1

BIOCHEMICAL ANALYSES OF FREE, COMPLEXED,

AND TOTAL PROSTATE SPECIFIC ANTIGEN IN

BENIGN PROSTATIC HYPERPLASIA AND

PROSTATE CANCER IN THE UNIVERSITY OF

CALABAR TEACHING HOSPITAL, CALABAR.

BY

DR. EYAM SUNDAY EYAM

DEPARTMENT OF CHEMICAL PATHOLOGY

UNIVERSITY OF CALABAR TEACHING HOSPITAL,

CALABAR

A DISSERTATION SUBMITTED TO THE FACULTY

OF PATHOLOGY OF THE NATIONAL

POSTGRADUATE MEDICAL COLLEGE OF NIGERIA

IN PARTIAL FULFILMENT OF THE REQUIREMENTS

FOR THE AWARD OF FMCPath FELLOWSHIP.

NOVEMBER, 2016.

2

TOPIC

BIOCHEMICAL ANALYSES OF FREE, COMPLEXED,

AND TOTAL PROSTATE SPECIFIC ANTIGEN IN

BENIGN PROSTATIC HYPERPLASIA AND

PROSTATE CANCER IN THE UNIVERSITY OF

CALABAR TEACHING HOSPITAL, CALABAR.

3

DECLARATION

This research project entitled “Biochemical Analyses of Free, Complexed and

Total Prostate Specific Antigen in Benign Prostatic Hyperplasia and Prostate Cancer in

University of Calabar Teaching Hospital, Calabar” is the original work of Dr. Eyam

Sunday Eyam of the Department of Chemical Pathology, University of Calabar Teaching

Hospital, Calabar. All cited works are properly acknowledged in the references.

Candidate Sign.:……………………………………

Dr. Eyam Sunday Eyam

Department of Chemical Pathology,

University of Calabar Teaching Hospital, Calabar.

4

DEDICATION

This work is dedicated to my beautiful and loving wife, Lilian Eyam, and my

children, Daniel, Gideon and Nathan, for their sacrifice, perseverance, and prayers

during my sojourn in Lagos in the course of training.

5

ACKNOWLEDGEMENT

I thank God Almighty for His grace and mercy without which am worthless and

this research project impossible. My gratitude goes to my teachers especially Professor

Debayo M. Bolarin who has been a father and a friend, and Professor E.C. Azinge for

her supervision of this work and my entire training. Her readiness to listen and give

motherly advice all the time will not be forgotten in a hurry. I cannot continue without

acknowledging my erudite teachers, Dr. O.O. Soriyan, Dr. T.A. Oshodi, Dr. I.C. Udenze,

Dr. O.I. Ajie. They were always there for me and inculcated in me the spirit of hard work

and quality.It is note-worthy to appreciate Dr. H.C Okpara, my H.O.D. in Calabar, for all

his support and mentoring. I cannot forget my colleagues in training, Dr. I.D. Osegbe,

Dr. N.I. Okoro, Dr. O.A. Dada, Dr. C.P. Oyenekwu, Dr. U.C. Adizua, Dr. O Akimola,

Dr. N.S. Nwosu, Dr. N. Obiora and above all Dr. E.U. Egbuagha for their inspiration

and motivation to learn. I also want to thank Dr. L.O. Okunowo, Dr. Uduak A. Ikpegbu,

Dr. E.M. Adekiitan, Dr. O.R. Ibrahim, Dr. A.D. Lafenwa, Dr. A.A. Etuk, and all others

too numerous to mention, for their support and friendship. This joy and happiness will

not be complete without due recognition to the management and staff of both my centre,

the University of Calabar Teaching Hospital, Calabar and Lagos University Teaching

Hospital, Idi-Araba, Lagos, for granting me the platform and opportunity to be trained. I

am short of words to express my appreciation to all those who assisted me in this work in

one way or the other including Mr. Zibril A.O , Dr. Henry Efobe, Dr. Kenneth Oqua, Dr.

G. Ushie, Dr. Y.M. Ogarekpe, Dr. Asuquo Ene, Dr. L. Ekpe, Dr. O. Inaku, Dr.

O.A.Ayodele and Dr. K. Babatope. Finally, to my beautiful and loving wife, my

children, mum and siblings for their endless prayers, I appreciate all of you. To God is

the glory. Amen.

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TABLE OF CONTENTS

PAGE

Title Page i

Declaration ii

Attestation iii

Dedication iv

Acknowledgement v

Table of Contents vii

List of Abbreviations ix

Abstract xi

CHAPTER ONE

Introduction 1

CHAPTER TWO

Aim and objectives 4

CHAPTER THREE

Literature review 5

CHAPTER FOUR

Materials and method 17

Participants and study location 17

Inclusion criteria 17

Exclusion criteria 18

Sample size determination 18

7

Study design 19

Study population 19

Patient preparation 20

Sample collection and storage 20

Precision and controls 21

Anthropometric data 21

Analytical Methodology 22

Statistical analysis 30

CHAPTER FIVE

Results 31

CHAPTER SIX

Discussion 42

Limitations of the study 44

Recommendation 44

Conclusion 45

REFERENCES 46

Consent form 57

Questionnaire 60

Ethical clearance

8

LIST OF ABBREVIATIONS

1. ACT Alpha 1 Antichymotrypsin

2. A2M Alpha 2 Macroglobulin

3. AUC Area Under the Curve

4. BPH Benign Prostatic Hyperplasia

5. DRE Digital Rectal Examination

6. DHT Dehydroxytestosterone

7. EIA Enzyme ImmunoAssay

8. ELISA Enzyme Linked Immunosorbent Assay

9. HRP Horseradish Peroxidase

10. H2SO4 Sulphuric Acid

11. 125I Iodine – 125 Isotope

12. IRMA ImmunoRadiometric Assay

13. IPSS International Prostate Symptom Score

14. LHRH Luteinizing Hormone-Releasing Hormone

15. LUTS Lower Urinary Tract Symptoms

16. LUTH Lagos University Teaching Hospital

17. MEIA Microparticle-Based Enzyme Immunoassay

18. PCa Prostate Cancer

19. phi Prostate Health Index

20. PSA Prostate Specific Antigen

21. bPSA BPH Associated Prostate Specific Antigen

22. iPSA Intact Prostate Specific Antigen

23. pPSA Pro Prostate Specific Antigen

24. fPSA Free Prostate Specific Antigen

9

25. cPSA Complexed Prostate Specific Antigen

26. RIA RadioimmunoAssay

27. ROC Receiver Operating Characteristics

28. TMB Tetramethyl-Benzidine

29. tPSA Total Prostate Specific Antigen

30. TRUS Transrectal Ultrasound Scan

31. UCTH University of Calabar Teaching Hospital

32. USA United States of America

33. UTI Urinary Tract Infections

34. WHO World Health Organization

35. XSD Mean Standard Deviation

10

ABSTRACT

Background

The study exploited adjunct tests of molecular components of prostate specific antigen

(i.e. free and complexed) to establish their clinical usefulness in the diagnosis of Benign

Prostatic Hyperplasia (BPH) and Prostate Cancer (PCa).

Method

It was a cross sectional descriptive study involving 120 male participants between the

ages of 40 and 80 years. They were consecutively recruited from the Urology Clinic of

the University of Calabar Teaching Hospital. The study lasted for a period of twelve

months. Participants were stratified into two groups of 60 each with histologically

confirmed diagnosis of PCa and BPH respectively. Their sera were analysed for free,

complexed, and total PSA using ELISA kits and read off with AWARENESS stat fax

2100 model microplate reader.

Results

Total Prostate Specific Antigen (tPSA) values were significantly lower in BPH than in

PCa with median values of 8.4 ng/ml and 19.3 ng/ml respectively. Levels of fPSA were

significantly higher in BPH with median value of 6.0 ng/ml compared to PCa with

median value of 2.6 ng/ml. cPSA on the other hand had significantly higher values in

PCa than BPH, with median values of 15.8 ng/ml in PCa and 0.85 ng/ml in BPH. The

ROCs of fPSA/tPSA ratio compared to cPSA/tPSA ratio was statistically not different

having AUC of 0.667 for fPSA/tPSA and 0.678 for cPSA/tPSA ratios. The sensitivity

and specificity were 80.3% and 52.5% respectively for fPSA/tPSA ratio and 57.6% and

71.2% respectively for cPSA/tPSA ratio. P = .001.

