Journal of Forensic Medicine

Postmortem Interval Estimation Using Protein Chip Technology Combined with Multivariate Analysis Methods

Xu-dong ZHANG, Yao-ru JIANG, Xin-rui LIANG, Tian TIAN, Qian-qian JIN, Xiao-hong ZHANG, Jie CAO, Qiu-xiang DU, Jun-hong SUN

2023, 39(2): 115-120. DOI: 10.12116/j.issn.1004-5619.2022.420407

Abstract ( 383 )

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Objective To estimate postmortem interval （PMI） by analyzing the protein changes in skeletal muscle tissues with the protein chip technology combined with multivariate analysis methods. Methods Rats were sacrificed for cervical dislocation and placed at 16 ℃. Water-soluble proteins in skeletal muscles were extracted at 10 time points （0 d, 1 d, 2 d, 3 d, 4 d, 5 d, 6 d, 7 d, 8 d and 9 d） after death. Protein expression profile data with relative molecular mass of 14 000-230 000 were obtained. Principal component analysis （PCA） and orthogonal partial least squares （OPLS） were used for data analysis. Fisher discriminant model and back propagation （BP） neural network model were constructed to classify and preliminarily estimate the PMI. In addition, the protein expression profiles data of human skeletal muscles at different time points after death were collected, and the relationship between them and PMI was analyzed by heat map and cluster analysis. Results The protein peak of rat skeletal muscle changed with PMI. The result of PCA combined with OPLS discriminant analysis showed statistical significance in groups with different time points （P<0.05） except 6 d, 7 d and 8 d after death. By Fisher discriminant analysis, the accuracy of internal cross-validation was 71.4% and the accuracy of external validation was 66.7%. The BP neural network model classification and preliminary estimation results showed the accuracy of internal cross-validation was 98.2%, and the accuracy of external validation was 95.8%. There was a significant difference in protein expression between 4 d and 25 h after death by the cluster analysis of the human skeletal muscle samples. Conclusion The protein chip technology can quickly, accurately and repeatedly obtain water-soluble protein expression profiles in rats’ and human skeletal muscles with the relative molecular mass of 14 000-230 000 at different time points postmortem. The establishment of multiple PMI estimation models based on multivariate analysis can provide a new idea and method for PMI estimation.

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Cytotoxicity of 4 Wild Mushrooms in a Case of Yunnan Sudden Unexplained Death

Wu LONG, Peng-fei QU, Lin MA, Rui WANG, Yan-mei XI, Yu-hua LI, Sheng-jie NIE, Ting DUAN, Jin-liang DU, Xue TANG, Jing-feng ZHAO, Pu-ping LEI, Yue-bing WANG

2023, 39(2): 121-128. DOI: 10.12116/j.issn.1004-5619.2021.310802

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Objective To explore the cytotoxicity of four wild mushrooms involved in a case of Yunnan sudden unexplained death （YNSUD）, to provide the experimental basis for prevention and treatment of YNSUD. Methods Four kinds of wild mushrooms that were eaten by family members in this YNSUD incident were collected and identified by expert identification and gene sequencing. Raw extracts from four wild mushrooms were extracted by ultrasonic extraction to intervene HEK293 cells, and the mushrooms with obvious cytotoxicity were screened by Cell Counting Kit-8 （CCK-8）. The selected wild mushrooms were prepared into three kinds of extracts, which were raw, boiled, and boiled followed by enzymolysis. HEK293 cells were intervened with these three extracts at different concentrations. The cytotoxicity was detected by CCK-8 combined with lactate dehydrogenase （LDH） Assay Kit, and the morphological changes of HEK293 cells were observed under an inverted phase contrast microscope. Results Species identification indicated that the four wild mushrooms were Butyriboletus roseoflavus, Boletus edulis, Russula virescens and Amanita manginiana. Cytotoxicity was found only in Amanita manginiana. The raw extracts showed cytotoxicity at the mass concentration of 0.1 mg/mL, while the boiled extracts and the boiled followed by enzymolysis extracts showed obvious cytotoxicity at the mass concentration of 0.4 mg/mL and 0.7 mg/mL, respectively. In addition to the obvious decrease in the number of HEK293 cells, the number of synapses increased and the refraction of HEK293 cells was poor after the intervention of Amanita manginiana extracts. Conclusion The extracts of Amanita manginiana involved in this YNSUD case has obvious cytotoxicity, and some of its toxicity can be reduced by boiled and enzymolysis, but cannot be completely detoxicated. Therefore, the consumption of Amanita manginiana is potentially dangerous, and it may be one of the causes of the YNSUD.

