diabetes - a project work carried in Ghana

CHAPTER ONE

INTRODUCTION

1.1. BACKGROUND

Diabetes mellitus (Greek diabetes, “a compass, and siphon”; Latin mellitus, “honey”) is a syndrome characterized by presence of chronic hyperglycaemia (raised blood sugar) due to defective insulin secretion, insulin action or both. It is estimated to afflict over 140 million people world wide (Wokoma, 2002). The long term effect of diabetes which include progressive development of the specific complication of retinopathy, nephropathy, neuropathy, neuropathy with microvascular disorders such as atherosclerosis are recognized major cause of mortality in the diabetic population and it is implicated in the circulatory disturbance seen in diabetes (Wokoma, 2002).

Most cases of diabetes mellitus falls into three broad categories: type1, type 2 and gestational diabetes. Type 1 Diabetes mellitus is characterized by loss of the insulin-producing beta cells of the islets of Langerhans in the pancreas leading to insulin deficiency. Majority of the type 1 diabetes is of the immune medicated nature, where beta cell loss is a T-cell mediated autoimmune attack (Rother, 2007) Type 1 diabetes can affects children or adults but was traditionally termed “juvenile diabetes” because it represents a majority of the diabetes causes in children.

Type 2 Diabetes mellitus is characterized by insulin resistance which may combine with relative reduced insulin secretion. Type 2 Diabetes is the most common type. Gestational diabetes involves a combination of relatively inadequate insulin secretion and responsiveness. It occurs in about 2 – 5% of all pregnancies and may improve or disappear after delivery (Lawrence et al., 2008).

The cause of diabetes type 2 is due primarily to lifestyle factors and genetics (Riserus et al., 2009). The classical symptoms of diabetes are polyuria (frequent urination), polydipsia (increased thirsty) and polyphagia (increased hunger) symptoms of what may develop rapidly (weeks or months) in type 1 diabetes while in type 2 diabetes, they usually develop much more slowly and may be subtle or absent (Cooke et al., 2008).

In 2002, the prevalence of Diabetes mellitus among all age groups worldwide was estimated at 2.8% and projected to be 4.4% by 2030 (Rheeder, 2006). In 2010, the international Diabetes Federation (IDF) reported the prevalence of DM to be 3.6% of 13 million Ghanaians between the ages of 20 – 79 years (Kengne et al., 2005).

Platelets are derived from fragmentation of precursor megakaryocles in the bone marrow. They play a fundamental role in haemostasis and are natural source of growth factors. Platelet disorders or thrombocytopathy may be either thrombocytopenia (decrease in number of platelets), thrombosthemia (decrease in function) or thrombocytosis (increase in number of platelets) (Toryila et al., 2009).

Platelets are heterogeneous with respect to their size, density and reactivity. It is suggested that changes in platelet size are determined at thrombopoiesis in the negakaryocytes and those changes may precede acute cardiac events (Toryila et al., 2009). The large platelets (Giant platelets) were thought to be young because it had previously been suggested that platelets decrease in size as the age during their lifetime in the circulation (Toryila et al., 2009).

Platelets form the platelets plug, playing a pivotal role in haemostasis with platelets membrane, glycoproteins mediating binding to subendothetical tissue and aggregation into haemostatic plugs (George, 2000). Reduced platelet will result in a bleeding tendency; while stimulation of platelets can lead to thrombosis (Ruttmann, 2006). In addition, aggregated, activated platelets provide procoagulated phospholipid equivalent surfaces upon which the complex-dependent reactions of the blood coagulation cascade are localized (Ruttmann, 2006).

Normal blood clots are part of the body’s healing process. Platelets which are small blood cells are held together by a glue-like substance that is made when proteins known as clotting factors triggered. The clot seals a cut or break that may be outside or inside the body. Once the damage is healed, natural processes go to work to break up and dissolved the clot. Diabetes patients could die from heart and blood vessel complications, which include clot fragments that have broken free (Ruttmann, 2006).

Cardiovascular disease, particularly coronary artery disease (CAD), often presenting as an acute coronary syndrome (ACS), is the leading cause of morbidity and mortality in patients with Diabetes mellitus (Ferreiro et al., 2010). Of note, Diabetes mellitus patients without any history of CAD have the same cardiac mortality risk as non-Diabetes mellitus patients with a history of myocardial infarction (MI) cardiovascular disease has also worse prognosis in patients with Diabetes mellitus as they have a higher risk of complications and recurrent atherothrombotic event than non-Diabetes mellitus patients (Lüscher et al., 2003).

In fact, Diabetes mellitus is a strong independent predictor of short and long term recurrent ischaemic events, including mortality, in and ACS scenario. (Malmberg et al., 2000; Roffi et al., 2001). Further, the concomitant presence of cardiovascular risk factors and comorbidities that negatively impact the outcomes of ACS is higher in Diabetes mellitus patients (Brogan et al., 2006).

Several factors contribute to the prothrombotic conditions that characterized patients with Diabetes mellitus, such as increased coagulation, impaired fibrinolysis, endothelial dysfunction and platelet hyperreactivity (Creager et al, 2003; Osende et al, 2001). The latter is of particular interest since platelets play a key role in the formation, development and sustainment of thrombi, which are platelet-driven processes (Davì and Patrono, 2007). Platelets of patients with Diabetes mellitus are characterized by dysregulation of several signaling pathways and have been proven to be hyperreactive with intensified adhesion, activation and aggregation (Creager et al., 2003; Stratmann and Tschoepe, 2005; Angiolillo et al., 2005; Vinik et al., 2001; Ferroni et al., 2004).  Such a hyperreactive platelet phenotype may contribute to the higher proportion of Diabetes mellitus patients with inadequate response to antiplatelet agents compared with non- Diabetes mellitus subjects (Angiolillo, 2009; Natarajan et al., 2008).

Multiple mechanisms caused by metabolic and cellular abnormalities have been suggested to play a role in the increased platelet reactivity observed in patients with Diabetes mellitus. These mechanisms can be grouped together into the following aetiopathogenic categories a) hyperglycaemia, b) insulin deficiency and resistance c) associated metabolic conditions and d) other cellular abnormalities (Ferreiro and Angiolillo, 2010).

