Pulmonary hypertension in non-transfusion-dependent thalassemia: Correlation with clinical parameters, liver iron concentration, and non-transferrin-bound iron

Abstract

Background

Pulmonary hypertension is a major cardiac complication in non-transfusion-dependent thalassemia (NTDT). Several clinical and laboratory parameters, including iron overload, have been shown to have a positive correlation with the incidence of pulmonary hypertension. Non-transferrin-bound iron (NTBI) is a form of free-plasma iron that is a good indicator of iron overload.

Objectives

The aim of this study was to determine the prevalence of pulmonary hypertension in patients with NTDT and to investigate its correlation with the clinical parameters, liver iron concentration (LIC) and NTBI.

Methods

Patients with NTDT were evaluated using echocardiography, and magnetic resonance imaging for cardiac T2* and LIC. Pulmonary hypertension was defined as peak tricuspid regurgitation velocity ≥2.9 m/s measured using trans-thoracic echocardiography. Clinical parameters and the status of iron overload as determined by LIC, serum ferritin, and NTBI level were evaluated for their association with pulmonary hypertension.

Results

Of 76 NTDT patients, mean age 23.7 ± 8.5 years, seven patients (9.2%) had pulmonary hypertension. Previous splenectomy (71.4 vs. 24.6%, P-value 0.019), higher cumulative red blood cell (RBC) transfusions (received ≥10 RBC transfusions 85.7 vs. 33.3%, P-value 0.011), higher nucleated RBCs (353 ± 287 vs. 63 ± 160/100 white blood cells, P-value <0.001), and a high NTBI level (5.7 ± 3.0 vs. 3.3 ± 2.8 µmol/l, P-value 0.034) were associated with pulmonary hypertension. There was no significant correlation between LIC or serum ferritin and pulmonary hypertension.

Conclusion

Pulmonary hypertension in NTDT is common, and is associated with splenectomy and its related factors. NTBI level shows a significant correlation with pulmonary hypertension.

Keywords:

• Iron overload
• Non-transferrin-bound iron
• Non-transfusion-dependent thalassemia
• Pulmonary hypertension

Introduction

Thalassemia is an inherited red blood cell (RBC) disorder caused by mutations of globin genes leading to a decrease in or absence of globin chain production. The phenotypic spectrum varies from asymptomatic to severe anemia and can be classified according to the transfusion requirement as transfusion-dependent and non-transfusion-dependent thalassemia (TDT and NTDT).¹ NTDT patients do not require lifelong regular transfusions for survival, although they may require occasional transfusions in particular scenarios, such as when they have anemia associated with fever and infection. NTDT is mainly comprised of three patient groups: beta-thalassemia intermedia, hemoglobin E/beta-thalassemia (mild and moderate forms), and alpha-thalassemia intermedia (hemoglobin H disease).^2,3

Although NTDT patients have mild-to-moderate anemia and do not require regular transfusion for survival as in TDT, they may develop many disease-related complications, including pulmonary hypertension, thrombosis, osteoporosis, extramedullary hematopoiesis, and leg ulcers.^4–6 The prevalence of and mechanisms leading to complications are somewhat different between NTDT and TDT.^4,7 Pulmonary hypertension leading to right-sided heart failure is a major cardiac complication in cases of NTDT, compared to myocardial siderosis and left ventricular dysfunction in TDT.^7–12 The overall prevalence of pulmonary hypertension in patients with thalassemia ranges from 10 to 75%.^{8–10,13–20} The pathogenesis of pulmonary hypertension is a combination of several mechanisms.^{4–7,14–31} One of the key pathogenetic mechanisms is chronic hemolysis which results in chronic anemia leading to tissue hypoxia and compensatory high cardiac output.^4,7,17 The chronic hemolysis depletes nitric oxide and results in pulmonary vasoconstriction and vascular remodeling.^7,17,30 Another important mechanism is the hypercoagulable state which results from cumulative effects of an increase in pathologic RBCs with membrane phosphatidylserine exposure, increased nucleated RBCs (nRBCs), and activated platelets.³¹ All of which leads to increased thrombin generation, resulting in pulmonary thromboembolism and pulmonary hypertension. Splenectomy aggravates pulmonary hypertension by the increase of pathologic RBCs, nRBCs, and platelet counts.^19,22 Iron overload is another key pathogenetic mechanism in pulmonary hypertension.^5,6 NTDT patients accumulate iron from increased intestinal absorption and occasional blood transfusion. Iron causes free radical formation and lipid peroxidation leading to cellular damage. Known predictors of pulmonary hypertension are asplenia, more severe hemolytic status, and higher numbers of platelet and nRBCs.^{14,20,22,25,27} A higher degree of iron overload, advancing age, degree of anemia and higher numbers of transfusions are also identified as risk factors for pulmonary hypertension in NTDT patients.^5,6,23,29

