Second Trimester Serum Markers

Second Trimester Serum Markers Jacob A. Canick, PhD,* and Andrew R. MacRae, PhD† Prenatal screening for Down syndrome in the early second trimester with multiple maternal serum markers has been available for more than 15 years. The multiple marker combination with the highest screening performance currently available is alpha-fetoprotein (AFP), unconjugated estriol (uE3), human chorionic gonadotropin (hCG), and inhibin A, together with maternal age (so-called quad marker test). With this combination, a detection rate of 80% at a 5% false positive rate is achieved. Inhibin A, the newest addition to second trimester serum screening, is an alpha-beta subunit hormone of placental origin, and is measured using a monoclonal two-site ELISA validated for use in prenatal screening. Quality control parameters for inhibin A measurement are acceptable and are monitored through the proficiency testing program administered by the College of American Pathologists. Research into other possible second trimester screening markers has included studies on the maternal urine and serum levels of an hCG variant, hyperglosylated hCG (h-hCG; invasive trophoblast antigen). Recent data indicate that h-hCG is similar to hCG itself, although its measurement in maternal urine may improve the performance of the established serum marker combinations. With the introduction of first trimester screening markers and their use in an integrated first and second trimester marker approach to screening, and with the fact that many women do not seek prenatal care until the early second trimester, prenatal screening for Down syndrome using second trimester serum markers remains a major resource in obstetrical care. Semin Perinatol 29:203-208 © 2005 Elsevier Inc. All rights reserved. KEYWORDS prenatal screenig, serum markers, Down syndrome, trisomy 21, inhibin A, hyperglycosylated hCG

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creening for fetal Down syndrome in the early second trimester using multiple markers measured in maternal serum has become a routine part of prenatal care. Alphafetoprotein (AFP) measurement, already increasing in use by the mid-1980s as a screening test for open neural tube defects, was reported by Irwin Merkatz in 1984 to be low in Down syndrome pregnancy.1 Within a few years, the measurement of human chorionic gonadotropin (hCG) and unconjugated estriol (uE3) was also shown to be informative of Down syndrome risk.2-4 In 1988, Wald and colleagues proposed that a multiple marker test, incorporating the measurement of all three second trimester markers and maternal age (the so-called triple test), would provide a major improvement in prenatal screening performance.5 By the mid-1990s, the American College of Obstetricians

*Department of Pathology and Laboratory Medicine, Women and Infants Hospital, Brown Medical School, Providence, RI. †The Research Institute at Lakeridge Health, Oshawa, Ontario. Address reprint requests to Jacob A. Canick, PhD, Department of Pathology, Women and Infants Hospital, 101 Dudley Street, Providence, RI 02905. E-mail: [email protected]

0146-0005/05/$-see front matter © 2005 Elsevier Inc. All rights reserved. doi:10.1053/j.semperi.2005.05.011

and Gynecologists (ACOG) had published Committee Opinions and Educational Bulletins recommending that multiple marker screening should be offered to all pregnant women under the age of 35, and that women of advanced maternal age who were reluctant to accept invasive testing (amniocentesis) might want to refine their risks by first having multiple marker screening.6,7 The uptake of serum screening by pregnant women in the United States is high, according to a survey published in 1997.8 Based on responses of clinical laboratories offering maternal serum screening, the Foundation for Blood Research in collaboration with the College of American Pathologists reported that between 1992 and 1995 there was an overall increase in second trimester screening from 51% to 65% of the approximately 4 million pregnancies annually. In 1992, most women who were having screening had the single marker AFP screen only, with only 26% of women screened using multiple markers. By 1995, these proportions were reversed; 78% of women who were having screening used multiple markers, and only 22% using AFP alone (Fig. 1A). The survey also found that, by 1995, 69% of the clinical laboratories offering screening were offering triple marker 203

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Figure 1 (A) The proportion of pregnant women in the United States having various types of second trimester maternal serum screening tests in 1992 and 1995. (B) The proportion of 264 clinical laboratories offering various screening tests in 1995.8 (Color version of figure is available online.)

screening exclusively, 6% were offering a choice of either double or triple markers, 14% were offering double markers exclusively, and only 11% of laboratories were offering single marker AFP alone (Fig. 1B). Based on the relative sizes of the laboratories reporting, it was estimated that by 1995 more than 95% of the women in the United States who were having multiple marker screening were having the triple test.

