Enhancement Reagent The Antihuman Globulin Test AHG

1. drmsaiem Enhancement Reagent & The Antihuman Globulin Test (AHG) Dr MH Saiemaldahr BB Med Tech

2. drmsaiem Enhancement Reagent Low Ionic Strength solution (LISS) decrease the ionic strength of a reaction medium and thus, reduce the zeta potential The reduction in the zeta potential leads to an increase in the attraction between positively charge Ab molecules and negatively charge red cells. LISS is used to increase the rate of uptake during sensitization with an incubation time of 5-15 mins instead of 30-60 mins.

3. drmsaiem Enhancement Reagent Albumin, enhances Ab detection test by reducing the zeta potential thereby promoting agglutination of sensitized red cells Antihuman Globulin Reagent can be used to promote agglutination of red cells sensitized with IgG or complement.

4. drmsaiem Zeta Potential Is an expression of the difference in electrostatic potential at the surface of the red cell and the ionic cloud of positive cations that are attracted to the negative charges on the surface. The net negative charge surrounding red cells is part of the force that repels red cells from each other. Reducing the Zeta potential should have the effect of allowing RBCs agglutination by IgG molecules.

5. drmsaiem ENZYMES Can enhance or suppress the reaction of certain blood group Ags and Abs There are certain enzymes can be used; Ficin (Fish), Trypsin (Pig stomach), Papain (papya), and Bromelin (Pineapple)

6. drmsaiem ENZYMES Action of Enzyme 1- The treatment of red cells with enzyme results in the release of sialic acid from the membrane of RBC with subsequent decrease in the negative charge of the cells which lead to reduction in the zeta potential. 2- Removes hydrophilic glycoprotein from the membrane of red cells, causing the membrane to become more hydrophobic, allowing the cells to come closer together.

7. drmsaiem ENZYMES Due to removal of the glycoprotein from the membrane, Ab molecules can be easily reacting with the Ag on the membrane surface Abs reactivity which can be enhanced by enzyme treatment are; Rh, Kidd, P1, Lewis, and I Ags Ags that can be destroy or depress by enzyme treatment are Fya, Fyb, M, N, and S.

8. drmsaiem AHG Basic Concepts of Antiglobulin Testing Two major classes of antibodies react with red blood cell antigens; (1) Complete or saline agglutinins will agglutinate red blood cells suspended in saline (these are usually IgM) (2) Incomplete agglutinins which do not react in saline and require special techniques to agglutinate red blood cells (these are IgG)

9. drmsaiem AHG Antiglobulin tests are used to detect IgG or complement sensitized red blood cells. For complement detection, because some blood group antibodies after combining to their corresponding antigen, they have the ability to activate and bind complement to the red blood cell membrane Sensitization can occur either on vivo or in vitro. The antiglobulin test can detect the presence of allo-antibodies, auto-antibodies, and complements components.

10. drmsaiem AHG Types of AHG There are two types of antiglobulin test (AHG); 1-Indirect antiglobulin (IDAT, IYT) test which formed in vitro 2- Direct antiglobulin (DAT) test which formed in vivo. The Direct antiglobulin test does not require the incubation phase, because the antigen- antibody complexes have formed in vivo.

11. drmsaiem AHG For Indirect antiglobulin test (in vitro) antigen-antibody reactions the following is required; 1- incubate red cells with anti-sera to allows time for antibody molecules attachment to red cells antigen. 2- perform 3 saline washing to remove free globulin molecules. 3- add antiglobulin reagent forms red cell agglutinates

12. drmsaiem AHG 4- centrifuge, to accelerate agglutination by bringing cells closer together. 5- examine for agglutination to interprets test as positive or negative 6- grade agglutination to determines the strength of the reaction 7- add antibody coated red blood cells to those with negative reaction, to checks for neutralization of anti-human sera by free globulin molecules (Coombs control cells are D-positive red blood cells that are coated with anti-D).

13. drmsaiem Indirect (IDAT) Indirect AHG Test Applications. This test determines in vitro sensitization of red cells. There are many applications for this test including; 1-Detection of incomplete antibodies to potential donor red cells (compatibility testing) or to screening cell (antibody screen) in serum. 2- Identification of antibody specificity using a panel of red cells with known antigen proteins. 3- Determination of red cell phenotype using known anti-sera for example (Kell typing, Du testing). 4- Titration of incomplete antibodies.

14. drmsaiem AHG Principles of the Antiglobulin Test 1- Antibody molecules and complement components are globulins. 2-Injecting an animal with human globulin stimulates the animal to produce antibody to the foreign protein. 3- AHG will react with human globulin molecules either bound to red blood cells or free in serum. 4- Washed red blood cells coated with human globulin are agglutinated by AHG.

15. drmsaiem Direct AHG Test (DAT). Principle and Application. Direct antiglobulin test is used to detect in vivo sensitization of red blood cells. Clinical conditions that can result in vivo coating of red blood cells with antibody or complement or both are; 1- haemolytic disease of the new born (HDN). 2- haemolytic transfusion reaction (HTR) 3- auto-immune and drug-induced hemolytic anemia.

16. drmsaiem DAT Direct antiglobulin Testing Panel (DAT). Initial (DAT) should include both anti-IgG and anti-C3d reagents or poly-specific (anti-IgG-C3d) reagent. A DAT panel using poly-specific and mono-specific reagents characterizes the type of protein sensitization that has occurred in vivo A DAT panel is useful in determining whether the patient’s red cells are coated with antibody, complement, or both.

17. drmsaiem DAT Significance of a positive DAT Result. Interpreting the significance of a positive DAT results requires knowledge of the patient’s diagnosis, drug therapy, and recent transfusion history.

18. drmsaiem DAT Evaluation of A positive DAT Result. Answering the following questions before investigating a positive DAT results for patient other than neonate will help determine what further testing is appropriate. Is there evidence of in vivo hemolysis, has the patient been transfused recently, does the patient’s serum contain unexpected antibodies, is the patient receiving any drugs, has the patient received blood products or components containing ABO-incompatible plasma, is the patients receiving antilymphocyte globulin (ALG) or antithymocyte globulin (ATG).