new clsi document for the validation of methods preformed ... · new clsi document for the...

New CLSI Document for the

Validation of Methods Preformed by Flow Cytometry –

Sneak Peek and Update

Virginia Litwin, Ph.D.

Vice President, Immunology

2

CLSI H62 – Validation of Methods Performed by Flow Cytometry

Sneak Peek Overview

Document Writing Committee

Process and Timelines

Content Highlights

H62 Document Writing Committee

Leadership• Virginia Litwin, Chair

• Teri Oldaker, Vice Chair

• Raul Louzao, Secretary

• Dave Sterry, CLSI Standards Director

Voting MembersDavid Barnett, Jacqueline, Cleary, Tom Denny, Cherie Green, Wolfgang

Kern, Natalia Kokorina, Jennifer Stewart, Lili Wang

Contributors and ReviewersElena Afonina, Ahmad Al Samman, Tony Bakke, Fiona Craig, Bruce Davis,

Lorella Di Donato, Steve Eck, Nancy Fine, Ben Hedley, Shuguang Huang,

Jerry Hussong, Andrea Illingworth, Cassie Jiang, Mike Keeney, Natalia

Kokorina, Sarah Maremont, Laura Marszalek, Kathy Muirhead, Andy

Rawstron, John Schmitz, Alan Stall, Maryalice Stetler-Stevenson, Horacio

Vall, Alessandra Vitaliti-Garami, Paul Wallace, Brent Wood, Yuanxin Xu

3

Document Writing Committee Composition

Affiliations

• Academia

• Biopharmaceutical

• CRO

• Clinical Laboratories

• Reagent/Instrument

Manufacturers

• Government

- FDA

- NIST

4

Scientific Societies

• AAPS

• CAP

• ESCCA

• ICCS

• ISAC

Provenance• Canada

• Germany

• Switzerland

• UK

• USA

Special Reviewers

ICCS, Advocacy Committee

AAPS, Flow Cytometry Action Program Committee

5

Ruth Barnard, Steve Eck, Catherine Fleener, Fiona

Germaschewski, Christele Gonneau, Cherie Green, Chris

Groves, Michael Hedrick, Shuguang Huang, Shibani Mitra-

Kaushik, David Lanham, Virginia Litwin, Thomas

McCloskey, Thomas McIntosh, Maxime Moulard, Sam Pine,

Kruti Shah, Ulrike Sommer, Soren Sonder, Jennifer Stewart,

Yongliang (Steve) Sun, Alessandra Vitaliti, Dave Williams,

Sam Witherspoon, Yuanxin Xu, Chelsea Xue

Thomas Denny, Pranav Dorwal, Jeannine Holden, Jerry

Hussong, Wolfgang Kern, Virginia Litwin, Sara

Monaghan, Teri Oldaker, Andy Rawstron, Stephanie

Toney, Christopher Trindade, Paul Wallace

Process and Timelines

Document publication (Word/InDesign), December 19

Final Draft, August 19 Final CLSI vote (20 days), September 19

Circulate right to appeal (30 days), July 19

Prepare responses and finalize comments , June 19

Circulate Draft / Open Comment (60 days), February 19

Final Draft Approved by Voting Members November 2018

Face-to-face Kick-off (Kansas City, MO) September 2017

6

Impact

• Extensive review process

• American National Standards Institute (ANSI)compliant

• Alignment with International Organization for Standardization (ISO)

- CLSI serves as the ANSI-appointed Secretariat for the ISO

Technical Committee 212 (ISO/TC 212)

• Regulatory agencies often recognize CLSI guidelines

Document Outline

Chapter 1 Scope

Chapter 2 Quality System Essentials

Teri Oldaker

Chapter 3 Fit for Purpose / Iterative Approach

Fiona Craig

Chapter 4 Instrument Qualification, Setup, and Standardization

Cherie Green

Chapter 5 Assay Development and Optimization

Ben Hedley

Chapter 6 Assay Validation

Steve Eck

Chapter 7 Examination Phase/ Post-Examination Phase

Raul Louzao

Pre

-Exa

min

atio

n P

ha

se

Chapter 1Scope

Scope

• Recommendations and Practical Instructions- One-stop shopping

- Current best practices

- Summarize recent white papers and scientific advances

• Target Audience- Basic research laboratories (non-regulated)

