This invention relates primarily to the field of clinical reference
solutions -- quality control reagents and calibrators. More specifically it relates
to methods of preparing multi-analyte reference solutions that have stable oxygen
partial pressure (pO2) in zero headspace containers, preferably in flexible
foil laminate containers. The solutions are stable at room temperature and have
long shelf and use lives.
BACKGROUND OF THE INVENTION
Clinical laboratories employ a variety of instrument systems for the
analysis of patient samples. For example, pH/blood gas instruments measure blood
pH, pCO2 and pO2. CO-Oximeter instruments typically measure
the total hemoglobin concentration (tHb), and the hemoglobin fractions -- oxyhemoglobin
(O2Hb), carboxyhemoglobin (COHb), methemoglobin (MetHb), reduced hemoglobin
(HHb) and sulfhemoglobin (SHb)(collectively referred to as "CO-Ox fractions"). Ion
selective electrode (ISE) instruments measure the content of blood electrolytes,
such as, Na+, Cl-, Ca++, K+, Mg++
and Li+. Also, a variety of other parameters such as, metabolites, e.g.,
glucose, lactate, creatinine and urea, can be measured in clinical laboratories
by related instrument systems.
Instrument systems currently available may combine the measurement
of blood pH, gases, electrolytes, various metabolites, and CO-Ox fractions in one
instrument for a comprehensive testing of the properties of blood. For example,
all such analytes are measured by the Rapidlab™ 865 critical care diagnostics
system from Chiron Diagnostics Corporation [Medfield, MA (USA)].
A calibrator is used to set the response level of the sensors. A control
is used to verify the accuracy and reliability of such an instrumentation system.
A control is a solution having a known concentration of an analyte
or analytes contained in the same, or a similar matrix in which the samples to be
analyzed exist. The assay results from the control product are compared to the expected
assay results to assure that the assay technique is performing as expected.
Commercial blood gas analysis systems have been available since the
1960s. The earliest reference materials were gas mixtures in pressurized cylinders,
and those materials are still commonly used. In the 1970s, the development of liquid
reference solutions began, leading to products in which reagents have been equilibrated
with precision gas mixtures and packaged in flexible containers with zero headspace,
requiring either refrigeration to maintain stability or the resort to calculations
to compensate for the expected pO2 changes during storage.
Most quality control materials for such analyzers consist of tonometered
aqueous solutions (a solution containing dissolved gases) in glass ampules. The
typical gas headspace above the liquid in those ampules provides a reserve of oxygen
against any potential oxygen-consuming reactions which may occur within the solution
during the shelf life of the product.
In the absence of a gas headspace within their containers, reference
solutions for oxygen determinations are particularly difficult to make and maintain
stable. The inventors determined that the sources of said instability could be several.
First, the instability may be due to reactivity between the dissolved
oxygen and the other components of the calibrator or quality control reagent. The
other components might either react with the dissolved oxygen, reducing its concentration,
or, alternatively, the other components may react with each other to generate oxygen,
thus also changing the oxygen concentration. Second, the solution might be contaminated
with microorganisms which, due to their metabolism, might change the oxygen content.
Third, the oxygen might permeate through, or react with, the packaging material,
also affecting the oxygen content of the reference material.
Reference materials that are manufactured for distribution in commerce
must be made to withstand the various conditions encountered in the distribution
chain and must be sufficiently stable to provide good performance within the time
frame in which they are expected to be used by the customer, which is usually at
least about six months, preferably for about nine months, and more preferably approximately
1 year for the typical calibrating or quality control solution distributed to commercial
laboratories and hospitals. In addition, reference solutions, as with other reagents,
should be packaged in containers which are easy to handle, convenient to use and
which meet other design requirements of their intended usage. This is particularly
true of reagents which are used in conjunction with various analytical instruments.
The users of instruments which determine the oxygen partial pressure of blood and
other body fluids have a need for such reference materials and would benefit from
liquid materials over the more conventional precision gas mixtures in cylinders
with regulators. Liquid reference solutions are inherently less expensive, safer,
and easier to manipulate than high-pressure gas tanks.
Although reference solutions used in instruments measuring pO2
have been made in the past, they have suffered from being unstable and having expensive,
complicated, or unreliable means to access their contents. Some reference solutions,
when used on analytical instruments, have extended their usefulness by allowing
the instrument to calculate the expected oxygen level, said level being calculable
from the age of the product, given the fact that the rate of decrease in oxygen
level can be predicted based on historic performance [Conlon et al., Clin. Chem.,
42: 6 -- Abstract S281 (1996)]. Several developers have included inner layers
of plastic materials selected because of their heat sealability (e.g., US 5,405,510
- Betts) or low gas permeability (US 4,116,336 - Sorensen) or gas tightness (US
4,163,734 - Sorensen). Some have disclosed that the inner layer should be inert,
but have not provided enablement as to how to select such an inner layer (US 4,643,976
- Hoskins) and/or weren't capable of maintaining oxygen at a precise level appropriate
for blood gas purposes.
Most blood gas/electrolyte/metabolite/CO-Oximetry/hematocrit quality
controls (QCs) on the market today are provided in glass ampules which must be manually
broken and manually presented to the analyzer. Rüther, H., U.S. Patent No. 5,628,353
(issued May 13, 1997) describes an automated device which breaks open glass ampules
by forcing a metal tube with thick walls and a small inner diameter, into the bottom
of an ampule, and then aspirates the contents of the ampule into an analyzer. Such
an automated ampule breaker is mechanically complex, requiring moving parts that
are subject to wear and risk of failure, and could be subject to jamming and clogging
from small bits of broken ampule glass.
In the 1980s, Kevin J. Sullivan disclosed an alternative to glass
ampules -- the first commercial product with a blood gas reagent in a flexible,
zero headspace package [U.S. Patent Nos. 4,266,941; 4,375,743; and 4,470,520]. Coated
aluminum tubes were filled with 40-50 mL of blood gas QC solutions without any headspace.
The tubes were enclosed in pressurized cans, to prevent outgassing and to supply
a source of force to cause the QC solutions to flow into the sample path of a blood
gas analyzer. One container of Sullivan's packaging design replaced about 30 glass
ampules. Sullivan's packaging relieved the user of the task of opening many glass
ampules and of the attendent risks of broken glass. The disadvantages of Sullivan's
packaging included a need to refrigerate, a shelf life of less than a year, a menu
of only three analytes, and the complexity and cost of a spring-loaded valve.
The instant invention not only overcomes the limitations of glass
ampules, such as sensitivity of gas values to room temperature due to the headspace
above the liquid, and complications resulting from the sharp edges which form upon
breaking them open, or from the small, sharp glass pieces which can break off during
ampule opening, but also overcomes the limitations of Sullivan's zero headspace
packaging described above. The multi-analyte reference solutions with stable pO2
of the instant invention are packaged in containers with zero headspace, preferably
in flexible foil laminate containers, and are stable at room temperature for a shelf
life of from about one to three years.
An additional shortcoming of storage devices for reference solutions
for oxygen determinations (oxygen reference solutions) has been the opening or valve
required to access the fluid for use, while maintaining the integrity of the fluid
during storage. The materials available for valve construction and the need to breach
the barrier layer to incorporate the valve may have compromised fluid stability.
The access device disclosed herein for the preferred foil laminate containers used
in the methods of the invention solves that problem. The simplicity of the one-piece
valve should result in cost savings and greater reliability.
WO 97/16309 discloses a flexible package for an oxygen reference solution,
which is made from a laminated film comprising preferably polypropylene a the inner
layer, aluminium foil as the middle layer, and polyester as the outer layer. The
seams are heat sealed, whilce an optional access device for allowing access to the
solution after the storage period, is attached to the inside wall of the bag without
breaching the middle barrier layer.
