Note: Descriptions are shown in the official language in which they were submitted.
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ME~HOD AND APPARATUS TO DETECT BACTERIAL
CONTAMINATION OF TRANSFUSABLE BLOOD
This is a continuation-in-part of U.S. Patent
Application Serial No. 07/638,481, filed January 4,
1991; which is in turn a continuation-in-part of U.S.
Patent Application Serial No. 07/609,278, filed
November 5, 1990.
Field of the Invention
This invention relates to a noninvasive
method and apparatus to detect the presence or
determine the concentration of microorganisms in a
container of transfusable blood prior to transfusion.
Back~round of the Invention
Microorganisms present in bodily fluid can be
detected using a culture bottle. Generally, a culture
bottle is a flask allowing positive cultures to be
detected rapidly. The flask is generally a
transparent closed container filled with nutrient that
promotes the growth of the organism. In particular,
bacteria in blood can be detected in culture. U.S.
Patent No. 4,772,559!.-(Hammann).
Many different qualitative and quantitative
detection means are used to monitor the growth of
microorganisms in a culture bottle. The
~- 25 microorganisms in a culture bottle have been detected
by use of external detectors such as a magnifying
lens, U.S. Patent No. 4,543,907 (Freudlich).
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Additionally, internal detectors such as liquid level
indicators can show bacterial growth as a function of
increased pressure in the vessel Swaine et al., EPA
124,193. Additionally, microorganisms can be detected
by measuring changes in pH caused by bacterial growth,
Mariel, G.B. Patent No. 1,601,689.
Still another method to detect microorganisms
involves the use of a culture media that contains a
compound which changes color or appearance according
to the growth of microorganisms. The change in the
media can be detected with a spectrophotometer. There
are many examples of reactions used in Microbiology
that rely on a color change. Bascomb, Enzvme Tests in
Bacterial Identification, Meth. Microbiol. 19, 105
(1987). For example, a variety of organisms can be
classified in large part by their pattern of
fermentation, oxidation or assimilation of carbon
sources. Fermentation of carbohydrates results in the
production of acid which causes a decrease in pH.
This drop in pH can be easily detected by including a
pH indicator like bromthymol blue or phenol red. With
both indicators, acid conditions representing the
fermentation of a particular carbohydrate result in a
yellow color (changing from blue-green for bromthymol
25 ,blue or pink/red for phenol red). The same approach
can be adopted for a variety of carbohydrates, ranging
from monosaccharides like glucose to polysaccharides
` like inulin. In an analogous fashion, increasing pH
can be also be monitored. Assays for detecting the
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presence of decarboxylase and urease, and the ability
to use malonate are based on an increase in pH, as
indicated by a color change in the indicator. Turner,
et al. U.S. Patent No. 4,945,060 discloses a device
S for detecting microorganisms. In this device changes
in the indicator medium resulting from pH changes in
C2 concentration in the medium are detected from
outside the vessel.
Chemical and enzymatic reactions are used to
detect or quantitate the presence of certain
substances in microbiological or other assays. Many
of these tests rely on the development or change of
color or fluorescence to indicate the presence or
quantity of the substance of interest.
lS Another approach to determine if an organism
can degrade a particular substrate, is to use a
reagent which is capable of reacting with one or more
of the intermediates or final products. For example,
the detection of the reduction of nitrate to nitrite.
If nitrite is formed, then a pink to deep red color
; will result when sulfanilic acid and
alpha-naphthylamine are added to the reaction mixture.
; In contrast to the indirect detection of an
~ enzymatic reaction illustrated by the nitrate/nitrite
,test, it is possible to use a,synthetic analog of a
natural substrate to directly indicate the presence of
an enzyme. For example, methylene blue can be reduced
under certain conditions by the action of reductase,
resulting in a shift from blue to colorless. In
another test, the oxidase assay relies on the
interaction of cytochrome oxidase with N, N, N',
N'-tetramethyl-p-phenylene-diamine producing a
blue color.
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Another example is the ability of
microorganisms to degrade sulfur-containing amino
acids as indicated by the production of H2S.