Assessment of liver iron concentration (LIC) is the gold standard for measuring total body iron in NTDT.^1,32 Magnetic resonance imaging (MRI) using either R2 or T2* is a reliable and non-invasive methods for assessing LIC.^33,34 Increased LIC has been shown to be associated with increased complications including thrombosis, pulmonary hypertension, hypothyroidism, osteoporosis, and hypogonadism in NTDT patients.⁶ A recent study demonstrated that NTDT patients with LIC ≥5 mg/g have a significantly higher prevalence of several vascular and endocrine/bone morbidities than patients with LIC <5 mg/g.⁵

Non-transferrin-bound iron (NTBI) is a form of plasma iron that is bound to ligands other than transferrin.³⁵ It is detected in a condition of iron overload when transferrin has become saturated. NTBI causes free radical formation and lipid peroxidation. A study in patients with NTDT demonstrated that NTBI was detectable above the reference range, and had a strong correlation with LIC and serum ferritin.³⁶ NTBI is also useful for monitoring the response to desferrioxamine chelation therapy in patients with thalassemia.^37,38 Thalassemia major patients with heart complications were shown to have significantly higher NTBI levels than those without.³⁹ However, there has been limited data published using NTBI as a marker for predicting the iron-related morbidities in patients with NTDT.

This study aimed to find the prevalence of pulmonary hypertension in patients with NTDT and to define the clinical and iron-overload parameters that are associated with pulmonary hypertension in these patients.

Patients and methods

Patients with NTDT, as defined by having thalassemia disease and receiving not more than three RBC transfusions per year in the last 5 years, age 10–50 years, were enrolled onto the study. Exclusion criteria were patients who had clinical evidence of other secondary causes of pulmonary hypertension, including human immunodeficiency virus infection, hepatitis virus infection, collagen vascular diseases, cirrhosis, chronic obstructive airway diseases, and acquired heart disease associated with pulmonary hypertension. All patients were treated at Chiang Mai University Hospital. The study period was September 2013–May 2014. The study protocol was approved by the institutional ethics committee. All patients and their parents in case of minorities gave written informed consent.

Patient charts were reviewed for thalassemia diagnosis and genotypes, transfusion history, history of splenectomy, and comorbidities. The patients were examined to record body weight, height, and liver size. Laboratory data collected were mean hemoglobin levels in the last 12 months and mean ferritin levels in the last 5 years.

Laboratory investigations including complete blood count, liver transaminase enzymes, serum ferritin, and NTBI level were performed. All patients were evaluated using echocardiography for cardiac function and pulmonary hypertension, and MRI for cardiac T2* and LIC.

