Antigenicity Preservation Is Related to Tissue Characteristics and the Post-Mortem Interval: Immunohistochemical Study and Literature Review

The main aim of this study was to investigate the post-mortem proteolytic degradation process of selected tissue antigens and correlate it to the post-mortem interval. During the autopsy of 12 cadavers (time interval ranging 1 day–2 years after death) samples of skin, liver, kidney, and spleen were collected. All samples were formalin-fixed and paraffin-embedded. Four µm paraffin sections were used for hematoxylin–eosin staining and immunohistochemical analysis (Ki67, Vimentin, Pan cytokeratin, and CD20). Data reported here show that immunohistochemical reactivity preservation was related to the characteristics of the tissues. In particular, the most resistant tissue was the skin, where the autolysis phenomena were not appreciable before 5 days. On the contrary, the liver and the spleen underwent early autolysis, while the kidney displayed an early autolysis of the tubules and a late one of the glomeruli. As concerns specific antigens, immunoreactivity was lost earliest for nuclear antigens as compared to cytoplasmic ones. In conclusion, our results demonstrate that immunohistochemical detection of specific antigens may be useful in estimating the post-mortem interval, especially when we need to know whether the post-mortem interval is a few days or more than 7–10 days.


Introduction
The identification of the post-mortem interval (PMI) in cadavers involved in judicial investigations is of particular relevance in the daily practice of forensic pathologists. The elements on which the medico-legal expert must base judgement are given by both the objectification of the cadaveric phenomena and tanatochronology science. The study of these parameters contributes to the construction of a time-dependent curve whose characteristics (e.g., slope) and initial point (time of death) can be influenced by internal, external, ante-or post-mortem factors [1]. The methods used in PMI estimation are based on the evaluation of post-mortem changes due to physical (cooling and hypostasis), metabolic (concentrations of particular metabolites, enzymatic activities), autolytic (loss of cell membrane selectivity and morphological changes), physiological and chemical (supravital reactivity, rigor mortis), and bacterial (putrefaction) processes [1]. The methods to estimate the time since death are not only different in nature but also have a widely differing scientific value concerning, for example, the validation of the method [2]. In addition, it has been shown that the Washings were performed using PBS/Tween20 pH 7.6. Reactions were detected using an HRP-conjugated compact polymer system HRP-DAB kit (UCS diagnostics, Rome, Italy).
The following antigens were used as markers for immunohistochemical analysis: (a) Vimentin Vimentin, derived from latin word vimentum (flexible twig bunch), is an intracellular protein and is a type III intermediate filament protein. This protein is expressed in fibroblasts, endothelial cells, and lymphocytes [8] in which they polymerize to form the basis of the cytoskeleton, allowing for the maintenance of cellular structure in addition to contributing to cell signaling and proliferation [9]. This protein is found in various non-epithelial cells, especially mesenchymal cells [10], and stains melanocyte cells and dermal vessels. Structurally, the vimentin monomer contains a non-helical amino head, the central structure of the alpha helix domain, and a carboxyl tail. Subsequently, the monomers combine to form dimers that represent the basic subunits of vimentin [11].

(b) Ki 67
Ki-67 is a nuclear DNA-binding protein constitutively expressed in cycling mammalian cells [12]. It exists in two human isoforms with a maximum expression in G2 phase or during mitosis [12]. It prevents chromosomes from collapsing into a single chromatin mass by forming a steric and electrostatic charge barrier: the protein has a high net electrical charge and act as a surfactant, dispersing chromosomes and enabling independent chromosome motility [13]. Ki-67 represents a useful marker of cell proliferation [14].

