Duke Human Vaccine Institute

Transcription

1 Duke Human Vaccine Institute

2 OUTLINE Immunology Quality Assessment Purpose Grading Criteria Proficiency Testing (PT) Cryopreservation Thawing Troubleshooting Challenges Technique/Tips

3 Immunology Quality Assessment

4 IQA Global Laboratory Participants in the Cryopreservation Proficiency Testing Program (94 participating laboratories as of June 2011) 65 in North America Currently there are 65 domestic and 28 international laboratories enrolled in the program. 13 in Central and South America 11 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 Quarterly Reminders Yearly Calendar (IQA website) Participation Reminder Collection Notification Shipment Notification Results Report Notification Investigational Report (IR) Notification Continuous communication via /phone from IQA and network personnel

8 Grading Criteria

9 Grading Criteria Performance Evaluation established by the IQA PBMC Cryopreservation PT Advisory Group (ICAG) Scoring System for One Sample % Viability % Viable Recovery Score/sample: % % % 50-69% % 1 < 65% < 50 % 0 - > 150% 0 No Submission 0

10 Grading Criteria Combined % Viability Score (both samples) Combined Scoring System for 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 2 PA 2 PA 0-1 OP 0-1 OP lower of the %viability/ %viable recovery statuses. A: Approved PA: Provisionally Approved OP: On Probation Example: % Viability Status: A % Viable Recovery Status: PA Overall Status: PA

11 Grading Criteria Site Vial ID Tech Viability (%) Viability Score Viability Status Viable Recovery (%) Viable Recovery Score Viable Recovery Status Combined status XXX 213V110000X BL A XXX 213V110000X BL PA PA 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

12 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

13 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

14 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 backup 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)

15 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)

16 March 2011 Results Data Status: Overall % Viability Overall % Recovery Combined Status # of labs with an Approved (A) Status Labs Enrolled 91 Participated March 2011 # of labs with a Provisionally Approved (PA) Status # of labs with an On Probation (OP) Status # of labs with an on Hold (Hold) Status NA NA 4-2 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

17 Proficiency Testing

18 Quarterly IQA PT Preparation PBMC Processing Shipment to IQA

19 Preparation Obtain IRB Consent Form Select/schedule two Donors (HIV +/-) Collect blood from each donor (ACD, EDTA or NaHep )

20 PBMC Processing Dilute Whole blood/ficoll Isolate buffy coat/pbmc Wash Count cells Adjust/re-suspend cells in Cryopreservation Solution

21 PBMC Processing 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 Ficoll

22 PBMC Processing Isolate Buffy Coat/PBMCs POST Centrifugation

23 PBMC Processing Isolate the Buffy Coat (PBMC) Remove plasma layer Isolate and harvest buffy coat (PBMC layer) plasma/platelets Buffy Coat (PBMC) separating medium granulocytes erythrocytes

24 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

25 Manual Ficoll Methods Buffy Coat (PBMC) (Underlay) Buffy Coat (PBMC) (Overlay)

26 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 Frit Barrier Centrifuge: (800 x g for 15 minutes, brake off) *Accuspin/Sigma-Aldrich

27 Cell Separation Tube With Frit Barrier (CSTFB) Plasma Layer Buffy Coat (PBMC) Ficoll Frit Barrier

28 BD Vacutainer CPT Cell Preparation Tube With Sodium Citrate* Centrifuge: 1,500 to 1,800 RCF for a minimum of 20 minutes, brake off 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 *Becton, Dickinson and Co.

29 BD Vacutainer CPT Cell Preparation Tube With Sodium Citrate Plasma Layer Buffy Coat (PBMC) Erythrocytes

30 Comparison of 3 Methods Factors: Manual Ficoll (overlay/underlay) Cell Separation Tube w/ Frit Barrier Cell Preparation Tube (CPT) Cost/Sample* $3.55/ 20mL whole blood $13.10/ 20mL whole blood Advantages Most cost effective Less time consuming $8.25/ 8mL whole blood Convenient (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

31 Review: Ficoll carefully (clean interface) Precise buffy coat/pbmc isolation/pipetting Attention to detail during set up (centrifuge settings, reagents, volumes)

32 PBMC Processing Dilute Whole blood/ficoll Isolate buffy coat/pbmc Wash ( x g for 10 minutes) Count cells Adjust/Re-suspend cells in Cryopreservation Solution

33 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

34 Cell Counting (manually on a hemacytometer)

35 Cell Counting Dead/ Non-Viable Cells Live/ Viable Cells

36 Cell Counting - Problems Dirty Sample -platelets/debris -more difficult to read Clean Sample -mostly PBMCs (viable/non-viable) -easy to read/decipher

37 Cell Counting - Problems Dead Cells/Poor Viability

38 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

39 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: 11 total cells: 237 (226/237) x 100 = 95.4% viable Cells

40 Formulas For Cell Counting 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) (10⁴) = 11.3x10 6 cells

41 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

42 PBMC Processing Dilute Whole blood/ficoll Isolate buffy coat/pbmc Wash Count cells Adjust/Re-suspend cells in Cryopreservation Solution

43 Cryopreservation

44 Why do we use DMSO and FBS as Cryoprotective Agents? 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%

45 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)

46 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

47 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

48 Freezing Containers Stratagene StrataCooler Cryo: Holds up to 32 cryovials ~$159.00/unit

49 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 ~$ /unit (+isopropanol)

50 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

51 Freezing Containers Factors: StataCooler Mr. Frosty CoolCell # Cryovials Cost/unit* $ $ (+isopropanol) $ Advantages Large sample-size, No Isopropanol Cost No Isopropanol Disadvantages Cost Up-keep, proper waste 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

52 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

53 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 5x10⁶cells/vial to the IQA during designated dates

54 Thawing

55 Thawing Process for IQA PT Thaw/Dilute Wash Assess Viability/Viable Recovery Report Results Lab Communication

56 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.

57 Troubleshooting

58 Challenges

59 Processing Challenges Granulocyte Contamination Centrifuge not set at correct speed/time 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: Count sample at higher magnification Careful attention during PBMC isolation/set-up Fix/Prevention: Extra wash Careful/accurate counting

60 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

61 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

62 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)

63 Technique/Tips

64 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

65 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