Screening Test

Overview of Prenatal
Screening Tests
04/ 04/ 2014
LabGenomics Clinical
Laboratories
Sung Eun Cho, M.D., Ph.D.
Prenatal Screening for Fetal Defects
It is now recommended by the American
College of Obstetricians and
Gynecologists (ACOG) to offer screening
to women of all ages.
Prenatal Screening for Fetal Defects
1.
2.
3.
4.
5.
6.
Terminology and Method of Risk Calculation
Second-Trimester Screening Tests
Newer Screening Algorithms
Follow-Up Testing of Screen Positive Results
Epidemiologic Monitoring
External Proficiency Testing
Screening Markers
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Human Chorionic Gonadotropin
Alpha-fetoprotein
Unconjugated Estriol
Inhibin A
Pregnancy-Associated Plasma Protein-A
Nuchal Translucency
Human Chorionic Gonadotropin
• Secreted by trophoblastic placental cells
• Secreted by a variety of tumors
• Two subunits : alpha & beta
joined noncovalently
• Biologically active αβ heterodimer (hCG)
• Free α (hCGα) & Free β (hCGβ) : detected in serum
and urine from pregnant women
Most Common Assays of hCG
• Intact hCG assays :
- hCG dimer (α and β subunit combined)
- negligible cross-reactivity against free subunits
• Total hCG assays :
- hCG dimer and the free β subunit (hCGβ)
- negligible cross-reactivity against the free
α subunit (hCGα)
• Free hCGβ assays : free β subunit (hCGβ)
- negligible cross-reactivity against intact hCG
and the alpha subunit (hCGα)
- dilution is not required
- unstable than intact forms
HCG during Pregnancy
• Intact or total hCG : < 5 IU/L
in nonpregnant female
• During pregnancy, hCG
double every 1-2 days
• Peak at 10 weeks of GA :
100000 – 200000 IU/L (100200 IU/mL)
• In early second trimester :
fall to plateau 20000 IU/L
(20 IU/mL) at 18 weeks’
gestation
Alpha-fetoprotein
• Major protein of embryonic plasma
• 68kDa, single polypeptide chain composed of 590
amino acid residues
• Belongs to the family of albumin-like proteins that
includes human serum albumin and vitamin Dbinding protein
• Produced by both the yolk sac and fetal liver
Maternal Serum
Alpha-fetoprotein
• Used in the second trimester to aid in
the Dx. of open NTD and DS screening
• In nonpregnant women : < 10 ng/mL
• MSAFP assays are optimized for the
concentration range of 1 to 500 ng/mL
• Reference Materials : 1st WHO IS for
AFP (72/225)
MSAFP levels are elevated
•
•
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•
•
•
•
•
•
Fetal demise
Fetal blood contamination
Underestimated gestational age
Twin gestation
Premature outcomes
Congenital nephrosis
Cystic hygroma
After amniocentesis
Preeclampsia
Unconjugated Estriol
• Produced by fetal adrenal tissue, fetal liver, placenta
• Low level - DS
- IUGR
- fetal death>24 weeks
- pregnancy loss
- oligohydramnios
• Very low level (undetectably low)
- fetal chromosome anomalies
- fetal structural anomalies
- fetal death
- steroid sulfatase deficiency
- congenital adrenal hypoplasia
- SLO syn.
- placental aromatase deficiency
Unconjugated Estriol during Pregnancy
• Original use : test for fetal
well-being in later pregnancy
• uE3 for MSS : recalibrated
at the lower concentrations
required for DS screening in
the second trimester
• Reference Materials : Not
exist
uE3 levels rise during the
midtrimester at approximately 25%
per week.
Inhibin A
• Inhibins : dimeric glycoprotein hormones, disulphidelinked subunits
- α and βA (dimeric inhibin A or DIA)
- α and βB (dimeric inhibin B)
• Both forms
- suppress FSH production by the anterior pituitary
gland
- have local modulating effects on gonadal
steroidogenesis
• Main source of inhibin : testicular and ovarian cells
Inhibin A
inhA levels increase during
the first trimester until week
10, then decline to stabilize
from week 15 to week 25, and
rise again peaking at term with
plateau.
