Transcript Antibody Detection

Practical
Blood Bank
Antibody Screening
Antibody Detection
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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
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Unexpected antibodies are a result of red
cell stimulation (transfusion, HDN)
Unexpected antibodies may be:
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Clinically significant (IgG)
Not clinically significant (IgM)
Antibody Screen
Clinically significant antibodies
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Usually IgG
React best at 37° 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
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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
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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 won’t 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
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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
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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
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Homozygous antigens will react stronger
Heterozygous antigens will react weaker
Examples
Fya
Fyb
SCI
+
0
4+
SCII
0
+
0
SCI
Fya
+
Fyb
+
2+
SCII
0
+
4+
If patient’s serum contains
anti-Fya, there will be a
stronger reaction because SCI
is homozygous for the Duffy
antigen
In this case, the person has
anti-Fyb. The antibody reacts
weaker with SCI
(heterozygous) and stronger
with SCII (homozygous)
Screening Cells
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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 screen…depends on the hospital
 However, the AC should be run with the antibody
panel…we’ll discuss this later
 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 patient’s cells or transfused cells (allo or
autoantibody).
Antibody
Panel (w/AC)
If Positive
Screen
If Positive
DAT
Potentiators
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Used in antibody detection and
identification to enhance antigen-antibody
reaction
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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)
Potentiators
Serum/cell mixture should incubate at least
20 - 30 minutes;
doesn’t enhance warm autoantibodies
Incubation time of 10 minutes;
LISS
lowers ionic strength allowing better reaction;
sensitive and quick!
Enhances warm autoantibodies;
PEG
Polyethylene glycol does not react well with insignificant
antibodies (IgM)
Albumin
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.
Testing Techniques – 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.
Testing Techniques – 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.
 Adding additional serum will increase ionic
strength, must not be done.
 MUST adhere to manufacturer’s instructions.
Testing Techniques – PEG Tube
 Polyethylene Glycol (PEG) is a water soluble, neutral
polymer which is an effective potentiator of antigenantibody 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.
Testing Techniques – 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. American Association of Blood
Banks Standards requires that these tests detect clinically
significant antibodies and that they include a 37°C 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 37°C for 15 to 30 minutes, according to the
manufacturer's recommendation for the
7. Incubate at 37°C 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.
8. Centrifuge, then gently resuspend the cell button and read
for agglutination or hemolysis. Record results.
9. Fill all tubes with saline, centrifuge, and discard supernatant.
This is called washing, and it removes unbound IgG that
neutralizes the AHG reagent.
10. Repeat step 8 two or three times to remove unbound
antibody completely.
11. Add 2 drops of AHG to each tube (polyspecific or anti-IgG).
11. 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.
12. Add 1 drop of Coombs control cells (or "check cells") to all
negative tests.
13. 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(s) did the reaction(s) 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.
Interpretation
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. Evaluation of samples with positive autologous control
or DAT results is often complex and may require a lot of time
and experience on the part of the investigator.
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.
Figure provides several examples of antibody screen results
with possible causes.
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. Mixedfield 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 (see Color Plate ).
b.
Rouleaux is observed in all tests containing the patient's
serum, including the autologous control and the reverse
ABO typing.
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
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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.
Limitations
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antibody levels decrease over time when the individual
is no longer exposed to the corresponding antigen. If the
level of an RBC antibody drops too low, results of
antibody screening tests and crossmatches will appear
negative and may lead to transfusion of donor units that
carry the corresponding antigen.
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
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GET THE HISTORY!!
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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
Cell
I
IS
37°C
AHG
CC*
0
0
0
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II
0
0
2+
ND
• IgG antibody
• Single specificity
• CC: Coombs Control Red Blood Cells
• ND: Not Done
Example 2
Screening Cell
IS
37°C
AHG
I
II
0
0
0
2+
3+
3+
• IgG antibody
• Multiple specificities
CC
Example 3
Screening Cell
IS
37°C
AHG
CC
I
II
1+
3+
0
0
0
0
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• IgM antibody
• Single specificity showing dosage
Neg AHG, add CC
Example 4
Screening Cell
IS
37°C
AHG
I
II
0
0
0
0
2+
2+
• IgG antibody
• Allo or autoantibody?
(don’t know without further testing)
CC