* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download Kassahun Tesfaye final2
Survey
Document related concepts
Hemolytic-uremic syndrome wikipedia , lookup
Blood sugar level wikipedia , lookup
Autotransfusion wikipedia , lookup
Blood transfusion wikipedia , lookup
Schmerber v. California wikipedia , lookup
Plateletpheresis wikipedia , lookup
Blood donation wikipedia , lookup
Jehovah's Witnesses and blood transfusions wikipedia , lookup
Hemorheology wikipedia , lookup
Men who have sex with men blood donor controversy wikipedia , lookup
Transcript
Frequency of ABO and RH Blood Groups Genotypic and Allelic Distribution among Students of Silti Secondary and Preparatory School, Siltie Zone, Ethiopia M.Sc. Thesis KASSAHUN TESFAYE OCTOBER 2012 Haramaya University Frequency of ABO and RH Blood Groups Genotypic and Allelic Distribution among Students of Silti Secondary and Preparatory School, Siltie Zone, Ethiopia A Thesis Submitted to the School of Graduate Studies, Department of Biology HARAMAYA UNIVERSITY In Partial Fulfillment of the Requirements for the Degree of MASTERS OF SCIENCE IN GENETICS By Kassahun Tesfaye October 2012 Haramaya University SCHOOL OF GRADUATE STUDIES HARAMAYA UNIVERSITY As Thesis Research Advisor, I hereby certify that I have read and evaluated this Thesis prepared, under my guidance, By Kassahun Tesfaye entitled “Frequency of ABO and RH Blood Groups Genotypic and Allelic Distribution among Students of Silti Secondary and Preparatory School, Siltie Zone, Ethiopia”. I recommend that it be submitted as fulfilling the Thesis requirements. Yohannes Petros (PhD.) Major Adivisor _______________ Signature _______________ Date Mebasellasie Andarige (PhD.) Co-Advisor _______________ Signature _______________ Date As member of the Board of Examiners of the M.Sc Thesis Open Defense Examination, We certify that we have read, evaluated the Thesis prepared by Kassahun Tesfaye and examined the candidate. We recommended that the Thesis be accepted as fulfilling the Thesis requirement for the Degree of Master in Genetics. _____________________ Chairperson _____________________ Signature _____________________ Date _____________________ Internal Examiner _____________________ Signature _____________________ Date _____________________ External Examiner _____________________ Signature _____________________ Date ii DEDICATION I dedicate this thesis manuscript to my wife Genet Mitiku, and my Mother Tsehay Woldemichael for their due effort for the success of my life. iii STATEMENT OF THE AUTHOR I declare that this thesis is my bonafide work and that all the sources of materials used for this thesis have been duly acknowledged. This thesis has been submitted in partial fulfillments of the requirements for an advanced MSc Degree at Haramaya University and is deposited at the University Library to be made available to borrowers under rules of the Library. I solemnly declare that this thesis is not submitted to any other institution anywhere for the reward of any academic degree, diploma or certificate. Brief quotations from this thesis are allowed without special permission provided that accurate acknowledgement of source is made. Requests for permission for extended quotation from or reproduction from this manuscript in whole or in part maybe granted by the head of major department or the Dean of School of Graduate Studies when, in his or her judgment, the proposed use of the material is in the interests of scholarship. In all other instances, however, permission must be obtained from the author. Name (Kassahun Tesfaye) Signature______________ Place: Haramaya University, Haramaya Date of Submission:________________ iv BIOGRAPHICAL SKETCH The author was born at Hawassa, SNNPR, Ethiopia, on 12th April, 1982. He attended his elementary school at Ethopia Tekedem Primary School and secondary school at Addis Ketema and Tabor Senior Secondary School from 1989 to 2001.Then he joined Haramaya University in 2002 and graduated with B.Ed. degree in Biology in July 2005. Soon after graduation, he was employed by SNNPR Regional Education Bureau, as Biology teacher at Silti Senior Secondary School. He joined Haramaya University in July 2010 to pursue his postgraduate study in Genetics. v ACKNOWLEDGEMENTS First of all, I would like to thank the Almighty God who helped me in all aspects of my life. I would also like to express my heartfelt appreciation and incalculable thanks to my thesis Advisors, Dr.Yohannes Petros and Dr. Mebasilasie Andarige for their inexorable instruction, guidance, and encouragement throughout the implementation of the research. Without the encouragement, insight and professional expertise of my advisors, the completion of this work would not have been possible. My thanks are extended to Hossana Health College for providing ethical clearance for the research study. I am also very grateful to Silti Secondary and preparatory School for allowing me to conduct laboratory test for blood group and for providing me related materials. My heartfelt gratitude also goes to Silti Wereda Health Office for their expert cooperation especially to Ms. Nuriya Mustafa for her assistance in giving ideas. My special thanks go to Silti Aynage Family Development Association for their unforgettable encouragement and financing per diem for lab technicians and others who have participated in testing students’ blood types and providing anti-sera for the blood typing. A special word of thanks also goes to the staff of Silti Secondary and Preparatory School for their all rounded support in many ways. I wish to express my deep, heartfelt gratitude to my mother and my relatives specially Alem Wolde and Mintiwab Furi, for their support and encouragement during the course of the study. It is also my pleasure to acknowledge my friends, Mr.Abyneh Gongeba, Mr.Delelgen Goshu, Mr.Thomas Demere, Mr. Francis Mthioes, Mr.Thomas Bekele, Mr.Aklilu Aberham, Mr.Yosef Denbu, Mr.Alemu Abate, Mr.Mengesha Kebede, Mr.Wondiye Thilahun, Mr.Brook Solomon, Mr Abebe Gebre, Ms.Flagot Estifanos, and all my friends in and outside the Campus for their friendship and for supporting this study. I am grateful for Mr Addisu Fekadu for his unforgettable encouragement, motivation and support during the period of the teaching learning process and also during proposal development. Finally, I would like to thank Dr. Ameha Kebede for his valuable comments on the study and for assistance in giving ideas. vi LIST OF ACRONYMS AND ABBREVIATIONS ELISA Enzyme Linked Immunosorbent assay FMC Flinders Medical Center HDN Hemolytic Disease of the Newborn IgM Immunoglobulin ISBT International Society of Blood Transfusion PCR Polymerase Chain Reaction RBCs Red Blood Cells Rh Rhesus SNNPR Southern Nations Nationalities and People Regional State SSPS Silti Secondary and Preparatory School WHO World Health Organization vii TABLE OF CONTENTS Page STATEMENT OF THE AUTHOR IV BIOGRAPHICAL SKETCH V ACKNOWLEDGEMENTS VI LIST OF ACRONYMS AND ABBREVIATIONS TABLE OF CONTENTS LIST OF TABLES LIST OF TABLES IN APPENDIX LIST OF FIGURES IN THE APPENDIX ABSTRACT VII VIII X XI XII XIII 1. INTRODUCTION 1 2. LITERATURE REVIEW 4 2.1. HISTORY OF ABO BLOOD GROUPING 2.2 BLOOD GROUP SYSTEMS 2.3 ABO BLOOD GROUP SYSTEM 2.4 RH BLOOD GROUP SYSTEM 2.5 CLINICAL SIGNIFICANCE OF BLOOD GROUP TYPING 2.5.1 Blood transfusion 2.5.2. Hemolytic disease of the newborn (HDN) 2.5.3. Blood products 2.5.4. Red blood cell compatibility and Universal donors and universal recipients 2.5.4.1 Red blood cell compatibility 2.5.4.2 Universal donors and universal recipients 2.5.6. Molecular method of blood group genotyping 2.5.7. Blood group and nutrition 2.6 THE HARDY-WEINBERG GENETIC EQUILIBRIUM 2.6.1 Extension of the Hardy – Weinberg law to Loci with more than two Allele 3. MTERIALS AND METHOD 3.1. DESCRIPTION OF THE STUDY AREA 3.2 SUBJECTS OF THE STUDY 3.3. BLOOD SAMPLE COLLECTION 3.4. BLOOD SAMPLE COLLECTION PROCEDURE viii 4 5 5 9 12 12 13 14 14 14 15 17 17 18 18 21 21 21 21 22 3.5 METHOD OF DATA ANALYSIS 22 4. RESULTS AND DISSCUSION 24 4.1 FREQUENCY OF ABO AND RH BLOOD GROUPING FOR EACH OF THE THREE ETHNIC GROUPS 24 4.2. ESTIMATION OF GENOTYPIC AND ALLELIC FREQUENCY DISTRIBUTION 28 4.2.1. Estimation of ABO blood group and Rh(D) alleles among students of each ethnic group 29 4.2.2. The Chi-square Test for each Ethnic group 31 4.2.2.1 The chi-square test in ABO group distribution 31 4.2.2.2 The chi-square test in Rh blood group 33 6. RECOMMENDATION 36 7. REFERENCES 37 8. APPENDICES 42 ix LIST OF TABLES Tables Pages Table 1. Racial and ethnic distribution of ABO (without Rh) blood types................................ 6 Table 2 Phenotypic Frequency of Rh blood groups s in different populations. ....................... 10 Table 3. Punnet Square showing Hardy-Weinberg frequencies for three autosomal alleles .. 19 Table 4. Frequency of ABO blood types among students of the three ethnic groups at Silti Secondary and Preparatory School .......................................................................................... 24 Table 5. Rh frequency among students of Silti Secondary and Preparatory School .............. 25 Table 6. ABO blood group frequency among students of each ethnic group based on Rh .... 26 Table 7. Allelic frequency of ABO and Rh blood groups in students of the three ethnic groups. ...................................................................................................................................... 29 Table 8. Chi-square test for Sodo ethnic group with Ethiopia’s ABO blood group frequency .................................................................................................................................................. 