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A control experiment with a nonsyphilitic blood may be necessary in case only one blood
is examined at a time. When several bloods are examined at the same time only one set of the
controls 6 and 7 for all may be used and there may not be the necessity of doing a control
with a nonsyphilitic serum, as any one of the bloods examined may give a nonsyphilitic reaction.
At the end of the second incubation the tubes are kept in ice over night to allow the corpuscles
not lysed to settle and then the result is read and recorded. There are many difficulties in carrying
out this method efficiently and with constant success:—(1) The chief trouble is with reference
to the manufacture of the antigen. The selection of a suitable liver involves vexatious delay
and dependence on a Maternity Hospital. Sometimes for several months no such liver is
procurable. (2) Granting that it is received in a fairly aseptic condition it is very difficult to
keep it so during the prolonged period of desiccation. (3) The saline extract of the dry powder
does not keep for a long time. It has to be kept in a cold store, or it has to be made frequently,
in which case maintaining the same strength of the extract is difficult. (4) A liver rich in
spirochætes gives different results than one less rich in the parasites. A constant strength of
the extract cannot therefore be guaranteed by using the same quantities of materials. Some
suggestions to minimise some of these defects in the above process are made below and it is
intended to try them during the current year. (1) If spirochætes can be consistently detected
in fresh syphilitic specimens by dark ground illumination, much delay, which is caused by the
elaborate method of staining for spirochætes, may be avoided. (2) Alcoholic extracts of the
fresh liver may be substituted for the saline extract of dry liver. This will certainly keep
better but whether it will react as well is not known.
Levaditi's modification.—In this method, the author has substituted an alcoholic extract of a
normal ox liver instead of the antigen (syphilitic liver extract) used in the original Wassermann.
The organ extract can be prepared by extracting finely minced liver with 4 volumes of alcohol
for 3 or 4 days and then filtering. All other reagents and the technique of the process remain
the same. This has many advantages over the original method. (1) All the reagents are pro-
curable at any time. (2) The alcoholic extract is a pure extractive of the principle without any
adventitious bacterial products. (3) The reaction is definite. Unlike what happens in the
original method, the fixation of complement is either entire or negative and results in giving
rise to complete lysis or no lysis. By direct observation the reaction can be readily seen. In
the original method, lysis takes place in such a varied degree that it has to be compared every
time with the controls as to the amount of deposit of unlysed red corpuscles or by the colour of
the partially lysed solution in the tubes. This method of observing the hæmolytic reaction is
not very satisfactory. (4) The advantages of this modification over the Hecht-Fleming, the
description of which will follow, consist in the separate addition of a standard amount of com-
plement and hæmolytic immune serum.
The Hecht-Fleming method is a much simplified process. In human blood there is both
complement and a substance which will lyse the corpuscles of the sheep or other animal. These
are taken advantage of and the complicated preparation of these substances is done away
with. The serum suspected for syphilis is not heated to 56°C. to remove the complement but
is added to different percentages of an organ extract, thereby supplying complement and
hæmolytic immune body to sheep cells, along with the syphilitic principle in positive cases;
so that three reagents are combined in one. Salt solution, patient's serum, alcoholic organ
extract and 5 per cent. emulsion sheep cells are all that is required for the test. Different
percentages of the extract (instead of the amount of the serum as in the original method)
e.g. , 10, 5, 2½ and 1¼ per cent. solutions in saline are taken for experiment to gauge the
degree of reaction. A series is put up with normal non-syphilitic serum as a control. For the
organ extract a rabbit's heart is taken. This is freed from blood clots by washing, minced
finely and put in alcohol (5 c.c. to every gramme). The whole is heated to 60°C. for one hour,
following which it is allowed to sediment for 24 hours. The clear supernatant fluid is stored
in glass capsules in ice. By a series of experiments with syphilitic and non-syphilitic sera that
strength of the extract is chosen which will completely prevent hæmolysis with the syphilitic
serum but which will have no effect on normal serum. This preliminary standardisation is
the most important part in the preparation of the extract. The following is a table showing the
technique of testing blood by the Hecht-Fleming method.