Final- 3. Direct Antiglobulin Test (DAT) Case Study.

Principle of the Anti-Human Globulin Test ____ antibodies readily cause agglutination. ____ antibodies do not cause agglutination. In order to detect the presence of ____ antibodies, we must add an antibody to the Fc portion of the antibody we wish to detect. the antibody that we add goes by many names: anti-human globulin, AHG, Coombs antibody, Coomb's serum, or the green stuff. After Centrifugation, coated red cells (or ___ RBCs) form an antibody-antigen bridge via the ____ antibody resulting in visible agglutination. - correct answer IgM antibodies cause agglutination. IgG do not IgG antibodies coated red cells = sensitized RBCs AHG antibody When do we use AHG technique? - correct answer In antibody screening and identification, antigen typing, and in direct antiglobulin testing. In DAT, where does the sensitization step occur? - correct answer in the body Direct antiglobulin tests determine what? Is IgM coating detected with this form of testing? Is IgA coating detected? What is in the control tube of this test? What results do you look for in the control and what do they mean? - correct answer DAT determines *in vivo* coating of red blood cells by IgG antibody, by complement, or by both IgG and complement. IgM coating is not detected, except by complement that may have been activated. IgA coating is not detected. The control tube consists of the patient's RBCs and saline. The control tube should be negative. If the control tube is positive it suggests polyagglutination and we cannot really trust anything going on in the DAT tube if the control is positive. Briefly list the steps in direct antiglobulin testing. - correct answer Collect EDTA sample. (antigen-antibody reaction occurs in the body) Wash the cells to remove free plasma antibody. Add AHG (polyspecific). If the cells were coated in vivo, then the test will be positive (agglutination will occur). In a positive DAT test with polyspecific AHG, you would need to continue testing to a monospecific AHG to determine if the cells were coated with IgG or with complement (C'). What type of tube is used in direct antiglobulin test? Why do we use this tube? - correct answer Sample is drawn in a lavender top tube. The EDTA will not allow blood to clot, if the tube allows blood to clot, there could be a false positive DAT because clotting can cause the complement cascade to start. [in more specific terms- EDTA chelates Ca++ and Mg++. Chelated Mg++ prevents C3 formation. This helps to verify that any C3 activity seen happened in vivo, before the sample was drawn.] DAT Technique Start with a ____ reagent. If Positive, set up again using ____ reagents. (make a fresh cell suspension for these tests)