A CLINICAL STUDY OF SERUM CYSTATIN C LEVELS IN CASES OF COPD AND ITS CORRELATION WITH SPIROMETRY AND IMPACT ON THEIR QUALITY OF LIFE

A CLINICAL STUDY OF SERUM CYSTATIN C LEVELS IN CASES OF COPD AND ITS CORRELATION WITH SPIROMETRY AND IMPACT ON THEIR QUALITY OF LIFE Dr. RAVI.APPORVA Dissertation submitted to BLDE (Deemed to be University) Vijayapur, Karnataka In partial fulfillment of the requirements for the degree of DOCTOR OF MEDICINE IN RESPIRATORY MEDICINE Under the guidance of Dr.SATISH.G.PATIL PROFESSOR DEPARTMENTOF RESPIRATORY MEDICINE BLDE (Deemed to be University) SHRIB.M.PATILMEDICALCOLLEGE HOSPITAL & RESEARCH CENTRE, VIJAYAPUR KARNATAKA 2020 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 1 "A CLINICAL STUDY OF SERUM CYSTATIN C LEVELS IN CASES OF COPD AND ITS CORRELATION WITH SPIROMETRY AND IMPACT ON THEIR QUALITY OF LIFE" DOCTOR OF MEDICINE IN RESPIRATORY MEDICINE DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 2 TABLE OF CONTENTS S. No. TOPIC PAGE NO. 01. INTRODUCTION 20 02. AIMS AND OBJECTIVES 22 03. REVIEW OF LITERATURE 23 04. MATERIALS AND METHODS 66 05. RESULTS 69 06. DISCUSSION 98 07. LIMITATIONS 113 08. CONCLUSION 114 09. SUMMARY 115 10. RECOMMENDATIONS 118 11. BIBLIOGRAPHY 119 ANNEXURES I : ETHICAL COMMITTEE APPROVAL LETTER 130 II : PATIENT CONSENT FORM 131 12. III : PROFORMA 135 IV : MASTERCHART 138 V: PLAGIARISM REPORT 139 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 3 LIST OF FIGURES S. No. FIGURES PAGE NO. 01. Refined ABCD assessment tool 38 02 Chest x ray 40 03 Computed Tomography 41 04. Representative flow-volume loops in normal subjects (red) and COPD (blue) 45 05. Smoking induces endogenous ROS production and destruction of lung 48 06. Correlation between age and Cystatin C 53 07. Relationship between age and Cystatin C concentrations across the age spectrum 54 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 4 LIST OF TABLES S. No. TABLES PAGE NO. 01. Common patterns of disease on PFTs 44 02. Severity Assessment according to spirometry 46 03. Severity of airflow limitation according to GOLD criteria 67 04. Distribution of serum Cystatin C (ng/ml) according to age 69 05. Distribution of serum Cystatin C ng/ml levels according to sex 70 06. Distribution of patients according to BMI in respect to serum Cystatin C ng/ml 71 07. Distribution of patients according to BMI domain of bode index in respect to serum Cystatin C level ng/ml 72 08. Distribution of patients according to pack years of smoking 72 09. Distribution of patients according to pack-years of smoking with respect to serum Cystatin C ng/ml 73 10. Distribution of patients according to serum Cystatin C levels ng/ml 75 11. Distribution of cases based on the severity of airflow limitation based on post bronchodilator fev1% predicted in respect to serum Cystatin C level ng/ml 75 12. Comparison of means of serum Cystatin C with respect to stages of airflow limitation according to gold guidelines of COPD. 76 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 5 13. Distribution of patients according to FEV1 domain of bode index in respect to serum Cystatin C level ng/ml 77 14. Distribution of patients according to redefined ABCD tool 77 15. Distribution of cases based on ABCD tool in respect to serum Cystatin C level ng/ml 78 16. Distribution of cases based on duration of disease in respect to serum Cystatin C level ng/ml 79 17. Distribution of patients according to MMRC scale 79 18. Distribution of patients according to MMRC scale in respect to serum Cystatin C level ng/ml 80 19. Distribution of patients according to six minute walk domain of bode index in respect to serum Cystatin C level ng/ml 81 20. Distribution of patients according to BODE index in respect to serum Cystatin C level ng/ml 81 21. Distribution of patients according to CCQ scores in respect to serum Cystatin C level ng/ml 82 22. Distribution of patients according to CAT scores in respect to serum Cystatin C level ng/ml 83 23. Distribution of patients according to SGRQ scores in respect to serum Cystatin C level ng/ml 84 24. Distribution of patients according to symptoms domain of SGRQ in respect to serum Cystatin C level ng/ml 84 25. Distribution of patients according to impact domain of SGRQ in respect to serum Cystatin C level ng/ml 85 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 6 26. Distribution of patients according to activity domain of SGRQ in respect to serum Cystatin C level ng/ml 85 27. Distribution of patients according to c-reactive protein levels 86 28. Distribution of patients according to c-reactive Protein in respect to serum Cystatin C level ng/ml 86 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 7 LIST OF GRAPHS S. No. GRAPHS PAGE NO. 01. Decline in lung function with age 27 02. Distribution of patients according to age 69 03. Distribution of patients according to gender 70 04. Distribution of patients according to BMI 71 05. Distribution of cases based on the severity of airflow limitation according to gold guidelines of COPD 73 06. Receiver operating curve indicating sensitivity and specificity of serum Cystatin C to diagnose COPD 74 07. Distribution of means of serum Cystatin C with respect to stages of airflow limitation according to gold guidelines of COPD. 76 08. Distribution of patients according to duration of disease 78 09. Distribution of patients according to 6-minute walk test 80 10. Distribution of patients according to clinical copd questionnaire score 82 11. Distribution of patients according to CAT scores 83 12. Correlation between post bronchodilator FEV1 % and serum Cystatin C (ng/ml) 87 13. Correlation between duration of disease and serum Cystatin C (ng/ml) 88 14. Correlation between CRP and serum Cystatin C (ng/ml) 89 15. Correlation between CCQ score and serum Cystatin C (ng/ml) 90 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 8 16. Correlation between total SGRQ score and serum Cystatin C (ng/ml) 91 17. Correlation between symptoms domain of SGRQ and serum Cystatin C (ng/ml) 92 18. Correlation between impact domain of SGRQ and serum Cystatin C (ng/ml) 93 19. Correlation between activity domain of SGRQ and serum Cystatin C (ng/ml) 94 20. Correlation between CAT score and serum Cystatin C (ng/ml) 95 21. Correlation between MMRC scale and serum Cystatin C (ng/ml) 96 22. Correlation between six minute walk test and serum Cystatin C (ng/ml) 97 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 15 The purpose of this study is to determine whether Cystatin C levels have any correlation with the clinical severity of COPD and determine its association with lung function in those patients. This study also aims to assess the impact on the quality of life of COPD patients. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 16 AIM AND OBJECTIVES OF THE STUDY AIM: The aim of the study is to correlate serum Cystatin C level with spirometry and also to evaluate its impact on the quality of life in chronic obstructive pulmonary disease patients. OBJECTIVES: 1. To determine levels of serum Cystatin C in COPD patients. 2. To perform pulmonary function tests in chronic obstructive pulmonary disease patients. 3. To assess the impact of quality of life using MMRC grading, CAT scoring, SGRQ-C, CCQ, 6 Minute walk test and Refined ABCD assessment tool as per Gold 2020 Report. 4. To correlate the quality of life with Cystatin C levels. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 17 REVIEW OF LITERATURE HISTORY Since the period of Laennec, chronic obstructive pulmonary disease has been extensively investigated. The relationship between the Disease and smoking was not made until the beginning of the first half of the 20th century.(7) By OPIE, et al. in 1905, the concept of an enzyme imbalance and the presence of antienzymes as a potential cause of emphysema was first documented. (8) There were also other more emphysema research studies. Emphysema most frequently impacted the heart, according to a study done in 1934 by Kountz and Alexander. A vascular atrophy model of the illness was also put up by Liebow et al. in the 1950s. Proteinase and anti-proteinase hypothesis in emphysema was developed in 1960 as a result of Gross and his co-workers' discovery that patients with emphysema had a 1 antitrypsin deficit. In its publication, the Medical Research Council introduced the phrase "chronic bronchitis" for the first time in 1956 to describe an illness characterised by a persistent cough and expectoration when other causes, such as pulmonary tuberculosis and bronchiectasis, were ruled out. (9) In the year, Higgins discovered a connection between smoking and persistent coughing and sputum production in 1959. (10) Owen and Campell discovered the pathological abnormalities brought on by smoking in 18 airways in the late 1960.(11) Dr. William Briscoe is thought to be the first person to use the term “chronic obstructive pulmonary disorder” at the 9thAspen Emphysema Conference in June of 1965. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 18 In 1971, 108 people with chronic obstructive pulmonary disease were investigated by Boushy SF et al. The pulmonary function test and hemodynamic data were linked with the ECG. (12) A series of publications on chronic obstructive pulmonary disease prognostic factors were published in 1973 by Bougly and colleagues mainly includes Lung function test prognostic values in Chronic Obstructive Pulmonary Diseases and pulmonary disease. Due to the long-term nature of this disease's course, it puts a significant strain on the resources of the healthcare system. Since COPD is a condition that may be prevented, it is extremely important for public health. PREVALANCE: Chronic obstructive pulmonary disease (COPD), which is now the fourth leading cause of mortality worldwide but is anticipated to become the third most common reason by 2020 (1), is a significant public health issue that is both curable and preventable. In 2012, COPD claimed the lives of more than 3 million individuals (1), or 6% of all fatalities worldwide. A significant public health issue that is both preventable and curable is COPD. It affects middle-aged and older people, and people under the age of 35 are less likely to contract it. Due to their higher smoking prevalence than women, men are the most frequently impacted. Due to an increase in environmental trigger factors throughout the winter, COPD exacerbations are more common. Previous research from other regions of the country claimed that among people over 40, the prevalence of chronic bronchitis was as high as 16% in North India(13) than in South India because of the climatic fluctuations there. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 19 Another study by Bhattacharya et al. (14) revealed that 57 per 1000 people in a rural population had chronic bronchitis by the age of 30 or older. The incidence of the disease increased in direct proportion to age and the number of pack-years of smoking. PREVALENCE IN WESTERN COUNTRIES: Around 16 million people in the USA are thought to have COPD, of which 14 million have chronic bronchitis and another 2 million have emphysema. The male-to-female ratio varies between 4 and 6% and 1 and 3%. With almost 1 lakh deaths each year, it is the third most common cause of mortality in the US. Prospective research conducted in the UK including 40,000 medical professionals revealed that chronic bronchitis was higher in smokers and proportional to pack years. Chronic Obstructive Pulmonary Disease (COPD) COPD is characterized by persistent respiratory symptoms and airflow limitation that is due to airway or alveolar abnormalities usually caused by significant exposure to noxious particles or gases. Nearly all patients have both the air space destruction associated with emphysema as well as the pathologic airway changes that are consistent with chronic bronchitis. However, subsets of the COPD population can differ in terms of natural history and response to therapeutic intervention. Daily cough and sputum for 3 months for two or more years is the qualifying definition for chronic bronchitis. Common, preventable, and treatable pulmonary disease generally caused by significant longterm cumulative exposure to noxious particles or gases (such as cigarette smoking) combined with other factors including hyperresponsiveness of airway, genetics and abnormal DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 20 development of lung, ultimately resulting in airflow limitations due to chronic airway inflammation and/or parenchymal destruction (emphysema)(15) Terms "emphysema" and "chronic bronchitis" not included in Global Initiative for Chronic Obstructive Lung Disease (GOLD) definition of COPD i. Chronic bronchitis - cough and excess sputum production for ≥ 3 months per year in each of 2 consecutive