Microvillar enzyme analysis in amniotic fluid and the prenatal diagnosis of cystic fibrosis

The activities of two microvillar enzymes, gamma-glutamyltranspeptidase (GGTP) and alkaline phosphatase (ALP) have been determined in amniotic fluid (AF) samples from 39 pregnancies with a 1-in-4 risk of cystic fibrosis. Seventeen of these were investigated prospectively. A reduced proportion of the fetal specific intestinal ALP isoenzyme was found in 7 of a total of 13 pregnancies with cystic fibrosis and in one pregnancy of confirmed normal outcome. Eight of the affected pregnancies were tested for AF GGTP activity and depressed levels were found in 15. None of the 3 liveborn cystic fibrosis cases in the prospective series was identified by the ALP assay although 2 had significantly reduced GGTP activity. There were several amniotic fluid samples from cases of cystic fibrosis, trisomy 18 and normal outcome which had discordant GGTP and ALP results. Four of the 6 cases of cystic fibrosis misclassified by the ALP assay had amniocentesis at 15 or 16 weeks gestation. Evidence is presented which confirms a previous suggestion that amniocentesis after 17 weeks gestation improves the predictability of the ALP isoenzyme assay for the prenatal diagnosis of cystic fibrosis.     

Prenatal diagnosis of cystic fibrosis using a monoclonal antibody specific for intestinal alkaline phosphatase

A monoclonal antibody (AAP-1), specific for the intestinal isoenzyme of alkaline phosphatase (ALP), has been used to develop an immunoassay for amniotic fluid samples. Values in the immunoassay correlated closely with those obtained by direct determination of phenylalanine-inhibitable ALP. A panel of 124 control second-trimester amniotic fluids and 21 fluids with a 1 in 4 risk of a cystic fibrosis fetus were examined in the immunoassay. Eight of 10 affected cases had values below an arbitrary cut-off of one third median, while all the non-affected cases were above this level. Almost identical results were obtained by enzymatic determination of phenylalanine-inhibitable ALP. However, in both systems the false positive rate (control fluids with values below one third median), was unacceptably high. It is pointed out that at present the most effective system for the prenatal diagnosis of cystic fibrosis is achieved by measuring the ratio of intestinal to total ALP in amniotic fluid supernatants. This is probably best effected by enzymatic assay in the presence of phenylalanine and homoarginine inhibition.     

A comparative study of microvillar enzyme activities in the prenatal diagnosis of cystic fibrosis

The potential of four enzyme-based analytical systems has been compared in the secondtrimester prenatal diagnosis of cystic fibrosis (CF). Direct activity measurements were made of γ-glutamyltranspeptidase (GGTP), aminopeptidase M (APM) and the intestinal isoenzyme of alkaline phosphatase (ALP). In the fourth system the proportions of total ALP inhibited by phenylalanine and homoarginine, respectively, were assessed. Each system was applied to amniotic fluid samples from 94 pregnancies with al in 4 risk of CF, divided into retrospective (36) and prospective (58) series. No system gave an absolute separation of affected from unaffected cases. Measurement of APM and intestinal ALP (phenylalanine-inhibitable ALP) gave a better detection rate for CF (35 of 41 cases, 85 per cent) than did measurement of GGTP (63 per cent) or assessment of ALP proportions (76 per cent). APM had a lower false positive rate (4 per cent) than intestinal ALP (8 per cent). For both the latter systems the detection rate of CF rose to 96 per cent (25 of 26), if gestations less than 17 weeks were excluded.     

Amniotic fluid microvillar enzyme activities in the early detection of fetal abnormalities

Measurement of the microvillar enzymes, γ-glutamyltranspeptidase (GGTP), aminopeptidase M (APM) and alkaline phosphatase (ALP), in amniotic fluid supernatant has been proposed as a method for the early prenatal diagnosis of cystic fibrosis. The activities of these enzymes in a series of other fetal abnormalities have now been examined. GGTP activities were below the 5th percentile in 28 out of 54 cases of trisomy 21 and 9 of 14 cases of trisomy 18, while APM values were below this cut-off in 26 of 54 cases of trisomy 21 and 8 of 14 cases of trisomy 18. Abnormal ALP isoenzyme ratios were found in 6 of 54 cases of trisomy 21 and 4 of 14 cases of trisomy 18. If prenatal cytogenetic studies are routinely carried out on amniotic fluid cells, the occasional confounding effect of abnormal microvillar enzymes associated with fetal trisomies rather than with cystic fibrosis should be avoided.     

