The Prevalence of Rh Phenotypes and the Distribution of the Major Rhesus antigens…

The Prevalence of Rh Phenotypes and the Distribution of the Major Rhesus antigens among Saudi Blood Donors in Aseer Region

The Rh system is the most significance blood group system after ABO in the field of transfusion medicine, and the primary cause of the hemolytic disease of fetal and newborns. In addition, Rh antibodies are the most prevalent RBCs alloantibodies among multitransfused patients in Saudi Arabia and other parts of the world. Several studies shows that the distribution of the Rhesus antigens varies from one population to another. Therefore, due to lack of relevant studies among Saudi donors, the aim of this research was to determine the frequencies of Rh phenotypes and the distribution of the major Rhesus antigens (D, C, E, c, e) among Saudi blood donors in Aseer region. The results of this research will help blood banks specifically in Aseer region and generally all over the kingdom as a reference to the frequencies of Rh antigens and Rh phenotypes in Saudi blood donors. Which, assist to avoid Rh alloimmunization and help in finding compatible units for previous immunized patients; therefore, minimize searching time and workload. This research conducted in the blood bank of Aseer central hospital on 734 healthy Saudi male donors, done by the antigen-antibody agglutination test by using a fully automated Blood Bank analyzer TANGO’ optimo. The results showed eight Rh phenotypes in the study samples, R1r (35.3%) was the most common phenotype followed by: R1R1 (25.9%), R1R2 (12.6%), r r (7.8%), R0r (7.8%), R2r (7.5%), R2R2 (2.6%) and r” r (0.5%). While the distribution of the major Rh antigens was D (91.7%), C (74.4%), E (22.8%), c (74.1%) and e (97.3%). The present research concludes that the frequencies of Rh antigens and Rh phenotypes displayed broad variance among Saudi blood donors and other ethnic groups. Therefore, the consistent population-based frequency data of Rh antigens and their phenotypes in any given population has a significance role in transfusion practice, instead of relying on international published ratios for other ethnic groups, as is the case now. It is recommended to conduct further studies in different regions of Saudi Arabia, in order to examine geographical and regional variance in Rh antigens and Rh phenotypes frequencies.

AABB American Association of Blood Banks
ACH Aseer Central Hospital
AHG Anti-Human Globulin
IHTR Immediate Hemolytic Transfusion Reaction
DAT Direct Antiglobulin Test
DHTR Delayed Hemolytic Transfusion Reaction
EDTA Ethylene Diamine Tetra acetic Acid
EVH Extra-Vascular Hemolysis
FC Fragment Crystallizable region
HDFN Hemolytic Disease of the Fetal and Newborn
HTR Hemolytic Transfusion Reaction
IAT Indirect Antiglobulin Test
IgG Immunoglobulin G
IgM Immunoglobulin M
ISBT International Society of Blood Transfusion
IVH Intra-Vascular Hemolysis
MLB Modified LISS Biotest
Q.C Quality Control
RBCs Red Blood Cells
Rh Rhesus blood group
RhAG Rh-Associated Glycoprotein


Chapter I
General Introduction
1.1 Overview of Human Blood Groups
The term blood groups are usually restricted to blood cells surface antigens and generally to RBCs surface antigens. These antigens are molecules present on the red blood cells membrane, which can trigger the immune response if they are foreign to the body.
Landsteiner discovered the first blood group system (ABO) in 1901, when he observed that plasma from some individuals directly agglutinated the red cells from others. Only those antibodies that directly agglutinate RBCs at room temperature could be studied, until 1945 when Coombs and others, developed the anti-humanglobulin test (AHG) the non-agglutinating antibodies was able to be detected, therefore many other blood group systems (including Rh system) have been discovered, and the science of blood groups serology evolved (1).
The International Society of Blood Transfusion (ISBT) now recognizes 36 human blood group systems and over 300 different blood-group antigens have been identified (2). Whereas each blood group system composed of one or more antigens that must be encoded by a single gene locus or by a complex of two or more very closely linked homologous genes, with no recombination occurring between them.
Biochemical structure of blood group antigens differs depending on the blood group system, and they fall into two main types, either protein molecules or carbohydrate molecules on glycoproteins and glycolipids.

1.2 Historical Background of Rh Blood Group System
Rh blood group system has not discovered until 1939, when Levine and Stetson reported sever Hemolytic Transfusion Reaction (HTR) for an obstetrical patient who had transfused with an ABO compatible blood from her husband, after delivery of a stillborn child. Levine and Stetson identified an alloantibody in the mother serum, which react at room temperature and 37”C with husband RBCs and with 80% of Caucasians RBCs. They postulated that this antibody had produced against a specific factor in both fetus and father RBCs that the mother lack. Levine suggested that this antibody had developed during the mother pregnancy after her exposure to a foreign factor from her fetus (3).
A year later Landsteiner and Wiener, discovered an antibody in the rabbits and guinea pigs sera after being injected with Rhesus Macacus monkeys’ RBCs, they noticed that the antibody agglutinates with 85% of human RBCs. This antibody named Rh after Rhesus monkeys. Another study carried by Levine demonstrates that Rh antibody which was described by Landsteiner and Wiener has the same specificities of the former antibody, which was discovered in 1939, and they thought it was belong to the same blood group. Years later, many investigations have showed that it was not true, and each of the two antibodies belong to different blood group. However, anti-Rh name retained to the human-produced antibody and the other antibody renamed anti-LW in the honor of Landsteiner and Wiener (4).
After the ABO, Rhesus blood group system is the most clinically significant in the field of transfusion medicine, and the primary cause of hemolytic disease of the fetus and newborn (HDFN) (5).
By the mid-1940s, five major Rh antigens have been identified (D, C, E, c, and e). Currently the Rh blood group system contain 54 different antigenetic specificities (2), expressed by two genes in chromosome one. Making this blood group system as the most complex and the highest polymorphic antigens compared to the other blood group systems. Furthermore, their importance to the field of transfusion medicine and obstetric-neonatal medicine Rh blood group antigens are relevant in many general studies such as population genetics and forensics (6).

