Analysis of sensitivity and specificity:precise recognition of neutrophils during regeneration of contused skeletal muscle in rats

In this report,we applied the TissueFAXS 200 digital pathological analysis system to rapidly and accurately identify neutrophils during regeneration of contused skeletal muscle,and to provide information for follow-up studies on neutrophils to estimate wound age.Rat injury model was established,and skeletal muscle samples were obtained from the control group and contusion groups at 1,1.5,2,3,4,and 6 h,as well as at 1,3,5,and 15 d post-injury(n紏5 per group).The expression of nuclei and neutrophils was detected by hematoxylin and eosin(HE)staining and immunohistochemical(IHC)staining.A total of 20 injury site areas of 0.25mm^(2)(0.5mm0.5mm)were then randomly selected at all time points.A TissueFAXS 200 digital pathological analysis system was used to identify the positive and negative numbers.Knowledge of five professional medical workers were considered the gold standard to measure the false positive rate(FPR),false negative rate(FNR),sensitivity,specificity,and area under the curve(AUC)of receiver operating characteristic(ROC)curves.As a result,with a staining area of neutrophils from 8 μm^(2) to 15 μm^(2),the FPR was 4.28%–12.14%,the FNR was 12.42%–64.08%,the sensitivity was 35.92%–87.58%,the specificity was 87.86%–95.72%,the Youden index was 0.316–0.754,the accuracy was 82.80%–88.30%,and the AUC was 0.771–0.826.The AUC was largest when the cut-off value of the staining area was 12 mm^(2).Our results show that this software-based method is more accurate than the human eye in evaluating neutrophil infiltration.Based on the sensitivity and specificity,neutrophils can be accurately identified during regeneration of contused skeletal muscle.The TissueFAXS 200 digital pathological analysis system can also be used to optimize conditions for different cell types under various injury conditions to determine the optimal cut-off value of the staining area and provide optimal conditions for further study.Furthermore,it will provide evidence for forensic pathology cases.

Vitality and wound-age estimation in forensic pathology:review and future prospects

Determining the age of a wound is challenging in forensic pathology,but it can contribute to the reconstruction of crime scenes and lead to arrest of suspects.Forensic scholars have tended to focus on evaluating wound vitality and determining the time elapsed since the wound was sustained.Recent progress in forensic techniques,particularly high-throughput analyses,has enabled evaluation of materials at the cellular and molecular levels,as well as simultaneous assessment of multiple markers.This paper provides an update on wound-age estimation in forensic pathology,summarizes the recent literature,and considers useful additional informa-tion provided by each marker.Finally,the future prospects for estimating wound age in foren-sic practise are discussed with the hope of providing something useful for further study.

Screening criteria of mRNA indicators for wound age estimation

Wound age estimation is a crucial and challenging problem in forensic pathology.Although mRNA is the most commonly used indicator for wound age estimation,screening criteria are lacking.In the present study,the feasibility of screening criteria using mRNA to determine injury time based on the adenylate-uridylate-rich element(ARE)structure and gene ontology(GO)categories were evaluated.A total of 78 Sprague-Dawley male rats were contused and sampled at 4,8,12,16,20,24,28,32,36,40,44,and 48 h after inflicting injury.The candidate mRNAs were classified based on with or without ARE structure and GO category function.The mRNA expression levels were detected using qRT-PCR.In addition,the standard deviation(STD),mean deviation(MD),relative average deviation(d%),and coefficient of variation(CV)were calculated based on mRNA expression levels.The CV score(CVs)and the CV of CV(CV’CV)were calculated to measure heterogeneity.Finally,based on classic principles,the accuracy of combination of candidate mRNAs was assessed using discriminant analysis to construct a multivariate model for inferring wound age.The results of homogeneity evaluation of each group based on CVs were consistent with the MD,STD,d%,and CV results,indicating the credibility of the evaluation results based on CVs.The candidate mRNAs without ARE structure and classified as cellular component(CC)GO category(ARE-CC)had the highest CVs,showing the mRNAs with these characteristics are the most homogenous mRNAs and best suited for wound age estimation.The highest accuracy was 91.0%when the mRNAs without ARE structure were used to infer the wound age based on the discrimination model.The accuracy of mRNAs classified into CC or multiple function(MF)GO category was higher than mRNAs in the biological process(BP)category.In all subgroups,the accuracy of the composite identification model of mRNA composition without ARE structure and classified as CC was higher than other subgroups.The mRNAs without ARE structure and belonging to the CC GO category were more homogenous,showed higher accuracy for estimating wound age,and were appropriate for rat skeletal muscle wound age estimation.