A Study on the Estimation of Postmortem Interval Based on Environmental Temperature and Concentrations of Substance in Vitreous Humor

A method to determine postmortem interval (PMI) based on environmental temperature and the concentrations of vitreous humor (VH) molecules were explored. Rabbit carcasses were placed in a chamber at 5, 15, 25, or 35°C, and 80–100 μL of VH was collected with the double‐eye alternating micro‐sampling method every 12 h. A Roche DPPI biochemical analyzer was used to measure the concentrations of six substances in VH samples. The interpolation function model and mixed‐effect model were employed for data fitting to establish equations for PMI estimation. The concentrations of K⁺, P, Mg²⁺, creatinine (CRE), and urea nitrogen (UN) exhibited an upward trend with increasing PMI in all temperature groups, while the concentration of Ca²⁺ showed a downward trend. Validation results using K⁺ and Mg²⁺ ions revealed that the mixed‐effect model provided a better estimation than the interpolation function model using the data from our experiment. However, both models were able to estimate PMI using temperature and VH molecule concentrations.     

Determination of potassium/sodium ratio in heart tissue. Evaluation of its use as an index of myocardial ischaemic damage. Comparison with the nitro-BT test

The succinate dehydrogenase method of staining and the determination of the K⁺/Na⁺ ratio of heart tissue have been applied to forensic material as a tool for demonstrating early signs of myocardial infarctions. Both methods are controlled by a series of forensic cases dying from causes other than arteriosclerosis of the coronary arteries. Based on an analysis of normal material some factors influencing the K⁺/Na⁺ ratio are defined. It is concluded that the determination of the K⁺/Na⁺ ratio shows more infarcts than the formazan method; however, the evaluation of the K⁺/Na⁺ ratio is difficult. The K⁺/Na⁺ determination is not recommended for routine use. A possible indication for the use of the K⁺/Na⁺ determination together with the formazan test could be cases of instantaneous death by accidents suspected to have been provoked by a fatal heart attack.     

The Role of Vitreous Magnesium Quantification in Estimating the Postmortem Interval

The use of magnesium as a parameter to estimate the time of death is controversial. In 32 traumatic deaths with known postmortem intervals (PMIs), small quantities of vitreous humor (VH) were sampled repetitively every 3 h until 24 h postmortem. The bodies were kept at the constant ambient temperature of 20°C (68°F). The concentrations of magnesium were in the range of 0.47–1.15 mM. A statistically significant correlation of the concentration of magnesium with the PMI was observed (r = 0.453, p < 0.01), but with small predictive value—coefficient of variation for regression was 45.5%; the average of the paired differences between the estimated and actual PMIs was 5.24 + 3.19 h. Although useful results might be expected due to the large transmembrane gradient for magnesium, the results of this study strongly disprove the usefulness of measuring magnesium in VH to estimate the time of death.     

The Application of Flow Cytometry as a Rapid and Sensitive Screening Method to Detect Contamination of Vitreous Humor Samples and Avoid Miscalculation of the Postmortem Interval

Research into maximizing the speed, precision, and reliability of estimating the postmortem interval (PMI) has been a recurring object of investigation and methodologies based on the vitreous humor (VH) have provided good results. However, contamination from causes not readily apparent, such as blood, can occur, and thus lead not only to an erroneous estimation of PMI, but also interfere with the correct identification of other substances in the VH. We have developed a flow cytometry method which quantifies blood contamination and is able to detect erythrocytes in 1:750,000 dilution of contaminated VH which affects the results of hypoxanthine. It is an improvement on the previous more complex mass spectrometry method, being faster, more sensitive, and readily available. As such, it could be proposed for the rapid screening of appropriate samples by detecting and eliminating blood contaminated samples from PMI estimation.     