(c) Cytokeratin
CKs are a heterogeneous group of proteins that contribute to the structure of intermediate filament that function as the supporting cytoskeleton in the epithelial cells [15]. The type I CKs consist of acidic proteins, arranged in pairs of heterotypic keratin chains, and the type II are basic or neutral proteins which are arranged in pairs of heterotypic keratin chains co-expressed during differentiation of simple and stratified epithelial tissues [16]. It has been demonstrated that keratin expression can be found in subsets of several types of mesenchymal cells [17].
CD20 is a B-cell marker expressed starting from late pre-B lymphocytes [18]; its expression is lost in terminally differentiated plasmablasts and plasma cells. It is a nonglycosylated 33-37 kDa protein member of the MS4A family. Structurally it consists of four hydrophobic transmembrane domains, one intracellular and two extracellular domains [18]. The extracellular portion of CD20 is 44 amino acids in length and provides the docking site for anti-CD20 MAbs binding. CD20 expression is initiated at the pre-B cell stage of development and remains present until terminal differentiation into a plasma cell [19].
Positive controls were performed by using multi-organ tissue micro arrays (TMA). Negative controls were performed without using the primary antibody.

Morphological Analysis
Hematoxylin-eosin staining allowed us to evaluate the morphological characteristics and conservation status of all tissues.

Skin
The skin showed a good preservation of its structure in all samples taken up to 15 days after death ( Figure 1A,B). The different layers of the epidermis and the annexal structures of the dermis were well recognizable, while the morphological structure of the skin was no longer recognizable in samples taken two years after death ( Figure 1C). keratin chains co-expressed during differentiation of simple and stratified epithelial tissues [16]. It has been demonstrated that keratin expression can be found in subsets of several types of mesenchymal cells [17].
CD20 is a B-cell marker expressed starting from late pre-B lymphocytes [18]; its expression is lost in terminally differentiated plasmablasts and plasma cells. It is a nonglycosylated 33-37 kDa protein member of the MS4A family. Structurally it consists of four hydrophobic transmembrane domains, one intracellular and two extracellular domains [18]. The extracellular portion of CD20 is 44 amino acids in length and provides the docking site for anti-CD20 MAbs binding. CD20 expression is initiated at the pre-B cell stage of development and remains present until terminal differentiation into a plasma cell [19].
Positive controls were performed by using multi-organ tissue micro arrays (TMA). Negative controls were performed without using the primary antibody.

Morphological Analysis
Hematoxylin-eosin staining allowed us to evaluate the morphological characteristics and conservation status of all tissues.

Skin
The skin showed a good preservation of its structure in all samples taken up to 15 days after death ( Figure 1A,B). The different layers of the epidermis and the annexal structures of the dermis were well recognizable, while the morphological structure of the skin was no longer recognizable in samples taken two years after death ( Figure 1C).

Kidney
Kidneys taken 1 day after death displayed a perfectly recognizable structure of the parenchyma, the glomeruli, and tubules ( Figure 2). At 3 days, there was a progressive autolysis of tubular cells with partial visualization of isolated tubular structures in contrast to the glomeruli, which were still preserved (Figure 2A). At 5 days, it was possible to observe partial autolysis of the glomerular cells. Lastly, at 15 days no cellular structures of the tubules and glomeruli were recognizable ( Figure 2B).

Kidney
Kidneys taken 1 day after death displayed a perfectly recognizable structure of the parenchyma, the glomeruli, and tubules ( Figure 2). At 3 days, there was a progressive autolysis of tubular cells with partial visualization of isolated tubular structures in contrast to the glomeruli, which were still preserved (Figure 2A). At 5 days, it was possible to observe partial autolysis of the glomerular cells. Lastly, at 15 days no cellular structures of the tubules and glomeruli were recognizable ( Figure 2B). Healthcare 2022, 10, x 5 of 15

Liver
The structure of the liver was well preserved in the sample taken at one day after death, in which the hepatocytes were clearly identifiable. Rare focal autolytic phenomena of the hepatocytes were observed in specimens taken 3 days after death ( Figure 3A). Autolysis phenomena were slightly increased in the 5-day specimen collection, while at 15 days, tissue autolysis was completed ( Figure 3B).

