Seham M Ragab1, Waleed M Fathy2, Walaa FAbd El-Aziz3 and Rasha T Helal2
Departments of Pediatrics1, Clinical pathology2 and Cardiology3, Faculty of Medicine, Menoufia University. Naser street, Shebeen El-koom, Menoufia, Egypt.
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Background: Cardiac iron toxicity is the leading cause of death among β-thalassaemia major (TM) patients. Once heart failure becomes overt, it is difficult to reverse.
Objectives: To investigate non-overt cardiac dysfunctions in TM patients using pulsed wave Tissue Doppler Imaging (TD I) and its relation to iron overload and brain natriuretic peptide (BNP).
Methods: Thorough clinical, conventional echo and pulsed wave TDI parameters were compared between asymptomatic 25 β-TM patients and 20 age and gender matched individuals. Serum ferritin and plasma BNP levels were assayed by ELISA.
Results: TM patients had significant higher mitral inflow early diastolic (E) wave and non significant other conventional echo parameters. In the patient group, pulsed wave TDI revealed systolic dysfunctions, in the form of significant higher isovolumetric contraction time (ICT), and lower ejection time (E T), with diastolic dysfunction in the form of higher isovolumetric relaxation time (IRT), and lower mitral annulus early diastolic velocity E` (12.07 ±2.06 vs 15.04±2.65,P=0.003) compared to the controls. Plasma BNP was higher in patients compared to the controls. Plasma BNP and serum ferritin had a significant correlation with each other and with pulsed wave conventional and TDI indices of systolic and diastolic functions. Patients with E/E` ≥ 8 had significant higher serum ferritin and plasma BNP levels compared to those with ratio < 8 without a difference in Hb levels.
Conclusion: Pulsed wave TDI is an important diagnostic tool for latent cardiac dysfunction in iron-loaded TM patients and is related to iron overload and BNP.
Thalassemia is one of the most
common genetic disorders. Thus, it is considered a global health
problem. Worldwide, about 5% of the population carry globin variants.
Beta (β)–Thalassemia is caused by the reduced synthesis of β-globin
chains, which leaves an erythrocyte excess of unopposed α-chains
resulting in ineffective erythropoiesis and chronic hemolytic anemia.
According to the severity, β-thalassemias are classified into: transfusion dependent β thalassemias (TDT) or β-thalassemia major (TM), non transfusion dependent β-thalassemia (NTDT) or β- thalassemia intermedia (TI) and β -thalassemia trait (asymptomatic carriers). β–TM is the severest form that develops during the first year of life and requires lifelong transfusion therapy for survival.
Although improving survival, repeated blood transfusion regimen causes iron overload and iron toxicity in different organs including the heart.
Despite the progress in iron chelation therapy, congestive heart failure due to iron accumulation
Is still the leading cause of death in β-TM patients.[4,5]
Iron overload in combination with other inflammatory and immunogenetic factors can cause left ventricular systolic dysfunction, dilatation and failure, whereas the sole iron overload may result in left ventricular diastolic dysfunction with myocardial restriction and subsequent pulmonary hypertension and right ventricular dilatation.
Patients with TM may remain asymptomatic and global left ventricular (LV) function may be preserved until late in the disease process.[7,8] So, early detection of myocardial dysfunction may be useful in the management plan.
Echocardiography is an essential imaging modality for diagnosis of ventricular function, that allows exclusion of overt LV systolic dysfunction (left ventricular ejection fraction < 50%).
However, changes of segmental wall motion – the early sign of myocardial dysfunction in thalassemia patients - may be subtle and could be missed by conventional echocardiographic examination which may remain normal until late stages during this disease process.
Tissue Doppler Imaging (TDI) is a relatively new Doppler ultrasound modality that records regional systolic and diastolic velocities within the myocardium. It allows quantitative measurement of both systolic and diastolic velocities directly from the ventricular myocardium with the determination of the extent of mitral annular displacement in systole and diastole.
This new technique can show additional information compared with other echocardiography techniques, detecting even minor changes before the occurrence of abnormal indices of global ventricular dysfunction.
