NationalLibraryOfScotland/medical-history-of-british-india huggingface dataset:
136
cells has supposedly taken place.But this is unsatisfactory owing to the ex-
traordinary persistence of the most resistant cells as shown by the following series
of observations :—
1 1 vol. blood 2 vols. distilled water:
Hæmolysis incomplete.
2
”
3
,,
,,
”
,,
3
”
4
”
,,
,,
”
4
”
5
”
”
”
”
5
,,
10
”
”
”
complete.
In, each case although there was an increasing amount of hæmolysis observable
this was incomplete until ten volumes of distilled water had been used.
11.—Wide limit of error.—Since the question of a fall in, value of 1 per cent.
salt in the osmotic tension of the plasma may be an important point, as
suggested by McCay's observations, it appeared highly necessary that we should
be able to measure the hæmosozic value within known limits with accuracy,
otherwise the uncertainty attaching to our observations would prevent con-
clusions being based upon them. This has been extremely difficult even in the
case of variations as great as .2 per cent. salt, by the use of either McCay's
or Wright's methods. An example of the wide limit allowed by Wright's method
will be apparent from the following instance :-
Suppose serum (a)/6 behaves as would an N/28 salt solution
and that serum (b)/6
Then the estimation will give serum (a)
....
1.248 per cent.
serum (b)
..
..
1.040 per cent.
Difference due to one dilution extra..
. .
. . •208 per cent.
It is of course possible to make intermediate dilutions ; but, taken together
with want of fixidity of the index, it is clear that there is much to be desired
in regard to delicacy and accuracy. By using a volume of blood with 2 volumes
of the different salts, centrifuging and taking the first distinct tint a greater power
of detecting differences in the same blood is given, but the most satisfactory and
simplest of all methods tried has been a modification of the original method of
Hamburgher.
In, the isotonic point of one's own blood we possess an in,dex'capable of being
accurately determined at any moment. Whether one takes the first trace of
liberated hæmoglobin or a more marked hæmolysis is immaterial so long as
measured quantities of blood and. solution are used and the tints compared.
137
By the use of a sharp spear-pointed lancet or other suitable pricker it is easy
to obtain one, two, or even more cubic centimeters of the blood to be tested. This
blood is allowed to clot in a suitable tube under conditions which preclude any
drying action and the serum is used in, preparing a series of dilutions. Of these
d i l u t i o n s 1 / 2 c . c . a r e a r r a n g e d i n s ma l l t u b e s a n d 1 0 c . m. o f o n e ' s o wn b l o o d a d d e d
After centrifugalisation one has a series comparable with another series of one's
own blood put up in the ordinary way for the estimation of the isotonic point
thus
1 / 2 c . c . o f
N / 1 2 N/13 N/14 N/15
isotonic series.
1 / 2
c . c . o f
8 / 2 8/ 24 8/24 8/24
serum series.
The index in this case is absolutely fixed ; and as one is using a considerable
quantity of serum, dilutions of any degree can be made.
Hœmosozic value in Black-water Fever.
The following are estimations of the hæmosozic value in several Black-water
Fever cases.
The exact relation to the period of onset, etc., will best be seen by reference
to the cases given in the appendix.
per cent.
Case XIX—End of mild attack.Wright's method..
1.06
Case XX—Onset of severe attack
..
..
..
...
1.12
Case XX—Next day..
..
. .
1.258
Case XX—Day following
. .
..
. .
..
1.25
Case XXI—After onset of severe attack
..
. .
..
1.258
Case XXV—Middle of attack
..
. .
..
1 . 1 4
Case XXVIII—Middle of severe attack. Serum rosy red
..
1.05
In Black-water Fever even quite early in the attack notably in case XX there
is no marked if any lowering of the hæmosozic value of the serum below the normal,
and certainly no such lowering as could conceivably result in hæmolysis by osmotic
tension. That there is not some raising of the hæmosozic value during the attack
is less certain.
Neither in the isotonic point nor in the hæmosozic value do we find variations
such as might suggest that hæmolysis was due to alterations in the osmotic
relation of the corpuscles and the plasma.
227 H. D.
2m
138
Hœmosozic value after quinine administration.
In our earlier estimates we used Wright's method. In many cases no change
was obtained, in others there appeared to be some reduction in the .value after
quinine sulphate administration. Since we dare base no conclusions on these
earlier data it is unnecessary, to give them.
The following estimations have been made with an unlimited supply of serum,
and the results rendered absolutely certain by the greatest care in dilutions and
the repetition of the observations.
