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Antenatal Screening

Antenatal Screening for Downs Syndrome

and Open Neural Tube Defects

The Quadruple Test

Information for Health Professionals

The Wolfson Institute of Preventive MedicineBarts and The London School of Medicine and Dentistry


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Antenatal Screening

The purpose of screening is to identify women with an increased risk ofhaving a pregnancy with Downs syndrome or an open neural tube defectso that they can be offered a diagnostic test.

The quadruple test is a method of screening involving the measurement offour substances in the maternal serum (serum markers). These are alpha-fetoprotein (AFP), unconjugated oestriol (uE

3), total human chorionic

gonadotrophin (hCG) and inhibin-A. The test is performed at about

16 weeks of pregnancy.

The four serum markers are used together with the womans age toestimate the risk of having a pregnancy with Downs syndrome. Women

Down's syndrome and offered a diagnostic test, usually an amniocentesis.

pregnancy.

The level of AFP alone is used to screen for open neural tube defects.

Women with a raised AFP level (equal to or greater than two and a halftimes the normal median) are interpreted as screen-positive and offeredfurther tests such as an ultrasound scan. About 1 in 100 of all womenscreened fall into the screen-positive group for an open neural tube defect,and about 1 in 15 women with screen-positive results have an affectedpregnancy.

and AFP measurement identifies 4 out of 5 cases of open spina bifida and

a diagnostic test.

Measurements used as part of the quadruple test can also identifypregnancies at high risk of Edwards syndrome (trisomy 18). The testidentifies about 6 out of 10 cases of Edwards syndrome.

SUMMARY

with a risk of 1 in 150 or greater are interpreted as screen-positive for

and about 1 in 15 women with screen-positive results will have an affected

The quadruple test identifies about 4 out of 5 cases of Downs syndrome

About 1 in 27 of all women screened fall into the screen-positive group

nearly all cases of anencephaly. About 4% of women screened are offered


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CONTENTSSummary ...........................................................................2

Downs syndrome (trisomy 21) ..........................................4

Open neural tube defects ..................................................4

Edwards' syndrome (trisomy 18) .......................................4

Timing of the test ...............................................................4

Interpretation of the test ....................................................5

Action following a screen-positive result ..........................5

Reporting of results ...........................................................5

Calculation of the risk of Downs syndrome ......................7

Factors affecting the test ...................................................9

Effect of maternal age on screening performance ..........10

Comparison with other Downs syndromescreening tests ................................................................ 11

Diagnostic tests ...............................................................12

Patient information ..........................................................13

References ......................................................................14

Useful telephone numbers ..............................................15

Performance of the Quadruple test ....................................6


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Antenatal Screening

DOWNS SYNDROME(TRISOMY 21)

Downs syndrome is the mostcommon cause of severe learningdisability in children. It arises from anextra copy of chromosome 21 in thecells of the fetus. In the absence ofantenatal screening, about 1 in 500babies born would be affected.

People with Downs syndromehave varying degrees of learningdisability, but usually the disability issevere. Some people will lead semi-independent lives while others will becompletely dependent. About 40%of Downs syndrome pregnancieswill miscarry between 11 weeks andterm, but nine out of ten affectedbabies who reach term will survivetheirfirst year. About 40% of babieswith Downs syndrome are born witha serious heart defect. The averagelife expectancy of a person withDowns syndrome is now about 60years, although most will developpathological changes in the brainassociated with Alzheimers diseaseafter the age of 40.

OPEN NEURAL TUBEDEFECTS

Neural tube defects are one of themost common serious congenitalmalformations. In the absence ofantenatal screening, about 1 in every650 babies born would be affected,or less than 1 in 1200 among women

immediately before becomingpregnant.

Anencephaly, one type of open neuraltube defect, is fatal at, or within hoursof birth, but the natural history ofspina bifida is variable. Babies bornwith open spina bifida, the other maintype of open neural tube defect, areoften severely handicapped and canrequire several surgical procedures

and hospitalisation. Disability typicallyconsists of weakness or paralysisof the legs, urinary and faecalincontinence, hydrocephaly, and, lessoften, learning disability.

EDWARDS SYNDROME(TRISOMY 18)

Edwards syndrome is a rare (birthprevalence about 1 in 4,500) and

usually fatal abnormality which arisesfrom an extra copy of chromosomenumber 18 in the cells of the fetus.

