METHODS AND SYSTEMS TO PREDICT PROSTATE CANCER RISK AND PROSTATE GLAND VOLUME

专利摘要:
methods and systems for predicting prostate cancer risk and prostate gland volume. The present invention relates to methods and apparatus for predicting prostate cancer risk and/or prostate gland volume. more particularly, the present invention relates to methods and apparatus for providing models and employing the models to predict prostate cancer risk and/or to predict prostate gland volume. methods and apparatus for predicting prostate cancer risk and/or prostate gland volume are provided using, at least in part, information from a panel of kallikrein markers.
公开号:BR112014021776A2
申请号:R112014021776-9
申请日:2013-03-05
公开日:2021-09-08
发明作者:Andrew J. Vickers；Peter T. Scardino；Hans Lilja；Vincent Linder；David Steinmiller
申请人:Oy Arctic Partners Ab；Opko Diagnostics, Llc；
IPC主号:

专利说明:

[001] [001] The present invention relates to methods and apparatus for predicting prostate cancer risk and/or prostate gland volume. More particularly, this description pertains to methods and apparatus for providing the models and employing them to predict prostate cancer risk and/or predict prostate gland volume. BACKGROUND
[002] [002] Most men with an elevated blood level of total prostate specific antigen (PSA) — the most common trigger for biopsy in men in the US — do not have prostate cancer. As a result, an estimated 750,000 unnecessary prostate biopsies are performed each year in the US. There is considerable evidence that measuring PSA isoforms separately rather than combining them into a single total PSA measure can help predict the presence of prostate cancer. These data include studies showing that cancer is predicted by free PSA, BPSA, or -2proPSA. Of course, free PSA is often measured separately, with urologists receiving data in terms of total PSA and the ratio of free PSA to total, with an estimated 10 million free PSAs measured per year. There is also evidence that hK2, the molecule that converts PSA from its proactive form, is informative of prostate risk. However, none of these markers by themselves is a good predictor of the prostate biopsy result.
[003] [003] There have been several attempts to build predictive models for prostate cancer, most notably the "Risk Calculator for the Prostate Cancer Prevention Assay", the "Sunnybrook" and the risk calculator for the European Randomized Trial Trial - gem to Prostate Cancer (ERSPC). The problem with these models is that they require more or less extensive and thorough clinical examinations, ie the patient needs to visit a urologist. For example, the risk calculator for the ERSPC requires data on prostate volume, which is obtained by inserting an ultrasound probe into the rectum. Consequently, new methods and devices to predict prostate cancer risk and/or prostate gland volume would be beneficial. SUMMARY OF THE INVENTION
[004] [004] Methods and apparatus are provided for predicting prostate cancer risk and/or prostate gland volume. More particularly, this description pertains to methods and apparatus for providing the models and employing them to predict prostate cancer risk and/or predict prostate gland volume. In some embodiments, methods and apparatus for predicting prostate cancer risk and/or prostate gland volume are provided using, at least in part, information from a panel of kallikrein markers. The purpose of this patent application involves, in some cases, related methods, alternative solutions to a particular problem and/or a plurality of different uses of the systems and devices.
[005] [005] An object of the present invention is to provide a method for obtaining a probability of an event using a logistic regression model to predict a man's risk of developing prostate cancer.
[006] [006] In a set of modalities, a computer is provided for determining a probability of an event associated with prostate cancer. The computer includes an input interface
[007] [007] In a set of modalities, a system is provided for determining a probability of an event associated with prostate cancer. The system includes a detector configured to measure a plurality of values for blood markers, wherein the plurality of blood markers include free prostate specific antigen (fPSA), total PSA (tPSA) and intact PSA (iPSA). The system also includes at least one processor in electronic communication with the detector. At least one processor is programmed to evaluate a logistic regression model based, at least in part, on the measured values for fPSA, tPSA, and iPSA to determine a probability of an event associated with prostate cancer of high degree in a person. The evaluation of the logistic regression model comprises the determination of the cubic slot terms for tPSA, where the determination of the cubic slot terms for tPSA comprises the determination of the cubic slot terms for tPSA based on a first cubic slot that has a first inner node between 4-5 and a second inner node between 6-8, the determination of the cubic slot terms for fPSA, wherein the determination of the cubic slot terms for fPSA comprises the determination of the cubic slot terms for fPSA based on a second cubic slot that has a third internal node between 0.25 and 1 and a fourth internal node between 1.0 and 2.0, determining a first value for tPSA based at least in part on on the received tPSA value and the given cubic slot terms for tP-SA, the determination of the second value for fPSA based at least in part on the received fPSA value and the given cubic slot terms for fPSA , the determination of p probability of the event associated with prostate cancer based, at least in part, on the first value and the second value and the issuance of an indication of the probability of the event associated with prostate cancer.
[008] [008] In a set of modalities, a method is provided for
[009] [009] In one set of embodiments, a computer-readable storage medium is provided encoded with a plurality of instructions which, when executed by a computer, execute a method for determining a probability of a event associated with prostate cancer.
[0010] [0010] In one set of modalities, a computer is provided for determining a probability of an event associated with prostate cancer. The computer includes an input interface configured to receive information from a plurality of blood markers, wherein the information for the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a PSA value total (tPSA), an intact PSA (iPSA) value and a human kallikrein 2 (kK2) value. The computer also includes at least one processor programmed to evaluate a logistic regression model based, at least in part, on the information received, for determining a probability of an event associated with prostate cancer in a person. The evaluation of the logistic regression model comprises the determination of the probability of the event associated with prostate cancer based, at least in part, on the tPSA value, the iPSA value, the hK2 value and a ratio between the of fPSA and the value of tPSA. The computer also includes an output interface configured to output an indication of the probability of the event associated with prostate cancer.
[0011] [0011] In a set of modalities, a method is provided for determining a probability of an event associated with prostate cancer. The method comprises receiving, via an input interface, information for a plurality of blood markers, wherein the information for the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a value of total PSA (tPSA), an intact PSA value (iPSA) and a human kallikrein 2 (kK2) value, to evaluate, using at least one processor, a logistic regression model based, at least in part, on the information received, for determining a probability of an event associated with prostate cancer in a person. The evaluation of the logistic regression model comprises the determination of the probability of the event associated with prostate cancer based, at least in part, on the tPSA value, the iPSA value, the hK2 value and a ratio between the fPSA value and the tPSA value and the issuance of an indication of the probability of the event associated with prostate cancer.
[0012] [0012] In one set of embodiments, a computer-readable storage medium is provided encoded with a plurality of instructions which, when executed by a computer, execute a method of determining a probability of an event associated with the prostate cancer. The method comprises receiving, through an input interface, information for a plurality of blood markers, wherein the information for the plurality of blood markers includes a free prostate specific antigen (fPSA) value. , a total PSA value (tPSA), an intact PSA value (iPSA) and a human kallikrein 2 (kK2) value, to evaluate, using at least one processor, a logistic regression model based on at least in part, on the information received, for determining a probability of an event associated with prostate cancer in a person. The evaluation of the logistic regression model comprises the determination of the probability of the event associated with prostate cancer based, at least in part, on the tPSA value, the iPSA value, the hK2 value and a ratio between the fPSA value and the tPSA value and the issuance of an indication of the probability of the event associated with prostate cancer.
[0013] [0013] In one set of modalities, a computer is provided for determining a probability of an event associated with prostate cancer. The computer includes an input interface
[0014] [0014] In a set of modalities, a method is provided for determining a probability of an event associated with prostate cancer. The method comprises receiving, via an input interface, information for a plurality of blood markers, wherein the information for the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a value of Total PSA (tPSA), an intact PSA (iPSA) value and a human kallikrein 2 (kK2) value. The method further comprises evaluating, using at least one processor, a logistic regression model based at least in part on the information received, for determining a probability of an event associated with prostate cancer in a person. The evaluation of the logistic regression model comprises the determination of a nonlinear term for tPSA by raising the value of tPSA to a first exponent, the determination of a nonlinear term for fPSA by raising the value of fPSA to a second exponent. and determining the probability of the event associated with prostate cancer based, at least in part, on the tPSA value, the fPSA value, the iPSA value, the hK2 value, the nonlinear term for tPSA, and the term nonlinear for fPSA. The method further comprises issuing an indication of the probability of the event associated with prostate cancer.
