INVITATION

1. INVITATION The joint Working Group “Personalized laboratory Medicine” of the EFLM and ESPT societies, has planned the Questionnaire: “Is Laboratory Medicine ready to the era of Personalized Medicine?” Pleaseletusknowyourpoint of view, and visit: https://it.surveymonkey.com/s/WG-PLM-questionnaire Questions: Contact me at: mario.pazzagli@unifi.it

2. Standardization of the pre‐analytical phase for molecular methods in blood Mario Pazzagli University of Florence

3. Standardization…of the pre‐analytical phase for molecular methods in blood NOTES FROM THE EuropeanCommittee for Standardization (www.cen.eu) • European Standards (ENs) are based on a consensus, which reflects the economic and social interests of 33 CEN Member countries channelled through their National Standardization Organizations. • Besides European Standards, CEN produces other reference documents, which can be developed quickly and easily: Technical Specifications, Technical Reports, and Workshop Agreements. • European Standard – EN • Goal: Development of normative specifications reflecting the current state of technology • European Technical Specification – CEN/TS • Goal: Specifications which aid market development and growth • European Technical Report – CEN/TR • Goal: Specifications of a recommendatory and explanatory nature • CEN Workshop Agreement – CWA • Goal: Special specifications developed with the rapid consensus of expert stakeholders

4. CEN-Technical Specifications(definition from www.cen.eu)  • A Technical Specification (TS) is a normative document, the development of which can be envisaged when various alternatives that would not gather enough as to allow agreement on a European Standard (EN), need to coexist in anticipation of future harmonization, or for providing specifications in experimental circumstances and/or evolving technologies.

5. Standardization of the pre‐analytical phase for molecular methods in blood preanalytical phase pre-examination processes preanalytical workflow processes that start, in chronological order, from the clinicians' request and include the examination request, preparation and identification of the patient, collection of the primary sample(s), temporary storage, transportation to and within the analytical laboratory, aliquoting, retrieval, isolation of analytes, and end when the analytical examination begins. SOURCE: EN ISO 15189:2012, definition 3.15, modified

6. Standardization of the pre‐analytical phase for molecular methods in blood Whichone? Genomic DNA Cellular RNA Circulatingcell free DNA Others notconsidered by the CEN/TC 140 WG3... Pathogen DNA/RNA Circulatingcell free RNA …..

7. BACKGROUND • Recent discoveries have revealed that RNA or DNA in blood can serve as biomarkers for early and reliable molecular diagnosis of important diseases. • Further discoveries have shown that the cellular profiles of these molecules in blood samples can change after collection during transport and storage, thus making clinical assay results and pharmaceutical research unreliable or even impossible. • It is therefore a decisive prerequisite for future and current diagnostic assays to develop standards (and eventually new technologies, tools and devices) that eliminate the errors in the pre-analytical steps.

8. SPIDIA • SEVENTH FRAMEWORK PROGRAMME • THEME 1 – HEALTH • Collaborative Project – 17 Partners • Project acronym: SPIDIA • Project full title: Standardisation and improvement of generic pre-analytical tools and procedures for in vitro diagnostics • Budget: 9 Million euro • Starting Date: October 1, 2008 • Duration: 4 years • Web site: www.spidia.eu

9. THE EU SPIDIA PROJECT: activities for blood specimens • AIM: Identification of critical steps in the pre-analytical phase of blood samples and evaluate solutions • Analytical Targets: • 1. Genomic DNA from whole blood, • 2. Circulating Cell-free DNA, from plasma • 3. Cellular RNA from whole blood • Experimental Model: • Implementation of Pan-europeanring trials (SPIDIA-DNA, SPIDIA-RNA, SPIDIA-DNAplas) with EFLM support, and time courses experiments. • SPIDIA contribution to Standardization: • THE RESULTS OF SPIDIA AVAILABLE TO CEN/TC 140 WG3 TO BE CONSIDERED FOR THE DEVELOPMENT OF OFFICIAL DOCUMENTS

10. GENOMIC DNA in BLOOD • CEN/TC 140 • Date:  2014-05 • TC 140 WI 00140096 • Molecular in-vitro diagnostic examinations — Specifications for pre-examination processes for blood — Genomic DNA

11. Scope This Technical Specification recommends the handling, documentation and processing of blood specimens intended for genomic DNA analysis during the preanalytical phase before a molecular assay is performed. This Technical Specification is applicable to molecular in-vitro diagnostic examinations (e.g., in-vitro diagnostic laboratories, laboratory customers, in-vitro diagnostics developers and manufacturers, institutions and commercial organizations performing biomedical research, biobanks, and regulatory authorities).

