LABORATORY: IMMUNOHEMATOLOGY (BLOOD BANK)

National University​
Bachelor of Science in Medical Technology 3rd year/MED233/Term 3
PRE-TRANSFUSION TESTING

INTRODUCTION performed to identify any irregular antibodies

Pre-transfusion testing refers to the tests performed before in the patient’s serum.
blood is transfused to a patient. This process involves
validation and double, triple, or even quadruple checking to d.​ Antibody identification
ensure accuracy and patient safety. In the blood bank, a If the antibody screen yields a positive result,
minimum of two personnel and sometimes up to three the next step is antibody identification to
personnel verify the procedure to minimize errors. Once the determine the specific type of antibody
blood is transfused, it is already beyond the medical present. This is important for selecting
technologist’s control. compatible blood units and preventing
transfusion reactions.
Pre-transfusion testing is performed to prevent adverse
transfusion reactions. These reactions may range from mild e.​ Comparison of current and previous test
cases, such as fever and allergic reactions, to severe results
complications like anaphylactic shock, circulatory overload, We perform quality control or delta checking
and even fatal hemolytic transfusion reactions. across the laboratory by comparing a
patient’s current results with previous
STEPS IN PRE-TRANSFUSION TESTING records such as blood group, transfusion
history, medications, and other relevant data
1.​ Request for transfusion using laboratory information systems. This
Patients may require blood transfusion for different helps detect discrepancies or significant
reasons. One common indication is low hemoglobin changes, ensuring accuracy, improving
or hematocrit levels, such as in patients with anemia. patient safety, and saving time and
However, even patients who are not anemic may still resources.
need transfusion, especially before scheduled
surgical procedures like amputations, fracture repairs, 4.​ Donor RBC unit testing:
or cases involving severe tissue tears and blood loss. a.​ ABO group confirmation and Rh type
In emergency situations, such as accidents that result confirmation for Rh negative RBC units
in massive bleeding, transfusion may also be This pertains to the unit of blood
necessary. Ultimately, the physician determines components. Even if the unit is already
whether a patient requires transfusion based on their labeled, it is still best practice to verify
condition and clinical needs. everything thoroughly to ensure complete
accuracy and patient safety.
2.​ Identification of transfusion recipient and blood
specimen collected 5.​ Donor red cell unit selection:
Correctly identify the patient, just as in routine blood a.​ Selection of blood components of ABO
collection procedure, and perform the complete and group and Rh type that are compatible with
proper venipuncture steps to ensure accurate the transfusion recipient and with any
specimen collection and patient safety. unexpected allogeneic antibodies
After testing the patient and rechecking the
3.​ Testing of transfusion recipient’s blood specimen: requisition form, the appropriate donor unit is
a.​ Blood group acceptability selected based on the patient’s needs. If
b.​ ABO group and Rh type antibody screening or identification shows
Even if the patient’s blood type is already that the patient has a specific antibody, a
known, it is still protocol to perform ABO compatible donor unit that lacks the
grouping and Rh typing because these are corresponding antigen is chosen to prevent a
essential parts of patient identification. hemolytic transfusion reaction. This process
Unexpected errors can still occur, so pertains to the donor unit, which is the blood
healthcare professionals should never bag containing the required blood
assume that everything is already correct component.
based on previous records or routine
workflow. Since errors are always possible, 6.​ Compatibility testing (Crossmatch):
ABO and Rh grouping must always be tested a.​ Serologic
to ensure accuracy and patient safety. b.​ Computer or electronic

c.​ Antibody detection testing/screening 7.​ Labeling of blood or blood components with the
This is done to detect the presence of recipient’s identifying information and issue
unexpected antibodies. In some blood The donor unit is labeled with the patient’s details so it
groups, antibodies are not naturally can be identified as already reserved for that specific
occurring and may develop due to exposure patient. Blood bag tags are also attached to units that
to foreign antigens, such as through previous have been crossmatched to indicate compatibility and
transfusions or pregnancy, or they may be reservation status. This reservation is time-limited; for
influenced by genetic factors. Because of example, a blood unit may be reserved for up to three
this possibility, antibody screening is days from the date of crossmatching, after which it

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 may need to be reprocessed or re-evaluated if not correction is then written clearly beside it, followed by the
transfused. appropriate initials or signature.