[0015] [0015] In one set of embodiments, a computer-readable storage medium is provided encoded with a plurality of instructions which, when executed by a computer, execute a method of determining a probability of an event associated with the prostate cancer. The method comprises receiving information for a plurality of blood markers, wherein the information for the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a total PSA (tPSA) value, an intact PSA (iPSA) value and a human kallikrein 2 (kK2) value. The method additionally comprises the evaluation of a logistic regression model based, at least in part, on the information received, to determine a probability of an event associated with prostate cancer in a person. The evaluation of the logistic regression model comprises the determination of a nonlinear term for tPSA when raising the value of tPSA to a first exponent, the determination of a nonlinear term for fPSA when raising the value of fPSA to a second exponent. exponent and determining the probability of the event associated with prostate cancer based, at least in part, on the tPSA value, the fPSA value, the iPSA value, the hK2 value, the nonlinear term for tPSA, and the term nonlinear for fPSA. The method further comprises issuing an indication of the probability of the event associated with prostate cancer.
[0016] [0016] In one set of modalities, a computer is provided for determining a probability of an event associated with prostate cancer. The computer includes an input interface configured to receive information for a plurality of blood markers, wherein the information for the plurality of blood markers includes a free prostate specific antigen (fPSA) value and a PSA value. total (tPSA), an intact PSA (iPSA) value and a human kallikrein 2 (kK2) value. The computer also includes at least one processor programmed to evaluate a logistic regression model based, at least in part, on the information received, for determining a probability of an event associated with prostate cancer in a person. The evaluation of the logistic regression model comprises the determination of linear slot terms for tPSA, determination of linear slot terms for fPSA, determination of a first value for tPSA based, at least in part, on the value of received tPSA and the linear slot terms determined for tPSA, determining a second value for fPSA based at least in part on the received fPSA value and linear slot terms determined for fPSA and determining of the probability of the event associated with prostate cancer based, at least in part, on the first value and the second value. The computer also includes an output interface configured to output an indication of the probability of the event associated with prostate cancer.
[0017] [0017] In a set of modalities, a method is provided for determining a probability of an event associated with prostate cancer. The method comprises receiving, via an input interface, information for a plurality of blood markers, wherein the information for the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a value of Total PSA (tPSA), an intact PSA (iPSA) value and a human kallikrein 2 (kK2) value. The method further comprises evaluating, using at least one processor, a logistic regression model based at least in part on the information received, for determining a probability of an event associated with prostate cancer in a person. The evaluation of the logistic regression model comprises determining linear slot terms for tPSA, determining linear slot terms for fPSA, determining a first value for tPSA based at least in part on the value of tPSA received and the linear slot terms determined for tPSA, determining a second value for fPSA based at least in part on the received fPSA value and linear slot terms determined for fPSA, and determining the probability of the event associated with prostate cancer based, at least in part, on the first and second values. The method further comprises issuing an indication of the probability of the event associated with prostate cancer.
[0018] [0018] In one set of embodiments, a computer-readable storage medium is provided encoded with a plurality of instructions which, when executed by a computer, execute a method of determining a probability of an event associated with the prostate cancer. The method comprises receiving information for a plurality of blood markers, wherein the information for the plurality of blood markers includes a free prostate specific antigen value.
[0019] [0019] In a set of modalities, a system for determining a high-grade cancer risk is provided. The system includes an input interface configured to receive information from a plurality of blood markers, wherein the information for the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a of total PSA (tPSA), an intact PSA (iPSA) value and an hK2 value. The system also includes at least one processor programmed to input the received values into a logistic regression model, where at least the tPSA value and the fPSA values are incorporated into the logistic regression model using both linear and nonlinear terms. and to evaluate the logistic regression model to determine the risk of high-grade cancer.
[0020] [0020] In a set of modalities, a system is provided for determining a probability of an event associated with prostate cancer in a person.
[0021] [0021] In a set of modalities, a method is provided for determining a probability of an event associated with prostate cancer in a person. The method involves providing a microfluidic sample analyzer comprising a housing, an opening in the housing configured to receive a cassette having at least one microfluidic channel, wherein the housing includes a component configured to interface with a component. coupling in the cassette to detect the cassette within the cassette and a pressure control system positioned within the casing, whose pressure control system is configured to pressurize at least one microfluidic channel in the cassette to move the sample. - tracing through at least one microfluidic channel. The microfluidic sample analyzer also includes an optical system positioned within the housing, which optical system includes at least one light source and at least one detector spaced from the light source, wherein the light source is configured to pass light through the cassette. when the cassette is inserted into the sample analyzer and wherein the detector is positioned opposite the light source to detect the amount of light passing through the cassette, and a user interface associated with the housing to receive at least the age of a person. The method involves determining the information for a plurality of blood markers using the microfluidic sample analyzer, wherein the information for the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a total PSA value (tPSA) and an intact PSA value (iPSA) and the evaluation, using at least one processor,
[0022] [0022] In a set of modalities, a system is provided. The system includes a device comprising a first analysis region comprising a first binding partner and a second analysis region comprising a second binding partner, wherein the first binding partner is adapted to bind to at least one of free prostate-specific antigen (fPSA), intact prostate-specific antigen (iPSA) and total PSA (tPSA) and wherein the second binding partner is adapted to bind at least one of the fPSA , iPSA and tPSA. The system includes a detector associated with the first and second analysis regions and a processor programmed to evaluate a logistic regression model based, at least in part, on information received from the detector, to determine a probability of occurrence. an event associated with prostate cancer in a person, in which the logistic regression model evaluation comprises grading each of a plurality of variables by a different coefficient value to produce graded variables, and the sum of the values for the graded variables used to produce the probability of the event associated with prostate cancer in a person, where the plurality of
[0023] [0023] In a set of modalities, a method is provided. The method comprises introducing a sample into a device which comprises a first analysis region comprising a first binding partner and a second analysis region comprising a second binding partner, wherein the first binding partner is adapted. to bind at least one of free prostate-specific antigen (fPSA), intact prostate-specific antigen (iPSA) and total PSA (tPSA) and wherein the second binding partner is adapted to bind to at least one other among fPSA, iPSA and tPSA. The method involves allowing anyone among the sample's fPSA, iPSA and/or tPSA to bind to the first and/or second binding partners in the first and second analysis regions, determining a characteristic of the fPSA, iPSA and /or tP-SA using one or more detectors associated with the first and second analysis regions, inputting the fPSA, iPSA and/or tPSA characteristics into a processor programmed to evaluate a logistic regression model with based, at least in part, on information received from at least one detector, for the determination of a probability of an event associated with prostate cancer in a person, where the evaluation of the logistic regression model comprises grading each of a plurality of variables by a different coefficient value to produce graded variables, and summing the values for the graded variables used to produce the probability of the event associated with prostate cancer in a person, where the plurality of variables includes age and at least two variables included in the information received from the detector and is selected from the group consisting of fPSA, iPSA and tPSA, and the determination of the probability of the event associated with cancer of prostate.
[0024] [0024] In a set of modalities, a device is provided. The device includes a microfluidic system comprising a first microfluidic channel that includes at least an inlet and an outlet, a first reagent stored in the first microfluidic channel, a seal that covers the inlet of the first microfluidic channel, and a seal that covers the first microfluidic channel. the outlet of the first microfluidic channel for storing the first reagent in the first microfluidic channel and a second microfluidic channel that includes at least an inlet and an outlet. The device also includes a first analysis region, a second analysis region, and a third analysis region, wherein each of the analysis regions includes one of an anti-iPSA specific capture antibody, an anti-fPSA specific capture antibody. and an anti-tPSA specific capture antibody, wherein one or more of the first, second and third analysis regions are in fluid communication with the second microfluidic channel. The device also includes a fluidic connector that can be connected to the microfluidic system, wherein the fluidic connector comprises a fluid passageway that includes an inlet for the passage of fluid and an outlet for the passage of fluid, wherein upon connection , the inlet to the fluid passageway connects to the outlet of the first microfluidic channel to allow fluid communication between the fluid passageway and the first microfluidic channel, and the outlet to the fluid passageway connects to the inlet of the second microfluidic channel to allow fluid communication between the fluid passage and the second microfluidic channel, wherein where the first and second microfluidic channels are not in fluid communication with each other an absent connection through the fluidic connector. The device also includes a source of a metal colloid conjugated to an antibody that binds to anti-PSA.
