A CROSS SECTIONAL STUDY OF ANTIPHOSPHOLIPID LEVELS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS IN ENUGU, SOUTH-EAST NIGERIA.

BY

DR. OBINECHE AGWU DEPARTMENT OF HAEMATOLOGY AND IMMUNOLOGY, UNIVERSITY OF NIGERIA TEACHING HOSPITAL (UNTH), ITUKU-OZALLA ENUGU STATE, NIGERIA.

A DISSERTATION SUBMITTED TO THE NATIONAL POST-GRADUATE MEDICAL COLLEGE OF NIGERIA IN PART FULFILMENT OF THE FMCPATH EXAMINATION REQUIREMENT

NOVEMBER 2017

DEPARTMENT OF HAEMATOLOGY AND IMMUNOLOGY UNIVERSITY OF NIGERIA TEACHING HOSIPAL (UNTH), ITUKU – OZALLA, ENUGU, NIGERIA.

TOPIC: A CROSS SECTIONAL STUDY OF ANTIPHOSOPHOLIPID ANTIBODY LEVELS IN PATIENTS WITH TYPE 2 DIABETES MELLITUS IN ENUGU, SOUTH-EAST, NIGERIA.

BEING A DISSERTATION SUBMITTED TO THE NATIONAL POST – GRADUATE MEDICAL COLLEGE OF NIGERIA, IN PART FULFILMENT OF THE REQUIREMENT OF THE FMCPATH, EXAMINATION FACULTY OF PATHOLOGY.

BY DR OBINECHE AGWU AF/008/02/004/455 SUPERVISOR: DR OBIKE G IBEGBULAM. FWACP (Lab MED) CONSULTANT HAEMATOLOGIST, UNTH ITUKU – OZALLA, ENUGU.

CO-SUPERVISOR: DR THERESA U. NWAGHA (MPH, FMCPath) CONSULTANT HAEMATOLOGIST, UNTH ITUKU – OZALLA, ENUGU.

DECLARATION

I, Dr Obineche Agwu, do declare that this work is original. The work has neither in whole nor in part been presented to any other college for fellowship or submitted elsewhere for publication.

………………………………………… DR OBINECHE AGWU

CERTIFICATION BY THE SUPERVISING CONSULTANTS

We certify that this dissertation was done by Dr Obineche Agwu under our supervision. We have also supervised the writing of this dissertation, in keeping with the requirement of the Faculty of Pathology, National Postgraduate Medical College of Nigeria.

……………………...... …………………………...... DR OBIKE G IBEGBULAM. FWACP (Lab MED) DR THERESA U. NWAGHA (MPH, FMCPath) CONSULTANT HAEMATOLOGIST, UNTH CONSULTANT HAEMATOLOGIST, UNTH ITUKU – OZALLA, ENUGU. ITUKU – OZALLA, ENUGU.

DEDICATION

This dissertation is dedicated to God Almighty who made everything about this work possible and, to my father –Late Johnson N. Agwu.

ACKNOWLEDGEMENT

I wish to express my profound gratitude to my supervising consultants; Dr Obike G Ibegbulam and Dr Theresa U Nwagha for their immeasurable guidance, encouragement and contribution during the course of this dissertation and to other consultants in my department namely: Prof Iheanyi Okpala, Dr Sunday Ocheni and Dr Madu A.

I am greatly indebted to Dr Chuku Abali (Medical Director, Federal Medical Center (FMC), Umuahia) and the entire management of FMC, Umuahia and my wife, Mrs Johnson-Obineche N.Stella. I am also indebted to my children; Johnson- Obineche Nkenna Success and Chukwuemeka Destiny. My beloved mother, Nneoma Roseline J N. Agwu deserves acknowledgement for her concern. My co- residents are also acknowledged and appreciated.

Finally I wish to praise and glorify God, who is the source of all good things.

………………………………………… DR OBINECHE AGWU

TABLE OF CONTENTS:

Declaration……………………………………………………. iv

Certification by supervising consultant………………………. v

Dedication……………………………….……………………. vi

Acknowledgment……………………………………………... vii

Glossary……………………………………………………….. vx

Summary……………………………………………………….. xvii

Chapter One- Introduction.………………………………… 1

Rational of study……………………………………………… 5

Chapter Two- Literature review……………………………. 6

The historical background of Antiphospholipid (APLS) 6

The Prevalence of Antiphospholipid Antibodies (APLS)……... 6

The Aetiopathogenesis of Antiphospholipid Antibodies (APLS) ...... 8

Diagnosis of Antiphospholipid Antibodies (APLS) and Antiphospholipid Syndrome (APS)………………………...... 14

Management of Antiphospholipid Syndrome (APS)...... 15 Antiphospholipid Antibodies (APLS) and Type- 2-diabetes mellitus (T2DM)………………………………………………………... 16

Antiphospholipid Antibodies (APLS), and other Disease conditions…………………………………………………….. 20

Chapter Three- Aim and Objectives of the study…………. 23

Chapter Four- Subjects and Methods………………………… 24

Study area………………………………………………………. 24

Study design…………………..………………………………… 24

Study population……………..………………………………… 25

Sample size………………………………………………….…. 25

Selection criteria………………………………………………. 25

Ethical and consent considerations………………………….… 26

Laboratory investigations………………………..….…………. 26

Sample collection………………………………..……....…….. 27

Handling and transportation of specimen……………..……….. 28

Preparing and storage of sample……………………………….. 29

Procedures …………………………………………………….. 29

Lupus (LA) assay…………………………..….. 29

Interpretation of LA result…………………………………… 33

IgG- β2GP1- dependent-ACA assay…………………………. 34

Interpretation of IgG- β2GP1- dependent-ACA result……… 35

Statistical analysis…………………………………………. 36

Chapter Five- results……………………………………. 37

General study characteristics of respondents……………… 37

Socio- demographic characteristics of respondents………… 39

Some relevant medical evaluation of respondents……………… 41

Prevalence of Antiphospholipid Antibodies of respondents 42

The comparison of LA of respondents…………… 44

IgG-β2GP1-dependent-ACA of respondents...... 45

Distribution of vascular complications in T2DM subjects. 46

Relationship/association between LA and vascular complication in T2DM. 47

Relationship/association between 1gG-ACA and vascular complication in T2DM 48 The mean count among respondents 49

The mean glycated haemoglobin (HbA1C) among respondent 50 Correlations between Antiphospholipid Antibodies, Platelet count and Glycated haemoglobin 1C (HbA1C) among respondents…………… 52

Chapter Six…………………………………………………. 62

Discussion ……………………………………………………. 62

Limitation of this study……………………………………….. 65

Conclusion…………………………………………………….. 66

Recommendation…………………………………………….. 66

References…………………………………………………… 67

Appendix I…………………………………………………... 83

Information forms……………………………… 83

Appendix II...... 85

Consent form……………………………………. 85

Appendix III…………………………………………………… 86

Questionnaire…………………………………… 86

Appendix IVa 89 Raw data for social demographic characteristics for T2DM with complications 89

Appendix IVb 92 Raw data for social demographic characteristics for T2DM without complications 92

Appendix IVc 96 Raw data for social demographic characteristics for healthy control subjects 96

Appendix Vai 99 Raw data for LA among T2DM with complication (first time) 99

Appendix Vaii 102 Raw data for LA among T2DM with complication (12 weeks) 102

Appendix Vbi 103 Raw data for IgG-β2GP1-ACA T2DM with complication (first time) 103

Appendix Vbii 105 Raw data for IgG-β2GP1-ACA T2DM with complication (12 weeks) 105

Appendix VIai 106 Raw data for LA among T2DM without complication (first time) 106

Appendix VIaii 109 Raw data for LA among T2DM without complication (12 weeks) 109

Appendix VIbi 110 Raw data for IgG-β2GP1-ACA T2DM without complication (first time) 110

Appendix VIbii 112 Raw data for IgG-β2GP1-ACA T2DM without complication (12 weeks) 112

Appendix VIIai 113 Raw data for LA among healthy control subjects 113

Appendix VIIbi 116 Raw data for IgG-β2GP1-ACA among healthy control subjects 116

Appendix VIIIai 118 Raw data for platelet counts for T2DM with complication (first time) 118

Appendix VIIIaii 120 Raw data for platelet counts for T2DM with complication (12 weeks) 120

Appendix VIIIbi 121 Raw data for platelet counts for T2DM without complication (first time) 121

Appendix VIIIbii 123 Raw data for platelet counts for T2DM without complication (12 weeek) 123

Appendix VIIIc 124 Raw data for platelet counts for healthy control subjects 124

FIGURES AND TABLES

Figure 1: Dysregulation of the protein C activation, with the resultant APC- Resistant (APC-R) effect……………………………… 9

Figure 2: Antibody mediated disruption of the Annexin- V antithrombotic shielding inhibitory effect……….....………… 11

Figure 3: cascade, showing the point of action of RVV 31

Table 1: Socio-demographics of respondents………………… 38

Table 2: Some relevant medical history of respondents……… 40

Table 3: Prevalence of APLS 42

Table 4: Comparison of LA of respondents 43 Table 5:IgG – ACA of respondents 44 Table 6: Distribution of Antiphospholipid Antibodies of respondents 45

Table 7: Relationship/association between LA and vascular complication in T2DM 47

Table 8: Relationship/association between 1gG-dependent-β2-GP1-ACA and vascular complication in T2DM 48

Table 9: The mean platelet count among respondents. 49

Table 10: The mean glycated haemoglobin (HbA1C) among respondents 50

Table 11: Correlation between APLS, platelet count and glycated haemoglobin. 51

Figure A1: LA confirmatory average time (AveT) versus platelet count in complicated T2DM 53

Figure A2: IgG-β2GPl ACA versus platelet count in complicated T2DM 54

Figure A3: Glycated haemoglobin 1C (HbA1C) versus platelet count in complicated T2DM 55

Figure B1: LA confirmatory average time (AveT) versus platelet count in uncomplicated T2DM 56

Figure B2: IgG-β2GPl ACA versus platelet count in uncomplicated T2DM 57

Figure B3: Glycated haemoglobin 1C (HbA1C) versus platelet count in uncomplicated T2DM 58

Figure C1: LA confirmatory average time (AveT) versus platelet count in healthy control 59

Figure C2: IgG-β2GPl ACA versus platelet count in healthy control 60

Figure C3: Glycated haemoglobin 1C (HbA1C) versus platelet count in healthy control 61

GLOSSARY

aβ2GP1 -- Anti – Beta 2 Glycoprotein 1 antibody

ACA -- Anticardiolipin antibody

APS -- Antiphospholipid Syndrome

APLS -- Antiphospholipid antibodies

APTT -- Activated Partial Thromboplastin Time

β2GP1 -- Beta 2 Glycoprotein 1

CF -- Calibrator Factor

DM -- Diabetes Mellitus

DRVVT -- Dilute Russell’s viper Venom Time

EDTA -- Ethylene diamine tetra acetic acid

ELISA -- – Linked immunosorbent assay

HbA1C -- Glycated Haemoglobin 1C

Ig -- Immunoglobulin

IgA -- Immunoglobulin A

IgG -- Immunoglobulin G

IgM -- Immunoglobulin M

INR -- International Normalized Ratio.

ISTH -- International Society of Thrombosis and Haemostasis

KCT -- Kaolin Clotting Time

LA --

NGO -- Non – Governmental Organisation

NIDD -- Non – Insulin Dependent Diabetes

OD -- Optic Density

PL -- Phospholipid

PPP -- Platelet Poor Plasma

TIA -- Transient Ischaemic Attack

T1DM -- Type 1 Diabetes Mellitus

T2DM -- Type 2 Diabetes Mellitus

UNTH -- University of Nigeria Teaching Hospital

CI -- Coefficient Interval

SD -- Standard Deviation

SUMMARY

BACKGROUND: Antiphospholipid antibodies (APLS) are group of acquired, heterogeneous, autoimmune antibodies that bind to phospholipid(s), phospholipid – binding proteins or both. Antiphospholipid antibodies are associated with thrombosis (arterial, venous or both). Type 2 – diabetes mellitus is the commonest type of diabetes mellitus, characterised by hyper-glycaemia due to relative insulin deficiency or insulin resistance, or both. Type 2 – diabetes mellitus predisposes to arterial thrombosis, with wide range of ischaemic thrombosis, with the manifestation of TIA and stroke, nephropathy, retinopathy, neuropathy, platelet abnormalities, hypertension and other complications.

OBJECTIVE: To detect and assess the prevalence of antiphospholipid antibodies in subjects with type 2 diabetes mellitus. METHODOGY: This was a cross –sectional study carried out on subjects with T2DM attending the diabetic clinic of University of Nigeria Teaching Hospital (UNTH), Ituku – Ozalla, Enugu, South – East Nigeria. The healthy control subjects were recruited from consented blood donors, in the blood transfusion unit of Department of Haematology and Immunology of UNTH; medical students and hospital (UNTH) workers. Ethical clearance was obtained from the ethical committee of UNTH. Two hundred and ten (210) subjects were recruited for this study. They were grouped into three (complicated T2DM, uncomplicated T2DM and healthy control) and, each had 70 subjects, matched for sex and age between complicated T2DM and uncomplicated T2DM. The (LA) was assayed using commercially Dilute Russell’s Viper Venom Time (DRVVT) reagent for the screening and confirmatory tests, manufactured by Technoclone GmbH, Austria, Vienna. The IgG –β2GP1 – dependent – Anticardiolipin antibody (ACA), was assayed using ELISA test kit (Anticardiolipin 1gG ELISA kit GWB – 521211, by GenWay Bio-tech, San Diego, USA). The other laboratory tests done apart from LA and IgG-β2GP1-dependent-ACA, included platelet count using TM Haematology Analyser-Mythic 22; glycated haemoglobin 1C (HbA1C) using the D-10 Haemoglobin Testing System Analyser. RESULTS: The Lupus anticoagulant (LA) showed a prevalence of 7.1% in complicated T2DM, 4.3% in uncomplicated T2DM and healthy control subjects respectively, while prevalence of IgG-β2GP1-ACA was 4.3% in all the groups. Analysis of Variance (ANOVA), showed significant statistical difference in the mean platelet count for complicated T2DM 208.5(52.30) and healthy control subjects 277.5(23.90), (F=77.993, P<0.001). There was significant difference in mean glycated haemoglobin 1C (HbA1C) between complicated T2DM and healthy control subjects (p<0.001, 95% CI: -3.0 to -2.1) and in uncomplicated T2DM and healthy control subjects (p<0.001, 95% CI:-2.8 to - 2.0), using Post Hoc statistical analysis. Pearsons statistical analysis, showed significant correlation for APLS and platelet count in complicated T2DM subjects (r=0.316, P=0.008).

CONCLUSION Lupus anticogulants (LA) were found in both subjects and controls, but the prevalence was significantly higher than in controls, only in subjects with complicated T2DM.

Anticardiolipin antibodies were found and were equally prevalent in both T2DM subjects and in normal controls.HbA1C levels were found to be significantly higher in subject with complicated T2DM. Significantly lower platelet counts were found in subjects with complicated and uncomplicated T2DM , compared to normal controls. There was no significant correlation found between prevalent of LA or ACA and platelet counts in

T2DM. However, there was significant correlation between HbA1C levels and platelet counts in both complicated and uncomplicated T2DM. There was no significant association between LA or ACA prevalence and occurrence of vascular or other complications in T2DM. In view of the findings in this study, presence of antiphospholipid antibodies may not caused by, or directly related to the T2DM, and cannot be regarded as a complication of T2DM.

RECOMMENDATION In addition to the usual biochemical tests, regular evaluation of HbA1C levels and platelet counts should be done in the routine monitoring of patients with T2DM. Since

APLS may be associated with vascular thrombosis, as may T2DM. However, regular evaluation of APLS may not be required in the management, or routine monitoring of patients with T2DM. Although, there is no causal or significant statistical association of

APLS and the risk of T2DM patients developing thrombosis was observed in this study, there is need for multicentre robust studies to evaluate the relationship of APLS and thrombosis in persons with complicated T2DM in our environment.

CHAPTER ONE INTRODUCTION Antiphospholipid antibodies (APLS) are a group of acquired, heterogeneous, autoimmune antibodies that react with negatively charged phospholipid (s) (PL(s)) or 1-4 proteins associated with phospholipids or both. Beta - 2 - glycoprotein 1 (β2GP1), prothrombin and annexin V are the most characterized antigenic targets5-7. Others include activated protein C, and protein S8-10. The most important APLS in thrombosis include lupus anticoagulants (LA), anticardiolipin antibodies (ACA), anti-Beta -2- glycoprotein 1 (aβ2GP1) antibodies and anti-Annexin V antibodies10-12.

Antiphospholipid antibodies (e.g. LA, ACA and aβ2GP1) have immunoglobulin G, M and A subtypes12,13. Immunoglobulin G isotype is the most sensitive in ischaemic thrombosis14-16. LA is associated mainly with ischaemic thrombosis in -vivo but in- vitro, it prolongs phospholipid dependent coagulation (clot-based) assays. Antiphospholipid antibodies can be transient as seen in infectious conditions (e.g. malaria, hepatitis C) and some drugs (e.g. fansidar, propranolol, hydralazine, chlorpromazine), it may also be persistent10,17. For example, dilute (dPT)18, activated partial thromboplastin time (APTT)19-21 and kaolin clotting time (KCT)22,23, may be used to screen and / or detect LA, however, the preferred method is dilute Russell’s Viper Venom 12. Time (DRVVT) test or LA sensitive APTT Anticardiolipin antibodies and aβ2GP1 can be identified by immunoassays (e.g the enzyme – linked immunosorbent assay (ELISA) or Western and dot blot assay)24 but preferably, the enzyme – linked immunosorbent 12. assays (ELISA) The combined positivity of LA, ACA, and aβ2GPI is associated with the highest risk of ischaemic thrombosis12.

Antiphospholipid antibodies have been associated with varying clinical features (arterial, venous or both) of thrombosis3,5,12 that affect virtually every tissue and organ of the body. Antiphospholipid antibodies (APLS) are associated with various clinical manifestations including thrombocytopenia, diabetes mellitus, adrenal insufficiency, nephropathy, neuropathy, hypertension, and retinopathy3,5,7,25,26. Other features include myocardial infarction, transient ischaemic attack (TIA), stroke, and pregnancy morbidities and mortalities27-29.

The persistent level of APLS in association with clinical features describe the phenomenon of antiphospholipid syndrome (APS)30, which is diagnosed by the presence of a clinical feature and one or more of the APLS persisting on ≥ 2 occasions at least 12 weeks apart12,31.

Insulin is produced by the Beta cells of the Islet of Langerhan in the . Diabetes mellitus (DM) is a metabolic disorder characterised by hyperglycaemia due to relative or absolute insulin deficiency or both32. Clinical manifestations include polyuria, polydipsia, and polyphagia among others. Diabetes mellitus (DM) has four types32. The type 1

Diabetes Mellitus (T1DM) is associated with absolute insulin deficiency. It accounts for < 10% of the DM and commoner in the younger age group. The type 2 Diabetes Mellitus

(T2DM) is another important type of DM. It is due to relative insulin deficiency or insulin resistance or both. It is the most common type33. At present the global burden of DM is 34. centred on T2DM, mostly in those from 20-79 years of age Other types of DM are the gestational diabetes mellitus, which is noticed during pregnancy, and the group of DM due to specific causes (e.g drugs, chemicals, genetic defects in insulin action and disease of the exocrine pancreas). Diabetes mellitus is characterised by abnormal plasma glucose levels. It is diagnosed by fasting plasma glucose ≥ 126mg/dl (≥ 7.1mmol/l) on more than one occasion, or random / casual plasma glucose of ≥ 200mg/dl (≥ 11.1mmol/l) with symptoms or 2 hour post glucose load with 75gm glucose in 250ml water ≥ 200mg/dl 32,35. (≥ 11.1mmol/l ), or glycated haemoglobin 1C (HbA1C), ≥ 6.5% Diabetes Mellitus has been associated with hypercoagulable and prothrombotic state36. It is associated with acute and chronic complications. These include diabetes ketosis, coronary artery and peripheral vascular disease, stroke, diabetic neuropathy, diabetic nephropathy, retinopathy, frequent infections, gangrene which may result in amputations, renal failure requiring dialysis, hypertension, pregnancy complications, transient ischaemic attack (TIA), cognitive dysfunction and dementia. It has been estimated that 40% and 60% of DM patients will die of kidney and cardiovascular /diseases respectively33. Type 2 diabetes mellitus (T2DM), a subtype of diabetes mellitus (DM) is the commonest type and accounting for greater than 85% of cases33. It is insidious in onset, affects both the young and adults, although the incidence increases with age. It is associated with obesity, positive family history of DM and a long latent asymptomatic period. Newly referred cases already have evidence of micro-vascular (retinopathy, neuropathy and 37. nephropathy) complications Colwell reported that T2DM is a disorder with an increased pro-coagulant state38. Adediran et al39 showed evidence of thrombotic events in these subjects. T2DM has also been associated with wide range of ischaemic thrombosis with manifestation of TIA and stroke, nephropathy, retinopathy, neuropathy, platelet abnormalities, hypertension, and other cardiovascular complications40-47.

Although, there is no established pathophysiology for APLS in predisposing to thrombosis, several hypotheses have been made.

