Antibody Screen – Anti-IgG Gel Card
1.0Principle
An antibody screen consists of testing the patient plasma against a set of screening cells of known antigenic composition.
The antibody screen test is used to detect unexpected blood group antibodies. In this test, the reagent red blood cells in a hypotonic buffered saline solution are combined with patient plasma to allow antigen/antibody interaction in the upper chamber of the microtube. This results in promoting antibody uptake. The detection of this antibody occurs when the sensitized red blood cells react with the Anti-IgG gel in the microtube during centrifugation.
2.0Scope and Related Policies
Note:ABO grouping, Rh typing and antibody screen together make up the Type and screen procedure.
2.1Antibody screening shall be done at 37°C and include an indirect antiglobulin procedure which has been shown to have good sensitivity. Alternative test methods may be used provided there is appropriate documentation of sensitivity and the supplier’s instructions are followed. The use of an antiglobulin reagent that contains only anti-IgG is acceptable when performing an antibody screen.9.1
2.2A minimum of two reagent red cells which express a wide variety of blood group antigens shall be used for antibody screening. Red cells with a double expression of antigens should be used. 9.1
2.3Reagent red cells for prenatal and pre-transfusion antibody screening shall not be pooled.9.1
2.4For requirements regarding antibody screening on neonatal patients see RT.005 – Antibody Screen.
2.5Patients who have been recently transfused or are pregnant may develop antibodies within three days of exposure to foreign antibodies. It is recommended that a sample for antibody screening be tested within three days of collection.
2.6A prewarm technique is usually done when a cold reactive, clinically insignificant antibody is present or suspected in a patient’s specimen. See GM.004 – Antibody Identification.
3.0Specimens
EDTA anticoagulated whole blood within five days of collection
If the patient has been recently transfused, or is pregnant, the sample should be within three days of collection.
Hemolyzed and grossly icteric specimens may cause difficulty in interpretation.
Grossly lipemic specimens containing particles that clog the gel, as indicated by diffuse blotches of red cells, may be clarified by centrifugation or filtration and re-tested.
4.0Materials
Equipment:ID – Micro Typing System:
Centrifuge
Incubator
Pipettor
Dispenser
Set-up workstation, optional
Serologic centrifuge
Supplies:ID-Tips (pipette tips)
Test tubes – 10 x 75 mm
Serologic pipettes
Package insert
Reagents:MTS Anti-IgG Card, Anti-IgG (Rabbit) suspended in gel
Antibody screen cells comprised of three vials of human red blood cells as:
0.8%, ready for use in MTS Anti-IgG Gel testing, or3%, to be prepared in-house for use in MTS Anti-IgG testing
MTS Diluent 2, a hypotonic buffered saline solution (for in-house preparation only)
Do not use beyond expiration date. Store cards at 2 to 25ºC. Store diluent and red cells at 2 to 8ºC. Bring reagents to room temperature (18 to 25°C) prior to use.
5.0Quality Control
5.1To recognize reagent deterioration, the reagents must be tested daily with appropriate controls.
5.2MTS Diluent 2 must be visually checked to ensure that the liquid is not discolored, turbid or showing any signs of bacterial contamination.
5.3To 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 cells. Reactivity must be present with the positive sample only.
5.4Do not freeze or expose cards to excessive heat. Store upright at 2 to 25°C. If the cards have not been stored in an upright position, centrifuge the cards before use.
5.5Do not use cards that show signs of drying. A liquid layer should appear on top of the gel in each microtube.
5.6Do not use cards in which the microtubes show discoloration, bubbles or crystals.
5.7Do not use the microtube cards where the seal to the microtube appears to be damaged or opened.
5.8Do not remove the foil seal to the microtubes until ready to use.
5.9The 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.
5.10Rouleaux caused by plasma with abnormally high concentration of protein or from patients who have received plasma expanders of high molecular weight can cause false positive results.
6.0Procedures
6.1Check the suitability of specimen(s). See PA.002 – Determining Specimen Suitability.
6.2Centrifuge specimen for 5 minutes at 3500 rpm or equivalent.
6.3Perform a patient history check. See PA.003 – Patient History Check
6.4Antibody Screen Cell Preparation, if necessary:
6.4.1Method 1(For 60 tests, from 3% cell suspensions)
6.4.1.1Label three test tubes 1, 2, and 3; include lot number, date and time of preparation.
6.4.1.2With an appropriate pipette, dispense 1.0mL of each antibody screen cell sample into its appropriately labeled tube and centrifuge one (1) minute to pack the red blood cells.
6.4.1.3Remove the supernatant and then add 3.0mL of MTS Diluent 2 to each tube. Mix gently. The final cell suspension should be approximately 0.8% and is stable for 24 hours. For best results, the suspension should not be less than 0.6% or exceed 1.0%.
6.4.2Method 2(For 20 tests, from packed cells)
6.4.2.1Label three test tubes 1, 2, and 3; include lot number, date and time of preparation. Prepare a volume of cells sufficient to provide 10L of packed red blood cells of each of the antibody screen samples.
6.4.2.2In separate labeled tubes, dispense 1.0mL of MTS Diluent 2. Add 10L of each of the packed
antibody screen cell samples to the appropriately
labeled tube.
6.4.2.3Mix gently. Final cell suspension should be approximately 0.8% and is stable for 24 hours. For best results, the suspension should not be less than 0.6% or exceed 1.0%.
6.4.2.4Bring specimens and reagents to room temperature (18-25C).
6.5Retrieve the patient specimen from the centrifuge and check for abnormal appearance. See PA.002 – Determining Specimen Suitability step 6.5.
