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Duke Human Vaccine Institute
OUTLINE
Immunology Quality Assessment Purpose Grading Criteria
Proficiency Testing (PT) Cryopreservation Thawing
Troubleshooting Challenges Technique/Tips
Immunology Quality Assessment
Currently there are 65 domestic and 28 international laboratories enrolled in the program.
IQA Global Laboratory Participants in the
Cryopreservation Proficiency Testing Program(94 participating laboratories as of June 2011)
65 in North America
13 in Central and South America
11 in Africa
5 in Asia
Purpose
What is the purpose of the IQA Cryopreservation Proficiency Testing (PT) Program?
1. Assess a laboratory’s ability to consistently process, cryopreserve
and ship viable peripheral blood mononuclear cells (PBMCs)
2. Troubleshoot and work with individual labs via conference calls, site visits or wet lab sessions performed at the IQA
3. Communicate with the networks to verify each lab’s status and collaborate to determine necessary corrective action
Quarterly Reminders Yearly Calendar (IQA website)
Participation Reminder
Collection Notification
Shipment Notification
Results Report Notification
Investigational Report (IR) Notification
Continuous communication via email/phone from IQA and network personnel
Grading Criteria
Grading Criteria Performance Evaluation
established by the IQA PBMC Cryopreservation PT Advisory Group (ICAG)
Scoring System for One Sample
% Viability % Viable Recovery Score/sample:
80-100% 70-120% 2
65-79% 50-69% 1
- 121-150% 1
< 65% < 50 % 0
- > 150% 0
No Submission 0
Grading Criteria
Combined Scoring System for Both Samples
Combined% Viability Score (both samples)
% Viability STATUS
Combined % Viable Recovery Score
(both samples)
% Viable Recovery STATUS
OVERALL STATUS (both viability and
viable recovery)
3-4 A 3-4 A Overall status is determined by the
lower of the %viability/ %viable recovery statuses.
2 PA 2 PA
0-1 OP 0-1 OP
Example: % Viability Status: A
% Viable Recovery Status: PA
Overall Status: PA
A: Approved
PA: Provisionally Approved
OP: On Probation
Grading CriteriaSite Vial ID Tech Viability (%)
Viability
Score
Viability
Status
Viable
Recovery
(%)
Viable
Recovery
Score
Viable
Recovery
Status
Combined
status
XXX 213V110000X BL 100 2A
95 2PA PA
XXX 213V110000X BL 97.5 2 68 1
An Investigation Report must be submitted to the IQA within 5 working days of receiving results report:
•If your site received a viability or percent viable recovery scores of less than 2 on one or both samples you are required
to complete and submit an IR and are advised to work within your lab and with the IQA.
Your lab must submit samples within 4 weeks of on probation status notification:
•If your site received a viability or percent viable recovery status of “On Probation” (OP), you are required to not only
complete and submit an IR but you must also process another set of samples to be tested by the IQAC
Grading Criteria APPROVED – A Status
If a score of 3 is received, an Investigational Report (IR) must be completed (even though A status earned)
Eligible to perform PBMC cryopreservation for network protocols
Eligible to start new network protocols
Eligible to serve as an IQA back-up lab
Not exempt from future proficiency testing
Grading Criteria PROVISIONALLY APPROVED – PA Status
Required to complete an IR
Eligible to perform PBMC cryopreservation for network protocols
