Detection and isolation of fetal cells from maternal blood using the flourescence-activated cell sorter (FACS)

The presence of fetal cells in the maternal circulation during pregnancy has been suggested by repeated observations of small numbers of cells containing Y chromatin or a Y chromosome in the blood of pregnant women. With the fluorescence-activitated cell sorter (FACS), we have used antibodies to a paternal cell surface (HLA) antigen, not present in the mother, to select fetal cells from the lymphocyte fractions of a series of maternal blood samples, collected as early as 15 weeks of gestation. These sorted cells have been examined for a second paternal genetic marker, Y chromatin. Y chromatin-containing cells were found among the sorted cells from prenatal maternal blood specimens in 8 pregnancies subsequently producing male infants whose lymphocytes reacted with the same antibodies to paternal antigen used for sorting with the FACS. In each of 17 pregnancies resulting in male infants who failed to inherit the antigen detected by the antibodies used for cell sorting, Y chromatin-containing cells were not found prenatally. The use of two paternal genetic markers, a cell surface antigen and nuclear Y chromatin, to identify fetal cells in maternal blood permits us to conclude that these cells are present in the mother's circulation, as early as 15 weeks gestation. Further development of the techniques reported here could lead to widespread screening of maternal blood samples during pregnancy for detection of fetal genetic abnormalities.     

Screening of monoclonal antibodies recognizing oncofetal antigens for isolation of trophoblasts from maternal blood for prenatal diagnosis

Many monoclonal antibodies have been produced against tumour-associated cell surface antigens for cancer therapy. They have therefore been selected for minimal reactivity with normal tissues and in particular for lack of binding to blood cells or serum components. Many of the antigens recognized are of fetal origin. These monoclonal antibodies may therefore be ideal candidates to recognize and sort fetal trophoblasts from maternal blood for prenatal diagnosis of genetic abnormalities. A panel of 19 anti-tumour antibodies were therefore screened for reactivity with early trimester placenta and two, 340 and 154, were shown to stain trophoblasts. If MAb 340 is linked to magnetic beads, it can efficiently sort trophoblast cell lines from whole blood.     

Erythroid-specific antibodies enhance detection of fetal nucleated erythrocytes in maternal blood

Fetal nucleated erythrocytes (NRBC) in maternal blood are a non-invasive source of fetal DNA for prenatal genetic screening. We compared the effectiveness of three monoclonal antibodies for the separation of fetal cells from maternal blood by flow sorting. Mononuclear blood cells from 49 healthy pregnant women were incubated with antibody to CD 71, CD 36, and/or glycophorin A (GPA), employed singly or in combination with each other. These monoclonal antibodies recognize surface antigens on haematopoietic precursor cells. Successful isolation of fetal cells was defined as detection of Y chromosomal sequences in maternal blood from women carrying male fetuses, with absence of Y sequences when female fetuses were carried. Thus, gender prediction accuracy was used as a measure of fetal cell separation. Using anti-CD 71 to isolate fetal cells, gender prediction was 57 per cent correct; with anti-CD 36, it was 88 per cent correct. Anti-GPA, an erythrocyte-specific antigen, used alone or in combination with anti-CD 71 or 36, improved gender prediction to 100 per cent. We conclude that antibody to GPA improves the retrieval of fetal NRBC from maternal blood, permitting genetic analysis by the polymerase chain reaction.     

Prenatal diagnosis of congenital cytomegalovirus infection in 189 pregnancies with known outcome