US 4,116,336 discloses a package containing a reference liquid for
the calibration and /or quality control of blood gas analyzers. The reference liquid
is enclosed in a flexible, gas-tight container without any gas bubbles in the container,
and the gas pressure in the liquid is kept below 600 mm Hg at 37 degrees Celsius-
The container is preferably a laminate bag og aluminium foil with interior layer
of heat sealable plastic of low gas permeability, preferably a polyacrylonitrile
SUMMARY OF THE INVENTION
One object of this invention was to overcome the shortcomings of glass
ampules as storage containers for QCs and calibrators used with whole blood analyzers,
while allowing for automation of QC and calibrator delivery. In one aspect, the
instant invention overcomes problems presented by glass ampules as storage containers
for oxygen reference solutions used as controls for instruments that measure blood
analytes. Disclosed herein is a novel flexible package for oxygen reference solutions.
The package is made from a laminated film comprising an inner layer
with low or no oxygen reactivity, preferably polypropylene, aluminum foil as the
middle layer, and an outer layer that protects the aluminum foil layer from physical
damage, e.g., abrasion or corrosion. The seams are heat sealed, while an optional
access device for allowing access to the solution after the storage period, is attached
to the inside wall of the bag without breaching the laminated layers. The foil laminate
packaging allows for mechanical simplicity.
Preferred tubing for conveying a multi-analyte reference solution
with stable pO2 from a container to a blood analyzer is also disclosed.
Such tubing is flexible and relatively gas impervious, having a durometer (Shore
D scale) in the range of 10 to 100, preferably from 70 to 94 and more preferably
from 80 to 84. Preferred for such tubing are polyamide condensation polymers, more
preferred are polyester/polyether block co-polymers or polyester elastomers, and
especially preferred are Nylon™ [DuPont; Wilmington, DE (USA)] and Hytrel™
The lining of the preferred foil laminate packaging of this invention
that contains the multi-analyte reference solutions with stable pO2 of
this invention is selected for its low reactivity to oxygen. The preferred polypropylene
lining of the foil laminate package, preferably a foil laminate pouch, was chosen
as it is essentially inert to oxygen.
Further, source materials, particularly organic source materials,
for the other components of the multi-analyte reference solutions with stable pO2
of this invention are also screened for low oxygen reactivity. It was found that
some source materials contain impurities that are oxygen reactive enough to destabilize
the pO2 of such multi-analyte reference solutions.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1a is a side view of a four-sided multilayer package of this invention.
Fig. 1b is a cross-sectional view showing three layers of the packaging. Fig. 1c
is a first end view of the package of Fig. 1a. Fig. 1d is a frontal view of a three-sided,
center seam package.
Fig. 2 is a side view of an access device used in the methods of this invention.
Fig. 3 is a side view of a probe which pierces the foil laminate and fits into
the access device of Fig. 2.
Fig. 4a is a diagram of a clamp and locating device that can be used in conjunction
with the foil laminate containers of this invention. Fig. 4b is a top view of the
device of Fig. 4a. Fig. 4c is a side view of the device of Fig. 4a.
Fig. 5 is an Arrhenius diagram showing the predicted shelf life of a typical
formulation contained in the novel packaging of this invention.
Fig. 6 graphically demonstrates a use life study wherein pO2 of a
representative automated quality control formulation over time was measured, wherein
the tubing used to convey solutions from the pierce probe to the fluidic selection
valve of the foil laminate pouch was either Nylon™ [DuPont; Wilmington, DE,
USA] or Hytrel™ 6356 [Dupont].
ABBREVIATIONS AND BRAND NAMES
automated quality control reagent
Brij 700™ -
polyoxyethylene 100 stearyl ether with 0.01% BHA and 0.005% citric acid as preservatives,
[surfactant from ICI Americas, Inc., Wilmington, DE, USA]
CO-Oximeter or CO-Oximetry for instrument and method, respectively of measuring
total hemoglobin and hemoglobin fractions, such as, O2Hb, MetHb, COHb,
SHb and HHb
Cosmocil CQ™ -
polyhexamethylene biguanide hydrochloride, 20% [biocide from Zeneca Biocides,
Wilmington, DE (USA)]
32% 1,3-bis(hydroxymethyl)-5,5-dimethylhydantoin and 7.5% hydroxymethyl-5,5-dimethylhydantoin,
in water [biocide from Lonza, Inc., Fair Lawn, NJ, (USA)]
ethylene diamine tetraacetate
2-[4-(2-hydroxyethyl)-1-piperazinyl] ethanesulfonic acid [pKa of 7.31 at 37°C]
linear low-density polyethylence
Model 288 Blood Gas Analyzer [Chiron Diagnostics Corporation; Medfield, MA (USA)]
methylisothiazolone [a biocide from Boehringer-Mannheim GmBH, Indianapolis,
3-(N-morpholino)propanesulfonic acid [pKa of 7.01 at 37°C]
M. Yellow 7 -
Mordant Yellow 7
P.B. Violet -
Patent Blue Violet
partial pressure of carbon dioxide
partial pressure of oxygen
ProClin 300™ -
2.3% of 5-chloro-2-methyl-4-isothiazolin-3-one and 0.7% of 2-methyl-4-isothiazolin-3-one
with 3% alkyl carboxylate in 94% of a modified glycol [biocide from Rhom & Haas
Co., Spring House, PA (USA)]
pounds per square inch
polyvinylidene chloride [Dow Chemical Company, Midland, MI (USA)]
sulforhodamine B (dye; CAS #3520-42-1)
time to failure
DESCRIPTION OF INVENTIONFoil Laminate Packaging
In one aspect, this invention concerns novel flexible packaging for
oxygen reference solutions. Typical oxygen reference solutions used in whole blood
analyzers comprise sodium, potassium, and calcium chloride salts, pH buffer, sodium
bicarbonate, calcium chelating agent, surfactant, and biocide, which are equilibrated
under partial vacuum with a carbon dioxide/oxygen gas mixture before filling. The
typical oxygen partial pressures are from about 30 up to about 700 mmHg, but partial
pressures as high as 2000 mmHg (i.e., greater than ambient) can be used, as well
as partial pressures as low as zero (no oxygen present).
The packaging described herein stabilizes the oxygen reference solutions
via the use of a multilayered film as the packaging material. In addition, the package
incorporates an unusual access device for removing the solution. The access device
is not exposed to the outside of the container. Instead it is sealed within the
container and, as a result, does not provide an opportunity for there to be leakage
around the seal during the pre-use storage as opposed to having the access device
sealed within the package seam or through the wall of the container, where one would
ordinarily expect it to be sealed.
The foil laminate packaging described herein is novel. First, the
packaging material is selected because of the non-reactivity of its inner layer
with oxygen. Second, the thickness of its layers are different from those of previous
flexible packages. Third, the package described herein has an optional, novel valve
or access device, which reduces the amount of leakage and better maintains the integrity
of the contents of the container. Fourth, all prior art in this area of technology
was based on 4-sided bags with the security of one continuous seal around the entire
perimeter of the package; whereas disclosed herein is a 3-sided, center-seal pouch
having in places two, in other places four, layers of laminate to seal through,
and six stress points per bag where laminate is folded at 360° and where one might
therefore expect that a thin channel allowing gas exchange might result.
The foil laminate packaging of this invention is filled under vacuum
without any headspace of gas above the oxygen reference liquid in order to make
the contents insensitive to temperature and barometric pressure changes. A suitable
fill volume would be between 10 and 1000 mL, and preferably about 20 to 250 mL.
Below under the heading Film, the multilayered foil laminate
packaging is described in detail. The access device is similarly described in detail
below under the heading The Access Device.
Multi-analyte Reference Solutions with Stable pO2
In another aspect, this invention concerns methods of preparing multi-analyte
reference solutions with stable pO2 in zero headspace containers, preferably
in the flexible foil laminate packaging described herein. The phrase "multi-analyte
reference solution with stable pO2" is herein defined to mean a reference
solution used as a calibrator or as a control for pO2 plus one or more
other analytes, wherein the pO2 of said reference solution is maintained
within a predetermined range. Exemplary of such a range is at a specified value
± 4mmHg, alternatively at a specified value ±2%, preferably ±1%.