Corneal‐Smart Phone: A novel method to intelligently estimate postmortem interval

The changes of postmortem corneal opacity are often used to roughly estimate the postmortem interval (PMI) in forensic practice. The difficulty associated with this time estimate is the lack of objective means to rapidly quantify postmortem corneal changes in crime scenes. This study constructed a data analysis model of PMI estimation and implemented an intelligent analysis system for examining the sequential changes of postmortem corneal digital images, named Corneal‐Smart Phone, which can be used to quickly estimate PMI. The smart phone was used in combination with an attachment device that provided a darkroom environment and a steady light source to capture postmortem corneal images. By segmenting the corneal pupil region images, six color features, Red (R), Green (G), Blue (B), Hue (H), Saturation (S), Brightness (V) and four texture features Contrast (CON), Correlation (COR), Angular Second Moment (ASM), and Homogeneity (HOM), were extracted and correlated with PMI model. The results indicated that CON had the highest correlation with PMI (R² = 0.983). No intra/intersubject variation in CON values were observed (p > 0.05). With the increase in ambient temperature or the decrease in humidity, the CON values were increased. PMI prediction error was <3 h within 36 h postmortem and extended to about 6–8 h after 36 h postmortem. The correct classification rate of the blind test samples was 82%. Our study provides a method that combines postmortem corneal image acquisition and digital image analysis to enable users to quickly obtain PMI estimation.     

Analysis of Δ-tetrahydrocannabinol in oral fluid samples using solid-phase extraction and high-performance liquid chromatography–electrospray ionization mass spectrometry

An analytical method using solid-phase extraction (SPE) and high-performance liquid chromatography–mass spectrometry (LC–MS) has been developed and validated for the confirmation of Δ⁹-tetrahydrocannabinol (THC) in oral fluid samples. Oral fluid was extracted using Bond Elut LRC-Certify solid-phase extraction columns (10 cm³, 300 mg) and elution performed with n-hexane/ethyl acetate. Quantitation made use of the selected ion-recording mode (SIR) using the most abundant characteristic ion [THC + H⁺], m/z 315.31 and the fragment ion, m/z 193.13 for confirmation, and m/z 318.00 for the protonated internal standard, [d₃-THC + H⁺]. The method proved to be precise for THC, in terms of both intra-day and inter-day analyses, with coefficients of variation less than 10%, and the calculated extraction efficiencies for THC ranged from 76 to 83%. Calibration standards spiked with THC between 2 and 100 ng/mL showed a linear relationship (r² = 0.999). The method presented was applied to the oral fluid samples taken from the volunteers during the largest music event in Portugal, named Rock in Rio-Lisboa. Oral fluid was collected from 40 persons by expectoration and with Salivette^®. In 55% of the samples obtained by expectorating, THC was detected with concentration ranges from 1033 to 6552 ng/mL and in 45% of cases THC was detected at concentrations between 51 and 937 ng/mL. However, using Salivette^® collection, 26 of the 40 cases had an undetectable THC.     

Clarifying the complex chemistry of cobalt(II) thiocyanate-based tests for cocaine using single-crystal X-ray diffraction and spectroscopic techniques

Cobalt(II) thiocyanate-based tests are routinely used to screen cocaine products, with the formation of a blue species interpreted as a positive response. An array of other organic bases has been identified as false positives – including well-documented cocaine product adulterant lidocaine and its salt. False positives prompt continued test development, though improvements are hindered by unresolved product structures and reaction pathways. Toward greater clarity, cobalt(II) thiocyanate reactions with cocaine hydrochloride, along with lidocaine and its salt, were investigated using multiple analytical techniques. Reactions involving cocaine hydrochloride yielded glassy, amorphous blue material while reactions of lidocaine hydrochloride monohydrate produced larger, needle-like crystals whose structure was determined via single-crystal X-ray diffraction to be an ion pair (Hlidocaine⁺)₂([Co(SCN)₄]²⁻)·H₂O. While the blue precipitate isolated from reactions involving cocaine hydrochloride was unsuitable for crystallographic structure determination, comparative ultraviolet–visible, attenuated total reflectance infrared, and Raman spectroscopic analysis – along with elemental analysis – supports that this solid is comprised of a comparable ion pair (Hcocaine⁺)₂[Co(SCN)₄]²⁻. Pink crystals isolated from lidocaine reaction vessels were identified as coordination compounds cis-[CoL₂(SCN)₂] and trans-[CoL₂(SCN)₂] where L = lidocaine, while pink crystals from both cocaine hydrochloride and lidocaine hydrochloride monohydrate reaction vessels were the coordination polymer trans-[Co(H₂O)₂(SCN)₂]·H₂O. The results presented herein enable reaction optimization to favor a desired product, whether ion pair or coordination species.     