• During pregnancy, the placenta
secretes inhibin A in later
pregnancy, maternal serum
inhibin A is placental in origin
• Function : unknown during
pregnancy
• Immunoassays : limited number
of asssays available
• Reference Materials : WHO 1st
International Reference Standard
for Human Inhibin A (91/624)
Pregnancy-Associated Plasma
Protein-A (PAPP-A)
• Zinc-containing metalloproteinase glycoprotein, at low
levels in human tissues, much higher concentrations in
the placenta
• During pregnancy, produced by trophoblast
• 1% of PAPP-A : free, active form
• The correlation was found to be significant at weeks 10
to 14 of pregnancy
• First-trimester screening
Pregnancy-Associated Plasma
Protein-A (PAPP-A)
• Reduced concentration in pregnancy :
chromosome abnormalities, T21, T13, T18
• Low levels in the first trimester : ass. with
spontaneous fetal loss at <24 weeks’ GA,
IUGR, pre-eclampsia, gestational HTN,
preterm delivery, fetal death at >24 weeks’
GA, preterm PROM, placental abruption
• Nonisotopic noncompetitive immunoassays,
ELISA, CLIA
• Reference Material : IRP 78/610  New IRP
Terminology &
Method of Risk Calculation
in Prenatal Screening
Calculation Procedure
1. Marker Concentration
2. MoM calculation
- Conc./Regressed (Expected) Median at GA
- MoM adjustment (Weight, Twin, IDDM,
Race, Smoking, IVF, etc.)
3. LR : Multivariate algorithm
- 4 parameter set
- Truncation (very low / very high)
- Log (MoM) : overlapping log Gaussian
distribution
4. Age Risk (a priori risk) X LR
Median values vs MoM
• The initial step in calculating an MoM
: develop a set of median values for each
week (or day) of gestation, using the
laboratory’s own assay values measured
on the population to be screened.
• Multiple of the Median (MoM)
= individual test results / median for
the relevant gestational week
Assay-Specific & Population-Specific
Median Values
• Difference of median values
-
reliability of gestational dating
race
maternal weight
differences in assay instrumentation
lot numbers of reagents or calibrators
Assay-Specific & Population-Specific
Median values
• CAP requires : “documentation that the
laboratory has established its own normal
median AFP values or verified the
manufacturer’s package insert or other
source for the population being screened
and that these medians are updated at
least annually.”
Assay-Specific & Population-Specific
Median Values
• Median values : routinely monitored & updated as
necessary
• At least 100 values for each week of gestation
 alternatives : 300-500 patient specimens
(unaffected singleton pregnancies : not necessary)
 median values for each completed week of gestation
 weighted regression analysis
 regression equation : to predict “smoothed”
medians for each week of gestation
Assay-Specific & Population-Specific
Median Values
• Measurements of the concentrations AFP,
uE3, and PAPP-A increase by a constant
proportion
- 15% per week for AFP
- 25% per week for uE3
- 50% per week for PAPP-A
 log-linear model best fits this relationship
[log of the measurements (y-axis) is
regressed against gestational age (x-axis)]
Assay-Specific & Population-Specific
Median Values
• Regression model for CG versus gestational age fits
an exponential model
• Regression model for inhA versus gestational age :
cubic model, with a minimum found between 16 and
18 weeks’ gestation
Day specific medians rather
than weekly medians
•
•
Observed and Regressed Day-Specific Medians for Unconjugated Estriol
during the 17th Week of Gestation
Calculate medians separately for gestational ages based on “dates” :
time from LMP or using US (crown-rump or biparietal diameter)
Gestational Age,
Week, day
No. of
Observations
uE3, ng/mL,
observed
uE3, ng/mL,
regressed
16,0
195
0.67
0.68
16,1
225
0.69
0.70
16,2
228
0.71
0.72
16,3
219
0.76
0.74
16,4
186
0.78
0.76
16,5
191
0.80
0.79
16,6
175
0.78
0.81
Concentration of Markers as MoM
• MoM : now universally used as a common factor for
converting analyte values into an interpretative unit and
serves as the starting point for calculating screening
results for NTD, Down syndrome, Trisomy 18
• The convenience of the MoM :
1) unitless (ratio of two concentrations)
2) directly providing information on whether a
given value is high or low, and by how much
3) readily adjusted to take into account the
other variables (maternal weight, IDDM, ethnicity)
4) used to calculate individual patient-specific risks
Adjustment Factors
• Gestational age : main factor
that affects changes in the
concentrations of serum markers
1) AFP : increase with gestational
age 15% per week
2) uE3 : increase with gestational
age 25% per week
3) hCG : decrease exponentially
4) DIA : changes little
Adjustment Factors
• Maternal weight : dilutional effect related
to increased blood volume with increasing
weight
• Ethnic differences : African descent have
higher MSAFP levels than in Caucasian
women
• IDDM : lower MSAFP levels (20%)
• Assisted Reproduction (Ovulation Induction
and In Vitro Fertilization) : higher hCG,
lower uE3
The Effect of Maternal Weight on
uE3, hCG, and DIA
• Inverse relationship between
maternal weight and serum
marker levels
• hCG, DIA, AFP : similar in
magnitude
• uE3 - lesser magnitude
- shorter half-life of uE3
• Overall effect of maternal
weight correction on
multiple marker screening
performance : slight
CLSI I/LA25-A2 Vol.31 No.8
Detection Rate (DR) &
False Positive Rate (FPR)
The likelihood ratio is determined
by calculating the ratio of the
heights of the affected and
unaffected overlapping population
distributions for any specified MoM
value.