Conclusion

The molecular forms of PSA i.e. fPSA and cPSA individually have a statistically

significant ability to differentiate between BPH and PCa compared to tPSA. While

fPSA/tPSA ratio showed high specificity for differentiating BPH, cPSA/tPSA ratio

outperformed fPSA/tPSA ratio by showing a higher specificity to discriminate PCa from

BPH.

CHAPTER ONE

INTRODUCTION

11

The prevalence of prostate cancer (PCa) vary across different races globally.1-3

This is thought to be the result of hereditary, environmental, social lifestyle and dietary

influences.4-8 It is the most commonly diagnosed cancer in African men, majority of

whom, are diagnosed at an advanced stage with consequent treatment complications and

poor outcome.4 The incidence rate among Nigerian men has been shown to be as high as

127/100, 000 males.9 This is in conformity with recent rise in hospital and cancer

registry data in a region hitherto considered low incidence area.10-13 Benign prostatic

hyperplasia (BPH) on the other hand constitutes a lot of health burden for adult males

with a prevalence of 25.35% in a studied population in South-Eastern Nigeria.14 There is

however, paucity of data on the prevalence of these diseases in the study area and how

they are biochemically differentiated.

For over 2 decades since its discovery, the serine protease, prostate specific

antigen (PSA) has been established as a useful tool for early detection and monitoring of

PCa.2, 15-17 Though, the performance characteristics of most PSA studies were

determined largely from Caucasian and African-American populations they are not

necessarily applicable to other ethnicities especially indigenous African populations18.

This has led to the use of less accurate decision limits of biomarker concentrations to

discriminate between BPH and PCa in our environment. Consequently, huge numbers of

unnecessary biopsies are being performed.19 Knowledge of PSA levels in men with

healthy prostate, in men with BPH and in men with PCa led to the adoption of decision

limits of: 0.0-4.0 ng/ml, 4.0-10.0 ng/ml, and 10.0 ng/ml and above; for normal prostate,

BPH, and PCa respectively in men 50 years and above.20 However there is considerable

overlap in PSA values.21 Some studies have shown that among Nigerian patients who

had PCa, PSA values below 10.0 ng/ml (< 10.0 ng/ml) were found in 20% of them while

12

10% had values below 4.0 ng/ml.22, 23 Distinguishing between patients with BPH and

those with PCa is difficult because of the overlap in PSA values in the two groups.22-24

In spite of the considerable impact of PSA assay on detection of prostate cancer,

it lacks specificity as a marker.15, 25 This is because using the conventional PSA cutoff of

4.0 ng/ml as the distinctive limit between prostate cancer and non-malignant prostate

diseases, showed significant false positive finding of 65% with increased serum PSA

concentration also seen in non-malignant prostate diseases.2, 26 Therefore, PSA test

though sensitive, is not sufficiently specific to differentiate between BPH and PCa ,

hence, requires additional approaches to enhance its specificity.27

Various attempts of enhancing PSA’s performance based on patient’s age, race,

and prostate volume have also been used as diagnostic indices.25 They were established

to be significantly different in men with BPH and those with PCa, but showed little

clinical relevance since they are highly subjective and outcome is thus a reflection of the

examiner’s perception. Other techniques like the Prostate Health Index (phi) have been

demonstrated to be useful in early detection of PCa but involves the use of 3 different

assays ([-2]proPSA, fPSA, and tPSA) with cost implications.28, 29

However, the differentiation between BPH and PCa can be improved by the

separate determination of different molecular forms of PSA in serum.2, 25, 30-32 Currently,

PSA have been characterized into distinct molecular forms of free PSA (fPSA) and

complexed PSA (cPSA), which together make up total PSA (tPSA) associated with BPH

and PCa.15, 32-34

In view of the confounding variables of using PSA as a marker, the overlap in values, its

lack of specificity and the relative paucity of information on the biochemical

differentiation of BPH from PCa in Nigeria and particularly in the study area, further

13

studies are necessary to enhance the differentiation of BPH from PCa 23, 35 Also, the

understanding that race, environment, life-style, and social factors influence the

development of prostate diseases,6, 7, 24, 36, 37 and because PSA performance

characteristics used in screening, diagnosis, and management of prostate diseases are

mostly based on studies of PSA in Caucasian and African-American populations it is

pertinent to carry out more research on the Nigerian population.18 This work determined

and compared the levels of the different molecular forms of PSA, and determined and

compared the ratios of free PSA to total PSA and complexed PSA to total PSA, with a

view to improve biochemical differentiation and diagnosis of BPH and PCa early in

patients in the University of Calabar Teaching Hospital, Calabar, Nigeria.

CHAPTER TWO

AIM AND OBJECTIVES

14

2.1 AIM:

To establish the usefulness of the molecular forms of PSA (fPSA, cPSA, and

tPSA) in the diagnosis of BPH and PCa in UCTH, Calabar.

2.2 SPECIFIC OBJECTIVES

A) To determine the level of fPSA, cPSA, and tPSA in patients with BPH

and those with PCa in UCTH, Calabar.

B) To compare the levels of fPSA, cPSA, and tPSA in BPH and PCa patients

in UCTH Calabar.

C) To determine the ratios of fPSA to tPSA and cPSA to tPSA in

discriminating between BPH patients and PCa patients in UCTH, Calabar.

D) To compare the result obtained from this study with those found in

literature.

CHAPTER THREE

LITERATURE REVIEW

15

3.1 PROSTATE GLAND.

The prostate gland, the site for two common diseases in aging men, BPH and

PCa38 is a pyramidal-shaped fibro muscular and glandular organ that measures averagely

3.5 cm x 2.5 cm long. It resembles the size and shape of a chestnut and weighs about 18

– 26 g. It is the largest male accessory reproductive organ that is endocrine in nature.36 It

synthesizes and secretes numerous hormones and growth factors including PSA.39 The

prostate is retroperitoneal, encircling the bladder neck and urethra, and lacks a capsule.8,

40 The development, growth, and differentiation of the normal prostate are regulated by

androgens acting via the androgen receptors.41 In adults the prostate can be divided into

four biologically and anatomically glandular zones (regions) namely: peripheral zone,

central zone, transitional zone, and pre-prostatic zone.6, 8, 36, 40, 42 The peripheral zone lies

mainly posterior and is where 70% of prostate cancers arise and does not contribute to

BPH, while the transitional zone is a small zone that forms less than 5% of the normal

glandular prostate tissue. It lies adjacent to the urethra and is the site of BPH.8, 36, 40

3.2 PROSTATE SPECIFIC ANTIGEN (PSA)

Prostate Specific Antigen (PSA) is a 33kD single chain serine protease of the

kallikrien gene family produced at high concentrations by normal, hyperplastic, and

diseased prostatic epithelium.26, 43, 44 Therefore PSA is organ rather than disease

specific.6, 43-45 It is mainly secreted into the seminal fluid and functions to cleave and

liquefy the seminal coagulum formed in freshly ejaculated semen.6, 26, 43, 45 Due to its

ability to bind with serum protease inhibitors such as alpha1-antichymotrypsin (ACT),

and alpha2-macroglobulin molecules, PSA exist in two (2) main forms: free PSA, and

complexed PSA, which together make up total PSA.30, 33, 43-45 Only small amount of PSA

16

leak out into circulation from the normal prostate tissue but this is increased in prostatic

diseases.43, 46 This makes PSA an important analyte used in the screening, early

diagnosis, treatment, and follow–up of patients with PCa because of its high sensitivity

(sensitivity of stage 1 disease is up to 67% using a cut-off of 4.0 ng/ml) and because PSA

levels have been demonstrated to be an independent variable to predict the presence of

