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PCH Donath-Landsteiner Test

The Donath-Landsteiner test is a confirmatory test that subjects the antibody in serum/plasma to appropriate cells at 4°C followed by 37°C incubations. The presence of hemolysis after these successive incubations confirms the presence of the biphasic hemolysin. If there is no hemolysis noted, the biphasic hemolysin is not present.
In this test, the patient's sample must be maintained at 37°C after collection. The test is illustrated in the image on the right.
Patient's serum is added to three sets of three tubes.
  1. Set #1 contains only patient serum.
  2. Set #2 contains patient's serum and fresh normal serum, which acts as a source of complement.
  3. Set #3 contains only fresh normal serum.
P-positive cells are then added to all tubes. One tube from each set is kept in an ice bath for 90 minutes (1A, 2A, 3A). Another tube from each set is kept at 37°C for 90 minutes (1B, 2B, 3B). The last tube from each set is put in an ice bath for 30 minutes and then incubated at 37°C for 60 minutes (1C, 2C, 3C).
After incubation, the tubes are examined for hemolysis. The Donath-Landsteiner test is positive when the patient's serum (with or without the normal serum) demonstrates hemolysis in tubes incubated in ice then incubated at 37°C. Hemolysis should not be present in any of the other tubes. Again, this serves to prove the biphasic nature of the hemolysin and its ability to lyse RBCs.