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Journal
of
Automatic
Chemistry
Vol.
10,
No.
4
(October--December
1988),
pp.
171-174
Temperature
control
and
volume
measurement
in
clinical
analysers
C.
Franzini,
Ospedale
di
Rho,
Corso
Europa
250,
20017
Rho,
Milano,
Italy
W.
Giinther,
Gebrfider
Haake
GmbH,
Karlsruhe,
FR
Germany
U.
Hagelauer
Institute
of
Biomedical
Engineering,
University
of
Stuttgart,
FR
Germany
and
P.
Bonini
Istituto
Scientifico
San
Raffaele,
Via
Olgettina
60,
20132
Milano,
Italy
Temperature
control
and
volume
measurement
are
two
instrumental
factors
that,
at
different
stages
of
the
analytical
cycle
(both
in
manual
and
automatic
work),
have
a
significant
impact
on
the
quality
(accuracy
and
precision)
of
the
analytical
result.
This
paper
reports
work
carried
out
by
two
committees
which
prepared
guidelines
on
the
definition
and
control
of
these
two
instrumental
factors
of
analytical
reliability.
"Temperature
control"
is
the
subject
of
an
"ad
hoc’
committee
on
temperature
control
(designated
AHCTC)
set
up
under
the
auspices
of
the
Standing
Action
Committee
on
Instrumentation
(SA
CI),
a
technical
committee
of
the
European
Committee
for
Clinical
Laboratory
Standards
(ECCLS).
The
membership
of
the
AHCTC
includes
C.
Franzini
(Chairman,
Italy),
W.
Giinther
(FR
Germany),
U.
Hagelauer
(FR
Germany)
and
A.
von
Klein
Wisenberg
(FR
Germany).
The
AHCTC’s
starting
point
was
an
attempt
to
define
the
parameters
which
describe
temperature
control
and
to
establish
the
requirements
for
such
parameters
in
the
clinical
laboratory.
The
time-course
of
the
temperature
in
a
liquid
mass
(of
temperature
To),
which
is
transferred
in
an
environment
thermostated
[1]
at
a
set-point
temperature
(Ts),
is
shown
schematically
in
figure
1.
The
AHCTC
expressed
the
opinion
[2]
that
the
periodical
temperature
variation
which
occurs
after
the
set-point
has
been
approached
is
better
defined
in
terms
of’permissible
deviation
[from
the
set-point]’
than
in
terms
of
accuracy
and
precision.
The
’equilibration
time’
and
the
’permissible
deviation’
(figure
1)
are
the
parameters
which,
together
with
a
third,
’temperature
uniformity’,
describe
temperature
control
in
clinical
analysis.
The
latter
refers
not
only
to
the
temperature
uniformity
among
different
tubes
and/or
cuvettes
in
a
rack,
in
a
block
or
in
a
rotor,
but
also
to
temperature
uniformity
in
a
single
cuvette
(or
tube)
[3].
The
next
step
was
to
define
the
requirements
for
temperature
control,
and
the
permissible
deviation
was
regarded
as
the
most
critical
parameter
for
this.
Consid-
ering
that
multi-purpose
analysers
are
used
to
perform
different
tests,
and
assuming
that
the
narrower
tempera-
REACTI
ON
OR
DATA-ACQUISITION
T
0
:
-’
TIME
EQUILIBRATION
TitlE
Figure
1.
Time-course
of
the
temperature
in
a
liquid
mass
at
the
temperature
’To;
transferred
in
an
environment
thermostated
at
the
set-point
temperature
"Ts’.
The
equilibration
time
and
the
permissible
deviation
(from
the
set-point)
are
the
two
parameters
selected
for
describing
the
temperature
variation.
Table
1.
Criteria
for
defining
permissible
deviation
of
temperature
in
conversion
rate
measurements.
Influence
ofthe
temperature
on
the
reaction
rate
as
a
function
of
the
energy
of
activation
of
enzyme.
Contribution
of
the
’temperature
error’
to
the
overall
error
of
the
assay.
ture
limits
are
required
for
enzyme
activity
determina-
tion,
the
AHCTC
attempted
to
define
the
permissible
temperature
deviation
for
this
kind
of
measurement.
Two
main
criteria
were
considered
[2]--see
table
1.
Theor-
etical
considerations
concerning
these
two
criteria
led
to
the
formulation
of
the
following
equation:
Tm.CVp
ATm
Kt
which
relates
the
permissible
deviation
(ATm)
to
the
assay
temperature
(Tm),
to
the
partial
error
due
to
temperature
variation
(CVp)
and
to
a
factor
(Kt),
which
is
a
function
of
the
energy
of
activation
of
the
enzyme.
