Research into non-invasive prenatal testing (NIPT) and circulating tumor DNA (ctDNA) testing based on circulating cell-free DNA (ccfDNA) is rapidly expanding. However, detection and quantification of ccfDNA is compromised by the release of genomic DNA (gDNA) from lymphocytes due to mechanical lysis or apoptosis during blood collection, storage and transport. PreAnalytiX has developed the PAXgene Blood ccfDNA System, consisting of the PAXgene Blood ccfDNA Tube, a plastic blood collection tube with a unique, non-crosslinking chemistry that preserves extracellular levels of ccfDNA and prevents the release of intracellular DNA from cells into the plasma, and the QIAsymphonyPAXgene Blood ccfDNA Kit for automated ccfDNA extraction from up to 5 ml of plasma. In this slidedeck, this new technology development is presented in comparison to other existing technologies.

2. PAXgene® Tissue System (RUO)
Two worlds in one sample
PAXgene is a tradmark of PreAnalytiX GmbH. All other brands are trademarks of theire respective owners. © 2010-2016 PreAnalytiX.
06.06.2016 2
New workflows &
applications
Dr. Tomasz Krenz

3. 06.06.2016 3
PAXgene Tissue System* (RUO)
Agenda
About PreAnalytiX
PAXgene Tissue System
Workflow 1: PF-tissue
Workflow 2: PFPE-tissue
Workflow 3: PFCE-tissue
Laser capture microdissection
References / Web resources
*The PAXgene Tissue applications presented here are for research use only.
Not for use in diagnostic procedures. No claim or representation is intended to provide information for
the diagnosis, prevention, or treatment of a disease

4. 06.06.2016 4
About PreAnalytiX GmbH
A QIAGEN/BD Joint Venture since 1998
BD
• Specimen collection & stabilization
• Storage & transport
QIAGEN
• Isolation of nucleic acids & other
biomolecules
• Automation
• Analysis

5. 06.06.2016 5
PreAnalytiX GmbH
Our Mission
Preanalytical errors cost ~
$460,000 / year in a typical
German hospital
Frost & Sullivan, 2011 survey on behalf of BD
Postanalytics
Preanalytics
68%
13%
19%
Analytics
“Preanalytical errors still account for nearly 60–70% of all problems occurring
in laboratory diagnostics, … attributable to mishandling procedures during
collection, handling, preparing or storing the specimens.”
Lippi, G. et al. (2011) Preanalytical quality improvement: from dream to reality. Clin Chem Lab Med 49,
1113−1126. Epub 2011 Apr 25.

6. 06.06.2016 6
PAXgene Tissue System* (RUO)
Agenda
PAXgene Tissue System

7. 06.06.2016 7
Morphology Nucleic Acid Preservation Protein Preservation
PAXgene Tissue
System √ √ √
Competitor A
Morphology destroyed or
compromised √ -
Competitor B
Morphology destroyed or
compromised
√ √
Formalin √
Crosslinking of biomolecules;
Lack of standardization;
no stabilization of biomolecules
PAXgene Tissue: Product Positioning
Simultaneous preservation of morphology and
biomolecules
 PAXgene Tissue enables combined histological and multimodal analysis of biomolecules
from the same sample

8. 06.06.2016 8
PAXgene Tissue System
System components
Collection, fixation and stabilization
PAXgene Tissue Container (10) containing
PAXgene Tissue FIX and STABILIZER
dual chamber: 1 histocassette
PAXgene Tissue FIX Container (10 x 50 ml)
+ PAXgene Tissue STABILIZER concentrate
(8 x 150 ml for 4 liters of STABILIZER reagent each)
single chamber: up to 4 histocassettes or larger tissue sample
+
RNA RNA & miRNA DNA Protein
Qproteome FFPE Tissue KitPAXgene Tissue Kits
Extraction

