1. Duke Human Vaccine Institute Immunology Quality Assessment Cryopreservation Proficiency Testing Program Presented by: Kyle Liebl July 16, 2012 www.iqa.center.duke.edu

2. OUTLINE  Immunology Quality Assessment  Purpose  Criteria  Proficiency Testing  Cryopreservation  Cell Counting  Troubleshooting  Challenges  Tips

3. Immunology Quality Assessment

4. Currently there are 65 domestic and 28 international laboratories enrolled in the program. IQA Global Laboratory Participants in the Cryopreservation Proficiency Testing Program (100 participating laboratories as of June 2012) 66 in North America 16 in Central and South America 13 in Africa 5 in Asia

5. Purpose

6. 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

7. Grading Criteria

8.  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 RecoveryScore/sample: 80-100%70-120%2 65-79%50-69%1 -121-150%1 < 65% < 50 % 0 -> 150%0 No Submission0

9. 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) 3-4A A Overall status is determined by the lower of the %viability/ %viable recovery statuses. 2PA2 0-1OP0-1OP Example: % Viability Status: A % Viable Recovery Status: PA Overall Status: PA A: Approved PA: Provisionally Approved OP: On Probation

10. Grading Criteria

11. SiteVial IDTechViability (%) Viability Score Viability Status Viable Recovery (%) Viable Recovery Score Viable Recovery Status Combined status XXX 213V110000XBL100 2 A 95 2 A A XXX 213V110000XBL97.5 2 68 1 An Investigation Report must be submitted to the IQA within 5 working days of receiving results report:Investigation 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

12. Proficiency Testing

13. Quarterly IQA PT OVERVIEW PreparationPBMC ProcessingShipment to IQA

14. Preparation Obtain IRB Consent FormSelect/schedule two Donors (HIV +/-) Collect blood from each donor (ACD, EDTA or NaHep )

15. PBMC Processing Dilute Whole blood/FicollIsolate buffy coat/PBMCWashCount cells Adjust/re-suspend cells in Cryopreservation Solution

16. 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

17. Cell Counting (manually on a hemacytometer)

18. Cell Counting Live/ Viable Cells Dead/ Non-Viable Cells

19. Cell Counting “Clean” Sample -mostly PBMCs (viable/non-viable) -easy to read/decipher “Dirty” Sample -platelets/debris -more difficult to read

20. Cell Counting Dead Cells/Poor Viability

21. 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%

22. 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

23. 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

24. Thawing Process for IQA PT Thaw/DiluteWashAssess Viability/Viable RecoveryReport ResultsLab Communication

25. Troubleshooting

26. Challenges Cell Counting Calculations Freezing Equipment Error Inadequate Mixing Reagents Sample Integrity

27. 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

28. 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

29. 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

30. Calculations Total Viable Cell Count (Concentration): total viable cells X dilution factor x re-suspension volume (mL) x 10 4 squares (4) Ex: total viable cells: 226 dilution factor: 10 volume: 2mL 10 4 : hemacytometer factor/chamber volume (226/4) (10) (2mL) (10e ⁴ ) = 11.3x10 6 cells

31. 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)

32. Calculations # of cells/vial: (Total cell concentration/Final CPS volume) x Freezing volume* = # cells (10 ⁶) /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

33. 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

34. 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

35. Processing Challenges Granulocyte Contamination  Centrifuge not set up correctly  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

36. 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

37. 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…

38. Tips

39.  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

40. 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

41. IQA Key Personnel Daniella Livnat Project Officer, IQA Thomas Denny Principal Investigator Raul Louzao IQA Program Manager Brooke Liebl Laboratory Research Analyst Kyle Liebl Laboratory Research Analyst

42. Thanks to: DENNY LAB!

43. Questions?