# Patent

## Title
Detector arrangement for blood culture bottles with colorimetric sensors

## Abstract

A detector arrangement for a blood culture bottle incorporating a colorimetric sensor which is subject to change of color due to change in pH or CO2 of a sample medium within the blood culture bottle. The detector arrangement includes a sensor LED illuminating the colorimetric sensor, a reference LED illuminating the colorimetric sensor, a control circuit for selectively and alternately activating the sensor LED and the reference LED, and a photodetector. The photodetector measures reflectance from the colorimetric sensor during the selective and alternating illumination of the colorimetric sensor with the sensor LED and the reference LED and generates intensity signals. The reference LED is selected to have a peak wavelength of illumination such that the intensity signals of the photodetector from illumination by the reference LED are not substantially affected by changes in the color of the colorimetric sensor.

## Background
CROSS REFERENCE TO RELATED APPLICATION
This application claims the benefit of U.S. Provisional Patent Application No. 61/400,001, entitled “Detector Arrangement for Blood Culture Bottles With Colorimetric Sensors”, filed Jul. 20, 2010, which is incorporated herein.


BACKGROUND
Bottles for culturing of blood for the presence of microorganism and related instruments for analyzing such bottles in a noninvasive manner are known in the art and described in the patent literature. See U.S. Pat. Nos. 5,858,769; 5,795,773; 4,945,060; 5,094,955; 5,164,796; 5,217,876; and 5,856,175. The bottles and instruments of the above-listed patents have been commercialized with success by the present assignee under the trademark BacT/ALERT.
The bottles described in these blood culture instruments utilize colorimetric sensors placed in the bottom of the bottle and in contact with the sample media to determine the presence/absence of bacterial growth. Once a clinical/industry sample is added to the liquid growth media present in the bottle and incubation occurs, the concentration of carbon dioxide increases as the number of microorganisms increase; carbon dioxide is a respiration by-product of bacterial growth. Alternatively, changes to the media pH that are related to the growth of microorganisms can also be monitored by the sensor. The basic operation of the BacT/ALERT sensor and monitoring electronics is described in U.S. Pat. No. 4,945,060 and also in an article by Thorpe et. al. in “BacT/Alert: an Automated Colorimetric Microbial Detection System” which was published in the Journal of Clinical Microbiology, July 1990, pp. 1608-12. The '060 patent and the Thorpe et al. article are incorporated by reference here.
The basic colorimetric sensing system described in the '060 patent is shown in FIG. 1 of the appended figures. A red Light Emitting Diode (LED) (4) shines onto the bottom of the BacT bottle (1). A colorimetric sensor (2) is deposited onto the bottom of the bottle (1). The LED light impinges on the sensor at a 45 degree angle relative to the bottom surface of the bottle (1). The majority of the light penetrates the structure of the bottle and impinges on the colorimetric sensor (2). Part of the light will reflect off the plastic bottle material and sensor (2) at 45 degrees to the bottom surface of the bottle, but in an opposite direction to the impinging light (e.g. the angle of reflection is equivalent to the angle of incidence). Much of the remaining light is scattered from the surface and interior of the sensor. The sensor (2) changes its color as the percentage of CO2 in the bottle varies from 0% to 100%; the color varies from blue to yellow, respectively. A silicon photodetector (5) “stares” (i.e., continuously monitors the scattered intensity signal) at the region in the sensor (2) where the light from the LED interacts with the sensor. The intensity of the scattered light that is detected by the photodetector is proportional to the CO2 level within the bottle (1). FIG. 1 also shows the associated electronics including a current source (6), current-to-voltage converter (7) and low pass filter (8).
FIG. 2 is a plot of the signal received by the photodetector (5) of FIG. 1. The data was collected using a fiber optic probe in place of the photodetector (5) in FIG. 1. The fiber optic probe is routed to a visible light spectrometer, which shows the scattered light as a function of intensity (Reflectance Units) and wavelength. The shape of each curve is the convolution of the LED intensity distribution with the reflectivity of the colorimetric sensor (2) at a specified CO2 level.
When the silicon photodetector (5) of FIG. 1 is substituted for the fiber optic probe, a photocurrent is generated by the photodetector that is proportional to the integrated wavelength signal shown in FIG. 2. In other words, the silicon photodetector integrates the spectral response into a photocurrent. In turn, this photocurrent is converted into a voltage signal using a transimpedance amplifier.
While the BacT/ALERT sensing system of FIG. 1 is robust and has been used in blood culture systems successfully for many years, it does have a few areas for improvement. First, if the blood culture bottle (1) moves in the cell (e.g. displacement in the z-axis so that it shifts away from the position of the photodetector), the system (as it is currently implemented) detects this movement as a reduction in intensity. However, this reduction in intensity is interpreted by the instrument as reduction in CO2 level in the bottle, which may not in fact be occurring. Since this effect is counter to the effect of a bottle's reflectivity increasing as carbon dioxide content increases (signifying bacterial growth), it is possible that the system would treat a translating bottle as having no growth (i.e., a false negative condition).
