Antihuman Globulin Testing Flashcards
what is the principle of antihuman globulin test
this test is used to detect the presence of human globulin molecules either bound to the RBC or free in serum
AGT makes use of reagents that reacts to human antibodies produced by?
rabbits
whats the use of DAT
DAT is used to detect antibody binding to antigen on the surface of RBCs that happens in vivo
what is the use of IAT
IAT is used to detect for the in vitro sensitization of red blood cells with blood group antibodies
happens in vivo
DAT (Direct Antihuman Globulin Testing)
happens in vitro
IAT (Indirect Antihuman Globulin Testing)
materials used for AGT
- test tubes
- centrifuge
- AHG reagent
- NSS
- microscope
reagents must be stored at what temp
2-8 deg centigrade, must be room temp prior to use
DAT: Step 1
prepare 3-5% RBC suspension of the patient’s red cell
DAT: STEP 2
In an appropriately labeled test tube, place 1-2 drops of cell suspension and wash it with saline 3-4 times
DAT: Step 3
add 2 drops of the AHG reagent to the cell button and resuspend the cells
DAT: Step 4
centrifuge at 3200 rpm for 15 seconds
DAT: Step 5
read and grade the reactions
DAT: Step 6
record the results
DAT: Step 7
If negative, add 1 drop of check cells to the negative test tube. This should give a positive result
what to do if the results of DAT is negative
add 1 drop of check cells to the negative test tube
IAT: Step 1
prepare 3-5% rbc suspension of the patient’s cells
IAT: Step 2
into a properly labeled test tube, place 2 drops of the prepared red cell suspension
IAT: Step 3
add 2 drops of patient’s serum into the same test tube
IAT: Step 4
incubate the tube at 37 deg centigrade in a water bath for 15 minutes
IAT: Step 5
remove the tube from the water bath, centrifuge and resuspend and observe for agglutination or hemolysis
IAT: Step 6
If no agglutination, wash the serum-cell mixture 3 times by NSS.
IAT: Step 7
After the third washing, add 2 drops of AHG reagent, mix and centrifuge at 3200 rom for 15 seconds.
IAT: Step 8
Read and grade the reactions.
IAT: Step 9
Record your results.
IAT: Step 10
If negative, add 1 drop of check cells to the negative test tube. This should aive a positive result.
what does Agglutination in the DAT procedure indicate
positive result.
whats the cause of the agglutination in DAT
this is due to in vivo sensitization of antibodies coating to the patient’s RBCS.
Antihuman globulin test is also known as ____ in honor of the person who discovered such test.
Coomb’s test
DAT is used to diagnose??
hemolytic disease of the newborn, autoimmune hemolytic anemia, and hemolytic transfusion reaction
IAT is used to demonstrate in vitro reactions between red cells and coating antibodies as in?
antibody detection, antibody identification, blood grouping, and compatibility testing.
what is the purpose of an antihuman globulin test
for detecting nonagglutinating (coating) Rh antibodies in serum
false negative results may arise in the ff circumstances:
- inadequate or improper washing of RBCs
- deterioration of reagent or neutralization
- AHG reagent not added
- serum not added in the indirect test
- serum not reactive because of complement inactivation
- inadequate incubation condition in the indirect test
- cell suspension either too heavy or too weak;
- undercentrifugation
- poor reading technique
false positive causes
• Improper specimen (refrigerated or clotted) → complement attaches in vitro
• Autoagglutinable cells
• Bacterial contamination (cells or saline used for washing)
• Positive DAT cells (used in IAT)
• Saline contaminated with heavy metals or colloidal silica
• Dirty glassware
• Overcentrifugation or overreading
• Polyagglutinable cells
• Preservative-dependent antibody in LISS reagents
• Contaminating antibodies in AHG reagent
factors affecting the sensitivity of the IAT
- temperature
- ionic strength
- ratio of serum to cells
- pH of the reaction mixture
- incubation time
who and in what year did they described a test for detecting nonagglutinating (coating) Rh antibodies in serum
Coombs, Mourant, and Race (1945)
