Variations in the procedures for HA and HI tests are practised in different laboratories. The following
recommended examples apply in the use of V-bottomed microwell plastic plates in which the final volume for
both types of test is 0.075 ml. The reagents required for these tests are isotonic PBS (0.01 M), pH 7.0–7.2,
and RBC taken from a minimum of three SPF chickens and pooled in an equal volume of Alsever’s solution.
(If SPF chickens are not available, blood may be taken from unvaccinated birds monitored regularly and
shown to be free from antibodies to NDV.) Cells should be washed three times in PBS before use as a 1%
(packed cell v/v) suspension. Positive and negative control antigens and antisera should be run with each
test, as appropriate.