NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
7
Here blood-stains are of necessity exposed to a higher temperature and brighter
sunlight than in Europe, but even stains that were exposed for three weeks to the
summer air and light, when treated with saline solution, soon yielded satisfactory
extracts, and it is not likely that the stains in a criminal case here would be sub-
jected to the influence of the weather for so long a period as three weeks. The
reader does not need to be told that when blood-stains have been thoroughly
washed out of the stained material, no satisfactory extract can then be obtained
by treating the material.
Later I shall give fuller information as to the process and results . of immuni-
sation ; here I would mention the animals that I worked with. Rabbits, which are
the animals of choice in European and American laboratories, I worked with
although they are neither cheap nor easy to get in the Calcutta market ; fowls are
cheap and plentiful, and, as my previous work had led me to believe that they
are good sources of antisera, I used a number of fowls in my experiments ; geese
I tried, encouraged by the results of my work with fowls, but, probably because I
did not push the doses of antigen —the substance injected, for which it is desired
to obtain an antiserum—I did not obtain a satisfactory result from those that
were treated ; goats have been found to be unsatisfactory in Europe, but never-
theless It r i e d t h e m h e r e , a s , i f b y a n y c h a n c e I m a n a g e d t o i m m u n i s e a g o a t
to a high degree, I would have a large stock of antiserum at my disposal ; monkeys
I tried, although I had previously failed to attain anything like the success reported
b y Uh l e n h u t h . 1 Wi t h f o wl s a n d r a b b i t s I o b t a i n e d s a t i s f a c t o r y r e s u l t s ; wi t h
goats and monkeys I had only failures.
The antihuman sera that I obtained reacted very well, but, as is the case in
Europe, the antiruminant sera were only to a certain extent specific : with goat's,
sheep's, buffalo's and ox' blood they gave reactions which differed only in degree.
For more exact differentiation many expedients have been suggested as of service
when an antiruminant serum has reacted with the extract of the suspected blood-
st ai n. Hambur ger 2 has r ecommended t hat t he st ai n- ext r act be di vi ded i nt o
several portions, each of which is then treated with a different antiruminant
serum : that antiserum with which the most rapid and most marked reaction is
obtained gives us the clue. Thus, if we have a stain, which on being extracted
gives a reaction with an antiruminant serum, we treat separate portions of the ex-
tract with anti-ovine, -bovine, -hircine, etc., serum, and observe in which tube the
reaction is most marked and rapid. That Hamburger's method of differentiation
is of the highest value, I am convinced : the results of the examination of stains
1 UHLENHUTH :—Ein Vcrfahren zur biologischen Unterscheidung von Blut verwandter Tiere.
Deutsche med. Wochenschr. 1905, 1673.
2 HAMBURGER :—Gerechtlijk onderzoek van bloed en andere lichaamsvochten. Tijdschr voor straf-
recht, 1904, xvii. 82.
C 2
9
added to the contents of the test tube some undilute antigen. If then the
precipitate cleared up this would show that it was really a dilution of the antigen
with which we had to deal in the first instance.
I have carried out many experiments on this point, and am of opinion that
Dehne's suggestion is of no value for Indian medico-legal work. With a dilution
of goat's serum a reaction was obtained with anti-ovine serum AO, and also with
anti-hircine serum AH: Into one tube were put (1) dilute goat's serum, (2) AO
and, after the reaction had occurred, (3) some sheep's serum; into another tube
were put (1) dilute goat's serum, (2) AH, and, after the reaction had occurred (3
) some goat's serum. Next morning the two tubes, that had been kept in the ice-
chest overnight ; presented identical appearances.
Again, an antibubaline serum AB reacted well with a high dilution of its
antigen, and less markedly with a similar dilution of ox' serum. Into one tube were
put (1) AB, (2) dilute buffalo's serum, and, after the reaction had occurred,(3) some
buffalo's serum; into another tube were put (1) AB, (2)dilute ox' serum and after the
reaction, (3) some ox' serum. The tubes were left in the ice-chest overnight
and next morning were found to present identical appearances. No differentia-
tion of kindred sera can be made on the results of these experiments.
Uhlenhuth has laid it down that ape's blood may be mistaken for human
blood, so it was of great importance to investigate this point, although, so far as I
am aware, it has not yet been alleged in an Indian murder trial that the blood-
stains in question had been caused by blood that had come from an ape, and not
from a man, as alleged by the prosecution. Two reasons for this occur to me.
