Demonstration of spontaneously dividing male fetal cells in maternal blood by negative magnetic cell sorting and fish

We investigated a case of massive feto-maternal bleeding by using negative magnetic cell sorting (MACS) and fluorescent in situ hybridization (FISH). A 37-year-old pregnant woman had an uncomplicated amniocentesis for advanced maternal age at 16 weeks' gestation. The fetal karyotype was 46, XY. At 19 weeks' gestation, she had a minor car accident and slight vaginal bleeding. A subsequent Kleihauer-Betke test showed a 140 ml feto-maternal haemorrhage. Serial sonographic examinations indicated a normal fetus and placenta. We performed FISH analysis on maternal peripheral blood at 25 weeks. Anti-CD45 and MACS were used to deplete maternal leucocytes, enriching the proportion of fetal nucleated erythrocytes present. The isolated cells were analysed by using dual-colour FISH with X and Y specific probes. Approximately 65 800 nucleated cells were obtained after MACS depletion. A total of 234 cells were analysed by FISH. The results revealed that 70 of the nucleated cells (30 per cent) were male with one X and one Y signal. Among these cells, six male metaphases were observed in spontaneously dividing cells.     

Chorionic villus metaphase chromosomes and interphase nuclei analysed by chromosomal in situ suppression (CISS) hybridization

Metaphase chromosomes and interphase nuclei of chorionic villus samples (CVS) in five cases were studied after treatment with trypsin and post-fixation in formaldehyde by chromosomal in situ suppression (CISS) hybridization. Our modified protocol enables the use of in situ hybridization. techniques on CVS preparations after 42 h ofculture. A balanced translocation and trisomy 13 were identified with the aid of CISS hybridization.     

Fluorescent in situ hybridization (FISH): A new application in the delineation of true vs. pseudomosaicism in prenatal diagnosis

Metaphase chromosomes and interphase nuclei from nine amniotic fluid cultures were studied with fluorescence in situ hybridization (FISH). The samples were initially analyzed with routine G-banding and were diagnosed as having true mosaicism (five patients) or pseudomosaicism (four patients). In our study, FISH analysis could provide additional information to distinguish pseudo– from true mosaicism by allowing interphase studies and analysis of an increased number of metaphase spreads. These results suggest a multilinear origin of ‘in situ’ colonies of cells.     

Role of amniotic fluid interphase fluorescence in situ hybridization (FISH) analysis in patient management

We retrospectively reviewed 309 amniotic fluid interphase fluorescence in situ hybridization (FISH) analyses performed from October 1995 to June 1999 to assess the role of interphase FISH in the management of patients at increased risk for fetal aneuploidies. Gestational age and indications for amniocentesis, clinical interventions after FISH results, as well as interventions after final culture reports were analyzed. There were 244 (79%) normal, 50 (16%) abnormal and 15 (5%) inconclusive FISH results. There were no false-positive or false-negative results, but there were nine (3%) clinically significant chromosomal abnormalities not detectable by FISH. Of the 50 women with abnormal FISH results, 26 (52%) elected to terminate the pregnancy prior to the availability of the standard chromosome analysis. In two of the fetuses with trisomy 21 no abnormalities were reported by ultrasound examination. Our experience indicates that interphase FISH results played an important role in decision making, especially for pregnancies close to 24 weeks' gestation. Standard karyotype analysis is still required for detection of chromosome abnormalities not detectable by interphase FISH techniques and for clarification of unusual or inconclusive FISH results. Copyright © 2001 John Wiley & Sons, Ltd.     

Detection of Phaeocystis globosa using sandwich hybridization integrated with nuclease protection assay (NPA-SH)

Phaeocystis globosa Scherffel is one of the common harmful algae species in coastal waters of the southeastern China. In this study, sandwich hybridization integrated with nuclease protection assay (NPA-SH) was used to qualitatively and quantitatively detect P. globosa. Results showed that this method had good applicability and validity in analyzing the samples from laboratory cultures and from fields. The linear regression equation for P. globosa was obtained, and the lowest detection number of cells was 1.8 × 10⁴ cells. Statistics showed that there was no distinct difference between the results of detecting the microalgae by NPA-SH and traditional microscopy. This technique has good reliability, accuracy, and can give a remarkably high sample processing rate. Sandwich hybridization integrated with nuclease protection assay will provide an efficient alternative to microscopic method for monitoring and investigating the bloom of P. globosa.     