Spleen
The morphostructure of the spleen in samples taken one day after death was well preserved, and cellular components in the red pulp and white pulp were clearly visible. In the 3-day sample, the lymphatic structures in the white pulp were still clearly visible and preserved, while a progressive autolysis of the red pulp cells was observed. Progressive autolysis of white pulp lymphocytes was also observed in specimens taken 5 days after death ( Figure 4).

Liver
The structure of the liver was well preserved in the sample taken at one day after death, in which the hepatocytes were clearly identifiable. Rare focal autolytic phenomena of the hepatocytes were observed in specimens taken 3 days after death ( Figure 3A). Autolysis phenomena were slightly increased in the 5-day specimen collection, while at 15 days, tissue autolysis was completed ( Figure 3B).

Liver
The structure of the liver was well preserved in the sample taken at one day after death, in which the hepatocytes were clearly identifiable. Rare focal autolytic phenomena of the hepatocytes were observed in specimens taken 3 days after death ( Figure 3A). Autolysis phenomena were slightly increased in the 5-day specimen collection, while at 15 days, tissue autolysis was completed ( Figure 3B).

Spleen
The morphostructure of the spleen in samples taken one day after death was well preserved, and cellular components in the red pulp and white pulp were clearly visible. In the 3-day sample, the lymphatic structures in the white pulp were still clearly visible and preserved, while a progressive autolysis of the red pulp cells was observed. Progressive autolysis of white pulp lymphocytes was also observed in specimens taken 5 days after death ( Figure 4).

Spleen
The morphostructure of the spleen in samples taken one day after death was well preserved, and cellular components in the red pulp and white pulp were clearly visible. In the 3-day sample, the lymphatic structures in the white pulp were still clearly visible and preserved, while a progressive autolysis of the red pulp cells was observed. Progressive autolysis of white pulp lymphocytes was also observed in specimens taken 5 days after death ( Figure 4).

Anti-Vimentin Antibody
Staining was positive and specific in all examined samples up to day 5 after death, although it slightly lost intensity. In the liver, the small portal vessels were stained ( Figure  5A). In the kidney, the cytoplasm of some tubule cells and some glomerular endothelial cells were stained for the vimentin and were specific and intense until the third day after death ( Figure 6B). Starting from the fifth day after death, only some glomerular endothelia were stained, while the tubules were completely negative. In the two-year sample, the vimentin signal was completely lost.
In the spleen, vimentin stains the endothelium of the chordae of the red pulp, and the staining was specific and abundant at day one, while it was completely negative on day 3-5 due to the presence of autolytic processes ( Figure 6C).

Anti-Vimentin Antibody
Staining was positive and specific in all examined samples up to day 5 after death, although it slightly lost intensity. In the liver, the small portal vessels were stained ( Figure 5A). In the kidney, the cytoplasm of some tubule cells and some glomerular endothelial cells were stained for the vimentin and were specific and intense until the third day after death ( Figure 6B). Starting from the fifth day after death, only some glomerular endothelia were stained, while the tubules were completely negative. In the two-year sample, the vimentin signal was completely lost.
In the spleen, vimentin stains the endothelium of the chordae of the red pulp, and the staining was specific and abundant at day one, while it was completely negative on day 3-5 due to the presence of autolytic processes ( Figure 6C).

Anti-Ki67 Antibody
The skin showed a strong staining in the one-day specimens ( Figure 6A); with the increase of the PMI, a decrease in the percentage of positive cells was appreciated. At the same time, specimens showed slight aspecific signal (background) ( Figure 6B).
In the spleen, the positivity was closely related to the degree of autolysis. The ki67 stain maintained a good ratio until the third day after death, where non-specific signals were observed ( Figure 6C,D).

Anti-Pan-Cytokeratin Antibody
In the skin, CK reactivity was perfectly maintained both on the epidermis and on the adnexal structures in all the samplings taken up to 3 days ( Figure 7A,B). Keratin remained expressed with a specific localization in the epidermis with a concomitant partial diffusion in the other tissues in the two-year sampling.
In the liver, hepatocyte staining was maintained in all preparations, although as a result of autolytic phenomena, positivity tended to become less specific. In the kidney, the antibody stained the tubules and the parietal cells of the glomerulus as well as the Bowmann's capsule. The positivity was maintained in the ducts that do not present autolytic phenomena even in 3-5 days but became aspecific by also staining the nuclei of the cells in autolysis ( Figure 7C,D).

