Quantitative computerized image analysis of immunostained lymphocytes. A methodological approach.
A methodological approach by computerized image analysis to quantify immunostained objects in histological sections is described. We have investigated antibodies against CD4, CD8, CD20, CD23 and CD25 in frozen sections of human nasal mucosa; however, the methodology of standardization is of general validity. The study was designed particularly to investigate 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 and the area of objects. Furthermore, random sampling and determination of 6) the area per section, and 7) the number of histological sections to be measured per biopsy. Finally, a study of reproducibility of immunostaining intensity was performed. The influence of the different parameters mentioned above was studied and the values (eg. threshold value) for our particular setting of microscope, image analysis equipment, 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 correlation of 0.99, both concerning number of immunopositive objects and immunopositive area. We emphasize the importance of a highly standardized methodology if the numeric data obtained from computer assisted image analysis are to be more accurate than semiquantitative assessments by experienced observers. With a thorough standardization as described in this method it is possible to obtain numeric values, and data with low deviations, which are two obvious and important advantages.
Department of Pathology, Orebro Medical Center Hospital, Sweden.
M G
MG
Karlsson
A
A
Davidsson
H B
HB
Hellquist
D015703:Antigens, CD / Q000032:analysis*; D006801:Humans; D007091:Image Processing, Computer-Assisted / Q000379:methods*; D007124:Immunoenzyme Techniques; D009297:Nasal Mucosa / Q000166:cytology; D015203:Reproducibility of Results; D013601:T-Lymphocytes / Q000276:immunology*
D015703:Antigens, CD