Alkaline phosphatase activity in amniotic fluid in pregnancies with fetal disorders

Alkaline phosphatase (ALP) activity was determined in 255 amniotic fluid samples collected by amniocentesis between 15 and 39 weeks of gestation. The samples were originally used for chromosomal analysis and/or alpha-fetoprotein measurements. The mean ALP activity in early amniotic fluid from pregnancies with fetal trisomy 18 and 21 syndromes was half of that found in the controls. Highly elevated ALP activity (over 10 times the median level) was found in 14 samples. Two of these pregnancies had normal outcome. Three samples were from pregnancies with intrauterine fetal death. Fetal disorders, including abdominal wall defect (four cases), Meckel's syndrome (two), hydrops fetalis syndrome (two) and genital anomaly (one), were observed in nine cases. Moderately elevated ALP activity (over three times the median) was found in 10 cases, including five pregnancies with a preterm labour shortly after the sample collection. The results indicate that elevated ALP activity in the third trimester amniotic fluid is often associated with fetal disorders.     

Prenatal diagnosis of cystic fibrosis: I. Prospective study of 51 pregnancies

A prenatal diagnosis was performed in 51 pregnancies with a 1-in-4 risk of having a child with cystic fibrosis. The criteria for determining an affected fetus were based on the results of alkaline phosphatase (ALP) residual activity after inhibition by phenylalanine and by homoarginine, of total ALP activity, and of gamma-glutamyltranspeptidase (GGTP) activity in the amniotic fluid taken between 16 and 19 weeks of pregnancy. The chromosomal analysis of amniotic fluid cells showed trisomy 13 in one case which was excluded from the analysis of biochemical assays. The biochemical assays were in the normal ranges in the amniotic fluid of 35 pregnancies: 26 have reached term and a normal infant has been born, 9 are still in progress. A deficiency of the ALP phenylalanine-inhibitable form, depressed values of total ALP and GGTP were observed in the amniotic fluid of 15 pregnancies: one pregnancy went to term and the infant had CF, in 14 cases the pregnancy was terminated, and meconium ileus was observed in ten of these cases. It was observed that the changes towards abnormal values became more significant with advancing gestational age and that 18 weeks appeared to be the optimum time for diagnostic amniocentesis.     

Amniotic fluid disaccharidases in the prenatal detection of cystic fibrosis

Intestinal disaccharidases in amniotic fluid were studied in 41 pregnancies with a recurrence risk for cystic fibrosis (CF). In 11 out of 13 pregnancies with CF fetuses the maltase and sucrase activities were either below the control range (8 cases) or below the 10th percentile of control values (3 cases). Trehalase and lactase were slightly less informative indicators of CF. Of the other 28 pregnancies 3 had low amniotic fluid activities of several intestinal enzymes and were terminated, 12 resulted in the birth of a healthy child and 13 are continuing. The findings in fetal CF suggest an impairment of the defaecation of intestinal contents into the amniotic fluid. Reduced or low amniotic fluid disaccharidase activities were also found in other fetal disorders with demonstrated or presumed intestinal anomalies: e.g. anal atresia (2 cases), anencephaly (3 our of the 7 cases), trisomy 13 (5 cases), trisomy 18 (3 of the 5 cases) and trisomy 21 (19 of the 22 cases). Reduced amniotic fluid disaccharidase activities, although not specific for CF, are highly informative in pregnancies at high risk for CF. Using the 10th percentile of the normal range for amniotic fluid disaccharidase activities as an action line, the sensitivity of CF detection is estimated at 80 to 90 per cent, which could in high risk pregnancies reduce the risk of having another affected child from 1 in 4 to 1 in 20.     

