BB LEC: ANTIBODY SCREENING AND
IDENTIFICATION
Green = Blaney’s
Blue = Powerpoint from upperbatch
Red= Harmening
Immune Alloantibodies
 Primarily important unexpected antibodies
 Produced in response to RBC stimulation through
transfusion, transplantation, or pregnancy
*Other unexpected Abs may be naturally occurring (without RBC
stimulation)
Naturally Occurring Abs
1. Are formed as a result of exposure to environmental sources:
pollen, fungus, bacteria [they have structures similar to some
RBC antigens)
Passively Acquired Antibodies
2. Third category of Abs
3. Abs produced in one individual and then transmitted to another
via plasma-containing blood components or derivatives such as
Intravenous Immunoglobulin (IVIG)
Clinically Significant Antibodies
 Cause decreased survival of RBCs possessing the target
antigen
 Are typically IgG antibodies that react at 37*C or that react
in AHG phase of IAT
Antibody Screening Test
 Application of Indirect Antiglobulin Test
 Required as part of pre-transfusion compatibility testing
 May be included when evaluating the compatibility of
hematopoietic progenitor cell (HPC) and bone marrow
donors with the intended trans- plant recipient
 Included in standard prenatal testing for obstetric patients
to evaluate the risk of HDFN in the fetus and to assess the
mother’s candidacy for Rh-immune globulin (RHIG)
prophylaxis.
 Perform both major and minor crossmatch
 Use patient’s and donor’s sera to determine the
presence of unexpected antibodies or clinically
significant antibodies
 If a person with an antibody is exposed to donor
cells with the corresponding antigen, serious
side effects can occur
 Determines whether an antibody to a red cell
antigen has been made.
Patient serum
 Performed to detect antibodies in the following
people
1. Patients requiring transfusion
2. Women who are pregnant or following
delivery
3. Patients with suspected transfusion
reactions
4. Blood and Plasma Donors
especially done in women with
previous pregnancies and patients
who have undergone multiple
transfusions
 In blood banking, we test “knowns” with
“unknowns”
 When detecting and/or identifying antibodies,
we test patient serum (unknown) with reagent
RBCs (known)
 Donors may be used as a source of antigen-typing sera
and antigen-negative RBC units
 Involves reaction between patient’s serum
(preferred)/plasma with 2 or 3 reagents whose
phenotypes were already determined with
multiple antigens incubated at 37⁰C and
performing an Indirect Antihuman globulin Test
(IAT) for the detection of IgG antibodies.
 Antibody screening cells are group “O” reagent
red cells.
 Purpose: To detect unexpected antibodies (no
Anti-A and Anti-B)
 Importance: For Selection of appropriate units
for transfusion (safe transfusion) and
investigation of Hemolytic Disease of Newborn
(HDN), Imuune Hemolytic Anemia, and
Transfusion Reactions
 Whatever you do in Antibody Screening, you
also do in Antibody Identification
 There are many possible unexpected antibodies
that you can detect and using screening cells
can help limit those antibodies for detection
 Antibody Panel vs. Screen
 Screening Cells and Panel Cells are the same
with minor differences:
1. Screening cells
 Antibody detection
 Sets of 2 or 3 vials
 packaged in sets of two or three cell
suspensions, each having a unique
Autocontrol combination of antigens. Within the set,
+ there should be one cell that is positive
for each of the following antigens: D, C,
c, E, e, K, k, Fya, Fyb, Jka, Jkb, Lea,
Leb, P1, M, N, S, and s.
2. Panel cells
 Antibody identification
 At least 10 vials per set
 An antibody panel is just an extended version of
an antibody screen
Group 6 | 3AMT 16’-17’
 Performed after Ab detection to determine the specificity of  2 drops of serum + 1 drop of SC-
the antibody/antibodies 1, SC-2 to respective tubes
 The screen only uses 2-3 cells: 2. AHG reagent
 Adding AHG reagent allows for the
agglutination of incomplete antibodies.
 Polyspecific AHG reagent or Polyvalent or Broad
Spectrum Coombs’ serum
- contains antibodies to both IgG and complement
 An antibody panel usually includes at least 10 components, either C3 and C4 or C3b and C3d
panel cells: - Abs to C3 especially to C3d are more desirable in the
reagent as these are more abundant on RBC surface
during complement activation and lead to fewer false-
positive reactions
** Jka react with complement alone
 Monospecific AHG reagent
- contains IgG only (clinically significant lang) to avoid
time-consuming investigation of insignificant antibodies

