THE TUNEL ASSAY IN THE DIAGNOSIS OF GRAFT-VERSUS-HOST DISEASE: CAVEATS FOR INTERPRETATION KEITH R. JEROME * , CLAUDIO VALLAN† AND ROLF JAGGI† * Department of Laboratory Medicine, University of Washington, Seattle, Washington, USA, and †Department of Clinical Research, University of Bern, Switzerland Summary 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 keratino- cytes (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 micros- copy 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 morphol- ogy. Although these criteria clearly miss some cells in the early stages of apoptosis, they provide the highest specificity for apoptotic cell death. Key words: TUNEL, apoptosis, graft-versus-host, artifact, laser confocal scanning microscopy Abbreviations: LCSM, laser confocal scanning microscopy; GVHD, graft- versus-host disease; TUNEL, Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling. Accepted 20 February 2000 Acute graft-versus-host disease (GVHD) remains a sig- nificant cause of morbidity and mortality following bone marrow transplantation (BMT). The pathology of GVHD appears to be multifactorial in origin, resulting from both a direct attack upon recipient tissues by immunocompetent cells from the donor as well as derangements of immune regulatory mechanisms with release of inflammatory cyto- kines (the so-called “cytokine storm”). These mechanisms most likely work together to cause the pathology of GVHD, which is most pronounced in the skin, GI tract, and liver. GVHD is treated most effectively when detected early, and in most institutions early diagnosis is based on skin biopsy. Unfortunately, the histopathological features of GVHD overlap with other skin pathology frequently seen in BMT recipients, such as drug reactions or alterations secondary to pre-transplant conditioning regimens. Among the criteria useful to distinguish GVHD from these pro- cesses, some authors have focused on the presence of “dyskeratotic bodies”. We and others have shown that the presence of such bodies, together with other findings such as an activated lymphomonocytic infiltrate, supports the diagnosis of GVHD. 1–3 With the recent realisation that these dyskeratotic bodies actually represent apoptotic kera- tinocytes, 4,5 attention has focused on methods such as the TUNEL reaction (terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling) which in theory might allow more sensitive detection of apoptotic cells in the skin. However, our experience with the TUNEL assay suggests that there are a number of commonly occurring artifacts that can lead to potential misinterpreta- tions in the evaluation of skin biopsies for GVHD. The TUNEL assay must be interpreted with caution in regard to these potential artifacts to prevent erroneous diagnosis of GVHD. In this report we illustrate artifacts that can lead to erroneous interpretation of TUNEL results, re-create these artifacts in experimental systems, and describe the appear- ance and causes of previously undescribed artifacts. A large number of biochemical and other tests now exist to study the process of apoptosis in cultured cells, including assays based on morphological changes, oligonucleosomal DNA fragmentation, cell membrane phospholipid distribu- tion, caspase activation, mitochondrial transmembrane potential and many others. However, there has been a clear need among pathologists and other scientists for methods capable of detecting apoptosis in intact tissue specimens. In 1992, Gavrieli et al. described a procedure for the detection of DNA fragmentation in situ, by end labeling of DNA strand breaks using terminal deoxyribonucleotidyl transfer- ase (TdT). 6 Since double-stranded DNA breaks occur during apoptosis, TdT would be expected to preferentially interact with such cells. At the broken DNA ends, TdT catalyses template-independent addition of labeled deoxyur- idine triphosphates (dUTPs), which can then be detected by immunohistochemical or other means. Since the introduc- tion of the TUNEL assay a wide variety of commercial kits have been introduced, based on this or similar technology, and there has been increased interest in the possible clinical utility of TUNEL-based testing. The TUNEL assay can be ISSN 0031–3025 printed/ISSN 1465–3931 online/00/030186– 05 © 2000 Royal College of Pathologists of Australasia Pathology (2000) 32, pp. 186– 190