One is that the Hindus revere the lungoor (semnopithecus entellus). The other and
more generally operative reason is that, even in India, the ape is not a domestic
animal, and therefore is not at hand when most needed—by the defence: for Cicero's
advice causam mendaciunculis adspergere is scarcely needed here in the East.
Through the kindness of the Superintendent of the Calcutta Zoological
Garden, and the Principal Medical Officer of the Royal Colonial Army of the
Netherlands-Indies, I have been able to examine blood-stains due to the blood of
the following varieties of the apes of the Old World:—
O r a n g
u t a n
— S i m i a s a t y r u s ,
Hooluck—Hylobates niger,
Siamang— Hylobates syndactylus,
Lungoor—Semnopithecus entellus,
Bonnet monkey —Semnopithecus mitratus,
Simpai—Semnopithecus melalophus.
Macacque —Macacus cynomolgus.
Pig-tailed monkey—Macacus nemestrinus.
Slender loris—Nycticebus tardigradus.
Rhesus monkey—Macacus rhesus.
10
The stains were extracted in the usual way, and the extracts were diluted
until they corresponded to 1-1000 dilutions of blood-sera. They were then all
tested in a row, with many tubes of 1-1000 dilutions of human sera and stain-
extract as controls. The rapidity and degree of reaction obtained in each tube
were noted, when to its contents was added some antihuman serum of known
specificity and potency. It was found that even in the case of the orang's blood-
stain the reaction obtained was not " human", as it was not visible till after the
expiry of twenty minutes; the human blood-stain extracts and dilute sera in each
case reacted well before five minutes had elapsed. These experiments were
repeated over and over again, the results being shown to those who did me the
honour of visiting the laboratory, and in no case was a " human" reaction
obtained with the extract of a simian blood-stain.
Uhlenhuth has had considerable success in the immunisation of monkeys
with human blood, and in thus obtaining antihuman simian sera which, he says,
react with human, but not with simian blood-dilutions, being thus of considerable
value for differentiation1. I have tried to immunise several monkeys, but with
no success, which is somewhat strange, seeing that India is the home of the
Rhesus, and that there was no fault in my technique, as I learned from correspon-
dence with the distinguished originator of " cross-immunisation ". The tabular
statements given hereunder will show what was done with monkeys. I am in a
measure consoled for this want of success by the results of the work, described
above, on the differentiation of simian from human blood.
There remained the interesting question as to whether, by means of the
precipitin test, one could differentiate the blood of one race of mankind from that
of another. By the kindness of the undermentioned gentlemen I was able to
experiment with the blood of the races mentioned:—
Sent by Prof. Surveyor of the Grant Medical College, Bombay: —Parsis,
Gujaratis, Iranis, Goanese, Marathas of low caste.
Sent by Dr. Leask, Singapore:—Chinese, Japanese, Malays.
Sent by Dr. Denier of the Pasteur Institute of Indo-China: —Annamites.
Received from the Eden Hospital, Calcutta, by the courtesy of Lieut.-Colonel
C. R. M. Green, I.M.S.—Europeans, Eurasians, Bengalis, Jews.
In all cases the serum of the race represented was diluted 1,000-fold
and treated with antihuman sera obtained by injecting into fowls the sera of the
various races mentioned as furnished by the Eden Hospital.
The results obtained were in all cases so similar that no difference of any
medico-legal importance could be said to exist.
1 UHLENHUTH: —Ein Verfahren zur biologischen Unterscheidung von Blut verwandter Tiere.
Deutsche med. Wochenschr. 1905, 1673.
Where no racial difference was detected by the precipitin test, it would have
been futile to seek for individual or sexual differences.
In order to test the medico-legal value of precipitating antisera in Indian
practice it was necessary to have at my command antisera for the bloods of the
f ol l owi ng ani mal s: — man, hor se, dog, cat , sheep, goat , ox, buf f al o and pi g : t he
camel might be classed as a domestic animal of Asia, but its red blood-corpus-
cles differ from those of the other mammals by their shape, and therefore it was
not necessary to have an antiserum for its blood.
From the Eden Hospital I got all the human placental blood that I needed,
and from the Bengal Veterinary College, through the kind offices of Colonel F.
