Government of Newfoundland and Labrador

Department of Health and Community Services

Provincial Blood Coordinating Program

INDIRECT ANTIGLOBULIN NLBCP-017

CROSSMATCH TUBE METHOD

Office of Administrative Issuing Authority

Responsibility
Medical Advisor to the Provincial Blood Dr. Lucinda Whitman
Coordinating Program
Manager, Provincial Blood Coordinating Daphne Osborne
Program

Author Melissa Leonard

Effective Date 2017-06-13

Version 3.0

Review Due Date 2019-06-13

 Indirect Antiglobulin Crossmatch Tube Method

Overview
An indirect antiglobulin crossmatch is performed to assure compatibility of red cell units for
transfusion in certain patients by incubating recipient’s plasma or serum and donor red cells
(obtained from a segment of tubing that was originally attached to the unit to be transfused)
then adding anti-human globulin reagent (AHG) to detect any antibody coating of the donor
red cells.

Policy
1. Compatibility testing shall be performed before red cells are transfused.
2. When an antibody screen indicates the presence of a clinically significant red cell
antibody, or the recipient has a previous history of clinically significant antibodies the red
cell donor unit shall be crossmatched using an antiglobulin technique.
3. The red cell donor unit(s) selected for crossmatch shall lack the corresponding antigen(s)
to any clinically significant antibodies detected in recipient’s serum or plasma.
4. No delay shall occur in initiating the centrifugation step or reading the reaction after
centrifugation is complete. Delay can lead to false-negative results and failure to detect
incompatibility.
5. Following transfusion of the first unit of blood, the original recipient blood sample may be
used to crossmatch additional units up to 96 hours.

Guidelines
1. Monospecific anti-IgG (which contains only anti-IgG) can also be used in place of AHG.
2. Patient transfusion records shall be reviewed and this review shall be documented. The
following information shall be reviewed:
2.1. Previous ABO and Rh typing;
2.2. Previous transfusions;
2.3. Difficulties in blood typing;
2.4. Previously identified clinically significant red cell antibodies;
2.5. Adverse reactions to previous transfusions; and
2.6. Special transfusion requirements.
3. Documentation to link donor unit to recipient shall include:
3.1. Recipients name;

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 Indirect Antiglobulin Crossmatch Tube Method

3.2. Recipient’s identification number;

3.3. ABO and Rh groups of the recipient;
3.4. ABO and Rh groups of blood component;
3.5. Name of the component;
3.6. Donor unit number;
3.7. Expiry date of donor unit;
3.8. Compatibility status of the donor unit; and
3.9. Date and time of crossmatch.

Materials
1. Isotonic saline
2. Test tubes (10x75mm or 12x75mm)
3. Transfer pipettes`
4. Test tube rack
5. Serological centrifuge
6. Donor unit(s)
7. Patient EDTA whole blood sample or patient serum (Do not use sample drawn into tubes
with neutral gel separators)
8. Polyspecific anti-human globulin (AHG) or monospecific anti-IgG
9. IgG sensitized red cells (check cells)
10. 22% albumin or other potentiating solution
11. Cell washer (if available)
12. Waterbath or heating block at 37°C ± 1°C
13. Interval timer
14. Microscope
15. Microscope slides

Procedure
Procedure as per AABB Technical Manual, 18th Edition.
1. Determine specimen suitability.
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 Indirect Antiglobulin Crossmatch Tube Method

