Haematological Indices and Antioxidant Enzyme Activity in Ghanaian Stroke Patients

Background Stroke is a cardiovascular disorder causing mortality globally and long-lasting harm worldwide. The disease occurs when the blood flow to the brain is either interrupted or blocked. This disruption leads to the increase in reactive oxygen species (ROS), especially superoxide free radicals, resulting in oxidative stress. The superoxide radicals are removed by superoxide dismutase (SOD), a key antioxidant enzyme. In this work, we investigated haematological indices and superoxide dismutase enzyme activity in Ghanaian patients with stroke and healthy control participants. Materials and Methods Thirty stroke patients attending a stroke clinic and thirty apparently healthy control participants were recruited into the study. Blood samples were collected to determine haematological indices and SOD enzyme activity in red blood cells. Results The stroke patients had significantly high blood parameters such as white blood cell (p < 0.001), neutrophil (p < 0.001), lymphocyte (p = 0.003), and eosinophil (p < 0.001) comparing with study participants without stroke, who were the control group in the study. Other blood parameters such as red blood cell, (p < 0.001), haemoglobin (p < 0.001), and haematocrit (p < 0.001) levels and mean cell haemoglobin concentration (p = 0.030), platelet (p = 0.010), and plateletcrit (p = 0.027) were high in stroke patients comparing with study control participants and statistically significant. Blood lymphocyte levels observed in stroke patients correlated negatively and significantly with SOD activity levels. SOD activity levels were significantly lower in stroke patients compared with the control group (p < 0.001). Low values of the antioxidant enzyme SOD activity levels, lymphocytes, and high values of plateletcrit were significant predictors of stroke. Conclusion Haematological parameters such as WBC, lymphocyte, platelet levels, and red cell indices were significantly different in the stroke patients being studied. There was negative correlation between lymphocyte significantly with SOD activity and high oxidative stress in stroke patients compared with the control group. Lymphocytes and plateletcrit levels were also good predictors of the occurrence of stroke.


Introduction
Stroke is a cardiovascular disorder causing mortality globally and long-lasting harm worldwide [1,2]. The disease is a leading cause of adult disability [3], the second cause of dementia [4], and the third cause of mortality in most developed countries such as sub-Saharan Africa [5]. These conditions result in about 70 percent of death and 87% of disabilities in low-and middle-level-income countries [1]. According to the World Health Organization, stroke is the second leading cause of death among people over 60 years of age and the fifth leading cause among those 15 to 59 years of age [6]. In Africa, especially sub-Saharan Africa, currently, there is an increase in stroke cases in middle-age lives [7,8]. The reported annual incidence of stroke in Africa was reported as 316 per 100,000 individuals [1]. Stroke is classified mainly into two types: ischaemic and haemorrhagic [9]. There are reports of high-level small-vessel disease-(ischaemic-) related stroke and intracerebral lesion on the continent of Africa than elsewhere in the world [10]. Hypertension remains the most important risk factor in Africa including other such as diabetes, stress, dyslipidaemia, cardiac disease, and obesity [11]. There are gaps in the general understanding of stroke care, practice, and policy in Africa [1]. Research insights into the disease would offer useful comprehension into stroke prevention and care.
Reactive oxygen species produced during ischaemic and perfusion phases in acute ischaemic stroke can lead to brain injury [12]. Also, unstable haemostasis in the brain causes the increased production of free radicals, especially reactive oxygen species such as superoxide. Increasing evidence suggests that reactive oxygen molecules are closely linked to tissue injury during stroke [13]. Elevation in reactive oxygen species can be injurious and also lead to adverse changes to biomolecules such as lipids, proteins, DNA, RNA, and enzymes in cells [14]. These changes include cell protein denaturation, DNA breaks, and alterations of cell membrane fluidity and eventually may lead to cell death. Furthermore, an increase in reactive oxygen species also leads to adverse oxidative stress situations in the stroke patients [15]. Biological functions such as apoptosis, necrosis, and phagocytosis are mediated by reactive oxygen species. These reactive metabolites are selectively neutralized by the body's defensive mechanism. Principal defensive agents are antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase [16].
The imbalance caused by oxidative stress results in red blood cell dysfunction, platelet destruction, and tissue injury. In stroke patients, such abnormalities may affect blood cell function as well as the coagulation system, leading to anaemia, infection, and hypercoagulability [17]. In the pathogenesis of acute ischaemic stroke, low-molecular weight antioxidants are involved in the reactions with free radicals generated during physiological cellular reactions resulting in oxidative stress [18]. The antioxidant enzyme superoxide dismutase and its derivatives cause vasodilation by opening potassium channels as well as deterioration of vascular reactivity [19]. Cerebrovascular thrombosis and subsequent cerebral perfusion injury in stroke necessitates constant monitoring of haemostatic parameters in stroke patients for effective management and care. In this study, we investigated haematological indices and superoxide dismutase activity in Ghanaian patients with stroke and study participants without stroke.

