Flow Cytometry in 

Translational and Clinical 

Science—Gap Analysis

NIST-FDA	Flow	Cytometry	Workshop:	Building	measurement	assurance	

in	flow	cytometry

Gaithersburg,	MD

October	25,	2017

Virginia Litwin, Ph.D.

Vice President, Immunology
Presentation Overview

Translational	and	Clinical	Science

Best	Practices	to	Get	to	High	Confidence	Data	

Contracting	Gaps

Expanding	Gaps	and	Concern
Translational and Clinical Science
Translational and Clinical Science

Pathway to accelerate the progression of scientific 

advances from the bench-to-the-bedside

Image:	VLitwin
Stakeholders
Translational Science – The Good News

- To increase the success rate of bringing new therapies to patients

- To decrease the timelines and costs of developing new therapies

- To allow for more informed decision making along the drug development pathway

- To build therapeutic potential and drug labeling claims

ROUTINELY APPLIED TO THE DRUG DEVELOPMENT PROCESS
Translational Science – The Bad News

- Martin Wehling, Journal of Translational Medicine 2008, 6:31

Translational processes need to be scientifically backed up by robust methods 

- Francis Collins, Science Translational Medicine 2011, 3:1

- The Case for Standards in Life Science Research

- Putting Translational Science on to a Global Stage

Nature Reviews Drug Discovery 2016, 15:217

- What does it mean when cancer findings can’t be reproduced?

Richard Harris, NPR January 18, 2017
Translational Science – Opportunities

The application of robust analytical method validation will, without 

question, lead to more success in the translational space
Best Practices to Get to High Confidence Data 

Contracting Gaps
Sources of Variability

Reducing Sources of Variability

1. Flow Cytometry Method Validation

2. Instrument Standardization

3. Reference Material

Figure from Maecker et al, Nature Immunology 11:975

Pre-analytical Instrumentation Analytical Post-analytical
Contracting Gaps

Method Validation
Flow Cytometry Method Validation

• No Official Guidance from Regulatory Agencies 

- Currently!

- But we are getting much closer

•New CLSI Guideline in preparation

-H62- Validation of Assays Performed by Flow Cytometry

• Impact

- Regulatory agencies often recognize CLSI guidelines
Target Audience

• Research laboratories (academic and non-academic)

• Clinical Laboratories

• Reagent/Instrument Manufactures

• Drug discovery, development, and manufacturing

• Regulatory Agencies
H62 Document Writing Committee

• CAP representation

• FDA representation

• NIST representation

• AAPS representation

• ICCS and ESCCA representation

• Members from USA, Canada, UK, Germany, Switzerland

• Members from biopharmaceutical, CRO, clinical 

laboratories, reagent/instrument manufacturers, 

regulatory agencies
The	Dream	Team

Leadership

Virginia	Litwin,	Chair

Teri	Oldaker,	Vice	Chair

Raul	Louzoa,	Secretary

Dave	Sterry,	CLSI	Standards	Director

Voting	Members

David	Barnett,	Jacqueline,	Cleary,	

Tom	Denny,	Cherie	Green,	

Wolfgang	Kern,	Natalia	Kokorina,	

Jennifer	Stewart,	Lili	Wang

Contributors

Elena	Afonina,	Ahmad	Al	Samman,	Tony	Bakke,	Fiona	Craig,	Steve	Eck,	Anka	Ehrhardt,	

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,	Soria	Tugulea,	Horacio	

Vall,	Alessandra	Vitaliti-Garami,	Paul	Wallace,	Brent	Wood
Document Outline

•Scope-- Recommendations and practical instructions

•Quality System Essentials

üFit for Purpose / Iterative Approach 

üInstrument Qualification, Setup, and Standardization 

üAssay Development and Optimization

üAssay Validation

•Examination Phase 

•Post-Examination Phase
Contracting Gaps

Instrument Standardization
Instrument Standardization

• 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

• Inter-instrument variation

- Major source of variability

o Within the same lab

o Between experiments 

o Multicenter clinical trials
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

o Vendor derived process

• Automated algorithms for compensation

• Fluorescence beads for compensation
Instrument Standardization

Conclusions

• Inter-instrument variability is reduced when hard dyed beads 

are used for standardization

• Inter-instrument variability is FURTHER reduced when 

instruments are standardized with covalently linked 

fluorochrome beads 

• Hard dyed beads are not optimal for monitoring between 

instrument variability

• Covalently linked fluorochrome beads or comp beads are 

better for monitoring between instrument variability

Mark	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
Instrument Standardization

