Part 31

_Variations in the Blood of different Animals._--If we contrast the blood of different animals of the vertebrate class we find striking differences both in microscopic appearances and in chemical properties. In the first place, the corpuscles vary in amount and in kind. Thus, whilst in a mammal the corpuscles form 40 to 50% of the total volume of the blood, in the lower vertebrates the volume is much less, e.g. in frogs as low as 25% and in fishes even lower. The deficiency is chiefly in the red corpuscles, the ratio of white to red increasing as we examine the blood from animals lower in the scale. The corpuscles themselves are also found to vary, especially the red ones. In the mammal they are biconcave disks with bevelled edges, they do not contain a nucleus so that they are not cells. In the bird they are larger, ellipsoidal in shape and have a large nucleus in the centre of the cell. In reptiles and amphibia the red corpuscles are also nucleated, but the _stroma_ portion containing the haemoglobin is arranged in a thickened annular part encircling the nucleus. When seen from the flat they are oval in section. In fishes the corpuscles show very much the same structure. A further very significant difference to be observed between the bloods of different vertebrates is in the amount of haemoglobin they contain; thus in the lower classes, fishes and amphibia, not only is the number of red corpuscles small but the amount of haemoglobin each corpuscle contains is relatively low. The concentration of the haemoglobin in the corpuscles attains its maximum in the mammal and the bird. Since the haemoglobin is practically the same from whatever animal it is obtained and can only combine with the same amount of oxygen, the oxygen-capacity of the blood of any vertebrate is in direct proportion to the amount of haemoglobin it contains. Therefore we see that as we ascend the scale in the vertebrate series the oxygen-carrying capacity of the blood rises. This increase was a natural preliminary condition for the progress of evolution. In order that a more active animal might be developed the main essential was that the chemical processes of the cell should be carried out more rapidly, and as these processes are fundamentally oxidative, increased activity entails an increased rate of supply of oxygen. This latter has been brought about in the animal kingdom in two ways, first by an increase in the concentration of the haemoglobin of the blood effected by an increase both in the number of corpuscles and in the amount of haemoglobin contained in each, and secondly by an increase in the rate at which the blood has been made to pass through the tissues. In the lower vertebrates the blood pressure is low and the haemoglobin content of the blood is low, consequently both rate of blood-flow and oxygen-content are low. In contrast with this, in higher vertebrates the blood pressure is high and the haemoglobin content of the blood is high, consequently both rate of blood-flow and oxygen-content are high. We must associate with this important step in evolution the means employed for the more rapid absorption of oxygen and for its increased rate of discharge to the tissues, the most important features of which are a diminution in the size of the corpuscle and the attainment of its peculiar shape, both resulting in the production of a relatively enormous corpuscular surface in a unit volume of blood.

Variations are also found in the white corpuscles as well as in the red, but these differences are not so striking and lie chiefly in unimportant details of structure of individual cells. Enormous variations are to be found in different species of mammals, but the cells generally conform to the types of secreting cells or phagocytes.

The platelets also differ in the different species. In the frog, for instance, many are spindle-shaped and contain a nucleus-like structure. Birds' blood is stated to contain no platelets. The variations in number of these bodies have not been satisfactorily ascertained on account of the difficulties involved in any attempt to preserve them and to render them visible under the microscope.

Differences are also found in the chemical composition of the plasma. The chief variation is in the amount of protein present, which attains its maximum concentration in birds and mammals, while in reptiles, amphibia and fishes it is much less. The bloods of the latter two classes are much more watery than that of the mammal. Moreover, it has been proved that there are specific differences in the chemical nature of the various proteins present even between different varieties of mammals. Thus the ratio of the globulin fraction to the albumin fraction may vary considerably, and again, one or other of the proteins may be quite specific for the animal from which it is derived.

_Clotting._--If a sample of blood be withdrawn from an animal, within a short time it undergoes a series of changes and becomes converted into a stiff jelly. It is said to _clot_. If the process is watched it is seen to start first from the surfaces where it is in contact with any foreign body; thence it extends through the blood until the whole mass sets solid. A short time elapses before this process commences--a time dependent upon two chief conditions, viz. the temperature at which the blood is kept and the extent of foreign surface with which it is brought into contact. Thus in a mammal the blood clots most quickly at a temperature a little above body temperature, while if the blood be cooled quickly the clotting is considerably delayed and in the case of some animals altogether prevented. For example, human blood kept at body temperature clots in three minutes, while if allowed to cool to room temperature the first sign of clotting may not make its appearance until eight minutes after its removal from the body. The process of clotting is also considerably accelerated by making the blood flow in a thin stream over a wide surface. The full completion of the process occupies some time if the blood be kept quiet, but ultimately the whole mass of the blood becomes converted into a solid. At this stage the containing vessel may be inverted without any drop of fluid escaping. A short time after this stage has been reached drops of a yellow fluid appear upon the surface and, increasing in size and number, run together to form a layer of fluid separated from the clot. This fluid is termed _serum_; its appearance is due to the contraction of the clot, which thus squeezes out the fluid from between its solid constituents. Contraction continues for about twenty-four hours, at the end of which time a large quantity (one-third or more of the total volume) of serum may have been separated. The clot contracts uniformly, thus preserving throughout the same general shape as that of the vessel in which the blood has been collected. Finally the clot swims freely in the serum which it has expressed.

The cause of the clot formation has been found to be the precipitation of a solid from the liquid plasma of the blood. This solid is in the form of very minute threads and hence is termed _fibrin_. The threads traverse the mass of blood in every possible direction, interlacing and thus confining in their meshes all the solid elements of the blood. Soon after their deposition they begin to contract, and as the meshwork they form is very minute they carry with them all the corpuscles of the blood. These with the fibrin form the shrunken clot.

If the rate at which blood clots be retarded either by cooling or by some other process the corpuscles may have time to settle, partially or completely, in which case distinct layers may form. The lowermost of these contains chiefly the red corpuscles, the second layer may be grey owing to the high percentage of leucocytes present, while a third, marked by opalescence only, may be very rich in platelets. Above these a clear layer of fluid may be found. This is _plasma_. The formation of these layers depends solely upon the rate of sedimentation of these elements, the rate depending partly upon differences in specific gravity, and partly upon the tendency the corpuscles have to run into clumps. Horse's blood offers one of the best instances of the clumping of red corpuscles, and in this animal sedimentation of the red corpuscles is most rapid.

If now such a sedimented blood is allowed to clot the process is found to start in the middle two layers, i.e. in those containing the white corpuscles and platelets. From these layers it spreads through the rest of the liquid, being most retarded, however, in the red corpuscle layer, and particularly so if the sedimentation has been very complete. Not only does the clotting process start from the layers containing the leucocytes and platelets, but in them it also proceeds more quickly. These observations clearly indicate that the clotting process is initiated by some change starting from these elements.

The object of the clotting of the blood is quite clear. It is to prevent, as far as possible, any loss of blood when there is an injury to an animal's vessels. The shed blood becomes converted into a solid, and this, extending into the interior of the ruptured vessel, forms a plug and thus arrests the bleeding. It is found that clotting is especially accelerated whenever the blood touches a foreign tissue, for instance, the outer layers of a torn blood-vessel wall, muscle tissue, &c., i.e. in exactly those conditions in which rapid clotting becomes of the greatest importance. Yet another very pregnant fact in connexion with clotting is that if an animal be bled rapidly and the blood collected in successive samples it is found that those collected last clot most quickly. Hence the more excessive the haemorrhage in any case, the greater becomes the onset of the natural cure for the bleeding, viz. clotting.

When we begin to inquire into the nature of clotting we have to determine in the first place whence the fibrin is derived. It has long been known that two chemical substances at least are requisite for its production. Thus certain fluids are known, e.g. some samples of hydrocele or pericardial fluid, which will not clot spontaneously, but will clot rapidly when a small quantity of serum or of an old blood-clot is added to it. The constituent substance which is present in the first-named fluids is known as fibrinogen, and that present in the serum or the clot is known as fibrin-ferment or _thrombin_.

Fibrinogen is present in living blood dissolved in the plasma; it is also present in such fluids as hydrocele or pericardial effusions, which, though capable of clotting, do not clot spontaneously. Thrombin, on the other hand, does not exist in living blood, but only makes its appearance there after blood is shed. It is not yet certain what is the nature of the final reaction between fibrinogen and thrombin. The possibilities are, that thrombin may act--(1) by acting upon fibrinogen, which it in some way converts into fibrin, (2) by uniting with fibrinogen to form fibrin, or (3) by yielding part of itself to the fibrinogen which thus becomes converted into fibrin. The experimental study of the rate of fibrin formation, when different strengths of thrombin solutions are allowed to act upon a fibrinogen solution, leads us to the probable conclusion that the first of these three possibilities is the correct one, and that thrombin therefore exerts a true ferment action upon fibrinogen. It is known that in the reaction, in addition to the formation of fibrin, yet another protein makes its appearance. This is known as fibrinoglobulin, and apparently it arises from the fibrinogen, so that the change would be one of cleavage into fibrin and fibrinoglobulin. It is very noteworthy that although the amount of fibrin formed during the clotting appears very bulky, yet the actual weight is extremely small, not more than 0.4 grms. from 100 cc. of blood.

Having ascertained that the clotting is due to the action of thrombin upon fibrinogen, we now see that the next step to be explained is the origin of thrombin. It has been shown that the final step in its formation consists in the combination of another substance, termed prothrombin, with calcium. Any soluble calcium salt is found to be effective in this respect, and conversely the removal of soluble calcium (e.g. by sodium oxalate) will prevent the formation of thrombin and therefore of clotting.

In the next place it can be proved that prothrombin does not exist as such in circulating blood, so that the problem becomes an inquiry as to the origin of prothrombin. Experiment has shown that in its turn prothrombin arises from yet another precursor, which is named thrombogen, and that thrombogen also is not to be found in circulating blood but only makes its appearance after the blood is shed. The conversion of thrombogen into prothrombin has been proved to be due to the action of a second ferment which has been named thrombokinase, and this latter is again absent from living blood. Hence the question arises, whence are derived thrombogen and thrombokinase? In the study of this question it has been found that if the blood of birds be collected direct from an artery through a perfectly clean cannula into a clean and dust-free glass vessel, it does not clot spontaneously. The plasma collected from such blood is found to contain thrombogen but no thrombokinase. A somewhat similar plasma may be prepared from a mammal's blood by collecting samples of blood from an artery into vessels which have been thoroughly coated with paraffin, though in this instance thrombogen may be absent as well as thrombokinase. If plasma containing thrombogen but no thrombokinase be treated with a saline extract of any tissues it will soon clot. The saline extract contains thrombokinase. This ferment can therefore be derived from most tissues, including also the white blood corpuscles and the platelets. Thrombogen is produced from the leucocytes, but it is not yet certain whether it is also formed from the platelets. The discovery of the origin of the thrombokinase from tissue cells explains a fact that has long been known, namely, that if in collecting blood, it is allowed to flow over cut tissues, clotting is most markedly accelerated. The fact that birds' blood if very carefully collected will not clot spontaneously tends to prove that thrombokinase is not derived from the leucocytes, and makes probable its origin from the platelets, for it is known that birds' blood apparently does not contain platelets, at any rate in the form in which they are found in mammalian blood. When examining the general properties of platelets, attention was drawn to the remarkably rapid manner in which they undergo change on coming into contact with a foreign surface. It is apparently the actual contact which initiates these changes, changes which are fundamentally chemical in character, resulting in the production of thrombokinase and possibly also of thrombogen.

Thus as our knowledge at present stands the following statement gives a recapitulated account of the changes which constitute the many phases of clotting. When blood escapes from a blood-vessel it comes into contact with a foreign surface, either a tissue or the damaged walls of the cut vessel. Very speedily this contact results in the discharge of thrombogen and thrombokinase, the former from the white blood corpuscles and also possibly from the platelets, the latter from the platelets or from the tissue with which the blood comes in contact. The interaction of these two bodies next results in the formation of prothrombin, which, combining with the calcium of any soluble lime salt present, forms thrombin or fibrin-ferment. The last step in the change is the action of thrombin upon fibrinogen to form fibrin, and the clot is complete.

The intrinsic value to the animal of these changes is quite plain. The power of clotting and thus stopping haemorrhage is of essential importance, and yet this clotting must not occur within the living blood-vessels, or it would speedily result in death. That the tissues should be able to accelerate the process is of very obvious value. That the inner lining of the blood-vessels does not act as a foreign tissue is possibly due to the extreme smoothness of their surface.

Further, an animal must always be exposed to a possible danger in the absorption of some thrombin from a mass of clotted blood still retained within the body, and we know that if a quantity of active ferment be injected into the blood-stream intravascular clotting does result. Under all usual conditions this is obviated, the protective mechanism being of a twofold character. First, it is found that thrombin becomes converted very quickly into an inactive modification. Serum, for instance, very quickly loses its power of inducing clotting in fibrinogen solutions. Secondly, the body has been found to possess the power of making a substance, antithrombin, which can combine with thrombin forming a substance which is quite inactive as far as clotting is concerned. Finally, there is evidence that normal blood contains a small quantity of this substance, antithrombin, and that under certain conditions the amount present may be enormously increased. (T. G. Br.)

_Pathology of the Blood._

The changes in the blood in disease are probably as numerous and varied as the diseases which attack the body, for the blood is not only the medium of respiration, but also of nutrition, of defence against organisms and of many other functions, none of which can be affected without corresponding alterations occurring in the circulating fluid. The immense majority of these changes are, however, so subtle that they escape detection by our present methods. But in certain directions, notably in regard to the relations with micro-organisms, changes in the blood-plasma can be made out, though they are not associated in all cases with changes in the formed elements which float in it, nor with any obvious microscopical or chemical alterations.

Immunity.

The phenomena of immunity to the attacks of bacteria or their toxins, of agglutinative action, of opsonic action, of the precipitin tests, and of haemolysis, are all largely dependent on the inherent or acquired characters of the blood serum. It is a commonplace that different people vary in their susceptibility to the attacks of different organisms, and different species of animals also vary greatly. This "natural immunity" is due partly to the power possessed by the leucocytes or white blood corpuscles of taking into their bodies and digesting or holding in an inert state organisms which reach the blood--phagocytosis,--partly to certain bodies in the blood serum which have a bactericidal action, or whose presence enables the phagocytes to deal more easily with the organisms. This natural immunity can be heightened when it exists, or an artificial immunity can be produced in various ways. Doses of organisms or their toxins can be injected on one or several occasions, and provided that the lethal dose be not reached, in most cases an increased power of resistance is produced. The organisms may be injected alive in a virulent condition, or with their virulence lessened by heat or cold, by antiseptics, by cultivation in the presence of oxygen, or by passage through other animals, or they may first be killed, or their toxins alone injected. The method chosen in each case depends on the organism dealt with. The result of this treatment is that in the animal treated protective substances appear in the serum, and these substances can be transferred to the serum of another animal or of man; in other words the

## active immunity of the experimental animal can be translated into the

passive immunity of man. According to the nature of the substances injected into the former, its serum may be antitoxic, if it has been immunized against any particular toxin, or antibacterial, if against an organism. Familiar examples of these are, of the former diphtheria antitoxin, of the latter anti-plague and anti-typhoid sera. An antitoxin exerts its effects by actual combination with the respective toxin, the combination being inert. It is probable that the ultimate source of the antitoxin is to be found in the living cells of the tissues and that it passes from them into the blood. The action of an antibacterial serum depends on the presence in it of a substance known as "immune-body," which has a special affinity and power of combining with the bacterium used. In order that it may exert this power it requires the presence of a substance normally present in the serum known as "complement." The development of these "anti-bodies," though it has been studied mainly in connexion with bacteria and their toxins, is not confined to their

## action, but can be demonstrated in regard to many other substances, such

as ferments, tissue cells, red corpuscles, &c. In some animals, for example, the blood serum has the power of dissolving the red corpuscles of an animal of different species; e.g. the guinea-pig's serum is "haemolytic" to the red corpuscles of the ox. This haemolytic power (haemolysis) can be increased by repeated injections of red corpuscles from the other animal, in this case also, as in the bacterial case, by the production and action of immune-body and complement. The antiserum produced in the case of the red corpuscles may sometimes, if injected into the first animal, whose red corpuscles were used, cause extensive destruction of its red corpuscles, with haemoglobinuria, and sometimes a fatal result.

Opsonic action depends on the presence of a substance, the "opsonin," in the serum of an immunized animal, which makes the organism in question more easily taken up by the phagocytes (leucocytes) of the blood. The opsonin becomes fixed to the organisms. It is present to a certain extent in normal serum, but can be greatly increased by the process of immunization; and the "opsonic index," or relation between the number of organisms taken up by leucocytes when treated with the serum of a healthy person or "control," and with the serum of a person affected with any bacterial disease and under treatment by immunization, is regarded by some as representing the degree of immunity produced.

Agglutinative action is evidence of the presence in a serum of a somewhat similar set of substances, known as "agglutinins." When a portion of an antiserum is added to an emulsion of the corresponding organism, the organisms, if they are motile, cease to move, and in any case become gathered together into clumps. In all probability several different bodies are concerned in this process. This reaction, in its practical applications at least, may be regarded as a reaction of infection rather than of immunization as ordinarily understood, for it is found that the blood serum of patients suffering from typhoid, Malta fever, cholera, and many other bacterial diseases, agglutinates the corresponding organisms. This fact has come to be of great importance in diagnosis.

The precipitin test depends on a somewhat analogous reaction. If the serum of an animal be injected repeatedly into another animal of different species, a "precipitin" appears in the serum of the animal treated, which causes a precipitate when added to the serum of the first animal. The special importance of this fact is that it can be utilized as a method of distinguishing between human blood and that of animals, which is often of importance in medical jurisprudence.