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Background: Approximately two million pregnancies in the United States are screened for Down syndrome annually by use of second-trimester maternal serum markers. At present, a combination of four markers can identify 75% of affected pregnancies when 5% of screened women are classified as candidates for amniocentesis. Although not currently included in screening panels, invasive trophoblast antigen (ITA) is a promising screening marker in serum or urine in both the second and first trimesters. This study aims at better defining the screening performance of serum ITA in the second trimester. Methods: In an earlier study, serum samples from an unbiased sampling of 45 Down syndrome (cases) and 238 unaffected (control) pregnancies between 14 and 20 weeks of gestation were collected from various centers in the United States. Samples were aliquoted and stored at −20 °C for 8 years. We measured ITA in these samples and determined the screening performance both univariately and in combination with other screening markers. Results: The median ITA in Down syndrome pregnancies was >3.00 multiples of the median, higher than that found for human chorionic gonadotropin (hCG) or free β-hCG. At a 5% false-positive rate, ITA univariately detected 38% and 40% of Down syndrome pregnancies, respectively, when assigned by date of last menstrual period or ultrasound date. Modeling yielded rates of 45% and 48%. ITA correlated strongly with hCG and free β-hCG. When substituted for either of these in a multiple marker panel, ITA performed comparably. Conclusions: This study indicates that serum ITA is an effective marker for Down syndrome. It is highly correlated with both hCG and free β-hCG and could replace either of them in a multiple marker panel.

Background: Down syndrome screening is commonly performed in the US using maternal age and three or four second-trimester maternal serum markers that can identify up to 75% of affected pregnancies by offering diagnostic studies to 5% of women. Invasive trophoblast antigen [ITA; hyperglycosylated human chorionic gonadotropin (hCG)] is a promising marker that can be measured in urine or serum in the first or second trimester. We report preliminary results for urinary ITA in an ongoing observational study.Methods: Women undergoing second-trimester amniocentesis for reasons not associated with biochemical testing provided consent and a urine (and possibly serum) sample that was tested within a few days. Demographic and pregnancy-related information was collected, along with karyotype. Screening performance was modeled for ITA alone and in combination with serum markersResults: Twelve recruitment centers collected urine from 2055 women with singleton pregnancies between 15 and 20 weeks of gestation (2023 unaffected, 28 Down syndrome, and 4 pregnancies with other chromosome abnormalities). After correction for gestational age, urine concentration, and maternal race and weight, the ITA measurements were higher in women with a Down syndrome pregnancy (median ITA, 4.33 multiples of the median). At a 75% detection rate, the false-positive rate could be reduced by substituting ITA for hCG measurements (from 5.6% to 2.6% for the triple test) or by adding ITA measurements to existing combinations (from 3.3% to 2.0% for the quadruple test).Conclusions: Our data provide preliminary confirmation of the potential usefulness of urinary ITA measurements in detecting Down syndrome in a setting that simulates routine usage.

Background: In the United States, Down syndrome screening is still performed mainly in the second trimester, using 3 or 4 markers. Moving screening into the first trimester has the advantage of earlier diagnosis. Currently, first-trimester screening typically includes maternal serum pregnancy-associated plasma protein-A (PAPP-A), the free β-subunit of human chorionic gonadotropin (free β), and ultrasound measurement of nuchal translucency thickness (NT). The current report describes a case–control study of serum invasive trophoblast antigen (ITA) and its possible inclusion in first-trimester screening for Down syndrome. Methods: As part of an earlier observational study, serum samples from 54 Down syndrome and 276 matched unaffected pregnancies were collected between 9 and 15 weeks of gestation. Samples had been aliquoted and stored at −20 °C for 8 years. ITA was measured and converted to weight-adjusted multiples of the median (MoM). The distributions of other first-trimester markers are from a single published study. Results: Median ITA MoM in Down syndrome pregnancies increase as gestational age increases (2.02 MoM at 11 and 2.44 MoM at 13 completed weeks). At 75% detection, maternal age in combination with ITA and PAPP-A measurements have an 8.0% false-positive rate, slightly lower than the 8.8% found for the free β and PAPP-A combination; adding NT measurements reduces false positives for the 2 combinations to 2.0% and 1.8%, respectively. Conclusion: Serum ITA appears to be a useful first-trimester Down syndrome marker that could replace free β measurements while maintaining performance.

Creatinine and specific gravity (relative density) measurements both allow differences in urine concentration to be taken into account in determining urine cotinine concentrations. In this study we demonstrate that the variance of urine cotinine measurements is reduced comparably when either creatinine or specific gravity measurements are used for correction. This reduction in variability improves the correlation between urine cotinine measurements and clinical endpoints. In this study, the clinical endpoints were pulmonary function in a population of nonsmoking children with asthma, 42% of whom were reported to have been exposed to environmental tobacco smoke. When corrected by either creatinine measurements or specific gravity values, the urine cotinine measurements performed as well or better than reported exposure (and comparably with each other) in assessments of lung function. A dose-response relationship was also more consistently apparent. Specific gravity values can be used reliably in place of creatinine values to adjust urine cotinine measurements for both research and clinical purposes.