Fetal erythroblasts from maternal blood identified with 2,3-bisphosphoglycerate (BPG) and in situ hybridization (ISH) using Y-specific probes

Different types of fetal nucleated cells can be found in maternal blood, providing the possibility of non-invasive prenatal diagnosis. For this purpose, we have studied fetal erythroblasts. We discovered that haemoglobin-containing cells treated with 2,3-bisphosphoglycerate (BPG) can be visualized by a peroxidase reaction, which at the same time visualizes an in situ hybridization (ISH) signal, specific for the X, Y or 21 chromosome. In order to prove that the BPG-positive cells were erythroid, an anti-glycophorin A (GPA) antiserum combined with a staphylococcal rosette technique was used. To enrich for erythroblasts, leukocytes were depleted from maternal blood by treatment with anti-CD45 monoclonal antibody and passage over an anti-mouse IgG-coated glass bead column. To evaluate the potential of the method for clinical use, we studied maternal blood samples from 18 women referred to us for prenatal diagnosis between 6 and 19 weeks of gestation. Erythroblasts were found in 13 out of 14 normal pregnancies. Erythroblasts with a Y-signal were found as early as 9 weeks of gestation, but at 6 weeks the Y-signal was seen in BPG-negative cells only. These cells showed an epithelioid morphology indicating that they were cytotrophoblasts. The BPG-ISH method provides a simple technique for identifying erythroblasts and simultaneously visualizing a desired probe.     

Cytogenetic analysis of trophoblasts by comparative genomic hybridization in embryo-fetal development anomalies

Cytogenetic studies of spontaneous abortions or intrauterine fetal death depend on conventional tissue culturing and karyotyping. This technique has limitations such as culture failure and selective growth of maternal cells. Fluorescent in situ hybridization (FISH) using specific probes permits diagnosis of aneuploidies but is limited to one or a few chromosomal regions. Comparative genomic hybridization (CGH) provides an overview of chromosomal gains and losses in a single hybridization directly from DNA samples. In a prospective study, we analyzed by CGH trophoblast cells from 21 fetuses in cases of spontaneous abortions, intrauterine fetal death or polymalformed syndrome. Six numerical chromosomal abnormalities including one trisomy 7, one trisomy 10, three trisomies 18, one trisomy 21 and one monosomy X have been correctly identified by CGH. One structural abnormality of the long arm of chromosome 1 has been characterized by CGH. One triploidy and two balanced pericentromeric inversions of chromosome 9 have not been identified by CGH. Sexual chromosomal constitutions were concordant by both classical cytogenetic technique and CGH. Contribution of trophoblast analysis by CGH in embryo-fetal development anomalies is discussed. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal application of fluorescent in situ hybridization (FISH) for identification of a mosaic Y-chromosome marker,IDIC(Yp)

An amniocentesis was performed at 13.3 weeks' gestation for advanced maternal age. A mosaic sex chromosome pattern was found: of 50 cells examined, 34 had a 45,X karyotype. In 14 cells with a modal number of 46, a recognizable Y was substituted by a small non-fluorescent marker. C-banding identified the marker as an isodicentric in 12 cells. In two cells, the non-fluorescent marker appeared to be monocentric and looked like a non-fluorescent del (Yq), but could have been an isodicentric Y with inactivation of one of the centromeres. Two cells with a modal number of 47 showed two copies of the monocentric marker. Fluorescent in situ hybridization with an alpha satellite Y-specific centromeric probe confirmed the Y-chromosome origin of the markers and allowed for more accurate prenatal diagnostic information.     