These included: the impairment of interaction between anticoagulant factors such as protein C and the phosphatidylserine (PS) surface, with the resultant activated protein C (APC) resistance formation, that has poor anticoagulation activity and subsequent tendency to fibrin formation that is resistance to fibrinolysis, and also prone to the formation of thrombosis; annexin A5 antithrombotic shielding inhibition effect, through the binding of APLS to the annexin A5 that result in the inhibition of annexin A5 anticoagulant role on the vascular endothelial cells of the placenta, thus exposing the thrombogenic phospholipid to blood proteins in circulation that are capable of forming thrombosis, ischaemia on the trophoblasts of the placenta with the resultant effect of foetal loss, and still birth; dysregulated activation of and endothelial cells, with the resultant formation of endothelial and platelets-microparticles (EMP) which procoagulant activity could predispose to thrombosis formation; and the complement activation that results in complement activation products that are thrombogenic in activity as observed in APS individuals48-56.

There are impairment of fibrinolysis as a result of annexin A2 receptor forming plasmin with endothelial plasminogen. The binding of APLS to the annexin A2 results in the inhibitional effect to forming plasmin, with the resultant clot formation that is resistant to fibrinolysis in APS subjects; antiprothrombin antibodies specific pathway, whereby there is binding competition between activated protein C (APC) and APLS on the epitopes of phosphatidylserine (PS), resulting in the reduction in APC anticoagulant role and enhancement of thrombrosis and the inhibition of antithrombin pathway due to APLS binding to sulfate/heparan epitopes, that result in the inhibition of heparan sulfate endothelial anticoagulant function, which predispose to the formation of procoagulant state and thrombosis57-61.

Similarly, sustained hyperglycaemia can lead to a series of inter-related alteration that can cause evident endothelial dysfunction and vascular lesions in diabetic subjets62. Consequently, there are formation of advanced glycated end products (AGEs), which accumulate to cause endothelial injury; loss of endothelial natural anti-aggregating agents such as nitric oxide (NO) and prostacyclin, which in turn predisposes to adhesion, aggregation, hyper-coagulability and thrombotic states; glycation of protein molecules such as fibrinogen and fibrin which are resistant to fibrinolysis and enhancing thrombosis formation, and increased platelet reactivity via the non-enzymatic glycation of proteins on platelet surfaces, resulting in decrease platelet membrane fluidity and increased propensity to activation, secretion of intracellular organelles such as thrombin, thromboxane A2 and ADP with pro-aggregation and pro-coagulant activities that can cause thrombotic vascular complecations36,63-73.

RATIONALE FOR THE STUDY Antiphospholipid antibodies (APLS) have been implicated in vascular (arterial, venous or both) thrombosis3,5,12. Diabetes mellitus (DM), as a disease entity has been associated with hyper-coagulable and pro-thrombotic states36, with studies showing an increased 38,39. pro-coagulant state and thrombotic events in Type 2 Diabetes Mellitus (T2DM) Locally in Nigeria, studies have been done to show the association of APLS in healthy state19, pregnancy22,23 and some disease conditions such as Human-immunodeficiency virus,74 and Sickle Cell Anaemia (SCA)75.This study, therefore, seek to know if DM and APLS occur together in the same individual, the thrombotic sequelae of DM may be aggravated by APLS which also produces thrombosis. Thus, providing the much needed data as well as platform to recommend screening for APLS in T2DM. This will aid in proactive management of APLS and APS, as well as reducing the accompanying incidence of ischaemic thrombotic complications in T2DM.

CHAPTER TWO LITERATURE REVIEW

THE HISTORICAL BACKGROUND OF ANTIPHOSPHOLIPID ANTIBODIES. The first antiphospholipid antibody was detected in a patient with syphilis in 1906 with cardiolipin being the antigenic target76. Similarly, in 1952, Moore and Mohr detected lupus anticoagulant in a patient with syphilis25. Conley and Hartmann, in 1952 first detected the presence of circulating anticoagulant in two patients with systemic Lupus erythematosus (SLE)77. In 1963, these APLS were correlated with their clinical thrombosis78. Feinstein and Rappaport79, introduced in 1972, the term “Lupus anticoagulant’’ which was used to describe the presence of acquired circulating agent that inhibit in vitro coagulation test despite thrombotic property in vivo. The triad of lupus anticoagulant (LA), recurrent abortion and thrombotic episode was described by Hughes in 198380. Solid phase immuno- assay for anticardiolipin antibodies (ACA) detection was developed in 1983, and ACA detected was strongly associated with LA, and thrombosis81.

Thiagarajan at el82, were the first to describe the use of a modified Russell’s Viper Venom time (RVVT) for the detection of LA. Beta- 2 - glycoprotein 1 (β2GP1) and prothrombin cofactors for ACA binding to cardiolipin was discovered in the early 1990s. At present, studies are focusing on phospholipids binding proteins rather than phospholipids themselves82.

THE PREVALENCE OF ANTIPHOSPHOLIPID ANTIBODIES (APLS). Depending on the type of APLS assayed and method for detection, varying degrees of prevalence were observed. There are two main types of Antiphospholipid Syndrome (APS) namely:

1. The Primary APS: There is no cause. It is associated with permanent ischaemic thrombosis. The immunologlobulin G isotype of APLS usually predominant in the primary APS. 2. The Secondary APS: There is accompanying cause or predisposing factor. It is associated with transient ischaemic thrombosis. Immunologlobulin M or A isotype of APLS are detected in this type of APS

There is a rare but severe form of APS, called the catastrophic antiphospholipid syndrome, CAPS, (Asherson Syndrome)83.

CAPS occur in less than one percent of APS subjects. It involves both small and large blood vessels occlusion, unlike the primary and secondary APS that are associated with large blood vessels mainly. Triggering factors include- infection, trauma, neoplasia, anticoagulant withdrawal during pregnancy or peurperium, surgery and lupus flares. It is acute in onset (less than one week). It involves multiple organs or systems84 with ischaemic thrombosis.

Miesbach et al85 found a prevalence rate of 2% APLS in apparently normal adults, with only 0.2% having high titers. Primary APLS occurs equally in both sexes, while that of secondary form is more frequent in women86. Von LP et al87 reported a prevalence of 2% among healthy controls. Rand and Senzel, reported a range of 1 – 5% in apparently healthy population25. The prevalent rate of 4% for APLS in patients with cerebral infarction was reported by Hart et al88.

In Nigeria, Awodu OA et al reported a prevalent rate for APLS 4.3% in women with recurrent abortion89 and 8.0% in multiparious90 population of Nigerians. Antiphospholipid antibodies (APLS) prevalent rate of 2.9% was reported in Human immunodeficiency virus (HIV) patients by Ndakotsu MA et al74. Olayemi E et al75, studied Nigerian children with homozygous sickle cell disease and showed a prevalent rate of 1.8% for APLS while 4.8% in healthy Nigerian adults in Ibadan was reported by Kotila TR et al19.

THE AETIOPATHOGENESIS OF ANTIPHOSPHOLIPID ANTIBODIES. There is no known established cause and mechanism for APLS pathogenesis. Some factors have been associated with APLS formation such as: genetic factor – the human leucocyte antigen (HLA) , viruses (Human immunodeficiency virus, hepatitis C, parvovirus B19); bacteria (leprosy, syphilis, tuberculosis, post-streptococcus, rheumatic fever), parasitic infections (malaria, visceral leishmaniasis), drugs (quinine, amoxicillin, hydralazine, oral contraceptive, propranolol, chlorpromazine, chlorthiazide, alpha interferon, phenytoin), malignant neoplasia (lung, colon and kidney cancers, leukaemias, Hodgkin and non-Hodgkin lymphomas)91-93. Several hypotheses on the aetiopathogenesis of APLS have been proposed and suggested and they include:

Impairment of interaction between anticoagulant factors and the phosphatidylserine (PS) surface 1. Dysregulation of the protein C activation pathway: Protein C is activated (APC) by the complex formation on the endothelial cells surfaces of the thrombin- thrombomodulin. APC in the presence of cofactor (protein S) down regulates factors Va and Vlla on the surfaces of platelet, endothelial cells and monocytes etc. LA disrupt this path way by:  Inhibiting the formation of thrombin  Decreasing protein C activation by the thrombin– thrombomodulin complex Binding to activated factors V ( FVa ) and VIII ( FVIIIa) in a manner to inhibit their proteolysis by APC, thus causing APC Resistance ( APC–R ) and subsequent poor anticoagulation and thrombosis. This is illustrated below68. Thrombomodulin PC = Protein C

Thrombin APC = Activated Protein C LA = Lupus anticoagulant PLT= Platelet .

Figure 1: DysregulationPLT = Platelet of the protein C activation, with the resultant APC … 48. Resistance (APC-R), effect … . . Annexin A5 antithrombotic shielding inhibitory effect.

Annexin A5, an anionic phospholipid-binding protein with potent anticoagulant activity in maintaining placenta integrity with possible thromboregulatory role at the vascular-blood interface through the formation of two dimentional crystals that shield anionic phospholipids from complexing with coagulation proteins in circulation. Antiphospholipids antibodies (APLS) and anti-β2 – GP1 / β2 – GP1 complex disturb the protective anticoagulant shielding on the vascular cells by annexin A5 / phospholipid (PL) complex by increasing the quantity of thrombogenic phospholipids exposed to the circulating blood proteins resulting in thrombosis and ischaemia on the trophoblast of the placenta and subsequent foetal loss. See the illustrated diagramme below49,50 (A and C normal but B and D abnormal)

Figure 2: Antibody mediated disruption of the annexin-V antithrombotic shielding effect49,50.

Dysregulated activation of platelet and endothelial cells

The submaximal pre -stimulation of platelets by agonist (thrombin or collagen) in vitro, exposes the phosphatidylserine (PS) to the outer surface membrane51,52. It is suggested that the anti- β2-GP1 Ab / β2 – GP1 complex has to initially form on the exposed PS, before interacting with specific platelet receptor, to enhance further activation52. Annexin

A2 has been suggested to interact with anti- β2-GP1 Ab / β2 – GP1 complex on the

53 endothelial cells surface, for further activation . β2-GP1 has been demonstrated in vitro to bind non-stimulated endothelial cells ,which enhances anti- β2-GP1 antibodies to bind the cells and to induce a procoagulant and proinfammatory phenotypes.39 The endothelial cells is a target of APLS, which can stimulate a procoagulant and proinfammatory functions, such as production of endothelial micro particles (EMP), at injury or dysfunction. The finding of elevated EMP in circulation of APS patients which was also detected by a positive Dilute Russell’|s Vieper Venom Time (DRVVT), Supported the clinical evidence of endothelial dysfunction and thrombosis.40

Complement activation. Davis and Brey56 demonstrated the possible role of complement activation in APS pathogenesis with increased complement activation products in the plasma of patients with APS who have had a thrombotic event, as opposed to non – APS- related subjects.

Impairment of fibrinolysis.

The Annexin A2 receptor roles in co-localizing tissue- plasminogen- activator (t -PA) and plasminogen on endothelial cells, results in plasmin generation57. Subsequent in vivo receptor disturbance had been reported to be associated with poor clot dissolution57. This could be in agreement with the presence of APLS (Annexin A2 antibodies, tissue plasminogen activator antibodies and antiplasmin antibodies) in the APS plasma subjects. They may contribute to the impairment of fibrinolysis in APS individuals Antiprothrombin antibodies specific pathway. There is binding of low affinity of antiprothrombin antibodies to prothrombin in APS patients58. Suggested pathway involve antiprothrombin-prothrombin complex competing for the same epitopes of the phosphatidylserine (PS), with APC, thereby reducing the APC anticoagulant role, in APS patients,and predisposing to thrombosis 59. Oxidation of Low Density Lipoprotein (LDL).

The cross – reactivity of APLS (e.g. ACA) against oxidized low density lipoprotein may be associated with increased risk of atherosclerosis60.

Inhibition of Antithrombin pathway.

Heparan Sulfate (HS) is a normal endothelial surfsce modulator of normal anticoagulation that binds to antithrombin and heparin. Shibata et al61, hypothesized that APLS could bind to heparin sulphate/heparin epitopes, rsulting in the inhibition anticoagulation function of heparin, thus promoting a procoagulant state and thrombosis.

DIAGNOSIS OF ANTIPHOSPHOLIPID ANTIBODIES (APLS) AND ANTIPHOSPHOLIPID SYDROME (APS).

The diagnosis of APS is based on the international consensus statement on update for diagnosis of APLS and APS12,31. It is based on the presence of clinical related features and laboratory detection for APLS12,31.

Clinical criteria include:

1. Vascular Thrombosis: At least one clinical event of arterial, venous or small vessel thrombosis, occurring within any tissue or organ in the body. 2. Pregnancy Complications: At least one unexplained death of morphologically normal foetus ≥ 10 weeks of gestation OR at least one premature birth of morphologically normal neonate ≤ 34 weeks of gestation OR at least three unexplained consecutive spontaneous abortion ≤ 10 weeks of gestation.

Laboratory citeria include: 1. “Lupus anticoagulant (LA) detected in accordance to the guidelines of the International Society of Thrombosis and Haemostasis (ISTH) preferable using DRVVT or LA – sensitive APTT assay. 2. ACA of isotype IgG and /or IgM present in medium or higher titre measured by a standardized ELISA.

3. Anti-Beta 2–glycoprotein 1 (aβ2GP1) antibody of isotype IgG and /or IgM, measured by a standardized ELISA. Diagnosis of definitive Antiphospholipid Syndrome (APS) requires the presence of at least one clinical criterion and at least one laboratory criterion present on ≥ 2 occasions at least 12 weeks apart in accordance with ISTH12,.

MANAGEMENT OF ANTIPHOSPHOLIPI SYNDROME (APS)

The management of APS addresses four (4) areas: (1) Prevention of APS. (2) Treatment of APS. (3) Treatment of catastrophic APS. (4) Management of pregnancy associated with APLS. Prevention of APS by eliminating any factor predisposing to thrombosis (e.g. immobilization, oestrogen therapy) and to modify secondary risk factors for atherosclerosis. Hydroxychloroquine may be protective against thrombosis in patients with secondary APS. The treatment of thrombosis in APS is long term or lifelong anticoagulation using with a targeted INR 2.0-3.0. Heparin, antiplatelet and anti – inflammatory therapy e.g. interferon alpha (INF-α) may be used. In resistant cases, Rituximab (anti –CD20), hydroxychloroquine, thrombin inhibitors (e.g. lepirudin, ximelagatran), statins and even autologous haematopoietic stem cell transplant (HSCT) may be beneficial. The treatment of catastrophic APS (CAP) and those patients with INR >3.5 involves the use of heparin, steroid, plasmaspharesis, intravenous immunoglobulin, fibrinolytic agents or cyclophosphamide. The treatment of obstetric complication involves the use of heparin (low-molecular weight or unfractionated heparin) and anti- inflammatory therapy e.g. INF- α blocker91.

ANTIPHOSPHOLIPID ANTIBODIES (APLS) AND TYPE 2 DIABETES MELLITUS.

Hyperglycaemia is the biochemical diagnostic hallmark of diabetes mellitus (DM)94. Since the introduction of glycated haemoglobin A1C (HbA1c) in 1976, it has slowly become the cornerstone for screening and diagnosis of diabetes in routine clinical practice, assessing the degree of metabolic control in diabetic patients as well as in risk prediction of vascular complication, and has a normal range cut – off point of 94-96. 48mmol/mol (6.5%) HbA1c reflects average plasma glucose over the previous 8-12 weeks97.

32. Type 2 Diabetes Mellitus (T2DM) is the commonest type of DM Colwell reported that 38. 39, T2DM is a disorder with an increased procoagulant state While Adediran et al showed evidence of thrombotic events in T2DM subjects, in another study. Type 2 diabetes mellitus has been reported with varying clinical manifestations. Kolawole and colleagues recorded stroke with a higher death rate among hypertensive T2DM subjects, with male preponderance40. Cardiovascular disease has been reported to be a common complication in T2DM and accounted for 52% of morbidity and mortality of all deaths 98. from DM. Diabetic retinopathy accounted for 1.6% blindness among T2DM individuals Global data survey support that Diabetic retinopathy (DR) will be one of the important 99. cause of blindness in future Furthermore, studies using the sera from T2DM subjects have shown a significant correlation with diabetic neuropathy and greater induction of apoptosis, due to autoimmune immunoglobulin that induces complement-independent, calcium-dependent apoptosis in neural cells, as opposed to those patients with neuropathy

100. and control individuals, too

In diabetes mellitus, platelet hyper-activation is multifactorial, involving biochemical factors such as hyperglycaemia, hyperlipidaemia, insulin resistance, an inflammatory and oxidant state, increased expression of glycoprotein receptors and growth factors11,16-19. Hyperglycaemia can increase platelet reactivity by inducing non-enzymatic glycation of proteins on the surface of the platelet, which can result to decrease membrane fluidity and increases the propensity of platelets to activate16-18. In inflammatory state, there is generation of superoxide which limits the biologic activity of nitric oxide (NO) as a result of the oxidative stress impairment on endothelial function that reduces production of NO and prostacylin, thus increasing platelet reactivity, aggregation, thrombosis formation, and other mitogenic substances such as platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF) that accelerate progression of local vascular lesions such as neovascularization of lens in diabetic retinopathies16-18. Triolo G et al101, study of APLS, T2DM, and platelets observed the co-existence of APLS and platelets in

T2DM patients, resulting in thrombosis and also suggested that APLS may play a role in the reduction of the thromboresistant function of the vascular endothelium, hence propagating platelet aggregation that were associated with the pathogenesis of diabetic complications.

Similarly, hyperglycaemia under the contributing factors of increased reactive oxidant stress, low grade inflammation and hypertension causes endothelial injury, dysfunction 102-104. and platelet hyper-reactive and dysfunction in patients with T2DM Consequently, there is glycation of lipids, fibrinogen, platelet protein membrane, collagen and sub- endothelial cells and antithrombin (AT), resulting in the formation of advanced glycated end products (AGEs), which in turn may accumulate to cause structural and functional disturbance in the blood vessels, causing atherosclerotic complications 104-110. In addition, there are decreased endothelial natural anti-aggregating agents such as nitric oxide and prostacyclin, which in turn have decreased sensitivity to the hyper-reactive platelets95. There are decreased endothelial thrombomodulin (TM), with relative increase in plasma TM, which indirectly causes a reduction in the protein C natural anticoagulant pathway; increase in plasma adhesion molecules such as P- selectin which mediates platelet binding to increased vWF to form aggregation; increased in plasma thrombin – antithrombin complex, prothrombin fragment 1 and 2, micro-particles and fibrinogen

36, that contribute to the prothrombotic and hyper-coagulability states in T2DM subjects 110 -117.

Studies on T2DM subjects showed increased plasma plasminogen activator inhibitor – 1 (PAI – 1), which inhibited both tissue type and urokinase – type plasminogen activator (t – PA and u – PA) resulting in the generation of less plasma118. Again, plasminogen activator inhibitor – (PA1 – 1) from hyper-reactive platelets enhances the formation of 118,119. platelet rich clots that are resistance to fibrinolysis in T2DM subjects Furthermore, increased glycated fibrin clots that are resistant to fibrinolysis had been observed in 120. T2DM individuals In addition, these glycated fibrin clots bind less to both t – PA and plasminogen, generating less plasmin but bind more to alpha 2 – antiplasmin, which in turn acts directly on plasmin to impair its catalytic function, with the resultant effect of 118,121. hypofibrinolysis in T2DM subjects Erkan D and colleague reported the occurrence of medium or high APLS levels (> 40 units) in individuals with ischaemic thrombotic (arterial or venous) events in T2DM122. Mattila K et al123 and McCulled DK124 in separate studies reported a prevalent rate of 19.5% for anticardiolipin antibodies (ACA) in uncomplicated T2DM while apparently healthy control subjects had 4.6%. Levine et al76, using isotypes IgG 125, and IgA ACA found a prevalent rate of 51.0% in T2DM subjects. Rana et al attributed higher prevalence rate of ACA in T2DM to the pathogenesis and / or progression of 13, complication in T2DM. Shahim et al reported a prevalence rate of 17.7% for ACA with greater percentage due to the presence of IgG isotype in patients with retinopathy. Shahim and colleague study did not find any significant correlation in age, sex, duration 126, of T2DM or type of T2DM. Becarevic M et al reported a significant correlation between adinonectin concentration, IgM anti –annexin A5 and T2DM in the premature atherosclerosis without any micro- and / or macro-vascular complication. Calvo-Romero 127, and Lima-Rodriguez prospective study on 56 patients with T2DM with arterial thrombosis showed that 1.8% had a titre of IgM ACA > 15 MPL units. There was no titre of IgG ACA > 15 GPL units. There was also no significant difference in the IgM and / or IgG ACA titers in patients with complicated and uncomplicated DM with and without nephropathy and retinopathy as well as cardiovascular complications. Calvo-Romero and Lima-Rodriguez study likewise other studies mentioned above did not assay for ≥ 2 APLS as recommended by the ISTH12,31, as such this could have contributed to their study out come23. This reiterates the fact that assay of ≥ 2 APLS could result in accurate detection of APLS12,23,31. The study by Triolo G and colleague101, reported the highest precalence rate of 86% for APLS, in T2DM patients with macrovascular complication and suggested that APLS may play a significant role in the pathogenesis of the complication. Contrarily, Tarku I et al128 study, which aimed at evaluating the association between microvascular and macrovascular complication of diabetes and antiphospholipid antibodies, showed that although, diabetic patients with complication recorded significant higher titres of APLS levels than those without complication (P<0.001), however, none of the patients had APLS level higher than 20GPL, which was accepted as a clinically significant value. Thus, this association of APLS and T2DM complication may not be clinically important, nor associated with T2DM complication, and do not have a major role in the pathogenesis of T2DM complications. The need for multicentre studies to explore this relationship of APLS and T2DM in causing vascular thrombosis become very important and necessary.

