T-cell IHC and image analysis

We had previously shown from MCP counter analysis [9] a small subset of TCHL patient samples that increased levels of T-cells were associated with response to TCHL-based therapy [10]. We had sufficient material from 13 patients who had matched pre and on-treatment biopsies to perform T-Cell IHC and image analysis. 3 µm serial tissue sections were cut using a Leica RM2135 microtome. IHC analysis was carried out on a Bond-III immunostainer (Leica Biosystems, Newcastle, UK). Primary antibodies for CD3 (Leica, NCL-L-CD3-565), CD4 (Leica, NCL-CD4-368) and CD8 (Leica, NCL-CD8-4B11) were diluted in Bond Primary Antibody Diluent (Leica, AR9352) at 1/40, 1/100 and 1/100, respectively. Pre-treatment of samples was carried out on the Bond-III using Bond Epitope Retrieval Solution I (Leica, AR9961) for 20 min (CD3, CD8) and using Bond Epitope Retrieval Solution II (Leica, AR9640) for 20 min (CD4). Detection and visualization of stained cells was achieved using the Bond Polymer Refine Detection Kit (Leica, DS9800) with Bond DAB Enhancer (Leica, AR9432). Tissues were counterstained with haematoxylin and cover slipped. The CD3, CD4 and CD8 stained slides for 13 cases (pre-treatment and on-treatment) were scanned at 40X using a Philips 2.0 scanner and the whole section analysed using the open access image analysis software QuPath [11]. The positive cell detection tool was used to measure the number of positive cells per square millimeter of tissue and compared against the assessment of a Histopathologist. Two comparisons were made using both QuPath and the Histopathologist: Firstly, for each antibody, the number of positive cells in the pre-treatment biopsy was compared to the post-treatment biopsy and secondly the number of CD4 + and CD8 + cells were compared between the pre-treatment biopsy and the post-treatment biopsy. Due to the large number of positive cells in most samples the pathologist score could not be given as a numerical value but was noted as a comparative statement between the samples being analyzed. The QuPath results for all samples were then compared to the pathologist score to ensure accuracy of the software, and QuPath results were then used for quantitative analysis.