Complete blood counts were performed using an automated hematological analyzer (Beckman Coulter A^C.T 5diff, Coulter Corp., Miami, FL, USA). The peripheral blood smears were reviewed for the nRBC count and corrected white blood cell (WBC) count. Platelet count was corrected as per a previously described method.⁴⁰

NTBI level was measured by a high performance liquid chromatography (HPLC) technique as described previously.⁴¹ In brief, 450-µl plasma was incubated at room temperature for 1 hour with 50 µl of 800-mM nitril-otriacetic acid (NTA) (a final concentration of 80 mM) solution pH 7.0. The mixture was further incubated for 30 minutes at room temperature to produce a ferric–nitrilotriacetate complex, Fe³⁺–(NTA)₂. After incubation, the Fe³⁺–(NTA)₂ was separated from plasma proteins by spinning the plasma mixture through the membrane filter (30-kDa cutoff, polysulfone type, 0.5-ml capacity) at 12 000g, 15°C for 45 minutes. Ultra-filtrate was injected into a non-metallic 50-µl loop and analysed by using HPLC. HPLC was composed of a glass analytical column (ChromSep-ODS1, 100 mm × 3.0 mm, 5 µm) and mobile-phase solvent (3 mM CP22 in 19% acetronitrile buffered with 5 mM MOPS pH 7.0). The mobile-phase solvent had a flow rate of 1.0 ml/min. NTBI as Fe³⁺–(NTA)₂ was fractionated on the column, then immediately on-column derivatized with CP22 to form a Fe³⁺–(CP22)₃ complex. The resulting orange-colored Fe³⁺–(CP22)₃ product was detected at 450 nm with the SpecMonitor^® 2300 flow-cell detector (LDC Milton-Roy, Inc., FL, USA). The NTBI peak height was integrated and recorded for further determination of NTBI concentration from the calibration curve. The calibration curve was produced by plotting obtained peak height values on the y-axis against iron concentration on the x-axis. An equation of linear regression line was used to calculate plasma NTBI concentration.

Complete standard two-dimensional, M mode, and Doppler trans-thoracic echocardiography was performed at rest in all enrolled patients with Philips iE33 (Philips Healthcare, Bothell, WA, USA). Pulmonary hypertension was defined as a peak tricuspid regurgitation velocity (TRV) ≥2.9 m/s according to the European Society of Cardiology (ESC) and the European Respiratory Society guidelines for diagnosis and treatment of pulmonary hypertension 2009, suggested to consider pulmonary hypertension ‘possible’ for TRV of 2.9–3.4 m/s, pulmonary artery pressure of 37–50 mmHg with or without additional signs of pulmonary hypertension.⁴² Patients would be excluded if there was inadequate holosystolic tricuspid regurgitation flow.

Magnetic resonance acquisition was performed using a 1.5 T MR scanner (GE Signa HDxt, GE Healthcare, Milwaukee, WI, USA), using 8-channel body array. For the measurements of myocardial iron overload, a T2* gradient-echo multiecho sequence (flip angle 25°, matrix: 192 × 128 pixels, field of view 38 × 28.5 cm, bandwidth 125 kHz, slice thickness 10.0 mm, number of excitations 1, view per segment 4, repetition time 18.5 ms) was used. A single mid-ventricular short-axis view of the left ventricle was acquired at eight echo times (TEs) (1.7 ms, which increased in 2.0–2.1 ms increments) in a single end-expiratory breath-hold. For the measurement of liver iron overload, a T2* gradient-echo multiecho sequence (flip angle 25°, matrix 128 × 64 pixels, field of view 48 × 36 cm, bandwidth 125 kHz, slice thickness 10.0 mm, number of excitations 1, repetition time 200 ms) was used. A single transverse slice through the liver was obtained at eight different TEs (1.1 ms, which increased in 1.5 ms increments) in a single end-expiratory breath-hold. Both cardiac and hepatic T2* analysis and hepatic iron quantification were carried out on a workstation (advantage Window 4.6, GE healthcare) using a commercial analysis software (StarMap 4.0, GE Healthcare).

Continuous data were reported as mean and standard deviation, and categorical data were reported as frequency and percentages. To compare characteristics between the groups with and without pulmonary hypertension, independent Student's t-test was used for the analysis of continuous data, and a Pearson's Chi-square test or Fisher's Exact test was used for categorical data. A univariate binary logistic regression analysis was carried out and the results are presented as odds ratios (ORs) and their 95% confidence interval (CI). Statistical analysis was carried out using Stata^® version 12 (StataCorp LP, College Station, TX, USA). A P-value of less than 0.05 was considered as statistically significant.