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Sex Estimation of Medial Aspect of the Ischiopubic Ramus in Adults Based on Deep Learning

Yong-gang MA, Yong-jie CAO, Yi-hua ZHAO, Xin-jun ZHOU, Bin HUANG, Gao-chao ZHANG, Ping HUANG, Ya-hui WANG, Kai-jun MA, Feng CHEN, Dong-chuan ZHANG, Ji ZHANG

2023, 39(2): 129-136. DOI: 10.12116/j.issn.1004-5619.2022.220505

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Objective To investigate the reliability and accuracy of deep learning technology in automatic sex estimation using the 3D reconstructed images of the computed tomography （CT） from the Chinese Han population. Methods The pelvic CT images of 700 individuals （350 males and 350 females） of the Chinese Han population aged 20 to 85 years were collected and reconstructed into 3D virtual skeletal models. The feature region images of the medial aspect of the ischiopubic ramus （MIPR） were intercepted. The Inception v4 was adopted as the image recognition model, and two methods of initial learning and transfer learning were used for training. Eighty percent of the individuals’ images were randomly selected as the training and validation dataset, and the remaining were used as the test dataset. The left and right sides of the MIPR images were trained separately and combinedly. Subsequently, the models’ performance was evaluated by overall accuracy, female accuracy, male accuracy, etc. Results When both sides of the MIPR images were trained separately with initial learning, the overall accuracy of the right model was 95.7%, the female accuracy and male accuracy were both 95.7%; the overall accuracy of the left model was 92.1%, the female accuracy was 88.6% and the male accuracy was 95.7%. When the left and right MIPR images were combined to train with initial learning, the overall accuracy of the model was 94.6%, the female accuracy was 92.1% and the male accuracy was 97.1%. When the left and right MIPR images were combined to train with transfer learning, the model achieved an overall accuracy of 95.7%, and the female and male accuracies were both 95.7%. Conclusion The use of deep learning model of Inception v4 and transfer learning algorithm to construct a sex estimation model for pelvic MIPR images of Chinese Han population has high accuracy and well generalizability in human remains, which can effectively estimate the sex in adults.

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Correlation between Elbow Flexor Muscle Strength and Needle Electromyography Parameters after Musculocutaneous Nerve Injury

Dong GAO, Pei-pei ZHUO, Dong TIAN, Dan RAN, Qing XIA, Wen-tao XIA

2023, 39(2): 137-143. DOI: 10.12116/j.issn.1004-5619.2022.221104

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Objective To explore the changes of elbow flexor muscle strength after musculocutaneous nerve injury and its correlation with needle electromyography （nEMG） parameters. Methods Thirty cases of elbow flexor weakness caused by unilateral brachial plexus injury （involving musculocutaneous nerve） were collected. The elbow flexor muscle strength was evaluated by manual muscle test （MMT） based on Lovett Scale. All subjects were divided into Group A （grade 1 and grade 2, 16 cases） and Group B （grade 3 and grade 4, 14 cases） according to their elbow flexor muscle strength of injured side. The biceps brachii of the injured side and the healthy side were examined by nEMG. The latency and amplitude of the compound muscle action potential （CMAP） were recorded. The type of recruitment response, the mean number of turns and the mean amplitude of recruitment potential were recorded when the subjects performed maximal voluntary contraction. The quantitative elbow flexor muscle strength was measured by portable microFET 2 Manual Muscle Tester. The percentage of residual elbow flexor muscle strength （the ratio of quantitative muscle strength of the injured side to the healthy side） was calculated. The differences of nEMG parameters, quantitative muscle strength and residual elbow flexor muscle strength between the two groups and between the injured side and the healthy side were compared. The correlation between elbow flexor manual muscle strength classification, quantitative muscle strength and nEMG parameters was analyzed. Results After musculocutaneous nerve injury, the percentage of residual elbow flexor muscle strength in Group B was 23.43% and that in Group A was 4.13%. Elbow flexor manual muscle strength classification was significantly correlated with the type of recruitment response, and the correlation coefficient was 0.886 （P<0.05）. The quantitative elbow flexor muscle strength was correlated with the latency and amplitude of CMAP, the mean number of turns and the mean amplitude of recruitment potential, and the correlation coefficients were -0.528, 0.588, 0.465 and 0.426 （P<0.05）, respectively. Conclusion The percentage of residual elbow flexor muscle strength can be used as the basis of muscle strength classification, and the comprehensive application of nEMG parameters can be used to infer quantitative elbow flexor muscle strength.

