ix
APPENDIX. I.
THE SERUM DIAGNOSIS OF SYPHILIS AS CONDUCTED IN 481 CASES DURING
THE YEARS 1911 AND 1912 INCLUDING A BRIEF COMPARATIVE STUDY
OF THE DIFFERENT MODIFICATIONS OF THE ORIGINAL WASSERMANN
TEST AND THE EFFECT OF SALVARSAN TREATMENT ON THE RE-
ACTION.
By Civil Assistant Surgeon T. Sitapati Iyer, L.M. & S.
On perusing an article by Captain Stevenson, I.M.S., in the report of the Bombay Bacteriolo
gical Laboratory for the year 1910, written on the Hecht-Fleming modification of the Wassermann
test, as adopted by him with some variations, I was struck with the simplicity of the method and
the reliability of the test claimed by the authors. I began testing a few bloods by this method but
before long found it was not giving satisfactory results in my hands. Thereupon, on the sug-
gestion of the Director, I took up the original Wassermann which was a little more complicated
but did give fairly satisfactory results. This method also had its own difficulties which I will
discuss later on but, on the whole, was of very satisfactory diagnostic value to the clinician.
Lastly I attempted Levaditi's modi fication and conducted a series of parallel experiments to
arrive at a comparison between the original method and it. I have also appended a table
(table VII) containing the results of the examination of different bloods by the different methods
with a brief history of the cases and the effect of Salvarsan on the reaction. Although it may
be redundant to discuss in detail the different methods fully described by their authors and
others in different works on bacteriology, still a brief description of the various methods and
the technique adopted in doing them may be essential in making this paper intelligible.
Two tests—(1) the Hæmolytic, and (2) the Bordet-Gengou fixation of complement, some-
times called complement deviation—need to be understood before the Wassermann or its modifica-
tions can be grasped, as they are preliminary processes in the technique leading to the serum tests
proper. The animals usually chosen for the hæmolytic test are the sheep, the rabbit and the guinea-
pig. Five cubic centimeters of blood are drawn from one of the jugular veins of the sheep by
means of a 10 c.c. sterile syringe containing about 1 c.c. of 10 per cent. sterile sodium citrate
solution, (Roux's pattern syringe, sterilised by oil or vaseline at 160°C and subsequently washed
with sterile saline solution, will do very well.) The usual antiseptic methods of cleaning the skin
over the vein of the sheep are of course adopted. The citrated blood is mixed with about 10 c.c.
of normal saline and centrifuged. The corpuscular deposit is again washed with 10 to 15 c.c. of
saline and again centrifuged. The deposit is then injected intraperitoneally into a rabbit. At
intervals of a week two similar intraperitoneally injections of washed corpuscles are given to the
same rabbit. A week after the third injection the rabbit is bled. About 30 to 40 c.c. of
blood can be easily taken from the rabbit's heart by means of a syringe without killing the
animal. The blood is allowed, to clot in a cool place and the serum separates out, and this
constitutes what is called rabbits hæmolytic immune serum. The centrifuge may be utilised
to hasten the process. This serum has a very high hæmolysing power towards the red cells of
the sheep. The serum is then filled into small glass capsules which are heated in a water-bath
at 56 to 57°C for half an hour on three successive days. I have found serum treated in this way
always sterile. This serum is devoid of complement which is an essential factor in hæmolysis.
Complement is a substance occurring in all normal freshly drawn blood, but it rapidly disappears
at room temperature and its presence cannot be counted on a few hours after drawing the blood.
It is therefore better to destroy the complement in all the other materials used, and to add a
standard quantity of serum, prepared from blood freshly drawn at the time the test is carried
out. Freshly prepared guinea-pigs serum is used for the purpose and 5 c.c. of blood may
readily be drawn from a guinea-pig's heart without injuring the animal. The results are thereby
rendered much more comparable as there is always approximately the same amount and kind.
of complement to be fixed. A 5 per cent. emulsion of well-washed sheep cells free from
t r a c e s o f s e r u m i s t h e n o b t a i n e d b y me a s u r i n g o u t 1 c . c . o f c o r p u s c u l a r d e p o s i t f o r e v e r y 1 9 c . c .
of saline. By a series of experiments the minimum amounts of rabbits hæmolytic immune
serum and guinea-pig complement containing serum, which are required to lyse a fixed amount
of the sheep's cell emulsion, should be first determined. This is effected by means of a gradu-
ated capillary pipette with one or two small bulbs in the centre worked by a rubber teat as is
used in Wrights opsonic work. In most cases it will work out to about two parts of 1 in 100
diluted rabbit's immune serum and 1 part of guinea-pig's serum for complement and ten parts
of 5 per cent. sheep red cell emulsion. Such a mixture is made up to 30 parts with saline and
put in a test tube 4″ by ½″ and incubated at 37° C for an hour. The three factors which consti-
tute a hæmolytic system are therefore (1) Hæmolytic immune body (derived from immunised
rabbit), (2) Complement (derived from a guinea-pig) and (3) Sheep's red cells. If any one of
these is absent or neutralised, (better still to use the bacteriological term fixed), lysis will not
3