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57 The Canadian Journal of Diagnosis / July 2001 Maternal Serum Screening: What Do The Results Mean? Maternal Serum Screening: What Do The Results Mean? By Bernard N. Chodirker, MD MSS can be a valuable screening test for fetal neural tube defects or chromosomal anomalies. As part of this, physicians should counsel their patients about the potential for false-positive or false-negative results. A lthough this article is being written in The Canadian Journal of Diagnosis, it must be emphasized that maternal serum screening (MSS) in not a diagnostic test. It is a screening test. The purpose of a screen- ing test is not to determine if a person has a particular condition, but rather to identify individuals who are at high risk for devel- oping that condition. In the case of MSS, the test is designed to identify women at increased risk for carry- ing a fetus with a neural tube defect, includ- ing spina bifida and anencephaly, or a chro- mosomal anomaly (i.e., Down syndrome or Trisomy 18). Although other maternal or fetal conditions may cause abnormal mater- nal serum screen results, these former con- ditions are the primary focus of MSS. Maternal serum alpha-fetoprotein (MSAFP) testing began in the 1970s as a means of screening for fetal neural tube defects. MSAFP is a protein of fetal origin that rises in the maternal serum with advancing gestation from 13 weeks to 32 weeks. MSAFP levels are first determined in biochemical units (e.g., µg/L). These are converted to multiples of the median (MOMs) by dividing this value by the Women’s Health Care Women’s Health Care

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57The Canadian Journal of Diagnosis / July 2001

Maternal Serum Screening:What Do The Results Mean?

Maternal Serum Screening:What Do The Results Mean?

By Bernard N. Chodirker, MD

MSS can be a valuable screening test for fetal neural tube defectsor chromosomal anomalies. As part of this, physicians shouldcounsel their patients about the potential for false-positive orfalse-negative results.

A lthough this article is being written inThe Canadian Journal of Diagnosis,

it must be emphasized that maternal serumscreening (MSS) in not a diagnostic test. Itis a screening test. The purpose of a screen-ing test is not to determine if a person has aparticular condition, but rather to identifyindividuals who are at high risk for devel-oping that condition.

In the case of MSS, the test is designed toidentify women at increased risk for carry-ing a fetus with a neural tube defect, includ-ing spina bifida and anencephaly, or a chro-mosomal anomaly (i.e., Down syndrome or

Trisomy 18). Although other maternal orfetal conditions may cause abnormal mater-nal serum screen results, these former con-ditions are the primary focus of MSS.

Maternal serum alpha-fetoprotein(MSAFP) testing began in the 1970s as ameans of screening for fetal neural tubedefects. MSAFP is a protein of fetal originthat rises in the maternal serum withadvancing gestation from 13 weeks to 32weeks. MSAFP levels are first determinedin biochemical units (e.g., µg/L). These areconverted to multiples of the median(MOMs) by dividing this value by the

Women’s Health CareWomen’s Health Care

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median for the gestational age at whichthe test was done.

A positive screen test for neural tubedefects occurs when the MSAFP is greaterthan a certain cut-off value. Most pro-grams use cut-offs between 2.0 and 2.5

MOMs. This cut-off is chosen as a com-promise to get the best detection rate rela-tive to the false-positive rate. As Figure 1demonstrates, if the only concern was ahigh detection rate, then a cut-off value of0.7 MOMs could be used. Althoughalmost all cases of fetal spina bifida andanencephaly would be detected, over 50%of pregnant women would have a positivetest. Conversely, if the major concern wasthe false-positive rate, a cut-off of 5.OMOMs could be used. There would bevery few false-positives, but the detectionrate would be very poor. With a cut-offbetween 2.0 and 2.5 MOMs, a detectionrate of 80% for spina bifida and 90% foranencephaly is expected, with a false-pos-itive rate of 2.5% to 4%.1

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Maternal Serum Screening

The Canadian Journal of Diagnosis / July 2001

Dr. Chodirker is medicalgeneticist and associateprofessor, department ofpediatrics and child health,biochemistry and humangenetics, University ofManitoba, and clinicaldirector, Manitoba MaternalSerum Screening Program,Winnipeg, Manitoba.

Figure 1. Distribution of MSAFP levels with fetal neural tube defects.

