Study On Micro-sampling Methodology Of Vitreous Humor And Postmortem Interval Estimating

Postmortem chemistry investigates the postmortem changes of chemical components in blood, cerebrospinal fluid, vitreous humor (VH) and some other body fluids. And vitreous humor has become the most utilized medium for postmortem chemistry analyze for reasons that it is anatomically isolated, well protected, less subject to contamination or putrefaction and its autolysis changes proceed slower compared to other fluids. Estimation of postmortem interval (PMI) is one of the most important aims in the field of forensic pathology. Since the late 60s, most investigations have focused on VH variety and its relativity with PMI, especially on the estimating precise. And the study field extends to sampling methods, elements selection and the establishment and evaluation of predictive equation, etc.This paper mainly explores micro-sampling method of VH and the confidence intervals of the predictive equation.Part I. Preliminary Study on Micro-sampling Methodology of VHVitreous humor is the most extensively studied medium in thanatochemistry investigation. According to eye physiological studies, the vitreous is non-even structured and the gradients established before death may be changed and new gradients may be formed after death. Thus, Coe recommended mandatory sampling of the whole vitreous humor to exclude gradient disturbance. Some micro-sampling method was reported in recent years but researchers haven't come to an agreement on these issues, such as electrolyte gradients in vitreous, concentration differences between left and right eyes and the bi-eyes changes post-mortem. The disputes effected VH sampling and research methods greatly, while no special studies were found in the literatures.In this part rabbit experimental models were employed to study the intraocular and binocular concentration gradients and difference between the repeated micro-extracted samples, the difference between the entire and micro sampling operations, and contributions of space micro-sampling to VH samples and their concentrations in functions of PMI. It is a systematic preliminary study on VH micro-sampling and spacemicro-sampling, which is a special methodology study on VH operation preparing forhuman specimen research.Experiment one. Study on Concentrations Variability of VH Samples[Objective] To research if there are significant intra-eye and between-eyesconcentration variability can be found in VH extract samples after death, and explorethe feasibility of repeated micro-sampling.[Methods] twenty-eight rabbits are divided into three groups. Animals of Group 1 werewholly extracted VH at 12h after death following the operation of Coe. Rabbits ofGroup 2 were subjected to multiple sampling fifteen times of 50μl at 12h after death andcarcasses of Group 3 were also multiple sampling fifteen times 50μl at 120h after death.Then measured the calcium, chlorine, potassium, sodium ion and inorganic phosphorusconcentrations and made statistic analysis of paired t-test and analysis of variance ofrepeated measurement and the two divided factors of time and between-eyes.[Results] The concentrations variability of such five inorganic ion between the two eyesand intra-eyes had no statistics meaning(P>0.05). The overall variance analysis ofmultiple sampling had no significant meaning too (P>0.05).[Conclusions] The material contents in vitreous humor between the tow eyes are quitesame after death. The reported intra-ocular local concentration differences could not befound in micro repeated VH samples.Experiment two. Comparative Study on wholly and Micro-sampling[Objective] On the basic of the previous study, this paper is for a compare of the whollyand micro sampling on VH contents.[Methods] Thirty-six rabbits were divided into four groups. Rabbits of group one werewholly sampled VH at 12h after death. Animals of group two were subjected to 50μlVH fifty times at 12h after death. As to group three, all left eyes were wholly sampledas group one and right eyes were micro-sampled same to group two. And all balls ofgroup four were extracted two times, with the first time of 50μl VH aspirated andresiduals extracted wholly at a next operation. Then measured the concentrations ofcalcium, chloride, potassium, sodium and phosphorus and made statistic analyze.