Body Iron Status and Its Complications in Patients With Beta Thalassemia Major: A Cross-Sectional Study

Introduction: Thalassemia is a genetic condition and is one of the commonest single-gene hereditary disorders. As it is among a group of hemoglobinopathies, it consists of a decrease in either alpha or beta globin chain synthesis. This eventually leads to the inappropriate ratio of alpha or beta globin chains causing ineffective erythropoiesis. Ineffective erythropoiesis thus causes chronic anemia and haemopoietic expansion to compensate for it. Aims & Objectives: This study aimed to investigate the serum ferritin levels of patients with beta thalassemia major presenting in a tertiary care hospital in Lahore, Pakistan and study the complications that occur with an abnormal iron load. Place and Duration of Study: It was conducted at CMH Hospital, Lahore, and completed over a period of one and a half year, from June 2021 to January 2023. Material & Methods: It was a descriptive cross-sectional study. The sample size of 32 was calculated using Raosoft calculator, with 90% Confidence Interval, 9% margin of error and 10.5% response distribution.Data was collected using an interviewer-administered questionnaire that compromised of 4 parts, demographics and socio-economic background of patient and caretakers; medical history and anthropometric measurement of the patient; details regarding serum ferritin levels and chelation; and lastly, laboratory (for example, TSH, T4, HbA1c and ALT for all patients, as well as FSH and LH for only those patients greater than 10 years of age), ultrasound, and cardiac echocardiography findings.Collected data was entered and analyzed in SPSS version 24. P value of <0.05 was taken as significant. Results: In this study, thirty-two patients with beta thalassemia major were included with mean age of 7.79 ± 4.57 years. Mean serum ferritin was 3410±2629 (cid:541) g/l and lack of compliance to chelation had significant association with serum ferritin values (p=0.05). Serum ALT levels showed that hepatic dysfunction was the most common endocrine complication in 17 (53.1%) patients. There was a significant association between compliance and echocardiography findings (p=0.04). No significant association of compliance was found with ultrasound findings, serum TSH and T4 levels, serum ALT levels and serum FSH and LH levels. Conclusion: This study concluded that high ferritin levels ultimately lead to significant complications.


INTRODUCTION
Thalassemia, one of the most common single gene hereditary disorders, is associated with significant mortality and morbidity worldwide 1 .In Pakistan, the prevalence of beta thalassemia major is estimated to be five to seven percent and approximately 50,000 patients are currently enrolled 1. 1 Department of Pediatrics, CMH Lahore Medical College, Lahore.2. 2 Department of Physiology, CMH Lahore Medical College, Lahore.3. 3  with different thalassemia centers throughout the country 2 .The carrier rate, that is persons with thalassemia minor, varies between five to seven percent; with almost 9.8 million carriers in the overall population 3 .Thalassemia is part of a group of disorders called hemoglobinopathies.It is characterized by a decrease in globin chain synthesis (alpha or beta) resulting in inappropriate ratio of alpha or beta globin chains, ineffective erythropoiesis, chronic anemia, and haemopoietic expansion to compensate for it 1 .In this study, we have focused on beta thalassemia only.The definitive cure is successful bone marrow transplant.If this is not possible, the patient has to remain on lifelong blood transfusions, leading to multiple complications.The complications are many and affect various organs throughout the body.Hemochromatosis (iron overload) due to repeated blood transfusions results in iron deposition in multiple organs especially the heart, pancreas, liver and kidney.Deposited iron contributes to cardiomyopathy, liver dysfunction, hypogonadism, hypothyroidism, hypoparathyroidism, diabetes

