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As you recall,
Antibody Screens use 2 or 3 Screening
Cells to detect if antibodies are
present in the serum
If antibodies are detected, they must
be identified
present
Not present
Why do we need to identify?
Antibody identification is needed for
transfusion purposes and is an
important component of
compatibility testing
It will identify any unexpected
antibodies in the patients serum
If a person with an antibody is
exposed to donor cells with the
corresponding antigen, serious side
effects can occur
Key Concepts
Known: Unknown:
Reagent RBCs + patient serum
Reagent antisera + patient RBCs
Panel cells
Antibody identification
At least 10 vials per set
Antibody Panel vs. Screen
Example: Panel Cell #10 has 9 antigens present: c, e, f, M, s, Leb, k, Fya, and Jka
Panel
An autocontrol should also be run
with ALL panels
Autocontrol
Patient RBCs
+
Patient serum
Panel
IS
37
AHG
Antibody ID Testing
1 2 3 4 5 6 7 8 9 10 11
AC
1 drop of each panel cell
+
2 drops of the patients serum
IS Phase
2+
0
0
Last
tube
(LISS) 37C Phase
2+ 0
0 0
0 0
2+
0
0
2+
0
2+
0
0
IAT Phase (or AHG)
Indirect Antiglobulin Test (IAT)
were testing whether or not
possible antibodies in patients
serum will react with RBCs in vitro
To do this we use the Anti-Human
Globulin reagent (AHG)
Polyspecific
Anti-IgG
Anti-complement
AHG Phase
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
0 0 0
And dont forget.
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
You have agglutinationnow what?
CC
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
0 0 0
??
Interpreting Antibody Panels
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
0 0 0
3. Consider antibodys usual reactivity
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
0 0 0
anti-
Lea
Guidelines
Again, its important to look at:
Autocontrol
Negative - alloantibody
Phases
IS cold (IgM)
Reaction strength
1 consistent strength one antibody
2+ 0 0
0 0 0
3 Positive 0 0 0
cells
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
3 Negative 2+ 0 0
cells 0 0 0
0 0 0
0 0 0
Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin
Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at immediate spin
What if the rule of three is not fulfilled?
ZZAP
Selected Cells
Selected cells are chosen from other
panel or screening cells to confirm
or eliminate the antibody
The cells are selected from other
panels because of their
characteristics
The number of selected cells
needed depends on how may
antibodies are identified
Selected Cells
#5 0 + 0 0 0 3+
#8 0 0 + 0 0 0
One-stage
Enzyme is added directly to the
serum/cell mixture
Two-stage
Panel cells are pre-treated with
enzyme, incubated and washed
Patient serum is added to panel cells
and tested
Enzyme techniques
Anti-K
A combination of proteolytic
enzymes and DTT
Denatures Kell, M, N, S, Duffy and
other less frequent blood group
antigens
Does not denature the Kx antigen
Good for adsorption techniques
frees autoantibody off patients cell, so that
autoantibody can then be adsorbed onto another
RBC
Autoantibodies.
Group A
individual with
cold autoanti-IH
RBC
Y
Y
Positive DAT Frees antibody Antibody ID
Elution
The eluate is a term used for the
removed antibodies
Testing the eluate is useful in
investigations of positive DATs
HDN
Transfusion reactions
Autoimmune disease
The red cells can also be used after
elution for RBC phenotyping if needed
When tested with panel cells, the eluate
usually remains reactive with all cells if a
warm autoantibody is present
Elution Methods
ABO
Heat (conformational change)
antibodies
Freeze-Thaw (lyses cells)
Adsorption
Adsorption procedures can be used
to investigate underlying
alloantibodies
ZZAP or chloroquine
diphosphate can be used to
dissociate IgG antibodies from the
RBC (may take several repeats)
After the patient RBCs are
incubated, the adsorbed serum is
tested with panel cells to ID the
alloantibody (if present)
Adsorption
Two types:
Autoadsorption
No recent transfusion
Autoantibodies are removed using patient
RBCs, so alloantibodies can be identified
Allogenic (Differential) adsorption
If recently transfused
Uses other cells with the patients serum
Remove
serum and
test for
2 alloantibody
tubes