[0025] [0025] In a set of modalities, a method is provided for obtaining a probability of an event using a logistic regression model to predict a man's risk of developing prostate cancer. The method comprises the steps of: a) provision of a logistic regression model obtained using the multivariable logistic regression of data from a series of male persons, whose said data comprise, for each male person of said series of male persons, prostate cancer status data and data, and precedent data of said prostate cancer status, comprising age; and determinations of blood markers, total prostate specific antigen (tPSA), free PSA (fP-SA), intact PSA (iPSA) and, optionally, human kallikrein 2 (hK2) from blood samples of said males, in which said logistic regression model is generated using the formula:  π  j log   = ∑ β i xi + c 1− π  i =1 where π is the probability of said event, βi is the coefficient for the variable xi for the variables of j that comprise age, tPSA, fPSA, iPSA and, optionally, hK2, respectively, to obtain said logistic regression model; b) provision of the age of a male in years; c) determining said blood markers i) tPSA, ii) fPSA, iii) iPSA, iv) optionally hK2, respectively, from a blood sample of said male; d) use of said logistic regression model using said age provided from step b) and said blood markers determined from step c) to obtain said probability of said event of said male to  π  i ) define using the formula: y = log   e  1− π   and y  ii) obtain said probability as π =  y   1+ e 
[0026] [0026] The feature for the method is that, in said logistic regression model, said risk for cancer is based on tPSA alone if tPSA is ≥ 15 ng/ml, preferably ≥ 20 ng/ml and with maximum preference ≥ 25 ng/ml.
[0027] [0027] Another objective of the present invention is to provide a method for predicting the volume of the prostate gland using a linear regression model.
[0028] [0028] Embodiments of the present invention provide a method for predicting the volume of the prostate gland using a linear regression model, wherein said method comprises the steps of: a) providing a linear regression model obtained using regression linear of data from a series of male persons, said data comprising, for each male person from said series of male persons: i) data on the volume of the prostate gland and ii) the data, the previous data on the volume of the prostate gland, which comprises age; and determinations of blood markers: total prostate specific antigen (tPSA), free PSA (fPSA), intact PSA (iPSA) and optionally, human kallikrein 2 (hK2), from blood samples from said persons in the male, in which said linear regression model is generated using the formula: j V = ∑ β i xi + ci =1 where V is the volume of the prostate gland, βi is the coefficient for the variable xi; for the variables of j comprising age, tPSA, fPSA, iPSA and, optionally, hK2, respectively, to obtain said linear regression model; b) provision of the age of a male in years; c) determining said blood markers, tPSA, fPSA, iPSA and optionally, hK2, respectively, from a blood sample from said male; d) employing said linear regression model using said age provided from step b) and said blood markers determined from step c) to obtain said predicted prostate volume of said male.
[0029] [0029] The feature for the method is that, in said linear regression model, said risk for cancer is based on tPSA alone if tPSA is ≥ 15 ng/ml, preferably ≥ 20 ng/ml and with the maximum preferably ≥ 25 ng/ml.
[0030] [0030] Other advantages and novel features of the present invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying figures. In cases where this descriptive report and a document incorporated by reference include conflicting and/or inconsistent descriptions, this descriptive report will prevail. If two or more documents incorporated by reference include descriptions that conflict and/or are inconsistent with each other, then the document that has the most recent effective date will prevail. BRIEF DESCRIPTION OF THE DRAWINGS
[0031] [0031] Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying figures, which are schematic and do not lend themselves to being drawn to scale. In the figures, each identical or nearly identical component illustrated is typically represented by a single numeral. For purposes of clarity, not every component is identified in each figure, as well as not every component of each embodiment of the invention is shown where illustration is not necessary, to enable the skilled person to understand the invention. In the figures:
[0032] [0032] FIG. 1 illustrates a flowchart of a process for determining a probability of a positive cancer biopsy in accordance with some embodiments of the invention;
[0033] [0033] FIG. 2 illustrates a flowchart of a process for conditionally selecting a logistic regression model in accordance with some embodiments of the invention;
[0034] [0034] FIG. 3 shows a schematic illustration of a computer system in which some embodiments of the invention may be implemented;
[0035] [0035] FIG. 4 illustrates an exemplary network environment within which some embodiments of the invention may be used;
[0036] [0036] FIG. 5 is a block diagram showing a microfluidic system and a variety of components that may form part of a sample analyzer that may be used for the determination of one or more blood markers in accordance with some embodiments of the invention;
[0037] [0037] FIG. 6 is a perspective view of a sample analyzer and cassette that can be used for the determination of one or more blood markers according to some modes.
[0038] [0038] FIG. 7 is a perspective view of a cassette that includes a fluidic connector that can be used for the determination of one or more blood markers in accordance with some embodiments of the invention;
[0039] [0039] FIG. 8 is an exploded assembly view of a fluidic connector that may be used for the determination of one or more blood markers in accordance with some embodiments of the invention;
[0040] [0040] FIG. 9 is an exploded assembly view of a cassette that may be used for the determination of one or more blood markers in accordance with some embodiments of the invention;
[0041] [0041] FIG. 10 is a schematic view of a cassette that includes a fluidic connector that can be used for the determination of one or more blood markers in accordance with some embodiments of the invention;
[0042] [0042] FIG. 11A is a schematic view of a cassette that may be used for the determination of one or more blood markers in accordance with some embodiments of the invention;
[0043] [0043] FIGS. 11B-11F are schematic views of cassettes formed from multiple components that can be used for the determination of one or more blood markers in accordance with a set of modalities;
[0044] [0044] FIG. 12 is a schematic view of a portion of a sample analyzer that may be used for the determination of one or more blood markers in accordance with some embodiments of the invention;
[0045] [0045] FIG. 13 is a block diagram showing a sample analyzer control system associated with a variety of different components that can be used to determine
[0046] [0046] FIG. 14 is a schematic diagram showing a microfluidic system of a cassette that can be used for the determination of one or more blood markers in accordance with some embodiments of the invention; and
[0047] [0047] FIG. 15 is a diagram showing the measurement of optical density as a function of time showing the determination of one or more blood markers in accordance with some embodiments of the invention. DETAILED DESCRIPTION OF THE INVENTION
[0048] [0048] As discussed above, many conventional techniques for predicting a probability of prostate cancer and/or prostate gland volume are based, at least in part, on a clinical examination (e.g., a digital rectal examination or DRE) of the patient. Some embodiments described herein relate to methods and apparatus for determining a predicted probability of prostate cancer and/or prostate gland volume based, at least in part, on a panel of blood markers, without the need for a thorough clinical examination. As discussed in more detail below, the predicted probability of prostate cancer on biopsy and/or prostate gland volume is a reliable metric that can be useful in aiding in prostate biopsy-related decisions. .
[0049] [0049] Some modalities are directed at a computer system that includes at least one processor programmed to assess a risk of prostate cancer, where the risk of prostate cancer is determined based, at least in part, on , in the values for a plurality of blood markers. In some embodiments, the computer system may be implemented as an integrated system (e.g., on an analyzer and/or a chip/cassette) with one or more detectors that determine a value for one or more of the blood markers described herein. In other embodiments, the computerized system may include a computer located remotely from one or more detectors, and values for one or more of the blood markers described herein may be manually entered using a user interface and/or values can be received through a network interface communicably coupled to a network (eg the internet). At least one processor in the computer system may be programmed to apply one or more templates to the inputs received for the assessment of a prostate cancer risk by biopsy, as discussed in more detail below.
[0050] [0050] The models used according to some embodiments of the invention help to integrate a plurality of information for the input factors. For example, input factors can be PSA, free-to-total PSA ratio, and/or digital rectal examination (DRE) status. Continuing with this example, a first patient might have a PSA of 3 ng/ml, a free/total PSA ratio of 15% and a negative DRE, a second patient might have a PSA of 9.5 ng/ml, a free to total PSA ratio of 50% and a negative DRE and a third patient may have a PSA of 1.5 ng/ml, a free to total ratio of 29% and a positive DRE. For the first patient, a urologist may want to know whether a low (but not extremely low) free-to-total PSA ratio is sufficient to warrant biopsy, given that PSA is moderate and DRE is negative. For the second patient, the elevated PSA value would normally warrant an immediate biopsy, but the very high free-to-total PSA ratio may be a strong indication that the PSA rise is benign. For the third patient, a positive DRE is normally a very worrying sign, but may be insufficient evidence that a biopsy is needed, given the low PSA and normal free-to-total PSA ratio. As can be appreciated from the above, when a physician looks at these factors in isolation, it can be difficult to determine when a biopsy is necessary. Furthermore, as the number of input factors increases, the decision to perform a biopsy based on numerical information for the various input factors becomes even more complex.
