Post on 15-May-2018
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OVERVIEW
• Reproducibility & Variability in Pre-Clinical Research
• Standards, Practices & Policies in Flow Cytometry (FCM)
• Case Study:
FCM for stem cell enumeration in Hematopoietic Stem Cell Therapy
FROM PRE-CLINICAL RESEARCH TO INDUSTRY
• Cellular Therapy recently entered biopharmaceutical sector • Cost-Benefit
• Quality Assurance & Control
• Product characterization for cell-based products in Cellular Therapy • Reliable
• Accurate
• Relevant characteristics
CELL (PRODUCT) CHARACTERIZATION
• Cell count • Viability • Cell surface markers • Intracellular markers • Cell cycle / DNA content
• Cell size • Gene expression • Enzymatic activity • Oxidative burst • MDR
STANDARDS, PRACTICES & POLICIES IN FCM, TODAY
• Reagents (antibodies)
• Data file format
• Reporting (publications)
• Training & Proficiency
• Calibration & Measurements • Instrument setup
• Immunophenotyping
• HLDA Workshops (1982)
• FCS files (1984)
• MIFlowCyt (2008)
• International Flow Cytometry Certification Examination (2011)
“(still) in progress”
VARIABILITY IN FLOW CYTOMETRY DUE TO:
• Sample preparation
•Reagents
• Instrument
• Instrument setup
•Data analysis
•Data interpretation
“What aspect of FCM contributes most to variability?”
Main concerns for FCM in Industry/Biotech:
- Analysis
- Instrument - Calibration
- Measurement
CASE STUDY
1995, ISHAGE: STEM CELL ENUMERATION CMTE
• CD34 antigen expressed on the surface of almost all hematopoietic stem cells
• Absolute number of CD34+ cells shown to correlate with engraftment potential in hematopoietic stem cell therapy
• An accurate CD34+ cell count can help optimize the timing of apheresis collection
• Flow cytometric enumeration of CD34+ cells – ANALYSIS, REAGENTS
EXISTING STANDARD REFERENCE MATERIAL IN FCM
• INSTRUMENT SETUP e.g. Rainbow Calibration Particles (RCP-30-5A)
• PROCESS CONTROL e.g. Stem-Trol (KG1 cells)
• DATA ANALYSIS e.g. Stem-Control (stabilized normal PBMCs)
• Provides a standard, single-platform flow cytometric method for enumerating CD34+ cells in PB, Leukapheresis, BM and CB
• Step-by-step instructions with figures for demonstrating data analysis according to ISHAGE guidelines
• RS is made from mobilized peripheral blood collected by apheresis of a G-CSF mobilized donor.
• Contains human leukocytes, erythrocytes and CD34+ cells that have been fixed and lyophilized
• Stable reference standard with CD34 and CD45 staining comparable to fresh G-CSF mobilized peripheral blood cells
• Well-defined cell count and range assigned from a multi-laboratory collaborative study
• Reconstitutes in water and is ready to use. No lysis steps required
USE OF USP CD34+ CELL ENUMERATION SYSTEM SUITABILITY REFERENCE STANDARD
• Used as a system suitability standard to assess the reagents used and ensure the correct gating during data (acquisition and) analysis for enumeration of CD34+ cells
• Results should fall within the range provided in the USP CD34+ Cell Enumeration System Suitability RS certificate.
MAIN POINTS
• Challenges for FCM in commercial cellular therapy • Calibration & Measurements
• Some reference materials for calibration, process control and analysis in use in FCM • but no ‘standard’
• CD34+ cell enumeration by FCM, now standard for commercial cell therapy • USP Chapter <127>
• USP CD34+ cell enumeration system suitability reference standard