[Results] In group one, the variation coefficients (CV) of calcium, chloride, potassium,sodium and phosphorus are 25.97%, 17.89%, 33.92%, 21.58% and 40.01%, which arelarger than that in group two as 10.08%, 4.76%, 11.35%, 5.30% and 17.70%. In group three, the average CVs of calcium between left and right eyes are 24.62% and 10.23%, chloride are 20.83% and 5.25%, potassium are 37.64% and 11.50%, sodium are 22.53% and 4.88%, phosphorus are 41.08% and 17.02%. As to group four, the CVs of the first and second aspirating are calcium 9.73% and 18.55%, chloride are 4.20% and 15.08%, potassium are 10.16% and 19.95%, sodium are 4.46% and 21.36%, and phosphorus are 17.71% and 35.12%. And the rates of between-eyes differences to averages are at ranges of 2.122% to 17.972% for first aspirating and 15.091% to 35.992% for second extracting.[Conclusions] We believe some variations may come from sampling operation. The wholly-sampling have some artificial influences on VH samples and made the between-eyes differences, variations and dispersion larger than micro-sampling. So the micro-sampling operation is more stability and repeatability, which is promising for forensic VH study and examination.Experiment three. Study on VH Micro-sampling with Intervals [Objective] This paper is for a further study on multiple micro-sampling with intervals to examine the effect on samples and PMI research.[Methods] Ninty-nine rabbits were divided into 7 groups as nine in group I to VI and fourty-five in group VII. The left eyes (of experimental group) in gropu I to VI were were multiple micro-sampled 50μl with the interval of 12h and ended at 24h, 48h, 72h, 96h and 120h postmortem, and the right eyes (of contrast group) were single micro-sampled 50μl at24h, 48h, 72h, 96h and 120h postmortem respectively. Measured the contents of calcium, chloride, potassium, sodium and phosphorus and made statistic Other nine rabbits in group VI were multiple micro-sampled alternatively between right and left eyes with the interval of 12h from 12h to 120h postmortem. Forty-five rabbits in group VII were single micro-sampled 10 eyes 50μl at every 12h to 120h postmortem. Detected the concentrations of calcium, potassium and sodium and made analysis of the PMI regress tendency.[Results] Only in group V, concentrations of potassium are different between left and right eyes (P=0.025), all others elements are same in each group (P>0.05). Difference t-test between group VI and VII on calcium, potassium and sodium prove no significant difference (P>0.05). The comparison analysis of linear regression equations of potassium and sodium in the two groups also have no difference in intercepts and slopes (P>0.05). The calcium performances of group VI and VII are accordant. [Conclusions] So we come to the conclusion that in the 96h postmortem period, multiple micro-sampling with intervals does not distort the contents and changes after death, and can be used for vitreous humor PMI forensic investigation.Part II. Detecting Content Variations of VH and DNA in Corneal Epithelium Basal Cell for PMI Function ResearchOne major subject of postmortem chemistry is to estimate the time since death performed on VH autolysis changes. But the value of the chemical methods for PMI is up to now very limited due to the different statistical parameters of the regress line and false estimation. Some researchers recommend differentiation according to death cause, ages, environment temperature, multiple gradients for multiple regression analysis, and PMI using as the independent variable, etc. And some foreign researchers were attracted by the formula confidence intervals.This study was performed on rabbits, by detecting the VH electrolytes following the variations with PMI, such as potassium, sodium, magnesium and ammonium. And the image analysis technology was engaged to investigate the DNA contents changes in the corneal epithelim basal cells, which is well studied in Forensic Science Department, Tongji Medical College. The forecast formulas and the confidence intervals of the 4 VH electrolytes and the 7 parameters of DNA were established respectively. And further multiple regression analyses were performed with the VH ions and DNA parameters. The confidence intervals of PMI forecast equations for estimation were discussed as an evaluating factor. The experiments will establish new assessment standards to the native PMI study and give suggestions to some essential reference for parameter election and multiple regression analysis.Experiment four. Evaluation of Potassium, Sodium, Magnesium and Ammonium Postmortem Changes and the Correlation to PMI by Micro-sampling VH with Intervals[Objective] In this experiment, we measured the postmortem changes of potassium, sodium, magnesium and ammonium contents by micro-sampling VH with the interval of 12h and investigated the correlations between changes and PMI to establish forecast equations for death time. By evaluating the speculate confidence interval, analysis the PMI