Evaluation of Serum Fructosamine As a Screening Test for The

Home , Fructosamine, Glucose tolerance test

EVALUATION OF SERUM FRUCTOSAMINE AS A SCREENING TEST FOR THE

DETECTION OF GESTATIONAL DIABETES MELLITUS

A dissertation submitted to the Faculty of Pathology, National Postgraduate Medical

College in partial fulfillment of the requirements for the award of Fellow of the National Medical

College in Pathology( FMCPath)

Dr. HALIMA HALADU

AF/008/13/115/1038

FACULTY: PATHOLOGY

SPECIALTY: CHEMICAL PATHOLOGY

DATE PASSED PART ONE: OCTOBER 2013.

TRAINING INSTITUTION:

AMINU KANO TEACHING HOSPITAL, KANO

NOVEMBER, 2016

SUPERVISOR:

Dr. Isah Adagiri Yahaya. FMCPath

Department of Chemical Pathology & Immunology,

Aminu Kano Teaching Hospital / Bayero University, Kano

PMB 3452.

Kano State, Nigeria

Signature……………………………

CERTIFICATION

I hereby certify that this Dissertation work titled “Evaluation of Serum Fructosamine as a

Screening Test for the detection of Gestational Diabetes Mellitus” was carried out in my

Department by Dr Halima Haladu.

Signature......

Dr. Mohammed Idris Yahaya . FMCPath

Consultant Chemical Pathologist,

Department of Chemical Pathology and Immunology,

Aminu Kano Teaching Hospital / Bayero University, Kano.

DECLARATION

I hereby declare that this work is original. It has not been presented to any other examining body for fellowship, nor has it been submitted elsewhere for publication.

......

Dr Halima Haladu

Department of Chemical Pathology

AKTH, Kano.

DEDICATION

I dedicate this work to my parents, Mr and Mrs Haladu Danjebou, my husband, Dr Ibrahim

Salisu, and my children for their care, support and patience throughout my period of residency training.

ACKNOWLEDGEMENT

My profound appreciation goes to the management of Aminu Kano Teaching Hospital, Kano for giving me the opportunity to undergo the residency training programme.

My special gratitude goes to my Head of Department, Dr Idris Yahaya Mohammed for his guidance and support.

No words can describe my appreciation to my supervisor, Associate Professor Isa Adagiri

Yahaya for his consistent interest, guidance, and mentoring. May God almighty give you strength and reward you abundantly.

I thank all my colleagues at the Department of Chemical pathology, Aminu Kano Teaching

Hospital, Kano for their contributions and support.

Dr Mohammmad Rayyan Garba of Community Medicine Department, Aminu Kano Teaching

Hospital, deserves a special appreciation. He helped me in statistical analysis and presentation of results.

LIST OF TABLES

TABLES PAGE

Table 2.1: Criteria for abnormal result for 100g, 3-hour OGTT in pregnant women 10

Table 2.2: Criteria for abnormal result for 75g OGTT in pregnant women 11

Table 3.1: Contingency table for calculating validity of a screening test 38

Table 5.1: Age Distribution of study participants 41

Table 5.2(i): Indications for oral glucose tolerance test 42

Table 5.2(ii): Frequency for indications of oral glucose tolerance test 43

Table 5.3: Anthropometric and clinical characteristics of study participants 44

Table 5.4: Serum Fructosamine Distribution of controls in Percentiles 46

Table 5.5: OGTT Results and Corrected Serum Fructosamine levels among Patients 47

Table 5.6: Comparison of Serum Fructosamine levels between patients and controls 48

Table 5.7: Patients’ (GDM and non-GDM) OGTT and Serum Fructosamine Results 49

Table 5.8: Serum Fructosamine, Albumin and Total Protein of the Study Participants 51

Table 5.9: Prevalence of GDM using OGTT and Serum Fructosamine 52

Table 5.10: Comparison of Prevalence Rates of GDM using Serum Fructosamine and OGTT 55

LIST OF FIGURES PAGE

Figure 1: Reaction between glucose and protein to form fructosamine 14

Figure 2: Pie chart showing prevalence of GDM using OGTT 53

Figure 3: Pie chart showing prevalence of GDM using serum fructosamine 54

ACRONYMS

ADA American Diabetes Association

AKTH Aminu Kano Teaching Hospital

BMI Body Mass Index

EDTA Ethylene Diamine Tetraacetic Acid

FN False Negative

FP False Positive

FPG Fasting plasma Glucose

GCT Glucose Challenge Test

GDM Gestational Diabetes Mellitus

GPF Glycated Protein Fragments

GSP Glycated Serum Protein

HAPO Hyperglycaemia Adverse Pregnancy Outcomes

HbA1c Glycated Haemoglobin

HMF 5-hydroxymethylfurfuraldehyde

HPLC High Pressure Liquid Chromatography

Min Minutes

NBT Nitroblue tetrazolium

NPV Negative Predictive value

OGTT Oral Glucose Tolerance Test

PG Plasma Glucose

PPV Positive Predictive Value

RBG Random Blood Glucose

SD Standard Deviation

TBA Thiobarbituric Acid

TN True Negative

TP True Positive

WHO World Health Organisation

TABLE OF CONTENTS page

Title page i

Supervision ii

Certification iii

Declaration iv

Dedication v

Acknowledgement vi

List of Tables vii

List of figures viii

Acronyms ix

Table of contents xi

Abstract 1

CHAPTER ONE

1.0 Introduction 3

1.1 Background 3

1.2 Statement of Research problem 4

1.3 Justification 5

1.4 Limitation 6

1.5 Hypothesis 6

CHAPTER TWO

2.0 Literature review 7

2.1 Gestational diabetes mellitus 7

2.1.1 Risk factors for Gestational Diabetes Mellitus 7

2.1.2 Pathophysiology of Gestational Diabetes Mellitus 7

2.1.3 Complications of Gestational Diabetes Mellitus 8

2.1.4 Screening for Gestational Diabetes Mellitus 8

2.1.5 Screening modalities for Gestational Diabetes Mellitus 9

2.2 Fructosamine 13

2.2.1 Determination of fructosamine 15

2.2.2 Clinical usefulness of fructosamine assay 17

2.3 Gestational Diabetes Mellitus and Fructosamine 18

CHAPTER THREE

3.0 Aim and Objectives 24

3.1 Aim 24

3.2 Specific Objectives 24

CHAPTER FOUR

4.0 Subjects and Methods 25

4.1 Study Area 25

4.2 Study Design 26

4.3 Study Population 26

4.4 Sampling Technique 26

4.5 Sample Size Determination 26

4.6 Inclusion Criteria 27

4.7 Exclusion Criteria 28

4.8 Ethical consideration 28

4.9 Data Collection 29

4.10 Sample Collection and Processing 30

4.11 Laboratory Methods 31

4.12 Data Processing and Statistical Analysis of Results 37

CHAPTER FIVE

5.0 Results 40

5.1 Age distribution of study participants 40

5.2 Indications for OGTT 40

5.3 Anthropometric and clinical characteristics of study participants 40

5.4 Serum Fructosamine Distribution of Controls in Percentiles 45

5.5 OGTT Results and Corrected Serum Fructosamine levels among Patients 45

5.6 Comparison of Serum Fructosamine levels between patients and controls 45

5.7 Patients’ (GDM and non-GDM) OGTT and Serum Fructosamine Results 45

5.8 Serum Fructosamine, Albumin and Total Protein of the Study Participants 50

5.9 Prevalence of GDM using OGTT and Serum Fructosamine 50

5.10 Comparison of Prevalence Rates of GDM using Serum Fructosamine and OGTT 50

CHAPTER SIX

6.0 Discussion 56

6.1 Conclusion 61

6.2 Recommendation 62

References 63

Appendices

Appendix I: Ethical Approval 73

Appendix II: Information Form 74

Appendix III: Informed Written Consent Form 75

Appendix IV: Questionnaire 76

Appendix V : Patients preparation form for OGTT 78

ABSTRACT

BACKGROUND: The use of serum fructosamine as a screening test for gestational diabetes mellitus (GDM) may provide a simpler alternative compared to oral glucose tolerance test which is tedious, requires multiple venepunctures, overnight fasting and may be associated with unpleasant side effects.

OBJECTIVE: This study evaluated the clinical usefulness of serum fructosamine as a screening test for the detection of GDM.

METHODS: This descriptive cross sectional study recruited a total of 313 participants, made up of 193 pregnant women with risk factors for GDM referred from antenatal clinic for oral glucose tolerance test (OGTT) and 120 apparently healthy non pregnant women as controls. A 75 g

OGTT, serum fructosamine, albumin and total protein assays were performed. Data generated were collated and analysed using SPSS version-22.0. The statistical significance of fructosamine values were assessed by student’s t-test and the validity of the test was assessed by calculating the sensitivity, specificity, positive predictive value and negative predictive value.

RESULTS: Forty one of the 193 pregnant women, tested positive for GDM based on the World

Health Organisation’s (WHO) criteria for OGTT. Although there was a statistically significant difference in the serum fructosamine levels across the 3 groups (GDM patients, non-GDM

17 patients and controls), there was no statistically significant difference (p=0.102) in the levels of fructosamine between the GDM and non GDM patients. In comparison with 75 g OGTT, single assay of serum fructosamine gave a sensitivity, specificity, positive and negative predictive values of 44%, 63%, 24% and 63% respectively with regard to detection of GDM.

