Antibody Detection and Identification

Antibody detection and identification are performed by testing patient serum or plasma with reagent red cells.
Agglutination or hemolysis indicates sensitization of the reagent red cells by an unexpected antibody in the patient's
serum. The reagent red cells come with an antigram or antigen profile sheet. The antigram shows the phenotype of
each reagent cell used.
Antibody detection is performed using an antibody screening test. An antibody screen consists of 2 or 3 group O
reagent red cells with known antigen phenotypes. A positive antibody screen means that an unexpected antibody is
present in the patient's serum.
If the antibody screen is positive, the antibody must be identified by performing an antibody panel. A panel consists
of 10 to 20 group O reagent red cells with known phenotypes.
The image below depicts an antibody screening antigram. A "+" indicates the antigen is present on the cell and an
"0" indicates the antigen is absent.

Indirect Antiglobulin Test (IAT)

IAT is used to detect and identify antibodies. The test uses antihuman globulin (AHG) to detect in vitro sensitization
of red cells. Patient serum or plasma is incubated with reagent red cells with known antigen phenotypes. If an
antibody is present in the serum, it will bind to the reagent red cells with the corresponding antigen. Because IgG
molecules are incapable of producing macroscopic agglutination, AHG is needed to act as a bridge. The AHG used
in the IAT is Anti-IgG. Anti-IgG will bind to the patient's IgG antibody if present and facilitate macroscopic
agglutination. IgG antibodies present in the patient's serum or plasma are considered clinically significant.

Test Methods
One of three methods is used to detect and identify antibodies:

Tube

Gel

Solid phase

The Tube method consists of three phases:

Immediate spin (IS)
Patient serum or plasma and reagent red cells are combined, then centrifuged, and observed for agglutination.
Enhancement media such as albumin, LISS or PEG, can be added.
37 C incubation
The tubes are incubated at 37C for 15-60 minutes, depending on the enhancement media used. After the incubation
period, the tubes are centrifuged and observed for agglutination. The cells are washed 3-4 times to remove any
unbound antibody. This is an important step in the tube method. Improper washing may lead to false-negative
results.
Indirect antiglobulin test or Anti-human globulin (AHG)
AHG is then added, the tubes are centrifuged, and are observed for agglutination. Check cells are added to all
negative tubes. Check cells are cells coated with IgG and should react positively with the AHG in the tube. If check
cells are negative, the procedure was not performed correctly and should be repeated.

Gel cards are used in the Gel method. The cards have microtubules filled with a dextran acrylamide gel containing
anti-IgG. The gel acts as a filter for agglutinates. Patient serum and reagent red cells are added to the microtubules.
The card is incubated at 37 C for 15 minutes and then centrifuged for 10 minutes. Washing and check cells are not
required. If no agglutinates are present, the red cells form a pellet at the bottom of the gel, indicating that no
clinically significant antibodies were detected. Agglutination indicates an antibody was detected. If large
agglutinates are present, the red cells form a layer at the top of the gel. Small agglutinates are dispersed throughout
the bottom of the gel. A gel card is depicted below.

In the Solid phase method, RBC antigens coat microtiter wells. Patient serum or plasma is added to each well along
with a low ionic strength solution (LISS). The wells are incubated at 37C, and washing is then required. Instead of
AHG, indicator cells (cells coated with anti-IgG) are added. If sensitization occurred, indicator cells react with the
antibody coating the well and form a diffuse pattern. This is a positive reaction indicating an antibody is present. If
no sensitization occurred, indicator cells form a pellet at the bottom of the well. This is a negative reaction indicating
no clinically significant antibodies are present. The image below illustrates negative and positive reactions.

Increased sensitivity in detection of antibodies is seen when using the tube method with PEG or the gel method
(especially for mixed field reactions). Solid phase testing has a better sensitivity than just using the test tube method
with LISS.
Significance of Reactions at Different Phases of Testing
Antibodies have optimum temperatures for reactivity. Reaction readings can be made at different phases: after
immediate spin, after incubation at 37 C, and after the addition of antihuman globulin (AHG) and centrifugation.
Reactivity in a certain phase will help to determine whether the antibody is cold reacting (IgM) or warm reacting
(IgG). It will also help to distinguish between antibodies that are clinically significant and not significant.
Clinically significant antibodies that are capable of causing acute and delayed hemolytic transfusion reactions (HTR)
or hemolytic disease of the newborn (HDN) are usually IgG and react best in the AHG phase.
Readings can be done at all three phases, if a tube method is used. If a gel method or solid phase is used, readings
are done only at AHG.