Results

Ninety-one patients were initially enrolled. Fifteen (16.5%) were excluded as there was inadequate holosystolic tricuspid regurgitation flow from echocardiography. Of the assessable 76 patients, there were 48 (63%) females. The mean age was 23.7 ± 8.5 years (range 10.2–45.5 years). Forty-three patients (56.6%) had hemoglobin E/beta-thalassemia, 4 patients (5.3%) had beta-thalassemia intermedia, and 29 patients (38.1%) had hemoglobin H disease or hemoglobin H/Constant Spring (CS) disease, with or without co-inherited Hb E carrier. Most of them (61.8%) had received less than 10 units cumulative of RBC transfusion. Twenty-two patients (28.9%) were previously splenectomized. Twenty-five patients (32.9%) received iron chelation; 22 received deferiprone, 2 desferrioxamine, and a patient received deferiprone and desferrioxamine combination.

Seven patients (9.2%) had pulmonary hypertension. All of them had a normal left ventricular ejection fraction (LVEF >55%). Patient characteristics and laboratory profiles according to their pulmonary hypertension status are shown in Table 1. Previous splenectomy and higher cumulative RBC transfusion (received 10 or more RBC transfusions) showed an association with pulmonary hypertension. There was no significant correlation between age or gender and pulmonary hypertension. Pulmonary hypertension was seen more frequently in beta-thalassemia than alpha-thalassemia, although the difference was not significant.

Table 1. NTDT patient characteristics and laboratory profiles according to their pulmonary hypertension status

Characteristics	TRV ≥2.9 m/s (n = 7)	TRV <2.9 m/s (n = 69)	P-value
Age (years)	21.9 ± 10.2	23.9 ± 8.3	0.539
Gender (%)			0.704
Male	3 (42.9)	25 (36.2)
Female	4 (57.1)	44 (63.8)
Body weight (kg)	46.0 ± 11.4	49.2 ± 9.1	0.387
Height (cm)	158.6 ± 11.4	159.2 ± 9.8	0.865
Liver size (cm)	6.0 ± 4.4	6.0 ± 4.7	0.988
Type of thalassemia (n, %)			0.241*
Hemoglobin E/beta-thalassemia	6 (85.7)	37 (53.6)
Beta-thalassemia intermedia	0	4 (5.8)
Hemoglobin H disease	0	8 (11.6)
EABart's disease	0	0
Hemoglobin H/CS disease	1 (14.3)	17 (24.7)
EABart's CS disease	0	3 (4.3)
Cumulative RBC transfusion (n, %)			0.011
Less than 10 transfusions	1 (14.3)	46 (66.7)
Ten transfusions or more	6 (85.7)	23 (33.3)
Splenectomy (n, %)			0.019
Yes	5 (71.4)	17 (24.6)
No	2 (28.6)	52 (75.4)
Echocardiographic findings
TRV (m/s)	3.2 ± 0.3	2.4 ± 0.3	<0.001
LVEF (%)	66.3 ± 8.2	63.9 ± 10.1	0.559

*Comparison between combined beta-thalassemia and alpha-thalassemia groups.

LVEF, left ventricular ejection fraction; NTDT, non-transfusion-dependent thalassemia; RBC, red blood cell; TRV, tricuspid regurgitation velocity.

Laboratory profiles are shown in Table 2. Higher nRBC count and NTBI level were found to be associated with pulmonary hypertension. Hemoglobin and mean hemoglobin levels in the previous 12 months, serum ferritin level, LIC, and MRI T2* were not significantly different between the groups with or without pulmonary hypertension. MRI T2* was normal (>20 ms) in all patients. Platelet count and mean serum ferritin level in the previous 5 years were higher in the pulmonary hypertension group, although the differences were not statistically significant. Summary of significant risk factors for pulmonary hypertension was as shown in Table 3.