Likelihood Ratio of
NTD vs Down Syndrome using MSAFP
Individualized Patient-Specific Risks
Using Multiple Markers
• When multiple tests are used, a single
likelihood ratio is calculated using the
overlapping distributions for each test but with
the correlation between tests taken into
account.
• The final risk should indicate the trimester for
which it was calculated (for example, 1 in 100,
first-trimester risk).
Parameter Sets
• Wald 1988
• Knight 1998
• Wald 2000
• SURUSS 2003
Risk of Down’s syndrome
according to maternal age
J Med Screen. 2005;12(4):202. Links Corrections to maternal age-specific
live birth prevalence of Down's syndrome.Morris J, Mutton D, Alberman E.
The Test Performance Using
Multiple Markers
• The test performance (detection and false-positive
rates) achievable depends on many factors, including
(1) the analyte combination chosen
(2) the risk cutoff chosen
(3) the method of dating used to establish
gestational age
(4) the maternal ages of the women being tested
Estimated Down Syndrome Detection Rates (DRs), False-Positive
Rates (FPRs), and Odds of Being Affected Given a Positive Result
(OAPR) at Three Risk Cutoff Values for Selected Combinations of
Maternal Serum Marker and Two Methods of Dating
Second Trimester
Risk Cutoff
(Term), 1 in
Maternal Age &
Serum Markers
DR, %
LMP
US
LMP
US
LMP
US
150(200)
AFP, uE3, CG
61
67
3.6
3.7
43
40
AFP, uE3,CG,inhA
68
71
2.8
2.9
30
29
AFP, uE3, CG
65
70
4.6
4.7
51
49
AFP, uE3,CG,inhA
71
74
3.5
3.7
36
36
AFP, uE3, CG
70
74
6.6
6.5
69
64
AFP, uE3,CG,inhA
75
78
5.0
5.1
48
48
190(250)
270(350)
FPR, %
OAPR, 1:N
Reporting Individual Results
1> maternal serum screening report
- concentrations and MoM values for measured analytes
- interpretation as screen positive or screen negative
- the Down syndrome risk estimate along with trisomy18
- recommendations for possible further action
Reporting Individual Results
2> Physician-provided information
- specimen collection date
- identification as a first or second specimen
- the date of LMP or gestational age confirmed by US,
and maternal birth date (or age)
- relevant pregnancy history
- number of fetuses (if known)
- maternal race
- the presence or absence of preconceptional maternal
DM requiring insulin therapy
Second-Trimester
Screening Tests
Neural Tube Defects
• Screening Test :
Maternal Serum AFP
- increased in the second
trimester
- useful only as an initial
screening test
• Diagnostic Procedure
Neural Tube Defects
• Optimal screening :
between 16-18 completed
weeks’ gestation
• Screening before 14 weeks :
difficult to justify
• 2.0 and 2.5 MoM : two most
commonly used AFP MoM
cutoffs
• 2.5 MoM : 70-75 % of
detection rate
• 2.0 MoM : 80-85 % of
detection rate
Down Syndrome
• In 1984, association between second trimester maternal
serumAFP and fetal Down syndrome was reported.
• Maternal serum AFP concentrations are about 25% lower
in Down syndrome than in unaffected pregnancies
• Unconjugated estriol (uE3), a product of the fetoplacental
unit, is about 25% lower in pregnancies with Down
syndrome
• Concentrations of CG were found to be about twice as
high
• In 1988, Triple test was proposed for combining maternal
age into a single Down syndrome risk estimate
• A fourth analyte, inhibin A (inhA) : introduced in the late
1990s  Quadruple test
Down Syndrome
• Triple test : AFP, uE3, CG
• Qudruple test : AFP, uE3, CG, InhA
Down Syndrome
Trisomy 18
• AFP : median 0.65 MoM (low)
• uE3 : median 0.43 MoM (low)
• CG : median 0.36 MoM (very low)
• Second-trimester risk cutoff 1:100
 DR 60%, IPR 0.5%
Newer Screening
Algorithms
First-Trimester Test for Down syn.