Assuming,
for
the
most
commonly
measured
enzyme
activities,
that
activation
energy
values
are
in
the
range
35-60
kj/mol
[4],
the
values
for
permissible
deviation
listed
in
table
2
were
calculated.
Following
this
approach,
temperature
requirements
are
not
given
in
fixed
terms
as
previously
[5],
but
in
relation
I71
C.
Franzini
et
al.
Temperature
control
and
volume
measurement
in
clinical
analysers
Table
2.
Values
for
permissible
temperature
deviation
(from
the
set-point)
as
a
function
of
the
(expected
or
required)
overall
error
and
of
the
assay
temperature.
Overall
Assay
Permissible
error
temperature
deviation
6%
30
C
0"38
C
6%
370C
0"390C
4%
30
C
0"25
C
4%
37C
0"27
C
1%
30
C
0"06
C
1%
37
C
0"07
C
to
the
expected,
or
desired,
overall
error
in
the
assay.
In
practical
work,
this
means
that
if
the
other
chemical
or
instrumental
factors
of
the
analytical
variability
(like
pH
control,
volume
measurement,
absorbance
measurement,
and
wavelength
calibration)
are
not
strictly
enough
controlled
to
allow
for
lower
than,
say,
4%
overall
error,
it
may
result
in
a
useless
effort
to
achieve
better
than
+0.2
K
(or
+0.2
C)
temperature
control.
A
tempera-
ture
control
within
+0.05
K
should
be
aimed
at,
which
will
give
an
overall
error
in
kinetic
measurements
of
1%.
The
verification
of
such
a
narrow
permissible
tempera-
ture
deviation
requires
a
high-quality
thermometer,
such
as
a
standard
platinum
resistance
thermometer
[6].
The
high
price
of
these
sophisticated
systems
leads
to
the
question
of
cost/benefit;
one
which
is
difficult
to
address.
The
verification
tools
(thermometers)
may
be
calibrated
with
reference
to
the
fixed
points
of
the
International
Practical
Temperature
Scale-1968
(IPTS-68)
or
to
some
suggested
fixed
points
for
the
life
sciences
[6],
as
listed
in
table
4.
Among
these,
the
rubidium
triple
point
standard
[8]
may
be
especially
useful
for
checking
the
accuracy
of
thermometers
intended
for
measuring
temperatures
near
to
37
C.
In
addition
to
those
listed
in
table
4,
the
second
or
third
order
fixed
points
(6,9)
listed
in
table
5
may
be
of
help
in
the
range
22.4-42.7C.
In
practical
work,
although
the
recommended
procedure
for
calibrating
thermistor
thermometers
includes
comparison
with
a
recently
calibrated
platinum
resistance
thermometer
[10],
comparison
at
three
temperature
values
with
a
certified
mercury-in-glass
thermometer,
in
a
well
equilib-
rated
water-bath
[11],
may
be
an
acceptable
procedure.
Table
4.
Fixed
points
for
the
calibration
of
thermometers.
Fixed
points
of
the
IPTS
(68)
(Ice
point
of
water):
0"000
C
Triple
point
of
water:
0.010
C
Liquid
and
vapour
phases
of
water:
100.000
C
Fixed
points
for
the
life
sciences
(SRMs
available
from
NBS)
Gallium
melting
point:
29"772
C
Rubidium
triple
point
39-265
C
Temperature
uniformity
should
be
controlled
within
the
same
limits;
equilibration
time
should
be
short
enough
to
allow
the
temperature
of
the
incubation
mixture
(Ts)
at
the
start
of
the
kinetic
measurement
(ts)
to
be:
Ts
Tm
+
A
Tm.
Further
work
of
the
AHCTC
will
involve
to
the
prepara-
tion
of
guidelines
to
verifying
how
the
expected
in-the-cell
temperature
control
is
being
achieved.
Basically,
for
this
operation,
appropriate
tools
and
feasible
methods
for
checking
calibration
are
needed.
The
tools
are
listed
in
table
3."
the
Pt-100
resistance
thermometer
may
represent
a
’reference
method’
[6],
but
well
calibrated
thermistors,
featuring
very
low
thermal
mass
and
thin
enough
probes
to
be
introduced
in
small-volume
cuvettes,
may
be
adequate
in
terms
of
accuracy
and
sensitivity.
