Transcript IMPAACT /ACTG Meeting

Duke Human Vaccine Institute
Immunology Quality Assessment
Cryopreservation Proficiency Testing Program
Presented by: Kyle Liebl
July 16, 2012
www.iqa.center.duke.edu
OUTLINE
 Immunology Quality Assessment
 Purpose
 Criteria
 Proficiency Testing
 Cryopreservation
 Cell Counting
 Troubleshooting
 Challenges
 Tips
Immunology Quality
Assessment
IQA Global Laboratory Participants in the
Cryopreservation Proficiency Testing Program
(100 participating laboratories as of June 2012)
Currently there are 65 domestic and 28 international
laboratories enrolled in the program.
66 in North
16 in Central and
13 in
America
South America
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
Grading Criteria
Grading Criteria

Performance Evaluation
 established by the IQA PBMC Cryopreservation PT Advisory Group (ICAG)
 ranges provided to account for subjective nature of cryopreservation
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
3-4
A
3-4
A
2
PA
2
PA
0-1
OP
0-1
OP
A: Approved
PA: Provisionally Approved
OP: On Probation
OVERALL STATUS
(both viability and
viable recovery)
Overall status is
determined by the
lower of the
%viability/ %viable
recovery statuses.
Example: % Viability Status: A
% Viable Recovery Status: PA
Overall Status: PA
Grading Criteria
The data obtained from the vials sent to the IQA Center by your site are shown below in Table 1.
Duke University School of Medicine
Immunology Quality Assessment Laboratory
Site
Vial ID
XXX
XXX
213V110000X
213V110000X
From: IQA Cryopreservation Proficiency Testing Advisory Group (ICAG)
Re:
LAB XXX
Date:
June 21, 2011
Table 1: Percent Viability and Viable Recovery
Viable
Viable
Viable
Viability
Viability Viability
Combined
Tech
Recovery Recovery Recovery
(%)
Score
Status
status
(%)
Score
Status
BL
100
95
2
2
PA
A
PA
BL
97.5
68
2
1
Figure 2 shows your site’s cumulative percent viability and Figure 3 displays the cumulative percent viable recovery for
the quarterly assessments in which your site has participated.
The leadership of the AIDS Clinical Trials Group (ACTG) and the International Maternal Pediatric Adolescent
AIDS Clinical Trials Group (IMPAACT) requires that Clinical Trial Units participate in a quarterly proficiency testing
program to evaluate the ability to reliably cryopreserve viable PBMCs. Performance in this program is currently reviewed
by the IQA Cryopreservation Proficiency Testing Advisory Group (ICAG).
Figure 2: % Viability From Quarterly Assessments
Figure 1: Distribution of the overall combined status of the percent viability and percent viable recovery.
100
90
80
70
60
50
40
30
20
10
0
140
Vial A
Vial B
120
100
Vial A
80
Vial B
60
40
20
0
Sept '10
Overall Combined Status For Participating
Laboratories
Figure 3: % Viable Recovery From Quarterly
Assessments
% Viable Recovery
The Immunology Quality Assessment Contract Laboratory (IQA) at the Duke Human Vaccine Institute received
two samples from two donors from 94 sites. Vials from each donor were thawed; viability and viable cell recovery were
tested and the results were compared to the information provided by each site.
% Viability
A scoring system will be used to evaluate laboratory performance and to determine whether the laboratory is
qualified to cryopreserve PBMCs for network protocols. The viability and viable percent recovery for each sample set is
assessed within each round of testing to determine the laboratory status.
Dec'10
March'11
Date
June '11
Sept '10
Dec'10
March'11
Date
June '11
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.
80
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
70
70
60
50
Filing an exception
If you would like to file an exception to your proficiency testing results with the IQA PBMC Proficiency Testing
Advisory Group (ICAG), please submit a memo describing the reasons for your exception and any supporting
documentation to Raul Louzao within ten working days of receiving the proficiency testing report. The exception
will be added to the agenda of the next ICAG conference call for consideration.
