Quantitation of staining for amyloid in histological sections: sources of variation, correlation analysis, and...

Five pathologists estimated with point counting the amount of amyloid in heart muscle autopsy samples from 19 patients who suffered from amyloidosis. The serial sections had been stained with 5 different methods. The different variation sources involved in a quantitative study on amyloid and the interstain reproducibility between the results given by these 5 stains were analyzed. The volume fractions of amyloid after different stains were subjected to correlation analysis. In our test system about a third (34%) of the variation was due to the point counting method. Sirius red had the lowest and Congo red the highest variation due to point counting, since Sirius red gave the most intense staining and highest volume fractions for amyloid. The volume fractions were lowest in Congo red. About 40% of the total variation was composed of the field variation. This variation includes the variation due to variation between fields, field selection and subjective intraobserver variation. Sirius red showed the smallest field variation in respect to the total variation. Congo red, on the other hand, had the highest variation. The variation caused by pure interobserver variation is presented by the difference between total variation and the calculated field variation. This was largest with Sirius red and smallest with Congo red. The pure interobserver variation accounted for about 26% of the total variation. The high reproducibility with Sirius stain is most probably due to the high volume fractions of positively stained material after this stain, not to low interobserver variation. Congo red seems to have low interobserver variation. The senior pathologists were more reliable in measuring the amyloid deposits than the junior pathologists as suggested by interstain reproducibility and variation.(ABSTRACT TRUNCATED AT 250 WORDS)