0.7 5.02.3

MSAFP levels in MOMs

Unaffected Spina Bifida Anencephaly

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In 1984, one study showed that MSAFPlevels in mothers whose babies had Downsyndrome were lower than the levels ofthose in control groups.2 This led to the useof MSAFP (combined with maternal age)as a screening test for fetal Down syn-drome. Since it was known that advancedmaternal age was a major risk factor forhaving a child with Down syndrome, theoriginal screening test for fetal Down syn-drome was to simply ask a woman how oldshe would be on her due date. If the answerwas 35 years or greater, she was consideredscreen-positive. Using this approach, about5% of women would be screen-positive(i.e., a 5% false-positive rate) and 20% ofbabies with Down syndrome would be bornto women who were screen-positive (i.e., a20% detection rate).3 Combining a riskbased on the MSAFP level with maternalage improved the screening performance toa 35% detection rate, with a similar false-positive rate.3

The most common form of MSS is tripletesting, which is based on MSAFP, humanchorionic gonadotropin (HCG) and estriol(E3). HCG is a placental hormone, the lev-els of which drop between 10 and 20 weeksgestation. E3 is made by the placenta fromprecursors from the fetal adrenal and liver.The levels of E3 continue to rise after nineweeks gestation.

Since MSAFP levels are lower in moth-ers carrying fetuses with Down syndrome, alow MSAFP level will statistically increasea woman’s risk of having a baby with Downsyndrome. The chance of this happening iscalculated by comparing the heights of theDown syndrome curve to the unaffected

curve for a given MOM. A MSAFP level ofabout O.5 MOMs will double the chance offetal Down syndrome (likelihood ratio =2.0) while a level of about 1.4 MOMs willdecrease the risk by one-half (Figure 2).

Maternal HCG levels tend to be higherand maternal E3 levels tend to be lower inmothers of Down syndrome fetuses.Likelihood ratios,therefore, are calcu-lated for HCG and E3in a similar fashion toMSAFP. The risk forfetal Down syndromethen is calculated bymultiplying thewoman’s risk, basedon her age, by thethree likelihoodratios. A computerprogram is requiredfor proper interpreta-tion. With triple testing, the detection ratefor fetal Down syndrome is about 60%,with a false-positive rate of 5%.4 Otherfirst- and second-trimester markers, includ-ing biochemical and ultrasound parameters,have been added by various groups to stan-dard triple testing. This has resulted in evenbetter screening performance.5

Fetal trisomy 18 is associated with lowlevels of all three biochemical parameters,and a similar calculation is made to deter-mine the fetal trisomy 18 risk.

The first step in MSS is to counsel thewoman patient. This test should be consid-ered strictly voluntary. She should beinformed that the test is associated with sig-nificant false-positive and false-negative

The Canadian Journal of Diagnosis / July 2001

Maternal Serum Screening

The mostcommon form of

MSS is tripletesting, which isbased on MSAFP,

human chorionicgonadotropin

and estriol.

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rates. She should be made aware that anabnormal test result does not mean that thefetus is affected, while a normal test result

does not guarantee anunaffected fetus.

Currently, the policyin Canada is that allwomen who will beover 35 years of age ontheir due date shouldbe offered an invasivetest for fetal chromoso-mal abnormalities (i.e.,an amniocentesis or

chorionic villus sampling). This policymay change in the future. If a womanchooses to have MSS, care must be takento send the sample at the appropriate gesta-

tion point. Some programs will requireultrasound dating before a sample is test-ed. Most Canadian centers prefer ultra-sound-derived dates, but will use gesta-tion calculations based on the patient’slast period, provided these dates are reli-able. The ideal time to send the bloodsample is between 15 and 18 weeks. Theauthor would not recommend sending asample at exactly 15 weeks, as the com-puter used by the MSS program maydetermine the gestation to be 14.9 weeks,and recommend repeat samples. Samplescould be drawn later—at up to 22weeks—but this can cause unnecessarydelays.

It is important that requisitions forMSS be filled in as completely and accu-

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Small errors inassignment ofgestational agecan cause majorerrors in riskdetermination.

Figure 2. Distribution of MSAFP levels with fetal Down syndrome.

MSAFP levels in MOMs

0.5 1.40.8

LR 2.0

LR 1.0

LR 0.5

Down syndrome

Unaffected

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rately as possible. Small errors in assign-ment of gestational age can cause majorerrors in risk determination. The interpre-tation also should take into considerationthe maternal age, maternal weight, diabet-ic status and the patient’s ethnic origin, allof which can influence the interpretation.

A series of six case presentations areprovided here to illustrate the possibleoutcomes after MSS. The reader is invitedto try and predict the outcome beforereading the actual outcome. All risks quot-ed are term risks, i.e., the chance of hav-ing a live child born with a specific condi-tion.

Case 1A 26-year-old gravida 2 (G2) para 1 (P1)woman has an MSS drawn at 16.2 weeksgestation. The MSAFP is 0.5 MOMs, theHCG is 2.0 MOMs and the E3 is 0.4MOMs. The gestational age is based onher last menstrual period (LMP). The riskfor fetal Down syndrome went from anage-related risk of 1/1,300 to 1/80.

