Antiglobulin Testing PDF

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Summary

The Antiglobulin Test- Also known as the Coombs test and serves to determine if the RBCs are coated with IgG or complement. IgG are non-agglutinating antibodies because they are small and cannot agglutinate RBCs directly. - The union of antigens to antibodies depends on charges and structures of mol...

Description

MLSC-3200, Transfusion Science The Antiglobulin Test -

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Also known as the Coombs test and serves to determine if the RBCs are coated with IgG or complement. IgG are non-agglutinating antibodies because they are small and cannot agglutinate RBCs directly. The union of antigens to antibodies depends on charges and structures of molecules. Pro Zone, more antibody than antigen. Post Zone, more antigen than antibody. Equivalence Zone, antibody and antigen are equal in number. This is the condition that is best for agglutination testing. Antibody-antigen reactions can be detected through: o Agglutination, macroscopic detection of agglutination caused by cross-bridging between RBC antigens and antibodies that are corresponding with each other. o Hemolysis, caused by antibody binding and activation of complement which results in the RBC membrane rupturing. o Solid Phase Testing, determines the presence of antibodies through automation and microplates coated with antibodies or antigens. Uses an adherence reaction to detect antibody-antigen reactions through adhesion and binding of antibodies and antigens. Agglutination reactions occur in two stages. o Sensitization, the antibody attaches to its corresponding antigen on the RBC membrane. These RBCs are considered sensitized. o Lattice Formation, involves the bridges forming between sensitized RBCs. We are usually interested in IgG and IgM. IgG has two antigen binding sites, while IgM has 5. The lattice formation will not occur with IgG because it is too small to bridge the gap between RBCs unlike the larger IgM that easily agglutinates RBCs. This is a problem in transfusion. Moreschi described the principle of the antiglobulin test and Coombs demonstrated that RBCs can combine with antibodies like IgG without actually agglutinating RBCs. Resulted in the formation of the antiglobulin test. Antiglobulin Test, principle is that negatively charged RBCs have a space of 30 nm between each other. IgM antibodies can bridge this space by spanning between 30-39 nm to form a lattice, unlike the smaller IgG. The Coombs “anti-antibody” reagent solved this problem. He injected rabbits with human IgG to cause the rabbits to make antibodies against human IgG (AHG serum). The AHG antibody serum binds to IgG to help them agglutinate RBCs using only IgG without IgM. Polyspecific AHG, contains antibody to human IgG and complement C3d. May also contain antibody to C3b, C4b and/or C4d. Monoclonal AHG is good because they are identical in structure and specificity, don’t require absorption of heterospecific antibodies and allow production of consistently pure reagent. Monospecific AHG contains only one antibody specificity, either IgG or C3d. The two tests used to detect sensitized RBCs are the direct antiglobulin test and the indirect antiglobulin test.

MLSC-3200, Transfusion Science Direct Antiglobulin Test -

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Used to detect in-vivo sensitization of RBCs with IgG and/or complement components before they are removed from the body for testing. AHG is added to the 5% RBCs from the patient and if they’re sensitized the AHG will allow the IgG to form a lattice and agglutinate in a positive reaction. DAT cannot tell us if IgG or IgG and complement or just complement is present. Only tells us one of those options are present. Further testing is needed to specify the combination. A saline control is needed as a negative control for the DAT test to identify random agglutination. Sensitized control check cells are also used. It serves to validate that the negative tests are truly negative. There is always a possibility of AHG contamination with unbound Ig. When we get a negative test, check cells are added (IgG coated RBCs) which react with the AHG present, meaning that all negative test tubes should be positive. All negative antiglobulin tests controlled in this manner must now be positive. Coombs check cells come from commercially prepared O positive cells coated with IgG or C3d for DAT negative tests. This test is helpful in diagnosing HDN, hemolytic transfusion reaction, AIHA and drug induced HA. A positive DAT must also be checked for drugs and recent transfusions that may cause a positive DAT. Elution Techniques, done when a DAT is positive due to IgG. IgG antibody complexes on RBCS are dissociated and placed into a solution to test the specificity. The recovered antibody is an elute. o Acid Elution, done using a test kit with an acidic eluting solution. The pH causes the antibodies to dissociate and come off into solution. The antibodies are then harvested for testing. Detects non-ABO antibodies. o Organic Solvent, they denature the antigen by dissolving the RBC membrane leading to the reversal of van der Waal forces. Safety issues have made this method obsolete. o Heat Method, simplest method for eluting antibodies. Heats up the antibodies until they fall off for harvesting. o Freeze Method, results in total elution and involve rapid thawing the RBCs causing them to burst and release the antibodies for harvesting.

