- EU SPIDIA Project Update -Standardization and Improvement of Generic

Preanalytical Tools and Procedures for In Vitro Diagnostics

5th Annual BRN SymposiumBethesda, February 22nd 2012

Dr. Uwe OelmuellerSPIDIA Coordinator (QIAGEN)

SPIDIA Project History and Goals

Results & StatusNew Technologies & ToolsPan-European GuidelinesBiospecimen 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 ~ 460,000 $ / year in an average German hospital caused by pre-analytical errors

Frost & Sullivan 2011 on behalf of BD

Pan-European guidelines for preanalytics (Molecular – Blood, Tissue)

New pre-analytical tools & technologies (Blood, Plasma, Tissue, Swabs)

Sample quality markers (Blood, Tissue)

Training and dissemination

Project Main Goals

Program European Commission FP7-HEALTH

Consortium 7 public research organizations8 companies1 standards organization (CEN)

Coordinator QIAGEN GmbH

Run Time October 2008 – September 2012 (prolongation request intended)

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

SPIDIA Project Project History and Goals

Results & StatusNew Technologies & ToolsPan-European GuidelinesBiospecimen Quality Markers

Agenda

New Tissue Fixation & StabilizationHistomorphology, IHC, RNA & DNA, Proteins

H&E Staining IDC of Breast

PAXg

ene

Form

alin

Estrogen Receptor a (clone 1D5) IDC of Breast

Kap M. et al., PLoS ONE 6(11): e27704 (2011) Viertler C. et al., submitted for publication Groelz D. et al., unpublished data.

PFPE revealed preservation of morphology and antigenicity comparable to FFPE

5

10

15

20

25

30

35

40

45

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CTN

NB

1C

OL1

A1

FN1

RH

OA

FOS

Gap

dhC

CN

D1

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BIA

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TP53

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2BW

NT1

genes (sorted by Ct cryo)

Ct

CRYOFFPEPFPE

MammacarcinomaTaqMan Array Gene Signature

Nucleic acid analysis superior to FFPE

FFPE

PFPE Cryo

fcNA Profiles in Whole Blood / PlasmaWhat is missing?

Studies for understanding fcDNAand fcRNA profile stability / changes in whole blood and in plasma

Development of fcDNA and fcRNA 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 AspiratesStabilization of morphology, antigenicity, DNA, RNA, proteome

Whole BloodStabilization of cell morphology and biomolecule profiles

SwabsStabilization 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 & StatusNew Technologies & ToolsPan-European GuidelinesBiospecimen 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 will use SPIDIA’s optimized workflows

Guidelines / Standards 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 fcDNA

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, fragmentationHigh 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

195 kb4.36 kb

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

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

L. Barraud et al. Unpublished data

Ref. DNA from UNFI (DIV 54%) Sample 38 (Poor quality) (DIV 32%)

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

Blood RNA Ring Trial Parameters

Purity

Interference substances (RT-qPCR)

Yield

Integrity

RNA Profile Stability / Changes

Human EDTA Blood stored at Room Temperature over 3 days

IL-1β mRNA

Changes of Transcripts Profiles in Blood Individual Samples React Differently

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

Empty bag

- Filled with 39 ml of EDTA solution under sterile condition

- Filled with 461 ml blood from phlebotomy

1 bag - 1 donor

Blood RNA Second Ring Trial Preparation of Blood Samples

BD EST Plastic Tubes

PAXgene Blood RNA Tubes

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) – in press.

Guenther K. et al. Clin Chim Acta. 2012, in press.

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 & StatusNew Technologies & ToolsPan-european GuidelinesBiospecimen Quality Markers

Agenda

Quality markers measuring RNA up- & down-regulation • >180 micro arrays (time course experiments)• 11 marker candidates (specific RNA degradation or gene down regulation,

specific RNA gene induction, random 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*10‐7p=3.17*10‐6

p=7.29*10‐7

p=0.33p=0.46

QIAGEN GmbH - CoordinatorMedical University of Graz (Prof. K. Zatloukal)University of Florence (Prof. M. Pazzagli)CIRMMP Florence, CERM (Prof. I. Bertini)TATAA BiocenterPreAnalytiX GmbHDIAGENIC ASAAros Applied BiotechnologyDako Denmark ACIESBiotechnology Inst. of Czech Academy of Science (Prof. M. Kubista)European Committee for Standardization (CEN)ImmunID TechnologiesErasmus 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 BoardProf. François Rousseau (Univ. Laval, Quebec. CanGeneTest Network)Dr. Roberta M. Madej (CLSI)

Project Ethics CommitteeDr. 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!