The value of noninvasive prenatal testing (NIPT).

A supplement for a genetic counselor’s flipbook.

NIPT uses cell-free DNA.

Maternal blood sample

Maternal cfDNA Fetal cfDNA

NIPT uses cell-free DNA.

• The maternal blood sample contains a mixture of both maternal and fetal cfDNA.

• Both maternal and fetal fragments are counted and analyzed in NIPT.

• Aneuploidies are detected by comparing the amount of chromosomal material against a set of reference chromosomes.

MPS—a deeper approach to sequencing.

Alignment and counting

cfDNA sequenced viamassively parallel sequencing (MPS)

Unaffected pregnancy

Affected pregnancy

Maternal and fetal cfDNA

MPS—a deeper approach to sequencing.

• Maternal blood sample contains a mixture of both maternal and fetal cfDNA.

• Massively parallel sequencing (MPS) , analyzes cfDNA fragments from a maternal blood sample.

• Sequences are aligned to genome to identify the chromosome origin of the fragment.

• Aneuploidies are detected by comparing the amount of chromosomal material for a specific chromosome in a patient sample against a set of reference chromosomes.

Detecting fetal chromosome abnormalities.

Chromosomes

2 3 4 5 61

8 9 10 11 127

14 15 16 17 1813

Trisomy 13

20 21 22 XX-female

or

XY-male19

Trisomy 21

Trisomy 18

Detecting fetal chromosome abnormalities.

• The verifi® prenatal test detects trisomies 21, 18, and 13 in both singleton and twin pregnancies.

Detecting sex chromosome abnormalities.

Chromosomes

2 3 4 5 61

8 9 10 11 127

14 15 16 17 1813

20 21 22 XX-female XY-male19

XXY XYYXXX Monosomy X

or

Detecting sex chromosome abnormalities.

• In singleton pregnancies, sex chromosome aneuploidies can also be detected. This test is optional based on patient and physician preference.

– Sex chromosome aneuploidies detected include 45, X, 47, XXX, 47, XXY, 47, XYY.

• In twin pregnancies, the presence or absence of Y is detected. This test is optional based on patient and physician preference.

Results

Aneuploidy detected

Aneuploidy suspected

No aneuploidy detected

How the verifi® test is reported.

How the verifi® test is reported.

There are three possible results for the autosomes:

• Aneuploidy detected, aneuploidy suspected (borderline value), aneuploidy not detected.

There are two possible results for the sex chromosome analysis:

• Aneuploidy detected and aneuploidy not detected.

– If aneuploidy is not detected, fetal sex will be reported.

– Patients who receive an aneuploidy detected or suspected result should be offered a follow-up discussion of results and options of invasive testing (CVS or amniocentesis) for confirmation. It is recommended that no irreversible action be made based on these results alone.

• For twin pregnancies, the presence or absence of Y will be reported.

PPV and NPV calculations.

To calculate PPV using a fixed sensitivity of 0.999 and specificity of 0.998, insert prevalence of the condition for your patient in decimal form.

PPV =

* 0.002 = 1 – specificity

0.999 x prevalence

(0.999 x prevalence) + (0.002* x (1 – prevalence))

GC calculations:Percentages calculated using sensitivity > 99.9% and specificity of 99.8%

Prevalence Positive predictive value Negative predictive value

1/10

1/100

1/250

1/500

1/1000

98.2%

83.5%

66.7%

50.0%

33.3%

> 99.9%

> 99.9%

> 99.9%

> 99.9%

> 99.9%* Positive predictive values and negative predictive values are dependent on sensitivity and specificity as well as prevalence of disease.

Definitions.

Definition Formula

Sensitivity The proportion of patients WITH a particular condition that test positive for that condition.

SpecificityThe proportion of patients WITHOUT a particular condition that test negative for that condition.

Positive Predictive Value (PPV)Probability of the condition among patients with a positive result.

Probability of no condition among patients with a negative result.Negative Predictive Value (NPV)

* Prevalence is equal to the proportion of people who have a particular condition at a specified point in time.

True negatives

True negatives + false positives

Sensitivity x prevalence*

Sensitivity x prevalence* + (1 – specificity) x (1 – prevalence*)

Specificity x (1 – prevalence*)

(1 – sensitivity) x prevalence* + Specificity x (1 – prevalence*)

True positives

True positives + false negatives

Typical prenatal testing sequence.

Serumscreening

NIPT(noninvasive)

NO

NONO

Is the serum screen result positive?

Is the verifi test result aneuploidy detected

or suspected?

(invasive)CVS/Amniocentesis

Diagnostic

Continue with pregnancy management according toyour practice’s protocols.

Continue with pregnancy management according to your practice’s protocols.

Continue with pregnancy management according to your practice’s protocols.

YES YES

YES

Patient elects prenatal testing

Typical prenatal testing sequence.

Pre-test counseling considerations

• Aneuploidy testing should be an informed choice.

• The verifi prenatal test screens for aneuploidies of chromosomes 21, 18 and 13. Sex chromosome testing is optional.