Immediate spin: Antibodies reacting in this phase tend to be cold reactive. They are usually IgM class and
not clinically significant (with the exception of the A and B antibodies).

37C: Antibodies that react in this phase include strong IgM or IgG antibodies. After incubation, the tubes
are examined for the presence of hemolysis. If complement was bound during incubation then hemolysis
could be seen. NOTE: This reaction would only occur in serum samples. If EDTA plasma samples are used
for testing, the complement cascade has been halted. Magnesium and calcium ions are not available for
complement to be activated.

AHG: Antibodies reacting in this phase are considered clinically significant. They are usually warm reactive
and IgG.

Antibodies in the Duffy, Kidd, and Kell blood group systems (anti-Fya, anti-Jka, and anti-K) react most commonly at
the AHG phase of testing. Antibodies in the Lewis, MNSs and P blood group systems (anti-Lea, anti-M, and anti-P1)

are commonly IgM antibodies and react optimally at room temperature and below. Antibodies in the Rh system
(anti-E, anti-D, and anti-c) generally react at both 37 C and AHG.
Products Used to Facilitate Antibody Identification
Monospecific anti-human globulin (IgG) enables sensitized red cells to cross-link so that agglutination is visible.
Enhancement media are sometimes used to further promote agglutination and reduce incubation time.

Low ionic strength saline (LISS) is the most common enhancement media. LISS reduces the ionic strength
in the testing sample and causes reduction of the zeta potential. It increases antibody uptake and decreases
incubation time. Gel technology, which is widely used, operates in a LISS environment.

Polyethylene glycol (PEG): brings red blood cells (RBCs) closer together and concentrates antibodies by
removing water molecules from the testing sample. It is the most sensitive of the enhancement media;
strengthening almost all clinically significant antibodies. However, it will also enhance some clinically
insignificant antibodies as well. Centrifugation should be avoided when PEG is used. PEG can cause
aggregates to form if the sample (red cell - serum mixture) with PEG added is centrifuged. Reaction readings
should only be done at the AHG phase. PEG is very effective for enhancing the reactions of antibodies in the
Kidd blood group system, which are typically weakly reactive.

22% albumin: reduces zeta potential, bringing the RBCs closer together and enhancing agglutination.
Although not widely used for routine testing, it is still an effective enhancement medium, especially with
antibodies in the Rh system.

Detection of some IgG antibodies can be enhanced with enzyme test methods.

Proteolytic enzymes (eg, papain and ficin): denature some RBC antigens and remove negative charges
from the RBC membranes. This reduces the zeta potential, bringing the cells closer together, enhancing some
blood group antibody-antigen reactions. Enzyme techniques are particularly useful in the identification of Rh
antibodies and antibodies in the Kidd, Lewis, P, and I systems. However, enzymes reduce or destroy the
reactivity of other antigens including Fya, Fyb, M, and N. The effect of proteolytic enzymes on the S and s
antigens are variable.

Technically, enzymes are categorized as chemicals rather than potentiating agents in most blood bank references.
A number of other chemicals are available commercially for use in antibody identification and other blood
banking procedures including: dithiothreitol (DTT), EDTA/glycine/acid (EGA), and chloroquine diphosphate.
Regardless of which reagents are chosen for blood bank serologic testing, it is important for the technologist to be
aware of the advantages and disadvantages of the reagent. Manufacturers directions must be followed and suitable
controls included as appropriate.
Effect of Enzymes and Dithiothreitol (DTT)