ANTIPHOSPHOLIPID ANTIBODIES (APLS), PREGNANCY AND OTHER DISEASE CONDITIONS Antiphospholipid antibodies (APLS) are a group of acquired heterogeneous antibodies that react with phospholipid, proteins associated with phospholipids or both. They have been associated with vascular (arterial, venous or both) thrombotic complications. These affect virtually every other tissue or organ in the body. The detection of APLS involves the combination of ≥ 2 different principles and techniques of assays (e.g immunoassay and the clot- based assay) 23,31. Antiphospholipid antibodies (APLS) have been studied in some clinical conditions. Antiphospholipid syndrome (APS) in pregnant women in relation to APLS, may be associated with foetal loss, pre-eclampsia, retarded foetal growth, placental insufficiency, still birth, repeated miscarriages and maternal death due to venous thromboembolism129-133. This may be related to the APLS inhibiting placental anticoagulant protein by binding to phospholipids, thereby leading to thrombosis134. The beta 2 glycoprotein, a natural anticoagulant, act as a cofactor in the binding of APLS to phospholipids, thus any interference in this natural anticoagulant role, may lead to thrombosis135. Spegiorin FJCL et al136. Study observed abnormal high ACA levels in 55.77% of women and LA in 2% with history of repeated miscarriages. Thus, this should be seen as a warning about the necessity to investigate hypercoagulability states during pregnancy especially in respect to APLS. Kotila TR et al19, studied one hundred and twenty five healthy adult Nigerians. Their study had fifty one males and seventy four females, with twenty-six nulliparous females, twenty-five multiparous females and twenty pregnant women with no history of habitual abortion, infertility or eclampsia. A combination of APTT and KCT assays was used to investigate the prevalence of LA in this study. Lupus anticoagulant was found in six subjects constituting a prevalent rate of 4.8%, which was found in two males (1.6%), one pregnant woman (0.8%) and three multi-parous females (2.4%). Further study was done by Akinbami A et al23. However, this study on the prevalence of APLS in pregnant women, involving one hundred and thirty one pregnant women, using the combination of KCT and ELISA for semi- quantitation of IgG and IgM, was 9.3% with subjects for the KCT and 2.3% prevalent rate for the ELISA23. This study by Akinbami and colleagues showed that many patients are wrongly diagnosed for APLS, when only screening test (e.g APTT, KCT, DPT, DRVVT) is used in making diagnosis23.

Again, Awodu and colleagues studied seventy six pregnant women, with twenty six pre- eclampsia and fifty apparent healthy pregnant women, serving as control22. The Lupus anticoagulant (LA) detection using KCT was done. This study found four (15.4%) of the twenty six pre-eclamptic subjects positive for LA against one (2.0%) of the controlled group. Similarly, Awodu et al89, investigated LA, in seventy three subjects, constituting of twenty three pregnant women with history of ≥ 2 recurrent abortion or ≥ 2 intra – uterine feotal death in previous , and fifty pregnant women without the above mentioned obstetric complications serving as control. The KCT clot based technique was used for the screening, mixing experiment in subjects for prolonged clotting screening time, and confirmatory of LA, if KCT ratio is ≥ 1.2. This study showed that one of the twenty three subjects had subnormal KCT and KCT ratio ≥ 1.2, confirming the presence of LA, with prevalence of 4.35% in subjects with recurrent abortion. One of the fifty pregnant women without complications (control subjects) was found positive for LA confirmatory test, constituting a prevalence rate of 2.0%. Furthermore, in a study of fifty multi-parous women for the prevalence of LA, using KCT clot based coagulation assay, Awodu and colleague found four subjects (8.0%) to be positive for LA90. However, some published investigation did not identify any association between APLS and repeated 137, 29,49,50. miscarriages but other studies did This demonstrates the importance for further studies using larger number of individuals and standardization of the investigational methods.

Olayemi E et al75, enrolled one hundred and nine subjects made up of fifty seven HbSS sickle cell disease (SCD) subjects and fifty two HbAA control subjects. There were thirty five males and twenty two females. These were subjected to laboratory screening, mixing studies and confirmatory test, using KCT, to detect the presence of Lupus anticoagulant (LA). Kaolin clotting time (KCT) ratio ≥ 1.2 was found to be diagnostic in one (1.8%) of the HbSS subjects for Lupus anticoagulant. This study showed none of the HbAA subjects was positive for LA and also no significant difference in the KCT ratio among the sexes.

Furthermore, Ndakotsu MA et al74, studied one hundred and fifty-five subjects, out of which, one hundred and four were highly active antiretroviral therapy-naive HIV patients, and fifty-one subjects were HIV-negative control participants. Lupus anticoagulant (LA) was the type of APLS investigated using activated partial thromboplastin time, prothrombin time and Kaolin clotting time for screening and diagnosis. This study showed that LA had prevalence of 2.9% in HIV infected subjects against 1.9% in the control participants. Although, there was slight difference in prevalence, this was not statistically significant (P =0.973).

CHAPTER THREE AIM AND OBJECTIVES OF STUDY AIM To detect and assess the prevalence of antiphospholipid antibodies in subjects with type 2 diabetes mellitus (T2DM). SPECIFIC OBJECTIVES

1. To detect and assess the prevalence of antiphospholipid antibodies in complicated

T2DM, uncomplicated T2DM and in healthy control subjects.

2. To determine the platelet count in complicated T2DM, uncomplicated T2DM and in healthy control subjects, as well as compare the platelet count among the respondent.

3. To determine the glycated haemoglobin 1C (HbA1C) in complicated T2DM,

uncomplicated T2DM and in healthy control subjects, as well as compare the

HbA1C among the respondent.

4. To determine if there is any correlation between antiphospholipid antibodies levels and platelet count in both complicated and uncomplicated type 2 diabetes mellitus and in healthy control subjects.

CHAPTER FOUR SUBJECTS AND METHODS STUDY AREA

The study was carried out on subjects with diagnosis of T2DM attending the diabetic clinic of UNTH, Ituku - Ozalla, Nigeria. The hospital (UNTH) is one of the tertiary health institutions in Enugu city, Enugu State. It is located 20km off Enugu – Port- Harcourt expressway. The Igbo ethnic group is in majority with other ethnic groups constituting the minority. The University of Nigeria Teaching Hospital (UNTH), Ituku – Ozalla, receives referrals from neighbouring states (Abia, Imo, Ebonyi, Anambra, Kogi and Benue).

STUDY DESIGN This was a cross sectional study, involving detailed medical history, physical examination, Administration of questionnaire and laboratory investigations on the known patients with complicated and uncomplicated T2DM attending diabetic clinic of UNTH, Ituku – Ozalla.

In addition to the application of questionnaires, the following were conducted: fundoscopy for dilated fundi and blood vessels, haemorrhagic spot(s), among others for diabetic retinopathy patients, a cotton wisp and pin-prick testing for diabetic neuropathy subjects; urinalysis for micro-albuminuria which was repeated twelve weeks later using combi-13 for the diabetic nephropathy individuals; viewed and read the radiological investigations such as Magnetic Resonance Imaging (MRI) and Computed Tomography (CT) of the brain of subjects diagnosed for Transient Ischaemic Attack(TIA) and diabetic ischaemic stroke, and blood pressure measurement for subjects with diabetic hypertension.

STUDY POPULATION Three groups of participants were recruited for this study. They included:

1. T2DM subjects with complications (The complications considered for this study were: retinopathy, neuropathy and nephropathy (grouped as microvscular); hypertension, ischaemic stroke and transient ischaemic attack (TIA), (grouped as macrovascular)), ( to serve as test subjects). 2. T2DM subjects without complications (patients without the above complications are referred to uncomplicated T2DM, ( to serve as disease control ). 3. Healthy subjects, to serve as control (to serve as normal control).

Subjects in groups one and two were recruited from the Endocrine Unit of the Department of Internal Medicine.The healthy control subjects were recruited from the blood transfusion unit of Department of Haematology and Immunology of UNTH, medical students and hospital (UNTH) workers and Random plasma glucose was done at point of care using glucometer machine (Accu-chekR Active Blood glucose (mg/dl) Monitoring System by Roche; REF: 06656897200;LOT: 06029556; Serial n0: GUO5701336), calibrated using the hexokinse method for blood glucose assay to rule out hyperglycaemia or asymptomatic diabetes mellitus.

SAMPLE SIZE Although the calculated minimal sample size were sixty-nine (69) 138,139, two hundred and ten (210) subjects (seventy (70) in each arm) were recruited to give allowance for possible attrition.

SELECTION CRITERIA INCLUSION CRITERIA:

 Consented adult (> 18 years) subjects with complicated or uncomplicated T2DM. The complications considered for this study were: retinopathy, neuropathy and nephropathy (grouped as microvscular); hypertension, ischaemic stroke and transient ischaemic attack (TIA), (grouped as macrovascular).  Consented apparently healthy adult (> 18 years) subjects to serve as control.

EXCLUSION CRITERIA:

 T2DM aged < 18 years.  Individuals on drugs or have taken drugs within 12 weeks period (fansidar, propranolol, procainamide, hydralazine etc), known to be associated with transient development of APLS.  Individuals on steroid therapy or have received steroid within 12 weeks period, as medication could normalize APLS activity giving a false negative result.  Subjects with infectious disease (HIV, sepsis and malignancy), who might have transient APLS.  Individuals on anticoagulation and / or on oral contraceptive therapy, as this might give a false positive result, due to interference with coagulation assay.  Individuals with history of autoimmune diseases and collagen diseases.  Non consenting individuals.

ETHICAL CLEARANCE AND CONSENT CONSIDERATIONS. Ethical clearance was obtained from the Ethical Committee of UNTH and consents obtained from subjects that had met the selection criteria.

LABORATORY INVESTIGATIONS: The following investigations were carried out: -

1. Lupus anticoagulant (LA) detection using DRVVT kit: - for screening, mixing tests and confirmatory studies.

2. Anticardiolipin antibody (ACA) assay, using IgG-β2GP1-dependent-ACA immunoassay (ELIZA) kit:

3. Full blood count (FBC) with emphasis on platelet count, using the Auto- Haematology Analyser-Mythic 22 machine.

TM 4. Glycated Haemoglobin 1C (HbA1c) using D-10 Haemoglobin Testing System Analyser. 5. Mid-stream-urine (MSU) for micro-albuminuria using combi-13A urinalysis reagent strips, for patients with diabetic nephropathy.

6. Fasting or random plasma glucose to rule out hyperglycaemia or asymptomatic diabetes mellitus in healthy control subjects.

SAMPLE COLLECTION The appropriate sample bottle was labelled using unique patient identifier (code serial number), thus:

 For the full blood count (FBC) and HbA1c, using test tube containing EDTA anticoagulant.

 For the LA assay, using plastic test tube containing 3.2% (0.109M) trisodium citrate solution, of anticoagulant- to -blood – ratio, 1:9.

 For the 1gG-β2GP1-dependent -ACA assay using plain plastic tube, to enable blood clot retraction.

A large vein identified on every subject’s forearm was selected for venepuncture (e.g. ante-cubital). Consequently, atraumatic phlebotomy using a 21 gauge of needle was done. Then a total of 10 mls of blood was collected from each consented subject, the inserted needle gauge removed immediately, and dry- clean cotton wool applied with pressure to maintain pressure haemostasis. Then needle gauge removed from the syringe, and blood drawn into the respective sample containers, thus:

- 2.5mls of blood into sample collecting tube for FBC and HbA1c then covered and inverted 3 to 4 times for proper mixing of anticoagulant.

- 3.0mls of blood into plain plastic tube for 1gG-β2GP1-dependent -ACA assay. The tube was covered and allowed to stand at room temperature (to enable blood clot retraction).

- 4.5mls of blood (nine parts) drawn into a plastic test tube containing 0.5ml (one part) of 3.2% (0.109M) trisodium citrate solution. The tube was covered immediately and inverted 3-4 times for proper mixing of anticoagulant. Universal sterile container, was given to each subject, for mid-stream-urine (MSU), which was analysed for micro-albuminuria immediately using Combi – 13 urinary kit (Micropoint Urinalysis Reagent Strips 13A (leucocytes, nitrite, urobilinogen, micro albumin, protein, pH, blood, specific gravity, ascorbate, , ketone, and glucose)). This was done and repeated 12 weeks later, for the T2DM subjects with nephropathy, especially.

HANDLING AND TRANSPORTATION OF SPECIMEN

Collected blood samples were put into racks. Agitation of samples was avoided to prevent the formation of variable sized protein micro particles that would falsely raise the automated platelet count. Similarly, exposure to heat (e.g sunlight) was avoided, to prevent denaturation of protein. Transportation and separation of collected specimen was done within 1-2 hours of collection, to prevent loss of labile factors ( and ) important in some clot –based coagulation assays e.g APTT and KCT.

PREPARATION AND STORAGE OF SPECIMEN

For LA study: The blood sample in the container for LA study was double centrifuged to obtain platelet poor plasma (PPP), thus: - the first centrifuge was done at 2000G for 15 minutes, followed by pipetting the plasma into another non-activating plastic container using plastic pipettes, and re-centrifuging at 2500G for 10 minutes. The citrated platelet poor plasma contained platelet count <10 x 109/L, that would prevent the activation of platelet factor 4, was controlled for platelet count equivocal result using Haematology Analyser-Mythic 22 machine. About 300µL aliquots of PPP was preserved on ice blocks and analysed immediately (1-2hrs) or frozen at ≤ 700C until LA is analysed in batches.

For 1g G - β2GP1 – dependent – ACA Assay: blood sample in the plain tube was allowed to stand at room temperature for clot retraction. This was followed by centrifuging at 1500G for 5minutes. The serum was pipetted into a plain container for use immediately (30-60minutes) or 300µl aliquots for storage at ≤ 700C until ACA is analysed in batches.

For the full blood count (FBC), with emphasis on platelet count investigation, hence, fresh sample was used: The 2.5mls blood sample was analysed using Haematology Analyser – Mythic 22. This was controlled by manual platelet count and peripheral blood film (PBF) examination for equivocal results. After, the same sample was used for HbA1c – assay using the D-10TM Haemoglobin Testing System Analyser immediately.

PROCEDURES: LUPUS ANTICOAGULANT (LA):

Assay was done using Dilute Russell’s Viper Venom Time (DRVVT) Kit:

A. LA-screening reagent kit: {Technoclot(R) LA screen (DRVVT reagent); Lot number: 6Y52B00.01; Reference number: 5343012; Expiring date: 02/10/2017; Technoclone GmbH, Austria Brunner Street 67, 1230 Vienna}.

B. LA-confirmatory reagent: {Technoclot(R) LA confirm (DRVVT reagent); Lot number: 6X51B00.01; Reference number: 5343016; Expiring date: 07/12/2016; Technoclone GmbH, Austria Brunner Street 67, 1230 Vienna}. C. Platelet Poor Plasma, (LA-negative) (PPP) reagent: {Technoclone Platelet Poor Plasma (reagent C); Lot number: IL52B00.01; Reference number: 5343022; Expiring date: 08/31/2017; Technoclone GmbH, Austria Brunner Street 67, 1230 Vienna}.

PRINCIPLE: The Russell’s viper venom (RVV) directly activates , which in the presence of phospholipids (PLs), prothrombin and calcium ions cleaves fibrinogen to fibrin. The DRVVT bypass factor VII of the extrinsic pathway and, the contact and antihaemophilic factors of the intrinsic pathway. In subjects with LA, there is prolongation of clotting time. The plasma of an individual that contains Lupus Anticoagulant (LA), LA binds to the PL, and prevents the action of RVV to form fibrin clot

RVV

-----● Point of action of RVV FIGURE 3: Coagulation cascade, showing the point of action of RVV

The Dilute Russell’s Viper Venom Time (DRVVT) contains a graded level of phospholipids (PLs), which can neutralise the action of LA. Consequently, clot formation proceeding relatively uninterrupted, achieving a lower clotting time, to prove the phospholipids dependence of the LA. Technoclot(R) LA screen was simplified DRVV reagent to screen for the presence of lupus Anticoagulants (LA). Therefore Technoclot(R) LA screen is more specific for LA than aPTT as it is not affected by contact factor abnormalities or by factor FVII deficiencies or antibodies. Technoclot LA confirm was a phospholipid rich DRVV reagent for the specific correction of lupus Anticoagulants(LA).Mixing tests may be useful to exclude factor II, V and X deficiencies, which may prolong LA screen and LA confirm results. This was done by mixing normal plasma with test plasma, which replenishes any factor deficiency in the test plasma. Prolonged mixing test, it indicated the presence of an inhibitor (such as LA) in the test plasma.

METHOD: The manufacturer kit’s standard operating procedure (S.O.P) was followed. This was a one step procedure. Thawed (200µl) or freshly made plasma was dispensed into a glass rimless test tube 75mm x 10mm in diameter and incubated in a water bath at 370c for one minute. Then, the pre-warmed 200µl of DRVV reagent was added and the stop watch started immediately. The clotting time was recorded. The test was done in duplicate and the average result recorded. The same procedure was done for the platelet poor plasma (LA-negative) (PPP) reagent, which served as the control / standard / reference plasma. Then, the dRVVT screening ratio was calculated as follows:

Clotting time of test DRVVT Screening = Clotting time of control The absence (negative) of LA was considered, when clotting time ratio of the test to that of the control specimen was < 1.2. But when the ratio was >1.2, the presence of LA and clotting factor deficiency (factors II, V, X, fibrinogen) was considered. This led to DRVVT mixing study.

DRVVT MIXING STUDY This involved adding equal volumes of test and control plasma in a glass test tube and clotting time recorded, as in the screening procedure. When the average clotting time of the test was <10 % longer than that of the control specimens, this may mean that the clotting factor deficiency was corrected. But when the clotting time of the test was ≥10% longer than of the control specimens, the presence of LA may be considered. Then the DRVVT confirmatory assay was performed.

DRVVT CONFIRMATORY ASSAY. The test and control (reference/standard) plasma were subjected to DRVV reagent with high phospholipid (PL) content, the clotting time determined and recorded as was done in the screening procedure. When the average clotting time of the test ratio to that of control specimens on subjection to DRVVT confirmatory reagent was > 10%, DRVVT confirmatory assay was considered positive. This is as recommended by the British Committee for standardization in Haematology (BCSH)17. This formula was used: Test DRVVT The (i) Percentage Correction = Test DRVVT + PL

And (ii) Percentage (Test DRVVT /Control DRVVT) – Test DRVVT + Control DRVVT +PL) = Correction ratio Test DRVVT / Control DRVVT.

INTERPRETATION OF RESULT. LA screen clotting time When LA confirmatory ratio = = > 1.2 LA confirmatory clotting time the presence of LA was considered, thus positive.

IG G- Β2GP1- DEPENDENT- ACA ASSAY

PROCEDURE: This was done using ELISA test kit (Anticardiolipin IgG ELISA kit GWB-521211; Lot number: ACARG-4032; Expiring date: 31/12/2016; Genway Biotech, Inc: 6777 Nancy Ridge Drive San Diego, CA 9212, U.S.A).

PRINCIPLE: The solid-phase ELISA is a heterogeneous, ligand-binding immunoassay with the ability to separate some component of analytical reaction mixture by adsorbing certain components onto immobilized solid phase. This involves the addition of liquid sample on to an immobilized solid phase with special binding abilities, followed by the addition of sequential multiple liquid reagents with specific enzyme, incubation, washing and colour development by the product of an enzymatic reaction (some optic change) in the final liquid in the well, from which the analyte or ligand investigated for, is quantified. Some specific are used in this enzymatic reaction. These enzymes generate specific amplified signals with some specific wavelength of light. The reading is usually based on the detection of intensity of transmitted light by spectrophotometer. METHOD: The manufacturer kit’s standard operating procedure (S.O.P) was strictly followed. Specimen and reagent were brought to room temperature (18-28 oC) and mixed gently. Then: 1. Coated strips were placed into the strips holder. 2. Test samples were diluted 1:21 with buffer by adding 10µl of the sample to 200µl of the sample diluents and mixed properly. Calibrators (So-S4 with respective concentration 0, 5, 10, 20, and 80 AU/mls), negative and positive controls were ready for use. 3. 100µl of diluted sera; calibrators and controls were dispensed into appropriate coated microtitre plate wells. 100µl of the reagent blank was dispensed into well position, as directed by the manufacturer.

4. Air bubbles were removed by tapping the holder gently. 5. This was incubated for 60mins at room temperature (22-28oC). Antibodies against this antigen if present in the cell room bind to the respective antigen. 6. The excess liquid was discarded from each well. 7. The wells were washed three times with 300µl of 10x diluted solution-wash buffer (prepared by adding 10x (25mL) solution to 475mL of distilled water) and, blotted on absorbent paper, to remove the unbound serum proteins. 8. 100µl of enzyme conjugate (anti-hIgG conjugate with horseradish peroxidase (HRP), was dispensed to each well and incubated for 60 minutes at room temperature to form a conjugate/antibody/antigen complex. 9. Excess enzyme conjugate was removed from each well, washed with 300µl of wash buffer three times and blotted on absorbent paper. 10. Then, 100µl of TMB (tetramethylbenzidine) substrate (chromogenic substrate), was added to each well, and incubated for 15 minutes in the dark at room temperature, to forming a blue colour as a result of hydrolysis of the formed complex. 11. The reaction was stopped, by adding 100µl of stop solution (sulphuric acid solution) to each of the microplate well, forming a yellow end product. 12. The microplate was sharken gently. 13. The absorbance (optical density) (OD) was read at 450 nm on ELIZA reader.

14. The amount of colour is directly proportional to the concentration of IgG- β2GP1- ACA in the test sample, 15. The optical density (OD) was analysed using “Excel Read out data”.