Improved estimation of postmortem interval based on differential behaviour of vitreous potassium and hypoxantine in death by hanging

Many formulae are available to estimate the relation between the potassium ([K+]) and hypoxantine ([Hx]) concentration in the vitreous humour and the postmortem interval (PMI). Typically these have been based on a correlation test and linear regression using the postmortal interval as the independent variable and [K+] or [Hx] as the dependent variable in order to estimate the confidence interval. However, a recent study has shown that a more precise measurement of PMI can be obtained if [K+] is used as the independent variable. The regression lines obtained from the most recent deceased subjects with forensic relevance received for autopsy in the Institute of Legal Medicine are [K+] = 5.589 + 0.174PMI and [Hx] = 26.459 + 3.017PMI, by changing the variables, we obtain PMI=3.967[K+] - 19.186 (R2 = 0.688, P < 0.001) and PMI = 0.172 [Hx] + 0.170 (R2 = 0.518, P < 0.001). In this paper we propose the cause of death as an extra factor which modifies the relationship and gives even greater precision in estimating PMI. In cases of death by hanging the results are considerably improved with [K+] = 5.224 + 0.225PMI and [Hx] = 15.161+4.957PMI, respectively, and consequently, PMI = 3.631[K+] - 17.334 (R2 = 0.818, P< 0.001) and PMI = 0.153[Hx] - 0.368 (R2 = 0.757, P < 0.001): the slope is less and the precision is obviously enhanced.     

外温变化下小鼠肝细胞18s rRNA降解与死亡时间关系研究

目的探讨在外界环境温度变化条件下,小鼠肝细胞18s rRNA降解与死亡时间的关系。方法小鼠断颈处死后置于10℃、15℃、20℃、25℃、和30℃温度下保存;从死后6h至72h,每6h分别提取肝细胞组织中总RNA。利用实时荧光定量PCR检测18s rRNA循环阈值(Ct值)的变化。应用插值函数进行分析拟合,建立死亡时间推断方程。结果各温度组Ct值随死亡时间延长均呈上升趋势,对所得数据进行插值拟合,得到一定温度变化区间内(10℃~30℃),Ct值变化与PMI关系的三元五次曲面方程:f(x,y)=-426.9+30.82x+44.48y-1.297x~2-1.837xy-1.388y~2+0.034 38x~3+0.038 17x~2y+0.038 67xy~2+0.028 77y3-0.000 612 9x~4-3.897e~(-7)x~3y-0.001 223x~2y~2+0.000256 6xy3-0.000 537 4y~4+3.606e~(-6)x~5-2.846e~(-6)x~4y+1.009e~(-5)x~3y~2-3.439e~(-6)x~2y3-2.556e~(-7)xy~4+2.664e~(-6)y~5(r2=0.999 4)。结论在外界温度变化条件下,小鼠肝细胞18s rRNA降解与死亡时间关系符合三元五次方程分布,利用插值函数拟合的方法可在外界环境温度变化条件下进行死亡时间推断。     

Application of MALDI‐TOF MS for Estimating the Postmortem Interval in Rat Muscle Samples

Estimating the postmortem interval (PMI) is very important in the forensic sciences. Although many approaches have been used for estimating the PMI, accurate PMI calculations are still difficult. In this study, four Sprague Dawley (SD) rats were sacrificed by suffocation, and muscle samples were collected by dissection at various time intervals (0, 48, 96, and 144 h) after death. All samples were probed using matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) to obtain molecular images and data for principal component analysis (PCA). The results showed that the peaks at m/z 1511, 1543, 1564, 1586 clearly decreased in intensity from 0 to 144 h postmortem and that the time groups were separated from each other on the PCA score plot. The prediction model showed high recognition capability (95.93%) and cross‐validation (83.72%). Our work suggests that MALDI‐TOF MS can be used to determine the PMI.     