Diabetes mellitus is associated with both metabolic and vascular abnormalities and although a causal link between the two has been suggested. This has not been fully established (Brogan et al., 2006). Patients with diabetes mellitus have an increased risk of cardiovascular diseases, especially myocardial infarction, cerebrovascular and peripheral vascular diseases. These complications account for 80% of the deaths in people with non-insulin dependent diabetes, with 60% attributable to ischemic heart disease. Thus much attention has been devoted to the pathogenic factors, altered haemostatic balance, including abnormalities in platelet function, increase in blood coagulability and altered fibrinolytic system (Schweigart et al., 2004.).

1.2 STATEMENT OF THE PROBLEM

The global burden of disease study of the World Health Organization (WHO) estimated that about 177 million people in the world had diabetes in the year 2000 (WHO, 2003). In the second edition of the International Diabetes Federation's Diabetes Atlas it is estimated that 194 million people had diabetes in the year 2003, and about two-thirds of these people lived in developing countries (IDF, 2003). Recent results from the general population of Tanzania, where a morbidity and mortality surveillance system has been set up, show that stroke mortality was three to six times that of England and Wales and that 4.4 percent of type 2 diabetic patients presented with stroke at the diagnosis of diabetes (Whiting et al., 2003). Part of this is attributed to the deep vein thrombosis and myocardial infarction conditions of diabetic patients. Therefore there is the need to research into the estimation of platelet level and clotting time in diabetic patients attending the Central Regional Hospital.

1.3. JUSTIFICATION

Adoption of Western lifestyles has been established as a consistent theme for the rise in diabetes in sub-Saharan Africa (Aspray et al., 2003). The result of this study would expose the prevalence of atherosclerosis among diabetic patients in setting and this could largely help in policy formulation in the monitoring and management of diabetes. Approximately 75% of patients with diabetes identified in community surveys in sub-Saharan Africa do not know that they have diabetes. They often have complications at the time of diagnosis with the estimated duration of type 2 diabetes mellitus from onset to diagnosis is 7 years (Nkegoum. 2002). However, targeted screening to identify individuals with high-risk characteristics should be undertaken and would be ideal in driving policy to help reduce incidence of diabetes in Ghana and the sub – regions.

1.4. HYPOTHESIS

Diabetes patients suffer from thrombocytopathy as a result of increased production level of platelet in their circulatory system and a minimum clotting time as compared with non diabetic patients. Diabetic patients are prone to thrombosis through a complex interplay of hyperlipidemia, platelets, fibrinolysis, thrombosis and endothelial injury. Diabetic patients also have increased level of plasminogen activator inhibitor, von Willebrand factors, factors VIII and VII fibrinogen and thrombin-antithrombin complexes. All these prolong the survival of clots on injured endothelium. Additional factors like decreased level of antithrombin III, protein C and protein S increase the predisposition to thrombosis (Sowers, 2003).

1.5.1 GENERAL OBJECTIVES

The main aim of the study is to determine the quantity of the platelets and the effectiveness of the clotting factors in general in diabetic patients at the Central Regional Hospital.

1.5.2        SPECIFIC OBJECTIVES

1.      To estimate the platelets level and clotting time in diabetic patients

2.      To estimate the platelets level and clotting time among the various age groups with diabetes.

3.      To compare the platelet level and clotting time of non diabetic and diabetic patients.

CHAPTER TWO

LITERATURE REVIEW

2.1. Definition Diabetes

Diabetes mellitus is a chronic metabolic disease characterized by hyperglycemia resulting from defects in insulin secretion, insulin action, or both. Uncontrolled chronic hyperglycemia results in long-term damage, particular dysfunction, and failure of the eyes, heart, blood vessels, nerves, and kidneys. The global burden of disease study of the World Health Organization (WHO) estimated that about 177 million people in the world had diabetes in the year 2000 (WHO 2003). In the second edition of the International Diabetes Federation's Diabetes Atlas it is estimated that 194 million people had diabetes in the year 2003, and about two-thirds of these people lived in developing countries (IDF 2003).

2.2. History of Diabetes

Diabetes mellitus describes a metabolic disorder of multiple aetiology characterized by chronic hyperglycaemia with disturbances of carbohydrate, fat and protein metabolism resulting from defects in insulin secretion, insulin action, or both (WHO, 2003). Diabetes is an ancient disease. Its symptoms, which include excessive drinking of water and frequent urination (to wash away the excess sugar in the blood), were noted on a scrap of Egyptian papyrus more than 3,500 years ago. The ancient Roman doctor Aretaeus of Cappadocia also gave a vivid description of diabetes,describing it as “a melting down of the flesh and limbs into urine” (Patlak, 2002).

Since then, many physicians have remarked on the sweet taste of diabetic’s urine. The combination of the Greek and Latin word diabetes mellitus means “sweet flow” or “siphon”. Because of this hall mark of diabetes, the disease was thought to be a disorder of the kidneys and bladder for more than two thousand years (Patlak, 2002). In 1776, Matthew Dobson confirms that the sweet taste was because of an excess of a kind of sugar in the urine and blood of people with diabetes (Dobson, 1776).

Diabetes has been recognized since antiquity and treatments of various efficacy have been know in various regions since the middle ages and in legend for much longer, pathogenesis of diabetes has only been understood experimentally since about 1990 (Patlak, 2002). The discovery of a role for the pancreas in diabetes is generally ascribed to Joseph von Mering and Oskar Minkowski in 1889 found that dogs whose pancreas was removed developed all the signs and symptoms of diabetes and died shortly afterwards (Patlak, 2002). In 1910, Sir Edward Albert Sharpey-Schafer suggested that people with diabetes were deficient in a single chemical that was normally produced by the pancreas he proposed calling this substance insulin from the Latin insula meaning island, in reference to the insulin-producing islets of Langerhans in the pancreas.

The remarkable success at using insulin to treat diabetes led to the notion that the disease was caused by a lack of insulin. But a series of observations in the 1930s by the British clinician Harry Himsworth lead to a startling new view of diabetes. Curious about how diet affects sensitivity to insulin, Himsworth conducted a series of experiments in both animals and people that led him to the discovery that the body’s used of sugar depends not only on how much insulin is present, but on how sensitive the body is to the effects of insulin. Diabetes could be caused not only by a lack of insulin but also by a lack of sensitivity to insulin (Patlak, 2002).