Prenatal detection of structural abnormalities of chromosome 18: associations with interphase fluorescence in situ hybridization (FISH) and maternal serum screening

We describe two cases of prenatally ascertained isochromosome 18. Case 1 included both an isochromosome 18p and an isochromosome 18q, while Case 2 involved only an isochromosome 18q. Both of these cases were associated with a positive maternal serum triple screen trisomy 18 risk (greater than 1 in 100 risk). In addition, fluorescence in situ hybridization (FISH) was performed on uncultured amniotic fluid interphase cells in both cases looking for aneuploidy for chromosomes 13, 18, 21, X and Y. The results of the interphase analyses support the common knowledge that careful interpretation of interphase FISH analysis is necessary and that results should be followed by full cytogenetic analysis. To our knowledge these are the first reported cases of structurally abnormal chromosomes 18 being associated with a positive maternal serum triple screen for trisomy 18. Copyright © 2002 John Wiley & Sons, Ltd.     

Rapid prenatal diagnosis of trisomy 18 and triploidy in interphase nuclei of uncultured amniocytes by non-radioactive in situ hybridization

Two biotinylated chromosome-specific DNA probes were used to quantify the number of chromosomes 18 and 1 in uncultured amniocytes. Thirty-three samples of uncultured amniocytes were hybridized with a chromosome 18-specific DNA probe. Uncultured cells from two of the 33 samples were also hybridized with a chromosome 1-specific probe. Thirty of the samples were disomic with respect to chromosome 18; two samples were trisomic with respect to chromosome 18, and one sample was trisomic with respect to chromosomes 1 and 18. The two cases of trisomy 18 and the single case of triploidy were identified on uncultured celis within 48-72 h after amniocentesis. They were found among five samples from pregnant women who had amniocentesis because of an ultrasonographically identified fetal malformation. A trisomic karyotype could be diagnosed with certainty in uncultured amniocytes because the majority of the responding nuclei exhibited three hybridization signals. In normal cells, the majority of nuclei exhibited two signals. In no cases was there discordance between the genotype as predicted by in situ hybridization and that determined by cytogenetic analysis.     

Significantly higher number of fetal cells in the maternal circulation of women with pre-eclampsia

Although the pathophysiology of pre-eclampsia is unknown, several studies have indicated that abnormal placentation early in pregnancy might play a key role. It has recently been suggested that this abnormal placentation may result in transfusion of fetal cells (feto-maternal transfusion) in women with pre-eclampsia. In the present study, fetal nucleated red blood cells were isolated from 20 women with pre-eclampsia and 20 controls using a very efficient magnetic activated cell sorting (MACS) protocol. The number of male cells was determined using two-color fluorescence in situ hybridization (FISH) for X and Y chromosomes. Significantly more XY cells could be detected in women with pre-eclampsia (0.61±1.2 XY cells/ml blood) compared to women with uncomplicated pregnancies (0.02±0.04 XY cells/ml blood) (Mann–Whitney U-test, p<0.001). These results suggest that fetal cell trafficking is enhanced in women with pre-eclampsia, and this finding may contribute to the understanding of the pathophysiology of the disease. Copyright © 2001 John Wiley & Sons, Ltd.     

Rapid sexing of human embryos by non-radioactive in situ hybridization: Potential for preimplantation diagnosis of X-linked disorders

Sixty spare human embryos at various stages of preimplantation development were prepared for cytogenetic analysis. Fluorescent staining of those with metaphases allowed scoring for the presence of a Y chromosome. In situ hybridization was then performed using a biotinylated Y-specific sequence, and the probe was detected by a standard streptavidinlinked alkaline phosphatase system. This enabled comparison of the chromosomal sex with that obtained after in situ hybridization in 28 embryos, and the sexing result obtained by the two methods was concordant in all cases. A further 21 embryos in which no metaphase chromosomes were obtained were sexed by biotinylated in situ hybridization only. Overall, 66 per cent of male interphase nuclei demonstrated a Y-specific hybridization signal. Results were obtained in under 24 h, which may permit the sexing of an embryo biopsied during cleavage and the transfer of sexed embryos at the blastocyst stage to the mother's uterus in the same cycle as oocytes are collected for in vitro fertilization.     

Rapid detection of aneuploidy in uncultured chorionic villus cells using fluorescence in situ hybridization

Rapid detection of aneuploidy using chromosome-specific repetitive DNA probes and the potential diagnostic accuracy of fluorescence in situ hybridization (FISH) on interphase cells of chorionic villus samples (CVS) are presented. Analyses demonstrated the ability to correctly identify aneuploidy using FISH in uncultured CVS. Our preliminary investigation suggests that this technique offers a significant clinical potential to circumvent problems of culture, time, and cost in cytogenetic analysis.     

