Transcript antigen - Global Healing

BASIC ANTIBODY
IDENTIFICATION
Jean Purcelli, MT (ASCP)SBB
Blood Centers of the Pacific
May 2010
Version 2 May 2012
There are two types of unexpected red blood cell
antibodies.
Alloantibodies which may occur as a result from:
Transfusion
Pregnancy
Transplantation
Injections of immunogenic material
I.V. or I.M. material such as IVIg or RhIg
(passively acquired)
Autoantibodies which occur with certain diseases or
medical treatments.
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Alloantibodies can be detected in about 0.3%-5% of the
general population.
The incidence increases if the transfused or previous
pregnancy populations are studied.
The incidence is affected by:
Antigenicity
Prevalence of the antigen in the population
Status of the immune system
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Anti-A and Anti-B are examples of naturally
occurring antibodies and they are expected.
Lewis (Anti-Lea, Leb), -P1, -M, -N are unexpected
antibodies, but can occur naturally.
Naturally occurring antibodies have resulted from
environmental, bacterial, or viral antigens that
are similar to blood group antigens.
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An Alloantibody is considered Clinically
Significant:
If it has caused hemolytic transfusion
reactions.
If it has caused marked decrease in the
survival of transfused red cells.
If it has caused hemolytic disease of the fetus
or newborn (HDFN).
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We concentrate on identifying 13 clinically significant
alloantibodies that are commonly found:
Rh system antibodies: D C E c e f
Kell system antibodies: K
Duffy system antibodies: Fya Fyb
Kidd system antibodies: Jka Jkb
MNSs system antibodies: S s sometimes M
(-M may be significant if it reacts at 37 C or by IAT)
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Clinically Insignificant
Several clinically insignificant alloantibodies and
autoantibodies are commonly found.
Alloanti-M (reacting only below 37ºC), -N, -P1, -Lea, Leb, generally react below 37ºC and usually are not
clinically significant.
It is usual to transfuse with pre-warmed IAT, crossmatch
compatible red cells. The IS crossmatch is dropped.
Autoanti-I and –IH are commonly found when testing is
conducted at room temperature or colder.
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Red Cell Panels and Master Sheets
A red cell panel is merely an expanded antibody detection set
with 8-10 cells designed to identify and rule out the
commonly found antibodies.
Make copies of both sides of the Master Lists when they arrive, a
new set will arrive every 2-4 weeks.
The panel sheets are used as work sheets.
The lot number of the panel should match the master sheet.
Use in-dated cells whenever possible.
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Panel Master Sheets
The panel is designed to have some homozygous expressions of
antigens, except P1. In addition K, Kpa, Jsa, and Lua antigens
rarely exist as homozygous expressions on a panel.
Next to the Vial Number is a column for Special Types.
There are columns for four High Frequency antigens: k, Kpb, Jsb,
Lub. Cells negative for these antigens rarely exist in panels.
There is a note in the lower left listing several more high
frequency antigens for which each cell was typed: I, Ge, Yta,
Tja, Vel, Coa, Dib.
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Panel Master Sheets
There are columns for five Low Frequency antigens: V,
Cw, Kpa, Jsa, Lua.
In addition, there is a note in the lower left corner
listing several more antigens of low frequency that
each cell has been typed and found negative for Mg,
Vw, Dia, Wra.
On the reverse side is another list of Extended Typings
and notations.
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PRE-TESTING PHASE
Obtain Medical and Transfusion History
• Age: may affect ABO typing.
• Sex
• Ethic origin: high prevalence antigens are lacking in certain
populations.
• Diagnosis: some diseases are associated with some
antibodies; warm and cold AIHA.
• Transfusions and Pregnancies: how many transfusions, when,
number of pregnancies
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PRE-TESTING PHASE
 Medications
 Intravenous solutions
 Check file for antibody history, difficulty in typing or
transfusion reaction or HDFN
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PLAN FOR TESTING PHASE
Each institution should develop a plan for antibody
identification.
A plan establishes consistency and a general guide to
antibody identification.
An example plan:
1. Use a specifically designed worksheet, panel
sheets, and selected cell panel sheets.
2. Obtain patient history.
3. Check files for previous work-up.
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PLAN FOR TESTING PHASE
Lab Testing
4.ABO-Rh
5.DAT
6.Initial Panel, use basic enhancement media
such as LISS.
7.A cold antibody screening.
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PLAN FOR TESTING PHASE
8. Establish identification and rule out other
possible antibodies in PEG/IAT.
