relation of the volume of the corpuscles to that of the plasma.
Mixtures of blood with physiological saline solution in various
proportions are made, in each the amount of nitrogen in the fluid which
is left after the corpuscles have settled is estimated. With the aid of
quantities so obtained they calculate mathematically the volume of the
serum and corpuscles respectively. Apart from the fact that a dilution
with salt solution is also here involved, this method is too complicated
and requires amounts of blood too large for clinical purposes. Th.
Pfeiffer has tried to introduce it clinically in suitable cases, but has
not so far succeeded in obtaining definite results. That, however, the
relations between the relative volume of the red corpuscles and quantity
of haemoglobin are by no means constant, is well shewn by conditions (for
example the acute anaemias) in which an "acute swelling" of the
individual red discs occurs (M. Herz), but without a corresponding
increase in haemoglobin. The same conclusion results from recent
observations of v. Limbeck, that in catarrhal jaundice a considerable
increase of volume of the red blood corpuscles comes to pass under the
influence of the salts of the bile acids.
As we have several times insisted, the quantity of haemoglobin affords
the most important measure of the severity of an anaemic condition. Those
methods which neither directly nor indirectly give an indication of the
amount of haemoglobin are only so far of interest that they possibly
afford an elucidation of the special pathogenesis of blood diseases in
particular cases. To these belong the ESTIMATION OF THE ALKALINITY OF
THE BLOOD, which in spite of extended observations has not yet obtained
importance in the pathology of the blood.
A value to which perhaps attention will be more directed than it has up
to the present time by clinicians is the RATE OF COAGULATION OF THE
BLOOD, for which comparative results may be obtained by Wright's handy
apparatus, the "Coagulometer." In certain conditions, particularly in
acute exanthemata, and in the various forms of the haemorrhagic
diathesis, the clotting time is distinctly increased, or indeed clotting
may remain in abeyance. Occasionally a distinct acceleration in the
clotting, compared with the normal, may be observed. Wright has further
ascertained in his excellent researches, that the clotting time can be
influenced by drugs: calcium chloride, carbonic acid raise, citric acid,
alcohol and increased respiration diminish the clotting power of the
blood.
Recently Hayem has repeatedly called attention to a condition, that is
probably closely connected with the coagulability of the blood. Although
coagulation has set in, the separation of the SERUM FROM THE CLOT occurs
only very slightly or not at all. Hayem asserts, that he has found such
blood in Purpura haemorrhagica, Anaemia perniciosa protopathica, malarial
cachexia: and some infectious diseases.
For such observations large amounts of blood are needed, which are
clinically not frequently available. Certain precautions must be
observed, as has been ascertained in the preparation of diphtheria
serum, so that the yield of serum may be the largest possible. Amongst
these that the blood should be received in longish vessels, which must
be especially carefully cleaned, and free from all traces of fat. If the
blood-clot does not spontaneously retract it must be freed from the side
of the glass with a flat instrument like a paper-knife without injuring
it. If no clot occurs in the cold, a result may perhaps follow at blood
temperature.
In spite however of all artifices and all care, it is here and there,
under pathological conditions, impossible to obtain even a trace of
serum from considerable amounts of blood. In a horse for example which
was immunised against diphtheria, and had before yielded an unusually
large quantity of serum, Ehrlich was able to obtain from 22 kg. of blood
scarcely 100 cc. serum, when the animal was bled on account of a tetanus
infection.
Perhaps a larger _role_ is to be allotted in the diseases of the blood
to these conditions. Hayem already turns the incomplete production of
serum to account, for distinguishing protopathic pernicious anaemia from
other severe anaemic conditions. A bad prognosis too may be made when for
example in cachetic states this phenomenon is to be observed.
A few methods still remain to be mentioned which test THE RESISTANCE OF
THE RED BLOOD CORPUSCLES to external injuries of various kinds.
Landois, Hamburger and v. Limbeck ascertain for instance the degree of
concentration of a salt solution, in which the red corpuscles are
preserved ("isotonic concentration," Hamburger) and those which cause an
exit of the haemoglobin from the stroma. The erythrocytes are the more
resistant, the weaker the concentration which leaves them still
uninjured.
