Discussion

The spleen generally plays a limited role in forensic diagnostics, except in cases of blunt splenic trauma, treatment-related injury, anaphylaxis, drowning, and sepsis [10]. This study focused on the post-mortem tissue mechanical characteristics of the human spleen, with particular emphasis on changes related to the PMI as well as factors derived from police records and autopsy findings. The aim was to explore the applicability of spleen biomechanics in routine forensic investigations and to expand the evidentiary value that can be derived from this organ.

The loss modulus of splenic tissue is a useful PMI marker

Within the investigated PMI range of 42 to 518 h, the G’’ was the only one of the three measured rheological parameters that showed a significant change. From a tissue mechanical perspective, G’’ reflects processes associated with energy dissipation during deformation [14]. For example, post-mortem dehydration and fluid redistribution result in reduced viscous resistance to deformation and, consequently, a decrease in G’’. In contrast, the G’ reflects the ability of tissue to store energy [14]. Energy storage is predominantly related to tissue architecture, including collagen and elastin networks. The absence of significant changes in G’ values within the investigated PMI range indicates that there is no biomechanical evidence of autolytic degradation of the splenic extracellular matrix during this period.

Based on receiver operating characteristic (ROC) curve analysis, G’’ values above 333 Pa can be used as a confirmatory threshold for PMIs of less than 200 h at 4 °C. Given a positive likelihood ratio of 12.75, this threshold constitutes strong diagnostic evidence. Compared with previously reported data for brain tissue [5], G’’ analysis of splenic tissue stored at 4 °C demonstrated superior confirmatory power for the 200 h threshold. In contrast, rheological analysis of brain tissue within a PMI range of 42 to 341 h showed optimal performance at thresholds of 150 and 250 h. Taken together, these findings demonstrate for the first time how biomechanical analyses of different tissue types may complement each other within the framework of forensic tissue mechanics.

In addition to its role in time since death estimation, the PMI must be considered a relevant confounding factor. Samples exhibiting microscopic signs of inflammation showed significantly lower G’’ and G* values compared with samples without inflammatory changes. However, the significantly longer PMI observed in samples with microscopic inflammation indicates that the observed differences were likely confounded by PMI rather than being directly attributable to inflammation.

Beyond its utility for time since death estimation, the present study demonstrates that PMI is a key factor influencing the biomechanical properties of splenic tissue more than other case variables. Accordingly, post-mortem intervals until autopsy should be kept as short as possible to preserve the biomechanical behavior of splenic samples. Within the PMI range of 42 to 518 h analyzed in this study, a marked decrease in G’’ values was observed beyond approximately 200 h post-mortem. Notably, the earliest investigated post-mortem time point was 42 h. In principle, biomechanical changes may begin immediately after death and progress during the first 42 h prior to the earliest data acquisition in this study. To date, no published biomechanical investigations have demonstrated significant changes in splenic rheometric properties within the first 42 h post-mortem at 4 °C. Kemper et al. reported that human splenic samples were tested within 48 h post-mortem to minimize adverse effects of tissue degradation; however, no correlation between biomechanical properties and PMI was performed [12]. Stingl et al. investigated critical tension and elastic modulus in 21 spleens with PMIs ranging from 24 to 36 h [13]. Due to the limited number of successfully tested samples, no statistical analysis was performed, and the relationship between biomechanical properties and PMI was not further addressed. In contrast, Taskent et al. investigated splenic stiffness in male Sprague-Dawley rats up to 36 h post-mortem at approximately 23 °C using shear-wave elastography and observed a progressive decline in stiffness over the analyzed PMI range [15]. At ambient temperatures of around 20 °C, it is anticipated that the PMI-related changes in G’’ detected here would occur sooner after death, as rheometric properties are known to be temperature-sensitive [9].

Several key parameters are unlikely to confound PMI-related rheological changes

Based on the results of this study, there is no evidence that the G’, G’’, or G* of the spleen depend on age at death, spleen weight, sex, cardiac resuscitation attempts, and microscopic organ features. Moreover, no significant difference was detected for microscopically congested and uncongested samples. One clear limitation of splenic tissue analysis is that samples could be prepared from only approximately 60% of spleens using the punching method applied in this study. This likely reflects advanced autolytic degradation or enzymatic destruction of splenic pulp leading to insufficient structural integrity for cylindrical sampling. In comparison, 95% of brain samples [5] and all liver samples [6] obtained at autopsy could be prepared and subsequently subjected to biomechanical testing. In practical terms, failure to prepare a splenic sample meant that it was not possible to punch a specimen that sufficiently retained its shape for loading into the testing apparatus using a biopsy punch. In routine practice, the number of available spleen samples may be further reduced because some individuals have previously undergone splenectomy, as the spleen - unlike the brain or liver - is a non-vital organ. In cases of splenic injury, it may nevertheless still be possible to punch a sample from the remaining (and ‘intact’) tissue, as the required test specimen is small relative to the size of the entire organ. The reasons why a substantial proportion of splenic samples could not be successfully punched are beyond the scope of this manuscript. However, no significant differences were detected between unsuccessful punching attempts and successfully tested samples with respect to age at death, spleen weight, or PMI. The number of unsuccessful punching attempts may be reduced in samples with PMIs shorter than 42 h, which represents the earliest time point investigated in this study.

Splenic tissue mechanics in literature and its value beyond forensic practice

Beyond forensic applications, there is a need for comprehensive characterization of the mechanical properties of splenic tissue for both basic research and clinical practice. High-quality biomechanical data on the spleen are essential for finite element modelling of blunt force injuries [16] and for the development of safety gear [17]. To date, post-mortem biomechanical properties of the spleen have been investigated predominantly in animal models [18–23], with only a few studies employing human tissue samples [12, 13].

In addition, biomechanical properties of the spleen are required for the implementation and refinement of haptic feedback for internal organs in surgical training simulators [24]. Beyond modelling and simulation, non-invasive measurements of splenic stiffness appear to be superior to liver stiffness measurements for monitoring portal hypertension in patients with advanced chronic liver disease [25, 26] and may serve as a predictor of survival following portosystemic shunt implantation [27].

The generally comparable viscoelastic properties suggest that, despite age-related differences in tissue composition and size, the mechanical response of pediatric spleen tissue falls within the range observed in adult samples. However, due to the small number of pediatric cases, no statistical testing was performed, and the observations are introduced as exploratory.

Notably, the spleens investigated in the present study were retrieved during autopsy and tested within two hours thereafter. This approach reflects the most common scenario in routine forensic practice, in which a body is discovered intact with an unknown time of death. From a practical standpoint, the scenario of an isolated human spleen being recovered after removal by a perpetrator remains largely theoretical. In routine casework, isolated internal organs presented for forensic evaluation are typically slaughter by-products submitted by laypersons for clarification of potential human origin, most frequently involving the heart.

Future research

In this study, human spleen samples with a mean PMI of approximately nine days stored at 4 °C were analyzed. The applied method was shown to be temperature sensitive in the animal model [9]. Hence, future investigations should investigate other forensically relevant storage temperatures including around 20 °C (room temperature) and 37 °C (body temperature). Moreover, rheometric data should be collected immediately post-mortem to gain “point zero” reference values and establish a baseline for the post-mortem changes.