CONCLUSION: Serum fructosamine may not be a useful screening test in the detection of

GDM.

CHAPTER ONE

1.0 INTRODUCTION

1.1 BACKGROUND

Gestational diabetes mellitus (GDM) is defined as a disorder of glucose tolerance with first onset or recognition during pregnancy.1 It is a common medical complication encountered during pregnancy. GDM is associated with adverse foetal and maternal outcomes that can be prevented by early diagnosis and management. Complications associated with GDM may include: foetal macrosomia, hypoglycaemia, polycythaemia, hyperbilirubinaemia, neonatal death and preeclampsia.2 The prevalence of GDM varies worldwide and may range from 1-14% depending on the study population and the diagnostic criteria employed.3 In the United States of America, the prevalence of GDM ranges from 4.6-9.2%.4 The prevalence of GDM in Africa ranged from 0% in Tanzania to 13.9% in Nigeria.5

GDM is a problem that affects a significant number of women during pregnancy. GDM can have lasting health impacts on both the mother and the foetus. In order to circumscribe and minimise potential complications to both the mother and the child, screening, diagnosis and management of hyperglycaemia are critical.

Routine screening for GDM during antenatal care is of paramount importance as it will help in early diagnosis and treatment thereby minimising complications associated with GDM. Glucose challenge test (GCT) has been widely used for screening for GDM.6 This is carried out in a two- step fashion using an initial 1-hour 50 g oral glucose challenge irrespective of the time and type of last meal of the patient and then followed by a diagnostic 75 g oral glucose tolerance test

(OGTT) if the GCT is positive. A positive result for GCT is defined as, blood glucose value of

≥140 mg/L (7.8 mmol/L) 1 hour post 50 g glucose load.7

Fructosamine is a generic name given to a compound known as plasma protein ketoamine. It is formed by a spontaneous non-enzymatic reaction between a carbonyl group of glucose molecule and an amino group of plasma protein, mainly albumin.8 It serves as an index of glycaemia over a period of 2-3 weeks which corresponds to the half life of albumin (17-20 days).8 Fructosamine may be particularly useful in monitoring diabetes in pregnancy in which hormonal changes cause greater short-term fluctuations in glucose concentrations.9

Determination of serum fructosamine using the nitroblue tetrazolium procedure is simple, fast reproducible and can be easily automated.8 Since fructosamine determines the average glucose over the past 2-3 weeks, the test is not affected by recent food intake hence patients are not required to fast before blood collection for its estimation in the laboratory.10

1.2 STATEMENT OF RESEARCH PROBLEM

Oral glucose tolerance test is considered worldwide as the gold standard for the diagnosis of

GDM.11

The procedure for OGTT is time consuming and inconvenient for most patients, as they are required to fast for a minimum of 8 hours and it also involves taking several blood samples over

20 a period of two to three hours. In addition, it may be associated with unpleasant side effects like nausea and vomiting.12 Glucose Challenge Test and OGTT therefore have limitations for use as screening tests, hence the need for simpler methods for the detection of GDM becomes imperative. Some researchers have suggested that measurement of fructosamine could serve as an alternative to the GCT and OGTT in screening for GDM, and it can also be used to monitor glucose control. 13, 14, 15

1.3 JUSTIFICATION FOR THE STUDY

Serum fructosamine assay has shown validity in monitoring recent glycaemic alterations and has been proposed to be useful in GDM patients, in whom short-term confirmation of the maternal glycaemic state is clinically warranted.16,17 In addition, due to the inherent sensitivity of fructosamine assay to monitor recent glycaemic fluctuations, there has been considerable interest in its application as screening test for GDM.18 The use of serum fructosamine for screening may provide a simpler and easier alternative when compared with OGTT which is tedious, associated with unpleasant side effects (mainly nausea and vomiting ) and requires multiple venipunctures.

Furthermore, fructosamine assay does not require prior fasting whereas 8-14 hours fasting is required if OGTT is to be used for screening. Also, second generation assays currently used for fructosamine assays are rapid, inexpensive and free from interferences by urates and triglycerides. In contrast, the first generation assays suffered from lack of specificity, lack of standardisation and susceptibility to interferences which lead to limited clinical use of fructosamine assays in the past.

There is a paucity of reports on the use of serum fructosamine in screening for diabetes mellitus in this environment. This study was therefore designed to assess the usefulness of serum fructosamine as an alternative method to OGTT in the detection of GDM among pregnant women in Kano.

1.4 LIMITATION OF THE STUDY

The information derived from the study may only be applicable to pregnant women and may not apply to men, non pregnant women and children.

1.5 HYPOTHESIS

NULL HYPOTHESIS: Serum fructosamine may not be useful as a screening test for the detection of GDM.

ALTERNATIVE HYPOTHESIS: Serum fructosamine may be useful as a screening test for the detection of GDM.

CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 GESTATIONAL DIABETES MELLITUS

Gestational diabetes mellitus is defined as a disorder of glucose tolerance with first onset or recognition during pregnancy.1

2.1.1 Risk factors

Risk factors for GDM include maternal age greater than 25 years, high body mass index (BMI), polyhydramnios, past history of GDM, members of ethnic groups known to have increased risk of developing type 2 diabetes mellitus (Africans, Native Americans, Hispanics, South or East

Asian or Pacific islanders), macrosomia in previous pregnancy, history of stillbirth, polycystic ovarian syndrome, metabolic syndrome and history of diabetes in first degree relatives.19

Pregnant women are said to be at high risk of GDM if they have two or more risk factors for

GDM. No known risk factors are identified in 50% of patients.20

2.1.2 Pathophysiology

The precise mechanisms involved in the development of gestational diabetes mellitus remain unknown. However, the possible explanations might include: insulin resistance, reduced insulin secretion, increased insulin degradation, increased secretion of hormones with anti-insulin effect

(glucagon, human placental lactogen, estrogens, progesterone and cortisol), or a combination of two or more of these mechanisms.21 Insulin resistance usually begins in the second trimester and progresses throughout pregnancy. Insulin normally promotes entry of glucose into most cells and therefore, insulin resistance prevents proper entry of glucose into cells leading to hyperglycaemia. More insulin is therefore required to overcome the resistance and about 1.5-2.5 times more insulin is produced in individuals with GDM than in normal pregnancy,22 hence women that develop GDM have a higher degree of insulin resistance when compared with women with normal pregnancies. Women who have a deficit in this additional insulin secretory capacity (in particular, first–phase insulin secretion) develop GDM. This deficit may be a marker of deterioration of β-cell function.23 The possible causes of β-cell dysfunction in women diagnosed with GDM could be autoimmune, monogenic, or most commonly occur due to background insulin resistance.24

2.1.3 Complications of GDM

GDM is associated with both fetal and maternal complications. Foetal complications include: neonatal hypoglycaemia, macrosomia, perinatal mortality, congenital malformation, hyperbilirubinaemia, polycythaemia, hypocalcaemia, and respiratory distress syndrome.25, 26

Long-term complications to the offspring are an increased risk of glucose intolerance, obesity and diabetes.7 Maternal complications include hypertension, preeclampsia, and an increased risk of caesarean delivery. Another complication is that women with a history of GDM have an

24 increased risk of developing diabetes after pregnancy compared to the general population, having a conversion rate of up to 3% per year.23

2.1.4 Screening for GDM

The need for screening and diagnosis of GDM is universally accepted but controversy exists about the most suitable method of screening among various populations. Most studies recommend screening for GDM between 24-28 weeks.27, 28, 29 There are many specific guidelines and recommendations available to screen GDM. The American Congress of Obstetricians and

Gynaecologists (ACOG) recommended selective screening up till 1994 but now recommends universal screening in certain high-risk settings.30 The American Diabetic Association (ADA) recommended universal screening in 1996 but then revised their recommendations in 1997, suggesting selective screening of women at high risk of GDM.31 The Australian Diabetes in

Pregnancy Society recommended that all pregnant women should be considered for screening depending on resources available.32

2.1.5 Screening modalities

Three screening tests with generally accepted criteria are frequently used for the diagnosis of

GDM in a one or two-step fashion. These are typically performed between 24 and 28 weeks gestation and are stated as follows:

A) 50 g Glucose Challenge Test: A two-step method using an initial 1-hour 50 g GCT

followed by either a diagnostic 75g or 100 g OGTT if the GCT is positive. A positive result

for GCT is defined by either of the following criteria depending on the level of sensitivity

desired:

 ≥130 mg/dL (7.2 mmol/l) , this identifies 90% of women with GDM.7

 ≥140 mg/dL (7.8 mmol/l) , this identifies 80% of women with GDM.7

B) 100 g OGTT: A one-step or a two-step method using a 3-hour 100 g diagnostic OGTT. This

test is considered positive if two or more of the hourly plasma glucose results exceed the

following values: (Table 2.1).

C) 75 g OGTT: A one-step or two-step method using a 75 g diagnostic OGTT. This test is

positive if two or more of the hourly plasma glucose levels meet or exceed the following

values (based on WHO or ADA recommendations) shown in table 2.2.