[0051] [0051] [35]Patients and clinicians vary in how likely they are to opt for biopsy, depending on differences in how they assess early cancer detection versus the risks, harm, and inconvenience of biopsy. It is often impractical to enter such preferences using strict decision rules (e.g., perform biopsy if PSA is > 4 ng/ml OR if free to total ratio is < 15%) or using risk (eg, a prostate health index (PHI) score of 29). For example, if a man is opposed to medical procedures, it can be difficult to determine how high enough a PSA and/or PHI score should be to warrant a biopsy.
[0052] [0052] Instead of using strict decision rules, according to some modalities, at least one processor is programmed to use one or more statistical models to process a plurality of inputs to guide decisions about prostate biopsy. OK. Inputs to statistical models may include, but are not limited to, blood marker values, patient characteristics (e.g., age), and other appropriate information, for determining a probability that a positive cancer biopsy will prostate is found. This probability represents an interpretable range that can be used to guide biopsy decisions given patient and clinician preferences.
[0053] [0053] FIG. 1 illustrates a flowchart of a process in accordance with some embodiments of the invention. At action 110, one or more values for blood markers are received by at least one processor for processing using one or more of the techniques described herein. As described in more detail below, the blood marker value(s) may be received in any appropriate manner including, but not limited to, through a local input interface such as a keyboard. , touch screen, microphone or other input device, a networked interface that receives the value(s) from a device located away from the processor(s) or directly from one or more detectors that measure the blood marker value(s) [for example, in an implementation where the processor(s) is(are) integrated with a measurement device that includes one or more detectors].
[0054] [0054] In response to receipt of the blood marker value(s), the process proceeds to action 120, where at least one logistic regression model is evaluated to determine a probability of a positive biopsy for prostate cancer, where the probability is based, at least in part, on the blood marker value(s) received. As described in more detail below, information other than the values received from the blood marker (eg age, cancer grade etc.) can optionally be used as factors in determining a particular model to use and /or be used as input factors for the evaluation of a selected model.
[0055] [0055] After having determined a probability of a positive biopsy for cancer, the process proceeds to action 130, where the probability is sent to a user (e.g., a doctor, a patient) to guide a decision process whether a biopsy is necessary. The probability can be sent in any appropriate way.
[0056] [0056] As discussed above, some modalities are directed at a method for obtaining a probability of an event using a logistic regression model to predict prostate cancer risk and/or prostate gland volume for a given time. male person. In some embodiments, the method involves the inclusion of information from one or more kallikrein markers, namely, total prostate specific antigen (tPSA), free PSA (fPSA), intact PSA (iPSA), and human kallikrein 2 (hK2). Any appropriate logistic regression model can be used, and the techniques described here are not limited in this regard. In some embodiments, the probability of the event is determined according to equation (I), reproduced below: Probability = __eL_ (I) 1 + eL where the logit (L) is determined using any one of a plurality of models of logistic regression. Non-limiting examples of nine different types of logistic regression models that can be used according to the techniques described here include:
[0057] [0057] In this model, the ratio between free PSA and total PSA is replaced by the term free PSA.  fPSA  L = β 0 + β1 ( Age ) + β 2 ( tPSA ) + β 3   + β 4 ( iPSA ) + β 5 ( hK 2 )  tPSA 
[0058] [0058] In this model, the log of tPSA is replaced by the term tPSA to clarify the increased contribution of this predictor.  fPSA  L = β 0 + β1 ( Age ) + β 2 ( log [ tPSA]) + β 3   + β 4 ( iPSA ) + β 5 ( hK 2 )  tPSA 
[0059] [0059] In this model, additional nonlinear terms for tPSA and fPSA are included. In the example equation provided below, the square of tPSA is used to emphasize the direct relationship between this term and prostate cancer risk, and the square root of the term free/total PSA is used to reflect the inverse association of this term with risk. It should be appreciated, however, that higher order polynomial terms (eg, cubic) may also be included in some embodiments.  fPSA  L = β 0 + β1 ( Age ) + β 2 ( tPSA ) + β 3 ( fPSA ) + β 4 ( iPSA ) + β 5 ( hK 2 ) + β 6 ( tPSA 2 ) + β 7   tPSA   
[0060] [0060] In this model, linear grooves are added, with a single node at the median value. The grooves can be determined using the following equations: sp1(x) = x if x < node sp1(x) = node if x ≥ node sp2(x) = 0 if x < node sp2(x) = x if x ≥ node with the model being represented as: L = β0 + β1 (age) + β2 (tPSA) + β3 (fPSA) + β4 (iPSA) + β5 (hK2) + β6 (sp1[tPSA]) + β7 (sp2[tPSA] ) + β8 (sp1[fPSA]) + β9 (sp2[fPSA]) + β10 (sp1[iPSA]) + β11 (sp2[iPSA]) + β12 (sp1[hK2]) + β13 (sp2[hK2])
[0061] [0061] In this model, linear grooves are included only for tPSA and fPSA, to reduce the number of variables and simplify the model. L = β0 + β1 (age) + β2 (tPSA) + β3 (fPSA) + β4 (iPSA) + β5 (hK2) + β6 (sp1[tPSA]) + β7 (sp2[tPSA]) + β8 (sp1[fPSA] ]) + β9 (sp2[fPSA])
[0062] [0062] In this model, cubic slots are included for each term. In the example provided below, a cubic groove with four nodes is described. It should be appreciated, however, that a cubic slot utilizing any appropriate number of nodes including, but not limited to, five nodes, six nodes, seven nodes and eight nodes, may alternatively be used. Grooves can be determined using the following equations: knot 4 − knot1 sp [ x ]1 = max ( [ x ] − knot1, 0 ) − max ( [ x ] − knot 3, 0 ) 3 3 knot 4 − knot 3 knot 3 − knot1 + max ( [ x ] − knot 4, 0 ) 3 knot 4 − knot 3 knot 4 − knot 2 sp [ x ] 2 = max ( [ x ] − knot 2, 0 ) − max ([ x ] − knot 3, 0 ) 3 3 knot 4 − knot 3 knot 3 − knot 2 + max ( [ x ] − knot 2, 0 ) 3 knot 4 − knot 3
[0063] [0063] where node1 and node4 are external nodes for the cubic slot and node2 and node3 are internal nodes for the cubic slot. In some embodiments, the internal nodes are specified within the range between about 2 to about 5 and between about 5 to about 8 for tPSA, between about 0.25 to about 1 and between about 1 .0 to about 2.0 for fPSA, between about 0.2 to about 0.5, and between about 0.4 to about 0.8 for iPSA, and between about 0.02 to about 0.04 and between about 0.04 and about 0.08 for hK2. For example, in one implementation, values of 3.89 and 5.54 are used for internal nodes for tPSA, values of 0.81 and 1.19 are used for internal nodes for fPSA, values of 0.81 and 1.19 are used for internal nodes for fPSA. values of 0.3 and 0.51 are used for the internal nodes of iPSA and values of 0.036 and 0.056 are used for the internal nodes of kK2.
[0064] [0064] In certain embodiments, one or more internal nodes for tPSA may independently be in the range between about 3 and about 5, between about 3 and about 6, between about 2.5 and about 6, between about 2.5 and about 6. from 2.5 to about 6.5, between about 5 and about 8, between about 5.5 and about 8, between about 5 and about 9, between about 5 and about 10, between from about 1 to about 5, from about 1 to about 4, and from about 1 to about 3. Other ranges are also possible.
[0065] [0065] In certain embodiments, one or more internal nodes for fPSA may independently be in the range between about 0.1 and about 1.0, between about 0.1 and about 1.2, between about 0, 3 and about 0.8, between about 0.4 and about 0.9, between about 0.5 and about 1.2, between about 0.7 and about 1.4, between about
[0066] [0066] In certain embodiments, one or more internal nodes for iPSA may independently be in the range between about 0.05 and about 0.5, between about 0.1 and about 0.5, between about 0, 2 and about 0.5, between about 0.1 and about 0.8, between about 0.2 and about 0.8, between about 0.4 and about 0.8, between about 0.4 to about 1.0, between about 0.3 and about 0.6, between about 0.5 and about 1.0, and between about 0.6 and about 0.8. Other tracks are also possible.
[0067] [0067] In certain embodiments, one or more internal nodes para in hK2 may independently be in the range between about 0.01 and about 0.03, between about 0.01 and about 0.04, between about 0 .01 to about 0.05, between about 0.02 and about 0.05, between about 0.02 and about 0.06, between about 0.03 and about 0.05, between about from 0.4 to about 0.07, from about 0.04 to about 1.0, from about 0.5 to about 1.0, and from about 0.6 to about 1.0. Other tracks are also possible.
