BLOOD-STAINS
It is important in medico-legal investigations to determine the nature
of stains found on clothes, weapons, articles of furniture, &c. In the
case of blood-stains note should be made of their incidence upon the
body or in its vicinity. Blood-stains may vary in their character,
incidence, and magnitude, as sprays, spirts, or jets, smears of various
forms, or pools of blood.
Notes should be made of the relation of the direction of a spray
of blood to the position of a wounded body when found. A plan with
the position of the stains should be sketched upon the spot, and
measurements taken carefully.
In the examination of blood-stains the purpose of the medico-jurist is
not to demonstrate all the properties of blood, but to identify it.
There is not much difficulty in ascertaining whether stains are due
to blood or not; but when the question arises as to whether the blood
be human or that of some other animal, the identification is more
difficult and less certain.
Blood-stains vary in colour, according to the age of the stain, the
quantity of blood in it--the thicker the stain the darker--and the
nature and colour of the material upon which it is. Recent stains are
reddish in colour, old stains brownish. This change of colour depends
upon the free access of air and the presence or absence of chemical
substances in the air, so that it is almost impossible to infer the
age of a blood-stain by its colour. On dark-coloured materials the
stains are rendered more visible by the aid of artificial light, such
as candle-light; on light-coloured materials, on leather, wood, iron,
and stone, they are more visible in good daylight. By reason of the
coagulation and the albuminous composition of blood, dry stains stiffen
the fabric when thin, and on thicker woollen materials the fine fibres
become matted. On metals, such as iron or steel, they appear as dark
shiny spots or smears, and when dried are often fissured or cracked.
Rust may so alter blood as to produce a difference between the stains
on the blade and handle of a knife. In quite recent blood-stains
the general appearances are sufficient to give rise to a conclusion
as to their nature, especially if the stains are large. The general
features as seen by the naked eye are such that one may often recognise
blood-stains as arterial by the _comet shape_ they retain when falling
slantwise on an object. Venous blood is not spurted in small jets like
arterial, but blood from veins may become splashed upon objects and
assume shapes similar to those produced from an arterial jet.
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.
[Illustration: 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).
(_c_) Normal saline solution.
(_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.
[Illustration: 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.
[Illustration: 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.
[Illustration: 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.
[Illustration: 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.)]
[Illustration: 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.
(_c_) By adding crystals of glycocholate or
taurocholate of soda to human blood.
(_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.
(_c_) A precipitate falls, dirty grey or brown in colour,
depending upon the amount of colouring matter present.
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).
[Illustration: Fig. 10.--Photo-micrograph of red blood corpuscles from
domestic fowl, × 250. (R. J. M. Buchanan.)]
[Illustration: Fig. 11.--Photo-micrograph of blood corpuscles of fish,
× 250. (R. J. M. Buchanan.)]
[Illustration: 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.
[Illustration: 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.
[Illustration: 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).
[Illustration: 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.
[3] Friedenthal, _Archiv für Anatomie und Physiologie_, 1900, p. 494.
[4] Deutsch, _Bulletin Medical_, Sept. 8, 1900; and _Centralblatt für
Bakteriologie_, Band xxix. p. 661.
[5] Uhlenhuth, _Deutsch Medicinische Wochenschrift_, 1901, Nos. 6, 17,
45.
[6] Wasserman and Schutze, _Berlin Klin. Wochenschr._, Feb. 21, 1901.
[7] Nuttall, _Jour. Hygiene_, vol. i., 1901, p. 367; _B. M. J._, vol.
i., 1901, p. 669.
[8] Tarchetti, _Gazz. degli Osped._, May 19, 1901; _B. M. J._, vol. i.,
1901.
[9] Grünbaum, _Lancet_, Jan. 18, 1902.
[10] Metchnikoff, _Centralblatt für Bakteriologie_, April 18, 1901.
[11] M‘Weeney, _Jour. State Med._, vol. ix. No. 7, p. 378. _Lancet_,
June 18, 1910.
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:
{ 1. Receives extract of known human blood-stain.
{ 2. “ “ “ ox “ “
{ 3. “ “ “ horse “ “
Set A { 4. “ “ “ sheep “ “
{ 5. “ “ “ pig “ “
{ 6. “ “ “ stain under observation.
Set B is now charged as follows:
{ 1. Receives normal rabbit serum 0.05 c.c.
{ 2. “ human antiserum 0.05 “
{ 3. “ ox “ 0.05 “
Set B { 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.
[12] M‘Weeney, _Lancet_, June 18, 1910.
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.