The tunel assay in the diagnosis of graft-versus-host disease: caveats for interpretation

Abstract

Acute graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality following bone marrow transplantation, and early detection is important to allow effective therapy. Since the presence of apoptotic keratinocytes (dyskeratotic bodies) has been suggested as a useful diagnostic criterion for GVHD, attention has focused on the use of the TUNEL assay to detect apoptosis in clinical specimens. We reviewed clinical specimens upon which TUNEL had been performed for possible artifacts that might interfere with accurate evaluation for GVHD. Several distinct types of artifact were found and could be re-created in experimental systems. Artifacts in TUNEL staining generally resulted from the lack of specificity of this reaction for apoptotic cell death. Artifacts were found resulting from inadequate fixation, over-exposure of the TUNEL reaction, and proximity to the section edge. In addition, a novel artifact, apparently resulting from DNA shearing during the sectioning process, was noted and confirmed using confocal microscopy of experimental specimens. The TUNEL assay must therefore must be interpreted with caution in the clinical setting. In our laboratory, we consider TUNEL-positive cells as apoptotic only when accompanied by apoptotic morphology. Although these criteria clearly miss some cells in the early stages of apoptosis, they provide the highest specificity for apoptotic cell death.

Caspase activation correlates with the degree of inflammatory liver injury in chronic hepatitis C virus infection

“…Several concerns have been expressed about pitfalls, lack of specificity, and difficulties to standardize the TUNEL technique. [32][33][34][35] Furthermore, it is a common experience that performance of TUNEL staining depends greatly on the tissue pretreatment and labeling procedures. 36 Another reason for the discrepancy in the number of TUNEL-positive cells and cells revealing caspase activation may be explained by the different time course of biochemical events in apoptosis.…”

The Prevalence of Apoptotic Keratinocytes in Equine Epidermis: A Retrospective Light‐microscopic Study of Skin‐biopsy Specimens from 253 Horses with Normal Skin or Inflammatory Dermatoses

“…Commercial immunohistochemical kits for use in human medicine were evaluated in very few domestic animal skin specimens (Andreoletti et al, 1997;Noli et al, 1998). The specificity of the TUNEL reaction was recently questioned (Grasl-Kraupp et al, 1995;Jerome et al, 2000). Baima and Sticherline (2002) compared three TUNEL kits for human skin specimens and found abnormally high non-specific staining with all three kits.…”

Automated TUNEL assay on tissue sections via robotics

“…3−5 Positive TUNEL staining has been observed in necrotic cells, 6 foci of DNA repair 7,8 and even as a result of mechanical damage during sectioning leading to DNA shearing. 9 Nevertheless, due to the relative ease and robustness of the protocol, the TUNEL assay remains one of the most accepted methods for the detection of late-stage apoptosis in tissue sections, and its popularity has led to the development of commercial staining kits by multiple manufacturers. The kits use a manual staining method on formalin-fixed, paraffin-embedded (FFPE) tissue involving a series of steps: section dewaxing, epitope recovery (usually protease treatment), incubation with TdT enzyme in a buffer containing labeled dUTPs, and the detection of the label.…”