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Technical Briefs |
1 Antenatal Diagnosis Unit, Department of Obstetrics and Gynaecology, and4 Department of Pathology and Laboratory Medicine, University Hospital, Groningen, The Netherlands;2 Institut für Humangenetik der Universität Göttingen, Göttingen, Germany;3 Quest Diagnostics Nichols Institute, San Juan Capistrano, CA;
aaddress correspondence to this author at: Department of Obstetrics and Gynaecology, University Hospital, PO Box 30.001, 9700 RB Groningen, The Netherlands; fax 31503611806, e-mail martinweinans{at}planet.nl
Hyperglycosylated human chorionic gonadotropin (hCG) is a variant of hCG with more asparagine (N)-linked triantennary carbohydrates and a serine (O)-linked tetrasaccharide core structure in the ß-subunit of hCG (1). Whereas nonhyperglycosylated hCG is secreted by differentiated syncytiotrophoblast cells, hyperglycosylated hCG is secreted solely by invasive cytotrophoblast cells and is therefore also called invasive trophoblast antigen (ITA) (2)(3). Before the sixth week of gestation, ITA appears to be the predominant form of hCG (2)(3)(4). In Down syndrome pregnancies, differentiation of the cytotrophoblast into a syncytiotrophoblast may be delayed, leading to increased production of ITA (5).
Urinary ITA is a promising candidate for use as a biochemical marker in Down syndrome screening. In one study conducted during the second trimester of pregnancy, ITA alone detected 78% of the Down syndrome cases at a 5% false-positive rate (6)(7). In a setting that simulated routine use, urinary ITA was reported to be the best single marker in the second trimester (8). In the first trimester of pregnancy, however, the performance of urinary ITA is lower, with a reported 63% Down syndrome detection rate at a 10% false-positive rate (9).
ITA is detectable in serum as well as in urine (10). Studies have not been performed with serum ITA because of concerns about the stability of ITA in serum, possible loss of ITA when blood is collected in gel barrier tubes, and possible aggregation. A recently developed automated immunochemiluminometric assay measures ITA in various sample types, including serum (11). In the present study, we investigated the Down syndrome screening performance of serum ITA before 12 weeks of gestation and compared it with the performance of pregnancy-associated plasma protein A (PAPP-A) and free ß-subunit in the same sample set.
Sera from 24 women with Down syndrome-affected pregnancies and 320 unaffected pregnant women were used in this retrospective study. Samples were collected from women at 9 weeks and 5 days (9 + 5) of gestation to 11 weeks and 4 days (11 + 4). These samples were collected between 1999 and 2002, with permission, before chorionic villus sampling. The primary indication for chorionic villus sampling was advanced maternal age (
36 years). All samples were collected at the Antenatal Diagnosis Unit of the University Hospital Groningen, The Netherlands, into non–gel-barrier Vacutainer Tubes. An Institutional Review Board-approved protocol was followed. Control samples were matched for gestational age, maternal age, and length of storage. The mean gestational age was 76.4 days for cases and 76.0 days for controls. The mean (SD) maternal age was 38.8 (2.36) years for cases and 37.2 (3.04) years for controls. The median duration of sample storage was 2 years and 1 month for cases and 2 years and 2 months for controls. Blood samples were allowed to clot for 1–3 h at room temperature and were centrifuged at 2500g and 10 °C for 10 min. Serum fractions were frozen immediately and stored at –20 °C and never thawed at room temperature except before being assayed. Serum samples were then thawed overnight at 4 °C and analyzed within 4 h.
ITA was measured by an immunochemiluminometric assay on the Nichols Advantage® platform (Nichols Institute Diagnostics) with an acridinium-ester–labeled, anti-hCGß monoclonal antibody (B207) and a biotinylated ITA-specific monoclonal capture antibody (B152) (11). The assay has a calibration range up to 300 µg/L, with automatic dilution at higher concentrations. The assay has <1% cross-reactivity with recombinant hCG (11). The reported intra- and interassay variations (as CV) are <8% and 12%, respectively.
PAPP-A and free ß-subunit were both measured by a fluoroimmunoassay (AutoDELFIA® PAPP-A and Free hCGß reagent sets; Perkin-Elmer). The detection limits of the assays are 5 mIU/L for PAPP-A and 0.2 µg/L for free ß-subunit. The intra- and interassay variations (CVs) for PAPP-A were <2% and 4%, respectively, at a concentration of 1500 mIU/L. For free ß-subunit, the CVs were <4% and <5% at 40 µg/L.
We used STATISTICA for Windows, Ver. 6 (StatSoft Inc). Multivariate discriminant analysis was performed to calculate the risks. The multiples of the median (MoM) were derived from regressed medians, with gestational days used as the independent variable. All parametric statistical procedures were based on the natural logarithms of the concentrations or MoM values. A Monte Carlo model was applied to adapt detection and false-positive rates to the present age-standardized population of The Netherlands. Each patient was assigned the mean of 10 randomized maternal age-related risks where the randomization was based on the proportion of maternal age rates according to the present birth frequencies in The Netherlands.
