- EU SPIDIA Project - Pre-analytical handling of biosamples; - - PowerPoint PPT Presentation

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• EU SPIDIA Project -

Pre-analytical handling of biosamples; optimising biobank sample quality for protein and nucleic acid studies

Symposium on Biosample Quality Guy´s Hospital, London, May 9th 2012

• Dr. Daniel Grölz

SPIDIA (QIAGEN)

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Agenda SPIDIA

Project Facts & Goals New Technologies & Tools Guidelines & Dissemination Evaluation of PAXgene Tissue

S Standardization tandardization and Improvement of Generic and Improvement of Generic P Pre re-

• analytical Tools and Procedures for

analytical Tools and Procedures for I In Vitro n Vitro Dia Diagnostics gnostics

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Clinical Results Patient Sample Pre-analytical Workflow Analytical Assays

Molecular Diagnostic Workflow

From Patients to Clinical Results

Patient

Sample Collection Sample Logistics Bioanalyte Preparation Anesthesia e.g. Drugs e.g. Arterial Clamp Time Patient Treatment Life Style

Time 0

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“Preanalytical errors still account for nearly 60%-70% of all problems occurring in laboratory diagnostics, most of them attributable to mishandling procedures during collection, handling, preparing or storing the specimens”.

Lippi G. et al.. Preanalytical quality improvement: from dream to reality. Clin Chem Lab Med. 2011 Jul; 49(7):1113-26. Epub 2011 Apr 25.

It is Real Problem

68 % 13 % 19 %

Preanalytics Analytics Postanalytics

Costs of ~ 460,000 $ / year in an average German hospital caused by pre-analytical errors

Frost & Sullivan 2011 on behalf of BD

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Understanding biomolecule profile changes during pre-analytical workflows New pre-analytical technologies preventing biomolecule profile changes New evidence based standards and guidelines Detailed description of biological / clinical samples histories for research, biobanking, diagnostics Ideally – sample quality markers

Improvements Needed

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New pre-analytical tools & technologies (Blood, Plasma, Tissue, Swabs) Sample quality markers (Blood, Tissue) Pan-European guidelines for preanalytics (Blood, Tissue) Training and dissemination

Project Main Goals

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• Program

European Commission FP7-HEALTH

• Consortium

7 public research organizations 8 companies 1 standards organization (CEN)

• Coordinator

QIAGEN GmbH

• Run Time October 2008 – September 2012

(prolongation requested)

• Budget

13 Mio € (9 Mio € EC contribution)

• Co-operations

NCI / OBBR, CLSI, EFCC, BBMRI and other international initiatives and organizations

• Web page

www.spidia.eu

• Newsletter

Project Facts

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Agenda SPIDIA

Project Facts & Goals New Technologies & Tools Pan-European Guidelines & Dissemination Evaluation of PAXgene Tissue

S Standardization tandardization and Improvement of Generic and Improvement of Generic P Pre re-

• analytical Tools and Procedures for

analytical Tools and Procedures for I In Vitro n Vitro Dia Diagnostics gnostics

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Tissue

Stabilization of morphology, antigenicity, DNA, RNA, proteome

Fine Needle Aspirates

Stabilization of morphology, antigenicity, DNA, RNA, proteome

Plasma

New stabilization technologies

Whole Blood

New stabilization technologies

Swabs

Stabilization and improved processing for molecular analysis

Stabilized Whole Blood

Integrated automated sample-to-result workflows (cellular RNA, ncRNAs incl. miRNAs)

New Technology & Tools Ongoing Developments

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Quality marker for blood and tissue

To monitor changes in clinical sample materials Ischemia time, storage time and temperature

Quality markers blood measuring RNA up- & down- regulation

>180 micro arrays (time course experiments) 17 marker candidates (specific RNA degradation or gene down regulation, specific RNA gene induction) Technical assay validation Next step: Performance validation within larger donor cohorts

