DETECTION, MONITORING AND TREATMENT OF ACUTE MYOCARDIAL INFARCTION

Abstract

Detection, monitoring and treatment of acute myocardial infarction (AMI) using a hybrid immunoassay to both Lp-PLA2 and ApoA1. Described herein are assays and method of performing them useful for the detection of AMI. Also described are methods of monitoring a patient at risk for, or suffering from, AMI. Also described herein are methods and assays for treating patient having or at risk of suffering from AMI.

Claims

1. A method of treating, detecting or monitoring acute myocardial infarction (AMI) comprising: exposing a blood sample from a patient to a solid phase support onto which an Lp-PLA2-binding molecule is coupled; incubating the blood sample with an ApoA1-binding molecule; and detecting the level of ApoA1 from the solid phase.

2. A method of treating, detecting or monitoring acute myocardial infarction (AMI) comprising: exposing a blood sample from a patient to a solid phase support onto which an Lp-PLA2-binding molecule is coupled; incubating the solid phase support with an ApoA1-binding molecule; washing the solid phase support; detecting the level of ApoA1 from the solid phase; and treating the patient for AMI if the amount of ApoA1 detected is below a threshold.

3. The method of claim 1, further comprising treating the patient for AMI if the level of ApoA1 is below a threshold.

4. The method of claim 1, further comprising treating the patient for AMI if the level of ApoA1 detected is below 0.7 ng/ml.

5. The method of claim 1, further comprising washing the solid phase support.

6. The method of claim 1, wherein exposing the blood sample from the patient to the solid phase support comprising adding the sample to a solid phase support to which an antibody or an antibody fragment specific for Lp-PLA2 is coupled.

7. The method of claim 1, wherein incubating the blood sample with the ApoA1-binding molecule comprises incubating with an antibody or an antibody fragment that binds ApoA1.

8. The method of claim 1, wherein detecting the level of ApoA1 from the solid phase support comprises reacting a horseradish peroxidase (HRP) bound to an antibody against ApoA1 to detect a signal and quantifying the detected signal.

9. A system for detecting or monitoring acute myocardial infarction (AMI), the system comprising: a first component comprising an Lp-PLA2 binding molecule coupled to a solid-phase support; a reagent comprising an ApoA1 binding molecule to which a detection moiety is coupled.

10. The system of claim 9, wherein the first component comprises a solid-phase support to which an antibody or antibody fragment that binds to Lp-PLA2 has been coupled.

11. The system of claim 9, wherein the reagent comprises a solution including an ApoA1 antibody or antibody fragment coupled to a detection moiety.

12. The system of claim 9, further comprising a set of standards configured to calibrate the level of ApoA1 detected by the detection moiety.

13. A system for detecting or monitoring acute myocardial infarction (AMI), the system comprising: a first component comprising an LpPLA2 binding molecule coupled to a solid-phase support; a first wash buffer; a reagent comprising an ApoA1 binding molecule to which a detection moiety is coupled; and a detection buffer.

14. The system of claim 13, wherein the first component comprises a solid-phase support to which an antibody or antibody fragment that binds to Lp-PLA2 has been coupled.

15. The system of claim 13, wherein the reagent comprises a solution including an ApoA1 antibody or antibody fragment coupled to a detection moiety.

16. The system of claim 13, further comprising a set of standards configured to calibrate the level of ApoA1 detected by the detection moiety.

17. The method of claim 2, further comprising treating the patient for AMI if the level of ApoA1 detected is below 0.7 ng/ml.

18. The method of claim 2, wherein exposing the blood sample from the patient to the solid phase support comprising adding the sample to a solid phase support to which an antibody or an antibody fragment specific for Lp-PLA2 is coupled.

19. The method of claim 2, wherein incubating the blood sample with the ApoA1-binding molecule comprises incubating with an antibody or an antibody fragment that binds ApoA1.

