Kurdistan Regional Government-Iraq Presidency of Ministerial Council Ministry of Higher Education and Scientific Research University of Sulaymaniyah Faculty of Medical Sciences School of Medicine Department of Pathology and Forensic Pathology __________________________________________ _________________________________________-
Phenotype Frequencies of Blood Group Systems (ABO, Rh, Kell, Kidd, Duffy, MNSs, Lutheran, Lewis and P) in Blood Bank Donors in Sulaymaniyah Province A Thesis submitted to the Council of the School of Medicine, Faculty of Medical Sciences at the University of Sulaymaniyah in partial fulfillment of the requirements for the degree of Master of Science in Hematology By Shaema Salih Amin M.B.Ch.B. /High Diploma Hematopathology
Supervised by
Dr. Hisham A. Getta M.B.Ch.B./FIBMS Lecturer in Hematopathology
January 2015 A.D
Rebandan 2715 K
من الرحي ْم َ ِِب ْس ِم هللا ِ الر ْح
" " و اءذا مرضت فهو يشفني
َ ص ظي ْم َ ُدَق هللا َ ِ ٲلع
(80)من سورة الشعراء االية
Linguistic Evaluation Certification This is to certify that Dr. Bakhtiar Sabir Hama has proofread this dissertation entitled “Phenotype Frequencies of Blood Group Systems (ABO, Rh, Kell, Kidd, Duffy, MNSs, Lutheran, Lewis and P) in Blood Bank Donors in Sulaymaniyah Province”, prepared by Shaema Salih Amin.
After marking and correcting the mistakes, the thesis was handed again to the researcher to make the corrections in this last copy.
Proofreader: Dr. Bakhtiar Sabir Hama Date: 07/12/2014 Department of English, School of Languages, Faculty of Humanities, University of Sulaymaniyah.
CERTIFICATE OF ORIGINAL WORK This is to certify that the dissertation entitled “Phenotype Frequencies of Blood Group Systems (ABO, Rh, Kell, Kidd, Duffy, MNSs, Lutheran, Lewis and P) in Blood Bank Donors in Sulaymaniyah Province” submitted in partial fulfillment of the requirements for the award of the degree of M.Sc. in Hematology from University of Sulaymaniyah, Faculty of Medical Sciences,
School of Medicine, Sulaymaniyah, Iraq is a record of bona fide work carried out by Shaema Salih Amin under my supervision and guidance and practical work was carried in the department of Hematology, Public Health Laboratory, Sulaymaniyah. The report embodies results of original work and the studies carried out by the student herself. No part of this dissertation has been submitted for any other degree.
Date: Place: Sulaymaniyah
Lecturer Dr. Hisham A.Getta Supervisor Department of Pathology and Forensic Pathology Sulaymaniyah, Iraq
In view of the available recommendation, we forward this thesis for debate by the examining committee.
Signature Asst. Prof. Dr. Sana D. Jalal M.B.Ch.B., FIBMS Head of-Department of Pathology and Forensic Pathology University of Sulaymaniyah Faculty of Medical Sciences School of Medicine Date:
CERTIFICATE OF ACCEPTANCE OF EVALUATION COMMITTEE
The dissertation entitled, “Phenotype Frequencies of Blood Group Systems (ABO, Rh, Kell, Kidd, Duffy, MNSs, Lutheran, Lewis and P) in Blood Bank Donors in Sulaymaniyah Province” has been prepared and submitted by Shaema Salih Amin in partial fulfillment of the requirements for the award of degree of M.Sc. in Hematology, Department of Pathology and Forensic Pathology, University of Sulaymaniyah. NAME
EVALUATION
SIGNATURE
Dr. Sana D. Jalal (Member) Assistant Professor Department of Pathology and Forensic Pathology
Dr. Chro N. Fatah (Member) Assistant Professor Department of Obstetric and Gynecology
Dr. Hisham A. Getta (Supervisor) Lecturer Department of Pathology and Forensic Pathology
The Dissertation has been examined by the Evaluation Committee and found………...
Date: Place:
Prof. Dr. Anwar Sheikha (Chairman) Evaluation Committee
SELF ATTESTATION I hereby declare that the research work embodied in this dissertation entitled “Phenotype Frequencies of Blood Group Systems (ABO, Rh, Kell, Kidd, Duffy, MNSs, Lutheran, Lewis and P) in Blood Bank Donors in Sulaymaniyah Province” has been carried out by me for the award of degree of M.Sc. Hematology in the department of Hematology, Public Health Laboratory, Sulaymaniyah. I also affirm that the work is original and has not been submitted in part or full for any degree or diploma to this or any other university.
Date: Place:
Shaema Salih Amin
Approved by the Council of the School of Medicine
Assist. Prof. Dr. Ari Sami Hussain Nadhim MBChB, FICMS, FRCS, FAANS Dean-School of Medicine Faculty of Medical Sciences University of Sulaymaniyah
Dedication
To: My great and supportive mother.
My beloved husband and sons.
All my family and friends.
I
Acknowledgments
Acknowledgements Thanks and praises to Al-MIGHTY ALLAH for giving me willingness and patience to complete this study. Special thanks and appreciation go to the Ministry of Higher Education and Scientific Research, University of Sulaymaniyah, School of Medicine for their support and facilities to continue my postgraduate study. All respect and appreciation is due to Asst. Prof. Dr. Ari Sami Hussain. Dean of School of Medicine/ University of Sulaymaniyah, for his continuous support and encouragement. I would like to express my thanks to Asst. Prof. Dr. Nabil AM Salmo, in the Department of Pathology and all teaching staff at the department for their support. My thanks and appreciations go to my supervisor Dr. Hisham A. Getta, for his role, advice and efforts in supervising this thesis. Special thanks and deepest gratitude to my dearest and best friend Dr. Aveen M. Raouf Abdulqader for her supports, continuous help and guidance during all the period of the study. I would like to express my thanks and appreciation to Ms. Kalsum Rasul Xdr, Manager of Sulaymaniyah-Blood Bank for her support and cooperation. I would like to express my deepest gratitude and special thanks to Mr. Aso Rahman Salih, Mr. Kaiwan Hasan Qadr, and Mr. Nasraden Muhamad Faraj, and all the other medical staff in the Blood Bank in Sulaymaniyah Province for their great help, support, and cooperation. I would also like to express my thanks to my husband Dr. Omed Hamed Ali for his patience and help during my study and to my merciful mother Maeeda Majeed and the rest of my loving family.
II
Abstract
ABSTRACT Background: Human blood groups are polymorphic and inherited integral structures of the red cell membrane. More than 300 red cell antigens have been identified and further categorized into 33 major discrete systems. Their distribution varies in different communities and ethnic groups. Identification of different blood group antigen in a population has various benefits in transfusion medicine. Most data in the literature have determined European, American and some Asian phenotype of blood groups, but none has been reported in Kurdistan Region of Iraq, except for ABO and Rh blood group systems.
Objectives: To determine the antigen and phenotype frequencies of various blood group systems in the local donor population and to create a donor database of these blood groups to improve transfusion services and for future multipurpose utilities: 1) Preparation of reagent panels cell to be used in antibody detection and identification. 2) Providing antigen compatible blood units for prevention of alloimmunization,
particularly
in
young
female,
pregnant
women
and
multitransfused patients. 3) Providing antigen negative blood for already alloimmunized multitransfused patients. 4) Donor registry for rare blood groups.
Subjects and Methods: This is a cross-sectional study in which a total of five thousands healthy voluntary blood donors in Central Blood Bank in Sulaymaniyah Province were typed for ABO and Rh (D) blood group system by gel technique. Out of these, 500 donors were typed for other Rh blood group antigens: Rh (C, c, E, e) and Kell (K) and 400 donors for extended antigen typing of other blood group systems: k (Cellano), Kidd, Duffy, MNSs, Lutheran, Lewis and P by conventional tube technique. Antigens and phenotype frequencies were expressed in percentages. III
Abstract
Results: In the ABO blood group, the most common phenotype was O (37%), followed by A (32.6%) and B (22.8%), whereas the lowest prevalent blood group was AB (7.6%). Among the Rh blood group antigens, e was the most common (95.2%) followed by D (91.3%), C (74.8%), c (69.4%), and E (30.6%) with DCe/DCe (R1R1) and dce/dce (rr) were the most common phenotypes among Rh (D) positive and Rh (D) negative groups, respectively. In the Kell blood group system, K was found to be positive in 5.8% of donors and no K+k- phenotype was found. For Kidd and Duffy blood group systems, Jk (a+b+) and Fy (a+b-) were the most common phenotypes (44.5% and 38.5%, respectively). In the MNS blood group system, M+N+S-s+ (40%) was the most common. Regarding the Lewis and Lutheran systems, the most common phenotypes were Le (a-b+) and Lu (a-b+) which were (54.5% and 92%, respectively). We found some rare phenotypes such as Fy (a-b-), Lu (a-b-), and Le (a+b+) which were (4%, 3.5%, and 10%, respectively). P1antigen was found in 76% of the donors.
Conclusion: In the ABO and Rh blood group systems, the most common blood groups were O, and e antigen, respectively. In the Kell, Kidd, Duffy, MNSs, Lutheran, Lewis, and P blood group systems, the most common phenotypes were [(K-k+), Jk (a+b+), Fy (a+b-), M+N+S-s+, Lu (a-b+), Le (a-b+), and P1, respectively]. The distribution of these blood group systems in our donor population was very close to previous studies in Kurdistan Region, other parts of Iraq, and Iran, with similar trends to the neighboring Arab countries, some Asian (India), and western Europeans (Caucasian), while it was different from that of the Black population. Some rare phenotypes such as Fy (a-b-), Lu (a-b-), and Le (a+b+) were also determined.
IV
Table of Contents Subject
Page
Dedication
I
Acknowledgments
II
Abstract
III
Table of Contents
V
List of Tables
XI
List of Figures
XIV
List of Abbreviations and Acronyms
XV
Introduction
1
Aims of the Study
6 Chapter One: Literature Review
1.1 Human Blood Group Systems
7
1.2 Antigens
8
1.2.1 The Blood Group Antigens
8
1.2.2 The Biological Significance of Blood Group Antigen
10
1.2.3 Model of Antigen Carrier Proteins
11
1.3 Antibodies
11
1.3.1 Classes of Immunoglobulin
12
1.3.2 Blood Group Antibodies
12
1.3.2.1 Naturally Occurring and Immune Antibodies
12
V
1.3.2.2 Cold and Warm Antibodies
13
1.3.2.3 IgM and IgG Antibodies
13
1.3.2.4 Clinical Significance of Red Cell Alloantibodies
14
1.4 Blood Group Antigen-antibody Reactions
15
1.4.1 Detection of Red Cell Antigen-Antibody Reactions
17
1.4.1.1 Principle of Agglutination Techniques
17
1.4.1.1.A Direct Agglutination
17
1.4.1.1.B Indirect Agglutination
17
1.4.1.2 Antiglobulin or Coombs Test
17
1.4.1.2.A Direct Antiglobulin Test (DAT)
18
1.4.1.2.B Indirect Antiglobulin Test (IAT)
18
1.4.1.3 Haemolysis
19
1.5 Techniques in Blood Group Serology
19
1.5.1 Slide Method
19
1.5.2 Tube Test
20
1.5.2.1 Reading Results of Tube Tests
20
1.5.2.1.A Macroscopic Reading
20
1.5.2.1.B Microscopic Reading
21
1.5.2.1.C Demonstration of Lysis
21
1.5.3 Microcolumn Test (gel and beads)
22
1.5.4 Microplate Technique
23
1.5.5 Automated Techniques
24 VI
1.5.6 Molecular Techniques for Blood Grouping
24
1.6 Hemolytic Transfusion Reaction
25
1.7 Alloimmune Haemolytic Disease of the Fetus & Newborn
26
1.7.1 Historical Overview
26
1.7.2 Spectrum of Hemolytic Disease of the Fetus and Newborn
27
1.7.2.1 ABO Hemolytic Disease of the Fetus and Newborn
27
1.7.2.2 Rh Hemolytic Disease of the Fetus and Newborn
27
1.7.2.3 Hemolytic Disease of the Fetus and Newborn due to other Antibodies
28
1.8 The ABO Blood Group System
29
1.8.1 History
29
1.8.2 Antigens of The ABO System
29
1.8.2.1 ABO Subgroups
30
1.8.2.2 H Antigen
31
1.8.2.3 Biochemistry and Biosynthesis of ABH Antigens
31
1.8.3 ABO Encoding Genes and H Genes
32
1.8.3.1 ABO Encoding Genes
32
1.8.3.2 H Genes
33
1.8.4 ABO Antibodies
34
1.8.4.1 Anti-A and Anti-B
34
1.8.4.2 Anti-A1 and Anti-H
34
1.8.5 Secretors and Non-Secretors
35
VII
1.9 Rhesus Blood Group System
36
1.9.1 Rh Antigens
36
1.9.1.1 Variants of D
38
1.9.1.1.A Weak D
39
1.9.1.1.B partial D
39
1.9.1.2 Other Rh Antigens
39
1.9.1.2.A Rh Null
39
1.9.1.2.B G, Cw and Cx, VS and V Antigens
40
1.9.2 Rh Encoding Genes
40
1.9.3 Antibodies of the Rh System
42
1.9.3.1 Naturally Occurring Rh Antibodies
42
1.9.3.2 Immune Rh Antibodies
42
1.10 The Kell Blood Group System
43
1.10.1 Antigens and Encoding Genes
43
1.10.2 Kell Antibodies
43
1.11 The Kidd Blood Group System
44
1.11.1 Kidd Antigens and Encoding Genes
44
1.11.2 Kidd Antibodies
44
1.12 The Duffy Blood Group System
45
1.12.1 Duffy Antigens and Encoding Gene
45
1.12.2 Duffy Antibodies
46
1.13 The MNS Blood Group System
46 VIII
1.13.1 The MNS Antigens and Encoding Genes
46
1.13.2 Antibodies of the MNS System
47
1.14 The Lutheran Blood Group System
47
1.14.1 Lutheran Antigens and Encoding Genes
47
1.14.2 Lutheran Antibodies
48
1.15 The Lewis Blood Group System
48
1.15.1 Lewis Antigens and Encoding Genes
48
1.15.2 Antibodies of the Lewis System
50
1.16 The P System and Globoside Collection
50
1.16.1 Antigens
50
1.16.2 Antibodies
51 Chapter Two: Subjects and Methods
2.1 Study Design
52
2.2 Donor Selection
52
2.3 Sample Collection
52
2.4 Performing ABO & Rh (D) Blood Grouping
52
2.4.1 Additional Reagents and Materials required
53
2.4.2 Preparation of Blood Samples
53
2.4.3 Test Procedure
53
2.4.4 Interpretation of Results for the Gel Test
54
2.5 Extended Red Cell Antigen Typing
54
IX
2.5.1 Preparation of Red Cell Suspension
54
2.5.2 The Reagents Required
54
2.5.2.A Additional Reagents & Materials Required
55
2.5.3 Test procedure
56
2.5.4 Interpretation of Test Results
57
2.6 Statistical Method
58 Chapter Three: Results
3. Results
65
3.1 The ABO Blood Group System
65
3.2 The Rh Blood Group System
66
3.3 The Distribution of Kell, Kidd, Duffy, MNSs, Lutheran, Lewis, and P 68
Blood Group Systems 3.3.1 Red Cell Antigens of Kell, Kidd, Duffy, MNSs, Lutheran, Lewis,
68
and P Blood Group Systems 3.3.2 Phenotype frequencies of Kell, Kidd, Duffy, MNSs, Lutheran,
70
Lewis, and P blood group systems Chapter Four: Discussion 4. Discussion
74
4.1 The ABO Blood Group System
75
4.2 The Rhesus Blood Group System
78
4.2.1 The Rhesus (D) Antigen
78
4.2.2 Other Rhesus Antigens (C, c, E, e)
81
X
4.2.3 The Rhesus Phenotypes
84
4.3 The Kell Blood Group System
85
4.4 The Kidd Blood Group System
87
4.5 The Duffy Blood Group System
89
4.6 The MNSs Blood Group System
91
4.7 The Lutheran Blood Group System
94
4.8 The Lewis Blood Group System
96
4.9 The P Blood Group System
98
Chapter Five: Conclusions and Recommendations 5.1 Conclusions
99
5.2 Recommendations
100
References
101
الخالصة ثوختة
List of Tables Table No.
Table Name
Page
1.1
Clinically important blood group systems
9
1.2
Putative functions of molecules containing blood group antigens
10
XI
1.3
Antibody specificities related to the mechanism of immune haemolytic destruction
14
1.4
Relative frequency of immune red cell alloantibodies
15
1.5
Scoring of results in red cell agglutination test
21
1.6
The ABO blood group system
30
1.7
Secretor status in the Caucasian population
35
1.8
Antigens of the Rh system
37
1.9
The Rh haplotypes in order of frequency (Fisher nomenclature) in Caucasian and the corresponding short notations
41
3.1
Frequency of ABO and Rh (D) blood groups in the present study
66
3.2
Distribution of Rh antigens in D+ve and D–ve donors (N=500)
67
3.3
Rh phenotype frequencies in blood donors
68
3.4
Frequency of red cell antigens of Kell, Kidd, Duffy,MNSs, Lutheran, Lewis and P blood group systems in (N=400)
69
3.5
Phenotype frequencies of Kell, Kidd, Duffy, Lutheran, Lewis, and P blood group systems
70
3.6
Phenotype frequency of MNSs blood group system
71
4.1
4.2
4.3
Antigen frequencies (%) of ABO blood group of the present study compared with other studies in Iraq, neighboring countries and some other populations Frequency of Rh (D) antigen in the present study compared with other studies in Iraq, neighboring countries and some other populations Antigen frequencies (%) of Rh (C, c, E, e) blood group in this study compared with published results XII
77
80
82
4.4
Rh phenotype frequencies of the present study compared with North Indian, northeast Iran, Caucasian and Black population
85
4.5
Antigen frequencies (%) of the Kell blood group system in this study compared with other published results
86
4.6
Phenotype frequencies of the Kell blood group system compared with other published results
87
4.7
Antigen frequencies (%) of the Kidd blood group system in this study compared with other published results
88
4.8
Phenotype frequencies (%) of the Kidd blood group system in this study compared with other published results
89
4.9
Antigen frequencies (%) of the Duffy blood group system in this study compared with other published results
90
4.10
Phenotype frequencies (%) of the Duffy blood group system in this study compared with other published results
91
4.11
Antigen frequencies (%) of the MNSs blood group system in this study compared with other published results
92
4.12
Phenotype frequencies (%) of the MNSs blood group system in this study compared with other published results
93
4.13
Phenotype frequencies of MNSs blood group system compared to other published data
94
4.14
Antigen frequencies (%) of the Lutheran blood group system in this study compared with other published results
95
4.15
Phenotype frequencies (%) of the Lutheran blood group system in this study compared with other published results
96
4.16
Antigen frequencies (%) of the MNSs blood group system in this study compared with other published results
97
4.17
Phenotype frequencies (%) of the MNSs blood group system in this study compared with other published results
98
XIII
List of Figures Figure No.