9. 06.06.2016 9
PAXgene Tissue System* (RUO)
Agenda
Workflow 1: PF-tissue

10. 06.06.2016 10
Analysis
Biomolecule
purification
Storage at
−20 to −80°C
PAXgene Tissue: Workflows
1. PF-Tissue
Tissue fixation,
stabilization
& transport
Tissue resection
Fixation
2–48 hours
(depending on tissue size)
Stabilization & storage
Up to 7 days at RT
Up to 4 weeks at 2–8°C
Up to 5 years at –20°C (studies
ongoing)
Up to 6 years at –80°C (studies
ongoing)
Biomolecule purification
PAXgene Tissue RNA kit
PAXgene Tissue miRNA kit
PAXgene Tissue DNA kit
Qproteome® FFPE kit &
supplementary protocol
PF-Tissue
≤ 15 x 15 x 4 mm
PAXgene Tissue
Container
PAXgene Tissue
FIX Container (50ml)
Cryo Vial
RNA, DNA, Protein

11. 06.06.2016 11
1. PF-Tissue Workflow
High-quality NA from different tissue types including
fibrous or fatty tissues
Φ RIN
- 21kb
RNA
DNA

12. 06.06.2016 12
PAXgene Tissue System* (RUO)
Agenda
Workflow 2: PFPE-tissue

13. 06.06.2016 13
Analysis
Biomolecule
purification
& Staining
Laser
Capture
Microdisection
Sectioning
Processing &
embedding
Storage at
-20 to -80°C
Tissue fixation,
stabilization
& transport
Paraffin-Embedding
PFPE-Tissue
RNA DNA Protein
H&E IHC ISH
PF-Tissue
PAXgene Tissue
Container
PAXgene Tissue
FIX Container (50ml)
Microtome
Cryo Vial
LCM
Processing and paraffin embedding
Position #1 of tissue processor:
STABILIZER or ≥ 80% EtOH
Low melting point paraffin (m.p. 54°C)
Inkubation in paraffin < 3 hours
Staining
Histochemical (H&E, PAS, …)
Immunohistochemical
In situ hybridization
PAXgene Tissue: Workflows
2. PFPE-Tissue
Biomolecule purification
PAXgene Tissue RNA kit
PAXgene Tissue miRNA kit
PAXgene Tissue DNA kit
Qproteome® FFPE kit &
supplementary protocol

14. 06.06.2016 14
2. PFPE-Tissue Workflow
Preservation of tissue morphology
Liver SpleenKidney Intestine Lung Brain
H&E staining

15. 06.06.2016 15
2. PFPE-Tissue Workflow
IHC & ISH with clinical samples
PFPEFFPE
IHC of human tonsil:
Ki-67, clone MIB-1
ISH of human breast cancer:
HER2 FISH

16. 06.06.2016 16
2. PFPE-Tissue Workflow
Evaluation of RNA quality with clinical samples
Viertler et al. (2012) J Mol Diagn 14, 458.
RNA integrity from human liver:
PFPE vs. FFPE vs. snap frozen
Gene expression from human liver:
qRT-PCR on a TaqMan array plate
92 cancer pathway genes
PFPE: Strong correlation (R² = 0.99)
to snap-frozen
FFPE: large gene-to-gene variation (R² = 0.81)

17. 06.06.2016 17
2. PFPE-Tissue Workflow
Evaluation of DNA quality with clinical samples
DNA integrity from human
colorectal cancer:
PFPE: High molecular mass, suited
for multiplex and long-range PCR
FFPE: No recovery of large DNA
molecules
Viertler et al. (2012) J Mol Diagn 14, 458.

18. 06.06.2016 18
2. PFPE-Tissue Workflow
Analytical protein techniques
Reverse-phase
protein arrays
Western blot
Maldi-imaging mass
spectrometry
2D-PAGE
ELISA (denatured
proteins)
Ergin, B. et al. (2010) J Proteome Res 9, 5188−5196.
Ergi, B. et al. (2010)
Gündisch, S. et al.(2013) PLoS One 8, e60638.
Gündisch, S. et al. (2013)
Gündisch, S. et al. (2013)

19. 06.06.2016 19
PAXgene-Tissue Workflow
Automated purification of genomic DNA and RNA from
PF & PFPE
QIAcube protocols available: significant savings of
hands-on time
RNA from sections of PFPE tissue
RNA from PF tissue
DNA from section of PFPE tissue
DNA from PF Tissue

