For my third rotation in the clinical laboratory, I was in the Transfusion Medicine laboratory. For those who are unfamiliar with this section of the lab, it is responsible for the testing of patients’ blood and blood products before transfusions occur.
One of the most common tests performed in this laboratory is ABO grouping, which consists of a forward grouping that tests for antigens present on red blood cells, and a reverse grouping that tests for antibodies present in plasma. The ABO blood group follows Landsteiner’s rule, which states that red blood cells contain antibodies against antigens they do not possess. Therefore, those who have group A blood will have A antigens present on their red blood cells, and anti-B in their plasma, and vice versa for group B blood. Group O blood does not have the presence of either A or B antigens, and therefore has both anti-A and –B present in the plasma. The forward grouping uses antisera with either anti-A or –B to detect the A and B antigens present on red cells. The reverse grouping uses reagent red blood cells that contain either the A or B antigen to detect anti-A or –B.
In addition to ABO grouping, an Rh grouping is also done, and consists of typing patients for the D antigen. Those patients that are Rh positive have the D antigen present, and those that are negative do not have it present. Unlike ABO antibodies, Rh antibodies do not follow Landsteiner’s rule, and are not inherently present in the absence of antigens. Therefore, antibodies are only produced whenever a stimulus is present, such as extraneous D antigen. This can occur by Rh negative patients being transfused with Rh positive blood, or Rh negative mother’s blood coming into contact with their Rh positive fetus’ blood. While most patients are either Rh positive or negative, there are some patients who present with weaker expressions of the D antigen. Therefore, whenever an Rh typing is preformed and a negative result is obtained, a weak D test is performed to detect these weaker reactions. An Rh control is included in this method, and both tubes are incubated at 37°C for 15 minutes, washed three times with saline, and have anti-human globulin added to increase their reaction strength. If throughout any of these steps a positive patient result is obtained with a negative control result, the patient is said to be weak D positive. It is very important to properly type patients as weak D positive, instead of Rh negative.
In routine testing in the transfusion medicine laboratory, antibody screens are performed along with blood groupings. In order to detect clinically significant antibodies present in patients’ blood, antibody screens are performed using two or three cells of group O reagent red cells against patient plasma. The reagent red cells should preferably be homozygous for high-prevalence antigens from other blood group systems in order to detect the weak reactions of antibodies. Some of the antigens present in these cells are other Rh antigens (C, c, E, and e), Duffy (Fya, Fyb), Kidd (Jka, Jkb), and MNSs (M, N, S, and s) to detect their associated antibodies. After a positive antibody screen is seen, an antibody panel is set up using ten or eleven cells containing varying strengths of antigens against patient plasma. Reactions against the different cells are graded and compared against an antigram that lists which antigens are present in each cell. Cells that show no reaction are first used in the exclusion process. Any antigens that are present in their homozygous form in negative cells are excluded as the possible antibody as there is no reaction against the strongest form of antigen. This process continues until as many cells as possible have been excluded. Then, reaction gradings are compared to the antigens that have not been excluded. Those antigens on the antigram that follow the same pattern as the patient results determines what antibody is present in the patient. In the case in which patients contain more than one antibody, it is helpful to use antigen panel cells with or without enzymes, as some antibodies are enhanced or destroyed in the presence of enzymes, and this helps in differentiating which antibodies are present. While performing an antibody panel, it is also common to include an autocontrol, which consists of patient red blood cells reacting with their own plasma. This control determines if there are autoantibodies present in the patient’s plasma.
Another common test performed in the transfusion laboratory is the Direct Antiglobulin Test (DAT). This test is performed to detect the presence of in vivo sensitization of red blood cells, and commonly used for the detection of autoantibodies. There are many different types of immunoglobulin antibodies present in the body, however, within transfusion medicine, we are mainly concerned with IgM and IgG antibodies. IgM antibodies are pentamers, consisting of five parts, and are very large in structure. IgG antibodies are only monomer structures, and are much smaller than IgM antibodies. Due to the large size of IgM antibodies, when reacting with red blood cells, they form a lattice and reactions are easily seen macroscopically. However, IgG antibodies are too small to form a lattice and are not seen macroscopically, nevertheless, their presence is still clinically significant. Another compound detected by the DAT are complement proteins. These proteins make up the complement system of the immune system. To perform this test, patients’ red blood cells with antibody are combined with anti-human globulin (AHG). This joins the antibodies to neighboring red blood cells to form a visible reaction. In the test procedure, polyspecific AHG is added to patients’ red blood cells to detect both IgG and complement coating the cells. Whenever this reaction is positive, monospecific reagents are used, such as anti-IgG (to detect IgG antibodies) and anti-C3d (to detect complement), to determine what exactly is coating the cells. For any negative results, Coombs control cells are added to validate that reagent was added to the tube. After the addition of Coombs control, a reaction should be 2+ or greater. If there is no reaction after the addition of Coombs control cells, the test is invalid and must be repeated. The DAT is positive in many clinical conditions in Transfusion Medicine, such as hemolytic disease of the newborn, hemolytic transfusion reactions, autoimmune hemolytic anemias, and drug-induced hemolytic anemias.
After a patient has been identified as having an IgG antibody coating their red blood cells, identification of this antibody needs to be performed. However, since the antibody is coating the patient’s red blood cells and not freely flowing in patient plasma, an elution needs to be performed to remove this bound antibody. Acid elution kits are commonly used that alter the pH of the red blood cell in order to dissociate the antibody from the cell. Then, the solution containing the unbound antibody, the eluate, can be tested against an antibody panel in order to determine its specificity.
The final test performed before red blood cells are given to a patient to be transfused is the crossmatch, and is performed in order to determine if the blood selected for transfusion will survive well in the patient. The crossmatch is performed by testing donor red blood cells against patient plasma. No agglutination should be seen between the reaction, and if so, it means that the patient has an antibody for an antigen present on the donor blood, and should not be transfused that blood. With a patient history of no clinically significant antibodies and a negative antibody screen, an immediate spin crossmatch can be performed, or an electronic crossmatch if two blood groupings for the patient are on file. However, whenever a patient has a positive antibody screen, a history of antibodies and/or ABO/Rh discrepancy, a full indirect antiglobulin crossmatch needs to be performed in order for blood to be chosen for the patient. All reactions of the crossmatch must be negative in order for the blood to be transfused to the patient. After a unit of blood has been chosen for a patient with an identified antibody, the blood chosen for transfusion must be negative for the antigen that the antibody would react with.