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Antibody Identification

Renee Wilkins, PhD, MLS(ASCP)cm


CLS 325/435
School of Health Related Professions
University of Mississippi Medical Center
The Basics..

As you recall,
Antibody Screens use 2 or 3 Screening
Cells to detect if antibodies are
present in the serum
If antibodies are detected, they must
be identified

present

Not present
Why do we need to identify?
Antibody identification is needed for
transfusion purposes and is an
important component of
compatibility testing
It will identify any unexpected
antibodies in the patients serum
If a person with an antibody is
exposed to donor cells with the
corresponding antigen, serious side
effects can occur
Key Concepts

In blood banking, we test knowns with


unknowns

Known: Unknown:
Reagent RBCs + patient serum
Reagent antisera + patient RBCs

When detecting and/or identifying


antibodies, we test patient serum
(unknown) with reagent RBCs (known)
Reagent RBCs

Screening Cells and Panel Cells are


the same with minor differences:
Screening cells
Antibody detection
Sets of 2 or 3 vials

Panel cells
Antibody identification
At least 10 vials per set
Antibody Panel vs. Screen

An antibody panel is just an


extended version of an antibody
screen
The screen only uses 2-3 cells:
Antibody Panel

An antibody panel usually includes


at least 10 panel cells:
Panel

Group O red blood cells


Panel
Each of the panel cells has been
antigen typed (shown on antigram)
+ refers to the presence of the antigen
0 refers to the absence of the antigen

Example: Panel Cell #10 has 9 antigens present: c, e, f, M, s, Leb, k, Fya, and Jka
Panel
An autocontrol should also be run
with ALL panels

Autocontrol
Patient RBCs
+
Patient serum
Panel

The same phases used in an


antibody screen are used in a panel

IS
37
AHG
Antibody ID Testing

A tube is labeled for each of the


panel cells plus one tube for AC:

1 2 3 4 5 6 7 8 9 10 11

AC
1 drop of each panel cell
+
2 drops of the patients serum
IS Phase

Perform immediate spin (IS) and


grade agglutination; inspect for
hemolysis
Record the results in the
appropriate space as shown:

2+
0
0

Last
tube
(LISS) 37C Phase

2 drops of LISS are added, mixed


and incubated for 10-15 minutes
Centrifuge and check for
agglutination
Record results
(LISS) 37C Phase

2+ 0
0 0
0 0
2+
0
0
2+
0
2+
0
0
IAT Phase (or AHG)
Indirect Antiglobulin Test (IAT)
were testing whether or not
possible antibodies in patients
serum will react with RBCs in vitro
To do this we use the Anti-Human
Globulin reagent (AHG)
Polyspecific
Anti-IgG
Anti-complement
AHG Phase

Wash cells 3 times with saline


(manual or automated)
Add 2 drops of AHG and gently mix
Centrifuge
Read
Record reactions
AHG Phase

2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
0 0 0
And dont forget.

.add check cells to


any negative AHG !
IS LISS AHG CC
37
2+ 0 0 All cells are
negative at
0 0 0
AHG, so
0 0 0 add
2+ 0 0 Check
0 0 0 Cells

0 0 0
2+ 0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
You have agglutinationnow what?

CC

2+ 0 0
0 0 0

0 0 0

2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0

2+ 0 0
0 0 0
0 0 0
0 0 0

??
Interpreting Antibody Panels

There are a few basic steps to


follow when interpreting panels
1. Ruling out means crossing out
antigens that did not react
2. Circle the antigens that are not
crossed out
3. Consider antibodys usual reactivity
4. Look for a matching pattern
Always remember:

An antibody will only react


with cells that have the
corresponding antigen;
antibodies will not react with
cells that do not have the
antigen
Heres an example:
1. Ruling Out

2+ 0 0
0 0 0

0 0 0

2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0

2+ 0 0
0 0 0
0 0 0
0 0 0

Cross out antigens that show NO REACTION in any phase; do


NOT cross out heterozygous antigens that show dosage.
2. Circle antigens not crossed out

2+ 0 0
0 0 0

0 0 0

2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0

2+ 0 0
0 0 0
0 0 0
0 0 0
3. Consider antibodys usual reactivity

2+ 0 0
0 0 0

0 0 0

2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0

2+ 0 0
0 0 0
0 0 0
0 0 0

Lea is normally a Cold-Reacting antibody (IgM), so it makes


sense that we see the reaction in the IS phase of testing;
The E antigen will usually react at warmer temperatures
4. Look for a matching pattern
E doesnt match and
its a warmer rx Ab

2+ 0 0
0 0 0

0 0 0

2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0

2+ 0 0
0 0 0
0 0 0
0 0 0

Yes, there is a matching pattern!


Interpretation

anti-
Lea
Guidelines
Again, its important to look at:
Autocontrol
Negative - alloantibody

Positive autoantibody or DTR (i.e.,alloantibodies)

Phases
IS cold (IgM)

37 - cold (some have higher thermal range) or


warm reacting
AHG warm (IgG)significant!!