The
use
of
thermochromic
solutions
[7]
may
allow
temperature
verification
in
fixed
or
rotating
spectrophotometric
cuvettes
which
are
unaccessible
to
the
probe,
but
the
inherent
accuracy
and
the
sensitivity
of
this
measuring
system
have
to
be
proved.
Other
methods,
listed
in
parentheses
in
table
3,
have
no
practical
application
at
the
moment.
Table
3.
Tools
for
monitoring
the
temperature
in
reaction
mixtures.
Metal-resistive-temperature-sensors
(Pt-
100).
Semiconductor-resistive-temperature-sensors
(thermistors).
Thermochromic
solutions.
(Radiation
thermometry)
(Liquid
crystal
thermography)
Table
5.
Calibration
or
verification
of
thermometers:
additional
fixed
points
in
the
range
22.36-42"7
C.
Second-order
fixed
points
Benzoic
acid
(tp):
22"36
C
Phenoxybenzene
(tp):
26"87
C
Na2SO4.10H20/Na2SO4
(trsp):
32"37
C
n-icosane
(tp):
34"49
C
1,3-dioxolan-2-one
(tp):
36"32
C
KF’2H20/KF
(trsp):
41"42
C
Third-order
fixed
points
LiNOa’3H20
(mp):
CaC12’6H20
(mp):
Ca(NO)2"4H20
(mp):
29"9
C
30.2
C
42.7
C
tp
triple
point;
trsp
transformation
point;
and
mp
melting
point.
Volume
measurement
is
another
critical
operation
in
analytical
procedures.
Problems
relating
to
this
operation
were
considered
by
the
Working
Party
on
Sampling,
Diluting
and
Dispensing
(WPSDD),
which
convened
at
the
suggestion
of
the
Expert
Panel
on
Instrumentation
(EPI)
of
the
International
Federation
of
Clinical
Chemistry
(IFCC).
Members
of
the
WPSDD
included
M.
Besozzi
(Italy),
P.
A.
Bonini
(Italy),
G.
Caminada
(Switzerland),
C.
Franzini
(Convenor,
Italy)
and
G.
Luise
(Italy).
Because
of
the
changed
structure
of
the
EPI,
the
WPSDD
ended
before
any
guideline
was
produced;
nevertheless,
collabora-
tive
efforts
produced
some
results
and
these
are
discussed
here.
The
first
step
in
the
WPSDD’s
work
was
to
define
and
classify
the
volumetric
operations
relevant
to
the
analy-
172
C.
Franzini
et
al.
Temperature
control
and
volume
measurement
in
clinical
analysers
a
b
c
Figure
2.
Three
different
types
of
POVA.
(a)
Air-displacement;
(b)
liquid-displacement;
(c)
positive-displacement.
tical
procedures,
as
shown
in
table
6.
Then,
the
equip-
ment
used
to
perform
the
different
types
of
volumetric
operations
were
grouped
into
three
main
categories,
as
shown
in
table
7.
The
most
commonly
used
devices,
the
’Piston
Operated
Volumetric
Apparatus’
(POVA)
are
described
in
DIN
norms
[12],
and
are
also
being
considered
in
draft
documents
by
ISO
[13].
Table
6.
Volumetric
operations
relevant
to
the
analytical
cycle.
Sampling:
A
stated
volume
of
the
specimen
is
transferred.
Diluting:
Either
a
stated
volume
of
specimen
plus
a
stated
volume
of
reagent
is
transferred
or
specimen
and
reagent
are
transferred
in
a
stated
volumetric
ratio.
Dispensing:
A
stated
volume
of
reagent
is
transferred.
Table
8.
Main
uses
of
the
doCferent
kinds
of
PO
VA.
Kind
of
POVA
Main
use
Air
displacement
Sampling
Diluting
Liquid
displacement
Dispensing
Positive
displacement
Sampling
Dispensing
and
through
the
tip,
and
distributed
from
the
tip
(figure
3[a]).
A
diluting
operation
is
obtained;
if
the
specimen
volume
is
set
to
0,
dispensing
is
achieved.
In
a
second
mode
(figure
3[b])
the
system
is
filled
with
a
washing
or
inert
fluid
and
both
the
reagent
and
the
specimen
are
aspirated
and
distributed
through
the
tip.
Here
again
diluting
and
dispensing
operations
can
be
performed.
These
two
operating
modes
include
the
presence
of
a
valve.