12. General Considerations • Blood genomic DNA can fragment or degrade after blood collection. • Special measures have to be taken to secure good quality blood samples for genomic DNA analysis. • This is particularly relevant for analytical test procedures requiring high molecular weight DNA. • Note: high molecular weight DNA HMW DNA, i.e. DNA larger than 50 kbp

13. Influence of preanalytical variables (blood storage time and temperature and DNA extraction methods) on high molecular weight (HMW) DNA integrity Panel A depicts the HMW DNA pattern of samples extracted by precipitation methods immediately after blood collection (To). M= ladder marker, a…g = Each line represents a DNA sample extracted immediately after blood collection Panel B shows the HMW DNA pattern of samples extracted by participating laboratories in the SPIDIA-DNA External Quality Assessment (EQA). All the participants received the same blood sample collected in K2EDTA tubes and performed DNA extraction using their own procedure, storing the blood following their own practise. The time storage range from to 2 days up to 30 days after blood collection and the methods used were based on magnetic beads, column based and precipitation. Blood was stored at different temperatures: room temperature and 4 °C. M= ladder marker, 1…84 = Each line represents the DNA extracted by each participant laboratory

14. Influence of High Molecular Weight (HMW) DNA integrity on an analytical test based on long PCR amplicons TCR rearrangement evaluated using a diagnostic kit [14] obtained in a subset of DNA samples (n = 15). of first SPIDIA DNA External Quality Assessment (EQA). The DNA samples were clustered depending on the HMW DNA profile as “fragmented”, “intermediate”, “intact” on the bases of Pulsed Field Gel Electrophoresis (PFGE) evaluation. The diagnostic test, based on long PCR amplicons, evaluates the T cell receptor rearrangement by multiplex-PCR using an upstream primer specific for a given Vx family and a downstream shared by Jy segment.

15. Primary blood sample storage requirements • When using blood collection tubes without stabilizers and no requirements on the storage conditions are available, the primary blood samples should be processed as soon as possible. • For the analysis of high molecular weight DNA the blood sample can be stored at room temperature or at 2 °C to 8 °C for up to two days.

16. CELLULAR RNA in BLOOD • CEN/TC 140 • Date:  2014-05 • TC 140 WI 00140092 • Molecular in-vitro diagnostic examinations — Specifications for pre-examination processes for blood — Cellular RNA

17. Scope This Technical Specification recommends the handling, documentation and processing of blood specimens intended for cellular RNA analysis during the preanalytical phase before a molecular assay is performed. This Technical Specification is applicable to molecular in-vitro diagnostic examinations (e.g., in-vitro diagnostic laboratories, laboratory customers, in-vitro diagnostics developers and manufacturers, institutions and commercial organizations performing biomedical research, biobanks, and regulatory authorities).

18. General Considerations • Due to the high instability of blood cellular RNA profiles in individual patients/donors, commercially available blood collection tubes containing blood cellular RNA profile stabilizers should be used. • Alternatively, during the design of the analytical test system, it is strongly recommended to investigate, if the specific blood cellular RNA molecule/s amount/s intended to be analysed in the analytical test is/are not affected by the entire preanalytical workflow.

19. Example of Stable genes (GAPDH) Expression of GAPDH [GAPDH: GAPDH Glyceraldehyde 3 –Phospohate dehydrogenase] stable gene in blood collected in tube with/without stabilizer. The same blood sample was collected in blood collection tubes without stabilizer (K2EDTA tube, EDTA, n = 78) or with stabilizer (PAXgene blood RNA tube), PAX, n = 28). The blood samples were stored at 4 °C and the extraction was performed within 24 h. The results were clustered depending on the blood collection tube: EDTA and PAX. PAXgeneblood RNA tube is the trade name of a product supplied by QIAGEN GmbH. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by CEN or CENELEC of the product named. Equivalent products may be used if they can be shown to lead to the same results.