Figure Name
Page
1.1
Model of RBC membrane components that carry blood group antigens
11
1.2
Structure of the basic immunoglobulin molecule
12
1.3
The antiglobulin test for antibody or complement on the surface of red blood cells (RBC)
18
1.4
Macroscopic appearances of agglutination in round-bottom tubes or hollow tiles
20
1.5
Results of a gel microcolumn test
22
1.6
Biosynthetic pathway of H antigen from its precursor, and of A and B antigens from H
32
1.7
Rh and related genes and the polypeptides they encode
40
1.8
Diagrammatic representation of H and Lewis antigens
49
2.1
A. and B. ABD gel card, ”DiaClon ABD-Confirmation for Patients” (Bio-Rad Laboratories, DiaMed Switzerland)
59
2.2
2.3
2.4
2.5
A. ABD gel card set ”DiaClon ABD-Confirmation for Patients” and diluents B. ID-Centrifuge 24. S (BIO-RAD, Switzerland) A. DiaClon ABD-confirmation for patients, (Bio-Rad Laboratories, DiaMed Switzerland) B. e.g. 49. An A+ve blood group donor e.g. 50. An O+ve blood group donor A. e.g. 9 and 10 a B+ve blood group donors B. e.g. 3. An O+ve blood group donor e.g. 4. An AB+ve blood group donor A. and B. Extended red cell antigen typing reagents (Rapid Labs Limited, England) C. Extended red cell antigen typing sets XIV
60
61
62
63
2.6
A. Centrifuge (Rotina 380 Hettich, Germany) B. Incubator (binder, Germany)
64
3.1
ABO blood group distribution in the present study (N=5 000)
65
3.2
Distribution of Rh (D) blood group in the present study (N=5 000)
66
List of Abbreviations and Acronyms Symbol
Detail
ABO
ABO Blood Group System
AHG
Antihuman Globulin
AIHA
Auto Immune Hemolytic Anemia
C3 DARC
Complement 3 Duffy Antigen Receptor for Chemokines
DAT
Direct Antiglobulin Test
DNA
Deoxyribonucleic Acid
EDTA FUT Fy
Ethylene Diamine Tetra Acetic acid Fucosyltransferase Duffy
GPA
Glycophorin A
GPB
Glycophorin B
HDFN HTR
Hemolytic Disease of Fetus and Newborn Hemolytic Transfusion Reaction XV
IAT
Indirect Antiglobulin Test
IgA
Immunoglobulin A
IgD
Immunoglobulin D
IgE
Immunoglobulin E
IgG
Immunoglobulin G
IgM
Immunoglobulin M
ISBT
International Society of Blood Transfusion
Jk
Kidd
K
Kell antigen
k
Cellano antigen
(KB)
Kilobases
kDa
Kilo Dalton
Le LISS
Lewis Low Ionic Strength Saline
Lu
Lutheran
LW
Landsteiner and Wiener
ml
Milliliter
ΜL
Microliter
PBS
Phosphate Buffered Saline
PCH
Paroxysmal Cold Hemoglobinuria
P value RBC
Probability value Red Blood Cell XVI
Rcf
Relative Centrifugal Force
Rh
Rhesus
RHAG Rpm Se
Rhesus Associated Glycoprotein Round Per Minute Secretor
SPSS
Statistical Package for Social Science
TTD
Transfusion-transmissible Disease
v 21
Version 21
+ve
Positive
-ve
Negative
XVII
Introduction
Introduction Human blood group antigens are integrated parts and unique structure of the red blood cells (RBCs) membrane, characterized by inherited polymorphisms and have many essential functions and different biochemical compositions. Since the discovery of the ABO blood group by Landsteiner in early twentieth century, different blood typing systems have been devised, and later they were found to be important determinants in transfusion medicine (Cartron & Colin, 2001; Thakral et al. 2010). According to The International Society of Blood Transfusion (ISBT), there are 287 antigens within the 33 blood group systems and 42 antigens in collections (low and high frequency antigens). Nine of the blood group systems (ABO, Rh, Kell, Kidd, Duffy, MNS, P, Lewis and Lutheran) are considered to be clinically significant (Tilley et al. 2010; Lӧgdberg et al. 2011). Each system represents either a single gene or a cluster of two or three closely-linked homologous genes (Reid, 2012). Nonetheless, ethnic differences are seen in the frequency of antigens (Barclay, 2001; Beadling & Cooling, 2007; M’baya et al. 2010). The blood group antigens are of clinical importance in blood transfusion, organ transplantation, autoimmue hemolytic anemia (AIHA), fetomaternal blood group incompatibility, paternity identification, and in forensic medicine (Daniels, 2007). The ABO blood group system was discovered by Karl Landsteiner, Decastello, and Sturli (Sealey et al. 1998; Ali et al. 2005). Its inheritance described by Bernstein in1924 (Crow, 1993), occurs from both parents through allelomorphic genes A, B, O resulting in different phenotypes A, B, AB, and O (Oka et al. 1982; Yazer et al. 2006). The A, B, and O antigens were originally found on the surface of red cells, but later they were also found on surface of various types of cells as well as in 1
Introduction
secretions. This includes platelets, lung tissues, intestinal mucosa, mucous cells, epidermis, nervous receptors and vascular epithelium (Hartman, 1941; Orial, 1992). The ABO blood group antigens are encoded by one genetic locus, the ABO locus, which has three alternative (allelic) forms A, B, and O, located on the long arm of chromosome 9 (Chester & Olsson, 2001). Anti-A and anti-B antibodies are usually naturally occurring IgM with immune forms produced by either transfusion or pregnancy. These antibodies are a potential cause of dangerous haemolytic transfusion reactions (HTR), if transfusions are given without regard to ABO compatibility (Schonewille et al. 2006). Hyperimmune IgG anti-A and/or anti-B from group O or group A2 mothers may cross the placenta and cause haemolytic disease of the fetus and newborn (HDFN) (Jeon H et al. 2000). The Rh system is the most polymorphic and the most clinically significant blood group system beside ABO system; currently it is composed of 50 antigens expressed by the genes on chromosome 1. The important antigens are: RhD, RhC, RhE, Rhc and Rhe. They are encoded by two closely linked genes RHD and RHCE. The Rh antibodies are considered potential agents of hemolytic disease of the fetus and newborn (HDFN), and hemolytic transfusion reaction (HTR) (Wagner & Flegel 2000; Sarkar et al. 2013). Regarding the Kell blood group system, it is composed of 34 antigens (K1K34); two major antigens K (Kell) and k (Cellano) are significant. The K and k genes are codominant alleles located on chromosome 7 that code for the antigens; well developed at birth, the K antigen is very immunogenic (second to the D antigen) in stimulating antibody production. Anti-K is an important antibody, it is nearly always immune, IgG and complement-binding. It causes severe hemolytic
2
Introduction
disease of fetus and newborn (HDFN) and hemolytic transfusion reaction (HTR) (Storry & Olsson, 2000). The Kidd blood group system has two alleles, Jka and Jkb, located on the chromosome 18. Anti-Jka is more common than Jkb; both are usually IgG. They are clinically significant because they are implicated in severe hemolytic transfusion reaction (HTR) and to a lesser extent, hemolytic disease of the fetus and newborn (HDFN). Kidd antibodies have often been implicated in delayed hemolytic transfusion reaction; they are IgG and predominantly complement fixing (Lucin et al. 2002). The Duffy locus is on chromosome 1; the locus has the following alleles: Fya, Fyb which code for the codominant Fya and Fyb antigens. Anti-Fya is much more common than Fyb, they are IgG, clinically significant, stimulated by transfusion or pregnancy (Denomme et al. 2000). The MNS system now contains 46 antigens; four of them are important antigens (M, N, S and s). M and n located on Glycophorin A while S and s located on Glycophorin B. The Glycophorin is a protein that carries many RBC antigens. Anti-M antibodies may be IgM or IgG; rare examples are reactive at 37˚c when they can give rise to HTR. Anti-N is nearly always a cold reactive IgM antibody and of no clinical significance. Anti-S and anti-s are usually IgG; both rarely have been implicated in HTR and HDFN (Placajornsuk, 2006). The Lewis antigens (Lea and Leb) are located on soluble glycosphingolipids found in saliva and plasma and are secondarily absorbed into the red cell membrane from the plasma. The Le gene at the LE locus is located on chromosome 19. When Secretor (Se) and Le are resent, the Le b antigen is produced; when Le but not Se is present, Lea is produced; and when Le is not present, neither Le a nor Leb is produced. The Lewis antigens are poorly developed at birth and red cells from cord blood are usually Le (a-b-). Lewis antibodies are naturally occurring in 3
Introduction
those who are Le (a-b-) and are usually IgM and complement binding. Anti-Lea is usually more hemolytic than anti-Leb. However only rare examples of anti-Lea that are strictly reactive at 37˚c give rise to HTR. Lewis antibodies do not cause HDFN as they are almost always IgM and do not cross the placenta and newborn have Le (a-b) red cells (Pourazar et al. 2004). Lutheran is a complex system comprising four pairs of allelic antigens: Lua and Lub, Aua and Aub, Lu6 and Lu9, Lu8 and Lu14. These represent single amino acid substitutions in the Lutheran glycoproteins. The antigens are encoded by the LU gene located at chromosome 19. Anti-Lua is uncommon and rarely of clinical significance, anti-Lub has been implicated in mild delayed HTRs (Inderbitzen & Windle, 1982). In the P blood group system, the most common phenotypes are P1 and P2. P1 consists of P1 and p antigens; P2 consists of only p antigen. Anti-P1 is naturally occurring IgM antibody of no clinical significance. Anti-p is produced in individual with paroxysmal cold hemoglobinuria (PCH); this PCH antibody is also called the Donath-Landsteiner antibody (Regan, 2012). Although blood transfusion can be life saving for a number of patients, they are not without risks. In addition to risks such as transfusion-transmissible diseases (TTD) caused by donor viruses, parasites, or bacterial contaminants of blood products, there is also a risk of alloimmunization due to donor-recipient antigen phenotype disparity (Eder & Chambers, 2007). In developing countries like Iraq, only ABO and Rh (D) status of blood donor and recipients are taken into account for compatibility testing. However, the phenotype of clinically significant blood group antigens on the donor red blood cells is required to be known at times when alloimmunization is particularly undesirable, such as in young females, pregnant women, and patients who are expected to require repeated transfusion in life, such as thalassaemia or sickle cell 4
Introduction
disease patients, cancer patients, patient on dialysis…..etc. When selecting blood for transfusion to such patients, it would be useful if we have access to already phenotyped RBCs of donor population so that particular antigen typed blood can be given to such patients to prevent alloimmunization. Furthermore, these are beneficial for already immunized patients if the transfusion is urgent and/or if clinically significant alloantibodies to particular antigen/antigens are present in the patient's serum. In such situations, corresponding antigen negative blood can be given to such recipients without much delay (Diedrich et al. 2001). Alloimmunization results from previous exposure to donor blood components. Even very small amount of donor antigens (especially on RBC) may elicit a significant alloimmune response. This will lead to difficulty in finding compatible blood, transfusion reactions, or refractoriness to platelets (Schonewille et al. 2006; Makroo, 2007). Racial differences in blood group antigen distributions are common and may result in striking and interesting findings. Most data on literature have determined European, American and some Asian phenotype of blood group systems. No information is available in the Kurdistan Region of Iraq. The present study was initiated to determine the extended red cell antigen and phenotype frequencies of various clinically significant blood groups amongst regular voluntary blood donors in Sulaymaniyah-Iraq and also to lay foundation of starting a donor database on RBC antigens.
5
Aims of the Study
Aims of the Study 1. To determine the antigens and phenotype frequencies of various blood group systems in the local donor population. 2. To establish a donor database of these blood groups to improve transfusion services and for future multipurpose utilities: Preparation of indigenous cell panels to be used in antibody detection and identification. Providing
antigen
compatible
blood
units
for
prevention
of
alloimmunization, particularly in young females, pregnant women, and multitransfused patients like thalassaemia, cancer patients, and patients on hemodialysis…….etc. Providing
antigen
negative
blood
for
already
alloimmunized
multitransfused patients for prevention of further alloimmunization. Donor registry for rare blood groups.
6
Chapter One
Literature Review
1.1 Human Blood Group Systems Human blood groups are unique surface membrane structures of red blood cells (RBCs), characterized by inherited polymorphisms. Since the discovery of the ABO system early in the twentieth century, they have been used as genetic markers of human polymorphism. Many blood group antigens and their genes have been identified, and their physiological roles uncovered (Storry, 2003), and later they were found to be important determinants in transfusion medicine (Daniels & Bromilow, 2007). The primary goal of any blood transfusion is to provide the patient with donor red blood cells that optimally survive after transfusion and serve their function and ensure that the patient actually benefits from the transfusion. To achieve this goal, donor red cells that are compatible with those of the patient’s blood are selected for transfusion (Chapman, 2004). Blood classification into groups is based on the presence or absence of inherited antigenic substances on the surface of red blood cells. Some of these antigens are also present on the surface of other types of cells and body secretions like saliva, sweats, semen, serum, tears, urine etc. which are used in forensic investigations (Brian et al. 2000). Several of these RBC surface antigens that stem from one allele (or very closely linked genes) collectively form a blood group system. Blood groups are genetically determined and exhibit polymorphism in different populations. They are inherited from both parents (Ganong, 2001). The main clinical importance of a blood group system in blood transfusion depends on the capacity of alloantibodies (directed against the antigens not possessed by the individual) to cause destruction of transfused red cells and cause haemolytic transfusion reaction or to cross the placenta and give rise to haemolytic disease in the fetus or newborn. This in turn depends on the frequency of the antigens and the alloantibodies and the characteristics of the latter: thermal range, immunoglobulin class and ability to fix complement. On 7
Chapter One
Literature Review
these criteria, the ABO and Rh systems are of major clinical importance (Hoffbrand & Moss, 2011). Hemolytic transfusion reactions (HTR) are premature destruction of transfused red cells reacting with antibodies in the recipient. Naturally occurring antibodies, such as ABO antibodies, are immunoglobulin M (IgM) and, if warm-reacting, can destroy red cells in vivo by complement fixation. Red cell alloantibodies are usually IgG and form in response to exposure, through previous transfusions or pregnancies. HTRs may occur immediately after the transfusion or may be delayed for anything up to 2 – 3 weeks (Schonewille et al. 1999; Eder & Chambers, 2007).
1.2 Antigens Originally, an antigen was defined as the part of a molecule that is bound by a specific antibody. More recently, it has become customary to define an antigen
as
a
substance
that
can
stimulate
an
immune
response
(immunogenicity). Immune response can be either positive or negative. Positive responses lead to the production of antibodies (humoral immunity) and/or proliferation of immunocompetent cells (cellular immunity) that can bind and eliminate their stimulatory antigen. In negative responses, the cells that mediate humoral and cellular immune responses are rendered non-responsive. This state is described as acquired immunological tolerance and is important in preventing autoimmune disease, as well as in establishing the ‘take’ or acceptance of transplanted syngeneic and allogeneic tissues ( Avent, 2009).
1.2.1 The Blood Group Antigens Human blood group antigens are unique, inherited polymorphisms on the extracellular surface of red blood cells. They have been used as genetically discrete markers of human polymorphism since the discovery of the ABO system in 1900. Since then, many blood group antigens have been identified, the 8
Chapter One
Literature Review
genes cloned, and their biological significance elucidated. Blood group antigens and antibodies play an important role in Transfusion Medicine (Jill, 2003). A total of 329 red blood cell antigens are now recognized by the International Society of Blood Transfusion (ISBT) and classified into systems, collection, low-frequency antigens, and high-frequency antigens. Of these 329 antigens, 287 of them are clustered into 33 major discrete blood group systems (Reid, 2012). Nine of which (ABO, Rh, Kell, Kidd, Duffy, MNS, P, Lewis and Lutheran) are considered to be clinically significant (Table 1.1), as these are known to cause hemolytic transfusion reaction (HTR) and hemolytic disease of the fetus and newborn (HDFN) (Smart & Armstrong, 2008; Daniels et al. 2009). Table 1.1: Clinically important blood group systems (Hoffbrand & Moss,2011).
Racial differences in blood group antigens distribution are common and may result in striking and interesting findings. These differences in blood group antigen distribution are important due to their influence on the clinical practice of transfusion medicine (Lamba et al. 2013).
9
Chapter One
Literature Review
1.2.2 The Biological Significance of Blood Group Antigens Blood group antigens are integral part of the red blood cell membrane and have many essential functions (membrane transporters and protein canals, ligand receptor, adhesion molecules, enzyme, and structural proteins) (Carton & Colin, 2001). The structure and functions of the membrane proteins and glycoproteins carrying blood group antigens have been reviewed by Daniels (2007). An illustration of the putative functions of molecules containing blood group antigens is provided in (Table 1.2).
Table 1.2: Putative functions of molecules containing blood group antigens (Regan, 2012).
10
Chapter One
Literature Review
1.2.3 Model of Antigen Carrier Proteins Blood group antigens are surface markers on the red cell, and consist of proteins and carbohydrates attached to lipids or proteins. A model of the membrane components carrying blood group antigens is shown in (Figure 1.1).
Figure 1.1: Model of RBC membrane components that carry blood group antigens (Reid ME et al. 1997).
1.3 Antibodies Antibodies are immunoglobulins produced by the B lymphocytes of the adaptive immune system in response to an antigen for which they exhibit specific binding. Depending on the origin of the antigenic stimulus, antibodies can be termed (i) alloantibodies, when produced by an individual against epitopes present in another individual of the same species; (ii) autoantibodies, when reactive with determinants present on the individual’s own antigens; and (iii) xenoantibodies (or heteroantibodies), when produced against antigenic determinants present on the cells of another species. The first two are the antibodies encountered routinely in the blood bank (Poel et al. 2002).
11
Chapter One
Literature Review
1.3.1 Classes of Immunoglobulin There are five classes of immunoglobulins: IgM, IgG, IgA, IgD and IgE. Antibodies with specificity for blood group antigens are found only in the IgG, IgM and, rarely, IgA classes. IgA antibodies play a minor role in blood group serology as they only appear as alloantibodies together with IgM and/or IgG (McPherson & Pincus, 2006). A diagram of the basic immunoglobulin molecule is provided in (Figure 1.2).
Figure 1.2: Structure of the basic immunoglobulin molecule (Contreras & Daniels, 2011).
1.3.2 Blood Group Antibodies 1.3.2.1 Naturally Occurring and Immune Antibodies Antibodies are naturally occurring when they are produced without any obvious immunizing stimulus, such as during pregnancy, transfusion or injection of blood. These antibodies are not present at birth and, in the case of anti-A and anti-B, start to appear in the serum at about 3 – 6 months of age. ABO antibodies are probably produced in response to antigens of bacteria, viruses and other substances that are inhaled or ingested; many Gram- negative organisms have antigens that are structurally similar to the A and B antigens. Despite this probable antigenic stimulus, the term ‘naturally occurring’ is 12
Chapter One
Literature Review
retained for these ‘non - red cell - induced’ antibodies. Immune blood group antibodies are only produced after pregnancy or following transfusion or injection of blood or blood group substances (Jeon et al. 2000).
1.3.2.2 Cold and Warm Antibodies Cold antibodies give higher agglutination titres at low temperatures (0– 4°C) and many of them will not agglutinate red cells at 37°C. Most naturally occurring antibodies are cold reacting. Some, such as naturally occurring anti A, B, have a wide thermal range and will still react at 37°C; at this temperature they will activate complement and lyse red cells, but the titre will be much higher at (0–4°C). Cold antibodies that fail to react above 30°C are of no clinical significance and can be ignored for blood transfusion purposes (Lee et al. 2000). The thermal optimum of warm antibodies is 37°C and this implies that higher titres are obtained at this temperature. Immune antibodies are warm reacting. Any red cell antibody reacting above 3°C should be considered potentially capable of destroying red cells in vivo (Daniels et al. 1995).
1.3.2.3 IgM and IgG Antibodies IgM or ‘complete’ antibodies agglutinate red cells when they are suspended in saline. They are often called saline or directly agglutinating antibodies in laboratory parlance. Conversely, ‘incomplete’ IgG antibodies will not agglutinate saline - suspended red cells. However, lack of agglutination does not mean that the antibodies have not bound to their antigen, and it can be shown that they have reacted by using antiglobulin reagents, which facilitate agglutination of antibody – coated cells. Most naturally occurring antibodies are cold reacting, complete and IgM. Immune antibodies are always warm reacting; most are partly IgG, but some may be IgM. Exceptionally, and when very potent, IgG complete antibodies are found (Goraya et al, 2001). 13
Chapter One
Literature Review
1.3.2.4 Clinical Significance of Red Cell Alloantibodies The clinical significance of different red cell antibodies depends partly on their destructive capacity and partly on their frequency. For example, anti-PP1P K (anti-Tja) is a very potent haemolysin, but it is of minimal importance in blood transfusion practice owing to its rarity. Conversely, ABO and D antibodies are by far the most significant, owing to their prevalence and destructive capacity (Contreras & Daniels, 2011). The significance of the alloantibodies described, with respect to the nature of the haemolytic transfusion reaction they produce, is provided in (Table 1.3).