- Clinical (regulated US and ex US)

- Drug discovery, development, and manufacturing (regulated and non-regulated)

- Reagent, assay, and instrument manufacturers

- Regulatory agencies

Out of Scope

• Out of Scope- Individual cell type-specific assay development

- The validation of flow cytometric assays for soluble analytes

- Third-party software and LIS interface validation

Chapter 4Instrument Qualification, Setup, and Standardization

Chapter 4 Outline

4 InstrumentQualification,Setup,andStandardization

4.1 InstallationQualificationandOperationalQualification(IQ,OQ)

4.2 Performancequalification(PQ)

4.2.1 LinearityandDynamicRange

4.2.2 ElectronicNoise

4.2.3 Resolution

4.2.4 Carryover

4.3 Cross-instrument,cross-sitestandardization

4.3.1 ExamplesofCross-standardization

4.4 Compensation:

4.4.1 Generalfactorstoconsiderforcalculatingcompensation:

4.4.2 Typesofcompensationcontrols

4.4.3 CompensationandLinearity

4.5 LongitudinalPerformance

4.6 Qualificationandverificationofinstrumentforintendedpurpose

Chapter 4--Take Home Message

• Instrument qualification is often neglected

• The foundation of good data

Goals of Instrument & Software System Qualification

Establish and maintain a controlled environment that can produce reliable data over a long period of time

Ensure integrity and reconstruction of data

Support lifecycle of the system by establishing procedures from installation to decommission

Installation Qualification

INSTALLATION

PARAMETER

PASS/FAIL

CRITERIA

DOCUMENTATION NOTES

Environment

Benchtop and

associated lab

space meet

vendor

specifications

Checklist with

vendor

requirements,

positive

notation of

Pass/Fail and

initial and date

Consider space

requirements for

instrument/computer

footprint and

additional clearance fo

future maintenance

Utilities

Temperature

and humidity

of lab space

meets vendor

specification

Checklist with

vendor

requirements,

positive

notation of

Pass/Fail and

initial and date

Equipment used to

perform verification

should be documented

in report appended to

the checklist

Electrical

Electrical

requirements

meet vendor

specifications

Checklist with

vendor

requirements,

positive

notation of

Pass/Fail and

initial and date

Equipment used to

perform verification

should be documented

in report appended to

the checklist

Hardware

Verify all

components

are installed

Document

instrument

specifications

(model, serial

number,

manufacturer

date)

Include all associated

components, if any,

including automated

sample acquisition

modules,

uninterrupted power

supplies, etc.

Operation Qualification

OPERATIONAL

PARAMETER PASS/FAIL CRITERIA DOCUMENTATION NOTES

Software

functionality

Perform automated

system functions

(startup, QC)

Screenshot and/or

report with positive

notation of Pass/Fail,

initial and date

Include automated

maintenance procedures

System alerts

Stress the system to

demonstrate that

system detects

problems and displays

appropriate warnings Document warnings

displayed with

screenshots, initial

and date

Visual cues can also be used

to prompt user to change

fluids

Example: Attempt to

acquire data with low

fluidics level or

disconnected

computer cable

Example: Fluidics icons

change color when low levels

are detected. System should

have warning and not allow

further acquisition until

fluidics issues are addressed

Optical

precision

Run calibration beads

to verify %CVs,

detector sensitivity

and laser power

output meets vendor

specifications

Checklist with

vendor

requirements,

positive notation of

Pass/Fail and initial

and date

Include any automated QC

report,

all testing reagents should be

documented in a report

attached to the checklist

Automated

sample

acquisition

Acquire triplicates of

testing material

(beads or cells) in

randomly distributed

locations in carousel

or plate

Checklist with

positive notation of

successful sample

acquisition, Pass/Fail

and initial and date

There is some overlap in PQ;

replicate samples could also

be used to demonstrate

precision. OQ can be

performed using beads

whereas PQ requires

intended use biological

samples.

