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1.
A monoclonal antibody with potential for aiding non-invasive prenatal diagnosis: utility in screening of pregnant women at risk of preeclampsia. 总被引:1,自引:0,他引:1
Alejandra Fernández Belén Prieto Ana Escudero Jack H Ladenson Francisco V Alvarez 《The journal of histochemistry and cytochemistry》2005,53(3):345-350
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. 相似文献
2.
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 相似文献
3.
Samura O Pertl B Sohda S Johnson KL Sekizawa A Falco VM Elmes RS Bianchi DW 《Human genetics》2000,107(1):28-32
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. 相似文献
4.
以18例孕7~25周的孕妇外周血为材料, 经Percoll不连续密度梯度离心初步富集胎儿有核红细胞。然后用Kleihauer抗酸染色法进行标记, 结果阳性胎儿有核红细胞的胞浆呈深红色, 而母亲的有核红细胞胞浆无色。显微操作法获取单个胎儿有核红细胞, 经全基因组扩增后, 产物进行性别鉴定及STR连锁分析检测, 验证有核红细胞的来源, 并完成9例杜氏肌营养不良(Duchenne muscular dystrophy,DMD)的无创性产前基因诊断。应用Kleihauer抗酸染色法标记胎儿有核红细胞, 它是一种快速、简单、直接的化学染色方法, 更易于推广到临床应用。 相似文献
5.
Diana W. Bianchi Anna Mahr Gretchen K. Zickwolf Timothy W. Houseal Alan F. Flint Katherine W. Klinger 《Human genetics》1992,90(4):368-370
Fetal cells were isolated from the peripheral blood of a pregnant woman at 19 weeks of gestation whose fetus had Down syndrome. An amniocentesis had been performed 2 weeks earlier because of abnormalities detected on an antenatal sonogram. Fetal cells were separated by fluorescence-activated cell sorting using monoclonal antibody to the transferrin receptor (TfR). Fluorescence in situ hybridization studies with probes for chromosomes Y and 21 revealed a small number of 47,XY,+21 cells in the TfR- sorted fraction. Although preliminary, the results of this study suggest the possibility that one day, fetal chromosome aneuploidy will be routinely diagnosed from maternal venous blood samples. 相似文献
6.
应用胎儿特异性抗体HbF(γ链)标记法无创性 产前基因诊断DMD 总被引:3,自引:1,他引:2
以孕8~26周孕妇外周血为材料,经过Percoll密度梯度离心初步富集,胎儿细胞特异性抗体—HbF标记、识别胎儿有核红细胞,母体和胎儿有核红细胞的精确区分是以胎儿和成人血红蛋白的组成差异为基础的。胎儿细胞胞浆黄染,而具有成人血红蛋白的母体细胞没有颜色。显微操作法获取全部阳性细胞后,以其全基因组扩增(PEP)的产物为模板,进行性别检测、DMD基因的多重PCR检测和STR连锁分析。结果,20名孕妇外周血中均发现与HbF呈阳性反应的胎儿NRBC。并完成7例DMD的产前基因诊断。HbF抗体标记法能有效识别胎儿有核红细胞,是无创性产前基因诊断中很有应用前景的标记方法。 Abstract: Maternal blood was obtained at 8-26weeks of gestation.After discontinuous density gradient centrifugation with Percoll ,HbF antibody was used to identify fetal NRBC.The precise differentiation between fetal and maternal NRBC is based on the constitutional difference between fetal and adult hemoglobin (Hb).Fetal cells appear yellow cytoplasmic staining,while adult cells colorless. NRBCs were collected by micromanipulation andwhole genome amplification was performed. DMD was prenatally diagnosed by using the combination of sex determination,multiplex PCR and linkage analysis of several STR sites of dystrophin. NRBCs stained with HbF were found in all of 20 maternal blood samples with gestations, and 7 fetuses with risk of DMD were diagnosed. We concluded that HbF antibody could identify fetal NRBC efficaciously,and this is a kind of more prospective application method. 相似文献
7.
