NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
17
known as the bacillus avisepticus. Dr. G. C. Chatterjee, Assistant Professor
of Pathology at the Medical College, kindly treated many of my fowls with a
vaccine of killed cultures of the microbe, and in some cases certainly this did
good; but there is no doubt in my mind that the plan of campaign against
this microbe should be based on rigid quarantine of all fowls bought in the open
market in Calcutta, as recommended by Colonel Raymond, who saw and
examined many of the fowls. Those fowls that have successfully passed the
time-test, isolated from each other in cages, may then be taken to the laboratory
and kept there while they undergo treatment with foreign serum.
It may be of interest to record my experience of treating goats with a view
to obtain from them precipitin-antisera.
A black goat weighing 14 kilos. received the following doses of hydrocele
fluid at three-day intervals—27.5 ; 30 ; and 35 c.c. ; six days after the last dose
of hydrocele fluid it was given 27.5 c.c. of human serum, then, after six days,
29.5 c.c. and after three days 15 c.c. All injections were made into the jugular
vein easily and without untoward results. The serum, on being titrated on the
tenth day after the last injection, was found to give a faint reaction with a dilution
of the antigen, weaker than a thousand-fold. After a month's rest the goat was
again treated, receiving at three-and-six-day intervals—30 ; 35 ; 33 and 30 c.c.
of human serum intravenously. Shortly after the last dose it died of serum
disease. A white goat weighing 14 kilos. received intravenously the following
quantities of human serum at three-day intervals—30 ; 30; 34 ; 30 c.c. Just
before its serum fell to be tested it contracted pneumonia and died.
A white goat weighing 13 kilos. received intravenously at three-day intervals
26, 26, and 30 c.c. of human serum. On the seventh day after the last dose its
serum was tested and found to be absolutely inert. Eleven days after this it
received 35 c.c. and six days thereafter 40 c.c. of human serum. Seven days
after the last dose its serum was found to be still quite inert when brought in
contact with a 1-100 dilution of human serum.
The following will give an idea of the work done with geese :—
151 Horse serum intraperitoneal
and intravenous.
65 c.c.
56 days
useless.
(two courses of treatment)
156 Sheep serum intraperitoneal
93
40
„
162 Human serum intravenous
(two courses)
52
„
32
163
„ „ „
died after 4th injection
164
„ „ „
30.5 c.c.
49 days
„
165
„ „ „
31.5
47 „
„
166
„ „ „
36.5
47 „
„
(One dose intraperitoneal.)
18
It did not appear to me to be at the time expedient to go on with my attempts
to immunise geese ; but whether these are or are not refractory is a question to
which the above data cannot be taken to give a definite answer. All that we
can at present say is that here geese are not easily immunisable.
Hydrocele fluid was first used by Schütze as antigen ; here it is very easily
obtainable, and therefore it was of importance to test its capabilities as a source of
antiserum. Unfortunately I have nothing good to say of it : its albumin-content is
far too low for one to hope to be able to immunise animals with it unless one were
to use relatively enormous doses, seeing that here the dose of serum as antigen
must be somewhat increased.
The intraperitoneal injections were given as described by me elsewhere, as
also the intravenous injections in the case of rabbits. For fowls I have found it
of service to give four injections, the dose being adapted to the size of the fowl :
first the right wing-vein, then the left, then the right cervical vein, and finally the
left are the sites of injection. As all who have worked much with fowls are
aware, it is not always easy to give an intravenous injection into the wing-vein,
and there is much more chance of causing a hæmatoma when doing this than
when giving, say, an intravenous injection in a rabbit even if the ear-vein be not
chosen as the site. Geese of course are much easier to treat intravenously, but
in them too the hæmatoma is almost the rule. I have not found that it entails a
bad result, however, in the case of fowls treated with serum.
Ever since Uhlenhuth pointed out the importance of starving one's animals
for a few hours before taking their blood for testing its titre, all observers have
followed his lead. The beautifully clear serum that one obtains from the blood,
allowed to clot in the cold—as described elsewhere—one can then pipette off
into small sterile tubes, which are then sealed in the flame, and kept frozen till
they are required.
