Quantitative peroxidase-antiperoxidase complex-substrate mass determination in tissue sections by a dual wavelength method.

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.