Typically, the organism is incubated with a high
concentration of a sulfur-containing substrate (e.g.
cysteine, cystine) in an acid environment. The
production of H2S is indicated by the formation of a
black precipitate in the presence of ferric ammonium
citrate.
Enzymes can usually act on more than one
substrate. This allows for the use of synthetic
enzyme substrates for the detection of enzyme
activities. Synthetic substrates contain a metabolic
moiety conjugated with a chromatic or fluorescent
moiety. The conjugated molecule usually has a
different absorption and/or emission spectrum from the
unconjugated form. Moreover, the unconjugated
chromatic or fluorescent moiety shows a considerably
higher absorption or fluorescence coefficients than
those of the conjugated molecule. This allows the
measurement of small amounts of products of enzyme
activities in the presence of the large amounts of
conjugated substrate required for maximal enzyme
;~ ~ activity. An example of a synthetic enzyme substrate ~ -
,is o-nitro-phenol-~-galactopyranoside used for the
detection of activity of the enzyme ~-galactosidase.
The conjugated substrate is colorless. The
~-galactosidase enzyme hydrolyzes the substrate to
yield ~-galactosidase and o-nitro-phenol.
o-nitro-phenol absorbs strongly at 405nm, and its
release can be measured by the increase in absorbance
~: at that wavelength. Bascomb, Enzvme Tests in
`~ Bacterial Identification, Meth. Microbiol. 19, 105
(1987), reviewed the synthetic moieties used for
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enzyme substrates and the enzymatic activities
measurable using this principle.
Presently, the monitoring of color or color
end-product in chemical and microbial reactions is
usually achieved in either of two ways; 1) the
detection of color or color end~product can be
achieved by visual observation and estimated
qualitatively, or 2) the detection of color
end-products or loss of color can be achieved by
measuring the intensity of color instrumentally.
Spectrophotometers that measure light absorbance are
commonly used for this purpose. When measuring the
concehtration of a number of substances it is
advantageous to use one instrument based on one
principle of measurement, otherwise cost is increased.
Although the use of colorimetric reactions is
widespread there are limitations, especially in the
sensitivity of detection. In order to improve
sensitivity and, in the case of identification of
microorganisms, thereby to decrease the time required
to obtain a result, fluorescence-based methods
frequently are used. Unfortunately, it may not be
possible to develop a fluorescent equivalent to every
~ assay. Additionally, the fluorescent reagents ~ -
-~ 25 ,themselves may be highly toxic and therefore difficult
to commercialize.
In such cases one might need to measure
activities of some enzymes fluorometrically, the
others colorimetrically However, most instruments are
suited to measure either absorbance or fluorescence,
and very few can be used to measure both.
The general principle of fluorescence
quenching has been accepted as a way to detect or
: deeermine enzymatic or cherical reactions. Por
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example, Fleminger et al. synthesized intramolecularly
quenched fluorogenic substrates for the assay of
bacterial aminopeptidase, P. Fleminger et al.,
Fluoroaenic Substrates for Bacterial Aminopeptidase P
and its Analoqs Detected in Human Serum and Calf Luna,
Eur. J. Biochem. 125, 609 (1982). In this case, the
fluorescence of the aminobenzoyl group is quenched by
the presence of a nitrophenylalanyl group. When the
enzyme is present, the nitrophenylalanyl group is
cleaved, with a concomitant increase in the sample's
fluorescence. A variety of enzymes have been assayed
by this type of procedure, including hydrolytic
enzymes, other amino- and carboxypeptidases and an
endopeptidase. Yaron et al., Intramolecularly
Ouenched Fluoroaenic Substrates for Hydrolytic
Enzvmes, Anal. Biochem. 95, 229 (1979); Carmel et al.,
IntramolecularlY - Ouenched Fluorescent Peptides as
Fluoroaenic Substrates of Leucine Aminopeptidase and
Inhibitors of Clostridial Amino~eptidase, Eur. J.
