Forensic%20Serology3

Forensic%20Serology3 - 8/11/10 Forensic
Serology


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Unformatted text preview: 8/11/10 Forensic
Serology
 Jeannette
M.
Perr,
Ph.D.
 Forensic
Serology
History
 •  1901
Karl
Landsteiner
discovered
blood
typing
 –  Blood
is
distinguishable
by
its
group
or
type
 –  A‐B‐O
system
 •  1937:
Rh
factor
discovered
(a.k.a.
D,
+/‐)
 •  Today:
many
different
blood
types
known
 •  Until
the
early
1990’s
forensic
scientist
used
blood
 typing
 •  High
frequency
of
blood
stains
at
crime
scenes
 –  Rape,
murder,
assaults
 •  The
more
factors;
the
more
individualized
 1 8/11/10 Forensic
Serology
Outline
 •  Origins
of
Modern
Serology
 •  Blood
Basics
 •  Immunoassay
Techniques
 •  Genetics
 •  Forensic
Characterization
of
Bloodstains
 •  Stain
Patterns
of
Blood
 •  Semen
and
Rape
Evidence
 DNA
changed
all
that…
 • Characterizing
blood
through
factors
 has
been
abandoned
characterizing
 specific
regions
of
our
genetic
code,
 deoxyribonucleic
acid
(DNA)
 • Before
DNA
though
was
Forensic
 Serology
 • Still
an
important
field
of
forensics!
Ex:
 used
to
find
basic
bodily
fluid
for
DNA
 testing
 2 8/11/10 The
DNA
Advantage
 •  DNA
does
have
its
advantages…
 –  DNA
can
be
extracted
from
dried
blood
 and
other
biological
fluids
 –  Highly
sensitive
analysis
 –  Individualizing
 What
is
Forensic
Serology?
 • Forensic
serologists…
 – Determine
type
and
characteristics
of
 blood
 – Blood
stain
and
pattern
analysis
 – Examination
of
semen,
saliva,
other
 body
fluids
 – Trial
testimony
 3 8/11/10 What
is
blood?
 •  Blood
is
a
mixture
of
cells,
enzymes
proteins,
and
 inorganic
compounds
 •  Plasma
is
the
fluid
portion
of
blood
(55%)
 •  Suspended
in
the
plasma
are
cells
(45%)
 –  Erythrocytes
(red
blood
cell)
 –  Leukocyte
(white
blood
cells)
 –  Platelets
 •  Blood
clots
when
fibrin
(a
protein)
traps
red
blood
cells
 •  Serum
is
the
liquid
that
is
separated
from
the
blood
 when
a
clot
forms
 Blood
Composition
 45% 4 8/11/10 Blood
 •  On
the
surface
of
red
blood
cells
are
 antigens
 –  Antigens
impart
blood
type
characteristics
 to
the
red
blood
cell
 •  15+
different
types
of
antigen
systems
have
 been
identified
 –  Ex:
A,
B,
O,
&
Rh
 –  Rh
is
also
known
as
antigen
D,
+/‐
 Typing
 •  Type
A
=
the
person
has
A
type
antigens
 •  Type
B
=
the
person
has
B
type
antigens
 •  Type
AB
=
both
antigens
 •  Type
O
=
has
neither
A
or
B
antigens
 –  O
is
the
universal
donor
(not
lucky);
AB
is
 universal
receiver
(lucky)
 •  Rh
(or
D)
=
positive
or
negative
 –  A+
=
type
A
with
D
 –  B‐
=
type
B
without
D
