Donna Fullerton

What is screening?

 Screening is a public health service in which members of

a defined population, who do not necessarily perceive
that they are at risk of, or are already affected by a
disease or its complications, are asked a question or
offered a test to identify those individuals who are more
likely to be helped than harmed by further tests or
treatments to reduce the risk of a disease or its
complications (NSC definition)
National Screening Programmes
 In England there are three categories of screening:

1) Antenatal and Newborn

-NHS Fetal Anomaly Screening Programme
-NHS Infectious Diseases Screening Programme
-NHS Sickle Cell and Thalassemia Screening Programme
-NHS Newborn Bloodspot Screening Programme
-NHS Newborn and Physical Examination Programme
-NHS Newborn Hearing Screening Programme

2) Young Person and Adult

-NHS Diabetic Eye Screening Programme
-NHS Abdominal Aortic Aneurysm Screening Programme

3) Cancer Screening Programmes

-NHS Breast Screening Programme
-NHS Cervical Screening Programme
-NHS Bowel Cancer Screening Programme
Down’s Syndrome
• Down’s Syndrome is a genetic condition that is caused by the presence of
three copies of Chromosome 21 (T21)

• Prevalence – 1 per 600-800 births

• Most common chromosomal disorder at birth

• Incidence increases with increasing maternal age

• Recurrence risk is 0.75%

• Typical life expectancy is 50-55 years

Standard T21 Karyotype
Aetiology of Down’s Syndrome
• 95% of cases are standard/regular T21
-Extra copy of chromosome 21 occurs by non disjunction
during cell division (failure of separation of the two copies of
chromosome 21 so one cell has two copies and one has no
copies.)

• 4% of cases are caused by translocation

-Part of chromosome 21 becomes attached to another
chromosome (the parts form one large chromosome)

• 1% of cases are caused by mosaicism

-Not all of the cells have an extra copy of chromosome 21.
-No problems with meiosis but an error occurs during mitosis
so that a variable number of cells have trisomy 21. In 15% of
cases the “extra” copy of chromosome 21 is contributed by
the father
Robertsonian Translocation

A reciprocal translocation occurs when a break in two chromosomes occurs and the segments are
exchanged to form new derivative chromosomes. This typically affects chromosomes 13, 14, 15, 21 and
22.
 Clinical Features of Down’s Syndrome

• Brushfield spots
(speckling of the iris)
• Simian crease (single
palmar crease)
• Clinodactyly (little fingers
are short and curved
inwards)
• Sandal gap (wide gap
between first and second
toes)

Image taken from www.emaze.com

Associated Problems
 Learning difficulties, reduced IQ

 Congenital Heart Defects (40-50%)

-Atrioventricular septal defects (AVSD) – 420 times more common
-Ventricular Septal defect (VSD)
-Patent Ductus Arteriosus (PDA)

 Hearing impairment (50%)

 Musculoskeletal problems and joint laxity

 Epilepsy

 Thyroid disorders

 Alzheimers disease
 Biochemical Screening for T21/T13/T18(1TS)
 First Trimester Screening (11+2 – 14+1 weeks)
-Nuchal translucency (NT measurement)
(Ideally measured at 11-14 weeks)

-Serum markers: PAPP-A and fβHCG

Image taken from www.studiolift.com

1TS Biochemistry Markers

 Pregnancy Associated Protein A (PAPP-A)

-Originates from placental syncytiotrophoblasts
-Concentration increases with gestation
-Most reliable at 8-11 weeks
-Lower levels observed in T21 and T13/T18 affected pregnancies

• Free Beta Human Chorionic Gonadotrophin (fβhCG)

-Higher levels observed in T21 affected pregnancies
-Lower levels observed in T13/T18 affected pregnancies
Biochemical Screening for T21 in the Second
Trimester (2TS)
 Second Trimester Screening (14+2 – 20+0)

 Alpha Fetoprotein (AFP)

-Glycoprotein of fetal origin synthesised initially in the embryonic yolk
sac and then by the fetal liver.
-AFP typically increases with gestation
-On average AFP = 0.72MoM in DS pregnancies (Lower levels)

 Human Chorionic Gonadotrophoin (hCG)

-Dimeric glycoprotein (α and β subunits secreted by fertilised ovum
and then by the placental tissue).
-Primary function is to maintain the corpus luteum.
-Typically decreases (post 10 weeks) during increasing gestation
-On average hCG = 2.2 MoM in DS pregnancies (Higher levels)
Biochemical Screening for T21 in the Second
Trimester (2TS)
 Unconjugated Estriol (uE3)
-Derived from fetal adrenal DHEAS, hydroxylated in fetal liver and
cleaved by steroid sulphatase in the placenta where the unconjugated
fraction is converted to uE3.
-uE3 typically increases with gestation
-On average uE3 = 0.64MoM in DS pregnancies (Lower levels)