INTERPRETATION of IgG- β2GP1-ACA Result. Anti-cardiolipin IgG antibody (AU/mls) <10 is said to be normal (negative). But anticardiolipin IgG antibody >10 is elevated, and said to be positive.

STATISTICAL ANALYSIS Data was analysed using SPSS (Statistical Package for Social Science ) version 18. Data was presented in tables and graphs. Qualitative variables such as sex, marital status, occupation, educational level, tribe, family history, smoking and distribution of antiphospholipid antibodies (lupus anticoagulant (LA) and IgG-β2GP1-depentent-ACA) was summarized using proportion and percentages. Quantitative variables such as age, duration of alcohol and smoking, blood pressure (BP), plasma glucose and body mass index (BMI) were summarized using mean and standard deviation. Chi-square was used to compare differences in distribution of antiphospholipid antibodies. Analysis of Variance (ANOVA) was used to compare if there was difference in mean between variables (LA, HbA1c, platelet count, IgG-β2GP1- depentent-ACA), for complicated T2DM, uncomplicated T2DM and healthy control subjects. When significant, Post Hoc analysis was done to establish the existing differences in mean. Pearson correlation coefficient was used to establish relationship between platelet count with LA screening ratio, LA mixing test, LA confirmatory test,

IgG-β2GP1-depentent-ACA and HbA1c for complicated T2DM, uncomplicated T2DM and healthy control. The bio-data, was used to socio-demographic characterised the respondents, chronic illness and drug histories were used to rule-out transient infection which would have affected the study, family and social histories helped to compare any correlation with the disease state and complications, stated complications were the people targeted for the study and helped to classify as being complicated and uncomplicated

T2DM, random or fasting plasma glucose helped to rule-out hyperglycaemia or asymptomatic diabetes mellitus in healthy control subjects respectively, and blood pressure measurement, was done for patients with diabetic hypertension.

CHAPTER FIVE RESULTS GENERAL STUDY CHARACTERISTICS:

For each study arm namely; complicated T2DM, uncomplicated T2DM and healthy control, seventy (70) subjects respectively were recruited. Table 1: socio – demographics of respondents

Socio-demographics Complicated T2 DM Uncomplicated T2 DM Healthy Control n = 70 n = 70 n = 70 Age (years) Mean(SD*) 59.5(10.0) 48.8(6.7) 31.1(10.5)

Freq Percent Freq Percent Freq Percent (%) (%) (%) Sex Female 41 58.6 36 51.4 14 20.0 Male 29 41.4 34 48.6 56 80.0

Marital status Married 69 98.6 70 100.0 31 44.3 Single 1 1.4 - - 39 55.7

Occupation Civil/public servant 20 28.6 45 64.3 17 24.3 Trading 16 22.9 14 20.0 16 22.9 Student/pupil - - - - 33 47.1 Others# 34 48.6 11 15.7 4 5.7

Educational level Primary and below 3 4.3 9 12.8 1 1.4 Secondary 30 42.9 30 42.9 23 32.9 Tertiary 14 20.0 31 44.3 46 65.7

Tribe Igbo 70 100.0 68 97.1 70 100.0 Others(Ibibio&Urobo) - - 2 2.9 - - SD*- Standard Deviation Others# - artisan, clergy, driver, housewife, unemployed etc

Table 1: Socio-demographics of respondents

SOCIO-DEMOGRAPHIC CHARACTERISTICS:

The mean age in years for complicated T2DM, uncomplicated T2DM and healthy control in years were 59.5(10), 48.8(6.7) and 31.1(10.5) respectively. They were predominantly females for both complicated T2DM 41 (n = 70, 58 .6%) and uncomplicated T2DM 36 (n= 70, 51.4%) but more of males for healthy control 56(n= 70, 80.0%). Respondents were mostly married for complicated T2DM 69 (n = 70, 98.6% and uncomplicated T2DM 70 (n = 70, 100.0%) respectively, but were single for healthy control 39(n = 70, 55,7%). About 33 (n = 47.1%) of them were students for healthy controls but none for both complicated and complicated T2DM subjects respectively. Most of the studied subjects had at least secondary education as the only form of education; and complicated T2DM with 3(n = 70, 4.3%), uncomplicated T2DM having 9(n = 70, 12.8%) and healthy control arm recording 1(n = 70, 1.4%) had primary education and below. They were predominantly Igbos with only 2 (n = 70, 2.9%) of uncomplicated T2DM being from other tribes (Ibibio and Urobo). (See table 1 and raw data in appendix IVa - IVc).

Table 2: Some relevant medical evaluation of respondents

Medical history Complicated T2 DM Uncomplicated T2 DM Healthy Control n=70 n=70 n=70 Freq Percent Freq Percent Freq Percent (%) (%) (%) Family History None 31 44.3 18 25.7 53 75.7 Diabetes 12 17.1 22 31.4 7 10.0 Hypertension 9 12.9 1 1.4 5 7.1 Diabetes & Hypertension 18 25.7 29 41.4 5 7.1

Alcohol intake No 48 68.6 59 84.3 53 75.7 Yes 22 31.4 11 15.7 17 24.3

Smokes/smoking No 61 87.1 65 92.9 63 90.0 Yes 9 12.9 5 7.1 7 10.0

Alcohol intake Mean(SD) duration ( years) 21.7(11.1) 7.1(2.3) 6.4(4.7) Mean(SD) quantity ( Bottles) 2.1(1.5) 1.9(0.7) 2.5(2.2)

Smokes/smoking Mean(SD) duration ( years) 10.2(6.1) 8.2(2.5) 7.0(4.2) Mean(SD) quantity ( 12.8(5.7) 10.2(7.1) 7.2(3.7) sticks/day)

Mean(SD) BP Systolic (mmHg) 145.5(20.3) 122.4(8.7) 120.2(9.6) Diastolic(mmHg) 85.8(10.6) 71.1(7.3) 74.4(7.8)

Mean(SD) BMI (kg/m2) 26.4(3.5) 25.3(3.5) 23.5(5.1)

Mean(SD) Plasma 177.2(68.1) 201.4(76.4) 114.4(10.2) Glucose(mg/dl) (Fasting plasma glucose for T2DM subjects and Random plasma glucose for healthy control individuals)

Table 2: Some relevant medical evaluation of respondents

SOME RELEVANT MEDICAL EVALUATION OF RESPONDENTS:

It was observed that 18 (n = 70, 25.7%) of respondents with complicated T2DM,

29(n = 70, 41.4%) uncomplicated T2DM and 5 (n = 70, 7.1%) healthy control had family history of diabetes and hypertension respectively. Twenty two, 22(n=70, 31.4%) complicated T2DM, 11(n=70, 24.3%) healthy controls have consumed alcoholic beverage (beer), with the mean number of bottles and duration in years 2.1(1.5) and 21.7(11.7) for complicated T2DM, 1.9(0.7) and 7.1(2.3) for uncomplicated T2DM and, 2.5(2.2) and

6.4(4.7) for healthy control respectively. Equally 9(n=70, 12.9%) complicated T2DM, 5(n=70, 10.0%) healthy controls gave a past history of cigarettes smoking with mean duration in years and quantity (sticks of cigarettes /day) 10.2(6.1) and 12.8(5.7) for complicated T2DM and 7.0(4.2) and 7.2(3.7) for healthy control. The mean blood pressure (BP) measured in millimetre / mercury were (systolic/diastolic)

145.5/85.8(20.3/10.6) for complicated T2DM, 122.4 /71.1(8.7/7.3) for uncomplicated

T2DM and, 120.2/74.4(9.6/7.8) for healthy control. Respondents mean body mass index (BMI) measured in kilogramme/meter squared (kg/m2) were 26.4(3.5) for complicated

T2DM, 25.3(3.5) for uncomplicated T2DM and, 23.5(5.1) for healthy control. The mean plasma glucose measured in milligramme / deciliter (mg/dl) were 177.2(68.1) for complicated T2DM, 201.4(76.4) for uncomplicated and 114.4(10.2) for healthy control subjects. (See table 2).

Table 3: Distribution of Anti-phospholipids of respondents.

Anti-phospholipids Complicated T2 DM Uncomplicated T2 DM Healthy Control Freq Percent Freq Percent Freq Percent

LA Conclusion remark Negative 65 92.9 67 95.7 67 95.7 Positive 5 7.1 3 4.3 3 4.3 χ2 = 0.767 p = 0.681

IgG-β2 GP1-ACA Negative 67 95.7 67 95.7 67 95.7 Positive 3 4.3 3 4.3 3 4.3 χ2 = 0.000 p = 1.000

Table 3: Distribution of Anti-phospholipids of respondents

PREVALENCE OF ANTIPHOSPHOLIPID ANTIBODIES OF RESPONDENTS. The prevalence of Lupus anticoagulants (LA) were 5(n=70, 7.1%) for complicated

T2DM, 3 (n = 70, 4.3%) for both uncomplicated T2DM and healthy control respectively, while prevalence for immunoglobulin G-β2-glycoprotein 1 – anticardiolipin(1gG –

β2GP1 – ACA) antibodies was 3(n=70, 4.3%) each for all groups (complicated T2DM, uncomplicated T2DM and healthy control) elevated and described positive. (See table 3 and appendix Vai, Vaii, Vbi, Vbii, VIai, VIaii, VIbi, VIbii, VIIai and VIIbi for raw datas).

Table 4: Comparison of LA of respondents

Test statistics Variables Mean SD ANOVA (F ratio) P value (seconds) LA screening ratio Complicated T2DM 1.1 0.1 Uncomplicated T2DM 1.4 1.6 1.328 .267 Healthy Control 1.4 1.8

LA mixed average T Complicated T2DM 146.3 94.2 Uncomplicated T2DM 164.3 86.4 0.378 .692 Healthy Control 114.8 76.2

LA confirmatory average T Complicated T2DM 42.8 2.2 Uncomplicated T2DM 47.0 2.0 6.658 .020 Healthy Control 41.0 2.0

Mean Sig. Difference 95% Confidence Interval Lower Bound Upper Bound LA screening ratio Complicated T2DM 0.3 0.187 -0.2 0.8 Uncomplicated T2DM .

Complicated T2DM 0.4 0.136 -0.1 0.8 Healthy Control

Uncomplicated T2DM 0.1 0.860 -0.4 0.5 Healthy Control

LA mixed average T Complicated T2DM -18.0 0.742 -97.2 133.2 Uncomplicated T2DM

Complicated T2DM -31.5 0.539 -138.7 75.8 Healthy Control

Uncomplicated T2DM -49.5 0.415 -175.6 76.7 Healthy Control

LA confirmatory average T Complicated T2DM -4.2 0.025 -7.7 -0.7 Uncomplicated T2DM

Complicated T2DM -1.8 0.271 -5.3 1.7 Healthy Control

Uncomplicated T2DM -6.0 0.008 -9.9 -2.1 Healthy Control

THE COMPARISON OF LA OF RESPONDENTS

Table 4 shows that there were no significant differences in means of LA

screening ratio for complicated T2DM, uncomplicated T2DM and healthy control (F = 1.328, p = 0.267) as well as LA mixed average

T (F = 0.378, p = 0.692). However there were significant differences in

means of LA confirmatory average T for complicated T2DM,

uncomplicated T2DM and healthy control (F = 6.658, p = 0.020). The

differences in mean were between complicated and uncomplicated T2DM

(p = 0.025, 95% CI: -7.7 to -0.7) as well as uncomplicated T2DM and healthy control (p = 0.008, 95% CI: -9.9 to -2.1)

Table 5: IgG- β2 GP1 – dependent - ACA of respondents

Test statistics Variables Mean SD ANOVA (F ratio) P value (Au/mls) Ig G ACA Complicated T2DM 3.6 8.2

Uncomplicated T2DM 3.2 5.7 0.281 0.755 Healthy Control 2.7 5.0

Mean Sig. Difference 95% Confidence Interval Ig G ACA Lower Bound Upper Bound Complicated T2DM -0.4 0.733 -1.8 2.5 Uncomplicated T2DM

Complicated T2DM -0.8 0.455 -3.0 1.3 Healthy Control

Uncomplicated T2DM -0.4 0.685 -2.6 1.7 Healthy Control

Mean Rank Kruskal Wallis P value Ig G ACA Complicated T2DM 101.32 Uncomplicated T2DM 110.84 0.896 0.639 Healthy Control 104.34

Table 5, shows that there were no significant differences in means of IgG ACA for complicated T2DM, uncomplicated T2DM and healthy control (F = 0.281, p = 0.755).

Table 6: Distribution of Vascular complications in complicated T2DM subjects

Variable Frequency Percent (100%) (n=70)

Macrovascular Absence of features 3 4.3 1 feature present 61 87.1 2 features present 3 4.3 3 features present 3 4.3

TIA Absent 66 94.3 Present 4 5.7

Stroke Absent 64 91.4 Present 6 8.6

Hypertension Absent 4 5.7 Present 66 94.3

Microvascular Absence of features 17 24.3 1 feature present 27 38.6 2 features present 24 34.3 3 features present 2 2.9

Retinopathy Absent 34 48.6 Present 36 51.4

Nephropathy Absent 56 80.0 Present 14 20.0

Neuropathy Absent 36 51.4 Present 34 48.6

Table 6 shows majority of patients that had macrovascular complication, 61(87.1%) had one complication. Of the patients that had complications, 66(94.3%) had Hypertension, 6(8.6%) had stroke while 4(5.7%) had TIA.

Majority of patients that had microvascular complication, had similarly a complication 27(38.6%) or two complications 24(34.3%). Of the patients that had complications, 36(51.4%) had Retinopathy, 14(20.0%) had Nephropathy while 34(48.6%) had Neuropathy.

Table 7: Relationship/association between LA and vascular complication in complicated T2DM subjects.

Variable LA Χ2 p value Positive Negative n(%) n(%) Macrovascular TIA Absent 3(75.0) 1(25.0) FT 0.800 Present 1(100.0) 0(0.0)

Stroke Absent 2(100.0) 0(0.0) FT 0.600 Present 2(66.7) 1(33.3)

Hypertension Absent 0(0.0) 1(100.0) FT 0.200 Present 4(100.0) 0(0.0)

Microvascular Retinopathy Absent 3(100.0) 0(0.0) FT 0.400 Present 1(50.0) 1(50.0)

Nephropathy Absent 3(75.0) 1(25.0) FT 0.800 Present 1(100.0) 0(0.0)

Neuropathy Absent 4(100.0) 0(0.0) FT 0.200 Present 0(0.0) 1(100.0)

Table 7 shows that there were no significant association between LA and macrovascular complication, TIA (p =0.800), stroke (p =0.600) and Hypertension (p =0.200). Similarly there were no significant association between LA and microvascular complication, Retinopathy (p =0.400), Nephropathy (p =0.800) and Neuropathy (p =0.200).

Table 8: Relationship/association between Ig G-ACA and vascular complication in complicated T2DM subjects.

Variable Ig G-ACA Χ2 p value Positive Negative n(%) n(%) Macrovascular TIA Absent 6(85.7) 1(14.3) FT 0.875 Present 1(100.0) 0(0.0)

Stroke Absent 4(80.0) 1(20.0) FT 0.625 Present 3(100.0) 0(0.0)

Hypertension Absent 1(100.0) 0(0.0) FT 0.875 Present 6(85.7) 1(14.3)

Microvascular Retinopathy Absent 3(75.0) 1(25.0) FT 0.500 Present 4(100.0) 0(0.0)

Nephropathy Absent 6(100.0) 0(0.0) FT 0.250 Present 1(50.0) 1(50.0)

Neuropathy Absent 4(100.0) 0(0.0) FT 0.500 Present 3(75.0) 1(25.0)

Table 8 shows that there were no significant association between IgG-ACA and macrovascular complication, TIA (p =0.875), stroke (p =0.625) and Hypertension (p =0.875). Similarly there were no significant association between Ig G - ACA and microvascular complication, Retinopathy (p =0.500), Nephropathy (p =0.250) and Neuropathy (p =0.500).

Table 9 :The mean platelet counts among respondents

Test statistics Variables Mean(×109/ SD ANOVA (F ratio) P value L) Platelet Complicated T2 T2DM 208.5 52.3 Uncomplicated T2 T2DM 198.7 40.7 77.99 0.000 Healthy Control 277.5 23.9

Mean Sig. 95% Confidence Interval Difference Lower Bound Upper Bound Platelet Complicated T2 T2DM -9.8 0.155 -3.7 23.4

Uncomplicated T2 T2DM

Complicated T2 T2DM 69.0 0.000 55.4 82.5 Healthy Control

Uncomplicated T2 T2DM 78.8 0.000 65.2 92.3 Healthy Control

THE MEAN PLATELET COUNT AMONG RESPONDENTS: 9 The mean platelet count (×10 /L) for complicated T2DM, uncomplicated T2DM and healthy control were 208.5(52.30), 198.7(40.7) and 277.5(23.9) respectively. Analysis of Variance (ANOVA), showed significant statistical differences in the mean platelet count for complicated T2DM 208.5(52.30), and healthy control 277.5(23.9), (F=77.993, P < 0.001). Post Hoc statistical analysis showed the differences in mean were between complicated T2DM and healthy control (P < 0.001, 95% CI: 55.4 to 82.5) as well as uncomplicated T2DM and healthy control (P< 0.001, 95% CI: 65.2 to 92.3%). (See table 9 and see appendix VIIIai, VIIIaii, VIIIbi, VIIIbii and VIIIc for the raw data).

Table 10:The mean glycated haemoglobin (HbA1C) among respondents

Test statistics Variables Mean(%) SD ANOVA (F ratio) P value HbA1C Complicated T2 T2DM 8.2 1.5 Uncomplicated T2 T2DM 8.0 1.7 83.747 0.000 Healthy Control 5.6 0.38 Mean Sig. 95% Confidence Interval Difference Lower Bound Upper Bound HbA1C Complicated T2 T2DM -0.2 0.379 -0.2 0.6 Uncomplicated T2DM

Complicated T2 T2DM -2.6 0.000 -3.0 -2.1 Healthy Control

Uncomplicated T2 T2DM -2.4 0.000 -2.8 -2.0 Healthy Control

THE MEAN GLYCATED HAEMOGLOBIN AMONG RESPONDENTS:

The mean glycated haemoglobin 1C (HbA1c) for complicated T2DM, uncomplicated

T2DM and healthy control were 8.2(1.5), 8.0(1.7) and 5.6(0.38) respectively. Analysis of

Variance (ANOVA), showed significant statistical differences in mean HbA1c among complicated T2DM, uncomplicated T2DM and healthy control respectively, (F=83.75,

P<0.001). The Post Hoc statistical analysis showed the differences in mean HbA1c between uncomplicated T2DM and healthy control (P<0.001, 95% CI: -3.0 to 2.0) as well as in complicated T2DM and healthy control (P<0.001, 95% CI: -2.8 to -2.0) (see table 10 and see appendix VIIIai, VIIIaii, VIIIbi, VIIIbii and VIIIc for the raw data).

Table 11: Correlation between Antiphospholipid antibodies, platelet count and glycated haemoglobin 1C among respondents. Test statistic Variables r (pearsons) P value Complicated T2 T2DM Platelet 0.080 0.524 LA screening Ratio

Platelet 0.081 0.849 LA mix average T

Platelet 0.598 0.286 LA confirmatory average T

Platelet 0.224 0.062 IgG-β2 GP1-ACA

Platelet 0.316 0.008* HbA1C Uncomplicated T2 T2DM Platelet 0.032 0.792 LA screening Ratio

Platelet 0.603 0.397 LA mix average T

Platelet 0.798 0.411 LA confirmatory average T

Platelet 0.172 0.154 IgG-β2 GP1-ACA

Platelet 0.502 0.000* HbA1C Healthy control Platelet 0.076 0.533 LA screening Ratio

Platelet 0.552 0.336 LA mix average T Platelet 0.737 0.472 LA confirmatory average T

Platelet 0.137 0.258 IgG-β2 GP1-ACA Platelet 0.201 0.095 HbA1C

CORRELATIONS OF ANTIPHOSPHOLIPID ANTIBODIES, PLATELET

COUNT AND GLYCATED HAEMOGLOBIN (HBA1C) OF RESPONDENTS:

There were significant correlation between glycated haemoglobin 1C (HbA1c), 1gG-

β2GP1-ACA and platelet count for complicated T2DM (r=0.316, P=0.008) and uncomplicated T2DM, (r=0.316, P≤0.001) respectively using Pearsons statistical analysis. However, there was no significant correlation for healthy control. (See table 11 and graphs A1 - A3, B1- B3, C1-C3).

GRAPH A2: IgG-β2GP1-ACA versus platelet count in complicated T2T2DM.

Platelet count

COMPLICATED T2DM FIGURE 1A: LA confirmatory average time (AveT) versus platelet count in complicated

T2T2DM.

Platelet count

FIGURE A2: IgG-β2GP1-ACA versus platelet count in complicated T2T2DM. Platelet count

FIGURE A3: Glycated haemoglobin 1c (HbA1C) versus platelet count in complicated T2T2DM.

Platelet count

UNCOMPLICATED T2DM

FIGURE B1: LA confirmatory average time (AveT) versus platelet count in uncomplicated T2T2DM.