Antibody

Effect of Enzymes

Effect of DTT

Anti- D

Ficin and Papain Enhanced

DTT Resistant

Anti-C

Ficin and Papain Enhanced

DTT Resistant

Anti-c

Ficin and Papain Enhanced

DTT Resistant

DTT Resistant
Anti-E

Ficin and Papain Enhanced

Acid Resistant

Anti-e

Ficin and Papain Enhanced

DTT Resistant

Anti-Cw

Ficin and Papain Enhanced

DTT Resistant

Anti-K

Ficin and Papain Resistant

DTT Sensitive

Anti-k

Ficin and Papain Resistant

DTT Sensitive

Anti-Kpa

Ficin and Papain Resistant

DTT Sensitive

Anti-Jsa

Ficin and Papain Resistant

DTT Sensitive

Anti-Fya

Ficin and Papain Sensitive

DTT Resistant

Anti-Fyb

Ficin and Papain Sensitive

DTT Resistant

Anti-Jka

Ficin and Papain Enhanced

DTT Resistant

Anti-Jkb

Ficin and Papain Enhanced

DTT Resistant

Anti-Lea

Ficin and Papain Enhanced

DTT Resistant

Anti-Leb

Ficin and Papain Enhanced

DTT Resistant

Anti-P1

Ficin and Papain Enhanced

DTT Resistant

Anti-M

Ficin and Papain Sensitive

DTT Resistant

Anti-N

Ficin and Papain Sensitive

DTT Resistant

Anti-S

Usually Ficin and Papain Sensitive; Some Variable

DTT Resistant

Anti-s

Usually Ficin and Papain Sensitive; Some Variable

DTT Resistant

Initial Steps for Identifying an Antibody

Patient history can provide valuable information when trying to identify an antibody. For example, some antibodies
are associated with a particular race or ethnicity. Transfusion or pregnancy history are helpful to determine if the
antibody is naturally occurring or immune.
The antibody screen can provide sufficient data to make initial hypotheses regarding the likely antibody specificities
and may be useful to presumptively rule-out some antibody specificities. When analyzing the antibody screen data,
the strength/characteristics of reactions (for example, mixed-field or weak versus strong), the phase of testing (for
example, room temperature versus AHG), and the pattern of reactivity (which cells react and which do not) are all
important factors that will provide clues about the possible identification of the antibody(ies). Many antibodies
exhibit dosage, that is, they react more strongly with homozygous cells than with heterozygous cells.
If a tube method is used, reactions are usually read at immediate spin, 37C and AHG phase. If a gel method is used,
reaction readings are done only at AHG phase.

Reactions occurring only at immediate spin phase could indicate a possible IgM antibody, cold agglutinin, or
rouleaux.

Reactions occurring in the 37C and AHG phase could indicate a possible IgG antibody.

Reactions occurring in both reaction phases could indicate a combination of both IgM and IgG antibodies or
a strong IgM antibody that carries through to AHG phase.

The presence of multiple antibodies should be considered if reactions vary in strength or there are two separate
reaction patterns in the IS and AHG phases.

Initial Observations of Antibody Panel

Look at the phase in which reactions are occurring. Reactions at immediate spin (IS) usually are not
clinically significant. Reactions at AHG are clinically significant.

Look at the strengths of reactions. Varying strengths of reactions could indicate dosage. In the example
below, the heterozygous (Jka Jkb) cells react weaker with a 2+ reaction. The homozygous cells react stronger
with a 3+ reaction.

Varying strengths may also mean more than one antibody is present.

Rule-Out Procedures
Rule-out (also referred to as exclusion or cross-out) is a process by which antibodies are identified as being unlikely
in a given sample due to the absence of an expected antigen-antibody reaction. In other words, the absence of a
reaction is noted with a cell that is positive for the corresponding antigen. Antibodies that could not be responsible
for the reactions seen are excluded or ruled-out.

Rule-out, while very useful, can lead to error. Ruling out an antibody should be combined with other supporting data
to increase confidence in the solution; the more data collected, the higher the probability that the final solution is
correct.
Non-reactive cells are selected for rule-out. To be classified as non-reactive, a cell must NOT have reacted in any
phase of testing in a given panel or screen.
In the case of cold antibodies: if reactions are only occurring at immediate spin and are negative in the AHG phase,
then that panel cell can be used as a rule out cell for IgG reactive antibodies but not for antibodies that react at
immediate spin (IgM).
If there is no reaction with a panel cell then it is possible that antibodies to the antigens on that panel cell are not
present in the sample being tested.
Rule-Out Procedures, continued
In the antibody screen shown below, cell 3 meets the criteria to be used as a rule-out cell.