[0068] [0068] As discussed above, cubic grooves that incorporate any appropriate number of interior nodes (e.g., three, four, five, six interior nodes) can be used, and the example of a cubic groove including two interior nodes is provided for illustration only and not as a limitation. In embodiments that include more than two internal nodes, the nodes can be placed within one or more of the ranges discussed above or in some other appropriate range. For example, in some embodiments, nodes may be specified such that the length of the slot segments between each of the neighboring node pairs is essentially equal.
[0069] [0069] The model can be represented as:
[0070] [0070] In this model, cubic slots are included only for tPSA and fPSA, to reduce the number of variables and simplify the model.
[0071] [0071] In certain embodiments, the internal nodes for tPSA and fPSA are specified using one or more of the ranges described above with respect to the cubic slot model for all four tests. For example, internal nodes can be specified within the range from about 2 to about 5 and from about 5 to about 8 for tPSA and from about 0.5 to about 1 and from about 1.0 to about 1. about 1.5 for fPSA. For example, in one implementation, values of 3.89 and 5.54 are used for internal nodes for tPSA and values of 0.81 and 1.19 are used for internal nodes for fP-SA. It should be appreciated, however, that other values and/or ranges may alternatively be used. Furthermore, it should be appreciated that any number of nodes (eg, with the exception of four nodes) may alternatively be used in some embodiments, as discussed above with respect to the cubic slot model for all four trials.
[0072] [0072] The model can be represented as: L = β0 + β1 (age) + β2 (tPSA) + β3 (fPSA) + β4 (iPSA) + β5 (hK2) + β6 (sp1[tPSA]) + β7 (sp2) [tPSA]) + β8 (sp1[fPSA]) + β9 (sp2[fPSA])
[0073] [0073] In this model, cubic slots are applied to a data series in two parts to generate different coefficients (β) for use with patients who are less than or greater than or equal to a particular age (e.g. , 65 years old). Con-
[0074] [0074] The model can be represented as:
[0075] [0075] If age < 65: L = β0 + β1 (age) + β2 (tPSA) + β3 (fPSA) + β4 (iPSA) + β5 (hK2) + β6 (sp1[tPSA]) + β7 (sp2[ tPSA]) + β8 (sp1[fPSA]) + β9 (sp2[fPSA])
[0076] [0076] If age ≥ 65: L = β0 + β1 (age) + β2 (tPSA) + β3 (fPSA) + β4 (iPSA) + β5 (hK2) + β6 (sp1[tPSA]) + β7 (sp2[ tPSA]) + β8 (sp1[fPSA]) + β9 (sp2[fPSA])
[0077] [0077] Each of the above-described logistic regression models includes a plurality of input factors, including age and blood marker values for one or more of total PSA (tPSA), free PSA (fPSA) , intact PSA (iPSA) and human kallikrein 2 (hK2). In some cases, blood marker values are concentrations of blood markers in a patient sample. In some of the logistic regression models described above, the linear or cubic slots for the nonlinear terms are determined. It should be appreciated that higher order grooves may alternatively be used, as the techniques described herein are not limited in this regard.
[0078] [0078] For the logistic regression models described above, each of the terms is multiplied by a value of the corresponding coefficient (β). Coefficients can be determined in any appropriate way. For example, each of the models can be applied to a range of data including patient information, serum assay results, and biopsy results. A best fit of each of the models to the information in the data series for cancer prediction can be determined, and the coefficients that correspond to the best fit result can be used according to the techniques described here.
[0079] [0079] It should be appreciated that the particular coefficients used in an implementation of the techniques described herein may differ from those described in Table 1, as the values in Table 1 are provided merely as an illustration. Furthermore, in some modalities, different coefficients may be used for different patient populations and/or to determine probabilities of different outcomes. For example, different coefficients can be used for patients with different age groups, as described above for the age-stratified cubic slot model. Different coefficients can also be used to determine probabilities of a positive biopsy for different grades of cancer. For example, in the modalities used for determining a probability of a high-grade cancer (eg, Gleason score = 7), a positive biopsy may use different coefficients for one or more of the models than the modalities used for the determination. a probability of a positive low-grade biopsy for cancer. In addition, different coefficients can be used based, at least in part, on the fact that one or more of the blood marker values were determined from serum or plasma.
[0080] [0080] In some modalities, a first logistic regression model can be used when a value for one or more of the markers is above a certain threshold and a second logistic regression model can be used when the value is - low limit. FIG. 2 illustrates a process for selecting a logistic regression model based on a threshold in accordance with some embodiments of the invention. At action 210, a value for the blood marker total PSA (tPSA) is received. While the illustrative process of FIG. 2 use tPSA as a blood marker value to determine which logistic regression model to use, it should be appreciated that any other blood marker value, combination of blood marker values, or any other appropriate information may be alternatively used. Consequently, in some embodiments, at least one processor may be programmed to execute and select from a plurality of models based, at least in part, on one or more input values.
[0081] [0081] After having received the value for the tPSA, the process proceeds to action 212, where a logistic regression model is selected based, at least in part, on the value of tPSA received. For example, in one implementation, when the tPSA value is ≥ ng/ml, preferably ≥ 20 ng/ml, and most preferably ≥ 25 ng/ml, the logistic regression model can be based on tPSA alone ( for example, the "Simple Model (tPSA only)" described above can be used). For this implementation, when the tPSA value is less than a particular threshold (eg, less than 15 ng/ml), one or more of the other logistic regression models can be selected.
[0082] [0082] Continuing with the process of FIG. 2, after a model has been selected, the process proceeds to action 214, where it is determined whether the selected model is a complete model (e.g., includes all four kallikrein markers) or a partial model. which includes less than all markers in a kallikrein panel. If it is determined that the selected model is not a complete model, the process proceeds to action 216, where the probability of cancer is determined based solely on the tPSA value received, as described above. If the selected model is determined to be a complete model, the process proceeds to action 218, where the probability of cancer is determined based on the selected model using multiple blood markers. Regardless of the particular model that is selected, after the cancer probability is determined, the process proceeds to action 220, where the cancer probability is emitted, as discussed above with respect to FIG. 1.
[0083] [0083] In some embodiments of the invention, said event for which said probability is obtained is evidence of prostate cancer of the prostate extracted from an asymptomatic male person or a male person with lower urinary tract symptoms.
[0084] [0084] In some embodiments of the invention, the event for which said probability is obtained is evidence of high-grade prostate cancer, i.e., a Gleason score of 7 or more, on prostate biopsy taken from a male sex. asymptomatic male or a male with lower urinary tract symptoms. Typically, prostate cancer progression or prostate cancer status is defined as (i) Gleason score 7 or greater, (ii) Gleason grade 4 + 3 or greater, or (iii) Gleason score 8 or greater. most.
[0085] [0085] In many preferred embodiments, the male series data comprises one or more biopsy data selected from the group consisting of reason for biopsy, year of biopsy, number of cores from biopsy, number of cores positives, percentage of positives in each nucleus, and any possible combination of these.
[0086] [0086] As discussed above, in many preferred embodiments, blood markers are included in a logistic regression model employing up to two non-linear terms for at least one blood marker. In certain embodiments, blood markers are included in a logistic regression model employing up to three nonlinear terms for at least one blood marker. In certain embodiments, blood markers are included in a logistic regression model employing up to four non-linear terms for at least one blood marker. In certain embodiments, blood markers are included in a logistic regression model including up to five non-linear terms for at least one blood marker.
[0087] [0087] In some modalities, the logistic regression model can be recalibrated when the anticipated event rate in a representative of the target male population for which the event probability is to be obtained differs from the event rate. to the series of males for which the data were used to obtain the logistic regression model by definition according to equation (II):  P / (1 − P )  k =    p / (1 − p )  (II), where p is the event rate in said data from said series of males and P is the anticipated event rate in said target population, and the definition accordingly with equation (III): π Odds = 1 − π (III), where π is the original probability of the model and the definition according to equation (IV): Recalibrated odds = Oddsxk (IV) and obtaining a probability recalibrated, according to the formula (V):  Oddsrecalibrated  π recalibrated =    1 + Oddsrecalibrated  (V), where πrecalibrated is the probability of said event.
