NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
25
this end in view. Since then the merits of the anaphylaxis test have attracted
much attention, but out here it is hard to come by the literature, so I have
had to trust to excerpts in the various journals, instead of to the original reports
of the observers mentioned in the following lines.
It is now matter of common knowledge that guinea-pigs (and rabbits) are
easily supersensitised by an injection of a foreign albumin. The condition does
not make its appearance at once, but if after the 12-14th day the animal receives
a second injection it will, in the majority of cases, react violently and rapidly.
The supersensitive condition, according to Rosenau and Anderson, who have
done a large number of experiments, may last for three years, that is to say,
during the whole life of the guinea-pig.1 It begins after the 10-12th day,
if the first injection have been made into the brain, in so small a dose as
0.001 c. c.; for intraperitoneal injection 0.01 c.c. is enough, even out here.
The why of the phenomena caused by the second dose of serum has been
much discussed by competent observers. Richet, to whom we owe the term Ana-
phylaxis, believes 2 that a new substance is formed in the animal's blood. Rosenau
and Anderson call this substance anaphylactin, and, since they found that the blood
of a supersensitised animal causes a previously-untreated animal to become at once
supersensitised, if the blood be drawn after the period of incubation has elapsed, it
would appear that their idea has much in its favour. If the blood be drawn before
the incubation period has elapsed, the animal into which it is injected does not
become immediately supersensitive to an injection of the original substance.
On the other hand Friedberger believes that it is because there is produced
an antiserum for the substance injected, and this combines with the substance at
the second injection, forming a combination of antiserum and antigen, that the
anaphylaxis occurs: the real state of affairs being an absorption of the comple-
ment of the animal's serum by the combination. This view is supported by the
fact that Doerr and Russ found when working with dogs —as Friedberger and
Hartoch had found in the case of guinea-pigs—that actively-supersensitised
animals, when they show symptoms of anaphylaxis, have much less complement
in their serum than normal, and passively-supersensitised animals at this time
may have entirely lost their complement. Complement is not absolutely neces-
sary for the maintenance of health, if it be true that during the first months of
life babies have none in their blood; but it is easily conceivable that when once it
is present its sudden diminution, not to speak of its total disappearance, will have
a profound effect on the organism.
1 ROSENAU and ANDERSON :—Bull. No. 5o.
2 RICHET :—Physiologie : travaux du laboratoire de M. Charles Richet. Tome vi. Anaphylaxie, ali-
mentation, toxicologie. Paris. 1909, cited in Brit. med. J ourn. 1909, i, 1254.
26
T s u r u 1 i s o f o p i n i o n t h a t i t i s b e c a u s e af o r e i g n
s e r u m h a s b e e n i n j e c t e d t h a t
the complement becomes less: he found that when guinea-pigs receive single
doses of dog's or rabbit's serum, their complement becomes markedly decreased
in amount, and further he found that the lessening of complement in actively-
supersensitised animals is always very slight, as also in passively-supersensitised
animals who have received a dose of blood from a sensitised animal of like
species. On the other hand, if the sensitised animal be of another species, the
loss of complement is marked.
Richet and Friedemann, and also Doerr and Russ, found that the injection
of a serum mixed with its antiserum causes symptoms of anaphylaxis to appear.
Friedberger2 desired to remove the interference of the foreign serum, and to attain
this end he allowed sheep's serum to react with anti-ovine serum, derived from a
treated rabbit, in vitro. The resulting precipitate was most carefully washed, to
get rid of the rabbit's and sheep's sera, and digested with fresh guinea-pig's serum,
the mixture being then centrifugalised. The clear supernatant liquid was then
injected into healthy, untreated, guinea-pigs, who all showed symptoms of anaphy-
laxis. If, on the other hand, the washed precipitate was digested with heated
guinea-pig's serum, which as a consequence of being heated had lost its comple-
ment, and then the mixture, after being centrifugalised, was injected into guinea-
pigs, these exhibited no signs of anaphylaxis. If these experiments are confirmed
we may conclude that Friedberger's original contention is well-founded. In this
case the phenomena of precipitation, complement-deviation, and anaphylaxis are
all brought into line, as being primarily dependent on the formation in the serum
of the treated animal of precipitins. Doerr and Moldovan, 3 to judge from the
short note on their work to which I have access, have found that where the preci-
pitinogen is used up by the animal, no supersensitisation occurs.
