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 Table of Contents  
ORIGINAL ARTICLE
Year : 2017  |  Volume : 42  |  Issue : 2  |  Page : 52-57

Serum visfatin in sickle cell disease: association with frequency of vaso-occlusive crises


1 Hematology Unit, Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, Egypt
2 Department of Pediatrics, Faculty of Medicine, Ain Shams University, Cairo, Egypt

Date of Submission13-Apr-2017
Date of Acceptance27-Apr-2017
Date of Web Publication6-Oct-2017

Correspondence Address:
Deena M.M. Habashy
Hematology Unit, Department of Clinical Pathology, Faculty of Medicine, Ain Shams University, Cairo, 11759
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ejh.ejh_21_17

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  Abstract 

Background Hypercoagulability in sickle cell disease (SCD) is multifactorial, involving abnormalities in platelet function, thrombin generation, fibrinolysis, and other multiple mechanisms. Visfatin, an adipocytokine with proinflammatory potential, can have a negative impact on the vascular endothelium in SCD.
Objective We aimed to evaluate the association between serum visfatin level in SCD patients and the frequency of vaso-occlusive crises (VOC)/year in those patients.
Patients and methods Sixteen sickle cell anemia (SCA) and 14 sickle β-plus thalassemia pediatric patients were studied in steady state. Twenty age-matched and sex-matched healthy individuals, who served as the control group, were evaluated with respect to serum visfatin levels by means of enzyme-linked immunosorbent assay.
Results Hemoglobin (Hb) level was higher in the control group than in the SCA group (P<0.001), whereas total leukocyte count and serum visfatin were higher in the SCA group than in the control group (P=0.02 and <0.001, respectively). Hb level and platelet count were higher in the control group than in the sickle β thalassemia group (P<0.001 and 0.04, respectively). Serum visfatin was higher in the sickle β thalassemia group than in the control group (P<0.001). HbS%, serum visfatin, and frequency of VOC/year were higher in the SCA group than in the sickle β thalassemia group (P=0.002, <0.001, and 0.002, respectively). Serum visfatin was positively correlated with total leukocyte count, serum ferritin level, and frequency of VOC/year (P=0.005, 0.01 and 0.03, respectively) in the SCA group.
Conclusion Serum visfatin is increased in SCD patients compared with that in healthy children and is associated with the frequency of VOC; it can be used as a predictive index for VOC occurrence and follow-up in those patients.

Keywords: sickle cell disease, vaso-occlusive crises, visfatin


How to cite this article:
Habashy DM, Shams MA. Serum visfatin in sickle cell disease: association with frequency of vaso-occlusive crises. Egypt J Haematol 2017;42:52-7

How to cite this URL:
Habashy DM, Shams MA. Serum visfatin in sickle cell disease: association with frequency of vaso-occlusive crises. Egypt J Haematol [serial online] 2017 [cited 2017 Oct 20];42:52-7. Available from: http://www.ehj.eg.net/text.asp?2017/42/2/52/216115


  Introduction Top


Sickle cell anemia (SCA) is a known hematological disorder that arises from a single point mutation in codon 6 of the β-globin gene resulting in glutamic acid to valine substitution [1]. The polymerization of deoxygenated hemoglobin S (HbS) together with the formation of irreversibly sickled erythrocytes ending with vaso-occlusion underlies the pathophysiology of sickle cell disease (SCD) [2] and leads to the two hallmarks of the disease: recurrent episodes of acute pain and chronic organ damage [3]. The severe pain is ischemic in origin and is the most common reason for patients seeking medical advice. Hemolysis also plays a crucial role in the pathophysiology, leading significantly to anemia, vasculopathy, nitric oxide deficiency, and inflammation [4].

