Cell-Based Noninvasive Prenatal Diagnosis I: Recovery of...

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

10

20

30

40

50

60

70

80

90

100

0 40 80 120 160 200

Fetal signal (1)

Ma

tern

als

ign

al (

1)

April09(2)

0

10

20

30

40

50

60

70

80

90

100

0 40 80 120 160 200

Fetal signal (2)

Mate

rnal sig

nal (2

)

11

April09(1)

0

10

20

30

40

50

60

70

80

90

100

0 40 80 120 160 200

Fetal signal (1)

Ma

tern

als

ign

al (

1)

April09(2)

0

10

20

30

40

50

60

70

80

90

100

0 40 80 120 160 200

Fetal signal (2)

Mate

rnal sig

nal (2

)

12

April09(1)

0

10

20

30

40

50

60

70

80

90

100

0 40 80 120 160 200

Fetal signal (1)

Ma

tern

als

ign

al (

1)

April09(2)

0

10

20

30

40

50

60

70

80

90

100

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