Part-1122CP09.15-A

TRANSFERRING CELL COUNTING METHODS –

BEST PRACTICES

04/10/2017 NIST

Lena Lee

Global Product Manager – Vi-CELL
Outline

• Introduction

• Case studies

• Recommendations

• Beckman Coulter Life Sciences
Vi-CELL XR

Fully automated, 

computer-operated image 

analyzer that uses the 

Trypan Blue Dye Exclusion 

Method for Viability, Cell

Counting and Total Cell 

Concentration.
Who are our Vi-CELL XR customers?

 Tissue Culture Facilities: user may be growing many types of cells 

and needs accurate knowledge of count and viability.

 Biopharma Lab: user performs cell based assays and needs 

accurate count and viability to quantify results from assays.

 Biopharma Production Facilities: utilizing yeast, insect cells and 

animal cells to produce biological therapies. User needs to monitor 

cell health for maximum production and harvest time.

 Clinical Research Labs: isolating cells from human or non-human 

samples.

▪ Blood

▪ Bone Marrow (Stem Cells)

▪ Spleen

▪ Lymph Node
Trypan Blue Dye Exclusion Method

• Trypan blue is a vital stain used to selectively color dead 

tissues or cells blue.

• The trypan blue dye exclusion test is used to determine the 

number of viable cells present in a cell suspension. It is based 

on the principle that live cells possess intact cell membranes 

that exclude certain dyes, such as trypan blue, whereas dead 

cells do not. A viable cell will be clear in the center whereas a 

nonviable cell will have a blue center.
Traditional Method

• Cell viability (Trypan Blue Dye Exclusion Method) 

determinations traditionally have been performed using a light 

microscope and hemacytometer. 

• Unfortunately, this technique has numerous major 

shortcomings. 

– The hemacytometer has a significant repeatability error. 

– Different technicians analyzing the same cell sample obtain 

variations in results. 

– Manual method is tedious and quite time consuming for today’s 

busy laboratory environment.
Applications in Research and Manufacturing

• Vi-CELL XR analyzes majority of mammalian cell types, insect 

cells and yeast.

– Cells in the range of 2-70 microns.
Features

 Automation of the Trypan Blue Assay Method

 % Viability

 Total Cell Concentration

 Total Viable Cell Concentration

 Mean Cell Size

 Real Time Cellular Images

 Calculates Bio-process Growth Rate and Doubling Time

 Convenient Reagent Packs

 Validated Reagents

 Reanalyze data
Easy to Use
Automatically Classifies Live Cells (Green Circles)

Automatically 

Classified

Dead cells

(Red Circles)

Instrument

Status and

Control

Results

Size 

Distribut

Plot

Vi-CELL Software

Main Window
Bioprocess Feature

• On the Vi-CELL XR, the user can monitor a bioprocess over 

time.

– Excellent for characterizing growth rate and doubling time

• Individual runs are automatically appended together.
Reagent Pack

 Contains all reagents required to run samples and clean system.

 System monitors reagent consumption.

Green: Buffer Solution

Red: Disinfectant

Yellow: Cleaning Agent

Blue: Trypan Blue Reagent

Reagent 

Pack
Concentration Control

• Beckman Coulter Vi-CELL concentration control standards are 

beads used to confirm the overall system performance.

– Control is recommended to be run daily

• Note: Viability standards are available through Bangs 

Laboratory
Reasons to Change

• Processes rarely remain the same over time. 

• Many factors drive change

– Obsolescence of equipment or materials

– Increased throughput 

– Need to decrease variation

– Process Improvement
Change sometimes requires Studies and Validation
Another Cell 

Counter

Manual Process

Changing the Cell Counting Process
Typical Criteria for new Method

• Correlate to previous method

– More important to have the same answer than an accurate 

answer

• Reliability 

• Repeatability
Case Study

• Dr. Iveta Bottova is a Process Development 

Specialist at SOTIO, a biotechnology company 

developing a next generation Active Cellular 

Immunotherapy drug. 

• Share SOTIO’s Validation study to change from the 

manual counting process of dendritic cells to an 

automated counting process using the Vi-CELL XR.
Dendritic Cells (DC)

• Dendritic cells are antigen-presenting cells of the 

mammalian immune system. Their main function is 

to process antigen material and present it on the 

cell surface to the T cells of the immune system. 

• SOTIO develops new medical therapies using an 

immunotherapy platform based on activated 

dendritic cells

• The correct cell count and adequate viability of DC 

are one of the quality control criteria for the final 

product release.
Validation Study

• Evaluated Accuracy and Precision against manual 

method using beads.

– Two Operators

– Concentration Control (latex beads) 1x10^6 beads/mL

– Triplicate measurements 

– Side by Side

Pretty good 

repeatability
Validation Study

• Assessed the cell diameter for optimal DC measurement

– SOTIO product contains DC and lymphocytes. 

– Evaluate whether Vi-CELL can use size to identify DC and ignore 

lymphocytes

• DC size 11 – 30 mm and lymphocytes 5 – 12.5 mm, partial overlapping but 

only ~4%

– 19 Lots used in study

Note: Size determined on 

20X microscope and Vi-CELL

Cell Type setting 

provides size limits
Size plot of 40 analyses with different size cut-off
Comparison against manual method with DC

• Compared Vi-CELL analysis of 11.5 or 12 mm as the lower 

size limit against manual counts.

