Estimation Of Postmortem Interval Using The Ambient Temperature And The Concentration Of Substance In Vitreous Humor

Background The estimation of the time since death（TSD）,known as postmortem interval（PMI）,is a main issue in the field of forensic science and legal medicine.TSD is important in both criminal cases as well as civil cases.So estimate TSD is one of the most difficult and hot problems for forensic pathologists.The traditional estimation methods mainly based on some postmortem changes,such as corpse,livor mortis,rigor mortis,which effected by many external factors and could only give an approximate inference.Since Sturner firstly established a linear regression equation between Vitreous Humor（VH）K⁺ concentration and PMI,lots of forensic scholars began to apply the blood,cerebrospinal fluid and vitreous fluid to estimate PMI.Since VH is located in the vitreous body which is at the back of the eye,it is not vulnerable to be damaged and decays slowly after death.The changes of concentrations of VH substances are relatively slow,with an apparent pattern.Therefore,VH has been a focus for the application of forensic pathology in the study on the PMI estimation and postmortem chemical analysis.Like other body fluid,the pattern of changes in the VH substances is affected by the external environmental factors,in which the temperature has the most significant impact.Objective Explored the relationship between the concentration of potassium（K⁺）,phosphorus（P）,magnesium(Mg²⁺),calcium(Ca²⁺),sodium（Na+）,chlorine（Cl-）,creatinine （Cr）and urea nitrogen（UN）eight substances in vitreous humor and the time of death（PMI）was under different ambient temperature conditions.To achieve determine PMI under the ambient temperature changes,improve the accuracy of PMI estimation.Try new data fitting models to improve the accuracy of PMI estimation.Material and Methods Forty healthy adult male and female rabbits with a weight of 1600²500 g（provided by the Experimental Animal Center of Tongji Medical College,Huazhong University of Science and Technology）were randomly divided into four experimental groups A,B,C,D of ten each.The rabbits were executed by air embolism in ear vein,and the carcasses were stored at 5℃,15℃,25℃,and 35℃,respectively.Another 18 rabbits（the same conditions as above）were randomly divided into three validation groups E,F,and G of six each.They were executed using the same method and were placed in environmental chambers at 10℃,20℃,and 30℃,respectively.The double-eye alternating micro-sampling method reported by Qian Liu et al was adopted for the rabbits in experimental groups,and 80-100 μl of samples were collected from the left eye at 0 h,24 h,48 h,72 h,96 h,and 120 h after death,respectively,and from the right eye at 12 h,36h,60 h,84h,and 108 h after death,respectively.For the validation group,80-100 μL of VH was collected at 20 h,40h and 65 h after death,respectively.The turbid samples or the samples mixed with blood were discarded,and the qualified samples were numbered and placed into the Eppendorf（EP）tubes that were stored at-80℃ and the measurement was performed within two weeks.Due to fast decay rate,the sampling for high temperature group（25℃ and 35℃）was only performed up to 72 hours after death.After the thawing at room temperature,60 μl of the sample was placed into a test vial and diluted with 1:4 deionized water.The contents of the six substances in VH of the experimental groups and the K⁺ and Mg²⁺ concentrations in VH of validation group were measured by Roche DPPI biochemical analyzer（Modular DPPI,Roche,China）.All experimental data are expressed as mean ± standard deviation（sx±）.The interpolation analysis and equation fitting were performed using Metlab16.0（METLAB Inc.,Natick,MA）,and the numerical analysis and equation fitting of the mixed-effect model were carried out using SAS9.3（SAS9.3,SAS,America）.Results The concentrations of K⁺,P,Mg²⁺,Cr and UN in each temperature group increased with increasing PMI,and the changes in the ion concentrations were more significant with increasing temperature.The concentration of Ca²⁺ in each temperature group decreased with increasing PMI,and the decrease of Ca²⁺ concentration was more significant with increasing temperature.The concentration of Na+,Cl-in each temperature group was irregular trend.Both the mixed-effect model and the interpolation function can be used for determine PMI.Conclusion The concentration of K⁺,P,Mg²⁺,Ca²⁺,Cr and UN in VH changes with time after death and is a good indicator for PMI estimation.The concentration of Na+,Cl-in VH changes irregular with time after death and is not a good indicator for PMI estimation.The use of double-eye alternating micro-sampling method could reduce the number of experimental individuals,control individual differences,and was worthy of promotion in the study of VH.The mixed-effect model can be used for determine PMI,and a new data analysis method has been opened for determine PMI.Both the interpolation function and the mixed-effect model for repeated measures could be used to estimate PMI with two parameters–temperature and VH substance concentrations.From the experimental results of this experiment,the estimation by the mixed-effect model was more accurate.In the future,the VH micro-sampling method can be adopted within creased temperature grouping,denser sampling time points,and reasonable selection of time span and VH detection indicators,to explore other suitable fitting models,accumulate human VH test data,and provide new methods for PMI estimation in forensic practice.