Raymond, A.V.S., I got most of the mammalian bloods that I required. Although
the air of Calcutta, being warm and very moist, is highly laden with bacteria, and
has been condemned by Cunningham and Rogers a great hindrance to bacterio-
logical work, yet the nursing staff of the Eden Hospital, to whom in the nature of
things the collection of placental blood had to be entrusted, were able by unremit-
ting care to send me blood of which only a few specimens had undergone bac
-terial contamination.
From the blood-clot the serum was collected in the manner described by me
elsewhere, and kept in the ice-chest. The ordinary type of American refrigerator
will, I found, keep up a temperature of 3.5-4.5ºC. (38.3-40.1º F.) in the lower of
its compartments, if the shelf between this and the upper compartment be perforated
to permit the cooled air to descend. For the conservation of the antisera in full
potency I tried various devices. The " dry method ", by which the serum is dried
over sulphuric acid or in the thermostat, and then sealed in glass tubes, which
may contain an atmosphere of hydrogen, is a method which has been found to
work well in the case of antitoxic sera. On being re-dissolved the dried serum
yields an opalescent solution, and for this reason is of no use for forensic work with
precipitin antisera. The " wet method," whereby an antiseptic is added to the
antiserum which is then sealed in glass tubes and kept in the cold and dark, was
at one time highly praised by Uhlenhuth; but I agree with Nuttall in thinking that
the antiseptic can well be left out, if sufficient care is taken to keep the anti-
serum frozen in the dark until it is needed. This is the method that Ehrlich pro-
posed years ago and no efficient substitute has been devised since then. Undoubt-
edly for Indian forensic work it is the method καт' έξοХήν
Exposure to light and
warmth very soon renders antisera of high potency quite inert : exposure to warmth
alone, or light alone, acts in a similar way but with less rapidity. Of this I had
proof in several cases in which I divided an antiserum into three portions, after
titrating it with known dilutions of its antigen in order to fix its potency. One
portion was kept frozen in the dark, another was kept in a drawer in the laboratory,
and a third was kept hanging up in the laboratory exposed to light as well as
12
warmth. The frozen portion of the antiserum would be found to be as good as
ever after many weeks—if time had permitted we might have kept it so for years ;
the portion which was exposed to light and warmth would be useless in two or
three days, and that protected from light useless in three or four days, whatever
high potency the serum might have originally possessed. When I say useless I
do not mean only useless from the forensic standpoint, but quite inert.
The thawing of the frozen antisera is apt, if it be carried out hurriedly, to
give rise to occasion for grave error : a rapidly-thawed antiserum will look flaky
and will cause a precipitate to appear when it is brought in contact with no matter
what antigen-dilution. Anti-equine antiserum will under these conditions react
with a dilute extract of a human blood-stain. The remedy for this is simple—
never to do things in a hurry, and to standardise the antiserum each time it is used
for a test, as to its potency and specificity. A slowly-thawed antiserum is clear
and sure in its reactions. I am not aware that much stress has been laid on this
point by other observers, but I am sure that workers in the tropics require this
word of caution.
III.—The Precipitin Test — continued.
Uhlenhuth reported 1 that from two Rhesus apes he had obtained anti-
human sera of 1-1600 titre, and from three Cercopithecus apes sera of 1-2000
titre. In his latest publication 2he writes on this point, " Es muss allerdings.
hervorgehoben werden, dass diese Antisera wegen der Artverwandtschaft nicht
sehr hochwertig waren "; but no one else has obtained antihuman simian sera of
anything like this strength, so far as I am aware.
That his luck was great appears from the fact that Professor Sachs of
Frankfort on Maine, whose competence as a serologist is well known, has often
tried to immunise monkeys with human antigen and as often failed to do so. 3
Working in the native country of the Macacus rhesus, I have had no success, as
the following tabular statement shows:—
No.
Total dose
c.c.
Injected.
Treatment
lasted days.
Potency of anti-serum obtained.
1
42.5
Subcutaneously . .
28
1-100.
2
. . .
......
...
Died of pneumonia after 8th
injection.
3
44
Intraperitoneally
24
......
1 UHLENHUTH :—Ein Verfahren zur biologischen Unterscheidung von Blut verwandter Tiere. Deuts-
che med., Wochenschr. 1905. 1673.
