Detection of patients with cancer by monoclonal antibody directed to lactoneotetraosylceramide (paragloboside).

A hybridoma producing monoclonal antibody (H11) directed to lactoneotetraosylceramide (paragloboside) has been established from spleen cells of a mouse immunized with paragloboside. The monoclonal antibody H11 (immunoglobulin M type) was selected from five clones showing different reactivities with paragloboside. The monoclonal antibody was highly specific to paragloboside and lacked reactivity with other glycolipids including glucosylceramide, lactosylceramide, globotriaosylceramide, globotetraosylceramide, gangliotriaosylceramide, gangliotetraosylceramide, and GalNAc beta 1-4[NeuAc alpha 2-3]Gal beta 1-4Glc beta 1-1Cer. However, the monoclonal antibody (H11) was found to bind to lactosamine-containing glycolipids at their terminals, such as i- and I-type glycolipids as well as paragloboside. A two-step sandwich radioimmunoassay method for paragloboside antigen in serum was established by using the monoclonal antibody. The mean paragloboside antigen concentration in the sera from 20 normal individuals was 25.3 ng/ml. If the cutoff value was set at 80.9 ng/ml [25.3 + 2 x 27.8 (SD)], only 1 of 20 healthy controls had an elevated paragloboside value in the serum, whereas sera from 9 of 12 (75.0%) hepatoma, 4 of 10 (40%) pancreatic cancer, 16 of 40 (40.0%) stomach cancer, and 6 of 10 (60%) lung cancer patients had elevated paragloboside values. Sera from 3 of 8 hepatitis patients and 7 of 10 liver cirrhosis patients were estimated to be positive but sera from 16 patients with benign disease had paragloboside levels lower than the cutoff value. A larger amount of the antigen was found in liver metastases from colorectal carcinoma compared to the normal counterpart. The antigen was also detected in the medium of various human cancer cells and meconium. However, the antigen in the sera, medium, meconium, and cancer tissue seemed to be associated with glycoprotein or lipoprotein, because most of the antigen activity was eluted in the void volume fraction on high-performance liquid chromatography with a gel filtration column.

Chemical fingerprinting of glycosphingolipids in meconium of a human blood group O Le(a-b+) secretor.

Studying blood group polymorphism, as expressed in intestinal tissue of single individuals, total non-acid glycosphingolipids of meconium of individual human newborns have been prepared. Silicic acid column chromatography of the acetylated derivatives were used for a stepwise separation into four groups of glycolipids from each individual meconium. By the combined use of mass spectrometry and NMR spectroscopy of permethylated and LiA1H4-reduced, permethylated derivatives and by immunology of the native fractions all the major glycolipids were identified, although in mixtures. The interest was focused on fucolipids known to be strictly regulated by the ABO, H, Le and Se genes. The fucosylated glycolipids of an O Le (a-b+) secretor child were dominated by blood group H-active and Lewis-active mon- and difucosyl compounds with 5-6 sugar residues and having a core lactotetraosyl structure. The lipophilic part was dominated by 2-hydroxy fatty acids with 16 and 20-24 carbon atoms bounds to either sphingosine of phytosphingosine.

Chemical fingerprinting of non-acid glycosphingolipids in meconium of a human individual of blood group B Le(a-b+) and secretor.

In an attempt to elucidate blood group polymorphism systematically in human intestinal tissue, glycosphingolipids of meconia of single individuals have been studied. In this report we describe the major glycosphingolipids in meconium of a newborn typed as a B Le(a-b+) secretor. The glycolipids were analysed as four subfractions obtained after deacetylation of fractions from silicic acid column chromatography of the peracetylated total non-acid glycolipid extract. Chemical characterization (mass spectrometry and 1H-NMR spectroscopy of permethylated and LiA1H4-reduced permethylated derivatives) and immunological characterization (haemagglutination-inhibition of native glycolipids in the liposome form) revealed a very complex pattern of fucosyl-containing glycolipids, probably the result of several blood group glycosyltransferase activities. The major fucolipids, all based on lactotetraosylceramide, had a characteristic ceramide of mainly phytosphingosine and 2-hydroxy fatty acids with 16 and 20-24 carbon atoms.