Prenatal diagnosis of ornithine transcarbamylase deficiency by using a single nucleated erythrocyte from maternal blood 总被引:7,自引:0,他引:7
A. Watanabe Akihiko Sekizawa Atsushi Taguchi Hiroshi Saito Takumi Yanaihara Mitsunobu Shimazu Ichiro Matsuda 《Human genetics》1998,102(6):611-615
We have developed a method that allows the prenatal DNA diagnosis of ornithine transcarbamylase (OTC) deficiency by using
a single fetal nucleated erythrocyte (NRBC) isolated from maternal blood. OTC gene analysis of a male patient (TF) with early
onset OTC deficiency was performed by single-strand conformation polymorphism (PCR-SSCP) and DNA sequencing. To investigate
the possible prenatal diagnosis of OTC deficiency, maternal blood was obtained at 13 weeks of gestation of a subsequent pregnancy,
from the mother of patient TF. NRBCs in the maternal blood were separated by using the density gradient method and then collected
with a micromanipulator. The entire genome of a single NRBC was amplified by primer extension preamplification (PEP). The
human leukocyte antigen (HLA)-DQ alpha genotype and sex were determined from small aliquots of the PEP product. The HLA-DQ
alpha genotype of each of the parents of the male patient was also determined. Once a single NRBC had been identified as being
of fetal origin, the OTC gene was analyzed by using the restriction fragment length polymorphism (RFLP) method. DNA analysis
revealed a point mutation in exon 9 of the OTC gene in the OTC-deficient patient (TF). All NRBCs retrieved from maternal blood
were successfully identified as being of fetal origin by HLA-DQ alpha genotyping and sex determination. RFLP analysis demonstrated
that the fetal OTC gene was normal. This is the first study to successfully diagnose OTC deficiency prenatally, by using a
single fetal NRBC from the maternal circulation. Such prenatal DNA diagnosis is non-invasive and can be applied to other genetic
diseases, including autosomal and X-linked diseases.
Received: 19 December 1997 / Accepted: 14 February 1998 相似文献
8.
The fetal erythroblast is not the optimal target for non-invasive prenatal diagnosis: preliminary results. 总被引:1,自引:0,他引:1
Steen K?lvraa Britta Christensen Lene Lykke-Hansen John Philip 《The journal of histochemistry and cytochemistry》2005,53(3):331-336
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. 相似文献
9.
Hennerbichler S Schmied R Petek E Kroisel PM Pertl B Tiran B Dohr G Wintersteiger R Sedlmayr P 《Cytometry》2002,48(2):87-92
BACKGROUND: Fetal nucleated red blood cells (NRBC) present in the peripheral blood of pregnant women at low frequency are a potential target for noninvasive prenatal diagnostics. METHODS: CD71-enriched cells from male cord blood (CB) were stained for the gamma chain of HbF (Hb-gamma) and cytocentrifuged. Fluorescence in situ hybridization (FISH) was done for the Y chromosome. Following staining of the nucleus with TO-PRO-3, laser scanning cytometry was performed. Artificial mixtures of small volumes of male CB and blood drawn from nonpregnant females were analyzed. RESULTS: In CB, 59% of events double positive for Hb-gamma and TO-PRO-3 were identified as CB-NRBC. In contamination studies, male fetal CB-NRBC were identified perfectly on the basis of morphologic characteristics and FISH reactivity following relocation and visual assessment. Mean recovery was 8.7%. CONCLUSIONS: Laser scanning cytometry of preenriched fetal NRBC may offer a promising way for noninvasive prenatal diagnostics. This is because it provides a virtual enrichment step and the position on the slides of cells visually confirmed to correspond to fetal NRBC is known. Further experimental procedures on well-defined and located target cells may be feasible. 相似文献
10.
Evaluation of bidirectional transfer of plasma DNA through placenta 总被引:11,自引:0,他引:11
Sekizawa A Yokokawa K Sugito Y Iwasaki M Yukimoto Y Ichizuka K Saito H Okai T 《Human genetics》2003,113(4):307-310
To clarify the origin of cell-free fetal DNA in maternal plasma, we analyzed bidirectional transfer of plasma DNA between fetus and mother. We analyzed maternal and fetal plasma DNA obtained from 15 pregnant women at the time of Cesarean section. The subjects were five patients with preeclampsia and 10 gestational-age-matched normal controls. DNA was extracted from 1.5-ml plasma samples and the cellular fraction of maternal and umbilical blood. Seven polymorphic marker genes were analyzed. The relative concentration of fetal DNA in maternal plasma and maternal DNA in cord blood were evaluated. The relative concentration of maternal DNA in fetal circulation (median, 0.9%; range, 0.2–8.4%) was significantly lower than that of fetal DNA in maternal blood (14.3%, 2.3–64%), with
P=0.007. The relative concentration of maternal DNA in fetal blood was not affected by preeclampsia. These findings indicate that cell-free DNA is unequally transferred through the placenta. The structural characteristics of the placenta suggest that the majority of cell-free fetal DNA in maternal plasma is derived from villous trophoblasts. 相似文献
11.
