Transcript IMPAACT /ACTG Meeting

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
IQA Global Laboratory Participants in the
Cryopreservation Proficiency Testing Program
93 participating
laboratories
Currently there are 65 domestic and 28 international
laboratories enrolled in the program.
65 in North
11 in Central and
11 in
America
South America
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 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 during
handling of frozen samples.
• Wear appropriate personal protective equipment.
• Process in Class II biological safety cabinet.
Peripheral Blood
Mononuclear Cells (PBMC)
Isolation
Plasma Isolation
Centrifuge EDTA , Heparin or ACD blood at 400 x g for 10
minutes.
Buffy Coat
Plasma
Basic PBMC Isolation Procedure
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:
Diluted anticoagulated
whole blood
Ficoll®
At the end of the centrifugation step, the following layers are
visually observed from top to bottom.
plasma/platelets
buffy coat (PBMC)
separating medium
granulocytes
erythrocytes
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 Preparation
Tube 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
Mononuclear Layer
Ficoll®
Frit Barrier
Cell Preparation Tube (CPT)
Plasma Layer
Mononuclear Layer
Erythrocytes
Manual 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
Number of squares counted
X Dilution factor x resuspension volume x 104= __ # cells
• Calculate cell yield per mL of whole blood:
(QC check)= (Cell Concentration/ Whole Blood Volume)
Cryopreservation
Why do we use DMSO and FBS as
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
PBMC
Ice 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 Containers
• Cells are gradually cooled at a rate of
approximately 1°C per minute using a ratecontrolled 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
Cells
Thawing 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
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
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.
Troubleshooting
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.
Processing Errors
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 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 (1530°C) room temperature.
LDMS Computational Error
Example 1.
Shipping Manifest
Cell concentrations
do not match.
Processing Report
How does a Clerical Error affect
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)
5.66
5.66
Total percent
viable recovery
Final performance
9.7
58%
Unsatisfactory
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 Error
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 Viability
Processing Report
Unexpected Viability
Freshly isolated PBMC
viability should be >95%.
Viability
is too low
Yields outside the expected
ranges may indicate:
•Long processing time
•Poor technique
IQA Personnel
Daniella Livnat
Project Officer for the IQA
Contract# HHSN27220070054C
Thomas Denny
Principal Investigator
[email protected]
IQA Personnel
Raul Louzao
IQA Program Manager
[email protected]
Tania Garrelts
[email protected]
Special Thanks To:
Questions?