Analytical Characterization of Erythritol Tetranitrate,an Improvised Explosive

Erythritol tetranitrate (ETN), an ester of nitric acid and erythritol, is a solid crystalline explosive with high explosive performance. Although it has never been used in any industrial or military application, it has become one of the most prepared and misused improvise explosives. In this study, several analytical techniques were explored to facilitate analysis in forensic laboratories. FTIR and Raman spectrometry measurements expand existing data and bring more detailed assignment of bands through the parallel study of erythritol [¹⁵N₄] tetranitrate. In the case of powder diffraction, recently published data were verified, and ¹H, ¹³C, and ¹⁵N NMR spectra are discussed in detail. The technique of electrospray ionization tandem mass spectrometry was successfully used for the analysis of ETN. Described methods allow fast, versatile, and reliable detection or analysis of samples containing erythritol tetranitrate in forensic laboratories.     

不同温度下兔玻璃体液K~+浓度与死亡时间的关系研究

目的探索在不同环境温度条件下,玻璃体液中钾离子浓度变化规律与死亡时间的关系。方法家兔处死后分别置于5℃、15℃、25℃和35℃温度下保存,在0~120h内每12h双眼交替微量提取玻璃体液80~100μL;应用罗氏DPPI生化分析仪检测玻璃体液中K~+的浓度;应用插值函数进行分析拟合,建立死亡时间推断方程。结果各温度组家兔玻璃体液中K~+浓度随死亡时间延长均呈上升趋势,对所得数据进行插值拟合,在5℃~35℃的温度变化区间内,K~+浓度变化与PMI关系的三元五次曲面方程:f(x,y)=-1.998e~(14)+1.345e~(12)x+5.902e~(13)y+0.005 585x~2-4.509e~(11)xy-3.876e~(12)y~2-0.000 286 8x~3+0.003 545x~2y+4.406e~(10)xy~2-1.746e~(10)y~3+2.669e~(-6)x~4-1.568e~(-5)x~3y-0.000 177 1x~2y~2-1.64e~9xy~3+6.669e~9y~4-8.672e~(-9)x~5+4.467e~(-8)x~4y+2.354e~(-7)x~3y~2+2.459e~(-6)x~2y~3+2.05e~7xy~4-1.214e~8y~5(R~2=0.995 6),其中温度为自变量x,K~+浓度值为自变量y,PMI为因变量f(x,y)。结论在5℃~35℃的环境温度区间内,家兔眼玻璃体液中K~+浓度变化与死亡时间关系符合三元五次方程分布,利用插值函数拟合的方法可将环境温度作为参数,实现在环境温度变化条件下进行死亡时间推断。     

The Influence of Preburial Insect Access on the Decomposition Rate

Abstract: This study compared total body score (TBS) in buried remains (35 cm depth) with and without insect access prior to burial. Sixty rabbit carcasses were exhumed at 50 accumulated degree day (ADD) intervals. Weight loss, TBS, intra‐abdominal decomposition, carcass/soil interface temperature, and below‐carcass soil pH were recorded and analyzed. Results showed significant differences (p << 0.001) in decomposition rates between carcasses with and without insect access prior to burial. An approximately 30% enhanced decomposition rate with insects was observed. TBS was the most valid tool in postmortem interval (PMI) estimation. All other variables showed only weak relationships to decomposition stages, adding little value to PMI estimation. Although progress in estimating the PMI for surface remains has been made, no previous studies have accomplished this for buried remains. This study builds a framework to which further comparable studies can contribute, to produce predictive models for PMI estimation in buried human remains.     