The efficacy of the antisera obtained by me may be judged on perusal of the
following tabular statement, which sets forth the results obtained by me when I
examined blood-stains of whose origin at the time of examination I was ignorant.
As in actual practice, the question that I desired to solve in each case was this :
Is this stain due to human blood ? If not, to what mammalian blood is it due,
assuming that the appropriate antiserum is at hand?—
No.
Stains sent by
Opinion expressed by
me after examination
of the stain.
Information subsequently
furnished by sender.
1. Dr. C. L. Bose, Rai Bahadur,
Assistant Chemical Exami-
ner, Calcutta.
Not human*
. Stain on blade of dao, on which
human hair was found.
2.
Ditto .
.
. Human
.
. Stain on clothes of a man accused
of murder.
9
Stains sent by
Opinion expressed by
me after examination
of the stain.
Information subsequently
furnished by sender.
3. Dr. C. L. Bose, Rai Bahadur,
Assistant Chemical Exami-
ner, Calcutta.
Human
. On clothes of murdered woman.
4.
Ditto .
. .
,, . .
On sari of murdered woman.
5.
Ditto .
.
. Not human*
. Another stain in case of 4.
6.
Ditto
. . .
,,
*
. Stain on quilt in case of 4 and 5.
7.
Ditto .
.
. Human
. .
Stain on bedding on which corpse
was found lying.
8.
Ditto .
.
.
,,
. .
Stain on pillow-case, an exhibit in
a murder trial.
9.
Ditto .
.
.
,,
.
. Stain on a sheet in this case.
10.
Ditto .
.
.
,,
.
. Stain on a man's dhoti in this
case.
11.
Ditto .
.
.
,,
. .
Stain on the woman's clothes in
this case.
12.
Ditto .
.
. Not human
. Stain made by cow's blood, 27
months old.
13.
Ditto .
.
. Human
.
. Human blood-stain 14 months old.
14.
Ditto .
.
. Not human
. Stain made by blood of pig from.
Orissa.
15.
Ditto .
.
.
,,
.
. Stain caused by blood of pig from
Bengal.
16.
Ditto .
.
.
,,
.
.
Stain of Calcutta pig's blood.
17.
Ditto
.
.
.
,,
. Buffalo's blood-stain.
18.
Ditto
.
.
.
,,
. .
Sheep's blood-stain.
19.
Ditto
.
.
.
,,
. .
Pillow-case stained with birds'
blood.
20.
Ditto
.
.
. Human
.
Stained cloth from a case of rape.
21.
Ditto
.
.
. Not human
. Cow's blood-stain.
22.
Ditto
.
.
. Not human, rumi-
nant—probably
buffalo.
Buffalo's blood-stain.
23.
Ditto
.
.
. Not human, may
be sheep or
goat or buffalo.
Sheep's blood-stain.
24.
Ditto
.
. Not human
. Pig's blood-stain.
25.
Ditto
.
.
. Human
.
. Human blood-stain.
26.
Ditto
.
. .
,, . .
Blood-stained garment from a
rape case.
27.
Ditto
.
.
. Human (examined
first microscopi-
cally).
Blood mixed with feathers.
28.
Ditto
.
. .
Human
. .
Stain on drawers from a rape case.
29.
Ditto
.
.
.
,,
.
. Stain on trousers from a rape case.
30.
Ditto
.
. .
,,
. .
Stain on skirt from a rape case.
31.
Ditto
.
. .
,,
. .
Stain on chemise from a rape case.
32.
Ditto
. . .
Not human; cow
or buffalo, pro-
bably cow.
Stain alleged to be of cow's blood.
* The real origin of this stain was not worked out, antisera for domestic animals' bloods not being at
the time available.
20
No.
Stains sent by
Opinion expressed by
me after examination
of the stain.
Information subsequently
furnished by sender.
33. Dr. C. L. Bose, Rai Bahadur,
Assistant Chemical Exami-
ner, Calcutta.
Not human ; cow
or buffalo, pro-
bably cow.
Another stain in this case.
34. Lieut.-Colonel Vaughan,
I.M.S., Calcutta.
Human ; not feline
nor ovine.
Stain on paper that had been rub-
bed on cut muscle of dissected
corpse.
35.
Ditto
. .
. Human
.
