The Tunel Assay in the Diagnosis of Graft-Versus-Host Disease: Caveats for Interpretation

Abstract

SummaryAcute 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.Key words: TUNEL, apoptosis, graft-versus-host, artifact, laser confocal scanning microscopy Abbreviations: LCSM, laser confocal scanning microscopy; GVHD, graftversus-host disease; TUNEL, Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling. Accepted 20 February 2000Acute graft-versus-host disease (GVHD) remains a significant 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 cytokines (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 processes, 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 keratinocytes, 4,5 attention has focused on me...

Apoptosis in the cortex of the developing mouse kidney

“…Indeed, our counts suggest that, by comparing propidiumstained frozen sections stained with and without TUNEL, it is possible to overestimate the amount of apoptosis by a factor of three or more, although TUNEL can give false positives (Jerome et al 2000). Even with this caveat, it is not clear why Coles et al (1993) obtained values for apoptosis in embryonic kidneys that were so much greater than ours.…”

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.…”

What is the role and nature of programmed cell death in phytoplankton ecology?

“…'Senescence' in multicellular plants involves PCD. from necrosis is debated (Malmusi & Ackerman, 1999;Ratel et al, 2001;Kanduc et al, 2002), since the specificity of molecular markers (e.g. TUNEL labelling, see below) that are claimed to be diagnostic for apoptosis may not be as specific as claimed (Labat-Moleur et al, 1998;Jerome et al, 2000). What is not questioned however, is that throughout all domains of life, cellular pathways exist that can lead to a biochemical cascade resulting in cell death (Aravind et al, 2001;Ameisen, 2002;.…”

“…(iii) Low molecular weight DNA fragmentation, detectable by the comet assay and later by TUNEL/ISEL assays, is thought to be an early marker of apoptosis and thus PCD (Patel et al, 1995) in animal cells. The specificity of this marker to apoptosis is questioned (Graslkraupp et al, 1995;Jerome et al, 2000;Kanduc et al, 2002).…”