1.3 Inheritance and Nomenclature of the Rh System
Due to the complexity of the genetic and inheritance nature of Rh blood group system in the beginnings. Numerous theories and studies tried to describe the inheritance pattern of this system, which lead to several nomenclatures and notations have been existed to describe Rh antigens, proteins and genes.
Two of these terminologies based on genetic theories of Rh inheritance. The third terminology describes only the presence or absence of a given antigen. The last nomenclature is the result of the combined efforts of the International Society of Blood Transfusion (ISBT).

1.3.1 Fisher ‘ Race Theory
The first theory of Rh inheritance established by Fisher and Race (1940s), they proposed three pairs of closely linked genes (now known to be two genes) encode the five major Rh antigens (D, C, E, c and e). Each gene produces one antigen D/d, C/c and E/e; while ‘d’ represent an amorphous allele ‘silent allele’, (Fig. 1.1 illustrates the gene expression according to this theory). Same letter designation were given for both gene and its corresponding antigen (Capital letters and italics refers to the gene to distinguish them from their antigens). The authors postulated each person inherits a set of genes from each parent, which form Rh haplotype. This theory lists eight possibilities of Rh alleles (haplotypes) (Dce, DCe, DcE, DCE, dce, dCe, dcE, and dCE). The combination of maternal and paternal haplotypes determines one’s genotype. Fisher and his colleague believed that the order of the genes on their chromosome was DCE, thus, it has become common practice to refer to them in this order, despite that the correct order is CDE.

Figure 1.1 Fisher-Race theory of inheritance (4)

1.3.2 Wiener’s Theory
The second theory of Rh inheritance proposed by Wiener in 1943, who suggested that there is only one gene with multiple alleles (R1, R2, R0, RZ ‘..), at a single locus encodes all Rh antigens. Author proposed that Rh gene is responsible for the production of one agglutinogen (antigen), that carry at least three factors (serological determinants) (Fig.1.2), whereas each factor could be determined by an antibody. Table 1.1 lists the major agglutinogens and their respective factors, along with the shorthand term that has come to represent each agglutinogen.
TABLE 1.1 Rh-Hr Terminology by Wiener

Rh0 Rh0 Rh0 hr” hr” R0
Rh1 Rh1 Rh0 rh” hr” R1
Rh2 Rh2 Rh0 hr” rh” R2
Rhz Rhz Rh0 rh” rh” Rz
rh rh hr” hr ” r
Rh” rh” rh” hr” r”
rh” rh” hr” rh” r”
rhy rhy rh” rh” ry
Figure 1.2 Wiener’s agglutinogen theory.

1.3.3 Tippett’s Two-Locus Model
In 1986, Tippett (7), suggested a new model of Rh genes, which based on serological data, he postulated that only two Rh genes controlling the expression of the Rh antigens, one encoding D antigen, and the second encoding the other four antigens (C, c, E and e).
The modern molecular biology techniques disclosed the accuracy of Tippett’s two-locus theory, and they have confirmed that neither of Fisher nor Wiener theories was completely correct. However, Fisher’s and Wiener’s notations still commonly used interchangeably in many scientific papers and by most blood banks, due to its convenient way to explain and communicate Rh phenotypes and genotypes (8-10).

1.3.4 Rh Numeric Terminologies:
As the Rh blood group system expanded, since many Rh antigens have been discovered, it is became more difficult to give names to new antigens using the previous nomenclatures. Therefore, numeric nomenclatures have been emerged, which were not depend on the genetic basis, nor on a theory of Rh inheritance.
In the 1962, Rosenfield (11), initiates the first numeric terminology, they approach a numeric system that allocate a serial number to each Rh antigen in order of its discovery date. According to this nomenclature, the phenotype is demonstrating by the presence or absence of the antigen on the RBC. Where a minus sign precede number for the negative antigen. The five major antigens (D, C, E, c and e) are assigned as (Rh1 Rh2, Rh3 Rh4, and Rh5) respectively.
Years later, International Society of Blood Transfusion (ISBT) (2), developed numeric terminology for all red blood cells systems including the Rh system, which based on the nomenclature described by Rosenfield. ISBT numeric system gave each RBCs antigen a specific number consist of sex digital numbers. The first three numbers refer to the blood group system, whereas (004) assigned for the Rh blood group system. While the remaining three digital numbers represent the antigenic specificities. Symbols of the five major antigens in different nomenclatures are summarized in the (Table 1.2).