 Inhibin A
-Placental heterodimeric glycoprotein
-Typically decreases (until 17 weeks) and then levels increase.
-On average Inhibin A = 1.79 MoM in DS pregnancies (Higher levels)
-Levels 40% higher in smokers
 Possible Screening Strategies
Test (all include Measurements FPR for 85% DR
maternal age)

Integrated test NT + PAPP-A at 10 weeks, 1.2

plus quadruple test at 14-20
weeks

Serum Integrated test Integrated test without NT, 2.7

PAPP-A at 10 weeks

Combined test NT, fbHCG and PAPP-A at 10 6.1

weeks

Quadruple test AFP, uE3, fbhCG and Inhibin- 6.2

A at 14-20 weeks

Triple test AFP, uE3, fbHCG at 14-20 9.3

weeks
Double test AFP and fbHCG at 14-20 13.1
weeks
NT measurement NT at 12-13 weeks 20.0

Current standard = 90% DR for 2%SPR for 1TS, 75% DR for 3% SPR for 2TS
Mathematics of Down’s Syndrome Screening: Terminology

MoM = Multiple of the Median

Median = The value where 50% of results are above and 50% of
results are below that value

Mean = Average result

Mode = Most common result

 Why use MoMs for Down’s Syndrome Screening?

 If the mean was used this can easily skew results.

 The median is a better way to evaluate a non-Gaussian distribution.
 BUT marker levels change through the pregnancy and so we need a
statistical marker to compare results at different/specific gestational
ages…the MoM!

17 weeks = 60 = 1MoM
60 14 weeks = 40 = 1MoM
80 = 2MoM
AFP 40
(IU/L) How far away is the measured value
away from the median?
-The use of MoMs allows us to
14 17 answer this question regardless of the
gestational age!
GA (weeks)
But its not that simple……..
 At any gestational age there will be a scatter of values around the
median value.

x x
x x
x x
AFP
(IU/L) x x
x

GA (weeks)
 How can we “adjust” the values to reduce the scatter?

 Answer = Linear Regression

 Linear Regression
 A regression line is the “line of best fit” (straight line) through a data set.
 We want to plot the median at each gestational age (days) and draw a line
though the data but the data is typically skewed.
Median = Mean
SD of Log10 AFP is
approximately constant
x
x x
x x
No. of x xxx Log AFP
x x (IU/L)
values x x x x
x x x
x xx
x x x x
x x x x x xx x
GA (weeks)
AFP (IU/L)
Skewed, Non-Gaussian distribution Log10 AFP values are more
One way to “normalise” the data is to symmetrical and the mean and
use algorithms eg, Log the data. median values are identical.
 Smoothed Mean Log10 (AFP)
Residual = distance
between the fitted line
0.3 and data point
x
x x x 0.2
Log AFP x SD Log
x
x AFP
x x
0.1
x
x
0
GA (days) GA (days)

As the SD for the log10 AFP is fairly constant for all gestational ages the
distribution of log10 AFP around the smoothed baseline is the same –
therefore we can combine the residual values.

If the smoothed regression line is a “good fit” to the data then the residual
values should have a mean value of 0 so if you plot the residual values
from the regression line you should now have a Gaussian distribution.
 Smoothed Mean Log10 (AFP)

If the smoothed regression line is a “good fit” to the data then the residual
values should have a mean value of 0 so if you plot the residual values from
the regression line you should now have a Gaussian distribution.

200

SD = 0.16
Frequency

Mean = 0.00
100
A good fit!

0
-1.0 1.0
Residual values
Multiple of the Median (MoM)
 Analyte values are expressed as a multiple of the smoothed median AFP
value for the specific gestational age (GA).

 MoM AFP = (AFP value) / (Median AFP for any specific GA)

log10 MoM AFP = log10 AFP - log10 (smoothed AFP median)

= log10 AFP – regression value
=Residual from regression line

Therefore, the residuals are the log10 MoM values.

regressionline (log data)

0.5
3

MoM AFP values

2
Residuals from

0 1
0
-1

-0.5 16 17
15 16 17 15
GA (weeks) GA (weeks)

If the smoothed medians are correct the log10 MoM values should be
centered on “0” and the MoM values centered on 1 for all gestations.
Regression Equation
 Calculation of the regression equation is nowadays done by statistical
software.

