NIST-FDA Cell Counting Workshop:

Challenges in Cell Counting

Sumona Sarkar

National Institute of Standards and Technology

sumona.sarkar@nist.gov

April 10, 2017
Cell Count

Cell count is a fundamental measurement in biotechnology, 

biomanufacturing, and regenerative medicine.

Cell number can effect:

• Potency and efficacy of a cell therapy treatment (e.g. dosing)

• Manufacturing process for cells and biopharmaceutical products

• Rate of growth of regenerated tissue in a biomaterial scaffold

• Bioassays that are normalized by activity per cell

• Functional assays based on cell count (e.g. proliferation)

Research Product 

Development

Process 

Development

Clinical 

Manufacture

Quality 

Assurance

Clinical 

Diagnostics
Workshop Participants

How often do you use cell counting for the following 

purposes?

% Respondents

Release 

Assay

Manufacturing

Control

Monitoring 

Control

Routine Cell 

Culture

Academic/ 

Basic 

Research

Part of a Other

Bioassay
Wide range of cell counting needs and methods

Budding Cells (Yeast 

Cells)

Irregularly Shaped 

Cells (Primary 

Hepatocytes)

Clumpy Cells (MCF7 breast cancer cell 

line)

Cell Suspensions 

with Debris 

(PBMCs)

Cells of varying size 

(MSCs)

Range of Counting 

Methods

Range of Sample 

Properties

Count estimates for 

passaging and culture

Monitoring cell 

response to stimuli

In-process count 

estimate for feed rate

Count and Viability as an 

indicator of potency

Accurate counts for 

cell therapy dosing

Range of Intended 

Purposes

Images: nexcelom.com and NIST

What strategies can we 

implement to assure confidence 

in cell counts across these broad 

needs?
How often do you use the following cell count 

methods?

Workshop Participants

Brightfield

Imaging

Fluorescent 

Imaging

Impedance Flow 

Cytometry

Metabolic 

Activity

DNA 

Quantity

Confluency Other

% Respondents
Workshop Participants

What do you consider to be the gold standard 

(or reference method) for cell counting?
Measurement Process for Cell Counting

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.
Many components of the cell sample can influence an analytical method

What are the samples properties to considered when conducting a cell counting method?

Cell Sample Composition Can Affect Cell Counting

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

Cytotherapy, 18(10), 1241-1244.
Appropriate Data for 

Statistical Analysis

Outlier Determination

Variability in 

a Cell 

Counting 

Assay 

Consumables

Pipette Tips

Pipettes/Pipettors

Refrigerators/ 

Freezers Stability

Water Baths 

(Fill Volume/Temp)

Equipment/ 

Consumables

Reagents/Raw 

Materials

Electrical Issues

Disruptions/Distractions

Cleanliness

Temperature

Humidity

Environment

Protocol Consistency Control/Reference 

Material

Sample Collection

Thaw Procedures

Sample Preparation

Dilution Procedure

Time

Analyst Training

Procedures Data Acquisition Data Analysis

Budding

Formulation

Morphology

Concentration

Subsampling

Homogeneity

Stability 

Clumping

Sample 

Preparation

Biological Sample Instrument

Cause

Effect

Reference 

Material Stability

pH

Lot-to-Lot Consistency

Osmolality

GMP Grade

Age 

(Expiration)

Light 

Sensitivity

Stability

Temperature 

Sensitivity

Toxicity

Purity/

Impurity Data Recording/

Recovery

Settings/Set-up

Instrument Variability 

(Trending)

Linearity/LOD

Cell Specific

Algorithm Instability

Clumping

Imaging Artifacts

Debris

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.

Sources of Variability in a Cell Counting 

Measurement Process
Evaluating Cell Counting Methods

Cell Concentration 

(Cells/mL)

• Comparability 

between methods

• Accuracy

• Precision

• Linearity 

(proportionality)

• Reproducibility

Experimental Designs to Evaluate the Quality of a Cell Counting Method

Proportionality

Proportionality Index (PI)

Dilution Fraction

Pooled CV

Comparability Precision

Dilution Fraction

Average Cell Concentration (cells/mL)

ISO/WD 20391-2 [Under development: 20.60]

Biotechnology -- Cell Counting -- Part 2: Experimental design and statistical analysis to quantify counting method performance
Workshop Participants

List guides, documentary standards, best practices etc that 

you used when establishing your cell counting measurement

In te rn a l S O P

IC H

U S P 1 0 3 4 L ite ra tu re G lo b a l P ro c e d u re fo r L a b In v e s tig a tio n s G M P g u id a n c e fo r m e th o d

IQ /O Q

F D A G u id a n c e M a n u fa c tu re r P ro to c o ls C o m p a ra b ility re p o rt

0

2

4

6

# R e s p o n d e n ts
Workshop Participants

How much time was spent developing a 

validated cell counting measurement?

% Respondents
Designing a Cell Counting Measurement Process 

that is Fit-for-purpose

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.
Measurement Assurance Strategies to 

Keep in Mind
Workshop Participants

List measurement assurance strategies for 

cell counting that you currently apply

D a ily In s tru m e n t V e rific a tio nS O P O rth o g o n a l M e th o d s R e p lic a te s a m p le s (P re c is io n ) R e fe re n c e R e a g e n ts R e fe re n c e C e l m a te ria l R e fe re n e B e a d s R e p ro d u c ib ility D ilu tio n in te g rity

IQ /O Q

Im a g e /D a ta R e v ie w

L in e a rity /R a n g e /A c c u ra c y e tc

0

2

4

6

8

1 0

# R e s p o n d e n ts
Quality Attribute

Characteristic of the 

sample

(e.g. Viable cell count)

Measurand

What is being measured

(e.g. count of un-stained 

objects per volume)

Concepts to Keep in Mind

Biological Response

Measurement Variability
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?