Table 1.3: Antibody specificities related to the mechanism of immune haemolytic destruction (Regan, 2012).
The significance of blood group antigens other than those of the ABO system and D by looking at the prevalence of transfusion-induced red cell alloantibodies, excluding anti-D, -CD and -DE (Table 1.4). Rh antibodies, 14
Chapter One
Literature Review
mainly anti-c or anti-E, accounted for 53% of the total and anti-K and anti-Fya accounted for a further 38%, leaving only about 9% for all other specificities (Mollison et al. 1997). Table 1.4: Relative frequency of immune red cell alloantibodies (Regan, 2012).
1.4 Blood Group Antigen-antibody Reactions The red cell is a convenient marker for serological reactions. Agglutination or lysis (owing to complement action) is a visible indication (endpoint) of an antigen–antibody reaction. The reaction occurs in two stages: in the first stage the antibody binds to the red cell antigen (sensitization) and the second stage involves agglutination (or lysis) of the sensitized cells (Regan, 2012). The first stage (i.e. association of antibody with antigen-sensitization) is reversible and the strength of binding depends on the ‘exactness of fit’ between antigen and antibody. This is influenced by the following: 1. Temperature: Cold antibodies (usually IgM) generally bind best to the red cell at a low temperature (e.g. 4°C), whereas warm antibodies (usually IgG) bind most efficiently at body temperature (i.e. 37°C).
15
Chapter One
Literature Review
2. PH: There is relatively little change in antibody binding over the pH range 5.5–8.5, but to ensure comparable results, it is preferable to buffer the saline in which serum or cells are diluted to a fixed pH, usually 7.0. Some antibody elution techniques depend on altering the pH to <4 or >10. 3. Ionic strength of the medium: Low ionic strength increases the rate of antibody binding. This is the basis of antibody detection tests using low ionic strength saline (LISS).
The second stage depends on various laboratory manipulations to promote agglutination or lysis of sensitized cells. Agglutination is brought about by antibody crosslinking between cells. The agglutination of red cells coated by either IgM or IgG antibodies is enhanced by centrifugation. However, it is standard procedure to promote agglutination of IgG-sensitized red cells by the following: 1. Reducing intercellular distance by pretreatment of red cells with protease enzymes (e.g. papain or bromelin), which reduce the surface charge of red cells. 2. Adding polymers (e.g. albumin), although the mechanism by which albumin or other water-soluble polymers enhance agglutination is uncertain. 3. Bridging between sensitized cells with an anti-globulin reagent in the antiglobulin test (Regan, 2012).
Some complement-binding antibodies (especially IgM) may cause lysis in vitro (without noticeable agglutination), which can be enhanced by the addition of fresh serum as a source of complement. However, complement activation may only proceed to the C3 stage; in these circumstances cell-bound
16
Chapter One
Literature Review
C3 can be detected by the antiglobulin test using an appropriate anticomplement reagent (Shaz, 2009). 1.4.1 Detection of Red Cell Antigen – Antibody Reactions 1.4.1.1 Principles of Agglutination Techniques There are various ways of detecting antigen–antibody reactions in vitro. In manual methods, tubes, microplates or gels can be used. The most widely used methods employ the following techniques:-
1.4.1.1.A Direct Agglutination Most IgM antibodies will directly agglutinate the appropriate red cells suspended in saline. This method is used routinely for ABO and Rh (D) grouping using monoclonal antibodies (Knowles, 2001).
1.4.1.1.B Indirect Agglutination Apart from ABO, antibodies against most blood group antigens are IgG and generally will not produce direct agglutination of red cells. Such antibodies can be detected with the aid of agents that enhance agglutination, for example proteases, albumin and other colloids, and aggregating agents such as polybrene (Knowles, 2001).
1.4.1.2 Antiglobulin or Coombs Test The antiglobulin test (Coombs test) was introduced by Coombs and colleagues in 1945 (Coombs et al. 1945). It is a fundamental and widely used test in both blood group serology and general immunology. It is used to detect IgG antibodies that do not cause direct agglutination of red cells carrying the corresponding antigen when suspended in saline. When antihuman globulin (AHG) is added to human red cells coated with immunoglobulin or complement components, agglutination of the red cells indicates a positive test (Figure 1.3). 17
Chapter One
Literature Review
The antihuman globulin (Coombs’) reagent may be broad spectrum or specific for immunoglobulin G (IgG), IgM, IgA or complement (C3). Direct and indirect antiglobulin tests can be carried out (Hoffbrand & Moss, 2011).
Figure 1.3: The antiglobulin test for antibody or complement on the surface of red blood cells (RBC) (Hoffbrand & Moss, 2011).
1.4.1.2.A Direct Antiglobulin Test (DAT) It is used to test directly, with an antiglobulin reagent, for the presence of antibodies or complement components that are bound to the red cells in vivo. The anti-human globulin (AHG) reagent is added to washed red cells and agglutination indicates a positive test. A positive test occurs in hemolytic disease of the fetus and newborn (HDFN), autoimmune hemolytic anemia (AIHA), and hemolytic transfusion reactions (HTR) (Simpson & Hall, 1999).
1.4.1.2.B Indirect Antiglobulin Test (IAT) The IAT is used to detect antibodies that have coated the red cells in vitro. It is a two-stage procedure: the first step involves the incubation of test red cells with serum; in the second step, the red cells are washed and the AHG regent is added. Agglutination implies that the original serum contained antibody which has coated the red cells in vitro. This test is used as part of the 18
Chapter One
Literature Review
routine antibody screening of the recipient’s serum before transfusion and for detecting blood group antibodies in a pregnant women, it is also used with many reagent antibodies for determining blood group phenotypes (Simpson & Hall, 1999).
1.4.1.3 Haemolysis Red cell lysis indicates a positive antigen–antibody reaction mediated by IgM complement-fixing antibodies. A pink or red colored supernatant after settling or centrifugation of red cell–antibody mixtures is an indication of red cell lysis (Rowley et al. 2012).
1.5 Techniques in Blood Group Serology The most important technique is based on the agglutination of red blood cells. Agglutination tests are usually carried out in tubes, microtitre plates or column agglutination (gel) technology. Slide tests are rarely used for emergency ABO and D grouping.
1.5.1 Slide Method Slide or tile techniques are widely used in under-resourced countries for ABO and D grouping. An emergency, rapid ABO grouping may be carried out on slides or tiles. Because of evaporation, slide tests must be read within about 5 min. Reagents that produce strong agglutination within 1–2 min are normally used for rapid ABO and RhD grouping. Because the results are read macroscopically, strong cell suspensions should be used (35–45% cells in their own serum or plasma). The method is satisfactory if potent grouping reagents are used. An immediate spin-tube test is preferable (Mohan, 2007)
19
Chapter One
Literature Review
1.5.2 Tube Tests In blood group serology, tube tests are generally done at 37°C, room temperature or both. Sedimentation tube tests are usually read after 1–2 h. Strong agglutination will, however, be obvious much sooner than this. In spintube tests, agglutination can be read after only 5–10 min incubation if the cell– plasma mixture is centrifuged. It can be performed with no enhancement reagents. More often an enhancement media is used to increase the sensitivity; these media include albumin and low ionic strength solution (LISS), which decrease the zeta potential (the repulsive electric potential between RBCs that prevent their aggregation) and bring the RBCs closer together (Yazer, 2006).
1.5.2.1 Reading Results of Tube Tests 1.5.2.1.A Macroscopic Reading A gentle agitation tip-and-roll ‘macroscopic’ method is recommended. A good idea of the presence or absence of agglutination can often be obtained by inspection of the deposit of sedimented cells: a perfectly smooth round button suggests no agglutination, whereas agglutination is shown by varying degrees of irregularity, ‘graininess’, or dispersion of the deposit (Figure 1.4) (Regan, 2012).
Figure 1.4: Macroscopic appearances of agglutination in round-bottom tubes or hollow tiles. Agglutination is shown by various degrees of ‘graininess’; in the absence of agglutination, the sedimented cells appear as a smooth round button, as on the extreme right (Regan, 2012).
20
Chapter One
Literature Review
Macroscopic reading thus gives lower titration values than does microscopic reading, but the former is recommended. The system of scoring is clarified in (Table 1.5). Table 1.5: Scoring of results in red cell agglutination test (Regan, 2012).
1.5.2.1.B Microscopic Reading It is essential that a careful and standardized technique be followed. Carefully drawing up a column of supernatant about 1 cm in length and then, without introducing an air bubble, drawing up a 1–2 mm column of red cells by placing the tip of the pipette in the button of red cells. Gently expel the supernatant and cells onto a slide over an area of about 2 × 1 cm (Regan, 2012).
1.5.2.1.C Demonstration of Lysis Many blood-group antibodies lyse red cells under suitable conditions in the presence of complement. This is particularly true to anti-A and anti-B, antiP, anti-Lea and Leb, anti- PP1Pk (anti-Tja) and certain autoantibodies (Rowley et al. 2012).
21
Chapter One
Literature Review
1.5.3 Microcolumn Tests (gel and beads) The principle of microcolumn tests is the separation of agglutinated from non - agglutinated red cells by centrifugation through a miniature filtration column. For blood grouping, red cells are layered on microcolumns impregnated with blood
grouping sera; for antibody screening and
identification, phenotyped panel red cells are mixed with patient's sera within the incubation chamber of the microcolumn. After centrifugation, agglutinated red cells are retained towards the top of the microcolumn because the agglutinates are trapped by the column matrix, whereas unagglutinated red cells form a button at the bottom of the column (Figure 1.5) (Lapierre et al. 1990).
Figure 1.5: Results of a gel microcolumn test. The subject is group B D negative. Red cells remaining at the top of the gel represent a positive result; red cells collected at the bottom of the tube represent a negative result (Regan, 2011). Positive and negative results are discriminated by the appearance of cells trapped within the matrix or at the bottom of the microcolumn respectively. The advantages of column agglutination technology are as follows:
22
Chapter One
Literature Review
1. Ease of use and reading, and theoretically it can be performed by relatively unskilled staff. 2. There is less chance of aerosol contamination from infected samples because of no cell washing before IATs. 3. The cards can be kept for up to 24 h, enabling the results to be reviewed by experienced staff. 4. Ease of automation and positive sample identification. In the Diamed ID system, Sephadex gels are used. The Ortho Biovue system uses glass beads rather than gels. Both systems can be automated and the agglutination results evaluated by image analysis (Novaretti et al. 2004).
1.5.4 Microplate Techniques Semi-automated blood grouping and antibody screening can be performed in microtitre plates, which can also be used for extended phenotyping of red cells, antibody identification and large - scale screening for rare red cells and antibodies. A single microplate is equivalent to 96 short test tubes and the same basic principles of discrete analysis of agglutination are applied. The advantages of these techniques include enhanced sensitivity, speed of performance,
reduced
reagent
requirements,
simplicity
and
reduced
requirements for laboratory space and expensive equipment (Rumsey & Ciesielski, 2000). Solid-phase systems, involving microplates containing red cells adhered to the surface of the plastic, help to reduce the variability between tests that is inherent in liquid-phase systems when undertaken by different operators, and easily lend themselves to automated reading of results. Blood grouping (e.g. for ABO and D groups) by solid phase can be accomplished by the use of U shaped microplate wells coated with the relevant antibody (e.g. anti- A, - B, D); suspensions of patients’ or donors’ red cells are added to the wells and then centrifuged. Positive results appear as a carpet of cells coated over the bottom of 23
Chapter One
Literature Review
the well. Negative results appear as a tight pinhead of unattached cells in the centre of the well (Ching, 2012).
1.5.5 Automated Techniques Fully automated blood grouping and antibody screening, using microcolumn techniques or microplates, are carried out in transfusion centres, where large numbers of donor samples are tested daily and increasingly in hospital transfusion laboratories (Garratty, 2010). In automated systems such as the Olympus PK, test samples are mixed with typing sera or screening cells in individual wells of a special microplate that are constructed to have a terraced surface at the bottom of the well. After incubation and settling of the red cells in the reaction mixtures, agglutination patterns are distinguished either on the basis of light transmission or by image analysis with the aid of a computer-controlled CCD camera. Other automated systems are available for use with microcolumns, for example ID - GelStation (Diamed) and AutoVue systems (Ortho). These are fully automated walk-away systems using bar codes to identify samples, reagents and test cards (Butch, 2008).
1.5.6 Molecular Techniques for Blood Grouping Almost all the genes for human blood groups have now been cloned and the molecular bases for all the clinically important blood group polymorphisms have been determined. Consequently, it is now possible to predict blood group phenotypes from DNA with a high degree of accuracy. This is usually performed when a blood group phenotype is required but a suitable red cell sample is not available. The most important application is the determination of fetal blood groups. When a pregnant woman has a blood group antibody with the potential to cause HDFN, it is beneficial to be able to determine whether her
24
Chapter One
Literature Review
fetus has the corresponding antigen and consequently whether it is at risk from HDFN (van der Schoot et al. 2004). Another use of molecular methods for blood grouping is for transfusiondependent patients, where serological methods are not possible because of the presence of transfused red cells that are always present in the patient’s blood (Daniels, 2004). Another application is blood grouping of patients with autoimmune haemolytic anaemia, whose red cells are coated with immunoglobulin, making serological typing difficult (Daniels, 2004).
1.6 Hemolytic Transfusion Reaction This is premature destruction of transfused red cells reacting with antibodies in the recipient. Naturally occurring antibodies, such as ABO antibodies, are IgM and, if warm - reacting, can destroy red cells in vivo by complement fixation. Red cell alloantibodies are usually IgG and form in response to exposure, through previous transfusions or pregnancies. HTRs may occur immediately after the transfusion or may be delayed for anything up to 2– 3 weeks (Goraya et al. 2001). Immediate
life-threatening
reactions
associated
with
massive
intravascular hemolysis result from complement-activating antibodies of IgM or IgG classes, usually with ABO specificity. Reactions associated with extravascular hemolysis (e.g. immune antibodies of the Rh system that are unable to activate complement) are generally less severe but may still be lifethreatening. The cells become coated with IgG and are removed in the reticuloendothelial system (Harris et al. 2007). In mild cases, the only signs of a transfusion reaction may be a progressive unexplained anemia with or without jaundice. In some cases where the pre-transfusion level of an antibody was too low to be detected in a crossmatch, a patient may be reimmunized by transfusion of incompatible red cells 25
Chapter One
Literature Review
and this will lead to a delayed transfusion reaction (most often caused by anti-c or anti-JK) with accelerated clearance of the red cells. There may be rapid appearance of anemia with mild jaundice (Brecher, 2009).
1.7 Alloimmune Hemolytic Disease of the Fetus and Newborn Hemolytic disease of the fetus and newborn (HDFN) is a condition in which the lifespan of the fetal/neonatal red cells is shortened due to maternal alloantibodies against red cell antigens inherited from the father. Maternal IgG can cross the placenta, and thus IgG1 and IgG3 red cell alloantibodies can gain access to the fetus. If the fetal red cells contain the corresponding antigen, then binding of antibody to red cells will occur, the implicated antibody could be naturally occurring (anti-A, anti-B) or immune antibodies which develop following a sensitizing event like transfusion or pregnancy (Roberts, 2008). When the antibody is of clinical significance e.g. anti-(D, c, E, K, Jka), and of sufficient potency, the coated cells will be prematurely removed by the fetal mononuclear phagocytic system. The hemolytic process may result in anemia or hyperbilirubinemia or both; thereby affecting fetal / neonatal morbidity and mortality (Basu et al. 2011).
1.7.1 Historical Overview Thirty years ago, HDFN was almost synonymous with Rh (D) alloimmunizati0n and was a common neonatal problem. The introduction of postnatal immunoprophylaxis in 1970 reduced the incidence of maternal D alloimmunization
from
(14%
to
1-2%).
Subsequently,
antenatal
immunoprophylaxis was also started which further reduced Rh (D) alloimmunization to further 0.1% (Chavez et al. 1991; Eder, 2006). In the Western world, ABO incompatibility is now the single largest cause of HDFN. However in many developing nations, anti-D is still one of the common antibodies found in pregnant women. Besides the anti-D alloantibody, 26
Chapter One
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moderate-severe HDFN cases attributed to other alloantibodies have been described from Asians countries in the last decade (Wu et al. 2003; Thakral et al. 2007). 1.7.2 Spectrum of Hemolytic Disease of the Fetus and Newborn ABO-hemolytic disease, Rh-hemolytic disease and hemolytic disease due to alloantibodies other than anti-D comprise the complete spectrum of HDFN.
1.7.2.1 ABO Hemolytic Disease of the Fetus and Newborn In 20% of births, the mother is ABO incompatible with her fetus. In A and B subjects, the anti-B and anti-A are predominantly IgM and do not enter the fetus. ABO HDFN is usually restricted to group O mothers possessing IgG anti-A, B, in addition to IgM antibodies. In 15% of all pregnancies of white mothers, a group O mother carries a group A or B fetus, but the overall incidence of ABO HDFN requiring treatment is extremely low (Jeon et al. 2000). The lack of severity of ABO HDFN can be accounted for by the widespread occurrence of A and B antigens, not only on red cells but also in plasma and on other cells, which will partially neutralize maternally derived ABO antibodies. Furthermore, the A and B antigens are not fully developed in the infant and the number of ABO sites is much smaller than in adults. Yet description of unusually severe disease necessitating exchange transfusions and active intervention has been documented in the literature (Marwaha et al. 2009).
1.7.2.2 Rh Hemolytic Disease of the Fetus and Newborn Until the early 1970s (when Rh immunoprophylaxis was introduced), (0.5 – 0.75%) of all births gave rise to infants affected by Rh HDFN. Anti-D was accounted for over 90% of all cases. Although anti - D HDFN has decreased significantly as a cause, it remains the most important. Of all infants affected by
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Rh HDFN, 10 – 20% died in utero or in the early neonatal period before effective therapy was possible (Judd, 2001). The disease due to anti-D is more severe than that due to most other alloantibodies (e.g. anti-c, -E, -C, -e) except for some cases of anti-K. Early detection of maternal alloantibodies, regular fetal monitoring and assessment of rises in antibody titres are prerequisites to a successful outcome (Prasad et al. 2006). Clinically significant allo-antibodies other than anti-D such as anti-E, anti-c and anti-K occur in 1:300 pregnancies, and risk of hemolytic disease of the fetus and newborn (HDFN) caused by these antibodies is 1:500 (Koelewijn et al. 2008).
1.7.2.3 Hemolytic Disease of the Fetus and Newborn due to Other Antibodies After anti-D, the antibodies encountered most commonly as a cause of HDFN are anti-c and anti-K (the former usually due to previous pregnancy and the latter to previous maternal blood transfusions). The disease is generally less severe than that caused by anti-D, but may sometimes be serious enough to warrant early delivery and/or exchange transfusion, and occasionally requires treatment of the fetus. Anti-K of high titre may cause severe fetal anaemia, because the K antigen is present in early red cell precursors (Daniels et al. 2003). Other IgG antibodies (e.g. anti-Fya and anti-Jka) uncommonly give rise to fetal hemolysis of sufficient severity to merit antenatal intervention. Failure of alloantibodies of some antigen systems, e.g. Le, Lu to cause HDFN may be due to paucity of antigen sites on fetal red cells or absorption of antibodies by fetal antigen in the placenta (Reid et al. 1996; Thakral et al. 2007).
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1.8 The ABO Blood Group System 1.8.1 History ABO was the first system to be discovered by Austrian Scientist, Karl Landsteiner in 1900, for which he received the Nobel Prize 30 years later, and remains the most important among 30 blood group systems in transfusion and transplantation (histo-blood group system), because ABO antigens are also expressed on most endothelial and epithelial membranes and are important histocompatibility antigens (Eastlund, 1998).