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Structural Analysis and Characterization of 4-F-α-PVP Analog 4-F-3-Methyl-α- PVP Hydrochloride

Hao-yang WANG, Jian WU, Qian ZHANG, Xin-feng MIN, Xiu-yan LIU, Yin-long GUO

2023, 39(2): 144-150. DOI: 10.12116/j.issn.1004-5619.2021.311002

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Objective To identify 1-（4-fluorophenyl）-2-（1-pyrrolidinyl） pentan-1-one （4-F-α-PVP） analog 1-（4-fluoro-3-methyl phenyl）-2-（1-pyrrolidinyl） pentan-1-one （4-F-3-Methyl-α-PVP） hydrochloride without reference substance. Methods The direct-injection electron ionization-mass spectrometry （EI-MS）, GC-MS, electrospray ionization-high resolution mass spectrometry （ESI-HRMS）, ultra-high performance liquid chromatography-high resolution tandem mass spectrometry （UPLC-HRMS/MS）, nuclear magnetic resonance （NMR）, ion chromatography and Fourier transform infrared spectroscopy （FTIR） were integrated utilized to achieve the structural analysis and characterization of the unknown compound in the sample, and the cleavage mechanism of the fragment ions was deduced by EI-MS and UPLC-HRMS/MS. Results By analyzing the direct-injection EI-MS, GC-MS, ESI-HRMS and UPLC-HRMS/MS of the compound in the samples, it was concluded that the unknown compound was a structural analog of 4-F-α-PVP, possibly with one more methyl group in the benzene ring. According to the analysis results of ¹H-NMR and ¹³C-NMR, it was further proved that the methyl group is located at the 3-position of the benzene ring. Since the actual number of hydrogen in ¹H-NMR analysis was one more than 4-F-3-Methyl-α-PVP neutral molecule, it was inferred that the compound existed in the form of salt. Ion chromatography analysis results showed that the compound contained chlorine anion （content 11.14%-11.16%）, with the structural analysis of main functional group information by FTIR, the unknown compound was finally determined to be 4-F-3-Methyl-α-PVP hydrochloride. Conclusion A comprehensive method using EI-MS, GC-MS, ESI-HRMS, UPLC-HRMS/MS, NMR, ion chromatography and FTIR to identify 4-F-3-Methyl-α-PVP hydrochloride in samples is established, which will be helpful for the forensic science laboratory to identify this compound or other analog compounds.

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Analysis of Forty-Two Psychoactive Substances in a Single Hair by Micro-Segmental Technique

Jiao-jiao JI, Duo-qi XU, Ping XIANG, Hui YAN, Min SHEN

2023, 39(2): 151-160. DOI: 10.12116/j.issn.1004-5619.2022.321101

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Objective To establish an LC-MS/MS method based on single hair micro-segmental technique, and verify the detection of 42 psychoactive substances in 0.4 mm hair segments. Methods Each piece of single hair was cut into 0.4 mm segments and extracted by sonication and the segments were immersed in dithiothreitol-containing extraction medium. Mobile phase A was the aqueous solution containing 20 mmol/L ammonium acetate, 0.1% formic acid, and 5% acetonitrile. Mobile phase B was acetonitrile. An electrospray ionization source in positive ion mode was used for data acquisition in multiple reaction monitoring （MRM） mode. Results The 42 psychoactive substances in hair had a good linear relationship within their respective linear ranges （r>0.99）, the limits of detection were 0.2-10 pg/mm, the limits of quantification were 0.5-20 pg/mm, the intra-day and inter-day precisions were 1.5%-12.7%, the intra-day and inter-day accuracies were 86.5%-109.2%, the recovery rates were 68.1%-98.2%, and the matrix effects were 71.3%-111.7%. The method was applied to hair samples collected from one volunteer at 28 d after a single dose of zolpidem, with zolpidem detected in 5 hairs was 1.08-1.60 cm near the root tip, and the concentration range was 0.62-20.5 pg/mm. Conclusion The micro-segmental technique of single hair analysis can be applied to the investigation of drug-facilitated sexual assault cases.

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Animal DNA Typing and Its Research Progress in Forensic Medicine

Hong-yan GAO, Guang-fu LIU, Jian WU, Peng-yu CHEN

2023, 39(2): 161-167. DOI: 10.12116/j.issn.1004-5619.2021.511210

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With the advance of molecular biology, DNA analysis technology has been widely applied in forensic science. Non-human DNA analysis can be used in some special cases and has unique forensic value to provide investigation clues and trial basis. Animal DNA typing plays a more prominent role in the detection of all kinds of non-human DNA related cases and is the main content of forensic non-human DNA analysis. This paper reviews the development history, present situation, advantages and disadvantages of animal DNA typing according to its technology, characteristic, challenges facing forensic science application scenarios, and also its future development.

