Document ID: EPA-HQ-OPP-2004-0405-0032
Agency: epa
Document Type: Supporting & Related Material
Title: 
Posted Date: 2005-02-08T05:00Z

NHEERL­
NTD­
CMTB­
SP/
98­
002­
000
February
10,
1998
Page
1
Procedural
Section
1.0
Scope
and
Application
1.1
The
Hitachi
911
Automatic
Analyzer
(
Boehringer
Mannheim,
Indianapolis,
IN)
is
used
to
run
a
wide
range
of
chemistries,
e.
g.,
cholinesterase.

1.2
The
Hitachi
911
is
a
spectrophotometer
equipped
with
sophisticated
robotics
that
automatically
dispense
samples
and
appropriate
reagents
into
cuvettes
suspended
in
a
37

C
water
bath.
Absorbance
readings
are
taken
and
enzyme
activity
is
calculated
by
the
analyzer.

1.3
These
analyzers
are
used
extensively
in
many
testing
and
clinical
laboratories
to
ensure
reproducibility
and
increase
sample
throughput.
However,
these
instruments
are
not
appropriate
to
use
in
the
determination
of
cholinesterase
activity
in
carbamate­
treated
tissues,
e.
g.,
carbaryl,
due
to
reactivation
of
cholinesterase
activity
that
occurs
during
the
analyzer's
performance
of
the
assay.

2.0
Prerequisites
2.1
Equipment
and
Supplies
Hitachi
911
Automatic
Analyzer*
Sample
Cups*
Reagent
Bottles*
Pipet,
200

l
Laboratory
Glassware
Analytical
Balance
Ice
*
Analyzer
and
its
supplies
obtained
from
Boehringer
Mannheim,
Indianapois,
IN
All
other
equipment
and
supplies
are
considered
standard
laboratory
devices
or
items
and
do
not
need
to
meet
critical
specifications.

2.2
Chemicals
Acetylthiocholine
Iodide
(
Sigma,
A­
5751)
5,5'­
dithio­
bis(
2­
nitrobenzoic)
Acid
(
DTNB)
(
Sigma,
D­
8130)
0.2M
Sodium
Phosphate,
monobasic
(
Sigma,
S­
9638):
27.6
g
NaH2PO4
in1000
ml
ddH2O
0.2M
Sodium
Phosphate,
dibasic
(
Sigma,
S­
9763):
28.4
g
Na2HPO4
in
1000
ml
ddH2O
0.1M
Sodium
Phosphate
Buffer,
pH
8.0:
380
ml
0.2M
Na2HPO4
(
dibasic)
+
20
NHEERL­
NTD­
CMTB­
SP/
98­
002­
000
February
10,
1998
Page
2
ml
0.2M
NaH2PO4
(
monobasic),
adjust
pH
to
8.0,
double
volume
by
adding
ddH2O
and
refrigerate
0.1M
Sodium
Phosphate
Buffer,
pH
8.0
+
1%
Triton
(
Sigma,
T­
6878)
0.1M
Sodium
Phosphate
Buffer,
pH
7.0:
61
ml
0.2M
Na2HPO4
(
dibasic)
+
39
ml
0.2M
NaH2PO4
(
monobasic),
adjust
pH
to
7.0,
double
volume
by
adding
ddH2O
and
refrigerate
DTNB
Stock
Solution:
118.8
mg
DTNB
45.0
mg
Sodium
Bicarbonate
(
Sigma,
S­
233)
30.0
ml
0.1M
Sodium
Phosphate
Buffer,
pH
7.0
Freeze
Saline­
Laboratory
grade
2.3
Biological
Samples
should
be
stored
at
­
80

C
in
an
alarm
equipped
freezer
Tissues
will
be
received
already
appropriately
diluted
and
homogenized
in
0.1M
Sodium
Phoshate
Buffer,
pH
8.0
containing
1%
Triton
Keep
samples
on
ice
during
preparation
2.4
Personnel
Training
Personnel
must
complete
the
manufacturer's
8­
day
Hitachi
911
Basic
Operator
Course
at
the
Boehringer
Mannheim
Training
Center,
Indianapolis,
IN
to
become
knowledgable
in
the
operation
of
the
automatic
analyzer.

3.0
Special
Considerations
3.1
Because
tissues
need
to
be
more
dilute
to
accomodate
the
robotics
of
the
analyzer
and
the
analyzer
employs
a
manufacturer's
set
preincubation
period,
tissues
that
have
been
treated
with
a
carbamate
pesticide
should
not
be
assayed
using
an
automatic
analyzer.
These
situations
lead
to
reactivation
of
cholinesterase
activity
and
thus,
results
will
not
be
accurate.

