Biology Reference
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(a) (b) (c)
Figure 3.5 In the presence of haem and hydrogen peroxide the chemical colour change can be
seen. To perform a Kastle-Mayer (KM) test a piece of filter paper is folded in half and then half
again to make a corner (b). This is rubbed gently against the blood, transferring a trace of the
dried stain to the filter paper. A drop of KM solution (should give no change by itself) followed by
adropofH 2 O 2 leads to a pink/purple colour reaction in the presence of haemoglobin (c)
phenolphthalein (Kastle - Meyer reagent) (colourless to pink) and tetramethylbenzi-
dine (TMB) (colourless to green) [16 - 19] (Figure 3.5).
Any biological material that contains peroxidase activity, such as some plant
extracts, or any material that leads to the hydrolysis of hydrogen peroxide can also
result in a false positive [20, 21].
Confirmatory tests
The Teichman and Takayama crystal tests, which are based on the formation of
haem-derived crystals, were developed in 1853 and 1912, respectively [17], but,
along with other microscopic and spectroscopic techniques, are rarely used now
[22]. Two relatively new confirmatory tests offer some advantages over previous
tests: messenger RNA (mRNA) analysis allows the origins of several different types
of biological material to be identified; and flow immunochromatographic strip tests
offer a simple and sensitive test for human blood.
mRNA expression analysis has been shown to be a viable confirmatory test
[23 - 27], even in bloodstains that are several months old [28]. The targets that are
amplified are blood-specific, such as the
-spectrin (SPTB) [23, 25, 27], porpho-
bilinogen deaminase (PBGD) [27], δ -aminolevulinate synthase (ALAS2) [25] and
the alpha locus 1 (HBA) marker [29]; the methodology can also be used to differ-
entiate between menstrual and vascular blood, targeting the menstrual blood-specific
matrix metalloproteinase-7 (MMP-7) transcript [24, 30, 31]. The major drawback of
mRNA technology is that it requires specialist techniques and is a more complex
process than DNA profiling itself; however, the extra probative value of identifying
the origin of the blood will be very valuable in some cases.
An antibody-based lateral flow immunochromatographic strip test is relatively easy
to perform. The test involves reacting the suspected bloodstain with a glycophorin A
antibody (glycophorin A is found in the membranes of red blood cells). The mixture
is applied to and migrates along a membrane; if blood is present a visible complex
is formed with an immobilized capture antibody (Figure 3.6) [32].
β
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