Forensic Biology Screening Workshop
Blood
What Is Blood?
• Slightly alkaline fluid made up of water, cells,
enzymes, proteins, glucose, hormones, organic
and inorganic substances
• Circulates throughout body
– Supplies nutrients and oxygen to body
– Removes waste
Blood Cells
• Blood cells are made in the bone marrow
– Hemopoiesis
– Starts as a “stem cell” (blasts)
• Some immature blood cells stay in the
bone marrow to mature
• Others travel to other parts of the body to
mature (lymph nodes, spleen, liver)
Blood Cells
• Cells mature and differentiate into several
classes of cells:
– Red blood cells
– White blood cells
– Platelets
Red Blood Cells (Erythrocytes)
• Have no nucleus
– Not useful for DNA analysis
• 6-8 µm in size
• ~45% total volume of blood
• Most abundant cell in the blood
Red Blood Cells
– Disk shaped cells which make up 99% of the
cells in the blood
– Principal carriers of the red colored
hemoglobin molecules.
• Hemoglobin is an iron containing protein
and binds about 97% of all oxygen in the
body.
Red Blood Cells
Hemoglobin
• Contains 4 subunits
• Globular proteins with heme
• Heme groups contain iron which binds O2
• Arterial Blood: bright red when hemoglobin
carries O2
• Venous Blood: dark red (veins appears blue in
color due to optical effects of light when
hemoglobin lacks O2)
Hemoglobin
Heme With Iron
White Blood Cells (WBC) (Leukocytes)
• Produced in bone marrow
• WBCs have a nucleus
– Useful for DNA analysis
• Vital source of defense against external
organisms
• White blood cells also clean up dead cells and
tissue debris that would otherwise accumulate
and lead to problems.
White Blood Cells
• Five classes of leukocytes:
– Neutrophil
– Eosinophil
– Basophil
– Monocyte
– Lymphocyte
Red and White Blood Cells
image courtesy of the U.S. National Institutes of Health
Platelets
• Irregularly-shaped, colorless bodies produced in
the bone marrow
• Their sticky surface lets them, along with other
substances, form clots to stop bleeding
• Only active when damage occurs to the
circulatory system walls
Hemostasis
Plasma
• Liquid portion of blood
– Composed of water, proteins, electrolytes
– blood cells and platelets are suspended in plasma
• Regulates osmotic pressure
• The transport medium for:
• Glucose
• Lipids
• Hormones
• Oxygen (not as much as heme)
• Clotting factors
• Waste
Serum
• Clear liquid that is left after blood coagulates
• Plasma without the clotting factors
Forensic Significance Of Blood
• Hemoglobin (RBC)
• Peroxidase-like activity can cleave H2O2
• Blood Group Antigen (RBC)
• Bound to RBC membrane (ABO groups)
• DNA (WBC)
• Found in cells with nucleus
• Proteins (PLASMA)
• Serum used in species testing
Forensic Testing
• Presumptive tests
– Indicates a substance is present
– Not specific
• Confirmatory tests
– Confirm a substance is present
– Specific
Presumptive Tests
• Kastle-Meyer (Phenolphthalein)
• Leucomalachite Green (LMG)
• Hemastix
• Luminol
• Other tests:
• Tetramethylbenzidine (TMB)
• Benzidine
• Ortho-tolidine
• Ortho-toluidine
Presumptive Tests
CATALYTIC TESTS
– Based on the fact that hemoglobin (and some of its
derivatives) exhibit a peroxidase activity
• Tests based on this property are generally named
after the compound undergoing oxidation
(benzidine, TMB, etc.) or the discoverer (Kastle-
Meyer, etc.)
– Oxidant (hydrogen peroxide) oxidizes a colorless
reagent to a colored reagent
– Heme catalyzes this oxidation by cleaving an oxygen
from hydrogen peroxide (H2O2)
Kastle-Meyer (KM) Reaction
Kastle-Meyer Test
HOW TO MAKE:
• Phenolphthalein + Potassium Hydroxide + Zinc
• Reflux – boil / condense
• Reduces phenolphthalein to phenolphthalin
– Oxygen removed and combined with OH
from potassium hydroxide – boils off as
water
– Becomes a colorless solution
• Working solution
• Phenolphthalin + ethanol + zinc pellets
Kastle-Meyer Test
HOW TO STORE:
– Amber bottle
• Light affects stability
– Zinc pellets
• Binds free oxygen, prevents oxidation
– Room temp (working solution)
– Refrigerate (stock solution)
Kastle-Meyer Test
HOW TO PERFORM:
• Place a small cutting, swabbing, or extract of the
suspected bloodstain on filter paper
- OR -
• Swab the stain using a slightly moistened swab
Kastle-Meyer Test
3 STEP TEST
STEP 1:
Add 2-3 drops of ethanol to the stain or
swabbing
Note: This will increase sensitivity by cleaning the
area around the hemoglobin, better exposing the
heme
Kastle-Meyer Test
STEP 2:
Add 2 drops of reagent
•Wait for ~5 seconds
Note: This step aids in ruling out false positives
due to the presence of chemical oxidants such as
rust.
