Unit 7
Detection and Identification of RBC
ALLOantibodies & AUTOantibodies
Rutgers School of Health Professions
Medical Laboratory Science Program
MLSC 2239 Immunohematology I
Objectives
1.
2.
For the antibody screening, antibody panel procedures:
1.Explain the purpose and indications for testing
2. List requirements for testing
3.Explain the principle and procedure 4.
Explain the antigenic composition of screening and panel reagent red cells
5. Explain the quality control procedures used
6. State the significance of a positive test
7. State the appropriate follow up for positive and negative test results
Given test results for an unknown single antibody:
1.Indicate the presence of alloantibody and/or autoantibody
2.Describe the criteria needed for identification of an antibody with 95% probability (rule of 3).
3.Evaluate results of an antigram to correctly identify the antibody.
4. Explain how phenotyping of patient’s cells is used to confirm the identity of the antibody and in
investigations of multiple antibodies.
5. Select appropriate panel cells as positive and negative controls for phenotyping patient cells for
the corresponding antigen
6. Recognize the dosage phenomenon in panel cell reactions with patient serum
7. Correctly identify the selected cells required to satisfy a rule of three if the rule cannot be
satisfied on the original panel
3. For detection of antibodies on red blood cells
1. List conditions that can cause a positive direct antiglobulin test
2. Interpret results of the direct antiglobulin test
3. State the indications for performing an elution
4. Explain types of elution procedures
4. For removal of autoantibodies from serum:
1. Describe the steps in an autoadsorption
2. Detail the next steps after an autoadsorption
Antibody Screen (Detection)
• Detection of “atypical” or “unexpected” antibodies in the
screen of a patient or donor
• Atypical/Unexpected refers to antibodies other thanABO
antibodies
• These antibodies can be made in response to previous
transfusion or pregnancy and are directed to non-self
antigens = Alloantibodies
• Or antibodies can be made in response to a patient’s disease and
are directed to self-antigens = Autoantibodies
Antibody Screen
 Antibody Screens use 2 or 3 Screening Cells
 If antibodies are detected, they must be identified!
 When detecting and/or identifying antibodies, we
test patient serum (unknown) with reagent RBCs
(known)
 Warm or Cold reacting
 Allo- and/orAutoantibody
 Single or Multiple
present
Not present
Why do we need to identify?
Antibody screens are performed to detect antibodies in:
1- patients requiring transfusion
2- patients with suspected transfusion reactions
3- women who are pregnant
4- blood donors
 Antibody identification is needed for transfusion purposes and is
an important component of compatibility testing (crossmatch XM)
 If a person with an antibody is exposed to donor cells with the
corresponding antigen, serious side effects can occur
Antigram
 The antigram lists the antigens present on the reagent screening
cells
 A reaction to one or both of the screening cells demonstrates the
presence of an unexpected antibody
 Some labs use a 2 or 3 cell screen
 3 cell screen: R1R1, R2R2, rr
 2 cell screen: R1R1, R2R2
Antigram
IS 37 AHG CC
Reagent RBCs
 Screening Cells and Panel Cells are made of group O
donors with known antigen phenotyping. They are primarily
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
Pre-Antibody ID
• Before beginningABID, it is essential to obtain complete patient
•
•
•
•
•
history
Mixed RBC populations from a previous transfusion can remain for up
to 3 months
Patient may have come from another hospital
Diagnosis, race, and age should be noted, because they offer additional
“clues” to the nature of the antibody problem
Some diseases are associated with the development of certain
antibodies
For example, a patient with lupus or carcinoma is frequently associated
with a warm autoantibody; whereas pneumonia may result in a cold
autoimmune process.
Antibody Identification
• The purpose of antibody identification is to identify the clinically significant
antibody present in the patient’s serum (plasma); this represents the patient’s
immune system.
• Testing patient serum or plasma against a panel of reagent cells with known
antigenic identity; is the protocol after the antibody detection test is positive
• The panel cells (like screening cells) are group O reagent red cells
• Panel cells are configured by the manufacturer of either 10, 12, 16 or 20
cells on one panel
• Each panel is different and vary by Lot# since the donors may change
Antibody Panel
 An antibody panel usually includes at least 10 panel cells:
Antibody Panel
 Some cells are Rh pos, some are Rh neg
Antibody 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
Antibody Panel
 An autocontrol should also be run withALL panels if
not performed with the antibody screen
Autocontrol (A/C)
• TheA/C tests the patient’s plasma with patient’s red cells and
employs the IAT phase, just like the antibody screen.
• The antibody screen: screens patient’s plasma for alloantibodies
• TheA/C: screens the patient’s plasma for autoantibodies
• Both follow the
IAT phases of testing:
• IS – 37 – AHG – CC
Antibody Panel
 The same phases used in an antibody screen are used in a
panel
•
IS
• 37°
• AHG
• CCC
Potentiators
 Used in antibody screening and antibody detection (ABID)
 Increase speed and sensitivity of antibody attachment to red
cell antigen
 LISS –Low Ionic Strength Solution
 BSA – Bovine serum albumin
 PEG- Polythene glycol
 See chart in Blaney page 162
Potentiators
Bovine Serum Albumin
•
•
•
•
•
Reduces zeta potential;
Affects stage II of
agglutination, between
sensitized cells and
forming lattices
Longer Incubation
Not sensitive for most
antibodies except in Rh
blood group
Does NOT enhance warm
AutoAb
Low Ionic Strength Soln
(LISS)
• LISS lowers the salt conc;
• Affects stage I of
agglutination; both the rate
and quantity of antibody
uptake
• Amount of serum in the
test is critical, since
increased sensitivity and
shortened incubation
time depend on ionic
conditions
• Enhances Cold AutoAb
Polyethylene Glycol (PEG)
• Exclusion of water
molecules; which results in
greater antibody uptake
• Affects stage I of
agglutination
• Tests are washed
immediately after 37c
incubation and Anti-IgG is
added (use monospecific
AHG)
• May require extra wash
• Enhances warm AutoAb
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
2 drops of the patients serum
+

