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48
titration proved that with normal serum at least 2.6 doses were required for
complete lysis, the excess in the control tubes was 1.3 dose and that in the diagnostic
tubes was 3.9 minus 3.3, or .6 dose. In view of these excesses the laking could be
expected to proceed at a reasonable rate in the control tubes, a little more rapidly
in the back row tubes (with the nominal 5 doses), and with a little delay in the
middle row tubes (containing the nominal 3 doses). This indeed actually did occur.
The middle row tubes contained an excess of .6 dose which is below the allowable
amount of one dose, and in the back row tubes the excess was 2.6 doses above that
contained in the middle row tubes. Therefore the total excess in the back row tubes
over the amount required for laking was 2.6 plus .6, or 3.2 doses, instead of the
allowable 3 doses, and thus these latter tubes contained approximately their usual
amount of 2 doses more than the diagnostic tubes.
The above figures show that by using 1:60 as the working titre only the
standard excesses of complement were used, and why the test proper gave reliable
results and thus justified the adoption of the modified titre.
5. Other possible sources of abnormal results.—An excessive degree of inactivation
of complement in the mixtures with antigen may sometimes be due to the use of
too much antigen, and so in such cases the titre of the antigen should be redetermined.
The titre of the antigen used in the above titrations was re-determined,
and in consequence was re-adjusted from 1:30 to 1:40. It was then found that
with the greater dilution of antigen subsequent titrations of complement gave normal
readings; that is, the working titre as found from the readings of the third and
back rows of tubes was identical with the true titre as determined by the front row
tubes of the titration. Thus the back row of tubes showed that with a normal
serum something less than 2 doses of complement were required to lake one volume
of sensitised red cells, and therefore, by using 3 doses of complement in the control
tubes of the test proper, there would be an excess of at least one dose. Also the
third row tubes showed that such serum and antigen would require not more than
2 or 2.1 doses to lake the red cells completely, so that by using 3 doses of complement
in the diagnostic tubes of the test proper there would again be a sufficient excess of
complement to make the results of the test reliable.
Abnormal results may arise from using a control serum which has deteriorated,
by becoming infected or otherwise, and this should be suspected if the degree of
inactivation of complement in the back row of the titration is excessive. Otherwise,
too low a reading may be chosen for the working titre which would cause too
much complement to be used in the test proper, and so the detection of some of the
doubtful sera would fail. If the correct working titre has been chosen, this will be
proved by any negative sera in the batch being tested laking within 10 minutes, but
the diagnostic tube of the bad normal (?) serum used as the control will probably
require 15 minutes or even more.
The condition of the sheep's red cells and of the amboceptor used to sensitise
them has also to be considered.
Summary.
This paper shows the necessity for the extra rows of tubes set up in titrating
guinea-pig serum so as to check the reading of the front row tubes for the titre.
These extra rows of tubes also act as a measure of the influence of the different
ingredients used in the test proper, both singly and when acting together. This
enables one to modify the reading of the front row tubes and assists in choosing the
titre best suited to the prevailing conditions. A table is outlined to facilitate the
conversion of dilutions of guinea-pig serum into definite units or doses of complement
per volume. A titration is discussed with these units to show how a modified titre
was justified. The advisability of re-estimating the titre of the antigen and
consideration of the quality of the normal control serum, the sheep's red cells and the
amboceptor are mentioned.
Reference.
1 Price, I.N.O., London County Council publication, 1933, No. 2995.