years ii. Emphysema - pathological term describing destruction of gas exchanging surfaces of lung (alveoli) COPD exacerbation defined as acute worsening of respiratory symptoms requiring additional therapy (15) Epidemiology(15) 90% of COPD deaths reported to occur in lowand middle-income countriesprevalence increases with age, but unclear if healthy ageing leads to COPD or if age reflects sum of cumulative exposures throughout life. Graph 1:Decline in lung function with age(1) DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 21 An estimated 175 million adults worldwide are thought to have COPD, an adult disease that typically manifests in the sixth or later decade of life. COPD is the third most common cause of death in the world, with an estimated 3.2 million deaths each year. (16) Given the time lag between starting to smoke and the manifestation of clinical illness, the frequency of cigarette smoking worldwide and the average population age predict that the number of cases will continue to climb. The projected yearly cost of COPD in the United States is $50 billion, of which $30 billion is spent on direct medical expenses and $20 billion on indirect expenses. The idea that smoking cigarette is the main risk factor for the onset of COPD is supported by epidemiologic data. The "dose" of smoking, expressed in pack-years is inversely correlated with FEV 1 at the population level. Lower peak lung function in adulthood is caused by factors that prevent full lung development, such as childhood respiratory illnesses; subsequent decrease, whether or not it is accelerated, can lead to the development of COPD. The primary cause of COPD is unquestionably cigarette smoking, but other exposures are also linked, particularly when biomass fuels are burned in poorly ventilated areas where they are used for heating and cooking. Less is known about the function of environmental air pollution. People who are exposed to workplace dust in mines, grain-handling facilities, and cotton mills may also have coughing, sputum, and a permanent loss of lung function. Research showing that higher pollution levels are temporally related to higher mortality in individuals with existing COPD. It is unknown if previous respiratory infections have any lasting consequences on adult lung function. Risk factors  Factors associated with COPD development and progression i. Tobacco smoke (active smoking or passive environmental exposure) DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 22 ii. other particle exposures such as a) outdoor air pollution such as from urban environments b) Indoor air pollution such as animal dung, crop residues, burning wood, or coal c) occupational exposures including organic and inorganic dusts, chemical agents, and fumes iii. genetic factors and family history of COPD iv. demographic factors a) older age b) male sex c) low socioeconomic status v. Respiratory features including a) suboptimal lung development during gestation or childhood (including leading to reduced maximal attained lung function) b) asthma and airway hyperreactivity c) infections such as a. history of severe childhood respiratory infection b. Pseudomonas aeruginosa infection c. Tuberculosis d) chronic bronchitis vi. HIV DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 23 Associated Conditions(15)  COPD frequently co-occurs with comorbidities (due to causal or exacerbating relationship and/or shared risk factors) which may affect prognosis and management strategies i. shared symptoms and/or signs between COPD and multiple frequent comorbidities necessitate careful evaluation and consideration ii. COPD may occur as part of multi-morbidity (≥ 2 chronic conditions)  significant comorbidities include(15) i. cardiovascular diseases, including ii. hypertension iii. coronary artery disease (CAD) iv. atrial fibrillation v. peripheral vascular disease vi. heart failure with reduced ejection fraction or heart failure with preserved ejection fraction vii. increased subclinical cardiovascular disease assessed by surrogate markers (carotid intima-media thickness and arterial stiffness measured by pulse wave velocity)  lung cancer(15) i. risk factors for lung cancer in patients with COPD include a) age > 55 years b) smoking history > 30 pack years c) emphysema on computed tomography scan d) airflow limitation on forced expiratory volume in 1 second/forced vital capacity < 0.7 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 24 e) body mass index < 25 kg/m2 f) family history of lung cancer Etiology and pathogenesis: Causes:-(15) • persistent airway inflammation and/or parenchymal destruction brought on by prolonged cumulative exposure to noxious particles or gases (such as cigarette smoking) and other factors like heredity and faulty lung development (emphysema) • People who never smoked can also get COPD, while it is uncertain what causes the inflammatory response in these circumstances. Pathogenesis(15):- According to data, the pulmonary vasculature, lung parenchyma, and airways all play significant roles in COPD patients. The relative relevance of these processes differs between patients, which affects how the disease manifests and how it responds to treatment. The protease-antiprotease theory is the most widely recognized idea for how emphysema develops. According to this idea, emphysema happens when there is an excess of elastolytic protease activity compared to antiprotease levels. This theory's foundation is the finding that those who are lacking in 1 -antiprotease have a higher likelihood of acquiring emphysema. 1 - Antiprotease is a significant inhibitor of neutrophil elastase and a member of the serpin (serine protease inhibitor) superfamily. Emphysema can result from the degradation of elastin by proteases. Emphysema can also be brought on by macrophage elastases in addition to neutrophil elastase. Smoking cigarettes cause an influx of inflammatory cells, such as neutrophils and macrophages, into the lung. In terms of quantity, macrophages make up the majority of this DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 31 However, it is important to note that at the individual patient level, FEV1 loses precision and thus cannot be used alone to determine all therapeutic options. Furthermore, in some circumstances, such as during hospitalization or urgent presentation to the clinic or emergency room, the ability to assess patients based on symptoms and exacerbation history, independent of the spirometric value, allows clinicians to initiate a treatment plan based on the revised ABCD scheme alone. This assessment approach acknowledges the limitations of FEV1 in making treatment decisions for individualized patient care and highlights the importance of patient symptoms and exacerbation risks in guiding therapies in COPD. The separation of airflow limitation from clinical parameters makes it clearer what is being evaluated and ranked. This facilitates more precise treatment recommendations based on parameters that are driving the patient’s symptoms at any given time. Fig:1 REFINED ABCD ASSESSMENT TOOL(1) ACUTE EXACERBATION OF COPD:- An increase in dyspnea beyond the patient's typical level, a coughing fit, or an increase and modification in the nature of mucus production are all considered signs of an acute exacerbation of COPD. Roughly 60% of the direct medical expenses associated with COPD DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 32 are attributable to acute exacerbations, which have an independent negative impact on quality of life and have a short-term death rate of about 10%. The most prevalent mechanism for an exacerbation, regardless of the initial stimulus, is an inflammatory response that comprises airway inflammation, mucus hypersecretion, and airway smooth muscle constriction, which combined produce in the classic triad of dyspnea, cough, and sputum. It is possible to find viral or bacterial infections in 75–80% of COPD acute exacerbations. Rhinovirus, influenza, parainfluenza, respiratory syncytial virus, coronaviruses, and adenovirus are the viruses that are most commonly implicated. The bacterial species that have been linked to exacerbations in a number of individuals with chronic bronchitis include Streptococcus pneumoniae, Haemophilus influenza, Moraxella catarrhalis, and, to a lesser extent, Pseudomonas aeruginosa. Other environmental exposures most certainly have a role as well, especially in the 20 to 25 percent of cases when no pathogen or viral exposure was found. For instance, people with COPD who visit the emergency room more frequently for respiratory symptoms do so when ambient air pollution is higher. Clinical Manifestations and Diagnosis: The symptoms of acute exacerbations of COPD are typically comparable to those of acute bronchitis because they are frequently accompanied by fever or happen after contact with ill people. Questions concerning the patient's recent history of exacerbations, concomitant conditions including heart disease, previous viral exposures, baseline functional state, past treatment responses (if any), and the presence of somnolence or disorientation that might indicate hypercarbia should be asked. In addition to acute COPD exacerbations, decompensated heart failure, pneumonia, pulmonary embolism, and pneumothorax should also DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 33 be taken into account as potential causes. A big airway tumour is less probable. The signs and symptoms of acute exacerbations of COPD are typically comparable to those of other illnesses since they frequently come with fever or happen after contact with sick people. Vital signs evaluation and detection of respiratory distress symptoms should be part of the physical examination. symptoms of left or right heart failure should all be looked for during the cardiac examination to determine whether there is tachycardia, irregular rhythm, or any of these conditions. Unusual mental state should prompt worries about hypercarbia. An arterial blood gas should be performed on individuals who are experiencing respiratory distress or who have other causes to suspect hypercarbia as part of the laboratory examination. Measuring the circulating amount of brain natriuretic peptide in patients may assist identify those with substantial left heart failure since tachycardia, increased jugular venous pressure, and peripheral edema can occur in both left and right heart failure. Antibiotic prescriptions can be decreased with no evidence of damage by using point-of-care C-reactive protein testing to direct dosing. Only around 5% of patients will likely have their medication changed as a result of a chest radiograph, which shows abnormalities in roughly 15% of individuals. Any patient with chest discomfort, leukocytosis, a history of heart disease, or another aggravating condition should always have a chest radiograph taken. A sputum culture is often unlikely to have an impact on treatment. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 34 Imaging Studies Fig:2 chest-xray17 Signs of hyperinflation such as(18) A Hyperlucency of the lungs B Increased rib space C. Obtuse costophrenic angle D. Low diaphragm (considered low if the border of the right hemidiaphragm in the midclavicular line lies at or below anterior end thethe of seventh rib)  Diaphragmatic flattening a) Seen best on lateral films b) Perpendicular height < 1.5 cm indicates flattening Other findings include:  Saber-sheath trachea (trachea normal to level of thoracic inlet, then narrows in coronal plane)  Increased retrosternal airspace (> 2.5 cm between sternum and ascending aorta)  Increased length of lung (> 30 cm) A B C D DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 35  Prominent vessels (large central pulmonary arteries if pulmonary hypertension has developed)  vascular markings get tapered rapidly Computed Tomography (CT) Scan:- Fig:3 computed Tomography17 A. Emphysematous lesions in the parenchyma adjacent to the pleural surfaces - Paraseptal emphysema Other uses:- i. Planned surgical procedure such as lung volume reduction or transplant, or prior to bronchoscopic lung volume reduction ii. For detecting comorbidities or differential diagnoses A DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 36 Diagnostic Testing: The diagnosis is established by spirometry showing airflow obstruction When the FEV 1 / FVC ratio drops to less than 0.7, obstruction is present. Lower ratios, which are typically stated as a percentage of expected determined from reference data gathered for a normal population, indicate severe blockage (standardised by age, sex, height, and ethnic background). The lung volumes may show residual volume (RV) and total lung capacity.