20. 06.06.2016 20
PAXgene Tissue System* (RUO)
Agenda
Workflow 3: PFCE-tissue

21. 06.06.2016 21
Analysis
Biomolecule
purification
& staining
Laser
Capture
Microdissection
Sectioning
Processing &
embedding
Storage at
−20 to −80°C
PAXgene Tissue: New Workflows
PFCE: PAXgene fixed, cryo-embedded
Tissue fixation,
stabilization
& transport
Cryo-Embedding
PFCE-Tissue
RNA DNA Protein
PF-Tissue
PAXgene Tissue
Container
PAXgene Tissue
FIX Container (50ml)
Cryotome
Cryo Vial
LCM
Processing and cryo embedding
Wash in 30% [w/v] sucrose
at 2-8°C
Overlay Tissue with cryo-
embedding media (e.g. Leica
FCS22)
Freeze in pre-chilled Isopentane
or liquid N2
H&E IHC ISH
Biomolecule purification and Staining
Supplementary Protocols

22. 06.06.2016 22
3. PFCE-Tissue Workflow
A. Preparation in isopentane on dry ice
< 2 x 15 x 15 mm
1. Tissue fixation and sucrose incubation
Tissue resection
Tissue fixation &
stabilization
ON incubation in
30% [w/v] sucrose
Freezing in isopentane
cooled on dry ice
2. Snap-freezing in isopentane and embedding in cryo-media
Frozen tissue added to
cryomold filled with cryo
-embedding medium
Specimen disk
placed on top
Cryomold submerged
into isopentane
PFCE specimen

23. 06.06.2016 23
< 2 x 15 x 15 mm
1. Tissue fixation and sucrose incubation
Tissue resection
Tissue fixation &
stabilization
ON incubation in
30% [w/v] sucrose
2. Embedding in cryo-media and snap-freezing in liquid nitrogen
Tissue placed in cryomold filled
with cryo-embedding media
Specimen disk
Placed on top
PFCE specimen
Snap freezing in
liquid nitrogen
3. PFCE-Tissue Workflow
B. Preparation with liquid nitrogen

24. 06.06.2016 24
3. PFCE-Tissue Workflow
Kits & Protocols
Object PAXgene Tissue Supplementary Protocols Kits / Products
Workflow -
Preparation
of PFCE
samples
Cryo-embedding tissue specimens fixed and
stabilized with the PAXgene® Tissue System
PAXgene Tissue Container
PAXgene Tissue FIX Container +
Stabilizer
DNA
Purification of genomic DNA from sections of
PAXgene® Tissue fixed, cryo-embedded (PFCE)
tissue
PAXgene Tissue DNA Kit
RNA
Purification of total RNA from sections of
PAXgene® Tissue fixed, cryo-embedded (PFCE)
tissue
PAXgene Tissue RNA Kit
miRNA
Purification of total RNA, including miRNA, from
sections of PAXgene® Tissue fixed, cryo-embedded
(PFCE) tissue
PAXgene Tissue miRNA Kit
Protein
Purification of full-length proteins from sections of
PAXgene® Tissue fixed, cryo-embedded (PFCE)
tissue
Qproteome FFPE Kit

25. Date Text, Location25
Liver Spleen
Heart muscleIntestine
3. PFCE-Tissue Workflow
Histomorphology
H&E staining

26. 06.06.2016 26
3. PFCE-Tissue Workflow
High-quality NA from different tissue types
RIN: 8.5 RIN: 7.6RIN: 8.9 RIN: 9.1RIN: 8.6
RNA
- 21kb
DNA
Liver Spleen Intestine Heart muscle Lung

27. 06.06.2016 27
Data kindly provided by Karl-Friedrich Becker,
Technical University of Munich, Germany
Liver Stomach
Section of PFCE tissue
mounted on slide
Cryo-medium removed
Area of interest removed manually
or by laser microdissection
Collected into Qproteome FFPE
buffer EXB Plus
Incubated for 10 min @95°C
3. PFCE-Tissue Workflow
Full-length (phospho-)proteins from PFCE tissue
SDS-PAGE & western blot
12.5 µg protein per lane

28. 06.06.2016 28
PAXgene Tissue System* (RUO)
Agenda
Laser capture microdissection

