NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
148
The injection of a specific serum is except in very large doses not immediately
followed by symptoms of blood destruction.
The following are observations of Celli, Casagrande, and Carduc.
Dog of six kilogrammes.
25th August.—Injected 5 c.m.c. of serum.
26th August.—Dog remains well.
27th August.—Injected another 5 c.m.c. of serum.
28th August—Sick, pallor, etc.
Dog of 5.5 kilogrammes.
25th August.—Injection of 1 c.m.c of serum under conjunctiva
.
29th August. —Injection of 1 c.m.c. of serum.
1st September.—Death.
Our own experiments show :-
Dog I.
6 p.m., 10th February 1908.—Received 4 c.c. goat's serum.
Morning, 11th February 1908.—No distinct pallor of tongue.
8 a.m., 11th February 1908.—Received 2.5 goat's serum.
8 a.m ., 12th February 1908.—Tongue paid, conjunctiva pale.
Dog IV.
10 a.m. , 2nd March 1908.—Received 15 c.c. goat's serum.
1 p.m. , 2nd March 1908.—Dog shivering. Anæmia present.
5 p.m. , 2nd March 1908.—Tissues pale, blood watery, etc.
Dog VI.
10 a.m., 2nd March 1908.—Received 3 c.c. goat's serum.
10 a.m., 3rd March 1908.—Passing hæmoglobinous urine.
Dog VII.
10 a.m., 2nd March 1908.—Received 1 c.c. goat's serum.
11 a.m., 5th March 1908.—Dog has been gradually getting more and more seedy.
Progressive anæmia now with weakness of hind legs as
in piroplasma.
Dog VIII.
11
a.m., 5th March 1908.—Received 3 c.c. goat's serum.
4-30 p.m., 5th March 1908.—Isotonic point at .83 per cent, salt.
8 a.m., 6th March 1908.—Isotonic point at 1 per cent. salt.
149
Dog IX.
11 a.m., 6th March 1908.—Received 6 c.c. goat's serum.
5 p.m., 6th March 1908.—Dog shows no symptoms.
10–30 a.m., 8th March 1908:—Dog quiet, anæmia marked but not profound.,
received 1.5c.c. goat's serum.
1–30 p .m., 8th March 1908.—Pallor increased, Dog profoundly ill.
Dog X.
5 p.m., 13th March 1908.—Received 3 c.c. goat's serum.
10 a. m., 14th March 1908.—No noticeable anæmia.
15th March 1908.—Marked pallor of tongue and a considerable amount of
anæmia.
Dog XI.
5 p. m., 13th March 1908.—Received 6 c.c. goat's serum.
10 a. m ., 14th March 1908.—Some pallor of tongue and some anæmia.
1 p.m.,14th March 1908.—Hæmoglobinuria.
Dog XII.
5 p. m., 13th March 1908.—Received 3 c.c. goat's serum and 10 c.c. normal goat's
serum.
10 a. m., 4th March 1908.—Moderate anæmia.
4 p . m ., 14th March 1908.—Extremities cold.
5 p.m., 15th March 1908.—Received 3 c.c. goat's serum.
16th March 1908.—Blood very anæmia, dog very seedy.
There is thus a variable period, during which the action of the poison, is not
apparent or slight, followed many hours or even, days later by intense blood
destruction.
To bring the facts relating to hæmolysis in the living body into harmony with
the results of hæmolysis in vitro is not easy. We have seen in Part III that the
injection of a hæmolytic serum does not necessarily lead to solution of the red cells
but only to their engulfment. Whether engulfment takes place before there
has been, time for solution, or whether the hæmolysin acts at all under these cir-
cumstances we do not know.
Barrat (88) has suggested that red cell phagocytosis is often, stimulated as a
result of the action, of substances of the nature of opsonins often present in, immune
sera.
We think that the changes are to a certain, extent due to agglutinins or possibly
other bodies which accompany specific sera such as we are dealing with. It is how
ever the conditions which give rise to the lysæmic form of blood destruction, the
ch aracteristic feature of Black-water Fever and paroxysmal hæmoglobinuria that
more especially demand our attention,.
227 H. D.
2P
150
What determines this condition we can, as yet only surmise. The most
important consideration as a basis for further work is the possible concerning of
complement in the process.