(TLC). Emphysema or pulmonary hypertension patients may have lower DLCOs, depending on how many pulmonary capillary vessels involved in gas exchange have been lost. Pulmonary Function Tests Pulmonary function test (PFTs) are diagnostic tests to evaluate lung function clinical role of PFTs includes  Aiding in the diagnosis of lung diseases by determining presence and severity of lung function defects  Monitoring of lung function in patients with known lung disease PFT measurements include  Capacities i. total lung capacity (TLC) - volume of air in the lungs at maximum inhalation ii. vital capacity (VC) - volume of air from maximal inspiratory effort to maximal expiratory effort iii. inspiratory capacity (IC) - volume of air from resting expiratory level to maximal inspiratory effort iv. functional residual capacity (FRC) - volume of air in the lungs after unforced tidal volume expiration DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 37 v. maximal voluntary ventilation (MVV) - maximal inspiration and expiration over 12-15 seconds  volumes i. tidal volume (Vt) - volume of air from nonforced expiratory level to normal nonforced inspiratory level ii. expiratory reserve volume (ERV) - volume of air that can be exhaled after resting expiration iii. inspiratory reserve volume (IRV) - volume of air that can be inhaled after normal inspiration iv. residual volume (RV) - volume of air left in the lungs after maximal expiratory effort v. forced expiratory volume in 1 second (FEV1) - maximal amount of air exhaled  Ratios during forceful expiration in first second from a point of maximal inspiration i. FEV1/FVC ratio - ratio of the volume of air expelled during the first second of forced expiration from maximal inspiration to the total volume of forced expiration from maximal inspiration ii. FEV1/VC ratio - similar to FEV1/FVC ratio, but the largest VC determination is used which can be obtained by slow inspiration, forced inspiration, slow expiration, or forced expiration  Flow rates and flow-volume loops i. peak expiratory flow (PEF) - maximum flow rate of air during forced expiration from maximal inspiration ii. forced expiratory flow25%-75% (FEF25-75) - flow rate in the middle half of forced expiration from maximal inspiration DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 38 iii. (flow-volume curve) - graphic display of the flow of air during forced expiration and forced inspiration i. bronchoprovocation testing is a method that measures bronchial hyperresponsiveness to a stimulant Table :1Common patterns of disease on PFTs (1) Measurement DISEASE CATEGORY Obstructive Restrictive FVC Normal/decreased Decreased FEV 1 Decreased Decreased FEV/FVC Decreased Normal  obstructive pattern common in i. asthma (reversible) ii. COPD iii. bronchiolitis obliterans iv. constrictive bronchiolitis obliterans v. sarcoidosis vi. bronchiectasis vii. cystic fibrosis (CF) viii. alpha-1 antitrypsin deficiency i. sarcoidosis  increased DLCO common in i. obesity ii. pulmonary alveolar hemorrhage DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 39 iii. asthma iv. polycythemia Fig :4 (15) Representative flow-volume loops in normal subjects ( red ) and COPD ( blue). Spirometry  Spirometry reported to be most reproducible, objective, noninvasive, and readily available measurement of airflow obstruction  Use spirometry to diagnose airflow obstruction in patients with respiratory symptoms but not to screen for airflow obstruction in patients without respiratory symptoms  Spirometry demonstrating forced expiratory volume in 1 second (FEV1)/forced vital capacity (FVC) ratio < 0.7 confirms diagnosis DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 40  In addition to diagnosis of COPD, spirometry also useful for COPD severity assessment and follow-up evaluations to inform therapeutic decisions and to identify worsening conditions. Table 2: Classification of Airflow limitation Severity in COPD (1) STAGE AND SEVERITY DEFINITION I: Mild FEV 1 /FVC <0.70, FEV 1 ≥80% of predicted II: Moderate FEV 1 /FVC <0.70, 50% ≤FEV 1 <80% of predicted III: Severe FEV 1 /FVC <0.70, 30% ≤FEV 1 <50% of predicted IV: Very severe FEV 1 /FVC <0.70, FEV 1 <30% of predicted or FEV 1 <50% of predicted plus chronic respiratory failure Mechanisms of Oxidant Injury and Antioxidants Oxidant stress and injury resulting from reactive oxygen species (ROS) is the ultimate trigger for the three major pathologic changes (mucus abnormalities, emphysema, and pulmonary microvascular changes) culminating in distinct COPD clinical phenotypes. The principal ROS are superoxide anion, hydrogen peroxide, and the hydroxyl radical, the most damaging ROS.ROS arise either exogenously, from air pollution, or endogenously. Mainstream CS contains high concentrations of oxidants (1014 molecules/puff) and 3000 ppm nitric oxide/puff, and over 4700 chemical compounds.(19) ROS in CS range from short-lived oxidants, such as the superoxide radical and nitric oxide, to long-lived organic radicals, such as semiquinones. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 47 Factors that affect serum Cystatin C levels : In a sample of the general population, variables linked to the synthesis and/or catabolism of serum Cystatin C may have a greater impact on serum Cystatin C levels than GFR. Thus, it is crucial to remember that advanced age, male gender, larger weight, and height In comparison to the healthy controls, the emphysema group's mean CysC levels were considerably greater. AGE: FIG :6(50) The graphic shows how participants without kidney disease risk factors distributed their Cystatin C concentrations by decade of age. With each additional decade of age, the mean and variance increase. When those without any clinical risk factors for renal disease had a range of Cystatin C values, we saw a substantial correlation between age and Cystatin C. (Figure 4). With an almost normal distribution, the mean Cystatin C concentration and the variation increased with each decade of higher age. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 48 Fig:7(50) Age and Cystatin C over the age range for participants with and without clinical risk factors for kidney disease, shown in a smoothed manner. Clinical risk factors included coronary heart disease, cerebrovascular disease, peripheral arterial disease, diabetes, hypertension, obesity, smoking, and heart failure. We found that participants with and without clinical risk factors for renal disease had a substantial, non-linear relationship between age and Cystatin C concentrations across the age spectrum those without risk elements It was found that participants with and without clinical risk factors for renal disease had a substantial, non-linear relationship between age and Cystatin C concentrations across the age spectrum (Figure 5). When compared to people without risk factors, the rate of rise in Cystatin C concentrations appeared to accelerate with age, especially in people who had clinical risk indicators for renal disease.(50) Sex:- According to several research, the demographics, obesity, thyroid function, and inflammation all had an impact on Cystatin C levels (51-53). Male gender was independently related to greater blood Cystatin C levels, according to Eric L. Knight et al.(53) DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 49 BODY MASS INDEX:- Males and females in the overweight and obese groups have substantially higher blood Cystatin C levels than those in the control group, indicating a possible influence of BMI. Since all nucleated cells, including adipocytes, are known to generate Cystatin C, overweight and obese people are likely to have greater blood Cystatin C levels. Serum Cystatin C levels (54) do seem to be affected by a number of variables, including higher weight and height. Serum Cystatin C and BMI have also been found to significantly correlate in other studies.(55,56). PATHOPHYSIOLOGY:- The imbalance between protease and anti-protease is a component of the pathophysiology. (2) Based on their chemical makeup, proteases are divided into four groups: Serine, Metallo, Cysteine, and Aspartic. The major function of the cathepsin family of papain-like cysteine proteases is to degrade of protein and peptides in the lysosomes. Lung extracellular matrix, particularly lung elastin, is broken down by extracellular cathepsins released by neutrophils and macrophages in the alveoli. The breakdown products of elastin fibres exert chemotactic effects on monocytes, which encourage their infiltration and worsen the lung's inflammatory response. When lung elastic tissue is more severely damaged due to COPD, transforming growth factor beta-1 is released from proteoglycan storage sites, which increases Cystatin C synthesis.(2) Inflammatory cells secrete Cystatin C, one of the main human extracellular cathepsins inhibitors, into the bloodstream.(3) Protein released from alkali having a 133kda molecular weight.(4) Cyc regulates protease production or leakage from lysosomes of sick cells by forming DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 50 complexes with cathepsins.(5) The activity of cathepsin may be indirectly reflected in Cystatin C levels. (6) Studies on COPD and its association with Cystatin C In 2012, Nowinski et al.(57) conducted a study to see the relationships regarding COPD severity and serum Cystatin C levels in prospective study. Study reported that COPD patients with more severe GOLD stages have higher serum Cystatin C levels but not creatinine or calculated creatinine clearence levels. In 2014, Zhang Y et al.(58) conducted a study on the possible biomarker of serum Cystatin C for the assessment of COPD. Acute exacerbation of chronic obstructive pulmonary disease (AECOPD) (n = 93), stable COPD (n = 299), and healthy controls (n = 151) were the three groups in the research that had their serum Cystatin C levels measured. Further research was conducted on the effects of smoking on serum Cystatin C levels and the relationship between Cystatin C and lung function metrics. Patients with COPD had considerably higher serum Cystatin C levels than healthy controls. Smoking raised the blood level of Cystatin C in SCOPD patients but not in AECOPD or control patients. In the SCOPD group, serum Cystatin C levels were inversely connected with FEV1% predicted, FEV1%FVC, MMEF75/25% predicted, and MVV% predicted, and favourably correlated with RV%TLC. DLco% forecasted, etc. FEV1% predicted and age were revealed to be independent predictors of serum Cystatin C levels in multiple line analysis, but not smoking status, sex, or BMI. Serum Cystatin C levels were greater in COPD, but smoking merely elevated Cystatin C levels in SCOPD. The predicted FEV1% was inversely linked with serum Cystatin C levels. These findings imply that Cystatin C may serve as a potential biomarker for the severity of COPD and the destruction of lung tissue. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 51 In 2016, Nakajima et al.(59) performed a study to investigate the potential role of plasma levels of cathepsin S or Cystatin C as COPD biomarkers. According to the study, the plasma levels of cathepsin S and Cystatin C were noticeably greater in the COPD and AT Risk (AR) groups than in the NS and HS groups. Plasma cathepsin S levels and cathepsin S/Cystatin C ratios were adversely correlated with severe airflow restriction (% FEV1 predicted 50%) and severe emphysema as measured by low attenuation area (LAA) score on chest CT scans in COPD patients and AR subjects, but not with Cystatin C levels (LAA 8.0). In 2016, Hu G et al. (6) carried out research to look at Cystatin C predictive value for in-hospital mortality in patients with a COPD exacerbation. Measurements were made on 477 participants in the study who had COPD exacerbations. Additionally, clinical traits were noted. The Cystatin C concentration that distinguished survivors from those who did not survive was determined using a receiver operating characteristic curve analysis. The risk variables for inhospital death were found using both univariate and multiple logistic regression models. Subgroup analyses were carried out based on the comorbidities, such as heart failure, renal dysfunction, pH, PaCO2, and PaO2 levels, in order to lessen the impact of confounders. According to the study, 418 participants had a full recovery whereas 59 subjects passed away while they were hospitalised . Lower pH (7.27 0.17 vs. 7.38 0.06, P .001), greater Cystatin C (2.21 1.05 mg/L vs. 1.39 0.54 mg/L, P .001), higher PaCO2 (77 39 mm Hg vs. 48 14 mm Hg, P .001), and lower PaO2 (74 32 mm Hg vs. The Cystatin C prediction of mortality has an area under the receiver operating characteristic curve of 0.77 (95% CI: 0.70-0.84). A substantially increased in-hospital death rate was linked to Cystatin C levels below 1.59 mg/L (relative risk = 5.49, 95% CI 3.24-9.32, P .001). In-hospital mortality was independently predicted by pH 7.20, Cystatin C 1.59 mg/L, and heart failure, according to multiple logistic regression analysis. The subgroup analysis revealed that the findings that Cystatin C was a mortality risk factor for those with COPD exacerbations were unaffected by the coexisting conditions of renal DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 52 dysfunction, congestive heart failure, and values of pH, PaCO2, and PaO2. exacerbation was unaffected by the values of pH, PaCO2, and PaO2. In 2016, Zhang et al.