Note: Descriptions are shown in the official language in which they were submitted.
wo 93/2153~ tUS'~3/~36(~
CALlBRAnON REAGENT HAVING A STABILIZED
CONCENTR~TION OF DISSOLVED GAS
Field of the Invention
This invention relates generally to the
calibration of analysis equipment. More specifically, the
present invention relates to calibration reagents including
dissolved gases therein. Most specifically, the present
invention relates to reagents for calibrating blood gas
analysis systems.
Backaround of the Invention
Automated chemical analyses are rapidly
supplanting manual techniques, particularly in the health
care field. The rapid and accurate analysis of blood
chemistry is particularly important in operating rsoms,
critical care facilities and clinical environments.
It is necessary to calibrate analysis systems in
order to assure the accuracy and reliability of their
results. Calibration is typically carried out by the use
of reagents which are facsimiles of the materials being
analyzed and which include known quantities of the target
analytes therein. For example, equipment for blood gas
analysis is typically calibrated by utilizing a blood
facsimile which includes ~nown concentrations of dissolved
oxygen and carbon dioxide therein and which may further
include bicarbonate, calcium, sodium and potassium ions, as
well as other ionic species and organic species such as
glucose.
WO93/2153~ ~ rCT/U~9~/~)36~
2~
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There are many problems associated with the use
of liquid based calibration reagents which contain
dissolved gases. The concentration of a given gas in a
liquid will vary depending upon the ambient temperature and
pressure conditions to which the liquid is exposed.
Furthermore, atmospheric gases can dissolve into the liquid
thereby changing the concentration of particular species
therein.
one approach to the problem of providing accurate
calibration reagents for blood gas analysis equipment
involves the on site preparation of the reagents by the
tonometery of liquid with a known concentration of gas.
In this process, a gas is bubbled th~ough a liquid under
controlled conditions; the concentration of gases in the
reagent may be calculated after compensating for
temperatuxe and barometric pressure, and the solution is
ready for use in a calibration procedure. Solutions thus
prepared must be used very promptly since tAeir gas
concentration can change rapidly.
Another approach to the problem of calibration
solutions involves the use of previously prepared ~olutions
which are stored in a flexible, relatively gas-impermeable
package having zero headspace. By zero headspace is meant
that the liquid occupies the entirety of the package and
there is no free gas therein. In preparing reagents of
this type, care must be taken to avoid the introduction, or
formation during storage, of any bubbles since they can
change the concentration of gases in the reagent and can
also interfere with the analysis itself. Typically,
W~93/2153~ 8 ~ 2 ~ PCT/US93/036(
-- 3
solutions of this type are packaged at total gas pressures
substantially less than atmospheric` and at elevated
temperatures. One package of this type is disclosed in
U.S. Patent No. 4,116,336 of Sorensen et al. The reagent
described therein is a blood facsimile which is packaged in
a laminate foil-polymer bag, at a total gas pressure of
less than 600 and preferably 500-550, mmHg at 37 C.
A major problem encountered with the flexible bag
approach is that the package is responsive to ambient
temperature and pressure changes and hence storage a~ high
altitudes, air transport or thermal cycling can cause
outgassing and bubble formation. In order to minimize such
problems, the total gas pressure is kept substantially
below atmospheric, typically in the range of 500-550 mmHg.
This very low pressure gives rise to various problems.
Most importantly, the low pressure within the bag
facilitates the diffusion of ambient atmospheric gases
therethrough, hence causing a change in the composition of
the reagent during storage. Although great care is taken
in selecting gas-impermeable packaging materials, it has
been found very difficult to provide a total barrier to
di~fusion. Also, filling of the bags at low dissolved gas
pressures necessitates additional care and increases the
cost of production. Another approach involves filling the
bags at higher pressures, typically 650 mm or more. While
~illing is simplified, problems of outgassing during
~torage are exacerbated. This approach is disclosed, for
example, in U.S. Patent 4,871,439.
W()93/~l~3~ l~CT/US93/(~3~
2 ~ 2 ~ ' :. . .
~, _
It is desirable to have a storage-stable package
of calibration reagent for blood gas analyzers and other
dissolved gas analysis systems. The calibration reagent
should resist outgassing even when stored under extreme
ambient temperature and pressure conditions and it should
be simple and economical to prepare. ~
"' ''
Brief Description of the Invention -~
As will be disclosed in greater detail
hereinbelow, the present invention provides an improved
calibration reagent and method for its preparation. The
reagent comprises a liquid, such as a blood facsimile,
having one or more gaseous materials dissolved therein.
