Cell-Based Noninvasive Prenatal Diagnosis I: Recovery of Cells Steen Kolvraa, M.D., Chief Scientific Officer, ARCEDI Biotech ApS, Denmark 1
Cell-Based Noninvasive Prenatal
Diagnosis I: Recovery of Cells
Steen Kolvraa, M.D.,
Chief Scientific Officer,
ARCEDI Biotech ApS, Denmark
1
BackgroundFetal cells in maternal blood have been known for many
decades
For more than three decades research groups and
diagnostic companies have tried to develop NIPT based
on these fetal cells originally by:
• Initial enrichment based on fetal cell markers
• Smearing on slides
• Identification of fetal cells on slides based on fetal cell
markers
• Demonstration of genomic abnormalities by FISH/PCR2
Background
In general limited success mainly due to the
fact that there are very few fetal cells in
maternal blood and due to lack of optimal
markers.
3
Identify a frequent fetal cell type:
Strategy
4
Identify a frequent fetal cell type:
Blood drawn from women pregnant with a male
fetus
”Unspecific” enrichment of fetal cells
- Lysis of red blood cells
- Depletion using CD45
cont….
Strategy
5
Smearing of pellets on slide
Find fetal cells using X and Y chromosome FISH
and automated scanning
Collect fetal cells by laser capture microdisection
Isolate mRNA from fetal and maternal cells to
make cDNA
Expression profiles
Strategy
6
Identification of fetal cells
MetaSystem system – in-house developed classifier
Results
7
Three expression profile experiments
• 23 fetal cells
• 100 fetal cells
• 2 x 98 fetal cells
10x maternal blood cells
Laser Capture Microdisection (LCM)
mRNA and cDNA synthesis
Results
8
RNA quality
Results
9
Three expression profile experiments
• 23 fetal cells
• 100 fetal cells
• 2 x 98 fetal cells
10x maternal blood cells
Laser Capture Microdisection (LCM)
mRNA and cDNA synthesis
PIQOR TM stem cell microarrays
Results
10
April09(1)
0
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0 40 80 120 160 200
Fetal signal (1)
Ma
tern
als
ign
al (
1)
April09(2)
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0 40 80 120 160 200
Fetal signal (2)
Mate
rnal sig
nal (2
)
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April09(1)
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0 40 80 120 160 200
Fetal signal (1)
Ma
tern
als
ign
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1)
April09(2)
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0 40 80 120 160 200
Fetal signal (2)
Mate
rnal sig
nal (2
)
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April09(1)
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90
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0 40 80 120 160 200
Fetal signal (1)
Ma
tern
als
ign
al (
1)
April09(2)
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0 40 80 120 160 200
Fetal signal (2)
Mate
rnal sig
nal (2
)
13
Array 1: 78 overexpressed genes
Array 2: 75 overexpressed genes
14
39 39 36
Array 1: 78 overexpressed genes
Array 2: 75 overexpressed genes
15
39 Common Genes in Array1 and Array2
16
ResultsThree expression profile experiments
• 23 fetal cells
• 100 fetal cells
• 2 x 98 fetal cells
Increasingly focused enrichment
10x maternal blood cells
Laser Capture Microdisection (LCM)
mRNA and cDNA synthesis
PIQOR TM stem cell microarrays
Bioinformatics17
• 56% were expressed in placenta
• 33% were expressed in endovascular
trophoblasts
• Expression of both ectodermal and mesodermal
proteins
Results
18
• 56% were expressed in placenta
• 33% were expressed in endovascular
trophoblasts
• Expression of both ectodermal and mesodermal
proteins
Endovascular Trophoblasts
Results
19
Front. Genet. 26 September 2013
What are endovascular trophoblasts?
20
BLOOD, 3 NOVEMBER 2011 VOLUME 118, NUMBER 18
What are endovascular trophoblasts?
Extravillous invading trophoblasts
Endovascular trophoblasts
21
Anchoring villus Decidua
What are endovascular trophoblasts?
22
Anchoring villus Decidua
What are endovascular trophoblasts?
23
Journal of Pregnancy 10, 2011.
What are endovascular trophoblasts?
