Bedeutung des EU Spidia Projekts fr Biobanken Standardization and - - PowerPoint PPT Presentation

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Bedeutung des EU Spidia Projekts fr Biobanken Standardization and Improvement of Generic PreanalyticalTools and Procedures for In Vitro Diagnostics 11. Jahrestagung Sektion Molekulare Diagnostik der DGKL Tutzing, 10. May 2012 Dr. Uwe

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SLIDE 1

Bedeutung des EU Spidia Projekts für Biobanken

Standardization and Improvement of Generic PreanalyticalTools and Procedures for In Vitro Diagnostics

  • 11. Jahrestagung

Sektion Molekulare Diagnostik der DGKL Tutzing, 10. May 2012

  • Dr. Uwe Oelmüller

QIAGEN GmbH (Koordinator)

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SLIDE 2

SPIDIA Project History and Goals

Results & Status

New Technologies & Tools Pan-European Guidelines Sample Quality Markers

Agenda

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SLIDE 3

Clinical Results Patient Sample Pre-analytical Workflow Analytical Assays

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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SLIDE 4

“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 ~ 347,000 € / year in an average German hospital caused by pre-analytical errors

Frost & Sullivan 2011 on behalf of BD

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SLIDE 5

1. Pan-European guidelines (Molecular) 2. New tools & technologies 3. Sample quality biomarkers 4. Training and dissemination 5. Co-work with other international initiatives

  • NCI / OBBR, CLSI, EFCC etc.

Project Main Goals

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SLIDE 6

Consortium 7 public research organizations 8 companies 1 standards organization (CEN) Coordinator QIAGEN GmbH October 2008 Kick Off Meeting Duration 4 years (6 months prolongation intended) Budget 13 Mio € EC Contribution 9 Mio € Web page www.spidia.eu Newsletter

SPIDIA Project Facts

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SLIDE 7

SPIDIA Project Project History and Goals

Results & Status

New Technologies & Tools

Pan-European Guidelines Sample Quality Markers

Agenda

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SLIDE 8

DNA RNA miRNA IHC H&E Western Blot ISH

> 1.500 compounds and mixtures screened (3 years) > 8.000 tissue samples processed to date Fixation & Stabilization (non-cross linking) Linked RNA, DNA, protein isolation procedures Allows histomorphology and molecular testing from the same specimen

For Research Use Only

Process Stabilize Fix Resect & Gross

New Tissue Collection & Stabilization

PAXgene Tissue

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SLIDE 9

Histomorphology and IHC

Research Study - PFPE vs. FFPE

H&E Staining IDC of Breast

PFPE FFPE

Estrogen Receptor a (clone 1D5) IDC of Breast

Groelz D. et al., unpublished data. Cap M. et al., PLoS ONE 6(11): e27704 (2011) Viertler C. et al., Journal of Molecular Diagnostics 2012, accepted for publication.

PFPE revealed preservation of morphology and antigenicity comparable to FFPE

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SLIDE 10

Viertler et al., Journal of Molecular Diagnostics 2012, accepted for publication). Groelz et al., unpublished data

5 10 15 20 25 30 35 40 45 50

18s CTNNB 1 COL1A1 FN1 RHOA FOS Gapdh CCND1 SP P1 E RB B2 IGF1R CDH1 NFKBIA A KT1 CDKN1B B CL2L1 CDK4 SHC1 ITGAV TP 53 A KT2 GSK 3B B AX CDKN1A ITGB1 TGFB 1 A BL1 HP RT1 M AP K14 TCF3 SRC NFK B1 GUS B E LK1 VE GFA K RA S GRB2 B CA R1 RA C1 B CL2 SM A D4 M DM 2 CRK NFK B2 BRAF M AP K3 FADD PTEN CCND3 JUN RE LA P TK 2 DVL1 PIK3R1 M Y C CDK2 NRA S TGFBR2 CDKN2A M AP K1 M AP 2K1 RB1 HRA S TGFBR1 PTK2B E2F1 AP C FZD1 CAS P9 SOS1 CAS P8 ITGB3 BID KDR BCL2L11 M AP K8 RAF1 P IK 3CA CCND2 M AP 3K5 FA S CCNE1 FY N LEF1 FGF2 E GFR IGF1 CYCS HGF FAS LG ITGA2B K IT CDC42 M AX CDKN2B W NT1

genes (sorted by Ct cryo)

Ct CRYO FFPE PFPE

TaqMan Array Gene Signature 96-Well Plate: Human molecular mechanism of cancer

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SLIDE 11

ccfNA Profiles in Whole Blood

What is missing?

Studies for understanding fcDNA and ccfRNA profile stability / changes in whole blood and in plasma Development of ccfDNA and ccfRNA profile preservation technologies

Horlitz M. et al., unpublished data

EDTA blood was incubated for up to 6 days at room temperature. Blood fcDNA pattern stability was determined by separating the purified plasma DNA on a 2100 Agilent Bioanalyzer

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SLIDE 12

Fine Needle Aspirates

Stabilization of morphology, antigenicity, DNA, RNA, proteome

Whole Blood

Stabilization of cell morphology and biomolecule profiles

Swabs

Stabilization and improved processing of respiratory and samples for molecular analysis

Stabilized Whole Blood

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

Ongoing Technology & Tools Developments for Other Sample Types

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SLIDE 13

SPIDIA Project Project History and Goals

Results & Status

New Technologies & Tools

Pan-European Guidelines

Sample Quality Markers

Agenda

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SLIDE 14
  • Phase 1 Trials - Laboratories used their workflows & tools
  • Let by Prof. Pazzagli (Univ. Florence), supported by the EFCC
  • Guidelines / Standards Concepts - CEN
  • Phase 2 Trials – Laboratories use SPIDIA’s optimized workflows
  • Guidelines / Technical Reports Developments - 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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SLIDE 15

Blood storage time before DNA extraction

  • 39 labs:

≤ 6 days

  • 60 labs:

6 – 10 days

  • 53 labs:

≥ 10 days

Blood storage temperature before DNA extraction

  • 18 labs:
  • 20 ˚C
  • 129 labs:

+4 ˚C

  • 9 labs:

ambient temp.