Kastle-Meyer Test
STEP 3:
Add 2-3 drops of 3% H2O2
• If immediate color change to PINK – the test is
POSITIVE for the possible presence of blood
• If no color change – blood is not present or is in
too limited quantity for the test to detect.
Note: swab will eventually turn pink (even if negative) over
time due to nature of oxidation reactions.
Kastle-Meyer Test
LIMITATIONS
• Sensitivity
– 1 in 105 to 1 in 106 on dried stains
• Specificity
– Can weed out false positives between steps 2 and 3
– Chemical oxidants, vegetable peroxidases
– Will not detect differences in animal or human blood
• Stability
– Relatively stable if the reagents are stored separately
and refrigerated.
Kastle-Meyer Video
Leucomalachite Green (LMG) Reaction
Leucomalachite Green
HOW TO MAKE:
• Malachite green + acetic acid + water + Zinc
• Reflux – boil / condense
– Becomes a colorless solution
• Working solution
• LMG reagent + zinc pellets
Leucomalachite Green
• HOW TO STORE:
– Amber bottle
– Zinc pellets
– Relatively stable at room temp
Leucomalachite Green
HOW TO PERFORM:
• Place a small cutting, swabbing, or extract of the
suspected bloodstain on filter paper
- OR -
• Swab the stain using a slightly moistened swab
Leucomalachite Green
HOW TO PERFORM:
STEP 1:
Add 1-2 drops of LMG reagent
Note color change, if there is a color change, the
test is considered inconclusive
Leucomalachite Green
HOW TO PERFORM:
STEP 2:
Add 1-2 drops of H2O2
Note results
– If color change to deep green-blue, the test is
positive for the possible presence of blood
– If no color change the test is negative
Leucomalachite Green
LIMITATIONS:
• Sensitivity
– ~1:1000
• Specificity
– Chemical oxidants, vegetable peroxidases
– Will not detect differences in animal or human
blood
• Stability
– Similar to KM
Leucomalachite Green Video
Hemastix
• Reagent strips
• Bottle of 50 reagent
strips
– Store at room temp
• Test is based on the
peroxidase activity of
hemoglobin
Hemastix
• Reagent on hemastix is diisopropylbenzene
dihydroperoxide and
3,3’,5,5’-tetramethylbenzidine (TMB)
• Color change ranges from orange to green
– Possibly blue with higher concentrations of
blood
Hemastix
HOW TO PERFORM:
STEP 1:
Slightly moisten the pad on the tip of the strip with water
STEP 2:
Rub the damp pad on the stain in question
Note color change within 60 seconds and compare to the
chart on the bottle
• more green indicates more hemoglobin
Hemastix
LIMITATIONS
• Sensitivity = 0.015-0.062 mg/dL free hemoglobin
• Specificity
– Chemical oxidants, vegetable peroxidases
– Will not detect differences in animal or human
blood
Stability
– Stable for ~ 1 year – date stamped expiration
date on bottle
Hemastix Video
Luminol
Luminol
• HOW IT WORKS:
– The iron in hemoglobin acts as a catalyst to
cause a reaction between the luminol and
H2O2.
– Luminol loses nitrogen and hydrogen and
gains oxygen.