20. The method of claim 2, wherein detecting the level of ApoA1 from the solid phase support comprises reacting a horseradish peroxidase (HRP) bound to an antibody against ApoA1 to detect a signal and quantifying the detected signal.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0021] FIG. 1 is a schematic overview of one variation of a hybrid LpPLA2/ApoA1 assay for use in monitoring, detecting and/or treating a cardiac disorder such as (but not limited to) AMI. FIG. 1 shows an ELISA Format Design for an Lp-PLA.sub.2/ApoAI Hybrid Assay.

[0022] FIG. 2 (which includes FIGS. 2A-2F) shows results of a hybrid LpPLA2/ApoA1 assay, examining both control and AMI patients. FIG. 2A shows a one-way analysis of APoA1 (ng/ml) by AMI for the Lp-PLA.sub.2/ApoAI Hybrid Assay-ANOVA Analysis. FIG. 2B shows the summary of fit for the one way ANOVA, FIG. 2C shows the t-test results, FIG. 2D shows the analysis of variance, FIG. 2E shows the means for the one way ANOVA, and FIG. 2F shows the means comparisons.

[0023] FIG. 3 (which includes FIGS. 3A-3D) shows a logistic fit for the results of the hybrid LpPLA2/ApoA1 assay of FIG. 2. FIG. 3A shows a logistic fit of AMI by ApoA1 (ng/ml) of the Lp-PLA.sub.2/ApoAI Hybrid Assay-Logistic Fit. FIG. 3B shows a whole-model test, FIG. 3C shows Parameter estimates, and FIG. 3D shows the receiver operating characteristic (ROC) curve.

[0024] FIG. 4 (which includes FIGS. 4A-4F) shows results of an LpPLA2 assay, examining both control and AMI patients. FIG. 4A shows a one-way analysis of PLC mass by AMI for the Lp-PLA.sub.2 Mass Assay (PLAC)-ANOVA Analysis. FIG. 4B shows the summary of fit for the one way ANOVA, FIG. 4C shows the t-test results, FIG. 4D shows the analysis of variance, FIG. 4E shows the means for the one way ANOVA, and FIG. 4F shows the means comparisons.

[0025] FIG. 5 (which includes FIGS. 5A-5D) shows a logistic fit for the results of the LpPLA2 assay of FIG. 4. FIG. 5A shows a logistic fit of AMI by PLAC mass of the Lp-PLA2 Mass (PLAC) Assay-Logistic Fit. FIG. 5B shows a whole-model test, FIG. 5C shows Parameter estimates, and FIG. 5D shows the receiver operating characteristic (ROC) curve.

[0026] FIG. 6 (which includes FIGS. 6A-6F) shows results of an ApoA1 assay, examining both control and AMI patients. FIG. 6A shows a one-way analysis ApoA1 (mg/dL) by AMI for the ApoA1 Assay-ANOVA Analyses. FIG. 6B shows the summary of fit for the one way ANOVA, FIG. 6C shows the t-test results, FIG. 6D shows the analysis of variance, FIG. 6E shows the means for the one way ANOVA, and FIG. 6F shows the means comparisons.

[0027] FIG. 7 (which includes FIGS. 7A-7D) shows a logistic fit for the results of the ApoA1 assay of FIG. 6. FIG. 7A shows a logistic fit of AMI by ApoA1 (ng/ml) of the ApoAI Assay-Logistic Fit. FIG. 7B shows a whole-model test, FIG. 7C shows Parameter estimates, and FIG. 7D shows the receiver operating characteristic (ROC) curve.

[0028] FIG. 8 (including FIGS. 8A-8C) is a comparison of the logistic fits of the hybrid Lp-PLA2/ApoA1, LpPLA2 and ApoA1 assays. FIG. 8A is a logistic fit of AMI by ApoA1 (ng/ml). FIG. 8B is a logistic fit of AMI by PLAC mass. FIG. 8C is a logistic fit of AMI by ApoA1.