. Cloth soaked in blood of putre-
fying corpse.
36. Dr. E. H. Hankin, Chemical
Examiner, Agra.
Not human ; rumi-
nant.
Stain from case in which a man
was accused of having stolen
and killed a goat.
37. Dr. C. L. Bose .
.
. Human
.
. Stain on coat of accused man.
38.
Ditto
.
.
. „
.
. Stain from bludgeon used by this
man.
39.
D i t t o
.
.
. Not human
. Stains stated by police to be pro-
bably of cow's blood.
40. Lieut.-Colonel Nott, I.M.S.,
Howrah.
Not human nor
ovine nor equine.
Stain of dog's blood.
41. Dr. C. L. Bose .
.
. Human
.
. Stain from a cot on which an in-
jured man had lain.
42. Captain F. P. Connor, I.M.S.,
Calcutta.
„
.
. Stain from operation case.
43.
Ditto
.
.
. Not human
. Dog's blood-stain.
44.
Ditto
.
.
.
„
.
. Stain of bird's blood.
45.
Ditto
.
.
. Human
.
. Human blood-stain.
46. Captain H. B. Steen, I.M.S.,
Calcutta.
Not human
. Stain made by raw beef-juice.
47.
Ditto
.
.
. Human
.
. Human blood-stain.
48.
Ditto
.
.
. Not human
. Bird's blood-stain.
49.
Ditto
.
.
. „
.
.
„
50.
Ditto
.
.
.
„
.
. Sheep's blood-stain.
51.
Ditto
.
.
. Human
.
. Human blood-stain.
The accuracy of the diagnosis made by me in each case shows that the
antisera that I employed were of excellent quality, and that the technique of the
examinations left nothing to be desired.
In most of the above-detailed cases, which were taken from actual medico-
forensic practice, the Court before which the case was being tried would have been
aided in coming to a decision by the information afforded by the precipitin test.
In no other way could it be put beyond a doubt that the stains in question had, or
had not, been caused by human blood.
Here, where advocacy knows no limits, it might be alleged that in a labora-
tory in which blood-stains of all kinds of animals, including man, are examined,
the air might become so vitiated by minute particles of stain that the results
obtained by means of the test could not be relied upon.
21
Nuttall long ago pointed out that when blood-stains that have not become
completely dried are in close contact one with the other, their extracts may give
fallacious reactions, by reason of the contamination induced by close contact.
But stains that come up for examination are dry, and such, if left in close contact
with other stains for months, do not become in any way modified by contact.
It is conceivable —and it is a fact—that human sweat, when it comes in con-
tact with, say, a blood-stain due to horse's blood, might modify the extract of that
stain so that it would give the human reaction: but, and it is a big but—when the
stain extract is diluted up to the proper limits no such interference with the nor-
mal reaction of its constituents can occur: countless experiments by many
observers have set this question at rest for good. If the extract were only a 1-20
dilution then errors might creep in; it must be fifty times as weak as this before
it is tested in forensic practice.
IV.—The Complement=deviation Test.
Neisser and Sachs, who introduced this test, considered1 t h a t i t m a y
be profitably used as a confirmation of the results obtained by means of the
precipitin test. Uhlenhuth, however, has all along been of opinion 2 that if
the results yielded by the precipitin test are not confirmed by those obtained
when the complement-deviation test is carried out, they should nevertheless
be accepted. He found that he obtained deviation of the complement, and
consequently a " positive reaction " when he examined by means of antihuman
serum the following articles, taken from the collection in his laboratory:—jute
sacking; military foot-bandages, new and old; hemp-dust; earth; gravel; bark;
hay; straw; bread; leather; hair of various kinds; woollen stockings; linen;
various kinds of trouserings; felt hats; coats made of various materials; urine;
pepton; pepton-bouillon; various undiluted sera. Neisser and Sachs replied
that boiling of the extract would be found to simplify the test: if in one set
of tubes unboiled, and in another boiled extract were tested, then if complement-
deviation occurred in both it must be due not to the presence of the antigen,
but to some vitiating influence being present. 3 F u r t h e r t h e y i n s i s t e d o n
the extreme delicacy of the reaction being utilised to get rid of disturbing
factors: if the unboiled extract be very highly diluted only the specific antigen-
reaction will be observed. So far as I can gather Uhlenhuth is not enthusiastic
about the merits of the test, but admits that it is of some use.
