Ultrasound Measurements Of Facial Soft Tissue Thickness And RNA Analysis Of Bloodstain And Their Applications On Facial Soft Tissue Reconstruction And Postmortem Interval Deduction

Personal identification and postmortem interval deduction are two important parts in forensicinvestigation when skeletalized remains were found at the scene. If there are some clues about thedeceased, DNA analysis, dental analysis and superimposing technique can be used to identify thebody, however, these methods can’t be used into the clueless remains.For the identification of the remains of no clue, some scholars began to use craniofacialreconstruction technique since19th century. Craniofacial reconstruction are integrated techniquesto reproduce the face of the dead by means of painting, sculpture and computer imagingtechnology taking the skull as a framework and using facial anatomical knowledge. Craniofacialreconstruction includs facial soft tissue reconstruction and facial organ reconstruction, facial softtissue reconstruction is the basis of facial organ reconstruction. Facial soft tissue reconstructionneed to determine the Facial Soft Tissue Thickness (FSTT) at the selected landmarks (landmarkcan also be called point). However, in the past, the selection of the landmarks depended on theattention of researchers and varied greatly, and the FSTT data are more from the dead body,inevitable error will be caused by tissue dehydration, muscle relaxation, rigor mortis and someother postmortem changes and the impact of embalming. Furthermore, since the bodies aredifficult to reach, so the data acquisition is limited. Also, due to indentation and subsequent resetof the indentation, the stabbing or needling method to collect FSTT data will be deviated to somedegree. Recently, X-ray, CT, MRI and ultrasound has been tried to collect FSTT data fromsubjects in vivo. Ultrasonic detection is free of radiation, and cost less, making data collection of alarger sample size easier.Therefore it is considered to be an ideal way to get FSTT data. Existingin vivo studies of Caucasians, Nigroids are more than Mongoloids.The FSTT data for Chinese isalso scarce, and most come more from methods like needling, X-ray.The impact factors of FSTTsuch as gender, age and race is still not clear.In addition to identification of clueless skeletal remains with craniofacial reconstructiontechnique, the judgement of postmortem interval is also very important issue. Until now, there isno accurate method for postmortem interval deduction of skeletal remains, some methods usednow including judging from the corruption degree or skeletalization degree,insect activity, growthof surrounding plants and some remnants at the scene such as watch. From our view point,postmortem interval can be deduced from bloodstain age. There are a variety of methods forbloodstains detection under research, such as high performance liquid chromatographic analysis ofhemoglobin degradation product, Bloodstain color change detecting using spectrum,oxyhemoglobin detection with oxygen electrode, electricity paramagnetic ultrasonic testing of the spin state of the iron in hemoglobin, red blood cell flexibility change analysis with atomicforce microscopy, plasma protein electrophoresis, UV absorption method for the determination ofplasma enzyme activities, RNA degradation detection of bloodstains using real-time quantitativePCR. About the analysis of RNA degradation to speculate bloodstains age, some researchers havealready confirmed rRNA and mRNA degradation rate in bloodstains are different, rRNA degradeslowly and mRNA decay relatively quickly.The ratio of both detected by real-time quantitativePCR show increase with bloodstain age, i.e. the postmortem interval can be presumed according tothe bloodstain RNA ratio.In this work, in order to try to use ultrasound technology to collect Chinese FSTT data andanalyze the impact factors, we established landmarks system with both the skeletal position andsoft tissue position, and collected FSTT data of153volunteers and investigated the influences ofright and left side of face, sex, BMI, race and etc. In addition, in order to study the relationshipbetween bloodstains RNA ratio and the time of death, we explored the bloodstains18S rRNA andβ-actin mRNA ratio changes over time of16volunteers.The main results are as follows:1The establishment of FSTT landmark systemIn order to collect the FSTT data of Chinese for craniofacial reconstruction, we need to createa facial landmark system. For landmark selection and positioning, Aulsebrook WA (1996) hasproposed several principles: similar with the choice of other authors in order to facilitatecomparison; landmark positionings are the same for all subjects; best at a relatively flat surface ofthe bone; try to select the bone bump like Glabella and Gonion, or in sunken area like Nasion andMentolabial sulcus. In accordance with the above principles and landmarks selected by otherauthors, we have established a landmark positioning system with both skeletal description and softtissue discription.2Northern Han Chinese young people’s FSTTWe collected FSTT data from153healthy college student volunteers, all aged21-25yearsold, of which113belong to Northern Han population (The individuals with deformity of the facehave been excluded). In the present study, the volunteers were photographed and FSTT data werecollected from the19landmarks