The distinction between what is now known as type 1 diabetes and type 2 diabetes was clearly made by Sir Harold Percival (Harry) Himsworth and published in January 1936 (Himsworth, 1936). Himsworth tested out this theory by giving diabetic patients sugar and insulin simultaneously and then checked to see how well the insulin fostered their use of the sugar. If they were relatively insensitive to the effects of insulin, their blood sugar levels shot up. These experiments showed that there were two types of diabetes: type 1 and type 2. People with type 1 diabetes were sensitive to insulin and had a history of suddenly developing the disease at a young age; those with type 2 diabetes were relatively insensitive to insulin and tended to gradually develop a milder form of the disease at middle age of older (Patlak, 2002)

In 1960, Yalow and Berson used their new techniques to measure and compare the insulin response to sugar in those with type 2 diabetes to those without the disease. They discovered that instead of producing less insulin after being given sugar, people with type 2 diabetes  often generated more insulin than did those without diabetes. This perplexing finding was totally unexpected and jotted the diabetes research community.

Other researchers then discovered that although people in the early stages of type 2 diabetes produce more than the normal amounts of insulin, over time, their insulin levels fall until eventually they dip below that seen in normal individuals and their diabetes become severe (Patlak, 2002)

The net result of all these findings was the hypothesis that to compensate for their lack of sensitivity to insulin (insulin resistance), people with type 2 diabetes initially produces excess insulin. That excess allows them to sufficiently convert the sugar in their diet to energy their tissues can use. But eventually the insulin-producing cells in the pancreas deteriorate and can’t keep up with the need for insulin. At this point, these people’s diabetes becomes severe, requiring insulin treatment (Patlak, 2002).

In 2000, according to the World Health Organisation, at least 171 million people world wide suffer from diabetes, of 2.8% of the population (Wild et al, 2004). Its incidence is increasing rapidly and it is estimated that by 2030, this number will almost double (Wild et al, 2004). The greatest increase in prevalence is, however; expected to occur in Asia and Africa, where most patients will probably be found by 2030 (Wild et al., 2004). The increase incidence of diabetes in developing countries follows the trend of urbanization and lifestyle changes, perhaps most importantly a “Western-style” diet. This suggested an environmental (i.e. dietary) effect, but there is little understanding of the mechanism(s) at present, though there is much speculation some of it most compellingly presented (Wild et al., 2004).

2.3. Classification of Diabetes

Diabetes mellitus can be classified into four principal types (WHO, 2003). This includes type 1 diabetes, type 2 diabetes, other specific types of diabetes, and gestational diabetes mellitus. The most common types of diabetes seen in Sub-Saharan Africa are type 2 and type 1 diabetes mellitus.

Type 1 diabetes results from autoimmune destruction of the pancreatic beta cells, causing the loss of insulin production. Children are usually affected by this type of diabetes, although it occurs at all ages and the clinical presentation can vary with age. Patients with this type of diabetes require insulin for survival (Mbanya and Ramiaya, 2006).

Type 2 diabetes is characterized by insulin resistance and abnormal insulin secretion, either of which may predominate but both of which are usually present. The specific reasons for the development of these abnormalities are largely unknown. Type 2 is the most common type of diabetes. Type 2 diabetes can remain asymptomatic for many years, and the diagnosis is often made from associated complications or incidentally through an abnormal blood or urine glucose test (Mbanya and Ramiaya, 2006).

Other specific types of diabetes include those due to genetic disorders, infections, diseases of the exocrine pancreas, endocrinopathies, and drugs. This last type of diabetes is relatively uncommon (Mbanya and Ramiaya, 2006).

Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance with onset or first recognition during pregnancy. The definition applies whether insulin or only diet modification is used for treatment and whether the condition persists after pregnancy. It does not exclude the possibility that unrecognized glucose intolerance may have antedated or begun concomitantly with the pregnancy. Approximately 7 percent of all pregnancies are complicated by GDM. The prevalence may range from 1 to 14 percent of all pregnancies, depending on the population studied and the diagnostic tests employed (Mbanya and Ramiaya, 2006).

The prevalence diabetes in blacks follows a Westernization gradient, with that of rural Africa generally below 1 percent but that of urban Africa between 1 and 6 percent. In general the prevalence of type 2 diabetes is low in both rural and urban communities of West Africa except in urban Ghana, where a high rate of 6.3 percent was recently reported (Amoah et al., 2002).  The prevalence of undiagnosed diabetes, which accounted for 60 percent of those with diabetes in Cameroon (Mbanya et al., 2001), 70 percent in Ghana (Amoah et al., 2002), and over 80 percent in the recent study in Tanzania (Aspray et al., 2000). It would therefore appear that in Sub-Saharan Africa, for every diagnosed person with diabetes, there are one to three undiagnosed cases.

2.4.1. Haemostasis

Normal haemostasis is the ability of the haemostatic system to control activation of clot formation and clot lysis in order to prevent haemorrhage without causing thrombosis. It classically involves vasoconstriction, platelet adhesion and aggregation at the site of injury, leading to a plug formation. This is followed by fibrin formation consolidating the plug and rendering it stable (Ruttmann, 2006). Coagulation is divided into two major systems: the primary and secondary systems of hemostasis. The primary system comprises platelet function and vasoconstriction. The secondary system involves coagulation proteins and a series of enzymatic reactions (Davis and Patrono, 2007).

2.4.2. Platelet production

Platelets are small (2 μm diameter), non-nucleated blood cells, which are activated rapidly after blood vessel injury or blood exposure to artificial surfaces, providing haemostasis by four interconnected mechanisms: (i) adhering to sites of vascular injury or artificial surfaces; (ii) releasing compounds from their granules; (iii) aggregating together to form a haemostatic platelet plug; and (iv) providing a procoagulant surface for activated coagulation protein complexes on their phospholipid membranes (O’Connell et al., 2008).

Reduced platelet activity will result in a bleeding tendency, while stimulation of platelets can lead to thrombosis (Ruttmann, 2006). Platelets are extremely small and discoid, 3.0 X 0.5µm in diameter, with a mean volume 7 – 11 fL. The platelets have glycoproteins on the surface coat which are important in their reaction of adhesion and aggregation which are the initial events leading to platelet plug formation during haemostasis. Adhesion to collagen is facilitated by glycoprotein Ia (GPIa). Glycoproteins Ib and IIb/ IIIa are important in the attachments of platelets to von Willebrand factor (vWF) and hence to vascular subendothelium where metabolic interactions occur (Collins et al, 2007).