Recently Pfeiffer 4has reported that when a guinea-pig has been rendered
supersensitive to a given albumin, on receiving the second injection of this it
shows, even when all other symptoms of anaphylaxis are absent, a marked lower-
ing of the body-temperature—as much as 6.5°C. This lowering of temperature
he and Mita have found to be a constant symptom, and in their opinion it may
therefore be taken to be the sign when the test is performed in forensic medical
practice.
One precaution must always be taken—the substance (serum) injected for
the test must be heated to 56°C.—Uhlenhuth recommends 6o°C.—for half an hour
1 TSURU :—Die Komplementabnahme bei der aktiven und passiven Anaphylaxie. Zeitschr. f.
Imnunitätsforschung u. exper. Therapie. 1909. 4 Bd. Heft 5, cit. in Münchener med. Wochenschr. 1910 , 806.
2 FRIEDBERGER :—Eiweissanaphylaitie. ibidem cit. in eodem loco.
3 DOERR and M0LDOVAN :—Analyse des Präzipitationsphänomens mit Hilfe der anaphylaktischen
Reaktion. Ibidem. 1909. 5 Bd. Heft 1. cit. in eodem, 1205.
4 PFEIFFER :—Ibidem. 19o9. 4 Bd. Heft 4, cit. in eodem, 705.
27
before it is used. Foreign unheated sera often cause untoward symptoms when
injected into guinea-pigs, and thus the anaphylactic reaction might be simulated,
especially in a case in which the test-serum was injected intracardially. Three
of my guinea-pigs died soon after receiving comparatively small doses of
sheep's serum (unheated). All observers insist on the necessity of preliminary
heating of the test-serum, which when used should be warmed to near the body-
heat of the guinea-pig. Time and again I have seen my rabbits and fowls suffer
apparently severely, when they received a dose of unwarmed foreign serum
intravenously, even if it had been previously heated to 56°C. for half an hour.
In precipitin work this does not matter much; but it might absolutely mislead
one when performing the anaphylaxis test.
So f a r a s I wa s a bl e t o c a r r y out wor k on t he t e s t I found it to be confirma-
tory of the results obtained by means of the precipitin test. Not all guinea-pigs
showed signs of anaphylaxis—I had not then heard of Pfeiffer's work on the
temperature-loss of otherwise apparently unaffected animals.
Thomsen, to whom belongs the credit of pointing out the medico-legal
value of the phenomena of anaphylaxis, found that the reaction is specific for
a group. Working with blood-stains of 2-3 months' age, that had been caused by
the blood of man, pigeons, fowls, sheep, goats and monkeys, he found that while a
supersensitised guinea-pig would react to a second dose of the substance to which
it was supersensitive, or of a similar substance—a humanised guinea-pig would
react to human or simian serum—it would not react to a substance far removed
from the sensitising substance. Thus a humanised guinea-pig would not react
to sheep's or goat's or fowl's serum. The following I take from my own notes:—
Four guinea-pigs supersensitised to human serum, by doses of 0 .0 1 c.c.,
were given injections of 4 c.c. intraperitoneally after t h e
p e r i o d o f i n c u b a t i o n
had elapsed: one died after 30 minutes; two were very ill, but recovered, and
the fourth was apparently but little, if at all, affected—its temperature was not
specially noted.
Two guinea-pigs supersensitised with 0 .0 1c . c . o f h u m a n s e r u m r e c e i v e d
intraperitoneally 4 c.c. of human serum on the 14th day: one died within 25
minutes, the other seemed to have recovered after 30 minutes, but was found
dead next morning.
A guinea-pig that had received 3 c.c. of human serum, was on the 14th day
treated with 1.5 c.c. of simian serum: it showed no signs of discomfort, but when it
received, on the 118th day, 3 c.c. of human serum, it promptly reacted and died.
So far we may conclude that here, as in Europe and America, the condition
of supersensitiveness persists for months; that it is a group-specific condition
is likely, and that, apart from temperature-lowering, sometimes anaphylactic
28
symptoms are not elicited in previously sensitised animals is evident. More work
requires to be done on this point.
The work done by Uhlenhuth and Haendel1 shows that the age of the
material makes no difference: they obtained the characteristic reaction in animals
that had received doses of extracts made from a cow's placenta that was 30
years old; from a frost-bitten limb 53 years old, and from a collection of blood,
14 years old, that was completely decomposed, and, owing to its long exposure
to light, had lost the power of reacting, by its extract, to a precipitin-antiserum.