31 Table 9. Chi-square test for Silte in ABO blood group with Ethiopia’s frequency ................ 32 Table.10 . Chi-square test for Meskan in ABO blood group with Ethiopia’s frequency........ 33 Table 11. Chi-square test for Sodo ethnic group with Ethiopia’s Rh blood group distribution .................................................................................................................................................. 33 Table 12. Chi-square test for Silte ethnic group with Ethiopia’s Rh blood group distrbution 34 Table 13. Chi-square test for Meskan ethnic group with Ethiopia’s Rh blood group distrbution .................................................................................................................................................. 34 x LIST OF TABLES IN APPENDIX Table Page 1. Probability Values for Chi-Square Analysis ....................................................................... 43 2. Consent form .........................................................................Error! Bookmark not defined. 3. Ethical clearnce for the research .......................................................................................... 47 xi LIST OF FIGURES IN THE APPENDIX Page Appendix Figure Figure.1. Pictures of Students during blood typing. ................................................................ 44 Figure 2. Pictures of technicians while determining blood types of the students. ................... 45 xii Frequency of ABO and RH Blood Groups Genotypic and Allelic Distribution among Students of Silti Secondary and Preparatory School, Siltie Zone, Ethiopia ABSTRACT The ABO and Rh blood groups are the most important blood groups despite the long list of several other blood groups discovered so far. The ABO and Rh blood groups frequency varies worldwide and are not found in equal frequency even among ethnic groups. Therefore, this study was aimed at determining the frequency of ABO and Rh blood groups among Students of Silti Secondary and Preparatory School, SNNPR, Ethiopia. A total of 441 students were randomly selected among the students of SSPS. The students were divided into 3 ethnic groups i.e., Sodo, 147 (83 males, 64 females), Meskan, 147 (86 males, 61females) and Silti, 147 (86 males, 61 females) students. Blood samples were collected by Open Slide test method between February 22 and 26/2012G.C. A drop of each of the antisera, anti- A, anti -B and anti- D was added and mixed with each blood sample and rocked gently for 60 sec to observe agglutination. There are frequency differences among the ABO blood types among students of the ethnic groups of the students. Blood group O and Rh-positive has highest frequency while blood group AB and Rh-negative has lowest frequency in the three ethnic groups. In this study, the frequency distribution of blood group O is 36.74%, 42.86% and 49.66% followed by blood group A, 31.97%, 28.57% and 23.81% and blood group B, 25.85%, 23.13% and 21.09% in Sodo, Silte and Meskan respectively and the least percentage frequency is that of blood group AB in the three ethnic groups which is 5.44% in all ethnic group. Whereas, that of Rh of the three studied ethnic groups was 91.16% Rh positive in Sodo and 8.84% were Rh negative. Similarly in Silte 93.20% were Rh positive and 6.8% were Rh negative and in Meskan 91.84% were Rh positive and 8.6% were Rh negative. However, apart from the importance of ABO and Rh blood groups in blood transfusion practice, it is therefore imperative to have information on the distribution of these blood groups in any population group that comprise different ethnic groups. Key words: ABO blood group, blood groups, Meskan, Rh blood group, Sodo, Silte. xiii 1. INTRODUCTION Blood is the most important body fluid, which is responsible for circulation of important nutrients, enzymes, and hormones all across the body, besides the most critical substance, oxygen. The human red blood cell membrane contains different types of polysaccharide antigens, called agglutinogen (Ganong, 1995). The antigenic substances are capable of inducing a specific immune response so that specific response results in the production of plasma cells that produce antibodies (Novak, 1995). Blood carries several antigens in it, which form the basis of its reactivity and hence it is not possible to mix the blood of different humans without initiating an immune reaction. Only the blood samples, which share the same antigenic identity, do not initiate an immune response, and hence are termed as compatible. The utility of these antigens is not only for blood transfusion or organ transplantation, but have also been utilized in genetic research, anthropology and tracing of ancestral relation to human beings (Khurshid,1992). Furthermore, the discovery of ABO and Rh blood groups has contributed immensely to blood banking services and transfusion medicine in order to avoid morbidity and mortality in both adults and children. The blood group of a person does not change within one’s own life time and so it is considered as a unique genetic marker for research. The blood group is determined by the genetic make-up of the alleles of a system (Gupta, 1999). According to Mourant et.al,(1976) the ABO blood groups and Rhesus (Rh) D blood group antigens are the most frequently studied genetic markers in a large number of populations worldwide. The ABO and Rh blood group alleles vary worldwide and are not found in equal numbers even among the same ethnic groups. For example, Among African-Americans the frequency of ABO blood group is O(46%), type A,(27%); type B, (20%); and type AB; (7%). Among Caucasians in the United States, the frequency of type O, (47%); A,( 41%); B,(9%), AB, (3%). Also, among Western Europeans, the frequency of O, (46%); A,( 42%); B, (9%); and AB, (3%) (Garratty,2000). 1 The human blood groups have been studied extensively for their involvement in incompatibility selection. Various studies on ABO incompatibility have produced evidences of high frequency of prenatal death among incompatible mating. Red blood cells contain a series of glycoprotein’s and glycolipids on their surface which constitute the blood group antigens. Production of these antigens is genetically controlled (Srikumari et al., 1987). ABO and Rhesus (Rh) blood group antigens are hereditary characters and are useful in population genetic studies, researching population migration patterns, as well as resolving certain medicolegal issues, particularly of disputed paternity and more importantly in compatibility test in blood transfusion practice. The need for blood group prevalence studies is multipurpose, as besides their importance in evolution, their relation to disease and environment is being increasingly sought in modern medicine (Green et al., 1995). Estimates of gene frequencies provide very valuable information on the genetic similarity of different populations and to some extent on their ancestral genetic linkage, despite the cultural and religious differences of the populations, (Meade et. al, 1994). Blood grouping has improved with the advent of monoclonal antibodies and the automation of tests. Although different advanced techniques, such as micro plate method, PCR based typing, FMC based typing, mini sequencing analysis, fluorescent immuno micro plate technique, sandwich ELISA method, etc., are available for ABO genotyping, the manual method has its own significance not only in blood typing but also measuring its genotypic frequency by Hardy-Weinberg Law,(Rai et.al,2009) ABO and Rh blood group systems in humans are two important genetic markers that are routinely analyzed prior to blood transfusion and medical treatment. The ABO blood group system is governed by a single gene with three alleles (IA, IB and IO), of which IA and IB alleles are co-dominant but both of them are dominant over the recessive allele IO in intra-allelic interaction in diploid condition. The gene for ABO blood group is located on chromosome 9 of human genome where as that of Rh is located on the short arm of chromosome 1 (Murphy et. al, 2003). 2 To the knowledge of this researcher of the study, there is no similar study reported in the literature regarding the distribution of ABO alleles in the population of Siltie Zone, but in Nigeria, India, Saudi Arabia, Pakistan and in other countries in the world, there are a number of literatures that report the frequency of ABO and Rh blood group alleles. Thus, this study is aiming to investigate the distribution of the blood group alleles of the ABO and Rh(D) among students of different ethnic groups in Silti Secondary and Preparatory School, SNNPR, Ethiopia and to make comparative population-genetic analysis with ethnic groups in the school (Silte, Meskan, and Sodo Gurage) and also with other populations to establish certain specific features in the genetic structure. The significance of the research is with a view to generate data with multipurpose future utilities for the health planners and also to see the common trend of the prevalence of various blood groups among the students. Therefore, the general objective of the research is to Compare the three ethnic groups with regard to ABO and Rh blood group system frequencies among the Silte,Sodo and Meskan ethnic groups. The specific objectives are: To determine the allelic frequency of ABO and Rh blood group of the three ethnic groups. To compare the ABO and Rh blood types and the allelic frequency of the ethnic groups with corresponding frequencies in Ethiopian population. To determine if the studied ethnic groups are at Hardy-Weinberg with regard to allelic and phenotypic frequency of ABO and Rh blood groups. . 