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THE NUMBER OF NEUTROPHIL IN SPUTUM INDUCTION OF ASYMPTOMATIC SMOKERS AND
SMOKERS WITH PULMONARY EMPHYSEMA BASED ON RADIOLOGIC FINDINGS
FINAL ASSIGNMENT
To fulfill the requirements for Degree of Bachelor of Medicine
By :
Uthaya Kumar NallayanNIM : 0810714039
MEDICAL PROGRAMMEFACULTY OF MEDICINE
UNIVERSITY OF BRAWIJAYAMALANG
2011
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CERTIFICATION PAGE
FINAL PROJECT
THE NUMBER OF NEUTROPHIL IN SPUTUM INDUCTION OF
ASYMPTOMATIC SMOKERS AND SMOKERS WITH PULMONARY
EMPHYSEMA BASED ON RADIOLOGIC FINDINGS
By:
UTHAYA KUMAR NALLAYAN
SRN : 0810714039
Has been examined on:Day: Friday
Date: 20 January 2012
and declared to pass by:
Examiner I,
Dr.dr.Retty Ratnawati,M.ScNIP: 19550201 198503 2 001
Examiner II / Supervisor I, Examiner III / Supervisor II,
dr.Triwahju Astuti Sp,P,MKes dr.Maimun ZulhaidahA,Mkes,SpPK NIP: 19632210 199601 2001 NIP: 19700526 199702 2005
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ACKNOWLEDGEMENT
First of all, I would like to thank God for blessing me to finish up my Final
Assignment to fulfill the precondition to achieve Medical Degree in Medical Faculty,
Brawijaya University. My title of final assignment is “THE NUMBER OF NEUTROPHIL
IN SPUTUM INDUCTION OF ASYMPTOMATIC SMOKERS AND SMOKERS WITH
PULMONARY EMPHYSEMA BASED ON RADIOLOGIC FINDINGS”.
Taking this opportunity, I would like to thank everyone whom always gives me
support and encouragement throughout my Final Assignment. I would like to thank :
1. Dr.dr Karyono Mintaroem, SpPA, as the Dean of Medical Faculty, Brawijaya
University for providing the facilities in Medical Faculty, Brawijaya University.
2. dr.Triwahju Astuti Sp,P,MKes as my first facilitator who spent her precious time
despite of her busy schedule helping and always supporting, advising and
correcting to make my research better.
3. dr.Maimun Zulhaidah A,Mkes,SpPK as my second facilitator who is full of
graciousness and willing to spend precious time for my final assignment and
providing necessary corrections.
4. My examiner, Dr.dr.Retty Ratnawati,M.Sc. who made me think out of the box
with her interesting questions and ideas relating to my thesis, and examining
my research with a smile.
5. My research advisor, dr Andreas Infianto, for helping and correcting my
mistakes despite of his busy schedule.
6. All the staffs in Respiratory Department and Pathology Clinic Laboratory of
Saiful Anwar who really helped a lot.
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7. My wonderful group mate, Kaviprathaa for always being there for me and
lending her help in completion of this thesis.
8. My family for their support, undying love and sacrifices. My heartfelt gratitude
goes to my parents Nallayan and Kaliammal as well as my three siblings,
Suresh Kumar, Balasubramaniam, Thunesh Kumar.
9. My dearest brother, Thunesh for the constant support and encouragement
through the thick and thin of my research.
10. All my friends whom never failed to lend a helping hand when I needed them the
most.
11. Final Assignment Team.
12. Those whom helped me directly and indirectly in completing this study.
Last but not least, I hope that my research will provide a great beneficial contribution
to society in the future. To accomplish that, I need critics and comment from everyone
who read my final assignment. Thank you very much.
Malang, February, 2012
Uthaya Kumar Nallayan
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ABSTRAK
Nallayan, Uthaya Kumar. 2011. Perbedaan jumlah neutrofil dalam induksi sputum perokok non symptomatis dan perokok emfisema berdasarkan gambaran radiologis.Tugas akhir Fakultas Kedokteran Universitas Brawijaya. Pembimbing: (1) dr.Triwahyu Astuti Sp,P.MKes (2) dr.Maimun Zulhaidah A,Mkes,SpPK
Rokok dengan kandungan radikal bebasnya dapat menyebabkan berbagai kerusakan di paru dan saluran nafas. Keadaan inflamasi yang terus menerus dapat berpengaruh pada keseimbangan neutrofil di alveoli pada perokok dan seterusnya dapat melandasi patogenesis terjadinya emfisema paru. Penelitian ini bertujuan untuk menentukan jumlah neutrophil dalam sputum pada perokok non simptomatis dan perokok dengan emfisema paru yang dipilih dengan menggunakan gambaran radiologis.Desain penelitian adalah Observational Cross Sectional dilakukan secara in vivo pada manusia. Terdapat 4 kelompok yang masing-masing terdiri dari 10 subyek yang dibahagi menjadi perokok ringan, perokok sedang, perokok berat dan untuk perokok simptomatis peserta yang dipilih adalah perokok berat dengan emfisema paru berdasarkan gambaran radiologis. Pada setiap subyek dicatat data klinisnya (darah lengkap, EKG, foto thoraks, spirometri) dan diambil sputum dan sampel darahnya sebanyak 5 ml untuk mengetahui kondisi badan peserta. Hasil penelitian menunjukkan bahawa hanya kelompok perokok simptomatis dengan emfisema paru yang memberi hasil signifikan (p<0.05) dan rerata jumlah neutrofil dalam kelompok lain tidak memberikan perbedaan yang bermakna. Di samping itu, berdasarkan Pearson test menunjukkan bahawa derajat merokok tidak mempengaruhi jumlah neutrofil pada sputum induksi perokok. Oleh yang demikian, kesimpulan daripada penelitian ini adalah, merokok akan meningkatkan jumlah neutrophil di alveolar tetapi derajat merokok tidak mempengaruhi jumlah neutrophil di alveolar yang ada di dalam sputum seseorang perokok.
Kata kunci : Rokok, neutrofil di alveolar, emfisema paru
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ABSTRACT
Nallayan, Uthaya Kumar. 2011.The Number of Neutrophil in Sputum induction of Asymptomatic Smokers And Smokers With Pulmonary Emphysema Based on Radiologic Findings Final assignment Fakultas Kedokteran Universitas Brawijaya. Pembimbing: (1) dr.Triwahyu Astuti Sp,P.MKes (2) dr.Maimun Zulhaidah A,Mkes,SpPK.
Cigarette contains various substances and free radicals that may be harmful to the smoker. Continous and progressive state of inflammation cause the recruitment of Neutrophil in alveolar in the smoker and this induces the pathogenesis of the pulmonary emphysema in the smoker.The study was aimed to determine the correlation of smoking with the number of neutrophil in alveolar that are recruited in the smokers. This study was a Cross Sectional Observational study which was carried out in vivo in humans. There were four groups each consisting of 10 patients where the asymptomatic smokers are grouped into three different groups which were classified as mild, moderate and severe smoker and the fourth group was the smoker with pulmonary emphysema based on radiologic findings. In each patient recorded the clinical data (complete blood count, ECG, chest X-ray, spirometry), sputum and 5ml of blood samples were taken. Based on the study, only the smoker with pulmonary emphysema group gave a significant result (p<0.05) whereas the other groups have an average distribution of neutrophil approximately the same. Despite of that, based on the Pearson test that was done to identify the correlation of smoking with the number of neutrophil, it revealed that, the stages of smoking are not proportional to the number of neurophil in the sputum induction of the smoker.Therefore, based on this study, it can be concluded that, smoking will increase the number of neutrophil in alveolar in the smoker but the stages of smoking has no effect on the number of neutrophil in alveolar from the sputum induction of the smoker.
Keywords: Cigarette, neutrophil in alveolar, pulmonary emphysema
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TABLE OF CONTENTS
Pages
Title ……….....................................................................................................................…. i
Certification page................................................................................................................ii
Preface..............................................................................................................................iii
Abstract ……......................................................................................................................v
Table of Contents.............................................................................................................vii
List of Figures.....................................................................................................................x
List of Tables.....................................................................................................................xi
List of Appendixes............................................................................................................xii
List of Abbreviation…………………………………………………………………………….xiii
CHAPTER I INTRODUCTION
1.1 Background............................................................................................................1
1.2 Statement of Study Problem...................................................................................4
1.3 Objectives of the Study...........................................................................................4
1.3.1 General Purpose............................................................................................4
1.3.2 Specific Purpose............................................................................................4
1.4 Significance of the Study........................................................................................5
CHAPTER II REVIEW OF RELATED LITERATURE 2.1 Smoking................................................................................................................6
2.1.1 Smoker Classification...................................................................................6
2.1.2 The Composition of Cigarette.......................................................................7
2.1.3 Factors that influence a person to smoke.......................................................7
2.1.4 Complication of Smoking...............................................................................8
2.1.4.1 Chronic Diseases...............................................................................8
2.1.4.2 Chronic Obstructive Pulmonary Disease..........................................10
2.1.4.2.1 Emphysema......................................................................12
2.1.4.2.2 Chronic Bronchitis............................................................14
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2.1.5 Management of Smokers……………………………………………………. .15
2.1.5.1 Drug Therapy…………………………………………………………...15
2.1.5.2 Non Drug Therapy ……………………………………………………..18
2.1.5.3 Combination Therapy……………………………………………….....18
2.2 Neutrophils…………………………………………………………………………….19
2.2.1 Introduction……………………………………………………………………..19
2.2.2 Classification……………………………………………………………………20
2.2.3 Mechanism of Production and Regulation………………………………….21
2.2.4 Biological Function of Neutrophil……………………………………………24
2.2.5 The Role of Neutrophil In Pulmonary Emphysema………………………..28
CHAPTER III CONCEPTUAL FRAMEWORK AND HYPOTHESIS3.1 Conceptual Framework........................................................................................31
3.2 Hypothesis of the Study.......................................................................................32
CHAPTER IV METHODOLOGY4.1 Study Design........................................................................................................33
4.2 Population and Sample of Study..........................................................................33
4.2.1 Population....................................................................................................33
4.2.2 Sample Size.................................................................................................33
4.2.3 Sample Size Estimation...............................................................................34
4.2.4 Sample Statistic...........................................................................................35
4.3 Location and time of study...................................................................................35
4.3.1 Place of Study..............................................................................................35
4.3.2 Time of Study...............................................................................................35
4.4 Variable................................................................................................................35
4.4.1 Dependent Variable....................................................................................35
4.4.2 Independent Variable.................................................................................36
4.5 Inclusion and Exclusion criteria of the Study........................................................36
4.6 Operational Definition...........................................................................................37
4.7 Instruments...........................................................................................................38
4.7.1 Studies Tools and Substances...................................................................38
4.8 Study Work Plan...................................................................................................39
4.8.1 Sputum Induction.......................................................................................39
4.8.2 Sputum Decontamination and Centrifugation..............................................40
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4.8.3 Neutrophil Counting....................................................................................40
4.9 Study Framework.................................................................................................42
CHAPTER V RESEARCH RESULT5.1 Subject and Study Location.................................................................................43
5.2 Characteristic of the Subject................................................................................43
5.3 Characteristic Of Supportive Examination Data of Subject..................................45
5.4 Neutrophil Counting in the sputum induction of the Subjects................................46
5.5 Data Analysis........................................................................................................47
5.5.1 Normality Test..............................................................................................47
5.5.2 Neutrophils Comparative hypothesis with using Mann Whitney U Test........47
5.5.3 One way Anova Test....................................................................................48
5.5.4 Correlation Test of Different Stages of Smoking..........................................48
CHAPTER VI DISCUSSION
6.1 Characteristic of Study Subjects...........................................................................50
6.2 Neutrophils Counting in the Sputum Induction......................................................51
6.3 The Correlation between smoking stages and number of Neutrophils..................52
6.4 The Weakness of The Study.................................................................................53
CHAPTER VII CONCLUSION AND SUGGESTION7.1 Conclusion............................................................................................................54
7.2 Suggestion...........................................................................................................55
LIST OF REFERENCES…..............................................................................................56
APPENDIXES……………………………………………………………….………................59
STATEMENT OF ORIGINALITY…………………...............................………….……...…66
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LIST OF FIGURES
Pages
Figure 2.1 The common adverse effect of smoking……………………………...…….10
Figure 2.2 A thin section of lung tissue stained with hematoxylin and eosin……….. 13
Figure 2.3 Histopathology Sample of chronic bronchitis………………………………15
Figure 2.4 Bacterial phagocytosis and destruction by a neutrophil …………………28
Figure 2.5 Pathogenesis of emphysema and smokers .........................................30
Figure 3.1 Conceptual Framework of Pathogenesis of emphysema and
association of Neutrophil………………………………………………………31
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LIST OF TABLES
Pages
Table 5.1 Characteristics of study subjects in each group............................................44
Table 5.2 Characteristics Of Supportive Examination Data of Subject.........................46
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LIST OF APPENDIXES
Table 1 Test of Normality………………………………………………………………...59
Table 2 Test of Homogeneity of Variances……………………………………………60
Table 3 One Way Anova Analysis………………………………………………………60
Table 4 Mann-Whitney Test……………………………………………………………...61
Table 5 t-Test………………………………………………………………………………62
Table 6 Correlations………………………………………………………………………63
Table 7 Raw Data………………………………………………………………………….64
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List of Abberviation
SGOT : Serum Glutamic Oxaloacetic Transaminase
SGPT : Serum Glutamate Pyruvate Transaminase
ANOVA : Analysis Of Variance
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CHAPTER 1
INTRODUCTION
1.1. Background
Tobacco use is responsible for more than 5 million deaths per year and is the
leading preventable cause of premature death worldwide. Tobacco companies have
gradually shifted their market from high-income to low-income countries, where many
people are poorly informed about the health risks of tobacco use and anti-smoking
policy is relatively weak.Few examples of such countries are Indonesia,India,and
Vietnam (Charlesworth et al., 2010).
