RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES, KARNATAKA BANGALORE 41
PROFORMA FOR REGISTRATION OF SUBJECT FOR DISSERATION.
NAME OF THE CANDIDATE KASHINATH N.
POST GRADUATE (M. Sc. M.L.T.) DEPARTMENT OF BIOCHEMISTRY, ST. JOHN’S MEDICAL COLLEGE AND ADDRESS HOSPITAL, SARJAPUR ROAD, BANGALORE – 560034
ST. JOHN’S MEDICAL COLLEGE, NAME OF THE INSTITTUTE BANGALORE.
COURSE OF STUDY AND SUBJECT M. Sc. M.L.T. (BIOCHEMISTRY)
DATE OF ADMISSION TO THE COURSE SEPTEMBER 1ST 2009
ASSOCIATION OF C-REACTIVE PROTEIN WITH CORONARY HEART DISEASE ALONG WITH OTHER BIOMARKERS TITLE SUCH AS FIBRINOGEN, ALBUMIN AND TOTAL LEUKOCYTE COUNT. NEED FOR THE STUDY:
Various epidemiological studies have reported association between coronary heart disease and blood levels of C-reactive protein ,fibrinogen ,albumin ,total leukocyte count 1. The studies reported by John Danesh et.al. indicates moderate but highly statistically significant association of these factors with coronary heart disease .1
Most of the published studies related coronary heart disease risk only to measurements of these factors made at baseline, even though levels of each factor fluctuate markedly within individuals over time. There are not much data available on particular study from India .So this study will be done to determine the nature of associations between coronary heart disease and C-reactive protein, fibrinogen, albumin and total leukocyte count
REVIEW OF LITERATURE:
Coronary Heart Disease:
Coronary heart disease refers to the failure of coronary circulation to cardiac muscle and surrounding tissue. It is the most common form of disease affecting heart and important cause of premature death in western countries2.
Coronary disease can be due to coronary vasospasm3.
Classification of coronary heart disease
It may be of various types:
Anuerysm
Angina
Atherosclerosis
Cerebrovascular accident
Cerebrovascular disease
Congestive heart failure Coronary artery disease
Myocardial Infarction
Peripheral Vascular disease
C reactive protein:
C reactive protein is an acute phase protein, the serum or plasma levels of which rise during a general, unspecific response to infections and non infectious inflammatory process such as rheumatoid arthritis, cardiovascular disease and peripheral vascular disease. It is synthesized in liver and present as trace constituent of serum or plasma. C reactive protein consists of 5 identical non glycocelated polypeptide subunits, non covalently linked to form a disc shaped cylindrical with a molecular weight of approximate 115kDa. It is important in the non specific host defense against inflammation, especially infection. The levels may rise up to 2000 folds after myocardial infarction, stress, infection, inflammation or surgery. Levels are much higher in bacterial infection than viral infection.4
Determination of CRP is clinically useful for screening the activity of an inflammatory disease such as rheumatoid arthritis, for detecting infection in systematic lupus erythmatous, in leukemia or after surgery. Epidemiologic studies have demonstrated that increased serum CRP concentration are positively associated with risk of future coronary events, such as coronary artery disease, cerebrovascular disease, peripheral arterial disease and levels may be combined with cholesterol markers in estimating risk. 1. When using CRP to assess risk of cardiovascular disease, measurement should be compared with previous values. Measurement of CRP by high sensitivity assays may add to the predictive value of other marker used to assess risk of cardiovascular disease.5-10.
Albumin:
Albumin is the protein of the highest concentration in plasma. Albumin is formed exclusively in the liver and serves as a transport and binding protein for calcium fatty acids, bilirubin, hormones, vitamins, trace elements and drugs. It is also of prime importance in maintaining the colloidal osmotic pressure in both the vascular and extra-vascular spaces.11. Decreased serum albumin concentration can result from liver disease. It can also result from Kidney disease, which allows albumin to escape into the urine. Decrease serum albumin may also be explained by malnutrition or low protein diet. Lower serum albumin concentration, even within the normal range (>38g/l), are associated with morbidity and mortality. Lower concentration has been associated with higher risk of myocardial infarction, coronary heart disease12.
Fibrinogen:
Fibrinogen is a fibrous protein involved in the clotting blood and is non globular. Fibrinogen is a soluble plasma glycoprotein that is synthesized by liver. Fibrinogen level can be measured in venous blood. Normal levels are about 1.5 to 3.0g/ltr, depending on the method which is used. Highest levels are amongst others associated with cardiovascular disease. (> 4.6grm/ltr) may be elevated in any form of inflammation and acute phase protein13.Results of epidemiological studies have indicated that fibrinogen is an important primary cardiovascular risk factor14. CHD patients were reported to have higher fibrinogen levels than healthy individuals14.
