Emerging technology: How the embryology lab is helping tovalidate an innovative approach to NIPT using Circulating

Fetal Nucleated Cells (CFNCs)

Gina Davis, MS, LCGCPacGenomics

28222 Agoura Road, Suite 200/201Agoura Hills, CA 91301

www.PacGenomics.com

Conflict of interest disclosure

I am an employee of PacGenomics Clinical Genetics Laboratory

Prenatal testing

Rowley, P. T., Loader, S. & Kaplan, R. M. Am. J. Hum. Genet. 63, 1160–1174(1998).

Toward the “holy grail” of prenatal testing

1970s

Invasive testingCVS and amniocentesis

2011

Noninvasive ScreeningCell-Free DNA (cfDNA)

2016?

Noninvasive ScreeningCirculating Fetal

Nucleated Cells (CFNCs)

Chorionic Villus Sampling (CVS)

Noninvasive TestsNo Risks to the Fetus

Invasive TestsRisks of Miscarriage

Amniocentesis Circulating Fetal Nucleated Cells

Cell-free DNA

SAMPLE

COVERAGE

Fetal cells

ACCURACY

TIMING

PlacentalTissues

Mixtures of Fetal and

Maternal DNAFetal Cells

High High T13, T18, T21, sexand more

High

High High High High

16 weeks 10 weeks >9 weeks>7 weeks

(potential to be > 5 weeks)

History of Prenatal Testing

History of risk modification by screening

90-94% detection

rate;

3-5% false positive

rate

Sequential Screen

80-85% detection

rate;

5% false positive

rate

1st Trim Combined

70% detection

rate;

5% false positive

rate

Triple Screen

43% detection

rate;

5% false positive

rate

AFP

25% detection

rate;

9% false positive

rate

Maternal Age

*Detection rate and false positive information for Down syndrome screening alone

Problems:1. Risk associated with

the invasive procedures

2. Psychological impact

on patients

3. A late timing that

makes intervention

difficult

The gold standard is to obtain fetal cells in

which the fetal genomic

DNA remains intact.

Diagnosis via invasive procedures

Amniocentesis

(>16 week GA)

Chorionic Villus Sampling (CVS)

(10 and 13 WGA)

Cordocentesis

Screening via non-invasive procedures

Combined test: Nuchal translucency (NT) + PAPPA + hCG

Quadruple test: AFP, hCG, Estriol, and Inhibin-A

Limitation: high false-positive rate

cfDNA-Based Non-Invasive Prenatal Testing (NIPT)

(as early as 9-10 WGA)

Limitation: Limited to common

aneuploidies & small panel of microdeletions/

increasing false positive rate with additional tests

An idealized solution is to develop Non-Invasive Prenatal

Diagnostics (NIPD) capable of

isolation and analysis of fetal cells in peripheral blood

Today – Prenatal Diagnosis vs. Screening

New genomic innovations at a

crossroads in clinical practice

Noninvasive prenatal screening

becoming ever more sensitive

and specific for select

aneuploidies

Microarray use during prenatal

diagnosis becoming ever more

sensitive and useful for a more

comprehensive genetic risk

assessment

Nomenclature: an ongoing discussion

NIPT: NonInvasive Prenatal Testing

NIPS: NonInvasive Prenatal Screening

NIPD: NonInvasive Prenatal Diagnosis

cffDNA: Cell Free Fetal DNA

NIDS: NonInvasive DNA Screening

Prevalence of chromosome

anomalies 16 population based registries in 11 European countries

Anomalies diagnosed prenatally or before 1 year of age,

delivered between 2000 and 2006

Included live births, fetal deaths from 20 weeks of gestation,

and terminations of pregnancy for fetal anomaly.

10,323 cases with a chromosome abnormality, out of

2,354,668 pregnancies, for total birth prevalence of

43.8/10,000 births.

Prevalence of chromosome

abnormalities

Adapted from Wellesley, D. et al. Rare chromosome abnormalities, prevalence and

prenatal diagnosis rates from population-based congenital anomaly registers in

Europe. Eur J Hum Genet 20, 521–526 (2012).

Breakdown of “Other Rare”

Chromosome Abnormalities

Clinically significant dels/dups 4282 women with prenatal diagnostic samples suitable for

simultaneous microarray and karyotype analysis

Overall, a total of 96 of the 3822 fetal samples with normal

karyotypes (2.5%) had a microdeletion or duplication of

clinical significance.

