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ANTIBODY SCREENING

SAJEESH PATTAMMADATH
Antibody Detection
The test used to detect antibodies is called
an antibody screen
Antibody screens are used for:
Patients needing a transfusion
Pregnant women
Cases of transfusion reactions
Blood and plasma donors
Uses patients plasma/serum against reagent red
cells to detect unexpected antibodies
Unexpected antibodies are found in addition to the
expected anti-A and/or anti-B
Unexpected antibodies are a
result of red cell stimulation
(transfusion, HDN)
Unexpected antibodies may be:
Clinically significant (IgG)
Not clinically significant (IgM)
Clinically significant antibodies
Usually IgG
React best at 37 C and AHG phase (IAT)
Clinically significant antibodies are
associated with hemolytic transfusion
reactions (HTR) and hemolytic disease of the
newborn (HDN)
Performing an antibody screen
Patients plasma/serum is incubated with
screening cells
After incubation, an IAT is performed (indirect
antiglobulin test) using AHG reagent
This will detect any IgG antibodies.
Screening Cells
Screening cells are single or pooled donor
group O cells, however, single-donor vials
offer increased sensitivity
Why group O? so anti-A and anti-B wont
react
Screening cells come in sets of 2 or 3 vials
each
Each vial (donor) has been phenotyped for
each antigen
18 antigens are required on at least one of the
vials: D, C, E, c, e, M, N, S, s, P1, Lea, Leb, K, k,
Jka, Jkb, Fya, Fyb
Screening cells
Screening cells come with a sheet of paper called an
antigram
Screening cells are an already prepared 2-5% RBC
suspension
An antigram (2 or 3 cells) will list the antigens present
in each vial
A reaction to one or more cells indicates the
presence of an unexpected antibody
2 Cells Antigram
The technologist should be aware that some
antigens demonstrate dosage
An attempt should be made to used
screening cells that are homozygous for the
clinically significant antigens (Rh, Duffy,
Kidd). Just be aware that different strengths
can occur
Homozygous antigens will react stronger
Heterozygous antigens will react weaker
Examples
If patients serum contains
Fya Fyb anti-Fya, there will be a
SCI + 0 4+ stronger reaction because SCI
is homozygous for the Duffy
SCII 0 + 0 antigen

Fya Fyb In this case, the person has


anti-Fyb. The antibody reacts
SCI + + 2+
weaker with SCI
SCII 0 + 4+ (heterozygous) and stronger
with SCII (homozygous)
Screening Cells
Screening cells may also contain low
incidence antigens like V, Cw, and Kpa
The presence of these antigens is not
required for screening cells
Pretransfusion Screening
Screening for antibodies is normally performed prior
to blood transfusion to detect antibodies that react
at body temperature (37)
Colder reacting antibodies (RT and below) are
therefore considered insignificant and just cause
interference when performing lab testing
The only important thing to remember concerning
cold antibodies is that they may bind complement
if a persons body temperature becomes low
Open-heart surgery
Hypothermia
Autocontrol
Tests patient serum with their own red cells
Some labs may or may not perform an autocontrol
(AC) with the screendepends on the hospital
However, the AC should be run with the antibody
panel.
AC is incubated with the antibody screen (or
antibody panel)
If a lab uses an AC with the screen and it is positive,
they may run a DAT (patient cells + AHG) to detect
in vivo coating
Autocontrol
The AC and DAT can help in determining
whether the antibodies are directed against
the patients cells or transfused cells (allo or
autoantibody).

Screen
If Positive

Antibody
If Positive DAT
Panel (w/AC)
Potentiators
Used in antibody detection and
identification to enhance antigen-antibody
reaction
Saline (may only enhance if incubated long
time)
Low-ionic strength solution (LISS)common
Bovine serum albumin (BSA)
Polyethylene glycol (PEG)
Proteolytic enzymes (can destroy some antigens)
Albumin Serum/cell mixture should incubate at least
20 - 30 minutes;
doesnt enhance warm Autoantibodies
LISS Incubation time of 10 minutes;
lowers ionic strength allowing better
reaction; sensitive and quick!
PEG Enhances warm Autoantibodies;
Polyethylene glycol does not react well with insignificant
antibodies (IgM)
Testing Techniques Saline Tube