Brain natriuretic peptide (BNP) is one of the natriuretic peptide system that is stored in the myocardial cells as pre- proBNP. It is secreted from the heart as a result of direct wall stress, caused by either stretch or pressure affecting cardiocytes. Once released, BNP has pronounced natriuretic, diuretic and vasodilating properties, working to dramatically reduce volume overload and hypertension. BNP level is useful for the diagnosis of left ventricular systolic and diastolic dysfunctions and is correlated with the severity and prognosis.[16,17]
So, the aim of this work was to investigate the utility of pulsed wave DTI to detect latent or non-overt cardiac dysfunctions in asymptomatic TM patients and its relation to the iron overload assayed by serum ferritin and to BNP as a biomarker of cardiac dysfunction.
Materials and methods
This is a cross-sectional study, performed upon 45 subjects
(patients and controls); 25 β-TM patients and 20 age and sex matched
healthy individuals as controls.
The patient group included 25 multi-transfused β-TM patients (14 males and 11 females). Their ages ranged from 4 to 20 years with mean age of 12 ± 5.79 years and median of 12 years. These patients were kept on a regular blood transfusion regimen (every 3-4 weeks) since infancy to maintain pre-transfusion Hb above 7 gm/dl and post-transfusion Hb above 10gm/dl. They were on long-term chelation therapy for at least one year either by Deferoxamine (DFO) monotherapy, 30–50 mg/kg body weight by subcutaneous infusion with an infusion pump for 8–12 h, 5 days per week (16/25=64%), oral Deferasirox monotherapy, 20-30 mg/kg/day, daily (4/25=16%) or combined therapy with DFO and Deferasirox (DFO at the dosage of 40 mg/kg/day for 3 days/week and daily Deferasirox at the dosage of 30 mg/Kg/day, 5/25=20%).
The study included cardiac asymptomatic TM patients with ejection fraction >55% and a normal resting 12-lead electrocardiogram (ECG).
Patients on cardiovascular treatment, with any cardiovascular complaints, documented arrhythmia, hypertension, renal disease, diabetes mellitus, congenital or rheumatic heart disease, use of medications altering myocardial functions or a history of smoking were excluded. Also, those who developed transfusion associated circulatory overload (TACO) or transfusion-related acute lung injury (TRALI) were excluded.
The control group consisted of 20 healthy age and gender-matched subjects (10 males and 10 females).Their ages ranged from 4 to 18 years with mean age of 10.9 ± 4.86 years and median of 10 years. They were free from acute (especially viral illness) or chronic illness (including cardiac diseases) with no family history of chronic hemolytic anemia. All controls had normal complete blood count (CBC), Hemoglobin (Hb) electrophoresis with normal ECG and conventional Echocardiographic findings. They had been randomly selected from children presented to our general outpatient clinic for routine check up especially for growth, or for non-specific complaints like non-specific abdominal pain.
This study had been carried out at the Hematology Unit, Pediatric Department in collaboration with Clinical Pathology and Cardiology Departments, Faculty of Medicine, Menoufia University, Egypt, in the period of time between January 2012 and September 2013.The study was approved by the ethical committee of Menoufia Medical School, and informed consent was obtained from the patient or his or her legal guardian.
Methods: All participants in the study were subjected to a full history taking and comprehensive clinical examination including a cardiac examination.
For the patient group, a special emphasis was given to the age of the disease manifestations, time of the first blood transfusion, frequency of blood transfusion with calculation of red blood cells transfusion index (RBCsTI) during the last year, chelation therapy details, hepatic, renal, histories and history of splenectomy.
For all included children (patients and controls) weight and height were measured by the standard methods and plotted against age and sex specific centiles.
The participants were investigated by the following:
I- Conventional Echocardiography
Echocardiography, in the form of complete two-dimensional, continuous and pulsed wave echocardiographic examination, was done using ultrasound machine (vivid 9, General Electric Medical Systems, Horton, Norway), equipped with 5MHz variable frequency harmonic –phased array transducer with simultaneous ECG monitoring, performed without sedation, during normal respiration in the left lateral decubitus. Images were recorded in the standard parasternal long axis, apical four and two chamber views.
Conventional Echo-Doppler Measurements
Routine M-mode, two-dimensional continuous wave Doppler recordings were obtained for each subject. The left atrial (LA), aortic (AO) diameters and left ventricular (LV) internal cavity dimensions including left ventricular end systolic diameter (LVESD) and left ventricular end diastolic diameter (LVEDD) were determined. LV ejection fraction (EF) and LV fractional shortening (FS) were measured using Teichholz’s M-mode formula.
