Quantification of relative chromatin content in flow cytometry standards using 3D OPTM imaging technique

A potential biomarker for early diagnosis of cancer is assessment of high nuclear DNA content. Conventional hematoxylin staining is neither stoichiometric nor reproducible. Although feulgen stain is stoichiometric, it is time consuming and destroys nuclear morphology. We used acidic thionin stain, which can be stoichiometric and also preserve the nuclear morphology used in conventional cytology. Fifty chicken erythrocyte nuclei singlets (CENs), diploid trout erythrocyte nuclei (TENs) and Triploid TENs were stained for 15 and 30 minutes each. After imaging with optical projection tomography microscope (OPTM), 3D reconstructions of the nuclei were processed to calculate chromatin content. The mean of ratios of individual observations was compared with standard ratios of DNA indices of the flow cytometry standards. Mean error, standard deviation and 97% confidence interval (CI) was computed for the ratios of these standards. At 15 and 30 minutes, the ratio of Triploid TEN to TEN was 1.72 and 1.76, TEN to CEN was 1.27 and 2.01 and Triploid TEN to CEN was 2.11 and 3.39 respectively. Estimates of DNA indices for all 3 types of nuclei had less mean error at 30 minutes of staining; Triploid TEN to TEN 0.349±0.04, TEN to CEN 0.36±0.04 and Triploid TEN to CEN 0.64 ± 0.07. In conclusion, imaging of cells with thionin staining at 30 minutes and 3D reconstruction provides quantitative assessment of cell chromatin content. The addition of this quantitative feature of aneuploidy is expected to add greater accuracy to a classifier for early diagnosis of cancer based on 3D cytological imaging.