Lennart Friis-Hansen Dept. of Clinical Biochemistry Rigshospitalet Univeristy of Copenhagen Denmark First trimester screening The Danish Experiences Vårmötet

Lennart Friis-HansenDept. of Clinical Biochemistry

RigshospitaletUniveristy of Copenhagen

Denmark

First trimester screeningThe Danish Experiences

Vårmötet 2007

Borås, May 11th, 2007page 2

First trimester screening in Denmark

Historical overview of thePrenatal screening in Denmark

– Early 1970s First antenatal diagnosis of DS– 1978 Introduction of national guidelines for when offer

karyotyping All women > 35 years Women with known risk factors:

Previous birth of a child with karyotype anomalies Carrier state of a known disease, e.g. thalassemia, CF,

– 1980 Screening for NTD using MS-AFP and ultrasound– 1990 Screening for DS using MS-AFP, HCG and uE3 as

part of trials in parts of Denmark



DS epidemiology – problems with the old screening policy

In the late 1990s and early 2000s the overall invasive rate reaches 10-12 % for all pregnancies.

– 40% detection rate– 60% of the Downs being

born by women < 35 years of age.



Types of tests

1st trimester– Combined test (“Double test”

+ NT) free β-hCG, PAPP-A, NT and maternal age

2nd trimester– Triple test

AFP, uE3, total hCG/free β-hCG and maternal age.

– Quadruple test AFP, unconjugated oestriol

(uE3), free β-hCG/total hCG, inhibin-A and maternal age.

1st + 2nd trimester– Integrated test

a single test result = the integration of NT and PAPP-A in the first trimester + the quadruple test in the second.

– Serum integrated test A variant of the integrated

test using serum markers only

Suruss, 2003

1st trimester 2nd trimesterWeek 8 - 14 11 – 14 15-18 PAPP-A/hCG NT AFP, uE3, βhCG/hCG, inhibin-A



Performans af forskellige screeningsstrategier

Screen positive (%)

Detection rate (%)Integreret (1+2 trim)

NT + doubletest(1 trim)

NT (1 trim)

Doubletest (1 trim)

Quadrupletest (2 trim)

Tripletest(2 trim)

Alder > 34 år(1 trim)

Ingen test

Simulation by H. Cuckle



Types of tests

Why 1st trimester screening is preferred– Faster result -> the pregnant women have less time in which

to worry– If needed abortion can be performed almost within limits for

“free abortion”– High % of attendance because: Pregnant women want to see

the baby – they accept the NT as part of “seeing the baby”. However, once they’ve seen it, they are less likely to come back.

– (Programs combining 1st and 2nd trimester screening suffer from patients not attending the 2nd trimester screening.)

1st trimester 2nd trimesterWeek 8 - 14 11 – 14 15 - 20 PAPP-A/hCG NT AFP, uE3, βhCG/hCG, inhibin-A



Initial questions from the Danish National board of health

– Can foreign programs be copied?– can their performance be achieved?



1st Trimester screening trials in Denmark

Copenhagen first trimester study (HS) (1997-2001) (Wøjdeman et al., 2005)

Intervention study – by NT only– NT was measured in 8622 singleton pregnancies (97.5% of the

screened cases) GA 10 + 3 and 13 + 6 weeks. DR for DS with NT alone 75%, FPR 1.8%.

– beta-hCG and PAPP-A were analyzed in 6441 cases. DR 73%, FPRs of 8.8%

– the combined test (NT + biochemistry) DR 91%, FPR 2.1%– Low beta-hCG and PAPP-A values (below 0.4 MoM) were observed in

0.5% of the women including all cases of triploidy and trisomy 18 and 13.

Skejby study (Schøidt et al., 2006)Intervention study by Double + NT– 881 had the full test. – Screen positive = 34. – CVS with aneuploidy = 11 (6 trisomy-21, 5 others).– FPR = 3.2%. Positive Predictive Value (PPV) = 17.6% for T-21.

