Phospho-cPLA₂ expression in IE-CTLs of active celiac patients. (A) Representative examples of double staining immunohistochemistry of CD3 (brown)/phospho-cPLA₂ (blue) in duodenal biopsies from patients with active celiac disease and controls (magnification ×200). Intraepithelial lymphocytes were randomly selected from the pool of CD3⁺ cells and automatically numbered as shown. Expression of phospho-cPLA₂ was measured within each outlined CD3⁺ IE-CTLs. In the left images, before overlay, phospho-cPLA₂—positive appears in blue. After overlay, in the right images, specific phospho-cPLA₂ staining as determined by threshold values is highlighted in red. (insets) IE-CTL #23 and #24 before and after applying the overlay tool. Note that double-positive IE-CTL #23 falls within the threshold values for phospho-cPLA₂—positive staining and thus is covered with solid red in overlaid image. In contrast, IE-CTL #24 falls below the threshold values for phospho-cPLA₂—positive staining and thus remains covered with white in overlaid image. Finally, increased phospho-cPLA₂—positive epithelial cells (colored in red after overlay) are present in active celiac patients compared with controls. Bars: (a) 40 µm; (b) 10 µm. (B) A summary with mean percentage of phospho-cPLA₂⁺ IE-CTLs ± SD is presented after manual and automated count. For manual count, four control and six celiac patients were analyzed. For automated count, >100 IE-CTLs in two control and two celiac slides were analyzed. P value was determined with the exact Wilcoxon rank sum test. Note that manual and automated count give similar percentage of phospho-cPLA₂⁺IE-CTLs.