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Sp. gravity	Quantity of Hæmoglobin
	(Fleischl's method)
1033–1035	25–30%
1035–1038	30–35%
1038–1040	35–40%
1040–1045	40–45%
1045–1048	45–55%
1048–1050	55–65%
1050–1053	65–70%
1053–1055	70–75%
1055–1057	75–85%
1057–1060	85–95%

In a paper which has quite recently appeared Diabella has investigated these relations very thoroughly, and his results partly correct, and partly confirm those of Hammerschlag. Diabella found from his comparative estimations that differences of 10% hæmoglobin (Fleischl) correspond in general to differences of 4.46 per thousand in the specific gravity (Hammerschlag's method). Nevertheless with the same amount of hæmoglobin, differences up to 13.5 per thousand are to be observed; and these departures are greater the richer the blood in hæmoglobin. Regular differences exist between men and women; the latter have, with the same amount of hæmoglobin, a specific gravity lower by 2 to 2.5.

Should the parallelism between the number of red blood corpuscles and the amount of hæmoglobin be considerably disturbed, the influence of the stroma of the red discs on the specific gravity of the blood will then be recognisable. Diabella calculates, that with the same amount of hæmoglobin in two blood testings, the stroma may effect differences of 3–5 per thousand in the specific gravity.

Hence the estimation of the specific gravity is often sufficient for the determination of the relative amount of hæmoglobin of a blood. It is only in cases of nephritis and in circulatory disturbances, and in leukæmia, that the relations between specific gravity and quantity of hæmoglobin are too much masked by other influences.

The physiological variations which the specific gravity undergoes under the influence of the taking in and excretion of fluid do not exceed 0.003 (Schmaltz). From what has been said, it follows that all variations must correspond with similarly occurring variations in the factors that underlie the amount of hæmoglobin and the number of corpuscles.

More recent authors, in particular Hammerschlag, v. Jaksch, v. Limbeck, Biernacki, Dunin, E. Grawitz, A. Loewy, have avoided an omission of many earlier investigators; for besides the estimation of the specific gravity of the 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 variations in the specific gravity of the total blood; partly by variations in number, or changes in their distribution; partly by their chemical instability; loss of water and absorption of water, and variations in the amount of iron.

The plasma of the blood on the contrary—and there is no essential difference between plasma and serum (Hammerschlag)—is much more constant. Even in severe pathological conditions, in which the total blood has become much lighter, the serum 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 chronic kidney diseases, and disturbances of the circulation. E. Grawitz has lately recorded that in certain anæmias, especially posthæmorrhagic and those following inanition, the specific gravity of the serum undergoes perceptible diminutions[3].

There are still therefore many contradictions in these results, and it is evidently necessary in a scientific investigation always to give the specific gravity of the serum and of the corpuscles, in addition to that of the total blood.

A method closely related to the estimation of the specific gravity is the direct estimation of the dried substance of the total blood, "Hygræmometry"; the clinical introduction of which we owe to Stintzing and Gumprecht. This method is really supplementary to those 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 weighed again. The figures so obtained for the dried substance have a certain independent importance; for they do not run quite parallel with those of the specific gravity, amount of hæmoglobin or number of corpuscles. The normal values are, for men 21.26%, for women 19.8%.

A further procedure for obtaining indirect evidence of the amount of hæmoglobin is the determination of the volume of blood corpuscles in 100 parts of total blood. For this estimation a method is desirable, which allows of the separation of the corpuscles from plasma in blood, 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 addition of sodium oxalate or other substances which prevent coagulation. The separation of the two constituents can be effected by simply allowing the blood to settle, or with the centrifugal machine, specially constructed for the blood by Blix-Hedin and Gärtner ("Hæmatocrit").

For these methods various diluting fluids are used, such as physiological saline solution, 2.5% of potassium bichromate and many others. According to H. Kœppe they are not indifferent as far as the volume of the red blood corpuscles is concerned; and a solution which does not affect the cells must be previously ascertained for each specimen of blood. For this reason attention may be called to the proceeding of M. Herz, in which the clotting of the blood in the pipette is prevented by rendering the walls absolutely smooth by the application of cod-liver oil. Kœppe 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 constructed a very convenient variation, the oil as the lighter body is completely removed from the blood; and the plasma is also separated from the corpuscles. Three sharply defined layers are then visible, the layer of oil above, the plasma layer, and the layer of the red blood corpuscles. In as much as the apparatus is calibrated, the relation between the volumes of the plasma and corpuscles can be read off. No microscopical alterations in the corpuscles are to be observed.

Though this procedure seems very difficult of execution, it is nevertheless the only one, which has really advanced clinical pathology. The results of Kœppe—not as yet very numerous—give the total volume of the red corpuscles as 51.1–54.8%, an average of 52.6%.

M. and L. Bleibtreu have endeavoured indirectly to ascertain the 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 hæmoglobin are by no means constant, is well shewn by conditions (for example the acute anæmias) in which an "acute swelling" of the individual red discs occurs (M. Herz), but without a corresponding increase in hæmoglobin. 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 hæmoglobin affords the most important measure of the severity of an anæmic condition. Those methods which neither directly nor indirectly give an indication of the amount of hæmoglobin 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 hæmorrhagic 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 hæmorrhagica, Anæmia 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 rôle 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 anæmia from other severe anæmic 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 hæmoglobin 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: anæmia, hæmoglobinuria, and after many intoxications, the resistance, as measured by the methods above indicated, is considerably lowered.