– This results in 3-aminopthalate which is
energized and emits light
Luminol
• HOW TO MAKE:
– Reagents needed:
• Luminol (3-aminophthalhydrazide)
• Sodium Perborate
• Distilled water
• Sodium Carbonate
Luminol
• HOW TO STORE:
– Spray bottle works best for testing
– Make each time you use it (daily)
Luminol
HOW TO PERFORM:
STEP 1:
Spray the luminol directly onto the stain in question
Note: This test needs to be done in the dark to see the
luminescence reaction which can last for approximately
15 seconds
–If the stain emits a light then the test result is
POSITIVE for the possible presence of blood
–If there is no reaction the result is NEGATIVE
Luminol
LIMITATIONS:
• Sensitivity
– 10-6 to 10-8 – most sensitive presumptive test
• Specificity
– Many false positives – bleach, metals, chemical
oxidants, vegetable peroxidases
– Will not detect differences in animal or human blood
• Stability
– Very unstable ~8 hour limit
• Mostly used at crime scene
– Can dilute out stain (possibly too much for DNA
analysis)
– Used more for blood spatter, crime scene
reconstruction
Tetramethylbenzidine (TMB)
Tetramethylbenzidine (TMB)
HOW TO MAKE:
• 3,3’, 5,5 ‘-tetramethylbenzidine (TMB) + glacial
acetic acid
• Easy to make compared to KM and LMG
reagents
Tetramethylbenzidine (TMB)
HOW TO STORE:
– Amber bottle
• Light affects stability
– Refrigerate between uses – only good for ~ 1
week.
Tetramethylbenzidine (TMB)
HOW TO PERFORM:
• Place a small cutting, swabbing, or extract of the
suspected bloodstain on filter paper
- OR -
• Swab the stain using a slightly moistened swab
Tetramethylbenzidine (TMB)
HOW TO PERFORM:
STEP 1:
Add one drop of TMB solution
STEP 2:
Add one drop of 3% H2O2
Detect color change:
• If the stain turns blueish green, the test result is
POSITIVE for the possible presence of blood
• NEGATIVE if no color change
Tetramethylbenzidine (TMB)
LIMITATIONS
• Sensitivity
– 1:10,000 on dried stains
• Specificity
– Not as specific as KM test
– False positives to vegetable peroxidases,
bleach, potassium permanganate
– Will not detect differences in animal or human
blood
Tetramethylbenzidine (TMB)
LIMITATIONS
• Stability
– Very unstable – 1 week maximum
– Loses sensitivity by a factor of 10 after 1 day
• Safety
– Mutagen
Other Tests
• Benzidine
• Ortho tolidine (o-tolidine) - 3,3'-
Dimethylbenzidine
– Increased sensitivity, decreased specificity,
and same stability when compared to Kastle-
Meyer
– Rarely used due to safety concerns-
carcinogenic
Species Testing
Definitions:
• Antigen (Ag) – a soluble protein that causes the
production of an antibody. Usually foreign to the
body.
• Antibody (Ab) – A “Y” shaped immunoglobulin
produced by B-lymphocytes in the bone marrow.
– Produced in response to an antigen.
Species Testing
• Ag and Ab bind together to create a lattice
network of molecules
• This becomes insoluble and precipitates out of
solution
Species Testing
To make the antibodies:
–Host animal
– Goat, rabbit
–Inject with human serum
–Makes antibodies in response
–Collect heart blood and test for Ab
Sample extracts are made from stained areas of interest,
and from nearby unstained areas (substrate controls).
Note that the use of unstained controls is a fundamental
principle in forensic immunologic testing.
Tube Method
• The original precipitin reaction was carried out
by layering a solution of antibody on top of a
solution of stain extract in a tube
• This mixture was left for a period of time to allow
the development of a precipitin band at the
interface.
• This is referred to as the tube method, and is still
used in a few laboratories today
Ouchterlony
Method published in 1948 and uses radial diffusion of the
antigen and antibody through agar gel.
How to perform:
•Extract the stain in a microcentrifuge tube with saline.
Extract should be straw colored.
•Stain and controls samples are loaded in the outer wells
and a drop of anti-human antiserum is loaded into the
center well. The process is repeated for antisera to other
species, such as dog, cat, and cow; this may include the
species from which the antiserum was obtained (e.g.,
rabbit).
Ouchterlony
• The plates are left at room temperature or 37°C for a
suitable period (which can range from a few hours to
overnight) and the serum proteins and antibody
molecules diffuse outward from the wells.
• A precipitin band is formed when the diffusing stain
contains proteins that are recognized by IgG molecules
in the diffusing antiserum.
• The precipitin band is sometimes clearly visible to the
naked eye, but it is normal to stain the plates with amido
black, Coomassie Blue, or other general protein stain, to
enhance sensitivity and clarity.
