SPECTROSCOPIC ANALYSIS OF A BIOLOGICAL FLUID REACTED WITH AN

ENZYME

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 60/683,688, filed May 23, 2005, titled SPECTROSCOPIC ANALYSIS OF A BIOLOGICAL FLUID REACTED WITH AN ENZYME, the contents of which are hereby incorporated herein by reference and made a part of this specification.

BACKGROUND OF THE INVENTION FIELD OF THE INVENTION

[0002] Certain embodiments disclosed herein relate generally to determining concentrations in material samples, and more particularly to a method and system for performing measurements on bodily fluids.

DESCRIPTION OF RELATED ART

[0003] The ability to monitor the condition of hospital patients depends, in part, in the ability to obtain timely and accurate information on the patient's status. Some information, such as heart rate and blood oxygen level, are continuously monitored and displayed bedside. Other information is monitored by analyzing samples at a remote location, for example by drawing blood at regular intervals, analyzing the blood at a remote location, and then reporting the results to the hospital staff.

[0004] Recently, it has been found that certain measurements, such as the measurement of glucose levels, can be used to greatly improve patient care. For example, some Intensive Care Unit (ICU) patients have glucose levels that are high, that vary greatly with time, or that do not stabilize to safe values. Glucose levels having unsafe values or that vary greatly with time may hinder the recover of the patient and, in extreme cases, the patient may die. Currently there are no commercially available devices that automatically provide real-time or near-real-time

measurement of glucose levels.

[0005] Thus there is a need in the art for a method and apparatus that provides timely information about the patient and in particular blood glucose levels and timely measurements of other characteristics of the blood that might prove useful in treating patients.

BRIEF SUMMARY OF THE INVENTION

[0006] It is one aspect of the presently disclosed technologies to provide an apparatus for accepting a material sample having an initial analyte concentration. The apparatus includes: an enzyme for reacting with the accepted material sample; an optical system to measure an optical property of the reacting material sample at at least two wavelengths; and a processor programmed to determine the initial analyte concentration from the measured optical properties. In a first embodiment, the apparatus further includes a passageway having a surface comprising an immobilized enzyme. In a second embodiment, the enzyme is in solution, and the apparatus further includes a mixing chamber for admixing the enzyme and the accepted material sample. In a third embodiment, the apparatus of further includes a test strip, the enzyme is an immobilized enzyme is on the test strip, and the test strip is insertable into the optical system.

[0007] In one embodiment, the analyte being measured is glucose, and the enzyme is glucose oxidase or glucose dehydrogenase. In another embodiment, the analyte is lactate, and the enzyme is lactate dehydrogenase, hydroxybutyrate dehydrogenase, or alanine transaminase.

[0008] In one embodiment, the optical system measures the optical density of the material sample at two or more wavelengths. In another embodiment, the optical system measures at one or more predetermined times.

[0009] It is another aspect to provide a method for measuring the presence of an analyte in a sample. The method includes: reacting the sample with an enzyme that is reactive with the analyte; measuring a reacting sample spectrum, where the reacting sample spectrum is a spectrum of the sample during the reacting; and determining the concentration of the analyte in the sample from the reacting sample spectrum. In one embodiment, the measuring the reacting sample spectrum includes measuring the reacting sample spectrum at a fixed time and at two or more wavelengths. In another embodiment, the determining includes comparing a linear

combination of the spectrum at the two or more wavelengths to a calibration of the concentration as function of the linear combination.

[0010] It is one aspect of the presently disclosed technologies to provide a method for measuring the presence of an analyte in a sample. The method includes: reacting the sample with an enzyme that is reactive with the analyte; measuring a reacting sample spectrum, where the reacting sample spectrum is a spectrum of the sample during the reacting; and determining the concentration of the analyte in the sample from the reacting sample spectrum. In one embodiment, the measuring the reacting sample spectrum includes measuring before completion the reaction of the enzyme and the analyte. In another embodiment, the measuring the reacting sample spectrum includes measuring after completion the reaction of the enzyme and the analyte.

[0011] It is yet another aspect of the presently disclosed technologies to provide a method for measuring the presence of an analyte in a sample. The method includes: reacting the sample with an enzyme that is reactive with the analyte; measuring a reacting sample spectrum, where the reacting sample spectrum is a spectrum of the sample during the reacting; and determining the concentration of the analyte in the sample from the reacting sample spectrum. In one embodiment, the measuring is at two or more wavelengths and the determining includes applying a calibration relating the analyte concentration to the reacting sample spectrum at the two or more wavelengths, where the calibration relates a linear combination of the reacting sample spectrum at the two or more wavelengths. In another embodiment, the calibration relates a ratio of the reacting sample spectrum at two or more wavelengths.

[0012] It is one aspect of the presently disclosed technologies to provide a method for measuring the presence of an analyte in a sample. The method includes: determining an unreacted sample spectrum; reacting the sample with an enzyme that is reactive with the analyte; measuring a reacting sample spectrum, where the reacting sample spectrum is a spectrum of the sample during the reacting; determining the concentration of the analyte in the sample from the reacting sample spectrum. In one embodiment, the determining an unreacted sample spectrum includes measuring the sample spectrum prior to the reacting. In another embodiment, the determining an unreacted sample spectrum includes extrapolating the reacting sample spectrum to a zero reaction time.

[0013] It is another aspect of the presently disclosed technologies to provide a method for measuring the presence of an analyte in a sample. The method includes: obtaining a blood sample from a patient; reacting the sample with an enzyme that is reactive with the analyte; measuring a reacting sample spectrum, where the reacting sample spectrum is a spectrum of the sample during the reacting; and determining the concentration of the analyte in the sample from the reacting sample spectrum. In one embodiment, obtaining automatically obtains the blood sample from a patient-connected catheter. In another embodiment, the enzyme is on a test strip, and where the obtaining includes obtaining blood from a pin prick.

[0014] It is yet another aspect of the presently disclosed technologies to provide a method for measuring the presence of an analyte in a sample. The method includes: reacting the sample with an enzyme that is reactive with the analyte; measuring a reacting sample spectrum, where the reacting sample spectrum is a spectrum of the sample during the reacting; and determining the concentration of the analyte in the sample from the reacting sample spectrum. In one embodiment the analyte is glucose, and where the enzyme is glucose oxidase or glucose dehydrogenase. In another embodiment, the analyte is lactate, and where the enzyme is lactate dehydrogenase, hydroxybutyrate dehydrogenase, or alanine transaminase. In one embodiment, the reacting includes reacting the sample by contacting the sample with an immobilized enzyme. In another embodiment, the reacting includes reacting the sample by admixing the sample with an enzyme solution.

[0015] These features together with the various ancillary provisions and features which will become apparent to those skilled in the art from the following detailed description, are attained by the presently disclosed technologies, preferred embodiments thereof being shown with reference to the accompanying drawings, by way of example only, wherein:

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING

[0016] FIGURE 1 is a schematic of a fluid handling system in accordance with one embodiment;

[0017] FIGURE IA is a schematic of a fluid handling system, wherein the fluid handling and analysis apparatus is shown in a cutaway view;

[0018] FIGUEE IB is a cross-sectional view of a bundle of the fluid handling system of FIGURE IA taken along the line IB-IB;

[0019] FIGURE 2 is a schematic of an embodiment of a sampling apparatus;

[0020] FIGURE 3 is a schematic showing details of an embodiment of a sampling apparatus;

[0021] FIGURE 4 is a schematic of an embodiment of a sampling unit;

[0022] FIGURE 5 is a schematic of an embodiment of a sampling apparatus;

[0023] FIGURE 6A is a schematic of an embodiment of gas injector manifold;

[0024] FIGURE 6B is a schematic of an embodiment of gas injector manifold;

[0025] FIGURES 7A-7J are schematics illustrating methods of using the infusion and blood analysis system, where FIGURE 7A shows one embodiment of a method of infusing a patient, and FIGURES 7B-7J illustrate steps in a method of sampling from a patient, where FIGURE 7B shows fluid being cleared from a portion of the first and second passageways; FIGURE 7C shows a sample being drawn into the first passageway; FIGURE 7D shows a sample being drawn into second passageway; FIGURE 7E shows air being injected into the sample; FIGURE 7F shows bubbles being cleared from the second passageway; FIGURES 7H and 71 show the sample being pushed part way into the second passageway followed by fluid and more bubbles; and FIGURE 7 J shows the sample being pushed to analyzer;

[0026] FIGURE 8 is a perspective front of an embodiment of a sampling apparatus;

[0027] FIGURE 9 is a schematic front view of one embodiment of a sampling apparatus cassette;

[0028] FIGURE 10 is a schematic front view of one embodiment of a sampling apparatus instrument;

[0029] FIGURE 11 is an illustration of one embodiment of an arterial patient connection;

[0030] FIGURE 12 is an illustration of one embodiment of a venous patient connection;

[0031] FIGURES 13A, 13B, and 13C are various views of one embodiment of a pinch valve, where FIGURE 13 A is a front view, FIGURE 13B is a sectional view, and FIGURE 13C is a sectional view showing one valve in a closed position;

[0032] FIGURES 14A and 14B are various views of one embodiment of a pinch valve, where FIGURE 14A is a front view and FIGURE 14B is a sectional view showing one valve in a closed position;

[0033] FIGURE 15 is a side view of one embodiment of a separator;

[0034] FIGURE 16 is an exploded perspective view of the separator of FIGURE 16;

[0035] FIGURE 17 is one embodiment of a fluid analysis apparatus;

[0036] FIGURE 18 is a top view of a cuvette for use in the apparatus of FIGURE 17;

[0037] FIGURE 19 is a side view of the cuvette of FIGURE 18;

[0038] FIGURE 20 is an exploded perspective view of the cuvette of FIGURE 18;

[0039] FIGURE 21 is a schematic of an embodiment of a sample preparation unit;

[0040] FIGURE 22A is a perspective view of another embodiment of a fluid handling and analysis apparatus having a main instrument and removable cassette;

[0041] FIGURE 22B is a partial cutaway, side elevational view of the fluid handling and analysis apparatus with the cassette space from the main instrument;

[0042] FIGURE 22C is a cross-sectional view of the fluid handling and analysis apparatus of FIGURE 22A wherein the cassette is installed onto the main instrument;

[0043] FIGURE 23A is a cross-sectional view of the cassette of the fluid handling and analysis apparatus of FIGURE 22A;

[0044] FIGURE 23B is a cross-sectional view of the cassette of FIGURE 23A taken along the line 23B-23B of FIGURE 23 A;

[0045] FIGURE 23C is a cross-sectional view of the fluid handling and analysis apparatus having a fluid handling network, wherein a rotor of the cassette is in a generally vertical orientation;

[0046] FIGURE 23D is a cross-sectional view of the fluid handling and analysis apparatus, wherein the rotor of the cassette is in a generally horizontal orientation;

[0047] FIGURE 23E is a front elevational view of the main instrument of the fluid handling and analysis apparatus of FIGURE 23C;

[0048] FIGURE 24A is a cross-sectional view of the fluid handling and analysis apparatus having a fluid handling network in accordance with another embodiment;

[0049] FIGURE 24B is a front elevational view of the main instrument of the fluid handling and analysis apparatus of FIGURE 24A;

[0050] FIGURE 25A is a front elevational view of a rotor having a sample element for holding sample fluid;

[0051] FIGURE 25B is a rear elevational view of the rotor of FIGURE 25 A;

[0052] FIGURE 25C is a front elevational view of the rotor of FIGURE 25 A with the sample element filled with a sample fluid;

[0053] FIGURE 25D is a front elevational view of the rotor of FIGURE 25C after the sample fluid has been separated;

[0054] FIGURE 25E is a cross-sectional view of the rotor taken along the line 25E-25E of FIGURE 25 A;

[0055] FIGURE 25F is an enlarged sectional view of the rotor of FIGURE 25E;

[0056] FIGURE 26A is an exploded perspective view of a sample element for use with a rotor of a fluid handling and analysis apparatus;

[0057] FIGURE 26B is a perspective view of an assembled sample element;

[0058] FIGURE 27 A is a front elevational view of a fluid interface for use with a cassette;

[0059] FIGURE 27B is a top elevational view of the fluid interface of FIGURE 27 A;

[0060] FIGURE 27C is an enlarged side view of a fluid interface engaging a rotor;

[0061] FIGURE 28 is a cross-sectional view of the main instrument of the fluid handling and analysis apparatus of FIGURE 22A taken along the line 28-28;

[0062] FIGURE 29 is a schematic illustration of another embodiment of the analyte detection system;

[0063] FIGURE 30 is a plan view of one embodiment of a filter wheel suitable for use in the analyte detection system depicted in Figure 29;

[0064] FIGURE 31 is a partial sectional view of another embodiment of an analyte detection system;

[0065] FIGURE 32 is a detailed sectional view of a sample detector of the analyte detection system illustrated in Figure 31;

[0066] FIGURE 33 is a detailed sectional view of a reference detector of the analyte detection system illustrated in Figure 31;

[0067] FIGURE 34A is a schematic of a portion of a first embodiment of a sampling system useful for reacting a sample with an enzyme;

[0068] FIGURE 34B is a sectional view of an enzyme coated passageway of the embodiment of Figure 34A;

[0069] FIGURE 35 is a schematic of a portion of a second embodiment of a sampling system useful for reacting a sample with an enzyme;

[0070] FIGURE 36 is a schematic of a portion of a third embodiment of a sampling system useful for reacting a sample with an enzyme;

[0071] FIGURE 37 is a schematic of a portion of a fourth embodiment of a sampling system

useful for reacting a sample with an enzyme;

[0072] FIGURE 38 is a schematic of a portion of a fifth embodiment of a sampling system useful for reacting a sample with an enzyme;

[0073] FIGURE 39 is a schematic of an alternative embodiment test strip;

[0074] FIGURE 40 is a graph of the spectra of a blood plasma and GOx-reacted blood plasma;

[0075] FIGURE 41 is a graph of the spectra of a blood plasma and GOx-reacted blood plasma at several times after the beginning of reaction;

[0076] FIGURE 42 is a graph of the spectra of a blood with different initial amounts of glucose at one minute after the commencement of reaction; and

[0077] FIGURE 43 is a graph showing a calibration curve using the data of FIGURE 42.

[0078] Reference symbols are used in the Figures to indicate certain components, aspects or features shown therein, with reference symbols common to more than one Figure indicating like components, aspects or features shown therein.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0079] Although certain preferred embodiments and examples are disclosed below, it will be understood by those skilled in the art that the inventive subject matter extends beyond the specifically disclosed embodiments to other alternative embodiments and/or uses of the disclosed invention(s) and obvious modifications and equivalents thereof. Thus it is intended that the scope of the invention(s) herein disclosed should not be limited by the particular disclosed embodiments described below. Thus, for example, in any method or process disclosed herein, the acts or operations making up the method/process may be performed in any suitable sequence, and are not necessarily limited to any particular disclosed sequence. For purposes of contrasting various embodiments with the prior art, certain aspects and advantages of these embodiments are described where appropriate herein. Of course, it is to be understood that not necessarily all such aspects or advantages may be achieved in accordance with any particular embodiment. Thus, for

example, it should be recognized that the various embodiments may be carried out in a manner that achieves or optimizes one advantage or group of advantages as taught herein without necessarily achieving other aspects or advantages as may be taught or suggested herein.

OVERVIEW OF EMBODIMENTS OF FLUID HANDLING SYSTEM

[0080] Disclosed herein are fluid handling systems and various methods of analyzing sample fluids. FIGURE 1 illustrates an embodiment of a fluid handling system 10 which can determine the concentration of one or more substances in a sample fluid, such as a whole blood sample from a patient P. The fluid handling system 10 can also deliver an infusion fluid 14 to the patient P.

[0081] The fluid handling system 10 is located bedside and generally comprises a container 15 holding the infusion fluid 14 and a sampling system 100 which is in communication with both the container 15 and the patient P. A tube 13 extends from the container 15 to the sampling system 100. A tube 12 extends from the sampling system 100 to the patient P.

[0082] The sampling system 100 can be removably or permanently coupled to the tube 13 and tube 12. In some embodiments, the sampling system 100 is a modular unit that can be removed and replaced as desired. The sampling system 100 can include, but is not limited to, fluid handling and analysis apparatuses, connectors, passageways, catheters, tubing, fluid control elements, valves, pumps, fluid sensors, pressure sensors, temperature sensors, hematocrit sensors, hemoglobin sensors, colorimetric sensors, and gas (or "bubble") sensors, fluid conditioning elements, gas injectors, gas filters, blood plasma separators, and/or communication devices (e.g., wireless devices) to permit the transfer of information within the sampling system or between sampling system 100 and a network. The illustrated sampling system 100 has a patient connector 110 and a fluid handling and analysis apparatus 140, which analyzes a sample drawn from the patient P. The fluid handling and analysis apparatus 140 and patient connector 110 cooperate to control the flow of infusion fluid into, and/or samples withdrawn from, the patient P.

[0083] As shown in FIGURE IA, the fluid handling and analysis apparatus 140 preferably includes a pump 203 that controls the flow of fluid from the container 15 to the patient P and/or the flow of fluid drawn from the patient P. The pump 203 can selectively control fluid flow

rates, direction(s) of fluid flow(s), and other fluid flow parameters as desired. The pump 203 can include, but is not limited to, a reversible peristaltic pump, two unidirectional pumps that work in concert with valves to provide flow in two directions, a unidirectional pump, a displacement pump, a syringe, a diaphragm pump, roller pump, or other suitable pressurization device.

[0084] The illustrated fluid handling and analysis apparatus 140 can also have a sampling unit 200 configured to analyze the drawn fluid sample. The sampling unit 200 can include, but is not limited to, separators, filters, centrifuges, sample elements, and/or detection systems. The sampling unit 200 (see FIGURE 3) can include an analyte detection system for detecting the concentration of one or more analytes in the body fluid sample. In some embodiments, the sampling unit 200 can prepare a sample for analysis. If the fluid handling and analysis apparatus 140 performs an analysis on plasma contained in whole blood taken from the patient P, filters, separators, centrifuges, or other types of sample preparation devices can be used to separate plasma from other components of the blood. After the separation process, the sampling unit 200 can analyze the plasma to determine, for example, the patient P's glucose level.

[0085] With continued reference to FIGURES 1 and IA, fluid 14 in the container 15 can flow through the tube 13 and into a fluid source passageway 111. The fluid can further flow through the passageway 111 to the pump 203, which can pressurize the fluid. The fluid 14 can then flow from the pump 203 through a patient connection passageway 112 and catheter 11 into the patient P. To analyze the patient's P blood, the fluid handling and analysis apparatus 140 can draw a sample of whole blood from the patient P through the catheter 11 to a patient connector 110. The patient connector 110 directs the fluid sample into a sampling passageway 113 which leads to the sampling unit 200. The sampling unit 200 can perform one or more analyses on the sample. The fluid handling and analysis apparatus 140 can then output the results obtained by the sampling unit 200 on a display 141.

[0086] In some embodiments, the fluid handling system 10 can draw and analyze body fluid samρle(s) from the patient P to provide real-time or near-real-time measurement of glucose levels. Body fluid samples can be drawn from the patient P continuously, at regular intervals (e.g., every 5, 10, 15, 20, 30 or 60 minutes), at irregular intervals, or at any time or sequence for desired measurements. These measurements can be displayed bedside with the display 141 for

convenient monitoring of the patient P.

[0087] The illustrated fluid handling system 10 is mounted to a stand 16 and can be used in hospitals, ICUs, residences, and the like. In some embodiments, the fluid handling system 10 is an ambulatory device for convenient transport and monitoring of a patient. The ambulatory fluid handling system 10 can be coupled (e.g., strapped, adhered, etc.) to a patient, and may be smaller than the bedside fluid handling system 10 illustrated in FIGURE 1. In some embodiments, the fluid handling system 10 is an implantable system sized for subcutaneous implantation and can be used for continuous monitoring.

[0088] In some embodiments, the fluid handling system 10 is a disposable fluid handling system and/or has one or more disposable components. As used herein, the term "disposable" when applied to a system or component, such as a cassette or sample element, is a broad term and means, without limitation, that the component in question is used a finite number of times and then discarded. Some disposable components are used only once and then discarded. Other disposable components are used more than once and then discarded. For example, the fluid handling and analysis apparatus 140 can have a main instrument and a disposable cassette that can be installed onto the main instrument, as discussed below. The disposable cassette can be used for predetermined length of time, to prepare a predetermined amount of sample fluid for analysis, etc. In some embodiments, the cassette can be used to prepare a plurality of samples for subsequent analyses by the main instrument. The reusable main instrument can be used with any number of cassettes as desired. Additionally or alternatively, the cassette can be a portable, handheld cassette for convenient transport. In these embodiments, the cassette can be manually mounted to or removed from the main instrument. In some embodiments, the cassette may be a non disposable cassette which can be permanently coupled to the main instrument, as discussed below.

[0089] Disclosed herein are a number of embodiments of fluid handling systems, sampling systems, fluid handling and analysis apparatuses, analyte detection systems, and methods of using the same. Section I below discloses various embodiments of the fluid handling system that may be used to transport fluid from a patient for analysis. Section II below discloses several embodiments of fluid handling methods that may be used with the apparatus discussed in Section

I. Section III below discloses several embodiments of a sampling apparatus that may be used with the apparatus of Section I or the methods of Section II. Section IV below discloses various embodiments of an analyte detection system that may be used to detect the concentration of one or more analytes in a material sample. Section V below discloses embodiments for performing a spectroscopic analysis of a biological fluid reacted with an enzyme.

SECTION I - FLUID HANDLING SYSTEM

[0090] Figure 1 is a schematic of the fluid handling system 10 which includes the container 15 supported by the stand 16 and having an interior that is fillable with the fluid 14, the catheter

II, and the sampling system 100. Fluid handling system 10 includes one or more passageways 20 that form conduits between the container, the sampling system, and the catheter. Generally, sampling system 100 is adapted to accept a fluid supply, such as fluid 14, and to be connected to a patient, including, but not limited to catheter 11 which is used to catheterize a patient P. Fluid 14 includes, but is not limited to, fluids for infusing a patient such as saline, lactated Ringer's solution, or water. Sampling system 100, when so connected, is then capable of providing fluid to the patient. In addition, sampling system 100 is also capable of drawing samples, such as blood, from the patient through catheter 11 and passageways 20, and analyzing at least a portion of the drawn sample. Sampling system 100 measures characteristics of the drawn sample including, but not limited to, one or more of the blood plasma glucose, blood urea nitrogen (BUN), hematocrit, hemoglobin, or lactate levels. Optionally, sampling system 100 includes other devices or sensors to measure other patient or apparatus related information including, but not limited to, patient blood pressure, pressure changes within the sampling system, or sample draw rate.

[0091] More specifically, FIGURE 1 shows sampling system 100 as including the patient connector 110, the fluid handling and analysis apparatus 140, and a connector 120. Sampling system 100 may include combinations of passageways, fluid control and measurement devices, and analysis devices to direct, sample, and analyze fluid. Passageways 20 of sampling system 100 include the fluid source passageway 111 from connector 120 to fluid handling and analysis apparatus 140, the patient connection passageway 112 from the fluid handling and analysis apparatus to patient connector 110, and the sampling passageway 113 from the patient connector

to the fluid handling and analysis apparatus. The reference of passageways 20 as including one or more passageway, for example passageways 111, 112, and 113 are provided to facilitate discussion of the system. It is understood that passageways may include one or more separate components and may include other intervening components including, but not limited to, pumps, valves, manifolds, and analytic equipment.

[0092] As used herein, the term "passageway" is a broad term and is used in its ordinary sense and includes, without limitation except as explicitly stated, as any opening through a material through which a fluid may pass so as to act as a conduit. Passageways include, but are not limited to, flexible, inflexible or partially flexible tubes, laminated structures having openings, bores through materials, or any other structure that can act as a conduit and any combination or connections thereof. The internal surfaces of passageways that provide fluid to a patient or that are used to transport blood are preferably biocompatible materials, including but not limited to silicone, polyetheretherlcetone (PEEK), or polyethylene (PE). One type of preferred passageway is a flexible tube having a fluid contacting surface formed from a biocompatible material. A passageway, as used herein, also includes separable portions that, when connected, form a passageway.

[0093] The inner passageway surfaces may include coatings of various soils to enhance certain properties of the conduit, such as coatings that affect the ability of blood to clot or to reduce friction resulting from fluid flow. Coatings include, but are not limited to, molecular or ionic treatments.

[0094] As used herein, the term "connector" is a broad term and is used in its ordinary sense and includes, without limitation except as explicitly stated, as a device that connects passageways or electrical wires to provide communication on either side of the connector. Connectors contemplated herein include a device for connecting any opening through which a fluid may pass. In some embodiments, a connector may also house devices for the measurement, control, and preparation of fluid, as described in several of the embodiments.

[0095] Fluid handling and analysis apparatus 140 may control the flow of fluids through passageways 20 and the analysis of samples drawn from a patient P, as described subsequently. Fluid handling and analysis apparatus 140 includes the display 141 and input devices, such as

buttons 143. Display 141 provides information on the operation or results of an analysis performed by fluid handling and analysis apparatus 140. In one embodiment, display 141 indicates the function of buttons 143, which are used to input information into fluid handling and analysis apparatus 140. Information that may be input into or obtained by fluid handling and analysis apparatus 140 includes, but is not limited to, a required infusion or dosage rate, sampling rate, or patient specific information which may include, but is not limited to, a patient identification number or medical information. In an other alternative embodiment, fluid handling and analysis apparatus 140 obtains information on patient P over a communications network, for example an hospital communication network having patient specific information which may include, but is not limited to, medical conditions, medications being administered, laboratory blood reports, gender, and weight. As one example of the use of fluid handling system 10, which is not meant to limit the scope of the present disclosure, FIGURE 1 shows catheter 11 connected to patient P.