1 NEISSER and SACHS:—Ein Verfahren zum forensischen Nachweis der Herkunft des Blutes Berl.
klin. Wochenschr. 1205, 1388, 1906, 67.
2 UHLENHUTH:—Komplementablenkung and Blut-Eiweissdifferenzierung. Deutsche med. Wochen
-schr. 1906, 1244.
3 NEISSER and SACHS :—Bemerkungen zu der Arbeit von Professor Uhlenhuth über Komple-
mentablenkung and Blut-Eiweissdifferenzierung. Ibid. 1906, 1580.
22
The very delicacy of the test, however, is held to be against it by such
competent authorities as Muir and Martin, 1 and Friedberger.2 Muir and
Martin state that, in their opinion, it is only when one has at one's disposal
antisera that are capable of inhibiting hæmolysis when such a small quantity
as the one hundred-thousandth of a cubic centimetre of the antigen is present,
that one should use the test in medico-forensic practice. Antisera of this very
high potency are obtainable with difficulty in Europe. It remained to be seen
whether they could be obtained here.
The test is based on the following facts:—If an animal be treated by means
of injections of the blood, or—better--of the washed red blood-corpuscles of an
animal of another species, it will, if it be immunisable, have its blood so modified
that, when its serum is brought in contact with the red blood-cells of the species
in question, these become dissolved. If this modified serum, however, be heated
to 56° C. for half an hour it loses its power of dissolving these cells, to regain it if
there be added to it some fresh serum—either that of the animal itself or that of
certain other species. The heat had destroyed one of the constituents of the modi-
fied serum— the complement, but left unaffected another constituent, the hæmolytic
antibodies that had been called into being by the injection of the foreign blood-
cells. According as we follow the reasoning of the French or German schools of
serology, whose protagonists are Ehrlich and Bordet respectively, we may
regard the immune bodies as binders of the complement to the cells, or as
rendering the cells liable to its action. In any case, for dissolution of the cells
to take place we require complement and immune bodies to be present. We
can by experiment fix the minimal dose of the treated animal's serum that is
required to dissolve a given quantity of the red corpuscles in a given time: say
that 0.001 c.c. of the heated serum of a rabbit, that has been treated with sheep's
cells, will just dissolve 1 c.c. of a 5 per cent. suspension of these cells in saline
solution within two hours, when to it has been added 0.1 c.c. of fresh guinea-
pig's serum. Now, if we put into a test-tube the required quantity of guinea-
pig's serum with some precipitin antiserum, and the sheep's cells-suspension, and
the heated hæmolytic antiserum in the required quantities, we shall find that
hæmolysis occurs—the cells are dissolved. If in another tube, in addition to
the above-mentioned materials, we have put some serum of an animal of a
species other than that for which the precipitin-antiserum is specific in its re-
action, hæmolysis will also occur. But in a third tube, into which there has
1
MUIR and MARTIN :—On the deviation of complement by a serum and its antiserum, and its
relation to the precipitin test. fourn. of Hygiene, 1906, 265.
2 FRIEDBERGER :—Zur forensischen Eiweissdifferenzierung auf Grund der hämolytischen Methode
mittelst Komplementablenkung nebst Bernerkungen der Bedeutung des Präzipitats für dieses Phänomen.
Deutsche med. Wochenschr. 1906, 578.
23
been put the serum of the species for which the precipitin-antiserum is specific
(the antigen of this antiserum, in other words) there will be no hæmolysis.
The complement (fresh guinea-pig's serum) has in this tube been taken up
by the mixture of the precipitin-antiserum and its antigen, and therefore has
not been able to act with the immune bodies of the heated hæmolytic anti-
serum, which without it are inert.
The reader who desires further—and perhaps clearer—information on this
point is referred to my monograph, in which I have endeavoured to set forth
the various processes involved in as simple language as the highly technical
nature of the subject allows one to use.
