Two methods of determining ABO and Rh groups - Essay Example

?Introduction Human red blood cells have many surface markers, which are important for the way in which they interact with other molecules. Two particularly important markers are the Rh and ABO. These two markers are used to define blood groups, and become especially relevant when the blood of two people mix, such as through blood transfusions. Because of this, it is important to be able to chemically identify the different blood types. In the ABO blood group system there are two types of marker that are present on the blood cells. These are type A and type B. If an individual has neither of these markers then they are considered to be type O. Individuals can be of blood type A, B, O or AB, as they inherit one type of marker from each parent. The human body does not produce antibodies for the markers that it contains, but does so for the ones that are not present. This is because the immune system sees the foreign marker as an invader and consequently defends itself against it . Thus, a person with type AB blood does not have antibodies against either A or B markers, and can consequently receive blood from any blood type. However, they also cannot give to any other blood type. In contrast, someone with type O blood can donate blood to any blood type as no antibodies will be raised, and but can receive blood only from other type O donors. Another factor that is present in the blood of humans is known as the Rh or rhesus system. This was first discovered through immunization of rabbits with blood that had been obtained from rhesus monkeys. It was found that the antibodies in the rabbit caused the blood to cogulate. Although the Rh system contains around 50 different antigens, five of which are considered to be the most important (D, C, c, E and e), and of these the D antigen is the most relevant. It is often thought to be the most polymorphic blood group system in humans . The term Rhesus factor is used to describe the Rh state of an individual, either they are Rh negative (does not have the D antigen) or Rh positive (the D antigen is present). The aim of this study was to elucidate the blood group of four unknown samples both in terms of the Rh (D) and the ABO group. This was undertaken using grouping reagents that work to show the whether antigens for ABO or Rh (D) are present. Secondly, the study used antibody screening on two plasma samples to determine the presence of antibodies. Materials and Methods Tube Grouping: Rh (D) and ABO antigens in unknown samples Four agglutinin reagents were to used in this experiment, Anti-A, Anti-B, Anti-A,B and Anti-D Alpha. These reagents react directly with the antigens present in red blood by making the cells clump together. Thus, they could be used to determine the blood type of each of the four patients. Sixteen clean test tubes were taken and labelled with patient name (Patient 1, 2, 3 or 4) and one of the four reagents so that for each patient there was a total of four tubes, each labelled with the name of a different reagent. Two drops of the labelled reagent were added to each tube. For each of the four patients, the cell sample was inverted several times to ensure the cells were thoroughly mixed, and then one drop of cells was placed in each of four test tubes for that patient. The cells were incubated at room temperature for 15 minutes and then examined for agglutination. Ortho ABD and Reverse Cassettes: Rh (D) and ABO antigens in unknown samples For this section of the experiment, the same four patient samples were used. The samples were inverted to mix them and then they were loaded into the cassettes. One cassette was used for each patient and these were labelled. Each cassette had four marked wells, A, B, D and control. In each well 10 µl of the respective patient sample was placed. The cassettes were then placed in the Ortho Centrifuge and spun for five minutes, and then the results read. Antibody Screening An Ortho Poly AHG cassette was provided. This had six wells that contained Poly Specific Anti – Human Globulin. Three antibody-screening cells were also used, labelled S1, S2 and S3. The cassette was visibly divided in half by drawing a line between the third and fourth well with a marker. On each side the wells were labelled as S1, S2 and S3, with the sections being labelled as Plasma 1 and Plasma 2 respectively (Figure 1). To each of the wells labelled as S1 50 µl of the S1 screening cell was added, S2 to the S2 wells and S3 to the S3 wells. Following this, 40 µl of test plasma 1 was added to the wells labelled as Plasma 1, and 40 µl of test Plasma 2 was added to those marked as Plasma 2. The card was then incubated for 15 minutes in the Ortho Incubator and then centrifuged for five minutes using the Ortho centrifuge. Figure 1: Schematic diagram of the cassette used for antibody screening Results Evidence of agglutinin was taken to be a positive result, and scored as a plus in the table below. Free red blood cells mixed with reagent were taken as a negative result and marked as a negative in the table. Patient one showed clumping in the test-tubes which contained Anti-A, Anti-A-B and Anti-D Alpha. Thus, the patient had the antigen for type A but not type B, indicating the patient’s blood type was type A. The patient showed a clumping response for the Anti-D Alpha antigen, so he was Rh positive. Patient two exhibited clumping responses for all reagents except Anti-A, indicating that his blood type was B and he was Rh positive. As patient three showed no agglutination responses for any of the A or B reagents, thus he was of blood type O. However, he was Rh positive due to a clumping result from the Anti-D Alpha