PCa. However, its specificity is limited by a high frequency of falsely elevated values in

men with BPH (as high as 25% - 50% of patients with BPH have values of PSA greater

than 4.0 ng/ml) and in prostatitis.47, 48 PSA secretion is also increased by a number of

physical factors including prostate manipulation during digital rectal examination (DRE),

recent sexual activity and the interval since ejaculation as well as some pharmacological

agents.26, 45, 47-50 PSA is expressed at high levels in the tall columnar epithelial cells of the

lumina of the prostate, while basal cells express very little and the intermediate

amplifying cells a small amount of PSA. These levels may seriously be altered if there is

alteration of cell differentiation.45

The homeostasis of PSA is tightly regulated and maintained within narrow limits

and its enzymatic activity sustained constantly. These functions are possible through the

secretion of PSA at apical side of the prostatic epithelium hence PSA can only reach the

serous side of the epithelium and the circulation through defects in the basement

membrane. Therefore, PSA concentration in the prostatic fluid is in the micromolar

range, and in the serum, in the absence of cancer, it is in the nanomolar range 45

In both localized and metastatic PCa, the expression of PSA per cell is reduced

but there is an increase in the level of systemic PSA, a phenomenon that is in keeping

with the stem cell model for the hierarchical pathway that leads to the development of

the adult prostate gland.45

17

The cellular basis of PSA in the circulation varies between men with healthy

prostate, those with BPH, and those with PCa. In men with PCa, systemic PSA is

primarily an effect of tissue degradation whereas in men with BPH, circulating PSA is

associated with increase in epithelial cell numbers. Again, as men become older above

the age of 40, their prostate tends to enlarge with consequent “normal” increase in serum

PSA.41, 44, 47, 48 Therefore, the use of serum PSA in men with BPH should be

discriminated from its use as a tumour marker with reference to level in serum. In the

process of this differential diagnosis, suggestions made by some researchers that an

increase of less than 0.8 ng/ml per year may be considered ‘‘normal’’ while an increase

greater than 0.8 ng/ml per year is a pointer to presence of PCa. Hence, PSA may serve as

a marker for BPH and as an indicator for endocrine intervention.45, 47, 48

3.3 FREE PROSTATE SPECIFIC ANTIGEN (fPSA)

Free prostate specific antigen (fPSA) is the unbound form of PSA in the blood

and constitutes about 10-30% of total PSA. fPSA in serum is composed of three distinct

forms namely: pro-prostate specific antigen (p-PSA), intact-prostate specific antigen (i-

PSA), and BPH associated-prostate specific antigen (b-PSA). b-PSA is relatively

localized in the transition zone of the prostatic tissue and is known to contribute mostly

to fPSA in BPH, whereas, pPSA is localized in the peripheral zone of the prostate gland

and contributes to fPSA in cancer serum. i-PSA component of free PSA is denatured

PSA.2, 44, 45, 51 It has been found that the level of fPSA is decreased in PCa compared to

benign prostatic conditions. Generally, a test result of less than 10% fPSA is suggestive

of cancer and this is known to be most helpful when the usual PSA test level is between

4.0 ng/ml and 10.0 ng/ml 15, 17, 25, 52, 53

18

3.4 COMPLEXED PROSTATE SPECIFIC ANTIGEN (cPSA)

Complexed PSA is the bound form of PSA consisting predominantly (70 – 90%)

of PSA complexed with the protease inhibitor α1-antichymotrypsin (ACT), and with α2-

macroglobulin molecule (A2M). Trace amounts of PSA (<5%) are also known to be

bound to inter-α-trypsin inhibitor, or protein C-inhibitor. The large size of A2M makes it

to completely encapsulate the smaller PSA molecules and because no free epitope sites

of the A2M are accessible to antibodies, it cannot be detected.2, 44, 45, 51 Levels of

complexed PSA are known to be higher in PCa than in BPH.43

3.5 FREE/ TOTAL PSA RATIO

This is the ratio of free PSA to total PSA. When this ratio is multiplied by 100

(fPSA/tPSA x 100), it is also known as percent fPSA. Although prostatic cancer cells do

not necessarily secrete more PSA than benign prostatic tissue,45, 47, 48 the cancerous cells

PSA product tends to evade the enzymatic cleavage of the bond that binds PSA and its

binding proteins, hence, men with PCa have a greater fraction of complexed serum PSA

and a lower percentage of free PSA compared to men with benign conditions of the

prostate.30, 45, 54 Thus, free PSA/total PSA ratio can be used to differentiate between PSA

elevations secondary to benign prostatic diseases versus PCa. This concept has found

particular relevance in patients with tPSA level of 4.0-10.0 ng/ml with a negative DRE.

This realization corroborates a study that found prostate cancer in 56% of men with

fPSA/tPSA of less than 0.10 and only 8% of PCa in men with fPSA/tPSA value greater

than 0.25.30, 45, 52, 53

19

3.6 PSA ASSAY METHODS

Assay methods for PSA analysis may be described as standard (traditional) and

ultrasensitive methods.45, 54-56The traditional methods use isotopic and non-isotopic

labels.45, 54-56 Isotopic traditional assays are decreasingly being used today and include

radioimmunoassay (RIA), and immunoradiometric assay (IRMA). These were also the

earliest standard commercial PSA assays.24, 55On the other hand; non-isotopic traditional

assays are frequently being used and include microparticle-based enzyme immunoassay

(MEIA) which principle is used in chemiluminescense and electrochemiluminescence

and enzyme immunoassay (EIA) which principle is used in enzyme-linked

immunosorbent assay method. RIA procedure utilized a polyclonal antibody in a

traditional competitive-binding assay format (between PSA and 125I-PSA for antibody-

binding sites), while in IRMA which has been replaced with immunometric assays

employing non-isotopic labels, PSA in the patient’s sample binds to antibody linked to

solid phase and second antibody labeled with 125I binds to solid phase antibody-PSA

complex to form a sandwich.24, 45, 54-56 The MEIA is currently the most commonly used

procedure for PSA measurement and employs monoclonal antibody coated onto inert

microparticles which are directed towards PSA. When PSA in the patient’s specimen

binds to the microparticles, the microparticles get trapped on a glass fibre matrix where a

second antibody labeled with enzyme binds to the PSA forming a sandwich complex. An

enzyme substrate is added which reacts with the bound enzyme and the product which is

directly related to PSA concentration is measured.54 Another popular immunoassay

procedure is an enzyme immunoassay version of IRMA procedure in which the label is

alkaline phosphatase (ALP) instead of 125I. The reaction of p-nitrophenyl phosphate with

ALP results in p-nitrophenol production which is monitored bichromatically at 405 and

450nM. Other non-radioisotope immunometric procedures for PSA include fluorescent

20

enzyme immunoassays that use enzyme-labeled antibody directed against PSA linked to

glass fibre paper (Baxter-Dade, Miami, Florida) or magnetic beads (Tosoh, San

Francisco, California) and a bioluminescence assay (Ciba-Corning). Using these

traditional assay methods, PSA detection above the biological detection limit of less than

0.4μg/L was possible.45, 54-56

Ultrasensitive methods of PSA measurement are assays with detection limits of

0.01 to 0.001μg/L. They are significantly less than the traditional PSA assays and can

detect an increase in PSA 1-2 years before traditional methods.54 The ultrasensitive assay

methods of detecting PSA unlike the traditional methods are capable of accurately

measuring less than 0.1 μg/L of PSA. This provides advantages over the standard

methods such as:

(a) early detection of residual disease after radical prostatectomy;

(b) assessment of PSA doubling time within the ultrasensitive range to

evaluate virulence of residual disease; and

(c) assuring that persistent low concentration of PSA in patients is a

probability of not developing a progressive disease.45, 54-56

3.7 BENIGN PROSTATIC HYPERPLASIA (BPH)

This is the increasingly hyperplastic development of the prostate occurring in

men after 40 years of age.8, 14, 36, 40 Prevalence statistics are well documented in the

21

developed world but not so well in African populations especially in Nigeria.14

Alteration in the size of the prostate could affect the urinary bladder or could constrict

the urethra, resulting in lower urinary tract symptoms that characterize the clinical setting

of BPH.14 BPH is divided into microscopic (i.e. histological) BPH, which is the

proliferation of epithelial and stromal elements seen on microscopy; macroscopic BPH,

which is enlargement of the gland detected by digital rectal examination (DRE) or

radiologically; and clinical BPH, which presents with lower urinary tract symptoms

(LUTS) such as incomplete emptying, retention, recurrent urinary tract infections (UTI),

haematuria and renal insufficiency.8, 57, 58 The LUTS are divided into obstructive