[0029] FIG. 9A shows comparisons of the ROC curves for the hybrid Lp-PLA2/ApoA1 (FIG. 9A1), LpPLA2 (FIG. 9A2) and ApoA1 assays (FIG. 9A3).

[0030] FIGS. 9B-9D provide further detail on the data illustrated in FIG. 9A. FIG. 9B shows the ROC curve analysis of the Lp-PLA2 Mass/ApoA1 hybrid assay; FIG. 9C shows the ROC curve analysis of the Lp-PLA2 (PLAC) mass assay; and FIG. 9D shows the ROC curve analysis of the ApoA1 assay.

[0031] FIG. 10 (including FIGS. 10A-10E) illustrates the results of an ApoA1 assay for CVD (cardiovascular disease). FIG. 10A graphically depicts a one-way analysis of ApoA1 by CVD. FIG. 10B shows the one-way analysis, including the summary of fit, t-test, analysis of variance, and means for one-way ANOVA. FIG. 10C shows the means comparison. FIG. 10D shows a logistic fit of CVD by ApoA1. FIG. 10E shows the ROC curve.

[0032] FIG. 11 (including FIGS. 11A and 11B) is a comparison of the logistic fits of the hybrid LpPL2/ApoA1 assay for AMI and the ApoA1 assay for CVD (of FIG. 10). FIG. 11A shows the logistic fit of AMP by ApoA1 using plasma for the Lp-PLA.sub.2 Mass/ApoAI Hybrid Assay. FIG. 11B shows a logistic fit of CVD by ApoA1 for an Lp-PLA.sub.2 Mass/ApoAI Hybrid Assay using serum.

[0033] FIG. 12A (including FIGS. 12A1 and 12A2) shows an ANOVA Analysis of another set of control patients compared to the AMI patients and previous control data. FIG. 12A1 shows the one-way analysis of ApoA1 conc. by group. FIG. 12A2 shows the details of the one-way analysis, including the summary of fit, analysis of variance, means for the one-way ANOVA, and means comparison.

[0034] FIG. 12B (including FIGS. 12B1-12B3) shows a logistic fit for the results of the hybrid LpPLA2/ApoA1 assay of FIG. 12A. FIG. 12B1 shows the logistic fit of AMI by ApoA1 conc. FIG. 12B2 shows the whole model test data and parameter estimates. FIG. 12B3 shows the ROC curve.

[0035] FIG. 13 (including FIGS. 13A and 13B) shows the ROC curve for the hybrid (LpPLA2/ApoA1) assay (FIG. 13A), and indicates the Youden cutoff criterion (e.g., 0.803 ng/ml) calculated from the ROC (FIG. 13B).

DETAILED DESCRIPTION

[0036] A hybrid Lp-PLA.sub.2/ApoAI Hybrid Assay was designed and constructed to compare blood samples from both acute myocardial infarction (AMI) and non-symptom control (Caucasian patients). In general the assay systems described herein are configured to sequentially pull down first an LpPLA2 binding component of a biological sample (e.g., blood, plasma, serum, whole blood, etc.) and then probe for ApoA1 from the bound LpPLA2 component.

[0037] The assays described herein my typically include a solid phase component that includes specific binding to LpPLA2 from a body fluid (e.g., blood, etc.). The assay may also include one or more wash buffers. Further, the assay may then include a labeled probe for ApoA1.

