Estimating time of death from measurement of the electrical excitability of skeletal muscle.

Post-mortem contraction was measured using sensitive force transducers inserted into human muscle. The muscle was stimulated using rectangular impulses of 1 sec duration and known current intensity. After some time, a noticeable change of the muscular contraction occurred, from a two-peak to a one-peak shape. The maximum force of the muscular contraction using a stable current intensity decreased with time since death, but relaxation time increased. The decrease of the maximum force and the increase of the relaxation time were used as criteria for extrapolating the time since death in a random sample of 50 bodies. The calculated 95% limits of confidence were 2.85 h (decrease of the maximum force) and 2.7 h (increase of relaxation time) up to 13 hours post mortem. The calculated 95% limits of confidence were checked on an independent sample of 21 bodies and proved to be reliable.     

Electrical excitability of skeletal muscle postmortem in casework

The paper gives a review of the development of methods of postmortem electrical excitability of skeletal muscle for determining the time since death. For practical application in casework, the following method can be proposed: position of electrodes in the orbicularis oculi muscle, stimulation by rectangular impulses of 10 ms duration, 30 mA in a repetition rate of 50/s. The muscular reaction on excitation is graded in 6 degrees according to the spread of movement and the intensity of contraction. To each degree a time information is corresponding (95%-limits of confidence). These 95%-limits of confidence were proved to be valid for cases of sudden natural or traumatic deaths. Exclusions: deaths after chronic lingering disease (shorter times), fatal hypothermia, haematomas, emphysemas of the eyelid (longer times). The practical application in casework together with the temperature method (Henssge 1988, 1) is described. Applying both methods the death time estimation in the early postmortem interval may be much more precise and accurate than using one method alone.     

Estimation of the time since death

The main principle of the determination of the time since death is the calculation of a measurable date along a time-dependent curve back to the start point. Characteristics of the curve (e.g. the slope) and the start point are influenced by internal and external, antemortem and postmortem conditions. These influencing factors have to be taken into consideration quantitatively in order to improve the precision of death time estimation. It does not make any sense to study the postmortem time course of any analyte without considering influencing factors and giving statistical parameters of the variability. Comparison of different methods requires an investigation of the same postmortem interval. For practical purposes, it must be concluded that the amount of literature on estimating the time since death has a reverse correlation with its importance in practice.     

Light microscopical investigations on structural changes of skeletal muscle as artifacts after postmortem stimulation

Samples of skeletal muscle were taken from 20 human corpses where mechanical or electrical stimulation had been carried out up to 8h postmortem (hpm) in order to estimate the time since death. The stimulation had caused an idiomuscular bulge or tetanic contraction of the muscle tissue at this location. The muscle samples were examined for structural changes of the fibers by light microscopy. A comparison with control muscle samples taken contralaterally from the same corpse, showed that the findings previously interpreted as being of intravital origin, e.g. destruction of fiber integrity, invagination and contraction bands, could also be due to postmortem alterations. It is hypothesized that structural changes to the muscle fibers can, in general, be produced as long as the manifestation time is shorter than the supravital phase after the time of force impact.     

Linearization of the relationship between postmortem plasma chloride concentration and postmortem interval in rats

A potentiometric titration procedure was employed for measurement of plasma chloride concentrations during the 0-96 h postmortem period in rats. The data revealed antemortem absolute values and postmortem rate of decrease in plasma chloride concentration (PCl) which were almost identical to those in dogs and in man. Expression of the data in the form of a double logarithmic plot of PCl versus postmortem interval (PMI) yielded a linear relationship of high correlation (r = -0.97; P less than 0.001). Attention is drawn to the possibility of utilizing postmortem rate of change in plasma chloride concentration as a means of estimating time elapsed since death.     

Interleukin-6 and C-reactive protein serum levels in sepsis-related fatalities during the early postmortem period

