1. V950-001 Overview of PBMC, Serum, Plasma and CSF Samples Holly Lash, Research Biochemist Jennifer Talaty, Medical Program Coordinator

2. PBMC Samples PBMC – Peripheral blood mononuclear cells  Assays  PBMC Network and Non-network  Supplies  Patient Scheduling

3. PBMC Assay  Immunological assay performed by Merck Vaccines and Biological Research to evaluate vaccine responses: ELISPOT Assay: interferon-gamma (IFN-γ) to assess cytokine secretion by T-cells

4. Cell Requirements for IFN-  ELISPOT Assay  High quality peripheral blood mononuclear cells (PBMC) Fresh-frozen equivalent to fresh cell preps. Fresh-frozen better than cells processed from overnight-shipped blood. Cycling of temperatures during storage or exposure to suboptimal (e.g. -20C) during frozen storage is detrimental to sample quality. Careful attention to handling during storage and shipment

5. Degradation in ELISPOT Response During Blood Storage

6. PBMC Labs  PBMC Network Local processing labs provided by Merck through LabCorp Teleconferences with sites in the Network will be conducted by LabCorp  PBMC Non-Network Sites and laboratories processing PBMCs will receive training from Merck

7. PBMC Processing  Blood is collected at all sites  Blood is sent at ambient temperature to: LabCorp Local PBMC lab Site laboratory  The labs will isolate, count and freeze PBMCs  Frozen cells are sent to Merck  Draw to freeze time is <12 hours

8. PBMC Supplies  All supplies will be sent to the site/lab LabCorp will provide supplies to in Network sites Non-Network sites will receive supplies from Merck and Quest  Supplies include: blood draws shipping materials specimen labels and inventory (IN) forms

9. Inventory List (IN) Form

10. Patient Scheduling  Send a schedule to your respective PBMC lab  If changes occur, send those changes to your PBMC lab as soon as possible  Discuss with your PBMC lab the level of detail in and frequency of sending the patient schedule  Communication is key!

11. Serum Samples  Assays  Supplies  Collection and Processing  Packaging  Shipping

12. Serum Assay Serological assays are performed at Merck Vaccines and Biological Research  LUMINEX assay: to assess serum Aβ IgG antibody titers

13. Serum Supplies  Blood draw Red-top tubes and phlebotomy supplies  A list of other acceptable tubes are in the Procedures Manual  Preparation 2 mL Sarstedt tubes (4), 1 mL pipette  Shipping Cell box, STP-320 diagnostic shipping container, dry ice, shipping labels  Specimen labels and IN forms Provided by Merck

14. Serum Collection and Processing  Obtain ~2 mL of blood from patient at each time point into appropriate tube (types of tubes allowed are indicated in the Procedures Manual)  Allow blood to clot at room temperature 30-60 minutes (depending on tube)  Centrifuge at 1000-1300 x g for 10-15 minutes (depending on tube) within 2 hours of clotting  Aliquot into plastic tubes  Freeze samples at -20°C or colder

15. Serum Packaging  Place Sarstedt tubes in cell box  Place cell boxes into clear plastic bag with absorbent material; remove excess air and seal  Place bag into white tyvek envelope and seal  Insert sealed envelope into inner STP-320 box  Place inner box into recessed area of STP-320 insulated shipper  Fill inner core of insulated shipper with (~5-10 kg) of dry ice  Place lid on shipper; place copy of inventory form (INs) inside plastic ziploc bag and tape to top of shipper  Seal shipping box with packaging tape

16. Serum Packaging (cont’d)  Record information on the outside of the shipping box: Primary Investigator Name, Address Vaccine/Study Number (V950-001) Consignee Information Check off “Blood Sample” in Clinical Specimen Box  Affix shipping labels: Biohazard label UN Number/Quantity of Dry Ice Label (record quantity of dry ice here) Air Eligibility *Dry Ice does NOT require a Dangerous Goods Shippers’ Declaration Form.

17. Plasma Samples  Assays  Supplies  Collection and Processing  Packaging  Shipping

18. Plasma Samples Plasma assays are performed at Merck.  Aβ testing to evaluate the AD related biomarkers such as Aβ42, Aβ40

19. Plasma Supplies  Wet Ice  Vacutainer tubes containing EDTA as anticoagulant  3.6 mL NUNC cryotubes (Nalgene Part #366524)  Dry ice

20. Plasma Collection and Processing  Collect 3 mL blood into a Vacutainer tube containing EDTA as the anticoagulant.  Place tube on ice and centrifuge within 30 minutes of collection at 1500 x g for 10 minutes at 4°C. If centrifuge has a breaking feature, make sure that break is turned off before spinning.  Transfer plasma to a prelabeled 3.6 mL Nunc cryotube.  Mix plasma and transfer half (~0.5 mL) to a 2nd prelabeled 3.6-mL Nunc cryotube.  Aliquots of plasma samples should be stored frozen at –70°C until shipment.

21. Cerebrospinal Fluid (CSF) Samples  Assays  Supplies  Collection and Processing  Packaging  Shipping

22. CSF Assay CSF assays for biomarkers will be performed at Merck.  Assay to measure: Aβ40, 42 Tau, P-Tau APP (sAPPα, sAPPβ)

23. CSF Supplies  Wet Ice  Polypropylene conical tubes  Aliquots  3.6 NUNC cryotubes (Nalgene Part #366524)  Dry Ice

24. CSF Collection and Processing  CSF (~15 mL) will be collected from each lumbar puncture directly into polypropylene conical tubes and gently mixed. [One aliquot (~2 mL) will be removed to another polypropylene tube for albumin and cell count analysis (with differential) at a local laboratory].  The remaining CSF will be placed on ice until centrifuged (within 30 min) at 2000 x g for 10 minutes at 2 to 4°C.  Transfer CSF supernatant (do not disturb pellet at bottom of the tube - leave approximately 0.5 mL CSF on pellet) to 6 other polypropylene tubes, mix, and transfer 0.5 mL aliquots to labeled, 3.6 mL Nunc cryotubes.  Freeze aliquots upright and store at -70°C until shipment.

25. Sample Labeling  Bar-coded labels for all samples will be provided by Merck Labels will be color-coded  Ensure all specimens are accurately labeled with the provided labels  Use fine black permanent marker when completing information on the labels

26. Sample Shipping (Serum, Plasma, CSF)  Samples should be kept on dry ice during shipment  Ship only Monday and Tuesday  Fax a copy of the INs form to Merck (Justin Cronk and MPCS)