How is a direct antiglobulin test (DAT) performed?

Updated: Jun 25, 2020
  • Author: Julie Katz Karp, MD; Chief Editor: Jun Teruya, MD, DSc, FCAP  more...
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The direct antiglobulin test is performed by tube agglutination, as follows: [4, 8]

  1. Patient RBCs are acquired from an ethylenediaminetetraacetic acid (EDTA)–anticoagulated blood sample

  2. One drop of a 2%-5% suspension of patient RBCs (in saline or native plasma) is dispensed into each of 4 test tubes

  3. The patient RBCs are washed 3-4 times with saline, and the final wash supernate is completely decanted

  4. Polyspecific AHG reagent is added to 1 tube

  5. Similarly, tests with the anti-IgG and anti-C3 reagents are set up

  6. Two drops of saline are added to the fourth tube

  7. The contents of each tube are gently agitated

  8. Centrifuge (Note: For anti-complement, some manufacturers may recommend a delay before centrifugation; others may recommend that initial negative results with anti-C3 be re-evaluated following incubation at room temperature for 5 minutes and then recentrifugation)

  9. The RBCs are examined for agglutination; the results are graded and recorded

  10. IgG-coated RBCs are added to all negative tests with anti-IgG or polyspecific AHG; complement-coated RBCs are added to all negative tests with anti-C3; recentrifuge and examine the tests macroscopically for agglutination

  11. All tests are repeated when IgG-coated RBCs and/or complement-coated RBCs are nonreactive

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