INTRODUCTION
Globally, acute generalized peritonitis ranks among the top surgical emergencies 1 .It is more common in Third World nations.The prevalence of perforation is low (0.6% -4.9%) in developed nations but high (33% -63%) in West Africa 2 .554 persons were discovered to have peritonitis in a study that took place over three years in India 3 .Researchers in Pakistan have conducted studies with similar methods, with one study reporting 650 cases in a just 9 months 4 .Most cases of peritonitis are caused by a gastrointestinal perforation or anastomotic leak 5 .In the case of peritonitis, anaerobes and gramnegative organisms are mostly responsible for sepsis and morbidity due to the overactive inflammatory cascade brought on by the release of endotoxins 5 .Clinical evidence is used to identify peritonitis.Diagnosis can be achieved via upright plain x-ray of the abdomen, USG, or CT scan.This is often done through diagnostic laparoscopy nowadays 6 .Resuscitation, diagnosis, prompt exploration, treatment of the underlying cause, and extensive surgical peritoneal lavage have always been the cornerstones of peritonitis therapy regimens (IOPL) 7,8 .Regular IOPL is performed to lessen bacterial contamination and burden.Even though large volumes of normal saline are used in IOPL, the rates of sepsis, wound infection, and mortality remain alarmingly high.Another method mellitus and short stature [4][5][6][7] .These complications affect quality of life, and can lead to mortality, with cardiac complications being the most common cause of death 8 .To prevent iron overload, all blood transfusion dependent thalassemia patients should be on iron chelation therapy.This can be effectively done by administration of iron chelating drugs.Deferoxamine (DFO), which is the first line chelator, is administered subcutaneously over a period of 8 hours, as an infusion for 6 out of 7 days, usually given during night hours 9 .The other drugs are orally administered.Despite its effectiveness in preventing life threatening complications, given the cumbersome administration, compliance can be low.Serum ferritin level measurements are easily available and relatively inexpensive, is used to monitor total body iron levels.Specific investigations to screen for organ dysfunction should also be carried out regularly 10 .Multiple studies have been done in various countries, including Sri Lanka and India to demonstrate a relationship between iron overload and various organopathies encountered in beta thalassemia patients, butdata from Pakistan is scarce.We believe this study was crucial, as it will add to the meager statistics present in Pakistan.Given the scanty data from Pakistan on this topic, this study is very important in helping us understand and manage complications from this disease better.Theprimary objective of this study was to determine the prevalence of organ dysfunction in patients with beta thalassemia and its association with serum ferritin levels.The secondary objective was to assess the compliance to chelation therapy.

MATERIAL AND METHODS
This descriptivecross-sectional study was conducted at the Thalassemia Center, CMH Lahore.The study received approval of the Ethical Review Committee of CMH Lahore Medical College, with the number 764/ERC/CMH/LMC.Sample size of 32 was calculated using the Raosoft calculator, with 90% confidence interval, 9% margin of error and 10.5% response distribution.Inclusion criteria were children with beta thalassemia major (diagnosed by hemoglobin electrophoresis), age up to 18 years, and receiving blood transfusions.Exclusion criteria included patients with thalassemia minor, and patients receiving blood transfusion for any other hematological disorder.Prior to enrollment in the study, informed written consent was obtained.The primary team members carried out interviews.Data was collected using an intervieweradministered questionnaire that compromised of 4 parts.The first part covered demographics and socio-economic information.The second part comprised of medical history, including history ofco-morbidities, Hepatitis B and/or Hepatitis C, any known disabilities and family history of blood disorders.Information was also gathered about age at which blood transfusions were started and frequency of transfusions.In the third part, serum ferritin levels were recorded, and compliance to chelation was assessed.The last section was used to record laboratory, ultrasound abdomen and cardiac echocardiogram (echo) findings.The lab tests and abdominal ultrasounds were done from CMH Diagnostics Centre, while echocardiograms were performed at the Army Cardiac Center, CMH Lahore.For all patients, laboratory investigations included serum TSH as well as free T4 to identify thyroid functioning, serum HbA1C for diabetes mellitus, and serum ALT to determine liver dysfunction.Serum FSH and LH were only obtained in patients older than ten years to assess gonadal development.The data collection tool was designed for the study (appendix) by the primary team, based on available literature.Two pediatricians, independent of the study, prior to starting the study, reviewed it.

Statistical Analysis:
Collected data was entered in SPSS version 24.Quantitative data was presented as mean and standard deviation while qualitative data as frequency and percentages.P value of <0.05 was taken as significant.

