blood Flashcards
detection of blood
color, solubility, catalytic test
visual examination
blood has a red, red/brown, or brown color
depends on the age of the stain
color change is due to the hemoglobin breaking into one of its derivatives
enviro factors will aid in the bread down of hemoglobin
heat light humidity
blood is water soluble
hemoglobin responsible for red color
moisten swab with di water and swab stain lightly
color transfer = water soluble
blood may not always appear red brown
sometimes blood can absorb color properties of substrate or chemicals it has been exposed to
leuco-crystal violet is used to enhance impressions (prints) in blood and blood will appear purple
LCV does not affect DNA analysis
this may affect your ability to report blood detected for blood indicatied
based on agency policy and procedure
LASD - red brown color, water soluble, positive KM test
light colored items
easiier to locate and document bloodstains
dark colored items
can be difficult to see
use diff light sources at different angles
ALS, flashlights
note any additional lights used to help locate stains
light sources can also help with very small stains (spatter)
use what you have, microscope
oblique lighting is about 45 deg
blood does not fluoresce in ALS, but substrate blood is on my fluoresce, blood is in gap of fluorescence
chem presumptive tests for blood
quick and easy
sensitive, can detect blood down to 1:10,000 (direct)
somewhat specific
reacts with human and non-human blood
always perform positive and negative controls, document
not an identification test, not specific to human blood
catalytic test for blood
used on visible blood stains
based on redo rxns
in biochemical reactions, oxidation often coincides with loss of hydrogen
hydrogen peroxide is usually employed as an oxidant, taking hydrogens
heme serves as a catalyst
has peroxidase-like activity
in the presence of heme, a colorless substrate is oxidized to a product with color, chemiluminescence, or florescence
3 part test can make more specific
add drop ethanol, add colorless substrate, ensure no color change, add peroxide
colorless AH2 + H2O2 => colored A + 2H2O
=> is heme
phenolphthalein ( Kastle-Meyer, KM) pink
leucomalachite green (LMG) blue-green
benzidine - not used anymore, carcinogenic
O-tolidine - not used any more carcinogenic
tetramethylbenzidine (TMB) - tetramethyl derivative of benzidine - tests for blood in urine, strips
hemastix - TMB based-kit
Kastle-Meyer, phenolphthalein
moisten swab and apply to stain
add couple drops ethanol
add couple drops KM reagent
add couple drops H2O2
if swab turns pink then presumptive positive for blood, based on agency
color change after KM, test is not valid, use another form of testing
if pink after swabbing (magenta bedsheet) use another test for better visualization
Leucomalachite green (LMG)
performed same as KM,
could be good to have another test if substrate color is the same as catalytic test
Hemastix
TMB based assay kit
used to detect blood in urine as well
strips
ways to perform color catalytic tests
direct method
cutting or scraping of bloodstain is placed into a tube where reagents are added directly to stain
most sensitive
indirect method, most preferred
bloodstain is transferred to a swab or filter paper, reagents added to that
aged bloodstains are less soluble and could require a direct test
false positives
turns pink without H2O2
sensitive tho tests are not specific
chemical oxidants and catalysts
copper and nickel salts, rust, some bleaches, potassium permanganate, pus and other leucocyte containing secretions
plant tissues and plant peroxidase
apple, apricot, bean, blackberry, Jerusalem artichoke, horseradish, potato, turnip, cabbage, onion, dandelion root
you increase specificity by the 3 step method, ethanol
chemiluminescence and fluorescence tests
in the chemiluminescence assay the light is emitted as a product of a chem rxn, luminal
oxidation of luminal catalyzed by heme produces an intense light
can only be observed in a dark enviro
no need to add light
in a fluorescence assay requires exposure of oxidized product to a particular wavelength of light, the fluorescence is then emitted at a longer wavelengths, fluorescin
when oxidized and catalyzed be heme, it demonstrates fluorescent properties
usually exposed to ALS between 425-485 nm will emit a yellowish-green fluorescent light
one advantage of these tests are that they can be sprayed over large areas where bloodstains are suspected
positive reaction identifies blood and also reveals patterns such as footprints, spatter, etc
these methods are very sensitive and can pinpoint locations of even small traces of blood
also they are useful for detecting blood at crime scenes that have been cleaned and show no visible staining
one disadvantage is that by spraying a small stain you may dilute a sample and thus lead to a low DNA yield
luminol
reagents,
0.1gm luminol
0.5gm sodium carbonate, provides the alkaline condition needed for oxidation
0.7 gm sodium perborate
100 ml DI water
poor specificity
cross reacts with chemical oxidants and catalysts
metals, iron, copper, cobalt compounds
penny as a positive control b/c known size
strong oxidants (bleaches and potassium permanganate)
plant sources
peroxidases
great sensitivity
can detect blood diluted one million times
always follow up with a catalytic color test
can also be used for dark clothing
species determination: Ouchterlony and CIE
used to detect human proteins in a bloodstain
both methods involve interaction of antigens and antibodies
positive results were obtained by the formation of a [recipitin band between the antisera well and sample well
CIE more sensitive than ouchterlony
replaced by quicker and faster tests
RSID blood, detects red blood cell antigen (cross reacts with higher primates)
Hematrace and hexagon OBTI- detects human hemoglobin antigen also reacts with higher primates
ouchterlony
double immunodiffusion assay
