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 = 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 0.25 mm² (0.5 mm × 0.5 mm) 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 µm² to 15 µm², 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 µm². 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.

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