DETECTION AND IDENTIFICATION

OF ANTIBODIES
Antibody screen
 Only 0.3-2% of people tested will have unexpected
antibodies in their serum
 To detect irregular or unexpected antibodies in the
serum/plasma of an individual
 Population groups requiring an antibody screen:
 Prenatal patients
 Pre-transfusion candidates
 Donor blood
Antibody screen
 Patient’s serum or plasma (unknown antibody) is tested
against red cells (known antigens)
 Typically 2-3 sets of cells are tested
 Method is an application of the IAT
 If all cells tested yield negative reaction in all phases of
testing, the screen is interpreted as negative
 A positive reaction with one or more cells during any
phase is interpreted as a positive antibody screen
 If antibodies are detected, they must be identified
Why 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 (antibodies) 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 that
are Group O red blood cells
 Antibody panel
 Each of the panel cells has been antigen typed
 + 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, Le a, k, Fya, Jka

 Antibody panel
 An autocontrol should also be run with ALL panels

Autocontrol: patient RBCs + patient serum

Antibody panel
 The same phases used in an antibody screen are used in a
panel

IS
37oC
AHG
 Antibody ID Testing
 A tube is labelled 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 shown:

2+
0
0
(LISS) 37oC Phase
 2 drops of LISS are added, mixed and incubated for 10-15
minutes
 Centrifuge and check for agglutination
 Record results
(LISS) 37oC Phase

2+ 0
0 0
0 0
2+
0
0
2+
0
2+
0
0
IAT Phase (AHG)
 Indirect Antiglobulin test (IAT) – we’re testing whether or
not possible autoantibodies in patient’s serum will react with
RBCs in vitro
 Uses the Anti-Human Globulin (AHG ) reagent
 Polyspecific
 Anti-IgG
 Anti-complement
IAT Phase (AHG)
 Wash cells 3 times with saline
 Add 2 drops of AHG and gently mix
 Centrifuge
 Read
 Record reactions
IAT (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
Check cells

 Are added to any negative AHG!!!

IS LISS AHG CC
37°
2+ 0 0 
All cells are
0 0 0  negative at
0 0 0  AHG, so 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
 “Ruling out” means crossing out antigens that did not react
 Circle the antigens that are not crossed out
 Consider antibody’s usual reactivity
 Look for a matching pattern
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.
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!

Phase Antibodies Detected

IS Lewis, Lua, M. N, & P1

Cold auto antibdoies (I, H, IH)
37oC May see D, E, & K
Hold over strong cold antibodies
AHG Rh, Kell, Kidd, Duffy, Ss, Lub, & Xga
May see cold antibodies, if they activate
complement, and if the AHG reagent
contains anti-complement
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
0 0 0 
cells
2+ 0 0 
0 0 0 
0 0 0 
2+ 0 0 
0 0 0 
3 Negative
2+ 0 0 
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
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
 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 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 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)…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
Elution
Sensitized
Y
Y

RBC
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
alloantibody
2 tubes

Wash x3 after Centrifuge after incubating;

incubation 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!!