Table 2.1: Criteria for abnormal result for 100 g, 3-hour OGTT in pregnant women.7, 33

Blood sample National Diabetes Data Carpenter and Coustan

Group Criteria Criteria

Fasting 105 mg/dL (5.8 mmol/L) 95 mg/dL (5.3 mmol/L)

1-hour 190 mg/dL (10.5 mmol/L) 180 mg/dL (10.0 mmol/L)

2-hour 165 mg/dL (9.2 mmol/L) 155 mg/dL (8.6 mmol/L)

3-hour 145 mg/dl (8.0 mmol/L) 140mg/dL (7.8 mmol/L)

Table 2.2: Criteria for Abnormal Result on 75-g OGTT in Pregnant Women.7, 34

Blood sample ADA mg/dL ADA mmol/L WHO mmol/L

Fasting 95 5.3 7.0

1-hour 180 10.0

2-hour 155 8.6 7.8

D) HAPO Diagnostic Criteria

The screening and diagnostic criteria for GDM have recently been modified extensively. The

Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study was a large prospective, multinational epidemiologic study to assess adverse outcomes as a function of maternal glycaemia.35 The study reported a strong correlation between increasing maternal glucose levels at 24-32 weeks of gestation and a range of adverse maternal and fetal outcomes. Subsequent consideration by the International Association of Diabetes and Pregnancy Study Groups

(IADPSG) in the year 2010 resulted in the formulation of a new consensus guideline for the testing and diagnosis of GDM.36 An expert consensus panel appointed by the IADPSG recommended that all pregnant women not previously known to have diabetes should be evaluated by a 75 g OGTT for GDM at 24–28 weeks of gestation. Diagnostic cut points for fasting, 1-hour and 2-hour plasma glucose concentrations have been established.

A diagnosis of GDM is made if one or more of the following glucose levels are elevated: 36

Fasting glucose ≥ 5.1 mmol/L

1‐hr glucose ≥ 10.0 mmol/L

2‐hr glucose ≥ 8.5 mmol/L

E) Random plasma glucose (RPG) and Fasting Plasma Glucose (FPG) measurements have been suggested as screening options for GDM because they are more economical and more tolerated than glucose challenge tests.37, 38 The cut off value for FPG is 5.3 mmol/L while those of RPG depend on the time of last meal (5.8 mmol/l if the last meal is more than 2 hours and 6.4 mmol/l if less than 2 hours). However, it has been found that the sensitivity of RPG and FPG is around 40% and 19.4% respectively; hence both cannot be regarded as suitable screening tests for GDM.39, 40

F) Serum fructosamine: The use of serum fructosamine for the screening of GDM has been widely reported.15,41,42 This is based on the high sensitivities and specificities reported in the studies. Robert et al reported a sensitivity, specificity, positive predictive value and negative predictive value of 86%, 95%, 66% and 98% respectively. Similarly, Hughes and colleagues achieved a sensitivity and specificity of 79.4% and 77.3% respectively.

2.2 FRUCTOSAMINE

Fructosamine is a product of non-enzymatic reaction between glucose and a protein (usually albumin). The reaction between an amino acid and a reducing sugar to form stable ketoamine adduct was first described by Maillard in 1912.8

When milk and other foods are heated, carbonyl groups of sugars combine with amino groups of proteins to form aldimine (Schiff bases) which progresses to form stable ketoamines, fructosamine (figure 1). Albumin was found to be glycated at multiple sites and mainly at the epsilon-amino groups of lysine residues, as it occurs with haemoglobin.43

Glycation of albumin and other plasma proteins known as Glycated Serum Protein (GSP) or

Glycated Albumin (GA) is increased in diabetes as compared with normal subjects.44

Fructosamine may thus be used in a way similar to glycated haemoglobin to estimate the average concentration of blood glucose over an extended period of time (2 to 3 weeks). Since albumin is the most common protein in blood, fructosamine levels typically reflect albumin glycation.13

Albumin has a half-life of approximately 20 days hence the plasma fructosamine concentration reflects relatively recent (2-3 weeks) changes in blood glucose.8

Figure 1: The reaction between glucose and protein to form fructosamine. (Culled from

Ambruster DA).8

2.2.1 Determination of Serum Fructosamine

Methods for measuring glycated proteins include:8

1. Colorimetric method using nitroblue tetrazolium (NBT)

Serum is added to carbonate buffer containing NBT (pH 10.8, 37˚C). The assay is based

on the reducing properties of fructosamine under alkaline conditions.

2. Colorimetric method using 2‐thiobarbituric acid (TBA)

Serum is heated with oxalic or acetic acid at 1000C for 18-24 hours to form

5‐hydroxymethylfurfuraldehyde (HMF); protein is precipitated with trichloroacetic acid;

HMF in the supernatant is heated with TBA at 40˚C for 30 minutes to form a derivative

measured at 443 nm.

3. Colorimetric method using phenylhydrazine

Phenylhydrazine reacts with fructosamine to form a phenylhydrazone adduct with

absorption at 350 nm. The absorbance is directly proportional to concentration of

fructosamine.

4. Enzymatic Glycated Serum Protein Assay which uses proteinase K to digest

glycosylated serum protein into low molecular weight glycated protein fragments (GPF),

and uses specific fructosaminase, to catalyze the oxidative degradation of Amadori

product GPF to yield Protein fragment or amino acids, glucosone and H2O2. The H2O2

released is measured by a colorimetric Trinder end-point reaction. The absorbance

measured at 546-600 nm is proportional to the concentration of glycated serum protein

The reactions are: Glycated protein → (proteinase K, EC 3.4 21.64) → glycated protein

fragments.

Glycated protein fragments → (ketoamine oxidase, EC not assigned) →amino acids +

H2O2

H2O2 + chromogens→ (horseradish peroxidase, EC 1.11.1.7) → colour +H2O

5. Affinity chromatography using immobilised phenylboronic acid.

6. High perfomance liquid chromatography (HPLC).

Fructosamine is hydrolysed with 6 M HCl at 95 ˚C for 18 hours producing lysine (50%),

furosine (30%) and pyridosine (10%). Furosine is quantified by HPLC using a reverse-

phase column with ultra violet detection at 254 nm and 280 nm.

Among all the above procedures employed in fructosamine assay, the most widely

accepted is the NBT method.8

2.2.1.1 Principle of NBT

The NBT method is based upon the reducing ability of fructosamine in alkaline solution. 8 Under alkaline conditions, Amadori rearrangement products such as fructosamine have reducing activities that can be differentiated from those of other reducing substances like glucose and N- glycosylamine derivatives of labile Schiff bases. 8 In the NBT procedure, serum sample is added to carbonate buffer (at basic pH, originally pH 10.8, and 37oC) containing NBT which is subsequently reduced. The absorbance is measured at 530nm after 10-15 minutes.45

The precise mechanism of the reduction of NBT by fructosamine is not known, but Jones et al 46 has presented evidence that it involves a superoxide radical intermediate. Fructosamine values will be falsely increased if a pre-incubation time of less than 10 minutes is used, due to the presence of interfering substances in serum that reduce NBT within the first 10 minutes of the assay.47 The NBT assay have been adapted to a variety of automated chemistry analysers.48, 49

2.2.1.2 Interfering substances in fructosamine assay.50

1. EDTA and heparin plasma samples give lower fructosamine results than serum samples in the

NBT colorimetric assay method.

2. Urate and glutathione produce artificially high results in the NBT assay method.

3. Serum concentrations of Vitamin C greater than 227 μmol/L, interfere significantly with the

NBT assay method.

4. Cysteine, methyldopa, dobesilate calcium and oxytetracycline can cause artificially low fructosamine results.

5. Serum bilirubin concentration greater than 34.2 μmol/L has been shown to cause false elevations of fructosamine result.

6. Haemolysis can also cause falsely low results.

2.2.1.3 Limitations of fructosamine assay

Measurements may be invalid when there are significant abnormalities of plasma protein concentrations e.g. in nephrotic syndrome, liver cirrhosis, paraproteinaemias, during an acute phase response and in untreated thyroid disease. However, fructosamine values appear to be unaffected when serum protein fluctuates within reference intervals.51

2.2.2 Clinical usefulness of serum fructosamine assay

Fructosamine serves as an index of short-term glycaemic control (2-3weeks) as compared to

glycated haemoglobin which takes longer time (6-8 weeks) hence fructosamine assay can alert the physician to deteriorating control much earlier. In patients with diseases that reduce red blood cell lifespan, such as haemolytic anaemia or haemoglobinopathies, HbA1c test can be misleadingly low. Furthermore, the HbA1c results may be abnormally high or low because

34 abnormal haemoglobin variants can interfere with the analysis. In these cases, fructosamine measurement can be used as a marker of blood sugar levels, as its measurements are based on the serum concentration of albumin instead of haemoglobin. In addition to being a measure of short- term diabetic control, fructosamine assay may be useful as a screening test for diabetes mellitus and for managing GDM. Some studies have found fructosamine to be effective in screening and detecting diabetes in pregnancy.15, 28

2.3 GDM AND FRUCTOSAMINE

A prospective study involving 165 Saudi Arabian pregnant women attending the antenatal care clinics at the Armed Forces Hospital, Riyadh during the year 2003-2004 was conducted by

Haseeb Khan et al.52 Venous blood samples were collected from all the subjects after at least 8 hours of fasting for the analysis of FPG; whereas the blood samples from the non-fasted subjects were collected for the analysis of RPG and serum fructosamine. The actual fructosamine levels were corrected for serum protein to give corrected-fructosamine (cFruct) for more precise presentation. Two cut-off values of FPG (>5.3 mmol/L and >7.0 mmol/L) and RPG (>7.8 mmol/L and >11.0 mmol/L) were used to classify hyperglycaemic subjects. The results of the study showed that FPG levels were significantly correlated with RPG (Pearson correlation =

0.597, p < 0.001). Significant correlations were also observed between cFruct and FPG (Pearson correlation = 0.673, p < 0.001) or RBG (Pearson correlation = 0.641, p < 0.001). It was also found out that among the study group, only 24 (14.5%) cases were classified as hyperglycaemic on the basis of FPG >7.0 mmol/L or RPG >11.0 mmol/L. However, the use of lower cut-off values resulted in higher frequencies of hyperglycaemia. A combined criteria of FPG >5.3 mmol/L and cFruct > 2.5 mmol/L predicted 35 patients as the most probable hyperglycaemic

35 patients when compared to 32 patients identified using the criteria of RPG >7.8 mmol/L and cFruct > 2.5 mmol/L. These criteria were associated with 4.8% and 3.6% false-positives at the expense of 3.6% and 3.0% false-negative outcomes, respectively. It was concluded that paired value of cFruct and FPG or RPG can be utilised for screening GDM thereby reducing the number of patients being subjected to OGTT.

