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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 43  |  Issue : 2  |  Page : 55-62

Alteration of trace elements and T-cell subsets in patients with β-thalassemia major: influence of high ferritin level


1 Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut; Department of Clinical and Chemical Pathology, Qena Faculty of Medicine, South Valley University, Qena, Egypt
2 Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt
3 Department of Microbiology and Immunology, Faculty of Medicine, Assiut University, Assiut, Egypt
4 Department of Biochemistry and Molecular Biology, Faculty of Medicine, Minia University, Minia, Egypt
5 Department of Pediatrics, Qena Faculty of Medicine, South Valley University, Qena, Egypt

Date of Submission26-Feb-2018
Date of Acceptance05-Mar-2018
Date of Web Publication7-Aug-2018

Correspondence Address:
Asmaa Nafady
Department of Clinical Pathology, Faculty of Medicine, Assiut University, Assiut 71515
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_3_18

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  Abstract 


Introduction Oxidative damage is believed to be found in transfusion-dependent patients with β-thalassemia. Oxidative damage may trigger an immune response and affect the trace elements.
Objective The aim of this study was to investigate the effect of iron transfusional overload on lymphocytes and on the serum trace elements.
Patients and methods A total of 53 patients with thalassemia were divided into two groups according to the ferritin level (≥1000 and <1000). The patients’ levels of zinc, copper, calcium, magnesium, and phosphorous, as well as the proportion of T cells, B cells, T-helper cells, cytotoxic T cells, and natural killer cells, were compared with 40 healthy volunteers (control).
Results Our finding showed that although the levels of hemoglobin, hematocrit, mean corpuscular volume, and mean corpuscular hemoglobin were significantly lower in all patients compared with control, their levels were comparable among patients with different ferritin level. Moreover, although serum zinc, calcium, and magnesium levels were significantly lower in patients, the serum level of phosphorous was significantly higher in patients, and serum copper showed an insignificant difference, and their levels did not differ among patients with different ferritin level. The proportion of total T cells and cytotoxic T cells was significantly increased in patients with higher ferritin level compared with control. On the contrary, the percentage of T-helper cells was lower in all patients regardless of the ferritin status. The percentages of B cells and natural killer cells were comparable among the study group.
Conclusion Despite the slight effect of high ferritin on trace elements and lymphocyte subsets, insignificant findings necessitate further expanded study with larger sample size.

Keywords: β-thalassemia major, ferritin, lymphocyte subsets, trace elements


How to cite this article:
Nafady A, Nasreldin E, Nafady-Hego H, Nasif KA, Abd-Elmawgoud EA, Sayed MM. Alteration of trace elements and T-cell subsets in patients with β-thalassemia major: influence of high ferritin level. Egypt J Haematol 2018;43:55-62

How to cite this URL:
Nafady A, Nasreldin E, Nafady-Hego H, Nasif KA, Abd-Elmawgoud EA, Sayed MM. Alteration of trace elements and T-cell subsets in patients with β-thalassemia major: influence of high ferritin level. Egypt J Haematol [serial online] 2018 [cited 2018 Oct 21];43:55-62. Available from: http://www.ehj.eg.net/text.asp?2018/43/2/55/238763




  Introduction Top


β-Thalassemia is one of the most frequent inherited single-gene disorders affecting children throughout the world especially in the Mediterranean region countries, for example, Egypt [1],[2]. β-Thalassemia major is a severe form of β-thalassemia in which the mutation or deletion of the β-globin gene causes reduced or absent β-globin protein. This inherited defect accelerates red blood cells (RBC) turnover owing to ineffective hemoglobin (Hb) synthesis. The effective treatment for β-thalassemia includes maintaining the concentration of blood Hb above 10 by regular transfusion. Despite the crucial role of regular blood transfusions to maintain normal growth and development and to suppress the extramedullary hemopoiesis and consequently decrease erythroid hyperplasia and correct anemia [3], these repeated transfusions cause precipitation of excess iron in body tissues and lead to oxidative damage and oxidative stress. This oxidative damage may cause growth impairment and several hepatic, cardiovascular, endocrine, and neurological disorders [4],[5]. In trial to prevent these complications, iron chelation therapy is usually introduced in transfusion-dependent patients with β-thalassemia major. Trace elements have a critical role in homeostasis, growth, and development, and several reports showed their affection in patients with β-thalassemia [6],[7],[8],[9],[10],[11]; nevertheless, the exact mechanism underlying trace element disorders in patients with β-thalassemia remains elusive. With respect to the immune system, several abnormalities have been reported in transfusion-dependent patients with β-thalassemia major [12], ranging from a nonspecific immune response to defects in lymphocyte subsets population and cytokine production [2],[13],[14]. Many hypotheses have been postulated to explain the defects in trace elements and immune cells in transfusion-dependent patients with β-thalassemia major, for example, immunosenescence acceleration as a result of iron overload-associated oxidative damage, trace element defect, and/or as a complication of iron chelation therapy [3],[15],[16],[17]. As the excess iron storage in the tissues is under the control of ferritin [18], the concentration of serum ferritin can be used for the assessment of iron stores [11] and prediction of oxidative damage. Herein, we studied the effect of ferritin on the serum levels of trace elements [copper (Cu), zinc (Zn), calcium (Ca), magnesium (Mg), phosphorous (P)] and on the lymphocyte subsets in patients with β-thalassemia major and compared the data with that of age-matched healthy volunteers (control).


