IN IN CELLULAR THERAPY STANDARDS IN FLOW CYTOMETRY OVERVIEW - - PowerPoint PPT Presentation

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Jan 05, 2024 197 likes •379 views

PRODUCT CHARACTERIZATION IN IN CELLULAR THERAPY STANDARDS IN FLOW CYTOMETRY OVERVIEW Reproducibility & Variability in Pre-Clinical Research Standards, Practices & Policies in Flow Cytometry (FCM) Case Study: FCM for stem

SLIDE 1

PRODUCT CHARACTERIZATION IN IN CELLULAR THERAPY

STANDARDS IN FLOW CYTOMETRY

SLIDE 2

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

SLIDE 3

FROM PRE-CLINIC ICAL RESEARCH TO IN 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

SLIDE 4 Kindly provided by

SLIDE 5

CELL (P (PRODUCT) CHARACTERIZATION

Cell count
Viability
Cell surface markers
Intracellular markers
Cell cycle / DNA content
Cell size
Gene expression
Enzymatic activity
Oxidative burst
MDR

SLIDE 6

CELL CHARACTERIZATION BY FLOW CYTOMETRY

EXTENSIVE
COMPLEX
EXPENSIVE
VARIABLE

SLIDE 7

STANDARDS, , PRACT CTICES & POLIC ICIES IN 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”

SLIDE 8

VARIA IABIL ILITY IN IN FLOW CYTOMETRY DUE TO:

Sample preparation
Reagents
Instrument
Instrument setup
Data analysis
Data interpretation

SLIDE 9

“What aspect of FCM contributes most to variability?”

Main concerns for FCM in Industry/Biotech:

Analysis
Instrument
Calibration
Measurement

SLIDE 10

CASE STUDY

1995, IS 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

SLIDE 11

EXIS ISTING STANDARD REFERENCE MATERIAL IN 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)

SLIDE 12

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

SLIDE 13

CD34+ Cell Enumeration System Suitability Reference Standard

SLIDE 14

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

SLIDE 15

USE OF USP CD34+ CELL ENUMERATION SYSTEM SUIT ITABILITY 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.

SLIDE 16

MAIN IN POIN INTS

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