Platelet aggregation itself also involves positive feed backs through the release from platelets of platelet agonists such as adenosine diphosphate (ADP) and thromboxane A2 (Ruttmann, 2006).

Nitric oxide (NO) is constitutively released from endothelial cells and also from macrophages and platelets. It has a short half-life of 3 – 5 seconds. It inhibits platelet activation and promotes vasodilatation. Prostacyclin synthesized by endothelial cells also inhibits platelet function and causes vasodilatation by raising cyclic guanosine monophospate (GMP) levels. The transmembrane protein PECAM-1 is expressed also on endothelial cells. It is its own ligand and inhibits platelet activation by collage (Ruttmann, 2006).

2.4.3. Coagulation cascade

Coagulation factors are produced in the liver, except for factor VIII, which is believed to be produced in the endothelial cells. The initiation of clotting begins with the activation of two enzymatic pathways that will ultimately lead to fibrin formation: the intrinsic and extrinsic pathways (Davi and Patrono, 2007).

The ‘extrinsic pathway’ is the primary route of thrombin formation, through which the physiological haemostatic response occurs. It occurs through contact with a non-blood protein source. The physiological initiator of coagulation is tissue factor, which comes into contact with the blood at the site of vascular injury and combines with plasma serine protease, factor VII (Ruttmann, 2006). The tissue factor/factor VIIa (TF/VIIa) complex activates factor X to factor Xa. It can, however, also activate factor IX to IXa (Ruttmann, 2006).  Factor VII forms a complex with tissue thromboplastin and calcium. This complex converts factors X and Xa, which in turn converts prothrombin to thrombin. Thrombin then converts fibrinogen to fibrin. This process takes between 10 and 15 seconds (Davis and Patrono, 2007).

The ‘intrinsic pathway’ starts with the formation of factor XIIa This can cleave prekallikrein to provide kallikrein, which in turn reciprocally activates factor XII. Factor XIIa, in the presence of highmolecular- weight kininogen, converts factor XI to factor XIa, which in turn converts factor IX to factor IXa. Factor IXa binds with its cofactor protein factor VIIIa, in the presence of calcium and the appropriate membrane surface, and activates factor X to factor Xa (Ruttmann, 2006). The reaction then enters the common pathway where both systems involve factors I, II, V, and X. This results in a fibrin monomer polymerizing into a fibrin clot. Factor XIII, or fibrin stabilizing factor, follows activation by thrombin. This will convert initial weak hydrogen bonds, cross-linking fibrin polymers to a more stable covalent bond (Davis and Patrono, 2007).

2.5. Disturbances of haemostasis in Diabetes mellitus

There is increasing evidence that diabetes mellitus is associated with several defects of coagulation and fibrinolysis that lead to a procoagulant, thrombogenic predisposition. Raised concentrations of fibrinogen, von Willebrand factor and other endothelium-derived mediators increase blood viscosity and promote platelet activation and adhesion. In addition, fibrinolysis is impaired by raised concentrations of plasminogen activator inhibitor-1 (Petrauskiene et al., 2005). The fibrinolytic system is natural defense against thrombosis. A balance exists between plasminogen activators and inhibitors, and impairment of this balance can caused either by diminished release of tissue plasminogen activator (t-PA) or increased levels of plasminogen activator inhibitor 1 (PAI-1). PAI-1 is a serine protease inhibitor and evidence suggests that it is the major regulator of the fibrinolytic system. It binds and rapidly inhibits from an inactive irreversible complex (Carr, 2001).

Diabetic patients are considered to be at increased risk of thromboembolic disease.  These patients are particularly prone to the classic presentations of thrombophilic diathesis, such as deep vein thrombosis or pulmonary embolism, but are at high risk for atherosclerosis and its vaso-occlusive complications (Wokoma, 2002). Fibrinolytic activity has been reported to be normal, elevated or low in diabetic patients. Alterations in factors such as fibrinogen concentration and turnover, fibronectin, and platelet adhesiveness and aggregation are found in non-insulin dependent diabetic patients and can lead to excess accumulation of fibrin and platelet on vascular walls (Schweigart et al., 2005).

Regarding fibrinogen level, high significant level is demonstrated in diabetes. Increase plasma fibrinogen may contribute to a hypercoagulable state in non-insulin dependent diabetes mellitus. Fibrinogen may induce thrombus formation by affecting platelets and erythrocytes to aggregation and by promoting increased blood viscosity. Plasma fibrinogen is often elevated in diabetes which has been identified as a major vascular risk factor (Schweigart et al., 2004).

In diabetic state, there is increased platelet derived nitric oxide destruction, contributing to increased platelet aggregation and adhesion to endothelial cells (Shammas, 2007). Other platelet abnormalities in diabetic include decreased platelet survival, increased platelet generation of vasoconstrictor prostanoids, reduced platelet generation of prostacyclin and increased glycosylation of platelet proteins. The major clinical manifestations are thrombosis and hemorrhage, probably reflecting both qualitative and quantitative abnormalities in platelets (Shammas, 2007).

2.6. Information and knowledge associated with diabetes

Decision makers need to be fully informed with clear and up-to-date evidence about the burden and the impact of diabetes mellitus and its complications. In addition, the advocacy base for diabetes awareness needs to be expanded. Furthermore, gaps in our knowledge about diabetes control in sub-Saharan Africa should be addressed using a "grand challenges initiative" (Varmus et al., 2003).

The role of government is critical to the development and implementation of well-grounded risk-factor control programs such as the WHO’s Framework Convention on Tobacco Control, which also has implications for food policies (Yach et al., 2003).  Approximately 75% of patients with diabetes identified in community surveys in sub-Saharan Africa do not know that they have diabetes. They often have complications at the time of diagnosis; the estimated duration of type 2 diabetes mellitus from onset to diagnosis is 7 years (Krauss and Siri, 2004). Widespread screening in the general population cannot be encouraged in sub-Saharan Africa. However, targeted screening to identify individuals with high-risk characteristics should be undertaken.

Health systems should be realigned to accommodate the diagnosis and primary and secondary prevention of diabetes and its complications in sub-Saharan Africa. Governments need to support this transformation if they are to realize significant health gains. The potential for secondary prevention of cardiovascular disease and diabetes in developing countries has recently been highlighted (WHO, 2002; Beaglehole and Yach, 2003), but policy needs to be translated into action.