3 2. LITERATURE REVIEW 2.1. History of ABO Blood Grouping The history of blood group antigens is characterized by important landmarks. Landsteiner in 1901 named the first 2 blood groups antigens A and B, using the first 2 letters of the alphabet while red blood cells (RBCs) not reacting with anti-A and anti-B were called type C. In 1902, Von Decastello and Sturli described RBCs reacting with both anti-A and anti-B, but did not give these types a name, but continued calling RBCs that did not react with anti-A and Anti-B type C. (Garratty,2000) In 1911, von Dungern and Hirszfeld were the first to use the term O to describe RBCs not reacting with anti-A and anti-B and the term AB for RBCs reacting with both anti-A and antiB (Mollison, 1994). Phenotypically, there are 4 blood groups namely, A, B, O, and AB determined by 3 allelic genes located near the tip of the long arm of chromosome 9. These alleles code for 2 glycosyltransferase enzymes, that transfer a terminal sugar unit to the precursor H chain giving either A or B antigenic properties to cell membranes. Although the Mendelian inheritance of the 2 glycosyltranferases is simple, the genetic control of A and B antigen expression is more complex, due to differential tissue expression and secretor gene control of A and B antigens of different tissues (Eastlund,1998) When blood transfusions from one person to another were first attempted, they were successful in some instances, but in many more, immediate or delayed agglutination and haemolysis of the red blood cells occurred. Soon it was discovered that the bloods of different persons usually have different antigenic and immune properties, so that antibodies in the plasma of one blood react with antigens on the surface of the red cells of another. Two particular groups of antigens are more likely than the others to cause blood transfusion reaction. These are the ABO blood group system antigens and those of the Rh system. Frequencies of ABO & Rh blood groups vary throughout the world, (Firkin et al., 1989), as it is shown in Tables 1 and 2. 4 2.2 Blood Group Systems A complete blood type would describe a full set of 30 substances on the surface of RBCs, and an individual's blood type is one of the many possible combinations of blood-group antigens. Across the 30 blood groups, over 600 different blood-group antigens have been found, but many of these are very rare, some being found mainly in certain ethnic groups (ISBT, 2008). Almost always, an individual has the same blood group for life, but very rarely an individual's blood type changes through addition or suppression of an antigen in infection, malignancy, or autoimmune disease. Another more common cause in blood-type change is a bone marrow transplant. Bone-marrow transplants are performed for many leukemia’s and lymphomas, among other diseases. If a person receives bone marrow from someone who is a different ABO type (e.g., a type A patient receives a type O bone marrow), the patient's blood type will eventually convert to the donor's type (Matsushita et al., 1983). Some blood types are associated with inheritance of other diseases; for example, the Kell antigen is sometimes associated with McLeod syndrome. Certain blood types may affect susceptibility to infections, an example being the resistance to specific malaria species seen in individuals lacking the Duffy antigen. The Duffy antigen, presumably as a result of natural selection, is less common in ethnic groups from areas with a high incidence of malaria (Kwiatkowski, 2005). 2.3 ABO Blood Group System The ABO system is the most important blood-group system in human-blood transfusion. The associated anti-A and anti-B antibodies are usually Immunoglobulin M, abbreviated IgM, antibodies. ABO IgM antibodies are produced in the first years of life by sensitization to environmental substances such as food, bacteria, and viruses. The O in ABO system is often called 0 (zero, or null) in other languages (Khurshid et al., 2008). In Table.1 shows the distribution of the ABO blood types along racial and ethnic lines. Blood group B has its highest frequency in Northern India and neighboring Central Asia, and 5 its incidence diminishes both towards the west and the east, falling to single digit percentages in Swiss. It is believed to have been entirely absent from Native American and Australian Aboriginal populations prior to the arrival of Europeans in those areas. Blood group A has high frequencies in Europe, especially in Scandinavia and Central Europe, although its highest frequencies occur in some Australian Aborigine populations and the Blackfoot Indians of Montana, (Encyclopedia Britannica, 2002). Table 1. Racial and ethnic distribution of ABO (without Rh) blood types People group O (%) A (%) B (%) AB (%) Aborigines(Australia) 61 39 0 0 Ethiopia (Abyssinian) 43 27 25 5 Ainu (Japan) 17 32 32 18 Albanians 38 43 13 6 Grand Andamanese 9 60 23 9 Arabs 34 31 29 6 Armenians 31 50 13 6 Asian (in USA—general) 40 28 27 5 Austrians 36 44 13 6 Bantus 46 30 19 5 Basques 51 44 4 1 Belgians 47 42 8 3 Blackfoot (N. Am. Indian) 17 82 0 1 Bororo (Brazil) 100 0 0 0 Brazilians 47 41 9 3 Bulgarians 32 44 15 8 Burmese 36 24 33 7 Buryats (Siberia) 33 21 38 8 Bushmen 56 34 9 2 Chinese-Canton 46 23 25 6 6 Chinese-Peking 29 27 32 13 Chuvash 30 29 33 7 Czechs 30 44 18 9 Danes 41 44 11 4 Dutch 45 43 9 3 Egyptians 33 36 24 8 English 47 42 9 3 Eskimos (Alaska) 38 44 13 5 Eskimos (Greenland) 54 36 23 8 Estonians 34 36 23 8 Fijians 44 34 17 6 Finns 34 41 18 7 French 43 47 7 3 Georgians 46 37 12 4 Germans 41 43 11 5 Greeks 40 42 14 5 Gypsies (Hungary) 29 27 35 10 Hawaiians 37 61 2 1 Hindus (Bombay) 32 29 28 11 Hungarians 36 43 16 5 Icelanders 56 32 10 3 Indians (India—general) 37 22 33 7 Indians (USA—general) 79 16 4 1 Irish 52 35 10 3 Italians (Milan) 46 41 11 3 Japanese 30 38 22 10 Jews (Germany) 42 41 12 5 Jews (Poland) 33 41 18 8 Kalmuks 26 23 41 11 Kikuyu (Kenya) 60 19 20 1 7 Koreans 28 32 31 10 Lapps 29 63 4 4 Latvians 32 37 24 7 Lithuanians 40 34 20 6 Malaysians 62 18 20 0 Maori 46 54 1 0 Mayas 98 1 1 1 Moros 64 16 20 0 Navajo (N. Am. Indian) 73 27 0 0 Nicobarese (Nicobars) 74 9 15 1 Norwegians 39 50 8 4 Papuas (New Guinea) 41 27 23 9 Persians 38 33 22 7 Peru (Indians) 100 0 0 0 Filipinos 45 22 27 6 Poles 33 39 20 9 Portuguese 35 53 8 4 Romanians 34 41 19 6 Russians 33 36 23 8 Sardinians 50 26 19 5 Scots 51 34 12 3 Serbians 38 42 16 5 Shompen (Nicobars) 100 0 0 0 Slovaks 42 37 16 5 South Africans 45 40 11 4 Spanish 38 47 10 5 Sudanese 62 16 21 0 Swedes 38 47 10 5 Swiss 40 50 7 3 Tatars 28 30 29 13 8 Thais 37 22 33 8 Turks 43 34 18 6 Ukrainians 37 40 18 6 USA (US blacks) 49 27 20 4 USA (US whites) 45 40 11 4 Vietnamese 42 22 30 5 Mean 43.91 34.80 16.55 5.14 Standard deviation 16.87 13.80 9.97 3.41 Source: (http://www.bloodbook.com/world-abo.html) ABO blood groups provide the clearest example of simple Mendelian inheritance and offered new criteria of race (AL-Rubeai, 1975). The distribution of the blood groups (A, B, O and AB) varies all over the world according to the population (Wikipedia 2012). Also there are variations in blood type frequency between human sub-populations (Khan.et.al,2004, .Rai.et.al,2009 and Subhashini, 2007).The O blood type is very common around the world , about 63% of humans share it, (Gunson And Martlew, 1996). Type O is particularly high in frequency among the indigenous populations of central and South America where it approaches 100%. The lowest frequency of (O) is found in Eastern Europe and central Asia, where B is common. 2.4 Rh Blood Group System The Rh system is the second most significant blood-group system in human-blood transfusion with currently 50 antigens. The most significant Rh antigen is the D antigen, because it is the most likely to provoke an immune system response of the five main Rh antigens. It is common for D-negative individuals not to have any anti-D IgG or IgM antibodies, because anti-D antibodies are not usually produced by sensitization against environmental substances. However, D-negative individuals can produce IgG anti-D antibodies following a sensitizing event: possibly a fetomaternal transfusion of blood from a fetus in pregnancy or occasionally 9 a blood transfusion with D positive RBCs. Rh disease can develop in these cases (Moise, 2008). Rh negative blood types are much less in proportion of Asian populations (0.3%) than they are in White (15%) In Table 2, the presence or absence of the Rh antigens is indicated by the + or - sign, so that for example the A- group does not have any of the Rh antigens (Cummings, 2000). Of the Rhesus blood group system, the gene D which gives rhesus positive status is at its lowest in Europe. It increases in frequency east and south to approximately 80% over almost all of Africa south of the Sahara. In eastern Asia, Australia and Indonesia; it often attains 100% .The same holds for American indigenous populations in many of whom the D frequency is 100 % (Reddy, et.al, 2008) Table 2 Phenotypic Frequency of Rh blood groups s in different populations. Population Rh+ Rh- References 0.9740 0.0260 Abdulazeez et al,(2008) Britain 0.8300 0.1700 Khattak et al(2008) Benin (Nigeria) 0.9388 0.0603 Enosolease and Adymama (Nigeria) Bazuaye,(2008) Ethiopia 0.94644 0.05356 Seifu and Kifle(1985) Germany 0.9500 0.0500 Akbas et al. (2003) Ibadan (Nigeria) 0.9500 0.0480 Omotade,et al.