Bent Nørgaard-Pedersen and Michael Chistriansen, both Statens Serum Institut



Antenatal care in Denmark2004/5 ->

• Week 8-10 (-12) GP (confirmatory) pregnancy testing, general information about the pregnancy and information 1st trimester scrrening program for DS

• Week 8-10 (-12) Blood sampling for biochemistry performed at GP or at the hospital

• Week 11-13+6 NT (maybe one-stop)• If screen positive CVS/AC

• Week 18-20 malformation screening by ultrasound

”Fetal Diagnostics and Risk Evaluation” A report from Danish National Board of Health, April 1st

2003



National guidelines for quality assurance of the 1st trimester screening

– Ultrasound Certification of sonographers Continuous monitoring (>500 scan/year) Each centre should handle at least 1,000 deliveries per year

– Biochemistry Internal & external quality control Minimal number of test required

> 5,000 tests/year -> the laboratory can operate independently < 5,000 tests/year -> the laboratory can only operate if they

collaborate with a another laboratory on quality assurance– Screening program performance

Screen positive rates should be monitored False negative rates should be monitored A National center for monitoring the quality of the program

should be established



Implementation of 1. trimester screening NT + biochemistry

Copy the FMF program to the very point =>

– known screen positive rates

– Known detection rates




The sonography followed FMF guidelines– NT had already been established at several

centers Major Departments of Fetal medicine performing the NT

had all been trained by the Fetal Medicine Foundattion, London, all sonographers were already FMF-certified.

– NT QC All sonographers are FMF certified

– Software All Departments of Fetal medicine were using the

Astraia software (FMF approved)




Biochemistry – Exclusively performed at Depts. of Clinical

biochemistry It was decided to follow the FMF standards and only

use FMF certified platforms because only data generate using these platforms can be entered into the Astraia software

Brahms Kryptor Perkin Elmer Delphia Systems

– QC Internal and external controls. All Depts. Of Clinical biochemistry are participating in

the UK-NEQAS 1. trimester QC program Monitor screenpostive rate Monitor medians MoM




Risk estimation All centers performing NT sonography use the

Astraia software Biochemical data generated using FMF approved

platforms can be entered directly into Astraia Astraia could be used for risk estimation Astraia could be used for quality assurance.



Setup

Week 8 - 13+6•PAPP-A + hCG•Patient ID (age)

Week 11 - 13+6•NT•Weight•Gestational age•Additional risk factors

CALCULATE RISK



Challenges - 1

– Astraia The window for which biochemistry is accepted in

Astraia Expanded from 10+0 – 13+6 to 8+0 – 13+6 in 2005

Inability of Astraia to communicate with the Hospital Laboratory Information Management System

All data have to be manually entered into Astraia. This is associated with a great risk for typing mistakes.

Astraia are currently working on making the necessary changes – deadline?



Challenges - 2

– Default medians – can they be used Initial study 1500 samples analyzed at Hvidovre and

Rigshospitalet -> Yes Follow-up 20000 samples analyzed at Hvidovre and

Rigshospitalet -> Yes but ability to modify medians would be preferable



50 60 70 80 90 1000

10

20

30

40

50

60

70

80

90Astraia program (new)

SKS (56-69) + HH+RH (70-97)

a

Gestational age, days

hC

G , I

U/l

-10

-5

0

5

10 SKS (56-69) + HH+RH (70-97)


60 70 80 90

hCG median +/-5 %

b

hC

G , I

U/l



50 60 70 80 90 1000

1

2

3

4

5

6

SKS (56-69) + HH+RH (70-98) Astraia program

a


PA

PP

-A

, IU

/l

-0.4

-0.2

0.0

0.2

0.4

SKS (56-69) + HH+RH (70-98)


60 70 80 90

PAPP-A median +/- 5 %b

PA

PP

-A

, IU

/l



20

05

-12

00

5-2

20

05

-32

00

5-4

20

05

-52

00

5-6

20

05

-72

00

5-8

20

05

-92

00

5-1

02

00

5-1

12

00

5-1

22

00

6-1

20

06

-22

00

6-3

20

06

-42

00

6-5

20

06

-62

00

6-7

0.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20

1.25b-hCGPAPP-A

Måned

Me

dia

n M

oM

Monthly median MoM



Conclusion Medians

– Medians can be used during start up phase of a first triemster program.

