An Experimental Study Of Estimation Postmortem Interval Based On Virtopsy

Postmortem interval (PMI) estimation is one of the most important and most difficult tasks in the practice and scientific research of forensic pathology. PMI estimation might be a complex system, which must be based on many independent and relative sub-systems. In most early PMI cases, the common indicators such as rigor mortis, livor mortis, and the body's core temperature would achieve acceptable results. But in late PMI cases, the general pathologic method doesn't work because of autolysis and putrefaction. So it's a very difficult task of estimation PMI especially in late PMI. Since 1970s various chemical methods have been developed in order to ameliorate and extend PMI estimation, e.g., using blood and serum, cerebrospinal fluid, vireous humor, skeletal muscle and liver. By now, there aren't convenient, practical, objective, feasible, quantitative, credible and acceptable methods in late PMI estimation.Under natural condition, all kinds of physical, chemical and biological factors would impact body. Organs would autolysis and putrefy; the volume of them would decrease. But postmortem organs would keep a long time during putrefaction. Many metabolic change and morphologic change would happen in organs. Using traditional methods such as pathologic section examinaction could not be effective. But rudimentary organ samples would be effective to estimate PMI. And detected their metabolic changes and morphologic changes by modern detective methods, the rudimentary organ samples would become the breakthrough point of estimation late PMI and could extend the research time span of estimation late PMI.With the radiology's development, magnetic resonance spectroscopy (MRS), and computed tomography (CT) could be used to exminate morphologic change and the metabolic change in all kinds of organs. The radiologic digital methods could be used to apply quantitative and qualitative analysis. Because they are non-invasive, rapid, objective, and its data were stored and copied easily. Virtopsy (virtual autopsy) based on CT and MRS has been used in forensic examination. Overseas scholars had used the virtopsy technology to definite individuals. But virtopsy is a new research fields, there was no reports on estimation PMI. We planed to use virtopsy based on MRS and spiral CT to perform the study of estimation PMI especially late PMI.The study was divided into two parts, including virtopsy based on high-resolution magnetic resonance spectroscopy and spiral CT. High-resolution MRS was used to detect body's postmortem metabolic changes, and spiral CT was used to analyze body's postmortem morphologic changes. During the study, animal death model and detective methods were established, and the results were used to establish mathematic models to estimate PMI.The study of part one: we used high-resolution ³¹P-MRS,¹H-MRS to detect the postmortem metabolic changes in rat death model. The proton spectrum and phosphate spectrum of brain postmortem metabolites were analyzed. The feasibility of PMI estimation (especially in late PMI) was explored by using metabolites postmortem changes. The results showed that phosphate spectrum's change was during early PMI (≦ 24h); the spectrum mostly reflected the energy change during postmortem change. And its results could be used to estimate early PMI. Proton spectrum's change was during the whole study period (0～240h), which reflected the whole body's putrefaction process. There were many metabolites disappeared, e.g., Naa, lactate, etc. and there were many metabolites appeared, e.g. fTMA, butyrate, iso-butyrate, etc. By regressive analysis and curve fitting, the equation of them could be used to estimate PMI (especially late PMI). The detective of ¹H-MRS was more important than that of ³¹P-MRS in the research of late PMI estimation. The disappeared and new appeared metabolites could be used to estimate PMI and could ameliorate and extend PMI estimation.The study of part two: the virtopsy was performed based on spiral computed tomography by using New Zealand rabbit death model. In this part, the postmortem morphologic digital data were used to establish the method of estimation PMI(especially late PMI),e.g. CT value, the area of organs, reconstruction images etc. The postmortem morphologic changes were detected by virtopsy based on spiral CT form the beginning of death to sleletonized remains. The results showed that many vital organs could be used to estimate PMI, such as brain, liver, celiac cavity and so on. The regressive equations were established by the organs' CT value and area, which could be used to estimate PMI (especially late PMI).According to the results of high-resolution MRS and spiral CT, the feasibility of virtopsy based on MRS and spiral CT was confirmed. The preliminary archievement of virtopsy would become the experiment and theory base of other bigger animals and human bodys' virtopsy. It also could indicate virtopsy research's direction, and make a helpful explorement of establising complicate system on estimation PMI.The innovations of the study were as following. 