26 Phenotyping -“Direct” 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 for transfusion to a patient with a history of /or have 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 direct antigen typing, specific antisera will directly agglutinate red blood cells that have the corresponding antigen. Agglutination will occur after a short incubation period either at room temperature or 37^oC, depending on the antiserum being used (follow manufacturer’s instructions).

Related Policies

This procedure applies to all test protocols that require direct red blood cell antigen typing. Whether an antibody is reactive by direct agglutination or by the indirect antiglobulin technique (IAT) is dictated by the manufacturer’s instructions.

Reagents

1. Commercial anti-sera.
2. Test red cells.
3. Control cells, negative and single-dose positive, for specific anti-sera used.
4. Isotonic saline.

Procedure

1. 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 auto-control or DAT was previously performed on the same specimen and was found negative (see note # 4).

2. 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. Positive controls are ALWAYS single dose for the relevant antigen (weakest source of positivity). Negative controls are negative for the relevant antigen.

3. Prepare 2-5% red cell suspension of patient or donor.
4. Add reagent anti-sera to the appropriate labeled tube.
5. Add appropriate number of drops of patient/donor red blood cells to the tubes.
6. Mix well and complete testing according to the manufacturer’s directions.
7. Re-suspend and read for agglutination/hemolysis.
8. Grade and record test results immediately while holding tubes in your hand.
9. If performing donor unit typing, label the donor unit as positive or negative for antigens tested according to the policy of the lab.

Interpretation

1. 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.
2. The absence of agglutination is considered to be a negative reaction, indicating the absence of the corresponding antigen on the red blood cells.

Notes

1. 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.
2. If the patient’s red cells test positive for an antigen for which it appears he or she has the corresponding antibody, the auto control 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.
3. Mixed-field reactions may be caused by the presence of donor cells in patients who have been transfused in the previous 3 months.
4. Patient cells coated with immunoglobulin (ie, a positive direct antiglobulin test) may spontaneously agglutinate and cause a false-positive result.
5. False-negative reactions may occur if red blood cells used are older than what is described in the specimen requirements of the manufacture.
6. Follow manufacturer’s directions for specific limitations of antisera being used.