Amniotic fluid digestive enzymes: Diagnostic value in fetal gastrointestinal obstructions

The diagnostic value of amniotic fluid gamma-glutamyl-transpeptidase (GGTP) and intestinal alkaline phos-phatase (iALP) was evaluated in 55 patients who underwent amniocentesis for karyotyping because fetal gastric or small bowel dilatation had been detected by ultrasound. Gastrointestinal malformation was confirmed in 46 cases and there was no gastrointestinal anomaly in nine cases. Prenatal ultrasound was suggestive of gastroduodenal dilatation in 34 cases (group I) and small bowel dilatation in 21 cases (group II). In group I, amniotic fluid GGTP above the 99th percentile was 71 per cent sensitive and 100 per cent specific for a true anatomical defect of the digestive tract (mainly duodenal atresia). In group II, high levels of GGTP and/or iALP were 69 per cent sensitive and 83 per cent specific for a fetal digestive tract anomaly. In other words, when digestive tract dilatations were diagnosed by prenatal sonography, abnormal amniotic fluid enzyme activities were strongly suggestive of such an anomaly, the possibility of which was not precluded by normal amniotic fluid iALP and GGTP activities. But amniotic fluid digestive enzyme activities do not help in defining the prognosis.     

Prenatal diagnosis of intestinal obstruction in the third trimester using amniotic fluid bile acid analysis

Total bile acid concentration in third trimester amniotic fluid was determined using an enzymatic technique and fluorometry. The technique was successfully applied to the prenatal diagnosis of intestinal obstruction in a 33-week fetus from a pregnancy presenting with polyhydramnios.     

Detection and enumeration of colonic mucosal cells in amniotic fluid using a colon epithelial-specific monoclonal antibody

Since its introduction, prenatal diagnosis of chromosomal and metabolic disorder by mid-trimester amniocentesis has relied upon the use of a mixture of fetal cells obtained from amniotic fluid. Little knowledge has been gained in the sorting of these cells for diagnosis of tissue-specific disorders. In an attempt to determine the contribution of fetal colonic mucosal cells to the overall amniocyte population, we used the colonic epithelial-specific monoclonal antibody (MC-Ab) 7E₁₂H₁₂, IgM isotype. Specimens of the small intestine, colon, buccal mucosa, kidney, urinary bladder, and umbilical cord were obtained from electively aborted normal fetuses of 12–28 weeks' gestation. All of these specimens were examined with 7E₁₂H₁₂ by the immunoperoxidase technique. The MC-Ab reacted with the colonic epithelial cells but not with any of the other tissues. In addition, 40 amniotic fluid samples obtained from women between 16 and 18 weeks of gestation, who underwent amniocentesis because of advanced maternal age, were tested using a fluorescent activated cell sorter. Among the amniotic fluid specimens examined, 18·4 ± 10·3 percent cells reacted with 7E₁₂H₁₂. Double immunofluorescence studies revealed that all Mc-Ab-stained cells contained secretory component, confirming that they were epithelial in origin. All fetuses whose amniotic fluid was analysed had normal karyotypes and amniotic fluid alpha-fetoprctein levels that were also normal. This study demonstrates that cell-specific Mc-Ab can be used to detect colon cells in the amniotic fluid and that colon cells contribute significant numbers in the mixture of amniotic fluid cells. This technique could be helpful in the prenatal diagnosis of disorders in which the flow of amniotic fluid through the fetal intestine is impaired, such as cystic fibrosis, imperforate anus, Hirschsprung aganglionic megacolon, and intestinal atresia.     

藻毒素对鱼类肝脏的毒理学效应

以鱼类肝脏为靶器官,研究了藻毒素对鱼类的毒性影响.结果表明,藻毒素在注射和口服两种不同的染毒方式下,均可引起受试鱼类发生急性中毒死亡.对中毒鱼类进行解剖分析发现,受试鱼类的肝/体重比高于对照组.说明无论哪种给药方式,藻毒素都会迅速地、专一地作用于鱼类肝脏,引起鱼类肝脏充血红肿,出现血斑.通过对鱼类肝脏组织碱性磷酸酶(ALP)同功酶谱的分析,可以发现在两种不同染毒条件下藻毒素各剂量组的ALP同功酶谱与对照组相同,均有ALP₁1～ALP₅5条带,只是高剂量组较低剂量组ALP₁～ALP₃ 3条带的着色深,并可使ALP同功酶各组分的相对活性上升.试验结果显示,藻毒素具有极高的细胞选择性和专一生物活性,其在生物机体内所引发的许多细胞学变化主要是对生物体肝脏酶系统的破坏所致,必须引起人们的重视.     