Had one been able to supersensitise one's guinea-pigs with human or
other sera, and then, as soon as a stain fell to be tested, to make an extract of
the stain and use this as the test-substance, forensic work with the anaphylaxis
test would have been rapidly done: a given stain could have been tested by this
and the precipitin test on the day of its arrival at the laboratory. Unfortunately
we cannot do this. A delay of at least fourteen days is required for the guinea-
pigs, that have received injections of the stain-extract, to develop supersensitive-
ness. For these injections but a small amount of extract is needed, whereas
if it be used for the test-dose we need many cubic centimetres of a strong
extract, and for this a large stain is required. The cases in which we can use
extract for the test-injections are thus few in number, for it appears to be of the
utmost importance to sensitise a number of animals with the same stain-extract,
or to test them therewith if they have been sensitised with normal serum: at least
five animals would be required in a forensic case—three supersensitised with the
extract, one untreated, and at least one supersensitised with some other material.
The results obtained, if confirmatory of the results yielded by the precipitin
test, would be of value. It is probable that if exact measurements of the
temperature of the animals were made, the observer would be able to detect
specific reaction even when visible symptoms were slight.
Pfeiffer claims that he obtained excellent results when testing extracts that
gave no reaction with a precipitin-antiserum, and, as we have seen, in one such
case Uhlenhuth and Haendel obtained a good result. Much more work will have
to be done on the test here before it can be said that its results, in the absence
of confirmation by the precipitin test, are to be accepted.
VI.—Methods and results.
For carrying out these Biochemical tests in the tropics the first desiderata are
apparatus for keeping the collected blood cool until sufficient serum has become
separated from the clot, and for freezing the sera and antisera obtained: an ordinary
American refrigerator-cabinet suffices for the first mentioned purpose, but a freez-
ing-chamber is required for the second.
1 IJHLENHUTH and HAENDEL : in UHLEN HUTH and WEIDANZ : p. 177.
29
For extensive work, or work in which sera sealed in capillary tubes are used,
the " Frigo" apparatus, made by Lautenschläger of Berlin on the designs of
Morgenroth, does not answer well, although by its means a very low temperature
can be kept up at comparatively small cost, even in Calcutta. The freezing-chamber
in this apparatus is small and must be removed from its bed of insulating material
every time that the freezing-mixture of crushed ice and salt is renewed—twice daily,
as I found. At the Medical College, however, there is a large freezing-chamber,
with electric motor and piping for the circulation of the cooling medium,—
ammonia. The temperature of the chamber, when once lowered, is kept from
rising rapidly by the presence of a large brine tank in its interior. This chamber
is all that is needed to make serological work in the tropics feasible, if not so easy
as in more temperate climates.
The close proximity of the Eden Hospital to the Medical College is of
advantage since the large supplies of placental blood, that are needed to keep up
a sufficient stock of antihuman serum, may thus be obtained fresh daily. Un-
doubtedly clear placental serum is the material of choice for the treatment of
animals from which it is desired to obtain antihuman sera. Uhlenhuth at one time,
if not now, favoured the drying of blood and the storage of the dried blood, which
might then be re-dissolved in saline solution when it was required for injection.
This plan works well in Europe, but cannot be easily followed out here: the dried
blood, unless air be totally excluded from it, would in a week be full of moulds in
Calcutta, and—what is of just as much moment—the drying of the large
quantities of blood that would be required would take up a great deal of the time
of the observer's staff, and would be a work of supererogation where ice-chests
and freezing-chamber are available.
All instruments and apparatus used in the laboratory must be carefully
sterilised before use. This work was thoroughly carried out by my assistant, Dr.
G. C. Mitra. The salt used for the saline solution must be pure chloride of
sodium, and it must be dissolved in distilled water, the solution being boiled each
morning to ensure its sterility.
For the preparation of the animals for injections and for the abstraction of
blood an elaborate ritual is followed in some European laboratories. As this takes
up a lot of time, I endeavoured to simplify the process, and succeeded in cutting
it down to a thorough swabbing of the area that was to be the site of operation
with pledgets of absorbent cotton-wool soaked in ether. None of the animals
showed untoward results from this method of preparation, nor did the blood
collected become more easily contaminated than when the directions of Nuttall
were followed. In the vicinity of the College the tetanus-germ abounds, but none
of my animals developed tetanus, although many were operated upon again and
again. The site of the puncture, made when the injection was given, was imme-
diately sealed with flexile collodion.
