Serum Hepcidin and Growth Differentiation Factor 15 In Patients with β-thalassemia and Its Relation to Blood Transfusion

INTRODUCTION

Thalassemia occurs due to defective hemoglobin protein spatial shapes due to imbalanced protein chains representation, specifically β-globin deficiency, due to genetic defect. ¹  β-thalassemia become more sever with more decreased formation or absence of β- chain.  ²

Regarding geographical distribution of β-thalassemia, it prevails more in countries on the Mediterranean, South east of Asia and in the east of Europe. In Egypt, it is the most common cause of chronic blood loss: One thousand cases are recorded annually for every 1.5 million live births the disease prevalence is equal to1000 cases per 1.5 million live births.³

The clinical manifestations of beta-thalassemia varies greatly from mild form of  chronic  anemia  to  severe recurrent anemia and infections and this is influenced by many different factors like gene level and function, levels of different types of globin chains, presence of modifiers and environmental factors. ⁴

In our study, we classified thalassemic cases into two groups according to frequency of blood transfusion. 1-TDT (transfusion dependent thalassemia), those cases require blood infusion as the cornerstone of treatment because they suffer severe red blood cell hemolysis like cases with homozygous beta globin chain loss and 2-NTDT (non-transfusion dependent thalassemia). ⁵

Increased serum Iron is the main cause of the manifestations of thalassemia even in patients with occasional blood transfusion. Iron absorption, transport and storage is regulated by the hormone hepcidin. Hepcidin is hepatic peptide. ⁶

Hepcidin is inhibited by erythropoiesis by a negative feedback mechanism. And this explains Hepcidin deficiency in cases maintained on blood transfusion and receives no oral iron because the anemic state and increased bone morrow red blood synthesis will inhibit Hepcidin production and consequently hyper oral iron absorption. ⁷

However, the exact pathophysiology of Hepcidin deficiency in thalassemia is still unknown. The theory of messenger molecule secreted from bone marrow to suppress Hepcidin remains the best theory. GDF-15, is increased in thalassemia and has inhibitory effect on hepcidin secretion in vitro.⁸

Previous studies showed a relation between pre- and post-transfusion hepcidin levels and anemia, RBCs synthesis, serum iron level and inflammation. In other words, blood transfusion in thalassemia cases lead to higher Hepcidin level. Transfusion mediates erythropoietic hyperplasia, which in turn raises Hepcidin production. ⁹

PATIENTS AND METHODS

The study was designed to be of a non-randomized observational study. Where 50 adult individuals who came to the hematology section in Maadi military hospital in the period from the 1st of June 2019 to the 1st of June 2020.

They have been classified into: Group (1):  includes 30 adult patients with TDT and divided into two subgroups: Group 1A: included 30 adult patients with TDT immediately prior to transfusion& Group 1B: included the same 30 adult patients with TDT eight days following transfusion. Group (2): includes 20 non β-thalassemia patients that will be selected randomly from presumably healthy population, sex and age matched. All subjects were informed and gave written consents.

Inclusion criteria: adult patients of both sexes with transfusion-dependent β-thalassemia. Exclusion criteria patients suffering from: Other types of anemia other than beta-thalassemia.

Complete history was taken and clinical assessment was done to all participants. Laboratory investigations including: (serum ALT – serum AST), (serum Urea – serum Creatinine), CBC, Hemoglobin electrophoresis, Reticulocyte count, Iron profile (Iron – Ferritin – TIBC) and C-reactive protein. And special tests: Serum Hepcidin and Serum GDF-15.

From each control and patients, 10 ml of venous blood was withdrawn, 4 ml blood in EDTA vacutainer tube for CBC, reticulocyte count and hemoglobin electrophoresis. 6 ml blood in plain vacutainer tube was left for clotting, then centrifuged, separated serum divided in 2 aliquots, 3 ml for routine chemical investigations and Iron profile (Iron – Ferritin – TIBC) and C-reactive protein and 3 ml frozen at -80C and used for hepcidin and GDF-15 assay using (ELISA) kits.