Addition of Inhibin A Inhibin is a family of alpha-beta subunit glycoproteins named in the 1930s for their ability to selectively inhibit the secretion of the anterior pituitary gonadotropin, follicle stimulating hormone. In 1992, van Lith and coworkers, using an immunoassay that measured all forms of inhibin and its alpha subunit, reported that second trimester maternal serum total inhibin levels tended to be elevated in Down syndrome pregnancy.9 By 1994 through 1996, with the advent of a specific

immunoassay that measured only dimeric inhibin A, the levels of inhibin A, specifically, were reported to be more elevated in Down syndrome pregnancy than the levels of total inhibin.10-16 By 1996, Wald and coworkers estimated that inhibin A measurement, added to the triple marker test, would increase screening performance significantly and recommended that a four marker “quad” approach be introduced.12 Various studies have reported their estimates of screening performance using various combinations of second trimester markers. Estimates from the two most recent such studies, SURUSS,17 conducted primarily in the United Kingdom, and FASTER,18 conducted in the United States, are remarkably similar (Table 1). The incremental gain in detection by moving from the triple to the quad marker test is in the range of 6 to 11 percentage points, so that a detection rate of approximately 80% for a 5% false positive rate can be achieved with quad marker testing. In an earlier study, Haddow and colleagues examined the

Second trimester serum markers

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Table 1 Second Trimester Test Performance Detection Rate @ 5% False Positive Rate Double markers: AFP ⴙ hCG Triple markers: AFP ⴙ hCG ⴙ uE3 Quad markers: AFP ⴙ hCG ⴙ uE3 ⴙ inh A

SURUSS17

FASTER18

66%

—

74%

70%

81%

81%

Results of two population-based studies. All gestations dated by crown-rump-length ultrasound measurement.

effect of substituting inhibin A measurement for either uE3 or hCG, to determine whether inhibin A was an effective marker in place of, rather than in addition to, established markers.19 They found that a triple marker combination of AFP, uE3, and inhibin A, or AFP, hCG, and inhibin A would provide a detection rate (at a fixed 5% false positive rate) of 75% or 74%, respectively, in comparison to a detection rate of 71% with the traditional triple test (AFP, uE3, hCG). When inhibin A was added to the traditional triple marker test, the resulting quad marker test had a detection rate of 78% for a 5% false positive rate. The distribution of the second trimester quad markers in

Down syndrome pregnancy is illustrated by examining the MoM values seen in the FASTER Trial (Fig. 2). On a log scale of MoM values, all four markers appear to be normally distributed in Down syndrome pregnancies, with the medians seen in FASTER very similar to the consensus medians derived from meta-analysis of the world’s literature. In FASTER, the AFP, hCG, and inhibin A medians in the Down syndrome population were as expected, whereas the median uE3 was lower than expected (0.61 MoM in FASTER, compared with 0.72 MoM from the published consensus). Other than chance, there is no explanation for this difference. Rather than using a statistical outlier, the consensus median for uE3 was used in all calculations of modeled performance in FASTER. The use of inhibin A in second trimester serum screening is increasing at a fast pace. Data in the 2004 FBR/CAP Survey show that 83 (40%) of the 209 subscribing laboratories are measuring inhibin A, and since most of the largest reference laboratories in the United States are among those 83, it can be surmised that considerably more than 40% of pregnant women having serum screening are having inhibin A as part of their test. Another independent estimate of the current use of inhibin A in prenatal screening in the United States can be derived from data on the number of inhibin A test kits that were being sold as of the

Figure 2 MoM values for the second trimester maternal serum quad markers (AFP, uE3, hCG, inhibin A) from 73 of the cases of Down syndrome identified during the FASTER Trial.18 Medians from SURUSS are provided for comparison at the bottom.17 (Color version of figure is available online.)

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Figure 3 Examination of the consistence of control values measured using the inhibin A ELISA from Diagnostic Systems Laboratories, Inc., according to well position in a 96-well microtiter array. (A) Low pool control, inhibin A mean level ⫽ 169 pg/mL, across-plate CV ⫽ 3.6%. (B) High pool control, inhibin A mean level ⫽ 327 pg/mL, across-plate CV ⫽ 2.8%. (Color version of figure is available online.)

end of 2004 (data provided by Diagnostic Systems Laboratories, Inc.). From this source, the estimate is 1.6 million (64%) of the 2.5 million women currently having prenatal serum screening are having inhibin A measured as part of their screening test.

Reliability of the Inhibin A Assay The assay currently available to measure inhibin A is in one format, an enzyme-linked immunosorbent assay (ELISA) produced by Diagnostic Systems Laboratories, Inc. (DSL), Webster, TX. The monoclonal antibodies used in the assay (one directed against the alpha-subunit and one against the beta-subunit) were developed by Nigel Groome at Oxford Brookes University, UK. The assay can be performed manually or by using an automated immunoassay platform. The DSL assay has been validated for use in Down syndrome screening in comparison with the original ELISA method, originally marketed by Serotec, Ltd, Oxford, UK. The DSL inhibin A assay has been checked for precision, assay drift, interassay reproducibility, and interlaboratory variability. Duplicate determinations, as a measure of precision, are rarely above a 10% coefficient of variation (CV). In a study of 400 samples run in 12 separate assays, the mean CV for patient sample duplicates was 2.3%, with 92% of duplicates lower than 5% CV, and 98% of duplicates lower than 10% CV. Within-assay tendency of sample values to increase or decrease, depending on their position on the 96well microtiter plate, was not observed for two different samples tested (Fig. 3). Interassay reproducibility was determined by using sample pools at two different inhibin A concentrations. The interassay CVs for the two sample pools were 9.5% (185 pg/mL) and 6.1% (563 pg/mL). Epidemiological studies have shown that CVs of 10% have no material effect on screening performance.20 Data on interlaboratory variability for inhibin A was obtained from a recent FBR/CAP survey report (FP-B, 2004). For 83 laboratories reporting, the interlaboratory CV on the mass unit of