– The bigger Danish centres would like to be able to individualise the Kryptor medians (scheduled to take place with the next Astraia update)

– The Delphia Express medians needed a major adjustment (which has taken place)



Challenges - 3

– Pre-analytical – can samples be sent by mail to the laboratories Pilot experiment Compare medians



Summer temperatures in Denmark



Temperatures, July 1st-4th, 2005



free-beta PAPP-A0.90

0.951.00

1.05

1.10

1.151.20

1.25

Rat

io a

fter

72

hrs

in

mai

l v

s 72

hrs

at

4C



50 60 70 80 90 1000

102030405060708090

100

Astraia program

HH juni05-maj06

SKS feb05-aug05RH juli05-maj06

a

gestational age, days

hC

G , IU

/l

50 60 70 80 90 1000

1

2

3

4

5

6 Astraia program

HH juni05-maj06

RH juli05-maj06

SKS feb05-aug05

b


PA

PP

-A

, IU

/l

No differences between medians– No differences

between medians obtained at a center that receive samples by mail and centers collecting and analyzing samples in house.



Conclusions transport

– Sending the samples to the laboratories using mail does not affect medians.

– However, care should be taken to avoid exposing samples to high temperatures.



Danish centers performing 1. trimester screening

23

1. Trimester NT only, externalsource for Biochemistry

1. Trimester NT + Biochem (Delphia)1. Trimester NT + Biochem (Brahms)



2003

County NT NT BC Malform. NT+BC Malform.

Northern Jutland all yes yes Only yes yes

Viborg County >35 + wish yes yes indication yes yes

Western Jutland >35 + wish april '05 yes yes yes yes

Ribe County >35 + wish > 35 yes yes yes yes

Vejle County >35 + IVF + wish yes yes yes yes yes

Southern Jutland few few tripletest no no no

Århus >35 ja > 35 no yes yes

Funen >35 March '05 March '05 ? yes yes

Western Zealand 0 March '05 No indication yes no

Northern Zealand 0 Yes Yes indication yes yes

Roskilde >35 January '05 yes indication yes yes

Southern Zealand >35 No No indication no no

Copenhagen County >35 + wish January '05 January '05 yes yes yes

Bornholm 0 yes yes no yes yes

Copenhagen City all yes yes yes yes yes

Fall 2004 Fall 2005

K Sundberg, unpublished data



The expected volumne at Danish centres performing the biochemistry (-hCG and PAPP-A)

– North Jutland, Aalborg 4000 tests/year

– Western Jutland, Holstebro 3500 tests/year

– Mid Jutland, Skejby 12000 tests/year

– Southern Jutland, Sønderborg 4000 tests/year

– Funen, Vejle 6000 tests/year

– National Serum Institute ? tests/year

– Northern Zealand, Hillerød 5000 test/year Copenhagen

County Gentofte 2000 tests/year Herlev 2000 tests/year Glostrup 2000 tests/year

– Copenhagen City Hvidovre 6000 tests/year Rigshospitalet 5000 tests/year

– Roskilde 3000 tests/year

– Western Zealand, Slagelse 2000 tests/year

– Southern Zealand, Næstved 3000 tests/year



Regimes at the centers

– One stop Samples drawn and NT performed at the same day at

the hospital in weeks 11-13+6 (Copenhagen County)

– Two stop Samples drawn at the GP in weeks 8-10, NT 11-13+6

at the Hospital (most hospitals) Samples drawn at the hospital in weeks 8-13+6, NT

11-13+6 at the Hospital (Hvidovre and Rigshospitalet)

– Risk calculation Only the final risk is calculated. An independent risk based on PAPP-A and -hCG is

NOT calculated



How the pregnant women chooseAccept to be screened

Yes > 80% RH

Accept having a CVS if screen positive

Yes - CVS

Wants a diagnostic despite a low risk estimate

Few

Choses abortion after the fetus has been found to carry trisomi 21

All but one!



Number of test per month in 2005 at Rigshospitalet

0

100

200

300

400

500

600

1 2 3 4 5 6 7 8 9 10 11 12

total




Percentage not enroling 6% - 8% • Late bookers • Ethnic minorities • Unaware of the screening program• Don’t wish to be screened (few)


Reasons for not enroling in the first trimester screening



Invasive procedures in Denmark

0

1000

2000

3000

4000

5000

6000

7000

8000

2000 2001 2002 2003 2004 2005 2006

12.2% 5.0%



Number of invasive tests i Copenhagen City

0

200

400

600

800

1000

1200

1400

Total Age DS

20012002200320042005




Newborns with Down syndrome

0

10

20

30

40

50

60

70

2000 2001 2002 2003 2004 2005 2006

Tabor, 2007



1st trimester questionaire, Jan 04 - April 05

Number of children? – 59% - 72% nullipara

From where did you get the information about the 1. trimester screening?

