Challenges for Product Evaluation by Flow Cytometry for Cellular Therapy Product Processing Laboratories

Transcription

1 Challenges for Product Evaluation by Flow Cytometry for Cellular Therapy Product Processing Laboratories C A R O LY N A. K E E V E R - TAY L O R, P H D M E D I C A L C O L L E G E O F W I S C O N S I N B L O O D A N D M A R R O W T R A N S P L A N TAT I O N P R O G R A M I S C T R E G I O N A L W O R K S H O P M E M P H I S, T N

2 Facility/Program Information Support for combined pediatric and adult FACT & CAP/CLIA accredited for processing and testing Processing and transplant activities for 2015 include: Transplants Total 299 Autologous 173 Allogeneic 126 DLI 18 Total Procedures 1300 Marrow harvests 26 Cryopreservation 389 Thawing (Dex/Alb Wash) 332 Fresh Infusions 127 Cell Separations (CliniMACS) 12 Additional Activities Peripheral Blood CD Product Flow 544 Inoculate Sterility Cultures Materials Management Quality Program Personnel Director 1 Medical Director 1 Assistant Director 1 Laboratory Manager 1 QA/QC Specialist 1 Cellular Therapy Specialists 5 Research Technologist 1

3 Objectives of Presentation Describe types of product flow analysis required by/for laboratory Identify specific challenges unique to analysis of HPC products Identify challenges of thawed product testing and very rare event analysis

4 Types of Product Analysis Products HPC, Apheresis HPC, Marrow HPC, Cord MNC, Apheresis Whole Blood Stage of assessment Fresh Thawed Cultured Reason for assessment Product release Need for additional collections Common manipulations Subset enriched MNC CD34+ cells NK cells or other subsets Subset reduced CD3+ cells, B cells or other subsets Procedure or process validation Stability studies or expiration time

5 Product Testing and Analysis Challenges Availability of flow services Within the laboratory External to the laboratory Clinical flow lab Research flow lab On or off site Hours of service Technical issues Dead cells Contaminating cells High background staining Rare events Panels Antibodies Fluorochromes Combinations per tube Custom trial specific Methods Staining Use of kits Wash steps Lyse steps Analysis methods Automated Manual Single platform Dual platform

6 Most Common Assessments Peripheral Blood CD34 Prior to first collection, or prior to each collection Viability high, hematology analyzer cells counts accurate Lysis required Washes not required Dual or single platform methods give similar results Fresh Product CD34- Same as above except for: Every collection Lysis not required when CD45 in every tube Both assays require rapid turnaround time due to clinical decisions regarding initial or subsequent collections. PB CD34 may be rare event analysis.

7 Example PB-CD34 Low vs High Events:300,000 Events:24 CD34/µL=4 Events:300,000 Events:100 CD34/µL=65

8 Example CD34 Product Events:100,000 Events:77 CD34/µL=191 Events:100,000 Events:256 CD34/µL=999

9 Validation Studies Fresh Products Viability Range of detection Effect of storage Frozen Products Staining Methods Acquisition Methods Stability and expiration

10 MCF 7-AAD+ %Viability Trypan Blue SSC-H: Side Scatter Example Method Validation-Viability by 7-AAD Determine conditions to kill but not lyse 100% of cells PBMC.001 alive Minutes at 56oC FL3-H: 7AAD Determine when to add and how much to add AAD Mean Channel Fluorescence AAD Concentration µg/ml

11 7-AAD sensitivity Cross titer dead into living cells Dilution Series % Dead % Living 98.0% 2.0% 80.0% 20.0% 60.0% 40.0% 35.0% 65.0% 20.0% 80.0% 12.0% 88.0% 7.0% 93.0% 4.0% 96.0% 2.0% 98.0% 1.5% 98.5% 1.0% 99.0% 0.5% 99.5% 0.1% 99.9% Whole Curve

12 Example Validation- CD34 sensitivity 1.00% Dilution of mobilized HPC(A) into nonmobilized PBMC CD34 Linearity & Fit to expected R² = % 0.00% 0.00% 0.20% 0.40% 0.60% 0.80% 1.00% 0.30% CD34 Linearity & Fit to expected R² = % 0.10% 0.00% 0.00% 0.10% 0.20% 0.30%

