International Journal of Medicine and
Pharmaceutical Science (IJMPS)
ISSN(P): 2250-0049; ISSN(E): 2321-0095
Vol. 5, Issue 6, Dec 2015, 29-38
© TJPRC Pvt. Ltd.
COMPARISON OF HEAMATOLOGICAL PARAMETERS AND SERUM ENZYMES
IN β-THALASSEMIA MAJOR PATIENTS AND HEALTHY CONTROLS
SARA FADHIL, ANWAR A. ABDULLA & MOHAMMED.A.JEBOR
Department of Biology, College of Sciences, Babylon University, Iraq
ABSTRACT
The aim of the present cross-sectional study was to assess specific hematological parameters and serum
enzymes in a group of people diagnosed with Thalassemia and compared with similar parameters from healthy controls.
Blood samples were collected from randomly selected seventy β- thalassemia patients, representing most regions of
Babylon governorate. The study subjects included 31 males and 39 females with age range of (1-40 years). All patients
were ethnic Arabs, blood transfusion dependent, who were diagnosed and registered at thalassemic center of Babylon
pediatric hospital. Additionally, blood samples were collected from 70 healthy individuals (without hemoglobinopathy)
as a control group that included 42 males and 28 females with age range of (3-40 years). Hematological parameters and
serum enzymes that were assessed included: white blood cell, hemoglobin concentration, platelets, ferritin, urea,
Aspartate Aminotransferase(AST) or Glutamic Oxaloacetic Transaminase (GOT) and Alanine Aminotransferase (ALT)
lower in blood samples of the thalassemic patients than the controls (P <0.05), while WBC, platelets, ferritin, urea, GPT
and GOT were significantly increased in blood samples of thalassemic patients compared to controls (P <0.05). Levels of
serum superoxide dismutase activity (SOD) were significantly increased in β-thalassemia patients compared to controls
(967.43± 115.6 U/ml vs. 170.7± 40.2 U/ml), while catalase (CAT) activity was significantly lower in thalassemic patients
than controls (144.77± 17.3 U/ml vs. 194.95± 47.2) P-value<0-05). High levels of antioxidant enzyme SOD with decrease
Original Article
or Glutamate Pyrovate Transaminase (GPT). The results indicated that values for Hb concentration were significantly
of antioxidant enzyme catalase, were associated with thalasemic patients compared to controls suggesting that
assessment of hematological parameters and serum enzymes are valuable tools to predict thalassemia in Iraqi
population
KEYWORDS: β- Thalassemia, Antioxidants, Serum Enzymes
Received: Nov 10, 2015; Accepted: Nov 19, 2015; Published: Nov 23, 2015; Paper Id.: IJMPSDEC20154
INTRODUCTION
β-thalassemia is an autosomal hematological disorder resulting in a genetically deficient synthesis of the
β-globin chain in hemoglobin [ 1]. The β-thalassemias refer to that group of inherited hemoglobin disorders which
are characterized by a reduced synthesis (β+-thalassemia) or absence (β0-thalassemia) of β -globin chain
production which causes anemia [ 2].It is most prevalent around the Mediterranean Sea, that is, countries like
Greece, Italy, Turkey, North African, Middle East and South East Asian countries. It is mostly caused by point
mutations. Transfused thalassemic patients may develop complications related to iron overload. Complications of
iron overload in children include growth retardation and failure or delay of sexual maturation. Later iron overloadrelated complications include involvement of the heart (dilated myocardio pathy or rarely arrhythmias), liver
(fibrosis and cirrhosis), and endocrine glands (diabetes mellitus, hypogonadism and insufficiency of the
parathyroid, thyroid, pituitary, and, less commonly, adrenal glands [ 3].Other complications are hypersplenism,
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30
Sara Fadhil, Anwar A. Abdulla & Mohammed. A. Jebor
chronic hepatitis(resulting from infection with viruses that cause hepatitis B and/or C), HIV infection, venous thrombosis,
and osteoporosis [ 4].
MATERIALS AND METHODS
Study Subjects
•
Patients Group
The study was conducted between 1/10/2014 – 1/10/2015. Seventy randomly-selected β-Thalassemia major
patients (clinically and laboratory confirmed) were selected from thalassemic center pediatrics hospital in Babylon city,
Iraq, including 31 males and 39 females with age range of (1-40) year. They were asked to fill a questionnaire during their
visiting periods for hematological examination and before blood transfusion when necessary from thalassemic center.