In summary, Patients with diabetes mellitus (DM) have accelerated atherosclerosis, which is the main underlying factor contributing to the high risk of atherothrombotic events in these patients. Atherothrombotic complications are the leading cause of morbidity and mortality in patients with DM. Among factors contributing to the prothrombotic condition which characterise patients with DM, platelet hyperreactivity plays a pivotal role. Platelets of DM patients are characterised by dysregulation of several signalling pathways leading to intensified adhesion, activation and aggregation. Multiple mechanisms are involved in platelet dysfunction of patients with DM, which can be categorised as follows: a) hyperglycaemia, b) insulin deficiency and resistance, c) associated metabolic conditions, and d) other cellular abnormalities.

Diabetic patients are therefore prone to thrombosis through a complex interplay of hyperlipidemia, platelets, fibrinolysis and endothelial injury.

CHAPTER THREE

METHODOLOGY

3.1.0 STUDY AREA

The study was in Cape Coast in central region of Ghana, which was historically part of western region until 1970 when it was carved out just before the 1970 population census. It occupies an area of about 9,826 square kilometres or 4.1 percent of Ghana’s land area. The research was targeted at diabetic patients, who visited the Central Regional Hospital’s Diabetic Clinic for medical review.

3.1.1 STUDY PERIOD

The study was conducted between the months of December, 2010 and February, 2011.

3.2.0 ETHICAL CLEARANCE

With the assistance of Head of the Diabetic Clinic in the Central Regional Hospital, informed consents were obtained from the administration of the hospital and the patients after reading and listening to the verbal description of the study’s objectives with the assurance that all information obtained would be treated with utmost confidentiality and used for the purpose of the research only.

3.3.0 SAMPLING AND DATA COLLECTION

A sample size of two hundred and seventy seven was used for the study and sampling includes volunteer diabetic patients who visited the Diabetic Clinic at the Central Regional Hospital.

3.3.1 SAMPLING

The whole blood clotting time was determined using the Lee – White method (Lewis et al, 2006). The haematological analysis from the Abbott Cell – Dyne product line (CELL DYNE 1800) was used to determine the platelet counts based on manufactures protocol and laboratory procedure.

3.4.0 LIMITATIONS

a. Only patients who have already been diagnosed were sampled.

b. The unwillingness of some patients to be added to the sample size.

3.5.0 DELIMITATION

The study covered patients with type 2 Diabetes mellitus irrespective of the age and the sex. Both the platelet counts and the clotting time were carried out for the patients whose samples were collected.

3.6.0 DATA ANALYSIS

Data from the samples taken were present in tables and graphs using Microsoft Excel and SPSS was use to test for the ANOVA.

CHAPTER FOUR

RESULTS

A total of 209 diabetic patients and 68 non – diabetic patients were sampled for platelet count and clotting time.

4.1 Distribution of sexes among the sampled population

Out of the 277 sampled, 51 (24.4%) and 158 (75.6%) were diabetic males and females respectively and 26 (38.2%) and 42 (61.8%) were non – diabetic males and females.

4.2 Age distribution among the sampled population

Majority 70 (33.5%) of the diabetics were aged between 56–65yrs, followed by 46–55yrs 62 (29.7%), and with a least of 8 (3.8%) aged above 75yrs, whilst the majority of non–diabetic patients were aged below 35yrs, 41 (60.29%), and least of 2 (2.94%) aged 46–55yrs and 66–75yrs.

4.3 Distribution of platelets among sampled populations

The overall distribution of platelets in diabetics were 31 (14.8%), 147 (83.3%), and 4 (1.9%), for thrombocytopenia, normal and thrombosis respectively (fig. 4.3).

4.4 Distribution of clotting time among the sampled population 

Also the overall distributions of coagulation in the diabetics were 13 (6.2%), and 196 (93.8%) for the hypercoagulation and normal respectively (fig. 4.4).

Fig. 4.4 Distribution of Clotting Time among the Diabetics

4.5 Distribution of platelets among the aged groups of the sampled population.There was a little decrease in platelets in all the age groups but those that fall within 35 – 65years also show slight increased in platelets above the normal range. The distribution of thrombopenia among the age groups were; below 35years, 1 (0.48%), 35 – 55 years, 6 (2.87%), 56 – 75years, 9 (4.31%), above 75, 1 (0.48%). This is shown in fig. 4.5

Fig. 4.5 Distribution of Platelets among the Age Groups with Diabetes

4.6 Distribution of clotting time among the age groups of the sampled population

All the age groups had a normal coagulation. There were slight hypercoagulation among 35 – 45 years, 1 (0.48%), 46 – 55 yrs, 5 (2.39%), 56 – 65yrs, 2 (0.96%), and 66 – 75yrs, 4 (1.91%), above 75, 1 (0.48%).

4.7 Distribution of clotting time among the sex of the sampled population

It can be observed that females suffer from diabetics than the males and there is a slight decrease in coagulation in females 9 (4.30%) than in males 4 (1.91%). The rest were within the normal range of clotting time males 47 (22.49%) and females 149 (71.29%).

4.8 Distribution of platelets among the sex of the sampled population

Fig. 4.8 shows that many females 20 (9.57%) slight decrease in platelet compared with the males 12 (5.74%). Both sexes show the same percentage of thrombosis 2 (0.96%) while the normal platelet counts in males were about 37 (17.70%) and females 136 (65.07%). 

4.9 Distribution of platelets among the state of diabetes in the sampled population

Fig. 4.9 indicates that diabetic patients in the state of hyperglycemia 2 (0.96%) and normal 1 (0.49%) experience slight thrombosis while those in the state of hyperglycemia 16 (7.66%) and normal 16 (7.66%) experience thrombopenia.  Normal platelet count was observed in hypoglycemia 5 (2.32%), normal 61 (29.19%) and hyperglycemia 108 (51.67%).

4.10 Distribution of clotting time among the state of diabetes in the sampled population

Fig. 4.10 also indicates that none of the diabetic condition state experienced hypercoagulation but the hyperglycemia 7 (3.35%) and the normal 6 (2.87%) experience hypocoagulation. The hypoglycemia 5 (2.39%), normal 72 (34.45%) and hyperglycemia 119 (56.94%) also indicated a normal clotting time in so of the diabetics.

