Quadruple Marker Research Articles

Clinical utility of PregascreenTM reflex genetic testing for pre-natal screening in Indian population: A new diagnostic approach

Prenatal screening with maternal biochemical dual and quadruple markers, along with reflex testing using karyotyping, and non-invasive prenatal testing via next-generation sequencing (NIPT-NGS) were evaluated to determine the clinical validity of Metropolis Pregascreen reflex testing approach among Indian women. Retro-prospective data of 51574 Indian women undergoing maternal marker screening from January 2021 to March 2022 were analysed at Metropolis Healthcare Limited, India. First and second-trimester prenatal screening were performed using Roche and Siemens platforms. Risk calculated using SSDW and PRISCA software, USG findings and biochemical values were incorporated. NIPT reflex testing was carried out using Thermo Ion torrent S5 NGS systems, while karyotyping on chronic villus sampling or amniocentesis. Total 51574 women opted for the combined biochemical markers test (dual and quadruple), 1394 cases (2.70%) and 50180 cases (97.28%) were screened as high-risk and low-risk, respectively. Of the total high-risk cases, 483 women (34.65%) opted for NIPT, while 25 (1.79%) opted for karyotyping reflex testing. Dual marker 92% and quadruple marker 94% of high risk cases were reclassified as low risk post NIPT, while dual marker 91% and quadruple marker 93% of high risk cases were reclassified as low risk post karyotyping. Possibility of ruling out false positive is almost equal with NIPT and karyotyping. Hence, invasive screening could be avoided as first line of investigation.Metropolis Pregascreen reflex testing with NIPT assisted in the delineation of actual high risk cases for accurate and safer diagnosis.

Assessment of indications and peri-procedural complications of amniocentesis

Aim: Assessment of indications and peri-procedural complications of amniocentesis.Materials and Methods: Cases were analyzed with some indications such as intermediate or high risk in dual and quadruple marker test, abnormal NT scan or target scan, history of child with congenital anomaly, abnormality in ultrasonography (cystic hygroma, omphalocele, diaphragmatic hernia, etc.) and to which amniocentesis will be applied. Patient and family were informed about the amniocentesis procedure, the role of this method in diagnosis and also its complications before the application. Results: The age and gestational age of the participants were 28.25±2.69 and 16.89±2.15 respectively. Most of the patients 17.14% have Hypothyroidism co morbidity. 65.71% patients show high risk assessment with dual marker & quadruple marker & abnormal NT scan & target scan indication for amniocentesis, followed by h/o child with congenital anomaly (14.2%), couple has thalassemia (11.4%), genetic disorder in family (2.9%) and chromosomal abnormality in previous child (2.9%). There was no complication of amniocentesis. Conclusion: we concluded that the genetic amniocentesis has a high success rate when performed by experienced obstetrician or foetal medicine specialist in 15-20 weeks of gestation. It should be offered to the patients when they have correct indication.

Management of Intra-Uterine Growth Restriction by Ayurvedic Regime: A Case Report

Maternal nutrition and congenital malformation are considered as very important factors for intra uterine growth restriction. Present case deals with these two factors in parallel way. A 32 years young lady second gravida, G2P0A1, housewife, residence of Jaipur, came to NIA OPD with complaint of amenorrhea since a month. According to patient her last menstrual period was on 3/3/20. Her antenatal care was continued in NIA OPD. First fetal wellbeing scan reveals mild ventriculomegaly so she was advised for second level scan and quadruple marker but due to low socioeconomic condition patient was not able to do second level anomaly scan and she continued her pregnancy on her own risk. After 7 months she came to us with the complaints of decreased fetal movements. Fundal height corresponding to period of gestation and fetal heart sound was present which was regular. A color doppler was advised which shows normal fetoplacental and uteroplacental circulation, fetal weight was normal according to period of gestation. On the eighth month the fundal height was found decreased along with maternal weight decreased by 1kg. She was suspected for IUGR and planned for Ksheerbasti. After four episodes Ksheer basti on the 36 weeks of gestation, mother had significant weight gain of 2 kg along with increase in fundal height. USG was done which reveals Single live intra uterine pregnancy of 33 weeks 2 days with lumbar spina bifida with meningomyelocele, CHIARI-II brain malformation, EFW– 2282gm. She delivered a live female child of 2300gm through normal vaginal delivery at maternity hospital. Child was having severe form of myelomeningocele with brain malformation.