Another prospective study was conducted by Cefalu et al.53 in which 97 pregnant women, aged

8-41 year were recruited. Patients were randomly selected based on a weekly review of the clinic roster. All pregnant women at 26-28 weeks of gestation had a formal 3-hour 100 g OGTT performed after an overnight fast following 3 days of unrestricted diet. Blood samples for FPG,

HbA1c and serum fructosamine were collected simultaneously. Gestational diabetes was diagnosed if two or more values exceeded the criteria established by the National Diabetes Data

Group. Among the 97 women studied, 13 were found to have GDM by the screening criteria established. Nine among the diagnosed GDM patients agreed to perform self-monitoring of blood glucose and were instructed in the use of a home glucose monitor (Accu-Chek II blood glucose metre). The monitored patients performed capillary blood glucose determinations before meals and at bedtime daily and returned at 2-week intervals at which time venipuncture was performed in the fasting state for glucose, HbA1c, and fructosamine. The study revealed that there was a significant difference at baseline and at each hour of the 3-h OGTT between gestational diabetic pregnant women and non-diabetic pregnant women (P < 0.005 at each point in time). There were no significant differences in the fructosamine levels (2.02 ± 0.08 mmol/L versus 1.98 ± 0.02 mmol/L) and HbAlc (4.42 ± 0.2% versus 4.6 ± 0.3%) at screening between gestational diabetic patients and non-diabetic women, respectively. The p-values were reported as non-significant. Using 2-SD and 95% confidence interval, the sensitivity of serum

36 fructosamine and HbA1c were 15.4% and 23% respectively. The specificity of fructosamine and

HbA1c were found to be 97.6% and 86.9% respectively.The serum fructosamine concentration obtained every 2 weeks during the last trimester in diabetic women correlated significantly to the fasting blood glucose obtained at that time (r = 0.81, p < 0.001). In addition, the mean blood glucose obtained at 2-week intervals with out-patient monitoring correlated significantly to the fructosamine level taken at that time (r = 0.62, p < 0.001). The HbAlc determined at 2-week intervals did not correlate to the FBG (r = 0.11) or to the average blood glucose (r = -0.12) recorded at 2-week intervals during the last trimester. The p-values were also reported as non- significant. It was concluded that, the serum fructosamine assay, as a single value, is a poor screening test for gestational diabetes. However, the fructosamine assay showed considerable potential in its ability to longitudinally monitor the maternal glycaemic state, as assessed with fasting blood glucose and mean out-patient blood glucose in the third trimester in contrast to

HbA1c. However, the fructosamine assay method was not clearly stated and the exact p-values were also not mentioned but only reported as insignificant.

A total of 161 pregnant women receiving antenatal care at the Peking University First Hospital were enrolled into a randomised prospective study by Kui and his colleagues.54 The pregnant women were divided into three groups according to gestational age (16-20 weeks group, 56 patients; 28-34 weeks group, 72 patients and 37-41 weeks group, 33 patients). Each group was subdivided into normal and abnormal glucose tolerance sub-groups. The glucose tolerance was evaluated through the glucose challenge test. The women with abnormal glucose tolerance were diagnosed with GDM based on the results of GCT and OGTT. The cut-off values were set at

GCT 7.8 mmol/L, and OGTT fasting 5.8 mmol/L, 1-hour 10.6 mmol/L, 2-hour 9.2 mmol/L, and

3-hour 8.1 mmol/L. Patients with abnormal fasting glucose level or ≥ abnormal OGTT values

37 were diagnosed with GDM. Levels of serum fructosamine were also measured. Out of the total subjects, 94 had abnormal glucose tolerance. In the 28-34 weeks group, the mean level of fructosamine in women with abnormal glucose tolerance was significantly higher than in those with normal glucose tolerance (P < 0.05). However, no significant difference in the level of serum fructosamine was observed among the other two study groups. It was hence concluded that serum fructosamine can be used to monitor glucose level of pregnant women with abnormal glucose tolerance, and to identify the pregnant women with high risk of GDM but cannot be used to predict GDM in early stage of pregnancy. Some of the drawbacks of the study include: In the methodology, there was no mention of HbA1c measurement rather it was only highlighted while discussing the study results. Likewise, the time when serum fructosamine was measured was not clearly stated.

Similarly, Weerasekera and co-workers28 conducted a cross-sectional, descriptive study involving 210 pregnant women at 28 weeks of gestation, attending antenatal clinics at Colombo

South Teaching Hospital. A 75 g OGTT and serum fructosamine assay in the fasting state and 2 hours after ingestion of oral glucose were performed. The upper normal level (265 μmol/L) of fructosamine was used as the cut-off point for diagnosis of GDM. Gestational diabetes mellitus was defined as fasting plasma glucose of more than 144 mg/dL or 2-hour plasma glucose level

(after taking 75 g oral glucose) between 144 mg/dL and 198 mg/dL. The OGTT was positive in

64 mothers, out of whom 56 had serum fructosamine level above the cut-off value, demonstrating a sensitivity of 87.5%. Out of the 146 subjects who tested negative by the OGTT,

138 were correctly identified by the fructosamine test with a specificity of 94.5%. This study also indicated that there was no significant difference (P < 0.01) in the serum fructosamine values in fasting and the 2 hours after ingestion of glucose. It was therefore concluded that a

38 single assay of serum fructosamine at any time of the day can be recommended as a screening test for detection of GDM.

In an attempt to evaluate the role of fructosamine/albumin ratio as an alternative screening parameter for GDM, Bor et al29 conducted a research at Gazi University School of Medicine,

Ankara, Turkey. Serum fructosamine, albumin, total protein, fructosamine/albumin ratio, and oral glucose tolerance were measured in 56 non-pregnant control healthy subjects (group one), and in 96 pregnant women at 24-28 weeks of gestation who screened positive after a 50 g glucose challenge-test (group two). Oral glucose tolerance test identified 12 of 96 pregnant women as having GDM. Fructosamine concentration of 1.98 ± 0.32 mmol/L and fructosamine/albumin ratio of 47 ± 10 μmol/g were obtained in non-pregnant control subjects.

During the second trimester a lower fructosamine level (1.84 ± 0.29 mmol/L, p < 0.05) and a higher fructosamine/albumin ratio (62 ± 15 μmol/g, p < 0.001) occurs in pregnant women, when compared to non-pregnant healthy control subjects, most likely due to the low serum albumin concentration (30 ± 6 g/L). The serum fructosamine levels and fructosamine/albumin ratio were only slightly higher in the pregnant women with GDM than in normal pregnant women (2.05 ±

0.47 mmol/L versus 1.84 ± 0.29 mmol/L, 67 ± 16 μmol/g versus 62 ± 15 μmol/L, respectively) but the differences were not statistically significant. The exact p-value was not stated. The sensitivity, specificity, positive predictive value and negative predictive value for fructosamine were 41.7%, 85.7%, 29.4% and 91%, and for fructosamine/albumin ratio were 25%, 79.8%, 15% and 88% respectively. The researchers concluded that both fructosamine and fructosamine/albumin ratio have low sensitivity as predictors of GDM and can therefore not be used as screening tests. However, the study design and sampling technique were not clearly stated.

Similarly, Ronald Comtois et al 55 measured serum fructosamine levels and fructosamine/protein among 100 pregnant women who had undergone glucose tolerance tests because of clinical risks.

Among the study participants, 13 were dignosed to have GDM. The study participants with

GDM had significantly higher fructosamine/protein levels (39 ± 3.9 μmol/g versus 37 ± 3.2

μmol/g, p < 0.05), fasting serum glucose levels (107 ± 13.7 mg/dL versus 82 ± 8.6 mg/dL, p <

0.001), and area under curve of glucose tolerance test (36 ± 5 g × min × dl−1 versus 22 ± 3.6 g × min × dl−1, p<0.001) compared with the normal pregnant women respectively. However, the serum fructosamine levels were not significantly different between the GDM and non-GDM groups (2.3 ± 0.26 mmol/L versus 2.2 ± 0.17 mmol/L) respectively; 10 out of the 13 women with

GDM had a fructosamine/protein ratio within 2 SD of the mean of the groups of the normal pregnant women. The study conclusion was that serum fructosamine and fructosamine/protein ratio levels should be considered insensitive as a screening test in pregnant patients with clinical risk of gestational diabetes. However, this study was prone to bias, for it was not blinded.

CHAPTER THREE

3.0 AIM AND OBJECTIVES

3.1 AIM: To evaluate the clinical usefulness of serum fructosamine as a screening test for the detection of GDM in at risk pregnant women.

