Abstract
A clonogenic assay is a biological technique for calculating the Surviving Fraction (SF) that quantifies the anti-proliferative effect of treatments on cell cultures: this evaluation is often performed via manual counting of cell colony-forming units. Unfortunately, this procedure is error-prone and strongly affected by operator dependence. Besides, conventional assessment does not deal with the colony size, which is generally correlated with the delivered radiation dose or administered cytotoxic agent. Relying upon the direct proportional relationship between the Area Covered by Colony (ACC) and the colony count and size, along with the growth rate, we propose MF2C3, a novel computational method leveraging spatial Fuzzy C-Means clustering on multiple local features (i.e., entropy and standard deviation extracted from the input color images acquired by a general-purpose flat-bed scanner) for ACC-based SF quantification, by considering only the covering percentage. To evaluate the accuracy of the proposed fully automatic approach, we compared the SFs obtained by MF2C3 against the conventional counting procedure on four different cell lines. The achieved results revealed a high correlation with the ground-truth measurements based on colony counting, by outperforming our previously validated method using local thresholding on L*u*v* color well images. In conclusion, the proposed multi-feature approach, which inherently leverages the concept of symmetry in the pixel local distributions, might be reliably used in biological studies.
Highlights
IntroductionClonogenic assays represent a well-established technique commonly used in biological laboratories for estimating the ability of a single cell, which underwent a specific treatment (e.g., irradiation, chemotherapy or monoclonal antibody administration), to self-duplicate and create a colony
Clonogenic assays represent a well-established technique commonly used in biological laboratories for estimating the ability of a single cell, which underwent a specific treatment, to self-duplicate and create a colony
Clonogenic assay evaluation represents a technique widely used in biological research, aimed at quantifying the effect of a specific treatment with antitumoral/
Summary
Clonogenic assays represent a well-established technique commonly used in biological laboratories for estimating the ability of a single cell, which underwent a specific treatment (e.g., irradiation, chemotherapy or monoclonal antibody administration), to self-duplicate and create a colony. The current manual clonogenic assay procedures, still commonly used, suffer from quantification errors related to subjectivity of CFU count, as well as to the difficulties to detect colonies. Some solutions require specific hardware, sometimes ad-hoc designed and implemented— using 3D printing technologies—mostly to acquire the cell colony images [18,19,20,21]. These devices are intended to ensure correct positioning (because Regions of Interest (ROIs) detection approaches used fixed positioning masks) and uniform lighting. Barber et al [18] developed an automated colony counter, tested on four cell types, relying on a specific hardware apparatus coupled with a Charge-Coupled Device (CCD) camera enabling high-quality image acquisition of the culture flasks
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