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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)