Examples of multi-analyte reference solutions with stable pO2
include the following: (1) a blood gas reference solution with a stable pO2
which calibrates or controls for pO2, pH and pCO2; (2) a blood
gas and electrolyte reference solution which calibrates or controls for pO2,
pH, pCO2 and electrolytes, such as, Na+, Cl-, K+,
Ca++, Li+ and Mg++; (3) a blood gas/electrolyte
and metabolite reference solution which calibrates or controls for pO2,
pH, pCO2, electrolytes, and metabolites, such as, glucose, lactate, bilirubin,
urea and creatinine; (4) a blood gas/electrolyte/metabolite and tHb reference solution;
(5) a blood gas/electrolyte/metabolite/tHb and CO-Ox fraction reference solution;
(6) reference solutions used for oxygen determination and to control or calibrate
for one or more other analyte(s) selected from pH, CO2, electrolytes,
metabolites, tHb, CO-Ox fractions, and Hct.
Exemplary of pO2 ranges calibrated or controlled by the
multi-analyte reference solutions with stable pO2 of this invention are
those between 0 to 1000 mmHg, 20 to 700 mmHg and 30 to 500 mmHg. Exemplary pCO2
ranges calibrated or controlled by the multi-analyte reference solutions of this
invention that test for blood gas are those between 0 to 150 mmHg, 5 to 100 mmHg
and 15 to 75 mmHg.
Described below under the heading Methods of Preparing Multi-Analyte
Reference Solutions with Stable pO2 are methods for maintaining
the pO2 of an multi-analyte reference solutions with stable pO2
within a predetermined range for a desirable shelf life of from one to about three
Described below under the sub-heading Analyte Levels and Formulations
of Representative QC and Calibrator Reagents, are exemplary and preferred five
level QC reagents of this invention. Parameters of a key all-inclusive level (exemplified
by level 3 below) are set forth under that sub-heading.
Methods of Preparing Multi-Analyte Reference Solutions with Stable pO2
The most unstable component of a multi-analyte clinical reference
solution in a zero headspace container used for oxygen determinations, among other
determination(s), is usually pO2. Methods are provided to maintain the
pO2 of multi-analyte reference solutions in a zero headspace container
within a predetermined range, that is, e.g., at a specified value ± 4 mmHg, alternatively
± 2%, preferably at ± 1%.
Central to the methods of maintaining the stability of pO2
in multi-analyte reference solutions in zero headspace containers is minimizing
the contact of the oxygen in such a reference solution with materials that are oxygen
reactive. As detailed infra, the lining of the foil laminate packaging for multi-analyte
reference solutions with stable pO2 of this invention is selected for
its low reactivity to oxygen. PP is the preferred lining material for the flexible
zero headspace packaging of this invention.
Further the methods of this invention for preparing multi-analyte
reference solutions with stable pO2 comprise preparing such reference
solution formulations with components that have been screened for low or no oxygen
reactivity. A representative raw material screening process is provided below. Particularly
important is the screening of organic materials for low or no oxygen reactivity.
It was found, as shown below, that some source materials may contain impurities
that are oxygen reactive enough to destabilize the pO2 of such a multi-analyte
reference solution in a zero headspace container.
Further are provided methods of preparing multi-analyte reference
solutions with stable pO2 in the least number of zero headspace containers
for detecting as many critical care analytes as practicable. Set forth below are
examples of such formulations. Again low oxygen reactivity is critical to preparing
stable formulations. It is important to formulate an all-inclusive level, wherein
the pO2 is low, for example, at 30 mmHg, 40 mmHg or at 50 mmHg, at a
low pH, for example, at pH 7.13 or 7.15, and at a low glucose concentration, for
example, at 46 or 50 mg/dL, and at a low dye concentration.
Further in regard to other levels of such a reagent, it is important
to separate the formulations used to test for mid-pO2 and high-pO2
from glucose and from the dyes needed to simulate tHb and CO-Ox fractions. Exemplary
formulations are provided below.
Analyte Levels and Formulation of Representative QC and Calibrator Reagents
It is desirable to prepare a minimum number of formulations for the
multi-analyte reference solution panels of this invention, [i.e., preferred quality
control (QC) reagents] so that, test time on analyzers is maximized and costs are
minimized. However, the lack of headspace in the packaging of this invention renders
that goal of minimizing the number of formulations to test a maximum number of analytes
difficult in that unlike the conventional glass ampule packaging which has on a
volume-to-volume basis, roughly 32 times more oxygen in the headspace than in solution,
the packaging of the instant invention has no oxygen reserve. Without an oxygen
reserve, organic materials in the solutions, such as, glucose and the dyes used
to simulate hemoglobin, or impurities in such source materials, react with the oxygen
present in the solutions, thereby reducing the pO2 of the solutions.
Key to combining so many critical analytes in as few containers as
practicable are (1) using a low pH/low pO2/low glucose/low tHb formulation
as an all-inclusive level (exemplified by level 3 herein); and (2) separating the
mid-pO2 and high-pO2 reference solutions from glucose and
from dyes. Exemplary formulations for a five level quality control reagent are provided
below. Such a five level QC combines from about 5 to about 20 analytes, preferably
from about 12 to about 20 analytes including pH, pO2, pCO2,
electrolytes, metabolites, hematocrit, tHb, and CO-Ox fractions. The all-inclusive
level of such a QC reagent controls for the following analyte levels:
(1) a low pH, from about 6.4 to about 7.4, more preferably from about 6.8 to
about 7.3, still more preferably from about 7.1 to about 7.2;
(2) a pO2 of from about 20 mmHg to about 75 mmHg, more preferably
from about 25 mmHg to about 70 mmHg, and still more preferably from about 30 mmHg
to about 60 mmHg; and
(3) a low glucose concentration of from about 10 mg/dL to about 80 mg/dL, more
preferably from about 30 mg/dL to about 60 mg/dL; and
(4) contains a low dye concentration corresponding to a hemoglobin concentration
of about 5 g/dL to about 11 g/dL, preferably from about 6 g/dL to about 10 g/dL,
more preferably from about 7 g/dL to about 9 g/dL.
Table 1 below shows exemplary analyte levels for a representative
5 level automatic quality control reagent ("5 Level AQC") of this invention.
It is further preferred that analyte levels of the reference solutions
include not only tHb as an analyte, but also the other CO-Ox fractions -- O2Hb,
COHb, MetHb SHb and HHb as shown in Table 1. Therefore, 16 analytes are controlled
by the representative all-inclusive level (Level 3) as follows:
Table 2 sets forth representative formulations that could be used
to prepare a 5-level AQC. It is preferred that Hct, creatinine and urea only be
monitored at two levels, whereas the other analytes are monitored at three levels
in five formulations.
Representative Formulations for 5-Level AQC12345MOPS:mmol/L3030271030NaOH"2928271226NaHCO3"21212166NaCl"11595751432KCl5.73.444Citric Acid"1.52.02.52.02.0CaCl2"126.96.36.199.42.4Mg++ (Acetate-)2"0.91.22.01.21.2Li+Lactate-"7.93.01.012.0Glucose:g/L0.501.002.00SRB (red dye)"0.4900.9241.104M. Yellow 7"0.2491.7700.786FD&C Blue #1".0027.0259P.B. Violet"0.103Creatinine".0100.0700Urea"0.2571.50Brij 700™".05.05.05.05.05MIT"" .40.40.400.400.40Tonometry gas % CO2/% O2/Bal.N26/4810/2517/56/486/48
A preferred all-inclusive level (designated Level 3 herein) formulation
of a 5-Level AQC would control from about 5 to about 20 analytes, preferably from
about 12 to about 18 analytes, more preferably from about 14 to about 16 analytes.
The following is an exemplary preferred formulation which includes 14 components:
Bags from throughout the lot were randomly selected and stressed at
elevated temperatures for appropriate time intervals in order to perform an accelerated
stability study and generate an Arrhenius plot to predict shelf life at room temperature.
The methods used were similar to those described infra. Results for pO2,
the least stable analyte, are shown in Table 3.