(2) conducted a research on patients with exacerbation and recovery from COPD using serum Cystatin C as an indicator of inflammation. According to the study, serum Cystatin C was inversely connected with predicted FEV1% and FEV1/FVC in convalescent COPD patients and positively correlated with hsCRP in both the exacerbation and convalescence phases of COPD. In conclusion, serum Cystatin C in COPD patients is a positive acute-phase reactant and may signify systemic inflammation during the development of COPD. In 2018, Telo S et al.(3) conducted a study on the possible diagnostic significance of serum Cystatin C levels and respiratory functioning in COPD. Study results 50 healthy participants and 126 people with COPD participated in the research (68 in stable times and 58 during exacerbation periods). The study discovered that serum Cystatin C levels were considerably higher in both COPD groups than the control group despite the fact that there was no statistically significant difference between the COPD groups (p>0.05) (p0.001 for both). Cystatin C levels showed a negative link with forced expiratory volume in one second (FEV1) and a positive correlation with C-reactive protein (CRP) levels in people with stable COPD. Blood Cystatin C levels and serum urea, creatinine, and CRP levels were significantly correlated in individuals with COPD exacerbation (r=0.333, p=0.011; r=0.260, p=0.049). When stable COPD and control groups were evaluated, serum Cystatin C showed an area under the curve (AUC) in the receiver operating characteristic (ROC) curve of 0.951 (0.909-0.994). In 2019, Diago et al.(60)conducted a study on Glomerular filtration rate estimated using serum creatinine and Cystatin C, and Creatinine Cleareance measured in COPD patients. Study reported that 44 COPD patients 65+/-6 years, FEV1 55,2 +/-17% Exacerbations during DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 53 previous year: 1 (0-2), mMRC dyspnea score 1 (0-2) CAT score 14,3+/-8,7, Charlson 1,39+/- 0,6. Cystatin C 97,18+/-17 mL/min, and both creatinine an Cystatin C 91,94+/-11,5 mL/min. In 2020, Chai Limin et al.(4) comprehensive meta-analysis of the research on the association between Cystatin C and COPD. This study's meta-analysis included 5949 controls, 4079 COPD patients, and a total of 15 studies. The results showed that blood Cys C levels were significantly greater in AECOPD patients than in SCOPD patients and statistically different from controls in COPD patients The relationship between serum Cystatin C levels FEV1%pre and FEV1/FVC were below normal values (Z = 0.45, 95%CI = -0.58--0.32, P = 0.011 and 0.006, respectively), while FEV1%pre was also below normal. Ethnicity, study methodology, or test technique had no impact on the serum Cystatin C levels. FEV1/FVC (Z = 0.32, 95%CI = 0.50, P = 0.006). The serum Cystatin C levels were unaffected by test technique, research design, or ethnicity. In 2021, Nishiki et al.(61) conducted a research to see if the blood Cr level adjusted for serum Cystatin C may be used to predict lung function and disease severity in COPD patients. Serum Cr and Cystatin C levels were evaluated in 201 individuals with COPD and 99 patients without COPD who had smoked more than 10 pack-years before to enrollment. Low attenuation area (LAA%) ( 960 Hounsfield units (HU).According to the study, there is a substantial association between the Cr/CysC ratio and the ESMCSA. Forced vital capacity (FVC) and forced expiratory volume in one second (FEV1) values were substantially correlated with the Cr/CysC ratio, notably in ex-smokers. In 2021, Hirai et al.(62) conducted a study on Serum creatinine/Cystatin C ratio as a surrogate marker for sarcopenia in patients with chronic obstructive pulmonary disease. Study included 234 male outpatients with COPD. Serum Cr/Cystatin C was shown to substantially correlate with both muscle mass and handgrip strength (r = 0.44, P 0.01) in the study. Serum Cr/Cystatin DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 54 C ratio had a considerably greater area under the sarcopenia curve than the other biomarkers (Cr/Cystatin C: 0.87, Cystatin C: 0.63, Cr: 0.61, albumin: 0.57). The incidence of acute exacerbations between patients in the lowand high-Cr/Cystatin C group, as determined by the cutoff value of 0.71, did not differ substantially according to multivariate analysis, although the frequency of severe acute exacerbations was considerably greater in the low-Cr/Cystatin C group. According to the study's findings, the serum Cr/Cystatin C ratio may be utilised to assess sarcopenia in male COPD patients with reasonable cost and ease. In 2021, Amado et al.(63) conducted a study on the Ratio Serum Creatinine/Serum Cystatin C (a Surrogate Marker of Muscle Mass) as a Predictor of Hospitalization in Chronic Obstructive Pulmonary Disease Outpatients. The study comprised 65 stable COPD outpatients and 18 healthy control participants with stable COPD. After enrolling in the trial, patients were prospectively observed for a period of one year. According to a study, COPD patients had less muscle mass and a lower SI than controls. Furthermore, compared to patients without these features, patients with a high risk of exacerbation, patients with a COPD Assessment Test score of 10, and patients with a modified Medical Research Council dyspnea score of 2 had lower levels of SI. SI had a significant inverse relationship with FEV1 (r = 0.491, p 0.001), the 6minute walking test (r = 0.560, p 0.001), and the fat-free mass index (r = 0.431, p = 0.017). A low SI is an independent predictor of, as demonstrated by univariate and multivariate Cox proportional risk analysis, healthy control participants with stable COPD. Hospitalization of COPD outpatients was monitored for a year after (HR 5.16, p = 0.025). An essential factor to consider when assessing the effects of chronic illnesses is quality of life (QOL). Measuring HRQOL makes it easier to assess the success of medical therapies and identify populations at risk for psychological or behavioural issues. [64] DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 55 Patients with COPD may delay seeking medical attention or present with little or no symptoms in the early stages, which might cause them to be overlooked in hospital settings and result in a worse QOL overall. St. George's Respiratory Questionnaire (SGRQ) SGRQ is a standardised, self-administered questionnaire used to assess HRQOL perception and diminished health in patients with airways illness. The St. George's Respiratory Questionnaire (SGRQ) was developed into the SGRQ-C, a condensed form, after extensive data analysis from significant research on COPD. [65] In place of the initial 50 items, it now has 40 items with 76 weighted responses that span three domains: symptoms, activities, and effect. [66] It also calculates a total score in addition to the domain scores. There is an empirical weight assigned to each item. SGRQ-C scores range from 0 to 100, with 0 being the highest HRQOL. Among COPD patients, this tool has been shown to be valid, reliable, and responsive. [66] Included studies Assessment of patient-reported outcome measures, such as the health-related quality of life (HRQL), is critical for determining how chronic illnesses affect patients. The St. George's Respiratory Questionnaire (SGRQ) is a respiratory-specific instrument that was initially developed to evaluate patients with COPD's health-related quality of life. However, the SGRQ has grown to be one of the most widely used instruments for evaluating patients with other chronic lung diseases, such as lymphangioleiomyomatosis (LAM), and interstitial lung disease linked to connective tissue disease (CTD-ILD). These studies shown that the SGRQ total score was a very significant predictor of mortality and that the SGRQ scores were related to the outcomes of the pulmonary function test (PFT). 65 stable control patients and 18 healthy. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 56 Clinical COPD questionnaire: The Clinical COPD Questionnaire (CCQ) has three categories and ten items with scores ranging from 0 to 6. (symptoms, functional, mental). (67) Adding together the individual item scores and dividing them by ten yields the total score, which ranges from 0 to 6, with higher scores denoting lower HRQoL. The adoption of a quicker health status questionnaire could result in more effective service delivery and subsequent cost savings, according to several recent studies that have proposed the CAT as a simpler, more practical alternative HRQoL measure to time-consuming questionnaires like the CRQ and SGRQ (68,69,70). Compared to more well-known surveys, the CCQ takes far less time and staff involvement to complete(71) , but it could possibly give more information due to the inclusion of domain and overall scores. Recent studies also seem to indicate that there is a modest preference for the CCQ over the CAT at the patient and clinician level. (71,72,73) COPD ASSESSMENT TEST: Eight items make up the CAT, which concentrates on respiratory symptoms like cough, sputum production, tightness in the chest, and dyspnea as well as non-respiratory symptoms like fatigue or sleep disturbances and additional indicators like difficulty performing tasks at home or lack of confidence leaving the house [74] According to Jones and colleagues, the item "breathlessness" has the most power of discrimination for milder patients, whereas the item "confidence leaving home" discriminates more well for more severe patients (74) CAT total score determines the GOLD classification of patients with COPD (classifying patients into low (A/C) and high (B/D) symptom groups) and, in turn, the recommended respiratory pharmacological treatment strategy (1) DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 63 RESULTS AGE DISTRIBUTION: The distribution of patients according to different age groups is depicted in Graph 2. The mean age was 57.95±3.66 years. TABLE 4: DISTRIBUTION OF SERUM CYSTATIN C (ng/ml) ACCORDING TO AGE There is a positive association between age and serum Cystatin C where the levels of serum Cystatin C increased with increasing age and it is statistically significant is represented in table 4. Serum Cystatin C level ng/ml <45-49 years 50-54 years >55years Mean±SD age in years MANN Whitney test p-value ≤532.3ng/ml 1 4 18 56.8±4 150 0.004 >532.3ng/ml 0 0 19 59±1 GRAPH2 DISTRIBUTION OF PATIENTS ACCORDING TO AGE 100.00% 88.10% 80.00% 60.00% 40.00% 20.00% 2.40% 9.50% 0.00% < 45 50 - 54 55+ No. of patients(%) DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 64 GENDER DISTRIBUTION It was observed that the gender inclination was towards Males with 64.3% of patients (27 in number) while 35.7% of patients were Females (15 in number) (Graph 3). TABLE:5 DISTRIBUTION OF SERUM CYSTATIN C ng/ml LEVELS ACCORDING TO SEX There is a positive association between sex and serum Cystatin C where the levels of serum Cystatin C increased in males and it is statistically significant (table 5) Gender N Mean±SD p value Females 15 444.49±237.57 0.04 Males 27 624.48± 293.3 GRAPH 3 DISTRIBUTION OF PATIENTS ACCORDING TO GENDER 35.70% 64.30% Males Females DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 65 GRAPH 4 DISTRIBUTION OF PATIENTS ACCORDING TO BMI 25 21 20 15 15 10 5 0 Under weight Normal weight BMI Over weight BMI DISTRIBUTION - Of the 42 patients, 21 were of Ideal body weight, whereas 6 were overweight, 15 were Underweight. The distribution of cases is shown in Graph 4. 