Simplest to perform.
Mix serum or plasma with saline suspended RBCs,
centrifuge and read, incubate at RT or 37C.
Used in crossmatching to detect ABO
incompatibility.
In antibody tests used to detect IgM antibodies
which react preferentially at RT: anti-M, -N, -P1, -Le
and I.
Rare examples of antibodies of other specificities
may be observed at RT but more often will be
reactive at 37C and/or AHG as well.
Bovine Albumin Tube
Utilized to enhance agglutination of IgG antibodies
since 1945.
Decreases amount of time required for incubation.
Controversy: Decrease zeta potential (affects
second stage of agglutination) or due to function
of ionic strength of albumin diluents does it
increase uptake of antibody onto cells?
Many antibodies have enhanced reactivity when
albumin is added to test system.
LISS Tube
Low Ionic Strength Saline shortens
incubation time.
Increases antibody uptake onto cell,
enhancing agglutination.
Several important factors to consider:
Incubation time and sensitivity
subsequent to AHG depends upon
desired ionic conditions.
PEG Tube
Polyethylene Glycol (PEG) is a water soluble, neutral
polymer which is an effective potentiator of antigen-
antibody reactions.
Advantages over albumin include:
Increases rate of detection of clinically significant
antibodies.
Decreases detection of clinically insignificant
antibodies.
May decrease need for other enhancement
techniques.
Procedure
Serum or plasma added to RBCs, perform IS.
Add PEG and incubate at 37C IS NOT READ AFTER 37C
Wash and add AHG.
Enzymes Tube
More appropriate for antibody ID than routine
testing.
GREATLY enhance reactivity of Rh antibodies.
CANNOT be only method used as M, N, S, Fy and
other antigens are destroyed, those antibody
specificities would not be detected.
Enzymes used include
Papain
Bromelain
Trypsin
Ficin MOST POPULAR
Procedure
Antibody screening tests using a test tube method are
performed in a variety of ways. AABB Standards requires
that these tests detect clinically significant antibodies and
that they include a 37C incubation and an AHG test.
Generally, testing includes the following steps:
1. Appropriately label each tube.
2. Add 2 drops of patient serum to each tube.
3. Add 1 drop of appropriate screening cells to each tube.
4. Centrifuge, then gently resuspend the cell button and
read for agglutination or hemolysis. Record results. It
should be noted that this step is optional because most
significant antibodies are IgG and do not cause
agglutination of saline-suspended RBCs.
5. Add 2 drops of enhancement reagent to each tube (may vary
with enhancement reagent used).
6. Incubate at 37C for 15 to 30 minutes, according to the
manufacturer's recommendation for the enhancement
reagent being used. During the incubation, antibody in the
patient serum will bind to antigens on the reagent RBC. This is
called the sensitization phase.
7. Centrifuge, then gently resuspend the cell button and read for
agglutination or hemolysis. Record results.
8. Wash 3-4 times, last step remove any excess of supernatant.
9. Add 2 drops of AHG to each tube (polyspecific or anti-IgG).
10. Centrifuge, then gently resuspend the cell button and read for
agglutination or hemolysis. Tests that are macroscopically
negative are usually checked for microscopic agglutination.
Record results.
11. Add 1 drop of Coombs control cells (or "check cells") to all
negative tests.
12. Centrifuge and read for agglutination. Repeat test if
agglutination is not observed.
Grading Reactions
Interpretation
Agglutination or hemolysis at any stage of testing is a
positive test result, indicating the need for antibody
identification studies. However, evaluation of the
antibody screen and autologous control results can
provide clues and give direction for the identification and
resolution of the antibody or antibodies.
The investigator should consider the following questions:
1. In what phase(s) did the reaction(s) occur?
2. Is the autologous control negative or positive?
3. Did more than one screening cell sample react, and, if
so, did they react at the same strength and phase?
4. Is hemolysis or mixed-field agglutination present?
5. Are the cells truly agglutinated, or is rouleaux present?
1. In what phase did the reaction occur?

