Association of Coronary Artery Disease With Lewis and ABO Blood Group Phenotypes- A Case Control Study
Conditions: Coronary Artery Disease
Sponsor: Jubilee Mission Medical College and Research Institute

 
1 
 Association of coronary artery disease with 
Lewis and ABO blood group phenotypes- A case 
control study   
 
Investigator 
Dr. Sree Raj.V 
Junior resident 
Department of Immuno Hematology & Transfusion Medicine 
Jubilee Mission Medical College & Research Institute, Thrissur 
 
 
Guide 
Dr. Susheela Jacob Innah 
Professor and Head 
Department of Immuno Hematology & Transfusion Medicine. 
Jubilee Mission Medical College & Research Institute, Thrissur 
 
 
Co-guide 
Dr. Bino Benjamin 
Associate Professor 
Department of Cardiology 
Jubilee Mission Medical College & Research Institute, Thrissur 
 
 
 
 
 
COMMUNICATION ADDRESS 
Sreeragam 
Mulankunnathukavu.P.O. 
Thrissur. 680581 
:9495018785 
Email id: sreerajvijay@gmail.com 
 
 

 
2 
From, 
Dr. Sree Raj.V 
Junior Resident 
Department of Immunohematology & Transfusion Medicine 
Jubilee Mission Medical College & Research Institute 
Thrissur 
 
To, 
The Chair Person  
Institutional Ethics Committee 
Jubilee Mission Medical College & Research Institute 
Thrissur 
 
Sir, 
Sub: Application for institutional ethics committee approval: reg 
 
I am enclosing herewith the detailed protocol of my research work entitled 
“Association of coronary artery disease with Lewis and ABO blood group phenotypes- A 
case control study” for the favour of approval from Institutional Ethics Committee. 
                                                                                           
Yours faithfully  
 
 
Place :Thrissur 
 
 
 
 
 
 
Date :14/08/2019  
 
 
 
 
 
 
Dr. Sree Raj.V 
 
 

 
3 
APPLICATION FOR ETHICS COMMITTEE APPROVAL 
1. Title of the research 
project 
: Association of coronary artery disease with Lewis 
and ABO blood group phenotypes- A case control 
study  
 
 
2. Name, Designation, 
Qualification & Experience 
of the principal investigator 
: Dr. Sree Raj.V,  
M.B.B.S 
Junior Resident 
Department of Immunohematology & 
Transfusion Medicine 
Jubilee Mission Medical College & Research 
Institute, Thrissur 
 
 
 
3. Name, Designation, 
Qualification & Experience 
of the guide 
: Dr. Susheela Jacob Innah 
MBBS, MD (Pathology), DCP 
Professor and Head 
Department of Immuno Hematology & 
Transfusion Medicine. 
Jubilee Mission Medical College & Research 
Institute, Thrissur 
20yrs 
 
 
 
4. Name, Designation, 
Qualification & Experience 
of the co-guide 
: Dr. Bino Benjamin 
MBBS, MD, DM (Cardiology) 
Associate Professor 
Department of Cardiology 
Jubilee Mission Medical College & Research 
Institute, Thrissur 
12 yrs  
 
 
 
5. Name & address of the 
funding agency  
: Jubilee Mission Medical College & Research 
Institute, Thrissur 
 
 
 
6. Duration of the scheme 
: One and a half year 
 
 
Place : Thrissur 
Date : 14/08/2019 
 

 
4 
ETHICS COMMITTEE CONCERNS 
1. Are the risks to the subjects minimised by using procedures which are consistent with 
sound research design and which do not unnecessarily expose the subjects to the risk 
and whenever appropriate, by procedures already being performed on the subjects for 
diagnostic purpose? 
- 
N/A 
 
2. Are the risk to the subjects reasonable in relation to  
a) The benefits that might be reasonably be expected as an outcome to the 
subjects/others.  
 
- N/A 
 
b) Provision for free treatment for research related injury. 
 
-N/A 
 
c) Compensation of subjects for disability or death resulting from such injury 
 -N/A 
 
d) The importance of the knowledge that may reasonably be expected to result from 
the research project 
 - N/A 
 
3. Will informed consent be sought from each positive subject /legally authorised 
representative? 
- 
YES 
 
 
4. Does the research plan make adequate provisions for confidentiality and monitoring the 
data collected to ensure the safety of subjects? 
- 
YES 
 
 
 
 

 
5 
DECLARATION OF THE PRINCIPAL INVESTIGATOR 
 
I hereby agree to abide by the rules and regulations of the institutional ethics 
committee and those of Jubilee Mission Medical College & Research Institute, Thrissur. I 
will not modify any aspect of the research project without prior approval from the ethics 
committee. 
 