牙髓细胞DNA含量与死后经过时间的相关性

目的探讨死后不同环境温度下离体牙的牙髓细胞平均DNA含量变化与死后时间(PM I)的相关性。方法采用细胞图像分析系统(PIPS-2020),测定离体即刻至15d牙齿在低温环境(10~15℃)和高温(30~35℃)环境下牙髓细胞DNA含量变化值,并对其数据进行统计学分析。结果在不同的环境温度下,牙髓细胞DNA降解的速度有所不同,温度的升高有加速牙髓细胞DNA降解的趋势,且DNA降解存在一个平台期。低温组牙髓细胞平均DNA含量与PM I之间的相关系数r=0.953,相关指数R2=0.917;高温组的相关系数r=0.991,相关指数R2=0.971。结论牙髓细胞平均DNA含量与PM I之间具有高度相关性,测定牙髓细胞的DNA变化情况对推断3d(高温环境)或6d(低温环境)后的PM I有参考价值。     

On Improving the Legislation on Territorial and Regional Soviets of Working People's Deputies

The Report of the Central Committee of the CPSU to the Twenty-fifth Party Congress advanced the proposal of adopting laws to define the jurisdiction of territorial [krai], regional [oblast'], and area [okrug] soviets of working people's deputies. This proposal was adopted unanimously by the delegates to the congress. In the course of the work begun to draft these laws, which will define the content and form of activity at the present stage of the building of communism of such an important link in the system of soviets as the regional and territorial soviets, many questions have arisen demanding profound and comprehensive scientific study. In this connection the editors invite legal scholars and practical workers to share in the pages of the journal the results of their research and generalizations from the experience of the work of the soviets and from application of the prevailing legislation, and to express judgments, proposals, and recommendations on the paths to be followed in improving the legislation on territorial, regional, and area soviets.     

不同温度下离体人静脉血ATP含量与死亡时间的关系

目的探讨在环境温度变化条件下,人体静脉血ATP降解与死亡时间的关系。方法健康志愿者48名,随机分为6组,肱静脉取静脉血,置于10℃、15℃、20℃、25℃、30℃和35℃下保存;每4h应用ATP检测仪对不同温度下的ATP含量进行检测;应用SPSS统计软件进行回归分析,MATLAB软件进行差值函数分析拟合。结果各温度组ATP值随死亡时间延长均呈下降趋势,从取血即刻的1 573.683 E-13mol/L,降至6.00 E-13mol/L左右,所经历的时间分别为236h(10℃)、163h(15℃)、124h(20℃)、92h(25℃)、72h(30℃)和64h(35℃),对所得数据进行回归分析,得到各温度组下ATP含量变化与PMI关系的二元三次曲线方程(R2范围为0.976～0.990);进行差值拟合,得到10～35℃范围内ATP含量变化与PMI关系的三元四次曲面方程。结论在不同温度下,人体静脉血ATP降解与PMI关系符合三元四次方程分布,利用差值函数拟合的方法可在温度变化条件下进行死亡时间推断。     

The effect of sodium fluoride preservative and storage temperature on the stability of 6-acetylmorphine in horse blood, sheep vitreous and deer muscle

This study examined the in vitro stability of 6-acetylmorphine (6AM) in horse blood, sheep vitreous humour (VH) and homogenised deer muscle stored under different storage conditions. The stability of 6AM in horse blood is of interest because many toxicological laboratories utilise this matrix for the preparation of blood calibration and check standards and the latter are typically stored during routine use. Data on the storage stability of 6AM in human VH is extremely limited and no data has been reported in muscle. In the absence of human samples, 6AM stability was demonstrated in sheep vitreous and deer muscle. Blood and VH were stored with and without NaF at room temperature (RT), 4 and -18°C for 84 days. Muscle tissue homogenates were prepared in water with and without NaF and also in phosphate buffer (pH 6.0) containing NaF. Homogenates were stored for 31 days at RT, 4 and -18°C. Morphine and 6AM were extracted using SPE and quantified by GC-ion trap-MS/MS. In the absence of NaF, 6AM could not be detected after 7 and 14 days in blood stored at RT and 4°C, respectively. Although at -18°C 6AM was stable for 7 days (12% loss), only 54% was detected by day 84. The addition of NaF to horse blood increased 6AM stability substantially at every temperature. Further, the rate of degradation was found to be significantly slower in blood preserved with 2% NaF compared with 1% NaF (p=.05). 6AM was stable for the study period in preserved blood (1 and 2% NaF) stored at -18°C. For laboratories utilising horse blood in the preparation of standards, preservation with 1% NaF (minimum) and storage at -18°C is recommended. The addition of NaF to VH was essential for 6AM stability. Irrespective of temperature substantial losses (≥ 42%) were observed in unpreserved sheep VH by day 7. In preserved VH the concentration declined by only 22% on day 7 following storage at RT and no loss observed in VH stored at 4 and -18°C at the same time. In muscle, 6AM was stable for 7 days in preserved samples stored at RT and in all samples stored at 4°C and below. The addition of NaF increased the stability of 6AM substantially in muscle. The increased stability of 6AM in VH and muscle preserved with fluoride was attributed to inhibition of bacterial action and the subsequent reduction in the rate of putrefaction of these tissues.     