Frequency of nucleated red blood cells in maternal blood during the different gestational ages 总被引:4,自引:0,他引:4
L. P. Shulman Owen P. Phillips Elizabeth Tolley David Sammons S. S. Wachtel 《Human genetics》1998,103(6):723-726
We wished to determine the time of pregnancy at which optimal numbers of nucleated red blood cells (NRBC) are present in maternal
blood. Because 30% of the NRBC in maternal blood are fetal, there are implications for prenatal screening and diagnosis. Samples
of whole blood were collected from each of 225 women at various times during pregnancy. The samples were processed by charge
flow separation (CFS), the NRBC enumerated, and the numbers compared on a week-to-week basis. To quantify the relationship
between week of pregnancy and actual and log-transformed numbers of NRBC recovered, Pearson product moment and Spearman correlation
coefficient were estimated for each of four CFS instruments and for the four instruments combined. When the data were analyzed,
we found no relationship between stage of pregnancy and numbers of NRBC recovered. Even after logarithmic transformation,
variability among the women, estimated by standard deviation, was large and relatively stable across the different stages
of pregnancy. The number of NRBC recoverable by CFS appears to be constant between 7 and 25 weeks.
Received: 26 August 1998 / Accepted: 26 October 1998 相似文献
12.
Sitar G Brambati B Baldi M Montanari L Vincitorio M Tului L Forabosco A Ascari E 《Experimental cell research》2005,302(2):153-161
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. 相似文献
13.
A noninvasive approach to studying fetal cells for prenatal diagnosis of chromosomal aneuploidies 总被引:1,自引:0,他引:1
Zolotukhina TV Shilova NV Zamulaeva IA Smirnova SG Orlova NV Saenko AS 《Genetika》1999,35(10):1422-1430
Fetal cells isolated from maternal peripheral blood during the second trimester of pregnancy were analyzed. Blood samples were centrifuged in a Ficoll-Paque gradient, the mononuclear cell fraction was isolated and stained with fluorescent monoclonal antibodies against glycophorine A (GPA + PE), transferrin (CD71 + FITC), and Hoechst 33342. Fluorescence-activated cell sorting (FACS) was conducted on a Vantage flow cytofluorimeter (Becton Dickinson). Fluorescence in situ hybridization (FISH) with Y chromosome-specific DNA probe revealed fetal cells that exhibited Y signal in all 20 blood samples obtained from women pregnant with healthy male fetuses. The concentration of these fetal cells averaged about 1.34% and ranged from 0.1 to 4.2% in different blood samples. In six cases, blood samples were obtained from pregnant women, in which prenatal cytogenetic analysis revealed various fetal aneuploidies. Using FISH with DNA probes specific for chromosomes X, 18, and 13/21, Fetal cells with chromosomal aberrations were detected in these six maternal blood samples at a concentration from 1.5 to 5.6% (on average 3.7%). These results indicate the possibility of a new noninvasive approach, which is safe for both mother and fetus when used for isolation of fetal cells from pregnant women's blood samples and prenatal diagnosis of a broad spectrum of fetal cell chromosomal aberrations. 相似文献
14.
