V950-001Overview of PBMC, Serum, Plasma and CSF Samples

Holly Lash, Research BiochemistJennifer Talaty, Medical Program Coordinator

PBMC Samples

PBMC – Peripheral blood mononuclear cells

Assays PBMC Network and Non-network Supplies Patient Scheduling

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

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. -20C) during frozen storage is detrimental to sample quality.

Careful attention to handling during storage and shipment

Degradation in ELISPOT Response During Blood Storage

Time (Hr) of Initiation of Blood Processing*

VZV

(CD4)

VE peptide pool

(CD8)

PHA

(mitogen)

SEB

(super antigen)

0 0.00% 0.00% 0.00% 0.00% 4 22.87% 9.03% 10.68% 9.84% 6 32.27% 13.24% 15.58% 14.39% 8 40.52% 17.25% 20.22% 18.72% 10 47.76% 21.08% 24.60% 22.82% 12 54.12% 24.73% 28.74% 26.72%

24** 78.95% 43.34% 49.22% 46.30%

Avg Loss/Hr 95% CI

6.29%

(4.24%,8.29%)

2.34%

(1.23%,3.43%)

2.78%

(1.88%,3.68%)

2.56%

(1.76%,3.35%)

Values are means from 5 blood donors. * Time from initiation of blood processing to cell freezing was approximately 2.5 hr. ** Extrapolated Time Point.

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

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

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

Inventory List (IN) Form

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!

Serum Samples

Assays Supplies Collection and Processing Packaging Shipping

Serum Assay

Serological assays are performed at Merck Vaccines and Biological Research

LUMINEX assay: to assess serum Aβ IgG antibody titers

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

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

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

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.

Plasma Samples

Assays Supplies Collection and Processing Packaging Shipping

Plasma Samples

Plasma assays are performed at Merck.

Aβ testing to evaluate the AD related biomarkers such as Aβ42, Aβ40

Plasma Supplies

Wet Ice Vacutainer tubes containing EDTA

as anticoagulant 3.6 mL NUNC cryotubes (Nalgene

Part #366524) Dry ice

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.

Cerebrospinal Fluid (CSF) Samples

Assays Supplies Collection and Processing Packaging Shipping

CSF Assay

CSF assays for biomarkers will be performed at Merck.

Assay to measure: Aβ40, 42 Tau, P-Tau APP (sAPPα, sAPPβ)

CSF Supplies

Wet Ice Polypropylene conical tubes Aliquots 3.6 NUNC cryotubes (Nalgene Part

#366524) Dry Ice

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.

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

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)