Protocol for prenatal diagnosis of cystic fibrosis based on studies of alkaline phosphatase isoenzymes

Amniotic fluid analysis of microvillar enzymes, including alkaline phosphatase (ALP) total activity and ALP isoenzymes, has been widely experimented with and used for the prenatal diagnosis of cystic fibrosis in the second trimester of gestation. Since the development of cystic fibrosis molecular analysis, interest in these biochemical tests has been maintained for those instances in which the pregnancy is not fully informative by restriction fragment length polymorphism analysis or DNA is not available from the index-affected child. However, recommended biochemical protocols do not provide clear-cut diagnostic results in a minority of cases. We have tested the reliability of cystic fibrosis biochemical prediction by ALP high-resolution electrophoresis and ALP kinetic studies after inactivation by urea. With this approach, all the amniotic fluid samples that had not been unambiguously classified as affected or unaffected by standard microvillar enzymes analysis were definitely categorized. The proposed method seems to improve the diagnostic accuracy in pregnancies with a one in four risk of resulting in a child with cystic fibrosis.     

Characteristics of maltase activity in amniotic fluid

The nature and origin of maltase activity present in amniotic fluid, and used as a marker enzyme in the prenatal monitoring of cystic fibrosis, has been studied. Using monoclonal antibodies against human intestinal disaccharidases and via heat inactivation experiments it is shown that the maltase activity found in amniotic fluids from pregnancies of 16-24 wk of gestational age originates completely from sucrase-isomaltase; no maltase-glucoamylase could be detected. With various monospecific antibodies the possible contribution of non-intestinal brush border enzymes to the total maltase pool could be excluded: neither renal nor lysosomal maltase appeared to be present.     

Systematic study of the hydrolysis of 4-methylumbelliferylguanidinobenzoate in plasma from patients with cystic fibrosis and controls

In contrast to the original reports by others, the hydrolysis of 4-methylumbelliferylguanidinobenzoate ((MUGB), and active-site titrant of serine proteases, was found not to be significantly different in plasma samples from patients with cystic fibrosis (CF), obligate heterozygotes, and normal controls. Since the steady-state turnover of substrate is the dominant term of MUGB hydrolysis, the assay in its present form does not meet the requirements of an active-site titration. The reproducibility and reliability of the assay are hampered by (1) high blank values due to non-specific hydrolysis, (2) the absence of a defined endpoint of the incubation, (3) the non-linearity of the assay with respect to plasma concentration, (4) the temperature-dependent evolution of esterase activity during incubation, and (5) most importantly the loss of activity during storage of plasma samples. Cleavage of MUGB catalysed by the imidazole ring of histidine accounts for a significant portion of the activity in plasma.     

Fetal echogenic bowel: parameters to be considered in differential diagnosis.

OBJECTIVES: To evaluate the extent that associated findings aid in the differential diagnosis and/or prognosis of fetal echogenic bowel. METHODS: Medical history, obstetric records and outcome details were examined for 131 consecutive pregnancies with fetal hyperechogenic bowel. RESULTS: In 62 (47%) cases, there were no visible anomalies other than hyperechogenic bowel and no evidence of growth restriction. This group included four (7%) pregnancies with Down syndrome, 15 (24%) with infection or a recent episode of influenza and eight (13%) with blood staining of amniotic fluid. In the remaining 69 (53%) cases, hyperechogenic bowel was accompanied by hydrops or nuchal edema (n = 16, 12.2%), growth restriction (n = 9, 6.9%), other markers for chromosome anomalies (n = 33, 25.2%) or multiple structural anomalies (n = 11, 8.4%). In this group, the prevalence of Down syndrome was 12%, infection or influenza was reported in 14 (20%) cases and there was blood staining of amniotic fluid in seven (10%). Cystic fibrosis screening was performed in 65 (50%) pregnancies; the results were negative in all cases and clinical assessment did not indicate cystic fibrosis in any of the 91 infants who were born alive. Maternal serum screening was performed in 41 (31%) pregnancies. High alpha-fetoprotein levels were associated with multiple abnormalities or severe growth restriction. CONCLUSIONS: In many pregnancies with fetal hyperechogenic bowel, there are multiple factors that may explain these findings. Thus identification of one potential underlying cause should not preclude further testing. Once chromosome defects, cystic fibrosis, structural abnormalities, infection and growth restriction have been excluded, parents can be counseled that the prognosis is good, irrespective of the presence or absence of blood stained amniotic fluid.     