• Selected microdeletion testing is optional.

• Any test offering should be based on clinical context.

• CVS and amniocentesis remain options for all pregnant women.

Post-test counseling considerations

• 3 possible results for each chromosome tested.

– No aneuploidy detected—This result is reassuring but does not ensure an unaffected pregnancy.

– Aneuploidy suspected/Borderline value: This result is suggestive of aneuploidy. There is a small possibility that the results might not reflect the chromosomes of the fetus but may reflect changes to the placenta or the patient.

– Aneuploidy detected: This result is consistent with aneuploidy. There is a small possibility that the results might not reflect the chromosomes of the fetus but may reflect changes to the placenta or the patient.

– Genetic counseling and CVS/amniocentesis are recommended for suspected and detected results.

• Open neural tube defect screening is not a part of NIPT testing and should be offered separately.

• All results should be reviewed in the context of the clinical, medical, and family history.

• It is recommended that no irreversible action should be made based on these results alone.

NIPT profile: the verifi® test by the numbers.

What you can’t put a number on— the reassurance of knowing

As early as weeks into pregnancy

10

vial of blood 1

Tests for the most commonautosomal aneuploidies andadditional sex aneuploidies

3

lowest test failure rate

0.1%

Results reported to lab in justbusiness days

3-5

How is the verifi® prenatal test different?

• The verifi test is an easy, in-office blood test—only one tube of blood is required.

• It can be performed as early as 10 weeks in pregnancy.

• It tests for trisomies 21, 18, and 13.

• Options for sex chromosomes aneuploidies and select microdeletions are available.

• Test reports are usually available in 3 – 5 business days after sample receipt. (Time to report may vary based on the partner laboratory providing the test.)

• It has the lowest failure rate among NIPTs (0.1%).

Because of this, the verifi test is able to provide reliable answers about the most common chromosome abnormalities.

What is mosaicism?

Normal cells Cells with genetic change

Cell division

Mosaic tissue

What is mosaicism?

• Mosaicism is a biological phenomenon which results from the presence of cells with differing genetic compositions in the same individual. It usually occurs during the process of mitotic cell division.

• Mosaicism can be an underlying biological cause of discordant NIPT results.

Types of mosaicism.

Disomy

Generalized mosaicism

Confined placental mosaicism

Fetal mosaicism

Trisomy

Types of mosaicism.

There are three types of mosaicism:

• Generalized mosaicism: Presence of two or more karyotypically different cell lines in both the placenta and the fetus. In cases of generalized mosaicism, there is a possibility of a false positive or a false negative NIPT result depending on the origin of the cfDNA.

• Confined placental mosaicism: Presence of two or more karyotypically different cell lines that are confined to the placenta and not present in the fetus. In cases of confined placental mosaicism, there is a possibility of a false positive NIPT result.

• Fetal mosaicism: Presence of two or more karyotypically different cell lines that are present in the fetus but not present in the placenta. In cases of fetal mosaicism, there is a possibility of a false negative NIPT result.

Confined placental mosaicism.

Cytotrophoblastic cell column

Syncytiotrophoblast

CytotrophoblastMesenchymal core

CVS

Mesenchymal core

Laboratory techniques used for chorionic villus sampling (CVS)

Villus culture

Cytotrophoblast suspension

Direct preparation

Confined placental mosaicism (CPM).

• CPM is defined as the presence of two or more karyotypically different cell lines that are confined to the placenta and not present in the fetus.

• The cfDNA that is analyzed in NIPT arises from the cytotrophoblasts; thus an NIPT result is most similar to a CVS direct preparation result.

• Type 1 CPM: Mosaicism is confined to trophoblastic cells.

• Type 2 CPM: Mosaicism is confined to the cells from chorionic villus stroma.

• Type 3 CPM: Mosaicism is present in both: trophoblastic and stroma cells.

• Type 1 and Type 3 CPM could result in false positive NIPT results.

Microdeletion panel.

Telomere

p arm

Centromere

q arm

Telomere

Telomere

p arm

Centromere

q arm

Telomere

Telomere

p arm

Centromere

q arm

Telomere

p arm

Centromere

q arm

Telomere

Microdeletion panel.

• Microdeletions are chromosomal disorders caused by small missing pieces of chromosome material.

• In singleton pregnancies, the verifi® microdeletion panel is optional based on patient and physician preference.

Conditions included on the panel:

– 22q11.2 deletion syndrome

– 1p36 deletion syndrome

– Prader Willi/Angelman syndrome (15q11.2 deletion)

– Wolf-Hirschhorn syndrome (4p-)

– Cri du Chat syndrome (5p-)

• Similar to NIPT for whole chromosome aneuploidies, positive predictive value is dependent on the prevalence of the microdeletion syndrome.

© 2015 Illumina, Inc. All rights reserved. Illumina, verifi®, and the pumpkin orange color are trademarks of Illumina, Inc. and/or its affiliate(s) in the US and/or other countries. PRESENTATION-0098 Rev Ewww.illumina.com/NIPT