Prediction of Low Birth Weight by Quadruple Parameters in High-Risk Pregnancies.

Aneuploidy screening is done in the early second trimester of pregnancy among all pregnant women as compulsory, with a special focus on those who had abnormal ultrasound parameters, higher dual marker risk, or other comorbidities. Recently, all individual quad markers of conventional trisomy screening have been suggested as useful in predicting adverse pregnancy outcomes (APO) such as preeclampsia, preterm labor, small for gestational age, and placental abruptions. However, similar studies on Indian pregnant women are limited. Hence, this study was intended to find the relation of quadruple markers with any other APO than aneuploidy. A retrospective study was conducted in a Tertiary Care multi-specialty hospital in North India. Data from 252 pregnant women's quadruple test was analyzed. The association of abnormal value of quadruple markers (human chorionic gonadotropin [HCG]/alpha-fetoprotein/uE3/Inhibin A) with adverse outcomes was evaluated. Multiple logistic regression analysis and classification and regression tree were used to predict the significant risk factor in high-risk pregnancies. In the study, a total (n = 252) of pregnant women, 190 were screened as high-risk pregnancies, whereas the remaining 62 were reported as low-risk using trisomy screening in the quadruple test. Baby birth weight was observed to be significantly associated with Inhibin-A, and HCG (P < 0.001), whereas Corrected (Corr)-multiple of median (MoM)-HCG (>1.415) and Inhibin-A Corr-MoM (>364.175) were the suitable predictor for the LBW. Both parameters were significantly higher in the high-risk group as compared to the low-risk group (each P < 0.05). Abnormal deviation of biochemical markers from aneuploidy screening assessment could help predict other perinatal adverse outcomes such as low birth weight babies.

Quadruple Screening in the Age of Cell-Free DNA: What are We Losing?

Cell-free DNA has emerged as the most reliable, non-invasive prenatal screening tool for fetal aneuploidies. It has come to replace the previously widely used quadruple screen offered in the second trimester of pregnancy. This change comes with improved detection for aneuploidy but also presents potential gaps in prenatal diagnosis including detection of open fetal defects and emerging data on prediction of adverse pregnancy outcomes. This review article provides a historical summary of the quadruple marker screen and evaluates the intersection of this screen with cell-free DNA. Furthermore, it discusses points to consider as providers trend toward cell-free DNA testing alone and reviews potential options to remedy any disparities.

Prenatal serum screening - a summary of our experience with high risk reporting

Objective The spectrum of pregnancy tests for screening of chromosomal disorders in the unborn child includes maternal serum testing through double, triple and quadruple marker as well as the emerging cell free fetal DNA analysis based noninvasive prenatal test. The aim of this short communication is to summarize our finding’s pertaining to high risk serum screening cases with reference to clinical indications and history. Methods Data of cases reported high-risk by serum screening including a total of 3368 women with median age of 32 years was included. Serum testing was done in the laboratory using the technology of Chemiluminescence micro particle two-step immunoassay for sensitive quantification of hormones routinely tested for in the double, triple and quadruple marker. Risk estimation using values of hormone levels and the resulting MoM was done using the PRISCA 5.0.2.37. Results A total of 16,608 samples were tested for serum screening in the study period and specific high risk analysis detected a total of 20% reported as high risk. Trisomy 21 was detected to be the most common finding at 86% followed by neural tube defects at 9%. Advanced maternal age (≥35 years) accounted for a total of 34% of the reported high risk cases. Conclusion The gap between absence of a nation-wide screening mandate, and guidelines for agencies involved in prenatal screening tests can be reduced with studies which focus on trends in recommendation and developing an understanding of the clinical backdrop leading to high risk screening results.

