Session 3: Needs and Approaches for overcoming 

gaps in obtaining sufficient measurement assurance 

for cell counting

Regulatory considerations for cell counting

Steven Bauer / FDA 

Measurement assurance strategies

John Elliott / NIST 

Round Table Discussion

• Common methods/best practices

• Reference materials

• In process controls for the measurement process

• Other strategies for measurement assurance

• Documentary standards
Round Table Discussion

Moderator: Sumona Sarkar, NIST
Some Considerations

What are the gaps in the existing resources for cell counting?

What are the best practices for establishing validated cell 

counting methods?

How can we improve our confidence in cell count 

measurements?

How can we assure comparability of cell counting 

measurements?

Are their pre-competitive activities the community can engage in 

to assure the quality of cell counting measurements?
Identifying 

appropriate 

control 

materials

Dealing with 

sample artifacts 

(bubbles, debris 

etc.)

Gating 

Practices

Sources of 

variability

Influence of 

Count on 

Bioassays

Approaches 

for limiting 

user variability

What set of 

cell types for 

instrument 

validation

Method 

acceptance 

criteria

Method 

Transfer

Control 

Experiments 

prior to a run

Collaboration 

to draft 

consensus 

practices

Standard for 

live/dead 

assessment 

and counting

Instrumentinstrument 

variability

Counting 

irregular 

cells

Method 

Comparability

Improving 

variability in 

cell counts

Sample Stability 

during Counting

Good Study 

Designs

Accuracy/ 

True Counts

What are 

common 

practices 

across cell 

counting
Additional Information
Workshop Goals

• Raise awareness of the importance and challenges 

associated with cell counting measurements 

• Develop and document best practices for cell 

counting

• Discuss options to address measurement challenges 

through collaborative studies (NIIMBL)

• Workshop outcomes to support the development of 

international standards and more specific 

measurement challenges 
Workshop Participants: Resources

• Bartlow, P. Importance of a Cell Counting Method to Develop a Robust Cell Therapy Process. Podium presentation, Cell Therapy 

Bioprocessing and Commercialization. Annual Meeting in Alexandria, VA, October 2015.

• Cytometry B Clin Cytom. 2007 Sep;72(5):427-32. CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY,May 1997, p. 309–313

• Accurate measurement of peripheral blood mononuclear cell concentration using image cytometry to eliminate RBC-induced counting error

• Morphological observation and analysis using automated image cytometry for the comparison of trypan blue and fluorescence-based 

viability detection method

• A high-throughput AO/PI-based cell concentration and viability detection method using the Celigo image cytometry

• Biotechnology Report 7 (2015) 9-16. Tran, SL, et al. PLoS One September 2011 6(9): e22876 Warren Strober, Current Protocols in 

Immunology, 1997, A.3B.1

• Cell Gene Therapy Insights 2016;2(6),663-673.

• USP 1034 Analysis of Biological Assays

• A Comparison of Assay Performance Measures in Screening Assays: Signal Window, Z' Factor, and Assay Variability Ratio ARTICLE in

JOURNAL OF BIOMOLECULAR SCREENING · MAY 2006

• Global Procedure for Lab Investigations - Phase 2b Cryopreserved Drug Product (CDP) - Phase 2b Working Cell Bank (WCB). - GMP guidance 

for method: accuracy, intermediate precision, repeatability precision, linearity, range, and stability indicating properties.

• FDA guidance on method validations: https://www.fda.gov/downloads/drugs/guidances/ucm386366.pdf 

• Validation of three viable-cell counting methods: Manual, semi-automated, and automated: Cadena-Herrera, D. etal.

• ICH Validation of Analytical Procedures: Text and Methodology Q2(R1)

• ICH Guidance for Industry Q2B Validation of Analytical Procedures: Methodology

• Simon, C. G., Lin-Gibson, S., Elliott, J. T., Sarkar, S., & Plant, A. L. (2016). Strategies for Achieving Measurement Assurance for Cell Therapy 

Products. Stem Cells Translational Medicine, 5(6), 705-708.

• Lin-Gibson, S., Sarkar, S., & Ito, Y. (2016). Defining quality attributes to enable measurement assurance for cell therapy products. 

Cytotherapy, 18(10), 1241-1244.

• Lin-Gibson, S., Sarkar, S., Elliott, J. T., & Plant, A. L. (2016). Understanding and managing sources of variability in cell measurements. Cell 

Gene Therapy Insights 2016;2(6),663-673.

• ISO/WD 20391-1 [Under development] Biotechnology -- Cell Counting -- Part 1: General guidance on cell counting methods

• ISO/WD 20391-2 [Under development] Biotechnology -- Cell Counting -- Part 2: Experimental design and statistical analysis to quantify 

counting method performance
Establishing 

written 

consensus 

standards

Collaborative/ 

Inter-lab 

studies

Protocols/ Best 

Practices 

Documents

White Paper

Case studies 

that exemplify 

cell counting 

method 

evaluation

Workshop Participants

What Types of Follow-on Activities/ Projects 

would be valuable from this workshop?
Aggregation

Sample 

Stability

Concentration

Sample 

volume

Culture format 

(microcarriers, 

planar)

Scale

Viability/ 

senescence

Debris

Source Sample 

stability

Morphology

Execution 

time

Sample prep 

(lysis)

Non-target 

cells

Cell Settling

Cell Optical 

Properties

Reagents
Cost

Ease of use

Assay timing

Range of 

detection

Measurement 

confidence

Precision

High 

throughput

GMP 

approved

Accuracy

Instrumentinstrument 

variability

Supports 

broad cell 

types

OperatorOperator 

Variability

Transferable

Autosampling

Trust in 

method

Reagent/ dye 

selection

Comprability