Chromosome 18 analysis by fluorescence in situ hybridization (fish) in human blastomeres of abnormal embryos after in vitro fertilization (ivf) attempt

We performed fluorescence in situ hybridization (FISH) with a chromosome 18-specific probe on human abnormal cleaved embryos, fertilized either by two spermatozoa and exhibiting three pronuclei (3 PN) or normally fertilized and exhibiting two pronuclei (2 PN) with subsequent severe fragmentation and/or blocking. The aim of the study was to evaluate the incidence of chromosome 18 anomalies among these embryos, in order to evaluate the FISH efficiency on such material and to obtain more precise and complete data than those obtained with classical cytogenetic analysis. For the 3 PN cleaved embryos, FISH confirmed the frequent regulation towards diploidy (25 per cent) and the high frequency of mosaics (53 per cent). For the 2 PN blocked or damaged embryos, FISH permitted chromosome evaluation, which was otherwise impossible with classical cytogenetic techniques: we also found a high mosaic frequency (45 per cent) with these embryos. If this frequency were the same for normally developing embryos, it would be a major obstacle to the reliability of either chromosomal or genetic preimplantation diagnosis.     

Fluorescence in situ hybridization (FISH) for rapid detection of aneuploidy: experience in 911 prenatal cases†

Fluorescence in situ hybridization (FISH) was performed with probes specific for chromosomes 13, 18, 21, X and Y on 911 of 11123 (8.2%) amniotic fluid samples submitted to the present authors' laboratory for cytogenetic analysis over an 8-year period. Altogether 3516 hybridizations were performed with an interpretable FISH result on all chromosomes requested in 884/911 (97%) of cases. An uninformative FISH result occurred in 44 hybridizations among 27 cases (3%). Of a total of 89 karyotypically proven cases with aneuploidy that might have been detected by FISH, the overall detection rate was 84%. An inconclusive or incomplete FISH result occurred in 9/89 (10%) of these proven aneuploid cases. In the remaining 80 informative proven aneuploid cases, correct detection of aneuploidy was accomplished in 75/80 (94%) of samples. A false-negative result occurred in the remaining 5/80 (6%) of such informative cases. Eighteen cases had karyotypically proven abnormalities that could not have been detected by the targeted FISH. Aside from these 18 cases, FISH allowed correct detection of normal disomy in 785/804 (98%) of such cases. An incomplete FISH result occurred in 18 normal disomic cases. There was a single possible ‘false-positive’ FISH result for chromosome 21. Interphase FISH analysis of uncultured amniotic fluid cells has been shown to be a useful laboratory tool for rapid fetal aneuploidy screening during pregnancy. As with all clinical laboratory diagnostic tests, incomplete or inconclusive results (or even interpretive errors) occur in a small percentage of cases. Nevertheless, FISH results accompanied by other data and by appropriate counseling provide clinicians and patients with valuable information for clinical decision-making surrounding family planning and pregnancy management. Copyright © 2001 John Wiley & Sons, Ltd.     

Enrichment of fetal cells from maternal blood by high gradient magnetic cell sorting (double MACS) for PCR-based genetic analysis

For simple and effective isolation of fetal cells from peripheral maternal blood, we combined depletion of maternal cells and enrichment of fetal cells by high-gradient magnetic cell separation (MACS). First CD45⁺ and CD14⁺ cells were depleted from maternal peripheral blood mononuclear cells by MACS. From the depleted fraction, CD71⁺ erythroid cells were enriched up to 80 per cent by MACS. This ‘double-MACS’ procedure yielded an average depletion rate of 780-fold and an average enrichment rate of 500-fold, with approximate recovery rates of 40–55 per cent. For paternity testing, cells from unseparated blood and the various fractions were analysed for polymorphism of the HLA-DQ-A1 locus and D1S80 locus by the polymerase chain reaction (PCR). In CD45⁻/CD71⁺ sorted cells from maternal blood, but not in unfractionated cells from maternal blood or CD45⁻/CD14⁻ cells, paternal alleles could be detected. In the CD45⁻/CD71⁺ fraction, the relative frequency of paternal alleles compared with maternal alleles ranged from 1 in 20 to 1 in 200 (determined by titration and depending on the quality of separation and biological variation). In 7 out of 11 cases, between weeks 12 and 25 of gestation, we could identify paternal alleles by PCR, either HLA-DQ-A1 or D1S80. This double-MACS procedure is simple, fast, efficient, and reliable for non-invasive prenatal diagnosis.     