Performance Qualification

Optical alignment

Linearity and dynamic range

Detection efficiency (Q)

Electronic noise (SDen)

Background signal (B)

Overall resolution of the detection system, which is impacted by efficiency, background, electronic noise

Acquisition carryover

Look What’s New!

19

• The National Institute for Standards and Technology (NIST)

o Fluorescence calibration beads with traceable equivalent

number of reference fluorophores

• Enable us to speak the same language

Traceable ERF Value Assignment to Commercial Microparticlesnt to Commercial Microparticles

FC Bead Ex Laser (nm) SRM 1934

FITC 488 Fluorescein

PE 488 Fluorescein

BB515 488 Fluorescein

PerCP 488 Nile Red

PerCP-Cy5.5 488 Nile Red

PE-Cy7 488 Nile Red

APC 633 APC

APC-R700 633 APC

APC-H7 633 APC

APC-Cy7 633 APC

V450 405 Coumarin 30

BV421 405 Coumarin 30

V500-C 405 Coumarin 30



Six Peak Hard

Dyed Micro-

particles

Ex Laser (nm) SRM 1934

Intensity 2-6 488 Fluorescein

Intensity 2-6 488 Nile Red

Intensity 2-6 633 APC

Intensity 2-6 405 Coumarin 30

Cytometry Part A ●●●● 73A: 279-288, 2008; Flow Cytometry Protocols: Third Edition, p53-65, 2011Current Protocols in Cytometry, 75:1.29.1-14, 2016; Flow Cytometry Protocols: Fourth Edition (in press)

Traceable ERF Value Assignment to Calibration Beads. Flow Cytometry Quantitation Consortium 81 Federal Register 136 (15 July

2016), pp. 46054-46055 ERF Value Assignment to Cytometer Calibration Beads Submitted by Consortium Members

Two step process:

Aim: Provides evidence of linear

range/proportionality and resolution, provides

evidence of comparability within experiment

and between experiments on single

instrument

Aim: Transforms fluorescence scale to ABC

scale, provides reasonable instrument

independent transferable scale

1. Establish linear range in

fluorescence scale using beads

assigned with “Equivalent number

of Reference Fluorophores (ERF)

values

2. Anchor the fluorescence scale

(FS) to a benchmark cell material

with a known protein expression in

the unit of Antibodies Bound per

Cell (ABC)B

en

ch

ma

rk s

ca

le

Lili Wang

Building Measurement Assurance in Flow Cytometry

CYTO Workshop 13

ERF for Benchmarking Instrument Scale

Instrument Standardization

• Why standardize?- Inter-instrument variation

- Major source of variability

o Within the same lab

o Between experiments

o Multicenter clinical trials

• Goal of instrument standardization- Reproducibly set gains (PMT voltages) to achieve equivalent

fluorescence measurements (MFIs)

o Experiment to experiment

o Instrument to instrument

o Lab to lab

o Platform to platform

- Accurately measure / assign fluorescence spillover values which are used for fluorescence compensation

- Maintain consistent longitudinal fluorescence measurements

21

Instruments Compatible for Standardization

• Similar excitation lasers and collection optics

• Have stable fluidics

• Be sufficiently sensitive to discriminate dim fluorescence signals

• Give reasonably low background (photon and/or electronic noise)

• Produce linear signal across dynamic range for intended use

• Produce data conforming to the current Flow Cytometry Standard (FCS) data format

22

Instrument Standardization

Recent Advances

• New instrumentation- Built-in, automated processes for setup and between

instrument standardization

• Existing instruments- Processes for reducing between instrument/platform variability

o Peer reviewed publications Cytometry Part A 73:279, 2008

Cytometry A 81:567, 2012

Cytometry Part A 85:1037, 2014

Cytometry B 90:159, 2015

o Vendor derived process M. Ettinger. A New Method For QUANTITATIVE STANDARDIZATION of Flow