 5 8/11/10 Blood
Type
 A
 A+
 A‐
 B+
 B‐
 AB+
 AB‐
 O+
 O‐
 X
 X
 Antigens
on
RBC
 B
 Rh(D)
 X
 X
 X
 X
 X
 X
 X
 X
 X
 X
 More
Blood
 •  Antibody:
a
protein
that
destroys
or
inactivates
a
 specific
antigen
 –  For
every
antigen
there
exists
a
specific
antibody
 •  Agglutination
 –  Clumping
together
of
red
blood
cells
by
the
 action
of
an
antibody
with
its
antigen
 –  Caused
when
antibody
combines
with
its
 antigen,
cross‐linking
red
blood
cells
(e.g.
A
 antigen
with
anti‐A
antibody)
 •  Test
A+
blood
with
anti‐A
antibody
=
clump
 6 8/11/10 Putting
it
together…
 Blood
 Antigens
 Antibodies
in
 Reaction
w/
 Reaction
w/
 Type
 on
RBC
 serum
 Anti‐A
serum
 Anti‐B
 serum
 A
 A
 Anti‐B
 +
 ‐
 B
 AB
 O
 B
 Anti‐A
 ‐
 +
 ‐
 +
 +
 ‐
 A
and
B
 Neither
Anti‐ A
nor
Anti‐B
 Neither
 Anti‐A
and
 A
nor
B
 Anti‐B
 Clump Clump •  AB
has
two
clumps
 •  B
clumps
with
anti‐B
 •  A
clumps
with
anti‐A
 •  O
clumps
with
nothing
 Clump Clump 7 8/11/10 Blood
Type
Statistics
 Genetics
of
Blood
Types
 •  Traits
are
demonstrated
through
an
allele
 –  Allele:
alternative
form
of
a
gene
located
at
the
 same
point
(locus)
on
a
particular
pair
of
 chromosomes
 •  In
the
A‐B‐O
system,
there
are
three
alleles:
A,
B,
 and
O
 •  In
the
Rh
system,
there
are
two
alleles:
Rh+
and
Rh‐
 •  A
genotype
contains
all
inherited
alleles
while
a
 phenotype
is
the
allele
that
is
actually
being
 expressed
 –  Ex:
brown
eyes
versus
blue
eyes
 8 8/11/10 Visualized
 •  Two
alleles
at
a
loci
 •  A
and
B
are
dominant;
 O
is
recessive
 •  Rh+
is
dominant;
Rh‐
is
 recessive
 Genotype
 AA
 BB
 AB
 AO
 BO
 OO
 Rh+/Rh+
 Rh+/Rh‐
 Rh‐/Rh‐
 Phenotype
 A
 B
 AB
 A
 B
 O
 Rh+
 Rh+
 Rh‐
 Heredity
and
Blood
Types
 What
blood
type
will
baby
be?
 Father’s Genotype O A Mother’s Genotype O B Punnet square 9 8/11/10 Problems
 •  Variation
in
agglutination
titer
–
sensitivity
 can
be
poor
 •  Works
best
with
blood
less
than
five
days
old
 •  Dried
stains
require
a
different
procedure
 •  Require
a
different
antibody
for
every
 antigen
 –  Over
100
antigens
 •  What
about
mixtures?
 Forensic
Application?
 •  Paternity
Testing!!!
 –  Suppose
Mom
is
type
AB
and
Baby
is
type
 AB,
could
the
male
from
the
last
example
 have
fathered
the
child?
(Dad
was
O,O)
 –  Suppose
the
Baby
was
type
A
or
type
B…
 –  What
can
we
say
about
the
male
from
the
 last
example?
 10 8/11/10 Forensic
Characterization
of
 Blood
Stains
 1.  Is
it
blood?
 2.  From
what
species
is
the
blood?
 3.  If
it’s
human
blood,
how
well
can
we
 associate
it
with
a
particular
individual?
 Is
it
Blood?
 Hemoglobin
tests:
 1.
Kastle‐Meyer
color
test
 