[0096] As discussed subsequently, fluid handling system 10 may catheterize a patient's vein or artery. Sampling system 100 is releasably connectable to container 15 and catheter 11. Thus, for example, FIGURE 1 shows container 15 as including the tube 13 to provide for the passage of fluid to, or from, the container, and catheter 11 as including the tube 12 external to the patient. Connector 120 is adapted to join tube 13 and passageway 111. Patient connector 110 is adapted to join tube 12 and to provide for a connection between passageways 112 and 113.

[0097] Patient connector 110 may also include devices that control, direct, process, or otherwise affect the flow through passageways 112 and 113. In some embodiments, one or more lines 114 are provided to exchange signals between patient connector 110 and fluid handling and analysis apparatus 140. The lines 114 can be electrical lines, optical communicators, wireless communication channels, or other means for communication. As shown in FIGURE 1, sampling system 100 may also include passageways 112 and 113, and lines 114. The passageways and electrical lines between apparatus 140 and patient connector 110 are referred to, with out limitation, as a bundle 130.

[0098] In various embodiments, fluid handling and analysis apparatus 140 and/or patient connector 110, includes other elements (not shown in FIGURE 1) that include, but are not

limited to: fluid control elements, including but not limited to valves and pumps; fluid sensors, including but not limited to pressure sensors, temperature sensors, hematocrit sensors, hemoglobin sensors, colorimetric sensors, and gas (or "bubble") sensors; fluid conditioning elements, including but not limited to gas injectors, gas filters, and blood plasma separators; and wireless communication devices to permit the transfer of information within the sampling system or between sampling system 100 and a wireless network.

[0099] In one embodiment, patient connector 110 includes devices to determine when blood has displaced fluid 14 at the connector end, and thus provides an indication of when a sample is available for being drawn through passageway 113 for sampling. The presence of such a device at patient connector 110 allows for the operation of fluid handling system 10 for analyzing samples without regard to the actual length of tube 12. Accordingly, bundle 130 may include elements to provide fluids, including air, or information communication between patient connector 110 and fluid handling and analysis apparatus 140 including, but not limited to, one or more other passageways and/or wires.

[0100] In one embodiment of sampling system 100, the passageways and lines of bundle 130 are sufficiently long to permit locating patient connector 110 near patient P, for example with tube 12 having a length of less than 0.1 to 0.5 meters, or preferably approximately 0.15 meters and with fluid handling and analysis apparatus 140 located at a convenient distance, for example on a nearby stand 16. Thus, for example, bundle 130 is from 0.3 to 3 meters, or more preferably from 1.5 to 2.0 meters in length. It is preferred, though not required, that patient connector 110 and connector 120 include removable connectors adapted for fitting to tubes 12 and 13, respectively. Thus, in one embodiment, container 15/tube 13 and catheter 11/tube 12 are both standard medical components, and sampling system 100 allows for the easy connection and disconnection of one or both of the container and catheter from fluid handling system 10.

[0101] In another embodiment of sampling system 100, tubes 12 and 13 and a substantial portion of passageways 111 and 112 have approximately the same internal cross-sectional area. It is preferred, though not required, that the internal cross-sectional area of passageway 113 is less than that of passageways 111 and 112 (see FIGURE IB). As described subsequently, the difference in areas permits fluid handling system 10 to transfer a small sample volume of blood

from patient connector 110 into fluid handling and analysis apparatus 140.

[0102] Thus, for example, in one embodiment passageways 111 and 112 are formed from a tube having an inner diameter from 0.3 millimeter to 1.50 millimeter, or more preferably having a diameter from 0.60 millimeter to 1.2 millimeter. Passageway 113 is formed from a tube having an inner diameter from 0.3 millimeter to 1.5 millimeter, or more preferably having an inner diameter of from 0.6 millimeter to 1.2 millimeter.

[0103] While FIGURE 1 shows sampling system 100 connecting a patient to a fluid source, the scope of the present disclosure is not meant to be limited to this embodiment. Alternative embodiments include, but are not limited to, a greater or fewer number of connectors or passageways, or the connectors may be located at different locations within fluid handling system 10, and alternate fluid paths. Thus, for example, passageways 111 and 112 may be formed from one tube, or may be formed from two or more coupled tubes including, for example, branches to other tubes within sampling system 100, and/or there may be additional branches for infusing or obtaining samples from a patient. In addition, patient connector 110 and connector 120 and sampling system 100 alternatively include additional pumps and/or valves to control the flow of fluid as described below.

[0104] FIGURES IA and 2 illustrate a sampling system 100 configured to analyze blood from patient P which may be generally similar to the embodiment of the sampling system illustrated in FIGURE 1, except as further detailed below. Where possible, similar elements are identified with identical reference numerals in the depiction of the embodiments of FIGURES 1 to 2. FIGURES IA and 2 show patient connector 110 as including a sampling assembly 220 and a connector 230, portions of passageways 111 and 113, and lines 114, and fluid handling and analysis apparatus 140 as including the pump 203, the sampling unit 200, and a controller 210. The pump 203, sampling unit 200, and controller 210 are contained within a housing 209 of the fluid handling and analysis apparatus 140. The passageways 111 extends from the connector 120 through the housing 209 to the pump 203. The bundle 130 extends from the pump 203, sampling unit 200, and controller 210 to the patient connector 110.

[0105] In FIGURES IA and 2, the passageway 111 provides fluid communication between connector 120 and pump 203 and passageway 113 provides fluid communication between pump

203 and connector 110. Controller 210 is in communication with pump 203, sampling unit 200, and sampling assembly 220 through lines 114. Controller 210 has access to memory 212, and optionally has access to a media reader 214, including but not limited to a DVD or CD-ROM reader, and communications link 216, which can comprise a wired or wireless Internet connection.

[0106] As described subsequently in several embodiments, sampling unit 200 may include one or more passageways, pumps and/or valves, and sampling assembly 220 may include passageways, sensors, valves, and/or sample detection devices. Controller 210 collects information from sensors and devices within sampling assembly 220, from sensors and analytical equipment within sampling unit 200, and provides coordinated signals to control pump 203 and pumps and valves, if present, in sampling assembly 220.

[0107] Fluid handling and analysis apparatus 140 includes the ability to pump in a forward direction (towards the patient) and in a reverse direction (away from the patient). Thus, for example, pump 203 may direct fluid 14 into patient P or draw a sample, such as a blood sample from patient P, from catheter 11 to sampling assembly 220, where it is further directed through passageway 113 to sampling unit 200 for analysis. Preferably, pump 203 provides a forward flow rate at least sufficient to keep the patient vascular line open. In one embodiment, the forward flow rate is from 1 to 5 ml/hr. In some embodiments, the flow rate of fluid is about 0.05 ml/hr, 0.1 ml/hr, 0.2 ml/hr, 0.4 ml/hr, 0.6 ml/hr, 0.8 ml/hr, 1.0 ml/hr, and ranges encompassing such flow rates. In some embodiments, for example, the flow rate of fluid is less than about 1.0 ml/hr. In certain embodiments, the flow rate of fluid may be about 0.1 ml/hr or less. When operated in a reverse direction, fluid handling and analysis apparatus 140 includes the ability to draw a sample from the patient to sampling assembly 220 and through passageway 113. In one embodiment, pump 203 provides a reverse flow to draw blood to sampling assembly 220, preferably by a sufficient distance past the sampling assembly to ensure that the sampling assembly contains an undiluted blood sample. In one embodiment, passageway 113 has an inside diameter of from 25 to 200 microns, or more preferably from 50 to 100 microns. Sampling unit 200 extracts a small sample, for example from 10 to 100 microliters of blood, or more preferably approximately 40 microliters volume of blood, from sampling assembly 220.

[0108] In one embodiment, pump 203 is a directionally controllable pump that acts on a flexible portion of passageway 111. Examples of a single, directionally controllable pump include, but are not limited to a reversible peristaltic pump or two unidirectional pumps that work in concert with valves to provide flow in two directions. In an alternative embodiment, pump 203 includes a combination of pumps, including but not limited to displacement pumps, such as a syringe, and/or valve to provide bi-directional flow control through passageway 111.

[0109] Controller 210 includes one or more processors for controlling the operation of fluid handling system 10 and for analyzing sample measurements from fluid handling and analysis apparatus 140. Controller 210 also accepts input from buttons 143 and provides information on display 141. Optionally, controller 210 is in bi-directional communication with a wired or wireless communication system, for example a hospital network for patient information. The one or more processors comprising controller 210 may include one or more processors that are located either within fluid handling and analysis apparatus 140 or that are networked to the unit.

[0110] The control of fluid handling system 10 by controller 210 may include, but is not limited to, controlling fluid flow to infuse a patient and to sample, prepare, and analyze samples. The analysis of measurements obtained by fluid handling and analysis apparatus 140 of may include, but is not limited to, analyzing samples based on inputted patient specific information, from information obtained from a database regarding patient specific information, or from information provided over a network to controller 210 used in the analysis of measurements by apparatus 140.

[0111] Fluid handling system 10 provides for the infusion and sampling of a patient blood as follows. With fluid handling system 10 connected to bag 15 having fluid 14 and to a patient P, controller 210 infuses a patient by operating pump 203 to direct the fluid into the patient. Thus, for example, in one embodiment, the controller directs that samples be obtained from a patient by operating pump 203 to draw a sample. In one embodiment, pump 203 draws a predetermined sample volume, sufficient to provide a sample to sampling assembly 220. In another embodiment, pump 203 draws a sample until a device within sampling assembly 220 indicates that the sample has reached the patient connector 110. As an example which is not meant to limit the scope of the present disclosure, one such indication is provided by a sensor that detects

changes in the color of the sample. Another example is the use of a device that indicates changes in the material within passageway 111 including, but not limited to, a decrease in the amount of fluid 14, a change with time in the amount of fluid, a measure of the amount of hemoglobin, or an indication of a change from fluid to blood in the passageway.

[0112] When the sample reaches sampling assembly 220, controller 210 provides an operating signal to valves and/or pumps in sampling system 100 (not shown) to draw the sample from sampling assembly 220 into sampling unit 200. After a sample is drawn towards sampling unit 200, controller 210 then provides signals to pump 203 to resume infusing the patient. In one embodiment, controller 210 provides signals to pump 203 to resume infusing the patient while the sample is being drawn from sampling assembly 220. In an alternative embodiment, controller 210 provides signals to pump 203 to stop infusing the patient while the sample is being drawn from sampling assembly 220. In another alternative embodiment, controller 210 provides signals to pump 203 to slow the drawing of blood from the patient while the sample is being drawn from sampling assembly 220.

[0113] In another alternative embodiment, controller 210 monitors indications of obstructions in passageways or catheterized blood vessels during reverse pumping and moderates the pumping rate and/or direction of pump 203 accordingly. Thus, for example, obstructed flow from an obstructed or kinked passageway or of a collapsing or collapsed catheterized blood vessel that is being pumped will result in a lower pressure than an unobstructed flow. In one embodiment, obstructions are monitored using a pressure sensor in sampling assembly 220 or along passageways 20. If the pressure begins to decrease during pumping, or reaches a value that is lower than a predetermined value then controller 210 directs pump 203 to decrease the reverse pumping rate, stop pumping, or pump in the forward direction in an effort to reestablish unobstructed pumping.

[0114] FIGURE 3 is a schematic showing details of a sampling system 300 which may be generally similar to the embodiments of sampling system 100 as illustrated in FIGURES 1 and 2, except as further detailed below. Sampling system 300 includes sampling assembly 220 having, along passageway 112: connector 230 for connecting to tube 12, a pressure sensor 317, a colorimetric sensor 311, a first bubble sensor 314a, a first valve 312, a second valve 313, and a

second bubble sensor 314b. Passageway 113 forms a "T" with passageway 111 at a junction 318 that is positioned between the first valve 312 and second valve 313, and includes a gas injector manifold 315 and a third valve 316. The lines 114 comprise control and/or signal lines extending from colorimetric sensor 311, first, second, and third valves (312, 313, 316), first and second bubble sensors (314a, 314b), gas injector 315, and pressure sensor 317. Sampling system 300 also includes sampling unit 200 which has a bubble sensor 321, a sample analysis device 330, a first valve 323a, a waste receptacle 325, a second valve 323b, and a pump 328. Passageway 113 forms a "T" to form a waste line 324 and a pump line 327.

[0115] It is preferred, though not necessary, that the sensors of sampling system 100 are adapted to accept a passageway through which a sample may flow and that sense through the walls of the passageway. As described subsequently, this arrangement allows for the sensors to be reusable and for the passageways to be disposable. It is also preferred, though not necessary, that the passageway is smooth and without abrupt dimensional changes which may damage blood or prevent smooth flow of blood. In addition, is also preferred that the passageways that deliver blood from the patient to the analyzer not contain gaps or size changes that permit fluid to stagnate and not be transported through the passageway.

[0116] In one embodiment, the respective passageways on which valves 312, 313, 316, and 323 are situated along passageways that are flexible tubes, and valves 312, 313, 316, and 323 are "pinch valves," in which one or more movable surfaces compress the tube to restrict or stop flow therethrough. In one embodiment, the pinch valves include one or more moving surfaces that are actuated to move together and "pinch" a flexible passageway to stop flow therethrough. Examples of a pinch valve include, for example, Model PV256 Low Power Pinch Valve (Instech Laboratories, Inc., Plymouth Meeting, PA). Alternatively, one or more of valves 312, 313, 316, and 323 may be other valves for controlling the flow through their respective passageways.

[0117] Colorimetric sensor 311 accepts or forms a portion of passageway 111 and provides an indication of the presence or absence of blood within the passageway. In one embodiment, colorimetric sensor 311 permits controller 210 to differentiate between fluid 14 and blood. Preferably, colorimetric sensor 311 is adapted to receive a tube or other passageway for detecting blood. This permits, for example, a disposable tube to be placed into or through a reusable

colorimetric sensor. In an alternative embodiment, colorimetric sensor 311 is located adjacent to bubble sensor 314b. Examples of a colorimetric sensor include, for example, an Optical Blood Leak/Blood vs. Saline Detector available from Introtek International (Edgewood, NJ).

[0118] As described subsequently, sampling system 300 injects a gas - referred to herein and without limitation as a "bubble" - into passageway 113. Sampling system 300 includes gas injector manifold 315 at or near junction 318 to inject one or more bubbles, each separated by liquid, into passageway 113. The use of bubbles is useful in preventing longitudinal mixing of liquids as they flow through passageways both in the delivery of a sample for analysis with dilution and for cleaning passageways between samples. Thus, for example the fluid in passageway 113 includes, in one embodiment, two volumes of liquids, such as sample S or fluid 14 separated by a bubble, or multiple volumes of liquid each separated by a bubble therebetween.

[0119] Bubble sensors 314a, 314b and 321 each accept or form a portion of passageway 112 or 113 and provide an indication of the presence of air, or the change between the flow of a fluid and the flow of air, through the passageway. Examples of bubble sensors include, but are not limited to ultrasonic or optical sensors, that can detect the difference between small bubbles or foam from liquid in the passageway. Once such bubble detector is an MEC Series Air Bubble/ Liquid Detection Sensor (Introtek International, Edgewood, NY). Preferably, bubble sensor 314a, 314b, and 321 are each adapted to receive a tube or other passageway for detecting bubbles. This permits, for example, a disposable tube to be placed through a reusable bubble sensor.

[0120] Pressure sensor 317 accepts or forms a portion of passageway 111 and provides an indication or measurement of a fluid within the passageway. When all valves between pressure sensor 317 and catheter 11 are open, pressure sensor 317 provides an indication or measurement of the pressure within the patient's catheterized blood vessel. In one embodiment of a method of operation, the output of pressure sensor 317 is provided to controller 210 to regulate the operation of pump 203. Thus, for example, a pressure measured by pressure sensor 317 above a predetermined value is taken as indicative of a properly working system, and a pressure below the predetermined value is taken as indicative of excessive pumping due to, for example, a

blocked passageway or blood vessel. Thus, for example, with pump 203 operating to draw blood from patient P, if the pressure as measured by pressure sensor 317 is within a range of normal blood pressures, it may be assumed that blood is being drawn from the patient and pumping continues. However, if the pressure as measured by pressure sensor 317 falls below some level, then controller 210 instructs pump 203 to slow or to be operated in a forward direction to reopen the blood vessel. One such pressure sensor is a Deltran IV part number DPT-412 (Utah Medical Products, Midvale, UT).

[0121] Sample analysis device 330 receives a sample and performs an analysis. In several embodiments, device 330 is configured to prepare of the sample for analysis. Thus, for example, device 330 may include a sample preparation unit 332 and an analyte detection system 334, where the sample preparation unit is located between the patient and the analyte detection system. In general, sample preparation occurs between sampling and analysis. Thus, for example, sample preparation unit 332 may take place removed from analyte detection, for example within sampling assembly 220, or may take place adjacent or within analyte detection system 334.

[0122] As used herein, the term "analyte" is a broad term and is used in its ordinary sense and includes, without limitation, any chemical species the presence or concentration of which is sought in the material sample by an analyte detection system. For example, the analyte(s) include, but not are limited to, glucose, ethanol, insulin, water, carbon dioxide, blood oxygen, cholesterol, bilirubin, ketones, fatty acids, lipoproteins, albumin, urea, creatinine, white blood cells, red blood cells, hemoglobin, oxygenated hemoglobin, carboxyhemoglobin, organic molecules, inorganic molecules, pharmaceuticals, cytochrome, various proteins and chromophores, microcalcifications, electrolytes, sodium, potassium, chloride, bicarbonate, and hormones. As used herein, the term "material sample" (or, alternatively, "sample") is a broad term and is used in its ordinary sense and includes, without limitation, any collection of material which is suitable for analysis. For example, a material sample may comprise whole blood, blood components (e.g., plasma or serum), interstitial fluid, intercellular fluid, saliva, urine, sweat and/or other organic or inorganic materials, or derivatives of any of these materials. In one embodiment, whole blood or blood components may be drawn from a patient's capillaries.

[0123] In one embodiment, sample preparation unit 332 separates blood plasma from a whole blood sample or removes contaminants from a blood sample and thus comprises one or more devices including, but not limited to, a filter, membrane, centrifuge, or some combination thereof. In alternative embodiments, analyte detection system 334 is adapted to analyze the sample directly and sample preparation unit 332 is not required.

[0124] Generally, sampling assembly 220 and sampling unit 200 direct the fluid drawn from sampling assembly 220 into passageway 113 into sample analysis device 330. FIGURE 4 is a schematic of an embodiment of a sampling unit 400 that permits some of the sample to bypass sample analysis device 330. Sampling unit 400 may be generally similar to sampling unit 200, except as further detailed below. Sampling unit 400 includes bubble sensor 321, valve 323, sample analysis device 330, waste line 324, waste receptacle 325, valve 326, pump line 327, pump 328, a valve 322, and a waste line 329. Waste line 329 includes valve 322 and forms a "T" at pump line 337 and waste line 329. Valves 316, 322, 323, and 326 permit a flow through passageway 113 to be routed through sample analysis device 330, to be routed to waste receptacle 325, or to be routed through waste line 324 to waste receptacle 325.

[0125] FIGURE 5 is a schematic of one embodiment of a sampling system 500 which may be generally similar to the embodiments of sampling system 100 or 300 as illustrated in FIGURES 1 through 4, except as further detailed below. Sampling system 500 includes an embodiment of a sampling unit 510 and differs from sampling system 300 in part, in that liquid drawn from passageway 111 may be returned to passageway 111 at a junction 502 between pump 203 and connector 120.

[0126] With reference to FIGURE 5, sampling unit 510 includes a return line 503 that intersects passageway 111 on the opposite side of pump 203 from passageway 113, a bubble sensor 505 and a pressure sensor 507, both of which are controlled by controller 210. Bubble sensor 505 is generally similar to bubble sensors 314a, 314b and 321 and pressure sensor 507 is generally similar to pressure sensor 317. Pressure sensor 507 is useful in determining the correct operation of sampling system 500 by monitoring pressure in passageway 111. Thus, for example, the pressure in passageway 111 is related to the pressure at catheter 11 when pressure sensor 507 is in fluid communication with catheter 11 (that is, when any intervening valve(s) are open). The

output of pressure sensor 507 is used in a manner similar to that of pressure sensor 317 described previously in controlling pumps of sampling system 500.

[0127] Sampling unit 510 includes valves 501, 326a, and 326b under the control of controller 210. Valve 501 provides additional liquid flow control between sampling unit 200 and sampling unit 510. Pump 328 is preferably a bi-directional pump that can draw fluid from and into passageway 113. Fluid may either be drawn from and returned to passageway 501, or may be routed to waste receptacle 325. Valves 326a and 326b are situated on either side of pump 328. Fluid can be drawn through passageway 113 and into return line 503 by the coordinated control of pump 328 and valves 326a and 326b. Directing flow from return line 503 can be used to prime sampling system 500 with fluid. Thus, for example, liquid may be pulled into sampling unit 510 by operating pump 328 to pull liquid from passageway 113 while valve 326a is open and valve 326b is closed. Liquid may then be pumped back into passageway 113 by operating pump 328 to push liquid into passageway 113 while valve 326a is closed and valve 326b is open.

[0128] FIGURE 6A is a schematic of an embodiment of gas injector manifold 315 which may be generally similar or included within the embodiments illustrated in FIGURES 1 through 5, except as further detailed below. Gas injector manifold 315 is a device that injects one or more bubbles in a liquid within passageway 113 by opening valves to the atmosphere and lowering the liquid pressure within the manifold to draw in air. As described subsequently, gas injector manifold 315 facilitates the injection of air or other gas bubbles into a liquid within passageway 113. Gas injector manifold 315 has three gas injectors 610 including a first injector 610a, a second injector 610b, and a third injector 610c. Each injector 610 includes a corresponding passageway 611 that begins at one of several laterally spaced locations along passageway 113 and extends through a corresponding valve 613 and terminates at a corresponding end 615 that is open to the atmosphere. In an alternative embodiment, a filter is placed in end 615 to filter out dust or particles in the atmosphere. As described subsequently, each injector 610 is capable of injecting a bubble into a liquid within passageway 113 by opening the corresponding valve 613, closing a valve on one end of passageway 113 and operating a pump on the opposite side of the passageway to lower the pressure and pull atmospheric air into the fluid. In one embodiment of gas injector manifold 315, passageways 113 and 611 are formed within a single piece of material (e.g., as bores formed in or through a plastic or metal housing (not shown)). In an alternative

embodiment, gas injector 315 includes fewer than three injectors, for example one or two injectors, or includes more than three injectors. In another alternative embodiment, gas injector 315 includes a controllable high pressure source of gas for injection into a liquid in passageway 113. It is preferred that valves 613 are located close to passageway 113 to minimize trapping of fluid in passageways 611.

[0129] Importantly, gas injected into passageways 20 should be prevented from reaching catheter 11. As a safety precaution, one embodiment prevents gas from flowing towards catheter 11 by the use of bubble sensor 314a as shown, for example, in FIGURE 3. If bubble sensor 314a detects gas within passageway 111, then one of several alternative embodiments prevents unwanted gas flow. In one embodiment, flow in the vicinity of sampling assembly 220 is directed into line 113 or through line 113 into waste receptacle 325. With further reference to FIGURE 3, upon the detection of gas by bubble sensor 314a, valves 316 and 323a are opened, valve 313 and the valves 613a, 613b and 613c of gas injector manifold 315 are closed, and pump 328 is turned on to direct flow away from the portion of passageway 111 between sampling assembly 220 and patient P into passageway 113. Bubble sensor 321 is monitored to provide an indication of when passageway 113 clears out. Valve 313 is then opened, valve 312 is closed, and the remaining portion of passageway 111 is then cleared. Alternatively, all flow is immediately halted in the direction of catheter 11, for example by closing all valves and stopping all pumps. In an alternative embodiment of sampling assembly 220, a gas-permeable membrane is located within passageway 113 or within gas injector manifold 315 to remove unwanted gas from fluid handling system 10, e.g., by venting such gas through the membrane to the atmosphere or a waste receptacle.

[0130] FIGURE 6B is a schematic of an embodiment of gas injector manifold 315' which may be generally similar to, or included within, the embodiments illustrated in FIGURES 1 through 6A, except as further detailed below. In gas injector manifold 315', air line 615 and passageway 113 intersect at junction 318. Bubbles are injected by opening valve 316 and 613 while drawing fluid into passageway 113. Gas injector manifold 315' is thus more compact that gas injector manifold 315, resulting in a more controllable and reliable gas generator.

SECTION II - FLUID HANDLING METHODS

[0131] One embodiment of a method of using fluid handling system 10, including sampling assembly 220 and sampling unit 200 of FIGURES 2, 3 and 6A ₃ is illustrated in Table 1 and in the schematic fluidic diagrams of FIGURES 7A-7 J. In general, the pumps and valves are controlled to infuse a patient, to extract a sample from the patient up passageway 111 to passageway 113, and to direct

F = Forward (fluid into patient), R = Reverse (fluid from patient), O = Open, C = Closed Table 1. Methods of operating apparatus 10 as illustrated in FIGURES 7A-7J

the sample along passageway 113 to device 330. In addition, the pumps and valves are controlled to inject bubbles into the fluid to isolate the fluid from the diluting effect of previous fluid and to clean the lines between sampling. The valves in FIGURES 7A-7J are labeled with suffices to indicate whether the valve is open or closed. Thus a valve "x," for example, is shown as valve "x-o" if the valve is open and "x-c" if the valve is closed.