2 UHLENHUTH and WEIDANZ:—Praktische Anleitung zur Ausfuhrung des biologischen Eiweiss-
differenzierungsverfahrens u.s.w. Jena. 1909.
3 Private communication.
13
No.
Total dose
c.c.
Injected.
Treatment
lasted days.
Potency of anti-serum obtained.
4
52 Intraperitoneally
.
32
1-400.
5
104
„
.
32
1-50 about.
6
. . .
„
.
. . .
Died after third injection.
7
. . .
„
.
. . .
Died of serum disease after 7th
injection.
8
1 1 3
„
.
45
. . . . . .
9
52
„
.
33
......
10
. . .
„
.
. . .
Died after second injection.
1 1
164
„
.
45
......
12
66.5 Subcutaneously .
.
46
......
13
79.5 Intraperitoneall
.
54
......
14
60
„
.
47
......
15
7 1
„ (one
dose intraven).
37
......
16
168
„
.
35
. . . . . .
17
190
„
.
43
......
18
141
„
.
32
. . . . . .
It might be argued that some of these apes received far too high doses of
the antigen for one to hope for any result from the treatment : but here large
doses and prolonged treatment are necessary, and the largest doses were given to
big old apes. Frequent titration of their sera showed that the majority had not
manufactured any antibodies ; but the question of the immunisability of monkeys
with the human sera obtainable here is scarcely definitely answered by these
results, though the prospects of general success do not appear to be bright.
That the work of the medico-legist here renders it absolutely necessary to
have at one's command a sufficient supply of antihuman simian serum, is not
apparent in the light of the results obtained by me when testing simian and human
blood-stains. Uhlenhuth is of opinion that one should have antihuman simian
sera ready for use in cases in which the possibility of the presence of simian blood
cannot be excluded. With diffidence I advance the opinion that such antisera
D
are not necessary, and that without them one can do all that is needed to further
the ends of Justice.
Bruck's findings as to the differentiation of the blood of one race from that of
another do not agree with mine 1 : h e u s e d t h e c o m p l e m e n t - d e v i a t i o n t e s t a s
well. However, as my findings agree with those of such able observers as
Linossier and Lemoine 2 at Home, and Marshall and Teague 3 in the Far East, I
think that the question of racial differences may be considered to be answered in
the negative for good.
I have not found that the tubes which were introduced for precipitin work by
Uhlenhuth have any advantages over tapering tubes which are used in Frankfort
on Maine. These have the merit of being easy to make from a length of glass-
tubing, though sometimes one comes across a bad glass here, which frosts as
soon as it is heated and is therefore useless for the work. Tube-stands of various
shapes have been devised by ingenious workers, but I have found that the ordinary
and time-honoured pattern of test-tube stand is all that is required.
The capillary method, devised by Hauser, 4 of carrying out the test I
have used several times, and found it easy enough to carry out. Carnwath's
modification of it 5 I have not used, but am of opinion that it has advantages
where a very small stain has to be tested. It may in the end supersede Hauser's
method.
Here in India the stains that fall to be examined would usually be of fair size.
In Europe they pride themselves on testing pin-point-sized stains, which in my
opinion is a work of supererogation, when larger stains are present. In a recent
note 6 I have shown how often cutting weapons are used by murderers in this
country, and how the murderer is not content with striking one blow, but makes
sure of his victim while he is about it. It will easily be understood that in such
cases the victim bleeds freely.
In order to give the reader a good idea of what is meant by a positive re-
action I asked Babu Behari Lal Das to draw the appearances in two tubes which
I had used in an experiment. In one of these the reaction was positive, in the
other negative, an antihuman fowl serum having been added to a dilution of a
stain-extract, made by me from a human blood-stain in the one case, and from an
1 BRUCK. :— Die biologische Differenzierung von Affenarten und menschlichen Rassen durch spezifische
Blutreaktion. Berliner klin. Wochenschr. 1908, 793.
2 LINOSSIER and LEMOINE :—Essai de différenciation des albumines du sérum chez les animaux de
même espèce, mais de races différentes. C. R. Soc. de biol. 1907. lxii, 4.
3 MARSHALL and TEAGUE :—A study of the precipitin and complement-fixation reactions. Philip-
pine journ. of sci. November 1908.