Gaps

•Processes for standardization are complex, expensive, 

time consuming

-Opportunity to streamline the process with add-on software 

tools

•We can’t all trade in our instruments for the newer ones
Contracting Gaps

Reference Material
Importance of Reference Material

• The lack of cellular reference material contributes to 

the challenges in validating flow cytometric methods

• Cellular reference material would facilitate the 

validation of analytical accuracy

• Cellular reference material is a critical part of overall 

quality monitoring

üInstrument performance qualification

üDaily run acceptance criteria

üInter-assay variation

üInter-instrument variation

üInter-analyst variation

üInter-laboratory variation

üLongitudinal assay performance

üLongitudinal instrument monitoring
Available Reference Material

Preserved Whole Blood

• Pros

- Good overall matrix control

- Good evaluating reagent lots

- Many subsets are detectable

• Cons

- Established ranges from the manufacture are only for the major 

lymphocyte subsets

o Very broad

o Not useful for accuracy

o No ranges for ”off-label” cell types

- High and Low QC material usually calibrated to CD4 T cell counts

o Values of other subsets in the High and Low QC may be the same

o Values in the Low Level may be higher than the High Level

- Relatively short shelf-life

o Continuously assessing mean values in new lots

o Several lots of material are used in longitudinal studies

- Loss of resolution of labile markers

- Decreased resolution of dim markers
Available Reference Material

Lyophilized Lymphocytes

• Pros

- Long shelf-life

- Good control for assays using PBMC

- Good evaluating reagent lots

• Cons

- Not a good matrix control for whole blood assays

- Limited to lymphocyte assays

- May or may not have established ranges
Advances with Reference Material

• Dried leucocytes

- 1 year shelf-life

Menilou Franzblau, Nga Bui, Uyen Tran, Chandana Korde, Daniel Santos, Aaron 

na, Marybeth Sharkey 

strate the Convenience, Consistency, Stability and 

mance of BD HorizonTM Dri Leukocyte Controls

are results against introduced 

nd procedural controls for 

d lymphoma, immune 

convenience, stability and 

on. Meanwhile, the majority 

clude short shelf life, 

on of dim populations. BD 

Biosciences proprietary 

d offer convenience, 

ow cytometry end users. 

ized and encapsulated with 

ences proprietary drying 

emperature. The marker 

200 antibody clones using a 

Leukocytes was tested using 

ain light scatter comparable 

ations including small 

er 150+ surface and 

s. Accelerated stability study 

Immunofluorescence Staining Performance

Scatter Profiles

Cell Surface and Intracellular Staining

Dendritic Cells (DCs) and Baso

Small populations like Dendritic Cells (DC) and B

be identified with BD Horizon Dri Leukocytes usin

color assay. The reagents include Lineage Cockta

CD3, CD14, CD16, CD19, CD20, and CD56) FITC

APC, Anti–HLA-DR V450, CD123 PE-CyTM 7 

Leukocytes: 

 access

 months

cy

rmance to Fresh Blood

Antibody panel #1 Fluorophore Marker

FITC CD3

PE CD16/56

PerCPCy5.5 CD45

PE-Cy7 CD4

APC CD19

APC-H7 CD8

Fluorophore Marker

FITC CD8+Lamda

PE CD56+Kappa

PerCP-Cy5.5 CD5

PECy7 CD19+TCRgd

Fluorophore Marker

FITC cyMPO

PE cyCD79a

Fluorophore Marker

FITC Lambda

PE Kappa

PerCP-Cy5.5 CD20

Fresh Blood BD Horizon Dri Leukocytes

Antibody panel #2

Antibody panel #3

Antibody panel #4

Chandana Korde, Daniel Santos, Aaron 

nsistency, Stability and 

Leukocyte Controls

escence Staining Performance

Dendritic Cells (DCs) and Basophils

Small populations like Dendritic Cells (DC) and Basophils can 
Advances with Reference Material