Biochem. 73, 617 (1977); Carmel et al., An
Intramolecularlv Ouenched Fluorescent Tri~e~tide as a
Fluoroqenic Substrate of Anaiotensin-I-Convertina
Enzvme and of Bacterial DipeptidYl Carboxv~eptidase,
~ Eur. J. Biochem. 87, 265 (1978); Florentin et al., _
,Hiahlv Sensitive Fluorometric Assay for
"EnkeDhalinase". a Neutral Metalloendo~eptidase that
Releases Tyrosine-GlYcine-Glvcine from Enkephalins,
Anal. Biochem. 141, 62 (1984). In each of the
previous approaches, a synthetic substrate containing
a quenching group and a fluorescing group was
generated in order to detect the activity of the
enzyme.
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An alternatlve to this approach would involve
the synthesis of a resonance energy transfer pair of
fluorescing groups on a substrate molecule. In this
method, cleavage by the enzyme of one of the groups
would result in a decrease in fluorescence, since the
critical distance would be exceeded, eliminating the
transfer of energy. However, the previously discussed
approaches are limited to specifically designed
substrates.
Still another approach involves the
estimation of a chromophore by fluorescence
- measurement. See W. Blumberg et al., Hemoqlobin
Determined in Whole Blood "Front Face" Fluorometry,
Clin. Hemo. 26, 409 (1980). Blumberg disclosed an
assay based on attenuation of fluorescence of a dye,
whose excitation wavelengths overlap with the
absorption wavelengths of the chromophore.
Subsequently, M. Shaffer, U.S. Patent No.
4,495,293 (hereinafter Shaffer) filed a patent
application disclosing a method to fluorometrically
determine a ligand in an assay solution using
conventional fluorometric techniques. In Shaffer the
intensity of the fluorescence emitted by the assay
~ solution is related to the change in transmissive
,properties of the assay solution produced by the
interac7tion of the ligand to be determined and a
reagent system capable of producing change in the
transmissive properties of the assay solution in the
presence of the ligand. More particularly, Shaffer
discloses a method to monitor absorbance using a
fluorophore in solution with the chromophore. In this
method the fluorophore may interact with the assay
cocktail and produce changes in fluorescence intensity
which are unrelated to the change being measured. The
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selection of the fluorophores is also restricted, in
that pH dependent or environment sensitive
fluorophores cannot be utilized. Additionally, when
the fluorophore is in solution, less than accurate
measure of absorbance may be obtained because light is
- absorbed exponentially through the chromophore sample.
Similarly, Beggs & Sand, EPA 91,837 disclosed
a solution based method for determination of
tryptophan-deaminase activity by measuring the
reduction in fluorescence in the presence of a
chromophore produced by the interaction between indole
pyruvic acid and metal ions using a fluorophore "whose
fluorescence is capable of being quenched by the
indole pyruvate-metal ion complex, the ions of the
fluorophore being present throughout the incubation
period".
Also, Sands, U.S. Patent No. 4,798,788
discloses a process to detect a nitrate reducing
microorganism by measuring reduction of fluorescence
in solution by causing the diazotization of the
fluorophore. In all these cases a specific
fluorophore needs to be chosen for each test to ensure
that it will fluoresce under the conditions of the
; ~ tèst, e.g. only few fluorophores fluoresce at pH of
2S ,less than 2Ø
In addition to blood culture tests, a need
exists to develop a noninvasive means to determine
bacterial contamination of blood in a collection bag
immediately prior to transfusion. Although, the
previously discussed blood culture test can be used to
determine bacterial contamination of transfusable
blood, these test may result in errors. First, the
transfusion bag must be later matched with a separate
blood culture bottle sent to a test center to make a
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determination of potential microbial contamination
prior to transfusion of the blood. This requirement
for subsequent matching could result in errors.
Additionally, blood culture bottles are cultured at
higher temperature than the temperature that blood is
normally stored; as such blood culture bottle tests
yield an accelerated picture of bacterial
contamination, while a test that simulates actual
blood storage conditions may yield more accurate
results.
.