Estimation by Wrights' method modified by blowing out tubes into hypertonic
citrate solution centrifuging and comparing tints of the two series.
(1) European. Previously no quinine.
Hæmosozic value before taking quinine 1.168 per cent. salt.
H æ m o s o z i c v a l u e 5 h o u r s a f t e r a d m i n i s t r a t i o n
15 grs. quinine sulph.
15 m. acid sulph. dil. 1.168 per cent. salt.
(2) European. Has not taken quinine for 15 days.
H æ m o s o z i c v a l u e b e f o r e t a k i n g q u i n i n e 1 . 4 p e r c e n t .
Hæmosozic value 8 hours after administration of-
15 grs. quinine sulph.
15 m. acid sulph. dil. 1.35 per cent.
(3) Eur opean. Has not t aken qui ni ne f or some t i me.
H æ m o s o z i c v a l u e b e f o r e t a k i n g q u i n i n e 1 . 2 8 p e r c e n t .
Hæmosozic value next morning after taking night previous-
10 grs. quinine sulph.
10 m. acid sulph. dil. 1.28 per cent.
Estimation by a modification of Hamburger's method.
Native. Previously no quinine. Blood drawn at 9 A.M. Shortly after 9 A. M. quinine
sulphate grains ten administered. Blood again taken at 5 P. M.
.5 c.c. dilutions of serum.
. 5 c . c . s a l t s o l u t i o n s .
S / 2
S
/ 2 . 1 2
S
/ 2 . 2 5
.475%
.45%
.425%
.4%
9 A.M.
. .
0
trace
distinct
0
0
trace
distinct
5 P.M. ..
0
trace
distinct
0
0
trace
distinct
139
Next morning observations repeated with different blood as an indicator
gave exactly the same results with several trials.
Therefore the hæmosozic value equals 2 (45 per cent.) or 9 per cent. salt.-
European. No quinine for several months previously. Blood taken at 10 A. M. Shortly
after, quinine sulphate grains 15 with minims 15 dilute sulphuric acid was administered. Blood
taken at 12 A.M., 5 p.m. and next morning.
10 A.m.
.5 c.c. serum dilutions.
.5 c. c. salt solutions.
S
/ 2 . 2
S
/ 2 . 3
S
/ 2 . 5
.475
.45
. 4 2 5
.4
Nil
trace
distinct
Nil
Nil
trace.
distinct
Hæmosozic value equals 2.2 ( .45 per cent. ) or .99 per cent. salt.
At
12 A. M.
5 P. M.
Next morning serum gave
S
/ 2 . 2
S/ 2. 2
S/ 2. 2
S
/ 2 . 3
Nil
Nil
Nil
trace
Hæmosozic value therefore equals 2.2 (.45 per cent.) or .99 per cent. salt.
We have not therefore been able to find the reduction in the hæmosozic values
noted by McCay after the administration of quinine sulphate. These obser-
vations are, however, at present few in number, and further work will be required
before it can be decided with certainty whether some reduction does not take
place. In the meantime there are strong reasons for disbelieving that this sup-
posed action of quinine sulphate has the slightest influence in the causation of
Black-water Fever.
That an attack may follow upon administration of the hydrochloride as well
as of the sulphate of quinine is shown by reference to the literature. Vedy (96)
gives two cases where Black-water Fever occurred after administration of the
hydrochloride and three out of the six cases investigated by Werner (58) were after
1 gramme .4 gramme and .6 gramme respectively of quinine hydrochloride.
Again if slight alterations in the salt contents of the plasma are shown some-
times to take place as a result of the ingestion of sulphates, we have no reason to
140
believe that the alteration can extend to such a range of osmotic tension as separates
the isotonic point and hæmosozic value in Black-water Fever ; nor are we justified
in concluding that such a reduction bears any causal relationship to an attack.
It is of interest to note in this connection that when in one of the experi-
ments on dogs the isotonic point as a result of the injection of specific serum had
been raised nearly to the hæmosozic value ( i.e., an isotonic point of 83 per cent.
salt solution and a hæmosozic value of about .9 per cent. salt) a dose of quinine
sulphate equivalent in amount to a 30 grain dose in man dissolved in dilute sulphuric
a c i d d i d n o t p r e c i p i t a t e t h e o n s e t o f h æ m o g l o b i n u r i a (v
i d e
d e g 7 )
BLACK-WATER FEVER NOT DUE TO CHEMICO-TOXIC ACTION.