Likewise, as the instrument ages in the clinical laboratory, the optical system may collect dust or optical materials experience reduced transmissivity as a function of time. For example, as plastics age, their transmissivity can be reduced by the effects of light, particulate buildup (dust) or repeated use of cleaning agents. These effects would not affect readings but would manifest as a drift in the response of the system. Periodic calibration checks could compensate for this drift. Thus, there is a long-felt but unmet need to have a real-time monitor of the transmission in the optical system and the capability to adjust or compensate for some of these sources of error, particularly the situation where the bottle is not fully installed in the receptacle and is not at the nominal or home position (has some Z-axis displacement away from the optical detector arrangement).
Other prior art of interest includes the following U.S. Pat. Nos. 7,193,717; 5,482,842; 5,480,804; 5,064,282; 5,013,155; 6,096,2726; 6,665,061; 4,248,536 and published PCT application WO 94/26874 published Nov. 24, 1994.
SUMMARY
An improved detection arrangement for blood culture bottle incorporating colorimetric sensors is disclosed.
The detection arrangement includes photodetector, a sensor LED and a reference LED, and a control circuit for selectively and alternately activating the sensor LED and the reference LED to illuminate the colorimetric sensor. The sensor LED functions like the LED of FIG. 1 and is used to determine the change in the colorimetric sensor color. The photodetector monitors the reflectance from the sensor when illuminated by the sensor LED by monitoring intensity changes. The reference LED is selected to have a wavelength such that the intensity readings of the photodetector from illumination by the reference LED are not affected by changes in the color of the colorimetric sensor. As such, the reference LED can be used as a reference, with the photodetector readings during illumination by the reference LED unaffected by changes in CO2 concentration within the bottle. It has been found that wavelengths in the near infra-red (peak λ for the LED between 750 and 950 nm) are suitable for the reference LED.
The reference LED is useful to indicate if the distance between the bottle and the detector subassembly changes, ambient lighting conditions change, or anything within the physical optical path between the sensor LED, the bottle and the photodetector changes. Since a change in the reference LED is not dependent on the state of the colorimetric sensor, the reference LED can provide information about changes in the optical system that are not related to microorganism growth so that such non-growth related changes from the system can be discriminated from growth-related changes. This feature helps reduce the false-positive rate in the system and improves sensing accuracy and reliability.
In use, the sensor LED and reference LED are illuminated alternately and repeatedly, e.g., in a time division multiplexed manner. The photodetector signals from such sequential illuminations are fed to a computer. The computer monitors changes in the photodetector signal when the reference LED illuminated; these changes would indicate a change in the bottle position or the optical system. The computer can compensate the sensor LED signals according to derived calibration relationships between the sensor LED and reference LED signals, e.g., due to offset of the bottle position in the detection system from a home or nominal position.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is an illustration of a known sensor and detector arrangement for blood collection bottles as described in U.S. Pat. No. 4,945,060.
FIG. 2 is plot of reflectance of a colorimetric sensor on a spectrometer in place of the photodetector of FIG. 1 as a function of wavelength and CO2 concentration.
FIG. 3 is a sensor and detector arrangement for blood collection bottles in accordance with the present disclosure.
FIG. 4 is a plot of intensity signals from the photodetector of FIG. 3 for sensor LED and reference LED illumination of the colorimetric sensor over 0-100% CO2 range present within the bottle.
FIG. 5 is a graph of photodetector intensity signals for the sensor LED and reference LED as a function of bottle displacement from nominal or home position in which the bottle is in its designed position proximate to the detection system of FIG. 3.
FIG. 6 is a plot of photodetector intensity signals for the sensor LED and reference LED as a function of time during conditions of microbial growth with the bottle.
FIG. 7 is a block diagram of the electronics operating the sensor arrangement of FIG. 3.
FIG. 8 is a graph of the duty cycle of the reference and sensor LED of FIG. 3, showing the time division multiplexing method of operation. The width of the pulses representing the duty cycle is not to scale; in one possible embodiment the duty factor is 33 percent: 1⁄3 of the time the reference LED is illuminated, 1⁄3 of the time the sensor LED is illuminated, and 1⁄3 of the time neither LED is illuminated to enable a “dark” measurement to be made.