Turns
pink
 2.
Hemastix®
 
Turns
green
 3.
Luminol
test
 
Gives
off
a
bluish‐green
 glow
under
UV
light
 (luminescence)
 11 8/11/10 General
Notes
on
GLP
and
PPE
 •  Wear
gloves
and
use
techniques
to
avoid
blood
spatter
 •  Assume
everything
is
infected
with
 –  Hepatitis
 –  AIDS
 –  TB
 •  Wash
 –  after
exposure
 –  before
eating
 –  when
leaving
the
scene
or
laboratory
 •  Never
 –  eat
in
the
laboratory,
 –  wear
your
lab
clothing
outside
the
laboratory
 –  wear
contact
lenses
in
the
laboratory
 •  Avoid
being
poked
by
sharps
 •  Follow
posted
warning
signs
 •  Decontaminate
work
area
 Presumptive
Tests
 •  Do
you
have
blood?
 –  Preliminary
tests
 • Benzidine
 • Phenolphthalein
(Kastle
Meyer)
 –  Both
tests
rely
on
the
fact
that
blood
reacts
with
 hydrogen
peroxide
(H2O2)
in
the
presence
of
an
 indicator
to
produce
a
deep
pink
color
 –  Interferences
include
horseradish,
potatoes,
or
 any
other
substance
with
peroxidase
activity
 12 8/11/10 Hemastix/Hematrace
tests
–
originally
for
blood
in
urine
 Hemastix
Blood
Reagent
Strips
 Hemastix
are
small
3”
plastic
test
strips
with
a
square
of
blood
reagent
 material
at
the
tip.
Unknown
substances
may
be
absorbed
onto
a
swab
 moistened
with
sterile
water,
and
then
transferred
through
contact
to
the
 square
of
blood
reagent
material.
The
yellow
reagent
material
will
then
turn
 shades
of
green
if
blood
is
present.
Used
commonly
to
detect
blood
in
urine.

 Sold
in
a
vial
of
50
test
strips.
 Luminol
 •  Sprayed
on
a
bloodstain
and
will
omit
light
 that
can
be
observed
in
the
light
 –  Interferences
with
metal
 –  Extreme
sensitivity
 –  Needs
darkness
 13 8/11/10 Now
that
its
blood…
 •  Is
it
human
blood?
 –  Human
anti‐serum
reacts
with
(antigens
in)
 human
blood
 –  Gives
a
precipitate
at
the
interface
 Not me! •  What
is
anti‐serum
and
how
is
it
made?
 –  Blood
serum
that
contains
certain
 antibodies,
in
this
case
human
 –  Rabbit
injected
with
human
blood
and
anti‐ sera
is
collected
 Ring
Precipitan
Test
 •  Blood
is
placed
in
a
capillary
tube
containing
 human
anti‐serum
 –  Ring
=
human
blood;
no
ring
=
not
human
blood
 14 8/11/10 Can
we
associate
the
blood?
 •  Can
human
blood
be
narrowed
down
to
a
 particular
human?
 •  Kinda…
 –  Prior
to
DNA
analysis
 • Blood
typing
and
blood
factors
 • Use
the
multiplication
rule……
 –  Now:
DNA
analysis
 Another
aspect…blood
spatter
 •  Surface
texture
 –  Harder,
smoother,
less‐ porous
=
less
splatter
 •  Direction
of
travel
 –  Pointed
end
=
direction
of
 travel
 •  Impact
Angle
 –  90°
=
circle.



As
angle
 decreases,
stain
elongates
 •  Impact
Splatter
 –  Intersection
of
lines
along
 long‐axis
=
emanation
point
 Read pages 359 – 364 in your book!!!! 15 8/11/10 Semen
Evidence
 1.  Locate
stain
 2.  Characterize
stain
 •  Presumptive
test
for
seminal
stain
 –  acid
phosphatase
enzyme
color
test

 •  Confirmatory
tests
for
semen
 –  microscopic
examination
of
spermatozoa
 –  p30
(prostate
specific
antigen,
PSA)
 Acid
Phosphatase
test
 •  Seminal
fluid
residue
can
be
detected
with
either:
 –  Sodium
alpha
naphthylphosphate
and
Fast
Blue
B
dye:
 turns
blue
 –  4‐methyl
umbelliferyl
phosphate
(MUP):
which
 fluroresces

under
UV
light
 •  However,
false
positives
may
occur
from
 –  –  –  –  Cauliflower
and
watermelon
 Fungi
 Contraceptive
creams
 Vaginal
secretions
 16 8/11/10 Semen
Stains
 •  Can
be
recovered
from
garments
years
later
 –  Can
be
visualized
as
a
white
or
yellowish
stain
 –  Can
fluoresce
under
UV
light
 Microscopic
confirmation
 •  •  •  •  Immerse
stained
material
in
small
amt
of
water
 Transfer
drop
to
microscope
slide
and
dry

