The Research Of Iron Overload-related Indicators In Non-transfusion-dependent Tha Lassemia Patients And The Establish Of Iron Overload Animal Model

THE FIRST PART OF THESIS THE STUDY OF CORRLATION INDEX IN IRON OVERLOAD IN PATIENTS WITH NON-TRANSFUSION-DEPENDENT THALASSEMIAObjective:This study aimed at investigating the conditions of the whole body iron overload,the cardiac iron overload and hepatic iron overload,the correlation between iron overload and clinically relevant detection index in non-transfusion-dependent thalassemia(NTDT)patients.Methods:We studied 110 patients who were aged 10 or older with NTDT patients from June 2013 to December 2013.We analyzed the clinical data,the Serum ferritin(SF)was measured by Electrochemiluminescence immunoassay(ECLIA),the liver R2 value and T2*cardiac magnetic resonance(CMR)in all patients.We compared the clinically data and inspection results.Results:1.The number of NTDT patients who were aged 10 or older was 110(65 males and 45 females)cases.The median age was 24 years(rang 10-63 years).The median age was 8 years(rang 1-45 years)for the first time with a blood transfused.29 cases(26.4%)have a history of remove iron treatment.The median age was 24 years(rang 3-59 years)for the first time of removing the iron.87 patients(79.0%)received blood transfused treatment.The median number of blood transfused was 6 U(rang 0-132 U),the mean level of hemoglobin(Hb)was 87.7 ± 15.6 g/L.Hepatomegaly and splenomegaly were found in 35 patients(31.8%)and 34patients(30.9%),respectively.49 patients(44.5%)received splenectomy treantment.The median number of SF was 1105(rang 36-19704 ng/ml).43 patients(39.1%)had mild and moderate iron overload(1000<SF<2500 ng/ml),and 16 patients(14.5%)had severe iron overload(SF≥2500 ng/ml),and 51 patients(46.4%)had normal iron(SF<1000 ng/ml),respectively.The level of SF in β-thalassemia and HbE/β-thalassemia were higer than that of HbH patients(P=0.001).2.The mean concentration of liver iron was 13.4±11.7 mg Fe/g dry weight.Among them,93 patients(84.5%)had hepatic iron overload,28 patients had mild hepatic iron overload(3≤LIC<7 mg Fe/g dw),and 29 patients(26.4%)had moderate hepatic iron overload(7≤LIC<15 mg Fe/g dw),and 36 patients(32.6%)had severe hepatic iron overload(LIC>15 mg Fe/g dw).The concentration of liver iron in(3-thalassemia and HbE/β-thalassemia were higer than that of HbH patients(P<0.001).3.Mean value of cardiac T2*value from 110 patients is 33.8±10.4 ms.Among them,light and moderate degree(10 ms≤cardiac T2*value<20 ms)5 cases(4.5%),severe degree(cardiac T2*value<10 ms)1 case(1.0%),normal degree(cardiac T2*>20 ms)104 cases(94.5%).The cardiac T2*value of HbH patients showed no statistical difference with that of(3-thalassemia and HbE/β-thalassemia respectively.4.SF level of patients was positively correlated with age,ALT,AST(R=0.413,P<0.001;R=0.715,P<0.001;R=0.538,P<0.001,respectively).Liver iron concentration was positively correlated with age,ALT,AST(R=0.410,P<0.001;R=0.608,P<0.001;R=0.531,P<0.001,respectively),SF level of patients was positively correlated with liver iron concentration(R=0.808,P<0.001).Mutiple linear regression analysis confirmed the positive correlation between serum ferritin and age(β=0.523,P<0.001),total of blood transfusion(β=0.281,P=0.001),type of disease(β=0.298,P=0.001)and splenectomized(β=0.347,P<0.001).Multivariate analysis also confirmed the positive correlation between liver iron concentration and age(β=0.483,P<0.001),total of blood transfusion(3=0.289,P=0.002),type of disease(β=0.388,P<0.001).Conclusions:1.Heavy liver iron overload were observed in NTDT patients.The liver iron overload degree of β-thalassemia and HbE/β-thalassemia were higer than that of HbH patients.2.The SF level did not reflect liver iron overload status accordingly,or more accurately,the level of SF maybe underestimate the risk of iron overload and could not be taken as measure marker of iron content level.3.The age,type of disease and blood transfusion are the common influence factors of serum ferritin and liver iron concentration for non-transfusion-dependent thalassemia patients.Among them,the age as the main influence factor.4.Liver iron overload may lead to liver damages and liver function abnormalities.THE SECOND PART OF THESIS SERUM MARKERS OF LIVER FIBROSIS IN PATIENTS WITH NON-TRANSFUSION-DEPENDENT THALASSEMIA AND RELATIONSHIP TO IRON OVERLOAD:A CONTROLLED