The smoking exacerbates the effects of poverty, as expenditures for tobacco
may divert household income from food, clothing, housing, health and education. The
amount of money spent on tobacco is especially problematic in low-income countries.
For example, in Vietnam in 1996, smokers spent an average of $US 49.05 on cigarettes
per year, which was 1.5 times that spent on education, five times that spent on health
care and about one-third that spent on food per capita in the household each year. In
the poorest households in Indonesia, more money was spent on tobacco than on
education and health care combined. Indonesia is the fifth largest market for tobacco in
the world, with 182 billion sticks consumed per year. The absolute domestic
consumption of tobacco increased by 159% between 1970 and 1980, coincident with
the mechanisation of the cigarette industry in Indonesia in the early 1970s (Semba et
al., 2006).
According to the World Health Organization (WHO) Tobacco Atlas of the year
2004,, smoking can be classified into few lucid types such as manufactured cigarettes
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consist of shredded or reconstituted tobacco processed with hundreds of chemical,
Bidis consist of a small amount of tobacco, hand-wrapped in dried temburni leaf and
tied with string ,Cigars are made of air-cured and fermented tobaccos with a tobacco
wrapper, and come in many shapes and sizes, from cigarettesized cigarillos, double
coronas, cheroots, stumpen, chuttas and dhumtis. Kreteks are clove-flavoured
cigarettes. Pipes are made of briar, slate, clay or other substance and tobacco is
placed in the bowl and inhaled through the cigarettes, sometimes through water and
lastly sticks are made from sun-cured tobacco known as brus and wrapped in cigarette
paper.
Smoking is known to have a major impact on human health, adversely affecting
almost every organ. Exposure to cigarette smoke increases the risk of many diseases,
including a wide range of cancers (from lung to pancreatic cancer), cardiovascular
diseases (including atherosclerosis and coronary heart disease), a range of respiratory
diseases (including chronic obstructive pulmonary disease and pneumonia), as well as
various other adverse health effects such as increased risk of cataracts, infection and
poor wound healing, and is generally detrimental to the overall health of individuals who
smoke (Semba et al., 2006).
Emphysema is defined pathologically as an abnormal permanent enlargement
of air spaces distal to the terminal bronchioles, accompanied by the destruction of
alveolar walls and without obvious fibrosis. Inflammatory response is normally amplified
in emphysema The inflammation is further amplified by oxidative stress and protease
production and this suggest the increase of macrophages in smokers. Oxidants are
produced from cigarette smoke whereas proteases are produced by macrophages. This
leads to a protease-antiprotease imbalance that leads to destruction of elastin and other
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structural elements. This is believed to be central in the development of emphysema
(Demirjian, 2011).
Neutrophils play a major role in defense mechanism in a person’s immune
response. Neutrophils are considered to be central to the pathogenesis of most forms
of acute lung injury (ALI). For the sake of clarity, neutrophil involvement in ALI can be
conceptualized as consisting of sequential stages, beginning with their sequestration in
the pulmonary microvasculature, followed by adhesion and activation, and culminating
in the production of a microbicidal or effector response, such as the generation of
reactive oxygen species or release of proteolytic enzymes. Great strides have been
made in elucidating these various stages of neutrophil involvement. Recent studies
have focused on the intracellular signaling pathways that govern neutrophil activation
and have elucidated complex cascades of kinases and other intracellular signaling
molecules that allow for amplication of the neutrophil response, yet simultaneously
confer specificity of a response. Inflammatory response in ALI may initially be adaptive,
such as the pivotal role played by neutrophils in a bacterial or fungal infection.
Ultimately, it is the persistence or the dysregulation of neutrophil activation that may
lead to ALI (Lee and Downey, 2001).
Smoking tobacco has done a vast majority of harmful effect to the mankind.
Research about smoking and adverse effect of smoking should be encouraged and
done more frequently from now on because it may save millions of lives. I am very
intrested to be a part of the research which might help to find the corelation of smoking
and adverse effect which will be useful in curing and decreasing the death toll because
of the smoking.
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1.2.1 Statement of Study Problem
The problems to be solved in this study are,
1) Does smoking increase the number of neutrophil in alveolar of the smokers?
2) Are there any differences in the number of neutrophil in alveolar according
to different stages of smokers?
3) Are there any differences in the number neutrophil in alveolar of smoker with
pulmonary emphysema and smoker without pulmonary emphysema?
1.3.1 Objective of the Study
1.3.2 General purpose
Generally, this study is conducted to determine the influence of smoking
towards the number of neutrophil in alveolar of the smokers.
1.3.3 Specific purpose
1. To determine the number of neutrophils in mild, moderate and severe
smokers.
2. To find out the difference in neutrophils number in pulmonary emphysema
smoker and non-pulmonary emphysema smoker.
3. To determine the correlation of stages of smoking to the body mass index
(BMI) of the smokers.
4. To determine the correlation of stages of smoking to the age of the smokers.
1.4 Significance of the Study
Benefits of this study are:
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1. Can be used to give information to the smokers about the risk factor of
smoking to the development of emphysema and eventually can promote
smoking cessation to the smokers.
2. Can be used to study the long term effect of smoking in chronic obstructive
pulmonary disease such as, emphysema and chronic bronchitis.
3. Can be used to compare the difference in neutrophil count in smoker with
pulmonary emphysema and smoker without pulmonary emphysema.
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CHAPTER 2
LITERATURE REVIEW
2.1 Smoking
2.1.1 Smoker Classification
Smokers can be classified according to the Brinkman index (BI), which is
defined as numbers of cigarette smoked per day times smoking years which can be
categorized into mild,moderate and severe smokers. A mild smoker has Brinkman
Index value of 1 to 199, moderate smokers with a value of 200 to 399 and severe
smokers with a value of 400-599 (Kume et al., 2009).
Besides that, smoker also can be classified according to Indrayan’s smoking
index where it is measured according to the number of cigarretes per day and duration
of smoking. A person’s amount of smoking may also vary from time to time. A measure
could be the total number of cigarettes smoked so far in life. This number is given by
S1 = n1x1 + n2x2 + ... +nK xK,
where nk (k = 1, 2, …, K) cigarettes per day (intensity) are smoked for xk years
(duration). This is more exact than pack-years generally used for smoking. For an
example,S1 suffers from the same demerit as the pack-years, namely that smoking 10
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cigarettes a day for 25 years is the same as smoking 25 cigarettes per day for 10 years
(Indrayan, 2008).
2.1.2 The Composition of Cigarette
The tar, nicotine, and carbon monoxide content of cigarettes varies markedly
according to the different brands and types of cigarettes. For example, carbon
monoxide content in cigarettes can vary from less than 0.05 to 3.0 mg per cigarette.
This difference would affect the intravascular levels of carbon monoxide and
carboxyhemoglobin and would therefore affect the degree of any presumed
pathophysiologic effect on the arterial walls but, this effect is difficult to quantify, since
tar, carbon monoxide, and nicotine levels determined by the amount of smoke that the
smoker takes in. Therefore it depends on how they smoke their cigarettes. The amount
of tar and nicotine a smoker actually gets can also increase if the smoker blocks tiny
ventilation holes in cigarette filters that are designed to dilute smoke with air. In
addition, many smokers of low tar or light cigarettes compensate by taking deeper,
longer, or more frequent puffs from their cigarettes and causes more harm to the
smokers eventually (Schillinger et al., 2004).
2.1.3 Factors that influence the person to smoke
There are many reason for a person to smoke, but the main reason a person
tend to smoke throughout his life is because of dependency or in other word, addiction.
The cigarette-dependence process, like other pathogenically induced diseases, is
influenced by host or individual factors, environmental factors, and the level of exposure
xx
to the pathogen. Initiation is often mediated by a variety of social and cultural factors.