we selected, taking into account the body feature of the Chinesepeople, we classified the data according to " Overweight and obesity prevention guide for Chineseadults "BMI criteria (low weight BMI <18.5, normal weight BMI18.5-23.9, overweightBMI24-29.9, obese BMI>30).For male, normal BMI group are34people,underweight are17, lowweight are2. For female, normal BMI group are38people, overweight group are2,underweightgroup are20. From BMI grouped FSTT data, only men Nasion, Alare curvature, Infra-canine andGonion didn’t increase with BMI,women Mid-lateral orbit, Rhinion and Mid-philtrum pointthickness did not increase with increasing BMI. However, taking into account the BMI groupingincluding the small sample like2people in male underweith group, and2in female overweithtgroup, the small group may lead to the deviation of the result, so we excluded the two groups of data and found that except the women Rhinion, all landmarks FSTT increased with the increasingBMI. This confirmed the BMI impact on FSTT. Overall, two landmarks with largest FSTT areSupra-M2and Infra-M2, of the cheek, and the variability of these two landmarks are also thelargest, which shows a greater difficulty of the buccal soft tissue reconstruction.3The inflences on FSTTIn order to clarify the influences on FSTT, we compared the detection results of the sittingposition and the supine position, the left and right side symmetry of FSTT, and gender, BMI andracial differences and etc.,the results are as follows:3.1Position impact on FSTTWe compared the ultrasound measurement results of12female and16male volunteers insitting and supine position, and did the t-test, found the sitting position measurement value ofSupra-canine, Infra-canine and Zygomatic bone are higher than supine positon (exceptInfra-canine point of women); while the measured values of Supra-M2, Infra-M2, Mid-mandibularand Gonion are lower in sitting position than in supine position. While at other landmarks themeasureing difference are not significant.This may due to the position change of subcutaneous fat,muscle and connective tissue with gravity. These results indicate that when the facial soft tissuereconstruction is carried out with the data obtained using different detection position, or whencomparing the data of different detection methods the positonal deviation of detection should benoted.3.2Bilateral symmetry of the FSTTFor the sake of symmetry study of human FSTT on left side and right side, we measuredFSTT on the left and right side by ultrasound of40volunteers (20males,20females), and carriedout statistical analysis t-test in FSTT data of the12side landmarks individually. Then we used thegeneral linear model analysis of the FSTT data of the whole landmarks of the left and right side.we didn’t find significant difference between the left and right FSTT measurement value, so theFSTT data of one side of the face is enough for facial reconstruction.3.3BMI impact on FSTTTo confirm the impact of BMI on FSTT, we first calculated the Pearson correlationcoefficient between the two, and found the FSTT of14landmarks correlated with BMIsignificantly in men, and the FSTT of11landmarks correlated with BMI significantly in women.There are9landmarks’ FSTT are significantly associated with BMI in both male and femalevolunteers.The landmarks of women which are significantly related to BMI are mainly distributedin the cheek, jaw and chin area.While the landmarks of men are more concentrated in the moutharea. Then we did the analysis of variance (ANOVA) between the different BMI groups and foundthe results are consistent with Pearson correlation results, the landmarks which show significantdifferences are the same with the significant landmarks in Pearson correlation test except Nasion.These results indicated that BMI had a significant effect on FSTT, grouping the FSTT dataaccording to BMI are necessary for facial soft tissue reconstruction. 3.4Gender impact on FSTTBecause all volunteers aged21-25years old, male and female age was not statisticallydifferent, and are all belong to the normal BMI group, so when we conduct the t-test we’vealready excluded the impact of age and BMI. The t-test results showed that FSTTmeasurements of men and women are obviously different in the following11landmarks:Mid-supraorbital, Mid-infraorbital, Mid-lateral orbital, Glabella, Alare curvature, Supra-canine,Infra-canine, Zygomatic bone, Zygomatic arch, Mid-mandibular, Pogonion.From the division ofareas, in addition to the three landmarks of the eye area, the nose area are Glabella and Alarecurvature, the mouth area are Supra-canine and Infra-canine, Zygomatic bone, Zygomatic arch ofthe cheek area and chin area Mentolabial sulcus and Menton. From this result, we found that thegender differences of FSTT landmarks are corresponding to skull gender differences at a certainextent, such as the male skull Glabella projections are often more than women, while the softtissue Glabella point FSTT are lower than women; men skull eyebrow protrusions are usuallyhigher than the female, and soft tissue thickness of the Mid-supraorbital point are lower than thefemale.3.5Racial differences of FSTTIn order to analyze the characteristics of FSTT in different races, we compared the reportedresults of different races. That is Northern Han Chinese (this paper) and Chinese-American the(Chan WN.et al,2011), African American (Manhein MH, et