4. Antihuman Globulin (AHG) Flashcards
AHG: Principle
- Human serum (globulin) is injected into rabbit
- Acts as Ag- stimulated rabbit to make Ab
- Anti-human globulin (AHG) harvested from rabbit - will bind to any human globulin (Ab) bound to red blood cells - will form lattice agglutination - complete ABSC
Overview of Antihuman Globulin Test
Test to detect globulin (antibody and/or complement) coating red blood cells
Coating of RBCs with globulin can occur:
In vivo (within the body) In vitro (in the laboratory)
Two routine BB antiglobulin tests
1) Direct Antiglobulin Test (DAT)
2) Indirect Antiglobulin Test (IAT)
AHG Test, Coombs Test: History
1) Direct - detects IgG or complement coating patient cells (DAT = Direct Antiglobulin Test)
2) Indirect - detects IgG or complement in patient serum or plasma that is capable of coating reagent (or donor) red blood cells (IAT = Indirect Antiglobulin Test or ABS/ABSC = Antibody Screen/ing)
Polyspecific Reagents
Polyspecific AHG reagent = broad spectrum
- Contains anti-IgG and anti-C3bC3d
- Will detect IgG Ab and/or C3bC3d bound to RBC
USES: DAT, IAT, can be neutralized
Monospecific Reagents
Anti-IgG - heavy chain specific
- No complement activity
- Mixture of 4 IgG subclasses
- Will detect only IgG Antibody bound to RBC
USES: DAT, IAT, can be neutralized
Anti-C3bC3d
- No anti-IgG activity
- Will detect only C3b or C3d bound to RBC Anti-C3bC3d vs. Anti-C3d
USES: DAT, not used for IAT, can be neutralized
Control cells: Negative result using polyspecific AHG reagent or Anti-IgG AHG reagent
How do we know the reagent was not neutralized?
Test all negative tubes with Coombs control cells - agglutination confirms NEG result with PS AHG or Anti-IgG (these are known as “check” cells)
Control cells: Negative result using anti-C3bC3d
How do we know if the reagent was not neutralized?`
Test all negative tubes with complement control cells - agglutination confirms NEG result with anti-C3bC3d
Direct Antiglobulin Test: Indications (looking for in-vivo Ag-Ab reaction)
- Hemolytic Disease of Fetus and Newborn (HDFN)
- Transfusion reaction
- Allo vs. Autoantibody
- Evaluation of Hemolytic Anemia
Direct Antiglobulin Test: less complicated than the IAT
- No need to try to attach antibody to red cells
- RBCs are already sensitized in the patient’s specimen (if positive)
Direct Antiglobulin Test: Whole blood collected in an EDTA (purple top) tube
- EDTA whole blood is used to reduce interference of the DAT caused by complement
- The EDTA binds calcium, prevents coagulation cascade
e. g. Red top tubes (clotted)= rich source of complement
DAT Procedure: Polyspeciific AHG Reagent
Two drops Polyspecific AHG reagent One drop 2-5% suspension washed RBC Spin and read immediately (IgG) 5 min RT incubation, spin and read immediately (C3) BOTH readings negative = negative interpretation Confirm with 1 drop of Coombs control cells, spin and read (pos) One and/or the other reading(s) positive = use monospecific reagents to determine cause of reactivity DAT
DAT Procedure: Anti-IgG Readent
Two drops Anti-IgG reagent
One drop 2-5% suspension washed RBC
Spin and read immediately
Negative = negative interpretation
Confirm with 1 drop of Coombs control
cells, spin and read (pos)
DAT Procedure: Anti-C3bC3d
Two drops Anti-C3bC3d reagent
One drop 2-5% suspension washed RBC
Spin and read immediately; if neg
5 min RT incubation, spin and read
immediately
BOTH readings negative = negative
interpretation
Confirm with 1 drop of Complement
control cells, spin and read (pos)
IAT Test: Indications
•Looking for “in vitro” Ag-Ab reaction
•The IAT is used to detect red cell antibodies in patient serum
•AKA the “antibody screen” or ABSC
•Part of the type and screen and type and crossmatch procedure
•Approximately 5% of patients have a positive IAT due to IgG antibodies, IgM antibodies, or both
•Most clinically significant alloantibodies are IgG antibodies that react best at 37C and are formed as a result of: Previous exposure via transfusion or pregnancy or transplant
e.g. of clinically significant include: antibodies to Rh, Kell, Kidd, and Duffy antigens
IAT: IgM antibodies
- IgM antibodies are usually not clinically significant (except for ABO antibodies) but are a source of in vitro serologic difficulty that may delay transfusion
e. g. of IgM antibodies include antibodies to the Lewis, M, N, I, and P1 red cell antigens (will come back to LMNOP’s) - IgM antibodies react best at cold temperatures (4C) and are usually naturally occurring in that they do not require a sensitizing event
IAT: Looking for “in-vitro” reaction
Testing for presence of antibody that could cause a transfusion reaction
Antibody Screening Test
If positive, identify
antibody(ies) present
Patient should receive Ag
NEG RBCs
Testing for compatibility of patient plasma with donor cells (crossmatch)
IAT: Indications – Phenotyping for presence of antigen on RBCs
Some phenotyping antisera require an IAT test to detect presence of antigen on RBCs
IAT Procedure for Antibody Detection (Screening): Immediate Spin (IS) Testing
If 3-cell antibody screening: Label 3 tubes I, II, III with patient ID Two drops patient plasma (or serum) One drop 2-5% suspension reagent RBC (I in 1st tube, II in 2nd tube, III in 3rd tube) Spin and read immediately (IgM) Record reaction
IAT Procedure for Antibody Detection (Screening): AHG Phase of Testing
Wash tubes 3x isotonic saline Dry pack Add 2 drops of AHG reagent (Anti-PS AHG or Anti-IgG) Spin and read immediately (IgG) Record reaction If neg, add Coombs control cells (1 dr) to each neg tube, spin, read, record
Sources of Error: False POSITIVES
BOTH DAT & IAT: - Saline stored in glass bottles (not done now) - Improperly cleaned glassware - Over-centrifugation - Improperly prepared AHG reagents
DAT: - Clotted specimens stored at 4oC - Clotted specimens in tubes with silicone gel - Specimens collected from IV lines - Polyagglutination
IAT: - When Phenotyping RBC coated "in vivo" - Pos DAT - Requires: Chloroquine treatment – cells
