Second Trimester Antenatal Screening for Down’s Syndrome

27CHAPTER

SUMMARY

Second trimester screening for Down’s Syndrome using maternal serum biomarkers such as a-Foetoprotein (AFP), human Chorionic Gonadotropin (hCG) and unconjugated Oestriol (uE3) iswell established.

Blood tests for second trimester screening are carried out between 14 and 20 weeks ofpregnancy. However, reliability of results depends on the analytical quality of theimmunoassays, precise knowledge of the age of the foetus, the median values of the serummarkers, reliability of the statistical coefficients, precise calculation of the multiple of medians(MoM) and the robustness of the models used for statistical calculation of the risk factor.

Screening today already achieves Down’s Syndrome detection rates of >80% for a 5% falsepositive rate. However, it is likely that scientific organisations will recommend a 90% detectionrate for a 1-2% false positive rate. This is achievable with new biomarkers e.g. dimeric inhibin Aand PAPP-A.

The purpose of Down’s Syndrome (DS) screening is to offer women a test that can identifythose with a high risk of having an affected pregnancy of chromosomal abnormality such asTrisomy 21 and Trisomy 18, so that they can be offered a diagnostic test. Prenatal tests such

Second Trimester Antenatal Screeningfor Down’s SyndromeThomas Brinkmann1, Jan van Nueten2 and Guido Vranken2

1Beckman Coulter Eurocentre, Nyon, Switzerland2Analis, Gent, Belgiume-mail: [email protected]

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as triple screening are performed during the second trimester of pregnancy, more specificallybetween the 14th and 20th week.

For triple test the following three biomarkers are measured in the maternal serum: alphafoetoprotein (AFP), human chorionic gonadotropin (hCG) and unconjugated oestriol (uE3). Tocalculate the risk, median values for normal pregnancies based on the stage of pregnancy mustbe determined beforehand. AFP, hCG and uE3 levels in maternal serum are dependent on theage of the foetus, which means that the exact date of the beginning of pregnancy is crucial andmust be precisely known.

Results of the screening are expressed in “multiples of median” (MoM). This explains whythe mean value is called “1 MoM”. AFP and uE3 values during pregnancies with Down’sSyndrome are lower than those associated with normal pregnancies, which means that valuesbelow the mean are below 1 MoM. The hCG value for Down’s Syndrome is above 1 MoM. TheMoMs and the maternal age are used to calculate the possible risk of chromosomalabnormalities such as trisomy 21. Specific computer programmes used for this calculation havebeen developed.

With the “Triple Test”, it’s possible to calculate the risk of giving birth to a child withtrisomy 21. Combination of these 3 markers offers a 50 to 80% chance of detecting pregnancieswith trisomy 21. AFP alone was previously used as a marker, with only a 30% chance ofdetection.

The risk of giving birth to a child with Down’s Syndrome increases with the age of themother. Thus, based on the model of Cuckle [1], the risk of Down’s Syndrome in a pregnantwoman aged 35, based solely on age, is, estimated at 1 out of 384. Before the “Triple Test”existed, a prenatal diagnostic test of amniocentesis was proposed to women over the age of 35.The same method is still used today with the “Triple Test”. When the calculated age-independent risk of the mother is equal to that of a woman around the age of 35, confirmatoryprenatal diagnosis is proposed. As amniocentesis is not without risk, the “triple test risk” mustbe reliably calculated.

Reliability of results of the “Triple Test” depends on a certain number of factors:• analytical quality of the immunoassays used to quantify the serum markers• precise knowledge of the beginning of the pregnancy• median values of the serum markers must be representative for the population studied• the reliability of the statistical coefficients (intercept and slope) describing the linear

relationship between the logarithm of the median value of the serum markers and thestage of pregnancy

• precise calculation of the MoM value (multiple of medians)• robustness of the models used for statistical calculation of the risk factor

ααααα-FOETOPROTEIN (AFP TEST)AFP is a single-chain glycoprotein mainly produced by the foetal liver and vitelline membrane.The medians increase significantly between the 15th and 22nd weeks. In the case of trisomy 21,lower values are observed. The analytical range required for study of the risk is between 5 and1,000 IU/mL. The Access is linear up to 2,490 IU/mL.

320 GONADAL AND NONGONADAL ACTIONS OF GONADOTROPINS

Figure 1 Maternal age is a risk factor for Down’s Syndrome pregnancy

CHORIONIC GONADOTROPIN (hCG TEST)hCG is a two-chain molecule (alpha and beta) produced by the placenta. The beta chain isunique to this glycoprotein. The medians decrease between the 15th and 22nd weeks. In the caseof trisomy 21, the values are higher than the norm. The measurement range required forscreening is between 3 and 200 IU/mL. The Access is equipped with an automatic dilutionsystem for hCG and the measurement range is between 0.5 and 200 IU/mL.

