Diagnosing and preventing inherited disease: Nucleated erythrocytes in maternal blood: quantity and quality of fetal cells in enriched populations

The discovery of nucleated erythrocytes in maternal circulationprovides a potential source for non-invasive prenatal diagnosis.We have evaluated the use of a three-stage procedure to determinethe number of cells that are of fetal rather than maternal origin.First, monoclonal antibodies specific for CD45 and CD14 wereused in conjunction with a magnetic (MACS) column to depleteunwanted leukocytes from maternal blood. This was followed bya positive MACS enrichment for nucleated erythrocytes, usingan anti-CD71 (transferrin receptor) monoclonal antibody. Todiscriminate between fetal nucleated erythrocytes and thoseof maternal origin, enriched fractions were simultaneously stainedwith an anti-fetal haemoglobin (HbF) antibody and hybridizedwith probes specific for X and Y chromosomes. Samples were thensubjected to blind analysis along with negative control samplesfrom non-pregnant volunteers. Using this dual analysis, we wereable to determine that less than one nucleated erythrocyte perml of maternal blood was of fetal origin. Small numbers of thesefetal cells were found in 87.5% of pregnancies, ranging from6 to 35 weeks gestational age. Comparison of HbF and X/Y probedata also suggests that the fetal cells are less suitable forfluorescence in-situ hybridization (FISH) analysis than similarpreparations from other sources.     

Prenatal diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative magnetic cell sorting.

Fetal nucleated cells in the maternal circulation constitute a potential source of cells for the non-invasive prenatal diagnosis of fetal genetic abnormalities. We have investigated the use of the Magnetic Activated Cell Sorter (MACS) for enriching fetal nucleated erythrocytes. Mouse monoclonal antibodies specific for CD45 and CD32 were used to deplete leucocytes from maternal blood using MACS sorting, thus enriching for fetal nucleated erythrocytes which do not express either of these antigens. However, significant maternal contamination was present even after MACS enrichment preventing the accurate analysis of fetal cells by interphase fluorescence in situ hybridisation (FISH). To overcome this problem, we used simultaneous immunophenotyping of cells with the mouse antifetal haemoglobin antibody, UCH gamma, combined with FISH analysis using chromosome X and Y specific DNA probes. This approach enables selective FISH analysis of fetal cells within an excess of maternal cells. Furthermore, we have confirmed the potential of the method for clinical practice by a pilot prospective study of fetal sex in women referred for amniocentesis between 13 and 17 weeks of gestation.     

HCMV感染孕妇外周血中胎儿细胞的分离

目的运用不连续密度梯度离心及免疫磁珠分离法分选孕妇外周血中的胎儿有核红细胞。方法取20名人巨细胞病毒抗原血症(pp65)阳性的孕妇外周EDTA抗凝血,经不连续密度梯度离心初步富集、免疫磁珠法分选及胎儿细胞。结果 20名pp65阳性孕妇外周血中分选到胎儿有核红细胞的有16例,10份正常妊娠妇女外周血细胞涂片中发现胎儿有核红细胞数9例。10份未妊娠妇女外周血细胞涂片中均未发现胎儿有核红细胞。结论不连续密度梯度离心及免疫磁珠分离法结合特异性抗体-HbF标记、识别能有效地分选出母血中胎儿有核红细胞,应用于进一步的无创性产前诊断,将有着良好的应用前景。     

Maternal origin of transferrin receptor positive cells in venous blood of pregnant women

We studied the origin of transferrin receptor (CD71) positive cells in blood from seven women pregnant with a male fetus in order to explore if fetal cells could be detected among them. We used a technique that allows direct chromosomal analysis by in situ hybridization on immunologically and morphologically classified cells. Enrichment was performed by magnetic activated cell sorting (miniMACS)® using an anti-CD71 monoclonal antibody. The cells were immunophenotyped by alkaline phosphatase anti-alkaline phosphatase immunostaining with the same antibody. The origin of the immunophenotyped cells was studied by in situ hybridization using an X cosmid Y repeat chromosome specific probe cocktail. CD71 positive cells were found in six of the seven women at the range of 4 to 43 in respective samples. Over 90% of the CD71 positive cells were nucleated erythrocytes. None of the detected positive cells were shown to be fetal. Thus, the use of transferrin receptor antigen alone in combination with the miniMACS® may not be sufficient for enrichment of fetal cells.     