1) the results of virtopsy based on radiologic methods was firstly used to estimate PMI. High-resolution ³¹P-MRS,¹H-MRS and spiral CT were used in the study to detect the morphologic and metabolic chemical postmortem changes. There were no reports on systemic research of estimation PMI by using virtospsy, expect for individual cases reports. 2) The high-resolution MRS was put into the research of forensic research by which we explore to establish the detective method of postmortem metabolites. By quantitative analysis of the metabolites postmortem change, the regressive equations were established and could be used to estimate PMI. The study would indicate the direction of the virtopsy forensic research; and it could become the base of experiment and theory of virtopsy based on clinical MR. 3) The spiral CT scan and reconstruction were put into forensic research, which became a helpful exploration of virtopsy based on spiral CT scan. The CT value, organs' area and reconstructive images of brain, liver, celiac cavity etc could be used to estimate PMI especially late PMI. The results indicated the research direction of PMI estimation and would become the base of experiment and theory in human body by virtopsy based on spiral CT scan.The study was fund by Deparment of education doctor's fund (the study of estimation PMI based on magnetic spectroscopy, serial number:20040487049, 2004) . The five articles were published on relative issues.Part OneAn Experimental Study of Applying Magnetic Resonance Spectroscopy to Estimate Postmortem IntervalExperiment IEstablishing the Rat Death Model Fit for Magnetic ResonanceSpectroscopy Detective Objective To establish the rat death model in which there was quantitative cause of death without vital organs' mechanical injury and exogenous drugs' interference. The model could be fit for MRS detect(magnetic resonance spectroscopy). Methods The lateral tail veins were exposed, and the volume of 2 ml air was infused into them within 10 seconds to establish the rat death model of air embolism. Results The quantity of insufflated air and the velocity of infusion played a crucial role in the experiment. The procedure was simple and very successful (with success rate of 100%), caused different weight group rats' rapid death reliably and there were no mechanical injuries of vital organs. Conclusion The study could provide an ideal animal death model for the study of applying MRS to estimate PMI. Experiment IIAn Experimental Study of Applying ³¹P-MRS, ¹MRS to Detect Rat Brain Postmortem Change to Estimate Postmortem IntervalObjective To evaluate of ³¹P-MRS, ¹H-MRS postmortem metabolites changes in rat brain, use the results of MRS to explore the new methods of postmortem interval (PMI) estimation. Methods: the rat death model was made by air embolism. During 0～240h after death, the rats' whole brain was picked out and put into liquid nitrogen to deepfreeze. The frozen brain was grinded into tissue powder. The perchloric acid extract was made by using 1g brain tissue powder and lyophilized. The lyophilized powder dissolved in D₂O. Using DSS/H₃PO₄ as internal/external standard, the extract was detected by ³¹P-MRS, ¹H-MRS respectively. The metabolites' peaks were integrated; the relative values were used to estimation postmortem interval. Results: High-resolution MRS detective of postmortem brain tissue was achieved perfectly. ³¹P-MRS revealed ATP, ADP, phosphocreatine, inorganic orthophosphate, manifold aliphatic phosphates and sugar phosphates; the spectrum change of ³¹P-MRS was during 24h after death. The relative values of inorganic orthophosphate and phosphomonoeste could be used to estimate PMI within 24h. Other substances such as ATP, ADP, and PCr etc changed more rapidly; they could be used to estimate PMI during 0～12h. ¹H-MRS showed many resonance peaks, including lactate, N-acetylaspartate, choline, creatine, GABA, myo-inositol, succinate, acetate, butyrate, free trimethylammonium, iso-butyrate and other metabolic substances. During early postmortem interval, Naa and Cr decreased rapidly. After PMI=60h, But, iBut, fTMA appeared in the spectrum. They and Ace increased continuously till to the end of the research. During the whole research period, Lac,Asp went up then decreased till disappeared. The results showed that it was the important time point of PMI=60h, there were many new metabolic substances appeared and original substances in the brain would disappear mostly. For estimation PMI, there were significance of the new metabolic substances such as But,iBut,fTMA,Ace and of the original substances in brain such as Naa,Cho,Cr,Lac. There were closely relation between them and PMI. Conclusions High-resolution MRS could be used to detect the postmortem change of rat brain. The ³¹P-MRS result of rat brain could be used to estimate early PMI(≦24h). The ¹H-MRS result could be used to estimate PMI especially late PMI; it had more important forensic significance than that of ³¹P-MRS; it could ameliorate and extend the span of estimation PMI. It was an important time point at PMI=60h, which suggested