The great majority of the chemical hæmolytic bodies give rise to met-hæmo-
globinuria. A case of met-hæmoglobinuria in a human subject has been described
by Hayem (85).
In this instance the substance giving rise to the condition was unknown, but
it was some poison exerting a profound effect upon the red cells, causing met-
hæmoglobinæmia and the appearance of this substance together with oxy-hæmo-
globin in the urine.
To microscopical examination the red cells appeared small, deformed and
brownish in colour and were collected into groups. Decolourised elements were also
to be observed, and leucocytes were very abundant, many of them being of great
size. In dried films the alterations in the red cells appeared even more marked.
They showed fragmentation and abnormal colouration. Polymorphonuclear
cells were relatively diminished, but absolutely increased in number. The pro-
portion of mononuclears was notably increased, this increase being principally
due to elements of large size with clear protoplasm. At the same time the blood
as a whole was greatly diminished in spite of an absence of excretion. This reduction
was also associated with anæmia, the red cells being a little over 1,000,000 per c.m
The serum showed the spectrum of met-hæmoglobin together with that of
exyhæmoglobin.
It is evident that, though in Black-water Fever a certain amount of met-
hæmoglobin may be present in the urine, the process is one of a liberation of
oxy-hæmoglobin. Not only is the serum tinted with oxy-hæmoglobin, as shown by
the spectroscope, but, if the urine be examined shortly after it is passed, it is quite
common to find only the merest trace of met-hæmoglobin ; although oxy-hæmo-
globin is present in abundance.
141
We can therefore eliminate from among the possible causes of Black-water
Fever most chemico-toxic substances ; even did the clinical evidence not enable
us to do so.
The only substance that it is really necessary to consider in this relation is
quinine, which has often been credited as the cause of Black-water Fever.
The hæmolytic effect of quinine hydrochloride has been shown by Stephens
and Christophers, who found that it extended to four or five dilutions of a saturated
aqueous solution. We have repeated and confirmed this Obs rvation, and have
made other experiments both with the hydrochloride of quinine and the pure
alkaloid.
The latter is extremely insoluble but suspensions in .9 per cent. salt solution
appear to exert no hæmolytic effect upon canine or human red cells.
With regard to the action of saturated solution of quinine hydrochloride the
following procedure was adopted. A saturated solution was first prepared
containing approximately 3 per cent. (Attfield (86) states that this salt is soluble
in 34 parts of water.) This saturated solution was then made up by the addition
of sodium chloride until it contained .9 per cent.of the latter salt.
On the addition of 200 c.ms. of this solution to suspensions of 10 c.ms, of
human bl ood i n ½ c. c. . 9 per cent , sal t sol ut i on nearl y compl et e hæmol ysi s t ook
place, both in the case of a normal and a Black-water blood.
But when in the case of both normal and Black-water blood suspensions, the
addition of 150 c.ms. of (a) normal and (b) Black-water serum was made prior to that
of the quinine solution, a reaction occurred accompanied with the precipitation of
the quinine as a fine white powder. Prolonged centrifuging failed to entirely clear
the supernatant fluid which remained slightly clouded but showed no trace of
hæmolysis. In what form quinine may exist in the circulating blood we do not
know ; but Stephens and Christophers found that cinchonised serum taken from a
healthy subject shortly after the administration of 2 grms. of quinine when sub-
jective symptoms were present, exerted no hæmolytic action upon the blood of a
Black-water Fever case. A consideration of these facts, together with those already
referred to in Part I, seems to exclude the action of quinine in itself as being the
cause of the lysæmia of Black-water Fever.
The indirect action exerted by this substance in the production of this con-
dition must be left for future consideration.
227 H. D.
2N
142
BLACK-WATER FEVER DUE TO THE CTION OF A SPECIFIC HÆMOLYSIN.
The resemblance between the action of specific hæmolytic serum and Black-
water Fever is very striking.
It is easy to understand that in a crudely prepared serum (containing all
the elements of the blood) wherein especially powerful agglutinins existed, the pro-
minence of the erythro-katalytic process might be more accentuated than in
Black-water Fever.
But though lysæmia is the characteristic feature of Black-water Fever our
observations show that both processes of blood destruction, which seem charac-
teristic of the action of blood destroying toxins acting in different ways, are con-
cerned in Black-water Fever.
Parasitic, osmotic and chemical actions we have excluded as causes and it
seems to us therefore most probable that Black-water Fever is due to some specific
hæmolysin arising within the body as a result of the conditions we have pictured
in the earlier parts of this memoir.