The predictive value of maternal serum screening tests for adverse pregnancy outcomes

Aim: Free beta-human chorionic gonadotropin (Fβ-hCG), pregnancy-associated plasma protein-A (PAPP-A), alpha-fetoprotein (AFP), unconjugated estriol (uE3), and inhibin-A have been shown to be useful not only in identifying chromosomal abnormalities in the first and second trimesters, but also in predicting adverse pregnancy outcomes (APOs). In this study, we aimed to investigate the predictive value of maternal serum screening tests for various APOs including preeclampsia, intrauterine growth retardation (IUGR), preterm labor, and gestational diabetes mellitus (GDM).Material and Methods: The study was carried out at our clinics and included a total of 220 pregnant women who respectively underwent the double and quadruple marker testsduring the 11th to 14th and 15th to 22nd gestational weeksbetween January 2017 and December 2018. Patient data (maternal age, parity/gravidity, gestational age, history of infertility, use of in vitro fertilization, maternal/fetal complications, preterm labor, mode of delivery, birth weight, and length of stay in the neonatal intensive care unit) were reviewed retrospectively.Results: The AFP levels were statistically significantly higher in patients with IUGR (p=0.022),GDM (p=0.036), and preterm labor (p=0.021), compared to the control group with no APO. No significant correlation was found between preeclampsia and the serum markers.Conclusion: Our results show that elevated maternal serum AFP levels might be associated with various APOs,requiring closer patient follow-up for early diagnosis and treatment.

Maternal serum placental growth factor combined with second trimester aneuploidy screening to predict small-for-gestation neonates without preeclampsia

ObjectiveTo investigate the role of maternal serum placenta growth factor (PlGF) and quadruple test parameters in predicting the risk of small for gestational age (SGA) infants of mothers without preeclampsia. Materials and methodsWe prospectively enrolled 300 pregnant patients who underwent blood sampling at 15–18 weeks gestation and followed them until delivery. Cases with SGA neonate delivery (n = 100) were compared with matched AGA neonate controls (n = 200). The plasma PlGF and quadruple markers were measured by enzyme-linked immunosorbent assay. The results were analyzed with Mann–Whitney U tests, and regression analysis was used to develop a model for the prediction of SGA. ResultsWomen who delivered SGA neonates had decreased levels of PlGF (median 0.71 MoM versus 0.7 MoM; p < 0.01), hCG (median 0.97 MoM versus 1.06 MoM; p = 0.046) and uE3 (median 0.92 MoM versus 1.04 MoM) compared to the AGA group. AFP, hCG and inhibin-A levels did not differ significantly. A PlGF concentration <0.37 MoM had a sensitivity of 28.0% (95% CI: 19.5–37.9) and a specificity of 89.5% (95% CI: 84.4–93.4) for the prediction of SGA neonates without PE. ConclusionSGA neonates in the absence of PE could potentially be identified at 15–18 weeks of pregnancy.

Duo test and aneuploidy detection in women under 35 years of age with high-risk pregnancy at the Hospital General de México