  Patients and methods Top


Patients

Patients previously diagnosed with β-thalassemia major and coming for follow-up in Pediatric Hematology Outpatient Clinic at Pediatric Hospital, Assiut University Hospital, Assiut, Egypt, were enrolled in our case–control study. Healthy children who visit pediatric growth clinic at the same hospital for follow-up were used as a control. During the period from January to October 2017, a total of 53 patients with thalassemia major and 40 apparently healthy children with matching age and sex (control group) were included in this study. Any patient with acute inflammation, liver cirrhosis, malignancy, and/or a bone marrow transplant was excluded.

Referring to the clinicians, they usually consider ferritin level of 1000 µg/l or more an indication of initiating iron chelation therapy [19]; based on this, we divided our patients into two groups, group with ferritin level of 1000 µg/l or more and group with ferritin level less than 1000 µg/l, and then their laboratory data were compared with control group.

Blood sampling

Blood samples were withdrawn from patients with thalassemia just before a scheduled transfusion and from control during their visit for follow-up of their growth. All hematological (Hb levels and RBC indices), biochemical (serum Cu, Zn, Ca, P, and Mg), C-reactive protein, and ferritin, iron, and total iron-binding capacity (TIBC) parameters were within normal range in controls.

Approximately 2 ml of venous blood was withdrawn from both patients and controls under complete aseptic conditions and inserted into K3-EDTA anticoagulant tubes for complete blood counts, peripheral blood smears, and flow cytometric analysis (for lymphocyte subsets immunophenotyping).

Serum Cu, Zn, Ca, P, Mg, ferritin, iron, and TIBC measurements were done by a withdrawal of 3 ml of venous blood and insertion in a plain tube without any anticoagulant. The serum was then separated and stored at −70°C till used for laboratory methodology. Complete blood count was performed by using an automated blood counter (Cell-Dyne 3700 Abbott, GMI Inc., Illinois, USA) according to the manufacturer’s instructions. The lymphocytic count was done by peripheral blood smears stained by Leishman stain. Detection of ferritine level was done using in-vitro enzyme-linked immunosorbent assay kit (Abcam Inc., Cambridge, Massachusetts, USA). The assay was performed according to the manufacturer’s instructions. Ferritin values were expressed asmicrogramper liter [20],[21]. The colorimetric assay was used to measure the total iron and TIBC by using the Stanbio Iron and TIBC Kit, procedure no. 0370 (Stanbio Laboratory, San Antonio, Texas, USA) Stanbio iron (quantitative colorimetric determination of iron in serum) as manufacturer prescriptions, and the results were taken at a wavelength of 560 nm.

Trace elements assay’s detection

For estimation of serum Cu, Zn, Ca, P, and Mg, colorimetric method with Quimica Clinica Aplicada S.A. Company (Amposta, Spain) was performed according to the manufacturer’s recommendations.