First trimester prenatal diagnosis of Sandhoff's disease

Chorionic villus sampling was performed on two patients with a previous family history of Sandhoff's disease. Total β-hexosaminidase (Hex) activity in case 1 was within the normal range (case 1: 6365 μmol/h/g protein; control range: 3227-24 495/miol/h/g protein). The β-hexosaminidase isoenzyme pattern was found to be normal. These results were confirmed on cultured amniotic fluid cells. In case 2, the total Hex activity was 672 μmol/h/g protein, i.e., 7 per cent of the control mean (10 085 μmol/h/g protein), and chromatography demonstrated that more than 50 per cent of this activity was due to the abnormal isoenzyme β-hexosaminidase S (Hex S). The fetus was predicted to be affected by Sandhoff's disease and this was confirmed on fetal tissues after termination of pregnancy. This study demonstrates that a fetus affected by Sandhof's disease can be reliably diagnosed during the first trimester of pregnancy.     

Amniotic fluid enzymes in pregnancies with trisomy 21

The activities of a range of microvillous enzymes in amniotic fluid from normal pregnancies (n = 213) and those complicated by trisomy 21 (n = 26) were compared in a prospective study. Using a centrifugal analyser, the activities of leucine aminopeptidase (LAP), gamma glutamyl transferase (GGT), aspartate transferase (AST), and isoenzymes of alkaline phosphate (ALP) were measured in amniotic fluid alongside alpha fetoprotein (AFP) levels. Of the markers studied, LAP was found to be the most reliable indicator of trisomy 21. Using levels below the 5th percentile, LAP showed sensitivity 73 per cent, specificity 94 per cent, and predictive value positive 63 percent. Although these tests would not replace karyotyping in all cases, the measurement of LAP could be useful as a rapid initial screening test, particularly when amniocentesis is performed for indications other than chromosomal abnormalities.     

Fetal intestinal microvilli in human amniotic fluid

The intestinal microvilli of fetal origin in human amniotic fluid were purified by Ca²⁺ precipitation of contaminating organelles followed by differential centrifugation of microvillar membranes. In the purified preparation, the specific activity of the microvillar marker-enzymes maltase and sucrase increased about 77-fold over that in cell-free amniotic fluid. Significant contamination of the purified preparation by endoplasmic reticulum (microsomes) and lysosomes was ruled out on the basis of a low content of the marker enzymes glucose-6-phosphatase (microsomes) and acid phosphatase (lysosomes). Amniotic fluid microvilli contain typical enzymes of the fetal intestine including maltase, sucrase, trehalase, alkaline phosphatase and γ-glutamyltransferase, and their morphology by electron microscopy resembles that of vesiculated intestinal microvilli. Prenatal detection of genetic diseases due to a deficiency of a protein expressed in these membranes or associated to abnormal microvilli seems feasible.     

Prenatal diagnosis of herlitz junctional epidermolysis bullosa by amniocentesis

Herlitz junctional epidermolysis bullosa (HJEB) is a severe blistering disorder which usually results in death during infancy. We have previously shown that the anchoring filament protein laminin-5 (kalinin/nicein), which mediates keratinocyte attachment and dermal–epidermal cohesion, is abnormally expressed in individuals with HJEB. Laminin-5 was detected by Western blot analysis in amniotic fluid from 44 consecutive normal secondtrimester control pregnancies, but was undetectable in second-trimester amniotic fluid from four pregnancies with fetuses affected by HJEB. In one case of severe non-Herlitz JEB, laminin-5 was detected in both amniotic fluid and skin. In human amniotic fluid, the laminin-5 a3 subunit was processed to a major 165 kD species and a minor 145 kD species and the β2 subunit was partially processed to 105 kD. Although laminin-5 was covalently associated with laminin-6 (K-laminin) in amniotic membrane, no covalent interaction was detected in amniotic fluid. Laminin-5 from amniotic fluid strongly supported keratinocyte attachment. These results suggest that Western blot analysis of second-trimester amniotic fluid is useful in determining the prenatal diagnosis of HJEB and that laminin-5 may serve a physiologically important function in amniotic fluid.     

