Low-budgetary scheme for differentiation and DNA quantity investigation in blood lymphocytes of patients with chronical tonsillitis

Boris V.Shilov1, Dmitry A.Dolgun2
1bvshilov@hotbox.ru, SSMU; 2, SSMU

Chromatin distribution and organization of DNA in nuclei of some atypical lymphocytes from patients with chronic tonsillitis can give important cellular diagnostic and prognostic information. A complex approach to investigating it has been used in the given study. The aim of the study is to reveal karyological and karyometrical aspects of chromatin distribution in chronization of the disease. The lymphocytes of patients with chronic tonsillitis were studied using on peripheral blood smears prepared in a traditional way and stained with the help of two methods: a unified one (according to Romanovsky-Gimza method) and according to Feulgen reaction. (The quantitative study of cell DNA content and characteristic of cell cycle phases was carried out using cytophotometry of Feulgen reaction) After staining according to Feulgen, it was possible to determine the stage of vital cycle but if was difficult to determine the nucleus belonging to any type of the cells. Computer analysis made it possible not only to estimate the morphometric indices of atypical lymphocytes in patients with chronic tonsillitis but to analyze the vital cycle of cell elements on the base of classification of transformed cells stained according to Romanovsky-Gimza. Cell images were obtained using the standard light microscope and digital camera. Traditional approach to a two-step image segmentation process was adapted to condition of low-budgetary science. In the first step nuclei areas were distinguished from the surrounding cells. The second step consisted of identifying a specific pattern defined as necessary for next work under the basis of the Canny Deriche algorithm. During the analysis, discriminant functions that allow dividing samples into the groups given above have been determined. When determining such functions images of the cells stained by Romanovsky-Gimza method were studied and later these functions were used for separating of nuclei images stained according to Feulgen. Blood specimens for staining using both methods were taken from the same patients. The data obtained were statistically processed. Step-by-step discriminant analysis was used to assign pathological mononuclear cells stained by Feulgen method to the appropriate group of cells. The lymphocytes like blasttransformed once were found in patients with compensated (I) and decompensated (II) forms of chronic tonsillitis. The research of these cells using of computer analysis imaging showed that the main part of the cells from the first group of patients contains DNA in a 4c (86%). The majority of abnormal lymphocytes contained DNA in a 2c (61%) in the second patientís group. The reduction of 4c-nucleus atypical lymphocytes amount in patients with decompensated tonsillitis testifies that synthetic process in a cellular nucleus are delayed or broken because of prolonged infection. On the basis of the received data it is possible to assume that patients with chronic decompensated tonsillitis are under the risk of accumulation of an adverse genetic background. More detailed molecular investigations could give the final answer.