Summarv of the Invention
This invention relates to a multi-layer body
fluid culture sensor comprised of a pH sensitive
absorbance based dye spectrally coupled to a pH
insensitive, or pH sensitive dye that is highly
buffered, fluorescence based dye. The pH sensitive
absorbance based dye is encapsulated ar isolated in a
polymeric layer that is permeable to CO2 and water,
but impermeable to protons. The pH insensitive
fluorophore is encapsulated or isolated in the second
polymeric layer that may or may not be permeable to
C2 and water. This type of sensor may be used to
detect or determine the concentration of
,microorganisms in bodily fluid. The spectral
criterion required to make this determination are such
~ that the absorption spectrum of the chromophore must
-` overlap the excitation and/or emission spectrum of the
fluorophore, thereby allowing the change in
fluorescence to be related to the change in the
reaction and consequently related to the presence or
quantity of the substance of interest.
Further, this sensor is used to monitor
microbial growth in collected transfusable blood. In
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particular, this sensor can be used to monitor
bacterial growth in a collection bag of bodily fluid
that is to be transfused into a patient. As bacteria
grow they generate CO2. The CO2 generated by the
bacteria diffuses into the polymeric layer that is in
direct contact with a hydrated pH sensitive absorbance
based dye. The CO2 reacts with the aqueous
environment to form carbonic acid (H2CO3), which
lowers the pH of the absorbance dye environment.- This
results in a concomitant chanqe in the pH sensitive
spectrum of the dye. Typically, as the absorbance of
an absorbance based dye decreases more light reaches
the fluorophore for excitation which results in a
larger amount of emitted fluorescence.
The sensor is attached to a blood collection
bag or separate sampler test bag. If a separate
sampler bag is used this bag may contain microbial
growth media or an inert substance such as a saline.
With this system microbial contamination of
transfusable blood in a collection bag can be
determined immediately prior to transfusion. In one
embodiment of this invention a detector, such as a
handheld fluorescence detector, is used to monitor the
emitted fluorescence.
.~ . .
Brief Description of the Fiaures
Fig. 1 shows a schematic diagram of a
- multi-layer blood culture sensor.
Fig. 2 shows a blood culture growth curve
detected by a xylenol blue-rhodamine 101 sensor.
Fig. 3 shows a blood culture growth curve
detected by xylenol blue in silicone-rhodamine B in
acrylic sensor.
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Fig. 4 shows a blood culture growth curve for
a xylenol blue in silicone-6213 acrylic sensor.
Fig. 5 shows a blood culture growth curve for
a bromthymol blue in silicone-rhodamine 101 in
silicone sensor.
Fig. 6 shows a sample test blood collection
bag and fluorescent detector for monitoring growth of
microorganisms in blood.
Fig. 7 shows the percent change of
fluorescent intensity versus time for two blood
samples.
.
Detailed Description - Best Mode
In this approach, fluorescence from a
fluorophore embedded in an inert light-transparent
matrix, is modulated by a pH sensitive absorbance dye
embedded in a polymeric gas permeable, but proton
impermeable matrix. The assay is carried out in a
blood collection bag or sampler test blood collection
bag.
In a fluorometric based colorimetric assay
the fluorescence intensity is regulated by changes in
absorbance of an interfering chromophore. As a pH
change occurs the chromophoric material alters the
,amount of emitted light reaching the fluorophore
and/or the amount of emitted light reaching the
detector. Spectrally compatible fluorescent and
colorimetric indicators are selected so that as the pH
changes due to the production of Co2 by
microorganisms present in the blood, the coIorimetric
indicators regulate the amount of light reaching the
fluorophore and/or photodetector and, thus cause a
change in the excitation and/or emission of the
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fluorescent dye. This change is detected with a
fluorescent reader and can be correlated with the
presence or concentration of microorganisms in the
blood.
A bodily fluid culture sensor is comprised of
a pH sensitive absorbance based dye in or isolated by
a polymeric gas permeable, but proton impermeable
matrix, and a fluorescent dye in a second polymeric
matrix.