30
In my monograph, and in the foregoing pages, I have described all that one
needs to know about the way in which the injections were given, and the blood
abstracted. I shall now give some extracts from my notes to show how the
animals were treated with antigen, and what results were obtained from the plan
of treatment adopted.
Fowl 43 received injections of human serum, given intraperitoneally
On 12th March
10.5 c.c.
17th "
1 0
"
22nd "
10
"
27th "
10
"
On the 8th day after the last injection its serum was found to have a titre of
1-2,000 over, i.e. , it was more than powerful enough to cause a reaction in a
2,000-fold dilution of human serum within a few minutes, and did not react with
any other antigen-dilution.
Rabbit 44: Feline serum intravenously:—
On 13th March
2
c . c .
16th "
2 "
19th "
2 "
22nd „
1 . 7 5
"
On the 8th day when its serum was titrated it was found to be 1-3000 over.
Rabbit 71 was treated intravenously with ass' serum.
It received on 17th April a dose of 2 c.c.
20th
"
2 "
23rd
"
2 "
26th
"
2 "
4th May
2 "
On the 7th day thereafter its serum was found to have a titre of 1-9000.
Rabbit 72 was treated in the same manner with human serum, receiving on
24th April 2 c.c., on 27th April 2 c.c., on 30th April 2 c.c., and on 6th May
1.5 c.c. Its serum on the 7th day thereafter was found to be inert.
Fowl 78 was treated with human serum as follows :—
On 8th May 1.5 c.c. subcutaneously.
11th "
1.75 " intravenously.
14th "
2
"
"
17th "
3
"
"
20th "
3
"
"
26th "
3
"
"
On 4th June its serum was found to have a titre of 1-3000.
Fowl 83 received doses of hircine serum as follows :—
On 12th May 7 c.c. intraperitoneally.
17th " 7 "
"
22nd " 3.5 " subcutaneously.
27th " 2.5 " intravenously.
31
On the 7th June it yielded a serum of 1-4000 over titre.
Fowl 91 received human serum in the following doses :—
On 1st June 2 c.c. intravenously.
4th " 2.5
"
7th " 2.5 "
1 0 t h " 2.5 "
14th " 2
"
Its serum-titre on the 25th June was 1-12000.
Fowl 135 received ovine serum thus:—On 19th July 2 c.c., on 23rd 1.5 c.c.,
on 26th 2.5 c.c., and on 30th 2.5 c.c., all intravenously. On 9th August its serum
was found to be inert.
Rabbit 144 received intravenous injections of human serum as under:—
On 26th July
1.25 c.c.
29th "
1.25 "
2nd August 1.5 "
6 t h
"
1.5 "
On the 16th August its serum was found to be inert.
Rabbit 34 received intravenous injections of human serum as follows:—
On 10th February 2 c.c.
1 3 t h "
2 "
16th
"
1.5 "
On 22nd February its serum was found to have a titre of 1-2000, so it was
given another injection—2 c.c., and on 1st March the titre had risen to 1-4000.
Rabbit 35 received doses of human serum intravenously as under:—
On 10th February 2 c.c.
13th
"
1.5 "
16th "
2 "
Its serum-titre on 22nd February was almost nil. It was given an injection
of 2 c.c. then and the serum was again tested on 1st March and found to be very
weak, its titre being less than 1-500.
Two fowls—139 and 140—received hircine serum intravenously as under :—
139
22nd July 2.5 c.c. same date
140
2 c.c.
26th "
2.5 "
2.5 "
30th "
2.5 "
2.5 "
3rd August 2.5 "
2.5 "
Titre 13th August 1-4000.
Nil
F 2
32
Rabbit 99 received washed sheep's red blood-corpuscles intravenously as
under :—
On 9th June
1.5 c.c.
15th " 2 "
21st "
2 "
26th "
2 "
These amounts represent, of course, the indicated quantities of a suspension
of corpuscles in saline solution, the saline being added in the original amount of
the serum that it replaced.
On 6th July the serum was tested and found to be capable of causing com-
plete dissolution of the standard amount of sheep's corpuscles—1 c.c. of a 5 per
cent. suspension in saline solution within 2 hours at 37° C.—when it had been
heated for half an hour at 56° C. and reactivated by the addition of fresh guinea-
pig serum. The working hemolytic dose of this antiserum was 0.085 c.c. of a
1-50 dilution, the minimal hæmolytic dose being 0.0085 c.c.