Sample Quality Marker Discovery

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Agenda SPIDIA

Project Facts & Goals New Technologies & Tools Pan-European Guidelines & Dissemination Evaluation of PAXgene Tissue

S Standardization tandardization and Improvement of Generic and Improvement of Generic P Pre re-

• analytical Tools and Procedures for

analytical Tools and Procedures for I In Vitro n Vitro Dia Diagnostics gnostics

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• Let by Prof. Pazzagli (Univ. Florence), supported by the EFCC
• Phase 1 Trials - Laboratories used their workflows & tools
• Phase 2 Trials - Laboratories use SPIDIA’s optimized workflows
• Guidelines / Technical Reports Development - CEN

92 % 276 299 Total 93 % 62 67 Plasma DNA 93 % 121 130 Blood DNA 91 % 93 102 Blood RNA Percentage of NA samples sent back Participants who sent NA samples back

• No. of

Participants (29 countries) SPIDIA Trials

Evidence Based Guidelines

Examples Blood DNA & RNA, Plasma ccfDNA

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Individual Report for Each Laboratory

2

48,5 145,5 23,1 9,42 6,55 97,0 194,0 4,36 [kb] 2,32 2,03

Maximum (kb)

Lab ID: L134

C.2 ImageJ data of your lab

19.139 101.034 1.011 Peak (Kb) Maximum (Kb) Minimum (Kb)

Peak (kb)

DNA4

warning

AL=0.70; WL=1.15 DNA4

in control

AL=36.29; WL=53.21 DNA4

in control

AL=14.15; WL=17.83

C.1 Your lab ( ) - Pulse field gel electrophoresis image Minimum (kb)

• C. Integrity of DNA4 (DNA extracted from blood)

C.3 Your lab ( ) versus overall distribution (N=157) – ImageJ data

In the figure the blue line represents the Action Limit (AL) and the gray line represents the Warning Limit (WL).

SPIDIA – DNAplas: Laboratory report

5 10 15 20 25 30 35 40 1 2 3 4 5 6 7 8 9 10 50 100 150 200 250 300 350

1

10 20 30 40 50 10 20 30 40 50 1.5 2.0 2.5 1.0 1.5 2.0 2.5

DNA4 Lab (N=167)

• B. Purity and Quantity of DNA4 (DNA extracted from blood)

B.1 Spectrophotometric data provided by your lab and by SPIDIA lab

AE-buffer Qiagen 200 200 40 25.1 2.049 28 2 . 0.007 0.014 Buffer Elution

• vol. (ul)

Extraction

• vol. (ul)

Dilution factor Quantity (ng/µl) Spidia Purity Spidia Quantity (ng/µl) Lab Purity Lab 320nm Lab 280nm Lab 260nm Lab Time interval (hours) Temperature of DNA storage Spectrophotometer Extraction 1 h 92.5 h 20° C

• 20°

C GeneQuant Pro GE healtcare QiaAmp Blood Mini kit (51106) Qiagen extraction to analysis arrival to extraction extraction to analysis arrival to extraction supplier producer supplier producer

B.2 Additional information provided by your lab

Lab ID: L134

B.4 Your lab ( ) versus overall distribution - Quantity Purity

DNA4 Spidia (N=176) In the figures the blue line represents the Action Limit (AL) and the gray line represents the Warning Limit (WL).

in control in control

Lower AL=1.26; Upper AL=2.12 Lower WL=1.55; Upper WL=1.96 Lower AL=1.57; Upper AL=2.28 Lower WL=1.76; Upper WL=2.14 AL=2.62; WL=10.12 AL=2.91; WL=9.79 DNA4 Spidia (N=174) DNA4 Lab (N=168)

Purity

zoom zoom zoom zoom

warning in control Quantity (ng/µl blood) Quantity (ng/µl blood) B.3 Your lab ( ) versus overall distribution – Purity

In the figures the blue lines represent the Action Limits (ALs) and the gray lines represent the Warning Limits (WLs).