[0038] For example, FIG. 1 schematically illustrates one possible principle for operation of the assays described herein. In this example, an LpPLA2-binding molecule is coupled to a solid phase support (e.g., a well or plate). The Lp-PLA2 binding molecule is an antibody (e.g., 2C10) to Lp-PLA2 is linked/absorbed to a solid phase surface (e.g., a well or wells of a microtiter plate, etc.). Any appropriate antibody capable of binding to Lp-PLA2 may work, including, for example, those described in U.S. patent application no. US-2014-0283157-A1, herein incorporated by reference in its entirety. A biological solution (e.g., blood) is then added to the solid phase and LpPLA2 from the sample is allowed to bind. The solid phase may then be washed, then probed with a detectable molecule that binds to ApoA1 (e.g., ApoA1-binding molecule); in this example, the Apo-A1 binding molecule is an antibody specific to Apolipoprotein A-I (ApoA1). ApoA1 is the major protein component of high density lipoprotein (HDL) in plasma. Thus, in some variations, other components of HDL (or other lipoproteins) may be used. The ApoA1 binding molecule may then be detected by either direct detection (e.g., where the APOA1 binding molecule is labeled, e.g. fluorescently labeled, HRP labeling, etc.) or detected by a secondary marker that specifically targets the ApoA1 binding molecule.

[0039] The amount of HDL is then assayed. For example, the ApoA1 binding molecule may be labeled, marked, or coupled to a marker/label, as mentioned. In FIG. 1, the ApoA1 antibody to the HDL component includes an indicator, in this example, horseradish peroxidase (HRP), that may be reacted to detect a signal. In some variations the marker may be directly visualized (e.g., fluorescent, etc.).

[0040] In one specific example, the Lp-PLA2/ApoAI Hybrid Assay protocol included: an LpPLA2 assay (e.g., commercial PLAC mass assay kit was manufactured by diaDexus, Inc.) modified for use with an ApoAI assay kit manufactured by AlerCheck, Inc. (15 Oak St., Ste 302, Springvale, Me. 04083).

[0041] In use, 20 l each of human plasma samples were loaded onto a 2C10 coated 96-well (from PLAC mass assay) and incubated at room temperature for 10 minutes. As a calibration/control, 4 strips of PLAC mass assay kit were replaced with that from the ApoA1 ELISA kit and 20 l of the ApoA1 standard (with series dilution from 36 ng/ml to 0 ng/ml in 13 points) in duplication were added. 100 l of HRP conjugated affinity purified goat anti-ApoAI (from AlerCheck ApoAI ELISA kit) were added into each well and incubated at room temperature for 2 hr. After washed the plate with TBST (tris buffered saline, pH 7.2, with 0.005% Tween-20 from PLAC mass assay kit), the plate was incubated with 100 l of TMB (from PLAC mass assay kit) for 30 minutes at room temperature and stopped with 100 l of 1 M HCl (from PLAC mass assay kit). Signal was read at 450 nm.

[0042] The plasma samples used included both acute myocardial infarction (AMI) patient's (positive) and non-symptom controls; all patients and controls were Caucasian. For all of the AMI patients, the AMI events are first time episode. Blood were collected between 7 and 12 hr. after symptom onset. All plasmas are EDTA plasmas. AMI cases were confirmed by electrocardiogram and the elevation of troponin-1 level (AQT90FLEXimmuno-chemical analyzer/Radiometry). AMI plasmas were collected during May to June, 2010 and control plasmas were collected during January to May, 2013.

TABLE-US-00001 TABLE 1 Information of AMI Plasma and Control Information on blood samples for AMI and control samples Sample ID Female Male Age Height Weight BMI Control 13 (62%) 8 (38%) 73 171 79 27 AMI 15 (60%) 10 (40%) 73 168 75 27

[0043] Results from the analysis, comparing signals between control and AMI patients for LpPLA2 alone, ApoA1 alone, and the hybrid LpPLA2/ApoA1 assay are shown in data (including graphs) in FIGS. 2-11.

[0044] For example, FIGS. 2-3 show preliminary characterization of the results of the hybrid LpPLA2/ApoA1 assay. A clear distinction in the control versus AMI groups may be seen. FIGS. 4-5 illustrate the results of just the LpPLA2 assay of the control and AMI samples groups. FIGS. 6-7 illustrate the results characterizing just the ApoA1 assay in the same AMI and control individuals.

