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Objective Intact fetal cells isolated from maternal blood can be used for non-invasive gender determination and genetic diagnosis. Recent studies demonstrating a large amount of cell-free fetal DNA in maternal plasma suggest that the circulating fetal DNA may result from fetal cells undergoing apoptosis. In the present study we evaluated the potential role of Fas and Fas ligand (FasL) cell surface expression with respect to apoptosis induction in fetal cells isolated from maternal blood. Methods We flow sorted candidate fetal cells that were gamma chain-positive and Fas- or FasL-positive or -negative, and subsequently analysed them by fluorescence in situ hybridization (FISH) analysis using X and Y chromosome-specific probes. Results Among all gamma hemoglobin-positive cells, there was a significant difference in the percent of cells expressing Fas versus FasL (4.4 and 12.3, respectively). We found no significant correlation between the total number of fetal nucleated red blood cells (NRBCs) and gestational age or the presence of Fas- and FasL-positive cells. From approximately 7 ml of maternal peripheral blood, most of the confirmed fetal (XY) cells were found in the Fas- and FasL-negative sorted population; the average numbers were 12.8 and 15.7, respectively. Conclusion We conclude that fetal NRBCs express FasL more than Fas, although most fetal NRBCs in first trimester maternal blood samples do not express Fas or FasL. This suggests the absence of a functional Fas/FasL apoptotic system in fetal NRBCs, and that programmed cell death in these cells, which may lead to circulating fetal DNA in maternal plasma, probably occurs by another pathway. Copyright © 2002 John Wiley & Sons, Ltd.

The nucleated erythrocyte (NRBC) is one of the target fetal cell types for noninvasive genetic diagnosis using maternal peripheral blood. However, it is now known that pregnancy can stimulate the production of maternal NRBCs. When isolating female gamma-positive NRBCs, fluorescence in situ hybridization (FISH) analysis may show two X chromosome signals per nucleus, and therefore it cannot be conclusively determined whether the isolated cells are fetal or maternal in origin. The purpose of this study was to develop a means of verifying that a female cell is fetal on the basis of polymorphic short tandem repeat markers. Peripheral blood samples were obtained from women who had just undergone termination of pregnancy. Nucleated candidate fetal cells were isolated by flow-sorting using antibody to the gamma-chain of fetal hemoglobin and Hoechst 33342. FISH analysis was performed using X and Y chromosome specific probes. Female gamma-positive cells and leukocytes were micromanipulated separately and subjected to fluorescent polymerase chain reaction amplification of chromosome 21 and/or 18 STR markers (D21S11, D21S1411, D21S1412, and D18S535). In all ten cases analyzed, the gamma-positive female candidate fetal cells were determined to be fetal in origin by the presence of shared and nonshared DNA polymorphisms when compared with maternal leukocytes. These results show that genetic analysis can be performed on all fetal NRBCs, including female fetal cells that cannot be distinguished from maternal cells based on FISH analysis alone.

The purpose of this study was to improve recovery of fetal nucleated erythrocytes (NRBCs) from maternal blood for non-invasive prenatal diagnosis. Peripheral blood samples were obtained from 27 women who had just undergone pregnancy termination at 6 to 23 weeks. Samples were split and mononuclear cells were isolated using Histopaque gradient at densities of 1.090 g/ml and 1.119 g/ml. CD45 depletion using magnetic activated cell-sorting, followed by flow-sorting with antibody to γ-globin and fluorescence in situ hybridization (FISH) analysis, were used to evaluate the number of fetal NRBCs recovered. In samples separated with the 1.119 g/ml density gradient, the yield of true anti-γ haemoglobin positive cells (median, 14.9; range, 0–717.5) was significantly higher than that with the 1.090 g/ml density gradient (median, 4.9; range, 0–532.5). After FISH analysis, in the 14 samples in which the fetal karyotype differed from the mother, the median number of fetal NRBCs separated by the 1.119 g/ml density gradient was 22.9 (2–717.5), which was significantly higher than that by the 1.090 g/ml gradient (median, 11.5; range, 0–532.5, p=0.022). Increased density of the gradient used for the initial enrichment of fetal cells results in improved fetal cell recovery in fresh post-termination blood samples, which may permit better non-invasive detection of fetal cells in maternal blood. Copyright © 2000 John Wiley & Sons, Ltd.

BACKGROUND Fluorescence activated cell sorting (FACS)-based anti-gamma (γ) positive selection and magnetic activated cell sorting (MACS)-based anti-CD45 depletion followed by anti-γ positive staining have been two of the most frequently used methods to isolate fetal cells from maternal blood. To date, there has been no direct comparison of fetal cell recovery by these two methods. This study was designed to address this issue. METHODS Fluorescence in situ hybridization (FISH) was performed on nucleated anti-γ positive cells using X and Y probes. Twenty-four maternal blood samples were obtained immediately after elective termination of pregnancy to ensure a detectable number of fetal cells. RESULTS The yield and purity of fetal nucleated erythrocytes (FNRBCs) was statistically higher in FACS sorted samples (P < 0.01). The specificity of staining for FNRBCs was statistically higher in MACS sorted samples (P < 0.01). CONCLUSIONS The data from this study demonstrate that both techniques have benefits and limitations. FACS has the advantage of having higher yield, higher purity, higher FISH efficiency and ease in microscope analysis, and MACS has the advantage of having higher specificity and less cell loss during FISH. Cytometry 39:224–230, 2000 © 2000 Wiley-Liss, Inc.