[0132] FIGURE 7A illustrates one embodiment of a method of infusing a patient. In the step of FIGURE 7A, pump 203 is operated forward (pumping towards the patient) pump 328 is stopped, valves 313 and 312 are open, and valves 613a, 613b, 613c, 316, 323a, and 323b are closed. With these operating conditions, fluid 14 is provided to patient P. In a preferred embodiment, all of the other passageways at the time of the step of FIGURE 7A substantially contain fluid 14.

[0133] The next nine figures (FIGURES 7B-7J) illustrate steps in a method of sampling from a patient. The following steps are not meant to be inclusive of all of the steps of sampling from a patient, and it is understood that alternative embodiments may include more steps, fewer steps, or a different ordering of steps. FIGURE 7B illustrates a first sampling step, where liquid is cleared from a portion of patient connection passageway and sampling passageway passageways 112 and 113. In the step of FIGURE 7B, pump 203 is operated in reverse (pumping away from the patient), pump 328 is stopped, valve 313 is open, one or more of valves 613a, 613b, and 613c are open, and valves 312, 316, 323a, and 326b are closed. With these operating conditions, air 701 is drawn into sampling passageway 113 and back into patient connection passageway 112 until bubble sensor 314b detects the presence of the air.

[0134] FIGURE 7C illustrates a second sampling step, where a sample is drawn from patient P into patient connection passageway 112. In the step of FIGURE 7C, pump 203 is operated in reverse, pump 328 is stopped, valves 312 and 313 are open, and valves 316, 613a, 613b, 613c, 323a, and 323b are closed. Under these operating conditions, a sample S is drawn into passageway 112, dividing air 701 into air 701a within sampling passageway 113 and air 701b within the patient connection passageway 112. Preferably this step proceeds until sample S extends just past the junction of passageways 112 and 113. In one embodiment, the step of FIGURE 7C proceeds until variations in the output of hemoglobin sensor 311 indicate the

presence of a blood (for example by leveling off to a constant value), and then proceeds for an additional set amount of time to ensure the presence of a sufficient volume of sample S.

[0135] FIGURE 7D illustrates a third sampling step, where a sample is drawn into sampling passageway 113. In the step of FIGURE 7D, pump 203 is stopped, pump 328 is on, valves 312, 316, and 323b are open, and valves 313, 613a, 613b, 613c and 323a are closed. Under these operating conditions, blood is drawn into passageway 113. Preferably, pump 328 is operated to pull a sufficient amount of sample S into passageway 113. In one embodiment, pump 328 draws a sample S having a volume from 30 to 50 microliters. In an alternative embodiment, the sample is drawn into both passageway 112 and 113. Pump 203 is operated in reverse, pump 328 is on, valves 312, 313, 316, and 323b are open, and valves 613a, 613b, 613c and 323a are closed to ensure fresh blood in sample S.

[0136] FIGURE 7E illustrates a fourth sampling step, where air is injected into the sample. Bubbles which span the cross-sectional area of sampling passageway 113 are useful in preventing contamination of the sample as it is pumped along passageway 113. In the step of FIGURE 7E, pump 203 is stopped, pump 328 is on, valves 316, and 323b are open , valves 312, 313 and 323a are closed, and valves 613a, 613b, 613c are each opened and closed sequentially to draw in three separated bubbles. With these operating conditions, the pressure in passageway 113 falls below atmospheric pressure and air is drawn into passageway 113. Alternatively, valves 613a, 613b, 613c may be opened simultaneously for a short period of time, generating three spaced bubbles. As shown in FIGURE 7E, injectors 610a, 610b, and 610c inject bubbles 704, 703, and 702, respectively, dividing sample S into a forward sample Sl, a middle sample S2, and a rear sample S3.

[0137] FIGURE 7F illustrates a fifth sampling step, where bubbles are cleared from patient connection passageway 112. In the step of FIGURE 7F, pump 203 is operated in a forward direction, pump 328 is off, valves 313, 316, and 323a are open, and valves 312, 613a, 613b, 613c, and 323b are closed. With these operating conditions, the previously injected air 701b is drawn out of first passageway 111 and into second passageway 113. This step proceeds until air 701b is in passageway 113.

[0138] FIGURE 7G illustrates a sixth sampling step, where blood in passageway 112 is returned to the patient. In the step of FIGURE 7G, pump 203 is operated in a forward direction, pump 328 is off, valves 312 and 313 are open, and valves 316, 323a, 613a, 613b, 613c and 323b are closed. With these operating conditions, the previously injected air remains in passageway 113 and passageway 111 is filled with fluid 14.

[0139] FIGURES 7H and 71 illustrates a seventh and eighth sampling steps, where the sample is pushed part way into passageway 113 followed by fluid 14 and more bubbles. In the step of FIGURE 7H, pump 203 is operated in a forward direction, pump 328 is off, valves 313, 316, and 323a are open, and valves 312, 613a, 613b, 613c, and 323b are closed. With these operating conditions, sample S is moved partway into passageway 113 with bubbles injected, either sequentially or simultaneously, into fluid 14 from injectors 610a, 610b, and 610c. In the step of FIGURE 71, the pumps and valves are operated as in the step of FIGURE 7E, and fluid 14 is divided into a forward solution Cl, a middle solution C2, and a rear solution C3 separated by bubbles 705, 706, and 707.

[0140] The last step shown in FIGURE 7 is FIGURE 7 J, where middle sample S2 is pushed to sample analysis device 330. In the step of FIGURE 7J, pump 203 is operated in a forward direction, pump 328 is off, valves 313, 316, and 323a are open, and valves 312, 613a, 613b, 613c, and 323b are closed. In this configuration, the sample is pushed into passageway 113. When bubble sensor 321 detects bubble 702, pump 203 continues pumping until sample S2 is taken into device sample analysis 330. Additional pumping using the settings of the step of FIGURE 7J permits the sample S2 to be analyzed and for additional bubbles and solutions to be pushed into waste receptacle 325, cleansing passageway 113 prior to accepting a next sample.

SECTION III - SAMPLING SYSTEM

[0141] FIGURE 8 is a perspective front view of a third embodiment of a sampling system 800 which may be generally similar to sampling system 100, 300 or 500 and the embodiments illustrated in FIGURES 1 through 7, except as further detailed below. The fluid handling and analysis apparatus 140 of sampling system 800 includes the combination of an instrument 810 and a sampling system cassette 820. FIGURE 8 illustrates instrument 810 and cassette 820 partially removed from each other. Instrument 810 includes controller 210 (not shown), display

141 and input devices 143, a cassette interface 811, and lines 114. Cassette 820 includes passageway 111 which extends from connector 120 to connector 230, and further includes passageway 113, a junction 829 of passageways 111 and 113, an instrument interface 821, a front surface 823, an inlet 825 for passageway 111, and an inlet 827 for passageways 111 and 113. In addition, sampling assembly 220 is formed from a sampling assembly instrument portion 813 having an opening 815 for accepting junction 829. The interfaces 811 and 821 engage the components of instrument 810 and cassette 820 to facilitate pumping fluid and analyzing samples from a patient, and sampling assembly instrument portion 813 accepts junction 829 in opening 815 to provide for sampling from passageway 111.

[0142] FIGURES 9 and 10 are front views of a sampling system cassette 820 and instrument 81O ₃ respectively, of a sampling system 800. Cassette 820 and instrument 810, when assembled, form various components of FIGURES 9 and 10 that cooperate to form an apparatus consisting of sampling unit 510 of FIGURE 5, sampling assembly 220 of FIGURE 3, and gas injection manifold 315' of FIGURE 6B.

[0143] More specifically, as shown in FIGURE 9, cassette 820 includes passageways 20 including: passageway 111 having portions Ilia, 112a, 112b, 112c, 112d, 112e, and 112f; passageway 113 having portions 113a, 113b, 113c, 113d, 113e, and 113f; passageway 615; waste receptacle 325; disposable components of sample analysis device 330 including, for example, a sample preparation unit 332 adapted to allow only blood plasma to pass therethrough and a sample chamber 903 for placement within analyte detection system 334 for measuring properties of the blood plasma; and a displacement pump 905 having a piston control 907.

[0144] As shown in FIGURE 10, instrument 810 includes bubble sensor units 1001a, 1001b, and 1001c, hemoglobin sensor unit 1003, a peristaltic pump roller 1005a and a roller support 1005b, pincher pairs 1007a, 1007b, 1007c, 1007d, 1007e, 1007f, 1007g, and 1007h, an actuator 1009, and a pressure sensor unit 1011. In addition, instrument 810 includes portions of sample analysis device 330 which are adapted to measure a sample contained within sample chamber 903 when located near or within a probe region 1002 of an optical analyte detection system 334.

[0145] Passageway portions of cassette 820 contact various components of instrument 810 to form sampling system 800. With reference to FIGURE 5 for example, pump 203 is formed from

portion Ilia placed between peristaltic pump roller 1005a and roller support 1005b to move fluid through passageway 111 when the roller is actuated; valves 501, 323, 326a, and 326b are formed with pincher pairs 1007a, 1007b, 1007c, and 1007d surrounding portions 113a, 113c, 113d, and 113e, respectively, to permit or block fluid flow therethrough. Pump 328 is formed from actuator 1109 positioned to move piston control 907. It is preferred that the interconnections between the components of cassette 820 and instrument 810 described in this paragraph are made with one motion. Thus for example the placement of interfaces 811 and 821 places the passageways against and/or between the sensors, actuators, and other components.

[0146] In addition to placement of interface 811 against interface 821, the assembly of apparatus 800 includes assembling sampling assembly 220. More specifically, an opening 815a and 815b are adapted to receive passageways 111 and 113, respectively, with junction 829 within sampling assembly instrument portion 813. Thus, for example, with reference to FIGURE 3, valves 313 and 312 are formed when portions 112b and 112c are placed within pinchers of pinch valves 1007e and 1007f, respectively, bubble sensors 314b and 314a are formed when bubble sensor units 1001b, and 1001c are in sufficient contact with portions 112a and 112d, respectively, to determine the presence of bubbles therein; hemoglobin detector is formed when hemoglobin sensor 1003 is in sufficient contact with portion 112e, and pressure sensor 317 is formed when portion 112f is in sufficient contact with pressure sensor unit 1011 to measure the pressure of a fluid therein. With reference to FIGURE 6B, valves 316 and 613 are formed when portions 113f and 615 are placed within pinchers of pinch valves 1007h and 1007g, respectively.

[0147] In other embodiments, a pair of pinch valve pinchers acts to switch flow between one of two branches of a passageway. FIGURES 13 A and 13B are front view and sectional view, respectively, of a first embodiment of a pinch valve 1300 in an open configuration that can direct flow either one or both of two branches, or legs, of a passageway. Pinch valve 1300 includes two separately controllable pinch valves acting on a "Y" shaped passageway 1310 to allow switch of fluid between various legs. In particular, the internal surface of passageway 1310 forms a first leg 1311 having a flexible pinch region 1312, a second leg 1313 having a flexible pinch region 1314, and a third leg 1315 that joins the first and second legs at an intersection 1317. A first pair of pinch valve pinchers 1320 is positioned about pinch region 1312 and a second pair of pinch valve pinchers 1330 is positioned about pinch region 1314. Each pair of pinch valve pinchers

1320 and 1330 is positioned on opposite sides of their corresponding pinch regions 1312, 1314 and perpendicular to passageway 1310, and are individually controllable by controller 210 to open and close, that is allow or prohibit fluid communication across the pinch regions. Thus, for example, when pinch valve pinchers 1320 (or 1330) are brought sufficiently close, each part of pinch region 1312 (or 1314) touches another part of the pinch region and fluid may not flow across the pinch region.

[0148] As an example of the use of pinch valve 1300, FIGURE 13B shows the first and second pair of pinch valve pinchers 1320, 1330 in an open configuration. FIGURE 13C is a sectional view showing the pair of pinch valve pinchers 1320 brought together, thus closing off a portion of first leg 1311 from the second and third legs 1313, 1315. In part as a result of the distance between pinchers 1320 and intersection 1317 there is a volume 1321 associated with first leg 1311 that is not isolated ("dead space"). It is preferred that dead space is minimized so that fluids of different types can be switched between the various legs of the pinch valve. In one embodiment, the dead space is reduced by placing the placing the pinch valves close to the intersection of the legs. In another embodiment, the dead space is reduced by having passageway walls of varying thickness. Thus, for example, excess material between the pinch valves and the intersection will more effectively isolate a valved leg by displacing a portion of volume 1321.

[0149] As an example of the use of pinch valve 1300 in sampling system 300, pinchers 1320 and 1330 are positioned to act as valve 323 and 326, respectively.

[0150] FIGURES 14A and 14B are various views of a second embodiment pinch valve 1400, where FIGURE 14A is a front view and FIGURE 14B is a sectional view showing one valve in a closed position. Pinch valve 1400 differs from pinch valve 1300 in that the pairs of pinch valve pinchers 1320 and 1330 are replaced by pinchers 1420 and 1430, respectively, that are aligned with passageway 1310.

[0151] Alternative embodiment of pinch valves includes 2, 3, 4, or more passageway segment that meet at a common junction, with pinchers located at one or more passageways near the junction.

[0152] FIGURES 11 and 12 illustrate various embodiment of connector 230 which may also form or be attached to disposable portions of cassette 820 as one embodiment of an arterial patient connector 1100 and one embodiment a venous patient connector 1200. Connectors 1100 and 1200 may be generally similar to the embodiment illustrated in FIGURES 1-10, except as further detailed below.

[0153] As shown in FIGURE 11, arterial patient connector 1100 includes a stopcock 1101, a first tube portion 1103 having a length X, a blood sampling port 1105 to acquire blood samples for laboratory analysis, and fluid handling and analysis apparatus 140, a second tube 1107 having a length Y, and a tube connector 1109. Arterial patient connector 1100 also includes a pressure sensor unit 1102 that is generally similar to pressure sensor unit 1011, on the opposite side of sampling assembly 220. Length X is preferably from to 6 inches (0.15 meters) to 50 inches (1.27 meters) or approximately 48 inches (1.2 meters) in length. Length Y is preferably from 1 inch (25 millimeters) to 20 inches (0.5 meters), or approximately 12 inches (0.3 meters) in length. As shown in FIGURE 12, venous patient connector 1200 includes a clamp 1201, injection port 1105, and tube connector 1109.

SECTION IV - SAMPLE ANALYSIS SYSTEM

[0154] In several embodiments, analysis is performed on blood plasma. For such embodiments, the blood plasma must be separated from the whole blood obtained from the patient. In general, blood plasma may be obtained from whole blood at any point in fluid handling system 10 between when the blood is drawn, for example at patient connector 110 or along passageway 113, and when it is analyzed. For systems where measurements are preformed on whole blood, it may not be necessary to separate the blood at the point of or before the measurements is performed.

[0155] For illustrative purposes, this section describes several embodiments of blood separators and analyte detection systems which may form part of system 10. Section IV. A below discloses a filter for use as a blood separator in the apparatus disclosed herein. Section IV.B below discloses an analyte detection system for use in the presently disclosed apparatus. Section IV.C below discloses a sample chamber for use in the presently disclosed apparatus. Section

IV.D below discloses a centrifuge and sample chamber for use in the presently disclosed apparatus.

SECTION IV. A - BLOOD FILTER

[0156] Without limitation as to the scope of the present disclosure, one embodiment of sample preparation unit 332 is shown as a blood filter 1500, as illustrated in FIGURES 15 and 16, where FIGURE 15 is a side view of one embodiment of a filter, and FIGURE 16 is an exploded perspective view of the filter.

[0157] As shown in the embodiment of FIGURE 15, filter 1500 that includes a housing 1501 with an inlet 1503, a first outlet 1505 and a second outlet 1507. Housing 1501 contains a membrane 1509 that divides the internal volume of housing 1501 into a first volume 1502 that include inlet 1503 and first outlet 1505 and a second volume 1504. FIGURE 16 shows one embodiment of filter 1500 as including a first plate 1511 having inlet 1503 and outlet 1505, a first spacer 1513 having an opening forming first volume 1502, a second spacer 1515 having an opening forming second volume 1504, and a second plate 1517 having outlet 1507.

[0158] Filter 1500 provides for a continuous filtering of blood plasma from whole blood. Thus, for example, when a flow of whole blood is provided at inlet 1503 and a slight vacuum is applied to the second volume 1504 side of membrane 1509, the membrane filters blood cells and blood plasma passes through second outlet 1507. Preferably, there is transverse blood flow across the surface of membrane 1509 to prevent blood cells from clogging filter 1500. Accordingly, in one embodiment inlet 1503 and first outlet 1505 may be configured to provide the transverse flow across membrane 1509.

[0159] In one embodiment, membrane 1509 is a thin and strong polymer film. For example, the membrane filter may be a 10 micron thick polyester or polycarbonate film. Preferably, the membrane filter has a smooth glass-like surface, and the holes are uniform, precisely sized, and clearly defined. The material of the film may be chemically inert and have low protein binding characteristics.

[0160] One way to manufacture membrane 1509 is with a Track Etching process. Preferably, the "raw" film is exposed to charged particles in a nuclear reactor, which leaves "tracks" in the

film. The tracks may then be etched through the film, which results in holes that are precisely sized and uniformly cylindrical. For example, GE Osmonics, Inc. (4636 Somerton Rd. Trevose, PA 19053-6783) utilizes a similar process to manufacture a material that adequately serves as the membrane filter. The surface the membrane filter depicted above is a GE Osmonics Polycarbonate TE film.

[0161] As one example of the use of filter 1500, the plasma from 3 cc of blood may be extracted using a polycarbonate track etch film ("PCTE") as the membrane filter. The PCTE may have a pore size of 2 μm and an effective area of 170 millimeter ² . Preferably, the tubing connected to the supply, exhaust and plasma ports has an internal diameter of 1 millimeter. In one embodiment of a method employed with this configuration, 100 μl of plasma can be initially extracted from the blood. After saline is used to rinse the supply side of the cell, another 100 μl of clear plasma can be extracted. The rate of plasma extraction in this method and configuration can be about 15-25 μl/min.

[0162] Using a continuous flow mechanism to extract plasma may provide several benefits. In one preferred embodiment, the continuous flow mechanism is reusable with multiple samples, and there is negligible sample carryover to contaminate subsequent samples. One embodiment may also eliminate most situations in which plugging may occur. Additionally, a preferred configuration provides for a low internal volume.

[0163] Additional information on filters, methods of use thereof, and related technologies may be found in U.S. Patent Application Publication No. 2005/0038357, published on February 17, 2005, titled SAMPLE ELEMENT WITH BARRIER MATERIAL; and U.S. Patent Application No. 11/122,794, filed on May 5, 2005, titled SAMPLE ELEMENT WITH SEPARATOR. The entire contents of the above noted publication and patent application are hereby incorporated by reference herein and made a part of this specification.

SECTION IV.B - ANALYTE DETECTION SYSTEM

[0164] One embodiment of analyte detection system 334, which is not meant to limit the scope of the present disclosure, is shown in FIGURE 17 as an optical analyte detection system 1700. Analyte detection system 1700 is adapted to measure spectra of blood plasma. The blood

plasma provided to analyte detection system 334 may be provided by sample preparation unit 332, including but not limited to a filter 1500.

[0165] Analyte detection system 1700 comprises an energy source 1720 disposed along a major axis X of apparatus 1700. When activated, the energy source 1720 generates an energy beam E which advances from the energy source 1720 along the major axis X. In one embodiment, the energy source 1720 comprises an infrared source and the energy beam E comprises an infrared energy beam.

[0166] The energy beam E passes through an optical filter 1725 also situated on the major axis X, before reaching a probe region 1710. Probe region 1710 is portion of apparatus 322 in the path of an energized beam E that is adapted to accept a material sample S. In one embodiment, as shown in FIGURE 17, probe region 1710 is adapted to accept a sample element or cuvette 1730, which supports or contains the material sample S. In one embodiment, sample element 1730 is a portion of passageway 113, such as a tube or an optical cell. After passing through the sample element 1730 and the sample S, the energy beam E reaches a detector 1745.

[0167] As used herein, "sample element" is a broad term and is used in its ordinary sense and includes, without limitation, structures that have a sample chamber and at least one sample chamber wall, but more generally includes any of a number of structures that can hold, support or contain a material sample and that allow electromagnetic radiation to pass through a sample held, supported or contained thereby; e.g., a cuvette, test strip, etc.

[0168] In one embodiment, sample element 1730 forms a disposable portion of cassette 820, and the remaining portions of system 1700 form portions of instrument 810, and probe region 1710 is probe region 1002.

[0169] With further reference to FIGURE 17, the detector 1745 responds to radiation incident thereon by generating an electrical signal and passing the signal to controller 210 for analysis. Based on the signal(s) passed to it by the detector 1745, the controller computes the concentration of the analyte(s) of interest in the sample S, and/or the absorbance/transmittaiice characteristics of the sample S at one or more wavelengths or wavelength bands employed to analyze the sample. Controller 210 computes the concentration(s), absorbance(s),

transmittance(s), etc. by executing a data processing algorithm or program instructions residing within memory 212 accessible by controller 210.

[0170] In the embodiment shown in FIGURE 17, the filter 1725 may comprise a varying- passband filter, to facilitate changing, over time and/or during a measurement taken with apparatus 322, the wavelength or wavelength band of the energy beam E that may pass the filter 1725 for use in analyzing the sample S. (In various other embodiments, the filter 1725 may be omitted altogether.) Some examples of a varying-passband filter usable with apparatus 322 include, but are not limited to, a filter wheel (discussed in further detail below), an electronically tunable filter, such as those manufactured by Aegis Semiconductor (Woburn, MA), a custom filter using an "Active Thin Films platform," a Fabry-Perot interferometer, such as those manufactured by Scientific Solutions, Inc. (North Chelmsford, MA), a custom liquid crystal Fabry-Perot (LCFP) Tunable Filter, or a tunable monochrometer, such as a HORIBA (Jobin Yvon, Inc. (Edison, NJ) H 1034 type with 7-10 μm grating, or a custom designed system.

[0171] In one embodiment detection system 1700, filter 1725 comprises a varying-passband filter, to facilitate changing, over time and/or during a measurement taken with the detection system 1700, the wavelength or wavelength band of the energy beam E that may pass the filter 25 for use in analyzing the sample S. ^" When the energy beam E is filtered with a varying- passband filter, the absorption/transmittance characteristics of the sample S can be analyzed at a number of wavelengths or wavelength bands in a separate, sequential manner. As an example, assume that it is desired to analyze the sample S at N separate wavelengths (Wavelength 1 through Wavelength N). The varying-passband filter is first operated or tuned to permit the energy beam E to pass at Wavelength 1, while substantially blocking the beam E at most or all other wavelengths to which the detector 1745 is sensitive (including Wavelengths 2-N). The absorption/transmittance properties of the sample S are then measured at Wavelength 1, based on the beam E that passes through the sample S and reaches the detector 1745. The varying- passband filter is then operated or tuned to permit the energy beam E to pass at Wavelength 2, while substantially blocking other wavelengths as discussed above; the sample S is then analyzed at Wavelength 2 as was done at Wavelength 1. This process is repeated until all of the wavelengths of interest have been employed to analyze the sample S. The collected absorption/transmittance data can then be analyzed by the processor 210 to determine the

concentration of the analyte(s) of interest in the material sample S. The measured spectra of sample S is referred to herein in general as C _s (λj), that is, a wavelength dependent spectra in which C ₈ is, for example, a transmittance, an absorbance, an optical density, or some other measure of the optical properties of sample S having values at or about a number of wavelengths λj, where i ranges over the number of measurements taken. The measurement C _s (λj) is a linear array of measurements that is alternatively written as Csj.

[0172] The spectral region of system 1700 depends on the analysis technique and the analyte and mixtures of interest. For example, one useful spectral region for the measurement of glucose in blood using absorption spectroscopy is the mid-IR (for example, about 4 microns to about 11 microns). In one embodiment system 1700, energy source 1720 produces a beam E having an output in the range of about 4 microns to about 11 microns. Although water is the main contributor to the total absorption across this spectral region, the peaks and other structures present in the blood spectrum from about 6.8 microns to 10.5 microns are due to the absorption spectra of other blood components. The 4 to 11 micron region has been found advantageous because glucose has a strong absorption peak structure from about 8.5 to 10 microns, whereas most other blood constituents have a low and flat absorption spectrum in the 8.5 to 10 micron range. The main exceptions are water and hemoglobin, both of which are interferents in this region.

[0173] The amount of spectral detail provided by system 1700 depends on the analysis technique and the analyte and mixture of interest. For example, the measurement of glucose in blood by mid-IR absorption spectroscopy is accomplished with from 11 to 25 filters within a spectral region. In one embodiment system 1700, energy source 1720 produces a beam E having an output in the range of about 4 microns to about 11 microns, and filter 1725 include a number of narrow band filters within this range, each allowing only energy of a certain wavelength or wavelength band to pass therethrough. Thus, for example, one embodiment filter 1725 includes a filter wheel having 11 filters with a nominal wavelength approximately equal to one of the following: 3 μm, 4.06 μm, 4.6 μm, 4.9 μm, 5.25 μm, 6.12 μm, 6.47 μm, 7.98 μm, 8.35 μm, 9.65 μm, and 12.2 μm.

[0174] In one embodiment, individual infrared filters of the filter wheel are multi-cavity,

narrow band dielectric stacks on Germanium or Sapphire substrates, manufactured by either OCLI (JDS Uniphase, San Jose, CA) or Spectrogon US, Inc. (Parsippany, NJ). Thus, for example, each filter may nominally be 1 millimeter thick and 10 millimeter square. The peak transmission of the filter stack is typically between 50% and 70%, and the bandwidths are typically between 150 nm and 350 nm with center wavelengths between 4 and lOμm. Alternatively, a second blocking IR filter is also provided in front of the individual filters. The temperature sensitivity is preferably <0.01% per degree C to assist in maintaining nearly constant measurements over environmental conditions.