 As the log10 AFP is linear the equation can be written in the form of y=mx+c

eg. Mean log10 AFP = 0.00807x GA(days) + 0.553

Mean log10 AFP = (0.00807 x 115) + 0.553 = 1.481

Antilog 1.481 = 30.3

At 115 days the mean AFP is 30.3 IU/L

The antilog of the regression fit for log10 AFP is smoother than the original
median data prior to transformation.
Factors Affecting Screening Results
 Weight: Markers get diluted in larger women

 Multiple pregnancies “Pseudo risk” calculated

 IVF: hCG higher in IVF (also higher in DS)

 IDDM: Higher incidence of DS in diabetic mothers

 Smoking: 23% reduction in hCG, 40% increase in Inhibin A

 Bleeding: Increases AFP

 Ethnicity
How do we “adjust” MoM values?
1) Marker values are adjusted for gestational age first

2) MoMs are plotted as a function of the variable eg. Maternal weight and another
regression equation is established
a) Plot log10 AFP values against GA
b) Plot SD log10 AFP values against GA
c) Calculate residuals
d) log10 MoM AFP = Residual from regression line
e) Calculate regression equation
f) If the expected and observed values are different this suggests that a
correction factor is required
(Use statistical tests to calculate if significantly different. Eg. Mann Whitney U-
test for continuous data. If p <0.05 data is statistically significantly different)

3) Values derived from the 2nd regression equation are the “adjusted” MoM values
for that variable.

4) Check adjustment by plotting Adjusted log10 MoM against GA and against

variable (eg. weight )– if MoMs centered on “0”then the adjustment is correct.
 Multiple Marker Adjustment
 Each marker is adjusted individually
 The Mean and SD’s of log10 (Adjusted MoM) are determined.
 To determine the effect (linear association) between each pair of marker
values the correlation coefficient is calculated.

 Correlation coefficient values lie between -1 and 1.

Positive correlation Zero correlation Negative correlation

(Values increase together) (One value decreases as
the other increases)
Terminology (1)

 Sensitivity = TP/(TP + FN) Proportion of people with a disease who

have a positive result.
(Identifies condition correctly)

 Specificity = TN/(TN + FP) Proportion of people without the disease

who have a negative result.
(Ability to exclude disease)
Terminology (2)
 Positive Predictive Value (PPV) = TP/(TP + FP)

 Negative Predictive Value (NPV) = TN/(TN + FN)

 Screen positive rate = No of women screened with high risk result

(Includes TP and FP)

 Detection rate = How many women with DS-affected pregnancies

have a positive screening result
 Bayes’ Theorem
Chance of having a Trisomy = Chance of a positive test x Chance of
affected pregnancy given a with an affected pregnancy affected pregnancy
positive screening result

Chance of a positive test x Chance of + Chance of a positive x Chance of an

with an affected pregnancy affected pregnancy test in unaffected pregnancy unaffected pregnancy

Formula can be simplified to:

Chance of having a Trisomy = Chance of a TP result

affected Pregnancy given a
positive screening result Chance of any positive result (TP +FP)
Calculation of Risk
 The posterior (final risk) is proportional to the Prior Risk (Prevalence) x Likelihood

1) List the outcomes (should be mutually exclusive and exhaustive)

2) Against each outcome, write down the prior probability (these should add up to 1)

3) Write down the Likelihood ratios (Likelihood ratio = No with condition/Unaffected)

4) Calculate the Likelihood x Prior

5) Divide by the total to get the posterior (final risk)

Example of Risk Calculation

Prior Risk Likelihood Prior x Posterior

Likelihood Risk

Condition X 0.002 0.99 0.00198 0.2841

Unaffected 0.998 0.005 0.00499 0.7159

Total 1.00 0.00697 1.0000

Risk of Condition X given a positive screening result = 1/Posterior Risk

Posterior Risk = 0.00198/0.00697 = 0.2841

Risk = 1/0.2841 = 1 in 4
Application to Screening
Maternal Age = 40, CRL = 65mm, NT 2.8mm, PAPP-A MoM = 0.3, βhCG MoM = 0.3
Likelihood ratio (Probabilities of observed Biochem and NT given the outcome)

