Cultured amniotic fluid cells for prenatal diagnosis of lysosomal storage disorders: a methodological study

The influence of culture conditions on the ultrastructure and enzyme activities of amniotic fluid cells are reported. Morphological changes were determined as a function of the number of lysosomal-like inclusion bodies per cell, and these results correlated to the activity of beta-hexosaminidase, alpha-mannosidase, beta-glucuronidase, arylsulphatase C and 5' nucleotidase. The parameters examined were pH of the culture media, type of media, increasing cell passage and day of harvest. Our results indicate that enzyme activities are less sensitive to changes in culture conditions as compared to ultrastructural changes. We therefore recommend that in order to obtain reliable ultrastructural results for the diagnosis of storage disorders, cultures should be grown in MEM as the culture medium, the pH of the medium carefully monitored to remain below pH 7.4, examining the cultures no later than the eighth cell passage and no later than the 10th day after subculture.     

Study of the choice between sampling of the chorionic villi and the amniotic fluid in prenatal diagnosis

We are reporting the results of a 21-month study during which 653 couples were seen in consultation at the prenatal diagnostic center of the University Hospital in Amiens, referred by their physician. 171 patients presented a theoretical term under 11 weeks of amenorrhea, for whom the choice between chorionic villi biopsy or amniotic fluid tap was possible. The different situations and results are compared for each method. The rate of fetal death was 5.4 per cent for chorionic villi biopsy and 1.5 per cent for amniotic fluid tap.     

The diagnostic performance of cytogenetic investigation in amniotic fluid cells and chorionic villi.

First-trimester chorionic villus sampling has not reached the popularity of second-trimester amniocentesis in prenatal cytogenetic diagnosis, in contrast to initial expectations. We investigated whether a difference in the diagnostic performances of cytogenetic investigation in amniotic fluid (AF) cells and chorionic villi in favour of AF-cells might justify this. Diagnostic performance was measured as laboratory failure rate, karyotype quality (G-band score, rate of follow-up samples, rate of wrong diagnoses), and karyotype representativity (rate of follow-up samples, rate of wrong diagnoses). From 1993-1999, 11 883 AF-samples were investigated (AF-cells). In chorionic villi, short term culture preparations solely were karyotyped from 1993-1996 (n=3499) (STC-villi), short and long-term culture preparations simultaneously provided a sufficient amount of tissue being available from 1997 onwards (n=1829) ((STC+LTC)-villi). Laboratory failure rates were the same after amniocentesis (0.40%) and chorionic villus sampling (0.50%). G-band scores (mean+/-SD) were equal in AF-cells (373+/-38.1) and LTC-villi (364+/-32.6) but significantly lower in STC-villi (311+/-34.6) (p=0.001). Follow-up sampling rates because of quality reasons were the same in AF-cells (0.14%), STC- villi (0.13%) and (STC+LTC)-villi (0.11%). Two wrong diagnoses turned up among AF-cells. Follow-up sampling rates because of representativity reasons differed significantly between AF-cells (0.10%), (STC+LTC)-villi (1.31%), and STC-villi (1.99%) (p<0.001). However, the ratios of the total numbers of follow-up samples and uncertain or abnormal cytogenetic results in STC, and (STC+LTC)-villi at cytogenetic risks > or =3% (0.132 and 0.160, respectively) were equal to that in AF-cells at risks <3% (0.155). Two wrong diagnoses were made in STC-villi. Diagnostic performance improved in the rank order of STC-villi, (STC+LTC)-villi and AF-cells. At cytogenetic risks > or =3%, (STC+LTC)-villi showed a diagnostic performance equal to that in AF-cells. This might justify a selective use of chorionic villus sampling.     

改良羊水原位培养法进行产前诊断的研究

目的 :探讨改良羊水原位培养法在产前诊断中的价值。方法 :抽取 138例孕16～ 30周孕妇的羊水 ,用全营养培养基 4ml+羊水细胞混悬液 5ml接种培养 ,适时改良法原位收获制片、分析。结果 :138例羊水培养均获成功 ,成功率 10 0 % ;平均培养时间为 7天 ;平均可分析核型数 38个 ,发现染色体异常 4例。结论 :用此法培养羊水成功率高 ,培养时间短 ,可分析核型多 ,培养适用范围宽 ,可满足临床产前诊断的要求     

Prenatal diagnosis of cystinosis by quantitative measurement of cystine in chorionic villi and cultured cells