DETAILED DESCRIPTION
The invention involves the use of secondary LED as a light source to compensate for non-Liquid Emulsion Sensor (LES) changes to the optical system. A block diagram of the optical configuration is shown in FIG. 3. The configuration is for testing a bottle 1 having a colorimetric LES 2 incorporated within the bottle 1. The configuration includes a sensor LED 4, an IR reference LED 10, and a photodetector 5 generating intensity signals. Both LEDs 4 and 10 are angled at 45 degrees in relation to the bottom surface of the bottle as shown in FIG. 3. The reflectivity of the bottle bottom and LES 2 is measured sequentially, by means of a control circuit (42, FIG. 7) which selectively and alternately activates the sensor LED and the reference LED. For example, the sensing or red LED 4 is turned on and the reflected signal is measured by the photodetector 5. The sensing LED 4 is then extinguished. The reference LED 10 is then illuminated and the same photodetector 5 measures the reflected light. Then it is extinguished, and the process is repeated. This approach is also referred to as a time-division multiplexed scheme, which is shown in FIG. 8 and will be described in further detail below.
As noted above, the LEDs 4 and 10 are oriented at a 45 degree angle relative to the bottom of the bottle. This is so that the reflection off of the bottom surface of the bottle is not strongly coupled into the photodetector 5. The angle of incidence=angle of reflection so that light striking the bottle bottom will exit off at 45 degrees and will not strongly affect the photodetector reading (since scattered light from the LES is only of interest). The LEDs have a spatial emission angle of 15-17 degrees; i.e., the LEDs emit light in a cone that is defined by Peak Emission and Full-Width angle at half maximum power; the angle of the cone is in the range of 15-24 degrees.
Testing was performed on a variety of LED colors, and it was found that near-infrared LEDs (peak wavelength from 750-950 nm) reflectivity were marginally effected by the LES color changes. All other wavelengths of light had a negative or positive change in reflectivity as the CO2 level was changed from 0% to 100%. This effect minimizes at wavelengths beyond about 750 nm (near-infrared LED) as is shown in Table 1.







TABLE 1







Photodetector output (volts) with CO2 spiked bottles


For sensing (RED) LED and reference (IR) LED










CO2

Sensing LED
Reference LED












Level
Samples
Mean
Std. Dev.
Mean
Std. Dev.