Lymphocyte immunophenotypic analysis

Using flow cytometry assay, blood samples were processed for analysis immediately; a total of 100 µl of blood sample was taken and incubated with an amount of 20 µl of various monoclonal antibodies for 20 min at 4°C in the dark. Then, lysing solution was used for RBC lysis, and then washing was done with PBS. Thereafter, the cells were resuspended in PBS for analysis using a FACS Calibur (Becton Dickinson Bioscience, CA, USA). Several monoclonal antibodies including fluoroisothiocyanate conjugated CD4 (Becton Dickinson Bioscience), phycoerythrin (PE)-conjugated CD8 (Becton Dickinson Bioscience), and peridinium–chlorophyllprotein-conjugated CD3 (Becton Dickinson Bioscience), PE-conjugated CD19 (Becton Dickinson Bioscience) and PE-conjugated CD56 (Becton Dickinson Bioscience) were used for analysis of T-helper lymphocytes, cytotoxic T lymphocytes, T lymphocytes, B lymphocytes, and natural killer lymphocytes, respectively. The proportion of lymphocyte cell was measured by Cell Quest software (Becton Dickinson Biosciences). For quality control, an isotype-matched negative control was used with each sample. Forward and side scatter histogram was used to define the lymphocyte population (R1). The absolute counts and percentages of CD3, CD4, CD8, CD 19, and CD56 subsets of lymphocytes were calculated.

Statistical analysis

The data analysis was made using the statistical package for the social sciences for Windows statistical software (version 16; IBM Corporation, Armonk, New York, USA). Data are presented as mean±SD, median, range, or percentage where appropriate. The Student t-test or one-way analysis of variance test was used to compare mean among quantitative data, whereas χ2-test or Pearson’s correlation coefficient was used to compare qualitative data. P values less than 0.05 were regarded as a significant result.

Ethical approval

The Human Research Ethics Committee of the Faculty of Medicine, Assiut University approved this study. Informed consent was provided for pediatric patients and their parents according to the Declaration of Helsinki.


  Results Top


Fifty-three patients with β-thalassemia major and 40 healthy children with matching age and sex as a control group were included in this study.

On analysis of all patients with β-thalassemia major, we found the mean ages in patients with β-thalassemia were comparable to those in group control. The mean ages of patients with β-thalassemia and control were 8.48±4.07 and 7.11±4.46 years, respectively (P=0.143). Sexes were comparable between the two study groups. Male/female ratio was 24/29 in patients with β-thalassemia compared with 23/17 in healthy controls (P=0.63) ([Table 1]).
Table 1 Demographic and hematological characteristics in patients with β-thalassemia major and controls

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The BMI was significantly lower in patients with β-thalassemia than that in the control, with 17.18±1.59 and 22.5±9.5, respectively (P=0.05).

Routine blood tests demonstrated that the Hb levels, hematocrit (HCT, l/l) level, the mean corpuscular volume (MCV, fl), and the mean corpuscular hemoglobin (MCH, pg) were significantly lower in patients with β-thalassemia than those in healthy group (Hb levels: 73.9±14.6 and 131.7±22.9 g/l, respectively, P<0.0001; HCT: 21.5±5.3 and 38±2.2 l/l, respectively, P<0.0001; MCV: 69.8±7.2 and 80.9±8.6 fl, respectively, P=0.028; and MCH: 22.5±2.8 and 30.2±3.4 pg, respectively, P=0.01); however, the MCH concentration and platelet count were comparable in patients with β-thalassemia and healthy group (MCH concentration: 328.0±13 and 332.0±12 g/l, respectively, P=0.471, and platelet count: 343.7±171.7 and 321.6±117.1×109/l, respectively, P=0.915) ([Table 1]).

Patients with β-thalassemia were divided into two groups according to the ferritin level taking a cutoff point of 1000 µg/l (patients with β-thalassemia with ferritin level 1000 µg/l or more and patients with β-thalassemia with ferritin level less than 1000 µg/l) to assess the effect of higher ferritin on demographic and hematological markers. The previous parameters were reassessed in the two patients’ groups. We found that the mean age was greater in patients with ferritin level of at least 1000 than patients with ferritin less than 1000. The mean BMI, Hb, HCT, MCV, and MCH were lower in patients with ferritin level of at least 1000 than patients with ferritin less than 1000, with no significant difference ([Figure 1]).
Figure 1 Effect of ferritin level on the hematological parameters and BMI in patients with β-thalassemia major and controls. Hb, hemoglobin; HCT, hematocrit; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume.

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Analysis of trace elements in all patients with β-thalassemia showed a significantly reduced Zn, Ca, and Mg levels in patients with thalassemia than the healthy control (Zn: 6.5±1.2 and 12.8±3.4 μmol/l, respectively, P<0.0001; Ca: 1.7±0.3 and 2.3±0.15 mmol/l, respectively, P<0.0001; and Mg: 75±0.2 and 0.95±0.2 mmol/l, respectively, P<0.05) ([Table 2]).
Table 2 Serum levels of some trace elements in patients with β-thalassemia major and controls

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However, P levels were significantly higher in patients with β-thalassemia than in the controls (1.97±0.2 and 1.26±0.23 mmol/l, respectively, P=0.014), and serum Cu levels were comparable between the two groups, with a mean of 16.2±3.5 μmol/l for patients and 15.8±4.2 μmol/l for controls (P=0.326).