Clinically significant CNV by indication:

Advanced maternal age (1.7%)

Positive aneuploidy screening test (1.6%)

Ultrasound anomaly (6.0%)

Wapner, R. J. et al. Chromosomal Microarray versus Karyotyping for Prenatal

Diagnosis. New England Journal of Medicine 367, 2175–2184 (2012).

Classification of CNVs 1399 of 3822 karyotypically normal pregnancies had CNV

1234 (88.2%) classified as common benign

35 (2.5%) predetermined pathogenic CNV

130 (9.2%) went to clinical geneticist for classification

36 (2.6%) considered likely benign

94 (6.7%) considered VUS

61/94 considered by Clinical Advisory Committee to have sufficient

clinical relevance to be reported as pathogenic—4.3% of total CNVs

33/94 considered by Clinical Advisory Committee to have insufficient

clinical relevance, not reported as pathogenic—2.4% of total CNVs

Classification of CNVs 1399 of 3822 karyotypically normal pregnancies had CNV

1234 (88.2%) classified as common benign

35 (2.5%) predetermined pathogenic CNV

130 (9.2%) went to clinical geneticist for classification

36 (2.6%) considered likely benign

94 (6.7%) considered VUS

61/94 considered by Clinical Advisory Committee to have sufficient clinical relevance to be reported as pathogenic—4.3% of total CNVs

33/94 considered by Clinical Advisory Committee to have insufficient clinical relevance, not reported as pathogenic—2.4% of total CNVs

Classification of CNVs

Toward more comprehensive

prenatal diagnosis

“Based on the increased detection of clinically relevant

abnormalities in both structurally normal and abnormal

pregnancies, chromosomal microarray analysis (CMA) should

be transitioned to become the first tier test for invasive prenatal

cytogenetic diagnosis.”

Wapner, et al. 2012

“In children with unexplained intellectual

disabilities, autism, or congenital anomalies, 15-20%

with have an abnormality detected by CMA,

compared to ~3% with karyotype”

Miller, D. T. et al. Consensus statement: chromosomal microarray is a first-tier

clinical diagnostic test for individuals with developmental disabilities or congenital

anomalies. Am. J. Hum. Genet. 86, 749–764 (2010).

Meanwhile,

cfDNA becomes reality

1997

2008

2011

Look Back the History of cfDNA-based NIPT

(14 Years – from Discovery to Commercial Products)

Circulating Cell Free Fetal DNA cfDNA represents ~10-15% of total DNA in maternal plasma (Lo

1997, Chiu 2011)

Much higher percentage than intact fetal cells (estimated at 1/billion)

cfDNA made up of short (<200 bp) DNA fragments (Chan, 2004)

Reliably detected after 7 wks gestation (Birch, 2005)

Higher concentrations late in gestation

Short half life (16 min), undetectable by 2 hrs postpartum (Lo, 1999)

Various methods used for

aneuploidy calls with cfDNA

Quantitative method using targeted sequencing or massively

parallel sequencing, followed by various methods of analysis

identifying whether the ratio of fetal DNA on involved

chromosomes is at the expected (disomic) level

SNP-based method using modeled parental alleles and

crossover frequency to generate risk scores

Bianchi, D. W. From prenatal genomic diagnosis to fetal personalized medicine:

progress and challenges. Nature Medicine 18, 1041–1051 (2012).

Meta Analysis of cfDNA screening

Trisomy 21 Trisomy 18 Trisomy 13Sex chromosome

aneuploidies

Unaffected 21608 21608 21608 21608

Affected 1051 389 139 233

Detection Rate 99.20% 96.30% 91.00% 90% 45,X/93% other

False Positive Rate 0.09% 0.13% 0.13% 0.37%

Adapted from Gil, M. M., Quezada, M. S., Revello, R., Akolekar, R. &

Nicolaides, K. H. Analysis of cell-free DNA in maternal blood in screening for

fetal aneuploidies: updated meta-analysis. Ultrasound Obstet Gynecol 45, 249–

266 (2015).

Microdeletion panels in cfDNA

Majority of deletions were modeled cell lines from few individuals rather than unique deletions identified in a prenatal population

They modeled the system beautifully, but time will tell if this is generalizable in terms of detection

PPV still very modest (most at ~5%) since there were a significant number of false positives (primarily in the prenatal cell lines, as opposed to the modeled deletion samples), and because of the low prevalence of the conditions in the population

Wapner, R. J. et al. Expanding the scope of noninvasive

prenatal testing: detection of fetal microdeletion

syndromes. American Journal of Obstetrics & Gynecology

212, 332.e1–332.e9 (2015).