1.8.2Antigens of The ABO System In the ABO blood group, individuals are divided into four major blood groups, A, B, AB and O, according to the presence of the antigens and agglutinins (Hoffbrand & Moss, 2011). Type A blood has type A antigens, type B blood has type B antigens, type AB blood has both types of antigens, and type O blood has neither A nor B antigens. In addition, plasma from type A blood contains type B antibodies, which act against type B antigens, whereas plasma from type B blood contains type A antibodies, which act against type A antigens. Type AB has neither type of antibody and type O blood has both A and B antibodies (Seeley et al. 1998) (Table 1.6).
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Table 1.6: The ABO blood group system (Regan, 2011).
1.8.2.1 ABO Subgroups Serologists have defined two common subgroups of the A antigen. Approximately 20% of group A and group AB individuals belong to group A2 and group A2B, respectively. The remainder belongs to group A1 and group A1B. These subgroups arise as a result of inheritance of either A1 or A2 alleles. The A2 transferase is less efficient in transferring N-acetyl-D galactosamine to available H antigen sites and cannot utilize Types 3 and 4 disaccharide chains. Consequently, A2 red cells have fewer A antigen sites (250,000) than A1 cells that carry more than (800,000) A antigens in a red cell and the plasma of group A2 and group A2B individuals may also contain anti-A1 (Olsson et al. 2001). The distinction between these subgroups can be made using the lectin Dolichos biflorus, which only reacts with A1 cells. The other subgroups of A are less frequently encountered, with the A3 subgroup being the next most frequent, occurring in 1 in 1,000 individuals. Similarly, weak subgroups of group B have been described due to mutations of the B gene (Yainamoto, 1995).
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1.8.2.2 H Antigen The H antigen is present to some extent on almost all red cells, regardless of the ABO group, but the amount of H antigen varies with the ABO group as follows: O > A 2 > A 2 B > B > A 1 > A 1 B. Group O cells have no antigens of the ABO system but do possess H antigen, the precursor upon which the products of the ABO genes act. The H gene (called FUT1) segregates independently from ABO and is on a different chromosome: ABO on chromosome 9, FUT1 on chromosome 19. Individuals with the rare Bombay phenotype are homozygous for inactive FUT1 alleles (h/h). Their red cells are not agglutinated by anti-A or anti-B, regardless of ABO genotype, but are not group O as they are also not agglutinated by anti–H (Joshi et al. 2000). The A, B and H antigens are detected early in fetal life but are not fully developed on the red cells at birth. The number of antigen sites reaches adult level at around 1 year of age and remains constant until old age, when a slight reduction may occur (Pourazar et al. 2004).
1.8.2.3 Biochemistry and Biosynthesis of ABH Antigens A, B and H antigens on red cells are predominantly glycoproteins, the majority being on the N-glycans of the anion exchanger (band 3) and the glucose transporter. Differences in the terminal sugars of the glycoproteins and glycolipids determine the specificity of these antigens: l - fucose (Fuc) for H; lfucose plus N - acetyl - d – galactosamine (GalNAc) for A; and l-fucose plus dgalactose (Gal) for B. A-and B-transferases synthesize the transfer of GalNAc and Gal, respectively, from their donor substrates UDP-GalNAc and UDP-Gal to the terminal galactosyl residue of type 1 H and type 2 H, creating A and B epitopes and masking H specificity (Contreras & Daniels, 2011) (Figure 1.6).
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Figure 1.6: Biosynthetic pathway of H antigen from its precursor, and of A and B antigens from H. H remains unconverted in the absence of A or B gene products (Contreras & Daniels, 2011). R, remainder of molecule. 1.8.3 ABO Encoding Genes and H Genes 1.8.3.1 ABO Genes There are three allelic genes in the ABO blood group system (A, B, and O) that are inherited in mendelian fashion. Both A and B are codominant alleles, whereas O is a recessive allele. Hence, these three genes result in four different phenotypes: A, B, AB, and O. An individual with the A phenotype can be homozygous for the A gene (AA) or heterozygous (AO). Similarly, the B phenotype can be the result of homozygous (BB) or heterozygous (BO) gene inheritance. The genotype of the AB phenotype is AB, and the group O phenotype is always genetically OO. Thus, there are four ABO group phenotypes (A, B, AB, and O) that arise from six possible genotypes (AA, AO, BB, BO, AB, and OO) (Webert et al. 2009). The ABO gene is located on the long arm of chromososme 9, comprises seven exons spanning over 18-20 Kilobases (Kb) in the DNA genome and encodes proteins with a structure characteristic of glycosyltransferases. A and B genes differ in seven nucleotides, resulting in different substrate specificity of 32
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the encoded enzyme. The difference in substrate specificity is mainly determined by four amino acids encoded by exon 7. The O gene is due to either a frameshift mutation leading to a stop codon or, rarely, a mutation producing a nonfunctional enzyme (Yamamoto & Hakomori, 1990; Yazer, 2005). The genes of the ABO system do not encode directly for the antigens but encode for enzymes called glycosyltransferases that add specific sugars to the red cell membrane. These sugars are the ABO red cell antigens that are detectable with serologic testing (Chester & Olsson, 2001). The A gene encodes for the transferase α (1,3) N-acetyl-galactosaminyltransferase, which adds an N-acetyl-galactosamine to the red cell membrane. The B gene encodes for the transferase α (1,3) galactosyl-transferase, which adds a galactose to the red cell membrane. In an individual with the AB phenotype, the A and B transferases coexist and compete for the same substrate. The O allele encodes for a nonfunctional transferase; hence, a specific sugar is not attached to the red cell membrane (Yamamoto et al. 1990).
1.8.3.2 H Genes Both the A and B transferases add sugar moieties to a substrate on the red cell membrane, which is encoded by the H gene. The H gene locus is located at chromosome 19q13.3, and the genes inherited at this locus are inherited in a mendelian manner. Two alleles have been identified at this locus: H and h. The allele H, most frequently inherited, encodes for an enzyme termed H transferase type II [α(1,2) fucosyl-transferases; FUT1], which adds an L-fucose to the terminal galactose molecule of oligosaccharide chains. This structure is called H substance, and it is to this structure that the A and B transferases add specific sugars resulting in A and B antigens. The rare allele sometimes inherited at the H locus is h. This h gene encodes for a nonfunctional transferase. If the h gene is inherited in the homozygous state (hh), L-fucose molecules (H substance) are not present on the red cell membrane (Oriol, 1995). 33
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Without the presence of H substance on the red cell membrane, the A and B transferases, even when present, are not able to add the specific sugars that give A and B antigen specificity. This hh genotype is known as the Bombay phenotype: Serologically, the red cells group as O; however, unlike the true O phenotype, which has large amounts of H antigen on the red cells; red cells from the Bombay phenotype lack H antigen (Watkins, 2001).
1.8.4 ABO Antibodies 1.8.4.1 Anti-A and Anti-B ABO antibodies, in the absence of the corresponding antigens, appear during the first few months after birth, probably as a result of exposure to ABH antigen-like substances in the diet or the environment (i.e. they are ‘naturally occurring’). The antibodies are a potential cause of dangerous haemolytic transfusion reactions if transfusions are given without regard to ABO compatibility (Eder & Chambers, 2007). Anti-A and anti-B are always, to some extent, immunoglobulin M (IgM). Although they react best at low temperatures, they are nevertheless potentially lytic at 37°C. Hyperimmune anti-A and anti-B occur less frequently, usually in response to transfusion or pregnancy, but they may also be formed following the injection of some toxoids and vaccines (Schwarting et al. 2005). They are predominantly of IgG class and are usually produced by group O and sometimes by group A2 individuals. Hyperimmune IgG anti-A and/or anti-B from group O or group A2 mothers may cross the placenta and cause haemolytic disease of the newborn (HDN) (Jeon et al. 2000; Goraya et al. 2001). 1.8.4.2 Anti-A1 and Anti-H Anti-A1 reacts only with A1 and A1B cells and is occasionally found in the serum of group A2 individuals (1–8%) and it is also common in the serum of group A2B subjects (25–50%). Anti-H reacts most strongly with group O and 34
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A2 red cells and also normally acts as a cold agglutinin. A notable, but rare, exception is the anti-H that occurs in the Oh Bombay phenotype, which is an IgM antibody and causes lysis at 37°C so that Oh Bombay phenotype blood would be required for transfusion (Marwaha et al. 2009).
1.8.5 Secretors and Non-Secretors The ability to secrete A, B and H substances in water-soluble form is controlled by FUT2 (dominant allele Se). The Sese genes, similar to the Hh gene, are located at chromosome 19q13.3; however, they are not part of the ABO system. The dominate Se gene codes for H transferase type 1 [α(1,2) fucosyl-transferase; FUT2]. Without the prior addition of a fucose to the oligosaccharide chains, A and B antigens would not be expressed in the body secretions, irrespective of the presence of A and B transferases. In a Caucasian population, about 80% are secretors (genotype SeSe or Sese) and 20% are nonsecretors (genotype sese) (Kelly et al. 1995) (Table 1.7).
Table 1.7: Secretor status in the Caucasian population (Regan, 2012).
Secretors have H substance in the saliva and other body fluids together with A substances, B substances or both, depending on their blood group. Only 35
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traces of these substances are present in the secretions of non-secretors, although the antigens are expressed normally on their red cells and other tissues. An individual’s secretor status can be determined by testing for ABH substance in saliva (Watkins, 2001).
1.9 Rhesus Blood Group System The Rh blood group system was discovered by Karl Landsteiner in cooperation with Alexander S Wiener in 1937 (Landsteiner & Wiener, 1940). The Rh system, formerly known as the Rhesus system, was so named because the original antibody that was raised by injecting red cells of rhesus monkeys into rabbits and guinea pigs reacted with most human red cells. Although the original antibody (now called anti-LW) was subsequently shown to be different from anti-D, the Rh terminology has been retained for the human blood group system (Avent, 2000). The Rh blood group system is the most polymorphic and next to ABO, is the most clinically significant blood group in transfusion medicine. This is not because Rh antibodies are usually present when the Rh antigen is absent, but because anti - RhD is formed readily when RhD – positive blood is transfused to an RhD - negative person. Moreover, as these immune antibodies are normally IgG, they are able to cross the placenta and cause HDFN (Anstee, 2009). 1.9.1 Rh Antigens The Rh proteins carry Rh antigens but are only expressed on the erythrocyte surface if RhAG is also present. The amino acid sequence homology (approximately 40%) of the Rh and RhAG proteins indicates an ancestral relationship, and collectively they are referred to as the “Rh protein family” (Avent et al. 2006). The Rh system now contains a total of 50 antigens (Table 1.8). The most common and important are D, C, E, c, and e. Although individuals can become 36
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alloimmunized to the C, c, E, and e antigens after red cell exposure through transfusion or pregnancy, these antigens are much less immunogenic than D, but D (RH1), because of its high immunogenicity, is by far the most important because it can induce the production of alloantibodies resulting in a hemolytic transfusion reaction (HTR) in alloimmunized patients or hemolytic disease of the fetus and newborn (HDFN) in nalloimmunized, D-negative pregnant women (Klein & Anstee, 2005). Between 82% and 88% of Caucasians, about 95% of black Africans and almost 100% of people from the Far East are D-positive (Daniels et al. 2002).
Table 1.8: Antigens of the Rh system (Contreras & Daniels, 2011).
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The D antigen is highly immunogenic after A and B antigens and induces an immune response in 80% of D-negative persons when transfused with 200 ml of D-positive blood (Mollison et al. 1997). For this reason, in most countries D typing is performed routinely on every blood donor and transfusion recipient so that D-negative patients will receive D-negative RBC products. An individual is considered to be Rh positive if his or her red cells express the D antigen. The term Rh negative refers to the absence of the D antigen. The absence of the D antigen occurs in approximately (15% - 17%) of individuals in white populations and is less frequent in other populations (Westhoff & Reid, 2003). In white populations, the absence of the D antigen is usually due to the deletion of the RHD gene. In Asian and Black populations, the absence of the D antigen is usually due to an inactive RHD rather than a gene deletion. Among the other common Rh antigens, c and e are more potent immunogenes. Antigens C, E and G prove themselves immunogenic mostly in relationship with D antigen (Singleton et al. 2000). Rh antigens appear early during erythropoietic differentiation, in the fetus and expressed on RBCs from the 6-week conceptus (Chown, 1955).
1.9.1.1 Variants of D There are numerous variants of D, which have, for convenience, been divided into two types, weak D (formerly Du ) and partial D. Most D - negative individuals lack the whole Rh (D) protein from their red cells and, when immunized by D - positive red cells, can make antibodies to an array of epitopes on the external loops of the Rh (D) protein. About 30 D epitopes have been defined using monoclonal antibodies (Daniels, 2002).
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1.9.1.1.A Weak D By definition, weak D (previously Du) red cells express all epitopes of D at a low level and individuals with weak D phenotype cannot make anti - D. Weak D red cells have fewer D sites per cell than normal D–positive cells, arise from a variety of mutant RHD genes. The weakest form of weak D, named DEL, can only be detected serologically by absorption and elution tests (Wagner & Flegal, 2000). 1.9.1.1.B Partial D Partial D individuals lack one or more epitopes of the D antigen, defined using panels of monoclonal reagents. DVI is perhaps the most important partial D phenotype because such individuals not infrequently make anti-D. Partial D phenotypes arise from DNA exchanges between RHD and RHCE genes and from other rearrangements (Daniels, 2007).
1.9.1.2 Other Rh Antigens 1.9.1.2.A Rh Null Rh null red cells lack all antigens of the Rh system and people with this rare phenotype can make an antibody, anti - RH29, that reacts with all red cells except those of the Rh null phenotype. Rh null subjects have a chronic haemolytic stomatocytic anaemia, which is usually compensated but may require splenectomy. There are two modes of inheritance of the Rh null phenotype: 1 Homozygosity for a deletion of RHD (characteristic of most D - negatives) plus homozygosity for an inactivating mutation in RHCE; this is termed amorph type of Rhnull (Hung et al. 2000). 2 Homozygosity for inactivating mutations (frameshift, splice site, missense) in RHAG, the gene encoding RhAG. In the absence of RhAG, no Rh antigens are expressed, despite the presence of normal Rh genes, this is termed regulator type Rh null (Hung et al. 2000). 39
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1.9.1.2.B G, Cw and Cx, VS and V Antigens G antigen is expressed when either D or C, or both, are present. Anti-G recognizes a determinant common to the products of RHD and the C allele of RHCE. Anti–G usually occurs with anti–D. C
w
and C x occur in about 2% and
0.2% of white people, respectively. Although substantially higher frequencies are found in Finns, they appear to have an allelic relationship. Anti-C
w
and
anti-C x have caused HDFN but in rare occasions, and it is usually mild. VS and V have frequencies of about 40% and 10%, respectively, in Africans, but are rare in other ethnic groups. Both are associated with an RHCE mutation. Neither anti - VS nor anti - V have caused HDFN or an HTR (Kruskall et al. 2000).
1.9.2 Rh Encoding Genes Rh antigens are encoded by two closely linked genes with 92% sequence homology. RHD encodes the D antigen and RHCE the Cc and Ee. Each consists of 10 exons and, unusually for homologous genes, the two genes are in opposite orientation on the chromosome (Denomme et al. 2000) (Figure 1.7).
Figure 1.7: Rh and related genes and the polypeptides they encode, showing the 10 exons of RHD and RHCE in reverse orientation on chromosome 1 and of RHAG on chromosome 6, and the RhD and RhCcEe polypeptides and RhAG glycoprotein crossing the membrane 12 times (Contreras & Daniels, 2011). 40
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Each gene encodes a 416 - amino - acid polypeptide of 30 – 32 kDa that is palmitoylated but not glycosylated. The polypeptides encoded by RHD and RHCE differ by 31 – 35 amino acids, depending on the RHCE genotype (Avent & Reid, 2000). The Rh haplotypes are named either by the component antigens (e.g. CDe, cde) or by a single shorthand symbol (e.g. R1= CDe, r = cde). Thus, a person may inherit CDe (R1) from one parent and cde (r) from the other and have the genotype CDe/cde (R1r). The haplotypes in order of frequency and the corresponding shorthand notation are given in (Table 1.9). Although two other nomenclatures are also used to describe the Rh system, namely, Wiener’s Rh-Hr terminology and Rosenfield’s numeric notation, the CDE nomenclature, derived from Fisher’s original theory, is recommended by a World Health Organization Expert Committee in the interest of simplicity and uniformity (WHO, 1977).
Table 1.9: The Rh haplotypes in order of frequency (Fisher nomenclature) in Caucasian and the corresponding short notations (Regan, 2012).
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The common Rh antigens D, C or c and E or e, were originally written in alphabetic order (CDE) but later, when it was recognized that C and E antigens are inherited enbloc, the order was changed to DCE. Fisher postulated that C/c locus lies between D/d and E/e loci (Daniels, 1995).
1.9.3 Antibodies of the Rh System Most Rh antibodies are IgG, although some may be IgM. They are usually not capable of activating complement. Anti-D is one of the most common Rh antibodies due to the high immunogenicity of the D antigen. AntiD can cause severe HDN and HTR. The frequency of anti-D has greatly decreased with the use of prophylactic Rh immune globulin administration to Rh (D) negative mothers during pregnancy and at delivery if the infant is Rh (D) positive. Antibodies against antigens of the Rh system including C, c, E, and e can be associated with mild HTR or HDN (Poole & Daniels, 2007).
1.9.3.1 Naturally Occurring Rh Antibodies Generally, Rh antibodies are only produced following immunization with red cells. However, anti-E is often naturally occurring; about half may occur without a history of pregnancy or transfusion. Rarely, naturally occurring anti-D and anti-C
w
are found. All such naturally occurring Rh antibodies react
optimally with enzyme - treated cells (Westhoff & Reid, 2003).
1.9.3.2 Immune RH Antibodies Immune Rh antibodies are predominantly IgG (IgG1 and/or IgG3), but may have an IgM component. They react optimally at 37°C, they do not bind complement and their detection is often enhanced by the use of enzyme treated red cells. Haemolysis, when it occurs, is therefore extravascular and predominantly in the spleen. Anti-D is clinically the most important antibody; it may cause haemolytic transfusion reactions and was a common cause of fetal 42
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death resulting from haemolytic disease of the newborn before the introduction of anti-D prophylaxis. D is considerably more immunogenic than the other Rh antigens, which have the following order of immunogenicity: D> c > E > e > C (Liumbruno et al. 2010). Of the non-D Rh antibodies, anti-c is most commonly found and can also give rise to severe haemolytic disease of the fetus and newborn. Anti-E is less common, whereas anti-C is rare in the absence of anti-D (Hadley, 2002).
1.10 The Kell Blood Group System 1.10.1 Antigens and Encoding Genes A total of 34 antigens have been identified (K1–K34), but three very closely linked sets of alleles are clinically important: K (KEL1) and k (KEL2); Kpa (KEL3), Kpb (KEL4) and Kpc (KEL21); and Jsa (KEL6) and Jsb (KEL7). These antigens are encoded by alleles at the KEL locus on chromosome 7, but their production also depends on genes at the KX locus on the X chromosome (Denomme et al. 2000). The K antigen is very immunogenic (second to the D antigen) in stimulating antibody production. The Kell protein is a single-pass glycoprotein and is believed to be complexed by a disulphide bridge to the Kx protein, (Storry & Olsson, 2000). In the McLeod phenotype, red cells lack Kx and there is a marked decrease in all Kell antigens, an acanthocytic morphology and compensated haemolysis. The McLeod syndrome is X-linked with slow progression to cardiomyopathy, skeletal muscle wasting and neurological defects (Redman et al. 1999; Danek et al. 2001). 1.9.2 Kell Antibodies Anti - K is an important antibody in white populations; it is nearly always immune, IgG and complement - binding. It causes severe HTRs and HDFN (Shaz, 2009). 43
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There is poor correlation with maternal anti-K titer and disease severity. Furthermore, HDN associated with anti-K tends to be less severe than HDN caused by anti-D. This is thought to occur because the Kell antigens are well expressed on fetal cells and appear on erythroid progenitor cells. It is postulated that anti-K, in addition to causing hemolysis, also causes a suppression of erythropoiesis (Wagner et al. 2004). The k antigen is also highly immunogenic. However, because only the individuals that are not expressing the k antigen (i.e., KK phenotype) produce anti-k, and because the k antigen is present in most individuals, anti-k is much less common. Anti-k has been associated with both HDN and HTR. The other Kell blood group system antibodies are much less common but are also clinically significant (Eder & Chambers, 2007).