(1999) Rh+ Rh10 References Population Ilorin (Nigeria) 0.9550 0.0450 Iyiola,(2011) Kenya 0.8030 0.1970 Lyko et al.,(1992) Lagos(Nigeria) 0.9400 0.0600 Adeyemo&Soboyejo,(2006) Bahauddin(Pakistan) 0.9140 0.0860 Anees et al,(2007) Nigeria 0.9430 0.0570 Falusi, et al,(1998) Ogbomoso (Nigeria) 0.9670 0.0330 Bakare et al,(2006) Portharcourt 0.9677 0.0323 Jeremiah , (2006) Red Indians(USA) 1.00 0 Reddy, et.al, (2008) Saudi Arabia 0.9300 0.0700 Khattak et al,(2008) U.S.A 0.8500 0.1500 Khattak et al,(.2008) Mandi (Nigeria) 11 2.5 Clinical Significance of Blood Group Typing 2.5.1 Blood transfusion Transfusion medicine is a specialized branch of hematology that is concerned with the study of blood groups, along with the work of a blood bank to provide transfusion services for blood and other blood products. Across the world, blood products must be prescribed by a medical doctor (licensed physician or surgeon) in a similar way as medicines (Daniels et al, 2006). Much of the routine work of a blood bank involves testing blood from both donors and recipients to ensure that every individual recipient is given blood that is compatible and is as safe as possible. If a unit of incompatible blood is transfused between a donor and recipient, a severe acute hemolytic reaction with heamolysis (RBC destruction), renal failure and shock is likely to occur, and death is a possibility. Antibodies can be highly active and can attack RBCs and bind components of the complement system to cause massive heamolysis of the transfused blood, (Nickel,et. al., 1999). Patients should ideally receive their own blood or type-specific blood products to minimize the chance of a transfusion reaction. Risks can be further reduced by cross-matching blood, but this may be skipped when blood is required for an emergency. Cross-matching involves mixing a sample of the recipient's serum with a sample of the donor's red blood cells and checking if the mixture agglutinates, or forms clumps. If agglutination is not obvious by direct vision, blood bank technicians usually check for agglutination with a microscope. If agglutination occurs, that particular donor's blood cannot be transfused to that particular recipient. In a blood bank it is vital that all blood specimens are correctly identified, so labeling has been standardized using a barcode system known as ISBT 128 (Bruce et.al., 2002) The blood group may be included on identification tags or on tattoos worn by military personnel, in case they should need an emergency blood transfusion. For example, Frontline 12 German Waffen-SS had blood group tattoos during World War II, (Bevel and Gardner., 1997). Rare blood types can cause supply problems for blood banks and hospitals. For example Duffy-negative blood occurs much more frequently in people of African origin, (Nickel et al., 1999).and the rarity of this blood type in the rest of the population can result in a shortage of Duffy-negative blood for patients of African race. Similarly, for RhD negative people, there is a risk associated with travelling to parts of the world where supplies of RhD negative blood are rare, particularly East Asia, where blood services may endeavor to encourage Westerners to donate blood (Bruce et al., 2002) 2.5.2. Hemolytic disease of the newborn (HDN) A pregnant woman can make IgG blood group antibodies if her fetus has a blood group antigen that she does not have. This can happen if some of the fetus' blood cells pass into the mother's blood circulation (e.g. a small fetomaternal hemorrhage at the time of childbirth or obstetric intervention), or sometimes after a therapeutic blood transfusion. This can cause Rh disease or other forms of hemolytic disease of the newborn (HDN) in the current pregnancy and/or subsequent pregnancies. If a pregnant woman is known to have anti-D antibodies, the Rh blood type of a fetus can be tested by analysis of fetal DNA in maternal plasma to assess the risk to the fetus of Rh disease (Daniels and Clarke, 2007). One of the major advances of twentieth century medicine was to prevent this disease by stopping the formation of Anti-D antibodies by D negative mothers with an inject able medication called Rh (D) immuno globulin. Antibodies associated with some blood groups can cause severe HDN, others can only cause mild HDN and others are not known to cause HDN (Cummings, 2000). 13 2.5.3. Blood products To provide maximum benefit from each blood donation and to extend shelf-life, blood banks fractionate some whole blood into several products. The most common of these products are packed RBCs, plasma, platelets, cryoprecipitate, and fresh frozen plasma (FFP). FFP is quickfrozen to retain the labile clotting factors V and VIII, which are usually administered to patients who have a potentially fatal clotting problem caused by a condition such as advanced liver disease, overdose of anticoagulant, or disseminated intravascular coagulation (DIC).Units of packed red cells are made by removing as much of the plasma as possible from whole blood units. Clotting synthesized by modern recombinant methods are now in routine clinical use for hemophilia, as the risks of infection transmission that occur with pooled blood products are avoided (Benjamin, et. al., 1996) 2.5.4. Red blood cell compatibility and Universal donors and universal recipients 2.5.4.1 Red blood cell compatibility it is a test that is used to check whether two individuals blood group compatible or not during blood transfusion or pregnancy, (Bruce et.al., 2002) Blood group AB individuals have both A and B antigens on the surface of their RBCs, and their blood plasma do not contain any antibodies against either A or B antigen. Therefore, an individual with type AB blood can receive blood from any group (with AB being preferable), but can donate blood only to another type AB individual. (Greenwalt, 1997). Blood group A individuals have the A antigen on the surface of their RBCs, and blood serum containing IgM antibodies against the B antigen. Therefore, a group A individual can receive blood only from individuals of groups A or O (with A being 14 preferable), and can donate blood to individuals with type A or AB (Simpkins and Williams, 1997). Blood group B individuals have the B antigen on the surface of their RBCs, and blood serum containing IgM antibodies against the A antigen. Therefore, a group B individual can receive blood only from individuals of groups B or O (with B being preferable), and can donate blood to individuals with type B or AB (Tamarin, 2004). Blood group O: individuals do not have either A or B antigens on the surface of their RBCs, but their blood serum contains IgM anti-A and anti-B antibodies against the A and B blood group antigens. Therefore, a group O individual can receive blood only from a group O individual, but can donate blood to individuals of any ABO blood group (i.e., A, B, O or AB). If anyone needs a blood transfusion in an emergency, and if the time taken to process the recipient's blood would cause a detrimental delay, O Negative blood can be issued (Griffiths, et al., 2008). Rh compatibility: If a woman is Rh negative and the father of the baby is Rh positive, there's a very good chance the baby will be Rh positive as well. There is a great potential for health problems for the baby in such a situation. If it is the mother's first pregnancy, the incompatibility problem is not so great because, barring an abnormality; the baby's blood does not enter the mother's circulatory system during pregnancy. However, during the birthing process, the baby's blood can mix with the mother's blood and this is where the problem begins. If the baby's Rh positive blood enters the mother's body, her body will recognize the Rh protein as a foreign, enemy substance and can being to produce antibodies against the Rh positive protein. Blood transfusions with Rh positive blood, miscarriage and ectopic pregnancies are other ways Rh negative pregnant women can be exposed to the Rh protein, (Tamarin, 2004). 2.5.4.2 Universal donors and universal recipients With regard to transfusions of whole blood or packed red blood cells, individuals with type O Rh D negative blood are often called universal donors, and those with type AB Rh D positive 15 blood are called universal recipients; however, these terms are only generally true with respect to possible reactions of the recipient's anti-A and anti-B antibodies to transfused red blood cells, and also possible sensitization to Rh D antigens. One exception is individuals with hh antigen system (also known as the Bombay blood group) who can only receive blood safely from other hh donors, because they form antibodies against the H substance (Fauci et al., 1998) Blood donors with particularly strong anti-A, anti-B or any atypical blood group antibody are excluded from blood donation. The possible reactions of anti-A and anti-B antibodies present in the transfused blood to the recipients RBCs need not be considered, because a relatively small volume of plasma containing antibodies is transfused (Daniels et al., 2006). Considering the transfusion of O Rh D negative blood (universal donor blood) into a recipient of blood group A Rh D positive, an immune reaction between the recipient's anti-B antibodies and the transfused RBCs is not anticipated. However, the relatively small amount of plasma in the transfused blood contains anti-A antibodies, which could react with the A antigens on the surface of the recipients RBCs, but a significant reaction is unlikely because of the dilution factors. Rh D sensitization is not anticipated (Avent, 2009). Additionally, red blood cell surface antigens other than A, B and Rh D, might cause adverse reactions and sensitization, if they can bind to the corresponding antibodies to generate an immune response. Transfusions are further complicated because platelets and white blood cells (WBCs) have their own systems of surface antigens, and sensitization to platelet or WBC antigens can occur as a result of transfusion, (Baloch and Ali, 2004). With regard to transfusions of plasma, this situation is reversed. Type O plasma, containing both anti-A and anti-B antibodies, can only be given to O recipients. The antibodies will attack the antigens on any other blood type. Conversely, AB plasma can be given to patients of any ABO blood group due to not containing any anti-A or anti-B antibodies (Anees, 2007) 16 2.5.6. Molecular method of blood group genotyping In addition to the current practice of serologic testing of blood types, the progress in molecular diagnostics allows the increasing use of blood group genotyping. In contrast to serologic tests reporting a direct blood type phenotype, genotyping allows the prediction of a phenotype based on the knowledge of the molecular basis of the currently known antigens. This allows a more detailed determination of the blood type and therefore a better match for transfusion, which can be crucial in particular for patients with needs for many transfusions to prevent allo-immunization (Anees, 2007). 