TIMING OF THE QUADRUPLETEST

The test can be performed on a 10mlblood sample taken between 14 and22 completed weeks of pregnancy (15and 22 for NTD interpretation). 16-

18 weeks is the best time to screenfor open neural tube defects andso 16 weeks is best. An ultrasoundestimate of gestation (based on headcircumference, crown-rump lengthor bi-parietal diameter) should beavailable prior to the test wheneverpossible, as this will improve theperformance of the test.who took folic acid supplements


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ACTION FOLLOWING ASCREEN-POSITIVE RESULT

If the result is screen-positive forDowns syndrome and the gestationalage has not been estimated using anultrasound scan one can beperformed. The result will only berevised if there is a large discrepancybetween the dates and scan estimateof gestation (at least 18 days

difference). This will reduce thepossibility of missing Downs syndromepregnancies in women whose resultschange from screen-positive toscreen-negative as a result of the scanrevision. If the result remains screen-positive, the women concerned areoffered a diagnostic amniocentesis.

If the result is screen-positive for openneural tube defects then a detailed

ultrasound scan at about 18-20weeks is offered and possibly an

amniocentesis.

REPORTING OF RESULTS

The screening results are usually readywithin 48 hours of receipt of the bloodsample and will be sent to the antenatalclinic or doctor who ordered the test.Screen-positive results are telephoned

and faxed directly to the antenatal clinicor doctor.

INTERPRETATION OF THEQUADRUPLE TESTThe test categorises women into twogroups: screen-positive with a high riskof having an affected pregnancy andscreen-negative with a lower risk ofhaving an affected pregnancy.

Screen positive

i)Screen-positive for Downs syndromeA woman is screen-positive if the riskof having a pregnancy with Down'ssyndrome based on her maternalage together with the serum levels ofAFP, uE

3, hCG and inhibin, is

in this group.

ii)Screen-positive for open neuraltube defects

If the serum AFP level is equal to, orgreater than, two and a half times thenormal median level (2.5 MoM), theresult is screen-positive. About 1 in100 women will be in this group .

Screen-negative

A screen-negative result means that(i) the risk of a pregnancy with Downssyndrome is below the specified riskcut-off or (ii) the AFP level is less than

2.5 MoM. A screen-negative resultdoes not exclude the possibility of anaffected pregnancy.

The quadruple test can identifypregnancies at high risk of Edwards'syndrome (trisomy 18). In caseswhere the risk is high this is reported.

estimated to be 1 in 150 or greater.About 1 in 27 screened women will be


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Antenatal Screening

PERFORMANCE OF THEQUADRUPLE TEST

The screening performance of a testis usually defined in terms of thedetection rate (also called 'sensitivity'),false positive rate and the oddsof being affected given a positiveresult (OAPR) which is the ratio oftrue positives to false positives. Thedetection rate (DR) is the proportionof affected pregnancies with screen-positive results and the false positiverate (FPR) is the proportion ofunaffected pregnancies with screen-positive results.

Down's syndrome

Open Neural Tube Defects

DR = 85% with open spina bifida (andnearly 100% with anencephaly) forabout a 1% FPR.

OAPR = 1:15 (i.e. among women witha screen-positive result for open neuraltube defects one will have an affectedpregnancy for every 15 that do not).

Maternal Risk of Maternal Risk of Maternal Risk of

age at Downs age at Downs age at DownsEDD* syndrome EDD* syndrome EDD* syndrome

20 1:1450 30 1:940 40 1:85

21 1:1450 31 1:820 41 1:70

22 1:1450 32 1:700 42 1:55

23 1:1400 33 1:570 43 1:45

24 1:1400 34 1:460 44 1:40

25 1:1350 35 1:350 45 1:35

26 1:1350 36 1:270 46 1:30

27 1:1200 37 1:200 47 1:30

28 1:1150 38 1:150 48 1:3029 1:1050 39 1:110 49 1:25

Table 1

* EDD =expected date of delivery Morris et al (2003) Ratio of affected to unaffected pregnancies

a screen-positive result for Down's

DR = 80% for a 3.5% FPR

syndrome one will have an affected

OAPR = 1:15 (i.e. among women with

pregnancy for every 15 that do not).


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CALCULATION OF THE RISKOF DOWNS SYNDROME

Maternal ageThe risk of having a pregnancy withDowns syndrome increases withmaternal age as shown in table

specific risk is the background riskof Downs syndrome that is usedto calculate a woman's screening

result based on the measurementof the screening markers.

The markersIn pregnancies affected by Down'ssyndrome, second trimester AFPand uE

3levels are, on average,

low (about three-quarters thatof unaffected pregnancies)and inhibin-A and hCGlevels are, on average, high(about double that of unaffectedpregnancies).