al.,2000) white Americans (ManheinMH, et al,2000), European white (De Greef S. et al.,2006).Overall, the FSTT of theChinese-American is relatively thin, and relatively thick in African Americans.From t-test resultswe found The FSTT different landmarks between Northern Han Chinese men andChinese-American are fewer,7landmarks, the FSTT different landmarks between Northern HanChinese men and white Americans are14, the FSTT different landmarks between Northern HanChinese men and African Americans are16, the FSTT different landmarks between Northern HanChinese men and European whites are11, and its distribution area are different. The differentlandmarks with Chinese Americans are mainly distributed in the mouth and chin area, the differentlandmarks with white Americans located in other regions outside the eye area, the differentlandmarks with African Americans located in every areas but less in eye, nose area, and thedifferent landmarks with European white are mainly distributed in other areas outside chin. TheFSTT different landmarks between Northern Han Chinese women and Chinese-American are9landmarks, the FSTT different landmarks between Northern Han Chinese women and whiteAmericans are10, the FSTT different landmarks between Northern Han Chinese women andAfrican Americans are10, the FSTT different landmarks between Northern Han Chinese womenand European whites are11, and its distribution area are different. The different landmarks withChinese Americans are mainly distributed in the mouth area and jaw area, the different landmarkswith white Americans located in mouth area, cheek area and jaw area,and the same for AfricanAmericans, and the different landmarks with European white are mainly distributed in mouth, nose and cheek area. These results indicate we should take into account racial differences duringfacial soft tissue reconstruction, and adopt the corresponding FSTT racial data.3.6The mutual relations of the19landmarksFor in-depth understanding of the links among the19landmarks，we did Pearson correlationcoefficient test for either two landmarks of the19. The correlation coefficients between the eithertwo points were filled in tabular form for both men and women. From the correlation coefficienttable for female, we can see the correlation coefficient between Mid-supraorbital, Infra-canine,Zygomatic bone, Zygomatic arch and Gonion and the other points are higher.At the same time, wealso observed the correlation coefficient between Rhinion, Alare curvature and Supra-canine andthe other points are generally lower. Compared with women, the correlation coefficient table formale show the correlation coefficient greater than0.3increased significantly, similar to women thecorrelation of Rhinion, Mid-philtrum and Supra-canine with the other points are also relativelysmall. From the analysis of the above data, it is found the points correlation coefficient smallerwith other point mark are located in the facial midline or near the midline, while the correlationcoefficient higher point is located at the side portion, i.e.the FSTT of the points at the side portionis easily impacted. Therefore when testing FSTT with ultrasonic method, the side point dataacquisition should be more careful, and requires the operator to have higher skills, the ultrasoundprobe as much as possible perpendicular to the potential bone surface, and wth the smallestpressure to soft tissue.In order to facilitate personal identification by craniofacial reconstruction, we collected theFSTT data of Chinese Han population with ultrasound and analyzed its influencing factors, theresults are described above. In order to determine the time of death of the deceased and providetime clues for personal identification, we choose to speculated the time of death according to thebloodstains age, so we carry out the following work to detect the bloodstain age: the observationof the color changes and the blood cell changes of the bloodstains, followed by RNA analysis ofdifferent aged bloodstains.4Bloodstain RNA analysisBloodstain color changed with time from bright red to dark brown irregularly, theerythrocytes and leukocytes in bloodstain didn’t show obvious change in half a year. To observebloodstain RNA ratio changes over time under room condition, we analyzed bloodstain18S rRNAand β-actin mRNA ratio of16healthy young Han (8men and8women) with real-timequantitative PCR, found the ratio of (18S: β-actin) gradually increased with time, and show linearrelationship with bloodstains age. Female ratio is slightly higher than the ratio of male.In summary, to reconstruct the face of the skeletal remains we established FSTT landmarksystem including skeletal positioning and soft tissue positioning, collected the FSTT ultrasonicdata of selected landmarks in northern Chinese Han youth. From FSTT data analysis, we foundthat the body position of detection, BMI, gender and race had a significant effect on the FSTTdetection results. By studying bloodstain aging degrees, we found bloodstain18S rRNA and β-actin mRNA ratio rise in28days with time, and the ratio of women was slightly higher than thatof their male counterparts. The results of this study suggest that the combination of bloodstainsRNA analysis and craniofacial reconstruction technology will improve the reliability of personalidentification of skeletal remains of no clue by time cues and restoration the face of the deceased.