The serum iron and ferritin levels in patients with β-thalassemia were significantly higher than those in control (iron: 25.7±7.3 and 19.8±5.6 μmol/l, respectively, P=0.04, and ferritin: 1087±519.6 and 27±12 µg/l, respectively, P<0.0001). On the contrary, the TIBC levels were significantly lower in patients with β-thalassemia than in the controls (29.34±12.5 and 55.26±0.18 μmol/l, respectively, P=0.01) ([Table 2]).

To assess the effect of higher ferritin on trace elements, the previous parameters were reassessed in the two patient groups (patients with β-thalassemia with ferritin level 1000 µg/l or more, and patients with β-thalassemia with ferritin level less than 1000 µg/l). The results showed that the mean Zn, Ca, Mg, Cu, and TIBC levels were lower in patients with ferritin level of at least 1000 than patients with ferritin less than 1000, whereas the mean P level is the same in patients with ferritin level of at least 1000 and patients with ferritin less than 1000, and the mean iron level is comparable in patients with ferritin level of at least 1000 and patients with ferritin less than 1000 ([Figure 2]).
Figure 2 Effect of ferritin level on the serum trace elements in patients with β-thalassemia major and controls. TIBC, total iron-binding capacity.

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On analysis of lymphocyte subsets in all patients with β-thalassemia, the results showed that although total leukocytic count and lymphocyte percentage were comparable between patients and controls (total leukocytic count: 10.63±7 and 9.2±4.3×109/l, respectively, P=0.266, and lymphocyte percentage: 37.1±9.5 and 33.32±16.71%, respectively, P=0.178), the lymphocyte count was higher in patients with thalassemia compared with controls (4±3.19 and 2.75±1.49×109/l, respectively, P=0.028).

On analysis of percentage and absolute count of total T cells (CD3-positive) and cytotoxic T cells (CD8-positive), they were significantly higher in patients with β-thalassemia than controls (CD3-positive cells%: 69.5±11.5 and 58.3±14.6%, respectively, P=0.002; CD3-positive absolute count: 2675.6±1907.4 and 1361.5±666.5/l, respectively; P=0.008; CD8-positive cells%: 34.9±12.4 and 28.3±8%, respectively, P=0.036, and CD8-positive absolute count: 1392±1311.8 and 656±289.4/l, respectively, P=0.029) ([Table 3]).
Table 3 Percentage and number of lymphocyte subsets in patients with β-thalassemia major and controls

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However, the percentage of T-helper cells (CD4-positive) was significantly reduced in patients with β-thalassemia than controls (CD4-positive cells%: 33.5±12.4 and 44.9±11.8%, respectively, P<0.0001), and the absolute counts of CD 4-positive cells were comparable between the two groups (CD4-positive absolute count: 1236.8±875.7= and 1074.1±559.6/l, respectively, P=0.34). The number and percentage of B cells (CD19-positive cells) and natural killer (CD56-positive cells) did not differ between patients and control (CD19-positive cells%, P=0.703 and CD19-positive cells absolute; P=0.102), (CD56-positive cells%, P=0.21, and CD56-positive cells absolute; P=0.812) ([Table 3]).

On dividing the patients according to the ferritin level taking a cutoff of 1000 µg/l, no significant difference was found among patients in the lymphocyte subsets with different ferritin level ([Figure 3]).
Figure 3 Effect of ferritin level on the percentage of lymphocyte subsets in patients with β-thalassemia major and controls. NK, natural killer.

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  Discussion Top


β-Thalassemia major is considered to be one of the most common inherited hemolytic anemia in Egypt. Our study showed that the patients were underweight as their BMI was significantly lower compared with controls, and this underweight is in accordance with previous reports, which can be explained by disorder in the endocrine system as a result of iron overload and prolonged use of iron chelating therapy [22],[23]. The present significant reduction in Hb and red blood indices such as HCT, MCV, and MCH in our patients compared with controls may be attributed to defect in the erythropoiesis associated with β- thalassemia. Similar data were also reported by Simsek et al. [24]. The higher the serum level of iron and ferritin, and the lower the serum level of TIBC found here and in other studies [24],[25] are consequences of the absence of β-globin chains and accumulation of unpaired alpha globin that causes iron overload and hence cellular oxidative damage [25].