CHAPTER FIVE

DISCUSSION, CONCLUSION AND RECOMMENDATION

5.1 DISCUSSION

Type 2 diabetes and its associated long-term complications continue to accelerate among patients who reside in developing countries. With the modernization of cultures taking place all over the world, the standard of living and lifestyle in many sub-Saharan African countries, particularly in urban areas, resembles those of many Western countries, with related epidemiological changes (Amoah, 2003). Chronic health problems such as cardiovascular diseases and diabetes have therefore become at least as important as infectious diseases.

Most circulatory disturbances seen in diabetes are compounded by alteration in platelet count and activity, coagulopathy, fibrinolytic aberration and changes in endothelial metabolism (Omar and Ayesha, 2002). Thrombosis can result from a wide range of conditions with several of them being diabetic condition. However, majority of the diabetics with signs of thrombosis appear to be healthy but were accidentally detected by routine blood testing.

Of the 209 diabetic patients in this study, 6.2% had a fast coagulation time, 93.8% had normal coagulation time. Meanwhile most of the diabetics had normal platelet count (83.3%) with a few showing abnormal high platelet (1.9%) and low platelet (14.8%) count. Comparing hypercoagulation between the diabetics and non–diabetics, 16.2% of the diabetics had abnormal coagulation time compared with none (0%) in non-diabetics. The result of the diabetics can be attributed to the fact with increased activation of platelets and plasma fibrinogen; they are prone to cardiovascular disease such as obesity and hypercholesterolemia (Muscari et al., 2008). This also confirms the work of Kengne et al., (2005) carried out in sub-Saharan Africa stating that 4%-28% of type 2 diabetes have coagulopathy as a result of reduced coagulation time which put the diabetics at risk of myocardial infarction. Studies done by Walker et al., (2000) indicated that 4.4 percent of type 2 diabetic patients in Tanzania presented fast coagulation time and stroke at the diagnosis of diabetes.

More females (75.6%) attended the diabetic clinic than the males (24.4%) and yet more females (4.30%) show fastness in their coagulation time. This could be due to the various physiological changes which occur in females as a result of pregnancy, delivery and the use of oral contraceptives which affect the production of antithrombin III (natural anticoagulant) in regulating coagulation. Normal flow of blood prevents the accumulation of procoagulant material. This mechanism is enhancing when the diabetics engage in physical activities. Research conducted by Edwards et al., (2000) reveals that low physical activity was normal for 22 percent of men and 52 percent of women with diabetes in urban Tanzania, whereas it was usual for only 10 percent of men and 15 percent of women with diabetes living in rural areas. This also gives another reason confirming why the females have fast coagulation time.

Findings of this study indicates that there was significant difference in platelet count and clotting time between the diabetic patients and non diabetic patients (p=0.000). This is in line with the study of Fattah et al., (2004) stating that platelets from DM patients have high levels of fibrinogen, a2- antiplasmin, & PAI and lower level of protein C. Since the platelets are slightly reduced below the normal average platelet count, there is increased platelet reactivity caused by hyperglycemia as a result of osmotic effect of glucose that activates platelet GP IIb/IIIa and P-selectin expression. This indicates that diabetic can develop cardiovascular disease as a result of this coagulopathy.

Majority of the diabetics fall between the ages of 56 – 65 years (33.5%) and 46 – 55 years (29.7%). Their clotting time and platelet count indicates that there was no significant difference among the various age groups that were affected with the diabetes (p=0.450). This result is in agreement with the studies of Fattah et al., (2004) who also suggested that any differences in the generation of thrombin activity in plasma of diabetic patients are likely to be insignificant and the blood coagulation is not enhanced in any diabetic patients. This is due to the normal production of coagulation factors II, VII and X in diabetic patient.  

Findings of this study reveals that most of the diabetics had hyperglycemia (60.3%) with the rest been normal (37.3%) and very few in hypoglycemic state (2.4%). It is worth mentioning that hyperglycaemia reduce the coagulation time in diabetic patients. Hyperglycaemia activates coagulation by raising concentra­tions of procoagulant factors (e.g. tissue factor, von Willebrand factor) and inhibits fibrinolysis by increasing concentrations of plasminogen activator inhibitor (Boden and Rao, 2007). This is in agreement with the work done by in Sudan which shows that 26% of diabetes/hyperglycemia patients were at risk with Coronary Artery Diseases (CAD) since they turn out to have least coagulation time (Kingue et al., 2000).

It was observed that all the diabetics under study did not have any significant difference in their clotting time or platelet count irrespective of the age, sex or the state of the glycemia (P=0.490). Platelet counts from patients with type-2 Diabetes mellitus were within the normal range (150-400 X10⁹/L). This may indicate that the bone marrow is functioning as expected due to the regular fragmentation of the cytoplasm of megakaryocytes. Also, the normal productions of platelets show that the thrombopoietin produced by the liver and kidneys in regulating the platelet production is not affected by the complication in diabetes.

High levels of plasma fibrinogen, a2- antiplasmin, plasminogen activator inhibitor with low plasma protein C activity in diabetics induces thrombus formation by affecting platelets and erythrocytes to aggregate and promote blood viscosity (Fattah et al., 2004). From these observations it may be suggested that hypercoagulation can be considered as an additional risk factor for development of thrombosis in diabetic patients.

5.2 CONCLUSION

Patients with Diabetes mellitus (DM) have platelet count within the normal range needed in the normal haemostic response to vascular damage. There was significance difference between the diabetics and the non-diabetics. The non-diabetics have shown an elevated platelet counts more than the diabetics who happen to show normal platelet count and slight thrombopenia.

The coagulation time measures the intrinsic system of coagulation. The diabetics happen to have a reduced clotting time compared with the non-diabetics. This reveals that there is elevation of plasma fibrinogen that may contribute to the hypercoagulable state of diabetes mellitus but there was no significant difference between the age groups and the gender of type 2 diabetes.

5.3 RECOMMENDATION

Diabetics are prone to thrombosis due to the fact that they do not partake in early diagnosis and monitoring of the glucose level in their blood stream. I therefore recommend the education on the importance on regular checking of the glucose level in the blood and the function of the pancreas and insulin level that is been produced. The patients should be taught the essence of engaging in active physical activity so that the blood will always have a normal flow.

Coagulation profile should be added to routine test that the diabetics undertake to check parameters that are deficient or abnormally produce which could contribute to hypercoagulation so that the necessary medication could be given to the patients to correct the abnormality.

Further study on the various coagulation assays can be carried on the diabetics to find out which of the coagulation factors is abnormally produced or deficient in production.