1.11 The Kidd Blood Group System 1.11.1 Kidd Antigens and Encoding Genes The Kidd blood group system consists of three antigens: Jk a, Jkb, and JK3. The Kidd blood group system gene is located at chromosome 18q12.3, and it has two alleles, Jk a and Jk b, which represent a single amino acid change in the Kidd glycoprotein. The Kidd glycoprotein is a urea transporter in red cells and in renal endothelial cells (Lucin et al. 2002). A Kidd null phenotype, Jk (a − b −), results from homozygosity for inactivating mutations in the Kidd gene. It is very rare and is found primarily in the Polynesian population. These null red cells have been demonstrated to be resistant to lysis by 2M urea; however, this phenotype is not associated with shortened red cell survival or clinical symptoms (Heaton & McLoughlin, 1982).
1.11.2 Kidd Antibodies Anti-Jka is more common than anti-Jkb; they may both cause severe transfusion reactions and, to a lesser extent, HDFN; anti-Jk3 is produced by 44
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individuals of the rare Jk (a-b-) phenotype. The Kidd antibodies are usually IgG and predominantly complement-fixing up to C3b, but occasionally are IgM. These antibodies tend to be short lived; therefore, they are frequently not detected before transfusion. However, they are capable of a rapid anamnestic response and have been associated with severe delayed HTRs (Thakral et al. 2006). 1.12 The Duffy Blood Group System The Duffy blood group system was discovered in 1950 in the serum of a multiply transfused male patient with hemophilia, Mr. Duffy (Cutbush et al. 1950; Howes et al. 2011).
1.12.1 Duffy Antigens and Encoding Genes The Duffy (Fy) locus is on chromosome 1; it has the following alleles: Fya, Fyb, which code for the co-dominant Fya and Fyb antigens, respectively, Fyx, which is responsible for a weak Fyb antigen, and Fy, which is responsible when homozygous for the Fy (a-b-) phenotype in black races. This Fy gene is identical to the Fyb gene in its structural region but has a mutation in the promoter region, resulting in the lack of production of red cell Duffy glycoprotein (Pogo & Chaudhuri, 2000). The Duffy gene encodes for a glycoprotein (also known as Duffy antigen receptor for chemokines, DARC) that is found on red cells as well as other tissues including brain, heart, lung, kidney, and spleen. On red cells, the glycoprotein has been identified as a receptor for various chemokines and may contribute to chemokine-induced leukocyte migration to sites of inflammation (Rot, 2005). The Duffy glycoprotein is also the receptor essential for penetration of Plasmodium vivax merozoites into erythroid cells; therefore, individuals who do not express Fya or Fyb on their red cells are not susceptible to this form of malaria (Horuk et al. 1993; Hadley & Peiper, 1997; Hamblin et al. 2002). 45
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The Duffy null phenotype has the Fyb allele with a mutation in the promoter region which abolishes the expression of the protein in erythrocytes only (Sellami et al. 2008).
1.12.2 Duffy Antibodies The Duffy antibodies are usually IgG. Anti-Fya is a common alloantibody and considered clinically significant as it has been associated with HDN and HTR. Anti-Fyb is uncommon and associated with mild HTR and only rarely with HDN (Goodrick et al. 1997). The other Duffy antibodies are much less common apart from anti-Fy3 (to both Fya and Fyb), which occurs in some African/Afro-Caribbean patients, in whom Fy(a-b-) antigen status is common; they are predominantly IgG1 and are sometimes complement binding (Vescio et al. 1987).
1.13 The MNS Blood Group System MNS was discovered by Landsteiner and his colleagues in 1927 and the MNS genes were the first blood group genes to be cloned, in 1986 and 1987 (Landsteiner & Levine, 1927).
1.13.1 The MNS Antigens and Encoding Genes The MNS system now contains 46 antigens; the major ones being M, N, S, s, and U, while the others are low-frequency antigens resulting from either amino acid substitutions or rearrangement between glycophorin A (GPA) and glycophorin B (GPB). The MN antigens are carried on GPA, which is encoded by the GYPA gene on chromosome 4. M and N differ by amino acids at positions 1 and 5 of the external N - terminus of GPA. GPB carries the S and s determinants, which represent an amino acid substitution at position 29. GPB is encoded by GYPB , which is closely linked and homologous to GYPA. S−s−U− red cells lack GPB (Palacajornsuk, 2006). 46
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The functions of glycophorin A and B are not clear. Glycophorin A has been demonstrated to be a receptor for certain malarial parasites as well as for bacteria and viruses. Other proposed functions of glycophorin A include regulation of transport of band 3 to the red cell membrane and complement regulation. However, the rare null phenotypes are not associated with any apparent health defects (Reid, 2009).
1.13.2 Antibodies of the MNS System Anti-M and anti-N antibodies are typically IgM and are reactive at cold temperatures, they are naturally occurring (environmentally stimulated). They are not generally considered to be clinically significant. Rarely, anti-M has been implicated in cases of HDN and HTR (Sancho et al. 1998). Anti - S, the rarer anti-s and anti-U are usually immune, IgG and can cause HDFN. They have also been implicated in HTRs. Anti-U only occurs in S−s− black people and reacts with all cells that have the S or s antigens and up to 50% of cells that are S−s−. Finding compatible blood for a patient with anti-U can prove difficult (Guastafierro et al. 2004; Poole & Daniels, 2007).
1.14 The Lutheran Blood Group System 1.14.1 Lutheran Antigens and Encoding Genes Lutheran is a complex system comprising four pairs of allelic antigens: Lua and Lub, Aua and Aub, Lu6 and Lu9, Lu8 and Lu14. These represent single amino acid substitutions in the Lutheran glycoproteins. There are another 11 antigens of very high frequency in this system. The extremely rare Lu null phenotype, in which no Lutheran antigens are expressed, results from homozygosity for an inactive Lutheran gene; anti - Lu3 may be produced ( Daniels, 1995). The antigens are encoded by the LU gene located at chromosome 19q13.32.The Lutheran glycoproteins bind the extracellular matrix glycoprotein 47
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laminin and might function as adhesion molecules with a role in erythropoiesis (Telen, 2000).
1.13.2 Lutheran Antibodies The Lutheran antigens are not well-developed at birth and as a consequence there are no documented cases of clinically significant haemolytic disease owing to Lutheran antibodies. Anti-Lua is uncommon and rarely of clinical significance, anti-Lub has been implicated in mild delayed HTRs (Inderbitzen & Windle, 1982; Dube & Zoes, 1982).
1.15 The Lewis Blood Group System The Lewis system differs from all other blood group systems in that it is primarily a system of soluble antigens present in secretions and in plasma (Grubb, 1951). The Lewis antigens on red cells are adsorbed passively from the plasma, and a constant presence of plasma is needed to maintain Lewis antigen on the red cells (Sneath, 1955).
1.15.1 Lewis Antigens and Encoding Genes There are two basic Lewis antigens: Lea and Leb. Expression of either requires the presence of an active Lewis gene, but Lewis phenotypes are also governed by the gene controlling H secretion (FUT2). Lewis antigens in saliva and plasma are glycoproteins and glycolipids, respectively. The Lewis antigens are poorly developed at birth and red cells from cord blood are usually Le (a−b−). Thereafter, Lea develops first, followed by Leb if the relevant Lewis and secretor genes are present. The definitive adult Lewis phenotype may not be reached until the age of 4 – 5 years (Pourazar et al. 2004). The Le (FUT3) gene is located on chromosome 19 (19p13.3) and is closely linked to the Hh and Sese genes, encodes an α 1,4- fucosyltransferase
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Literature Review
that catalyses the addition of l - fucose in 1 → 4 linkage to the subterminal GlcNAc of type 1 chains (Figure 1.8) (Cutbush et al. 1956).
Figure 1.8: Diagrammatic representation of H and Lewis antigens. Le a requires the action of the Lewis α 1,4 - fucosyltransferase, H the action of the H α 1,2fucosyltransferase, Leb the action of both Lewis and H fucosyltransferases, and A Leb and B Leb the action of Lewis and H fucosyltransferases and the A or B glycosyltransferases (Contreras & Daniels, 2011). If the type 1 core structure has been unmodified, Lea antigen is produced. If the secretor α 1,2-fucosyltransferase has already modified the type 1 chains to produce type 1H, the action of the Le–transferase leads to the formation of Leb (Pourazar et al. 2004).
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1.15.2 Antibodies of the Lewis System Lewis antibodies are generally made only by individuals with the Le (a − b −) red cell phenotype. Anti - Lea occurs fairly frequently. Anti - Leb commonly accompanies anti - Lea. Pure anti - Leb is uncommon and made by people who are non - secretors and whose red cells are Le (a−b−) (Dracker et al. 1991). Lewis antibodies are usually not hemolytic because the antibody does not react at 37C°. Furthermore, Lewis antigens in the donor’s plasma neutralize the antibody, and the Lewis antigens elute from RBCs into the plasma. The antibodies do not cause HDN because they are IgM and do not cross the placenta. Furthermore, Lewis antigens are poorly developed on fetal red cells (Kissmeyer-Nielsen, 1965).
1.16 The P System and Globoside Collection 1.16.1 Antigens The P blood group system consists of a single antigen: P1, it is the product of a galactosyl-transferase encoded by the gene P1, which is located at chromosome 22q11.2-qter (Denomme et al. 2000). The P1 antigen of the P system and the P and Pk antigens of the globoside collection are related. P1 was discovered by Landsteiner and Levine in 1927, who used suitably adsorbed sera of rabbits injected with human red cells. About 75% of subjects tested were positive for P1, which is inherited as a Mendelian dominant character. P1 frequency varies in different populations and the P1 - negative phenotype is called P2. P1 is weakly expressed at birth and its strength varies considerably in adults. The P antigen is the receptor for the B19 parvovirus, which causes erythema infectiosum and occasionally more severe disease, such as RBC aplasia. Individuals with the p phenotype lack P and P k, and are therefore resistant to parvovirus B19 infection (Brown et al. 1994).
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1.16.2 Antibodies Anti-P 1 is a naturally occurring antibody commonly found in the serum of P 2 individuals. Unlike anti-A and anti-B, anti-P1 rarely causes transfusion reactions because it is usually a cold-reacting IgM antibody, often not reactive above 30°C. Anti-P1, as well as anti-Pk, can be neutralized with P1 substance, hydatid cyst fluid (Echinococcus cyst fluid) or pigeon egg white (Cameron & Stavely, 1975). Alloanti- P is also a naturally occurring antibody found in individuals with the rare Pk phenotype. Auto-anti-P is the specificity attributed to the Donath–Landsteiner antibody; it is a potent biphasic haemolysin, responsible for paroxysmal cold haemoglobinuria (PCH), which is a rare autoimmune hemolytic anemia but typically occur in young children following a viral infection. These children have a biphasic IgG antibody. The term biphasic antibody is used because the antibody binds to the RBCs at low temperature, but RBC lysis does not occur until the RBC with the antigen-antibody complex warms up. The Donath - Landsteiner test is used to diagnose PCH; a positive test result is when hemolysis does not occur if the specimen is kept at 4°C or 37°C, but occurs only when the specimen is first placed at 4°C and then warmed to 37°C (Shaz, 2009). Anti-PP1Pk is a naturally occurring high-titre IgM or IgG antibody and it is found only in individuals with the rare p phenotype. It is reactive at 37°C and is capable of causing intravascular haemolysis and HDN. It is also associated with spontaneous miscarriage in early pregnancy (Westhoff & Reid, 2003).
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Subjects & Methods
2.1 Study Design This is a cross-sectional study, carried out from 1st of June 2014 to 30th of September 2014 in the Central Blood Bank of Sulaymaniyah province. The study was conducted after taking approval from ethical and scientific commodities of medical school, blood bank and informed consent from blood donors for their participation in the present study.
2.2 Donor Selection A total of five thousands healthy repeated regular voluntary blood donors aged between 18-60 years were included for red cell antigen typing of ABO (A, B, AB & O) and Rh (D) blood group system. Out of them, 500 donors were randomly selected for red cell antigen typing of other Rh blood group system antigens : Rh (C, c, E, e) & Kell (K), and 400 donors for extended antigen typing of other blood group systems: k (Cellano), Kidd (Jka, Jkb), Duffy (Fya, Fyb), MNSs (M, N, S, s), Lewis (Lea, Leb), Lutheran (Lua, Lub) and P (P1).
2.3 Sample Collection A 2.5 milliliter of peripheral blood sample was drawn from the anti-cubital vein in the anti-cubital fossae from each blood donor in a disposable syringe and transferred immediately to an Ethylene Diamine Tetra Acetic Acid (EDTA) tube for the blood group typing.
2.4 Performing ABO and Rh (D) Blood Grouping ABO and Rh (D) grouping was performed during less than 24 hours of blood collection on freshly prepared 5% red cell suspension from 5 000 blood samples by dextran acrylamide gel technique using ID-Card ”DiaClon ABDConfirmation for Patients” which contains monoclonal anti-A [cell line LM 297 / 52
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Subjects & Methods
628 (LA-2)], anti-B [cell line LM 306 / 686 (LB-2)] and anti-D (cell lines TH-28, RUM-1, LDM1] within the gel matrix, (Bio-Rad Laboratories, DiaMed Switzerland).
2.4.1 Additional Reagents and Materials required: 1- ID-Diluent 2: modified LISS for red cell suspensions. 2- ID-Dispenser. 3- ID-Tips (pipetor tips). 4- Suspension Tubes. 5- ID-Working table. 6- ID-Centrifuge 24 S (BIO-RAD, Switzerland). 2.4.2 Preparation of Red Cell Suspension In ID-Diluent 2, (5%) red cell suspension was prepared as following: 1- Dispensing 0.5 ml of ID-Diluent 2 into a clean tube. 2- Adding 50 microliter of whole blood and mixing gently. 2.4.3 Test Procedure: 1- Identifying the ID-Card with the unique donor number. 2- Removing the aluminium foil from the micro tubes by holding the ID card in the upright position. 3- Adding 10 micro liter of the red cell suspension of the first sample to the first 3 micro tubes of the ID-Card. 4- Centrifuging the ID-Card for 10 minutes in the ID-Centrifuge. 5- Reading & recording the results.
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2.4.4 Interpretation of Results for the Gel Test: 1- Positive: Agglutinated cells formed a red line on the surface of gel in the micro tube or were dispersed throughout the gel. These were graded from 4+ to 1+ and indicated the presence of the corresponding antigens. 2- Negative: A compact button of cells on bottom of the micro tube indicated the absence of the corresponding antigen. 2.5 Extended Red Cell Antigen Typing 2.5.1Preparation of Red Cell Suspension A 3-5% suspension of test red cells in isotonic saline was prepared.
2.5.2 The Reagents Required 1- Anti-C, anti-c, anti-E and anti-e reagents contain monoclonal human IgM antibodies diluted in a buffer containing macromolecular chemical potentiators, (Rapid Labs Limited, England). 2- Human anti-Kell blood grouping reagents are prepared from human serum diluted in a sodium chloride solution, (Rapid Labs Limited, England). 3- Anti-Jka and anti-Jkb are monoclonal IgM blood grouping reagents contain human monoclonal antibodies diluted in a phosphate buffer, (Rapid Labs Limited, England). 4- Anti-Fya and anti-Fyb are monoclonal IgG blood grouping reagents contains human monoclonal antibodies diluted in a phosphate buffer, (Rapid Labs Limited, England). 5- Anti-M blood grouping reagent is prepared from human serum diluted in a sodium chloride solution, (Rapid Labs Limited, England). 54
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6- Anti- N Lectin blood grouping reagent is prepared from an extract of Vicia graminae seeds, diluted with a sodium chloride solution, (Rapid Labs Limited, England). 7- Human Anti-S and anti-s blood grouping reagents are prepared from human serum diluted in a sodium chloride, (Rapid Labs Limited, England). 8- Human anti-Lua and anti-Lub blood grouping reagents are prepared from human serum diluted in a sodium solution, (Rapid Labs Limited, England). 9- Anti-Lea and anti-Leb reagents contain mouse monoclonal IgM antibodies diluted in a phosphate buffer, (Rapid Labs Limited, England). 10- Monoclonal IgM anti-P1 blood grouping reagent contains mouse monoclonal IgM antibodies prepared from the cell line, (Rapid Labs Limited, England).
2.5.2.A Additional Reagents and Materials Required: 1- Anti-human globulin (AHG), Rapid Labs Ltd. Poly specific AHG (Cat BGAHG10). 2- Isotonic saline. 3- Glass test tubes (10×75 mm). 4- Centrifuge (Rotina 380 Hettich, Germany). 5- 37°c incubator (binder, Germany). 6- Timer. 7- Slides. 8- Automated pipette/Slamed. 9- Tips. 10- Microscope (Olympus, Germany). 55
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2.5.3 Test procedure The blood grouping for the above antigen types were done by conventional tube technique following manufacturer’s instructions. The antisera and the antihuman globulin reagents used in the study were from Rapid Labs Limited, England. The typing of Rh (C, c, E & e), Lewis (Lea, Leb), Kell (K), Kidd (Jka, Jkb), MNSs (M, N) and P1 antigens were done by direct tube technique using monoclonal IgM antisera, as following: 1- Washing all the blood samples 3 times with isotonic saline before being tested. 2- Preparing a 2-3% suspension of washed test red cells in isotonic saline. 3- Placing in an appropriately labeled test tube: 1 volume (50Microliter) of Rapid Labs Ltd. monoclonal reagent and 1 volume (50Microliter) of test red cell suspension. 4- Mixing well and incubate at different temperature according to the type of antisera, as follows: For Lewis (Lea & Leb), incubation at room temperature for 15 minutes. For M & P1 antigens incubation at 4c° ± 2c°. For N antigen incubation at 37c° for 15 minutes. While for Rh (C, c, E, e), Kell (K) & Kidd (Jka, Jkb) antigens, no incubation required. 5- Centrifuging all tubes for 20 seconds at 1000 relative centrifugal force (rcf). 6- Gently resuspending red cell button and reading macroscopically for agglutination.
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Typing of k (Cellano), Duffy (Fya, Fyb), Lutheran (Lua, Lub), & MNSs (S, s) antigens were done by Indirect Antiglobulin Technique (IAT), using monoclonal IgG antisera, as following: 1- Washing all the blood samples 3 times with isotonic saline before being tested. 2- Preparing a 2-3% suspension of washed test red cells in isotonic saline. 3- Placing in an appropriately labeled test tube: 1 volume (50Microliter) of Rapid Labs Ltd. Monoclonal IgG reagent and 1 volume (50Microliter) of test red cell suspension. 4- Mixing thoroughly and incubation at 37c° for 15 minutes. 5- Washing test red cells 3 times with isotonic saline. Decanting saline after last wash completely. 6- Adding 2 volumes of anti-human globulin to each tube. 7- Mixing thoroughly & centrifuging all tubes for 20 seconds at 1000 rcf. 8- Gently resuspending red cell button and reading them macroscopically for agglutination. Positive and negative control cells and coombs' control cells were used for quality controls
2.5.4 Interpretation of Test Results Positive: agglutination of the test red cells constitutes a positive test result and within accepted limitations of test procedure, indicates the presence of the appropriate antigen on the test red cells. Agglutination reactions were recorded using agglutination viewer and were graded as 1+ to 4+.
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Negative: No agglutination of the test red cells constitutes a negative test results and within accepted limitations of test procedure, indicates the absence of the appropriate antigen on the test red cells.
2.6 Statistical Methods Calculation of red cell antigen and phenotype frequencies of the various blood group systems was performed using SPSS (v21) software. Results were expressed in a percentage.
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A
B Figure 2.1: A. and B. ABD gel card, ”DiaClon ABD-Confirmation for Patients” (Bio-Rad Laboratories, DiaMed Switzerland).
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A
B Figure 2.2: A. ABD gel card set,”DiaClon ABD-Confirmation for Patients” and diluents. B. ID-Centrifuge 24. S, (BIO-RAD, Switzerland).
