Identification of Blood Group in Terms of ABO and Rh - Essay Example

ABO and Rh Typing A blood group is usually an inherited character of the erythrocyte (red blood cell). Researchers and scientists have identified 29 blood group systems with a total of 262 antigens in humans. Antigens may be defined as molecules that are found on the red cell surface and are capable of producing an antibody. The red cell antigens can be of various forms; they can be glycolipids, carbohydrates, proteins and glycoproteins. Thus, its presence on the membrane of the red cell ensures that the cell integrity is maintained and improves on the functions of the membrane. The first blood group system that was discovered by Karl Landsteiner in 1901 was the ABO system and this blood group system play a major role in transfusion medicine (Daniels, 2005). The ABO system can further be grouped into four major groups: A, B, AB, and O. The antigens that are found in the ABO system are usually made of type II oligosaccharides that H transferase adds a fucose that results in the formation of the H substance. Thus, the H substance contributes in the formulation of antigens A and B. Under the condition of the inherited gene, the transferase of A and/or B will then include a terminal sugar to the H substance. Hence, the A transferase will add a D-N-acetyle-galactosamine (Gal-NAc) while the B transferase will add the D-galactose (Gal). Not only can the ABH antigens can be found on the blood cell but can be found in many cells in the body and especially the body fluids. Even though the ABH antigens are found in body fluids and various cell membranes, the normal physiologic function of the glycoprotein is not clear. For example, those individuals who do not have any ABH antigens commonly referred to as Bombay phenotype usually have normal functions and survival of the red cell. The antigen(s) that are present on the membrane of the red blood cells (phenotype) determines the name of the blood group as illustrated in Table 1. The benefit of the ABO system in transfusion medicine lies in the fact that the antibodies against the ABH antigens are usually present in the plasma or serum of a health person and thus does not require and form of red cell stimulation; it is usually produced because of environmental stimulation. Moreover, the individuals who lack A or B antigen in their plasma, these individuals will have IgM antibody in their plasma. In most instances, these antibodies through activation of the classic complement pathway can cause intravascular haemolysis, which may result in fatal transfusion reactions due to incompatible blood transfusion or may result to hyperejection in case of transplantation of body organ. Thus, this illustrates the importance of ABO cross matching before any transfusion can take place. Determination of the presence or absence of the antigens and antibodies can be achieved by laboratory techniques such as serological blood grouping. The test for antigens (testing red cells) is forward test while for the antibodies (testing antiserum) is reverse test, which in most cases is a confirmatory test. Another important aspect in the blood group after the ABO is the Rh blood group system. Rh blood group system is the most polymorphic blood group system. This blood group system has five major antigens D, C, c, E and e that are encoded by two genes RHCE and RHD that are found in chromosome 1. The RhD is a crucial antigen since its absence will lead into the formation of the antibodies that will be against the D antigen. It is capable of inducing immune responses in 80% of D-negative persons who are transfused with 200 ml of D-positive blood (Learoyd, 2005). Thus, it is important for RhD typing to be done before any transfusion can take place; for example, D-negative patients receive only D-negative blood products. The production of the Rh antibodies may be attributed to immune response that is attributed to pregnancy state. RH antibodies can cross the placenta since it is usually IgG and may contribute in causing haemolytic disease of the foetus; hence, it is paramount to determine the Rh phenotype of D-negative mothers’ foetus. Aim The goal of this practical was to determine the Rh blood groups and ABO of members of a family utilizing four different assays. The methods that were used were then compared in terms of simplicity, time required and sensitivity. These methods that were used are based on the principle of hemagglutination, which includes: U Well Microplate Method 6 whole blood sample (which were separated in red cells and plasma) of a family of six members was provided to each student. The antibodies were removed from the samples by washing the samples 3 times with PBSS. It was followed by preparation of 3-5% red blood cell suspension in PBSS for each of the six samples to provide an antigen that was optimum; optimum antigen is the ration of the antibody for the agglutination reaction. On the Microplate, the antisera were added followed by the 3-5% patient red cells in PBSS. The antisera contained monoclonal antibodies, which enables the detection of specific blood antigen. Patient serum was added to the last columns for the reverse method followed by A1 and B cells respectively. The A1 group were used because they do not have antibodies for A2 antigens rather than A2 group; A2 group possess antibodies to A1 antigens. The microplate was incubated for 1hour at room temperature and then it was agitated for 1min and then the results were read. The positive result was indicated by a clot that was at the bottom of a well while a homogenous sample in the well showed the negative test (Fig 1). * A2 groups have antibodies to A1 antigens. Hence, the sample 2 may belong to an A1 group (individual) ** Usually, babies have a low antibody subgroup. Thus, it is likely that Sample 6 belongs to a baby. Column Agglutination (Gel card by Diamed) For column agglutination technique, the Gel card by Diamed was used. The structure of the gel card can easily support this experiment because it has 6 microcolumns, in which the lower part is narrow and there is a matrix of sephadex gel micro-beds. The matrix of each microcolumn differed e.g. the first microcolumn contains anti-A, the second contains anti-B, the third contains anti-D while the fourth contain AB serum. 