In the absence of complement from the plasma, whether as a result of non-
production (Metchnikoff we have seen denies that it exists free in the plasma) or of
absorption, amboceptor specific for the red blood cells if produced within the
body, though it might combine with red cells could not bring about their extra-
cellular solution.
If now under certain circumstances complement is suddenly produced and set
free in the plasma whether within an organ, or the general circulation, it will at once
combine with the amboceptor laden red cells, producing their rapid dissolution.
Whether such a combination of amboceptor with the red cell ever occurs or
indeed what exactly happens in the case of a hæmolysin in the body we shall see
to be an extremely difficult matter to determine.
The condition, which we have turned extra-vascular lysæmia is also of extreme
interest.
We have already seen, that the condition, generally described as hæmo-
globinæmia does not mean the presence of hæmoglobin dissolved in the plasma
during life. None the less the auto-solution, of hæmoglobin in the serum during
clotting of the blood may be of great significance. It is essentially the hæmolysis
of a certain number of corpuscles consequent upon, the act of coagulation ; and this
process we know to be associated with the formation, of complement, a substance
whose existence in the circulating blood is problematical.
If we conceive then of the liberation of complement in the presence of ambocep-
tor-bound red cells within the body there might result lysæmia or true hæmoglobi-
æmia but in the absence of complement from the plasma we may have the state
of affairs giving rise to extra-vascular lysæmia.
Some support of this view is given by certain, clinical facts. It has been fre-
quently noted that in paroxysmal hæmoglobinuria the so-called hæmoglobinærmia
was present when no attack was in progress. Bensuade (63) describes one case in,
which the serum was more coloured between than during the attacks, an observa-
tion which suggests that the red colouration of the serum is due to a condition
characteristic of the blood in this disease, rather than to the liberation,
of hæmoglobin responsible for hæmoglobinuria.
In syphilis Justus (79) has shown that hæmoglobinæmia can be caused by
producing stagnation of the blood in the vessels ; and that blood removed from the
median basilic vein after lightly constricting the arm for a few minutes may yield a
reddish serum.
151
CHAPTER II.
THE DEMONSTRATION OF AUTOLYSINS.
The demonstration of isolysins is not fraught with any particular difficulties.
It is essential only that a fitting complement and certain other conditions be
present, and that there should be no anti-hæmolysin present. In the demonstra-
tion of autolysins on the other hand the greatest difficulty is encountered.
In the investigation of certain diseases in which hæmolytic action appears to
be concerned, many observations have been made regarding the power supposed to
be exerted upon normal red corpuscles by the pathological serum. Evidence of hæ-
molytic action has been considered an indication of the presence of the hæmo-
toxins which have initiated the blood changes in the patient.
Ehrlich (78) endeavouring to gain some insight into the nature of hæmolytic
bodies which may arise within the body as a result of morbid processes, injected
certain goats with large quantities of the blood of other goats. As a result he was
able to produce hæmolysins within the bodies of the inoculated goats, but these
hæmolysins had no action upon the corpuscles of the goats in whose bodies they
arose ; they were isolysins therefore and not autolysins.
From these experiments Ehrlich concludes that the demonstration that the
serum of a patient possesses the power to dissolve the red cells of another person,
does not prove that this action has had any relation in the patient to the occurrence
of anæmia or any blood destruction. On the contrary it only demonstrates the
presence of what is probably only an isolysin.
It is clear then that if we are to ascertain the exact mechanism of the
blood destroying process in Black-water Fever, we must especially study autoly-
sins.
But before approaching the subject of autolysins it will be advantageous to
study conditions that have resulted from the introduction of artificially produced
hæmolysins in the body, and to see by what means apart from the recognition of its
effects we may be enabled to demonstrate the presence of a hæmolysin that has
once been introduced and absorbed into the system.
THE DEMONSTRATION OF HÆMOLYSINS ARTIFICIALLY INTRODUCED
IN THE BODY.
Celli, Cassagrandi, and Carlucci (77), using dogs injected with the serum of
rabbits previously treated with dogs' blood, were the first to attempt to re-isolate or
demonstrate the presence of the hæmolysin under these conditions. At the outset
they determined that the serum of a hæmoglobinuric dog has no action either upon
a healthy dog's corpuscles or on those of a hæmoglobinuric dog.
