Detection and

Identification
of Antibodies
Bea Angelli D. Laude, RMT
MLS Faculty
San Pedro College
What is antibody screening?
Blood donor Recipient

Clinically
significant
antibodies
 ✓Tube Method
Methods ✓Gel Method
of ✓Solid Phase Adherence
Antibody •Interpretations
Screening •Limitation
•GROUP O typed red cells (2%-5% suspension
with preservative diluent)
▪Comes in a package in sets of two or three cell
suspensions, each having a unique combination of
Reagent
antigens
▪Within each set, there should be one suspension Red
positive for each of the following Ags:
D, C, c, E, e, K, k, Fya, Fyb, Jka, Jkb, Lea, Leb, P1, M, N,S Cells
and s (Screen cells)
•Each set of screen cells is with an antigen profile
sheet
•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
•22% ALBUMIN
•LISS (Glycine+ Albumin solution)
•Polyethylene Glycol (PEG) Enhancement
• AHG Reagents agents
(Potentiators)
• Polyspecific has IgG and complement
components

• Control: COOMB’S CHECK CELLS

 TUBE
METHOD
 ADVANTAGES:
•Flexibility of the test system
•Use of common lab equipments

Tube •Relative low cost

DISADVANTAGES:
Method •Instability of reactions
•Subjective manner of grading
•Time-consuming
•Failure to follow steps
 ADVANTAGES:
•Sensitive
•Washing and coombs control cells are

Gel omitted
•Reaction is stable for 24 hours
•Standardized grading of reactions
Method DISADVANTAGES:
•Need of incubators and centrifuges that can
accommodate gel cards
 ADVANTAGES:
SOLID •Ease of use (no predilution is
required)
PHASE •Ability to test hemolyzed, lipemic or
icteric samples
ADHERENCE •Enhanced sensitivity

METHOD DISADVANTAGES:
•Need of special equipments
1. In what phase(s) did the reaction(s) occur?
• Anti-N, anti-I and anti-P1 (IgM)
• Abs to Rh, Kell, Kidd, Duffy, Ss (IgG)
• Lewis and M (Mixed)
2. Is the autologous control neg or pos?
• (+) Ab screen and (–) autologous ctrl = alloantibody
3. Did more than one screen cell sample react? If so, did
they react at the same strength and phase?
• Multiple Abs: abs react at different phases or strengths
• Single Abs: abs react at same phase or strengths
 Interpretation Of Results

4. Is hemolysis or mixed field agglutination present?

• Anti- Lea, Anti-Leb, anti- PP1PK and anti-Vel (in vitro
hemolysis)
• Anti- Sda and Lutheran antibodies (mixed field
agglutination)

5. Are the cells truly agglutinated or is rouleaux present?

LIMITATIONS
• Cell to Serum Ratio
• 2 drops of serum: 1 drop of screening cells
• Temperature and Phase of Reactivity
• Length of incubation
• 30 mins to 1 hour
• with potentiators: 10 minutes
• pH
• neutral pH (6.8-7.2)
• Anti-M (6.5)
 •Antibody identification is needed for
Why do we transfusion purposes and is an
important component of compatibility
testing
need to •It will identify any unexpected
antibodies in the patient’s serum
identify? •If a person with an antibody is exposed
to donor cells with the corresponding
antigen, serious side effects can occur
•Patient History •Diagnosis
•Age •Transfusion
•Sex •Pregnancy
•Race History
•Reagents
▪Antibody Identification Panel
✓collection of 11 to 20 group O
RBCs with various antigen
expression

•Exclusion (Rule Out)

•An antibody panel usually includes at least 10 panel cells (Group O 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 Example: Panel Cell #10 has 9 antigens present: c, e, f, M, s, Leb, k, Fya, and Jka
absence of the antigen
•An autocontrol
should also be
run with ALL
panels.
AUTOCONTROL =Patient RBCs + Patient serum
•The phases
used in an
antibody
screen and
panel
Antibody Panel 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 Testing
•A tube is labeled for each of the panel cells plus one tube for AC
AC

 1 drop of each panel cell +   2 drops of the patients serum

• Perform immediate spin (IS) and grade agglutination; inspect for hemolysis
• Record the results in the appropriate space
 °
•2 drops of LISS are added, mixed and
incubated for 10-15 minutes
•Centrifuge and check for agglutination
•Record results
°
•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
•Wash cells 3 times with saline
(manual or automated)
•Add 2 drops of AHG and gently mix
•Centrifuge
•Read
•Record reactions
 add COOMB’S
….
CHECK CELLS to
any negative
AHG!
IS LISS AHG CC
37°
2+ 0 0  All cells are
0 0 0 
0 0 0  negative at
2+ 0 0  AHG, so add
0 0 0 
0 0 0 
“Check” Cells
2+ 0 0 
0 0 0 
2+ 0 0 
0 0 0 
0 0 0 













Interpreting Antibody Panel
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
 An antibody will only
REACT with cells that have
the corresponding antigen;
antibodies will NOT REACT
with cells that do not
have the antigen.
’

:


























 ’
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.
E doesn’t match and it’s a warmer rx Ab















…Yes, there is a matching pattern!

Anti-
Le a
 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
GUIDELINES
•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
 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)
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 T HREE
• 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 T HREE 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 result…Why?
Remember:
’
Individuals DO NOT make allo-antibodies against
antigens they have.
•Multiple antibodies may be more
of a challenge than a single
antibody
•WHY?
•Reaction strengths can vary
•Matching the pattern is difficult
•Several procedures can be performed to
identify multiple antibodies
•Selected Cells
•Neutralization
•Chemical treatment
• Proteolytic enzymes
• Sulfhydryl reagents
• ZZAP
•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 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 S Jka P1 IS LISS 37° AHG
#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.
•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)
•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
•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
•Sd substance derived from human or guinea pig urine
a
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 (Proteolytic)
•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

• Duffy
antigens are
destroyed Anti-K

• Kell antigens
are not
affected
Perfect match for anti-Fya
 sulfhydryl agents
•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