Outcome Further review determined that she hashad only one period since her last preg-nancy due to breast feeding. An ultra-sound examination showed that she wasonly 12.5 weeks pregnant when the bloodwas drawn. The original blood test was,therefore, drawn too early. A repeat bloodsample was drawn at 15.5 weeks by ultra-sound dates, and was screen-negative. Noamniocentesis was done and the mothergave birth to a healthy baby.

CommentThis case illustrates the importance of anaccurate determination of gestation. SinceAFP and E3 levels rise and HCG levelsfall with advancing gestation, overestima-tion of the gestational age will show anMSS pattern identical to that expectedwith fetal Down syndrome. Conversely,the underestimation of dates will result inelevated MSAFP levels. Inaccurate esti-mation of the gesta-tion is one of themost common expla-nations for false-pos-itive test results.

Case 2A 24-year-oldwoman has an MSSdrawn at 15.5 weeks,according to herLMP. Her periodshave been regular,occurring every 28 days, and the last peri-od was normal. Her MSAFP level was 2.5MOMs, the HCG was 1.2 MOMs and theE3 level was 0.9 MOMs. The Down syn-drome risk was 1/14,000. The MSS wasdone as part of her routine pregnancyblood work without any explanation orconsent. She is extremely anxious, as shebelieves her baby has spina bifida.

OutcomeThe woman was told that, although an ele-vated MSAFP level is a risk factor for var-ious problems, including fetal spina bifi-da, this MSAFP level is associated most

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Maternal Serum Screening

Elevated MSAFPlevels can be a

sign of placentalcompromise, and

may indicatelater problems,

such as maternalhypertension.

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often with a normal outcome. A fetalassessment showed no abnormality andconfirmed the gestational age. She wasseen again at 31 weeks gestation and theassessment was again normal. Her babywas born healthy.

CommentThis case illustrates the worst aspects ofMSS. The patient was not properly coun-seled in advance of testing or about themeaning of a positive result. Only aboutone in 50 women with an elevatedMSAFP level will have a baby with spinabifida.6 Elevated MSAFP can be seen withother fetal anomalies, including ventralwall defects. Elevated MSAFP levels alsocan be a sign of placental compromise,and can be indicators of later problems,such as maternal hypertension, intrauter-ine growth retardation, oligohydramniousand subsequent fetal demise. Although anelevated MSAFP level is a risk factor for

all these conditions, mostwomen with elevated MSAFPlevels—especially those withminimal elevations—go on tohave normal pregnancies andhealthy babies.

Case 3An 18-year-old woman fromNorthern Manitoba had anMSAFP drawn at 16 weeks bysure dates. The MSAFP levelswas 7.8 MOM, the HCG was1.1 MOM and the E3 was 0.1MOM.

OutcomeExtremely high MSAFP levels in associa-tion with very low E3 levels strongly sug-gest fetal anencephaly. The E3 levels arelow because the fetal adrenals are under-developed. The primary-care physicianwas notified about this and an ultrasoundexamination was arranged in NorthernManitoba, rather than in Winnipeg. Theultrasound confirmed fetal anencephaly.The woman chose to have a pregnancytermination in her hometown. She wassubsequently seen for genetic counselingin Winnipeg.

CommentExtremely high MSAFP levels (i.e., > 5.0MOM) are usually associated with pooroutcomes. In this case, the low E3 levelsuggested anencephaly. One cause of anextremely elevated MSAFP level that hasa good prognosis is gastroschisis, whichcan be corrected post-natally.

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Case 4A 36-year-old woman had an MSS at 16.5weeks gestation, as determined by anearly ultrasound examination. The MSAFPis 0.8 MOM, the HCG is 1.3 MOM and theE3 is 0.9 MOM. The risks for fetal Downsyndrome went from 1/340 to 1/370. Thefetal assessment confirms the gestation, andno abnormalities suggestive of Down syn-drome are seen.

OutcomeAfter appropriate counseling, the womandecided to have an amniocentesis. Thefetal karyotype was 47,XY, +21, confirm-ing Down syndrome. After much delibera-tion, the woman decided to terminate thepregnancy.

CommentThis is an example of a true-positivescreen. Although the risks of fetal Downsyndrome were lowered by the MSS, theresults were considered screen-positivesince the risks were greater than the cut-offused by the MSS program. The ManitobaMSS program uses a risk of 1/384 as a cut-off. This is the risk of an average 35-year-old having a live-born child with Downsyndrome. Some programs base their riskcut-off on the risk of diagnosing fetalDown syndrome at amniocentesis. A mid-trimester risk of 1/270 is equivalent to aterm risk of 1/384, since about one-third offetuses with Down syndrome will diebefore term. This case also illustrates thatapplying a second screening test, such asultrasound, to a positive test is inappropri-ate. Since less than 70% of fetuses with

Down syndrome have any detectable signson a second-trimester ultrasound,7 relyingon the ultrasound as a second layer screenwould decrease the overall detection rate to60% times 0.7, or42%.