However, over time the reinforcing effects of the drug strengthen and the individual's
control over use weakens. Although other factors continue to operate, cigarette
dependence is powerfully and critically driven by the positively and negatively
reinforcing effects of nicotine .Like other drug dependencies, nicotine dependence is a
"progressive," "chronic," "relapsing" disorder. Mean age of cigarette smoking onset is
13-14 years. The level of nicotine dependence in adults is inversely related to the age
of smoking initiation according to the diagnostic criteria of the American Psychiatric
Association (Henningfield et al., 1999).
According to the WHO Tobacco Atlas year 2002, the teenage smoker, in
contrast to the middle aged chronic smoker,experiences primarily the pleasant effects
of smoking. In fact,impairment of pulmonary function can be demonstrated in teenage
smokers but only by rather subtle tests . By middle age and older these differences are
more pronounced .The pathological changes induced by smoking in various parts of the
body slowly accumulate over time measured in years and decades and if death occurs
in middle age or later these can be fairly easily identified by the pathologist. Every child
should know that smokers’ lungs are darker than non-smokers’ because smoke
residues accumulate there.
2.1.4 Complication of smoking
2.1.4.1 Chronic Diseases
Several components in tobacco smoke contribute to its cardiovascular harm.
Substances such as carbon monoxide reduce the oxygen carrying capacity of red blood
cells, thus forcing the circulatory system to increase its efforts to deliver needed oxygen
to all cells of the body while also predisposing the heart to rhythm disturbances.
xxi
Oxidizing chemicals, such as polycyclic aromatic hydrocarbons, cause inflammation
and can lead to atherosclerosis. These same oxidizing chemicalscan cause endothelial
dysfunction and promote vascular damage. Other toxins in tobacco smoke are
thrombogenic and can increase platelet adhesiveness, predisposing to clot formation
within the vessel. Nicotine itself has some modest physiologic effects on pulse, blood
pressure, and vascular tone. However, these are mild in comparison with the other
cardiovascular effects of the numerous other toxins . However, despite the degree to
which smoking leads to coronary artery disease and cerebrovascular disease,
cessation treatment is still not widely implemented (Burton et al., 2007).
Besides that,smoking also has high relation with kidney disease. Cigarette
smoking has been reported to exacerbate existing diabetic and non-diabetic kidney
disease and may also be an etiologic factor triggering the onset of proteinuria and
reduced renal function. Despite the recognized relationship between cigarette smoking
and kidney disease, it is unclear whether smoking cessation or reduction can attenuate
the progression of renal injury in CKD (Burton et al., 2007).
xxii
Figure 2.1 Common adverse effect of smoking (Morrow, 2007).
2.1.4.2 Chronic Obstructive Pulmonary Disease ( COPD )
Chronic obstructive pulmonary disease (COPD) is a leading cause of disability
and death worldwide. Many research and studies have indicated that long term use of
smoking is highly related to serious respiratory complications such as chronic
obstructive pulmonary disease ( COPD ). Chronic obstructive pulmonary disease
(COPD) is estimated to affect 32 million persons in the United States and is the fourth
leading cause of death in this country (Vogelmeier et al., 2011).
Patients typically have symptoms of chronic bronchitis and emphysema, but the
classic triad also includes asthma which reversible compared to the first two diseases
mentioned earlier which cannot be reversed. Exacerbations of COPD indicate
instability or worsening of the patient’s clinical status and progression of the disease
and have been associated with the development of complications, an increased risk of
subsequent exacerbations, a worsening of coexisting conditions, reduced health status
xxiii
and physical activity, deterioration of lung function, and an increased risk of death.The
prevention of exacerbations therefore constitutes a major goal of treatment (Mosenifar,
2011).
Pathophysiology of the development of COPD occurs in the large (central)
airways, the small (peripheral) bronchioles, and the lung parenchyma. The normal
inflammatory response is amplified in persons prone to COPD development. The
pathogenic mechanisms are not clear but are most likely diverse. Increased numbers of
activated polymorphonuclear leukocytes and macrophages release elastases in a
manner that cannot be counteracted effectively by antiproteases, resulting in lung
destruction. Increased oxidative stress caused by free radicals in cigarette smoke, the
oxidants released by phagocytes, and polymorphonuclear leukocytes all may lead to
apoptosis or necrosis of exposed cells. Accelerated aging and autoimmune
mechanisms have also been proposed as having roles in the pathogenesis of COPD.
Cigarette smoke causes neutrophil influx, which is required for the secretion of MMPs;
this suggests, therefore, that neutrophils and macrophages are required for the
development of emphysema (Mosenifar, 2011).
Most patients with chronic obstructive pulmonary disease (COPD) seek medical
attention late in the course of their disease. Patients often ignore the symptoms
because they start gradually and progress over the course of years. Patients often
modify their lifestyle to minimize dyspnea and ignore cough and sputum production.
With retroactive questioning, a multiyear history can be elicited.Patients typically
present with a combination of signs and symptoms of chronic bronchitis, emphysema,
and reactive airway disease. These include cough, worsening dyspnea, progressive
exercise intolerance, sputum production, and alteration in mental status. Symptoms of
COPD are such as productive cough or acute chest illness, breathlessness, wheezing.
xxiv
Systemic manifestations (decreased fat-free mass, impaired systemic muscle function,
osteoporosis, anemia, depression, pulmonary hypertension, cor pulmonale, left-sided
heart failure. A productive cough or an acute chest illness is common. The cough
usually is worse in the mornings and produces a small amount of colorless sputum
(Mosenifar, 2011).
2.1.4.2.1 Emphysema
Emphyesema is defined as a chronic disease characterized by destruction of
the alveolar walls, with subsequent abnormal permanent enlargement of the respiratory
air spaces. Progressive breakdown of elastin in the lung parenchyma is a key feature in
the pathogenesis of emphysema. The major known cause of emphysema is cigarette
smoking, but the initiating elastinolytic factor in smoking-induced emphysema is still not
clear. The other known cause of emphysema is a genetic deficiency of a1-proteinase
inhibitor, an inhibitor of neutrophil elastase. In a1-proteinase inhibitor deficiency-
associated emphysema, lung elastin breakdown is undoubtedly triggered by the
unopposed action of neutrophil elastase due to the insufficient levels of its inhibitor in
the bronchoalveolar epithelial lining . In cigarette smoking-associated emphysema,
there is debate whether increased macrophage and/or neutrophil elastinolytic activity
within the alveolar matrix, resulting from a smoking-induced accumulation of
macrophages and neutrophils in the lung, may be responsible for the elastinolytic
damage (Foronjy et al., 2010).
The 3 described morphological types of emphysema are centriacinar, panacinar,
and paraseptal. Centriacinar emphysema begins in the respiratory bronchioles and
spreads peripherally. Also termed centrilobular emphysema, this form is associated with
long-standing cigarette smoking and predominantly involves the upper half of the lungs.
xxv
Panacinar emphysema destroys the entire alveolus uniformly and is predominant in the
lower half of the lungs. Panacinar emphysema generally is observed in patients with
homozygous alpha1-antitrypsin (AAT) deficiency. In people who smoke, focal panacinar
emphysema at the lung bases may accompany centriacinar emphysema. Paraseptal
emphysema, also known as distal acinar emphysema, preferentially involves the distal
airway structures, alveolar ducts, and alveolar sacs. The process is localized around
the septae of the lungs or pleura. Although airflow frequently is preserved, the apical
bullae may lead to spontaneous pneumothorax. Giant bullae occasionally cause severe
compression of adjacent lung tissue (Demirjian, 2011).
Figure 2.2 A thin section of lung tissue stained with hematoxylin and eosin The
individual suffers from emphysema (Demirjian, 2011).
2.1.4.2.2 Chronic Bronchitis
Chronic bronchitis is defined clinically as cough with sputum expectoration for at
least 3 months a year during a period of 2 consecutive years. Chronic bronchitis is
associated with hypertrophy of the mucus-producing glands found in the mucosa of
large cartilaginous airways. As the disease advances, progressive airflow limitation
xxvi
occurs, usually in association with pathologic changes of emphysema. This condition is
called chronic obstructive pulmonary disease. Chronic bronchitis is a clinical syndrome
defined by chronic sputum production,and it is associated with periodic exacerbations in
which the patient experiences a worsening of respiratory symptoms (Saetta et al.,
1994).
Chronic bronchitis is associated with excessive tracheobronchial mucus
production sufficient to cause cough with expectoration for 3 or more months a year for
at least 2 consecutive years. The alveolar epithelium is both the target and the initiator
of inflammation in chronic bronchitis. A predominance of neutrophils and the
peribronchial distribution of fibrotic changes result from the action of interleukin 8,
colony-stimulating factors, and other chemotactic and proinflammatory cytokines.
Airway epithelial cells release these inflammatory mediators in response to toxic,
infectious, and inflammatory stimuli, in addition to decreased release of regulatory
products such as angiotensin-converting enzyme or neutral endopeptidase. Chronic
bronchitis can be categorized as simple chronic bronchitis, chronic mucopurulent
bronchitis, or chronic bronchitis with obstruction. Mucoid sputum production
characterizes simple chronic bronchitis. Persistent or recurrent purulent sputum
production in the absence of localized suppurative disease, such as bronchiectasis,
characterizes chronic mucopurulent bronchitis (Fayyaz, 2011).
xxvii
Figure 2.3 Histopathology sample taken from a chronic bronchitis patient
showing goblet cell hyperplasia (Demirjian, 2011).
2.1.5 Management of smokers
Smokers move through stages in relation to quitting: of precontemplation,
contemplation, readiness then action, followed by maintenance or relapse. Many move
through this cycle several times before they finally quit, while others report they found it
easier to quit than they expected. These stages are influenced by increased costs from
tax increases or reduction of smuggling, illness in the smoker, family or friends dying
from tobacco, the media, health profession, bans on promotion, creation of smokefree
areas and, while most smokers still quit on their own, availability of support and
treatment. There are now techniques to assist those who want to quit smoking,
although these are not available in all parts of the world: social support, clinics,
quitlines, internet sites; skills training; nicotine replacement therapy (NRT) and other
pharmaceutical treatments (Crapo et al., 2004).
2.1.5.1 Drug Therapy (Pharmacotherapy)
Drug therapy can be further categorized into pharmacotherapy, which comprises
four nicotinic group such as, nicotinic gum, inhaler, nasal spray and patch, whereas,
non-nicotinic agent is Sustained- Release Bupropion( bupropion SR) (Crapo et al.,
2004).
Nicotinic gum, it is given in a flexible dosage according to specific cravings of
the smokers. An individual who smokes 1 pack per day should use 4-mg pieces. The 2-
mg pieces are to be used by individuals who smoke less than 1 pack per day. Instruct
the patient to chew hourly and also to chew when needed for their initial cravings for 2
xxviii
weeks. Gradually reduce the amount chewed over the next 3 months. Proper
information should be given about the usage of the gum such as avoid drinking liquids
while chewing the gum and using gum within 15 minutes of drinking acidic
beverages(e.g., cola, coffee, citrus juice) may reduce the effect by decreasing the
absorption of the nicotine (Crapo et al., 2004).
