Antibody Detection &
Identification
The Basics…..
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
A positive AC could
indicate an
autoantibody OR
alloantibody to
recently transfused
cells
Autocontrol 0 0 2+
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
Antibody Panel steps
After reagent cells and patient
plasma or serum are added, several
phases of testing are conducted on
each tube:
Immediate Spin (IS)
LISS/37°C (10-15 min.)
Wash X3
AHG
Coombs’ control cells if indicated
Step 1: 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,
including AC
Step 2: 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 (example)
2+
0 0
0 0
2+
0
0
2+
0
2+
0
0
What do you do next?
Step 3: AHG
Indirect Antiglobulin Test (IAT) – this
tests whether or not antibodies in
patient’s serum will react with RBCs in
vitro (i.e., in the tube)
To do this we use the Anti-Human
Globulin reagent (AHG)..the green stuff!
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
2+
0 0 0
0 0 0
2+
0 0 0
2+
0 0 0
0 0 0
0 0 0
And don’t forget….
….add Coombs’ control
cells to any negative AHG !
IS LISS AHG CC
37°
2+
0 0 0 All cells are
0 0 0 negative at
AHG, so
2+ add
0 0 0 “Coombs”
0 0 0 Cells
2+
0 0 0
2+
0 0 0
0 0 0
You have agglutination…now what?
CC
2+
0 0 0
0 0 0
2+
0 0 0
0 0 0
2+
0 0 0
2+
0 0 0
0 0 0
0 0 0
??
An antibody
is present
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 (neg result)
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…
…let’s get to work
RULING OUT
Skip 2+ 0 0
0 0 0
0 0 0
Skip 2+ 0 0
0 0 0
0 0 0
Skip 2+ 0 0
0 0 0
Skip 2+ 0 0
0 0 0
0 0 0
0 0 0
Highlight cells that show NO REACTION in ALL phases; Moving from left to
right, Cross out antigens from the highlighted area only; do NOT cross out
heterozygous antigens that show dosage [Rh(not D), Kidd, Duffy, MNSs]
CIRCLE ANTIGENS NOT ELIMINATED
2+
0 0 0
0 0 0
2+
0 0 0
0 0 0
2+
0 0 0
2+
0 0 0
0 0 0
0 0 0
CONSIDER USUAL REACTIVITY
2+
0 0 0
0 0 0
2+
0 0 0
0 0 0
2+
0 0 0
2+
0 0 0
0 0 0
0 0 0
Anti-Lea is normally a Cold-Reacting antibody (IgM), so it
makes sense that we see the reaction in the IS phase of testing;
The anti-E will usually react at higher temperatures
LOOK FOR MATCHING PATTERN
E doesn’t match and
it’s a warmer rx Ab
2+
0 0 0
0 0 0
2+
0 0 0
0 0 0
2+
0 0 0
2+
0 0 0
0 0 0
0 0 0
…Yes, there is a matching pattern!
Interpretation
anti-
Lea
Additional Considerations
Again, it’s important to look at:
Autocontrol
Negative - alloantibody
Positive – autoantibody or alloantibody to recently
transfused cells (RBCs have a life span of 120 days)
Phases
IS – cold (IgM)
37° - cold (some have higher thermal range) or
warm reacting
AHG – warm (IgG) or complement…significant!!
If there is a reaction in ALL cells…may be autoantibody
Reaction strength
1 consistent strength – one antibody
Different strengths – multiple antibodies or dosage
Considerations: Dosage
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 cause a reaction too
weak to be seen
Considerations
Matching the pattern
Single antibodies usually show a
pattern that matches one of the
antigens (see previous panel example)
Multiple antibodies are more difficult to
match because they often show
variable reaction strengths
Confirming the antibody
To confirm the antibody identified
from a panel, it is important to
Use the rule of three
Phenotype the patient’s RBCs
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
3 Positive 0 0 0
cells
2+
0 0 0
0 0 0
2+
0 0 0
3 Negative 2+
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 (or screening
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 result…Why?
Remember Landsteiner’s Rule?
Individuals DO NOT make
allo-antibodies against
antigens they have
MULTIPLE ANTIBODIES
Identifying 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
Adsorption
Chemical treatments
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
to separate antibodies or confirm
the presence of an antibody
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 (nothing is being neutralized)
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
Adsorption
Adsorption procedures can be used to
investigate underlying alloantibodies especially
when the patient has an autoantibody to
common antigens (e.g., I, H)
ZZAP or chloroquine diphosphate can be
used to dissociate IgG antibodies from the
RBC (may take several repeats)
After RBCs are incubated with patient serum
(to adsorb the autoantibody), the residual
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 in serum can be
identified using a panel
Allogenic (Differential) adsorption
If recently transfused (past 3 months)
Uses other cells with the patients serum
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 initial agglutination disappears
after treatment, then it is assumed
the enzymes destroyed the antigen
Duffy, M, N, S, s
If agglutination is stronger, the
enzymes enhanced the antigen
Rh, Kidd, Lewis, P
Some antigens are not affected
Kell, U, Lutheran
Other chemicals:
Sulfhydryl Reagents
Cleave the disulfide bonds of IgM
molecules and help differentiate
between IgM and IgG antibodies
Good to use when you have an IgM
and need to identify underlying IgG,
usually after enzyme treatment
Both reagents denature K antigens
Jsa and Jsb are particularly vulnerable
Dithiothreitol (DTT) — a thiol
2-mercaptoethanol (2-ME)
Effect of enzymes and DTT
Enzyme reaction DTT reaction Possible antibody
0 (destroyed) + M, N, S, s, Duffy
+ (no effect) Weak + Lutheran
+ (no effect) 0 (destroyed) Kell
+ (enhanced) + P, Rh, I, Lewis, Kidd
+ (no effect) enhanced Kx
Other Chemicals:
AET
2-aminoethylisothiouronium
bromide (2-AET)
Removes activity of Kell system
antigens from RBCs (except Kx)
Other Chemicals:
ZZAP
A combination of proteolytic
enzymes (papain) and DTT
Destroys Kell, M, N, S, Duffy and
other less frequent blood group
antigens
Does not denature Kx antigen
Dissociates IgG bound to RBCs
Good for adsorption techniques
Used more when warm autoantibodies
are present
Other Chemicals:
CDP and Glycine acid EDTA
Both of these chemicals dissociate
IgG from RBCs
They render DAT+ red cells DAT-
Keeps the RBC intact for
phenotyping (using indirect AHG
phase)
Other Chemicals:
Chloroquine
diphosphate: Glycine Acid EDTA:
Dissociates IgG from
Dissociates IgG from RBC
RBC
Takes only a few
May weaken Rh minutes compared to
antigens an hour to treat RBCs
Denatures HLA Destroys Kell,
antigens (Bg) on RBCs Cartwright (Cw), Er,
RBCs should be Bg
treated for about an Useful in denaturing
hour (no more than 2) antigens for select cell
panels
Special Considerations in
Antibody ID
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 or complement (in the
body)
AHG is added to washed patient red
cells to determine if IgG or
complement are coating the RBCs
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 recently 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
A positive DAT may also occur when a red
top is used (in vitro bound complement)
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 screen
may be negative, meaning
something is coating the RBC
Identifying autoantibodies
Auto-antibodies can sometimes
“mask” clinically significant allo-
antibodies, so it’s important to
differentiate between auto- and
allo-antibodies
READING ASSIGNMENT :
ANTIBODY SCREENING &
IDENTIFICATION
Pages 76-77
BB LAB MANUAL