RESULTS
The study enrolled thirty-two patients (6 months -18 years of age) with beta thalassemia major.Mean age was 7.79 ± 4.57 years; 15 (46.9%) patients were males.Blood transfusion was required once monthly in 27 (84.4%),and twice monthly for five (15.6%) patients.For all patients, the primary caregiver was the mother, all of whom defined themselves as a homemaker.Most mothers, 13 (40.6%)had no formal education, 10 (31.3 %) were educated up to Matriculation, whereas nine (21.8%) were educated up to Intermediate level and above.In 21 (65.6%)families, the monthly income was less than PKR 50,000.In 30 (93.8%) patients, parents had a consanguineous marriage.Mean serum ferritin was 3410±2629 g/l.Only three (9.37%)children had serum ferritin levels less than 1000 g/l.However, 13 (40.62%)had serum ferritin between 1000 and 2499 g/l and 16 (50%) patients had serum ferritin levels at or above 2500 g/l.(Table - These, two were less than one year of age with ferritin levels less than 1000 g/l, while one of them was more than a year old but had serum ferritin levels less than 1000 g/l; four(30.7%)identified lack of awareness as the reason for poor or no compliance (Fig- 1).Lack of compliance was significantly associated with serum ferritin (p=0.055)(Table -2).In 31 (96.9%)patients, there was no co-morbidity, and no complications of beta thalassemia were reported.Similarly, 28 (87.5%)patients complained of no known disabilities.However, laboratory reports and radiological evidence proved otherwise.Serum ALT levels showed that hepatic dysfunction was the most common biochemical complication in 17 (53.1%)patients.Hypothyroidism was found in three (9.4%)patients and hyperthyroidism in only one (3.1%);five(15.6%) patients had diabetes mellitus.
Only seven (21.9%) participants who were above the age of 10 years were tested for gonadal function with serum FSH and LH levels.Serum FSH and LH was low in three (9.4%), while serum FSH was high in four (12.5%) and LH was high in one (3.1%).Hepatitis B was positive in one (3.1%),Hepatitis C in four (12.5%), while both were positive in one (3.1%)patient.Hepatomegaly and splenomegaly were found in six (18.8%) patients had both on ultrasound, while two (6.3%) had hepatomegaly and four (12.5%) had splenomegaly individually.Isolated valvular dysfunction was found on cardiac echo in 16 (50%) patients, while valvular dysfunction, along with abnormalities of chambers was found in only one (3.1%)patient.There was a significant association between high serum ferritin levels, which was mostly due to lack of compliance, and echo findings (p=0.047)(Table -3).However, there was no significant association of compliance with ultrasound findings; serum TSH and T4 levels; serum ALT levels and serum FSH and LH levels.

