20
7·1 per cent.
G.
1·5 cc. plasma plus 3·75 cc. of 7·5 per cent.
sodium citrate in 0·85 per cent. NaCl.
H.
1·5 cc. plasma plus 3·5 cc. of 3 per cent.
sodium chloride in 0·85 per cent. NaCl.
10·. 5 per cent.
I. 2 cc. plasma, plus 3 cc. of 10 per cent. sodium
citrate in 0·85 per cent. NaCI.
J. 2 cc. plasma plus 3 cc. of 4 per cent. sodium
chloride in 0·85 per cent. NaCl.
The final solutions A to J have an equal content of amboceptor. 0·5 cc. of sheep
erythrocytes were in each case sensitized in 5 cc. of mixture for 30 minutes at 37ºC,
then washed, so that no citrate appeared in the tubes put up.
Each tube contained 0·05 cc. erythrocytes and its total volume was made up to
2 cc. with 0·85 per cent. NaCl.
0·25 cc. fresh sheep serum sufficed in each series to produce complete lysis of the
sensitized erythrocytes, so that it is clear that sensitization of the sheep erythrocytes
resulted in all concentrations of sodium citrate up to 10·5 per cent.
The presence of sodium citrate in a sensitizing mixture is no bar to complete
sensitization.
(b) Complement—
As a hæmolytic system human erythrocytes, sensitized in heated sheep serum,
and fresh horse serum for complement were used. Horse serum by itself has little or
no lytic action on human erythrocytes, whereas it is able to some extent to act as
complement to sheep amboceptor.
0·5 cc. human erythrocytes A were sensitized in 5 cc. heated sheep serum for 30
minutes at 37ºC and washed as usual. Another quantity B was sensitized in citrated
sheep serum.
A and B were put - up with fresh horse serum, citrated and uncitrated, as comple-
ment. Lysis occurred in the tubes complemented with uncitrated horse serum whether
the erythrocytes bad been sensitized in heated sheep serum or in citrated sheep serum.
There was no lysis in any of the tubes complemented with citrated horse serum, though
the tonicity was in no case higher than the equivalent of 1·2 per cent. NaCl, nor in
the controls.
The citrate either as a chemical or by virtue of its added tonicity entirely
inhibited complement action.
This experiment was repeated and modified in many ways, invariably with a
similar result.
Summary.
(1) Raising the tonicity of a lytic serum to 1·2 per cent. NaCl by adding NaCl
reduces the degree of lysis which it is able to produce.
(2) If the quantity of lytic serum in the system be increased twice or thrice com-
plete lysis may occur even when the tonicity is equivalent to 1·6 per cent. NaCl, but
if the high tonicity is due to the presence of sodium citrate even six times the needful
amount of serum will not secure complete lysis.
(3) The relative tonioities of sodium chloride and sodium citrate are in the pro-
portion of 1 to 0·3 or 0·4.
(4) If this relative tonicity of sodium chloride to sodium citrate is correct it
appears as if the citrate has some effect on blood in the way of hindering lysis apart
from the tonicity of the mixture.
(5) The lytic power of citrated blood can be to some extent, but only slightly,
restored by dilution.
(6) The presence of sodium citrate in a mixture does not interfere with the com-
plete sensitization of erythrocytes.
(7) The action of complement on sensitized erythrocytes is inhibited by sodium
citrate. It is possible that, as in the case of the coagulation of blood, lysis of erythro-
cytes by amboceptor and complement is dependent on the presence in solution of
calcium salts.