Table 2.

Post-mortem histomorphological and immunohistochemical investigation as a tool for the PMI estimation: a review of the literature on this topic

Ref.	No. of cases	Biological samples	Immunohistochemical/histological exams	PMI investigated	Results	Impact on the scientific community
[22]	147 corpses Studied with external factors (temperature and humidity), environmental factors (closed, open or in water) and factors related to the body (clothes or body) differing in each case	Colloid and follicular cells of the thyroid gland	DAKO EPOS-System: monoclonal mouse antihuman thyroglobulin antibody with diaminobenzidine	From 1 to 21 days after death	Positive immunoreaction for thyroglobulin for up to 5 days, in all cases No reaction after 13 days	Positive correlation between post-mortem changes in the structure of thyroglobulin and the colourability of the same are time-dependent: the denaturation of the protein makes it unsuitable for immunohistochemical staining Immunohistochemical detection of thyroglobulin can be considered a useful method for delimiting the PMI, especially in cases of longer PMI
[23]	128 corpses Studied with external factors (temperature and humidity), environmental factors (closed, open, in water) and factors related to the body (clothes, body) differing in each case	Cells B pancreas	Guinea pig polyclonal antibody (DAKO Company) used for the detection of insulin The biotinylated anti-rabbit-F (ab9) 2 fragment was used as a secondary antibody. Detection was carried out with the avidin–biotin-peroxidase method with 3,39-diaminobenzidine	From 1 to 45 days after death	Positive immunoreaction to insulin for up to 12 days after death; no reaction for longer than 30 days	Positive correlation between post-mortem changes in the structure of insulin and its colourability over time: the denaturation of the protein makes it unsuitable for immunohistochemical staining However, the time limits resulting from the study may change in the event of a significant variation in the environmental conditions related to the discovery of the corpse
[24]	136 corpses Studied with external factors (temperature and humidity), environmental factors (closed, open, in water) and factors related to the body (clothes, body) differing in each case	Thyroid C cells	Immunohistochemical staining with the DAKO-Epos system. Detection of calcitonin with a prediluted rabbit anti-human calcitonin antibody. Detection of antibody binding was carried out with diaminobenzidine	From 1 to 21 days after death	Positive immunoreaction to calcitonin up to 4 days after death. No reaction 13 days after death	Positive correlation between the post-mortem changes in the structure of calcitonin and its colourability over time: the denaturation of the protein makes it unsuitable for immunohistochemical staining However, the time limits resulting from the study may change in the event of a significant variation in the environmental conditions related to the discovery of the corpse
[25]	214 corpses Studied with external factors (temperature and humidity), environmental factors (closed, open, in water) and factors related to the body (clothes, body) differing in each case	Pancreas	Rabbit polyclonal antibody (DAKO Co) for glucagon. Anti-rabbit biotinylated secondary antibody F (ab0) 2. Detection of the avidin–biotin peroxidase complex by method	From 1 to 21 days after death	Positive immunoreaction to glucagon in all cases for up to 6 days. No immunoreactions were found in the cases analysed 14 days after death	Possible use of the immunohistochemical detection of glucagon at different time intervals to estimate PMI
[26]	105 corpses Cases with diabetes mellitus, thyroid disease, or gross pancreatic disease were excluded. External factors varied from case to case	Pancreas and thyroid	Antibodies against insulin, glucagon, thyroglobulin and calcitonin (Kit: universal LSABTM2 Kit/HRP, Rb/Mo, Dako K0675, antibodies: Polyclonal Guinea Pig Anti-Insulin, Dako A0564, Rabbit Monoclonal [EP3070] to Glucagon, Abcam ab92517. Kit: EnVisionTM + Dual Link System/ HRP, Dako K4061, antibodies: Polyclonal Rabbit Anti-Human Calcitonin, Dako A0576, Polyclonal Rabbit Anti-Human Thyroglobulin, Dako A0251. All kits were sourced from Dako North America Inc., Carpinteria, CA, USA. Immunohistochemical staining followed the protocols of Wehner et al	From a few hours to 22 days after death	In comparison with the studies by Wehner et al.: earlier negativization of immunoreactions for insulin and glucagon	Immunohistochemical detection of different antigens promising in forensic practice