Cytometry Instruments. Contract Pharma. 2015

I. Athanasiadou and C. Gonneau. Challenges of flow cytometry for global clinical trials. ESCCA, 2017

23

Case Study

Inter-instrument variability is reduced when instruments are standardized

Standardization ProcessTwo Identical Instruments

Fluorescence intensity readout

Best Detector Worst Detector

None

Manufacturer’s setup process

Daily Setup passed

17.9 %CV 37.4 %CV

Standardized Instrument

(hard dyed beads)9.36 %CV 0.93 %CV

Standardized Instrument

(true fluorescence)<5%CV <5%CV

M. Ettinger. A New Method For QUANTITATIVE STANDARDIZATION of Flow Cytometry Instruments. Contract Pharma.

2015

I. Athanasiadou and C. Gonneau. Challenges of flow cytometry for global clinical trials. ESCCA, 2017

Want More Details???

Validation the Key to Translatable Flow Cytometry a Three Part Series:

Instrument Qualification

29 October 2018

cytou.org

Chapter 5Assay Development and Optimization

Chapter 5 Outline

Chapter5:Pre-examinationAssayDevelopmentandOptimization

5.2 Assay Evaluation

5.2.1 Steric hindrance 5.2.2 Tandem Fluorochrome interactions

5.2.3 Differences in affinity

5.2.4 IgG dependence

5.2.5 Fc Binding

5.3 Assay Optimization

5.3.1 Premade Cocktailed Antibody Combinations

5.3.2 Preliminary Stability: Reagent/Cocktail stability

5.3.3 Specimen Stability

5.3.4 Controls 5.3.5 Data Acquisition

5.3.6 Data Analysis

5.3.7 Documentation

5.1.1 Assay Development

5.1.2 Define the Assay

5.1.3 Considerations

5.1.4 Matrix 5.1.5 Viability

5.1.6 Antigen and Antibody Selection

5.1.7 Fluorophore Selection

5.1.8 Other Reagents 5.1.9 Sample Cell Concentration

5.1.10 Sample Lysis 5.1.11 Antibody titrations

5.1.12 Blocking

5.1.13 Fixatives

5.1.14 Gating Strategies

5.1.15 Incubation Temperature

Process Map

Assay Objectives

Assay Design

Optimization

Initial Characterization

• Precision

• Stability

Initial Considerations

• What do you want to measure?

• How will you define your population of interest?

• Positive selection antigens

• Negative selection antigens

• In what matrix?

Establish the Assay Objective

• Number and type of lasers

• Number of detectors

• Number of markers

• Number of tubes

• Filter configuration

• Fluorophore selection

What instrument do you have?

Assay Types

• Immuno-phenotyping assays- mAb

- Multimers

• Leukemia/Lymphoma diagnostic assays

• Minimal residual disease (MRD) monitoring

• Phos flow assays

• Receptor occupancy (RO) assay

• Functional assays - Intra-cellular cytokine detection

• Pharmacokinetics (PK) assays - CAR-T

- Other cell-based therapies

Data Output

• Relative percentage of a parent population

• Cell concentration (cells/unit volume)

• Fluorescence intensity

• Percent bound, percent free for RO assays

• Phenotypic description

Matrix

• Whole blood (anticoagulant choice)

• Bone marrow aspirates/cores (anticoagulant choice)

• Other body fluids (CFS, sputum)

• PBMC/BMMC

• Tissues

• TILs

• Cell lines

• Other (marine, bacteria, ….)

Blood Collection Materials

Anticoagulant Description Pros Cons

EDTA- -

Sodium Heparin - - -

ACD- - -

Sodium Citrate- - -

Stabilization Tubes-

-

Assay Design

• Reagents- Antigen/Fluorophore pairing

- mAb clone evaluation

- Reagent titration

Goals of Titration

• Determine specificity and staining intensity of an antibody

• Minimize compensation requirements

• Determination of quantity of new antibody to be used

- Signal should provide adequate room for evaluation of dim or

bright expression

• Quality control of new antibody lots

- Antibody performance should compared to known lot

1 mL 2 mL 3 mL 5 mL 8 mL 10 mL

Optimal Titer

Volume MFI+ MFI- SDneg S/N SI%

Gated

10uL 160.088 2.178 1.215 73.5 100.3 70

8uL 127.259 1.537 1.055 82.8 113.0 70

5uL 92.462 1.075 0.859 86.0 117.4 70

2.5uL 52.466 0.61 0.502 86.0 117.4 70

1uL 32.016 0.492 0.492 65.1 88.8 70

0.5uL 19.258 0.422 0.274 45.6 62.3 70

Staining Index =(�� − �� )