Using the logic that if the rule-out cell is positive for a given antigen, it should have reacted with the corresponding
antibody, the technologist can rule-out antibodies that correspond to antigen-positive cells. For example, cell 3 in the
screen above is positive for big K. If anti-K were present, it should have reacted. It did not react. Therefore, anti-K is
most likely NOT present and can be presumptively ruled-out.
To increase the probability that rule-out will not mistakenly eliminate a weakly-reacting antibody that exhibits
dosage, most laboratories will rule-out using only cells that are homozygous for the corresponding antigen for those
systems that generally show dosage. Generally these include: C, c, E, e, Fya, Fyb, Jka, Jkb, M, N, S, and s. For
example, in the screen above, cell 3 is homozygous positive for c (c positive, C negative). Since the cell did not
react, anti-c can be presumptively ruled-out. Exemptions are usually made for low-prevalence antigens, which are
rarely expressed as homozygous (K, Kpa, Jsa, Lua).
Using these principles, in the antibody screen example above, the antibodies highlighted in red are presumptively
ruled-out:

Ruling Out Example

The first step for ruling out is to choose the cells appropriate for exclusion. When using the tube method, all phases
should be non-reactive to rule out. If using a gel or solid phase method, only the AHG phase is examined.
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. This is why non-reactive cells are chosen for ruling out.
The following example is an abbreviated panel performed with a gel method. With each non-reactive cell, a rule out
is performed. Below, the first non-reactive cell is examined and D, C, e, k, and s are ruled out on homozygous cells.

The second non-reactive cell is examined and c and E can be ruled out. The cell is heterozygous Ss so even though S
is present on the cell, it cannot be ruled out.

Ruling Out Example, continued

The process continues until all non-reacting cells have been examined for possible rule-outs. On cell #5, no
additional rule-outs can be performed. The technologist should move on to the next cell.

On cell #7, S can be ruled out.

Once rule out is complete, there should be one or more antigens that are not crossed out. The antigen should be
present on cells that reacted with the patient serum and absent on cells that did not react. In this example, the pattern
matches perfectly with K. This pattern strongly supports that the patient has Anti-K. If other antibodies could not be
ruled out, a selected cell panel would need to be performed. (Selecting additional cells are dicussed later in this
course.)
NOTE: In cases of multiple antibodies , a perfect matching pattern will be difficult to obtain.

Conclusive antibody identification is attained when the patient's serum is reactive with at least 3 antigen-positive
cells and non-reactive with 3 antigen-negative cells. Another confirmation of an alloimmune antibody is the patient's

red cell phenotype is negative for the corresponding antigen. In the picture below, the positive reactions are circled
in pink and the negative reactions are circled in green. This patient's red cells should also be K negative.

Case Study 1: Immune Alloantibody

A 42-year-old male received six units of Red Blood Cells during open heart surgery six months ago. His antibody
screen was negative at that time. He has returned for a follow up surgery and his antibody screen is now positive
with both screen cells at the AHG phase.
(IS = Immediate Spin; AHG = Antihuman Globulin Phase; CC = Check Cells; AC = Auto Control; ND= Not done)

Refer to Case Study 1 panel below to see reactions of antibody panel.

Observations:

Reactions are occurring at the AHG phase. This indicates an IgG clinically significant antibody.

Reactions are multiple strengths indicating either multiple antibodies or a single antibody showing dosage.

The autocontrol is negative which indicates the patient has an alloantibody.

he next step is to rule out. The cells used for ruling out are highlighted in yellow. Only cells that had negative
reactions in all three phases can be used. Remember to only rule out on homozygous cells, with the exception of K.
As demonstrated in the panel below, cell#2 is the first cell selected for ruling out. D, C, e, CW, K, k, KpbJsb, Jkb, Leb,
N, P1, and Lub can all be excluded on this cell. The cells highlighted in pink are heterozygous and are not accepted
for rule outs. The exclusion process should continue using all applicable cells.

The image below demonstrates the completed exclusion process. The antigens without highlighting could not be
ruled out: Kpa, Jsa, Jka, and Lua. The following antigens are not indicated on the panel: Kpa, Jsa, and Lua. The serum
reactions cannot be occurring because of these antigens. The next step is to look for a matching pattern.

