Surface-exposed calreticulin (ecto-CRT) plays a crucial role in the phagocytic removal of apoptotic cells during immunotherapy. Ecto-CRT is an immunogenic signal induced in response to treatment with chemotherapeutic agents such as doxorubicin (DOX) and mitoxantrone (MTX), and two peptides (KLGFFKR (Integrin-α) and GQPMYGQPMY (CRT binding peptide 1, Hep-I)) are known to specifically bind CRT. To engineer CRT-specific monobodies as agents to detect immunogenic cell death (ICD), we fused these peptide sequences at the binding loops (BC and FG) of human fibronectin domain III (FN3). CRT-specific monobodies were purified from E. coli by affinity chromatography. Using these monobodies, ecto-CRT was evaluated in vitro, in cultured cancer cell lines (CT-26, MC-38, HeLa, and MDA-MB-231), or in mice after anticancer drug treatment. Monobodies with both peptide sequences (CRT3 and CRT4) showed higher binding to ecto-CRT than those with a single peptide sequence. The binding affinity of the Rluc8 fusion protein–engineered monobodies (CRT3-Rluc8 and CRT4-Rluc8) to CRT was about 8 nM, and the half-life in serum and tumor tissue was about 12 h. By flow cytometry and confocal immunofluorescence of cancer cell lines, and by in vivo optical bioluminescence imaging of tumor-bearing mice, CRT3-Rluc8 and CRT4-Rluc8 bound specifically to ecto-CRT and effectively detected pre-apoptotic cells after treatment with ICD-inducing agents (DOX and MTX) but not a non-ICD-inducing agent (gemcitabine). Using CRT-specific monobodies, it is possible to detect ecto-CRT induction in cancer cells in response to drug exposure. This technique may be used to predict the therapeutic efficiency of chemo- and immuno-therapeutics early during anticancer treatment.

Background: Bleeding and thrombosis are major perioperative complications in liver transplantation (LT), with mortality rates up to 30%. Bleeding occurs in 0–10% of cases and is 2–3 times more frequent in children than in adults. This study aims to determine the diagnostic significance of specific laboratory tests in predicting adverse outcomes due to thrombohemorrhagic complications in LT. Methods: This prospective observational analytical study employed a case-control design involving 30 liver recipients. Patients were categorized into two groups based on outcomes: favorable or unfavorable (thrombosis/bleeding leading to fatal outcomes in the early postoperative period). Results: Among 30 patients, 17 (56.7%) had favorable outcomes without complications, while 13 (43.3%) experienced complications early postoperatively. Specifically, portal vein thrombosis (PVT) occurred in 7 (23.3%) cases, and fatal outcomes in the early postoperative period were observed in 5 (16.7%) cases. However, complications were successfully managed in 8 patients. Receiver operating characteristic (ROC) analysis demonstrated prognostic significance in predicting fatal thrombohemorrhagic complications post-LT for the following parameters: fibrinogen level below 160.1 mg/dL within the first day post-surgery showed a sensitivity of 89% and specificity of 96%, with an area under the ROC curve (AUC) of 0.896 (P<0.05), antithrombin-III levels below 15%, protein C levels below 17%, and plasminogen activity below 21% within the first day post-intervention. Intraoperative blood loss exceeding 1,200 mL was identified as a risk factor for hemorrhagic complications post-LT. Conclusions: Key predictors of fatal thrombohemorrhagic complications following LT include antithrombin-III levels below 15%, protein C levels below 17%, and plasminogen activity below 21% on the first day post-surgery. A fibrinogen level under 160.1 mg/dL on the first day post-surgery, demonstrating 89% sensitivity and 96% specificity, strongly predicts fatal complications. Intraoperative blood loss over 1,200 mL represents a significant risk factor for post-LT hemorrhagic complications.