TABLE 1.2 Symbols of Five Major Rh Antigens in Different Nomenclatures.

Fisher & Race Weiner Rosenfield ISBT
D Ro RH-1 004001
C rh’ RH-2 004002
E rh” RH-3 004003
c hr’ RH-4 004004
e hr” RH-5 004005

1.4 Genetic Basis of Rhesus Genes
In 1990s, several molecular studies on RH genes (8-10), confirmed that there are only two genes (RHD, RHCE) encode Rh proteins. RHCE gene was discovered and cloned in 1990, two years later the RHD gene was cloned, these genes reside in close proximity on the short arm of chromosome one, and inherited together as codominant alleles. RHD gene encodes RhD protein, while RHCE gene encodes the Cc and Ee antigens in various combinations (Rhce, RhCe, RhcE, or RhCE).
An important feature to note about RHD and RHCE genes is that they are precisely the same in terms of 97% of their form; both RHD and RHCE genes have 10 exons each and they engage in the encoding of proteins that are different within the range of 32-35 of 417 amino acids (4). It is notable that this characteristic is not a feature of the majority of other blood group antigens, which are encoded by single genes with alleles that vary by just one or a small number of amino acids (5). The extensive variance among amino acids explains why exposure to RhD antigen can lead to a strong immune response in those people who do not display RhD.
Ridgewell et al. (1992) found an additional gene called the Rh-associated glycoprotein gene (RHAG), situated on chromosome six. The scholarly research revealed that the gene is crucial for the effective expression of Rh antigens (12). The product of this gene closely resembles to Rh proteins except it is glycosylated (glycoprotein), and it is found to be a corepressor for all Rh proteins that forms a complex with them within RBCs membrane. Nevertheless, this glycoprotein does not express any Rh antigens by itself, but its presence in the red cell membrane is very important for the expression of Rh antigens. Thus, when mutations in the RHAG gene occur, it is significantly affect and altered the expression of Rh proteins, which known as Rhnull phenotype that has a very rare frequency (approximately 1 in 6 x 106 individuals), where the RBCs of those people lack of the all Rh antigens. This phenotype associated with RBCs membrane abnormalities, causing a shortened in vivo survival, and mild compensated hemolytic anemia. Thus, it is also known as (Rhnull syndrome and Rhnull disease) (1).

1.5 Haplotypes, Genotypes and Phenotypes of Rh Blood Group System:-
A haplotype is a group of genes within one chromosome that inherited together from a single parent. There are eight different Rh haplotypes have been identified (listed in Table 1.3), which vary in their proportions in different populations (13).
These eight Rh haplotypes can paired to make 36 different Rh genotypes. Although, by applying anti-D, -C, -c, -E, and -e only, with individuals’ red blood cells, 18 phenotypes can be found. Only eight of these phenotypes represent a single genotype, the other 10 represent two or more possible genotypes (14).
TABLE 1.3 List of the Eight Rh Haplotypes.

Rh Haplotypes
Fisher-Race Notation Dce DCe DcE DCE dce dCe dcE dCE
Wiener’s Designation R0 R1 R2 Rz r r” r” ry
1.5.1 Determining Rh Phenotypes by Serological Reaction
American Association of Blood Banks (AABB) confirmed that it is possible to determine the Rh phenotypes (14), by determine the presence or absence of the five major Rh antigens (D, C, E, c, and e). Therefore, the assortment of antigens detected on a person’s red cells constitutes the person’s Rh phenotype, (Table 1.4).

Table 1.4 Determination of the Rh Phenotypes From the Results of Tests with the Five Principal Rh Blood Typing Reagents (5, 14).
Reagent Antigens Present Phenotype
Anti-D Anti-C Anti-E Anti-c Anti-e Fisher-Race Weiner
+ + 0 + + D, C, c, e DCe/dce R1 r
+ + 0 0 + D, C, e DCe/DCe R1 R1
+ + + + + D, C, c, E, e DCe/DcE R1 R2
+ 0 0 + + D, c, e Dce/dce R0 r
+ 0 + + + D, c, E, e DcE/dce R2 r
+ 0 + + 0 D, c, E DcE/DcE R2 R2
+ + + 0 + D, C, E, e DCe/DCE R1 Rz
+ + + + 0 D, C, c, E DcE/DCE R2 Rz
+ + + 0 0 D,C,E DCE/DCE Rz Rz
0 0 0 + + c, e dce/dce r r
0 + 0 + + C, c, e dCe/dce r’ r
0 0 + + + c, E, e dcE/dce r’ r
0 + + + + C, c, E, e dCe/dcE r’ r’
0 + – – + C, e dCe/dCe r’ r’
0 0 + + 0 c, E dcE/dcE r’ r’
0 + + 0 0 C, E dCE/dCE ry ry
0 + + 0 + C, E, e dCe/dCE r’ ry
0 + + + 0 C, c, E dcE/dCE r’ ry