Postmortem interleukin-6 (IL-6) and C-reactive protein (CRP) serum levels were investigated prospectively in sepsis-related fatalities and non-septic fatalities by using a linear regression model. At least three blood samples were collected between 0.3 and 139 h postmortem from sepsis-related fatalities (n=8) and non-septic fatalities (n=16). In addition, one antemortem blood sample was collected shortly before death from the septic patients. Antemortem and postmortem IL-6 and CRP levels were highly elevated in all individuals included in the sepsis group. An excessive postmortem increase of IL-6 serum levels associated with progressive time after death was observed in five out of the eight septic patients. Both, IL-6 and CRP serum concentrations seem to be suitable biochemical postmortem markers of sepsis. The determination of IL-6 serum levels above 1500 pg/ml in peripheral venous blood obtained in the early postmortem interval can be considered as a diagnostic hint towards an underlying septic condition. A more precise postmortem discrimination between sepsis and non-septic underlying causes of death is provided by the postmortem measurement of serum CRP in peripheral venous blood: on condition that at least two postmortem CRP values have been determined at different time points postmortem, the CRP level of a deceased at the time of death can be calculated by using linear regression analysis. When assessing postmortem IL-6 and CRP concentrations as biochemical postmortem markers of sepsis, various clinical conditions, such as a preceding trauma or burn injury going along with elevated IL-6 and/or CRP levels prior to death as a result of the systemic inflammatory response syndrome (SIRS) should be taken into consideration, thus adding relevant information for the practical interpretation of the results.     

Supravital electrical excitability of muscles

A new method of postmortem electrical stimulation of muscles, measuring the force of the reaction against time, is presented. Preliminary investigations have yielded the following results: The maximum force of reaction in response to a definitive stimulation, measured by a sensitive force transducer, decreases in proportion to the post mortem interval. Using this method, the mechanical reactions of muscles by electrical stimulation can be demonstrated over a much longer postmortem period as these movements are visible. The duration of postmortem reaction of different muscles seems to be much more uniform than was previously assumed. Occasionally, reactions of the M. orbicularis oculi can be seen for the longest time. The basal muscular tonus changes relative to time. The time course of a single force reaction also changes relative to the postmortal interval: the reaction becomes slower. It has been theoretically established that measurement of influencing factors such as temperature and pH in the tested muscle will enable the time of death to be more precisely estimated.     

Amount of postmortem bleeding: an experimental autopsy study

An experimental autopsy study was performed on 64 cases (55 male, 9 female; average age 51.5 +/- 16.2 years) of sudden natural (38 cases) and asphyxic deaths (26 cases). The study objective was the amount of postmortem bleeding from postmortem cutting of the thoracic aorta, related to the time since death. The amount of postmortem bleeding ranged from 100 to 1300 cm, 440.6 +/- 268.1 cm on average. The time since death up to the autopsy time ranged from 4 to 72 hours, 19.4 +/- 12.9 in average. A statistically significant correlation between the amount of postmortem bleeding and postmortem time interval was stated: Pearson correlation test value r = -0.461 (P = 0.000): the shorter the time interval, the larger the amount of bleeding. The formula of linear regression was estimated according to this correlation: amount of postmortem bleeding (cm) = -9.571 x time since death (h) + 626.659. This proves that the amount of postmortem bleeding (eg, from aortic blunt rupture) could be about 620 cm.     

家兔死后的角膜厚度变化及其与死亡时间的关系

目的探讨家兔死后不同时间角膜厚度变化及其与死亡时间的关系。方法将25只家兔采用空气栓塞的方法处死后,用角膜超声测厚仪分别于死后即刻、2h、4h、8h、12h和24h测量角膜的厚度,所得数据进行统计学处理。结果即刻死亡家兔角膜厚度平均值为（357.10±6.41）μm,死亡24h角膜厚度平均增加（187.52±11.38）μm;4h内,眼睑开启与闭合对角膜厚度的增加无明显影响（F=3.5290,P〉0.05）,8h后随时间延长,眼睑闭合角膜厚度为（429.24±8.80）μm,明显超过眼睑开启角膜平均厚度（421.40±9.53）μm,其差异有显著性意义（F=14.4480,P〈0.05）。眼睑闭合的角膜厚度与死亡时间关系为y=8.2418x＋357.33（相关系数r1=0.8732）;眼睑开启的角膜厚度与死亡时间关系为y=7.3737x＋357.23（r2=0.8578）。结论在一定条件下,家兔死后24h内角膜厚度与死亡时间呈线性相关。     

Practical experiences with the determination of cadaver age by evaluation of bacterial metabolic products

