Bedeutung des EU Spidia Projekts für Biobanken

Standardization and Improvement of Generic PreanalyticalTools and Procedures for In Vitro Diagno stics

11. JahrestagungSektion Molekulare Diagnostik der DGKL

Tutzing, 10. May 2012

Dr. Uwe OelmüllerQIAGEN GmbH (Koordinator)

� SPIDIA Project History and Goals

� Results & Status� New Technologies & Tools� Pan-European Guidelines� Sample Quality Markers

Agenda

Clinical Results

Patient Sample

Pre-analytical Workflow

Analytical Assays

Diagnostic WorkflowFrom Patients to Clinical Results

Patient

Sample Collection

SampleLogistics

BioanalytePreparation

Anesthesiae.g. Drugse.g. Arterial Clamp Time

Patient Treatment

Life Style

Time 0

“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

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

� Consortium 7 public research organizations8 companies1 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

� SPIDIA Project Project History and Goals

� Results & Status� New Technologies & Tools� Pan-European Guidelines� Sample Quality Markers

Agenda

DNA

RNA

miRNA

IHC

H&E

WesternBlot

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 testingfrom the same specimen

For Research Use Only

ProcessStabilizeFixResect& Gross

New Tissue Collection & StabilizationPAXgene Tissue

Histomorphology and IHCResearch Study - PFPE vs. FFPE

H&E Staining IDC of Breast

PF

PE

FF

PE

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 antige nicity comparable to FFPE

Mammacarcinoma CaseRNA Profiling – Cryo vs FFPE vs PFPE

Viertler et al., Journal of Molecular Diagnostics 2012, accepted for publication).Groelz et al., unpublished data

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genes (sorted by Ct cryo)

Ct

CRYOFFPEPFPE

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

ccfNA Profiles in Whole BloodWhat is missing?

� Studies for understanding fcDNAand 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 fcDNApattern stability was determined by separating the purified plasma DNA on a 2100 Agilent Bioanalyzer

� 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

� SPIDIA Project Project History and Goals

� Results & Status� New Technologies & Tools

� Pan-European Guidelines� Sample Quality Markers

Agenda

� 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 %276299Total

93 %6267Plasma DNA

93 %121130Blood DNA

91 %93102Blood RNA

Percentage of NA samples sent back

Participants who sent NA samples back

No. ofParticipants (29

countries)SPIDIA Trials

Evidence Based Guidelines Examples Blood DNA & RNA, Plasma ccfDNA

� 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

� 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 resultsPazzagli M. et al., manuscript in preparation

195 kb4.36 kb

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)

Human EDTA Blood stored at Room Temperature over 3 da ys

IL-1ββββ mRNA

Individual Samples React DifferentlyChanges of Transcripts Profiles in Blood

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

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

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.

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

Blood Sample Shipment - RNA Profile ChangesStabilized 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.

� SPIDIA Project Project History and Goals

� Results & Status� New Technologies & Tools� Pan-european Guidelines� Sample Quality Markers

Agenda

� 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

0 2 6 24 48 72

hLMNA_SFw

p=4.28*10-7

p=3.17*10-6

p=7.29*10-7

p=0.33p=0.46

Clinical Results

Patient Sample

Pre-analytical Workflow

Analytical Assays

Diagnostic WorkflowFrom Patients to Clinical Results

Patient

Sample Collection

SampleLogistics

BioanalytePreparation

Anesthesiae.g. Drugse.g. Arterial Clamp Time

Patient Treatment

Life Style

Time 0

� 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)

AcknowledgementSPIDIA 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)

SPIDIA ConsortiumBi-Annual Meeting Berlin November 2011

Questions ?

Thank you!