Using Bland–Altman Analysis to Identify Appropriate Clonogenic Assay Colony Counting Techniques

Objective: Determine the interchangeability of various methods utilized for counting colonies in clonogenic assays. Methods: Clonogenic assays of 2 head and neck cancer cell lines were counted through 4 different counting modalities: Manual counting pen, via microscope, 1 publicly available automated algorithm, and a semiautomated algorithm presented by the authors. Each method counted individual wells (N = 24). Pen and microscopic counts were performed by 2 observers. Parameters included both low-growth (<150 colonies/well) and high-growth (>150 colonies/well) cell lines. Correlational and Bland–Altman analyses were performed using SPSS software. Results: Interobserver manual pen count correlation R² value in both growth conditions was 0.902; controlling for only low-growth conditions decreased R² to 0.660. Correlation of microscopic versus pen counts R² values for observers 1 and 2 were 0.955 and 0.775, respectively. Comparing techniques, Bland–Altman revealed potential bias with respect to the magnitude of measurement (P <.001) for both observers. Correlation of microscopic counts for both interobserver (R²= 0.902) and intraobserver (R²= 0.916) were analyzed. Bland–Altman revealed no bias (P =.489). Automated versus microscopic counts revealed no bias between methodologies (P =.787) and a lower correlation coefficient (R²= 0.384). Semiautomated versus microscopic counts revealed no bias with respect to magnitude of measurement for either observer (P =.327,.229); Pearson correlation was 0.985 (R²= 0.970) and 0.965 (R²= 0.931) for observer 1 and 2. Semiautomated versus manual pen colony counts revealed a significant bias with respect to magnitude of measurement (P <.001). Conclusion: Counting with a manual pen demonstrated significant bias when compared to microscopic and semiautomated colony counts; 2 methods were deemed to be interchangeable. Thus, training algorithms based on manual counts may introduce this bias as well. Algorithms trained to select colonies based on size (pixels²) and shape (circularity) should be prioritized. Solely relying on Bland–Altman or correlational analyses when determining method interchangeability should be avoided; they rather should be used in conjunction.