1. Antiglobulin Test (Coomb’s Test)

2. The antiglobulin test
The antiglobulin test was first introduced into clinical medicine in 1945 by R.R. Coombs
He showed that it could be used to detect non-agglutinating red cell antibodies or sensitized red cells
Most non-agglutinating (incomplete) antibodies are IgG,
These antibodies do not spontaneously cause agglutination due to a strong electronegative charge on the red cell surface that prevents the cells from coming into close proximity.
The antiglobulin reagent is able to bridge these negative forces.

3. Antihuman Globulin Definition:
Antihuman: antibodies against human antigens
Globulin: all antibody molecules are globulins
Therefore: Antihuman Globulin is antibody directed against the Fc portion of human antibodies and/or complement components.

4. AHG Technique: What is the importance?
Some very clinically significant unexpected antibodies (eg. Kidd, Duffy) attach to red cell but do NOT cause agglutination at immediate spin or 37o phase.
Yet, these antibodies are capable of causing severe hemolytic transfusion reactions or hemolytic disease of the newborn.

5. ANTIGLOBULIN TEST
Detection of non-agglutinating Abs, especially IgG or complement components (C3) attached to RBCs in vivo or in vitro.
Direct antiglobulin test (DAT) - detection of sensitization of RBC s (coated with Abs and/or complement components) in vivo
Indirect antiglobulin test (IDAT) - detection of sensitization of RBCs (coated with Abs and/or complement components) in vitro

6. Antiglobulin Test
Principle - Antihuman globulins (AHG) from immunized animals bind to human globulins either free in serum or attached to RBCs
Pentameric IgM Abs are so large that, when bound to RBC Ags, the RBCs agglutinate (usually at RT)
IgG Abs usually need a little help, a bridge molecule, to agglutinate RBCs
AHG acts as a bridge molecule

7. Reagent Preparation Polyclonal or monoclonal sources
Polyclonal - Animals immunized with human globulins (IgG & complement (C3); bled for antisera to obtain high titered
Monoclonal - Hybridoma cells
To produce monoclonal antibodies, B-cells are removed from the spleen of an animal that has been challenged with the relevant antigen. These B-cells are then fused with myeloma tumor cells that can grow indefinitely in culture (myeloma is a B-cell cancer). This fusion is performed by making the cell membranes more permeable

8. Reagent Preparation Polyspecific AHG Monospecific AHG
Abs to human IgG, and
Abs to human C3d (C3b breaks down to C3c and C3d)
Advantage is that polyspecific AHG may detect complement-dependent Abs on RBCs
Disadvantage - more nuisance positives
Monospecific AHG
Abs to human IgG only or human C3d only
Fewer nuisance positives; may miss an important Ab

9. Antiglobulin Test

10. Direct Antiglobulin Test
Detects in vivo sensitization of RBCs
The DAT is used to detect IgG or C3 bound to the surface of the red cell.
In patients with hemolysis, the DAT is useful in determining whether there is an
Immune etiology
OR Non-immune etiology of hemolysis

11. Direct Antiglobulin Test

12. Non-immune causes of hemolysis
Mechanical hemolysis such as those due to artificial valves or burns,
Hemoglobinopathies (sickle cell, thalassemia),
Red cell enzyme deficiencies (G6PDP, pyruvate kinase),
and red cell membrane defects (hereditary spherocytosis, PNH)
Have a negative DAT
PNH = Paroxysmal nocturnal hemoglobinuria

13. DAT Uses Have a positive DAT Immune causes of hemolysis
Autoimmune hemolytic anemias,
Drug induced hemolysis,
Delayed or acute hemolytic transfusion
Hemolytic Disease of the new born
Have a positive DAT

14. Indirect Antiglobulin Test (IAT)
Detection of in vitro sensitization of RBCs
Most clinically significant alloantibodies are IgG antibodies that react best at 37oC and are formed as a result of previous exposure via transfusion or pregnancy.
Examples include antibodies to Rh, Kell, Kidd, and Duffy red cell antigens.
Same as DAT, except:
Step 1 entails incubating RBCs (reagent or unknown) with antisera (reagent or unknown) allowing time for in vitro attachment of Abs to RBCs

15. Indirect Antiglobulin Test

16. IAT Uses When the unknown is sera:
Ab Screening: The patient’s serum is incubated with red cells of known phenotype to detect Ab in patient’s serum against a specific red cell Ag
Phenotyping: Ab of known specificity is incubated with patient’s RBC to identify specific blood group Ag on the cells
Crossmatch: The patient’s serum is incubated with donor red cells to detect Abs that might reduce the survival of transfused red cells

17. Modifications of IAT
Modifications can be done to increase sensitivity & to decrease time to perform IAT
Modification of suspending media
Modification of RBCs
Each modification has advantages and disadvantages

18. Modification of suspending medium
Mechanism of action
Advantages
Disadvantages
Saline
Not applicable
Inexpensive
Long incubation time required
LISS
Facilitates sensitization but diminishes agglutination
Short incubation time
expensive
Albumin
Facilitates sensitization and agglutination
Short incubation time, enhances certain clinically significant Abs
Enhances the reaction caused by cold autoantibodies

19. Modification of RBCs RBCs used in the IAT can be treated with enzymes
This increases sensitivity of test for detection of certain auto & allo-antibodies including Rhesus & Kidd
Enzyme treatment destroy certain Abs including M, N, S & Duffy

20. Importance of Anti-complement in antiglobulin reagents
Anticomplement increases detection sensitivity for certain Abs (Anti-Kidd)
Approx. 200 molecules of cell bound IgG required for +ve antiglobulin test
Ab sensitization below this level will not be detected by anti-IgG alone
If antiglobulin reagent contains both anti-IgG & anti-C3b will increase probability of detection of sensitization

21. False Positive Reactions
Mechanism
Cold Autoantibody
“in vitro” complement fixation & auto-agglutination
Technical
Dirty tubes, colloidal silica
Anti-species Ab
Heterologous Ab in the antiglobulin reagent
Polyagglutinable red cells
Presence of anti-T or anti-Tn in the antiglobulin reagent
neuraminidase-treated red blood cells

22. False Negative Technical problems Failure to add reagent
Neutralization of the antiglobulin reagent due to insufficient washing of the test cells
Contamination or neutralization of antiglobulin reagent
False –ve test can be detected by addition of IgG sensitized cells to –ve antiglobulin tests. These cells should be agglutinated by the anti-IgG