40
30
20
10
14
7
3
0
Number of labs with an Number of labs with an Number of labs with an Number of Labs On Hold
Approved (A) status
Provisionally Approved On Probation (OP) status
(PA) status
The next round of submissions will take place on August 29, 2011. Sites should initiate IRB review for these
quarterly submissions to ensure continuing participation in this program. The staff at the IQA Center is available
for technical consultation concerning laboratory techniques for these procedures. You may contact Raul Louzao at 919684-5861 [email protected] or Brooke Liebl at 919-613-4469 [email protected].
Thank you for your participation,
IQA Cryopreservation Proficiency Testing Advisory Group
Grading Criteria
Site
Vial ID
Tech
Viability (%)
Viability
Score
XXX
213V110000X
BL
100
2
XXX
213V110000X
BL
97.5
2
Viability
Status
A
Viable
Recovery
(%)
Viable
Recovery
Score
95
2
68
1
Viable
Recovery
Status
Combined
status
A
A
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
Proficiency Testing
Quarterly IQA PT
OVERVIEW
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
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
Dead/
Non-Viable Cells
Live/
Viable Cells
Cell Counting
“Dirty” Sample
-platelets/debris
-more difficult to read
“Clean” Sample
-mostly PBMCs (viable/non-viable)
-easy to read/decipher
Cell Counting
Dead Cells/Poor Viability
Cryopreservation
 DMSO helps dehydrate the cells prior to freezing preventing
the formation of ice crystals that could cause cells to lyse
during thawing.
 FBS contains an abundance of proteins, which helps nourish
the cells during the freezing and thawing processes when
cells are deprived of nutrients.
Cryopreservation Solution (CPS) – Freezing Media
Fetal Bovine Serum (FBS)
Dimethyl sulfoxide (DMSO)
90%
10%
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
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 Process for IQA PT
Thaw/Dilute
Wash
Assess Viability/Viable Recovery
Report Results
Lab Communication
Troubleshooting
Challenges
•
•
•
•
•
•
•
Cell Counting
Calculations
Freezing
Equipment Error
Inadequate Mixing
Reagents
Sample Integrity
Cell Counting
 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
Calculations
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
Calculations
Percent viable cells:
# live cells /# total cells (viable + non-viable) x 100
Ex: total viable cells: 226
total non-viable cells: 11
total cells: 237
(226/237) x 100 = 95.4% viable Cells
Calculations
Total Viable Cell Count (Concentration):
total viable cells
squares (4)
X dilution factor x re-suspension volume (mL) x 104
Ex: total viable cells: 226
dilution factor: 10
volume: 2mL
104 : hemacytometer factor/chamber volume
(226/4) (10) (2mL) (10e⁴) = 11.3x106 cells
Calculations
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)
Calculations
# 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
 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
Equipment Error,
Inadequate Mixing, Reagents
 Calibrate daily
 Check settings before every run/use
 Attention to detail during set up
(centrifuge settings, reagents, volumes)
 Mix samples thoroughly before and after each step
 Check expirations dates, temperatures, and volumes
Processing Challenges
Granulocyte Contamination
 Centrifuge not set up correctly
 Ficoll® not room temperature
 Collected from below the
PBMC band (Ficoll®)
Platelet Contamination
 Could cause cell
clumping/misleading counts
 Sample quality
 Collected from above the
PBMC band (plasma)
Fix/Prevention:
Fix/Prevention:
 Count sample at higher
magnification
 Extra wash
 Careful attention during PBMC  Careful/accurate counting
isolation/set-up
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, re-ficoll, careful handling and setup
Processing Challenges
Cell 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…
Tips
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
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
IQA Key Personnel
Daniella Livnat
Thomas Denny
Project Officer , IQA
Principal Investigator
Raul Louzao
IQA Program Manager
Brooke Liebl
Kyle Liebl
Laboratory Research Analyst
Laboratory Research Analyst
Thanks to: DENNY LAB!
Questions?