Fig. 3

Comparison of quantitative and automatic IF-FISH scoring method with the standard HER2 assessment for tissue and cell line samples. a Correlation between HER2 overexpression (HER2/CK ratio obtained from the microfluidic staining protocol and automated IF image-processing) and HER2 amplification (HER2/CEP17 ratio obtained from standard FISH scoring). The threshold 0.25 for positivity of HER2/CK obtained by IF is defined as the lower bound of the 95% confidence interval obtained from a t-test on the mean HER2/CK scores of three HER2-positive cell line samples. The threshold 2 for positivity of HER2/CEP17 obtained by FISH is obtained from the ASCO/CAP 2013 guidelines. b Correlation between the HER2 loci number per cell obtained from our automated counting algorithm and the standard FISH technique. The threshold for positivity for the variable HER2 loci/cell is taken as 6 (dotted lines), obtained from the ASCO/CAP 2013 guidelines. c Correlation between the HER2/CEP17 ratio obtained from our automated counting algorithm and the standard FISH technique. In our automatic method, the HER2/CEP17 ratio is calculated as the mean of the HER2/CEP17 ratios in all CK-positive cells of a tissue, while in the standard method it is calculated as the ratio of mean HER2 and CEP17 signals in one or several clusters of 20 cells chosen by the pathologist. The threshold for positivity for the automatic HER2/CEP17 ratio (horizontal dotted line) is 2.4 (see text)