This study was a cross-sectional study conducted from March to May 2024, in the Therapy and Thalassemia Clinics, Universitas Indonesia Hospital. Study participants were adult transfusion-dependent thalassemia patients.

Clinicals data obtained from medical records included age, body weight, height, mean Hb levels over the past two months, packed red cells volume per kg body weight received for previous two months, and average ferritin levels over six months. The measurement of cIMT was performed after blood transfusion, performed by internist-cardiovascular consultant, using techniques in line with the 2012 Mannheim Carotid Intima-Media Thickness and Plaque Consensus. The carotid intima-media thickness was visualized as a double-line pattern between the intima-lumen and media-adventitia interfaces of the carotid artery in a longitudinal view using B-mode ultrasound. The measurement was taken near the carotid bifurcation at a plaque-free area in the distal common carotid artery segment. The artery wall was examined at a lateral probe position with a 90-degree insonation angle. Measurements were recorded for both the left and right distal common carotid artery walls, 5 mm proximal to the bifurcation. Data about cIMT diameter were collected for each wall and mean thickness values from both sides.^{[5, 6]}

Data analysis was performed using SPSS v. 28. Categorical data were shown in proportions, while numerical data were presented as mean or median. The Mann-Whitney test was used to assess rank differences of cIMT by age group, sex, body mass index (BMI), and packed red cell volume per kg body weight over the last two months. Spearman correlation tests examined relationship between Hb, ferritin, and cIMT (both maximum and mean diameter).

Ethical approval for the study was obtained from the Ethics Committee of FMUI, Ethics number KET-263/UN2.F1/ETIK/PPM.00.02/2024.

In this study, most subjects were male (60.0%) with a median age of 25 years (18.0–68.0), and an underweight nutritional status (52.0%). The mean pre-transfusion Hb level in this study was 8.55±0.76 g/dl, consistent with reports of thalassemia in Indonesia, which still faces a challenge in implementing the pre-transfusion Hb target of >9 g/dl, as well as poor adherence to iron chelation therapy.^[7] The mean ferritin level in this study was 5535.88±2792.67 ng/mL, which aligns with literature indicating that the proportion of subjects with ferritin levels >4000 ng/mL is higher in the Asia-Pacific and Middle East regions compared to Europe.^[8]

The median maximum diameter of cIMT observed in this study was 0.5 mm, which was larger than the 75th percentile diameter for populations under 30 years of age, both male (right carotid 0.48 mm, left carotid 0.49 mm) and female (right carotid 0.43 mm, left carotid 0.47 mm).‌^[9] Thalassemia has been associated with increased cIMT. Intravascular hemolysis in thalassemia would lead into increased free heme levels in the circulation. Free heme, a pro-atherogenic free radical, increases endothelial permeability, smooth muscle proliferation, and LDL oxidation.^[10] In thalassemia patients, the hemopexin levels, a glycoprotein that binds free heme, are also reduced, leading to higher free heme levels.^[11]

In this study, 52% of patients had underweight BMI, which is a higher proportion compared with other studies that reported 40% of subjects were underweight. Underweight in thalassemia patients had shown due to low percentage of body fat. Low body fat had been associated also with low bone mass in thalassemia patients.^[12] The pathogenesis of low BMI and altered body composition had multiple risk factors such as nutritional deficiency, chronic anemia and hypoxia, iron overload, inadequate use of chelating agents, and endocrinopathies.^[13]

This study found that subjects with an underweight BMI had thicker cIMT compared with those with a normal BMI. This finding aligns with the conclusions of previous studies, which indicated that patients with malnutrition exhibit thicker intima-media layers in comparison to those with adequate nutritional status.^[14–16] The relationship between malnutrition and increased cardiovascular risk, including cIMT, is associated with heightened systemic inflammation. Previous study reported that systemic inflammation was linked to malnutrition, marked by decreased prealbumin and albumin levels.^[16] Increased oxidative stress markers in thalassemia patients had been associated with elevated resting energy expenditure and eventually with being underweight. Meanwhile, oxidative stress had been associated with increased cIMT.^{[13, 17]}

Increased transfusion frequency has been linked to iron overload. Each unit of packed red cells is estimated to contain 200-250 mg of iron. Inadequate transfusion volume leads to more frequent transfusions. Studies have shown that patients with lower pre-transfusion Hb targets are associated with more frequent transfusions and increased iron levels compared with patients with higher pre-transfusion Hb targets. A lower pre-transfusion hemoglobin (Hb) level has been shown to be associated with hypersplenism, which has the potential to complicate the achievement of the pre-transfusion target level.^[18] Transfusions cause a temporary increase in serum iron and transferrin saturation within the first 24 hours, persisting up to 36 hours post-transfusion.^[19] Conversely, frequent blood donors had been shown thinner tunica intima-media thickness, as blood donation lowers body iron levels, and also ferritin and hepcidin level.^{[20, 21]}

Higher ferritin levels in thalassemia patients have been associated with increased intima-media thickness in previous studies.^{[10, 22]} Ferritin is a marker of iron overload in thalassemia patients. The iron overload condition has been known to be linked to increased free iron radicals, which heighten oxidative stress. Increased iron followed with increased reactive oxygen species formation, especially hydroxyl radicals. Hydroxyl radicals could oxidize low-density lipoprotein, which would initiate foam cell formation, and eventually atherosclerosis.^{[20, 21]} Elevated free iron radicals cause endothelial injury and accelerating atherosclerosis.^[21] Jindal et al.^[17] had reported that lipid peroxidation product malondialdehyde (MDA) increased in thalassemia patients. The study also reported correlation between MDA and cIMT in thalassemia subjects.

Increased ferritin also followed with increased hepcidin which caused iron retention in macrophages. The process of iron retention within the macrophages has been demonstrated to result in an increase in oxidative stress and lipid oxidation.^[23] Macrophage iron content had been known as a determinant factor in atherosclerotic plaque progression.^[24]