 Examine
under
a
compound
microscope
at
400X
 Potential
issues:
 –  Spermatozoa
brittle
when
dry
 –  Bind
tightly
to
cotton
 –  Low
sperm
count
 17 8/11/10 Acid
Phosphatase
Test
 This
test
offers
the
possibility
to
determine,
on
the
spot,
whether
the
traces
 found
originate
from
spermatozoa.
Take
a
small
piece
of
the
suspect
material
(a
 few
square
millimeter)
and
soak
for
about
one
minute
in
water
or
physiological
 saline
solution.
Remove
only
as
many
test
papers
as
are
required
and
reseal
the
 container
immediately
after
use.
Expose
the
test
paper
by
pulling
apart
the
ends
 of
the
plastic
tape.
Do
not
touch
the
test
paper
zone.
Place
the
moist
test
 material
on
the
test
paper,
which
after
a
few
seconds
will
show
a
clear,
violet
 coloration
in
the
presence
of
acid
phosphase.

After
the
test,
it
can
be
resealed
 and
stored
as
proof.


 Specifity
 Phosphatesmo
KM
is
specific
for
acid
phosphatase.
Control
tests
with
saliva,
 mucus,
or
female
genital
secretions
give
negative
results.
The
reaction
is
not
a
 suitable
substitute
for
the
microscopic
determination
of
live
spermatozoa.
 http://www.bvda.com/EN/prdctinf/en_phosp.html
 Why
PSA?
 •  Prostate
specific
 antigen
(PSA)
is
an
 enzyme
produced
in
 the
ducts
of
the
 prostate
and
absorbed
 into
the
bloodstream
 •  It
is
proof
of
human
 sperm
 •  Will
be
found
even
in
 vasectomized
males
 18 8/11/10 Rape
Evidence
 •  Victim
rape
collection
kit
 –  Pubic
combings
 –  Pubic
hair
and
head
hair
reference
samples
 –  Vaginal
/
rectal
/
oral
/
skin
swabs
 –  Blood
sample
(DNA
and
tox)
 –  Fingernail
scrapings
 –  All
clothing
 –  Urine
sample
(tox)
 Rape
Evidence
 •  Suspect
collection
 –  All
clothing
 –  Pubic
hair
combings
 –  Head
and
pubic
hair
reference
samples
 –  Penile
swab
 –  Blood
sample
or
buccal
swab
(DNA)
 19 8/11/10 Immunoassay
Techniques
 •  Enzyme‐multiplied
immunoassay
technique
(EMIT)
 –  Detecting
drugs
in
urine
 •  i.e.,
marijuana
(THC‐9‐carboxylic
acid)
 •  How
does
it
work?
 –  1.
Protein‐drug
complex
(antigen)
injected
in
animal.
Recovered
serum
 contains
antibodies.
 –  2.
Subject’s
urine
is
mixed
with
antibodies.
Any
drug
present
binds
to
 antibodies.
 –  3.
Enzyme
labeled
drug
is
added
in
excess.
It
binds
with
free
antibodies.
 –  4.
Unbound
enzyme
labeled
drug
is
measured
and
related
to
original
 concentration
of
drug
in
urine.

 Testing
for
semen
 •  Membrane
contains
two
monoclonal
anti‐PSA
 antibodies.

One
of
these
antibodies
is
 immobilized
at
the
test
region.
 •  A
second
fiber
pad
contains
the
dried
and
gold
 labeled
second
monclonal
murine
anti‐PSA
 antibody.

 •  PSA
at
the
sample
will
bind
to
the
re‐mobilized
 gold‐labeled
antibody
and
form
a
PSA‐gold‐ labeled‐anti‐PSA‐antibody‐complex.
Through
the
 capillary
action,
the
reaction
mixture
is
carried
 upwards
with
the
fluid.
If
the
sample
contains
 PSA
the
complex
will
bind
to
the
immobilized
 monoclonal
antibody
of
the
test
region
that
 recognizes
another
epitope
on
the
PSA
molecule.
 The
binding
is
indicated
by
the
formation
of
an
 additional
line.
 •  
Thus
a
PSA
positive
sample
will
show
three
 colored
lines
in
the
result
well.
The
line
in
the
 middle
(internal
standard)
correlates
with
an
 amount
of
4
ng/ml
PSA.
In
some
cases
it
might
be
 helpful
to
estimate
the
amount
of
PSA
inthe
 sample
by
comparison
of
the
result
line
with
the
 internal
standard
line.