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A
B Figure 2.3: A. DiaClon ABD-confirmation for patients, (Bio-Rad Laboratories, DiaMed Switzerland). B. e.g. 49. An A+ve blood group donor. e.g. 50. An O-ve blood group donor.
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A
B Figure 2.4: A. e.g. 9 and 10 a B+ve blood group donors. B. e.g. 3. An O+ve blood group donor. e.g. 4. An AB+ve blood group donor.
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A
B
C Figure 2.5: A. and B. Extended red cell antigen typing reagents (Rapid Labs Limited, England). C. Extended red cell antigen typing sets.
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A
B Figure 2.6: A. Centrifuge (Rotina 380 Hettich, Germany). B. Incubator (binder, Germany).
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Chapter Three
Results
3. Results A total of five thousand healthy regular voluntary blood donors were typed for ABO (A, B, O, AB), and Rh (D) blood group system antigens. Out of these, 500 donors were typed for other Rh antigens (C, c, E, e) and Kell (K) antigen; among them 400 donors were randomly selected and typed for other blood group system antigens: k (Cellano), Kidd (Jka, Jkb), Duffy (Fya, Fyb), MNSs (M, N, S, s), Lewis (Lea, Leb), Lutheran (Lua, Lub) and P (P1) antigens.
3.1 The ABO Blood Group System The frequency of ABO phenotypes are shown in (Figure 3.1). The most common blood group was found to be O (37.0%), followed by A (32.6%), and B (22.8%). Blood group AB occurred at the lowest frequency (7.6%).
37.0% 40% 32.6%
35% 30%
O
22.8%
25%
A
20%
B AB
15% 7.6% 10% 5% 0%
O
A
B
AB
Figure 3.1: ABO blood group distribution in the present study (N=5 000).
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The frequency of the ABO phenotypes linked with Rh (D) phenotype were O+ve (33.2%), followed by A+ve (29.8%), B+ve (21.1%), and AB+ve (7.1%). The lowest frequent was AB-ve (0.5%) (Table 3.1).
Table 3.1: Frequency of ABO and Rh (D) blood groups in the present study Rh (D) positive
Rh (D) negative
Total donors
Number (%)
Number (%)
Number (%)
N = 4563 (91.3)
N = 437 (8.7)
N = 5000
O
1662 (33.2)
190 (3.8)
1852 (37.0)
A
1491 (29.8)
137 (2.7)
1628 (32.6)
B
1055 (21.1)
84 (1.7)
1139 (22.8)
AB
355 (7.1)
26 (0.5)
381 (7.6)
ABO blood group
3.2 The Rh Blood Group System Among 5 000 blood donors, Rh (D) positive was found in (91.26%), and Rh (D) negative in the remaining (8.74%) of donors (Figure 3.2).
8.7%
91.3%
D+ve
D-ve
Figure 3.2: Distribution of Rh (D) blood group in the present study (N=5 000).
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Frequencies of the main antigens in the Rh blood group system are summarized in (Table 3.2).
Table 3.2: Distribution of Rh antigens in D+ve and D–ve donors (N=500). AF* in D+ve
AF* in D-ve
Total donors
donors, Number
donors, Number
Number (%)
(%), N= 447
(%), N= 53
N= 500
C
365 (81.7)
9 (17.0)
374 (74.8)
E
153 (34.2)
0 (0.0)
153 (30.6)
c
294 (65.8)
53 (100)
347 (69.4)
e
423 (94.6)
53(100)
476 (95.2)
Antigens
AF* Antigen frequency
Among these antigens, the e antigen was found to have the highest frequency (95.2%), followed by D, C, c & E (91.26%, 74.8%, 69.4% & 30.6%, respectively). The phenotype frequencies of Rh (D) positive and Rh (D) negative groups are shown in (Table 3.3). Eight probable phenotypes were found to be present in our population. The DCe/DCe (R1R1), and dce/dce (rr) were the most common phenotypes among Rh (D) positive and Rh (D) negative groups, respectively.
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Table 3.3: Rh phenotype frequencies in blood donors Phenotype
Number
Percent (%)
Total (%) N=500
Rh (D) positive donors (N=447) R1R1 (DCe/DCe)
152
34
30.4
R1r (DCe/dce)
132
29.6
26.4
R1R2 (DCe/DcE)
82
18.3
16.4
R2r (DcE/dce)
47
10.5
9.4
R2R2 (DcE/DcE)
24
5.4
4.8
R0 r (Dce/dce)
10
2.2
2
Rh (D) negative donors (N=53) rr (dce/dce)
44
83
8.8
rʹr (dCe/dce)
9
17
1.8
3.3 The Distribution of Kell, Kidd, Duffy, MNSs, Lutheran, Lewis, and P Blood Group Systems 3.3.1The frequency of Red Cell Antigens (Kell, Kidd, Duffy, MNSs, Lutheran, Lewis, and P) Blood Group Systems The frequency of red cell antigens of Kell, Kidd, Duffy, MNS, Lutheran, Lewis and P blood group systems are shown in (Table 3.4).
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Table 3.4: Frequency of red cell antigens of Kell, Kidd, Duffy, MNSs, Lutheran, Lewis and P blood group systems in (N=400). Antigens
Number
Percent (%)
K
29/500
5.8
k
400/400
100
Jka
308/400
77
Jkb
270/400
67.5
Fya
280/400
70
Fyb
230/400
57.5
M
318/400
79.5
N
266/400
66.5
S
216/400
54
s
354/400
88.5
18/400 382/400
4.5
Kell
Kidd
Duffy
MNSs
Lutheran Lua Lub
95.5
Lewis Lea
174/400
43.5
Leb
258/400
64.5
P1
304/400
76
P
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3.3.2 Phenotype frequencies of Kell, Kidd, Duffy, MNSs, Lutheran, Lewis, and P blood group systems The phenotype frequencies of the above blood group systems are shown in (Tables 3.5 and 3.6).
Table 3.5: Phenotype frequencies of Kell, Kidd, Duffy, Lutheran, Lewis, and P blood group systems Phenotype
Donors (%)
K-k+
94.2
K+k+
5.8
Jk (a+b+)
44.5
Jk (a+b-)
32.5
Jk (a-b+)
23
Fy (a+b-)
38.5
Fy (a+b+)
31.5
Fy (a-b+)
26
Fy (a-b-)
4
Lu (a-b+)
92
Lu (a+b+)
3.5
Lu (a-b-)
3.5
Lu (a+b-)
1.0
Le (a-b+)
54.5
Le (a+b-)
33.5
Le(a+b+)
10
Le (a-b-)
2
P1
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Table 3.6: Phenotype frequency of MNSs blood group system. Phenotype
Donors (%)
M+N+
46
M+N-
33.5
M-N+
20.5
S-s+
46
S+s+
42.5
S+s-
11.5
M+N-S+s-
5.5
M+N-S+s+
19
M+N-S-s+
18
M+N+S+s-
22.5
M+N+S+s+
17.5
M+N+S-s+
40
M-N+S+s-
0.5
M-N+S+s+
6
M-N+S-s+
14.5
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In the Kell blood group system, 5.8% (29/500) of the donors were typed as K antigen positive. Out of the 500 donors, 400 donors were randomly selected and typed for k (Cellano) antigen and found to be positive in all the donors (100%). Accordingly, the K-k+ phenotype was the most common in the donors (94.2%). No K+k- & K-k- phenotypes were observed in any donors. In the Kidd blood group system, Jk (a+b+) was the most common phenotype seen in about 44.5% of donors. No Jk (a-b-) was observed in any donors. Jka and Jkb antigens were determined in 77% and 67.5% of donors, respectively. In the Duffy blood group system, Fy (a+b-) was the most common phenotype seen in about 45% of subjects. The Duffy null or Fy (a-b-) phenotype was observed in 4% of donors. Fya and Fyb antigens were observed in 72% and 51.5% of donors, respectively. In the MNSs blood group system, 33.5% of donors were homozygous for M antigen (M+N-), and only 20.5% were homozygous for N antigen (M-N+). Furthermore, M antigen was positive in 79.5% and N antigen in 66.5% of donors. S and s antigens were positive in 54% and 88.5% of donors, respectively. M+N+ and S-s+ were the most common phenotype observed in the MNS blood group system, they were 46% for both. No S-s- phenotype was found in the donors of the present study. Out of nine possible phenotypes , M+N+S-s+ (40%) was the most common phenotype whereas M-N+S+s- (0.5%) was the least common phenotype observed in the MNS blood group system. In the Lutheran blood group system, the most common phenotype was Lu (a-b+) which was found in 92% of the donors, Lua and Lub antigens were observed in 4.5% and 95.5% of donors, respectively. Null phenotype Lu (a-b-) was determined in 3.5% of the donors.
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The most common phenotype in the Lewis blood group system was Le (ab+) which was about 54.5%. Lea and Leb antigens were observed in 43.5% and 64.5% of donors, respectively. Approximately 76% of donors were positive for P1 antigen.
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Discussion
4. Discussion In this study, we examined the composition of RBC antigens and phenotype frequencies of various clinically significant blood group systems in local donor population in Central Blood Bank of Sulaymaniyah Province. All the donors were males, even though females do not expect to have a different pattern; this is due to the fact that blood group inheritance is autosomal and the frequencies are not different in the two sexes. The knowledge of prevalence of various blood group antigens and phenotype frequencies in the local donor population is important in day to day work in a transfusion service in areas such as antenatal serology, paternity testing, and selecting compatible blood in problem transfusions (Brecher, 2005). Beside ABO and Rh antibodies, antibodies to other clinically significant antigens are also known to cause HTR, HDFN, or shortened survival of transfused red cells (Poole & Daniels, 2007). Multiply transfused patients such as those with thalassaemia, sickle cell anemia, patients on hemodialysis, cancer patients, etc. are likely to develop antibodies against these clinically significant minor blood group antigens (Thakral et al. 2010). Finding compatible blood units for such patients without having any knowledge of the prevalence of the implicated antigens in the local donor population is a difficult task, more so if the patient has developed more than one antibody. In these situations, antigen-negative donor units for such cases can be easily retrieved from the donor database of various blood groups available with a blood transfusion center. For this particular reason, all blood banks should have the donor database on red cell antigen frequency of other blood group systems in their local donor population (Sarkar et al. 2013; Lamba et al. 2013; Kahar & Patel, 2014).
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Chapter Four
Discussion
For the best of our knowledge, most data on literature have determined European, American and some Asian phenotype of blood group systems. In this study, for the first time we have determined extended red cell antigen and phenotype frequencies of various blood groups in the Kurdistan Region of Iraq. The results of the study were compared with that of other studies done in Iraq, neighboring countries, Asian (India), White and Black populations.
4.1 The ABO Blood Group System It is well established that ABO and rhesus (Rh) genes and phenotypes vary widely across races and geographical boundaries, (Dacie & Lewis, 2001; Reid & Lomas-Frances, 1997). Some variations may even occur in different areas within one small country (Kolmakova & Kononova, 1999; Musa et al. 2012), despite the fact that the antigens involved are stable throughout life. The resultant polymorphism remains important in population genetic studies, estimating the availability of compatible blood, evaluating the probability of hemolytic disease in the newborn, resolving disputes in paternity/maternity and for forensic purposes (Polesky, 1996; Calhoun & Petz, 2001). The four phenotypes: A, B, O and AB are present in all human populations, but their frequencies differ substantially throughout the world, (Daniels & Bromilow, 2007). In the population studied here, the blood group O (37.0%) was the most frequently encountered phenotype followed closely by the blood group A (32.6%), and then by the blood group B (22.8%), and the lowest was AB (7.6%). The achieved results closely approach to a previous study in Erbil, Kurdistan Region of Iraq, in which 53,234 Kurdish donors were included for ABO and Rh (D) typing, and they were (37%, 32%, 24%, 7%, respectively) (Jaff, 2010), and in agreement with other studies in other parts of Iraq (e.g. Salih (2009) conducted a research in 75
Chapter Four
Discussion
Babylon in which 7317 donors were involved, and Mashaali (2014) also conducted a study in Baghdad. The frequency of O, B, and AB in Babylon were (35.8%, 28.2%, 8.3%, respectively) (Salih, 2009), and were (39%, 26%, 11%, respectively) in Baghdad (Mashaali, 2014). Blood group A in the current study was higher than in the two later studies, which was (32.6% vs. 27.7% and 26%, respectively). This difference most probably can be attributed to the difference in ethnicity between our donor population (Kurds) and their population (Arabs), and/or to the smaller sample size of Mashaali study, in which only 100 donors were involved. Furthermore, our results were very close to studies done in Iran including Boskabady et al. study (35%, 33%, 23%, 9, respectively) (Boskabady et al. 2005) and Keramati et al. study (34%, 30%, 28%, 8%, respectively) (Keramati et al. 2011). In this study we have documented some similarity, but with less close trends to some of the neighboring Arab countries, e.g. Kuwait (Al-Bustan et al. 2002), Saudi Arabia (Sarhan et al. 2009), and Jordan (Hanania et al. 2007). These trends were again observed in populations of other ethnicity like Caucasian (Guyton & Hal, 2005), Blacks (Enosolease & Bazuaye, 2008) and Europeans (Mollison et al. 1997). On the other hand in many Asian populations, there is an increase in the prevalence of group B, e.g. India and Malaysia (Ali et al. 2005; Agrawal et al. 2013). Our results were not comparable to that reported in the neighboring Turkey (Kaya et al. 1999), and Syria (Sakharov & Nofal’Kh, 1996), in which higher prevalence of group A was reported (Table 4.1).
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Discussion
Table 4.1: Antigen frequencies (%) of ABO blood group of the present study compared with other studies in Iraq, neighboring countries and some other populations. People groups
O%
A%
B%
AB%
Sulaymaniyah-Iraq
37
32
23
8
present study
Erbil-Iraq
37
32
24
7
Jaff, 2010
Babylon-Iraq
36
28
28
8
Salih, 2009
Baghdad-Iraq
39
26
24
11
Mashaali, 2014
Iran
35
33
23
9
Boskabady et al. 2005
Iran
34
30
28
8
Keramati et al. 2011
Kuwait
44
27
24
5
Al-Bustan et al. 2002
Saudi Arabia
51
26
19
4
Sarhan et al. 2009
Jordan
37
38
18
7
Hanania et al. 2007
Caucasian
47
41
9
3
Guyton & Hal, 2005
53
24
20
3
European
43
40
12
5
Mollison et al. 1997
Asians (e.g. India)
39
23
33
5
Ali et al. 2005
32
22
37
9
Agrawal et al. 2013
Turkey
32
46
15
7
Kaya et al. 1999
Syria
38
46
13
3
African blacks (e.g. Nigeria)
Asian (e.g. North India)
77
References
Enosolease & Bazuaye, 2008
Sakharov & Nofal’Kh, 1996
Chapter Four
Discussion
The frequency of the ABO phenotypes linked with Rh (D) phenotype was O+ve (33.2%), followed by A+ve (29.8%), B+ve (21.1%), and AB+ve (7.1%). The lowest frequent phenotype was that of AB-ve (0.5%). These results are again very close to Jaff study in Erbil, Kurdistan Region of Iraq (Jaff, 2010), in which the frequency of O+ve, A+ve, B+ve, and AB+ve were (34.0%, 30.0%, 21.7%, and 6.0%, respectively), and the lowest phenotype was that of AB-ve (0.5%).
4.2 The Rhesus Blood Group System The Rh blood group system is the most polymorphic, and next to ABO, is the most clinically significant blood group in obstetric and transfusion medicine (Avent & Reid, 2000; Anstee, 2009).
4.2.1 The Rhesus (D) Antigen The worldwide prevalence of D positive antigen differs in different ethnic groups; it being 85% in Whites and 92% in Blacks (Beadling & cooling, 2007; Barclay, 2001). In the present study we found D positive antigen frequency to be (91.3%), and D negative found in (8.7%). The results were in agreement with studies in other parts of Iraq, e.g. Erbil, Kurdistan Region of Iraq (91.7%, 8.3%, respectively) (Jaff, 2010), and in Babylon- Iraq (90.1%, 9.9%, respectively) (Salih, 2009). They are also in accordance with results reported in some neighboring countries, e.g. Saudi Arabia (91.2%, 8.9%, respectively) (Al-Himaidi & Umar, 2002), Bahrain (91.1%, 8.9%, respectively) (Jenan, 2012), Arians in Pakistan (91.7%, 8.3%, respectively) (Ali et al. 2005), Kuwait (92.5%, 7.5%, respectively) (Al-Bustan et al. 2002), Iran (90.2%, 9.8%, respectively) (Keramati et al. 2011), and with India (93.4%, 6.6%, respectively) (Thakral et al. 2010). On the other hand, there is a marked difference with a study in BaghdadIraq (80%, 20%, respectively) (Mashaali, 2014), most probably due to the smaller 78
Chapter Four
Discussion
sample size of the later study as mentioned previously in comparison to our study. Compared with racial groups, the results gained in this study were similar to that of Blacks (92%, 8%, respectively) (Beadling & Cooling, 2007; Barclay, 2001), but with marked differences to those of Whites (85%, 15%, respectively), including European and their descents (Beadling & cooling, 2007; Barclay, 2001), and Asians (99%, 1%, respectively) (Reid & Lomas-Frances, 1997). This suggests that the expected frequency of Rh (D) alloimmunization in patients receiving blood transfusion, and among pregnant women, would be lower in our population, neighboring countries and much lower in Asian than that encountered among Europeans and White Americans (Table 4.2).
79
Chapter Four
Discussion
Table 4.2: Frequency of Rh (D) antigen in the present study compared with other studies in Iraq, neighboring countries and some other populations. Rh (D)
Rh (D)
+ve %
–ve %
Sulaymaniyah-Iraq
91.3
8.7
Present study
Erbil-Iraq
91.7
8.3
Jaff, 2010
Babylon-Iraq
90.1
9.9
Salih, 2009
Saudi Arabia
91.2
8.9
Al-Himaidi & Umar, 2002
Bahrain
91.1
8.9
Jenan, 2012
Arians (Pakistan)
91.7
8.3
Ali et al. 2005
Kuwait
92.5
7.5
Al-Bustan et al. 2002
Iran
90.2
9.8
Keramati et al. 2011
India
93.4
6.6
Thakral et al. 2010
Baghdad-Iraq
80
20
Mashaali, 2014
Blacks
92
8
Whites
85
15
Asians
99
1
People groups
80
References
Beadling & Cooling, 2007; Barclay, 2001 Beadling & Cooling, 2007; Barclay, 2001 Reid & Lomas-Frances, 1997
Chapter Four
Discussion
4.2.2 Other Rhesus Antigens (C, c, E, e) Regarding the frequency of other Rh antigens (C, c, E and e), very limited number of studies are available for comparison in Iraq; for our knowledge the only single study that is available for comparison is that of Mashaali in Baghdad-Iraq. In this study we found that the e antigen has the highest frequency (95.2%), followed by D (91.3%), C (74.8%), c (69.4%) and the lowest frequent antigen was E (30.6%). Apart from D antigen frequency, the results are in agreement with results reported by Mashaali in Baghdad in which the frequency of e, C, c and E were (94%, 77%, 67% and 32 %, respectively) (Mashaali, 2014). In comparison to neighboring countries, the results of the current study are in accordance with other studies in northeast of Iran, which were (97.9%, 75.9%, 73.9%, 29.5%, respectively) (Keramati et al. 2011), and Bahrain (97.3%, 73.2%, 71%, 21%, respectively) (Jenan, 2012). Again the achieved results in this study were to some extend similar to the frequencies reported in white population in US, particularly for e, C and E (98%, 78%, 80% and 29%, respectively) (Beadling & Cooling, 2007; Barclay, 2001). However, apart from e antigen, the results were markedly different from that reported in north Indian (98.1%, 90.2%, 49.5%, 18.9%, respectively) (Thakral et al. 2010), and black population (98%, 32%, 99%, 2%, respectively) (Beadling & Cooling, 2007; Barclay, 2001) (Table 4.3).
