COLLEGE OF HEALTH – HAIL
Medical laboratory Dept.- Second term
THIRD YEAR – Blood banking
Compatibility test
PRACTICE -5
Purpose of compatibility testing
The purpose of compatibility testing (crossmatching) is to prevent a transfusing
reaction by ensuring:
● The ABO group of the blood to be transfused is compatible with the patient’s
ABO group.
● There are no detectable irregular antibodies in the patient’s serum that will
react with the donor’s red cells, causing their destruction
or reducing their normal survival. blood should only be requested when it is
absolutely necessary. The request must be in writing using a Blood Transfusion
Request Form and it must be signed by the medical officer with responsibility for
the patient’s care. The following is an example of a Request Form suitable for use
in district hospitals.
Note: When the patient has been transfused before, laboratory staff should check
the patient’s notes and laboratory records, noting the reason for the trans
fusion, whether any antibody was detected and whether any adverse reaction
occurred during or following the previous transfusion. blood transfusion work
involve patient misidentification often due to incorrect labelling of samples or
collecting blood from the wrong patient.
Blood Transfusion Request Form
Patient’s name . . . . . . . . . . . . . . . Age . . . . . . . . . . . . . . . .
Hospital Number . . . . . . . . . . . . . Ward. . . . . . . . . . . . . . .
Reason for transfusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
..................................................
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Requirement
Concentrated red cells, amount. . . . . . . . . . .
Whole blood, amount . . . . . . . . . . . . . . . . . . .
Date and time required . . . . . . . . . . . . . . . . .
History
Has the patient been transfused before? . . . . . . . . . . . . . . .
Blood group (when known) . . . . . . . . . . . . . . . . . . . . . . . . . .
Previous pregnancies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Signed . . . . . . . . . . . . . . . . . . . . . . Date . . . . . . . . . . . . . . .
Patient’s blood sample
-For blood grouping and compatibility testing, collect blood into EDTA
anticoagulant and
5–7 ml into a dry clean glass tube (not plastic).
-Label the containers clearly with the patient’s name, hospital number, ward,
and date. The EDTA blood is used to obtain red cells for cell grouping and to
measure the patient’s haemoglobin or packed cell volume. Serum from the
clotted blood sample is
used for serum grouping and compatibility testing.
When blood is collected in advance, the serum used to perform the compatibility
test must not be more than 48 hours old. Serum used for compatibility
testing should be kept by the laboratory for 3 days following a transfusion in case
it is needed for re-testing in the unlikely event of a transfusion
reaction. When blood is held for a patient who has been transfused, it must be recrossmatched when more than 48 hours have passed since the first
transfusion.
Group of Choice of blood
patient 1st 2nd 3rd 4th
Group A Gp A GpO – –
Group B Gp B GpO – –
Group O GpO – – –
Group AB Gp AB Gp A* Gp B GpO
*Group A is the second choice of blood because anti-B in GpA blood is likely to be
weaker than anti-A in GpB blood.
Compatibility testing
A compatibility test is a final check on the compatibility of blood for a patient,
particularly ABO compatibility. Several techniques are available for
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crossmatching blood to detect ABO incompatibility and clinically important
irregular antibodies in a patient’s serum. Compatibility testing is essential
when prior antibody screening is not performed
In district laboratories, a simple to perform single tube technique comprising the
following three stages is recommended:
_ Detection of ABO incompatibility in saline at room \temperature:
Donor’s cells are mixed with patient’s serum at room temperature
Centrifuged and examined visually for agglutination.
When there is no agglutination or haemolysis, proceed to next stage.
_ Detection of antibodies agglutinating at 37 _C in saline. The tube is incubated at
37 _C for 20 minutes, recentrifuged and examined for agglutination.
When there is no agglutination or haemolysis, proceed to the third stage.
_ Detection of immune IgG antibodies that have sensitized donor’s cells and are
only agglutinated by using antiglobulin (AHG) reagent:* An indirect antiglobulin
test (IAT) is performed in which red cells after being incubated in patient’s serum
are tested after washing for bound antibody.
An albumin technique will detect most anti-D antibodies, some other Rhesus
antibodies and irregular antibodies, but it is not as sensitive as the AHG technique
and will miss some antibodies.
Single tube compatibility technique using
AHG reagent
The following are required:
● Patient’s serum
● Donor’s washed 3% red cell suspension prepared as follows:
– Transfer 0.2–0.5 ml of red cells from the pilot tube of the donor blood into
about 5 ml of physiological saline and mix.
– Centrifuge at high speed (e.g. 1 000 g) for about 2 minutes. Discard the
supernatant fluid and resuspend the cells in a further 5 ml of saline. Mix,
centrifuge, and discard the supernatant fluid.
– Prepare a 3% red cell suspension by adding1 volume of packed cells to 30
volumes of saline.
● Antiglobulin polyspecific reagent, usually colour coded green. AHG
reagent will agglutinate red cells sensitized with antibodies and/or coated with
detectable levels of complement components.
● Anti-D serum to make AHG control cells.
● AHG control IgG sensitized red cells, prepared as follows:
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– Wash group O Rh positive red cells three times in saline. Discard the final saline
supernatant fluid.
– Add an equal volume of IgG anti-D to the packed red cells and mix.
– Incubate at 37 _C for 30 minutes. Wash the cells four times in saline. Remove
the final supernatant fluid.
– Suspend the packed cells in saline to make a 5% red cell suspension. When
added to AHG reagent, the sensitized cells should show visible agglutination–
Store the sensitized cells at 2–8 _C. They can be kept for 2–3 days.
Method
1 Label a small (e.g. 75 _ 12 mm) clean glass tube with the number of the donor
blood and write this number also on the patient’s blood
transfusion request form.