DISCUSSION
This study points out the extent of iron overload and its associated complications in patients with beta thalassemia major in an urban setting inPakistan.
As the definitive treatment of bone marrow transplantation is only available to a small proportion of patients, most depend on regular blood transfusions, leading to iron overload.Several known complications of iron overload can be prevented by adequate chelation 11 .The most significant finding in this study was that 29 (90.7%)patients had higher than normal serum ferritin levels.Regular blood transfusions were being carried out to sustain hemoglobin levels of patients with beta thalassemia major.Because of poor compliance to iron chelation therapy, iron overload has been demonstrated in various organ systems.In our study, from the organs studied, hepatic dysfunction was found to be the most common, in 17 (53%).This was followed by cardiac valvular dysfunction in 17 (53.1%),diabetes mellitus in five (15.4%), hypothyroidism in three (9.4%), and hypogonadism in three (9.4%).Assessing iron overload in patients with beta thalassemia major receiving regular blood transfusions is an essential part of their management.Although protocols may differ slightly, it is accepted to begin chelation therapy after a certain number of blood transfusions have been administered (10-12 at our center), or after serum ferritin has surpassed an acceptable normal limit (1000 g/l at our center).Blood transfusions are given with the intention to keep the mean hemoglobin at 12g/dl.For this, pre-transfusion hemoglobin is taken at 9-10.5 g/dl and posttransfusion hemoglobin to be less than 14-15 g/dl 12 .There are numerous methods for assessment of body iron status.Specific tests target the organs; for example, T2 MRI to assess for cardiac status or a biopsy for liver involvement.Despite these often complicated and expensive specific tests, serum ferritin remains the most common entity to assess body iron status due to its low cost and availability 13 .In this study, a dismal two (6.2%) had normal serum ferritin level.Mean serum ferritin levels was 3410 g/l; half of them had values greater than 2500 g/l.Ferritin values are considerably higher those reported in Sri Lankan and Indian studies, where mean serum ferritin level was 2992 g/l and 2767 g/l, respectively 7,14 .Although it is difficult to understand why this difference exists, it could be attributed to higher literacy in both these countries compared with Pakistan, where higher literacy might lead to better understanding of the disease process and chelation 15 .Formal education had not been attained by 13 (40.6%)mothers in this study.When we compare this with a similar study conducted in Karachi, ferritin levels in this study were lower than their reported level of 4236.5±2378.3 ng/ml 16 .Being a single centered study, we cannot completely explain these discrepancies.It could be attributed to availability of chelation agents, which are given free of cost to patients at our center.This combined with awareness regarding the disease and its treatment might have contributed to lower ferritin levels than in other local studies.
There is a paucity of more local studies for us to compare this with.
The most common complication from iron overload reported in our study was hepatic dysfunction (17, 53.1%) determined by abnormal ALT levels;six (25%) of whom had ultrasonographic evidence of hepatomegaly.There is no local data to compare this with.In Sri Lanka and Iran, delayed puberty was found to be most prevalent organ dysfunction 13,17 .There was no statistical association between serum ferritin levels and hepatic dysfunction (or hepatomegaly) in our study.Hepatic dysfunction, although mainly caused by iron overload in our patients, has also shown to be attributed side effect of chelating agents 18 .Hepatitis B and C, which was reported in 18% patients, can also contribute to hepatic dysfunction 19 .This supports the literature than liver involvement in persons with beta thalassemia major could be multifactorial 20 .Cardiac valvular dysfunction was noted in 17 (53.1%) of our study population, on cardiac echo.A statistically significant relationship was found between high serum ferritin and echocardiographic findings of valvular and chamber dysfunction of heart, indicating sensitivity of cardiac tissue to toxic effects of iron overload.However, out of 382 subjects enrolled in a study in Dubai, only seven (1.8%) suffered from iron overload cardiomyopathy, based on T2 MRI 6 .
Given that cardiac complications are the most common cause of mortality in children with beta thalassemia major in Pakistan and worldwide, protocols should focus not only on adequate chelation, but also evaluation of cardiac status in thalassemia patients.Although the gold standard is T2 MRI, electrocardiography and echocardiography can be used in resource-limited settings 8,21 .Besides liver and cardiac complications, this study also looked at the prevalence of diabetes mellitus, hypothyroidism and gonadal dysfunction.Other complications like parathyroid disorders were not tested for at because of cost constraints, which is probably true for most centers seeing patients with beta thalassemia major in Pakistan.Diabetes mellitus, assessed by HbA1C was found in five (15.6%) patients; hypothyroidism in three (9.4%), and hyperthyroidism in only one (3.1%)patient.Unfortunately, no local study was found in the last five years for comparison of our findings.In a Sri Lankan study, none of the 54 patients had diabetes or hypothyroidism 13 .Another parameter assessed in our study was gonadal function by evaluating FSH and LH values.Gonadal dysfunction was found in only nine (21.9%) of children.On the contrary, among 21 patients in Sri Lanka, nine (42.8%) suffered from pubertal delay 13 .Likewise, Saffariet.al.evaluated gonadal development by assessing serum FSH, LH andtestosterone levels, along with carrying out Tanner staging to estimate breast and testicular development.Impaired pubertal development was the commonest endocrine finding in their study 17 .Tanner staging was not carried out, and we had a small proportion of patients around pubertal age; hence, we cannot draw conclusions on the significance of gonadal dysfunctionfound in our results.More studies are required to judge the influence of elevated body iron status on pubertal growth in patients with beta thalassemia major.It is well established that effective chelation has a significant effect in reducing iron overload.Although a serum ferritin of less that 2,500 g/l is usually aimed for, some studies suggest that lower levels of about 1000 g/l might confer further benefit 22 .Despite this, serum ferritin levels often remain high, as in this study and in similar studies in developing countries 5,7,16,17,21 .Not surprisingly, a statistically significant association was found between compliance to chelation and serum ferritin levels in this study.This corresponds to results from a systemic review conducted in 2022 on 37 articles from different parts of world 23 .This emphasizes the need to create awareness among caretakers regarding the importance of adherence to iron chelating therapy.In this study, 13(40.6%)reported poor compliance.Although it was hypothesized that most would report discomfort and difficulty in administration, surprisingly four(30.7%) reported a lack of knowledge about its importance.Sidhuet.al. in India, found only 11% non-adherent to chelation, mainly because of difficulty in administration 24 .There were some limitations of this study.It was a single center study with a small sample size.Given the location in an urban setting in the second largest city of Pakistan, the authors feel that the results of this study are generalizable to children with beta thalassemia major in Pakistan.Due to financial constraints, the authors were unable to consistently use gold standard testing to evaluate iron overload.

CONCLUSION
This single center study adds to the data related to beta thalassemia major patients in an urban center of Pakistan.We concluded that patients of beta thalassemia major suffered from elevated serum ferritin levels, which was in turn found to be related to compliance to chelation.Therefore, we realized that iron overload is a significant problem in children living with beta thalassemia major in our setting.Compliance to chelation needs to be addressed by doctors and health policy makers.Access and affordability of chelating agents, along with education is the order of the day.

Fig- 1 :
Fig-1: Reasons for lack of compliance Department of Community Medicine, CMH Lahore Medical College, Lahore.

Table - 1: Serum Ferritin Values Among Patients With -Thalassemia Major (n = 32) Compliance to Iron Chelation
1) On assessment of compliance to chelation, 19 (59.3%) patients were compliant to therapy, while three (23%) were not taking any chelation.