2 � (�� )

�� = ��!� − ��

Assay Design

• Wash/lyse/fix sequence and buffer evaluation

• Acquisition Templates- Number of events to acquire

- Thresholds

- Voltage Settings

• Gating Strategy- The population of interest should be included in the gate

- Other cell subsets/non-specific events should be excluded

Lymph PC Blasts Monos Grans Debris

Lyse A 62% 0% 27% 0% 6% 3%

Lyse A 60% 0% 30% 0% 3% 5%

Lyse C 27% 0% 15% 0% 3% 54%

Lyse D 11% 0% 6% 0% 1% 81%

Lyse E 29% 0% 17% 0% 2% 51%

Other Reagents

• 7-amino-actinomycin D (7AAD)

• 4’6-diamidino-2-phenylindole (DAPI)

• Propidium Iodide (PI)

• Detergents and Solvents

• Lysis Reagents

• Fixatives

• Blocking Reagents


Step-by-Step Multi-parameter Panel Design

Jennifer Wilshire

Tomas Baumgartner

15 October 2018

cytou.org

Chapter 3 Outline

Chapter3:FitforPurposeApproachtoAnalyticalMethodValidationfor

FlowCytometricMethods

3.1 Considerations

3.1.1 BioanalyticalDataCategoriesandCalibrationCurves

3.1.2 ReferenceStandardsforFlowCytometry

3.2 ApplicationofStandardValidationParametersforFlowCytometricMethods

3.2.3 Accuracy/Trueness

3.2.4 Linearity

3.2.5 SpecificityandSelectivity

3.2.6 Sensitivity

3.2.7 Precision

3.2.8 Stability

3.2.9 Assaycarryover

3.2.10 ReferenceIntervals

3.3 Fit-for-PurposeApproach

Fit-for-Purpose Validation Concept

• Fit- All data must be reliable

• Purpose- Any purposes

• Fit-for-Purpose- Analytical validation requirements

o Specific to the current intended use of the data

o Specific to the regulatory requirements, if any, associated with that use

• Practical, iterative approach

Lee et al. Pharmaceutical Research, 22:499, 2005

Iterative Validation Approach

Assay Development /Optimization

InitialValidation

• Assess required parameters for INITIAL intended use

Assay Implementation

Extended Validation

• Assess additional parameters appropriate to NEW intended use

42

Challenges for Validation in Flow Cytometry

• The complexity of cellular analytes/measurands

- Increase complexity of cellular analytes in disease state samples

• The technology

- Highly complex

- Highly flexibility

• The reagents

- Highly complex

- mAb, fluorescent tags, tandem dyes

• The rate of technological advances

- Technology

o New instruments

o New software

- Reagents

o New fluorophores

• The rapid rate of biological discoveries

- New subsets identified

- Phenotypic definitions of existing subsets change

• The lack of TRUE reference material

• The fact that data are not derived from a calibration curve43

Validation Parameters and Flow Cytometry

ALWAYS

• Specificity

• Precision/Robustness

• Sensitivity

• Limit of Detection

• Limit of Quantitation

• Stability

• Reference Intervals

SOMETIMES

• Interference (Matrix, Drug)

IT'S COMPLICATED

• Accuracy

• Linearity

• Selectivity

“NEVER”

• Range of Quantification

• Incurred Sample Reanalysis

• Normal Signal Distribution

• Prozone Effect

What are the Validation Parameters?

Can they be evaluated in Flow Cytometry?