Reaction strength
1 consistent strength one antibody

Different strengths multiple antibodies or dosage


About reaction strengths
Strength of reaction may be due to
dosage
If panel cells are homozygous, a strong
reaction may be seen
If panel cells are heterozygous,
reaction may be weak or even non-
reactive
Panel cells that are heterozygous
should not be crossed out because
antibody may be too weak to react
(see first example)
Guidelines (continued)

Matching the pattern


Single antibodies usually shows a
pattern that matches one of the
antigens (see previous panel example)
Multiple antibodies are more difficult to
match because they often show mixed
reaction strengths
Rule of three

The rule of three must be met to


confirm the presence of the antibody
A p-value 0.05 must be observed
This gives a 95% confidence interval
How is it demonstrated?
Patient serum MUST be:
Positive with 3 cells with the antigen
Negative with 3 cells without the antigen
Our previous example fulfills the
rule of three

2+ 0 0
0 0 0
3 Positive 0 0 0
cells
2+ 0 0
0 0 0
0 0 0
2+ 0 0
0 0 0
3 Negative 2+ 0 0
cells 0 0 0
0 0 0
0 0 0

Panel Cells 1, 4, and 7 are positive for the antigen and gave a reaction at immediate spin
Panel Cells 8, 10, and 11 are negative for the antigen and did not give a reaction at immediate spin
What if the rule of three is not fulfilled?

If there are not enough cells in the


panel to fulfill the rule, then
additional cells from another panel
could be used
Most labs carry different lot
numbers of panel cells
Phenotyping

In addition to the rule of three,


antigen typing the patient red cells
can also confirm an antibody
How is this done?
Only perform this if the patient has NOT
been recently transfused (donor cells
could react)
If reagent antisera (of the suspected
antibody) is added to the patient RBCs, a
negative reaction should resultWhy?
Remember Landsteiners Rule

Individuals DO NOT make


allo-antibodies against
antigens they have
Multiple antibodies

Multiple antibodies may be more of


a challenge than a single antibody
Why?
Reaction strengths can vary
Matching the pattern is difficult
So what is a tech to do?

Several procedures can be


performed to identify multiple
antibodies
Selected Cells
Neutralization
Chemical treatment
Proteolytic enzymes
Sulfhydryl reagents

ZZAP
Selected Cells
Selected cells are chosen from other
panel or screening cells to confirm
or eliminate the antibody
The cells are selected from other
panels because of their
characteristics
The number of selected cells
needed depends on how may
antibodies are identified
Selected Cells

Every cell should be positive for


each of the antibodies and negative
for the remaining antibodies
For example:
Lets say you ran a panel and identified
3 different antibodies: anti-S, anti-Jka,
and anti-P1
Selected cells could help
Selected Cells

Selected S Jka P1 IS LISS AHG


cells 37
#1 + 0 0 0 0 2+

#5 0 + 0 0 0 3+

#8 0 0 + 0 0 0

These results show that instead of 3 antibodies, there


are actually 2: anti-S and anti-Jka
Neutralization

Some antibodies may be neutralized


as a way of confirmation
Commercial substances bind to
the antibodies in the patient serum,
causing them to show no reaction
when tested with the corresponding
antigen (in panel)
Neutralization
Manufacturers directions should be
followed and a dilutional control
should always be used
The control contains saline and serum
(no substance) and should remain
positive
A control shows that a loss of reactivity
is due to the neutralization and not to
the dilution of the antibody strength
when the substance is added
Neutralization
Common substances
P1 substance (sometimes derived from hydatid
cyst fluid)
Lea and Leb substance (soluble antigen found
in plasma and saliva)
I substance can be found in breast milk
Sda substance derived from human or guinea
pig urine

**you should be aware that many of these


substances neutralize COLD antibodies; Cold
antibodies can sometimes mask more clinically
significant antibodies (IgG), an important reason
to use neutralization techniques
Enzymes (proteolytic)
Can be used to enhance or destroy
certain blood group antigens
Several enzymes exist:
Ficin (figs)
Bromelin (pineapple)
Papain (papaya)
In addition, enzyme procedures
may be
One-step
Two-step
Enzymes

Enzymes remove the sialic acid


from the RBC membrane, thus
destroying it and allowing other
antigens to be enhanced
Antigens destroyed: M, N, S, s,
Duffy
Antigens enhanced: Rh, Kidd,
Lewis, I, and P
Enzyme techniques

One-stage
Enzyme is added directly to the
serum/cell mixture
Two-stage
Panel cells are pre-treated with
enzyme, incubated and washed
Patient serum is added to panel cells
and tested
Enzyme techniques

If there is no agglutination after


treatment, then it is assumed the
enzymes destroyed the antigen
Enzyme

Enzyme treatment treament

Anti-K

Perfect match for anti-Fya

Duffy antigens destroyed


Kell antigens not affected
Sulfhydryl Reagents

Cleave the disulfide bonds of IgM


molecules and help differentiate
between IgM and IgG antibodies
Good to use when you have both
IgG and IgM antibodies (warm/cold)
Dithiothreitol (DTT) is a thiol and will
denature Kell antigens
2-mercaptoethanol (2-ME)
ZZAP

A combination of proteolytic
enzymes and DTT
Denatures Kell, M, N, S, Duffy and
other less frequent blood group
antigens
Does not denature the Kx antigen
Good for adsorption techniques
frees autoantibody off patients cell, so that
autoantibody can then be adsorbed onto another
RBC
Autoantibodies.

