Transcript Antibody Detection

Antibody Detection
Mohammed Jaber
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
Antibody Screen
 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° 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
screening cells
is
incubated
with
 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 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
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-Cell Antigram
IS 37°
Some manufacturers will use
“+” instead of “1” to
indicate the presence of the
antigen
AHG
Screening Cells I & II
CC
Screening cells
 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
Fya
Fyb
SCI
+
0
4+
SCII
0
+
0
Fya
Fyb
SCI
+
+
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
 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)
 In this hospital:
Screen
If positive
Antibody
Panel (w/AC)
If positive
DAT
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)
Potentiators
Albumin
LISS
PEG
Polyethylene
glycol
Serum/cell mixture should incubate at least
20 minutes, 30 minutes preferred; doesn’t
enhance warm autoantibodies
Incubation time of 10 minutes; lowers
ionic strength allowing better reaction;
sensitive and quick!
Enhances warm autoantibodies; 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.
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 diluent does it increase uptake of
antibody onto cells?
 Many antibodies have enhanced
albumin is added to test system.
reactivity
when
Testing Techniques – LISS Tube
 Low Ionic Strength Saline shortens incubation time.
 Increases
antibody
agglutination.
uptake
onto
cell,
enhancing
 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,
manufacturer's recommendation for the
according
to
the
Procedure
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.
7. Centrifuge, then gently resuspend the cell button and read for
agglutination or hemolysis. Record results.
8. Fill all tubes with saline, centrifuge, and discard supernatant.
This is called washing, and it removes unbound IgG that
neutralizes the AHG reagent.
9. Repeat step 8 two or three times to remove unbound antibody
completely.
10.Add 2 drops of AHG to each tube (polyspecific or anti-IgG).
Procedure
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
questions:
should
consider
the
following
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?
Interpretation
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.
Interpretation
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.
Interpretation
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.
Interpretation
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 highmolecular-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.
Interpretation
 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
 Very effective in detecting antibodies
 If negative, then the crossmatch should be
compatible
Limitations
 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.
 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.
Limitations
 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
races‫عرق‬
 Get diagnosis, age, race, etc…
Example 1
Screening
Cell
I
IS
37°C
AHG
CC*
0
0
0

II
0
0
2+
ND
Not Done
• IgG antibody
• Single specificity
*Coombs Control Red Blood Cells (CC)
Example 2
Screening
Cell
IS
37°C
AHG
I
0
0
3+
II
0
2+
3+
• IgG antibody
• Multiple specificities
CC
Example 3
Screening
Cell
IS
37°C
AHG
CC
I
1+
0
0

II
3+
0
0

Neg AHG, add CC
• IgM antibody
• Single specificity showing dosage
Example 2
Screening
Cell
IS
37°C
AHG
I
0
0
2+
II
0
0
2+
• IgG antibody
• Allo- or autoantibody?
(don’t know without further testing)
CC