For trace elements, although some studies showed the increased serum Cu levels in patients with β-thalassemia major [26],[27],[28], others found reverse results [29],[30],[31]. We and those who found hypercupremia in patients with β-thalassemia major related that finding to hemochromatosis associate with β-thalassemia major.

Serum Zn levels were found to be significantly lower in patients than controls, which may be owing to either an excessive release from hemolyzed red cells, desferrioxamine therapy, or undernutrition in those patients [32],[33]. Although several studies revealed lower serum Zn in patients with thalassemia [32],[33], others revealed higher serum Zn in the patients with thalassemia, with no relation to ferritin level [34]. Our patients also showed a significantly lower serum Ca and significantly higher P levels that are in agreement with previous studies [8],[9],[10]. We think that the reason for this might be delay in starting iron chelating therapy and poor compliance with the therapy in some of our patients, and this further explains the underweight in those patients. However, other reports showed no changes in mean Ca and P levels [35],[36]. Furthermore, serum Mg levels were significantly reduced in patients with β-thalassemia than in controls. This finding was in agreement with Karim et al. [37] who explained the presence of hypomagnesemia as a result of lower thyroid hormones resulted from iron overload. On the contrary, opposite result was described in the study by Al-Samarrai et al. [27]. Herein, most patients had a high ferritin levels (mean: 1087±519.6 µg/l), with more than 50% of patients having more than 1000 µg/l, which is a result of repeated blood transfusion, being the main feature of patients with β-thalassemia [38],[39],[40],[41]. So, we tried to correlate our findings in patients after dividing them into two groups depending on the clinical cutoff of 1000 µg/l, and the results did not differ significantly with ferritin level of at least 1000 µg/l. This new finding may be owing to either the cutoff of 1000 µg/l is very high, so the pathology is present in both groups equally.This study found T cells but not B cells or natural killer cells are raised in patients with β-thalassemia. This indicates activation of the cell-mediated immune cells in those patients. Furthermore, this increase in the total T-lymphocyte pool is mainly related to increase in the cytotoxic T-lymphocytes and not in T-helper lymphocytes, which in fact were reduced in our patients. This is in accordance with the concept described by many other researchers [42],[43],[44]. Whether this specific increase in CD8 T cells and the decrease in CD4 is owing to continuous alloantigenic stimulation of the immune system with autoimmune hemolysis as a result of iron overload remains elusive. This is different to the finding by others who found either increase in the proportion of both CD8-positive cells and CD4-positive cell or no change in T-lymphocyte population [12],[45]. The discrepancy between our results and theirs may be related to the changes in the immune system being time related and depend on the disease duration and iron overload amount. The absence of changes in the other immune arms (humoral and innate) reported here is in agreement with the findings by Ahmadiafshar et al. [46], who found no abnormalities in cellular and humeral system, and unlike those who found alteration of immunoglobulins [47]. Further analysis of the effect of ferritin on immune markers depending on the cutoff 1000 µg/l could reveal significant difference between patients with ferritin level of at least 1000 and patients with ferritin less than 1000 when compared with the controls, as the level of T cells and CD8-positive cells is higher in patients with ferritin level of at least 1000 but not in patients with ferritin less than 1000. In agreement with this, a recent work of Gharagozloo and colleagues [43],[44] found a significant negative correlation between serum ferritin and CD3 and CD4, and positive correlation with CD8. Our results were in contrast with Noulsri et al. [48],[49] who reported insignificant differences in T-cell subsets CD3, CD4, and CD8 between patients and controls, and they concluded that high iron levels in patients with β-thalassemia have a more significant effect on the function and activity of T cells rather than on their number [45],[48]. Variation among results can be contributed to several factors such as the clinical heterogeneity among patients with β-thalassemia, frequency of blood transfusion, splenectomy, serum iron status, and iron chelation therapy.


  Conclusion Top


Although the alteration in the level of trace elements and cell-mediated immunity did not significantly differ among patients with β-thalassemia with high ferritin level (≥1000) and those with low ferritin level (<1000), the impaired levels of these molecules and cells in the whole patients indicate oxidative stress and damage even in patients with ferritin level (<1000). Initiating chelation therapy regardless of the level of ferritin can be a more realistic approach in β-thalassemia major. In addition, further large-scale studies are recommended.

Acknowledgements

A.N., E.N., E.A., M.M. and H.N. participated in research design and carried out the research; A.N., H.N., E.A., M.M. and K.N. conducted the data analysis and wrote the paper.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

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    Tables

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