Outcome Prior Risk Biochem NT LR x Prior Posterior

Risk

T13 0.0004 25 4 0.04 0.0359

T18 0.001 40 1.5 0.06 0.0538

T21 0.01 0.5 5.0 0.025 0.0224

Unaffected 0.9886 1 1.0 0.9886 0.8877

Total 1.0 1.1136

Risk of T13 or T18 = 0.0359+0.0538 = 0.08980

1/0.08980 = 11.1 or 1 in 11
Risk Calculation Software Requirements
 Audit Trail
 Validation – able to facilitate checks on MoM values and risks
 CE marked
 Calculation of GA from CRL or HC measurements
 Calculation of maternal age
 Able to adjust risks for: Ethnicity: 5 levels
Smoking: 5 levels
Twins: Mono and dichorionic
Diabetes: 5 levels
Method of conception: 5 levels
 Inclusion of regression fitting software is NOT a requirement for NHS UK
 Risk calculation
 Twin-specific risks
 Export data
Trisomy Screening: T13/18
• Implemented on1st April 2016

• Trisomy 13 (Patau’s Syndrome) /Trisomy 18 (Edward’s Syndrome) Screening

First Trimester: Women will have four possible choices:

1) T21/T13/T18 screening
-T21 risk
-T13/T18 risk

2) T13/T18 screening only

3) T21 screening only

4) Decline all screening

Second Trimester:
T21 screening only
Reproduced with permission from D. Wright (DQASS)
Pataus’ Syndrome (T13)
• Genetics
-75-90% cases caused by the presence of a whole extra
copy of Chromsome 13 (Trisomy 13).
-5-10% cases due to chromosomal translocation
-5% cases due to mosacism
-Occasionally due to partial T13 (only part of
chromosome 13 is involved)

In the majority of cases T13 is caused by non-

disjunction during maternal meiosis.

There is a high spontaneous loss of pregnancy (64%

loss rate from the Second Trimester) with very limited
survival rates (Median survival time is 10 days).
 Pataus’ Syndrome (T13)
• Clinical Features
-Fetal/Congenital malformations include:
-holoprosencephaly
-microphthalamia
-cleft lip and palate
-polydactyly
-cardiac malformations (>80% of cases)
-severe growth retardation
-mental retardation
-kidney malformations
-low birth weight

• Prognosis
->50 % die within one month
-8-10% survive beyond 1yr but have severe learning
difficulties
-Long term survival is associated with mosacism or
partial T13.

• Treatment
Image taken from www.prezi.com Supportive treatment only
Edward’s Syndrome (T18)
Genetics:
-Caused by a full “extra” copy of chromosme
18 due to non-disjunction during maternal
meiosis.
-Very occasionally can be due to translocations
involving Chromosome 18.

A significant proportion of T18-affected

pregnancies spontaneously abort (36% loss
rate from the 2nd Trimester).
Edward’s Syndrome (T18)
• Clinical Features
-Antenatal features include:
-Increased NT
-Growth retardation
-Choroid plexus cysts
-Polyhydroamnios
-Multiple congenital abnormalities

-Congenital abnormalities (usually multiple and occur together with growth retardation):
-Cardiac defect (80-90%)
-Characteristic fixed, flexed positioning of fingers
-Renal anomalies, oesphageal atresia, diaphragmatic hernia, abdominal wall
defects
-Cleft palate and lip
-Convex “rocker bottom” shape to sole of the feet.

• Prognosis
->50 % die within 2 weeks of birth (usually due to apnoea or congenital abnormalities)
-8% survive beyond 1yr but have severe learning difficulties
-Median life expectancy is 14 days

• Treatment
Supportive treatment only/Nasogastric feeding
Reproduced with permission from D. Wright (DQASS)
 PAPP-A levels are
reduced in T13 and T18
(Same direction as T21)

Reproduced with permission from D. Wright (DQASS)

 Free βhCG levels are
reduced in T13 and T18
(Opposite direction to T21)

Reproduced with permission from D. Wright (DQASS)

Reproduced with permission from D. Wright (DQASS)
Twin Pregnancies
Monochorionic vs Dichorionic Twin Pregnancies

• Twins can have separate placentas and separate amniotic sacs or share a single
placenta and sac.