Laker tests the red blood corpuscles as regards their resistance to the
electric discharge from a Leyden jar, and measures it by the number of
discharges up to which the blood in question remains uninjured.
Clinical observation has not yet gained much by these methods. So much
only is certain, that in certain diseases: anaemia, haemoglobinuria, and
after many intoxications, the resistance, as measured by the methods
above indicated, is considerably lowered.
FOOTNOTES:
[1] For the estimation of the numbers of white corpuscles, relatively to
the red, and of the different kinds relatively to each other, see the
section on the morphology.
[2] In Roy's method, mixtures of glycerine and water are used. By means
of a curved pipette, the drop of blood is brought into the fluid, and
its immediate motion observed. Lazarus Barlow has modified this method.
He employs mixtures of gum and water, and instead of several tubes, one
only; and into this the mixtures are introduced, those of higher
specific gravity being naturally at the bottom. The alternate layers are
coloured, and remain distinguishable for several hours.
[3] In conditions of shock experimentally produced, the specific gravity
of the total blood is increased, that of the plasma, however, is
diminished (Roy and Cobbett).
THE MORPHOLOGY OF THE BLOOD.
A. METHODS OF INVESTIGATION.
A glance at the history of the microscopy of the blood shews that it
falls into two periods. In the first, which is especially distinguished
by the work of Virchow and Max Schultze, a quantity of positive
knowledge was quickly won, and the different forms of anaemia were
recognised. But close upon this followed a standstill, which lasted for
some decades, the cause of which lay in the circumstance that observers
confined themselves to the examination of fresh blood. What in fact was
to be seen with the aid of this simple method, these distinguished
observers had quickly exhausted. That these methods were inadequate is
best shewn by the history of leucocytosis, which after the precedent of
Virchow was in general referred to an increased production on the part
of the lymphatic glands; and further by the imperfect distinction
between leucocytosis and incipient leukaemia, which was drawn almost
exclusively from purely numerical estimations. It was only after Ehrlich
had introduced the new methods of investigation by means of stained dry
preparations, that the histology of the blood received the impulse for
its second period.
We owe to them the exact distinction between the several kinds of white
blood corpuscles, a rational definition of leukaemia, polynuclear
leucocytosis, and the knowledge of the appearances of degeneration and
regeneration of the red blood corpuscles, and of their degeneration in
haemoglobinaemic conditions. The same process, then, has gone on in the
microscopy of the blood that we see in other branches of normal and
pathological histology: by advances in method, advances in knowledge
full of importance result. It is therefore little comprehensible, that
an author quite recently should recommend a reversion to the old
methods, and emphatically announce that he has managed to make a
diagnosis in all cases, with the examination of fresh blood. At the
present time, after the most important points have been cleared up by
new methods, in the large majority of cases, this is not an astonishing
achievement. For any difficult case (for instance the early recognition
of malignant lymphoma, certain rare forms of anaemia, etc.) as the
experienced know, the dry stained preparation is indispensable. The
object of examining the blood, is certainly not to make a rapid
diagnosis, but to investigate exactly the individual details of the
blood picture. To-day, we can only take the standpoint, that everything
that is to be seen in fresh specimens--apart from the quite unimportant
rouleaux formation, and the amoeboid movements--can be seen equally
well, and indeed much better in a stained preparation; and that there
are several important details which are only made visible in the latter,
and never in wet preparations.
As regards the purely technical side of the question, the examination of
stained dry specimens is far more convenient than that of fresh. For it
leaves us quite independent of time and place, we can keep the dried
blood with few precautions for months at a time, before proceeding to
further microscopic treatment; and the examination of the preparation
may last as long as required, and can be repeated at any time. On the
contrary, the examination of the wet preparation is only possible at the
bedside, and must be conducted within so short a time, on account of the
changeability of the blood, clotting, destruction of white corpuscles
and so forth, that a searching investigation cannot be undertaken. In
addition the preparation and staining of dried blood specimens is
amongst the simplest and most convenient of the methods of clinical
histology. In the interest of its wider dissemination, it will be
justifiable to describe it more in detail.