The nicotine inhaler, also nicknamed "the puffer" is a thin, plastic cartridge that
contains a porous nicotine plug in its base. By puffing on the cartridge, nicotine vapor is
extracted and absorbed through the lining of the mouth. Each cartridge delivers up to
400 puffs of nicotine vapor. It takes at least 80 puffs to obtain the equivalent amount of
nicotine delivered by one cigarette. The inhaler mimics the habitual hand-to-mouth
action but without combustion, the smoker is not exposed to carbon monoxide, tar or
other carcinogens that can be found in tobacco smoke. Few reported side effects from
the inhaler are,coughing,rhinitis,and local irritation of the mouth and throat (Crapo et al.,
2004).
The patch is a nicotine delivery system that was developed in part because of
the difficulty in the patients to optimize the usage of nicotine gum. It is applied on the
non hairy surface of the skin and absorbed readily through the skin and distributed
throughout the body, reducing withdrawal symptoms and the craving for tobacco.
Transdermal nicotine patches are available readily for replacement therapy. Long-term
success rates are 22-42%, compared with 2-25% with a placebo. These agents are well
tolerated, and the adverse effects are limited to localized skin reaction. Nicotine
replacement therapy patches are sold under the following trade names such as
NicoDerm, Nicotrol, and Habitrol. Each of these products is dosed with a scheduled
graduated decrease in nicotine over 6-10 weeks (Crapo et al., 2004).
xxix
The nicotine nasal spray is the strongest form of nicotine replacement therapy,
which is particularly useful and effective for highly dependent heavy smokers who
cannot give up by any other means. The reason that the nasal spray is so much more
effective is because of its fast action. Once the nicotine has been administered, it enters
the bloodstream and reaches the brain within 10 minutes. Other methods take much
longer. This method also most realistically mimics the fast "hit" obtained when smoking
a cigarette. This makes it much easier to control and satisfy cravings if they suddenly
arise.However it has highest level of side effect compared to other nicotine therapies
which about 94% of the users reporting nasal irritation of moderate to severe intensity
during initial use (Crapo et al., 2004).
Bupropion SR is the only nonnicotine pharmacotherapy to be approved by Food
and Drug Administration (FDA) to date. This atypical antidepressant will block the
reuptake of dopamine or norepinephrine in the brain.This therapy is initiated 1 to 2
weeks before the the quiting date and started with a dosage of 150mg in the morning
for 3 days and then increased into 150mg twice a day for up to 3 months following the
quit date.Bupropion is contraindicated to patients with seizure or eating disorder,who
took monoamine oxidase inhibitor within the previous 14days.Commonly reported side
effect are insomnia and dry mouth.It is also sometimes be beneficial in treating patients
with depression history (Crapo et al., 2004).
2.1.5.2 Non Drug Therapy
There are a wide variety of non drug therapy can be given such as affect
management, diet programs, hypnosis, acupuncture, social support, and sensory
deprivation. Self-help materials such as pamphlets, manual , and audiotapes and
videotapes were found to be a marginal effective method. Individual counseling from a
xxx
smoking cessation specialist may help smokers to make a successful attempt to stop
smoking by doing intra treatment such as communicating encouragement and concern
to persons quitting while allowing them to speak openly about their experience and
extra treatment social support, where, the clinician identify friends or family members
who may be sources of support (Crapo et al., 2004).
2.1.5.3 Combination therapy
The best therapy to those who motivated to make a serious quit attempt would
be a combination of drug therapy and basic counseling which may give favorable result
by increasing long term cessation to three to four times from what they would without a
treatment (Crapo et al., 2004).
2.2 Neutrophils
2.2.1 Introduction
Neutrophil is a large numbers of polymorphonuclear neutrophil (PMN)
granulocytes are rapidly recruited from the bloodstream to the site of infection or injury
via transmigration through the vascular endothelium. Neutrophils constitute the ’’first
line of defense’’ and are considered as primary effector cells in infectioninduced acute
inflammatory reactions where they serve to destroy invading pathogens. Neutrophils
are inherently short-lived cells with a half life of only z6-10 h in the circulation and
rapidly undergo spontaneous apoptosis . In infected tissues their apoptosis can be
delayed both by microbial constituents and by proin- flammatory stimuli . Finally,
however, tissue neutrophils die in large numbers. Because uncontrolled release of toxic
substances from dead neutrophils can propagate the inflammatory response leading to
tissue destruction, recognition of dying inflammatory neutrophils has a critical function
xxxi
for the resolution of the in- flammatory response. It leads not only to the removal of the
inflammatory cells themselves, along with anything they have ingested, but also to the
generation of anti-inflammatory mediators that shut down the on going inflammation
(Esmann et al., 2010).
Though neutrophils are short lived, with a half-life of four to ten hours when not
activated and immediate death upon ingesting a pathogen, they are plentiful and
responsible for the bulk of an immune response. They are the main component of pus
and responsible for its whitish color. Neutrophils are present in the bloodstream until
signaled to a site of infection by chemical cues in the body. They are fast acting,
arriving at the site of infection within an hour (Esmann et al., 2010).
Before ingesting invasive bacteria, neutrophils can release a net of fibers called
a neutrophil extracellular trap (NET), which serves to trap and kill microbes outside of
the cell. When neutrophils ingest microbes, they release a number of proteins in
primary, secondary, and tertiary granules that help kill the bacteria. They also release
superoxide, which becomes converted into hypochlorous acid, or chlorine bleach, which
is theorized to play a part in killing microbes as well (Esmann et al., 2010).
2.2.2 Classification
Neutrophil granulocytes are subdivided into segmented neutrophil(segs) and
banded neutrophil (band). The segmented neutrophil term is derived from the
multilobed nature of the cell’s nucleus. Generally, neutrophil nuclei exhibit 3-5
segmented lobes (Witko et al., 2000).
xxxii
A second characteristic of functional importance is the large number of
cytoplasmic granules. Three types of granules are present in the cytoplasm of
neutrophils :
- Small, specific granules (0.1 µm in diameter)
- Larger azurophilic granules (0.5 µm in diameter)
- The newly discovered tertiary granules
Specific granules contain various enzyme and pharmacological enzymes and
pharmacological
agents tahat aid the neutrophil in performing its antimicrobial functions. Azurophilic
granules are lysosomes, containing acid hydrolases, myeloperoxidase, the antibacterial
agent lysozyme, bactericidal permeability increasing protein, cathepsin G, elastase, and
nonspecific collagenase. Tertiary granules contain gelatinase and cathepsins as well as
glycoproteins that are inserted into plasmalemma (Esmann et al., 2010).
Besides that, like segmented neutrophil, the term banded neutrophil is derived
from the cell’s characteristic nuclear staining. The nuclear material is in simple U-
shaped pattern. The banded is an immature cell on the way to becoming a mature
segmented neutrophil. When there is demand for neutrophils because of an infection or
chronic cell damage, the bone marrow is stimulated to release its supply of
mature(segmented) and some immature(banded) cells. Generally, in the presence of a
long term bacterial infection or chronic tissue necrosis. The level of “bands” reported in
CBC increases (Anderson, 1999).
2.2.3 Mechanism of production and regulation
xxxiii
Neutrophils are produced in the marrow, where they arise from progenitor and
precursor cells by a process of cellular proliferation and maturation. They differentiate
from the pluripotential stem cell through a series of progressively more committed
progenitor or colony forming units, including the granulocyte-monocyte colony forming
unit and the granulocyte colony forming unit, which give rise to neutrophils. The early
progenitor cells cannot be recognized under the microscope but can be identified by
marrow culture. The earliest microscopically recognizable neutrophil precursor is the
myeloblast. From there, the formal sequence of precursor development is myeloblast
promyelocyte myelocyte metamyelocyte band neutrophil segmented neutrophil. The
term granulocyte often is loosely used to refer to neutrophils but strictly speaking
includes eosinophils and basophils. Eosinophilic and basophilic granulocytes develop
from progenitors in a manner analogous to the neutrophils, although commitment to
neutrophilic, eosinophilic, or basophilic development probably is established at an early
progenitor stage (Williams, 2007).
The normal human neutrophil production rate is 0.85 to 1.6 x 109 cells/kg/day.
Mature neutrophils are stored in the marrow before they are released into the blood.
They leave the circulation randomly, with a half-disappearance time of approximately 7
hours. The cells then enter the tissues and probably function for 1 or 2 days before their
death or loss into the gastrointestinal tract through mucosal surfaces (Williams, 2007).
The humoral regulators involved in granulopoiesis have been defined by in vitro
culture systems. Originally identified by their ability to stimulate colony formation from
marrow progenitor cells, the hemopoietins (cytokines) came to be called colony
stimulating factors (CSF). With regard to neutrophil production, at least four human
CSFs have been defined. Granulocyte-monocyte colony stimulating factor (GM-CSF) is
a 22,000 relative molecular mass (Mr) glycoprotein that stimulates the production of
xxxiv
neutrophils, monocytes, and eosinophils. Granulocyte colony stimulating factor (G-CSF)
has an Mr of 20,000 and stimulates only the production of neutrophils. Interleukin-3 (IL-
3), or multi-CSF, also has an Mr of 20,000 and acts relatively early in hematopoiesis,
affecting pluripotential stem cells. Finally, stem cell factor (also known as c-kit ligand or
steel factor), with an Mr of 28,000, acts in combination with IL-3 and/or GM-CSF to
stimulate the proliferation of the early hematopoietic progenitor cells. In addition to their
effects on neutrophil precursors, G-CSF and GM-CSF act directly on the neutrophil,
enhancing its function. These cytokines regulate the production, survival, and functional
activity of neutrophils. The mature neutrophil lacks IL-3 receptors and thus is not
affected by IL-3. However, IL-3 receptors are present on mature eosinophils and
monocytes. IL-3 is produced by activated T lymphocytes and thus is expected to have a
physiologic role in circumstances of cell-mediated immunity. GM-CSF also is produced
by activated lymphocytes. However, like G-CSF, it also is elaborated by mononuclear
phagocytes and endothelial and mesenchymal cells when these cell types are
stimulated by certain cytokines, including IL-1 and tumor necrosis factor, or bacterial
products, such as endotoxin. Stem cell factor is secreted by a variety of cells, including
marrow stromal cells, and affects the development of several kinds of tissues (Williams,
2007).
