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Duke Human Vaccine Institute
OUTLINE• IQA Proficiency Testing Program
– Purpose– Workflow– Reminders
• PBMC Processing Methods– Isolation – Cell Counts– Freezing– Thawing
• Troubleshooting– Processing Errors– Common Clerical Errors– Poor Technique For Thawing PBMC
How Does The Cryopreservation Proficiency Program Work?
• To assess a laboratory’s ability to process, cryopreserve and ship viable PBMC specimens.
• Troubleshooting efforts would include conference calls, site visits or wet lab sessions performed at the IQA laboratory.
Website: www.iqa.center.duke.edu
Currently there are 65 domestic and 28 international laboratories enrolled in the program.
IQA Global Laboratory Participants in the Cryopreservation Proficiency Testing Program
93 participating laboratories
65 in North America
11 in Central and South America
11 in Africa
6 in Pacific Rim
IQA Sample Workflow
• Laboratories obtain and process samples quarterly.
• Frozen PBMC samples are shipped overnight to the IQA and tracking number is provided to IQA.
• IQA receives shipments, cryo samples are unpacked and inspected.
• LDMS information is uploaded.
IQA Sample Workflow
• Vials are stored in a vapor phase liquid nitrogen tank.
• Empty boxes are returned to labs if prepaid return label was provided to IQA.
• Samples are removed from vapor phase liquid nitrogen storage for evaluation.
• Vials that do not meet network criteria are confirmed by testing the second vial.
IQA Sample Workflow• Create/distribute summary spreadsheet.
• Laboratories are emailed to retrieve performance report on the IQA website (iqa.center.duke.edu).
• One week later the IQA begins troubleshooting unsatisfactory laboratory performances.
How Does The IQA Remind Labs To Participate In The Program?
• Laboratories remindersLaboratories reminders– Yearly Calendar (IQA website)– Email Participation Reminder (10 weeks prior to
shipping )– Email Collection Notification (1 week prior to
sample collection)– Email Shipment Notification (1-2 weeks prior to
due date)
Safety• Treat all human-derived specimens as infectious
using universal safety precautions.
• Wear a full-face shield and cryo-gloves duringhandling of frozen samples.
• Wear appropriate personal protective equipment.
• Process in Class II biological safety cabinet.
Peripheral Blood Peripheral Blood Mononuclear Cells (PBMC) Mononuclear Cells (PBMC)
IsolationIsolation
Plasma Isolation
Centrifuge EDTA , Heparin or ACD blood at 400 x g for 10 minutes.
Buffy Coat
Plasma
Basic PBMC Isolation Procedure
Diluted anticoagulated whole blood
Ficoll®
PBMC can be isolated from whole blood samples using different density gradient centrifugation procedures such as:
• Manual Ficoll®• Cell Separation Tube with Frit Barrier (CSTFB) • CPT tubes
Anticoagulated whole blood is layered over the separating medium:
plasma/platelets
separating medium granulocytes
erythrocytes
buffy coat (PBMC)
At the end of the centrifugation step, the following layers are visually observed from top to bottom.
The PBMC layer is then removed and washed twice to get rid of contaminants before cell type and cell viability can be confirmed.
Manual Ficoll® Methods
Overlay Underlay
Cell Separation Tube With Frit Barrier (CSTFB)
1. Load the Ficoll into a polypropylene tube with a frit (porous polyethylene barrier).
2. Centrifuge to settle the Ficoll below the frit.
3. Layer whole blood on top of the frit.
4. Centrifuge the tube.
Frit Barrier
BD Vacutainer® CPT™ Cell PreparationTube With Sodium Citrate
8 mL Draw Capacity(16 x 125mm silicone glass
coated tube )1.0 mL of 0.1 Molar
Sodium Citrate Solution (Top Fluid Layer)
Polyester Gel Barrier
FICOLL™ HYPAQUE™ density medium
Buffy Coat Isolation
Comparison of Manual Ficoll® Methods
Underlay Method Mononuclear layer
Overlay Method Mononuclear layer
Cell Separation Tube With Frit Barrier (CSTFB)
Plasma Layer
Ficoll®
Mononuclear Layer
Frit Barrier
Cell Preparation Tube (CPT)
Mononuclear Layer
Plasma Layer
Erythrocytes
Manual Cell CountsManual Cell Counts
What to Count on the Hemacytometer?
