Prenatal Screening and Diagnosis

Indications for Testing

Diagnostic testing should be offered to all women but should be discussed in detail with those at high risk for either a chromosome abnormality or ONTD as defined by the following
- Previous pregnancy with chromosome disorder (eg, DS or T18)
- Either parent is a known carrier of a genetic translocation or inversion
- Family history of ONTD
- Maternal age ≥35 at estimated date of delivery (EDD)
- Patient taking valproic acid or carbamazepine
- Patient is an insulin-dependent diabetic
Amniocentesis or chorionic villus sampling (CVS) confirms diagnosis suggested by screening tests
- CVS – 10-13 weeks
- Amniocentesis – at or after 15 weeks
Serum PAPP-A levels
- Extensively studied for possible prediction of pregnancy complications
- Decreased levels correlated with intrauterine growth restriction, preterm labor, preeclampsia and fetal demise

Screening can be done between 10–24 weeks gestation; diagnosis is available at ≥10 weeks gestation
Screening involves various combinations of PAPP-A, hCG, uE3, AFP, DIA and/or ultrasound (US) for nuchal translucency (NT) with blood draws in the first or second trimester or both
ACOG screening recommendations, 2007
- All women, regardless of age, should have the option of invasive testing
- Maternal age of 35 years alone should no longer be used as a cutoff to determine who is offered screening vs. who is offered invasive testing
- Patients seen early in pregnancy should ideally be offered aneuploidy screening that combines first- and second-trimester testing (integrated or sequential)
- Options for women seen first during the second trimester are limited to quadruple (or “quad”) screening and ultrasound exam
Recommend any of the following screening tests based on patient and physician preference (see Test tab and Prenatal Screening table below for more details about these tests)
- If patient presents in the first trimester and both CVS and NT-certified sonographer are available
  - First-trimester (combined) screen for patient who accepts higher risk of a screen positive test (SPR)
  - Sequential screen for patient who prefers lower SPR, closer to that of the integrated test, but wants to know early if she is high risk
  - Integrated test for those patients who want the best possible detection rate (DR) coupled with the lowest SPR
    - Also appropriate for patients who want to avoid invasive testing
- If patient presents in the first trimester and either no CVS or no NT-certified sonographer is available
  - Integrated screen
    - Available even without an NT measurement (serum integrated)
    - Results available in the second trimester, so availability of CVS is not an issue
- If patient presents in the second trimester
  - Quad screen
  - US
Screening follow-up
- Abnormal for DS or high AFP
  - US to look for twins and/or fetal/placental abnormalities and to confirm dates
    - If EDD changes by ≥10 days based on US – recalculate
      - Normal – low risk for DS, T18, and ONTD; no further testing recommended
      - Abnormal – offer genetic counseling and diagnostic testing
      - If recalculated gestational age is <14 weeks (15 weeks at some labs) when sample was drawn, redraw sample and repeat test
    - If EDD is correct within 10 days, offer genetic counseling and diagnostic testing
- If screen negative for ONTD, DS and T18 – low risk for ONTD, DS, T18, no further testing recommended
  - If EDD changes at scheduled second trimester US (18-22 weeks) by >10 days, recalculate
Ultrasound NT alone is inadequate for aneuploidy detection
- If NT ≥3.5 mm
  - Patient is at high risk for fetal aneuploidy and additional screening is not indicated
    - Offer CVS or amniocentesis as well as targeted US and fetal echocardiogram in the second trimester
- If NT ≥2.5 multiples of median (MoMs) after screening
  - Patient should be offered targeted ultrasound and fetal echocardiogram in the second trimester even if the screening results suggest a low risk for aneuploidy
- Sonographers must be certified to perform NT measurements by the Fetal Medicine Foundation in England (FMF) or the Nuchal Translucency Quality Review Board in the United States (NTQR)
- For more information, refer to ARUP's First- and Second-Trimester Screening Options
Prenatal screening test information (see table below)

Prenatal Screening

Test

Recommended for

Purpose

First trimester (combined) screen

Maternal Screen, First Trimester

Order during the first trimester (between 11w0d and 13w6d gestation)