Content of chorionic gonadotropin in human fetal tissues.

As part of a study on the physiological role of hCG in the human fetus, the hCG concentrations in homogenates of various fetal tissues were measured using a hCG beta subunit RIA. The mean concentrations (picograms of hCG per mg wet tissue +/- SEM; n greater than 10, unless otherwise indicated) found in human fetuses of 12-20 weeks were: ovary, 46.9 +/- 4.3; testis, 8.2 +/- 1.7; kidney, 20.3 +/- 2.8; thymus, 11.5 +/- 1.2; adrenal, 2.6 +/- 0.4; lung, 3.4 +/- 0.7; liver, 1.8 +/- 0.2; spleen, 1.4 +/- 0.4 (n = 5); muscle, 2.4 +/- 0.8 (n = 6); and meconium, 356 +/- 104. That the immunoreactive material measured behaved like hCG was determined by RIA of the supernatants. Parallelism was demonstrated between dilution curves for the tissue homogenates and the hCG standard for all tissues except meconium. A rat Leydig cell in vitro bioassay was used to demonstrate that there was hCG biological activity in the supernatants in ovarian, thymic, and renal tissues. The mean ratios of biological to immunological activities were 5.3 in kidney (n = 4), 1.6 in thymus (n = 3), and 1.3 in ovary (n = 2). Blood content of the tissues was determined from measurements of hemoglobin levels and it was found that for the ovary, testis, kidney, and thymus, hCG concentrations were higher than could be explained by the presence of circulating hCG in the tissues. These results, together with our previous results of the binding and effects of hCG in the human fetal testis, support the fact that the fetal testis is a target organ for hCG in the stimulation of steroidogenesis. The presence of high levels of hCG in the ovary, thymus, kidney, and meconium poses questions for further study of the possible physiological role of hCG.

Selective terminal alpha 2-3 and alpha 2-6 sialylation of glycosphingolipids with lacto-series type 1 and 2 chains in human meconium.

Human meconium was found to contain two kinds of gangliosides with the same carbohydrate sequence belonging to the lacto-series. They were detected by TLC-immunostaining with monoclonal antibodies directed to the NeuAc alpha 2-6Gal and Lc4Cer structures. One of these two gangliosides, a major one, which migrated on TLC to a position below that of standard IV3NeuAcnLc4Cer from human erythrocytes, reacted with the antibody to NeuAc alpha 2-6Gal. The other minor one, which migrated on TLC to a position corresponding to standard IV3NeuAcnLc4Cer, was detected with the antibody to Lc4Cer only when the plate, on which the individual gangliosides were separated, was subjected to prior treatment with Vibrio cholerae sialidase. The structures of the gangliosides, each identified by means of permethylation anaylsis with Vibrio cholerae sialidase. The structures of the gangliosides, each identified by means of permethylation anaylsis and enzyme treatment after isolation with antibody monitoring, were shown to be IV6NeuAcnLc4Cer for the former and IV3NeuAcLc4Cer for the latter, indicating that the lacto-series type 2 (nLc4Cer) and 1 (Lc4Cer) chains are sialylated at different linkages, alpha 2-6 and alpha 2-3, respectively. IV6NeuAcLc4Cer and IV3NeuAcnLc4Cer were not detected, even in trace amounts, on TLC-immunostaining with the monoclonal antibodies. The concentrations of IV6NeuAcnLc4Cer and IV3NeuAcLc4Cer were 448 and 18 nmol/g dry wt of human meconium.

Cystic fibrosis detection by means of a test-strip.

The effectiveness of meconium screening for albumin as an indication of cystic fibrosis is examined. BM-Test Meconium was applied to 69,000 investigations. In 60 positive tests, cystic fibrosis was confirmed later. No increased albumin content was observed in four cases of cystic fibrosis.

Should we screen all newborns for cystic fibrosis?