The concentrations of the markers

vary with gestational age. In thesecond trimester AFP and uE

3

hCG decreases, and inhibin-Adecreases before 17 weeks andincreases after 17 weeks. Also, themeasurement of serum markersmay vary between laboratories.In order to take account ofthese sources of variation, the

concentration of each marker isexpressed as a multiple of themedian for pregnancies of thesame gestational age (MoM).

The figure below illustrates theconcept. A hypothetical marker has

iu/mL at 10 weeks her level would

iu/mL at 14 weeks this would be

Calculating MoM values (Multiples of the Median)

1 5

2 5

1 0 0

5 0

1 0 0

1 0 1 1 1 2 1 3 1 4

G e s ta t i o n ( we e ks )

0 . 5 M o M1 . 0 M o M2 . 0 M o M

Serum

m

arkerlevel(

iu/m

L)

1 on page 6. The maternal age-

increase with gestational age,

weeks, 50 iu/mL at 12 weeks and100 iu/mL at 14 weeks. If a womanwere found to have a level of 50

be twice the median (50/25) or 2.0MoM. Similarly if the level were 50

half the median (50/100) or 0.5 MoM.

a median level of 25 iu/mL at 10


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Antenatal Screening

AFP (MoM)

Downs syndrome Unaffected

uE3

(MoM)

UnaffectedDowns syndrome

Inhibin-A (MoM)

Downs syndromeUnaffected

Risk of Down's syndrome inrelation to serum marker levels

The graphs above show theoverlapping relative frequencydistributions of AFP, uE

3, inhibin

detceffanudnadetceffaniGChdnapregnancies. The points ofintersection are the values at

which the risk of Downs syndromeis the same as the backgroundrisk in the population. From thesegraphs, it can be seen that AFPand uE

3values below 0.86 MoM

and 0.83 MoM respectively, andinhibin and hCG values above1.54 MoM and 1.45 MoMrespectively will each tend to

increase the risk of Downssyndrome above the backgroundrisk while values in the oppositedirections will tend to decrease therisk.

Gestational ageError in the estimate of gestational

age will give inaccurate MoMvalues of the four serum markers.Since these MoM values are usedin calculating the risk of Downssyndrome, gestational age errorwill result in an inaccurate estimateof the risk. It is therefore importantto obtain as precise an estimate ofgestational age as possible, ideallyby an ultrasound scan.

Unaffected Downs syndrome

0.125 0.25 0.5 1 2 4 8 16

0.125 0.25 0.5 1 2 4 8 16 0.125 0.25 0.5 1 2 4 8 16

0.125 0.25 0.5 1 2 4 8 16

total hCG (MoM)


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FACTORS AFFECTINGTHE TEST

Maternal weight, ethnic group, InVitro Fertilisation (IVF), InsulinDependent Diabetes Melli tus(IDDM) and smoking

All four marker levels tend to be

increased in lighter women.

AFP levels tend to be about 20%higher and hCG levels about10% higher in Afro-Caribbeanwomen than in Caucasian women.

hCG levels tend to be about10% higher and uE levels

3

about 10% lower in women whohave become pregnant as a resultof IVF compared with non-IVF

pregnancies.

AFP and uE3levels tend to be

low (about 8% and 6% lowerrespectively) in women with insulindependent diabetes mellitus.

hCG levels tend to be about 20%lower and inhibin levels about60% higher in women who smoke.

Appropriate adjustments of the MoM

values are made for these factors.

TwinsThe serum marker levels are raisedin twin pregnancies. Adjustments aremade to take account of this.

Screening in twin pregnanciesposes a difficulty because of thepossibility that one fetus may be

affected and the other may not.Because of the presence of twofetuses the amniocentesis is a slightlymore complex procedure in a twinpregnancy. If one twin is found to beaffected and the other unaffected,selective feticide can be offered. Thisprocedure poses a substantial risk tothe unaffected twin. The presence of atwin pregnancy may therefore be seen

by some women as a reason to avoidscreening.

Previous affected pregnanciesIf a previous pregnancy with Downssyndrome or open neural tube defectis reported, the result will be classifiedas screen-positive regardless of thelevel of the screening markers so thatfurther testing can be discussed withthe woman. A risk is calculated which

takes account of a womans previouspregnancy with Downs syndrome.

The woman's age at the time of herprevious pregnancy with Down'ssyndrome affects the recurrence riskand this is taken into account in therisk calculation.

Taking account of screening in aprevious pregnancyIf a woman has been screened for

Downs syndrome or open neural tubedefects in a previous pregnancy thelevels of the screening markers in thatpregnancy can be used to adjust themarker levels in the current pregnancy.