Antibodies of the IgM class react best at low


temperatures and are capable of causing
agglutination of saline-suspended RBCs (immediate
spin reading). Antibodies of the IgG class react best
at the AHG phase. Of the commonly encountered
antibodies,
anti-N. anti-I, and anti-PI are frequently IgM,
whereas those directed against Rh. Kell. Kidd, and
Duffy antigens are usually IgG.
Lewis and M antibodies may be IgG, IgM, or a
mixture of both.
2. Is the autologous control negative or
positive?
A positive antibody screen and a negative
autologous control indicate that an
alloantibody has been detected.
A positive autologous control may indicate
the presence of autoantibodies or
antibodies to medications.
If the patient has been recently transfused,
the positive autologous control may be
caused by alloantibody coating circulating
donor RBCs.
3. Did more than one screening cell sample react, and, if
so, did they react at the same strength and phase?

More than one screening cell sample is positive


when the patient has multiple antibodies, when the
antibodies' corresponding antigen is found on more
than one screening cell, or when the patient's serum
contains an autoantibody. A single antibody
specificity should be suspected when all cells react
at the same phase and strength. Multiple antibodies
are most likely when cells react at different phases
and strengths, and autoantibodies are suspected
when the autologous control is positive.
4. Is hemolysis or mixed-field
agglutination present?
Certain antibodies-such as anti-Lea, anti-
Leb, anti P+P1+Pk, and anti-Vel are known
to cause in vitro hemolysis.
Mixed-field agglutination is associated with
anti-Sda and Lutheran antibodies.
5. Are the cells truly agglutinated, or is
rouleaux present?
Serum from patients with altered albumin-to-globulin ratios
(e.g., patients with multiple myeloma) or who have
received high-molecular-weight plasma expanders (e.g..
dextran) may cause nonspecific aggregation of RBCs,
known as rouleaux.
Rouleaux is not a significant finding in antibody screening
tests, but it is easily confused with antibody-mediated
agglutination.
Knowledge of the following characteristics of rouleaux
helps in differentiation between rouleaux and
agglutination:
a. Cells have a "stacked coin" appearance when viewed
microscopically.
c. Rouleaux does not interfere with the AHG phase of testing
because the patient's serum is washed away prior to the
addition of the AHG reagent.
d. Unlike agglutination, rouleaux is dispersed by the addition
of 1 to 3 drops of saline to the test tube.
Limitations
Very effective in detecting antibodies
If negative, then the crossmatch should be
compatible
Antibody screening tests are designed to detect
significant RBC antibodies, but they cannot detect all
such antibodies. Antigens with frequencies of less
than 10 percent (e.g., Cw. Lu-, Kpa) are not usually
represented on screening cells, and, as a result, their
corresponding antibodies are not detected in routine
screening tests.
Antibody screening tests may also yield negative
results when the titer or concentration of antibody
drops below detectable limits.
Re exposure to the RBC antigen will elicit a secondary
immune response, resulting in a dramatic increase in the
antibody titer and possible immunologic destruction of
the transfused RBCs. this is called a delayed hemolytic
transfusion reaction (DHTR) because it occurs days or
weeks after the transfusion.
The student should keep in mind that proper
performance and interpretation of antibody detection
tests minimize the risk of DHTRs.
Patient History
GET THE HISTORY!!
Mixed red cell populations from a previous
transfusion can remain for up to 3 months
Patient may have come from another hospital
Some diseases are associated with antibodies
Some antibodies occur at a higher frequency
in some races
Get diagnosis, age, race, etc
Example 1

Screening IS 37C AHG CC*


Cell
I 0 0 0
II 0 0 2+ ND

IgG antibody
Single specificity

CC: Coombs Control Red Blood Cells


ND: Not Done
Example 2
Screening Cell IS 37C AHG CC

I 0 0 3+
II 0 2+ 3+

IgG antibody
Multiple specificities
Example 3
Screening Cell IS 37C AHG CC

I 1+ 0 0
II 3+ 0 0
Neg AHG, add CC
IgM antibody
Single specificity showing dosage
Example 4
Screening Cell IS 37C AHG CC

I 0 0 2+
II 0 0 2+

IgG antibody
Allo or autoantibody?
(dont know without further testing)

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