Antibody Detection

Dr. Ali Ibrahim

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 is incubated

with screening cells
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, singledonor vials offer increased sensitivity
Why group O? so anti-A and anti-B wont
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 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

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 Examiner 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

4+

SCII

Fya

Fyb

SCI

2+

SCII

4+

If patients 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,
Jsa ,Lua 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 screendepends
on the hospital
However, the AC should be run with the
antibody panel
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 patients
cells or transfused cells (allo-or
autoantibody)

Potentiators

Used in antibody detection and

identification to enhance antigenantibody 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

Serum/cell mixture should incubate at least

20 minutes, 30 minutes preferred; doesnt
enhance warm autoantibodies

LISS

Incubation time of 10 minutes; lowers

ionic strength allowing better reaction;
sensitive and quick!

PEG

Enhances warm autoantibodies; does not

react well with insignificant antibodies
(IgM)

Limitations

Very effective in detecting

antibodies
If negative, then the crossmatch
should be compatible

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 races
Get diagnosis, age, race, etc

Example 1
Screening
Cell
I

IS

37C

AHG

CC

II

2+

ND
Not
Done

IgG antibody
Single
specificity

Example 2
Screening
Cell

IS

37C

AHG

3+

II

2+

3+

IgG antibody
Multiple specificities

CC

Example 3
Screening
Cell

IS

37C

AHG

CC

1+

II

3+

Neg AHG, add

CC

IgM antibody
Single specificity showing
dosage

Example 2
Screening
Cell

IS

37C

AHG

2+

II

2+

IgG antibody
Allo- or autoantibody?
(dont know without
further testing)

CC

Antibody Identification

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:
Reagent RBCs
+
Reagent antisera +

Unknown:
patient serum
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

These are 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

Autocontrol
Patient RBCs
+
Patient serum

An autocontrol should also be run

with ALL panels

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:
3

1 drop of each panel cell

+

2 drops of the patients serum

10

11

AC

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

0
0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0

And dont forget.

.add check cells to

any negative AHG !

IS
2+
0
0
2+
0
0
2+
0
2+
0
0

LISS AHG
37
0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0

CC

All cells are

negative at
AHG, so
add
Check
Cells

You have agglutinationnow what?

CC

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

??

0
0
0
0
0
0
0
0
0
0
0
0

0
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.

2.

3.

4.

Ruling out means crossing out

antigens that did not react
Circle the antigens that are not
crossed out
Consider antibodys usual reactivity
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
2+
0
0
2+
0
2+
0
0
0

0
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.

0
0
0
0
0
0
0
0
0
0
0
0

2. Circle antigens not crossed out

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

0
0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0

3. Consider antibodys usual reactivity

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

0
0
0
0
0
0
0
0
0
0
0
0

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

Yes, there is a matching pattern!

0
0
0
0
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0

Interpretation
antiLea

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 nonreactive

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

3 Positive
cells

3 Negative
cells

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

0
0
0
0
0
0
0
0
0
0
0
0

0
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

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
cells

Jka

P1

IS

LISS AHG
37

#1

2+

#5

3+

#8

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 treatment

Enzyme
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

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 alloantibodies, 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-C3b 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
Y

Y
Positive DAT

Sensitized
RBC

Elution

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)

Organic solvents (ether, chloroform)

ABO
antibodies

Most common
Lowers pH, causing antibody to
dissociate
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
alloantibody

2
tubes
Wash x3 after
incubation

Centrifuge after
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!!