Note: Responses reflect Canadian practices, which vary from location to location.

Question 1: Why is dosage an important concept in pretransfusion antibody screening tests?

Dosage is important in pretransfusion testing because it is a potential cause of false negatives in antibody screen tests. False negative may occur if the patient has a weak antibody that will not react with red cells from heterozygous donors, since such cells have fewer antigens than red cells from homozygous donors.

This is why it is important to use sensitive antibody screen methods, as well as screen cells in which at least one donor is homozygous for (genes that control) antigens that exhibit dosage.

Question 2: Which follow-up tests are routinely done when investigating possible delayed hemolytic transfusion reactions (DHTR)?

Once a suspected transfusion reaction has been identified and evaluated as a possible DHTR, the following are examples of tests that are routinely performed in accordance with a laboratory's policies and procedures on the patient's post-transfusion specimen

Routine tests

  1. check for visible hemolysis
  2. direct antiglobulin test (DAT)
  3. ABO and Rh
  4. antibody screen

Many labs perform only the check for hemolysis and the DAT and do no further serologic testing* if results are negative. This abbreviated protocol assumes that patient and donor unit clerical checks have eliminated a misidentification error and that clinical symptoms and other tests do not indicate hemolysis.

* Similar symptoms may occur in several types of transfusion complications. When beginning an investigation, all possibilities must be investigated. As patient history, clinical presentation, and laboratory results accumulate, a differential diagnosis is  assessed from which the ultimate diagnosis emerges.

Additional tests

Depending on the results, the following tests may also be done:

If DAT is Positive

If the DAT done on the patient's post-transfusion EDTA blood sample is positive:

  • DAT on the pre-transfusion blood sample (for comparison)

    If the DAT on the pre-transfusion specimen is negative, a more complete investigation may be done, an example of which follows.

    NOTE: The DAT on the patient's post-transfusion specimen may be negative - even though a hemolytic transfusion reaction has occurred - if at the time of testing most or all transfused donor red cells have been removed from circulation.

  • Monospecific DATs.

    If the post-transfusion DAT is positive with polyspecific antiiglobulin serum, the DAT can be repeated with monospecific anti-IgG and anti-C3b/-d to determine the substances sensitizing the patient's red cells. The purpose is to assess if an elution is worthwhile to identify antibodies that may be sensitizing the patient's cells.

  • Elution

    If IgG is sensitizing the patient's red cells, an eluate may be prepared from the DAT-positive red cells. An antibody identification is done on the eluate to identify the antibodies sensitizing the cells.

If Antibody Screen is Positive

  • Antibody identification

    If a new or unexpected antibody is detected, it is identified, with subsequent antigen phenotyping of patient's pre-transfusion red cells and implicated donors.

Question 3: Why should donor units that are crossmatch-compatible NOT be released, unless life-threatening, before the antibody is identified?

Because weak antibodies may give false negatives with red cells that have a weak expression of the corresponding antigen, the risk is that seemingly crossmatch-compatible donors may be positive for the corresponding antigen. If transfused, they can cause a hemolytic transfusion reaction.

Question 4: Why can a patient with a clinically significant antibody sometimes have a negative antibody screen while experiencing a delayed hemolytic transfusion reaction in which antibody levels are rising?

A temporary decrease in antibody level occurs at the time of a 2o response. The decrease occurs when antibody attaches to transfused red cells and the transfused cells adsorb (mop up) the patient's antibody. During this period, the antibody screen may be negative until antibody production reaches the level at which it can be detected.

Question 5: Provide six best practices for preventing age: delayed hemolytic transfusion reactions.

Best practices include:

  1. Records. Checking records for prior history of transfusion and presence of known antibodies. For patients; with clinically significant antibodies, antigen-negative donor units must be crossmatched even if the antibody is currently undetectable.
  2. Communication. Information about known antibodies should be communicated to the transfusion service.
  3. Sensitive antibody screen cells. At least one screening cell should be homozygous for any antigens that show dosage.
  4. Sensitive antibody detection methods. LISS, PEG, gel, and solid phase adherence assays are sensitive methods. If the less sensitive methods such as saline or albumin are used, a larger plasma to cell ratio and longer incubation times increase test sensitivity.
  5. Fresh patient specimens (less than 3 days). Fresh specimens will increase the likelihood of detecting that a 2° immune response in a patient with a pre-existing weak antibody.
  6. Periodic re-identification of antibodies. If a patient with a known antibody is being transfused regularly, re-identification should be done periodically to detect the presence of new antibodies that may have formed.
  7. IAT crossmatch for patients with antibodies. If a patient has a previously identified clinically significant antibody, antigen-negative red cells must be selected for crossmatch and the crossmatch must be performed by the antiglobulin (IAT) method.
  8. Antigen typing of crossmatch compatible donors. If the antibody screen is positive, seemingly compatible donors should NOT be issued (except for life-threatening emergencies) until the antibody is identified and the donors are antigen typed and found to be antigen-negative.