Comparison of methods for erythroblast selection: application to selecting fetal erythroblasts from maternal blood.

BACKGROUND: Many methods have been employed to obtain fetal cells from maternal blood for prenatal diagnostics, but there has been little work done that compares the efficacy of different methods. This study presents a comparison of two commonly used methods for selecting erythroblasts with selection directly from whole blood. METHODS: Erythroblasts were isolated from maternal blood by either differential lysis or density separation, followed by selection with an antibody to the transferrin receptor. These methods were compared with antibody selection directly from whole blood. The total yield of erythroblasts was determined for each method. RESULTS: Red cell lysis is not recommended because the lysis step cannot be well controlled. Density separation followed by antibody selection works well. However, a faster and simpler method, antibody selection directly from whole blood using Immunicon Ferrofluid and magnetic separators, works as well and has the potential to yield even more cells. CONCLUSIONS: Considering the need for a simple and quick method for selecting fetal cells from maternal blood, we suggest selection directly from whole blood.     

The use of non-physiological conditions to isolate fetal cells from maternal blood

Fetal cells are always present in maternal blood starting in the first trimester of pregnancy, however a rapid, simple, and consistent procedure for their isolation for prenatal non-invasive genetic investigation is still lacking. Sensitivity and recovery of fetal cells is jeopardized by the minute amount of circulating fetal cells and their loss during the enrichment procedure. We report here a single-step approach to isolate fetal cells from maternal blood which relies on the use of non-physiological conditions to modify cell densities before their separation in a density gradient and in a newly developed cell separation device. Isolated fetal cells have been investigated using cytochemistry, Soret band absorption microscopy, monoclonal antibodies for epsilon- and gamma-chain-Hb, monoclonal antibody for i-antigen, and by fluorescence in situ hybridization (FISH). Fetal cells were always detected in all 105 maternal blood samples investigated and fetal aneuploidies were correctly diagnosed by FISH, in a pilot study of pathological pregnancies, in fetal cells isolated from maternal blood obtained either before or after invasive procedure.     

Fetal nucleated erythrocyte recovery: fluorescence activated cell sorting-based positive selection using anti-gamma globin versus magnetic activated cell sorting using anti-CD45 depletion and anti-gamma globin positive selection

BACKGROUND: Fluorescence activated cell sorting (FACS)-based anti-gamma (gamma) positive selection and magnetic activated cell sorting (MACS)-based anti-CD45 depletion followed by anti-gamma 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-gamma 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.     

Measurements of the dielectric properties of peripheral blood mononuclear cells and trophoblast cells using AC electrokinetic techniques

The separation of trophoblast cells from the maternal circulation could provide a valuable diagnostic tool for prenatal diagnosis of genetic abnormalities. This has been attempted using antibody methods, but due to non-specificity of the antibodies, maternal cell contamination remains a problem. We have investigated the potential of dielectrophoretic separation methods as a means of isolating trophoblast cells from mixed peripheral blood mononuclear cells. To determine the potential of this method the dielectric properties of trophoblast cells and mixed peripheral blood mononuclear cells were measured using dielectrophoretic crossover and single cell electrorotation methods. Both dielectrophoretic crossover data and electrorotation data gave an average specific membrane capacitance of the peripheral blood mononuclear cells of 11.5 mF m(-2). Trophoblast cells prepared using three different methods had a higher average specific membrane capacitance in the range 13-18 mF m(-2). The differences in capacitance between the cell types could be exploited as the basis of an AC electrokinetic-based system for the separation of trophoblast cells from peripheral blood mononuclear cells.     

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 semiautomated scanning microscopy to search for candidate cells. We conclude that erythroblasts form only a small fraction of fetal cells present in maternal blood.     