UNCONJUGATED OESTRIOL (uE3 TEST)

uE3 is a steroid hormone produced by the placenta from precursors synthesised in the foetaladrenal gland. The median values increase between the 15th and 22nd weeks. In the case oftrisomy 21, the values are lower. Screening requires very high sensitivity. The measurementrange of the Access is 0.06 to 24 nmol/L.

MEDIAN VALUESA study designed to determine the median values was performed at the Women’s Clinic inBirmingham, England. The Access immunoassay system was used to test the samples ofmaternal serum collected from a population from the West Midlands and North-West Thames.A total of 1,140 samples from pregnant women between their 14th and 20th week of pregnancywere tested for AFP, hCG and uE3. The median values were determined for completed weeks.The median values for AFP were calculated by log-linear regression analysis. An exponentialmodel was used for hCG and uE3.

GEOGRAPHICAL DIFFERENCES AND VARIATIONSIn a retrospective study, AFP, hCG and uE3 values for the second trimester were determined ina Belgian population of 862 pregnant women with normal pregnancy [2]. The absolute

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Figure 2 Weighted regressed medians of AFP, hCG and uE3 in relationship to gestational week

Tabel 1 The values are provided as an indication. Each laboratory must determine the median values for its ownpopulation

Beckman Coulter Weighted Regressed Medians

Weeks n AFP hCG uE3 uE3IU/mL IU/mL ng/mL nmol/mL

14 40 23.61 39.96 0.65 2.2615 275 26.97 33.91 0.82 2.8416 472 30.82 27.60 10.3 3.5817 236 35.22 22.94 1.30 4.5118 78 40.24 19.07 1.64 5.6719 20 45.98 15.85 2.06 7.1420 19 52.53 13.17 2.59 8.99

concentration of medians differ significantly from the medians published for othergeographical regions.

In addition the variation of the estimations of the risk of Down’s Syndrome, especiallyduring weeks 14 and 19, is dependent, among other reasons, on comparison of the methodsused to convert the analytical results of the serum markers into MoM values. Therefore, alsomethods used to calculate the intermediate results must be similar in order to reduce possibleinter-laboratory variations to a minimum.

WEIGHT CORRECTION OF MOM VALUES

In prenatal Down’s Syndrome screening programmes, correction of maternal serum markersfor maternal weight is considered to be standard practice. However, the choice of model for


Table 2 Medians from the second trimester for Access serum markers determined from serum of 862 pregnantBelgian women with normal pregnancy

Gestational age N AFP hCG uE3Week/days (IU/ml) (IU/ml) (nmol/L)

14/3 93 22.1 37.3 2.1515/3 373 25.8 30.2 2.816/3 207 30.2 24.5 3.6617/3 94 35.3 19.9 4.7718/3 61 41.2 16.1 6.2219/3 32 48.2 13.0 8.11

taking maternal weight into account is rarely explicitly evaluated. The relationship betweenmaternal weight and the prenatal serum markers alpha foetoprotein (AFP), human chorionicgonadotropin (hCG) and unconjugated oestriol (uE3), determined with Beckman CoulterAccess reagents, was studied in a Belgian cohort of 752 pregnant women screened for Down’sSyndrome. Two different models (log-linear equation and linear-reciprocal equation) wereused to determine the relationship between the serum markers and maternal weight [3, 4].

A significant relationship between the log10 multiples of median (MoM) and weight (kg)was observed for the three markers and the log-linear model was associated with slightlyhigher coefficients of determination than those with the linear-reciprocal model. Table 3 showsthe MoM values for the serum markers in the so-called triple test by weight category.

Tabel 3 Access MoM medians of serum AFP, hCG, and uE3 as a function of maternal weight

Weight in kg N AFP hCG uE3

–49 33 1.39 1.18 1.0950 - 59 184 1.12 1.00 1.0760 - 69 259 1.01 1.12 1.0070 - 79 157 0.96 0.91 1.0180 - 89 54 0.81 0.94 0.9390 - 99 37 0.76 0.88 0.90> 100 28 0.66 0.83 0.83

All weight categories 752 0.99 1.00 1.01

Correction of weight with the two models achieved the optimal effect that the correctionfactor for a woman with a median weight of 65.5 kg was not significantly different from 1.Simulated weight-corrected MoM values calculated using the two methods were comparedand variation was estimated. The mean difference between the weight-corrected MoM valuescalculated by the two methods was 7.8% (SD 4.3%) for AFP, 14.0% (SD 4.4%) for hCG and 5.9%(SD 3.2%) for uE3. Estimation of the risk of Down’s syndrome for a woman with median weightresulted in a 1.7% - 5.3% difference depending on the correction algorithm used.


Figure 3 a-c Relationship between maternal weight and the serum markers during the second trimester. Maternalweight (kg) is shown on the X-axis and the serum markers on the Y-axis (represented by MoM values). The solid linerepresents fitting of the linear-reciprocal model and the dashed line is fitting of the log-linear model. The verticallines are the 95% confidence intervals of the median values. This figure clearly shows that both models allow goodadjustment of the data points, are equivalent and are appropriate for prenatal screening programmes. The lastmedian value was not used for fitting given the small number of data points.