29. 06.06.2016 29
Biomolecule
Purification
Laser Capture MicrodissectionStaining (H&E)
Sectioning/
removal of
embedding medium
Processing &
embedding
Paraffin-Embedding
Cryo-Embedding
PFPE-Tissue
PFCE-Tissue
Microtome
Cryotome
RNA DNALMD (e.g. Leica 6500)H&E
PFPE workflow
PFCE workflow
PAXgene Tissue: New Workflows
Laser Capture Microdissection (LCM)

30. 06.06.2016 30
MicrodissectionSet Up
4. Laser Capture Microdissection (LCM)
Procedure
Sample
Processing
Slide
preparation
PFPE/PFCE tissue sections Microscope + Sample
LCM Software
Collection tubesstain
Definition of region
Cutting
Collection
Collection in Lysis buffer
Purification of genomic DNA
Purification of total RNA
Purification of total RNA,
including miRNA

31. 06.06.2016 31
Object PAXgene Tissue Supplementary Protocols Kits / Products
Workflow -
Preparation
of slides
Preparation of sections from PAXgene® Tissue fixed,
paraffin-embedded (PFPE) and PAXgene Tissue
fixed, cryo-embedded (PFCE) tissues for manual or
laser microdissection (LMD)
PAXgene Tissue Container
PAXgene Tissue FIX Container +
Stabilizer
DNA
Purification of genomic DNA from microdissected
PAXgene® Tissue-fixed, paraffin-embedded (PFPE)
and PAXgene Tissue-fixed, cryo-embedded (PFCE)
tissues
PAXgene Tissue DNA Kit
RNA
Purification of total RNA from microdissected
PAXgene® Tissue fixed, paraffin-embedded (PFPE)
and PAXgene Tissue fixed, cryo-embedded (PFCE)
tissues
PAXgene Tissue RNA Kit
miRNA
Purification of total RNA, including miRNA, from
microdissected PAXgene® Tissue fixed, paraffin
embedded (PFPE) and PAXgene Tissue fixed,
cryoembedded (PFCE) tissues
PAXgene Tissue miRNA Kit
4. Laser Capture Microdissection (LCM)
Kits & Protocols

32. 06.06.2016 32
liver
spleen
colon
liver
spleen
colon
liver
spleen
colon
PFPE PFCE FFPE[nt] High-quality and high-molecular−weight RNA
Agilent Bioanalyzer: Gel and corresponding
RIN values of total RNA from an area of 1
mm² (4,000−12,000 cells)
ß-act RT PCR
4. Laser Capture Microdissection (LCM)
RNA from PFPE & PFCE tissue
# cells

33. 06.06.2016 33
PFPE PFCEliver
spleen
colon
liver
spleen
colon
High-quality miRNA
Agilent Bioanalyzer Small RNA: Gel
with calculated miRNA content and
Electropherograms
Area of 1 mm² (4,000−12,000 cells)
selected
[nt]
4. Laser Capture Microdissection (LCM)
miRNA from PFPE & PFCE tissue

34. 06.06.2016 34
Total yield and performance
(ß-act PCR)
Area of 0.005−1 mm² (20−12,000 cells)
selected
4. Laser Capture Microdissection (LCM)
DNA from PFPE & PFCE tissue

35. 06.06.2016 35
PAXgene Tissue System* (RUO)
Agenda
References / Web resources

36. 06.06.2016 36
References
Projects / Collaborations
SPIDIA
STRATFix UK
Genotype Tissue
Expression Project
(GTEx)
CBmed Austria
Barret Syndrome
Project
Genesis
Enabling stratified medicine with novel fixatives for improved pre-
analytical pathology workflows
Next generation sequencing in molecular pathology diagnostics
Identification of Biomarkers for the Development of Adenocarcinoma
from Barrett-Esophagus
Genetic Biopsy for Prediction of Surveilance Intervals after
Endoscopic Resection of Colonic Polyps
Standardization and Improvement of Generic Pre-analytical Tools and
Procedures for In Vitro Diagnostics
7 research organizations, 8 companies, 1 standards organization
(CEN), Coordinated by QIAGEN
8 clinical sides, 1 company, Coordinated by QIAGEN
Establish a resource database and tissue bank to study the
relationship between genetic variation and gene expression
Over 700 donors, >21,000 collected tissue samples
Up to 2000 patients
220 samples, 100 patients; follow up project with ~10.000 patients