25 ul of packed red cells of a patient was added to a tube that contains 0.5 ml of cell stabilizer. The contents were then mixed and incubated at room temperature. With the help of the Gilson pipette, 25 µL of patient cell suspension was then pipette into the first 4 wells of the Gel card. For microcolumns 5 and 6, 50 ul sample plasma was added followed by the addition of 25 µL of A1 group was added to cell 5 and in cell 6, B 25 µL of B cells were added. The card was centrifuged with the help of the S24 Diamed centrifuge and then the data was read (see Fig 2 below). For a positive result (graded as 4), is observed when the red cells agglutinate and forms a clot on the surface of the gel while a negative result (graded as 0) is illustrated by the unagglutinated cells reaching the bottom of the column. The data that was obtained is shown in table 5. Tube Technique The group reagents were put into appropriate glassed tubes and were followed by patient plasma. Consequently, red cell reagents and 3% in PSB cell suspension were added to the appropriate glass test tubes to allow for back grouping. This method has various weaknesses, which include time consuming, and weak agglutination can be missed, when the reagents are mixed very hard. Moreover, the glass tubes are usually hazardous material when used in a clinical laboratory setting. The results were then recorded from the glass tubes. The picture below illustrates the positive and negative reaction. The use of the tube technique was successful since the results that were observed correlated with the results that were obtained from the Rh groups and ABO when the microplate technique and Gel card was utilised. Hence, this illustrates the efficiency of the test. Card Tile Technique The nature of the card tiles is smooth and has pre-printed cards, which allows for the incubation of antibodies and red cells to occur on the surface of the card tiles. Two drops of corresponding antisera was added to each and appropriate square of the card layout. It was followed by the addition of 3-5% cell suspension, mixed and incubated for 1 min at room temperature and then the results were recorded. According to the strength of the agglutination, the results were graded from 0 to 5. Even though this method is subjective, the technique is cheap and simple. The results that were obtained are illustrated in figure 4. Discussion: By combination of the results that were obtained from the tests that were carried out, the family tree of the ABO and Rh phenotype and genotype can be completed. The blood group of the mother is B and can only possess BO genotype since the antigen B she inherited from the grandmother while the O gene was inherited from the grandfather. On the other hand, the blood group of the father is A and cannot be homozygous for A since there is no child that belongs to AB blood group. This is because the blood group O of child A implies that each parent contributed O gene. The Rh genotype of the grandfather is dce/dce (rr). Moreover, the grandmother, mother and child B have Rh antigens D, C, c, e with 3 genotypes: DCe/dce, DCe/Dce and Dce/dCe. On the other hand, the father has 2 possible genotypes: Dce/Dce and Dce/dCe. Thus, the genotype of child A is dCe/dce, and this means that the child inherited dCe from the father and dce from the mother. From this perspective, the possible genotypes of the father and the mother are reduced to Dce/dCe for the father and Dce/dce for the mother. Conclusion The experiments that were carried out with the help of different techniques enabled the identification of blood group in terms of ABO and Rh of six family members. The use of tube technique on ABO and Rh typing is simple and easy to perform but the technique is prone to laboratories hazards e.g. breakage. The use of microplate technique provides accurate results since it is sensitive and produces efficient results within a short time. Moreover, the results can be analysed automatically reducing the chances of errors associated with manual handling. Additionally, this method provides additional information on Rh antigens: C, E, c and e. Nevertheless, the method is expensive and consumes a lot of time. The use of gel card is efficient, simple and stable. Moreover, the results can easily be read and graded. The weakness of this method is that it is very expensive and thus many laboratories cannot use this method in routine tests. The card tile technique was the most convenient way to carry out the experiment. However, the results that are obtained through this technique are subjective and are prone to errors; the errors may originate from wrong reading and grading. Moreover, the method is subjective to cross contamination. Thus, the experiment that was carried out with the help of various techniques illustrates the advantages and disadvantages of the different techniques that were used. Hence, the choice of the technique to be used depends on availability of the resources and preferences that are in place. Read More