• Monochorionic (shared placenta)

-Monochorionic Monoamniotic (MC, MA)

Both babies share a placenta and amniotic sac

-Monochorionic Diamniotic (MC, DA)

Both babies share a placenta but have separate amniotic sacs

• Dichorionic
-Each baby has a separate placenta
Twin Pregnancies

www.babymed.com
Twin Pregnancies and the Standards
 First Trimester
Monochorionic: Both fetuses are either affected or unaffected so a single
risk is reported :
1) Risk for T21 and a separate risk for T13/T18
2) Risk for T21 only
3) Risk for T13/T18 only

Dichorionic: Twin specific risk for each fetus is reported for:

1) Risk for T21 and a separate risk for T13/T18
2) Risk for T21 only
3) Risk for T13/T18 only

 Second Trimester
Previously Second Trimester screening was not available for twin pregnancies.
A single (twin-specific risk not available) T21 only risk is provided for women presenting at the first
time for Second Trimester screening or for women where the Nuchal Translucency (NT) measurement
could not be attained.

Monochorionic: 80% DR for 3% SPR

Dichorionic: 40-50% DR for 3% SPR

Therefore aim to screen all dichorionic twin pregnancies in the First Trimester.
Problems
 -Vanishing Twin
Screening not completed if there is evidence of fetal material in the
second pregnancy sac. A risk based on maternal age and NT is
offered.

 Unusual results
If the results are not indicative of a Trisomy affected pregnancy but
“unusual” (eg. High hCG could indicate a tumour) what should we do?

The standards stipulate that this is a screening programme for Trisomy

only and that the laboratory should take no further action.
Standards
 Fetal Anomaly Screening Programme Laboratory Handbook (2015)
-To be a stand alone screening laboratory must have an annual
workload of 8,000 per year (per Trimester).

-If <8,000 must be part of a “managed network” with each

laboratory having a minimum of 2,000 screens per year.

-Submit screening data to DQASS twice a year.

-Management mechanism and fail-safe processes in place.

-Key Performance Indicator (KPI)

-97% of DS reports must be issued within 3 working days of
receipt of sample at laboratory.
Role of a Clinical Scientist in Trisomy Screening
 Ensure that the laboratory is compliant with all FASP standards (Form Review)

 Be responsible for the day-to-day running of the laboratory

 Be responsible for the risk algorithm (factor adjustment calculation and implementation)

 Take responsibility for DQASS/FASP/KPI/NCARDS data submission

 Business case development and validation of equipment/software

 Liaise with the Antenatal teams (answer queries, solve problems, develop failsafe
procedures)

 Attend local quality meetings, attend Antenatal Programme Board meetings, DQASS
workshops, FASP workshops

 Liaise with instrument/software suppliers

 Incident investigation and associated documentation

 Peer Assessor Reviews/UKAS inspections

NIPT (Non-Invasive Prenatal Testing)
 Relies on Next Generation Sequencing of cell free fetal DNA (cffDNA)

 cffDNA is shed from the placenta into the bloodstream and is detectable from
4 weeks gestation

 The proportion of cffDNA increases with increasing gestation

 Up to 10% of total maternal circulating DNA is cffDNA

 Cleared rapidly, within 30 minutes post-delivery and so is pregnancy specific

NIPT (Non-Invasive Prenatal Testing)
Blood Draw and Centrifugation

cffDNA extraction from plasma

Preparation of DNA Library

Whole Genome Sequencing

Data analysis

Report

99% detection rate, 0.09% FP rate

NIPT Proposed Standards

 Women with high-risk (1:150 cut-off) serum screening result will be offered
NHS-funded NIPT testing. NHS-funded NIPT will screen for T21/T13/T18
only and will not include fetal sex determination

 NIPT high-risk result confirmed with CVS/amniocentesis

If NIPT fails woman will be offered one further NIPT, CVS/amniocentesis

 Two genomic centres will be awarded tender for NHS-funded NIPT work
-ITT closes Jan 2018, Contract awarded March 2018 with October 2018 “Go-Live” date

 5-7 day turn-around time stipulated

 CE/IVD marked equipment only can be used for NIPT screening

Problems with NIPT
• NIPT will never be 100% accurate:
-Confined placental mosaicism
-May detect maternal chromosomal rearrangements
-Effect of twin demise
-Maternal malignancy issues

• Unsuitable for cases of maternal malignancy, blood transfused patients and organ
transplanted patients.

• Cost (£400-£900 per test)

• Increased information - what do we report? Loss of informed choice?

• Could lose additional screening (IUGR/ Pre-eclampsia)

• Failure rate

• Insufficient cffDNA yield from ladies with large BMI

• Not able to complete NIPT until 10 weeks gestation due to low level of ccfDNA

• Still need confirmation of positive results with invasive prenatal testing

The Future

 Link to “Out-come” data

 Continuous improvement for marker adjustments (eg. Diabetes)

 Pre-eclampsia screening

 UKAS inspections using Peer-Assessor Review Standards (QA

teams)