Glial fibrillary acidic protein in amniotic fluids from pregnancies with fetal neural tube defects

The glial fibrillary acidic protein (GFAP) is the subunit protein of intermediate filaments in astrocytes and closely related cell types. By means of an enzyme immunoassay we have determined the concentration of GFAP in amniotic fluids from normal pregnancies and from pregnancies complicated by various fetal malformations. The group of 20 cases of fetal anencephaly had a significantly higher mean amniotic fluid GFAP concentration (115 μg/1±133.6 (S.D.), range 6–378 μg/1) than the control group of 117 normal pregnancies (13 μg/1k±5.5 (S.D.), range 0–31 μg/1), (P<0.001). Two cases of fetal encephalocele likewise had very high amniotic fluid GFAP concentrations. None of the other cases of fetal malformations investigated, including 12 cases of spina bifida, had increased amniotic fluid GFAP concentrations. We conclude that determination of the amniotic fluid GFAP concentration may give additional information in the prenatal diagnosis of fetal nervous system malformations.     

Maternal contamination of amniotic fluid demonstrated by DNA analysis

DNA from 16 sets of samples comprising DNA from uncultured amniotic fluid cells, cultured amniotic fluid cells, fetal tissue, and maternal blood was analysed by the polymerase chain reaction (PCR) with AC-repeat primers. The analysis was performed to investigate the presence of contaminating maternal cells in amniotic fluid which would affect the reliability of DNA studies for prenatal diagnosis. In three sets, maternal contamination of uncultured amniotic fluid cells was detected. In one of the three sets, maternal contamination was present in both uncultured and cultured amniotic fluid cells. The use of amniotic fluid cells as a source of DNA for prenatal diagnosis should be limited to cases where the purity of the DNA can be demonstrated prior to the diagnostic test being performed. This limitation in the use of amniotic fluid DNA also extends to other forms of diagnosis relying on the purity of amniotic fluid samples, particularly the new in situ hybridization methods currently being developed.     

HUMAN CHORIONIC GONADOTROPHIN AND ALPHA-FETOPROTEIN LEVELS IN MATCHED SAMPLES OF AMNIOTIC FLUID,EXTRAEMBRYONIC COELOMIC FLUID,AND MATERNAL SERUM IN THE FIRST TRIMESTER OF PREGNANCY

Separately identified samples of amniotic fluid and extraembryonic coelomic fluid obtained by high resolution transvaginal ultrasound-guided amniocentesis from 32 women between 7 and 12 weeks of pregnancy were analysed for human chorionic gonadotrophin (hCG) and alpha-fetoprotein (AFP). There was a highly significant difference between the hCG levels in amniotic fluid (median level 6.3 U/ml; range 1.6–310.0 U/ml) and those in extraembryonic coelomic fluid (median level 400.0U/ml; range 135.0–2250.0U/ml) (p<0.001; Mann-Whitney U/–test). The levels of AFP were very similar in amniotic fluid (median 26.0 kU/ml; range 10.0–116.5 kU/ml) and extraembryonic coelomic fluid (median level 24.1 kU/ml; range 12.4–94.4 kU/ml).     

First-trimester diagnosis of hypophosphatasia. Importance of gestational age and of purity of CV samples

Prenatal diagnosis of hypophosphatasia was made by alkaline phosphatase (ALP) assay on a chorionic villous sample taken in the first trimester. Very low activities of the LBK isoenzyrnes indicated an affected fetus. Diagnosis was confirmed at 12 weeks of gestation by measurement of LBK isoenzyme activities in fetal bone tissue. In control chorionic villous samples an inverse relation was observed between LBK and placental ALP percentage during gestational age. High LBK ALP activities are observed in decidual tissue. Chorionic villous tissue must not be sampled after 12 weeks of gestation and decidual tissue must be excluded from the sample.     

Correct prenatal diagnosis of a hurler fetus where amniotic fluid cell cultures were of maternal origin

Contamination of amniotic fluid cell cultures by maternal cells can be expected to lead to misdiagnosis of fetal genotype in 0·1 to 0·5/100 cultures, when assays are carried out directly on cultured cells. Chemical analysis of the cell-free amniotic fluid supernatant may overcome this source of error and has the added advantages of speed and independence from amniotic cell culture failure. We describe a pregnancy at risk for Hurler's disease where amniotic cells cultured at amniocentesis had a female karyotype and an α-iduronidase activity towards both phenyl and 4-methylumbelliferyl substrates at the lower end of the normal range, suggesting a heterozygous fetus. An affected fetus was predicted, however, because of a high concentration of dermatan sulphate in the amniotic fluid. The discrepancy between these findings was shown to be due to maternal cell contamination of amniotic fluid cell cultures by the birth of a male infant with Hurler's disease.