c. Donath-Landsteiner Test.
(a) STEP 1. Blood from the patient is allowed to clot, undisturbed, at
37C and the serum separated (preferably by centrifugation at 37C).
STEP 2. Pipette 10 volumes of patient's serum into two 10- X
(c) STEP 3. As patients with PCH may have low serum complement
levels leading to false-negative results, it is recommended that tests with added
complement be included. Pipette five volumes of fresh compatible normal serum as a
source of complement into two 10- X 75-rnm tubes. Add five volumes of patient's
serum to one tube. The tube containing complement alone acts as a negative control.
(d) STEP 4. To all four tubes add 1 volume of a 50 percent
suspension of washed normal group O, P-positive RBCs.
(e) STEP 5. One of the tubes from step 2 and both tubes from step 3
are incubated, in melting ice for 1 hour.
(f) STEP 6. The other tube from step 2 is incubated in a 37C water
bath, to act as a negative control.
(g) STEP 7. After 1 hour, the tubes in the melting ice are gently mixed
and moved to 37C for 30 minutes.
(h) STEP 8. All tubes are gently mixed and centrifuged.
(2) Lysis of cells. Lysis visible to the naked eye indicates a positive test.
The control tubes containing normal serum only, and the tube left at 37C (step 6)
throughout the test should be negative.
(3) Alternative method. The test can also be performed by placing samples
of the patient's blood into tubes prewarmed to 37C. One sample is left to clot at 37C.
The other is placed immediately in melting ice and left undisturbed for 1 hour. It is then
moved to 37C for 30 minutes, centrifuged, and inspected for hemolysis. This is a
simple way of performing the test, but rather wasteful of blood.