inhibin A (pg/mL) was in the range of 11% to 13%. This is somewhat higher than the interlaboratory CVs seen for the other protein markers, AFP and hCG, but similar to those of the steroid marker, uE3. The high interlaboratory CVs seen for inhibin A do not reflect the actual CVs within a single laboratory, which are considerably smaller. For example, within our laboratories, the average CV of inhibin A measurement is routinely under 5%. The extra variability in the interlaboratory CVs reflects the use of different lots of inhibin A kits being used at the same time by the various laboratories. The lot-to-lot variability of inhibin A kits is currently often more than 10% to 15% and is in the process of being reduced through more stringent quality control measures by the manufacturer, with feedback from a panel of outside experts. Notwithstanding the issue of lot- to-lot variability, within an individual laboratory, lot-to-lot variability is easily accounted for by adjusting the inhibin A medians when a new kit lot is used.

Hyperglycosylated HCG A more recent research finding of some interest has been the observation that a specific variant of hCG, hyperglycosylated hCG (h-hCG), also called invasive trophoblast antigen (ITA), has potential value as a screening marker for fetal Down syndrome. H-hCG is a variant with an increased carbohydrate content, making it structurally more complex. Rather than having predominantly biantenary (two branches) carbohydrate side chains, h-hCG has a proportion of triantenary (three branched) side chains. This hCG form is commonly found in choriocarcinoma and is more common in the first trimester of normal pregnancy than in later pregnancy. Cole and colleagues were the first to provide evidence that h-hCG levels, measured in maternal urine and in maternal serum in the second trimester, tend to be elevated in Down syndrome pregnancy, and that the elevation is greater than that seen with hCG itself.21-23 The initial findings on h-hCG levels in both maternal urine and serum samples were very encouraging, with Down syn-

Second trimester serum markers drome medians in the range of 3.5 to 4.5 MoM and with detection estimates of 60% for a 5% false positive rate using h-hCG univariately (ie, without maternal age). Larger studies have been published recently, and the results are less promising. A study by Palomaki and colleagues, published in 2004, examined maternal serum samples from 45 Down syndrome pregnancies, matched with samples from 238 unaffected pregnancies.24 While the median h-hCG value in Down syndrome cases was high (3.15 MoM), the distribution of values was quite broad (SD of log h-hCG MoM ⫽ 0.31 and 0.46 for controls and cases, respectively), offsetting the effect of the high case median. Substituting h-hCG for hCG itself in the quad marker test had no effect; both quad tests had a detection rate of 79% for a fixed 5% false positive rate. The conclusion of that study was that h-hCG was comparable to hCG as a second trimester serum marker. Two recent studies on h-hCG in maternal urine indicate that, in that sample source, h-hCG provides an incremental improvement to the existing serum quad marker test. SURUSS showed that the median h-hCG in maternal urine from Down syndrome pregnancies in the second trimester was 3.51 MoM, and that addition of maternal urine h-hCG to the serum quad test reduced the false positive rate needed to achieve a fixed 85% detection rate by 26% (from 6.2% to 4.6% false positive rate for the quad test and the quad plus urine h-hCG, respectively).17 Similarly, a study by Palomaki and colleagues found a median h-hCG of 4.33 MoM in second trimester urine from Down syndrome pregnancies, and that the addition of maternal urine h-hCG to the serum quad test reduced the false positive rate needed to achieve a 75% detection rate by almost 40% (from 3.3% to 2.0% false positive rate for the quad test and the quad plus urine h-hCG, respectively).25 The incremental gain provided by the addition of a maternal urine sample is offset, perhaps, by the need to obtain a second type of sample, in addition to a blood sample, preferably at the same prenatal visit. More evidence is needed to determine whether h-hCG will have a clinical role in prenatal screening.

Conclusions In the United States and Canada, second trimester serum screening is transitioning from the measurement of triple markers to quad markers. Maternal serum inhibin A, measured in the second trimester, is an important clinical marker, and the available assay performs properly. Recent studies on the clinical utility of hyperglycosylated hCG as a second trimester marker have not replicated the impressive results seen in the initial reports. However, maternal urine measurement of h-hCG may improve existing screening performance, with the trade-off of a second specimen type needed for analysis. Prenatal screening using maternal serum markers in the second trimester is a mature clinical process and maintains an important role in obstetrical care. With the advent of first trimester ultrasound and serum markers, it is probable that a

207 proportion of pregnant women will seek earlier screening, and that, depending on their choice, the volume of second trimester screening might decline. However, other new tests which integrate first and second trimester markers into a single risk estimate that is reported in the second trimester show major promise and provide the best possible screening performance now available. For women having integrated testing and for those women who present for prenatal care later than the first trimester, second trimester markers will continue to be a major resource.

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