The great interest of the phenomena described lies in the fact that it is
the presence of the antigen of the precipitin-antiserum that causes the comple-
ment to be deviated. If we are using an antihuman serum, then we know that
its antigen must be present, if hæmolysis does not occur when we have put into
the tube twice the minimal dose of the heated hæmolytic serum, and have very
highly diluted the extract of the stain that we are testing. Using boiled and
unboiled extract in parallel series of tubes we may safely conclude that it is no
vitiating influence present in the material stained that has led us astray. If the
precipitin test has already indicated the presence of human material in the stain
extract we are now quite sure that our complement-deviation test has confirmed
this observation.
But in the tropics the animals that are immunisable require large doses of
antigen and prolonged treatment, and in the end do not yield antisera of very
high potency. I have utterly failed to obtain the high-potency antisera that
are, in Muir and Martin's opinion, needed to ensure accuracy. From the work
that I had the privilege of doing in Frankfort on Maine I am sure that the high
dilution of the stain-extract is a conditio sine qua non of accurate work with
this test, and this entails the use of a very powerful antiserum. Here the most
powerful antiserum that I obtained was able to cause deviation of complement
when a ten-thousandth part of a cubic centimetre of its antigen was present,
which is only a tenth of the strength demanded by Muir and Martin. And be
it noted that this antiserum was the only one that I obtained of this strength
—the others varied from 1-5000 upwards.
Here too we have another obstacle in the path that leads to success. For
the accurate performance of the test the complement must be standardised:
it must be known what is the smallest dose of the fresh guinea-pig's serum that
is required to render active the ascertained minimal dose of the heated hæmoly-
tic antiserum. This standardisation of the complement entails its exposure to
light and heat, do what one will to avoid these causes of deterioration. Conse-
quently it is hard to tell exactly how much the complement has lost power
E 2
24
between the time of its standardisation and the moment of its being used for the
test. Of course, as it is so delicate it would be so used at once when its power
had been fixed; but there is always a deterioration to be taken into account.
Naturally I endeavoured to find out whether the process of standardisation
could be omitted, and the full power of the complement thus utilised for the test
itself. I had no great hopes of success, since in Europe the sera of different
guinea-pigs have been found to vary so much, but the point was well worthy of
investigation here. The results obtained by me after doing numerous experi-
ments show that, for the attainment of that accuracy which forensic work
demands, one must not neglect to standardise the guinea-pig's serum just before
using it for the test. The usual dose is 0.1 c. c. and in one case I found that this
quantity was of itself able to cause dissolution of the standard quantity of sheep's
red cells, while in several cases as much as 0.3 c. c. was required to reactivate
the ascertained minimal dose of the hæmolytic serum: in other words, without ex-
periment one cannot say what is the power of any given guinea-pig's serum.
With the comparatively weak antisera which I used the results obtained by
me when doing the complement-deviation test were interesting, but by no means
conclusive from the forensic point of view, and I much doubt the likelihood of this
test ever becoming a matter of regular routine in India. For it to become so we
should have to be able to obtain in large quantity very highly potent antisera,
and this will, in my opinion, be always the crux, even if the problem of satisfac-
tory conservation of the complement be solved.
For the determination of the fact that a patient is syphilitic the very
similar test proposed by Wassermann, Bruck and Neisser is of the greatest value,
and can, as I have found, be carried out here as well as in Europe. Clinical tests
may lead one into error, but the error is easily rectified; in forensic work error
must be absolutely excluded.
Even at its best the Neisser-Sachs test is, as its distinguished originators
claimed, only confirmatory. I agree with Uhlenhuth in holding that it cannot be
held to be corrective of the results yielded by the precipitin test. And that these
are accurate here in India the reader will have already gathered.
V.—The Anaphylaxis Test.
The reports made by Rosenau and Anderson1 led me to believe that
the peculiar phenomena of anaphylaxis might well be turned to account in
medico-legal practice, but before I was able to do any work on the subject I
learned that Thomsen 2 had already carried out a number of experiments with
1 ROSENAU and ANDERSON: — Studies upon hypersusceptibility and immunity. Bull. No. 36 Hyg
Lab. U. S. Pub. H. and Mar. Hosp. Serv. Further studies upon Anaphylaxis. Ibid. Bull. No. 45.
2 THOMSEN :—loc. cit. page 4 supra.