PROCESS MAP COLLECTION OF PATIENT SAMPLES

1.​ Avoid hemolyzing the sample.

Since hemolysis is considered a positive reaction in
blood bank testing, a sample that is already
hemolyzed upon collection will result in a
false-positive interpretation. In such cases,
compatibility testing cannot be reliably performed, as
hemolysis may interfere with the results and no
appropriate donor unit can be determined if the issue
persists.

2.​ Serum or plasma may be used for pre-transfusion

testing.

When a transfusion request is received, the process begins 3.​ About 10 mL of blood is usually sufficient for all
with proper patient identification and specimen collection. ABO testing procedures.
and Rh typing are then performed, followed by antibody Whole blood is collected so that both red cell and
screening. If the screen is positive, antibody identification is plasma components are available. The red cell
carried out to determine the specific antibody present. A history portion is used for tests such as forward ABO and Rh
review or delta check is also done, considering factors such as grouping, as well as autocontrol. The plasma portion
previous transfusions, pregnancy, or certain drugs that may is used for reverse ABO grouping, antibody
lead to antibody formation. screening, and, if the screening is positive, antibody
identification using the same plasma sample. Plasma
Once the patient’s blood type and required component are is also used for major crossmatching.
determined, the appropriate donor unit is selected. Even if the
unit is already labeled, all details are rechecked, and ABO and 4.​ Sample should not be taken from IV tubing lines.
Rh typing may be repeated for confirmation. Compatibility
testing is then performed through crossmatching, where patient 5.​ The phlebotomist must initial or sign the label.
and donor samples are mixed to check for agglutination or
hemolysis. A positive reaction indicates incompatibility due to COLLECTION OF DONOR SAMPLES
antigen–antibody interaction, while no reaction indicates
compatibility. 1.​ RBCs for donor pretransfusion testing can be
prepared from the segmented tubing through which
If compatible, the blood unit is labeled with the patient’s the donor blood was collected.
information, stored or released for transfusion, and once
transfused, it can no longer be reversed or recalled. a.​ Make sure this is not exposed to an open
system. A sealer is used where the tubing is
IDENTIFICATION, COLLECTION, AND PREPARATION OF placed and sealed, allowing a segment to be
SAMPLES separated without exposing the whole unit.
This is where the donor sample is taken.
1.​ Proper identification and collection of the patient
sample. b.​ One technique is to cut the RBC end of the
segment tubing with scissors and use an
2.​ Clerical error is a major cause of applicator stick to remove or squeeze the
transfusion-associated fatalities. tubing to express a drop.

a.​ Incorrect ABO groupings and transfusion of DONOR SAMPLE TESTING

ABO incompatible blood
1.​ ABO grouping and Rh typing (including a test for
3.​ The recipient's identification must always be weak D) must be performed on a sample of blood
compared with the blood request form. donor taken at the time of collection

a.​ The recipient’s full name and hospital ID a.​ Even if the ABO type is already labeled on
number the blood bag, the ABO and Rh typing
should still be tested again for verification.
b.​ Age, date of birth, sex, and name of
requesting physician b.​ Weak D donors are labeled as Rh positive
When it is a donor, even in cases of weak D,
REMEMBER! mosaic D, or even small amounts of D
When an error occurs during labeling in the blood bank, antigen, the antigen is still considered
correction tape or correction fluid must not be used. Instead, present. Therefore, it should be labeled as
the incorrect entry is neatly marked with a single line, and the Rh positive.

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 2.​ AABB Standards requires a screening test for cells are used because some antigens may be absent
unexpected antibodies to RBC antigens on samples in one cell but present in another, allowing better
from donors who have a history of transfusion or detection of unexpected antibodies.
pregnancy.
5.​ Only IAT is required.
3.​ Confirm the ABO cell grouping on all units and Rh
typing on units labeled Rh negative. Repeat weak D 6.​ To each test cell, a patient's serum is added followed
testing is not required. by AHG.

PATIENT SAMPLE TESTING 7.​ An AC is run parallel (but not required) with the test
ABO Grouping cells (AHG is added to patient’s RBC and serum)
1.​ ABO grouping can be performed in slides or in tubes,
using solid-phase RBC adherence or column gel 8.​ The antibody screen may be carried out at room
technology. temperature and 37 degrees, or at 37 degrees only.