Enrichment of fetal trophoblasts and nucleated erythrocytes from maternal blood by an immunomagnetic colloid system

The aim of this study was to isolate fetal trophoblasts and nucleated erythrocytes from maternal blood using the immunomagnetic colloid system. About 25 ml of maternal blood was collected from pregnant women between of 14 and 20 weeks gestation. Nucleated erythrocytes (NRBCs) were isolated from 5 ml of maternal blood and a nested polymerase chain reaction for the Y chromosome was used to determine fetal origin. The sensitivity of the fetal gender diagnosis was 80% and the specificity was 86%. Both fetal trophoblasts and NRBCs were isolated from the remaining 20 ml of maternal blood. The fetal gender of the trophoblast-enriched fraction was determined using fluorescence in situ hybridisation (FISH) with dual-colour XY-specific DNA probes. XY-specific signals were observed in 0.38% of cells sorted from all pregnant women carrying male fetuses (n = 10). Simultaneous immunophenotyping for the fetal haemoglobin and FISH using XY probes were used to evaluate the fetal origin of cells enriched with anti-CD71. The mean percentage of male fetal erythroblasts was 0.24% and the number of fetal erythroblasts was estimated to be about 672 in 20 ml of maternal blood. The number of fetal erythroblasts detected in our study was greater than that detected by most other separation techniques. Our study shows that it would be feasible to use the immunomagnetic colloid system for the isolation of both trophoblasts and NRBCs from the same maternal blood sample with relatively good efficiency. Received: 17 December 1998 / Accepted: 9 February 1999     

Fetal DNA in maternal serum: does it persist after pregnancy?

Fetal DNA and cells present in maternal blood have previously been used for non-invasive prenatal diagnosis. However, some fetal cells can persist in maternal blood after a previous pregnancy. Fetal rhesus status and sex determination have been performed by using amplification by real-time polymerase chain reaction (PCR) of fetal DNA sequences present in maternal circulation; no false-positive results related to persistent fetal DNA from a previous pregnancy have been reported. This idea has recently been challenged. An SRY real-time PCR assay was performed on the serum of 67 pregnant women carrying a female fetus but having previously given birth to at least one boy and on the serum of 30 healthy non-pregnant women with a past male pregnancy. In all cases, serum was negative for the SRY gene. These data suggest that fetal DNA from a previous pregnancy cannot be detected in maternal serum, even by using a highly sensitive technique. Therefore, non-invasive prenatal diagnosis by fetal sex determination for women at risk of producing children with X-linked disorders, and fetal RHD genotyping is reliable and secure as previously demonstrated.     

Presence of fetal DNA in maternal plasma decades after pregnancy

Cells of fetal origin and cell-free fetal DNA can be detected in the maternal circulation during pregnancy, and it has recently been shown that fetal cells can persist long after delivery. Given the various biological and clinical implications of this fact, we tested the hypothesis that cell-free fetal DNA can be present in maternal plasma decades after pregnancy. We extracted DNA from plasma samples and nucleated blood cells of 160 healthy women with male offspring at different time intervals after delivery (range 1-60 years). All of the samples were tested by means of a real-time quantitative PCR assay for a specific Y chromosome sequence (the SRY gene). Y chromosome-specific DNA was detected in 16 peripheral blood cell samples (10%) and 35 plasma samples (22%). The women with male sequences in the cell fraction had significantly greater total parity ( P=0.018). The proportion of women with detectable Y sequences in the plasma or cell samples was not related to the time since delivery. The fetal DNA concentrations in the genomic material extracted from plasma samples were significantly higher than those extracted from the Y-positive cell samples (149+/-140 vs 20+/-13 genome-equivalents/ml; P<0.001). There was no relationship between the concentration of fetal DNA and the time since delivery. Not only fetal cells, but also fragments of fetal DNA can be present in the maternal circulation indefinitely after pregnancy. This finding has practical implications for non-invasive prenatal diagnoses based on maternal blood, and may be considered for possible pathophysiological correlations.     

Method for isolation of floating fetal RBCs from maternal circulation based on their inherent magnetic property

Isolation of floating fetal cells from maternal circulation has immense potential in diagnosing of various genetic alterations in the developing fetus. Currently, non-invasive fetal cell isolation methods include fluorescence/magnetic activated cell sorting that use antibodies specific to fetal cells. Apart from being complex and expensive the biggest challenge associated with these cell sorting methods is low concentration of fetal cells in maternal peripheral blood. In order to make the complete process much simpler and effective, we propose a novel method for isolation of floating fetal cells that uses a continuous flow of maternal blood for effectively harvesting a higher fetal cell volume compared to any other existing method. The isolation mechanism is based on the difference in the magnetic susceptibility of fetal hemoglobin (HbF-α₂γ₂) and maternal hemoglobin (HbA-α₂β₂). HbF has high oxygen saturation capacity (diamagnetic) compared to HbA (paramagnetic), and this difference in saturation is further enhanced by presence of 2,3-bisphosphoglycerate (2,3-BPG). When placed in magnetic field, these cells get separated based on the difference (p ≤ 0.001) in their magnetophoretic mobility. This separation method may also be used for detection of fetomaternal hemorrhages and also treatment of Rh incompatibility.     