In an experiment a corpse had been kept at room temperature (16 degrees-23 degrees C) for 1163 h. At regular intervals brain samples were taken and the content of free amino acids and related compounds was determined by column chromatography. It could be demonstrated that in a period of 4-20 days postmortem the age of the corpse could be calculated from the concentrations of alpha-aminobutyrate (ABU), gamma-aminobutyrate (GABA), and glutamic acid (GLU) in brain employing the previously [5] presented formula: (formula; see text) T is the postmortem time lapse (days). ABU, GABA, and GLU are the concentrations (mumol/g) wet tissue of the corresponding amino acids. During this postmortem interval there is nearly a linear correlation; from this interval a correct assignment between concentrations and time cannot be given. The determination method is in the range of about 15 degrees-25 degrees C (guarantees bacterial growth and metabolism) independent of ambient temperature. The brain samples (cortex or parts of the putrified brain mush) can be taken without any special precaution during normal autopsy. The results of the experiment (in Fig. 1) were supported by some practical cases where the time lapse since death was well (in Fig. 1) or reasonably (in Fig. 1) known. These results suggest that this method allows in many cases the determination of the age of a corpse found in a warm environment (approximately 15 degrees-25 degrees C) approximately 4-20 days after death.     

Postmortem investigation of serum myoglobin levels with special reference to electrical fatalities

The serum myoglobin levels of 58 fatalities were investigated with special reference to correlations of the myoglobin-concentrations with the postmortem interval, the site of blood sampling and the cause of death (especially concerning death caused by electric current). An increase of the myoglobin values with the passage of postmortem time is obvious. The topographical sampling site plays an important role; the highest concentrations are found adjacent to striated muscles (i.e. in blood from the heart or the femoral vein). There was no significant correlation of myoglobin values and death by electric current.     

The changes of fourier transform infrared spectrum in rat brain

Estimation of the time since death (postmortem interval [PMI]) is one of the most difficult problems in forensic investigations, and many methods currently are utilized to estimate the PMI. The goal of this study was to investigate the changes of attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra of rat brain from postmortem time 0-144 h. The intensity ratios of major absorbance bands were examined (I(1066)/I(1392), I(1168)/I(1392), I(1234)/I(1454), I(1301)/I(1392), I(1647)/I(2956), I(2921)/I(2850), and I(1647)/I(1539)). The spectra of rat brain displayed prominent changes with increasing PMI. The band at 2871 and 1737 per cm became weak with the time increasing and even disappeared at postmortem 96 and 72 h, respectively. A close linear correlation was shown between the relative absorption intensity and the PMI, and the I(1234) /I(1454) offered a stronger correlation (r = 0.973). Our results indicate that ATR-FTIR spectroscopy may be a useful technique for estimating the PMI.     

Estimation of postmortem interval using the protein marker cardiac Troponin I

The importance of determining the time since death is crucial to criminal, civil and forensic cases. A technique exploiting the degradation of a protein, cardiac Troponin I (cTnI), to estimate the postmortem interval (PMI) was investigated. Cardiac Troponin I is a basic regulatory protein found as part of a ternary complex responsible for calcium dependent muscle contraction. An efficient extraction protocol to analyze the banding pattern of cTnI in postmortem tissue was developed. The analysis involves extraction of the protein, separation by denaturing gel electrophoresis (SDS-PAGE) and visualization by Western blot using cTnI specific monoclonal antibodies. A bovine model was used to develop and optimize the protocol. The homology of cTnI amongst mammalian species allows for the cross-reaction of human anti-cTnI antibodies with bovine cTnI. The results indicate a characteristic banding pattern amongst human cadavers (n=6), a pseudo-linear relationship between percent cTnI degraded and the log of the time since death (r>0.95), and a qualitative degradation band pattern that in a simple comparative analysis with a standard human heart (known time since death) can be used to estimate the postmortem interval. The degradation-banding pattern of tissue cTnI is useful in the determination of the early postmortem (pm) interval (0-5 days). Overall, this technique offers advantages such a wide postmortem interval, measurable degradation pattern, a temporal semiquantitative relationship and manageable temperature dependence over direct temperature methods.     

Cathepsin activity in the myocardium and skeletal muscles as a sign of the time of death

Determination of postmortem interval according to the time course of cathepsins activity within 5 days since death was carried out using myocardium and skeletal muscles of white breedless rats and cadavers of people who died of craniocerebral trauma. Natural time course of changes in cathepsins activity was determined which makes it possible to use these values as one of criteria in determination of postmortem interval.     

Microscopical study on estimation of time since death in skeletal remains

For the purpose of estimating time since death in skeletal remains, postmortem changes in human compact bones were examined by microradiography and electron microscopy. The UV-fluorescence of the peripheral zone of compact bone was also examined by microscopic spectrophotometry. Microradiographic examination revealed no morphological changes in bones left in the open air for long periods, except one of 15 years since death. In bones left in the soil, vacuoles of 5-10 microns diameter, which contained a honeycomb-like structure formed by small vacuoles of 0.5-1 microns diameter, were found in the peripheral zone of the substantia compacta approximately 5 years since death, and in bones of 6 years or more, this change extended to the mid-zone. In bones left in the sea for 4-5 years, vacuoles of 5-10 microns diameter were observed in the outer peripheral zone of the substantia compacta. The relative intensity of UV-fluorescence in bones dwindled with time since death and the correlation coefficient was considerably high.     

