Lessons Learned from Measuring Cell Response by Quantitative Automated Microscopy FDA Workshop, Potency Measurements for Cellular and Gene Therapy Products, Feb 2006 John T. Elliott , Alex Tona, Kurt Langenbach and Anne Plant NIST, Biochemical Science Division, Gaithersburg, MD 20899
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Lessons Learned from Measuring Cell Response by Quantitative Automated Microscopy FDA Workshop, Potency Measurements for Cellular and Gene Therapy Products,
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Lessons Learned from Measuring Cell Response by Quantitative Automated Microscopy
Lessons Learned from Measuring Cell Response by Quantitative Automated Microscopy
FDA Workshop, Potency Measurements for Cellular and Gene Therapy Products, Feb 2006FDA Workshop, Potency Measurements for Cellular and Gene Therapy Products, Feb 2006
John T. Elliott, Alex Tona, Kurt Langenbach and Anne PlantNIST, Biochemical Science Division, Gaithersburg, MD 20899
John T. Elliott, Alex Tona, Kurt Langenbach and Anne PlantNIST, Biochemical Science Division, Gaithersburg, MD 20899
• Founded in 1901, NIST is a non-regulatory federal agency within the U.S. Department of Commerce
• NIST's mission: To develop and promote measurement, standards, and technology to enhance productivity, facilitate trade, and improve the quality of life.
Z-factor as a Metric for Assay QualityZ-factor as a Metric for Assay Quality
•Average means and standard deviations are obtained from positive and negative control replicates.•Z-factors can be used to establish an assay robustness specification.
C e l l r e s p o n s e
+ C t r l- C t r l
D y n a m i c r a n g e o f a s s a y
9 5 % C o n fi d e n c e I n t e r v a l s ( ± 3
M e a n - M e a n -
(3-+ 3+)
|m-- m+|1-Z=
Reference: Zhang, et al. (1999) J. Biomol. Screen. 4, 67.
KS Test and the D-StatisticKS Test and the D-Statistic
•The KS test is a non-parametric test for statistically comparing The KS test is a non-parametric test for statistically comparing distributions of data.distributions of data.•The The D-statisticD-statistic is the maximum absolute vertical distance is the maximum absolute vertical distance between two cumulative distributions.between two cumulative distributions.•It is sensitive to changes in distribution position and shape.It is sensitive to changes in distribution position and shape.•It varies from 0 to 1.It varies from 0 to 1.
SummarySummary• Cells exhibit a distribution of responses
• A valid measurement of the distribution of cellular responses requires sampling an adequate number of cells.
• Internal positive and negative controls during assay measurement can be used to evaluate assay quality and robustness.
• Alternative methods to measure differences in cell response can take advantage distribution shape information.
• Statistical analysis requires measurements with uncertainty values. It is most useful for determining the significance of small measurement differences.
Advantages: -Unbiased data collection -Sample large number of cells -Multi-fluorophore imaging -Live cell imaging -Evaluate cells in real culture conditions
Advantages: -Unbiased data collection -Sample large number of cells -Multi-fluorophore imaging -Live cell imaging -Evaluate cells in real culture conditions