Formulas For Cell Counts
• Percent viable cells :
# live cells _______ /# total cells (live+dead) _________x 100 = _______ %
• Total Viable Cell Count (Concentration):Total number of live cells counted X Dilution factor x resuspension volume x 104= __ # cellsNumber of squares counted
• Calculate cell yield per mL of whole blood:
(QC check)= (Cell Concentration/ Whole Blood Volume)
Cryopreservation
Why do we use DMSO and FBS as Why do we use DMSO and FBS as
Cryoprotective Agents?Cryoprotective Agents?
• DMSO helps dehydrate the cells prior to freezing therefore preventing the formation of ice crystals that will cause cells to lyse during thawing.
• During the freezing and thawing processes cells are deprived of nutrients and therefore, FBS contains an abundance of proteins.
Effects of Freezing Rates on Mononuclear Cells
PBMCIce Crystals
Slow cooling rate.•Cells dehydrate and shrink but they survive.•Less intracellular ice.•More osmotic imbalance.
Fast cooling rate.•Intracellular ice crystals form. •Less osmotic imbalance.• Cell death.
Cell Freezing ContainersCell Freezing Containers
• Cells are gradually cooled at a rate of approximately 1°C per minute using a rate-controlled freezer, or they are placed in a “Mr. Frosty”, “StrataCooler”, “Cool-Cell” in a -80°C freezer overnight.
• Latent heat released from the cooling cells is absorbed by the freezing chamber preventing the heat from damaging cells.
Thawing of Cryopreserved Thawing of Cryopreserved CellsCells
Thawing of Cryopreserved PBMCThawing of Cryopreserved PBMC
• If PBMC are not thawed properly, viability and cell recovery can be compromised; cells may not perform optimally in functional assays.
• Cells should be thawed quickly but diluted
slowly to remove DMSO. Cells that have DMSO permeated into their membranes are very fragile and must be pelleted and handled gently.
Why Are The Cryopreserved Cells Why Are The Cryopreserved Cells Diluted?Diluted?
• The gradual dilution of DMSO avoids the osmotic shock, and the warm temperature from the media assures that the cells can actively compensate the decreasing osmotic pressure.
Why use Benzonase® and RPMI Why use Benzonase® and RPMI during the thawing procedure?during the thawing procedure?
• Benzonase will remove the contaminated cellular DNA/RNA and RPMI (rich nutrient) is used to compensate from the stress that cells undergo during thawing.
• Use Benzonase when thawing cells for assays.
TroubleshootingTroubleshooting
Processing Errors Processing Errors Red blood cell contamination
• Density gradient media or centrifuge not (15-30°C) room temperature.
• Centrifugation time is too short.
Excess of platelets• Not removing most of the plasma above the interface.
No defined or distinct mononuclear layer – Centrifugation time is too short.
– Centrifuge not calibrated.
– The brake was left on.
– The centrifugation speed was too high.
– The centrifuge arms and buckets were not properly greased and oiled.
– Hyperlipemic sample.
Processing ErrorsProcessing Errors
Processing Errors Processing Errors Granulocyte contamination
– Removing excess amounts of the separation media with the PBMC band.
– Centrifuge was not set up at the appropriate speed.
– Density gradient media or centrifuge not (15-30°C) room temperature.
LDMS Computational ErrorLDMS Computational ErrorExample 1.Example 1.
Shipping Manifest Processing Report
Cell concentrations do not match.
How does a Clerical Error affectHow does a Clerical Error affectthe Results?the Results?
Total viable cell count performed by
the IQA (x10^6 cells per vial)
Sample cell concentration
reported by processing site
(x 10^6 cells per vial)
Total percent viable recovery Final performance
5.66 9.7 58% Unsatisfactory
5.66 5.4 104.8% Satisfactory
Formula Percent Viable Recovery:
Number of cells counted by IQA divided by number of cells reported by site.
Results multiplied by 100.
LDMS Computational ErrorLDMS Computational ErrorExample 2.Example 2.
Shipping Manifest Processing Report
Low cell yield:0.58 x106 cells/ml of
blood
Lab froze 3 vials at 10 million cells each, possibly
3.5 x106 cell/vial
Cell ViabilityCell Viability
Viability is too low
Processing Report Unexpected Viability
Freshly isolated PBMCviability should be >95%.
Yields outside the expected ranges may indicate:
•Long processing time•Poor technique
IQA PersonnelIQA Personnel
Daniella Livnat Project Officer for the IQAContract# HHSN27220070054C
Thomas DennyPrincipal Investigatorthomas.denny@duke.edu
IQA PersonnelIQA Personnel
Raul LouzaoIQA Program Managerraul.louzao@duke.edu
Tania GarreltsTania.garrelts@duke.edu
Special Thanks To:
Questions?Questions?
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