尸体胸骨骨髓细胞核DNA含量变化及其与死后经过时间的关系

目的探讨推断腐败尸体死亡时间的新方法。方法应用计算机图像分析技术,对以改良的Feu lgen法染色,死后经过不同时间(<1~14d)的胸骨骨髓细胞核DNA含量进行观测,并对所得数据进行相关分析。结果胸骨骨髓细胞核DNA含量随死亡时间延长逐渐下降,死后2周DNA量达最低值;死后DNA降解速率与死亡时间呈直线相关。结论测定胸骨骨髓细胞核DNA含量有助于腐败尸体的死亡时间推断。     

Postmortem biochemical examination of synovial fluid--a preliminary study

Vitreous humor chemistry is used for postmortem analysis since serum values of many components are thought to be reflected in vitreous humor and to be stable for a prolonged postmortem interval (pmi). A similar isolated compartment to vitreous humor is synovial fluid which up to now was hardly used for postmortem chemistry.The aim of the present examination was to compare the values of various analytes in both fluid compartments to get first hints for the reliability of the examination of synovial fluid. Therefore, in 74 cases of sudden death both fluids were taken and analysed for natrium, potassium, calcium, chloride, urea, creatinine, and glucose. The results show that the examination of synovial fluid as compared to vitreous humor is a reliable method to get hints on the premortal metabolic status since most analytes are stable postmortem. The range of all values is comparable in both fluids. The time course of glucose concentrations and, much more important, potassium concentrations is nearly similar. If no vitreous humor is available synovial fluid may be used with all precautions in diagnosis known from vitreous humor.     

Postmortem interval estimation by creatinine levels in human psoas muscle.

A new method was tested for estimating time after death in the middle range of postmortem interval (weeks). Creatinine muscular concentration in human cadaver is positively correlated to postmortem estimation. Temperature should be mainly related to the creatinine transformation rate. A stronger correlation was found at 11 degrees C up to 30 days and at 20 degrees Celsius up to 15 days.     

Preliminary quantitative investigation of postmortem adipocere formation

The accurate determination of postmortem interval (PMI) using the formation of adipocere presents a significant challenge to forensic scientists interested in determining the time of death. Several attempts have been made to determine the time since the occurrence of death. However, up to date, this has been difficult because previous approaches have been mainly qualitative, focusing on the later stages of degradation processes. This work presents preliminary results of an experimental model of postmortem adipocere formation using liquid chromatography. Three pig cadavers were submerged in distilled water, chlorinated water, and saline water. Fresh specimens resulting from the degradation in the subcutaneous fat were obtained from the pigs at two-week intervals for a period of ten weeks, and were subjected to chromatographic analysis. By correlating the ratio of the disappearance of hydrolyzed fatty acids with the formation of hydroxystearic and oxostearic acids after death, a simple, quantitative analytical method was developed for the determination of PMI. Experimental observation of the chemistry of adipocere formation indicated that adipocere can be formed only a few hours after an incidence of death and this continues until the saturation of oleic acid degradation after several weeks. Different time courses were obtained for cadavers immersed in distilled, chlorinated, and saline water, respectively. This work has not in any way solved the time since death problem. But it may be an approach to the problem that has not been adequately explored.     

Experimental studies of the development of linear electric current marks

By means of reconstructing electric current pathways electrophysical conditions were explored causing electrocution death in the bathtub, according to many authors quite often indicated by linear electric lesions. In our experimental settings we observed appearance of circumscribed linear marks of pallor similar to electric lesions in the region of postmortem lividity of corpses at the same level as bathtub water. None of typical histological indicators could be seen in skin samples. So no evidence was given for electric burns even under conditions of high density and extended duration of current. Similar linear marks and equivalent histological results were found when corpses were put into the bathtub filled with cold water without any experimental electric contact. CONCLUSIONS: Linear marks of pallor may occur at water level even without any electric contact to corpse or water neither bathtub. IMPLICATIONS: According to specific conditions mentioned above linear so called electric lesions at water level do not indicate electric current yet lethal electric damage.