Prenatal diagnosis: progress through plasma nucleic acids

Over the past 40 years, much effort has been spent on developing non-invasive prenatal diagnostic methods. Since 1997, the progress of this field has been accelerated by the unexpected finding of extracellular fetal nucleic acids in maternal plasma. These developments have been translated into many novel genetic, epigenetic and gene-expression markers, and are expected to have a fundamental impact on the future practice of prenatal diagnosis.     

母体外周血中胎儿有核红细胞的分离和富集方法的研究进展

母体外周血中分离的胎儿有核红细胞（fNRBCs）包含胎儿完整的遗传信息,可用于无创产前诊断。fNRBCs的分离和富集方法主要分为三类：物理分选法、抗原-抗体结合分离法和增殖法。不同的方法获得的fNRBCs的数量和纯度不同,多种方法联合使用可以提高富集产物中fNRBCs的纯度和数量。本文就母体外周血中fNRBCs的分离和富集方法进行综述。     

Human fetal mesenchymal stem cells

Stem cells have been isolated at all stages of development from the early developing embryo to the post-reproductive adult organism. However, the fetal environment is unique as it is the only time in ontogeny that there is migration of stem cells in large numbers into different organ compartments. While fetal neural and haemopoietic stem cells (HSC) have been well characterised, only recently have mesenchymal stem cells from the human fetus been isolated and evaluated. Our group have characterised in human fetal blood, liver and bone marrow a population of non-haemopoietic, non-endothelial cells with an immunophenotype similar to adult bone marrow-derived mesenchymal stem cells (MSC). These cells, human fetal mesenchymal stem cells (hfMSC), are true multipotent stem cells with greater self-renewal and differentiation capacity than their adult counterparts. They circulate in first trimester fetal blood and have been found to traffic into the maternal circulation, engrafting in bone marrow, where they remain microchimeric for decades after pregnancy. Though fetal microchimerism has been implicated in the pathogenesis of autoimmune disease, the biological role of hfMSC microchimerism is unknown. Potential downstream applications of hfMSC include their use as a target cell for non-invasive pre-natal diagnosis from maternal blood, and for fetal cellular and gene therapy. Using hfMSC in fetal therapy offers the theoretical advantages of avoidance of immune rejection, increased engraftment, and treatment before disease pathology sets in. Aside from allogeneic hfMSC in utero transplantation, the use of autologous hfMSC has been brought a step forward with the development of early blood sampling techniques, efficient viral transduction and clonal expansion. Work is ongoing to determine hfMSC fate post-transplantation in murine models of genetic disease. In this review we will examine what is known about hfMSC biology, as well as discussing areas for future research. The implications of hfMSC trafficking in pregnancy will be explored and the potential clinical applications of hfMSC in prenatal diagnosis and fetal therapy discussed.     

Search for the optimal fetal cell antibody: results of immunophenotyping studies using flow cytometry

Fetal nucleated cells circulating in maternal peripheral blood are a noninvasive source of fetal DNA for prenatal genetic diagnoses. The successful isolation of fetal cells from maternal blood depends upon identification of differences between fetal and maternal cell surface antigen expression. To our best knowledge, a monoclonal antibody that binds only fetal blood cells has not yet been identified. We studied antigens recognized by six different monoclonal antibodies for their biologic expression on fetal blood cells as a function of gestational age, and compared their ability to bind fetal but not maternal cells. The results suggest a relationship between gestational age and nucleated cell surface antigen expression. The monoclonal antibodies FB3-2, H3-3, CD71 and 2-6B/6 are suitable reagents for first or early second trimester fetal cell isolation, although FB3-2 and H3-3 are more specific for fetal cells due to significantly lower expression of these antigens on maternal mononuclear cells. The observation that samples from fetuses with chromosome abnormalities or multiple structural anomalies express higher levels of these antigens indicates that these reagents will potentiate the detection of abnormal fetal cells in maternal blood samples. Received: 23 November 1996 / Accepted: 13 February 1997     

A monoclonal antibody with potential for aiding non-invasive prenatal diagnosis: utility in screening of pregnant women at risk of preeclampsia.

