Standard Operating Procedure

Subject Crossmatch Procedure – Gel Method

Index Number Lab-8769
Section Laboratory
Subsection Regionals/Affiliates
Category Departmental
Contact Rachel Blum
Last Revised 12/5/2018

References
Required document for Laboratory Accreditation by the College of American Pathologists (CAP), Centers
for Medicare and Medicaid Services (CMS) and/or COLA.

Applicable To
Employees of the Gundersen St. Joseph’s Health Services Laboratory, Gundersen Tri-County Hospital
Laboratory, Gundersen Boscobel Area Hospitals and Clinics laboratories and Gundersen Palmer Lutheran
Hospital and Clinic Laboratories and Moundview Hospital and Clinic laboratories.

Detail
PRINCIPLE:
Patient serum is mixed with donor cells, centrifuged, and observed for macroscopic agglutination or
hemolysis. Agglutination or hemolysis of the donor cells is indicative of an antibody in the recipient’s
serum that is directed against an antigen on the donor cells, hence an incompatible crossmatch. No
agglutination can be interpreted as a compatible crossmatch if the patient has a negative antibody
screen.

CLINICAL SIGNIFICANCE:
The crossmatch is performed to provide donor blood compatible with the recipient in the medical or
surgical situation where transfusion is indicated. The compatible crossmatch effectively, though not
completely, rules out the possibility of an antigenic reaction by the patient to the donor cells. The
crossmatch cannot prevent however the possibility of sensitization of the recipient to an antigen in the
donor blood that the recipient may lack.

SPECIMEN:
An EDTA sample should be used for the procedure. Use of serum (from any tube) may result in false
positives due to fibrin in the serum. Avoid hemolysis as this could mask a hemolytic type of
incompatibility reaction. The patient should be banded with an identifying blood bank number, and this
number should also be written on the tubes of blood drawn for the blood bank testing. (See Lab-8654)
The tubes of blood should be labeled with the patient’s name, date, and the first initial and last name of
the person drawing the blood, and the patient’s blood bank number.

REAGENTS/MATERIALS:
Donor red blood cells
Patient EDTA plasma
MTS™ Buffered Gel Card
MTS™ Anti-IgG Gel (Anti-IgG, Rabbit) Card

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MTS™ Diluent 2 Plus

MTS™ Diluent 2
MTS™ A/B/D Monoclonal Grouping Card

EQUIPMENT INSTRUMENTATION:
Ortho MTS™ Workstation
MTS™ Digital Preprogrammed Pipettor
12 x 75 test tubes

QUALITY CONTROL:
See Lab-1514 Quality Control of MTS Gel System

Implementation
PROCEDURE:
Preliminary Procedure:
1. Check the blood bank file, inactive blood bank file and Gladiator to determine if the patient has
had blood bank work at this hospital before. Prepare a file if necessary.
2. Confirm or establish the ABO and Rh type of the patient by performing the ABO, Rh procedure.
3. Perform an antibody screen using the gel method with the IgG card.
4. If the antibody screen is negative and there is no history of antibodies for the patient, proceed
with the crossmatch using the appropriate type of units of blood on hand.
5. If the antibody screen is positive, the patient specimen must be sent to the Blood Center
reference lab in Milwaukee, LifeServe of Des Moines, IA for Palmer Lutheran samples, or to the
Gundersen Health System in LaCrosse, for antibody identification and selection of the
appropriate antigen negative units.
6. If the patient has a previously identified antibody, but the antibody screen is negative, order
antigen negative units from the Blood Center reference lab and proceed with the crossmatch
when the units arrive.

Note: It is acceptable to have the Blood Center or LifeServe send you crossmatch compatible units if
you cannot do the antibody identification and crossmatch for a patient on site.

Crossmatch Procedure (immediate spin):

Note: Can only be performed after preliminary procedure has been completed. Immediate spin
crossmatching can only be performed if patient has a negative antibody screen and no history of
previous antibodies. Patient specimen should be drawn within 3 days unless a patient history of no
prior transfusion within previous 3 months. If repeated transfusions are given, a new specimen is
needed every three days.

1. Select the number of appropriate type units ordered for crossmatching from the blood bank
refrigerator.
2. Obtain a segment from each unit and place in a 12X75 tube labeled with the unit number.
3. Return the units to the refrigerator to hold while the crossmatch is performed.
4. Make a 0.8% suspension of the donor red blood cells.
a. Using the segment puncture device squeeze several drops of cells into the labeled tube.

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b. Label another 12X75 tube with the unit number/0.8%.

c. Dispense 1.0 mL of MTS™ Diluent 2 Plus into labeled tube from b.
d. Using P6 on the electronic pipette, take up 100 µl diluents from tube in c, add airspace,
take up 10 µl donor cells in pipette, dispense all into tube with diluent from c and mix.
5. Visually inspect each gel card before use. Each microtube should have a clear liquid layer on top
of opaque gel.

CAUTION: Do not use gel cards if the gel matrix is absent or if the liquid level in the microtube is at or
below the top of the gel matrix. Do not use gel cards that show signs of drying, discoloration, bubbles,
crystals, or other artifacts. Do not centrifuge cards that have failed the visual inspection. The use of
these cards may lead to erroneous test results. Do not use cards if foil seals appear damaged or opened.

