- EU SPIDIA Project Update - Standardization and Improvement of - - PowerPoint PPT Presentation

• EU SPIDIA Project Update -

Standardization and Improvement of Generic Preanalytical Tools and Procedures for In Vitro Diagnostics

5th Annual BRN Symposium Bethesda, February 22nd 2012

• Dr. Uwe Oelmueller

SPIDIA Coordinator (QIAGEN)

SPIDIA Project History and Goals

Results & Status

New Technologies & Tools Pan-European Guidelines Biospecimen Quality Markers

Agenda

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

“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 organizations 8 companies 1 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 & Status

New Technologies & Tools

Pan-European Guidelines Biospecimen Quality Markers

Agenda

New Tissue Fixation & Stabilization

Histomorphology, IHC, RNA & DNA, Proteins

H&E Staining IDC of Breast

PAXgene Formalin

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of Breast Kap M. et al., PLoS ONE 6(11): e27704 (2011) Viertler C. et al., submitted for publication Groelz D. et al., unpublished data.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 50

1 8 s C T N N B 1 C O L 1 A 1 F N 1 R H O A F O S G a p d h C C N D 1 S P P 1 E R B B 2 I G F 1 R C D H 1 N F K B I A A K T 1 C D K N 1 B B C L 2 L 1 C D K 4 S H C 1 I T G A V T P 5 3 A K T 2 G S K 3 B B A X C D K N 1 A I T G B 1 T G F B 1 A B L 1 H P R T 1 M A P K 1 4 T C F 3 S R C N F K B 1 G U S B E L K 1 V E G F A K R A S G R B 2 B C A R 1 R A C 1 B C L 2 S M A D 4 M D M 2 C R K N F K B 2 B R A F M A P K 3 F A D D P T E N C C N D 3 J U N R E L A P T K 2 D V L 1 P I K 3 R 1 M Y C C D K 2 N R A S T G F B R 2 C D K N 2 A M A P K 1 M A P 2 K 1 R B 1 H R A S T G F B R 1 P T K 2 B E 2 F 1 A P C F Z D 1 C A S P 9 S O S 1 C A S P 8 I T G B 3 B I D K D R B C L 2 L 1 1 M A P K 8 R A F 1 P I K 3 C A C C N D 2 M A P 3 K 5 F A S C C N E 1 F Y N L E F 1 F G F 2 E G F R I G F 1 C Y C S H G F F A S L G I T G A 2 B K I T C D C 4 2 M A X C D K N 2 B W N T 1

genes (sorted by Ct cryo)

C t CRYO FFPE PFPE

Mammacarcinoma TaqMan Array Gene Signature Nucleic acid analysis superior to FFPE

FFPE PFPE Cryo

fcNA Profiles in Whole Blood / Plasma What is missing?

Studies for understanding fcDNA and 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 fcDNA pattern 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

Biospecimen 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 % 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 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, 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

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)

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 β β β β 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 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.

SPIDIA Project Project History and Goals

Results & Status

New Technologies & Tools Pan-european Guidelines Biospecimen 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 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

• 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)

SPIDIA Consortium

Bi-Annual Meeting Berlin November 2011

Questions ? Thank you!