Indirect Antigen Typing – Anti-IgG Gel Card
1.0Principle
Red Cells are tested with specific antisera to determine the presence or absence of blood group antigen(s).
In this test, the red blood cells, in a hypotonic buffered saline solution, are centrifuged through a gel microtube containing anti-IgG. The detection of IgG sensitization occurs when the sensitized red blood cells react with the Anti-IgG in the gel of the microtube during centrifugation.
Bone Marrow Transplant
When performing antigen typing for Bone Marrow Transplant (BMT) patients, the patient history should always be considered. Post BMT patients may exhibit chimerism due to presence of red cell antigens because of the persistence of the recipient’s B lymphocytes.
2.0Scope and Related Policies
2.1If a patient has been transfused within the last three months phenotyping should not be performed on the current sample. If a pre-transfusion sample is available it should be used for phenotyping
2.2Previous transfusion records shall be reviewed. Previous results must be compared with current results.9.1
2.3All reagents shall be used and controlled according to the supplier’s recommendations and procedures.9.1
2.4If upon inspection the gel/reagent in the cards do not appear to be evenly distributed the cards may be centrifuged prior to use at 895 rpm for 10 minutes to ensure optimal distribution of the gel/reagent.
3.0Specimens
EDTA anticoagulated sample drawn within five days of testing
Donor cells collected in AS3, CPDA-1 or CPD may be tested up to the expiration date of the unit. Some blood samples, e.g., cord blood, can develop fibrin clots when diluted. These samples may be washed prior to dilution in MTS Diluent 2.
4.0Materials
Equipment:Centrifuge
Incubator
Pipettor
Dispenser
Set-up workstation, optional
Serologic centrifuge
Supplies:Pipette tips
Test tubes – 10 x 75 mm
Serologic pipettes
Package insert
Reagents:MTS Diluent 2, a buffered hypotonic saline solution
MTS Anti-IgG Card, Anti-IgG (Rabbit) suspended in gel
Saline
Anti-sera (for antigen typing)
Do not use beyond expiration date. Store cards at 2 to 25°C. Store diluent and red cells at 2 to 8°C. Bring reagents to room temperature (18 to 25°C) prior to use.
5.0Quality Control
5.1When using commercially prepared blood grouping reagents not labeled for use in Gel card testing the laboratory must verify the test performance of the reagent before using this procedure. The cell and Lot number of cells used must be documented.
5.2Cells known to be positive and negative for the antigen being tested must be set up and tested at the same time as the patient/donor cells, e.g., when testing for the K1 antigen both a K1+ and a K1- cell must be included. The expected reactions must be obtained for the test to be valid.
5.3To recognize reagent deterioration, the reagents must be tested on day of use with appropriate controls. MTS Diluent 2 must be
visually checked to ensure that the liquid is not discolored, turbid or showing any signs of bacterial contamination.
5.4To confirm the specificity and reactivity of the MTS Anti- IgG card, it is recommended that each lot be tested on each day of use with
known positive and negative antibody samples with the appropriate red cell. Reactivity must be present with the positive sample only.
5.5The manufacturer recommends that, following centrifugation, results should be read immediately. Results may be affected by drying of
the gel, hemolysis of the red cells and slanting of the reaction patterns due to storage in a non-upright position.
6.0Procedures
6.1Patient/Donor/Control 0.8% Cell Preparation, using packed red blood cells from patient sample or donor unit segments.
6.1.1Label a test tube
6.1.1.1For each donor to be tested
6.1.1.2For patient being tested
6.1.1.3For each positive and negative control
(not required if using 0.8% commercially prepared screen cells).
6.1.2Dispense 1.0 mL of MTS Diluent 2 into the labeled tube(s). Add 10µL of the patient/donor packed red blood cells to the appropriately labeled tube.
6.1.3Mix gently. Final cell suspensions should be approximately 0.8% and are stable for 24 hours. For best results, suspensions should not be less than 0.6% or exceed 1.0%.
6.2Test Procedure
6.2.1Label the MTS Anti-IgG Card with the appropriate identification and test information.
6.2.1.1Patients family name
6.2.1.2Last 4 digits of the donor number
6.2.1.3The reagent being used and the lot # (e.g., anti-FyaLot 123)
6.2.2Remove the foil seal from the microtubes to be used.
6.2.3To the correct microtube, using an appropriate pipette, add 50L of a 0.8% red blood cell suspension of:
- Patient cells
- Positive/negative control cells
- Each donor unit.
Do not touch pipette to gel card. A positive and negative control should be set up with each batch incubated.
6.2.4Using an appropriate pipette, add 25µL of antisera to the correct microtube.
6.2.5Incubate at 372C for 15 minutes. Refer to package insert for comment on extending incubation times.
6.2.6Centrifuge the gel card at the preset conditions of 895rpm for 10 minutes.
6.2.7Read the front and the back of each microtube and record reactions as described in the interpretation section of the corresponding MTS Gel Card package insert. See also PA.007 Reading and Recording Gel reactions Using MTS Columns.
7.0Reporting
7.1No agglutination of the red cells is a negative test result and indicates the absence of an antigen/antibody reaction and that the patient/donor is negative for the antigen being tested.
7.2Agglutination of the red cells is a positive result and indicates that the patient/donor is positive for the antigen being tested.
8.0Procedural Notes
8.1Interpretation of mixed-field reactions must be done with caution. The presence of fibrin, clots or particulates may result in some cells layering at the top of the gel. Mixed-field reactions are generally only observed in tests containing a dual population of red cells, such as a transfused patient, bone marrow recipient or when a pooled cell
sample is used for testing. However, not all mixed cell situations have a sufficient minor population to be detected.
8.2Clotted segments on a donor unit:
8.2.1If clotted segments are found on a donor unit, open subsequent segments attached to the unit until a segment is found that is not clotted.
8.2.2If all segments are clotted the blood supplier should be notified. See IM.005 – Final Disposition of Blood, Blood Components and Other Related Products Not Suitable for Transfusion Manual Procedure.
8.3Hemolyzed segments may only be noticeable while washing the segment (i.e., “reddish” supernatant).
8.3.1If hemolyzed segment is found, open subsequent segments until a segment is found that is not hemolyzed.
8.3.2If all segment are hemolyzed the blood supplier should be notified. See IM.005 – Final Disposition of Blood, Blood Components and Other Related Products Not Suitable for Transfusion Manual Procedure.
8.4LIMITATIONS:
8.4.1False-positive results may occur if antibodies to components of the preservative solution are present in the anti-sera.
8.4.2Significant variations in red blood cell suspensions (<0.6 or >1.0%) may result in false-positive or false-negative reactions.
8.4.3Adherence to the test procedure is critical to test performance.
9.0References
9.1Current package insert: Anti-Human Globulin Anti-IgG (Rabbit) MTS
Anti-IgG Card. Pompano Beach, FL: Micro Typing Systems, Inc.
9.2Current package insert: MTS Diluent 2 Red Blood Cell Diluent.
Pompano Beach, FL: Micro Typing Systems, Inc.
9.3Malyska H, Weiland D. The gel test. Laboratory Medicine,
1994;25:81-5.
9.4Standards for blood bank and transfusion services. 18th ed. Bethesda, MD: American Association of Blood Banks, 1997.
9.5Current anti-sera product insert.
/ Ontario Regional Blood Coordinating NetworkStandard Work Instruction Manual / GM.008
Page 1 of 6