(+) Positive reaction. (0) Negative reaction
1.7 Biochemistry of Rh Proteins
Rh antigens are located on two Rh proteins (RhD and RhCE) expressed on RBCs membrane, both are transmembrane, highly hydrophobic and nonglycosylated proteins. Each protein composed of 417 amino acids, that span membrane 12 times, made up 6 extracellular loops (responsible for the immune response), and 7 intracellular loops, The N-terminal and C-terminal portions are intracellular (15).
The RhD protein expresses the D antigen, while the RhCE protein carries either C or c antigens on its second extracellular loop, together with E or e antigens on the fourth extracellular loop on the same protein. The C and c antigens only differ in one amino acid at position 103; the same applies to E and e antigens that differ in the amino acid located at position 226 in the RhCE protein (16) (Fig. 1.3).
Figure 1.3 Schematic representation of RHD, and RHCE genes and their RhD, RhCE proteins. Has been generated by (5).

1.8 Variations of D Antigen Expression
Most of the all Rh (D)-positive RBCs show strong positive macroscopic agglutination after centrifugation with anti-D reagents, and readily classified as Rh (D) positive, while negative results required further investigation to confirm their D-antigen status, thus indirect antiglobulin test (IAT) performed for all the preliminary negative results. Negative results for IAT directly reported as Rh (D) negative red blood cells. While, positive IAT results for D-antigen, are classified as D-positive, but it should be described as “weak D’, since Rh (D) antigen of those individuals was altered due to a mutation in their RHD gene, and some of these individuals may produce anti-D if they exposure to positive-D red blood cells.
Based on the locus and type of mutation, altered D antigens are categorized into three main phenotypes, weakened D due to C in trans to RHD, weak D and partial D.

1.8.1 C in Trans to RHD
Position effect or gene interaction effect is the first mechanism that may lead to weakened expression of D antigen that results of a translocation of the RHD allele to the allele carrying C gene in the opposite haplotype, which causes weakened expression of D-antigen in the RBCs membrane. The D Ag is structurally complete, thus these individuals can receive D-positive RBCs with no adverse effects (4).

1.8.2 Weak D
The second mechanism results in weakened expression of the D antigen called weak-D (20), which results of a mutation in RHD gene causing change in amino acids present in the intracellular region of the RhD protein. Therefore, there is no change in the extracellular loops of the D-antigen, which appear to be complete and normal in its structure but few in numbers. As results, this mutation causes weak expression of the D-antigen in those individuals and the majority of them do not produce anti-D after espousing to D-antigen red blood cells.

1.8.3 Partial D
The third mechanism know as partial D (20), that caused weak expression of D-antigen, due to a mutation in one or more nucleotides in the RHD gene that encode part of the extracellular loop of the RhD protein, which then result in altered or missed in one or more D epitopes within the entire D protein.
Individuals with partial-D antigen have different reaction results when there red blood cells mixed with commercial anti-D, which depends on the type and site of mutation. Some individuals give weaker reaction than expected, others may not react at all when routine procedures are used with most commercial anti-D reagents.
Partial-D individuals may produce allo D-antibody against the missed epitopes when they receive RhD positive cells, thus those individuals must conceded as RhD negative when they intended to receive a blood transfusion, and as RhD positive when they are donating blood, because RhD negative patients may produce D-antibody to the present part of the partial-D antigen .

1.9 Rh Antibodies
Rh antibodies are IgG immunoglobulin, react only either at 37”C or after adding antihuman globulin (AHG) and do not fix complement. These antibodies are not naturally occurring antibodies, and they are only produced after exposure to foreign red blood cells, through either transfusion or pregnancy.
Rh antibodies may show dosage effect, wherefore react best with RBCs possessing double-dose of their correspond antigens. Moreover, all Rh antibodies are enhanced when testing with enzyme-treated RBCs.
Rh antigens are very immunogenic antigens, and the most potent antigen in the Rh immune system is the D antigen (3). Thus, exposure to (100 ”L) of D-positive RBCs can stimulate antibody production in a D-negative person.
Whereas the D antigen is the most immunogenic, it is followed by (c, E, C, and e), respectively in order of their immunogenicity and their significance in transfusion medicine and obstetrics’ fields.
All types of IgG immunoglobulins subclasses have been reported (IgG1, IgG2, IgG3, and IgG4). However, IgG1 and IgG3 are of the considerable clinical significance due to their ability to clear RBCs rapidly from the circulation (4).
Following the first exposure of Rh antigen to Rh-negative individual IgM class of Rh antibodies are formed initially, replaced by a transition to IgG, and once they produced, they remain in the circulation for years. Some of those individuals may have Low-titer of the produced antibody that can not be detected by pre-transfusion tests, therefore, they may encounter an anamnestic (secondary) antibody response if they exposed to the same sensitizing antigen. Thus, careful checking of the patient history to determine whether an Rh antibody has been identified previously is essential to avoid an anamnestic response.