POVAs
can
also
be
operated
in
a
without-valve
mode,
as
shown
in
figure
3(c).
This
last
operating
mode
also
allows
sampling
operations
to
be
easily
performed.
REAGENT
WASHING
OR
I
INERT
FLUID
b
Table
7.
Devices
for
sampling,
diluting
and
dispensing
which
are
commonly
used
in
mechanized
analysis.
(1)
Piston-operated-volumetric-apparatus
(POVA).
(2)
Peristaltic
pumps.
(3)
Other
systems:
Pressure/time
operated
systems.
Sampling
valves.
POVAs
can
be
classified
into
three
different
types,
as
shown
schematically
in
figure
2.
In
the
air-displacement
type
(figure
2[a]),
the
piston
and
the
fluid
being
measured
move
in
different
zones,
and
the
movement
is
transmitted
from
the
piston
to
the
liquid
by
an
air
cushion.
In
the
liquid-displacement
type
(figure
2[b])
the
piston
and
the
liquid
again
move
in
different
zones,
but
the
movement
is
transmitted
by
liquid:
this
can
be
either
a
reagent
or
a
diluent
to
be
added
in
a
stated
volume
to
the
measured
volume
of
specimen,
or
water
or
an
inert
fluid.
In
the
positive-displacement
type
(figure
2[c])
the
piston
and
the
liquid
being
measured
move
in
the
same
zone.
The
main
uses
of
the
different
types
of
POVA
in
the
above
mentioned
volumetric
operations
are
summarized
in
table
8.
In
automated
work,
liquid-displacement
systems
are
most
commonly
used;
they
can
be
operated
in
three
main
modes
(figure
3).
The
system
may
be
filled
with
the
reagent
(or
diluent):
the
desired
amounts
of
reagent
and
of
specimen
are
aspirated,
respectively,
from
a
reservoir
Figure
3.
Three
main
different
modes
for
operating
liquid-
displacement
POVAs.
(a)
and
(b)
With-valve
operation;
(c)
without-valve
operation.
Since
the
reagent-to-sample
volume
ratio
is
frequently
high,
the
systems
operate
with
two
pistons
(syringes)
of
different
diameter,
but
single-piston
instruments
may
also
be
operated
in
three
modes
cited
above.
POVAs’
pistons
are
driven
in
different
ways,
see
table
9.
Significant
improvements
have
recently
been
achieved
in
this
by
the
introduction
of,
microprocessor-controlled
stepper
motor
driven
pistons;
the
additional
monitoring,
via
external
sensors,
of
the
fluid’s
level
may
lead
to
further
reliability
of
volume
measurements
in
the
zl
range.
Table
9.
Operation
of
the
piston
in
PO
VAs.
Manual,
with
mechanical
stops.
Pneumatic,
with
mechanical
stops
and
valves.
Motor-driven,
mechanical
or
electrical
stops.
Microprocessor-controlled
stepped
motor.
Microprocessor-controlled
stepped
motor
with
additional
monitoring
(sensors)
of
the
fluid’s
level.
The
performance
characteristics
of
volumetric
apparatus
may
be
defined
by
precision
and
accuracy
of
volume
173
C.
Franzini
et
al.
Temperature
control
and
volume
measurement
in
clinical
analysers
Table
10.
Methods
for
the
verification
of
the
performance
of
volumetric
apparatus.
’Primary’
method
of
verification:
gravimetry.
’Secondary’
method
of
verification:
spectrophotometry
of
dye
solutions,
in
comparison
with
gravimetrically
checked
vol-
umetric
pipettes
and
flasks.
measurements,
and
by
carry-over
and
dead
volume.
These
characteristics
may
be
verified
by
means
of
’primary’
and
’secondary’
methods
[14],
as
shown
in
table
10.
Since
the
secondary
methods
are
based
upon
comparison
with
gravimetrically
verified
volumetric
equipment,
the
assessment
ultimately
relies
upon
grav-
imetry.
Specifications
concerning
balance
performance,
and
calibration
by
means
of
NBS
standards,
procedures
and
statistical
calculations
have
been
published
14].
In
the
secondary
methods,
the
use
of
potassium
dichromate
[14]
and
of
Evans
blue
[15]
solutions
has
been
suggested.
For
the
secondary-method
verification
of
samplers
inten-
ded
for
serum
measurement,
the
use
of
iso-viscosity
dye
solution,
such
as
Evans-blue-dyed-serum,
has
been
suggested
16]:
this
may
be
particularly
important
for
the
verification
of
air-displacement
POVAs.
