45 ABID by Micro Typing Systems (MTS)- extended

Principle

The antibody identification test is used to identify red cell antibodies detected during the antibody screen. Column agglutination uses gel or glass beads to trap agglutinated red cells. The commercially prepared systems use a card or strip of several microtubes of reactants that allows several tests to be performed at once. In the gel test to detect or identify antibodies, the reagent red cells are combined with sample serum/plasma in the upper reaction chamber of the microtube of an anti-IgG gel card. The card is incubated at 37 degrees to enhance the antigen/antibody interaction in the chamber at the top of each column. The card is then centrifuged and the red cells move into the column medium; sensitized red cells react with the anti-IgG incorporated in the gel of the microtube during the centrifugation step. The column medium separates agglutinated red cells (which are too large to enter the gel matrix and thus remain at the top of the gel) from non-agglutinated cells that move through the pores in the gel to the bottom of the column. Agglutinated red  cells, agglutinates, become trapped in the microtube gel at various levels  based on their size. Free, non-agglutinated, red cells pass through the gel and form a button on the bottom of the microtube. Agglutination indicates the presence of an antigen/antibody reaction while lack of agglutination indicates the absence of an antigen/antibody reaction. One advantage of this technology is that results are permanent and do not require washing or control cells.

Specimen

Serum or plasma may be used. Specimen age must comply with policies and procedures of the institution. CAN/CSA Z902 Blood and Blood Components standards indicate “the sample shall be collected within 96 h prior to the scheduled transfusion if:

• The recipient has been transfused with a blood component containing red cells within the previous 3 months
• The recipient has been pregnant within the previous 3 months; or
• The recipient’s history is questionable or unavailable

For repeat recipients, the original sample may be used to crossmatch additional units within the 96 hour period following transfusion of the first unit of blood.”

Hemolyzed and grossly icteric blood samples may cause difficulty in interpretation, and test results should be used with caution.  Grossly lipemic samples containing particulates that clog the gel, as indicated by diffuse blotches of red blood cells in the microtube, may be clarified by centrifugation or filtration and retested.

Reagents

1. Anti-Human Globulin anti-IgG MTS anti-IgG card
2. 0.8% Panel Cells ( Can be prepared from 2-5%  Panels)
3. MTS diluent 2 (a hypotonic buffered saline solution used for in-house red blood cell preparation only)
4. Do not use reagents beyond expiration date
5. Store gel cards upright at 2°C to 8°C
6. Store diluents and red blood cells at 2°C to 8°C
7. Bring reagents to room temperature (18°C to 25°C) prior to use.

Quality Control

Gel Card

To confirm the specificity and reactivity of the MTS anti-IgG card, it is best practice to test each lot number “in use”, on the day of use, with known positive and negative antibody samples and appropriate red cells. Reactivity must be present with the positive sample only.

Diluent

MTS Diluent 2 should be visually checked to ensure that the liquid is not discolored, turbid or showing any signs of bacterial contamination.

Procedure for Preparation of 0.8% Suspension of Panel Identification Cells

For 20 tests, from packed red blood cells

1. Label two sets of 12X75 test tubes for the panel cells (1-10/TC).
2. In one set of labeled tubes; Place at least 6 drops of the respective manufacturer’s 2-5% panel red cell suspension in the properly labeled tube. Spin for 45 seconds and remove the supernatant from the cells by tipping the tube to the side and using a glass pasteur pipet being careful not to leave any diluent behind.
3. In the second set of labeled tubes; dispense 1000 microliters of MTS Diluent 2ä into each tube. Add 10 µL of the appropriate packed red blood cell sample to the diluent.
4. Mix gently to re-suspend. The final red blood cell suspensions should be approximately 0.8%. For best results, the red blood cell suspensions should be used on the day of dilution.

Preparation of the Auto Control (if desired)

1. Place 1000 microliters of MTS Diluent 2ä in a test tube labeled with the test sample identification
2. Add 10 µL of packed red blood cells from the sample to be tested.
3. Mix gently to re-suspend. The final red blood cell suspensions should be approximately 0.8%. For best results, the red blood cell suspensions should be used on the day of dilution.

Procedure for setting up Gel cards

1. Visually inspect each gel card before use. Each microtube should have a clear liquid layer on top of opaque gel (LISS layer). 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 use cards if foil seals appear damaged or opened.
2. Label the MTS Anti-IgG Cardä with the appropriate identification (I, II, III, auto for screens or 1-10/TC for panels and patient name).
3. Remove the foil seal from the gel card or from the individual microtubes used for testing. Note: Foil should be removed immediately before testing or within one hour of testing. Once opened, the gel may begin to dry out which could affect test results.
4. Add 50 µL of the respective 0.8% antibody screen red blood cell suspension to the appropriately labeled microtube. Caution: The pipette tip should not touch the gel card. Erroneous results due to carryover may occur.
5. Add 25µL of the patient’s serum or plasma to the microtubes. Caution: The pipette tip should not touch the gel card. Erroneous results due to carryover may occur.
6. Incubate the MTS Anti-IgG Cardä for 15 minutes at 37± 2 degrees C.
7. After incubation, centrifuge the gel card(s) in the MTS Centrifuge™ at the pre-set conditions.
8. For manual readings, observe the front and the back of each microtube macroscopically and record reactions as described in the interpretation section of the corresponding MTS Gel Card package insert.

Interpretation

1. Negative Result – No agglutination and no hemolysis of the red blood cells is a negative test result. Red cells will fall to the bottom of the microtube.
2. Positive Result – Agglutination and/or hemolysis of the red blood cells is a positive test result. Red blood cells may remain suspended on the top of the gel or are dispersed throughout the gel in varying degrees. A few red blood cells may form a button in the bottom of the microtube in some positive reactions.

Comments

1. Identification of the antibody present in the serum/plasma may be made by matching the reactions obtained with the antigen profiles of red blood cells on the panel sheet. Additional red blood cells may be needed to identify multiple antibodies.
2. A room temperature or 37°C panel procedure using the MTS Buffered Gel Card may be helpful in identification of some antibodies, especially when more than one antibody specificity is suspected.
3. Reactivity of the serum/plasma with the autologous control red blood cells may indicate the presence of an autoantibody. Clinical history regarding recent red blood cell transfusion may be helpful.
4. Interpretation of mixed-field reactions must be done with caution. The presence of fibrin, clots, or particulates may result in some red blood cells layering at the top of the gel. Mixed-field reactions should only be observed in tests containing a dual population of red blood cells, such as a transfused patient, bone marrow recipient or when a pooled cell sample is used for testing. However, not all mixed red blood cell situations have a sufficient minor population to be detected.

Notes

1. Variations in the red blood cell concentration can markedly affect the sensitivity of test results. If red blood cell suspensions are too concentrated, they can give weaker results due to the increase in the antigen/antibody ratio. In addition, red blood cells may fail to completely migrate to the bottom of the microtube and could cause a false positive interpretation. When the red blood cells are too low in concentration, they become difficult to visualize and, in extreme cases, a weak positive can fail to be detected.
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. Optimal reaction conditions vary across antibody specificities. No single test method will detect all antibodies. In some low ionic strength test systems, certain antibodies, such as Anti-E and Anti-K, have been reported to be nonreactive.
5. There is the potential for IgM antibodies to react in this test. Some patient antibodies that are IgM in nature may react with corresponding antigens in the upper portion of the microtube and be trapped in the top portion of the gel at the time of centrifugation resulting in a positive reaction.
6. False positive results may occur if a card that shows signs of drying is used in testing.
7. The Anti-H of Para-Bombay individuals may not be detectable in gel.