AUTOMATION OF THE COLONY-FORMING CELL ASSAY FOR MEASURING HEMATOPOIETIC PROGENITORS IN CORD BLOOD

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1 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)

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

3 HEMATOPOIETIC CFC-ASSAY 1. Prepare Cells 2. Add cells to semisolid medium 3. Plate and Incubate 14 days in most media Total CFC, BFU-E, CFU-GM, CFU-GEMM 7 days in selected media Total CFC only 4. Count Colonies

4 VARIABILITY OF CFC-ASSAY RESULTS Intra-laboratory Coefficient of Variation: 10-20% (STEMCELLL: in-house data) Inter-laboratory Coefficient of Variation: ~30% (Spellman et al: Cytotherapy Mar 30) Manual Colony Counting is one Source of Variability in the CFC-Assay Goal: Standardize the CFC-assay on Cord Blood and reduce variability by automating colony identification and enumeration

5 AUTOMATION OF COLONY IDENTIFICATION AND SCORING 7-day CFC-assay: Total colonies 14-day CFC-assay: Total, Erythroid and Myeloid colonies

6 AUTOMATED COLONY DETECTION IN 7-DAY CFC-ASSAY

7 AUTOMATED COLONY DETECTION AND CLASSIFICATION IN 14-DAY CFC-ASSAY Erythroid Myeloid

8 MANUAL AND AUTOMATED COUNTS SHOW STRONG CORRELATION 7-DAY CFC-ASSAYS ON CB Regression analysis: y=1.02x R 2 = 0.92 N=294 Bias* = -4.0% Data contributing laboratories: StemCell Technologies n=102 3 NMDP-associated CB Banks n=192 Assays were performed in MethoCult Express * Bias = % average difference between manual and automated counts

9 MANUAL AND AUTOMATED COUNTS SHOW STRONG CORRELATION 14-DAY CFC-ASSAYS ON CB Total CFCs Regression analysis: y=0.98x R 2 = 0.83 N=167 Bias* = -9.0 % Data contributing laboratories: StemCell Technologies n= 62 4 NMDP associated CB Banks: n=105 Assays were performed in MethoCult H4034, H4434 or H4435. * Bias = % average difference between manual and automated counts

10 MANUAL AND AUTOMATED COUNTS SHOW STRONG CORRELATION 14-DAY CFC-ASSAYS ON CB Erythroid Colonies Myeloid Colonies

11 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 7-day CFC-assay : Overall CV= 11% N=36

12 AUTOMATED COLONY COUNTS ARE MORE REPRODUCIBLE THAN MANUAL COLONY COUNTS Coefficient 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 7-day CFC-assay : Overall CV= 11% : Overall CV= 5% N=36

13 AUTOMATED COLONY COUNTS ARE MORE REPRODUCIBLE THAN MANUAL COLONY COUNTS Variability of pooled replicate data Manual Automated Total CFC (7 day assay) Total CFC (14 day assay) 11% 5% 12% 5% BFU-E 11% 10% CFU-GM+CFU- GEMM 17% 9%

14 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)

15 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

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