[27]	3 corpses. Information was collected relating to age, height, weight, date and cause of death, time elapsed and environmental conditions of conservation of the body (refrigerated or not) from death to arrival at the facility. The bodies were placed, 2 in a prone position and 1 supine, undressed, outside in a dry and shaded wooded area with a cool to temperate climate (38–77°F)	Skin collected from 4 sites: scalp, sole of the foot, hypostatic region of the trunk and non-hypostatic region of the trunk	Histology: hematoxylin/eosin stain No immunohistochemical investigation	Up to 7 days (samples collected each 12 h in the first 2 days, then every 24 h for the following 4 days)	Macroscopically, no significant differences were highlighted. The three major histological changes were: focal dermal-epidermal separation, necrosis of the eccrine duct (appearing between 4 and 7 days), and degeneration of the dermis (in non-truncal skin biopsies on day 2)	Although the study presents the limitation of the small number of subjects studied and the presence of uncontrolled extrinsic factors, the histological results from selected skin biopsies can be useful in estimating early PMI (necrosis of the eccrine glands and dermal degeneration)
[28]	500 corpses with known times of death and different environmental conditions and causes of death. Cases with pancreatic or cerebral pathologies diagnosed in life were excluded	Pancreas Brain	Anti-somatostatin and anti-glial fibrillar acid protein antibodies (monoclonal mouse anti-human GFAP)	From 1 to 23 days after death	Somatostatin: positive staining within 2 days after death; negative immunoreaction after 11 days GFAP: colourable in the frontal cortex within 3 days of death; constant negative immunoreaction after 14 days	The accuracy, practicability, and cost-effectiveness of immunohistochemical methods for delimiting the time of death make them likely candidates for routine forensic practice, especially when longer time since death is suspected
[29]	10 corpses	Gingival tissues	Types I and III anti-collagen antibodies: Type 1 anti-human collagen antibody (Millipore, Merck S.p.A., Milan, Italy) and with the anti-human collagen type III antibody (Sigma-Aldrich, St. Louis, Missouri, USA). Subsequent incubation with secondary antibody conjugated with peroxidase for 1 h at room T (Histofine immunohistochemical staining kit, Nichirei Biosciences INC, Tokyo, Japan). Signal detection with DAB following the manufacturer’s protocol (Histofine Immunohistochemical staining kit, Nichirei Biosciences INC, Tokyo, Japan)	Samples collected at: 1–3 days, 4–6 days, 7–9 days after death	Gradual degradation of the extracellular matrix in suboral connective tissue. PMI was related to increased nuclear chromatin condensation and cytoplasmic vacuolization in both epithelial and connective tissues	Potential for morphological characteristics (histological and ultrastructural) and immunohistochemical markers (type I and II collagen) of gingival tissues to estimate the time of death more accurately
[30]	10 corpses	Gingival tissues: maxillary gingiva adjacent to the first incisors	Immunohistochemical distribution and mRNA expression of hypoxia inducible factor (HIF-1a) Primary antibody against HIF-1a (Invitrogen, Thermo Fisher Scientific, Monza, Italy) Secondary anti-rabbit antibody, followed by diaminobenzidine tetrachloride (DAB)	Different time frames studied in an overall PMI ranging from 1 to 10 days: 1–3 days, 4–5 days, 8–9 days	Time-dependent correlation of the HIF-1° protein and its mRNA with the different time intervals from death: 1–3 days: High signal of the HIF-1° protein mainly localized in the basal layer of the oral mucosa 4–5 days: the signal gradually decreases 8–9 days: the signal is not detected	Interesting potential utility of techniques based on immunohistochemistry as important complementary tool to be used in forensic investigations
