QUANTITATIVE COMPUTERIZED IMAGE-ANALYSIS OF IMMUNOSTAINED LYMPHOCYTES

A methodological approach by computerized image analysis to quantify i mmunostained objects in histological sections is described. We have in vestigated antibodies against CD4, CD8, CD20, CD23 and CD25 in frozen sections of human nasal mucosa; however, the methodology of standardiz ation is of general validity. The study was designed particularly to i nvestigate the following points: 1) light intensity, 2) the grey level for counter staining intensity, 3) the grey level threshold value for positive objects, 4) the minimal acceptable size of a positive object , 5) the influence of the brightness of the light on both the number a nd the area of objects. Furthermore, random sampling and determination of 6) the area per section, and 7) the number of histological section s to be measured per biopsy. Finally, a study of reproducibility of im munostaining intensity was performed. The influence of the different p arameters mentioned above was studied and the values leg. threshold va lue) for our particular setting of microscope, image analysis equipmen t, computer software etc, were defined. The method was then tested for intra- and interindividual variation which was found to be less than 5%. Correlation analysis of the reproducibility gave coefficients of c orrelation of 0.99, both concerning number of immunopositive objects a nd immunopositive area. We emphasize the importance of a highly standa rdized methodology if the numeric data obtained from computer assisted image analysis are to be more accurate than semiquantitative assessme nts by experienced observers. With a thorough standardization as descr ibed in this method it is possible to obtain numeric values, and data with low deviations, which are two obvious and important advantages.