Accelerated Stability of an Exemplary Level 3 FormulationTemperature, °CTime, wksΔpO2 v. control, mmHg551-4.32-6.4502-1.86-4.0456-3.310-4.4Allowable Change±4
The table below shows the Arrhenius calculations used to derive the
estimated shelf life.
Arrhenius Calculations for a Preferred Level 3 FormulationTemperature, °C1/KTime-to-Failure, wksLog(ttf)55.00304881.10.05550.00309605.80.7745.00314478.50.9325.00335578752.94
The projected room temperature shelf life of 875 weeks, or 17 years,
for the representative Level 3 formulation was estimated using 0.94 as the correlation
coefficient. A more conservative estimate can be made using a rule-of-thumb which
relies on the fact that the minimum change in reaction rate per 10°C increase in
reaction temperature is an increase of two times. Based on failure in 2 months at
45°C, the inventors would estimate that failure will not occur at 25°C until at
least 8 months. However, the inventors consider it highly unlikely that the reaction
rate increase per 10°C increase would be any less than three times. Therefore, the
inventors consider that a realistic but still conservative estimate of the shelf
life of the representative Level 3 formulation would be at least 18 months.
To prepare the formulations of this invention, all solutions require
tonometry with the appropriate gases to achieve the gas levels listed above. Although
gas values are not always listed above for levels 4 and 5, tonometry is still desirable
in order to achieve gas levels which minimize hysteresis and drift effects on the
The tonometry can be performed at temperatures such as 25°C or 37°C
or even 50°C, and of course the choice of temperature will affect the composition
of the tonometry gas. More importantly, tonometry should be performed at sub-atmospheric
pressures, preferably in the 300-500 mmHg range, so that outgassing will not occur
if the solutions are used at high altitudes where the barometric pressure is below
normal, or in warm environments. Obviously, the higher the tonometry temperature,
the higher the pressure allowed in the tonometer. An example of a suitable condition
is 37°C at 450 mmHg, where the gas composition for a level 2 QC would be 10% CO2,
25% O2 and 65% N2.
Exemplary preferred dyes for the formulations of this invention are
listed in Table 2, supra. Those dyes are disclosed in Li, J., EP 0 743 523 A2 (published
November 20, 1996).
HEPES and MOPS are preferred buffers for the formulations of this
invention. MOPS is a particularly preferred buffer. Other suitable buffer systems,
including the sodium salt derivatives, are described by Good et al., Biochemistry,
5: 467-477 (1966) and Ferguson et al., Analytical Biochemistry, 104:
Shelf Life and Use Life
One object of this invention is to increase the shelf life and use
life of the QC and calibrator formulations of this invention. An acceptable shelf
life (i.e., closed package) would be about one year. A preferred shelf life would
be from about one year to two years, and still more preferred from about one to
An acceptable use life (i.e., open package) would be about two weeks,
preferably from about two weeks to about a month, and more preferably from about
two weeks to about two months. The use life is extended by appropriate selection
of tubing material to conduct reference solutions from the access device to the
blood analyzer as described infra.
The inventors discovered a critical element in how the formulations
impact shelf life by de-stabilizing pO2. One study compared a very simple
formulation, containing only sodium bicarbonate to neutralize the CO2
in the tonometry gas, and Brij 700 surfactant, to create appropriate surface tension,
such that the solution behaves normally in the tonometer and filler, to a complete
10-ingredient formulation. The data are summarized in Tables 5 and 6.
Accelerated Stability of 2- vs 10-Ingredient Formulation: ΔpO2,
mmHg from controlTempTime, wksOnly Brij + Bicarb+ 8 Other Chemicals60°C1-5.3-14.055°C1-2.4-7.72-5.6-15.450°C1-2.6-3.32-2.7-12.445°C2+0.2-4.7Allowable Change±4.4±4.4
Arrhenius Calculation Based on pO2 Data for Formulations in Table
XVIBicarb + Brij+8 ChemicalsTemp1/KTime-to-failureLog(ttf)Time-to failureLog(ttf)60°C.00300305.8 days0.7632.2 days0.34255°C.003048811.3 days1.0544.0 days0.60250°C.003096022.8 days1.3587.1 days0.85445°C.003144713.1 days1.11725°C.00335571042 days3.018185 days2.268
The correlation for the Arrhenius prediction for the 2-component formulation
was 0.99999, and for the 10-component formulation, 0.9999 (r). It can be seen that
addition of eight additional chemicals, the inorganic compounds NaCl, KCl, CaCl2,
NaOH, and the organic compounds citric acid, glucose, MOPS (pH buffer),and ProClin
300 (biocide), caused the pO2 to be less stable by 5-6 times as compared
to the simple, 2-component formulation. The shelf life estimate for 25°C decreased
from 34 months for the 2-component formulation to 6 months for the 10-component
formulation. Thus, some or all of the eight added chemicals reacted with oxygen
in the aqueous solution in the flexible bag, causing premature loss of shelf life.
Therefore, studies by the inventors have shown that it is difficult
to achieve stable pO2 in a zero-headspace package with formulations having
many ingredients, each potentially capable of reacting with oxygen, and realizing
that interactions among ingredients could also be de-stabilizing. Specifically,
test results suggest that glucose and the dyes used to simulate hemoglobin can react
with oxygen. The oxygen reactivity of those chemicals is one reason the inventors
prefer to separate those chemicals in QC levels 4 and 5 from QC levels 1 and 2.
However, the inventors realize that the QC all-inclusive level (level 3) includes
those 3 analytes along with the other nine analytes, but determined that that all-inclusive
level 3 formulation should work because:
1. at pH 7.15, glucose is more stable than at the two higher pH levels;
2. the levels of glucose and Hb-simulating dye are all low; and
3. the pO2 is low. In fact, the true pO2 at the low level
is roughly half of the measured pO2.
Thus, the inventors discovered that the unique properties of level
3 allow the packaging of a QC in 5 containers rather than 6, provides the advantage
to the customer of more patient samples to be assayed in a given time period.
Direct Comparison of pO2Stability in Zero
Headspace Packaging v. Ampules
A study was performed to compare a conventional multi-analyte QC formulation,
similar to the formulation in Table A of U.S. Patent No. 5,637,505, in glass ampules
to that same formulation in a zero headspace foil laminate package of this invention.
To achieve roughly the same pCO2 and pO2 values in the foil
laminate packaging process, as occur in the ampuling process, the foil laminate
pouches were filled with QC solutions that were tonometered under partial vacuum
with the appropriate gases, and then the solutions were pumped into zero-headspace
foil laminate pouches, and pasteurized as set forth below. A limited accelerated
stability study was then performed in accordance with the method described above.
The two studies allowed us to make the following comparison:
Comparison of Packages with and without Headspace Values below are ΔpO2,
mmHg (except for factors)ConditionLevel 2Level 3Temp, °CTime, wksAmpulesBagsFactorAmpulesBagsFactor452-1.2-3630X-0.9-1214X502-1.0-4242X551-2.6-5320X-1.3-2116X602-1.9-5730X
It can be seen that there is a considerable range among the six factors,
from a low of 14X to a high three times as great, 42X. What can be concluded from
the data is that maintaining pO2 stability in a relatively inert, zero
headspace package is at least an order of magnitude more difficult than maintaining
the same degree of pO2 stability in a package with a headspace at least
half as large as the solution volume.
Raw Material Screening Test
A representative screening test for the components of formulations
of this invention is demonstrated by the study of this section. Ten solutions with
the same defined level of pO2, were prepared simultaneously by equilibrating
deionized water in glass containers at 50°C in a water bath. The temperature of
the water bath must be at least as high as the temperature intended to be used for
the accelerated test which is to follow, so as to avoid outgassing of oxygen during
the stress cycle at elevated temperature.
