For Research Use Only. Not for use in diagnostic procedures

For up-to-date licensing information and product-specific disclaimers, see the respective QIAGEN kit handbook or user manual.QIAGEN kit handbooks and user manuals are available at www.qiagen.com or preanalytix.com or can be requested from QIAGEN Technical Services or your local distributor.

Andrea Ullius1,2, Thorsten Voss1,2, Joachim Bonnet3, Wera Hofmann3, Markus Stumm4, Nadine Dettmann1,2, Katharina Pfaff1,2, Franziska Heese1,2, Daniel Grölz1,2

1 QIAGEN GmbH, Hilden, Germany; 2 PreAnalytiX GmbH, Hombrechtikon, Switzerland; 3 LifeCodexx AG, Konstanz, Germany; 4 Centre for Prenatal Diagnostics and Human Genetics, Berlin, Germany

BackgroundIntroduction: Circulating cell-free DNA (ccfDNA) has become an emerging tool in non-invasive prenatal testing (NIPT) and

in cancer diagnostics. PreAnalytiX has developed the PAXgene® Blood ccfDNA System, consisting of a blood collection tube,

which stabilizes whole blood during transport and storage, and the QIAsymphony® PAXgene Blood ccfDNA workflow, for

automated extraction of ccfDNA from plasma. Here, we demonstrate the performance of this system.

Methods: Whole blood was drawn into PAXgene Blood ccfDNA tubes, Streck Cell-Free DNA BCT® or spray-dried EDTA tubes

and stored for up to 10 days. Genomic DNA (gDNA) was purified from stored blood using the QIAamp® DNA Mini Kit and

analyzed by gel electrophoresis. ccfDNA was extracted from plasma using the QIAamp Circulating Nucleic Acid Kit or the

QIAsymphony PAXgene Blood ccfDNA protocol and the obtained ccfDNA was analyzed using an Agilent Bioanalyzer and

quantitative real-time PCR. Fetal ccfDNA from pregnant donors was analyzed by PrenaTest® (LifeCodexx AG).

Results: The PAXgene Blood ccfDNA stabilization reagent prevented cell lysis and release of gDNA and hemoglobin into

the plasma. The ccfDNA fraction in blood stored for up to 10 days in EDTA tubes showed a strong increase of 18S rDNA

fragments. By contrast, copy numbers of these fragments remained constant in PAXgene Blood ccfDNA stabilized blood.

An initial feasibility study of a small pregnant cohort revealed that the prototype PAXgene Blood ccfDNA tube performs

comparably with the Cell-Free DNA BCT in the NGS-based PrenaTest.

Conclusion: The PAXgene Blood ccfDNA tube enables whole blood stabilization needed for NIPT. The fully automated

QIAsymphony PAXgene Blood ccfDNA workflow is an efficient method for extraction of ccfDNA.

Handling Benefits of PAXgene Blood ccfDNA Tube• PAXgene Blood ccfDNA stabilization

reagent integrated into a BD plastic

Vacutainer® Tube.

• Plastic material mitigates risk of tube

breakage during transport and centrifugation.

• Safety BD Hemogard® closure helps

protecting laboratory personnel from

contact with blood.

• PAXgene Blood ccfDNA tube allows distinct

separation of plasma from buffy coat.

• PAXgene Blood ccfDNA tube and automated

QIAsymphony kit: Integrated collection-

stabilization-preparation (CSP) system.

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A New Blood Collection Tube and AutomatedExtraction Method for Stabilization and Analysisof ccfDNA

Trademarks: QIAGEN®, Sample to Insight®, QIAamp®, QIAsymphony® (QIAGEN Group); PAXgene® (PreAnalytiX GmbH), BD Hemogard™, Vacutainer® (Becton, Dickinson and Company), PrenaTest®, QuantYfeX® (LifeCodexx AG), Cell-Free DNA BCT® (Streck, Inc.). Registered names, trademarks, etc. used in this document, even when not specifically marked as such, are not to be considered unprotected by law. © 2015 QIAGEN, all rights reserved.

BCT with blood from pregnant women3 days after collection. Blood was transported from physician to testing laboratory andcentrifuged once. (Picture kindly provided by LifeCodexx.)

StreckBCT

PAXgeneBlood ccfDNA tube

• PAXgene Blood ccfDNA stabilization reagent prevents the hemolysis of erythrocytes.

• Blood stored in EDTA BCT shows a strong increment of DNA in plasma.

• Blood stabilized with PAXgene Blood ccfDNA stabilization reagent can be stored for 10 days at room temperature without

increase in ccfDNA levels.

