Transcript ACTG Cryo Presentation June 2009

Duke University School of Medicine
Immunology Quality Assessment
Center
Tania Garrelts / Raul Louzao
ACTG Meeting
June 23,2009
OUTLINE
• Role of IQA
• Frequently Asked Questions
1) Reagents
2) Equipment
3) Cell counts
• Site Visit Findings
What is the role of the IQA?
• To assess the ability of laboratories to
process and store viable peripheral blood
mononuclear cells (PBMC) for use in various
immunology and virology based assays.
• To provide assistance to faltering
laboratories.
• Currently there are 63 domestic and 21
international laboratories enrolled in the
program.
How is the assessment accomplished?
• The assessment is accomplished by
comparing the viability of PBMCs and the
viable yield before freezing and after thawing.
• Proper processing and freezing is a critical
component of the process for storage of
viable PBMCs.
How often do participating sites should submit
samples?
• Every three months (March, June,
September and December), frozen PBMCs
must be shipped from the participating
laboratory to the NIAID Immunology Quality
Assessment Program (IQA)
• Shipping schedule can be found at our
website (iqa.center.duke.edu)
What should the participating labs achieve?
Network*
Minimum
Viability (%)
Minimum Viable
Recovery (%)
ACTG
80
80
IMPAACT
75
70
*Laboratories who serve both the ACTG and
IMPAACT must conform to the more stringent
criteria of the ACTG (80% viability and 80% viable
cell recovery)
Frequently Asked Questions About
The Cryopreservation Of
Peripheral Blood Mononuclear Cells
Are Ficoll–Hypaque and
Histopaque-1077 the same?
Similarities:
Both are mixture of Ficoll
(polysucrose)
and Hypaque
(sodium diatrizoate).
Light Sensitive.
Ficoll-Paque Plus
Vendor is GE
Healthcare
Unknown density gradient
Store between 4°C and
30°C
Histopaque –
1077
Vendor is SigmaAldrich
A known density of 1.077
g/ml.
Store in refrigerator (2-8°C)
Are Ficoll–Hypaque and
Histopaque-1077 the same?
• IQA currently uses Histopaque-1077 by
Sigma Aldrich because of the known density
gradient, 1.077 ± 0.001 gram/ml which is
between that of plasma and red blood cells.
• One part of blood to one of part of buffer,
equal part of density gradient.
What is the difference between Ficoll
Ovelayering and Undelayering
Technique?
Before and After Density Gradient
Video of Overlaying Technique
Manual Ficoll using Overlay and Underlay Methods
Click on the link bellow
http://youtu.be/-SqlIMLUfcI
Video of Overlaying Technique
Click on Link
http://youtu.be/andvFWRzQ0c
Insufficient amount of density gradient
Buffy Coat Isolation
Good versus Poor Mononuclear cell isolation
Why do I have to remove the supernatant
(HBSS-Plasma layer)?
• Removing the PBMC band with the
supernatant will promote contamination by
platelets and plasma proteins.
Leave Platelets + proteins= cell clumps
Why I can’t remove all the density
gradient?
• Removing the excess amount of Ficoll with the
mononuclear layer will increase a chance of
granulocyte contamination.
Granulocyte contamination = misleading cell counts
How much supernatant should I leave
before isolate the PBMC band?
• Remove the plasma-HBSS fraction down to
within approximately 1-2 cm of the PBMC
band.
Common used techniques to isolate the
Mononuclear Layers.
Should I remove the cells stuck to the
side of the test tube?
• From previous experience the cells attached
to the inner walls of the tubes are
erythrocytes, cell debri and platelets.
• The density gradient will separate the viable
lymphocytes and monocytes.
What should I do if I accidently mix the
layered blood with ficoll?
• Don’t Panic.
• Finish Mixing the blood with ficoll. (New
mixture)
• Set up new ficoll tubes and overlay or
underlay them with the new mixture.
Why do we need to use a centrifuge with
swinging -bucket rotor?
