Procedure number: 213 Page 1 of 6

(Blood Bank Name and address)

Subject: Crossmatch Test

CROSSMATCH TEST

PURPOSE

To test the serum or plasma of a blood recipient for antibody directed against donor RBCs,
including most instances of donor and recipient ABO incompatibility (exception: rare weak
A and B subgroups).

PRINCIPLE

"Compatibility testing" encompasses all of the steps in pretransfusion testing including

recipient and donor ABO and Rh typing, the antibody (screening) test, the crossmatch, and
the search of blood bank records for previous patient history. This procedure concerns
only the technical details of performing a crossmatch. Policies and procedures for
compatibility testing are outlined in procedure #116.

The major crossmatch and the antibody screen both are intended to detect recipient
antibody directed against donor RBCs, and are redundant, at least for detection of
antibodies directed against the common antigens present on the screening cells. With
appropriate validation of the antibody detection test the AHG phase of the crossmatch
may be eliminated if it is negative, with the immediate spin phase being retained in order
to detect ABO typing errors that might lead to immediate hemolytic transfusion reactions.

The major crossmatch is carried through the AHG phase when the patient has a positive
antibody screen, even when the antibody has been identified and the donor's RBCs are
shown to lack the corresponding antigen, because such individuals are regarded as being
"responders" and to have an increased risk of immunization against low frequency
antigens. Similarly, an AHG crossmatch is performed on all individuals with a history of a
clinically significant blood group antibody, even when the patient's antibody is no longer
detectable. (See procedure #116 for specific policies on performing a crossmatch).

This sensitivity of the procedure depends in part on the serum/cell ratio and incubation
time employed. The procedure below calls for 4 drops of serum or plasma and a 30’
incubation period at 30oC, but many laboratories would use only 2 drops of serum.

MATERIALS AND SPECIMENS

See General Guidelines for serologic testing, procedure # 201

Donor blood "segment" or washed RBC suspension from a donor unit of type specific or
compatible RBCs
Patient Blood Bank Record, appendix 210.1 or other patient worksheet

PROCEDURE

Determine the Type of Crossmatch

If the patient has, or is expected to have, a positive antibody screen or a history

of clinically significant blood group antibodies (see procedure #116 for exact
requirements) perform a crossmatch using a saline indirect antiglobulin test.
 Procedure number: 213 Page 2 of 6
(Blood Bank Name and address)
Subject: Crossmatch Test

If the patient has a negative antibody screen and there is no ABO typing
discrepancy, perform a crossmatch looking at the immediate spin phase of testing only.

Note: It is often convenient to start crossmatches at the same time as the antibody screen.
In this case the tubes can be incubated while the antibody screen is completed as outlined
in step #6 below.
 Procedure number: 213 Page 3 of 6
(Blood Bank Name and address)
Subject: Crossmatch Test

Perform the Crossmatch(s)

1. Properly label two 10x75 or 12x75 mm test tubes for each unit crossmatched, one
with the patient identifier and unit # (for crossmatch) and the other with the unit #
only (for preparation of a cell suspension).

2. Cut off one tubing segment from a type specific or compatible donor unit. Cut open
the segment and add one drop of red cells into the tube labeled only with the donor
number. Inspection the donor unit at this time. Any unit which appears
contaminated (i.e., cloudy or discolored) is not to be used. The unit and any available
components must be quarantined and the supervisor notified. After separation of the
segment(s), archive them according to procedure #203.

3. Prepare a washed 3-5% red cell suspension of each unit of donor RBCs (see procedure
#202).

4. If only an "immediate spin crossmatch" is to be done, add 2 drops of patient serum

and 1 drop of the donor cell suspension to the tube marked with the patient identifier
and unit #. If crossmatches are to be carried through to the AHG phase, start with 4
drops of patient serum.

5. Centrifuge the test tube at the appropriate speed and time for the serofuge in use.

6. After centrifugation, examine the tube(s) for agglutination or hemolysis. Grade and
record the reaction(s) on the patient worksheet in the “IS” column.

If only an immediate spin crossmatch was to be performed and there is no evidence

of agglutination, interpret the crossmatch as "Comp" for compatible, in the
“Interp[retation]” column. If the antibody screen is not complete, place the tubes in
the 37o incubator. They can be discarded if the screen is negative or completed if it is
positive.

If agglutination is identified, proceed with the IAT crossmatch as follows, and

investigate the cause of the unexpected positive test as listed below.