2.​ Forward and reverse typing. 9.​ If no reactions take place with any of the test cells,
then the antibody screen is negative and one may
3.​ If the patient’s ABO group cannot be satisfactorily proceed to crossmatch.
determined and immediate transfusion is required, And also after testing with Coombs control cells, if
group O packed RBCs should be used. positive agglutination is observed at the end, the
sample is reported as antibody screen negative
Weak D because the positive reaction validates the test
1.​ The test for weak D is unnecessary when testing procedure.
transfusion recipients.
10.​ If any of the cells react in the antibody screen, then
a.​ Weak D recipients are considered Rh the responsible antibody must be identified.
negative
In patient samples, weak D due to
incomplete or positional effects should be
treated as Rh negative because the patient
may develop anti-D antibodies. If a blood
unit containing the D antigen is transfused, it
may cause a major transfusion reaction.

2.​ If the Rh type of the recipient cannot be determined

and transfusion is essential, Rh negative blood should
be given.

3.​ Female patients whose RBCs type as weak D are

considered Rh positive and may receive Rh positive
blood during transfusion Autocontrol is used to check for the presence of
autoantibodies, which react with the patient’s own red cells
Antibody Screening rather than with screening cells.
1.​ Test for so-called unexpected antibodies
CONVENTIONAL TUBE METHOD
2.​ Detect as many clinically significant antibodies as
possible
a.​ HDN: anti-AB, anti-D, anti-kell, anti-C
b.​ IHTR: anti-A, anti-Kell, anti-Jkª, anti-Fyª
c.​ DHTR: anti-Jkª, anti-E, anti-D, anti-C

These can cause hemolytic transfusion reactions,

which may be immediate or delayed. In an immediate
reaction, adverse effects or signs of hemolysis may
appear within minutes to a few hours after
transfusion. In delayed hemolytic transfusion
reactions, symptoms may appear several days later,
usually after 7 days or more, as seen with notorious
antibodies such as Kidd antibodies.

3.​ Alloantibodies are almost always acquired (i.e.,

transfusion and pregnancy).
There are three phases in antibody screening: the immediate
spin phase (optional), the 37°C phase or thermophase, and the
4.​ Performed with 2 or 3 test/screen cells, all type O
AHG phase. In this procedure, only the patient’s sample is
negative.
tested against the antibody screening cells.
Screen cells are red cell suspensions made from
Group O, Rh-negative blood, and each screening
cell contains different antigens. Three screening

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 1.​ Immediate Spin Phase (Optional) – Two drops of
the patient’s plasma and one drop of screening cell
reagent are added into a tube, mixed, and
centrifuged. After centrifugation, the cell button is
gently dislodged and observed for agglutination or
hemolysis. If agglutination is present, the test is
stopped and reported as antibody screen positive,
indicating the presence of cold-reacting antibodies
that may cause hemolytic reactions.​

2.​ 37°C Phase (Thermophase) – If there is no

agglutination in the immediate spin phase, a
potentiator or enhancement reagent is added to
enhance antigen-antibody reactions and reduce
incubation time. The mixture is then incubated at
37°C; if an incubator is unavailable, a water bath may
be used. After incubation, the tube is centrifuged
OTHER METHODS
again, gently dislodged, and examined for
agglutination or hemolysis. If a reaction occurs, the 1.​ Gel Technology – The patient’s plasma and antibody
result is reported as antibody screen positive, and the screening cells are added into the gel matrix. If
next step is antibody identification to determine the antibodies are present and react with the antigens on
specific antibody present.​ the screening cells, agglutination occurs and the red
cells become trapped within the gel.
3.​ AHG Phase – If no reaction is observed in the 37°C
phase, the procedure proceeds to the AHG phase.
The cells are washed three times with normal saline
solution to remove unbound antibodies. Unlike red
cell suspension preparation, where aspiration is used
to avoid disturbing the cells, the tubes in this
procedure are completely decanted to ensure all
saline is removed. The final result after washing
should be a dry cell button. Two drops of anti-human
globulin (AHG) reagent are then added. The AHG
reagent helps detect antibodies attached to red cells
by linking sensitized cells together, making
agglutination visible. The tube is centrifuged, gently
dislodged, and observed for agglutination. If positive,
the test is reported as antibody screen positive and
proceeds to antibody identification.

