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Ab Identification Renee Wilkins
Ab Identification Renee Wilkins
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
An antibody panel is just an
extended version of an antibody
screen
The screen only uses 2-3 cells:
Antibody Panel
An antibody panel usually includes
at least 10 panel cells:
Panel
Group O red blood cells
Panel
Each of the panel cells has been
antigen typed (shown on antigram)
+ refers to the presence of the antigen
0 refers to the absence of the antigen
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
The same phases used in an
antibody screen are used in a panel
IS
37
AHG
Antibody ID Testing
A tube is labeled for each of the
panel cells plus one tube for AC:
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
Perform immediate spin (IS) and
grade agglutination; inspect for
hemolysis
Record the results in the
appropriate space as shown:
2+
0
0
Last
tube
(LISS) 37C Phase
2 drops of LISS are added, mixed
and incubated for 10-15 minutes
Centrifuge and check for
agglutination
Record results
(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
Wash cells 3 times with saline
(manual or automated)
Add 2 drops of AHG and gently mix
Centrifuge
Read
Record reactions
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
There are a few basic steps to
follow when interpreting panels
1. Ruling out means crossing out
antigens that did not react
2. Circle the antigens that are not
crossed out
3. Consider antibodys usual reactivity
4. Look for a matching pattern
Always remember:
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?
Chemical treatment
Proteolytic enzymes
Sulfhydryl reagents
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
Every cell should be positive for
each of the antibodies and negative
for the remaining antibodies
For example:
Lets say you ran a panel and identified
3 different antibodies: anti-S, anti-Jka,
and anti-P1
Selected cells could help
Selected Cells
#5 0 + 0 0 0 3+
#8 0 0 + 0 0 0
Anti-K
Group A
individual with
cold autoanti-IH
RBC
YY
Y
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
Acid elutions (glycine acid)
Most common
Lowers pH, causing antibody to
dissociate
Organic solvents (ether, chloroform)
Dissolve bilipid layer of RBC
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