VGFV 8/31/17

BB - Antibody Identification to some RBC antigens.

Purpose:  But most unexpected antibodies are formed
as part of the immune response after
 Detection of atypical or unexpected exposure to foreign antigens during
antibodies (other than ABO) pregnancy or transfusion
 This could be made in response to:  These antibodies can cause acute and
a. Transfusion of red cells or fetal cells delayed hemolytic transfusion reactions.
during pregnancy and delivery – these  It is therefore important to conduct test to
antibodies are directed to a non-self screen for and identify unexpected
antigen (alloantibodies) antibodies before transfusion.
 Autoantibodies – formed from disease
process or medication – made from
person’s own red cells Clinically Significant Antibodies
 Clinically significant antibodies are of the
Antibody Screening is used to test:
Group I Group II Group III Group IV
1. Donor plasma to make sure NO unexpected Clinically Benign Clinically Antibodies
antibodies will be transfused Significant antibodies insignificant if that are
2. Patient serum before transfusion to make sure antibodies not reactive at sometimes
patient has no unexpected antibodies to react 37oC; possibly clinically
significant when significant
with donor cells
reacting at 37oC
3. Maternal serum to make sure pregnant ABO Chido/Rogers Lewis (Lea, Leb) Yta
mothers has no antibodies to react with fetal (Cha, Rga)
cells Rh Xga M, N Vel
(D, C, c, E, e)
Kell (K, k) Bg P1 Ge
Unexpected Antibodies Duffy HTLA Lutheran Gya
 The unexpected antibodies of primary (Fya, Fyb) (Lua, Lub)
Kidd Csa A1 Hya
importance are the immune alloantibodies,
(Jka, Jkb)
which are produced in response to red blood S,s Kna Sda
cell (RBC) stimulation through transfusion, McCa, Yka
transplantation or pregnancy. JMH
 Unexpected antibodies are often referred as IgG class and react in vitro, preferentially at
IRREGULAR antibodies because their 37C or at the anti-human globulin phase of
existence and type are unknown before testing
conducting an antibody screening test  Thse antibodies were determined to be
 This category of antibodies includes most clinically significant if they have been
blood type antibodies excluding ABO and responsible for hemolytic transfusion reactions
some P blood type antibodies like D, C, E, or hemolytic disease of the newborn.
c, e, M, N, S, s, P, Lea, Leb, K, k, Fya, Fyb, Clinically siginifcant antibodies are those
and Jkb that cause decreased survival of RBCs.