study values of the four electrolyte elements in rabbits VH. [Methods] Twelve rabbits were divided into 2 groups. Nine animals in experiment group were micro-sampled 50μl alternately between-eyes with the interval of 12h from 12h to 96h postmortem, and measured the contents of potassium, sodium, magnesium and ammonium. By statistics analysis, we investigated the correlation between postmortem changes and death time, evaluated the equations confidence interval and the PMI values respectively. Six balls of three rabbits in testing group were extracted 50μl at a random selected time from 12h to 96h as 12h, 24h, 60h, 78h, 84h and 96h. The contents of testing group were used to appraising the forecast equations making from experiment group.[Results] From the experiments we found the contents of potassium, magnesium and ammonium increased, while sodium decreased after death. The four element changes all have significant correlations with PMI, and the regress coefficients all are above 0.95. The PMI forecast equation of potassium(X₁) is Y₁=12.038-1.272X₁+0.123X₁² (r²=0.99700), sodium(X₂) is Y₂=438.346-3.177X₂ (r²=0.9864), magnesium(X₃) is Y₃=259.158X₃-53.455X₃²-216.430 (r²=0.9388) and ammonium(X₄) is Y4=0.072X4-40.826 (r²=0.9566). But as to the confidence interval analysis, sodium and magnesium forecast equations have a large average forecast error of 15.25h and 13.52h, and the potassium and ammonium are small of 8.89h and 8.04h. The 95% confidence interval of potassium forecast equation widens out with the PMI, and when to 72h PMI it is about to ±14 hours. This debases potassium PMI value in some distance. The 95% confidences of the other elements are about the same as ±6h, ±13h and ±15h respectively. In testing Group, the average calculated errors from the sampling time are potassium 6.48h, magnesium 7.13h, ammonium 9.29h and sodium 19.34h. [Conclusions] From the data analysis, the four electric elements postmortem variations all have significant correlation with death time, and may be utilized for PMI calculative equations in 96h. A proper evaluating way on PMI forecast equation including confidence interval and blind sample testing. As to our study, the PMI values of sodium and magnesium shrike for the wide 95% confidence intervals about 13 hours to 15 hours. The confidence of potassium enlarges with the postmortem time, which is independent of sample size. So potassium may be limited in 72h PMI and ammonium is proper for 24h to 96h postmortem time study. Experiment five. Study on Corneal Epithelium Basal Cell DNA Contents and VH Potassium, Sodium for the Relations with PMI[Objective] Using Image analysis technology(IAT), to research the DNA content variety in the corneal epithelium basal cell after death and the relations with PMI. And make a tentative discuss for combined forecast with VH potassium and sodium. [Methods] Thirty-six rabbits were divided into twelve groups and were subjected at 01k 6h 12h 24h 36h 48h 60h 72h 84h 96h 108h and 120h postmortem respectively. The corneal sections were Feulgen stained, and gathered DNA content information with IAT as area(A), mean diameter(MD), index of density(ID), average optical density(AOD), integral optical density(IOD), light density coefficient(LDC) and average gray(AG). The parameters date were managed to discuss the variations along with postmortem time. Some multivariate regression analyses were made for advanced calculated time.[Results] All the seven parameters performed significant correlation with PMI (P<0.001), and had the correlation coefficients of 0.9511 to 0.9929. A cursory PMI forecast equation for every parameter was established and its predicting confidence was estimated. The average forecast warp of A predict equation is 11.00lh, MD is 55.452h, ID is 76.687h, AOD is 48.405h, IOD is 3.165h, LDC is 35.197h and AG is 8.918h. In 96h postmortem, the multivariate regression equation of sodium(X₈) and AG(X₇) is Y₁=451.10267-2.41185X₈-0.57622X₇ (r²=0.9975), its 95% confidence interval is less than single sodium or AG and the average forecast is more close to the time. While the equation of potassium(X₉) and AG(X₇) is Y₂=7.42380X₉ +1.85974X₇-457.65495 (r²=0.9812), it does no contribution to confidence nor prediction precise. [Conclusions] The postmortem changes of DNA content in rabbit corneal epithelium basal cell have significant relationship with PMI. Considering estimating confidence interval, Area, Integral Optical Density and Average Gray parameter have some values for PMI investigation. Confidence interval analysis is essential for any multivariate regression equation. And a combine estimation of DNA content AG and VH sodium may be a way for veracity.