0%
390
0.65838
0.00045
2.32539
0.00045


2%
390
0.84627
0.00048
2.25763
0.00048


15%
390
1.29105
0.00047
2.40419
0.00048


100%
390
1.92822
0.00063
2.29345
0.00050









FIG. 4 shows the graphical equivalent of Table 1. The photodetector readings for the reference sensor are plotted as line 20 and the photodetector readings for the sensor LED are plotted as line 22. A large increase in the red LED signal 22 is seen in the graph (it changes from about 0.6 volt to almost 2 volts) as the carbon dioxide level in the bottle is increased from 0% CO2 to 100% CO2. At the same time, the Reference LED signal 20 changes from 2.32 volts to 2.29 volts (a change of 30 mV), so it is very stable over the course of the LES changing color.
In order to study the changes in the optical signal as a function of the bottle position in relation to the optical system, a calibration/test fixture was constructed consisting of a digital micrometer that is attached to the BacT/ALERT bottle. The bottle is first placed in the normal (home) position in the BacT/ALERT rack assembly so that it is as close to the optical system as is possible. Readings of the reflectance are taken, then the bottle is displaced by adjusting the micrometer. The micrometer provides precise small adjustments to the z-axis displacement (i.e. it moves the bottle further from the optical system) so that the effects of displacement can be quantified. The normalized change in optical signal as a function of the displacement is shown graphically in FIG. 5, again with photodetector signal for illumination of the reference LED plotted as line 20 and the photodetector signal for the sensor LED plotted as line 22. It is seen that the displacement causes a linear shift in the signals received by the photodetector. While the sensor LED signal 22 and the reference LED signal 20 have different slopes of change, each is linear, so that a relationship can be developed to compensate for changes in the signal LED as a function of changes in the reference LED detector output, e.g., due to displacement of the bottle from a home or nominal position. Equations were computed for the graphs in FIG. 5; the equations are listed below in table 2 along with the goodness of fit parameter (R2).










TABLE 2









Detector_output (Signal) = 0.2652 − 0.2554x
R2 = 0.9963



Detector_output(Reference) = 0.5621 − 0.2384x
R2 = 0.9999







Where x = the linear displacement distance (in inches)






Accordingly, by mapping the change in intensity of the reference LED's output, a displacement value can be determined. Applying that value to the signal LED's output, the amount of intensity reduction can be quantified and compensated for.
A further test of the capabilities of the detector arrangement of FIG. 3 was performed by injecting a inoculum of Saccharomyces cerevisiae into the blood culture bottle and monitoring the colorimetric sensor using the sensor LED and reference LED optics while the yeast grows in the bottle. FIG. 6 shows the growth curve of the yeast growth—lag, exponential and stationary growth phases are shown. During the growth (and changes in the response of the LES sensor), it is seen that the reference LED signal 20 is unchanging, whereas the sensor LED signal 22 changes due to change in CO2 concentration as a result of microbial growth. The flatness of the curve 20 verifies the insensitivity of the photodetector readings during illumination of the reference LED to changes in the LES color. It further verifies its ability to monitor changes in the optical system while not being affected by bacterial growth.
FIG. 7 is a block diagram of the electronics 30 for the embodiment of FIG. 3. The electronics 30 includes an “optical nest” 32 consisting of the sensor LED 4, the reference LED 10, and the photodetector 5. The output of the photodetector is converted into a digital signal in an A/D converter 34 and fed to a data acquisition system 36. The data acquisition system sends signals to an LED control board 42 which includes control circuits and LED drivers which send signals over the conductors 44 and 46 to cause the LEDs 4 and 10 to illuminate in a time division multiplexed manner. Photodetector signals from the data acquisition system are sent to a computer 38, which may be part of the instrument incorporating the optical nest 32 of FIGS. 3 and 7. (Incidental electronics such as filters and current-to-voltage converter are omitted in the Figure but may be present in the electronics).
Memory 40 stores the calibration constants and relationships between the reference and signal LED outputs, derived from curves such as FIG. 5 and explained above in Table 2. For example, the memory 40 stores a calibration relationship between intensity signals for the sensor LED as a function of distance of the bottle from the home position (plot 22 in FIG. 5); the computer 38 compensates for a drop in intensity signals from the sensor LED due to the bottle being positioned a distance away from the home position in accordance with calibration relationships for the sensor LED and the reference LED.
FIG. 8 is a graph of the duty cycle of the reference LED 10 and sensor LED 4 of FIG. 3, showing the time division multiplexing method of operation. The sensor LED on and off states are shown on line 50; the reference LED on and off states are shown in line 42. The width of the pulses representing the duty cycle is not to scale and can vary. In one possible embodiment the duty factor is 33 percent: 1⁄3 of the time the reference LED is illuminated, 1⁄3 of the time the sensor LED is illuminated, and 1⁄3 of the time neither LED is illuminated to enable a “dark” measurement to be made.
Compensation for dust, drift, changes in the optical system, and aging of the optical materials in the beam path are also possible with the arrangement of FIG. 3. Since these occur over an extended time (expected to be in the duration of months), they would be very slow changing. Compensation is achieved by saving data points from the initial calibration (e.g., derived from FIG. 5) and compare the photodetector signals for the IR LED 10 emission levels to initial values to compensate for degradation mechanisms in the optical system. This change would also be applied to the sensor LED 4. For shorter time period drift events, changes are monitored in the IR LED 10 which should be very steady over the growth cycle of bacteria; any changes in the IR LED performance cause adjustments in the sensor LED photodetector readings accordingly, e.g., using stored calibration relationships.
The appended claims are further statements of the disclosed inventions.