37. 06.06.2016 37
References
Scientific articles in peer-reviewed journals
Author Titel Journal Content
Yamaguchi, et al. Comprehensive DNA Methylation and Extensive Mutation Analyses of HER2-Positive Breast
Cancer.
Oncology. 2015 Jan;
[Epub ahead of print]
409 cancer-related genes/ bench-top NGS/ DNA methylation
statuses/ bead array with 485,512 probes
Keen J and Moore H The Genotype-Tissue Expression (GTEx) project: Linking Clinical Data with Molecular Analysis
to Advance Personalized Medicine.
J.Pers. Med. 2015 Feb;
5:22-29
Whole genome and RNA sequencing
Andersen GB, et al. Improved reproducibility in genome-wide DNA methylation analysis for PAXgene® fixed
samples compared to restored FFPE DNA.
Anal Biochem. 2014 Sep; 468C:50-58. Genome-wide DNA methylation analysis/ Illumina 450K BeadChip
Gündisch S, et al. Evaluation of colon cancer histomorphology: a comparison between formalin and
PAXgene tissue fixation by an international ring trial.
Virchows Arch. 2014 Nov;
465(5):509-19.
Blinded, randomized ring trial by 20 pathologists; virtual
microscopy; histological grading and quality parameter
Kap M, et al. Inactivation of Influenza A virus, Adenovirus, and Cytomegalovirus with PAXgene
Tissue Fixative and Formalin.
Biopreservation and Biobanking.
2013 Aug; 11(4): 229-234
Viral inactivation characteristics by PAXgene Tissue fixative
Staff S, et al. Preservation of nucleic acids and tissue morphology in paraffin-embedded clinical samples:
comparison of five molecular fixatives.
J Clin Pathol. 2013 Sep;
66(9):807-10
Comparison study Z7, RCL2, PAXgene Tissue, Allprotect, RNAlater,
Formlin; DNA, RNA, Morphology, IHC, FISH, CISH
Oetjen J, et al. MRI-compatible pipeline for three-dimensional MALDI imaging mass spectrometry using
PAXgene fixation.
J Proteomics. 2013 Sep;
90:52-60
Computational pipeline for molecular analysis combining 3D
MALDI imaging, magnetic resonance imaging and H&E
Lonsdale, et al. The Genotype-Tissue Expression (GTEx) project. Nature Genetics 2013 Jun;
45, 580-585
Description of GTEx project
Gündisch S, et al. The PAXgene Tissue System Preserves Phosphoproteins in Human Tissue
Specimens and Enables Comprehensive Protein Biomarker Research.
PLoS One. 2013;
8(3):e60638
Proteome including phosphoproteome analysis; fresh frozen,
PFPE, FFPE, western blotting, 2D-PAGE, ELISA
Belloni B, et al. Will PAXgene substitute formalin? A morphological and molecular comparative study
using a new fixative system.
J Clin Pathol. 2013 Feb;
66(2):124-35
Melanoma fresh frozen, PFPE, FFPE, morphology, IHC, DNA, RNA
Groelz D, et al. Non- Formalin Fixative versus Formalin-fixed Tissue: A Comparison of Histology and
RNA Quality.
Exp Mol Pathol. 2013 Feb;
94(1):188-94
Morphology and RNA preservation; comparison PFPE and FFPE;
RT-qPCR
Viertler C, et al. A New Technology for Stabilization of Biomolecules in Tissues for Combined Histological and
Molecular Analyses.
J Mol Diagn. 2012 Sep;
14(5):458-66
RNA, DNA, miRNA preservation, fresh frozen, PFPE, FFPE,
expression profiling
Trede D, et al. Exploring three-dimensional matrix-assisted laser desorption/ionization imaging mass
spectrometry data: three-dimensional spatial segmentation of mouse kidney.
Anal Chem. 2012 Jul;
84(14):6079-87.
3D MALDI imaging, 3D spatial segmentation
Baker M Biorepositories: Building better biobanks. Nature 2012 Jun;
486(7401):141-6.
Efforts to improve biobanking
Kap M, et al. Histological Assessment of PAXgene Tissue Fixation and Stabilization
Reagents.
PLoS ONE. 2011;
6(11)
Quality assessment PFPE vs FFPE, 20 different human
tissue types, H&E, IHC, 4 pathologists, histological grading
and quality
Ergin B, et al. Proteomic analysis of PAXgene-fixed tissues. J Proteome Res. 2010 Oct;
9(10):5188-96
Proteome analysis from fresh frozen, PFPE and FFPE;
western blotting, reverse-phase microarrays, MALDI imaging
MS
Loibner et al. (2016) Pathogen Inactivating Properties and Increased
Sensitivity in Molecular Diagnostics by PAXgene,
a Novel Non-Crosslinking Tissue Fixative.
PLoS One. 2016 Mar
14;11(3)
Oberauner-Wappis
et al. (2016)
Protocol for HER2 FISH determination on
PAXgene-fixed and paraffin-embedded tissue in
breast cancer
Accepted in
International Journal
of Experimental
Pathology
Carithers et al.
(2015)
A novel approach to high-quality postmortem
tissue procurement: the GTEx project
Biopreserv Biobank.
2015 Oct;13(5):311-9.
Hara et al. (2015) Surgical Specimens of Colorectal Cancer Fixed
with PAXgene Tissue System Preserve High-
Quality RNA
Biopreserv Biobank.
2015 Oct;13(5):325-
34.
Gillard, M. et al.
(2015)
Next-gen tissue: preservation of molecular and
morphological fidelity in prostate tissue.
Am J Transl Res
7:1227-1235
Yamaguchi, T. et al.
(2015)
Comprehensive DNA Methylation and Extensive
Mutation Analyses of HER2-Positive Breast
Cancer.
Oncology.
2015;88(6):377-84
Loibner et al. (2016) Pathogen Inactivating Properties and Increased
Sensitivity in Molecular Diagnostics by PAXgene,
a Novel Non-Crosslinking Tissue Fixative.
PLoS One. 2016 Mar
14;11(3)
Oberauner-Wappis
et al. (2016)
Protocol for HER2 FISH determination on
PAXgene-fixed and paraffin-embedded tissue in
breast cancer
Accepted in
International Journal
of Experimental
Pathology
Carithers et al.
(2015)
A novel approach to high-quality postmortem
tissue procurement: the GTEx project
Biopreserv Biobank.
2015 Oct;13(5):311-9.
Hara et al. (2015) Surgical Specimens of Colorectal Cancer Fixed
with PAXgene Tissue System Preserve High-
Quality RNA
Biopreserv Biobank.
2015 Oct;13(5):325-
34.
Gillard, M. et al.
(2015)
Next-gen tissue: preservation of molecular and
morphological fidelity in prostate tissue.
Am J Transl Res
7:1227-1235
Yamaguchi, T. et al.
(2015)
Comprehensive DNA Methylation and Extensive
Mutation Analyses of HER2-Positive Breast
Cancer.
Oncology.
2015;88(6):377-84