Limitations of cfDNA Accuracy seems to depend on proportion of extracted DNA

derived from fetus

Results are based on (powerful) statistical methods rather

than direct biological measurements

Unable to determine mosaicism if present

Degree of extension of analyses beyond aneuploidy yet to be

determined and validated

Additional conditions beyond aneuploidy will raise false

positive rate

cfDNA vs. sequential screening

cfDNA vs. sequential screening

The rate of "other" abnormalities detected with sequential

screening is 53.8%, which ranges from 24.2% for

Robertsonian translocations to 91.0% for triploidy

False-positive rates were 4.5% for sequential screening,

5.1% for cfDNA if no-result cases were considered high risk

and referred for follow-up, and 1.0% for cfDNA if no-results

cases received no follow-up

Differing opinions “Overall, when considering all chromosome abnormalities and including

cases with no test result, sequential screening has better test performance than cfDNA. . . Disorders other than trisomy 21 are important considerations in prenatal testing. . . Consideration of all outcomes is important.”

-Mary Norton, MD, University of California, San Francisco

“At this point, we don't see an advantage to the universal use of cfDNA. ACOG recommends it be used only in high-risk patients, not the vast majority of women; this is what we do in our practice.”

-Vincenzo Berghella, MD, Thomas Jefferson University, president of the SMFM

“For prenatal testing, cfDNA has 99.0% accuracy for detecting Down syndrome and 98.9% accuracy for detecting trisomy 13. And patients do not want a risk score, they want a yes or no result.”

-Laxmi Baxi, MD, New York University Langone Medical Center and Columbia University

Innovations in genomic

technologies at crossroads

Test Detects #Abnormals

cfDNA T21, T18, T13 1/272

plus sex chrom abnl

Invasive testing T21, T18, T13, sex chr. 1/60

With CMA plus other abnl

>500 kb in size

Microarray vs. cfDNAIF

aCGH detects an abnormality in 1.7% of cases (about 1/60 pregnancies)

AND

cfDNA detects T13,18, 21 plus sex chromosome abnormalities (about 1/272 pregnancies)

THEN

If cfDNA is the routine screening test, it will detect only about 22% of diagnosable chromosomal abnormalities

Isolation of fetal cells for noninvasive

access to full fetal genome

Circulating fetal

nucleated cells

Placental

trophoblasts

Fetal nucleated red

blood cells (fNRBCs)Leukocytes

Mesenchymal

stem cells

? ?

Isolation of circulating fetal nucleated cells

Feto-maternal cellular trafficking is the

bidirectional passage of cells that results in the

presence of CFNCs in maternal circulation.

Several types of CFNCs e.g., placental

trophoblasts, fetal nucleated red blood cells

(fNRBCs), leukocytes, and mesenchymal stem

cells, are found to traverse the placental barrier

into maternal circulation, offering promising

targets for implementing CFNC-based NIPD.

Expectations: Early timing (5 WGA) // Minimum Risk

Cell Type Properties Challenges

Placental

trophoblasts

(1-3 per mL)

• No need to cross

placenta

• Early gestation (4W)

• Mosaicism

• No established surface markers

for separation

Fetal

nucleated red

blood cells

(fNRBCs)

(1-3 per mL)

• Transient, ONLY from

current pregnancy (6W)

• Reflect fetal genome

• Established surface markers

for separation and

identification (e.g., CD71,

CD147 and g-Hg)

• Fragility

Leukocytes

(? per mL)

• Long life time • Persistence

• Carry over from previous

pregnancy

• No established surface markers

for separation

Mesenchymal

stem cells

(1-3 per mL)

• Potential for

engraftment

• Persistence

• No established surface markers

for separation

Different Types of Circulating Fetal Nucleated Cells

History of CFNC-Based NIPT/NIPD – 46 Years of Frustration

1119

. TABLE IV-PREGNANCIES IN PATIENTS TAKING CHLORMADINONE

ACETATE, 0-5 mg. DAILY, ACCORDING TO DURATION OF TREATMENT

a failure-rate 2 means two pregnancies in 1200 cycles. In

this trial the failure-rate based on use-effectiveness is 9-5,

or, if the pregnancies occurring in women who recordederrors in tablet-taking are omitted, 6-5. This failure-

rate is in excess of that of the intrauterine device (2-4) andsimilar to, or in excess of, that of condom or cap used in

conjunction with a spermicidal jelly or cream.Have our results any deficiencies which may have led

to an unjustifiably pessimistic view of chlormadinone ?The numbers of participants and total cycles are com-

paratively small, for it is accepted that for 90% confidencein a pregnancy-rate 4500 cycles must be completed-butthe failure-rate would not fall below 3 even if no further

pregnancies occurred before that number was reached.Another possible defect in the design of the trial is that

day-marked tablet packs, probably the best aid to regular

dosage, were not available when the investigation was

started. However, care exercised in compiling the diary-cards was impressive. Patients understood the import-ance of a daily tablet taken at 24-hour intervals, and wehave no reason to disbelieve women who stated that

tablets had been taken precisely according to instructions.This view is supported by the findings of Elstein et al.