The purpose of this study was to determine if apoptosis occurs in fetal cells that have crossed into the maternal circulation, which would potentially explain the difference between the number of intact fetal cells and the amount of fetal DNA detectable in maternal plasma. We flow-sorted fetal nucleated erythrocytes (FNRBCs) using antibody to the gamma chain of fetal haemoglobin and confirmed them to be fetal in origin by FISH analysis using chromosome-specific probes. Fetal cells were then analysed microscopically for the presence of terminal UdTP nuclear end labelling (TUNEL) staining. Apoptotic change was observed in 42.7% of fetal NRBCs (106/246) and 3.5% of maternal cells (29/818). Results of this study indicate that a significant number of fetal cells in maternal blood are undergoing apoptosis at the time of sampling. Apoptosis may be one mechanism by which fetal cells are cleared by the maternal circulation.

Prenatal diagnosis of fetal chromosomal abnormalities using interphase fetal nucleated erythrocytes (FNRBCs) separated from maternal peripheral blood can be technically challenging due to the limited number of FNRBCs available for analysis, the limited number of probes that can be used simultaneously, and low FISH efficiency on the formaldehyde-fixed and immunohistochemically stained interphase FNRBCs. We developed a technique of sequential FISH analysis that involves removal of the previous hybridized probe under denaturing conditions, and rehybridization with different probes to improve FISH efficiency. This technique facilitates the analysis of multiple chromosome-specific probes on the same nuclei. Results from our experiments show that FISH can be performed at least nine times on the same interphase nucleus and at least three different probes can be used simultaneously. Thus, theoretically, at least 24 different chromosomes can be analysed on a single interphase fetal cell isolated from maternal blood. We have termed this technique 'Poly-FISH', and have successfully diagnosed trisomy 21, triploidy, and other chromosome abnormalities in FNRBCs using this technique.

A pregnant woman who was a carrier for a balanced chromosome translocation [46,XX, t(1;6) (p31;q14)] and who had had six miscarriages, declined invasive testing but agreed to non-invasive prenatal diagnosis by analysis of fetal cells in maternal blood. Monoclonal antibody (Mab) against the zeta (z) and gamma (gamma) chains of embryonic and fetal haemoglobin were used to identify fetal nucleated erythrocytes (FNRBC). There were no FNRBC detected at 7 weeks, one anti-z-positive FNRBC was detected at 11 weeks, and 12 anti-gamma-positive FNRBC were detected at 20 weeks. Fluorescent in-situ hybridization was performed using probes for chromosomes X, Y, 1 and 6 to identify fetal gender and the presence of an unbalanced chromosomal translocation. A tentative prenatal diagnosis was made of a female fetus disomic for chromosomes 1 and 6. A female infant with a 46,XX karyotype was born at term. This is the first attempt of exclusion of a chromosome translocation using fetal cells isolated from maternal blood. There is an advantage of using fetal cells isolated from maternal blood for non-invasive prenatal diagnosis in couples who have a history of multiple miscarriages due to a parental translocation, and who decline invasive testing in a pregnancy that continues to the second trimester.

Objective: Our aim was to develop a new technique, which we have termed fetal cell recycling, that combines the 2 powerful methods of fluorescence in situ hybridization and polymerase chain reaction to maximize the genetic information available from a small number of fetal nucleated erythrocytes obtained noninvasively from the blood of pregnant women. Study Design: Blood samples were obtained from 4 Rh-negative women after elective termination of pregnancy at 7 to 17 weeks' gestation. Fetal nucleated erythrocytes were separated by flow sorting with antibody to the γ chain of fetal hemoglobin. Fluorescence in situ hybridization with chromosome-specific probes was used to diagnose fetal gender. After fluorescence in situ hybridization analysis the fetal nucleated erythrocytes were recycled by a micromanipulation technique and deoxyribonucleic acid diagnosis was performed with polymerase chain reaction amplification of the RhD gene. Results: Among the 4 case patients we detected a total of 101 fetal nucleated erythrocytes. All targeted cells were successfully retrieved with a micromanipulator. In each case we successfully performed both fluorescence in situ hybridization and polymerase chain reaction analysis. The predicted fetal gender and Rh status corresponded to the results obtained from fetal tissue. Conclusions: Fetal cell recycling combines the powers of highly sensitive molecular methods to maximize the genetic information available from a single fetal cell. This technique will permit noninvasive diagnosis of recessively inherited single-gene disorders. (Am J Obstet Gynecol 1999;181:1237-42.)

Objective: To improve the recovery of fetal nucleated erythrocytes (NRBCs) from maternal blood for noninvasive prenatal genetic diagnosis. Methods: Blood samples were obtained from 10 women at 8–22 weeks of gestation. Samples were split and mononuclear cells were isolated using 1.083 and 1.090 g/ml of Percoll solution. Flow sorting with antibody to fetal hemoglobin and fluorescence in situ hybridization (FISH) analysis were used to evaluate the number of fetal cells recovered. Results: In samples separated with the 1.090 density gradient, the yield of true γ-hemoglobin-positive cells (median 21.0, range 2.2–303.8) was 1.9 times higher than that in the 1.083 density (median 11.1, range 1.1–87.5), although it took 2.1-fold longer time to flow sort the γ-hemoglobin-positive cells. In 7 out of 10 cases, the number of γ-hemoglobin-positive cells recovered from the 1.090 density gradient was 3 times or greater than that from 1.083 gradient. After FISH analysis, we detected a median of 13.3 (range 2.2–98.8) fetal NRBCs per 10-ml maternal blood in the 1.090 density gradient, whereas a median of 11.0 fetal NRBCs were detected in the 1.083 gradient (range 1.1–35.0). The number of fetal NRBCs in the 1.090 density was significantly higher than that in the 1.083. Conclusion: Increased Percoll density results in improved fetal cell recovery in fresh posttermination maternal samples. The increased yield of fetal cells using this gradient may permit better noninvasive detection of fetal chromosome as well as DNA abnormalities in maternal blood.