孕妇外周血胎儿有核红细胞的分离与检测

目的从孕妇血循环中分选出有核红细胞（ＮＲＢＣｓ），并证实其是胎儿来源的。方法选用红细胞特异单克隆抗体ＧｌｙｃｏｐｈｏｒｉｎＡ（ＧＰＡ）结合流式细胞术成功地从３例妊娠１６～２４周的孕妇血中分离出ＮＲＢＣｓ，并用Ｙ－染色体特异重复序列ＤＮＡ探针（ＰＹ３．４）荧光原位杂交及聚合酶链反应技术检测Ｙ－染色体特异序列（ＳＲＹ）结果ＦＩＳＨ结果，３例分选出的ＧＰＡ阳性细胞中ＰＹ３．４杂交阳性率分别是３１．４％、０、３８％；其ＰＣＲ结果，分别为阳性、阴性、阳性，并在引产后得到性别证实。结论孕妇外周血中确实存在胎儿有核红细胞，采用非创伤性方法可以分离并检测它们     

Non-invasive exclusion of fetal aneuploidy in an at-risk couple with a balanced translocation

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.     

Isolation of fetal erythroid cells from maternal blood based on expression of erythropoietin receptors

Fetal nucleated red cells which pass into the maternal circulation during pregnancy are a potential cell source for non-invasive prenatal genetic diagnosis. To sort these rare cells with a high degree of specificity, we focussed our attention on the erythropoietin receptor, a strictly erythroid-specific antigen. We first labelled these receptors with biotin-(sialyl)-erythropoietin, then isolated the erythroid cells by magnetic beads conjugated with streptavidin in a MiniMACS (magnetic cell separator). The effectiveness of this strategy for the enrichment of fetal cells was evaluated by assessing its accuracy for gender prediction in 18 male-bearing pregnancies. Polymerase chain reaction (PCR) results on maternal blood samples sorted for Epo-r and CD71 antigens displayed similar sensitivity (55% Epo-r, 61% CD71) in detecting Y-specific sequences while immunocytochemical studies on four maternal blood samples, sorted after increasing the binding time of the ligand to Epo-r (8 h), showed a substantial improvement in fetal cell recovery and purity. We conclude that sorting by Epo-r/biotin-(sialyl)-erythropoietin provides effective enrichment of fetal nucleated red cells allowing the possibility of direct prenatal cytogenetic analysis by multiprobe fluorescent in-situ hybridization (FISH).      

Noninvasive prenatal diagnosis of chromosomal aneuploidies by isolation and analysis of fetal cells from maternal blood

The isolation and analysis of nucleated fetal cells (NFCs) from maternal blood may represent a new approach to noninvasive prenatal diagnosis. Although promising, these techniques require highly accurate separation of NFCs from nucleated cells of maternal origin; the two major problems limiting these techniques are the relative rarity of fetal cells in maternal blood and the need to establish their fetal origin. We now report a novel procedure that has allowed accurate separation of NFCs from maternal cells. The technique reported involves direct micromanipulator isolation of histochemically identified hemoglobin F‐positive nucleated cells to obtain fetal nucleated red blood cells (FNRBCs) of high yield and purity. Using this technique, followed by cell‐by‐cell multicolor fluorescence in situ hybridization (FISH) analysis of purified FNRBCs, we were able to detect some of the most common human aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in 33 pregnant women referred for amniocentesis. The procedure used, which can be completed in <72 hrs, produced complete concordance with the results of amniocentesis. We also confirm findings of prior studies suggesting that the number of FNRBCs in maternal circulation is remarkably higher in abnormal pregnancies than in normal pregnancies, especially in women carrying a fetus with trisomy 21. © 2001 Wiley‐Liss, Inc.     

Detection of fetal erythroid cells from maternal blood using fluorescence in situ hybridization and liquid culture

Fetal nucleated erythrocytes circulating in maternal blood are a potential source of fetal DNA for noninvasive prenatal genetic diagnosis. However, the estimated ratio of fetal to maternal cells is extremely small. In order to enrich these cells, we performed direct culture using a two-phase liquid system. Mononuclear cells were obtained from maternal blood samples at 8-10(+3) weeks of gestation and cultured in the first phase. After 4-5 days, the nonadherent cells were harvested and recultured with erythropoietin in the second phase for another 3-5 days. We examined cellular morphology, and counted the number of benzidine- positive cells and the percentage of glycophorin A/CD71 positive erythroid cells. We also did Kleihauer-Betke stain for Hb F, polymerase chain reaction (PCR) for SRY/DYZ1, chromosome analysis, and fluorescence in situ hybridization (FISH). The number of total erythroid cells reached about 0.1 x 10(6)-1.0 x 10(6)/mL with a purity of 84.0-97.3%. Hb F stain showed total erythroid cells of approximately 0.4 x 10(4)-9.8 x 10(4)/mL. Male DNA was detected in one case by PCR. In this case, the XY karyotype was confirmed by FISH and amniocentesis. This approach provides enriched source of fetal cells for further prenatal genetic analysis without complicated separation or sorting procedures.     