SPIDIA – DNAplas: Laboratory report

1.0 1.5 2.0 2.5 3.0 3.5 1.0 1.5 2.0 2.5 3.0 3.5 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

3

10 20 30 40 50 60

1 2 3 4 5 6 7 8 9 10 50 100 150 200 250 300

Lab ID: L134

Quantity(ng/µl blood) D.1 Your lab ( ) versus overall distribution Quantification of RNaseP Kineret distance

In the figure the blue line represents the Action Limit (AL) and the gray line represents the Warning Limit (WL).

• D. Quantification of RNaseP by real-time PCR on DNA4 and evaluation of interferences

In the figure the two lines represent the two Kineret threshold for

• utliers identification: 5.99 (weak outlier) and 9.21 (strong outlier).

DNA4 (N=167) AL=11.72; WL=52.80

Interferences

DNA4 (N=172)

zoom zoom

in control in control

SPIDIA – DNAplas: Laboratory report

50 100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 850 900 950 1000

Pazzagli et al.. University of Florence – manuscript in preparation Statistical analysis by Verderio P. et al. (IRCCS, Milano)

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• Trainings, Workshops
• Newsletter (subscribe at www.sidia.eu)
• Scientific publications – 3 papers published, 6 manuscript submitted
• Co-operation with international initiatives and organizations

NCI / OBBR

• Biospecimen Research Network (BRN)
• Cancer Human Biobank (caHUB)

CLSI EFCC BBMRI CD-Society (Austria) m4 Cluster (Munich)

Dissemination

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Agenda SPIDIA

Project Facts & Goals New Technologies & Tools Pan-European Guidelines & Dissemination Evaluation of PAXgene Tissue

PAXgene Tissue System & Workflow Evaluation studies

S Standardization tandardization and Improvement of Generic and Improvement of Generic P Pre re-

• analytical Tools and Procedures for

analytical Tools and Procedures for I In Vitro n Vitro Dia Diagnostics gnostics

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Tissue Preservation

Stabilization of Morphology and Biomolecules

• Development began in 2007:

>1,500 compounds and combinations screened

• Technology requirements

Histomorphology must be equivalent to FFPE tissue RNA, DNA, miRNA must be preserved and of high quality

• Two-reagent system finalized in 2009

Fixation and stabilization reagents, both formalin-free Evaluation within Spidia ongoing >8,000 tissue samples tested to date

• First collection device

Available as *PAXgene Tissue Container Container with two chamber one closure

* For Research Use Only

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*PAXgene Tissue Workflow

Analytic Analyte Purification Processing Stabilization for Transport & Storage

** PAXgene T. Fixed, Paraffin- Embedded

DNA RNA IHC H&E PAS Proteins ISH miRNA

* For Research Use Only

DNA RNA miRNA Proteins

Fixation Resection

4 mm thickness 2 - 24 hours 7 days @22° C 4 wks @4° C 2 yrs @-20° C 3 yrs @-80° C studies

• ngoing

**PFPE

• Start with ≥ 80%

EtOH

• Formalin free

reagents

• Low melting

paraffin

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PAXgene Tissue System Components

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.

PAXgene Tissue Container

• Tissue Fixation
• Tissue Stabilization
• Process Standardization
• Formalin-Free

RNA&miRNA

PAXgene Tissue Kits

• PAXgene Tissue Supplementary Protocols

RNA/DNA or miRNA purification from microdisected tissue Purification of full-length proteins from PFPE samples DNA RNA

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Morphology & RNA Preservation in Rat PFPE Tissue Samples

Liver Liver Spleen Spleen Kidney Kidney Intestine Intestine Lung Lung Brain Brain

ø RIN 7.3 ø RIN 6.6 ø RIN 7.9 ø RIN 8.4 ø RIN 7.9 ø RIN 7.2

4 hours fixation, 7 days stabilization at 22 4 hours fixation, 7 days stabilization at 22° °C C

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Morphology Preservation in Human Samples

26 human tissue types Mirrored samples FFPE and PFPE Evaluation by virtual microscopy 4 pathologists from three institutes Scoring system -2 to +2

• nuclear details
• cytoplasmic details
• membrane details
• contrast
• verall impression

Histological Assessment of PAXgene Tissue Fixation and Stabilization Reagents Kap M. et al., PLoS ONE 6(11): e27704 (2011)

Kap et al.