Statistics:

Data were examined and interpreted using (SPSS) software. Many tests were done: Independent-samples t-test of significance, Chi-square test and Pearson's correlation coefficient (r) test. Quantitative data were expressed as mean ± standard deviation (SD). Qualitative data were expressed as number and percentage.

Probability (P-value): P-value 0.05 was considered insignificant.

RESULTS

The characteristics for the studied group cases (19 males and 31 females) are shown in table (1). This table shows: No statistically significant difference (p-value > 0.05) between studied groups (group I and group II) as regard age & sex; Statistically significant difference (p-value < 0.05) between studied groups (group I and group II) as regard height; and Highly statistically significant difference (p-value < 0.001) between studied groups (group I and group II) as regard weight. As regard days since last transfusion, the mean in studied patients’ group was 20.73 ± 1.2 days with minimum days of 18 days and maximum days of 24 days. As regard mean age of transfused units (days), the mean in studied patients’ group was 11.8 ± 2.2 days with minimum days of 10 days and maximum days of 16 days.

As regard S. ferritin, there was highly significant increase in S. Ferritin in group (I) patients with a mean of (1613.8 ± 994.9) in comparison to control group with a mean of (43.4 ± 16.9). No statistically significant difference (p-value > 0.05) between studied groups (group I and group II) as regard Urea, Creat, and CRP.

As regard serum hepcidin, there was no significant difference in S. hepcidin level in group (I) patients with a mean of (25.2 ± 14.7) in comparison to control group with a mean of (20.8 ± 2.1).

As regard GDF-15, there was highly significant increase in S. GDF-15 in group (I) patients with a mean of (6945.7 ± 1035) in comparison to control group with a mean of (398.2 ± 180.8).

Table 1: Comparison between studied groups as regard demographic data. (T: independent sample T test; HS: p-value < 0.001 is considered highly significant; NS: p-value > 0.05 is considered non-significant).

Table 2: Comparison between studied groups (I & II) as regard. (T: independent sample T test; HS: p-value < 0.001 is considered highly significant; NS: p-value > 0.05 is considered non-significant).

To evaluate the direct effect of transfusion on hepcidin level, iron indices, laboratory investigations were determined in group (1A) patients (pre-transfusion) and compared with those of group (1B) patients (post-transfusion) as shown in table (3).

Serum hepcidin levels were significantly increased from (25.2 ± 14.7) to (54.6 ± 31.03) with P-value < 0.001 HS. Serum GDF-15 were decreased from (6945.7 ± 1035) to (4417.5 ± 1363) with significant improvement of serum ferritin from (1613.8 ± 994.9) to (2463.6 ± 1919). (Figure 1, 2).

Also, serum iron was significantly increased from (236.2 ± 49.3) to (264.1 ± 54). There was significant improvement of Hb concentration from (6.4 ± 0.5) to (7.8 ± 0.6).

The study showed that there were statistically significant negative correlations (p-value > 0.05) between hepcidin and weight, height, reticulocyte count, TIBC, and GDF-15 in thalassemia patients. Statistically significant positive correlations (p-value < 0.05) between hepcidin and Hb, Iron, and Ferritin in thalassemia patients

Table 3: Comparison between studied groups (I & II) as regard. (T: independent sample T test;S: p-value < 0.05 is considered significant; HS: p-value < 0.001 is considered highly significant; NS: p-value > 0.05 is considered non-significant).

Fig 1:. Comparison between studied groups as regard GDF-15 and Hepcidin.

Table 4: Correlation study between Hepcidin and other studied parameters in studied groups. ((r): Pearson correlation coefficient.;S: p-value < 0.05 is considered significant; HS: p-value < 0.001 is considered highly significant; NS: p-value > 0.05 is considered non-significant).