Transmittal flow patterns were obtained by pulsed-wave Doppler echocardiography from apical four-chamber view. The peak of early diastolic flow velocity (E), the peak of late diastolic flow velocity (A) and the ratio of E/A were measured.
II- Pulsed Wave Tissue Doppler Imaging (TDI) Measurements
The pulsed wave TDI was performed using the same machine. To display tissue velocities; from the apical 4 and 2-chamber views, the Doppler sample volume was placed at four different sites of the mitral annulus: anterior, lateral, septal and inferior walls in order to record major velocities. The following parameters were registered: mitral annulus systolic velocity (S`), mitral annulus early diastolic velocity (E`), mitral annulus late diastolic velocity (A`) and time intervals; isovolumetric contraction time (ICT), isovolumetric relaxation time (IRT) and ejection time (ET).
Then calculation of the mean E/E`(mitral inflow E wave/ E` mitral annulus velocity) ratio was done. According to the E/E` ratio, patients were classified into: patients with E/E`≥ 15 (diastolic dysfunction), patients with E/E`≥ 8 but less than 15 (suspected diastolic dysfunction) and those with E/E`< 8 (without diastolic dysfunction).[19,20]
All pulsed-wave Doppler and PW-TDI parameters were measured at the end of expiration, at a sweep speed of 100 mm/s on three consecutive heart beats and the average for each was taken.
All data were obtained according to the recommendations of the American Society of Echocardiography.
III- Laboratory investigations including:
1- Complete blood count (CBC): using Beckman 750, Int, U.S.A, Auto-counter after calibration.
2- Serum ferritin level was measured by Enzyme Linked Immune Sorbent Assay (ELISA) technique (ELISA, GenWay Biotech, Inc, NP 000137, Swiss) on Microplate reader (Bio-Rad 680 Hercules, California, USA).The mean yearly serum ferritin level in the previous year was considered (on the average of 4 determinations) for patients and at the time of sampling for the controls.
3- Plasma BNP: Three ml venous blood samples were drawn by sterile vein-puncture on EDTA tube. Blood samples were immediately centrifuged for 15 minutes at 3000 rpm; plasma samples were separated then were stored at –20°C until analysis. Plasma BNP level was measured by ELISA using kits supplied by Ray Biotech, Inc. GA 30092.
For the patient group, the echocardiographic examination and the blood sampling for CBC and BNP assay were performed on the fourth day following blood transfusion.
Statistical analysis: The data were processed on an IBM-PC compatible computer using SPSS version 16 (SPSS Inc., Chicago, IL, USA).Continuous parametric variables were presented as means± SD while for categorical variables numbers (%) were used.Chi-square test was used for qualitative variables. The difference between 2 groups was performed by student’s t-test for parametric continuous variables and Man Whitney (U) test for non-parametric variables. Pearson correlation (r): was the test used to measure the association between two quantitative parametric variables and Spearman correlation coefficient was applied for non-parametric data. Receiver Operating Characteristic curve (ROC curve) analysis is a graph of sensitivity against 1- specificity at different cutoff points. The optimal cutoff point is that gives the highest sensitivity and specificity. Two-sided p value of < 0.05 was considered statistically significant.
For the patient group, their ages at diagnosis ranged from 0.5–1.5
years with a mean of 0.77± 0.24 years. The mean age of first blood
transfusion was 0.69 ± 0.21 years with a range of 0.5–1 year. The mean
duration of transfusion treatment was 10.1 ± 5.11 years, that of the
number of the transfusions /year was 11.2 ±1.22 (median of 11
Comparison between the studied groups regarding clinical and laboratory data were represented in Table 1. The studied groups were matched regarding age, sex, the mean body weight, the mean height, and pulse rate. History of splenectomy was documented in 12 (48%) of TM patients. The patient group had a significantly lower post-transfusion Hb level with significantly higher mean yearly serum ferritin and plasma BNP levels.
