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Fig. 1 | Diagnostic Pathology

Fig. 1

From: PD1hi cells associate with clusters of proliferating B-cells in marginal zone lymphoma

Fig. 1

Workflow for immunohistochemistry and image analysis. a Diagram of the immunohistochemical workflow. b Quantification of the efficiency of elution for three antibodies: anti-PD1, anti-FOXP3 and anti-CD4. Black bars represent antibody staining intensity (mean ± SEM) and gray bars represent staining intensity following elution. Each experiment was carried out three times. The dotted line is the background intensity produced by an unstained tissue section. c Flow diagram to show Visiopharm methodology (www.visiopharm.com) used to analyse tissue sections stained sequentially with anti-CD4, anti-PD1 and anti-FOXP3 antibodies. The algorithm first identified cell nuclei by haematoxylin counterstain following which CD4 expressing cells were marked green. Next CD4 expressing cells were interrogated for co-expression of FOXP3. There was then a branch point in the algorithm. If FOXP3 expression was detected but there was no co-expression of PD1 at a high level (PD1hi), as previously defined by comparison with tonsillar Tfh cells, then the cell was determined to be Treg and the nucleus was labelled yellow. However, if the cell co-expressed CD4, FOXP3 and was PD1hi then it was determined to be a Tfr cell and the nucleus was labelled turquoise. Cells that were CD4 expressing and PD1hi, but not FOXP3 expressing were determined to be Tfh cells and the nuclei were labelled orange. Cells of each subset were then counted digitally. d Identification of specific T-cell subsets by multiplex immunohistochemistry. Tissue sections were stained with anti-CD4, anti-PD1 (EuroMabNet, clone NAT105) and anti-FOXP3 (EuroMabNet, clone 236A). Example Tfh cell (CD4+PD1+FOXP3) painted orange, Treg cell (CD4+PD1FOXP3+) painted yellow and Tfr cell (CD4+PD1+FOXP3+) painted turquoise are presented

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