The activities of exogenously administered biosynthetic (recombinant) human G-
CSF and GM-CSF in humans are well documented. G-CSF administration rapidly
induces neutrophilia, whereas GM-CSF causes an increase in neutrophils, eosinophils,
and monocytes. GM-CSF cannot be detected easily in normal plasma; thus, its role as
a day-to-day, long-range modulator of neutrophil production is uncertain. Mice in which
the GM-CSF gene is "knocked out" have generally normal hematopoiesis but show
macrophage abnormalities, pulmonary alveolar proteinosis, and decreased resistance
xxxv
to microbial challenge. However, G-CSF appears to be a critical regulator of neutrophil
development, as giving an animal an antibody to G-CSF leads to profound
neutropenia.The G-CSF knockout mouse shows severe neutropenia.Neutropenia that
results from a production disturbance, such as exposure to cytotoxic drugs, is
associated with high circulating serum concentrations of G-CSF (Williams, 2007).
2.2.4 Biological Function of Neutrophils
Neutrophils phagocytose and destroy the bacteria by the help of the their
various granules. Neutrophil interact with chemotactic agents to migrate to site of
infection. They accomplish this by entering postcapillary venules in the region of
inflammation and adhering to the various selectin molecules of endothelial cells of this
vessels by use of their selectin receptors. The interaction between the neutrophil’s
selectin receptor and the selectin of the endothelial cells cause neutrophils to roll slowly
along the vessels endothelial lining. As the neutrophil are slowing their migrations,
Interleukin-1 (IL-1) and tumor necrosis factor (TNF) induce the endothelial cells to
express intercellular adhension molecule type 1 (ICAM-1), to which the integrin
molecules of neutrophils avidly bind (Kerrigan et al., 2009).
When binding occurs, the neutrophils stop migrating in prepartion fot their
passage through the endothelium of postcapillary venule to enter connective tissue
compartment. Once there, they destroy the infected cells by phagocytosis and by
release of hydrolytis enzymes (and respiratory burst). In addition, by manufacturing
and releasing leukotrienes, neutrophil assist in the initiation of the inflemmatory process
(Doan et al., 2008).
The sequence of event as follows:
xxxvi
1. The binding of neutrophil chemotactic agents to the neutrophil’s plasmalemma
facilitates the release of the content of tertiary granules into extracellular
matrix
2. Gelatinase degrades the basal lamina, facilitating neutrophil migration.
Glycoproteins that become inserted in the cell membrane aid the process of
phagocytosis.
3. The content of the specific granules are also released into the extracellular
matrix, where they attack the invading matrix and aid neutrophil migration.
4. Microorganisms, phagocytosed by neutrophils, become enclosed in
phagosomes. Enzymes and pharmacological agents of the azurophilic
granules are released into the lumina of these intracellular vacuoles, where
they destroy the ingested microorganisms. Because of their phagocytic
functions, neutrophils are also known as microphages to distinguish them from
the larger phagocytic cells, the macrophages.
5. Bacteria are killed not only by the actions of enzymes but also by formation of
reactive oxygen compounds within the phagosomes of neutrophils. These are
superoxide, formed by the action of NADPH oxidase on oxygen in a
respiratory burst; hydrogen peroxide, formed by action of superoxide; and
hydrochlorus acid(HOCL), formed by the interaction of myeloperoxidase(MPO)
and chloride ions with hydrogen peroxide.
6. Occasionally, the contents of the azurophillic granules are released into
extracellular matrix, causing tissue damage, but usually catalase and
gluthione peroxidase degrade hydrogen peroxide.
xxxvii
7. Once neutrophils perform their function of killing microorganisms, they also
die, resulting in formation of pus, the accumulation of dead leukocytes,
bacteria, and extracellular fluid.
8. Not only do neutrophils destroy bacteria, they also synthesize leukotrienes
from arachidonic acids in thier cell membranes. These newly formed
leukotrienes aid the initiation of the inflammatory process (Doan et al., 2008).
Phagocytosis of neutrophils involve cell surface receptors associated with
specialized region called clathrin coated pits. The mechanism of phagocytosis
involves :
a) Recognition and attachment of microbes by phagocytes.
Phagocytosis is initiated when a phagocyte binds a cell or molecule that has
penetrated the body’s barrier. The binding occurs at various receptors on
phagocyte surface. These include PRRs(including TLRs) that recognize microbe
related molecule, complement receptors(CR) that recognize certain fragments of
complements (especially C3b) that adhere to microbial surfaces, Fc receptors
that recognize immunoglobins that have bound to microbial surfaces or other
particles, scavenger receptors, and others.
b) Ingestion of microbes and other materials :
Following attachment to the cell membrane, a microorganism or foreign particle
is engulfed by extensions of the cytoplasm and cell membrane called
pseudopodia and is drawn into the cell internalization or endocytosis. In addition
to phagocytosis, dendritic cells can extend plasma membrane projections and
encircle large amounts of extracellular fluids to form cytoplasmic vesicles
independent to cell surface attachment. Once internalized, the bacteria are
trapped within phagocytic vacuoles or cytoplasmic vesicles within the cytoplasm.
xxxviii
The attachment and ingestion of microbes trigger changes within the phagocyte.
It increases in size, becomes more aggressive in seeking additional microbes to
bind and ingest, and elevates production of certain molecules. Some of these
molecules contribute to destruction of the ingested microbes; others act as
chemotactic agents and activators for other leukocytes.
c) Destruction of ingested microbes and other materials:
Phagosomes, the membrane bound organells containing the ingested
microbes/materials, fuse with lysosomes to form phagolysosomes. Lysosomes
employ multiple mechanisms for killing and degrading ingested matter. These
include
Lysosomal acid hydrolase, including protease and nucleases.
Several oxygen radicals, including superoxide radicals,
hypochlorite, hydrogen peroxide, and hydroxyl radicals, that are
highly toxic to microbes. The combined action of these
molecules involves a period of heightened oxygen uptake known
as the oxidative burst.
- Nitrous oxide (NO)
- Decrease pH
- Other microbial molecules
d) Secretion of cytokines and chemokines :
Once activated, phagocytes secrete cytokines and chemokines that attract and
activate other cells involved in innate immune responses. Cytokines or chemical
messengers such as interleukin-1(IL-1) and interleukin-6(IL-6) induce the
production of proteins that lead to elevation of body temperature. Other
cytokines, such as tumour necrosis factor-α(TNF- α), increase the permiability of
xxxix
local vascular epithelial to increase its permiability and enhance the movement
of cells and soluble molecules from the vasculature into tissues. Still others,
such as interleukin-8(IL-8) and interleukin-12(IL-12) attract and activate
leukocytes such as neutrophils and NK cells (Doan et al., 2008).
Figure 2.4 Bacterial phagocytosis and destruction by a neutrophil (Doan et al.,
2008).
2.2.5 The Role Of Neutrophil in Pulmonary Emphysema
When oxidant from cigarrete smoke is exposed to the lungs, macrophage will be
activated causing, histone deacetylase-2 to be inactivated, shifting the balance toward
acetylated or loose chromatin, exposing nuclear factor B sites and resulting in
transcription of matrix metalloproteinase-9, proinflammatory cytokines interleukin 8 (IL-
8), and tumor necrosis factor(TNF). This will lead to neutrophil recruitment (Longo et al.,
2011).
CD8+ T-cells are also recruited in response to cigarette smoke and release
interferon inducible protein-10 (IP-10, CXCL-7) that in turn leads to macrophage
xl
production of macrophage elastase [matrix metalloproteinase-12 (MMP-12)]. Matrix
metalloproteinases and serine proteinases, most notably neutrophil elastase, work
together by degrading the inhibitor of the other, leading to lung destruction. Proteolytic
cleavage products of elastin also serve as a macrophage chemokine, fueling this
destructive positive feedback loop that lead to emphysema (Longo et al., 2011).
Collagen turnover in emphysema is complex. The three collagenases (MMP-1,
MMP-8, and MMP-13) that initiate the cleavage of interstitial collagens are also induced
in both inflammatory cells and structural cells in emphysema. While collagen is
disrupted as alveolar units are obliterated, overall there is a net increase in collagen
content in the emphysema , with prominent accumulation in the airway submucos
(Longo et al., 2011).
xli
Figure 2.5 Pathogenesis of emphysema and association of neutrophil (Longo et
al, 2011).
xlii
CHAPTER 3
CONCEPTUAL FRAMEWORK AND HYPOTHESIS
Figure 3.1 Conceptual Framework of Pathogenesis of emphysema and association
of macrophage
xliii
SMOKING
DEACYTELASE-2 INACTIVETED
TRANSCRIPTION OF MM9
INTERLEUKIN-8
TUMOR NECROSIS FACTOR-α
ALVEOLAR MACROPHAGE
OXIDANTS
PROTEASE
PARENCYMAL LUNG DAMAGE
COPD
EMPHYSEMA
1-AT deficiency
Related To Macrophage Profile
Indirectly related to Study
CD 8+ Cells
NEUTROPHIL
3.1 Conceptual Framework of the Study
Long term smoking is directly related to emphysema. Under normal
condition,alveolar macrophage will patrol the lower airspace but when chronic exposure
to smoking occurs, it will activate the alveolar macrophages and
subcequenly,macrophages will accumulate in the alveolar area.This is due to the
oxidants produced by cigarettes. Activated macrophages will release several chemical
substances such as tumor necrosis factor-alpha1 (TNF-1), interleukin-8 ( IL-8), and
protease.This will lead to the neutrophil recruitment. Accumulated neutrophils will play
its role by further activating macrophages to produce excessive protease.This will
cause protease and anti protease imbalance. that leads to destruction of elastin and
other structural elements which cause recurrent inflammation.This mechanism will lead
to irreversible enlargement of the air spaces distal to the terminal bronchioles. This
parenchymal lung damage will cause the breakdown of elasticity and loss of fibrous
and muscle tissue, making the lungs less compliant or in other word,emphysema is
occured in the person.
3.2 Hypothesis of the Study
Based on the summary of problem and study purpose, the hypothesis is,
1) Smoking will increase the number of neutrophil in alveolar in the smokers.
2) The number of neutrophil in alveolar will be increased by the three degree of
smokers, that are mild, moderate and severe smokers.
3) There is a difference in neutrophil count in the alveolar between
asymptomatic smoker and smoker with pulmonary emphysema.
CHAPTER 4
xliv
STUDY METHOD
4.1 Study design
This study was a laboratory observational study with a study design of cross
sectional of the group’s posttest only on smokers without any other systemic
complication (mild, moderate, severe) and smokers with pulmonary emphysema
without any other systemic complication. The purpose of the study is to observe
the effect of smoking on the level of neutrophil among different groups of
smokers.
4.2 Population and Sample of Study
4.2.1 Population
The study populations were smokers from 4 groups which composed of 3 types of
smokers and have the following requirements:
Mild smokers ( Brinkman Index : 1 to199 )
Moderate smokers ( Brinkman Index : 200 to 399 )
Severe smokers ( Brinkman Index : 400 to 599 )
Smokers with pulmonary emphysema
4.2.2 Sample size
The sample used was sputum collected from the four groups. The groups were
asymptomatic mild, moderate and severe smokers according to the Brinkman
Index and the fourth group is smokers with pulmonary emphysema. The criteria
for the fourth group were chest x-ray with a posteroanterior (PA) position.
xlv
4.2.3 Sample size estimation
Explanation
= sample size
= standard deviation
= absolute accuracy of the different levels mean value (0.5)
= z value at alpha 5% is 1.96
Accordingly,
(Hamilton, 2009).