9. Perform partial or extended phenotype if there
were no recent transfusions.
10.Other tests as necessary. Based on other
findings.
Steps 4-7 may be performed simultaneously.
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TEST SYSTEM
Most test systems include enhancement media
and antiglobulin reagent. Testing with panel
cells is conducted at 37ºC, examined for
agglutination or hemolysis and converted to
the antiglobulin test.
A separate cold screen with Screening Cells and
an auto control (in saline) offers valuable
information.
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THE ANALYTICAL PHASE
Exclude specificities with negative reactions using a cross-out method.
A single antibody usually produces a clear pattern.
• Both positive and negative reactions are important.
• Results should correspond with reactions found with the antibody
screening cells.
• Rule out other possible antibodies.
• A selected cell panel may be necessary.
• When the antibody(s) have been identified, the next step is to confirm the
identity by typing the patient’s red cells for the antigens.
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If the patient’s plasma is reactive with 2 cells positive
for the antigen and non-reactive with 2 cells negative
for the antigen the minimum requirement for
identification has been met.
Additional reactive antigen + cells and non-reactive
antigen = cells increase the probability of correct
identification of the antibody.
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To exclude other possible antibodies, at least
two cells with a homozgous expression of the
antigen should be non-reactive, whenever
possible.
The exclusion method by crossing out is a
tentative and imperfect method to use until
criteria have been met with a selected cell panel
using the most sensitive enhancement media.
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Rules for exclusion: Exceptions
1. Not all antigens exist as a homozygous
expression. P1,Cw, V, VS.
2. Many low frequency antigens are not readily
available as homozygous expressions: K, Jsa,
Kpa, Lua, etc.
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• More exceptions to exclusion with
homozygous cells:
• Anti-C when anti-e is present. Conclusion:
“cannot exclude possible anti-C”
• Anti-E when anti-c is present. Conclusion:
“cannot exclude possible anti-E”
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More exceptions to exclusion with homozygous
expression
• Anti-C when anti-D is present. OK to exclude C with r’r cells.
• Anti-E when anti-D is present. OK to exclude E with r”r cells.
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A possible antibody can also be ruled out by
typing for the antigen.
If a person’s cells type positive for the antigen,
that person will not make the corresponding
antibody.
Exceptions: There are many instances of D+
people producing anti-D and many autoantibodies show specificities in the Rh system,
particularly -e.
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• Monoclonal anti-Rh antisera show discrepant
results when the antibody is made from
different clones or sources.
• A C+ person may make antibody appearing to
have anti-C specificity. Is it an autoantibody or
is the patient C negative?
• It is advantageous to have more than 1 source
of antisera.
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In general a person who types negative for an
antigen is capable of producing the antibody.
Lewis system antibodies are an exception: Only
Le(a-b-) persons can make Lewis antibodies of
any specificity.
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If no discernable pattern is observed consider:
Multiple antibodies: D+C, or D+E, or E+K
An antibody that demonstrates dosage:
anti-M,-N,-S,-s,-Jka,-Jkb, -C, -E,-c,-e
Some antigens have variable expression in different
individuals: P1, I, “HTLA”
Unwanted reactions from cold or warm autoantibodies.
Refer to the DAT or autocontrol and the cold
antibody screen for clues.
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Multiple Antibodies
There are several different approaches to
resolving multiple antibodies :
•Phenotyping
•Selected Cell panel
•Neutralization
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Multiple antibodies
• Enzymes
• Adsorption
We will concentrate on the most efficient way:
a combination of phenotyping and using
selected cell panels.
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Multiple Antibodies
Extended phenotyping is expensive and
should be used sparingly.
From a phenotype, you learn which antibodies
are possible and which can be ruled out.
Then a selected cell panel is created and
tested.
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Selected Cell Panels
1. Use a selected panel worksheet.
2. Selected cells are chosen from other panel
or screening cells to confirm or eliminate
possible antibodies.
3. Chosen cells should be positive (+) for the
suspected antigen and negative (0) for
others.
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Selected Cell Panels
1. Choose homozygous expressions whenever
possible.
2. Record the (+) and (o) antigens and include
the panel lot number, the donor number and
the vial number for each cell chosen.
3. Test with PEG or LISS. Analyze results.
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Proteolytic Enzymes
Two stage techniques using either ficin or papain to
pre-treat red cells are the most useful.
Enzymes are used to enhance or destroy certain
blood group antigens.