3.2 SPECIFIC OBJECTIVES

1. To conduct a 2- hour OGTT and measure serum fructosamine, albumin and total protein in pregnant women attending the antenatal clinic at Aminu Kano Teaching Hospital (AKTH),

Kano, Nigeria.

2. To measure serum fructosamine, fasting plasma glucose, albumin and total protein in non- pregnant apparently healthy age-matched women volunteers attending immunization clinic at

AKTH.

3. To determine the prevalence of GDM among the study subjects using the World Health

Organisation’s criteria for OGTT

4. To determine the prevalence of GDM among the study subjects using serum fructosamine

5. To assess the validity of serum fructosamine in the detection of GDM against OGTT.

CHAPTER FOUR

4.0 SUBJECTS AND METHODS

4.1 STUDY AREA

The study participants were recruited from the antenatal clinic (ANC) of AKTH, while the investigations were done in the hospital’s Chemical Pathology Department. The Hospital is located in Kano, North Western Nigeria. It is a tertiary health centre established in 1998 primarily for Kano, Katsina and Jigawa states. It also serves neighbouring states like Zamfara,

Bauchi and Gombe. It has 17 clinical Departments and functional laboratories in the four sub- specialities of Pathology, namely: Chemical Pathology & Immunology, Haematology and Blood

Transfusion, Medical Microbiology and Histopathology.

The Department of Chemical Pathology is equipped with automated and semi automated analysers like Cobas Integra 400 plus, routine chemistry analysers, Elecsys 2010 immunology analyser, Ion Selective Electrode (ISE) analysers and Microplate reader and washer. The

Department provides both routine and specialised laboratory services to other Departments of the hospital, other Health centres within and outside Kano State. Dynamic function tests such as

OGTT, dexamethasone suppression test, short synacthen test and water deprivation test are performed routinely in the Department.

The Antenatal Clinic is run by the Department of Obstetrics and Gynaecology from Mondays to

Thursdays by 4 teams comprising of Consultants and Resident Doctors. The ANC receives an average of 200 pregnant women per clinic day. Pregnant women with risk factors for GDM during booking or during subsequent visits are usually referred to the Department of Chemical

Pathology for OGTT, which is conducted on Mondays and Thursdays with an average number of

10 patients per week.

4.2 STUDY DESIGN

The study was a descriptive cross sectional study.

4.3 STUDY POPULATION

The study was conducted among two groups of women. Group I involved 193 pregnant women at 24 to 28 weeks of gestation (with risk factors for GDM) who attended antenatal clinic at

AKTH, Kano. Group II were the controls made up of 120 non-pregnant, apparently healthy women that attended immunization clinic at AKTH, Kano.

4.4 SAMPLING TECHNIQUE

Systematic sampling technique was used to select the patients while controls were recruited consecutively until the desired sample size of 120 apparently healthy, non-pregnant women was achieved.

4.5 SAMPLE SIZE DETERMINATION

The sample size was determined using Fisher’s statistical formula as follows:56 n=Z2pq/d2

Where, n= Minimum sample size

Z=1.96 (confidence interval) which is a constant.

P = Prevalence rate from a previous study (13.9%).4

Therefore, P = 13.9 = 13.9/100 = 0.139 q (Complimentary probability) = 1-P = 1- 0.139 = 0.861 d= Precision (margin of error) at 95% confidence limit = 0.05 n= (1.96)2 x 0.139 x 0.861/ (0.05)2 = 183.9

From the above, 184 was the approximate minimum sample size for the study. A sample size of

193 was therefore used as the study group. One hundred and twenty non-pregnant women volunteers were recruited as the control group. Hence, a total of 313 participants were recruited for the study.

4.6 INCLUSION CRITERIA

The study participants were grouped as patients and controls.

Inclusion Criteria for Patients: Pregnant women at 24 to 28weeks of gestation with risk factors for GDM (glycosuria, intrauterine foetal deaths, family history of diabetes mellitus, booking weight > 90 kg, macrosomia, previous history of neonatal deaths, history of recurrent miscarriages etc) receiving antenatal care in AKTH who have consented to participate in the study.

Inclusion Criteria for Controls: Non-pregnant, apparently healthy women at immunisation clinic who consented to participate in the study.

4.7 EXCLUSION CRITERIA

Exclusion Criteria for Patients:

1. Pregnant women with established GDM

2. Known diabetes mellitus patients

3. Patients with disorders that can affect serum albumin level like nephrotic syndrome, liver cirrhosis and monoclonal gammopathy.

Exclusion Criteria for Controls:

1. Pregnancy

2. Known diabetic women.

3. Women within the puerperal period.

4.8 ETHICAL CONSIDERATION

Ethical approval (Appendix-I) was obtained from the Research Ethical Committee of AKTH.

Informed consent was obtained from all the subjects. Participants were adequately informed

(Appendix-II) about the aims and benefits of the study which included early detection of GDM, recommendations for treatment and monitoring. In line with the Helsinki Declaration, informed written consent (Appendix-III) was obtained from the participants before collecting data and blood samples from them. All data collected from the participants were kept confidential. The results of the study were in no way used to the detriment of the participants. Participation was voluntary and refusal was not associated with any negative consequence. Also, the study did not add any financial cost to the participants beyond what the standard of care requires.

4.9 DATA COLLECTION

4.9.1 TOOLS

Questionnaires, Weighing scale (RGZ -120 Health scale), Stadiometre (RGZ -120 Health scale),

Accoson Mercury Sphygmomanometre, Cobas Integra 400 plus (Roche), Vacutainer venepuncture set.

4.9.2 COLLECTION METHODS

Participants that consented were recruited for the study and their biodata was obtained using a structured pre- tested questionnaire that was administered by the interviewer (Appendix IV).

Their Blood Pressure was determined using Accoson mercury sphygmomanometer. Korotkoff’s sounds phases I and V were used to determine the systolic and diastolic blood pressure respectively.

Weight of each participant was measured in kilogram and approximated to 1 decimal point using

RGZ-120 Health weighing scale. Participants were politely told to remove their shoes, have minimal clothing and keep their bags on a nearby table after which they were requested to stand steadily on the scale facing the investigator. The weight was then determined by reading from the screen of the weighing scale.

The height of each participant was determined using a stadiometre (RGZ- 120 Health scale).

Participants were requested to remove their shoes and head gear to stand erect facing the investigator. The scale was adjusted until it touched the superior part of the scalp of the participant and the reading was obtained in metres approximated to 1 decimal point by direct reading of the scale.

Their body mass index (BMI) was determined as weight in kilogram divided by height in metre square (kg/m2).

The patients were prepared for OGTT according to the departmental guidelines attached as appendix V. On the day of the test, they were counselled on the protocols of the study and blood samples were collected between 8 and 9 am for fasting plasma glucose. The patients were then given a 75 g load of anhydrous glucose dissolved in 300 ml of drinking water and blood samples for glucose estimation were collected at 30 minutes interval for 2 hours. Samples were also collected for serum fructosamine, albumin and total proteins.

4.10 SAMPLE COLLECTION AND PROCESSING

A tourniquet was applied to the upper arm above the cubital fossa, the most prominent peripheral vein was selected and the site was cleaned with an alcohol-soaked cotton wool. The vein was then punctured using a vacutainer system and whole blood was collected into fluoride oxalate tube for fasting plasma glucose. Whole blood was also collected simultaneously for fructosamine, albumin and total protein assays. Blood samples for serial glucose estimation during the OGTT were also collected in fluoride oxalate tubes. The serum from plain tube and plasma from fluoride oxalate tube were separated by centrifugation at a relative centrifugal force

(RCF) of 1200X g for ten minutes. The serum and the plasma were then transferred into appropriately labelled containers, and frozen at -200C. Samples were analysed in batches within

4 weeks of collection.

4.11 LABORATORY METHODS

4.11.1 FRUCTOSAMINE (Nitroblue tetrazolium (NBT) method, Johnson RN, Metcalf PA,

Baker JR: 1982).57

Quantitative measurement of serum fructosamine was done using nitroblue tetrazolium method on Cobas Integra 400 plus (Roche) automated chemistry analyser.

4.11.1.1 Test Principle

This test is based on the ability of ketoamines to reduce nitroblue tetrazolium in alkaline medium. The rate of formation of formazane (chromogen) is directly proportional to the fructosamine concentration and was measured photometrically at 522 nm.58 Measurement was done against Roche fructosamine calibrator.

4.11.1.2 Test procedure

Using automatic process, 60 μL of reagent 1, 12 μL of reagent 2 and 24 μL of the diluent were mixed with 6 μL of sample and fructosamine was measured automatically by the cobas integra auto-analyser.

4.11.1.3 Quality Control

Precinorm fructosamine and precipath fructosamine which are fructosamine quality control samples used by cobas integra plus were used for quality control. Analyses were done in batches and controls were run with each batch, and values that are > ± 2 SD were repeated.

Precision: The within-run coefficient of variation (CV) of this method for frutosamine using level 1 and level 2 controls were 0.92% and 0.65% respectively (n = 20). The between-run CV using level 1 and 2 controls were also found to be 2.8% and 2.5% respectively (n = 20).

4.11.1.4 Calculation

Cobas Integra analyser automatically calculated the analyte concentration of each sample in

µmol/L.

Corrected fructosamine: After obtaining the results from cobas integra, fructosamine levels were corrected (for total proteins) in the pregnant women using the formula below:59

Fructosaminecorr = measured fructosamine X 72/measured total protein (g/L).