Clinical significance of Rh blood group system

Chapter II
Clinical significance of Rh blood group system
2.1 Rh and Hemolytic Transfusion Reaction
Hemolytic transfusion reaction (HTR) is the destruction of donors’ RBCs by the recipient immune system after incompatible transfusion. There are two types of HTR, acute (HTR) which occur immediately within the transfusion process characterized by sever intravascular hemolysis (IVH) this type of reaction usually accompanied with ABO incompatibility and very rare with other blood groups. The second type is delayed hemolytic transfusion reactions (DHTR) occurs after seven days of transfusion and results in extravascular hemolysis (EVH) (21).
Rh-mediated hemolytic transfusion reaction usually occurs as DHTR. The recipient may have an unexplained fever, mild bilirubin elevation, and decrease in hemoglobin and haptoglobin. The direct antiglobulin test (DAT) is usually positive due to RBCs sensitization, and the antibody screen may or may not demonstrate circulating antibody. When the DAT indicates the recipient’s RBCs are coated with IgG, elution studies may be helpful in defining the offending antibody specificity. If antibody is detected in either the serum or eluate, the only way to prevent the occurrence of DHTR is to select blood lacking the relevant antigens to the involved antibody for each subsequent transfusions process (22).
2.2 Rh and Hemolytic Disease of the Newborn
Hemolytic Disease of Fetal and Newborn (HDFN) is the destruction of fetal and newborn RBCs by the maternal IgG antibodies, which produced by previously alloimmunized mother, these antibodies cross the placenta to the fetal circulation, lead to anemia and frequently caused fetal brain damage due to increased levels of bilirubin (kernicterus) and even death (erythroblastosis fetalis) (21).
Published studies (23,24) show that Rh antigens represent the major cause of maternal alloimmunization cases. Therefore, feto-maternal Rh incompatibility consider as the primary cause of HDN, and the most clinical significant blood group system in this field.
Prophylactic Rh-D immunoglobulin is used to reduce the incidence of maternal alloimmunization of D antigen. However, a signi’cant number of women still become alloimmunized during pregnancy for different reasons, including non-administration of Rh immunoglobulin, unrecognized miscarriage, and leakage of fetal RBCs into the maternal circulation late in pregnancy (25).
Depend on the severity of RBCs destruction, variety of treatments are used to treat HDN, include ultraviolet phototherapy, exchange transfusion or even intrauterine transfusion may be required.

2.3 Incidence and Prevalence of Rh Alloimmunization among Transfusion-Dependent Patients.
Rh alloantibodies were found to be the most common alloantibodies overall alloimmunized individuals either by transfusion or pregnancy, and the highest frequent alloantibody among transfusion-dependent patients. In recent years, increase amount of literatures (26-29), reveal a significant association between Rh alloimmunization and transfusion-dependent patients.
Woldie et al. 2015 (26), conduct a study on sickle cell patients, to estimate the risk of RBCs alloimmunization, due to chronic transfusion therapy. They applied retrospective analysis of 121 patients at Detroit Medical Center, California. To determine the presence of alloantibody and to identify its specificity. The results showed (56.2%) of the total samples were alloimmunized. Overall, Rh system antibodies were the most common (39%) of the identified antibodies.
Another study performed by Chou ST, et al (27). Reported (45%) among patients receiving chronic transfusion therapy were Rh immunized and, one-third of them developed delayed hemolytic transfusion reactions (DHTR).
The above finding is consistent with study done by Hassab AH et al (28), they detected high incidence of anti-E in 23.8% in alloimmunized patients. Anti-E was the highest frequency in cases who developed clinically significant antibodies, followed by anti-D and anti-e respectively.
In addition, according to other study conducted in 2007 by Bashrawi LA, in Saudi Arabia at King Faisal University Hospital (29), the results also showed anti-E (18.8%) as the most common allo-antibodies detected among sickle cell patients.
The findings of the previous studies (26-29) provide evidence of the significance of the Rh antigens and the importance of determining the prevalence of Rh phenotypes and the distribution of the major Rh antigens (D, C, c, E and e) among Saudi population.

2.4 Aim of the Work
The present research aims to determine the frequencies of Rh phenotypes and the distribution of the major Rhesus antigens (D, C, E, c, e) among Saudi blood donors in Aseer region, by using a fully automated blood bank analyzer system, TANGO’ optimo, which approved by FDA for automating all routine immunohematology procedures.
The findings of this research will help blood banks specifically in Aseer region and generally in all over the kingdom as a reference to the frequencies of Rh antigens and Rh phenotypes in Saudi blood donors. Which help to avoid Rh alloimmunization. In addition, the findings of this research will going to assist in finding compatible units for Rh alloimmunized patients therefore, minimize waiting time for the patients and to reduce workload.


Chapter III
Materials and Methods
3.1 Study Area
This research was conducted at Aseer Central Hospital (ACH), Abha. (South-west of Saudi Arabia).

3.2 Study Duration and Study Sample
Seven hundred thirty four (734) healthy Saudi male blood donors were involved in this research after obtained their written consent. This research continues for six months from August 1st 2015 (16/10/1436 H) to January 31st 2016 (20/4/1347 H). Samples were collected from donors reporting to the Blood Bank, ACH. All donors have met the ACH standard operating procedures (SOPs) (30) of donors’ selection criteria, according to (age, weight, height, Hemoglobin level’).

3.3 Sample Collection
3 ml of whole blood from each blood donor was collected into EDTA anticoagulated tube, following general blood sampling guidelines (30). The specimens were stored at (2”C to 8”C), and tested within 7 days of collection, according to the manufacturer’s instructions (31).