Crown-rump length (CRL) must be between 42-85 mm and a nuchal translucency measurement must be obtained

Do not round gestational age to nearest week; use the estimated due date (EDD) to avoid clerical errors

Use when mother accepts higher SPR and wants results in the first trimester

Does not detect ONTD

Sequential screen combines first and second trimester screening results

First specimen drawn between 11w0d and 13w6d gestation

CRL must be between 42-85 mm and a nuchal translucency measurement must be obtained

Second specimen drawn between 15w0d and 22w6d

Do not round gestational age to nearest week; use the estimated due date (EDD) to avoid clerical errors

Specimen 1 measures PAPP-A and total hCG

Specimen 2 measures AFP, uE3, hCG, and DIA

An interpretation is provided after the first draw so that pregnancies at very-high risk for DS can be identified in the first trimester

Patients who are at intermediate or low risk after the first draw go on for the second draw and the complete screen

Integrated screen, combines first and second trimester screening results

First specimen drawn between 10w3d and 13w6d gestation

CRL must be between 36-85 mm (an NT measurement is optional for this test)

Second specimen drawn between 15w0d and 22w6d

Do not round gestational age to nearest week; use the estimated due date (EDD) to avoid clerical errors

Specimen 1 measures PAPP-A

Specimen 2 measures AFP, uE3, hCG, and DIA

When combined with a first trimester certified ultrasound for NT, this test yields the best detection rate and lowest false-positive rate of all prenatal screens

Can be run without an NT (serum integrated) yielding the same detection rate with a slightly higher false-positive rate

Single Screen

(Maternal Serum Screen, Alpha Fetoprotein Only)

Women who have had early amniocentesis, CVS or first trimester screening

Ideal time period is 16-18 weeks gestation; however, reference medians are available from 14w0d to 24w6d

Do not round gestational age to nearest week; use the estimated due date (EDD) to avoid clerical errors

Screen for fetal risk of ONTD

Triple Screen

Maternal Serum Screen, Alpha Fetoprotein, hCG, & Estriol

This panel is obsolete

ACOG recommends the Quad for second trimester aneuploidy screening

Do not round gestational age to nearest week; use the estimated due date (EDD) to avoid clerical errors

Triple screen for fetal risk of DS, T18 and ONTD

Quad Screen

Maternal Serum Screen, Alpha Fetoprotein, hCG, Estriol, & Inhibin A

Ideal time period is 16-18 weeks gestation; however, reference medians are available for 14w0d and 24w6d

ACOG recommends the Quad for second trimester aneuploidy screening

Offer to patients who

Present to care in the second trimester
Do not wish to have first trimester screening
Did not have access to first trimester screening

Quad is the most economical prenatal screening test for aneuploidy

Ideal time period is 16-18 weeks gestation; however, reference medians are available from 14w0d to 24w6d

Do not round gestational age to nearest week; use the estimated due date (EDD) to avoid clerical errors

Quad screen for fetal risk of DS, T18 and ONTD

Better detection rate at a lower false-positive rate than the triple screen

Best second trimester screen available

Prenatal diagnosis – amniotic fluid and chromosome analyses (see table below)

Prenatal Diagnosis – Amniotic Fluid and Chromosome Analyses
Test	Recommended for	Purpose
Chromosome Analysis, Chorionic Villus Sampling (CVS)	Indications include Advanced maternal age (patients ≥35 at EDD) Abnormal first trimester screen for DS or T18 Fetal ultrasound abnormalities Family history of chromosome abnormality or genetic disorder	Prenatal diagnosis in pregnant patient at 10-13 weeks gestation
Chorionic Villus, FISH	Rapid detection of aneuploidy involving chromosomes 13, 18, 21, X and Y Preliminary results usually available within 48 hours of sample receipt by lab	Order in conjunction with chromosome analysis, chorionic villus sampling (CVS)
Chromosome Analysis, Amniotic Fluid	Indications include Advanced maternal age (patients ≥35 at EDD) Abnormal maternal screen for ONTD, DS or T18 Fetal ultrasound abnormalities Family history of chromosome abnormality or genetic disorder	Prenatal diagnosis in patient >14 weeks gestation Amniocentesis is discouraged <15 weeks gestation due to high rates of fetal loss, leakage of amniotic fluid and increased risk of club foot
Chromosome Analysis, Prenatal FISH	Rapid detection of aneuploidy involving chromosomes 13, 18, 21, X and Y Preliminary results usually available within 48 hours of sample receipt by lab	Order in conjunction with chromosome analysis, amniotic fluid testing
Amniotic Fluid AFP with Reflex to Acetylcholinesterase	Indications include Abnormal MSAFP screen Family history of ONTD Patient taking valproic acid or carbamazepine Patient is insulin-dependent diabetic or diabetic in poor control	Prenatal diagnosis for ONTD at 14-25 weeks gestation Amniocentesis is discouraged <15 weeks gestation due to high rates of fetal loss, leakage of amniotic fluid and increased risk of club foot