The serum of a hæmoglobinuric dog plus the spleen of a healthy dog plus the
corpuscles of a healthy dog was also negative. But the serum of a hæmoglobinuric
dog plus the spleen of a healthy dog plus the corpuscles of a hæmoglobinuric dog
, g a v e m a r k e d h æ m o l y s i s .
Applying these facts to the demonstration of a malarial hæmolysin in man,
these observers got no result.
Approaching the subject a fresh we sought in our own experiments on dogs to
demonstrate the presence of the hæmolysin that we had ourselves injected. The
corpuscles of the dog are very fragile, and if left standing for a few hours they are
apt even in the case of the normal animal to show a certain amount of laking. This
we obviated by the use of the solution of salt and. sugar recommended by Blasi (90).
The results of our experiments were as follows—
(1) The serum of dogs. suffering from intoxication and hæmoglobinuria
was not hæmolytic to the washed corpuscles from a healthy dog.
There was therefore no evidence of free hæmolysin.
(2) The addition of normal goat serum to the above did not bring about
hæmolysis.
Therefore the absence of hæmolysis was not due to lack of complement but to absence
of amboceptor, for normal goat serum contained fitting complement for the goat
derived amboceptor we were using.
For a priori reasons also it appears hardly conceivable that any amboceptor
could remain free in the presence of red cells which possessed suitable receptors for
its cytophile group or affinity.
( 3 ) T h e w a s h e d c o r p u s c l e s o f d o g s s u f f e r i n g f r o m i n t o x i c a t i o n w e r el
e f t
at 37° Cent. suspended in salt and sugar solution. No hæmolysis or
agglutination took place.
( 4 ) W a s h e d c o r p u s c l e s f r o m t h e h e p a t i c , p o r t a l , r e n a l , a n d h e a r t b l o o d w e r e
left at 37° Cent. in sugar and salt solution. No hæmolysis or agglu-
tination resulted.
The red cells in neither the peripheral nor the visceral circulation were bound
with hæmolysin (amboceptor and complement) for had they been so bound they
should have dissolved after a short time.
153
5. Pulp scraped from the spleen and washed to remove free hæmoglobin, and
then left in .9 per cent. salt solution, showed marked hæmolysis, but when placed
in the sugar and salt solution no hæmolysis occurred. Some agglutination ap-
peared to be present. It is doubtful if even the red cells collected in the spleen
were on the point of hæmolysis such as occurs in vitro.
6. Washed red blood cells from the portal circulation plus crushed liver sub-
stance from a treated dog, showed very marked agglutination but no hæmolysis.
Agglutination of red cells was frequently very marked in the blood from the
hepatic veins. There was also a similar tendency observed in blood from the kid-
neys, but an agglutinating effect of the injected dog's serum upon normal dog's
corpuscles was never obtained. Agglutinins were, however, readily demonstrated
in the oedema fluid about the site of the injection.
The demonstration of an injected hæmolysin or even the presence of agglutinins
is therefore fraught with great difficulty. Nevertheless in spite of the absence of
h æ m o l y t i c p h e n o m e n a i t i s t o b e n o t e d t h a t t h e r e d c e l l s o f t h e t r e a t e d d o g c o u l d
be shown to be abnormal by their behaviour on examination as regards—
( 1 ) C h a n g e i n t h e i s o t o n i c p o i n t .
( 2 ) C h a n g e i n a p p e a r a n c e a n d s t a i n i n g r e - a c t i o n .
( 3 ) A p p e a r a n c e o f a g g l u t i n a t i o n .
THE DEMONSTRATION OF AUTOLYSINS IN CHRONIC MALARIA.
Innumerable attempts have been made to demonstrate the haemolytic agent
in malaria, but with results either quite negative or unconvincing.
Celli, Casagrandi, and Carducci and later Blasi (91) testing the action of the
serum of malaria patients against normal corpuscles failed to find evidence of
hæmolytic power.
Similarly Capogrossi (94) ascertaining the agglutinating power of malarial
serum against healthy corpuscles came to the conclusion that the appearance of
agglutination was not diagnostic of malaria.
More recently Casagrandi claims to have discovered a Hæmolysin mask-
ed by an ante-hæmolysin in the blood of a case of malarial hæmoglobinuria.