The development of a non-invasive method of prenatal diagnosis in maternal blood has been the goal of our investigations during the last years. We have developed several anti-CD71 monoclonal antibodies and optimized a protocol for the isolation of nucleated red blood cells (NRBC) from peripheral maternal blood. The enhanced traffic of fetal erythroblasts into the maternal circulation in preeclampsia has been investigated by several groups. In this study, we compared one of our antibodies, 2F6.3, with a commercial anti-CD71 antibody in blood samples from pregnant women suffering pregnancy-induced hypertension (PIH) and in a control group of pregnant women without clinical features suggestive of an increased risk of developing preeclampsia. The mAb 2F6.3, developed by our group, has succeeded in isolating a significantly higher number of erythroblasts with less maternal cell contamination than the commercial antibody in both women with PIH and in the control group (p<0.01; Wilcoxon Signed Ranks Test). Fluorescence in situ hybridization analysis also demonstrated that 2F6.3 is a better antibody for the isolation of fetal NRBC in maternal blood than the commercial anti-CD71 antibody.     

Cellular origin and induction of pregnancy-associated alpha 2-glycoprotein synthesis in pregnant rats

The synthesis of alpha 2-PAG was measured and compared in tissues and cells from normal non-pregnant females, and maternal and fetal rats in vitro to define the target cells hormonally regulated during pregnancy. Synthesis was measured by [L-14C]leucine incorporation into immunochemically isolated alpha 2-PAG and confirmed by radioimmunodiffusion. alpha 2-PAG synthesis was demonstrated in maternal peripheral blood leucocytes, placenta, breast, spleen, liver and fetal peripheral blood leucocytes and liver. Maternal and fetal liver were the most active tissue producers and fetal liver synthesized 4 times more alpha 2-PAG than did maternal liver. Furthermore, fetal peripheral blood leucocytes synthesized 2 times more alpha 2-PAG per cell than did these same maternal cells. A direct comparison of synthesis by cells from pregnant and non-pregnant female rats revealed that (1) maternal peripheral blood leucocytes synthesized 5 times more alpha 2-PAG per cell than did normal leucocytes, although these same cells synthesized approximately equal amounts of total cell protein per cell, (2) maternal peritoneal exudate macrophages also synthesized 5 times more alpha 2-PAG per cell than did macrophages obtained from normal female rats, and total protein synthesis by these cells also closely paralleled each other, (3) maternal and fetal plastic-adherent peripheral blood monocytes synthesized 22 and 58 times more alpha 2-PAG per cell respectively than did normal monocytes, (4) maternal and fetal non-adherent lymphocytes synthesized 8 and 16 times more alpha 2-PAG per cell respectively than did normal lymphocytes and (5) fetal monocytes and lymphocytes synthesized 3 and 2 times more alpha 2-PAG per cell than did maternal monocytes and lymphocytes.(ABSTRACT TRUNCATED AT 250 WORDS)     

hESCs/hiPSCs体外诱导产生红细胞的研究进展及其临床应用的展望

人类胚胎干细胞和多功能诱导性干细胞的诞生,标志着干细胞研究已经跨入了全新的应用时代。干细胞研究领域的一个重要方向是特定谱系成熟细胞的定向诱导分化。在诸多的血细胞中,成熟红细胞因为无核而携带着最小量的遗传物质,可能作为最早的干细胞治疗产品而应用于输血替代治疗。最近,干细胞向造血细胞（包括红细胞）的研究正方兴未艾。但由于方法学上的偏差,诱导产生的红细胞的成熟度各有所不同。该文在结合了作者实验室的工作经验的基础上,对目前人类多潜能干细胞向红细胞特定诱导分化的方法做了综合的描述,并提出了该研究领域亟需解决的重大科学问题。     

Winnowing DNA for rare sequences: highly specific sequence and methylation based enrichment

Rare mutations in cell populations are known to be hallmarks of many diseases and cancers. Similarly, differential DNA methylation patterns arise in rare cell populations with diagnostic potential such as fetal cells circulating in maternal blood. Unfortunately, the frequency of alleles with diagnostic potential, relative to wild-type background sequence, is often well below the frequency of errors in currently available methods for sequence analysis, including very high throughput DNA sequencing. We demonstrate a DNA preparation and purification method that through non-linear electrophoretic separation in media containing oligonucleotide probes, achieves 10,000 fold enrichment of target DNA with single nucleotide specificity, and 100 fold enrichment of unmodified methylated DNA differing from the background by the methylation of a single cytosine residue.     