Materials and Methods
2.1. Study Design and Site. This cross-sectional study was conducted between May and July 2017 recruiting 60 study participants following informed consent by random sampling. Of these, thirty were diagnosed with stroke and were on admission at the stroke clinic of Korle-Bu Teaching Hospital (KBTH). The control group (n = 30) comprised of persons without clinical evidence of stroke or any history of the disease which were recruited from relatives of patients in the out-patient clinic of the healthcare facility. Structured questionnaire was used to collect data about subject demographic, life style, alcohol intake, disease condition, treatment history, and family history of stroke.
2.2. Inclusion Criteria. Inclusion criteria: patients (males and females) between the ages of 20 and 80 who were clinically diagnosed with stroke and who are in recovery during the first three to four months, were included in the study. Apparently, healthy subjects between the ages of 20 to 80, without any clinical evidence of stroke, served as control participants.

Clinical Evaluation.
The clinical history of all participants including demographic data, diagnosis, treatment history, and family history of stroke was recorded.

Blood Sample
Collection. Three millilitres of venous blood was collected from each study participant into an ethylenediaminetetraacetate (EDTA) tube for full blood count analysis. The blood samples were then centrifuged at 4000 rpm for 10 minutes at 4°C. Red blood cells obtained were stored by refrigeration for further analysis, while plasma and buffy coat were aliquoted into separate microcentrifuge tubes and stored at −20°C until use.

Superoxide Dismutase (SOD)
Assay. Superoxide dismutase activity was determined using a commercial kit, SOD Assay Kit-WST (Sigma-Aldrich, St. Louis, MO, USA), following the manufacturer's protocol. Red blood cell lysate of the study participants was used. Prepared briefly as follows, 50 μl of RBC pellets from each participant was lysed on ice for 5 min using double-distilled ice-cold sterile water followed by centrifugation at 13000 g for 15 min at 4°C. The supernatant was diluted 100x using double-distilled ice-cold sterile water. The manufacturer's protocol required that twenty microlitres of the supernatant from each sample processed is added to the wells of a 96-microtitre plate. Three wells on the microtitre plate are designated as blanks 2 BioMed Research International 1, 2, and 3. Two hundred microlitres of water-soluble formazan (WST) working solution was added to each well and mixed well. Also added was 20 μl of sample dilution buffer to blanks wells, labeled 2 and 3. Twenty microlitres of enzyme working solution was added to samples and blank 1 except blanks 2 and 3. The microtitre plate was mixed thoroughly by incubating briefly on a rocking platform. The reaction mixture was incubated at 37°C for 20 min and absorbance was read at 450 nm using a spectrophotometer (LabSystems Multiskan MS, LabSystems, Finland). SOD activity was calculated per formula described by the kit manufacturer. The SOD activity assays were done in duplicates [20,21]. , and plateletcrit (PCT)) in predicting stroke. The ROC curve, area under the curve, sensitivity, specificity, and accuracy of prediction were the criteria used. Sensitivity measures the probability that a person has stroke given that the person actually has stroke, whilst specificity measures the probability that a person does not have stroke given that the person does not have stroke [22].
2.9. Ethics. Ethical clearance was sought from the ethics and protocol review committee of the School of Biomedical and Allied Health Sciences (SBAHS), University of Ghana, prior to the commencement of the study and enrollment of subjects. Also, permission was sought from the head of the stroke unit of Korle-Bu Teaching Hospital before the start of the study. Written informed consent was obtained from the study participants.