(1969) in a series closely comparable in numbers of

patients and cycles in which a similar number of preg-nancies occurred despite the use of day-marked packs.Another disturbing feature is that pregnancies seemed

to be more common with increasing duration of treatment:two pregnancies occurred in the first 3 months of treat-

ment, four between the 4th and 6th months, three

between the 7th and 9th months, and three more between

the 10th and 12th months despite increasing accuracy oftablet taking, and a decrease in the number of women

completing each successive 3 months of treatment

(table iv). The margin for error seems to be small; if onetablet is taken late, missed, or not absorbed because of a

gastrointestinal upset, pregnancy may result.

Against this apparent risk of pregnancy must be set theevidence which is gradually accumulating to suggest thatmetabolic disturbances and thrombotic incidents are

less common than with conventional pills.This trial is continuing, but participants have been told

that the pregnancy-rate seems to be greater than originallyanticipated, and that in view of this they may wish towithdraw. Since women who use contraceptives and

become pregnant are increasingly requesting termination,it is important to ensure that only those prepared to acceptanother pregnancy should continue to use a method which

on present evidence may be less efficient than others. In

the light of this experience chlormadinone acetate, in a

daily dose of 0-5 mg., should be restricted to women forwhom a pregnancy earlier than intended will not come

amiss, particularly if they are highly fertile.

We thank F.P.A. doctors participating in this trial for under-

taking the clinical care of these patients, and Mrs. Ursula Watkinsand Mrs. Waltraud Bounds for technical assistance. ImperialChemical Industries, Pharmaceutical Division supplied the chlorma-dinone tablets. This work was supported by a grant from the OliverBird Trust and was supervised by Dr. G. 1. M. Swyer, chairman,clinical trials committee, Council for the Investigation of FertilityControl.

Requests for reprints should be sent to C. B., Margaret PykeHouse, 27-35 Mortimer Street, London WIN 8BQ.

REPERENCES

Elstein, M. (1969) Personal communication.&mdash; Howard, G. M., Blair, M. J. (1969) Personal communication.

Poller, L., Thomson, J. M., Tabiowo, A., Priest, C. M. (1969) Br. med. J.i, 554.

Rudel, H. W., Martinez-Manautou, J., Maqueo-Topete, M. (1965) Fert.Steril. 16, 158.

PRACTICAL AND THEORETICAL

IMPLICATIONS OF FETAL/MATERNALLYMPHOCYTE TRANSFER

JANINA WALKNOWSKA FELIX A. CONTE

MELVIN M. GRUMBACH

FROM THE DEPARTMENT OF PEDIATRICS,

UNIVERSITY OF CALIFORNIA, SAN FRANCISCO MEDICAL CENTER,

SAN FRANCISCO, CALIFORNIA 94122

Summary Lymphocyte cultures were prepared from

peripheral-blood samples taken from thirtypregnant women. In twenty-one cultures one or more

euploid metaphase figures with 5 small acrocentric

chromosomes interpreted as 46/" XY " were found.

Nineteen of the twenty-one women gave birth to male

infants, and two gave birth to females (P=0&middot;0001). Thetwo "false-positive " results were in women in whom

only 1 cell with 5 small acrocentric chromosomes had been

found, whereas all ten patients in whom 2 or more cellswere found gave birth to males. Artefact or chim&aelig;rism for

fetal 46/XY cells persisting from an earlier pregnancy mayaccount for the two false-positive results. In nine other

women, no "

XY "

cells were found. Six gave birth to

female infants while three gave birth to males. Cells with

46/XY karyotype in the maternal circulation were detectedas early as the 14th week of gestation (the earliest agestudied). The data suggest that the fetomaternal transferof lymphocytes is common, happens at least as early as the14th week of gestation, and may be a consequence of

transplacental migration of circulating fetal lymphoid cells,as well as leakage of blood. The antenatal diagnosis ofa male fetus can be made by karyotypic analysis of

lymphocytes in maternal blood. Similarly, it should be

possible to identify fetal chromosome abnormalities bythis procedure. Transfer of fetal lymphocytes to the

mother may play a part in the acceptance of the fetus asa homograft.