Note: Refer to ID-Micro Typing System™ Interpretation Guide for additional information related to the
visual inspection of gel cards before use.

6. Label the MTS™ Buffered Gel microtubes with the appropriate identification.
7. Remove the foil seal from the gel card or from the individual microtubes used for testing.
8. Pipette 50uL of 0.8% donor red blood cell suspension into a labeled microtube of the MTS
Buffered Gel Card.
9. Pipette 50uL of patient plasma into microtube.
10. Centrifuge the gel card.
11. Read the gel card for agglutination. Record results on blood bank log.
12. If there is any question about the compatibility of a unit, do not use it. Refer to the Investigation
of Blood Type/Crossmatch Problems policy for direction on how to proceed.

Crossmatch Procedure (through IgG)

Note: Can only be performed after preliminary procedure has been completed.

1. Select the number of appropriate type units ordered for crossmatching from the blood bank
refrigerator.
2. Obtain a segment from each unit and place in a 12X75 tube labeled with the unit number.
3. Return the units to the refrigerator to hold while the crossmatch is performed.
4. Make a 0.8% suspension of the donor red blood cells.
a. Using the segment puncture device squeeze several drops of cells into the labeled tube.
b. Label another 12X75 tube with the unit number/0.8%.
c. Dispense 1.0 mL of MTS™ Diluent 2 into labeled tube.
d. Using P6 on the electronic pipette or appropriate other pipette, take up 100 µl diluents
from tube in c, add airspace, take up 10 µl donor cells in pipette, dispense all into tube
with diluents from c and mix.
e. Palmer: take 10 µL of donor cells and dispense into tube from c.
5. Visually inspect each gel card before use. Each microtube should have a clear liquid layer on top
of opaque gel.
6. Label the MTS™ Anti-IgG microtubes with the appropriate identification.
7. Remove the foil seal from the gel card or from the individual microtubes used for testing.
8. Pipette 50uL of the 0.8% donor red blood cell suspension into a labeled microtube of the MTS
Anti-IgG card.

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9. Pipette 25uL of patient plasma into microtube.

10. Incubate the card in the MTS Incubator at 37° ± 2°C for 15 minutes.
11. Centrifuge the gel card.
12. Read the gel card for agglutination.
13. Record results on blood bank log.
14. If there is any question about the compatibility of a unit, do not use it.

Preparation of unit for potential transfusion:

1. Affix to each compatible unit a unit tag containing the recipient’s name, medical record number,
blood bank number, blood type, the unit number and type.
2. Write the blood bank number from the patient wristband on the unit.
3. Complete required information for release on the unit tag.
4. Place the unit and corresponding unit tag in the blood bank refrigerator.

Release of Crossmatched Blood:

Refer to the Routine release of Crossmatched Blood Policy.

Releasing of Units Not Transfused:

1. A type and screen is good for 3 days post draw (provided the Blood Bank armband stays on the
patient). Day zero (0) is the draw day. The workup is good for three MORE days and would
expire on the day three (3) at 2359. Without a negative or reliable patient interview (regarding
pregnancy and/or transfusion history), this may be extended slightly to include the hours from
midnight up until day four (4) begins (at the time of the draw). With a negative patient interview
(provided the patient has NOT been transfused at any facility, or pregnant within the last 3
months) the workup can be extended until a transfusion occurs and for 3 days beyond
transfusion.
2. Once that time frame has been exceeded or the order transfusion is canceled, the units are to
be released and returned to the available blood supply.
3. Remove all patient identification stickers from the unit.

Procedure Notes:
Proper documentation of all work performed should be logged into the blood bank worksheet.
Comments in regards to the special testing done, need to be documented on the patient’s blood bank
folder. Additional documentation of testing preformed, description of the discrepancy and resolution
including verbal consult from the Blood Center staff, must be added to the patient’s blood bank folder.

SELECTION OF BLOOD FOR ROUTINE CROSSMATCHING:

ABO Selection: If possible, choose blood of the recipient’s ABO and Rh.
Emergency- type unknown-
Recipient 1st Choice 2nd Choice 3rd Choice 4th Choice active bleeding
A+ A+ A- O+ O- O-
B+ B+ B- O+ O- O-
O+ O+ O- O-
AB+ AB+ A+, B+, AB- * O+ O- O-
A- A- O- O-

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B- B- O- O-
O- O- O-
AB- AB- A-, B- * O- O-

* In this order, depending on product availability. When substituting an ABORH compatible red cell type
for a type AB patient, switch the type you are most likely to be able to continue to supply i.e. AB to A.
Group O blood can be used for A, B, or AB patients without prior permission of blood bank director or
attending physician if:
1. ABO specific blood is not available.
2. Type A or AB patient with anti-A1 antibody must receive group O, IgG crossmatch compatible
RBCs (See Lab-3444 ABO+RH Testing.)
3. Time does not permit blood typing of patient. (Emergency blood request)
4. ABO cannot be accurately determined.
5. Antigen negative or irradiated blood is requested and not readily available. (This has been
approved by blood bank director for adults (over age 15). Any additional requirements (Rh, CMV
status, Leukoreduced) would still have to be met. For any recipient age 15 or younger, consult
with medical director or attending physician.

