Evaluating the Role of Red Blood Cell Lifespan in Transfusion-Dependent β-Thalassemia and Impact of Thalidomide Treatment

β-Thalassemia is characterized by ineffective erythropoiesis (IE), anemia, and iron overload. It involves both intramedullary apoptosis and the destruction of red blood cells (RBCs) owing to membranes developing abnormalities as a result of an excess of unpaired globin chains [1]. RBC destruction caused by IE or hemolysis shortens the lifespan of these cells. Although laboratory indicators can detect increased RBC destruction and compensatory hyperplasia in the bone marrow, studies performed on this to date have primarily relied on surrogate markers instead of direct measurements. Direct quantitative assessment of RBC lifespan is therefore essential for advancing thalassemia research and evaluating treatment strategies.

To enhance the interpretation of studies in which surrogate markers of RBC survival in β-thalassemia were used, the correlations between these markers and directly measured data should be clarified. Toward this goal, this report presents a prospective study examining the use of carbon monoxide (CO) breath tests to quantify RBC lifespan in patients with transfusion-dependent β-thalassemia (TDT). We determined the correlations of the obtained data with markers of hemolysis, erythropoiesis, iron regulation, and oxidative stress, and discussed the effects of thalidomide treatment on RBC lifespan in these patients.

RBC lifespan was assessed using an automatic device (ELS TESTER; Seekya Biotec Co. Ltd., Shenzhen, China). CO breath tests were conducted at least 2 weeks post-transfusion, after ensuring that the participants had not smoked within 24 h and had an empty stomach. The majority of TDT patients in this study were treated with thalidomide. Patients were informed of its benefits and side effects and warned against becoming pregnant or impregnating a woman while taking the drug. Thalidomide was administered daily at a dose of 100 mg/day for 3 months. Blood transfusion was recommended to maintain hemoglobin levels of > 9.0 g/dL during the treatment. The hematological responses to thalidomide were defined as follows: major response, transfusion independence, and maintenance of hemoglobin level > 9.0 g/dL; minor response, ≥ 50% reduction in transfusion requirement and maintenance of hemoglobin level > 9.0 g/dL; and no response, < 50% reduction in transfusion requirement to maintain a pretransfusion hemoglobin level of 9.0 g/dL.

The baseline characteristics of our cohort, consisting of 33 patients with TDT (18 β0/β0, 12 β+/β0, 3 β+/β+), are detailed in Table S1. The median age was 16 years (range 12–37), and 51.5% were male, 36.4% had undergone splenectomy, and 12.1% had co-inherited α-thalassemia. Our findings indicated that RBC lifespan was significantly shorter in patients with TDT than in normal controls, being nearly eight times longer in the latter group (median 15 vs. 119 days, Table S2). Univariate logistic regression analysis revealed no significant factors affecting RBC lifespan, including sex, age, genotype, splenectomy status, transfusion timing, and transfusion interval (p > 0.05).

We employed linear regression to explore the correlations between RBC lifespan and various markers of hemolysis, erythropoiesis, iron regulation, and oxidative stress. The main markers were as follows: reticulocytes, total bilirubin (TBIL), indirect bilirubin (IBIL), lactate dehydrogenase (LDH), haptoglobin, plasma free hemoglobin, erythropoietin (EPO), soluble transferrin receptor (sTfR), growth differentiation factor-15, hepcidin, serum ferritin, serum iron, total iron-binding capacity, unsaturated iron-binding capacity, transferrin, transferrin saturation, reactive oxygen species, malondialdehyde, thiobarbituric acid–reactive substances, reduced glutathione, oxidized glutathione, glutathione peroxidase, catalase, glutathione reductase, and superoxide dismutase (Table S3). In terms of the identified correlations, RBC lifespan showed negative correlations with the indirect hemolysis markers TBIL (r = −0.570, p = 0.001), IBIL (r = −0.602, p < 0.001), and LDH (r = −0.529, p = 0.002), and a positive correlation with haptoglobin (r = 0.517, p = 0.002). RBC lifespan was also negatively correlated with EPO (r = −0.467, p = 0.006) and sTfR (r = −0.642, p = 0.001), but positively correlated with hepcidin (r = 0.351, p = 0.045). No other factors were significantly correlated with RBC lifespan (p > 0.05). Multiple linear regression including these significantly correlated variables confirmed that IBIL, EPO, and sTfR were significantly inversely correlated with RBC lifespan (Table S4).

Twenty-five patients received thalidomide treatment. After this treatment, their RBC lifespan increased from a median of 15 days (range 9–30) to 20 days (range 11–44; p = 0.001), reflecting a median increase of 3 days (range −10–31; Figure 1H). This increase mainly occurred in patients demonstrating a hematologic response (p = 0.001, n = 20; Figure 1I). In contrast, non-responders exhibited no significant change in RBC lifespan (p = 0.688, n = 5; Figure 1J). The RBC lifespan was prolonged by 5 (−10–31) days in patients with a major response (15 patients) and by 3 (−1–11) days in those with a minor one (5 patients). Overall, patients with a hematologic response [5 (−10–31) days] had significantly more prolonged RBC lifespans than those with no response [1 (−2–3) days; p = 0.037]. Furthermore, those with a hematologic response had a significantly longer RBC lifespan after treatment than at baseline (p = 0.003) and than in non-responders (p = 0.048), although it remained shorter than that in normal controls (p < 0.001; Figure S1). The RBC lifespans at baseline and during follow-up are shown in Table S5.

