Peripheral blood lymphocyte subpopulations as a marker of severe mechanical soft tissue injury in rats: an immunocytochemical study and ROC analysis

BACKGROUND: Injuries and burns are accompanied by changes in lymphocyte subpopulations, reflecting the immune system's response. ROC (receiver operating characteristic) analysis is a classification method that uses ROC curves. An ROC curve is a graph that evaluates the quality of binary classification; it shows the relationship between the proportion of true positives (sensitivity) and the proportion of false positives (1 – specificity). Using an ROC curve, one can identify the main diagnostic criteria for trauma severity and determine the post-traumatic stage based on the total lymphocyte count and the percentage of various subpopulations (CD3+, CD4+, CD8+, CD19+, CD56+). This approach enables prediction of the main pathogenetic mechanisms of wound healing. AIM: To study, using immunocytochemical methods, the responses of different peripheral blood lymphocyte types (T cells, B cells, and NK cells) to mechanical soft tissue injury of the thigh in rats. METHODS: The study was performed on outbred male white rats weighing 180–200 g. Using a special device (under ether anesthesia), a mechanical thigh injury comparable in kinetic energy to that caused by a 5.6 mm bullet was induced in the animals. The animals were euthanized by decapitation at 3 and 14 days after injury in the morning; blood was collected from the tail vein beforehand. Quantitative assessment of peripheral blood lymphocyte subpopulations was performed using a panel of monoclonal antibodies; the relative and absolute counts of the following subpopulations were determined: CD3+ (all T lymphocytes); CD4+ (T-helpers); CD8+ (cytotoxic T lymphocytes); CD19+ (B lymphocytes); and CD56+ (NK cells). Statistical analysis (ROC analysis) was performed using STATISTICA v.7.0 (StatSoft Inc., USA). RESULTS: ROC analysis accurately evaluates the quality of binary classification – the effect of a given factor on the state of the components of the system under study. Here, using ROC analysis, we demonstrated the effect (yes/no) of high-kinetic soft tissue injury of the thigh and the effect of time (3 and 14 days after injury) on the relative counts of individual lymphocyte subpopulations in the peripheral blood of rats. CONCLUSION: On day 3 post-injury, the relative counts of all lymphocyte subpopulations had changed in response to the injury. By day 14, changes in ROC curves were observed only for B lymphocytes and NK cells. These data indicate that the addition of the time factor primarily affects B lymphocytes and NK cells but does not affect T lymphocytes.