CALCULATIONS: N/A

INTERPRETATIONS:
1. If the crossmatch is compatible and antibody screen is negative, no known history of antibodies
are found, the blood is generally safe to transfuse.
2. If the crossmatch is positive and the antibody screen is negative with no known antibody on file,
the incompatibility could be due to:
a. Positive DAT of donor cells.
b. Antibody to a low incidence antigen
c. Antigen not represented on screening cells or showing dosage due to zygosity or low
titer.
d. Anti-Bg’s or Sda.
e. Anti-A1
3. Donor blood must be antigen negative if the following antibodies are present or on file: D, C, E,
c, e, S, s, K, k, Fya, Fyb, Jka, Jkb. If the patient has antibodies to M,N,P, Lea, or Leb, crossmatches
must be AHG compatible. (Antigen negative not required.)
4. Agglutination or hemolysis at any stage of testing indicates incompatibility. However, the testing
should be continued because evaluation of the reaction of the antibody in all phases of the
crossmatch will help identify the antibody.
5. If the clinical circumstances or other laboratory findings indicate additional testing is needed,
the routine Immediate Spin Crossmatch should be expanded to the AHG crossmatch. See Lab-
3450 Antibody Identification (Panels).
6. If the antibody screen is positive, discuss with the patient's physician how many units are
appropriate to be crossmatched and available. Blood will be available immediately to any
patient with a negative antibody screen and the immediate spin crossmatch will be performed
immediately.

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7. The tech will do an immediate spin crossmatch instead of the AHG crossmatch if the patient has
no present or previous clinically significant antibodies.
8. The use of plasma (EDTA or Heparin) and anti-IgG may result in the failure to detect rare
complement dependent antibodies.
9. Separate procedures for ABO, Rh and antibody screen and/or identification are found in the
procedure manual.
10. Recipient's blood specimen and sample of donor blood must be kept at 1-6oC for at least 7 days
after transfusion.

LIMITATIONS:
CAUTION: Adherence to the manufacturer’s Instructions for Use is critical to test performance.
1. Proper centrifuge calibration is particularly important to the performance of the MTS™ Buffered
Gel Card. The MTS™ Centrifuge has been exclusively designed to provide the correct time,
speed, and angle.
2. False positive or false negative test results can occur from bacterial or chemical contamination
of test materials, inadequate incubation time or temperature, improper centrifugation,
improper storage of materials, or omission of test samples.
3. Anomalous results may be caused by fresh serum, fibrin, or particulate matter in serum or
plasma, or red blood cells that stick to the sides of the microtube. Anomalous results (i.e., a line
of red blood cells on the top of the gel) may be observed with serum samples and can be
minimized by the use of EDTA plasma.
4. Rouleaux caused by serum or plasma with abnormally high concentrations of protein (such as in
patients with multiple myeloma or Waldenstrom’s macroglobulinemia or from patients who
have received plasma expanders of high molecular weight) may infrequently cause difficulties in
Gel Test interpretation. False positive results or hazy reactions may occur with these samples
but are rare. If false positive reactions (e.g. rouleaux, cells coated with immunoglobulins, etc.)
occur in the control gel, the blood group cannot be established. Additional testing will be
necessary to resolve this false positive reaction. If the control test is positive, the test cells
should be washed several times in warm saline and retested. If the control test again gives a
positive reaction, a valid interpretation of the results obtained cannot be made.
5. Red blood cells must be diluted in the appropriate MTS™ Diluent at the proper concentration
before the addition to the MTS™ Buffered Gel Card.
6. Cold agglutinin testing may be done by pre-chilling and incubating cards at 2-8oC. However,
these reactions will be warmed during the centrifugation, which may result in the weakening of
cold agglutination reactions.
7. Antibodies to preservatives, medication, disease states, Wharton’s jelly, and/or cross-
contamination of reaction tubes may cause false positive reactions.
8. False positive results may occur if a card that shows signs of drying is used in testing.

The crossmatch test will not:

1. Ensure normal survival of donor red cells.
2. Prove that recipient and/or donor serum is free of irregular antibodies.
3. Prevent immunization of the recipient.
4. Detect all ABO grouping errors.
5. Detect errors in RH typing of either recipient or donor.
6. Detect all errors of identification

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REVIEW AND CHANGES:

This document and all attached forms should be reviewed optimally on an annual basis, with 2 years as
the maximum review date. Review will be done by the Technical Leader, Supervisor, Manager, Medical
Director or designated person. Changes require retyping document or form and review by the Medical
Director.

REFERENCES:
1. AABB Technical Manual 16th Edition 2008, American Association of Blood Banks, Bethesda, MD.
ID-MTS Quick Reference Card, Ortho-Clinical Diagnostics, © 2008.
2. Instructions for use: MTS™ Anti-IgG Card.
3. Instructions for use: MTS™ Buffered Gel Card.
4. Instructions for use: Diluent 2.
5. Instructions for use: Diluent 2 Plus

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