that brain would go into rapid putrefaction period. The appeared and disappeared metabolites were effective index to be used estimate PMI especially in late PMI. There were wide application prospects and important forensic significance of applying virtopsy based on magnetic resonance spectroscopy to estimate PMI. Part twoAn Experimental Study of Virtopsy to Estimate Postmortem Interval Based on Spiral CTExperiment IAn Experimental Study of Applying New Zealand WhiteRabbit's Brain Postmortem Change Detected by Spiral CTto Estimate Postmortem IntervalObjective Investigate the postmortem changes of New Zealand white rabbit's brain by spiral CT scan, and establish effective methods of estimating PMI by using brain CT value and the area rate of brain /cranial cavity. Methods The rabbit's death model was established by venous air embolism. Virtopsy based on spiral CT scan was performed to investigate the brains' postmortem change after death 0～120h. Results The values of brain tissue increased, the rate of brain area/cranial area decreased after death. The regression equations: CT value=47.4780-0.1768T+0.0049T² ( T/h , CT value/HU),area rate =99.7368+0.3098T-0.0118T²/100(T/h). Conclusion CT scan could show brain postmortem changes, the CT value and area rate were effective index to estimate PMI before brain disappearance. Experiment IIAn Experimental Study of Applying New Zealand WhiteRabbit's Celiac Cavity Postmortem Change Detected by SpiralCT to Estimate Postmortem Interval Objective Investigate the postmortem changes of New Zealand white rabbit's celiac cavity by virtopsy badsed on spiral CT scan, and establish effective methods of estimating PMI by using the area rate of celiac cavity /vertebra. Methods Virtopsy based on spiral CT scan was performed on rabbit's death model to investigate the celiac cavity's postmortem change after death 0～240h. The slice image of lumbar 4(L4) was chosen to analyze the postmortem changes of the area rate of celiac cavity/vertebra. Results the L4 image of CT scan showed that the volume of gas increased in the celiac cavity from the beginning of death to the abdomen wall's perforation. The area rate of celiac cavity /vertebra increased continuously from Oh to 108h after death. At PMI=120h, the abdomen wall perforated and putrefaction gas went out, the rate decreased greatly. But At PMI=144h, the rate went up a little then went down. Before abdomen wall perforation (PMI=0～108h), the regression equation: of the area rate could be used to estimate PMI. (the area rate=19.758+0.1951T+0.0012T²,T/h , R²=0.993 F=482.42, PMI=0～108h) Conclusions the image of L4 could reflect the New Zealand white rabbit's celiac cavity postmortem change, obtained by virtopsy (spiral CT scan). The area rate of celiac cavitya/vertebra (L4) could be used to estimate PMI during 0～108h. Experiment IIIAn Experimental Study of Applying New Zealand White Rabbit's Liver Postmortem Change Detected by Spiral CTto Estimate Postmortem IntervalObjective Investigate the postmortem changes of New Zealand white rabbit's liver by virtopsy based on spiral CT, and establish effective methods of estimating PMI by using liver CT value and the area rate of liver /vertebra. Methods Virtopsy based on spiral CT was performed on rabbit's deathmodel to investigate the liver's postmortem change after death 0²40h. The sliceimage of lumbar 1(L1) was chosen to analyze the postmortem changes of liver'sCT values and the rate area of liver/vertebra (L1). CT value-PMI and arearate-PMI characteristic curve were fitted on detected data, the best regressionequations were established to estimate PMI. Results the results of virtopsy basedon spiral CT showed that liver's CT value changed firstly during putrefaction.During the whole experiment, the liver's CT values decreased continuously; andat PMI=108, the CT value reach the lowest point. The value increased a little,and then the value went down till it could not be detected by CT at PMI=180h.The area rate of liver /vertebra didn't changed during 0～48h. After PMI=48h, therate of area deceased continuously; but at PMI=132h, the rate went up a littleand then went down till liver disappeared at PMI=180h. Regression equationswere established between CT value, the area rate and PMI: liver's CTvalue=100.773-1.6026T+0.0018T²(CTvalue/HU, T/h, F=41.18, R²=0.882) ,the area rate of liver/ vertebra(L1)=18.9310-0.1181T+0.0002T²(T/h, F=50.68,R²=0.894, ). An important CT sign (intra-hepatic bile duct dilation filled with air)was found at PMI=48h. After the sign appeared, the livers' area decreasedgreatly. When the putrefaction gas appeared in the liver, the sign disappearedgradually. Conclusions virtopsy based on spiral CT could obtain all informationof liver's postmortem changes. The liver's CT value and the area rate ofliver/vertebra (L1) could be used to estimate PMI and the research span ofestimation PMI was extended. The CT sign(intra-hepatic bile duct dilation fillwith air) would be important significance for suggesting liver's rapidputrefaction's beginning. Virtopsy based on spiral CT could be morecomprehensive than traditional autopsy.