2 Pipette 3 volumes of patient’s serum into the tube.
3 Add 1 volume of donor’s washed 3% red cell suspension and mix.
4 Centrifuge at slow speed e.g. at 150 g for 1 minute or 500 g for 10 seconds.
5 Tilting the tube back and forth, examine for haemolysis or agglutination.
Important
Haemolysis or agglutination means that the donor blood is ABO incompatible. The
blood MUST NOT BE GIVEN TO THE PATIENT. When there is haemolysis or
agglutination, recheck the ABO group of the patient and
donor blood, and also check that the correct patient’s blood sample has been
tested.
6 When there is no agglutination, mix the contents of the tube and incubate at 37
_C for 20–30 minutes.
7 Centrifuge at slow speed. Tilting the tube back and forth, examine for
haemolysis or agglutination. Haemolysis or agglutination indicate that the blood
is incompatible and must not be given to the patient.
8 When there is no haemolysis or agglutination, perform an indirect AHG test. Fill
the tube with saline, centrifuge (high speed), and remove the supernatant fluid.
Wash the cells a further 3 times. At the end of the final wash remove all the
supernatant fluid.
9 Resuspend the cells by tapping the bottom of the tube. Add 2 drops of AHG
reagent and mix.
10 Centrifuge at slow speed, e.g. at 150 g for 1 minute or at 500 g for 10–15 sec.
11 Tilting the tube back and forth, look for agglutination. When no agglutination
is seen, transfer a few of the cells to a slide and check for agglutination
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microscopically using the 10_ objective. When there is no agglutination, check
that the AHG has not been neutralized by adding 1 drop of AHG control sensitized
cells to the tube. Repeat steps 10–11. The control cells will show agglutination,
providing the AHG is active and the test has been performed correctly. When
there is agglutination after adding AHG reagent, this means that the patient’s
serum contains an immune IgG antibody reactiveagainst the donor’s cells which
may cause a transfusion reaction.
12 Enter the test results in the Blood Transfusion Records Book.
Single tube compatibility technique using AHG reagent and
LISS to reduce incubation time
The technique is performed as described above except that at step 6, before the
incubation stage, add 3 drops of LISS to the tube. This has the advantage that the
incubation time can be reduced to 15 minutes.
Compatibility testing using a saline and
albumin technique
A saline and albumin technique is appropriate to use
when it is not possible to obtain AHG reagent. The
following are required:
● Patient’s serum
● Donor’s washed 3% red cell suspension,prepared as described previously
● Bovine albumin 20%*
Method
1 Label two small tubes with the number of the donor blood and write this
number also on the patient’s transfusion request form.
2 Pipette 1 volume of patient’s serum into each tube. Add 1 volume of donor’s 3%
cell suspension to each tube and mix.
3 Incubate one tube at 37 _C for 45 minutes.
4 Centrifuge the second tube at slow speed, e.g. 150 g for 1 minute or 500 g for
10–15 seconds. Tilting the tube back and forth, examine for haemolysis or
agglutination.
5 At the end of 45 minutes incubation, add carefully 1 drop of 20% albumin to the
second tube. Do not mix. Incubate for a further 10–15 minutes.
6 Look for agglutination in the albumin tube by carefully tilting the tube back and
forth. When no agglutination is seen, transfer some of the cells toa slide and
check for agglutination microscopically. When there is agglutination in the
albumin tube this means that the patient’s serum contains an immune IgG
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antibody reactive against the donor’s cells which may cause a transfusion
reaction.
7 Enter the test results in the Blood Transfusion Records Book.
Emergency compatibility testing
In an emergency, blood is issued after completion of the first stage of a standard
crossmatch, i.e. after centrifuging the saline room temperature tube and
checking for haemolysis or agglutination to exclude ABO incompatibility. This
takes about 10 minutes and therefore even when blood is urgently required it
should be possible to perform this important part of a crossmatch.
Note: The risk to a patient when blood is transfused that has only been ABO
checked is low when the patient has never been transfused previously or
been pregnant.
Note: When blood is issued after excluding ABO incompatibility only, this must be
indicated on the label attached to the blood pack and the crossmatch must be
completed.
Difficulties in crossmatching
Most of the problems which can occur when crossmatching are those which will
have already been met when blood grouping, providing both cell and
serum grouping have been performed. They include rouleaux and cold reacting
autoagglutinins. Bacterially contaminated donor cells can also cause difficulties
when crossmatching.
STANDARD CROSSMATCH
Patient . . . . . . . . . . . . . . . . . . . . . . Ward. . . . . . . . . . . . . . .
Hospital Number . . . . . . . . . . . . . Date . . . . . . . . . . . . . . .
Group of patient. . . . . . . . . . . . . .
Crossmatch
This blood has been found compatible by a standard
crossmatch
Unit number . . . . . . . . . . . . . . . . . Group. . . . . . . . . . . . . .
Expiry date . . . . . . . . . . . . . . . . . .
Signed . . . . . . . . . . . . . . . . . . . . . .
EMERGENCY CROSSMATCH
Patient . . . . . . . . . . . . . . . . . . . . . . Ward. . . . . . . . . . . . . . .
Hospital Number . . . . . . . . . . . . . Date . . . . . . . . . . . . . . .
Group of patient. . . . . . . . . . . . . .
Crossmatch
This blood has been found compatible by an
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EMERGENCY Crossmatch which excludes an ABO
incompatibility
Unit number . . . . . . . . . . . . . . . . . Group. . . . . . . . . . . . . .
Expiry date . . . . . . . . . . . . . . . . . .
Signed . . . . . . . . . . . . . . . . . . . . . .
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