Introduction

FETAL sex has been assessed by examination of the

sex-chromatin pattern and by karyotypic analysis of cellsin the amniotic fluid (Makowski et al. 1956, Shettles 1956,Riis and Fuchs 1960, Serr and Margolis 1964, Amaroseet al. 1966, Steele and Breg 1966).

While investigating the cytogenetic features of circu-

lating lymphocytes of pregnant women, we noted a smallnumber of euploid cells which contained 5 small acro-centric chromosomes. The karyotype of these cells wasconsistent with an XY, rather than the expected XX

LANCET, June 7th, 1969

The antenatal diagnosis of a male fetus can be made by karyotypic

analysis of lymphocytes in maternal blood. Similarly, it should be

possible to identify fetal chromosomal abnormalities by this procedure.

1120

sex-chromosome complement. This finding suggested the

development of chimaerism, transient or persistent, in

pregnant women as a result of fetomaternal passage of cells

capable of responding to phytohasmagglutinin (P.H.A.).We have extended these observations, and correlated our

findings with the sex of the fetus.

Methods

Samples of peripheral venous blood were obtained from

thirty healthy pregnant women attending the obstetric clinic ofthe University of California, San Francisco Medical Center.The study gioup included twenty primiparae and ten multipartbetween 14 and 37 weeks of gestation.

10 ml. of blood was drawn from a peripheral vein into a

heparinised syringe and allowed to sediment for 30 minutes.The leucocyte-rich plasma was transferred to a sterile tube and

centrifuged at 500 r.p.m. for 5 minutes. The supernatant

plasma was discarded and the leucocyte button was resuspended

PREVALENCE OF 46/XY METAPHASE FIGURES IN THE BLOOD OF PREGNANTWOMEN AND ITS CORRELATION TO WEEKS OF GESTATION, GRAVIDITY,

AND THE SEX OF THE FETUS

* 2 patients with 1 metaphase figure which contained 5 small acrocentricchromosomes who gave birth to female infants.

t 3 patients with no detected XY metaphase figures who gave birth to males.

in 3 ml. of Eagle’s minimum essential media (M.E.M.). The

leucocytes were counted and added to 10 ml. of media in a finalconcentration of 106 cells per ml. The culture media contained

8 ml. M.E.M., 1-5 ml. fetal calf serum, 0-15 ml. (0-3 mM)L-glutamine, 0- 15 ml. P.H.A. M., 2000 units potassium penicillinG, and 2 mg. streptomycin sulphate. After 70 hours at 37&deg;C,0.5 ml. (20 fLg.) of colchicine was added. At 72 hours the

cultures were harvested, treated with hypotonic solution, fixed,and stained with Giemsa. 10-15 slides were prepared fromeach blood-sample. Each metaphase figure was examined; onlyintact, well-spread euploid metaphase plates were scored.

Those cells with 5 small acrocentric chromosomes were

karyotyped.Mixed leucocyte cultures utilising equal proportions of

maternal and fetal leucocytes were set up by Hirschhorn’s

(1968) method. The number of mitotic figures per 1000 cellswas determined.

Results

Chromosome analysis (see table) of lymphocytes from

Fig. 1-Karyotype of 46/XY cell of mother subsequently deliveredof a male infant.

The 5 small acrocentric chromosomes are pairs 21 and 22 and theY chromosome.

the venous blood in twenty-one of the thirty pregnantwomen showed one or more metaphase figures with 46chromosomes which included 5 small acrocentrics; thesecells were interpreted as having a 46/XY karyotype (fig. 1).Nineteen of the twenty-one women in whom cells with

5 small acrocentric chromosomes were found gave birth to

male infants (P=0-0001); the other two gave birth to

female infants. There was no evidence of an XY sex-

chromosome constitution in the metaphase cells of the

remaining nine women, of whom six delivered female

infants and three male infants (not significant).Fig. 2 shows the percentage of metaphase figures with

an XY sex-chromosome constitution in relationship to the

length of gestation in the nineteen women harbouringthese cells. From the 14th week (the earliest stage studied)to mid-gestation, 0- 14-1-5% (mean 0-6%) of the total cellsin metaphase contained an XY sex-chromosome consti-

tution ; in the second half the percentage of fetal cells was

0-2-0-8% (mean 0-4%).Four mixed-lymphocyte cultures of maternal and

umbilical-vein blood lymphocytes in the absence of P.H.A.

