Histology of the Blood, Normal and Pathological

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LINICAL METHODS OF INVE

acteristic of the an?mic condition; pallor of the skin, a diminution of the normal redness of the mucous membranes of the eyes, lips, mouth, and pharynx. From the pres

ptoms will bring into line much that is unconnected, and will perhaps omit what it should logically

fully to define its extent. For this purpose the symptoms above mentioned are litt

may be "general" and affect the whole organism; or "local" and limited to a particular r

the blood may be quite independent of the amount of blood, and must primarily express itself in a diminution of the physiologically important constituents. Hence we distinguish the f

of h?moglobin can be recognised; in by far the greater number of cases, if not in

h?matology bear directly or indirectly

take the latter as a measure of the total amount of blood, isolated vessels, visible to the naked eye, e.g. those of the sclerotic, may be observed. Most suitable is the ophthalmoscopic examination of the width of the vessels at the back of the eye. R?hlmann has shewn that in 60% of the cases of chronic an?mia, in which the skin and mucous membranes are very white, t

y of the blood. Anyone who has made frequent blood examinations will have observed that in this respect extraordinary variations occur. In some cases scarcely a

ty of blood in the body has always been recognised as important, and its solution would constitute a real advance. The methods which have so far been proposed for clinical purposes originate from Tarchanoff. He suggested that

practicable in special cases and is open to several theoretical errors. First, it depends upon the relative number of red blood corpuscles in the blood; inasmuch as the transfusion of normal blood into normal blood, for example, would produce no alteration in the count. This consideration is enough to shew that this proceeding can only be used in special cases. It has indeed been found th

per cubic millimetre of blood. The convenience of the counting apparatus, and the apparently absolu

methods of their use are known. A number of fluids are used to dilute the blood, which on the whole fulfil the requirements of preserving the form and colour of the re

tion. Hydrarg

chlo

erin

stillat

ion. Hydrarg.

sulp

chlo

stillat

diluting fluid which destroys the red blood corpuscles, but which brings out the nuclei of the white corpuscles, so that the latter are more easily recognised. F

frequently confirmed observations of R. Thoma and I. F. Lyon, only a small error. In a count of 2

pplication of these methods, which in other

ple, in a one-sided paralytic, the capillary blood is different on the two sides; and congestion, cold, and so forth raise the number of red blood corpuscles. Hence, for purposes of enumeration, the rule is to take blood only from those parts of the body which are free from

e of children) the big toe. For the puncture pointed needles or specially constructed instruments, open or shielded lancets, are unnecessary: we recommend a fine steel pen, of which one ni

o be quite unsurveyable. According to the new and complete compilation of Reinert and v.

e

Minimu

4,000,00

me

Minimu

4,500,00

the number of red corpuscles only in so far that in the newly-born, polycyth?mia (up to 8-1/2 millions during the first days of life) is observed (E. Schiff). After the first occasion on which food is taken a decrease can be observed, and gradually (though by stages) the normal figure is reached

uscles is to be ascribed to the taking in of water, and is so insignificant, that th

, pregnancy, lactation, do not alter the number of blood corpuscles to any

els, locally diminish the number of red blood corpuscles; excitation of the vasodilators brings about the opposite effect. Hence it follows, that the normal variations of the number contain

chr?der, Miescher, Kündig and others, shew, the number of red blood corpuscles in a healthy man, with the normal average of 5,000,000 per mm.3, begins to rise immediately after reaching a height considerably above the sea-level. With a rise proceeding by stages, a new average figure is reached in 10 to 14 days, considerably larger than the old one, and indeed the g

e to which the number of blood corpuscles may vary a

Height above sea

G?rbersdorf 561

e Reiboldsgrün

sa 1800 "

erillas 439

e morphological phenomena produced during acclimatisation to high altitudes, has shewn, that in the increase of the number of red corpuscles two mutually independent and distinct processes are to be distinguished. He observed that, although the number of red corpuscles was raised so soon as a few hours after arrival at Reiboldsgrün, numerous poikilocytes and microcytes make their appearance at the same time. The initial increase is therefore to be explained by budding and division of the red corpuscles already present in the circulating blood. K?ppe sees in this process, borrowing Ehrlich's conception of poikilocytosis, a physiological adaptation to the lower atmospheric pressure, and the resulting greater difficulty of oxygen absorption. The impediment to the function of the h?moglobin is to a certain e

on of the blood, due to the greater loss of water from the body at these altitudes. The blood of laboratory animals which Grawitz allowed to live in correspondingly rarefied air underwent similar changes. Von Limbeck, as well as

th of the vessels and the rate of flow in them. If one reflects how multifarious are the merely physiological influences at the bottom of which these two factors lie, one will not interpret alterations in the number of the red corpuscles without bearing them in mind. In residence at high altitudes various factors bring about alterations in the w

thors, finds its solution in the fact that the causes of altered distribution of the blood, and of loss of water, play a large p

that after long residence in elevated districts the number of red blood corpuscl

en tested. Eykmann as well as Glogner found no deviation from the normal, although the almost constant pallor of the European in the tropic

rally speaking all the red cells are of the same size and contain the same amount of h?moglobin. In the former the red corpuscles, as we shall shew later, differ c

normal blood corpuscles. Hence the necessity of closely correlating the result of the count of red blood corpuscles with the h?moglobinom

uscles individually. This is effected by direct measurement with the ocular micrometer; and can be performed on wet (see below

n this difference as follows. In the thick layers the red discs float in plasma before drying, whilst in the thinner parts they are fastened to the glass by a capillary layer. Desiccation occurs here nearly instantaneously, and star

umann and others, 8.5 μ for men and women (max. 9.0 μ. min. 6.5 μ.) In an?mic blood the differences between the individual elements become greater, so that to obtain the average value, the max

the decisive measure of the degree of the an?mia. A number of clinical methods are in use for this estimation; first direct, such as the colorimetric estimation of the amount of h?moglo

to distribute itself in a thin layer. In this manner one can recognise the difference between the colour of an?mic and of healthy blood more clearly than in the drop as it comes from the finger prick. After a few trials one can in this way draw conclusions as to the degree of the existing an?mia. Could this simple method which is so convenient,

colour can be calculated. As a normal solution Langemeister uses a glycerine solution of meth?moglobin prepared from pig's blood. To our knowledge this method has not yet been applied clinically. Its introduction would be valuable, for in practice we must at present be content with methods that are less exact, in which coloured glass or a stable coloured solution serves as a measure for the depth of colour of the blood. There are a number of instruments of this kind, of which the "h?mometer" of Fleischl, and amongst others, the "h?moglobinometer" of Gowers, distinguished by its low price, are specially used for clinical purposes. Both instruments give the percentage of the h?moglobin of normal blood whic

led a "ferrometer;" which renders possible an accurate valuation of the iron in small amounts of blood. However for pathological cases this method of h?moglobin estimation from the iron present is not to be recommended. For if one tests the blood of an an?mic patient under the microscope for iron one finds the iron reaction in numerous red blood corpuscles. This means the presence of iron which is not a normal constituent of h?moglobin. Other iron may be contained

example, amongst other things, he found the iron in two cases of mild, and one of severe chlorosis quite normal. He concludes that chlorosis, and other an?mias, shew no diminution, but even a relative increase of h?moglobin: but that other proteids of the blood on the contrary are reduced. These difficult iron estimations stand out very sharply

s have come into use which require only a small quantity of material, and do not appear to be too complicated for practical clinical purposes. One of these has been worked out by R. Schmaltz, in which small amounts of blood are exactly weighed in capillary glass tubes (the capillary py

32-on the average 1030. From which it at once follows that the red corpuscles must be the chief cause of the great weight of the blood. If their number diminishes, or their number remaining constant, they lose in h?moglobin, or in volume, the specific gravity

gravity and the amount of h?moglobin is much closer than between the specific gravity and the

Quantity o

chl's

1035

1038

1040

1045

1048

1050

1053

1055

1057

1060

differences of 10% h?moglobin (Fleischl) correspond in general to differences of 4.46 per thousand in the specific gravity (Hammerschlag's method). Nevertheless with the same amount of h?moglobin, differences up to 13.5 per thous

ence of the stroma of the red discs on the specific gravity of the blood will then be recognisable. Diabella calculates, that with

of h?moglobin of a blood. It is only in cases of nephritis and in circulatory disturbances, and in leuk?mia, t

of fluid do not exceed 0.003 (Schmaltz). From what has been said, it follows that all variations must correspond wi

total blood, they have carried out that of one at least of its constituents, either of the corpuscles or of the serum. The red blood corpuscles have consistently shewn themselves as almost exclusively concerned with variatio

preserves its physiological constitution, or undergoes but relatively slight variations in consistence. Considerable diminutions in the specific gravity of the serum are much less frequently observed in primary blood diseases, than in chron

ently necessary in a scientific investigation always to give the specific grav

so far mentioned, and like them can be carried out with the small amounts of blood obtainable at the bedside without difficulty. Small quantities of blood are received in weighed glass vessels: which are then weighed, dried at 65°-70° C. for 24 hours and then weighe

lood, that is as far as possible unaltered. The older methods do not fulfil this requirement; since they recommend either defibrination of the blood (quite impossible with the quantities of blood which are generally clinically available); or keeping it fluid by the additi

is prevented by rendering the walls absolutely smooth by the application of cod-liver oil. K?ppe has slightly varied this method; he fills his handily constructed pipette, very carefully cleaned, with cedar wood oil, and sucks up the blood, as it comes from the fingerprick into the filled pipette. The blood displaces the oil, and as it only comes into contact with perfectly smooth surfaces, it remains fluid. By means of a centrifugal machine, of which he has construc

e, which has really advanced clinical pathology. The results of K?ppe-not as yet very n

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 h?moglobin are by

ndirectly give an indication of the amount of h?moglobin are only so far of interest that they possibly afford an elucidation of the special pathogenesis of blood diseases in part

tions, particularly in acute exanthemata, and in the various forms of the h?morrhagic diathesis, the clotting time is distinctly increased, or indeed clotting may remain in abeyance. Occasionally a distinct acceleration in the clotting, compared with the nor

. Although coagulation has set in, the separation of the serum from the clot occurs only very slightly or not at all. Hayem asserts,

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 doe

rom considerable amounts of blood. In a horse for example which was immunised against diphtheria, and had before yielded an unusually large quant

ncomplete production of serum to account, for distinguishing protopathic pernicious an?mia from other severe an?mic

ich test the resistance of the red blood corp

red corpuscles are preserved ("isotonic concentration," Hamburger) and those which cause an exit of the h?moglobin fro

the electric discharge from a Leyden jar, and measures it by the num

in, that in certain diseases: an?mia, h?moglobinuria, and after many intoxications,

TNO

s, relatively to the red, and of the different kinds rel

ion 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

specific gravity of the total blood is increased, that

OLOGY OF

S OF INVE

y 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

prehensible, 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 an?mia, etc.) as the experienced know, the dry stained preparati

d 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 corpu

he important relation between the number of the red and of the white corpuscles

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 por

ucocytes is effected by the simple "typing" of several hundred cells, a count

on of the d

icient 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 fr

asily 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 i

latter, the smaller the surface over which it has to be spread. Large drops are quite us

n easy and sure mastery over it. We have felt compelled to describe the method minutely, since preparations so often come under our notice wh

t. 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

n of the d

ht 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 al

eans of fixatio

ry H

erature of the plate is reached, the part nearest to the flame is hottest, that farther away is cooler. By dropping water, toluol, xylol,

purpose, with a roof of thin copper-plate, perforated for the opening of the vapour tube. Small quantities of toluol

at about 110° C. for one half to two minutes. For differential staining mixtures, for instance the eos

mical

iforoff, in a mixture of absolute alcohol and ether of equal parts, for two hours. Th

enzinsky's fluid, or h?matoxylin-eosin solution. It is an advantage in many cases, especially when rapid

ions. The fixation is complete in one minute, and the granulations can be demonstrated.

ial purposes, for instance, the demonstration of mitoses, blood platelets, etc., other harden

g of the d

sified according to the purp

or this it is sufficient to use such solutions as stain h?moglobin

anner, a special kind of cell, e.g. the eosinophils, mast cells, or bacteria. Sing

of elements. Although we must use high magnifications with these stains, we are compensated by a knowledge of the blood conditio

s used this method knows how difficult it is to get constant results, howev

there is much obscurity with regard to the principle on which it r

onium 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

al staining processes are concerned, and therefore the p

inations are possi

osin-aurantia-nigrosin mixture, in which the h?moglobin takes on an

n fuchsin, methyl green, methyl violet, methylene blue. On the other hand, mixtures of three bases are fairly difficult to prepare, and the

rom the time that they were first introduced by Ehrlich into the histology of th

dye, and so the preparation of solutions of the required strength, readily kept, was made possible. Among the basic dyes which are suitable for this purpose are those particularly which contain the ammonium group, especially methyl green, methylene blue, amethyst violet[5] (tetraethylsafraninchloride), and to a certain extent pyronin and rhodamin also. In contradistinction to these, the members of the triphenylmethan series, such as fuchsin, methyl violet, bismarck brown, phosphin, indazine, are in general less suited for the purpose, with the

he further addition of the orange. No more orange should be added than is necessary for complete solution. This is the type of a simple neutral staining fluid. Chemically the above-ment

ining, which can only be attained by mixing together two simple neutral mixtures, each consisting of two components. A chemical decompositio

ures of 1 acid a

-amethyst-m

yronin-me

ronin-meth

d 1 base, in particular the mixtur

id fuchsin-m

n-acid fuchsin

ons with methylene blue, and am

hat they pick out definite substances, which would not be demonstrate

f the basic dye; acidophil elements in that of one of the two acid dyes; whilst those portions of tissue which from their constitu

recipitates are very easily produced, which render the preparation quite useless. This danger is particularly great in freshly prepared solutions. In solutions, such as Chenzinsky's, which can be kept active for a longer time, it is less. Hence fresh solutions stain far more intensely and more variously th

nd iodine-eosine solution described below (s

solution with e

(cryst

oxyli

hol

. d

ine aa

acetic a

in

eating, stain in from half-an-hour to two hours. The h?moglobin and eosinophil granules are red

phil, were frequently observed in the white blood corpuscles, particularly in the region of the nucleus. They were not recognised, even by practised observers (e.g. Neusser) as artificial,

irst prepared, and cleared by standing for some co

. Orange-

cid fuchsi

. Aqu.

c. Al

c. Meth

c. Al

c. Gl

reen onwards the fluid is thoroughly shaken. The solution can be used at once, and keeps indefinitely. The staining of t

ion copper red, the neutrophil violet. The mast cells stand out by "negative sta

ded for good general preparations; it is indispensable in all c

ted, watery methyl-green solutio

the nuclei green, the red blood corpuscles red, the protoplasm of the leucocytes fuchsin c

ue mixtures, for exam

ry methylene blue

ution in 70% a

est. 4

utes in absolute alcohol. The staining takes 6-24 hours (in air-tight watch-glasses) at blood temperature. The nuclei and the

lei, the baso and eosinophil granulations, and it is used by

e are mixed, and used at once, see page 41. Time of staining 1, at most 2 minutes. The staining is characteristic only in preparatio

in methyl alcohol of the precipitate fo

uble eosine, yellow

ethylene blue

ol (Merck). The stain may be obtained from R. Kanthack, 18, Berners Street, London, ready for use. It is

the dried blood preparation is employed directly, without previous fixation: 1. the recognition

on of glycog

preparation into a drop of thick cleared iodine-indiarubber solution under the mic

n a few minutes it takes on a dark brown colour, and is then mounted in a saturated l?vulose solution, whose index o

white blood corpuscles, or extracellular, are characterised by a beautiful mahogany brown colour. The second modification of this method is specially to be recommended on account of the strong clearing action of the l?vulose syrup. In using the iodine-indiarubber solution a small qu

t of the distribution

id, which is precipitated by acidifying solutions of the salt, is very sparingly soluble in water. It is, on the contrary, very easily soluble in organic solvents, so that by shaking, it completely passes over into an etherial solut

ed their shape completely. The plasma shews a distinct red colour, whilst the red corpuscles have taken up no colour. The protoplasm of the white corpuscles is red, the nuclei appear as spaces, because unstained (negative nuclear staining). The disintegrated corpuscles and the fibrin which is produced, shew an intense red stain. These stains are peculiarly instructive, and shew many details which are not visible in other methods. The study of these preparations is really of the highest value, since they allow the products of manipulation of the dry preparation and every error of production to stand out in

TNO

Berlin, supply these af

and Soda manufact

Anilin-dye Company of Berlin have prepar

pus always shews a considerable glycogen reaction of the pus cells. It is found, moreover, in ce

THOLOGICAL HISTO

stain, so that for a practised observer the depth of stain gives a certain indication of the h?moglobin equivalent of each cell, and a better one than the natural colour of the h?moglobin in the fresh specimen. Corpuscles poor in h?moglobin are easily recognised by their fainter staining, especially by the still greater brightness of the central zone. When somewhat more marked, they present appearances which from the isolated staining of the peri

cially in posth?morrhagic, secondary and chlorotic cases. On the contrary, as Laache first obs

p and replaced by new. Every drop of blood contains, side by side, the most various stages of life of fully formed erythrocytes. For this reason influences which affect the blood-provided their intensity does not ex

?mic constitution of the blood as such, the effect of which in this

e observe characteristic c

olychromatophi

our. For instance, the red corpuscles are pure red in preparations of normal blood, stained with h?matoxylin-eosine mixture. But in preparations of blood of a chromic an?mia stained with the same solution, in which possibly all stages of the degeneration in question are present, one s

oplasm. The latter takes up, as is the case in coagulation necrosis, the proteids of the blood, and acquires thereby the power of combining with nuclear stains. At the same time the discoplasm loses its

others. The polychromatophil discs are not, they say, dying forms, but on the contrary represent young individuals. The circumstance,

hes to this subject, the grounds for regarding this cha

By the notching of their margins they appear to eyes practised in the judgment of morpholog

ce in large numbers in the blood can be produced. That is, precisely in condition

hin so short a time as the first 24 hours. Whilst in our observations, which are very numerous upon this point, embracing several

egeneration, are as a rule free from polychromatophil degeneration, gave the key for the interpretation of this appearance. And similarly for the nucleated red blood corpuscles of lower animals. Askanazy asserts that the nucleated red blood corpuscles of the bone-marrow, which he was able to investigate in a case of empyema, shew, immediately after the resection of the ribs, compl

ration. To explain the presence of erythroblasts which have undergone these changes we must suppose that in severe injuries to the life of the blood these elements

in the red blood corpuscles of

us an?mia, as Laache first observed, and as has since been generally confirmed. On the contrary in all other severe or moderate an?mic conditions, the red corpuscles shew a diminution in volume, and in their amount of h

d in the early days of the microscopic investigation of the blood, a special significance for the severe an?mias. They are however nothing but contraction forms of the poikilocytes, as the crenated are of the normal corpuscles. Consequently microcytes are but seldom found in dried specimens, whilst in wet preparations they are easily seen after some time. It is further of importance to know, that in fresh blood the poikilocytes

duced in any blood, forces one to the assumption that the poikilocytes are products of a fragmentation of the red blood corpuscles ("schistocytes," Ehrlich). And correspondingly the smallest fragments s

them complete functional power, and regard their production as a purposeful reaction to the diminished number of corpuscles. For by