STUDYObjective:To study the expression of serum markers of liver fibrosis in patients with non-transfusion-dependent thalassemia(NTDT),as well as explore the relationship between these markers and iron overload.Methods:Serum levels of serum collagen type Ⅳ(C Ⅳ),precollagen typeⅢ(PIIINP),hyaluronic acid(HA),AST and ALT were measured prospectively in 105 patients with NTDT and 139 with β thalassemia major(TM),as well as in 120 healthy controls.None of the study subjects had a history of infection with hepatitis B or C virus.Levels of serum fibrosis markers were determined using a time-resolved fluoroimmunoassay.The serum levels of AST and ALT were determined by biochemical method.In addition,serum ferritin levels were determined using an electrochemiluminescence immunoassay,and liver iron concentration was assayed using R2 magnetic resonance imaging.Results:Serum levels of C Ⅳ and HA were significantly higher in patients with NTDT or TM than in healthy controls.C Ⅳ levels were elevated above control levels in 94 of 105(89.5%)patients with NTDT and in all 139(100%)of patients with TM(P<0.05).HA levels were above control levels in 70 of 105(66.7%)patients with NTDT and 137 of 139(98.6%)patients with TM(P<0.05).PIIINP content was significantly higher in patients with TM than in controls,but PIIINP levels were similar between patients with NTDT and controls.PIIINP levels were above control levels in 76 of 105(72.4%)patients with NTDT and 127 of 139(91.4%)patients with TM.Serum ferritin levels correlated positively with serum levels of C Ⅳ(R=0.194,P=0.002),PIIINP(R=0.183,P=0.004),HA(R=0.435,P<0.001),ALT(R=0.681,,P<0.001)and AST(R=0.565,P<0.001).Liver iron concentration also correlated positively with serum levels of C IV(R=0.175,P=0.039),PIIINP(R=0.334,P<0.001),HA(R=0.0.528,P<0.001),ALT(R=0.693,P<0.001)and AST(R=0.604,P<0.001).Conclusions:1.Values for serum markers of liver fibrosis in NTDT patients were significantly lower than TM patients,but higher than controls.Serum markers of liver fibrosis correlated positively with iron overload.Iron overload may contribute to liver fibrosis in NTDT.2.C IV,PIIINPC and HA may be clinically useful for predicting risk of iron overload-related liver fibrosis in Chinese patients with NTDT.THE THIRD PART OF THESIS THE RELATIONSHIP BETWEEN IRON OVERLOAD AND GENOTYPE OR CLINICAL PHENOTYPE IN HEMOGLOBIN H DISEASE PATIENTObjective:The aim of this study was to assess the relationship between genotype and iron overload,and between linical phenotype and iron overload.Methods:We studied 130 patients with hemoglobin H disease(HbH)patients from June 2013 to December 2013.Clinical parameters including age starting blood transfusion,age of attack,height,weight and so on.We analyzed hematology and serum ferritin(SF)in 130 HbH patients.The liver iron concentration(LIC)was measured in 77 HbH patients.The cardiac T2*was measured in 65 HbH patients.Results:1.The number of HbH patients was 130(72 males and 58 females)cases.The median age was 23 years(range:2-63 years).Hepatomegaly and splenomegaly were found in 27 patients(20.8%)and 55 patients(42.3%),respectively.31 patients(23.8%)received splenectomy treantment.The median number of blood transfused was 5 U(range:0-88 U).The median number of serum ferritin was 533 ng/ml(range:27-19704 ng/ml).35 patients(26.9%)had serum ferritin>1000 ng/ml,6 patients(4.6%)had serum ferritin>2500 ng/ml.77 patients underwent hepatic R2 MRI.The mean value of liver iron concentration was 6.6 mg Fe/g dry weight.Among them,56 patients(72.7%)had hepatic iron overload(LIC≥3 mg Fe/g dry weight),and 20 patients(26.0%)had mild hepatic iron overload,21 patients(27.3%)had moderate hepatic iron overload,and 15 patients(19.4%)had severe hepatic iron overload.The minimum age of serum ferritin or liver iron overload was 4 years old.The incidence of liver iron overload was significantly higher than serum ferritin overload(72.7%VS 26.9%,P<0.001).2.According to the clinical phenotype dividing the 130 patients.59(45.4%)patients had mild HbH(serum ferritin 465 ng/ml,range:27-3544 ng/ml),41(31.5%)patients had moderate HbH(serum ferritin 573 ng/ml,range:36-6128 ng/ml),30(23.1%)patients had severe HbH(serum ferritin 720 ng/ml,range:132-19704 ng/ml),respectively.There were significant differences in serum ferritin among mild,moderate and severe HbH(P<0.05).The Spearman correlation analysis reveals that the