Maternal serum cell-free fetal DNA levels are increased in cases of trisomy 13 but not trisomy 18 总被引:16,自引:0,他引:16
Wataganara T LeShane ES Farina A Messerlian GM Lee T Canick JA Bianchi DW 《Human genetics》2003,112(2):204-208
Cell-free fetal DNA in the maternal circulation is a potential noninvasive marker for fetal aneuploidies. In previous studies with Y DNA as a fetal-specific marker, levels of circulating fetal DNA were shown to be elevated in women carrying trisomy 21 fetuses. The goal of this study was to determine whether cell-free fetal DNA levels in the serum of pregnant women carrying fetuses with trisomies 13 or 18 are also elevated. Archived maternal serum samples from five cases of male trisomy 13 and five cases of male trisomy 18 were studied. Each case was matched for fetal gender, gestational age, and duration of freezer storage to four or five control serum samples presumed to be euploid after newborn medical record review. Real-time quantitative polymerase chain reaction amplification of DYS1 was performed to measure the amount of male fetal DNA present. Unadjusted median serum fetal DNA concentrations were 97.5 GE/ml (genomic equivalents per milliliter; 29.2-187.0) for the trisomy 13 cases, 31.5 GE/ml (18.6-77.6) for the trisomy 18 cases, and 40.3 GE/ml (3.7-127.4) for the controls. Fetal DNA levels in trisomy 13 cases were significantly elevated ( P=0.016) by analysis of variance of the ranks of values within each matched set. In contrast, fetal DNA levels in trisomy 18 cases were no different from the controls ( P=0.244). Second trimester maternal serum analytes currently used in screening do not identify fetuses at high risk for trisomy 13. Fetal DNA may facilitate noninvasive screening for trisomy 13 provided that a gender-independent fetal DNA marker can be developed. 相似文献
15.
Strategies for rare-event detection: an approach for automated fetal cell detection in maternal blood. 总被引:3,自引:0,他引:3
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J C Oosterwijk C F Knepfl W E Mesker H Vrolijk W C Sloos H Pattenier I Ravkin G J van Ommen H H Kanhai H J Tanke 《American journal of human genetics》1998,63(6):1783-1792
This article explores the feasibility of the use of automated microscopy and image analysis to detect the presence of rare fetal nucleated red blood cells (NRBCs) circulating in maternal blood. The rationales for enrichment and for automated image analysis for "rare-event" detection are reviewed. We also describe the application of automated image analysis to 42 maternal blood samples, using a protocol consisting of one-step enrichment followed by immunocytochemical staining for fetal hemoglobin (HbF) and FISH for X- and Y-chromosomal sequences. Automated image analysis consisted of multimode microscopy and subsequent visual evaluation of image memories containing the selected objects. The FISH results were compared with the results of conventional karyotyping of the chorionic villi. By use of manual screening, 43% of the slides were found to be positive (>=1 NRBC), with a mean number of 11 NRBCs (range 1-40). By automated microscopy, 52% were positive, with on average 17 NRBCs (range 1-111). There was a good correlation between both manual and automated screening, but the NRBC yield from automated image analysis was found to be superior to that from manual screening (P=.0443), particularly when the NRBC count was >15. Seven (64%) of 11 XY fetuses were correctly diagnosed by FISH analysis of automatically detected cells, and all discrepancies were restricted to the lower cell-count range. We believe that automated microscopy and image analysis reduce the screening workload, are more sensitive than manual evaluation, and can be used to detect rare HbF-containing NRBCs in maternal blood. 相似文献
16.
Susanne Mergenthaler Tatiana Babochkina Vivian Kiefer Olaf Lapaire Wolfgang Holzgreve Sinuhe Hahn 《The journal of histochemistry and cytochemistry》2005,53(3):319-322
Current cytogenetic approaches in noninvasive prenatal diagnosis focus on fetal nucleated red blood cells in maternal blood. This practice may be too restrictive because a vast proportion of other fetal cells is ignored. Recent studies have indicated that fetal cells can be directly detected, without prior enrichment, in maternal blood samples by fluorescence in situ hybridization (FISH) analysis for chromosomes X and Y (XY-FISH). In our blinded analysis of 40 maternal blood samples, we therefore examined all fetal cells without any enrichment. Initial examinations using conventional XY-FISH indicated a low specificity of 69.4%, which could be improved to 89.5% by the use of two different Y-chromosome-specific probes (YY-FISH) with only a slight concomitant decrease in sensitivity (52.4% vs 42.9%). On average, 12-20 male fetal cells/ml of maternal blood were identified by XY- and YY-FISH, respectively. 相似文献
17.
The participation of interleukin-6 (IL-6) in the pathophysiology of normal and abnormal human parturition was evaluated by determining IL-6 concentrations in amniotic fluid (AF). Biologically active IL-6 was determined (in U/ml) using the B9 hybridoma growth factor assay, while the concentrations of immunoreactive IL-6 species (in pg/ml) were assessed using a monoclonal antibody (moAb)-based ELISA. Two hundred and twenty-seven AF samples from women in normal labor and from those presenting with a clinical diagnosis of premature rupture of membranes (PROM) were assayed. In selected instances, IL-6 levels were evaluated simultaneously in AF and in maternal and fetal plasma. Women with a normal pregnancy had low titers of biologically active IL-6 in AF both at midtrimester (group 1, n = 27; median IL-6 concentration = 16 U/ml) and at term (group 2, n = 33; median = 15 U/ml). There was an increase in the IL-6 bioactivity in AF from women in normal labor at term (group 3, n = 40; median = 74 U/ml; p less than 0.001). In order to distinguish between the relative contributions of parturition per se and of intrauterine infection to the elevation of biologically active IL-6 levels in AF, IL-6 titers were compared in four different groups of women with PROM.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
18.