Non-invasive procedures for prenatal diagnosis are the most frequently used methods in the first trimester of pregnancy in pregnant women under 35 years of age because they do not involve risk of pregnancy loss; however, they are not considered to be a definitive diagnosis method. During the first trimester the duo test (PAPP-A and free βhGC), cell-free fetal DNA in maternal blood and structural ultrasound scans are the principal tools used; the quadruple marker test (αFP, E3, β-hCG, inhibin A) is used in the second trimester. However, the definitive diagnosis is performed by cytogenetic analysis through amniocentesis. MethodsThirty women, under 35 years of age with high-risk pregnancy, were studied with duo test and structural ultrasound in the first trimester and amniocentesis in weeks 15–18. ResultsOnly five duo tests were positive: three showed risk of trisomy 18 and one of Turner syndrome, they all corroborated with the cytogenetic study; the fifth showed a risk of Down's syndrome, however it was a chromosomally normal product. Three patients with a negative duo test cytogenetically detected with karyotypes with structural abnormalities, which were: deletion of the short arm of chromosome 18 [46,XY, del(18)(p11)], Robertsonian translocation between chromosome 13 and 14 [45,XY, rob(13:14)] and a chromosome derived from X [46,X, der(X)]. The duo test is a very useful tool for the diagnosis of numerical chromosome abnormalities, but not for detecting structural chromosome aberrations. However, it is essential to perform amniocentesis to definitively rule out chromosomal aberrations in products of conception.

Socioeconomic Disparities in Diagnostic Testing After Positive Aneuploidy Screening

Approximately 20% to 50% of pregnant women who test positive in screening for fetal chromosomal abnormalities forego diagnostic testing procedures, including chorionic villus sampling and amniocentesis. Many factors influence the decision for screening and diagnostic testing. Previous studies examining the relationship between sociodemographic characteristics and willingness to undergo diagnostic testing after positive screening found mixed results. This study investigated the possible existence of a socioeconomic disparity, specifically, the relationship between insurance type (Medicaid or private) and the refusal of diagnostic testing after positive screening for aneuploidy. The retrospective cohort study took place from 2008 to 2011 and included women pregnant with a singleton who tested positive for increased risk of aneuploidy and gave birth at Northwestern Memorial Hospital. Screening methods included first-trimester screening, sequential screening, or quadruple marker screening. Clinical and demographic data on the patients were collected, as well as whether the patient had received an ultrasound before making a decision about diagnostic testing. Diagnostic testing after positive aneuploidy screening is covered by Medicaid, so testing was equally accessible to all patients. There were 499 women included in the study, 75 of whom were insured through Medicaid. From the remaining 424, 75 women who had private insurance were randomly selected for the control group. The Medicaid group was younger (33.8 ± 6.9 years at delivery vs 36.9 ± 3.7 years, P < 0.001), more likely to be nonwhite (88% vs 27%, P < 0.01), more likely to have a first language other than English (12% vs 0%, P < 0.01), and more likely to be unmarried (65% vs 19%, P < 0.01). The timing of screening was similar between the 2 groups (28% vs 31% for first-trimester screening, 72% vs 69% for second-trimester screening). In the Medicaid group, 35% of women underwent diagnostic testing, whereas in the private insurance group, 57% did so. Insurance by Medicaid was associated with a lower likelihood of electing for diagnostic testing, even after adjusting for variables that differed between the 2 groups (odds ratio, 0.26; 95% confidence interval, 0.09–0.80). The authors of the study believe that factors such as lower household income, lower educational attainment, and health literacy were responsible for the differences between the 2 groups in the use of diagnostic testing and that the women in the Medicaid group may not fully understand the implications and purpose of the screening test. These results likely speak to a difference in patient-physician communication in regard to the choice to undergo diagnostic testing. Further investigation on the topic and interventional strategies are needed.

Topography of Lymphatic Markers in Human Iris and Ciliary Body.