To assess the value of detecting albumin in meconium as a screening procedure for cystic fibrosis [CF] 68,000 meconium samples were examined by BM Meconium Test, single radial immunodiffusion and benzidine reaction. The specificity and sensitivity of this combination of tests were 99.67% and 78.57% respectively. The prevalence of CF at birth was confirmed as 1:3600 in this country. This low prevalence resulted in a relatively high number of false positives. Therefore, a positive test result has a low predictive value [3.39%] and this is a serious drawback of the method. The experiences and opinions of 37 local paediatricians about the screening programme were evaluated by a simple questionnaire. Gold's decision rule was applied. The least relative cost of misclassification justifying a mass-screening programme was 3 times higher than the actual relative cost as suggested by the aggregate opinion of paediatricians in the region. These results support the view that with the methods used screening may have more disadvantages than not screening.

Assay of serum immunoreactive trypsin in dried blood spots and the early detection of cystic fibrosis.

Immunoreactive trypsin (IRT) content in blood spots from 17 cystic fibrosis (CF) children born between 1975-80 were assayed with reagents from two commercial radioimmunoassay kits. The manufacturers' procedures were modified to allow assay of blood spot IRT. Half of the CF samples had a significantly raised IRT content compared to normal blood spots. Of the blood spots from five children whose meconium screening gave negative results, three or five (depending on the kit used) had a raised IRT content. On the other hand, less than 50% of children initially detected by meconium screening had raised blood spot IRT values. In our hands, analysis of blood spots for IRT does not consistently identify the newborn with CF and offers no advantage over other screening methods.

Satisfactory screening for cystic fibrosis with the BM meconium procedure.

The Boehringer Mannheim test (BM test) for cystic fibrosis has been applied to 15 734 neonates. A positive result occurred in 130, seven of whom were subsequently shown to have the disease. To our knowledge no cases have been missed. Tests have been performed centrally by experienced laboratory staff and the false-positive rate of 0.83% is considered acceptable. It is important to follow up weak as well as strongly positive reactions to the BM test strip.

Immunologic and biological properties and clinical significance of placental proteins PP5 and PP12.

Among the number of newly isolated placental proteins, PP5 and PP12 share some common characteristics: Both are present in the syncytiotrophoblast of normal placenta and hydatidiform mole, but less frequently, if at all, in choriocarcinoma. The levels in heparinized plasma of both proteins are lower than those in serum, and both are heat-labile. The function of PP12 is completely unknown, whereas PP5 appears to be related to the blood coagulation and fibrinolytic systems at the placental site through its antiplasmin activity. Many exciting avenues of research have been opened to uncover the biological role of these proteins in fetal development and cancer. We are pursuing this research with the immediate goal of assessing the role of PP12 in the blood coagulation system and of studying the expression of both proteins in various forms of cancer.

Meconium staining of amniotic fluid at midtrimester amniocentesis.

Meconium-stained fluid was found in six of the first 234 amniocenteses performed at the University of Washington Prenatal Diagnosis Center. The taps were done between 16 and 20 weeks from the last menstrual period. Although in each case there was a factor that could have produced fetal distress, in no instance did intrauterine demise occur. All the pregnancies have terminated in the births of healthy children. Therefore, we believe that meconium staining of midtrimester amniotic fluid may in fact reflect a transient episode of fetal compromise but that the finding cannot be used to prognosticate either impending fetal death or the presence of congenital malformations in the newborn.

Admission test: a screening test for fetal distress in labor.

The usefulness of a short electronic fetal heart rate recording at admission of patients in labor (admission test) was investigated in low-risk patients in two prospective studies. The admission test was done in a concealed manner, and the result of the test was evaluated after delivery so as not to influence the clinical management. In part I of the investigation, the test was performed in 130 patients monitored during labor with pH determinations in scalp blood and in cord blood at birth. Patients with reactive admission tests had a low rate of intrauterine asphyxia in labor (0.9%), whereas half of the patients with ominous traces had intrauterine fetal asphyxia with a low scalp blood pH and neonatal depression. Similar results were obtained in part II, when the admission test was used as a screening procedure involving 1041 patients. The test was reactive in 94.3%, and in this group fetal distress (cesarean section, or forceps on that indication, or an Apgar score less than 7 at five minutes) occurred in 1.3%. Ten patients (1.0%) had ominous tests; four of these had fetal distress, and one of these fetuses died in utero three hours after admission, during which time stethoscopic auscultation failed to detect the fetal compromise. It is concluded that the admission test can detect fetal distress already present at admission and unnecessary delay in intervention can be avoided in such a case. The test seems also to have some predictive value for the fetal well-being for the next few hours of labor. The test is simple and convenient for screening purposes.

