Totally 50.9% (114) of healthy people and 50.9% patients were females. Also, the mean age of subjects between two groups was no different (p= 0.633, Table 1). Among the thalassemia group, 56.5% (65) of non-splenectomized subjects and 45.0% splenectomized subjects were female (p=0.083). The splenectomized subjects (6.85 ± 1.69 years) were younger than non-splenectomized subjects (7.37 ± 1.71 p= 0.023).

Serum ferritin level in healthy subjects was much lower than patients (p<0.001, Table 1), but there was no significant difference between the two patient groups, splenectomized (832.11 ± 568.08) and non-splenectomized (981.37 ± 592.99, p= 0.056). Serum anti-diphtheria antibody level in the healthy group was higher than in patients group (p< 0.001, Table 1).

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Although, serum antibody level against diphtheria was higher in splenectomized patients (1.60 ± 1.76) than in non-splenectomized ones (1.43 ± 1.43), there were no significant difference (p=0.448).

All participants had protective serum anti diphtheria antibody level (partial or completely protected). One hundred and ninety-seven (87.9%) of healthy individuals were completely protected against diphtheria which was significantly higher than patients (71.0%, p< 0.001) (Table 2).

Although, the percent of complete anti-diphtheria protection was higher (75.2%) in splenectomized patients than in non-splenectomized patients (73.9%), there was no significant difference (p= 0.821).

Table 3 shows the relation between diphtheria antibody level with age and serum ferritin by backward linear regression analysis. In this model, age did not affect the relationship.

This analysis showed that only 24.1% anti-diphtheria antibody (IgG) alterations was dependent to serum ferritin level in the patients group (p<0.001), while, in healthy subjects only 1.8% of anti-diphtheria antibody was dependent to serum ferritin (p=0.045). The level of anti-diphtheria antibody decreased with intensification of ferritin level. Thus, when serum ferritin increased 1 ng/ml among patients, serum diphtheria antibody decreased 0.001 IU/ml. This model is better predicted in non- splenectomized patients; with about 31% of the serum anti-diphtheria antibody which is predictable by knowing the serum ferritin level (p<0.001).

DISCUSSION

Although survival of subjects with beta thalassemia major has improved, both treatment modalities and the underlying disease may effect in secondary immunodeficiency. Thus these patients are at risk of attaining a variation of infectious diseases. We therefore assessed the serologic immunity against diphtheria in patients with beta thalassemia major and compared to healthy subjects.

Our study found that the mean of serum diphtheria antibody was lower in patients with beta thalassemia major than age and sex-adjusted healthy subjects. But for ferritin level it was the reverse, in other words, the level of serum ferritin was much higher in healthy subjects than in patients. Also, less percent of patients had complete antibody protection against diphtheria as compared to healthy subjects.

From the 1980s serological research showed that a high percentage of subjects become susceptible to diphtheria with advancing age. This may be due to a decrease in the level of antibody in individuals over time. Chatchatee et al in the Thai population demonstrated that subjects aged between 5 and 9 years had the highest titer of tetanus antibody, and subjects above 60 years of age had the lowest titer (24). In the present study to eliminate the effect of time and sex on the antibody levels; we used sex and age-adjusted healthy subjects as controls for patients.
We found no similar research that compared diphtheria antibody among patients with beta thalassemia major and healthy subjects.

Our study showed that the diphtheria antibody level and the percent of complete protection antibody were lower in patient groups than controls. Jahromi and Rahmanian (25) reported that mean anti-tetanus antibody titers (1.53 ± 1.71 vs 2.02 ± 2.05) and the complete protective level of anti-tetanus antibody (71% vs 87.9%) were lower in patients with beta thalassemia major in comparison to healthy persons. Also Modarresi et al reported that the patients on dialysis had less protective levels of anti-diphtheria than normal populations (19).

In the present study seventy-one percent of beta thalassemia patients were completely protected against diphtheria. Our finding is in contrast with other published results. Kown et al (17) reported that 31.5% of 146 Korean children with hematologic malignancies aged 1-17 years were completely protected against diphtheria. Also, van der Hardt et al (18), Ek et al (20) and Small et al (26) found that less than 50% patients against diphtheria, 17.0% of ALL patients against diphtheria and less than 70% of peripheral blood stem cell transplantation recipients against pertussis had complete immunity, respectively. Similarly, complete protection anti-diphtheria antibody was found in our study to be more than anti tetanus titers that were reported by Aminzade (27), Modarresi et al (19) and Kruger et al (28) in dialysis patients; but less than that Zengin and Sarper (83.3-100%) in patients with acute lymphoblastic leukemia (16).

One of the reasons for this difference in patients with beta thalassemia major may be related to high level of serum iron. Iron overload, a primary complication of both thalassemia itself and transfusion therapy, is thought to be the main causing mechanism of immune incompetence in beta thalassemia major (3). Patients with thalassemia major who had serum ferritin level more than 3000 ng/ml had lower C4 and CH50 levels (10). Recent studies on immune competence in beta-thalassemia have revealed numerous quantitative and functional defects, involving T and B lymphocytes and immunoglobulin production (3). Also, Alavi et al (29) indicated that chemotherapy has independent adverse effects on vaccine-induced antibody protection against diphtheria. In the present study, serum ferritin levels were found to be much higher in patients than in healthy subjects.

In our study, only 25.4% of the thalassemia patients had a partial protective level of IgG against diphtheria and they may susceptible to infection. Adversely, in one study conducted in beta thalassemic patients (aged 5-17 years) who were submitted for bone marrow transplantation, a high percentage (83%) of subjects had anti-diphtheria antibody levels below the protective levels (30). Zengin and Sarper (16) in their study showed that 11.1% of subjects with acute lymphoblastic leukemia had protective level for diphtheria after chemotherapy. Also, Alavi et al (29) reported that chemotherapy in hematologic malignancies caused failure to achieve protective levels of antibodies against diphtheria. In another study, Aminzadeh et al found a non-protective level of IgG against tetanus in most of the hemodialysis patients (27). This alteration seems to be due to difference in mean age of study participants.

CONCLUSION

Our study indicated that increased serum iron levels in beta thalassemia patients decreased the level of antibody against diphtheria. Therefore, lowering the serum levels of iron may prevent further reduction of antibody levels as compared to healthy people over time. It is suggested that further studies are done.

Acknowledgement
We thank patients and personnel of thalassemia ward of hospitals in Jahrom and Babdar Abbas, Iran. Also, the present study supported by Deputy of research, Jahrom University of Medical Sciences.