[0175] In one embodiment, the detection system 1700 computes an analyte concentration reading by first measuring the electromagnetic radiation detected by the detector 1745 at each center wavelength, or wavelength band, without the sample element 1730 present on the major axis X (this is known as an "air" reading). Second, the system 1700 measures the electromagnetic radiation detected by the detector 1745 for each center wavelength, or wavelength band, with the material sample S present in the sample element 1730, and the sample element and sample S in position on the major axis X (i.e., a "wet" reading). Finally, the controller 1700 computes the concentration(s), absorbance(s) and/or transmittances relating to the sample S based on these compiled readings.

[0176] In one embodiment, the plurality of air and wet readings are used to generate a pathlength corrected spectrum as follows. First, the measurements are normalized to give the transmission of the sample at each wavelength. Using both a signal and reference measurement at each wavelength, and letting Si represent the signal of detector 1745 at wavelength i and R; represent the signal of the detector at wavelength i, the transmittance, Tj at wavelength i may computed as Tj = Si(wet) / Si(air). Optionally, the spectra may be calculated as the optical density, ODj, as - Log(Ti). Next, the transmission over the wavelength range of approximately 4.5 μm to approximately 5.5 μm is analyzed to determine the pathlength. Specifically, since water is the primary absorbing species of blood over this wavelength region, and since the optical density is the product of the optical pathlength and the known absorption coefficient of water (OD = L σ, where L is the optical pathlength and σ is the absorption coefficient), any one of a number of standard curve fitting procedures may be used to determine the optical pathlength, L from the measured OD. The pathlength may then be used to determine the

absorption coefficient of the sample at each wavelength. Alternatively, the optical pathlength may be used in further calculations to convert absorption coefficients to optical density.

[0177] Blood samples may be prepared and analyzed by system 1700 in a variety of configurations. In one embodiment, sample S is obtained by drawing blood, either using a syringe or as part of a blood flow system, and transferring the blood into sample chamber 903. In another embodiment, sample S is drawn into a sample container that is a sample chamber 903 adapted for insertion into system 1700.

[0178] FIGURE 29 depicts another embodiment of the analyte detection system 1700, which may be generally similar to the embodiment illustrated in FIGURE 17, except as further detailed below. Where possible, similar elements are identified with identical reference numerals in the depiction of the embodiments of FIGURES 17 and 29.

[0179] The detection system 1700 shown in FIGURE 29 includes a collimator 30 located between source 1720 and filter 1725 and a beam sampling optics 90 between the filter and sample element 1730. Filter 1725 includes a primary filter 40 and a Filter wheel assembly 4420 which can insert one of a plurality of optical filters into energy beam E. System 1700 also includes a sample detector 150 may be generally similar to sample detector 1725, except as further detailed below.

[0180] As shown in FIGURE 29, energy beam E from source 1720 passes through collimator 30 through which the before reaching a primary optical filter 40 which is disposed downstream of a wide end 36 of the collimator 30. Filter 1725 is aligned with the source 1720 and collimator 30 on the major axis X and is preferably configured to operate as a broadband filter, allowing only a selected band, e.g. between about 2.5 μm and about 12.5 μm, of wavelengths emitted by the source 1720 to pass therethrough, as discussed below. In one embodiment, the energy source 1720 comprises an infrared source and the energy beam E comprises an infrared energy beam. One suitable energy source 1720 is the TOMA TECH ™ IR-50 available from HawkEye Technologies of Milford, Connecticut.

[0181] With further reference to FIGURE 29, primary filter 40 is mounted in a mask 44 so that only those portions of the energy beam E which are incident on the primary filter 40 can

pass the plane of the mask-primary filter assembly. The primary filter 40 is generally centered on and oriented orthogonal to the major axis X and is preferably circular (in a plane orthogonal to the major axis X) with a diameter of about 8 mm. Of course, any other suitable size or shape may be employed. As discussed above, the primary filter 40 preferably operates as a broadband filter. In the illustrated embodiment, the primary filter 40 preferably allows only energy wavelengths between about 4 μm and about 11 μm to pass therethrough. However, other ranges of wavelengths can be selected. The primary filter 40 advantageously reduces the filtering burden of secondary optical filter(s) 60 disposed downstream of the primary filter 40 and improves the rejection of electromagnetic radiation having a wavelength outside of the desired wavelength band. Additionally, the primary filter 40 can help minimize the heating of the secondary filter(s) 60 by the energy beam E passing therethrough. Despite these advantages, the primary filter 40 and/or mask 44 may be omitted in alternative embodiments of the system 1700 shown in FIGURE 29.

[0182] The primary filter 40 is preferably configured to substantially maintain its operating characteristics (center wavelength, passband width) where some or all of the energy beam E deviates from normal incidence by a cone angle of up to about twelve degrees relative to the major axis X. In further embodiments, this cone angle may be up to about 15 to 35 degrees, or from about 15 degrees or 20 degrees. The primary filter 40 may be said to "substantially maintain" its operating characteristics where any changes therein are insufficient to affect the performance or operation of the detection system 1700 in a manner that would raise significant concerns for the user(s) of the system in the context in which the system 1700 is employed.

[0183] In the embodiment illustrated in FIGURE 29, filter wheel assembly 4420 includes an optical filter wheel 50 and a stepper motor 70 connected to the filter wheel and configured to generate a force to rotate the filter wheel 50. Additionally, a position sensor 80 is disposed over a portion of the circumference of the filter wheel 50 and may be configured to detect the angular position of the filter wheel 50 and to generate a corresponding filter wheel position signal, thereby indicating which filter is in position on the major axis X. Alternatively, the stepper motor 70 may be configured to track or count its own rotation(s), thereby tracking the angular position of the filter wheel, and pass a corresponding position signal to the controller 210. Two suitable position sensors are models EE-SPX302-W2A and EE-SPX402-W2A available from Omron

Corporation of Kyoto, Japan.

[0184] Optical filter wheel SO is employed as a varying-passband filter, to selectively position the secondary filter(s) 60 on the major axis X and/or in the energy beam E. The filter wheel 50 can therefore selectively tune the wavelength(s) of the energy beam E downstream of the wheel 50. These wavelength(s) vary according to the characteristics of the secondary filter(s) 60 mounted in the filter wheel 50. The filter wheel 50 positions the secondary filter(s) 60 in the energy beam E in a "one-at-a-time" fashion to sequentially vary, as discussed above, the wavelengths or wavelength bands employed to analyze the material sample S. An alternative to filter wheel 50 is a linear filter translated by a motor (not shown). The linear filter may be, for example, a linear array of separate filters or a single filter with filter properties that change in a linear dimension.

[0185] In alternative arrangements, the single primary filter 40 depicted in FIGURE 29 may be replaced or supplemented with additional primary filters mounted on the filter wheel 50 upstream of each of the secondary filters 60. As yet another alternative, the primary filter 40 could be implemented as a primary filter wheel (not shown) to position different primary filters on the major axis X at different times during operation of the detection system 1700, or as a tunable filter.

[0186] The filter wheel 50, in the embodiment depicted in FIGURE 30, can comprise a wheel body 52 and a plurality of secondary filters 60 disposed on the body 52, the center of each filter being equidistant from a rotational center RC of the wheel body. The filter wheel 50 is configured to rotate about an axis which is (i) parallel to the major axis X and (ii) spaced from the major axis X by an orthogonal distance approximately equal to the distance between the rotational center RC and any of the center(s) of the secondary filter(s) 60. Under this arrangement, rotation of the wheel body 52 advances each of the filters sequentially through the major axis X, so as to act upon the energy beam E. However, depending on the analyte(s) of interest or desired measurement speed, only a subset of the filters on the wheel 50 may be employed in a given measurement run. A home position notch 54 may be provided to indicate the home position of the wheel 50 to a position sensor 80.

[0187] In one embodiment, the wheel body 52 can be formed from molded plastic, with each

of the secondary filters 60 having, for example a thickness of 1 mm and a 10 mm x 10 mm or a 5 mm x 5 mm square configuration. Each of the filters 60, in this embodiment of the wheel body, is axially aligned with a circular aperture of 4 mm diameter, and the aperture centers define a circle of about 1.70 inches diameter, which circle is concentric with the wheel body 52. The body 52 itself is circular, with an outside diameter of 2.00 inches.

[0188] Each of the secondary filter(s) 60 is preferably configured to operate as a narrow band filter, allowing only a selected energy wavelength or wavelength band (i.e., a filtered energy beam (Ef) to pass therethrough. As the filter wheel 50 rotates about its rotational center RC, each of the secondary filter(s) 60 is, in turn, disposed along the major axis X for a selected dwell time corresponding to each of the secondary filter(s) 60.

[0189] The "dwell time" for a given secondary filter 60 is the time interval, in an individual measurement run of the system 1700, during which both of the following conditions are true: (i) the filter is disposed on the major axis X; and (ii) the source 1720 is energized. The dwell time for a given filter may be greater than or equal to the time during which the filter is disposed on the major axis X during an individual measurement run. In one embodiment of the analyte detection system 1700, the dwell time corresponding to each of the secondary filter(s) 60 is less than about 1 second. However, the secondary filter(s) 60 can have other dwell times, and each of the fllter(s) 60 may have a different dwell time during a given measurement run.

[0190] From the secondary filter 60, the filtered energy beam (Ef) passes through a beam sampling optics 90, which includes a beam splitter 4400 disposed along the major axis X and having a face 4400a disposed at an included angle θ relative to the major axis X. The splitter 4400 preferably separates the filtered energy beam (Ef) into a sample beam (Es) and a reference beam (Er).

[0191] With further reference to FIGURE 29, the sample beam (Es) passes next through a first lens 4410 aligned with the splitter 4400 along the major axis X. The first lens 4410 is configured to focus the sample beam (Es) generally along the axis X onto the material sample S. The sample S is preferably disposed in a sample element 1730 between a first window 122 and a second window 124 of the sample element 1730. The sample element 1730 is further preferably removably disposed in a holder 4430, and the holder 4430 has a first opening 132 and a second

opening 134 configured for alignment with the first window 122 and second window 124, respectively. Alternatively, the sample element 1730 and sample S may be disposed on the major axis X without use of the holder 4430.

[0192] At least a fraction of the sample beam (Es) is transmitted through the sample S and continues onto a second lens 4440 disposed along the major axis X. The second lens 4440 is configured to focus the sample beam (Es) onto a sample detector 150, thus increasing the flux density of the sample beam (Es) incident upon the sample detector 150. The sample detector 150 is configured to generate a signal corresponding to the detected sample beam (Es) and to pass the signal to a controller 210, as discussed in more detail below.

[0193] Beam sampling optics 90 further includes a third lens 160 and a reference detector 170. The reference beam (Er) is directed by beam sampling optics 90 from the beam splitter 4400 to third lens 160 disposed along a minor axis Y generally orthogonal to the major axis X. The third lens 160 is configured to focus the reference beam (Er) onto reference detector 170, thus increasing the flux density of the reference beam (Er) incident upon the reference detector 170. In one embodiment, the lenses 4410, 4440, 160 may be formed from a material which is highly transmissive of infrared radiation, for example germanium or silicon. In addition, any of the lenses 4410, 4440 and 160 may be implemented as a system of lenses, depending on the desired optical performance. The reference detector 170 is also configured to generate a signal corresponding to the detected reference beam (Er) and to pass the signal to the controller 210, as discussed in more detail below. Except as noted below, the sample and reference detectors 150, 170 may be generally similar to the detector 1745 illustrated in FIGURE 17. Based on signals received from the sample and reference detectors 150, 170, the controller 210 computes the concentration(s), absorbance(s), transmittance(s), etc. relating to the sample S by executing a data processing algorithm or program instructions residing within the memory 212 accessible by the controller 210.

[0194] In further variations of the detection system 1700 depicted in FIGURE 29, beam sampling optics 90, including the beam splitter 4400, reference detector 170 and other structures on the minor axis Y may be omitted, especially where the output intensity of the source 1720 is sufficiently stable to obviate any need to reference the source intensity in operation of the

detection system 1700. Thus, for example, sufficient signals may be generated by detectors 170 and 150 with one or more of lenses 4410, 4440, 160 omitted. Furthermore, in any of the embodiments of the analyte detection system 1700 disclosed herein, the controller 210 and/or memory 212 may reside partially or wholly in a standard personal computer ("PC") coupled to the detection system 1700.

[0195] FIGURE 31 depicts a partial cross-sectional view of another embodiment of an analyte detection system 1700, which may be generally similar to any of the embodiments illustrated in FIGURES 17, 29, and 30, except as further detailed below. Where possible, similar elements are identified with identical reference numerals in the depiction of the embodiments of FIGURES 17, 29, and 30.

[0196] The energy source 1720 of the embodiment of FIGURE 31 preferably comprises an emitter area 22 which is substantially centered on the major axis X. In one embodiment, the emitter area 22 may be square in shape. However the emitter area 22 can have other suitable shapes, such as rectangular, circular, elliptical, etc. One suitable emitter area 22 is a square of about 1.5 mm on a side; of course, any other suitable shape or dimensions may be employed.

[0197] The energy source 1720 is preferably configured to selectably operate at a modulation frequency between about 1 Hz and 30 Hz and have a peak operating temperature of between about 1070 degrees Kelvin and 1170 degrees Kelvin. Additionally, the source 1720 preferably operates with a modulation depth greater than about 80% at all modulation frequencies. The energy source 1720 preferably emits electromagnetic radiation in any of a number of spectral ranges, e.g., within infrared wavelengths; in the mid-infrared wavelengths; above about 0.8 μm; between about 5.0 μm and about 20.0 μm; and/or between about 5.25 μm and about 12.0 μm. However, in other embodiments, the detection system 1700 may employ an energy source 1720 which is unmodulated and/or which emits in wavelengths found anywhere from the visible spectrum through the microwave spectrum, for example anywhere from about 0.4 μm to greater than about 100 μm. In still other embodiments, the energy source 1720 can emit electromagnetic radiation in wavelengths between about 3.5 μm and about 14 μm, or between about 0.8 μm and about 2.5 μm, or between about 2.5 μm and 20 μm, or between about 20 μm and about 100 μm, or between about 6.85 μm and about 10.10 μm. In yet other embodiments, the energy source

1720 can emit electromagnetic radiation within the radio frequency (RF) range or the terahertz range. All of the above-recited operating characteristics are merely exemplary, and the source 1720 may have any operating characteristics suitable for use with the analyte detection system 1700.

[0198] A power supply (not shown) for the energy source 1720 is preferably configured to selectably operate with a duty cycle of between about 30% and about 70%. Additionally, the power supply is preferably configured to selectably operate at a modulation frequency of about 10Hz, or between about 1 Hz and about 30 Hz. The operation of the power supply can be in the form of a square wave, a sine wave, or any other waveform defined by a user.

[0199] With further reference to FIGURE 31, the collimator 30 comprises a tube 30a with one or more highly-reflective inner surfaces 32 which diverge from a relatively narrow upstream end 34 to a relatively wide downstream end 36 as they extend downstream, away from the energy source 1720. The narrow end 34 defines an upstream aperture 34a which is situated adjacent the emitter area 22 and permits radiation generated by the emitter area to propagate downstream into the collimator. The wide end 36 defines a downstream aperture 36a. Like the emitter area 22, each of the inner surface(s) 32, upstream aperture 34a and downstream aperture 36a is preferably substantially centered on the major axis X.

[0200] As illustrated in FIGURE 31, the inner surface(s) 32 of the collimator may have a generally curved shape, such as a parabolic, hyperbolic, elliptical or spherical shape. One suitable collimator 30 is a compound parabolic concentrator (CPC). In one embodiment, the collimator 30 can be up to about 20 mm in length. In another embodiment, the collimator 30 can be up to about 30 mm in length. However, the collimator 30 can have any length, and the inner surface(s) 32 may have any shape, suitable for use with the analyte detection system 1700.

[0201] The inner surfaces 32 of the collimator 30 cause the rays making up the energy beam E to straighten (i.e., propagate at angles increasingly parallel to the major axis X) as the beam E advances downstream, so that the energy beam E becomes increasingly or substantially cylindrical and oriented substantially parallel to the major axis X. Accordingly, the inner surfaces 32 are highly reflective and minimally absorptive in the wavelengths of interest, such as infrared wavelengths.

[0202] The tube 30a itself may be fabricated from a rigid material such as aluminum, steel, or any other suitable material, as long as the inner surfaces 32 are coated or otherwise treated to be highly reflective in the wavelengths of interest. For example, a polished gold coating may be employed. Preferably, the inner surface(s) 32 of the collimator 30 define a circular cross-section when viewed orthogonal to the major axis X; however, other cross-sectional shapes, such as a square or other polygonal shapes, parabolic or elliptical shapes may be employed in alternative embodiments.

[0203] As noted above, the filter wheel 50 shown in FIGURE 31 comprises a plurality of secondary filters 60 which preferably operate as narrow band filters, each filter allowing only energy of a certain wavelength or wavelength band to pass therethrough. In one configuration suitable for detection of glucose in a sample S, the filter wheel 50 comprises twenty or twenty- two secondary filters 60, each of which is configured to allow a filtered energy beam (Ef) to travel therethrough with a nominal wavelength approximately equal to one of the following: 3 μm, 4.06 μm, 4.6 μm, 4.9 μm, 5.25 μm, 6.12 μm, 6.47 μm, 7.98 μm, 8.35 μm, 9.65 μm, and 12.2 μm. (Moreover, this set of wavelengths may be employed with or in any of the embodiments of the analyte detection system 1700 disclosed herein.) Each secondary filter's 60 center wavelength is preferably equal to the desired nominal wavelength plus or minus about 2%. Additionally, the secondary filters 60 are preferably configured to have a bandwidth of about 0.2 μm, or alternatively equal to the nominal wavelength plus or minus about 2%-10%.

[0204] In another embodiment, the filter wheel 50 comprises twenty secondary filters 60, each of which is configured to allow a filtered energy beam (Ef) to travel therethrough with a nominal center wavelengths of: 4.275 μm, 4.5 μm, 4.7 μm, 5.0 μm, 5.3 μm, 6.056 μm, 7.15 μm, 7.3 μm, 7.55 μm, 7.67 μm, 8.06 μm, 8.4 μm, 8.56 μm, 8.87 μm, 9.15 μm, 9.27 μm, 9.48 μm, 9.68 μm, 9.82 μm, and 10.06 μm. (This set of wavelengths may also be employed with or in any of the embodiments of the analyte detection system 1700 disclosed herein.) In still another embodiment, the secondary filters 60 may conform to any one or combination of the following specifications: center wavelength tolerance of ± 0.01 μm; half-power bandwidth tolerance of ± 0.01 μm; peak transmission greater than or equal to 75%; cut-on/cut-off slope less than 2%; center-wavelength temperature coefficient less than .01% per degree Celsius; out of band

attenuation greater than OD 5 from 3 μm to 12 μm; flatness less than 1.0 waves at 0.6328 μm; surface quality of E-E per Mil-F-48616; and overall thickness of about 1 mm.

[0205] In still another embodiment, the secondary filters mentioned above may conform to any one or combination of the following half-power bandwidth ("HPBW") specifications:

[0206] In still further embodiments, the secondary filters may have a center wavelength tolerance of ± 0.5 % and a half-power bandwidth tolerance of ± 0.02 μm.

[0207] Of course, the number of secondary filters employed, and the center wavelengths and other characteristics thereof, may vary in further embodiments of the system 1700, whether such further embodiments are employed to detect glucose, or other analytes instead of or in addition to glucose. For example, in another embodiment, the filter wheel 50 can have fewer than fifty secondary filters 60. In still another embodiment, the filter wheel 50 can have fewer than twenty secondary filters 60. In yet another embodiment, the filter wheel 50 can have fewer than ten secondary filters 60.

[0208] In one embodiment, the secondary filters 60 each measure about 10 mm long by 10 mm wide in a plane orthogonal to the major axis X, with a thickness of about 1 mm. However, the secondary filters 60 can have any other (e.g., smaller) dimensions suitable for operation of the analyte detection system 1700. Additionally, the secondary filters 60 are preferably configured to operate at a temperature of between about 5 ⁰ C and about 35 °C and to allow transmission of more than about 75% of the energy beam E therethrough in the wavelength(s) which the filter is configured to pass.

[0209] According to the embodiment illustrated in FIGURE 31, the primary filter 40 operates as a broadband filter and the secondary filters 60 disposed on the filter wheel 50 operate as narrow band filters. However, one of ordinary skill in the art will realize that other structures can be used to filter energy wavelengths according to the embodiments described herein. For example, the primary filter 40 may be omitted and/or an electronically tunable filter or Fabry- Perot interferometer (not shown) can be used in place of the filter wheel 50 and secondary filters 60. Such a tunable filter or interferometer can be configured to permit, in a sequential, "one-at-a- time" fashion, each of a set of wavelengths or wavelength bands of electromagnetic radiation to pass therethrough for use in analyzing the material sample S.

[0210] A reflector tube 98 is preferably positioned to receive the filtered energy beam (Ef) as it advances from the secondary filter(s) 60. The reflector tube 98 is preferably secured with respect to the secondary filter(s) 60 to substantially prevent introduction of stray electromagnetic radiation, such as stray light, into the reflector tube 98 from outside of the detection system 1700. The inner surfaces of the reflector tube 98 are highly reflective in the relevant wavelengths and preferably have a cylindrical shape with a generally circular cross-section orthogonal to the major and/or minor axis X, Y. However, the inner surface of the tube 98 can have a cross-section of any suitable shape, such as oval, square, rectangular, etc. Like the collimator 30, the reflector tube 98 may be formed from a rigid material such as aluminum, steel, etc., as long as the inner surfaces are coated or otherwise treated to be highly reflective in the wavelengths of interest. For example, a polished gold coating may be employed.

[0211] According to the embodiment illustrated in FIGURE 31, the reflector tube 98 preferably comprises a major section 98a and a minor section 98b. As depicted, the reflector tube 98 can be T-shaped with the major section 98a having a greater length than the minor section 98b. In another example, the major section 98a and the minor section 98b can have the same length. The major section 98a extends between a first end 98c and a second end 98d along the major axis X. The minor section 98b extends between the major section 98a and a third end 98e along the minor axis Y.

[0212] The major section 98a conducts the filtered energy beam (Ef) from the first end 98c to the beam splitter 4400, which is housed in the major section 98a at the intersection of the

major and minor axes X, Y. The major section 98a also conducts the sample beam (Es) from the beam splitter 4400, through the first lens 4410 and to the second end 98d. From the second end 98d the sample beam (Es) proceeds through the sample element 1730, holder 4430 and second lens 4440, and to the sample detector 150. Similarly, the minor section 98b conducts the reference beam (Er) through beam sampling optics 90 from the beam splitter 4400, through the third lens 160 and to the third end 98e. From the third end 98e the reference beam (Er) proceeds to the reference detector 170.

[0213] The sample beam (Es) preferably comprises from about 75% to about 85% of the energy of the filtered energy beam (Ef). More preferably, the sample beam (Es) comprises about 80% of the energy of the filtered energy beam (Es). The reference beam (Er) preferably comprises from about 10% and about 50% of the energy of the filtered energy beam (Es). More preferably, the reference beam (Er) comprises about 20% of the energy of the filtered energy beam (Ef). Of course, the sample and reference beams may take on any suitable proportions of the energy beam E.

[0214] The reflector tube 98 also houses the first lens 4410 and the third lens 160. As illustrated in FIGURE 31, the reflector tube 98 houses the first lens 4410 between the beam splitter 4400 and the second end 98d. The first lens 4410 is preferably disposed so that a plane 4612 of the lens 4410 is generally orthogonal to the major axis X. Similarly, the tube 98 houses the third lens 160 between the beam splitter 4400 and the third end 98e. The third lens 160 is preferably disposed so that a plane 162 of the third lens 160 is generally orthogonal to the minor axis Y. The first lens 4410 and the third lens 160 each has a focal length configured to substantially focus the sample beam (Es) and reference beam (Er), respectively, as the beams (Es, Er) pass through the lenses 4410, 160. In particular, the first lens 4410 is configured, and disposed relative to the holder 4430, to focus the sample beam (Es) so that substantially the entire sample beam (Es) passes through the material sample S, residing in the sample element 1730. Likewise, the third lens 160 is configured to focus the reference beam (Er) so that substantially the entire reference beam (Er) impinges onto the reference detector 170.

[0215] The sample element 1730 is retained within the holder 4430, which is preferably oriented along a plane generally orthogonal to the major axis X. The holder 4430 is configured to

be slidably displaced between a loading position and a measurement position within the analyte detection system 1700. In the measurement position, the holder 4430 contacts a stop edge 136 which is located to orient the sample element 1730 and the sample S contained therein on the major axis X.

[0216] The structural details of the holder 4430 depicted in FIGURE 31 are unimportant, so long as the holder positions the sample element 1730 and sample S on and substantially orthogonal to the major axis X, while permitting the energy beam E to pass through the sample element and sample. As with the embodiment depicted in FIGURE 29, the holder 4430 may be omitted and the sample element 1730 positioned alone in the depicted location on the major axis X. However, the holder 4430 is useful where the sample element 1730 (discussed in further detail below) is constructed from a highly brittle or fragile material, such as barium fluoride, or is manufactured to be extremely thin.

[0217] As with the embodiment depicted in FIGURE 29, the sample and reference detectors 150, 170 shown in FIGURE 31 respond to radiation incident thereon by generating signals and passing them to the controller 210. Based these signals received from the sample and reference detectors 150, 170, the controller 210 computes the concentration(s), absorbance(s), transmittance(s), etc. relating to the sample S by executing a data processing algorithm or program instructions residing within the memory 212 accessible by the controller 210. In further variations of the detection system 1700 depicted in FIGURE 31, the beam splitter 4400, reference detector 170 and other structures on the minor axis Y may be omitted, especially where the output intensity of the source 1720 is sufficiently stable to obviate any need to reference the source intensity in operation of the detection system 1700.

