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

Maternal age Gestational age Screening for trisomy 21 Biochemical Markers; 2nd Trimester 1st Trimester AFP free  hCG free  hCG uE3 Papp-A Inhibin A Other Markers; Nuchal Translucency Maternal age Gestational age

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 120 IU/L = 2 multiples Median 60 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)

Background Risk Maternal age 0.0001 0.001 0.01 0.1 1 10 20 25 30 35 40 44 Years Risk % Trisomy 21 Trisomy 18 Trisomy 13 xxx/xxy/xyy 45x Triploidy Nicolaides et al., The 11-14-week scan, London 1999 Snijders et al 1995

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 11-14-week scan, London 1999 Snijders et al 1995

Previous Chromosomal Abnormality Assessment of Risk Previous Chromosomal Abnormality Trisomy 21 Trisomy 18 Trisomy 13 } + 0.50 - 0.75% } 45XO 47XXY/XXX Triploidy

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

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

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

SAMSAS©

LRaff = h2/h1 Unaffected Affected h2 Detection h1 Rate False -ve Y= 0.425*ln_beta_MoM – 0.631*ln_Papp-a_MoM +0.761*ln_NT_MoM Unaffected Affected h2 Detection Rate h1 False -ve False +ve’s

DS Risk =Mat. Age Risk × LR SAMSAS© DS Risk =Mat. Age Risk × LR 20 yrs = 1 in 1600 × 2 = 1 in 800 30 yrs = 1 in 1100 × 2 = 1 in 550 35 yrs = 1 in 500 × 2 = 1 in 250 40 yrs = 1 in 156 × 2 = 1 in 78 45 yrs = 1 in 40 × 2 = 1 in 20

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

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. 276-280. Median MoMs free B-hCG = 2.15 and Papp-A = 1.93 weight corrected.

Biochemistry and Screening for trisomy 21 Maternal serum free ß-hCG & PAPP-A at 11-13+6 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 11-14 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

Biochemistry and screening for trisomy 21 OSCAR: Fetal NT, maternal serum free ß-hCG & PAPP-A at 11-13+6 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

Diandric Digynic

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

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%

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

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 http://www.nelh.nhs.uk/screening/dssp/model_bestpractice.pdf http://www.phgu.org.uk/info_database/diseases/downs_syndrome/downs.html#published http://www.ncchta.org

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

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

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

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.

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

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;39:567-76. -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 2003 40;219-31. 8.8 4.5 6.9 9.1 10.4 8.5 4.6 ß-hCG t-hCG NORMAL PAPP-A

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 2.50MoM PAPP-A 0.55MoM Marker CV LR CV 1 2.5% 7% 2 1.5% 4% 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.

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