(voiding) and irritative (storage) symptoms. Obstructive (voiding) symptoms result from

direct urinary flow obstruction. Dynamic obstruction secondary to contraction of the

smooth muscles of the prostate, urethra, and bladder neck could also cause obstructive

symptoms which include: incomplete emptying, intermittency, weak stream,

straining/hesitancy. Irritative (storage) symptoms on the other hand are secondary to

bladder dysfunction. They may also result from bladder wall hypertrophy and collagen

deposition in the bladder. Storage symptoms include urinary frequency during the day,

urgency/ urge incontinence, and nocturia.14, 58

The precise mechanisms that lead to the development of BPH and its associated

symptoms are yet to be fully elucidated, however, there are views that hyperplastic

growth of the prostate may result from molecular, environmental and hormonal interplay

in the gland.57 Meanwhile, some factors implicated in BPH have been identified to

include the presence of functioning testes and normal androgen levels. Others are

positive familial and genetic factors, elevated levels of dihydroxytestosterone (DHT),

increase in 5α-reductase activity, oestrogen imbalance, increase epithelial cell

hyperplasia, decrease cell death of epithelial and stromal cells, increase activity of

22

growth factors such as fibroblast growth factor, transforming growth factor, epidermal

growth factor, keratinocyte growth factor, insulin-like growth factor, endothelial growth

factor, epithelial mitogens, and non-androgenic testicular substances.6, 8, 36, 59

BPH affects both glandular epithelium and connective tissue stroma. Typically,

the sub-mucous group of glands in the transitional zone is affected resulting in the

formation of nodular enlargement.36

Several methods have been used to screen and diagnose BPH including the

international prostate symptom score (IPSS) index but are not very sensitive and

specific.42 PSA estimation is becoming more and more acceptable as a screening and

diagnostic tool for BPH. Management of BPH involves full clinical history, physical

examination, DRE, laboratory estimation of PSA, urea, creatinine, electrolytes; peak

flow rate, post void residual urine and treatment that may include (a) watchful waiting

(b) medical therapy (c) minimally invasive treatment (4) surgery.6, 8, 36, 40, 51

3.8 PROSTATE CANCER (PCa)

Prostate cancer (PCa) has evolved as a major health problem in the male

population. It is the most commonly diagnosed malignancy and the second leading cause

of death from cancer in males.3, 6, 60In many African countries, PCa is the leading cause

of cancer among men.56,57 It is the most common malignant tumour in men over the age

of 50 years. It is rare before 50 years but after this age there is a steady rise in incidence.

From postmortem specimen, it has been noted that 14% of men over age 50 and 80% of

men over age 70 have the disease.3, 6, 8, 36, 60 PCa occurs commonly at the peripheral zone

hence prostatectomy does not confer protection against PCa subsequently. There is

23

varied prevalence and incidence worldwide.60 In the year 2000 in Wales and England,

21,000 men were registered and 9000 died from PCa. The corresponding figures in the

United States of America (USA) were 260,000 men and 35000 died of PCa.3, 60, 61

Another report in 2008, noted that 237,800 men in Europe were diagnosed with PCa in

2004 out of which 85,200 died of the disease.62 In Nigeria, PCa is the most common

urological malignancy in males accounting for 70% of the urological cancers. This view

is supported by some researchers who stated that PCa is increasing in incidence in

Nigeria (127 cases per 100,000 population) and the West-African sub-region.3, 60, 63 Other

investigators corroborated this assertion by reporting that in Nigeria, studies have shown

age adjusted PCa incidence to approach that of African Americans and Jamaicans at 300

per100000 population.3, 60, 63 Therefore, in men with high risk of the disease, screening

for PCa is recommended to begin at 40 years of age. Screening is also encouraged for

men at age 40 and at 45 years to detect uncommon cases of young men with PCa before

the disease becomes incurable. In Africa however, there are generally no screening

programmes put in place, maybe, due to perceived low prevalence by world health

organization (WHO) put at 4% and perhaps the relative unavailability and cost of PSA

measurement.3, 6, 7, 63, 64 Several factors including age, race, family history, and hormone

levels, as well as presence of testes, lifestyle and environmental influences have been

implicated as causative factors of PCa.6-8, 36, 37

3.9 CLINICAL USEFULLNESS OF PSA AND ITS MOLECULAR FORMS.

Two main types of prostate diseases are known to occur in adult males, namely

Benign Prostatic Hyperplasia (BPH) and PCa. These two conditions share several

24

characteristics in common, some of which are: increase incidence in both conditions with

advancing age, increase in serum PSA, and the requirement of androgens for

development of the diseases.42, 65

One of the means of diagnosing BPH and PCa is by DRE but its accuracy is

limited and subjective, also, prostate volume, gland size estimation using transrectal

ultrasonography (TRUS) have been used but are all subjective and relatively

inaccurate.49, 57 Therefore, an alternative biochemical, endocrine or simple physiological

end point such as PSA estimation is preferred to more invasive methods.49, 57 Substitution

of PSA level for prostate volume may also be an effective strategy for more effective

identification of patients, since level of PSA correlate with both prostate volume and

BPH.57 Recent research has shown that PSA may be associated with the development of

prostatic conditions other than PCa, such as BPH and may serve as a predictive index of

therapeutic response following medical intervention. PSA therefore is not only a

screening and monitoring tool for PCa but also an index of response in patients with

BPH.2, 15, 49

Normal, enlarged and malignant prostatic growth is governed by a range of

molecular factors and interrelationships.41, 42 PSA expression may be increased by

activation or sensitization of androgen receptors (AR) by nonandrogenic compounds

such as interleukin-4 (IL-4). IL-4 increases the AR-mediated genetic expression of PSA

in PCa cells by activating the AR and sensitizing it to lower level of androgens.41, 42, 57, 61

Also, in localized and metastatic PCa, the expression of PSA per cell is reduced but there

is an increase in the systemic levels of PSA.41 The cellular basis of PSA in the circulation

varies between men with healthy prostate, men with BPH and those with PCa. In PCa,

systemic PSA is primarily an effect of tissue degradation, whereas, amongst men with

BPH circulating PSA is associated with epithelial cell numbers.6, 41 Hence, serum PSA

25

can serve both as a marker for epithelial hyperplasia and as an indicator for endocrine

intervention,57 but its use in BPH should be discriminated from its use in PCa as a tumor

marker. The acceptance of the conventional PSA value above 4.0 ng/ml as a predictive

value for PCa diagnosis led to a noticeable increase in detection of the disease.66 High

levels of PSA have over the years been associated with the diagnosis and management of

BPH and PCa, lower values of PSA of 4.0-10.0 ng/ml are associated with lower rates of

positive biopsy2, 44Overall, when PSA value of 4.0 ng/ml is used as threshold for biopsy

there is an average lead time of 5-6 years of PCa detection.44 But there is some degree of

age dependency for this threshold with lower PSA levels of 3.5-4.0 ng/ml for men 50-70

years and 2.0-2.5 ng/ml for men 40-50 years generally considered abnormal.46 Although,

PSA level rise with advancing age in the blood, in clinical practice PSA of 4.0 ng/ml

may be used as threshold for further diagnostic evaluation e.g. prostate biopsy. This is

common when there are two consecutive rises in PSA level of a patient.44 PSA level of

4.0-10.o ng/ml also occur in BPH and PCa is present only in 25% of patients with PSA

level in this range. Although PSA is a sensitive test, it is not sufficiently specific to

discriminate between BPH and PCa at intermediate values. Additional approaches are

therefore required to enhance its specificity for PCa and BPH diagnosis without reducing

the sensitivity.27, 44

In the diagnosis of BPH and PCa variants of PSA have been investigated based

on the understanding that PSA can bind with proteins in the blood to form complexes.

