Hematological profile of children with sickle cell Disease in special reference to body iron stores

Introduction: Sickle cell anemia is a genetic disorder commonly seen in rural population of western and central India. Being an important cause of hemolytic anemia, repeated blood transfusions are frequently required for the sickle cell anemia cases. Few studies have exclusively addressed hematological profile of sickle cell disease patients. This study was planned to study the hematological profile of children with special reference to body iron stores. Methods: This was a prospective observational study carried out in department of pediatrics S.B.K.S MIRC. All the confirmed cases of children diagnosed with sickle cell disease or sickle β thalassemia were subjected to complete hematological analysis in special reference to body iron store by evaluation of serum iron, TIBC, % transferrin saturation and serum ferritin to find out total body iron store. Results: About 110 patients were included in the study. In our study low MCV values were seen in 60.4% of patients, low MCH values in 84.9% of patients while MCHC was normal in 94.3% of patients. Serum iron levels showed normal values in majority of patients (94.3%) while transferrin saturation was low in 62.3% patients thus showing the discrepancy on correlating both values for diagnosis of iron deficiency. Conclusion: Out of various parameters for diagnosis of iron status in SCD-SS patients, low MCV, MCH, Serum iron level and transferrin saturation more consistently associated with iron deficient state. Key wordsSickle cell disease, Serum iron, % transferrin saturation, TIBC, Anemia .......................................................................................................................................


Introduction
Sickle Cell Disease (SCD) is one of the most common childhood-onset, single-gene disorder affecting approximately 80,000 people in India. Worldwide approximately 300,000 infants are born with Sickle Cell Disease. Sickle cell trait is most common in individuals originating from Africa, South America, Central America, the Caribbean islands, the Mediterranean, India, and Saudi Arabia [1].
Sickle cell disease results from an inherited hematological disorder involving defects in normal haemoglobin production. Sickle cell syndromes include sickle cell anaemia (SCD-SS), Sickle cell disease (SCD-SS) is the most common and severe variant. It results when an individual inherits a substitution of valine for the normal glutamic acid in the β-globin chain of haemoglobin from both parents [2]. This substitution alters the haemoglobin molecule so it crystallizes and deforms the red cell into a sickle shape when the haemoglobin loses oxygen. Both parents must be carriers of the defected gene in order for the transmission of the disease to occur. Children who only inherit one sickle cell gene have an SCD-AS pattern, the A representing a normal haemoglobin gene. This is also referred to Pediatric Review: International Journal of Pediatric Research Aailable online at: www.pediatricreview.in 371|P a g e as sickle cell trait. Overt symptoms rarely occur in this variant [3]. The clinical manifestations of sickle cell anemia result from increased blood viscosity and vascular obstruction by deformed sickled red cells. These clinical difficulties are related to the loss of oxygen to necessary tissue areas, in that the flexibility of the red blood cells is lessened causing a decrease in their ability to carry oxygen.
This causes vascular occlusions, hemorrhages, infarctions and ischemic necrosis of tissues and organs throughout the body causing several complications including recurrent vaso-occlusive crises and associated stroke, splenic sequestration crisis, aplastic crisis, infections, bone damage (i.e. hip necrosis), jaundice, leg ulcers, priapism, delayed growth, fatigue and painful episodes [2,4].
With the realization that patients with sickle cell anemia may lead relatively normal lives with Hb levels between 7.0 and 9.0 g/dl, regular blood transfusions are no longer used in the management of these patients. However, blood transfusions may be necessary for complications such as an 'aplastic' or 'sequestration' crisis. Among the hazards of repeated blood transfusion is accumulation of iron as the human body has no effective physiological mechanism for excreting excess iron [5]. Therefore, in conditions where transfusions are frequently indicated, exogenous iron can accumulate, circulate as non -transferrin bound iron, enter tissues, form reactive oxygen species and result in end organ damage.
It has been reported that iron deficiency anemia is uncommon in individuals with sickle cell disease because of availability of an adequate iron source potentially from increased red cell turnover and from blood transfusion [6,7]. It is believed that iron released by hemolysis is available for reutilization and that iron deficiency is uncommon in these conditions. However, contrary to the previous belief few studies reported that iron deficiency was common than expected in untransfused sickle cell anemia cases [8,9]. Iron deficiency anemia often goes unnoticed because the sickle cell disease patients are already anemic. Iron deficiency in sickle cell patients may result in lowering the intracellular haemoglobin concentration and this may ameliorate sickling [5]. The patients with sickle cell disease do not acquire excessive iron burden during the first two decades of life.
The gold standard for assessing liver iron stores in the absence of cirrhosis is the hepatic iron content determined by liver biopsy and quantification with atomic absorption spectrometry. Noninvasive methods include blood tests (Serum Iron, ferritin, Percentage Transferrin saturation and Total Iron binding Capacity) and imaging techniques (MRI based techniques).Sickle cell disorder has remained a neglected field of research in our country and magnitude of the problem has never been appreciated. This study was carried out with the main purpose to assess the hematological profile of children in special reference to body iron status of patients with sickle cell disease.

