AUTOMATION OF THE COLONY-FORMING

CELL ASSAY FOR MEASURING

HEMATOPOIETIC PROGENITORS

IN CORD BLOOD

Bert Wognum

Oliver Egeler

Caren Grande

Ning Yuan

Steven Woodside

Terry Thomas

Allen Eaves

STEMCELL Technologies Inc. (Vancouver, Canada)

HEMATOPOIETIC COLONY FORMING

CELL/UNIT (CFC or CFU) ASSAY

Applications in Hematopoietic Stem and Progenitor Cell Transplantation:

Assessing Frequency and Quality of hematopoietic Progenitors in Cell

Products

Quality Control of Cell Therapy Laboratory Procedures

Potential Use for Graft Selection

CFC content of grafts correlates with :

• Time to neutrophil engraftment

• Time to platelet engraftment

• Overall survival

Prasad et al., Blood 112:2979-89. 2008;

Page et al. J., Biology of Blood and Marrow Transplantation (2011), in press.

HEMATOPOIETIC CFC-ASSAY

1. Prepare Cells

3. Plate and Incubate

4. Count Colonies

2. Add cells to semisolid medium

14 days in most media

Total CFC, BFU-E, CFU-GM, CFU-GEMM

7 days in selected media Total CFC only

• Intra-laboratory Coefficient of Variation: 10-20% (STEMCELLL: in-house data)

• Inter-laboratory Coefficient of Variation: ~30% (Spellman et al: Cytotherapy. 2011 Mar 30)

Goal:

Standardize the CFC-assay on Cord Blood and reduce

variability by automating colony identification and

enumeration

VARIABILITY OF CFC-ASSAY RESULTS

Manual Colony Counting is one Source of

Variability in the CFC-Assay

AUTOMATION OF COLONY IDENTIFICATION

AND SCORING

7-day CFC-assay:

Total colonies

14-day CFC-assay:

Total, Erythroid and Myeloid colonies

AUTOMATED COLONY DETECTION

IN 7-DAY CFC-ASSAY

AUTOMATED COLONY DETECTION AND

CLASSIFICATION IN 14-DAY CFC-ASSAY

Erythroid

Myeloid

* Bias = % average difference between manual and automated counts

Data contributing laboratories:

• StemCell Technologies n=102

• 3 NMDP-associated CB Banks n=192Assays were performed in MethoCult Express

Regression analysis:

y=1.02x + 0.89

R2 = 0.92

N=294

Bias* = -4.0%

MANUAL AND AUTOMATED COUNTS SHOW

STRONG CORRELATION7-DAY CFC-ASSAYS ON CB

* Bias = % average difference between manual and automated counts

Data contributing laboratories:

• StemCell Technologies n= 62

• 4 NMDP associated CB Banks: n=105Assays were performed in MethoCult H4034, H4434 or

H4435.

Regression analysis:

y=0.98x + 5.8

R2 = 0.83

N=167

Bias* = -9.0 %

MANUAL AND AUTOMATED COUNTS SHOW

STRONG CORRELATION14-DAY CFC-ASSAYS ON CB

Total CFCs

MANUAL AND AUTOMATED COUNTS SHOW

STRONG CORRELATION14-DAY CFC-ASSAYS ON CB

Erythroid Colonies Myeloid Colonies

REPRODUCIBILITY OF COLONY COUNTING:

which method has lower variability?

Coefficient of Variation (CV) of:

Manual (microscopic) counts by 3-5 people counting the same cultures

N=36

: Overall CV= 11%

7-day CFC-assay

AUTOMATED COLONY COUNTS ARE MORE

REPRODUCIBLE THAN MANUAL COLONY

COUNTSCoefficient of Variation (CV) of:

Manual (microscopic) counts by 3-5 people counting the same cultures

Replicate automated counts of the same cultures imaged repeatedly on 3-5

instruments

N=36

: Overall CV= 11%

: Overall CV= 5%

7-day CFC-assay

AUTOMATED COLONY COUNTS ARE MORE

REPRODUCIBLE THAN MANUAL COLONY

COUNTS

Variability of pooled replicate data

Manual Automated

Total CFC(7 day assay)

11% 5%

Total CFC (14 day assay)

12% 5%

BFU-E 11% 10%

CFU-GM+CFU-

GEMM17% 9%

CONCLUSIONS AND FUTURE DIRECTIONS

Automated Colony Identification and Counting:

1. Shows good correlation with manual colony counting in 7 day and 14

day CFC-assays on Cord Blood

2. Reduces variability relative to manual colony counting

3. Provides a permanent image record of CFC-assay results

4. Can be modified for specific applications, e.g.,

• CFU-GM enumeration in media without Erythropoietin

• CFC-assays on other cell sources (BM, MPB, patient samples)

ACKNOWLEDGEMENTS

STEMCELL Technologies:

• Research and Development Dept.

• Instrumentation Group

• Quality Control Dept.

• Education & Contract Assay Services

Collaborators:• Puget Sound Blood Center, Seattle, Washington, USA

• MD Anderson Cancer Center, Houston, Texas, USA

• Duke University Medical Center, Durham, North

Carolina, USA

• St. Louis Cord Blood Bank, St. Louis, Missouri , USA

• Anthony Nolan Trust, Nottingham, UK

Vancouver, British Columbia, Canada