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NIST-FDA Cell Counting Workshop: 

Sharing practices in cell counting measurements 

April 10, 2017

Mike O’Grady

ThermoFisher Scientific
2 Proprietary & Confidential

• Manual 

• Variable 

• Personal bias affects 

count

Cell Counting| Counter Considerations

• Automated 

• 104-107 Concentration 

and 3-25 μM sizem

range accuracy

• Brightfield count only

• Automated 

• 104-107 Concentration and 

3-25 μM size range 

accuracy

• Brightfield and 

Fluorescence capabilities

Intended purpose affects choice
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• Test and retest to users gold standard

• Automated faster over larger area

• Options matter for users- “Trust but 

verify” cell count

Cell Counting| Measurement Considerations

• Reusable Hemacytometer

• Dedicated machine slides

Users need validation to their standard
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Cell Counting| Cell prep and variability 

• Higher variability with manual counting

• Sample prep continues to be an issue

• Size variability within sample

• Autofocus and counting algorithms 

improve accuracy and repeatability

PBMCs

NIH 3T3

Ongoing need as users push what they need counted
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Cell Counting| User Needs and Variability 

Focusing - Get what you “auto focus” for Light intensity- Difficult to predict what users will do

• Users need flexibility-But 

introduces variability

• Gating on size, circularity, 

brightness, and fluorescent 

intensity

• Training and examples

• Applications and White Papers

• Field Support

• Ability to customize and save 

protocols

Gating in or out of cell populations

Flexibility allows for options and better cell counts

Flexibility and merging of counting algorithms across platforms
6 Proprietary & Confidential

• Difficult for users because of 

sample variability and 

differences between preps

• Precious sample with limited 

quantity

• Provide “Flow-Like” gating 

capability for inclusion/exclusion

• Cross validate between 

instruments

Cell Counting| PBMC User Issues

Automated Counter Flow Cytometer

User wants/needs evolve- Agility in pre & post development essential
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• Counting for original Seeding of iPSCs critical 

to Cardiomyocyte differentiation

• 40K in 12 well plate gives optimal results

Cell Counting| Stem Cell User- iPSC to Cardiomycte

Flow Data

Imaging of TnT

User verifies across counting and assay platforms for end result
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• Leverage established assays in automated counting

Cell Counting| Cell Health 

• Cell health important for downstream uses

• Need accurate cell #s and accurate picture of cell 

health

• Traditional imaging and flow cell health reagents 

can be used

Live/Dead - Metabolism/Membrane Integrity

Fixable Live/Dead - Membrane Integrity

Flow Cytometry
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Cell Counting| Cell Health is a Continuum 

Where are the cells being counted on continuum?
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• Cells apoptotic?

• Cell Therapy – Need to know 

• Untreated cells show some apoptosis

• Normal for functional cells, but how much?

Cell Counting| Apoptosis

What amount of cells being counted are apoptotic?
11 Proprietary & Confidential

Cell Counting| Oxidative Stress & Mitochondrial Function

• Reagents for Pre Lethal readouts

• Combine in counting with gating 

for desired assay?

• Control population with User 

defined protocol

What does cell therapy user need to test?
12 Proprietary & Confidential

• Sample prep and user variability

• Cell type and form (Spheroids/Organoids) constantly changing

• Best way to count for desired outcome difficult to predict

• Counting platforms becoming more of hybrid 

• Imaging

• Flow

• Classic counter

• When using limited sample how representative of entire population?

• User training and developer agility necessary

• Needs/Wants make it hard to establish standard across uses

• Adaptability of instrument and user saved protocols help

• Continuum of Cell Health has to be considered with counting needs

• Cell Therapy will need more than cell # and whether Live/Dead

Cell Counting| Major challenges and recommendations