1. Screening
“the systematic application of a test procedure to identify individuals at sufficient risk to warrant diagnostic investigations”
CVS 12wks
Amniocentesis 16wks
Morphology Ultrasound 18wks
Aim is to maximise detection of affected pregnancies and minimise false +ves

2. Maternal age Gestational age
Screening for trisomy 21
Biochemical Markers;
2nd Trimester 1st Trimester
AFP
free  hCG free  hCG
uE Papp-A
Inhibin A
Other Markers; Nuchal Translucency
Maternal age
Gestational age

3. Marker Levels Change Significantly with Gestation
Measured levels are converted to Multiples Of the Population Median or MoM values.
Reference is therefore 1 MoM
beta hCG at 10 wks GA
Patient IU/L = 2 multiples
Median IU/L
MoMs are independent of gestational age and concentration Units
LogMoM values are used in calculations as they exhibit a Gaussian distribution ( Mean +/- SD)

4. Background Risk Maternal age 0.0001 0.001 0.01 0.1 1 10 20 25 30 35 4044
Years
Risk %
Trisomy 21
Trisomy 18
Trisomy 13
xxx/xxy/xyy
45x
Triploidy
Nicolaides et al., The week scan, London 1999
Snijders et al 1995

5. Gestational age Background Risk 20 40 60 80 100 10 15 25 30 35
65% Trisomy 21 20 40 60 80
100 10 15 25 30 35
15% Trisomy 13
12% Trisomy 18
<1% Triploidy
95% 47xxx/xxy/xyy
20% 45x %
Nicolaides et al., The week scan, London 1999
Snijders et al 1995

6. Previous Chromosomal Abnormality
Assessment of Risk
Previous Chromosomal Abnormality
Trisomy 21
Trisomy 18
Trisomy 13 } + % }
45XO
47XXY/XXX
Triploidy

7. Effectiveness of different methods of screening
Screening for trisomy 21
Effectiveness of different methods of screening
100,000 pregnancies
Method of screening
Number detected
Detection rate
Screen positive 5%
N=5,000
Trisomy 21
N=200
Maternal age 60
30%
Serum biochemistry at 16 wks
130
65%
Nuchal translucency (NT) at 12 wks
75%
150
Fetal NT & ß-hCG & PAPP- A at 12 wks
180
90%
Nicolaides KH. Fetal nuchal translucency. Am J Obstet Gynecol 2004

8. SAMSAS©
Maternal Age Screening alone
Second trimester biochemical screening
First trimester combined screening
Amniocenteses performed per case detected
250 40 20
Fetal loss per case of Down syndrome detected
1 : 1
1 : 5
1 : 10

9. SAMSAS©
1st trimester Risk Odds show better separation between Unaffected and Affected pregnancies when compared to 2nd trimester Risk Odds.

10. SAMSAS©

11. LRaff = h2/h1 Unaffected Affected h2 Detection h1 Rate False -ve
Y= 0.425*ln_beta_MoM – 0.631*ln_Papp-a_MoM *ln_NT_MoM
Unaffected
Affected h2
Detection
Rate h1
False -ve
False +ve’s

12. DS Risk =Mat. Age Risk × LR
SAMSAS©
DS Risk =Mat. Age Risk × LR
20 yrs = 1 in × 2 = 1 in 800
30 yrs = 1 in × 2 = 1 in 550
35 yrs = 1 in × 2 = 1 in 250
40 yrs = 1 in × 2 = 1 in 78
45 yrs = 1 in 40 × 2 = 1 in 20

13. SAMSAS©
Noveux 1996, Graaf/Cuckle 2000, SAMSAS/Murdoch 2002

14. SAMSAS© Increasing Incidence of Twins (1990–2003) 1:70 to 1:55
* Assisted reproduction
* Rate of twinning increases with age.
>17% of pregnancies are now to women 35yrs or over, up from <9% in 1990.
FMF software uses data as published by Spencer, Br J Obstet Gynaecol March 2003, Vol. 110, pp Median MoMs free B-hCG = 2.15 and Papp-A = 1.93 weight corrected.

15. Biochemistry and Screening for trisomy 21
Maternal serum free ß-hCG & PAPP-A at wks 2 4 6 8 10 12 14 16 18 20
-3.5
-2.5
-1.5
-0.5
0.5
1.5
2.5
3.5
Free ßhCG (SD) %
Trisomy 21 20 2 4 6 8 10 12 14 16 18
-3.5
-2.5
-1.5
-0.5
0.5
1.5
2.5
PAPP-A (SD) %
Trisomy 21
Normal
Normal
In trisomy 21 pregnancies at weeks, maternal serum free ß-hCG is increased and PAPP-A is decreased
The alterations in maternal serum biochemistry are independent of fetal NT thickness
Screening by fetal NT and serum free ß-hCG and PAPP-A identifies 90% of cases for FPR of 5%
ß-hCG: higher in Africans & IVF pregnancy, lower in smokers; PAPP-A: higher in Africans, lower in IVF & smokers
Brizot et al 1994; 1995; Liao et al 2001; Noble et al 1995; Spencer et al 1999; 2000; 2001; 2002; 2003; 2004; 2005

16. Biochemistry and screening for trisomy 21
OSCAR: Fetal NT, maternal serum free ß-hCG & PAPP-A at wks
Singleton pregnancies n=75,821; Maternal age 31 (13-49) yrs
Normal
Risk >1 in 300
3,909/75,277 ( 5.2%)
Trisomy 21
301/325 (92.6%)
Trisomy 18/13
108/122 (88.5%)
Other defects
83/97 (85.6%)
Nicolaides et al 2005

