Detection of apoptotic cells in cytology specimens: An application of TdT‐mediated dUTP‐biotin nick end labeling to cell smears

Abstract

We applied TdT-mediated deoxyuridine triphosphate (dUTP)biotin nick end labeling (TUNEL) to cytologic smears in order to detect the cells undergoing apoptosis. These smears were obtained by scraping the cut surface of 9 cases of carcinoma, including renal-cell carcinoma (3 cases), esophageal squamous-cell carcinoma (3 cases), and gastric adenocarcinoma (3 cases), and were fixed and prepared by different methods. The results were also compared with those of tissue sections. TUNEL in smears was generally associated with higher background nuclear stain than in tissue sections. Smears that were fixed in 4% or 8% paraform aldehyde or absolute methanol exhibited results comparable with those of tissue sections, with minimum background in all cases examined. There were no significant differences in TUNEL labeling index among tissue sections and smears fixed in 4% or 8% paraformaldehyde or in absolute methanol. Smears treated in Carnoy's fixative (3:1 methanol:acetic acid) and air-dried smears demonstrated a higher background. TUNEL positivity could not be detected in slides decolorized from May-Grünwald-Giemsa stain. Markedly high background, which may occur as a result of artifactural DNA breaks, was also observed in slides decolorized from Papanicolaou stain, in which TUNEL-positive cells could be evaluated only in 3/8 cases. Application of the TUNEL method to cytology specimens has disadvantages or limitations compared to its application to histological sections, but the method is considered the most suitable one for detecting cells undergoing apoptosis in cytology materials.

Use of a Fluorescently Labeled Poly-Caspase Inhibitor for in Vivo Detection of Apoptosis Related to Vascular-Targeting Agent Arsenic Trioxide for Cancer Therapy

“…Figure 1A summarizes the tissue damage and vascular effects of ATO treatment observed within the window chamber tumor growth model. Observation of tumor tissue within the window chamber validated our previous work with ATO in hind-limb tumor models that suggested vascular damage as a primary cause of the anti-tumor effects of ATO (1)(2)(3)12). Total clearance of unbound FLIVO has been found to occur by 30-50 min in mice and, therefore, we chose 30 min after injection to assess binding of the reagent in the tumor (unpublished observation).…”

“…Unfortunately, there are few, if any, reagents currently available to assess or monitor the occurrence of apoptosis in living tissue. TUNEL (t-UTP nick end labeling) has been arguably the most common label used for the identification of apoptotic cells, even though it has a relatively low level of specificity for apoptotic cells as nicked DNA can occur in cells dying by either necrosis or apoptosis (1)(2)(3). Another major drawback is that TUNEL cannot be used on living cells or animals since it requires permeabilization of the cell membrane.…”

Evaluation of apoptosis in cytologic specimens

“…5 Techniques of staining cytological samples such as bronchoalveolar lavage (BAL), fine needle aspiration (FNA) biopsy, and smears with trypan blue, TUNEL and Fas have been described. [13][14][15][16][17] An example of TUNEL-positive cells (arrows) seen in a smear of renal cell carcinoma is present in Figure 2. 14 IHC can allow visualization of apoptotic pathway constituents including p53, Bcl-2, death receptors and ligands TNF, FasL, and TRAIL.…”

Comparison of cytological and immunocytochemical methods for detecting apoptotic epithelial cells in cervicovaginal smears

“…In this study, we used CC3 antibody to detect this newly formed epitope, and this antibody cannot bind procaspase form of caspase 3 and other caspases. Along with CC3, loss of membrane asymmetry, cleavage of anti‐apoptotic Bcl‐2, cleavage of caspase substrates, analysis of DNA fragmentation by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were also used to detect apoptosis …”