Rounded to the nearest value it will be 10.
The total samples were taken from ten persons in each group and we had 4
groups as a sample. So, the total sample for the study would be forty.
4.2.4 Sample statistic
The statistic that was used for this observational study is Anova, because it uses
logical extension of T test. Since we have data from four independent groups.
Anova would be the best method to calculate the statistic. The observations was
independent which means the value of one group is not correlated with the other
xlvi
group, therefore, the observation in each group was normally distributed and the
variance of each group was equal to the that of any other group (homogeneity of
variances).
4.3 Location and time of study
4.3.1 Place of study
The study was carried out from the smokers around the RSSA and also smokers
with emphysematous lung disease who came to RSSA as outpatient.
The research was done in Respiratory Department and Central Laboratory of
RSSA.
4.3.2 Time of study
Research was done at the estimated time of thirty days.
4.4 Variable
4.4.1 Dependent variable
Dependent variable in this study was the level of neutrophil in the sputum that
was collected from four different types of comparison groups.
4.4.2 Independent variable
The independent variables of this study consist of four groups that were the
following:
Group 1 : Mild smoker without any other systemic complication.
Group 2 : Moderate smoker without any other systemic complication.
Group 3 : Severe smoker without any other systemic complication.
xlvii
Group 4 : Smoker with pulmonary emphysema without any other systemic
complication.
4.5 Inclusion and exclusion criteria of the study
Inclusion Criteria :
- Male
- Smoker
- 45- 75 years old
-Smoker with pulmonary emphysema is chosen based on a radiologic findings
- ECG in a normal range
Exclusion Criteria :
- COPD patient with acute exacerbation
- Female
- With a sign of cor pulmonale
- Heart attack, tuberculosis, Asthme bronchitis, Hypertension
4.6 Operational Definition
i. Neutrophil is a a polymorphonuclear granular leukocyte having a nucleus
with three to five lobes connected by threads of chromatin, and cytoplasm
containing very fine granules which involves in phagocytic process.
ii. Smoker is defined as the person who knowingly smoke the cigarette by
inhaling and exhaling the cigarette smoke with minimal cigarette smoking
one per day.
xlviii
iii. Mild smoker is categorized according to the Brinkman Index by multiplying
number of cigarettes smoked per day with years of smoking and the
score should be 0 – 199.
iv. Moderate smoker is categorized according to the Brinkman Index by
multiplying number of cigarettes smoked per day with years of smoking
and the score should be 200 – 599.
v. Severe smoker is categorized according to the Brinkman Index by
multiplying number of cigarettes smoked per day with years of smoking
and the score should be more than 599.
vi. Brinkman Index is multiplying number of cigarettes smoked per day with
years of smoking.
vii. Non symptomatic smoker is a person that inhales and exhales cigarette
smoke purposely without any clinical findings in the respiratory system.
viii. Non smoker is a person who does not smoke and neither family member
nor working colleague smokes and exposed to cigarette smoke less than
eight hours per week.
ix. Positive control smoker is a person that inhales and exhales cigarette
smoke purposely and has x-ray findings of pulmonary emphysema.
x. Passive smoker is a person that exposed to cigarettes smoke emitted
from cigarettes smoke by other person for more than four hours per day.
xi. Pulmonary emphysema is described radiologically as an abnormal
permanent enlargement of air spaces distal to the terminal bronchioles,
that the x-ray image shows an appearance of hyperlucent and
hyperinflation at both the lungs.
xii.
xlix
4.7 Instruments
4.7.1 Studies Tools and Substances
40 pieces of sterile disposable plastic pots for sputum collection
Sticker labels
1 unit spirometry equipment (nebulizer ultrasonic)
Salbutamol
2 fl 3% Nacl
NaOH 4%
Water bottles
Laboratory request form
Gloves
Mask
10 ml syringe
15ml centrifuge tube
Tube rack
Pasteur pipette
Biosafety Cabinet class II
Vortex mixer
Bacti-cinerator
Bio contained centrifuge 3000g
Sysmex XT-2000i Hemoanalyzer
Computer with monitor
Printer
Tissue paper
4.8 Study Work Plan
l
4.8.1 Sputum Induction
i. Detailed information and instruction to the subject were given and
consent to the procedure was taken.
ii. The safety of the equipment was checked and the ultrasonic nebulizer
was prepared (output approximately 1ml/ min)
iii. Subjects were asked to rinse their mouth using hypertonic saline solution
(NaCl 3%) and asked to breathe through the nose in order to avoid
contamination of saliva. Saliva removed prior to begin the sputum
induction procedure.
iv. Subject was asked to cough and throw up the sputum, firstly in the fifth
minute and the secondly at tenth minute.
v. Sputum collected in container with subject’s name label on it.
4.8.2 Sputum Decontamination and Centrifugation
i. Collected sputum was mixed with sodium hydroxide (NAOH) 4% in a ratio of
(1: 2) and fastened firmly.
ii. The sample was mixed gently with vortex mixer and placed in temperature
of 15 °C room temperature to stimulate homogenization.
iii. Centrifugation was done at 3000 x g for 15 minutes.
iv. The aerosol was left to become sediment for 10 minutes.
v. Supernatant was removed.
vi. Distilled water was added up to the 15ml.
vii. The solution was centrifuged again at 3000 x for 15 minutes
viii. The aerosol was left to become sediment for 10 minutes.
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ix. Supernatant was removed and the homogenized sample kept to do
neutrophil counting through sysmex-XT hemoanalyzer.
4.8.3 Neutrophil Counting
i. Sysmex XT-2000i hemoanalyzer was turned on.
ii. Computer CPU and printer were turned on too.
iii. QC *QC analysis click * select a QC file to be executed, press OK * Enter
the e - check that has been in homogenization into the sample probe. *
Press Start. * Make sure the QC results in target and click Accept. * To see
the QC chart, graph and select the desired type.
iv. The sample was ran according to the instruction below:
Perform first order on the work list
Enter Number and type of test sample
Enter the patient ID and patient data (if any)
Click Save
Click Manual
Type of sample Number (adjust to the work list)
Press OK
Enter been homogenizing samples into the sample probe
Press START
v. Results can be viewed by clicking on the Explorer or the Data Browser
vi. Switch off the appliance:
Click shut down on-screen menu
Enter Cell clean the sample probe into
Press the START 1 (one) time
lii
Wait until the process is complete, then turn off the equipment and
programs
Click START on the Windows program
Click shut down
Turn off the monitor and printer
liii
Figure 4.1 Study Framework
4.8 Study Framework
CHAPTER 5
liv
Mild Smokers
(10 Subjects)
Moderate Smokers
(10 Subjects)
Severe Smokers
(10 Subjects)
Smokers with pulmonary
emphysema based on radiologic findings
(10 Subjects)
Sputum samples were collected from each group using sputum pot
Collected sputum Mixed with NAOH 4% in a ratio of (1 :2) for decontamination
Centrifugation is done at 3000 x g for 15 minutes
Supernatant is removed, add distilled water up to the15ml
Centrifugation is done at 3000 x g for 15 minutes
Sediment
Sysmex XT-2000i instrument
NeutrophilCounting
Result been recorded according
to the group
(A) Smokers with Pulmonary Emphysema
(B)Mild Smokers
(C) Moderate Smokers
(D) Severe Smokers
STUDY RESULTS
5.1. Subject and Study Location
There were 42 subjects been given informed consent about the purpose,
procedures and the approval letters to be signed. Two subjects were excluded, one due
to pulmonary tuberculosis lesion from radiological study and the other due to lysis of
blood plasma obtained. So, we used 40 subjects to study and they have been divided
into 4 groups. Where 10 subjects of light smoker, 10 subjects of moderate smokers, 10
subjects of severe smokers and lastly 10 severe smokers with pulmonary emphysema.
5.2. Characteristic of the subjects
The numbers of subjects of this study are 40 people and all are male. The mean
age of the subjects of the study was 60.78 + 7.82 years with the youngest age range 46
years old and the oldest is 77 years of age as shown in the table.
lv
Table 5.1 Characteristics of study subjects in each group
Characteristic Mild Smokers (n = 10)
Moderate Smokers (n = 10)
Heavy Smokers (n = 10)
Smokers with Pulmonary Emphysema (n = 10)
Age (Years) 53,60+6,68
58,30+5,90
63,80+ 6,61
67,40+ 4,29
Occupation1. Retired PNS2. Retired TNI/Polri3. Private Lecturer4. Farmers5. Enterpreneur6. Salesman7. BUMN (PDAM/PLN)8. Security Guard
111-5-2-
32--3-11
7---2--1
61-111--
Jenis Rokok1. Clove Cigarettes2. Filter and Filter Clove Cigarettes3. Hand-rolled Cigarettes4. Clove Cigarettes and Filters
26-2
2--8
26-2
7111
Brinkman Index (Cigarettes/year) 145,8+63,3
366,8 +87,9
819,8 + 224,97
845,40+ 213,18
Body Mass Index1. Underweight (< 18,5 kg/m2)2. Normal (18,5-24,9 kg/m2)3. Overweight (> 25-29,9 kg/m2)
BMI (kg/m2)
-73
23,50+ 3,86
136
24,70+ 3,38
172
22,70+ 3,84
451
20,20+ 4,07
Data on the total and the mean (SD)
From the characteristic of the subjects of the study, the average age of the mild
smoker is between 46 to 63 and moderate ranges from 48 to 67, followed by severe at
57 to 77 and pulmonary emphysema group which has a age range of 61 to 73 years
old.
Most of the candidates taken for the research are retired PNS. Most usual type
of cigarette smoked by study subjects are a mixture of clove cigarettes and
filters (alternate) as much as 80% (as shown in the figure) the mean of the
lvi
Brinkman index, in the smokers with pulmonary emphysema group is at a number of
845.40+ 213.18 cigarettes / year, in the group of the mild smokers is
about 145.8+ 63.3 cigarettes /year, in the group of moderate smokers is about
366.8 + 87.9 cigarettes/year, in the group of severe smokers is
819.8 224.97 + cigarettes/year. The mean Brinkman index in all study subjects
are 544.45 + 344.90 cigarettes/year.
From the subjects of the study Brinkman index from the mild smokers
range from 10-192 cigarettes / year (p <0.05), moderate smokers in the group with
a range of 230-468 cigarettes / year (p <0.05), a group of severe smokers with a range
of 630-1152cigarettes / year (p <0.05) and in the fourth group with a range of 640-
1120 cigarettes /year (p <0.05) as shown below.
