Table 5.

Crossmatching protocol. Major crossmatching should be compatible at 37° and 24°C (cold agglutinins) and minor at 37°C (Adapted from Fox [41]).

1. Collect 2 mL of blood into EDTA from tom/kitten and queen.

2. Centrifuge 3400 × g 1 minute, separate plasma from red blood cells. Keep plasma.

3. Wash red blood cells two times, into at least twice its volume, with isotonic saline solution.

Discard supernatant and keep red blood cells.

4. Dilute red blood cells at 2% : 10 μL washed red blood cells plus 490 μL isotonic saline solution.

5. Major crossmatching:

2 drops of (50 μL) tom/kitten's red blood cell dilution

2 drops of (50 μL) queen's plasma

6. Minor crossmatching:

2 drops of (50 μL) queen's red blood cell dilution

2 drops of (50 μL) tom/kitten's plasma

7. Negative control:

2 drops of (50 μL) tom/kitten's red blood cell dilution

2 drops of (50 μL) tom/kitten's plasma

8. Incubate 30 minutes at 25°C and also at 37° and 24°C.

9. Centrifuge 3400 × g 1 minute.

10. Examine the supernatant for any haemolysis. Any haemolysis indicates also incompatibility.

11. Rotate tubes between the fingers to mix and examine for agglutination. The presence of agglutination indicates a positive test and

tom or kitten/queen incompatibility.