Compatibility testing,Cross match-major and minor,LISS,Procedure.

1. Compatibility
testing
Dr.Sumitha.J
M.Sc.,M.Phil.,PGDBI,Ph.D.

2. Learning objectives
Compatibility testing
Cross match-major and minor
LISS
Procedures of cross match methods
Factors affecting compatibility
testing.

3. The aim of Blood transfusion 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.Compatibility Testing.
4.Accurate completion of paperwork and
labels
Compatibility testing -
Blood transfusion

4. 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.

5. The purposes of compatibility testing
are:
To select blood components that will not cause
harm to the recipient and will have acceptable
survival when transfused
If properly performed, compatibility tests will
confirm ABO compatibility between the
component and the recipient and will detect the
most clinically significant unexpected antibodies
To detect irregular antibodies in the recipient
serum that are directed against the donor’s cells.

6. Components of CT
Proper specimen collection
Reviewing patient transfusion
history
ABO, Rh, and antibody testing
(screen/ID)
Crossmatching
Actual transfusion

7. Specimen collection
The sample should also have the full patient name, hospital
number, and physician
Date and time of collection, phlebotomist’s initials
All of this should be on the request form and the sample
Collected in tube with EDTA or no additives
If the venipuncture causes hemolysis, the sample may be
rejected
If the sample is drawn from an IV line, the IV infusion
should be stopped 5-10 minutes prior to blood drawing
and the first 10 mL discarded
Testing should be performed on samples less than 72
hours or else complement dependent antibodies may
be missed (complement can become unstable

8. Cross matching
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.

9. Cross matching
The two main functions of the cross-match
test can be cited as,
It is a final check of ABO compatibility
between donor and patient.
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.

10. 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.

11. The major crossmatch involves testing the patient's
serum with donor cells to determine whether the patient
has an antibody which may cause a hemolytic transfusion
reaction or decreased cell survival of donor cells.
Of greatest importance is detection of ABO
incompatibilities. ABO problems quickly become
apparent when the mixture of patient's serum and donor
cells is examined after centrifugation (immediate spin
crossmatch). Unexpected cold reacting antibodies may
also become apparent at this step, but once identified as
such, they are usually considered insignificant.
Basic Principles

12. The crossmatch is incubated at 37C to
detect clinically
significant agglutinating or hemolyzing
antibodies. After incubation and
washing, antiglobulin serum (Coomb's
serum) is employed to detect antibodies
that have attached to cells without
causing agglutination (sensitization).

13. Additional solutions such as albumin,
polymerized albumin or low ionic salt
solutions (LISS) are frequently employed to
increase the sensitivity of the crossmatch or
to reduce the incubation time. By lowering
the ionic strength or increasing the dielectric
constant of the test medium, these solutions
increase antibody uptake and enhance the
strength of the antigen-antibody reaction.

14. The minor crossmatch involves testing the
patients cells with donor plasma to determine
whether there is an antibody in the donor's
plasma directed against an antigen on the
patient's cells. Since all donors are routinely
screened at the collection facility for irregular
antibodies this procedure is no longer routinely
performed. There may be rare instances when
this procedure may need to be performed so be
familiar with it.

15. Major cross matching-
Procedure
1.Collect one tube from each recipient and
possible donor(s).
2. Centrifuge tube(s) at 1000rpm for 5 min. to
separate plasma from red blood cells (RBCs).
3. Remove plasma from each sample with a
clean pipette and transfer to clean, labeled glass
or plastic tubes.
4. Wash RBCs 3 times with a normal saline
solution; resuspend to make a 3-5% RBC
suspension.

16. 5. Prepare for each donor 3 tubes labeled with Major,
Minor, and Recipient control.
6. Add to each tube 2 parts of plasma and 1 part of RBC
suspension as follows:
Major BCM(Blood Cross match): recipient plasma +
donor cells
Minor BCM(Blood Cross match): donor plasma +
recipient cells
Recipient control: recipient plasma + recipient cells
7. Mix gently and incubate for 15 min. at room
temperature.

17. 8. Centrifuge for 15 sec. at 1000 x 9.
9. Examine supernatant for hemolysis.
10. Gently resuspend button of cells by tapping
tube with a finger and examine for macroscopic
agglutination.
11. If macroscopic agglutination is not observed,
transfer a small amount onto a glass slide and
examine for microscopic agglutination.
12.The absence of hemolysis or agglutination
indicates compatibility.

18. cross matching
methods
Saline method-Open Slide
method,Tube method-Complete cold
antibodies.
Immediate spin (IS) cross-match-
Complete warm antibodies
Albumin Tube method-incomplete
immune antibodies(Rh)
Coomb’s Cross matching-
incomplete immune antibodies(Rh)

19. Saline method
Saline suspension of cells is mixed
with serum.this is done at RT.
Saline technique is designed to detect
compatibility of IgM antibody(ies) in
patient’s serum against antigens on
donor’s red cells. Only Complete,
saline active, cold antibodies will
be detected.

20. Method
Label 1 tube for each donor sample to be tested.
Put 2 drop of patient’s serum in labeled tube.
Add 1 drop of 2-5% saline suspended red cells of
donor
Mix and incubate for 5-10 min. (spin method) or
incubate for 30-60 min (sedimentation method)
at RT.
Centrifuge at 1000 rpm for 1 min. in spin method
(after 5-10 min. incubation);centrifugation is
optional in sedimentation method.

21. Read the result, observe for hemolysis and agglutination.
Negative result should be confirmed under microscope.
Interpretation
Agglutination or hemolysis indicates a positive result
(incompatible)
Note: In emergency spin technique is acceptable.
Saline technique is inadequate as a complete
compatibility test because it is inadequate to detect
clinically significant IgG antibodies.

22. Is method
In Immediate Spin method ,the
patient’s serum with donor cell
are centrifuged at 500-1000
rpm for 2 minutes,Incubate at
37degrees for 60 minutes

23. Albumin tube method
Set up the tubes as in other methods and
incubate the cells for 60 minutes at 37
Allow a drop of BSA to each of the tube so
that it forms a layer between the cells and
the serum
Incubate for an additional 30 mins
Look for agglutination microscopically.

24. Coomb’s cross match
The procedure begin in the same
manner as the IS cross-match,
continues to 37 C incubation and
cells are washed thrice in saline and
combos serum is added, Centrifuged
at 500-1000 rpm for 2 minutes.

25. Minor cross match -
procedure
1. Label a test tube with donor number and
recipient's initials.
2. Add one drop of 2-5% suspension Recipient
cells.
3. Add 2 drops of Donor serum and 1 drop of 22%
bovine albumin to the tube.
4. Centrifuge immediately 1 min at 1000 rpm.
5. Read macroscopically for Haemolysis and
agglutination.
6. Incubate at 37o C for 30 minutes.
7. Centrifuge 1 min at 1000 rpm.

26. 1. Read macroscopically for Haemolysis
and agglutination.
2. Wash the tube 3 times with saline.
3. Add 2 drops of anti human globulin
serum to the dry cell button.
4. Centrifuge 1 min at 1000 rpm.
5. Read macroscopically for Haemolysis
and agglutination.
6. Add Check Cells to all negative tests;
spin, read and record results.

27. Factors leading to
false results
Auto-agglutination
Cold antibodies
Bacterial contamination
Drying

28. Questions??