Enzyme analysis of amniotic fluid for prenatal diagnosis of cystic fibrosis in high-risk pregnancies

We determined the activity concentrations of alkaline phosphatase (ALP), ALP isoenzymes, gamma-glutamyltransferase (GGT), and alpha-glucosidase (AGL) in 1200 unselected amniotic fluids and in amniotic fluids from 40 pregnancies at high risk for cystic fibrosis (CF). From the results we established the normal range and CF-predictive cutoff values for these enzymes in the second trimester of pregnancy. In all predicted normal pregnancies that went to term, normal children were born. Among the predicted affected pregnancies, 14 were terminated and two went to term, one resulting in a CF-affected child and the other in a healthy child. Evidence for CF was found in all 13 aborted fetuses examined (the parents of one refused to allow autopsy). We noted no differences in the amniotic fluid enzyme activities for the Arab and various Jewish ethnic groups living in Israel. We conclude that prenatal diagnosis of CF among the Israeli population at risk for CF is feasible by means of a reliable, fast, and economic test in the second trimester of pregnancy.     

Proton magnetic resonance spectroscopy of human amniotic fluids sampled at 17–18 weeks of pregnancy in cases of decreased digestive enzyme activities and detected cystic fibrosis

Low digestive enzyme activities in human amniotic fluid can be observed in normal and disease-affected pregnancies: cystic fibrosis, trisomy 21, intestinal atresia. Amniotic fluids were analyzed by proton nuclear magnetic resonance (NMR) spectroscopy in order to specify prenatally the etiology of low digestive enzyme activities observed at 17–18 weeks of amenorrhea. A total of 114 amniotic fluid samples were collected at 17–18 weeks of amenorrhea. Karyotyping and assays of digestive enzyme activities were performed in all cases. Samples were divided into six groups according to enzyme activities and pathology. Proton spectra were retrospectively recorded. Many compounds, such as amino acids and carboxylic acids, were detected by NMR. The same resonance intensities (normalized to creatinine) were observed in the six groups. Nevertheless, an unidentified resonance at 1.05 ppm was detected in seven out of 13 cases of cystic fibrosis affected fetuses. The NMR spectra demonstrated the stability of the amniotic fluid composition at 17–18 weeks of amenorrhea, even when the fetus was affected by a disease such as trisomy 21 or intestinal atresia. The resonance associated with most cases of cystic fibrosis should be further investigated.     

Fetal neural tube defects detected by rocket-on-line immunoelectrophoresis of amniotic fluids

Immunization of rabbits with human fetal brain membranes evoked antisera with reactivity against D2-protein from brain and cerebrospinal fluid. In amniotic fluids from pregnancies with fetal neural tube defects the presence of D2-protein could be demonstrated by rocket-on-line immunoelectrophoresis. In a prospective study on 72 amniotic fluids with increased alphafetoprotein concentration neural tube defects were correctly predicted in 26 pregnancies. One false-negative and four false-positive pregnancies were encountered.D2-protein determination may thus have a role in antenatal diagnosis of neural tube malformations as a supplement to alphafetoprotein analysis.     

Differences in the carbohydrate moieties of plasma and amniotic fluid fibronectins as revealed by crossed immunoaffinity electrophoresis with concanavalin A

Human fibronectins isolated from pooled human plasma and amniotic fluid were studied as to differences in their carbohydrate moieties. The chemical analyses showed that amniotic fluid fibronectin is different from adult plasma fibronectin in carbohydrate content and composition while there seems to be no significant differences in amino acid composition. Crossed immunoaffinity electrophoresis with free concanavalin A, as well as rocket immunoelectrophoresis with immobilized concanavalin A intermediate gel, indicated that amniotic fluid fibronectin has little or no reactivity with this lectin while adult plasma fibronectin is strongly reactive. Fetal cord plasma fibronectin apparently interacted with concanavalin A, but its reactivity was weaker than that of adult plasma fibronectin. Fibronectin isolated from ascitic fluid of an ovarian cancer patient which was examined in additional experiments showed much weaker Con A-reactivity than fetal cord plasma fibronectin. These results suggest that fibronectins from various body fluids differ in their carbohydrate structures.     

