Chapter 5 Preparation of AHG

The Antiglobulin Test Polyclonal AHG production

Introduction
Antihuman globulins (AHGs) bind to human
globulins.
Source of AHGs
IgG or complement
Free in serum or attached to RBC
antigens
IgM and IgG blood group antibodies
IgG antibodies nonagglutinating/incomplete
History of the AHG Test (contd)
AHG Test may be used to detect RBCs
sensitized with
IgG alloantibodies
IgG autoantibodies
Complement components
History of the AHG Test
Prior to AHG Test, only IgM antibodies
detected
IgG antibodies nonagglutinating or incomplete
IgG monomeric structure, size
19451946, Coombs and coworkers
First used for Rh antibody detection
Later for other blood groups
Coombs procedure for AHG production
AHG able to detect in vivo and in vitro
sensitization of RBCs
In vivo: Direct Antiglobulin Test (DAT)
One-stage procedure
In vitro: Indirect Antiglobulin Test (IAT)
Two-stage procedure
Advantages and disadvantages
< Insert Figure 5-1>
Monoclonal AHG production
Advantages and disadvantages
< Insert Figure 5-2>
Antibodies Required in AHG
Anti-IgG activity must be present
Mixture of IgG1 and IgG3 subclasses
Other nonagglutinating antibodies
encountered rarely
Anti-complement activity
Complement components detected by AHG
Polyspecific vs. Monospecific AHG in the Indirect
Antiglobulin Test (IAT)
Most clinically significant antibodies are IgG
Subject of ongoing debate: advantages and
disadvantages in both approaches
Influence of use of LISS and albumin
Influence of complement activity
Principles of the Antiglobulin Test
Five basic principles of the Direct
Antiglobulin Test (DAT)
RBCS coated in vivo with IgG and/or
complement
Applications in: HDN, HTR, and AIHA
Sample should be collected in an
anticoagulant such as EDTA
The DAT Panel
Evaluation of a Positive DAT
The AABB Technical Manual relevant sections
Need for further testing in various clinical states

AHG Reagents The Indirect Antiglobulin Test (IAT)

Polyspecific AHG Detects in vitro sensitization of RBCs
Specificities Clinical applications in
Monospecific AHG Compatibility testing
Anti-IgG RBC phenotyping
Anti-Complement Titration of incomplete antibodies
 Factors Affecting the Antiglobulin Test
The DAT can detect a level of 100 to 500 IgG
molecules per RBC and 400 to 1100 molecules
of C3d per RBC.
For the IAT there must be between 100 and 200
IgG or C3 molecules on the cell to obtain a
positive reaction.
Ratio of serum to cells
Reaction medium: albumin, LISS, PEG
Temperature
Incubation time
Washing of RBCs
Saline for washing
Addition of AHG
Centrifugation for reading

Most Common Sources of Error in the AHG Test

Inadequate washing
Nonreactive AHG reagent
Failure to add AHG reagent
All negative antiglobulin test reactions must be
checked by the addition of IgG-sensitized cells.

Modified and Automated Antiglobulin Test

Techniques
Low ionic polybrene technique
Enzyme-linked antiglobulin test
Solid phase
Gel test

Comparison of AHG Methodologies

Goal is to detect all clinically significant
antibodies, both DAT and IAT types, and none
of the clinically insignificant antibodies, such
as warm- and cold-reacting autoantibodies.

Comparison of AHG Methodologies (contd)

DAT Methods
Comparison of tube and gel testing
techniques
IAT Methods
Comparison of tube and gel testing
techniques
Use of LISS and PEG