OBJECTIVE: Prenatal diagnosis of cystinosis has been available for over 30 years by the incubation of cultured amniotic cells, intact chorionic villi and cultured chorionic cells with [35S]-cystine followed by thin layer chromatography and visual inspection of autoradiographs of the chromatograms for cystine. This method has proved highly reliable but because of the short half-life of [35S]-cystine, its cost and the length of the assay procedure, an alternative method of diagnosis was investigated. METHOD: Cystine was quantitatively measured in chorionic villi directly, in cultured chorionic villi and cultured amniotic cells using a cystine-binding protein from Escherichia coli. RESULTS: Twelve pregnancies at risk for cystinosis were monitored by both the [35S]-cystine uptake method and the new quantitative method in uncultured chorionic villi. There was no discrepancy between the results obtained with the two methods and subsequently 15 pregnancies have been monitored by the quantitative assay only--13 in chorionic villi directly, 1 in cultured chorionic villi cells and 1 in cultured amniotic cells. Grossly elevated levels of cystine were found in seven pregnancies. CONCLUSION: An unequivocal diagnosis of cystinosis can be made within 24 h of sampling by the quantitative measurement of cystine in uncultured chorionic villi.     

Discriminant analysis for assessing the value of amniotic fluid microvillar enzymes in the prenatal diagnosis of cystic fibrosis.

We have analysed the sensitivity, specificity, and reliability of biochemical diagnosis based on microvillar membrane enzyme assay and using discriminant analysis in amniotic fluid samples obtained from 54 pregnancies at high risk for cystic fibrosis and 125 normal pregnancies. Our results show that amniotic fluid trehalase, alkaline phosphatase, alkaline phosphatase isoenzymes and gamma-glutamyltransferase enzyme activities measured during 16-20 gestational weeks, in spite of their non-specificity for cystic fibrosis, have a very good predictive value for fetal cystic fibrosis or exclude the possibility of the disease. Overall enzyme activity analysis provided over 90 per cent reliability of the method.     

Mucolipidosis type IV: accumulation of phospholipids and gangliosides in cultured amniotic cells. A tool for prenatal diagnosis.

Cultured amniotic fluid cells from two mucolipidosis type IV (MLIV)-affected fetuses demonstrated accumulation of phospholipids and gangliosides when compared with normal controls. Like cultured skin fibroblasts from MLIV patients, cultured amniotic cells from the affected fetuses accumulated primarily lyso phospholipids and this could be demonstrated by radioactive labelling with appropriate precursors, either inorganic phosphate or oleic acid. Furthermore, like cultured skin fibroblasts, there was significant retention of exogenously supplied GD1A ganglioside in the affected amniotic cells. This storage was previously demonstrated to be unique to MLIV and thus can be used at present as a specific procedure for prenatal diagnosis of MLIV.     

羊水细胞培养用于染色体病产前诊断64例分析

目的探讨用羊水细胞培养对孕中期孕妇进行产前诊断的可行性及必要性,防止染色体病患儿出生。方法2003年3月至2004年12月北京大学深圳医院采用羊水细胞培养G显带技术,对64例具有产前诊断指征的孕妇进行检查。结果发现1例世界罕见染色体异常核型46,XY,der(11)t(8;11)(q24;q25),58例正常核型,5例核型为多态,诊断结果与随访情况一致。结论羊水细胞培养进行产前诊断是十分安全而可靠的。     

Extraction of DNA from amniotic fluid cells for the early prenatal diagnosis of genetic disease

Ten-ml samples of amniotic fluid were taken from pregnancies being terminated at 8-14 weeks' gestation. DNA was extracted from the amniotic cells by sequential centrifugation and analysed using the polymerase chain reaction (PCR). Fifteen samples were analysed for evidence of maternal contamination using Mfd5 oligonucleotide primers for repeat polymorphisms. Ten amniotic fluid samples were tested for the Delta-F508 deletion characteristic of cystic fibrosis to demonstrate a diagnostic application for the technique. In each case, DNA extracted from fetal tissue from the same pregnancy was included in the controls. In 14 of the 15 cases tested with the Mfd5 primers, both the amniotic fluid DNA and the fetal DNA showed no evidence of contaminating DNA. In one case, neither the amniotic fluid cells nor the fetal cells yielded results. In nine of the ten cases tested with the Delta-F508 primers, the amniotic fluid cell DNA provided accurate information about the genetic status of the fetus; in the tenth, the fetal DNA failed to amplify. The results indicate that adequate DNA can be extracted from amniotic fluid from 8 weeks' gestation onward and these samples are suitable for prenatal diagnosis using PCR.     