6 TABLE:6 DISTRIBUTION OF PATIENTS ACCORDING TO BMI IN RESPECT TO SERUM CYSTATIN C NG/ML There is a No association between BMI and serum Cystatin C levels and it is statistically not significant ( table 6) Serum Cystatin C level ng/ml Over weight Normal weight Under weight Mean BMI Chi square test P value ≤532.3ng/ml 3 12 8 20.49 0.1153 0.94 >532.3ng/ml 3 9 7 20.47 NO OF PATIENTS DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 66 TABLE :7 DISTRIBUTION OF PATIENTS ACCORDING TO BMI DOMAIN OF BODE INDEX IN RESPECT TO SERUM CYSTATIN C LEVEL ng/ml There is a No association between BMI Domain of BODE index and serum Cystatin C levels and it is statistically not significant ( table 7). BMI Domain of BODE index No of patients with mean serum Cystatin C level >532ng/ml No of patients with mean serum Cystatin C level ≤532ng/ml CHISQUARE VALUE P value ODDS RATIO ≤21 11 15 0.237 0.31 0.733 C.I (0.20992.563) >21 8 8 TOTAl 23 19 TABLE NO:8 DISTRIBUTION OF PATIENTS ACCORDING TO PACK YEARS OF SMOKING Of the 27male patients, maximum no of patients have pack years of smoking more than 10 years The distribution of cases is shown in Table 8 Pack years No of patients ≤ 10 years 7 >10 years 20 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 67 TABLE:9 DISTRIBUTION OF PATIENTS ACCORDING TO PACK-YEARS OF SMOKING WITH RESPECT TO SERUM CYSTATIN C NG/ML There is a positive association between pack-years and serum Cystatin C. patients with more than 10 pack years have odds of 5.8 times higher chance of increased serum Cystatin C ng/ml levels and was statistically significant (table 9). Pack years Serum Cystatin C level ≤532ng/ml Serum Cystatin C level 532ng/ml Odds ratio P value >10 years 6 14 5 .833 (CI 0.74-38.9) 0.03 ≤ 10years 5 2 TEST USED – MID P EXACT TEST GRAPH 5: DISTRIBUTION OF CASES BASED ON THE SEVERITY OF AIRFLOW LIMITATION ACCORDING TO GOLD GUIDELINES OF COPD Out of 42 patients, 31% had moderate airflow limitation whereas 38.1% were with severe air limitation and 31% were with very severe airflow limitation GRAPH 5 DISTRIBUTION OF PATIENTS BASED ON SEVERITY OF AIRFLOW LIMITATION ACCORDING GOLD GUIDELINE OF COPD 20 38.1 15 31% 31% 10 5 0 MODERATE SEVERE VERYSEVERE severity of airflow limitation according to Gold guidelines No of patients DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 68 GRAPH :6 RECEIVER OPERATING CURVE INDICATING SENSITIVITY AND SPECIFICITY OF SERUM CYSTATIN C TO DIAGNOSE COPD Area Under the Curve is 0.942 Serum Cystatin C had an area under the curve in the receiver operating characteristic curve of 0.942. A Cystatin C level of 532.3 ng/ml was accepted as the cut-off value. Cystatin C has a highest sensitivity of 100% and specificity of 79.3% This point represents serum Cystatin C value of 532.3ng/ml. Has highest sensitivity of 100% and specificity of 79.3% DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 69 TABLE10: DISTRIBUTION OF PATIENTS ACCORDING TO SERUM CYSTATIN C LEVELS ng/ml Of 42 patients, 54.7% had a serum Cystatin C level equal to less than 532ng/ml whereas 45.3% had a serum Cystatin C level ng/ml more than 532ng/ml. (table 10) SERUM CYSTATIN C LEVEL (ng/ml) NO OF PATIENTS PERCENT ≤532.3ng/ml 23 54.7 >532.3ng/ml 19 45.3 TOTAL 42 100 TABLE 11: DISTRIBUTION OF CASES BASED ON THE SEVERITY OF SPIROMETRY BASED ON POST BRONCHODILATOR FEV1% PREDICTED IN RESPECT TO SERUM CYSTATIN C LEVEL ng/ml The association between the severity of spirometry and serum Cystatin C levels(ng/ml) was found to be statistically significant. (table 11). Odd ratio cant be done as values are missing SEVERITY OF SPIROMETRY BASED ON POSTBRONCHODILATOR FEV1% PREDICTED ACCORDING COPD GOLD GUIDELINES SERUM CYSTATIN C LEVEL (≤532.3ng/ml) SERUM CYSTATIN C LEVEL (>532.3ng/ml) CHI – SQUARE TEST P VALUE ≥80FEV1%PREDICTED 0 0 26.8 0.0001 50%≤FEV1 <80% PREDICTED 13 0 50%≤FEV1≥30% PREDICTED 10 6 FEV1 <30% PREDICTED 0 13 TOTAL 23 19 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 70 GRAPH :7 DISTRIBUTION OF MEANS OF SERUM CYSTATIN C WITH RESPECT TO GOLD CLASSIFICATION 861.25 567.6 250.02 copd classification according to GOLD GRAPH:7 DISTRIBUTION OF MEANS OF SERUM CYSTATIN C WITH RESPECT TO STAGES OF SPIROMETRY ACCORDING TO GOLD GUIDELINES OF COPD. Mean serum Cystatin C levels ng/ml in moderate, severe, and very severe stages were 250.02ng/ml, 567.6ng/ml and 861.2ng/ml respectively. TABLE:12 COMPARISON OF MEANS OF SERUM CYSTATIN C WITH RESPECT TO STAGES OF SPIROMETRY ACCORDING TO GOLD GUIDELINES OF COPD. Mean serum Cystatin C levels increases as airflow limitation increases and were found to be statistically signification as in table 12 SEVERITY OF SPIROMETRYBASED ON POSTBRONCHODILATOR FEV1% PREDICTED ACCORDING COPD GOLD GUIDELINES Mean±SD ANOVA P VALUE ≥80%FEV1PREDICTED 0 51.5 0.001 50%≤FEV1<80% PREDICTED 250.02±57.25 50%≤FEV1≥30% PREDICTED 567.61±163.67 FEV1 <30% PREDICTED 861.25±199.56 Cystatin c level ng/ml DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 71 TABLE:13 DISTRIBUTION OF PATIENTS ACCORDING TO FEV 1 DOMAIN OF BODE INDEX IN RESPECT TO SERUM CYSTATIN C LEVEL ng/ml There is an association between FEV 1 Domain of BODE index and serum Cystatin C levels and it is statistically significant ( table 13) FEV1 (% PREDICTED) OF BODE INDEX No of patients with mean serum Cystatin C level ≤532ng/ml No of Patients with mean serum Cystatin C level >532ng/ml CHISQUARE P value ≥65 5 0 19.11 0.0001 50-64 8 0 36-49 7 0 ≤35 3 13 TOTAL 23 19 TABLE 14: DISTRIBUTION OF PATIENTS ACCORDING TO REDEFINED ABCD TOOL Out of 42 patients 3 belongs to a group, 5 belong to B group and 34 belong to D group as represented in the table 14 ABCD Tool NO of patients A 3 B 5 C 0 D 34 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 72 TABLE 15: DISTRIBUTION OF CASES BASED ON ABCD TOOL IN RESPECT TO SERUM CYSTATIN C LEVEL ng/ml The association between the severity of disease and serum Cystatin C levels(ng/ml) was found to be statistically significant. (table 15). Odds ratio cant be done as values are missing REDEFINED ABCD TOOL SERUM CYSTATIN C LEVEL <532.3ng/ml SERUM CYSTATIN C LEVEL >532.3ng/ml CHISQUARE VALUE P VALUE A 3 O 8.164 0.017 B 5 0 C 0 0 D 15 19 TOTAL 23 19 GRAPH :8 DISTRIBUTION OF PATIENTS ACCORDING TO DURATION OF DISEASE Out of 42 patients, maximum no of patients were with duration of disease less than equal to 4 years of duration as depicted in (GRAPH 8). DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 79 There was a positive association between sgrq symptom score and serum Cystatin C ng/ml and patient with St George symptom score more than 50 have odds of 9 times chance of increased levels of serum Cystatin C levels and found to be statistically significant shown in the (table 24) STGEORGE SYMPTOM SCORE No of patients with mean serum Cystatin C level ≤532ng/ml No of patients with mean serum Cystatin C level >532ng/ml P value ODDS RATIO >50 11 17 0.001 9.27 CI 1.73-49.6 ≤50 12 2 TEST USEDMID P EXACT TEST TABLE:25 DISTRIBUTION OF PATIENTS ACCORDING TO IMPACT DOMAIN OF SGRQ IN RESPECT TO SERUM CYSTATIN C LEVEL ng/ml There was a positive association between sgrq impact score and serum Cystatin C ng/ml and patient with St George total score more than 50 have odds of 7.03 times chance of increased levels of serum Cystatin C levels and found to be statistically significant shown in the (table 25) STGEORGE IMPACT SCORE No of patients with mean serum Cystatin C level ≤532ng/ml No of patients with mean serum Cystatin C level >532ng/ml P value ODDS RATIO >50 8 15 0.002 7.03 CI(1.73-28.4) ≤50 15 4 TEST USEDMID P EXACT TEST DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 80 TABLE :26 DISTRIBUTION OF PATIENTS ACCORDING TO ACTIVITY DOMAIN OF SGRQ IN RESPECT TO SERUM CYSTATIN C LEVEL ng/ml There was a positive and statistically significant association between Activity domain of sgrq and serum Cystatin C ng/ml as shown in the (table 26). Odd ratio can’t be calculated as one value is missing STGEORGE ACTIVITY SCORE No of patients with mean serum Cystatin C level ≤532ng/ml No of patients with mean serum Cystatin C level >532ng/ml CHISQUARE P value >50 15 19 7.969 0.002 ≤50 8 0 TABLE :27 DISTRIBUTION OF PATIENTS ACCORDING TO C-REACTIVE PROTEIN LEVELS Out of 42 patients, maximum no of patients (38) had C – Reactive protein more than 10 CRP (mg/dl) No of patients <10 4 >10 38 Total 42 TABLE :28 DISTRIBUTION OF PATIENTS ACCORDING TO C-REACTIVE PROTEIN IN RESPECT TO SERUM CYSTATIN C LEVEL ng/ml There is a positive association between C -Reactive protein and serum Cystatin C level No of patients No of patients CHIP value with mean with mean SQUARE CRP serum Cystatin serum Cystatin C level C level ≤532ng/ml >532ng/ml DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 81 <10 4 0 5.62 0.04 >10 19 19 TOTAl 23 19 GRAPH :12 CORRELATION BETWEEN POST BRONCHODILATOR FEV1 % AND SERUM CYSTATIN C (ng/ml) There is a negative correlation between serum Cystatin C level ( ng/ml )and post bronchodilator FEV1% and it is statistically significant DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 82 CORRELATION BETWEEN POSTBRONCHODILATOR FEV1 % AND SERUM CYSTATIN C (ng/ml) POSTBRONCHODILATOR FEV1 % SERUM CYSTATIN C VALUES r co-efficient -.802 P value 0.0001 GRAPH :13 CORRELATION BETWEEN DURATION OF DISEASE AND SERUM CYSTATIN C (ng/ml) There is a positive correlation between serum Cystatin C level ( ng/ml )and Duration of disease and it is statistically significant. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 83 DURATION OF DISEASE SERUM CYSTATIN C VALUES r co-efficient 0.615 P value 0.001 GRAPH :14 CORRELATION BETWEEN CRP AND SERUM CYSTATIN C (ng/ml) There is a positive correlation between serum Cystatin C level ( ng/ml )and Creactive protein and it is statistically significant. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 84 CRP SERUM CYSTATIN C VALUES r co-efficient 0.0554 P value 0.0001 GRAPH :15 CORRELATION BETWEEN CCQ SCORE AND SERUM CYSTATIN C (ng/ml) There is a positive correlation between serum Cystatin C level (ng/ml )and CCQ sore and it is statistically significant. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 85 CCQ SCORE SERUM CYSTATIN C VALUES r co-efficient 0.743 P value 0.001 GRAPH :16 CORRELATION BETWEEN TOTAL SGRQ SCORE AND SERUM CYSTATIN C (ng/ml) There is a positive correlation between serum Cystatin C level (ng/ml )and TOTAL SGRQ score and it is statistically significant. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 86 SGRQ SERUM CYSTATIN C VALUES r co-efficient 0.767 P value 0.001 GRAPH :17 CORRELATION BETWEEN SYMPTOMS DOMAIN OF SGRQ AND SERUM CYSTATIN C (ng/ml) There is a positive correlation between serum Cystatin C level (ng/ml) and symptoms domain of SGRQ score and it is statistically significant DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 87 SYMPTOMS DOMAIN OF SGRQ SERUM CYSTATIN C VALUES r co-efficient 0.747 P value 0.001 GRAPH :18 CORRELATION BETWEEN IMPACT DOMAIN OF SGRQ AND SERUM CYSTATIN C (ng/ml) DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 88 There is a positive correlation between serum Cystatin C level (ng/ml) and impact domain of SGRQ score and it is statistically significant IMPACT DOMAIN OF SGRQ SCORE SERUM CYSTATIN C VALUES r co-efficient 0.741 P value 0.001 GRAPH :19 CORRELATION BETWEEN ACTIVITY DOMAIN OF SGRQ AND SERUM CYSTATIN C (ng/ml) DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 95 In our study CrossELISA 57.95±3. 64.3 Indian 560.1± No NIL section KIT 66 285.5 al In our study Mean serum Cystatin C level is 560.1± 285.5ng/ml which is less and contrast in cases and even in controls when compared to the other studies done by Zhang et al.(2) which was 1.31± 0.30 mg/l, Selda et al.(3) was 0.97 ± 0.29mg/l, Shu-Hong Fu et al.(5) was 115.45µg/l and Yonghongzhnag et al.(77) was 1.09± 0.22mg/l. Hence we performed ROC analysis to find specific cut-off values to determine normal and pathological levels. A serum Cystatin C level of 532.1 ng/L was accepted as the cut-off with the highest sensitivity of 100% and specificity of 79.3% as depicted in the graph6. The receiver operating characteristic (ROC) curve's area under the curve (AUC) is 0.942. Cystatin C concentration of 0.69 mg/ml is selected as the cut-off value in the Selda et al. investigation with a 91percentage sensitivity and 80percentage specificity. The cut-off value findings in this study are lower than those reported in other studies. This might be because of the different assay methods used for measuring serum Cystatin C, study design, ethnicity, age, subjects, (%men), BMI, and Presence of co-morbidities. Cystatin C levels have been studied in kidney and cardiovascular disease and are valuable in assessing renal function and predicting cardiovascular mortality, particularly in the elderly[78,32]. Cystatin C is strongly associated with renal function, so renal dysfunction might affect the concentration of COPD. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 96 Increased serum Smoking, advanced age, male gender, C-reactive protein (CRP), and cardiovascular risk factors such hypertension, poor high-density lipoprotein cholesterol, and an elevated body mass index have all been linked to Cystatin C [32] . Serum Cystatin C levels do seem to be impacted by a number of variables, including increased weight and height, according to Knight et al. According to Rubina Bashir et al.,(54) both males and females in the overweight and obese groups had substantially higher blood Cystatin C levels than those in the control group, indicating a possible connection to BMI. Since all nucleated cells, including adipocytes, are known to generate Cystatin C, overweight and obese people are likely to have greater blood Cystatin C levels. Muntner P et al.