Identification of biomarkers for hepatocellular carcinoma by semiquantitative immunocytochemistry

Figure 3 Representative images and fluorescence intensities of cells obtained from patients. A: Representative image of circulating tumor cells (CTCs) with DAPI-stained nuclei (blue), positive staining of ASGPR/GPC3/CK (green), and negative staining for CD45. The scale bar is 5 μm. A total of 7.5 mL of peripheral blood was collected from each individual, mixed with red blood cell lysis buffer, and incubated with CD45 microbeads to deplete the white blood cells. The remaining cell pellets were fixed on SuperFrost Plus slides for immunofluorescence staining; B: The bar graph of ASGPR/GPC3/CK (from left to right) fluorescence intensities in 30 HCC patients, 7 chronic HBV-infected patients, and 15 healthy controls. Fluorescence intensities were measured using NIH Image J software. Biomarker relative intensities were calculated as the difference between the biomarker staining intensity of the cell and the background intensity. The comparisons between HCC patients and healthy controls were analyzed by the Mann-Whitney test (^bP < 0.01 vs control).