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PrenaTest
Setting standards in NIPT in Europe
What is NIPT?
According to International Society for Prenatal Diagnosis (ISPD) NIPT is an advanced screening
method for women at increased risk of common fetal aneuploidies
Numerous publications with first experiences in the clinical application
Random MPS is the best validated method with the vast majority of NIPT data
Method Overview
1. Blood collection
2. Plasma preparation
3. Extraction of cell free DNA
4. Quantification of fetal DNA
(QuantYfeX)
5. Preparation of a genomic
library
6. next generation
sequencing
Method Overview
1. Blood collection
2. Plasma preparation
3. Extraction of cell free DNA
4. Quantification of fetal DNA
(QuantYfeX)
5. Preparation of a genomic
library
6. next generation
sequencing
Method Overview
1. Blood collection
2. Plasma preparation
3. Extraction of cell free DNA
4. Quantification of fetal DNA
(QuantYfeX)
5. Preparation of a genomic
library
6. next generation
sequencing
11
Method Overview
1. Blood collection
2. Plasma preparation
QuantYfeX
Proprietary qPCR-Assay
6. next generation
sequencing
4. Add on: determination of
fetal sex
Method Overview
1. Blood collection
2. Plasma preparation
3. Extraction of cell free DNA
4. Quantification of fetal DNA
(QuantYfeX)
5. Preparation of a genomic
library
6. next generation
sequencing
13
Method Overview
1. Blood collection
2. Plasma preparation
Illumina Sequencing
Technology:
HiSeq2500
Single-reads
PraenaTest
express
6. next generation
sequencing
High output
run 48h
128 samples
Quantification
Examples:
Karyotype
% Chr
13
% Chr
18
% Chr
21
46XX / 46XY*
3.61 %
2.97 %
1.27 %
47XX+13 / 47XY+13
3.65 %
2.97 %
1.27 %
47XX+18 / 47XY+18
3.61 %
3.02 %
1.27 %
47XX+21 / 47XY+21
3.61 %
2.97 %
1.32 %
* Median calculations of chromosomal percentages of more than 400 samples having normal karyotype
z-score
3 T21 positive
< 3 T21 negative
EVS 2012
SCS 2013
total
Correct
classified
(total)
466/468*
340/340
806/808
Trisomy 13 (n)
5/5
3/3
8/8
Trisomy 18 (n)
8/8
6/6
14/14
Trisomy 21 (n)
40/41
34/34
74/75
Total detection
rate
98.11%
100%
98.97%
False-positive
rate
1/414
0.24%
0/297
0%
1/711
0.14%
Within the EVS, one result was false-negative for trisomy 21 and one result was false-positive for trisomy 18 (a sample with an
aberration of chromosome 10).
Referenz
T21
T18
T13
12
27
275
9*
1
* one T21 not detected
PraenaTest Validation
Groemminger et al. 2014
62**
Generally increased applications of prenatal diagnosis methods (+3,6%) after FTS, primarily in
an intermediate risk (1:300 1:50) cohort (+10,7%)
Insuspicious results:
Affected cases:
Trisomy 13:
Trisomy 18:
Trisomie 21:
Turner Syndrome:
Klinefelter Syndrome:
47XYY Syndrome:
TripleX Syndrome:
Twins:
Discordant results :
False positive: 1 x T13, 16 x T18, 5 x T21, 1x Klinefelter Syndrome
False negative: 1 x T13, 1x T18, 1x T21
General PrenaTest performance parameter:
Overall false positive rate (FPR): 0.13%
Overall detection rate: 98.7%
Failure rate due to low cffDNA fraction in first blood samples: 1.2%
Overall no call rate: 0.6%
NIPT is much more sensitive in detecting CPM then CVS. by Rava et al. 2013
More precisely: investigate cell-free placental DNA
Vanishing twins
Case report 2
Hungary: Vanishing Twin
Singleton pregnancy
Borderline z-score of chr. 21: 3.4
cffDNA fraction 13.44%
Gender not clearly determined using QFX; 3% based on Y reads for chr. Y
Interpretation: consider possibility of a vanishing twin with trisomy 21
Confirmation: started as a multiple pregnancy (using ART); Sex of the living fetus is female
Grmminger et al. J. Clin. Med. 2014
Case report 3
Germany: Confined Placental Mosaicism (CPM)
positive z-score of chr. 18: 4.6
cffDNA fraction 10.1%
Case report 4
Germany: T21 mosaicism
Examined as part of R&D
Karyotyp using CVS:
47,XX,+21[10]/46,XX [ 5]
Confirmed:
positive z-score of chr. 21 is 7.4
cffDNA 16.68%
In clinical study:
T21 mosaicism 47,XY,+21[6]/46,XY[34]
z-score: 0.5
cffDNA 7%
Case report 5
Germany: T18 deletion
z-score of chr. 18: -6.3
inconspicuous for trisomy 18
Indication for (partial) deletion
of chr.18
Case report 6
Germany: Triple X
z-scores of chr. 13/18/21 < -3
39
Backup
Specific enzymes digest only non-methylated DNA methylated DNA remains intact.
http://www.mdpi.com/2077-0383/3/3/679
a)
b)
sensitive marker
(fetal DNA)
fetal DNA
maternal DNA
methylation sensitive restriction sites
Gender determination
sensitive marker
(fetal DNA)
fetal DNA
maternal DNA
methylation sensitive restriction sites
Gender determination
https://www.neb.com/products/e6040-nebnext-dna-library-prep-master-mix-set-for-illumina