Platelet count

FIGURE B2: IgG-β2GP1-ACA versus platelet count in uncomplicated T2T2DM. Platelet count

FIGURE B3: Glycated haemoglobin 1c (HbA1C) versus platelet count in uncomplicated

Platelet count

FIGURE C1: LA confirmatory average time (AveT) versus platelet count in healthy control

Platelet count

FIGURE C2: IgG-β2GP1-ACA versus platelet count in healthy control

Platelet count

FIGURE C3: Glycated haemoglobin 1c (HbA1C) versus platelet count in healthy control CHAPTER SIX Discussion Antiphospholipid antibodies have been associated with vascular (arterial, venous or both) thromboses3,5,12. Diabetes mellitus, most especially type 2 diabetes is associated also with vascular (arterial) thrombosis38,39. However, there is no known established mechanism for antiphospholipid antibodies (APLS) in the pathogenesis of vascular thrombosis in type 2 diabetes mellitus (T2DM). Depending on the selection criteria of the subjects, the type of APLS investigated and the methods for APLS detection, several prevalent rates of Antiphospholipid antibodies have been reported in patients diagnosed of type 2 diabetes mellitus. In this study, the prevalence of lupus anticoagulant (LA) was 7.1% for complicated

T2DM and 4.3% for both uncomplicated T2DM and healthy control subjects respectively.

The prevalence of IgG-β2GP1-anticardiolipin antibodies (ACA) was 4.3% for all groups too. Though, there were detectable APLS in the three groups, in this study, these antibodies showed no statistical significant difference to infer Antiphospholipid antibodies as an independent risk factor for vascular thrombosis in T2DM. Palomo and Colleagues140, found 14.3% type 2 diabetes mellitus patients were positive for anticardiolipin antibodies. They also observed that the T2DM patients were lupus 141, anticoagulant (LA) negative. In another study, Palomo IG et al observed 25.2% T2DM individuals were positive for ACA and 13.7% for anti-prothrombin antibodies (aPT). This was supported by many investigators, who failed to find significant association 112,128,142. between vascular complications in T2DM and APLS But in comparison to Palomo et al earlier study, this present study showed that APLS was statistically significant and, ACA and aPT were the predominant antibodies in patients with thrombosis. Again, an increased tendency to venous and arterial thrombosis in the 143. presence of APLS in T2DM subjects had been reported These APLS might play a role in the impairment of thrombo-resistant function of vascular endothelium and enhancement of platelet activation and aggregation, ischaemia as well as thrombosis143. Thus, might play a role in the pathogenesis and progression of diabetic complications101,143. These differences in prevalence rate could be attributed to the selection criteria for the T2DM subjects, the type of APLS investigated and the methods for APLS detection, compared to the International Society of Thrombosis and Haemostasis ISTH recommendation12,31.

The distribution pattern of T2DM complication subjects in this study was: macrovascular complications recorded 66(94.3%), ischaemic stroke 6(8.6%) and TIA 4(5.7%); while that of the microvascular complications had retinopathy 36(51.4%), nephropathy 14(20.0%) and neuropathy 34(48.6%). The study by Triolo G and colleagues101, reported a prevalence rate of 86% for APLS in T2DM patients with macrovascular complication. Asherson RA et al144, recorded 50% cases of TIA and ischaemic stroke in subjects younger than 50yrs with IgG anticardiolipin antibodies (ACA). Shahin M et al13, reported a prevalence rate of 17.7% for patients with diabetic retinopathy. Platelets are small discoid blood cells (the un-activated form) that circulate and participate in haemostasis. In response to stimuli generated by the endothelial blood vessels damage, platelets change shape, adhere to the sub-endothelial surface, secrete the intracellular organelle contents, and aggregate to form a thrombus145. Chronic hyperglycaemia causes endothelial and platelet dysfunction146,147. Therefore, platelet may assume an important role in the development of advanced atherosclerosis in diabetes mellitus (DM)148-151 Although, the platelet counts among the three arms of the subjects for this study were within the normal reference range, lower platelet counts range had been observed in patients with complicated T2DM. This may be attributed to increased platelet adhesion, aggregation and activation in responses to vascular endothelial injury145. Nagwa MM et al151, found platelet count was within normal reference range in all studied groups of subjects. Umadevi SV and colleagues152, study was in agreement with the finding by Nagwa and colleagues too.

This study showed statistical significant differences in mean platelet count between complicated T2DM and healthy control subjects (P<0.001. 95% CI: 55.4 to 82.5) and in uncomplicated T2DM and healthy control subjects (P<0.001, 95% CI: 65.2 to 92.3).

Some studies have reported the mean platelet count to be higher in T2DM group (complicated and uncomplicated) than those in non-diabetic healthy control subjects. Therefore, platelet count could be dependent on several variables, such as mean platelet 153. survival, platelet production and, turnover rate in T2DM The low mean platelet count in T2DM with complicated, could be centred on the rapid consumption of activated platelet, in response to endothelial injury, as they bind to the sub-endothelial surface145,153,154. In the non-diabetic individual, the normal range of glycated haemoglobin 1C is 4.0 to 6.0%, while that of diabetic subject is at ≥ 6.5%32,35. Higher values > 6.5% were observed in complicated T2DM subjects, compared to the uncomplicated T2DM, and healthy control subjects with normal values in this study. This was in agreement with the study 151, by Nagwa MM et al that glycated haemoglobin 1C (HbA1C) was higher in T2DM patients with complication compared to T2DM without complication and healthy control subjects. This could be attributed to dysfunction of insulin, resulting in relatively less functional low insulin and relatively more resistant insulin for adequate glucose 113 by the Antiphospholipid antibodies in T2DM with complication . Consequently, there is development of raised glycated haemoglobulin 1C, increased hyper-activity of the platelet and enhancement of diabetic vascular complications151. This raised glycated haemoglobulin 1C observed in T2DM with complication could also be attributed to lack of knowledge regarding the diet, poor dietary practices, poor drug compliance and poverty. In this study, though there was no significant correlation for healthy control subjects with respect to the Antiphospholipid antibodies (LA and IgG-β2GP1-ACA), Glycated haemoglobin 1C (HbA1C) and platelet count, however, significant correlation was observed for LA, IgG-β2GP1-ACA and platelet count in complicated T2DM (r=0.316,P=0.008). This could be attributed to increase in consumption of activated platelet as the Antiphospholipid antibodies (LA and IgG-β2GP1-ACA) bind to phospholipid epitopes of platelet membrane155,156, causing adhesion and aggregation of 145, activated platelet in T2DM subjects with complication. The study did not observe any significant correlation between family history of some relevance medical history such as hypertension and diabetes mellitus to the development 33. of T2DM as reported in some study This could be attributed to low level of education, as majority of the diabetic patients had primary or attempted secondary educational level as compared to healthy control subjects with tertiary level of education and aware of the complications of diabetes mellitus.

LIIMITATION OF THIS STUDY 1. The study did not assay for laboratory evidence of anticoagulant drug effect, although, thorough clinical history and the application of the questionnaire helped to rule-out the anticoagulant medication, which would have had affected the outcome of my result, as indicated for the exclusion criteria. However, the commercially DRVVT reagents that was used have the potential to neutralise up to 1 unit /ml unfractionated or low molecular weight heparin, which reduces the potential for false positive lupus Anticoagulant ( LA) test abnormalities due to heparin in the DRVV test system, for most samples152. Similarly, with high clinical suspicion of vascular thrombosis and the panel of tests using DRVVT reagent with potential of neutralising heparin, LA presence was confirmed. 2. The study did not assay for factor inhibitors, although the DRVVT reagent bypasses the factor VIII inhibitor, to factor X; the evidence for factor inhibitor creates a room for further study and research breakthrough. 3. The population size of two hundred and ten (210) subjects, evenly distributed into three groups, showed a significant statistical result. However, increasing the population size will improve the statistical power.

CONCLUSION Lupus anticogulants (LA) were found in both subjects and controls, but the prevalence was significantly higher than in controls, only in subjects with complicated T2DM.

Anticardiolipin antibodies were found and were equally prevalent in both T2DM subjects and in normal controls.HbA1C levels were found to be significantly higher in subject with complicated T2DM. Significantly lower platelet counts were found in subjects with complicated and uncomplicated T2DM , compared to normal controls. There was no significant correlation found between prevalent of LA or ACA and platelet counts in

T2DM. However, there was significant correlation between HbA1C levels and platelet counts in both complicated and uncomplicated T2DM. There was no significant association between LA or ACA prevalence and occurrence of vascular or other complications in T2DM. In view of the findings in this study, presence of antiphospholipid antibodies may not be caused by, or directly related to the T2DM, and cannot be regarded as a complication of T2DM.

RECOMMENDATION In addition to the usual biochemical tests, regular evaluation of HbA1C levels and platelet counts should be done in the routine monitoring of patients with T2DM. Since

APLS may be associated with vascular thrombosis, as may T2DM. Presence of APLS should be excluded in patients with T2DM. However, regular evaluation of APLS may not be required in the management, or routine monitoring of patients with T2DM. Although, there is no causal or significant statistical association of APLS and the risk of

T2DM patients developing thrombosis as was observed in this study, there is need for further multicentre robust studies to evaluate the relationship of APLS and thrombosis in persons with complicated T2DM in our environment.

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122. Erkan D, Lockshin MD. Antiphospholipid Sydrome. Curr Opin Rheumatol 2006; 8: 242 - 8. 123. Mattila K, Vaarala O, Palosuo T. Serological response against common antigen in young patients with acute Myocardial infarction. Clin Immunol Immunopathol 1989; 51: 418 – 428. 124. McCulled DK. Patient information: Diabetes Mellitus, type 2. 2008; www.Update.Com/patients. 125. Rana SA, Haider JM, May KI. Relationship between ACA and T2DM patients type 2. Journal of AL-Nahrain University 2009; 12 (2): 113– 119. 126. Becareric M, Seferovic J, Ignjatovic S, Singh S, Singh – Majkic N. Adiponectin, non – esterified antibodies in type 11 diabetes mellitus. J. Med. Biochem 2012; 31 (3):199 – 204. 127. Calvo – Romero MJ and Lima – Rodriguez ME. Anticardiolipin Antibodies in patients with type 2 Diabetes Mellitus. CM&R Rapid Release. Published online ahead of print February 26, 2009 as doi:10.3121/cmr. 2008.828 pp 1-10. 128. Tartun I, Hacihanefioglu A, Tarkun P, Cetinarslan B, Canturk Z. Anticardiolipin and anti-β2 glycoprotein 1 antibody concentration in patients with type 2 diabetes mellitus. Diabetes Research and Clinical Practice. June 2005; 68(3): 181-187. 129. Heilmann L, von Tempelhoff GF, Pollow K. Antiphospholipid syndrome in obstetrics. Clin Appl Thromb Hemost 2003; 9(2): 143- 50. 130. Branch DW, Dudlwy DJ, Scott IR, Silver JM. Antiphospholipid antibodies and fetal loss. N Engl J Med 1992; 326: 951-2. 131. Love PE, Santoro SA. Antiphospholipid antibodies: anticardiolipin and lupus anticoagulant in systemic lupus erythematosus (SLE) and in non-SLE disorders; prevalence and clinical. Ann Intern Med 1990; 112: 682-98. 132. Cowchock S. The role of antiphospholipid antibodies in obstetric medicine. Curr Obstet Med 1991; 1: 229-47. 133. The ministry of Welfare, Children and Families Bureau Maternal and Child Heath Division Maternal and Child Heath Statistic of Japan Tokyo: The Ministry of Welfore, 1999, pp. 76-8. 134. Sammaritano LR, Gharavi AE, Soberano C, Levy RA, Lockshin MD. Phospholipid binding of antiphospholipid antibodies and placental anticoagulant protein. J Clin Immunol 1992; 12: 27-35. 135. Bu C, Zhang C, Li Z, Gao L, Xie Z, Cai G. Autoantibodies to plasminogen and tissue plasminogen activator in women with recurrent pregnancy loss. Clin Exp Immunol 2007; 149(1): 31-9. 136. Spegiorin FJCL, Galao AL, Bagarelli BL, Oliani HA, Godoy-de PMJ. Prevalence of anticardiolipin Antibodies in Pregnancies with History of Repeated Miscarriages. The Open Rheunmatology Journal, 2010; 4, 28-30. 137. Boos CJ, Lip GY. Assessment of mean platelet volume in coronary arterydisease- what does it mean? Thromb Res 2007;120:11-3. 138. Onwuasigwe C. Determination of sample size in Medical Research Project: A practical guide. Eldemak publication Enugu, 2004;111-118. 139. Richard S, Jonathan S, Paul Z. The global Burden: Diabetes and Impaired Glucose Tolerance. Baker IDI Heart and Diabetes Institute Publisher, 4th Ed. Pg 4.an Association for the study of Diabetes, International Federation. 140. Palomo IG,. Mujica VE, Alarcon ML, Pereira JG, Vasquez MR Prevalence of antiphospholipid antibodies is not different in Chilean diabetic patients and normal individuals. J Diabetes complications 2005, 19 (3): 1 33 -7.

141. Palomo IG, Pereira JG, Alarcon ML, Vasquez MR, Pinochet C, Velez TM, et al. Prevalence and isotype distribution of antiphospholipid antibodies in unselected Chilean patients with Venous and arterial thrombosis. Clin Rheumatol 2004; 23 (2): 129 – 33. 142. Cojocaru IM, Popescu AN, Tanasescu R. Studying of Antiphospholipid antibodies to Type 2 Diabetes Mellitus with and without Diabetes retinopathy. Rom. T. Intern. Med, 2009; 47(3), 267-271. 143. Galtier-Deveure F, Biron C, Vis M. Vascular complications of diabetes mellitus. What role for phospholipid-binding antibodies Lupus, 1998; 7:469-474. 144. Asherson RA, Khamoshta MA, Ordi-Ros J, Derksen RH, Machin ST, Barquinero J et al. The Primary antiphospholipid syndrome: Major Clinical and Serological features. Medicine (Baltimore), 1989; 68: 366-374. 145. Mitchell RN. Hemodynamic Disorder, Thromboembolic Disease and Shock. In: Kumar V, Abbas AK, Fausto N, Aster JC, editors. Robbins and Cotran Pathologic Basis of Disease. 8th ed. New Dethi: Elsevier, 2010.p 111-134. 146. Ouvina SM, La Greca RD, Zanaro NL, Palmer L, Sassetti B. Endothelial dysfunction, nitric oxide and platelet activation in hypertension and diabetic type II patients. Thromb Res 2001; 102(2): 107-114. 147. Meigs JB, Mittleman MA, Nathan T2DM, Tofler GH, Singer DE, Murphysheehy PM, et al. Hyperinsulinemia, hyperglycaemia, and impaired hemostasis: the Framingham Offspring Study. JAMA 2000; 283(2): 221-228.

148. Bae SH, Lee J, oh KH, Klim J. Platelet activation in patients with diabetic retinopathy. Korean J Ophthalmol 2003; 17:140-144.

149. Angiolillo DJ, Fernadez-Ortiz A, Bernardo E, Ramirez C, Sabate M, Jimenez Quevedo P et al. Platelet function profile in patients with type 2 diabetes and coronary artery disease on combined aspirin and clopidogrel treatment. Diabetes 2005; 54:2430-2435.

150. Colwell JA, Nesto RW. The platelet in diabetes-focus on prevention of ischemic events. Diabetes care2003; 26:2181-8 151. Nagwa MM, Ebrahim M, Biomy MH, Mahmoud MB. A study of the values of some platelet parameters in patients with type II diabetes mellitus. AL-Azhar Assiut Medical Journal 2015; 13(1): 13-18.

152. Umadevi SV, John J, John N. Platelet count, Mean Platelet volume, serum creatinine and delayed clotting Times as surrogate marker of type 2 diabetes mellitus. Int J Biol Med Res 2014; 5 (2): 3970 – 3974. 153. Hekimsory Z, Payzinb B, Ornek T, Kandogam G. Mean platelet volume in Type 2 diabetic patients. J Diabetes Complications 2004;18:17 3 – 6. 154. Demirtunc R, Duman D, Basar M Bilge M, Teomete M, Garip T. The Relationship between glycemic control and plateled activity in type 2 diabetes mellitus. J Diabetes Complications 2009; 23: 89 – 94. 155. Arfors L, Winiarski J, Lefvert AK. Prevalence of antibodies to cardiolipin in chronic ITP and reactivity with platelet membranes. Eur J Haematol 1996; 56:230-234 156. McNeil HP, Simpson RJ, Chesterman CN, Krillis SA. Antiphospholipid antibodies are directed against a complex antigen that includes a lipid-binding inhibition of

coagulation, β2-glycoprotein (apolipoprotein H). Proc Natie Acad Sci USA 1990; 87:4120-4124.

APPENDIX 1 INFORMATION FORM

I Dr. OBINECHE AGWU, of the Department of Haematology and Immunology, University of Nigeria Teaching Hospital (UNTH), Enugu, humbly request your consent to participate in the study aimed at evaluating the presence of Antiphospholipid antibody

(APLS) levels in subjects with type 2 Diabetes Mellitus (T2DM) in Enugu, South-East, Nigeria

IMPORTANCE OF THE STUDY

The information obtained will help physicians and researchers involved in the management of diabetes mellitus (T2DM), especially T2DM. It will highlight the need to introduce screening tests and appropriate management for patients at risk of ischaemic thrombotic events and improve outcome.

VOLUNTARY NATURE OF PARTICIPATION

Participation is purely voluntary and is at no cost to you. Your declining to participate in this study will not affect your management.

STUDY PROCEDURE

You will be required to answer some questions about yourself. Your hospital folder (file) will be looked into for additional information. About 10 millilitre of blood will be collected from you, finger prick for glucose estimation will be done, under aseptic measures with minimal discomfort to you at the commencement of the study by the researcher, and you will be expected to collect your urine in a sterile container (to be provided by the researcher), for urinalysis. These will be repeated at least 12 (twelve) weeks later. I, (the researcher) will be responsible for the cost of the tests. Your weight, height and blood pressure will be measured too.

CONFIDENTIALITY

All information obtained in this study will be confidential. Your identity will be duly concealed by the use of numbers rather than names. The results of the test will be communicated to you on request.

SIDE EFFECTS OF THE PROCEDURE

The procedure is not accompanied with any side effect, except that the 10mls of blood that will be collected from you and pain at venipuncture site.

RENUMERATION FOR THE PARTICIPANTS

Participation will be strictly voluntary. There will be no payment to the participants, except token transport fare in indigent cases.

For further clarification, you can contact me on 08033739848 or Department of Haematology & Immunology, UNTH Ituku Ozalla.

Attached is the consent form.

------

Name and Signature of Researcher

------Date

APPENDIX II

CONSENT FORM

I…………………………….. have read the information consent form or have been educated about this study. Aimed at evaluating the presences of Antiphospholipid antibody (APLS) levels in subjects with type 2 Diabetes Mellitus (T2DM) in Enugu, South-East, Nigeria

I understand the information given for the above study.

I hereby consent to participate in this study.

………………………………………………………………

Name and Signature/Thumbprint of subject

……………………………………..

Date

…………………………………………………………………

Name and signature of Researcher

………………………………….

