Quantifying tumour-infiltrating lymphocyte subsets : a practical immuno-histochemical method

Background: Efficient histological quantification of tumour-infiltrating T and B lymphocyte (TIL) subsets in archival tissues would greatly facilitate investigations of the role of TIL in human cancer biology. We sought to develop such a method. Methods: Ten ×40 digital images of 4 μ sections of 16 ductal invasive breast carcinomas immunostained for CD3, CD4, CD8, and CD20 were acquired (a total of 640 images). The number of pixels in each image matching a partition of Lab colour space corresponding to immunostained cells were counted using the ‘Color range’ and ‘Histogram’ tools in Adobe Photoshop 7. These pixel counts were converted to cell counts per mm2 using a calibration factor derived from one, two, three or all 10 images of each case/antibody combination. Results: Variations in the number of labelled pixels per immunostained cell made individual calibration for each case/antibody combination necessary. Calibration based on two fields containing the most labelled pixels gave a cell count minimally higher (+ 5.3%) than the count based on 10-field calibration, with 95% confidence limits − 14.7 to + 25.3%. As TIL density could vary up to 100-fold between cases, this accuracy and precision are acceptable. Conclusion: The methodology described offers sufficient accuracy, precision and efficiency to quantify the density of TIL sub-populations in breast cancer using commonly available software, and could be adapted to batch processing of image files

Quantitative imaging of tissue sections using infrared scanning technology

Quantification of immunohistochemically (IHC) labelled tissue sections typically yields semi‐quantitative results. Visualising infrared (IR) ‘tags’, with an appropriate scanner, provides an alternative system where the linear nature of the IR fluorophore emittance enables realistic quantitative fluorescence IHC (QFIHC). Importantly, this new technology enables entire tissue sections to be scanned, allowing accurate area and protein abundance measurements to be calculated from rapidly acquired images. Here, some of the potential benefits of using IR‐based tissue imaging are examined, and the following are demonstrated. Firstly, image capture and analysis using IR‐based scanning technology yields comparable area‐based quantification to those obtained from a modern high‐resolution digital slide scanner. Secondly, IR‐based dual target visualisation and expression‐based quantification is rapid and simple. Thirdly, IR‐based relative protein abundance QIHC measurements are an accurate reflection of tissue sample protein abundance, as demonstrated by comparison with quantitative fluorescent Western blotting data. In summary, it is proposed that IR‐based QFIHC provides an alternative method of rapid whole‐tissue section low‐resolution imaging for the production of reliable and accurate quantitative data

Role of the galanin-1 receptor gene (GAL1R) and its regulation in infectious diarrhea.

Role of the galanin-1 receptor gene (GAL1R) and its regulation in infectious diarrhea