Managing Specimen Stability for Robust Flow Cytometric Clinical Biomarker Assays

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1 Managing Specimen Stability for Robust Flow Cytometric Clinical Biomarker Assays Dianna Wu, Richard Wnek Molecular Biomarkers & Diagnostics Merck Co., Inc 2014 AAPS Annual Meeting San Diego, CA

2 (Fluorescent Intensity) CD4-APC Side Scatter (granulation) Flow Cytometry Technology Specimen collection, preparation & mab Staining Cell Acquisition Data Analysis Major specimen types Whole blood Frozen PBMC Bone marrow aspirate Y Y Y Granulocytes Monocytes Lymphocytes Forward Scatter (size) CD8-PE (Fluorescent Intensity) 2

3 Biomarker Assay Types Assay output Frequency of cells with certain phenotype (%) Absolute number of cells with certain phenotype (cells per ul) Protein expression level on cells (MFI) Biomarker assessment Immune phenotyping o Leukocyte subsets and their differentiation states o Progenitor cells o Hematopoietic malignant cells, circulating tumor cells Cell function o Biomarker expression on/in cells o Cytokine production (Intra-cellular cytokine assay) o Cytotoxicity (Granzyme B assay, CD107 assay) o Antigen-specific T cells (Tetramer/Pentamer assay) o Phospho-protein o Proliferation, Cell Cycle, Apoptosis Target engagement o Receptor occupancy o Receptor competition o Receptor internalization 3

4 Managing Specimen Stability Challenge: Limited post-collection specimen stability Cell-based assay with stability of hours to days Develop and validate assay with extend stability Choice of specimen type: whole blood vs. frozen PBMC Specimen collection tube Assay panel design: markers, mab clones, flurochromes Method optimization and standardization to improve robustness Handle specimens properly at clinical sites Specimen collection/handling/shipping instruction On-site training Analyze specimens within stability window Location of analytical lab FTE resource and analysts qualification Instrument redundancy 4

5 Choice of Specimen Type Whole blood Advantage Closely mimics in vivo state of cell phenotype and function Frozen PBMC Could be cryopreserved for months: Analyze specimens in batches Relatively easy to train lab scientists on PBMC processing Disadvantage Limited specimen stability: Must be processed and analyzed upon receiving Demands FTE and instrument time to perform analysis at real time May lose cell phenotype and/or function during PBMC isolation/cryopreservation/thaw Consideration Use whole blood-based assay for exploratory biomarker testing in early stage/small clinical studies Use frozen PBMC-based assay, if feasible, to verify biomarker in late stage/large clinical studies Perform assay qualification to ensure biomarker expression when changing specimen types 5

6 Choose Specimen Collection Tube EDTA Na-Heparin ACD Cyto-Chex BCT ~24hr stability ~48-72hr stability ~48-72hr stability ~48hr -7 days stability Relatively short stability Often used for BMA Ship specimen at ambient temperature Can be shipped at 4 o C Need to adjust dilution factor for calculation of absolute cell count May not suitable for functional assays Costly 6

7 Treg (%) Change from D0 (%) Design Assay Panel Regulatory T cells: CD3 + CD4 + CD25 hi CD127 -/lo IC-FOXP3 + Collection Tube: Na-Heparin FoxP3 + Tregs % Change from Day Day 0 Day 1 Day 2 Day 3-60 Day 0 Day 1 Day2 Day3 Posy-collection Time Point Posy-collection Time Point 7

8 Re-design Assay Panel 8

9 Treg (%) Change from D0 (%) Re-design Assay Panel Regulatory T cells: CD3 + CD4 + CD25 hi CD127 -/lo CCR4 + Collection Tube: Cyto-Chex BCT CCR4 + Tregs % Change from Day Day 0 Day 1 Day 2 Day 3 Day 4-60 Day 0 Day 1 Day2 Day3 Day4 9

10 % Change from Day 0 Optimize Assay for Signal Robustness Lyse-stain-wash Stain-lyse-wash BMx on CD4 T cells BMx on CD4 T cells Lyse-stain-wash Stain-lyse-wash Stain-lyse-no wash No lyse-no wash Post-collection Stability Day 0 Day 1 Day 2 Day 3 Day 4 Day 7 10

11 BMx pstat3 expression MESF (PBS Subtracted) (MES) Reduce Handling Steps at Clinical Site Background Biomarker: Phospho-protein expression in monocytes after in vitro stimulation Assay with ~4-hr stability On-site specimen processing is required Fresh 24hrs 48hrs Post-collection Blood Storage Time time (4 C) point 11

12 Original Assay Flow Chart Blood Collection <4hr Stimulation 30 min 37º C Fixation 15 min 37º C Performed at Clinical Study Site Permeabilization 30 min Ice 2x mab. Staining Fixation Ship at RT 2x Flow Cytometry Data Acquisition 12

13 Modified Assay Flow Chart After assay optimization and validation Blood Collection <4hr Stimulation 30 min 37º C Fixation Freeze Performed at Clinical Study Site Ship on Dry Ice Thaw 2x Permeabilization 30 min Ice 2x mab. Staining Fixation 1hr. Ambient Temp. 2x Flow Cytometry Data Acquisition 13

14 On-site Training and Proficiency Test Background: Biomarker with <24-hour stability On-site specimen processing is required On-site training Training plan: trainer, trainees Providing clinical site with specimen processing procedure, reagents Demonstration run by trainer and practice runs by trainees Proficiency test Pre-defined acceptance criteria Retraining and re-testing Proficiency Test I: Proficiency Test II: P = PASS F = FAIL Lessons-Learned Sufficient time, travel budget and FTE resource shall be allocated for on-site training Re-training and a second qualification assessment may be required prior to clinical specimen analysis 14

15 Use Appropriate Shipping Package Maintain specimen integrity by minimizing temperature fluctuation during shipping (18-25 o C) Corrugated Shipping Carton Cooler Gel Packs (2) 95kPa Pressure bag Absorbent Sleeve 15

16 Deviations 16

17 Summary Limited post-collection specimen stability is one of the key challenges in implementing flow cytometric biomarker assays in clinical trials. During assay development and analytical validation stage, one of the goals is to extend assay stability. This could be achieved by choosing proper collection tube, optimizing assay panel/method and improving assay robustness. During clinical specimen analysis stage, minimizing preanalytical and analytical deviation is critical to maintain specimen integrity and biomarker data quality. 17

18 Useful References Recommendations for the validation of flow cytometric testing during drug development: Section II assays. O Hara D, et al, JIM 2011; 363:120 Flow Cytometry in Drug Discovery and Development. Litwin V, Marder P, John Wiley & Sons, 2011 Standardizing Immunophenotyping for the human Immunology project. Maecker HT, et. Al, Nature Rev Imm 2012; 12:191 Validation of cell-based fluorescence assays: Practice guidelines from the ICSH and ICCS o Part I-rational and aims. Davis BH, et al, Cytometry Part B Clin Cytom 2013; 84:282 o Part II-Preanalytical issues. Davis BH et al, Cytometry Part B Clin Cytom 2013; 84:286 o Part III-Analytical issues. Tanqri S et al, Cytometry Part B Clin Cytom 2013; 84:291 o Part III-Post-analytical considerations. Barnett D et al, Cytometry Part B Clin Cytom 2013; 84:309 o Part V-Assay performance criteria. Wood B, et al, Cytometry Part B Clin Cytom 2013; 84:315 Flow cytometric devices: Draft guidance for industry and FDA staff

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