Aperio scanscope at turbo

Eighteen (86%) of the 21 cases of EMPD were subjected to evaluation of CD3, CD8, and PD-1 in the immune infiltrate by automated image analysis. Three cases were excluded from automated image analysis because of metastatic disease in a lymph node (n = 1), a scant immune infiltrate (n = 1), or a small biopsy specimen (n = 1). The slides chosen were scanned at 20× magnification (Aperio ScanScope AT Turbo; Leica Biosystems). With the help of image analysis software (Aperio ImageScope), three 1 mm² squares were designated in the areas of highest density of immune cells positive for the IHC marker in question. The number of cells positive for the marker in each of the 3 squares was then counted using a modified nuclear algorithm, as described in our previous study [18 (link)]. For each tumor, the mean number of cells positive per square was then calculated to obtain a final number of positive cells per mm² for each case.

Slides were digitised using slidescanners (AperioScanScope AT Turbo, Leica Biosystems; 3DHistech) at 20× and 40× magnification and stored on a server (msdlt‐slide.dpag.ox.ac.uk). The regions of interest were outlined using the imagescope programme (Aperio, v11.2.0.780) and the longest diameter of every neuroserpin‐immunopositive (neuroserpin‐ip) cell body in the telencephalic wall was manually measured. Heatmaps were visualised based on the coordinates of neuroserpin‐ip cells using qgis 3.2.2 software (Ic6 line).

Kidney histopathology was conducted as previously described (22 (link)). Briefly, formalin-fixed, paraffin-embedded sections of 3 µm thickness were stained with hematoxylin and eosin or Periodic Acid-Schiff (PAS) for histopathological analysis. PAS-stained slides were imaged by Aperio Scanscope AT Turbo (Leica Biosystems). Mean glomerular cross-sectional area was quantified by polygon tracing using ImageJ software (NIH, 1.52d). Mean disease score criteria were assessed blinded as follows; 0 – Normal glomerular cellularity and morphology; 1 – Mild cellular expansion and/or early-stage disrupted glomerular morphology which includes lobularity and/or Bowman’s space enlargement; 2 – Advanced cellular expansion with distinct disruption to glomerular morphology, pyknosis and/or karyorrhexis; 3 – Severe cellular expansion/consolidation with advanced lobularity or fragmentation and/or enlargement of the Bowman’s space with cellular infiltration (with or without crescent formation) and/or peri-glomerular cellular expansion and/or evidence of segmental necrosis; 4 – End-stage glomerular destruction, progressive or complete loss of cellularity and/or distinct glomerular morphology. At least 30 glomeruli were assessed for each kidney.

Slides were scanned at 20× magnification (Aperio Scanscope AT Turbo; Leica Biosystems Wetzlar, Germany using Perio eSlide Manager software version 12.3.3.5049, Leica Biosystems Wetzlar, Germany). Image analysis software (Aperio ImageScope version 12.3.2.8013, Leica Biosystems Wetzlar, Germany) was used to calibrate and measure the degree of staining within designated areas in the images. An algorithm was set for each antibody in order to minimize the detection of background positive staining. These measurements were used to calculate a positivity score using the formula positivity = (number of positive pixels)/(total number of pixels).