Project
protocol
—
Contents
Workflow
and
sampling
Equipment
and
supplies
Reagents
and
solutions
Procedure:
Mouse
densitometry
using
the
DXA
system
Definitions
&
formulas
Data
Reference
Workflow
and
sampling
Workflow
Step
|
Procedure
accomplished
|
Data
collected
|
1
|
Top
loading
scale
for
body
weight
|
bw
|
2
|
Euthanasia:
cervical
dislocation
or
CO2
asphyxiation
|
- |
3 |
Mouse
body
is
propped
for
scanning
|
- |
4
|
|
- |
5
|
Whole
mouse
body
is
scanned
using
PIXImus
Mouse
Densitometer
|
X-ray
image
of
the
mouse
in
2-dimension
|
6
|
Region
of
interest
(ROI)
is
outlined
in
red,
yellow
or
blue;
the
head
is
excluded
and
outlined
in
green
|
BMC,
lean
tissue
mass,
body
fat
mass,
%
body
fat
|
Equipment
and
supplies
- Top
loading
balance:
Ohaus
Navigator,
Model
N12120
with
internal
calibration
(Ohaus
Corp.,
Pine
Brook,
NJ).
Mouse
densitometer
dual
energy
X-ray
absorptiometry
(PIXImus
small
animal)
- DXA
system
(LUNAR,
Madison,
WI),
software
version
1.43.036.008):
The
PIXImus
mouse
densitometer
has
been
reconfigured
with
lower
x-ray
energy
than
in
human
- DXA
machines
in
order
to
achieve
optimal
contrast
in
small
specimens.
- The
Lunar
PIXImus
for
rodents
is
a
fully
integrated
densitometer
designed
for
the
estimation
of
bone
mineral
density
(BMD)
and
body
composition.The
resolution
of
the
PIXImus
is
0.18
x
0.18
mm
pixels
with
a
usable
scanning
area
of
80
x
65
mm,
allowing
for
measurement
of
a
single
mouse
or
collections
of
isolated
specimens.
- The
PIXImus
has
been
calibrated
with
a
phantom
utilizing
known
values,
and
a
QA
is
performed
daily
with
this
same
phantom.
- The
precision
for
BMD
is
less
than
1%
coefficients
of
variations
(CV)
for
whole
body,
approximately
1.5%
CV
for
specialized
regions
(Nagy
et
al,
2000).
Correlation
with
pQCT
values
for
614
isolated
spinal
vertebrae
is
significant
(p<0.001;
r=.704).
- Assessment
of
accuracy
for
the
PIXImus
is
done
with
a
set
of
hydroxyapatite
standards
(0-2,000
mg),
yielding
a
correlation
of
0.999
between
standards
and
PIXImus
measurement
of
mineral.
- For
additional
information:
PIXImus
- Lily
tubs:
64-oz
paper
bucket
(Sweetheart
Cup
Company,
Owings
Mills,
MD).
- Disposable
specimen
trays
with
sticky
immobilizing
tape:
Lunar
PIXImus
Corporation
Headquarters,
726
Heartland
Trail,
Madison,
WI
53717.
Figure
A:
Lunar
PIXImus2
densitometer
with
integrated
PC
computer.
Figure
B:
Close-up
detail
of
the
Lunar
PIXImus2
densitometer
with
specimen
tray.
Reagents
and
solutions
Detergent
and
cleaning
solutions
Acclimation
to
test
conditions
Mice
are
obtained
at
8
and
16
wks
of
age
(DOB
+/-
3
days)
from
production
rooms
and
are
acclimated
to
test
conditions
for
at
least
half-hour.
Procedure:
Mouse
densitometry
using
the
DXA
system
Collecting
image
scans:
a.
The
PIXImus
densitometer
apparatus
is
first
calibrated
with
a
"phantom
mouse"
according
to
manufacturer's
protocol.
b.
Then
each
mouse
is
placed
on
the
specimen
sticky
tray
(body
must
be
within
blue
line
on
the
tray)
under
the
PIXImus
beam
path.
The
tail
is
placed
alongside
the
body,
the
front
legs
are
extended
to
the
side,
and
the
neck
and
spine
are
gently
straightened.
c.
Trays
are
positioned
so
that
the
area
of
the
head
is
always
oriented
toward
the
left
from
the
investigator's
point
of
view,
and
the
mice
are
position
dorso-ventral
so
that
the
entire
body
and
tail
are
measured
in
the
scan.
d.