• In swinging bucket rotors, the sample
tubes are loaded into individual buckets
that hang vertically while the rotor is at
rest. When the rotor begins to rotate the
buckets swing out to a horizontal
position This rotor is particularly useful
when samples are to be resolved in
density.
Can a vertical rotor be used with Ficoll?
• In vertical rotors, sample tubes are held
in vertical position during rotation. This
type of rotor is not suitable for pelleting
applications.
Difference between Vertical versus
Swinging-Bucket Rotor
What temperature should I use during
centrifugation?
• During separation the centrifuge temperature
should be control between 18-25 °C because the
density components changes with temperature.
For Example: Centrifugation at 4 °C may result in
poor cell recovery due to cell clumping.
What speed and time should I use in my
centrifuge?
• If using Sigma Ficoll centrifuge at 800 x g for
30 minutes. GE Ficoll centrifuge at 400 x g for
40 minutes.
• If using Cell Separation tube with Frit Barrier
(CSTFB) , centrifuge at 800 x g for 15
minutes or 1000 x g for 10 minutes.
• No Brake, if brake is on it will disrupt the
layers.
What should I check if I do not get a
PBMC layer?
Check:
• The centrifuge speed and time.
• The centrifuge brake was off.
• The centrifuge buckets are not sticking.
• If the centrifuge buckets are balanced
properly.
• Maintenance record for last calibration.
How much buffer do I need for my cell
wash and why?
• For the 1st cell wash, measure your isolated
PBMC layer and the balanced salt solution
volume should be add at least 3 times the
volume of the harvest. Do not exceed 45 ml.
• Centrifuge at 300 x g for 10 minutes.
Low centrifugation + QS buffer= removes
platelets and plasma contamination
What is the total number of cell washes?
• Minimum two cell washes, depending on the
tech buffy coat isolation technique.
• Decant or aspirate the medium solution.
• Resuspend your pellet by gently pipetting
them up and down, or finger flick.
• Gently mix the solution by inverting the test
tube.
Video of incorrect pellet re-suspension
Click on Link
http://youtu.be/4wuSfHmPZ-k
Video of gentle pellet re-suspension
Click on Link
http://youtu.be/4WObBqpJD0I
How much buffer is left in my test tube
after I decant the supernatant?
• The amount of balanced buffer left over with
the pellet after the HBSS is decanted is
approximately 200 to 400uls.
• You have enough buffer to gently finger flick your
cells.
What volume of buffer should I use to
re-suspend my pellets?
• If you have multiple tubes combine the pellets
by adding 1ml of buffer on each tube.
• Depending on the size of the cell pellet, the
re-suspension volume would range from 10%
to 50% of the usable whole blood.
• For example:
Starting volume of whole blood is 17 mls.
IQA would re-suspend cells in 5 mls ~30%
What type of pipettes and tips do
I need to have?
What is the Forward Pipetting
Technique?
What are the effects of how I pipette?
What are some common pipetting
technique errors?
— Human Factor, working too quickly.
— Removing the pipette tip before sample aspiration
is complete.
— Angle pipettes takes up too much liquid. Keep
vertical.
— Releasing the plunger too rapidly.
— Not pre-wetting a new tip.
What type of pipette aid should I use?
What type of plasticware should I use?
• Avoid using high binding plastics such
as polystyrene to prevent monocyte
adhesion.
• Use Polypropylene
Are powered gloves acceptable for
processing PBMCS?
• Powered gloves will contaminate the process
by activating monocytes which will cause a
lower percent recovery.
What is the pH, solution storage requirement,
and length of time that cells can stay in
trypan blue?
• pH: 7.0 – 7.4
• Storage requirement: 15-30 °C. Some of vendors
recommend that it be protected from light and filter
after prolonged storage.
• Cells should be count within 3-10 minutes of mixing
with trypan blue, longer incubation will result in cell
death and low viability counts.
What are some common problems with a
Hemacytometer?