24
Endovascular trophoblasts undergo EMT:
Ectodermal markers (in the cytoplasm)
Mesodermal markers (endothelial)
25
Endovascular trophoblasts undergo EMT:
Ectodermal markers (in the cytoplasm)
Mesodermal markers (endothelial)
ARCEDI Marker Combination
26
cbNIPT Procedure
Pregnant
women
GA: 10 to
13 weeks
Blood
Sampling
(30mL of
whole
blood)
cbNIPT Procedure
Selection
and
Staining
using
ARCEDI
markers
cbNIPT Procedure
Pregnant
women
GA: 10 to
13 weeks
Blood
Sampling
(30mL of
whole
blood)
Scanning and
Identification of
Fetal Cells
cbNIPT Procedure
Selection
and
Staining
using
ARCEDI
markers
Pregnant
women
GA: 10 to
13 weeks
Blood
Sampling
(30mL of
whole
blood)
Positively
Identified
Fetal Cell
cbNIPT Procedure
Scanning and
Identification of
Fetal Cells
Selection
and
Staining
using
ARCEDI
markers
Pregnant
women
GA: 10 to
13 weeks
Blood
Sampling
(30mL of
whole
blood)
Approx no. of maternal cells
(enrichment factor)
30 ml whole blood
retrieved from a
pregnant woman (GA 11-
13)
“Blood processing”
– fixation followed by RBC lysis
After “magnetic
cell separation,
staining and
smearing”
After “Scanning”
After “Visual
inspection”
0
32
1.5 x 108 120,000 200
Example of fetal cell found with ARCEDI marker set
33
Example of fetal cell found with ARCEDI marker set
34
Approx no. of maternal cells
(enrichment factor)
30 ml whole blood
retrieved from a
pregnant woman (GA 11-
13)
“Blood processing”
– fixation followed by RBC lysis
After “magnetic
cell separation,
staining and
smearing”
After “Scanning”
After “Visual
inspection”
1.5 x 108 120,000 200 0
35
143 cells from blood samples drawn from women
carrying male fetuses were scored as fetal and
subsequently FISHed with probes specific for X and
Y chromosomes
Specificity of ARCEDI marker combination
(male pregnancies)
XY: 116
XX: 0
No FISH signal 19
Lost 8
36
30 ml whole blood
retrieved from a
pregnant woman (GA 11-
13)
Blood processing
fixation followed by RBC lysis
Magnetic cell
separation, staining
and smearing
Smearing and Drying the cells on
slides
Mounting and
Scanning
Visual inspection
ARCEDI Workflow
0 2 5 12 12.511
Processing time in hours (continuous)
37
38
No. samples 37
No. fetal cells 487
Mean 13.2 fetal cells/30 ml
Range 2-45 fetal cells/30 ml
Number of fetal cells using ARCEDI technology
(male and female pregnancies/30 ml blood each
Fetal Cell Distribution
39
Positively
Identified
Fetal Cell
cbNIPT Procedure
Scanning and
Identification of
Fetal Cells
Selection
and
Staining
using
ARCEDI
markers
Pregnant
women
GA: 10 to
13 weeks
Blood
Sampling
(30mL of
whole
blood)
Pregnant
women
GA: 10 to
13 weeks
Blood
Sampling
(30mL of
whole
blood)
Selection
and Staining
using
ARCEDI
markers
Scanning and
Identification of Fetal
Cells
Positively
Identified Fetal
Cell
‘Picking’ the
Fetal CellWhole Genome
Amplification
(WGA)
cbNIPT
using
CMA/NGS
cbNIPT Procedure
Whole Genome Amplification
• Arthur L. Beaudet, M.D. Professor of Molecular and Cellular Biology
Professor of Molecular and Human Genetics Baylor College of Medicine
42
Picking of Fetal Cells using
ALS CellCelector RareCyte CytePicker
43
* Loss of X
* Gain of Y
Gender Result: Male
* Loss of X
* Gain of Y
Performed at Baylor College of Medicine
CASE1: Normal Male Pregnancy
44
X chromosome plot
Y chromosome plot
Moving window is
at 20 Mb
CASE1: Normal Male Pregnancy
45
Performed at Baylor College of Medicine
CASE2: Trisomy21 Female Pregnancy
Gender Result: FemalePerformed at Baylor College of Medicine
Cell 337 Cell 450
CASE2: Trisomy21 Female Pregnancy
CHR21
Gender Result: FemalePerformed at Baylor College of Medicine
CHR21
Most of q arm called
AR HR33 cells 337&450 (T21 female) vs. NL male ctrl
Gain of 21 and X
Loss of Y
CASE2: Trisomy21 Male Pregnancy
Gender Result: MalePerformed at Baylor College of Medicine
Cell 447 Cell 478
CASE3: Trisomy21 Male Pregnancy
Gender Result: MalePerformed at Baylor College of Medicine
AR HR15 cells 447&478 (T21 male) vs. NL female ctrl slide 252206053662_3
Most of q arm called
Gain of 21 and Y
Enroll 100 women pregnant in week 10–13 coming for CVS
due to an increased risk of trisomy 21
Blood sample before invasive procedure and isolation of fetal
cells
Isolation of white blood cell DNA (maternal)
Isolation of DNA from a small sample of the CVS (fetal)
Pick single fetal and maternal cells from the slides followed by
WGA of both
Perform array CGH on both the WGA amplified and genomic
DNA, and compare the profiles
Validation study: in progress
50
ARCEDI Technology.
• Offers high specificity marker combination for
isolation and identification of fetal endovascular
trophoblasts
• Fast enrichment of fetal cells from every blood
sample collected from pregnant women in
gestation week 10-12
• Opens possibilities for subsequent genetic analysis
on circulating fetal cells using aCGH, NGS etc.
Conclusions
51
Niels Uldbjerg
Ida Vogel
Acknowledgement
Arthur L. Beaudet
Amy Breman
Elizabeth Normand
Sadeem Qdaisat
Ignatia Van den Veyver
Palle Schelde
Ripudaman Singh
Lotte Hatt
Peter Schelde Hoey
Simon Taby Arrey
52
ALL THE PREGNANT WOMEN FOR PROVIDING BLOOD SAMPLES