– 58% - 81% GP– 12% - 14% friends– 8% - 10% media– 7% - 10% internet– 4% - 5% previous contact with health services– 1% - 3% Midwifes– 8% - 16% unanswered / other– – 12% - 8% Acknowledge more than one source




Future tasks

– Establishment of a national QC database which will pair data from Astraia

NT data Biochemistry data

The Danish Cytogenetic Central register (DCCR) National patient register (LPR) which includes data

from the Danish newborn register and the Danish abortion register

– Better information to the pregnant women



New markers and new strategies

– ADAM12– Contingent testing– Repeated measurement





Christiansen, 2002



PAPP-A MoM

1Time

First Trimester

Second Trimester

D Wright, 2006

-hCG MoM

Time

1

First Trimester

Second Trimester





Conclusions

– It has been possible to offer all pregnant women first trimester screening in Denmark by Duplicating an existing well proven program (for

instance the FMF program) Having well educated staff

Nuchal translucency Biochemistry

Adhering to the strict quality control rules (e.g. the rules set up by the FMF)

Nuchal translucency Biochemistry

Choosing components that work well together Software Analytical platform



Acknowlegdements

– Karin Sundberg, Rigshospitalet– Ann Tabor, Rigshospitalet– Connie Jørgensen, Rigshospitalet

– Niels Tørring, Skejby Hospital– Steen Sørensen, Hvidovre Hospital



Thank you for your attention



PAPP-A66

-166

-266

-367

-167

-267

-368

-168

-268

-369

-169

-269

-370

-170

-270

-371

-171

-271

-372

-172

-272

-373

-173

-273

-374

-174

-274

-375

-175

-275

-376

-176

-276

-377

-177

-277

-378

-178

-278

-379

-179

-279

-3

0.80

0.85

0.90

0.95

1.00

1.05

1.10

1.15

1.20Hospital 1Hospital 2Hospital 3Hospital 4Hospital 5

DK-ratio

QC shipment

Rat

io

Danish QC values



50 55 60 65 70 75 80 85 90 95 1000

1

2

3

4

5

6

PAPP-A i Astraia program (new)

Hosptial 1 Median

Hosptial 2 Median

Hosptial 3 Median


PA

PP

-A,

IU/l



Outline

– How it was Prevention oriented, age determined

– The new policy Informed Choice

– Implementation Choice of program to chose Choice of analytical platform

– Status 2006 Where are we in Denmark

– New markers and new strategies



Outline

– How it was Prevention oriented, age determined

– The new policy Informed Choice

– Implementation Choice of program to chose Choice of analytical platform

– Status 2006 Where are we in Denmark

– New markers and new strategies



Danish centers performing 1. trimester screening

23

1. Trimester NT only, externalsource for Biochemistry

1. Trimester NT + Biochemistry (Delphia)1. Trimester NT + Biochemistry (Brahms)

1. Trimester NT + Biochemistry (Brahms)starting up



2003

County NT NT BC Malform. NT+BC Malform.

Northern Jutland all yes yes Only yes yes

Viborg County >35 + wish yes yes indication yes yes

Western Jutland >35 + wish april '05 yes yes yes yes

Ribe County >35 + wish > 35 yes yes yes yes

Vejle County >35 + IVF + wish yes yes yes yes yes

Southern Jutland few few tripletest no no no

Århus >35 ja > 35 no yes yes

Funen >35 March '05 March '05 ? yes yes

Western Zealand 0 March '05 No indication yes no

Northern Zealand 0 Yes Yes indication yes yes

Roskilde >35 January '05 yes indication yes yes

Southern Zealand >35 No No indication no no

Copenhagen County >35 + wish January '05 January '05 yes yes yes

Bornholm 0 yes yes no yes yes

Copenhagen City all yes yes yes yes yes

Fall 2004 Fall 2005




Performance

– 4% screen positive rate– Due to the low number of undected DS, the

detection rate is difficult to estimate at present. However it seems to be between 80-95%, but numbers are still to small to allow for a proper evaluation at the different centers.