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Progress in the Application of Magnetic Nanoparticles in Forensic Trace Analysis

Zhong-ping CHENG, Yan-fei LIU, Xing-min XU, Yao-nan MO

2023, 39(2): 168-175. DOI: 10.12116/j.issn.1004-5619.2022.320202

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Given the complexity of biological samples and the trace nature of target materials in forensic trace analysis, a simple and effective method is needed to obtain sufficient target materials from complex substrates. Magnetic nanoparticles （MNPs） have shown a wide range of application value in many research fields, such as biomedicine, drug delivery and separation, due to their unique superparamagnetic properties, stable physical and chemical properties, biocompatibility, small size, high specific surface area and other characteristics. To apply MNPs in the pretreatment of forensic materials, maximize the extraction rate of the target materials, and minimize interference factors to meet the requirements of trace analysis of the target materials, this paper reviews the application of MNPs in the fields of forensic toxicological analysis, environmental forensic science, trace evidence analysis and criminal investigation in recent years, and provides research ideas for the application of MNPs in forensic trace analysis.

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Research Progress on Biological Matrix Reference Materials in Forensic Toxicology

Hang CHEN, Jing HU, Zheng QIAO, Hong-xiao DENG, Min LÜ, Wei LIU

2023, 39(2): 176-185. DOI: 10.12116/j.issn.1004-5619.2022.320801

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Biological matrix reference material is a reference material that combines the target material with the biological matrix. The biological matrix reference material has higher consistency with the authentic specimens in forensic toxicology, and its application has a positive effect on improving the accuracy of test results. This paper reviews the research on the matrix reference materials corresponding to three common biological test materials （blood, urine and hair）. In order to provide reference for the development and application of biological matrix reference materials in forensic toxicology, this paper mainly introduces the research progress of preparation technology of biological matrix reference materials and some existing products and their parameters evaluation.

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Survey on the Construction Status of Forensic Virtual Autopsy Laboratory and the Applicability of Laboratory Accreditation

Jun-wei GAO, Yang LU, Yan-jun LI, Dong-hua ZOU, Guang-long HE, Yan-bin WANG

2023, 39(2): 186-192. DOI: 10.12116/j.issn.1004-5619.2022.421109

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Objective To survey the development status and actual needs of virtual autopsy technology in China and to clarify the applicability of forensic virtual autopsy laboratory accreditation. Methods The questionnaire was set up included three aspects：（1） the current status of virtual autopsy technology development; （2） the accreditation elements such as personnel, equipment, entrustment and acceptance, methods, environmental facilities; （3） the needs and suggestions of practicing institutions. A total of 130 forensic pathology institutions were surveyed by online participation through the Questionnaire Star platform. Results Among the 130 institutions, 43.08% were familiar with the characteristics of virtual autopsy technology, 35.38% conducted or received training in virtual autopsy, and 70.77% have establishment needs （including maintenance）. Relevant elements were suitable for laboratory accreditation. Conclusion Virtual autopsy identification has gained social recognition. There is a demand for accreditation of forensic virtual autopsy laboratory. After the preliminary assessment, considering the characteristics and current situation of this technology, China National Accreditation Service for Conformity Assessment （CNAS） can first carry out the accreditation pilot of virtual autopsy project at large comprehensive forensic institutions with higher identification capability, and then CNAS can popularize the accreditation in a wide range when the conditions are suitable.

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Exploration and Practice of the “One Combination， Two Highlights， Three Combinations， Four in One” Innovative Talents Training Mode in Forensic Medicine

Jiang-wei YAN, Jun-hong SUN, Hong-xing WANG, Zhi-wen WEI, Xiang-jie GUO, Ji LI, Cai-rong GAO, Geng-qian ZHANG, Xin-hua LIANG, Qiang-qiang ZHANG, Hong-wei WANG, Si-jin LI, Ying-yuan WANG, Ke-ming YUN

2023, 39(2): 193-199. DOI: 10.12116/j.issn.1004-5619.2023.430101

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Talent is one of the basic and strategic supports for building a modern socialist country in all aspects. Since the 1980s, the establishment of forensic medicine major and the cultivation of innovative talents in forensic medicine have become hot topics in higher education in forensic medicine. Over the past 43 years, the forensic medicine team of Shanxi Medical University has adhered to the joint education of public security and colleges, and made collaborative innovation, forming a training mode of “One Combination, Two Highlights, Three Combinations, Four in One” for innovative talents in forensic medicine. It has carried out “5+3/X” integrated reform, and formed a relatively complete talent training innovation mode and management system in teaching, scientific research, identification, major, discipline, team, platform and cultural construction. It has made a historic contribution to China’s higher forensic education, accumulated valuable experience for the construction of first-class major and first-class discipline of forensic medicine, and provided strong support for the construction of the national new forensic talent training system. The popularization of this training mode is conducive to the rapid and sustainable development of forensic science, and provides more excellent forensic talents for national building, regional social development and the discipline construction of forensic science.

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