Normal karyotype in cultured chorionic villus cells, but mosaicism in amniotic and fetal cells.

A case with a normal male karyotype in cultured chorionic villus cells, but 46,XY/45,X/46,X,i(Yq) mosaicism in amniotic and fetal tissue is reported. The fetus was a phenotypic male. Pathological examination revealed discrete features, which might indicate a syndrome, and histological examination showed large, bright cells in the tubules of the testes. Possible explanations for discordance between the karyotype of embryonic and extraembryonic tissue are discussed.     

Early prenatal diagnosis of 21-hydroxylase deficiency using amniotic fluid 17-hydroxyprogesterone determination and DNA probes

The results of early prenatal diagnoses of congenital adrenal hyperplasia are reported. The determination of 17-hydroxyprogesterone values in amniotic fluid taken transabdominally at 11 weeks of gestation enabled prenatal diagnosis of congenital adrenal hyperplasia due to 21-hydroxylase (21-OH) deficiency. There is a clear-cut difference between normal and pathological values at that time of pregnancy. This method of diagnosis can be combined with genotyping of the fetus by HLA-DNA probes on chorionic villus sampling or can be used alone. Prenatal diagnosis with a 21-OH probe is possible when a preliminary study has demonstrated that the index case is homozygous for the deletion.     

Quality aspects of prenatal cytogenetic diagnosis: determining the effect of various factors involved in handling amniotic fluid and chorionic villus material for cytogenetic diagnosis

OBJECTIVES: To investigate the effect of factors involved in cell culturing and slide preparation of amniotic fluid (AF) and chorionic villus biopsies (CVB) for prenatal cytogenetic diagnosis. METHODS: The effect on the outcome of our standard AF cell culture procedure of volume and appearance of the submitted AF specimen, gynaecologist performing the amniocentesis, week of gestation in which the specimen was taken and culture medium was retrospectively investigated. In a prospective study controlled experimental variation was introduced in composition of fixative, relative humidity, temperature and airflow during slide preparation from primary CVB and AF in situ cultures. For evaluation, analysis of regression or variance was used. RESULTS: Provided that at least 0.8 mL AF per culture dish was admitted, none of the investigated factors appeared as critical resulting in unacceptable variation in outcome. Variation in appearance of the AF had a relatively major impact: bloody or brown AF resulted in a 3 days longer culture time.To a limited degree, metaphase quality of AF and CVB cells was affected by composition of fixative, relative humidity, ambient temperature and airflow during slide preparation. CONCLUSION: Current prenatal cytogenetic practice as described here appears in general to be robust and reliable. The investigated conditions are not critical within the investigated range. Expensive measures for fine control of these conditions are, therefore, not required.     

Early genetic amniocentesis and chorionic villus sampling. Expanding the opportunities for early prenatal diagnosis

At centers where chorionic villus sampling (CVS) programs are operational, first-trimester prenatal diagnosis has been shown to have many advantages, both medical and psychologic. However, most medical centers do not have CVS capability, nor are all patients candidates for CVS. We investigated the feasibility of performing very early genetic amniocentesis (9-13 weeks' gestational age). The results from those amniocenteses were compared to our own CVS data. In experienced hands, (1) CVS can be performed safely at 8-13 weeks, with the most technical ease at 9-11 weeks; (2) CVS or amniocentesis can be performed on many patients at 12-13 weeks or perhaps even earlier, although no accurate loss rates are available yet; (3) when technically feasible, CVS may be advantageous because of the much faster time period for cytogenetic results from direct preparation or short-term culture; and (4) in those patients on whom CVS cannot be performed, early amniocentesis in selected patients may offer the benefits of early diagnosis.     

Ultrasound and amniotic fluid alpha-fetoprotein in the prenatal diagnosis of spina bifida

Amniotic fluid alpha-fetoprotein (AFP) assays and detailed ultrasound examinations were performed in 376 prenatal patients at risk for a neural tube defect (high-risk group). In addition, 2436 patients who underwent amniocentesis for other indications underwent preamniocentesis ultrasound screening and amniotic fluid AFP assays (low-risk group). There were 10 neural tube defects in the high-risk group (7 open and 3 closed) and 3 in the low-risk group (all open). Two of the 3 closed defects were detected prenatally. The predictive value of an elevated AFP level for an abnormal fetus was much higher in the high-risk (6 of 6, 100%) than in the low-risk group (1 of 6, 17%). When both ultrasound and AFP assay results were normal, the chance of a normal outcome was very high in both the high- and low-risk groups (99.7 and 100%, respectively). It was of particular interest that in the low-risk group, the likelihood of an abnormal outcome in women with elevated AFP and a normal ultrasonogram was low (0 of 5).     

