1. Cryopreservation Proficiency Testing Program
Duke Human Vaccine Institute
Immunology Quality Assessment
Cryopreservation Proficiency Testing Program
Tania Garrelts
IMPAACT/ACTG Meetings
June 2010

2. 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

3. 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:

4. 93 participating laboratories 11 in Central and South America
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 America
11 in Central and South America
11 in Africa
6 in Pacific Rim

5. 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.

6. 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.

7. IQA Sample Workflow Create/distribute summary spreadsheet.
Laboratories are ed to retrieve performance report on the IQA website (iqa.center.duke.edu).
One week later the IQA begins troubleshooting unsatisfactory laboratory performances.

8. How Does The IQA Remind Labs To Participate In The Program?
Laboratories reminders
Yearly Calendar (IQA website)
Participation Reminder (10 weeks prior to shipping )
Collection Notification (1 week prior to sample collection)
Shipment Notification (1-2 weeks prior to due date)
This calendar also serves as a timeline to aid in the identification and selection of appropriate donors in a efficient manner.
On a quarterly basis participants in the IQA Program cryopreservation program are reminded via participation notice. The reminder notice contains detailed instructions about collection requirements, sample processing and shipping information along with deadlines. Cryopreserved PBMC samples are submitted by U.S and non U.S. laboratories on a quarterly cycle to the IQA Program. The submitting site notifies the IQA Program of the shipment’s tracking number via or fax, and the shipped box is tracked from origin to final delivery at the IQAC. The express delivery format of samples is essential to this program and measures are developed to ensure the timely delivery of shipments. Shipments that are sent within the US are shipped by overnight express service. International laboratories are further instructed to ship samples with provided LN2 dry shippers using Federal Express. International sites will contract with World Courier services for the express delivery of their shipments. The IQAC will provide instructions to a site in the use of LN2 dry shipper. For EQAPOL, we propose to use a similar process with appropriate customization to meet the EQAPOL site/program needs.
Upon receipt of shipment at the IQAC, the samples are unpacked, inspected for signs of premature thawing, and stored in the vapor phase of liquid nitrogen for a period of no less than one week prior to thawing and testing. If there are any indications of thawing, the site will be notified immediately and allowed the option to send replacement samples. Sites are instructed to ship to the IQAC two aliquots from each donor, and store two aliquots from each donor in their lab’s -70C freezers for a maximum of five weeks for possible future testing or troubleshooting purposes. The sample identification along with the shipping manifests is reviewed for accuracy and the information is recorded. The cryopreserved samples are evaluated in accordance with the NIAID-ACTG consensus protocol

9. 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.

10. Peripheral Blood Mononuclear Cells (PBMC) Isolation

11. Centrifuge EDTA , Heparin or ACD blood at 400 x g for 10 minutes.
Plasma Isolation
Centrifuge EDTA , Heparin or ACD blood at 400 x g for 10 minutes.
Buffy Coat
Plasma

13. 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®

14. At the end of the centrifugation step, the following layers are visually observed from top to bottom.
plasma/platelets
separating medium
granulocytes
erythrocytes
buffy coat (PBMC)
The PBMC layer is then removed and washed twice to get rid of contaminants before cell type and cell viability can be confirmed.

16. Manual Ficoll® Methods
Overlay
Underlay

18. Cell Separation Tube With Frit Barrier (CSTFB)
Load the Ficoll into a polypropylene tube with a frit (porous polyethylene barrier).
Centrifuge to settle the Ficoll below the frit.
3. Layer whole blood on top of the frit.
Centrifuge the tube.
Frit Barrier

20. 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

21. Buffy Coat Isolation

22. Comparison of Manual Ficoll® Methods
Underlay Method
Mononuclear layer
Overlay Method
Mononuclear layer

24. Cell Separation Tube With Frit Barrier (CSTFB)
Plasma Layer
Mononuclear Layer
Ficoll®
Frit Barrier

26. Cell Preparation Tube (CPT)
Plasma Layer
Mononuclear Layer
Erythrocytes

28. Manual Cell Counts

30. What to Count on the Hemacytometer?

31. 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 X Dilution factor x resuspension volume x 104= __ # cells
Number of squares counted
Calculate cell yield per mL of whole blood:
(QC check)= (Cell Concentration/ Whole Blood Volume)

32. Cryopreservation

33. 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.

34. 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.

36. Cell Freezing Containers
Cells 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”, “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.

37. Thawing of Cryopreserved Cells

38. 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.

39. 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.

41. 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.

42. Troubleshooting

43. Processing Errors Red blood cell contamination Excess of platelets
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.

44. 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.

45. Granulocyte contamination
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 (15-30°C) room temperature.

46. LDMS Computational Error Example 1.
Shipping Manifest
Processing Report
Cell concentrations do not match.

47. 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)
Total percent viable recovery
Final performance
5.66
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.

48. 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

49. Cell Viability Processing Report Unexpected Viability
Freshly isolated PBMC
viability should be >95%.
Yields outside the expected ranges may indicate:
Long processing time
Poor technique
Viability is too low

52. IQA Personnel Daniella Livnat Project Officer for the IQA
Contract# HHSN C
Thomas Denny
Principal Investigator

53. IQA Personnel Raul Louzao IQA Program Manager raul.louzao@duke.edu
Tania Garrelts

54. Special Thanks To:

55. Questions?