44

Bioanalytical Data Categories

Definitive Quantitative

Relative Quantitative

Quasi-quantitative

Qualitative


45

Definitive Quantitative Data

• Calibration curve

- Reference Standards

o Well defined

o Fully representative of the endogenous analyte

• Numeric results are interpolated from the calibration curve

• Intended use of the data

- Determine the absolute quantitative values for unknown samples

• Example

- LC-MS assay for PK

• Accuracy demonstrated by spike/recovery with well defined standard

Talanta 70.4 (2006): 678-690 46

Relative Quantitative Data

Image from proteintech

• Calibration curve

- Reference Standards

o “Less” well defined

o Not fully representative of the endogenous biomarker

• Numeric results are interpolated from the calibration curve


- Estimate the quantitative values for unknown samples

- Emphasis on temporal changes in concentrations rather than absolute concentrations

• Examples

- Cytokine enzyme immunoassays

• Accuracy demonstrated by spike/recovery with standards

47

Quasi-Quantitative Data


- Estimate the quantitative values

- Emphasis on temporal changes in concentrations rather than absolute concentrations

• Examples

- Flow cytometric assays

o Population frequency

o MRD

• Quasi

- Having some resemblance to

- Possession of certain attributes of

• Does not use calibration curve

- No reference standards

• Numeric data are reported

- Results are expressed in terms of a characteristic of the test sample

Image from Clinical Laboratory News, 12: 8, 2013

CD4CD4CD4

48

Qualitative Data

• No calibration standards

• Non-numeric data are reported

- Results are expressed in terms of a characteristic of the test sample

- Categorical data are reported

o nominal (yes/no) format

o ordinal (+, ++, +++) format (semi-quantitative) (EP12)


- Characterization of the samples

• Examples

- Leukemia/Lymphoma characterization for diagnosis

- Anti-nuclear Antibodies

- Genetic marker/SNP

AML example from Paul Wallace

49

Impact of Type of Data on Validation Design

Validation Parameter

Definitive Quantitative

Relative Quantitative

Quasi-Quantitative Qualitative

Accuracy √ √ - -

Precision √ √ √ -

Sensitivity √ LLOQ √ LLOQ √ √

Specificity √ √ √ √

Dilutional Linearity √ √ - -

Matrix Stability √ √ √ √

Bioanalytical Data Category vs Validation Parameter


50

Assay Risk Categories

Clinical risk Purpose / Intended use of assay

Low Basic research assay

Drug discovery assay

Clinical trial biomarker assay (exploratory end point)

Moderate Laboratory developed test used as an aid to diagnosis

Clinical trial biomarker assay (secondary endpoint)

High

Clinical trial biomarker assay (primary endpoint)

Clinical trial biomarker assay (enrollment criteria endpoint)

Complementary diagnostic

Combination product / Companion diagnostic



cytou.org

Method Validation Overview, Concepts

13 August 2018

Chapter 6 Outline

Chapter6:AnalyticalMethodValidation

6.1 ValidationPlanningPhase(SayIt!)

6.1.1 ValidationPlan

6.1.1.1 AcceptanceCriteria

6.1.2 QuantitativeData/Methods

6.1.3 QualitativeData/Methods

6.2 ValidationExecution(DoIt!)

6.3 ValidationReports(ProveIt!)

6.4 Fit-for-PurposeValidationPlans

Quantitative Data/ Methods

• Accuracy/Trueness • Specificity and Selectivity • Sensitivity

- Sensitivity – Analytical (LOB/LOD)

- Sensitivity - Functional (LLOQ)

• Precision - Experimental Design

- Precision acceptance criteria

• Linearity - Linearity for Relative Quantitative Data

- Linearity for Receptor Occupancy (RO) Assays

- Linearity for Quasi-Quantitative Data

• Stability- Specimen Stability

- Processed Sample Stability

• Assay carryover (instrument)• Reference Intervals

Qualitative Data/ Methods

• Accuracy/Trueness

• Specificity

• Sensitivity

• Precision

• Stability

• Assay carryover (instrument)

• Reference Intervals

Validation Test Menus

*International Medical Device Regulatory Forum

Regulatory

Setting Intended Use of Data Assay Type

Validation

Recommendation

Non-regulated Basic research Novel assay Fit-for-Purpose

Validation Level 1

Non-regulated Drug discovery Novel assay Fit-for-Purpose

Validation Level 1

Non-regulated Exploratory endpoint in clinical development

Novel assay Fit-for-Purpose Validation Level 1

Non-regulated Secondary endpoint in clinical

development Novel assay

Fit-for-Purpose

Validation Level 2

Clinical

Laboratory Patient care/treatment

IVD/CE Approved

Kit

Verification per

CLIA

Clinical

Laboratory

Patient care/treatment clinical

risk LDT

CLIA/IMDRF*

Validation

GLP??? Primary endpoint in clinical

development Novel assay

Full Validation

Level 1

Manufacturing

(GMP)