Reports of lymphatics in the anterior human uvea are contradictory. This might be caused due to a certain topography, which has not been considered yet. Therefore, here we systematically analyze iris and adjacent ciliary body with immunohistochemistry by combining various lymphatic markers. Human iris and ciliary body were obtained from cornea donors and prepared for cryosectioning. Cross sections of tissue blocks at 12/3/6/9 o'clock position and at corresponding intersections (1:30/4:30/7:30/10:30) were processed for immunohistochemistry of LYVE-1, PDPN, PROX1, FOXC2, VEGFR3, and CCL21, and when necessary, these lymphatic markers were combined with CD31, α-smooth muscle-actin, CD68, and 4',6-diamidino-2 phenylindole dihydrochloride (DAPI). Double, triple, and quadruple marker combinations were documented using confocal microscopy. Numerous podoplanin+ cells were mainly located at the anterior border of the iris while LYVE-1+ cells were distributed throughout the nonpigmented part. Both cell populations were PROX1/FOXC2/CCL21/VEGFR3-. Blood vessels, iris smooth muscles, and individual cells were VEGFR3+. While PDPN+ cells were rarely detected posteriorly of the iris root, many LYVE-1+ cells were present within the ciliary body muscle and villi. Within the muscle, occasionally PDPN+ vessel-like structures were detectable, but these were never colocalized with LYVE-1. Similar vessel-like structures were VEGFR3+/PROX1-/CCL21-, but CD31+. Further, ciliary muscle fibers and ciliary epithelium were immunoreactive for VEGFR3/CCL21, but were LYVE-1/PDPN-. A certain topography of structures at the various uvea-positions investigated was not obvious. The majority of LYVE-1+ cells displayed immunoreactivity for CD68. Lymphatic vessels colocalizing for at least two lymphatic markers were not detectable. Therefore, if present, putative lymphatic channels of the anterior uvea might display a different marker panel than generally presumed.

Role of second‐trimester ultrasound in screening for Down syndrome

Role of second‐trimester ultrasound in screening for Down syndrome

First trimester versus second trimester down's syndrome screening in the west of Scotland

During 2010, we compared performance of first versus second trimester Down's syndrome screening in 2 hospitals 4 miles apart, serving the same population in West of Scotland. As the same...

Role of Second-Trimester Genetic Sonography After Down Syndrome Screening

To estimate the effectiveness of second-trimester genetic sonography in modifying Down syndrome screening test results. The First and Second Trimester Evaluation of Risk (FASTER) aneuploidy screening trial participants were studied from 13 centers where a 15- to 23-week genetic sonogram was performed in the same center. Midtrimester Down syndrome risks were estimated for five screening test policies: first-trimester combined, second-trimester quadruple, and testing sequentially by integrated, stepwise, or contingent protocols. The maternal age-specific risk and the screening test risk were modified using likelihood ratios derived from the ultrasound findings. Separate likelihood ratios were obtained for the presence or absence of at least one major fetal structural malformation and for each "soft" sonographic marker statistically significant at the P<.005 level. Detection and false-positive rate were calculated for the genetic sonogram alone and for each test before and after risk modification. A total of 7,842 pregnancies were studied, including 59 with Down syndrome. Major malformations and 8 of the 18 soft markers evaluated were highly significant. The detection rate for a 5% false-positive rate for the genetic sonogram alone was 69%; the detection rate increased from 81% to 90% with the combined test, from 81% to 90% with the quadruple test, from 93% to 98% with the integrated test, from 97% to 98% with the stepwise test, and from 95% to 97% with the contingent test. The stepwise and contingent use of the genetic sonogram after first-trimester screening both yielded a 90% detection rate. Genetic sonography can increase detection rates substantially for combined and quadruple tests and more modestly for sequential protocols. Substituting sonography for quadruple markers in sequential screening was not useful. II.

Second-trimester Down's syndrome serum screening: double, triple or quadruple marker testing?