[0088] [0088] Some modalities are directed to methods and apparatus to predict the volume of the prostate gland using a linear regression model, in which said method comprises an action of a) provision of a linear regression model obtained using linear regression of data from a series of male persons, said data comprising, for each male person of said series of male persons: (i) data on the volume of the prostate gland and (ii) the data, the preceding data on the volume of the prostate gland, which comprises age; and determinations of blood markers including tPSA, fP-SA, iPSA and, optionally, hK2, from blood samples from said males. Said linear regression model can be generated using the formula (VI): j V = ∑ β i xi + c i =1 (VI),
[0089] [0089] where V is the volume of the prostate gland, βi is the coefficient for the variable xi for the variables of j that comprise age, tPSA, fPSA, iPSA and, optionally, hK2 , respectively, to obtain said linear regression model. The method further comprises an action of b) providing the age of a male in years, c) determining said blood markers tPSA, fPSA, iPSA and, optionally, hK2, respectively, from a sample of blood of said male and d) employing said linear regression model using said age provided from step b) and said blood markers determined from step c) to obtain said predicted prostate volume from said male person. In some modalities, the statistical model of said cancer risk is based on tPSA alone if tP-SA is ≥ 15 ng/ml, preferably ≥ 20 ng/ml, and most preferably ≥ 25 ng/ml. ml.
[0090] [0090] It should be appreciated that any appropriate logistic regression model that includes, but is not limited to, the models described above for determining a probability of prostate cancer
[0091] [0091] In some embodiments, the data from step a) (ii) to provide the logistic regression model or the linear regression model and the determination of blood markers of said male person comprise human kallikrein two.
[0092] [0092] In many preferred embodiments of the method of the invention in which the volume of the prostate gland is predicted, the volume of the prostate gland is provided as defined by transrectal ultrasound.
[0093] [0093] In many preferred embodiments of the method of the present invention, the data for each male of said male series to provide the logistic regression model or the linear regression model additionally includes the results of the digital rectal exam (DRE) and, consequently, the DRE is performed in the male person and the result obtained is used using the logistic regression model or the linear regression model, respectively, to obtain the said probability. DRE results are preferably expressed as binary values, ie normal = 0 and nodularity present = 1 with or without a second value for the volume estimate, ie small = 0, medium = 1 and big = 2.
[0094] [0094] In some preferred embodiments of the method of the present invention, the data from the series of males for obtaining the model comprise only the data of males with high levels, defined as the specific median of the age or older, tPSA and, consequently, the probabilities of the event or predicted prostate volume being obtained only for males with said elevated tPSA levels.
[0095] [0095] In preferred embodiments of the method of the present invention,
[0096] [0096] In some preferred embodiments of the method of the present invention, the logistic regression model or the linear regression model is provided, employing data from a series of males aged 40 to 75 years and, hence, the probability that the event or the predicted prostate volume was obtained from a male person aged 40 to 75 years.
[0097] [0097] In some preferred embodiments of the method of the present invention, the logistic regression model or the linear regression model is provided, employing data from a series of male subjects with a blood tPSA ≥ tertile of the maximum age, ≥ quartile of maximum age, ≥ quintile of maximum age, or ≥ decile of maximum age and, consequently, the probability that the event or predicted prostate volume was obtained from a male with tPSA in blood ≥ tertile of maximum age, ≥ quartile of maximum age, ≥ quintile of maximum age or ≥ decile of maximum age, respectively. As an example, for a sixty-year-old male, the corresponding total PSA values might be: 1.5 ng/ml, for ≥ maximum age tertile, 1.9 ng/ml, for ≥ quartile of maximum age, 2.1 ng/ml, for ≥ quintile of maximum age and 3 ng/ml, for ≥ decile of maximum age. Exemplifying computer system
[0098] [0098] An illustrative implementation of a computer system
[0099] [0099] To perform any of the functionalities described herein, the processor(s) 310 may execute one or more instructions, such as program modules, stored on one or more storage media. computer-readable (e.g., memory 320), which may serve as the non-transient computer-readable storage media that store instructions for execution by the processor 310. In general, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or perform particular abstract data types. The modalities can also be implemented in distributed computing environments where tasks are performed by remote processing devices that are linked through a communications network. In a distributed computing environment, program modules can be placed on local and remote computer storage media including memory storage devices.
[00100] [00100] Computer 300 may be operated in a networked environment that uses logical connections to one or more remote computers. One or more remote computers may include a computer
[00101] [00101] When used in a LAN-type network environment, the computer 300 can be connected to the LAN through a network interface or adapter. When used in a WAN-type networking environment, computer 300 typically includes a modem or other means for establishing communications over the WAN, such as the Internet. In a networked environment, program modules or portions thereof can be stored on the remote memory storage device.
[00102] [00102] Various inputs described herein for assessing a prostate cancer risk and/or determining a prostate gland volume may be received by the computer 300 over a network (e.g. LAN, WAN or some other network ) from one or more remote computers or devices that store data associated with the entries. One or more of the remote computers/devices may perform analysis on the remotely stored data before sending the analysis results as input data to the computer 300. Alternatively, the remotely stored data may be sent to the computer 300 as input. stored remotely, without any remote analysis. In addition, inputs may be received directly by a user of computer 300 using any of a number of input interfaces (e.g., input interface 340) that may be incorporated as components of computer 300.
[00103] [00103] Various outputs described here, including the output of a prostate cancer risk probability and/or prostate gland volume, can be provided visually on an output device (e.g. a screen) connected directly to the computer 300 or the output(s) may be provided to a remotely positioned output device connected to the computer 300 via one or more wired or wireless networks, once that the modalities of the invention are not limited in this respect. The outputs described here may be additionally or alternatively provided with the exception of using visual presentation. For example, the computer 300 or a remote computer to which an output is provided may include one or more output interfaces that include, but are not limited to, loudspeakers and vibrating output interfaces, for the provision of an indication of the exit.
[00104] [00104] It should be appreciated that while the computer 300 is illustrated in FIG. 3 as a single device, in some embodiments, the computer 300 may comprise a plurality of communicably coupled devices to perform some or all of the functionality described herein, and the computer 300 is only an illustrative implementation of a computer. which can be used in accordance with the embodiments of the invention. For example, in some embodiments, computer 300 may be integrated with and/or in electronic communication with the system shown in FIG. 5.
[00105] [00105] As described above, in some embodiments, the com-
[00106] [00106] Detector 420 can be configured to determine values for one or more of the blood markers used to determine a probability of prostate cancer and/or prostate gland volume, in accordance with one or more of the techniques described herein. Although detector 420 is illustrated in FIG. 4 as a single detector. It should be appreciated that detector 420 may be implemented as multiple detectors, each configured to determine one or more of the blood marker values used in accordance with one or more of the techniques described herein. Additional examples of detectors and detection systems are provided in more detail below (eg in FIG. 12).
[00107] [00107] In some embodiments, information corresponding to values for blood markers determined from detector 420 may be stored prior to sending values to computer 300. In such embodiments, information corresponding to values can be stored locally in local storage 420 communicably coupled to detector 420 and/or stored in network-connected central storage 440.
[00108] [00108] As described herein, in some embodiments, a system may include a processor or computer programmed to evaluate a logistic regression model in electronic communication with an analyzer to determine a probability of an event associated with breast cancer. prostate (for example, risk of prostate cancer and/or prostate gland volume). The analyzer can be adapted and arranged for the determination of one or more characteristics of blood markers to be received in the logistic regression model. In some embodiments, the analyzer is a microfluidic sample analyzer; for example, the analyzer can be adapted and arranged for the determination of a sample processed in a microfluidic device/cassette. It should be appreciated, however, that other types of analyzers may also be used (eg, analyzers for microwell ELISA-type assays) and that the systems described herein are not limited in this regard.
[00109] [00109] An example of such a system includes, in a set of embodiments, a microfluidic sample analyzer comprising a housing, an opening in the housing configured to receive a cassette having at least one microfluidic channel, in which the housing cro includes a component configured to interface with a docking component on the cassette to detect the cassette within the housing. The analyzer may also include a pressure control system positioned within the housing, which pressure control system is configured to pressurize at least one microfluidic channel in the cassette to move a sample through at least one microfluidic channel. . An optical system is positioned within the housing, which optical system includes at least one light source and at least one detector spaced from the light source, wherein the light source is configured to pass light through the cassette when the cassette is introduced into the sample analyzer and where the detector is positioned opposite the light source to detect the amount of light that passes through the cassette. The system may also include a user interface associated with the wrapper to receive at least a person's age and/or other information to receive in the linear regression model.
