Cellular and biochemical heterogeneity contributes to the phenotypic diversity of transfusion-dependent beta thalassemia

• Red cells in TDT exhibit unique ultrastructural distortions, susceptibility to oxidants, and reduced proteasome activity

• The wide heterogeneity in RBC features, EVs, and plasma metabolome is a function of patient and transfusion-related variables

Transfusion-dependent thalassemia (TDT) is a type of protein aggregation disease. Its clinical heterogeneity imposes challenges in effective management. Red blood cell (RBC) variables may be clinically relevant as mechanistic parts or tellers of TDT pathophysiology. This is a cross-sectional study of RBC and plasma physiology in adult TDT subjects versus healthy control. TDT plasma was characterized by increased protein carbonylation, antioxidants, and larger than normal extracellular vesicles. RBCs were osmotically resistant but prone to oxidative hemolysis. They overexposed phosphatidylserine and exhibited pathologically low proteasome proteolytic activity (PPA), that correlated with metabolic markers of the disease. RBC ultrastructure was distorted, with splenectomy-related membrane pits of 300-800 nm. Plasma metabolomics revealed differences in heme metabolism, redox potential, short-chain fatty acids, and NO bioavailability, but also in catecholamine pathways. According to coefficient of variation assessment, hemolysis, iron homeostasis, PPA and phosphatidylserine exposure were highly variable among patients, as opposed to RBC fragility, and plasma antioxidants, amino acids, and catecholamines. Sex-based differences were detected in hemolysis, redox and energy variables, while splenectomy-related differences referred to thrombotic risk, RBC morphology, and plasma metabolites with neuroendocrine activity. Hepcidin varied according to oxidative hemolysis and metabolic markers of bacterial activity. Subjects with higher (>10 g/dL) pretransfusion Hb levels presented mildly distorted profiles and lower membrane-associated PPA, while classification by severity of mutations revealed different levels of hemostasis, inflammation, plasma epinephrine, hexosamines, and methyltransferase activity markers. The currently reported heterogeneity of cellular and biochemical features probably contributes to the wide phenotypic diversity of TDT at clinical level.