Historically, there has been confusion relating to the diagnostic nomenclature for individual cell death. Toxicologic pathologists have generally used the terms ‘‘single cell necrosis’’ and ‘‘apoptosis’’ interchangeably. Increased research on the mechanisms of cell death in recent years has led to the understanding that apoptosis and necrosis involve different cellular pathways and that these differences can have important implications when considering overall mechanisms of toxicity, and, for these reasons, the separate terms of apoptosis and necrosis should be used whenever differentiation is possible. However, it is also recognized that differentiation of the precise pathway of cell death may not be important, necessary, or possible in routine toxicity studies and so a more general term to indicate cell death is warranted in these situations. Morphological distinction between these two forms of cell death can sometimes be straightforward but can also be challenging. This article provides a brief discussion of the cellular mechanisms and morphological features of apoptosis and necrosis as well as guidance on when the pathologist should use these terms. It provides recommended nomenclature along with diagnostic criteria (in hematoxylin and eosin
- Use necrosis and apoptosis as separate diagnostic terms.
- Use modifiers to denote the distribution of necrosis (e.g., necrosis, single cell; necrosis, focal; necrosis, diffuse; etc.).
- Use the combined term apoptosis/single cell necrosis when (a) There is no requirement or need to split the processes, or (b) When the nature of cell death cannot be determined with certainty, or (c) When both processes are present together.
- The diagnosis should be based primarily on the morphological features in H&E-stained sections. When needed, additional, special techniques to identify and characterize apoptosis can also be used.
This guidance is for the toxicologic pathologist to use for the routine evaluation of H&E-stained histological sections to distinguish apoptosis and necrosis. This document will serve as current guidance for the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) Organ Working Groups and the toxicologic pathology community at large. Any previously published INHAND documents that do not align with this guidance will be revised. Importantly, apoptosis and single cell necrosis are not synonyms although previous guidance has indicated otherwise and toxicologic pathologists have often used them interchangeably. We recognize that not all situations demand the same degree of precision with regard to distinguishing apoptotic from necrotic cell death, so the combined term apoptosis/single cell necrosis is available. This term can be used when the distinction between the different types of cell death is not desired or when it cannot be determined by either H&E or other stains that were employed. Recommendations for the use of the terms apoptosis and necrosis as well as the combined term apoptosis/single cell necrosis are discussed in this document and summarized in Figure 1.
Previous guidance on cell death nomenclature was provided in 1999 with the publication of ‘‘The Nomenclature of Cell Death: Recommendations of an ad hoc Committee of the Society of Toxicologic Pathologists’’ by Levin et al. (1999). This Committee recommended that the term ‘‘necrosis’’ be used to describe any morphological findings of cell death in histological sections, regardless of the pathway by which the cells died. Furthermore, ‘‘apoptotic,’’ ‘‘oncotic,’’ or ‘‘mixed apoptotic and necrotic’’ could be used as modifiers to specify the predominant cell death pathway, if appropriate. The overarching goal was that ‘‘pathologists use terms that accurately and concisely convey the level of information appropriate to the study’s needs.’’
However, these terms were not widely accepted in the toxicologic pathology community, and this may have been due to the intense focus of apoptosis research within the scientific community and a global understanding that these two forms of cell death occurred by different mechanisms. To address the issue of cell death nomenclature in the scientific community, an international group of scientists first published ‘‘Cell Death and Differentiation’’ in 2005 and then provided the updated ‘‘Classification of Cell Death: Recommendations of the Nomenclature Committee on Cell Death 2009’’ 4 years later (Kroemer et al. 2005; Kroemer et al. 2009). They proposed unified criteria for the definition of cell death and its different morphologies. Definitions were provided for apoptosis, necrosis, autophagy and cornification, and tentative definitions of atypical cell death modalities (mitotic catastrophe, anoikis, Wallerian degeneration, paraptosis, pyroptosis, and pyronecrosis) were discussed. They recommended that specific biochemical analyses (i.e., DNA ladders) should not be used as exclusive tests for apoptosis. Likewise, proteolytically active caspases or cleavage products of their substrates are not sufficient to define apoptosis. However, it was stated that these tests, along with other features, may be helpful in the diagnosis of apoptosis. Part of the conclusions stated that there are 3 distinct routes of cellular catabolism that can be defined by morphological criteria: autophagy, necrosis, and apoptosis. The use of terms such as necroapoptosis and aponecrosis to describe cell death, where apoptosis and necrosis occur simultaneously, was discouraged and instead they recommended using necrosis and apoptosis as separate terms.
