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.
2.
3.
Use a specifically designed worksheet, panel
sheets, and selected cell panel sheets.
Obtain patient history.
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 nonreactive 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
auto-antibodies 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 Pre-warmed 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 Glycine-HCI-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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