This led to the characterization of PSA to its molecular forms of free, complexed and

total PSA.2, 15, 33, 34, 49 The separate determination of the molecular forms of PSA have

improve the sensitivity and specificity of discriminating between BPH and PCa

especially in the intermediate range of PSA values.2, 22, 25, 30-32

26

Determination of fPSA and the calculation of percent fPSA have increased the

performance of early detection of PCa thus reducing unnecessary biopsies for patients

with BPH.22, 67 Although different studies have quoted different values of percent fPSA,

they all agree that higher percent fPSA values are associated with BPH while low values

are associated with PCa. Commonly quoted %fPSA values are >23% for BPH and <6%

for PCa.22, 67 The fPSA-to-tPSA ratio increased specificity of PCa detection in patients

with a tPSA between 4.0-10.0 ng/ml with a non-suspicious DRE therefore resulting in a

reduction of useless biopsy rate and defining more relevant indications for biopsies.68 In

order to diminish biopsy rate in men 70 years and above, a cutoff of 16% fPSA ratio

should be used. A cutoff of 20% fPSA ratio is recommended in men younger than 70

years to increase sensitivity in that age group.69

Complexed PSA on the other hand represents the major proportion of measurable

PSA in serum and has been suggested to be an alternative to the use of percent fPSA.21, 70

cPSA alone has been observed to perform better than tPSA or the fPSA/tPSA ratio and

obviates the need for a second analyte determination. It is believed that this marker may

offer significant enhancement in PSA testing with significant economic benefits.21

Again, the calculation of cPSA/tPSA ratio has been shown to perform better compared to

fPSA/tPSA ratio in classifying disease status of the prostate especially in the diagnostic

gray range of 2.0-10.0 ng/ml.71, 72

CHAPTER FOUR

MATERIALS AND METHOD

27

4.1 PARTICIPANTS AND STUDY LOCATION

A total of one hundred and twenty (120) participants, Sixty (60) patients with

Benign Prostatic Hyperplasia (BPH) and 60 patients with Prostate Cancer (PCa) between

the ages of 40-80 years were recruited in the urology clinic of the University of Calabar

Teaching Hospital, Calabar. This is the major hospital in Calabar, Cross River State,

South-South of Nigeria that offers specialist services in urology.

4.2 ETHICAL CONSIDERATION

Approval was sought and obtained from the Ethics and Research Committee of

the University of Calabar Teaching Hospital, Calabar.

Informed consent (written and verbal) was obtained from patients or relatives of

patients.

4.3 INCLUSION CRITERIA

1. Patients between the ages of 40 and 80 years

2. Fresh patients without medical treatment with drugs known to affect PSA levels

such as androgen inhibitors, 5-phosphodiesterase inhibitors, luteinizing hormone-

releasing hormone (LHRH) agonist

3. Patients with histologically diagnosed Prostate cancer.

4. Patients diagnosed histologically with BPH and are not yet on medications

5. Patients without prostatectomy

6. Patients who gave informed consent and were eligible

28

4.4 EXCLUSION CRITERIA

1. Patients on cytotoxic drugs.

2. Immunosupressed patients

3. Patients not histologically diagnosed.

4. Patients on androgen inhibitors and other PSA enhancing medicines

5. Patients that have had prostatectomy.

6. Patients with prostatitis.

7. Patients with urinary retention.

8. Patients on urethral catheter

9. Patients with urinary tract infections

4.5 SAMPLE SIZE DETERMINATION

The sample size was determined using the formula below:

N=Z2PQ / δ2 73, 74

Where N = sample size (the minimum number of partcipants required for the

study)

Z = the standard deviation corresponding to 95% level of confidence at 1.96.

P = prevalence rate of PCa obtained from previous studies = 4% ≡ 0.043, 64

Q = 1-P = 1 - 0.04 = 0.96

29

δ = precision at 5% ≡ 0.05

:. N = (1.96)2 x 0.04 x 0.96 / (0.05)2

= 3.8416 x 0.04 x 0.96 / 0.0025

=3.8416 x 0.0384 / 0.0025

= 59.007

= 60 participants x 2

= 120 participants for the 2 groups, BPH and PCa.

4.6 STUDY DESIGN

It was a cross sectional descriptive study that used participants with histological

diagnosis of PCa and BPH. Participants were consecutively recruited from the Urology

Clinic of the University of Calabar Teaching Hospital, Calabar.

4.7 STUDY POPULATION

The study population included male participants between the ages of 40 and 80

years newly diagnosed histologically with PCa and BPH. They were recruited with the

help of the urologists, from the urology clinic of the University of Calabar Teaching

Hospital, Calabar. Recruitment was done within the period of twelve (12) months.

4.8 PATIENT PREPARATION

30

Pre-analytical conditions observed included:

1. Three days of abstinence from sexual activity or ejaculation prior to sample

collection.

2. DRE was avoided before sample collection or sample collected 72 hours after

DRE.

3. Transrectal ultrasonography was avoided before sample collection.

4. Prostate manipulative procedures (transurethral resection, transrectal biopsy and

urethral catheterisation) were avoided prior to sample collection.

4.9 SAMPLE COLLECTION AND STORAGE

Blood samples of patients with preliminary diagnosis of BPH or PCa were

collected, after initial clinical assessment by the Urologist. This was done before

invasive procedures were performed and before any PSA enhancing drugs were

commenced. Blood samples were collected using multi-sample needles and vacutainers.

Asceptic procedures and universal precautions were maintained.

Participants were in the sitting position and relaxed before blood sample

collection from the antecubital vein into plain vacutainers and allowed to clot before

separation. This was done by the investigator.

Clotted blood samples were spun at 3000 revolutions per minute for 5 minutes in

a centrifuge.

The supernatant serum was aliquoted (3 aliquots / sample) into cryotubes and

stored at -80oc until analysis was done by the investigator.

Samples were pooled and analysed in batches after every six (6) months of storage.

31

4.10 PRECISION AND CONTROLS

Precision studies were carried out using control sera obtained from ACCUBIND.

Within-run, and within batch precision studies were done.

4.11 ANTHROPOMETRIC DATA

1. The weight was measured with a “HANSON” bathroom scale placed on a flat

surface. The participants removed their shoes and heavy objects like phones and

keys and extra thick clothing before mounting the scale after adjusting to zero.

2. The weight was read to the nearest 0.1Kg

3. The height was measured in meters using a stadiometer and read to the nearest

0.1M

4. The BMI was calculated using the formula:

BMI = weight (Kg) / height2 (m2)

5. Waist circumference in centimeters (cm) was measured with a tape using the

mid-point of the iliac crest and the umbilicus as landmarks.

4.12 ANALYTICAL METHODOLOGY

A) FREE PROSTATE-SPECIFIC ANTIGEN (fPSA)

i) PRINCIPLE OF ASSAY FOR fPSA.75-81

32

The fPSA enzyme linked immunosorbent assay (ELISA) kit obtained from

ACCUBIND, with lot number EIA-23Q1B4 and expiry date of 2017-02 was used. The

assay was a quantitative sandwich immunoassay method where the microtiter plate had

been pre-coated with a monoclonal antibody specific for fPSA. The standards, controls

and test samples were then added to the microtiter plate wells and fPSA was bound to the

antibody pre-coated on the wells. Another preparation of horseradish peroxidase-

conjugated PSA antibody was also added to each well in order to quantify the amount of

fPSA in the samples. The conjugated antibody was bound to which is a substrate for

horseradish peroxidase (HRP) was added to each microtiter well. To stop the reaction, a

stop solution of sulphuric acid (H2SO4) was added and the colour change was then

measured and read with a microtiter plate reader at 450nm wavelength.

ii) MATERIALS

1. Single channel precision pipettes and tips

2. Reagent containers

3. Test tubes and racks

4. Polypropylene tubes (25mL)

5. EllenMeyer flasks (100mL, 1L)

6. Incubator

7. Microtiter plate reader

8. Squirt bottle

9. Sodium hypochlorite solution

10.Distilled water

33

11.Plastic plate cover

12.Absorbent paper

13. Vortex mixer

iii) ASSAY PROCEDURE.

1. All reagents were brought to room temperature before use

2. Frozen serum was allowed to thaw at room temperature before analysis.

3. All fPSA standards were prepared before starting assay procedure.

4. Desired numbers of strips were secured from the coated microtiter plate to the

holder.

5. 50μL of standard or sample was added to the appropriate wells

6. 100µl of fPSA enzyme reagent was added and mixed for 20 seconds with a

vortex mixer

7. Plate was covered and incubated at room temperature for 60 minutes

6. All wells were decanted and washed three times with wash buffer solution.

7. Plates were inverted and blotted dry by hitting onto absorbent paper until no

moisture was visible.