•
Control Group
Seventy of healthy people without hemoglobinopathy disorders from Babylon population were randomly selected
to compare with patients group including 28 females and 42 males with ages ranging from (3-40) years.
Blood Sampling
Three milliliters of blood are collected by vein puncture in EDTA tubes from all patients and control groups and
were transported within 2-24 hours using cooling container and stored in refrigerator (4ºC) for DNA extraction, molecular
analysis and antioxidant analysis.
Hematological and Serum Enzymes Examination
The following hematological parameters were measured in this study:
•
PCV (Packed Cell volume )
•
Platelet count
•
W B C (White Blood Cells ) count
•
ABO blood grouping
•
●Ferritin ● S.GOT
•
● S.Urea ●S.GPT
MEASUREMENT OF ANTIOXIDANTS
•
Catalase
Human Catalase ELIZA kit (Cat No : E-EL_5408 E. Lab Science. Korea) which is based on a sandwich- ELIZA
as the method, was used as per the manufacturer’s instruction. The micro ELIZA plate had been coated with antibody
specific to Catalase. Then biotenylated detection antibody specific for Catalase and Avidin-Horseradish peroxidase (HRP)
conjugate were added to each micro plate well successively and incubated. Free components were washed away. The
substrate solution was added to each well.Only those wells that contain catalase biotinylated detection antibody and
Avidin-HRP conjugate will appear blue in color. The enzyme – substrate reaction was terminated by the addition of a
Impact Factor (JCC): 5.4638
NAAS Rating: 3.54
Comparison of Heamatological Parameters and Serum
Enzymes in β-thalassemia Major Patients and Healthy Controls
31
sulphuric acid solution and the colors turns yellow. The Optical density (OD)was spectrophometrically at a wave length
450nm.The concentration of the samples were calculated by the comparing O.D. of samples to the standard curve.
•
Superoxide Dismutase Measurement
Human SOD ELIZA kit (cat No:E-EL-H1113. E. Lab Science. Korea) is based on a sandwich –ELIZA as the
method. followed The micro ELIZA plate had been pre-coated with an antibody specific to SOD. During the reaction,
Human SOD in the sample or standard competes with fixed amount of SODI on the solid phase supporter for sites on the
biotinylated Detection Ab specific to Human SODI. Excess conjugate and unbound sample or standard are washed from
the plate and Avidin-conjugate to Horseradish peroxidase (HRP) was added to each well. the enzyme – substrate reaction
was terminated by the addition of sulphuric acid solution and the color change and then measured spectrophotometrically
at wavelength 450nm. The concentration of the samples were calculated by the comparing O.D of samples to the standard
curve.
Biostatistical Analysis
The calculation of percentages, standard deviation estimation and t-test statistical analysis were applied for the
analysis of results. Differences were considered significant at the probability of (P value <0.05) [5].
Ethical Consideration
This study was conducted in strict adherence to the ethical guideline for conducting research on human subjects.
RESULTS
Estimation of WBC, Hb, platelets and Ferritin
The results of hematological examination of 70 β-thalassemia major patients are shown in table (1)
Table 1: Hematological Characteristics of the Study Groups
Control
Patients
n=70
n=70
Mean ±SD
Mean ±SD
WBCX 109/L
5.79± 1.5*
8.97± 6.4
Hb (g /dL)
13.19± 0.95*
7.05± 1.43
Platelets109/L
197.5± 36.7*
258.03± 207.02
Ferritin ( ng/ml) 141.89± 83.8* 2854.89± 1923.8
(Significantly different from control value (P < 0.05).
Parameters
p- value
<0.001
<0.001
<0.001
<0.001
Blood Grouping
Blood grouping was determined for patients as demonstrated in figure (1). The results indicated that the most
frequent blood group were blood group A (43.6%), while the lowest frequent was blood group B (15%).
Figure 1: Distribution of Blood Grouping of the Patients
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Sara Fadhil, Anwar A. Abdulla & Mohammed. A. Jebor
Demographic Distribution of the Samples
Gender Distribution
The results indicated that the sample distribution according to gender was(63%) were females and (37%) were
males as demonstrated in figure (2).