The calibration reagent is prepared at total dissolved gas
pressures which are near, or only slightly below,
15 atmospheric; however, it is resistant to bubble formation -;~;
during storage. The reagent retains its s~ated composition
during storage at high altitudes and at temperature -~
extremes.
There is more specifically disclosed herein a
calibration reagent for a gas analyzer. The reagent
comprises a liquid vehicle having a known concentration of
a first gaseous material dissolved in the vehicle and
further including helium dissolved therein. Helium has
the uni~ue property of being more soluble in warm liquid
than in cold liquid. The first gaseous material may
comprise oxygen or carbon dioxide and in particular
embodiments, the reagent may include a plurality of gaseous
materials dissolved therein. `~
w~3/2~53~ 8 ~ 2 ~ PCT/US93/0360
-- 5
The reagent may comprise a calibration reagent -
for a blood gas analyzer and in such instance the reagent
may include oxygen in a range of concentrations sufficient
to create a partial pressure of 50-300 mmHg. The reagent
may also include a range of concentrations of carbon
dioxide sufficient to produce a partial pressure of 10-100
mmHg therein. In yet further embodiments, the reagent may
include dissolved ionic materials such as bicarbonate ion,
sodium ion, potassium ion and the like.
The reagent is preferably supplied in a flexible
package having zero head space. The package is fabricated
from a material having a low permeability to gases and
particularly from a material having a higher permeability
for helium than for oxygen, carbon dioxide and nitrogen. ~-
Brief Description of the Drawinqs
FIGURE 1 is a graph depicting the pressure of
dissolved gas in reagent package of the present invention
as a function of storage time; and
FIGURE 2 is a cross sectional view of a flexible
package of calibration reagent structured in accord with
the principles of the present invention.
~;
Detailed Description of the Invention
The calibration reagents of the present invention
include a liquid vehicle together with a known
25 concentration of one or more gaseous materials therein and ;
further include helium dissolved in the vehicle. As a
result o~ this formulation, the calibration reagents retain
W093/2~33 ~ rCT/US93/~36(~
- 6 -
their compositional integrity over relatively long periods
of time and are not detrimentally affected by changes in
pressure or temperature during storage. Helium is an
extremely inert substance which is generally not included
in reagents for chemical analyses; however, helium
manifests unique properties and the present invention
recognizes that these properties make helium an
advantageous additive for stabilizing the compositional
integrity of solutions having gases dissolved therein.
Because of its inertness to all commonly
encountered reagents, helium will not adversely interfere
with calibration or analytical procedures. Helium also
posse~es unigue solubility properties. In
contradistinction to most gases, helium is more soluble in
hot water than in cold water. At atmospheric pressure
approximately 9.4 cc of helium dissolves in 1 liter of cold
(0 C.) water while approximately 10.5 cc of helium dissolves
in 1 liter of hot (50 C.) water. These figures also make
. . .
clear the fact that helium has a very low solubility in
water and hence relatively small amounts of dissolved
helium can create a relatively large partial pressure.
Finally, helium has an extremely high mobility and is far
more volatile than any other gases present in calibration
reagents. All of these unique properties of helium
synergistically interact to stabilize dissolved-gas-
containing calibration reaqents toward compositional
change. Bubble formation occurs in a liguid when the
total pre~sure of the gases dissolved therein exceeds the
atmospheric pressure. Bubble formation can occur in
'
WO93/21533 ~ I1 X'l 2 4 PCT/US93/036
-- 7 --
calibration reagents when they are heated s ince most gases
become less soluble in hot liquids. Since helium is more
soluble in warm liquids than in cold liquids, inclusion of
helium in a gas-containing calibration reagent tends to
inhibit bubble formation at elevated temperatures. As the
reagent is warmed, there is tendency for most dissolved
gases to increase their partial pressures and if the total
gas pressure becomes to high, bubble formation will occur;
however, if helium is included in the reagent, it will
decrease its partial pressure as the temperature increases
thereby preventing bubble formation. -
The fact that helium has a very low solubility in
aqueous based liquids further operates to its advantage
since relatively small amounts of dissolved helium will
exert a fairly large partial pressure; hence, the effects
of helium are magnified by its low solubility. If the --~
ambient pressure become~ sufficiently low so as to be less
~ ..
than the total dissolved gas pressure in the liquid, bubble
formation will still occur; however, the generation of an
extremely small helium bubble will significantly lower the
total pressure of dissolved gas thereby precluding further
bubble fo~mation. Thus it will be seen that helium tends
to inhibit bubble formation by decreasing the total
pressure of dissolved qases in a calibration liquid as the
Z5 temperature increases and further acts to mitigate the
e~fects of any possible bubble formation that may occur.