Sickle β thalassemia, a type of SCD, is an inherited autosomal recessive condition that affects the Hb concentration. Patients have a different mutation in each copy of their HbB gene: one that causes red blood cells (RBCs) to form a crescent shape and a second that is associated with β-thalassemia. Depending on the β-thalassemia mutation, people may have no normal Hb (called sickle β-zero thalassemia) or a reduced amount of normal Hb (called sickle β plus-thalassemia). The presence of sickle-shaped RBCs, which often break down prematurely and get stuck in blood vessels, together with a reduction of mature RBCs, leads to the presentation of sickle β-thalassemia. Features, including anemia, repeated infections, and frequent episodes of pain, vary in severity according to the amount of normal Hb produced [5].

Adipose tissue is not only a storage and release depot for lipids but also an active endocrine organ that produces various cytokines called adipocytokines, including tumor necrosis factor-α, interleukin-6, leptin, adiponectin, resistin, visfatin, and ometinin, which have multiple biological activities [6]. Visfatin can exert various deleterious effects on vascular cells, including inflammation and proliferation [7].

We aimed in this study to compare the expression of visfatin in SCD patients that in healthy children and evaluate the association between serum visfatin level in SCD patients and the frequency of vaso-occlusive crises (VOC)/year encountered in those patients for possible usage of that marker as a predictive index for VOC occurrence and follow-up.


  Patients and methods Top


Patients

This study was conducted on 30 SCD patients in steady state (16 SCA and 14 sickle β-plus thalassemia) who were attending the Hematology Clinic of Ain Shams University Pediatric Hospital during the period October 2016 to February 2017. Patients were diagnosed as per the results of Hb electrophoresis. Steady state was defined as a crisis-free period for at least 4 weeks after the previous clinical event. SCA patients comprised 11 boys and five girls, with a male-to-female ratio of 2.2 : 1. Their ages ranged from 4 to 18 years, with a median of 10 years. Sickle β thalassemia patients comprised seven boys and seven girls, with a male-to-female ratio of 1 : 1. Their ages ranged from 4 to 17 years, with a median of 10.5 years. Twenty age-matched and sex-matched healthy volunteers served as the control group. They comprised eight boys and 12 girls, with a male-to-female ratio of 1.5 : 1. Their ages ranged from 4 to 15 years, with a median of 8.5 years.

Written informed consent was obtained from parents or guardians. The procedures applied in this study were approved by the Ethical Committee of Human Experimentation of Ain Shams University and are in accordance with the Helsinki Declaration of 1975, as revised in 1983.

Exclusion criteria

Patients were excluded from the study if they had any of the following:
  1. Acute illness or fever.
  2. Malignancy.
  3. Hepatic or renal dysfunction.
  4. Autoimmune disease or cardiopulmonary disease.
  5. Surgery or trauma within a month before the study.
  6. C-reactive protein (CRP) titre greater than or equal to 6.


Methods

Sampling

Blood samples were collected between 8 and 10 a.m. from the antecubital vein. Blood samples of 1.8 ml were anticoagulated with sodium citrate 3.2 mg/ml at a ratio of 9 : 1 (blood : anticoagulant) and were immediately centrifuged at 2500g for 20 min at 4°C. Plasma was frozen in small portions and stored at −20°C until the assay was performed. Two milliliters of venous blood were collected into a sterile dry vacutainer. Fresh serum was obtained by centrifugation of clotted blood for CRP detection.

Procedure

C-reactive protein estimation

CRP titre was calculated using the CRP Latex (Diagnostics Automation/Cortez Diagnostics INC., USA) Test kit, which depends on the occurrence of agglutination between latex particles coated with anti-human CRP antibodies and CRP antigen in tested serum. Presence of agglutination indicated a CRP level greater than or equal to 6 mg/l in the tested sample, which is considered the lowest concentration of clinical significance.

Serum visfatin measurement

Serum visfatin level was detected using the NAmPRTase ELISA kit (Wuhan EIAab Sicence Co., Ltd, Wuhan, China). Briefly, 100 µl of plasma or standard was introduced into a microplate well coated with a biotin-conjugated antibody specific for serum visfatin antigen; thereafter, following a washing step, 200 μl of avidin conjugated to horseradish peroxidase was added to each microplate well and incubated. Following a new washing step, 200 ul of the peroxidase substrate tetramethylbenzidine in the presence of hydrogen peroxide was introduced and a blue color developed. The color turned yellow when the reaction was stopped with 50 µl of 0.45 mol/l sulfuric acid. Absorbance was read at 450 nm. The amount of color developed was directly proportional to the concentration of serum visfatin in the tested sample. The mean absorbance value was calculated for each set of reference standards, controls, and samples. A standard curve was constructed and the concentration of serum visfatin was determined from the curve.