Examining the blood of a number of persons suffering from malaria and
malarial anæmia we found the serum to possess no action upon healthy
• corpuscles, producing neither hæmolysis nor agglutination.
It seemed to us also that such a possibility as the occurrence of free ambo-
ceptor circulating in the plasma was very remote ; for if both amboceptor and
227 H. D.
2Q
154
complement were present they would at once combine with the red cells; and
hæmolysis would result.
But the possibility that in malaria as in trypanosomiasis complement might
be so far absorbed, and that thus failing the completion of the hæmolysin,
amboceptor might be bound by the red cells suggested to us the idea of
searching for the presence of amboceptor bound red cells in malaria.
The observations described by Metchnikoff, previously referred to, in which
guinea pig spermatozoa became bound with amboceptor within the living body
in the absence of complement appeared to indicate the condition which we might
possibly find in the red cells of malarial patients.
Ehrlich suggests the complements can disappear during certain pathological
conditions in consequence perhaps of a more rapid destruction or a slower pro-
duction.
In the constant blood destruction of malaria the using up of complement
appeared an extremely likely phenomenon. Under such circumstances the red
cells might become bound with amboceptor though to all appearances normal.
If such a condition were present it would only be necessary to add to wash-
ed malarial corpuscles a little normal human serum, and hæmolysi would occur.
When we came to examine a number of malarial patients we found that as
a rule their red cells exhibited a great susceptibility to the action of normal serum
so much so that rapid hæmolysis took place in some cases when only 50 c.m. of
serum from certain normal subjects was added to ½ c.c. of a 5 per cent suspen-
sion of their red cells in .8 per cent salt solution. This hæmolysis was also in-
variably associated with a marked agglutination.
A reference to the literature showed that a similar susceptibility had been
noted in the case of the blood of anæmic subjects in Italy by Ascoli (89).
On further testing the action of the serum of the malarial subjects we found
that it not only failed to produce any hæmolysis of healthy blood corpuscles,
but even in very small quantities inhibited the action of powerfully acting
normal serum upon susceptible red cells.
OBSERVATION ON THE BLOOD IN BLACK-WATER FEVER.
Our results with the blood of Black-water Fever cases have been as follows.
Further details of the cases are given in the appendix :—
Black-Water Fever serum
+ Washed normal cor- = No hæmolysis.
puscles.
No agglutination
Black-Water Fever serum
+ Normal corpuscles and = No hæmolysis.
normal serum.
No agglutination.
•
155
That is neither alone, nor with the undoubted presence of ccmplemont, had
the Black-water Fever serum any action on the normal corpuscles used. (Class
A., vide next chapter.)
Washed Black-water Fever cor-
puscles.
+ Allowed to stand in
.9% Salt at room temp.
= No hæmolysis.
No agglutination.
Washed Black-water Fever cor-
puscles.
+ Black-water Fever serum = No effect.
These cells had therefore neither been already acted upon by an active hæmo-
lysin, nor did the patient's own serum have any action upon them.
Black-water Fever corpuscles
+ Black-water Fever serum
and normal serum (com-
plement).
= No hæmolysis.
Black-water Fever corpuscles
+ Serum of malarial
patient.
= No hæmolysis.
No agglutination.
Black-water Fever serum
+ Washed corpuscles of
malarial patient.
= Marked agglutination.
Case XX. —
Black-water Fever serum:
+ Washed normal corpus-
cles, S.R.C.
= No hæmolysis.
No agglutination.
Black-water Fever serum
+ Washed normal corpus-
cles, C.A.B.
= No hæmolysis.
No agglutination.
Black-water Fever serum
+ Washed normal corpus-
les and normal serum
(complement).
= No hæmolysis.
No agglutination.
Black-water Fever serum
+ Washed Black-water Fever
corpuscles and normal
serum (complement).
= No hæmolysis.
No agglutination.
Washed Black-water Fever cor-
puscles.
+ Allow to stand in .9
per cent. salt at room
temperature.
= No hæmolysis.
No agglutination.
Black-water Fever. serum
+ Washed corpuscles of
malarial patient.
= Slight hæmolysis.
Marked agglutination.
Normal serum, S.R.C.
+ Washed corpuscles of
malarial patient.
= Marked agglutination.
Normal serum, C. A. B.