The recommendations of the UK National Screening Committee (NSC) are based on the findings of the Serum, Urine and Ultrasound Screening Study (SURUSS). Our study sought to establish if the SURUSS findings, in terms of detection rates (DR) and false-positive rates (FPR) for various second-trimester marker combinations, could be replicated in a local setting. We investigated the effects of adding inhibin-A and unconjugated oestriol to our existing double test protocol. This retrospective study examined 1000 control pregnancies and 128 affected pregnancies. The inhibin-A method was associated with considerable assay drift and very marked within-batch imprecision (intrabatch percent coefficient of variation [CV] = 17%). At a cut-off of 1 in 250, the quadruple test showed a DR of 72%, the triple test 70% and the double test 63%. There were no significant differences between the FPRs for any of the combinations, which were all between 6.6% and 7.0% for a 1 in 250 cut-off. In our view, the current inhibin-A assay is unacceptable as a screening marker due to poor assay performance. We have reviewed the NSC benchmark programme outcomes, and would suggest that the 2005 target of a DR of at least 60% with an FPR of less than 5% is achievable using triple testing in the second trimester in conjunction with universal scan dating. Our results suggest that the April 2007 target of a DR of 75% with an FPR of less than 3% is unachievable using current second-trimester maternal serum screening.

Practical strategies in contingent sequential screening for Down syndrome

To design and assess the performance of protocols for contingent sequential Down syndrome screening that can be implemented in practice. Protocols were designed in which all women received first-trimester measurement of nuchal translucency (NT) together with maternal serum pregnancy-associated plasma protein-A (PAPP-A) and either free beta- or total human chorionic gonadotrophin (hCG). Those women with borderline Down syndrome risks received follow-up second-trimester maternal serum involving double, triple, or quadruple serum screening markers: alpha-fetoprotein, free beta-hCG or total hCG, unconjugated estriol and inhibin-A. Specific ranges of risks were used to define the borderline group. Separate protocols were developed for the United Kingdom and the United States to reflect differences in commonly used tests, cut-offs, and the gestational age at testing. Detection rates and false-positive rates were estimated by multivariate Gaussian modelling with Monte Carlo simulation. Proposed protocols based on first-trimester NT, PAPP-A and free beta-hCG or total hCG, followed by selective use of second-trimester quadruple markers can result in a 91% detection rate and 2.1% false-positive rate for the United Kingdom and a detection rate of 89% and false-positive rate of 3.1% for the United States. For both countries, over 60% of affected pregnancies would be detected in the first trimester and less than 20% of women would require a second-trimester Down syndrome risk assessment. Use of alternative cut-offs to define those with borderline risks or different combinations of second-trimester markers also yielded high detection rates and low false-positive rates. With appropriate patient counselling, it should be possible to provide highly effective Down syndrome screening using contingent sequential protocols.

Minimal residual disease monitoring by flow cytometry

In patients with acute leukaemia, studies of minimal residual disease (MRD) provide powerful and independent prognostic information. Multiparameter flow cytometry is a widely applicable and reliable approach for monitoring MRD. Using triple or quadruple marker combinations, aberrant or uncommon phenotypic profiles can be identified in about 80% of patients with acute myeloid leukaemia (AML) and 95% of patients with acute lymphoblastic leukaemia (ALL). These profiles can reveal leukaemic cells even when these are not evident by morphological analysis. Thus, one leukaemic cell among 1000–10 000 normal bone marrow or peripheral blood cells can be routinely detected. In this chapter we discuss technical aspects of MRD detection by flow cytometry and summarize results of correlative studies between MRD, clinical and biological features of leukaemia and treatment outcome. Current knowledge indicates that MRD studies using well-tested methodologies are clinically useful and should be incorporated into the clinical management of patients with acute leukaemia.

Immunologic monitoring in adults with acute lymphoblastic leukemia.

Investigation of minimal residual disease (MRD) by immunophenotyping and molecular techniques has proven to be a powerful approach for disease monitoring in patients with acute leukemia. Multiparameter flow cytometry, through the use of triple or quadruple marker combinations, identifies aberrant or uncommon phenotypic profiles in more than 90% of adult patients with acute lymphoblastic leukemia (ALL) at diagnosis. These profiles allow identification of residual leukemic cells in bone marrow or peripheral blood once morphologic complete remission is achieved. Until now, most immunophenotypic MRD studies in ALL have focused on children. In contrast, information on the value of MRD in adults with ALL is scanty and usually restricted to polymerase chain reaction studies. In this review, we focus on technical aspects of MRD detection by flow cytometry and on the clinical data concerning the value of immunologic MRD studies as a tool for relapse prediction in adult ALL. Although prospective studies are needed, we assert that immunophenotypic MRD studies are clinically useful. Such studies should be incorporated into the routine management of adult ALL patients for identification of those at high risk of relapse, who could benefit from new alternative therapeutic approaches, and to distinguish these patients from others who could be cured with more conventional approaches.