Date

APPENDIX III

QUESTIONNAIRE

BIODATA

Participant identification number (ID)

Folder (File) Number

Age (Nearest Future)

Sex: Male ( ) Female ( )

Occupation:-

Tribe:- Marital Status:-

Level of Education (LOE)

None ( ) Primary ( ) Secondary ( ) Tertiary ( )

CHRONIC ILLNESS

Leprosy ( ) Tuberculosis ( ) HIV /AIDS ( )

Rheumatoid /Osteo - arthritis ( ) Cancer ( ) Leukemia ( ) Lymphoma ( )

Seizure ( )

DRUG HISTORY

Corticosteroid ( ) Fansidar ( ) Methyldopa ( ) Quinine ( )

Hydralazine ( ) Amoxicillin ( ) Heparin ( ) Warfarin ( ) Propranolol ( ) Oral contraceptive ( ) Chlorpromazine ( ) Quinidine ( )

FAMILY HISTROY

Hypertension ( ) Obesity ( ) Diabetes Mellitus (T2DM) ( ) SOCIAL HISTORY

Alcohol Intake:

(i) Yes ( ) or No ( )

(ii) How long:

(iii) Quantity:

Smoking /Snuff :

(i) Yes ( ) or No ( )

(ii) How long:

(iii) Number of sticks:

DURATION OF T2DM (NEAREST FUTURE) ( )

COMPLICATIONS (COMORBIDITY) / DURATION (NEAREST YEAR)

Stroke ( ) TIA ( )

Hypertension ( ) Retinopathy ( )

Neuropathy ( ) Nephropathy ( )

PHYSICAL EXAMINATION

Blood Pressure: Systole-

(mm /Hg) Diastole-

Height (Nearest Meter) =

Weight (Nearest Kilogramme) = Body Mass Index (BMI) = Weight (Kg) / Height (M2)

LABORATORY INVESTIGATIONS First Time At Least 12 weeks Final Result apart Lupus Anticoagulant (LA) IgG β2 GP1 dependent ACA

Full Blood Count (FBC) – Platelet count (x109/l)

HbA1c (%)

Fasting Plasma Glucose (FPG) mg/dl or Random Plasma Glucose (RPG) mg/dl Micro-albuminuria (Spot collection analysis using Combi – 13)

APPENDIX IVa RAW DATA FOR SOCIAL DEMOGRAPHIC CHARACTERRISTICS FOR T2DM WITH COMPLICATIONS. T2DM WITH COMPLICATIONS

BIODATA, FAMILY HISTORY, SOCIAL HISTORY, DURATION OF T2DM, COMPLICATIONS AND PHYSICAL EXAMINATION,

)

2

),

N

),

O

pathy

TIA

Stroke

Neuropthy

), None(),

Retino

Nephropathy

Hypertension

Weight Weight

DM

), Obesity(

DM (Nearest year) (Nearest DM

Married (M) or or Married (M)

2

H

Blood Presure: Blood Presure:

S/N

Tribe

Single (S)

(Nearest Kilogramme) (Nearest

Height (Nearest Meter)Height (Nearest

Systole/Diastole (mmHg) Complications Body Mass index (Kg/M Mellitus(

Family History: Family

Level of Education Age (Nearest Age year) (Nearest (Comorbidity) / Duration

how long: (Nearest year) how long: (Nearest

Number of Sticks per per dayNumber of Sticks

how long: (Nearest howyear) long: (Nearest bottlesper Day Quantity:

Sex Sex Female(F), Male(M)

Alcohol intake: Yes( ) No( ) Yes( Alcohol ) intake: (Nearest year)

Smoking /Snuff Yes( ) No( ) Smoking Yes( ) /Snuff

Trader(T), Servant(CS) Civil

Duration of T Duration of

Hypertension(

Occupation: Housewife(H/W), Occupation: Housewife(H/W),

Marital Marital Status: Physical Examination:

Diabetes SOCIAL HISTORY 1 58 F Trader Igbo Married Primary H, DM No No 16 17 7 5 150/90 1.60 66 25 2 44 M Physcian Igbo Married Tertiary H, DM Yes 20 1 Yes 9 10 5 5 154/104 1.73 99 33 3 55 M Trader Igbo Married Tertiary H Yes 20 2 No 15 2 2 1 140/90 1.80 81 24 .2 4 61 M CS Igbo Married Secondary H, DM Yes 27 1 No 2 16 160/100 1.60 70 26 5 65 F R. Teacher Igbo Married Tertiary H,O,DM No No 1 1 160/100 1.70 81 27 6 50 F Trader Igbo Married Primary DM No No 6 6 1 1 150/100 1.60 81 31 7 53 F CS Igbo Married Tertiary DM No No 9 3 9 2 110/80 1.67 92. 33 4 8 73 F H/W Igbo Married Primary DM No No 15 5 1 1 200/100 1.60 56 23 9 52 F H/W Igbo Married Primary N No No 13 23 3 5 110/70 1.60 62 24 10 60 F H/W Igbo Married Primary H No No 14 7 2 120/80 1.70 60 20 11 47 F CS Igbo Married Tertiary H,O,DM No No 4 4 2 1 150/90 1.70 82 28 12 60 F H/W Igbo Married Primary O No No 13 13 3 2 120/80 1.60 65 29 13 53 M Lecturer Igbo Married Tertiary N No No 8 4 1 5 150/90 1.70 74 24 89

14 42 F CS Igbo Married Secondary N No No 11 11 6 11 160/90 1.60 73 28 15 63 M Engr Igbo Married Tertiary DM No No 1 21 130/90 1.80 95 29 16 68 M Trader Igbo Married Primary N Yes 33 3 No 3 11 2 2 170/90 1.70 82 28 17 72 M Farmer Igbo Married None N Yes 30 2 Yes 10 10 2 2 1 140/80 1.70 75 25 .5 18 64 M Trader Igbo Married Primary N Yes 15 1 No 5 5 120/80 1.67 91 32 19 50 F CS Igbo Married Tertiary N No No 11 10 6 5 140/80 1.50 58 25 20 50 F Trader Igbo Married Primary DM No No 4 4 120/70 1.70 71 24 21 60 M CS Igbo Married Tertiary N No No 5 5 5 5 2 150/90 1.70 75 26 22 60 F Farmer Igbo Married Primary N No No 3 3 1 3 170/100 1.70 87 29 .5 23 62 F H/W Igbo Married Primary N No No 12 8 150/80 1.60 70 27 24 55 M CS Igbo Married Secondary N No No 10 10 10 110/70 1.60 79 30 25 70 F Farmer Igbo Married Primary N No No 12 4 2 1 160/80 1.50 50 25 26 54 F Teacher Igbo Married Tertiary H, DM No No 15 5 1 5 160/80 1.70 78 26 27 31 F CS Igbo Single Tertiary H, DM Yes 8 2 No 8 8 140/90 1.70 97 34 28 56 F Trader Igbo Married Primary H, DM No No 14 4 5 14 2 150/80 1.60 50 19 29 59 M CS Igbo Married Secondary H,O,DM No No 16 11 10 8 150/90 1.80 83 25 30 72 F R. Teacher Igbo Married Tertiary DM Yes 30 1 No 10 2 7 2 152/100 1.70 65 22 31 49 F Trader Igbo Married Secondary DM No No 11 2 4 3 110/70 1.70 86 29 32 60 F CS Igbo Married Tertiary H No No 20 2 10 160/90 1.70 64 22 33 57 F Caterer Igbo Married Primary N No No 20 11 10 8 10 124/78 1.60 55 21 34 62 M Farmer Igbo Married None H No No 25 1 5 5 130/80 1.60 63 24 35 62 M CS Igbo Married Primary N Yes 4 2 No 2 2 2 150/90 1.60 52 20 36 70 F R.CS Igbo Married Secondary H, DM No No 12 10 12 170/80 1.60 70 27 37 51 M Trader Igbo Married Secondary H,O,DM No No 5 4 140/90 1.80 108 33 38 63 F Caterer Igbo Married Primary H, DM No No 27 9 1 1 168/70 1.70 80 27 39 53 M Driver Igbo Married Primary N Yes 30 1 No 8 3 2 150/90 1.70 76 25 - 2 40 84 F H/W Igbo Married Primary N No No 5 3 3 2 160/70 1.40 65 33 41 63 F R. Teacher Igbo Married Tertiary N No No 8 7 2 140/70 1.50 49 22

90

42 69 F Trader Igbo Married Primary N No No 15 10 130/80 1.60 64 25 43 78 M R. Lecturer Igbo Married Tertiary H, DM No No 25 7 8 8 7 140/90 1.70 84 29 44 68 F Farmer Igbo Married Primary H, DM No No 10 28 28 120/80 1.60 68 26 45 60 F H/W Igbo Married Primary N No No 30 30 10 180/100 1.70 86 29 46 63 F R.Secetary Igbo Married Tertiary DM Yes 15 1 No 15 11 11 1 1 210/90 1.60 74 28 47 58 M T. Rular Igbo Married Tertiary H, DM No No 5 3 3 3 140/90 1.80 75 23 48 62 M Lecturer Igbo Married Tertiary H No No 5 28 3 130/80 1.70 88 30 49 70 M Artisan Igbo Married Tertiary N Yes 53 6 Yes 5 10 3 3 2 1 150/100 1.60 67 26 50 57 F Trader Igbo Married Primary H Yes 20 1 No 2 2 5 2 120/70 1.60 78 30 51 67 F H/W Igbo Married Secondary N No No 11 10 2 2 140/80 1.70 77 26 52 50 M Driver Igbo Married Secondary N Yes 25 4 Yes 12 10 1 1 1 150/90 1.70 76 26 53 53 M CS Igbo Married Tertiary H Yes 23 6 Yes 6 20 1 2 5 160/100 1.80 89 27 54 49 F Trader Igbo Married Secondary N No No 10 7 7 3 150/80 1.70 96 33 55 52 M CS Igbo Married Secondary H Yes 5 2 Yes 5 10 8 2 3 140/100 1.60 68 28 56 53 F Lecturer Igbo Married Tertiary O, DM Yes 20 1 No 16 15 10 160/88 1.65 65 24 57 48 M Trader Igbo Married Primary N No No 12 2 8 7 100/60 1.60 70 27 58 82 M R.CS Igbo Married Secondary N Yes 25 2 Yes 20 20 16 1 30 1 120/80 1.70 70 24 - 3 59 55 F Trader Igbo Married Primary DM No No 8 15 3 120/70 1.60 65 25 60 70 M R.CS Igbo Married Tertiary H, DM Yes 20 2 Yes 20 20 15 10 8 160/100 1.60 57 22 61 60 F H/W Igbo Married Primary N No No 1 1 1 155/90 1.60 65 25 62 65 F H/W Igbo Married None N No No 14 7 160/70 1.70 58 20 63 56 M Farmer Igbo Married Primary N No No 12 1 3 4 160/90 1.70 66 23 64 50 F H/W Igbo Married Tertiary N Yes 20 1 No 3 6 1 3 160/95 1.60 80 31 65 60 M Trader Igbo Married Secondary DM Yes 30 2 No 11 11 130/80 1.64 70 26 66 78 F Farmer Igbo Married Primary H No No 14 7 5 8 150/70 1.57 70 29 .6 67 62 F H/W Igbo Married Primary N No No 6 2 160/100 1.65 72 26 68 36 M Trader Igbo Married Primary DM Yes 5 2 Yes 5 5 4 2 150/100 1.68 71 25 69 75 F H/W Igbo Married Primary N No No 20 3 8 150/90 1.64 67 25 70 65 M R.CS Igbo Married Secondary H, DM No No 20 3 6 10 145/100 1.72 69 23 91

APPENDIX IVb RAW DATA FOR SOCIAL DEMOGRAPHIC CHARACTERRISTICS FOR T2DM WITHOUT COMPLCATIONS. T2DM WITHOUT COMPLICATIONS

BIODATA, FAMILY HISTORY, SOCIAL HISTORY, DURATION OF T2DM AND PHYSICAL EXAMINATION,

),

N

),

)

O

2

), None(),

DM

), Obesity(

S/ orMarried (M)

H

History:

DM (Nearest year) (Nearest DM N Tribe

2

(mmHg)

Single (S)

(Nearest year) (Nearest

how long:

Social

Family History: Family

Level of Education

Age

Physical Examination:

Sex Sex Female(F), Male(M)

Height (Nearest Meter)Height (Nearest

Number of Sticks per per dayNumber of Sticks

Body Mass index (Kg/M

Quantity: bottles per bottlesper Day Quantity:

how long: (Nearest howyear) long: (Nearest

Trader(T), Servant(CS) Civil

Alcohol intake: Yes( ) No( ) Yes( Alcohol ) intake:

Hypertension(

Weight (Nearest Kilogramme)

Occupation: Housewife(H/W), Occupation: Housewife(H/W), No( ) Smoking Yes( ) /Snuff

Marital Marital Status:

Blood Presure: Systole/Diastole Blood Systole/Diastole Presure:

Duration of T Duration of

DiabetesMellitus( 1 48 F H/W Igbo Married Tertiary N No No 2 130/70 1.70 54 18.5 2 56 F T Igbo Married Primary N No No 5 130/80 1.70 64 22 3 54 F CS Igbo Married Secondary N No No 6 120/60 1.66 72 25 4 31 M Driver Igbo Married Secondary N Yes 5yrs 2-3 Yes 5yrs 15 4 130/80 1.70 72 25 5 53 F T Igbo Married Secondary N No No 4 100/60 1.65 66 24 6 53 M T Igbo Married Secondary DM Yes 10yrs 1-2 No 6 135/60 1.70 72 25 7 52 F Teacher Igbo Married Tertiary H No No 8 130/80 1.68 53 18 8 52 M CS Igbo Married Secondary DM Yes 5yrs 1-2 No 6 120/70 1.70 70 24 9 55 M Artisan Igbo Married Primary H, DM Yes 10yrs 3 Yes 10yr 20 5 130/70 1.70 73 25 s 10 35 M T Igbo Married Secondary DM No No 4 110/70 1.66 70 25 11 55 M CS Igbo Married Secondary N No No 6 130/80 1.72 78 26 12 50 M Cs Igbo Married Secondary DM No No 5 110/70 1.60 75 29 13 52 M CS Igbo Married Secondary O, DM No No 7 130/80 1.70 82 28 14 52 M CS Igbo Married Secondary H, DM No No 6 110/70 1.58 56 23.6 92

15 35 M Trader Igbo Married Secondary DM No No 6 120/60 1.60 64 25 16 49 M Trader Igbo Married Secondary DM No No 5 130/70 1.70 66 23 17 53 F Trader Igbo Married Secondary H, DM No No 4 120/70 1.65 67 21 18 60 M Trader Urhob Married Secondary N No No 6 120/80 1.70 55 19 o 19 37 F Caterer Igbo Married Primary N No No 11 120/80 1.60 47 18.2 20 53 M Farmer Igbo Married Primary N No No 4 130/70 1.70 56 19 21 55 F Trader Igbo Married Primary N No No 5 125/58 1.60 70 27 22 53 M Clergy Igbo Married Tertiary N No No 6 128/72 1.80 65 20 23 55 F H/W Igbo Married None N No No 6 130/70 1.60 68 26 24 60 F H/W Igbo Married Primary N No No 7 130/60 1.60 58 22 25 46 F CS Igbo Married Tertiary DM No No 6 110/60 1.50 55 24 26 48 M CS Igbo Married Secondary DM No No 5 130/60 1.70 72 24.5 27 53 M Trader Igbo Married Primary N No No 4 130/85 1.71 76 25 28 50 F Teacher Igbo Married Tertiary N No No 4 120/80 1.65 69 25 29 56 M CS Igbo Married Secondary DM Yes 5yrs 1-2 No 7 110/80 1.63 67 25 30 53 M CS Igbo Married Secondary H, DM No No 4 100/60 1.57 66 26 31 46 F CS Igbo Married Secondary DM No No 4 130/80 1.69 71 24 32 55 F Trader Igbo Married Secondary DM Yes 5yrs 3 No 6 100/70 1.67 68 24 33 45 F Cs Igbo Married Secondary H, DM No No 4 130/70 1.63 65 24 34 44 M Clergy Igbo Married Tertiary DM No No 5 110/80 1.70 70 24 35 53 F CS Igbo Married Secondary DM No No 6 130/70 1.60 70 27 36 50 M CS Igbo Married Secondary DM No No 3 120/70 1.67 76 27 37 43 F CS Igbo Married Tertiary H, DM No No 2 120/70 1.62 63 24 38 42 M CS Igbo Married Secondary DM No No 2 125/70 1.56 65 26 39 45 F CS Igbo Married Secondary H,O,D No No 4 130/70 1.63 79 29 M 40 55 F Trader Igbo Married Secondary DM No No 3 120/60 1.61 68 26 41 41 F CS Igbo Married Tertiary DM Yes 6yrs 1 No 2 130/70 1.68 75 21 42 52 M CS Igbo Married Tertiary H,O,D No No 4 127/80 1.68 69 24 93

M 43 42 F CS Igbo Married Tertiary H,O,D No No 3 120/70 1.61 74 28 M 44 52 M CS Igbo Married Tertiary H,O,D Yes 10 2 Yes 10 5-8 4 130/80 1.69 86 29.5 M 45 42 M CS Igbo Married Tertiary H, DM No No 3 125/70 1.72 93 31 46 50 M Lecturer Igbo Married Tertiary H,O,D Yes 10 1-2 Yes 10 5-7 4 125/80 1.74 95 31 M 47 50 M Lecturer Igbo Married Tertiary H, DM No No 4 120/70 1.64 81 29 48 47 M CS Igbo Married Secondary H,O,D No No 4 110/70 1.53 77 32 M 49 43 M CS Igbo Married Tertiary H, DM Yes 6 1-2 No 3 130/70 1.72 89 29 50 45 F Trader Igbo Married Secondary DM No No 4 127/70 1.68 74 26 51 44 F Trader Igbo Married Secondary DM No No 3 115/60 1.52 73 31 52 44 F CS Igbo Married Tertiary H, DM No No 3 124/70 1.61 75 28 53 43 F CS Igbo Married Tertiary H, DM No No 3 132/80 1.61 75 28 54 52 F CS Igbo Married Tertiary H, DM No No 3 115/70 1.62 79 26 55 49 F CS Igbo Married Tertiary H, DM No No 3 125/72 1.68 83 29 56 48 M CS Igbo Married Tertiary DM Yes 6 1-2 Yes 6 3 4 120/70 1.72 89 29 57 50 F CS Igbo Married Secondary N No No 4 110/70 1.70 58 20 58 47 F CS Igbo Married Tertiary H,O,D No No 3 110/70 1.65 77 28 M 59 42 F CS Igbo Married Tertiary H,O,D No No 3 122/70 1.66 92 33 M 60 44 F CS Igbo Married Tertiary H,O,D No No 3 120/70 1.67 95 34 M 61 42 F CS Igbo Married Tertiary H,O,D No No 3 125/70 1.66 73 26 M 62 31 F CS Igbo Married Tertiary H, DM No No 2 120/60 1.70 68 23 63 47 M CS Ibibi Married Tertiary N No No 6 110/60 1.63 73 27 64 50 M CS Igbo Married Tertiary H, DM No No 4 130/80 1.70 71 24 65 46 F CS Igbo Married Tertiary DM No No 4 130/80 1.60 58 22 94

66 53 F CS Igbo Married Tertiary DM No No 3 120/84 1.60 61 23 67 57 M CS Igbo Married Tertiary H, DM No No 5 135/85 1.63 67 25 68 44 M CS Igbo Married Tertiary N No No 3 120/60 1.70 68 23 69 66 M Retiree Igbo Married Secondary H, DM No No 5 130/70 1.60 58 22 70 62 F H/W Igbo Married Primary H, DM No No 4 130/70 1.65 65 27

95

APPENDIX IVc RAW DATA FOR SOCIAL DEMOGRAPHIC CHARACTERRISTICS FOR HEALTHY CONTROL SUBJECTS. HEALTHY CONTROL SUBJECTS BIODATA, FAMILY HISTORY, PHYSICAL EXAMINATION,

(mmHg)

abetes abetes

)

2

),

N

), Di

O

), None(

Tribe

DM

), Obesity(

Married (M) or Single (S) orMarried Single (M)

how long:

H

Servant(CS)

Social History:

Family History: Family

Level of Education

Physical Examination:

Sex Sex Female(F), Male(M)

Mellitus(

Height (Nearest Meter)Height (Nearest

Number of Sticks per per dayNumber of Sticks

Body Mass index (Kg/M

Quantity: bottles per bottlesper Day Quantity:

how long: (Nearest howyear) long: (Nearest

Alcohol intake: Yes( ) No( ) Yes( Alcohol ) intake:

Weight (Nearest Kilogramme)

Smoking /Snuff Yes( ) No( ) Smoking Yes( ) /Snuff

Hypertension(

Marital Marital Status:

Blood Presure: Systole/Diastole Blood Systole/Diastole Presure:

Occupation: Housewife(H/W), Trader(T), Civil Occupation: Housewife(H/W), F CS Igbo Married Tertiary H No No 130/70 1.50 61 27 F CS Igbo Single Secondary N No No 120/80 1.66 56 20 F CS Igbo Married Secondary N No No 130/80 1.70 99 34 F CS Igbo Married Tertiary N No No 110/70 1.64 82 30 F Business Igbo Married Secondary H, DM No No 120/60 1.60 130 50 F Student Igbo Single Tertiary DM No No 110/70 1.70 59 20 M Student Igbo Single Tertiary N No No 100/60 1.70 58 20 M Trader Igbo Married Tertiary N No No 130/80 1.70 87 29 M CS Igbo Married Secondary DM No No 140/80 1.71 68 23 M Student Igbo Single Tertiary DM No No 130/80 1.74 72 23 M Trader Igbo Married Secondary H, DM Yes 3yrs 1-2 Yes 3yrs 3 120/70 1.80 89 27 F Student Igbo Single Tertiary N No No 120/70 1.71 60 20 96

F Student Igbo Single Tertiary N No No 120/80 1.74 64 23 M Trader Igbo Married Secondary H No No 120/80 1.70 64 22 M Trader Igbo Married Secondary H, DM Yes 5yrs 3 Yes 5yrs 10 120/70 1.73 68 22 M Student Igbo Single Tertiary H No No 100/80 1.75 70 23 M Business Igbo Married Secondary H Yes 7yrs 1-2 Yes 7yrs 10 100/70 1.70 79 25 M Student Igbo Single Tertiary N No No 130/60 1.74 67 22 M Student Igbo Single Tertiary N No No 130/60 1.85 25 22 M Trader Igbo Married Secondary N No No 120/80 1.71 64 22 M Student Igbo Single Tertiary N No No 120/60 1.73 68 22 M Student Igbo Single Tertiary N No No 120/80 1.80 83 25 M Student Igbo Single Tertiary N No No 130/80 1.76 77 24 M Student Igbo Single Tertiary N No No 120/70 1.73 63 21 M CS Igbo Married Tertiary N No No 130/80 1.71 87 29 M CS Igbo Married Tertiary N No No 110/70 1.80 76 23 M Trader Igbo Married Secondary N No No 110/80 1.70 72 24 M Student Igbo Single Secondary N Yes 2yrs 1 No 120/70 1.80 72 22 M Driver Igbo Single Secondary N Yes 3yrs 3-4 No 110/70 1.80 65 20 M CS Igbo Single Tertiary N Yes 6yrs 3 No 110/70 1.80 66 20 M Student Igbo Single Tertiary N No No 120/60 1.80 66 20 M Business Igbo Married Secondary N Yes 10yrs 2 Yes 4yrs 2 130/90 1.70 53 18.1 M Trader Igbo Married Secondary N No No 130/80 1.80 95 29 M Student Igbo Single Tertiary N No No 120/70 1.73 63 22 M Student Igbo Single Secondary N No No 120/80 1.80 66 20 M Business Igbo Married Tertiary N No No 120/80 1.80 88 27 M Student Igbo Single Tertiary N Yes 3yrs 3 No 120/70 1.80 65 20 M Student Igbo Single Secondary N No No 140/70 1.80 69 21 M Trader Igbo Married Secondary N Yes 10yrs 1 No 115/80 1.70 70 24 F CS Igbo Married Tertiary O No No 140/95 1.70 115 39.4 M ENGR Igbo Single Tertiary N No No 110/70 1.70 63 23 M Student Igbo Single Secondary DM No No 130/70 1.80 63 19 F Student Igbo Single Tertiary DM Yes 5yrs 1 No 110/80 1.60 59 22.5 97