+ Washed corpuscles of
malarial patient.
= Marked agglutination.
156
Washed Black-water Fever cor-
puscles.
+ Black-water Fever serum
and extract of dog's
spleen.
= Some hæmolysis.
Normal corpuscles
+ Normal serum and extract
of dog's spleen.
= Some hæmolysis.
Case XXII
—
Washed Black-water Fever cor-
puscles.
+ Normal serum
= hæmolysis.
½ c.c. washed Black-water Fever
corpuscles emulsion,
+ (50 c.m. Black-water Fever =
normal serum) (at 37 for 15 min.)
No hæmolysis.
Agglutination.
½ c.c. washed corpus cles native
+ 150 c.m. Black-water
=
Fever serum.
No hæmolysis.
No agglutination.
½ c.c. washed corpuscles very anæmi
c
native.
+ 150 c.m. Black-water Fever =
serum.
Agglutination.
Case XXIV—
½ c.c. washed Black-water Fever
corpuscles emulsions.
+ 150 c.m. normal serum = Rapid hæmolysis.
½ c.c. washed Black-water Fever
corpuscles emulsions.
+ (150 c.m. normal serum, =
.50 c.m. Black-water Fever
serum (at 37 for 15 min.)
No hæmolysis.
Agglutination.
Washed Black-water Fever
corpuscles
+ Allowed to stand in .9 =
per cent, salt.
No hæmolysis.
Case XXV—
½ c.c. washed Black-water Fever
corpuscles emulsions.
+ 150 c.m. normal serum, S.R.C. =. No hæmolysis.
No agglutination.
Washed Black-water Fever
corpuscles.
+ Undiluted normal serum,
= S.R.C.
No hæmolysis.
No agglutination.
Case XXVI
—
Washed Black-water Fever
corpuscles.
+ Allowed to stand in .9
=
per cent salt room temperature
No hæmolysis.
Black-water Fever corpuscles
+ Black-Water Fever serum = No hæmolyisis.
No agglutination.
½ c.c. Washed Black-water corpus-
cl es emul si ons.
+ 150 c.m. normal serum
= Complete hæmolysis.
½ c.c. washed Black-water Fever
corpuscles emulsions.
+ 150 c.m. normal serum,
=
50 c.m. Black-water Fe-
ver serum (at 37 for 15 min.)
Hæmolysis slightly re-
tarded.
157
½ c.c. washed Black-water Fever
corpuscles emulsions.
+ 150 c.m. normal serum
100 c.m. Black-water
Fever serum(at 37for 15 min.)
= No immediate hæmolysis.
Slight after some hours.
Agglutination.
Case XXVII
—
Washed Black-water Fever
c o r p u s c l e s .
+ Allowed to stand in .9
per cent, salt at room temp.
= No hæmolysis.
Washed normal corpuscles
+ Undiluted Black-Water
Fever serum.
=
No hæmolysis.
No agglutination.
Washed normal corpuscles
+ Undiluted Black-water
Fever serum (at 37 for 30 min.)
centrifuged normal serum.
= No hæmolysis.
No agglutination.
½ c.c. washed Black-water Fever
corpuscles emulsions.
+ 150 c.m. normal serum
= No hæmolysis.
No agglutination.
Washed Black-water Fever
corpuscles.
+ Undiluted normal serum
= No agglutination.
½ c.c. washed corpuscles, native
(class B). ( Vide next chapter.)
+ 300 c.m. Black-water Fe-
ver serum
= No hæmolysis.
Marked agglutination.
½ c.c. washed corpuscles native
(class B.)
+ 150 c.m. normal serum, S.R.C. = Complete hæmolysis.
½ c.c. washed corpuscles native
(class B)
+ 50 c.m. normal serum
50 c.m. Black-water Fever
serum at 37 for 15 min.)
= No hæmolysis.
No hæmolysis.
½ c.c. washed corpuscles, native
(Class B).
+ 150 c.m. normal serum
20 c.m. Black-water Fever
serum (at 37 for 15 min.)
= Hæmolysis.
In this case there was therefore anti-body, in spite of the fact that the
blood was a modified class A. (vide next chapter).
These results were intelligible only after we had gained the further know-
ledge detailed in the next chapter.
227 H. D.
159
CHAPTER III.
HUMAN ISOLYSINS.
