Preparation and Standardization of Anti Human Globulin AHG Reagent

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ANTI-HUMAN GLOBULIN REAGENT (Coombs’ Sera)
• This is used to detect incomplete antibodies (IgG antibodies)
which are capable of sensitising red cells but cannot
agglutinate the same cells when suspended in saline as
against to IgM antibodies.
• The technique can be used to detect the presence of
antibodies or complement components that have been bound
to the red cells in vivo, i.e. the direct antiglobulin test (DAT,
direct Coombs’ test).
• The test can detect IgG antibodies in the patient’s serum.
Incubate the serum with ‘O’ cells. The antibodies or comple-
ment will coat on these cells. Wash the cells and continue
the procedure similar to DAT. This is indirect antiglobulin
test (IAT, indirect Coombs’ test).
PREPARATION OF AHG
Poly-specific AHG: Contains IgG and complement (C3b and
C3d).
Anti-human globulin is prepared by injecting laboratory
animals with human globulins or complements. Rabbits are
the most suitable animals for immunization. Sheep and goats
also can be used. This type of response produces polyclonal
antibodies to IgG and complements. Polyclonal antibodies are
mixture of antibodies produced from different clones of plasma
cells. The resulting antibodies recognize different antigenic
determinants (epitopes) week young rabbits with low titer of
heteroagglutinins are ideal. Purified human IgG and C3 are
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Preparation and Standardization
of Anti-Human Globulin (AHG)
Reagent
63

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64
Essentials of Blood Banking and Transfusion Medicine
injected. Alum precipitation method is commonly used.
Animals are bled after 9–10 days after last injection. The
antibodies produced are harvested.
Monospecific AHG: Contains only one antibody, may be anti-
IgG, anti-C3d or anti-C3b.
The monoclonal antibodies are produced from hybridoma
technology. Monoclonal antibodies are derived from one clone
of plasma cells and can recognize only single epitope.
This is prepared by immunizing mice with purified human
globulin or C3. After suitable immune response, the mouse
splenic lymphocytes which are capable of secreting immuno-
globulins are fused with myeloma cells. The hybridoma cells
are screened for antibodies with required specificity and
affinity.
Standardization of anti-human globulin reagent
• If IAT is undertaken with red cells suspended in normal ionic
strength saline (NISS), maximum antibody is up-taken and
hence maximum sensitivity is achieved in 60–90 minutes
incubation period.
• The incubation period can be reduced to 10–15 minutes by
using low ionic strength solutions (LISS). LISS–IAT is less
sensitive than NISS
– IAT for identifying antibodies like anti-K as the incuba-
tion period is reduced. LISS–IAT if used in the cold,
insignificant antibodies like anti-Pl, anti-Le can be
detected because of their increased uptake at low ionic
strength conditions. Hence while using LISS manu-
facturer’s instructions are followed strictly.
LISS solution: This consists of 0.3 M glycine in 0.03 M
sodium chloride. No buffer is added. The pH is 6.0.
• When IAT and DAT are performed in tubes, thorough
washing (3 to 4 times) with large volume of saline is required
before adding the antiglobulin reagent, as any free IgG or
complement will neutralise the antiglobulin reagent.
• Prozone phenomena can give negative results and this can
be eliminated by washing the cells after incubation with
AHG or doing the test after diluting the serum.

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Preparation and Standardization of AHG Reagent
65
• For the rare cases where the test is negative with poly-specific
sera, it is necessary to test with monospecific sera having
specific subclasses of anti-IgG, anti-C3b or anti-C3d.
Monospecific sera against IgM and subclasses of IgG also
can be prepared.
• In AHG anti-C4d should be avoided. So also, IgM antibodies
should not be present. Each batch of reagents undergoes
rigorous quality control at various stages of manufacture
for its specificity, avidity and titre.
Poly-specific antiglobulin reagent containing blend of anti-
IgG, anti-C3b and anti-C3d (anti-C3b and anti-C3d at
controlled levels) are suitable for pre-transfusion testing,
including crossmatch are recommended as per ISBT/ICSH
guidelines.
Reagent storage and stability
a. Store the reagent at 2–8°C. DO NOT FREEZE.
b. The shelf life of the reagent is as per the expiry date
mentioned on the reagent vial label.
The reagent contains sodium azide 0.1% as preservative.
Avoid contact with skin and mucosa. On disposal, flush with
large quantities of water.
Extreme turbidity may indicate microbial contamination or
denaturation of protein due to thermal damage. Such reagents
should be discarded.
Reagents are not from human source, hence contamination
due to HBsAg and HIV is practically excluded.
Sample collection and storage
No special preparation of the patient is required prior to sample
collection by approved techniques. Do not use haemolysed
samples.
For direct antiglobulin test blood drawn into EDTA is
preferred but oxalated or citrated blood also can be used. The
blood sample should be tested as soon as possible after
collection and should not be stored. Stored blood can have
C3d and Coombs’ test can be positive.