There have been numerous advances in the field of cell death since the Levin et al. (1999) publication. Although the mechanisms and morphologies of apoptosis and necrosis differ, there is evidence that they share a similar biochemical network, described as the ‘‘apoptosis–necrosis continuum’’ (Zeiss 2003). As an example, a decrease in the availability of caspases and intracellular adenosine triphosphate (ATP) will convert an ongoing apoptotic process into a necrotic process (Denecker et al. 2001; Leist et al. 1997). Although the two processes can overlap due to available substrates and energy, there is and has been clear evidence that necrosis and apoptosis occur via different pathways.
This article provides guidance on the identification and diagnosis of apoptosis and necrosis. This guidance document also includes discussion of the cellular mechanisms of apoptosis and necrosis, when and where apoptosis occurs, descriptions of morphological features of apoptosis and necrosis in H&E-stained tissue sections, additional techniques that can be used to confirm apoptosis, and some organ-specific examples of when distinguishing between apoptosis and single cell necrosis may be challenging.
There are other forms of cell death that have been described and discussed in the literature such as autophagy, necroptosis, eryptosis, anoikis, and pyroptosis (Bergsbaken, Fink, and Cookson 2009; S. A. Elmore 2015; Gilmore 2005; Lang, Lang, and Foller 2012; Linkermann and Green 2014; Newton 2015; Ryter, Mizumura, and Choi 2014). These other forms of cell death are beyond the scope of this article; therefore, they will not be discussed here.
Recommendations for Cell Death Nomenclature
Review of cellular mechanisms of apoptosis. Apoptosis, also described as ‘‘programmed cell death,’’ was first described by Kerr, Wyllie, and Currie (1972) and is a process of cell death that is mediated by a genetically controlled, energyrequiring program. The 3 main pathways of apoptosis are (1) targeting mitochondria functionality (mitochondrial or intrinsic pathway), (2) direct transduction of the signal via adaptor proteins (death receptor or extrinsic pathway), and (3) the perforin/granzyme pathway (S. Elmore 2007). One of the key features of apoptosis is the cleavage of cytoskeletal proteins by aspartate-specific proteases, which results in the collapse of subcellular components. This can be seen histologically as cytoplasmic and nuclear condensation and nuclear fragmentation (Figure 2). Importantly, for all 3 pathways, plasma membrane integrity persists until late in the process. This prevents release of cellular components into the cytosol and thus the lack of inflammation. Eventually, apoptotic cells and fragments (apoptotic bodies) will externalize phosphatidylserine on the cell membrane, which results in recruitment and engulfment by macrophages (tingible body macrophages).
The mechanisms of apoptosis are highly complex and sophisticated, involving an energy-dependent cascade of molecular events (S. Elmore 2007). The intrinsic pathway is characterized by increased permeability of the mitochondria, release of cytochrome c, formation of a multiprotein complex called the ‘‘apoptosome,’’ and initiation of the caspase cascade through caspase 9. The extrinsic pathway involves proapoptotic ligands (i.e., FasL/FasR, tumor necrosis factor [TNF]-α/ TNFR1, etc.), binding with transmembrane death receptors (members of TNF receptor gene superfamily), transduction of intracellular signals, binding of adaptor proteins, deathinducing signaling complex formation, and then initiation of the caspase cascade through caspase 8. The perforin/granzyme pathway is involved in T cell-mediated cytotoxicity and is characterized by secretion of the transmembrane poreforming molecule perforin, exophytic release of cytoplasmic granules containing serine proteases (granzymes) through the pore and into the target cell, and then either initiation of the caspase cascade through caspase 10, direct initiation of caspase 3, or a caspase independent DNA cleavage via a SET complex (nucleosome assembly protein SET, apurinic/apyrimidinic endonuclease 1 [Ape1], protein phosphatase 32 [pp32], High mobility group protein 2 [HMG2]). Factors that determine which apoptotic pathway is activated include the stage of the cell cycle, the type and magnitude of stimuli, and, for immune cells, the stage of cellular activation. Different apoptotic pathways may occur concomitantly. Unlike necrosis, apoptosis can be, under certain circumstances, reversible. One example is the reversibility of p53-induced apoptosis (Geske et al. 2001). The tumor suppressor p53 is activated by a variety of cellular insults such as damaged DNA, nucleotide depletion, hypoxia, and heat shock. Geske and colleagues have shown that p53-induced apoptotic cells can be rescued from the early stages of the apoptotic process. DNA repair was shown to be activated early in p53-induced apoptosis and could modulate the cell death process if the apoptotic stimulus was removed.
When and where apoptosis occurs. In multicellular organisms, apopt