Prenatal detection of trisomy 21 in uncultured amniocytes by fluorescence in situ hybridization: A prospective study

A prospective study was undertaken to evaluate the use of fluorescence in situ hybridization (FISH) for the detection of trisomy 21 in interphase nuclei of uncultured amniotic fluid cells. Five hundred cases were analysed in situ and classified as normal or abnormal; the results were subsequently checked against the cytogenetic findings. Four hundred and ninety-three were correctly identified as normal with an 86·6 per cent average frequency of scored nuclei exhibiting two signals; six cases were correctly identified as trisomic for chromosome 21 with 81·7 per cent of scored nuclei exhibiting three signals; and one abnormal case involving an unbalanced chromosome 21·21 translocation was falsely scored as normal due to poor hybridization/detection efficiency. The method has been substantially improved and simplified so that it is suitable for the rapid detection of trisomy 21. As aneuploidy detection in interphase does not identify structural chromosome aberrations, it is not a substitute for fetal chromosome analysis.     

Characterization of marker chromosomes by microdissection and fluorescence in situ hybridization

We characterized by microdissection and fluorescence in situ hybridization (FISH) two marker chromosomes: (1) a de novo, acrocentric marker chromosome detected in 88 per cent of the amniotic fluid cells of one of two physically and developmentally normal twins; and (2) a metacentric marker chromosome present in a phenotypically normal female. Analysis of FISH probes developed from the marker chromosomes indicated that the marker chromosomes in cases 1 and 2 were del(14)(q11) and a derivative chromosome from a Robertsonian translocation, respectively. Microdissection in combination with FISH may prove to be a valuable technique in determining the chromosomal origin of de novo marker chromosomes and unbalanced structural rearrangements detected during prenatal diagnosis.     

Mid-trimester fetal sex determination from maternal peripheral blood by fluorescence in situ hybridization without enrichment of fetal cells

To determine the fetal sex on 30 women who were 16–20 weeks pregnant, about 100 000 maternal blood nucleated cells were analysed by means of fluorescence in situ hybridization (FISH) with a Y-chromosome-specific DNA probe. Cells with the hybridization signal were detected in 12 of the 30 women. All the 12 mothers gave birth to a male child. Of the other 18 women who had no Y-positive cells in the peripheral blood, 14 gave birth to a female child and four gave birth to a male child. These false-negative results probably occurred because the number of cells examined was inadequate. The data obtained in this study suggest that fetal sex determination using maternal peripheral blood with FISH is possible and that this diagnostic method will be clinically useful when more cells are analysed.     

Fetal granulocytes in maternal venous blood detected by in situ hybridization

Fetal male cells from maternal venous blood were detected by a non-radioactive in situ hybridization method using the biotinylated Y-specific DNA probe pY431. The hybridizations were performed on Ficoll-Paque-isolated nucleated blood cells obtained from 11 pregnant women in the seventh to 31st week of gestation. A Y-specific signal was detected in both granulocytes and lymphocyte-like cells in seven of the 11 women studied. These women gave birth to boys. In one of the four remaining cases, a Y-specific signal was detected in the lymphocyte-like cells but not in the granulocytes. This woman gave birth to a girl. The other three women had no cells with a Y-specific signal and all three gave birth to girls. Altogether, 83 500 nucleated cells were analysed. One hundred and three cells showed a Y-specific signal. Of these Y-specific cells, 62 per cent were granulocytes and 38 per cent lymphocyte-like cells. Our results suggest that fetomaternal transfer of granulocytes is common and that it occurs as early as in the seventh week of gestation. None of the ten non-pregnant female control samples showed positive cells with the Y-chromosome-specific probe; approximately 97 per cent of the cells from the five adult male controls showed a Y-specific signal. Our results indicate that in situ hybridization using a Y-specific DNA probe performed on granulocytes in maternal blood can be used for fetal male sex determination.