[31]	Rats: 40 adult male albino rats (weight 230–260 g), sacrificed after 1 week of acclimatization in a cage with 50 mg/kg pentobarbital Man: 40 bodies from court cases with known times of death (within the first 24 h). 23 males and 17 females ranging in age from 18 to 50 years	HMGB1 in serum and full thickness skin	Biochemical, histological and immunohistochemical study on blood and skin Histology with hematoxylin–eosin Immunohistochemistry with HMGB1 rabbit polyclonal antibody (Abcam, Cambridge, MA, USA; dilution 1:200) and secondary antibody conjugated with peroxidase	Rats: samples at 0.3, 12, 24 h Corpses: I: 0 h II: < / = 3 h III: 4–6 h IV: 7–12 h V: 13–24 h	Significant increase as a function of time in the serum levels of HMGB1, together with its overexpression in the skin. In parallel, histological changes in the epidermis, dermis and hypodermis were analysed	HMGB1 could be a post-mortem marker to provide a precise and feasible method for estimating PMI. However, further studies are needed to analyse different time intervals and other physical factors that can influence post-mortal levels of HMGB1 in different tissues

[32]	42 adult albino rats weighing 200–220 g (12 weeks of age) were sacrificed by spinal dissection	Heart Kidneys Testicles	Gene expression analysis of MDA, SOD, GSH, HMGB1; immunohistochemistry and histopathology of bcl2	Samples at: 0, 12, 24, 48, 72, 96 and 120 h after death	PMI is correlated with different tissue levels of MDA, SOD, GSH. HMGB1 exhibited increased post-mortem gene expression, peaking at 48 h post death. Obvious time-dependent histopathological changes were observed in all organs until 5 days after death. Oxidizing agents and antioxidants are related to PMI up to 120 h after death. BCL2 began to decline at 24 h and became negative 96 h after death	Expression of the HMGB1 gene can be used for estimating the PMI, as it shows time-dependent changes in the form of a progressive increase. Oxidizing agents and antioxidants and the immunohistochemical expression of BCL2 show variations that can be correlated with the passage of time since death
[33]	24 adult albino rats weighing 150.55 ± 8.56 g were sacrificed by cervical dislocation	Skin Muscles	Histology: hematoxylin / eosin stain No immunohistochemical investigation	8 groups: 0.8, 16, 24, 32. 40, 48 and 72 h after death	Significant differences were found in epidermal nuclear chromatin. The highest intensity was detected at 40 h The nuclei of the sebaceous glands and the condensation of nuclear chromatin showed dramatic decrease Striation of subcutaneous muscles can be identified between 8 and 16 h after death, while initial skeletal muscle degradation processes begin at 16 h	Histopathological changes in the subcutaneous muscles can be considered a tool for the determination of the PMI. The histopathological changes in the skin are similar to previous studies but appeared later than the others. This diversity can be attributed to different environmental factors This study proposes the use of morphometric parameters of the skin and underlying muscles to evaluate the time of death (early and late), while also considering various environmental factors and different tissues
[34]	31 corpses	Gingival tissues	Histology exams No immunohistochemical investigation	3 subgroups: 0–8 h (n 10); 8–16 h (n 10); 16–24 h (n 11)	The initial epithelial changes were homogenization and eosinophilia, with cytoplasmic vacuolation, epithelial crushing, ballooning, loss of nuclei and suprabasilar cleavage in the late interval (16–24 h). Nuclear changes such as vacuolisation, anorexia, pyknosis and karyolysis became increasingly evident with the lengthening of PMI. Homogenization of collagen and vacuolation of fibroblasts also appeared. The onset of decomposition at the cellular level appeared within 24 h of death, and other features of decomposition were observed thereafter	The histological changes that occur post-mortem in human gingival tissues seem to be a useful method for estimating the time of death in the first PMI (0–24 h). However, further studies are needed to verify, refine and expand these initial findings. Despite some constraints, such as the limited number of subjects and the relatively short time period of the post-mortem period, the present study demonstrated the potential of this method as a tool to be used in forensic practice Further research with larger samples and an extended time frame might help forensic professionals in the estimation of a precise hour of death