3.4 Sample Processing
Solid phase Hemagglutination technique was performed by using TANGO’ optimo system (described below), to determine the ABO blood group, as well as Rh (D, C, c, E and e) phenotyping and weak-D testing.

3.4.1 TANGO’ optimo
In this research, all techniques have been performed by using TANGO’ optimo system (Bio-Rad, USA) (Fig. 3.1). Which is a fully automated blood bank analyzer system that has been approved by FDA (4), for automating all routine immunohematology-testing procedures including ABO blood grouping, Rh phenotyping, weak-D testing, antibody screening and cross matching with very little operator support, thus decreasing the opportunities for human errors. Besides, it provides the level of quality assurance required by AABB (14). Bar-coding reduces identification errors by providing accurate samples and reagent identification and standardized techniques that reduce testing errors to provide accurate, precise and reproducible results (32).
Figure 3.1 TANGO’ optimo (Bio-Rad, USA), automated blood bank analyzer.

3.4.2 Quality Control
Quality control (QC) tests have been performed for each run, to ensure the validity and reliability of the reagents, plates and analyzer. Positive and negative QC samples for each reagent was provided by the manufacturer (Bio-Rad, USA).
Positive control samples contain heterozygous weakened antigen and negative control samples contain antigen-negative RBCs, to verify positive and negative reactions. Each reagent is satisfactory for use if it reacts only with antigen-positive RBCs (31).
In addition, random samples have been run by the manual tube technique to compare it with the results extracted from the automated machine, as internal control.

3.4.3 Solid Phase (Erytype” s) for Rh Phenotyping Principle
A hemagglutination technique for ABO and Rh (D, C, E, c and e) phenotyping, has been applied by using erytype” plates. The erytype” plates are composed of 12 test strips (8 wells each) that contain dried IgM monoclonal antisera for antigen determination. The antigens on the red cells will form agglutination in the presence of the corresponding antibody. To achieve the optimum reaction, bromelin enzyme has been used to split off polypeptide chains from the surface of the red cells membrane. Therefore, the removal of the polypeptide chains decreases the negative charge between the red cells. Which leads to a stronger attraction of the red cells in the presence of the corresponding antibody. Method
1% of RBCs suspension was prepared in bromelin solution, then 50 ”l of the cell suspension were pipetted into the microplate’s wells containing the dried antisera. The microplate was moved to the linear shaker, which shake the strips to dissolve the dried antisera and mix reagents. Then the strips were incubated at room temperature for approximately 10 minutes. After incubation, the strips were centrifuged for one minute and resuspension of the cells which done by moving the strips to the orbital shaker for approximately three minutes. Finally, the strips were moved to the measurement chamber where the reaction is evaluated as positive reaction indicated by RBCs agglutination and no agglutination for negative reaction.

3.4.4 Weak’D Testing by Solidscreen II Technology
The Solidscreen II assay, also known as (Solid-Phase Protein A Technology) is a new methodology that has been approved by FDA (4). Used to detect IgG antibodies, by using microplate wells, which are coated with (protein A). Protein A is a component of the cell wall of Staphylococcus aureus that has a very high affinity for the (FC) portion of most immunoglobulin classes.
All samples that have shown negative D-antigen results by conducting the hemagglutination technique with IgM anti-D reagents were subjected to determine the weak-D status (formerly known as Du) by using Solidscreen II Technology. Method
50 ”L of Anti-D reagent (blend of two IgG monoclonal Abs) and 50 ”L of the donors’ RBCs suspension of are added to the Protein-A coated microwell. Sensitization of the RBC occurs if weak D-antigen is present on the RBC. Following incubation for 20 minutes at 37”C, and two wash processes to remove unbound protein, 100 ”L of Anti-Human Globulin (AHG) was added to the well, which acts as a link between the antibodies coating of neighboring RBCs, and induces solid phase. Uncoated RBCs will form a red blood cell button. Following centrifugation for one minute, the well is evaluated, a smooth monolayer of RBCs is indicative of a positive reaction while a compact button of cells in the middle of the well is indicative of a negative reaction (Fig. 1.4)
Figure 3.2 Interpretation the results of Solidscreen II assay.

3.5 Statistical Analysis
Statistical analysis of data was performed using SPSS software.



Chapter IV
Seven hundred and thirty-four Saudi male blood donors’ samples were collected in Aseer Central Hospital (ACH); the mean age was 32.6 ” 9.5 years, between 18 and 57 years old. Each sample was examined for ABO grouping and Rh phenotyping for the major Rh antigens (D, C, c, E, and e).

4.1 Frequencies of ABO’Rh (D) Antigens
For ABO grouping, the present research demonstrated that the blood group with the highest prevalence was O (49.7%), followed by A (37.7%), B (11.5%) and AB (1.3%). While the results for the prevalence of ABO in respect with Rh (D) antigen, revealed ‘O Positive’ group as the highest blood group (44.8%). The second group was ‘A Positive’ (35.7%), followed by ‘B-Positive’ (10.1%) then O negative (4.9%). However, the least prevalent ABO-Rh blood group was AB positive and AB-Negative’ (1% and 0.27%) respectively. The following Table 4.1 demonstrates the distribution of the ABO-Rh (D) blood groups across the current research.
TABLE 4.1: Frequencies and Percentages of ABO-Rh (D) Blood Groups.