Isolated DNA storage before analysis

  • 20 labs:
  • 20 ˚C
  • 111 labs:

+4 ˚C

  • 27 labs:

ambient temp.

Blood DNA Trial 1 - Examples for Pre-analytical Workflow Variations

Pazzagli M. et al., manuscript in preparation

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SLIDE 16

High molecular weight DNA integrity: degradation, fragmentation High variability among samples

48,5 145,5 23,1 9,42 6,55 97,0 194,0 4,36 [kb] 3 17 63 82 88 103 118 120 130 138 175 183 207 1 14 49 52 65 78 100 124 125 169 195 203

DNA Length Variation – Pulse Field Gel Electrophoresis

Hartmann C. et al., unpublished results Pazzagli M. et al., manuscript in preparation

195 kb 4.36 kb

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SLIDE 17

Impact of DNA quality on Immune T cell Repertoire Analysis (ImmunID Technologies)

Lost of all long V–J rearrangements Lost of part of intermediate length rearrangements

  • L. Barraud et al. Unpublished data. Pazzagli M. et al., manuscript in preparation
  • Ref. DNA from UNFI (DIV 54%)

Sample 38 (Poor quality) (DIV 32%)

V contribution for each J gene – Research Trial (ImmunID Technologies, France)

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SLIDE 18

Human EDTA Blood stored at Room Temperature over 3 days β β β β mRNA

Individual Samples React Differently

Changes of Transcripts Profiles in Blood

Guenther K. et al.. AMP Poster (2005)

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SLIDE 19

Learning from Blood RNA Ring Trial 1

No pooling of different donors’ blood

  • Accept that only sub-groups of ring trial participating

laboratories get the same blood samples

No usual blood collection bags

  • Use dedicated EDTA bags

Immediate cooling of blood bags

  • Artificial gene induction and down regulation to be avoided

Use of intracellular RNA markers

  • External markers will behave differently
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SLIDE 20

Proficiency Testing for Preanalytical Workflows used for Blood RNA Analysis

  • K. Günther, F. Malentacchi, P. Verderio, S. Pizzamiglio, C. M. Ciniselli, A. Tichopad, M. Kubista, R. Wyrich, M.

Pazzagli, S. Gelmini. Implementation of a proficiency testing for the assessment of the preanalytical phase of blood samples used for RNA based analysis. Clin Chim Acta. 2012 Apr 11;413(7-8):779-86.

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SLIDE 21

Guenther K. et al.. Clin Chim Acta. 2012 Apr 11;413(7-8):779-86.

Blood Sample Shipment - RNA Profile Changes

Stabilized vs. EDTA Blood

Box plots reflecting the mRNA expression of GAPDH (Panel A), IL1B (Panel B), IL8 (Panel C), and FOS (Panel D) measured in the three sample types REF, RNA A (PAXgene Blood RNA) and RNA B (EDTA). Each box indicates the 25th and 75th percentiles. The horizontal line inside the box indicates the median, and the whiskers indicate the extreme measured

  • values. The dotted

horizontal line indicates the median value of the REF samples (prior shipment) and serves for comparison.

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SLIDE 22

SPIDIA Project Project History and Goals

Results & Status

New Technologies & Tools Pan-european Guidelines Sample Quality Markers

Agenda

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SLIDE 23

Quality markers measuring RNA up- & down-regulation

  • >150 micro arrays & RT-PCR studies (time course experiments)
  • 17 candidates (gene induction, gene down regulation, RNA degradation)
  • Technical assay validation
  • Next step: Performance validation within larger donor cohorts

Blood RNA Quality Marker Discovery

Rian E. et al., unpublished data

  • 1.0

0.0 1.0 2.0 3.0 4.0 5.0 2 6 24 48 72

hLMNA_SFw

p=4.28*10-7 p=3.17*10-6 p=7.29*10-7 p=0.33 p=0.46

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SLIDE 24

Clinical Results Patient Sample Pre-analytical Workflow Analytical Assays

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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SLIDE 25
  • 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
  • ACIES
  • 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)

Acknowledgement SPIDIA Consortium Members

Scientific Advisory Board

  • Prof. François Rousseau

(Univ. Laval, Quebec. CanGeneTest Network)

  • Dr. Roberta M. Madej (CLSI)

Project Ethics Committee

  • Dr. Anne Cambon-Thomsen

(CNRS, INSERM, Tolouse, France)

  • Dr. Ruth Chadwick (ESRC

Centre, Cardiff University, UK)

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SLIDE 26

SPIDIA Consortium

Bi-Annual Meeting Berlin November 2011

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SLIDE 27

Questions ? Thank you!