CROSSMATCH TECHNIQUE (Indirect Antiglobulin Test/ Indirect Coomb’s test)

PRINCIPLE
The patient's ABO and Rh type is determined and the serum is screened for the presence of
clinically significant alloantibodies. If the antibody screen is negative, and there is no record of such
antibodies having been detected in the past, the compatibility test may be abbreviated to include only
the immediate spin phase, which demonstrates ABO incompatibility.
The complete crossmatch with a 37oC incubation and antiglobulin phase MUST be performed
on all patients whose antibody screen is positive in Coombs, or whose records show a previously-
detected clinically significant antibody, even though it may not be currently detectable.
The complete crossmatch should also be done on patients whose Immediate Spin crossmatch is
positive.

SAMPLE
One 10 ml red top (clotted) tube is preferred. A 4 or 5 ml red top is satisfactory if enough serum
is available to crossmatch the number of units requested. The sample MUST be dated and signed by the
phlebotomist, and must be tested within 24 hours of the time it was drawn to maintain adequate
complement activity.
A new sample must be obtained and tested after 72 hours if the patient has received blood or
has been pregnant within the past three months, or if the transfusion or pregnancy history is unknown.

REAGENTS, EQUIPMENT, AND SUPPLIES

Material used in ABO/Rh TYPING AND ANTIBODY SCREEN
Donor packed red cells
Lancet

PROCEDURE
TYPE AND SCREEN:
Fill out a worksheet. Minimum information should include:
Patient name (legibly printed, last name first).
Medical record number
Name of phlebotomist
Date and time sample drawn
Other helpful information would include gender, birthdate, diagnosis or surgical procedure, and nursing
unit
Make sure the sample is thoroughly clotted, then centrifuge two minutes at high speed in the serofuge.
Remove the serum to a labeled 12 x 75 mm tube.
Verify that the patient information on the sample matches information on the worksheet.
Label the following 11 tubes with the patient's initials and:
WC (Washed Cells)
A, B, a1, b, I, II, III, D,DC
1. Place 2-3 drops patient cells into the WC tube and make a washed 3% suspension. One wash is
usually sufficient.
2. Place two drops patient serum into the a1,b, I, II, and III Screening Cells
3. Place one drop of Screening Cell I into the I tube, one drop Screening Cell II into the II tube, and
one drop Screening Cell III into the III tube.
 4. Place one drop of the patient's washed 3% cells into the AUTO tube.
5. Centrifuge the I, II, III, at a 15 second saline spin.
6. Read, grade, and record reactions on the worksheet.
7. Add two drops PEG to each tube and incubate at 37C 15-30 minutes.
8. While the screen is incubating, add the ABO/Rh reagents and patient cells to the appropriate
tubes and perform the ABO/Rh. No need to use anti-A,B when typing recipient blood.
9. Read, grade and record ABO/Rh reactions on the worksheet. Resolve any ABO discrepancies
before setting up donor blood.
10. Wash the antibody screen 4 times, blot the last wash, and add one drop anti-human globulin.
11. Centrifuge the length of time required for the Coombs reaction on your serofuge.
12. Read, grade and record results. Check all negative reactions with Coombs Control Cells.
13. Look up the patient in the master file.
14. If there is a file on the patient, be sure the ABO/Rh matches your results, and check to see if any
antibodies have been identified in the past.
15. If there is no file on the patient, start one. Information should include:
16. Name, hospital number, birth date
17. ABO/Rh
18. Any crossmatch problems (antibodies, ABO discrepancies)
19. Donor units crossmatched, date
20. Donor units transfused, date
21. If one or both screening cells were positive in Coombs, and there is no record of an antibody in
the patient's file, identify the antibody using a panel. See ANTIBODY IDENTIFICATION.

CROSSMATCHING:

1. Select the requested number of donor red cells of the appropriate ABO/Rh.
2. In general, select units that will outdate soonest.
3. Exceptions are units of blood for sickle cells patients and newborns.
4. Arrange the units in order of outdate, and enter the unit numbers in the appropriate place on
the worksheet, oldest units first.
5. Label a 12 x 75 mm tube for each donor unit, using the last 3 digits of the unit number.
6. Use either Seg-Safe ® or a Hematype® cap to puncture a segment from each donor unit and
allow a drop of blood to run into the appropriate tube.
7. Place the punctured segment into a 12x75 test tube and save till the procedure is complete.
8. Prepare a washed 3% suspension of each donor unit. One wash is generally sufficient, unless the
supernatant is grossly hemolyzed.
9. Re-check the ABO type of each donor unit by performing an ABO test (forward grouping only).
You may use anti-A,B instead of anti-A and anti-B for group O units. If the donor unit is Rh
negative, also perform an Rh test with control, including 15 minute incubation.
10. Record donor blood typing results in the appropriate spot on the worksheet.
11. Label a second tube for each donor unit, using the last 3 digits of the unit number, the patient's
initials, and XM.
12. Place two drops of patient serum into each XM tube.
13. Place one drop of washed 3% donor cells into the appropriate tube, shake to mix, and centrifuge
the length of time for the saline calibration of the serofuge.
 14. Gently resuspend and examine macroscopically for agglutination. Record results under the
Immediate Spin (IS) column of the worksheet.
15. If the crossmatch results are negative, save the crossmatch tubes and donor cell suspensions
until the antibody screen is complete and the records have been checked.
16. The units may be reported as compatible at this point if the patient has no current or previous
antibodies. See COMPATIBLE IMMEDIATE SPIN CROSSMATCH, NO ANTIBODIES under
INTERPRETATION.
17. If the patient has an antibody or a record of an antibody, the crossmatch must be carried
through the antiglobulin phase, and the donor units typed for the corresponding antigen. See
CURRENT ANTIBODY or HISTORY OF AN ANTIBODY under INTERPRETATION.
18. To carry the crossmatch through Coombs, add two drops PEG to each donor tube after the
immediate spin reading, incubate 15-30 minutes, wash 4 times and add AHG. Spin and read).

INTERPRETATION

COMPATIBLE IMMEDIATE SPIN CROSSMATCH, NO ANTIBODIES

- Many institutions choose to end the crossmatch after the immediate spin reading. This is
appropriate if:
the crossmatch is compatible upon immediate spin, AND
the patient has no clinically significant antibodies, AND
the patient has no record of clinically significant antibodies in the past.
- If the antibody screen is negative, with results confirmed by Coombs Control Cells, and the
records check shows no previously detected clinically significant antibodies, and the immediate
spin crossmatch is compatible, the test is complete. Record the units as "compatible" on the
worksheet, and sign your name and the date.

HISTORY OF AN ANTIBODY (BUT ANTIBODY SCREEN IS NEGATIVE)

- Frequently a patient may have developed an antibody in the past, which is recorded in his file,
but the titer may have dropped and it is no longer detectable in the antibody screen.
- If this is the case, it is not necessary to set up any panel cells. Merely carry the crossmatch
through the LISS/37C/AHG phase (see step 33) and type the donor units for the corresponding
antigen, if antiserum is available. Be sure to run positive and negative controls. The donor units
must be negative for the antigen before they can be reported as compatible for the patient. See
manufacturer's directions for specific commercial antisera.
- If no typing serum is available, and the antibody is clinically significant, notify your Donor
Reference Center (American Red Cross in the Madison area), that you need antigen-negative
blood, or give them the donor numbers of your compatible units and have them antigen-type
the segment retained at the center.
- Label the donor unit with the results of the antigen typing, and also record the results on the
worksheet.

CURRENT ANTIBODY (ANTIBODY SCREEN IS POSITIVE)

If the identity of the antibody is known and part of the patient's records, it is not necessary to set up an
entire antibody identification panel. Only set up enough selected antigen-negative cells to rule out
formation of any new antibodies.
If the identity of the antibody is not in the patient's file, identify the antibody and continue with the
LISS/37C/AHG phase of the crossmatch (step 33). Identification of antibodies reacting only at room
temperature is optional. Be sure to record the antibody in the patient's file, once it is identified.

Type the donor units for the corresponding antigen, if antiserum is available. Be sure to run positive and
negative controls. The donor units must be negative for the antigen before they can be reported as
compatible. See manufacturer's directions for specific commercial antisera.

NOTE: It is not necessary to specially type donor units for D when a patient has anti-D. D-typing is always
done as part of the routine re-checking of Rh-negative donor units.

If no typing serum is available, and the antibody is clinically significant, notify your Donor Reference
Center (American Red Cross in the Madison area), that you need antigen-negative blood, or give them
the donor numbers of your compatible units and have them antigen-type the segment retained at the
center.

Label the donor unit with the results of the antigen typing, and also record the results on the worksheet.

NO ANTIBODIES, INCOMPATIBLE IMMEDIATE SPIN CROSSMATCH

If the immediate spin crossmatch results are positive, double-check the ABO types of both patient and
donor. If they are incompatible, select donor units of the proper ABO type. If they are compatible,
continue with the LISS/37C/AHG phase of the crossmatch. This is performed exactly like the LISS/37
oC/AHG phase of the antibody screen, performed earlier under TYPE and SCREEN.

If the crossmatch is compatible in Coombs, report the units as compatible.

NOTES
You may stop after the immediate spin phase of the crossmatch if the patient has no current antibodies;
no previous record of antibodies, and the immediate spin crossmatch is negative.

If multiple antibodies are present, the donor units must be typed for all corresponding antigens.

It is not necessary to do the complete crossmatch when the cause of an Immediate Spin reaction is
rouleaux, if the reaction can be made negative with the saline replacement technique.