Enzyme technique is useful in sorting out multiple
antibodies, enhancing weak antibodies, providing
clues to the identity of certain antibodies to high
prevalence antigens, and can be used in certain
adsorption procedures.
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Proteolytic Enzymes
Pre-treating panel cells takes about 10 minutes,
then the serum is added and placed at 37º for
30 minutes.
With papain, “pan-agglutination” is often seen
at the 37º stage and is disregarded if it is seen
in all cells, including the auto control.
Unwanted reactions due to cold allo or
autoantibodies and warm autoantibdies may
be enhanced.
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Antigens destroyed: M, N, S, Fya, Fyb.
Small s is variable and
should be QC’d with
anti-s.
Antigens enhanced: Rh: D, E, c, e, f, and
all Rh antigens. Jka,
Jkb. Lewis, I.
Antigens unchanged: K, k and all Kell
antigens.
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DAT POSITIVE
• The red cells were coated in vivo.
• In a transfusion reaction, the transfused cells have
become coated with immunoglobulins and the reactions
can be quite weak.
• In Warm Auto Immune Hemolytic Anemia, the red cells
are coated with IgG only or with both IgG and C3, and
the reactivity is often very strong.
• In Cold Agglutinin Disease, the red cells are coated with
C3.
• In HDFN, the mother’s antibody has crossed the
placenta and coated the fetus’ red cells.
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• IgG coated cells (positive DAT) cannot be typed with
antisera requiring the antiglobulin test.
• Monoclonal, direct agglutinating reagents reagents
can be used reliably.
• To test for Fya, Fyb, Jka, Jkb, S, s; the cells must be
treated first with glycine-HCI-EDTA (EgA) to remove
the bound IgG.
• Glycine-HCI-EDTA destroys all Kell antigens. To type
for K, a direct agglutinating monoclonal reagent can
be used on untreated red cells.
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An elution is performed and eluate tested if the
patient was recently transfused.
If the patient was not recently transfused, an
elution is not performed, unless requested by
a physician.
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AUTOANTIBODIES – COLD AND WARM
Cold autoantibodies are usually non-pathological and considered
“normal” if they react only at 4ºC.
Normal cold autoantibodies may show anti-I or anti-IH
specificity, but identity is not important.
Pathological cold autoantibodies have a wider thermal range,
reacting at RT, 37º, and at AHG.
Patients with Cold Agglutinin Disease have red cells coated with
complement components (C3).
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Pathological cold agglutinins may present
difficulty with proper cell typing. Warm a
bottle of saline to 37º in a water bath. Prewash the red cells several times with warmed
normal saline before typing.
Cold agglutinins with a wide thermal range may
require Cold Autoabsorption and use of Prewarmed saline techniques.
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Warm autoantibodies may cause slow or rapid
hemolysis.
Patients become severely anemic and may require
transfusions.
The DAT is positive for IgG or both IgG and C3.
The plasma reacts with all panel cells.
Warm autoantibodies may “mask” or obscure
underlying alloantibodies.
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DAT positive red cells can only be typed with monoclonal
reagents. To type antiserum requiring the antiglobulin
test, the red cells first have to be altered with GlycineHCI-EDTA.
Warm auto adsorption is used to remove autoantibody.
There are several methods available.
PEG Autoadsorption is the most efficient, most effective,
and least expensive.
Autoadsorption can be used only if the patient has not
been recently transfused.
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Post Identification
• Whenever allo- or autoantibody is detected or
identified, the full IAT crossmatch should be
performed at the hospital.
• At the blood center, screen red cells that are
“antigen negative” by performing full IAT
crossmatches first. (Optional, saves antisera).
• Select compatible units and type for the antigen
using manufactured antiserum of known potency.
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Post Identification
The unit(s) are labeled as antigen negative at
the blood center and shipped to the hospital.
When the antibody has been identified as
anti-P1, -Le , -Le, -M, -N it is usually not
necessary to type units for the antigen. A
prewarmed saline IAT crossmatch is
performed at the hospital.
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Post Identification
• Patient’s with warm autoantibodies will not
have compatible IAT crossmatches and the
units should be labeled as Incompatible at the
hospital.
• Physicians should be informed by the
laboratory Medical Director of a possible
increased risk of a transfusion reaction.
• Most patients respond well to transfusion and
do not have complications.
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Results of the antibody identification, difficulty
in typing, transfusion reactions, and special
needs should be kept on file in the hospital
Transfusion Service and the Blood Center
Antibodies may become undetectable over time,
but antigen negative units should continue to
be provided.
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