a.​ If the AHG phase is negative, quality control

is performed using Coombs control cells or
check cells. These reagent red cells are
coated with IgG antibodies. Since the AHG
reagent remains unused in a true negative
reaction, adding Coombs control cells should
produce a positive agglutination reaction.
This validates the earlier negative result and
confirms the antibody screen as negative.
However, if no agglutination occurs after
adding Coombs control cells, it indicates
invalid testing, possibly due to improper
washing, failure to add AHG reagent, or use 2.​ Solid Phase Technology – The solid phase is
of expired or nonviable AHG reagent. already pre-coated with antigens. Upon addition of the
Therefore, antibody screen negative results patient’s plasma, corresponding antibodies will bind to
must still show positive agglutination upon the antigens and produce a visible positive reaction,
the addition of Coombs control cells to indicating antigen-antibody interaction.
validate the test.

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 EXAMPLE!

REMEMBER! SCREEN CELLS!

Weak or mixed-field reactions may indicate possible
discrepancies. In crossmatching, even a 1+ reaction is
considered positive and reported as incompatible. Even
minimal reactions are significant because they can still cause
transfusion reactions.

POTENTIATORS

POTENTIATORS INCUBATION TIME

None (Saline) 30-60 min

LISS 10-15 min

PEG 15 min
INTERPRETATION OF ANTIBODY SCREEN RESULTS
“A positive test result at any phase of testing indicates the
Bovine albumin 15-30 min need for antibody identification studies.”

ANTIBODY ANTIGRAM IN WHAT PHASE(S) DID THE REACTION(S) OCCUR?

An antigram contains screening cells, and each screening cell
has specific antigens present. These screening cells are red 1.​ Antibodies of the IgM class react best at room
cell suspensions made from Group O, Rh-negative blood with temperature or lower and are capable of causing
known antigen profiles. agglutination of saline-suspended RBCs (immediate
spin reaction).
In the example below, the screening cells contain antigens
such as D, C, e, Cw, K, k, Kpᵇ, Fyᵇ, Jkᵇ, Leᵇ, P, M, N, S, s, and 2.​ Antibodies of the IgG class react best at the AHG
Luᵇ. If the patient’s plasma contains corresponding antibodies phase.
against any of these antigens, a reaction will occur. ○​ Immediate spin: IgM
○​ 37°C and AHG phase: IgG

IS THE AUTOLOGOUS CONTROL NEGATIVE OR

POSITIVE?

1.​ If the antibody screen (+) but the autocontrol (−), it

suggests the presence of an alloantibody.

If the antibody screen (+) and the autocontrol (+), it is

most likely due to an autoantibody. This may be
associated with autoimmune disease or medications
that trigger the formation of autoantibodies.

a.​ Evaluate patient’s history

b.​ Possible indications: Presence of
autoantibodies or antibodies to medications
c.​ If recently transfused, may be caused by
alloantibodies coating the circulating donor
RBCs.

2.​ Autologous control

Patient’s RBCs tested against the patient’s serum in
the same manner as antibody screen

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 3.​ Direct Antiglobulin Test (DAT)
b.​ From those who have received high
DID MORE THAN ONE SCREEN CELL SAMPLE REACT? IF molecular weight plasma expanders
SO, DID THEY REACT AT THE SAME STRENGTH AND
PHASE? 2.​ Rouleaux formation:
a.​ Cells have a “stacked coin” appearance
1.​ Single antibody when viewed microscopically.
a.​ All screen cells yielding a positive reaction
react at the same phase(s) and strength b.​ Rouleaux is observed in all tests containing
the patient’s serum, including the autologous
2.​ If the patient has multiple antibodies: control and the reverse ABO grouping.
a.​ More than one screen cell may be positive
b.​ Screen cells react at different phases or c.​ Rouleaux does not interfere with the AHG
strengths phase of testing in tube method or
solid-phase technology because the patient’s
Based on the reaction, determine the strength, serum is washed away prior to the addition
whether the reactions are uniform or not, and the of the AHG reagent.
phase in which they occurred. If all screening cells
show the same reaction strength in the same phase, d.​ Unlike agglutination, rouleaux disperses by
this suggests the presence of a single antibody. adding one to three drops of saline to the
However, if the screening cells react with varying test tube when performing tube testing.
strengths and in different phases, this indicates the
presence of multiple or unexpected antibodies. SAMPLES OF ANTIBODY SCREEN RESULTS WITH
These findings serve as important indicators during POSSIBLE CAUSES
antibody identification.