Prolactin in amniotic fluid: its correlation with maternal plasma prolactin.

Prolactin concentrations in amniotic fluid from 319 women with normal pregnancies and 29 women with complicated pregnancies were determined by radioimmunoassay. Prolactin levels varied from 36 ng/ml to 1800 ng/ml mean +/- S.D. = 408 +/- 297) in the normal pregnancy group but showed no definite pattern of rise or fall during pregnancy. No difference in levels was found in complicated pregnancies. Prolactin concentrations in the plasma from 203 of these women were also assayed. The levels in the amniotic fluid were about 9 fold higher than those in the plasma. There was no significant correlation between amniotic fluid and plasma levels of prolactin.     

Evaluation of a commercial immunoenzymometric assay for alpha-fetoprotein

A two-site immunoenzymometric assay (Abbott Diagnostics) for alpha-fetoprotein (AFP) in maternal serum and amniotic fluid has been evaluated for its suitability as a screening test for open neural tube defects. In a retrospective study based on 190 pregnancies of known outcome, performance of the kit in measuring both serum and amniotic fluid AFP correlated well with that of an in-house radioimmunoassay. Of 39 pregnancies associated with open neural tube defects, only four would not have been detected by the use of sequential measurement of serum and amniotic fluid AFP (also essentially in agreement with results obtained by the RIA). We conclude that this immunoassay could form the basis for a screening program for antenatal detection of open neural tube defects.     

Study of soluble proteins from human amniotic fluid by fraction chromatography (author's transl]

The nature and origin of soluble proteins from human amniotic fluid have been investigated by gel filtration chromatography on Sepharose 6B and ion-exchange chromatography on Ecteola-cellulose. Each fraction has been studied by immunoelectrophoresis. Specific antisera against serum proteins, amniotic fluid proteins, fetal proteins and salivary proteins have been used. These various antisera showed that the amniotic fluid contains maternal proteins, but also more specific proteins from fetal origin such as beta2-microglobulin, urinary mucopolysaccharides, salivary proteins and carcinoembryonic antigen. These results not only confirm that amniotic proteins are essentially from maternal origin, but prove the important fetal contribution and emphasize the importance of the fetoplacental unit in the monitoring of high risk pregnancies.     

Isoenzymes of alkaline phosphatase in amniotic fluid: implications in prenatal screening for cystic fibrosis

Utilizing their differential susceptibilities to inhibitors and heat, we determined the amounts of the placental, liver, and fetal-intestinal isoenzyme forms of alkaline phosphatase in 143 samples of normal amniotic fluid obtained at 14 to 18 weeks' gestation (1). For reliable results, it was necessary to standardize inhibition profiles for each pure isoenzyme in amniotic fluid. Total activity and the absolute amounts of placental and fetal-intestinal activities were significantly related to gestational age (p less than 0.05). These relationships that were absent when activities were expressed as percentages of the total. The mean isoenzyme composition of the 143 samples, expressed as a percentage of total alkaline phosphatase activity, was: placental, 3.4%; liver, 9.8% (maximum, 47%); and fetal intestinal, 87% (minimum, 53%). The presence of phosphate in the assay medium (13.5 mmol/L) profoundly and differentially inhibited the isoenzymes of alkaline phosphatase and changed the inhibition profiles of the tissue-specific enzymes; thus, it would not be feasible to use inhibitors to differentiate the forms. We therefore propose a phosphate-free technique for quantifying the isoenzymes of alkaline phosphatase in amniotic fluid obtained at 14 to 18 weeks' gestation, to achieve the highest predictive values in a prenatal diagnostic test for cystic fibrosis.     

Accuracy of ultrasonography in evaluating amniotic fluid volume at less than 24 weeks' gestation.

