Feline Neonatal Isoerythrolysis and the Importance of Feline Blood Types

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]).

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

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2. Centrifuge $3400\times \text{g}$ 1 minute, separate plasma from red blood cells. Keep plasma.

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

 Discard supernatant and keep red blood cells.

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4. Dilute red blood cells at $2\%:10\hspace{0.17em}\mu \text{L}$ washed red blood cells plus 490 $\mu$L isotonic saline solution.

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5. Major crossmatching:

    2 drops of (50 $\mu$L) tom/kitten’s red blood cell dilution

    2 drops of (50 $\mu$L) queen's plasma

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6. Minor crossmatching:

    2 drops of (50 $\mu$L) queen’s red blood cell dilution

    2 drops of (50 $\mu$L) tom/kitten’s plasma

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7. Negative control:

    2 drops of (50 $\mu$L) tom/kitten’s red blood cell dilution

    2 drops of (50 $\mu$L) tom/kitten’s plasma

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8. Incubate 30 minutes at ${25}^{°}$C and also at ${37}^{°}$ and ${24}^{°}$C.

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9. Centrifuge $3400\times \text{g}$ 1 minute.

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

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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.

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