1. Vanessa L. Peachee, MS, PhD Director, Immunotoxicology March 14, 2016
Flow Cytometry: Applications and Specialized Method Development in Preclinical Toxicology
Vanessa L. Peachee, MS, PhD
Director, Immunotoxicology
March 14, 2016

2. Flow Cytometry in Immunotoxicology
Immunotoxicology is a rapidly expanding field with new methodologies constantly being developed in drug discovery.
Flow cytometry is a powerful analytical tool used at different stages in drug development.
Validation basics
Overview of importance and challenges of flow cytometry in preclinical toxicology

3. Validation Basics in Flow Cytometry
Quantitative: LC-MS/MS
Absolute quantification of unknown samples: calibration curves
Reference standard: well defined & output is representative of endogenous analyte
Relative Quantitative: Cytokine immunoassays (ELISA or flow cytometry)
Temporal changes in concentrations rather than absolutes: calibration curves
Reference standard: not well defined & output is not representative of endogenous analyte
Quasi-Quantitative: Flow cytometry (immunophenotyping)
No reference standards or calibration curves
Quantitative: Genetic marker
Lacks proportionality to the amount of analyte
Results are non-numeric:+, ++, yes/no or positive/negative
Lee JW, Pharmaceutical Research, Vol. 22, No. 4

4. Critical Parameters in Validation
Accuracy
Closeness to target
Precision
Repeatability
Specificity
Sensitivity
Stability
High accuracy
High precision
Low accuracy
High precision
High accuracy
Low precision
Low accuracy
Low precision
Viginia Litwin and Philip Marder, Flow Cytometry in drug development and discovery, Edition 2011

5. Critical Parameters in Validation
Accuracy
Closeness to target
Precision
Repeatability
Specificity
Sensitivity
Stability
Assay Design
High accuracy
High precision
Development
Low accuracy
High precision
Validation
Implementation
High accuracy
Low precision
Re-Validation
Low accuracy
Low precision
Viginia Litwin and Philip Marder, Flow Cytometry in drug development and discovery, Edition 2011

6. Critical Parameters in Validation
Precision
Closeness of agreement between independent assay results
Expressed as co-efficient of variation (%CV)
Acceptance criteria
<20% CV for cell frequencies >10% of parent population
<30% CV for cell frequencies <10% of parent population (rare populations)
Types
Intra-assay precision
Between replicates
Inter-assay precision
Between experiments
Requirement: Stability
Between analysts
Between instruments
Between laboratories
Thymus

7. Critical Parameters in Validation
Specificity
The ability to correctly identify the target in the
presence of other substances
Marker, antibody and fluorochrome selection
Gating strategy and compensation
Backgating
Fluorescence Minus One (FMO) controls
Differential gating
Peer reviewed literature helpful
Assay Design
Development
Validation
Implementation
Re-Validation

8. Critical Parameters in Validation
Specificity: Backgating
Myeloid or Lymphoid (blood)
T cells
B cells
Myeloid cells
T cells
B cells
Myeloid cells
FSC
SSC
FSC
CD45
T cells
B cells
Myeloid
CD3
B220
B220
CD11b/c

9. Live cells without L/D stain Non-specific staining
Critical Parameters in Validation
Specificity: viability stain
700C for 30min
Thymus
Live cells without L/D stain
Live cells with
L/D stain
Dead cells with
L/D stain
Dead cells
Live cells
99.3
Live cells
95.3
Live cells
1.39
Live dead
FSC
Non-specific staining

10. Critical Parameters in Validation
Specificity: FMO control
L/D stain
CD45
CD4
CD8
Thymus
Gated on L/D-CD45+ leukocytes
CD4
CD8

11. Critical Parameters in Validation
Stability
Thymus
CD4
CD4
CD8
Acceptance criteria : 20% from base line
Freeze thaw caused detection of higher number mature thymic T cell phenotype

12. Critical Parameters in Validation
Sensitivity
Assay sensitivity is the measure of assay performance with known negative samples used in determining the lowest limit of measurement in known positive samples.
Assay setup requires target cells at very low frequencies
Difficult under general immunophenotyping
Reagent sensitivity is important in developing a flow cytometric assay to find the minimum staining intensity above background fluorescence with acceptable precision.
Sensitivity assessment should determine both the maximum fluorescence intensity of a positive population and the maximum separation between the positive and negative populations.