 
 
Place : Thrissur 
 
 
Signature : 
Date  : 14/08/2019  
 
  
 
Dr. Sree Raj.V 
Junior Resident 
Department of Transfusion Medicine 
J.M.M.C & R.I, Thrissur 
 
 
 
 
 
 
 
 
 
 
 
 

 
6 
DECLARATION OF GUIDE 
 
I hereby agree to guide the principal investigator throughout the period of research 
as per the protocol submitted for approval. I have necessary qualification and experience 
to guide this project. 
 
 
Place : Thrissur 
 
 
Signature : 
Date  : 14/08/2019  
 
  
 
Dr. Susheela Jacob Innah 
Professor & Head 
Department of Transfusion Medicine 
J.M.M.C & R.I, Thrissur                     
 
 
 
            
 
 
 
 
   
 
 
 
 

 
7 
DECLARATION OF CO-GUIDE 
 
I hereby agree to guide the principal investigator throughout the period of research 
as per the protocol submitted for approval. I have necessary qualification and experience 
to guide this project. 
 
 
Place : Thrissur 
 
 
Signature : 
Date  : 14/08/2019  
 
  
 
Dr. Bino Benjamin 
Associate Professor 
Department of Cardiology 
J.M.M.C & R.I, Thrissur         
 
 
 
 
 
 
 
 
 
 
 
 
 

 
8 
Introduction 
Epidemiologic studies have found the Lewis negative phenotype to be independently 
associated with a two-fold higher prevalence of coronary artery disease and four-fold fatal 
coronary events [1]. Lewis (a-b-) blood group has been reported to be associated with 
hypercholesterolemia, diabetes mellitus and insulin resistance conditions which eventually 
lead to CAD [2]. Many studies have also shown the relationship between blood groups and 
different manifestations of cardiovascular disease [3]. 
Different clinical studies have shown that blood A phenotypes are more susceptible to 
coronary artery disease [3]. The major one being the Framingham study[2]. 
In contrast, Garg P et al. reported a significant association between CAD and blood group 
B. while Sahita P et al. from Gujarat, India, did not observe any association between blood 
group and CAD. Cakir B et al reported a non- significant association between Lewis 
genotype and subclinical atherosclerosis and Saloma V et al. observed that carotid intima-
media thickness was slightly higher among persons with Lewis negative phenotype than 
among persons with Lewis positive phenotype. 
There is no literature from Kerala correlating Lewis antigen and ABO blood group with 
CAD, hence the present study to focus on the pattern and association of ABO and Lewis 
blood group phenotypes in clinically confirmed CAD patients. 
Rationale 
The present study aims to evaluate the association between Lewis antigen, ABO blood 
group, and coronary artery disease. This may allow identifying subjects at increased risk 
for coronary artery disease (CAD) and subsequently leading to more focused preventive 
measures and eventually to a specific treatment. 
 
 
Review of literature 
Coronary artery disease in Kerala is progressive and degenerative resulting in death at a 
very young age. The prevalence of definite CAD in Kerala increased nearly three times 
since 1993 without any difference in urban and rural areas. Most risk factors of CAD are 
highly prevalent in the state[4] Hence early detection of these risk factors is highly 
essential to curtail CAD. 
The ABO and Lewis blood group systems have both been linked to ischaemic heart disease 
(IHD) [5]. 
The Lewis Antigens play a well-defined role in selectin-mediated cell adhesion events, and 
in inflammation and defense against infection. In the absence of these antigens, as in 
Le(a-b-), inflammation and chronic infection, in turn contribute to the multifactorial 
pathogenesis of coronary disease events[2]. Like the ABO blood group, the Lewis blood 
group reflects the actions of two distinct glycosyltransferases, FUT2/Secretor and 
Lewis/FUT3, an α1,3/4-fucosyltransferase[5]. 

 
9 
 
T. Alexy et al. reported hypertriglyceridemia, insulin resistance, and higher prevalence of 
non-insulin dependent diabetes mellitus in the Le(a-b-) population [1]. M.N.Hasan et al 
revealed a higher prevalence of CAD in patients with blood group B [3]. 
 