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Multiples of Median values can be influenced significantly by weight. The log-linearcorrection factor – to correct for the difference between 65 kg (median weight) and 95 kg forAFP – is equal to 1.34, or 34%. Various algorithms are presented in the literature to correct formaternal weight. In the cohort studied, the log-linear weight correction model proposed wasslightly more effective than the linear-reciprocal model, based on an analysis of statisticaladjustment. The differences between the weight-corrected MoM values by the two modelswere highly significant (P < 0.001, Wilcoxon signed-rank test), but the effect on estimation ofrisk was non significant. The changes in the risk were found to increase in significance thefarther the MoM correction factors deviated from 1. It is recommended to apply the weightcorrection to all analytes in Down’s Syndrome screening programmes. Data from the literatureindicates that correction of weight improves detection of Trisomy 21 pregnancies.

COMPUTER SOFTWARE TO CALCULATE THE RISK OF DOWN’S SYNDROMEBeckman Coulter’s Access prenatal biomarkers were caculated with Analis RiskCalcprogramme developed by Professor Dr. Tim Reynolds, Queen’s Hospital Burton upon Trent,UK. The calculation method used in Reynolds’ software is based on the “likelihood” method(probability method) [5]. This method is based on the principle that a Gaussian distribution canbe defined both for normal and abnormal values of a biochemical marker. It has beendemonstrated that the distribution of serum values can be normalised using the logarithm ofthe MoM value [6].

After having established this Gaussian distribution, the probability that a givenconcentration is in the normal or abnormal population can then be determined. The ratio ofthese two probabilities can be used to adapt the risk of Down’s Syndrome to the stage ofpregnancy in order to compare the resulting risk with a risk calculation threshold. A concreteexample illustrates this principle well. The risk of Down’s Syndrome in a 25 year-old woman is1:1,351. This risk can be calculated using the following formula [1]:

Risk = [0.999373 – e((0.286x Age) – 16.2395)]/[0.000627 + e((0.286x Age) –16.2395)]

The normal and abnormal Gaussian values of the individual Multiples of Medians (MoM)of the serum markers are then calculated. Figure 4 shows two possibilities: in the first case, theratio of the values of the normal and abnormal curves (N1/A1) is more or less equal to 4.5.

In the second case, N2/A2 is approximately 0.37. The age-specific risk is multiplied by thisratio to determine the definitive risk. Under scenario 1, the definitive risk for a 25 year-oldwoman is 1:6,080. Under the second scenario, the risk is 1:500.

The risk threshold for Down’s Syndrome can be entered by the user. In accordance with theliterature, a risk threshold of 1:250 is used as a default.

The following information must be completed in order to evaluate the risk:• sample date• mother’s date of birth• stage of pregnancy (number of weeks and days)• marker values (AFP, hCG and uE3)


Figure 4 Basis for the “likelihood method (probability method)

Optional data:• Scan data• Name of the mother• Identification no.• Weight/smoker/diabetic

Figure 5 RiskCalc Software data entry screen

The result screen shows the MoM, the corrected MoM and the calculated risks of trisomy21 and 18 calculated with 2 and 3 serum markers. A colour code indicates the significance of therisk: green means that there is no calculated risk, yellow indicates an intermediary risk and redis the sign of a high risk. The software provides a comment for NTD, Trisomy 21 (Down Risk)and Trisomy 18 and a typicality.

Basis of Likelihood ratios

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Figure 6 Results screen for a sample with intermediary risk (yellow) for the double test and high risk (red) oftrisomy 21 for the triple test

REFERENCES1. Cuckle HS, Wald NJ & Thompson SG: Estimating a woman’s risk of having a pregnancy

associated with Down’s syndrome using her age and serum alpha-fetoprotein. Br J ObstetGynaecol 1987; 94: 387-402.

2. Vranken G, Reynolds TM & van Nueten J: Medians for second-trimester maternal serummarkers: geographical differences and variation caused by median multiples-of-medianequations. J Clin Pathol 2006; 59: 639-644.

3. Reynolds TM, Vranken G, van Nueten J & Aldis J: Evaluation of Down’s syndrome screeningpopulation data sets by simulation: analyser-specific parameters may be superior to meta-analysis-derived parameters. Int J Clin Practice 2007; 59: 639-44.

4. T.M. Reynolds, G. Vranken and J. van Nueten: Weight correction of MoM values: whichmethod? J Clin Pathol 2006; 59: 753-8.

5. Palomaki GE & Haddow JE: Maternal, serum alfa-fetoprotein, age and Down’s syndrome risk.Am J Obstet Gynecol 1987; 156: 1557-9.

6. Wald NJ, Cuckle HS, Densem J et al: Maternal serum screening for Down’s syndrome in earlypregnancy. Br Med J 1988; 297: 883-8.

Second Trimester Antenatal Screening for Down’s Syndrome

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