1 drop of each panel cell
10
11
AC
IS Phase


Perform immediate spin (IS) and inspect for hemolysis;
grade agglutination
Record the results in the appropriate space as shown:
2+
0
0
37°C Phase
• Add potentiator and incubate for 15-30 mins. Read after
incubation (except if using PEG)
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 antibodies in patient’s serum will react
with RBCs in vitro
 To do this we use theAnti-Human Globulin reagent (AHG)
 Polyspecific (Anti-IgG andAnti-C3)
 Anti-IgG (monospecific)
 Anti-complement C3 (monospecific)
AHG Phase
 Wash cells 3 times with saline (manual or automated)
 Blot tube to absorb all excess liquid
 Add 2 drops of AHG and gently mix
 Centrifuge
 Read
 Record reactions
AHG Phase
2+
0
0
2+
0
0
2+
0
0
0
0
0
0
0
0
0 0
0 0
0 0
0 0 0
2+ 0 0
0 0 0
0 0 0
0 0 0
And don’t forget….
….add “check” cells to
any negative AHG !
IS
ALB
37°
AHG
CC
2+
0
0
2+
0
0
2+
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
2+
All cells are
negative at
AHG, so
add
“Check”
Cells
You have agglutination…now what?
CC
2+
0
0
2+
0
0
2+
0
2+
0
0
0
??
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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