8. 100μL of working substrate was added to each well and incubated at 37oc for 15

minutes without shaking.

9. 100μL of stop solution was then added to each well and was thoroughly mixed.

34

12. Absorbance was read at 450nm using microtiter plate reader (AWARENESS,

STAT 2100) within 10 minutes.

iv) CALCULATION

The calibration standards that were assayed alongside the samples were used by

the microtiter plate reader to produce a standard curve of absorbance versus fPSA

concentration (ng/mL). The concentration of the fPSA in the samples was then

determined by comparing the absorbance of samples to the standard curve.

B) TOTAL PROSTATE-SPECIFIC ANTIGEN (tPSA)

i) PRINCIPLE OF tPSA ASSAY.75-81

The tPSA enzyme linked immunosorbent assay obtained from ACCUBIND had

lot number EIA-CV4B5 and expiry date of 2018-02. The assay was a quantitative

sandwich immunoassay technique in which the microtiter plate was pre-coated with a

monoclonal antibody specific for PSA. Standards, controls or samples were then added

to the microtiter plate wells and PSA, bound to the antibody pre-coated on the wells. In

order to quantify the amount of PSA present in the sample, a standardized preparation of

horseradish peroxidase (HRP)-conjugated monoclonal antibody, specific for PSA was

added to each well to “sandwich” the PSA immobilized on the plate. The microtiter plate

was then incubated, followed by thorough washing of the wells to remove all unbound

components. Next, 3,3’,5,5’tetramethyl-benzidine (TMB) substrate solution was added to

each well. The enzyme HRP and the substrate solution were allowed to react over a short

incubation period. Only wells that contain PSA and enzyme-conjugated antibody

exhibited a change in colour. The enzyme-substrate reaction was terminated by the

35

addition of a sulphuric acid solution and the colour change was measured and read by a

microtiter plate reader at a wavelength of 450nm.

ii) MATERIALS

1. Precision pipettes with disposable tips (10-100μL and 50-200μL)

2. Pipette reservoir

3. Test tubes and racks.

4. Erlenmeyer flasks: 100mL, 400mL, 1L.

5. Incubator (37oc).

6. Microtiter plate reader (AWARENESS, STAT 2100)

7. Microtiter plate washer (squirt bottle).

8. Deionized or distilled water.

9. Plastic plate cover.

10. Absorbent paper.

11. Vortex mixer.

iii) ASSAY PROCEDURE.

1. All PSA standards were prepared before starting assay procedure.

36

2. Frozen serum was kept at room temperature to thaw before analysis.

3. The desired numbers of coated wells were secured in the holder.

4. 25μL of standards and samples were then added in the appropriate wells pre-

coated with antibody.

5. 100µL of tPSA enzyme reagent was then added to the wells, covered, mixed

and incubated for 30 minutes at room temperature.

6. All the wells were decanted and washed with wash buffer solution three

times.

7. Plate was inverted and blotted dry by hitting the plate onto absorbent paper

towels until no moisture appeared.

8. 100μL of working substrate solution was added to each well and incubated

for 15 minutes at 37oc.

9. 50μL of stop solution was then added to the wells and properly mixed.

10. Absorbance was read with the microtiter plate reader within minutes at

450nm.

iv) CALCULATION:

37

The calibration standards were assayed the same time as the samples. This

allowed the microtiter plate reader to produce a standard curve of absorbance versus PSA

concentration. The concentration of tPSA in the samples was then determined by

comparing absorbance of the samples to the standard curve.

C) COMPLEXED PSA

i) PRINCIPLE OF ASSAY: 75-81

The cPSA enzyme linked immunosorbent assay (ELISA) kit obtained from

WKEA with lot number 20150706 and expiry date of 2016-01-05 was used. The assay

was a quantitative sandwich immunoassay technique where the microtiter plate has been

coated with a monoclonal antibody specific for human cPSA. Standards, controls and

test samples were then added to the microtiter plate wells and cPSA was bound to the

antibody pre-coated on the wells. Another preparation of horseradish peroxidase-

conjugated PSA antibody was also added to each well in order to quantify the amount of

cPSA in the samples. The conjugated antibody was then bound to cPSA immobilized on

the plate after incubation. 3,3’,5,5’-tetramethyl-bendizine (TMB) which is a substrate for

horseradish peroxidase (HRP) was added to each microtiter well and the reaction

stopped be the addition of sulphuric acid (H2SO4) solution after 15 minutes of

incubation. The colour change was measured and read with a microtiter plate reader at

450nm wavelength.

ii) MATERIALS:

38

1. Calibrated pipettes

2. Pipette reservoir

3. Test tubes and racks.

4. Polypropylene tubes (25mL).

5. Erlenmeyer flasks: 100mL, 400mL, 1L.

6. Incubator (37oc).

7. Microtiter plate reader.

8. Microtiter plate washer (squirt bottle).

9. Sodium hypochlorite solution, 5.25% (household liquid bleach).

10. Deionized or distilled water.

11. Plastic plate cover.

12. Absorbent paper.

13. Vortex mixer.

iii) ASSAY PROCEDURE.

1. All cPSA standards were prepared before starting assay procedure.

2. Frozen serum was kept at room temperature to thaw before analysis.

3. The desired numbers of coated wells were secured in the holder.

39

4. Serial dilution of standards was done in duplicates using 50μL of

standard diluent to add to the wells, and samples added to the

corresponding wells, covered and incubated at 37oc for 30 minutes.

5. All the wells were washed with distilled water five times.

6. Plate was inverted and blotted dry by hitting the plate onto absorbent

paper towels until no moisture appears.

7. 50μL of conjugate was added to each well and incubated for 30

minutes at 37oc.

8. Wash procedure as above was repeated five times.

9. 100μL of substrate solution was added to each well, covered and

incubated at 37oc for 15 minutes.

10. 50μL of stop solution was then added to the wells and properly mixed.

11. Absorbance was read with the microtiter plate reader within 30

minutes at 450nm.

iv) CALCULATION:

The calibration standards were assayed the same time as the samples. This

allowed the microtiter plate reader to produce a standard curve of absorbance

versus cPSA concentration. The concentration of cPSA in the samples was then

determined by comparing absorbance of the samples to the standard curve.

40

4.13 STATISTICAL ANALYSIS

Statistical analysis was done using SPSS version 22.0 Armonk NY: IBM. The

Kolmogorov-Smirnov test was used to test for normality of continuous variables such as

age, fPSA, cPSA, and tPSA. Descriptive statistics were presented as means and standard

deviations for normally distributed data and medians and interquartile ranges for skewed

data. Comparison of means was done using t-test for normally distributed data and

Mann-Whitney U-test for skewed data. Receiver operating characteristic (ROC) curve

were generated to compare the diagnostic performance of fPSA, cPSA, and tPSA. Tests

of hypothesis were considered to be two-tailed and P = .05.

41

CHAPTER FIVE

RESULTS

DEMOGRAPHIC STATUS

The total number of study participants was 120, out of which 60 had

histologically confirmed Benign Prostatic Hyperplasia (BPH) and 60 had histologically

confirmed prostate cancer (PCa). Mean age of participants was 64.8±7.8 years with the

age range being between 40 - 80 years as shown in table 1. The participants were age

matched, P = .490.

Table1:Shows age distribution of total participants

Variable Age (years)

Minimum 48

Maximum 80

Mean 64.8

42

The median age of participants with histologically confirmed Benign Prostatic

Hyperplasia (BPH) was 64 years with a minimum age of 50 years and maximum age of

80 years compared to that of histologically confirmed prostate cancer (PCa) cases which

was 65 years with minimum and maximum ages of 48 years and 80 years respectively.

They were age matched with P = .490. This is illustrated in table 2 below.

TABLE 2: Mean and age range of participants with BPH and PCa.

Variable BPH PCa

N=60 N= 60 P-value

Age range (Years) 50 – 80 48- 80

Mean ±SD 64.2±7.8 65.2±8.0 0.490

43

There were four categories of age groups of participants as shown in table 3 below. The

group of 41-50 years had a frequency of 3 representing 2.5 percent. 51-60 years age

grouping had a frequency of 39 and a percentage of 32.5 while 61-70 years age grouping

had a frequency of 51 which represented 42 percent. A frequency of 27 which

represented 22.5 percent was seen in the 71-80 years age grouping.