4
HAUSER :—Ueber einige Erfahrungen bei Anwendung der sero-diagnostischen Methode für
gerichtliche Blutuntersuchungen. Münchener med. Wochenschr. 1904, 289.
5
CARNWATH :—Zur Technik der Untersuchung kleinster Blutspuren. Arbeiten a. d. kais. Gesund-
heitsamte, 1907, 27 Bd. Heft 2.
6 SUTHERLAND :—Medico-legal practice in the Mofussil. Indian med. gaz. 1910, 47.
15
ovine blood-stain in the other. The clear colourless appearance of the extracts in
the tubes, to the taper bottoms of which the clear straw-coloured antiserum has
B. L. DAS del.
THE PRECIPITIN-REACTION.
The contrast between the contents of the first tube, in which the antiserum has reacted with the stain-
extract dilution, and those of the second, which remain clear at the point of contact of extract and anti-
serum, is well brought out in this plate.
sunk, is well brought out in the plate. It is not every case that gives such a well-
marked reaction as that depicted here, which, however, is fairly typical of what
one obtains when using a powerful antiserum and a highly diluted stain-extract.
When we know that our extract is at least diluted to the same strength as a
thousand-fold dilution of the blood that caused the stain, or—to speak more
accurately—of the serum of that blood, and a reaction does not occur within
twenty minutes with the diluted extract, while with a thousand-fold dilution of the
antigen of the antiserum that we are using we obtain a marked reaction well
within this period, we are justified in concluding that in the stain before us there is
not the antigen of our antiserum.
The task of the serologist here is somewhat harder than it is in a temperate
climate: the animals require larger doses and more prolonged treatment, and at
the end of this more of them will be found to be refractory. About 33 per cent.
are either refractory or only slightly immunisable in Europe; here the percentage
D 2
16
of unsatisfactory animals is higher, and the disappointments of the observer more
frequent. There a high-potency antiserum which will detect the presence of its
antigen in a hundred-thousand-fold dilution—something like a wineglassful in a
caskful—is not rarely obtained, and antisera of half this strength are easily
enough obtained. Here the only antiserum that approached this strength was
one of 1-40000 titre that I obtained from a fowl that had been treated with human
(placental) serum. The following extracts from my notes show the difficulties
which I met with in Calcutta:—
Animal.
Antigen.
Injected.
Total
amount c.c.
Duration in
d a y s .
Titre of
Antiserum.
Fowl 23
. Canine .
. Intraperitoneal .
48.5
29
1-4000
„ 24
. Human .
.
„
.
5 0
28
1-2000
„ 25
. Cat's serum
.
„
.
Died on
20th day
.
Epizootic.
„ 26
. Horse's .
.
„
.
„ 23rd „
.
„
„ 2 7
. Dog's
.
.
„
.
46.5 c. c.
29 days
.
Useless.
„ 28
. Human .
.
„
.
41
29 „
.
1-3000
„ 29
. „
.
.
„
.
Died on
22nd day
.
Epizootic.
„ 30
. Sheep's .
.
„
.
40.5
23 days
.
1-3000
„ 31
. Goat's
.
.
„
.
40
23 „
.
1-3000
„ 88
. Human .
. Intravenous
.
Died on
8th day .
.
Epizootic.
„ 89
.
„ .
.
„
.
„ 9th „ .
.
„
„ 90
.
„ .
.
„
.
„ 12th „ .
.
„
„ 91
.
„ .
.
„
.
11.5 c. c.
24 days
.
1-12000
„ 92
.
„ .
.
„
.
12
25 „
. .
1-40000
„ 93
.
„ .
.
„
.
Died on
6th day .
.
„ 94
. Camel's .
.
„
.
„
9th „
.
.
Rabbit 44
. Cat's
.
.
„
.
775 c. c.
18 days
.
1-3000
„ 45
. Dog's
.
.
„
.
8
18 „ .
.
less than
1-1000.
„ 46
. Horse's .
.
„
.
8.25
18 „ .
.
1-2000
„ 47
. Sheep's .
.
„.
6.8
18 „ .
.
less than
1 - 2 0 0 0 .
„ 48
. Human*
.
„
.
1 1 . 5
26 „ .
.
l e s s t h a n
1-2000.
* Titre showed no improvement between 12th and 26th day.
The epizootic which carried off so many of the fowls—those that had received
injections of foreign serum succumbed most easily—was caused by the pasteurella