Sensitivity and initial positive rates for Down syndrome (see table below)

Sensitivity and Initial Positive Rates for Down Syndrome
Screening Test	% DS Detection	% Initial Positive	DS cutoff
Triple	75-80	5-6	1/190
Quad	81	4-5	1/150
Integrated – serum only	85	3-4	1/110
Integrated – with NT	87	1.0	1/110
Sequential	63 (1st) 23 (2nd) 86 (total)	0.6 (1st) 1.0 (2nd) 1.6 (total)	1/25 (1st) 1/110 (2nd)
First Trimester	85	5-6	1/230

Amniotic fluid AChE specificity and sensitivity rates for open neural tube defects (see table below)

Amniotic Fluid AChE Specificity and Sensitivity Rates for Open Neural Tube Defects (Alpha Fetoprotein [Amniotic Fluid] with Reflex to Acetylcholinesterase)
Testing for	Sensitivity
Open Neural Tube Defects	95%
Anencephaly	97%
Open Spina Bifida	99%
Abdominal Wall Defects	40-79%

Targeted ultrasound test results follow-up (see table below)

Targeted Ultrasound Test Results Follow-Up
Test Result	Next Action
Anomaly detected on ultrasound	Confirm with follow up tests: Alpha Fetoprotein (Amniotic Fluid) with Reflex to Acetylcholinesterase and Fetal Hemoglobin Chromosome Analysis, Amniotic Fluid Chromosome Analysis FISH, Prenatal or Chromosome Analysis, Chorionic Villus Sampling (CVS) Chorionic Villus, FISH Maternal Serum Screen, Alpha Fetoprotein (only)
No anomaly on ultrasound, but MSAFP MoM* 2.5-3.0	Repeat MSAFP 2 weeks after initial draw to see if AFP level is increasing or decreasing; if increasing, treat patient as below If decreasing, probable transient maternal-fetal bleed; monitor pregnancy Review or repeat Level II ultrasound (including examination for signs of placental bleeding) or Treat patient as below
No anomaly on ultrasound, but MSAFP MoM* >3.0	Confirm with amniotic fluid tests Alpha Fetoprotein (Amniotic Fluid) with Reflex to Acetylcholinesterase and Fetal Hemoglobin Chromosome Analysis, Amniotic Fluid If AF-AFP is normal, patient is at risk for poor pregnancy outcome (prematurity, small-for-gestational-age infant, still birth) Offer counseling Monitor pregnancy
No anomaly on ultrasound, but hCG MoM* >3.5	Patient is at increased risk for poor pregnancy outcome (preeclampsia, imminent fetal death, small-for-gestational-age infant) Offer counseling Monitor pregnancy
*MoM measures are multiples of the median, calculated as the value of the substance divided by the median value based on gestational age of the fetus. Adjustments to MoM values are made for maternal weight, race, number of fetuses and maternal insulin-requiring diabetes.