2.5.7. Blood group and nutrition The genetic history of a person can be known by studying the blood groups (Sokolov,1993). For instance, type O blood is the oldest blood and shows a connection to the hunter-gatherer cultures. This blood type is strongly aligned with high animal protein consumption; individuals generally produce higher stomach acids and experience more incidence of gastric ulcer disease than the other groups. Blood group A is primarily associated with vegetarian food sources and individuals in this group secrete smaller amounts of stomach acid and have lesser chances for gastric ulcers, heart diseases, cancer and diabetes (Viola and Carolyn, 1991) There has been extensive scientific research over the past 30 years that shows evidence that your individual blood type determines your predisposition toward getting certain diseases, such as cancer, heart disease, diabetes, lupus, muscular sclerosis, allergies, etc. Our blood type also determines what type of biochemistry our digestive systems are made of. "Your blood type is a powerful genetic fingerprint that identifies you as surely as your DNA". (Sokolov, 1993) There are four blood type groups: O, A, B, and AB. The majority of people are Blood Type O. Next comes Blood Type A, then Blood Type B; and, Blood Type AB is very rare and has only been around for about 1000 years. Less than 5% of the world's population has Blood Type 17 AB. Have you ever noticed that some people can eat a variety of foods with no problems, while others suffer from gas, bloating, indigestion and heartburn? The reason for this is that people with different blood types cannot eat or digest the same foods equally. The following is a brief overview of the peculiarities of each blood type group (. D'Adamo, 1996) 2.6 The Hardy-Weinberg genetic equilibrium The Hardy-Weinberg principle provides the solution to how variation is maintained in a population with Mundelein inheritance. According to this principle, the frequencies of alleles (variations in a gene) will remain constant in the absence of selection, mutation, migration and genetic drift. The Hardy-Weinberg "equilibrium" refers to this stability of allele frequencies over time, ( James, 1999) A second component of the Hardy-Weinberg principle concerns the effects of a single generation of random mating. In this case, the genotype frequencies can be predicted from the allele frequencies. For example, in the simplest case of a single locus with two alleles: the dominant allele is denoted A and the recessive a and their frequencies are denoted by p and q; frequency (A) = p; frequency (a) = q; p + q = 1. If the genotype frequencies are in HardyWeinberg proportions resulting from random mating, then we will have frequency (AA) = p2 for the AA homozygotes in the population, frequency (aa) = q2 for the aa homozygotes, and frequency (Aa) = 2pq for the heterozygotes, (Russell, 2005) p2 (AA): 2pq (Aa): q2 (aa) 2.6.1 Extension of the Hardy – Weinberg law to Loci with more than two Allele When two alleles are present at a locus (with the frequency of p and q), the Hardy-Weinberg law tells us that at equilibrium the frequencies of the genotype is p2 + 2pq + q2, which is the square of allelic frequencies (p+q)2. This is the simple binomial expansion, and this principle of probability theory can be extended to any number of alleles that are sampled two at a time into a diploid zygote (Daniel et al., 2007) 18 Table 3. Punnet Square showing Hardy-Weinberg frequencies for three autosomal alleles (ABO blood group) Male gamete Female gamete Allele Frequency Allele Frequency IA IB IO p q r IA IA IA IB IAIO p2 Pq Pr IA p IA IB IB IB IBIO IB q Pq q2 Qr IO r IAIO IOIB IOIO Pr Rq r2 Extension of the Hardy-Weinberg principle to multiple alleles of a single autosomal gene can be illustrated by a three-allele case. Table.3 shows the results of random mating in which three alleles are considered. The alleles are designated p, q, and r, where the uppercase letter represents the gene and the subscript designates the particular allele. The allele frequencies are p, q, and r, respectively. With three alleles (as with any number of alleles), the allele frequencies of all alleles must sum to 1; in this case, p + q+ r, = 1.0. As in Table 3, the entry in each square is obtained by multiplying the frequencies of the alleles at the corresponding margins; any homozygote (such as AA) has a random-mating frequency equal to the square of the corresponding allele frequency (in this case, p2). Any heterozygote (such as AO) has a random-mating frequency equal to twice the product of the corresponding allele frequencies (in this case, 2pr). The extension to any number of alleles is straightforward. 19 Frequency of homozygote = the square of allelic frequency (p2) Frequency of heterozygote = 2×product of allelic frequency (2×pq) Frequency of homozygote = the square of allelic frequency (q2) Frequency of heterozygote = 2×product of allelic frequency (2pr) Frequency of heterozygote = 2×product of allelic frequency (2qr) Frequency of homozygote = the square of allelic frequency (r2) 20 3. MTERIALS AND METHOD 3.1. Description of the Study Area The study was conducted in the Southern Nation Nationalities and People’s Regional State, Silte Zone, Silti Woreda, Kibet town, (Silti Secondary and Preparatory School) which has got over 2000 students belonging to different ethnic groups. The town is located at an altitude of 2095 meter above sea level and 144km south of Addis Ababa on the way to Hossana where the three ethnic groups are living together and the town is situated at 805΄N,38019´E. 3.2 Subjects of the Study According to Vice principal, Silti Secondary and Preparatory School had 1841 students enrolled for the present academic year 2011G.C. The study was conducted on 441 sample students comprising approximately 24% of the student population in the school. The study sample include 147 students aged 18 and above from each of the three major ethnic groups, Siltie, Meskan and Sodo Gurage, this is so, because the number of Sodo and Meskan ethnic group in the school much less than that of Silte ethnic groups. Thus, the sample was divided in to three groups each consisting of 147 students and stratified along ethnic lines. The information about the ethnic group was provided by the students themselves when filling their personal profile form, before conducting blood group test. 3.3. Blood Sample Collection Blood typing was conducted during February 22-26/2012 G.C from each sample population after they signed in the consent form that assures their willingness. The ABO and Rh blood group test was performed by using sterilized needle, to obtain a drop of blood from a sterilized finger. 21 3.4. Blood Sample Collection Procedure Blood samples were taken from finger pricks, and open slide method of testing ABO blood types and Rh (D) factor following (Bhasin and Chahal 1996). Then, the blood was placed on a clean slide in three places and a drop of one of the, Anti A, Anti B and Anti D [manufactured by Tulip Diagnostics (P) Limited, Old Goa, India] was added to each of an individual’s blood samples and mixed using a glass rod. Blood group was determined on the basis presence or absence of agglutination and recorded, as blood type A+, B+, AB+ O+ or A-, B-, AB- and O-. The blood samples were collected by qualified laboratory technicians using the standard clinical procedure with sterilized lancet blade, slides, and anti- A, Anti-B and Anti-D. 3.5 Method of Data Analysis The genetic structure of a population is determined by the total of all alleles (the gene pool).In the case of sexually interbreeding individuals, the structure is also characterized by the distribution of alleles into genotypes. The genetic structure can be described in terms of phenotypic, allelic and genotypic frequencies (Russell, 2005).For the present study, the frequency of the blood group phenotypes was used to calculate the frequency of the ABO blood group alleles by using the extension of Hardy-Weinberg principle as employed by (Griffith et.al., 2008) For this study three alleles are computed (A, B and O), with frequencies equal to p, q and r, respectively. The frequencies of the genotypes at equilibrium are computed by trinomial expansion (p+q+r) 2. (p + q + r)2 = p2(AA) + 2pq(AB) + q2(BB) + 2pr(AO) + 2qr(BO) + r2(OO) (Griffith et.al, 2008 ) 22 A chi-square test was used to compare the allelic frequencies (ABO) students of each ethnic group with the allelic frequency of Ethiopia population as reported in the literature in Table 1. Pearson's chi-square goodness of fit test statistic is or X2=(O-E)2÷E - Where Oj are observed counts, Ej are corresponding expected count and C is the number of classes for which counts/frequencies are being analyzed. 