WEEKS Of GESTATION

Fig. 2-% of 46/XY metaphase figures found in the blood of pregnawomen who delivered male infants in relation to week of gestatio

Confirmation in 1979

A circulating fetal cell isolated by FACS from

maternal blood. Arrow indicates Y-chromatin.

Disappointment in 2002

Thus technological advances are needed before fetal

cell analysis has a clinical application…

Many People Tried… but Failed to Create a Reliable Solution

August 21st, 2013

Challenges to progress An acknowledged rarity of intact fetal cells in maternal circulation

The fragility of target cells that makes delays between blood draw and analysis difficult

The relatively low efficiency of enrichment methods leading to loss of fetal samples, sample to sample

The persistence of white cells from prior pregnancies in maternal circulation for considerable periods of time

The difficulty of identifying markers that are sufficiently sensitive and distinct/differentially expressed to provide a pointer to a fetal cell consistently

The DNA amplification of a fixed cell

CFNC-Based NIPD Is Implemented via Two Steps

Blood Draw

10-30 mL blood

Gradient

centrifuge

immunomagnetic

separation

Single CFNC

picking

1. Enrichment / purification

Single-CFNC

WGA & QC

CGH Microarray

8x60K, Agilent

2. Whole Genome Amplification+ Genotyping

Next

Generation

Sequencing

1-3 CFNC

per billion

blood cells

Single-cell array CGH has been

used to conduct pre-implantation genetic diagnosis

(PGD) in IVF clinicS since 2012

Single-cell

WGA & QC

CGH Microarray

8x60K, Agilent

The unmet need is to exploit the use of

rare-cell sorting technologies to enrich/isolate pure CFNCs

with intact genomic DNA

A brief overviewon

the major players who have been working on the development of Non-Invasive Prenatal Diagnostics (NIPD)

based on circulating fetal nucleated cells (CFNCs)

Promising NIPD Technology-ONE

Arthur Beaudet, M.D.Baylor College of Medicine

Baylor Miraca Genetics Laboratories

Steen Kølvraa, M.D.

ARCEDI Biotech, Denmark

Blood Draw

30-mL blood

1st Enrichment

by gradient

centrifuge

immunomagnetic

separation using

anti-HLA-G

Single CFNCs

picking on glass

slides

Single-CFNC

WGA & QC

CGH Microarray

8x60K, Agilent

Required time for CFNC isolation = 12 h

** 1-3 Extravillous trophoblasts (EVTs) per 1-mL blood are found by this approach.

1.5 x 108

cells

120,000 cells 200 cells 3-10 cells

Extravillous trophoblasts (EVTs)

are defined as (HLA-G+/CK7+/CD45-)

Throughput = 6 samples / day

Promising NIPD Technology-TWO

Arthur Beaudet, M.D.Baylor College of Medicine

Baylor Miraca Genetics Laboratories

Blood Draw

15-mL blood

1st Enrichment

by AccuCyte®

System

PBMCs

smeared on

30 glass slides

Single CFNCs

picking by

CytePicker®

Single-CFNC

WGA & QC

CGH Microarray

8x60K, Agilent

Required time for CFNC isolation = 12 h

** 1-3 Extravillous trophoblasts (EVTs) per 1-mL blood are found by this approach.

Extravillous trophoblasts (EVTs)

are defined as (CK7+/CD45-)

Throughput = 2 samples / day

Promising NIPD

Technology-THREE

Brynn Levy, M.Sc.(Med)., Ph.D.Columbia University

Fetal Nucleated Red

Blood Cells (fNRBCs)

are defined as

events

(4B9+/Epsilon+)

Blood Draw

30-mL blood

1st Enrichment

by gradient

centrifuge

Flow cytometry

separation

(4B9+/Epsilon+)

Single CFNCs

picking on glass

slides

Single-CFNC

WGA & QC

CGH Microarray

8x60K, Agilent

** 1-3 Fetal Nucleated Red Blood Cells (fNRBCs) per 1-mL blood are found by this approach.

Toward a novel approach in

fetal cell isolation One in a billion?

3-6 fetal cells/mL whole maternal blood1

Amplification of the genome of one or a few putative fetal

cells has been problematic for researchers

Multidisciplinary team with expertise in rare cell

isolation and clinical genetic analysis of single cells

needed

1. Emad, A. et al. Validation of automatic scanning of microscope slides in

recovering rare cellular events: application for detection of fetal cells in maternal

blood. Prenat. Diagn. 34, 538–546 (2014).