3. Coombs’ Control/Check Cells

4. Enhancement reagents
 Potentiators
 Group O red blood cells  Added to the cell/serum mixture before
the 37°C incubation phase to increase
the sensitivity of the test system.
 Allows for a shortened incubation time
 BSA, PEG, and LISS
A. 22% Albumin
- In an electrolyte solution, negatively charged
RBCs are surrounded by cations, which in turn
are surrounded by anions. The effect is to
produce an ionic cloud around each RBC, forcing
the cells apart
- reduces the zeta potential and disperses the
 Panel charges, thus allowing RBCs to approach each
other and increasing the chances of agglutination
1. Each of the panel cells has been
antigen typed (shown on antigram) *Zeta Potential
 + refers to the presence of the - difference in electrical potential between the
antigen surface of the RBC and the outer layer of the
 0 refers to the absence of the ionic cloud
antigen
B. Low Ionic Strength Solution (LISS)
2. An autocontrol should also be run - contains glycine in an albumin solution
with ALL panels - lowers zeta potential
3. The same phases used in an antibody - increases the uptake of antibody onto the RBC
screen are used in a panel during the sensitization phase.
 IS
 37⁰ C. Polyethylene Glycol (PEG)
- PEG in a LISS solution removes water from the
 AHG test system, thereby concentrating any
antibodies present, increasing the degree of RBC
 Requirements for antibody screening (also the sensitization.
requirements for antibody identification): - NO centrifugation after the 37*C incubation as
1. Reagent RBC PEG can cause non specific aggregation of cells
- generally, more sensitive than LISS, albumin, or
 For screening, use screening
saline systems BUT in Px with elevated plasma
cells (SC-1, SC-2, SC-3; all are protein (Multiple Myeloma), PEG is not used due
type O Red Cells whether for to increased precipitation of proteins.
screening or identification)

Group 6 | 3AMT 16’-17’

METHODS PERFORMED:  Omission of washing and Coombs;
control steps result in fewer hands-on
 TUBE METHOD steps for MTs
 Rxns are stable for yp to 24 hrs and
 Traditional method for detecting Abs is an IAT
may be captured electronically leading
performed in a test tube
to standardized grading of rxns
 RBC agents, enhancement reagents, and AHG
 Mixed-field rxns are more apparent
reagents are used to sensitize the reagent
 Ability to automate many of the
RBCs with the patient’s antibodies, followed by
pipetting and reading steps thereby
formation of visible RBC agglutinates.
allowing increased productivity (One of
the greatest adv)
Method:
 Disadvantage:
o The patient’s serum or plasma is mixed with RBCs that
 Need for incubators and centrifuges
have known antigen content.
that can accommodate the gel cards
o The test may include an immediate spin phase to detect
antibodies reacting at room temperature.
 Immediate Spin Phase - not required and  SOLID PHASE ADHERENCE METHOD
may lead to the detection of clinically  Commonly used to perform the antibody screen
insignificant cold antibodies  One example: Immucor’s Capture-R where RBC
o Must include a 37°C incubation phase during which IgG antigens coat microtiter wells rather than being
molecules sensitize any RBCs that possess the target present on intact RBCs
antigen, coating those RBCs with antibody.  Rather than traditional AHG reagent, indicator red
o Enhancement media may be added to increase the blood cells that have been coated with anti-IgG are
degree of sensitization. added
 Depending on the enhancement added,  Advantages:
centrifuge tube and observe for hemolysis  May perform pipetting steps and
and agglutination after incubation determine the degree of reactivity by
o Tubes are then washed with 0.9% saline a minimum of taking multiple readings of light
three times to remove all antibodies that remain unbound. transmission through each well
o AHG reagent is added to each tube. Centrifuge and  Smaller sample size making it ideal in
observe for hemolysis. In this phase, hemolysis may pediatric setting
appear as a loss of cell button mass.  LISS rgt that changes color when
 If the RBCs are coated with IgG antibodies, added to serum or plasma ensuring
the anti-IgG antibody in the AHG reagent adequate sample is present in the test
will create a bridge between sensitized system
RBCs, resulting in observable agglutination.  Disadvantage: Need for careful pipetting due to small
sample and rgt volume

INTERPRETATION

 Agglutination or Hemolysis at any stage of testing is a

positive test result, indicating the need for antibody
identification.
 Evaluation of the antibody screen results can provide clues
and give direction for the identification and resolution of the
antibody or antibodies.