38. 06.06.2016 38
Web Resources
Technical Support
Technical notes
PI3K mutation analysis
IHC assay optimization
(Ki67)
Formalin contamination
during processing
Vacuum sealing for fixed
and stabilized tissues for
transport
RNA yield, purity and
integrity from PFPE rat
tissue
KRAS and BRAF
mutational status using
Pyrosequencing technology
…
Supplementary protocols
Manual processing
Deparaffinization with
Deparaffinization Solution
Purification of proteins from
sections of PFPE tissue
Allprep: Simultaneous purification
of RNA and DNA from sections of
PFPE tissue
Purification of RNA/DNA from
microdissected PFPE and PFCE
tissue
IHC application list
Manual staining
Benchmark XT
Microwave
Leica Bond Max
Dako Autostainer
Other
Handbooks
Posters
Brochures
List of references in
peer reviewed journals
FAQ List
Safety data sheets
Constantly growing list of technical support:
http://www.preanalytix.com/products/tissue/fixation-stabilization/paxgene-tissue-container

39. 06.06.2016 39
Web Resources
Tissue Atlas
http://www.preanalytix.com/products/tissue/tissue-atlas
Human large intestine Rat retina Rat tongue
Rat kidney Human large intestine Human stomach
H&E
PAS
IHC
Human tonsil Human colorectal cancer Human breast cancer Human tonsil
Human liver
Rat skin sebaceous glands

40. Q&A session
06.06.2016 40
Thank you for your
attention!
For up-to-date licensing information and product-specific
disclaimers for QIAGEN products, see the respective QIAGEN
kit handbook or user manual. QIAGEN kit handbooks and user
manuals are available at www.qiagen.com or
www.preanalytix.com or can be requested from QIAGEN
Technical Services or your local distributor.