Spectrally compatible fluorescent and
colorimetric indicators are selected so that when an
organism is present in blood, the colorimetric
indicator will regulate the amount of light reaching
the fluorophore thereby causing a change in the
emission intensity from the fluorescence dye reaching
the photodetector. The change, indicating the
presence of bacteria, is detected with a fluorometric
reader.
More particularly, spectrally compatible
fluorescence and absorbance dyes are selected dyes are
selected so that as carbonic acid is produced (CO2
and H2O), the absorbance of the dye will change
: thereby regulating the amount of light reaching the
~fluorophore and/or photodetector, thus producing a
change in the measured fluorescence. This change is
detected with a fluorescence reader. Spectrally
compatible dyes are rhodamine B and xylenol blue.
Additionally, bromthymol blue and rhodamine 101 are
also spectrally compatible.
For example this can be illustrated by
inoculating a bag containing the appropriate growth
media with Yersinia enterocolitica. As the organism
grows, it produces CO2 gas. The silicone is
permeable to the CO2. The CO2 diffuses to the
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absorbance layer and reacts with water to produce
carbonic acid (H2C03). The carbonic acid causes a
drop in the pH in the absorbance dye environment
resulting in a change in measured absorbance. For
5 example, as the pH drops in an absorbance layer
containing the dye xylenol blue, the absorbance of
xylenol blue decreases, thereby allowing more light to
reach the fluorophore to excite it and thus increase
the amount of fluorescence emitted at 590nm. A
10 positive culture using xylenol blue as the absorbance ?
dye is detected by increase the amount of fluorescence
emitted at 590nm. A positive culture using xylenol
blue as the absorbance dye is detected by a measured
increase in fluorescence as the xylenol blue decreases
15 in absorbance See Fig. 7.
The pH sensitive absorbance based dye is
encapsulated in or isolated by a polymeric matrix that
is gas permeable, but proton impermeable. The
polymeric matrix must be optically transparent in the
20 visible region, permeable to gas, autoclavable, stable
for at least six months, and proton impermeable. In s
particular, silicone may function as the polymeric
matrix used to encapsulate or isolate the absorbance
~ based dye. Silicones found to meet these criteria
25 ,were Dow, Rhone Poulenc, G.E. and Wacker.
Similarly, the fluorescence based dyes can
; also be encapsulated in a polymeric matrix. The
polymeric matrix used for the fluorophore does not
have to meet all of the above requirements listed for
30 the matrix used to encapsulate or isolate the
absorbance dye. The similar features that it must ~- -
possess are that it must be optically transparent in
the visible region, autoclavable and stable for at
least six months.
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The polymeric matrix containing or isolating
the absorbance based dye must be coupled to the
polymeric matrix containing the fluorescent dye. It
should be noted that the polymeric matrices must be in
close proximity so that light that has been regulated
by the absorbance layer will have an effect on the
emission intensity of the fluorophore as received by
the photodetector. This can be accomplished by
applying the same polymeric material to one side of
each polymeric matrix and curing these matrices. Once
the matrices containing the dyes have been adhered
together they must be rehydrated. The clarity of the
sensor upon rehydration is also a factor in matrix
selection.
In particular, in the present invention, a
microorganism growth monitoring system for collected
transfusable blood is shown in Fig. 6. The monitoring
system shown in Fig. 6 is comprised of a sampler test
blood collection bag 20. A bar code 28 can be
attached to the bag to record data for later
inspection. Blood from the blood collection bag to be
transfused is expressed through tube 24 to sampler
` test blood collection bag 20. The wall of the blood
~ collection bag contains a multi-layer sensor 22
,comprising a pH sensitive dye in a light transmissive,
- gas permeable, proton impermeable matrix and a pH
- insensitive fluorescence dye in inert light
transparent matrix, said first and second matrices
being spectrally coupled. The blood collection or
- 30 blood storage bag can contain whole blood, plasma,
serum, erythrocytes, red blood corpuscles, leukocytes,
white blood corpuscles, thrombocytes and blood
platelets, collectively referred to as blood storage.
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In the present invention the sensor can be
located on the interior wall of the blood collection
bag or a separate sampler test blood collection bag to
which blood can be shunted for assessment. These
various types of bags are heretofore collectively
referred to as blood collectlon bag.
If a separate blood collection bag is
employed the bag may contain a growth media or an
inert substance such as saline.
In the present invention the two-layer sensor
is mounted inside a blood collection bag such that one
layer, of the sensor is positioned facing outside the
- bag. The second layer which is fluorescent is
positioned facing the interior of the bag. The sensor
may be formed integrally with the wall of the bag.
The invention, then, is comprised of the two-layer
sensor outlined abovej mounted inside a blood
collection container in such a way that by utilizing a
fluorometer to excite the fluorescent sensor and
detect the emitted fluorescent light, a determination
can be made as to the presence of a threshold level of
microorganisms contained within the blood collection
container.
An additional feature of this invention is
i 25, that the bag is stored at normal blood storage
temperatures, i.e. 4~C. In this environment certain
microorganisms are not affected by the cold: Yersinia
enterocolitica and Enterobacter aaalomerans.
Additionally, the system allows for the determination
of other bacteria that would not normally grow rapidly
in the cold but might be present in such high
concentrations over time that it would be unsafe to
transfuse into a patient: Citrobacter freundii;
Pseudomonas aeruainosa; Staphvlococcus aureus; and
Staphvlococcus e~idermidis.
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An alternative approach would follow the same
design but would consist solely of an absorbance
sensor. In this case the increase is CO2 produced
by the growing microorganisms and subsequent drop in
pH would result in a visible color change in the
sensor. This change in color visible to the blood
handler, or detected by a colorimeter would signal
that the blood contained a threshold level of
microorganisms.
Another feature of this invention involves
covering the sensor with a gas permeable membrane to
prevent naturally fluorescing substances in the blood
from interacting with the fluorescent measurement.
In and alternative embodiment shown in Fig.
1, a bodily fluid culture sensor, is comprised of a pH
sensitive absorbance based dye encapsulated in or
isolated by a polymeric gas permeable, but proton
impermeable matrix 4 and a fluorescent dye in a second
polymeric matrix 2. Reflectiv~ surface 6 can be
included to facilitate the transmission of light to
the detecting element 12. In Fig. 1 interrogation
light enters the sensor and is regulated by pH
sensitive matrix 2 which in turn causes a change in
~ the fluorescence emission 10 of the fluorophore in
- 25 ,matrix 4. This sensor offers the advantage of maximal
surface area.
A measurement is taken by first reading
reference light intensity. Next the reading from the
sensor disk is measured. The data is plotted by
taking the ratio of reference, excitation light, to
sample. In particular, as CO2 levels increase in
the blood collection bag, the absorbance of the
absorbance dye changes, thereby changing the amount of
light reaching the fluorescence layer and/or
photodetector. This causes a change in emitted
fluorescence that is detected.
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The following examples serve to illustrate
the method of the present invention. The
concentration of reagents and other variable
parameters are only shown to exemplify the methods of
the present invention and are not to be considered ~ .
limitation thereof.
Example 1
At the time a unit of blood is drawn, an
additional amount (lOml) is collected in such a manner
as to be subsequently sectioned off from the unit to
be transfused. This additional blood is then
expressed through a tube into a flexible bag, attached
to the blood collection bag. This side bag is
supplied containing a microorganism growth media and
an attached multi-layer sensor. The sensor is capable
of detecting microorganism growth by measuring an
increase in CO2 production through a change in
fluorescence emitted from the sensor.
After adding the additional blood to the bag
containing media and the sensor, and waiting a
predetermined amount of time (2 - 6 hours) for the
sensor to equilibrate, an initial reading of the
sensor is made using a portable handheld fluorometer
` ,to produce a baseline fluorescence level. This level
can be ~anually recorded for latter comparison or a
bar code can be provided and attached to the bag. The
; blood and additional monitoring bag are then stored in
-~ a normal manner (4C).