A. In what phase(s) did the rxn occur?

o IgM antibodies
- best react at room temperature or lower
- capable of causing agglutination of saline-suspended
 GEL METHOD RBCs (immediate spin rxn)
 performed using a microtubule filled with a dextran - anti-N, anti-I, and anti-P1
acrylamide gel
o IgG antibodies
 screen cells used for this technique meet the same
- best react at AHG phase
criteria as for the tube test but are suspended in LISS
- Rh, Kell, Kidd, Duffy, and Ss
to a concentration of 0.8%.
o Lewis and M antibodies may be IgG, IgM or a mixture of
 Patient’s serum or plasma specimen and screen cells
both
are added to a reaction chamber that sits above the
gel
B. Is the autologous control negative or positive?
 Advantages:
o Autologous control
 As sensitive as PEG test tube method

Group 6 | 3AMT 16’-17’

 - patient’s RBCs tested against the patient’s serum or
plasma in the same manner as the antibody screen
- (+) autologous control or direct antiglobulin test (DAT)
may indicate presence of autoantibodies or antibodies to
medications (recent transfusion; previous 3 months)
- (+) antibody screen and (-) autologous control:
alloantibody has been detected

C. Did more than one screen cell sample react? If so,

did they react at the same strength and phase?
o More than one screen cell sample positive when:
 Px has multiple Abs
 Single Ab’s target antigen is found on more than
one screen cells
 Px serum contains an autoantibody
o Multiple antibodies are most likely when screen cells react
at different phases or strengths, and autoantibodies should
be suspected when the autologous control is positive.

D. Is hemolysis or mixed-field agglutination present?

o In-vitro hemolysis: anti-Lea, anti-Leb, anti-PP1Pk, anti-Vel
o Mixed-field agglutination: anti-Sda and Lutheran antibodies

E. Are the cells truly agglutinated or is rouleaux

present?
o Rouleaux: Px with altered albumin:globulin (seen in
multiple myeloma); Px who have received HMW plasma
expanders (dextran) causing nonspecific aggregation
o Characteristics of Rouleaux:
 “Stacked coin” appearance microscopically
 Observed in all tests containing the Px serum,
including the autologous control and the reverse
ABO grouping
 Does not interfere with the AHG phase because
Px serum is washed away before adding the AHG
rgt
 Dispersed by adding 1-3 drops of saline, unlie
agglutination

LIMITATIONS
**When using a three-cell screen set, a negative result with all three
cells gives the technologist 95% confidence that there are no
clinically significant antibodies are present.

1. The screen will not detect antibodies when the antibody titer has
dropped below the level of sensitivity for the screening method
employed.
2. Antibodies formed in response to transfusion found that two-
thirds of antibodies were no longer detectable 5 years after
formation.
3. The screen also cannot detect antibodies directed against low-
prevalence antigens that are not present on any of the RBCs in
the screen cell set
4. Antibodies showing dosage may not be detected if none of the
screen cells have homozygous expression of the target antigen
FACTORS INFLUENCING SENSITIVITY OF ANTIBODY
SCREEN:
1. Cell:Serum
– 2 drops serum: 1 drop RCS (proper balance)
- Altered depending on the test employed (if Ab is weak,
increase amount of serum 4-10 drops to provide more Abs
to react with available antigens; only done when potentiators
have not been included in the test system)
 Antibody in excess: False negative (Prozone)
 Antigen in excess: False negative (Postzone)

2. Temperature and Phase of Reactivity

- In pretransfusion compatibility testing, the focus is on
clinically significant Abs, which generally react at 37*C or