3.​ Demonstrate dosage

Rh (except D)
Kidd
Duffy
MNSs
Lutheran

Duffy and NoD, the Rich Kidds had a dose of MNS

“NT” means not tested

1.​ The Coombs control was performed in Cells I and III

since they yielded negative results across all phases.
It was not performed in Cell II because it was already
a.​ Homozygous antigens will react stronger positive in the AHG phase. This is interpreted as an
b.​ Heterozygous antigens will react weaker antibody screen positive result.

These antigens are coded at the same locus of an In the autocontrol, the polyagglutination testing was
allele and compete for expression at that locus. If both negative, which indicates the presence of an
antigens are present, the reaction may appear alloantibody.
weaker. For example, if both Jkᵃ and Jkᵇ are present,
they compete at the same locus, resulting in a weaker Since the reaction occurred only in the AHG phase
reaction. However, if only one antigen is present in a and only in one screening cell, this suggests a
homozygous state, either Jkᵃ or Jkᵇ, the reaction presence of an IgG antibody with single specificity.
becomes stronger since there is no dosage effect
from a competing antigen. This principle can be used 2.​ In this example, a Coombs control was not performed
during antibody identification and rule-out since all cells showed reactions. There was a reaction
procedures. in the 37°C phase, and in the AHG phase, all cells
reacted with varying strengths.
ARE THE CELLS TRULY AGGLUTINATED, OR IS
ROULEAUX PRESENT? In the autocontrol, the polyagglutination testing was
negative, indicating the presence of an alloantibody.
1.​ Can cause rouleaux
a.​ Serum from patients with altered Since the reactions occurred in both the 37°C and
albumin-to-globulin ratios AHG phases, this suggests the presence of an IgG
This may be caused by a washing problem. antibody with multiple specificities.
If the sample is not washed properly due to
an insufficient albumin-to-globulin ratio, it
may yield a false-positive reaction.

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 screening cells, antibody identification uses 11 to 20 reagent
cells to determine the specific antibody present.

SCREENING CELLS PANEL CELLS

Antibody detection Antibody identification

Sets of 2 or 3 vials 11 to 20
An antibody panel is just an extended version of an antibody
screen. The screen only uses 2 to 3 cells.

RECALL
3.​ It reacted in all screening cells only in the immediate
spin phase and showed uniform strength. At this 1.​ Antibody screens use 2 or 3 screening cells to
point, it should be reported as antibody screen “detect” if antibodies are present in the serum. If
positive. antibodies are detected, they must be identified.

In the autocontrol, the polyagglutination testing was 2.​ Once an antibody has been detected in the antibody
negative, which indicates the presence of an screen, additional testing/review is necessary to
alloantibody. identify the antibody and determine its clinical
significance.
Since the reaction occurred only in the immediate
spin phase, this suggests the presence of an IgM SIGNIFICANCE OF ANTIBODY SCREENING
antibody with single specificity because it reacted in
only one phase and with uniform strength. 1.​ Antibody identification is needed for transfusion
purposes and is an important component of
compatibility testing.
4.​ It reacted in all screening cells only in the immediate
spin phase and showed uniform strength. 2.​ It will identify unexpected antibodies in the patient’s
serum.
In the autocontrol, it reacted in polyagglutination
testing and C3. Since the autocontrol is positive, this 3.​ If a person with an antibody is exposed to donor cells
indicates the presence of an autoantibody. with the corresponding antigen, serious side effects
can occur.
Since the reaction occurred only in the immediate
spin phase, this suggests the presence of an IgM REAGENT RBCS
antibody with single specificity. This is a cold These contain panel cells, with at least 10 vials per set.
autoantibody since it reacted at room
temperature/immediate spin phase.