Total Leukocyte Count:
Leukocytes are cells of the immune system defending the body against both infectious disease and foreign material. The number of WBC in blood is often an indicator of disease. There are normally between 4 X 109 and 1.1 X 1010 by blood white blood cells in a liter of blood making up approximately 1% of blood in a healthy adult. Epidemiological evidence is accumulating that a moderately increased WBC count as a risk factor CHD. Although observed effect for the most part has been accreted to smoking. 15 Biological mechanism for the association has also been described15.In general there is much current debate regarding the importance of conventional risk factors of CHD in the elderly likewise it can be questioned whether WBC continues to predict CHD with advancing age16.
OBJECTIEVES:
1. To estimate blood levels of C-reactive protein, fibrinogen, albumin and total leukocyte count in patient with coronary heart disease.
2. To correlate the level C-reactive protein, fibrinogen, albumin and total leukocyte Count with coronary heart disease. DESIGN OF STUDY:
Source of data:
It is a prospective study of sample size 30. Samples to be collected from the patients attending the Cardiac OPD or Inpatient department of St. John’s Medical College and Hospital with CHD, according to criteria of Cardiologist.
Inclusion criteria:
Patient with coronary heart disease between the age of 18-60 years.
30 normal individuals without history of coronary heart disease.
Exclusion Criteria:
Patient with inflammatory diseases such as rheumatic fever, Kawasaki disease, renal failure, rheumatoid arthritis, SLE and sepsis.
Method:
Sample Collection
For C-reactive protein, serum sample is collected by venipuncture. 20,21
For albumin, normal procedure for collecting and storing serum is used.20,21
For fibrinogen, plasma anti coagulated with 3.12% sodium citrate is used. A ratio of 9 parts blood to 1 part anticoagulant is used.20,21
For leukocyte estimation, EDTA sample is used.20,21
C-Reactive Protein:
The C reactive protein Extended range (RCRP) method is used on the dimension clinical chemistry system.
Principle
The RCRP method is based on particle enhanced turbidometric immunoassay (PETIA) technique. Synthetic particle coated with antibody to CRP (ABPR) aggregate in presence CRP in the sample. The increase turbidity which accompanies aggregation is proportional to the CRP concentration.
CRP + ABPR------ aggregate absorbs at 340 nm
Albumin Estimation:
The albumin method used on the dimension (R) clinical chemistry system is an invitro diagnostic test intended for quantitative determination of albumin in human serum and plasma. The albumin method is an adaptation of bromocresol purple dye-binding method reported by Carter and Louderback.17
Principle:
In the presence of the solubilizing agent bromocresol purple binds to albumin at pH 4.9. The amount of albumin BCP complex is directly proportional to the albumin concentration. The complex absorbs at 600 nm and is measured using polychromatic end point technique.
Albumin + BCP dye----pH4.9------ albumin –BCP complex
Leukocyte count:
Non –cyanide hemoglobin method is used
Principle:
Blood sample is aspirated measured to a pre determined volume, detected at the specific ratio, than fed into each transducer. The transducer chamber has minute hole called aperture, on both side of which there are electrodes between which flows direct current. Blood cells suspended in the diluted sample pass through this aperture causing direct current resistant to changes between the electrodes. As direct current resistant changes blood cell sings is detected as electric pulses. Blood Cell count in calculated by using counting pulses and histogram of blood cell signs is plotted by determining the pulse sign.
Reagent and equipments: Automated cell counter sysmex kx/2 Fibrinogen:
Principle:
It is based on method described by Clauss18, 19. The Kit uses excess of thrombin to convert fibrinogen to fibrin in diluted sample. A high thrombin and low fibrinogen concentration the rate of reaction is function of fibrinogen concentration. This fibrinogen test based on clot monitoring method.