In a study conducted by Enevold et al, no significant associations were observed between 
Lewis blood group/AB0 blood groups and hypertension, IHD, or obesity [5]. The meta-
analysis by Zhuo Chen et al suggests that blood group A and non-O are associated with an 
increased risk of CAD [6]. A study by Sharif S et al showed that subjects with blood group A 
had a significantly higher risk of developing IHD compared to other blood groups [7]. 
  
 
Materials and methods 
 
Research question: 
 
Does ABO and Lewis blood group phenotypes have an association with Coronary Artery 
Disease? 
 
 
Aim and Objectives: 
 
Primary objective 
 To study the association of Lewis antigen and ABO blood group phenotypes 
with CAD patients attending the tertiary care hospital in Kerala. 
Secondary objective 
 To find the association of Lewis antigen and ABO blood group phenotypes with 
smoking, alcohol use, diabetes, hypertension and dyslipidemia.  
 
Study design  
Hospital-based case-control analytical study 
 
Study setting and duration:  
The Study will be conducted over a period of eighteen months in the Department of 
Transfusion Medicine in association with Department of Cardiology at Jubilee Mission 
Medical College and Research Institute, Thrissur, a tertiary care multispecialty teaching 
hospital in central Kerala. 
 

 
10 
Sampling: 
 Sample size 
Based on the proportion of Le(a-b-) observed in an earlier publication[2] with 95% 
confidence level and 90% power, minimum sample size comes to 70 in cases and 140 
in control group 
 
 
 
 
 
 Inclusion criteria: 
 Patients in the age group of 30-55 years at the time of data collection 
 Diagnosed with coronary artery disease on the basis of positive changes in 
coronary angiogram (More than 70% coronary stenosis)  
 
 Exclusion Criteria: 
 Patients with other systemic illnesses like malignancy, autoimmune 
diseases.  
 
 Patients who are not willing to participate in the study.  
 
 Sampling procedure: 
First 70 cases attending the Cardiology department which satisfy the inclusion and 
exclusion criteria will be in included in the study. 
 
 Method of data collection 
70 cases attending the Cardiology department which satisfy the inclusion and 
exclusion criteria will be taken as cases. 140 age and gender matched healthy blood 
donors who are eligible to donate blood as per the inclusion and exclusion criteria 
of Drugs and Cosmetics Rules, 1945 are taken as controls. 
 
After taking written, informed consent from the patients and blood donors satisfying 
the inclusion criteria, 3 mL blood is collected from cubital vein in Ethylene Diamine 
Tetra Acetic acid (EDTA) tubes under aseptic precautions. Samples are collected 
separately from each CAD patient as well as from blood donors.  
n = [Z1-α/2.√{2p(1-p)} + Z1-β √{p1(1-p1)+p2(1-p2)}]2 
(p1 - p2)2 

 
11 
Collection of blood samples repeatedly from the same subjects are avoided by 
personally verifying the subject’s demographic profile such as full name, age, 
address, including hospital registration number and by collecting the blood sample 
in the required quantity. 
Lipid profile values, fasting and post prandial blood sugar values will be collected 
from the laboratory information system. Blood pressure values will be obtained 
from the medical record and donor forms. 
 
Diabetes mellitus is defined as fasting serum glucose >126 mg/dL and or the 
reported use of diabetic medications[8]. Hypertension is defined as systolic blood 
pressure (BP) >140 or diastolic BP >90 mmHg, or reported use of antihypertensive  
medications[8]. 
 
Alcohol use pattern is categorised as none, low/moderate use (1–14 drinks/week), 
and heavy use (>14 drinks/week) [9]. 
Smoking pattern is categorised as Smoker (An adult who has smoked at least 100 
cigarettes in his or her lifetime, and who now smokes every day), Ex smoker (An 
adult who has smoked at least 100 cigarettes in his or her lifetime but who had quit 
smoking), Never smoker: An adult who has never smoked, or who has smoked less 
than 100 cigarettes in his or her lifetime[9]. 
ABO blood grouping is performed according to department's Standard Operational 
Procedures (SOP). Commercially available monoclonal blood group antisera are 
used for forward grouping; for reverse grouping 5% pooled suspension of A, B and 
O cells are used which are prepared according to SOP. 
Lewis antigen typing is done by using monoclonal antisera as per manufacturer’s 
instructions.  
 