– )– NT– Biochemical markers– Nick Wald introduces the MoMs (multiples of

meadian







The general idea

– Chromosome aberrations and/or developmental abnormalities lead to altered secretions of peptides/proteins from the placenta

– The maternal plasma concentration of these analytes can be measured

– These results can be used for Screening Diagnosis



PAPP-A

– IGF4BP protease– Secreted by the trophoblast– Released directly into maternal serum (not

present in amniotic fluid)– Detectable at ~6 wks of pregnancy– Doubles every 6 days– Increases to plateau at 14 wks – Decreased levels in Downs (0.3 -0.4 MoM)



Low Levels of PAPP-A– Trisomy 21– Other chromosome abnormalities

Trisomy 18 Trisomy 13

– Aneuploidy– Impending fetal death– Impaired fetal wellbeing

Preterm labour Low birth weight



Free -subunit hCG

– Glycoprotein hormone– Secreted by placental trophoblast subunit derived by elastase activity on total

HCG– Detectable 10 days post ovulation– Initially doubles every 1.5 – 2 days– Peaks at 8-10 weeks (FTS performed at 10-14

weeks)– Increased levels in Downs (2.0 -2.5 MoM)



MoM (analyte A) =Measured concentration of A

Median concentration of A at gest.age

__________________________________

MoMs are independent of gestational age

and concentration units

MoM ~ ”fraction of normal”

Multiples of median MoMs



-hCG

AB

C

D

A B

C

D

Total HCG

Alpha Subunit

Beta Subunit



LogMoM values are used in calculations

as they exhibit a Gaussian distribution

Barnes, 2005



Factors influencing marker performance

– Maternal weight– Smoking– Diabetes mellitus– Twins– Ethnicity– Mode of conception– Parity



Bivariate Risk Calculation

f x y

x y x y

x y

x

x

y

y

x

x

y

y

( , )

exp .

1

2 1

1

12

2

2

2 2

H Cuckle




The sonography follows FMF guidelines– NT performed at centers with trained

sonographers and doctors Major Departments of Fetal medicine performing the

NT had all been trained by the Fetal Medicine Foundattion, London, all sonographers were already FMF-certified.

– Software All Departments of Fetal medicine were using the

Astraia software. (All FMF approved)

– NT QC All sonographers are FMF certified



The challanges

– Pre-analytical– Astraia

The window for which biochemistry is accepted in Astraia

Inability of Astraia to communicate with the Hospital Laboratory Information Management System

Medians



The biochemists 10 wishes

1. Reproducibility of values over time (no drift)2. Reproducibility of values over time (no drift)3. Reproducibility of values over time (no drift)4. Reproducibility of values over time (no drift)5. Reproducibility of values over time (no drift)6. Reproducibility of values over time (no drift)7. Precision8. Robustness9. High specificity – no interference10.Documentation



Number of women not enroling in the first trimester screening: 6% - 8%

Reasons• Late bookers • Ethnic minorities • Unaware of the screening program• Don’t wish to be screened (few)


Reasons for not enroling in the first trimester screening



Transport of blood samples to the laboratories – unexpected help from the Royal Danish Mail

Most counties in Denmark are currently implementing systems where the samples will be picked up at the GPs

(This is not because the National Health Care wants to reduced the pre-analytical errors but because they can save money – sending blood samples by mail has become too expensive).



How to handle the changes in the medians

– The Astraia database generates a new set of medians from data collected in it. Fourthermore there is a mimgenererer nye medianer baseret på de data som er indtastet i Astraia databasen ud fra de krav vi har opstillet (f.eks 200 pr. uge i de forudgående 6 måneder. Det vil gøre det umuligt at dele medianer mellem 2 centre fordi medianen udelukkende kan genereres ud fra data i den enkelte database.

– At Astraia genererer medianer ud fra indtastede medianer, som et var i den gamle version. Dvs. at man blot indtaster en medianværdi for hver enkelt gestationsuge 8+3, 9+3, 10+3 etc. Og at Astraia så selv genererer den nye median. Det er et ”åbent system” hvor der ikke er krav til datamængde, og hvor medianer kan deles mellem centre. Og hvor det også vil være muligt at generere DPC Immulite medianer hvis det er det som man har lyst til!!

– Data is sent to London (Kevin) or Germany (Roland). From these data they generate a new set of medians.

– Probelms FMF has the rights to the risk module in Astraia. What will they accept.

Documents

Lennart Friis-Hansen Dept. of Clinical Biochemistry Rigshospitalet Univeristy of Copenhagen Denmark First trimester screening The Danish Experiences Vårmötet