[0045] Individually, ApoA1 and LpPLA2 may provide some distinctions between the two groups, however, the combined/hybrid assay provides a remarkably robust separation of AMI and control (non-AMI) groups. For example, FIG. 8 compares the logistic fit between the hybrid (far left), LpPLA2 alone (middle) and ApoA1 (right). The strong separation of AMI and non-AMI groups is even more pronounced when looking at the receiver operator characteristic (ROC) curves and cutoff comparison in FIGS. 9A-9D. As shown in FIG. 9A1, the assay results in an almost idea ROC result (showing both high sensitivity and high specificity) compared to the ROC for Lp-PLA2 mass (PLAC) assay alone or APoA1 assay alone as shown in FIGS. 9A2 and 9A3, respectively. The details of these results are provided in FIGS. 9B-9D. FIG. 10 shows (for comparison) an analysis of ApoA1 by cardiovascular disease. FIG. 11 shows a comparison of plasma and serum samples examined (on left) as described above for the hybrid Lp-PLA2/ApoA1 assay for AMI, compared with a serum assay for cardiovascular disease (CVD) using ApoA1.

[0046] In comparison to the ApoA1 assay and/or the Lp-PLA2 assays alone, the hybrid Lp-PLA2 and ApoA1 assay shows an extremely high degree of specificity and sensitivity. The reliability of the results was confirmed by the ANOVA analysis and logistic fits. In FIGS. 2 and 12, one way ANOVA analysis of the LpPLA2/ApoA1 hybrid assay is shown. The ANOVA results show a good separation between the control (or first control and second control) groups and the AMI group, both within groups and between groups, when looking at the within group error and the between group error. In these cases the error with the groups was smaller than the error between the groups, suggesting a reliable level of separation between the control and AMI groups. Similarly, the logistic fit between the groups suggests that the hybrid (LpPLA2/ApoA1) assay reflects a high degree of separation between the AMI and control groups; the nearly vertical line separating the AMI group from the control (e.g., FIG. 3 Logistic fit of AMI by ApoA1, for the ApoA1 pulled down by the LpPLA2 in the hybrid assay) indicates a high degree of separation. Compare this to the less robust correlation for either LpPLA2 or ApoA1 alone, as shown in FIGS. 4-5 and 6-7, as well as FIG. 8.

[0047] In the data shown in FIGS. 2-8, the control samples were more recent than the AMI samples. To confirm that the results were not an artifact due to the age of the control versus the AMI samples, the assay was re-run using controls taken at about the same time (from age/gender matched control patients). The results are shown in FIGS. 12A-12B. In this example, 20 ul of each CVD or control plasma (ProteoGenex) were incubated with 2C10 plate for 10-15 min at room temperature, as described above. Next, 100 ul of anti-ApoAI-HRP conjugate (AlerCHEK ELISA kit, Cat# A70101, Lot# K103414) was added, mixed and the plates were incubated at room temperature for 3 hr. Plates were then washed with PLAC kit wash buffer and detected with reagents of the PLAC test. In addition, plates were incubated with 100 ul/well TMB for 60 min (Plate 4) or overnight (Plate 3) and stopped with 100 ul/well of 1 M HCl.

[0048] The results of this second set of experiments confirm the earlier data (e.g., shown in FIGS. 2-8). Specifically, the control groups (control 2010 and contro12013) were nearly identical to each other, and significantly different from the AMI group.

[0049] Thus, the results of these experiments indicate that an assay that includes a solid phase linked LpPLA2 binding molecule and a probe for ApoA1 to determine the level of ApoA1 associated with LpPLA2 pulled out of a fluid (e.g., blood) sample from a patient may reliably indicate acute myocardial infraction. Further, this assay may be surprisingly more robust (both specific and sensitive) than either LpPLA2 or ApoA1 alone.

