2. Turn on blower in hood. 3. While wearing gloves, clean the hood with sterile gauze pads and 70% ethanol. 4. Lay out the sterile field in the hood. 5. Open the packages of sterile gauze packets. Allow gauze to fall onto sterile field. 6. Lay all other required items inside the hood (not on the sterile field). 7. Remove the protective covers to expose the stop pers on the sterile water, 12-MP and Tet vials. 8. Wipe the rubber stopper of the 12-MP, Tet and st erile water using a new alcohol swab for each vial. 9. Place a 19 gauge needle on a plastic 5 mL syringe. Uncap the needle and withdraw 1.5 ml of sterile water. 10. Inject the 1.5 ml of sterile water into the 12-MP vial. Swirl until dissolved. This solution is now at 100 mcg/ml. 11. Using the same 5 mL plastic sy ringe and needle, withdraw all of the 12-MP solution (1.5 ml). 12. Inject the 1.5 ml of the 12-MP solution into the Tet vial. Swirl until dissolved. This now contains 100 mcg/ml of each 12MP plus 200 mcg/ml of tetanus peptide. 13. Using the same 5 mL plastic syri nge and needle, withdraw all of the peptide solution (1.5 ml). Remove any air from the syringe and remove the needle. 14. Remove a cap from the I-connector. Attach the sy ringe containing the pep tide solution to the I- connector. 15. Lay the 5 mL syringe containing the peptide solution and I-connector on the sterile field. Montanide Preparation – 16. Wipe the top of the vial of Mont anide ISA-51 with an alcohol swab. 17. Place a new 19 gauge needle on the second 5 mL syringe. Uncap the needle and withdraw 1.5 ml of Montanide ISA-51. 18. Remove the other cap from the I-connector. Atta ch the syringe containing the Montanide ISA- 51 to the I-connector. Mixing and Testing 19. Hold the syringe/connector/syringe system firmly to guarantee a constant connection. Thumbs will be used to push the plungers apart. (To avoid any leak, do not push with both of the thumbs simultaneously). 20. Push completely on the plunger of one of the syringes in order to get both phases in one syringe. 21. Start to emulsify by transferring alternatively the formulation from one syringe to the other very slowly. NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 90 22. The first 20 cycles (one cycle corresponds to the passage of th e entire formulation from one container to the other through th e connector and back) are done at slow rhythm. A complete cycle requires an average of 4 seconds. The 20 cycles should take 1 minute and a half. 23. Once 20 cycles are complete, the speed is dramat ically increased for 40 remaining cycles. This speed is as fast as possible. A timer can be set for 40 seconds to avoid losing count. 24. Transfer the entire emulsion into one syringe. 25. Disconnect the syringe from the connector. 26. Twist a sterile 25 gauge needle onto the full sy ringe and drop 1-2 drops of the emulsion to a container of de-ionized water. If the drops disperse in the water, mix for an additional 40 seconds and recheck as described. Th is test may be repeated twice. Preparing the vaccine for injection 27. Inject excess emulsion (down to 2 ml in syringe) onto a sterile gauze pad. Tap on the syringe to void any bubbles. This 2 ml now contains 100 mcg of e ach of the 12MP plus 200 mcg of tetanus peptide plus 1 ml Montanide ISA-51 VG. 28. Recap the needle and pull back on the piston slightly. 29. Affix the patient information label to the syringe an d add an additional label inside the Ziploc bag. 30. Place the syringe into the Ziploc bag for transpor t and attach an additional label to the bag for identification Documentation & Clean-up 31. Dispose of the syringes, needles, and vials in a cont aminated materials container. Dispose of all other packaging in a regular trash can. 32. Complete paperwork. The remaining vaccine should be discarded. The prepared vaccine should be refrigerated until just before administration. Ideally, the vaccine should be administered within 1–2 hours after mixing. The vaccine must be administered within 4 hours of mixing. If the vaccine is not administered within 4 hours after mixing, it should be discarded. NCI Protocol #: CITN-04 Version Date: 27 Jan 2016 91 APPENDIX F PATIENT MEDICATION DIARY Patient ID: ____________________ Site ID: _______________________ Cycle: ________________________ STUDY MEDICATION: INCB024360 Date Time A.M. Time P.M.