7. Mix tubes and incubate at 37oC for 30 minutes.

8. Centrifuge at the appropriate speed and time for the serofuge in use.

9. After centrifugation, examine each tube for agglutination or hemolysis. Grade and
record the reaction(s) the "37o" column.

10. Wash the tube with normal saline 4 times. This may be done manually or with the
automatic cell washer.

11. Add 1 or 2 drops of anti-human globulin (typically 1 drop for monoclonal anti-IgG and
1-2 drops for rabbit anti-IgG) to the tube and mix.

12. Centrifuge at the appropriate speed and time.

 Procedure number: 213 Page 4 of 6
(Blood Bank Name and address)
Subject: Crossmatch Test

13. After centrifugation, examine each tube for agglutination or hemolysis. If the result is
negative or equivocal, examine it microscopically. Grade and record the reactions in
the "AHG" column.

14. To any tube showing a negative AHG result, add one drop of Coombs' control cells.

15. Centrifuge, read, and record the result in the "CC" column.

16. Record the interpretation, "Comp" for compatible or "Inc" for incompatible, in the
“Interp” column.

INTERPRETATION

Negative reactions at all test phases indicate that the unit is serologically compatible with
the patient and may be reserved for the patient if other criteria are met.

A positive reaction at any test phase indicates an incompatibility between donor red cells
and patient's serum. Further investigation is necessary and, except in emergencies, units
may not be released until the problem is resolved (see procedure # 116 for compatibility
requirements). It may be acceptable to transfuse a unit with a positive in vitro crossmatch
if the responsible antibody has been identified and is known to be clinically insignificant as
defined in procedure #116.

Causes of an incompatible crossmatch in the presence of a negative antibody

screen

1. Error in ABO grouping of either donor or patient.

2. Presence of an antibody in the patient's serum directed against a low incidence
antigen or another antigen not present on the screening cells (e.g. anti-Kp a or anti-A1
respectively).
3. Donor has a positive DAT.
4. An antigen present in double dose (homozygous donor) on the donor cells but single
dose on the antibody detection (screening) cells. In this case the patient's antibody
may not be strong enough to react with the single dose screening cells ("dosage
effect").
5. Rouleaux
6. A cold allo- or autoantibody.

Resolution of Incompatibility

The most likely cause of a positive IS crossmatch after a negative antibody screen is a cold
allo- or autoantibody. However, an ABO incompatibility due to a patient typing or unit
labeling error must be immediately ruled out. If the IS crossmatch is positive in a
patient with a negative antibody screen proceed as follows:
1. Perform a clerical check of the unit and patient specimen, and then confirm the unit
typing and repeat the patient ABO typing, both forward and reverse (consider
incubating the reverse type if anti-A1 is a possibility).
2. Examine the tube for rouleaux and, if negative, complete the crossmatch through to
the AHG phase. Initiate a second pre-warmed AHG crossmatch(s) (see procedure #
263).
 Procedure number: 213 Page 5 of 6
(Blood Bank Name and address)
Subject: Crossmatch Test

3. Perform a DAT (procedure #215) on the donor RBCs. If the donor’s DAT is positive,
repeat the crossmatch with a new unit.
If there is no discrepancy in the ABO, the DAT on the donor cells is negative, there is no
rouleaux (see procedure #269 for resolution), proceed with investigation as for a positive
antibody screen (procedure #221) including tests to identify cold antibodies (see procedure
#221).

NOTE: If an ABO incompatibility is ruled out and both the antibody detection test and an IS
crossmatch with pre-warmed elements is negative, units may be issued prior to
completion of the workup without requiring an Emergency blood Request to be
signed.

If the antibody screen is positive after an immediate spin crossmatch (started

concurrently) was performed, resolve the antibody problem (see procedure #221)
and complete all crossmatch(s) on the patient through the antiglobulin phase (see
step #6 above).
 Procedure number: 213 Page 6 of 6
(Blood Bank Name and address)
Subject: Crossmatch Test

If an IAT crossmatch(s) is positive in a patient with a blood group antibody using

donor RBCs lacking the corresponding antigen:
1. Review the antibody identification procedure for errors.
2. Initiate an investigation for an additional antibody directed against a low incidence
antigen.

After the above investigations are complete, select antigen negative units and/or an
appropriate crossmatch technique (e.g. use of autoadsorbed specimen, or pre-warmed
technique), and repeat the crossmatch(s) as required in procedure #116.

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