Creatinine and specific gravity (relative density) measurements both allow differences in urine concentration to be taken into account in determining urine cotinine concentrations. In this study we demonstrate that the variance of urine cotinine measurements is reduced comparably when either creatinine or specific gravity measurements are used for correction. This reduction in variability improves the correlation between urine cotinine measurements and clinical endpoints. In this study, the clinical endpoints were pulmonary function in a population of nonsmoking children with asthma, 42% of whom were reported to have been exposed to environmental tobacco smoke. When corrected by either creatinine measurements or specific gravity values, the urine cotinine measurements performed as well or better than reported exposure (and comparably with each other) in assessments of lung function. A dose-response relationship was also more consistently apparent. Specific gravity values can be used reliably in place of creatinine values to adjust urine cotinine measurements for both research and clinical purposes.

We welcome the opportunity to clarify the points made by Hallahan and his colleagues concerning our report of maternal serum invasive trophoblast antigen (ITA) measurements and Down syndrome screening in the first trimester (1). Findings from the one other independent report of serum ITA in the first trimester are consistent with ours (2). With any new marker, such as ITA, the process of evaluation begins with reports …

Staphylococcus aureus cells coated with rabbit anti-goat immunoglobulin G were compared with soluble second antibody in a radioimmunoassay for human serum alphafetoprotein. Recovery of primary immune complexes with use of the bacterial immunoadsorbant (a) allowed second-antibody incubation time to be shortened from 3 h to 20 min at 37 degrees C, (b) decreased by 80% the amount of second antibody required per assay, thereby significantly reducing assay costs, and (c) provided a convenient and reproducible method for separation of free and bound antigen. The ease of preparation and the uniformity and stability of the S. aureus second-antibody reagent are additional advantages that may be useful in other radioimmunoassay procedures.

We describe a polyclonal-antiserum-based 125I-radioimmunoassay for cotinine that is suitable for measuring nonsmokers' passive exposure to tobacco smoke in the environment. The standard curve ranged from 0.25 to 12.0 micrograms/L, with an estimated lower limit of sensitivity of 0.2 microgram/L (95% B/Bo = 0.2 microgram/L; 50% B/Bo = 4.0 micrograms/L). The median within-assay CVs for patients' samples with cotinine values from 0.4 to 1.3, 1.4 to 2.4, 2.5 to 4.6, and 4.7 to 15.6 micrograms/L were 13.9%, 7.2%, 5.1%, and 5.7%, respectively. Between-assay CVs for two quality-control sera with average values of 1.53 and 3.68 micrograms/L were 14.3% and 7.8%, respectively. Analytical recoveries of cotinine from smokers' sera diluted in zero calibrant ranged from 91% to 116%. Cotinine values determined on 79 paired sera and urines from nonsmokers showed significant correlation with self-reported exposure to environmental tobacco smoke (r = 0.49, P less than 0.001 for sera; r = 0.57, P less than 0.001 for urine). The log of the values for serum and urine cotinine were also significantly correlated (r = 0.85, P less than 0.001). Evidently, polyclonal antiserum can be used to develop a cotinine assay for measuring exposure to environmental tobacco smoke that compares well with that described for monoclonal-based assays.

We describe an 125I-based RIA for cotinine, the major metabolite of nicotine. The slope of the dose-response curve was quite shallow (6-8% change in binding per doubling dose), resulting in between-assay CVs of 15 to 20%. This effect occurred because the radioligand formed by linking a cotinine derivative to tyramine manifested greater affinity for the anti-cotinine antibodies than did cotinine itself. We absorbed the serum with a derivative of nicotine coupled to the carrier protein via a chemical bridge similar to that used to form the cotinine/carrier protein immunogen. An RIA in which we used such absorbed serum showed a significantly increased slope of the dose-response curve (11-13% change in binding per doubling dose), and between-assay CVS were only 6 to 8%. We suggest that this improvement results because absorption removes anti-bridge antibodies directed against the chemical-bond common to the cotinine/carrier-protein immunogen and to the cotinine/tyramine radioligand.

A radioimmunoassay and a radioenzymatic assay for netilmicin, a new aminoglycoside, were developed in our laboratories to assist in the study of the pharmacology of the drug and to establish values for use in its monitoring. The assays are sensitive, precise, and rapid, giving results that correlate (r = 0.90) with each other and with those of a microbiological assay in which Klebsiella pneumoniae is used as the test organism. Preliminary pharmacological studies show the drug to have a biological half-life of 135 min. which is comparable to that for other aminoglycosides.