The very high mobility of helium still further
enhances the stability of gas-containing calibration
reagents. The permeability of a large number of materials
w~)s3/2ls3~ PCTtUS93/~)36
-- 8
to helium is far higher than it is for most other gases.
In a typical calibration reagent, such as one which might
be employed for a blood gas analyzer, there is present a
partial pressure of oxygen which is generally no more than
300 mm Hg and a partial pressure of carbon dioxide which
usually does not exceed 100 mm Hg. In prior art reagents
the remainder of the atmosphere was made up of nitrogen and
the total gas pressure was usually kept below 550 mm Hg to
avoid bubble formation. Even though great care is taken to
lo employ packaging materials which have very low gas
permeabilities, some leakage will occur during storage and
since the total pressure in the package is relatively low,
atmospheric gases will tend to leak into the package. By
the use of helium, as was set forth hereinabove, total gas
pressures within the reagent package can be near, or only
slightly less than, atmospheric and leakage into the
package is minimized. Furthermore, the greater
permeability of the package to helium in contrast to oxygen
and nitrogen further stabilizes the composition.
Figure 1 illustrates this phenomenon. Shown in
the figure is a graph depicting pressure of the dissolved
gases as a function of time. Curve A depicts the total gas
pressure in a calibration reagent which includes helium and
a known concentration of dissolved oxygen. The reagent is
contained in a package which has a higher permeability for
helium than for oxygen or nitrogen. It will be noted that
the total gas pressure manifests an initial drop followed
by a slow rise. This corresponds to helium leaking out of
the package and being replaced by nitrogen which leaks in.
WO 93/21533 rcr/uss:~s/036(~
211X~29
g
The two processes occur simultaneously, but the rate of
helium leakage is faster. The low point on Curve A
represents the depletion of helium and the slow rise in
total gas pressure is attributable to continued inward
diffusion of nitrogen. The package is initially filled at
a total gas pressure which is near atmospheric and because
of the solubility properties of helium, bubble formation is
initially inhibited. As the total gas pressure decreases,
the tendency to bubble formation is further inhibited. As
the helium is being replaced by nitrogen, the tendency to
bubble formation does increase; however, bubble formation
becomes a real concern only when the total gas pressure of
the nitrogen containing solution becomes a significant
fraction of atmospheric pressure.
Curve B represents the concentration of dissolved
oxygen and throughout the entire process it will be noted
~hat the concentration is essentially stable. Since the
concentration of oxygen in the solution is approximately
that of a solution equilibrated with ambient air, and since
the package has a low permeability to oxygen, leakage of
oxygen into or out of the package is fairly minimal. The
time scale for diffusion of the helium and nitrogen will
depend upon the permeability of the package, the nature of
the reagent and the actual pressure of the gases. It has
been found that a typical calibration solution of the type
which will be described in greater detail hereinbelow,
reaches a helium depletion point after approximately four
~onths and manifests a sufficiently long pressure rise time
that a total useful life of approximately two years is
...,, .~ ,...
W~93~2153~ PC~/US93~036(~
- 10 -
achieved before bubble formation becomes a significant
concern.
As mentioned hereinabove, the calibration reagent
of the present invention is most advantageously packaged in
a flexible package having zero head space. By æero head
space is meant that a liquid entirely fills the package
without any bubbles or voids. Referring now to Figure 2
there is shown, in cross section, one particular reagent
package 20 structured in accord with the principles of the
present invention. The package is fabricated from a
relatively low gas permeability material comprising a
laminate of metallic foil 22 interposed between layers of
thermo plastic polymer 24,26. The packet is ~abricated by
laminating the interior layers 26 of polymeric material by
a heat sealing process so as to provide a packet which
contains the calibration reagent 28 of the present
invention therein. It is to be understood that the reagent
of the present invention may be packaged in other manners
than as is shown herein. ~or example, the packaging may be
totally, or partially rigid, with zero head space.