Potential Forensic Application of Receptor for Advanced Glycation End Products (RAGE) as a Novel Biomarker for Estimating Postmortem Interval

Studies which estimate the postmortem interval (PMI) based on diatoms or bacterial communities have been well‐conducted. However, predicting the PMI based on microorganisms has limited accuracy. The objective of this study was to discover specific biomarkers for estimating the PMI for drowning. The in vivo experimental model consisted of rats classified into nine groups (seven drowned groups from Day 1 to Day 7 and two control groups). Their lung tissues were analyzed for the receptor for advanced glycation end products (RAGE) mRNA expression determined by qRT‐PCR. RAGE protein levels were determined by Western blotting and immunohistochemical (IHC) staining. RAGE mRNA and protein levels gradually degraded over time. Immunostaining analysis revealed that RAGE in the Day 1 sample was significantly higher than that in the Day 6 or Day 7 samples. These results indicate that RAGE in drowned lungs could be a potential biomarker for the determination of the PMI after drowning.     

Evaluation of postmortem biochemical markers: Completeness of data and assessment of implication in the field

Throughout the years an increase has been observed in research output on biochemical markers for determining the postmortem interval (PMI). However, to date, a complete overview is missing on the results of postmortem biochemical markers (PBM's) for PMI estimation. In this paper, literature was reviewed in order to identify the knowledge lacunae of PBM research from a practical point of view. A three-step approach was undertaken in order to achieve the set goal. Literature was collected, the PBM's were evaluated for completeness by means of a scorings index based on set criteria, and PBM's were subsequently evaluated in light of the Daubert & Frye criteria for scientific evidence in court. Seven PBM's were found to be well investigated, from which potassium had the highest completion score. However, none of these PBM's could be qualified as suitable for court evidence. Further, this study revealed that the majority of PBM's (94%) is not well investigated. Consequently, these PBM's did not meet Daubert & Frye criteria. In order to improve the assessment for use of PBM's as evidence in court regarding PMI estimation, PBM's should be investigated more thoroughly and data should be made readily available.     

A new perspective in the estimation of postmortem interval (PMI) based on vitreous

The relation between the potassium concentration in the vitreous humor, [K+], and the postmortem interval has been studied by several authors. Many formulae are available and they are based on a correlation test and linear regression using the PMI as the independent variable and [K+] as the dependent variable. The estimation of the confidence interval is based on this formulation. However, in forensic work, it is necessary to use [K+] as the independent variable to estimate the PMI. Although all authors have obtained the PMI by direct use of these formulae, it is, nevertheless, an inexact approach, which leads to false estimations. What is required is to change the variables, obtaining a new equation in which [K+] is considered as the independent variable and the PMI as the dependent. The regression line obtained from our data is [K+] = 5.35 + 0.22 PMI, by changing the variables we get PMI = 2.58[K+] - 9.30. When only nonhospital deaths are considered, the results are considerably improved. In this case, we get [K+] = 5.60 + 0.17 PMI and, consequently, PMI = 3.92[K+] - 19.04.