The Clinical Respiratory Journal
ORIGINAL ARTICLE
Effects of blood transfusion on cytokine profile and pulmonary function in patients with thalassemia major Bora Gülhan1, Ebru Yalçın1, S¸ule Ünal2, Berna Og˘uz3, Ug˘ur Özçelik1, Deniz Dog˘ru Ersöz1, Fatma Gümrük2 and Nural Kiper1 1 Department of Pediatric Pulmonology, Hacettepe University, Ankara, Turkey 2 Department of Pediatric Hematology, Hacettepe University, Ankara, Turkey 3 Department of Pediatric Radiology, Hacettepe University, Ankara, Turkey
Abstract Background and Aims: Thalassemia major (TM) is characterized by abnormal hemoglobin synthesis, which results in decreased oxygen delivery to the tissues, ineffective erythropoiesis and iron overload. The purpose of this study was to find out the predominant type of lung mechanical abnormalities in TM patients, the prevalence of the change in pulmonary diffusing capacity and to search the association of cytokines with pulmonary function tests (PFTs). Methods: TM patients (n = 26) who were on regular transfusion program were included in the study. Pretransfusional and posttransfusional PFTs were performed. Before transfusion, blood samples were collected for complete blood count, serum ferritin, zinc, transforming growth factor-β1 (TGF-β1), interleukin (IL)-8 and IL-10 levels. Results: Obstructive lung pattern was the predominant pulmonary sequela. Male gender was the main risk factor for pulmonary function abnormality. Because of very low values, serum IL-10 levels of all patients were undetectable, and serum IL-8 levels could be measured only in six patients. Median serum IL-8 level was higher in patients with abnormal PFT. Pretransfusional single breath diffusion of carbon monoxide (DLCO) correlated negatively with serum TGF-β1 and ferritin levels. After transfusion, there was a statistically significant decrease in forced expiratory volume in 1 s, forced expiratory flow between 25% and 75% of vital capacity, and airway conductance but significant increase in DLCO. Conclusions: Although they are mostly asymptomatic, TM patients have important PFT abnormalities. Blood transfusion may have an acute deleterious effect on pulmonary functions. The disturbed pulmonary functions were found to correlate with IL-8 and TGF-β levels. Relations of different cytokines with different PFT parameters suggest that the immune system is effective pulmonary dysfunction. Please cite this paper as: Gülhan B, Yalçın E, Ünal S¸, Og˘uz B, Özçelik U, Ersöz DD, Gümrük F and Kiper N. Effects of blood transfusion on cytokine profile and pulmonary function in patients with Thalassemia major. Clin Respir J 2014; ••: ••–••. DOI:10.1111/crj.12193.
Key words cytokine – ferritin – pulmonary function tests – thalassemia major Correspondence Bora Gülhan, MD, Hacettepe University, I˙hsan Dog˘ramacı Çocuk Hastanesi, Çocuk Gög˘üs Hastalıkları Ünitesi, 06100 Sıhhiye/Ankara, Türkiye. Tel: +90 536 4613759 (mobile), +90 312 3051224 (office) Fax: +90 312 309423 email: [email protected] Received: 03 February 2014 Revision requested: 02 June 2014 Accepted: 22 July 2014 DOI:10.1111/crj.12193 Authorship and contributorship Dr. Bora Gülhan: performed the study and wrote the paper. Prof. Dr. Ebru Yalçın: performed the study and analyzed data. Assoc. Prof. Dr. S¸ule Ünal: collected data. Assoc. Prof. Dr. Berna Og˘uz: collected data. Prof. Dr. Ug˘ur Özçelik: contributed important reagents. Prof. Dr. Deniz Dog˘ru Ersöz: contributed important reagents. Prof. Dr. Fatma Gümrük: designed the study. Prof. Dr. Nural Kiper: designed the study. Ethics The study was approved by the Hacettepe University Ethics Committee (LUT 08/10-41). Conflict of interest The authors have stated explicitly that there are no conflicts of interest in connection with this article.
The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
1
Pulmonary function in thalassemia patients
Gülhan et al.