Eligible to start new network protocols
NOT eligible to serve as an IQA back-up lab
Grading Criteria ON PROBATION – OP Status
Required to complete an IR and submit the equivalent of one round of IQA PT within 4 weeks (to improve score)
Eligible to perform PBMC cryopreservation for network protocols
NOT eligible to start new network protocols OR serve as an IQA back-up lab
If lab remains OP for 2 consecutive rounds, re-certification* and network involvement must take place
(*Fast Track: submitting the equivalent of two rounds of samples;
with satisfactory results, lab becomes PA)
Grading Criteria ON HOLD/Not Approved – Hold/NA Status
Lab is enrolled in the IQA PT program, but currently not participating
NOT eligible to perform PBMC cryopreservation, take on new PBMC cryopreservation protocols, or serve as an IQA back-up lab
Lab must re-certify* when they wish to start the IQA PT program again
(*Fast Track: submitting the equivalent of two rounds of samples;
with satisfactory results, lab becomes PA)
March 2011 Results Data
Status:Overall %
Viability
Overall %
Recovery
Combined
Status
# of labs with an
Approved
(A) Status91 84 81
# of labs with a
Provisionally Approved
(PA) Status0 7 9
# of labs with an
On Probation
(OP) Status0 0 0
# of labs with an
on Hold
(Hold) StatusNA NA 4
94 Labs Enrolled
91 Participated March 2011
-2 Labs participated in the Fast Track
(received satisfactory results (A), but
are PA for the next round of testing)
-1 Lab was placed on Hold status
(by network)
-0 Labs are currently OP
Proficiency Testing
Quarterly IQA PT
Preparation
PBMC Processing
Shipment to IQA
Preparation
Obtain IRB Consent Form
Select/schedule two Donors (HIV +/-)
Collect blood from each donor
(ACD, EDTA or NaHep )
PBMC Processing
Dilute Whole blood/Ficoll
Isolate buffy coat/PBMC
Wash
Count cells
Adjust/re-suspend cells in Cryopreservation Solution
PBMC Processing
Ficoll®
Dilute Whole-blood and Layer over Density Gradient
PBMC can be isolated from whole blood samples using different density gradient centrifugation procedures:
Manual Ficoll® Cell Separation Tube with Frit Barrier (CSTFB) Cell Preparation Tubes (CPT)
Anti-coagulated whole blood is layered over the separating medium
Diluted anti-coagulated whole blood (manual)
Ficoll®
PBMC ProcessingIsolate Buffy Coat/PBMCs – POST Centrifugation
plasma/platelets
separating medium granulocytes
erythrocytes
Buffy Coat (PBMC)
PBMC ProcessingIsolate the Buffy Coat (PBMC)
Remove plasma layer Isolate and harvest buffy coat (PBMC layer)
Manual Ficoll® Methods
Overlay Underlay
Overlay: diluted blood is layered OVER room temperature Ficoll® Underlay: room temperature Ficoll® is added UNDER diluted blood
Centrifuge: 400 x g for 30 minutes, brake off
Manual Ficoll® Methods
Buffy Coat (PBMC)
(Underlay)
Buffy Coat (PBMC)
(Overlay)
Cell Separation Tube With Frit Barrier (CSTFB*)
Load room temperature Ficoll® into a polypropylene tube with a frit barrier (if necessary)
Centrifuge to settle the Ficoll® below the frit barrier(800 x g for 30 seconds)
Layer whole blood on top of the frit barrier
Centrifuge:(800 x g for 15 minutes, brake off)
*Accuspin/Sigma-Aldrich
Frit Barrier
Cell Separation Tube With Frit Barrier (CSTFB)
Plasma Layer
Ficoll®
Buffy Coat (PBMC)
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
Centrifuge: 1,500 to 1,800 RCF for a minimum of 20 minutes, brake off
*Becton, Dickinson and Co.
