Quality control of three methods for lung tumorigenesis studies

Many variables influence experimental results obtained from laboratory animal studies. One of the variables is tissue sampling for the detection of lesions. The contribution of different levels of sampling to the variability in reported tumour rates was evaluated in a tumorigenesis study using 1872 CBA/J mice. The number of lung neoplasms was estimated by three methods and the results compared. These methods were: 1. counting the macroscopically visible nodules, 2. microscopical examination of macroscopically-detected nodules and one histological section of each lung lobe, cut at the level of the bronchi (common method) and 3. microscopical examination as in method number 2 and additional microscopic examination of step-sections (200 microm interval) of the remaining lung tissue beginning at the level of the bronchi. Analysis using only macroscopic examination (method 1) showed that 40% (747/1872) of the animals had single or multiple nodules (i.e. tumour suspicious areas) in the lungs. When combined with microscopic examination (method 2), primary lung tumours were diagnosed in only 586 animals (31%). Evaluation by gross examination alone therefore gave an apparent overestimation of lung tumours compared to microscopic evaluation of grossly visible nodules. This was found to be due to a significant number of mice having nodules formed by processes other than primary lung tumours (i.e. non-specific inflammation, alveolar histiocytosis, focal hyperplasia of the alveolar epithelium, lymphoma infiltration or tumour metastases). On the other hand, in the more thorough sectioning of the lungs (method 3), primary lung tumours were detected in 712/1872 animals (38%). Additionally, these three different methods influenced the results with regard to the tumour multiplicity in each tumour-bearing animal.