[0218] FIGURE 32 depicts a sectional view of the sample detector 150 in accordance with one embodiment. Sample detector 150 is mounted in a detector housing 152 having a receiving portion 152a and a cover 152b. However, any suitable structure may be used as the sample detector 150 and housing 152. The receiving portion 152a preferably defines an aperture 152c and a lens chamber 152d, which are generally aligned with the major axis X when the housing 152 is mounted in the analyte detection system 1700. The aperture 152c is configured to allow at least a fraction of the sample beam (Es) passing through the sample S and the sample element

1730 to advance through the aperture 152c and into the lens chamber 152d.

[0219] The receiving portion 152a houses the second lens 4440 in the lens chamber 152d proximal to the aperture 152c. The sample detector 150 is also disposed in the lens chamber 152d downstream of the second lens 4440 such that a detection plane 154 of the detector 150 is substantially orthogonal to the major axis X. The second lens 4440 is positioned such that a plane 142 of the lens 4440 is substantially orthogonal to the major axis X. The second lens 4440 is configured, and is preferably disposed relative to the holder 4430 and the sample detector 150, to focus substantially all of the sample beam (Es) onto the detection plane 154, thereby increasing the flux density of the sample beam (Es) incident upon the detection plane 154.

[0220] With further reference to FIGURE 32, a support member 156 preferably holds the sample detector 150 in place in the receiving portion 152a. In the illustrated embodiment, the support member 156 is a spring 156 disposed between the sample detector 150 and the cover 152b. The spring 156 is configured to maintain the detection plane 154 of the sample detector 150 substantially orthogonal to the major axis X. A gasket 157 is preferably disposed between the cover 152b and the receiving portion 152a and surrounds the support member 156.

[0221] The receiving portion 152a preferably also houses a printed circuit board 158 disposed between the gasket 157 and the sample detector 150. The board 158 connects to the sample detector 150 through at least one connecting member 150a. The sample detector 150 is configured to generate a detection signal corresponding to the sample beam (Es) incident on the detection plane 154. The sample detector 150 communicates the detection signal to the circuit board 158 through the connecting member 150a, and the board 158 transmits the detection signal to the controller 210.

[0222] In one embodiment, the sample detector 150 comprises a generally cylindrical housing 150a, e.g. a type TO-39 "metal can" package, which defines a generally circular housing aperture 150b at its "upstream" end. In one embodiment, the housing 150a has a diameter of about 0.323 inches and a depth of about 0.248 inches, and the aperture 150b may have a diameter of about 0.197 inches.

[0223] A detector window 150c is disposed adjacent the aperture 150b, with its upstream

surface preferably about 0.078 inches (+/- 0.004 inches) from the detection plane 154. (The detection plane 154 is located about 0.088 inches (+/- 0.004 inches) from the upstream edge of the housing 150a, where the housing has a thickness of about 0.010 inches.) The detector window 150c is preferably transmissive of infrared energy in at least a 3-12 micron passband; accordingly, one suitable material for the window 150c is germanium. The endpoints of the passband may be "spread" further to less than 2.5 microns, and/or greater than 12.5 microns, to avoid unnecessary absorbance in the wavelengths of interest. Preferably, the transmittance of the detector window 150c does not vary by more than 2% across its passband. The window 150c is preferably about 0.020 inches in thickness. The sample detector 150 preferably substantially retains its operating characteristics across a temperature range of -20 to +60 degrees Celsius.

[0224] FIGURE 33 depicts a sectional view of the reference detector 170 in accordance with one embodiment. The reference detector 170 is mounted in a detector housing 172 having a receiving portion 172a and a cover 172b. However, any suitable structure may be used as the sample detector 150 and housing 152. The receiving portion 172a preferably defines an aperture 172c and a chamber 172d which are generally aligned with the minor axis Y, when the housing 172 is mounted in the analyte detection system 1700. The aperture 172c is configured to allow the reference beam (Er) to advance through the aperture 172c and into the chamber 172d.

[0225] The receiving portion 172a houses the reference detector 170 in the chamber 172d proximal to the aperture 172c. The reference detector 170 is disposed in the chamber 172d such that a detection plane 174 of the reference detector 170 is substantially orthogonal to the minor axis Y. The third lens 160 is configured to substantially focus the reference beam (Er) so that substantially the entire reference beam (Er) impinges onto the detection plane 174, thus increasing the flux density of the reference beam (Er) incident upon the detection plane 174.

[0226] With further reference to FIGURE 33, a support member 176 preferably holds the reference detector 170 in place in the receiving portion 172a. In the illustrated embodiment, the support member 176 is a spring 176 disposed between the reference detector 170 and the cover 172b. The spring 176 is configured to maintain the detection plane 174 of the reference detector 170 substantially orthogonal to the minor axis Y. A gasket 177 is preferably disposed between the cover 172b and the receiving portion 172a and surrounds the support member 176.

[0227] The receiving portion 172a preferably also houses a printed circuit board 178 disposed between the gasket 177 and the reference detector 170. The board 178 connects to the reference detector 170 through at least one connecting member 170a. The reference detector 170 is configured to generate a detection signal corresponding to the reference beam (Er) incident on the detection plane 174. The reference detector 170 communicates the detection signal to the circuit board 178 through the connecting member 170a, and the board 178 transmits the detection signal to the controller 210.

[0228] In one embodiment, the construction of the reference detector 170 is generally similar to that described above with regard to the sample detector 150.

[0229] In one embodiment, the sample and reference detectors 150, 170 are both configured to detect electromagnetic radiation in a spectral wavelength range of between about 0.8 μm and about 25 μm. However, any suitable subset of the foregoing set of wavelengths can be selected. In another embodiment, the detectors 150, 170 are configured to detect electromagnetic radiation in the wavelength range of between about 4μm and about 12 μm. The detection planes 154, 174 of the detectors 150, 170 may each define an active area about 2 mm by 2 mm or from about 1 mm by 1 mm to about 5 mm by 5 mm; of course, any other suitable dimensions and proportions may be employed. Additionally, the detectors 150, 170 may be configured to detect electromagnetic radiation directed thereto within a cone angle of about 45 degrees from the major axis X.

[0230] In one embodiment, the sample and reference detector subsystems 150, 170 may further comprise a system (not shown) for regulating the temperature of the detectors. Such a temperature-regulation system may comprise a suitable electrical heat source, thermistor, and a proportional-plus-integral-plus-derivative (PID) control. These components may be used to regulate the temperature of the detectors 150, 170 at about 35 ⁰ C. The detectors 150, 170 can also optionally be operated at other desired temperatures. Additionally, the PID control preferably has a control rate of about 60 Hz and, along with the heat source and thermistor, maintains the temperature of the detectors 150, 170 within about 0.1 °C of the desired temperature.

[0231] The detectors 150, 170 can operate in either a voltage mode or a current mode,

wherein either mode of operation preferably includes the use of a pre-amp module. Suitable voltage mode detectors for use with the analyte detection system 1700 disclosed herein include: models LIE 302 and 312 by IiifraTec of Dresden, Germany; model L2002 by BAE Systems of Rockville, Maryland; and model LTS-I by Dias of Dresden, Germany. Suitable current mode detectors include: InfraTec models LIE 301, 315, 345 and 355; and 2x2 current-mode detectors available from Dias.

[0232] In one embodiment, one or both of the detectors 150, 170 may meet the following specifications, when assuming an incident radiation intensity of about 9.26 x 10 ^"4 watts (rms) per cm , at 10 Hz modulation and within a cone angle of about 15 degrees: detector area of 0.040 cm (2 mm x 2 mm square); detector input of 3.70 x 10 watts (rms) at 10 Hz; detector sensitivity of 360 volts per watt at 10 Hz; detector output of 1.333 x 10 ^"2 volts (rms) at 10 Hz; noise of 8.00 x 10 ^"8 volts/sqrtHz at 10 Hz; and signal-to-noise ratios of 1.67 x 10 ⁵ mis/sqrtHz and 104.4 dB/sqrtHz; and detectivity of 1.00 x 10 ⁹ cm sqrtHz/watt.

[0233] In alternative embodiments, the detectors 150, 170 may comprise microphones and/or other sensors suitable for operation of the detection system 1700 in a photoacoustic mode.

[0234] The components of any of the embodiments of the analyte detection system 1700 may be partially or completely contained in an enclosure or casing (not shown) to prevent stray electromagnetic radiation, such as stray light, from contaminating the energy beam E. Any suitable casing may be used. Similarly, the components of the detection system 1700 may be mounted on any suitable frame or chassis (not shown) to maintain their operative alignment as depicted in FIGURE 17, 29, and 31. The frame and the casing may be formed together as a single unit, member or collection of members.

[0235] In one method of operation, the analyte detection system 1700 shown in FIGURE 29 or 31 measures the concentration of one or more analytes in the material sample S, in part, by comparing the electromagnetic radiation detected by the sample and reference detectors 150, 170. During operation of the detection system 1700, each of the secondary filter(s) 60 is sequentially aligned with the major axis X for a dwell time corresponding to the secondary filter 60. (Of course, where an electronically tunable filter or Fabry-Perot interferometer is used in place of the filter wheel 50, the tunable filter or interferometer is sequentially tuned to each of a

set of desired wavelengths or wavelength bands in lieu of the sequential alignment of each of the secondary filters with the major axis X.) The energy source 1720 is then operated at (any) modulation frequency, as discussed above, during the dwell time period. The dwell time may be different for each secondary filter 60 (or each wavelength or band to which the tunable filter or interferometer is tuned). In one embodiment of the detection system 1700, the dwell time for each secondary filter 60 is less than about 1 second. Use of a dwell time specific to each secondary filter 60 advantageously allows the detection system 1700 to operate for a longer period of time at wavelengths where errors can have a greater effect on the computation of the analyte concentration in the material sample S. Correspondingly, the detection system 1700 can operate for a shorter period of time at wavelengths where errors have less effect on the computed analyte concentration. The dwell times may otherwise be nonuniform among the filters/wavelengths/bands employed in the detection system.

[0236] For each secondary filter 60 selectively aligned with the major axis X, the sample detector 150 detects the portion of the sample beam (Es), at the wavelength or wavelength band corresponding to the secondary filter 60, that is transmitted through the material sample S. The sample detector 150 generates a detection signal corresponding to the detected electromagnetic radiation and passes the signal to the controller 210. Simultaneously, the reference detector 170 detects the reference beam (Er) transmitted at the wavelength or wavelength band corresponding to the secondary filter 60. The reference detector 170 generates a detection signal corresponding to the detected electromagnetic radiation and passes the signal to the controller 210. Based on the signals passed to it by the detectors 150, 170, the controller 210 computes the concentration of the analyte(s) of interest in the sample S, and/or the absorbance/transmittance characteristics of the sample S at one or more wavelengths or wavelength bands employed to analyze the sample. The controller 210 computes the concentration(s), absorbance(s), transmittance(s), etc. by executing a data processing algorithm or program instructions residing within the memory 212 accessible by the controller 210.

[0237] The signal generated by the reference detector may be used to monitor fluctuations in the intensity of the energy beam emitted by the source 1720, which fluctuations often arise due to drift effects, aging, wear or other imperfections in the source itself. This enables the controller 210 to identify changes in intensity of the sample beam (Es) that are attributable to changes in

the emission intensity of the source 1720, and not to the composition of the sample S. By so doing, a potential source of error in computations of concentration, absorbance, etc. is minimized or eliminated.

[0238] In one embodiment, the detection system 1700 computes an analyte concentration reading by first measuring the electromagnetic radiation detected by the detectors ISO, 170 at each center wavelength, or wavelength band, without the sample element 1730 present on the major axis X (this is known as an "air" reading). Second, the system 1700 measures the electromagnetic radiation detected by the detectors 150, 170 for each center wavelength, or wavelength band, with the material sample S present in the sample element 1730, and the sample element 1730 and sample S in position on the major axis X (i.e., a "wet" reading). Finally, the controller 180 computes the concentration(s), absorbance(s) and/or transmittances relating to the sample S based on these compiled readings.

[0239] In one embodiment, the plurality of air and wet readings are used to generate a pathlength corrected spectrum as follows. First, the measurements are normalized to give the transmission of the sample at each wavelength. Using both a signal and reference measurement at each wavelength, and letting Sj represent the signal of detector 150 at wavelength i and Rj represent the signal of detector 170 at wavelength i, the transmission, τ; is computed as Tj = Si(wet)/Rj(wet) / Si(air)/Rj(air). Optionally, the spectra may be calculated as the optical density,

[0240] Next, the transmission over the wavelength range of approximately 4.5 μm to approximately 5.5 μm is analyzed to determine the pathlength. Specifically, since water is the primary absorbing species of blood over this wavelength region, and since the optical density is the product of the optical pathlength and the known absorption coefficient of water (OD = L σ, where L is the optical pathlength and σ is the absorption coefficient), any one of a number of standard curve fitting procedures may be used to determine the optical pathlength, L from the measured OD. The pathlength may then be used to determine the absorption coefficient of the sample at each wavelength. Alternatively, the optical pathlength may be used in further calculations to convert absorption coefficients to optical density.

[0241] Additional information on analyte detection systems, methods of use thereof, and

related technologies may be found in the above-mentioned and incorporated U.S. Patent Application Publication No. 2005/0038357, published on February 17, 2005, titled SAMPLE ELEMENT WITH BARRIER MATERIAL.

SECTION IV.C - SAMPLE ELEMENT

[0242] FIGURE 18 is a top view of a sample element 1730, FIGURE 19 is a side view of the sample element, and FIGURE 20 is an exploded perspective view of the sample element. In one embodiment, sample element 1730 includes sample chamber 903 that is in fluid communication with and accepts filtered blood from sample preparation unit 332. The sample element 1730 comprises a sample chamber 903 defined by sample chamber walls 1802. The sample chamber 903 is configured to hold a material sample which may be drawn from a patient, for analysis by the detection system with which the sample element 1730 is employed.

[0243] In the embodiment illustrated in FIGURES 18-19, the sample chamber 903 is defined by first and second lateral chamber walls 1802a, 1802b and upper and lower chamber walls 1802c, 1802d; however, any suitable number and configuration of chamber walls may be employed. At least one of the upper and lower chamber walls 1802c, 1802d is formed from a material which is sufficiently transmissive of the wavelength(s) of electromagnetic radiation that are employed by the sample analysis apparatus 322 (or any other system with which the sample element is to be used). A chamber wall which is so transmissive may thus be termed a "window;" in one embodiment, the upper and lower chamber walls 1802c, 1802d comprise first and second windows so as to permit the relevant wavelength(s) of electromagnetic radiation to pass through the sample chamber 903. In another embodiment, only one of the upper and lower chamber walls 1802c, 1802d comprises a window; in such an embodiment, the other of the upper and lower chamber walls may comprise a reflective surface configured to back-reflect any electromagnetic energy emitted into the sample chamber 903 by the analyte detection system with which the sample element 1730 is employed. Accordingly, this embodiment is well suited for use with an analyte detection system in which a source and a detector of electromagnetic energy are located on the same side as the sample element.

[0244] In various embodiments, the material that makes up the window(s) of the sample element 1730 is completely transmissive, i.e., it does not absorb any of the electromagnetic

radiation from the source 1720 and filters 1725 that is incident upon it. In another embodiment, the material of the window(s) has some absorption in the electromagnetic range of interest, but its absorption is negligible. In yet another embodiment, the absorption of the material of the window(s) is not negligible, but it is stable for a relatively long period of time. In another embodiment, the absorption of the window(s) is stable for only a relatively short period of time, but sample analysis apparatus 322 is configured to observe the absorption of the material and eliminate it from the analyte measurement before the material properties can change measurably. Materials suitable for forming the window(s) of the sample element 1730 include, but are not limited to, calcium fluoride, barium fluoride, germanium, silicon, polypropylene, polyethylene, or any polymer with suitable transmissivity (i.e., transmittance per unit thickness) in the relevant wavelength(s). Where the window(s) are formed from a polymer, the selected polymer can be isotactic, atactic or syndiotactic in structure, so as to enhance the flow of the sample between the window(s). One type of polyethylene suitable for constructing the sample element 1730 is type 220, extruded or blow molded, available from KUBE Ltd. of Staefa, Switzerland.

[0245] In one embodiment, the sample element 1730 is configured to allow sufficient transmission of electromagnetic energy having a wavelength of between about 4 μm and about 10.5 μm through the window(s) thereof. However, the sample element 1730 can be configured to allow transmission of wavelengths in any spectral range emitted by the energy source 1720. In another embodiment, the sample element 1730 is configured to receive an optical power of more than about 1.0 MW/cm ² from the sample beam (Es) incident thereon for any electromagnetic radiation wavelength transmitted through the filter 1725. Preferably, the sample chamber 903 of the sample element 1730 is configured to allow a sample beam (Es) advancing toward the material sample S within a cone angle of 45 degrees from the major axis X (see FIGURE 17) to pass therethrough.

[0246] In the embodiment illustrated in FIGURES 18-19, the sample element further comprises a supply passage 1804 extending from the sample chamber 903 to a supply opening 1806 and a vent passage 1808 extending from the sample chamber 903 to a vent opening 1810. While the vent and supply openings 1806, 1810 are shown at one end of the sample element 1730, in other embodiments the openings may be positioned on other sides of the sample element 1730, so long as it is in fluid communication with the passages 1804 and 1808, respectively.

[0247] In operation, the supply opening 1806 of the sample element 1730 is placed in contact with the material sample S, such as a fluid flowing from a patient. The fluid is then transported through the sample supply passage 1804 and into the sample chamber 903 via an external pump or by capillary action.

[0248] Where the upper and lower chamber walls 1802c, 1802d comprise windows, the distance T (measured along an axis substantially orthogonal to the sample chamber 903 and/or windows 1802a, 1802b, or, alternatively, measured along an axis of an energy beam (such as but not limited to the energy beam E discussed above) passed through the sample chamber 903) between them comprises an optical pathlength. In various embodiments, the pathlength is between about 1 μm and about 300 μm, between about 1 μm and about 100 μm, between about 25 μm and about 40μm, between about 10 μm and about 40 μm, between about 25 μm and about 60 μm, or between about 30 μm and about 50 μm. In still other embodiments, the optical pathlength is about 50 μm, or about 25 μm. In some instances, it is desirable to hold the pathlength T to within about plus or minus 1 μm from any pathlength specified by the analyte detection system with which the sample element 1730 is to be employed. Likewise, it may be desirable to orient the walls 1802c, 1802d with respect to each other within plus or minus 1 μm of parallel, and/or to maintain each of the walls 1802c, 1802d to within plus or minus 1 μm of planar (flat), depending on the analyte detection system with which the sample element 1730 is to be used. In alternative embodiments, walls 1802c, 1802d are flat, textured, angled, or some combination thereof.

[0249] In one embodiment, the transverse size of the sample chamber 903 (i.e., the size defined by the lateral chamber walls 1802a, 1802b) is about equal to the size of the active surface of the sample detector 1745. Accordingly, in a further embodiment the sample chamber 903 is round with a diameter of about 4 millimeter to about 12 millimeter, and more preferably from about 6 millimeter to about 8 millimeter.

[0250] The sample element 1730 shown in FIGURES 18-19 has, in one embodiment, sizes and dimensions specified as follows. The supply passage 1804 preferably has a length of about 15 millimeter, a width of about 1.0 millimeter, and a height equal to the pathlength T. Additionally, the supply opening 1806 is preferably about 1.5 millimeter wide and smoothly

transitions to the width of the sample supply passage 1804. The sample element 1730 is about 0.5 inches (12 millimeters) wide and about one inch (25 millimeters) long with an overall thickness of between about 1.0 millimeter and about 4.0 millimeter. The vent passage 1808 preferably has a length of about 1.0 millimeter to 5.0 millimeter and a width of about 1.0 millimeter, with a thickness substantially equal to the pathlength between the walls 1802c, 1802d. The vent aperture 1810 is of substantially the same height and width as the vent passage 1808. Of course, other dimensions may be employed in other embodiments while still achieving the advantages of the sample element 1730.

[0251] The sample element 1730 is preferably sized to receive a material sample S having a volume less than or equal to about 15 μL (or less than or equal to about 10 μL, or less than or equal to about 5 μL) and more preferably a material sample S having a volume less than or equal to about 2 μL. Of course, the volume of the sample element 1730, the volume of the sample chamber 903, etc. can vary, depending on many variables, such as the size and sensitivity of the sample detector 1745, the intensity of the radiation emitted by the energy source 1720, the expected flow properties of the sample, and whether flow enhancers are incorporated into the sample element 1730. The transport of fluid to the sample chamber 903 is achieved preferably through capillary action, but may also be achieved through wicking or vacuum action, or a combination of wicking, capillary action, peristaltic, pumping, and/or vacuum action.

[0252] FIGURE 20 depicts one approach to constructing the sample element 1730. In this approach, the sample element 1730 comprises a first layer 1820, a second layer 1830, and a third layer 1840. The second layer 1830 is preferably positioned between the first layer 1820 and the third layer 1840. The first layer 1820 forms the upper chamber wall 1802c, and the third layer 1840 forms the lower chamber wall 1802d. Where either of the chamber walls 1802c, 1802d comprises a window, the window(s)/wall(s) 1802c/1802d in question may be formed from a different material as is employed to form the balance of the layer(s) 1820/1840 in which the wall(s) are located. Alternatively, the entirety of the layer(s) 1820/1840 may be formed of the material selected to form the window(s)/wall(s) 1802c, 1802d. In this case, the window(s)/wall(s) 1802c, 1802d are integrally formed with the layer(s) 1820, 1840 and simply comprise the regions of the respective layer(s) 1820, 1840 which overlie the sample chamber 903.

[0253] With further reference to FIGURE 20, second layer 1830 may be formed entirely of an adhesive that joins the first and third layers 1820, 1840. In other embodiments, the second layer 1830 may be formed from similar materials as the first and third layers, or any other suitable material. The second layer 1830 may also be formed as a carrier with an adhesive deposited on both sides thereof. The second layer 1830 includes voids which at least partially form the sample chamber 903, sample supply passage 1804, supply opening 1806, vent passage 1808, and vent opening 1810. The thickness of the second layer 1830 can be the same as any of the pathlengths disclosed above as suitable for the sample element 1730. The first and third layers can be formed from any of the materials disclosed above as suitable for forming the window(s) of the sample element 1730. In one embodiment, layers 1820, 1840 are formed from material having sufficient structural integrity to maintain its shape when filled with a sample S. Layers 1820, 1830 maybe, for example, calcium fluoride having a thickness of 0.5 millimeter. In another embodiment, the second layer 1830 comprises the adhesive portion of Adhesive Transfer Tape no. 947 ILE available from 3M Corporation. In another embodiment, the second layer 1830 comprises an epoxy, available, for example, from TechFilm (31 Dunham Road, Billerica, MA 01821), that is bound to layers 1820, 1840 as a result of the application of pressure and heat to the layers.

[0254] The sample chamber 903 preferably comprises a reagentless chamber. In other words, the internal volume of the sample chamber 903 and/or the wall(s) 1802 defining the chamber 903 are preferably inert with respect to the sample to be drawn into the chamber for analysis. As used herein, "inert" is a broad term and is used in its ordinary sense and includes, without limitation, substances which will not react with the sample in a manner which will significantly affect any measurement made of the concentration of analyte(s) in the sample with sample analysis apparatus 322 or any other suitable system, for a sufficient time (e.g., about 1-30 minutes) following entry of the sample into the chamber 903, to permit measurement of the concentration of such analyte(s). Alternatively, the sample chamber 903 may contain one or more reagents to facilitate use of the sample element in sample assay techniques which involve reaction of the sample with a reagent.

[0255] In one embodiment, sample element 1730 is used for a limited number of measurements and is disposable. Thus, for example, with reference to FIGURES 8-10, sample

element 1730 forms a disposable portion of cassette 820 adapted to place sample chamber 903 within probe region 1002.

[0256] Additional information on sample elements, methods of use thereof, and related technologies may be found in the above-mentioned and incorporated U.S. Patent Application Publication No. 2005/0038357, published on February 17, 2005, titled SAMPLE ELEMENT WITH BARRIER MATERIAL; and in the above-mentioned and incorporated U.S. Patent Application No. 11/122,794, filed on May 5, 2005, titled SAMPLE ELEMENT WITH SEPARATOR.

SECTION IV.D - CENTRIFUGE

[0257] FIGURE 21 is a schematic of one embodiment of a sample preparation unit 2100 utilizing a centrifuge and which may be generally similar to the sample preparation unit 332, except as further detailed below. In general, the sample preparation unit 332 includes a centrifuge in place of, or in addition to a filter, such as the filter 1500. Sample preparation unit 2100 includes a fluid handling element in the form of a centrifuge 2110 having a sample element 2112 and a fluid interface 2120. Sample element 2112 is illustrated in FIGURE 21 as a somewhat cylindrical element. This embodiment is illustrative, and the sample element may be cylindrical, planar, or any other shape or configuration that is compatible with the function of holding a material (preferably a liquid) in the centrifuge 2110. The centrifuge 2110 can be used to rotate the sample element 2112 such that the material held in the sample element 2112 is separated.

[0258] In some embodiments, the fluid interface 2120 selectively controls the transfer of a sample from the passageway 113 and into the sample element 2112 to permit centrifuging of the sample. In another embodiment, the fluid interface 2120 also permits a fluid to flow though the sample element 2112 to cleanse or otherwise prepare the sample element for obtaining an analyte measurement. Thus, the fluid interface 2120 can be used to flush and fill the sample element 2112.