 Depending on the type of antibody, some

develop NATURALLY in patients (may
form as a result of exposure to
environmental sources [e.g. pollen, fungus,
and bacteria]), which have structures similar
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Antibody Screen
 AABB Standards require the use of an
antibody screen to detect clinically
significant antibodies in the blood donor and
the intended recipient.
 An antibody screen is included in standard
prenatal testing to evaluate the risk of
HDFN in the fetus and to assess the mother’s
candidacy for Rh-immune globulin
prophylaxis (RhIG)
Test to be done
A. Compatibility testing
B. Antiglobulin testing (coomb’s test)
C. Elution techniques
D. Antibody titration test
E. Panel cell testing
Ab screen determines whether an antibody to a red
cell antigen has been made
 Ab screens are performed in the following
a) Patients requiring transfusion
b) Women who are pregnant or following
delivery
c) Patients with suspected transfusion
reactions
d) Blood and plasma donors
Compatibility testing
 All steps in the identification and testing
of a potential transfusion recipient and
donor blood before transfusion in an
attempt to provide a blood product that
survives in vivo and provides its
therapeutic effect in the recipient
Antiglobulin test
 Direct antiglobulin test: test used to
detect antibody bound to red cells in-vivo
(within the body)
 Indirect antiglobulin test: test used to
detect antibody bound to red cells in-vitro
Antibody screen: Tube Method
 The traditional method of detecting antibodies
 An indirect antiglobulin test performed in a
test tube
1. Serum or plasma mixed with RBCs
having known antigens
 Immediate spin phase (not required)
2. 37oC incubation phase
 Enhancement media may be added to
increase the degree of sensitization
3. Anti-human globulin reagent (AHG)
phase
 Coomb’s control cells (also known as
check cells)
 Depending on the enhancement
added, the tubes may be centrifuged
and observed for hemolysis and
agglutination following incubation
 The degree of reactivity at all phases
of testing is graded
Tube Method Reagents Needed
o RBC Reagents
 Screen cells, from group O individuals,
are packaged in sets of 2 or 3 cell
suspensions
 Each set of screen cells is accompanied
by an antigen profile sheet, detailing
which antigens are present in each vial
of cells.
o RBC Reagents
 Homozygous cells
 Heterozygous cells
 Antibodies that react more strongly with
cells having homozygous antigen
expression are said to show dosage.
Antibody screen: Tube Method
o Enhancement reagents
 May be added to the cell/serum mixture
before the 37oC incubation phase to
increase the sensitivity of the test system.
o 22% albumin
o Low ionic strength solution (LISS)
o Polyethylene glycol (PEG)
o Antihuman globulin (AHG) Reagents
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 Polyspecific AHG Reagent (also called  Measure volumes of red cells and
polyvalent or broad spectrum plasma/serum are added to reaction chamber
Coombs’ serum) of the microtube
 Monospecific AHG  Reaction chamber allows red cell
sensitization with antisera and gel particles
AABB Standards requirements for antibody
detection  Centrifugation separates + and –
 Coombs’ control cells agglutination results
 + results: agglutinated red cells are trapped
Analytical process
in gel at various levels
 - results: unagglutinated red cells pass
through the gel and form a button of the
microtube
Gel antibody screening

Antibody Screen: Gel Method

 The process involves a microtubule filled
with a dextran acrylamide gel
 The screen cells used for this technique meet
the same criteria as for the tube test but are
suspended in LISS to a concentration of 0.8%

 Gel Technology (column

agglutination/solid-phase red cell adgerence
(SPRCA), sensitive to tube testing
 Suitable for automation
Microplate Testing Methods
 It is hemagglutination reaction, red cells
migrate through the gel matrix to separate  microplate techniques apply the same
agglutinated from an unagglutinated red principle of hemagglutination as the tube
cells test
 in contrast, a solid phase technology
Procedure
reactions are opposite.
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 + reaction is adherence to the wells Optimal Temperature of Reactivity for Some Common
Antibodies
 - reaction is a red cell button
Phase Room 37oC Antiglobulin
Temperature incubation phase
(Immediate
spin)
Antibodies Cold Potent cold Rh antibodies
autoantibodies antibodies K
(I, H, IH) (especially
Duffy
M, N if causing
hemolysis) Kidd
P1
D, E S, s
Lea, Leb
K Lub
Lua
Xga
Immunoglobulin IgM IgG IgG
class
Antibody Detection Limitations Clinically No Yes Yes
significant
1. Cell to Serum Ratio
 Manipulations:
Prozone and Postzone Phenomena
 Decreasing the temperature 24oC or even
to 4oC enhances the reaction of IgM
antibodies
 Prewarming or maintaining a reaction
temperature of 37oC provides the optimum
temperature for detection of IgG
antibodies, while preventing most IgM
antibodies from reacting

3. Length of Incubation
Cell to Serum Ratio
 manipulations: increase the amount of
serum (4 drops) – if no potentiator used
4. PH
o use potentiator or enhancement medium
2. Temperature
o influences the equilibrium constant and
or reaction rate depending on the class
of antibody involved
o focus on a clinically significant antibody
which generally reacts at 37oC
 Different Ag/Ab reactions reach
equilibrium over different time periods
 Manipulation:
 Increasing the time of incubation in a
given test system may enhance the
detection of a weakly reactive antibody
 At a pH range of 6.5 – 7.5, chemical
groups on Ag and Ab are oppositely
charged.
 Manipulation:
 Changing the pH affects the
equilibrium constant for selected
antibodies that may react
preferentially in a more acidic
environment (e.g., anti-M, anti-Pr)