2. Centrifuge specimen (speed and time as recommended by manufacturer’s instructions.)

3. Check specimen for abnormal appearance after centrifuging (e.g. hemolysis.)
4. Create crossmatch worksheet.
5. Perform patient history check and check for special requirements (eg. irradiated
products).
6. Choose acceptable donor unit(s) from blood bank fridge.
Note: If antigen negative red cell units are not available in current inventory, perform
antigen typing on the red cells in current inventory or request antigen negative units
from blood supplier prior to beginning crossmatch.
7. Record donor unit number(s) on crossmatch worksheet.
8. Label two (2) test tubes for each donor red cell suspension being tested with patient’s
serum or plasma.
9. Remove a segment from each donor red cell unit.
10. Return donor unit(s) to fridge.
11. Wash the donor cells (from donor unit(s) segments) with isotonic saline and prepare a 2-
5% red cell suspension(s) in the corresponding labelled tube.
12. Add the following to each of the second labelled tubes:
12.1. Two (2) drops of patient’s serum or plasma;
12.2. One (1) drops of the donor red cell suspension;
12.3. Two (2) drops of potentiating solution (eg. 22% albumin, LISS)
NOTE: Not all facilities use potentiating solution.
13. Mix the contents of the tube(s).
14. Incubate tube(s) at 37°C for 30 to 60 minutes.
15. Centrifuge tube(s). (Speed and time as recommended by manufacturer’s instructions.)
16. Observe macroscopically for hemolysis and agglutination. Grade and record the results.
17. Wash the red cells three to four times with saline completely decant the final wash. Cell
washer can also be used.
18. Add two (2) drops of AHG or IgG to the dry red cell button.
19. Centrifuge tube(s).

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 Indirect Antiglobulin Crossmatch Tube Method

20. Resuspend the red cell button(s) while examining macroscopically then microscopically
for agglutination.
21. Grade and record results on crossmatch worksheet.
21.1. No agglutination or hemolysis indicates the donor unit(s) is/are compatible.
21.2. Agglutination or hemolysis indicated the donor unit(s) is/are incompatible.
22. Confirm the validity of negative results by adding IgG-coated red cells (check cells).
22.1. After check cells are added and centrifuged, agglutination should be present.
22.2. If agglutination is absent, crossmatch results are invalid. Repeat crossmatch.

Quality Control
1. All reagents used shall be controlled as required, before results are released.
2. An ABO and Rh type and antibody screen is performed on the patient sample to be
crossmatched. Any discrepancies shall be resolved or antibodies identified before
crossmatch is performed.
3. Incompatible results shall be investigated as per facility procedure.
4. IgG sensitized red cells must be added to all negative indirect antiglobulin tests to
confirm:
4.1. AHG or IgG was added; and
4.2. AHG or IgG added is working.

Key Words
Crossmatch, anitglobulin, AHG, IgG

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 Indirect Antiglobulin Crossmatch Tube Method

Supplemental Materials

No
Is the specimen Request new To second tube add: Yes
suitable? specimen 2 drops patient’s Further
Agglutination
serum/plasma, 1 drop investigation
present?
donor suspension, 2 required
Yes drops of 22% albumin
Centrifuge
specimen No

Mix tube(s)
Add 1 drop of IgG
coated red cells
Is specimen
hemolyzed?
Yes
Incubate tube(s) @
37°C for 30-60
No minutes
No Further
Agglutination
investigation
Create crossmatch present?
required
worksheet

Centrifuge tube(s)
Yes
Perform patient
history check Donor unit(s) are
compatible
Yes
Further
Is hemolysis
Choose donor investigation
present?
unit(s) required

No
Record donor unit
Wash red cells 3-4
number(s) on
times with saline,
worksheet
completely decant
the final wash
Remove segments
then return donor
unit(s) to fridge Add 2 drops of AHG
or IgG to dry red cell
button
Label 2 tubes for
each donor unit

Centrifuge tube(s)

Wash donor unit

segment 3 to 4
times
Resuspend and
examine
macroscopically
Prepare 3-5% then microscopiclly
suspension from
washed donor cells
in first labelled tube

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 Indirect Antiglobulin Crossmatch Tube Method

References

Canadian Society for Transfusion Medicine. (2017). Standards for Hospital Transfusion
Services. (Version 4.0). Markham, ON: Author.
Canadian Standards Association. (2015). Blood and blood components, Z902-15.
Mississauga, ON: Author.
Downes, Katherine A. MD & Shulman Ira A. MD. Pretransfusion Testing. In: Fung M,
Grossman BJ, Hillyer CD, Westhoff CM, eds. Technical manual. 18th edition.
Bethesda, MD: AABB, 2014:367-389.

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