BD Vacutainer® CPT™ Cell PreparationTube With Sodium Citrate
Buffy Coat (PBMC)
Plasma Layer
Erythrocytes
Comparison of 3 Methods
Factors:Manual Ficoll
(overlay/underlay)Cell Separation Tube
w/ Frit BarrierCell Preparation Tube
(CPT)
Cost/Sample*$3.55/
20mL whole blood$13.10/
20mL whole blood$8.25/
8mL whole blood
Advantages Most cost effective Less time consumingConvenient
(can ship after spin),very time effective
Disadvantages Time consuming Cost Cost, volume limitation
*Prices are for reagents only, are approximate and may vary
This information is provided to give you a general idea of what method might be best for your lab
Review:
Ficoll® carefully (clean interface)
Precise buffy coat/PBMC isolation/pipetting
Attention to detail during set up
(centrifuge settings, reagents, volumes)
PBMC Processing
Dilute Whole blood/Ficoll
Isolate buffy coat/PBMC
Wash (200-400 x g for 10 minutes)
Count cells
Adjust/Re-suspend cells in Cryopreservation Solution
Cell Counting Gently re-suspend cells
Thoroughly mix staining agent (0.4% trypan blue or 0.05% crystal violet) and cells together
Load hemacytometer (10uL) using correct cover slip
Let mixture stain; 8-12 seconds
Count viable and nonviable cells from outer 4 quadrants
Cell Counting (manually on a hemacytometer)
Cell Counting
Live/
Viable Cells
Dead/
Non-Viable Cells
Cell Counting - Problems
“Clean” Sample-mostly PBMCs (viable/non-viable)-easy to read/decipher
“Dirty” Sample-platelets/debris-more difficult to read
Cell Counting - Problems
Dead Cells/Poor Viability
Formulas For Cell Counting
Dilution Factor (DF):
final volume / aliquot volume
(final volume= aliquot + diluent)
Ex: (1mL cells + 9mL PBS) / 1mL cells = 10 DF
Ex: (20uL cells + 20uL trypan blue) / 20uL cells = 2 DF
Formulas For Cell Counting
Percent viable cells:
# live cells /# total cells (viable + non-viable) x 100
Ex: total viable cells: 226 total non-viable cells: 11total cells: 237
(226/237) x 100 = 95.4% viable Cells
Formulas For Cell Counting
Total Viable Cell Count (Concentration):
total viable cells X dilution factor x re-suspension volume (mL) x 104
squares (4)
Ex: total viable cells: 226
dilution factor: 10
volume: 2mL
104 : hemacytometer factor/chamber volume
(226/4) (10) (2mL) (10⁴) = 11.3x106 cells
Review:
Keep counts consistent
Outer four quadrants, top/left borders
Mix sample well for accurate representation
Check hemacytometer and cover slip
Don’t over fill chamber (only 10uL)
Double check calculations
Check yourself with automated cell counter
optional
PBMC Processing
Dilute Whole blood/Ficoll
Isolate buffy coat/PBMC
Wash
Count cells
Adjust/Re-suspend cells in Cryopreservation Solution
Cryopreservation
Why do we use DMSO and FBS as Cryoprotective Agents?
DMSO helps dehydrate the cells prior to freezing preventingthe formation of ice crystals that could cause cells to lyseduring thawing.
FBS contains an abundance of proteins, which helps nourishthe cells during the freezing and thawing processes whencells are deprived of nutrients.
Cryopreservation Solution (CPS) – Freezing Media
Fetal Bovine Serum (FBS) Dimethyl sulfoxide (DMSO)
90% 10%
Formulas for Freezing
Determining final CPS re-suspension volume:
Total Cell Concentration/Freeze down concentration
= actual volume of CPS need
Ex: Total Cell Concentration: 11.3 X 10⁶ cells
Freeze down concentration: 1mL/5x10⁶cells
11.3 X 10⁶ cells / (1mL/5x10⁶cells) = 2.26mL
(round down to nearest whole mL)
Formulas for Freezing# of cells/vial:
(Total cell concentration/Final CPS volume) x Freezing volume*
= # cells (10⁶)/vial (mL)
*Freezing Volume: usually 1mL
Ex: Total cell concentration: 11.3 x 10⁶ cells
Final CPS volume: 2mL
Freezing volume: 1mL
(11.3 x 10⁶ cells/ 2mL) x 1mL = 5.65 x 10⁶ cells/mL
Freezing Containers
Once cells have been aliquoted, they 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, or CoolCell in a -80°C freezer overnight.