Noninvasive prenatal diagnosis of chromosomal aneuploidies by isolation and analysis of fetal cells from maternal blood.

The isolation and analysis of nucleated fetal cells (NFCs) from maternal blood may represent a new approach to noninvasive prenatal diagnosis. Although promising, these techniques require highly accurate separation of NFCs from nucleated cells of maternal origin; the two major problems limiting these techniques are the relative rarity of fetal cells in maternal blood and the need to establish their fetal origin. We now report a novel procedure that has allowed accurate separation of NFCs from maternal cells. The technique reported involves direct micromanipulator isolation of histochemically identified hemoglobin F-positive nucleated cells to obtain fetal nucleated red blood cells (FNRBCs) of high yield and purity. Using this technique, followed by cell-by-cell multicolor fluorescence in situ hybridization (FISH) analysis of purified FNRBCs, we were able to detect some of the most common human aneuploidies (including Down syndrome, Klinefelter syndrome, and trisomy 13) in 33 pregnant women referred for amniocentesis. The procedure used, which can be completed in <72 hrs, produced complete concordance with the results of amniocentesis. We also confirm findings of prior studies suggesting that the number of FNRBCs in maternal circulation is remarkably higher in abnormal pregnancies than in normal pregnancies, especially in women carrying a fetus with trisomy 21.     

用荧光原位杂交从母血中检测胎儿细胞

目的 从母血中分离胎儿细胞并确定其来自胎儿。方法 从孕早、中期各２０名、分娩后１５名母血中富集并分离有核细胞。用Ｙ特异性探针（ＰＹ３．４）行荧光原位杂交,从中识别胎儿细胞。结果 孕早、中期孕妇各怀１５名男胎。阳性细胞比例是１：６５２８．０及１：２７３．８。与同期１０名女胎阳性细胞相比,差别有高度显著性。分娩１周内的３名,阳必民孕中期的差别没有显著性。分娩３个月后的阳性率与比,差别有高度显著性。分娩     

Strategies for automated fetal cell screening

Studies to date have demonstrated fetal sex determination and aneuploidy detection from maternal blood, but a clinical screening technique has not yet emerged. A key limiting factor is the small number of fetal cells, which makes detection specificity and reliability critical. Visual inspection of unsorted or sorted fetal cells is laborious, and cells can be easily missed. Moreover, it is impractical to examine manually all the separated cells. It is highly likely that automation may increase the number of cells inspected, resulting in higher detection sensitivities. Flow and image cytometry are two feasible approaches for automated detection of cells. This review details computerized microscopy (image cytometry) techniques for the automatic detection of fetal cells. Microscopy-based approaches used to identify fetal origin include: (i) immunocytochemical identification of fetal haemoglobin-specific cells (light or fluorescence microscopy); (ii) identification of sex chromosomes and/or aneuploidy using fluorescence in-situ hybridization; and (iii) morphological identification of nucleated red blood cells using light microscopy. The relevant instrumentation, including motorized stages and filters, cameras and digitizer boards are discussed, and software algorithms, including image enhancement, autofocusing, object detection and relocation, and features for operator review and data analysis, are outlined.     

Intact fetal cell isolation from maternal blood: improved isolation using a simple whole blood progenitor cell enrichment approach (RosetteSep)

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.     