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APPENDICE I

LABORATORY TEST PROCEDURE FOR CLOTTING TIME AND PLATELET COUNT

Blood (venous) samples were taken from patient into EDTA tubes using the evacuated tube and treated to standard methods for determining platelet count and a plain 12X75 mm glass tube for determining clotting time.

Procedure:

The venipuncture site of the of the patient was cleaned with an antiseptic (alcohol) which helps prevent microbial contamination of the specimen and the patient. The tourniquet was applied to the venipuncture site and the vein was anchored below the puncture site. The needle was inserted at an angle 15 to 30 degrees, smoothly and beveled up. The elevated tube was inserted into the venipuncture site and the blood was allowed to flow freely into the tube. The tourniquet was released, tubes were removed and mixed by inversion, and the glass tubes were placed in the water bath immediately. Gauze was placed over the puncture site.

Some of the laboratory test conducted on the samples included:

1.      Clotting time; which provides information on blood clot in a glass tube, measuring the intrinsic system of coagulation in patient using a manual technique.

Procedure:

Lee – White Method

a.       I used the syringe and needle method of clotting blood into 12 X 75mm plain and EDTA tubes. 3mls of venous blood was clotting into three 12 X 75mm plain tube and 1ml into the EDTA tube.

b.      A stopwatch was started soon as the blood entered the syringe. The needle was removed from the syringe and each of the three tubes was filled to the 1ml mark.

c.       The test tubes were put into the water at 37ºC. After 3 minutes the first tube was removed from the water bath and tilted gently to and angle of 45º to verify whether the blood has clotted. When the blood was not clotted, it was returned to the water bath and examines at 30 seconds intervals to see if it has clotted.

d.      The tube was tilted through an angle of 90º without the content spilling. As soon as the blood was clotted, the second and third tube was examined immediately.

e.       The stopwatch was stopped and the time taken from the three tubes. The average time was calculated and it indicates the coagulation time (Lewis et al, 2006).

2.      Platelet Count with the use of an improved Haussser Neubauer ruled Bright – line counting chamber. These chambers are the finest quality, optically ground, and polished milled glass chambers available. The chamber is diamond etched and has a double improved Neubauer Ruling, which has a worldwide reputation in hospitals an laboratories for unmatched reliability, meeting the most demanding of standards. The standard Hausser blood counting chambers are one piece construction (measuring 75mmX32mmX4.5mm) ensuring long term durability and absolute accuracy in measurement and count.

Reagents and equipment:

Ammonium oxalate 10g/l diluting fluid, Neubauer Counting Chamber, microscope, tubes (glass tubes).

Procedure:

Platelet count would be done within two hours of collection of blood.

0.38ml of filtered ammonium oxalate dilution of blood.

1.      0.38ml of filtered ammonium oxalate diluting fluid was obtained and dispensed into a small container or tube.

2.      20 µl of the well – mixed anticoagulanted venous blood was pipette and released into the tube containing the diluting fluid.

3.      The chamber was assembled and filled with the well – mixed sample after which it was left undisturbed for 2 minutes. The chamber will be placed in a plastic container on a blotting paper and covered with a lid.

4.       The underside of the chamber was dried and placed on a microscope stage. Using the 10X objective, the rulings of the grid was focused and the central square of the chamber was brought to view. The objective was changed to the 40X objective and the small platelets were focused. The number of platelets counted was reported in the litres of blood, thus the actual number of platelets counted X10⁹.

APPENDIX II: RESULTS

Table 1: Population Size
		Frequency	Percent	Valid Percent	Cumulative Percent
Valid	Diabetics	209	74.9	75.5	75.5
	non diabetics	68	24.4	24.5	100.0
	Total	277	99.3	100.0
Missing	System	2	.7
Total		279	100.0

Table 2: Sex of Diabetics
		Frequency	Percent	Valid Percent	Cumulative Percent
Valid	Male	51	24.4	24.4	24.4
	Female	158	75.6	75.6	100.0
	Total	209	100.0	100.0

Table 3: Age Groups for Diabetics
		Frequency	Percent	Valid Percent	Cumulative Percent
Valid	below 35	10	4.8	4.8	4.8
	35 to 45	23	11.0	11.0	15.8
	46 to 55	62	29.7	29.7	45.5
	56 to 65	70	33.5	33.5	78.9
	66 to 75	36	17.2	17.2	96.2
	above 75	8	3.8	3.8	100.0
	Total	209	100.0	100.0

Table 4: Category of Clotting for Diabetics
		Frequency	Percent	Valid Percent	Cumulative Percent
Valid	Lesser than normal	13	6.2	6.2	6.2
	Normal	196	93.8	93.8	100.0
	Total	209	100.0	100.0

Table 5: Category of Platelet for Diabetics
		Frequency	Percent	Valid Percent	Cumulative Percent
Valid	Thrombopenia	31	14.8	14.8	14.8
	Normal	174	83.3	83.3	98.1
	Thrombosis	4	1.9	1.9	100.0
	Total	209	100.0	100.0

Table 6: Category of FBS for Diabetics
		Frequency	Percent	Valid Percent	Cumulative Percent
Valid	Hypoglycemia	5	2.4	2.4	2.4
	Normal	78	37.3	37.3	39.7
	Hyperglycemia	126	60.3	60.3	100.0
	Total	209	100.0	100.0

Table 7: Statistics of Non – Diabetics
		Age Group	Sex of control subject	Category of clotting time	Category of platelets
N	Valid	68	68	68	68
	Missing	0	0	0	0
Mean		1.78	1.62	2.00	2.16
Median		1.00	2.00	2.00	2.00
Mode		1	2	2	2
Std. Deviation		1.325	.490	.000	.371

Table 8: Age Group of non – Diabetics
		Frequency	Percent	Valid Percent	Cumulative Percent
Valid	below 35	41	60.3	60.3	60.3
	35 to 45	17	25.0	25.0	85.3
	46 to 55	2	2.9	2.9	88.2
	56 to 65	3	4.4	4.4	92.6
	66 to 75	2	2.9	2.9	95.6
	above 75	3	4.4	4.4	100.0
	Total	68	100.0	100.0

Table 9: Sex of non – Diabetics
		Frequency	Percent	Valid Percent	Cumulative Percent
Valid	male	26	38.2	38.2	38.2
	female	42	61.8	61.8	100.0
	Total	68	100.0	100.0