Means, SD values based on logarithmic MoM values, and median MoM values for all 3 markers are summarized in Table 1
.
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The squared Mahalanobis distance was 1.42 for ITA, 1.73 for free ß-subunit, and 2.50 for PAPP-A.
The predicted detection rates for Down syndrome for the combination of ITA and PAPP-A at 3%, 5%, and 10% false-positive rates were 62%, 71%, and 83%, respectively. For the combination of free ß-subunit and PAPP-A, the predicted detection rates were 58%, 75%, and 79%, respectively.
The associations among ITA, PAPP-A, and free ß-subunit concentrations were calculated in both affected and control pregnancies. In controls, there was a significant correlation between ITA and free ß-subunit. The Pearson correlation coefficients (r) between the log-transformed MoM values were as follows: ITA/free ß-subunit, 0.63; ITA/PAPP-A, 0.15; and PAPP-A/free ß-subunit, 0.27.
The ROC curves for both ITA and free ß-subunit in combination with PAPP-A, including maternal age after modeling against the age-standardized population of The Netherlands in 2002 (12), are shown in Fig. 1
. From these curves, the predicted detection rate for a given screen-positive rate can be determined.
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We have shown that serum ITA is a useful first-trimester marker for Down syndrome screening. In the present study the combination of PAPP-A and ITA detected 71% of the Down syndrome cases at a 5% false-positive rate. The predicted detection rate for the combination PAPP-A/ITA, including maternal age after modeling against the age-standardized population of The Netherlands, was 63% at a 5% false-positive rate (Fig. 1
).
PAPP-A was the most powerful biochemical marker in this study, as evidenced by the highest Mahalanobis distance. Although the median MoM value in Down syndrome cases was slightly higher for ITA (2.6 MoM) than for free ß-subunit (2.2 MoM), ITA was not a better marker than free ß-subunit, perhaps because there was less variation in free ß-subunit concentrations in the control population (see the SDs in Table 1
). The higher Mahalanobis distance for free ß-subunit (1.7) compared with ITA (1.4) indicates that free ß-subunit was a slightly better marker in this study.
The ROC curves (Fig. 1
) indicate that at false-positive rates of 2%–7.5%, the PAPP-A/free ß-subunit combination outperforms the PAPP-A/ITA combination, whereas at false-positive rates <2% and between 7.5% and 10%, the PAPP-A/ITA combination performed better.
Because the gestational age window in the present study was only 2 weeks (9 + 5 to 11 + 4) and the sample size was small, we do not know whether ITA is a better marker than free ß-subunit during the very early first trimester (
9 weeks of gestation). In late first trimester (12–14 weeks of gestation), serum ITA might be a better marker than free ß-subunit because urinary ITA concentrations in affected pregnancies have been shown to be very high at those gestational ages (13). Moreover, because the correlation between ITA and PAPP-A is less than that of free ß-subunit and ITA (i.e., ITA is more independent of PAPP-A), an ITA/PAPP-A combination may be a more effective screen than the free ß-subunit/PAPP-A combination.
ITA should not be added as a third marker (i.e., added to free ß-subunit and PAPP-A) because of the high correlation between ITA and free ß-subunit in unaffected pregnancies.
The results of this study are comparable to the early first-trimester (10–11 weeks of gestation) study of ITA in maternal urine samples (9). Because urinary ITA studies during the second trimester of pregnancy show a greater discriminatory power (i.e., 78% detection at 5% false-positive rate) (6)(7), it is expected that serum ITA will also have a higher Down syndrome detection rate in the second trimester. Currently, studies are in progress to establish the role of ITA in the second trimester of pregnancy as a serum marker for Down syndrome.
Acknowledgments
We gratefully acknowledge Robert de Vrij and Jurjen IJlstra for excellent technical assistance. We also thank Nichols Institute Diagnostics for providing ITA reagents.
References
- and ß-subunits of human chorionic gonadotropin from normal and aberrant pregnancy and choriocarcinoma. Endocrine 1997;7:15-32.[Web of Science][Medline]
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The following articles in journals at HighWire Press have cited this article:
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  S.F. de Medeiros and R.J. Norman Human choriogonadotrophin protein core and sugar branches heterogeneity: basic and clinical insights Hum. Reprod. Update, January 1, 2009; 15(1): 69 - 95. [Abstract] [Full Text] [PDF]
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 G. E. Palomaki, G. J. Knight, L. M. Neveux, J. E. Haddow, and R. Pandian First-Trimester Down Syndrome Screening: Reply Clin. Chem., January 1, 2006; 52(1): 161 - 161. [Full Text] [PDF]
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