In addition to dissolved gases, it is generally
preferable that the calibration reagents include other
species such as known concentrations of potassium, calcium,
bicarbonate, sodium and other such ions. Additionally, the
reagent will preferably include a buffer to establish a
s~able pH, and may include other species such as glucose or
colorimetric standards. The specific quantities of each of
the foregoing species present in the solution will depend
upon a particular applications and the particular equipment
. .
WO93/2l~33 ~ i 2 4 PCT/US93/0360~
being calibrated. The following examples detail some -
specific compositions of calibration reagent for a blood-
gas analyzer and the methods by which they are
manufactured. i
ExamDle 1
This calibration reagent was prepared to 't`
approximate a blood sample and is intended for use in
calibrating a carbon dioxide sensor. The solution is water
based and Table 1 hereinbelow sets forth the concentrations
10 of the dry and liquid components of the solution. ;
AB~E 1
COMPOUND CONCENTRATION_ GRAMS/LITER
MOPS ACID 60 m~ol/l 12.588g
NaMOPS 25 mmol/l 5.672g
NaHCO3 10 mmol/10.840g
Na2SO3 42 mmol/l 5.294g
NaCl 57 mmol/l 3.33lg
XCl 2.0 mmol/l0.149g
CaCl2 2H2O 0.25 mmol/l0.037g
The calibration .e~lution was prepared by dissolving the
ingredients of Table 1 in distilled water. The solution
thus prepared was heated to 37 C. and tonometered at 700 mm;~
Hg absolute pressure with a gas mixture comprising 10%
carbon dioxide in helium. Tonometring was carried out
until saturation was achieved. The solution was then --
packaged in a flexible bag similar to that set forth i.
Figure 2.
The solution was analyzed and found to have the
following properties: pH 6.890 - 6.910; pressure of CO2, ;
',."` '
. ~'' ~" ."
W093/21533 ~ CT/US93/03
- 12 -
63-67 mmHg; pressure 2~ O.OmmHg; potassium concentration as ~:
measured by a Radiometer brand analyzer 1.8 -1.9 mmol/l; ;.
potassium ion concentration as measured by a Nova brand
analyzer 1.83 -1.98 mmol/l and a calcium ion concentration
of .18 - .22 mmol/l.
Example 2
A second calibration solution was prepared which
included both oxygen and carbon dioxide therein. The
liquid and solid components of the calibration solution are : -
set forth in Table 2.
TAB~ 2
COMPOUND _ CONCENTRATION GRAMS/LITER
:"
Buffer, NaHC03 20.0 mmol/l1.680 g ;.
NaCl 110 mmol/l6.430 g -~
KCl 6.0 mmol/l.447 g ~ -~
In order to prepare the calibration solution, the
ingredients set forth in the table were dissolved in
distilled water, pH was adjusted to approximately 7.3 by
the use of 1.0 N HCl and the resultant solution was
tonometered with a gas mixture comprising 21% 2 and 6.3%
~2 in helium. Tonometring was carried out at 37 C. and 700
mm Hg absolute pressure. As in the preceding example, the
resultant tonometered solution was sealed in an air tight;
zero head space package. Analysis of the calibration
solution of Example 2 indicated the following properties:
pH 7.330 - 7.345: pressure of C02 37.0 - 41.0 mm Hg;
W O 93/21533 ~ 1 1 8 ~ 2 4 PC~/US93/0360~
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pressure 2 135 -140 mm Hg; potassium ion concentration 5.6
- 5.8 mmol/l as measured by a Radiometer brand apparatus;
and a concentration of potassium ion of 5.60 - 5.75 mmol/l
as measured by a Nova brand apparatus.
It has been found that the solutions as prepared
herein a~ove exhibit a long shelf life. The concentration
of the various species, particularly the gas concentrations
are not changed if the packages are stored at high
altitudes or exposed to temperature extremes.
Various other compositions may be prepared in
accord with the principles of the present invention. For
ease of processing and handling, the total gas pressure
within the packages is most advantageously near, or
slightly below atmospheric.
Although the foregoing has primarily been
concerned with the preparation of calibration reagents
which are blood facsimiles used in calibration of blood gas
analysis equipment, it is to be understood that the present
invention is not so limited and that it may be employed to
prepare a variety of calibration reagents including
dissolved gases therein. Toward that end it will be
appreciated that the foregoing drawings, discussion and
description are merely illustrative of particular
Pmbodiments of the pre-sent invention and not limitations
upon the practice thereof. It is the following claims,
including all equivalents, which define the scope of tbe
invention.
.. ~,: ......
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