Prenatal genetic diagnosis from maternal blood: simultaneous immunophenotyping and FISH of fetal nucleated erythrocytes isolated by negative and positive magnetic activated cell sorting

Fetal nucleated red blood cells (nRBCs) are rare in maternal circulation, but their presence constitutes a potential source of non-invasive prenatal genetic diagnosis. This study was undertaken to establish a non-invasive prenatal genetic diagnosis method using isolated fetal nRBCs. A multi-step method including triple density gradient and magnetic activated cell sorting (MACS) using CD45 and CD71, cytospin centrifugation, K-B staining, and glycophorin A-immuno fluorescence in situ hybridization (GPA-immuno FISH) was performed. The study population included 65 patients from 8 to 41 weeks of gestation, and fetal nRBC was separated from all cases. The number of fetal nRBCs retrieved was 12.8 +/- 2.7 in 8 to 11 gestational weeks, 15.2 +/- 6.5 in 12 to 18 gestational weeks, 16.4 +/- 6.5 in 19 to 23 gestational weeks, 10.6 +/- 3.2 in 24 to 28 gestational weeks, and 5.5 +/- 1.9 in 35 to 41 gestational weeks: the mean number of nRBCs collected from 20 ml of maternal peripheral blood was 13.7 +/- 6.2. The highest value of yield was 45.6% from 12 to 18 weeks gestation. The fetal sex determination confirmed by amniocentesis or chorionic villus sampling showed 100% sensitivity and 91.7% specificity for males; 91.7% sensitivity and 100% specificity for females. We showed that fetal cells can be reliably enriched from maternal blood and that they can be used for detecting specific chromosomes by FISH with a specificity superior to current non-invasive methods.     

Hybridoma Monoclonal Antibodies to Human Fetal Haemoglobin

Monoclonal antibodies against human fetal haemoglobin (HbF) were obtained by fusion of mouse myeloma cells with spleen lymphocytes from mice immunized against HbF from human umbilical cord blood erythrocytes. Initial antibody screening was by indirect immunofluorescence assays on smeared cord blood and normal adult blood erythrocytes. The specificity of these antibodies was further studied by combined immunoprecipitation assays and direct or inhibition solid-phase enzyme immunoassays. The combined pattern enabled recognition of three types of antigenic determinants: (i) common to both HbF and HbA, by both immunofluorescence and enzyme immunoassays; (ii) common to both HbF and HbA by enzyme immunoassays, present on fixed cord blood erythrocytes and adult thalassaemic erythrocytes by immunofluorescence, but not found on fixed normal adult erythrocytes; and (iii) private HbF by both immunofluorescence and enzyme immunoassays.     

Quantification of all fetal nucleated cells in maternal blood between the 18th and 22nd weeks of pregnancy using molecular cytogenetic techniques

Different types of nucleated fetal cells (trophoblasts, erythroblasts, lymphocytes, and granulocytes) have been recovered in maternal peripheral blood. In spite of many attempts to estimate the number of fetal cells in maternal circulation, there is still much controversy concerning this aspect. The numbers obtained vary widely, ranging from 1 nucleated cell per 104 to 1 per 109 nucleated maternal cells. The purpose of our project was to determine the absolute number of all different types of male fetal nucleated cells per unit volume of peripheral maternal blood. Peripheral blood samples were obtained from 12 normal pregnant women known to carry a male fetus between 18 and 22 weeks of pregnancy. Three milliliters (3 ml) of maternal blood has been processed without any enrichment procedures. Fluorescence in situ hybridization (FISH) and primed in situ labeling (PRINS) were performed, and fetal XY cells were identified (among maternal XX cells) and scored by fluorescent microscopy screening. The total number of male fetal nucleated cells per milliliter of maternal blood was consistent in each woman studied and varied from 2 to 6 cells per milliliter within the group of normal pregnancies. The number of fetal cells in maternal blood, at a given period, is reproducible and can therefore be assessed by cytogenetic methods. This confirms the possibility of developing a non-invasive prenatal diagnosis test for aneuploidies. Furthermore, we demonstrate that it is possible to repeatedly identify an extremely small number of fetal cells among millions of maternal cells.     

Involvement of Hofbauer cells and maternal T cells in villitis of unknown aetiology

Aims:  The nature of villitis of unknown aetiology (VUE) is intriguing in terms of its aetiology, origin of inflammatory cells and immunophenotype of T cells involved. The aim was to determine the origin of macrophages and the immunophenotype of T lymphocytes in VUE associated with various complications of pregnancy.
Methods and results:  Placentas with VUE ( n  = 45) were studied by chromogenic in-situ hybridization (CISH) for Y chromosome (DYZ1) and immunohistochemistry for CD14, CD68, Ki67 ( n  = 10; all from male neonates) and a panel of T-cell antigens (CD3, CD4 and CD8) ( n  = 35). All of the placentas from male neonates showed CISH+ signals from Y chromosomes in the majority of macrophages, but not in lymphocytes, indicating that the macrophages were of fetal origin. Many macrophages of the affected chorionic villi were Ki67+, suggesting that they are hyperplastic Hofbauer cells. Among the lymphocytes, CD8+ T cells outnumbered CD4+ T cells in all placentas with different obstetrical conditions.
Conclusions:  We define primary components of VUE as maternal CD8+ T cells and hyperplastic Hofbauer cells. We propose that VUE is a unique inflammatory reaction where the leucocytes from two hosts are key partners, analogous to either allograft rejection or graft-versus-host disease.     