Table 2. Laboratory profiles of NTDT patients according to their pulmonary hypertension status

Characteristics	TRV ≥2.9 m/s (n = 7)	TRV <2.9 m/s (n = 69)	P-value
Complete blood count
Hb (g/dl)	7.7 ± 0.9	7.9 ± 1.5	0.801
Mean Hb in previous 12 months (g/dl)	7.7 ± 0.6	7.9 ± 1.3	0.611
Nucleated RBCs/100 WBCs	353 ± 287	63 ± 160	<0.001
Platelet count (×10^9/l)	673.0 ± 319.2	458.7 ± 268.9	0.052
Clinical chemistry
Ferritin (ng/ml)	839 ± 533	773 ± 750	0.821
Maximum ferritin in previous 5 years (ng/ml)	2088 ± 1402	1354 ± 1778	0.294
NTBI (µmol/l)	5.7 ± 3.0	3.3 ± 2.8	0.034
AST (U/l)	29.4 ± 10.0	35.7 ± 17.4	0.355
ALT (U/l)	17.7 ± 7.4	29.9 ± 25.2	0.007
MRI findings
LIC (mg Fe/g dry weight)	10.3 ± 5.5	8.2 ± 7.0	0.440
MRI T2* (ms)	46.09 ± 7.6	44.5 ± 6.3	0.334

ALT, alanine aminotransferase; AST, aspartate aminotransferase; Hb, hemoglobin; LIC, liver iron concentration; MRI, magnetic resonance imaging; NTBI, non-transferrin-bound iron; NTDT, non-transfusion-dependent thalassemia; RBC, red blood cell; TRV, tricuspid regurgitation velocity; WBC, white blood cell.

Table 3. Risk factors for pulmonary hypertension in NTDT patients

Risk factors	TRV ≥2.9 m/s	TRV <2.9 m/s	Odds ratio (95% CI)	P-value
Previous splenectomy (n, %)	5/7 (71.4)	17/69 (24.6)	7.65 (1.36–43.1)	0.021
Cumulative transfusion	6/7 (85.7)	23/69 (33.3)	12.00 (1.36–105.67)	0.025
Ten times or more (n, %)
Nucleated RBCs/100 WBCs	353 ± 287	63 ± 160	1.005 (1.002–1.008)	0.002
NTBI (µmol/l)	5.7 ± 3.0	3.3 ± 2.8	1.333 (1.004–1.769)	0.047

NTBI, non-transferrin-bound iron; NTDT, non-transfusion-dependent thalassemia; RBC, red blood cell; TRV, tricuspid regurgitation velocity; WBC, white blood cell.

NTDT patients who had previous splenectomy had significantly higher cumulative RBC transfusions (81.8 vs. 20.4% had received 10 or more transfusions, P-value <0.001), higher nRBC count (280 ± 276 vs. 13 ± 32/100 WBCs, P-value <0.001), higher platelet count (807.3 ± 235.4 vs. 344.5 ± 157.0 × 10⁹/l, P-value <0.001), and also higher serum ferritin levels (1226 ± 597 vs. 786 ± 626 ng/ml, P-value <0.001), NTBI level (4.6 ± 2.9 vs. 2.9 ± 3.0 µmol/l, P-value 0.027), and LIC (11.1 ± 6.4 vs. 7.3 ± 6.8 mg Fe/g dry weight, P-value 0.031).

