ANTIGLOBULIN TEST Flashcards
memorization
The antiglobulin test is also known as:
COOMBS TEST
TYPES OF AHG REAGENTS:
- Polyspecific AHG
- monospecific AHG
- Polyclonal AHG
- Monoclonal AHG
AHG reagent that contains Anti-IgG and Anti-C3d (2 antibodies)
Polyspecific AHG
AHG reagent that contain either Anti-IgG only; or Anti-C3d only
Monospecific AHG
AHG reagent that contains a pool of heterogenous Anti-IgG from many immunized rabbits (recognize multiple epitopes)
Polyclonal AHG
AHG reagent that contains a pool of Anti-IgG from a single clone of plasma cells; prepared by Hybridoma Technology; uses laboratory mice (recognizes single epitope; more specific)
Monoclonal AHG
TYPES OF ANTIGLOBULIN TEST:
DAT (Direct Antiglobulin Test)
IAT (Indirect Antiglobulin Test)
Antiglobulin test that detects bound antibodies to RBCs/ In vivo sensitization of RBCs
Direct Antiglobulin Test (DAT)
Antiglobulin test that detects serum antibodies/ In vitro sensitization of RBCs
Indirect Antiglobulin Test (IAT)
Specimen required for DAT:
Patients Washed RBCs (EDTA WB)/
Red Cell Suspension (RCS) method = 2-5%
Specimen required for IAT:
Patient’s serum (Ab) + Reagent red cells (Ag)
Does DAT require incubation at 37C?
NO
because the purpose of incubation is to sensitize RBC with IgG antibodies. In DAT, sensitization happens in vivo therefore there is no need for incubation in DAT
Does IAT require incubation at 37C?
YES
Sensitization of RBCs by IgG Abs; patient serum (Ab) + Reagent Red cells (Ag) + AHG + incubation = sensitization
Composed of Type O RBCs coated with IgG antibodies (anti-D)
Check cells or Coombs control
Note: Check cells/Coombs control is only done for TUBE METHOD
Check cells/Coombs control is only added to ______________ of AHG test for validation
NEGATIVE TUBES
Note: Check cells/Coombs control is only done for TUBE METHOD
Clinical conditions detected by DAT:
HTR - Px Abs coating the Donor’s RBCs
HDN - Maternal Abs coating the fetal RBCs
AIHA - Px Abs coating own RBCs
DIIHA - Presence of a drug causes Ab-binding/production of Abs
Clinical conditions/applications of IAT:
Crossmatching (Patient’s serum + Donor’s RBC)
Antibody screening
Antibody identification
Weak D test
Red cell phenotyping
Note: All these tests require incubation at 37C
Interpretation of Check cells/Coombs control
Agglutination (+) =
No agglutination (-)=
Interpretation of Check cells/Coombs control
Agglutination (+) = TEST IS VALID
No agglutination (-) = INVALID TEST RESULT (AHG rgt most likely neutralized); repeat procedure from the start
DAT Panel Reactions:
Rgt 1 (Anti-IgG) = positive
Rgt 2 (Anti-C3d) = positive
TYPE OF AIHA:
WAIHA (Warm AIHA) (67%)
DAT Panel Reactions:
Rgt 1 (Anti-IgG) = positive
Rgt 2 (Anti-C3d) = negative
TYPE OF AIHA:
WAIHA ( Warm AIHA) (20%)
DAT Panel Reactions:
Rgt 1 (Anti-IgG) = negative
Rgt 2 (Anti-C3d) = positive
TYPE OF AIHA:
CAS, PCH, WAIHA (13%)
CAS - Cold Agglutinin Syndrome
PCH - Paroxysmal Cold Hemoglobinuria
Ratio of serum to cells in antiglobulin test:
Ratio should be 40:1 or 2 drops of serum + 1 drop of 5% RCS
*in tube method
- 2 drops serum
- 1 drop 5% RCS
= 40:1 ratio
Reaction medium uses
Albumin:
LISS:
PEG:
Reaction medium uses
Albumin: Allow Ab-coated cells to come into closer with each other (reduce zeta potential)
LISS: Enhance Ab uptake and allow incubation time to be decreased
PEG: Action is to remove water thereby concentrating antibody
Optimum temperature for IgG activity and complement activation:
37C (Body temperature)
Incubation time
- using 22% albumin:
- using LISS:
Incubation time
- using 22% albumin: 15-60 minutes
- using LISS: 10-15 minutes (extended incubation = decreased sensitivity)
How many times do the RBCs have to wash before immediate addition of AHG:
3 times
*wash RBC 3 times before immediate addition of AHG
purpose of washing of cell:
Remove free or unbound globulins
Inadequate washing will result to __________ results:
FALSE-NEGATIVE; because of neutralization by free globulins
Excessive washing of cells:
NO EFFECT
Washing of cells is only applicable in:
TUBE (traditional) METHOD and SOLID PHASE TECHNOLOGY
Saline for washing should have a pH of _________
7.2 - 7.4 pH
Saline stored for long periods in plastic containers will:
Decrease in pH (will become acidic)
When should the lab tech add the AHG?
immediately after washing
Recommended centrifugation requirement for AHG:
1000 RCF for 20 seconds
(FALSE RESULTS IN AHG)
Clotted specimen (false pos/false neg)
Clotted specimen - false positive
(FALSE RESULTS IN AHG)
Non-reactive AHG/expired (false pos/false neg)
Non-reactive AHG/expired - false negative
(FALSE RESULTS IN AHG)
Cell suspension too weak or too heavy (false pos/false neg)
Cell suspension too weak or too heavy - false negative
(FALSE RESULTS IN AHG)
Bacterial contamination (false pos/false neg)
Bacterial contamination - false positive
(FALSE RESULTS IN AHG)
Undercentrifugation (false pos/false neg)
Undercentrifugation - false negative
(FALSE RESULTS IN AHG)
Metal contamination of cells (false pos/false neg)
Metal contamination of cells - false positive
(FALSE RESULTS IN AHG)
Dirty glassware (false pos/false neg)
Dirty glassware - false positive
(FALSE RESULTS IN AHG)
Serum/AHG not added (false pos/false neg)
Serum/AHG not added - false negative
(FALSE RESULTS IN AHG)
Old serum samples (false pos/false neg)
Old serum samples - false negative
(FALSE RESULTS IN AHG)
Overcentrifugation (false pos/false neg)
Overcentrifugation - false positive
(FALSE RESULTS IN AHG)
Overreading (false pos/false neg)
Overreading - false positive
(FALSE RESULTS IN AHG)
Contaminated AHG (false pos/false neg)
Contaminated AHG - false positive
(FALSE RESULTS IN AHG)
Inadequate incubation conditions (false pos/false neg)
Inadequate incubation conditions - false negative
(FALSE RESULTS IN AHG)
Inadequate washing of cells (false pos/false neg)
Inadequate washing of cells - false negative