20. Example of unstable genes (IL8 and FOS) NOTE: changes in expressionlevels of individual RNA molecules are NOT associated with RNA quality parameters evaluated by generally accepted physical, chemical and biochemical procedures (e.g., wavelength scan, A 260/A 280 ratio, cellular RNA integrity number (RIN) by electrophoresis, etc., Expression of 2 unstable genes in blood collected in tubes with or without cellular RNA stabilizer stored 48h at Room temperature (RT) or 4 °C. The same blood sample was collected in blood collection tubes without stabilizer (K2EDTA tube, EDTA, n = 78) or with stabilizer (PAXgene blood RNA tube), PAX, n = 28). The blood in PAXgene blood RNA tubes was stored at RT (PAX-RT, n = 28), the blood collected in K2EDTA tubes was stored at 4 °C (EDTA-4°C, n = 39) or RT (EDTA-RT, n = 39) and the extraction was performed at 48 h after blood collection. The results were clustered depending on the blood collection tube and temperature storage: PAX-RT, EDTA-4°C, and EDTA-RT. PAXgeneblood RNA tube is the trade name of a product supplied by QIAGEN GmbH. This information is given for the convenience of users of this International Standard and does not constitute an endorsement by CEN or CENELEC of the product named. Equivalent products may be used if they can be shown to lead to the same results.

21. Circulatingcell free DNA • CEN/TC 140 • Date:  2014-04 • TC 140 WI 00140091 • Molecular in-vitro diagnostic examinations — Specifications for pre-examination processes for blood — Circulating cell free DNA

22. Scope • This Technical Specification recommends the handling, documentation and processing of blood specimens intended for circulating cell free DNA (ccfDNA) analysis during the preanalytical phase before a molecular assay is performed. • This Technical Specification is applicable to molecular in-vitro diagnostic examinations (e.g., in-vitro diagnostic laboratories, laboratory customers, in-vitro diagnostics developers and manufacturers, institutions and commercial organizations performing biomedical research, biobanks, and regulatory authorities).

23. General Considerations • Blood ccfDNA profiles can change significantly after blood collection from the donor (e.g., release of genomic DNA from white blood cells) • Lack of Reference Materials and Methods for ccfDNAfragmentation and ccfDNA quantity analysis. No consensus in literature • Special measures have to be taken to secure good quality blood samples for ccfDNA analysis.

24. Influence of blood collection tubes and storage time on ccfDNA recovery amount • Adapted from: Norton SE, Lechner JM, Williams T, Fernando MR. A stabilizing reagent prevents cell-free DNA contamination by cellular DNA in plasma during blood storage and shipping as determined by digital PCR. (2013) Chinical Biochemistry. 46: 1561-1565

25. CONCLUSIONS • Three CEN official documents are under development by CEN/TC 140 WG3 on: Standardization of the pre‐analytical phase for molecular methods in blood (Genomic DNA, Cellular RNA, circulating cell-free DNA). • The important role of specifically designed blood collection tubes should be considered • IFCC (the Molecular Diagnostics Committee (C-MD) is also contributing to the development of these documents. • They should be available within 2014. • A proposal to ISO for thesedocumentsis under evaluation

26. Acknowledgements SPIDIA-EQAs participants (420 laboratories) SPIDIA WP 1.2 partners: - QIAGEN - UNIFI - IRCCS Milano - TATAA biocenter - Labonnet - ImmunID SPIDIA team UNIFI team This work has received funding from the European Union Framework Programme 7 under grant agreement no. 222916, SPIDIA project: www.spidia.eu 

27. INVITATION The joint Working Group “Personalized laboratory Medicine” of the EFLM and ESPT societies, has planned the Questionnaire: “Is Laboratory Medicine ready to the era of Personalized Medicine?” Pleaseletusknowyourpoint of view, and visit: https://it.surveymonkey.com/s/WG-PLM-questionnaire Questions: Contact me at: mario.pazzagli@unifi.it