13. Critical Parameters in Validation
Antibody concentration in flow cytometry
for flow cytometry
for quantification
Fluorescence
antibody
Antibody Concentration
autofluorescence
isotype
isotype
Fluorescence
Antibody dilution
High concentration of antibody preferred for flow cytometry

14. Complexity in Tissue Flow Cytometry
Challenges in immunophenotyping data Interpretation
Variation: inter- and intra- animals
Broad range in historical control animal data: Example NHPs
Frequency versus absolutes
Varying levels of information and data in peer reviewed publications

15. Absolutes gave more abnormal cell types than frequency
Flow Cytometry Data Interpretation – Case study 1
Challenges in data interpretation
Frequency versus absolutes
Thymus
Gated on CD3+ cells
B cells
Myeloid
γδ T
T cells
αβ T
Normal
Abnormal
Absolutes gave more abnormal cell types than frequency
CD4 T DP
CD4 T DP
CD4 DN
CD8 T DN
CD8 T
CD8

16. Antibody drug conjugate
Antibody Drug Conjugates
ADCs are a new class of biopharmaceuticals designed as a targeted therapy for the treatment of cancer.
Complex molecules composed of monoclonal antibodies conjugated to cytotoxic drugs through chemical linkers with labile bonds.
Internalization of ADC bound to specific cell leads to release of the cytotoxic drug and death of target cells.
Receptor Occupancy: Requires a specialized approach that addresses the biologic antibody, the drug and the linker.
Linker
Drug
Fab Fc
Death of target cell
Antibody drug conjugate
CH1 CL
CH2
CH3 VH VL

17. ADC Receptor Occupancy in NHPs – Case Study 2
Monocytes
Granulocytes
B cells
T cells
Linker
Drug
Fab Fc
ADC binding to leukocytes
T cells
B cells
Monocytes
Granulocytes
ADC isotype Ab
ADC Ab
Naked Ab
Isotype ADC
Antibody ADC
Naked ADC
ADC binding neutralization by specific peptide
Isotype ADC + Peptide
Antibody ADC + Peptide
ADC isotype Ab + Peptide
ADC Ab + Peptide
Examination of antibody binding to leukocytes within 2-4 hrs of blood collection

18. ADC Receptor Occupancy - NHPs
T cells
Vehicle
Low dose
Medium dose
High dose
CD4 T cells
B cells
CD8 T cells
Monocytes
Dose dependent ablation (day 2 and day 7) and gradual replenishment (day 21) of T and B cells
Monocytes increased subsequent to ADC treatment.

19. Summary
Critical to develop an implementation plan for assay qualification, validation, and sample analysis. Prior to validation the assay should be evaluated and qualified.
Specificity, precision and stability are critical in flow cytometry method validation.
Immunotoxicity of ADCs requires a specialized approach that addresses the binding of antibody (Fc or Fab), the drug and the linker to the target cell.

20. Acknowledgements WIL Research Pathology and Immunology Services:
George Parker, DVM, PhD, DACVP, DABT, Vice President, Global Pathology
Norbert Makori, PhD, Director, General Toxicology
Josely Figueiredo, DVM, MS, PhD, DACVP, Staff Pathologist
Tracey Papenfuss, DVM, PhD, DACVP, Staff Pathologist
Raghu Tadagavdi, BVSc, MVSc, PhD, DABT, RAC, Research Scientist, Immmunotoxicology
Gary Coleman, DVM, PhD, DACVP, DACVPM, Director, Pathology
Technical personnel
Julia England, MS, Flow Cytometry/Project Specialist
Lisa Manson, MT, MLT, Group Supervisor, Clinical Pathology

21. THANK YOU.