Warm & Cold Reacting


Autoantibodies
Autoantibodies can be cold or
warm reacting
A positive autocontrol or DAT may
indicate that an auto-antibody is
present
Sometimes the autocontrol may be
positive, but the antibody screening
may be negative, meaning
something is coating the RBC
Getting a positive DAT

We have focused a lot on the IAT


used in antibody screening and ID,
but what about the DAT?
The direct antiglobulin test
(DAT) tests for the in vivo coating
of RBCs with antibody (in the body)
AHG is added to washed patient red
cells to determine this
What can the DAT tell us?
Although not always performed in
routine pretransfusion testing, a
positive DAT can offer valuable
information
If the patient has been transfused, the
patient may have an alloantibody
coating the transfused cells
If the patient has NOT been transfused,
the patient may have an
autoantibody coating their own cells
Identifying autoantibodies

Auto-antibodies can sometimes


mask clinically significant allo-
antibodies, so its important to
differentiate between auto- and
allo-antibodies
Cold autoantibodies

React at room temperature with


most (if not all) of the panel cells
and give a positive autocontrol
The DAT is usually positive with
anti-C3 AHG (detects complement)
Could be due to Mycoplasma
pneumoniae, infectious mono, or
cold agglutinin disease
Cold autoantibodies

Mini-cold panels can be used to help


identify cold autoantibodies
Since anti-I is a common
autoantibody, cord blood cells (no I
antigen) are usually included
Group O
individual with
cold autoanti-I

Group A
individual with
cold autoanti-IH

Anti-IH is reacting weakly with the cord


cells (some H antigen present)
Avoiding reactivity
Cold autoantibodies can be a
nuisance at times. Here are a few
ways to avoid a reaction:
Use anti-IgG AHG instead of
polyspecific. Most cold antibodies react
with polyspecific AHG and anti-C AHG
because they fix complement
Skipping the IS phase avoids the
attachment of cold autoantibodies to
the red cells
Use 22% BSA instead of LISS
Other techniques
If the antibodies remain, then
prewarmed techniques can be
performed:
Red cells, serum, and saline are
incubated at 37 before being combined
Autoadsorption is another
technique in which the autoantibody
is removed from the patients serum
using their own red cells
The serum can be used to identify any
underlying alloantibodies
Warm autoantibodies

More common that cold


autoantibodies
Positive DAT due to IgG antibodies
coating the red cell
Again, the majority of panel or
screening cells will be positive
The Rh system (e antigen) seems to
be the main target although others
occur
Warm autoantibodies
Cause warm autoimmune hemolytic
anemia (WAIHA)H&H
How do you get a warm autoantibody?
Idiopathic
Known disorder (SLE, RA, leukemias, UC,
pregnancy, infectious diseases, etc)
Medications
Several techniques are used when
warm autoantibodies are suspected
Elution (whenever DAT is positive)

Elution techniques free


antibodies from the sensitized
red cells so that the antibodies
can be identified
Y
Elution
Sensitized
Y
Y

RBC
Y
Y
Positive DAT Frees antibody Antibody ID
Elution
The eluate is a term used for the
removed antibodies
Testing the eluate is useful in
investigations of positive DATs
HDN
Transfusion reactions
Autoimmune disease
The red cells can also be used after
elution for RBC phenotyping if needed
When tested with panel cells, the eluate
usually remains reactive with all cells if a
warm autoantibody is present
Elution Methods

Acid elutions (glycine acid)


Most common
Lowers pH, causing antibody to
dissociate
Organic solvents (ether, chloroform)
Dissolve bilipid layer of RBC

ABO
Heat (conformational change)
antibodies
Freeze-Thaw (lyses cells)
Adsorption
Adsorption procedures can be used
to investigate underlying
alloantibodies
ZZAP or chloroquine
diphosphate can be used to
dissociate IgG antibodies from the
RBC (may take several repeats)
After the patient RBCs are
incubated, the adsorbed serum is
tested with panel cells to ID the
alloantibody (if present)
Adsorption

Two types:
Autoadsorption
No recent transfusion
Autoantibodies are removed using patient
RBCs, so alloantibodies can be identified
Allogenic (Differential) adsorption
If recently transfused
Uses other cells with the patients serum
Remove
serum and
test for
2 alloantibody
tubes

Wash x3 after Centrifuge after


incubation incubating; and
transfer serum to 2nd
tube of treated cells;
incubate and
centrifuge again
More reagents.

Many of elution tests can damage


the antigens on the RBC
Choroquine diphosphate (CDP)
and glycine acid EDTA reagents can
dissociate IgG from the RBC without
damaging the antigens
Very useful if the RBC needs to be
antigen typed
Chloroquine diphosphate

Quinilone derivative often used as


an antimalarial
May not remove autoantibody
completely from DAT positive cells
Partial removal may be enough to
antigen type the cells or to be used
for autoadsorption of warm
autoantibodies
THE END!!