We must also mention here the use of the dry preparation in the
estimation of the important relation between the number of the red and
of the white corpuscles; and also of the relative numbers of different
kinds of white blood corpuscles.
For this purpose, faultless specimens, specially regularly spread, are
indispensable. Quadratic ocular diaphragms (Ehrlich-Zeiss) are
requisite, which form a series, so that the sides of the squares are as
1:2:3 ... :10, the fields therefore as 1:4:9 ... :100. The eye-piece
made by Leitz after Ehrlich's directions is more convenient, in which,
by a handy device, definite square fractions of the field can be
obtained. The enumeration is made as follows. The white blood corpuscles
are first counted in any desired field with the diaphragm no. 10, that
is with the area of 100. Without changing the field, the diaphragm 1,
which only leaves free a hundredth part of this area, is now put in, and
the red corpuscles are counted. The field is then changed at random, and
the red corpuscles counted in a portion of the area which represents the
hundredth of that of the white. About 100 such counts are to be made in
a specimen. The average of the red corpuscles is then multiplied by 100
and so placed in proportion with the sum of the white. If the white
corpuscles are very numerous, so that counting each one in a large field
is inconvenient, smaller sections of the eye-piece 81, 64, 49, etc. may
be taken.
The important estimation of the percentage relation of the various forms
of leucocytes is effected by the simple "typing" of several hundred
cells, a count which for the practised observer is completed in less
than a quarter of an hour.
[alpha] Preparation of the dry specimen.
To obtain unexceptionable preparations cover-slips of particular quality
are necessary. They should not be thicker than 0.08 to 0.10 mm., the
glass must not be brittle or faulty, and must in this thickness easily
allow of considerable bending, without breaking. Every unevenness of the
slip renders it useless for our purposes. The glasses must previously be
particularly carefully cleaned, and all fat removed. It is generally
sufficient to allow the slips to remain in ether for about half-an-hour,
not covering one another. Each one still wet with ether is then wiped
with soft, not coarse, linen rag or with tissue-paper. The slips now are
put into alcohol for a few minutes, are dried in the same manner as from
the ether, and are kept ready for use in a dust-tight watch-glass.
Bearing in mind, that these cover-slips are not cut out from a flat
piece but from the surface of a sphere, it is evident that only with
glasses thus prepared, can it be expected that a capillary space should
be formed between two of them, in which the blood spreads easily. For
with the smallest unevenness or brittleness of the glass it is an
impossibility for the one to fit every bend of the other. And it is only
then that the slips can be drawn away one from another, without using a
force which breaks them.
To avoid fresh soiling of the cover-slips, and above all the contact of
the blood with the moisture coming from the finger, the cover-glass is
held with forceps[4] to receive the blood. We recommend for the under
cover-glass a clamp forceps _a_, with broad, smooth blades; the ends may
be covered with leather or blotting-paper for a distance of about 1/2
in. For the other cover-slip a very light spring forceps _b_, with
smooth blades, sharp at the tips, is used, with which a cover-glass can
be easily picked up from a flat surface. The lower slip is now fixed by
one edge in the clamp forceps, and held ready in the left hand. The
right hand applies the upper glass with the forceps _b_ to the drop of
blood as it exudes from the puncture, and takes it up, without touching
the finger itself. The forceps _b_ is then quickly brought to _a_ and
the slip with the little drop of blood allowed to fall lightly on the
other. In glasses of the right quality the drop distributes itself
spontaneously in a completely regular capillary layer. With two fingers
of the right hand on the edge of the upper glass, it is now carefully
pulled from the lower, which remains fixed in the clamp, without
pressing or lifting. Frequently only one, the lower, shews a regular
layer, but occasionally both are available for examination. During the
desiccation in the air, generally complete in 10-30 seconds, the
preparations must naturally be protected from any dampness (for example
the breath of the patient).
The extent of surface which is covered depends on the size of the drop,
the smaller the latter, the smaller the surface over which it has to be
spread. Large drops are quite useless, for with them, the one
cover-glass swims on the other, instead of adhering to it.
Although a written description of these manipulations makes the method
seem rather intricate, yet but little practice is required to obtain an
easy and sure mastery over it. We have felt compelled to describe the
method minutely, since preparations so often come under our notice
which, although made by scientific men, who pursue haematological
investigations, are only to be described as technically completely
inadequate.
The specimens so obtained, after they are completely dried in the air,
should be kept between layers of filter-paper in well closed vessels
till further treatment. In important cases, preparations of which it is
desirable to keep for some considerable time, some of the specimens
should be kept from atmospheric influences by covering them with a
layer of paraffin. The paraffin must be removed by toluol before
proceeding further. The preparations must naturally be kept in the dark.
[beta]. Fixation of the dry specimen.
All methods of staining available for the blood require the fixing of
the proteids of the blood. A general formula cannot be given, since the
intensity of the fixation must be regulated in accordance with the kind
of stain that is chosen. Relatively slight degrees of hardening suffice
for staining in simple watery solutions, for example, in the triacid
fluid, and can be attained by a short, and not too intense action of
several reagents. For other methods, in which solutions that are
strongly acid or alkaline are employed, it is however necessary to fix
the structure much more strongly. But here, too, an excess as well as an
insufficiency must be guarded against. It is easy with the few staining
fluids that are in use to ascertain the optimum for each.
The following means of fixation are employed.
1. Dry Heat.
A simple plate of copper on a stand is used, under one end of which
burns a Bunsen flame. After some time a certain constancy in the
temperature of the plate is reached, the part nearest to the flame is
hottest, that farther away is cooler. By dropping water, toluol, xylol,
etc. on to it, one can fairly easily ascertain that point of the plate
which has reached the boiling temperature of the particular fluid.
Far more convenient is Victor Meyer's apparatus, used by chemists. This
consists of a copper boiler, modified for our purpose, with a roof of
thin copper-plate, perforated for the opening of the vapour tube. Small
quantities of toluol are allowed to boil for a few minutes in the
boiler, and the copper-plate soon reaches the temperature of 107 deg.-110 deg..
For the ordinary staining reagents (in watery fluids) it is enough to
place the air-dried preparation at about 110 deg. C. for one half to two
minutes. For differential staining mixtures, for instance the
eosin-aurantia-nigrosin mixture, a time of two hours is necessary, or
higher temperatures must be employed.
2. Chemical means.
_a._ To obtain a good triacid stain, the preparations may be hardened,
according to Nikiforoff, in a mixture of absolute alcohol and ether of
equal parts, for two hours. The beauty of specimens fixed by heat is
however not quite fully reached by this method.
_b._ Absolute alcohol fixes dried specimens in five minutes sufficiently
to stain them subsequently with Chenzinsky's fluid, or haematoxylin-eosin
solution. It is an advantage in many cases, especially when rapid
investigation is required, to boil the dried preparation in a test-tube
in absolute alcohol for one minute.
_c._ Formalin in 1% alcoholic solution was first used by Benario for
fixing blood preparations. The fixation is complete in one minute, and
the granulations can be demonstrated. Benario recommends this method of
fixing, especially for the haematoxylin-eosin staining.
These methods are described as the most suitable for
blood-investigation in general. For special purposes, for instance, the
demonstration of mitoses, blood platelets, etc., other hardening
reagents may be used with advantage: Sublimate, osmic acid, Flemming's
fluid, and so forth.
[gamma]. Staining of the dry specimen.
Staining methods may be classified according to the purpose to which
they are adapted.
We use first those which are suitable for a simple general view. For
this it is sufficient to use such solutions as stain haemoglobin and
nuclei simultaneously. (Haematoxylin-eosin, haematoxylin-orange).
Occasionally a stain is desirable which only brings out, but in a
characteristic manner, a special kind of cell, _e.g._ the eosinophils,
mast cells, or bacteria. Single staining is attained on the principle of
maximal decoloration. (Cp. E. Westphal.)
Finally, we have panoptic staining; that is, by methods which bring out,
as characteristically as possible, the greatest number of elements.
Although we must use high magnifications with these stains, we are
compensated by a knowledge of the blood condition that cannot be reached
in any other way. A double stain is generally insufficient, and at least
three different dyes are used.
Successive staining was formerly used for this purpose. But everyone who
has used this method knows how difficult it is to get constant results,
however careful one may be in the concentration and time of action of
the stain.
Simultaneous staining offers undoubted and important advantages. As
there is much obscurity with regard to the principle on which it rests
we may here shortly explain the theory of simultaneous staining.
We will begin with the simplest example: the use of picro-carmine, a
mixture of neutral ammonium carmine and ammonium picrate. In a tissue
rich in protoplasm, carmine alone stains diffusely, though the nuclei
are clearly brought out. But if we add an equally concentrated solution
of ammonium picrate, the staining gains extraordinarily in distinctness,
in as much as now certain parts are pure yellow, others pure red. The
best known example is the staining of muscle with picro-carmine, by
which the muscle substance appears pure yellow, the nuclei pure red. If,
however, instead of ammonium picrate we add another nitro dye which
contains more nitro-groups than picric acid, for example the ammonium
salt of hexa-nitro-diphenylamine, the carmine stain is completely
abolished, all parts stain in the pure aurantia colour. The explanation
of this phenomenon is obvious. Myosin has a greater affinity for
ammonium picrate than for the carmine salt, and therefore in a mixture
of the two combines with the yellow dye. Owing to this combination it is
not now in a condition to chemically fix even carmine. Further, the
nuclei have a great affinity for the carmine, and therefore stain pure
red in this process. If, however, nitro dyes be added to the carmine
solution, which have an affinity for all tissues, and also for the
nuclei, the sphere of action of the carmine becomes continually smaller,
and finally by the addition of the most powerful nitro body, the
hexa-nitro compound, is completely abolished. Connective tissue and bone
substance, however, behave differently with the picro-carmine mixture,
in as much as here the diffuse stain depends exclusively on the
concentration of the carmine, and is quite uninfluenced by the addition
of a chemical antidote. This staining can only be limited by dilution,
but not by the addition of opposed dyes. We must look upon the latter
kind of tissue stain not as a chemical combination, but as a mechanical
attraction of the stain on the part of the tissue. We may also say:
=chemical stains are to be recognised by the fact that they react to
chemical antidotes; mechanical stains to physical influences=; of course
always assuming, that purely neutral solutions are employed, and that
all additions, which alter the chemical relation of the tissues such as
alkalis and acids, or which raise or limit the affinity of the dye for
the tissues, are avoided. A further consequence of this view is, that
all successive double staining may be serviceably replaced by
simultaneous multiple staining, if the chemical nature of the staining
process is settled. In contradistinction, in all double stains, which
can only be effected by successive staining, mechanical factors are
concerned.
In the staining of the dry blood specimen, purely chemical staining
processes are concerned, and therefore the polychromatic combination
stain is possible in all cases.
The following combinations are possible for the blood:
1. Combined staining with acid dyes. The best known example is the
eosin-aurantia-nigrosin mixture, in which the haemoglobin takes on an
orange, the nuclei a black, and the acidophil granulations a red hue.
2. Mixtures of basic dyes. It is possible straight away to make mixtures
consisting of two basic dyes. As specially suitable we must mention
fuchsin, methyl green, methyl violet, methylene blue. On the other hand,
mixtures of three bases are fairly difficult to prepare, and the
quantitative relations of the constituents must be exactly observed. For
such mixtures, fuchsin, bismarck brown, chrome green, may be used.
3. Neutral mixtures. These have played an important part in general
histology, from the time that they were first introduced by Ehrlich into
the histology of the blood up to the present day; and deserve before all
others a full consideration.