M ENGR Igbo Married Tertiary DM No No 130/80 1.60 73 26 M Business Igbo Single Primary N No No 120/60 1.90 69 19 F CS Igbo Married Tertiary N No No 110/70 1.70 74 25 F CS Igbo Married Tertiary H,O,DM No No 128/80 1.70 78 27 M Student Igbo Single Tertiary N No No 103/70 1.73 52 17.2 M CS Igbo Married Tertiary N Yes 10yrs 3 No 130/80 1.90 86 23 M Business Igbo Married Tertiary N No No 130/80 1.80 88 22 M Business Igbo Married Secondary N No No 120/80 1.80 75 23 M CS Igbo Married Tertiary N Yes 20yrs 1 Yes 15yrs 10 130/80 1.70 65 22 M Student Igbo Single Tertiary N Yes 1yrs 1 No 110/70 1.80 63 19 F CS Igbo Married Tertiary H, DM No No 135/80 1.60 70 22 M Artisan Igbo Single Secondary N Yes 2yrs 10 No 130/65 1.90 80 22 M Artisan Igbo Married Tertiary N No No 120/80 1.90 102 28 F CS Igbo Married Tertiary H No No 110/80 1.63 93 34.5 M Student Igbo Single Tertiary N No No 120/70 1.80 64 20 M Student Igbo Single Tertiary N No No 120/75 1.90 70 19 M Student Igbo Single Tertiary N Yes 3yrs 1 No 110/70 1.81 66 20 M Student Igbo Single Secondary N No No 120/80 1.74 70 23 M Student Igbo Single Tertiary N No No 110/70 1.80 73 22 M Student Igbo Single Tertiary N No No 110/60 1.72 64 21 M Student Igbo Single Tertiary N No No 110/80 1.84 73 21 M Driver Igbo Married Secondary N Yes 5yrs 2-3 Yes 5yrs 5 130/90 1.64 71 21 M Trader Igbo Married Secondary N Yes 10yrs 3-4 Yes 10yrs 10 110/80 1.70 72 25 M Student Igbo Single Tertiary N No No 120/80 1.84 85 25 M Student Igbo Single Tertiary N No No 120/70 1.78 68 21 M Student Igbo Single Tertiary N No No 120/70 1.85 70 20 M Student Igbo Single Tertiary N No No 120/80 1.70 60 21

98

APPENDIX Vai RAW DATA FOR LA AMONG T2DM WITH COMPLCATIONS (FIRST TIME). T2DM WITH COMPLICATIONS (LA ASSAY) (FIRST TIME) SPECIMEN T1 T2 T1 T 2 AVERAGE (AVE) T 2

Control Plasma Platelet Poor plasma (PPP) 32 30 31

T2

T1

T2

Ave 'T'

T1

Ave"T"

LA ASSAY

T2

T1 CONCLUSION

S/N Ave 'T' (POSITIVE / LA LA MIXING TEST RATIO LA NEGAATIVE)

lotting time of test of time lotting

Screen clotting time clotting Screen LA Screem ratio LA Screem CONFIRMATORY clotting time of test clotting time of 50:50

clotting time clotting time of control

LA LA

Clotting time of control of time Clotting

(ProlongNormal)or (ProlongNormal)or

LA Screening Remark LA Screening Remark LA Screening Remark TEST

LA Confirm clotting time clotting Confirm LA

= C =

(TEST:CONTROL) = LA SCREENING 1 28 27 27.5 27.5 31 0.89 Normal Negative 2 36 34 35 35 31 1.13 Normal Negative 3 38 36 37 37 31 1.19 Normal Negative 4 36 34 35 35 31 1.13 Normal Negative 5 36 36 36 36 31 1.16 Normal Negative 6 31 29.5 30 30 31 0.97 Normal Negative 7 35 33 34 34 31 1.10 Normal Negative 8 33 31 32 32 31 1.03 Normal Negative 9 36 32 34 34 31 1.10 Normal Negative 10 37 35 36 36 31 1.16 Normal Negative 11 30 31 30.5 30.5 31 0.98 Normal Negative 12 33 35 34 34 31 1.10 Normal Negative 13 36 35 35.5 35.5 31 1.15 Normal Negative 14 36 34 35 35 31 1.13 Normal Negative 99

15 35 33 34 34 31 1.10 Normal Negative 16 39 37 38 38 31 1.23 Prolong 33 31 32 Normal Negative 17 29 28 28.5 28.5 31 0.92 Normal Negative 18 38 36 37 37 31 1.19 Normal Negative 19 29 27 28 28 31 0.90 Normal Negative 20 37 35 36 36 31 1.16 Normal Negative 21 31 29 30 30 31 0.97 Normal Negative 22 40 38 39 39 31 1.26 Prolong 34 32 33 Normal Negative 23 31 29 30 30 31 0.97 Normal Negative 24 28 26 27 27 31 0.87 Normal Negative 25 35 33 34 34 31 1.10 Normal Negative 26 32 30 31 31 31 1.00 Normal Negative 27 36 34 35 35 31 1.13 Normal Negative 28 35 33 34 34 31 1.10 Normal Negative 29 37 35 36 36 31 1.16 Normal Negative 30 28 26 27 27 31 0.87 Normal Negative 31 38 36 37 37 31 1.19 Normal Negative 32 34 32 33 33 31 1.06 Normal Negative 33 >300 >300 >300 >300 31 >9.68 Prolong 216 214 215 Prolong 43 41 42 >300/41 = >7.32 Positive 34 28 30 29 29 31 0.94 Normal Negative 35 34 32 33 33 31 1.06 Normal Negative 36 36 34 35 35 31 1.13 Normal Negative 37 35 33 34 34 31 1.10 Normal Negative 38 >300 >300 >300 >300 31 >9.68 Prolong 210 208 209 Prolong 46 44 45 >300/45 = >6.67 Positive 39 28 26 27 27 31 0.87 Normal Negative 40 34 32 33 33 31 1.06 Normal Negative 41 32 30 31 31 31 1.00 Normal Negative 42 33 31 32 32 31 1.03 Normal Negative 43 29 27 28 28 31 0.90 Normal Negative 44 32 30 31 31 31 1.00 Normal Negative 45 36 38 37 37 31 1.19 Normal Negative 100

46 34 32 33 33 31 1.06 Normal Negative 47 36 34 35 35 31 1.13 Normal Negative 48 31 29 30 30 31 0.97 Normal Negative 49 28 26 27 27 31 0.87 Normal Negative 50 36 34 35 35 31 1.13 Normal Negative 51 31 29 30 30 31 0.97 Normal Negative 52 35 33 34 34 31 1.10 Normal Negative 53 38 36 37 37 31 1.19 Normal Negative 54 35 33 34 34 31 1.10 Normal Negative 55 36 34 35 35 31 1.13 Normal Negative 56 35 33 34 34 31 1.10 Normal Negative 57 33 31 32 32 31 1.03 Normal Negative 58 34 33 33.5 33.5 31 1.08 Normal Negative 59 30 28 29 29 31 0.94 Normal Negative 60 35 33 34 34 31 1.10 Normal Negative 61 32 30 31 31 31 1.00 Normal Negative 62 >300 >300 >300 >300 31 >9.68 Prolong 230 228 229 Prolong 41 39 40 >300/40 = >7.50 Positive 63 32 30.5 31 31 31 1.00 Normal Negative 64 36 34 35 35 31 1.13 Normal Negative 65 34 32 33 33 31 1.06 Normal Negative 66 45 43 44 44 31 1.42 Prolong 35 33 34 Normal Negative 67 28 26 27 27 31 0.87 Normal Negative 68 34 32 33 33 31 1.06 Normal Negative 69 >300 >300 >300 >300 31 >9.68 Prolong 210 218 219 Prolong 44 46 45 >300/45 = >6.67 Positive 70 >300 >300 >300 >300 31 >9.68 Prolong 200 198 199 Prolong 43 41 42 >300/42 = >7.14 Positive

101

APPENDIX Vaii RAW DATA FOR LA AMONG T2DM WITH COMPLCATIONS (12 WEEKS LATER).

T2DM WITH COMPLICATIONS (LA ASSAY) (12 weeks later) SPECIMEN T1 T2 T1 T 2 AVERAGE (AVE) T 2

Control Plasma Platelet Poor plasma (PPP) 34 32 33

LA ASSAY

CONCLUSION

(POSITIVE /

T1 T2 NEGAATIVE)

T2

Ave 'T'

Remark Remark

T1

Ave"T"

T2

T1

S/N Ave 'T'

LA RATIO LA

lotting time of test of time lotting

LA Screem ratio LA Screem

clotting time of test clotting time of LA

clotting time clotting time of control LA MIXING TEST time clotting Screen LA Clotting time of control of time Clotting

(ProlongNormal)or (ProlongNormal)or

LA Screening Remark LA Screening Remark LA Screening

LA Confirm clotting time clotting Confirm LA = C = CONFIRMATORY

= 50:50 TEST LA SCREENING (TEST:CONTROL) 33 34 32 33 33 33 1.00 Normal Negative

38 >300 >300 >300 >300 33 >9.09 Prolong 180 179 180 Prolong 45 43 44 >300/44 = >6.82 Positive

62 >300 >300 >300 >300 33 >9.09 Prolong 200 198 199 Prolong 44 42 43 >300/43 = >6.98 Positive

69 >300 >300 >300 >300 33 >9.09 Prolong 210 208 209 Prolong 43 41 42 >300/42 = >7.14 Positive

70 >300 >300 >300 >300 33 >9.09 Prolong 170 168 169 Prolong 42 41 41.5 >300/41.5 = Positive >7.23

102

APPENDIX V bi RAW DATA FOR IgG - β2GPI T2DM WITH COMPLCATIONS (FIRST TIME).

T2DM WITH COMPLICATIONS

(IgG - B2GPI - DEPENDENT ACA ASSAY (FIRST TIME) S/N OPTICAL DATA 1 DATA 2 CONCENTRATION OF IgG DENSITY ACA (AU/mL) (OD) 1 0.087 29.22 2.86 -0.31786 2 0.100 29.22 2.86 0.062 3 0.397 29.22 2.86 8.74034 4 0.258 29.22 2.86 4.67876 5 0.181 29.22 2.86 2.42882 6 0.137 29.22 2.86 1.14314 7 0.089 29.22 2.86 -0.25942 8 0.844 29.22 2.86 21.80168 9 0.262 29.22 2.86 4.79564 10 0.102 29.22 2.86 0.12044 11 0.220 29.22 2.86 3.5684 12 0.133 29.22 2.86 1.02626 13 0.494 29.22 2.86 11.57468 14 0.118 29.22 2.86 0.58796 15 0.187 29.22 2.86 2.60414 16 0.220 29.22 2.86 3.5684 17 0.061 29.22 2.86 -1.07758 18 0.106 29.22 2.86 0.23732 19 0.096 29.22 2.86 -0.05488 20 0.106 29.22 2.86 0.23732 21 0.134 29.22 2.86 1.05548 22 0.119 29.22 2.86 0.61718 23 0.169 29.22 2.86 2.07818 24 0.091 29.22 2.86 -0.20098 25 0.185 29.22 2.86 2.5457 26 0.102 29.22 2.86 0.12044 27 0.137 29.22 2.86 1.14314 28 0.095 29.22 2.86 -0.0841 29 0.142 29.22 2.86 1.28924 30 0.294 29.22 2.86 5.73068 31 0.108 29.22 2.86 0.29576 32 2.271 29.22 2.86 63.49862 33 0.425 29.22 2.86 9.5585 34 0.208 29.22 2.86 3.21776 35 0.127 29.22 2.86 0.85094 103

36 0.142 29.22 2.86 1.28924 37 0.077 29.22 2.86 -0.61006 38 0.167 29.22 2.86 2.01974 39 0.239 29.22 2.86 4.12358 40 0.098 29.22 2.86 0.00356 41 0.235 29.22 2.86 4.0067 42 0.176 29.22 2.86 2.28272 43 0.129 29.22 2.86 0.90938 44 0.137 29.22 2.86 1.14314 45 0.177 29.22 2.86 2.31194 46 0.104 29.22 2.86 0.17888 47 0.101 29.22 2.86 0.09122 48 0.101 29.22 2.86 0.09122 49 0.131 29.22 2.86 0.96782 50 0.079 29.22 2.86 -0.55162 51 0.308 29.22 2.86 6.13976 52 0.098 29.22 2.86 0.00356 53 0.239 29.22 2.86 4.12358 54 0.083 29.22 2.86 -0.43474 55 0.142 29.22 2.86 1.28924 56 0.254 29.22 2.86 4.56188 57 0.116 29.22 2.86 0.52952 58 0.129 29.22 2.86 0.90938 59 0.397 29.22 2.86 8.74034 60 0.294 29.22 2.86 5.73068 61 0.099 29.22 2.764 0.12878 62 0.134 29.22 2.764 1.15148 63 0.254 29.22 2.764 4.65788 64 0.222 29.22 2.764 3.72284 65 0.293 29.22 2.764 5.79746 66 0.257 29.22 2.764 4.74554 67 0.129 29.22 2.764 1.00538 68 0.156 29.22 2.764 1.79432 69 0.391 29.22 2.764 8.66102 70 0.420 29.22 2.764 9.5084

104

APPENDIX V bii RAW DATA FOR IgG - B2GPI T2DM WITH COMPLCATIONS (12 WEEKS LATER). T2DM WITH COMPLICATIONS

(IgG - B2GPI - DEPENDENT ACA ASSAY (12 WEEKS LATER) S/N OPTICAL DENSITY DATA 1 DATA 2 CONCENTRATION (OD) OF IgG ACA (AU/mL)

3 0.397 29.22 2.764 8.83634

8 0.836 29.22 2.764 21.66392

13 0.489 29.22 2.764 11.52458

32 2.257 29.22 2.764 63.18554

33 0.449 29.22 2.764 10.35578

59 0.441 29.22 2.764 10.12202

69 0.46 29.22 2.764 10.6772

70 0.51 29.22 2.764 12.1382

105

APPENDIX VI ai RAW DATA FOR LA AMONG T2DM WITHOUT COMPLCATIONS (FIRST TIME). T2 DM WITHOUT COMPLICATION (LA ASSAY) (FIRST TIME) T1 T2 AVERAGE (AVE) T T1 T 2 SPECIMEN 2 Control Plasma Platelet Poor plasma (PPP) 35 33 34

LA

ASSAY

T2 T2

T1 T1

Ave 'T' CONCL

Ave"T"

T2 T1 USION S/N Ave 'T' (POSITI

LA RATIO LA VE /

lotting time of test of time lotting

clotting time of test clotting time of LA Screem ratio LA Screem LA MIXING TEST LA clotting time clotting time of control NEGAA

LA Screen clotting time clotting Screen LA

Clotting time of control of time Clotting

(ProlongNormal)or (ProlongNormal)or

LA Screening Remark LA Screening Remark LA Screening Remark

LA Confirm clotting time clotting Confirm LA

= C = 50:50 CONFIRMATORY

= TIVE) LA SCREENING (TEST:CONTROL) TEST 1 38 36 37 37 34 1.09 Normal Negative 2 35 33 34 34 34 1.00 Normal Negative 3 36 35 35.5 35.5 34 1.04 Normal Negative 4 34 32 33 33 34 0.97 Normal Negative 5 39 38 38.5 38.5 34 1.13 Normal Negative 6 35 34 34.5 34.5 34 1.01 Normal Negative 7 37 35 36 36 34 1.06 Normal Negative 8 35 34 34.5 34.5 34 1.01 Normal Negative 9 38 36 37 37 34 1.09 Normal Negative 10 32 30 31 31 34 0.91 Normal Negative 11 39 37 38 38 34 1.12 Normal Negative 12 38 36 37 37 34 1.09 Normal Negative 13 33 31 32 32 34 0.94 Normal Negative

106

14 35 34 34.5 34.5 34 1.01 Normal Negative 15 33 32 32.5 32.5 34 0.96 Normal Negative 16 36 35 35.5 35.5 34 1.04 Normal Negative 17 37 36 36.5 36.5 34 1.07 Normal Negative 18 36 34 35 35 34 1.03 Normal Negative 19 40 38 39 39 34 1.15 Normal Negative 20 39 37 38 38 34 1.12 Normal Negative 21 36 35 35.5 35.5 34 1.04 Normal Negative 22 35 33 34 34 34 1.00 Normal Negative 23 34 33 33.5 33.5 34 0.99 Normal Negative 24 36 35 35.5 35.5 34 1.04 Normal Negative 25 37 35 36 36 34 1.06 Normal Negative 26 38 36 37 37 34 1.09 Normal Negative 27 38 37 37.5 37.5 34 1.10 Normal Negative 28 38 36 37 37 34 1.09 Normal Negative 29 37 35 36 36 34 1.06 Normal Negative 30 34 32 33 33 34 0.97 Normal Negative 31 36 35 35.5 35.5 34 1.04 Normal Negative 32 38 36 37 37 34 1.09 Normal Negative 33 34 32 33 33 34 0.97 Normal Negative 34 35 34 34.5 34.5 34 1.01 Normal Negative 35 38 36 37 37 34 1.09 Normal Negative 36 35 33 34 34 34 1.00 Normal Negative 37 30 32 31 31 34 0.91 Normal Negative 38 38 37 37.5 37.5 34 1.10 Normal Negative 39 >300 >300 >300 >300 34 8.80 Prolong 215 213 214 Prolong 50 48 49 >300/49 = >6.12 Positive 40 37 36 36.5 36.5 34 1.07 Normal Negative 41 34 32 33 33 34 0.97 Normal Negative 42 34 32 33 33 34 0.97 Normal Negative

107

43 36 34 35 35 34 1.03 Normal Negative 44 34 33 33.5 33.5 34 0.99 Normal Negative 45 37 35 36 36 34 1.06 Normal Negative 46 >300 >300 >300 >300 34 8.80 Prolong 210 208 209 Prolong 46 44 45 >300/45 = >6.67 Positive 47 44 42 43 43 34 1.26 Prolong 36 34 35 Normal Negative 48 38 37 37.5 37.5 34 1.10 Normal Negative 49 38 35 36.5 36.5 34 1.07 Normal Negative 50 37 35 36 36 34 1.06 Normal Negative 51 33 31 32 32 34 0.94 Normal Negative 52 39 38 38.5 38.5 34 1.13 Normal Negative 53 34 32 33 33 34 0.97 Normal Negative 54 35 33 34 34 34 1.00 Normal Negative 55 38 37 37.5 37.5 34 1.10 Normal Negative 56 36 38 37 37 34 1.09 Normal Negative 57 37 36 36.5 36.5 34 1.07 Normal Negative 58 >300 >300 >300 >300 34 8.80 Prolong 200 198 199 Prolong 48 46 47 >300/47 = >6.38 Positive 59 40 38 39 39 34 1.15 Normal Negative 60 34 32 33 33 34 0.97 Normal Negative 61 37 36 36.5 36.5 34 1.07 Normal Negative 62 39 37 38 38 34 1.12 Normal Negative 63 36 35 35.5 35.5 34 1.04 Normal Negative 64 38 37 37.5 37.5 34 1.10 Normal Negative 65 38 36 37 37 34 1.09 Normal Negative 66 38 37 37.5 37.5 34 1.10 Normal Negative 67 37 35 36 36 34 1.06 Normal Negative 68 39 37 38 38 34 1.12 Normal Negative 69 37 33 35 35 34 1.03 Normal Negative 70 34 31 32.5 32.5 34 0.96 Normal Negative

108

APPENDIX VI aii RAW DATA FOR LA AMONG T2DM WITHOUT COMPLCATIONS (12 WEEKS LATER).

T2 DM WITHOUT COMPLICATION (LA ASSAY) (12 weeks later) SPECIMEN T1 T2 AVERAGE (AVE) T T1 T 2 2 Control Plasma Platelet Poor plasma (PPP) 35 33 34

LA ASSAY

CONCLUSION

(POSITIVE /

LA RATIO LA

T2 T2

T1 T1 NEGAATIVE)

Ave 'T'

Ave"T"

T1 T2

S/N Ave 'T'

LA Screen clotting time clotting Screen LA

lotting time of test of time lotting

clotting time of test clotting time of

LA Screem ratio LA Screem

LA MIXING TEST LA time clotting Confirm LA

clotting time clotting time of control

=

Clotting time of control of time Clotting

(ProlongNormal)or (ProlongNormal)or

LA Screening Remark LA Screening Remark LA Screening Remark

= C = 50:50 CONFIRMATORY LA SCREENING (TEST:CONTROL) TEST 19 38 36 37 37 34 1.09 Normal Negative

39 >300 >300 >300 >300 34 8.82 Prolong 24 212 213 Prolong 48 46 45 >300/45 = >6.66 Positive

46 >300 >300 >300 >300 34 8.82 Prolong 224 222 223 Prolong 44 42 43 >300/34 = >8.82 Positive

58 >300 >300 >300 >300 34 8.82 Prolong 212 210 211 Prolong 50 48 49 >300/49 = >6.12 Positive

59 38 36 37 37 34 1.09 Normal Negative

109

APPENDIX VI bi RAW DATA FOR IgG - β2GPI –ACA T2DM WITHOUT COMPLCATIONS (FIRST TIME).