A solid-phase enzyme immunoassay is described for measuring hepatitis B surface antigen in human serum or plasma. Immunologically purified antibody labeled with horseradish peroxidase was used as the indicator. In the assay system, antibody-coated controlled-pore glass is used as a solid support and there are three sequential incubations, totaling 2 h, at room temperature. Results for serially diluted positive and reference sera compare favorably to radioimmunoassay in sensitivity and specificity.

A solid-phase enzyme immunoassay is described for measuring hepatitis B surface antigen in human serum or plasma. Immunologically purified antibody labeled with horseradish peroxidase was used as the indicator. In the assay system, antibody-coated controlled-pore glass is used as a solid support and there are three sequential incubations, totaling 2 h, at room temperature. Results for serially diluted positive and reference sera compare favorably to radioimmunoassay in sensitivity and specificity.

We describe a polyclonal-antiserum-based 125I-radioimmunoassay for cotinine that is suitable for measuring nonsmokers' passive exposure to tobacco smoke in the environment. The standard curve ranged from 0.25 to 12.0 micrograms/L, with an estimated lower limit of sensitivity of 0.2 microgram/L (95% B/Bo = 0.2 microgram/L; 50% B/Bo = 4.0 micrograms/L). The median within-assay CVs for patients' samples with cotinine values from 0.4 to 1.3, 1.4 to 2.4, 2.5 to 4.6, and 4.7 to 15.6 micrograms/L were 13.9%, 7.2%, 5.1%, and 5.7%, respectively. Between-assay CVs for two quality-control sera with average values of 1.53 and 3.68 micrograms/L were 14.3% and 7.8%, respectively. Analytical recoveries of cotinine from smokers' sera diluted in zero calibrant ranged from 91% to 116%. Cotinine values determined on 79 paired sera and urines from nonsmokers showed significant correlation with self-reported exposure to environmental tobacco smoke (r = 0.49, P less than 0.001 for sera; r = 0.57, P less than 0.001 for urine). The log of the values for serum and urine cotinine were also significantly correlated (r = 0.85, P less than 0.001). Evidently, polyclonal antiserum can be used to develop a cotinine assay for measuring exposure to environmental tobacco smoke that compares well with that described for monoclonal-based assays.

A radioimmunoassay and a radioenzymatic assay for netilmicin, a new aminoglycoside, were developed in our laboratories to assist in the study of the pharmacology of the drug and to establish values for use in its monitoring. The assays are sensitive, precise, and rapid, giving results that correlate (r = 0.90) with each other and with those of a microbiological assay in which Klebsiella pneumoniae is used as the test organism. Preliminary pharmacological studies show the drug to have a biological half-life of 135 min. which is comparable to that for other aminoglycosides.

Maternal serum alpha-fetoprotein concentrations are influenced by maternal weight during the second trimester. Heavier pregnant women have lower median values, apparently as a result of a diluting effect of larger blood volume. This phenomenon is of clinical interest because alpha-fetoprotein concentration in a pregnant woman's serum is one of the factors considered in assessing risk of poor outcome. A revision of the reference interval for alpha-fetoprotein to take body weight into account might improve its use as a diagnostic aid, especially in heavier women.

Staphylococcus aureus cells coated with rabbit anti-goat immunoglobulin G were compared with soluble second antibody in a radioimmunoassay for human serum alphafetoprotein. Recovery of primary immune complexes with use of the bacterial immunoadsorbant (a) allowed second-antibody incubation time to be shortened from 3 h to 20 min at 37 degrees C, (b) decreased by 80% the amount of second antibody required per assay, thereby significantly reducing assay costs, and (c) provided a convenient and reproducible method for separation of free and bound antigen. The ease of preparation and the uniformity and stability of the S. aureus second-antibody reagent are additional advantages that may be useful in other radioimmunoassay procedures.

In this method for clinical measurement of beta 2-microglobulin, a 10 g/L agarose gel containing anti-beta 2-microglobulin is used, with subsequent staining with Coomassie Blue. Although the method is slow (requiring 30 h), it is inexpensive, reliable, and accurate over the range of 1 to 10 mg/L, and is useful in the determination of beta 2-microglobulin in serum and cerebrospinal fluid. A modified procedure in which staining with silver is used may be sufficiently sensitive for the clinical assay of urine.

Maternal serum alpha-fetoprotein concentrations are influenced by maternal weight during the second trimester. Heavier pregnant women have lower median values, apparently as a result of a diluting effect of larger blood volume. This phenomenon is of clinical interest because alpha-fetoprotein concentration in a pregnant woman's serum is one of the factors considered in assessing risk of poor outcome. A revision of the reference interval for alpha-fetoprotein to take body weight into account might improve its use as a diagnostic aid, especially in heavier women.