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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
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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