[35]	24 samples from patients undergoing extraction of third molars for orthodontic reasons	Dental pulp	Histology: hematoxylin/eosin stain and tricromica di masson No immunohistochemical investigation	The 24 samples were divided into 4 experimental groups: 1: group 1 ◊ 6 dental pulps 24 h after extraction 2: group 2 ◊ 6 dental pulps 3 months after extraction 3: group 3 ◊ 6 dental pulps 3 months after extraction 4: group 4 ◊ 6 dental pulps 6 months after extraction	Histological transformation of the pulp as the PMI increased Qualitative analysis: at 24 h, all structures were recognizable. With the increase in PMI, characteristic morphological changes were observed for each time interval. The quantitative analysis showed a significant decrease in nuclei as the PMI increased in contrast to the percentage of collagen fibres, which increased in relation to the PMI (significant differences, especially between 24 h and 6 months and between 3 and 6 months). No significant differences at 1 month	The study proposes the use of dental pulp in the estimating of PMI, considering a longer PMI than in previous studies, of up to a PMI of 6 months The qualitative and quantitative results support the use of this methodology in the PMI estimation to be applicable to forensic cases. Further research should focus on the use of a higher number of samples and in different environmental conditions to allow for a wider application of the methodology and possibly to help narrow down PMI time estimates
[20]	32 corpses	Dental pulp	Histology: hematoxylin/eosin stain No immunohistochemical investigation. Count of odontoblasts	Extraction of 1 tooth with only one root every 24 h from the jaw. Stored in an open plastic bag for 5 days Cases divided into 2 groups: 15 at room temperature (23 °C) and 17 at refrigerated temperature (4 °C)	The number of odontoblasts drops with the passage of time since death. The average drop in odontoblast density is 130 per square millimetre per hour at room temperature, and 120 per square millimetre per hour in refrigerated conditions. The pulp is free of odontoblasts 5 days after death The study also showed that lower temperatures do not significantly slow the breakdown of odontoblasts	The number of odontoblasts and their histological appearance may be an additional parameter in the estimation of the time of death. More cases are needed to provide other exploratory possibilities, for example, how odontoblasts degenerate and degrade as a function of time
[21]	74 corpses (adults aged 20 to 97: 40 men and 34 women) Biopsies: 22 patients aged 7 to 90 years of both sexes who donated bone marrow or who underwent biopsies	Bone marrow (BM)	TRAP detection as a marker of osteoclasts by formalin histochemistry	PMI from 0 to 5 days in cases of pathological anatomy and from 2 to 42 days in forensic autopsies	Osteoclasts remain TRAP-positive for 7 days post-mortem, albeit with markedly reduced staining intensity compared to biopsies Since TRAP-SI has not been correlated with the duration of PMI (individual variability of TRAP positivity in osteoclasts present in vivo), TRAP staining of osteoclasts in BM cannot serve as a tool to determine the time of death of a patient	TRAP staining is an appropriate tool for detecting medullary osteoclasts in autopsy specimens However, due to the individual variability of TRAP positivity in osteoclasts, staining alone cannot serve as a tool for determining the time since death
[22]	Man, 29 cadavers (17 males, 12 females) with an average age of 74 at death (range 54–94). In all cases, the death was due to natural causes	Glands of skin on the ventral surface of the limbs	Analyses: 1. Cytochemistry with hematoxylin and eosin 2. Immunohistochemistry (S-100, CEA, Cytokeratin, ASM) 3. Ultrastructural electron microscope	Withdrawals 3, 6, 9 and 12 h after death	Electron microscopy identified specific changes for each chronological phase: reduction of intracellular glycogen in light cells and secretory granules in dark cells are signs of the first stage after death (3 h); mitochondrial dilatation and rarefaction of the crypts in light and dark cells at the second stage (6 h); and rarefaction of microvilli in light and dark cells at the last stage (12 h) Cytochemistry and immunohistochemistry provide useful information not for the whole chronological stage considered, but for single phases: 3 h for hematoxylin–eosin and 6 h for alcian-PAS	Potential use of optical, electron and immunohistochemical microscopy techniques in the determination of post-fatal time intervals, using the morphology of the eccrine sweat glands. The study proposes the combined use of these methods on a larger number of samples to deepen their use in forensic practice