[0259] As shown in FIGURE 21, the centrifuge 2110 comprises a rotor 2111 that includes the sample element 2112 and an axle 2113 attached to a motor, not shown, which is controlled by the controller 210. The sample element 2112 is preferably generally similar to the sample

element 1730 except as described subsequently.

[0260] As is further shown in FIGURE 21, fluid interface 2120 includes a fluid injection probe 2121 having a first needle 2122 and a fluid removal probe 2123. The fluid removal probe 2123 has a second needle 2124. When sample element 2112 is properly oriented relative to fluid interface 2120, a sample, fluid, or other liquid is dispensed into or passes through the sample element 2112. More specifically, fluid injection probe 2121 includes a passageway to receive a sample, such as a bodily fluid from the patient connector 110. The bodily fluid can be passed through the fluid injection probe 2121 and the first needle 2122 into the sample element 2112. To remove material from the sample element 2112, the sample 2112 can be aligned with the second needle 2124, as illustrated. Material can be passed through the second needle 2124 into the fluid removal probe 2123. The material can then pass through a passageway of the removal probe 2123 away from the sample element 2112.

[0261] One position that the sample element 2112 may be rotated through or to is a sample measurement location 2140. The location 2140 may coincide with a region of an analysis system, such as an optical analyte detection system. For example, the location 2140 may coincide with a probe region 1002, or with a measurement location of another apparatus.

[0262] The rotor 2111 may be driven in a direction indicated by arrow R, resulting in a centrifugal force on sample(s) within sample element 2112. The rotation of a sample(s) located a distance from the center of rotation creates centrifugal force. In some embodiments, the sample element 2112 holds whole blood. The centrifugal force may cause the denser parts of the whole blood sample to move further out from the center of rotation than lighter parts of the blood sample. As such, one or more components of the whole blood can be separated from each other. Other fluids or samples can also be removed by centrifugal forces. In one embodiment, the sample element 2112 is a disposable container that is mounted on to a disposable rotor 2111. Preferably, the container is plastic, reusable and flushable. In other embodiments, the sample element 2112 is a non-disposable container that is permanently attached to the rotor 2111.

[0263] The illustrated rotor 2111 is a generally circular plate that is fixedly coupled to the axle 2113. The rotor 2111 can alternatively have other shapes. The rotor 2111 preferably comprises a material that has a low density to keep the rotational inertia low and that is

sufficiently strong and stable to maintain shape under operating loads to maintain close optical alignment. For example, the rotor 2111 can be comprised of GE brand ULTEM (trademark) polyetherimide (PEI). This material is available in a plate form that is stable but can be readily machined. Other materials having similar properties can also be used.

[0264] The size of the rotor 2111 can be selected to achieve the desired centrifugal force. In some embodiments, the diameter of rotor 2111 is from about 75 millimeters to about 125 millimeters, or more preferably from about 100 millimeters to about 125 millimeters. The thickness of rotor 2111 is preferably just thick enough to support the centrifugal forces and can be, for example, from about 1.0 to 2.0 millimeter thick.

[0265] In an alternative embodiment, the fluid interface 2120 selectively removes blood plasma from the sample element 2112 after centrifuging. The blood plasma is then delivered to an analyte detection system for analysis. In one embodiment, the separated fluids are removed from the sample element 2112 through the bottom connector. Preferably, the location and orientation of the bottom connector and the container allow the red blood cells to be removed first. One embodiment may be configured with a red blood cell detector. The red blood cell detector may detect when most of the red blood cells have exited the container by determining the haemostatic level. The plasma remaining in the container may then be diverted into the analysis chamber. After the fluids have been removed from the container, the top connector may inject fluid (e.g., saline) into the container to flush the system and prepare it for the next sample.

[0266] FIGURES 22A and 23B illustrate another embodiment of a fluid handling and analysis apparatus 140 which employs a removable, disposable fluid handling cassette 820. The cassette 820 is equipped with a centrifuge rotor assembly 2016 to facilitate preparation and analysis of a sample. Except as further described below, the apparatus 140 of FIGURES 22A- 22C can in certain embodiments be similar to any of the other embodiments of the apparatus 140 discussed herein, and the cassette 820 can in certain embodiments be similar to any of the embodiments of the cassettes 820 disclosed herein.

[0267] The removable fluid handling cassette 820 can be removably engaged with a main analysis instrument 810. When the fluid handling cassette 820 is coupled to the main instalment 810, a drive system 2030 of the main instrument 810 mates with the rotor assembly 2016 of the

cassette 820 (FIGURE 22B). Once the cassette 820 is coupled to the main instrument 810, the drive system 2030 engages and can rotate the rotor assembly 2016 to apply a centrifugal force to a body fluid sample carried by the rotor assembly 2016.

[0268] In some embodiments, the rotor assembly 2016 includes a sample element 2448 (FIGURE 22C) for holding a sample for centrifuging. When the rotor 2020 is rotated, a centrifugal force is applied to the sample contained within the sample element 2248. The centrifugal force causes separation of one or more components of the sample (e.g., separation of plasma from whole blood). The separated component(s) can then be analyzed by the apparatus 140, as will be discussed in further detail below.

[0269] The main instrument 810 includes both the centrifuge drive system 2030 and an analyte detection system 1700, a portion of which protrudes from a housing 2049 of the main instrument 810. The drive system 2030 is configured to releasably couple with the rotor assembly 2016, and can impart rotary motion to the rotor assembly 2016 to rotate the rotor 2020 at a desired speed. After the centrifuging process, the analyte detection system 1700 can analyze one or more components separated from the sample carried by the rotor 2020. The projecting portion of the illustrated detection system 1700 forms a slot 2074 for receiving a portion of the rotor 2020 carrying the sample element 2448 so that the detection system 1700 can analyze the sample or component(s) carried in the sample element 2448.

[0270] To assemble the fluid handling and analysis apparatus 140, the cassette 820 is placed on the main instrument 810, as indicated by the arrow 2007 of FIGURES 22A and 22B. The rotor assembly 2016 is accessible to the drive system 2030, so that once the cassette 820 is properly mounted on the main instrument 810, the drive system 2030 is in operative engagement with the rotor assembly 2016. The drive system 2030 is energized to spin the rotor 2020 at a desired speed. The spinning rotor 2020 can pass repeatedly through the slot 2074 of the detection system 1700.

[0271] After the centrifuging process, the rotor 2020 is rotated to an analysis position (see FIGURES 22B and 23C) wherein the sample element 2448 is positioned within the slot 2074. With the rotor 2020 and sample element 2448 in the analysis position, the analyte detection system 1700 can analyze one or more of the components of the sample carried in the sample

element 2448. For example, the detection system 1700 can analyze at least one of the components that is separated out during the centrifuging process. After using the cassette 820, the cassette 820 can be removed from the main instrument 810 and discarded. Another cassette 820 can then be mounted to the main instrument 810.

[0272] With reference to FIGURE 23A, the illustrated cassette 820 includes the housing 2400 that surrounds the rotor assembly 2016, and the rotor 2020 is pivotally connected to the housing 2400 by the rotor assembly 2016. The rotor 2020 includes a rotor interface 2051 for driving engagement with the drive system 2030 upon placement of the cassette 820 on the main instrument 810.

[0273] In some embodiments, the cassette 820 is a disposable fluid handling cassette. The reusable main instrument 810 can be used with any number of cassettes 820 as desired. Additionally or alternatively, the cassette 820 can be a portable, handheld cassette for convenient transport. In these embodiments, the cassette 820 can be manually mounted to or removed from the main instrument 810. In some embodiments, the cassette 820 may be a non disposable cassette which can be permanently coupled to the main instrument 810.

[0274] FIGURES 25A and 25B illustrate the centrifugal rotor 2020 which is capable of carrying a sample, such as bodily fluid. Thus, the illustrated centrifugal rotor 2020 can be considered a fluid handling element that can prepare a sample for analysis, as well as hold the sample during a spectroscopic analysis. The rotor 2020 preferably comprises an elongate body 2446, at least one sample element 2448, and at least one bypass element 2452. The sample element 2448 and bypass element 2452 can be located at opposing ends of the rotor 2020. The bypass element 2452 provides a bypass flow path that can be used to clean or flush fluid passageways of the fluid handling and analysis apparatus 140 without passing fluid through the sample element 2448.

[0275] The illustrated rotor body 2446 can be a generally planar member that defines a mounting aperture 2447 for coupling to the drive system 2030. The illustrated rotor 2020 has a somewhat rectangular shape. In alternative embodiments, the rotor 2020 is generally circular, polygonal, elliptical, or can have any other shape as desired. The illustrated shape can facilitates loading when positioned horizontally to accommodate the analyte detection system 1700.

[0276] A pair of opposing first and second fluid connectors 2027, 2029 extends outwardly from a front face of the rotor 2020, to facilitate fluid flow through the rotor body 2446 to the sample element 2448 and bypass element 2452, respectively. The first fluid connector 2027 defines an outlet port 2472 and an inlet port 2474 that are in fluid communication with the sample element 2448. In the illustrated embodiment, fluid channels 2510, 2512 extend from the outlet port 2472 and inlet port 2474, respectively, to the sample element 2448. (See FIGURES 25E and 25F.) As such, the ports 2472, 2474 and channels 2510, 2512 define input and return flow paths through the rotor 2020 to the sample element 2448 and back.

[0277] With continued reference to FIGURE 25B, the rotor 2020 includes the bypass element 2452 which permits fluid flow therethrough from an outlet port 2572 to the inlet port 2574. A channel 2570 extends between the outlet port 2572 and the inlet port 2574 to facilitate this fluid flow. The channel 2570 thus defines a closed flow path through the rotor 2020 from one port 2572 to the other port 2574. In the illustrated embodiment, the outlet port 2572 and inlet port 2574 of the bypass element 2452 have generally the same spacing therebetween on the rotor 2020 as the outlet port 2472 and the inlet port 2474.

[0278] One or more windows can be provided for optical access through the rotor 2020. The window 2460a proximate the bypass element 2452 can be a through-hole (see FIGURE 25E) that permits the passage of electromagnetic radiation through the rotor 2020. The window 2460b proximate the sample element 2448 can also be a similar through-hole which permits the passage of electromagnetic radiation. Alternatively, one or both of the windows 2460a, 2460b can be a sheet constructed of calcium fluoride, barium fluoride, germanium, silicon, polypropylene, polyethylene, combinations thereof, or any material with suitable transmissivity (i.e., transmittance per unit thickness) in the relevant wavelength(s). The windows 2460a, 2460b are positioned so that one of the windows 2460a, 2460b is positioned in the slot 2074 when the rotor 2020 is in a vertically orientated position.

[0279] Various fabrication techniques can be used to form the rotor 2020. In some embodiments, the rotor 2020 can be formed by molding (e.g., compression or injection molding), machining, or a similar production process or combination of production processes. In some embodiments, the rotor 2020 is comprised of plastic. The compliance of the plastic material can

be selected to create the seal with the ends of pins 2542, 2544 of a fluid interface 2028 (discussed in further detail below). Non-limiting exemplary plastics for forming the ports (e.g., ports 2572, 2574, 2472, 2474) can be relatively chemically inert and can be injection molded or machined. These plastics include, but are not limited to, PEEK and polyphenylenesulfide (PPS). Although both of these plastics have high modulus, a fluidic seal can be made if sealing surfaces are produced with smooth finish and the sealing zone is a small area where high contact pressure is created in a very small zone. Accordingly, the materials used to form the rotor 2020 and pins 2542, 2544 can be selected to achieve the desired interaction between the rotor 2020 and the pins 2542, 2544, as described in detail below.

[0280] The illustrated rotor assembly 2016 of FIGURE 23A rotatably connects the rotor 2020 to the cassette housing 2400 via a rotor axle boss 2426 which is fixed with respect to the cassette housing and pivotally holds a rotor axle 2430 and the rotor 2020 attached thereto. The rotor axle 2430 extends outwardly from the rotor axle boss 2426 and is fixedly attached to a rotor bracket 2436 which is preferably securely coupled to a rear face of the rotor 2020. Accordingly, the rotor assembly 2016 and the drive system 2030 cooperate to ensure that the rotor 2020 rotates about the axis 2024, even at high speeds. The illustrated cassette 820 has a single rotor assembly 2016. In other embodiments, the cassette 820 can have more than one rotor assembly 2016. Multiple rotor assemblies 2016 can be used to prepare (preferably simultaneously) and test multiple samples.

[0281] With reference again to FIGURES 25A, 25B, 25E and 25F, the sample element 2448 is coupled to the rotor 2020 and can hold a sample of body fluid for processing with the centrifuge. The sample element 2448 can, in certain embodiments, be generally similar to other sample elements or cuvettes disclosed herein (e.g., sample elements 1730, 2112) except as further detailed below.

[0282] The sample element 2448 comprises a sample chamber 2464 that holds a sample for centrifuging, and fluid channels 2466, 2468 which provide fluid communication between the chamber 2464 and the channels 2512, 2510, respectively, of the rotor 2020. Thus, the fluid channels 2512, 2466 define a first flow path between the port 2474 and the chamber 2464, and the channels 2510, 2468 define a second flow path between the port 2472 and the chamber 2464.

Depending on the direction of fluid flow into the sample element 2448, either of the first and second flow paths can serve as an input flow path, and the other can serve as a return flow path.

[0283] A portion of the sample chamber 2464 can be considered an interrogation region 2091, which is the portion of the sample chamber through which electromagnetic radiation passes during analysis by the detection system 1700 of fluid contained in the chamber 2464. Accordingly, the interrogation region 2091 is aligned with the throughhole 2460b when the sample element 2448 is coupled to the rotor 2020. The illustrated interrogation region 2091 comprises a radially inward portion (i.e., relatively close to the axis of rotation 2024 of the rotor 2020) of the chamber 2464, to facilitate spectroscopic analysis of the lower density portion(s) of the body fluid sample (e.g., the plasma of a whole blood sample) after centrifuging, as will be discussed in greater detail below. Where the higher-density portions of the body fluid sample are of interest for spectroscopic analysis, the interrogation region 2091 can be located in a radially outward (i.e., further from the axis of rotation 2024 of the rotor 2020) portion of the chamber 2464.

[0284] The rotor 2020 can temporarily or permanently hold the sample element 2448. As shown in FIGURE 25F, the rotor 2020 forms a recess 2502 which receives the sample element 2448. The sample element 2448 can be held in the recess 2502 by factional interaction, adhesives, or any other suitable coupling means. The illustrated sample element 2448 is recessed in the rotor 2020. However, the sample element 2448 can alternatively overlie or protrude from the rotor 2020.

[0285] The sample element 2448 can be used for a predetermined length of time, to prepare a predetermined amount of sample fluid, to perform a number of analyses, etc. If desired, the sample element 2448 can be removed from the rotor 2020 and then discarded. Another sample element 2448 can then be placed into the recess 2502. Thus, even if the cassette 820 is disposable, a plurality of disposable sample elements 2448 can be used with a single cassette 820. Accordingly, a single cassette 820 can be used with any number of sample elements as desired. Alternatively, the cassette 820 can have a sample element 2448 that is permanently coupled to the rotor 2020. In some embodiments, at least a portion of the sample element 2448 is integrally or monolithically formed with the rotor body 2446. Additionally or alternatively,

the rotor 2020 can comprise a plurality of sample elements (e.g., with a record sample element in place of the bypass 2452). In this embodiment, a plurality of samples (e.g., bodily fluid) can be prepared simultaneously to reduce sample preparation time.

[0286] FIGURES 26A and 26B illustrate a layered construction technique which can be employed when forming certain embodiments of the sample element 2448. The depicted layered sample element 2448 comprises a first layer 2473, a second layer 2475, and a third layer 2478. The second layer 2475 is preferably positioned between the first layer 2473 and the third layer 2478. The first layer 2473 forms an upper chamber wall 2482, and the third layer 2478 forms a lower chamber wall 2484. A lateral wall 2490 of the second layer 2475 defines the sides of the chamber 2464 and the fluid channels 2466, 2468.

[0287] The second layer 2475 can be formed by die-cutting a substantially uniform-thickness sheet of a material to form the lateral wall pattern shown in FIGURE 26A. The second layer 2475 can comprise a layer of lightweight flexible material, such as a polymer material, with adhesive disposed on either side thereof to adhere the first and third layers 2473, 2478 to the second layer 2475 in "sandwich" fashion as shown in FIGURE 26B. Alternatively, the second layer 2475 can comprise an "adhesive-only" layer formed from a uniform-thickness sheet of adhesive which has been die-cut to form the depicted lateral wall pattern.

[0288] However constructed, the second layer 2475 is preferably of uniform thickness to define a substantially uniform thickness or path length of the sample chamber 2464 and/or interrogation region 2091. This path length (and therefore the thickness of the second layer 2475 as well) is preferably between 10 microns and 100 microns, or is 20, 40, 50, 60, or 80 microns, in various embodiments.

[0289] The upper chamber wall 2482, lower chamber wall 2484, and lateral wall 2490 cooperate to form the chamber 2464. The upper chamber wall 2482 and/or the lower chamber wall 2484 can permit the passage of electromagnetic energy therethrough. Accordingly, one or both of the first and third layers 2473, 2478 comprises a sheet or layer of material which is relatively or highly transmissive of electromagnetic radiation (preferably infrared radiation or mid-infrared radiation) such as barium fluoride, silicon, polyethylene or polypropylene. If only one of the layers 2473, 2478 is so transmissive, the other of the layers is preferably reflective, to

back-reflect the incoming radiation beam for detection on the same side of the sample element 2448 as it was emitted. Thus the upper chamber wall 2482 and/or lower chamber wall 2484 can be considered optical window(s). These window(s) are disposed on one or both sides of the interrogation region 2091 of the sample element 2448.

[0290] In one embodiment, sample element 2448 has opposing sides that are transmissive of infrared radiation and suitable for making optical measurements as described, for example, in U.S. Patent Application Publication No. 2005/0036146, published February 17, 2005, titled SAMPLE ELEMENT QUALIFICATION, and hereby incorporated by reference and made a part of this specification. Except as further described herein, the embodiments, features, systems, devices, materials, methods and techniques described herein may, in some embodiments, be similar to any one or more of the embodiments, features, systems, devices, materials, methods and techniques described in U.S. Patent Application Publication No. 2003/0090649, published on May 15, 2003, titled REAGENT-LESS WHOLE-BLOOD GLUCOSE METER; or in U.S. Patent Application Publication No. 2003/0086075, published on May 8, 2003, titled DEVICE AND METHOD FOR IN VITRO DETERMINATION OF ANALYTE CONCENTRATIONS WITHIN BODY FLUIDS; or in U.S. Patent Application Publication No. 2004/0019431, published on January 29, 2004, titled METHOD OF DETERMINING AN ANALYTE CONCENTRATION IN A SAMPLE FROM AN ABSORPTION SPECTRUM, or in U.S. Patent No. 6,652,136, issued on November 25, 2003 to Marziali, titled METHOD OF SIMULTANEOUS MIXING OF SAMPLES. In addition, the embodiments, features, systems, devices, materials, methods and techniques described herein may, in certain embodiments, be applied to or used in connection with any one or more of the embodiments, features, systems, devices, materials, methods and techniques disclosed in the above-mentioned U.S. Patent Applications Publications Nos. 2003/0090649; 2003/0086075; 2004/0019431; or U.S. Patent No. 6,652,136. All of the above-mentioned publications and patent are hereby incorporated by reference herein and made a part of this specification.

[0291] With reference to FIGURES 23B and 23C, the cassette 820 can further comprise the movable fluid interface 2028 for filling and/or removing sample liquid from the sample element 2448. In the depicted embodiment, the fluid interface 2028 is rotatably mounted to the housing 2400 of the cassette 820. The fluid interface 2028 can be actuated between a lowered position

(FIGURE 22C) and a raised or filling position (FIGURE 27C). When the interface 2028 is in the lowered position, the rotor 2020 can freely rotate. To transfer sample fluid to the sample element 2448, the rotor 2020 can be held stationary and in a sample element loading position (see FIGURE 22C) the fluid interface 2028 can be actuated, as indicated by the arrow 2590, upwardly to the filling position. When the fluid interface 2028 is in the filling position, the fluid interface 2028 can deliver sample fluid into the sample element 2448 and/or remove sample fluid from the sample element 2448.

[0292] With continued reference to FIGURES 27A and 27B, the fluid interface 2028 has a main body 2580 that is rotatably mounted to the housing 2400 of the cassette 820. Opposing brackets 2581, 2584 can be employed to rotatably couple the main body 2580 to the housing 2400 of the cassette 820, and permit rotation of the main body 2580 and the pins 2542, 2544 about an axis of rotation 2590 between the lowered position and the filling position. The main instrument 810 can include a horizontally moveable actuator (not shown) in the form of a solenoid, pneumatic actuator, etc. which is extendible through an opening 2404 in the cassette housing 2400 (see FIG. 23B). Upon extension, the actuator strikes the main body 2580 of the fluid interface 2028, causing the body 2580 to rotate to the filling position shown in FIGURE 27C. The main body 2580 is preferably spring-biased towards the retracted position (shown in FIGURE 23 A) so that retraction of the actuator allows the main body to return to the retracted position. The fluid interface 2028 can thus be actuated for periodically placing fluid passageways of the pins 2542, 2544 in fluid communication with a sample element 2448 located on the rotor 2020.

[0293] The fluid interface 2028 of FIGURES 27A and 23B includes fluid connectors 2530, 2532 that can provide fluid communication between the interface 2028 and one or more of the fluid passageways of the apparatus 140 and/or sampling system 100/800, as will be discussed in further detail below. The illustrated connectors 2530, 2532 are in an upwardly extending orientation and positioned at opposing ends of the main body 2580. The connectors 2530, 2532 can be situated in other orientations and/or positioned at other locations along the main body 2580. The main body 2580 includes a first inner passageway (not shown) which provides fluid communication between the connector 2530 and the pin 2542, and a second inner passageway (not shown) which provides fluid communication between the connector 2532 and the pin 2544.

[0294] The fluid pins 2542, 2544 extend outwardly from the main body 2580 and can engage the rotor 2020 to deliver and/or remove sample fluid to or from the rotor 2020. The fluid pins 2542, 2544 have respective pin bodies 2561, 2563 and pin ends 2571, 2573. The pin ends 2571,

2573 are sized to fit within corresponding ports 2472, 2474 of the fluid connector 2027 and/or the ports 2572, 2574 of the fluid connector 2029, of the rotor 2020. The pin ends 2571, 2573 can be slightly chamfered at their tips to enhance the sealing between the pin ends 2571, 2573 and rotor ports. In some embodiments, the outer diameters of pin ends 2573, 2571 are slightly larger than the inner diameters of the ports of the rotor 2020 to ensure a tight seal, and the inner diameters of the pins 2542, 2544 are preferably identical or very close to the inner diameters of the channels 2510, 2512 leading from the ports. In other embodiments, the outer diameter of the pin ends 2573, 2571 are equal to or less than the inner diameters of the ports of the rotor 2020.

[0295] The connections between the pins 2542, 2544 and the corresponding portions of the rotor 2020, either the ports 2472, 2474 leading to the sample element 2448 or the ports 2572,

2574 leading to the bypass element 2452, can be relatively simple and inexpensive. At least a portion of the rotor 2020 can be somewhat compliant to help ensure a seal is formed with the pins 2542, 2544. Alternatively or additionally, sealing members (e.g., gaskets, O-rings, and the like) can be used to inhibit leaking between the pin ends 2571, 2573 and corresponding ports 2472, 2474, 2572, 2574.

[0296] FIGURES 23A and 23B illustrate the cassette housing 2400 enclosing the rotor assembly 2016 and the fluid interface 2028. The housing 2400 can be a modular body that defines an aperture or opening 2404 dimensioned to receive a drive system housing 2050 when the cassette 820 is operatively coupled to the main instrument 810. The housing 2400 can protect the rotor 2020 from external forces and can also limit contamination of samples delivered to a sample element in the rotor 2020, when the cassette 820 is mounted to the main instrument 810.

[0297] The illustrated cassette 820 has a pair of opposing side walls 2041, 2043, top 2053, and a notch 2408 for mating with the detection system 1700. A front wall 2045 and rear wall 2047 extend between the side walls 2041, 2043. The rotor assembly 2016 is mounted to the inner surface of the rear wall 2047. The front wall 2045 is configured to mate with the main

instrument 810 while providing the drive system 2030 with access to the rotor assembly 2016.

[0298] The illustrated front wall 2045 has the opening 2404 that provides access to the rotor assembly 2016. The drive system 2030 can be passed through the opening 2404 into the interior of the cassette 820 until it operatively engages the rotor assembly 2016. The opening 2404 of FIGURE 23B is configured to mate and tightly surround the drive system 2030. The illustrated opening 2404 is generally circular and includes an upper notch 2405 to permit the fluid interface actuator of the main instrument 810 to access the fluid interface 2028, as discussed above. The opening 2404 can have other configurations suitable for admitting the drive system 2030 and actuator into the cassette 820.

[0299] The notch 2408 of the housing 2400 can at least partially surround the projecting portion of the analyte detection system 1700 when the cassette 820 is loaded onto the main instrument 810. The illustrated notch 2408 defines a cassette slot 2410 (FIGURE 23A) that is aligned with elongate slot 2074 shown in FIGURE 22C, upon loading of the cassette 820. The rotating rotor 2020 can thus pass through the aligned slots 2410, 2074. In some embodiments, the notch 2408 has a generally U-shaped axial cross section as shown. More generally, the configuration of the notch 2408 can be selected based on the design of the projecting portion of the detection system 1700.