This is useful because markers usedin screening tend to 'track' betweenpregnancy (e.g. a hCG level that ishigh in one pregnancy tends to

decreased in heavier women and


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Antenatal Screening

be high in a subsequent pregnancy).So a woman with a false positiveresult in one pregnancy is likely tohave a false positive result again ina subsequent pregnancy. Adjustingmarker levels for those in a previouspregnancy can help avoid thisproblem of false-positives recurring indifferent pregnancies.

Vaginal bleeding

Vaginal bleeding immediately beforetaking the blood sample can affect thescreening result by increasing the

maternal serum AFP level and so,in these circumstances, it may beadvisable to delay collecting blood forthe screening test until a week afterbleeding has stopped.

Testing after amniocentesisIf an amniocentesis has beenattempted in the pregnancy prior totaking the blood sample, the resultcannot be interpreted. This is due

to the possibility of feto-maternaltransfusion which can increase thematernal serum AFP level.

age, the probability of a screen-positive result and the proportionof Downs syndrome pregnanciesdetected. Whatever the womans

age, the best estimate of her risk ofhaving an affected pregnancy is therisk obtained by using informationon her age in conjunction with hermarker values.

An older woman is more likely tohave a screen-positive result than ayounger woman as she starts witha higher age-specific risk of Downs

syndrome. For this reason, the testis more likely to detect a Downssyndrome pregnancy in an olderwoman than in a younger woman.

Table 2 below shows, according to

EFFECT OF MATERNAL AGE ON SCREENING PERFORMANCE

(early mid-trimester estimates)

Maternal Probability of a Proportion of Downs

age group screen-positive syndrome pregnancies

(years) result detected (%)

52rednU92-52

43-03

35-39

40-44

45 and over

llA

Table2

1 in 80

1 in 3

1 in 60

1 in 10

1 in

6062

70

85

93

1 in 35

96

801 in 27

5


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COMPARISON WITH OTHER DOWN'S SYNDROME SCREENINGTESTS

Table 3 below shows the estimated detection rate (DR) and odds of beingaffected given a positive result (OAPR) for various Down's syndrome screening

also using a 1% false positive rate. The estimates are based on a large UKstudy (Wald et al 2003) and apply to the early second trimester of pregnancy.

They are corroborated by results from other studies.

Table 3 Method of screening FPR(%) DR (%) OAPR

Maternal age alone

Triple test

(AFP, uE , hCG)3

Quadruple test(AFP, uE , hCG, inhibin)

Combined test

(Nuchal translucency [NT],

free -hCG. PAPP-A at 11 weeks)

Serum Integrated test

(PAPP-A at 11 weeks and

Quadruple markers at 14-22 weeks)

Integrated test

(NT and PAPP-A at 11 weeks and

Quadruple markers at 14-22 weeks)

1 85 1:4

(Gestational age estimated by ultrasound scan and marker levels adjusted for maternal weight)NB All tests include maternal age

methods using a 3% fixed false positive rate (FPR) and, for the integrated test,

25

76

86

82

92

1:45

1:15

1:14

1:12

1:13

1:11

69

3

3

3

3

3

3

3


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Antenatal Screening

DIAGNOSTIC TESTS

AmniocentesisAn amniocentesis is performed at about 15 to 16 weeks of pregnancy. Underultrasound guidance a sample of amniotic fluid is collected using a needleinserted through the abdominal wall. Cells from the sample can be used todiagnose Downs syndrome. The risk of miscarriage due to the procedure isabout 1%.

Downs syndrome is diagnosed using a technique called quantitativefluorescence polymerase chain reaction (QF-PCR). This provides a rapiddiagnosis of Downs syndrome, usually within 48 hours of the amniocentesis

being performed. It also detects trisomy 18, 13 and sometimes sexchromosome abnormalities. To diagnose other conditions the cells must growbefore they can be examined and so the final results can take up to 2-3 weeks.

Chorionic Villus Sampling (CVS)Occasionally this test may be offered as an alternative to amniocentesis. CVSinvolves taking a sample of placental tissue, by inserting a needle through theabdominal wall or a fine instrument through the cervix. As with amniocentesisQF-PCR is used to provide a rapid diagnosis for Downs syndrome, trisomy

18 and 13 and sometimes sex chromosome abnormalities. At this stage ofpregnancy the risk of miscarriage due to the procedure is thought to be aboutthe same as the risk following an amniocentesis.

With CVS there is a chance (about 1 in 100) that the test will not provide aconclusive result. In these circumstances an amniocentesis will need to beperformed to provide a definite diagnosis.

