The aim of this study was to develop a method of quantitating prostate-specific
acid phosphatase (PSAP) in histologic sections of prostate
tumor tissue labeled with the
peroxidase-antiperoxidase (
PAP) complex technique using diaminobenzidine (DAB) as a substrate. Studies of PAP-DAB- and
hematoxylin-stained prostate tissue sections were performed with a black-and-white, computerized microscope image system. The mass of brown reaction product generated in PAP-DAB staining was the
indicator of PSAP intensity. The mass of brown reaction product was determined by using a dual wavelength method in which two 10-nm bandpass filters, peaked at 450 and 510 nm in wavelength, were used. The wavelength-dependent ratio of mass absorptivity of PAP-DAB
stain (brown product) and that of
hematoxylin (blue product) were estimated at wavelengths of 450 and 510 nm by using slides stained with only PAP-DAB or
hematoxylin. The accuracy of the mass measurements, investigated by relating the measurement to the true mass of the brown PAP-DAB product, is reported. There was no significant difference between the measurements at magnifications of 10x, 20x, 40x or 60x in the reproducibility investigation. The PSAP
stain intensity was quantitatively determined by the difference of the PAP-DAB
stain mass per pixel between the
tumor and normal cell region. The relationship between the objective measurement and the conventional subjective grades is presented.