Acknowledgements
The
collaboration
of
the
members
of
WPSDD,
M.
Besozzi
(Ospedale
Del
Ponte,
Varese,
Italy),
G.
Cami-
nada
(Hamilton
Bonaduz
AG,
Bonaduz,
Switzerland),
and
G.
Luise
(Instrumentation
Laboratory
SpA,
Milano,
Italy)
is
gratefully
acknowledged;
and
the
frequent
advice
of
A.
von
Klein
Wisenberg
(Instand,
Freiburg,
FR
Germany),
a
member
of
AHCTC,
was
of
great
help.
References
1.
IFCC-EPI.
The
effect
of
instrumental
and
environmental
factors
on
the
thermal
regulation
of
the
temperature
of
incubation.
IFCC
Document,
Stage
2,
Draft
1
(1985).
2.
ECCLS.
Guidelines
for
Temperature
Control
in
Clinical
Chem-
istry.
Part.
I:
Requirements
for
temperature
control.
First
Draj?
(1978).
3.
HAC;ELAUER,
U.,
ARNADOV,
K.
and
FAUST,
U.,
International
Laboratory
(June
1986),
62.
4.
L.Na’NER,
C.
(Ed),
Tavole
Scientifiche
Geigy,
8th
edn
(Edi-
zione
italiana).
Ciba-Geigy
Ltd
(Basel,
1984).
5.
IFCC-EPE,
Approved
recommendations(1978)
on
IFCC
methods
for
the
measurement
of
catalytic
concentration
of
enzymes.
Re-
produced
in
Clinica
Chimica
Acta,
98
(1979),
165F.
6.
SI-IOOL.V,J.
F.,
Thermometry
(CRC
Press,
1986).
7.
O’LEARV,
T.
D.,
BAD.NOeI-I,
J.
L.
and
BAIS,
R.
Annals
of
Clinical
Biochemistry,
20
(1983),
153.
8.
MAGU,
B.
W.,
A
temperature
reference
standard
near
39.30
C.
NBS
S/ecial
Publication
260-87
(1983).
9.
vo
KL.I
WISBRG,
A.,
Personal
communication
(1987).
10.
NCCLS,
Guide
for
the
Selection
of
Accuracy
Class
of
Thermistor
Thermometers
and
the
Verification
,of
Their
Accuracy
(NCCLS,
1979).
11.
W,N,
S.,
International
Clinical
Products
Review,
4
(1985),
65.
12.
DIN,
Volumenmessgerate
mit
Hubkolben.
DIN
12650,
Tell
1
(1978),
Tell
2
(1981),
Tell
3
(1981),
Tell
4
(1981),
Tell
5
(1981).
13.
ISO,
Piston
and
Plunger
Operated
Volumetric
Apparatus
(POVA).
Part
1:
Definitions;
Part
2:
Operating
considerations:
Part
3:
Test
methods;
Part
4:
Specifications
(Drafted
documents).
14.
NCCLS,
Determining
Performances
of
Volumetric
Equipment.
Proposed
Guideline.
Vol.
4,
No.
6
(1984).
15.
GEARY,
T.
D.
and
FARRANCE,
I.,
Clinical
Biochemistry
Reviews,
3
1981
).
16.
DvcAn,
I.
W.,
MATH.R,
A.
and
CooP.R,
G.
R.,
The
Procedure
for
the
Proposed
Cholesterol
Reference
Method
(Center
for
Disease
Control,
Atlanta,
Georgia,
1982).
DIODE-ARRAY
SPECTROSCOPY
For
scientists
learning
about
or
using
UV/visible
spectrophotometers,
Hewlett-Packard
Company
has
published
a
gratis
64-page
book
by
Dr
A.
J.
Owen,
HP
scientist,
that
describes
the
advantages
of
diode-array
over
conventional
mechanical-scanning
technology
in
many
applications.
For
teaching
purposes,
there
are
sections
that
outline
the
history
and
theory
of
UV/visible
spectrophotometric
techniques.
Detailed
explanations
of
how
diode-array
spectrophotometers
work
and
how
to
get
the
best
results
for
different
applications
provide
a
clear
picture
of
when
to
use
a
particular
capability.
The
book
fully
explains
the
advantages
of
diode-array
technology,
including
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more
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and
precise
results;
higher
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greater
reliability;
and
lower
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of
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Copies
from
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Sales
Office
or
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150,
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CH-1217
Meyrin
2,
Geneva,
Switzerland.
174