Setting-Department pediatrics S.B.K.S MIRC rural teaching hospital
Inclusion & exclusion criteria-children between the age of 6 months to 18 years were eligible for the study. All diagnosed and suspected cases of sickle cell disease reporting to the hospital were included in the study. Prior consent was taken from parents/ guardian before enrollment and ascent was sought from adolescents. Children with age less than 6 months, with concomitant chronic infective diseases (like chronic osteomyelitis, HIV etc.), with history of blood transfusion in the preceding 3 months or patients not consenting for participation were excluded from the study.
All the patients with positive solubility test and strongly suspected clinically but sickle negative were subjected to high performance liquid chromatography (HPLC) for confirmation and to differentiate sickle cell disease from other variants such as sickle cell trait, Sickle Beta thalassemia etc. The patients showing the presence of band representing HbS in the Hb electrophoresis were further categorized as Sickle Cell Disease, Sickle cell Disease with thalassemia and sickle cell trait depending upon HbS and HbA2 levels. All the patients having HbS more than 50% were enrolled in the study for further analysis. Patients with sickle cell disease and sickle cell disease with Pediatric Review: International Journal of Pediatric Research Aailable online at: www.pediatricreview.in 372|P a g e thalassemia were further investigated for Serum Iron, Serum ferritin, Total iron Binding capacity (TIBC) and Percentage Transferrin Saturation (% TFR). All Children were investigated for complete blood picture (Hemoglobin, Total leucocyte count, Platelet Count, RBC Indices). All the tests were carried out in institution's laboratory. Institutional ethics committee's permission was taken before conducting the study.
Detailed history general examination as well as systemic examination was carried out and was recorded.
Statistical analysis -All the was collected data were analyzed using SPSS software and described in the form of percentages, mean and Standard deviation Result 200 patients were screened in the study out of which 110 (55%) who tested positive for sickling by the solubility test were subjected to hemoglobin electrophoresis. Out of the total 200 patients, 26 patients tested positive for SCD-SS, 27 for SCd-B thallsemia and 57 for sickle cell trait. Mean age of the enrolled patients were 10.2 years and 73.6% were males.
As patients with sickle cell trait remain asymptomatic most of the time or have minor manifestation only, patients with SCD-SS and SCD--thalassemia only were included in further analysis. Distribution of patients was studied with respect to hematological profile. (Table-1) and no significant difference was observed between the two groups. In both group the MCV and MCH was low suggestive of probable underlying iron deficiency specially in SCD -SS group. Distribution of various hemoglobin variants in our study population is described in Table 2. No significant difference was observed in the levels of HbF and HbS amongst the two groups but the HbA2 level was significantly different with higher levels being observed in Sickle thalassemia patients. On comparing parameters of body iron store in the two groups, serum iron was marginally more in SCD-SS (61.9±31.9) as compared to SCD- thal (58.7±40.1), whereas Serum ferritin was significant higher in SCD-thal (244.5) as compared to SCD-SS (61.9±31.9) Pediatric Review: International Journal of Pediatric Research Aailable online at: www.pediatricreview.in 373|P a g e Serum iron, TIBC were with in normal limits while % Transferin saturation were low. Few records of serum ferritin was too high probably it may be acting as acute inflammatory marker in patient admitted due to various reasons.

Discussion
Sickle cell disease is a chronic hemolytic disease that results from a single base pair change, thymine for adenine at the 6th codon of -globin chain leading to polymerization of deoxygenated hemoglobin S and distorting the shape of the cell.
A total of 53 patients, majority of whom were males (73.6%) were included in the study. Similar findings were seen by Khan et.al [10]. This could be explained by increased chances of severe sickness and crisis in males or gender bias in tribal community, resulting in health care seeking behavior favoring males.
Most of the patients were in the age group of 7-12 years as also seen in study by Sahu et al [11]. However Kar et.al. found minimum age of presentation to be 6 months. Probable reason for patients reporting at a later stage in our study might be initial treatment at primary level before reporting to our tertiary care hospital [12].  [14]. However, in a study done by Kaur et al, the mean HbF varied between 9.7-13.5%. the distribution of values could be attributed to inclusion of adult patients in their study [15].
All the patients were found to be anemic. The mean hemoglobin concentration was 8.2 gm% in patients with SCD-SS and 8.5gm% in patients with SCD-thal. The reticulocyte count was found to be high in majority of patients (81.1%) suggesting chronic response to hemolysis. Similar findings were seen in study done by Khan et al [10] and Juwah et al [16].  [14] concluded that serum ferritin was more sensitive indicator than serum iron, TIBC, and transferrin saturation.
Thus, based on present and previous studies available, out of various parameters for diagnosis of iron status in SCD-SS patients, low MCV and MCH are more consistently associated with iron deficient state. Such patients need to be further evaluated on long term basis after giving trial of iron therapy. The limitation of the study was the small study group which was not enough regarding the actual status of body iron among patients with sickle cell disease. The confounding effect of underlying illness of serum ferritin used to assess iron stores also added to problems.
We propose further studies with larger sample population in this area. More specific tests and serial estimations of iron status are required for better definition of iron deficiency status in sickle cell disease. Response to therapeutic iron trial can be used as a diagnostic criterion for iron deficiency in sickle cell disease.