In order to magnify the oxygen consumption of individual ingredients,
especially in cases where there may be several minor contributors as opposed to
one or two major contributors, it is desirable to increase the concentrations above
their normal use levels. In this study, the inventors increased concentrations by
The inventors isolated the eight chemicals added to the two component
formulations in the study described above under the heading Shelf Life and Use
Life. Those eight chemicals are the inorganic compounds NaCl, KCl, CaCl2,
NaOH, and the organic compounds, citric acid, glucose, MOPS (pH buffer) and ProClin
300 (biocide). However, in order to test in the neutral pH range (6-8) some chemicals
had to be tested together, namely, MOPS with NaOH, and citric acid with sodium bicarbonate.
For efficiency, the three chloride salts were tested together, based on our prediction
that the inorganic chemicals were unlikely to be significant contributors to slow
oxidation reactions. In addition to the eight chemicals already mentioned, we also
tested an alternative pH buffer, HEPES, and two dyes, SRB and Mordant Yellow 7.
Chemicals were added to the pre-warmed deionized water in glass bottles,
and mixed by inversion. When all chemicals in all bottles were dissolved, solutions
were poured into bags which had been sealed on 3 sides, followed immediately by
sealing the fourth side below the liquid level. After a 44hr/65°C pasteurization
step, half of the bags were left at room temperature while the other half were stressed
for twelve days at 50°C, followed by cooling to room temperature. Controls and stressed
bags were tested for pO2 in one run on two model 288s. The following
results were obtained:
Screening Test of Chemical Ingredients for Oxygen ReactivitySubstanceMean ΔpO2RangeWater blank-4mmHg4mmHgMOPS, Sigma-54MOPS, Research Organics-44Glucose, Sigma-143Glucose, Fluka-113ProClin 300, lot LA60507-72ProClin 300, lot LA64543-94Citric Acid, Bicarbonate, Brij-910NaCl, KCl, CaCl2-54HEPES (pH buffer)-63Sulforhodamine B (red dye)-68Mordant Yellow 7 (dye)-135
Those results show that:
1. glucose and Mordant Yellow 7 are the most significant oxygen reactives;
2. ProClin 300 is moderately reactive;
3. MOPS, HEPES, and the three chloride salts are relatively non-reactive; and
4. results for SRB and the citric/bicarb/Brij mixture were non-conclusive due
to excessive bag-to-bag variability. However, further substantially similar screening
showed that SRB was moderately reactive, and that citric acid, sodium bicarbonate
and Brij were relatively non-reactive.
In regard to Mordant Yellow 7, shown above to be significantly oxygen
reactive, it can be concluded that it would be preferred that another yellow dye
or Mordant Yellow 7 that is less oxygen reactive, e.g., from another source, be
used in the formulations of this invention. When tHb is the only CO-Oximetry analyte
to be tested, a red dye is sufficient. SRB is a red dye, and the particular SRB
screened was found to be moderately reactive. It may be preferred to screen SRBs
from other sources or other red dyes for an SRB or other red dye having lower oxygen
reactivity. However, the accelerated stability results in Table 4 show that the
level 3 formulation containing the above-screened SRB and Mordant Yellow 7 dyes
has significantly more than a year's shelf life. Shelf life of such a formulation
may be further prolonged by screening and incorporating therein dyes having lower
Effect of Glucose on pO2Instability
The strong destablizing effect of glucose on pO2 stability
was noted in the study described below. This study compared two sources of glucose,
used at 1.8 g/L - one from Fluka Chemical Corp. [Ronkonkoma, NY (USA)] and one from
Sigma Chemical Co. [St. Louis, MO (USA)] - in a 150 mmHg pO2 calibrator
at pH 6.8 to the same calibrator without any glucose added. A limited accelerated
stability test was conducted on those solutions, with the following outcome.
Effect on pO2 of Storing 150mmHg Calibrator at High Temperatures
for 2 wks
Mean difference from Non-heated solutions, mmHgTEMPNo Glucose addedFluka Glucose addedSigma Glucose added45°C-2.2-5.7-6.350°C-4.7-8.8-9.9
It can be seen that:
1. at both temperatures, both sources of glucose at least double the pO2
2. the differences between the two glucose sources are relatively minor.
Thus, those results fit very well with the results reported in the
section on screening raw materials above. Moreover, because the source appears to
play a relatively minor role, this suggests that the oxygen reactivity is inherent
in glucose, which was not obvious before we undertook this study.
There are at least three well-known degradation mechanisms for glucose:
1. reaction with oxygen, forming gluconic acid, if glucose oxidase is present;
2. reaction with ATP, forming glucose-6-phosphate, if hexokinase is present;
3. alkaline rearrangement, forming first fructose, later mannose.
The first two are widely used in clinical chemistry assays to measure the level
of glucose in blood. The third, occurring at even mildly basic pH, is the most common
route for glucose instability in quality controls used in conjunction with glucose
None of those three common reactions explain the presumed reaction
between glucose and oxygen in the formulations of this invention because only one
lists oxygen as a reactant, and in that
The film which is used for the container is multilayered and uses
a material having low or no oxygen reactivity, preferably polypropylene (PP), for
the inner layer, aluminum foil for the middle layer, and an outer layer that is
protective of the aluminum layer, preferably polyester. The outer layer merely provides
protection for the aluminum layer, preventing abrasion and corrosion. Thus, for
example, a nylon layer, or even a simple lacquer coating are suitable alternatives.
[Nylon is a family of high-strength, resilient synthetic materials, the long-chain
molecule of which contains the recurring amide group CONH. The term "nylon" was
coined by its inventors at E.I. duPont de Nemours & Co., Inc.] However, the
outer layer should have a melting point greater than PP's melting point which is
An important parameter of the aluminum layer is that it be thick enough
so that there are no pinholes, thus preventing physical leakage of oxygen, yet thin
enough so that it can be readily formed into pouches on automated machines and will,
after being filled, release its contents without undo force by readily collapsing
as the contents are removed.
The inner PP layer is important for several reasons. First, it must
melt and form the seal which closes the package. Second, it must be unreactive with
the oxygen. It is this second factor which distinguishes this packaging material
from those previously used for this purpose.
To the inventors' knowledge, this laminate has never been used commercially
for packaging products which contain high-precision solutions with dissolved gases
for scientific, medical, analytical purposes. The PP lined laminate is not known
to be used by others as an oxygen barrier for chemical products. A former manufacturer
of oxygen calibrators (Mallinckrodt Sensor Systems, Inc., Ann Arbor, MI) has used
laminated film to package a calibrator, but they used polyethylene as the inner,
sealing layer. The PP lined laminate has been used in the past mainly for food products,
and has been chosen for the high melting point of the polypropylene sealing layer,
which makes this material suitable for sterilization in a steam autoclave or similar
Films from various suppliers were evaluated for efficacy in maintaining
the dissolved gas concentrations of solutions stored within. Films were obtained
from Kapak Corp., Minneapolis, MN (part no. 50703), American National Can Co., Mount
Vernon, OH (part nos. M-8309, M-8359, M-8360), James River Corp., Cincinnati, OH
(part nos. JR 4123, JR 4400), Technipaq, Inc., Crystal Lake, IL ("Dull Foil Laminate"),
Lawson Mardon Flexible, Inc., Shelbyville, KY (spec nos. 13362 and 15392), Smurfit
Flexible Packaging, Schaumburg, IL (LC Flex 70459, 70464), and Rollprint Packaging
Products, Inc., Addison, IL (RPP #26-1045). 4-sided bags were either purchased with
3 sides pre-sealed or were formed using an impulse heat sealer from Toss Machine
Components, Inc., Bethlehem, PA, Model 01617. The 3-side sealed bags were filled
with various reference solutions and immediately sealed through the liquid, allowing
no headspace inside the package. In some instances, for enhanced stability of the
oxygen partial pressure in the reference solution stored within the bags, filled,
sealed bags were heat-treated at elevated temperatures between approximately 50°C
and 121°C for times ranging from 15 minutes to 7 days, depending on the temperature.
Fig. 1a shows a side view of a sealed bag 1, and one possible
location of the access device 5 in the interior of the bag is shown. The
sealed portion of the bag is also shown 6. Fig. 1b shows the 3 layers of
a preferred film, the inner polypropylene layer 2, the middle aluminum layer
3, and the outer polyester layer 4.