Evaluation of cell-free fetal DNA as a second-trimester maternal serum marker of Down syndrome pregnancy.

Second-trimester cell-free fetal DNA (studied only in pregnancies with male fetuses) is higher in maternal serum samples from women carrying Down syndrome fetuses than in unaffected pregnancies. In this study we evaluated the potential performance of fetal DNA as a screening marker for Down syndrome. Data on maternal serum fetal DNA concentrations and the corresponding concentrations of the quadruple serum markers were available from 15 Down syndrome cases, each matched for gestational age and length of freezer storage, with 5 control samples. Analyte values were expressed as multiple(s) of the control or population median. Screening performance of fetal DNA, both alone and when added to estimates of quadruple marker performance, was determined after modeling using univariate and multivariate gaussian distribution analysis. The median fetal DNA concentration in Down syndrome cases was 1.7 times higher than in controls. In univariate analysis, fetal DNA gave a 21% detection rate at a 5% false-positive rate. When added to quadruple marker screening, fetal DNA increased the estimated detection rate from 81% to 86% at a 5% false-positive rate. Cell-free fetal DNA, measured in maternal serum, can modestly increase screening performance above what is currently available in the second trimester. If and when maternal serum fetal DNA can be measured in pregnancies with both male and female fetuses, the utility and cost-effectiveness of adding it as a Down syndrome screening marker should be assessed.

Hyperglycosylated Human Chorionic Gonadotropin (Invasive Trophoblast Antigen) Immunoassay: A New Basis for Gestational Down Syndrome Screening

Serum human chorionic gonadotropin (hCG) and hCG free beta-subunit tests are used in combination with unconjugated estriol and alpha-fetoprotein in the triple screen test, and with the addition of inhibin-A in the quadruple marker test for detecting Down syndrome in the second trimester of pregnancy. These tests have a limited detection rate for Down syndrome: approximately 40% for hCG or free beta-subunit alone, approximately 60% for the triple screen test, and approximately 70% for the quadruple marker test, all at 5%, or a relatively high, false-positive rate. New tests are needed with higher detection and lower false rates. Hyperglycosylated hCG (also known as invasive trophoblast antigen or ITA) is a new test. It specifically detects a unique oligosaccharide variant of hCG associated with Down syndrome pregnancies. We evaluated this new Down syndrome-directed test in prenatal diagnosis. Hyperglycosylated hCG was measured in urine samples from women undergoing amniocentesis for advanced maternal age concerns at 14-22 weeks of gestation, 1448 with normal karyotype and 39 with Down syndrome fetuses. The median hyperglycosylated hCG value was 9.5-fold higher in Down syndrome cases (9.5 multiples of the normal karyotype median). The single test detected 80% of Down syndrome cases at a 5% false-positive rate. Urine hyperglycosylated hCG was combined with urine beta-core fragment (urine breakdown product of serum hCG free beta-subunit), serum alpha-fetoprotein, and maternal age-related risk. This urine-serum combination detected 96% of Down syndrome cases at a 5% false-positive rate, 94% of cases at a 3% false-positive rate, and 71% of cases at a 1% false-positive rate. These detection rates exceed those of any previously reported combination of biochemical markers. Hyperglycosylated hCG is a new base marker for Down syndrome screening in the second trimester of pregnancy. The measurement of hyperglycosylated hCG can fundamentally improve the performance of Down syndrome screening protocols.