Stabilization of Whole Blood and ccfDNA During Transport and Storage

Stabilization during transport and storage. Whole blood from 6 healthy donors was drawn in EDTA BCT. After draw, blood in EDTA tubes was eitherstored or directly transferred into tubes containing PAXgene Blood ccfDNA stabilization reagent (PAXgene). Stabilized blood was immediately processed (t0) or stored for up to 10 days at room temperature (t10) until plasma was generated. A Hemolysis was determined by measuring the absorption of freehemoglobin in plasma at 414 nm. Mean absorption and standard deviation of plasma from 8 donors is shown. B ccfDNA from 2.4 ml plasma was extracted using the PAXgene Blood ccfDNA QIAsymphony protocol and ccfDNA yield was quantified by real-time PCR (18S rDNA gene, 66 bp/500 bp amplicon).The ratio of copy numbers at day of storage (tx) to immediately processed plasma (t0) is shown.

A B

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t0 t1 t3 t6 t8 t10

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EDTA PAXgene

18S rDNA qPCR assay66 bp amplicon500 bp amplicon

t1 t3 t6 t8 t10 t1 t3 t6 t8 t10

• All specimens collected in prototype PAXgene Blood ccfDNA tubes fulfilled the quality criterion ‘fetal fraction >4%’.

• Absolute fetal ccfDNA yield and fetal-to-maternal DNA ratio were comparable with the current method.

• Fetal gender was concordant between collection tubes for all specimens.

• All 18 specimens stored in PAXgene Blood ccfDNA tubes then analyzed with NGS and the PrenaTest DAP.plus software were

concordant with the current method.

• All samples were negative for trisomies 13, 18 and 21.

NIPT From Stabilized Maternal Blood

Comparable performance between prototype PAXgene Blood ccfDNA tube and Streck Cell-Free DNA BCT. Whole blood specimens from pregnant women were collected into prototype PAXgene Blood ccfDNA tubes and Streck Cell-Free DNA BCT. Blood was transported to LifeCodexx and ccfDNA was extracted using the QIAamp Circulating Nucleic Acid Kit. A Circulating fetal DNA and fetal gender of 18 samples were determined using the QuantYfeX® assay. B Eluates were subjected to genomic library preparation and NGS (Illumina). With PrenaTest DAP.plus software analysis total reads of NGS wereanalyzed into percentage of mapped and unique mapped reads to determine chromosomal aberrations (n=18).

A B

30

25

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15

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5

0

ccfDNA, %

Fetal ccfDNA (QFX)

QuantYfeX

StreckPAXgene

5,E+07

5,E+07

4,E+07

4,E+07

3,E+07

3,E+07

2,E+07

2,E+07

1,E+07

5,E+06

0,E+00Total reads

NGS

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Reads, %

StreckPAXgene

StreckPAXgene

5,E+07

5,E+07

4,E+07

4,E+07

3,E+07

3,E+07

2,E+07

2,E+07

1,E+07

5,E+06

0,E+00Total reads

NGS

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10Mapped reads Unique reads

Reads, %

StreckPAXgene

StreckPAXgene

PAXgene Blood ccfDNA tube.

BD Hemogard safety closure

Proprietarystabilizationreagent

• PAXgene Blood ccfDNA stabilization reagent inhibits the apoptosis of blood cells.

• Release of genomic DNA into the plasma is prevented by stabilizing blood with PAXgene Blood ccfDNA stabilization reagent.

Prevention of Blood Cell Lysis and Release of gDNA

Blood cell lysis and genomic DNA release. Whole blood from 2 healthy donors was drawn in EDTA BCT. After draw, blood in EDTA tubes was eitherstored or directly transferred into tubes containing PAXgene Blood ccfDNA stabilization reagent (PAXgene). Stabilized blood was immediately processed (t0) or stored for up to 10 days at room temperature (t10). A Genomic DNA was extracted from stored blood using the QIAamp Blood Mini Kit and 400 ng DNA was separated by agarose gel electrophoresis. B Plasma was generated by double centrifugation after 6 and 10 days of storage and ccfDNA was extracted from 2.4 ml plasma using the PAXgene Blood ccfDNA QIAsymphony protocol. 1 µl eluate was analyzed using the Agilent High Sensitivity DNA Kit.

A B

EDTAt0 t10

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EDTA t0 PAX t0 PAX t6 EDTA t10 PAX t10

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0

PAXgene EDTA PAXgene

PAXgene Blood ccfDNA System Workflow

Blood collectionand stabilization

Blood sample Analytical assays ResultsPre-analytical workflow

Transport,storage,

plasma separationccfDNA extraction

Data analysis

Real-time PCR,NGS, others

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0 4010 20 30Cycles

QIAsymphony

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Tube