An Erratum has been published for this article in Prenatal Diagnosis 21(7) 2001, 605. Prenatal diagnosis (PD) of fetal cytomegalovirus (CMV) infection was performed in 242 pregnancies, with known outcome in 189 cases. In 141/189 pregnancies, PD was carried out on account of suspicious maternal CMV serology up to gestational week (WG) 23, and in 48 cases on account of abnormal ultrasonic findings detected between WG 18 and 39. Chorionic villus samples (n=6), amniotic fluid (AF, n=176) and/or fetal blood specimens (n=80) were investigated for detection of virus by cell culture, shell vial assay, PCR and/or CMV-specific IgM antibodies. Of 189 fetuses correctly evaluated by CMV detection either in fetal tissue following therapeutic abortion/stillbirth (n=24) or in urine of neonates within the first 2 weeks of life (n=33), 57 were congenitally infected. In women with proven or suspected primary infection, the intrauterine transmission rates were 20.6% (7/34) and 24.4% (10/41), respectively. Of the congenitally infected live-born infants, 57.6% (19/33) had symptoms of varying degree. The overall sensitivity of PD in the serologic and ultrasound risk groups was 89.5% (51/57). A sensitivity of 100% was achieved by combining detection of CMV-DNA and CMV-specific IgM in fetal blood or by combined testing of AF and fetal blood for CMV-DNA or IgM antibodies. There was no instance of intrauterine death following the invasive procedure. The predictive value of PD for fetal infection was 95.7% (132/138) for negative results and 100% (51/51) for positive results. Correct results for congenital CMV infection by testing AF samples can be expected with samples obtained after WG 21 and after a time interval of at least 6 weeks between first diagnosis of maternal infection and PD. In case of negative findings in AF or fetal blood and the absence of ultrasound abnormalities at WG 22–23, fetal infection and neonatal disease could be excluded with high confidence. Positive findings for CMV infection in AF and/or fetal blood in combination with CMV suspicious ultrasound abnormalities predicted a high risk of cytomegalic inclusion disease (CID). Furthermore, detection of specific IgM antibodies in fetal blood was significantly correlated with severe outcome for the fetus or the newborn (p=0.0224). However, normal ultrasound of infected fetuses at WG 22–23 can neither completely exclude an abnormal ultrasound at a later WG and the birth of a severely damaged child nor the birth of neonates which are afflicted by single manifestations at birth or later and of the kind which are not detectable by currently available ultrasonographic techniques. Copyright © 2001 John Wiley & Sons, Ltd.     

Isolating fetal nucleated red blood cells from maternal blood: The baylor experience—1995

In our previous work we have isolated fetal cells from maternal blood and used fluorescent in situ hybridization (FISH) for chromosome-specific probes to detect aneuploidy. Current efforts in the Baylor College of Medicine programme are focusing on obtaining consistency in flow-sorting methodology and on determining sensitivity and specificity. To this end, systematic evaluation of five glycophorin A (gly A) antibodies all produced agglutination, leading us to abandon the use of gly A antibodies for positive selection of fetal cells. Conversely, we have found LDS-751 to be useful for nuclear selection. CD45 negative selection can best be accomplished by the use of flasks coated with goat antibodies against mouse antibodies. Positive selection by flow sorting for either CD71⁺ cells or gamma-globin-positive cells seems to be successful. Using these two approaches, we have recently detected male (fetal) cells in pregnancies in which the fetus was 46, XY in 10 of 18 and in 12 of 14 cases, respectively.     

Prenatal diagnosis of severe combined immunodeficiency with defective synthesis of HLA molecules

The immunodeficiency associated with a defective expression of HLA molecules is an autosomal recessive disorder leading to death during childhood. We have performed prenatal diagnosis for six fetuses at risk for this disease by membrane immunofluorescence on blood lymphocytes and monocytes, using specific monoclonal antibodies for HLA class I and II molecules. Two pregnancies have been found to be affected. The diagnosis has been confirmed on each abortus by the study of the membrane expression of HLA class I and II molecules on blood lymphocytes and monocytes, and on thymic and splenic cells. The four other cases were found to be normal both during pregnancy and after birth. The detection of the defect as early as the 20th week of gestation allows selective termination.     