FIGURE 1

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Associations between red blood cell lifespan and indices of hemolysis, erythropoiesis, and iron regulation. (A–G) Red blood cell lifespan was negatively correlated with total bilirubin (r = −0.570, p = 0.001), indirect bilirubin (r = −0.602, p < 0.001), lactate dehydrogenase (r = −0.529, p = 0.002), erythropoietin (r = −0.467, p = 0.006), and soluble transferrin receptor (r = −0.642, p = 0.001), and positively correlated with haptoglobin (r = 0.517, p = 0.002) and hepcidin (r = 0.351, p = 0.045). Red blood cell lifespan before and after thalidomide treatment (H) in all patients, (I) in responders, and (J) in non-responders.

We also examined the relationships of changes in markers of hemolysis, erythropoiesis, and iron regulation with the prolonged RBC lifespan after thalidomide treatment. Prolonged RBC lifespan correlated positively with changes in hepcidin (r = 0.605, p = 0.001; Figure S2A) and negatively with changes in sTfR (r = −0.625, p = 0.001; Figure S2B). No other parameters were significantly correlated with prolonged RBC lifespan (Table S6).

Recent research has indicated that measuring RBC lifespan can help to guide treatment decisions, assess drug efficacy, and contribute to understanding the mechanisms behind anemia and related conditions [2]. The combination of a CO breath test and hemoglobin measurement provides a simple, rapid, and noninvasive method for determining RBC lifespan [3]. Previous studies have shown that Levitt's CO breath test produces results comparable to those obtained by the ¹⁵N glycine labeling technique for this purpose [4]. Our study confirmed the correlations between RBC lifespan and markers of hemolysis, erythropoiesis, and iron regulation in patients with TDT. The findings indicated that RBC lifespan not only reflected the severity of hemolysis but also was closely tied to erythropoiesis and iron regulation. Measuring RBC lifespan may thus enhance our understanding of thalassemia and inform treatment evaluations.

The primary causative mechanism of β-thalassemia is IE, with peripheral hemolysis as a secondary factor. This IE is generated by an imbalance of globin chains, resulting in anemia, and increased EPO production, which stimulates erythropoiesis and suppresses hepcidin in the liver [5]. This cascade of events causes increased intestinal iron absorption, contributing to iron overload. Indeed, it is the main cause of iron overload in patients with TDT, alongside blood transfusions. Most of the CO produced by the human body results from the destruction of RBCs; CO produced by other routes accounts for about 30% of the total, a proportion that is relatively fixed. In view of this, and given that RBC destruction in β-thalassemia is predominantly driven by IE, we propose that the concentration of expired CO can reflect the severity of IE. The current results suggest that RBC lifespan derived from the CO breath test is closely related to IE and iron regulation indicators in patients with TDT. RBC lifespan determined in this way could thus be a useful indicator for evaluating patient condition and treatment efficacy in cases of β-thalassemia.

Conventionally, studies on the efficacy of thalassemia treatments have focused on changes in hemoglobin levels and transfusion volume [6]. However, some patients in our cohort who responded to treatment did not exhibit significant changes in RBC lifespan, while a few even experienced decreases. Increases in RBC lifespan among non-responders mirrored the minimal improvements seen in responders. Although thalidomide treatment enhanced the overall lifespan of RBCs, the degree of increase was limited, implying that other mechanisms may be involved. In other words, an increased RBC lifespan could not fully explain the improved hemoglobin levels in responders. A retrospective analysis of these responders indicated that IBIL, LDH, EPO, and hepcidin levels did not improve significantly post-treatment, suggesting that the changes in hemoglobin may not accurately reflect hemolysis and erythropoiesis in responders. Monitoring RBC lifespan could serve as a valuable indicator of changes in hemolysis and may also be useful for assessing erythropoiesis and iron regulation. We observed that the post-treatment RBC lifespan was not only correlated positively with hemoglobin increases but also closely associated with changes in hepcidin and sTfR, indicating its potential value for assessing β-thalassemia severity and treatment effects, especially in clinical trials.

In summary, this study provided a straightforward and accessible method for assessing RBC lifespan in patients with thalassemia using expiratory CO. Our findings indicated that RBC lifespan not only reflected the severity of hemolysis but also provided insights into IE and iron regulation. Importantly, we observed that thalidomide treatment increased the RBC lifespan in patients with TDT, albeit to a limited extent. We believe that analyzing RBC lifespan will enhance our understanding of thalassemia and other anemias, thus facilitating the evaluation of new treatments.