1. Dirty hemacytometer or cover slip, clean with 70%
alcohol and lens paper.
2. The chamber may have been loaded incorrectly.
3. The cover slip may have been bumped.
4. Insufficient mixing of sample.
5. Using a wrong cover slip.
Definition for Dilution
• Dilutions are expressed as the ratio of the quantity
of a desired solute (serum, urine, chemical
solution, etc.) contained in a solvent (diluent).
For example:
A 1:10 dilution of serum was made by adding one part
serum to nine parts diluent to make a total of ten
parts.
volume of serum/volume of solution = [1.0 mL serum ]/[1.0 ml
serum + 9.0 mL H20]
Video of Counting Cells
Click on Link
http://youtu.be/GTlAdxUX9mU
How to perform an accurate count?
Example: IQA re-suspend pellet in 5 mls
• Mix cells with buffer by inverting the tube.
• Take 20 uls of cellular solution and mix with
20 uls of trypan blue. (1:2 dilution)
• Load in clean hematocytomer. Wait 10
seconds, for cells to settle and stain.
• Count 4 quadrants.
• Record Viable and Nonviable PBMCs.
What are the formulas for cell counts?
Example
Live (L)
Dead (D)
Dilution Factor
Final Volume
488
20
1:2
5 ml
Answer: 488/4x2x5x10^4=12.2 x10^6 (total cell
concentration)
Cell concentration per ml=488/4x2x10^4=2.44 cells/ml
Percent Viability= L/L+Dx 100
488/488+20=488/508
=0.96x100=96%
10X Objective with Cells
What is purpose of using DMSO?
Serves as a cryoprotective agent, it helps dehydrate
the cells prior to intracellular freezing.
How much DMSO do I need to add to
cryopreservation media?
• The cryopreservation solution (CPS) is composed
by 10% DMSO and 90% FBS.
• Make sure CPS is chilled prior to adding to cells.
DMSO is highly toxic and produces an exothermic
reaction.
Rate Control Freezer
Stratacooler versus Mr. Frosty
Cooling Rate for Mr. Frosty and Rate
Control Freezer
Amount of Cells per vial?
How do we know a reagent is stable?
• Deterioration of Ficoll and DMSO is indicated by
the appearance of a yellow color, cloudy or
particulate material in the clear solution.
• PBS is indicated by cloudiness in the clear solution.
• Fetal Bovine Serum is indicated by turbidity in the
bottle, or by microscopy.
Causes for Unsatisfactory Performance
•
Some reagents were not properly stored
according to manufacturer recommendation.
•
Several bottles of this item were found on the
shelf without receiving dates. Therefore IQA
could not determine how long reagents were
stored in that environment.
•
Heavy RBC contamination in their PBMC
samples due to inadequate blood and HBSS
dilution. The current HBSS has Phenol red which
makes it difficult to isolate buffy coat.
Causes for Unsatisfactory Performance
• Having trouble aspirating the cell layer
without mixing the density gradient media.
• Mr. Frosty had an insufficient amount of
isopropyl alcohol and it was filled with
wrong alcohol (Ethyl).
• Centrifuge and pipettes not calibrated. The
centrifuge was set at the wrong speeds.
• Wrong cryopreservation vials, no O-ring
and unknown material.
Work Cited
• Cross- Network PBMC Processing Procedure
• Duke University Standard Operating Procedures.
• Cryopreservation Technical Manual by Nalgene,
Nunc
• Reagent Manufacturer inserts.
• Beckman Coulter
• Guide to Pipetting by AccuTek Laboratories
Acknowledgments
IQA Center Laboratory
Tom Denny
Ambrosia Garcia-Louzao
Raul Louzao
John Wong
Eugene Urrutia
Tania Garrelts
Estelle Berengier
DHVI
Tony Moody
Josh Eudley
Dongning Wang
Kyle Liebl
Brooke Parker
Sherry Leonard
Sara Brown
Christie Brinkley
Jamilia Davis