38 Antibody Screening by LISS IAT
Principle
The antibody screen test is used in the detection of unexpected blood group antibodies. In this test, screening cells ( reagent red cells) are combined with patient serum/plasma. The test may include a potentiating medium (low ionic strength saline/solution or LISS) which promotes antibody/antigen reactions to occur quickly. Positive reactions at any phase of testing indicates the presence of an allo- or auto-antibody. An autologous control, is included in antibody investigations of positive antibody screens and tests patient plasma and red cells under the same conditions as the antibody screen test. Incubation and the presence of enhancement/potentiating media may cause reactivity in the autologous control that is only an “in-vitro” phenomenon; an auto-control is expected to be negative.
Related Policies
This procedure applies to all testing that requires antibody screening.
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 hr 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 hr period following transfusion of the first unit of blood.”
Reagents
- Isotonic saline.
- AHG reagent. (polyspecific or anti-IgG may be used)
- Screening cells.(commercially available un-pooled group O cells)
- LISS additive.
- CC’s for addition to negative AHG tests.
Procedure
- Label tubes with patient and screening cell number identification or “auto”.
- Add 2 drops of test serum/plasma to each clean, labeled tube.
- Add 2 drops of LISS to each tube and mix well.
- Add 1 drop of thoroughly mixed screening or patient cells (for the auto control) to their respective tubes.
- Incubate at 37°C for 10-15 minutes (as per manufacturer’s instructions).
- Centrifuge and observe for hemolysis.
- Gently re-suspend the cell button and examine macroscopically,(may use an optical aid), for agglutination.
- Grade and record results (37°C) immediately while holding tubes in hand. All positive and negative results should be carried on through to the end of the procedure.
- Wash the cells 4 times with isotonic saline and completely decant/blot the final wash.
- Add 2 drops of AHG to the dry cell button.
- Mix thoroughly but gently and centrifuge for 20 seconds.
- Gently re-suspend the cell button and examine macroscopically.
- Grade and record results immediately while holding tubes in hand.
- Confirm validity of negative tests by adding 1 drop of CCs. Centrifuge for 20 seconds, suspend, and read for agglutination; record results.
Interpretation
- The presence of agglutination/hemolysis in any of the phases may indicate the presence of an unexpected antibody. Hemolysis is a positive reaction when using a serum sample with polyspecific AHG.
- The absence of agglutination/hemolysis in all of the phases is a negative result. There must be agglutination in the last step with the IgG-sensitized red cells (CCs) confirming the presence of active antiglobulin reagent in the test mixture. If there is no agglutination or the results are indeterminate, testing must be repeated from the beginning. Record the strength of CC reaction as 1-4+.
Notes
- The Antiglobulin test must be completed without delays. Interruptions or extended delays may result in false negative and /or positive results depending on which phase of testing is impacted. Delays during the washing stage may cause IgG antibody to elute off the red cells if extended interruptions
- If tests with all reagent red cells are reactive, the possibility of spontaneous agglutination should be considered. A control of cells washed 4 times added to two drops of saline must be non-reactive.