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Out line
Collection and Preparation of Samples
Collecting Patient Samples
Donor Samples
Selection of Appropriate Donor Units
Testing of the donor sample
Testing of the patient sample
Summary of Pre-transfusion Testing


As the knowledge of new blood group system increased,

so did the search for more sensitive pre-transfusion
compatibility testing methods. Pioneer blood bankers
mixed the patient’s serum and the donor’s red cells and
observed for direct red blood cells lysis, agglutination, or
both. This became known as the major cross-match test.

The term compatibility test and cross-match are some

times used interchangeably, they should be clearly


The cross-match became part of a series of pre-

transfusion test known as compatibility testing. The
compatibility test includes an ABO and Rh grouping
performed on the donor and recipient samples, screening
of the donor’s and patient’s sera for unexpected
antibodies, and a cross-match.

The purpose of pre-transfusion or compatibility testing is

to ensure the best possible results of a blood transfusion.

The transfused red cells will have an acceptable survival

rate, and there will be no significant destruction of the
recipient’s own red cells.


Collection and Preparation of Samples.

Positive Patient Identification
The major cause of transfusion associated fatalities have
been clerical errors resulting in incorrect ABO grouping

The most common cause of clerical errors and thus

transfusion accidents is misidentification of the
patient involved in the transfusion. Confusion in
identification of the patient when the blood sample was
drawn a mixed up samples during handling in the lab,
and error in identification of the patient when the
transfusion was given.


To prevent collection of samples from the wrong patient,

the blood request form must be used to confirm the
patient’s identity before phlebotomy is performed. The
request form must state the intended recipient’s full
name, and unique hospital identification number.

Other information such as age and date of birth, address,

sex, and name of requesting physician can be used to
verify patient identity further but is not required on the


The patient’s wrist band identification must always be

compared with the requisition form.

If the patient does not have a wristband or if the patient’s

identity is unknown, some form of positive identification
must be attached to the patient before collection of


Collecting Patient Samples

Hemolyzed samples can not be used for testing
because hemolysis caused by activation of complement

Serum or plasma may be used for pre-transfusion testing.

Most blood bank technologist prefer serum because plasma
may cause small fibrin clots to form which may difficult to
distinguish from true agglutination.


When a specimen is received in the lab, a blood bank

technologist must confirm that the information on the
sample and requisition form agree.

All discrepancies must be resolved before the sample is

accepted, and if any doubt exists, a new sample must be


Donor Samples.
Donor testing samples must be taken when the full donor
unit is drawn. Depending on the method used for testing,
clotted sample, anti-coagulated samples, or both, are
Donor information and medical history card, the pilot
samples for processing, and the collection bag must all
be labeled with the same unique number code before
starting the phlebotomy, and the numbers must be
verified again immediately after filling.


Donor and recipient samples must be stored for a

minimum of 7 days following transfusion.

The samples should be stoppered and refrigerated at 1-

6°C, carefully labeled, and adequate in volume so that
they can be re-evaluated if the patient experiences an
adverse response to the transfusion.

Compatibility Testing Protocols.

Selection of Appropriate Donor Units.

In almost all cases, blood and blood components of the

patient’s own ABO and Rh group should be selected for

When blood and blood components of the patient’s type

are unavailable or when some other reason precludes
their use, units selected must lack any Ag against which
the patient has a significant Ab.

Selection of Appropriate Donor Units.

When transfusion of an ABO group different from the

recipient must be given, packed red cells must be used
rather than whole blood which contains plasma Abs that
are incompatible with the patient’s red blood cells.

Group O packed red blood cells can be safely used for all
patients, however, conservation of a limited supply of
group O blood should dictate its use for patients of other
AB types only in special circumstances.

Selection of Appropriate Donor Units.

If ABO-specific blood is not available or is in less than adequate

supply, alternative blood groups are chosen as summarized in the
following table;

Patient ‘s BG Alternative BG given as packed cells


AB A, B, O
only one of the three should be used for a given patient

Selection of Appropriate Donor Units.