ABO ‘Rh Frequency
(n) Percentage
( % )
O positive 329 44.8
O negative 36 4.9
A positive 262 35.7
A negative 13 1.8
B positive 75 10.1
B negative 10 1.4
AB positive 7 1
AB negative 2 0.3
Total 734 100

4.2 Distribution of the Major Rh Antigens (D, C, E, c and e)
Rh (D) typing, in combination with other major Rh antigens (C, E. c and e), was carried out for each single sample. The results demonstrated that, of the initial 734 participants, 91.7% (673) were Rhesus (D) positive while 8.3% (61) were Rh (D) negative (Fig 4.1), and 5 samples were reported as weak-D variant, these samples were included in Rh (D) positive group.
Figure 4.1 Percentage of Rh-D Positive donors.
The results of other Rh antigens included (C, E. c and e), illustrates that the most frequent Rh antigen in the context of the sample group was e (97.3%), followed by the C and c antigens, (74.4%, and 74.1%) respectively. However, the E Ag was recorded as the antigen with the lowest frequency, (22.7%). Table 4.2 shows the distribution of the major Rh antigens (D, C, E, c and e) in the present research in comparison with other studies.

TABLE 4.2 Comparison of the Frequencies and Percentages of the Major Rh Antigens (D, C, E, c and e) between the present research and other studies
Author Distribution of Major Rh antigens (%)
Rh (D)positive Rh (D) Negative C E c e
Present research 91.7 8.3 74.1 22.8 74.4 97.3
Junainah E. et al (33) 91 9 ND* ND* ND* ND*
Sarhan MA. et al (34) 92.8 7.2 ND* ND* ND* ND*
Elsayid M. et al (35) 96 4 ND* ND* ND* ND*
Hassan FM. et al (36) 93.1 6.9 62.3 42.7 41.7 91
Bahrain (34) 94.5 4.5 ND* ND* ND* ND*
Yemen (34) 92.9 7.1 ND* ND* ND* ND*
Indians (37) 92 8 87 26 51 98
Caucasians (38) 85 15 68 29 80 98
African Blacks (38) 92 8 27 22 96 98
Asians (38) 99 1 93 39 47 96

*ND = Not Determined
4.3 The Prevalence of Rh Phenotypes
The interpretation of Rh phenotypes was determined by referring to (Table 1.4, p10). Eight Rh phenotypes were observed in the present research, the highest frequency was DCe/dce (R1r) 35.3%, and the second most common was DCe/DCe (R1R1) 25.9%, followed by DCe/Dce (R1R2) 12.6%. However, the lowest frequency Rh phenotype was dCe/dce (r”r) 0.5%. Table 4.3 shows the results of the Rh phenotypes in the present research along with other ethnic groups.

TABLE 4.3 Comparison of the Frequencies and Percentages of the Rh Phenotypes in the present research and other ethnic groups.

Phenotype Percentage (%)
Antigens present Weiner’s notation Fisher-Race’s
notation Present Research Caucasians
(5) African Blacks (5)
DCcee R1 r DCe / dce 35.3 31.1 8.8
DCCee R1 R1 DCe / DCe 25.9 17.6 2.9
DCcEe R1 R2 DCe / DcE 12.6 11.8 3.7
ccee r r dce / dce 7.8 15 7
Dccee R0 r Dce / dce 7.8 3 22.9
DccEe R2 r DcE / dce 7.5 10.4 5.7
DccEE R2 R2 DcE / DcE 2.6 2 1.3
Ccee r” r dCe / dce 0.5 1 1


