosome in maternal blood from male pregnancies after Carnoy fixation, leaving the nuclei for hybridization with X-and Y-chromosome-specific probes. We found with a similar technique 28 fetal cells in 15 ml of maternal blood. The fetal origin of cells was confirmed by hybridizing the nuclei with X- and Y-chromosome-specific probes, using two consecutive hybridizations with the two probes in opposite colors (reverse FISH). Candidate fetal cells were inspected after each hybridization. Only cells that were found to change the color of both probe signals from first to second hybridization were diagnosed as fetal. To reduce the labor-intensive slide screening load, we used semi-automated scanning microscopy to search for candidate cells. We conclude that erythroblasts form only a small fraction of fetal cells present in maternal blood.

Epsilon Haemoglobin Specific Antibodies with Applications in Noninvasive Prenatal Diagnosis

Invasive procedures for prenatal diagnosis are associated with increased risk of abortion; thus, development of noninvasive procedures would be beneficial. Based on the observation that embryonic nucleated red blood cell (NRBC) crosses the placenta and enters the circulation of pregnant women, the ability to identify such cell would allow development of such procedures. Identification of NRBCs in blood samples would be possible provided that specific antibodies are available. Here we have isolated recombinant antibodies using phage display. From the panel of antibody fragments specifically recognising ε-Hb, one was chosen for further characterization, DAb1. DAb1 binds to ε-Hb both in Western blots and immunocytochemistry. Several ε-Hb positive cells were detected in a blood sample taken as postchorionic villus sampling (CVS). To evaluate the sensitivity of the method, K562 cells (which express ε-Hb) were spiked in a blood sample followed by staining in solution and FACS analysis.

Sensitivity and Specificity of the Identification of Fetal Cells in Maternal Blood by Combined Staining with Antibodies against Beta-, Gamma- and Epsilon-Globin Chains

Objectives: Antibodies against fetal and embryonic hemoglobins may identify fetal cells in maternal blood. Both γ- and Ε-globins are used as fetal cell markers. γ-Globin is not fetus specific. So far Ε-globin has been claimed to be fetus specific. In this communication, we compare the specificity of anti-Ε- and anti-γ-globin staining when combined with staining for β-globin. Methods: We applied single and double color immunofluorescent staining techniques in combination with XY chromosome hybridization. The blood sample was taken after chorion villus biopsy at 11 weeks of gestation from a woman carrying a male fetus. Results: By γ-globin staining alone, 21 fetal and 2 maternal nucleated red blood cells (NRBCs) were identified. Only 1 of the 2 maternally derived NRBCs expressed β-globin. By Ε-globin staining, 92 additional fetal NRBCs were identified. Conclusions: Ε-Globin antibody and combined Ε- and γ-globin antibody staining of a blood sample from a pregnant woman at 11 gestational weeks showed higher sensitivity but lower specificity for the fetal origin of erythroblasts with combined compared with separate staining. The final decision of the origin of cells was made by gender determination by FISH. Out of 2 γ-positive maternal cells 1 was β-globin antibody positive, 1 was β-globin negative, indicating that 100% specificity for fetal origin could not be obtained by combining all 3 hemoglobin types. Although only 1 blood sample was tested and only 2 γ-positive maternal NRBCs were identified, the result indicates that β-hemoglobin does not discriminate completely between γ-positive NRBCs of fetal and maternal origin.