4.0
Procedure
1.
Tissues
may
require
additional
dilution
using
0.1M
Sodium
Phosphate
Buffer,
pH
8.0
containing
1%
Triton
to
be
run
on
the
analyzer.
Based
on
historical
data
generated
in
the
laboratory
using
the
automatic
analyzer,
tissues
should
be
diluted
as
follows:
Plasma­
undiluted
Erythrocytes­
(
1:
25)
dilution
Brain­
(
1:
50)
dilution
Heart­
(
1:
50)
dilution
2.
Prepare
substrate
as
follows:
a.
2.444
mg
Acetylthiocholine
Iodide
+
1.0
ml
ddH2O
NHEERL­
NTD­
CMTB­
SP/
98­
002­
000
February
10,
1998
Page
3
b.
Each
cholinesterase
test
requires
50

l
of
substrate
c.
Prepare
the
proper
amount
of
substrate
according
to
the
following
formula:
X*
2=
Y
Y*
50

l=
Z
Where:
X=
single
#
of
samples
to
be
run
Y=
total
#
of
samples
Z=
ml
of
substrate
required
d.
Weigh
out
acetylthiocholine
iodide
and
divide
by
2.444
to
determine
the
volume
of
ddH2O
to
be
added.
Example:
32
samples
are
to
be
run.
32*
2=
64
64*
50=
3.2
ml
of
substrate
required
26.4
mg
of
acetylthiocholine
iodide
is
weighed
out
26.4
mg/
2.444=
10.8
ml
of
ddH2O
added
3.
Similarly
prepare
the
DTNB/
Buffer
solution
according
to
the
following:
a.
1
ml
DTNB
(
from
frozen
stock)
+
29
ml
0.1M
Sodium
Phosphate,
pH
8.0
Buffer
b.
Each
cholinesterase
test
and
its
blank
requires
300

l
of
DTNB/
Buffer
c.
Prepare
the
proper
volume
of
DTNB/
Buffer
according
to
the
following
formula:
X*
4=
Y
Y*
300

l=
Z
Where:
X=
single
#
of
samples
to
be
run
Y=
total
#
of
samples
Z=
ml
of
DTNB/
Buffer
required
Example:
Same
32
samples
to
be
run.
32*
4=
128
128*
300=
38.4
ml
DTNB/
Buffer
required
3
ml
DTNB
+
87
ml
0.1M
Sodium
Phosphate
Buffer,
pH
8.0
4.
Pour
substrate
into
a
barcoded
reagent
bottle.
Split
the
volume
of
DTNB/
Buffer
into
reagent
bottles.
Fill
a
third
reagent
bottle
with
saline
to
be
run
as
a
blank.
Place
reagent
bottles
into
their
assigned
positions
on
the
reagent
disk.
5.
Perform
a
calibration
of
the
analyzer
and
run
controls
6.
Pipet
200

l
of
sample
into
sample
cup
and
place
it
on
sample
disk
7.
When
sample
disk
is
filled,
place
on
analyzer
and
program
analyzer
8.
Analyzer
will
calculate
cholinesterase
activity
and
print
results
Quality
Control
Section
NHEERL­
NTD­
CMTB­
SP/
98­
002­
000
February
10,
1998
Page
4
1.0
All
samples
are
run
in
duplicate.

1.1
The
analyzer
uses
the
analysis
of
blanks.

1.2
The
analyzer
operator
runs
a
set
of
brains
at
the
following
dilutions:
(
1:
50),
(
1:
100),
(
1:
200)
and
(
1:
400)
to
be
used
as
controls
to
verify
that
reagents
were
prepared
correctly
and
the
machine
is
operating
properly
prior
to
any
samples
being
run.
Control
values
are
archived
bythe
analyzer
and
are
checked
in
the
QC
Parameter
function
to
verify
that
the
values
reported
for
each
activity
remain
in
a
tight
range
(

50
units).

1.3
A
calibration
of
the
analyzer
is
performed
each
day
that
the
machine
is
used.

1.4
Calculations:
a.
The
analyzer
reports
results
in
an
undefined
unit/
liter.
b.
Since
this
measurement
is
not
significant
in
the
science
field,
all
data
collected
from
the
analyzer
is
expressed
as
%
of
control.
c.
To
ensure
that
the
data
generated
from
the
automatic
analyzer
is
accurate,
a
subset
of
samples
are
run
on
both
the
analyzer
and
by
the
radiometric
method.
When
the
data
from
both
methods
are
calculated
as
%
of
control,
the
percentages
are
within

5%.

1.5
Recordkeeping
Requirements
All
sample
inventories,
correspondance,
etc.
for
a
study
are
kept
in
a
labeled
hanging
file
folder
in
the
laboratory's
file
cabinet.
As
assays
are
run,
notations
on
procedure,
including
any
problems,
are
documented
in
the
technician's
laboratory
notebook.
Resulting
data
is
organized
in
Excel
(
Microsoft
Office
`
97)
files.
Hardcopies
are
printed
and
placed
in
the
study
file.
The
printouts
generated
by
the
automatic
analyzer
are
labeled
at
the
top
with
the
study
name,
tissue
assayed,
tissue
dilution
and
assay
date
before
being
placed
in
the
study
file.