Throughout the period of the samples were taken, subjects did not show any
adverse effect based on the anamnesis, physical examination and additional
examination that was done here. Similarly, most of the vital signs such as blood
pressure, pulse rate and respiratory rate was normal during the time samples were
taken. The mean body mass index or Body Mass Index (BMI) of study subjects from
each group is a norm weight and its distribution can be seen in the picture.
5.3. Characteristics Of Supportive Examination Data of Subject
Routine blood test, SGOT, and SGPT that was done among all the subjects
from different types of group, showed normal value. The results of
electrocardiographic examination showed normal value for 12 respondents and only
one respondent showed right axis deviation. PA position of radiographic
examinations obtained from 13 study subjects had normal radiological picture, and
17 study, subjects had an increased bronchovascular pattern, while radiographical
lvii
picture in the fourth group gained from 10 subjects with radiographical studies with
pulmonary emphysema.
Tabel 5.2 Characteristics Of Supportive Examination Data of Subject
Characteristics Mild Smoker(n = 10)
Moderate Smoker(n = 10)
SevereSmoker(n = 10)
Smokers with Pulmonary Emphysema(n = 10)
Routine Blood Test1. Haemoglobin (gr/dl)2. Leukocytes(gr/dl)
14,03+ 0,916656+ 2368,6
14,26+ 1,597882+ 2915,1
13,94+ 1,637294+ 2602,4
13,8+ 1,288453+ 3475,5
ECG1. Normal2. RAD
10-
10-
91
10-
Chest X-Ray (PA)1. Normal2. Increase
Bronchovascular pattern
3. Emphysema
73
-
46
-
28
-
--
10
Neutrophil in Sputum (/mm3)
617.8 + 600.7
1369.4 +1362.2
953.9+ 749.9
789.44+ 571.5
Data on the total and the mean (+ SD) (source: study)
5.4 Neutrophil counting in the sputum induction of the subjects
The average counting of neutrophils from the sputum induction of the subjects
has the highest value in moderate group at a number of 1369.4 + 1362.2/mm3 (p<0,05),
and severe smokers about 953.9 + 749.9/mm3 (p>0,05) and then pulmonary
emphysema group at about 789.44 + 571.5/mm3 (p>0,05) and finally mild smokers at a
number 617.8 + 600.7/mm3 (p<0,05). Only severe smokers pulmonary emphysema
group and another group which is the severe smokers gave the significant result and
lviii
normally distributed compared to the other group which are the mild and moderate
smokers.
When comparing the non pulmonary emphysema group and pulmonary emphysema
group, it can be shown that the numbers are 981.0 + 376.3 /mm3 and 789.4 + 571.6
/mm3 that are shown in the picture.
5.5 Data Analysis
5.5.1 Normality Test
Normality test was done to identify whether neutrophils are distributed normally
in the different stages of smoking such as mild,moderate and severe and also the
pulmonary emphysema group.From the Shapiro –Wilik with Liliefors correlation test,
only pulmonary emphysema group and asymptomatic severe smokers group are
normally distributed with p > 0.05. Because the other stages do not give normal
distribution, so further test that is Mann Whitney test was done.
5.5.2 Neutrophils Comparative Hypotesis Test with Using Mann Whitney U Test
Based on the Mann- Whitney test that was done on the neutrophils counting by
taking pulmonary emphysema group as one group and the combination group of mild,
moderate and severe smokers as another group, the value obtained was p
>0.05.Therefore it can be concluded that there is no any differences between the
groups or in other word, there value is not significant.
lix
5.5.3 One Way Anova Test
This observation used variable that is the neutrophils counting in the sputum
induction from all the groups of smokers based on the different stages of smokers ( > 2
unpaired groups ) without involving the fourth group which is the severe smokers with
pulmonary emphysema.
With considering the result of the test of homogeneity of neutrophils or on the
other word the Levene test, with an arrangement of p >0.05, it can be concluded that
there is no variable differences of the data of the groups that are compared and can be
further continued with the one way Anova test.
Based on the Levene variant test it can be concluded there is no differences in
the neutrophils variable from the different groups according to the stages of smoking
with a distribution of p> 0.05. Since the data of the variants are the same, one way
Anova test is valid with a value of p >0.05 from the neutrophils variable and therefore
can be concluded there is no significant differences between the variable of all the three
groups.
5.5.4 Correlation Test Of Different Stages Of Smoking
Correlation test was done to identify the influence of the different stages of
smoking with the neutrophils variable by using normally distributed Product Momen
Pearson numerical correlative hypothesis test. It can be shown clearly that only BMI
and age give significant result with the value p < 0.05. The correlation of the stages of
smoking with the BMI value has negatively correlation value, therefore it can be said
that the bigger the stage of smoking, the lower the value of the BMI of the person.
Contrarily for this statement, the age of the smoker has positive correlation with the
lx
stages of smoking. So, the bigger the stage of smoking, the higher the age of the
smoker.
It was found out that, the stages of smoking with the counting of the neutrophils
from the sputum induction has positive correlation with p > 0.05, so therefore it is not
significant also.
lxi
Chapter 6
Discussion
6.1 Characteristic of the Research Subjects
Based on the characteristic of the respondent from all the groups, it can be
found out that all the respondents are male with an average age of 60,775 + 7,816
years old and most of them are retired PNS who have smoking habit currently. T.B.
Grydeland et al, (2009) concluded that emphysema is more frequently occurs in male
compared to female. The higher emphysema score in males could have several
explanations. Emphysema quantification is very sensitive to the level of inspiration, but
even though males have larger lung, there are no indications that males inspired
deeper. Environmental cause is another possible explanation, as males are more
exposed to occupational airborne agents, which are potentially harmful to the lungs.
The prevalence of smoking is more prominent in developing countries such as China
and India compared to developed countries such as United States and United
Kingdom. This is due to most of the people who practice routine smoking hailed from
low social-economical status society who are low in literacy and do not have proper job
and eventually have low per capital income.
The type of cigarettes smoked by the smokers are clove, filtered, hand rolled
and most of the respondent has the habit of smoking a combination of kretek and
filtered cigarettes. Smoking filtered cigarettes has many beneficial such as reduced
lavel of nicotine and tar compared to the non-filtered cigarettes, however the level of
carbon monoxide is stil the same in both filtered and non-filtered, so the amount of
hazardous toxic effect that are produced by both the cigarettes are the same. Hence
neither type will protect the airway of the person.
lxii
The weight of the respondents were taken to identify the level of Body Mass
Index (BMI).BMI is the index used to categorize the person into underweight, normal,
overweight, and obese category. From the studies it is identified that most of the
respondents have higher BMI level compared to the controlled group which is the
smoker with emphysematous lung. Similarly, Verbeken et al, (1992) also found out that
BMI level of the normal healthy smoker is higher compared to the level of
emphysematous patients. Hence, emphysema together with smoking increase the
workload of the lung, and the airway muscles, so much energy will be used to
compensate this process and this causes the smoker with pulmonary emphysema to be
thinner.
6.2 Neutrophil Counting In Sputum Induction
The counting of neutrophils of the sputum induction is higher in the fourth group
which is the severe smokers with pulmonary emphysema. Apparently Morrison et al,
(1998) also found out that neutrophils are normally higher in emphysematous patient
compared to healthy smoker. This is because the underlying mechanisms of
emphysema include inflammatory processes in the lung and airways. Cigarette smoke
and other irritants activate macrophages and airway epithelial cells in the respiratory
tract, which release neutrophil chemotactic factors, including IL-8 and leukotriene (LT)
B4. Neutrophils and macrophages then release proteases that break down the
connective tissue in the lung parenchyma, resulting in emphysema. Therefore the
higher number of neutrophil in emphysematous patient is because of increased activity
of inflammation in the lung compared to healthy smoker.
Based on Mann –Whitney test that was conducted on the neutrophils counts of
the two groups which are the pulmonary emphysema group and the non pulmonary
lxiii
emphysema group which is the combination group of mild, moderate and severe
smokers, it can be concluded that there is no any significant differences between the
both groups. Apparently, this result is quite parallel to the research done by Domagala
et al, (2003) where they counted the neutrophils level in smokers with emphysematous
lung and healthy ex-smokers and they showed that there are no significant differences
in the cellular profile of induced sputum of the samples between patients with COPD
who are active smokers and those who have ceased smoking.
Based on the one way Anova test, which was done among the mild, moderate
and severe smokers, there is no any difference between the groups and it can be
concluded that the neutrophils level is not significantly variable among the groups, so
the values are approximately the same. It also can be clearly identified that smoking
stages do not directly affect the variability of the observation subjects, unless subjects
that are studied are non-smoker or not smoking currently.
6.3 The correlation between smoking stages and number of neutrophil in
alveolar
Pearson test was used to identify the correlation of stages of smoking to the
level of neutrophils and it was found that there is no any correlation of the stages of
smoking that was characterized according to Brinkman Index as mild, moderate and
severe smokers to the quantity of neutrophils in the person. It is because the value of p
> 0.05, therefore, it is not significance. From the research, it can be concluded that, the
number of neutrophil increases significantly even in the mild stage and therefore, the
severity of smoking is the same regardless the number of cigarette smoked per day.
lxiv
6.4 The Weakness of the Study
The study was done in a population of 40 candidates and this can be referred as
a small population to do this study. Therefore, the result would have been more
accurate and reliable if the study population was bigger. Apart from that, the smokers
with pulmonary emphysema were chosen based on the solitary confirmation from x-ray
radiologic findings. This would have created lesser accuracy because not any further
diagnosis was done to confirm the candidates are having pulmonary emphysema.
Hence, if more examinations were done, then it would have increase the accuracy of
the study.
lxv
CHAPTER 7
CONCLUSION
7.1 Conclusion
From the study result, it can be concluded that:
1. Smoking does influence the level of macrophages in sputum induction of a
smoker compared to the healthy person
2. The numbers of neutrophils do not increase proportionally according to
Brinkman Index from mild, moderate and severe smoker, hence, there is a
significant increase of macrophage even in mild smoker
3. There neutrophil counts in sputum induction of smoker with pulmonary
emphysema or non-pulmonary emphysema smoker (mild, moderate,
severe) are approximately the same.
4. Different stages of smoking are inversely proportional to the level of BMI of
the smoker
5. Different stages of smoking are directly proportional to the age of the
smoker.
lxvi
7.2 Suggestions
From this study, suggestion or opinions in future study are:
1. Further study should be done to identify how actually stages of smoking
influence the recruitment of neutrophils in the sputum induction of the
smoker.
2. Further study should be done to identify the correlation of smoking to the
level of neutrophils in the sputum induction of the smoker.
3. The number of the candidates used should be increased to decrease the
unwanted error in the study.