Rabbit 82 received doses of a normal suspension of red corpuscles intra-
venously as follows:—On 11th May 3 c.c., 15th 3 c.c., 19th 2 c.c., 24th 2 c.c.
It aborted on 30th May, but was not at all ill, so on 4th June it was given 2.5 c.c.
and its serum, on being titrated on 18th June, was found to have a minimal
hæmolytic dose of 0.002 c.c.
We see, then, that every attempt at immunisation of an animal is to be
regarded as an interesting experiment here as in Europe: one cannot predicate of
any animal that it will, or will not, yield an antiserum. Even if it be not refrac-
tory, one cannot guess what the titre of its serum will be. All that one may with
confidence affirm is that if one uses care, is patient, does not strive to hurry
matters, and treats a number of animals with human or other serum, one will have
one's reward in the shape of a high-potency antiserum from some of these ani-
mals, and that the antisera thus obtained will be in every way satisfactory from
the point of view of actual forensic medical practice.
There is no royal road in serological work, and the pitfalls in the path of the
observer, especially in the tropics are—as I have indicated—many; but with care,
patience, and practice, which is said to make perfect, these may be avoided and
the desired goal may be reached at last.
There remains to be said a word as to the most expeditious method of
obtaining an answer to the questions that arise when a suspected stain comes
to be examined: is this a blood-stain ? and, if so, to what blood is it due ?
Uhlenhuth appears to consider that the guaiacum-test (van Deen's1 )
is of considerable value as a negative test. I do not think that it is
1 van DEEN: Tinctura guajaci and ei n Ozonträger, als Reagens auf sehr geringe Blutmengen. Arch.
f. d. holländ. Beitr. z. Nat. u. Heilk. 1861-64. iii. 228.
33
worth one's while to use this test, which has so much against it as a positive
test—as I have recounted in my monograph—when one has the peroxide of
hydrogen test (Zahn's)1 at one's command. If there be no froth developed by
the contact of the reagent with a small fragment of the stain, then one is not
inclined to believe that blood is likely to be present, and this belief will be much
strengthened if the application, to another fragment of the stain, of the leucomal-
achite-green (Adlers') 2 test, or of the hydrazin (Riegler's) 3 test, gives a negative
result. If either of these tests gives a positive result then the aid of the spec-
troscope should be sought, the spectrum of hæmochromogen being so easily
obtained and so characteristic. In the case in which this spectrum is obtained,
the employment of the Teichmann test (hæmin crystals) is not necessary. It
remains to fix the form of the blood-corpuscles present in the stain by examining
a fragment under the microscope. Then an extract, made in the usual way,
is tested with antihuman serum, if we have found in the stain cells which are
obviously mammalian red blood corpuscles. In case there be any doubt about
this,— it is not every stain made by bird's blood that can be at once diagnosed
as such,—the use of the antiserum will help us.
In a series of test-tubes we have :—
Stain-extract diluted to at least 1-1000, a similar dilution of the antigen of
our antiserum, a 1-1000 dilution of an extract of a stain known to have been
caused by the antigen-blood, similar dilutions of extracts of stains known to
have been caused by other bloods, similar dilutions of other antigens, saline
solution alone, and finally a tube in which is the diluted extract of the suspected
stain to which instead of the antiserum is added the serum of an untreated animal
of the species from which the antiserum was taken. If now we have within
the time-limit, a reaction in the tubes, treated with antiserum, in which are : stain-
extract that is being tested, known human serum dilution, and known human
blood-stain extract; but none in any of the other tubes, we have convincing
proof that we are dealing with a human blood-stain. The following protocol
will perhaps make this clearer:—
Tube 1, Stain-extract dilute.
Antihuman fowl serum
Reaction.
" 2, Human blood-stain extract dilute
"
"
" 3, Human serum 1-1000
"
"
"
"
4, Equine serum 1-1000
5, Canine serum 1-1000
"
"
No reaction,
"
1Z A H N : D i e A n w e n d u n g d e s Wa s s e r s t o f f d i o x y d s z u m N a c h w e i s v o n B l u t f l e c k e n . C o r r . - B l . f
schweizer Aerzte 1871, i, 322.
2 ADLER, 0. and R.: Ueber das Verhalten gewisser organischen Verbindungen gegenüber Blut, mil
besonderer Berücksichtigung des Nachweises von Blut. Zeitschr. f. physiol. Chemie. 1904, 59.
3 RIEGLER: Ref. in Pharm. Cent. Halle 1905, 355.