New trends in non-invasive prenatal diagnosis: applications of dielectrophoresis-based Lab-on-a-chip platforms to the identification and manipulation of rare cells

The isolation of rare cells, such as fetal nucleated red blood cells and trophoblasts, from maternal blood for non-invasive prenatal diagnosis is a new field of research exhibiting several difficulties since this strategy requires unresolved basic technological protocols for a successful outcome. However, several achievements in the field of Laboratory-on-a-chip (Lab-on-a-chip) technology have provided clear advancements in projects aimed at the isolation of rare cells from biological fluids. Among the most interesting approaches are those based on dielectrophoresis (DEP). DEP-based Lab-on-a-chip platforms have been demonstrated to be suitable for several applications in biotechnology and biomedicine. DEP-based arrays are able to manipulate single cells, which can be identified and moved throughout the DEP chip to recovery places. DEP buffers are compatible with molecular interactions between monoclonal antibodies and target cells, allowing integration of these devices with magnetic cell sorting (MACS). DEP treatment does not alter the viability of manipulated cells.     

Identification of fetal mesenchymal stem cells in maternal blood: implications for non-invasive prenatal diagnosis

Strategies for genetic prenatal diagnosis on fetal cells in the maternal circulation have been limited by lack of a cell type present only in fetal blood. However, the recent identification of mesenchymal stem cells (MSC) in first trimester fetal blood offers the prospect of targeting MSC for non-invasive prenatal diagnosis. We developed protocols for fetal MSC enrichment from maternal blood and determined sensitivity and specificity in mixing experiments of male fetal MSC added to female blood, in dilutions from 1 in 10(5) to 10(8). We then used the optimal protocol to isolate fetal MSC from maternal blood in the first trimester, using blood taken after surgical termination of pregnancy as a model of increased feto-maternal haemorrhage. In model mixtures, we could amplify one male fetal MSC in 2.5 x 10(7) adult female nucleated cells, yielding a 100% pure population of fetal cells, but not one fetal MSC in 10(8) nucleated cells. Fetal MSC were identified in one of 20 post-termination maternal blood samples and confirmed as fetal MSC by XY fluorescence in-situ hybridization (FISH), immunophenotyping and osteogenic and adipogenic differentiation. We report the isolation of fetal MSC from maternal blood; however, their rarity in post-termination blood suggests they are unlikely to have a role in non-invasive prenatal diagnosis. Failure to locate these cells routinely may be attributed to their low frequency in maternal blood, to sensitivity limitations of enrichment technology, and/or to their engraftment in maternal tissues soon after transplacental passage. We speculate that gender microchimerism in post-reproductive maternal tissues might result from feto-maternal trafficking of MSC in early pregnancy.     

Fetal cells in transcervical samples at an early stage of gestation

Several investigations are in progress with the aim of performing prenatal diagnosis of inherited disorders by noninvasive or minimally invasive techniques. The most important approaches are based on the detection of fetal nucleated cells in maternal blood, the analysis of fetal DNA present in maternal plasma, and the identification and isolation of fetal trophoblastic cellular elements shed into the uterine cavity and the endocervical canal. In this review, we discuss the methods that have been employed for the collection of the transcervical samples at an early stage of gestation and the techniques used for the identification of fetal cells. We also report the results of using endocervical cells for the detection of fetal chromosomal disorders by fluorescent in-situ hybridization and for performing prenatal diagnosis of fetal Rh(D) phenotypes. Recent investigations have also shown that — after the isolation of trophoblastic cells from maternal contaminants by micromanipulation — transcervical samples can be employed for the prenatal diagnosis of single gene defects, such as those causing thalassemia and sickle cell anemia. Although the present results are promising, further investigations are required to demonstrate the feasibility of performing accurate diagnosis of fetal diseases by this minimally invasive approach in all transcervical samples retrieved at an early stage of gestation. Received: November 7, 2000 / Accepted: November 27, 2000