Chapter V
This research is the first of its kind that produce data describe the prevalence of Rh phenotypes and the distribution of the major Rh Ags (D, C, E, c, and e) among Saudi blood donors in Asser region, Saudi Arabia.
Rh (D) antigen is the most clinically significant antigen after ABO antigens, in the field of transfusion Medicine and the primary causes of HDN (4). The results of this research showed (91.7%) of the samples were Rh (D) positive and the remaining (8.3%) were Rh (D) negative. This is in agreement with previous results (Junainah et al. 2016) (33), who conducted a study on 800 Saudi donors, which aimed to determine the distribution of ABO-D antigens by performing a conventional tube method. Authors have reported (91%) of their sample as Rh (D) positive. Similar results has been seen in the study performed by (Sarhan et al. 2009) (34), who performed a study to identify the prevalence of ABO-D in the Medical students, at King Khalid University in Abha. Authors applied slide method technique for 900 Saudi students and they concluded that (92.8%) where Rh (D) positive.
However a retrospective study performed by (Elsayid M. et al. 2015) (35), showed wide variation of the prevalence of Rh (D) antigen (96%) versus (91.7%) in the present research. This difference may be attributed to geographical variation or to the nature of the study’s subjects that they selected, since they only analyzed patients data who received blood or blood products, verses healthy blood donors in this research.
Although it is important to note, that the previous studies (33-35) focused only on the prevalence of ABO blood group along with the D antigen rather the other major Rh antigens (C, E, c and e). Moreover, most of all studies have been done in Saudi Arabia used manual techniques such as traditional slide and tube methods. For the slide technique performed by (Sarhan et al. 2009), they collected whole blood drops samples by finger-prick and slid technique method, which is an old technique that lack quality controls and not recommended for routine blood grouping, due to it is low sensitivity and accuracy and not reliable for weakly reactive antigens (14). Besides its inability to do reverse blood grouping or weak-D testing. While for the traditional tube method used by (Junainah et al. 2016), which based on manual labeling and visual inspections for RBCs agglutination that may lead to clerical and human errors (14), moreover authors did not perform weak-D testing for Rh-negative samples. In the other hand, the fully automated solid phase technique that has been conducted in the present research provides a high level of quality assurance and standardized methods, which insures high accuracy, precise results with very little operator support that decreases the opportunities for human errors (4) (see chapter 3).
When considering the results of Rh (D) in this research (91.7%), in relation to those reported from different countries and ethnic groups (37,38). It was found to be comparable with those of African blacks (92%) and Indian population (92%). Although, the present results was disagree with its prevalence in Caucasians (85%). Moreover, the prevalence of Rh (D) antigen across various samples groups from different ethnic populations can range from (60% to 99%), where the highest frequency is reported in Asians (99%), while the lowest prevalence is reported in Southern France and Northern Spain (37).
For the frequencies of the other Rh antigens (C, E, c, and e) they were found to be distinguish in their percentages among the present results in relation to other ethnic groups. Published data (37, 38), showed that C antigen frequency varied between (27%) in African blacks and (93%) in Asians, versus (74.4%) in the present research. While the c antigen found to be (96%) of African blacks and the lowest frequency of c antigen found in Indian and Asian populations (51% and 47%) respectively, verses (74.1%) in this research. E antigen found to be the lowest frequent Rh antigen in most ethnic groups, but in different percentages, with the highest frequency recorded in Asians (39%) followed by (29%) in Caucasians, and the lowest frequency was found in African blacks (22%) which agreed with the present results. On the other hand, the e antigen frequency in this research found to be comparable with its prevalence in Caucasian (98%), Black (98%) and Indian (98%), populations. Table 4.2 illustrates the variance of the prevalence of the Rh antigens among different populations and ethnic groups.
For the comparison of the present results of the major Rh antigens distribution with local studies in different regions of Saudi Arabia, Hassan et al. 2013 (36), conducted a study in Sakaka city to determine the frequency of Rhesus alleles, haplotypes and genotypes in Aljouf population. Authors applied manual ID-card technique on 412 subjects selected from Primary Health Centers (PHC). Their results revealed the distribution of major Rh antigens in descending order as follows: D (93.1%), e (91.0%), C (62.3%), c (41.7%) and E (42.7%). Which totally disagree with this research’s results where is the most prevalent Rh antigen was e (97.3%) followed by D (91.7%), C (74.4%), c (74.1%) and E (22.8%). Authors also have reported Rhnull phenotype in (5.2%) of their samples, which is in contrast of the prevalence of this extremely rare phenotype with a reported frequency of approximately 1 in 6 million individuals (1). Moreover, authors reported more than 30% of their results as one or more absence of antithetical antigens (C and c) or (E and e) which indicate for Rh genes deletion. This totally disagree with the present results where is no Rhnull phenotype was observed nor absence of antithetical antigens. These differences might be due to geographical variances between the two studies, or to different methodologies have been used since they used manual technique that may leads to technical and interpretation errors (14). It is worth mentioning that the research topic of their study pointed to determine the Rhesus alleles, haplotypes and genotypes, which are unable to be determined by serological tests as they used, Instead of that, they reported all of the probable genotypes which can not explain their population’s haplotypes, alleles or the actual genotypes.
The prevalence of the Rh Phenotypes in the present research have shown eight different Rh phenotypes. The phenotype with the highest prevalence was reported as R1r (35.29%) which comparable with its prevalence in the Caucasian population (31.1%), but in contrary with its lower percentage (8.8%) in African blacks (5). The second highest frequency phenotype in Saudi donors at Aseer region was R1R1 phenotype (25.9%) which has been observed as (17.6%) of Caucasians and (2.9%) of the African black populations. While the R0r phenotype frequency in the present research’s population was (7.3%), which disagree with its prevalence in Caucasians (3%), as well as African blacks (22.9%). Table 4.3 shows the variance of the prevalence of the Rh phenotypes among the present results and other ethnic groups.
The previous comparisons of the present findings of Rh antigens frequencies and the prevalence of Rh phenotypes with other groups locally and worldwide, showed a wide variation and significant differences among each population. Thus, the determination of Rh phenotypes and Rh antigens is clinical significance in any specific population. As with these results, it is possible to avoid Rh-mediated (DHTR). In addition, these findings will going to assist in finding compatible units for Rh alloimmunized patients, which reduce workload and minimize waiting time for the patients (4).
Therefore, the findings of this research will help blood banks specifically in Aseer and generally all over the kingdom as a reference to the frequencies of Rh antigens and Rh phenotypes in Saudi blood donors, instead of relying on international published ratios for other ethnic groups as the case now.

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