Second trimester maternal serum screening tests – result patterns (see table below)

Second Trimester Maternal Serum Screening Tests – Result Patterns
AFP	hCG	uE₃	DIA	Pattern
L	H	L	H	Normal, overestimated gestation, and DS
H	L	H	N	Normal, underestimated gestation
L	L	L	*	Trisomy 18, fetal death
H	H	H	H	Multiple fetuses
H	N	N	N	Spina bifida, fetal-maternal hemorrhage, ventral wall defect
VH	N	L	N	Anencephaly, fetal death
VL	H	VL	N	Mole or partial mole
L = low; H = high; N = normal; VL = very low; VH = very high; * = may be high, low, or normal; not taken into account for risk calculation

Because most families who have a child with an open neural tube defect (ONTD), Down syndrome (DS) or trisomy 18 (T18) have no prior family history of these disorders, prenatal screening should be discussed with all pregnant women.

Open neural tube defects (ONTD)

Epidemiology

Incidence – varies with racial background and geographical location
- Incidence has decreased from approximately 1/1,000 to 1/1,700, likely because many foods are fortified with folic acid

Pathophysiology

Most common types include spina bifida (a developmental defect of the spine and overlying skin) and anencephaly (developmental failure of the brain, skull and overlying skin)
- Lesions of spina bifida include the following
  - Simple meningocele
  - Lipomyelomeningocele
  - Diastematomyelia
  - Myelocystocele
  - Neuritic cyst
  - Intraspinal and intrapelvic meningoceles
  - True spina bifida occulta associated with spinal dysraphism
Spina bifida often results in the following sequela, but clinical severity depends on several factors, especially location and size of the lesion
- Paralysis of the lower limbs
- Difficulty with bowel and bladder control
- Cerebral ventriculomegaly requiring shunt placement
Anencephaly associated with limited lifespan
- 50% of infants are stillborn
- Remainder of newborns die within hours or days of birth

Down syndrome (DS)

Epidemiology

Incidence – 1/700 births regardless of race or geographical location

Risk Factors

Risk increases with maternal age in a sigmoid fashion
- 20s – risk for a child born with DS is ~1:1,500
- 30s – risk rises dramatically
- 40s – risk levels out to ~1:100

Pathophysiology

Caused by an extra chromosome 21 in every cell of the body
Mosaic DS
- Caused by an extra chromosome 21 in some, but not all, cells
- Clinical phenotype
  - Usually milder than non-mosaic DS
  - Can vary from normal to severely affected

Clinical Presentation

Moderate to severe developmental delay
Characteristic facial features
- Down-slanting palpebral fissures
- Epicanthic folds
- Depressed nasal bridge
- Flat mid-face
- Low-set ears
Cardiac abnormalities
- Ventricular septal defect (VSD)
- Endocardial cushion defect
Hypothyroidism
Leukemia

Trisomy 18 (T18)

Epidemiology

Incidence – 1/6,000 births
Survival
- 90% stillborn
- Only 5-10% of infants who survive delivery will be alive at 1 year

Pathophysiology

Caused by an extra chromosome 18 in every cell of the body

Clinical Presentations

Severe developmental delay
Heart defects
Failure to thrive
Clenched fists
Rocker bottom feet
Spina bifida
Nonambulatory
Inarticulate
- Survivors can learn sign language

Tests generally appear in the order most useful for common clinical situations

Test name: Maternal Serum Screen, First Trimester

ARUP #: 0081150

Methodology: Quantitative Chemiluminescent Immunoassay

Use:

Screening test for DS and T18 during the first trimester

Draw sample and obtain NT measurement* (required) when CRL measures 42-85 mm

If NT is unobtainable, order Maternal Serum Screening, Integrated, Specimen #1 in the first trimester and follow with Maternal Serum Screening, Integrated, Specimen #2 in the second trimester, which can be interpreted without an NT value

*NT can be obtained when CRL greater than or equal to 36mm

Limitations:

Requires NT measurement performed by an FMF- or NTQR-certified ultrasonographer

This test does not screen for ONTD

Follow-up:

Maternal Serum Screen, Alpha Fetoprotein (only) test is recommended in the second trimester to screen for ONTD

Test name: Maternal Screening, Sequential, Specimen #1

ARUP #: 0081293

Methodology: Quantitative Chemiluminescent Immunoassay

Use:

Screening test for DS, T18 and ONTD

Order test in the first trimester; must be followed by a Maternal Screening Sequential Specimen #2 test in the second trimester