Rh-negative blood can be given to Rh-positive patients,

however, good inventory management again should
conserve this limited resource for use in Rh-neg

If Rh-neg units is near expiration, the unit should be

given rather than wasted.

Selection of Appropriate Donor Units.

Rh-pos blood should not be given to Rh-neg women of

childbearing age.

Transfusion of Rh-neg male patients and female patients

beyond menopause with Rh-pos blood is acceptable as
long as no performed anti-D is demonstrable in the sera.

Compatibility Testing Protocols.

Testing of the donor sample.

According to the Code of Federal Regulation (CFR) and
the American Association of BB (AABB) standards, ABO
and Rh grouping (including a test for weak D) and tests
intended to prevent disease transmission must be
performed on a sample of blood taken at the time of
collection of the unit of blood from the donor.

A screening test for unexpected antibodies to red blood

cell Ags is required by AABB standards on samples from
donors revealing a history of prior transfusion or

Testing of the donor sample

The transfusing facility is required by AABB standards to

confirm the ABO cell grouping on all units and Rh
grouping on units labeled Rh-neg.

Tests for weak D (Du) are not required to be reported.

The transfusion facility does not need to repeat any other
testing procedure.

Testing of the patient sample.

A record of all results obtained in testing patient samples must be

Identification number should be assigned each time a patient is
admitted for treatment.
Any discrepancies between previous and current results must be
resolved before transfusion is initiated.
A new sample should be collected from the patient, if necessary to
resolve the problem.
ABO and Rh grouping results should be included in the file.
Also, notations concerning unusual serologic reactions and the
identity of unexpected Abs in the patient’s serum should be

Testing of the patient sample.

ABO and Rh grouping and Ab screening of the patient’s

serum can be performed in advance of or at the same
time as the cross-match.

If the patient has had a transfusion or has been pregnant

within the last 3 months or if the history is unavailable or
uncertain, the sample must be obtained from the patient
within 3 days of scheduled transfusion.

Testing of the patient sample.

ABO Grouping.
Determination of the patient’s correct ABO group is the
most critical pre-transfusion serologic test.

If the cell and serum grouping results do not agree,

additional testing must be conducted to resolve the
If the patient’s ABO group cannot be satisfactory
determined and immediate transfusion is essential,
group O packed red blood cells should be used.

Testing of the patient sample.

Rh Grouping.
Rh grouping is performed using anti-D blood grouping
serum. Tube or slide tests should be performed
according to the manufacture’s directions for the
reagent, which may or may not include the use of a
suitable diluents control.

Control must be run in parallel with Rh grouping tests

performed on patient’s samples, to avoid incorrect
designation of Rh neg, patient as Rh positive.

Testing of the patient sample.

Direct antiglobulin test (DAT) should be performed on

the patient’s red blood cells to determine whether uptake
of autoantibody, (alloantibodies, if the patient’s has been
recently transfused) is responsible for the positive
control result.

Testing of the patient sample.

If the Rh group of the recipient can not be determined

and transfusion is essential, Rh negative blood should be

The test for Du is unnecessary when testing transfusion

recipients. Individuals typing as Rh neg in direct testing
should receive Rh-neg blood and those typing as Rh pos
in direct testing should receive Rh pos blood.

As Du are considered Rh pos and may receive Rh pos

blood during transfusion.

Testing of the patient sample.

Antibody Screening.
The patient’s serum or plasma must be tested for
unexpected Abs.
The aim of the Ab screening test is to detect as many
clinically significant Abs as possible.
Clinically significant Abs refers to Abs that are reactive at
37°C or in the DAT or both and are known to have
caused a transfusion reaction or unacceptably short
survival of the transfused red blood cells.

Testing of the patient sample.

Abs Regarded as always being potentially clinically

ABO Rh Kell Duffy Kidd S s U

Abs that may sometime be clinically Significant

Lea p Lua Lub Cartwright.

Abs that rarely, if ever, are clinically significant

Leb Chido/Rodgers (Cha/Rha) York, Sd Xg& Bg

Testing of the patient sample.

Correct ABO grouping results are much more critical to

transfusion safety than Ab screening.