The
X-ray
process
to
obtain
a
single
full
scan
is
approximately
5
min;
specific
regions
of
interest
(ROI)
are
then
subsequently
defined.
e.
Disposable
plastic
trays,
with
sticky
tape
for
immobilizing
mice,
can
be
saved
and
re-used
after
a
thorough
cleaning
and
disinfections.
Measurement
acquisition
and
image
scan
analysis
a.
Based
on
PIXImus
validation
studies
(Nagy,
2000;
Johnston,
2005)
DXA-estimated
measurements
of
fat
tissue
correlate
well
with
measurements
obtained
from
chemical
extraction.
This
is
made
possible
by
developing
software
versions
with
equations
that
adequately
correct
raw
DXA
measurements.
b.
Following
the
completion
of
an
image
scan
the
DXA
system
automatically
implements
specialized
software
to
identify
bone
tissue
from
either
fat
tissue
or
from
lean
tissue
based
on
the
resulting
X-ray
densities
at
two
distinct
energy
levels
(Pietrobelli,
1996;
Johnston,
2005).
c.
Visually,
following
the
completion
of
a
scan,
the
mouse
sample
is
then
outlined
with
red
and
green
colored
circle
and
square
to
define
specific
regions
of
interest.
d.
By
using
the
screen
interactive
display,
F3
is
first
clicked
to
prompt
measurement
adjustments,
and
then
clicked
again
for
the
second
time
to
adjust
ROI
(region
of
interest).
e.
The
area
to
be
analyzed
is
defined
(red
box),
and
areas
to
be
excluded
from
the
calculations
are
defined
(green).
The
arrow
keys
are
used
to
adjust
to
the
desired
size,
in
addition
to
holding
the
control
key
down
to
enlarge
or
elongate
the
circle
or
square
areas.
f.
Once
the
desired
ROI
is
achieved,
the
Enter
key
is
clicked
and
resulting
data
measurement
is
displayed.
By
pressing
F5
a
hard
copy
of
the
image
and
the
scan
analysis
result
is
printed.
g.
To
prompt
the
computer
to
finish
the
session,
F8
or
Esc
key
is
clicked
once,
and
then
clicked
again
to
return
to
the
main
menu
screen
where
the
next
subject
to
be
tested
begin.
h.
Acquired
data
is
saved
on
the
hard
drive
and
on
a
zip
or
CD
disk
for
later
archiving.
Safety
For
safety,
gloves
must
be
worn
and
radiation
safety
guidelines
are
strictly
adhered
to,
such
that
technicians
must
be
behind
portable
shields
or
6
feet
away
from
the
PIXImus
machine
during
scanning.
A
dosimetry
badge
and
ring
must
also
be
worn.
Since
both
the
PIXImus
unit
and
the
computer
present
icons
that
indicate
the
"ON"
status
of
the
X-ray
tube
with
open
shutters,
precautions
and
any
possibility
of
exposure
should
be
avoided.
Definitions
&
formulas
Bone
area
measurement
is
generated
by
outlining
or
specifying
the
limits
or
dimensions
of
the
entire
skeletal
bone
regions
of
the
body
(limbs,
neck,
spine,
and
tail),
excluding
the
head,
as
regions
of
interest
(ROI)
following
a
full
body
X-ray
scan.
Bone
mineral
content
(BMC)
is
generated
from
PIXImus
density
scans
which
are
assessed
for
accuracy
using
a
set
of
0.0
mg
to
2,000
mg
of
hydroxyapatite
standards.
According
to
the
DXA
system,
bone
mineral
content
(measured
as
the
attenuation
of
the
X-ray
by
the
bones
being
scanned)
is
divided
by
the
area
(also
measured
by
the
machine)
of
the
site
being
scanned
to
obtain
bone
mineral
density
(BMD):
BMD
=
BMC
÷
bone
area
Lean
body
(non-fat)
tissue
weight
=
(Total
body
tissue
weight
–
body
fat
weight)
Body
fat
tissue
weight
=
(Total
body
tissue
weight
-
lean
body
tissue
weight)
Total
body
tissue
weight
=
(BMC
+
Body
fat
tissue
weight
+
Lean
body
tissue
weight)
Data
collected
by
investigator
PIXImus
data
are
obtained
from
the
entire
body
and
tail,
excluding
the
head
area:
reported
tissue
variables
included
lean
tissue
weight,
fat
tissue
weight,
total
body
composition,
and
percent
fat,
bone
mineral
density
(BMD),
bone
mineral
content
(BMC),
and
bone
area.
Test age:
8, 16 wks