4.11.2 ALBUMIN (Bromocresol green method, Dumas BT, Watson WA, Biggs HG : 1971) 60

Serum albumin was measured manually using Bromocresol green (BCG) method.

4.11.2.1 Test Principle

The measurement of serum albumin is based on its quantitative binding to the indicator, BCG.

The albumin –BCG- complex absorbs maximally at 578 nm, the absorbance being directly proportional to the concentration of albumin in the sample.60

4.11.2.2 Test Procedure

Blank Standard Specimen

Reagent 2.5 ml 2.5 ml 2.5 ml

Standard ------10 µl ------

Specimen ------10 µl

Mixed and incubated for approximately 5 minutes at 25 °C. Absorbance was measured at a wavelength of 578 nm against reagent blank within 60 minutes.

4.11.2.3 Calculation

Albumin Concentration (g/L) =

Absorbance of Sample Concentration of standard × Absorbance of standard

4.11.2.4 Quality Control

Randox assayed multisera levels 1, 2 and 3 were used for daily quality control. All levels of control were assayed with each analytical run and values that are > ± 2 SD were repeated.

Precision: The within-run coefficient of variation (CV) of this method for albumin using level 1 and level 2 controls were 2.06% and 0.75% respectively (n = 20). The between-run CV using level 1 and 2 controls were also found to be 2.18% and 0.74% respectively (n = 20).

4.11.2.5 Linearity

This method is linear up to 75.5 g/L. For concentrations that exceeded this value, the samples were diluted 1 in 1 with normal saline (0.9% NaCl) solution and reassayed. The results were multiplied by 2.

4.11.3 GLUCOSE (Glucose oxidase method, Howanitz PJ and Howanitz JH : 1984) 61

Glucose was estimated manually by glucose oxidase method.

4.11.3.1 Test Principle

Glucose is determined after enzymatic oxidation in the presence of glucose oxidase. The hydrogen peroxide formed reacts under catalysis of peroxidase with phenol and 4- aminoantipyrine (chromogen) to form red violet quinoneimine dye as indicator which was read at 546 nm.

4.11.3.2 Test procedure

Blank Standard Specimen

Reagent 1.0 ml 1.0 ml 1.0 ml

Standard ------10 µl ------

Specimen ------10 µl

Mixed and incubated for 10 minutes at 37°C. Absorbance was measured at a wavelength of

546nm against reagent blank within 30 minutes.

4.11.3.3 Calculation

Glucose Concentration (mmol/L)

Absorbance of Sample = Concentration of standard × Absorbance of standard

4.11.3.4 Quality Control

Randox assayed multisera levels 1, 2 and 3 were used for daily quality control. All levels of control were assayed with each analytical run and values that are > ± 2SD were repeated.

Precision: The within-run coefficient of variation (CV) of this method for glucose using level 1 and level 2 controls were 1.09% and 0.83% respectively (n = 20). The between-run CV using level 1 and 2 controls were also found to be 1.17% and 0.98% respectively (n = 20).

4.11.4 TOTAL PROTEIN (Biuret method, Gornall AG, Bardawill CJ, David MM: 1949) 62

Serum total protein was estimated using Biuret method.

4.11.4.1 Test Principle

This method is based on the principle that copper in alkaline solution reacts with the peptide bonds in protein to produce a violet color that was measured spectrophotometrically at 550 nm.

4.11.4.2 Test procedure

Blank Standard Specimen

Reagent 1.0 ml 1.0 ml 1.0 ml

Standard ------20 µl ------

Specimen ------20 µl

Mixed and incubated for 10 minutes at 25 °C. Absorbance was measured at a wavelength of

550nm against reagent blank within 30 minutes

4.11.4.3 Calculation

Total protein concentration (g/L) =

Absorbance of Sample Concentration of Standard × Absorbance of Standard

4.11.4.4 Quality Control

Randox assayed multisera levels 1, 2 and 3 were used for daily quality control. All levels of control were assayed with each analytical run and values that are > ± 2 SD were repeated.

Precision: The within-run coefficient of variation (CV) of this method for glucose using level 1 and level 2 controls were 2.47% and 2.2% respectively (n = 20). The between-run CV using level 1 and 2 controls were also found to be 2.53% and 2.4% respectively (n = 20).

4.12 DATA PROCESSING AND STATISTICAL ANALYSIS

Data obtained from the questionnaires were analysed using statistical package for social sciences

(SPSS) version 22.0. The serum fructosamine, plasma glucose (fasting plasma glucose and serial plasma glucose during OGTT), total proteins and albumin levels were presented as mean and standard deviation in tabular form. Analysis of variance (ANOVA) was used to compare the means between the three groups (GDM patients, non-GDM patients and controls), while t-test was used to compare means between the GDM and non-GDM patients.

The prevalence of GDM using OGTT was determined using the following formula:

Total number of women that tested positive using OGTT X 100

Total number of pregnant women in the study

The prevalence of GDM using serum fructosamine was determined using the formula below:

Total number of women that tested positive using fructosamine X 100

Total number of pregnant women in the study

The sensitivity, specificity, positive predictive value and negative predictive value were calculated from a 2 x 2 contingency table as shown in table 4.1.

Table 4.1: Contingency Table for Calculating Validity of a Screening Test

OGTT Positive Negative Total Serum Positive TP FP TP+ FP

Fructosamine Negative FN TN FN+ TN

Total TP + FN FP+TN TP+FP+FN+TN

TP – True Positive, FP- False Positive, TN – True Negative, FN- False Negative

Sensitivity = TP/ (TP+FN)

Specificity = TN/ (TN+FP)

Positive Predictive Value = TP/ (TP+FP)

Negative Predictive Value = TN/ (TN+FN).

Qualitative variables were compared using Chi-squared test. The alpha level of statistical significance was set at < 5% (0.05).

The reference interval for serum fructosamine in the controls was established by non parametric method by considering the reference interval to lie between the 2.5th to 97.5th percentiles.

4.13. OUTCOME MEASURES

1. The results of OGTT were interpreted using WHO criteria.34

2. The result of serum fructosamine was considered to be in the diabetic range when the value is

˃332 µmol/L (upper reference limit established from the distribution of serum fructosamine in the controls of this research work).

CHAPTER FIVE

5.0 RESULTS

A total of three hundred and thirteen participants were recruited for the study. The participants were categorised into two groups. Group I were the patients consisting of 193 pregnant women with risk factors for GDM. Group II were the controls made up of 120 non-pregnant, apparently healthy age-matched women.

5.1 Age distribution of the Study Participants

Table 5.1 shows the age distribution of the study participants. There was no statistically significant difference in the age distribution of the two study groups.

5.2 Indications for OGTT

The main indications for OGTT in the study participants are shown in table 5.2. Family history of diabetes mellitus was the most common indication (53.4%). About 70% of the participants had only one indication, while 24% had two indications and 6% had three indications. None of the participants had more than three indications for OGTT.

5.3 Anthropometric and clinical characteristics of the Study Participants

Table 5.3 shows the weight, height and blood pressure of the participants. There were statistically significant differences in the weight and BMI between the patients and controls but there were no statistical differences in their height, diastolic and systolic blood pressure.

Table 5.1: Age distribution of the study participants

Variables Group p -value Patients n (%) Controls n (%) Age Group

15-20 years 42 (21.8) 33 (27.5)

21-29 years 50 (25.9) 42 (35.0)

30-39 years 76 (39.3) 30 (25.0) 0.052

≥ 40 years 25 (13.0) 15 (12.5)

Total 193 (100) 120 (100)

Table 5.2 (i): Indications for OGTT

Indications Frequency Percentage (%)

(n = 193)

Glycosuria 40 20.7

Intrauterine Foetal Deaths 15 7.8

Family History of Diabetes Mellitus 103 53.4

Booking Weight > 90 Kg 34 17.6

Macrosomia 39 20.2

Previous History of Neonatal Deaths 14 7.3

History of Miscarriages 14 7.3

Previous history of GDM 8 4.1

5.2 (ii): Frequency for indications of OGTT

Indications Frequency Percentage (%)

(n=193)

Patients with only one indication 135 70

Patients with two indications 46 24

Patients with three indications 12 6

Total 193 100

Table 5.3: Anthropometric and Clinical Characteristics (mean ± SD) of the Study

Participants

Parameter Patients Controls p-value

( n= 193) (n=120)

Weight (Kg) 80.57 ± 18.4 64.78 ± 14.0 < 0.001

Height (m) 1.8 ± 0.28 1.71 ± 0.16 0.055

BMI (kg/m2) 30.01 ± 9.1 25.07 ± 5.5 < 0.001

Systolic Blood Pressure (mmHg) 125.6 ± 14.4 132 ± 14.0 0.065

Diastolic Blood Pressure (mmHg) 79.01 ± 10.5 73.22 ± 10.6 0.059

Gestational Age of Patients (weeks) 27.2 ± 0.4 ------

5.4 Serum Fructosamine distribution of controls in percentiles.

Table 5.4 shows serum fructosamine distribution among the controls in percentiles. The reference range for serum fructosamine concentration was established using values representing

2.5th to 97.5th percentiles (158.2 μmol/L to 332 μmol/L) respectively. The mean and median were found to be 221 μmol/L and 212 μmol/L respectively.

5.5 OGTT results and serum fructosamine distribution among patients (mean ± SD)

Table 5.5 shows OGTT results and serum fructosamine distribution among the patients presented as mean ± standard deviation.