Thyroid antibodies are not a risk factor for pregnancies with down syndrome

The observation that thyroid disease is frequent in mothers of children with Down syndrome (DS) has suggested that maternal thyroid antibodies could be a factor predisposing to trisomy 21 in their offspring. In this study, the incidences of thyroglobulin (Tg) and thyroid peroxidase (TPO) antibodies were analysed with a sensitive solid-phase immunosorbent radioassay in sera from 29 mothers giving birth to children with trisomy 21 and 87 control mothers. The serum samples were collected at delivery. There was no statistical difference regarding the proportion of thyroid antibodies (against Tg and/or TPO) in the two groups. Thyroid antibodies were detected in 6/29 (20.7 per cent) of the DS mothers and in 23/87 (26.4 per cent) of the control mothers. Among the women with thyroid antibodies, 4/6 (66.7 per cent) of the DS mothers and 12/23 (52 per cent) of the control mothers had antibodies against both Tg and TPO. There was no increase in the relative risk of having a child with DS if the titre of either Tg or TPO antibodies or both were positive, i.e. ≥ 1/5. The results indicate that the presence of thyroid antibodies in the serum of a pregnant woman has no prognostic value for the birth of an infant with DS.     

草丁膦人工抗原的合成与免疫效价的测定

用戊二醛作为交联剂合成草丁磷-卵清蛋白(oviparous,OVA)人工抗原,通过紫外吸收、31P核磁扫描的方法检测和鉴定草丁磷人工抗原,并用此合成抗原免疫小鼠,检测抗体的生成。结果表明,人工抗原紫外吸收光谱与OVA、草丁磷光谱相比发生了改变,人工抗原、草丁磷31P核磁光谱位移大致相同,通过计算人工抗原中草丁磷与卵清蛋白的偶联比是36.7∶1。免疫小鼠数周后在其血清中检测出抗体(其血清的效价为1∶320000)。草丁磷人工抗原合成及对小鼠的免疫成功,为制备草丁磷单克隆抗体打下了基础。     

五氯酚钠与二英对人外周血淋巴细胞的影响

将五氯酚钠(Na-PCP)与二(噁)英(2,3,7,8-TCDD)作为受试物,观察了人外周血淋巴细胞的彗星率和微核率.结果显示:Na-PCP与二噁英各质量浓度组的彗星率与微核率均比阴性对照组有明显的增高(P＜0.001),而且随着质量浓度的增加而增高,呈明显的剂量-效应关系;阴性对照组与助溶剂对照组没有明显差异(P＞0.05);Na-PCP与二(噁)英(2,3,7,8-TCDD)会对人外周血淋巴细胞的DNA造成损伤,而且随着质量浓度的增加,其对DNA的损伤将更加严重;Na-PCP对人外周血淋巴细胞造成的损伤为联合毒性所致.      

Possible effect of gestational age on the detection of fetal nucleated erythrocytes in maternal blood

Maternal venous blood samples, obtained from six pregnant women, were used as a source of fetal nucleated erythrocytes (NRBC). Fetal cell enrichment was potentiated by flow sorting with the monoclonal antibodies Tf R, Leu-4, and Leu-M3. Single copy Y chromosomal DNA sequences were detected in samples obtained from two women at 11 and 12 weeks' gestation. Y DNA sequences were absent in a subsequent sample from one of these women at 19 weeks and in two other women at 16 and 20 weeks. All four women delivered males. Y DNA sequences were not detected in two women who delivered females. By combining these results with prior data on the detection of Y chromosomal DNA sequences in maternal blood from male-bearing pregnancies, a relationship between gestational age and feta-maternal transfer of NRBC is suggested.     

Measurement of inhibin A: a modification to an enzyme-linked immunosorbent assay

Inhibin A is a useful prenatal marker of Down syndrome. Currently, the available enzyme-linked immunosorbent assays (ELISAs) for inhibin A are based upon the same paired monoclonal antibodies. In the present study we have confirmed for one of those ELISAs that short-term sample storage as whole blood leads to a significant decline in detectable inhibin A and that this is most likely due to erythrocyte catalase interference with a critical oxidation step in the assay. While this interference can be eliminated by heating the samples pre-assay, this process is labour intensive. In the present study we have demonstrated that the addition of 3-amino-1,2,4-triazole (AT), a catalase ‘suicide’ inhibitor, also prevents the decline of inhibin A levels in samples stored as whole blood. We suggest that the addition of AT to samples prior to assay is a simple modification to the inhibin A ELISA that affords optimum performance. Copyright © 2001 John Wiley & Sons, Ltd.     