• Lyophilized PBMC

- Customized preparations

• Novel materials

- Slingshot Biosciences

- Polymer droplets that can mimic the physical/optical cell type

- FlowCytesTM WBC Cell Mimics 

- Conceivably imbedded appropriate antigens in the polymer

CONFIDENTIAL

FlowCytesTMSynthetic White Blood Cells

FlowCytesTM WBC Mimics*Lysed Whole Blood WBC*

1 week shelf-life in solution at 4C

Multi-year shelf-life in solution at 

room temp + (freeze/boil)

1250 45th Street, Suite 150

Emeryville CA 94608

Tel: 646-450-7332

brandon@slingshotbio.com

Stability

slingshotbio.com

FlowCytesTMSynthetic White Blood Cells

FlowCytesTM WBC Mimics*Lysed Whole Blood WBC*

1 week shelf-life in solution at 4C

Multi-year shelf-life in solution at 

room temp + (freeze/boil)

1250 45th Street, Suite 150

Emeryville CA 94608

Tel: 646-450-7332

brandon@slingshotbio.com

Stability

slingshotbio.com

u FlowCytesTM scatter can be made to match white blood cells

u Plastic particles look nothing like cells (Panel C) but are the only current option

u Panels A and B were collected with no instrument adjustments, streamlined workflow. Parti

are identical in size with tuned refractive index and side scatter.

Purified White Blood Cells FlowCyteTM WBC Mimics

Spherotech

3um Polystyren

Panels A and B

Off Scale, requ

V/Gain Adj. to 

First product: FlowCytesTM WBC Cell 

Mimics

A) B) C)
Remaining Gaps Reference Material

• Leukemia/lymphoma controls 

- We need them!

- Useful for the validation of leukemia/lymphoma diagnositic

panels

- Critical for MRD panel validations
Expanding Gaps
Expanding Gap
Bench vs Bedside in Flow Cytometry

Bench 

• Constituents

- Basic research

- Clinical research

- Drug development

- Biotech

- Instrument and reagent 

vendors

• Funding

- Grants

- Investments

- Internal

Bedside

• Constituents

- Local hospital

- University Medical Centers

- Reference Labs

• Funding / Reimbursement

- Fee for service

- Medicare

- Insurance agencies
Flow Cytometry Reimbursement 

CPT Code Description Decrease	in	Payment	

2018	vs	2016

88184 1st marker	TC 80%

88185 Additional	markers	TC 66%

88187 Professional	2-8	markers 66%

88188 Professional	9-15	markers 71%

88189 Professional	16	and	greater	markers 78%

• Continued cuts from Centers for Medicare and Medicaid (CMS) for 

reimbursement for flow cytometry services for Medicare patients

- Physician Fee Schedule

- Clinical Laboratory Fee Schedule

• Medicare rates influence private insurance reimbursement rates

J.	Hussong.		ICCS	Advocacy	Committee	Chair
Dimensionality

Bench

• Cytof

• >20 Flow cytometry

• 12-18 Flow cytometry

Bedside

• FDA approved/ CE marked 

Instruments

- 4-10 color

• FDA approved/ CE marked 

leukemia panels

- 5-8 colors
Validation

Bench

• Extensive Validation

- Manufacturers

- Biopharma

o GLP/Toxicology

o Clinical Testing

· Exploratory

· Primary/Secondary endpoint

· Enrollment criteria

· Complementary diagnostic

• Maybe no validation

- Research environment

- Non-regulated biopharma (drug 

discovery) 

Bedside

• ???

• No official guidance

- Not clear what’s needed and when

- Wide range of intended-use of data

• Lack of staff/time

• Gap of understanding of 

validation principals and valueadded
Conclusions

• “It is the best of times, it is the of worst times”

• “It takes a village”

- Even greater collaboration between bench and bedside 

scientist is required

o Education

o Resource (information sharing)

o Application Tools from Vendors

- We need to make sure that the innovation from the Bench 

makes it to the Bedside

• Resource Gaps

- Bench- Greater funding is needed to fuel innovation

- Bedside- Better resources for patient care and treatment
Acknowledgements

• BD Biosciences

- Alan Stall

- Marybeth Sharkey

- Joan Batchelder

• ICCS

- Fiona Craig, Mayo Clinical Arizona, ICCS Past President

- Jerry Hussong, Sonic Healthcare, Chair ICCS Advocacy 

Committee