Jacob M?rup Schlütter Ida Kirkegaard Olav Bj?rn Petersen Nanna Larsen Britta Christensen David M. Hougaard Steen K?lvraa Niels Uldbjerg 《PloS one》2014,9(9)
Objective
To identify factors influencing the number of fetal cells in maternal blood.Methods
A total of 57 pregnant women at a gestational age of weeks 11–14 were included. The number of fetal cells in maternal blood was assessed in 30 ml of blood using specific markers for both enrichment and subsequent identification.Results
Participants carrying male fetuses had a higher median number of fetal cells in maternal blood than those carrying female fetuses (5 vs. 3, p = 0.04). Certain cytokines (RANTES, IL-2 and IL-5) were significantly associated with the number of fetal cells in maternal blood.Conclusion
The number of fetal cells in maternal blood is associated with certain cytokines and fetal gender. 相似文献19.
J. H. Steel Q. Hamid S. Van Noorden P. Jones P. Denny J. Burrin S. Legon S. R. Bloom J. M. Polak 《Histochemistry and cell biology》1988,89(1):75-80
Summary We have investigated the use of in situ hybridisation together with immunocytochemistry for the study of endocrine cell function, using as an example the expression of prolactin messenger RNA (mRNA) in pituitaries of rats under various endocrinological conditions. In situ hybridisation using a 32P-labelled cRNA probe for rat prolactin was carried out on sections of 4% paraformaldehyde-fixed pituitaries from prepubertal, pubertal, pregnant, lactating and ovariectomised rats and adjacent sections were immunostained for prolactin. Northern gel analysis was performed on total RNA extracts of pregnant, lactating and control pituitaries. While in ovariectomised rat pituitaries both prolactin immunoreactivity and prolactin mRNA were decreased, no differences in prolactin immunostaining were seen between prepubertal, pubertal, pregnant or lactating rats and controls, even when the supra-optimal dilution technique was used. However, using in situ hybridisation, prolactin mRNA signal was increased in prepubertal rats, and with hybridisation and northern gel analysis the signal was reduced in pregnant rats and markedly increased in lactating rats. The combined use of in situ hybridisation and immunocytochemistry provides morphological information concerning endocrine gene expression and protein synthesis in the pituitary gland. 相似文献
20.
Non-invasive fetal RHD and RHCE genotyping using real-time PCR testing of maternal plasma in RhD-negative pregnancies. 总被引:2,自引:0,他引:2
Ilona Hromadnikova Lenka Vechetova Klara Vesela Blanka Benesova Jindrich Doucha Radovan Vlk 《The journal of histochemistry and cytochemistry》2005,53(3):301-305
We assessed the feasibility of fetal RHD and RHCE genotyping by analysis of DNA extracted from plasma samples of RhD-negative pregnant women using real-time PCR and primers and probes targeted toward RHD and RHCE genes. We analyzed 45 pregnant women in the 11th to 40th weeks of pregnancy and correlated the results with serological analysis of cord blood after delivery. Non-invasive prenatal fetal RHD exon 7, RHD exon 10, RHCE exon 2 (C allele), and RHCE exon 5 (E allele) genotyping analysis of maternal plasma samples was correctly performed in 45 out of 45 RhD-negative pregnant women delivering 24 RhD-, 17 RhC-, and 7 RhE-positive newborns. Detection of fetal RHD and the C and E alleles of RHCE gene from maternal plasma is highly accurate and enables implementation into clinical routine. We recommend performing fetal RHD and RHCE genotyping together with fetal sex determination in alloimmunized D-negative pregnancies at risk of hemolytic disease of the newborn. In case of D-negative fetus, amplification of another paternally inherited allele (SRY and/or RhC and/or RhE positivity) proves the presence of fetal DNA in maternal circulation. 相似文献