5.6 Comparison of Serum Fructosamine levels between patients and controls

Table 5.6 shows there is a significant difference (p= 0.001) in the levels of serum fructosamine between the patients and controls.

5.7 Patients’ (GDM and non- GDM) OGTT and Serum Fructosamine Results

OGTT and serum fructosamine levels of the patients are presented in table 5.7 showing statistically significant difference (p < 0.001) in all the plasma glucose values between patients with GDM and those without GDM. The GDM patients had higher values of plasma glucose all through. However, there was no statistically significant difference in the serum fructosamine levels between the GDM and non-GDM patients.

Table 5.4: Serum Fructosamine Distribution amongst Controls in Percentiles

R.I

Percentiles 2.5th 25th 50th 75th 90th 95th 97.5th 2.5th-97.5th

Serum 158.2 192.2 212.0 245.0 277.6 320.9 332.0 158.2-332.0 fructosamine (μmol/L)

R.I –Reference Interval

Table 5.5 OGTT results and corrected Serum Fructosamine levels among patients (mean ±

SD)

Parameter Patients

(n=193)

FPG (mmol/L) 4.93 ± 1.4

30 min P mmol/L) 7.86 ±2.0

60 min PG (mmol/L) 8.54 ± 1.9

90 min PG (mmol/L) 8.29 ± 1.9

120 min PG (mmol/L) 7.53 ± 1.7

Corrected Serum

Fructosamine (µmol/l) 318.89 ± 135

Table 5.6 Comparison of Serum Fructosamine levels between patients and controls (mean ± SD)

Parameter Patients Controls p- value ( n= 193) ( n=120)

Corrected Serum Fructosamine 318.89±135 221.08 ± 41.8 <0. 001 (μmol/L)

Table 5.7: Patients’ OGTT and Serum Fructosamine (mean ±SD) Results

Parameter GDM Non GDM p-value

( n=41) (n= 152)

FPG (mmol/L) 5.87 ± 2.1 3.99 ± 0.7 <0.001

30 min P mmol/L) 9.61±2.9 6.11±1.1 ˂0.001

60 min PG (mmol/L) 10.83 ± 2.6 6.24 ± 1.2 ˂0.001

90 min PG (mmol/L) 10.75 ± 2.7 5.83 ± 1.1 ˂0.001

120 min PG (mmol/L) 9.82 ± 2.3 5.23 ± 1.0 <0.001

Corrected Serum

Fructosamine (µmol/l) 337.39 ± 146 300.39 ± 123 0.102

FPG – Fasting Plasma Glucose

PG – plasma Glucose

Min - minutes

5.8 Serum Fructosamine, Albumin and Total Protein Levels of the Study Participants

Table 5.8 shows that there was statistically significant difference in the serum fructosamine, albumin and total proteins levels between the two study groups. The table also showed that the control group had lower levels of serum fructosamine but higher levels of serum albumin and total proteins.

5.9 Prevalence of GDM using OGTT and Corrected Serum Fructosamine

Table 5.9 shows that the prevalence of GDM among the patients using the WHO cut off points for OGTT was found to be 21.2% while the prevalence of GDM using our healthy controls cut off points of 2.5th to 97.5th percentiles (equivalent to 158.2 – 332 μmol/L) for serum fructosamine was found to be 38.9% (figures 2 and 3).

5.10 Comparison of Serum Fructosamine with OGTT

Table 5.10 shows the performance of fructosamine as a screening test. The sensitivity, specificity, positive predictive value and negative predictive value calculated for the 193 patients were 44%, 63%, 24% and 81% respectively.

Table: 5.8 Serum Fructosamine, Albumin and Total Protein (mean ± SD) of the Study

Participants

Variable Patients Controls p-values GDM Non-GDM Serum Albumin 30.88 ± 5.6 33.43 ± 7.1 42.25 ± 3.8 < 0.001 (g/L) Serum Total Proteins 62.15 ± 6.3 61.75 ± 7.5 68.52 ± 4.3 < 0.001 (g/L) Corrected Serum 337.39 ± 146 300.39 ± 123 221.08 ± 41.8 < 0.001 Fructosamine (µmol/L)

SD – Standard Deviation

Table 5.9: Prevalence of GDM using OGTT and Serum Fructosamine

Parameter Frequency Percentage (%) (n=193) OGTT(using WHO cut off points)

Normal 152 78.8

GDM 41 21.2

Total 193 100.0

Serum Fructosamine(Using our locally established reference interval)

Normal 118 61.1

GDM 75 38.9

Total 193 100.0

21.20%

Non-GDM GDM 78.80%

Figure 2: Prevalence of GDM using OGTT

38.90% 41.10%

Non- GDM GDM

Figure 3: Prevalence of GDM using Serum Fructosamine

Table 5.10: Comparison of Prevalence Rates of GDM using Serum Fructosamine and OGTT

OGTT Positive Negative Total

Serum Positive 18 57 75

Fructosamine Negative 23 95 118

Total 41 152 193

Sensitivity = TP/ (TP+FN) = 18/41 = 0.439 = 44%

Specificity = TN/ (TN+FP) = 95/152 = 0.625 = 63%

Positive Predictive Value = TP/ (TP+FP) = 18/75 = 0.240 = 24%

Negative Predictive Value = TN/ (TN+FN) = 95/118 = 0.805 = 81%

CHAPTER SIX

6.0 DISCUSSION

The study has shown that the age distribution among the two study groups was not statistically different with p-value of 0.052l. This observation was desirable as the results obtained from the study were subject to comparison.

Family history of diabetes mellitus, past history of macrosomic baby and glycosuria were the most common indications for OGTT referral as shown in this study. This is relatively similar to the findings of Salami and co-workers in a study conducted at a tertiary hospital in Abuja,

Nigeria, where they observed that the three commonest GDM risk factors which warranted referral for OGTT were family history of diabetes mellitus, history of previous delivery of macrosomic baby and maternal weight greater than 90 kg.63 Solomon et al also reported that non modifiable risk factors for GDM include advanced maternal age ( >35years) a family history of type 2 diabetes and a personal history of GDM.64

In this study, previous history of GDM was found to be statistically significant (p=0.021). About

4.1% of the patients had previous history of GDM while none of the women in the control group had such history. This could be explained by the fact that previous history of GDM is a risk factor for the development GDM in subsequent pregnancies.65 Cihangir and colleagues in a study to establish the prevalence of GDM in Turkish women observed that the prevalence of GDM was strongly correlated with past history of GDM in the patients.66

There were statistically significant differences in the weight and BMI between the patients and the control group (p < 0.001). About 71% of the patients diagnosed to have GDM were obese while only 44% of the non-GDM patients had obesity. Higher values in weight and BMI could be explained by the fact that pregnant women are more likely to be obese due to gestational

73 weight gain. Obesity is strongly linked to the development of GDM.67 In a population-based cohort study of about 97,000 singleton births; it was found that obese women had a 3-fold higher risk of developing GDM than non-obese women.68

The mean concentrations of albumin and total proteins were significantly lower among the pregnant women (p < 0.001). This finding has further reaffirmed the fact that a fall in the concentration of total proteins and albumin occurs during pregnancy as reported by previous studies.69,70,71 There was also a statistically significant difference in the mean serum fructosamine between the patients and controls (p < 0.001) with the controls having lower mean serum fructosamine levels. This observation is expected as the controls were apparently healthy, non- pregnant, non diabetic women (mean FPG = 4.3 mmol/L) compared to the patients who had

GDM and some degree of abnormal plasma glucose levels. However, the mean serum fructosamine was not statistically different (p = 0.102) in the GDM and non-GDM patients. This finding is consistent with reportsfrom previous studies.53,55 Cefalu, Comtois and co-workers both found that there were no statistically significant differences in the levels of serum fructosamine between GDM and non-GDM patients, eventhough none of the researchers made mention of the p- values.

In this study, the prevalence of GDM using OGTT was found to be 21.2%. Several values for prevalence of GDM have been reported world-wide and could be attributed to the use of wide range of definitions and diagnostic test criteria in the various studies. A study conducted among

Saudi women by Alfadhli and colleagues reported a higher GDM prevalence of 39.4%.72 This could be attributed to the use of IADPSG criteria which employed a lower cut-off for the fasting plasma glucose (≥ 5.1 mmol/L) compared to the WHO criteria (employed by this study) that uses a cut-off fasting plasma glucose of ≥ 7.0 mmo/L. Studies conducted in Jos and Lagos, reported

GDM prevalence rates of 8.3% and 11.6% respectively.73,74 These lower prevalence values could be explained by the fact that the studies were respectively conducted about seven and twenty years ago; meanwhile prevalence of GDM and diabetes mellitus in general has been on the increase over the years.75

The prevalence of GDM using fructosamine was found to be 38.9% in this study. This is quite high compared to that obtained using OGTT (21.2%). This finding may be as a result of high false positives observed using the serum fructosamine. The cut off value of serum fructosamine used for diagnosis of GDM was the upper reference of serum fructosamine established from this study.

The validity of a screening test could be determined by the sensitivity, specificity, positive predictive value and negative predictive value.76 Sensitivity of a screening test refers to a test's ability to designate an individual with disease as positive. A highly sensitive test means that there are few false negative results, and thus fewer cases of disease are missed. It is desirable for a good screening test to have a high sensitivity. In this study, the sensitivity of serum fructosamine as a screening test was found to be 44% which means that serum fructosamine was only able to detect 44% of GDM cases as diagnosed by the gold standard test (OGTT). GDM being a disease associated with serious complications especially to the foetus, will be missed in about 6 out 10 pregnant women and this implies that the sensitivity is quite low for a test that detects GDM.