Figure 2: Prevalence of β-thalassemia in Male and Female
Regional Distribution
Patients of β- thalassemia have been divided to city and rural living patient (as mean), as show in figure (3).
Figure 3: Geographical Distribution of β-thalassemia
Figure 4: Consanguinity for Patients of β-thalassemia
Serum Enzymes
The examination of urea, GPT, GOT in serum of the patients as compared to the controls is presented in table (2):
Impact Factor (JCC): 5.4638
NAAS Rating: 3.54
Comparison of Heamatological Parameters and Serum
Enzymes in β-thalassemia Major Patients and Healthy Controls
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Table 2: Serum Enzymes and Urea of Patients with
β- thalassemia Compared with Control
Control
Patients
n=70
n=70
Parameters
Mean ±SD
Mean ±SD
Urea
3.3± 0.6*
4.8± 1.77
GPT
9.5± 2.66*
12.7± 13.01
GOT
7.6± 2.25*
12.7± 9.8
Level of significance at p value <0.05.
p- value
<0.001
<0.001
<0.001
Antioxidant Enzyme
Superoxide dismutase activity (SOD) was significantly increased in β-thalassemia patients compared to controls
(967.43± 115.6 U/ml vs. 170.7± 40.2 U/ml), while catalase was significantly less than controls (144.77± 17.3 U/ml vs.
194.95± 47.2),
Table 3: The Levels of SOD and Catalase in β-thalassemia Compared with Control
Control
Patients
n=70
n=70
Mean ±SD
Mean ±SD
Catalase
194.95± 47.2 144.77± 17.3
SOD
170.7± 40.2
967.43± 115.6
Level of significance at P Value <0.05
Parameters
p- value
0.001
0.001
DISCUSSIONS
The Hematological parameters measured in the present study revealed significant differences between patients
and controls except that for Hb.All patients have severe anemia as compared to the controls. The hemoglobin level in
patients is decreased of the level recorded in controls, (7.05± 1.43; 13.19± 0.95) g/dL with significant differences (P ˂0.05)
respectively, so they had needed to blood transfusion which was primary reason for iron overload [ 6]. Increased iron
might be increase the potential of oxidative injury to erythrocytes and cell organelles [ 7]. The significant increase of serum
iron and ferritin in Iraqi patients indicated an existing iron overload. The acute iron overload found in β-thalassemia can
lead to an iron intestinal hyper absorption [ 8].The frequency of transfusion is usually every two to four weeks.Wlile shows
high levels of W.B.C. as compared to the controls. The number of white blood cells may appear raised due to the presence
of a large number of immature (nucleated) red blood cells, which the cell counter may mistakenly identify as white blood
cells[ 9].While the platelets level in patients (258.03± 207.02)109/Lwith significant differences (P ˂ 0.05) compared with
control(197.5± 36.7), due to the iron chelators (deferasirox) had reduced the platelet level in β- thalassemia,so the patients
must stop of taken the iron chelators until the level of platelets became normal [ 10]. The reason for the low level of
platelets in patients with β-thalassemia major is the result of a secondary effect of the disease through splenomegaly, while
for the high level of platelet thalassemia is produced as a result of the removal of the spleen in some patients [ 11].The
most frequent blood group among β-Thalassemia were blood group B[ 9].
Previous studies, concerning the ABO group reported that the most frequent blood group were respectively AB
and B [12]. The β-thalassemia may affect patients of different blood group. Accordingly, there is no association between
blood groups and phenotypes; this may be due to gene polymorphism of ABO system, since it located on chromosome 9
whereas the β-thalassemia gene is located on chromosome 11 [ 13]. The incidence of thalassemia in females more than
males [ 14]. Whereas Al-Assadi [ 15] reported that there are no statistical significant differences at (P > 0.05) between both
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34
Sara Fadhil, Anwar A. Abdulla & Mohammed. A. Jebor
sex in patients and control groups. This is due to a fact that β-thalassemia is an autosomal recessive disease caused by
abnormalities in the β-globin gene located on chromosome 11; so there is no sex linked disease. Since this disease is
unaffected by sex variable, both sex are equally affected with β-thalassemia [16].
This result agreed with the findings of [17 ] who found the rate of incidence of β-thalassemia in rural more than
urban, may be due to a high frequency of consanguineous marriages in rural as compared to the urban. People living in
rural areas be likely to have higher levels of disease and infection than those in main urban. on average, people living in
rural do not have the same chances for good health than those living in major cities. For example, educational and
employment opportunities and income [18].
Bhardwaj et al, [ 19] reported a high degree of consanguinity among couples screened for β-thalassemia carrier
state and consanguineous marriages (first and second-degree)constituted more than 40% of the marriages in the Gaza Strip.
Since consanguineous individuals have a higher probability of carrying the same alleles than less closely or non-related
ones, therefore births from consanguineous marriages are more frequently homozygous for various alleles than those from
non-consanguineous marriages [ 20]. Although consanguineous marriage is the choice of more than 10 % of the world
population, however an overalldecline in its popularity has been reported particularly in developed nations. Some studies
showed that 71.4 % of β-thalassemia major birth sare from consanguineous marriages, and they concluded that
consanguinity increase the size of the β-thalassemia due to the limited health education of the parents [ 21].Figure(4)
shows consanguineous among patients of β-thalassemia, the relative parents was (86%) more than non-relative parents
(14%).
This study is agreed with research that found the genetic diseases like thalassemia and sickle cell anemia wide
spread in relative parents than non-relative parents[ 22].They indicated that marriage among the same family leads to
hereditary disease because of the accumulation of the genes responsible for this disease and becomes dominance in the
family Internal marriage will not change the repetition of genes but some small clan may be loss some alleles when ever
increasing internal marriage [ 23].
The results indicated that all parameters measured (urea, GPT, GOT), which markers for kidney and liver
functions in the present study revealed significant differences between patients and controls. High level of serum urea and
creatinine among patients of β-thalassemia. Severe anemia and chronic hypoxia shortened red cell lifespan and excess iron
causes functional and physiological abnormalities in various organ systems [24], in β-thalassemia patients have a high
prevalence of renal tubular abnormalities [ 25].The blood transfusion every (2-4) weeks to treated severe anemia which
result iron overload in various tissue including the liver, heart and endocrine tissue. The kidneys are another site of iron
accumulation in thalassemia.Unlike in the other organs, it is unclear whether kidney affection results solely from
intravascular hemolysis, chronic transfusion or as a complication of iron chelation therapy [ 26]. Increased levels of GPT
and GOT in β-thalassemia due to iron overload, a very low hemoglobin level with very high (GPT, GOT) level along with
spleenomegaly is suggestive of poor prognosis in these patients [ 9]. The important elevation of ferritin was a major risk
factor for myocardial infarction [ 27] The repeated transfusion of blood cause iron overload in the β-thalassemia without
chelation therapy that lead to many complication such spleenomegaly and influence on liver function that lead to the
increaseof(GPT,GOT,[9].
The increased activity of SOD in β-thalassemia may be involved in scavenging the superoxide radical(O2¯ ),
thereby producing more hydrogen peroxide in the erythrocytes [ 28].In addition, the increase of intracellular antioxidant
Impact Factor (JCC): 5.4638
NAAS Rating: 3.54
Comparison of Heamatological Parameters and Serum
Enzymes in β-thalassemia Major Patients and Healthy Controls
35
enzymes might be hypothesized to be a direct effect of increased intracellular iron on gene expression [ 29]. During the
course of metabolism, superoxide anion was converted to H2O2 by ubiquitous enzyme superoxide dismutase. Normally
H2O2 was converted to innocuous compounds by the action of catalase and peroxidase [ 30]. But if free iron was available,
it reacted with H2O2 to form hydroxyl radicals which were extremely reactive species leading to depolymerisation of
polysaccharide, DNA strand breakage, inactivation of functional proteins etc. [31].
CONCLUSIONS
The present results agree with other results that reported by(Kassab-Chekir et al., [29][;32;33] they observed that
iron, ferritin, SOD and GPX were increased in β-thalassemic patients. Increased red cell SOD values in thalassemic
patients have previously been explained as a reaction to, or compensation for, the increased production of superoxide
radicals[ 34]. While the result of catalase was significantly decreased in β-thalassemia patients compared to controls, this
result was agree with [ 34] showed that SOD level is increased while possible explanation for lower red cell catalase level
found in the more severe genotype of β- thalassemia is that the greater amount of hydrogen peroxide might cause direct
toxic damage to catalase. The antioxidants increased in β-thalassmia to protect the cells from damage of free radical that
result from iron overload [35].
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