Statistical analysis

IBM SPSS statistics (V. 24.0; IBM Corp., New York, NY, USA) was used for data analysis. Data were expressed as median and percentiles for quantitative nonparametric measures in addition to both number and percentage for categorical data. The Wilcoxon rank sum test (Z) was used to compare nonparametric data between two independent groups. The χ2-test was carried out to compare categorical data between two independent groups. Spearman’s ranked correlation test (r) was used to study the possible association between two variables in each group with regard to nonparametric data. The receiver operating characteristic curve was constructed to obtain the most sensitive and specific cutoff for serum visfatin level. To evaluate the most discriminating cutoff between the compared groups, the area under the curve was calculated. P value at 0.05 was considered significant, whereas 0.01 and 0.001 were considered highly significant.


  Results Top


Demographic, clinical, and laboratory data of the studied groups are shown in [Table 1]. A cutoff value of 9.5 was calculated for serum visfatin level by means of the receiver operating characteristic curve to discriminate the SCA group from the sickle β thalassemia group and controls with a sensitivity of 97.1%, specificity of 93.8%, and efficacy of 96%. A cutoff value of 5.3 was calculated for serum visfatin level to discriminate the sickle β thalassemia group from controls with a sensitivity of 100%, specificity of 78.6%, and efficacy of 91.2% ([Figure 1]). Median Hb level was higher in the control group compared with that in the SCA group (P<0.001), whereas median total leukocyte count (TLC) and serum visfatin level were higher in the SCA group compared with that in the control group (P=0.02 and <0.001, respectively) ([Table 2]). Medians Hb level and platelet (PLT) count were higher in the control group compared with that in the sickle β thalassemia group (P<0.001 and 0.04, respectively). Median serum visfatin level was higher in the sickle β thalassemia group than in the control group (P<0.001) ([Table 2]). Median HbS%, serum visfatin level, and frequency of sickling crises/year were higher in the SCA group compared with that in the sickle β thalassemia group (P=0.002, <0.001, and 0.002, respectively) ([Table 3]). Serum visfatin level was positively correlated with TLC, serum ferritin level (P=0.005 and 0.01, respectively) ([Table 4]), and frequency of sickling crises/year in the SCA group (P=0.03) ([Figure 2]), whereas there was no correlation between serum visfatin level and other studied parameters in the control group or the sickle β thalassemia group (P>0.05) ([Table 4]).
Table 1 Demographic, clinical, and laboratory data of the studied groups

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Figure 1 ROC curve analysis showing the diagnostic performance of visfatin for discriminating all studied groups from each other.

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Table 2 Comparison between the control group and the two patient groups regarding all studied parameters

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Table 3 Comparison between the sickle cell anemia group and the sickle β thalassemia group regarding all studied parameters

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Table 4 Correlation between serum visfatin level and all studied parameters in the control and patient groups

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Figure 2 Linear regression analysis showing positive correlation between serum visfatin and frequency of sickling crises/year in the sickle cell anemia group.

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  Discussion Top


Visfatin is a proinflammatory adipocytokine that is predominantly expressed in visceral adipose tissue [8]. Several experimental studies have indicated its role in inflammatory processes and chronic inflammatory diseases [9],[10]. The potential properties of visfatin make it as a target for novel therapeutic strategies in different inflammatory and metabolic diseases [11].

Vaso-occlusion is the most common outcome and the hallmark of SCD. It is responsible for many clinical complications of SCD, including osteomyelitis, osteonecrosis, stroke, splenic infarct, renal insufficiency, and acute chest syndrome [12].

