18
A tonicity of 1·4 due to NaCl necessitates the use of just twice as much of the
lytic serum to produce complete lysis as is required of the serum to which no NaCl has
been added. In table 2 it is seen that, whereas 0·45 cc. defibrinated blood secured
complete lysis, 1·05 cc. of citrated blood, more than twice as much, gave no lysis and
the tonicity was equivalent to only 1·2 per cent. NaCl.
In the other example given 0· 2 cc. defibrinated blood secured complete lysis,
while 1·25 cc. citrated blood, more than six times as much, gave incomplete lysis
with a tonicity equivalent to only 1·3 per cent. NaCl.
(c) On the effect of raising the tonicity of defibrinated blood to equal that of
the citrated blood.
The tonicities of rabbit citrated blood and defibrinated Mood having been deter
-
mined in terms of NaCl by a preliminary experiment, the tonicity of the defibrinated
blood was raised to that of the citrated by the addition of NaCl. Parallel series of
tubes were then put up with the two, using goat R.B.Cs. as an indicator. The final
tonicity of the two bloods was 1·63 per cent. NaCl. It was found that 0·3 cc. defibrinated
blood completely dissolved 0·05 cc. goat R.B.Cs. and that 1·95 cc. of the citrated blood
only partially dissolved a similar quantity.
The experiment was repeated using goat blood and rabbit R.B.Cs. The final
tonicity of the defibrinated and citrated bloods was 1·6 per cent. NaCl. Complete lysis
was obtained by 0·35 cc. defibrinated blood and no lysis by 1·95 cc. citrated blood.
(e) On the effect of lowering the tonicity of the citrated blood by dilution
An experiment showed that 0·65 cc. citrated rabbit blood of a tonicity of 1·08 per
cent. NaCl, and 1·95 cc. of a tonicity of 1·63 per cent. NaCl, gave an equal degree of
lysis with goat R.B.Cs., proving that the lytic power of citrated blood can be to some
extent, but only slightly, restored by lowering its tonicity by dilution.
Rabbit R.B.Cs. obtained from ( a) citrated, and (b ) defibrinated blood and washed
in the usual way were lysed by fresh sheep serum in exactly similar degrees, so citrate
has no permanent alterative influence on the R.B.Cs. themselves.
The inferences to be drawn from these experiments were either that the citrate
precipitates some substance the presence of which in solution is essential for lysis, or
that in the presence of citrate amboceptor and complement cannot combine with each
other, or that amboceptor is not bound to the red cells.
The next step was to ascertain whether the addition of citrate to blood after
its defibrination by whipping still inhibited its lytic power. Several experiments
united in showing that the addition of 1 per cent. citrate after defibrination causes con
-
siderable diminution of lysis but not to the same degree as when the citrate is added
to the whole blood. This holds good whether rabbit blood is used with sheep R.B.Cs.
or goat blood with rabbit R.B.Cs.
Attempts were made to remove the citrate from citrated blood by precipitation
with calcium chloride, but lysis instead of being restored was entirely abolished even
when the tonicity of the solution was so far diminished by dilution as to render
complete lysis possible. Other workers, however, have reported success in this
experiment.
Assuming for the sake of argument that the citrate precipitates something
essential to lysis from the blood no adequate explanation offers itself of the fact that
the citrate has greater inhibitory power when added to the whole blood than when
added to previously defibrinated blood. It can only be suggested that defibrinated
blood is richer in complement on account of the mechanical disintegration of a certain
proportion of leucocytes in the process of whipping, a factor of disturbance which is
absent from plasma obtained by citration.
The fluid obtained after citration was always opalescent even after prolonged
centrifugalisation whether the whole blood was used or whether the citrate was added
after defibrination, whereas the plasma obtained by defibrination was clear. This
opalescence may have been due to the precipitation of calcium citrate. It does not
appear, however, if sodium citrate is added to clear serum after clotting in any
proportion from 1 per cent. to 10 per cent.