41. Q&A session
06.06.2016 41
Supplement
For up-to-date licensing information and product-specific
disclaimers for QIAGEN products, see the respective QIAGEN
kit handbook or user manual. QIAGEN kit handbooks and user
manuals are available at www.qiagen.com or
www.preanalytix.com or can be requested from QIAGEN
Technical Services or your local distributor.

42. 06.06.2016 42
1. PF-Tissue Workflow
Full-length (phospho-)proteins from PF tissue
Data kindly provided by Karl-Friedrich Becker,
Technical University of Munich, Germany
12.5 µg protein per lane
Up to 100 mg of PF tissue
washed with PBS
Disrupted and homogenized
with TissueLyser
Incubated for 10 min @95°C
Collected into Qproteome
FFPE buffer EXB Plus
Liver Stomach
SDS-PAGE & western blot

43. 06.06.2016 43
References
SPIDIA
Main Facts
For “Standardization and Improvement of Generic
Pre-analytical Tools and Procedures for In Vitro Diagnostics”
Funded by FP7-HEALTH (EC)
7 public research organizations, 8 companies, 1 standards
organization (CEN)
Coordinated by QIAGEN
Run time from Oct. 2008 – March 2013
Main Goals
New preanalytical tools & technologies (blood, plasma, tissue, swabs)
Sample quality markers (blood, tissue)
Pan-European guidelines for preanalytics (blood, tissue)
Training and dissemination

44. 06.06.2016 44
References
The Genotype Tissue Expression Project (GTEx)
NIH common fund initiative
Aim: establish a resource database and tissue bank to study the relationship
between genetic variation and gene expression
Over 700 donors (>21,000 tissue samples) have been collected to date
PFPE-tissue for histological examination
RNA from PF-tissue for RNA sequencing
”PAXgene is at least as good as formalin for histology. It is now our only morphologic fixative.”
OBBR pathologists Philip Branton, John Madden James Robb, Leslie Sobin, Carolyn Compton
http://www.genome.gov/Pages/About/NACHGR/September2011AgendaDocs/NACHGR_Sep122011_%20GTExUpdate_Struewing.pdf
“Each tissue is preserved in PAXgene® tissue fixative …
sections are examined by a team of board certified pathologists …
images and histological data will be available to researchers …
PAXgene® tissue-fixed samples are then shipped to … for whole genome and RNA sequencing.”
Keen, J.C. and Moore, H.M. (2015) The Genotype-Tissue Expression (GTEx) Project: Linking clinical data with
molecular analysis to advance personalized medicine. J Pers Med 5, 22−29.

45. 06.06.2016 45
References
The Barret Syndrome Project
endoscopic tissue retrieval
PAXgene Tissue fixation & stabilization,
transport and processing
H&E, PAS – staining for diagnosis
IHC staining
DNA, RNA purification
Whole genome amplification
Next-generation sequencing
m4 Cluster Munich fund initiative — collaboration with Technical University Munich
Identification and Analysis of Biomarkers for the Development of Adenocarcinoma
from Barrett-Esophagus
Aims:
Optimization of pathogenesis, diagnosis and therapy of esophageal cancer
Identification of predictive biomarkers in Barret syndrome
Implementation of PAXgene Tissue for clinical application
Validation of tumor evolution theory (dysplasia caused by cardia stem cells)
Up to 2000 patients

46. 06.06.2016 46
References
The STRATFix Project
“Enabling stratified medicine with novel fixatives for improved preanalytical pathology workflows”
Formaldehyde free workflows in clinical routine
Solid tissue, fine needle biopsies, blood
Partners: