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47
4. Critical examination of the results obtained in the titration of guinea-pig
serum.—There was no apparent reason to doubt the accuracy of the reading of the
front row tubes (containing dilutions of guinea-pig serum and sensitised red cells
only), so it may be assumed that 1:78 was the true titre. Dilution of this serum 26
times should therefore give a solution containing 3 doses (M.H.D.) of complement
per volume.
Referring to table II, the mixture in the back row of tubes is similar to that
present in the front row (control) tubes of the test proper with normal sera. It will
also be seen that approximately 2.6 doses of complement would be inactivated by a
normal serum and by the action on the red cells, thus leaving only .4 dose, of the
3 doses used in the test proper, to allow for variations between different sera. This
is important, especially in those which are positive. Thus, laking in the control
tubes of strongly positive sera is often very noticeably delayed in comparison with
control tubes of normal sera. In addition, excess of complement is advisable to
ensure a rate of lysis which will enable the reaction to be completed within a
reasonable time. This also enables the end point to be judged more accurately.
An allowance of one dose over that required for the red cells and the serum is found
to be a sufficient excess to cover all the above points, and should be present when
3 doses of complement are used as the standard minimum amount chosen for the test.
Thus it is obvious that with only .4 dose available as the excess instead of one
dose, even if complete laking occurred in a control tube, it would proceed in the
final stages at a very slow rate, and also that laking would not be completed with
some of the strongly positive sera at the end of the test (30 minutes). Reliable
results could not be recorded with such controls, and what has been said of the
control tubes applies even more to the diagnostic tubes, to which the third row of
tubes in the complement titration corresponds, as these contain antigen as well as
serum.
Table II shows the third row of tubes required 3.3 doses of complement for
complete laking (when the true titre is used), and whereas complete laking might
occur in some of the control (front row) tubes of the test proper, it could not be
expected to occur in the diagnostic (middle row) tubes with only 3 doses of complement;
some negative sera in such cases would be read as false positives.
In the tubes with 5 doses of complement (back row of test proper) laking would
occur where the sera were negative or weakly positive, because the extra 2 doses of
complement over that in the diagnostic tubes would be more than sufficient to cause
complete laking of all the red cells.
The foregoing explains why the test gave unreliable results on the occasion
mentioned when the complement titre was read from the front row of tubes alone.
The results obtained by using the modified titre 1:60 can now be similarly
examined.
Since the serum and the red cells in the back row tubes of the complement
titration (see table II) usually require about 2 doses of complement for complete
laking, the reading 1:30 could be considered as an indication that a nominal
2 M.H.D. complement had been used in this tube. It follows, therefore, that the
corresponding working titre should be 1:60. The third row tube with 1:20
dilution would thus contain 3 M.H.D., so that the reading 1:24 would correspond
with a nominal 2.5 doses at which strength it was considered that lysis would have
been complete. Thus a titre of 1:60 should ensure complete lysis with normal
sera in the diagnostic tubes of the test proper.
But since the true titre was read as 1:78, the dilution 1:60 actually contained
1.3 dose per volume instead of one dose. The 1:20 dilution similarly contained
3.9 doses per volume, and the 1:12 dilution 6.5 doses instead of the standard
3 and 5 doses respectively (table I).
By using the working titre 1:60 in the test proper, the back row (quantitative
diagnostic) tubes had 6.5 minus 3.9, or 2.6 doses more than the middle row (diagnostic)
tubes, instead of the usual amount of 2 doses. The middle row tubes and the
control tubes contained 3.9 doses instead of 3 doses. But since the complement