Investigating the phenomenon of lesion vitality on human ribs: From untargeted proteomics to the application of immunostainings for specific proteins

Introduction. The research upon vital reactions is a hot topic in forensic pathology [1]. However, there are very few studies which focus on the bone tissue and scientific data are currently limited to conventional histopathology [2-3]. Therefore, the possibility to establish the timing of skeletal traumatic injury is difficult especially for short survival times. For these reasons, bone marrow may represent a potentially useful substrate for the identification of bone lesion vitality. Furthermore, novel omics techniques can improve the investigation of reliable forensic biomarkers [4]. This study provides the application of proteomics on human bone marrow of traumatized ribs with the purpose to a) define a significant pattern of vital reaction on broken ribs versus undamaged ones; b) evaluate the proteomic changes over different known survival times; c) assess proteomic differences among resuscitation fractures versus other types of rib traumas (e.g., vehicle and train crashes, falling from heights). Furthermore, the most statistically significant proteins undergo an immunofluorescence or immunohistochemistry antibody-immunostaining to test their sensitivity, specificity and accuracy, respectively Material and methods. This study enrolls prospectively 25 cases with rib fractures and different survival times, and 15 controls with different causes of deaths but no traumatic injuries. Specifically, the survival times of cases include (7) < 1 hour, (4) = 1 hour, (4) = 2 hours, (5) = 5-10 hours, (2) 12 <24 h, (3) t > 24 h up to 96 h; ratio men-to-women is 1,5:1 and the mean age 55,04. The selection of the case samples is based on the highest hemorrhagic infiltration which can be detected macroscopically; for controls, sampling is always conventionally taken on the 4th right rib. Then, soft tissues around each sample are mechanically removed and frozen at -80°C. Proteomic analyses are performed on the extracted bone marrows and results are expressed over log fold changes. Statistical tests include ANOVA applying the false discovery rate (q<0,05). Then, immunohistochemical analysis is performed on 4 µm-thick paraffin-embedded tissue sections for carbonic anhydrase 2 (CAII) and complement C9. For immunofluorescence, sections are incubated with an anti-fibrinogen antibody which is visualized using a fluorescently labeled secondary antibody. Results. The trauma group significantly (q<0,001) overexpresses 555 acute-phase inflammatory proteins, different extracellular matrix proteins, and bone-related specific proteins. Noteworthy, some of these proteins show very interesting patterns such as C9 which provides the best significant results over time, fibrinogen which statistically increases starting from 4 h after trauma, and carbonic anhydrase 2 which is linearly overexpressed in the first 12 h. There are no statistical differences between resuscitation fractures versus other types of rib blunt injuries. Immunohistochemical staining for C9 reveals sensitivity and specificity of 64% and 46%, respectively, and for CAII both sensitivity and specificity of 60%. Immunofluorescent staining for fibrinogen yields sensitivity, specificity and overall accuracy of 72%, 80%, and 75%, respectively, with a positive predictive value of 85%. Conclusion. Untargeted proteomics applied to bone marrow of injured ribs gives the opportunity to identify proteins which can be used in forensic field for the research upon lesion vitality.