25 Phenotyping – “Indirect” Method
Principle
After an alloantibody has been identified, it is necessary to test the patient’s pre-transfusion red blood cells to determine that they lack the corresponding antigen. Additionally, all red blood cells used for transfusion to a patient with a history of or with a current clinically significant antibody must be negative for the corresponding antigen. The presence or absence of the corresponding antigen is determined with the use of potent commercially available antisera. In indirect antigen typing, specific antisera will agglutinate red blood cells that have the corresponding antigen. Agglutination is demonstrated by the indirect antiglobulin technique (IAT) technique (follow manufacturer’s instructions).
Related Policies
This procedure applies to all test protocols that require indirect (IAT) red blood cell antigen typing.
Reagents
- Commercial antisera
- Test red cells
- Control cells, negative and single-dose positive, for specific antisera used.
- AHG (polyspecific or anti-IgG)
- CC’s
- Isotonic saline
Procedure
- If typing a patient specimen, verify that the patient has not been transfused with red blood cells during the 3 previous months. Also, check to see if an autocontrol or DAT was previously performed on the same specimen and was found negative (see note #5).
- Label tubes with patient name and MRN or donor unit number and anti-sera identification. Include a positive and negative control, if not already performed that same day.
- Prepare 2-5% red cell suspension of patient or donor.
- Add appropriate number of drops of reagent anti-sera to the appropriate labeled tube.
- Add appropriate number of drops of 2-5 % cells suspension of patient or donor red blood cells to the tubes.
- Mix well and incubate at 37°C for incubation time period indicated in the manufacturer’s directions.
- Wash all tubes 4 times with isotonic saline. Decant completely and blot after last wash.
- Add two drops of AHG reagent and spin for 20 seconds.
- Re-suspend and read for agglutination.
- Grade and record test results immediately while holding tubes in your hand.
- Control all negative antiglobulin tests by adding one drop of CC’s. Centrifuge for 20 seconds, re-suspend and read for agglutination. Record results.
- If performing donor unit typing, label the donor unit as positive or negative for antigens tested according to the policy of the lab.
Interpretation
- Agglutination of red blood cells in the presence of the specific antiserum is a positive reaction and indicates the presence of the corresponding antigen on the red blood cells.
- The absence of agglutination is considered to be a negative reaction, indicating the absence of the corresponding antigen on the red blood cells.
Notes
- There must be agglutination in the last step with the IgG-sensitized red cells (CCC’s) 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. Results that are 2+ or greater are recorded ONLY as a check mark (√)
- The positive control (single-dose cell) must have reaction strength of 1+ or greater and the negative control must be nonreactive, or the test is invalid and must be repeated.
- If the patient’s red cells test positive for an antigen for which it appears he or she has the corresponding antibody, the autocontrol might be positive and these results should be investigated. The patient may have been transfused in the previous 3 months or the antibody was not correctly identified originally.
- Mixed-field reactions may be caused by the presence of donor cells in patients who have been transfused in the previous 3 months or women having delivered a child in the previous 3 months.
- Patient cells coated with immunoglobulin or complement (ie, a positive direct antiglobulin test) may spontaneously agglutinate and cause a false-positive result.
- False-negative reactions may occur if red blood cells used are older than what is described in the specimen requirements of the manufacture.
- Follow manufacturer’s directions for specific limitations for antisera being used.