Antibody Identification

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 patient’s 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) 37°C Phase

• 2 drops of LISS are added, mixed and

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

2+ 0
0 0
0 0
2+
0
0
2+
0
2+
0
0
IAT Phase (or AHG)

• Indirect Antiglobulin Test (IAT) – we’re

testing whether or not possible antibodies
in patient’s 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 don’t 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”
Cells
0 0 0 
0 0 0 
2+ 0 0 
0 0 0 
2+ 0 0 
0 0 0 
0 0 0 
You have agglutination…now 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 antibody’s 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
Here’s 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 antibody’s 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 doesn’t match and it’s
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, it’s 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
cells 0 0 0 

2+ 0 0 

0 0 0 

0 0 0 

2+ 0 0 

0 0 0 
3 Negative
cells 2+ 0 0 

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
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:
– Let’s 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

• Manufacturer’s 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
treament

Enzyme treatment

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 patient’s 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 it’s
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)
• 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
Y
Sensitized
Y
Y
Y

RBC
YY
Y

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
• Heat (conformational change)
• 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
Providing Compatible Donor Units
 Once an antibody has been identified, the
next task is to provide appropriate units of
RBCs for transfusion.

 When clinically insignificant antibodies

are detected, use of crossmatch-
compatible RBCs is appropriate.
• No further testing is needed to confirm
compatibility when the antibody is anti-M,
anti-N, anti-Pi. Lea, or Leb.

• However, when a clinically significant

antibody is identified, the blood must be
cross-match compatible and confirmed as
antigen-negative with reagent antisera.
• Knowledge of the incidence of antigens is
useful for determining how many units of
blood to screen or cross­match for patients
with antibodies.
EXAMPLE:

• If a patient with an anti-Jka needed 4 units

of blood, how many units of red cells
would need to be tested to find compatible
units? Frequency of Jka+ donor is 77%.

FORMULA:
# unit screen = # unit requested
frequency of related Ag
*if 2 frequencies given, multiply the frequencies
unit screen = 4 units/0.23

Jka+ = 77% or 0.77

Jka- = 23% or 0.23

ANSWER: 17 OR 18 UNITS
EXAMPLE:

• Anti-K and Anti-c were found in a patient

with colon cancer. How many units of red
cells would need to be screened to find
two compatible units for surgery? The
antigen frequency for K and c are 0.91 and
0.20, respectively.
ANSWER: 27.7777 OR 28 UNITS
THE END!!