23 4. RESULTS AND DISSCUSION 4.1 Frequency of ABO and Rh Blood Grouping For Each of the Three Ethnic Groups For this study, 441 students were randomly selected and these consisted of 255 males and 186 females. The frequency of ABO blood groups and Rh among students of each ethnic group is presented in Table 4 & 5. Table 4. Frequency of ABO blood types among students of the three ethnic groups at Silti Secondary and Preparatory School Ethnic Blood type frequency distribution Total groups Type A Type B Type AB Type O Sodo 47(31.97%) 38(25.85%) 8(5.44%) 54(36.74%) 147(100%) Silte 42(28.57%) 34(23.13%) 8(5.44%) 63(42.86%) 147(100%) Meskan 35(23.81%) 31(21.09%) 8(5.44%) 73(49.66%) 147(100%) Total 124(28.11%) 103(23.35%) 24(5.44%) 190(43.08%) 441(100%) There are differences in frequency distribution of the blood group (ABO) among the ethnic groups of the students. Blood group O has the highest frequency while blood group AB has the lowest frequency in (Table 4). In this study, the frequency of blood group O is 36.74%,42.86% and 49.66% followed by blood group A, 31.97%, 28.57% and 23.81% and blood group B, 25.85%, 23.13% and 21.09% in Sodo, Silte and Meskan respectively and the least percentage frequency is that of blood group AB in the three ethnic groups which is 5.44% in all ethnic group as it is shown in Table.4. Blood group O is highly distributed in Meskan ethnic group than Silte and Sodo ethnic group. Blood group A frequncey is higher in 24 Sodo than Silte and Meskan ethnic group and blood group B is dominated in Sodo ethnic group than Silte and Meskan. Blood group AB has equal frequency in the three ethnic groups. It has also been reported in several studies that there are variation in ABO blood group among different ethnic groups (Nwauche and Ejele, 2004; Falusi et al., 2000). Many other studies have shown that blood group O was the most common blood group and blood group AB was the least common blood group in different ethnic groups (Nwauche and Ejele, 2004).Thus, the gene segregation for ABO systems always followed a particular pattern for its distribution in different ethnic group with exceptional cases. Among the Caucasians in the United States of America, the frequency of blood group O, A, B and AB are 47.0, 41.0, 9.0 and 3.0 %, respectively (Adeyemo and Soboyejo, 2006) which is in agreement with this study. However, the finding of this findings seem to deviate from the results obtained by Khan and his colleagues on the genotype frequencies of blood group antigens from Bannu region in Pakistan where ABO blood group frequency occurred in the order B>A> O> AB ( Khan et al., 2009 ).It also seem not to agree with the results obtained from Swat district in Pakistan where the percentage frequencies were A=27.92% , B= 32.40 %, O = 29.10% and AB=10.58% ( Khattak et al.,2008). Table 5. Rh frequency among students of Silti Secondary and Preparatory School Ethnic group Rh positive Rh negative Total Sodo 134(91.156%) 13(8.843%) 147 Silte 137(93.197%) 10(6.8%) 147 Meskan 135(91.84%) 12(8.16%) 147 Total 406(92.06%) 35(7.94%) 441 The frequency distribution of Rh blood group among each ethnic group of the students is shown in Table 5. The variations in the frequency distribution of Rh - positive and Rh negative among the three (3) ethnic groups followed the pattern shown in Table 5. 25 Table 6 shows the frequency distributions of ABO blood group in the three ethnic groups based on Rh blood group. Table 6. ABO blood group frequency among students of each ethnic group based on Rh Ethnic Rh groups blood Blood types Total A B AB O 39(26.53%) 33(22.47%) 8(5.44%) 54(36.73%) 134(91.16%) Negative 8(5.44%) 5(3.42%) 0 0(0%) 13(8.89%) Positive 32(21.77%) 7(4.76%) 59(40.14%) 137(8.89%) 2(1.36%) 1(0.682%) 4(2.72%) 10(6.8%) 29(19.732%) 8(5.44%) 68(46.26%) 135(91.84%) 2(1.36%) 5(3.40%) 12(8.16%) group Positive Sodo Silte 39(26.53%) Negative 3(2.04%) Meskan Positive 30(20.41%) Negative 5(3.4%) Total 0% Positive 108(24.49%) 94(21.32%) 23(5.22%) 181(41.042%) 406(92.06%) Negative 16(8.88%) 9(2.04%) 1(0.23%) 9(2.04%) 35(7.94%) 441(100%) The ABO blood group distribution based on Rh in Sodo and Silte is the same in blood group A with Rh positive (26.53%) but in Meskan the percentage of blood group A is reduced to be 20.41% of the total population. Blood group B with Rh positive of the three ethnic groups was found to be 22.47, 21.77 and 19.73% in Sodo, Silte and Meskan respectively. Blood group AB with Rh positive of the three ethnic groups obtained was 5.44% in Sodo, 4.76% in Silte 26 and 5.44% in Meskan, which is a small percentage distribution of blood group in the three ethnic groups. Blood group O with Rh positive of Sodo was 36.73% and that of the Silte was 40.14% which is higher than the Sodo ethnic group and 46.26% for Meskan ethnic group. So, blood group O with Rh positive is dominant in Meskan ethnic group. As compared to the other blood groups, blood group O with Rh positive percentage distribution varies significantly in the three ethnic groups. The percentage distribution of Rh negative is very small or rare in the three representative groups. Blood group A negative was 5.44%, 2.04%, 3.40%, and 3.40%, 1.36%, 1.36% in blood group B Rh negative, in blood group AB Rh negative 0%, 0.68%, 0%, and in blood group O it was 0, 2.72 and 3.40% in Sodo, Silte and Meskan ethnic group respectively. Blood group AB and O negative was not found in Sodo ethnic group which is 0% and so doe’s blood group AB negative in Meskan. Blood group A negative distribution is higher in Sodo (5.44%) than Meskan(3.40%) ethnic group and the least in Silte ethnic group which is 2.04%. Blood group B negative in Sodo is the highest of Silte and Meskan which is 3.4% and 0.68% each for the other two ethnic groups. Blood group AB negative was not found in Sodo and Meskan ethnic group during blood test but in Silte ethnic group there was one person that tested to became AB negative which is 0.68% of the total sample population. Finally, 3.40% of the Meskan sample population blood test resulted O negative which is the highest, 2.72% in Silte and 0% in Sodo ethnic group. In addition, the incidence of rhesus negativity in the study area (Silti Secondary and Preparatory School, SNNPR, Ethiopia) was found to be between 6.8 and 8.84%. It is 6.8, 8.16, and 8.84% among Silti, Meskan and Sodo, respectively. Similar pattern of frequency was also observed in and other studies (Khattak et. al 2008 and Anees et. al, 2007). Thus, apart from the importance of ABO and Rh blood group systems and the variations in these blood group systems among ethnic groups, there is a need to have information on these blood group systems in any population of different ethnic group. The relevance of having knowledge about the blood group systems among different ethnic groups in any population is enormous. The types of information obtained from the findings are useful for genetic 27 information, genetic counseling, medical diagnosis and general and physiological wellbeing of individuals in a population. 4.2. Estimation of Genotypic and Allelic Frequency Distribution An important application of the Hardy-Weinberg law is estimating the heterozygous frequencies in a population .The majority of the deleterious recessive genes in human population are carried in heterozygous condition. To calculate the frequency of individuals who have heterozygous recessive traits, we usually begin by counting the number of homozygous recessive individuals. These homozygous individuals can be distinguished from the rest of the population by clinical symptoms that indicate the defects. By using the HardyWeinberg law we can calculate the frequency of the heterozygous condition, (Cummings, 2000) For this study, the frequencies of the ABO blood group genotypes and alleles were calculated or estimated using the extension of the Hardy-Weinberg law as employed by (Griffith et.al, 2008) In other words , when you add up the frequency of the A,B and O alleles, you have accounted for 100% of the alleles for this gene that are present in the population .The genotypic frequencies are given by the following equation, when the allelic frequencies are p=A, q=B and r=O. (p + q + r)2 = p2(AA) + 2pq(AB) + q2(BB) + 2pr(AO) + 2qr(BO) + r2(OO) (Griffith et.al, 2008) Three alleles are computed (A, B and O), with frequencies equal to p, q and r respectively. The frequencies of the genotype at equilibrium will be computed by the square of the allelic frequencies. In this system, the alleles A and B are co-dominant and both are dominant to O. This system has six possible genotypic combinations but only four phenotypic blood groups. Homozygous AA individuals and heterozygous AO individuals are phenotypically identical, as are BB and BO individuals. This results in four phenotypic combinations, known as blood types A, B, 28 AB, and O. The frequency of AA genotype is predicted to be p2, AB individuals 2pq, AO individuals 2pr,BB individuals q2,2qr individuals BO, and OO individuals r2. 