Introduction
Materials and methods
Beta thalassemia major (TM) is a disorder of hemoglobin (Hb) synthesis. It is characterized by complete absence of β globin production, which results in decreased oxygen delivery to the tissues, ineffective erythropoesis and severe anemia (1). Patients usually develop complications of chronic anemia including growth retardation (2). Survival of TM patients has greatly improved during recent decades, mostly because of specialized treatment protocols. Conventional management of TM includes regular blood transfusion in order to suppress the bone marrow for synthesis of abnormal Hb and to prevent tissue hypoxia. Both regular blood transfusions and ineffective erythropoesis leading to increased iron absorption from gut will contribute iron overload (3). Although excessive iron overload can be prevented by iron chelators, iron is still increased in amount in many organs, especially the liver, heart and pancreas (4). Pulmonary iron deposition in the lungs has been observed on postmortem examination of TM patients receiving multiple blood transfusions (5, 6). As patients survive longer, iron deposition in the lungs leads to pulmonary dysfunction. However, studies about the nature of pulmonary function abnormality have conflicting results. Most studies on pediatric and adult patients show a predominant restrictive spirometric pattern (7–14); on the other hand, others indicate a predominant obstructive pattern (15–18). These contradictory results may be originated from the heterogenous nature of the studied population regarding the age, ethnical origin or treatment protocols. Another important result of multiple blood transfusions are functional alterations in the immune system of TM patients. Recent studies revealed numerous quantitative and functional defects involving lymphocytes, neutrophils and macrophages in this group of patients. These defects cause some changes in cytokine profile of TM patients (19). Decreased zinc levels have been reported among patients with TM, related to both hemolysis and dietary habits of these patients, because food rich in iron are usually good sources for zinc, as well, and avoidance of iron-rich food usually ends up with low zinc intake (3). The aims of the study were to evaluate both acute and chronic effects of blood transfusion on pulmonary function tests (PFTs), and to assess risk factors in patients with abnormal PFTs. We have also analyzed the association of cytokines with PFT parameters.
Study design
2
Patients were enrolled from Hacettepe University Department of Pediatric Hematology between June 2008 and June 2010. All TM patients who were under regular blood transfusion program and could cooperate PFT were included in the study. Exclusion criteria include history of lung operation, history of asthma or congestive heart failure, smoking, active upper or lower respiratory system infection at the time of the study, pulmonary hypertension and inability to cooperate PFT. Transcutaneous oxygen saturation of the patients while breathing room air was greater than 95%. Subjects were ascertained and enrolled in the study after obtaining informed consent, in accordance with research protocols approved by the Hacettepe University in Ankara (LUT 08/10–41). After assessing eligibility to the study and written informed consent from the patient and his/her parents, patients were included in the study. Data on demographic features (name, age, sex and birthplace), date of diagnosis of TM, frequency of blood transfusion, history of medication including iron chelating therapy, presence of splenectomy and history of any respiratory system problem were noted in a prepared form. Complete physical examination including height and weight measurements was performed. Body mass index (BMI) was calculated as the ratio (weight)/ (height)2 (kg/m2, Quatelet index). Before transfusion, blood samples were collected for complete blood count, serum ferritin, zinc, transforming growth factor-β1 (TGF-β1), interleukin (IL)-8 and IL-10 levels. For cytokines, serum samples were stored at –80°C until analysis. Serum TGF-β1, IL-8 and IL-10 levels were measured with enzyme-linked immunosorbent assay (Bender MedSystems GmbH, Vienna, Austria). Chest radiographs were obtained for all patients and were graded by an expert radiologist without any knowledge of the medical history or clinical findings, according to the study of Villani et al. (20). Pulmonary injury was assessed in four grades. These grades were as follows: grade 0, no lesion; grade 1, mild interstitial sclerosis but no paramediastinal retraction; grade 2, pulmonary hilus retraction or altered location of cardiovascular structures with interstitial retracting sclerosis; and grade 3, mediastinal shift with interstitial and alveolar retracting sclerosis.
PFTs PFTs were performed in all patients before scheduled blood transfusion and 1–24 h later after blood
The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
Gülhan et al.
transfusion. The chronic effects of blood transfusion on lung mechanics were investigated with pretransfusion PFT. All PFTs were studied in the morning and by the same respiratory technician and pediatric pulmonologist without knowing clinical status of the patient. Forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), forced expiratory flow between 25% and 75% of vital capacity (FEF25–75), FEV1/FVC and airway conductance (Gaw) were recorded collecting at least three technically acceptable values. Functional residual capacity, residual volume (RV) and total lung capacity (TLC) were measured by plethysmography (Viasys Vmax-Spectra; VIASYS Respiratory Care Inc.,Yorba Linda, CA, USA) and expressed in liters corrected for body temperature, atmospheric pressure and saturation with water vapor. Plethysmography results were reported as percentage of predicted normal values corrected to age, sex and height. Single breath diffusion of carbon monoxide (DLCO) was corrected for the Hb level and alveolar volume (DLCOAdj) (21). Z scores were calculated for each PFT parameter with the formula (z score = [observed value – predicted mean value)/standard deviation (SD) of the reference population]. Patients with abnormal PFTs were referred to pediatric pulmonology department.
