Anti Human Globulin Test ( Chapter 5 ) Flashcards
Anti Human Globulin Test is also known as
Coomb’s Test
This antibodies are also termed as nonagglutinating or incomplete antibodies
IgG antibodies
Where does the AHG binds ?
It binds to the Human Globulins such as IgG or Complement either in free serum or attached to the antigen of the RBC
In what year does Coombs and associate describe the use of the antiglobulin test for the detection of weak and nonagglutinating Rh antibodies in serum
1945
What is the purpose of Absorption in the procedure for AHG
To remove heterospecific antibodies
What is the purpose of Dilution in the procedure for AHG
to avoid prozone
First Blood Group System that were reported only weeks after Coomb’s had described the test
Kell Blood Group System
It is based on the principle that antihuman globulins obtained from immunized nonhuman species bind to human globulins
Anti-Human Globulin Test/Coomb’s Test
First to describe the principle of Coomb’s Test
Moreschi 1908
Early AHG reagents were prepared using a crude globulin fraction as the _____.
Immunogen
Presented the first indication that there might be another antibody activity present that influenced the final reaction in AHG test
Dacie 1951
The AHGT can be used to detect RBC sensitized with _____ , _______ and ___________
IgG alloantibodies , IgG autoantibodies and complement components.
In AHG reactivity cell sensitized with cold antibodies is resulted from what type of Globulin Activity
Anti-NonGamma Globulin activity
In AHG reactivity cell sensitized with warm antibodies is resulted from what type of Globulin Activity
Anti-Gamma Globulin activity
NonGamma Globulin component is believe to be what type of globulin
Beta Globulin
The use of AHG to detect in vitro sensitization of RBC’s in two-stage technique is referred as
Indirect Anti-Globulin Test
In vivo sensitization is detected by one stage procedure called
Direct Anti-Globulin Test
Give 3 AntiHuman Globulin Reagents that are Polyspecific
- Rabbit Polyclonal
- Rabbit/Murine monoclonal Blend
- Murine Monoclonal
Give 3 AntiHuman Globulin Reagents that are Monospecific
1,Rabbit Polyclonal
- IgG heavy chain specific
- Monoclonal IgG
A Polyspecifc AHG RGt. that contains rabbit polyclonal Anti-IgG and Murine monoclonal anti-C3d and anti-C3b
Rabbit/Murine monoclonal Blend
A monospecific AHG RGT that contains only antibodies reactive againts Human Gamma Chains
IgG Heavy Chain specific
What are the licensed monospecific AHG that are commonly use
Anti-IgG and anti-C3b-C3d
If Polyspecific AHG Rgt. reacts with IgA and IgM molecules what type of chain is responsible for this reaction
Light Chain Kappa and Lambda
Anti-IgG reagents contain antibodies specific to Fc fragment of what type of chain
Heavy Gamma Chain
A technology that can be used to produced monoclonal antiglobulin serum.
Hybridoma Technology
Are derived from one clone of plasma cells and recognize a single epitope
Monoclonal antibodies
Who devised the monoclonal antibody technique
Kohler and Milstein
The only IgG subclass that has no clinical significance in AHG test
IgG4
If large volume is needed in Polyclonal AHG technique what animal would you use?
Goat or Sheep
AHG reagents that contain only one antibody specificity either anti-IgG or antibody specific complement components such as C3b or C3d
Monospecific AHG RGT
In monoclonal AHG production antibody is collected as
Ascites
True or False . Monoclonal AHG production needs absorption to remove heteromorphic antibody
False it does not need Absorption
In monoclonal AHG production how can u give potent rgt. that give a fewer false positive reactions .
Monoclonal Antibodies to human complements Anti-C3b and Anti-C3d may be blended with POLYCLONAL anti-IgG from rabbits.
Animal use in Monoclonal AHG production
Mice
13 Most Antibody that fix complements
A-Le-Le-Sc1-Jk-Jk-Co3-Ge2-Ge3-li-Vel-P-PP1Pk
Considered to be the most important anticomplement component because of its limited capacity to cause nonspecific rxn.
Anti-C3c
AHG Rgt. should be dyed in what color for identification
Green
Serum to Cell ratio
2:1
Antibody to Antigen ratio
40:1
Indirect Antiglobulin Test is used in the ff. situation
Detection of incomplete(nonagglutinating) antibodies to potential donor RBCs ( compatibility testing ) or to screening cells ( antibody screen ) in serum
Determination of RBC phenotype using known anti sera
Titration of incomplete antibodies
Determinant of Immunoglobulin classes
CH3 portion of the heavy gamma chain
4 uses of Direct Antiglobulin Test
HDN
HTR
AIHA
How many times would you wash RBC
3 times
DAT can detect how many IgG and C3d
100-500 IgG molecules per RBC
400-1100 molecules C3d per RBC
IAT can detect how many IgG and C3d
100-200 IgG and C3 molecules per RBC
Factors affecting AGT
R R T I W S A C
Source of false positive in AGT
I A B C S D O P P C
3 types of Reaction mediums
22% Bovine Albumin
LISS
PEG
Recommended method for the evaluation of AHG centrifugation time
1000 RCF in 20seconds
Saline is buffered to a pH of _______
7.2 - 7.4
The usual incubation temperature for the IAT
37*C
The optimum temp for complement activation
37*C
Incubation time for LISS
10-15 mins
Incubation time using 22% bovine albumin
30 mins
are macromolecule additive used with LISS to bring sensitized RBCs closer to each other to facilitate cross-linking and agglutination rxn .
PEG and Polybrene
DAT incubation temperature
Room Temp 16-22*C
Is a process that detects RBC antigen-antibody rxn by means of using a chamber filled with polyacrylamide gel
Gel Test
It is a potent rouleaux forming reagent
Polybrene
enummerate 4 modified and automated AGT techniques
LIP
ELAGT
SPT
Gel Test
An anticoagulant used to collect samples for the DAT to avoid in vitro complement attachment associated with refrigerated clotted specimens
EDTA
In ELAGT what is the usual OD
405 nm
3 types of Gel Test
Neutral
Specific
Antiglobulin
IgG subclasses that strongly binds to the complement
IgG 1 and IgG 3
Drug induces Ab that binds to RBC only when drug is present in soluble form , unknown mechanis,
Type 3 (Drug Dependent)
Ab reacts with normal RBCs when soluble drug is present
Type3 (Drug Dependent)
Drug binds covalently to membrane proteins and stimulates hapten dependent Ab
Type 1 (Hapten Ab dependent)
Drug induces autoantibody specific for RBC membrane protein through unknown mechanism
Type 2 ( Autoantibody)
Type of Drug induce wherein Ab reacts with normal RBC in the absence of drug
Type 2
Autoantibody reacts with patients RBC in in vivo
WAIHA
Cold reactive IgM autoagglutinins binds to RBCs in peripheral circulation (32*C)
CAS
The IgG autoantibody reacts with colder part of body, causes complement to be bound irreversibly to RBCs and then elutes to warmer temp
PCH
IgM binds completely as RBCs return to warmer parts of the circulation
CAS
IgM dissociates leaving RBCs coated only with complement
CAS