143
PART IV.
NATURE OF THE HÆMOLYSIN IN BLACK-WATER FEVER.
CHAPTER I.
HÆMOLYSINS.
We have previously referred to sera which are able to destroy red cells of a
particular animal both in vitro and in vivo.
It is necessary before going further to study more closely these specific
hæmolytic bodies. Bordet (85) was the first to show that the serum of guinea pigs, after
they had been injected several times with 3 c.c. to 5 c.c. of defibrinated rabbit's
blood, acquired the property of dissolving rapidly and intensely, in a test tube,
the red cells of a rabbit ; whereas such action by normal guinea pig serum is
either absent or very feeble. This acquired property Bordet showed to be
a specific one, but based upon a principle of general application. An animal of
species A injected with the red cells of an animal of species B developes in, its blood
a, greatly increased or entirely new solvent power for red cells of species B.
The study of this specific hæmolytic action has since gone on hand in hand
with that of the bacteriolysins and other phenomena connected with immunity.
Bordet discovered that heating the hæmolytic serum to 55° Cent. for half an
hour destroyed its solvent action ; but that the addition of normal serum, itself
devoid of hæmolytic power, restored this lost property.
Since then there has grown up a large body of facts relating to the" immune
body," " sensibilisatrice " or amboceptor," the thermostable body in
hæmolytic sera and the " cytase," " alexine " or " complement " the thermolabile
substance, which in combination with the immune body or amboceptor forms the
complete and active hæmolysin.
In a literature so extensive as that relating to hæmolysis it is undesirable to
follow the increase in our knowledge step by step. But it is necessary, in order that
our own observations may be more easily followed, to give the chief facts known
regarding the action of hæmolysins. The principal fact, the existence of two
separate substances combining to form the active hæmolysin, has already been,
noted. According to Ehrlich's theory, which in its main propositions is now
generally accepted, a hæmolysin consisting of an amboceptor plus a suitable
complement can only act upon red cells with possess " side-chains " or " recep-
tors" which fit the " haptophore group " or cell combining affinity of the ambo
ceptor. We have thus to deal with three essential factors in hæmolysis by specific
hæmolysins.
145
Usually their cytophile affinity is stronger than their complementophile affi-
nity, for they can combine with cells in the cold at a temperature of 0° Cent. at
which temperature they will not combine with complement.
Amboceptors are as we have seen specific, they can enter into combination
with only one kind of cell, that is cells possessing like receptors. But ambo-
ceptor alone, although it may enter into combination, cannot effect any
definite change in the cell to which it becomes bound by its cytophile affinity
Its function then appears to be the linking up of a cell to complement or cytase'
in such a way that this latter body which has toxic or ferment-like properties,
can act and destroy the cell to which the amboceptor has united it.
We have seen therefore that amboceptors are not in themselves toxic, because
they possess no " zymotoxic group," and cells plus amboceptor remain apparently
undamaged and capable of performing all their normal functions.
We have also seen that amboceptor possesses such a strong affinity for cells
with suitable receptors that it will enter into combination with them at exceed-
ingly low temperatures.
Combination of red cells and amboceptor in vitro.
(1) To a suspension of red cells in .8 per cent. salt solution, specific hæmolytic
serum previously heated for ½ hour at 55° Cent. is added and the mixture is
allowed to stand for a time. The red cells remain apparently unaltered, but on
the addition of a little normal serum, which can be shown to have no action
upon similar red cells without treatment, rapid hæmolysis ensues.
(2) To a suspension of red cells, unheated specific serum, capable of producing
hæmolysis, is added and the mixture is maintained at a temperature of 0° Cent.
for a time. No hæmolysis occurs. The mixture is then centrifuged and the red
cells separated from the supernatant liquid. The red cells are apparently un-
altered, but, on the addition of a little normal serum, speedy hæmolysis takes
place. If the mixture of haemolytic serum with red cells has been made in suitable
amounts, the separated supernatant liquid will show, on further examination, that
all the amboceptor has combined and been removed with the red cells, but
that free complement still remains, its combination with the amboceptor having
been prevented by the low temperature, at which therefore no hæmolysis
occurred.
By a series of experiments such as these Ehrlich was able to show that red
cells possess the power of combining with far more specific amboceptor than is
sufficient to cause hæmolysis in the presence of complement. The amount taken up
227 H. D.
20
146
by them varies in different cases, but may be even one hundred times in excess
of that required in combination with complement for their complete destruction.