The value of amniocentesis in prolonged pregnancy.

A total of 2702 transabdominal amniocenteses performed at the Los Angeles County--University of Southern California Medical Center were reviewed, with particular emphasis on 392 samples performed beyong 41 weeks' gestation. A significant rise in the percent of amniocenteses with meconium staining was found to occur at an beyond 39 weeks. Meconium-stained fluid at amniocentesis was found to be associated with an increased incidence of babies weighing greater than 4000 g, maternal diabetes mellitus, and cesarean deliveries, in comparison to samples with clear amniotic fluid. Infants with meconium-stained fluid had an increased incidence of low 1-minute Apgar scores, but all 5-minute Apgar scores were 7 or greater. Ten perinatal deaths occurred after an amniocentesis with clear fluid in prolonged pregnancy, with four of these occurring within 7 days of amniocentesis. Lecithin/sphingomyelin (L/S) ratios less than 2.0 were found in 6% of amniocenteses performed beyond 41 weeks. However, none of the newborns with low L/S ratios develop subsequent neonatal respiratory distress syndrome. Amniotic fluid creatine values or blood-contaminated fluid were not found to be correlated with fetal outcome. No fetal mortality was attributable to amniocentesis. In view of the significant amount of false-positive and false-negative results, and the rare inherent danger associated with amniocentesis, its use solely to demonstrate the presence or absence of meconium staining appears to be of questionable value in the management of prolonged pregnancy.

Interrelationships among abnormal cardiotocograms in labor, meconium staining of the amniotic fluid, arterial cord blood pH, and Apgar scores.

A prospective study of the relationships among fetal heart rate pattern, meconium staining of the amniotic fluid, umbilical cord artery pH, and Apgar score was carried out in 1219 consecutive births. Interpretable cardiotocogram patterns and cord arterial pH and blood gas analysis were obtained in 698 cases. The sensitivity of an abnormal cardiotocogram at any time for acidosis (more than 1 SD below the mean, pH less than 7.17) was 80%, and for severe acidosis (more than 2 SDs below the mean, pH less than 7.085) was 83%. However, the predictive value was low, and 32% of fetuses had an abnormal cardiotocogram but no acidosis. If only cardiotocogram abnormality in the first stage of labor was considered, sensitivity was still 47% for acidosis and 67% for severe acidosis, and the false-positive rate was reduced to only 14%. We attempted to improve the prediction of acidosis by including meconium staining of the amniotic fluid, but 65% of the variation in umbilical cord artery pH and 72 and 86% of the variation in 1- and 5-minute Apgar scores, respectively, remained unexplained. In light of these poor correlations, the current practice of considering cardiotocogram abnormality, meconium staining of the amniotic fluid, acidosis, and low Apgar scores as indicating one single disorder, "fetal distress," is not valid.

Routine amnioscopy at term.

A controlled study for the management of antepartum meconium-stained amniotic fluid (MSAF) detected by amnioscopy performed in a total of 508 patients is presented. The incidence of antepartum MSAF was 2.2%, while intrapartum MSAF was found in 15% of cases. A significant incidence of depressed neonates was found in the latter group. Patients with antepartum MSAF had no perinatal losses, whether managed expectantly or by oxytocin induction. Immediate operative intervention (ie, cesarean section) is not warranted. Observations on FHR patterns, fetal pH, and Apgar scores of these patients are presented.

Meconium in the amniotic fluid and fetal acid-base status.

Of 323 pregnancies with meconium-stained amniotic fluid at 36-42 weeks' gestation, 68 (21%) had a pH less than 7.20 in umbilical arterial blood, 21 (7%) had a pH less than 7.15, and only three newborns (0.9%) had true metabolic acidemia. At birth, of the 74 newborns with normal electronic fetal heart rate (FHR) tracings, eight (11%) had an umbilical arterial pH less than 7.20. There was a significantly higher frequency of acidemia (defined as pH less than 7.20) in newborns with both baseline and periodic FHR abnormalities. Although there was a significant difference (P less than .05) in the frequency of meconium found below the cords in these neonates with an umbilical artery pH less than 7.20 compared with those with values exceeding 7.20, there was no significant difference in the frequency of clinical meconium aspiration syndrome. We conclude that meconium-stained amniotic fluid correlates poorly with infant condition at birth as reflected by umbilical cord acid-base measurements.