Table 3: Frequencies and percentages of Age groups of participants

Age range (years) Frequency (%)

41-50 3 (2.5)

51-60 39 (32.5)

61-70 51(42.5)

71-80 27 (22.5)

Total 120 (100)

44

The age group frequency of disease is shown in table 4. At a range of 41-50 years the

frequency was 1 for Benign Prostatic Hyperplasia (BPH) and 2 for Prostate Cancer

(PCa). 51-60 years age range had a frequency of 21 and 18 for BPH patients and PCa

patients respectively. A higher frequency of 28 was seen in BPH patients against 23 in

PCa patients at the age range of 61-70 years while the age range of 71-80 years had a

frequency of 10 patients with BPH and 17 patients with PCa. The P = .458.

Table 4: The frequency distribution table for disease type in the different age

groupings.

Variable BPH PCa

N= 60 N=60

Age group (years) frequency (%) frequency(%) P-value

41-50 1 (1.7) 2 (3.4)

51-60 21 (35) 18(30)

61-70 28 (46.7) 23 (38.3)

71-80 10 (16.7) 17 (28.3)

Total 60 (100) 60 (100) .458

45

LEVELS AND COMPARISON OF LEVELS OF fPSA, cPSA AND tPSA

The values of total prostate specific antigen (tPSA), free prostate specific antigen

(fPSA) and complexed prostate specific antigen (cPSA) are shown in table 5. Total PSA

levels were significantly lower in patients with benign prostatic hyperplasia (BPH) than

in patients with prostate cancer (PCa) with median values of 8.4 ng/ml (3.8-15.0ng/ml)

and 19.3ng/ml (10.6-72.5ng/ml) respectively, P < .001. Levels of free PSA were

statistically significantly higher in BPH patients with a median value of 6.0 ng/ml (2.6-

12.9ng/ml) than in PCa patients with a median value of 2.6 ng/ml (0.8-4.8 ng/ml), P =

.001. Complexed PSA on the other hand had significantly higher values in PCa than in

BPH with median values of 0.9 ng/ml(0.5-1.2 ng/ml) and 15.8 ng/ml(7.7-40.1 ng/ml) in

patients with BPH and PCa respectively, P = .001.

Table 5: Interquartile range (IQR) i.e. 25th-75thpercentile of tPSA, fPSA, and

cPSA.

Variable BPH PCa

N= 60 N=60

Median (IQR) Median (IQR) p-Value

tPSA 8.4 (3.8-15.0) 19.3 (10.6-72.5) < .001

fPSA 6.0 (2.6-12.9) 2.6(0.8-4.8) .001

cPSA 0.9 (0.5-1.2) 15.8 (7.7-40.1) .001

46

RATIOS AND COMPARISON OF RATIOS OF fPSA/tPSA AND cPSA/tPSA

The medians free prostate specific antigen to total prostate specific antigen ratio

(fPSA/tPSA) and complexed prostate specific antigen to total prostate specific antigen

ratio (cPSA/tPSA) are shown on table 6. The median fPSA/tPSA ratio was 0.74 for

benign prostatic hyperplasia (BPH) with interquartile range (IQR) of 0.63-0.88 and 0.16

with IQR of 0.03-0.26 for prostate cancer (PCa), P = .001. The median cPSA/tPSA ratio

for BPH was 0.14 with an IQR of 0.04-0.20 and 0.67 for PCa with an IQR of 0.53-0.85,

P = .001.

Table 6: Median interquartile ranges of ratios of fPSA/tPSA, cPSA/tPSA and

fPSA/cPSA.

Variable BPH PCa

N=60 N=60

Median IQR Median IQR P- value

fPSA/tPSA 0.74(0.63-0.88) 0.16(0.03-0.26) .001

cPSA/tPSA 0.14 (0.04-0.20) 0.67(0.53-0.85) .001

fPSA/cPSA 11.00 (3.60-16.00) 0.29 (0.06-0.43) .001

47

UTILITY Of tPSA AND cPSA IN THE DISCRIMINATION OF BPH AND PCa.

Figure1: ROC comparing performance characteristics of complexed PSA and

total PSA.

Receiver operating characteristic (ROC) curve analysis of tPSA and cPSA

showed that tPSA had area under the curve (AUC) of 0.735 at a cutoff above 11 ng/ml (>

11 ng/ml) with specificity and sensitivity of 64.4% and 75.7% respectively compared to

complexed PSA with AUC of 0.575 at a cutoff of greater than 1 ng/ml (> 1 ng/ml) and

specificity and sensitivity of 62.7% and 56.0% respectively as shown in figure 1.

48

TABLE 7: Table of ROC curve of tPSA and cPSA

Variable Sensitvity(%) Specitivity(%) Cutoff(ng/ml) AUC

cPSA 56.0 62.7 >1 0.575

tPSA 75.7 64.4 >11 0.735

49

UTILITY OF fPSA AND cPSA IN THE DISCRIMINATION OF BPH AND PCa.

Figure 2: ROC comparing the performance characteristics of free PSA and

complexed PSA.

Figure 2 shows ROC curve analysis of cPSA vs. fPSA. cPSA had AUC of 0.575

while fPSA had AUC of 0.541. Their specificity and sensitivity are 62.6% and 56.0%

respectively for cPSA. P = .147.

50

UTILITY OF fPSA/tPSA RATIO AND cPSA/tPSA RATIO IN

DISCRIMINATING BETWEEN BPH AND PCa.

Figure 3: ROC comparing performance characteristics of fPSA/tPSA ratio and

cPSA/tPSA ratio.

ROC curve analysis of fPSA/tPSA ratio compared with cPSA/tPSA ratio showed

AUC to be 0.66 at cutoff of less than/equal to 26% (≤ 0.26) with a sensitivity of 80.3%

and specificity of 52.5% for fPSA/tPSA ratio while cPSA/tPSA ratio had AUC of 0.68 at

a cutoff of less than/equal to 33% (≤ 0.33) with sensitivity and specificity of 57.6% and

71.2% respectively.

51

TABLE 8: ROC curve of fPSA/tPSA ratio and cPSA/tPSA ratio.

Variable Sensitivity(%) Specificity(%) Cutoff(%) AUC

fPSA/tPSA 80.3 52.5 ≤ 0.26 0.66

cPSA/tPSA 57.6 71.2 ≤ 0.33 0.68

52

CHAPTER 6

DISCUSSION

Prostate specific antigen (PSA) has been in use as a major biochemical tool for

the screening, diagnosis, and follow-up management of patients with benign prostatic

hyperplasia (BPH) and prostate cancer (PCa) for over two decades now. Due to its lack

of specificity several measures are being employed to enhance the specificity of

diagnosis of BPH and PCa.22 In this study the assay of total prostate specific antigen

(tPSA) and the different molecular forms of PSA i.e. free prostate specific antigen

(fPSA) and complexed prostate specific antigen (cPSA) in BPH and PCa patients was

carried out and revealed different patterns of performance characteristics (specificity,

sensitivity, area under the curve etc). There were elevated levels of total prostate specific

antigen (tPSA) in both BPH and PCa, however, levels where higher in PCa than in BPH

which was in conformity with previous studies. Generally, the receiver operating

characteristic curve (ROC) area under the curve (AUC) for tPSA was higher in both

groups of patients than with free prostate specific antigen (fPSA) and complexed prostate

specific antigen (cPSA). Also, tPSA had a high sensitivity for diagnosis of prostate

diseases but did not sufficiently differentiate between BPH and PCa which was in

conformity with other studies carried out in the past.2, 26, 43 The mean values of fPSA

were seen to be significantly elevated in patients with histologically confirmed BPH

when compared with patients who were histologically diagnosed with PCa in this study,

which was in agreement with other studies that had established the levels of fPSA to be

higher in BPH than in PCa previously.15, 25, 52, 53 On the other hand cPSA was seen to be

higher in patients who had histologically confirmed PCa with a high specificity and AUC

which agrees with most studies that had demonstrated a similar result in the past.2, 44, 45, 51

This is attributable to the fact that PSA product of cancerous cells tend to evade the

53

enzymatic splitting of the bonds that unite PSA with its binding proteins thereby causing

an increase level of complexing with various proteins which results in increased levels of

cPSA in PCa.31, 47, 48

In the grey zone when tPSA values are indeterminate, the free prostate specific

antigen to total prostate specific antigen (fPSA/tPSA) ratio has been used to increase the

specificity of differentiation between BPH and PCa.22 In this study, the ratio of

fPSA/tPSA and complexed prostate specific antigen to total prostate specific antigen

(cPSA/tPSA) ratio were determined and compared. It was shown that cPSA/tPSA ratio

was not statistically different from the fPSA/tPSA ratio in increasing the differentiating

between BPH and PCa which is similar to the results of the studies done in China and in

Germany.82, 83 This result was at variance with the study done by Okihira et al in which

the fPSA/tPSA ratio had the highest AUC in which a larger sample size was employed.84

This difference may be attributable to the small sample size that was used in this study.

