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Polyspecific AHG Reagent (also called Measure volumes of red cells and
polyvalent or broad spectrum plasma/serum are added to reaction chamber
Coombs’ serum) of the microtube
Monospecific AHG Reaction chamber allows red cell
sensitization with antisera and gel particles
AABB Standards requirements for antibody
detection Centrifugation separates + and –
Coombs’ control cells agglutination results
+ results: agglutinated red cells are trapped
Analytical process
in gel at various levels
- results: unagglutinated red cells pass
through the gel and form a button of the
microtube
Gel antibody screening
+ reaction is adherence to the wells Optimal Temperature of Reactivity for Some Common
Antibodies
- reaction is a red cell button
Phase Room 37oC Antiglobulin
Temperature incubation phase
(Immediate
spin)
Antibodies Cold Potent cold Rh antibodies
autoantibodies antibodies K
(I, H, IH) (especially
Duffy
M, N if causing
hemolysis) Kidd
P1
D, E S, s
Lea, Leb
K Lub
Lua
Xga
Immunoglobulin IgM IgG IgG
class
Antibody Detection Limitations Clinically No Yes Yes
significant
1. Cell to Serum Ratio
Manipulations:
Prozone and Postzone Phenomena
Decreasing the temperature 24oC or even
to 4oC enhances the reaction of IgM
antibodies
Prewarming or maintaining a reaction
temperature of 37oC provides the optimum
temperature for detection of IgG
antibodies, while preventing most IgM
antibodies from reacting
3. Length of Incubation
Different Ag/Ab reactions reach
equilibrium over different time periods
Manipulation:
Increasing the time of incubation in a
Cell to Serum Ratio
given test system may enhance the
manipulations: increase the amount of detection of a weakly reactive antibody
serum (4 drops) – if no potentiator used
4. PH
o use potentiator or enhancement medium
At a pH range of 6.5 – 7.5, chemical
groups on Ag and Ab are oppositely
2. Temperature charged.
o influences the equilibrium constant and Manipulation:
or reaction rate depending on the class Changing the pH affects the
of antibody involved equilibrium constant for selected
o focus on a clinically significant antibody antibodies that may react
which generally reacts at 37oC preferentially in a more acidic
environment (e.g., anti-M, anti-Pr)
Temperature
1. 22% albumin: reduces zeta potential
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○ Each of the panel cells has been antigen typed ○ A tube is labeled for each of the panel
(shown on antigram) cells plus one tube for AC:
● + refers to the presence of the antigen
● 0 refers to the absence of the antigen
IS Phase
○ Perform immediate spin (IS) and grade
agglutination; inspect for hemolysis
○ Record the results in the appropriate
space as shown:
○ 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
(LISS) 37°C Phase
○ 2 drops of LISS are added, mixed and
incubated for 10-15 minutes
○ Centrifuge and check for agglutination
○ Record results
Panel
○ The same phases used in an antibody
screen are used in a panel
Antibody ID Testing
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2+ 0
0 0
0 0
2+
0
0
2+
0
2+
0
0
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Interpretation: Lea
Guidelines
○ Again, it’s important to look at:
● Autocontrol
○ Negative - alloantibody
○ Positive – autoantibody or DTR
(i.e.,alloantibodies)
○ DTR – delayed transfusion reaction
(donor cells are sensitized with
patient’s antibody)
● Phases
○ IS – cold (IgM)
○ 37° - cold (some have higher thermal
range) or warm reacting
○ AHG – warm (IgG)…significant!!
● Reaction strength
○ 1 consistent strength – one antibody
○ Different strengths – multiple
antibodies or dosage
About reaction strengths……
○ Strength of reaction may be due to “dosage”
● If panel cells are homozygous, a strong
reaction may be seen
● If panel cells are heterozygous, reaction
may be weak or even non-reactive
○ Panel cells that are heterozygous should
not be crossed out because antibody may
be too weak to react (see first example)
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Idiopathic
Known disorder (SLE, RA, leukemias,
UC, pregnancy, infectious diseases, etc)
Medications
Avoiding reactivity
○ Several techniques are used when warm
○ Cold autoantibodies can be a nuisance at autoantibodies are suspected…
times. Here are a few ways to avoid a Elution (whenever DAT is positive)
Elution (whenever DAT is positive)
reaction:
Elution
○ Elution techniques
techniques “free” from
“free” antibodies
○ Use anti-IgG AHG instead of polyspecific. antibodies from the sensitized
the sensitized red cells so that the
Most cold antibodies react with polyspecific red cells so that the antibodies
antibodiescan
can be
be identified
identified
AHG and anti-C AHG because they fix
complement
Y
Y
○ Skipping the IS phase avoids the attachment Elution
Sensitized
Y
Y
RBC
of cold autoantibodies to the red cells YY
Y
Y
○ Use 22% BSA instead of LISS Y
Positive DAT Frees antibody Antibody ID
Remove
serum and
test for
2 tubes alloantibody
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