4.2.1. Estimation of ABO blood group and Rh(D) alleles among students of each ethnic group By using the extension of the Hardy-Weinberg law employed by (Griffith et.al. 2008) the genotypic and the allelic frequency distribution among students of each ethnic group calculated and listed in Table 7. Table 7. Allelic frequency of ABO and Rh blood groups in students of the three ethnic groups. Ethnic groups Gene(allele) Frequency Genotype Frequency Phenotype Frequency O(r) 0.6060 OO 0.3673 O 36.73% A(p) 0.223 AA 0.04973 A 4.97% B(q) 0.1712 AO 0.26997 A 26.99% BB 0.0293 B 2.93% BO 0.2293 B 22.93% AB 0.0544 AB 5.44% DD 0.49 Rh(D)+ve 49% Dd 0.422 Rh(D)+ve 42.2% Sodo D Silte 0.7 D 0.297 dd 0.09 Rh(D)-ve 9% O (r) 0.6546 OO 0.4285 O 42.85% A(p) 0.19 AA 0.0361 A 3.61% B(q) 0.1553 AO 0.2496 A 24.96% BB 0.0535 B 5.35% BO 0.1778 B 17.78% AB 0.0544 AB 5.44% 29 Silte D 0.715 DD 0.51 Rh(D)+ve 51% Dd 0.42 Rh(D)+ve 42% d 0.285 dd 0.082 Rh(D)-ve 8.2% O(r) 0.7047 OO 0.4966 O 49.66% A(p) 0.1524 AA 0.0232 A 2.32% B(q) 0.1429 AO 0.2148 A 21.48% BB 0.0204 B 2.04% BO 0.1905 B 19.05% AB 0.0544 AB 5.44% DD 0.51 Rh(D)+ve 51% Dd 0.401 Rh(D)+ve 42% dd 0.0812 Rh(D)-ve Meskan D d 0.715 0.285 8.2% As shown Table.7, most of the A and B blood types are heterozygous (dominant) in each of the ethnic groups. The genotypes AO makes 26.99%, 24.96% and 21.48% in Sodo, Silte and Meskan respectively. Whereas, genotype BO is 22.93%, 19.05% and 17.78% in Sodo, Meskan and Silte respectively. This study also agreed with the suggestion of Bakare et.al.2006), that the predominance of O allele may also be as a result of the fact that many A’s and B’s may have been heterozygous carrying O allele silently thereby maintaining O allele in the heterozygous population. Homozygous blood group A (AA) of the three ethnic groups calculated to be 4.97% in Sodo, 3.61% in Silte, 2.32% in Meskan. The result shows blood group AA is more frequent in Sodo ethnic group than in the other two (4.97%) and the least distribution in Meskan ethnic group (2.32%). Where as in homozygous blood group B the result obtained was 2.93% in Sodo, 5.53% in Silte and 2.04% in Meskan ethnic group. Homozygous blood group B is the highest in Silte and the least in Meskan ethnic group. 30 With respect to Rhesus blood grouping system, of the total 441sample population 92.06% of the population sampled were Rh (D) +ve while 7.94% were Rh (d)-ve. The frequency of heterozygous Rh +ve (Dd) also calculated using the the Hardy-Weinberg law and listed in Table 7.The heterozygous Rh +ve (Dd ) in each ethnic group found to be 42.2%,42% and 42% in Sodo, Silte and Meskan respectively. The percentage of heterozygous Rh+ve in Silte and Meskan is the same but there is a little deviation in Sodo ethnic group. 4.2.2. The Chi-square Test for each Ethnic group The chi-square test for each ethnic group were calculated using the result obtained from Table.7 and the corresponding report given for the ABO and Rh frequency distribution for Ethiopia to know whether the population of each ethnic group in Hardy-Weinberg equilibrium or not. 4.2.2.1 The chi-square test in ABO group distribution In this study the ABO blood group distribution of each ethnic group is compared with the population of Ethiopia by using the Chi-square test at P value <0.05, 95% confidence level. The ABO blood group distribution of Ethiopia is given in Table 1 which is blood group O 43%, A 27%, B 25% and 5% AB. Table 8. Chi-square test for Sodo ethnic group with Ethiopia’s ABO blood group frequency d2 d2/ Expected Blood Observed Expected Difference group number(O) number(E) d(O –E) O 54 63.21 -9.21 84.8241 1.571 A 47 39.69 7.31 53.4361 1.14 B 38 36.75 1.25 1.5625 0.4 AB 8 7.35 0.65 0.4225 0.053 Total 147 147 X2= 1.54 31 In Table 8 the calculated Chi-Square value is 1.54, which has the P value is between 0.7 and 0.5 with 3 degrees of freedom. This means that there is no significant difference between values obtained for Sodo ethnic group and the values reported for Ethiopia. In Silte ethnic group the Chi-square value obtained was 0.393, the P value is between 0.95 and 0.90 with 3 degrees of freedom, the result shows us that the ABO blood group distribution of the Silte population strongly agrees with that of the distribution of ABO blood group of Ethiopia in general as shown below in Table 9. Table 9. Chi-square test for Silte in ABO blood group with Ethiopia’s frequency Blood group d2 d2/E Observed Expected Difference(d) number(O) number(E) (O-E) O 63 63.21 0.21 0.0441 0.0007 A 42 39.69 2.31 5.336 0.1334 B 34 36.75 -2.75 7.5625 0.206 AB 8 7.35 0.65 0.4225 0.053 Total 147 147 X2 =0.393 Finally, the Chi-square test analysis for Meskan ethnic group was calculated to be 3.054, the P value is between 0.5 and 0.3 with 3 degrees of freedom. The result indicates that the ABO blood group distribution in Meskan ethnic group shows a greater deviation from the population of Ethiopia as a whole .However, the difference is not significant, it is accepted as it is given in table 10. 32 . Table.10 . Chi-square test Blood group for Meskan in ABO blood group with Ethiopia’s frequency d2 d2/E Observed Expected Difference(d) number(O) number(E) (O-E) O 73 63.21 9.79 95.84 1.313 A 35 39.69 -4.69 21.996 0.628 B 31 36.75 -5.75 33.1 1.06 AB 8 7.35 0.65 0.4225 0.053 Total 147 147 X2 =3.054 4.2.2.2 The chi-square test in Rh blood group The chi-square test for Rh for each ethnic group is compared with Ethiopia’s population in general at P value <0.05, 95% confidence level. The Rh blood group phenotypic distribution is given in Table 2 as Rh positive, 94.644% and Rh negative, 5.356%. Table 11. Chi-square test Blood group for Sodo ethnic group with Ethiopia’s Rh blood group distribution d2 d2/E Observed Expected Difference(d) number(O) number(E) (O-E) Rh+ 134 139.12 -5.12 26.2144 0.188 Rh- 13 7.88 5.12 26.2144 2.946 147 147 X2=3.134 In Table.11 the calculated chi-square is 3.134, which has the p value between 0.1 and 0.05 with 1 degree of freedom. This means that there is no significant difference between values obtained for Sodo ethnic group and values reported for Ethiopia. In Silte ethnic group the chi-square value obtained was 0.6026, which has the P value between 0.5 and 0.3 with 1 degree of freedom. The Silte ethnic group does not show significant difference in Rh distribution of Ethiopia. 33 Table 12. Chi-square test for Silte ethnic group with Ethiopia’s Rh blood group distribution Blood group d2 d2/E Observed Expected Difference(d) number(O) number(E) (O-E) Rh+ 137 139.12 -2.12 4.4944 0.0323 Rh- 10 7.88 2.12 4.4944 0.570 147 147 X2=0.6026 Finally, in Meskan ethnic the chi-square value obtained was 1.54, which has the P value between 0.3 and o.2 with 1 degree of freedom. The Meskan ethnic group does not show significant difference in Rh blood group distribution with that of Ethiopia. Table 13. Chi-square test Blood group for Meskan ethnic group with Ethiopia’s Rh blood group distribution d2 d2/E Observed Expected Difference(d) number(O) number(E) (O-E) Rh+ 135 139.12 -4.12 16.97 0.1257 Rh- 12 7.88 4.12 16.97 1.414 147 147 X2=1.54 34 5. CONCLUSION Phenotypic, genotypic and allelic frequency of ABO blood group system in the studied population of the three ethnic groups do not show significant differences compared to the corresponding frequency for the general Ethiopian population data. This study will have significant implications for the major blood banks of Silte Zone where certain blood groups are needed more than others in emergency conditions, for, instance blood group O is highly required and blood group AB required least by blood banks of Silte Zone or Woreda. Furthermore, the data generated in this study would be helpful to the researchers in the field of population genetics to explore the factors responsible for the observed distribution patterns of these genetic markers in this part of central Ethiopia or even to east Africa. The three ethnic groups are in Hardy-Weinberg equilibrium in general, but in the Silte ethnic group the equilibrium is stronger than the other two and the least in Meskan ethnic group. 35 6. RECOMMENDATION Studies of similar kind should be carried out in other populations too, so as to have better information about the distribution of ABO and Rh blood group alleles among different ethnic groups in the country. The sample size may not represent the three ethnic groups, further study with more sample size is needed. Further study at molecular level would definitely reveal the degree of genetic proximity of the three groups in quantitative terms. 36 7. REFERENCES Abdulazeez,A.A., E.B.Alo. and S.N.Rebecca,2008. Carriage rate of Human Immunodeficiency Virus (HIV)infection among different ABO and Rhesus blood groups in Adamawa state, Nigeria Biomedical Research; 19 (1): 41-44 Adeyemo, A.F and O.B,Soboyejo,. 2006. Frequency distribution of ABO, RH blood groups and blood genotypes among the cell biology and genetics students of University of Lagos, Nigeria. Afri J. of Biotechnol . 5 (22), 2062-2065 AL-Rubeai M.A.F.(1975) ."Taste sensitivity to phenylthiocarbamide and blood groups in the Iraqi population (sample from Baghdad) Bull. Coll. Sci. 16(2): 205 -215. Akbas, F.,M.,Aydin and A.,Cenani, 2003. AB0 blood subgroup allele frequencies in the Turkish population. Anthropol. Anz. 61:257-60. Anees, M, A.,Jawad and I.Hashmi., 2007 . Distribution of ABO and Rh blood group alleles in Mandi Bahauddin district of Punjab, Pakistan. Proc. Pakistan Acad. Sci. 44(4):289-294. Avent, N.D., 2009. Large-scale blood group genotyping: clinical implications. Br. J .Haematol 144 (1): 3–13 Bakare.A.A., M.A.Azeez. and J.O.Agbolade.,2006. Gene frequencies of ABO and rhesus blood groups and haemoglobin variants in Ogbomosho, South-West Nigeria. Afr. J. Biotechnol., 5: 224-229. Baloch .M.K and Ali, (2004)."An alysis of genotype frequencies of blood group antigens from Bannu region (NWFP) in Pakistan Gomal S. Medical Sci. 2(1) : 1-5 . Benjamini,E., G.Sunshine and S.Leskowitz, 1996. Immunology: a Short Course.3rd edition.Wileys –Liss. New York.P484 Bevel,T., and M. Gardner.,1997. Blood Stain Pattern Analysis. CRC Press Boca Raton, New York.P300 Bhasin,M.K., and S.M.Chahal. 