Ouchterlony
– If a white line occurs between the samples,
then the test sample is positive for human
antigen activity (or whatever species is being
tested)
– If no white line occurs then the test sample is
negative for human antigen activity (or
whatever species is being tested)
Ouchterlony
• Sensitivity
– 10-1
• Specificity
– False positives:
» Detergents
» Tannic Acid
– False negatives:
» Excess Ab or Ag – the lattice will not
form, no precipitate
Ouchterlony
Ouchterlony Video
Cross-over Electrophoresis
• A variant of the Ouchterlony test, which uses an
electric field rather than diffusion to move the
extract and antibody through the gel.
• Cross-over electrophoresis for species
identification is conducted using agar at a pH of
8.6.
• Stain extracts are loaded into wells arranged in
a line at the cathode end of the plate and the
antiserum is loaded into wells at the anode end.
Cross-over Electrophoresis
• During electrophoresis, the electric field drives
the serum proteins towards the anode, but the
IgG molecules, which are essentially neutral at
this pH, are driven to the cathode by the process
of electroendosmosis.
• The antigen-antibody precipitation occurs at the
interface between the two rows of wells.
Cross-over Electrophoresis
• Electroendosmosis occurs because the
supporting medium acquires a net negative
charge.
– If free, the negatively charged molecules
would migrate to the anode, but this is not
possible because the agar is immobilized on
the plate.
– Instead, the effect is countered by positively
charged water molecules migrating to the
cathode. The migrating water molecules
carry any dissolved neutral molecules (such
as IgG) with them.
ABAcard® Hematrace®
• Tests for human hemoglobin (Hb)
• If human Hb is present – reacts with a mobile
monoclonal anti-human HB antibody
• Forms a mobile Ag-Ab complex
• This migrates to the “T” zone
ABAcard® Hematrace®
• In the “T” zone – polyclonal antihuman HB
antibodies.
• Forms Ab-Ag-Ab complex
• Antibodies tagged with pink dye – upon
aggregation at “T” zone – pink line
• Control zone – has immobile anti-
immunoglobulin which binds excess antihuman
HB antibodies – form a pink line
ABAcard® Hematrace®
To perform:
• Extract cutting from stain in ~300 µl buffer
• Leave 1-5 minutes
• Add 150 µl of sample to the “S” well of the test
card
• Wait 10 minutes – read results
• Pink line in “T” and “C” zones = POSITIVE
• Pink line in only “C” zone = NEGATIVE
ABAcard® Hematrace®
Limitations:
• High Dose Hook Effect – can give a false negative
– Occurs with excess hemoglobin which binds to the
stationary antihuman HB Antibody in the “T” area
– This prevents the mobile Ab-Ag complex from binding
– Results in no pink line = NEGATIVE
– FALSE NEGATIVE
• Not a confirmatory test – gives positive result with ferret
blood
ABAcard® Hematrace®
Hematrace Video
Seratec HemDirect Hemoglobin Assay
Tests for human hemoglobin (Hb)
• Similar to the ABAcard® Hematrace® test
Hematin Crystals
• Teichmann published method in 1853
• Blood is treated with a solution of potassium bromide,
potassium chloride and potassium iodide in glacial acetic
acid, and is heated to react with hemoglobin. The
reaction first converts the hemoglobin to hemin, and then
the halides react with the hemin to form characteristic
brownish-yellow rhomboid crystals.
• The test went through many modifications
• Test did not always work even when the presence of
blood was known
Hemochromagen Crystals
• Many variations were used
• Takayama described his method in 1912, and it
became the preferred method. His name is
commonly used to describe the reagents and
procedure he devised and also the
hemochromogen crystals obtained
• Blood is treated with a saturated dextrose
solution, sodium hydroxide, pyridine and water.
Ferrous iron from hemoglobin reacts with
pyridine to produce red feathery crystals of
pyridine ferroprotoporphyrin
Takayama Crystals
image courtesy Steve Gilbert, M.F.S., PhD -- SUNY at Canton
Crystal Tests
– Crystals are unique to blood
– Not human specific
– Not widely used anymore
– Combo KM and ABA card
– Time/money constraints
– DNA testing is higher primate specific
– Use of qPCR
Controls
• Positive
• Negative
• Substrate
Positive Controls
• Used to determine if the tests are working
properly
– a quality control check
• This is documented in the case file
Example: Known blood on cloth or a swab
Negative Controls
• Used to determine if the reagents are
contaminant free and working properly
– a quality control check
• This is documented in the case file
Example: unstained cloth or a swab
Substrate Controls
• Used to determine if the substrate is interfering
with the test
– troubleshooting
• This is documented in the case file
Example: unstained area adjacent to the
questioned stain being tested