Discussion

Pulmonary hypertension is a major cardiovascular complication in thalassemia, especially in NTDT.^7–11,29 From our study, 7 out of 76 patients (9.2%) had pulmonary hypertension. The lower occurrence of pulmonary hypertension in this study than that seen in previous studies in Thailand (37.3 and 29%)^14,15 can be explained by the difference in the diagnostic criteria for pulmonary hypertension by echocardiography which is a stricter criteria in this study. The prevalence from this study is comparable to another study in Thai NTDT patients (10.9%) that uses the same limitation criteria.²⁰

The pathogenesis of pulmonary hypertension in NTDT is proposed to be from several mechanisms including hemolysis resulting in decreased nitric oxide, increased cardiac output associated with anemia, splenectomy leading to increased platelet aggregates and increased circulating RBC-derived microvesicles, abnormal RBCs and RBC precursors, liver dysfunction, and iron overload causing endothelial cell toxicity.^26,43 Our study demonstrates that splenectomy and its related factors including higher cumulative RBC transfusion, higher nRBCs, and higher NTBI level are significant risk factors for pulmonary hypertension. The results consolidate the previous studies, and add higher NTBI level as a significant risk factor.

Regarding the high platelet number which has been shown as a risk factor for pulmonary hypertension in thalassemia,²⁷ in our study NTDT patients with pulmonary hypertension were also shown to have higher platelet numbers than the patients without pulmonary hypertension, although the difference was not statistically significant (P-value 0.052) which is likely limited by the small numbers of patients with pulmonary hypertension.

Interestingly, our study shows that NTBI level is the only iron-overload parameter that shows a significant correlation with pulmonary hypertension. Serum ferritin level and LIC were not significantly different between the groups with or without pulmonary hypertension. The mean 5-year serum ferritin level was higher in the pulmonary hypertension group, but this was not statistically significant. This discrepancy may be explained by the fact that serum ferritin and LIC are a result of iron accumulation over time, while NTBI is a consequence of ineffective erythropoiesis and catabolic iron.³⁶ Moreover, NTBI is a free form of iron which is the catalyst of the formation of toxic free oxygen radicals, and therefore has a direct damage effect to tissues. Therefore, NTBI is a more sensitive index for iron-related complications. We propose that NTBI measurement should be considered as a non-invasive monitoring method for the iron status in NTDT patients.

Recent studies show that splenectomized patients have higher NTBI levels than non-splenectomized patients, suggesting that the spleen may be a storage site for overloaded iron and may contribute to the decrease in the free iron level.^36,44 Whether NTBI by itself is a risk factor of pulmonary hypertension, or is merely associated with splenectomy which is already a strong risk factor of pulmonary hypertension is unknown and will be a subject for further studies.

Gender is not found to be a significant risk factor for pulmonary hypertension in our study. This information is comparable to previous studies.^23,27,29 Several studies demonstrate that advanced age is a significant risk factor for pulmonary hypertension.^23,24,28,29 Our study does not show the significant differences in gender or age between the groups with and without pulmonary hypertension. On the other hand, four out of seven patients (57%) in the pulmonary hypertension group were younger than 18 years old. This finding emphasizes that even young NTDT patients may be at risk of pulmonary hypertension. Patients with NTDT should receive annual echocardiographic assessment as per the current guideline, which can contribute to earlier detection and proper intervention.¹

Our study does not show the effect of hemoglobin level on pulmonary hypertension. As the majority of patients in the pulmonary hypertension group were previously splenectomized, although hemoglobin levels were comparable in both groups, the degrees of hemolysis and ineffective erythropoiesis were likely to be different. Hemoglobin level needs to be interpreted along with splenectomy status and other factors for its association with pulmonary hypertension.

A recent study demonstrates that the beta-thalassemia genotype is an independent risk factor for pulmonary hypertension, which is explained by a greater disease severity in beta-thalassemia patients when compared to alpha-thalassemia patients in general.²⁰ In this study, pulmonary hypertension was seen in six beta-thalassemia patients and only one patient with hemoglobin H/CS disease, although the difference was not significant which may be limited by a small number of patients in the pulmonary hypertension group.

One limitation of this study is the small number of patients with pulmonary hypertension. Although the study could identify several significant risk factors for the occurrence of pulmonary hypertension by using univariate analysis, it is underpowered for a calculation of adjusted OR using multivariate analysis. Another limitation is the data collection of the exact amount of cumulative RBC transfusion which was not feasible in some cases who also received blood transfusions outside of the institute. Apart from the review of the hospital records, each patient was interviewed in detail to check for additional transfusions, and the cumulative transfusion was classified as none or less than 10 transfusions, and 10 transfusions or more, so this limitation is negligible.