Draw sample and obtain NT measurement* (required) when CRL measures 42-85 mm

If NT is unobtainable, order Maternal Serum Screening, Integrated, Specimen #1 in the first trimester and follow with Maternal Serum Screening, Integrated, Specimen #2 in the second trimester, which can be interpreted without an NT value

Specimen 1 measures PAPP-A and total hCG

*NT can be obtained when CRL greater than or equal to 36mm

Limitations:

Requires an NT measurement that has been performed by an FMF- or NTQR-certified ultrasonographer

Most expensive screening test (a combination of the required ultrasound, and the first and second trimester lab tests)

Follow-up:

Interpretation is provided after first draw so pregnancies at very high risk for DS can be identified in the first trimester

Patients at intermediate or low risk after first draw go on for second draw and complete screen

No risk estimate is provided after first sample unless patient is at very high risk for either DS or T18; all patients receive a risk estimate after second sample is received

Test name: Maternal Screening, Sequential, Specimen #2

ARUP #: 0081294

Methodology: Quantitative Chemiluminescent Immunoassay

Use:

Order if Maternal Screening Sequential Specimen #1 was ordered in the first trimester

Draw sample between 15w0d and 22w6d gestation

Specimen 2 measures hCG, AFP, uE3 and DIA

Limitations:

Requires a previously submitted first trimester specimen (Maternal Screening, Sequential Specimen #1)

Test name: Maternal Serum Screening, Integrated, Specimen #1

ARUP #: 0081062

Methodology: Quantitative Chemiluminescent Immunoassay

Use:

Screening test for DS, T18 and ONTD

Screen combines a first and second trimester blood specimen, with or without a first trimester NT measurement

Draw first sample between 10w3d and 13w6d gestation

CRL must be between 36-85 mm at time of NT measurement

First specimen of the Integrated Maternal Serum Screening is used to measure PAPP-A

Limitations:

Ultrasound (US) for nuchal thickness is optional for this test; if an NT measurement is submitted to the lab, the sonogram must be performed by an FMF- or NTQR-certified ultrasonographer

Sample may be drawn as early as 10w3d (CRL of 32mm) but NT, if done, must be measured when CRL is 36-85 mm)

Sample draw and US do not have to be done on the same day

Follow-up:

When NT is included, this test yields the best detection rate and lowest false-positive rate of all prenatal screens

Final results are available after the second trimester sample is received by the lab

Test name: Maternal Serum Screening, Integrated, Specimen #2

ARUP #: 0081064

Methodology: Quantitative Chemiluminescent Immunoassay

Use:

Screen combines first and second trimester blood specimens, with or without a first trimester NT measurement

Draw second sample between 15w0d and 22w6d gestation

Limitations:

Requires a previously submitted first trimester specimen, Maternal Serum Screening, Integrated, Specimen #1

Follow-up:

Final interpretive report available only when the second specimen test results are complete

Test name: Maternal Serum Screen, Alpha Fetoprotein (Only)

ARUP #: 0080434

Methodology: Quantitative Chemiluminescent Immunoassay

Use:

Screen for fetal risk of ONTD

Order single screening test for mothers who have had early amniocentesis, CVS or first trimester screening

Ideal time period is 16-18 weeks gestation; however, reference medians are available for 14w0d-24w6d

Follow-up:

If AFP is between 2.5 and 3.0 MoMs, repeat AFP test (no earlier than 2 weeks after original test was drawn) and offer targeted US and amniocentesis for chromosomes and AFAFP

If AFP greater than or equal to 3.0 MoMs, offer US and amniocentesis

Test name: Maternal Serum Screen, Alpha Fetoprotein, hCG, Estriol, & Inhibin A

ARUP #: 0080269

Methodology: Quantitative Chemiluminescent Immunoassay

Use:

Screening test for DS, T18 and ONTD

Ideal time for ordering quad screening test is 16-18 weeks gestation; however, reference medians are available for 14w0d-24w6d

Quad screen is the most economical prenatal screening test

Limitations:

Better detection rate and lower false-positive rate than triple screen

Best second trimester screen available

Follow-up:

Request recalculation only if ultrasound exam reveals a due date discrepancy >10 days

Test name: Chromosome Analysis, Chorionic Villus

ARUP #: 2002291

Methodology: Giemsa-Band Analysis

Use:

Prenatal diagnosis at 10-13 weeks gestation

Indications include

Advanced maternal age
Abnormal first trimester screen for DS or T18
Fetal ultrasound abnormalities
Family history of chromosome abnormality or genetic disorder

Limitations:

Time-sensitive test

Patient may have a higher rate of spontaneous fetal loss postprocedure than with amniocentesis

Follow-up:

Genetic counseling for abnormal results recommended

Test name: Chorionic Villus, FISH

ARUP #: 0040203

Methodology: Fluorescence in situ Hybridization

Use:

Offered in conjunction with fetal chromosome study

Provides rapid detection of aneuploidy involving chromosomes 13, 18, 21, X and Y

Preliminary results usually available within 48 hours

Limitations:

Does NOT detect structural chromosome abnormalities, mosaicism or aneuploidy involving chromosomes other than 13, 18, 21, X or Y

Follow-up:

Genetic counseling for abnormal results

Irreversible therapeutic action should not be initiated on the basis of FISH results alone

Test name: Chromosome Analysis, Amniotic Fluid

ARUP #: 2002293

Methodology: Giemsa-Band Analysis

Use:

Prenatal diagnosis after 14 weeks gestation

Indications include

Abnormal maternal screen results for DS, T18 or ONTD
Fetal ultrasound abnormalities
Family history of chromosome abnormality or genetic disorder

Limitations:

Time-sensitive test

Follow-up:

Genetic counseling for abnormal results

Test name: Chromosome FISH, Prenatal

ARUP #: 2002297

Methodology: Fluorescence in situ Hybridization

Use:

Offered in conjunction with fetal chromosome study

Provides rapid detection of aneuploidy involving chromosomes 13, 18, 21, X and Y

Preliminary results usually available within 48 hours

Limitations:

Does NOT detect structural chromosome abnormalities, mosaicism or aneuploidy involving chromosomes other than 13, 18, 21, X or Y

Follow-up:

Genetic counseling for abnormal results

Irreversible therapeutic action should not be initiated on the basis of FISH results alone

Test name: Alpha Fetoprotein (Amniotic Fluid) with Reflex to Acetylcholinesterase and Fetal Hemoglobin

ARUP #: 0080427

Methodology: Chemiluminescent Immunoassay/Electrophoresis

Use:

Prenatal diagnosis of ONTD at 14-25 weeks gestation

Indications include

Abnormal MSAFP screen
Family history of ONTD
Patient taking valproic acid or carbamazepine
Patient is an insulin-dependant diabetic

Do not round gestational age to nearest week; use the estimated due date (EDD) to avoid clerical errors

Limitations:

AFP amniotic fluid test results are confounded by contamination with fetal blood which occurs in approximately 8% of samples collected

AChE is much less affected by fetal blood

Cannot be performed until second trimester

Follow-up:

Positive tests will automatically reflex to testing that will check for the presence of ACHE and/or fetal blood

Genetic counseling for abnormal results

Additional Tests Available

Test name: Inhibin A (Dimer)

ARUP #: 0070137

Methodology: Quantitative Chemiluminescent Immunoassay

Comments:

Test name: Maternal Serum Screen, Alpha Fetoprotein, hCG, & Estriol

ARUP #: 0080108

Methodology: Quantitative Chemiluminescent Immunoassay

Comments:

Screening test for DS, T18 and ONTD

Ideal time for ordering triple screening test is 16-18 weeks gestation; however, reference medians are available for 14w0d-24w6d

ACOG recommends the Quad for second trimester aneuploidy screening

Request recalculation only if ultrasound exam reveals a due date discrepancy of more than 10 days

Test name: Acetylcholinesterase and Fetal Hemoglobin

ARUP #: 2002387

Methodology: Qualitative Gel Electrophoresis/Radial Immunodiffusion

Comments:

Test name: Aneuploidy Panel by FISH

ARUP #: 0040208

Methodology: Fluorescence in situ Hybridization

Comments:

Guidelines

General

ARUP Institute for Clinical and Experimental Pathology®

Comprehensive Review: December 2011
Last Update: December 2011

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