Most Abs, other than anti-A and anti-B do not cause

severe hemolytic transfusion reactions. Thus the vast
majority of patients would not suffer grave consequences
if transfused with blood from ABO group compatible
donor without the benefit of Ab screening tests.

Testing of the patient sample.

Detection of unexpected Abs is important, however, for

the selection of donor red blood cells that are likely to
survive maximally in the patient circulation.

Weakly reactive Abs that are capable of reacting with

their Ags at 37°C can cause decreased survival of
transfused incompatible red cells.

Testing of the patient sample.

Because large numbers of Ab molecules are present in

the patient’s circulation compared with the number of
red cells in a unit of blood, incompatible donor cells are
highly vulnerable to destruction by patient Abs.

Abs screening offers several advantages over direct

cross-matched testing for detection of Abs;
1- Testing is performed using selected group O red cells
that are known to carry optimal representation of
important blood group Ags.

Testing of the patient sample.

2- Testing can be performed well in advance of the

anticipated transfusion, allowing ample time for
identification of unexpected Ab and location of suitable
donor units lacking the corresponding Ag.

Methods used to detect Abs in patient’s sera must

demonstrate all significant coating, hemolyzing, and
agglutinating Abs active at 3 7°C.


The two main functions of the cross-match test can be

cited as,

I- It is a final check of ABO compatibility between donor

and patient.

2- It may detect the presence of an Ab in the patient’s

serum that will react with Ags on the donor RBCs but
that was not detected in the Ab screening because the
corresponding Ag was lacking from the screening cell.


Major and Minor cross-match tests

Major cross-match test, consisting of mixing the patient’s
serum with donor RBCs.

Minor cross-match test, consisting of mixing the donor’s

plasma with patient’s RBCs

The minor cross-match test has been completely

eliminated in most blood banks, because donor samples
are screened beforehand for the more common Abs.


Method for major cross-match tests.

Cross-match methods can be categorized by the test

phase in which the procedure ends.

Immediate spin (IS) cross-match (Abbreviated


When no clinically significant Abs are detected nor are

there previous record of such Abs, a serologic test to
detect ABO incompatibility is sufficient.


In IS (the patient’s serum with donor cell are centrifuge

immediately) absence of hemolysis or agglutination
indicates compatibility.

False reaction may be seen in the presence of other IS

reaction (auto -I). In patient with hyperimmune ABO
Abs, when the procedure is not performed correctly
(delayed in centrifugation or reading) when rouleauex is
observed, or when infant’s specimens are tested.


Antiglobulin Cross-match
The procedure begin in the same manner as the IS cross-
match, continues to 37°C incubation and finishes with
AHG test.


Is to provide safe, compatible blood for transfusion to

each individual patient. The steps necessary for safe
transfusion are:
1. Accurate ABO and Rh typing of the patient.
2. Accurate ABO and Rh typing of the donor.
3. Screening tests for antibodies in the donors and
patients serum.
4. In the presence of patient antibodies, selection of
appropriate units for each patient.
5. Compatibility Testing - (Major)
6. Accurate completion of paperwork and labels


Compatibility Testing
Each compatibility test is a unique experiment in which
an unknown (patient) serum and (donor) red cells are
tested for the detection of unexpected antibodies which
are directed against antigens found on the cells. Negative
results indicate compatibility. This is one of the most
important tests performed by a transfusion service.


The purposes of compatibility testing are:

1. To detect irregular antibodies in the recipient serum
that are directed against the donor’s cells.
2. To detect errors in ABO grouping.
3. To detect errors in labeling, recording, or identifying
donor’s or recipient’s samples.


Compatibility testing does not:

1. Ensure normal survival of donor red cells.
Prove that donor and or recipient serum is free of
antibodies. Prevent immunization of the recipient.
Detect ALL ABO typing errors.
Detect errors in Rh typing of either recipient or donor
unless the recipient’s serum contains an Rh antibody.
Detect ALL error of identification.
Pre-transfusion testing of the recipient must include an
ABO and Rh typing, antibody screen, and a cross-match
with all donor units.

Patient red cell with –D Ag.

If Rh –ve do