Detailed ultrasound scanThis is offered when women are reported as screen-positive due to anincreased risk of an open neural tube defect. Nearly all cases of anencephalyand open spina bifida can be detected.


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PATIENT INFORMATION

Points to remember when discussing the screening test with a woman considering

whether to be screened:

Obtain an explicit decision on whether to be screened.

Assess her knowledge of Downs syndrome and whether more informationis needed.

Satisfy yourself that she understands that the test does not give a definitiveanswer it divides women into a higher risk group (screen-positive) anda lower risk group (screen-negative). For Downs syndrome the result

defects the result is screen-positive if the AFP level is 2.5 MoMs or higher.

result for Downs syndrome and they will be offered an amniocentesis or aCVS, both of which carry a risk of miscarriage. Most women with a screen-positive result will not have affected pregnancies.

Check that she knows that the test will not detect all pregnancies with

Downs syndrome. Explain that in the few pregnancies in which Downs syndrome is

diagnosed, the woman will be offered a termination of pregnancy.

Women should have the opportunity to have time to consider whether tobe screened, and discuss this with others before making a decision. Whilescreening cannot provide complete reassurance and will cause anxiety,particularly if the screening test is positive, it provides the opportunity offindingout whether the pregnancy is affected with Down's syndrome. If women do notwant this information while pregnant screening is best avoided.

is screen-positive if the risk is 1 in 150 or greater. For open neural tube

Explain that about 1 in 27 women screened will have a screen-positive


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Antenatal Screening

REFERENCESReport of the RCOG Working party on biochemical markers and the detection

of Downs syndrome. RCOG press. J uly 1993.

Selikowitz M. Downs syndrome: The facts. Oxford University Press, 1991.

Wald NJ , Cuckle HS. Biochemical Screening. In: Prenatal Diagnosis and screening.

Ed Brock D, Rodeck C and Ferguson-Smith MA. Churchill

Livingstone: 563-577, 1992.

Wald NJ : Biochemical detection of neural tube defects and Downs syndrome.

Turnbulls Obstetrics 2nd Edition. Ed. Chamberlain G. ChurchillLivingstone: 195-209, 1995.

Wald NJ , Densem J W, George L, Muttukrishna and Knight G. Prenatal Screening

for Downs syndrome using inhibin-A as a serum marker. Prenatal diagnosis 16:143-

153 (1996).

Wald NJ , Rodeck C, Hackshaw AK, Walters J , Chitty L, Mackinson AM. First and

second trimester antenatal screening for Downs syndrome: the results of the

Serum, Urine and Ultrasound Screening Study (SURUSS). Health Technol Assess

2003;7:i-iv,1-88. pISSN: 13665278 and in J Med Screen 2003;10:56-104.

Wald NJ , Rodeck C, Hackshaw AK, Walters J , Chitty L, Mackinson AM, BestwickJ P. Corrections to SURUSS report. J Med Screen 2006;51:52

Morris J K, Wald NJ , Mutton DE, Alberman E. Comparison of models of maternal

age-specific risk for Down syndrome live births.Prenat Diagn. 2003 Mar;23(3):252-8


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USEFUL TELEPHONE NUMBERS AND WEBSITES

Antenatal Screening Service, Barts and The LondonSchool of Medicine and Dentistry ................................................ 020 7882 6293www.wolfson.qmul.ac.uk/epm/screening

Downs Syndrome Association .................................................... 0845 230 0372www.downs-syndrome.org.uk

Antenatal Results and Choices (ARC) ....................................... 020 7631 0285www.arc-uk.org

Association for Spina Bifida and Hydrocephalus .........................0845 450 7755www.asbah.org


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Antenatal Screening

Wolfson Institute of Preventive Medicine

For further information, please contact:

Antenatal ScreeningCentre for Environmental and Preventive Medicine

Wolfson Institute of Preventive MedicineBarts and The London School of Medicine and Dentistry

Charterhouse SquareLondon

EC1M 6BQTelephone: 020 7882 6293/4

e-mail: [email protected]

or find us at: www.wolfson.qmul.ac.uk/epm/screening

The Wolfson Institute of Preventive Medicine has played a leading role in thediscovery, development and implementation of antenatal screening methods. It is

committed to improving the efficacy and safety of screening. We use informationcollected as part of our screening programme, including measurements on stored

blood samples, to audit our screening programme and ensure that it is meeting ourexpected quality standards. Such information may also be used to help discover

and validate new tests that improve the quality of screening services.

October 2011

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