4. Another research should be done to identify how the mechanism of
recruitment of neutrophil in the sputum induction from the alveolar of a
smoker.
lxvii
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lxx
Kolmogorov-Smirnov Shapiro-Wilk
Group Statistic df Sig Statistic df Sig
Type Of Occupation Smoker with P.E .348 10 .001 .721 10 .010**
Mild Smokers .283 10 .023 .906 10 .315
Moderate Smokers .241 10 .103 .871 10 .111
Heavy Smokers .422 10 .000 .655 10 .010**
Age Smoker with P.E .263 10 .049 .877 10 .147
Mild Smokers .231 10 .140 .867 10 .096
Moderate Smokers .147 10 .200* .975 10 .922
Heavy Smokers .166 10 .200* .888 10 .212
BMI Smoker with P.E .180 10 .200* .948 10 .615
Mild Smokers .176 10 .200* .925 10 .423
Moderate Smokers .135 10 .200* .961 10 .767
Heavy Smokers .145 10 .200* .969 10 .863
Brinkman Index Smoker with P.E .289 10 .017 .775 10 .010**
Mild Smokers .305 10 .009 .778 10 .010**
Moderate Smokers .137 10 .200* .961 10 .770
Heavy Smokers .327 10 .003 .779 10 .010**
Type Of Cigarettes Smoker with P.E .406 10 .000 .623 10 .010**
Mild Smokers .410 10 .000 .708 10 .010**
Moderate Smokers .482 10 .000 .509 10 .010**
Heavy Smokers .410 10 .000 .708 10 .010**
Neutrophil Smoker with P.E .175 10 .200* .908 10 .323
Mild Smokers .247 10 .085 .791 10 .014
Moderate Smokers .299 10 .012 .763 10 .010**
Heavy Smokers .199 10 .200* .870 10 .0104
APPENDIXES
Table 1 : Test of Normality
Tests of Normality
**. This is an upper bound of the true significance
*. This is a lower bound of true significance
a. Lilliefors Significance Correction
Kolmogorov-Smirnov Shapiro-Wilk
lxxi
a
ANOVA
NEUTROPHIL
2831736 2 1415868.050 1.528 .235
25011816 27 926363.565
27843552 29
Between Groups
Within Groups
Total
Sum ofSquares df Mean Square F Sig.
Group Statistic df Sig Statistic df Sig
Macrophage Smokers With P.E .175 10 .200* .908 10 .323
Asymptomatic Smokers .201 30 .003 .774 30 .010**
Test of Normality
**. This is an upper bound of the true significance
*. This is a lower bound of true significance
a. Lilliefors Significance Correction
Table 2 : Test of Homogeneity of Variances
Table 3 : One Way Anova Analysis
Descriptives
95 % Confidence Interval
for Mean
N MeanStd.
Deviation Std. ErrorLower Bound
Upper Bound Minimum Maximum
Mild Smokers 10 617.8300 600.9134 190.0255 187.9625
1047.6975 126.70 2061.70
Moderate Smokers 10
1369.1400 1362.1994 430.7653 394.6813
2343.5987 228.00
4225.80
Heavy Smoker 10 955.9300 749.9378 237.1512 419.4568 1492.4032 236.40 2344.00
Total 30 980.9667 178.8969 178.8969 615.0815 1346.851
8 126.70 4225.80
lxxii
Test of Homogeneity of Variances
NEUTROPHIL
2.370 2 27 .113
LeveneStatistic df1 df2 Sig.
Table 4: Mann-Whitney Test
Table 5: t-TestGroup Statistics
lxxiii
Ranks
10 20.50 205.00
30 20.50 615.00
40
NEUTROPHIL
N Mean Rank Sum of Ranks
Asymptomatic SmokersSmoker with P.E
Group
Total
Test Statisticsb
150.000
615.000
.000
1.000
1.000a
Mann-Whitney U
Wilcoxon W
Z
Asymp. Sig. (2-tailed)
Exact Sig. [2*(1-tailedSig.)]
NEUTROPHIL
Not corrected for ties.a.
Grouping Variable: Groupb.
Group N Mean Std. DeviationStd. Error
Mean
Neutrophil Symptomatic Smokers 10 789.5000 571.5877 180.7519
Asymptomatic Smokers 30 980.9667 979.8584 178.8969
Independent Samples Test
Levene's Test for quality of
Variancet-test for Equality of Means
95% Confidence Interval of the
Difference
F Sig t df
Sig. (2-
tailed)Mean
DifferenceStd. Error Difference Lower Upper
Neutrophil
Equal Variance assumed 1.469 .233 -.583 38 .564 191.4667 328.6547 56.7933 73.8600
Equal Variance not assumed -.753 27.176 .458 191.4667 254.3135 13.1170 30.1836
Table 6: Correlations
Correlations
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Macrophage BMI AGEBrinkman Index
Type of Cigarettes
Type of Occupation
NeutrophilPearson Correlation 1.000 -.030 -.057 .099 -.015 .255
Sig. (2-tailed) - .855 .726 .545 .927 .113
N 40 40 40 40 40 40
BMIPearson Correlation -.030 1.000 -.289 -352* -.113 .034
Sig. (2-tailed) .855 - 0.71 .026 .489 .834
N 40 40 40 40 40 40
AGEPearson Correlation -.057 -.289 1.000 .667** -.099 -.516**
Sig. (2-tailed) .726 .071 - .000 .542 .001
N 40 40 40 40 40 40 Brinkman Index
Pearson Correlation .099 -.352* .667** 1.000 -.012 -.357*
Sig. (2-tailed) .545 .026 .000 - .943 .024
N 40 40 40 40 40 40 Type of Cigarettes
Pearson Correlation -.015 -.113 -.099 -.012 1.000 .323*
Sig. (2-tailed) .927 .489 .542 .943 - .042
N 40 40 40 40 40 40Type of Occupation
Pearson Correlation .255 .034 -.516** -.357* .323* 1.000
Sig. (2-tailed) .113 .834 .001 .024 .042 -
N 40 40 40 40 40 40
*. Correlation is significant at the 0.05 level (2-tailed)
**. Correlation is significant at the 0.01 level (2-tailed)
Table 7 : Raw Data
Type Pekerjaa
Usia BMI Indeks Brinkman
Type Rokok
C R PA WBC MACROPHAGE
NEUTROPHIL
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n n kg /
m2 106 /
ml106 /ml 106 /ml
A1 1 68 23 680 1 Emphysema 900.0
61.2 319.5
A2 1 61 23 740 4 Emphysema 200.0
11.8 59.6
A3 1 73 16.87
1120 1 Emphysema 4500.0
423.0 2079.0
A4 2 71 18 1100 1 Emphysema 800.0
95.2 333.6
A5 4 68 21.8 640 1 Emphysema 2400.0
249.6 513.6
A6 1 68 21.4 720 2 Emphysema 2800.0
114.8 596.4
A7 1 67 21.4 670 1 Emphysema 5400.0
540.0 901.8
A8 5 59 14.3 644 1 Emphysema 4600.0
501.4 1131.6
A9 1 69 28.75
1080 1 Emphysema 2400.0
223.2 964.0
A11 6 70 17.7 1060 5 Emphysema 2300.0
250.7 995.9
mean: 67.4 20.622
845.4 2630.00
247.09 789.50
B1 5 63 20.2 190 5 Bronkovaskular meningkat
1600.0
184.0 547.2
B2 5 60 23.05
190 2 Bronkovaskular meningkat
800.0
79.2 260.8
B3 7 53 22.6 192 2 Normal 2300.0
188.6 1143.1
B4 7 46 19.8 112 2 Normal 5300.0
519.4 2061.7
B5 5 48 22.4 192 2 Normal 2100.0
216.3 854.7
B6 2 46 24.65
180 1 Bronkovaskular meningkat
700.0
67.2 126.7
B7 5 60 25 180 1 Normal 1100.0
107.8 364.1
B8 5 47 30.96
68 2 Normal 1200.0
120.0 278.4
B9 9 53 23.3 144 5 Normal 1900.0
159.6 410.4
B10 1 60 27.4 10 2 Normal 800.0
66.4 131.2
mean: 53.6 23.936
145.8 1780 170.85 617.83
C1 7 61 27.4 310 2 Normal 8200.0
787.2 4225.8
C3 2 64 27.6 230 2 Bronkovaskular meningkat
1000.0
87.0 335.0
C4 5 53 22.5 390 2 Bronkovaskular meningkat
2400.0
242.4 943.2
C5 1 63 25.7 258 1 Bronkovaskular meningkat
2200.0
202.4 930.6
C6 5 59 24 490 2 Bronkovaskular meningkat
8000.0
704.0 3456.0
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C7 5 57 25 400 2 Bronkovaskular meningkat
4200.0
399.0 1419.6
C8 2 59 18 468 2 Bronkovaskular meningkat
1000.0
92.0 228.0
C9 1 67 30.4 450 1 Normal 2300.0
230.0 662.4
C10 1 52 22.7 336 2 Normal 2800.0
218.0 1027.6
C11 8 48 27.23
336 2 Normal 1200.0
110.4 463.2
mean:
58.3 25.053
366.8 3330 307.24 1369.14
D1 5 65 19.6 1082 5 Bronkovaskular meningkat
3100.0
229.4 1667.8
D2 5 59 22.4 1056 2 Bronkovaskular meningkat
4600.0
432.4 1826.2
D3 1 68 24.3 1152 1 Normal 1100.0
69.3 317.9
D4 1 57 26 648 2 Bronkovaskular meningkat
8000.0
768.0 2344.0
D5 1 57 21.8 684 2 Bronkovaskular meningkat
2700.0
288.9 820.8
D6 1 68 23.9 658 2 Normal 2000.0
174.0 564.0
D7 1 77 16.6 660 1 Bronkovaskular meningkat
1200.0
121.2 236.4
D8 1 68 23.62
1020 2 Bronkovaskular meningkat
2200.0
173.8 1097.8
D9 8 57 29.36
608 2 Bronkovaskular meningkat
1700.0
212.5 360.4
D10 1 62 24.69
630 5 Bronkovaskular meningkat
1500.0
156.0 324.0
mean: 63.8 23.227
819.8 2810 262.55 955.93
67.4 20.622
845.4
A 67.4 20.622
845.4 2630.00
247.09 789.50
B 53.6 23.936
145.8 1780 170.85 617.83
C 58.3 25.053
366.8 3330 307.24 1369.14
D 63.8 23.227
819.8 2810 262.55 955.93
TOTAL MEAN 60.775
23.2095
544.45 2637.5
246.9325
933.1
STATEMENT OF ORIGINALITY
The undersigned,
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Name : Uthaya Kumar Nallayan
NIM : 0810714039
Study Program : General Medicine, Faculty of Medicine, Brawijaya
University
Declares that this Final Project is an original research, not an acquisition of others
writing and thoughts which I claim as my own. If later it is revealed that this Final Project
contains partly or wholly plagiarized of others’ intellectual work of any kind, I will readily
accept the sanction established by the university on this matter.
Malang, January 2012
Sincerely,
(Uthaya Kumar Nallayan)
0810714039
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