(56) and Al-Wakeell JS et al.(55) have also reported. AGE DISTRIBUTION AND ITS ASSOCIATION WITH SERUM CYSTATIN C LEVELS (ng/ml):- The age of the patients being studied ranged from 44 years to 60 years & the mean age was 57.95±3.66. In our study, the mean age 56.8± 4, had serum Cystatin C levels less than or equal to 532.3ng/ml whereas the mean age 59±1, had serum Cystatin C levels more than 532.3 ng/ml. High serum Cystatin C levels were seen as age increases and the association between them was statistically significant ( P <0.05) as depicted in table 4. In other studies, Rokadia et al., (47) Zhang et al., (2) and Selda et al. (3) had a mean age of 52.4, 63 ± 7, and 64.19± 10.6 respectively & which correlated well with our study. In the study done by Shu-Hong fu et al. (5) mean age was 81 .78 ±6.6 which is in contrast to our study. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 97 The findings of the investigations by Shu-Hong Fu et al., (5) Gouping hu et al. (6) and Yonghong zhang et al. (58) showed a positive correlation between age and serum Cystatin C levels. Michelle et al. (50) concluded the mean and variance are greater with each ascending decade of age without risk factors for kidney disease. Cystatin C levels were 46% higher in participants 80 and older compared with those aged <40 years (P<0.001). GENDER DISTRIBUTION IN ASSOCIATION WITH SERUM CYSTATIN C LEVEL ( ng/ml: In our study, out of 42patients, 27 patients were males & 15 were females & the male: female ratio was 1.8:1 as depicted in graph 3. In other population-based studies of COPD like Rokadia et al. (47) male to female prevalence, the ratio was 1.04: 1 concurrence to our study. Studies done by & Zhang et al. (2) and Shu-Hong fu et al. (5) included only men whereas in the study done by Selda et al. (3) ratio was 5:1 which was in contrast to our study. Eric L knight et al. concluded male gender, was independently associated with higher serum Cystatin C levels. Among the 42 patients, the mean Serum Cystatin C levels distribution according to sex was 624±293 ng/ml in male patients and 444±237.5ng/ml in female patients as seen in table 5. Serum Cystatin C levels were notably increased in males and it was statistically significant ( P <0.001). BMI DISTRIBUTION AND ITS ASSOCIATION WITH SERUM CYSTATIN C LEVEL( ng/ml):- DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 98 In our study Mean BMI is 20±3 which is in the normal range, the distribution of patients according to BMI was mentioned in Graph 4. Among the 42 patients,23 patients with a mean BMI of 20.47 had serum Cystatin C levels ≤532.1ng/ml whereas 19 patients with a mean BMI is 20.49Serum Cystatin C levels >532.1ng/ml there was no association between them as seen in table 6. In other studies done by zhang et al., (2) Shu-Hong Fu et al., (5) and yonghong et al. (58) patients were in the normal BMI range (24 ±2). Whereas in the study by Selda et al.(3) patients were in the overweight BMI range (25.86± 7) and there was no association between serum Cystatin C levels and BMI which is concurrence with our study. ASSOCIATION OF PACK YEARS WITH SERUM CYSTATIN C LEVEL (ng/ml) Out of 42 patients, 27 patients were smokers & 15 patients were nonsmokers. Among the 15 non-smokers, 15 were females & 2 were males. In our study, the mean pack years was 12.1 ± 13.1 years. In other studies done by zhang et al.,(2) the mean pack year was 38.8±20.6, in Selda et al.,(3) the mean pack year was 19.06±10 which was higher and in contrast to our study. Among 27 Patients, 7 patients with pack years less than equal to 10 had serum Cystatin C levels <532.1ng/ml levels whereas 20 patients with pack years had serum Cystatin C levels >532.1ng/ml. In our study serum, Cystatin C levels increased as pack years increased. There is a positive association between pack years and serum Cystatin C. patients with more than 10 pack years have odds of 5.8 times increased chance of serum Cystatin C ng/ml levels which was statistically significant. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 99 These increased levels of Cystatin C may be related to pulmonary inflammation caused on by smoking. ASSOCIATION OF SERUM CYSTATIN C LEVEL ( ng/ml) WITH SPIROMETERY: Our study consists of 42 subjects who were divided into four categories depending on their FEV1/FVC ratio & Post-Bronchodilator FEV1 predicted percentage into mild, moderate, severe, and very severe (According to GOLD CRITERIA for classification of COPD) mentioned in the graph 5. Among the 42 patients,19 patients had serum Cystatin C levels above 532.1ng/ml and 23 patients had serum Cystatin C levels less than or equal to 532.1ng/ml. association between them is statistically significant as depicted in table 11. The mean Serum Cystatin C levels in patients with moderate airway limitation was 250.02 ng/ml, 567.60 in patients with severe airflow limitation, and 861.25 ng/ml Serum Cystatin C levels in patients with very severe airflow limitation. Serum Cystatin C was notably increased as airflow limitation (% FEV1 predicted) decreases and the association between them was statistically significant ( P<0.001). The serum concentration of Cystatin C was Negatively correlated with FEV1% predicted (r= -0.802, p=0.0001) as depicted in graph 12. Serum Cystatin C levels can be used as an alternative diagnostic tool in patients with poor or inadequate effort for performing pulmonary function tests for the diagnosis of COPD. In a study done by zhang et al. (2) the mean Serum Cystatin C levels distribution according to airway limitation was 1.10± 0.27 mg/l in the mild patient’s category 1.20± 0.24 mg/l in the moderate patient’s category, 1.43± 0.2 mg/l in severe patients category and1.59 ±0.25 mg/ml DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 100 Serum Cystatin C levels in the very severe patient category which were a contrast to our study and higher than our serum Cystatin C levels. Mean Serum Cystatin C was notably increased as airflow limitation (% FEV1 predicted) decreases and the negatively correlated between them was statistically significant ( P <0.001). In the studies by Zhang et al., (2) and Shu-Hong Fu et al., (5) Serum concentration of serum Cystatin C was negatively correlated with FEV1 predicted ( r= -0.475, p=0.0001). ASSOCIATION OF REDEFINED ABCD TOOL WITH SERUM CYSTATIN C LEVEL (ng/ml) According to the severity of the patients' symptom, H/O exacerbation, and hospitalisation, patients were staged in our study using the Redefined ABCD evaluation method. Out of 42 patients, 3 (7%) of them were in class A, 5(11%) of them were in class B and 34 (81%) of them were in class D as depicted in table 14. Serum Cystatin C levels were less than equal to 532.1 ng/ml in 8 patients belonging to the A and B classes. Among 34 patients in class D, 15 had serum cystatin levels less than or equal to 532.1 ng/ml. These patients had ≥1 exacerbations history whereas 19 patients had serum Cystatin C levels of more than 532.1 ng/ml and had exacerbation history ≥2 during the duration of the disease. Serum Cystatin C levels were increased with Disease severity and in patients with increased no of exacerbations and the association between them was statistically significant. (p<0.05). According to a study by Selda et al., (3) 58 COPD who are stable, 26 patients (38.4%) fell into class B, while 42 patients (42%) fall into class C. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 101 The mean serum Cystatin C level 1.05 ±0.56, 1.33 ±2.30 respectively and (P>0.05). and concluded that serum Cystatin C may be used only to determine and follow up on the severity of airflow limitation which is in contrast to our study. ASSOCIATION OF DURATION OF DISEASE WITH SERUM CYSTATIN C LEVEL (ng/ml):- In our study, the mean duration of illness is 4.9 ± 2.2 years. The distribution of patients according to the Duration of the disease was depicted in graph 8. There is a positive association between serum Cystatin C ng/ml and Duration of disease and patients with a duration of disease more than 4 years have odds of 6.5 times increased chance of serum Cystatin C levels found to be statistically significant as mentioned the table 16. There is a positive correlation between serum Cystatin C level (ng/ml) and Duration of disease and it is statistically significant as depicted in the graph13. ASSOCIATION OF MODIFIED MEDICAL RESEARCH COUNCIL SCALE WITH SERUM CYSTATIN C LEVEL (ng/ml):- Dyspnea is the most frequent symptom reported by patients suffering from COPD, and the Modified medical research scale is the most commonly used validated scale to assess dyspnea in patients. The distribution of patients according to the Modified medical research scale is depicted in table 17. The distribution of patients according to the Modified medical research scale concerning serum Cystatin C levels is depicted in table 18. Serum Cystatin C levels were higher in patients with mMRC score ≥3. There is an association between the Modified medical research scale and serum Cystatin C levels and it is statistically significant p<0.05 (table 18). DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 102 In the study done by Magno,F et al. (80) had mean serum Cystatin C level 550ng/ml with mMRC score 1.7±1.2. ASSOCIATION OFSIX-MINUTE WALK TEST AND SERUM CYSTATIN C LEVEL ( ng/ml):- The 6-min walk test (6MWT) is commonly used to evaluate the functional exercise capacity in COPD patients. The distribution of patients according to the six-minute walk test was depicted in graph 9. There was a positive association between the six-minute walk and serum Cystatin C levels and patients with a 6-minute walk test less than 250m have odds of 13 times increased chance of serum Cystatin C levels it was found to be statistically significant (table 19). There is a negative correlation between serum Cystatin C level (ng/ml) and the six-minute walk test it is statistically significant (r = -0.830, p= 0.001) as depicted in graph 2. ASSOCIATION OF BODE INDEX WITH SERUM CYSTATIN C LEVEL (ng/ml) The BODE index is a multidimensional index that incorporates: the body mass index (BMI), the degree of airflow obstruction assessed by the Forced Expiratory Volume in one second (FEV1), the modified Medical Research Council (mMRC) dyspnea scale, and the exercise capacity assessed by the 6-min walking distance (6MWD) test Bode Index ranged from 0-10 In our study among 42 patients, 26 patients had a BODE index score less than or equal to 6, whereas 16 patients’ BODE index score is more than 6. There is a positive association between BODE index and serum Cystatin C levels and patients with a BODE index of more than 6 have odds of 14 times increased chance of serum Cystatin C levels and it was statistically significant ( table 20). DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 103 Serum Cystatin C level had a statistically significant positive association across all domains of the BODE index except BMI. In the study done by Magno,F et al. (80) had mean serum Cystatin C level 550ng/ml with BODE index 4±2.1. ASSOCIATION OF CRP LEVELS WITH SERUM CYSTATIN C LEVEL (ng/ml) In our study as serum CRP levels increase with increased serum Cystatin C levels and are positively correlated and statically significant. (r=0.554, p,0.001). Due to a significant association of CRP and Cystatin C in our study population, it might be speculated that elevation of Cystatin C occurs secondary to inflammatory processes in the lung. Studies done rokadia et al.,(47) Selda et al.,(3) S-H Fu et al.,(5) and zhang et al., (2)also concluded, a progressive increase in the mean serum CysC level with serially increasing CRP concentrations and statically significant which is similar to our study. ASSOCIATION OF SERUM CYSTATIN C LEVELS WITH QUALITY OF LIFE IN COPD PATIENTS. In chronic diseases, health-related quality of life (HRQL) is an important patient-oriented measurement of the impact of health on well-being. Health-related quality of life was assessed using the St. George's Respiratory Questionnaire (SGRQ) and clinical COPD Questionnaire (CCQ) and CAT score. To our knowledge, this is the first study to evaluate the serum Cystatin C levels’ impact on the quality of life in COPD patients. The SGRQ is a standardized self-administered airways disease-specific questionnaire divided into three subscales: symptoms (eight items), activity (16 items), and impacts (26 items). For DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 104 each subscale and the overall questionnaire, scores range from zero (no impairment) to 100 (maximum impairment). ASSOCIATION OF SERUM CYSTATIN C LEVEL WITH A TOTAL SCORE OF ST. GEORGE QUESTIONNAIRE (I.E SYMPTOMS, ACTIVITY AND IMPACT). Among 42 patients, Distribution according to St. George Respiratory Questionnaire total score according to serum Cystatin C levels was shown in table 23. To our knowledge, this is the first study to evaluate the serum Cystatin C levels’ impact on the Total score of St. George questionnaire (i.e symptoms, activity and impact ). There was a positive association between SGRQ total score and serum Cystatin C ng/ml and patients with St George total score more than 50 have odds of 4.8 times chance of increased levels of serum Cystatin C levels and were found to be statistically significant shown in (table 23). There is a positive correlation between serum Cystatin C level (ng/ml) and SGRQ Total score and it is statistically significant as shown in Graph 16. In the study done by Magno,F et al. (80) had mean serum Cystatin C level was 550ng/ml with St. George total score 45.3±30.6. ASSOCIATION OF SERUM CYSTATIN C LEVEL WITH INDIVIDUAL DOMAIN OF ST. GEORGE QUESTIONNAIRE (I.E SYMPTOMS, ACTIVITY, AND IMPACT). Among 42 patients, the Distribution of patients according to the St. George Respiratory Questionnaire symptom score concerning serum Cystatin C levels was shown in table 24. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 111 10. Patients with a duration of disease of more than 4 years have odds of 6.5 times chance of increased serum Cystatin C levels found to be statistically significant (P<0.05). 11. Serum Cystatin C levels were higher in patients with mMRC score ≥3. There is an association between the Modified medical research scale and serum Cystatin C levels and it is statistically significant (P<0.05). 12. There is a positive association between the six-minute walk and serum Cystatin C levels and patients with a 6-minute walk test less than 250m have odds of 13 times chance of increased serum Cystatin C levels it was found to be statistically significant(P<0.05). 13. There is a positive association between BODE index and serum Cystatin C levels and patients with a BODE index of more than 6 have odds of 14 times chance of increased serum Cystatin C levels and it was statistically significant (p<0.05). 14. Serum Cystatin C level had a statistically significant positive association across all domains of the BODE index except BMI. 15.serum CRP levels increase with increased serum Cystatin C levels and are positively correlated and statically significant. 16. There was a positive association between SGRQ total score and serum Cystatin C ng/ml and patients with St George total score more than 50 have odds of 4.8 times chance of increased levels of serum Cystatin C levels and were found to be statistically significant. (P<0.05). 17. There was a positive association between SGRQ symptom score and serum Cystatin C ng/ml and patients with St George symptom scores more than 50 have odds of 9 times chance of increased levels of serum Cystatin C levels and were found to be statistically significant. (P<0.05). DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 112 18. There is a positive correlation between serum Cystatin C level (ng/ml) and impact domain of SGRQ score and it is statistically significant. (P<0.05). 19. There is an association between the clinical COPD questionnaire and serum Cystatin C levels and patients with Clinical COPD questionnaire scores more than or equal to 3 have odds of 4.7 times chance of increased serum Cystatin C levels and found it was statistically significant (p<0.05) 20. There is a positive association between cat score and serum Cystatin C levels and patients with CAT scores more than 20 have odds of 4.5 times chance of increased serum Cystatin C levels and found it was statistically significant (p <0.05). RECOMMENDATIONS: 1. Serum Cystatin C level does not have clinical management impact, further studies in different population are required as data is available only from China. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 113 2. Quality of life like BODE index, St.George Questionnaire to be used in all patients in every follow up to know the impact on patient. 3. CRP is also a good prognostic test and is easily available. BIBLIOGRAPHY: DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 114 1. Global Initiative for Chronic Obstructive Lung Disease. Global strategy for prevention,diagnosis and management of COPD. https://goldcopd. org/wpcontent/uploads/2018/11/GOLD-2019-v1. 7-FINAL-14Nov2018-WMS. pdf. 2019. 2. Zhang M, Li Y, Yang X, Shan H, Zhang Q, Ming Z, Xie Y, Chen H, Liu Y, Zhang J. Serum Cystatin C as an inflammatory marker in exacerbated and convalescent COPD patients. Inflammation. 2016 Apr;39(2):625-31. 3. Telo S, Kuluöztürk M, Deveci F, Kırkıl G, Öner Ö, Kaman D. Serum Cystatin C levels in COPD: potential diagnostic value and relation between respiratory functions. Journal of medical biochemistry. 2018 Dec;37(4):434. 4. Chai L, Feng W, Zhai C, Shi W, Wang J, Yan X, Wang Q, Zhang Q, Li M. The association between Cystatin C and COPD: a meta-analysis and systematic review. BMC Pulmonary Medicine. 2020 Dec;20(1):1-1. 5. Zhang M, Fu SH, Cui H, Zhu BP, Liu L, Wang DL. Serum Cystatin C and indices of lung function in elderly Chinese men with chronic obstructive pulmonary disease. Aging Clinical and Experimental Research. 2014 Apr 1;26(2):193-9. 6. Hu G, Wu Y, Zhou Y, Yu Y, Liang W, Ran P. Cystatin C as a predictor of in-hospital mortality after exacerbation of COPD. Respiratory care. 2016 Jul 1;61(7):950-7. 7. Laennec RT. A treatise on the diseases of the chest. Laennec RTH, trans. New York Academy of Medicine: the history of medicine series/1, 1962. 8. William MacNee “Chronic Bronchitis and emphysema”. Crofton and Douglass Respiratory Diseases Chapter 616, edited by Anthony Seaton, Douglas Seaton, fifth edition, Blackwell Sciences, Volume – 1, 650. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 115 9. Medical Research Council. 1965. “Definition and classification of chronic bronchitis for clinical and epidemiological purpose”, Lancet, 1: 775 10. Higgins IT. Tobacco smoking, respiratory symptoms, and ventilatory capacity. British medical journal. 1959 Feb 2;1(5118):325. 11. Owen CA, Campbell MA, Sannes PL, Boukedes SS, Campbell EJ. Cell surface-bound elastase and cathepsin G on human neutrophils: a novel, non-oxidative mechanism by which neutrophils focus and preserve catalytic activity of serine proteinases. The Journal of cell biology. 1995 Nov;131(3):775-89. 12. Taha RA, Boushy SF, Thompson Jr HK, North LB, Aboumrad MH. The electrocardiogram in chronic obstructive pulmonary disease. American Review of Respiratory Disease. 1973 Jun;107(6):1067-70. 13. Wig KL, Guleria JS, Bhasin RC, Holmes JR. E, Vasudeva YL, Singh H. Certain clinical and epidemiological aspects of chronic bronchitis as seen in Northern India. Indian J Chest Dis. 1964;6:183-94. 14. Bhattacharya SN. et al., 1975. “Chronic bronchitis in rural population”, Indian J Chest Dis, 1: 17. 15. Pauwels RA. GOLD Scientific Committee. Global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease. NHLBI/WHO Global Initiative for Chronic Obstructive Lung Disease (GOLD) Workshop summary. Resp Crit Care Med. 2001;163:1256-76. 16. Global Initiative for Chronic Obstructive Lung Disease (GOLD) : Global strategy for the diagnosis, management and prevention of COPD. http://goldcopd.org DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 116 17. Ho M, Yap J, Bell D, et al. Chronic obstructive pulmonary disease. Reference article, Radiopaedia.org (Accessed on 27 Dec 2022). 18. Shaker SB, Dirksen A, Bach KS, Mortensen J. Imaging in chronic obstructive pulmonary disease. COPD: Journal of Chronic Obstructive Pulmonary Disease. 2007 Jan 1;4(2):143-61 19. Church DF, Pryor WA. Free-radical chemistry of cigarette smoke and its toxicological implications. Environmental health perspectives. 1985 Dec;64:111-26. 20. Sussan TE, Gajghate S, Thimmulappa RK, Ma J, Kim JH, Sudini K, Consolini N, Cormier SA, Lomnicki S, Hasan F, Pekosz A. Exposure to electronic cigarettes impairs pulmonary anti-bacterial and anti-viral defenses in a mouse model. PloS one. 2015 Feb 4;10(2):e0116861. 21 Garcia-Arcos I, Geraghty P, Baumlin N, Campos M, Dabo AJ, Jundi B, Cummins N, Eden E, Grosche A, Salathe M, Foronjy R. Chronic electronic cigarette exposure in mice induces features of COPD in a nicotine-dependent manner. Thorax. 2016 Dec 1;71(12):1119-29. 22. Reinikovaite V, Rodriguez IE, Karoor V, Rau A, Trinh BB, Deleyiannis FW, TarasevicieneStewart L. The effects of electronic cigarette vapour on the lung: direct comparison to tobacco smoke. European Respiratory Journal. 2018 Apr 1;51(4). 23.Ghosh A, Coakley RD, Ghio AJ, Muhlebach MS, Esther Jr CR, Alexis NE, Tarran R. Chronic ecigarette use increases neutrophil elastase and matrix metalloprotease levels in the lung. American journal of respiratory and critical care medicine. 2019 Dec 1;200(11):1392-401. 24.Bhatta DN, Glantz SA. Association of e-cigarette use with respiratory disease among adults: a longitudinal analysis. American journal of preventive medicine. 2020 Feb 1;58(2):182-90. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 117 25. Simonsen O, Grubb A, Thysell H. The blood serum concentration of Cystatin C (γ-trace) as a measure of the glomerular filtration rate. Scandinavian journal of clinical and laboratory investigation. 1985 Jan 1;45(2):97-101. 26. Fan L, Levey AS, Gudnason V, Eiriksdottir G, Andresdottir MB, Gudmundsdottir H, Indridason OS, Palsson R, Mitchell G, Inker LA. Comparing GFR estimating equations using Cystatin C and creatinine in elderly individuals. Journal of the American Society of Nephrology. 2015 Aug 1;26(8):1982-9. 27Levey AS, Tighiouart H, Simon AL, Inker LA. Comparing newer GFR estimating equations using creatinine and Cystatin C to the CKD-EPI equations in adults. American Journal of Kidney Diseases. 2017 Oct 1;70(4):587-9. 28Rule AD, Teo BW. GFR estimation in Japan and China: what accounts for the difference?. American Journal of Kidney Diseases. 2009 Jun 1;53(6):932-5. 29. Grubb A, Löfberg H. Human gamma-trace, a basic microprotein: amino acid sequence and presence in the adenohypophysis. Proceedings of the National Academy of Sciences. 1982 May 1;79(9):3024-7. 30. Abrahamson M, Olafsson I, Palsdottir A, Ulvsbäck M, Lundwall Å, Jensson O, Grubb A. Structure and expression of the human Cystatin C gene. Biochemical journal. 1990 Jun 1;268(2):287-94. 31. Merz GS, Benedikz E, Schwenk V, Johansen TE, Vogel LK, Rushbrook JI, Wisniewski HM. Human Cystatin C forms an inactive dimer during intracellular trafficking in transfected CHO cells. Journal of cellular physiology. 1997 Dec;173(3):423-32. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 118 32. Köttgen A, Selvin E, Stevens LA, Levey AS, Van Lente F, Coresh J. Serum Cystatin C in the united states: The third national health and nutrition examination survey (NHANES III). American Journal of Kidney Diseases. 2008 Mar 1;51(3):385-94. 33. Knight EL, Verhave JC, Spiegelman D, Hillege HL, De Zeeuw D, Curhan GC, De Jong PE. Factors influencing serum Cystatin C levels other than renal function and the impact on renal function measurement. Kidney international. 2004 Apr 1;65(4):1416-21. 34. Rule AD, Bailey KR, Lieske JC, Peyser PA, Turner ST. Estimating the glomerular filtration rate from serum creatinine is better than from Cystatin C for evaluating risk factors associated with chronic kidney disease. Kidney international. 2013 Jun 1;83(6):1169-76. 35. Stevens LA, Schmid CH, Greene T, Li L, Beck GJ, Joffe MM, Froissart M, Kusek JW, Zhang YL, Coresh J, Levey AS. Factors other than glomerular filtration rate affect serum Cystatin C levels. Kidney international. 2009 Mar 2;75(6):652-60. 36. Liu X, Foster MC, Tighiouart H, Anderson AH, Beck GJ, Contreras G, Coresh J, Eckfeldt JH, Feldman HI, Greene T, Hamm LL. Non-GFR determinants of low-molecular-weight serum protein filtration markers in CKD. American journal of kidney diseases. 2016 Dec 1;68(6):892-900. 37. Foster MC, Levey AS, Inker LA, Shafi T, Fan L, Gudnason V, Katz R, Mitchell GF, Okparavero A, Palsson R, Post WS. Non-GFR determinants of low-molecular-weight serum protein filtration markers in the elderly: AGES-Kidney and MESA-Kidney. American Journal of Kidney Diseases. 2017 Sep 1;70(3):406-14. 38. Conti M, Moutereau S, Zater M, Lallali K, Durrbach A, Manivet P, Eschwege P, Loric S. Urinary Cystatin C as a specific marker of tubular dysfunction. Clinical Chemistry and Laboratory Medicine (CCLM). 2006 Mar 1;44(3):288-91. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 119 39. chaefer L, Gilge U, Heidland A, Schaefer RM. Urinary excretion of cathepsin B and cystatins as parameters of tubular damage. Kidney International Supplement. 1994 Nov 2(47). 