Microscopic identification of bloodstains

As liquid blood dries and forms a bloodstain, red blood cells, like other cells, dehydrate. If placed in
an environment with a higher solute concentrate, water leaves the cell by osmosis and the cells shrink
and change shape. If stains are relatively fresh, it is possible to reconstitute the stain and proceed with
microscopical identification of cellular components. A number of techniques have been reported for
microscopic examination of erythrocytes and leukocytes in bloodstains. The results obtained with these
methods are much affected by the conditions of the bloodstains. Aging, environmental factors, or heating
can considerably alter blood cells and make it difficult to produce interpretable and reliable results. In
addition to aiding in the identification of a sample as blood, the microscopic appearance of cells found in a
stain extract may also reveal other information. For example, sickle-shaped erythrocytes may indicate that
the blood sample originated from a person having sickle cell disease.
Reconstituting the blood cells with a solution to restore their original shape can be attempted with the
following techniques.
1. Put a fragment of fresh blood crust on the center of a clean slide.
2. Add a drop of a solution of albumin:glycerol: 0.85% saline (20:20:60 by vol.) on the crust and mix gently
until the crust is dissolved.
3. Place the slide in a moisture chamber for two hours at room temperature.
4. Prepare a thin film smear of the mixture and air dry.
5. Stain with Wrights stain or other polychromatic stain suitable for blood samples.
6. Examine the slide under the microscope for red blood cells and white blood cells.

Chemical identification of blood

When blood dries to form a bloodstain, the cells are destroyed and their contents released into the
surrounding environment. More than 250 proteins, enzymes, and other compounds have been found in
the red blood cell, mostly in the soluble portion of the erythrocytes. The predominant erythrocyte protein is
hemoglobin (Hb). More than 100 variants of hemoglobin have been described. Identification of blood in
stains by means of chemical methods is based on the detection of heme or its derivatives in the stain
sample. Such tests can be classified under one of two categories: catalytic tests and crystal tests.
Catalytic tests (screening or presumptive tests) All catalytic blood tests depend on an oxidation
reaction in which an oxidant, for example, hydrogen peroxide, oxidizes a colorless material, such as
phenol-phthalin or tetramethylbenzidine, to a colored one. Alternately, 3-amino-phthalhydrazide (luminol),
a colorless material, can be oxidized to a product which luminesces. The general presumptive test
reaction is:
H202 + reduced reagent (color 1) < H20 + oxidized reagent (color 2).
The heme group of hemoglobin exhibits a peroxidase-like activity which may catalyze the
breakdown of hydrogen peroxide. The majority of tests which have been devised for the forensic
identification of blood are based on the peroxide-mediated oxidation of leukomalachite green,
phenolphthalin, o-tolidine, luminol, tetramethylbenzidine, fluorescein, and other less commonly used
compounds. At one time, benzi-dine and its derivatives were widely used as the color reagent in
screening tests for blood. However, due to the carcinogenic nature of these compounds and the health

risks involved in their use, laboratories no longer use these types of chemical reagents. The tests most
commonly employed in modern crime scene procedures are phenolphthalin, leukomalachite green,
luminol and tetramethylbenzidine. Reaction schemes for some of these common chemical reagents are
shown in Fig. 2. All of these chemicals are highly sensitive to minute traces of hemoglobin and its
derivatives, but all suffer from the occurrence of false positive reactions with some of the following
materials: catalases, peroxidases, cytochromes, strong oxidizing agents and metallic salts.
Testing procedures Prior to testing, the nature, color and appearance of the stain should be noted.
These are important data which will assist the scientist in interpreting any positive reactions noted with the
test reagents. All efforts should be made to limit alteration of the stain or pattern while performing the
screening test for blood. The screening test for blood should be performed by scraping a small sample
from the stain or removing a small portion of stained material.
1. Color reagent is added to the stain material.
2. If no color develops within 30 s, a drop of 3% hydrogen peroxide is added. A resulting color change
indicates that blood may be present.
3. Alternatively, the stain area may be lightly rubbed with a clean cotton swab or filter paper moistened
with distilled water. Reagent and hydrogen peroxide are then added to the swab sample. This method is
preferred for crime scene work and when determining which stains warrant additional testing on items of
physical evidence. 4. Small samples of suspected blood mixed with other materials, such as soil, may be
dissolved and the resulting supernatant tested accordingly.

Spectrophotometry methods of blood identification

Spectrophotometric procedures are seldom used at the present time in forensic analyses. This
type of examination is based on the identification of hemoglobin and its derivatives through their specific
absorption spectra. Absorption spectroscopy of hemoglobin was first described by Hoppe in 1862 as a
means for blood identification. During the early days of the development of procedures, this method was
considered one of the most conclusive tests for the identification of bloodstains. The determination of near
ultraviolet and visible absorption spectra allows sufficient reliability and sensitivity for the identification of
hemoglobin and derivatives such as methemoglobin, oxyhemoglobin, carboxyhemo-globin, and
sulfhemoglobin.
In the near ultraviolet and visible regions of the spectrum, a complex system of absorption bands is
present due to the heme portion of the hemoglobin molecule. The visible region of the spectrum of the
heme derivatives differs substantially from derivative to derivative, but all have in common a strong
absorption band at 400-425 nm (the Soret band). Porphyrin compounds and their derivatives from other
animal or vegetable sources may share spectral characteristics with hemoglobin, hematin, or
hemochromogen. Therefore, the identity of bloodstains should never be inferred solely from a single
absorption spectrum.