Interpretation of PFT results Restrictive pulmonary disease was defined when TLC was below 80% of the predicted value. Obstructive pulmonary disease was defined when FEF25–75 was below 75% of the predicted value and/or normal or decreased FEV1 (
ORIGINAL ARTICLE
Effects of blood transfusion on cytokine profile and pulmonary function in patients with thalassemia major Bora Gülhan1, Ebru Yalçın1, S¸ule Ünal2, Berna Og˘uz3, Ug˘ur Özçelik1, Deniz Dog˘ru Ersöz1, Fatma Gümrük2 and Nural Kiper1 1 Department of Pediatric Pulmonology, Hacettepe University, Ankara, Turkey 2 Department of Pediatric Hematology, Hacettepe University, Ankara, Turkey 3 Department of Pediatric Radiology, Hacettepe University, Ankara, Turkey
Abstract Background and Aims: Thalassemia major (TM) is characterized by abnormal hemoglobin synthesis, which results in decreased oxygen delivery to the tissues, ineffective erythropoiesis and iron overload. The purpose of this study was to find out the predominant type of lung mechanical abnormalities in TM patients, the prevalence of the change in pulmonary diffusing capacity and to search the association of cytokines with pulmonary function tests (PFTs). Methods: TM patients (n = 26) who were on regular transfusion program were included in the study. Pretransfusional and posttransfusional PFTs were performed. Before transfusion, blood samples were collected for complete blood count, serum ferritin, zinc, transforming growth factor-β1 (TGF-β1), interleukin (IL)-8 and IL-10 levels. Results: Obstructive lung pattern was the predominant pulmonary sequela. Male gender was the main risk factor for pulmonary function abnormality. Because of very low values, serum IL-10 levels of all patients were undetectable, and serum IL-8 levels could be measured only in six patients. Median serum IL-8 level was higher in patients with abnormal PFT. Pretransfusional single breath diffusion of carbon monoxide (DLCO) correlated negatively with serum TGF-β1 and ferritin levels. After transfusion, there was a statistically significant decrease in forced expiratory volume in 1 s, forced expiratory flow between 25% and 75% of vital capacity, and airway conductance but significant increase in DLCO. Conclusions: Although they are mostly asymptomatic, TM patients have important PFT abnormalities. Blood transfusion may have an acute deleterious effect on pulmonary functions. The disturbed pulmonary functions were found to correlate with IL-8 and TGF-β levels. Relations of different cytokines with different PFT parameters suggest that the immune system is effective pulmonary dysfunction. Please cite this paper as: Gülhan B, Yalçın E, Ünal S¸, Og˘uz B, Özçelik U, Ersöz DD, Gümrük F and Kiper N. Effects of blood transfusion on cytokine profile and pulmonary function in patients with Thalassemia major. Clin Respir J 2014; ••: ••–••. DOI:10.1111/crj.12193.
Key words cytokine – ferritin – pulmonary function tests – thalassemia major Correspondence Bora Gülhan, MD, Hacettepe University, I˙hsan Dog˘ramacı Çocuk Hastanesi, Çocuk Gög˘üs Hastalıkları Ünitesi, 06100 Sıhhiye/Ankara, Türkiye. Tel: +90 536 4613759 (mobile), +90 312 3051224 (office) Fax: +90 312 309423 email: [email protected] Received: 03 February 2014 Revision requested: 02 June 2014 Accepted: 22 July 2014 DOI:10.1111/crj.12193 Authorship and contributorship Dr. Bora Gülhan: performed the study and wrote the paper. Prof. Dr. Ebru Yalçın: performed the study and analyzed data. Assoc. Prof. Dr. S¸ule Ünal: collected data. Assoc. Prof. Dr. Berna Og˘uz: collected data. Prof. Dr. Ug˘ur Özçelik: contributed important reagents. Prof. Dr. Deniz Dog˘ru Ersöz: contributed important reagents. Prof. Dr. Fatma Gümrük: designed the study. Prof. Dr. Nural Kiper: designed the study. Ethics The study was approved by the Hacettepe University Ethics Committee (LUT 08/10-41). Conflict of interest The authors have stated explicitly that there are no conflicts of interest in connection with this article.
The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
1
Pulmonary function in thalassemia patients
Gülhan et al.