13 Expected Effect of overnight product storage Validation Evaluated CD34, CD3, and MNC content at receipt and after overnight refrigerated storage Selected products with a range of MNC content (>70%, <70%). Duplicate tubes to measure inter and intra assay variation Results, inter assay cv <5% before and after storage. Good correlation of results over wide range of MNC content. Observed

14 Rare Event Analysis CD3 Reduction followed by CD56 enrichment in the setting of haploidentical transplantation T cells defined as CD3+CD56- Limit 1 x 10 4 T cells/kg Target CD56+ cells of 5.0 x 10 6 /kg Approach to improve sensitivity Acquire minimum of 150,000 debris free events Use a combination of CD14 and CD33 to remove monocytes and immature myeloid cells from analysis gate Use detection antibodies for CD3 and CD56 that recognize different epitopes than antibodies used for depletion or enrichment

15 NK Enrich Example Analysis Starting HPC, MNC NK Cells, Apheresis FS vs SS CD45 vs SS 7-AAD vs SS CD14/CD33 vs SS CD3 vs CD56

16 Validation of Methods to Assess Recovery in Thawed Products Reasons for performing flow cytometry on thawed products Assessment of cellular content post thaw when uncertain, e.g. CD34+ cells for cord blood, CD3+ cells for DLI products Validation of new or modified cryopreservation procedures Establishment of post thaw expiration Stability assessment of cryopreserved products Challenges performing flow cytometry assessment of thawed products include: Staining methods may introduce artifacts Technical issues such as dead cells or debris that can clog cytometer TNC from hematology analyzer may not be accurate when doing dual platform cytometry Handling of sample while still having it be representative of the product Approaches Single platform versus dual platform methods to avoid hematology analyzer problems Avoid using lysing reagents that may further damage cells Use no wash method (required if beads already in tube) Careful handling of product or product sample to reduce cell loss due to clumping

17 Wash/Lyse versus No Wash/No Lyse- Cord Blood Wash/Lyse Dual Platform 62.1 % 96.2 % 0.39 % 0.35 % 0.35 % TNC Frozen: 1.01 x 10 9 CD34 Frozen: 1.44 x 10 6 TNC Recovered: 4.3 x 10 8 CD34 Recovered: 1.67 x 10 6 %CD34 Recovered: 116% TNC Recovered: 3.5 x 10 8 CD34 Recovered: 0.89 x 10 6 %CD34 Recovered: 61.9% No Wash/No Lyse Single Platform 51.1 % 98.7 % 0.30 % 0.26 % 0.26 % 7-AAD vs SS CD45 vs SS CD34 vs SS CD45Dim vs SS LFS vs LSS

18 %CD34 Recovery Cells per ml Cord Blood CD34 Recovery- Dual vs Single Platform 300 Lyse & Wash No Lyse No Wash No Lyse/No Wash P value < Cord Dual Mean ± SEM of column A Mean ± SEM of column B Cord Single ± N= ± N= P value Dual Platform Single Improved (realistic) results with single platform method does not appear to be completely due to more accurate cell counts since counts are only marginally higher. Might be due to removing lysing and/or wash steps

19 %Recovery Viable Cells Stability Post Dex/Albumin Wash Validation Full Study on HPC(A) products Immediate vs 90 min HES vs Dex Study %MNC %TNC %CD34 N= Post Thaw Time (minutes) Flow assessment after a period of equilibrium seems to improve before stabilizing. Also seen in HES validation 90 min for CD34 recovery and viability, but not for TNC, MNC, and overall viability.

20 CD14 Single Platform Gating, Dextran 8.3%/4.3% HSA Arm Immediate Post Wash Non-Viable Gate CD34 FSC CD45 7-AAD CD45 CD34 CD45 FSC

21 Other Suggestions for Flow Analysis of Thawed Cells Include more events typically considered as debris Found that dead CD34 under-estimate since scatter with debris Our own findings do not support this as we get good agreement between total CD34 frozen and the sum of living and dead CD34 as gated in previous example Include anti-glycophorin Ab in Cord Blood assessments for removal of NRBC s and unlysed RBC from analysis Did not find significant differences from using only CD45 to remove RBC from gates in MCW experience For non-washed product stability, dilute samples in protein containing medium and maintain all reagents and tubes in the cold Could help. Since MCW washes all thawed products we validated only immediate post thaw samples encompassing the 15 minutes set for post thaw expiration Found that after the first staining period of 15 minutes, largely failed to get useful data when directly thawed products stored and resampled at later intervals without product or product sample dilution