A study for TUNEL assay as a method of detecting apoptotic cells

Since 1992, in situ TdT-mediated digoxygenin (biotin)-dUTP nick end labeling (TUNEL) assay has become prevalent as a method of detecting apoptotic cells. However, our first trial with such specimens using a commercially available TUNEL kit and its protocol was disappointing, because of the considerable number of false-positive and false-negative cells. Therefore, we realized that this assay requires more studies about sample preparation and staining conditions to obtain higher specificity and sensitivity, especially in its application to cytologic smears and paraffin-embedded sections. In the present study, as a definite control of apoptotic and non-apoptotic cells, human leukemia HL-60 cells treated with 0.15μM campthotecin for 3 h were used. This treatment is known to induce apoptosis in approximately 30% of HL-60 cells. Smears and paraffin-embedded sections of those cells were prepared before and after fixation, respectively, under several conditions. Then, they were stained with agents included in a TUNEL assay kit (ApopTag™, Oncor) at various concentrations of TdT enzyme and after various incubation times, with or without pretreatment with proteinase K. We regarded conditions resulting in 30% positive as optimal. We obtained good conditions for TUNEL assay for smears and paraffin-embedded sections of HL-60 cells. In addition, this method under those conditions fairly stained 25 surgically excised specimens prepared as touch smears and paraffin -embedded tissue sections. Notably, we also found that TUNEL staining is strongly influenced by fixation, proteinase K treatment, and TdT activity. Therefore, one should be very careful in evaluating results of TdT staining of materials whose preparatory conditions are unknown.