Temperature
1. 22% albumin: reduces zeta potential
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2. Low ionic strength solution (LISS) ○ Antibody detection

 Combination of albumin and glycerine ○ Sets of 2 or 3 vials
solution
 Increases the uptake of the antibody and ● Panel cells
enhances the agglutination ○ Antibody identification
3. Polyethylene Glycol (PeG)
○ At least 10 vials per set
 Removes the water from the test system
 More sensitive than LISS, albumin or Antibody Panel vs. Screen
saline system ○ An antibody panel is just an extended
version of an antibody screen
The Basics…..
○ The screen only uses 2-3 cells:
○ As you recall,
● Antibody Screens use 2 or 3 Screening
Cells to “detect” if antibodies are present
in the serum Antibody Panel
● If antibodies are detected, they must be ○ An antibody panel usually includes at least
identified… 10 panel cells:
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
Panel
Key Concepts
○ Group O red blood cells
○ In blood banking, we test “knowns” with
“unknowns”

○ 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:
Panel
● Screening cells
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○ Each of the panel cells has been antigen typed ○ A tube is labeled for each of the panel
(shown on antigram) cells plus one tube for AC:
● + refers to the presence of the antigen
● 0 refers to the absence of the antigen

IS Phase
○ Perform immediate spin (IS) and grade
agglutination; inspect for hemolysis
○ Record the results in the appropriate
space as shown:
○ 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
(LISS) 37°C Phase
○ 2 drops of LISS are added, mixed and
incubated for 10-15 minutes
○ Centrifuge and check for agglutination
○ Record results

Panel
○ The same phases used in an antibody
screen are used in a panel

Antibody ID Testing

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(LISS) 37°C Phase

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

IAT Phase (or AHG)

○ 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
AHG Phase
○ Wash cells 3 times with saline (manual or
automated) Interpreting Antibody Panels
○ Add 2 drops of AHG and gently mix ○ There are a few basic steps to follow when
● Centrifuge interpreting panels
● Read 1. “Ruling out” means crossing out antigens
● Record reactions that did not react
AHG Phase 2. Circle the antigens that are not crossed out
3. Consider antibody’s usual reactivity

2+ 0 0 4. Look for a matching pattern

0 0 0
0 0 0 Always remember:
2+ 0 0
0
0
0
0
0
0  An antibody will only react with cells that
2+ 0 0 have the corresponding antigen; antibodies
0 0 0
2+ 0 0 will not react with cells that do not have
0 0 0
0 0 0
the antigen
0 0 0

And don’t forget: add “check” cells to any

negative AHG!

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Interpretation: Lea
Guidelines
○ Again, it’s important to look at:
● Autocontrol
○ Negative - alloantibody
○ Positive – autoantibody or DTR
(i.e.,alloantibodies)
○ DTR – delayed transfusion reaction
(donor cells are sensitized with
patient’s antibody)
● 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)
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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
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?
o Reaction strengths can vary
o Matching the pattern is difficult
So what is a tech to do?
 Several procedures can be performed to
identify multiple antibodies
o Selected Cells
o Neutralization
o 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
○ Every cell should be positive for each of the
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antibodies and negative for the remaining guinea pig urine