In this study, the diagnosis of pulmonary hypertension was made by echocardiography instead of a gold standard right heart catheterization as the method is non-invasive and more practical.⁴² A previous meta-analysis study shows that the sensitivity and specificity of echocardiography for the diagnosis of pulmonary hypertension are 83 and 72%, respectively.⁴⁵

According to the Thalassaemia International Federation (TIF) guideline for management of pulmonary hypertension in NTDT, patients with NTDT are recommended to undergo annually echocardiographic assessment to monitor for pulmonary hypertension, especially for the subgroups with higher risks including patients with beta-thalassemia intermedia and hemoglobin E/beta-thalassemia, patients who are adults, splenectomized, minimally transfused, patients with elevated platelet or nRBC counts, lower hemoglobin levels, iron overload, a history of thrombosis, or having other risk factors for pulmonary hypertension.¹ Our study emphasizes the roles of the risk factors and we propose that NTBI should be measured along with serum ferritin and LIC by MRI. The cutoff criteria for NTBI level as a risk factor for pulmonary hypertension will need to be established in larger studies.

As per TIF guideline, patients who are likely to have pulmonary hypertension by clinical and echocardiographic criteria should be referred to a cardiologist to have the diagnosis confirmed by right heart cardiac catheterization, and then treated accordingly. Ventilation/perfusion lung scan to search for pulmonary thromboembolism is also recommended. The multimodality treatments include RBC transfusion, hydroxyurea, sildenafil citrate, iron chelation, and anticoagulant therapy.¹ RBC transfusion ameliorates anemia, decreases hemolysis, and decreases the hypercoagulable state. Hydroxyurea has been shown to decrease the incidence of pulmonary hypertension in beta-thalassemia. Hydroxyurea increases hemoglobin F production and therefore is postulated to improve oxygenation and decrease pulmonary vasoconstriction. It also decreases hypercoagulable state by lowering the platelet count and circulating myeloid cell series.⁴⁶ Sildenafil citrate is a phosphodiesterase-5 inhibitor which inhibits the degradation of cyclic guanosine monophosphate that mediates smooth muscle relaxation.⁴⁷ Iron chelation decreases excessed iron that causes tissue damage. Anticoagulant therapy ameliorates the hypercoagulable state.

In conclusion, pulmonary hypertension in NTDT is common, and can be seen in younger patients. Pulmonary hypertension is associated with splenectomy and its related factors including higher cumulative RBC transfusion, higher nRBCs, and a higher NTBI level. NTBI level is the only iron-overload parameter that shows a significant correlation with pulmonary hypertension. NTBI measurement should be considered as a non-invasive monitoring method for the iron status of NTDT patients.

Acknowledgements

The authors are grateful to Ms Suwakon Wongjaikam for her technical assistance.

Disclaimer statements

Contributors PC, KI, SS, AT, NC, and TS conceived the study and were responsible of the interpretation of results and the final preparation of the paper. PC, KI, AT, WC, and RR were responsible for recruitment of patients and collection of clinical and laboratory data. SS and AP were responsible for performing echocardiography and interpretation of results. CS and PV were responsible for obtaining and interpretation of T2* magnetic resonance imaging results. SS was responsible for measurement of non-transferrin-bound iron. All authors contributed to the final revision and approval of the paper.

Funding This work was supported by a Diamond Research Grant, Faculty of Medicine, Chiang Mai University, Chiang Mai, Thailand (DM 2555), and a Research Chair Grant from the National Science and Technology Development Agency (NSTDA) Thailand (NC).

Conflicts of interest The authors report no conflict of interest.

Ethics approval The study protocol was approved by the ethics committee of Faculty of Medicine, Chiang Mai University.