5.​ It only reacted in the AHG phase with uniform

strength.
This is an example of an antigram used for antibody
In the autocontrol, it reacted in both polyagglutination identification, which involves a larger number of reagents. In
testing and IgG. Since the autocontrol is positive, this this case, 11 panel cells are used; these are red cell
indicates the presence of an autoantibody. suspensions made from Group O, Rh negative blood with
known antigen profiles. The “+” indicates the presence of
Since the reaction occurred only in the AHG phase specific antigens on each panel cell in the corresponding row,
and shows uniform strength, this suggests the while the right side of the antigram is where the test reactions
presence of an IgG antibody with single specificity. are recorded. The patient typing section serves as the
This is a warm antibody because it reacted in the autocontrol in other antigrams. In this section, the patient’s
AHG phase. plasma is mixed with the patient’s red cell suspension to
determine if the autocontrol is positive.
ANTIBODY IDENTIFICATION
Anything that tests positive in antibody screening, even if only It follows the same process, which includes the immediate spin
one screening cell reacts, requires antibody identification to be phase, addition of a potentiator, incubation at 37°C, washing,
performed. Unlike antibody screening, which uses 2 to 3 and the AHG phase. The difference is that it uses different
reagents, consisting of at least 10 panel cells.

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PANEL

1.​ These are red cell suspensions made from Group O,

Rh negative blood with known antigen profiles.

2.​ Each of the panel cells has been antigen typed

(shown on antigram).
a.​ + refers to the presence of antigen
b.​ 0 or - refers to the absence of antigen

3.​ An autocontrol should also be run with ALL panels.

4.​ The same phases used in an antibody screen are First, review for negative reactions across all phases. If no
used in a panel. reaction is observed, this suggests that the patient likely does
not have antibodies against the antigens present on those
ANTIBODY IDENTIFICATION TESTING panel cells, allowing those corresponding antigens to be ruled
A tube is labeled for each of the panel cells plus one tube for out in the antibody identification process.
autocontrol:
However, antigens that exhibit dosage effects cannot be
completely excluded, as they may show weak or even
non-reactive results when only heterozygous expression is
present. Therefore, only antibodies corresponding to antigens
with no dosage effect and those expressed in homozygous
form (which should show stronger reactions if present) can be
reliably ruled out.

INTERPRETING ANTIBODY PANELS

There are a few basic steps to follow when interpreting panels

1.​ “Ruling out” means crossing out antigens that did not
react
2.​ Circle the antigens that are not crossed out
3.​ Consider antibody’s unusual reactivity
4.​ Look for a matching pattern

REMEMBER!
An antibody will only react with cells that have the After ruling out the non-reactive antigens, the remaining
corresponding antigen; antibodies will not react with cells that possibilities are highlighted and reassessed based on their
do not have the antigen reaction patterns. In this example, only E and Leᵃ remain. The
phases in which reactions occurred are reviewed; antibodies
RULING OUT that consistently react across the panel cells in the same
phase and with similar strength are more likely to be
significant. In this case, Leᵃ shows more consistent reactions
than E. Additionally, Leᵃ is typically an IgM antibody, which
aligns with its reactivity in the immediate spin phase.
Therefore, the most likely identification is anti-Leᵃ. If a reaction
is observed, it indicates that the corresponding antigen is
present on the reacting panel cell.

GUIDELINES

1.​ Autocontrol
a.​ Negative: alloantibody
b.​ Positive: autoantibody or DTR (i.e.,
alloantibodies)

2.​ Phases
a.​ IS: cold (IgM)
b.​ 37°C: cold (some have higher thermal range
or warm reacting)
c.​ AHG: warm (IgG), significant

3.​ Reaction strength

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 a.​ 1 consistent strength: one antibody What if the “rule of three" is not fulfilled?
b.​ Different strengths: multiple antibodies or a.​ If there are not enough cells in the panel to fulfill the
dosage rule, then additional cells from another panel could be
used.
4.​ Matching the pattern
a.​ Single antibodies usually shows a pattern b.​ Most labs carry different lot numbers of panel cells
that matches one of the antigens
ORDER OF SELECTION
b.​ Multiple antibodies are more difficult to
match because they often show mixed
reaction strengths