~- At the time the blood is to be used for
`; 30 transfusion a second reading of the sensor is made and
compared to the first reading. This is compared to
the original reading. If a bar code was produced, the
bar code and sensor are read. The instrument will
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compare the initial and final fluorescent values and
based upon an established threshold level of change
will signal negative or positive for growth. In this
particular embodiment, the instrument will signal
green for no growth or red for growth based on
differences in original and final sensor readings.
Exam~le 2 Xylenol Blue - Rhodamine 101 Sensor
Wacker silicone elastomer 3601 part A is
thoroughly mixed with Wacker 3601 catalyst part B in a
9:1 ratio, as recommended by the manufacturer. Next
- 5% w/w of a 50mM xylenol blue, dissolved in 5mM borate
buffer pH 11 containing 1% Tween 80, is added to the
silicone and homogenized to ensure a uniform
distribution of the dye. The absorbance layer mixture
is then poured into an aluminum square mold to a
thickness of 30/1000 of an inch and cured at 55C for
2 hours.
Wacker silicone is prepared, as described
above. Next 2~ w/w of 7.5mM Rhodamine 101, in 50mM
Tris-HCl buffer pH 8.5 in 95% ethylene glycol, is
added to the silicone. The mixture is poured over the
previously cured xylenol blue layer in the mold,
described above, and cured at 55C overnight. This
cured, dehydrated, double layer sensor consists of two
25 distinct layers, each 30/1000 of an inch thick. Disks
may now be punched out of the mold and adhered onto
~- the base of bottles using more silicone, ensuring that
the absorbance layer is face down. Finally, the
bottles are cured at 55C for 15 minutes, rehydrated
with normal saline and autoclaved on the wet cycle for
17 minutes. Saline is replaced with growth media and
inoculated with E. coli by injecting a suspension with
a sterile needle through the septum. The blood
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WO 92/19764 PCr/US92f i)3~
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culture bottle is placed in the instrument and
fluorescence emission is measured.
As the concentration of C02 increases in
the blood culture bottle, the pH sensitive absorbance
dye, Xylenol blue, the absorbance of the dye
decreases, thus allowing more light to reach the
fluorophore, Rhodamine 101, to thus increase the
amount of fluorescence emitted at 590nM. This
increase in fluorescence intensity v. time is shown in
the blood culture growth curve at Fig. 2.
ExamPle 3 Xvlenol Blue in Silicone/Rhodamine B in
AcrYlic
Rhone Poulenc silicone elastomer 141 part A
is thoroughly mixed with Rhone Poulenc 141 catalyst
part B in a 10:1 ratio, as recommended by the
manufacturer. Next 1% w/w of a lOOmM xylenol blue
solution pH #11, dissolved in lOmM borate buffer
containing 1% Tween 80, is added to the silicone and
mixed thoroughly with a tongue blade to ensure uniform
distribution of the dye. The absorbance layer mixture
is then poured into an aluminum square mold to a
thickness of 30/1000 of an inch. The mold is allowed
; ~ to sit out on the countertop at room temperature for
; , about one hour or until the bubbles have disappeared,
at which time the mold is placed in the incubator to
cure at 55~C for two hours.
Rhone-Poulenc silicone is prepared, as
described above. Next, a 40/1,000" thick acrylic disc
(Glasflex, Inc.), approximately 1 cm in diameter,
- 30 containing 0.2 grams/lb of rhodamine B (Sigma) is
glued onto the above absorbance layer using the
Rhone-Poulenc Silicone at the 10:1 ratio as glue. The
double layer sensor is then placed back in the 55C
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WO92/19764 PCT/US92/03637
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incubator for two hours to allow for adherence of the
two layers. Following the curing, the double layer
sensor is punched out with a cork borer, and glued
onto the base of a Wheaton bottle, ensuring that the
absorbance layer is face dow~, using the Rhone Poulenc
silicone as mentioned above. The bottle is placed in
the 55C incubator to cure for at least two hours.
The bottle is then rehydrated overnite and tested the
following day as described in Example 1.