Some filled bags were left at room temperature; others were stored
at elevated temperatures for various times. To simplify reporting of this and subsequent
trials, we used storage at 55°C for 1 week as a basis for comparison. After removing
test bags from the incubator, they were cooled to room temperature and tested on
two critical care analyzers [generally selected from the 200 Series Critical Care
Diagnostic Systems manufactured by Chiron Diagnostics Corporation; Medfield, MA
(USA); a 278 was often used with a 288] with control bags in the same run. In particular,
the pO2 results were examined in a series of six studies. Due to differences
in conditions such as reagent composition and package surface-to-volume ratios,
the pO2 differences are not directly comparable. Therefore, all results
were converted to relative scores where the most stable laminate was assigned a
score of 1.00, and all other laminates were assigned scores on the basis of ΔpO2
ratios. Using this convention, the following results were obtained:
MaterialNMean ScoreRange of ScoresPolyethylene40.140.10 - 0.16Polypropylene60.410.18 - 1.00Polyester20.280.26 - 0.30
The preferred and most preferred laminates have an inner PP liner
of the thickness shown below, a middle layer of aluminum as shown below, and an
outer polyester layer. (The thickness and material selection of the outer layer
is least critical and can vary somewhat.) Acceptable film thicknesses are also shown.
Approximate thicknesses of layers in mils (1/1000 inch):
Other acceptable layers include polyester at 0.5-2 mil for the inner
layer; for the outer layer either nylon with thickness of 0.2 - 2 mil or lacquer
coating. Polyethylene has not been found to be acceptable as an inner layer.
There are detrimental properties that result if any of the film layers
are too thick. Namely, the laminate becomes too rigid, making it difficult to form
and fill during manufacture, and difficult to pump out the liquid contents from
the pouch/bag during use. Furthermore, if the aluminum layer is too thin, there
is a higher probability of having pin-holes, which may lead to gas leakage. If the
sealing layer is too thin, it may be entirely displaced at the moment of heat-sealing
at the seal under high pressure required for strong seals, thereby exposing bare
aluminum which would react with oxygen.
Stability testing has shown that the PP lined film is preferred over
the polyethylene film. The Arrhenius method of predicting product shelflife is well-established
in the in-vitro diagnostics and pharmaceutical industries (Conners et al, "Chemical
Stability of Pharmaceuticals: A Handbook for Pharmacists", NY: Wiley, 1986; Porterfield
& Capone, MD&DI 45-50, Apr 1984; Anderson & Scott, Clin Chem,
37: 3, 398-402, 1991; Kirkwood, Biometrics, 33, 736-742, Dec 1977).
Products are stored at elevated temperatures for various times, following which
they are re-equilibrated at ambient temperature and tested against non-stressed
controls for critical properties such as activity of a component or measured analyte.
The rate of change or more conveniently, the time-to-failure, of a given analyte
is determined for each temperature, often by plotting log(C/Co) vs time, which is
a linear function for the most common, first-order reactions. Owing to the linear
relationship between log(time-to-failure) and the inverse of the absolute temperature
(1/K), a plot can be constructed from the elevated-temperature data, and the resulting
line can be extended to the maximum recommended storage temperature to predict the
time-to-failure at that temperature. In this manner, actual shelflife can be predicted
In an early predicted shelflife study using polyethylene-lined bags,
finished packages filled with an oxygen reference solution were stored at 35, 45,
and 55°C for times ranging from 4 days to 8 weeks, depending on the storage temperature,
using longer times with lower storage temperatures. Each test condition included
4 bags tested on two blood gas analyzers [200-series manufactured by Chiron Diagnostics
Corp. (CDC), supra]. Time-to-failure (TTF) was defined as a 2% change in
Regression analysis on the above data, based on plotting log(ttf)
as a function of 1/K, results in a predicted 25°C shelflife of 3 months for an oxygen
reference solution stored in the polyethylene-lined bag. The correlation coefficient,
r, is 0.98.
In the polypropylene study, finished packages containing an oxygen
reference solution were stored at 35, 40, 45, and 50°C for times ranging from 1
to 9 weeks, depending on the storage temperature, using longer times with lower
temperatures. Each test condition included 3 bags tested in singlicate on two blood
gas analyzers (200-series from CDC, supra). The first-order model was used
to determine time-to-failure (TTFs), where failure was defined as a 2% change in
Using the four TTFs, an Arrhenius plot was constructed (see Fig. 5),
where time to failure (in weeks) (TTF) is shown as a function of inverse temperature,
1/K (shown as T in Fig. 5). (1/K is the inverse of Kelvin temperature.) The linear
extrapolation to 25°C is 61 weeks or 14 months, for an average pO2 change
of -.066mmHg/wk. The reliability of the prediction is affirmed by the highly linear
relationship among the 4 points, with a correlation coefficient, r, of 0.99. A score
of 1.00 would indicate that all points fall on a straight line; a score of 0.00,
that no relationship exists between log ttf and 1/K. (Note that the equation for
the Arrhenius plot exemplified was found to be log y = -19.48 + 6339x.)
The resulting predicted shelflife of the oxygen reference solution
in polypropylene-lined bags represents a four-to-fivefold improvement over the shelflife
predicted for oxygen reference solution stored in the polyethylene-lined bags. It
also represents a nearly tenfold improvement over a recent state of the art product,
known as "Cal B" which was sold by Mallinckrodt Sensor Systems, Inc. [Ann Arbor,
MI (USA)]. The software in the GEM® Premier Analyzer that accompanies that system
automatically subtracts 0.58mmHg pO2 from the initial assigned pO2
for every week which has elapsed since manufacturing in order for the Cal B calibrator
to be useable for its expected commercial usage period. If not for this calculation,
using our 2% criterion, the useful shelflife would be only 7 weeks, clearly too
short a time for commercial use of the product. Moreover, note that the actual Cal
B shelflife, 6 months, limits the shelflife of the entire cartridge to only six
months, arguably the minimum practical shelflife for an in-vitro diagnostic product.
On the other hand, 14 months is clearly an acceptable shelflife.
Other factors which discourage use of PP-lined laminates are their
greater stiffness and higher melting points. PP durometer hardness, on the Shore
D scale (ASTM Designation: D 2240-91 American Society for Testing and Materials,
Philadelphia, PA), is 70-80 compared with only 44-48 for PE. Stiffness impedes high
surface:volume ratio, which improves shelflife, and makes automation on form/fill/seal
machines more difficult. The higher melting point for PP, 171°C compared to only
137.5°C for PE, requires more energy, time, or both to seal the bags.
Other variations in the packaging method are possible. For example,
other shapes of packages that reduce the ratio of surface area of package to volume
of solution and gas within the package (e.g., 2 circular pieces of film which are
sealed together), would reduce even further the exposure of the solution and gas
to the film, even further reducing the oxygen degradation. The packaging disclosed
herein is also effective in protecting tonometered solutions containing other gases
aside from oxygen. Furthermore, various configurations of package (e.g., three-sided
seal or side-seam; four-sided sealed; gusseted packages; or "stand-up" pouches)
can be used. (Compare, for example, Fig. 1c, which shows 4 sides sealed, to Fig.
1d, which shows a 3-sided seal.) These package variations affect utility of the
packaging method and are not simply design alternatives. Other variations will be
apparent to those with expertise in this technology area.
The Access Device
The access device is attached inside of the package. Attachment can
be achieved using any technique available, for example, via use of adhesive, heat-bonding,
ultrasonic welding, etc. This access device is an optional component of the package
and is particularly useful when the contents of the container are used over a period
of time after a prolonged storage interval. In previous approaches, a valve has
been sealed into the edge or through the wall of the container so that it would
be accessible from the outside of the container. However, in the package used herein,
the access device is sealed totally within the package on the inner wall, and does
not breach the seal or the walls of the container.
Figs. 1a, 1c and 1d show typical locations for the access device.