[0300] Although not illustrated, fasteners, clips, mechanical fastening assemblies, snaps, or other coupling means can be used to ensure that the cassette 820 remains coupled to the main instrument 810 during operation. Alternatively, the interaction between the housing 2400 and the components of the main instrument 810 can secure the cassette 820 to the main instrument 810.

[0301] FIGURE 28 is a cross-sectional view of the main instrument 810. The illustrated centrifuge drive system 2030 extends outwardly from a front face 2046 of the main instrument 810 so that it can be easily mated with the rotor assembly 2016 of the cassette 820. When the centrifuge drive system 2030 is energized, the drive system 2030 can rotate the rotor 2020 at a desired rotational speed.

[0302] The illustrated centrifuge drive system 2030 of FIGURES 23E and 28 includes a

centrifuge drive motor 2038 and a drive spindle 2034 that is drivingly connected to the drive motor 2038. The drive spindle 2034 extends outwardly from the drive motor 2038 and forms a centrifuge interface 2042. The centrifuge interface 2042 extends outwardly from the drive system housing 2050, which houses the drive motor 2038. To impart rotary motion to the rotor 2020, the centrifuge interface 2042 can have keying members, protrusions, notches, detents, recesses, pins, or other types of structures that can engage the rotor 2020 such that drive spindle 2034 and the rotor 2020 are coupled together.

[0303] The centrifuge drive motor 2038 of FIGURE 28 can be any suitable motor that can impart rotary motion to the rotor 2020. When the drive motor 2038 is energized, the drive motor 2038 can rotate the drive spindle 2034 at constant or varying speeds. Various types of motors, including, but not limited to, centrifuge motors, stepper motors, spindle motors, electric motors, or any other type of motor for outputting a torque can be utilized. The centrifuge drive motor 2038 is preferably fixedly secured to the drive system housing 2050 of the main instrument 810.

[0304] The drive motor 2038 can be the type of motor typically used in personal computer hard drives that is capable of rotating at about 7,200 RPM on precision bearings, such as a motor of a Seagate Model ST380011A hard drive (Seagate Technology, Scotts Valley, CA) or similar motor. In one embodiment, the drive spindle 2034 may be rotated at 6,000 rpm, which yields approximately 2,000 G's for a rotor having a 2.5 inch (64 millimeter) radius. In another embodiment, the drive spindle 2034 may be rotated at speeds of approximately 7,200 rpm. The rotational speed of the drive spindle 2034 can be selected to achieve the desired centrifugal force applied to a sample carried by the rotor 2020.

[0305] The main instrument 810 includes a main housing 2049 that defines a chamber sized to accommodate a filter wheel assembly 2300 including a filter drive motor 2320 and filter wheel 2310 of the analyte detection system 1700. The main housing 2049 defines a detection system opening 3001 configured to receive an analyte detection system housing 2070. The illustrated analyte detection system housing 2070 extends or projects outwardly from the housing 2049.

[0306] The main instrument 810 of FIGURES 23C and 23E includes a bubble sensor unit 321, a pump 2619 in the form of a peristaltic pump roller 2620a and a roller support 2620b, and valves 323a, 323b. The illustrated valves 323a, 323b are pincher pairs, although other types of

valves can be used. When the cassette 820 is installed, these components can engage components of a fluid handling network 2600 of the cassette 820, as will be discussed in greater detail below.

[0307] With continued reference to FIGURE 28, the analyte detection system housing 2070 surrounds and houses some of the internal components of the analyte detection system 1700. The elongate slot 2074 extends downwardly from an upper face 2072 of the housing 2070. The elongated slot 2074 is sized and dimensioned so as to receive a portion of the rotor 2020. When the rotor 2020 rotates, the rotor 2020 passes periodically through the elongated slot 2074. When a sample element of the rotor 2020 is in the detection region 2080 defined by the slot 2074, the analyte detection system 1700 can analyze material in the sample element.

[0308] The analyte detection system 1700 can be a spectroscopic bodily fluid analyzer that preferably comprises an energy source 1720. The energy source 1720 can generate an energy beam directed along a major optical axis X that passes through the slot 2074 towards a sample detector 1745. The slot 2074 thus permits at least a portion of the rotor (e.g., the interrogation region 2091 or sample chamber 2464 of the sample element 2448) to be positioned on the optical axis X. To analyze a sample carried by the sample element 2448, the sample element and sample can be positioned in the detection region 2080 on the optical axis X such that light emitted from the source 1720 passes through the slot 2074 and the sample disposed within the sample element 2448.

[0309] The analyte detection system 1700 can also comprise one or more lenses positioned to transmit energy outputted from the energy source 1720. The illustrated analyte detection system 1700 of FIGURE 28 comprises a first lens 2084 and a second lens 2086. The First lens 2084 is configured to focus the energy from the source 1720 generally onto the sample element and material sample. The second lens 2086 is positioned between the sample element and the sample detector 1745. Energy from energy source 1720 passing through the sample element can subsequently pass through the second lens 2086. A third lens 2090 is preferably positioned between a beam splitter 2093 and a reference detector 2094. The reference detector 2094 is positioned to receive energy from the beam splitter 2093.

[0310] The analyte detection system 1700 can be used to determine the analyte concentration

in the sample carried by the rotor 2020. Other types of detection or analysis systems can be used with the illustrated centrifuge apparatus or sample preparation unit. The fluid handling and analysis apparatus 140 is shown for illustrative purposes as being used in conjunction with the analyte detection system 1700, but neither the sample preparation unit nor analyte detection system are intended to be limited to the illustrated configuration, or to be limited to being used together.

[0311] To assemble the fluid handling and analysis apparatus 140, the cassette 820 can be moved towards and installed onto the main instrument 810, as indicated by the arrow 2007 in FIGURE 22A. As the cassette 820 is installed, the drive system 2030 passes through the aperture 2040 so that the spindle 2034 mates with the rotor 2020. Simultaneously, the projecting portion of the detection system 1700 is received in the notch 2408 of the cassette 820. When the cassette 820 is installed on the main instrument 810, the slot 2410 of the notch 2048 and the slot 2074 of the detection system 1700 are aligned as shown in FIGURE 22C. Accordingly, when the cassette 820 and main instrument 810 are assembled, the rotor 2020 can rotate about the axis 2024 and pass through the slots 2410, 2074.

[0312] After the cassette 820 is assembled with the main instrument 810, a sample can be added to the sample element 2448. The cassette 820 can be connected to an infusion source and a patient to place the system in fluid communication with a bodily fluid to be analyzed. Once the cassette 820 is connected to a patient, a bodily fluid may be drawn from the patient into the cassette 820. The rotor 2020 is rotated to a vertical loading position wherein the sample element 2448 is near the fluid interface 2028 and the bypass element 2452 is positioned within the slot 2074 of the detection system 1700. Once the rotor 2020 is in the vertical loading position, the pins 2542, 2544 of the fluid interface 2028 are positioned to mate with the ports 2472, 2474 of the rotor 2020. The fluid interface 2028 is then rotated upwardly until the ends 2571, 2573 of the pins 2542, 2544 are inserted into the ports 2472, 2474.

[0313] When the fluid interface 2028 and the sample element 2448 are thus engaged, sample fluid (e.g., whole blood) is pumped into the sample element 2448. The sample can flow through the pin 2544 into and through the rotor channel 2512 and the sample element channel 2466, and into the sample chamber 2464. As shown in FIGURE 25C, the sample chamber 2464 can be

partially or completely filled with sample fluid. In some embodiments, the sample fills at least the sample chamber 2464 and the interrogation region 2091 of the sample element 2448. The sample can optionally fill at least a portion of the sample element channels 2466, 2468. The illustrated sample chamber 2464 is filled with whole blood, although the sample chamber 2464 can be filled with other substances. After the sample element 2448 is filled with a desired amount of fluid, the fluid interface 2028 can be moved to a lowered position to permit rotation of the rotor 2020.

[0314] The centrifuge drive system 2030 can then spin the rotor 2020 and associated sample element 2448 as needed to separate one or more components of the sample. The separated component(s) of the sample may collect or be segregated in a section of the sample element for analysis. In the illustrated embodiment, the sample element 2448 of FIGURE 25C is filled with whole blood prior to centrifuging. The centrifugal forces can be applied to the whole blood until plasma 2594 is separated from the blood cells 2592. After centrifuging, the plasma 2594 is preferably located in a radially inward portion of the sample element 2448, including the interrogation region 2091. The blood cells 2592 collect in a portion of the sample chamber 2464 which is radially outward of the plasma 2594 and interrogation region 2091.

[0315] The rotor 2020 can then be moved to a vertical analysis position wherein the sample element 2448 is disposed within the slot 2074 and aligned with the source 1720 and the sample detector 1745 on the major optical axis. When the rotor 2020 is in the analysis position, the interrogation portion 2091 is preferably aligned with the major optical axis X of the detection system 1700. The analyte detection system 1700 can analyze the sample in the sample element 2448 using spectroscopic analysis techniques as discussed elsewhere herein.

[0316] After the sample has been analyzed, the sample can be removed from the sample element 2448. The sample may be transported to a waste receptacle so that the sample element 2448 can be reused for successive sample draws and analyses. The rotor 2020 is rotated from the analysis position back to the vertical loading position. To empty the sample element 2448, the fluid interface 2028 can again engage the sample element 2448 to flush the sample element 2448 with fresh fluid (either a new sample of body fluid, or infusion fluid). The fluid interface 2028 can be rotated to mate the pins 2542, 2544 with the ports 2472, 2474 of the rotor 2020. The fluid

interface 2018 can pump a fluid through one of the pins 2542, 2544 until the sample is flushed from the sample element 2448. Various types of fluids, such as infusion liquid, air, water, and the like, can be used to flush the sample element 2448. After the sample element 2448 has been flushed, the sample element 2448 can once again be filled with another sample.

[0317] In an alternative embodiment, the sample element 2448 may be removed from the rotor 2020 and replaced after each separate analysis, or after a certain number of analyses. Once the patient care has terminated, the fluid passageways or conduits may be disconnected from the patient and the sample cassette 820 which has come into fluid contact with the patient's bodily fluid may be disposed of or sterilized for reuse. The main instrument 810, however, has not come into contact with the patient's bodily fluid at any point during the analysis and therefore can readily be connected to a new fluid handling cassette 820 and used for the analysis of a subsequent patient.

[0318] The rotor 2020 can be used to provide a fluid flow bypass. To facilitate a bypass flow, the rotor 2020 is first rotated to the vertical analysis/bypass position wherein the bypass element 2452 is near the fluid interface 2028 and the sample element 2448 is in the slot 2074 of the aiialyte detection system 1700. Once the rotor 2020 is in the vertical analysis/bypass position, the pins 2542, 2544 can mate with the ports 2572, 2574 of the rotor 2020. In the illustrated embodiment, the fluid interface 2028 is rotated upwardly until the ends 2571, 2573 of the pins 2542, 2544 are inserted into the ports 2572, 2574. The bypass element 2452 can then provide a completed fluid circuit so that fluid can flow through one of the pins 2542, 2544 into the bypass element 2452, through the bypass element 2452, and then through the other pin 2542, 2544. The bypass element 2452 can be utilized in this manner to facilitate the flushing or sterilizing of a fluid system connected to the cassette 820.

[0319] As shown in FIGURE 23B, the cassette 820 preferably includes a fluid handling network 2600 which can be employed to deliver fluid to the sample element 2448 in the rotor 2020 for analysis. The main instrument 810 has a number of components that can, upon installation of the cassette 820 on the main instrument 810, extend through openings in the front face 2045 of cassette 820 to engage and interact with components of the fluid handling network 2600, as detailed below.

[0320] The fluid handling network 2600 of the fluid handling and analysis apparatus 140 includes the passageway 111 which extends from the connector 120 toward and through the cassette 820 until it becomes the passageway 112, which extends from the cassette 820 to the patient connector 110. A portion Ilia of the passageway 111 extends across an opening 2613 in the front face 2045 of the cassette 820. When the cassette 820 is installed on the main instrument 810, the roller pump 2619 engages the portion Ilia, which becomes situated between the impeller 2620a and the impeller support 2620b (see FIGURE 23C).

[0321] The fluid handling network 2600 also includes passageway 113 which extends from the patient connector 110 towards and into the cassette 820. After entering the cassette 820, the passageway 113 extends across an opening 2615 in the front face 2045 to allow engagement of the passageway 113 with a bubble sensor 321 of the main instrument 810, when the cassette 820 is installed on the main instrument 810. The passageway 113 then proceeds to the connector 2532 of the fluid interface 2028, which extends the passageway 113 to the pin 2544. Fluid drawn from the patient into the passageway 113 can thus flow into and through the fluid interface 2028, to the pin 2544. The drawn body fluid can further flow from the pin 2544 and into the sample element 2448, as detailed above.

[0322] A passageway 2609 extends from the connector 2530 of the fluid interface 2028 and is thus in fluid communication with the pin 2542. The passageway 2609 branches to form the waste line 324 and the pump line 327. The waste line 324 passes across an opening 2617 in the front face 2045 and extends to the waste receptacle 325. The pump line 327 passes across an opening 2619 in the front face 2045 and extends to the pump 328. When the cassette 820 is installed on the main instrument 810, the pinch valves 323a, 323b extend through the openings 2617, 2619 to engage the lines 324, 327, respectively.

[0323] The waste receptacle 325 is mounted to the front face 2045. Waste fluid passing from the fluid interface 2028 can flow through the passageways 2609, 324 and into the waste receptacle 325. Once the waste receptacle 325 is filled, the cassette 820 can be removed from the main instrument 810 and discarded. Alternatively, the filled waste receptacle 325 can be replaced with an empty waste receptacle 325.

[0324] The pump 328 can be a displacement pump (e.g., a syringe pump). A piston control

2645 can extend over at least a portion of an opening 2621 in the cassette face 2045 to allow engagement with an actuator 2652 when the cassette 820 is installed on the main instrument 810. When the cassette 820 is installed, the actuator 2652 (FIGURE 23E) of the main instrument 810 engages the piston control 2645 of the pump 328 and can displace the piston control 2645 for a desired fluid flow.

[0325] It will be appreciated that, upon installing the cassette 820 of FIGURE 23A on the main instrument 810 of FIGURE 23E, there is formed (as shown in FIGURE 23E) a fluid circuit similar to that shown in the sampling unit 200 in FIGURE 3. This fluid circuit can be operated in a manner similar to that described above in connection with the apparatus of FIGURE 3 (e.g., in accordance with the methodology illustrated in FIGURES 7A-7J and Table 1).

[0326] FIGURE 24A depicts another embodiment of a fluid handling network 2700 that can be employed in the cassette 820. The fluid handling network 2700 can be generally similar in structure and function to the network 2600 of FIGURE 23B, except as detailed below. The network 2700 includes the passageway 111 which extends from the connector 120 toward and through the cassette 820 until it becomes the passageway 112, which extends from the cassette 820 to the patient connector 110. A portion Ilia of the passageway 111 extends across an opening 2713 in the front face 2745 of the cassette 820. When the cassette 820 is installed on the main instrument 810, a roller pump 2619 of the main instrument 810 of FIGURE 24B can engage the portion Ilia in a manner similar to that described above with respect to FIGURES 23B-23C. The passageway 113 extends from the patient connector 110 towards and into the cassette 820. After entering the cassette 820, the passageway 113 extends across an opening 2763 in the front face 2745 to allow engagement with a valve 2733 of the main instrument 810. A waste line 2704 extends from the passageway 113 to the waste receptacle 325 and across an opening 2741 in the front face 2745. The passageway 113 proceeds to the connector 2532 of the fluid interface 2028, which extends the passageway 113 to the pin 2544. The passageway 113 crosses an opening 2743 in the front face 2745 to allow engagement of the passageway 113 with a bubble sensor 2741 of the main instrument 810 of FIGURE 24B. When the cassette 820 is installed on the main instrument 810, the pinch valves 2732, 2733 extend through the openings 2731, 2743 to engage the passageways 113, 2704, respectively.

[0327] The illustrated fluid handling network 2700 also includes a passageway 2723 which extends between the passageway 111 and a passageway 2727, which in turn extends between the passageway 2723 and the fluid interface 2028. The passageway 2727 extends across an opening 2733 in the front face 2745. A pump line 2139 extends from a pump 328 to the passageways 2723, 2727. When the cassette 820 is installed on the main instrument 810, the pinch valves 2716, 2718 extend through the openings 2725, 2733 in the front face 2745 to engage the passageways 2723, 2727, respectively.

[0328] It will be appreciated that, upon installing the cassette 820 on the main instrument 810 (as shown in FIGURE 24A), there is formed a fluid circuit that be operated in a manner similar to that described above, in connection with the apparatus of FIGS. 9-10.

[0329] In view of the foregoing, it will be further appreciated that the various embodiments of the fluid handling and analysis apparatus 140 (comprising a main instrument 810 and cassette 820) depicted in FIGURES 22A-28 can serve as the fluid handling and analysis apparatus 140 of any of the sampling systems 100/300/500, or the fluid handling system 10, depicted in FIGURES 1-5 herein. In addition, the fluid handling and analysis apparatus 140 of FIGURES 22A-28 can, in certain embodiments, be similar to the apparatus 140 of FIGURES 1-2 or 8-10, except as further described above.

SECTION V - SPECTROSCOPIC ANALYSIS OF A BIOLOGICAL FLUID REACTED WITH

AN ENZYME

[0330] This section describes embodiments where one or more enzymes are reacted with a material sample, and where one or more spectroscopic measurements of the reacted or partially reacted material sample (referred to herein and without limitation as the "reacted sample") are used to determine an analyte concentration. The embodiments include analyzing the spectrum of enzymatically reacted material samples to determine the concentration of analytes removed by the reaction. The enzymes are highly specific, analyte consuming enzymes.

[0331] In general, the material sample includes an analyte that is detectable by the analyte detection system, such as analyte detection system 334, and other "interfering" compounds in the unreacted material sample that are also detectable by the analyte detection system. The enzyme(s) discussed herein preferentially remove a specific analyte while leaving other

compounds in unreacted material sample. While the enzymatic reaction may result in the production of compounds that are detectable by the analyte detection system, these new or additional compounds are present in proportion to analyte concentration and can thus be accounted for. The enzyme(s) produce specific product(s), and the spectrum are analyzed in a manner that is insensitive to interferents, such as those of water or other compounds that are detectable by the analyte detection system.

[0332] As described below, a material sample of biological fluid is reacted with one or more enzymes. Spectra at two or more wavelengths are obtained from the reacted sample. Alternatively, a spectrum is obtained from an unreacted (original) material sample. The spectra are analyzed according the methods described below to determine an analyte concentration. In one embodiment, a few, predetermined wavelengths are selected for spectroscopic analysis.

[0333] In general, the methods include spectroscopically analyzing a material sample reacting with an enzyme. The enzyme preferentially removes a specific analyte that may be in the sample, and the spectroscopic analysis determines the presence, and preferably the concentration, of the analyte removed by the reaction. The method is thus capable of determining the amount of analyte in the original sample.

[0334] The embodiments described in this section thus combine the advantages of infrared spectroscopy-based methods with enzyme-based methods. In addition, by combining enzymatic reactions and spectroscopic techniques, the spectroscopic analysis may be made with fewer measurements (that is, at fewer wavelengths) than purely spectroscopic techniques due to the specificity of the technique to specific analytes. Thus, for example, an analyte concentration can be determined by measurements at a few wavelengths, where the number of wavelengths is determined, in part, by the analyte and background spectra and the accuracy required. In one embodiment, an analyte concentration is determined from spectra at two wavelengths. In another embodiment, the spectra are three or more wavelengths.

[0335] The embodiments described in this section are generally similar to the embodiments of the sampling system and methods as described in Sections I through IV above, except as further detailed below. Specifically, Section V.A below discloses apparatus for reacting a biological fluid with enzymes for spectroscopic analysis, Section V.B below discloses several

enzymes for reacting with a biological fluid, and Section V.C below discloses methods of determining analyte concentrations using the apparatus and methods of this section.

SECTION V. A - APPARATUS FOR REACTING A BIOLOGICAL FLUID WITH ENZYMES FOR SPECTROSCOPIC ANALYSIS

[0336] The scope of the disclosure includes an apparatus useful for spectroscopically analyzing a material sample, such as a biological fluid, that has reacted, or that is reacting with, an enzyme. Several embodiments, which are not limiting to the scope of this disclosure, are discussed in this section.

[0337] As described subsequently in greater detail ^' , one embodiment provides an apparatus in which the material sample reacts with one or more enzymes by contacting a surface having immobilized enzymes, or admixing the material sample with the enzymes. Alternatively, the apparatus also provides oxidizer if needed for enzymatic reactions to proceed.

[0338] The spectra may be obtained, for example and without limitation, by analyte detection system 334. Formation of the reacted sample occurs by contacting the original sample with an enzyme or an enzyme and an oxidant. Thus, for example, the material sample is reacted by admixing the enzyme and original sample, or by flowing the sample over a surface containing immobilized enzyme or enzyme and oxidant. In a preferred embodiment, the products of the analyte-enzyme reaction are not measurable by spectral analysis, and the spectroscopic analysis includes subtracting the reacted sample spectrum from the original sample spectrum, and comparing the subtracted spectrum with the analyte spectrum to obtain an analyte concentration. Optionally, the reacted sample is further reacted with a second analyte-specific enzyme to form a twice-reacted sample, and the spectrum of the twice-reacted sample is subtracted from the spectrum of the original sample and compared to the second analyte spectrum to determine the spectrum of the second analyte.

[0339] With regard to immobilized enzymes, the methods described here include enzymatic catalysis by surface immobilized enzymes. Immobilization of enzymes is applied to many areas of science and technology; such preparations are particularly useful for specific separations of single components out of complex mixtures as applied in the various chromatographic separation methods. The techniques for immobilizing enzymes are well known in the art.

[0340] FIGURES 34A and 34B are schematics of portions of a first embodiment of a sampling unit 3400 useful for reacting a sample with an enzyme. Sampling unit 3400 may be generally similar to sampling system 100, 300, 500, or 800, except as further detailed below.

[0341] Sampling unit 3400 includes a passageway 3401 having an inner volume 3403 through which a material sample flows between analysis device 330 and waste receptacle 325. As described subsequently, Sampling unit 3400 permits a material sample to analyzed in several different ways.

[0342] More specifically, passageway 3401 includes a tube 3405 having an inner surface 3407 that forms the material sample conduit. At least a portion of inner surface 3407 contains a reactive compound including, but not limited to, an enzyme. In one embodiment, inner surface 3407 includes an immobilized enzyme. Alternatively, at least a portion of tube 3405 is also gas permeable, permitting oxygen to diffuse inwards and participate in the reaction between the material sample and the reactive compound.

[0343] For analysis using a "total reaction amplitude" method, measurements are obtained and compared using sampling unit 3400 for both the material sample after fully reacted with the enzyme and the original material sample, where the original material sample measurement is made either before reaction has occurred or by extrapolating later measurements to a zero time. Thus, for example, sampling unit 3400 is operated to: measure a material sample in analysis device 330; mix the material sample with an enzyme; and then measured a the reacted sample a second time in the analysis device. In one embodiment of a sampling method, sampling unit 3400 is operated as follows. A material sample is obtained from a patient as described with reference to Figures 7A-7J. After analyzing the material sample in analysis device 330, the sample is pumped through passageway 3401 and towards, but not into, waste receptacle 325. After the material sample has contacted inner surface 3407 for a sufficiently long enough time for reactions to occur between the material sample and inner surface, the reacted material sample is drawn back into analysis device 330 for analysis. With reference to the embodiment of Figures 7A-7J, for example, pump 203 is reversed to pull the sample back into analysis device 330. After the reacted sample is analyzed, pump 230 is run forward, pushing the sample into waste receptacle 325.

[0344] In one embodiment, pump 203 pumps the material sample in analysis device 330 through passageway 3401 to contact immobilized enzymes of inner surface 3407. Pump 203 stops pumping for a predetermined amount of time, and reverses to draw the material sample back into analysis device 330. In an alternative embodiment, pumps 203 is alternately run forward and reverse to enhance mixing of the material sample and the enzyme, before drawing the reacted material sample back into analysis device 330.

[0345] FIGURE 35 is a schematic of a portion of a second embodiment of a sampling system 3500 useful for reacting a sample with an enzyme. Sampling unit 3400 may be generally similar to sampling system 100, 300, 500, 800, or 3400 except as further detailed below.

[0346] Sampling system 3500 includes two enzyme sources including a first source 3501, (illustrated as, though not limited to, a syringe pump) containing a first enzyme El and a second source 3503 (illustrated as, though not limited to, a syringe pump) of a second enzyme E2. Sampling system 3500 also includes a mixing chamber 3505 in fluid communication with analysis device 330 and waste receptacle 325 and adapted for receiving enzymes El and/or E2. Sources 3501 and 3503 are in communication with, and are operated by commands from, controller 210. Mixing chamber 3505 preferably has a volume sufficient to contain the material sample and enzymes. In one embodiment, mixing chamber 3505 and has an inner surface that does not participate in the reactions that take place therein.

[0347] Although Figure 35 illustrates an embodiment for injecting up to two enzymes in the material sample, it is to be understood that various embodiments of sampling system 3500 include a system having only one enzyme source, such as source 3501, or that has three or more enzyme sources.

[0348] Sampling system 3500 can be operated to analyze the material sample before reaction has started, at times after the reaction with the enzyme has commenced, or after reactions have completed. In one embodiment of a sampling method, sampling unit 3500 is operated as follows. A material sample is obtained from a patient as described with reference to Figures 7A-7J. After analyzing the material sample in analysis device 330, the sample is pumped into mixing chamber 3505. Mixing occurs from the injection of an enzyme, such as El and/or E2, with the material sample pumped into mixing chamber 3505. Alternatively, mixing chamber 3505 includes a

magnetically actuated stirrer that is also in communication with and controlled by controller 210. Alternatively, mixing chamber 3505 includes a stirrer that uses vibratory, ultrasonic, or other types of energy to stir the mixture.