[36]	Rats, 23 male mice (25–30 g), sacrificed with an intraperitoneal overdose of ketamine and xylazine	Skeletal and cardiac muscle	Histological study with hematoxylin–eosin and immunohistochemical for the research of caspase 9 and caspase 3 ◊ monoclonal rabbit anti-caspase 9 (Abcam, code ab222231) and monoclonal rabbit anti-caspase 3 (Abcam, code ab224271)	0, 4, 8, 12, 24 and 72 h after death 6 groups: -N. 4 a 0 h -N. 4 a 4 h -N. 4 a 8 h -N. 3 a 12 h -N 4 a 24 h -N 4 a 72 h	Skeletal muscle: Increase in the immunoreactivity of caspase 9 from 4 to 8 h, followed by stable levels, even if slightly decreased, for up to 24 h; significant reduction at 72 h. Immunoreaction for caspase 3 absent up to 4 h, the highest level at 8 and 12 h, and progressive decrease at 24–72 h The decrease of both at 24 and 72 h is in line with the appearance of increasing alterations in tissue morphology at hematoxylin and eosin. The apoptotic pathway was also confirmed: following initiator caspase 9 activation and then executing caspase 3 activation (statistically significant 4 h after death) Skeletal muscle matching matrix proteins degrade more consistently as PMI progresses than heart muscle	Usefulness of the combined immunohistochemical analysis of both caspases in the progression of time from death, in estimating the PMI
[26]	12 healthy horses	Brain, skeletal muscle, liver	Histology: hematoxylin/eosin stain No immunohistochemical investigation	0, 1, 2, 4, 6, 12, 24, 36, 48, 60 and 72 h after death The samples were stored at 22 °C and 8 °C	From 5 to 7 parameters associated with autolysis were identified for each tissue and quantified based on the percentage of the field involved under the microscope The changes were most noticeable in the liver and muscle tissues in the first 72 h post-mortem. The most evident characters at both temperatures in the first 72 h are: individualization of hepatocytes and separation of the bile duct epithelium from the basement membrane. In the interruption of the continuity of myofibers, hypereosinophilia and loss of striation The brain changes were highly variable	High statistical correlation between autolysis and the time/temperature variables, can be useful for establishing histological algorithms for identifying the PMI
[37]	30 corpses: 24 males and 6 females with ages between 15 and 64, died from various causes	Skin of the anterior thoracic region (epidermis and dermis with annexes)	Histology: hematoxylin/eosin stain No immunohistochemical investigation	3, 6, 9, 12, 15, 18, 21 and 27 h after death	The epidermis and dermis appeared normal for 6 h after death, and after this period the degenerative changes began 6 to 9 h after death, degeneration began in the dermis, and by the end of 18 h, the dermis began to disintegrate. The sweat glands appeared normal for about 3 to 4 h. For 18 h after death, the sebaceous glands and hair follicles appeared normal. After this period degeneration began	The histological changes found in the present study are similar to those described in previous literature. However, the changes seem to appear earlier due to environmental differences, which, as is well known, affect the decomposition. The analysis of the morphology and degeneration times of the sebaceous glands and hair follicles appeared useful in this PMI study. The parameters described can therefore represent a useful method for helping to estimate the time of death in the first post-mortem period
[31, 38]	8 cadavers aged between 22 and 33 years, including 2 women and 6 men, BMI 21.2–23.3	Full thickness skin with subcutis at sternum level	Histology: hematoxylin/eosin stain No immunohistochemical investigation	Autopsy performed in all cases within 4 h of death. A 10 cm × 2 cm piece of skin was taken from each corpse and refrigerated at 4–6 °C. Samples were taken from the piece at different PMIs: 4 h, 6 h, 12 h, 18 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, 6 d, 8 d, 10 d, 12 d, 16 d, 20 d, 24 d, 28 d and 32 d	The epithelial cell nucleus condensed within 24 h of death and cell lysis was exhausted after 20 days The changes in the dermis occurred later than those in the epidermis (72 h), but after the onset of the epidermal changes, the processes in the dermis were more rapid. At 16 d, the layers had homogenized. The epidermis and dermis had completely separated 24 days after death. The changes in the sweat glands appeared earlier (24 h) and disappeared later (32 days); the sebaceous glands and hair follicles began to undergo degenerative changes at 96 h after death, and at about 20 d, only their outline remained	Post-mortal histological changes in the skin occur at specific times. Therefore, they could be used to help deduce the time of death Comprehensive observation of changes in the composition/structure of the skin is important to fully analyse the possible times of death