Endothelial damage plays a crucial role in VOC occurrence in SCD. There is strong evidence that visfatin can directly promote endothelial dysfunction by directly inducing inflammation through the activation of the extracellular signal-regulated kinase 1/2–nuclear factor-κB–inducible nitric oxide synthase axis [13] and stimulation of vascular smooth muscle cell proliferation [14]. In human endothelial cells, visfatin promotes nuclear factor-κB activation, leading to the expression of vascular adhesion molecules, matrix metalloproteinase activation, and the release of cytokines and chemokines, including interleukin-6 or monocyte chemotactic protein-1 [15],[16],[17].

In this study, we measured the level of serum visfatin in SCD in order to prove an association between its expression and the frequency of occurrence of VOC in those patients.

We compared SCD patients (SCA and sickle β thalassemia groups) with healthy children with respect to demographic and laboratory data. The median Hb level was higher in the control group compared with that in the SCA group (P<0.001), whereas the median TLC was higher in the SCA group compared with that in the control group (P=0.02). Median Hb level and PLT count were higher in the control group compared with that in the sickle β thalassemia group (P<0.001 and 0.04, respectively). It was previously stated that the average Hb level is 8 g/dl in SCA [18] with elevated TLC [19] and an increased PLT count, which decreases during VOC [20].

Median HbS% and frequency of sickling crises/year were higher in the SCA group compared with that in the sickle β thalassemia group (P=0.002) ([Table 3]). It has been proven earlier that SCA is due to homozygosity for the abnormal Hb, HbS. The presence of HbS associated with other hereditary variants of Hb, such as Hb C, D and S/Sβ-zero and β-plus thalassemia, characterizes SCD [21],[22]. It was shown before that the frequency, severity, location, and duration of VOC can vary considerably, even within a specific disease subtype. Patients with homozygous sickle cell and sickle β-zero thalassemia have a higher frequency of VOC than do patients with the sickle β-plus thalassemia genotype [23].

Serum visfatin level was higher in the SCA group and in the sickle β thalassemia group compared with that in the control group (P<0.001) ([Table 2]); it was higher in the SCA group compared with that in the sickle β thalassemia group (P<0.001) ([Table 3]). Serum visfatin level was positively correlated with TLC, serum ferritin level, and frequency of sickling crises/year in the SCA group (P=0.005, 0.01, and 0.03, respectively) ([Table 4]). Serum visfatin was not studied before in SCD, but other adipocytokines were measured. In a study by Makis et al. [24], leptin level was measured and was lower in sickle β thalassemia patients when compared with that in controls, whereas adiponectin was significantly elevated. No correlations were found between adipocytokines and age, sex, Hb level, ferritin, and clinical events. Concerning adiponectin, a positive correlation was detected with TLC.

Other investigators measured serum visfatin in thalassemic patients. Serum visfatin level was proved to be higher in β-thalassemia major patients compared with that in controls, with no association with serum ferritin level [25]. Another study compared different subtypes of β-thalassemia and controls with respect to serum visfatin level, together with serum resistin and adiponectin. They found that resistin and visfatin concentrations were significantly higher in β-thalassemia minor patients than in controls. Adiponectin, resistin, and visfatin concentrations were significantly higher in both β-thalassemia intermedia and major patients than in controls [26].

In a study measuring leptin and adiponectin in β-thalassemia major, leptin was significantly lower in patients compared with that in controls, whereas adiponectin was elevated. For both adipocytokines, no correlations were found with age, gender, or Hb level. Only leptin was negatively correlated with ferritin [27].


  Conclusion Top


Serum visfatin is increased in SCD patients compared with that in healthy children and is positively correlated with the frequency of VOC; it can be used as a predictive index for VOC crisis occurrence and follow-up. Study of the expression of the rest of the adipocytokine group in SCD will provide additional information about its role in the pathogenesis of vasculopathy and will aid clinicians in planning a treatment strategy for controlling VOC crisis in SCD patients.

Acknowledgements

The authors greatly appreciate the facilities offered by the Clinical and Chemical Pathology Department, Hematology Unit of Ain Shams University Hospitals, Cairo, Egypt, which enabled them carry out this work.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

 
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