Regulatory submission for new

diagnostic test Novel assay

Full Validation

Level 2

Manufacturing (GMP)

CDx Novel assay Full Validation Level 2

Test Menu Structure

Parameter Comments Samples Replicates Analytical Runs

Accuracy/Trueness

Specificity

Selectivity

Sensitivity

LOD

LLOQ

Precision

Repeatability (Intra-assay)

Reproducibility (Inter-assay)

Inter-operator

Inter-instrument

Linearity

Stability

Specimen

Processed Sample

Carryover

Reference Intervals

Documentation Validation PlanValidation

ReportQA Review

✓ ✓ ✓

Parameter Comments Samples Replicates Analytical Runs

Accuracy/Trueness

Specificity

Selectivity

Sensitivity

LOD

LLOQ

Precision

Repeatability (Intra-assay)

Reproducibility (Inter-assay)

Inter-operator

Inter-instrument

Linearity

Stability

Specimen

Processed Sample

Carryover

Reference Intervals

Documentation Validation PlanValidation

ReportQA Review

✓ ✓ ✓



cytou.org

Method Validation

Planning and Execution

10 September 2018

Additional Resources

Guidelines for the use of flow cytometry and cell sorting in

immunological studiesVolume47, Issue10Special Issue: Featuring the Guidelines for

the use of flow cytometry and cell sorting in

immunological studies

October 2017

Pages 1584-1797


Special Issue: Validation of Cell‐‐‐‐Based Fluorescence Assays: Practice Guidelines from the International Council for Standardization of

Haematology and the International Clinical Cytometry Society

Volume 84, Issue 5Pages: 279-357

September/October 2013


• Recommendations for the Validation of Flow Cytometric Testing During Drug Development: I Instruments. JIM, 363:104-119, 2011.

• Recommendations for the Validation of Flow Cytometric Testing During Drug Development: II Assays. JIM 363:120-134, 2011.

• Validation of Cell-Based Fluorescence Assays: Practice Guidelines from the International Council for Standardization of Haematology and International Clinical Cytometry Society. Cytometry Part B: Clinical Cytometry Special Issue volume 84B:2013

• Recommendations for the Evaluation of Specimen Stability for Flow Cytometric Testing During Drug Development. JIM, 418:1, 2015.

• Recommendations for the development and validation of flow cytometry-based receptor occupancy assays. Special Issue: Receptor Occupancy by Flow Cytometry, Cytometry Part B: Clinical Cytometry, 90B; 141, 2016.

• Best practices in Performing Flow Cytometry in a regulated environment: feedback from experience within the EBF. Bioanalysis 9:1253, 2017.

• ISAC CYTO University Webinars (available for download at cytou.org)

- Validation, the Key to Translatable Flow Cytometry

o Part 1: Method Validation- Overview, Concepts, August 13

o Part 2: Planning and Executing, September 10

o Part 3: Instrument Qualification, October 29

Summary

• Flow cytometric methods provide high quality, reliable data

• Validation parameters appropriate for soluble analytes are not appropriate

• Validation guidelines….coming soon!

62

Acknowledgment

• CLSI H62 Document Writing Committee

• Teri Oldaker, Co-chair

• Slide Sharing- Cherie Green, Genentech

- Jennifer Stewart, FCS Labs

- Ben Hedley, LHSC

63

64

Figure courtesy of Ira Schieren, Columbia University

Questions?

64

Contact Information

Virginia Litwin, Ph.D.

Vice President, Immunology

Caprion Biosciences Inc.

Montreal, Quebec

Canada

[email protected]

WWW.CAPRION.COM

LinkedIn: https://www.linkedin.com/in/virginia-litwin-99869511/

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