Practical

Blood Bank

Antibody Identification
Antibody Presence
Presence of an antibody may be indicated by the
following serological tests:
1. A discrepancy in the results of cell and serum ABH
grouping.
2. A positive test for unexpected antibodies.
3. A positive direct Coombs test.
4. An incompatible major cross match.
The Basics..
As we said in the previous lecture,
Antibody Screens use 2 or 3 Screening Cells
to detect if antibodies are present in the
serum
If antibodies are detected, then they should
be identified
Why do we need to identify?
Antibody identification 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 (i.e. transfusion
reactions).
Key Concepts
In blood banking, we test knowns with
unknowns:-

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

When detecting/screening and/or identifying

antibodies, we test patient serum (unknown
that contain blood bank antibodies) with
reagent RBCs (known)
Reagent RBCs
Screening Red Cells and Panel Red Cells
are the same with minor differences:
Screening red cells
Antibody detection/screening
Sets of only 2 or 3 vials
Panel red cells
Antibody identification
At least 10 vials/set
Antibody Panel vs. Screen
An antibody panel is just an extended version of an
antibody screen
The screen only uses 2-3 red cells:
Antibody Panel
While antibody panel usually includes at least
10 panel cells: (8-16 group O RBCs)
Group O red blood cells obtained from donors
Panel Red Cells
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
Panel Red Cells
An autocontrol (AC) should also be run
with panels
Panel Red Cells
The same phases used in an antibody screen
are used in a panel
Antibody ID Testing
A tube is labeled for each of the panel cells
plus one tube for AC
IS Phase
Perform immediate spin (IS) and grade
agglutination; inspect for hemolysis
Record the results in the appropriate space as
shown.
(LISS) 37C Phase
2 drops of LISS are added, mixed and incubated
for 10-15 minutes.
Centrifuge and check for agglutination
Record results as previous but now fill the 37C
lane.

Agglutination Viewer
Grading Reactions
(LISS) 37C Phase
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 AHG
Monospecific Anti-IgG
Monospecific Anti-Complement
IAT (AHG) Phase
Wash red cells 3X with saline (manual or
automated (cell washer))
Add 2 drops of AHG and gently mix
Centrifuge
Read for agglutination
Record reactions
IAT (AHG) Phase
And dont forget.
.add check cells to any
negative AHG !
You have agglutinationnow what?
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
Matching the pattern
Single antibodies usually shows a pattern that matches one of the
Multiple antibodies are more difficult to match because they often show mixed
reaction strengths
Look for a matching pattern
Rule of three
The rule of three must be met to confirm the
presence of the antibody
How is it demonstrated?

Patient serum MUST be:

Positive with 3 panel cells with the antigen, +ve
reaction.
Negative with 3 cells without the antigen and
should not be reacting.
Our previous example fulfills the
rule of three
 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
Better to carry different lot numbers of panel cells
Patient Antigen Typing (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 (chimera)).
If reagent antisera (of the suspected antibody) is added
to the patient RBCs, a negative reaction should
resultWhy?
Remember Landsteiners Rule
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 we have to do?
Several procedures can be performed to
identify multiple antibodies
Selected Cells
Neutralization
Chemical treatment
Proteolytic enzymes
Sulfhydryl reagents
 1- Selected Red 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.
Every cell should be positive only for each of the antibodies
and negative for the remaining suspicious antibodies
For example:
Lets say you ran a panel and identified 3 different
antibodies (you cannot rule out): anti-S, anti-Jka, and
anti-P1
Selected cells could help
Selected Red Cells .. Contd
2- 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)
Common substances
P1 substance (derived from hydatid cyst fluid)
Lea and Leb substance (soluble antigen found in plasma and
saliva)
I substance can be found in breast milk

**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
3- Again: Proteolytic Enzymes
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

One-stage
Enzyme is added directly to the serum/panel cell mixture

Two-stage
Panel cells are pre-treated with an enzyme, and washed

Patient serum is added to treated panel cells and tested

If there is no agglutination after treatment, then it is

assumed the enzymes destroyed the antigen
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)
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
Avoiding reactivity
Cold Autoantibodies can be a trouble 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 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
Warm autoantibodies
Cause warm autoimmune hemolytic anemia
(WAIHA)
How do you get a warm autoantibody?
Idiopathic
Known disorder (SLE, RA, leukemias, 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
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
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
Summary
If an unexpected antibody is detected in a patients
serum or plasma it must be identified.
Once identified the clinical significance must be
determined.
Summary
If the antibody is clinically significant antigen
negative donors must be found and crossmatched
for the patient, a Coombs crossmatch must be
done.
If the antibody is not clinically significant it is not
necessary to provide antigen negative blood, but
the donors must be compatible by the Coombs
crossmatch.
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.
Providing Compatible Donor Units
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.
Example
Knowledge of the incidence of antigens is useful for
determining how many units of blood to screen or cross-
match for patients with antibodies.
If a patient with an anti-Jk(a-) needed 4 units of blood,
how many units would need to be tested to find them?
Jk (a+)= 0.77
Jk (a-) = 0.23

4 units Jk(a-) blood needed = 17.4 units 0.23 incidence of

Jk(a-)
In this case, testing 17 or 18 random units should yield 4
Jk(a-) units.
Example 2
The same calculations can be used when multiple
antibodies are present if the antigen frequencies are first
multiplied together.
E.g. a patient with an anti-K and anti-Jka, 10 random units
would need to be tested to find 2 that are compatible.
Jk(a+) = 0.77 K positive = 0.09
Jk(a-) = 0.23 K negative = 0.91
Jk(a-) (0.23) X K negative (0.91) = 0.20 Jk(a-) and K
negative

2 units needed = 10 units 0.20 Jk(a-) and Kell negative