 
Statistical analysis  
Statistical analysis will be carried out using SPSS version 25.0. Descriptive Statistics for the 
categorical variables are performed by computing the frequencies (percentages) in each 
category. Comparison of categorical data between association of ABO and Lewis 
phenotypes in clinically confirmed CAD patients is done using chi-square test. 
Estimated expenditure 
Establishment  
 Antisera   
 
 
 
: ₹ 50000/- 
 Vacutainers  
 
 
: ₹ 1700/- 
Equipment                         
 
: nil 
Research literature             
 
: ₹ 1000 
Cost of preparation of research papers  : ₹ 1000 
Expenditure will be met by the institution. 

 
12 
Ethical Considerations 
No ethical issues expected. Study will be conducted only after the approval of the ethical 
committee. 
 
 
 
 
 
 

 
13 
References  
1. Alexy T, Pais E, Wenby RB, Mack WJ, Hodis HN, Kono N, Wang J, Baskurt OK, Fisher TC, 
Meiselman HJ. Abnormal blood rheology and chronic low grade inflammation: possible risk 
factors for accelerated atherosclerosis and coronary artery disease in Lewis negative 
subjects. Atherosclerosis. 2015 Mar 1;239(1):248-51. 
 
2. Madithadu A, Rajendran A, Durgaprasad R, Jothibai S. Association of Coronary Artery 
Diseases with ABO and Lewis Blood Group Phenotypes at a Tertiary Care Teaching 
Hospital in Southern India. Journal of Clinical & Diagnostic Research. 2018 Jul 1;12(7). 
 
3. Hasan MN, Chowdhury AW, Islam LM, Safiuddin M, Haque M, Hoque H. Association of 
ABO blood group with CAD in patients undergoing CAG in Cardiology Department of 
Dhaka Medical College and Hospital. University Heart Journal. 2014;10(2):81-4. 
 
4. Krishnan MN, Zachariah G, Venugopal K, Mohanan PP, Harikrishnan S, Sanjay G, 
Jeyaseelan L, Thankappan KR. Prevalence of coronary artery disease and its risk factors in 
Kerala, South India: a community-based cross-sectional study. BMC cardiovascular 
disorders. 2016 Dec;16(1):12. 
 
5. Enevold C, Nielsen CH, Molbo D, Lund R, Bendtzen K, Fiehn NE, Holmstrup P. Lewis and 
AB0 blood group-phenotypes in periodontitis, cardiovascular disease, obesity and stroke. 
Scientific reports. 2019 Apr 18;9(1):6283. 
 
6. Chen Z, Yang SH, Xu H, Li JJ. ABO blood group system and the coronary artery disease: 
an updated systematic review and meta-analysis. Scientific reports. 2016 Mar 18;6:23250. 
 
7. Sharif S, Anwar N, Farasat T, Naz S. ABO blood group frequency in Ischemic heart disease 
patients in Pakistani population. Pakistan journal of medical sciences. 2014 May;30(3):593. 
 
8. Theodore A. Kotchen. Hypertensive vascular diseases. In:James f Shanahan, Kim J david, 
editors. Harrison’s principles of internal medicine. New York: McGraw-Hill; 2011. p. 2047-
59. 
 
9. Kelly SG, Plankey M, Post WS, Li X, Stall R, Jacobson LP, Witt MD, Kingsley L, Cox C, Budoff 
M, Palella Jr FJ. Associations between tobacco, alcohol, and drug use with coronary artery 
plaque among HIV-infected and uninfected men in the multicenter AIDS cohort study. PloS 
one. 2016 Jan 26;11(1):e0147822. 
 
 
 
 

 
14 
PROFORMA 
CASE 
 
 
CONTROL 
 
 
Name  
: 
 
 
 
Age:   
 
 
Sex: 
Hosp Number : 
Address 
: 
 
 
 
 
 
: 
 
Presenting complaints 
: 
Past history  
 
: 
Personal history 
 
: 
Tobacco use _____________________  
Alcohol _______________________ 
Physical Examination 
Height :_________ Kg 
 
       Weight: _________ cm  
            BMI: _________ 
Investigations 
Angiogram 
:  ___________________________________________________________________ 
________________________________________________________________________________ 
 Hypertension:    yes 
no   
 
 
On medication:  yes 
no  
 BP: ____________mmHg 
 Lipid profile:  
 
 
 
 
Diabetes: 
    yes 
no 
 Total Cholesterol : ____________ mg/dL  
On medication  yes 
no  
 TG 
: ____________ mg/dL  
PPBS : ____________  
mg/dL  
 HDL 
: ____________ mg/dL   
FBS 
: ____________  
mg/dL 
 LDL 
: ____________ mg/dL 
 
Blood Group 
 
Rh 
 
Lea 
 
Leb 
A 
B 
AB O 
 
+ 
- 
 
+ 
- 
 
+ 
- 
Sl No: ___________ 
Date : ___________ 

 
15 
PATIENT INFORMATION SHEET 
1. Name of the study: 
 Association of coronary artery disease with Lewis and ABO blood group 
phenotypes- A case control study.  
 
