A New Study On Estimation Of Postmortem Interval

Estimating the postmortem interval is one of the important and difficult points in the practical case detecting and research work in forensic pathology. For a long time, scholars from domestic and abroad tried to deduce PMI, especially early deduction, by applying lots of techniques and methods, such as various phenomena of posthumous cadaver, muscular supravital reaction, K~+ concentration of vitreous body liquid, biochemical changes in blood of posthumous and so on. However, the accuracy is often influenced by various factors outside, the errors are usually large. Till now, there is still no public received simple, practical, accurate and reliable deduction way, specifically for the middle and late PMI deduction. Therefore, we investigate the role of the magnitude and phase angle changes of musculature impedance in rat cadavers, in order to evaluate the significance of middle and late PMI estimation by Bio-impedance measurement, and try to develop a simple and accurate new method. We selected an unused index — HeadDNA% to detect the role of nuclear DNA degradation of both bone marrow and brain in rat cadavers followed by PMI by applying the single-cell gel electrophoresis, and to develop a new parameter of estimating early PMI by single-cell gel electrophoresis. Part IResearch on estimating of the postmortem interval by the magnitude and phase angle changes of musculature impedancein rat cadavers[Objective] To investigate the role of the magnitude and phase angle changes of musculature impedance in rat cadavers, to evaluate the significance of middle and late PMI estimation by Bio-impedance measurement, and try to develop a simple and accurate new methods to estimate the middle and late PMI. Meanwhile, we analyze the mechanism of the trend of impedance magnitude changes generated in terms of muscular morphology.[Methods ]Divided 20 healthy adult Wistar rats into 4 groups, 5 each correlated to 4 different detecting temperature(9±1℃, 14±1℃, 16±1℃, 19±1℃). Rats were sacrificed by cervical dislocation, and inserted the electrode into leg muscles and fixed it. Rats were put in the homeothermia incubator or refrigerator with the fixed temperature, and the magnitude and phase angle changes of musculature impedance in rat cadavers were measured by applying a Bio-impedance measurement system based on four-electrode method. The impedance and the environment temperature were recorded once for every 3 hours, since Oh of death. The experiment stopped when the time was long enough (impedance decreased to around 70% of the original one or over 15 days). The simple means and variance were analyzed based on the discrete data by excel method. The data were analyzed, the line was fitted, the graph was made , and the related coefficient was calculated.Pick out the 4 same rats (one as control before death, 3 as control after death), draw the materials from the leg subdermal muscular tissues in the site of electrical impedance measured at 12h, 24h and 72h, respectively. The tissues were fixed, sectioned, HE stained regularly, and the morphological changes were observed under microscope, and changes of impedance were comparatively analyzed. [Results]1. All endermic musculature impedance magnitude of rat cadavers presented the similar change, first rose, later decreased, and the downgrade part of middle and late phase shown some linear change tendency. The impedance magnitude at early stage of death(PMI = 1～ 4 day) upgraded quickly, and presented linearly downgraded tendency (PMI=4～15 day) after reached the peak value with the time passed, and finally to platform.2. All phase angle of endermic musculature impedance showed no similar regular change, the changes of which were in a great mess.3. The relative impedance could eliminate the specificity caused by different rats, and the dispersion from different rats at the same temperature was lower than that of absolute impedance.4. The higher the temperature, the faster it rose and faster it descended for the absolute or relative impedance; the lower the temperature, the slower it rose and slower it descended for the absolute or relative impedance. The slope rate of downgrade part of the absolute or relative impedance positively related with changes of temperature.5. Relative coefficients of the slope rate of downgrade part from each group at different temperature with PMI were all over 0.97, which showed that the dependability was quite high. So PMI at middle or late stage was supposed to estimate by use of this point.6. No change of nucleus was observed before death, the transverse striation of muscular bundles was clear; 10h after death, a few muscular bundles in surface layer were swelled, most of myofilament presented particle shape or homogenous shape, the transverse striation disappeared; 72h later, more muscular fiber swelled, particular like change, and the transverse striation