1. Biomarker Study Protocol WP 09, VHIR-Barcelona EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201326 slides

2. WP 09 - Biomarker Study OBJECTIVE Hypothermia proof of concept Hypothermia proof of concept Efficacy Safety Biomarkers analysis EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 20131 of 26

3. WP 09 - Biomarker Study PROTOCOL OVERVIEW EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 20132 of 26

4. Tube holder*Serum tubes*Plasma tubes*RNA tube Pipettes Serum capsPlasma capsDNA caps Blood collection Blood aliquotting *Kindly donated by Microtubes WP 09 - Biomarker Study MATERIAL EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 20133 of 26

5. Microtube storage RNA storage Blood storage EuroHYP.1 001:001 A1 PLASMA Patient numberCentre number Sample type Sample time All the material will be correctly packed and labeled WP 09 - Biomarker Study MATERIAL EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 20134 of 26

6. WP 09 - Biomarker Study PROTOCOL EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 20135 of 26

7. Remove the needle cap and perform the venipuncture 10 times Immediatly mix the tube by inverting it top-down Collect blood pressing the tubes to the botton of the holder Take the tube out when is correctlly filled REPEAT FOR EACH TUBE RNA PlasmaSerum *Try to avoid hemolysis during blood collection and mixing WP 09 - Biomarker Study BLOOD COLLECTION EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 20136 of 26

8. WP 09 - Biomarker Study BLOOD PROCESSING Store in the freezer at -80°C RNA tube EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 20137 of 26

9. Centrifuge at 1500g, 4°C for 15 minutes within 4 hours of blood collection Serum tubes Keep in the fridge at 4°C for at least 30 minutes Plasma tubes *Try to balance the centrifuge WP 09 - Biomarker Study BLOOD PROCESSING EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 20138 of 26

10. Serum tubes Plasma tubes WP 09 - Biomarker Study BLOOD PROCESSING EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 20139 of 26

11. In the Patient Sample Rack microtubes are distributed according to sample time and sample type WP 09 - Biomarker Study BLOOD ALIQUOTTING EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201310 of 26

12. WP 09 - Biomarker Study BLOOD ALIQUOTTING Aspirate serum, aliquot and cap *Please do not fill enterely the microtubes to avoid cap release during freezing Transfer to the Sample Storage Rack EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201311 of 26

13. Aspirate plasma, aliquot and capTransfer to the Sample Storage Rack WP 09 - Biomarker Study BLOOD ALIQUOTTING EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201312 of 26

14. WP 09 - Biomarker Study BLOOD ALIQUOTTING Aspirate buffy coat, aliquot and capTransfer to the Sample Storage Rack EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201313 of 26

15. WP 09 - Biomarker Study SAMPLE STORAGE Store in the freezer at -80°CSample Storage Rack EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201314 of 26

16. Complete the eCRF Biomarker Information for the corresponding patient WP 09 - Biomarker Study DATA RECORD EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201315 of 26

17. WP 09 - Biomarker Study DATA RECORD Complete the Sample Data Record sheet Complete Bioimarker Information on the Source Document EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201316 of 26

18. WP 09 - Biomarker Study DATA RECORD Obtain and securely store the Patient Informed Consent EuroHYP-1 Biomarker Study Informed Consent EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201317 of 26

19. WP 09 - Biomarker Study SAMPLE SHIPMENT Send stored samples to the EuroHYP-1 Biobank at Barcelona RNA Storage Rack Sample Storage Rack EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201318 of 26

20. WP 09 - Biomarker Study SAMPLE SHIPMENT Program the shipment wih the Study Coordination and send by way of the Study’s Courier Polystyrene boxDry ice Class 9 labelUN1845 labelUN3373 label EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201319 of 26

21. WP 09 - Biomarker Study SAMPLE SHIPMENT Sample Data Record sheetInformed Consent Custody Certificate Include important documents EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201320 of 26

22. WP 09 - Biomarker Study SAMPLE SHIPMENT *Non-EU countries Include important documents for sample shipment through customs Proforma invoice* EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201321 of 26

23. WP 09 - Biomarker Study SAMPLE SHIPMENT Record sample shipment on the eCRF EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201322 of 26

24. WP 09 - Biomarker Study SAMPLE SHIPMENT Complete the data for shipment registry and management EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201323 of 26

25. WP 09 - Biomarker Study SAMPLE SHIPMENT Ask for more Patient Sample Material EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201324 of 26

26. WP 09 - Biomarker Study SAMPLE SHIPMENT Complete the form for material consignment EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201325 of 26

27. Written and Video versions of the Biomarker Protocol will be available at: —EuroHYP-1 Training Portal —Milliarium Platform → Documents → SOPs —http://goo.gl/XpCfxhttp://goo.gl/XpCfx For further information regarding the Biomarker Study please contact Biomarker Study Coordination at info.lin.bcn@gmail.com info.lin.bcn@gmail.com Neurovascular Research Laboratory Vall d’Hebrón Research Institute Mediterrania Building Pg. Vall d’Hebrón, 119-129 08035 Barcelona Phone: +34 93 489 40 29 Fax: +34 93 489 40 15 WP 09 - Biomarker Study MORE INFO EudraCT-No.: 2012-002944-25Biomarker Study, Protocol Training, v1.0, June 201326 of 26