Group 6 | 3AMT 16’-17’

 with anti-IgG in the AHG rgt o Coombs’ control cell if indicated
- Immediate spin and RT phase may be omitted to limit the
detection of insignificant cold Abs  Step 1: IS Phase
- To identify Abs reacting at RT: incubate screen at 18*C or
4*C to enhance reactivity; autocontrol included to aid in the
1. Perform immediate spin (IS) and
detection of common cold autoAbs such as anti-I or anti-IH grade agglutination; inspect for
hemolysis
3. Length of Incubation 2. Record the results in the appropriate
- Dependent on the medium in which the reaction takes space
place: Saline (30 mins to 1 hr); Potentiators (10 mins)
 Step 2: LISS/37°C Phase
4. pH 1. 2 drops of LISS are added, mixed and
- most Abs react at neutral pH (6.8-7.2) incubated for 10-15 minutes
- some Anit-M: pH 6.5 (acidifying the test system may aid in 2. Centrifuge and check for
identifying anti-M from other Abs) agglutination
3. Record results
Antibody ID Testing  Step 3: AHG
– patient’s serum or plasma is tested against additional RBCs 1. Wash cells 3 times with saline
possessing known antigens. (manual or automated)
 Patient History 2. Add 2 drops of AHG and gently mix
 Race
- Anti-U is more frequently associated with persons of
 Centrifuge
African descent (mostly U negative individuals)  Read
 Transfusion and Pregnancy History  Record reactions
- Px who have been exposed to “non self” RBCs via
transfusion or pregnancy are more likely to have produced INTERPRETING ANTIBODY PANELS
immune Abs
- Naturally occurring antibodies (e.g., anti-M, anti-Leb)  There are a few basic steps to follow when
should be suspected in patients with no transfusion or interpreting panels
pregnancy history. 1. “Ruling out” means crossing out antigens that
- IVIG and RhIG may passively transfer Anti-A, Anti-B, Anti-
did not react (neg result) (Exclusion)
D
- Antilymphocyte Globulin may passively transfer 2. Circle the antigens that are not crossed out
Antispecies antibodies 3. Consider antibody’s usual reactivity
 Positive Autologous Control/DAT 4. Look for a matching pattern
- certain infectious and autoimmune disorders
- delaye hemolytic transfusion rxn (in px transfused within (BB Lab experiment)
the past 3 months)
 Information regarding recent transfusions  Make use of Antigram (table for elimination of
- Postive rxns may be caused by the presence of donor
unexpected antibodies that are not found in the
RBCS remaining in the px circulation showing mixed-field
agglutination serum)
 A tube is labeled for each of the panel cells plus 1. For both Screening (use of screening
one tube for AC: cells) and Identification (use of panel
cells, which are also type O red cells
that have been previously antigen
typed; expanded version of screening
test)
2. Both SC-1 and SC-2 cannot both have
positive AHG or negative AHG as you
will not be able to detect any possible
unexpected antibodies
 After reagent cells and patient plasma or serum 3. If negative AHG reaction, cross out all
are added, several phases of testing are the positives in the antigram,
conducted on each tube: meaning those that are not crossed
o Immediate Spin (IS) out are possible antibodies detected
o Incubation Period (10-15 minutes) in the serum (vice versa is also the
o Wash 3X same, it depends on which of the two
o AHG
Group 6 | 3AMT 16’-17’
 reactions [positive or negative] are clinically significant (if it can cause
fewer in the AHG phase) HDN and HTR, mostly IgG antibodies)
 (+) means the presence of or not (IgM antibodies)
antigen while (0) means the
absence of antigen REMEMBER:
 If, for example, AHG is positive, An antibody will only react with cells that have the
then there would be a specific corresponding antigen; antibodies will not react with
antibody that had reacted with cells that do not have the antigen
its corresponding antigen [thus
having a (+)]
4. After finding out unexpected
antibody through elimination using
antigram, you need to know if it is

EXAMPLE:
RULLING OUT

 Highlight cells that show NO REACTION in ALL phases; Moving from left to right, Cross out antigens from the
highlighted area only; do NOT cross out heterozygous antigens that show dosage [Rh(not D), Kidd, Duffy, MNSs]

CIRCLE ANTIGENS NOT ELIMINATED

Group 6 | 3AMT 16’-17’

CONSIDER USUAL REACTIVITY

 Anti-Lea is normally a Cold-Reacting antibody (IgM), so it makes sense that we see the reaction in the IS phase of
testing; The anti-E will usually react at higher temperatures