Anti-Ki67 Antibody
The skin showed a strong staining in the one-day specimens ( Figure 6A); with the increase of the PMI, a decrease in the percentage of positive cells was appreciated. At the same time, specimens showed slight aspecific signal (background) ( Figure 6B).
In the spleen, the positivity was closely related to the degree of autolysis. The ki67 stain maintained a good ratio until the third day after death, where non-specific signals were observed ( Figure 6C,D).

Anti-CD20 Antibody
To evaluate the immunoreactivity of CD20, a typical membrane protein that stains Blymphocytes, splenic tissue was used since in it numerous lymphoid follicles are normally present. The reactivity was intense and specific in the one-to two-day samples (Figure 8), whereas in the five-day sample, it became less specific (Figure 8).
Tables 2-5 summarize the results of immunohistochemistry in the tissues analyzed.

Anti-Pan-Cytokeratin Antibody
In the skin, CK reactivity was perfectly maintained both on the epidermis and on the adnexal structures in all the samplings taken up to 3 days ( Figure 7A,B). Keratin remained expressed with a specific localization in the epidermis with a concomitant partial diffusion in the other tissues in the two-year sampling.
In the liver, hepatocyte staining was maintained in all preparations, although as a

Anti-CD20 Antibody
To evaluate the immunoreactivity of CD20, a typical membrane protein that stains B lymphocytes, splenic tissue was used since in it numerous lymphoid follicles are normally present. The reactivity was intense and specific in the one-to two-day samples (Figure 8) whereas in the five-day sample, it became less specific (Figure 8).