However, the cPSA/tPSA ratio had a higher specificity for the diagnosis of prostate

cancer while fPSA/tPSA ratio had a higher specificity for identifying BPH than PCa. It

was also noted in this study that cPSA, and cPSA/tPSA ratio had better performances

than total PSA alone in differentiating between BPH and PCa disease conditions in the

intermediate zone and like fPSA/tPSA can reduce unnecessary prostate biopsies to the

barest minimum, which is in agreement with previous studies done elsewhere.85

Some studies previously demonstrated that the lower the fPSA/tPSA ratio the

more likelihood of the presence of prostate cancer21 which is in agreement with this

study. The present study has also shown that the higher the percent cPSA (%cPSA) i.e.

cPSA/tPSA ratio, the higher the likelihood of the presence of prostate cancer.

54

LIMITATIONS OF STUDY

The duration of disease was not taken into consideration.

There was inability to control information provided by participants.

The cross sectional nature of the study leaves some questions that need further research

work to be done.

Incessant industrial disputes within the health sector made sample collection difficult.

RECOMMENDATIONS

To reduce unneeded prostate biopsies and restore patients’ confidence in

clinicians and the healthcare system, it is recommended that evaluation of patients for

prostatic diseases should routinely include:

Total prostate specific antigen (tPSA) alongside

Free prostate specific antigen (fPSA) or

Complexed prostate specific antigen (cPSA) and

The calculation of the free to total PSA ratio or complexed to total PSA ratio

55

CONCLUSION

It can be concluded from this study that total prostate specific antigen remains a

useful biochemical maker for diagnosis of prostate diseases. However, the use of

molecular forms of prostate specific antigen and their ratios i.e. fPSA/tPSA and

cPSA/tPSA ratios, can complement the use of total prostate specific antigen in the

diagnosis of prostate diseases as well as enhance the specificity of diagnosis in

differentiating between benign prostatic hyperplasia and prostate cancer, even at

indeterminate levels of total prostate specific antigen. This will help to reduce

unnecessary prostate biopsies in patients with equivocal tPSA values and without

obvious clinical signs.

56

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67

APPENDIX I

RESPONDENT’S INFORMED CONSENT FORM

Title of the research: Biochemical analyses of free, complexed, and total

prostate-specific antigen in benign prostatic hyperplasia and prostate cancer in

University of Calabar Teaching Hospital, Calabar.

Name and affiliation of researcher: This study is being conducted by Dr. Eyam

Sunday Eyam of the Department of Chemical Pathology, University of Calabar

Teaching Hospital, Calabar.

Introduction: PSA levels are important in making diagnosis of prostate cancer or

BPH

The acceptance of PSA value above 4.0 ng/ml as a predictive value for presence

of prostate cancer has led to a noticeable increase in early detection of the

disease.

Sponsor: The study will be sponsored by the UCTH prostate cancer research

team.

Purpose of research: The study is to determine and compare the molecular

forms of prostate specific antigen in the diagnosis of benign prostatic hyperplasia

and prostate cancer.

Procedure of the research: 4-5mL of blood will be collected from the patient’s

vein with the patient sitting and relaxed. Sample will be analyzed for the levels of

three different molecular forms of PSA.

Expected duration of research and participant’s involvement:

This study is expected to be concluded in one year, but participants’ sample will

be collected just once during their clinic visits.

68

Potential Risk: There may be mild pain at the phlebotomy site. Transient

swelling and bleeding may also occur, but not enough to cause any harm.

Cost to participants: Your participation in this study is at no cost to you.

Potential Benefit(s): The goal of this research is to find a better way of making

diagnosis of benign prostatic hyperplasia and prostate cancer. Prospective

participants will have knowledge of their levels of the different forms of PSA.

Confidentiality: All information collected from you will be confidential and

used only for the purpose of this study and will not be used otherwise without

your permission. Code numbers will be assigned to samples and used in place of

names.

Voluntariness: Your participation in this study is entirely voluntary. You can

choose to withdraw from the study at any time and withdrawal will not deny you

of any medical attention.

Statement of person obtaining informed consent:

I have fully explained this research to the respondent in the best understood

language, and have given him sufficient information, including about risks and

benefits, to make an informed decision.

DATE…………………………….

SIGNATURE…………………………………….

NAME: ……………………………………………………………..

69

Statement of person giving consent:

I have read the description of the research/have had it translated into language I

understand. I have also talked it over with the doctor to my satisfaction. I understand that

my participation is voluntary. I know enough about the purpose, method, risks and

benefits of the research study to judge that I want to take part in it. I understand that I

may freely stop being part of this study at any time. I have received a copy of this

consent form and additional information sheet to keep for myself.

DATE…………..… SIGNATURE OR RIGHT THUMP PRINT………………

NAME: ………………………………………………………………………………

For further enquiry, please contact:

Researcher’s contact

Dr. Eyam S. Eyam

Mobile contact: 08035523407

E-mail contact: dr.eyam@yahoo.com

Department of Chemical Pathology,

UCTH, Calabar.

70

APPENDIX II

Questionnaire for the biochemical analyses of free, complexed, and total PSA

in BPH and PCa in University of Calabar Teaching Hospital, Calabar.

SECTION A: BIODATA

1) Serial number………………………………………………………………….

2) Initials………………………………………………………………………….

3) Age………………………………………………………

4) Sex ……………………………………..

5) Occupation………………………………………………………………………..

6) Hospital number ……………………………………………………

SECTION B: ANTHROPOMETRY

7) Weight………………Kg 8) Height……………m 9) BMI…………….. ..Kg/m2

10) Waist circumference……………….cm 11) BP…………………..mmHg

SECTION C: CLINICAL HISTORY

To the following questions tick YES or NO:

12) Do you have history of nocturia (waking up at night to urinate)? [Yes] [No]

13) Any history of urinary frequency (Urinating many times in the day)? [Yes] [No]

14) Any history of urinary urgency (not being able to hold urine)? [Yes] [No]

15) Any history of intermittency? [Yes] [No]

16) Any history of terminal dripping? [Yes] [No]

17) Any history of weak stream? [Yes] [No]

18) Any history of bloody urine? [Yes] [No]

19) Any history of straining / hesitancy? [Yes] [No]

71

Tick one of the following:

20) Have you been diagnosed before of enlarged prostate? [Yes] / [No] If “yes”

when? ......................................................

21) Have you been diagnosed before of PCa? [Yes] / [No] If “yes” when?

…………………………………………..

22) Has a biopsy of the prostate been taken? [Yes] / [No] if “yes”, what is the

diagnosis? …………………………………………Do you have a copy of the result?

[Yes] / [No]

23) Are you currently receiving treatment for your prostate condition? [Yes] / [No]

24) If yes state name of the

drug(s)……………………………………………………………………………………

25) Are you a known diabetes mellitus patient? [Yes] / [No]

26) Are you a known hypertensive? [Yes] / [No]

27) Other Medical conditions

indicate…………………………………………………………………………………..

SECTION D: SOCIAL HISTORY

28) Do you take tobacco in any form (cigarette or snuff)? [Yes] / [No]

29) Do you take alcohol? [Yes] / [No]: If yes, how much in a day?

30) When was the Last time you had sex / ejaculation? Tick as appropriate:-

A) A day ago

B) Two days ago.

D) Over three days

72

73