Prenatal ultrasonographic diagnosis of fetal cerebral ventriculitis associated with asymptomatic maternal cytomegalovirus infection

Cytomegalovirus is the most common cause of congenital viral infection. In utero infection is usually suspected in patients with growth-retarded fetuses or when maternal illness precipitates serological investigations. A case is presented where routine ultrasound examination at 30 weeks' gestation in an asymptomatic patient demonstrated mild fetal ventriculomegaly. Transvaginal ultrasound enabled the visualization of intraventricular adhesions and small periventricular cysts. The suspected diagnosis of in utero cytomegalovirus infection was confirmed by the presence of IgM antibodies in fetal blood and subsequently by isolation of the virus from the infant's urine. The presence of mild fetal ventriculomegaly should prompt transvaginal brain imaging.     

Phenotypic analysis of fetal blood leucocytes: Potential for prenatal diagnosis of immunodeficiency disorders

Recent technological advances allow the detection and quantitation of subsets of leucocytes using monoclonal antibodies. We have taken advantage of this to study the ontogeny of fetal blood leucocytes, using very small blood samples obtained at fetoscopy. By 14 weeks gestation T cells represent 35 per cent or more of fetal leucocytes and the distribution of the helper/inducer and suppressor/cytotoxic subsets is similar to that of adults. B lymphocytes before 161/2 weeks are low (4–20 per cent), but rise to a mean of 28 per cent in 17–26 week fetuses. Granulocytic cells, many of which are phenotypically immature, represent 18–34 per cent of total leucocytes. The methodology employed is very reliable and offers the opportunity for the prenatal diagnosis of some immunodeficiency disorders, since using the same reagents we have diagnosed children with severe combined immunodeficiency shortly after birth.     

Prenatal diagnosis of congenital Chagas' disease (American trypanosomiasis)

The prenatal diagnosis of congenital transmission of Chagas' disease in a pregnant woman with the indeterminate form of the disease is reported. Sonography revealed fetal hydrops at 31 weeks' gestation. Anti-Trypanosoma cruzi IgM and IgG antibodies were negative in the fetal blood sampled by cordocentesis, but T. cruzi trypomastigotes were found in its buffy coat. Owing to anemia, in utero exchange transfusion was undertaken, but fetal demise ensued. Labor was induced and a stillborn infant weighing 2030 g was delivered. The pathological examination revealed placentitis and meningoencephalitis, myocarditis and splenitis in the stillborn fetus. Amastigotes were found in the myocardium, brain and placenta. Copyright © 2004 John Wiley & Sons, Ltd.     

基于SCGE的五氯酚对稀有鮈鲫DNA损伤的研究

采用单细胞凝胶电泳(SCGE)技术,研究了在不同的染毒时间内不同浓度的五氯酚(PCP)对稀有鮈鲫(Gobiocypris rarus)血细胞和肝脏细胞DNA的损伤.结果显示,各PCP染毒组细胞彗星尾部DNA含量 (%DNAT) 和彗星尾长(TL) 显著增加,与空白对照组比较,差异极显著(P0.926,说明在实验浓度范围内, 存在显著的浓度-效应关系.在同一浓度下,随着暴露时间的增加,处理组%DNAT和TL逐渐增加,存在显著的时间-效应关系.由于PCP可引起稀有鮈鲫血细胞和肝脏细胞DNA的严重损伤,因此稀有鮈鲫血细胞和肝脏细胞DNA的损伤可作为PCP遗传毒性的指示.     

Trophoblast-like cells sorted from peripheral maternal blood using flow cytometry: A multiparametric study involving transmission electron microscopy and fetal dna amplification

Three monoclonal antibodies (MAbs) against trophoblast (GB17, GB21, and GB25) and flow cytometry were used to sort trophoblast-like cells (TLCs) from peripheral blood of pregnant women. Sorted TLCs were processed for electron microscopy and fetal DNA amplification of the Y-specific sequences from mothers carrying male fetuses. At the ultra-structural level, most of the nucleated cells had the morphology of leucocytes, suggesting maternal contaminants, and we did not find the characteristic features of the free inter-villous trophoblast cells. Nevertheless, polymerase chain reaction (PCR) analysis showed an amplification of Y-specific sequences in two out of three samples of sorted TLCs. These results suggest that besides the maternal leucocytes, sufficient trophoblast nucleated fetal cells can be obtained using cell enrichment by sorting. This sensitive method holds promise for non-invasive prenatal diagnosis of fetal sex and if sufficient Y(positive) nuclei are found, for the diagnosis of selected numerical chromosome abnormalities.     