17. Diandric
Digynic

18. Combined Integrated 11-13 weeks: NT NT PAPP-A PAPP-A  hCG
Biochemistry and Screening for Trisomy 21
Combined or Integrated Screening?
Combined Integrated
11-13 weeks: NT NT
PAPP-A PAPP-A
 hCG
15-20 weeks: hCG
AFP
UE3
Inhibin A

19. Biochemistry and Screening for Trisomy 21
Integrated Screening
FPR for DR 85% DR
FPR 5%
Malone
Wald
2004
DR (%)
100
Double test
71%
Integrated test 90
2.8%
61%
Quadruple test 80
Triple test
Triple test
77%
Double test 70
66% 60
Quadruple test
83% 50
75% 40
1.2%
Wald
2003
Wald
1999
1.0%
Nuchal translucency
34% 30 20
77% 10
NT & ß-hCG/PAPP-A
83%
90% 1 2 3 4 5 6 7 8 9 10
FPR (%)
Integrated test
93%

20. Biochemistry and Screening for Trisomy 21
Combined or Integrated Screening?
Combined
Integrated
High detection rate
Single visit
Earlier screening result
Early reassurance (for most)
Early Diagnosis / ToP
Multiphase anxiety
Increased default rate
Delayed screening result
Delayed diagnosis
Late termination

21. Biochemistry and screening for trisomy 21
Receiver Operator Curves for Down Screening Tests
0.0
20.0
40.0
60.0
80.0
100.0
2.0
4.0
6.0
8.0
10.0
False Positive Rate (%)
Detection Rate (%)
Comb / Int
NT / quad
triple
double
Wald et al. 2003; Nicolaides et al. 2005
UKNSC ~ benchmark by April 2007 ~ 3% FPR 75% DR

22. Biochemistry and screening for trisomy 21
Women’s attitudes towards screening
Monni: options of NT or triple test
information on CVS and Amnio
496 of 500 opted for NT
Lancet ‘98
Kornmann:
109 2nd trimester screen - 76% would prefer 1st trimester
49 declined 2nd trimester screening - 2 would consider earlier
79 had CVS - 32% would have had 1st trimester screening
Prenat Diagn ‘97

23. Biochemistry and screening for trisomy 21
Women’s attitudes towards screening
Hypothesis: when informed about the rate of miscarriage of Downs
pregnancies, most women would prefer to delay screening.
Results: the clear majority of women wanted the earliest possible
test, even if this only identified pregnancies destined to
miscarry
Mulvey et al. BJOG 2000

24. Rates of fetal death in Down syndrome pregnancies.
SAMSAS©
Rates of fetal death in Down syndrome pregnancies.

25. SAMSAS©
Assume 10% will abort before 2nd TR (90% progress to 2TR).
OBS prevalence of T21 in 1st TR = 1:347
17,600 pregnancies x 1:347 = 50 cases
45 cases progress to 2nd TR
61.9% Detected in 2nd TR = 27 cases
77.8% Detected in 1st TR= 35 cases or 8 more viable cases.

26. Analytical error in biochemical screening
Gestational Changes in BhCG and PAPP-A in T21
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0.1
0.2
0.3
0.4
0.5
0.6
Log10 PAPP-A or Free ß-hCG (MoM) 5 7 9 11 13 15 17 19 21
Gestation (wks)
ß-hCG in Tr21
PAPP-A in Tr21
NORMAL
At 10 wks PAPP-A is better
At 14 wks ß-hCG is better
Spencer et al. 2002

27. Detection Rates at 5% FPR (using correct variable separation model)
Analytical error in biochemical screening
Screening performance with difference types hCG
Spencer et al, Ann Clin Biochem 2002;39:
-0.7
-0.6
-0.5
-0.4
-0.3
-0.2
-0.1
0.1
0.2
0.3
0.4
0.5
0.6
Log10 PAPP-A or Free ß-hCG (MoM) 5 7 9 11 13 15 17 19 21
Gestation (wks)
56.3
65.1
Overall
55.6
60.1 14
55.7
62.6 13
55.8
64.9 12
56.5
66.9 11
58.4
68.8 10
62.1
70.6 9
67.9
72.5 8
Total
Free ß
GA (wks)
Detection Rates at 5% FPR
(using correct variable separation model)
Screening by PAPP-A & hCG (free ß vs total)
Spencer et al, Ann Clin Biochem ;
8.8
4.5
6.9
9.1
10.4
8.5
4.6
ß-hCG
t-hCG
NORMAL
PAPP-A

28. Analytical error in biochemical screening
Impact of analytical error in risk assessment
Kryptor
Delfia
Imm 2000 50
100
150
200
250
300
350 1 2 3 4
Risk: 1 in….
Age 24y
CRL 55mm NT 2.2mm
fB-hCG MoM
PAPP-A MoM
Marker CV LR CV
1 2.5% 7%
2 1.5% %
3 4-5% 15%
4 6-7% 25%
Spencer 2003 DS News
FMF risk calculation software has specific medians and distributions for Kryptor and Delfia. These systems meet the analytical imprecision criteria.

29. Risk Calculation
MoM values
Maternal Age
Gestational Age
Recurrence Risk
Singleton vs Twins
Maternal Weight
Ethnic Origin
Smoking
IVF
Analytical Imprecision
NT vs Biochemistry vs Combined vs Integrated
OUTCOME