The specificity of a test is its ability to designate an individual who does not have a disease as negative. A highly specific test means that there are few false positive results. It may not be feasible to use a test with low specificity for screening, since many people without the disease will be screened as positive, and potentially undergo unnecessary diagnostic procedures.

Specificity of serum fructosamine found in this study was 63% implying that serum fructosamine was able to identify less than two-third of those that do not have GDM as negative. It is desirable to have a test that is both highly sensitive and highly specific.

Positive predictive value (PPV) is the probability that a patient with a positive (abnormal) test result actually has the disease. The result of this study showed that fructosamine had a positive predictive value of 24% meaning that if a pregnant woman screens positive for GDM using serum fructosamine, there is only a 24% probability that she has GDM. Though, the PPV is dependent on the disease prevalence (higher in diseases with high prevalence irrespective of the sensitivity and specificity), it is quite low as the prevalence of GDM is relatively high from this study.

Negative predictive value (NPV) is the probability that a person with a negative (normal) test result is truly free of disease. This study reported an NPV of 81% which means that if a pregnant woman screens negative for GDM using serum fructosamine, there is 81% probability that she does not have GDM.

Findings relatively similar to those obtained in this study were reported by Cefalu et al53 who obtained sensitivity and specificity of 15.4% and 86.9% respectively. Similarly, a research conducted by Bor and co-workers29 concluded that serum fructosamine cannot be used as a screening test with sensitivity, specificity, positive predictive and negative values of 41.7%,

85.7%, 29.4% and 91% respectively. Comtois et al 55 also reported that fructosamine is an insensitive screening test in detecting GDM (in patients with risk factors) because they found that the serum fructosamine levels were not significantly different between the patients with

GDM and those without GDM. Corcoy.77 Salemans78 and co-workers identified serum

76 fructosamine as an insensitive parameter for GDM screening with sensitivities of 2.26% and

17% respectively. A study addressing the issue of serum fructosamine as a screening test for diabetes mellitus in a non-pregnant population also found it to be an insensitive parameter.79

Agrawal et al80 recruited 849 pregnant women for the evaluation of the value of serum fructosamine as a screening test for GDM. In their study, fructosamine achieved a sensivity of

85.8%, a poor specificity of 23.4% and a positive predictive value of only 14.7%. They concluded that despite all advances in technology, serum fructosamine is considered as a poor screening test for GDM. This further confirmed the finding of this study.

However, the results of this study were in contrast with those of Roberts and colleagues who reported sensitivity, specificity, positive predictive value and negative predictive value of 86%,

95%, 66% and 98% respectively.15 Ab-initio, the diagnostic criteria (NDDG) employed by these researchers for OGTT were different from that used by the current study (WHO). The high sensitivity of serum fructosamine reported by Roberts and colleagues might be explained by the fact that in four out of the nine GDM patients detected by OGTT, the abnormally high serum fructosamine values were obtained much later (after 8 weeks) following the diagnosis using

OGTT. Therefore, the long duration of hyperglycaemia probably made the serum fructosamine to be abnormal.

Weerasekera et al28 also demonstrated serum fructosamine sensitivity and specificity of 87.5% and 94.5% respectively and concluded that a single assay of serum fructosamine can be recommended as a screening test for GDM. However, the cut-off point used by these researchers

(265 µmol/) for serum fructosamine is much lower than that used by the current study (332

µmol/L) and serum fructosamine they used was not corrected for total proteins.

6.1 CONCLUSION

The following conclusions were made from this study:

1. The mean serum albumin concentrations of the patients and the controls were

found to be 32 g/L and 42 g/L respectively. Both means were within reference

limits for serum albumin. The mean serum total protein concentrations of the

patients and the controls were found to be 62 g/L and 68.5 g/L respectively.These

were also within reference limits for serum total proteins.

2. The mean corrected serum fructosamine in the GDM patients, non GDM patients

and healthy controls were found to be 337.39 μmol/L, 300.39 μmol/L and 221

μmol/L respectively. The reference interval for serum fructosamine was

established from the study as 158.2-332 μmol/L.

3. The prevalence of GDM using OGTT was found to be 21.2% while the

prevalence of GDM using serum fructosamine was 38.9% (with very high false

positives).

4. Serum fructosamine had low sensitivity, low specificity and low positive

predictive value thereby rendering it a poor screening test for the detection of

GDM.

6.2 RECOMMENDATIONS

1. The prevalence of GDM in this locality using both OGTT and corrected serum

fructosamine is quite high, therefore, screening for GDM at ANC should be intensified so

that diagnosis will be made early and prompt treatment instituted to prevent or minimise

the attendant complications of GDM.

2. In view of the findings from this study, serum fructosamine is not as useful as OGTT as a

screening tool in detecting GDM. Therefore because of the poor sensitivity, specificity

and positive predictive value of serum fructosamine, the use of OGTT for screening and

diagnosis of GDM should be maintained.

3. More extensive studies should be conducted on serum fructosamine with regards to GDM

and diabetes mellitus in general to ascertain the usefulness or otherwise of serum

fructosamine in the screening, diagnosis and management of diabetes mellitus.

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APPENDIX I: ETHICAL APPROVAL

APPENDIX II: INFORMATION FORM

Serial No:

Hospital No:

I am Dr. Halima Haladu, a resident doctor in the Department of Chemical Pathology and

Immunology of AKTH, Kano conducting a study titled: Evaluation of serum fructosamine as a screening test for the detection of Gestational diabetes mellitus. Results/outcome of this might help in providing a simpler method for detecting GDM.

Results of investigations carried out on you will be made available to you and the study shall not incur any additional cost to your hospital expens es. You are free to participate or decline without any consequences. Your safety shall be given utmost priority. This study will hopefully help your managing Doctors to serve you better. The information obtained from you in the course of the study will be treated with strict confidentiality. If you wish to participate in the study your cooperation to sign the informed consent form will be highly appreciated.

If you need further clarification, contact me at chemical pathology department of AKTH or via

07036114251 and [email protected].

Thank you.

APPENDIX III: INFORMED WRITTEN CONSENT

I……………………………………………………………………..of……………………………

…………………………………………… (Address) agree to participate in the study titled:

Evaluation of serum fructosamine as a screening test for the detection of Gestational Diabetes

Mellitus. The details of the research have been explained to me.

I understand that a sample of my blood will be taken for tests and that if I so wish the results will be communicated to me in confidence.

I sign this consent willingly without being subjected to any pressure.

Participant’s name…………………………………… signature…………………date...... …...

Witness name………………………………………… signature…………………date……......

Researcher’s name…………………………………….signature……………… date......

APPENDIX IV: QUESTIONNAIRE

RESEARCH QUESTIONNAIRE: EVALUATION OF SERUM FRUCTOSAMINE AS A

SCREENING TEST FOR THE DETECTION OF GESTATIONAL DIABETES

MELLITUS

1.0 BIODATA HOSPITAL NO: SERIAL NO: DATE: a. Age (years):

15-20 [ ] 21-29 [ ] 30-39 [ ] >40 [ ] b. Marital Status:

Single [ ] Married [ ] Divorced [ ] widowed [ ] Separated [ ] c. Occupation:

Civil Servant [ ] Private Sector [ ] Self Employed [ ] Full Time House wife [ ] Others [ ] d. Parity

Primipara( ) Multipara( ) Grand Multipara( ) e. Gestational Age by.....

LMP...... weeks

USS...... weeks

2.0 MEDICAL HISTORY

Gestational diabetes in previous pregnancy Yes [ ] No [ ]

Hypertension Yes [ ] No [ ]

Thyroid Disease Yes [ ] No [ ]

Sickle Cell Disease Yes [ ] No [ ]

Medication Use Yes [ ] No [ ]

History of Diabetes Mellitus in First Degree Relative......

Reason for Referral to undergo OGTT......

3.0 SOCIAL AND FAMILY HISTORY

Smoking Yes [ ] No [ ]

Alcohol Ingestion Yes [ ] No [ ]

4.0. PHYSICAL EXAMINATION a. Weight (kg):………………………. b. Height (m):………………………. c. BMI (kg/m2):……………………… d. Blood Pressure (mmHg):......

LABORATORY PARAMETERS

a. Oral Glucose Tolerance Test Result:

i) Fasting Plasma Glucose

ii) 30 minutes post glucose load (mmol/L)......

iii) 60 minutes post glucose load (mmol/L)......

iv) 90 minutes post glucose load (mmol/L)......

v) 120 minutes post glucose load (mmol/L)...... b. Serum Fructosamine (μmol/L)……………………………… c. Serum Albumin (g/L)………………………………………. d. Serum Total Protein (g/L)………………………………….. e. Corrected serum fructosamine (μmol/L) ......

APPENDIX V: GUIDELINES FOR PATIENT’S PREPARATION

1. Patient for OGTT should be on normal unrestricted diet and usual physical activity 3 to 4

days prior to the test.

2. Drugs that affect glucose metabolism like corticosteroids, oral contraceptives, thiazides

and sympathomimetic agents should be discontinued when possible before the test.

3. The test should be preceded by an overnight fast of 10-16 hours.

4. The test should begin between 7 A.M and 9 A.M.

5. Smoking is not permitted during the test.