The conventional echocardiography parameters were presented in Table 2. Compared to the controls, TM patients had significant higher AO, LA, LVEDD, LVESD diameters and the mitral inflow early diastolic wave velocity (E). No significant difference was found between the studied groups regarding the left ventricular EF, left ventricular FS, the mitral inflow late diastolic wave velocity (A) or the E/A ratio.
|Table 1. Comparison of the clinical and laboratory parameters between TM patients and controls.|
|Table 2. Comparison of conventional echocardiographic measures between TM patients and the controls.|
As regard to the pulsed wave TDI parameters (Table 3), TM patients had significant higher ICT and lower ET compared to the controls in all tested sites of the mitral annulus as well as in the mean values of these parameters. The mean IRT and its values at the septal and the lateral walls of the mitral annulus were significantly higher in TM compared to the controls. The mean E` as well as its values at the lateral, anterior and inferior walls of the mitral annulus were significantly lower in TM patients compared to the controls. No significant difference was found in the S` or A` between the studied groups at any of the tested mitral annulus walls or in the mean values. TM patients had non-significant difference in E/E` ratio in comparison to the controls. Abnormal E/E` (mitral inflow E wave/E` mitral annulus velocity) ratio (≥15) was not found in any of TM patients. According to E/E`, TM patients were classified into those with E/E` <8 (16 patients, 64% ) and those with this ratio ≥ 8 but less than 15 (9 patients, 36%). Patients with E/E` ≥ 8 exhibited significant higher RBCs TI , mean yearly serum ferritin and plasma BNP levels compared to those with E/E` ratio < 8 without difference in post-transfusion Hb levels (Figure 1 A and B).
|Table 3. Comparison of Pulsed wave TDI measures between TM patients and the controls.|
1A. Comparison of post transfusion Hb ( g/dl), plasma BNP (pg/ml) and
RBCs TI (ml/kg/year) in TM patients with E/E` ratio ≥8 compared to
patients with E/E` <8.
Figure 1B.Comparison of mean yearly serum ferritin ( ng/ml) in TM patients with E/E` ratio ≥8 compared to patients with E/E` <8.
among TM patients revealed that serum ferritin and plasma BNP were
positively correlated with each other and each of them had significant
positive correlation with the mean values of E, ICT, and IRT;
significant negative correlation with ET without significant
correlation with E/A or E/E` ratios (Table 4).
The data, obtained from the Roc Curve, showed that the best sensitivity of 100% and specificity of 81.9% for the plasma BNP were at cutoff point of 28.5 pg/ml in ruling out diastolic dysfunction (E/E< 8). Negative predictive value was 100 % while positive predictive value was 75%.The area under the curve was 0.86, P = 0.003 (95% CI = 0.71 – 1.01) (Figure 2A).
For the mean yearly serum ferritin , the data obtained from the Roc Curve revealed that serum ferritin level at cutoff point of 4790.5 ng /ml had the best sensitivity of 88.9% and specificity of 81.2% in ruling out diastolic dysfunction (E/E< 8). Negative predictive value was 92.9% while positive predictive value was 72, P =0.007. The area under the curve was 0.83 (95% CI = 0.64 – 1.02) (Figure 2B).
|Table 4. Correlations between BNP and serum ferritin with different conventional echo cardiography and TDI parameters .|
|Figure 2A. Receiver operator characteristic curve of BNP values for the identification of patients with E/E`≥8.
Figure 2B. Receiver operator characteristic curve of serum ferritin values for the identification of patients with E/E`≥8.
Comparison of the pulsed wave TDI mean parameters regarding the age and serum ferritin categories (age of <14 years and ≥ 14years ; serum ferritin ≤2500 ng/ml and > 2500 ng/ml ) revealed that the ET was significantly lower in TM patients ≥ 14 years compared to those < 14 years. TM patients with serum ferritin >2500 ng/ml had significantly higher IRT compared to those with serum ferritin level ≤ 2500 ng/ml. There was no significant difference in the age categories regarding E/E` ratio < 8 or ≥ 8, while there was a trend of prevalent E/E` ratio < 8 in those with serum ferritin ≤ 2500 ng/ml (P=0.052) (Table 5).
|Table 5. Comparison of Pulsed wave TDI mean measures among TM patients regarding the age and serum ferritin categories..|
Asymptomatic TM children under regular chelation therapy may have latent diastolic and or systolic dysfunctions that could not be detected by conventional echocardiography but could be highlighted by TDI. Hence, application of pulsed-wave TDI in these patients is appropriate. Integrated use of echocardiography, pulsed-wave TDI and BNP level for an accurate assessment of cardiac functions is highly recommended to help identifying subjects at risk and facilitates early intervention