Electrophoretic methods

Two electrophoretic approaches have been recommended for identifying bloodstains: (1)
separation and identification of hemoglobin by electrophoresis and (2) separation and identification of
serum proteins by immunoelectrophoresis.
Hemoglobins are conjugated proteins. After selection of an appropriate buffer pH, the charged
hemoglobin molecules are moved by electrophoresis through a support medium toward the electrode with
the opposite charge. Most of the substances which give false positives with chemical tests for blood are
either uncharged or have a different charge from hemoglobin and are thus eliminated by this method.
After electrophoretic separation, the hemoglobin fractions are visualized by staining with leu-komalachite
green solution or any other catalytic color test reagent. Banding patterns may then be compared with
known standards.

Immunoelectrophoresis involves the combination

of the techniques of immunodiffusion and electro-phoresis for the analysis of biological fluids. In this
procedure, bloodstain extract is placed in wells in agar on a glass slide and then subjected to electrophoresis by application of an electric current. A bloodstain extract contains hemoglobin as well as serum
proteins. Under these conditions, the individual proteins move at different rates. After electropho-resis,
antihuman serum is placed in a trough running the length of the slide and parallel to the path of migration.
The separated proteins and antiserum diffuse toward one another, permitting the corresponding human
serum proteins to undergo an antigen-antibody reaction with the antibodies and forming preciption lines at
the points where these complexes form. The hemoglobin will remain near the point of origin and give a
pinkish ring around the sample well. These white precipitin lines and the pinkish hemoglobin ring are a
positive indication of blood. There are no other substances besides blood that will give this pattern
combination. Another advantage of this method is that the species of origin of the bloodstain can be
determined at the same time.

Immunological (antihemoglobin) tests

Anti-hemoglobin precipitin sera have been used for the identification of human bloodstains. The
highly specific reaction obtained between human bloodstains and the antihuman hemoglobin serum
allows a stain to be identified in a single operation as blood of human origin. This test can be carried out
through either one-dimensional or two-dimensional diffusion techniques. A positive result with this test is
not only absolute identification of a stain as blood, but also shows the stain is from a human source.

Determination of Species of Origin

After a stain has been identified as blood, it is necessary for the forensic scientist to determine
whether that blood is of human origin. If it is not human, it may then be necessary to determine to what
species the blood does belong. Most methods in common use for determining the species of origin are
immunological in nature. If an animal is injected with a protein molecule from another species, it will
sometimes recognize this protein as a foreign substance (antigen) and will produce an antiserum
(antibody) which will react with such protein both in vivo and in vitro. The immunological precipitin test for
medicolegal species determination in bloodstains was first used in 1901.

The in vitro antibody-antigen reaction is detected by the formation of an antigen-antibody (Ag-Ab)

complex. The reaction requires the presence of three elements: antiserum, bloodstain extract (antigen)
and buffer. The temperature, pH, incubation time, and ionic strength at which a precipitin reaction is
performed have a direct influence on the precipitin band formation. For example, the most favorable
temperature is usually between 25C and 37C and the optimal pH is between 7 and 8. However, the
exact conditions which are optimal for a system must be determined for each new antigen-antibody
system under investigation.
The specificity of the antiserum plays the most important role in species determination. Traces of
contaminating antibodies in commercially prepared antisera could cause serious error. Therefore, the
precise specificity of the antiserum in use must be known. Antihuman sera can be produced by injecting
human serum or hemoglobin into various animals. The most commonly used antisera created by this
method are produced by rabbits, goats or sheep. These antisera produce a stable precipitate. Monoclonal
antibodies are also commercially available for species testing. To ensure the specificity of the anti-serum,
it is imperative that laboratory scientists select by direct testing for crossreactivity and determine the
strength of the antiserum by a titration method. During species determination, the same batch of tested
antiserum must always be used. Only by such strict controls can the forensic scientist maintain the
degree of certainty and reproducibility required for a reliable species determination.
Figure 3 depicts several methods for the determination of the Ag-Ab complex in species tests.
The following are the most commonly used methods for species determination in forensic laboratories.

Precipitin methods
Ouchterlony method: double diffusion in two directions This diffusion method was first described by
Ouchterlony in 1949. It involves the use of agar gel plates with wells for both antibodies and antigens. The
two reactants diffuse into the gel where the soluble antigens and antibodies form an insoluble complex -a
precipitate. The Ouchterlony method allows both qualitative and semiquantitative evaluation of the
reactants. Precipitin band formation gives the scientist considerable information regarding the identity,
partial identity or nonidentity of the antigen and antibody reaction. It also yields information on the
diffusion coefficients and concentrations of the reactants.
Crossed-over electrophoresis The crossed-over elec-trophoretic technique can be used for both
quantitative and qualitative determination of a blood sample.

Human DNA quantitation

A sample can be determined to be blood of human origin by reaction with a probe
specific for human DNA. Probes complementary to primate specific DNA sequences,
such as those found at the locus D17Z1, are readily available and used primarily to
determine the amount of human DNA extracted from a sample prior to DNA typing. DNA
extracted from a sample is spotted on a membrane along with known concentrations of
human DNA. After reaction with the human-specific probe, results obtained from the

unknown sample are compared to the signal intensity of the known standards; the
amount of human DNA in the sample can be estimated in this manner. The sensitivity of
the human DNA quantitation test is commonly in the 0.15-0.20 ng range when a color
reagent detection method is employed. One disadvantage of this technique is that
human DNA from any tissue or cells will produce a positive reaction. Thus, it is
necessary to determine that the DNA obtained is from blood by using one of the h