The differences were smaller 

and more centered around 

zero in the case of 11.5 mm 

as the lower limit.
Validation Conclusion

• The Vi-CELL method was found to be accurate and 

suitable for DC counting and comparable to the 

currently used quality control method Bürker

chamber (manual method). 

• There was no significant difference between DC 

counts values obtained by Vi-CELL and by Bürker

chamber, moreover the size range 11.5-30μm is 

important for DC recognition in the Vi-CELL.
OTHER REAL EXAMPLES
Dilution Study

• Dilution Study to move from Manual to Vi-CELL

– Customer was concerned about high variance from 

expected result.

Volume of 

concentration 

control stock 

(uL)

Volume of 

diluent (uL)

Reported Total 

cells/ml ( x 

10^6)

Diff from 

expected 

conc

Reported Total 

cells/ml ( x 

10^6)

Diff from 

expected 

conc

Dilution factor

Set 1 Set 2

20 980 1.7135 69.8% 1.4772 42.4% 50

50 950 1.5363 52.2% 1.489 43.5% 20

100 900 1.1935 18.3% 1.4063 35.5% 10

500 500 1.1038 9.4% 1.0612 2.3% 2

500 0 1.0092 1.0376 1
Dilution Study

• Dilution Study Problem

– Concentration of starting material is too low for the dilution 

study or dilution factors are not appropriate for the sample 

being used.

Volume of 

concentration 

control stock 

(uL)

Volume of 

diluent (uL)

Reported Total 

cells/ml ( x 

10^6)

Diff from 

expected 

conc

Reported Total 

cells/ml ( x 

10^6)

Diff from 

expected 

conc

Dilution 

factor

Estimated total 

bead count Count per image

Set 1 Set 2

20 980 1.7135 69.8% 1.4772 42.4% 50 20 0.4

50 950 1.5363 52.2% 1.489 43.5% 20 50 1

100 900 1.1935 18.3% 1.4063 35.5% 10 100 2

500 500 1.1038 9.4% 1.0612 2.3% 2 500 10

500 0 1.0092 1.0376 1 1000 20
Bridge Study

• Count Comparison to move from Manual to Vi-CELL

– Customer was concerned that Vi-CELL VCD had high 

variance with their manual method.

Viability (%)

Viable cells 

/ ml (x10^6)

Viability 

(%)

Viable cells 

/ ml (x10^6)

Difference 

in Viab

Difference in 

VCD

98.3 3.98 98 3.16 0.3% 21%

97.7 4.20 96 3.01 1.7% 28%

71.9 2.40 73.5 1.78 -2.2% 26%

56.6 1.75 53 1.10 6.4% 37%

41.2 1.21 37.5 0.70 8.9% 42%

Vi-Cell (N Std) Manual

Summary

Vi-Cell vs manual
Sample

Vi-CELL: 

Viability (%)

Total cells / 

ml (x10^6)

Viable cells / 

ml (x10^6)

1 58.1 3.85 2.24

2 60.1 3.99 2.40

3 60.6 3.98 2.41

4 61.6 4.12 2.54

5 62.3 3.63 2.26

6 63.4 3.75 2.38

7 62.9 4.04 2.54

8 63.1 3.61 2.27

9 62.4 3.70 2.31

Average 62 3.85 2.37

SD Std 1.7 0.19 0.11

RSD 2.8% 4.9% 4.7%

Min 58 3.6 2.2

Max 63 4.1 2.5

Bridge Study

• Count Comparison

Operator

Viability

Manual

VCD

Manual

A 62 1.12

B 70 1.31

C 62 1.42

D 64 1.51

E 55 0.97

F 51 1.43

G 53 1.2

H 53 0.97

I 60 1.59

Average 59 1.28

SD 6.3 0.228

RSD 10.7% 17.8%

Min 51 0.97

Max 70 1.59

Vi-CELL is 

more 

Repeatable
Which process is more repeatable?

Vi-CELL %CV = 3%

Manual %CV = 11% 

How can you correlate to a 

moving target?
Beckman Coulter Company Confidential

IMAGE FROM ANOTHER CELL COUNTER

Magnification 4x

Differences 

in resolution
VI-CELL IMAGE (1280 X 960 PIXELS)

Magnification 6.75x

Differences 

in resolution
Recommendations for Correlation Study

• Optimize instrument settings as close to previous method (size, 

brightness, cluster, ignore debris)

92.4% Viability

VCD = 6.56x10^6

90% Viability

VCD = 6.71x10^6
APPLYING DECLUSTERING

Same CHO Cell clump BUT due to the Medium 

Declustering, each cell within the clump is

differentiated thus increasing cell count
CHANGING VIABLE CELL SPOT AREA

For cells on the border of live/dead, adjust viable 

spot area to increase or decrease % viability
Recommendations for Correlation/Bridge Study

• Use concentration and viability standards (latex beads) to start 

study

• Practice good sample handing(mixing, pipetting, time, temp)

• Test side by side (same sample, same time)

• Practice good statistics – Ensure a representative sample

• Account for variables in the process – realistic tolerance ranges

– Some processes inherently have high variability 

• Account for differences in Technology (Resolution, parameters)
Thank you

Questions?

Contact me:

LenaLee@Beckman.com