[0050] In use, the hybrid (Lp-PLA2/ApoA1) assay may be used to determine if a patient has experienced an AMI. The assay may be performed quickly (e.g., within a few hours) and a single result may be indicative. Thus, for example, if the level of ApoA1 from a sample LpPLA2 bound biological (blood) material) is below a threshold value, the patient has likely experienced (or is experiencing) and acute myocardial infarct (AMI) event. Any appropriate threshold may be used. In particular, the threshold may be determined from the ROC curve, as illustrated in FIG. 13. In this example, a cutoff value (e.g., the Youden cutoff criterion) has been determined to be approximately 0.803 ng/ml. Thus, for example, if the level of ApoA1 from a sample of LpPLA2 bound material from a patient's fluid sample is less than or equal to the cutoff (e.g., approx. 0.8) then the patient is positive for AMI (e.g., likely experienced an AMI). For example, the cutoff may a level of ApoA1 detected from a sample pulled-down with Lp-PLA2 (using an Lp-PLA2 solid phase support) that is less than about 0.7 ng/ml, e.g., below about 0.72 ng/ml, below about 0.73 ng/ml, below about 0.74 ng/ml, below about 0.75 ng/ml, below about 0.76 ng/ml, below about 0.77 ng/ml, below about 0.78 ng/ml, below about 0.79 ng/ml, below about 0.8 ng/ml, below about 0.81 ng/ml, below about 0.82 ng/ml, below about 0.83 ng/ml, etc.).

[0051] In addition, the construction of the assay has also been found to be important in determining AMI risk and treatment. For example, when ApoA1 or other non-Lp-PLA2 components of the HDL were used to first tether the HDL, which was then probed for Lp-PLA2 (e.g., using any of the same components described herein, including 2C10), there was no or only weak correlation between detected signal and AMI (data not shown). This was both surprising and informative, given the strength of the signal when the hybrid assay is performed as described above. Thus, is important that the steps of performing the assays are executed in the proper order, so that the Lp-PLA2 is used to initially pull-down the HDL particle/fraction, which is thereafter probed for ApoA1. Note that it may be possible to label the loose (untethered) HDL particles (e.g., bound to labeled ApoA1) first, before tethering them with a solid-phase to Lp-PLA2, however, based on the current data it appears important that the Lp-PLA2 be used as the solid-phase (tethering) link.

Treatment

[0052] Morbidity and mortality from myocardial infarction are significantly reduced if recognized early, so that prompt attention may be provided, and to shorten the time to definitive treatment. It may also be beneficial to recognize that an AMI has occurred within the recent past (e.g., within 1-24 hours). As a general rule, initial therapy for acute myocardial infarction is directed toward restoration of perfusion as soon as possible to salvage as much of the jeopardized myocardium as possible. This may be accomplished through medical or mechanical means, such as PCI or CABG. Treatment may also include restoration of the balance between the oxygen supply and demand to prevent further ischemia, pain relief, prevention and treatment of any complications that may arise, and coronary collateral circulation.

[0053] Thus, in general, described herein are treatment methods that include performing a hybrid LpPLA2/ApoA1 assay (or any other hybrid LpPLA2/HDL assay) to determine if the amount of ApoA1 selectively pulled down by LpPLA2 solid phase is low compared to a threshold (e.g., is below a threshold, such as below 0.7 ng/ml, e.g., below 0.72 ng/ml, below 0.73 ng/ml, below 0.74 ng/ml, below 0.75 ng/ml, below 0.76 ng/ml, below 0.77 ng/ml, below about 0.78 ng/ml, below 0.79 ng/ml, below about 0.8 ng/ml, below about 0.81 ng/ml, below 0.82 ng/ml, below 0.83 ng/ml, etc.). If below this threshold, the patent may be experiencing (or may have recently experienced) AMI and should be treated for AMI, for example, by delivering reperfusion therapy.

