International Journal of Medical Laboratory

Thalassemia is the most common genetic disease in the world, especially in Iran [1]. The effects of iron overload can be seen on the scale of damage to the blood and bone [2]. Disease generates from a defect in the formation of the globin beta chain [3]. The main symptom of loss, defect or non-production of hemoglobin units is that it leads to impairment in the process of hematopoiesis which results to severe anemia [4]. Infact, it is the most common genetic defect has been reported in 60 countries including Cyprus, Italy and Iran [5]. Diagnosis is made before the age of two [6]. If blood hemoglobin concentration is more than 10 g/dl, it largely neutralizes the effect of anemia. Maintaining hemoglobin levels within the recommended range ensures survival and growth, but it increases the body iron. Over the years, it leads to a problem with iron overload and hemosiderosis in patients with major beta thalassemia. Ultimately, due to iron overload, heart and endocrine disorders cause death before the age of twenty-two [7]. Osteoporosis is a common complication in patients with major beta thalassemia, [8] as a result of osteoclastic activity increment due to endocrine dysfunction [9]. Some researchers report that Wnt route plays an important role in bone formation. This pathway contains a large protein complex that transmits receptors from the cell surface to the genome [10]. Other studies have shown that inhibitors of this pathway, including sclerostin and dkk1, contribute to inhibition of bone metabolism and even osteoporosis [11, 12]. Sclerostin is a protein encoded by the gene (SOST) and located in human chromosome 17 (17 q11.2). This protein is produced by osteocytes and has anti-anabolic effects on bone structure [13, 14]. Therefore the study was conducted to measure and compare sclerostin, serum iron, ferritin, and bone parameters related to osteoporosis in patients with major thalassemia in comparison to control group.
Materials and Methods
This case control study was performed in Shafa Hospital in Ahvaz, Iran. For each group of patients and control, 40 people were selected. The study was approved by the Ethics Committee of Jundishapur University of Medical Sciences, Ahvaz, Iran. Blood samples were taken from patients who had referred to the laboratory unit, and then transferred to the blood bank of the hospital. After serum separation, measurement was maintained at -70°C.
Selection criteria
Healthy people without thalassemia and osteoporosis, who are not at risk of osteoporosis, and who are more likely to be closer to the patient in terms of age and gender, were chosen as controls. Other pieces of information were extracted from the patients' medical records. Clinical variables including age, sex, presence or absence of osteoporosis, presence or absence of splenomegaly, blood leukocyte count, hemoglobin, and red blood cell count were recorded.
 
Laboratory tests
In this study, additional serum samples taken from patients were used for specific tests, therefore patients' costs will be reduced. Five ml whole blood was collected and the serum was separated and frozen until the test was conducted. The samples were stored at -70°C.
Serum samples were used for sclerostin examination in vitro. Also the latest laboratory results of patients (up to one month before sampling for recent cross-match) such as ferritin, white blood cells, hemoglobin, hematocrit, height, and weight were recorded from the patients' medical records. People with osteoporosis were diagnosed with bone mineral density test. Enzyme-linked immunosorbent assay (ELISA) test was used for measuring the sclerostin.The ELISA kit of 155440 anti-Human SOST abcam from the company of the USA was used for this assay. Osteoporosis parameters that were evaluated in this study were Z and T score. The Z-score is the comparison of the age-matched normal and is generally applied in cases of severe osteoporosis. T score is the related scale when screening for osteoporosis. It is the bone mineral density (BMD) at the area when compared to the young normal reference average. It is a comparison of a patient's BMD to that of a healthy 30 years old [15].
Statistical analysis
SPSS v. 19 was used for analyzing the data. P value less than 0.05 was considered significant.
Results
In our study, the number of case and control groups were equal. The case and control groups comprised of 10 males (25%) and 30 females (75%). Gender distribution was not different significantly in the two groups on the basis of the chi-square test (p= 0.23). The average, minimum and maximum age were 22, 15 and 34 years in the case group respectively. T-test did not show any significant differences between the groups (p= 0.06). The factors associated with osteoporosis in the case group are shown in Table 1. Serum sclerostin level in case and control groups were 100.07 and 143.1 pg/ml, respectively. The minimum, maximum, and mean±SD sclerostin levels in the case group were 13.4, 258.1 and 100.07±66.08, respectively; these values amounted to 27.4, 212.2 and 143.1+54.7 pg/ml in the control group, respectively (Table 2).
According to T-test, there was a significant difference between the patients and the control group in the amount of serum sclerostin (p=0.01). Sclerostin and ferritin showed association in the control group (p=0.04), but that was not seen in the case group (p=0.7). Figure 1 discloses two parameters correlations. Also, the relationship between sclerostin and hematocrit (p=0.05) as well as sclerostin and hemoglobin (p=0.03) was significant. There was no significant relationships between sclerostin and White blood cells count (p=0.5) and bone parameters such as T score of spine (p=0.45), Z score of spine (p=0.3), T score of femur (p=0.7) and Z score of femur (p=0.9). However, evaluating the statistical results revealed a significant difference between ferritin and bone parameters such as T score of spine (p=0.05), Z score of spine (p=0.02), T score of femur (p=0.04) and Z score of femur (p=0.03).

Table 1. Osteoporosis parameters in case group

 

Min	Max	SD	Mean	Parameter
5.0	10.0	1.03	8.05	Heamoglobin(g/dl)
14.2	30.1	3.22	23.4	Heamatocrit (%)
430	10000	2135.86	2770.33	Ferritin (mg/ml)
1200	26000	6297.53	9005.00	White blood cell x103 (µL)
13.4	258.1	66.08	100.07	Sclerostin (pg/ml)

Based on the results, there is no significant relationship between sclerostin, ferritin and osteoporosis parameters. It suggests that the sclerostin protein may play an important role in the pathogenesis of osteoporosis. For better results, it is suggested to examine a larger community.

 
 
References