Unique monoclonal antibodies specifically bind surface structures on human fetal erythroid blood cells

Background

Continuing efforts in development of non-invasive prenatal genetic tests have focused on the isolation of fetal nucleated red blood cells (NRBCs) from maternal blood for decades. Because no fetal cell-specific antibody has been described so far, the present study focused on the development of monoclonal antibodies (mAbs) to antigens that are expressed exclusively on fetal NRBCs.Methods: Mice were immunized with fetal erythroid cell membranes and hybridomas screened for Abs using a multi-parameter fluorescence-activated cell sorting (FACS). Selected mAbs were evaluated by comparative FACS analysis involving Abs known to bind erythroid cell surface markers (CD71, CD36, CD34), antigen-i, galactose, or glycophorin-A (GPA). Specificity was further confirmed by extensive immunohistological and immunocytological analyses of NRBCs from umbilical cord blood and fetal and adult cells from liver, bone marrow, peripheral blood, and lymphoid tissues.Results: Screening of 690 hybridomas yielded three clones of which Abs from 4B8 and 4B9 clones demonstrated the desired specificity for a novel antigenic structure expressed on fetal erythroblast cell membranes. The antigenic structure identified is different from known surface markers (CD36, CD71, GPA, antigen-i, and galactose), and is not present on circulating adult erythroid cells, except for occasional detectability in adult bone marrow cells.Conclusions:The new mAbs specifically bind the same or highly overlapping epitopes of a surface antigen that is almost exclusively expressed on fetal erythroid cells. The high specificity of the mAbs should facilitate development of simple methods for reliable isolation of fetal NRBCs and their use in non-invasive prenatal diagnosis of fetal genetic status.     

Fetal erythroblast isolation up to purity from cord blood and their culture in vitro

BACKGROUND: Erythroblasts have been the most encouraging candidate cell type for noninvasive prenatal genetic investigation. We previously showed that human erythroblasts can be recovered from bone marrow and blood bank buffy coats by a physical cell separation. In the present study, we modified our previous methodology, taking into account the peculiar behavior of erythroblasts in response to modifications of pH and osmolality of the separation medium. METHODS: Twenty to forty milliters of cord blood were initially centrifuged on Ficoll/diatrizoate (1.085 g/ml). The interphase cells were further separated on a continuous density gradient (1.040-1.085 g/ml). Two different gradients were initially compared: the first was iso-osmolar and neutral, whereas the second also contained an ionic strength gradient and a pH gradient (triple gradient). A subsequent monocyte depletion was performed by using magnetic microbeads coated with anti-CD14 monoclonal antibody (mAb), and erythroblasts were purified by sedimentation velocity. Purified cells were investigated by analyses with fluorescence-activated cell sorting (FACS) and fluorescence in situ hybridization (FISH) and immunocytochemistry with mAb against fetal hemoglobin and were cultured in vitro. RESULTS: When nucleated cells were spun on an iso-osmolar and neutral continuous density gradient, two separated bands of nucleated red blood cells (NRBCs) were obtained: a light fraction banding at 1.062 g/ml and an heavy fraction banding at 1.078 g/ml. Conversely, when cells were spun in the triple gradient, NRBCs were shifted to the low-density region. Monocyte depletion by immunomagnetic microbeads and velocity sedimentation provided a pure erythroblast population. FACS and FISH analyses and immunocytochemistry substantiated the purity of the isolated cell fraction, which was successfully cultured in vitro. CONCLUSIONS: We have shown that fetal erythroblasts can be purified up to homogeneity from cord blood, but further refinements of the isolation procedure are necessary before the same results can be obtained from maternal peripheral blood.