LOOK FOR MATCHING PATTERN

INTERPRETATION: anti- Lea

Group 6 | 3AMT 16’-17’

Another Example:

In the table below: reactions are caused by alloantibody, not by

autoantibody
 First, we perform exclusion. To perform
exclusions or “rule-outs,” the RBCs that gave a Is there sufficient evidence to prove the suspected
negative reaction in all phases of testing are antibody? The patient’s serum reacted with seven Fya-
examined. positive cells (1, 3, 4, 6, 7, 10, and 11) but did not react
 Cell numbers 1, 3, 4, 6, 7, 10, and 11 reacted with four Fya-negative cells (2, 5, 8, and 9). As a result,
positively (AHG phase) and cannot be used for additional cells need not be tested to increase the
exclusions. confidence level to 95%, and the identification of anti-
 Cell number 2 reacted negatively and can be
Fya is conclusive.
used to exclude D, C, e, Cw, K, Kpb, Jsb, Fyb,
Jka, P1, M, S, Lub, and Xga
 Cell number 5 can be used to exclude k, Jkb, and
Leb
 Cell number 8 is used to rule out c, Lea, and Lua
 Cell number 9 eliminates N and s.
 Leaving anti-E, anti-Kpa, anti-Jsa, and anti-Fya
as possible antibodies present.

 Do all of the positive cells react at the same

phase, or do any react at different or multiple
phases? All reactions occurred in the AHG
phase with strengths of both 2+ and 3+. Multiple
antibodies or a single antibody showing dosage
should be considered. 

 Does the serum reactivity match any of the
remaining specificities? 
 The serum reactivity
matches the Fya pattern exactly. The serum gave
uniform positive results with all Fya-positive
cells (1, 3, 4, 6, 7, 10, and 11) and negative
results with all of the Fya- negative cells (2, 5, 8,
and 9). 

 Is the autologous control (last row in panel
antigen pro- file) positive or negative? The auto-
control is negative, indicating that the positive
Group 6 | 3AMT 16’-17’
CONSIDERATIONS
Autocontrol
 tests the patient’s serum with his or her own
red cells and includes the potentiator used
 optional
 Negative - alloantibody
 Positive – autoantibody or alloantibody to
recently transfused cells (RBCs have a life span
of 120 days)
Direct antiglobulin test (DAT)
 performed on the patient’s cells without serum
and potentiator or an incubation step
Phases
 IS – cold (IgM)
 37° - cold (some have higher thermal range) or
warm reacting
 AHG – warm (IgG) or complement…significant!!
If there is a reaction in ALL cells, may be autoantibody
Reaction strength
 1 consistent strength – one antibody
 Different strengths – multiple antibodies or
dosage
Dosage
• 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 cause a
reaction too weak to be seen
Matching 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 variable reaction
strengths
To confirm the antibody identified from a panel, it is
important to
– Use the rule of three
– Phenotype the patient’s RBCs
RULE OF THREE
 at least three antigen-positive red cells that
react and three antigen-negative red cells that
do not react should be observed
 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
 This rule is applied separately when multiple Abs are
present
 How is it demonstrated?
Patient serum MUST be:
1. Positive with 3 cells with the antigen
2. Negative with 3 cells without the
antigen
 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 (or screening cells)
PHENOTYPING
 In addition to the rule of three, antigen typing
the patient red cells can also confirm an
antibody
 How is this done?
Group 6 | 3AMT 16’-17’
 o Only perform this if the patient has NOT - Directed against antigens expressed on one’s own RBCs
been recently transfused (donor cells • should be suspected if the autocontrol or DAT is
could react) positive.
o If reagent antisera (of the suspected • Techniques such as adsorption and elution to identify
antibody) is added to the patient RBCs, the antibody on the red cell and the alloantibody in
a negative reaction should result the serum.
• May mask the presence of clinically significant
alloantibodies because they react with all RBCs tested
PROBLEMS IN ANTIBODY DETECTION AND
IDENTIFICATION

1. Single antibody specificity

• pattern easily identifiable with a panel. Confirmation
that the patient is negative for the corresponding
antigen helps confirm the specificity. 