From cancer cells to fetal cells

Circulating Tumor Cells Primary

tumor

Cell Trafficking Circulating Tumor Cells (CTCs) as surrogate tumor biopsytoward cancer companion diagnostics

Circulating Fetal Cells

Circulating Fetal Nucleated Cells (CFNCs)toward non invasive prenatal diagnostics(NIPD)

Nanostructures-Embedded Microchips for Detection,

Isolation, and Characterization of Circulating Tumor

Cells, Acc. Chem. Res. 2014, 47: 2941-2950.

Foundation of Our Rare-Cell Capture Technology:

“NanoVelcro Chips” Exhibit Enhanced Cell Affinity

Working mechanismof Velcro straps

Harnessing Velcro-like interactions to achieve unprecedented cell-capture efficiency

Nanostructured cell surface components

Matching nanostructures on cell-affinity substrates

Enhanced Topographic Interactions

“Three-Dimensional Nanostructured Substrates toward Efficient Capture of Circulating Tumor Cells.” Angew. Chem. Int. Ed. 2009, 48, 8970-8973.

NanoVelcro Chip

NanoVelcro Substrates Can Be Fabricated byManufactory Processes Developed for Semiconductor Industry

Capture antibodies confersspecificity to silicon nanowire substrates

“Three-Dimensional Nanostructured Substrates toward Efficient Capture of Circulating Tumor Cells.” Angew. Chem. Int. Ed. 2009, 48, 8970-8973.

fNRBC

Trophoblast

CFNC Capture in Action (2 h per Sample)

Streptavidin

Capture antibody:fNRBC(CD-71)Trophoblast(HLA-G)

Device assembly

CFNC capture

Immunostaining

Imaging on a microscope

Capture agent coating30 min

10 min

10 min

30 min

CFNC identification

20 min

10 min

PDMS-based chaotic mixer

PatternedSiNW substrate

50

1st-Enrichemnt

3-layer centrifuge

(1 h)

General Operation Protocol

2nd-Enrichment

on NanoVelcro Chips

(1.5 h)

Single-fNRBC

isolation,

followed by analysis

(15 min + time)

Mononuclear

Cells

fNRBCs

Granulocytes

Mature

RBC’s

Plasma

c)

PLGA NanoVelcro

substrate on a commercial

LMD slide

PDMS component

with embedded

herringbone

micropatterns

Upper piece

Lower piece

Click-on unit

Syringe

Reservoirs

Rotary

valve

Blood

inlet

Waste

Pathologist evaluation

Immunostainingmicroscope imaging

CFNC isolation workflow

Isolation of captured CFNCs by LCM

iii) Confirmation of

nucleus iii) iv)ii)i) v)

54

CFNC isolation workflow and timeline

Blood Draw

& Shipment

(<24 h)

Bulk

Enrichment and

Banking (30 min)

CFC Capture

in NanoVelcro

Chips (60 min)

Single CFCs

Isolation by LCM

(30 min)

Single-CFC

WGA & QC (STR)

(3 h)

CGH Microarray

8x60K, Agilent

(5 h)

Turn-around time < 48 h (after sample arrival)Fetolumina

shipment

solution

** 10-20 CFCs (per 2mL blood)

are captured by Fetolumina’s

NanoVelcro Chip

Banked

blood

samples offer

a reliable

fallback plan

Pilot study

CFNCs isolated for genetic analysis (FISH, aCGH and

DNA fingerprinting)

Maternal DNA isolated for DNA

fingerprinting

Paternal DNA/PGS amplified DNA for DNA fingerprinting

20 maternal blood

samples from

IVF/PGS pregnancies

(8-14 weeks GA)

8 commercially obtained

maternal blood samples for

which the pregnancies had

a known abnormal fetal

karyotype

paternal blood

samples and

amplified DNA from

blastocyst biopsies

+

Results Sufficient capture of CFNCs was obtained for each expectant

mother

FISH studies confirmed the gender in the CFNC samples tested

Microarray studies (Agilent 8X60K oligo array) performed on all the CFNC samples were concordant to those obtained in the PGS studies, and demonstrated excellent quality control metrics

Genomic fingerprinting of STR loci on both the CFNC samples and corresponding WBCs from paternal and maternal blood confirmed the biological parental-fetal relationship

Genomic fingerprinting on amplified DNA from PGS samples confirmed that embryo transferred matched result from CFNC sample