6.6Compare the patient’s name and identification number on the specimen with the one on the card, the request form and computer screen, if applicable.
6.7Antibody Screen Test Procedure
6.7.1Label the MTS Anti-IgG Card with the appropriate identification and test information.
If using a 3-cell screen, 2 patients’ specimens may be tested on the same card:
I / II / III / I / II / IIIPatient #1 name
ID number / Patient #2 name
ID number
6.7.2Remove the foil seal from the microtubes to be used. See Procedural Notes 8.3.
6.7.3Using an appropriate pipette, add 50L of each of the 0.8% antibody screen cell suspensions to its labeled microtube. Do not touch pipette to gel card. Caution: The pipette tip should not touch the gel card.
6.7.4Using an appropriate pipette, add 25L of plasma to the labeled microtubes. Caution: The pipette tip should not touch the gel card.
6.7.4.1If the volume or appearance is not consistent, the microtubes should not be read and all of the tests must be repeated using new microtubes.
6.7.5Incubate at 37±2C for 15 minutes. Refer to the package insert for comment on extending incubation times and Procedural Notes 8.5.
6.7.6Centrifuge the gel card at the preset conditions of 895±25 rpm for 10 minutes.
6.7.7Read 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.
6.8Grade and record results. See PA.007 – Reading and Recording Gel Reactions.
6.9Interpret the antibody screen result. See 7.0 – Reporting.
6.10Initial or sign and record the completion time and date on the request form or in the computer.
6.11When the procedure is complete, perform a clerical check. For each antibody screen check that:
- The patient name and identification number are identical on the gel card and on the request form or computer screen (and on all specimens, if applicable)
- The reactions have been recorded on the appropriate patient’s request form or computer screen
- The test results have been interpreted correctly
6.12Report the result of the antibody screen. See 7.0 – Reporting.
7.0Reporting
7.1No agglutination or hemolysis of the screening cells in the gel card is a negative test result and indicates the absence of an antigen/ antibody reaction. If reactions are negative, unexpected antibodies
were not present or were undetected. Interpret and report the antibody screen as negative.
7.2Hemolysis in the absence of a hemolyzed sample or agglutination of any of the red cells in the gel card indicates the presence of an antibody directed against the corresponding antigen, which is present on the screening cells. If any or all reactions were positive, unexpected antibodies may be present. Interpret and report the antibody screen as positive. Perform antibody identification. See GM.005 - Antibody identification - Anti-IgG Gel Card. Or send to a reference laboratory as per established procedure.
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.
8.2False positive or false negative test results can occur from bacterial contamination of test materials, inadequate incubation time or temperature, improper centrifugation, improper storage of materials or omission of test samples.
8.3Unsealed microtube(s) should be used within 60 minutes. Reagent evaporation may occur if microtubes are left open for a longer period of time. Unused sealed microtubes, on a gel card that has been incubated and centrifuged, may be used up to the date of expiration of the particular MTS card.
8.4Addition of cells and plasma.
8.4.1Red cell suspension should be added before the plasma because the volume of red cell suspension is greater than the volume of plasma. Insufficient mixing may occur if the smaller volume of plasma is added before the red cell suspension.
8.4.2Plasma should be added within 10-15 minutes of adding the red cell suspension to the reaction chambers. Any red cells
that come in contact with the gel column prior to centrifugation may not have the opportunity to come in
contact with the plasma and may begin to migrate through the gel potentially giving a weaker reaction after centrifugation.
8.5Incubation times in low ionic strength solutions between 5 – 40 minutes have been recommended in the literature. No single incubation time will be optimal for all antibodies. If the incubation time is changed from the manufacturer’s recommendation, validation studies are required.
8.6LIMITATIONS:
8.6.1Antibodies specific for low-incidence antigens not represented on the test cells will not be detected.
8.6.2Antibodies below the threshold level may not be detected with this test.
8.6.3False-positive results may occur if antibodies to components of the preservative solution are present in the serum tested.
8.6.4Significant variations in red blood cell suspensions (<0.6 or >1.0%) may result in false-positive or false-negative reactions.
8.6.5Anomalous results may be caused by fresh serum, fibrin or particulate matter in serum or plasma, or red cells that stick to the sides of the microtube. Use of EDTA plasma will minimize the problem.
8.6.6Adherence to the manufacturer’s package insert is critical to test performance.
9.0References
9.1Heddle N, ed. Standards for transfusion medicine, 6th ed. Saskatoon, SK: Canadian Society for Transfusion Medicine, 1999: E6.00.
9.2Vengelen-Tyler, V, ed. Technical Manual. 12th ed. Bethesda, MD: American Association of Blood Banks, 1996:225-6, 331-43, 633 -5.
9.3Current package insert: Reagent Red Blood Cells SURGISCREEN. Raritan, NJ: Ortho-Clinical Diagnostics Inc.
9.4Current package insert: Anti-Human Globulin Anti-IgG (Rabbit) MTS Anti-IgG Card. Pompano Beach, FL: Micro Typing Systems, Inc.
9.5Current package insert: MTS Diluent 2 Red Blood Cell Diluent. Pompano Beach, FL: Micro Typing Systems, Inc.
9.6Malyska H, Weiland D. The gel test. Laboratory Medicine, 1994:25:81-5.
9.7Standards for blood bank and transfusion services. 18th ed. Bethesda, MD: American Association of Blood Banks, 1997.
/ Ontario Regional Blood Coordinating NetworkStandard Work Instruction Manual / GM.002
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