reagent. Finally, patient four showed agglutinin for all reagents. Consequently, he was blood type AB+ (Table 1). Table 1: Agglutinin results for the tube grouping experimental For the second part of the experimental, the same criteria was used to differentiate positive and negative results. For all patients, the first two wells of this part of the experimental used the same reagents and produced the same results as the first part of the experiment. Likewise, the Anti-D well in the second experimental has the same outcome as the first experimental. Consequently, this confirmed the conclusions of the first part of the experiment, that the patient’s blood groups were A+, B+, O+ and AB+ for patients one to four respectively (Table 2). The presence of the control well indicated that there was no contamination. If there had been, then some agglutinin would have been observed. Table 2: Agglutinin results for the Ortho ABD and reverse cassettes experimental Reagent Patient 1 Patient 2 Patient 3 Patient 4 Anti-A + - - + Anti-B - + - + Anti-D + + + + Control Well - - - - Blood Group A+ B+ O+ AB+ Table 3 details the reactions results that were observed as a result of the antibody screening. A ten-cell panel sheet was used to identify the two plasma samples. For Plasma 1, there were antibodies detected for Rh-hr CW and V, KELL Kpa and Jsa+ amd LUTHERAN Lua. Within Plasma 2 antibodies were detected for Rh-hr D, Duffy Fyb, KIDD Jka and Sex linked Xga. Table 3: Reaction results for Plasma 1 and Plasma 2 under each of the three serum screening cells Reagent Plasma 1 Plasma 2 S1 - + S2 - + S3 - - Discussion Within this experiment two methods of testing blood grouping were used. The first made use of grouping reagents within test tubes, while the second used reverse cassettes. The first technique was simpler and easier to carry out. Four reagents were used to determine blood group, although the blood group could have been determined without the use of the Anti-A,B reagent, as the Anti-A, and Anti-B provided the same information. However, this provided confirmation that the results were correct. While the cassette method was more complicated, the use of a control meant that it was possible to determine whether there was contamination. Pretransfusion testing is used to determine whether enough of the red blood cells that are being transferred will survive. For the most part, pretransfusion testing focuses on the ABO blood groups and Rh types, however these are not the only relevant factors. The tests that were used in the first part of this experimental represent some of the means, which can be used to determine the Rh and ABO types. The second part of this experimental was the screening of serum for red cell antibodies. This is a technique that is commonly used to ensure that there are no negative reactions when the blood transfusion takes place. A common-place technique in modern times is to screen all donor blood and determine not only the Rh and ABO types but also the antibodies. This information is then stored for future reference. This allows for fast matching of donor and recipient, which can be crucial on occasions where time is lacking. It has been found that antibodies can disappear following a transfusion, only to reappear at a later point and cause complications. . Because of this factor, it is important to keep records of the antibodies that are present in donor blood. Transfusions are not the only occasions where antibodies can cause severe negative effects. Antenatal care is the standard care that is given to a pregnant woman. If the woman’s blood and serum are screened at this point, it can be possible to prevent any severe complications from occurring. Hemolytic disease is a severe illness that occurs in the foetus and newborn as a consequence of an immune reaction. Here, antibody molecules produced by the mother are passed through the placenta, and some of these cells begin to attack the red blood cells of the foetus. In severe cases this can cause death of the foetus from heart failure. One way in which haemolytic disease can occur is through the differences between the Rh factors in the blood of the mother and the child. If the foetus has Rh positive blood while the mother has Rh negative, and the two mix, the mother can produce antibodies against her own child, and as a consequence her immune system will begin to attack the blood cells of the foetus. A direct antibody test examines the potential problems that could occur. A negative result is the normal value and it indicates that there are no antibodies attached to the red blood cells. Determining the antigens within the mothers blood make care and prevention effective, and because of this, screening methods such as the serum screening that was used in this experimental can be crucial. Conclusion Understanding the antibodies that are present in blood is an important factor when considering blood transfusion or antenatal care. This study examined two methods of determining ABO and Rh groups as well as a general method of screening for antibodies. Because of the numerous complications that can occur, these types of test are crucial prior to giving birth and prior to any blood transfusion. These many variations reveal that human blood types are not as simple as they first appear, and that substantial consideration needs to be given to them to avoid complications. References Mozzarelli, A. & Bettati, S. 2011. Chemistry and Biochemistry of Oxygen Therapeutics: From Transfusion to Artificial Blood, Wiley, 270-280. Reverberi, R. 2008. The Persistence of Red Cell Alloantibodies. Blood Transfusion, 6, 225. Starr, C. & McMillan, B. 2008. Human Biology, Belmont Brooks/Cole, 140-150.  Read More