 20 8/11/10 Tests
for
Saliva
 Saliva

consists
of
99%
water,
mucin
(protein

that
helps
in
swallowing),

and
 amylase
(enzyme
that
helps
digest
carbohydrates) 
 To
test
for
saliva
mix
starch,
iodine,
 and
the
stain
together.
\
Starch
mixed
 with
iodine
produces
a
blue
color.

If
 amylase
is
present
it
will
turn
a
dark
 blue‐purple.
The
color
will
fade
in
 about
15
minutes
at
37
°C. Tissue
and
Stain
Typing
by
RNA
 •  An
alternative
method
to
identify
body
fluids
under
 development
by
the
FBI
is
the
probing
of
messenger
RNA
 (mRNA)
species
that
are
selectively
expressed
in
cells
that
 collectively
comprise
a
particular
body
fluid
 •  Each
cell
type
in
the
human
body
has
a
unique
pattern
of
gene
 expression
that
is
manifested
by
the
presence
and
relative
 abundance
of
specific
mRNA
species,
the
molecular
 intermediate
between
genomic
DNA,
and
expressed
protein
 •  Candidate
tissue‐specific
genes
have
already
been
identified
for
 saliva
(statherin,
histatin
3,
PRB1,
PRB2,
and
PRB3),
but
tissue‐ specific
genes
for
other
body
fluids
of
forensic
importance
 have
yet
to
be
identified
 21 8/11/10 Rape
Evidence
 •  Victim
rape
collection
kit
 –  Pubic
combings
 –  Pubic
hair
and
head
hair
reference
samples
 –  Vaginal
/
rectal
/
oral
/
skin
swabs
 –  Blood
sample
(DNA
and
tox)
 –  Fingernail
scrapings
 –  All
clothing
 –  Urine
sample
(tox)
 Rape
Evidence
 •  Suspect
collection
 –  All
clothing
 –  Pubic
hair
combings
 –  Head
and
pubic
hair
reference
samples
 –  Penile
swab
 –  Blood
sample
or
buccal
swab
(DNA)
 22 8/11/10 Differential
Extraction
 Sperm
Cells
and
Non‐Sperm
Cells
 Regular
Cells
are
Broken
 •  Evidence
swab
is
placed
in
EDTA,
salt,
buffer,
 and
a
detergent
 •  Proteinase
K
is
added
and
heated
for
two
 hours
 •  Swab
is
centrifuged
and
removed
 •  Pellet
contains
the
sperm
and
the
fluid
 contains
the
broken
cell
DNA
 23 8/11/10 Get
out
the
Sperm
DNA
 •  Tris‐HCl,
EDTA,
salt,
buffer,
Sarkosyl,
 Proteinase
K,
DTT
 •  Wait
two
hours…
 Conclusions
 •  Forensic
serology
involves
the
detection
of
biological
stains
at
a
 crime
scene
 •  Serological
analysis
is
performed
prior
to
DNA
testing
and
 determines
the
type
of
stain
and
if
it
is
human
 •  Serological
tests
consist
of
presumptive
(chemical)
and
 confirmatory
(antigen/antibody)tests
 •  Presumptive
tests
for
blood
are
based
on
peroxidase
activity
of
 hemoglobin
 •  Confirmatory
tests
are
based
on
inherited
factors
in
the
blood
–
 ABO,
Rh,
PGM
and
are
highly
specific
 •  Serological
tests
exist
for
blood,
(see
above)
semen
(acid
 phosphatase
and

p
30)
and
saliva(amylase)
 •  Sperm
can
be
identified
microscopically
by
staining
 •  Victims
of
rape
are
processed
by
a
sexual
assault
nurse
examiner
 (SANE)
in
concert
with
a
police
officer
and
a
victim’s
advocate.

A
 rape
kit
is
used
in
the
collection
of
evidence.
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This note was uploaded on 03/06/2011 for the course CHS 3501 taught by Professor Perr during the Spring '09 term at FIU.

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