It is sometimes observed that the standard HI titre of the standard anti-serum tested on the same day with the same antigen preparation and protocol may vary. A difference in HI titre of one dilution that is one log base 2 (21) can be regarded as due to random errors and an inherent variability in biological responses. However if the titre of the standard positive serum differs by more than one dilution or log base 2, then the test is invalidated. In this case, fresh antigen must be prepared and tested and the HI test repeated.