Red blood cells (RBCs) stressed by high temperature are similar to senescent or damaged RBCs in pathological conditions. RBCs can be efficiently labelled with 18F-fluorodeoxyglucose (FDG). The aim of this study was to assess stressed RBCs erythrophagocytosis and organ distribution in vivo with the application of 18F-FDG PET/CT. RBCs were induced under high temperature (48 °C) to prepare stressed RBCs. Fluorescence-activated cell sorting (FACS) was used to analyse reactive oxygen species (ROS) generation, intracellular Ca2+ concentration and membrane phosphatidylserine (PS) externalization of RBCs. 18F-FDG was used to label RBCs and assess the erythrophagocytosis. Finally, 18F-FDG PET/CT was applied to reveal and measure the organ distribution of stressed RBCs in mice. Compared with untreated RBCs, stressed RBCs decreased in cell volume and increased in ROS level, intracellular Ca2+ concentration, and PS exposure. RBCs could be labelled by 18F-FDG. Stressed RBCs tended to be phagocytosed by macrophages via assessment of FACS and radioactivity. 18F-FDG PET/CT imaging showed that stressed RBCs were mainly trapped in spleen, while untreated RBCs remained in circulation system. Thus, stressed RBCs can be effectively labelled by 18F-FDG and tend to be trapped in spleen of mice as assessed by PET/CT.
Anemia is a common complication of hepatitis B-related acute-on-chronic liver failure (HB-ACLF). Eryptosis, a suicidal erythrocyte death characterized by phosphatidylserine (PS) externalization and red blood cell-derived microparticle (RMP) generation, decreases erythrocyte lifespan. Herein, we investigated whether enhanced eryptosis is involved in the anemia pathophysiology associated with HB-ACLF. PS exposure, cell volume, cytosolic Ca2+, and reactive oxygen species (ROS) production were determined using flow cytometry. RMPs were extracted using a polyethylene glycol (PEG)-based method. We found that hemoglobin (Hb) and hematocrit (Hct) were significantly lower in HB-ACLF patients than in healthy controls (HC), Chronic hepatitis B (CHB) and cirrhosis. The direct antiglobulin test positive rate was 75.9% in HB-ACLF patients, while its intensity associated with anemia. The ratio of abnormal erythrocytes was higher in HB-ACLF patients than in HC, CHB, and cirrhosis patients. The percentage of PS-exposed erythrocytes was higher in HB-ACLF patients (2.07% ± 0.11%) compared with HC (0.37% ± 0.05%), CHB (0.38% ± 0.03%), and cirrhosis (0.38% ± 0.04%). The cytosolic Ca2+ and ROS abundance were also higher in HB-ACLF patients compared with HC, CHB, and cirrhosis patients, and were inversely correlated with the anemia in HB-ACLF patients. PS exposure of erythrocytes collected from HC was significantly pronounced following incubation in plasma from HB-ACLF patients compared with incubation in plasma from HC. The protein concentration and RMPs size significantly increased in HB-ACLF patients compared with HC. Thus, the anemia in patients with HB-ACLF is associated with increased eryptosis, which is partially triggered by increased cytosolic Ca2+ and oxidative stress.
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