Validity of Test Report:
Correlated with clinical detail of PT APTT and TT.19
Reference Range:
220 -496 mg/dl.19
Reportable Range:
< 10 mg/dl -1000 mg/dl.19
Results and Analysis:
The quantitative data will be represented in the form of mean and standard deviation. The correlation between the biochemical parameters and coronary heart disease will be done by regression analysis. P value < 0.05 will be considered significant. REFERENCES:
1. John danesh, MBChB, MSC; Rory Collins, MBBS, MSc, Paul Appleby, MSc; Richard Peto, FRS, Association of fibrinogen, c- reactive protein, albumin or leukocyte count with coronary heart disease. Meta analysis of prospective studies
2. Boon NA, Colledge NR, Walker BR,Hunter JAA(2006) Davidson and principles and practice of medicine, 20 th edition churchil
3. William MJ ,Low CJ, (FEB 2008), Myocardial infarction in young people with normal coronary artery, heart 79(2) 191-4 AMID-9538315
4. Burtis CA,Ashwood ER,Bruns DE.Tietz textbook of clinical chemistry and molecular biology.
5. Hind CRK, Pepys MB. The role of serum C-reactive protein (PCR) measurement in clinical practice. Int med 1984; 5:112-151
6. Van Leeuwen M, van Rijswijk MH, acute phase proteins in monitoring of inflammatory disorders. Bailliere’s . clinical rheumatology 1994;8:531-552
7. Ridker PM, Cushman M, Stampfer MJ, Tracy RP, Hennekens CH. Plasma concentration of C – Reactive protein and risk of developing peripheral vascular disease. Circulation 1998; 97:425-428.
8. Ridker PM, Burring JE, Hennekens CH. Prospective study of C-reactive protein and risk of future cardiovascular events among apparently healthy women. Circulation 1998;98:731-733
9. Rifai N,Ridker PM ; Proposed cardiovascular risk assessment algorithm using high sensitivity c reactive protein and lipid screening.
10. Tracy RP, Lemaitre RN, Psaty BM, Ives DG, Evans RW, Cushman M, Meilahn EN, Kuller LH. Relationship of C-reactive protein to risk of cardiovascular disease in elderly: results from the cardiovascular health study and rural health promotion project. Arterioscler Thromb Vasc Biol. 1997; 17:1121-1127. 11. Burtis CA,Ashwood ER,Bruns DE.Tietz textbook of clinical chemistry and molecular biology,fourth edition,Elsevier Saunders,St.louis,MO 2006;547-548
12. Schalk BWM, Visser M, Bremmer MA, Penninx BMJH, Bouter LM, Deeg DJH. Change of serum albumin and risk of cardiovascular disease and all-cause moratality. American Journal of Epidemiology 2006; 164(10):969-977
13. Fibrin. [Online]. 2009 Nov 13; Available from:URL: http://en.wikipedia.org/.
14. Lee AJ, Lowe GDO, Woodward M, Tunstall-Pedoe H. fibrinogen in relation to personal history of prevalent hypertension diabetes stroke, intermittent claudication, coronary heart disease, and family history: the Scottish Heart Health Study. Br Heart J 1993;69:338-342.
15. White blood cell. [Online]. 2009 Nov 11; Available from: URL:http://en.wikipedia.org/
16. H.Toss,MD,PhD;B.Lindahl MD,PhD;A.Siegbahn, MD,PhD;L.Wallentin, MD,PhD;Prognostic influence of increased fibrinogen and C-reactive protein levels in unstable coronary artery disease.
17. Carter p. ultramicroestimation of human serum albumin; binding cationic dye 5,5`dibromo-o cresolsolfonaphthalein,Microchem J 1970;15:531-539.
18. ACL ELITE/ ELITE PRO OPERATORS MANUAL
19. Mitchell Lewis.S BSc MD FRCPath DCP FIBMS Dr , Barbara Bain.J FRACP FRCPath Professor , Imelda Bates MD FRCP FRCPath Dr; Dacie and Lewis Practical haematology.
20. Clinical and laboratory standards institute/NCCLS. Procedure for the collection of diagnostic blood specimen by venipuncture Approved Standard-fifth edition .
21. Clinical and laboratory standards institute/NCCLS. Procedure for the handling and processing of blood specimen; Approved guidelines-third edition. SIGNATURE
NAME AND DESIGNATION OF GUIDE DR.VINOD GEORGE THYKADAVIL ASSOCIATE PROFFESOR, DEPARTMENT OF BIOCHEMISTRY, ST.JOHN`S MEDICAL COLLEGE, BANGALORE-34.
REMARKS OF THE GUIDE
SIGNATURE OF GUIDE
NAME AND DESIGNATION OF DR.SHRILAKSHMI ASSISTANT PROFFESOR CO-GUIDE DEPARTMENT OF CARDIOLOGY, ST.JOHN MEDICAL COLLEGE BANGALORE-34.
SIGNATURE OF CO-GUIDE
HEAD OF THE DEPARTMENT DR.SULTANA FURRUQH, PROFFESOR AND HOD, DEPARTMENT OF BIOCHEMISTRY, ST.JOHN`S MEDICAL COLLEGE BANGALORE-34.
REMARKS
SIGNATURE OF THE HOD
SIGNATURE OF DEAN