The purpose of this investigation was to evaluate the accuracy of common sonographic techniques in assessing the amniotic fluid volume in pregnancies of less than 24 weeks' gestation. Patients at less than 24 weeks' gestation undergoing an amniocentesis for the placement of prostaglandin F2 alpha for termination (because of genetic or fetal anomalies, or both) were assessed for amniotic fluid volume. All fetuses were alive at the time of prostaglandin instillation. The amniotic fluid index and two-diameter pocket were used to determine the amniotic fluid volume. Prior to the prostaglandin instillation, the amniotic fluid volume was determined with para-aminohippurate using a diazo dye reaction with spectrophotometric analysis. The amniotic fluid volume was determined in 21 pregnancies between 15 and 24 weeks' gestation, yielding volumes ranging from 189 to 1840 ml. Using published standards for amniotic fluid volume in singleton pregnancies, oligohydramnios was present in three gestations, the volume was found to be normal in 15, and hydramnios complicated three pregnancies. The two-diameter pocket identified the amniotic fluid volumes correctly more often (18 of 21 [85.7%]) than the amniotic fluid index (10 of 21 [47.6%]) (P = 0.02). Normal amniotic fluid volume was identified in nine of 15 (60%) pregnancies by the amniotic fluid index and in 14 of 15 (93.3%) by the two-diameter pocket (P = not significant). Abnormal amniotic fluid volumes, oligohydramnios, and hydramnios were recognized more often by the two-diameter pocket (66.7%) than by the amniotic fluid index (1 of 6 [16.7%], P = not significant).     

An improved determination of total glycosaminoglycans in body fluids by formation of complexes with quinacrine: changes in amniotic fluid total glycosaminoglycans during normal pregnancies and in pregnancies at risk for mucopolysaccharidoses.

Acidic glycosaminoglycans form insoluble complexes with quinacrine and this has been exploited for their analysis in blood, urine and amniotic fluid. The method is specific for glycosaminoglycans including keratan sulphate and the samples do not have to be deproteinized. Values for normal urine, serum and amniotic fluid are presented. Urinary total glycosaminoglycans excreted by patients with mucopolysaccharidoses were also determined. The normal changes in amniotic fluid total glycosaminoglycans have been measured between 14 weeks' gestation and term, and values are given for amniotic fluid total glycosaminoglycans in several pregnancies at risk for mucopolysaccharidoses. It is suggested that this method is a potentially valuable analytical tool in the pre-natal diagnosis of mucopolysaccharidoses.     

Concanavalin A binding of alpha-fetoprotein in amniotic fluid as an aid in the diagnosis of neural tube defects

Concanavalin A nonreactive alpha-fetoprotein was determined in samples of amniotic fluid from 16 abnormal pregnancies complicated by anencephaly (7), open spina bifida (6), intra-uterine death (1), anencephaly with exomphalos (1), or open spina bifida with exomphalos (1), and in amniotic fluid from 50 normal pregnancies with gestational age between 13 and 24 weeks. In all 16 cases with fetal malformations, the proportion of nonreactive alpha-fetoprotein was significantly decreased (median 5.3%) as compared with amniotic fluid from pregnancies with a normal outcome (median 39.7%). The results confirm that this measurement is useful in the diagnosis of neural tube defects, especially when the concentration of alpha-fetoprotein in amniotic fluid is normal or only slightly above normal and gestational age is uncertain.     

Subjective versus objective evaluation of amniotic fluid volume of pregnancies of less than 24 weeks' gestation: how can we be accurate?

This study was undertaken to compare subjective versus objective ultrasonic evaluation of amniotic fluid volume in pregnancies of less than 24 weeks' gestation. Amniotic fluid volume was subjectively (visualization without ultrasonic measurements) and objectively (visual interpretation with ultrasonic measurements) evaluated in 42 singleton pregnancies undergoing termination. The actual amniotic fluid volume was then determined using a dye-dilution technique. The women evaluated were in their mid-20s, primarily African American, and between 15 and 23 weeks' gestation. There was no significant difference in the total number of correct estimates of amniotic fluid volume when the data were stratified by level of operator experience (P = .34), ultrasonic technique (P = .33), or the combined correct subjective versus combined correct objective estimates (P = .68). We have concluded that the accuracy of amniotic fluid volume assessment in pregnancies of less than 24 weeks is not influenced by the level of operator experience or the type of ultrasonic measurement.     

Immunoassay for the determination of surfactant apoprotein (SP-A) in human amniotic fluid: comparison with other indices of assessing foetal lung maturity.