No Evidence of Hepatitis E Virus Infection in Farmed Deer in Germany

Hepatitis E virus (HEV) is a zoonotic agent, which is mainly transmitted by consumption of undercooked meat products originating from infected animals. Domestic pigs and wild boars are the major animal reservoirs, but HEV infections have been also repeatedly described in wild deer species. However, farmed deer has been only sparsely investigated so far. Here, 108 blood and 106 liver samples from fallow deer, red deer, and sika deer strictly hold in game enclosures from 11 farms in Germany were analyzed for markers of HEV infection. Using a commercial double antigen sandwich ELISA, 3/108 (2.7%) serum samples were scored borderline for HEV-specific antibodies, whereas the remaining samples were negative. No HEV-RNA (0%) was detected in the 106 liver samples. The results suggest a low risk of HEV infection in farmed deer in Germany.     

汽车尾气对人精子运动能力影响探讨

采用［日］岛津AA－670型原子吸收分光光度计,四唑氮蓝（Nitro－BT）染色法,及图象分析仪（MIAS－300）分析了交通警察和男性师生两人群精液的精子运动能力参数和血铅水平。结果显示：与对照组比较,交警组精子活动能力降低（P＜0.05）,精子琥珀酸脱氢酶（SDH）活性明显抑制（P＜0.001）。而且SDH活性与血铅水平呈负相关。认为交警精子活动能力下降可能和铅干扰精子线粒体能量代谢有关。     

低剂量微囊藻毒素亚急性肝毒性及其机理

为了研究低剂量微囊藻毒素 (MC)的亚急性肝毒性及其作用机理 ,将SD大鼠随机分为 4组 ,每组雌雄各 1 0只 ,分别经腹腔注射MC 0 ,4,8,1 2 μg·(kg·d) - 1,3 5天后观察各组大鼠血清酶学和肝脏病理学变化 .用原位末端标记法 (TUNEL)观察肝细胞凋亡 ,并用ABC免疫组化技术检测肝脏增殖细胞核抗原 (PCNA) .结果表明染毒组大鼠血清γ 谷氨酰转移酶 (GGT)活力和全血谷胱甘肽 (GSH)浓度下降 ,血清乳酸脱氢酶 (LDH)和谷草转氨酶(AST)升高 ,谷丙转氨酶 (ALT)未见显著变化 .染毒组大鼠肝脏出现特征性的形态学变化 ,并有活跃的肝细胞凋亡和增生并存的现象 .说明微囊藻毒素引起的氧化损伤和肝细胞凋亡可能是其致肝脏毒性的原因     

珠江三角洲地区男性血液中多溴联苯醚暴露水平与精液质量的研究

采集了珠江三角洲地区103名成年男性的血液和精液样本,运用冷冻干燥-ASE萃取-GC-MS方法测定了血液中多溴联苯醚(PBDEs)含量,并按照WHO的精子质量检测标准及仪器操作规范检测精液质量,分析了血液中PBDEs与男性精液质量之间的相关性,同时,探讨了不同因素与血液中PBDEs含量之间的关系.结果表明,不同年龄对血液中PBDEs含量的影响未呈现一致的趋势,除BDE-47外,不同体重指数(BMI)人群组单个PBDEs含量差异不显著,吸烟者血液中PBDEs含量高于不吸烟者,饮酒者和不饮酒者则与之相反,随着学历升高,PBDEs含量水平未有递增或递减的一致规律;BDE-153、BDE-154和BDE-183含量与精液浓度显著负相关,而PBDEs与精子活动性不相关.因此,血液中PBDEs含量与珠江三角洲地区男性精液质量无显著相关性.