2. Expected duration of participation in the study: 
 Duration of hospital stay 
 
3. Expected benefits of the study: 
 Better detection and prevention of coronary artery disease 
 
4. Risks associated with the study: 
 Nil  
 
5. Confidentiality of documents: 
 All information regarding the patient will be kept confidential 
 
6. Treatment of research related injuries: 
 Not applicable 
 
7. Compensation of study related disability or death: 
 Not applicable 
 
8. Freedom to withdraw from the study without penalty: 
 Yes 
 
9. Expenses for collecting and testing samples: 
 No additional expense 
  
10. Research related expense:  
 No additional expense 
 
11. Place of study: 
 Jubilee Mission Medical College & Research Institute, Thrissur 
 
12. Name and address of the investigator: 
 Dr. Sree Raj.V 
Junior Resident 
Department of Transfusion Medicine 
Jubilee Mission Medical College & Research Institute, Thrissur 
 9495018785 

 
16 
PATIENT CONSENT FORM 
Research title: 
‘Association of coronary artery disease with Lewis and ABO blood group phenotypes- A 
case control study’  
 
I have read and understood the patient information sheet. I understand that I have the 
opportunity to ask questions. The investigator has explained the type, objective, scope, 
benefits, duration and other relevant details regarding the study. 
The investigator has clearly explained the rare complications that can arise during blood 
collection. I am willing to undergo medical examination and to provide necessary data and 
the required blood samples for the estimation of blood sugar, lipid profile, blood group and 
Lewis phenotyping.  
 
I know that I will have to bear no extra cost for participating in the study. I am agreeing 
to participate in this study at my own will and I understand that I don’t have any 
obligation to participate in this study. 
I also know that I can withdraw from this study at any time without providing any 
explanation and that my withdrawal from the study will not affect the treatment or rights 
given to me. 
 
I have been assured that my medical records will be kept confidential and no personal 
reference will be made in the study data. I hereby give permission to any authorized 
persons to analyse the data collected from me. 
 
After reading and understanding all the conditions given above I give my consent to 
participate in this study. 
  
Date : 
Place : Thrissur 
 
Name 
:  
 
 
 
 
Name of the investigator: 
Address  :  
Dr. Sree Raj. V  
 
 
Junior resident 
 
 
Department of Transfusion Medicine 
 
 
Jubilee Mission medical College 
 
 
Thrissur  
 
 
 9495018785 
   
Sign: 
 
 
 
 
 
Sign:                                    
 

 
17 
CONSENT FORM (controls) 
Research title: 
‘Association of coronary artery disease with Lewis and ABO blood group phenotypes- A 
case control study’  
 
The investigator has explained that the objective of this study is to investigate the 
association of coronary artery disease with Lewis and ABO blood group phenotypes. I 
understand that this study has two groups, Patients with coronary artery disease and 
people without the disease and that I am invited to be part of the second category. 
I am willing to undergo medical examination and to provide necessary data and the 
required blood samples for the blood grouping and Lewis phenotyping. The investigator 
has clearly explained the rare complications that can arise during blood collection.  
 
I know that I will have to bear no extra cost for participating in the study. I have been 
assured that my medical records will be kept confidential and no personal reference will be 
made in the study data. I hereby give permission to any authorized persons to analyse the 
data collected from me. 
 
I am agreeing to participate in this study at my own will and I understand that I don’t 
have any obligation to participate in this study. I also know that I can withdraw from this 
study at any and that my withdrawal from the study will not affect me in any ways. 
 
After reading and understanding all the conditions given above I give my consent to 
participate in this study. 
  
 
Date : 
Place : Thrissur 
 
Name 
:  
 
 
 
 
Name of the investigator: 
Address  :  
Dr. Sree Raj. V  
 
 
Junior resident 
 
 
Department of Transfusion Medicine 
 
 
Jubilee Mission medical College 
 
 
Thrissur  
 
 
 9495018785 
   
Sign: 
 
Sign: