EXAMINATION OF BLOOD-STAINS

The examination of blood-stains should be carried out in the following way:

Physical Examination

1. Examine the stains carefully with a good pocket lens or a low power microscope lens. A fabric will show matting of its fibres, red filaments, and minute coagula in its meshes. In old blood-stains coagula may be absent and the fabric appear as if dyed. The characters of any fibres or hairs adhering to the stain and the nature of the substance upon which the stain is should be noted.

Fig. 4.—Photo-micrograph of wool fibres, × 250.
(R. J. M. Buchanan.)

2. Make accurate notes of the position and shape of the stains on the material examined.

3. Take one stain, if there are several, or part if single, and note the solubility of it in water, or in a mixture of water and some other substance. The solubility of the colouring matter is greater if the stain be recent than if it be old. The older the stain the less soluble it becomes, as the hæmoglobin is gradually changed in course of time to insoluble hæmatin.

An endeavour should be made to obtain a solution for microscopical, chemical, and spectroscopical examination. The solvent, in order to obtain the blood corpuscles in as natural a form as possible, should approach in its specific gravity the liquor sanguinis.

The following solvents fulfil this purpose:

(a) Glycerine and water, 1 to 7 (sp. gr. 1030).

(b) Pacini‘s solution of chloral hydrate in water (1 in 10).

(d) Roussin‘s solution of glycerine 3 parts, sulphuric acid 1 part (by weight), and water so that the mixture shall have a sp. gr. of 1028.

(e) Saturated solution of borax in distilled water.

If distilled water alone be used, the red corpuscles lose their hæmoglobin and become “laked” or “phantom” corpuscles; if the solution be of higher sp. gr. than liquor sanguinis, then the corpuscles become crenated and irregular in shape.

Fig. 5.—Photo-micrograph of flax fibres, × 250.
(R. J. M. Buchanan.)

The technique of examination has to be varied in certain details, according to the material upon which the stain is. Stains may have to be examined upon cloth-fabrics, wood, plaster, metal, or leather. These will be taken separately, and the methods of examination described which will prove most reliable in each case.

1. Cloth-fabrics.—Cut out a stain, or part of one, and macerate it in a quantity of one of the solvents mentioned above, sufficient for the purpose. If the stain be very small, squeeze with fine forceps one or more drops upon microscope slides for microscopic, and keep the remainder of the solution for spectroscopic, examination. In dyed fabrics, which have been mordanted, the mordant may fix the blood-stain so as to prevent solution, and especially so when the attempts have been made to wash out the stain with soap and water.

To make a solution of the stain in such cases it is best to use distilled water to which a small quantity of ammonia or citric acid has been added; in one or other of these the colouring matter will dissolve.

2. Wood.—Note the kind of wood, cut off a thin shaving and treat with one of the solvents mentioned above. If on wood containing tannic acid, such as oak or elm, the best solvent is a two per cent. solution of hydrochloric acid.

3. Plaster.—Scrape off some of the stained plaster, and treat as for cloth or wood.

4. Metal.—If the stain be upon a clean and unrusted metal, e.g. the clean blade of a knife, then gently heat the metal on the side opposite to the stain, when the latter, if recent, will peel off or can be easily detached. This requires some care and dexterity. It is easy, however, to scrape the stain off into a watch-glass, and this procedure is necessary when the metal is rusted and the stain mixed with the rust, or when the stain is thin.

Fig. 6.—Photo-micrograph of silk fibres, × 250.
(R. J. M. Buchanan.)

If on iron and mixed with rust the borax solution may be used, with a drop or two of solution of ammonia; use a fine camel-hair pencil dipped in the solution, and brush the stain off into a watch-glass. Becker advises that stains mixed with rust should be digested with a weak solution of ammonia and common salt for a few hours; decant the solution and evaporate it upon a microscope slide to dryness, then test the residue by the “hæmin test.”

Ganttner‘s test should be used to a portion of a stain upon metal when thin or mixed with rust. It may be carried out upon the metal itself or upon a scraping of the stain in a watch-glass resting upon a black surface. Moisten the scraping in a watch-glass with a drop or two of distilled water rendered feebly alkaline, then add a minute drop of hydrogen peroxide. Wherever blood is present bubbles of gas develop, which give the material a white beaded appearance. The froth develops from the outside of the drop towards the centre when the stain is mainly composed of blood. In a scraping consisting of mixed particles of rust and blood, the reaction only appears upon the particles of blood, and rust to which blood adheres; it does not take place on those particles of rust free from blood. Before adding the peroxide of hydrogen it may be necessary to dissipate any air-bubbles which may cling to the scraping in the alkaline water by gentle agitation with the point of a fine glass rod. Should the above reaction with peroxide of hydrogen not take place, then one can rest assured that no blood is present. The test, however, is a negative one; it is not a positive test for blood only; other fluids and exudations from the body, such as saliva and pus, give the reaction. The reaction will take place with blood-stains of any age.

Fig. 7.—Photo-micrograph of cotton fibres, × 250.
(R. J. M. Buchanan.)

In examining a clasp-knife or any hinged weapon for blood-stains, the instrument should be taken to pieces and all the hinges and recesses carefully examined, for in these places blood may be found, although the weapon had previously been wiped clean, and appear free from stains.

5. On Leather.—The tannic acid in leather forms a compound with blood which is insoluble in the solvents generally used. A thin shaving of the stained portion should be taken and folded, with the stained surface outwards, in the form of a loop. If the outer surface of the loop with the stain be made to touch the surface of the glycerine and water solution, at the same time taking care that the leather itself be not moistened, a recent stain may yield a sufficient quantity of colouring matter for the purposes of examination. Failing this, the shaving should be digested in a small quantity of a two per cent. solution of hydrochloric acid in distilled water (Sorby).

Microscopical Examination

The microscopical examination of blood-stains, for the purpose of identifying the presence of the red blood corpuscles, is especially applicable to recent stains. In these the corpuscles may retain, to a great extent, their normal characters; but their condition varies with the age of the stain; they become altered in appearance and irregular in shape with increasing age, until a stage is reached when they become completely disintegrated and unrecognisable. Having obtained a solution of a stain by one of the methods recommended, a few drops should be placed upon a clean microscope slide and covered with a No. 1 cover-glass. In a recent stain, where minute coagula are present, one may be placed on a microscope slide and moistened by breathing upon it several times, and then covering it with a No. 1 slip, or a drop of the glycerine solution may be allowed to act upon it on the slide until it be sufficiently moistened, when it should be covered in the same way. The preparation should be examined through the microscope with a good lens (preferably a ¹/₁₂th oil immersion), magnifying 300 to 400 diameters, and if any corpuscles be found, their characters should be carefully observed and noted.

All such specimens should be carefully preserved and labelled with a description of the method of preparation, the case to which they belong, the date of preparation, and the signature of the individual who has made and examined them. It is essential that the preparation and examination of the specimen should be made by the same individual. In certain cases the conditions may be sufficiently favourable to allow of the production of stained specimens, which can be mounted so as to retain their original characters permanently. In every case it is advisable to pursue the investigation with this object in view.

This process is especially applicable to recent blood-stains, in which, from preliminary examination, the presence of blood corpuscles has been determined; where complete disintegration of the blood corpuscles has taken place it would not be of any value.

It may so happen that by means of stained specimens the identity of blood corpuscles may be more easily established, when the result of examination is uncertain in a specimen not so prepared.

By the action of certain dyes upon the corpuscles their special features are rendered easier of recognition. Any of the approved methods of preparing blood films for general clinical purposes, which will suit the circumstances, may be employed. An easy and reliable method is as follows. A drop of the solution of the blood-stain properly prepared as previously recommended, or if obtainable a small coagulum moistened with normal saline solution, is placed on a clean coverslip and spread evenly over its surface with the aid of a fine glass rod. The film is allowed to dry in the air, covered with a watch-glass for protection against dust. When dry it is passed three times through the flame of a Bunsen burner, or placed in a mixture of equal parts of absolute alcohol and ether, to fix it. After fixation it should be placed for a minute or more in an aqueous solution of eosin.

Any excess of stain should be removed by washing in distilled water, and the specimen allowed to drain by standing it on edge upon a piece of filter paper; it should then be allowed to dry, and then counterstained with a freshly prepared aqueous solution of methylene blue, hæmatoxylin solution, or other nuclear dye. Wash again in distilled water, allow to dry, and mount in Canada balsam. By this method the corpuscle will be stained pink, and if nucleated, the nucleus will be stained by the methylene blue, hæmatoxylin, or other nuclear stain which may have been used.

Leishman‘s stain may be used. This stain being a methyl-alcohol solution is used for fixing and staining at the same time. A few drops of the stain is placed on the dried film; after standing until evaporation is almost complete, distilled water is dropped on to the slide, and left to stand for two or three minutes, it is then drained off, and a few more drops of distilled water added until the film is pink in colour, then dried with filter paper. Red corpuscles are tinted red with the eosin, and nuclei of leucocytes or nucleated red cells violet or deep blue. The specimen may be examined direct with the oil immersion or mounted in Canada balsam.

When examining specimens prepared from blood-stains, it is necessary to search carefully for other cellular structures such as epithelial cells, spermatozoa, or fragments of hair.

It may be advisable, in certain cases where the amount of material submitted for examination is small, to centrifugalise some of the solution in a fine glass tube, in order to determine any cellular elements present to one spot. By making use of this concentrated portion containing the cellular elements for the preparation of a microscopic specimen, one not only facilitates the microscopical examination, but is able to place more reliance upon the results obtained.

The Results of Microscopical Examination of
Blood-Stains in Their Medico-Legal Relations.

As previously stated, the examination of alleged blood-stains from a medico-legal standpoint is pursued essentially for the purpose of testifying as to whether they have been produced by blood or not. Where the examination yields a negative result, further procedure is necessary with a view of identifying the true nature of the stain. Should, however, the result be positive, the question arises as to the possibility of distinguishing between human blood and the blood of other animals, and determining the exact animal from which the blood has been derived. Such an examination should be pursued in full recognition of its importance as a factor towards the establishment of truth essential to the administration of justice.

To fulfil this obligation the methods employed should be so selected as to produce results bearing testimony free from any possibility of doubt.

Certain differences exist, and may be detected by microscopical examination, between the red corpuscles of human blood and those of some other animals sufficiently well marked to render differentiation possible. The differences are those of form and structure.

(1) In man the red corpuscles appear as circular biconcave discs, averaging ¹/₃₅₀₀ of an inch in diameter, and are non-nucleated.

The red corpuscles of mammals present the same features, with the exception of the

(2) Camel tribe, in which the corpuscles are oval in form, but non-nucleated.

(3) In birds, fishes, reptiles, and amphibians the red corpuscles are oval in shape, and possess a nucleus.

Guided by the above facts, one is able to testify whether or not the corpuscles exhibit the characters of mammalian blood.

Many attempts have been made with a view to establishing a reliable means of differentiation between the red blood corpuscles of man and other mammals (the camel excepted), and with a certain degree of success, such as might be expected, under select conditions favourable to histological research, but which do not obtain in medico-legal practice. Differences in size of the red corpuscles, as revealed by micrometric measurement, have been suggested as a possible means of distinguishing between the blood of different mammals. Of the common animals, the red blood corpuscles of the sheep present the most marked difference in size compared with those of man. The following table of the dimensions of red blood corpuscles is derived from measurements made by Treadwell, and quoted by White (The Medico-Legal Journal, New York, 1895):

	μ.
Human	7.940
Dog	6.918
Rabbit	6.365
Ass	6.293
Pig	6.101
Horse	5.503
Cat	5.463
Ox	5.436
Sheep	4.745

Menstrual blood contains no fibrin, has an acid reaction due to the vaginal mucus which keeps it fluid, and contains squamous epithelial cells. None of these characters can be differentiated on fabrics, especially when contaminated with urinary stains in addition. Hence, in cases of alleged rape, no distinction can be drawn between blood-stains on the underclothing of the female, which may have arisen from hæmorrhage the result of violence to the sexual parts, and those which might have arisen from the ordinary menstrual flow or metrorrhagia. The detection of spermatozoa, however, would add considerable value to the observation.

Fig. 8.—Measurement of Blood Corpuscles.

Photo-micrograph of human red blood corpuscles, × 800. Each corpuscle in diameter covers two divisions of the scale. Compare with sheep‘s blood, [Fig. 9].

(R. J. M. Buchanan.)

Fig. 9.—Measurement of Blood Corpuscles.

Photo-micrograph of red blood corpuscles from the sheep, × 800. The diameter of the corpuscle covers one division of the scale. Compare with human blood, Fig. 8.

(R. J. M. Buchanan.)

Blood Crystals.—Professor Preyer of Jena pointed out many years ago that the hæmoglobin crystals from the blood of some animals differed in shape from those of man, and this fact has given rise to many attempts to trace the identity of the blood to the animal from which it has been derived. The results have not been of sufficient value to establish it as trustworthy for medico-legal purposes. Dr. Monckton Copeman (B. M. J., vol. ii. p. 190, 1889) has carefully investigated the subject, and his researches, partly confirmed by Professor Glaister of Glasgow, show that from the guinea-pig, rat, and squirrel, crystals of hæmoglobin may be easily obtained, but the solubility of human hæmoglobin renders it much more difficult to crystallise. Crystals may, however, be obtained in the following ways:

(a) By feeding leeches on human blood, crystals may be found, after some weeks, in the gastric dilatation of the alimentary canal.

(b) By diluting human blood with the fluid from hydrocele, ascites, or pleurisy when they have undergone decomposition.

(d) By adding a drop of cat‘s bile to human blood on a microscope slide, but the crystals are those of reduced hæmoglobin.

Crystals of human hæmoglobin appear in the form of rectangular plates, with a greenish or pale claret colour. On spectroscopic examination they exhibit the characters of reduced hæmoglobin, in contradistinction to the crystals derived from the lower animals, which produce the spectrum of oxyhæmoglobin.

The blood of the bullock, sheep, and pig is very difficult to crystallise. By the method adopted by Gamgee of adding to defibrinated blood one-sixteenth its volume of ether, shaking until the mixture becomes transparent, and allowing to stand in an ordinary temperature for 48 hours, crystals may be obtained from the blood of the following animals:

1.	Horse.
2.	Bullock.
3.	Sheep.
4.	Pig.
5.	Dog.
6.	Cat.
7.	Rabbit.
8.	Squirrel.
9.	Guinea-pig.
10.	Rat.
11.	Mouse.
12.	Chicken.
13.	Pigeon.

Crystals from human blood are not easily obtainable by this process, but when they are, they always give the spectrum of reduced hæmoglobin, whereas those from the animals mentioned above give the spectrum of oxyhæmoglobin.

Chemical Examination

Having obtained a coloured solution from a supposed blood-stain, if sufficient in quantity, apply the following chemical tests to separate portions:

1. Add a few drops of a weak solution of ammonia in distilled water. The colour may remain unchanged, or, at the most, a slight heightening may take place, if it be due to blood. If the solution of ammonia be too strong, a brown colour may be produced if blood be present.

2. Heat to boiling, when the following changes take place if blood be present:

(a) The colour may disappear.

(b) Coagulation follows.

On adding caustic potash to the precipitate it will dissolve, and the solution formed will appear greenish by transmitted and red by reflected light. This phenomenon is called the dichroism of blood. Authorities differ in opinion as to whether the colour is green by transmitted and red by reflected light, or vice versa. “As a matter of fact, the phenomenon is chameleon-like as regards colour, so that both sets of observers may be considered right or wrong” (Glaister).

Fig. 10.—Photo-micrograph of red blood corpuscles from domestic fowl, × 250.
(R. J. M. Buchanan.)

Fig. 11.—Photo-micrograph of blood corpuscles of fish, × 250.
(R. J. M. Buchanan.)

Fig. 12.—Photo-micrograph of blood corpuscles from a dried stain of the blood of a codfish, × 250.
(R. J. M. Buchanan.)

3. Add tincture of guaiacum, freshly prepared: an opaque, cream-coloured precipitate of the guaiac resin will form in the aqueous solution. On the addition of ozonic ether, turpentine, or peroxide of hydrogen, a blue colour will be produced at the junction of the fluids: proportionate to the amount of blood-colouring matter present, the blue colour will vary in intensity.

This test, known as Day‘s or Schönbein‘s, is extremely delicate, and reacts to no coloured substance except blood.

In cases where the blood-stain is small, the test may be applied as follows. Moisten a pure white filter paper with a drop of distilled water, or one of the solutions recommended in the section on physical examination, and touch the stain with the moistened portion. On adding a drop of tincture of guaiacum followed by a drop of ozonic ether to the wet filter paper the blue colour will be produced and easily recognised on the white surface.

The guaiacum test, although extremely delicate, can only be accepted as providing negative evidence. The absence of reaction proves the absence of blood, except in very old blood-stains, which may not respond to the test. The blue colour produced indicates that the substance may be blood, but it cannot be accepted without corroboration. Gluten, raw potato, milk, bile, sweat (Ogston), and other oxidising substances give a blue colour with guaiacum and ozonic ether; some substances give the blue colour with guaiacum alone.

With blood, however, the test is sufficiently delicate to detect one drop in six ounces of water.

4. Nitric acid added to a portion of the solution of blood in distilled water produces a whitish-grey precipitate.

Fig. 13.—Photo-micrograph of frog‘s blood showing oval nucleated red corpuscles, × 250.
(R. J. M. Buchanan.)

5. Hæmin Crystals.—Concentrate a portion of the solution upon a microscope slide, add to it a minute crystal of chloride of sodium and a few drops of glacial acetic acid. Heat gently to dryness or to a lesser degree under a coverslip; examine with the microscope; if blood be present, crystals of hæmin, or the hydrochloride of hæmatin, will be found. They are of a yellowish-red to brownish-black colour with a metallic lustre. They occur in rhomboidal prisms, or six-sided in shape, or in the form of “whetstones,” often in clusters; many of the crystals exhibit a lipped projection on one side. They are known as Teichmann‘s crystals. It is well to verify their origin from blood by placing upon them a drop of hydrogen peroxide, when they will give off bubbles of oxygen gas.

They are insoluble in water, alcohol, and dilute acetic and hydrochloric acids. They dissolve in boiling acetic or hydrochloric acids, and the caustic alkalies. They respond to the guaiacum test, and the ash produced by incineration shows the presence of iron by the red colour produced on the addition of a drop of hydrochloric acid and a solution of potassium sulphocyanide.

The production, by the methods described, of such crystals affords conclusive proof of the presence of blood.

Spectroscopic Examination

To a portion of the coloured solution, filtered if necessary, the spectroscopic tests should be applied. The following points must be remembered in carrying out a spectroscopic examination:

(a) The colouring matter of fresh blood is hæmoglobin, and it may exist in two states, according to the degree of its combination with oxygen.

In arterial blood it is present as oxidised hæmoglobin, and the same obtains in blood which has been exposed to the air under certain conditions and for a varying period of time.

Fig. 14.—Photo-micrograph of crystals of hæmin, × 250.
(R. J. M. Buchanan.)

In venous blood, especially when obtained under conditions preventing oxidation, as from the heart cavity of an animal newly asphyxiated, it is present as deoxidised hæmoglobin.

(b) In dry stains, especially if they have been subjected to the action of impure air containing the products of coal-combustion, the colouring matter becomes changed into methæmoglobin, or hæmoglobin in which its combination with oxygen has been altered in such a way that a current of a neutral gas, such as hydrogen or nitrogen, will not dissociate it, as it does with oxyhæmoglobin. Such stains have a brownish colour, and may give an acid reaction.

(c) In stains which have retained moisture, from having lain in damp places, the hæmoglobin becomes converted into hæmatin. The same change takes place in dry stains after a longer period of time.

On examining the solution of the colouring matter from a blood-stain with the spectroscope, the spectrum will vary according to its condition and the nature of the solvent used.

The spectra of hæmoglobin and its derivatives are characteristic, and afford conclusive evidence of the presence of blood. The spectra must be recognised, however, in more than one condition. Other substances may yield spectra very similar to that of oxyhæmoglobin, but when subjected to certain tests they do not alter in the same way. They cannot be made to give the spectra of reduced hæmoglobin and reduced hæmatin, and any colouring matter which may be made to yield the spectra of reduced hæmoglobin and reduced hæmatin is derived from blood.

Blood Spectra

1. Oxidised hæmoglobin (O₂Hb) is characterised by the presence in its solar spectrum of two absorption bands between the D and E lines. The first band commences at the D line and extends a short distance towards the E. The second commences at a little distance from it, and terminates at the E line; it is about twice the breadth of the first. The band at D is more defined than the other ([Fig. 15, 1]).

2. Deoxidised or reduced hæmoglobin presents one broad band occupying almost the whole of the space between D and E slightly to the left of these lines ([Fig. 15, 2]).

3. Methæmoglobin presents two bands between D and E, in the same position as those of O₂Hb, but in addition a third band between C and D and near to the former ([Fig. 15, 3]).

A solution of oxyhæmoglobin or methæmoglobin may be reduced by the addition of a reducing agent, such as Stokes‘ reagent, consisting of ferrous sulphate with a small quantity of tartaric acid dissolved in water and rendered alkaline at the time of using with ammonia, or, better still, by the addition of ammonium sulphide. The spectrum will change to that of reduced hæmoglobin.

4. Acid hæmatin presents a spectrum with a band between D and E, commencing at a little distance from D and ending at E, also a narrower band between C and D and commencing at C. It is a difficult spectrum to obtain.

5. Alkaline hæmatin presents a spectrum with a single band between C and D near to the D line. It is more difficult to obtain than the spectrum of acid hæmatin.

It is not necessary, however, to obtain these spectra, viz. 5 and 6, but it is necessary to reduce solutions of either acid or alkaline hæmatin in order to obtain the spectrum of reduced hæmatin. To do so proceed as follows. To some of the solution of colouring matter obtained from the stain add a small quantity of a 20 per cent. solution of sodium hydrate; the solution will alter in colour, and the spectrum of O₂Hb or MetHb will disappear. On adding to this solution of alkaline hæmatin a few drops of ammonium sulphide, or Stokes‘ fluid, it becomes claret-coloured, and on examination with the spectroscope the spectrum of reduced hæmatin will be seen. This is the most pronounced of all blood spectra. Its production can be hastened by gently warming the solution.

If the stain be old and already changed into hæmatin, its solution will yield the spectrum of acid hæmatin, and will give the spectrum of reduced hæmatin on the addition of ammonium sulphide or Stokes‘ fluid without previous alkalisation.

6. Reduced hæmatin presents a spectrum with a dark band about midway between D and E, and a broad but paler band commences near the E line and extends to the b line ([Fig. 15, 4]).

Fig. 15.—Blood Spectra.

1. Oxyhæmoglobin.	3. Methæmoglobin.	5. CO hæmoglobin.
2. Reduced hæmoglobin.	4. Reduced hæmatin.	6. CO hæmoglobin and
		reduced hæmoglobin.

In cases of death by asphyxia, in which the hæmoglobin is in combination with CO₂, the blood, if removed and examined immediately after death, gives a spectrum of reduced hæmoglobin, but on exposure to the air it rapidly changes to oxyhæmoglobin. The period after death at which blood is usually submitted for medico-legal examination is sufficiently late to allow of this change, and so prevents the possibility of determining death by asphyxia by spectroscopic examination of the blood. Where death has been caused by the action of carbon monoxide, the blood is of a cherry-red colour, and it will retain this colour unchanged for a long time, in fact for years, due to a stable combination of the CO and hæmoglobin, called carboxyhæmoglobin. Such blood yields a very characteristic spectrum, with two bands similar to those of O₂Hb, but nearer to the violet end ([Fig. 15, 6]). Their position should be assured by accurate measurement and comparison with a spectrum of O₂Hb. The CO hæmoglobin, however, cannot be reduced; on the addition of ammonium sulphide or Stokes‘ fluid the bands remain unaltered.

In cases where the amount of fluid obtainable for examination is very small, recourse must be had to the micro-spectroscope, using a Sorby‘s cell to hold the fluid, and substituting for the eye-piece of the microscope a specially constructed spectroscope arranged so as to throw the spectrum of a known solution of blood-colouring matter alongside of that yielded by the solution under examination. Artificial light should be used, and the D line located by placing in the flame a platinum wire carrying a salt of sodium.

Biological Tests for Blood

The results of the experimental investigations of Friedenthal,[3] Deutsch,[4] Uhlenhuth,[5] Wasserman and Schutze,[6] Nuttall,[7] Tarchetti,[8] Grünbaum,[9] Metchnikoff,[10] and M‘Weeney[11] into “blood relationships” have led to the suggestion of a new method—the “biological test,” by which different kinds of mammalian blood may be distinguished one from the other. Their experiments show in a general way that the “serum from an animal which has been injected intraperitoneally with any given organic fluid will, if mixed in small quantity with a dilute solution of the fluid used for the injection, produce a more or less definite precipitate.” If human defibrinated blood be injected into the peritoneal cavity of a rabbit, the serum obtained from the rabbit‘s blood, when mixed with a clear solution of the blood of man or ape, will produce a precipitate, and agglutinate the red blood corpuscles if present: the same reaction will not follow if such serum be added to a solution of the blood of any other animal.

The introduction of substances of albuminous or proteid nature into the body of an animal, and which can be taken up by its cells as a food, produces in the body of the animal a series of substances called antibodies, of which one is designated precipitin from its power of producing a precipitate with the substance introduced. The organic substance introduced capable of producing antibodies is called the antigen. The precipitin is formed and present in the blood serum of the inoculated animal.

Metallic poisons, alkaloids and carbohydrates, do not produce antibodies; some vegetable proteids do.

The serum containing the precipitin is called the antiserum.

The precipitin in the antiserum is specific, and produces the effect only with its own antigen, not with that derived from another species of animal, but will act with one derived from a closely allied species, e.g. man and the higher apes, sheep, and goat.

In order to eliminate this the antigen should be diluted to 1:1000.

The blood used as antigen may be dried and sterilised, thus it may be kept for a long time, and when required for use dissolved in normal saline.

The most convenient human antigen for general use is ascitic or pleuritic fluid, and may be kept in good condition when mixed with a small quantity of chloroform.

To produce the antiserum 3 to 10 c.c. of the antigen serum is injected into a vein or the peritoneal cavity of a rabbit, and repeated at an interval of four or five days until 25 c.c. have been injected. The serum of the rabbit is then tested with the antigen diluted 1:100. The injections are then continued until 70 to 80 c.c. have been administered. The animal is then killed, the blood collected, and stood in a refrigerator for the serum to separate. This is then drawn into sterile pipettes, sealed, and kept in the cold. For purposes of preservation one-tenth its volume of 5 per cent. phenol may be added before pipetting. The antiserum may be dried on slips of black paper for future use.

For medico-legal purposes, the antiserum should always be tested with its antigen to be sure of its efficacy.

The antigen to be tested should be diluted 1:1000 at least, as potent antisera may give reactions with strong solutions of antigens derived from other species of animals. Various antisera may be made by inoculating rabbits with the serum of different animals.

Metchnikoff has shown that intraperitoneal injection is not absolutely necessary in order to produce an antiserum, feeding a rabbit on the blood will act in the same way.

The procedure for testing a stain is as follows. First of all the stain must be proved to be blood by the usual methods; this because the antiserum will give reactions with other albuminous substances, e.g. mucus, pus, semen, milk, or albuminous urine derived from the animal providing the antigen.

Having proved the stain to be blood, a solution of it should be made in normal saline, sufficiently strong to give the HNO₃ reaction for albumin, or to foam when shaken up. If the amount of solution be small, the tests can be carried out in capillary tubes or pipettes. The solution of the stain must be cleared by filtration or the centrifuge.

The tests must be controlled by comparison with known human blood, and blood from several domestic animals. For this purpose the various antisera should be kept in stock.

Two sets, A and B, of six small tubes are used, into each of A is placed 0.05 c.c. of human antiserum from a sensitised rabbit. Into each tube of Set A is then added double the amount of diluted antigen, as follows:

Set A —	1.	Receives	extract	of known	human	blood	-stain.
	2.	“	“	“	ox	“	“
	3.	“	“	“	horse	“	“
	4.	“	“	“	sheep	“	“
	5.	“	“	“	pig	“	“
	6.	“	“	“	stain	under observation.

Set B is now charged as follows:

Set B —	1.	Receives	normal	rabbit serum	0.05	c.c.
	2.	“	human	antiserum	0.05	“
	3.	“	ox	“	0.05	“
	4.	“	horse	“	0.05	“
	5.	“	sheep	“	0.05	“
	6.	“	pig	“	0.05	“

To each tube in Set B is now added about 0.1 c.c. of the extract of the suspected stain.

It will thus be seen that in Set A known antigen is added to known precipitin, with the exception of tube 6, which receives suspected antigen, while in Set B the suspected antigen is added to known antisera, with the exception of No. 1, which contains normal rabbit serum.

The tubes are observed in an hour and the results noted. Should the stain be human blood, positive reactions will be present in tubes 1 and 6 of stand A, and tube 2 of B.

The negative reactions in the other tubes in stand A prove that the antiserum used is specific, and will only react with its own antigen; in stand B that the stain is composed of specific human antigen, reacting only with its own antiserum.

Should the stain suspected be other than human, and derived from one of the other animals used for antiserum in stand B, then the positive reaction will occur in the particular tube.[12] The test is now recognised as very sensitive and reliable, and is also applicable to the detection of the flesh or fluids of animals, and has thus been used for the detection of meat stuffs.

Tarchetti advises the following procedure in the examination of blood-stains: Dissolve the stain in a few drops of a 0.9 per cent. aqueous solution of sodium chloride, filter, and divide the filtrate into two portions; to one (a) add 0.5 c.c. of the prepared rabbit serum (the so-called antiserum); to the other (b) serum from a rabbit which has not been injected with human blood. Both are to be placed in an incubator at 37° C. for an hour. By this time, if the solution of the stain be of human or anthropoid origin, the contents of the tube (a) will have become turbid, the contents of tube (b) will remain clear. From a series of experiments with blood-stains of man and other animals on a variety of materials, Tarchetti states that this method is reliable. Prepared rabbit “human antiserum” has been shown to have no such reaction with the blood of the pig, ox, calf, mouse, or rat.

From the result of his investigations Grünbaum points out that these reactions must be looked upon rather as “special” than “specific,” in view of the fact that his “chimpanzee antiserum” gave a slight but distinct turbidity after a few hours with horse blood. He also suggests a method for the microscopical application of the “biological test,” by using a 1 per cent. blood solution with a drop of “antiserum.” This method has enabled him to distinguish between human and anthropoid blood, the reaction occurring earlier and being more complete when the “antiserum” is used on its own blood.

Vegetable and other Stains
which resemble Blood

Certain vegetable colouring matters give spectra which may be mistaken for blood, from their close similarity. Of these cochineal dissolved in a solution of alum gives two bands similar to O₂Hb. On the addition of boric acid the bands move to the violet end of the spectrum, but they are unaffected if the colouring matter be blood. Lac-dye, alkanet root, madder, and others also give spectra resembling O₂Hb, but they are changed or disappear on adding ammonia or sulphite of potassium, while the spectrum of blood remains unaltered.

As stated previously, spectra of colouring matters other than blood are not capable of being altered by reducing agents, so that, however similar they may be to O₂Hb, they cannot be accepted as derived from blood unless the spectra of reduced Hb and reduced hæmatin can be obtained in the way described.

Cochineal, colours of certain roots and wood, turn crimson on the addition of ammonia, logwood bluish-black.

The colour of the rose and certain flowers turn green on adding ammonia.

Fruit-stains from mulberry, currants, gooseberries, &c., turn bluish-green with ammonia.

Vegetable stains have their colour heightened by the action of dilute acids.

Chlorine bleaches fruit-stains, but turns the colour of blood-stains to an olive-green.

Red dyes fixed by a mordant are not influenced by ammonia.

Iron stains are usually blackened by ammonium sulphide.

Red paint may contain red oxide of iron; digest with hydrochloric acid and test for iron, by adding ferrocyanide of potassium to obtain the Prussian blue. Iron stains may be of a reddish-brown or orange colour, and insoluble in water, so that HCl is used to dissolve them.

Citrate and malate of iron stains are soluble in water; the addition of ammonia to an aqueous solution produces no change; guaiacum will give a blue reaction if a persalt of iron be present. The addition of hydrochloric acid and ferrocyanide of potassium will give the Prussian blue reaction. A drop of nitric acid added to the solution will oxidise the iron to the ferric state, and on the addition of a few drops of fresh-made aqueous solution of sulphocyanide of potassium the port-wine colour of sulphocyanide of iron will be produced.

A control test must be made with distilled water to prove the purity of the reagents, and the two results compared with each other.

Aniline stains resembling blood are changed to greenish-yellow or yellow on the addition of dilute nitric acid. Eosin stains produce a fluorescent solution when dissolved in water. Grease, tar, pitch, snuff, and paint may be mistaken for blood, especially on dark fabrics. They may be detected by two methods:

(a) The Wet Method.—Having failed to obtain a solution by the aid of the ordinary solvents for blood, other solvents must be used; ether or benzene for grease, paint, or tar. The solution obtained must be examined with the spectroscope.

(b) The Dry Method.—Place the cloth or other fabric stain down upon a clean white filter paper; then on pressing the cloth with a hot laundry iron, grease, tar, or pitch will stain the paper, paint or snuff will not.