Introduction
Materials and methods
Beta thalassemia major (TM) is a disorder of hemoglobin (Hb) synthesis. It is characterized by complete absence of β globin production, which results in decreased oxygen delivery to the tissues, ineffective erythropoesis and severe anemia (1). Patients usually develop complications of chronic anemia including growth retardation (2). Survival of TM patients has greatly improved during recent decades, mostly because of specialized treatment protocols. Conventional management of TM includes regular blood transfusion in order to suppress the bone marrow for synthesis of abnormal Hb and to prevent tissue hypoxia. Both regular blood transfusions and ineffective erythropoesis leading to increased iron absorption from gut will contribute iron overload (3). Although excessive iron overload can be prevented by iron chelators, iron is still increased in amount in many organs, especially the liver, heart and pancreas (4). Pulmonary iron deposition in the lungs has been observed on postmortem examination of TM patients receiving multiple blood transfusions (5, 6). As patients survive longer, iron deposition in the lungs leads to pulmonary dysfunction. However, studies about the nature of pulmonary function abnormality have conflicting results. Most studies on pediatric and adult patients show a predominant restrictive spirometric pattern (7–14); on the other hand, others indicate a predominant obstructive pattern (15–18). These contradictory results may be originated from the heterogenous nature of the studied population regarding the age, ethnical origin or treatment protocols. Another important result of multiple blood transfusions are functional alterations in the immune system of TM patients. Recent studies revealed numerous quantitative and functional defects involving lymphocytes, neutrophils and macrophages in this group of patients. These defects cause some changes in cytokine profile of TM patients (19). Decreased zinc levels have been reported among patients with TM, related to both hemolysis and dietary habits of these patients, because food rich in iron are usually good sources for zinc, as well, and avoidance of iron-rich food usually ends up with low zinc intake (3). The aims of the study were to evaluate both acute and chronic effects of blood transfusion on pulmonary function tests (PFTs), and to assess risk factors in patients with abnormal PFTs. We have also analyzed the association of cytokines with PFT parameters.
Study design
2
Patients were enrolled from Hacettepe University Department of Pediatric Hematology between June 2008 and June 2010. All TM patients who were under regular blood transfusion program and could cooperate PFT were included in the study. Exclusion criteria include history of lung operation, history of asthma or congestive heart failure, smoking, active upper or lower respiratory system infection at the time of the study, pulmonary hypertension and inability to cooperate PFT. Transcutaneous oxygen saturation of the patients while breathing room air was greater than 95%. Subjects were ascertained and enrolled in the study after obtaining informed consent, in accordance with research protocols approved by the Hacettepe University in Ankara (LUT 08/10–41). After assessing eligibility to the study and written informed consent from the patient and his/her parents, patients were included in the study. Data on demographic features (name, age, sex and birthplace), date of diagnosis of TM, frequency of blood transfusion, history of medication including iron chelating therapy, presence of splenectomy and history of any respiratory system problem were noted in a prepared form. Complete physical examination including height and weight measurements was performed. Body mass index (BMI) was calculated as the ratio (weight)/ (height)2 (kg/m2, Quatelet index). Before transfusion, blood samples were collected for complete blood count, serum ferritin, zinc, transforming growth factor-β1 (TGF-β1), interleukin (IL)-8 and IL-10 levels. For cytokines, serum samples were stored at –80°C until analysis. Serum TGF-β1, IL-8 and IL-10 levels were measured with enzyme-linked immunosorbent assay (Bender MedSystems GmbH, Vienna, Austria). Chest radiographs were obtained for all patients and were graded by an expert radiologist without any knowledge of the medical history or clinical findings, according to the study of Villani et al. (20). Pulmonary injury was assessed in four grades. These grades were as follows: grade 0, no lesion; grade 1, mild interstitial sclerosis but no paramediastinal retraction; grade 2, pulmonary hilus retraction or altered location of cardiovascular structures with interstitial retracting sclerosis; and grade 3, mediastinal shift with interstitial and alveolar retracting sclerosis.
PFTs PFTs were performed in all patients before scheduled blood transfusion and 1–24 h later after blood
The Clinical Respiratory Journal (2014) • ISSN 1752-6981 © 2014 John Wiley & Sons Ltd
Gülhan et al.
transfusion. The chronic effects of blood transfusion on lung mechanics were investigated with pretransfusion PFT. All PFTs were studied in the morning and by the same respiratory technician and pediatric pulmonologist without knowing clinical status of the patient. Forced vital capacity (FVC), forced expiratory volume in 1 s (FEV1), forced expiratory flow between 25% and 75% of vital capacity (FEF25–75), FEV1/FVC and airway conductance (Gaw) were recorded collecting at least three technically acceptable values. Functional residual capacity, residual volume (RV) and total lung capacity (TLC) were measured by plethysmography (Viasys Vmax-Spectra; VIASYS Respiratory Care Inc.,Yorba Linda, CA, USA) and expressed in liters corrected for body temperature, atmospheric pressure and saturation with water vapor. Plethysmography results were reported as percentage of predicted normal values corrected to age, sex and height. Single breath diffusion of carbon monoxide (DLCO) was corrected for the Hb level and alveolar volume (DLCOAdj) (21). Z scores were calculated for each PFT parameter with the formula (z score = [observed value – predicted mean value)/standard deviation (SD) of the reference population]. Patients with abnormal PFTs were referred to pediatric pulmonology department.
Interpretation of PFT results Restrictive pulmonary disease was defined when TLC was below 80% of the predicted value. Obstructive pulmonary disease was defined when FEF25–75 was below 75% of the predicted value and/or normal or decreased FEV1 (