[0054] In some variations, the patient may be hospitalized and may immediately receive one or more of: oxygen (e.g., by nasal prongs); sublingual nitroglycerin (e.g., unless systolic arterial pressure is less than 90 mm Hg or heart rate is less than 50 or greater than 100 beats per minute); analgesia (e.g., with morphine sulfate or meperidine); aspirin (e.g., 160 to 325 mg orally); and/or immediate reperfusion therapy, either by fibrinolysis or primary percutaneous transluminal coronary angioplasty (PTCA).

[0055] Treatment of AMI may therefore generally be directed to preventing ischemia, relieving pain, and preventing complications of AMI. Treatments may include pharmacological treatment (e.g., using drugs such as aspirin), procedural therapies (e.g., reperfusion therapies), and monitoring. For example, a patient may be monitored by electrodes (EEG) and/or by further blood tests (e.g., looking at other markers such as troponin).

[0056] Thus, for example, described herein are methods of treating patients for acute myocardial infarction (AMI) by taking a sample (e.g., of blood), and using a solid-phase assay that binds Lp-PLA2, and detecting the amount of an HDL marker, such as in particular ApoA1, from the material bound to the solid phase, and when the amount of HDL marker (e.g., ApoA1) is below a threshold, treating the patient for AMI.

[0057] Terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. For example, as used herein, the singular forms a, an and the are intended to include the plural forms as well, unless the context clearly indicates otherwise. It will be further understood that the terms comprises and/or comprising, when used in this specification, specify the presence of stated features, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, steps, operations, elements, components, and/or groups thereof. As used herein, the term and/or includes any and all combinations of one or more of the associated listed items and may be abbreviated as /.

[0058] Although the terms first and second may be used herein to describe various features/elements, these features/elements should not be limited by these terms, unless the context indicates otherwise. These terms may be used to distinguish one feature/element from another feature/element. Thus, a first feature/element discussed below could be termed a second feature/element, and similarly, a second feature/element discussed below could be termed a first feature/element without departing from the teachings of the present invention.

[0059] As used herein in the specification and claims, including as used in the examples and unless otherwise expressly specified, all numbers may be read as if prefaced by the word about or approximately, even if the term does not expressly appear. The phrase about or approximately may be used when describing magnitude and/or position to indicate that the value and/or position described is within a reasonable expected range of values and/or positions. For example, a numeric value may have a value that is +/0.1% of the stated value (or range of values), +/1% of the stated value (or range of values), +/2% of the stated value (or range of values), +/5% of the stated value (or range of values), +/10% of the stated value (or range of values), etc. Any numerical range recited herein is intended to include all sub-ranges subsumed therein.

[0060] Although various illustrative embodiments are described above, any of a number of changes may be made to various embodiments without departing from the scope of the invention as described by the claims. For example, the order in which various described method steps are performed may often be changed in alternative embodiments, and in other alternative embodiments one or more method steps may be skipped altogether. Optional features of various device and system embodiments may be included in some embodiments and not in others. Therefore, the foregoing description is provided primarily for exemplary purposes and should not be interpreted to limit the scope of the invention as it is set forth in the claims.

[0061] The examples and illustrations included herein show, by way of illustration and not of limitation, specific embodiments in which the patient matter may be practiced. As mentioned, other embodiments may be utilized and derived there from, such that structural and logical substitutions and changes may be made without departing from the scope of this disclosure. Such embodiments of the inventive subject matter may be referred to herein individually or collectively by the term invention merely for convenience and without intending to voluntarily limit the scope of this application to any single invention or inventive concept, if more than one is, in fact, disclosed. Thus, although specific embodiments have been illustrated and described herein, any arrangement calculated to achieve the same purpose may be substituted for the specific embodiments shown. This disclosure is intended to cover any and all adaptations or variations of various embodiments. Combinations of the above embodiments, and other embodiments not specifically described herein, will be apparent to those of skill in the art upon reviewing the above description.