Isolation of amniotic fluid erythrocytes and their possible use in prenatal diagnosis

Many of the components of amniotic fluid have been found to be valuable in prenatal diagnosis; however, the presence of erythrocytes is usually considered undesirable. The authors have used buoyant density centrifugation on 96 amniotic fluid specimens from 70 subjects to isolate small numbers of erythrocytes from a majority of these specimens. Through immunofluorescence, these specimens were found to have higher levels of fetal hemoglobin-containing cells than the adult, indicating that the erythrocytes were at least in part fetal in origin. Thus, erythrocytes present in amniotic fluid could also be used in prenatal diagnosis.     

羊水细胞染色体产前诊断3495例结果分析

目的 探讨各种细胞遗传学产前诊断指征与胎儿染色体异常的关系。方法 2011年1月至2013年4月于重庆医科大学附属第一医院妇产科在知情同意的前提下,由超声引导对3495例孕中期高危孕妇（孕16~21+6周）行羊膜腔穿刺术,抽取适量羊水进行细胞培养及染色体核型分析。比较不同产前诊断指征与胎儿染色体异常核型检出率的关系。 结果 羊水培养成功3494例,成功率99.97%。检出异常核型120例,异常率为3.43%（120/3494）,其中染色体数目异常70例,结构异常31例,其他异常19例。各种产前诊断指征中,单纯高龄（分娩时孕妇年龄≥35岁）1498例,检出异常核型47例,异常检出率为3.14%;母血清学筛查高风险1560例,异常核型38例,检出率2.44%;无创产前DNA检测高风险38例,异常核型30例,检出率78.95%,后者检出率分别与前两者相比差异有统计学意义（P<0.05)。结论 掌握好各种产前诊断指征,对高危孕妇进行羊膜腔穿刺及染色体核型分析可有效提高胎儿染色体病的检出率,减少出生缺陷的发生。     

False elevation of amniotic fluid enzymes of relevance for prenatal diagnosis: creatine kinase measurement in relation to Duchenne muscular dystrophy

The creatine kinase activity of amniotic fluid was measured in samples collected at fetoscopy. In our first study, the control sample range was 0.25 IU/l, although four samples had activities of 35-85 IU/l. Elevated values did not correlate with the activities in the fetal or maternal circulations. Electrophoresis revealed the presence of the BB isozyme of creatine kinase rather than just the MM form as expected. This suggested that the source of the elevated enzyme activity was from the myometrium, damaged by insertion of the trocar and cannula. In a further series the first 2 ml of amniotic fluid withdrawn yielded a much higher creatine kinase activity than a second aliquot. A control series of such second samples (first 2 ml discarded) gave an activity range of 0-7 IU/l with no spuriously high values. This compares favourably with a series from single samplings taken by amniocentesis. Normal creatine kinase activities were found in the amniotic fluids from 20 pregnancies at risk of Duchenne muscular dystrophy. We conclude that for accurate measurement of amniotic fluid enzyme activity the first portion withdrawn should be discarded. Amniotic fluid creatine kinase activity is of no value for the prenatal diagnosis of Duchenne muscular dystrophy.     

Rapid prenatal diagnosis by fluorescent in situ hybridization of chorionic villi: an adjunct to long-term culture and karyotype.

OBJECTIVE: This series was designed to assess in a pilot study the feasibility of using fluorescence in situ hybridization on chorionic villi. STUDY DESIGN: We constructed probes derived from specific subregions of human chromosomes 21, 18, 13, X, and Y that give a single copylike signal when used in conjunction with suppression hybridization. RESULTS: In a blind series of 47 samples all, including one trisomy 21, were correctly identified. The samples were correctly classified as disomic for five chromosomes. CONCLUSIONS: The combination of chromosome-specific probe sets composed primarily of cosmid contigs and optimized hybridization and detection allowed accurate chromosome enumeration in uncultured human chorionic villi; these results are consistent with those obtained by traditional cytogenetic analysis and suggest a use for fluorescence in situ hybridization as an adjunct to karyotyping when rapid results are needed.