Rate controlled freezing is important so that you avoid rapid intracellular freezing and excess extracellular ice formation, both lead to cell death
Freezing Containers
Stratagene StrataCooler® Cryo:
Holds up to 32 cryovials
~$159.00/unit
Freezing Containers
NALGENE ® Mr. Frosty
Must be stored at an ambient temperature (15-30°C)
Uses isopropanol, must be filled to correct level and replaced every fifth freeze/thaw cycle (with record log)
Holds up to 18 cryovials
~$75.00-80.00/unit
(+isopropanol)
Freezing Containers
BioCision ® CoolCell
Allow inner-ring and foam to reach ambient temperature (15-30°C) after each use
Holds up to 12 cryovials
~$100.00/unit
Freezing ContainersFactors: StataCooler® Mr. Frosty CoolCell
# Cryovials 32 18 12
Cost/unit* $159.00$75.00-80.00
(+isopropanol)$100.00
AdvantagesLarge sample-size,
No IsopropanolCost No Isopropanol
Disadvantages CostUp-keep, properwaste disposal
Small sample-size
Pre-chill each freezing container to 2-8°C prior to each use
(per HANC Cross-Network PBMC Processing SOP, draft)
*Costs are approximate and may vary. This information is provided to
give you a general idea of what method might be best for your lab
Review: Double check all calculations/dilutions
Perform the final centrifugation and re-suspension with CPS carefully
Keep cells chilled while adding CPS and freeze immediately after
Keep freezing container in a secure location within -80°C freezer, away from door/possible temperature fluctuation
Make sure freezers have been calibrated and are monitored
Shipment to IQAC
Prepare Shipment to Lab 213:
Enter specimen into LDMS/Label
Package Specimen
(Category B Biological Substance):
Include LDMS manifest, box report, batch file and completed return label
Ship 4 vials (2 from each donor) @ 5x10⁶cells/vial to the IQA during designated dates
Thawing
Thawing Process for IQA PT
Thaw/Dilute
Wash
Assess Viability/Viable Recovery
Report Results
Lab Communication
Thawing
If PBMCs are not thawed properly, viability and cell recovery can be compromised; cells may not perform optimally in functional assays.
Cells should be thawed quickly (to avoid the formation of ice crystals) but diluted slowly (drop-wise) to remove DMSO.
RPMI 1640 is nutrient rich and nourishes the cells as they undergo stress during the thawing process.
Troubleshooting
Challenges
Processing Challenges
Granulocyte Contamination
Centrifuge not set at correct speed/time
Ficoll® not room temperature
Collected from below the PBMC band (Ficoll®)
Fix/Prevention:
Count sample at higher magnification
Careful attention during PBMC isolation/set-up
Platelet Contamination
Could cause cell clumping/misleading counts
Sample quality
Collected from above the PBMC band (plasma)
Fix/Prevention:
Extra wash
Careful/accurate counting
Processing Challenges
No Distinct Buffy Coat/PBMC Layer
Could cause cellular contamination
Possible causes
Incorrect time, speed, temperature of centrifuge
Brake left ON
Layers disrupted/sample dropped or jarred
Poor Ficoll® technique, not room temperature
Fix/Prevention: Remix sample and repeat Ficoll® steps, careful handling and setup
Processing ChallengesCell concentration differ (after thawing procedure)
(inaccurate % Viable Recovery is the reason almost all labs fail!)
Contaminated sample = misleading counts
Calculation errors (fail to include DF, use incorrect DF, incorrect volume, etc)
Dilution errors Skip final centrifugation CPS made incorrectly Poor mixing (uneven distribution between vials)
VS…
Why use swinging buckets?As the rotor begins to rotate
the buckets swing out to a
horizontal position, allowing
the cells to pellet at the
bottom of the tubes.
A vertical rotor has no swing
and cells are forced to the
side of the tube, which is not
ideal for pellet applications.
This is why centrifugation
speed and time are important
factors (as it all affects the
way the cells are manipulated)
Technique/Tips
Technique/Tips
Read and follow the SOP
Check all reagents before processing (temperature, expiration dates, volumes)
Carefully inspect sample (before and throughout processing) to check for any irregularities (hemolysis, clumping, no pellet, poor layering, etc…)
Careful and precise pipetting/isolation
Handle sample gently, but make sure to mix well and re-suspend your pellet thoroughly
Technique/Tips
Mix sample well before each step
Work quickly, but not hastily
Count accurately
Double check all calculations/dilutions
Perform final re-suspension and aliquot samples into vials carefully
Freeze immediately
Remember to treat all human-derived specimen as infectious using universal safety precautions