REACTION STRENGTHS

1.​ Strength of reaction may be due to “dosage”

This is the order of selection based on the recipient’s blood
a.​ If panel cells are homozygous, a strong
type. The first choice is always a donor unit of the same ABO
reaction may be seen
and Rh type as the patient. If this is not available, alternative
compatible units are selected. For Group A and B patients,
b.​ If panel cells are heterozygous, reaction may
Group O Rh-negative is considered a universal donor option.
be weak or even non-reactive
For Group AB patients, the second choice is Group A, the third
choice is Group B, and the fourth choice is Group O.
2.​ Panel cells that are heterozygous should not be
crossed out because antibody may be too weak to
CROSSMATCHING
react
1.​ This usually refers to “major” crossmatch, showing
REMEMBER!
compatibility between recipient serum and donor
RBCs
In major crossmatching, donor red blood cells are
mixed with the recipient’s serum or plasma to check
for compatibility. In minor crossmatching, the patient’s
red blood cells are tested against the donor’s serum.
Major crossmatching is prioritized because it helps
detect antibodies in the recipient that may cause
hemolytic transfusion reactions. In contrast, minor
crossmatching is less commonly used since the
volume of donor plasma is much smaller, making the
risk of a hemolytic transfusion reaction relatively low.

2.​ Two main functions:

a.​ It is a final check of ABO compatibility
between donor and patient.
RULE OF THREE
b.​ It may detect the presence of an antibody in
The rule of three must be met to confirm the presence of the the patient’s serum that was not detected in
following antibody. This is used to confirm if the result is the screening
consistent.
3.​ This is required before transfusion of any product that
a.​ A p-value ≤ 0.05 must be observed contains at least 2 mL or RBCs
b.​ This gives 95% confidence in interval a.​ This is needed for the transfusion of:
c.​ How is it demonstrated? i.​ Whole blood
i.​ Patient serum must be: ii.​ Red blood cells
Positive with 3 cells with the antigen iii.​ Granulocyte concentrate
Negative with 3 cells without the antigen
b.​ But are not needed for transfusion of:
i.​ Plasma (FFP or FP24)
ii.​ Platelets (unless heavily
contaminated with RBCs)
iii.​ Cryoprecipitate

Anything that does not contain red blood

cells is not routinely crossmatched, and
type-specific components are generally
given instead. For example, for a patient with
blood group A, Group A plasma components
should be given.

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PROCEDURE

1.​ Collect blood samples from patients. Prepare serum

and red cell suspension.
2.​ Perform ABO and Rh typing
3.​ Choose the appropriate donor (the donor must be
ABO and Rh identical with that of the recipient)
4.​ Prepare the donor’s plasma and red cell suspension.
5.​ Repeat ABO and Rh typing on the donor’s sample.
6.​ Label 2 test tubes: Major and Minor

Saline/Immediate Spin Phase

CAUSES OF POSITIVE RESULTS IN THE SEROLOGIC
Incubation/37°C/Thermo Phase CROSSMATCH

AHG Phase 1.​ Incorrect ABO grouping of the patient or donor.

2.​ An alloantibody in the patient’s serum reacting with
the corresponding antigen on donor RBCs.
3.​ An autoantibody in the patient’s serum reacting with
SALINE/IMMEDIATE SPIN PHASE the corresponding antigen on donor RBCs.
4.​ Prior coating of the donor RBCs with protein, resulting
1.​ Add 2 drops of patient serum in a positive antihuman globulin test.
2.​ Add 1 drop of donor RCS 5.​ Abnormalities in the patient’s serum.
3.​ Mix gently and centrifuge 6.​ Contaminants in the test system.
4.​ Dislodge and examine
ANTI-HUMAN GLOBULIN (AHG)
If there is positive agglutination or hemolysis, report the result
as incompatible. If there is no reaction, proceed to the next 1.​ Direct Antihuman Globulin (DAT): In vivo coating of
phase. red cells with antibodies and complement.