1996. A Laboratory Manual for Human Blood Analysis. Delhi: Kamla-Raj Enterprise. Bruce,M.G, 2002. BCF - Members - Chairman's Annual Report.The Blood Care Foundation. http://www.bloodcare.org.uk/html/resources_chairman. Accessed on June,2011 37 Cummings, M.R, 2000.Human Heredity Principles and Issues.5th edition. University of Illinois, Chicago.P478. D'Adamo, P. (with additional material by Catherine Whitney) (1996). Eat Right 4 your Type. Putnam. ISBN 0-399-14255-X Daniels.G., K.Finning., P.Martin., and J.Summers., (2006). "Fetal blood group genotyping: present and future". Annals of the New York Academy of Sciences 1075: 88–95. Daniel, H.L. and A.G,Clark., 2007. Principle of Population Genetics.4th.Sinauer. Associates. Sunderland, Massachusetts.P633 Eastlund,T., The histo-blood group ABO system and tissue transplantation. Transfusion 1998; 38: 975-988. Enosolease, M.E., and G.N, 2008. Distribution of ABO and Rh-D blood groups in the Benin area of Niger-Delta: Implication for regional blood transfusion. Asian J Transfus Sci. 2008 January; 2(1): 3–5. doi: 10.4103/0973-6247.39502. The free encyclopedia . (2012 )."Human blood types" Wikipedia Falusi,A.G., O.G.Ademowo., C.A.Latunji., A.C.Okeke., P.O.Olatunji., T.O.Onyekwere., et al 2000. Distribution of ABO and Rh genes in Nigeria. Afr. J. Med. Sci. 29:23-6. Fauci, Anthony S., Eugene Braunwald., Kurt.J.Isselbacher, Jean .D.Wilson, Joseph.B. Martin., Dennis. Kasper, Stephen L. Hauser, Dan L. Longo1998. Harrison's Principals of Internal Medicine. New York: McGraw-Hill. pp. 719 Firkin,F., C.Chesterman., D.Penington., and B.Rush (editors), de Gruchy’s Clinical Hematology in Medical Practice 5th edition. Blackwell Scientific Publications. Oxford, London, Edinburh, Boston, Melbourne; 1989; 476-480. Ganong,WF.,1995 Review of Medical Physiology. 17th edition. Prentice Hall International Inc. London: pp. 487-9. Garratty,G., W.Dzik., PD.Issitt., DM., ME.Lublin Reid., and T.Zelinski, 2000. Terminology for blood group antigens and genes– historical origins and guideline in the new millennium. Transfusion; 40: 477-489. Green,D., O.Jarret, K.J.Ruth., A.R.Folsom., K.Liu, 1995. Relationship among Lewis phenotype, clotting factors and others cardiovascular risk factors in young adults. J Lab Clin Med. 125:334–9. Greenwalt,TJ., A short history of transfusion medicine. Transfusion. 1997;37(5):550-563. 38 Griffiths.,A.F, S.R,Wessler, R.C,Lewontin., and S.B.Carroll., 2008. Introduction to Genetic Analysis. 9th edition. W.H. Freeman and Company. New York. P838 Gunson, H.H. and V.J.Martlew, 1996 ., Blood transfusion. In Oxford Text Book of Medicine. D.J. Weatherall,J.G.G. Ledingham and D. A. Warrell. Oxford University Press, Oxford New York, Tokyo., 3687-3689. Gupta, P. K., 1999. Genetics. Rastogi Publications, Meerut, India. International Society of Blood Transfusion, 2008. http://ibgrl.blood.co.uk/isbt%20pages/isbt%20terminology%20pages/table%20of%20blood% 20group%20systems.htm. Accessed on June,2011 Iyiola, O.A., O.O. Igunnugbemi., U.A .Raheem. and A.T. Anifowoshe., 2011. Gene frequencies of ABO and Rh (D) blood group alleles in Ilorin, North-Central Nigeria. University of Ilorin, Ilorin Kwara state, Nigeria James. F. (1999). "Hardy, Weinberg and language impediments". Genetics 152 (3): 821– 825.. Jeremiah, Z.A., 2006. Abnormal haemoglobin variants, ABO and Rh blood groups among students of African descent in Port Harcourt, Nigeria, African Health Sciences 6 (3):177-181 Kwiatkowski. D.P. (2005). "How Malaria Has Affected the Human Genome and What Human Genetics Can Teach Us about Malaria". American Journal of Human Genetics 77 (2): 171–92. Khan, M.N., I. Khaliq, A. Bakhsh, M.S. Akhtar and M Amin-ud-Din (2009). Distribution of ABO and Rh D blood groups in the population of Poonch District, Azad Jammu and Kashmir. East Mediterr Health. J., 15: 717-721. Khattak,I.D., T.M.Khan., P.Syed ., A.M.Shah., S.T.Khattak., and A.Ali.,( 2008 ). Frequency of ABO and rhesus blood groups in district Swat, Pakistan. J. Ayub Med Coll Abbottabad; 20(4). Khurshid, B., M.Naz., M.Hassan., SF.Mabood.`1992, Frequency of ABO and Rh (D) blood groups in district Swabi N.W.F.P (Pakistan). J. Sc. Tech. Univ.Peshawar; 16:5-6. Lyko,J., H.Gaertner., J. N.Kaviti., M.W. Karithi, and B.Akoto., 1992. The blood groups antigens ABO and Rh in Kenyans. Hamdard Medicus35:59-67. 39 Matsushita, S., T.Imamura., T.Mizuta., and M.Hanada., (November 1983). "Acquired B antigen and polyagglutination in a patient with gastric cancer". The Japanese Journal of Surgery 13 (6): 540–2 Meade, T.W., J.A.Cooper, Y.Stirling, D.J.Howarth, V. Ruddock, & G. J. Miller 1994. Factor VIII, ABO blood group and the incidence of ischemic heart disease. Br. J .Haemat. 88:601– 7. Moise. K.J (July 2008). "Management of rhesus alloimmunization in pregnancy". Obstetrics and Gynecology 112 (1): 164–76 Mollison PL.1994, The genetic basis of the Rh blood group system transfusion; 34: 539-541. Mourant,A.E., AC.Kopec, K.Domaniewska-Sobczak.,1976. The Distribution of the Human Blood Groups and Other Polymorphisms. London: Oxford University Press. Murphy. W.J, L. Fronicke, S.J. O'Brien, R. Stanyon, 2003. "The Origin of Human Chromosome 1 and Its Homologs in Placental Mammals". Genome Res 13 (8): 1880–8. Nwauche, C.A. and O.A. Ejele, 2004. ABO and rhesus antigens in a cosmopolitan Nigeria population. Niger J. Med., 13(3): 263-266. Nickel,R.G., S.A.Freidhoff., and L.Robert, 1999. Determination of Duffy genotypes in three populations of African descent using PCR and sequence-specific oligonucleotides". Human Immunology 60 (8): 738–42 Novak,P.O.,1995. Dorland's Pocket Medical Dictionary, 25th edition. W .B. Saunders Company, Pennsylvania: 1995, pp. 351-75. Omotade, O.O., A.A. Adeyemo., C.M. Kayode., S.L. Falade., and S. Ikpeme,1999.Gene frequencies of ABO and Rh (D) blood group alleles in a healthy infant population in Ibadan, Nigeria. West Afr. J. Med. 18(4): 294- 297. Racial and ethnic distribution of ABO blood types. Bloodbook.com. http://www.bloodbook.com/world-abo.html Rai. V. ; R.P patel and P. Kumar. (2009) " Study of ABO and Rh(D) blood groups in Scheduled caste of Jaunpur District " Anthropologist, 11(2) : 151-152. 40 Reddy., V.M. ; M. Daniel ; E. Bright ; S.R. Broad and A.A. Moir. (2008) "Is there an association between blood group O and epistaxis". J. Laryngology & Otology , 122 : 366368 . Russell. J.P, 2005. iGenetics: A Mendelian Approach. Pearson Benjamin Cummings. New York. P842 Seifu Seyoum and Kifle Dagne, 1985, ABO and Rhesus Blood Type Frequencies In Data From Hospitals And The Red Cross In Ethiopia. Ethiopia.Med.J.23.1 Subhashini.A.B. (2007)." Distribution of ABOand Rh(D) blood groups among Irulas, a tribal population of Pondicherry, India " Anthropologist, 9(2): 163-164. Simpkins, J and J.I. William,1997. Advanced Biology, 3rd Edition Collins Educational, London.P760 Sokolov. R.,1993. Why We Eat What We Eat: How Columbus Changed the Way the World Eats. NewYork: Simon & Schuster. pp 1-50. Srikumari, C.R., J. Rajanikumari., & T. V. Rao., 1987. Acuity of selective mechanisms operating on ABO, Rh (D) and MN blood groups. Am. J. Phy. Anthrop. 72(1): 117-121. Tamarin, R.H, 2004. Principle of Genetics. 7th edition. McGraw-Hill Company , Inc. New York. P609 Viola.H.J. and Carolyn, M. 1991. Seeds of change: Five Hundred Yyears since Columbus. 2nd ed., Washington and London, Smithsonian Institution. pp. 110-153. 41 8. APPENDICES 42 Table 1. Probability Values for Chi-Square Analysis Probabilities df 0.95 0.90 0.70 0.50 0.30 0.20 0.10 0.05 0.01 1 .004 .016 .15 .46 1.07 1.64 2.71 3.84 6.64 2 .10 .21 .71 1.39 2.41 3.22 4.61 5.99 9.21 3 .35 .58 1.42 2.37 3.67 4.64 6.25 7.82 11.35 4 .71 1.06 2.20 3.36 4.88 5.99 7.78 9.49 13.28 5 1.15 1.61 3.00 4.35 6.06 7.29 9.24 11.07 15.09 6 1.64 2.20 3.83 5.35 7.23 8.56 10.65 12.59 16.81 7 2.17 2.83 4.67 6.35 8.38 9.80 12.02 14.07 18.48 8 2.73 3.49 5.53 7.34 9.52 11.03 13.36 15.51 20.09 9 3.33 4.17 6.39 8.34 10.66 12.24 14.68 16.92 21.67 10 3.94 4.87 7.27 9.34 11.78 13.44 15.99 18.31 23.21 Acceptable Unacceptable Note. From Statistical Tables for Biological and Medical Research (6th ed.), Table IV, by R.Fisher and F.Yates, Edinburgh: Longman Essex, 1963. 43 Figure.1. Pictures of Students during blood typing. 44 Figure 2. Pictures of technicians while determining blood types of the students. 45 Consent form I the undersigned have been informed and understand that the purpose of this particular research project is to find out the distribution of ABO and Rh blood group alleles among Students of Silti Secondary and Preparatory School, Silte zone, Ethiopia. I have also been informed that the information that is obtained from me will be treated confidentially. Furthermore, I have been told that I can refuse to participate in the study. Hence, with this understanding, I hereby agree to participate in this particular research voluntarily. Name of the student __________________ Age ___________________ Signature __________________ Date: __________________ 46 Ethical clearance for the research 47 48 49 50