Quantifying Leukemic Cell IGF2BP3 Expression

Slides were scanned with Hamamatsu Nanozoomer XR using 40× magnification. QuPath software (version 0.2.3) [23 (link)] was used to detect cytoplasmic IGF2BP3 positivity in TMA-sections from annotated areas with leukemic cells. A pathologist manually set detection parameters and thresholds using the cytoplasmic staining of IGF2BP3 in germinal center cells as a reference, and the nuclear staining in germinal centers and proliferating epithelium as a reference for Ki-67 staining. The stain vectors and intensity thresholds for the cell and antibody detection were adjusted according to the instructions of the QuPath software in visual control. Inadequate samples were removed from the analysis. Areas with artifacts caused by compression or folding of the tissue were disregarded by setting the proper threshold values for background intensity. With the IGF2BP3 and CD19/Ki-67- double-stained slides, stain vectors were adjusted for hematoxylin, 3,3′-diaminobenzidine (DAB), and alkaline phosphatase (AP) staining using a representative region of interest. Hematoxylin-stained cells were detected using the cell detection function in the QuPath, while the nuclear DAB of Ki-67-positive cells were recognized from the CD19-positive (AP) areas. Single intensity thresholds for IGF2BP3, CD19, and Ki-67 were used to assess the proportion of positive cells.