[00110] [00110] In certain embodiments, a processor is (or is adapted to be) in electronic communication with the microfluidic sample analyzer. In some cases, the processor is inside the analyzer housing. However, in other embodiments, the processor is not included within the analyzer housing, but can be accessed by an electronic device as described herein. The processor can be programmed to evaluate a logistic regression model based, at least in part, on information received from at least one detector, to determine a probability of an event associated with prostate cancer in a person, wherein the evaluation of the logistic regression model comprises grading each of a plurality of variables by a different coefficient value to produce graded variables, and summing the values for the graded variables used to produce the probability of the associated event with prostate cancer in a person, wherein the plurality of variables includes age and at least two variables included in the information received from the detector and is selected from the group consisting of fPSA, iPSA, and tPSA.
[00111] [00111] A method for determining a probability of an event associated with prostate cancer in a person may include, for example, the provision of a microfluidic sample analyzer. The microfluidic sample analyzer may comprise a housing, an opening in the housing configured to receive a cassette having at least one microfluidic channel, wherein the housing includes a component configured to interface with a coupling component in the cassette to detect the cassette inside the housing. The analyzer may additionally include a pressure control system positioned within the housing, which pressure control system is configured to pressurize at least one microfluidic channel in the cassette to move the sample through at least one microfluidic channel. An optical system is positioned within the housing, which optical system includes at least one light source and at least one detector spaced from the light source, wherein the light source is configured to pass light through the cassette when the cassette is inserted into the cassette. sample analyzer and in which the detector is positioned opposite the light source to detect the amount of light that passes through the cassette. The parser can also include a user interface associated with the wrapper to receive at least a person's age. The method may involve determining the information for a plurality of blood markers using the microfluidic sample analyzer, wherein the information for the plurality of blood markers includes an fPSA value, an iPSA value, a tPSA value. and, optionally, a value of hK2. The method may also involve the evaluation, using at least one processor, of a logistic regression model.
[00112] [00112] Another example of a system includes, in a set of embodiments, a device (e.g., a microfluidic cassette) that comprises a first analysis region comprising a first binding partner and a second analysis region comprising a second binding partner. The first binding partner is adapted to bind at least one of fPSA, iPSA and tPSA and the second binding partner is adapted to bind at least one of fPSA, iPSA and tPSA. In some embodiments, the device includes a third analysis region that includes a third binding partner adapted to bind the third among the fPSA, iPSA, and tPSA. Optionally, the device may include a fourth analysis region that includes a fourth binding partner adapted to bind hK2. The system includes a detector associated with the first and second analysis regions, and a processor programmed to evaluate a logistic regression model based, at least in part, on information received from the detector, to determine a probability of an event associated with prostate cancer in a person. The evaluation of the logistic regression model comprises the grading of each of a plurality of variables by a different coefficient value to produce graded variables, and the sum of the values for graded variables used to produce the probability of the associated event with prostate cancer in a person, where the plurality of variables includes age and at least two variables included in the information received from the detector and is selected from the group consisting of fPSA, iPSA and tPSA.
[00113] [00113] A method of determining the probability of the event associated with prostate cancer in such a system may include, for example, the actions of introducing a sample into a device (e.g., a microfluidic cassette) that comprises a first region analysis region comprising a first binding partner and a second analysis region comprising a second binding partner, wherein the first binding partner is adapted to bind at least one of the fPSA, iPSA and tPSA and wherein the second binding partner is adapted to bind to at least one other of fPSA, iPSA, and tPSA. In some embodiments, the device includes a third analysis region that includes a third binding partner adapted to bind to the third of fPSA, iPSA, and tPSA. Optionally, the device may include a fourth analysis region that includes a fourth binding partner adapted to bind hK2. The method may involve allowing any of the fPSA, iPSA and/or tPSA in the sample to bind with at least the first and/or second binding partner in the first and second analysis regions and determining of a feature of the fPSA, iPSA and/or tPSA using one or more detectors associated with the first and second analysis regions. The method involves inputting the fPSA, iPSA and/or tPSA characteristics into a processor programmed to evaluate a logistic regression model based, at least in part, on information received from at least one detector, to determine a probability of an event associated with prostate cancer in a person, wherein the logistic regression model assessment comprises grading each of a plurality of variables by a different coefficient value to produce graded variables, and the sum of the values for the graded variables used to produce the probability of the event associated with prostate cancer in a person, where the plurality of variables includes age and at least two variables included in the information received from the detector and is selected of the group consisting of fPSA, iPSA and tPSA. Consequently, the probability of the event associated with prostate cancer can be determined.
[00114] [00114] In certain embodiments, a device for determining blood markers (eg, fPSA, iPSA, tPSA and/or hK2) is provided. In some cases, the device may allow simultaneous determination of blood markers, for example, on a single cassette. The device may include a microfluidic system comprising a first microfluidic channel that includes at least an inlet and an outlet, a first reagent stored in the first microfluidic channel, and a seal that covers the inlet of the first microfluidic channel and a seal that covers the outlet of the first microfluidic channel, to store the first reagent in the first microfluidic channel. The device may additionally include a second microfluidic channel that includes at least an inlet and an outlet, a first analysis region, a second analysis region and a third analysis region. Each of the analysis regions can include one of an anti-iPSA-specific capture antibody, an anti-fPSA-specific capture antibody, and an anti-tPSA-specific capture antibody (and, optionally, an hK2-specific capture antibody). ). One or more of the first, second and third analysis regions may be in fluid communication with the second microfluidic channel. The device also includes a fluidic connector that can be connected to the microfluidic system, wherein the fluidic connector comprises a fluid passageway that includes an inlet for the passage of fluid and an outlet for the passage of fluid, wherein, upon connection, the inlet for the fluid passage connects to the outlet of the first microfluidic channel to allow fluid communication between the fluid passage and the first microfluidic channel, and the outlet for the fluid passage connects to the inlet of the second microfluidic channel to allow fluid communication between the fluid passage and the second microfluidic channel. The first and second microfluidic channels are not in fluid communication with each other without connection through the fluidic connector. The device may optionally include a source of a metal colloid conjugated to an antibody that binds to anti-PSA.
[00115] [00115] In some embodiments involving a device described herein, at least two (or at least three) of the first, second and third analysis regions are in fluid communication with the second microfluidic channel. In certain cases, each of the first, second and third (and optionally fourth) analysis regions is in fluid communication with the second microfluidic channel. In some examples, the first analysis region is in fluid communication with the second microfluidic channel, and the second analysis region is in fluid communication with a third microfluidic channel. The second and third analysis regions (as well as the second and third microfluidic channels) can, for example, be formed in the same layer of the substrate or in different layers of the substrate, as described herein. Furthermore, in some modalities, the third analysis region is in fluid communication with a fourth microfluidic channel. The third and fourth analysis regions (as well as the third and fourth microfluidic channels) can, for example,
[00116] [00116] Regardless of whether the analysis regions are formed in different layers of the substrate or in the same layer of the substrate, in some embodiments, the reagents can be stored and sealed in the first, second and/or third (and , optionally fourth) analysis regions, eg before using the device. Reagents can include, for example, an anti-iPSA specific capture antibody, an anti-fPSA specific capture antibody, and an anti-tPSA specific capture antibody (and, optionally, an hK2 specific capture antibody). When using the device (e.g. when connecting a fluidic connector to the microfluidic system) the first microfluidic channel can be placed in fluid communication with one or more of the first, second and third (and optionally, fourth) analysis regions. For example, the fluidic connector can connect to one or more inputs of the second, third and/or fourth microfluidic channels when connecting to the microfluidic system. Examples of device configurations are described in more detail below.
[00117] [00117] In certain devices described here, the analysis involves the use of a detection antibody that recognizes more than one of iPSA, fPSA, tPSA and hK2. For example, a detecting antibody can recognize PSA and hK2 and then a blocker can be used to interfere with PSA in such a way that only hK2 is detected. For example, in a particular embodiment, an analysis region may include an anti-hK2 capture antibody (which can also capture, for example, 5-10% tPSA and which can be stored in the analysis region prior to use). , as described herein), as well as blocking antibodies that block tPSA. An anti-hK2 detector antibody (which can also detect tPSA) can be used to detect the amount of hK2 binding. A different analysis region may include, for example, an anti-tPSA capture antibody (which can be stored in an analysis region prior to use, as described herein) that captures both fPSA and tPSA. Two different detector antibodies, for example an anti-tPSA detector antibody with a fluorescent tag for one wavelength and an anti-fPSA detector antibody with a fluorescent tag for a different wavelength, can be used. for detection. A different analysis region may include, for example, an anti-fPSA capture antibody and, optionally, an anti-iPSA capture antibody. Two different detector antibodies, for example an anti-fPSA detector antibody with a fluorescent tag for one wavelength and an anti-iPSA detector antibody with a fluorescent tag for a different wavelength, can be used. for detection.