Discussion
The economic impact of stroke causes financial cost to a country's health care system as well as loss of income and productivity on the part of those affected, either directly by the disease or indirectly as caregivers to those with stroke [23]. In this work, we investigated haematological indices and superoxide dismutase enzyme activity in Ghanaian patients with stroke and healthy control participants. White blood cells in the stroke patients and control participants were significantly different as seen in Table 2. Hu et al. [24] reported that high white cell indices were predictive of stroke severity and the results of the study was in agreement with the present one. In addition, the stroke patients with haemorrhagic stroke had elevated WBC. There are reports in a study that elevated levels of WBC and lymphocytes could be due to atherosclerosis, an inflammatory process, and could result in cerebral infarction [25]. There were      5 BioMed Research International significant differences in blood parameters such as mean cell haemoglobin concentration (MCHC) and haematocrit (HCT) levels (Table 3). There are reports that an increase in RBC above normal levels thickens blood and makes clots likely and increases the risk for stroke as this contributes to cerebrovascular blood viscosity [26]. This could possibly explain the high levels of RBC indices observed in the stroke patients in this study; increased levels of RBC indices are reported as risk factors of ischaemic stroke, cardiovascular disease, and venous thromboembolism [27]. There was significant difference in the platelet number, with stroke patients having high values (Table 2). Agreeing with reports that increase in platelet levels may imply augmented platelet aggregation and activity in early stroke, and the value of platelet counts may possibly be predictive of an event of stroke [28].
Reduced SOD activity levels in this study observed in the stroke patients ( Figure 1) agree with those reported by Cherubini et al. [29]. Additionally, there was also negative correlation between SOD activity levels and lymphocyte levels that was significant (Table 4). SOD activity levels weakly correlated with neutrophil, eosinophil, and monocyte levels. This could be explained by reports that leucocytes are endogenous sources of ROS and could possible explain the low SOD activity levels in the stroke patients [12]. The sensitivity and specificity of biological markers are useful in predicting stroke if their sum is greater or equal to 1.5 using receiver operator characteristic analysis [30]. We also assessed the stroke predictive ability of haematological indices and superoxide dismutase activity using the sensitivity, specificity, accuracy, and area under the curve (Table 5, Figures 2(a) and 2(b)). For biological markers where smaller test values indicate more positive test, the area under curve (AUC) values show that SOD enzyme activity was a better predictor of stroke. Lymphocytes were also a good predictor of stroke, looking at sensitivity, specificity, and AUC values. For markers where larger test values indicate a more positive test, the AUC values showed that PCT excellently predicts stroke, with very high sensitivity and specificity values. However, the study has limitations including the small sample size used and we could not analyze other antioxidant enzymes such as catalase and glutathione peroxidase.

Conclusion
Haematological parameters such as WBC, lymphocyte levels, and red cell indices were significantly different in the stroke patients being studied. There was negative correlation of lymphocytes significantly with SOD activity and high oxidative stress in stroke patients compared with the control group. Lymphocytes and plateletcrit levels were also good predictors of the occurrence of stroke. Further investigations of these parameters would allow better clinical management and care of stroke outcome.

Data Availability
All data generated or analyzed during the study are included in this article.  BioMed Research International