2. Combination in the living body.—In the absence of suitable complement
it is theoretically possible, though such a condition has not, so far as we are aware,
been described, for cells such as the red corpuscles to become laden with ambo-
ceptor in the body, such a combination producing no hæmolysis.
But a phenomenon of this nature has been recorded by Metchnikoff (87) in the
case of guinea pigs inoculated with guinea pig spermatozoa. As the result of this
inoculation, spermotoxin is formed in the blood serum ; and this is shown to be toxic
to the spermatozoa of the injected animal as well as to those of other guinea pigs.
Although living and active spermatozoa may be seen in the epididymis and testes
of the injected animal, they are no longer normal, for if placed in normal guinea
pig serum, in which healthy guinea pig spermatozoa will live for several hours,
they are immediately destroyed.
Thus although to all appearance normal, they have in reality become laden
with immune body (amboceptor) which exposes them to rapid destruction in the
presence of normal serum containing complement.
COMPLEMENT.
Complement is, as we have already seen, a constituent of normal serum. In
the presence of immune body (amboceptor) it can act upon various cells causing
bacteriolysis, hæmolysis, etc. A number of different conceptions as to its exact
nature have been put forward from time to time, some of which it will be necessary
to discuss in greater detail later, when we are considering the treatment of Black-
water Fever. But here we need only note that in its power of entering into com-
bination with amboceptor it displays far less specificity than does amboceptor
in combining with cells. Thus many different normal sera will provide complement
that will combine with one specific amboceptor.
Although complement can be demonstrated in most sera, its existence in the
plasma is a matter of discussion. Metchnikoff believes it to be derived from the
dissolution of leucocytes, and to be a constituent of living cells, not of the plasma.
Leaving the nature of these cells an open question, we may say that this view
seems to be generally admitted.
Absorption of complement.
Complement can be absorbed in vitro, either by specific combination with
amboceptor as in hæmolysis or bacteriolysis, or by the addition of a variety of
147
s ubs t a nc e s ; t hus by di ge s t i ng a s e r um wi t h ye a s t c e l l s ,i t s
c o m p l e m e n t m a y b e
entirely removed.
In specific absorption a fitting amboceptor must be present ; and use is made
of this reaction in testing for the presence of an amboceptor fitted to act against
any given organism. In certain diseases absence of complement in the serum
has been observed. Examples of this condition are syphilis and trypanosomiasis.
HÆMOLYSTS IN VITRO AND IN VIVO.
Hæmolysis in vitro has been the subject of much study, and has led to clear
conceptions regarding the mechanism of the reaction under artificial conditions ;
but the factors which regulate hæmolysis within the body are still very obscure.
There is often a notable want of relation betweer action in vitro and action
in vivo. Celli, Casagrandi and Craducci (77), while investigating experimental
Læmoglobinuria, found that a serum, possessing no action in vitro, may on
injection into an animal cause intense blood destruction hæmoglobinuria and
death. Lamb (72) notes that daboia venom is less hæmolytic i n v i t r o
t h a n c o b r a
venom, but more so in vivo.
At Major Lamb's suggestion we have tried in vivo the action of cobra-lecithid.
This substance has all the components of a complete hæmolysin (amboceptor
plus complement) and is in vitro strongly hæmolytic to rabbit's red cells.
But when injected into rabbits in doses of from .01 to .3 gramme it causes
no observable blood destruction and does not produce the raising in the isotonic
point of the corpuscles observed after the injection of daboia venom. Another
illustration of want of relation between action in vitro and in vivo is shown by
the injection into rabbits of a serum strongly hæmolytic in vitro. This serum we
produced by immunising a calf against rabbit's blood.
Injected into rabbits in doses of .15 c.c. it appeared to have no action. Possibly
the formation of anti-complements produced co-incidently with the amboceptor
during the process of immunising the calf with defibrinated rabbit's blood may
have prevented action in this case.
In the case of the cobra-lecithid it appears possible that its apparent want of
action in vivo may be due to its absorption by cells other than red cells possessing
perhaps greater affinity and thus switching off the hæmolysin and preventing
hmolysis.*
* Arrhenius states that " normal rabbit serum is itself a little antitoxic to cobra-leci hid "; a proper-
ty which it loses on heating for 30 minutes at 64° C.
(Immunochemistry by S. Arrhenius, page 213: New York, Macmillan Company, 1907.)