49 LISS Antihuman Globulin (AHG) Crossmatch

Principle

The serologic (AHG) crossmatch is used to detect blood group antibodies in the serum/plasma of a recipient to antigens on donor RCs. The RCs are combined with patient serum/plasma to allow antigen-antibody interaction.  The immediate-spin (IS) crossmatch provides serologic testing to detect ABO incompatibility only. The completion of the crossmatch through the antihuman globulin phase of testing permits detection of incompatibilities caused by antibodies (IgG)that react with cells at 37oC. The antihuman globulin crossmatch is used when a patient has an identified or previously identified red cell antibody.

Related Policies

This procedure applies to compatibility testing of all transfusion recipients with clinically significant antibodies.

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 h period following transfusion of the first unit of blood.”

Reagents

1. Isotonic saline.
2. AHG reagent. (polyspecific or anti-IgG may be used)
3. Donor RCs; from segments attached to the units to be transfused.
4. LISS or other potentiator(s) as needed.
5. CC’s for addition to negative AHG tests.

 Procedure

1. Label tubes with patient name, donor unit number and test identification (XM).
2. Prepare a 2-5% red cell suspension in isotonic saline from each unit segment.
3. Add 1 drop of donor cell suspension to the labeled tubes (in student labs you may need to use 2 or more drops of cells if there is a risk the “units” are hemolyzed or “old”). Instructor will provide you with the relevant directions for this lab..
4. Add 2 drops of patient serum to each of the tubes.
5. Add two drops of LISS to each tube and mix well.
6. Incubate at 37°C for 10-15 minutes (as per LISS instructions).
7. Perform a 37 ℃ spin/reading if ABS was positive at this temperature, if not proceed to 10.
   1. Centrifuge for 20 seconds and observe for hemolysis..
8. Gently suspend the cell button and examine macroscopically.
9. Grade and record results immediately while holding tubes in hand. All positive and negative results should be carried on through to the end of the procedure.
10. Wash the cells 4 times with saline and completely decant the final wash.
11. Add 2 drops of AHG to the dry cell button.
12. Mix thoroughly but gently and centrifuge for 20 seconds.
13. Gently suspend the cell button and examine macroscopically
14. Grade and record results immediately while holding tubes in hand.
15. Confirm validity of negative tests by adding 1 drop of CCs. Centrifuge for 20 seconds, re-suspend, and read for agglutination; record results.

Interpretation

1. The presence of agglutination/hemolysis in any of the phases may indicate the presence of a serologically incompatible crossmatch. This result is interpreted as “incompatible”. The cause of all positive results must be investigated before transfusion. Hemolysis is considered positive when using serum samples and polyspecific AHG.
2. The absence of agglutination/hemolysis in all phases is a negative result and indicates a serologically compatible crossmatch providing the CCs worked as follows: there must be agglutination in the last step with the IgG-sensitized red cells (CCs) confirming the presence of active antihuman globulin reagent in the test mixture. If there is no agglutination or the results are indiscriminate, the negative testing must be repeated from the beginning. Record the reaction strength of positive results (1-4+). After addition of CCs to a negative test, the presence of agglutination indicates that the AHG added was capable of reacting and that the antiglobulin test is valid. This result is interpreted as “compatible”.
3. Calculating the number of units to be tested when a patient has an antibody: Example: Patient has anti-K, require 2 compatible units

• • Use the negative frequency of the Ag (9% have “K” Ag therefore 91% do not have “K” Ag-negative frequency is 0.91)
  • Need 2 units. Divide the number of units required by the negative frequency for the antigen: 2/.91=2.2 (3) units need to be screened to get 2 compatible units
  • For multiple Ab’s: Multiply negative frequencies for ALL Ag’s and divide the number of units required by this combined frequency

Notes

1. The antihuman globulin test is sensitive to delays in testing. It is not permissible to interrupt or delay the test, especially during the washing stage as the sensitizing IgG antibodies may “fall” off the red cell.
2. The antihuman globulin crossmatch will not detect errors in Rh typing, prevent iso-immunization of the recipient or ensure normal red blood cell survival.