MLSC-3200, Transfusion Science Indirect Antiglobulin Test -

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Performed to determine in-vitro sensitization of RBCs. Patient unknown serum is mixed with known RBCs with or without certain antigens to check for antibodies in the patient using AHG Coomb’s antibodies. Compared to the DAT test, RBC sensitization occurs outside of the body when we incubated the known RBC/serum mixture to make IgG bind to the RBCs. This makes the IAT much longer since DAT RBCs come straight from the body where they may or may not have already undergone sensitization/binding with IgG. Patient plasma is collected, mixed with known RBCs and incubated at 37 degrees for 1530 minutes. Next, the cells are washed to remove unbound globulin and AHG is added to allow cross linking if possible. Centrifugation is done once the reagent is added to promote agglutination. After centrifugation the mixture is analyzed for agglutination. Finally, Coomb’s check cells are added to all negative tests to verify that the AHG is working. Check cells always react with AHG and should be positive for agglutination if the test worked. This test is frequently done in the lab as a typing/grouping screen. Blood type testing is often requested with the goal of transfusion, so we must also check for their antibodies along with their ABO group. The IAT allows us to use the patient serum and the donors RBCs as our known RBCs do see if the patient would react adversely to the transfusion. ABO antibodies are very potent and can bind to complement to cause hemolysis.

Sources of Error in Antiglobulin Testing -

False Positives, can occur due to: o Improper Samples, samples must not be clotted, usually by using EDTA tubes. o RBC/Plasma Bacterial Contamination, results in polyagglutination when the RBCs agglutinate all normal serum. o Serum Bacterial Contamination, will result in panagglutination when the serum agglutinates all RBCs. o Saline Bottle Types, plastic bottle should always be used for saline since glass can leak silica into the solution, causing false positives when it promotes random agglutination. o Dirty Glassware, always check to see that all glassware is clean. Particles in solution may result in false positives. o Contaminated AHG Reagent, usually very uncommon but still possible. This is why quality control is always done on the reagents (Coomb’s Check Cells). o Over-Centrifugation, can cause extreme packing of cells which will make a strongly connect button that could mimic agglutination in the tube. o DAT Positive Cells for IAT, can result in a double positive effect to occur resulting in a false positive.

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False Negatives, can occur due to: o Inadequate Washing, most common error. Only layering the saline on the top of the cells or not resuspending RBCs between washes can leaves some unbound globulin behind to bind randomly to AHG. Causes a false negative. o Failure to add AHG, not adding AHG means there is no chance of agglutination even for positive RBCs. o AHG Non-Reactive, due to reagent deterioration or neutralization. o Cell Suspension, too heavy or too light suspensions can cause false negatives. o Over/Under-Centrifugation o Prolonged Washing, if saline is left standing on the RBCs for too long, the antibodies tend to elute off and positive RBCs will become falsely negative. o Poor Reading Technique, does not allow for proper interpretation of results. Shaking the tube too harshly will break all truly positive agglutination. Results in true positives being missed as false negatives. o Improper Incubation Time, deviations in incubation time will result in less than optimal conditions for proper antibody binding. o Deterioration of Complement Levels, stored plasma has been known to lose significant amounts of complement if left at RT or 4 degrees for several days, causing a false positive. Always use fresh plasma for IAT testing. o Dirty Glassware, can cause issue especially if they are contaminated with dried serum.

Factors Affecting Antiglobulin Testing -

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Ratio of Serum to cells, increasing the Serum:Cell ratio increases the sensitivity of the test system. A ratio of 2 drops of serum per 1 drop of RBCs is used. Reaction Medium, the use of albumin, low ionic strength saline (LISS) and polyethylene glycol (PEG) will enhance the antibody uptake resulting in shortened incubation times. Albumin is not commonly used as clinically significant antibodies may be missed. Washing RBCs, both DAT and IAT testing requires three 1 minute washes. Removes the unbound globulins that will cause random agglutination. Saline must be completely decanted after last wash or it may dilute the AHG reagent decreasing the sensitivity. Addition of AHG, must be added immediately after washing to prevent antibody elution and subsequent AHG reagent neutralization. Centrifugation, relative centrifugal force is important. Higher RCFs provide more sensitive results but increase the chance of incomplete button resuspension (false positive) or clot breaking from harsh resuspension to overcome the tight button (false negative). Temperature, the incubator should be 36 to 38 degrees Celsius. Incubation Time, should be 30 minutes or 10-15 minutes with LISS. Saline, saline should be fresh or buffered to 7.2-7.4 pH. Monoclonal AHG has a narrow pH range for optimum reactivity and can cause false negatives if not in that range.

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Anti-CD38, Daratumumab and other anti-CD38 monoclonal antibodies such as isatuximab have been recognized as therapies that can interfere with blood bank testing by binding to RBCs to cause panagglutination on the IAT. Leads to redundant testing and significant delays in patient care....