Fig. 2 shows the detail of a typical access device, with 7 being the portion
of the access device sealed to the wall of the container, 8 being the outer
portion of the delivery channel, 9 being the inner portion of the delivery
channel, and 10 being the sealed portion of the delivery channel which is
punctured by the probe, which then makes a tight fit with the inner portion of the
delivery channel, thus preventing leakage from the container. Fig. 3 shows a typical
probe, which is used to puncture the bag and fit into the access device inside the
bag, with 11 representing the probe and 12 representing the sharp
end of the probe which punctures the sealed portion of the delivery channel. The
probe is incorporated in a clamping device 13 (see Fig. 4a, 4b and 4c) which
has a circular opening 14 which fits over the hemispherical back of the access
device 15 aligning the probe with the delivery channel. The probe is connected
to other components which allow the oxygen reference solution to flow to the apparatus
where it can be utilized in assays. When the package is punctured, the probe pierces
the wall and forms a tight seal with the delivery channel of the access device.
Before the package is punctured, the access device is totally isolated within the
(more or less) impermeable walls of the container. This approach has an advantage
over other valves and access devices in that it does not provide a diffusion pathway
to the outside environment. Obviously there can be variations in the design of the
access device and probe, which will be apparent to those with skill in the art.
The access device is also made of PP so that it seals well with the
wall of the container. The description of the access device should allow for some
variations of the preferred access device. For example, the access device might
be sealed to both walls of the package to provide an added benefit of stabilizing
the shape of the package. The access device can be sealed at any location inside
the container, for example, in a corner (for ease of attaching a clamp) or away
from the edge of the container. Furthermore, the access device does not need to
be attached to the container if there is some technique incorporated for locating
the access device. For example, if the access device were to contain an embedded
magnet, the application of an exterior magnet could be used to capture and position
the access device. Other shapes (cones, indents, etc.) might be used for the locating
feature. Rings can be molded into the inner wall of the delivery channel to improve
the seal after puncture. The travel distance of the probe can be limited to prevent
puncture of the adjacent wall of the container.
The access device of the packaging of this invention extends the use
life of oxygen reference solutions. Once the packaging is opened, the access device
is designed to minimize oxygen diffusion thereby increasing the use life of the
reference solution. Further, flexible and relatively gas impervious tubing is used
to minimize oxygen diffusion.
The tubing conveys the oxygen reference solution from the package
through the pierce probe (Fig. 3) to the analyzer. For example, in Fig. 3, such
tubing would have a diameter which fits tightly into the second of the three cylindrical
regions, wherein the third cylindrical region has the same diameter as the internal
diameter of the tubing (illustrated with broken lines in Fig. 3) that intersect
the pierce probe (11).
The durometer (Shore D scale) of such tubing is in a range of from
10 to 100, preferably from 70 to 94, and more preferably from 80 to 84. Condensation
polymers having the requisite durometer characteristics are preferred, particularly
preferred are polyamide condensation polymers, more preferred are polyester/polyether
block co-polymers or polyester elastomers. Especially preferred tubing is Nylon™
[DuPont; Wilmington, DE (USA)] and Hytrel™ 8238 [DuPont].
Representative experiments below are described wherein tubing materials
can be tested for suitability for use in the methods of this invention. Silicone,
fluoropolymers and plasticized polyvinylchloride were thereby determined not to
be suitable tubing materials.
Use Life -- Selecting Tubing Material
Similar to shelf life, which is often limited by pO2 due
to reaction of oxygen with packaging or contents, use life is also often limited
by pO2, but by a different mechanism -- diffusion. The effectiveness
of the access device design of the foil laminate packaging of this invention minimizes
pO2 diffusion. This study employed two flexible tubing materials -- Hytrel
6356 [DuPont] and Zytel 42 Nylon [DuPont]. That tubing was used to conduct the oxygen
reference solution from the probe of Fig. 3 (as discussed above) which fits into
the access device of the foil laminate pouch to the analyzer (M288 model from CDC,
An open bag use life test was run on the following formulation which
had a pO2 of 40 mmHg:
The pO2 equilibrium point is approximately 190 mmHg at 22°C when measured
at 37°C. The lower pO2 within the bag increases the driving force for
oxygen from room air to diffuse into the bag and thereby into the test solution.
Six bags were tested over a 28-day period using 2 M288s [CDC, supra]. Results
are summarized in the table below and in Fig. 6.
A reasonable tolerance limit for allowable pO2 change is
±4mmHg at this low pO2. It can be seen that all six bags performed within
this range, but the bags with Nylon tubing attached had, on average, less increase
in pO2 over the test period.
The inventors' best explanation for the greater stability of pO2
in the bags with Nylon tubing attached is that the Nylon has a higher durometer,
or hardness, than the Hytrel 6356. Using the Shore D scale (ASTM Designation,
supra), Zytel 42 Nylon (Dupont) is rated 82 compared with 63 for Hytrel 6356.
The higher durometer implies that the molecules of the nylon are packed more tightly
together both making the material more rigid and making it more difficult for gas
molecules to diffuse through the interstitial spaces. Therefore, Zytel 42 Nylon,
and presumably other nylons are preferred tubing materials. Also, Hytrel 8238 has
the requisite durometer and is a preferred tubing material.
Further experiments with tubing materials were performed, wherein
aqueous solutions were tonometered with a gas mixture containing no oxygen, aspirated
into a section of test tubing sufficient to contain 100uL using a syringe, held
in the tubing for 60 seconds, and then aspirated into a model 288 analyzer [CDC,supra]
beyond the segmentation valve by manually turning the pump roller. The resulting
pO2 readings served as indicators of the degree to which oxygen from
the tubing diffused into the aqueous solutions. More than 15 tubing materials were
tested in this manner. The results indicated that polyester/polyether block co-polymers,
notably Zytel 42 Nylon and Hytrel 8238, are preferred tubing materials. Another
preferred tubing material is Saran™ [polyvinylidene chloride; Dow Chemical
Company; Midland, MI (USA)]. Silicone, fluoropolymers and plasticized polyvinylchloride
were found not to be suitable as tubing materials.
Reactivity of Oxygen with Polypropylene
Oxygen is much less reactive with PP than it is with polyethylene.
It is this lower reactivity that makes PP a more desirable material to be used as
an inner layer of the foil laminate packaging of this invention. In the past, developers
were concerned with permeability of the inner layer to oxygen, but this turns out,
however, to be a less important attribute than the reactivity for this type of reference
Both PP and PE provide reasonable sealing, although the PP has a higher
melting temperature. In addition, both materials provide equivalent protection against
liquid leakage. However, in polyethylene, there is more reactivity between oxygen
and the polymer, thus reducing the oxygen level. It is not permeation through the
polyethylene film that was largely responsible for reducing the oxygen level. This
argument is based on the following numbered points.
1. Although the pO2 level in the oxygen reference solution seems
to be considerable, at roughly 200mmHg, in molar terms, it is only 0.27mmol/L. The
calculation to convert from mm Hg partial pressure to mmol/L oxygen concentration
is reasonably simple and straightforward, but oxygen is rarely described in the
literature in molar units. Rather, where it is not in partial pressure units such
as mm Hg or kPa, it is found in concentration units such as mg/L or mL/dL. However,
approaching the oxygen loss problem from the molar perspective teaches us that reaction
of only 0.005mmol/L (2%) would cause product failure. Ultraviolet (UV) spectroscopy
studies showed that at elevated temperatures, water-soluble, UV-absorbing substances
are extracted from the sealing layer into the bag contents. This is true for both
PP- and PE-lined bags. Finally, whereas only 0.005mmol/L reactant is required for
product failure (by pO2 decrease), with 100 mL reagent in a 4"x6" bag,
only 0.1% of an additive with a molecular weight of 500 in a 4 mil PP film would
provide 0.05mmol/L of oxidizable reactant, ten times the amount needed to explain
a 2% decline in pO2. Thus, the stoichiometry is reasonable, even assuming
an extraction efficiency of only 10%.
