- 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 9 th 2012 Dr. Daniel Grölz SPIDIA (QIAGEN)
Agenda Standardization tandardization and Improvement of Generic and Improvement of Generic S Pre re- -analytical Tools and Procedures for analytical Tools and Procedures for P In Vitro n Vitro Dia Diagnostics gnostics I � SPIDIA � Project Facts & Goals � New Technologies & Tools � Guidelines & Dissemination � Evaluation of PAXgene Tissue
Molecular Diagnostic Workflow From Patients to Clinical Results Patient Pre-analytical Clinical Patient Analytical Assays Sample Workflow Results Sample Sample Bioanalyte Patient Anesthesia Collection Preparation Treatment Logistics e.g. Drugs e.g. Arterial Life Style Clamp Time Time 0
It is Real Problem “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. Preanalytics Costs of ~ 460,000 $ / year in 68 % an average German hospital Analytics caused by pre-analytical errors 13 % Frost & Sullivan 2011 on behalf of BD 19 % Postanalytics
Improvements Needed � 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
Project Main Goals � 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 Facts � 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
Agenda Standardization tandardization and Improvement of Generic and Improvement of Generic S Pre re- -analytical Tools and Procedures for analytical Tools and Procedures for P In Vitro n Vitro Dia Diagnostics gnostics I � SPIDIA � Project Facts & Goals � New Technologies & Tools � Pan-European Guidelines & Dissemination � Evaluation of PAXgene Tissue
New Technology & Tools Ongoing Developments � 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)
Sample Quality Marker Discovery � 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
Agenda Standardization tandardization and Improvement of Generic and Improvement of Generic S Pre re- -analytical Tools and Procedures for analytical Tools and Procedures for P In Vitro n Vitro Dia Diagnostics gnostics I � SPIDIA � Project Facts & Goals � New Technologies & Tools � Pan-European Guidelines & Dissemination � Evaluation of PAXgene Tissue
Evidence Based Guidelines Examples Blood DNA & RNA, Plasma ccfDNA � 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 No. of Participants Percentage of Participants (29 who sent NA NA samples SPIDIA Trials countries) samples back sent back Blood RNA 102 93 91 % Blood DNA 130 121 93 % Plasma DNA 67 62 93 % Total 299 276 92 %
Individual Report for Each Laboratory SPIDIA – DNAplas: Laboratory report SPIDIA – DNAplas: Laboratory report SPIDIA – DNAplas: Laboratory report Lab ID: L134 Lab ID: L134 Lab ID: L134 B. Purity and Quantity of DNA4 (DNA extracted from blood) D. Quantification of RNaseP by real-time PCR on DNA4 and evaluation of interferences C. Integrity of DNA4 (DNA extracted from blood) B.1 Spectrophotometric data provided by your lab and by SPIDIA lab D.1 Your lab ( ) versus overall distribution C.1 Your lab ( ) - Pulse field gel electrophoresis image 260nm 280nm 320nm Purity Quantity (ng/µl) Purity Quantity (ng/µl) Dilution Extraction Elution Buffer Lab Lab Lab Lab Lab factor vol. (ul) vol. (ul) [kb] Spidia Spidia Quantification of RNaseP Interferences AE-buffer 194,0 In the figure the two lines represent the two Kineret threshold for 0.014 0.007 . 2 28 2.049 25.1 40 200 200 In the figure the blue line represents the Action Limit (AL) and Qiagen 145,5 the gray line represents the Warning Limit (WL). outliers identification: 5.99 (weak outlier) and 9.21 (strong outlier). B.2 Additional information provided by your lab 97,0 Temperature of DNA 1000 60 Extraction Spectrophotometer Time interval (hours) zoom zoom storage 950 300 10 48,5 900 arrival to extraction arrival to extraction 9 producer supplier producer supplier 850 extraction 250 50 extraction to analysis to analysis 23,1 8 800 Qiagen QiaAmp Blood Mini kit (51106) GE healtcare GeneQuant Pro -20° C 20° C 92.5 h 1 h Quantity(ng/µl blood) 750 7 200 700 Kineret distance 9,42 40 6 B.3 Your lab ( ) versus overall distribution – Purity 650 150 5 600 In the figures the blue lines represent the Action Limits (ALs) and the gray lines represent the Warning Limits (WLs). 6,55 550 4 500 100 30 3.5 3.5 4,36 zoom zoom 3 2.5 2.5 450 400 2 50 350 20 1 300 3.0 3.0 0 0 250 2.0 200 2,32 10 150 AL=11.72; WL=52.80 2,03 2.0 100 2.5 2.5 Purity Purity 50 0 0 1.5 DNA4 DNA4 2.0 2.0 (N=172) (N=167) C.2 ImageJ data of your lab in control in control 1.0 1.5 Minimum (Kb) Maximum (Kb) Peak (Kb) 1.5 1.5 1.011 101.034 19.139 Lower AL=1.26; Upper AL=2.12 Lower AL=1.57; Upper AL=2.28 Lower WL=1.76; Upper WL=2.14 Lower WL=1.55; Upper WL=1.96 C.3 Your lab ( ) versus overall distribution (N=157) – ImageJ data 1.0 1.0 In the figure the blue line represents the Action Limit (AL) and the gray line represents the Warning Limit (WL). DNA4 Lab DNA4 Spidia 10 350 40 (N=167) (N=176) 9 warning in control 35 300 8 30 B.4 Your lab ( ) versus overall distribution - Quantity 250 7 Maximum (kb) In the figures the blue line represents the Action Limit (AL) and the gray line represents the Warning Limit (WL). Minimum (kb) 25 Peak (kb) 6 1000 1000 200 zoom zoom 950 950 50 50 5 20 900 900 850 850 150 4 800 40 800 40 15 Quantity (ng/µl blood) Quantity (ng/µl blood) 750 750 3 700 700 100 30 30 10 650 650 2 600 600 50 550 550 5 20 20 1 500 500 450 450 0 0 0 400 400 10 10 350 350 DNA4 DNA4 DNA4 300 300 250 0 250 0 AL=0.70; WL=1.15 AL=36.29; WL=53.21 AL=14.15; WL=17.83 200 200 150 AL=2.62; WL=10.12 150 AL=2.91; WL=9.79 warning in control in control 100 100 50 50 0 0 DNA4 Lab DNA4 Spidia (N=168) (N=174) in control in control 1 3 2 Pazzagli et al.. University of Florence – manuscript in preparation Statistical analysis by Verderio P. et al. (IRCCS, Milano)
Dissemination � 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)
Agenda Standardization tandardization and Improvement of Generic and Improvement of Generic S Pre re- -analytical Tools and Procedures for analytical Tools and Procedures for P In Vitro n Vitro Dia Diagnostics gnostics I � SPIDIA � Project Facts & Goals � New Technologies & Tools � Pan-European Guidelines & Dissemination � Evaluation of PAXgene Tissue � PAXgene Tissue System & Workflow � Evaluation studies
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