2. Multiple antibodies
• necessitate the use of carefully selected cells and
antibody techniques. Distinguishing multiple
specificities necessitates understanding of antibody
characteristics. The determination of the patient’s red
cell phenotype is also useful. 


3. Antibodies to high-incidence antigens

• should be suspected if all panel cells are positive.
Identification depends on locating cells that are
negative for high-incidence antigens and determining
whether underlying antibodies exist. 


4. Low-frequency antibodies
• usually found with other antibodies. Identification
depends on the availability of additional cells for
testing. Transfusions should not be delayed to
determine specificity. 


5. Weak IgG antibodies

• can be enhanced by using a different potentiator,
increasing the serum-to-cell ratio, or increasing
incubation time. The detection of newly formed
alloantibodies in recently transfused patients is
especially important. 


6. Cold alloantibodies
 usually clinically insignificant. Avoiding the reactions
or using neutralization or prewarm techniques to
eliminate agglutination is sometimes necessary.

7. Autoantibodies
• either cold or warm type
- Complicate detection of clinically significant Abs

Group 6 | 3AMT 16’-17’

 What we did in the lab

CN Rh-Hr Kell Duffy Kidd Lewis P MNSs Lutheran S-L Rxn

D C E c e CW f V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga IS 37°C AHG CC
I + 0 + + + 0 0 0 + + 0 + + 0 0 + + + + 0 + + + + 0 0 + 0 0 0 0 +
II 0 + 0 0 0 0 + 0 0 0 + 0 0 + + 0 0 0 0 + 0 0 0 0 + + 0 + 0 0 +
1 + + + 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 0 + + 0 + 0 0 0 +
3 0 0 + + + + 0 + + + 0 0 + 0 + + 0 + 0 + + + 0 + + 0 + + 0 0 +
4 + + 0 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + 0 + + + 0 + 0 0 0 +
5 0 + + + 0 + + 0 0 0 + + + 0 + + + 0 0 + 0 + + 0 + 0 + + 0 0 +
6 0 + 0 + + 0 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 + 0 + 0 0 0 0 +
7 + 0 0 + + 0 + 0 + + 0 0 + + + 0 + + + 0 0 + + + + 0 + 0 0 0 +
8 + + + 0 + + + + + + + 0 + 0 0 + + 0 + + 0 + + 0 + + + + 0 0 0 +
9 0 + 0 + + + 0 0 0 0 + + 0 + + + 0 0 + 0 + + 0 + 0 + 0 0 0 0 +
10 0 + + + 0 0 0 0 + + + 0 + + 0 + 0 + + + 0 + + 0 + + 0 + 0 0 0 +
11 + 0 + + + 0 0 + + 0 0 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 0 0 0 +

Example:

SC-1 = 0, 0, 0, + ; SC-2 = 0, 0, + ; AB-C 1, 8, 10, 11 = 0, 0, 0, +

1) Antibody Screening Test

 In this test, the reagents used are SC-1 (I) and SC-2 (II)
 The above table is an antigram where we will be determining the possible unexpected antibodies present in the patient’s serum by eliminating and
narrowing down the possible antibodies
 First, look at the first two rows of the antigram. If the reaction in the AHG phase of either I or II (not both) is negative, then all reactions that are positive
in that row are eliminated/crossed out

Group 6 | 3AMT 16’-17’

CN Rh-Hr Kell Duffy Kidd Lewis P MNSs Lutheran S-L Rxn

D C E c e CW f V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga IS 37°C AHG CC
I + 0 + + + 0 0 0 + + 0 + + 0 0 + + + + 0 + + + + 0 0 + 0 0 0 0 +
II 0 + 0 0 0 0 + 0 0 0 + 0 0 + + 0 0 0 0 + 0 0 0 0 + + 0 + 0 0 +
1 + + + 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 0 + + 0 + 0 0 0 +
3 0 0 + + + + 0 + + + 0 0 + 0 + + 0 + 0 + + + 0 + + 0 + + 0 0 +
4 + + 0 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + 0 + + + 0 + 0 0 0 +
5 0 + + + 0 + + 0 0 0 + + + 0 + + + 0 0 + 0 + + 0 + 0 + + 0 0 +
6 0 + 0 + + 0 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 + 0 + 0 0 0 0 +
7 + 0 0 + + 0 + 0 + + 0 0 + + + 0 + + + 0 0 + + + + 0 + 0 0 0 +
8 + + + 0 + + + + + + + 0 + 0 0 + + 0 + + 0 + + 0 + + + + 0 0 0 +
9 0 + 0 + + + 0 0 0 0 + + 0 + + + 0 0 + 0 + + 0 + 0 + 0 0 0 0 +
10 0 + + + 0 0 0 0 + + + 0 + + 0 + 0 + + + 0 + + 0 + + 0 + 0 0 0 +
11 + 0 + + + 0 0 + + 0 0 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 0 0 0 +