2. PP sealing layers from different vendors differ markedly in the pO2
changes in oxygen calibrator sealed within them when they are subjected to elevated
temperatures, as demonstrated in Table 11 above. Yet the permeability of polypropylene
roll stock from any of the several vendors can be expected to be similar because
it should be a property of the bulk polymer, unless it has been modified into an
oriented polypropylene. (Oriented PP is not known to be laminated to aluminum foil.)
Thus, it is unlikely that permeability differences can explain the differences in
pO2 deltas shown in Table 11. However, since the various PP vendors are
known to use a considerable variety of additives to the basic PP resin (these additives
being nearly always proprietary), it is quite likely that differences in additives
among the various resins explain a considerable portion of the differences in pO2
deltas, as different additives or even the same additives in different concentrations
would react to a greater or lesser degree with the oxygen in the calibrator.
3. The most convincing evidence to support the importance of reactivity over
permeability is from an experiment which isolated the two effects. A uniform population
of 3-side-sealed PP-lined bags were filled with an oxygen calibration solution tonometered
such that oxygen partial pressure would be roughly 200 mmHg. A control group of
the same bags was filled normally and immediately sealed on the Toss impulse sealer.
Two test groups had five pieces, cut so as to just fit into the bag, of either polyethylene
or polypropylene added to the bags just before filling and sealing. As in the stability
tests described above, some bags from all three groups were left at room temperature,
while others, randomly selected, were stored at 55°C for 1, 2, and 3 weeks. Bags
were cooled to and allowed to equilibrate at room temperature for at least 24 hours,
and then tested in the usual manner, that is, in triplicate on two 200-series blood
gas analyzers [CDC, Medfield, MA (USA)], alternating during runs between control
and test conditions. The following results were obtained:
Test GroupStress ConditionpO2, mean (SD)ΔpO2Net ΔpO2ControlControl201(3) mmHg3 wks at 55°C191(1)-10 mmHg+ PolypropyleneControl219(3)3 wks at 55°C206(6)-13-3 mmHg+ PolyethyleneControl221(2)3 wks at 55°C179(6)-42-32
The effect of the polyethylene on pO2 is both dramatic,
being an order of magnitude more severe than polypropylene, and significant, with
the additional 29mmHg decrease being nearly five times the greatest SD, 6mmHg. Permeability
cannot explain this difference because the plastic sheets were contained entirely
within the bags.
Verfahren, um den Partialdruck von Sauerstoff in einer mehrere Analyten enthaltenden
Vergleichslösung in einem Behälter ohne Kopfraum bei Raumtemperatur auf einem vorgegebenen
Wert ±4 mmHg zu halten, um auf die mehrere Analyten enthaltende Vergleichslösung
zuzugreifen und um die mehrere Analyten enthaltende Vergleichslösung zu einem Analysator
zu leiten, worin der Behälter aus einem Vielschichtlaminat besteht, wobei das Verfahren
(a) Herstellung des Behälters durch
(i) Auswählen einer inneren Schicht für den Behälter, die geringe oder keine
(ii) Auswählen einer mittleren Schicht für den Behälter, die aus Aluminium besteht;
(iii) Auswählen einer äußeren Schicht für den Behälter, welche die mittlere
Schicht, die aus Aluminium besteht, vor physischem Schaden schützt,
(b) Bereitstellung einer Zugriffsvorrichtung, die sich vollständig innerhalb
des Behälters befindet und weder die Laminatschichten des Behälters durchbricht
noch die Kante des Behälters, an welcher der Behälter versiegelt ist, unterbricht,
(c) Bereitstellung einer Sonde zum Durchstoßen der Zugangsvorrichtung umfasst,
dadurch gekennzeichnet, dass die Sonde über Schläuche
mit einem Durometer-Wert (Shore D) im Bereich von 10 bis 100 mit dem Analysator
Verfahren nach Anspruch 1, worin die Schläuche einen Durometer-Wert im Bereich
von 70 bis 94 aufweisen.
Verfahren nach Anspruch 1, worin die Schläuche einen Durometer-Wert im Bereich
von 80 bis 94 aufweisen.
Verfahren nach einem der Ansprüche 1 bis 3, worin die Schläuche aus einem Polyamid-Kondensationspolymer
Verfahren nach einem der Ansprüche 1 bis 3, worin die Schläuche aus einem Polyester/Polyether-Blockcopolymer
oder einem Polyester-Elastomer bestehen.
Verfahren nach einem der Ansprüche 1 bis 3, worin die Schläuche aus Nylon™,
Zytel 42 Nylon, Hytrel™ 8238 oder Polyvinylidenchlorid bestehen.
A method of maintaining a partial pressure of oxygen in a multi-analyte reference
solution in a zero headspace container at a specified value ±4 mmHg at room temperature,
of accessing said multi-analyte reference solution and of conveying said multi-analyte
reference solution to an anlyzer, wherein said container is formed from a multi-layered
laminate, said method comprising
(a) preparing said container by
(i) selecting an inner layer for said container that has low or no oxygen reactivity;
(ii) selecting a middle layer for said container that is aluminum; and
(iii) selecting an outer layer for said container that protects the middle layer
that is aluminum from physical damage,
(b) providing an access device, which is located entirely within the container
and does not breach the container's laminated layers and does not interrupt the
container's edge at which the container is sealed,
(c) providing a probe for piercing said access device
characterized in that,
said probe is connected to said analyzer through tubing that
has a durometer (Shore D scale) in the range of 10 to 100.
A method according to Claim 1 wherein said tubing has a durometer in the range
70 to 94.
A method according to Claim 1 wherein said tubing has a durometer in the range
of 80 to 84.
A method according to one of the Claims 1 to 3 wherein said tubing is a polyamide
A method according to one of the Claims 1 to 3 wherein said tubing is a polyester/polyether
block co-polymer or a polyester elastomer.
A method according to one of the Claims 1 to 3 wherein said tubing is Nylon™,
Zytel 42 Nylon, Hytrel™ 8238 or polyvinylidene chloride.
Procédé pour maintenir une pression partielle d'oxygène à une valeur spécifiée
de ± 4 mm de Hg à la température ambiante dans une solution de référence multi-analytes
contenue dans un récipient à espace de tête nul, pour accéder à cette solution de
référence multi-analytes et pour transférer cette solution de référence multi-analytes
à un analyseur, dans lequel le récipient en question est formé d'un stratifié multicouche,
procédé qui comprend les étapes consistant
(a) à préparer le récipient
(i) en choisissant pour ce récipient une couche interne à réactivité faible
ou nulle envers l'oxygène ;
(ii) en choisissant pour ce récipient une couche intermédiaire en aluminium
(iii) en choisissant pour ce récipient une couche externe qui protège d'une
détérioration physique la couche intermédiaire en aluminium,
(b) à prévoir un dispositif d'accès qui est entièrement logé à l'intérieur du
récipient et qui n'ouvre pas de brèche dans les couches stratifiées du récipient
et n'interrompt pas le bord de ce dernier au niveau duquel il est scellé,
(c) à prévoir une sonde destinée à perforer le dispositif d'accès,
caractérisé en ce que
la sonde est reliée à l'analyseur par une tubulure qui a une
caractéristique durométrique (échelle Shore D) dans la plage de 10 à 100.
Procédé suivant la revendication 1, dans lequel la tubulure a une caractéristique
durométrique dans la plage de 70 à 94.
Procédé suivant la revendication 1, dans lequel la tubulure a une caractéristique
durométrique dans la plage de 80 à 84.
Procédé suivant l'une des revendications 1 à 3, dans lequel la tubulure est
en un polymère de condensation du type polyamide.
Procédé suivant l'une des revendications 1 à 3, dans lequel la tubulure est
en un copolymère séquencé polyester/polyéther ou en un élastomère du type polyester.
Procédé suivant l'une des revendications 1 à 3, dans lequel la tubulure est
en Nylon™, Nylon Zytel 42, Hytrel™ 8238 ou en poly(chlorure de vinylidène).