81
Chapter Four
Discussion
Table 4.3: Antigen frequencies (%) of Rh (e, C, c, E) blood group in this study compared with published results. People groups
e%
C%
c%
E%
Sulaymaniyah-Iraq
95.2
74.8
69.4
30.6
Baghdad-Iraq
94
77
67
32
Northeast-Iran
97.9
75.9
73.9
29.5
Bahrain
97.3
73.2
71
21
North-India
98.1
90.2
49.5
18.9
Whites
98
78
80
29
Blacks
98
32
99
2
References Present study Mashaali, 2014 Keramati et al. 2011 Jenan, 2012 Thakral et al. 2010 Beadling & Cooling, 2007; Barclay, 2001 Beadling & Cooling, 2007; Barclay, 2001
A significant difference was noted in the frequencies of C, E c, and e antigens when donors were segregated as D+ve and D-ve. The C and E antigens were detected in (81.7% and 34.2%, respectively) of D+ve donors while they were present in only (17% and 0%) of D-ve donors (P=0.02), suggesting that C and E antigens are more prevalent on D+ve red cells. These results are in accordance with a study in north Indian population in which the frequency of C and E antigens in D+ve donors were (91.2% and 18.9%, respectively), while they were present in only (8.5% and 3.7%, respectively) among D-ve donors (Thakral et al. 2010). This is strengthened by our observation of DCe/DCe (R 1R1) to be the most common phenotype (34%), followed by DCe/dce (R1r) and DCe/DcE (R1R2) (29.6%, and 18.3%, respectively) in our D+ve donors population. 82
Chapter Four
Discussion
Noteworthy, in this study the c and e antigens were detected in almost all Dve donors (100%) as compared to (65.8% and 94.6%, respectively) in D+ve donors (p=0.02). These results are again in agreement to a study in north Indian population in which the frequency of c and e antigens were (100%) in D-ve, while they were detected in (49.5% and 98.1%, respectively) among D+ve donors (Thakral et al. 2010). This is also strengthened by our observation that the dce/dce (rr) found to be the most common phenotype in our D-ve donors population (83%). This is relevant because in a study on red cell alloimmunization in 228 D-ve pregnancies in India, it was found that no any case of hemolytic disease of newborn due to anti-c was recorded (Thakur et al. 2006). In contrast to this, in another study on prevalence on alloimmunization in 531 multi-transfused patients, anti-c was found to be the most common antibody specificity (38.8%), followed by anti-E (22.2%) (Thakral et al. 2008), all of them are present in D+ve recipients. The knowledge of these antigen and phenotype frequency is clinically important as one can predict the common alloantibodies that could be formed in pregnant women and patients receiving blood transfusions. For instance, E antigen frequency in our donors population was the lowest (30.6%) which is quite low, followed by c antigen (69.4%) in ascending order of frequency. Thus, it can be assumed that the most common alloantibodies in Rh blood group system among pregnant women and in patients receiving blood transfusion would be anti-E and anti-c. This is in agreement with studies published in India which found that anti-E and anti-c are the most common alloantibodies detected in multitransfused thalassemics (Thakral et al. 2008; Chaudhary, 2011) and patients on hemodialysis (Shukla & Chaudhary, 1997). This finding is also in accordance with studies done in Malaysia, in which anti-E is the most clinically significant allo-antibody detected in the Malaysian blood recipients (Al-Joudi et al. 2011), and among pregnant women (Usman et al. 2013). These clinically significant allo-antibodies 83
Chapter Four
Discussion
(anti-E and anti-c), occur in 1:300 pregnancies, and the risk of hemolytic disease of the fetus and newborn (HDFN) caused by these antibodies is1:500 (Koelewijn et al. 2008). Another advantage of knowing antigen and phenotype frequency is that it helps in selection of antigen negative blood units for patients with presence of alloantibodies. For instance, if a patient in our population has alloantibody against C and needs two units of blood, a minimum of 8-10 units of ABO and Rh (D) matched blood units will need to be tested for C antigen to find two units of antigen negative blood (since C antigen negative donors form about 25% of all our donors population).
4.2.3 The Rhesus Phenotypes The distribution of Rh phenotypes, out of 500 randomly selected blood donors in the present study, the most common phenotype was found to be DCe/DCe (R1R1) 30.4%, followed by DCe/dce (R1r) and DCe/DcE (R1R2) (26.4% , and 16.4%, respectively). These results are comparable to studies done in India which were (35.2%, 30.7%, 8.1%, respectively) (Sarkar et al. 2013), and (43.8%, 30%, 8.22%, respectively) (Thakral et al. 2010). In contrast, the most common Rh phenotype in Bahrain (Jenan, 2012), Iran (Keramati et al. 2011) and among Caucasian was DCe/dce (R1r) (30.9%, 31.8%, and 34.9%, respectively), and that in Blacks was Dce/dce (R0r) 45.8% (Reid & Lomas-Frances, 2004), but Dce/dce (R0r) was detected in only2% of our donors. As shown in (Table 4.4) there is a lot of variation in the common Rh phenotypes in different world populations.
84
Chapter Four
Discussion
Table 4.4: Rh phenotype frequencies of the present study, compared with North Indian, Northeast Iran, Caucasian and Black population. Phenotypes
Present
*North-
study (%) India (%)
˚Northeast •Caucasian
•Black
Iran (%)
(%)
(%)
18.5
2.0
DCe/DCe (R1R1)
30.4
35.2
25
DCe/dce (R1r)
26.4
30.7
31.8
DCe/DcE (R1R2)
16.4
8.1
16.5
13.3
4.0
DcE/dce (R2r)
9.4
5.9
9.6
11.8
18.6
DcE/DcE (R2R2)
4.8
0.7
1.7
2.3
0.2
2
2.2
4.2
2.1
45.8
dce/dce (rr)
8.8
0.3
8.3
15.1
6.8
dCe/dce (rʹr)
1.8
2.5
1.3
0.8
Rare
Dce/dce (R0r)
* Sarkar et al. 2013
˚ Keramati et al. 2011
34.9
21.0
• Reid & Lomas-Frances, 2004
4.3 The Kell Blood Group System The K antigen is very immunogenic (second to the D antigen) in stimulating antibody production. Anti-K is an important antibody; it is nearly always immune, IgG and complement-binding. It causes severe HTRs and HDFN (Shaz, 2009). It's frequency in this study was (5.8%), which is similar to that of Thakral et al. study in north India (5.56%) (Thakral et al. 2010), and it occurs between the frequencies reported by Keramati et al. in northeast of Iran (Keramati et al. 2011) and Whites (8%, and 9%, respectively), and that of Blacks (2%) (Beadling & Cooling, 2007). The frequency of k (Cellano) antigen was detected in almost (100%) in our donor 85
Chapter Four
Discussion
population, which is similar to the results reported in north Indian 100% (Thakral et al. 2010), and black populations 100% (Beadling & Cooling, 2007), whereas 0.2% of Whites and 2.3% of northeast-Iran are k (Cellano) negative. This implies that while Whites and northeast Iranian population might occasionally develop anti-k (Cellano), the likelihood of finding this alloantibody in our population is negligible (Table 4.5).
Table 4.5: Antigen frequencies (%) of the Kell blood group system in this study compared with other published results. Kell (K)
Cellano (k)
(%)
(%)
Sulaymaniyah-Iraq
5.8
100
Present study
North-India
5.56
100
Thakral et al. 2010
Northeast- Iran
8.0
97.7
Keramati et al. 2011
Whites
9.0
99.8
Beadling & Cooling, 2007
Blacks
2.0
100
Beadling & Cooling, 2007
People groups
References
Regarding the distribution of the Kell phenotypes, the most common phenotype was found to be K-k+ (94.2%), followed by K+k+ (5.8%). None of the donor was found to be K homozygous (K+k-) or (K-k-). These results are similar to that reported in north Indian population in which K-k+, K+k+ found in (94.32%, 5.68%, respectively) (Thakral et al. 2010), and (95.96%, 4.04%, respectively) (Nanu & Thapliyal, 1997). Again our results were found to be intermediate
86
Chapter Four
Discussion
between that reported by Keramati et al. in northeast of Iran (Keramati et al. 2011) and White, and that of Black populations (Beadling & Cooling, 2007) (Table 4.6).
Table 4.6: Phenotype frequencies of the Kell blood group system compared with other published results. K-k+
K+k+
K+k-
K-k-
(%)
(%)
(%)
(%)
Sulaymaniyah-Iraq
94.2
5.8
0.0
0.0
Present study
North-India
94.3
5.7
0.0
0.0
Thakral et al. 2010
North-India
96.0
4.0
0.0
0.0
Northeast-Iran
92.0
5.7
2.3
0.0
Whites
91.0
8.8
0.2
0.0
Blacks
98.0
2.0
Rare
0.0
People groups
References
Nanu &Thapliyal, 1997 Keramati et al. 2011 Beadling & Cooling, 2007 Beadling & Cooling, 2007
4.4 The Kidd Blood Group System The frequencies of the Kidd blood group system antigens were (Jka = 77%, Jkb = 67.5%). Our results are similar to that reported by Keramati et al. in northeast of Iran (Jka =79.1%, Jkb=65.1%) (Keramati et al. 2011), and comparable to that of Whites (Jka = 77%, Jkb = 74%) (Beadling & Cooling, 2007), and north-Indian population (Jka = 82.65%, Jkb = 66.56%) (Thakral et al. 2010), while there is
87
Chapter Four
Discussion
marked difference with that of Black population (Jka = 92%, Jkb = 49%) (Beadling & Cooling, 2007) (Table 4.7).
Table 4.7: Antigen frequencies (%) of the Kidd blood group system in this study compared with other published results. Jka (%)
Jkb (%)
Sulaymaniyah-Iraq
77.0
67.5
Present study
Northeast-Iran
79.1
65.1
Keramati et al. 2011
North-India
82.6
66.6
Thakral et al. 2010
Whites
77
74
Blacks
92
49
People groups
References
Beadling & Cooling, 2007 Beadling & Cooling, 2007
The most common Kidd phenotype was Jk (a+b+) (44.5%), which is similar to results reported by Keramati et al. in northeast of Iran (44.4%) (Keramati et al. 2011), and comparable to that reported by Thakral et al. in north India (Thakral et al. 2010) and Whites (Beadling & Cooling, 2007) (49.21 % and 49%, respectively). While in Blacks it is much lower (34%), and Jk (a+b-) is the most common phenotype among the Blacks (57%) (Beadling & Cooling, 2007). No Jk (a-b-) phenotype was detected in any donor, which is also very rare in White and Black people, except for Polynesians (< 1%) (Beadling & Cooling, 2007) (Table 4.8).
88
Chapter Four
Discussion
Table 4.8: Phenotype frequencies (%) of the Kidd blood group system in this study compared with other published results. Jk People groups
Jk
Jk
(a+b+) (a+b-) (a-b+)
Jk (a-b-)
References
(%)
(%)
(%)
(%)
Sulaymaniyah-Iraq
44.5
32.5
23
0.0
Present study
Northeast-Iran
44.4
34.7
20.7
0.2
Keramati et al. 2011
North-India
49.2
33.4
17.3
0.0
Thakral et al. 2010
Whites
49
28.0
23.0
Blacks
34.0
57
9.0
Very
Beadling & Cooling,
rare
2007
Very
Beadling & Cooling,
rare
2007
4.5 The Duffy Blood Group System The frequencies of the Duffy blood group system antigens were (Fya =70%, Fyb=57.5%). The Fya antigen frequency is very close to that reported in northeast of Iran (73.8%) (Keramati et al. 2011), and it is intermediate between results reported in north Indian population (Thakral et al. 2010), and Whites (Beadling & Cooling, 2007), which are (86.75%, 66%, respectively). While the frequency of Fyb antigen is much closer to that reported by Thakral et al. (56.15%), and it is in between that of Keramati et al. study and Whites (49.2%, 83%, respectively). But our results were much more than that of the Blacks (Fya = 10%, Fyb = 23%) (Beadling & Colling, 2007) (Table 4.9).
89
Chapter Four
Discussion
Table 4.9: Antigen frequencies (%) of the Duffy blood group system in this study compared with other published results. People groups
Fya (%)
Fyb (%)
Sulaymaniyah-Iraq
70
57.5
Present study
Northeast-Iran
73.8
49.2
Keramati et al. 2011
North-India
86.7
56.1
Thakral et al. 2010
Whites
66
83
Blacks
10
23
References
Beadling & Cooling, 2007 Beadling & Cooling, 2007
In the Duffy blood group system, Fy (a+b-) was the most common phenotype in our study (38.5%), which is comparable to the results reported in north-India (Thakral et al. 2010) and northeast of Iran (Keramati et al. 2011), which are (43.9%, 47.4%, respectively). However, it is much higher than that of Whites (17%) and Blacks (9%) population (Beadling & Cooling, 2007). The most common phenotype in Whites is Fy (a+b+) 49%, and Fy (a-b-) in Blacks (68%) (Beadling & Cooling, 2007). Duffy antigen is postulated to be the receptor for entry of the plasmodium vivax on the red cells (Rayner, 2005; Langhi & Bordin, 2006). This probably explains high prevalence of Duffy null phenotype Fy (a-b-) in the endemic area of malaria such as among the black people (68%) (Reid & Lomas-Frances, 2004; Mohandas & Narla, 2005; Beadling & Cooling, 2007). The frequency of Duffy null phenotype Fy (a-b-) in our study was (4%), which is very close to that 90
Chapter Four
Discussion
reported by Keramati et al. in northeast of Iran (3.4%) (Keramati et al. 2011). While it is very rare in Whites (Beadling & Cooling, 2007) and (0%) in north Indian population (Thakral et al. 2010) (Table 4.10). This higher rate of null phenotype frequency in our donors may be related to the existence of endemic areas of malaria in the Kurdistan Region of Iraq in the past.
Table 4.10: Phenotype frequencies (%) of the Duffy blood group system in this study compared with other published results. Fy People groups
Fy
Fy
(a+b-) (a+b+) (a-b+)
Fy (a-b-)
References
(%)
(%)
(%)
(%)
Sulaymaniyah-Iraq
38.5
31.5
26
4.0
Present study
Northeast-Iran
47.4
26.4
22.8
3.4
Keramai et al. 2011
North-India
43.8
42.9
13.3
0.0
Thakral et al. 2010
Whites
17.0
49.0
34.0
Blacks
9.0
1.0
22.0
Very
Beadling & Cooling,
rare
2007
68.0
Beadling & Cooling, 2007
4.6 The MNSs Blood Group System The frequencies of the MNSs blood group system antigens M, N, S and s in our study were (79.5%, 66.5%, 54%, and 88.5%, respectively). These results are similar to the results reported in north Indian population which were (75.4%, 61.5%, 56.5%, and 87.4%, respectively) (Thakral et al. 2010), and comparable with that of Keramati et al. study in northeast of Iran (87%, 56.7%, 56.7%, and 91
Chapter Four
Discussion
84.5%, respectively) (Keramati et al. 2011), and in Whites (78%, 72%, 55%, and 89%, respectively), while they are different with that of Blacks, particularly for S and s antigens frequency (Beadling & Cooling, 2007) (Table 4.11).
Table 4.11: Antigen frequencies (%) of the MNSs blood group system in this study compared with other published results People groups
M (%)
N (%)
S (%)
s (%)
Sulaymaniyah-Iraq
79.5
66.5
54
88.5
Present study
North-India
75.4
61.5
56.5
87.4
Thakral et al. 2010
Northeast-Iran
87
56.7
56.7
84.5
Keramati et al. 2011
Whites
78
72
55
89
Blacks
74
75
31
93
References
Beadling & Cooling, 2007 Beadling & Cooling, 2007
Regarding the phenotype frequency, M+N+ and S-s+ were the most common phenotypes observed in the MNS blood group system in our study which was nearly equal (46%). These results are comparable to that of Keramati et al. study in northeast of Iran, which reveals M+N+ and S-s+ to be the most common phenotypes and found in (43.7% and 43.3%, respectively) (Keramati et al. 2011), and comparable to that reported in White and Black populations (Beadling & Cooling, 2007), while in Thakral et al. study the most common phenotype was M+N- and S+s+ (Table 4.12).
92
Chapter Four
Discussion
Table 4.12: Phenotype frequencies (%) of the MNSs blood group system in this study compared with other published results.
People groups
M+N+ M+N-
MN+
S-s+
S+s+
S+s-
%
%
%
%
%
46
33.5
20.5
46
42.5
11.5
Northeast-Iran
43.7
43.3
13
43.3
41.2
15.5
North-India
36.9
38.5
24.6
43.5
43.8
12.6
Whites
50
28
22
45
44
11
Blacks
44
26
30
69
28
3
Sulaimaniyah – Iraq
%
References
Present study Keramati et al. 2011 Thakral et al. 2010 Beadling & Cooling, 2007 Beadling & Cooling, 2007
Out of nine possible phenotypes found in our study, M+N+S-s+ (40%) was the most common phenotype; whereas, M-N+S+s- (0.5%) was the lowest common phenotype observed in the MNS blood group system. The frequency of M+N+Ss+, the most common phenotype in this study, is comparable to the results reported by Agarwal et al. in India (28.8%) (Agarwal et al. 2013), as well as in European (22.6%) and African-American (33.4%) (Lal et al. 2000; Cleghorn, 1960), while in Thakral study in north Indian population the most common phenotype was M+N+S+s+ (19.6%) (Table 4.13).
93
Chapter Four
Discussion
Table 4.13: phenotype frequencies of MNSs blood group system compared to other published data. Phenotype
Present study (%)
•India (%)
*European
*African
(%)
American (%)
M+N+S-s+
40
28.7
22.6
33.4
M+N+S+s+
17.5
20.9
22.4
13
M+N+S+s-
22.5
5.12
3.9
2.2
M+N-S-s+
18
13.8
10.1
15.5
M+N-S+s+
19
15
14
7
M+N-S+s-
5.5
7.1
5.7
2.1
M-N+S-s+
14.5
5.1
15.6
19.2
M-N+S+s+
6
3.1
5.4
4.5
M-N+S+s-
0.5
1.2
0.3
1.6
•(Agarwal et al. 2013)
* Lal et al. 2000; Cleghorn, 1960.
4.7 The Lutheran Blood Group System The frequencies of the Lutheran blood group system antigens were (Lua = 4.5%, Lub = 95.5%). The Lub antigen frequency was similar to the results reported by Keramati et al. in northeast of Iran (Lub = 94.8%) (Keramati et al. 2011), and to that of Thakral et al. study in north Indian population ( Lub = 96.8 %) (Thakral et al. 2010), while the frequency of Lua antigen is in the middle of the results achieved in the Keramati et al. and Thakral et al. studies (8.8 % and 0.9 %, respectively) (Table 4.14).
94
Chapter Four
Discussion
Table 4.14: Antigen frequencies (%) of the Lutheran blood group system in this study compared with other published results. People groups
Lua (%)
Lub (%)
Sulaymaniyah-Iraq
4.5
95.5
Present study
Northeast-Iran
8.8
94.8
Keramati et al. 2011
North-India
0.95
96.8
Thakral et al. 2010
References
Regarding the phenotype frequency, Lu (a-b+) with a frequency of (92%) was the most common phenotype in the Lutheran system in our study and it is similar to the results reported in Whites and Blacks (92.3%, 93%, respectively) (Beadling & Cooling, 2007), and it is intermediate between that of Keramati et al. study in northeast of Iran and Thakral et al. study in north Indian population which are (88.8%, 95.9%, respectively). A very rare phenotype Lu (a-b-) of Lutheran system in Whites and Blacks was found in (3.5%) of our donors and is comparable with that of Thakral et al. (3.15%) and Keramati et al. studies (2.7%). Lu (a+b+) is reported to be 7.5% in Whites, while in our study it was 3.5% which is lower than in the Whites and intermediate between that of Thakral et al. and Keramati et al. studies (0.95% and 6.3%, respectively) (Table 4.15).