clinical phenotype had positively correlated with serum ferritin degree(R=0.322,P<0.001),and had no correlation with liver iron concentration,cardiac T2*(R=-0.020,P=0.865;R=-0.101,P=0.423).3.Among 130 HbH patients,the number of deletional type is 30(23.1%),nondeletional type is 100(76.9%).The serum ferritin level and liver iron concentration of nondeletional type were significantly higher than that of deletional type(p<0.001,respectively).No statistical difference in cardiac T2*value were seen in both two groups(p>0.05).Clinical features,hematology indices and Iron overload indices from 100 nondeletional type patients were examined.Serum ferritin level increased obviously both in splenectomized group and iron chelation therapy group.Serum ferritin level was positively correlated with age,Hb level,ALT(R=0.383,P<0.001;R=0.294,P=0.003;R=0.514,P<0.001,respectively).Liver iron concentration increased in iron chelation therapy group comparing to splenectomized group(p<0.05).Liver iron concentration was positively correlated with age,ALT level and AST level(R=0.467,P<0.001;R=0.600,P<0.001;R=0.272,P=0.028,respectively).There was no statistically significant differences between cardiac T2*and parameters(p>0.05).4.The serum ferritin level in non-splenomegaly group,splenomegaly group,splenectomized group was 439 ng/mL(range:27-3774 ng/mL),409 ng/mL(range:132-229 ng/mL),1004 ng/mL(range:145.7-19704 ng/mL),respectively.There was statistically significant differences among the three groups(P<0.05).The hemoglobin level in non-splenomegaly group,splenomegaly group,splenectomized group was 93.5 g/L(range:77.3-118.9 g/L),81.6 g/L(range:33.8-117.0 g/L),98.0 g/L(range:40.9-117 g/L),respectively.There was statistically significant differences in the three groups(P<0.05).Conclusions:1.HbH patients have a high burden of liver iron overload and liver iron overload happened earlier.2.Patients with nondeletional type of HbH had more symptoms at a younger age,more severe hemolytic anemia,larger spleens and hepatics,and were more likely to appearance liver iron overload.In clinical,we should be mornitor the serum ferritin and liver iron concentration earlier for patients with HbH3.The clinical phenotype had positively correlated with serum ferritin degree in HbH patients.The higher level of serum ferritin,the severer of clinical phenotype.4.Splenectomy in HbH not only increased hemoglobin level but also increased iron deposition,so we should be pay more attention to mornitor the serum ferritin or liver iron concentration for HbH with splenectomized.THE FOURTH PART OF THESIS COMBINED HISTOLOGICAL AND HEMATOLOGICAL ASSESSMENT OF IRON-INDUCED ORGAN DAMAGE IN A GERBIL MODEL OF IRON OVERLOADBackground:Previous studies with gerbil models have suggested that excessive iron exposure causes cardiomyopathy and hepatic injury,but pathological analysis was not comprehensive,preventing a detailed understanding of how the metal induces this damage.Methods and results:Gerbils received single intraperitoneal injections of iron dextran(200 mg/kg)or saline and were then analyzed comprehensively for hematological and histological signs of organ damage.These tests included hematology parameters and determination of liver iron concentration,malondialdehyde levels and glutathione peroxidase activity;examination of heart and liver tissue stained with hematoxylin and eosin,Prussian-blue and Masson stain;and electron microscopy analysis of heart and liver ultrastructure.Iron-overloaded animals showed significantly different hematology parameters and significantly higher liver iron concentrations than saline-injected animals,as well as significantly higher malondialdehyde levels and significantly lower glutathione peroxidase activity.Histology analyses showed cellular damage,iron deposits,and both myocardial and liver fibrosis,while electron microscopy of heart and liver sections showed abundant iron deposition lysosomes,and disordered and swollen mitochondria.All these pathological changes increased with exposure time.Conclusions:This comprehensive assessment of iron overload in a gerbil model suggests that excessive iron deposition induces extensive cellular damage,particularly fibrosis in heart and liver.This damage may be the direct result of iron-mediated lipid peroxide damage and of iron deposition that cause compression of myocardial and liver cells,as well as vascular occlusion.