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DAT Negative Autoimmune Hemolytic Anemia

Most patients with autoimmune hemolytic anemia (AIHA) have a positive direct antiglobulin test (DAT) for IgG, complement or both. However, approximately 10% of patients presenting clinically with AIHA have a negative DAT. Physicians need laboratory evidence of an immune basis for hemolytic anemia before starting immunosuppressive therapy.

There are at least three potential causes of a negative DAT in a patient with AIHA:

  • RBC bound IgG below the threshold of detection of the routine DAT
  • Low affinity IgG that washes off RBCs during the washing phase of the tube test DAT
  • RBC bound IgA or IgM that is not detected by routine antiglobulin reagents

The routine tube DAT has a lower limit of detection of about 200 IgG molecules per red blood cell. A more sensitive method is needed to detect cases associated with lower levels of autoantibody. The most commonly available methods include: enzyme linked antiglobulin test (ELAT), flow cytometry, direct Polybrene test, solid phase and column agglutination.

Low affinity IgG can sometimes be retained on red blood cells by washing them with ice cold saline or low ionic strength saline (LISS) and then retesting using routine anti-IgG and anti-C3d reagents. Column agglutination tests may also detect low affinity IgG autoantibody because red cells are not washed prior to testing.

IgM autoantibodies usually fix complement, which can be detected by anti-C3d reagent. Anti-IgM and anti-IgA antisera are available commercially but are not approved for clinical use by the FDA in the United States. Approximately 2% of AIHA cases are caused by IgA autoantibodies.

Many of these more esoteric tests are not available in hospital transfusion services. Testing can be sent to reference laboratories that are often located within regional blood centers.

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