Antihuman Globulin Testing

Question 1

what is the principle of antihuman globulin test

Answer 1

this test is used to detect the presence of human globulin molecules either bound to the RBC or free in serum

Question 2

AGT makes use of reagents that reacts to human antibodies produced by?

Answer 2

rabbits

Question 3

whats the use of DAT

Answer 3

DAT is used to detect antibody binding to antigen on the surface of RBCs that happens in vivo

Question 4

what is the use of IAT

Answer 4

IAT is used to detect for the in vitro sensitization of red blood cells with blood group antibodies

Question 5

happens in vivo

Answer 5

DAT (Direct Antihuman Globulin Testing)

Question 6

happens in vitro

Answer 6

IAT (Indirect Antihuman Globulin Testing)

Question 7

materials used for AGT

Answer 7

• test tubes
• centrifuge
• AHG reagent
• NSS
• microscope

Question 8

reagents must be stored at what temp

Answer 8

2-8 deg centigrade, must be room temp prior to use

Question 9

DAT: Step 1

Answer 9

prepare 3-5% RBC suspension of the patient’s red cell

Question 10

DAT: STEP 2

Answer 10

In an appropriately labeled test tube, place 1-2 drops of cell suspension and wash it with saline 3-4 times

Question 11

DAT: Step 3

Answer 11

add 2 drops of the AHG reagent to the cell button and resuspend the cells

Question 12

DAT: Step 4

Answer 12

centrifuge at 3200 rpm for 15 seconds

Question 12

DAT: Step 5

Answer 12

read and grade the reactions

Question 13

DAT: Step 6

Answer 13

record the results

Question 14

DAT: Step 7

Answer 14

If negative, add 1 drop of check cells to the negative test tube. This should give a positive result

Question 15

what to do if the results of DAT is negative

Answer 15

add 1 drop of check cells to the negative test tube

Question 16

IAT: Step 1

Answer 16

prepare 3-5% rbc suspension of the patient’s cells

Question 17

IAT: Step 2

Answer 17

into a properly labeled test tube, place 2 drops of the prepared red cell suspension

Question 18

IAT: Step 3

Answer 18

add 2 drops of patient’s serum into the same test tube

Question 19

IAT: Step 4

Answer 19

incubate the tube at 37 deg centigrade in a water bath for 15 minutes

Question 20

IAT: Step 5

Answer 20

remove the tube from the water bath, centrifuge and resuspend and observe for agglutination or hemolysis

Question 22

IAT: Step 6

Answer 22

If no agglutination, wash the serum-cell mixture 3 times by NSS.

Question 23

IAT: Step 7

Answer 23

After the third washing, add 2 drops of AHG reagent, mix and centrifuge at 3200 rom for 15 seconds.

Question 24

IAT: Step 8

Answer 24

Read and grade the reactions.

Question 25

IAT: Step 9

Answer 25

Record your results.

Question 26

IAT: Step 10

Answer 26

If negative, add 1 drop of check cells to the negative test tube. This should aive a positive result.

Question 27

what does Agglutination in the DAT procedure indicate

Answer 27

positive result.

Question 28

whats the cause of the agglutination in DAT

Answer 28

this is due to in vivo sensitization of antibodies coating to the patient's RBCS.

Question 29

Antihuman globulin test is also known as ____ in honor of the person who discovered such test.

Answer 29

Coomb's test

Question 31

DAT is used to diagnose??

Answer 31

hemolytic disease of the newborn, autoimmune hemolytic anemia, and hemolytic transfusion reaction

Question 32

IAT is used to demonstrate *in vitro* reactions between red cells and coating antibodies as in?

Answer 32

antibody detection, antibody identification, blood grouping, and compatibility testing.

Question 33

what is the purpose of an antihuman globulin test

Answer 33

for detecting nonagglutinating (coating) Rh antibodies in serum

Question 34

false negative results may arise in the ff circumstances:

Answer 34

- 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

Question 35

false positive causes

Answer 35

• 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

Question 36

factors affecting the sensitivity of the IAT

Answer 36

- temperature - ionic strength - ratio of serum to cells - pH of the reaction mixture - incubation time

Question 38

who and in what year did they described a test for detecting nonagglutinating (coating) Rh antibodies in serum

Answer 38

Coombs, Mourant, and Race (1945)