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
Guidelines for the Interpretation of a
Panel
1
2
3
4
5
6
7
Autocontrol
Phases
Reaction Strength
Ruling Out
Matching the Pattern
Rule ofThree
Phenotyping the patient
1. Autocontrol
 A/C determines whether an alloantibody or an autoantibody
exists
 Usually a positive autocontrol or positive DAT indicates an
autoantibody or an antibody produced against recently transfused
red cells.
 Autoantibodies can be cold or warm, depending on the optimal
phase of reactivity
2. Phases of Reactivity
• The phase or reaction temperature at which agglutination
appears is an indication that the antibody is IgM or IgG
• IgM antibodies typically react at room temp(I.S.) or below
(18°C, 4°C)
• Common IgM:Anti-Lea, Leb, M, N, I and P1
• IgG react at 37c and/orAHG phase
3. Reaction Strength
• The strength of the reaction is a clue to the number of antibodies
present
• Whether one antibody or multiple antibodies
• Varying strengths 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
More on Dosage…
 Some antigens will react stronger with
the corresponding antibody when it is
inherited as homozygous
 For example:
Antigens M and N show dosage in these
ways:
M+N- = stronger
M+N+ = weaker
A little more on Dosage…
 When some antigens are inherited as
homozygous, and they belong to an
allele ‘pair’, the RBC will have more
antigens on it, so reaction is
stronger (M+N-)
 Heterozygous = having both alleles
expressed, which reduces the # of
antigens on the RBC for each
(M+N+)
So, which antigens show dosage?
•
•
•
•
•
Rh (E,e and C,c)
Duffy (Fya, Fyb)
Lutheran (Lua, Lub)
Kidd (Jka, Jkb)
MNS (M,N and S,s)
Rh and Duffy are Lutheran,
and their Kidds eat MNS (M&Ms)
4. Ruling Out
• Rule out: to eliminate the possibility that an antibody exists in
the serum based on non-reactivity with a particular antigen
• Panel cells that give negative (0) reactions with all tested phases
can be used to “rule out” antibodies
• See Blaney page 165 -166
1. Ruling Out- cross out antigens that
show NO REACTION in any 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
0 0 0
Do NOT cross out heterozygous antigens that show dosage.

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
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
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5. Matching the Pattern
• The next step in panel interpretation is to look at he reactions
that are positive and match the pattern.
• When a single antibody is present, the pattern of reactions
observed matches one of the antigen columns (easiestABID)
• See Blaney panel, page 166
Circle antigens not crossed out
2+
0
0
2+
0
0
2+
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
Consider antibody’s usual reactivity
2+
0
0
2+
0
0
2+
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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, plus it’s a warmer reacting Ab
2+
0
0
2+
0
0
2+
0
2+
0
0
0
…Yes, there is a matching pattern!
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
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6. Rule of Three
• Identifying antibodies involves performing tests and making a
conclusion based on reaction patterns
• To make a scientific conclusion, these reactions must be
statistically greater than those of a random event
• The (p) value or probability value, must be .05 or less
for identification to be considered valid (95%
confidence)
• To obtain this probability, at least 3 antigen positive red cells that
react and 3 antigen negative red cells that do not react should
be observed
Rule of Three
• p value: probability value; value that provides a confidence
limit for a particular event
• Rule ofThree: confirming the presence of an antibody by
demonstrating three cells that are positive and three cells that are
negative
• If there are not enough cells in this panel, additional or
“selected cells” can be hand picked from another lot number
of panel cells to be used to get to the “rule of three”
Our previous example fulfills the
“rule of three”
3 Positive
cells
3 Negative
cells
2+
0
0
2+
0
0
2+
0
2+
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0

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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
Interpretation
antiLea
What if the “rule of three” is not fulfilled
with cells from my panel?
 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
 Selected Cell Panel
Review
 Again, it’s important to look at:
 Autocontrol
 Negative - alloantibody
 Positive – autoantibody or DTR (alloantibodies)
 Phases
 IS – cold (IgM)
 37° - warm reacting
 AHG – warm (IgG)…significant!!
 Reaction strength
 1 consistent strength – one antibody
 Different strengths – multiple antibodies or one antibody
showing dosage
Review (continued)
 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 mixed reaction strengths