As the concentration of co2 increases in
the blood culture bottle, the absorbance of the pH
sensitive absorbance based dye xylenol blue decreases,
thus allowing more light to reach the fluorophore
(rhodamine B) doped acrylic, to thus increase the
amount of fluorescence emitted at 590nm. This
increase in fluorescence intensity v. time is shown in
the blood culture growth curve in Fig. 3.
Example 4 XYlenol Blue in Silicone/6213 Red Standard
AcrYl ic
Wacker silicone elastomer 3601 part A is
thoroughly mixed with Wacker 3601 catalyst part B in a
9:1 ratio, as recommended with Wacker 3601 catalyst
part B in a 9:1 ratio, as recommended by the
,manufacturer. Next 5% w/w of a 50mM xylenol blue,
dissolv$d in 5m~ borate buffer pH 11 containing 1%
- Tween 80, is added to the silicone and homogenized to
ensure a uniform distribution of the dye. The
absorbance layer mixture is then poured into an
aluminum square mold to a thickness of 30/1000 of an
inch and cured at 55C for two hours.
~ Next, a 40/1,000" thick acrylic disc
; (Glasflex, Inc.), approximately 1 cm in diameter,
" referred to as No. 6213 Red (Glasflex Standard
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WO92/19764 2~S6~ PCT/US92/~3637
21 ~
Product) is glued onto the above absorbance layer
using the Wacker silico~e at the 9:1 ratio as glue.
The double layer sensor is then placed back in the
55 C incubator for two hours to allow for adherence of
the two layers. Following the curing, the double
layer sensor is punched out with a cork borer, and
glued onto the base of a Wheaton bottle, ensuring that
the absorbance layer is face down, using the Rhone
Poulenc silicone as mentioned above. The bottle is
placed in the 55C incubator to cure for at least two
hours. The bottle is then rehydrated overnite and
tested the following day as described in Example l.
As the concentration of C02 increases in
the blood culture bottle, the absorbance of the pH
sensitive absorbance based dye xylenol blue decreases,
thus allowing more light to reach the fluorophore
(rhodamine B) doped acrylic, to thus increase the
amount of fluorescence emitted at 590nm. This
increase in fluorescence intensity v. time is shown in
the blood culture growth curve in Fig. 4.
ExamPle 5 Bromthvmol Blue in Silicone/Rhodamine lOl in
Silicone
Wacker silicone elastomer 3601 part A is
,thoroughly mixed with Wacker 3601 catalyst part B in a
9:1 ratlo, as recommended by the manufacturer. Next
5% w/w of 50mM bromthymol blue, dissolved in 5mM tris
buffer pH 12 in ethylene glycol, is added to the
silicone and homogenized to ensure a uniform
distribution of the dye. The absorbance layer mixture
is then poured into an aluminum square mold to a
thickness of 30/1000 of an inch and cured at 55C for
two hours.
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WO92~197~ PCT/US~2/03637
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Wacker silicone ls prepared, as described
above. Next 2% w/w of 7.5mM Rhodamine 101, in 50mM
Tris-HCl buffer pH 8.5 in 95% ethylene glycol, is
added to the silicone. The mixture is poured over the
previously cured xylenol blue layer in the mold,
described above to isolate the absorbance layer. This
sensor is then cured at 55C overnight. This cured,
dehydrated, double layer sensor consists of two
distinct layers, each 30/1000 of an inch thick. Disks
may now be punched out of the mold and adhered onto
the base of bottles using more silicone, ensuring that
the absorbance layer is face down. Finally, the
bottles are cured at 55C for 15 minutes, rehydrated
with normal saline and autoclaved on the wet cycle for
17 minutes. Saline is replaced with growth media and
inoculated with E. coli by injecting a suspension with
a sterile needle through the septum. The blood
culture bottle is placed in the instrument and
fluorescence emission is measured. The increase in
fiuorescence intensity v. time is shown in blood
culture growth curve in Fig. 5.
- Although this invention has been described
with respect to specific embodiments, the details
~thereof are not to be construed as limitations, for it
,will be apparent that various equivalents, changes and
modifications may be resorted to without departing
from the spirit and scope thereof and it is understood
-~ that such equivalent embodiments are intended to be
included herein.
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