## Claims

1. A detection system comprising:
a blood culture bottle comprising a sample medium;
a colorimetric sensor positioned in the blood culture bottle and subject to change of color due to change in pH or CO2 of the sample medium;
a sensor LED illuminating the colorimetric sensor;
a reference LED illuminating the colorimetric sensor;
a control circuit configured to selectively and alternately activating the sensor LED and the reference LED; and
a photodetector, the photodetector configured to measure reflectance from the colorimetric sensor during the selective and alternating illumination of the colorimetric sensor with the sensor LED and the reference LED and configured to generate intensity signals;
a computer receiving the intensity signals, the computer including a memory storing a calibration relationship between intensity signals for the reference LED as a function of distance of the bottle from a home position in relation to the detection arrangement;
wherein the reference LED has a peak wavelength of illumination between 750 and 900 nm.

2. The detection arrangement of claim 1, wherein the memory further stores a calibration relationship between intensity signals for the sensor LED as a function of distance of the bottle from the home position and wherein the computer comprises computer executable instructions configured to compensate for a drop in intensity signals from the sensor LED due to the bottle being positioned a distance away from the home position in accordance with calibration relationships for the sensor LED and the reference LED.

3. A method comprising the steps of:
providing a blood culture bottle comprising a sample medium and a colorimetric sensor positioned in the blood culture bottle, the colorimetric sensor subject to change of color due to change in pH or CO2 of the sample medium;
alternately and repeatedly illuminating the colorimetric sensor with a sensor LED and a reference LED;
measuring reflectance from the colorimetric sensor due to the illumination of the colorimetric sensor by the sensor LED and reference LED with a photodetector, the photodetector responsively generating intensity signals;
receiving the intensity signals at a computer, the computer including a memory storing a calibration relationship between intensity signals for the reference LED as a function of distance of the bottle from a home position in relation to the detection arrangement;
wherein the reference LED has a peak wavelength of illumination of between 750 and 900 nm.

4. The method of claim 3, further comprising the step of storing in computer memory a calibration relationship between intensity signals for the reference LED as a function of distance of the bottle from a home position in relation to the sensor LED, reference LED and photodetector.

5. The method of claim 3, further comprising the step of storing in computer memory a calibration relationship between intensity signals for the sensor LED as a function of distance of the bottle from a home position in relation to the sensor LED, reference LED and photodetector.

6. The method of claim 5, further comprising the step of compensating for a drop in intensity signals from the sensor LED due to the bottle being positioned a distance away from the home position in accordance with calibration relationships for the sensor LED and the reference LED.

7. The method of claim 6, wherein the step of compensating comprises the step of determining a displacement value for the bottle using the calibration relationship for the reference LED and using the calibration relationship for the sensor LED to adjust the intensity signal from the photodetector to correct for the displacement of the bottle by the displacement value.

8. The method of claim 6, further comprising determining bacterial growth in the bottle based at least in part on the reflectance from the colorimetric sensor due to the illumination of the colorimetric sensor by the sensor LED after compensating for the drop in the intensity signals.

9. The method of claim 3, further comprising determining bacterial growth in the bottle based at least in part on the reflectance from the colorimetric sensor due to the illumination of the colorimetric sensor by the sensor LED.

10. The method of claim 3, further comprising determining a calibration relationship between intensity signals for the reference LED as a function of distance of the bottle from a home position in relation to the sensor LED, reference LED, and photodetector.