Male-110 (XY), normal, DLRSD = 0.47

Female-97 (XX), normal, DLRSD = 0.35

Detecting Fetal Genders

DLRSD: Derivative Log Ratio Standard Deviation

Non-Invasive Prenatal Diagnosis of Trisomy 21 (14-20

WGA)

Non-Invasive Prenatal Diagnosis of Trisomy 13 and 18

Two Unique Samples

#7 47, XYY, +18

#9, XXXXY

Detecting Relatively Short Deletion and Duplication

Whole Genome Sequencing, FISH, and Microarray Data

Male (XY) / Normal

Male (XY) / T18

Whole Genome Sequencing Data of Trisomy Samples

Male (XYY) / T18

MB96_CFC-NIPD

MB96_Maternal Blood

MB96_Paternal BloodD

21

S1

1 (2

8)

TH

01

(7)

X vW

A(1

6)

TP

OX

(8)

D5

S8

18

(12

)

D1

3S

31

7 (1

1)

D7

S8

20

(9)

Y D2

1S

11

(29

)

CS

F1

PO

(10

)

TH

01

(6)

D1

3S

31

7 (1

2)

D1

6S

53

9 (1

2)

D7

S8

20

(10

)

TP

OX

(10

)

D5

S8

18

(13

)

vW

A(1

7)

D1

6S

53

9 (1

3)

CS

F1

PO

(11

)

D2

1S

11

(28

)

TH

01

(9)

XX

TP

OX

(8)

D5

S8

18

(12

)

D1

3S

31

7 (1

1)

D7

S8

20

(12

)

D2

1S

11

(29

)

CS

F1

PO

(10

)

D1

3S

31

7 (9

)

D1

6S

53

9 (1

2)

D7

S8

20

(10

)

TP

OX

(10

)

vW

A(1

7)

D1

6S

53

9 (1

3)

CS

F1

PO

(12

)

D2

1S

11

(28

)

TH

01

(7)

X vW

A(1

6)

TP

OX

(8)

D5

S8

18

(12

)

D1

3S

31

7 (1

1)

D7

S8

20

(9)

Y D2

1S

11

(30

)

TH

01

(6)

D1

3S

31

7 (1

2)

D1

6S

53

9 (1

2) X

2

D7

S8

20

(12

)

TP

OX

(11

)

D5

S8

18

(13

)

vW

A(1

7)

CS

F1

PO

(11

)

2000 2500 3000 3500 4000 45001500 5000

40000

30000

20000

10000

0

Forensic Evidence

STR Fingerprints Confirming Fetal-Parental Relationship

MB86_CFC-

NIPD

MB86_Maternal

blood

MB86_Umbilical

Cord Tissue

D2

1S

11

(28

)

TH

01

(7)

XX

TP

OX

(8)

TP

OX

(9)

D5

S8

18

(12

)

D1

3S

31

7

(8)

CS

F1

PO

(12

) X2

D7

S8

20

(12

)

D1

6S

53

9 (1

2)

X2

D2

1S

11

(27

)

vW

A(1

7) X

2

TH

01

(9.3

)

D1

3S

31

7

(12

)

D5

S8

18

(9)

D7

S8

20

(11

)

D2

1S

11

(28

)

XX

TP

OX

(8)

TP

OX

(9)

D5

S8

18

(11

)

CS

F1

PO

(12

) X2

D7

S8

20

(12

)

D1

6S

53

9 (1

2)

D2

1S

11

(27

)

vW

A(1

7) X

2

TH

01

(9.3

) X2

D1

3S

31

7

(12

)

D5

S8

18

(9)

D7

S8

20

(10

)

D1

6S

53

9 (1

1)

D2

1S

11

(28

)

TH

01

(7)

XX

TP

OX

(8)

TP

OX

(9)

D5

S8

18

(12

)

D1

3S

31

7

(8)

CS

F1

PO

(12

) X2

D7

S8

20

(12

)

D1

6S

53

9 (1

2)

X2

D2

1S

11

(27

)

vW

A(1

7) X

2

TH

01

(9.3

)

D1

3S

31

7

(12

)

D5

S8

18

(9)

D7

S8

20

(11

)

40000

30000

20000

10000

0

Forensic Evidence –

STR Fingerprints Confirming Fetal Identity

2000 2500 3000 3500 4000 45001500 5000

Next steps Optimize workflow to enable high throughput isolation of

CFNCs

Enroll patients for clinical validation of findings in a high

risk population undergoing clinical prenatal diagnosis or

termination of pregnancy for known genetic abnormality

Assess clinical utility in larger population; assess for

integration into clinical practice

Scientific Research Team of PacGenomics Laboratory

Thank you!