antibodies
**you should be aware that many of these
○ For example: substances neutralize COLD antibodies; Cold
○ Let’s say you ran a panel and identified 3 antibodies can sometimes mask more clinically
different antibodies: anti-S, anti-Jka, and significant antibodies (IgG), an important reason
anti-P1 to use neutralization techniques
○ Selected cells could help… Enzymes (proteolytic)
○ Can be used to enhance or destroy certain
Selected Cells
blood group antigens
○ Several enzymes exist:
Selected S Jka P1 IS LISS AHG ● Ficin (figs)
cells 37°
● Bromelin (pineapple)
#1 + 0 0 0 0 2+
● Papain (papaya)
#5 0 + 0 0 0 3+
○ In addition, enzyme procedures may be
#8 0 0 + 0 0 0✓ ● One-step
These results show that instead of 3 antibodies, there ● Two-step
are actually 2: anti-S and anti-Jka
Enzymes
Neutralization ○ Enzymes remove the sialic acid from the
○ Some antibodies may be neutralized as a way RBC membrane, thus “destroying” it and
of confirmation allowing other antigens to be “enhanced”
○ Commercial “substances” bind to the ○ Antigens destroyed: M, N, S, s, Duffy
antibodies in the patient serum, causing them
○ Antigens enhanced: Rh, Kidd, Lewis, I,
to show no reaction when tested with the
and P
corresponding antigen (in panel)
Enzyme techniques
○ Manufacturer’s directions should be followed
and a dilutional control should always be used ○ One-stage
● Enzyme is added directly to the
○ The control contains saline and serum (no serum/cell mixture
substance) and should remain positive
○ Two-stage
○ A control shows that a loss of reactivity is
● Panel cells are pre-treated with enzyme,
due to the neutralization and not to the
incubated and washed
dilution of the antibody strength when the
substance is added ● Patient serum is added to panel cells
and tested
○ Common substances
○ If there is no agglutination after treatment,
○ P1 substance (sometimes derived from
then it is assumed the enzymes destroyed the
hydatid cyst fluid)
antigen
○ 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
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VGFV 8/31/17
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
○ 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
○ 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
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 Idiopathic
 Known disorder (SLE, RA, leukemias,
UC, pregnancy, infectious diseases, etc)
 Medications
Avoiding reactivity
○ Several techniques are used when warm
○ Cold autoantibodies can be a nuisance at autoantibodies are suspected…
times. Here are a few ways to avoid a Elution (whenever DAT is positive)
Elution (whenever DAT is positive)
reaction:
Elution
○ Elution techniques
techniques “free” from
“free” antibodies
○ Use anti-IgG AHG instead of polyspecific. antibodies from the sensitized
the sensitized red cells so that the
Most cold antibodies react with polyspecific red cells so that the antibodies
antibodiescan
can be
be identified
identified
AHG and anti-C AHG because they fix
complement
Y
Y
○ Skipping the IS phase avoids the attachment Elution
Sensitized
Y

Y
RBC
of cold autoantibodies to the red cells YY

Y
Y
○ Use 22% BSA instead of LISS Y
Positive DAT Frees antibody Antibody ID

Other techniques Elution

○ 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
○ 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

○ Again, the majority of panel or screening ○ Acid elutions (glycine acid)

cells will be positive ● Most common
○ The Rh system (e antigen) seems to be the ● Lowers pH, causing antibody to
main target although others occur dissociate
○ Cause warm autoimmune hemolytic ○ Organic solvents (ether, chloroform)
anemia (WAIHA)…H&H
● Dissolve bilipid layer of RBC
○ How do you get a warm autoantibody?
ABO ○ Heat (conformational change)
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VGFV 8/31/17

○ Freeze-Thaw (lyses cells) More reagents….

○ Many of elution tests can damage the
antigens on the RBC
Adsorption
○ Choroquine diphosphate (CDP) and
○ Adsorption procedures can be used to
glycine acid EDTA reagents can dissociate
investigate underlying alloantibodies
IgG from the RBC without damaging the
○ ZZAP or chloroquine diphosphate can antigens
be used to dissociate IgG antibodies from
● Very useful if the RBC needs to be
the RBC (may take several repeats)
antigen typed
○ After the patient RBCs are incubated, the
adsorbed serum is tested with panel cells
to ID the alloantibody (if present) Chloroquine diphosphate
○ Two types: ○ Quinilone derivative often used as an
antimalarial
 Autoadsorption
○ May not remove autoantibody completely
○ No recent transfusion
from DAT positive cells
○ Autoantibodies are removed
○ Partial removal may be enough to antigen
using patient RBCs, so
type the cells or to be used for
alloantibodies can be identified
autoadsorption of warm autoantibodies
 Allogenic (Differential) adsorption
○ If recently transfused
○ Uses other cells with the patients
serum

Remove
serum and
test for
2 tubes alloantibody

Wash x3 after Centrifuge after

incubation incubating; and
transfer serum to 2nd
tube of treated cells;
incubate and
centrifuge again

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