Example

The correct response is Jka. This is the most likely antibody causing the reactions in the patient's serum. Kpa, Jsa, and
Lua are not indicated on this panel. While they cannot be fully excluded, their corresponding antibodies cannot be
causing the reactions seen on this panel. All other clinically significant antibodies have been ruled out.

Rule Out Procedures: Selecting Additional Rule-Out Cells

Once an antibody hypothesis is generated, additional cells may be selected to rule out any other commonly
encountered antibodies that could not be ruled out with the initial antibody screen and panel. Cells should be
selected that are negative for the antigen(s) that correspond to the hypothesized antibody and positive for the
antigen(s) to commonly encountered antibodies that have not been ruled out. If not ruled out, laboratories often
select cells for at least the following: anti-D, anti-C, anti-c, anti-E, anti-e, anti-K, anti-k, anti-Fya, anti-Fyb, Anti-Jka,
anti-Jkb, anti-Lea, anti-Leb, anti-P1, anti-M, anti-N, anti-S, and anti-s.
Antibodies to antigens of very low incidence (for example, anti-Jsa) are generally not eliminated in initial testing, but
in most settings it is not feasible to try and find rule-out cells. In these cases, it is important for the technologist to
understand that these antibodies HAVE NOT been ruled-out due to limitations in the test system.
Rule-Out Procedures: Selecting Additional Rule-Out Cells-- Example
If a patient is hypothesized to have an anti-Fya after the initial antibody screen and panels are completed but anti-E
could not be ruled out, a cell should be selected to do so. This rule-out cell should meet the following criteria:
1. Negative for Fya (so it will not react with the anti-Fya that has been identified in the serum of the patient).
2. Homozygous for big E (big E-positive and little e-negative, so that it will react most strongly with anti-E if
present).
In the panel shown below, four cells (36) meet the first criterion (Fya negative). Of these cells, three (4-6) are big Epositive, and of these, only one cell (4) is homozygous for E (big E positive and little e negative). Therefore, cell 4
would be a suitable rule-out cell in this case.

Based on initial serologic testing, a patient is hypothesized to have an anti-K, but anti-E cannot be ruled out. In this
case, which of the following is the BEST cell to select to rule-out anti-E?
You answered the question incorrectly.
The correct answer is highlighted below
Big K positive; big E heterozygous
Big K positive; big E homozygous
Big K negative, big E heterozygous
Big K negative, big E homozygous
Feedback
Rule-out cells should be negative for the antigen that corresponds to the antibody present in the serum of the patient
and positive (preferably homozygous) for the antigen that corresponds to the antibody that you are trying to rule-out.
In this case, the cell should be big K negative and big E homozygous positive.
Picking Selected Panel Cells Conservatively
Choose cells that can help rule out more than one antibody at a time in order to help decrease supply usage and tech
prep time.
Example: Ruling out C, Fyb, and M if you have a suspected Jka
c

Fya

Fyb

Jka

Jkb

Panel cell 9
Panel cell 10
Panel cell 11
Panel cell 12

0
0
+
+

+
+
+
+

0
+
0
+

+
+
+
+

0
0
0
0

+
+
+
+

+
0
+
+

0
+
0
0

Instead of running 3 separate cells to rule out the antibodies, you can choose one that is homozygous positive for M,
C, Fyb and negative for Jka. Panel cell 9 works in this case.
If the only antibody that is present is Jka, then your test results should be negative. If the results are positive then
further rule outs will be needed to determine what is present.
Rule-Out Procedure Summary Guidelines
These guidelines should be followed when performing initial rule-out procedures:

Only cells that did NOT react can be used to rule-out.

For antibodies that exhibit dosage, homozygous cells are preferred for rule-out.

Rule-out is not free of error and should be evaluated in the context of other data, such as the phase of testing.

Ungraded Practice Question

These antibody panel results were obtained on a patient sample. Which of the following antibodies could account for
all of the reactions? (A PDF of this antibody panel is available on this page and can be printed to use in working
through the rule-out process).
Please select the single best answer
Anti-D
Anti-Jka
Anti-Lua
Anti-M
page 23 antibody panel [click to view / print]
Adobe Acrobat PDF file