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Morphology Preservation

Kap M. et al., PLoS ONE 6(11): e27704 (2011) "morphology of PAXgene-fixed paraffin embedded tissue was well preserved" "in general results obtained with PAXgene-fixed tissue are comparable to those of formalin-fixed tissue"

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RNA Preservation Human Clinical Samples

Viertler C. et al., accepted for publication

Liver sample: Cryo, PAXgene tissue and formalin fixed

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RNA Preservation Human Clinical Samples

Viertler C. et al., accepted for publication Ct summary of more than 800 RT-qPCR assays 45 human malignant and non-malignant tissue samples from different organs Samples fixed with PAXgene Tissue for 3 to 120 hours

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DNA Preservation Human Clinical Samples

Viertler C. et al., accepted for publication

Colorectal cancer cases (CRC): Cryo, PAXgene tissue and formalin fixed

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Protein Preservation in PFPE

Proteins from PFPE tissues are non- degraded and immunoreactive

Ergin B. et al. Proteomic analysis of PAXgene fixed

• tissues. J Proteome Res. 9(10), 5188-96 (2010)

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Immunohistochemistry Assay Optimization

20 min at 98° C in citrate buffer pH 6

PFPE human tonsil Ki-67, clone MIB-1 Labelled streptavidin-biotin (LSAB) assay

10 min at 70° C in Tris/EDTA buffer pH 9 no retrieval 20 min at 98° C in Tris/EDTA buffer pH 9

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Immunohistochemistry Different Assays

pH6, 20´ ´ ´ ´ 98° ° ° °C | pH9, 10´ ´ ´ ´ 70° C

Pankeratin CK (AE1/AE3) CD5 (CD5/54/F6) Vimentin (V9)

pH9, 20´ ´ ´ ´ 98° ° ° °C | pH9, 10´ ´ ´ ´ 98° C pH9, 20´ ´ ´ ´ 98° ° ° °C | no HIER

ERα (1D5) ERα (1D5) ERα (1D5)

pH9, 20´ ´ ´ ´ 98° ° ° °C | pH9, 10´ ´ ´ ´ 98° C Human Breast Cancer Cases www.preanalytix.com

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• QIAGEN GmbH - Coordinator
• Medical University of Graz (Prof. K. Zatloukal)
• University of Florence (Prof. M. Pazzagli)
• CIRMMP Florence, CERM (Prof. I. Bertini)
• TATAA Biocenter
• PreAnalytiX GmbH
• DIAGENIC ASA
• Aros Applied Biotechnology
• Dako Denmark
• Biotechnology Inst. of Czech Academy of Science

(Prof. M. Kubista)

• European Committee for Standardization (CEN)
• ImmunID Technologies
• Erasmus Medical Center Rotterdam (Prof. P. Riegman)
• Technical University Munich (Prof. H. Hoefler, Prof. K. Becker)
• Fondazione IRCCS Istituto Nazionale dei Tumori (Dr. P. Verderio)
• Novamen

Acknowledgement SPIDIA Consortium Members

Scientific Advisory Board

• Prof. François Rousseau

(Univ. Laval, Quebec. CanGeneTest Network)

• Dr. Roberta M. Madej (CLSI)

Project Ethics Advisors

• Dr. Anne Cambon-Thomsen

(CNRS, INSERM, Tolouse, France)

• Dr. Ruth Chadwick (ESRC

Centre, Cardiff University, UK)

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SPIDIA Consortium

Bi-Annual Meeting Berlin November 2011