 Since the AHG phase for I produced a negative result, then all those who are negative in that row are possible antibodies detected in the patient, all
those that are positive are crossed out (black squares)
o We also cross out Cw and V since in the antigram, both SC-1 and SC-2 are negative for these antigens and thus detection of antibodies is not
possible
o We don’t have to cross out in SC-2 row because it would just be the same as in SC-1 (you will cross out all the negatives and it would just be the
same as crossing out the positives in SC-1 row)
o the possible unexpected antibodies detected are: Anti-C, Anti-f, Anti-Kpa, Anti-Jsb, Anti-Fya, Anti-Leb, Anti-s, Anti-Lua, Anti-Xga

Group 6 | 3AMT 16’-17’

2) Antibody Identification Test

 What we have done in the Antibody Screening Test, we also do in the Antibody Identification Test but instead of using SC-1 and SC-2 as reagent, we use
AB-C 1-11 (also called Panel Cells). Panel Cells are group O reagent red cells that have been antigen typed beforehand
o For example in AB-C 9, the antigens present on the red cell are C antigen, c antigen, e antigen, Kpa antigen, etc… (those with plus sign); thus, a
positive AHG means that the antibody for the corresponding antigen is present in the patient’s serum
 Since the number of negative AHG is lesser than the number of positive AHG, then we would use the rows with negative AHG to eliminate some of the
possible unexpected antibodies detected in the screening test

CN Rh-Hr Kell Duffy Kidd Lewis P MNSs Lutheran S-L Rxn

D C E c e CW f V K k Kpa Kpb Jsa Jsb Fya Fyb Jka Jkb Lea Leb P1 M N S s Lua Lub Xga IS 37°C AHG CC
I + 0 + + + 0 0 0 + + 0 + + 0 0 + + + + 0 + + + + 0 0 + 0 0 0 0 +
II 0 + 0 0 0 0 + 0 0 0 + 0 0 + + 0 0 0 0 + 0 0 0 0 + + 0 + 0 0 +
1 + + + 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 0 + + 0 + 0 0 0 +
3 0 0 + + + + 0 + + + 0 0 + 0 + + 0 + 0 + + + 0 + + 0 + + 0 0 +
4 + + 0 0 + 0 + 0 + 0 0 + + 0 + 0 0 + + 0 + 0 + + + 0 + 0 0 0 +
5 0 + + + 0 + + 0 0 0 + + + 0 + + + 0 0 + 0 + + 0 + 0 + + 0 0 +
6 0 + 0 + + 0 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 + 0 + 0 0 0 0 +
7 + 0 0 + + 0 + 0 + + 0 0 + + + 0 + + + 0 0 + + + + 0 + 0 0 0 +
8 + + + 0 + + + + + + + 0 + 0 0 + + 0 + + 0 + + 0 + + + + 0 0 0 +
9 0 + 0 + + + 0 0 0 0 + + 0 + + + 0 0 + 0 + + 0 + 0 + 0 0 0 0 +
10 0 + + + 0 0 0 0 + + + 0 + + 0 + 0 + + + 0 + + 0 + + 0 + 0 0 0 +
11 + 0 + + + 0 0 + + 0 0 0 + + 0 + + 0 + + + 0 0 + 0 + + 0 0 0 0 +

 yellow background represents the possible antibodies detected during the screening test (these are the only ones you need check and cross out)
 For Identification, cross out all positive reactions in the antigram for those tubes with a negative reaction for AHG and find the column with no cross out.
o In the antigram above, the only column without a cross out is column Fya, which means the unexpected antibody present in the patient is
Anti-Fya

Group 6 | 3AMT 16’-17’