• In the first trimester :
10-13 weeks
• PAPP-A : low
median 0.4 - 0.5 MoM
• CG (free beta subunit
or total) : high
median 1.7 - 2.2 MoM
• Maternal age, PAPP-A,
CG : DR 60%, FPR 5%
First-Trimester NT for Down syn.
• Nuchal Translucency (NT) :
subcutaneous space
between the skin and the
cervical spine
• NT : median 2.0 MoM
(high)
• NT alone : DR 60%, FPR 5%
First-Trimester Combined Test
for Down syn.
• Combined Test :
- NT & serum markers (PAPP-A, CG)
- DR 85%, FPR 5%
- Comparable or slightly better than the
second-trimester quadruple test for Down syn.
• Limitations of Combined Test
- DR & FPR are dependent on the Gestational age
- PAPP-A, free beta CG : not cleared by FDA
- NT must be performed by trained sonographers
- NTD by AFP : performed in the second trimester
Expected Down Syndrome False-Positve Rates (FPRs)
for a Detection Rate of 85% During the First Trimester
Test Combinations*
Weeks’ Gestation
11
12
13
11 to 13
Combined
NT
27
37
54
39
PAPP-A
43
51
60
51
Free CGβ
56
50
42
50
Total CG
72
61
45
60
NT, PAPP-A, and free CGβ
5.3
6.1
7.6
6.3
NT,PAPP-A, and total CG
6.8
7.2
7.3
7.1
* Maternal age is used with each test combination. CG, Chorionic
gonadotropin; NT, nuchal translucency; PAPP-A, pregnancy-associated
plasmaprotein-A. (Adv Clin Chem 2007;43:177-210.)
Integrated Test
• PAPP-A , NT : 1st trimester without interpretation
• Quadruple test (AFP, uE3, CG, inhA) : 2nd trimester
• Six tests results are then combined into a single
risk estimate
• For Down Syndrome : DR 85%, FPR 1%
• Cost-effective approach
• High risk women : amniocentesis recommended
Serum Integrated Test
• PAPP-A : first-trimester without interpretation
• Quadruple test (AFP, uE3, CG, inhA) : 2nd trimester
• Five tests results without NT results
are then combined into a single risk estimate
• For Down Syndrome : DR 85%, FPR 4%
• Cost-effective approach
• High risk women : amniocentesis recommended
Screening Performance of the Combined, Quad,
and Integrated Tests According to Maternal Age
Mater First-Trimester
-nal
Combined Test
age
Second-Trimester
Quad Test
First-and SecondTrimester Integrated
Test
DR (%) FPR(%) OAPR
DR (%) FPR(%) OAPR
DR (%)
FPR(%)
OAPR
74
1.9
1:29
70
2.8
1:44
81
0.8
1:11
25-29 75
2.3
1:27
73
3.3
1:42
83
0.9
1:10
30-34 81
4.0
1:26
80
5.7
1:37
86
1.6
1:9
35-39 90
11
1:20
90
14
1:26
92
4.2
1:8
40-44 95
28
1:15
96
32
1:16
96
11
1:6
≥ 45
45
1:11
98
46
1:11
97
18
1:4
<25
98
OAPR, odds of being affected given a positive result.
OAPR of the Tests
Wald NJ, et al.SURUSS in perspective. Semin Perinatol 2005; 29:225-235.
ROC curves for tests to screen Down syndrome
(SURUSS in perspective. Semin
Perinatol 2005;29:225-35)
Sequential Integrated Test
•
First Trimester
- Interpretation performed with first dual markers
- Women at the highest risk for Down Syndrome
 receive the results
 recommended the chorionic villus sampling
for diagnostic testing
•
Second Trimester
- Remaining women (about 98%)
- Integrated analysis with six tests
- High risk women : amniocentesis recommended
•
Overall DR 85%, FPR 2-3%
Contingent Screening Test
• Patients at very high and at very low risk
: receive results in the first trimester
• Those with an interim risk go on to have
the second part of the full integrated
screening test
• The protocol of this has yet to be evaluated
Follow-Up Testing for
Women with
Screening Positive Results
Screening Positive for NTD
1) Amniocentesis
- Amniotic fluid AFP : false-positive result if
contaminated with a small amount of fetal blood
- All abnormal amniotic fluid AFP : must confirmed by
acetylcholinesterase (AChE)
2) High-Resolution Ultrasound
•
•
So reliable that it is used for Dx. without amniotic fluid
measurement
Presence of two cranial signs (known as fruit signs)
- lemon sign
- banana sign
Screening Positive for DS
• Genetic counseling
• Most common reasons for screen positive :
overestimated gestational age
 verify gestational age by US examination
• Still at increased risk after the US exam.
 Amniocentesis to obtain fetal cells for karyotyping
• Amniocentesis Procedure-related rate of fetal loss :
as high as 1:200
• Screen-positive result in the first trimester :
- Chorionic Villus Sampling (CVS) in 10-12 week
- Slightly higher risk of fetal loss than amniocentesis
Epidemiologic Monitoring
Epidemiologic Monitoring of
Open NTD Screening
• MSAFP screening for open neural tube defects
• Initial Positive Rate (IPR) : the proportion of
women with values above the specified MoM
cutoff
• Screening cutoff of 2.5 MoM  IPR : 1 to 3 %
• IPR falls outside the expected range
 new median is incorrect
• Simultaneous monitoring of the IPR and the
results of quality control samples can facilitate
identification of shifts in assay values.
Epidemiologic Monitoring of
Down Syndrome Screening
• IPR : influenced by each of the analytes
• It is necessary to monitor each assay separately
• Grand MoM from 300-500 individuals screened
after the new set of medians is implemented
• Consider important, as a minimum, a consistent
deviation of 10% from 1.00 MoM (range 0.90 to 1.10)
• Stricter warning limits : 7% (0.93-1.07)
Epidemiologic Monitoring of
Down Syndrome Screening
• IPR screening cut-off : 5%
• Affected by various factors
- type of tests
- screening cut-off chosen
- maternal age distribution of the tests referred
to the laboratories
- performance of the obstetrician measuring
US markers
Epidemiologic Monitoring
LabGenomics Clinical Laboratories
Risk Cut-Offs of Screening Tests
Screening Tests
Cut-Off
Trisomy 21
Trisomy 18
NTD (MoM)
Triple Test
1:270
1:200
2.5
Quadruple Test
1:270
1:200
2.5
Integrated Test
1:495
1:150
2.5
First Trimester Dual Test
1:250
1:300
X
Sequential Test
(1st trimester)
1:41
X
X
Sequential Test
(2nd trimester)
1:450
1:150
2.5
LabGenomics Clinical Laboratories
External Proficiency Testing
• All laboratories performing prenatal screening should
participate in an external proficiency testing program
- concentrations of analytes
- MoM value
- patient-specific risks
- Interpretation
• CAP www.cap.org : 200 enrolled laboratories
Proficiency Samples
- Five second-trimester serum
- Two amniotic fluid
- Five first-trimester serum
- distributed three times a year
• New York State external proficiency testing
• www.ipmms.org
External Proficiency Testing of
NT Measurements
• To bring uniformity to NT measurements,
credentialing agencies have been
established to train and certify sonographers.
• Fetal Medicine Foundation and the Nuchal
Translucency Quality Review Program,
a subsidiary organization of the Society for
Maternal-Fetal Medicine (SMFM)
New Techniques
• Fetal DNA in maternal plasma  Massive sequencing
of DNA fragments in maternal blood with
chromosome quantification
• Selective amplification of placental methylated DNA
with quantification by mass spectrometry
• Calculation of haplotype ratios using tandem single
nucleotide polymorphisms
References
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•
•
•
•
•
•
Ashwood ER, Grenache DG. Lambert-Messerlian G. Pregnancy and its
Disorders. In : Tietz textbook of clinical chemistry and moleclar
diagnositcs. 5th ed. Philadelphia : Elsevier Saunders, 2012;1991-2044.
Borawski D and Bluth MH. Reproductive Function and Pregnancy. In :
Henry’s clinical diagnosis and management by laboratory methods.
22nd ed. Philadelphia : Elsevier Saunders, 2011;402-416.
CLSI guidelines I/LA25-A2-Maternal Serum Screening; Approved
Standard-Second Edition, 2011.
CLSI guidelines I/LA25-A-Maternal Serum Screening; Approved
Standard, 2004.
Wald NJ, Rodeck C, Hackshaw AK, Rudnicka A. SURUSS in Perspective.
Semin Perinatol 2005;29:225-35.
Wald NJ, Walters J, Rodeck C, Chitty L, Hackshaw AK, Mackinson AM.
First and second trimester antenatal screening for Down’s syndrome:
the results of the Serum, Urine and Ultrasound Screening Study
(SURUSS). Health Technol Assess 2003;7:1-77.
Morris J, Mutton D, Alberman E. Corrections to maternal age-specific
live birth prevalence of Down’s syndrome. J Med Screen 2005;12(4):202.

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