The detection ratefor Down syndromeincreases withadvancing maternalage, but so does thefalse-positive rate.The detection rate for20-year-olds is about40%, with a false-positive rate of 2.4%, ascompared to the detection rate of 75% anda false-positive of 16% for 36-year-olds.8

Case 5A 22-year-old has an MSS at 16.0 weeksby sure dates. The MSAFP is 2.5 MOM,the HCG is 2.1 MOM and the E3 is 1.7MOM. The Down syndrome risk is1/26,400.

OutcomeA fetal assessment shows that this is atwin pregnancy. The results are consid-ered normal for twins.

CommentMSAFP and HCG levels tend be be abouttwice as high in twin pregnancies. E3 lev-els are about 1.67 times higher. This MSSpattern should strongly suggest a twinpregnancy. Physicians should be carefulnot to rely on MSS as a screening test fortwins, as only about 50% to 60% of cases

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MSAFP and HCGlevels tend be be

about twice ashigh in twinpregnancies.

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of twins are associated with elevatedMSAFP levels.9 Occasionally, the elevat-ed HCG levels in twin pregnancies willcause the test to be screen-positive forDown syndrome. The MSS should not beconsidered reliable in predicting thechances of fetal Down syndrome whentwins are present. Some programs willcalculate a “pseudo-risk” estimate in thissituation.

Case 6A 31-year-old has an MSS at 16 weeks bycertain dates. The MSAFP is 0.3 MOM,the HCG is 0.2 MOM and the E3 is 0.3MOM. The Down syndrome risk is 1/736by age and 1/1,011 by MSS. The risk forfetal trisomy 18 is quoted as 1/11.

OutcomeThe women is counseled about the possi-bility of trisomy 18, and consents to anamniocentesis. The ultrasound, however,shows a fetal demise. The fetus appears tobe about 13 weeks size.

CommentsThe pattern of low levels of all three para-meters is suggestive of trisomy 18, butalso can be seen with a fetal demise aslevels return to the non-pregnant state.Since fetal demise is much more com-mon, it may be wise to suggest an ultra-sound examination be done locally beforethe woman travels long distances to thetertiary center for an amniocentesis. Lowlevels of E3 can be seen in a variety ofother conditions, including a relatively

benign skin disorder known as X-linkedichthyosis. A recent fetal demise willoften cause elevated MSAFP levelsbefore the levels begin to drop.

ConclusionMSS can be a valuable screening test forfetal neural tube defects or chromosomalanomalies. Physicians should counseltheir patients about the potential for false-positive or false-negative results. Thereare many causes for positive MSS results.The pattern of the MSS levels may sug-gest an underlying cause and may be use-ful in providing post-test counseling.

References1. Rose NC, Mennuti MT: Maternal serum screening for

neural tube defects and fetal chromosome abnormal-ities. West J Med 1993; 159:312-7.

2. Merkatz IR, Nitowsky HM, Macri JN, et al: An associationbetween low maternal serum alpha-fetoprotein andfetal chromosomal abnormalities. Am J ObstetGynecol 1984; 148:886-94.

3. Knight GJ, Palomaki GE, Haddow JE: Use of maternalserum alpha-fetoprotein measurements to screen forDown’s syndrome. Clin Obstet Gynecol 1988; 31:306-27.

4. Canick J, Knight GJ: Multiple marker screening for fetalDown syndrome. Contemporary Ob/Gyn 1992; 36:25-42.

5. Wald NJ, Watt HC, Hackshaw AK: Integrated screeningfor Down’s syndrome based on tests performed dur-ing the first and second trimester. N Engl J Med 1999;341:461-7.

6. Haddow JE, Palomaki G, Knight GJ, et al: The Foundationfor Blood Research Handbook. Volume 1. TheFoundation for Blood Research, Maine, 1990, pp. 5-8.

7. Smith-Bindman R, Homer W, Feldstein VA, et al: Second-trimester ultrasound to detect fetuses with Down syn-drome. JAMA 2001; 285:1044-55.

8. Haddow JE, Palomaki GE: Prenatal screening for Downsyndrome. In: Simpson JL, Elias S (eds.): Essentials ofPrenatal Diagnosis. Churchill Livingstone, New York,1993, p. 209.

9. Johnson JM, Harman CR, Evans JA, et al: Maternalserum a-fetoprotein in twin pregnancy. Am J ObstetGynecol 1990; 162:1020-5.

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