95
Chapter Four
Discussion
Table 4.15: Phenotype frequencies (%) of the Lutheran blood group system in this study compared with other published results. Lu People groups
Lu
Lu
(a-b+) (a+b-) (a+b+)
Lu (a-b-)
References
(%)
(%)
(%)
(%)
92
1.0
3.5
3.5
Present study
Northeast-Iran
88.8
2.5
6.3
2.7
Keramati et al. 2011
North-India
95.9
0.0
1.0
3.1
Thakral et al. 2010
White
92.3
0.2
7.5
Blacks
93
0.1
5
Sulaymaniyah-Iraq
Very
Beadling & Cooling,
rare
2007
Very
Beadling & Cooling,
rare
2007
4.8 The Lewis Blood Group System The frequencies of the Lewis blood group system antigens were (Lea =43.5%, Leb = 64.5%). Our results are similar to Keramati et al. study in northeast of Iran (Lea = 43.2%, Leb = 63.1%) (Keramati et al. 2011), and to that of Thakral et al. study in north India, particularly for Le b antigen (Leb = 60.6%) (Thakral et al. 2010) (Table 4.16).
96
Chapter Four
Discussion
Table 4.16: Antigen frequencies (%) of the Lewis blood group system in this study compared with other published results. People groups
Lea (%)
Leb (%)
Sulaymaniyah-Iraq
43.5
64.5
Present study
Northeast-Iran
43.2
63.1
Keramati et al. 2011
North-India
20.8
60.6
Thakral et al. 2010
References
The most common phenotype in the Lewis blood group system was Le (ab+) (54.2%), followed by Le (a+b-), Le (a+b+) and Le (a-b-) which were (33.5%, 10%, and 2.5%, respectively).
These results are similar to that reported by
Keramati et al. in northeast of Iran which are (55.2%, 35.3%, 7.9% and 1.6%, respectively) (Keramati et al. 2011). The frequency of Le (a-b+) phenotype in our study is also similar to that reported in Blacks (55%) (Beadling & Cooling, 2007), and Thakral et al. study (60.6%) (Thakral et al. 2010), but it is lower than that reported in Whites (72%) (Beadling & Cooling, 2007). The Le (a+b+) is rare in European and American populations (Blaney & Hovard, 2000; Beadling & Cooling, 2007; Shaz, 2009), yet it is observed in some Asian Populations (e.g. Polynesian, Japanese or Taiwanian ancestry) from 10-40 % (Reid & Lomas-Frances, 2004; Shaz, 2009), as well as, it is observed in about 10% of our donors and in (7.9%) in northeast of Iran (Keramati et al. 2011). In contrast, Le (a-b-) was seen in a lower frequency (2.5%) in our study, and in Keramati et al. study (1.6%) than that of White (6%), north Indian (18.6%), and Blacks populations (22%) (Table 4.17).
97
Chapter Four
Discussion
Table 4.17: Phenotype frequencies (%) of the Lewis blood group system in this study compared with other published results. Le People groups
Le
Le
(a-b+) (a+b-) (a+b+)
Le (a-b-)
References
(%)
(%)
(%)
(%)
Sulaymaniyah-Iraq
54.5
33.5
10
2.5
Present study
Northeast-Iran
55.2
35.2
7.9
1.6
Keramati et al. 2011
North-India
60.6
20.8
0.0
18.6
Thakral et al. 2010
Whites
72
22
Rare
6
Blacks
55
23
Rare
22
Beadling & Cooling, 2007 Beadling & Cooling, 2007
4.9 The P Blood Group System Approximately 76% of our donors were positive for P1 antigen, which is intermediate between results reported in Whites (79%) (Beadling & Cooling, 2007), and north Indian population (71.9%) (Thakral et al. 2010), but it is higher than that reported in Keramati et al. study in Iran (66.2%) (Keramati et al. 2011), while much lower than that in Blacks (94%).
The knowledge of antigen and phenotype frequency of different blood group systems in the given population is clinically important as one can predict the common alloantibodies that could be formed in pregnant women and patients receiving transfusions, and it helps in selection of antigen negative blood units for patients with presence of alloantibodies. 98
Chapter Five
Conclusions and Recommendations
5.1 Conclusions 1. In the ABO blood group system, O was the most common blood group, followed by A, B, and AB. In the Rh blood group system, the most common antigen was e, followed by D, C, c, E. In the Kell, Kidd and Duffy blood group systems, the most common phenotypes were [(K-k+, Jk (a+b+), Fy (a+b-), respectively]. In the MNSs, Lutheran and Lewis blood group systems, the most common phenotypes were, M+N+S-s+, Lu (a-b+), Le (a-b+), respectively. P1 antigen was observed in 76% of the donors.
2.The distribution of these blood group systems in general in our donor population, was very close to the previous studies conducted in Kurdistan Region, other parts of Iraq, and Iran, with similar trends to the neighboring Arab countries, some Asian (India), and western Europeans (Caucasian) populations, while it was different from that of Black population.
3. We determined some differences in the frequency of rare phenotypes compared with other studies. Higher frequency of Fy (a-b-), Lu (a-b-), and Le (a+b+), were observed which were (4%, 3.5%, 10%, respectively).
99
Chapter Five
Conclusions and Recommendations
5.2 Recommendations 1. Performing at least limited versus extended red cell phenotyping for ABO, Rh (D, C, c, E, e), and Kell (K) in donors and transfusion dependent patients before receiving their first blood transfusion. Taking into account the heavy financial burden of complete phenotyping on the blood bank.
2. Providing antigen compatible blood units for prevention of alloimmunization, and antigen negative blood for already alloimmunized multitransfused patients for prevention of further alloimmunization.
3. The necessity of introducing antibody screening and identification for pregnant women as part of the antenatal care to look for significant allo-antibodies other than anti-D, as well as in multitransfused patients with alloantibodies.
4. Intensive studies from different parts of Iraq need to be carried out to observe regional differences in antigen phenotype frequency taking into consideration the ethnic heterogeneity in our country.
5. Further studies are required to establish the genetic make-up of various blood group systems in our population. Molecular investigations are more informative to be done, since only serological investigation has been performed in the current study. .
011
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جامعة السليماني جمموعة العلوم الطبية كلية الطب الدراسات العليا ____________________________________________ ترددات النمط الظاهرى لفصائل الدم ( ,Rh ,ABOكيل ,كيد ,دافى ,MNSs ,اللوثرية ,لويس و (Pفى املتربعني بالدم فى مصرف الدم فى حمافظة السليمانية
رسالة مقدمة جمللس كلية الطب ,جمموعة العلوم الطبية ,يف جامعة السليمانية كجزء من متطلبات منح درجة املاجستري يف علم أمراض الدم (هيماتولوجي)
من قبل شيماء صاحل امني بكالوريوس يف الطب واجلراحةالعامة دبلوم عالي يف علم أمراض الدم (هيماتولوجي)
باشراف احملاضرالدكتور هشام عارف ططة M.B.Ch.B./FIBMS /Lecturer Hematopathology
كانون الثانى 5102م
ِِريَبةندان 5172ك
اخلالصة
اخلفليةة :ان فصائل الدم البشرية هي تراكةب موروثة خمتفلية تشكل جزاْ ال يتجزاْ من غشاء اخلفلةة احلمراء .وقد مت حتديد أكثر من 033مستضدات الكريات احلمر وتصنةيها إىل اكثر من 00أنظمة منيصفلة رئةسةة .خيتفلف توزيعها يف خمتفلف اجملتمعات واجلماعات العرقةة .ان التعرف عفلى خمتفلف املستضدات يف الدم يف جمموعة من السكان له فوائد عديدة يف طب نقل الدم .حددت معظم البةانات النمط الظاهري ليصائل الدم يف األوروبةني واألمريكةني وبعض اآلسةويني ،وفى كردستان العراق مل يتم حتديد غري ABOو.Rh األهداف :لتحديد ترددات املستضدات والنمط الظاهري ملختفلف جمموعات فصائل الدم بني املتربعني احملفلةني ،إلنشاء
بنك معفلومات ليصائل الدم لفلمتربعني لتحسني خدمات نقل الدم و لألغراض متعددة يف املستقبل منها :إعداد خاليا األلواح الكاشف الستخدامها يف الكشف عن األجسام املضادة و حتديدها ,وتوفري وحدات الدم املطابق يف املستضدات لفلوقاية من ،alloimmunizationوتوفري الدم املستضد سفلبةا لفلمرضى احملتاجني لنقل الدم بصورة متكررة و املصابني بال( .)alloimmunizedمت تسجةل امساء املتربعني ذوى فصائل الدم النادرة. مواد وطرق البحث :هذه دراسة استطالعةة ،واليت ما جمموعه مخسة آالف الصحةني املنتضمني املتكررين املتربعني بالدم يف مصرف الدم املركزي يف حمافظة السفلةمانةة ،مت حتديد ال )AB & O ،B ،A( ABOوالريس ( )Dيف نظام فصائل الدم بواسطة تقنةة اجلل .من ضمن هذه اجملموعة 033 ،من املتربعني مت حتديد املستضدات األخرى من فصةفلة الدم الريس )e ،E ،c ،C( :وكةل ( ،)Kومنها 033من املتربعني لتحديد املستضدات املمتدة جملموعة فصائل الدم األخرى:
كةل (ك ، )cellano-كةد ( ،)Jkb ، Jkaدايف ( ،)S ،s ،N ،M( MNSs )Fyb ،Fyaلويس ( )Leb، Leaوالفلوثرية ( Lua b
) Lu ،و ) ،)P (P1بواسطة تقنةة أنبوب التقفلةدية .وأعرب عن املستضدات وترددات النمط الظاهري كنسب مئوية. النتائج :يف جمموعة الدم ،ABOكان النمط الظاهري األكثر شةوعا ،)33%( Oتفلةها )0.23%( Aو ،(22.8%) Bيف حني كانت فصةفلة الدم األقل انتشارا .(7.6%) ABمن بني مستضدات فصةفلة الدم الريس e ،كانت األكثر شةوعا ()202.% تفلةها ،)3220%( c ، (74.8%( C , (91.3%) Dو (30.6%) Eمع ) DCE/DCE (R1R1و ( dce/dce )rrكان الظواهر األكثر شةوعا بني جمموعات الريس ( )Dاإلجيابةة و الريس ( )Dالسفلبةة ،عفلى التوالي .يف نظام فصةفلة الدم كةل ،وجد K أن تكون إجيابةة يف ٪ 025من املتربعني و النمط الظاهري K+k-مل يوجد .من بني فصائل الدم كةد ودايف Jk (a+b+) ،و ) Fy (a+b-الظواهر األكثر شةوعا ( ٪0020و ٪0520عفلى التوالي) .بالنسبة ليصةفلة الدم ،MNSكان M + N + S -S + األكثر شةوعا ( . (40%من بني فصائل الدم لويس والفلوثرية ،الظواهر األكثر شةوعا كانت ) Le (a- b+و(Lu (a- b + اليت كانت ( ٪0020و ٪2.عفلى التوالي) .وجدنا الظواهر النادرة ) Le (a+b+), Lu (a-b-),Fy (a-b-فى( 020 ، ٪ 0 ، ٪و ٪ 03عفلى التوالي) .مت العثور عفلى املسضد P1يف ٪63من املتربعني. االستنتاجات ِ:ضمن فصائل الدم ABOوالريس ،كانت اليصائل األكثر شةوعا ،Oواملستضد ،eعفلى التوالي ,ومن
ضمن فصائل الدم كةل ،كةد ،دايف ، MNSs ،الفلوثرية ،لويس و ، Pالظواهر األكثر شةوعا كانت [ ) ، (K –k+كةد ) ،(a+b+دايف ) ,(a-b+)، M + N + S -S + ،(a+b-الفلوثرية لويس ), (a-b+و P1عفلى التوالي ] .وكان توزيع هذه اليصائل لدى املتربعني لدينا قريبا جدا من الدراسات السابقة التى اجريت يف إقفلةم كردستان ومناطق أخرى من العراق و إيران ،و مماثفلالْ لدراسات اجريت يف الدول العربةة اجملاورة ،وبعض اآلسةويني ( اهلند ) ،واألوروبةني الغربةني ( قوقازي ) ،يف حني أنها ختتفلف عن دراسات اجريت عفلى السكان السود .كما مت احصاء بعض الظواهر النادرة مثل دافى (, )a-b- الفلوثرية ( ,)a-b-و لويس (.)a+b+
Chapter One
Literature Review
Chapter Two
Subjects and Methods
Chapter Three
Results
Chapter Four
Discussion
Chapter Five
Conclusions and Recommendations
References
ﺳﻟﻴﻤﺍﻧﻲ ﺯﺍﻧﻜﺅﻱ َ ﻛﺆﻤﺓﻟﺓﻱ ﺯﺍﻧﺴﺘﻲ ﺛﺯﻳﺸﻜﻲ ﺳﻜﻮﻟﻲ ﺛﺯﻳﺸﻜﻲ ﺧﻮﺻﻨدﻧﻲ باﻹ ___________________________________________ دووبارةبونةوةى فينؤتايثي طروثةكاني خويَين ( ,Rh ,ABOﻛﻴَ َل ,ﻛﻴد ,دﺍفى ,MNSs ,لوثريان ,ﻟﻮﻳس و (Pلة خويَن بةخشانى بانكى خويَن لة ثاريَزطاى سليَمانى ئةم تيَزة ثيَشكةشة بة ئةجنومةني سكولَي ثزيشكي ,كومةلَةي زانسيت ثزيشكي ,زانكوي سليَماني وةك بةشيَك لة داواكاريةكاني بة دةستهيَناني ثلةي ماستةر لة زانستى نةخٍوٍَشيةكاني خويَن (هيماتوَلوَجى)
لةاليةن
شيماء صاحل امني بةكالوريوس لة ثزيشكى و نةشتةرطةري طشتى
دبلوَمى باالَ لة زانستى نةخٍوٍَشيةكاني خويَن (هيماتوَلوَجى)
بةسةرثةرشيت
وانةبيَََََُُُُّّّّّّذ دكتوَر هشام عارف كطة M.B.Ch.B./FIBMS /Lecturer Hematopathology
كانونى دووةم 5102م
ريَبةندان 5102ك
ثوختة ثيَشةكى :طروثةكانى خويَنى مرؤظ شيَوازى جؤراو جؤرى هةية ,كة بةشيَوةيةكى بؤ ماوةيى لة ثيَكهاتةى ثةردةى خرِؤكة سورةكانداية .زياتر لة ( ) 033دذة ثةيدا كةرةى خرِؤكةى سوور ديارى كراوة و زياتر ثؤلني كراوة بؤ ( ) 00سيستمى جياوازى طةورة .بالَوبوونةوةيان دةطؤرِيَت لة كؤمةلَطا جياوازو طروثة نةذادييةكاندا .ناسينةوةى دذة ثةيداكةرةكانـى طروثى خويَن لـــة دانيشتواندا ضةند سووديَكى جياوازى هةية لــــة زانستى ثزيشكى خويَن طواستنةوةدا .زؤربــــةى زانياريةكان لة بالَوكراوةكاندا جؤرةكانى طروثى خويَنى ئةوروثيةكان و ئةمريكيةكان و هةنديَك لة ئاسياييةكانى ديارى كردووة ,هيض رِاطةياندراويَك نى ية جطة لة ABOو Rhلةهةرميى كوردستانى عيَرقدا . ئاماجنةكان :ديارى كردنى رِيَذةى بالَوبوونةوةو دوبارة بوونةوةى دذة ثةيداكةرةكانى طروثى خويَنى جؤراو جؤر لة خويَن بةخشانى دانيشتوانى خؤجىَ يدا .دروست كردنى بانكى زانيارى خويَن بةخشان لة طروثةكانى خويَن بؤ ئامادة كردنى خانةى دةستةى ناسةر كة بةكارديَت بؤ دؤزينةوةو ناسينةوةى دذةتةن .بؤ دةستةبةر كردنى يةكةى خويَنى شياوو هاوتا لة دذة ثةيدا كةرة بــــؤ خؤثاراسنت لــة ( )alloimmuiazationبؤ دةستةبةركردنى خويَنى دذة ثةيداكةرةى سلبى بــــؤ نةخؤشانى هةميشة خويَن وةرطر ى ) .)alloimmunizedتؤمار كردنى طروثى خويَنى دةطمةن لــــة خويَن بةخشان. بابةت و رِيَطاكانى ليَكؤلَينةوة :ئةمة ليَكؤلَينةوةيةكى برِطةثةرِةوةية كة تيايدا لة كؤى ( )0333ضةند جارة خويَن بةخشى خؤبةخشى لةش ساغ لة بانكى خويَنى ناوةندى لة شارى سليَمانى جؤراو جؤر كران بؤ طروثى RhDو ABOبةتةكنيكى جيَلَ .لةنيَوئةم ذمارةيةدا ( )033خويَن بةخش جؤراو جؤركران بؤ دذة ثةيداكةرةكانى ترى طروثى خويََنى )C,c,E,e( Rhو كيَلَ ( )033( , )kخويَن بةخش بؤ جؤرةكانى دذة ثةيدا كةرةى دريَذكراوةى طروثةكانى خويَنى تر (:كيَلَ ,كيد,دةفى ,MNS,لوثريان,لويس و )Pبةتةكنيكى تيوبى ئاسايى ,دووبارة بوونةوةى دذةثةيداكةرةو فينؤتايث بةشيَوةى سةدى دةردةبرِيَت. ئةجنامةكان :لة سيستمى طروثى ( )ABOدا ,باوترين فينؤتايث )%37( Oبةدوايدا )%32.6(Aو )%22.8(Bبةالَم كةمرتين طروثى خويَنى باو ABبوو ( .)%7.6لةنيَوان دذة ثةيداكةرةكانى طروثى خويَنى Rhدا e ,باوترين بوو (, )%95.2بةدوايدا c )%74.8( C,)%91.3( D ( )%30.6( E )%69.4لةطةلَ )R1R1,(DcelDceو )rr( dceldceباوترين فينؤتايث بوون لةنيَوان ( )Rh (Dاجيابى و ( Rh )Dسلبى, بةدواييةكدا .لةسيستمى طروثى خويَنى كيَلَ K ,دةركةوت كة اجيابى بوو لة ( )%5.8خويَن بةخشان و فينؤتايثى K+k-تياياندا دةرنةكةوت . لةسيستمةكانى طروثى خويَنى كيد و دةفى ( JK )a+b+و ( Fy )a+b-باوترين فينؤتايب بوون ( %38.5 ,%44.5بةدواييةكدا) لةسيستمى طروثى خويَنى MNSدا )%40( M+N+S-S+باوترين بوو ,دةربارةى سيستمى لويس ولوثريان باوترين فينؤتايث ( Le ) a-b+و ( Lu ) a-b+كة ( %54.5و )%92بةدواييةكدا .دةركةوتنى فينؤتايثى دةطمةن لة ( Lu )a-b-( ، Fy )a-b-و ( Le )a+b+كة ( %10,%3.5%,%4 بةدواييةكدا) .دذة ثةيداكةرةى P1دةركةوت لة %76خويَن بةخشان. دةرئةجنامةكان :لةسيستمى طروثى خويَنى ABOو Rhدا ,باوترين طروثى خويَن Oو دذة ثةيداكةرةى eبوون بةدواييةكدا .لةسيستمةكانى كيَلَ ,كيد ,دةفى ,MNS,لوثريان,لويس و Pطروثى خويَندا ,باوترين فينؤتايث ),Fy (a+b-), Jk (a+b+),)K-k+
M+N+S-S+
), P1, Le(a-b+) ,Lu(a-b+بةدواييةكدا.دابةشكردنى ئةم سيستمى طروثى خويَنانة لةخويَن بةخشانى دانيشتوانى خوجيَ يدا زؤر نزيك بوون لةتويَذينةوةكانى ثيَشووتردا لةهةرميى كوردستان و بةشةكانى ترى عيَراق و ئيَران ,هاوشيَوةبوو لةطةلَ والَتانى عةرةبى دراوسيَداو هةنديَك والَتانى ئاسيا (هيند)و ئةوروثاى رِؤذئاوا (قةوقازييةكان) بةالَم جياواز بو لةطةلَ دانيشتوانى رِةش ثيَستدا ,هةروةها هةنديَك فينؤتايثى دةطمةن دةركةوت ). Le(a+b+),Lu(a-b-) ,Fy (a-b-