The concentration of the major surfactant-associated protein SP-A (28-36 kDa) was determined in 73 amniotic fluid samples obtained from normal (n = 40) and complicated (n = 33) pregnancies. Lecithin/sphingomyelin (L/S) ratio and phosphatidylglycerol (PG) levels were also determined in all the samples by one-dimensional step-wise thin-layer chromatography. An enzyme-linked immunosorbent assay was used to determine human lung surfactant apoprotein SP-A. The amount of SP-A in human amniotic fluid increased as a function of gestational age from 8 mg l-1 at 36 weeks to 11.75 mg l-1 at 40-41 weeks of gestation. There was a significant difference (p less than 0.01) in amniotic fluid SP-A concentration from female (9.93 +/- 0.60 micrograms ml-1) compared to male (9.10 +/- 0.52 micrograms ml-1) foetuses. In amniotic fluid samples obtained from a group of complicated pregnancies, SP-A levels were significantly lower than in the normal group when adjusted for gestational age and sex of the foetus (p less than 0.05).     

The effects of maternal position on the amniotic fluid index.

OBJECTIVE: We investigated the influence of maternal positioning on the measurement of the amniotic fluid index (AFI) in third-trimester pregnancies. We wanted to determine whether a change in the position of the women, from supine-flat to supine-elevated, would influence the measurement of the AFI. METHODS: Each patient had AFI measurements obtained in both positions by the same investigator. RESULTS: We determined the values of the amniotic fluid index to be consistent in both positions for pregnancies with normal AFI measurements. CONCLUSIONS: Measurements of the amniotic fluid did not appear to be influenced by maternal position in the third trimester when the AFI was in the normal range.     

Binding of corticotropin-releasing hormone (CRH) in maternal and fetal plasma and in amniotic fluid

Placenta secretes corticotropin-releasing hormone (CRH) into the maternal and fetal circulation, but a CRH binding protein in plasma may decrease its biological activity. Using a charcoal adsorption method we found that 92% of added 125I-Tyr-CRH was bound to a binding protein in the nonpregnant plasma, 72% in the plasma at term pregnancy, 90% in umbilical cord plasma, 82% in the amniotic fluid in the second and 25% in the third trimester. CRH added to plasma inhibited the binding of 125I-Tyr-CRH over the concentration range of 0.1-8.8 nmol/l in plasma and of 0.1-2.2 nmol/l in amniotic fluid. There was a significant negative correlation (R = -0.80) between the binding capacity of the CRH-binding protein and CRH concentration in maternal plasma. Plasma or amniotic fluid was incubated with 125I-Tyr-CRH and subjected to gel filtration on Sephadex G-50. The bound radioactivity was eluted at the region of Mr 25-40 kDa and the unbound radioactivity at the location of synthetic CRH. Bound and unbound CRH concentrations were determined using charcoal adsorption method and gel filtration on Sephadex G-50 in ten maternal plasma samples at the third trimester of pregnancy. Following mean percentages were found to be bound: charcoal method 61.9 +/- 6.80% (SE) and gel filtration 62.8 +/- 6.33%. We conclude that the bulk of CRH is bound to a binding protein in maternal and fetoplacental circulation, whereas at term pregnancy the role of the binding is small in amniotic fluid.     

Evaluation and quality control of a monoclonal antibody based enzyme antigen immunoassay of acetylcholinesterase in amniotic fluid

We have evaluated a new monoclonal antibody based enzyme antigen immunoassay (EAIA) for acetylcholinesterase in amniotic fluid, intended for the detection of open fetal abnormalities, especially open neural tube defects. With a sample volume of 50 microliters, the detection limit is 0.7 nkat/l and linearity is found up to an amount 200-times the detection limit. CV's are less than or equal to 6.6% within assays and less than or equal to 10.3% between assays; recoveries averaged 103%. The upper limit of the reference range for amniotic fluids from non-affected pregnancies is 8.5 nkat/l for clear specimens and 25.0 nkat/l for haemolysed specimens. Amniotic fluid specimens from 1473 patients (1388 normal and 85 pathological pregnancies) were examined with both the new EAIA and the original procedure using a polyclonal rabbit antiserum. The two procedures showed identical sensitivities and specificities of about 100%, except for amniotic fluid samples containing haemolysed blood. For the latter the new EAIA showed a specificity of 100% compared to 55.6% for the original procedure. We conclude that the new EAIA is accurate and reproducible and shows, compared with the original procedure, an increased specificity in the analysis of amniotic fluid samples containing haemolysed blood.