T2DM WITHOUT COMPLICATION

(IgG - B2GPI - DEPENDENT ACA ASSAY (FIRST TIME) S/N OPTICAL DENSITY DATA 1 DATA 2 CONCENTRATION OF IgG (OD) ACA (AU/mL) 1 0.158 29.22 2.86 1.75676 2 0.181 29.22 2.764 2.52482 3 0.312 29.22 2.764 6.35264 4 0.086 29.22 2.764 -0.25108 5 0.127 29.22 2.764 0.94694 6 0.177 29.22 2.764 2.40794 7 0.118 29.22 2.764 0.68396 8 0.133 29.22 2.764 1.12226 9 0.184 29.22 2.764 2.61248 10 0.153 29.22 2.764 1.70666 11 0.217 29.22 2.764 3.57674 12 0.312 29.22 2.764 6.35264 13 0.112 29.22 2.764 0.50864 14 0.199 29.22 2.764 3.05078 15 0.083 29.22 2.764 -0.33874 16 0.107 29.22 2.86 0.26654 17 0.129 29.22 2.86 0.90938 18 0.208 29.22 2.86 3.21776 19 1.042 29.22 2.86 27.58724 20 0.114 29.22 2.86 0.47108 21 0.096 29.22 2.86 -0.05488 22 0.102 29.22 2.86 0.12044 23 0.118 29.22 2.86 0.58796 24 0.119 29.22 2.86 0.61718 25 0.258 29.22 2.86 4.67876 26 0.105 29.22 2.7645 0.3036 27 0.127 29.22 2.86 0.85094 28 0.139 29.22 2.86 1.20158 29 0.208 29.22 2.764 3.31376 30 0.312 29.22 2.764 6.35264 31 0.286 29.22 2.764 5.59292 32 0.15 29.22 2.764 1.619 33 0.102 29.22 2.764 0.21644 110

34 0.133 29.22 2.764 1.12226 35 0.211 29.22 2.764 3.40142 36 0.112 29.22 2.764 0.50864 37 0.155 29.22 2.764 1.7651 38 0.105 29.22 2.764 0.3041 39 1.026 29.22 2.764 27.21572 40 0.187 29.22 2.764 2.70014 41 0.113 29.22 2.764 0.53786 42 0.23 29.22 2.764 3.9566 43 0.208 29.22 2.764 3.31376 44 0.131 29.22 2.764 1.06382 45 0.111 29.22 2.764 0.47942 46 0.164 29.22 2.764 2.02808 47 0.181 29.22 2.764 2.52482 48 0.135 29.22 2.764 1.1807 49 0.112 29.22 2.764 0.50864 50 0.109 29.22 2.764 0.42098 51 0.133 29.22 2.764 1.12226 52 0.108 29.22 2.764 0.39176 53 0.181 29.22 2.764 2.52482 54 0.198 29.22 2.764 3.02156 55 0.166 29.22 2.764 2.08652 56 0.222 29.22 2.764 3.72284 57 0.128 29.22 2.764 0.97616 58 0.155 29.22 2.764 1.7651 59 1.123 29.22 2.764 30.05006 60 0.343 29.22 2.764 7.25846 61 0.256 29.22 2.764 4.71632 62 0.315 29.22 2.764 6.4403 63 0.218 29.22 2.764 3.60596 64 0.132 29.22 2.764 1.09304 65 0.116 29.22 2.764 0.62552 66 0.192 29.22 2.764 2.84624 67 0.133 29.22 2.764 1.12226 68 0.119 29.22 2.764 0.71318 69 0.131 29.22 2.764 1.06382 70 0.204 29.22 2.764 3.19688

111

APPENDIX VI bii RAW DATA FOR IgG - β2GPI –ACA T2DM WITHOUT COMPLCATIONS (12 WEEKS LATER).

T2DM WITHOUT COMPLICATION

(IgG - B2GPI - DEPENDENT ACA ASSAY (12 WEEKS LATER) S/N OPTICAL DATA 1 DATA 2 CONCENTRATION OF IgG ACA DENSITY (OD) (AU/mL) 19 1.034 29.22 2.86 27.35348

39 1.018 29.22 2.764 26.98196

46 0.211 29.22 2.764 3.40142

58 0.103 29.22 2.764 0.24566

59 1.103 29.22 2.764 29.46566

112

APPENDIX VII ai RAW DATA FOR LA AMONG HEALTHY CONTROL SUBJECT HEALTHY CONTROL SUBJECT (LA ASSAY) SPECIMEN T1 T2 T1 T 2 AVERAGE (AVE) T 2 Control Plasma Platelet Poor plasma (PPP) 32 30 31

LA

ASSAY

T2 T2

T1 T1

CONCLU

Ave 'T'

Ave"T"

T2

T1 SION

S/N Ave 'T' (POSITI

LA MIXING TEST LA RATIO LA VE /

lotting time of test of time lotting

Screen clotting time clotting Screen

LA Screem ratio LA Screem clotting time of test clotting time of 50:50 CONFIRMATORY NEGAAT

clotting time clotting time of control

LA LA

Clotting time of control of time Clotting

(ProlongNormal)or (ProlongNormal)or

LA Screening Remark LA Screening Remark LA Screening Remark

LA Confirm clotting time clotting Confirm LA

= C =

(TEST:CONTROL) TEST = IVE) LA SCREENING 1 34 32 33 33 31 1.06 Normal Negative 2 37 35 36 36 31 1.16 Normal Negative 3 34 32 33 33 31 1.06 Normal Negative 4 38 36 37 37 31 1.19 Normal Negative 5 37 35 36 36 31 1.16 Normal Negative 6 33 31 32 32 31 1.03 Normal Negative 7 35 33 34 34 31 1.10 Normal Negative 8 31 29 30 30 31 0.97 Normal Negative 9 35 33 34 34 31 1.10 Normal Negative 10 32 30 31 31 31 1.00 Normal Negative 11 >300 >300 >300 >300 31 > 9.68 Prolong 180 178 179 Prolong 46 40 43 >300/43 = >6.98 Positive 12 >300 >300 >300 >300 31 > 9.68 Prolong 150 148 149 Prolong 40 38 39 >300/39 = >7.69 Positive 13 34 32 33 33 31 1.06 Normal Negative 14 32 30 31 31 31 1.00 Normal Negative 15 37 35 36 36 31 1.16 Normal Negative

113

16 30.5 29.5 30 30 31 0.97 Normal Negative 17 28 30 29 29 31 0.94 Normal Negative 18 28 26 27 27 31 0.87 Normal Negative 19 34 32 33 33 31 1.06 Normal Negative 20 35 33 34 34 31 1.10 Normal Negative 21 32 30 31 31 31 1.00 Normal Negative 22 35 33 34 34 31 1.10 Normal Negative 23 37 35 36 36 31 1.16 Normal Negative 24 32 31 31.5 32 31 1.02 Normal Negative 25 28 30 29 29 31 0.94 Normal Negative 26 32 30 31 31 31 1.00 Normal Negative 27 36 34 35 35 31 1.13 Normal Negative 28 35 33 34 34 31 1.10 Normal Negative 29 28 30 29 29 31 0.94 Normal Negative 30 28 26 27 27 31 0.87 Normal Negative 31 40 38 39 39 31 1.26 Prolong 34 32 33 reduced Negative 32 38 36 37 37 31 1.19 Normal Negative 33 28 26 27 27 31 0.87 Normal Negative 34 39 37 38 38 31 1.23 Prolong 33 31 32 reduced Negative 35 35 33 34 34 31 1.10 Normal Negative 36 33 31 32 32 31 1.03 Normal Negative 37 30 28 29 29 31 0.94 Normal Negative 38 36 34 35 35 31 1.13 Normal Negative 39 32 30 31 31 31 1.00 Normal Negative 40 30 28 29 29 31 0.94 Normal Negative 41 34 32 33 33 31 1.06 Normal Negative 42 28 26 27 27 31 0.87 Normal Negative 43 33 32 32.5 33 31 1.05 Normal Negative 44 >300 >300 >300 >300 31 > 9.68 Prolong 180 182 181 Prolong 42 40 41 >300/41 = >7.32 Positive 45 36 34 35 35 31 1.13 Normal Negative 46 33 32 32.5 33 31 1.05 Normal Negative 114

47 30.5 29.5 30 30 31 0.97 Normal Negative 48 37 35 36 36 31 1.16 Normal Negative 49 28 26 27 27 31 0.87 Normal Negative 50 34 32 33 33 31 1.06 Normal Negative 51 33 31 32 32 31 1.03 Normal Negative 52 31 29 30 30 31 0.97 Normal Negative 53 33 31 32 32 31 1.03 Normal Negative 54 35 33 34 34 31 1.10 Normal Negative 55 33 32 32.5 33 31 1.05 Normal Negative 56 36 34 35 35 31 1.13 Normal Negative 57 30 29 29.5 30 31 0.95 Normal Negative 58 36 35 35.5 36 31 1.15 Normal Negative 59 36 37 36.5 37 31 1.18 Normal Negative 60 33 31 32 32 31 1.03 Normal Negative 61 35 33 34 34 31 1.10 Normal Negative 62 24 22 23 23 31 0.74 Normal Negative 63 37 35 36 36 31 1.16 Normal Negative 64 34 32 33 33 31 1.06 Normal Negative 65 33 32 32.5 33 31 1.05 Normal Negative 66 31 29 30 30 31 0.97 Normal Negative 67 35 34 34.5 35 31 1.11 Normal Negative 68 36 34 35 35 31 1.13 Normal Negative 69 36 35 35.5 36 31 1.15 Normal Negative 70 38 36 37 37 31 1.19 Normal Negative

115

APPENDIX VII bi

RAW DATA FOR IgG - β2GPI – ACA AMONG HEALTHY CONTROL SUBJECTS.

HEALTHY CONTROL SUBJECT

(IgG - B2GPI - DEPENDENT ACA ASSAY S/N OPTICAL DATA 1 DATA 2 CONCENTRATION DENSITY OF IgG ACA (OD) (AU/mL) 1 0.208 29.22 2.86 3.21776 2 0.122 29.22 2.86 0.70484 3 0.099 29.22 2.86 0.03278 4 0.139 29.22 2.86 1.20158 5 0.130 29.22 2.764 1.0346 6 0.181 29.22 2.764 2.52482 7 0.114 29.22 2.764 0.56708 8 0.261 29.22 2.764 4.86242 9 0.187 29.22 2.764 2.70014 10 0.715 29.22 2.764 18.1283 11 0.951 29.22 2.764 25.02422 12 1.121 29.22 2.764 29.99162 13 0.278 29.22 2.764 5.35916 14 0.233 29.22 2.764 4.04426 15 0.099 29.22 2.764 0.12878 16 0.179 29.22 2.764 2.46638 17 0.192 29.22 2.764 2.84624 18 0.116 29.22 2.764 0.62552 19 0.157 29.22 2.764 1.82354 20 0.099 29.22 2.764 0.12878 21 0.166 29.22 2.764 2.08652 22 0.061 29.22 2.764 -0.98158 23 0.306 29.22 2.764 6.17732 24 0.128 29.22 2.764 0.97616 25 0.254 29.22 2.764 4.65788 26 0.115 29.22 2.764 0.5963 27 0.167 29.22 2.764 2.11574 28 0.234 29.22 2.764 4.07348 29 0.308 29.22 2.86 6.13976 30 0.187 29.22 2.86 2.60414 31 0.083 29.22 2.86 -0.43474 32 0.134 29.22 2.86 1.05548 33 0.187 29.22 2.86 2.60414 34 0.137 29.22 1162.86 1.14314

35 0.162 29.22 2.764 1.96964 36 0.152 29.22 2.764 1.67744 37 0.089 29.22 2.86 -0.25942 38 0.162 29.22 2.764 1.96964 39 0.131 29.22 2.86 0.96782 40 0.115 29.22 2.86 0.5003 41 0.105 29.22 2.86 0.2081 42 0.157 29.22 2.764 1.82354 43 0.141 29.22 2.764 1.35602 44 0.147 29.22 2.764 1.53134 45 0.254 29.22 2.764 4.65788 46 0.216 29.22 2.764 3.54752 47 0.108 29.22 2.86 0.29576 48 0.094 29.22 2.86 -0.11332 49 0.114 29.22 2.86 0.47108 50 0.102 29.22 2.86 0.12044 51 0.092 29.22 2.86 -0.17176 52 0.107 29.22 2.764 0.36254 53 0.172 29.22 2.764 2.26184 54 0.123 29.22 2.764 0.83006 55 0.138 29.22 2.764 1.26836 56 0.087 29.22 2.86 -0.31786 57 0.208 29.22 2.86 3.21776 58 0.119 29.22 2.86 0.61718 59 0.091 29.22 2.764 -0.10498 60 0.178 29.22 2.764 2.43716 61 0.111 29.22 2.764 0.47942 62 0.095 29.22 2.764 0.0119 63 0.089 29.22 2.764 -0.16342 64 0.105 29.22 2.764 0.3041 65 0.216 29.22 2.764 3.54752 66 0.131 29.22 2.764 1.06382 67 0.147 29.22 2.764 1.53134 68 0.242 29.22 2.764 4.30724 69 0.316 29.22 2.764 6.46952 70 0.178 29.22 2.764 2.43716

117

APPENDIX VIII ai RAW DATA FOR PLATELET COUNTS FOR T2DM WITH COMPLICATIONS (FIRST TIME).

T2DM WITH COMPLICATIONS PLATELET COUNT,GLYCATED HAEMOGLOBIN, FASTING PLASMA GLUCOSE AND MICROALBUMINURIA (FIRST TIME) S/N PLATELET HbA1C (%) FASTING PLASMA MICROALBUMINURIA (SPOT COUNT (X109/L) GLUCOSE (mg/dL) COLLECTION ANALYSIS USING COMBI - 13) (POSITIVE OR NEGATIVE) 1 203 7.2 165 2 252 5.9 140 3 200 8.7 240 Positive 4 251 6.5 97 5 180 8.5 340 6 258 8.5 158 Positive 7 269 7.0 125 8 192 7.2 182 9 250 9.7 196 10 180 8.2 264 Positive 11 237 10.6 149 Positive 12 202 10.3 219 13 250 6.5 128 14 199 7.8 150 15 208 7.7 160 16 160 10.3 353 Positive 17 203 8.3 140 18 192 8.3 143 19 193 8.3 220 Positive 20 205 9.5 160 21 256 8.2 140 Positive 22 226 6.2 127 23 201 7.9 226 24 200 6.3 138 25 276 8.2 208 Positive 26 237 6.5 137 27 267 7.2 232 28 167 9.9 160 Positive 29 300 6.5 69 30 163 7.9 167 31 206 7.3 145 32 339 5.3 101 118

33 221 12.7 201 34 185 10.6 104 35 200 8.0 151

36 265 6.4 135 37 260 8.0 147 38 259 7.8 125 Positive

39 170 7.0 258 40 160 8.6 179 41 261 6.8 116 42 302 7.8 170 43 200 7.4 130 Positive

44 253 7.7 140 45 142 10.2 160 46 160 7.2 240 Positive

47 290 8.1 103 48 300 6.8 153 49 152 6.8 238 50 200 7.4 167 51 297 9.4 110

52 231 7.1 160 53 101 6.3 160 54 187 7.3 155

55 155 9.2 192 Positive 56 120 11 279 57 205 9.6 80 58 250 8.6 80 Positive 59 274 10.4 352 Positive

60 158 7.4 150 Positive 61 133 10.2 256 62 130 10.5 442 63 130 10.7 270 64 153 6.8 111 65 130 7.2 180 66 141 10.2 185 67 185 7.2 180

68 198 7.8 170 69 167 10.8 203 70 149 7.9 190

119

APPENDIX VIII aii RAW DATA FOR PLATELET COUNTS FOR T2DM WITH COMPLICATIONS (12 WEEKS LATER)

T2DM WITH COMPLICATIONS (12 WEEKS LATER) PLATELET COUNT,GLYCATED HAEMOGLOBIN, FASTING PLASMA GLUCOSE AND MICROALBUMINURIA S/N PLATELET HbA1C FASTING MICROALBUMINURIA (SPOT COUNT (%) PLASMA COLLECTION ANALYSIS USING (X109/L) GLUCOSE COMBI - 13) (POSITIVE OR (mg/dL) NEGATIVE) 3 206 9.9 187 Positive 6 236 9.3 151 Positive 8 153 9.4 230 10 202 6.8 215 Positive 11 206 8.6 102 Positive 13 220 6.3 114 16 102 8.9 230 Positive 19 156 9.5 150 Positive 21 270 7.0 130 Positive 25 318 6.8 337 Positive 28 198 8.9 253 Positive 32 265 6.0 140 33 230 9.0 224 38 286 8.5 101 Positive 43 175 13.3 261 Positive 46 135 6.8 137 Positive 55 160 8.4 117 Positive 56 230 9.2 97 Positive 59 146 8.5 240 Positive 60 162 8.5 129 Positive 62 152 9.5 327 69 139 9.8 177 70 153 8.3 200

120

APPENDIX VIII bi RAW DATA FOR PLATELET COUNTS FOR T2DM WITHOUT COMPLICATIONS (FIRST TIME)

T2DM WITHOUT COMPLICATION PLATELET COUNT,GLYCATED HAEMOGLOBIN AND FASTING PLASMA GLUCOSE (FIRST TIME) S/N PLATELET COUNT HbA1C (%) FASTING PLASMA (X109/L) GLUCOSE (mg/dL) 1 236 6.2 168 2 210 6.5 188 3 160 12.5 323 4 236 6.8 140 5 160 9.5 168 6 260 6.8 203 7 200 7.2 180 8 200 6.8 130 9 130 12.5 257 10 140 7.8 182 11 206 7.5 160 12 212 8.5 160 13 155 8.5 102 14 205 10.5 529 15 150 6.1 129 16 160 10.9 240 17 120 10.5 210 18 260 6.5 190 19 150 6.5 191 20 186 6.3 153 21 162 13 175 22 210 6.4 155 23 177 6.8 127 24 249 8.2 114 25 200 8.2 273 26 280 6.5 130 27 210 2.7 162 28 180 6.5 248 29 231 8.1 191 30 156 6.8 150 31 157 8.7 177 32 212 7.7 138 33 232 7.9 262 121

34 211 8.2 193 35 290 6.3 160 36 178 7.9 204 37 216 7.9 148 38 253 7.1 138 39 168 8.9 221 40 210 6.9 148 41 155 9.7 306 42 192 7.0 136 43 179 7.8 163 44 196 8.1 169 45 239 7.7 189 46 253 7.4 225 47 261 7.0 148 48 149 8.7 148 49 232 7.5 158 50 162 7.1 218 51 149 8.9 276 52 176 7.5 186 53 248 7.3 182 54 252 7.0 166 55 192 8.3 203 56 222 7.2 190 57 234 7.7 182 58 155 10.3 412 59 158 9.2 321 60 207 6.9 142 61 149 8.7 212 62 267 7.6 285 63 183 8.7 237 64 206 9.5 206 65 168 10.3 340 66 191 8.2 190 67 152 8.7 182 68 161 10.2 206 69 243 7.2 150 70 260 7.1 452

122

APPENDIX VIII bii RAW DATA FOR PLATELET COUNTS FOR T2DM WITHOUT COMPLICATIONS (12 WEEKS LATER)

T2DM WITHOUT COMPLICATION PLATELET COUNT,GLYCATED HAEMOGLOBIN AND FASTING PLASMA GLUCOSE (12 WEEKS LATER) S/N PLATELET COUNT HbA1C (%) FASTING PLASMA (X109/L) GLUCOSE (mg/dL)

19 170 7.0 146

39 158 8.7 184

46 267 7.2 245

58 167 8.7 306

59 185 7.8 218

123

APPENDIX VIII c RAW DATA FOR PLATELET COUNTS FOR HEALTHY CONTROL SUBJECTS.

HEALTHY CONTROL SUBJECTS PLATELET COUNT,GLYCATED HAEMOGLOBIN AND RANDOM PLASMA GLUCOSE S/N PLATELET COUNT HbA1C (%) RANDOM PLASMA (X109/L) GLUCOSE (mg/dL) 1 230 5.8 126 2 267 5.0 110 3 283 5.2 120 4 295 5.5 100 5 278 5.8 131 6 236 5.3 121 7 286 5.2 129 8 281 5.1 123 9 263 5.7 131 10 304 5.5 125 11 242 5.4 102 12 276 5.6 115 13 233 5.9 101 14 265 6.0 97 15 281 5.3 117 16 297 5.7 132 17 263 6.0 126 18 288 5.4 107 19 277 5.9 133 20 287 5.0 128 21 278 5.6 118 22 302 5.1 121 23 298 5.6 99 24 289 5.9 119 25 301 5.1 103 26 296 5.2 98 27 297 5.7 122 28 257 5.7 108 29 301 5.6 113 30 286 5.7 109 31 278 5.6 115 32 298 5.3 102 33 272 6.2 119 34 298 6.0 121

124

35 298 5.7 104

36 305 5.3 104 37 292 5.5 99 38 296 5.8 109 39 302 5.2 107 40 306 6.3 117 41 286 5.7 111 42 256 5.8 109 43 280 6.8 123 44 286 5.6 106 45 282 5.1 98 46 263 6.0 121 47 232 5.9 117 48 320 5.3 114 49 262 6.2 117 50 326 5.5 108 51 296 6.1 115 52 285 5.6 108 53 287 5.8 118 54 290 5.9 128 55 280 6.3 129 56 278 5.8 106 57 230 6.5 127 58 293 5.8 118 59 267 5.3 98 60 203 5.9 118 61 252 5.7 116 62 237 5.8 128 63 256 5.1 104 64 292 5.3 118 65 231 5.3 102 66 292 5.3 104 67 289 5.1 112 68 255 5.3 103 69 263 5.1 129 70 273 5.3 121

125