40..Donadio C. Serum and urinary markers of early impairment of GFR in chronic kidney disease patients: diagnostic accuracy of urinary β-trace protein. American Journal of Physiology-Renal Physiology. 2010 Dec;299(6):F1407-23. 41. Bökenkamp A, Ciarimboli G, Dieterich C. Cystatin C in a rat model of end-stage renal failure. Renal Failure. 2001 Jan 1;23(3-4):431-8. 42. Hossain MA, Emara M, Shoker A. Comparing measures of Cystatin C in human sera by three methods. American journal of nephrology. 2009;29(5):381-91. 43..Kyhse-Andersen J, Schmidt C, Nordin G, Andersson B, Nilsson-Ehle P, Lindström V, Grubb A. Serum Cystatin C, determined by a rapid, automated particle-enhanced turbidimetric method, is a better marker than serum creatinine for glomerular filtration rate. Clinical chemistry. 1994 Oct 1;40(10):1921-6. 44. 3. Chapman HA Jr, Reilly JJ, Yee R, Grubb A. Identification of Cystatin C, a cysteine proteinase inhibitor, as a secretory product of human alveolar macrophages in vitro. Am Rev Respir Dis 1990; 141: 698–705. 45. Lee YT, Chen SC, Shyu LY, Lee MC, Wu TC, Tsao SM, et al. Significant elevation of plasma cathepsin B and Cystatin C in patients with community-acquired pneumonia. Clin Chim Acta 2012; 413(5–6): 630–5. 46. Takeyabu K, Betsuyaku T, Nishimura M, Yoshioka A, Tanino M, Miyamoto K, et al. Cysteine proteinases and Cystatin C in bronchoalveolar lavage fluid from subjects with subclinical emphysema. Eur Respir J 1998; 12(5): 1033–9. 15. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 120 47. Rokadia HK, Agarwal S. Serum Cystatin C and emphysema: results from the National Health and Nutrition Examin ation Survey (NHANES). Lung 2012; 190(3): 283–90. 48. Warfel AH, Cardozo C, Zucker OH, Franklin DJ. Cystatin C and cathepsin B production by alveolar macrophages from smokers and nonsmokers. J Leukoc Biol 1991; 49: 41–7. 49. Balta S, Demirkol S, Ay SA, Cakar M, Sarlak H, Celik T. Serum cystatin-C levels correlate with endothelial dysfunction in patients with the metabolic syndrome. J Intern Med 2013; 274(2): 200–1. 50. Odden MC, Tager IB, Gansevoort RT, Bakker SJ, Katz R, Fried LF, Newman AB, Canada RB, Harris T, Sarnak MJ, Siscovick D. Age and Cystatin C in healthy adults: a collaborative study. Nephrology Dialysis Transplantation. 2010 Feb 1;25(2):463-9. 51. Macdonald J, Marcora S, Jibani M, Roberts G, Kumwenda M, Glover R, Barron J, Lemmey A. GFR estimation using Cystatin C is not independent of body composition. American journal of kidney diseases. 2006 Nov 1;48(5):712-9. 52. Fricker M, Wiesli P, Brändle M, Schwegler B, Schmid C. Impact of thyroid dysfunction on serum Cystatin C. Kidney international. 2003 May 1;63(5):1944-7. 53. Knight EL, Verhave JC, Spiegelman D, Hillege HL, De Zeeuw D, Curhan GC, De Jong PE. Factors influencing serum Cystatin C levels other than renal function and the impact on renal function measurement. Kidney international. 2004 Apr 1;65(4):1416-21. 54. BASHIR R, IMTIAZ S, YASIR M. Effect of Body Mass Index on Serum Cystatin C Level in Healthy. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 127 I have been explained the reason for conducting this study and selecting me/my ward as a subject for this study. I have also been given a free choice for either being included or not in the study. PROCEDURE: I understand that I will undergo a detailed history and clinical examination and investigations. RISKS AND DISCOMFORTS: I understand that I/my ward may experience hypersensitivity or anaphylaxis while doing the procedure. I understand that necessary measures will be taken to reduce these complications as and when they arise. BENEFITS: I understand that I/my ward's participation in this study will help find out a clinical study of serum Cystatin C levels in cases of copd and its correlation with spirometry and impact on their quality of life. CONFIDENTIALITY: I understand that this study's medical information will become a part of this hospital records and will be subjected to the confidentiality and privacy regulation of this hospital. Information of a sensitive, personal nature will not be a part of the medical records but will be stored in the investigator's research file and identified only by a code number. The code key connecting the name to numbers will be kept in a separate secure location. If the data are used for publication in the medical literature or for teaching purposes, no names will be used, and other identifiers such as photographs and audio or videotapes will be used only with my special written permission. I understand that I may see the photograph and videotapes and hear audiotapes before giving this permission. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 128 REQUEST FOR MORE INFORMATION: I understand that I may request more questions about the study at any time. Dr.RAVI APOORVA is available to answer my questions or concerns. I understand that I will be informed of any significant new findings discovered during this study, which might influence my continued participation. If during this study, or later, I wish to discuss my participation in or concerns regarding this study with a person not directly involved, I am aware that the social worker of the hospital is available to talk with me. And that a copy of this consent form will be given to me to keep for careful reading. REFUSAL OR WITHDRAWAL OF PARTICIPATION: I understand that my participation is voluntary, and I may refuse to participate or may withdraw consent and discontinue participation in the study at any time without prejudice to my present or future care at this hospital. I also understand that Dr. RAVI APOORVA will terminate my participation in this study at any time after she has explained the reasons for doing so and has helped arrange for my continued care by my physician or therapist if this is appropriate. INJURY STATEMENT: I understand that in the unlikely event of injury to me/my ward, resulting directly in my participation in this study, if such injury were reported promptly, medical treatment would be available to me, but no further compensation will be provided. I understand that I am not waiving any of my legal rights by my agreement to participate in this study. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 129 STUDY SUBJECT CONSENT STATEMENT: I confirm that Dr. RAVI APOORVA has explained the purpose of this research, the study procedure that I will undergo, and the possible discomforts and benefits that I may experience in my own language. I have been explained all the above in detail in my own language, and I understand the same. Therefore I agree to give my consent to participate as a subject in this research project. (Participant) Date (Witness to above signature) Date I have explained to , the purpose of this research, the procedures required and the possible risks and benefits, to the best of my ability in patient’s own language. DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 130 Date: Dr. RAMESH. S. BABAR Dr. RAVI APOORVA (Guide) (Investigator) DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 131 ANNEXURE III PROFORMA Name of the patient: Age: Address: Sex: IP no/OP no: Occupation: Presenting Complaints: History of Present Illness: Past history: Personal history: 1. DietVeg/Mixed: 2. Appetite: 3. Sleep: 4. Bowel and bladder habits: Family history: Menstrual History: GENERAL PHYSICAL EXAMINATION: DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 132 Conscious/ oriented/ co-operative: Built: Nourishment: Ht (cm): Wt (kg): BMI: Pallor Icterus Clubbing Cyanosis Lymphadenopathy Edema 6. Vital parameters: a. Temperature: b. Pulse: c. Respiratory rate: d. BP: e. SpO2: SYSTEMIC EXAMINATION: RESPIRATORY SYSTEM ABDOMEN EXAMINATION CARDIOVASCULAR SYSTEM CENTRAL NERVOUS SYSTEM INVESTIGATIONS: DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 133 Complete blood count: Total Count Neutrophils % Lymphocytes % Monocytes % Eosinophils % Basophils % Hemoglobin (gm/dl) Platelet count (per cu.mm) Serum creatinine Urea Serum Cystatin C level: Chest X-ray: ECG: Pulmonary Function Test: FINAL DIAGNOSIS: DATE SIGNATURE DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 134 ANNEXURE IV MASTERCHART DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 135 SGR Q S.NO NAME AGE SEX BMI SMOKING STATUS POST BRONCHODILATOR FEV1/FVC POSTBRONCHODILATOR FEV1% PREDICTED CRP SERUM CYSTATIN C VALUES 6MWT TOTALSCORE SYMPTOMS ACTIVITY IMPACT MMRC CAT SCORE CCQ SCORE ABCD TOOL DURATION OF DISEASE BODE INDEX pack years 1 SHEELA 55 FEMALE 19 ABSENT 69.4 51 10 188 340 26.28 32.41 44.8 13.14 2 16 1.5 B 0.5 4 0 2 SANGABASAPPA.KUMBAR 60 MALE 23.2 PRESENT 62.4 67 28 268.93 374 43.15 43.27 59.34 33.48 2 24 2.9 D 4 2 5 3 MAHADEVAPPA.MANDUR 60 MALE 21 PRESENT 68.3 60 28.3 229.78 384 38.71 51.17 40.65 33.38 3 24 2.4 B 5 4 4 4 SIDAMMA 52 FEMALE 22.2 ABSENT 57.2 63 21.2 309.71 352 56.85 52.6 67.63 51.64 3 29 2.7 D 5 3 0 5 SHANKAR 60 MALE 18.2 PRESENT 56.3 66 27 249.28 400 48.92 43.71 75.79 34.76 3 26 2.4 D 2 5 8 6 SOMNATHSOMNAL 60 MALE 18.7 PRESENT 67.2 56 15.5 276.43 330 67.55 63.16 75.79 64.15 3 28 3.3 D 4 4 7 7 MALLAPPA SAJJAN 60 MALE 18.4 PRESENT 45.8 31 22 444.07 220 61.4 76.62 74.91 48.82 3 23 2.4 D 8 8 12 8 LAMANI PARSU 60 MALE 21.1 PRESENT 59 70 5 172 440 9.83 16.73 22.48 0 1 4 0.9 A 0.5 1 3 9 ROKAMMA 60 FEMALE 20.7 ABSENT 49.7 55 22 362.75 320 62.85 71.62 68.6 56.43 3 26 2.5 D 7 6 0 10 BABANGOUDA 60 MALE 22.1 PRESENT 49.8 26 12 631.32 200 67.69 51.17 75.79 68.58 3 30 3.2 D 8 8 15 11 VAJABAI 60 FEMALE 20.1 ABSENT 64.4 53 21 234.3 340 25.49 23.83 44.87 14.53 2 18 1.8 B 3 3 0 12 RAMAPPA 60 MALE 18.1 PRESENT 46.5 29 12 729.84 200 89.54 92.41 84.03 91.84 3 22 2.3 D 6 8 24 13 RAMANGOUDA 60 MALE 24.2 PRESENT 62.9 39 44 692.46 220 59.82 67.13 60.44 56.94 3 25 3.3 D 6 6 18 14 MAHADEVAPPA.B 58 MALE 18 PRESENT 38.1 32 33 447.9 200 70.25 50 60.44 83.01 3 29 3.1 D 4 7 13 15 SHAKUNTALA 58 FEMALE 26 ABSENT 70.3 67 5 178 410 16.63 16.73 29.84 8.75 1 11 1.6 A 1 0 0 16 YALLAWAA 57 FEMALE 20.4 ABSENT 70.2 67 10 197.8 360 17.74 10.3 29.77 13.14 2 10 1.3 A 0.5 2 0 17 HARSING MADHU 50 MALE 15.6 ABSENT 56.3 40 20 456.95 260 47.7 60.4 52.16 39.57 3 22 2.8 D 5 6 16 18 DANAPPA NADASHETTI 60 MALE 17.2 PRESENT 51.8 47 25 410.5 300 41.29 32.41 52.16 37.88 3 22 2.4 D 6 6 12 19 MIRABAI 58 FEMALE 17.9 ABSENT 67.7 45 43 418.72 300 49.19 41.65 67.63 40.82 3 28 2.7 D 7 6 0 20 RANJANA 44 FEMALE 26.5 ABSENT 61.1 38 36 519.18 260 55.51 52.07 44.87 63.01 3 24 2.4 D 3 5 0 21 BASAPPA BIRADAR 60 MALE 18.4 PRESENT 53.2 44 20 520.07 280 64.71 60.4 67.63 64.45 3 30 2.8 D 5 6 18 22 DEVAKKI 55 FEMALE 25.8 ABSENT 56.8 28 36 609.71 180 78.38 67.13 75.79 83.77 3 32 3.3 D 7 7 0 23 KALYANSHETTI 60 MALE 21.8 PRESENT 53.4 60 12 295.49 390 25.15 25.98 44.87 13.14 2 19 1.8 B 2 2 8 24 BASAMMA 58 FEMALE 22.2 ABSENT 57.9 28 22 544.55 160 71.73 67.13 75.79 70.85 3 23 3.2 D 6 7 0 25 PADMAVATHI 50 FEMALE 18.1 ABSENT 65.7 32 17 480.15 240 57.65 60.95 51.7 60.05 3 26 3.6 D 5 8 0 26 SIDANNAGOUDA 60 MALE 29.3 PRESENT 57.3 36 18 552.46 300 43.55 52.74 53.14 34.69 3 24 2 D 4 5 22 27 SHIVASHARANAE 57 MALE 25.2 PRESENT 49.2 36 11.4 487.64 310 51.35 56.2 59.34 44.94 3 17 2.6 B 3 5 12 28 MALAPPA MUREGAPPA 60 MALE 14.9 PRESENT 44.4 29 301 974.43 150 84.17 97.05 75.79 84.74 3 29 3.6 D 8 8 25 29 SIDDARAY GOUDA BIRADAR 60 MALE 29.7 PRESENT 52.3 28 45 640.46 140 50.63 55.81 67.63 38.75 3 22 2.7 D 5 8 8 30 MALLAMMA 50 FEMALE 24.6 ABSENT 58.2 39 32 503.47 200 44.27 60.95 67.63 24.67 3 22 2.5 D 3 6 0 31 SAVITRI 59 FEMALE 19 ABSENT 41 50 12 287.81 325 58.6 45.11 60.44 62.02 3 20 2.1 D 8 5 0 32 SHIVAPPA 60 MALE 23.1 ABSENT 54 47 18 561.29 300 42.3 58.8 60.33 25.91 3 20 2.6 D 4 5 16 33 SANGAMMA MANNUR 60 FEMALE 13.8 ABSENT 50.5 26 37 965.6 170 81.93 67.13 84.03 85.98 3 23 3.1 D 6 8 0 34 SANGANGOUDA 56 MALE 19.5 PRESENT 41.3 21 107 1054.5 130 84.83 94.37 73.63 87.84 3 33 3.6 D 9 9 45 35 MALDEGARI 60 MALE 14.9 PRESENT 44.4 29 103 1053 150 84.17 97.05 75.79 84.74 3 29 3.6 D 8 8 40 36 CHANDRASHA 60 MALE 18.1 PRESENT 65.3 37 184 776.06 260 66.74 45.34 67.63 73.54 3 21 2.9 D 7 6 20 37 GOURABAI 60 FEMALE 21.4 ABSENT 68.3 38 43 867.63 270 49.5 60.4 52.6 44.19 3 21 2.9 D 5 5 0 38 MALLAPPA . IRAKAR 60 MALE 20.14 PRESENT 36.1 19 88 1058.3 140 85.9 97.05 91.58 78.75 4 27 4.2 D 6 10 40 39 ISHWARAPPA 60 MALE 15 PRESENT 33.6 26 25 872.34 200 64.48 62.38 67.63 63.33 3 20 3.2 D 5 8 24 40 MARTHANDARAO 58 MALE 21 PRESENT 52 49 180 943.19 270 58.72 62.38 60.44 56.45 3 25 3.2 D 5 6 25 41 SHARNAPPA KALAPPA 59 MALE 19.8 PRESENT 40.8 19 39 1009.2 150 73.42 49.63 75.79 80.15 3 25 2.9 D 6 8 34 42 SANGAPPA GURUBASAPPA 60 MALE 16 PRESENT 56.3 29 22 1053 160 89.32 92.41 82.76 91.84 3 29 4.6 D 7 8 36 DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67 DocuSign Envelope ID: BE53C82B-5CF4-4F80-AC92-52FE289E929A 136 ANNEXURE V PLAGIARISM REPORT DocuSign Envelope ID: 28B1C4CD-1140-4FEF-8700-B9E568F51E67