Detecting Blood

Detecting blood, in the field and in the lab, is an important part of forensic work, but it is
surprisingly difficult to establish unambiguously that a suspect stain is in fact blood. Even if
obvious splatters or pools of blood-like material are found at a crime scene, it can’t be assumed
that they are blood. More than one investigator has been fooled by paint, hydraulic fluid, or other
liquids that resemble blood, sometimes quite closely. Furthermore, bloodstains are by no means
always obvious. Bloodstains may have been washed away by the criminal, leaving only invisible
traces, or the blood may have been deposited on foliage or other materials that make it difficult
to see.
If the crime scene covers a large area, it may not even be immediately obvious where to
begin looking for latent bloodstains (also called occult bloodstains, where occult is used in the
sense of hidden). After taking specimens of any patent bloodstains, forensic technicians use one,
two, or all three of the following methods to “scan” the crime scene for possible latent
bloodstains. All three of these methods are presumptive. A positive result does not establish the
presence of blood, but provides a reasonable supposition that blood may be present.

Alternate light sources Unlike most body fluids, blood does not fluoresce at visible
wavelengths, but that doesn’t mean an alternate light source is entirely useless in locating blood
stains. Blood strongly absorbs light in the near UV and violet ranges, so illuminating a suspect
area with a black light may render bloodstains more visible because they are darkened relative to
the background.

Luminol
In a 1937 paper, the German chemist Walter Specht was the first to suggest the use of
luminol for forensic blood detection. An aqueous or alcoholic solution of luminol and an oxidizer
is catalyzed by the iron present in the hemoglobin component of blood to produce 3-
aminophthalate (3-APA) in an excited state. Excited 3-APA molecules quickly return to their
base state, emitting photons that are visible as a characteristic weak blue luminescence (not
fluorescence; in the presence of iron or another catalyst, luminol emits light directly). Luminol is
extraordinarily sensitive to the presence of blood, yielding positive results at dilutions as high as
100,000,000:1, and by some reports 1,000,000,000:1.
 Washing a surface thoroughly or even painting over a bloodstained surface often leaves
sufficient traces of blood to yield a positive test with luminol. Unfortunately, this high sensitivity
is accompanied by low selectivity. Many materials other than blood—including copper and other
metals, laundry bleach, and many food items—yield positive luminol tests that are
indistinguishable from positive results caused by actual blood. Interestingly, most body fluids
other than blood do not react with luminol. When luminol is used at a crime scene, cameras and
luminescent markers are set up first, the crime scene is darkened, and then luminol solution is
sprayed liberally on all surfaces that may have latent bloodstains. (It’s not uncommon for
forensic technicians to use what amount to garden sprayers to spray luminol solution literally by
the liter.) Walls are sprayed first to detect spatter patterns, followed by the ceiling to detect cast-
off patterns, followed by the floor to detect footprints, drag marks, and so on.
Luminol, at least in aqueous solution, is considered nondestructive. Although it may
interfere with some older serological tests, using aqueous luminol does not interfere with
subsequent PCR DNA analysis.

Fluorescein
Aqueous or alcoholic solutions of fluorescein are used in the same way as luminol to
detect latent bloodstains. The chief differences are that fluorescein is much less sensitive than
luminol—10,000:1 versus 10,000,000:1 or more—and must be illuminated by an ALS (alternate
light source) at about 445 nm (indigo) to induce fluorescence at 520 nm (green). The deep violet
visible light produced by most “black light” bulbs may produce faint fluorescence with
fluorescein; more intense forensic ALS units produce much brighter fluorescence. Like luminol,
fluorescein is subject to false positives caused by many common materials. With all of those
drawbacks, you might reasonably wonder why any forensic scientist would use fluorescein
instead of luminol. The answer is, they don’t. Fluorescein is used only if luminol fails to yield
usable results. At times, fluorescein may reveal latent bloodstains when luminol fails, because
materials are present that either yield false positives with luminol or suppress the luminescence
of luminol. In particular, fluorescein is used when the presence of bleach interferes with luminol.
If patent possible bloodstains are present, or if a positive result with any of the preceding
tests indicates that latent bloodstains may be present, the next step is to run a color-change test to
verify the result from the preliminary screening test. Colorchange tests are less sensitive than
luminol, but more selective.
Although a positive result with the color-change test is not confirmatory in a legal sense,
it adds weight to the presumption of the presence of blood and indicates that legally confirmatory
tests should be done in the lab. If the color-change test is negative, investigators know not to
waste scarce lab resources to investigate further. The first such color-change test, the guaiacum
test, was introduced in 1865. In the presence of blood, guaiacum and hydrogen peroxide react to
form the dye guaiacum blue.
Although it is relatively sensitive, about 50,000:1, the guaiacum test is extremely
unselective, yielding false positives with numerous materials. Despite its lack of selectivity, the
guaiacum test was commonly used until 1904, when the benzidine test was introduced. In the
presence of acetic acid and hydrogen peroxide, benzidine reacts with blood to form the dye
benzidine blue. The benzidine test is no more selective than the guaiacum test, but is
considerably more sensitive at about 250,000:1.
The next color-change test reagent, o-toluidine, was introduced in 1912. In the presence
of acetic acid and ethanol, o-toluidine reacts with blood to form a characteristic blue-green stain.
The o-toluidine test is no more selective than the guaiacum or benzidine tests, but is considerably
more sensitive, at about 5,000,000:1.
The benzidine and o-toluidine tests remained in use for decades, until it was learned that
both of these compounds are strongly carcinogenic. Fortunately, alternative methods are
available, including the following:

Kastle-Meyer test
The Kastle-Meyer (KM) test, introduced in 1901 by Kastle and improved in 1903 by
Meyer, uses an alkaline phenolphthalin solution (the reduced form of the phenolphthalein
commonly used as an acid-base indicator). Phenolphthalin in alkaline solution is colorless or a
very pale straw yellow, as opposed to phenolphthalein, which is bright pink in alkaline solution.
KM reagent reacts with the heme component of the hemoglobin in blood and hydrogen peroxide
to produce the oxidized phenolphthalein form, which turns bright pink.
The KM test can be run in two ways. If the phenolphthalein reagent and the hydrogen
peroxide are combined first, the test is extremely sensitive (10,000,000:1 or better) but is also
extremely unselective. Conversely, if the reagents are applied sequentially—phenolphthalin
reagent first, followed by hydrogen peroxide—the test is much less sensitive (~20,000:1) but
extremely selective. For that reason, if KM is to be used as a secondary test, the sequential
method is used for its higher selectivity. For that method, a small amount of the suspect stain is
transferred to a cotton swab moistened with water. A drop or two of alcohol is placed on the
swab to lyse the blood cells and release the catalase enzyme present in hemoglobin. A drop or
two of the alkaline phenolphthalin reagent is placed on the swab and observed for a few seconds.
An immediate pink stain is inconclusive, indicating the presence of an inorganic oxidizer
or a base. (Blood may also be present, but is screened by the oxidizer or base.) If the swab
remains colorless, a drop or two of drugstore 3% hydrogen peroxide is placed on the swab. If
blood is present, the catalase enzyme catalyzes the breakdown of the hydrogen peroxide into
water and oxygen, and the swab immediately assumes a pink color. (Atmospheric oxygen causes
the swab to turn pink within a minute or so even in the absence of blood.)
False positives can be produced by vegetable peroxidases, including those present in
horseradish, potato, and other vegetable matter. The KM test is still widely used because it
combines high sensitivity and selectivity with low cost. Well-funded departments often use the
more convenient (and more expensive) tetramethylbenzidine test (described in just a moment),
but the KM test remains a mainstream procedure.

Although, like all catalytic tests, the KM test is subject to false positives, those false
positives may be at least somewhat distinguishable from true positives. That is, in the
presence of interfering materials, many blood reagents yield color changes that are
indistinguishable from the color change that occurs in the presence of actual blood. KM
reagent reacts with many interfering materials to yield a color change, but that color often
has a yellowish, orangish, or reddish tint rather than the pure bright pink of a true
positive.

Leucomalachite green
The leucomalachite green (LMG) test works on the same principle as the KM test. In its
reduced form, LMG is colorless. When it reacts with the oxygen released by catalase catalysis of
hydrogen peroxide, LMG is oxidized to the blue-green dye malachite green. In use, a small
amount of the suspect stain is transferred to a cotton swab moistened with water. A drop or two
of LMG solution is placed on the swab and observed for a few seconds. An immediate blue-
green stain is inconclusive, indicating the presence of an inorganic oxidizer. If the swab remains
colorless, a drop or two of drugstore 3% hydrogen peroxide is placed on the swab. If blood is
present, the catalase enzyme catalyzes the breakdown of the hydrogen peroxide into water and
oxygen, and the swab immediately assumes a blue-green color. Although the LMG test is less
sensitive than the KM test and is subject to many of the same interferences, including vegetable
peroxidase enzymes, it is sometimes still used because it is less affected by the presence of
reducing agents like ascorbic acid (vitamin C), which produce false negatives with the KM test.

Tetramethylbenzidene
The tetramethylbenzidine (TMB) test was introduced in 1976 as a safer alternative to the
benzidine and o-toluidine tests. At about 1,000,000:1, the sensitivity of the TMB test is superior
to the sequential KM test, and its selectivity is similar. TMB is the most commonly used color
test because, in addition to its high sensitivity and selectivity, it is very convenient. A packaged
TMB test is available in the form of Hemastix, which are plastic test strips originally designed
for detecting blood in urine. The treated pad on these strips contains 3,3’,5,5’-
tetramethylbenzidine and diisopropylbenzene dihydroperoxide (an oxidizer similar to hydrogen
peroxide). When moistened with distilled water, these strips react with blood within a minute to
form a colored stain that typically ranges from orange through green. If blood is present in high
concentration, the stain may assume a blue color. Some forensics departments use Hematest
tablets rather than Hemastix strips. These tablets work similarly and provide essentially identical
results. In this group, we’ll explore the sensitivity and selectivity of KM reagent and use it to
detect latent bloodstains.