Table 10: Category of clotting time for non – Diabetics
		Frequency	Percent	Valid Percent	Cumulative Percent
Valid	Normal	68	100.0	100.0	100.0

Table 11: Category of platelets for non – Diabetics
		Frequency	Percent	Valid Percent	Cumulative Percent
Valid	Normal	57	83.8	83.8	83.8
	Thrombosis	11	16.2	16.2	100.0
	Total	68	100.0	100.0

Table 12a: Descriptive of the population subjects
		N	Mean	Std. Deviation	Std. Error	95% Confidence Interval for Mean		Minimum	Maximum
						Lower Bound	Upper Bound
platelets level	Diabetics	209	232.52	88.197	6.101	220.49	244.54	49	743
	non diabetics	68	297.19	99.414	12.056	273.13	321.25	151	586
	Total	277	248.39	95.074	5.712	237.15	259.64	49	743
clotting time	Diabetics	209	7.3100	1.61318	.11159	7.0901	7.5300	3.56	11.18
	non diabetics	68	9.3050	1.33110	.16142	8.9828	9.6272	6.33	12.45
	Total	277	7.7998	1.76950	.10632	7.5905	8.0091	3.56	12.45

Table 12b: ANOVA between the Diabetics and Non – diabetics
		Sum of Squares	df	Mean Square	F	Sig.
platelets level	Between Groups	214605.402	1	214605.402	25.883	.000
	Within Groups	2280154.706	275	8291.472
	Total	2494760.108	276
clotting time	Between Groups	204.193	1	204.193	85.080	.000
	Within Groups	660.004	275	2.400
	Total	864.197	276

Table 13a:  Descriptives of the sexes in the populated sample
		N	Mean	Std. Deviation	Std. Error	95% Confidence Interval for Mean		Minimum	Maximum
						Lower Bound	Upper Bound
Platelets	male	51	2.2657E2	125.33096	17.54985	191.3187	261.8185	63.00	743.00
	female	158	2.3444E2	72.71815	5.78515	223.0100	245.8635	49.00	434.00
	Total	209	2.3252E2	88.19734	6.10074	220.4895	244.5440	49.00	743.00
Clotting time	male	51	6.9367	1.69374	.23717	6.4603	7.4130	4.06	11.00
	female	158	7.4306	1.57296	.12514	7.1834	7.6777	3.56	11.18
	Total	209	7.3100	1.61318	.11159	7.0901	7.5300	3.56	11.18

Table 13b: ANOVA between diabetics males and females
		Sum of Squares	df	Mean Square	F	Sig.
Platelets	Between Groups	2386.814	1	2386.814	.306	.581
	Within Groups	1615597.377	207	7804.818
	Total	1617984.191	208
Clotting time	Between Groups	9.405	1	9.405	3.660	.057
	Within Groups	531.886	207	2.569
	Total	541.291	208

Table 14a: Descriptives of the age groups of the populated sample
		N	Mean	Std. Deviation	Std. Error	95% Confidence Interval for Mean		Minimum	Maximum
						Lower Bound	Upper Bound
Platelets	Below 35	10	2.3030E2	57.51145	18.18672	189.1588	271.4412	115.00	304.00
	35-45	23	2.1683E2	66.13392	13.78988	188.2276	245.4245	61.00	351.00
	46-55	62	2.4524E2	119.93478	15.23173	214.7842	275.6997	49.00	743.00
	56-65	70	2.3476E2	74.27864	8.87800	217.0460	252.4683	78.00	412.00
	66-75	36	2.2350E2	72.37620	12.06270	199.0114	247.9886	59.00	384.00
	Above 75	8	2.0275E2	59.03449	20.87185	153.3959	252.1041	135.00	284.00
	Total	209	2.3252E2	88.19734	6.10074	220.4895	244.5440	49.00	743.00
Clotting time	Below 35	10	7.4480	1.30633	.41310	6.5135	8.3825	6.06	10.18
	35-45	23	7.5796	1.58122	.32971	6.8958	8.2633	4.31	10.56
	46-55	62	7.0465	1.55974	.19809	6.6504	7.4426	3.56	9.47
	56-65	70	7.5611	1.59827	.19103	7.1800	7.9422	4.05	11.18
	66-75	36	7.1197	1.77850	.29642	6.5180	7.7215	4.06	10.41
	Above 75	8	7.0650	1.82027	.64356	5.5432	8.5868	4.20	10.14
	Total	209	7.3100	1.61318	.11159	7.0901	7.5300	3.56	11.18

Table 14b: ANOVA among the age groups
		Sum of Squares	df	Mean Square	F	Sig.
Platelets	Between Groups	26118.045	5	5223.609	.666	.650
	Within Groups	1591866.147	203	7841.705
	Total	1617984.191	208
Clotting time	Between Groups	12.367	5	2.473	.949	.450
	Within Groups	528.924	203	2.606
	Total	541.291	208

Table 15: ANOVA among the sugar level in diabetics
		Sum of Squares	df	Mean Square	F	Sig.
Clotting Time	Between Groups	3.735	2	1.868	.716	.490
	Within Groups	537.556	206	2.609
	Total	541.291	208
Platelet (x10^9)	Between Groups	9520.847	2	4760.424	.610	.545
	Within Groups	1608463.344	206	7808.074
	Total	1617984.191	208

Table 16a: Descriptives between the males and female Clotting Time and platelet Count
		N	Mean	Std. Deviation	Std. Error	95% Confidence Interval for Mean		Minimum	Maximum
						Lower Bound	Upper Bound
Category of Clotting	Male	51	1.90	.300	.042	1.82	1.99	1	2
	Female	158	1.95	.220	.017	1.91	1.98	1	2
	Total	209	1.94	.242	.017	1.90	1.97	1	2
Category of Platelet	Male	51	1.80	.491	.069	1.67	1.94	1	3
	Female	158	1.89	.349	.028	1.84	1.95	1	3
	Total	209	1.87	.389	.027	1.82	1.92	1	3

Table 18b: ANOVA among the sexes with Diabetes
		Sum of Squares	df	Mean Square	F	Sig.
Category of Clotting	Between Groups	.087	1	.087	1.482	.225
	Within Groups	12.105	207	.058
	Total	12.191	208
Category of Platelet	Between Groups	.302	1	.302	2.002	.159
	Within Groups	31.210	207	.151
	Total	31.512	208