INCUBATION/37 °C/THERMO PHASE There are no donor cells involved in this test. The
patient’s red blood cells may already be coated in vivo
1.​ Add 1-2 drops of potentiator due to autoantibodies directed against their own
2.​ Mix and incubate (based on the potentiator added) antigens, autoimmune conditions that lead to
3.​ Centrifuge autoantibody formation, medications that trigger
4.​ Dislodge and examine alloantibody or autoantibody production, or previous
transfusions that stimulated the formation of certain
If there is positive agglutination or hemolysis, report the result antibodies.
as incompatible. If there is no reaction, proceed to the next
phase. 2.​ Indirect Antihuman Globulin (IAT): Presence of
unexpected antibodies in serum that can coat the
AHG PHASE antigens in vitro.

1.​ Wash 3x (Decant last wash completely) This refers to the pre-transfusion testing performed to
2.​ Add 1 to 2 drops AHG determine whether the donor and patient are
3.​ Mix and centrifuge compatible. It is carried out as part of
4.​ Dislodge and examine crossmatching, antibody screening, and antibody
identification.
Note: All AHG negative results must be examined
microscopically. REAGENTS AND SAMPLES

5.​ Add check cells to any negative AHG. 1.​ Specimen: EDTA sample
2.​ NSS
a.​ Check cells are sensitized red blood cells 3.​ AHG reagent
pre-coated with IgG antibodies. Incomplete 4.​ Coomb’s control (Check cells)
abs do not show agglutination in saline
bodies. DIRECT ANTIHUMAN GLOBULIN (DAT)

If the AHG reagent has not been utilized across the phases, it 1.​ Label test tube as “U”
will bind to the Fc portion of IgG attached to the sensitized 2.​ Add 2 drops of EDTA blood
red blood cells (check cells). To validate the test result, a 3.​ Wash blood with NSS
positive agglutination reaction should be observed. If, after the 4.​ Decant completely the supernatant after last washing
addition of check cells, no agglutination occurs, it indicates that 5.​ Add 2 drops of AHG sera
the test is invalid. This may be due to AHG being neutralized 6.​ Centrifuge at 3,400 rpm for 15 seconds
from improper washing of the sample, the AHG reagent being 7.​ Gently dislodge, examine and grade.
deteriorated or expired, or the AHG not being added at all.

K.L.M | BSMT 10
 6.​ Centrifuge at 3,400 rpm for 15 seconds
+ DAT positive In vivo red cell
sensitization If there is a reaction, report the result as IAT positive. If the IAT
is specifically performed for crossmatching, report the result as
- Add 1 drop Coomb’s + = DAT negative incompatible. If the IAT is performed for antibody screening or
control (Check cells) and - = INVALID (repeat identification, report it as antibody positive.
centrifuge for 15 seconds the testing)

+ IAT positive Presence of atypical

antibodies in serum

- Add 1 drop Coomb’s + = IAT negative

control (Check cells) and - = INVALID (repeat
centrifuge for 15 seconds the testing)

8.​ Interpretation:
DAT (+) = red cells were sensitized in vivo

9.​ Clinical significance:

a.​ HDFN
b.​ Hemolytic transfusion reaction
c.​ Drug-induced hemolytic anemia
d.​ Autoimmune hemolytic anemia
7.​ Interpretation:
10.​ Antibody Screening and Identification: Elution IAT (+) = red cells were sensitized in vitro by the
After a positive DAT result, the antibodies are already unexpected antibodies in the patient’s serum
attached to the red blood cells. To identify the specific
antibody, elution is performed to separate the 8.​ Clinical significance:
antibody from the red blood cells. a.​ Compatibility testing
INDIRECT ANTIHUMAN GLOBULIN (IAT)

1.​ Label 2 tubes as “U” and “NC”

REFERENCES

Notes from the discussion by:

Mrs. Beatrice T. Sisayan

National University Powerpoint Presentation:

Mrs. Beatrice T. Sisayan
For crossmatching, add one drop of donor red cell
suspension. For antibody screening or identification,
add one drop of screening cells or panel cells.

2.​ Cover, mix and incubate at 37°C for 15 minutes

3.​ Centrifuge at 3,400 rpm for 15 seconds
4.​ Examine for agglutination

Note: If there is no agglutination, wash “U” tube 3x with NSS

and decant last washing.

5.​ Add two drops of AHG reagent to cross-link sensitized

red blood, allowing visible agglutination to occur.

K.L.M | BSMT 11