Discussion
Immunohistochemistry represents a promising application in the forensic and medicolegal field, and its use can be exploited, together with other methods, to reach multiple objectives. In fact, immunohistochemistry is currently used for the dating of lesions and to support the differential diagnostic workflow between vital and post-fatal lesions [20][21][22]. Some authors have instead investigated the feasibility of employing immunohistochemistry in the diagnosis of death, achieving in some cases promising results [23][24][25]. Since the 1990s, immunohistochemical studies have also been applied in the estimation of the PMI. In this field of application, along with entomological studies and high-resolution 1H magnetic resonance tomography, immunohistochemical methods are particularly suited for delimitating long PMI [3,[26][27][28][29][30]. A brief review of the literature revealed that only a few studies have been published based on the analysis of post-mortem protein degradation by immunohistochemistry to estimate the time of death (Table 6). In six studies, tissues from internal organs such as thyroid, pancreas, and brain were used [3,[27][28][29][30][31], while in the other two [26][27][28][29][30][31][32], the analysis was performed on samples of gingival and skin tissue. The main protein markers investigated were insulin and glucagon for pancreatic tissue, calcitonin and thyroglobulin for thyroid, collagen type I and III for gingival tissue, GFAP for brain, and CEA, S-100 protein, CK and smooth muscle actin for sweat glands. In all the examined cases, the PMI was known before the autopsy, and the analyzed samples were heterogeneous with regard to (a) the environmental conditions in which the corpses were found, (b) the body structure, and (c) the causes of death. Almost all mentioned studies show a progressive reduction of the positivity of the specific marker until the complete negativity as the PMI interval increases, except for the study conducted by Cingolani et al. [26]. This could be explained by considering the short PMI used by the authors.
Regarding thyroglobulin, used in two studies as a specific marker for thyroid tissue, the results showed a significant variability; in particular, according to Wehner et al. [30], all analyzed cases within the fifth day after death were positive, with progressive reduction of the number of positive cases until the twelfth day. A complete negativization of cases was observed starting from the 13th day. In the study conducted by Ortmann et al., [31] some tissues showed negativity for thyroglobulin already from the second day, with progressive reduction of the number of positive cases until the eighth day after death. Glucagon exhibited similar patterns, with positivity maintained in most cases up to 6-8 days and complete negativization from day 12-13 onwards. Among all studied markers, insulin showed the longest persistence, with positivity of all cases maintained until day 12 and complete negativity of samples only starting from day 30 after death. Our results show that the immunoreactivity for nuclear antigens is lost earlier as compared to the cytoplasmic ones. In addition, we observed that the preservation of immunohistochemical reactivity is related to the intrinsic characteristics of the analyzed tissues and their different ability to withstand autolysis at the same PMI.
The most resistant tissue seems to be the skin, since autolysis phenomena are not appreciable before 5 days' postmortem; the liver, but especially the spleen, undergo early autolysis, while the kidney presents a variable autolysis degree, showing an early alteration of the tubules. In this regard, our results confirmed the usefulness of vimentin and pan-CK staining for the assessment of tissue preservation status. The vimentin tends to become negative only when marked autolysis is occurring, such as in the spleen after the third day or in renal tubules on the fifth day, while it remains positive, even if with a slight decrease in intensity, in the skin. The results of staining with antibodies against pan-CK, a cytoplasmic marker, are like those obtained with vimentin, showing, however, a greater capacity for preservation. In fact, whereas vimentin was completely negative in skin samples at two years, CK staining persisted in the epidermis. Reactivity for antibodies against membrane antigens, such as CD20, demonstrates rapid degradation as observed in the spleen where the positivity becomes nonspecific or negative already 2 days after death.
Preservation of antigenic determinants is one of the most important methodological problems of immunohistochemical studies [33]. Fixation preserves cell and tissue morphology by preventing antigen destruction; inappropriate fixation can partially destroy antigenic determinants or alter their structure to such an extent that they are unrecognizable to the antibody. Conversely, an unfixed antigen may disappear altogether or may diffuse from the site of synthesis into the surrounding tissue.
Factors such as cold ischemia time in surgical settings, PMI, fixation time, paraffin, storage time in paraffin, storage temperature, age of cut sections, antigen retrieval technique, and detection systems have been reported to influence the outcome of immunohistochemistry [3]. Furthermore, post-mortem tissues are also subjected to specific variables such as the agonal state; this can influence the tissue characteristics and immunoreactivity by pH changes [34]. In addition, the strong influence that climatic and environmental factors seem to have on the reliability of immunohistochemical results should be kept in mind. In accordance with the observations of some authors, at the same PMI, markedly more negative immunoreactions are seen in the warmer months than in the cooler months [30]. These differences are obviously due to the influence that the temperature has on the proteolytic and autolytic processes. It is therefore necessary to take this into account for all mentioned variables that could significantly alter the tissue immunoreactivity.

Conclusions
The results of this preliminary study suggest that immunohistochemical detection of specific antigens may be useful in estimating the PMI. Our findings demonstrated that the study of post-mortem antigenic degradation, especially for vimentin, pan-CK and ki67, could be promising when we need to know whether the PMI is a few days or more than 7-10 days. Therefore, further studies on large sample sizes are necessary to investigate how the variables related to causes of death, environmental conditions in which the corpse remains before being found, timing of retrievals with respect to the time of death, and mode of preservation and fixation of tissues may affect the protein degradation. To make this method reliable for forensic practice, it would also be desirable to carry out studies aimed at analyzing the behavior of different antigens or to evaluate the simultaneous use of multiple parameters for improving the accuracy of PMI estimation. In this context, cadavers obtained from cadaveric donation programs for scientific research purposes that are becoming established worldwide could profitably be used as a study sample [35].

Institutional Review Board Statement:
The study was conducted according to the guidelines of the Declaration of Helsinki. The approval of the ethics committee was not necessary as all the procedures carried out are set by legislation (Presidential Decree 285/1990) as essential to ascertain the natural causes of death.

Data Availability Statement:
The data used to support the findings of this study are available on request from the corresponding author.

Conflicts of Interest:
The authors declare no conflict of interest.