Perinatal outcome after recent cocaine usage.

Eighty-eight neonates born to mothers with a history of cocaine use during pregnancy were divided into two groups based upon the detection of cocaine metabolites in the first neonatal urine. Forty neonatal urine samples were positive for cocaine and 46 were negative. Preterm labor, premature rupture of membranes, and meconium-stained amniotic fluid were significantly more frequent in those mothers whose neonates tested positive for cocaine metabolites than in those whose infants were negative (P less than .05). Neonates testing positive were more likely to exhibit signs and symptoms of acute cocaine intoxication. Low birth weight, growth retardation, and abruptio placentae were also more frequent than would be expected in the general population, but were not statistically different between the groups. These findings suggest that the differences noted in the cocaine-positive group may represent acute and chronic exposure, whereas the negative group reflects the problems associated with chronic usage alone.

Perinatal management of ventral wall defects.

Reported is the analysis of morbidity, mortality, and mode of delivery in 38 cases of ventral wall defects identified from among 128,500 consecutive live births in Maine (January 1975 to December 1982). Thirteen of the ventral wall defects were classified as gastroschisis, and only one had an additional defect not directly attributable to the ventral wall defect itself. By contrast, 16 of the 25 omphalocele cases had additional defects, including eight congenital heart lesions, four genitourinary malformations, two neural tube defects, and three trisomies. Ten cases of omphalocele and one of gastroschisis died, all as a result of independent defects or involvement of adjacent structures. Intrauterine growth retardation was prominently associated with gastroschisis. Vaginal delivery occurred in three of the six ventral wall defects diagnosed antenatally and in 28 of the 32 ventral wall defects not diagnosed until delivery. The only episode of birth trauma to ventral wall defect sac or abdominal viscera occurred during cesarean section in an undiagnosed case. The present data provide a basis for prognosis and management of antenatally diagnosed ventral wall defects and suggest that these defects are not, a priori, an indication for abdominal delivery.

Determination of phosphatidylglycerol in amniotic fluid by a simple one-dimensional thin-layer chromatography method.

Phosphatidylglycerol (PG) in amniotic fluid is the second important component of lung surfactant phospholipids and may be clinically useful in assessing fetal lung maturity in utero. Although methods for PG determination are available, there are shortcomings in clinical application. We developed an alternative reliable one-dimensional thin-layer chromatography(TLC) method for separating and quantitating PG in amniotic fluid. A mini-TLC plate (8 X 10 cm) was prepared from silica gel H containing 5% ammonium sulfate. The plate was first developed in tetrahydrofuron/dimethoxymethane/methanol/2N ammonium hydroxide (30.0:20.6:5.6:3.0 v/v) and then in chloroform/methanol (60.9, v/v) in the same direction. PG was clearly separated from other phospholipids and neutral lipids, even when large amounts of other phospholipids were present on the TLC plate. The density of the charred PG was directly proportional to the amount of PG up to 8 nmol. The content of PG in nmol in the specimen can be quantitated by comparing with a standard PG. Up to 10% of blood serum or 3% meconium showed no detectable PG, nor did these substances affect PG quantitation in amniotic fluid. This method is sensitive and accurate. It is also time-saving and economical.

A rapid qualitative method for detecting phosphatidylglycerol in amniotic fluid.

A fast, reliable and inexpensive procedure of the qualitative determination of phosphatidylglycerol (PG) in amniotic fluid, using one-dimensional thin layer chromatography and a phosphorus-specific spray, is described. The method detects amounts of PG exceeding 1 microgram or 1.7% of total phospholipids in amniotic fluid. Contamination with serum does not influence the results. Excessive amounts of meconium interfere with the chromatography. The presence of PG was always associated with a mature L/S ratio (greater than 2), whereas its absence did not necessarily exclude lung maturity.