الواقع والحياد
13-03-2007, 05:13 PM
Prewarmed Technique for Testing Serum Containing Cold Agglutinins
Application
The reactivity of IgM cold autoantibodies can be reduced or eliminated by performing prewarmed tests. Most problems encountered in compatibility testing, antibody detection, and identification tests that are caused by cold agglutinins can be resolved with the use of this technique. By preventing the reaction between the cold agglutinin and the RBC at room temperature (during centrifugation and testing), complement activation is prevented. The anti-C3 in polyspecific AHG reagent reacts with the remaining C3, making detection of significant AHG-reactive antibodies difficult. Prewarming of the serum, cells, and additive will hinder the binding of the cold autoantibody and the resulting complement activation.
Materials
1. Normal saline, warmed to 37°C
2. Patient's serum
3. Additive solution (bovine albumin, low ionic strength solu¬tion, polyethylene glycol, etc.), if any
4. Cells to be tested (screening or panel cells, donor cells)
5. Antihuman globulin (AHG) anti-IgG
Method
1. Label one tube for each reagent or donor cell sample to be tested.
2. Add one drop of the appropriate 296-4% cell suspension to each tube.
3. Place the tubes containing the RBCs, a tube containing a small amount of patient's serum, and a tube containing the additive solution, if any, at 37°C for 5-10 minutes.
4. Transfer two drops of prewarmed serum into each tube containing prewarmed RBCs; mix without removing the tubes from the incubator. Additive will be added according to manufacturer's insert.
5. Incubate at 37°C for at least 30 minutes with no additive, or the appropriate time for the additive used.
6. Without removing the tubes from the incubator, fill all tubes with prewarmed saline (37°C). Centrifuge and wash two to three more times with warm saline.
7. Add anti-IgG antiglobulin reagent, centrifuge, and record reactions.
Note: Do not prewarm the anti-IgG reagent Why???
Application
The reactivity of IgM cold autoantibodies can be reduced or eliminated by performing prewarmed tests. Most problems encountered in compatibility testing, antibody detection, and identification tests that are caused by cold agglutinins can be resolved with the use of this technique. By preventing the reaction between the cold agglutinin and the RBC at room temperature (during centrifugation and testing), complement activation is prevented. The anti-C3 in polyspecific AHG reagent reacts with the remaining C3, making detection of significant AHG-reactive antibodies difficult. Prewarming of the serum, cells, and additive will hinder the binding of the cold autoantibody and the resulting complement activation.
Materials
1. Normal saline, warmed to 37°C
2. Patient's serum
3. Additive solution (bovine albumin, low ionic strength solu¬tion, polyethylene glycol, etc.), if any
4. Cells to be tested (screening or panel cells, donor cells)
5. Antihuman globulin (AHG) anti-IgG
Method
1. Label one tube for each reagent or donor cell sample to be tested.
2. Add one drop of the appropriate 296-4% cell suspension to each tube.
3. Place the tubes containing the RBCs, a tube containing a small amount of patient's serum, and a tube containing the additive solution, if any, at 37°C for 5-10 minutes.
4. Transfer two drops of prewarmed serum into each tube containing prewarmed RBCs; mix without removing the tubes from the incubator. Additive will be added according to manufacturer's insert.
5. Incubate at 37°C for at least 30 minutes with no additive, or the appropriate time for the additive used.
6. Without removing the tubes from the incubator, fill all tubes with prewarmed saline (37°C). Centrifuge and wash two to three more times with warm saline.
7. Add anti-IgG antiglobulin reagent, centrifuge, and record reactions.
Note: Do not prewarm the anti-IgG reagent Why???