When we found that the red cells of people anæmie from malarial infection
were characterised by a marked susceptibility to hæmolysis on the addition of
normal serum we at first considered that this observation pointed to a binding
by them of amboceptor, which, on the addition of normal serum containing comple-
ment, resulted in their speedy hæmolysis.
We have since added to our observations with the result that our original
idea has been shown to be untenable. But our observations have served to point
to a condition we believe not generally recognised, and of which we can find no
indication in the work of Ehrlich and others. Isolysins in man have generally
been considered an occasional phenomenon due to some pathological con-
dition of the person whose serum was isolytic. That the red cells against
which such a serum was tested might be an equally important factor has not gen-
erally been taken into account.
If therefore in the serum of a Black-water Fever case we were to demon-
strate isolysins it might be thought that we had shown a point of pathological im-
portance.
Again we have seen that in malaria we appeared to have found red cells
bound with immune body (amboceptor) since on the addition of normal serum
they became rapidly hæmolysed.
But the real facts possess quite a different significance and make all work
on pathological isolysins undertaken in ignorance of them, open to constant
error.
And the same may be said of any attempt to demonstrate the presence of
autolysins in disease.
The following experiments will make this clear.
I.—Experiments to shew susceptibility of washed blood corpuscles of Class B bloods to
hæmolysis and agglutination by Class A serum and want of action either hæmo-
lytic or agglutinant of Class B serum upon washed corpuscles of Class A
or Class B blood.
SERIES I.
Case I.—Child; jaundice and anæmia.
Child's washed corps. + Small quantity normal = Marked agglutination.
serum, S. R. C,
160
Washed corps., S. R. C. + Small quantity child's
serum
=
Nil.
Washed corps. child in
.9 per cent. salt.
=
Nil.
Washed corps. child
+ child's serum
=
Nil.
Case II.—Adult ; extreme anæmia (malaria).
1 c.c. emuls. corps. No. 2 +
2 0 c . m . n o r m a l s e r u m ,
S.R.C.
= Marked agglutination.
1 c.c. emuls, corps. No. 2 + 120 c.m. normal serum,
S.R.C.
= rapid and complete hmolysis.
1 c.c. emuls. corps. No. 2
=
Nil
1 c.c. emuls. normal corps.
S.R.C.
+ 1 120 c.m. serum, No. 2 =
Nil.
Case III.—Adult ; fever and enlarged spleen, no parasites.
½ c.c. emuls. corps No. 3 + 1 c.m. normal serum,
S.R.C.
= Agglutination.
½ c. c. , , , , „
+ 5 c.m.
„
„ = Trace of hæmolysis and agglu-
tination.
½ c.c. ,, ,, ,,
+ 20 c.m. ,,
,,
=
, , , , , ,
½ c.c.
„
,,
,,
+ 40 c.m. „ ,, = Considerable hæmolysis.
½ c.c. „
+ 60 c.m. ,,
,, = Nearly complete hæmolysis.
½ c.c.
„
,, ,,
+ 100 c.m. „
„ = Complete hæmolysis in ½ hour.
½ c.c.
„
,,
+ 1 c.m. normal serum,
C.A.B.
=
Nil.
½ ' c.c., ,,
„ + 5 c . m. „
„ = Trace of hæmolysis.
½ c.c. ,, ,, „
+ 20 c.m. „
, ,
= Slight hæmolysis.
½ c.c.
„
,,
,,
+ 40 c.m. „
, ,
= Marked hæmolysis.
½ c.c. „ „ „
+ 60 c.m. „
„
=
,,
, ,
½ c.c. ,, normal corps., + 20 serum No. 3
S.R.C.
=
Nil.
½ c.c. „ normal corps.,
C.A.B.
+ 120 serum, No. 3 =
Nil.
Case IV .—Boy ; Simple Tertian infection parasites average 1 per field.
½ c.c. emuls. corps. No. 44 + 10 c.m. serum, S.R.C. = Agglutination.
½ c.c.•
,, ,,
, ,
+ 40 ,, .,,. „
,,
=
, ,
½ c.c. ,, ,, „
+ 200 c.m. „ ,,
= Almost complete hæmolysis.
½ c.c. corps., S.R.C.
+ 200 c.m. serum No. 4 =
Nil.