[0349] After the material sample is reacted with the enzyme it is drawn back into analysis device 330 for analysis. With reference to the embodiment of Figures 7A-7J, for example, pump 203 is reversed to pull the sample back into analysis device 330. After the reacted sample is analyzed, pump 230 is run forward, through mixing chamber 3505 and into waste receptacle 325.

[0350] Alternatively, the analyzed, reacted mixture is pumped into mixing chamber 3505 where a different enzyme is added and mixed. The twice reacted material sample is then drawn back into analysis device 330, and is then discharged in waste receptacle 3505.

[0351] In an other alternative embodiment, sampling unit 3500 is operated as follows. A material sample is obtained from a patient as described with reference to Figures 7A-7J. The sample is pumped into mixing chamber 3505. Mixing occurs from the injection of an enzyme, such as El and/or E2, with the material sample pumped into mixing chamber 3505. The reacting material sample is then drawn back into analysis device 330 for analysis. With reference to the embodiment of Figures 7A-7J, for example, pump 203 is reversed to pull the sample back into analysis device 330. Analysis device 330 then obtains spectral measurements on the reacting material sample at one or more times. After the reacted sample is analyzed, pump 230 is run forward, through mixing chamber 3505 and into waste receptacle 325.

[0352] FIGURE 36 is a schematic of a portion of a third embodiment of a sampling system 3600 useful for reacting a sample with an enzyme. Sampling unit 3600 may be generally similar to sampling system 3500 except as further detailed below.

[0353] Sampling unit 3600 includes a valve 3601 that is in communication with and operated by controller 210. Valve 3601 has a first position to provide fluid communication between analysis device 330 and a mixing chamber 3505, and a second position to provide fluid communication between analysis device 330 and waste receptacle 325. The operation of sampling unit 3600 is similar to that of sampling unit 3500, except that valve 3601 permits the flushing of the material sample to waste receptacle 325 without passing through mixing chamber

3505.

[0354] FIGURE 37 is a schematic of a portion of a fourth embodiment of a sampling system

3700 useful for reacting a sample with an enzyme. Sampling unit 3700 may be generally similar to sampling system 100, 300, 500, 800, 3400, 3500, or 3600 except as further detailed below.

[0355] Sampling system 3700 includes a first source 3701 of a third enzyme E3. Source

3701 is in communication with, and is operated by commands from, controller 210. While Figure 37 illustrates an embodiment for injecting one enzyme in the material sample, it is to be understood that various embodiments of sampling system 3700 include a system having two or more enzyme sources.

[0356] Source 3701 is adapted to inject enzyme E3 into the analysis device 330. As one example that is not meant to limit the scope of this disclosure, source 3701 injects an aliquot of enzyme into cuvette 1730, either before, during, or after a material sample has been provided to the cuvette.

[0357] In one embodiment of a sampling method, sampling unit 3700 is operated as follows. A material sample is obtained from a patient as described with reference to Figures 7A-7J. In one embodiment, after analyzing the material sample in analysis device 330, an enzyme is injected from source 3701 and the sample is analyzed at a predetermined time after the commencement of reaction. In a second embodiment, the reacting sample is analyzed as the reaction proceeds with time. The original spectra (of the unreacted sample) may either be recorded before an appreciable reaction has taken place, or may be extrapolated to a zero time from time resolved spectra. After the reacted sample is analyzed, pump 230 is run forward, through mixing chamber 3505 and into waste receptacle 325.

[0358] In one embodiment, a material sample is reacted with one analyte specific enzyme. In another embodiment, a material sample is reacted, either simultaneously or sequentially, with two analyte specific enzymes. In yet another embodiment, there are two analytes and a material sample is reacted, either simultaneously or sequentially, with an enzyme specific to a first analyte, followed by an enzyme specific to a second enzyme.

[0359] FIGURE 38 is a schematic of a portion of a fifth embodiment of a sampling system

3800 useful for reacting a sample with an enzyme. Sampling unit 3800 may be generally similar to sampling system 100, 300, 500, 800, 3400, 3500, or 3700 except as further detailed below. Sampling unit 3800 includes a test strip 3810 and analyte detection system 3820. Analyte detection system 3820 may be generally similar to analyte detection system 1700 except as further detailed below.

[0360] Analyte detection system 3820 includes energy source 1720 and detector 1745. Energy source 1720 and detector 1745 are arranged to illuminate and object and detect reflected radiation. That is, energy source 1720 is directed towards a surface and detector 1745 is directed to receive radiation from the surface. This arrangement is illustrated in FIGURE 38. In one embodiment, analyte detection system 3820 analyzes the reacting material sample during reactions. In an another embodiment, one analysis is after all reactions have occurred. In yet another embodiment, one analysis is before reactions occur.

[0361] In one embodiment, unit 3800 analyzes material samples contained within a disposable test strip. In this embodiment, a test strip 3810 is provided with a material sample from a pin prick, for example, and the material sample containing test strip is inserted into analyte detection system 3820. In an alternative embodiment, sampling unit 3800 is an automated sampling unit, such as sampling unit 100, where the sample preparation unit 332 includes test strip 3810.

[0362] In one embodiment, test strip 3810 includes a substrate 3811, a first layer 3813 and a second layer 3815. Substrate 3811 supports first layer 3813 and may be, for example, a thin layer of plastic or glass. First layer 3813 is a layer that is reflective to energy beam E from energy source 1720. Second layer 3815 is a porous material, or has a particular design geometry, that draws material samples into the layer by capillary action.

[0363] In another embodiment, first layer 3813 is a thin metal layer, which may be, but is not limited to, a vapor deposited gold or aluminum layer. Second layer 3805 is a porous material formed of inert particulate matter coated with enzyme(s), reagents, or additives. In one embodiment, the particles of second layer 3805 are bonded to first layer 3803 by the enzyme(s), reagents, or additives. Examples of inert particulate matter include, but are not limited to, TiO ₂ or silica particles in the range of from about 0.5 μm to about 5.0 μm in diameter. In one

embodiment, second layer 3815 is formed from TiO ₂ particles about 0.5 μm to about 5.0 μm in diameter that are coated with one or more enzymes for reacting with an analyte.

[0364] Enzymes coated on such particles are activated upon hydration from a wicked material sample, preferably after layer 3815 is uniformly filled. The design of such materials is known in the field of reagent strips for measuring glucose, to prevent washout during filling of reagent strips. See, for example, Zhang, H. and Meyerhoff, M.E.., Gold-coated magnetic particles for solid-phase immunoassays: enhancing immobilized antibody binding efficiency and analytical performance, Anal Chem. 2006, Jan 15;78(2):609-16.; Topcu, Sulak M., Gokdogan, O., Gulce, A., and Gulce, H., Amperometric glucose biosensor based on gold-deposited polyvinylferrocene film on Pt electrode, Biosens Bioelectron. 2006 Mar 15;21(9): 1719-26. Epub 2005 Sep 28., or Ren, X., Meng, X., Chen, D., Tang, F., and Jiao, J., Using silver nanoparticle to enhance current response of biosensor, Biosens Bioelectron. 2005 Sep 15;21(3):433-7. Epub 2004 Dec 22. It is preferred that the average distance between particles of layer 3815 is small enough to ensure complete mixing by passive diffusion.

[0365] FIGURE 39 is a schematic of an alternative embodiment test strip 3910. Test strip 3910 may be generally similar to test strip 3810 except as further detailed below.

[0366] Test strip 3910 includes a substrate forming a well of depth H, as indicated in FIGURE 39. In one embodiment, the thickness H is from about 10 μm to about 50 μm. (The thickness of first layer 3813 in the Figure is exaggerated.) The well thickness helps to control the distribution of material sample on test strip 3910.

[0367] In the embodiments of FIGURES 38 and 39, a patient provides a material sample as a blood sample obtained by pin prick. The blood is placed adjacent to second layer 3815, and the test strip, which may be test strip 3810 or 2810, is placed within analyte detection system 3820. In an alternative to the embodiments of FIGURES 38 and 39, a patient provides a material sample via a catheter 11, and as and one or more test strips, which may be test strip 3810 or 2810 and analyte detection system 3820 are located within fluid handling and analysis apparatus 140. Examples of an automated test strip analyzer may be found in Curme, H. and Rand, R. N., Clinical Chemistry. 1997;43: 1647-1652. In an alternative embodiment, the material sample is divided into multiple portions - one containing the original sample, and the others containing

samples for each reacting with an analyte-specific enzyme. The reactions may proceed in parallel, with the spectrum of each obtained sequentially or in parallel.

SECTION V.B - ENZYMES FOR THE SPECTROSCOPIC DETERMINATION OF ANALYTES

[0368] In general, the enzymes of this section are reacted with a material sample to remove specific analytes that may be present in the material sample. Enzymes are selected for their ability to react with the material sample and convert an analyte of interest into reaction product(s). Measurements of the reacting sample thus provide optimal specificity as determined by the specific enzymes. As described in subsequent sections, the spectrum of the reacted material sample is obtained for analysis to determine the analyte concentration. For illustrative purposes that are not meant to limit the scope of the present disclosure, embodiments are described below for the analyte glucose. Enzymes that are useful for glucose measurement include, but are not limited to, glucose oxidase (GOx) and glucose dehydrogenase (GDH). In alternative embodiments the enzyme is premixed with an oxidizer as may be required for certain reactions. Thus, for example, in one embodiment GDH is premixed with an oxidizing agent, such as nicotinamide adenine dinucleotide phosphate (NADP).

[0369] The reaction of glucose with GOx proceeds as follows:

/J'- D - glucose + enzyme - FAD ϊ=≡±ε,mjim - FADH. + S-O - gluconolβctoiB EϊIξTπI5 -FADH2 +02 » Enzyme -FAD + H2O2

Glucose oxidase (β -D-glucose:oxygen 1-oxidoreductase, EC 1.1.3.4) catalyses the oxidation of β-D-glucose to D-glucono-l,5-lactone and hydrogen peroxide, using molecular oxygen as the electron acceptor. The initial product of glucose oxidation is D-glucono-l,5-lactone, which hydrolyses spontaneously. The rate constant for this hydrolysis is pH dependent. At a pH of 8, the reaction proceeds with a half life of approximately 10 minutes.

[0370] The reaction of glucose with GDH proceeds as follows:

D-glucose + β-NADP -» D-glucono-l,4-lactone + β-NADPH + H+ ,

where β-NADP is β-Nicotinamide Adenine Dinucleotide, Phosphate Oxidized Form, and ^β -

NADPH = β-Nicotinamide Adenine Dinucleotide, Phosphate Reduced Form.

[0371] Although GOx has a higher degree of specificity for glucose than does GDH, oxygen is required for the GOx reaction to proceed. The GDH reaction has the advantage of not requiring oxygen, but it does require prior admixing of a selectable oxidizer such as oxygen or NADP. For higher specificity, GOx and GDH reactions may be completed, either separately or combined, before spectroscopic analysis. In order to utilize GOx as the glucose-specific enzyme, a sufficient supply of an oxidizer is needed for the reaction to go to completion. The oxidizer NADP may be provided by mixing or contacting with the sample. The oxidizer oxygen may be provided from the atmosphere by the use of gas-permeable tubing which allows oxygen to diffuse toward the enzyme.

[0372] In alternative embodiments, the analyte is lactate. Enzymes that are useful for lactate measurement include, but are not limited to, lactate dehydrogenase (LD); hydroxybutyrate dehydrogenase (HBD); and alanine transaminase (ATL).

[0373] It a preferred embodiment, a more than sufficient amount of enzyme is contacted with the material sample for completely converting the analyte of interest.

SECTION V.C - METHODS OF DETERMINING ANALYTE CONCENTRATIONS

[0374] This section discusses computational methods or algorithms which may be used to detect, or calculate the concentration of, the analyte(s) of interest in the sample S, and/or to compute other measures that may be used in support of calculations of analyte concentrations. Any one or combination of the algorithms disclosed in this section may reside as program instructions stored in the memory 212 so as to be accessible for execution by controller 210 of the fluid handling and analysis apparatus 140 or, without limitation, of analyte detection system 334 to compute the concentration of the analyte(s) of interest in the sample, or other relevant measures.

[0375] As one embodiment that is not meant to limit the scope of the present disclosure, consider the measurement of glucose in blood or blood plasma. According to one embodiment, blood plasma is analyzed in analyte detection system 334, the blood is reacted with a glucose enzyme, and the reacted blood plasma is analyzed in the analyte detection system. Either GOx or

GDH convert glucose to a different substance with a different infrared absorption spectrum. The spectral difference caused by such reaction is proportional to glucose and insensitive to the sample background; i.e. glucose in any body fluid of any composition will be converted quantitatively and give rise to the same, unique infrared difference spectrum.

[0376] By reacting a biological fluid with one or more of analyte-specific enzymes, such as glucose-specific or lactate-specific enzymes, the sample becomes essentially free of all interferences except those few carbohydrates that 1) the enzyme has a measurable activity for, and 2) that are also present in patients' body fluids. The few interfering carbohydrates that are present in significant amounts in a material sample can be detected and identified by direct IR absorbance. The enzymatic glucose spectrum difference can be corrected for the known presence of another interfering carbohydrate. A system based on these methods may be calibrated by measuring several material samples having a known analyte concentration in analyte detection system 334, and then curve fitting the results using linear and/or ratios of values measured by the analyte detection system. The resulting curve fit may then be used to estimate the analyte concentration in a material sample.

[0377] FIGURE 40 is a graph of the infrared spectra of blood containing lg/dL glucose and the same blood after the glucose has been removed by reaction of glucose to gluctone using GOx. Each spectrum in FIGURE 40 includes contributions from water and many other components of blood. The GOx-reacted blood spectra do not include the spectra of glucose, but does include a small quantity of enzyme reaction products. It is seen from this Figure that there is a measurable difference between the unreacted and reacted blood samples. This difference in spectra forms the basis for the analysis of enzyme reacted samples.

[0378] In one embodiment, the spectrum of a sample (the "original sample") is obtained, the original sample is reacted with an analyte-specific enzyme to form a reacted sample, and the spectrum of the reacted sample is obtained. In an alternative embodiment, two samples (a first sample and a second sample) are obtained at essentially the same time. The first sample is an original sample that is spectrally analyzed. The second sample is enzyme reacted and then spectrally analyzed, either before or after the first sample. In either of these embodiments, the spectral signatures of the interferents in the sample spectra are essentially the same, and the

difference between the spectrum of the original and reacted samples is principally due to the amount of analyte in the original sample.

[0379] FIGURE 41 is a graph of the change of reacting blood spectra with time. The data for this graph was obtained by reacting a glucose containing blood sample with 10 times the amount of glucose oxidase required to remove all glucose, and following the progression of the reaction. Specifically, a blood plasma sample having 500 mg glucose per dl was prepared, and a glucose oxidase solution was prepared and added to provide 139 units of GOx per 500 microliters of blood plasma. To obtain the data of FIGURE 41, a 5 ml test tube was sealed containing a 500 microliter sample of the blood plasma, a 6 microliters sample of an enzyme solution containing 139 units of GOx, 5 ml of oxygen gas. The test tube was rocked for 1 minute, and samples were pulled at 1, 8, and 20 minutes and analyzed in an FTIR (35 μm path length; temperature 23 C, 4000-700 cm-1 scan range; data averaged over 75 scans).

[0380] As is evident from FIGURE 41, there are several wavelengths that exhibit little change with time, e.g. wavelengths from about 8.0 μm to about 8.4 μm, and from wavelengths of about 8.8 μm, about 9.1 μm and about 9.26 μm. These wavelengths are useful for a baseline measurement - they create a glucose assay that is independent of time. In addition, there are also several wavelengths that exhibit some change with time, e.g. at about 9.45 μm and at about 9.70 μm.

[0381] FIGURE 42 is a graph of the spectra of a blood with different amounts of glucose with GOx at one minute after the commencement of reaction. The data of FIGURE 42 was obtained as follows: glucose spiked plasma was prepared at 70, 125, 250, 375, and 500 mg glucose per dl plasma. As before, the plasma was mixed with 10 times the 13.9 units of GOx to consume the glucose, and the spectra was measured one minute after the commencement of reaction.

[0382] The graph of FIGURE 42 illustrates that the change in spectra, even without full conversion of the glucose, is measurable as a function of the initial glucose concentration. This property of the spectra is then used to calibrate the measurement. FIGURE 43, for example, is a graph showing a calibration curve using the data of FIGURE 42, and illustrating one method of obtaining a glucose concentration from the enzyme reacted spectra. The graph of FIGURE 43

was obtained using data from FIGURE 42 at 8.5 μm, a baseline wavelength which showed little change with glucose concentration, and at 9.65 μm, a signal wavelength which showed a large change with glucose concentration when a series of glucose solutions at different glucose concentration levels was measured and analyzed. Specifically the method is a type of self- regression, where a least squares, linear regression analysis was performed of the difference between the optical density of the material sample at the signal wavelength to the optical density at the baseline wavelength versus the known initial glucose concentration. The resulting fit is then used as a calibration of the measurements. FIGURE 43 shows the fit using the calibration (the "computed glucose") versus the actual initial glucose concentration. As is seen in FIGURE 43, this method determines the glucose concentration at levels of less than 1 microgram/microliter independent of the presence of interferents. Alternatively, a self-regression is performed using a ratio of optical density at the signal wavelength to the baseline wavelength.

[0383] As noted above, the filter wheel 50 shown in FIGURE 31 comprises a plurality of secondary filters 60 which preferably operate as narrow band filters, each filter allowing only energy of a certain wavelength or wavelength band to pass there through. In one embodiment, a filters are selected which show 1) little change with glucose concentration; and 2) a large positive or negative change in concentration. In this way, a baseline may be established and measurements with a high sensitivity to glucose. Thus, in one embodiment, filter wheel 50 has only two secondary filters 60: one at 8.5 μm and the other at 9.65 μm.

[0384] Another alternative method uses more than one signal wavelength. A calibration of analyte detection system 334 is performed using multiple linear regression of the optical density of the material sample, or of ratios of signal to baseline optical densities. Multiple regression allows for optimizing sensitivity to the glucose and reducing the effect of interfering compounds in the reacting material sample.

[0385] In an embodiment that measures at more than one signal wavelength, four filters are used, 8.5 μm and 9.65 μm, one at 9.1 μm (which shows little change with glucose concentration) and one at 9.20 μm (which shows a negative change with glucose concentration). These measurements may be arithmetically combined in different combinations to obtain a measure of the glucose concentration. Thus for example, the sum of the measurements at 9.20 μm and 9.65

μm can be subtracted from the average of the measurements at 8.5 μm and 9.1 μm.

[0386] As is apparent from the spectra, as shown, for example in FIGURES 41 or 42, graph, that there are many wavelengths that may be combined as a measure of glucose. Thus, for example, one or more wavelengths have spectral intensities that vary as glucose is being converted to a metabolized form can be used as signal wavelengths and one or more wavelengths that have spectral intensities that do not greatly vary with glucose conversion can be used as baseline wavelengths. Choices of wavelengths are to be chosen ideally to represent the best compromise of sensitivity and specificity for the data collection sampling period of time and the mode of operation, i.e. amplitude or time-based assay.

[0387] Since every chemical reaction rate is temperature dependent, the accuracy of the methods that depend on the reaction rate can be improved by measuring or controlling the reaction temperature. In one embodiment, the temperature is controlled, for example, with heaters and a thermostat. In another embodiment, a measured temperature is used to correct for a reaction-dependent measurement, such as the spectroscopic measurements described herein. Regarding the analysis of measurements from test strip 3810, in one embodiment the temperature of the sample being measured can be determined by analysis of temperature-dependent absorption features. The determined temperature may then be used to temperature index the observed reaction rate of glucose and to correct for temperature variation during the measurement. In one embodiment, a system is calibrated using a range of known initial glucose concentrations and measured temperature. The resulting experimental results are then used as a look-up table to correct for the actual reaction temperature.

[0388] As an alternative to the methods described above include, but are not limited to, methods that utilize the slope of the reaction based assay; i.e. a short period, such as a few seconds, permitting the initial reaction time to be estimated from a linear or other function, to derive the reaction rate which is a linear function of the glucose concentration present in solution.

[0389] Except as further described herein, the embodiments, features, systems, devices, materials, methods and techniques described herein can, in some embodiments, be similar to or employed in connection with any one or more of the embodiments, features, systems, devices,

materials, methods and techniques described in U.S. Provisional Application No. 60/673,551, filed on April 21, 2005, titled APPARATUS AND METHODS FOR SEPARATING SAMPLE FOR ANALYTE DETECTION SYSTEM; or in U.S. Patent Application Publication No. 2005/0038357 Al, published on February 17, 2005, titled SAMPLE ELEMENT WITH BARRIER MATERIAL. The entirety of each of the above-mentioned Provisional Application No. 60/673,551 (less its Appendix) and Publication No. 2005/0038357 Al are attached hereto in an Appendix, and are hereby incorporated by reference herein and made a part of this specification.

[0390] In particular, any of the methods disclosed herein for determining the concentration of an analyte (e.g. glucose) in a biological fluid (e.g. blood or blood components) can be implemented as a method, algorithm or program instructions executable by (and stored within memory accessible by) any of the embodiments the system/instrument disclosed in the above- noted Provisional Application no. 60/673,551, or any of the embodiments of the analyte detection system disclosed in the above-noted Publication No. 2005/0038357 Al.

[0391] It will be understood that the steps of methods discussed are performed in one embodiment by an appropriate controller (or processors) of a processing (i.e., computer) system executing instructions (code segments) stored in appropriate storage. It will also be understood that the disclosed methods and apparatus are not limited to any particular implementation or programming technique and that the methods and apparatus may be implemented using any appropriate techniques for implementing the functionality described herein. The methods and apparatus are not limited to any particular programming language or operating system. In addition, the various components of the apparatus may be included in a single housing or in multiple housings that communicate by wire or wireless communication.

[0392] Further, the interferent, analyte, or population data used in the method may be updated, changed, added, removed, or otherwise modified as needed. Thus, for example, spectral information and/or concentrations of interferents that are accessible to the methods may be updated or changed by updating or changing a database of a program implementing the method. The updating may occur by providing new computer readable media or over a computer network. Other changes that may be made to the methods or apparatus include, but are not

limited to, the adding of additional analytes or the changing of population spectral information.

[0393] One embodiment of each of the methods described herein may include a computer program accessible to and/or executable by a processing system, e.g., a one or more processors and memories that are part of an embedded system. Thus, as will be appreciated by those skilled in the art, embodiments of the disclosed inventions may be embodied as a method, an apparatus such as a special purpose apparatus, an apparatus such as a data processing system, or a carrier medium, e.g., a computer program product. The carrier medium carries one or more computer readable code segments for controlling a processing system to implement a method. Accordingly, various ones of the disclosed inventions may take the form of a method, an entirely hardware embodiment, an entirely software embodiment or an embodiment combining software and hardware aspects. Furthermore, any one or more of the disclosed methods (including but not limited to the disclosed methods of measurement analysis, interferent determination, and/or calibration constant generation) may be stored as one or more computer readable code segments or data compilations on a carrier medium. Any suitable computer readable carrier medium may be used including a magnetic storage device such as a diskette or a hard disk; a memory cartridge, module, card or chip (either alone or installed within a larger device); or an optical storage device such as a CD or DVD.

[0394] Reference throughout this specification to "one embodiment" or "an embodiment" means that a particular feature, structure or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures or characteristics may be combined in any suitable manner, as would be apparent to one of ordinary skill in the art from this disclosure, in one or more embodiments.

[0395] Similarly, it should be appreciated that in the above description of exemplary embodiments, various features of the inventions are sometimes grouped together in a single embodiment, figure, or description thereof for the purpose of streamlining the disclosure and aiding in the understanding of one or more of the various inventive aspects. This method of disclosure, however, is not to be interpreted as reflecting an intention that any claim require more

features than are expressly recited in that claim. Rather, as the following claims reflect, inventive aspects lie in a combination of fewer than all features of any single foregoing disclosed embodiment. Thus, the claims following the Detailed Description are hereby expressly incorporated into this Detailed Description, with each claim standing on its own as a separate embodiment.

[0396] Further information on analyte detection systems, sample elements, algorithms and methods for computing analyte concentrations, and other related apparatus and methods can be found in U.S. Patent Application Publication No. 2003/0090649, published May 15, 2003, titled REAGENT-LESS WHOLE BLOOD GLUCOSE METER; U.S. Patent Application Publication No. 2003/0178569, published September 25, 2003, titled PATHLENGTH-INDEPENDENT METHODS FOR OPTICALLY DETERMINING MATERIAL COMPOSITION; U.S. Patent Application Publication No. 2004/0019431, published January 29, 2004, titled METHOD OF DETERMINING AN ANALYTE CONCENTRATION IN A SAMPLE FROM AN ABSORPTION SPECTRUM; U.S. Patent Application Publication No. 2005/0036147, published February 17, 2005, titled METHOD OF DETERMINING ANALYTE CONCENTRATION IN A SAMPLE USING INFRARED TRANSMISSION DATA; and U.S. Patent Application Publication No. 2005/0038357, published on February 17, 2005, titled SAMPLE ELEMENT WITH BARRIER MATERIAL. The entire contents of each of the above-mentioned publications are hereby incorporated by reference herein and are made a part of this specification.

[0397] A number of applications, publications and external documents are incorporated by reference herein. Any conflict or contradiction between a statement in the bodily text of this specification and a statement in any of the incorporated documents is to be resolved in favor of the statement in the bodily text.