disappeared. Self autopepsia splitting gradually developed with PMI passed, the tissue conductibility increased, consequently showed that the impedance continuously decreased[Conclusion]1. The endermic musculature impedance magnitude showed first upgraded then downgraded tendency with time passed in rats after death, there was linear relation between changes of the impedance magnitude in upgraded part with PMI. The changes of impedance were similar under the similar measurement circumstance.2. The environmental temperature is the main parameter which affects the impedance magnitude, and the higher the temperature the faster the changes. Thus, the measurement of impedance should pay attention to the optimal scale of temperature, both too high and too low temperatures are unavailable.3. Transfering the absolute impedance to relative one better reflects the objective law of changes of impedance when the magnitude of impedance is measured.4. The slope rate of impedance on downgraded part positively correlated with changes of temperature, and which highly related with PMI at different temperature. If the definite relation of the beginning point, peak value and slope rate on the upgraded part was determined, the linear relation on the downgraded part could be used to provide the evidence for estimating the middle and late stage of PMI.5. Since phase angle of impedance is strongly influenced by circumstance, which can't be used to estimate PMI.6. The result consistent with the theory of cellular biology is drawn from the preliminary study on the mechanism of the tendency of impedance magnitude changes generated in terms of muscular morphology.7. Compared with other PMI estimating techniques, impedance possess the advantages of easy to measure, curacy and reliability. However, with the limit of techniques and environmental factors, whether it is available in practical settings, or develops to a new PMI estimating technique should be further studied. Part IIResearch on estimating of the postmortem interval by determining nuclear DNA degradation of both bone marrow and brain in rat cadavers with single-cell gel electrophoresis[Objective] To detect directly nuclear DNA degradation of both bone marrow and brain in rat cadavers at different temperature by applying the single cell gel electrophoresis (SCGE), analyze the amount of DNA degradation followed by changes of PMI by selecting an unused index — HeadDNA%, to establish the regression function of both and compare with standard index TailLength(TL), Oliver TailMoment (TM) and be expected to develop a new parameter of estimating early PMI.[Methods] Rats were sacrificed by cervical dislocation, put in the experimental environment with the fixed temperature at 10℃±1℃ or 20℃±1℃. The brain and bone marrow were taken out for every 4h, during 0～40h after death, and single cell suspension was prepared; Then single cell gel electrophoresis was carried out and observed under fluorescence microscope and photographed. The pictures were analyzed by CASP1.22 comet graph essaying software, and an unused index-head DNA% parameter analysis was used to essay the role of DNA degradation with changes of PMI, Statistic analysis was conducted with SAS 8.0 and SPSS 12.0 software. Set up regression function of both and the results were compared with standard index TailLength(TL), Oliver TailMoment (TM). Meanwhile, the calculating function for PMI was inferred reversely based on the established function and headDNA% value within 95% confidence interval.[Results]1. Different degree of comet like tail was found in the electrophoresis pictures of brain cells and bone marrow cells in rats after death;2. The good linear relationship existed between decreasing of nuclear HeadDNA% in both brain and bone marrow with PMI during the early PMI, whatever at 10℃ or 20℃; four linear regression function was set up, their coefficients were high and could be used to estimate early PMI;3. The downgraded nuclear HeadDNA% was influenced with environment temperature and tissue kinds after death;4. The calculating function was established to deduce reversely PMI by a known HeadDNA%;5. HeadDNA% was better in fitting by linear regression function than the standard common used TL, TM and PMI in early study;6. The linear regression analysis fitted by using three parameters with PMI of brain tissues and bone marrow, the definite coefficients of former was higher.[Conclusion]1. SCGE can be used to estimate early PMI;2. The environment temperature and tissue disparity significantly affect nuclear DNA degradation after death; ;3. The new parameter HeadDNA% is more valuable for PMI estimation than the common used standard parameter TL, TM in early study. The reliability needs further investigation, especially for testing the human materials;4. The known HeadDNA% can be used to estimate reversely PMI scale;5. Brain tissues are more ideal as candidate materials than bone marrow.