37 Antibody Screening by Saline IAT (SIAT)

Principle

The antibody screen test is used in the detection of unexpected blood group antibodies. In this test, the antibody-screening reagent red blood cells are combined with patient serum/plasma. This is a “saline indirect antiglobulin test” (SIAT) test. Positive reactions at any phase of testing indicate the presence of alloantibody or autoantibody in the serum. The autologous control, in which serum and autologous red cells are tested under the same conditions as the plasma and reagent red cells, is an important part of antibody identification. Incubation and the presence of enhancement media may cause reactivity in the autologous control that is only an “in-vitro” phenomenon. The autocontrol should 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 hours 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

1. Normal saline
2. AHG reagent (polyspecific or anti-IgG may be used)
3. Screening cells (commercially available un-pooled group O cells)
4. CC’s for addition to negative AHG tests.

Procedure

1. Label tubes with patient name and MRN and screening cell number identification or “auto”.
2. Add 4 drops of serum to clean, labeled tubes.
3. Add 1 drop of thoroughly mixed screening or patient cells (for the auto control) to the respective tubes.
4.  Incubate at 37°C for 30 minutes (maximum incubation time is 60 minutes).
5. Centrifuge and observe for hemolysis and agglutination.
6. 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.
7. Wash the cells 4 times with saline and completely decant (blot) the final wash.
8. Add 2 drops AHG to the dry cell button.
9. Mix thoroughly but gently.
10. Centrifuge for 20 seconds.
11. Gently re-suspend the cell button and examine macroscopically.
12. Grade and record results immediately while holding tubes in hand.
13. Confirm validity of negative tests by adding 1 drop of CCs, centrifuge for 20 seconds, read and record.

Interpretation

1. The presence of agglutination/hemolysis in any of the phases may indicate the presence of an unexpected antibody. Hemolysis is considered a positive reaction when using a serum sample with polyspecific AHG.
2. 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 less than 2+ the negative testing must be repeated from the beginning of the procedure. Results that are 2+ or greater are recorded ONLY as a check mark(√)

 Notes

1. Once commenced, the antiglobulin test must be completed. It is not permissible to interrupt or delay the test, especially during the washing stage as the sensitizing IgG antibody may “fall off” if the testing is interrupted for any length of time.
2. 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 nonreactive.