[00118] [00118] In other embodiments, however, specific capture antibodies can be used for species detection. Each of the specific capture antibodies can be positioned in different analysis regions, as described herein. Advantageously, the use of specific capture antibodies and/or the placement of capture antibodies in different analysis regions may allow the use of the same detection antibody for the detection of each of the species. In some such embodiments, the same wavelength may be used to determine each of the species. This may allow the use of simplified detectors and/or optical components for detection. For example, in some embodiments, detection involves the accumulation of an opaque material in different analysis regions that can be determined at a particular wavelength, as described in more detail below.
[00119] [00119] For example, in a set of embodiments, an anti-iPSA-specific capture antibody, an anti-fPSA-specific capture antibody, and an anti-tPSA-specific capture antibody (and, optionally, an anti-tPSA-specific capture antibody). specific capture hK2) can be included in different analysis regions as described here, optionally together with the negative and positive controls. A detection antibody such as a gold-labeled antibody that is both anti-PSA and anti-hK2 can be used to detect each of iPSA, fP-SA, tPSA and/or hK2. In other embodiments, however, a mixture of gold-labeled antibodies, such as an anti-hK2 gold-labeled antibody, a gold-labeled anti-PSA antibody, and/or a gold-labeled anti-iPSA antibody, can be used for detection. In such a system, the same wavelength can be used to determine each of the species, and this can allow the use of simplified detectors and/or optical components for detection.
[00120] [00120] Examples of specific systems, devices and analyzers that can be used in combination with the modalities provided here are now described.
[00121] [00121] FIG. 5 shows a block diagram 510 of a microfluidic system and various components that may be included according to a set of embodiments. The microfluidic system may include,
[00122] [00122] In general, as used herein, a component that is "operatively associated with" one or more other components indicates that such components are directly connected to each other, in direct physical contact with each other without being connected. joined or joined or are not directly connected to each other or in contact with each other, but are interconnected mechanically, electrically (including through electromagnetic signals transmitted through space) or fluidly (for example, through channels such as piping) to cause or allow the components thereby associated to perform their intended functionality.
[00123] [00123] The components shown illustratively in FIG. 5, as well as other optional components such as those described herein, may be operatively associated with a 550 control system. In some embodiments, the control system may be used to control fluids and/or conduct control. of quality by using feedback from one or more events that occur in the microfluidic system. For example, the control system can be configured to receive input signals from one or more components, calculate and/or control various parameters, to compare one or more signals or signal patterns with signals pre-programmed in the control system. le and/or to send signals to one or more components to modulate fluid flow and/or control operation of the microfluidic system. The control system can also be optionally associated with other components such as an interface 554, an identification system 556, an external communication unit 558 (e.g. a USB) and/or other user components as described. in more detail below.
[00124] [00124] Cassette (eg, microfluidic device) 520 can have any configuration of channels and/or components suitable for performing a desired analysis. In one set of embodiments, cassette 520 contains stored reagents that can be used to perform a chemical and/or biological reaction (eg, an immunoassay), for example, as described in more detail in the present invention. The cassette may include, for example, an optional reagent inlet 562 in fluid communication with an optional reagent storage area 564. The storage area may include, for example, one or more channels and/or reservoirs which may, in some embodiments, be partially or completely filled with fluids (e.g., liquids and gases, including immiscible reagents such as reagent solutions and wash solutions, optionally separated by immiscible fluids, as described in more detail herein ). The cassette may also include an optional sample or reagent loading area 566, such as a fluidic connector that can be used to connect the reagent storage area 564 to an optional analysis region 568. The analysis region , which may include one or more areas for detecting a component in a sample (e.g. the analysis regions), may be in fluid communication with an optional waste area 570 and be coupled to output 572. In some cases, these and other features of the device may be formed on or in different components or layers of a cassette, as described in more detail herein. Thus, it should be appreciated that a cassette may include a single component or multiple components that are joined during use, such as a combination article with the fluidic connector joined, as described herein. In one set of embodiments, the fluid may flow in the direction of the arrows shown in the figure. Further description and examples of these and other components are provided herein.
[00125] [00125] In some embodiments, cassette sections 571 and 577 are not in fluid communication with each other prior to introducing a sample into the cassette. In some cases, sections 571 and 577 are not in fluid communication with each other prior to the first use of the cassette, and on the first use, the sections are brought into fluid communication with each other. In other embodiments, however, sections 571 and 577 are in fluid communication with each other prior to first use and/or prior to introducing a sample into the cassette. Other cassette configurations are also possible.
[00126] [00126] As shown in the exemplary embodiment illustrated in FIG. 5, one or more fluid flow sources 540 such as a pump and/or a vacuum or other pressure control system, valve system 528, detection system 534, temperature regulation system 541 and/or others components can be operatively associated with one or more of reagent inputs 562, reagent storage area 564, reagent or sample loading area 566, reaction area 568, waste area 570 , output 572 and/or other regions of cassette 520. Detection of processes or events in one or more regions of cassette can produce a signal or pattern of signals that can be transmitted to control system 550. Based on in the signal(s) received by the control system, this feedback can be used to manipulate the fluids within and/or between each of these regions of the microfluidic device, such as when controlling one or more of a pump, a vacuum, a valve system, a detection system, a temperature regulation system and/or other components.
[00127] [00127] Referring to FIG. 6, an embodiment of a microfluidic sample analyzer 600 is illustrated. As shown in the exemplary embodiment of FIG. 6, the analyzer includes a housing 601 that is configured to cover or retain the components of the analyzer which are discussed in more detail below. An opening 620 in the housing is configured to receive a cassette 520. As indicated in more detail below, the analyzer 600 may also include a user interface 650 positioned within the housing that is configured for a user to receive information. in the sample analyzer. In this particular embodiment, the user interface 650 includes a touch screen, but, as discussed below, the user interface may be configured differently.
[00128] [00128] In some embodiments, the analyzer may include a fluid flow source (e.g., a vacuum system) configured to pressurize the cassette, an identification reader configured to read information associated with the cassette, and a mechanical subsystem. nico which includes a component configured to interface with the cassette to detect the cassette within the housing. As mentioned above, an opening in the housing is configured to receive a cassette. Aperture 620 can be configured as an elongated notch. The aperture may be so configured to receive a substantially card-shaped cassette. It should be appreciated that in other embodiments, the opening may be differently formed and configured, as the invention is not thereby limited.
[00129] [00129] As mentioned above, the 600 microfluidic sample analyzer can be configured to receive a variety of 520 cassette types (eg, microfluidic devices). FIGS. 7-11F illustrate various exemplary embodiments of cassette 520 for use with analyzer 600. As shown, the cassette may be substantially card-shaped (i.e., similar to a card key), having a structure similar to a card. substantially rigid plate.
[00130] [00130] Cassette 520 can be configured to include a fluidic connector 720 which can grip one end of the cassette. In certain embodiments, the fluidic connector may be used to introduce one or more fluids (eg, a sample or a reagent) into the cassette.
[00131] [00131] In a set of modalities, the fluidic connector is used to fluidly connect two (or more) cassette channels during first use, which channels are not connected before first use. For example, the cassette may include two channels that are not in fluid communication prior to the first use of the cassette. Unconnected channels can be advantageous in certain cases,
[00132] [00132] As used herein, "prior to first use of cassette" means a time or time before the cassette is first used by an intended user after commercial sale. The first use can include any steps that require a user to manipulate the device. For example, the first use may involve one or more steps such as puncturing a sealed inlet to introduce a reagent into the cassette, connecting two or more channels to cause fluid communication between the channels, preparing the device (e.g., loading reagents into the device) prior to analyzing a sample, loading a sample into the device, preparing a sample in a region of the device, performing a reaction with a sample, detection of a sample etc. The first use, in this context, does not include manufacturing or other preparatory or quality control steps followed by the cassette manufacturer. Those skilled in the art are aware of the significance of first use in this context, and will be able to easily determine whether a cassette of the invention has already been used or not. In one set of embodiments, the cassette of the invention is disposable after first use (e.g., after completion of a trial) and this is particularly evident when such devices are used for the first time, as it is typically It is possible to use the devices again (eg to perform a second test) after the first use.
[00133] [00133] As shown in the exemplary embodiment illustrated in FIG. 8, fluidic connector 720 may include a substantially U-shaped channel 722, or a channel having any other suitable shape, which can store a fluid and/or reagent (e.g., a fluid sample and/or a or more detection antibodies) before being connected to the cassette. Channel 722 can be housed between two shield components that form the connector
[00134] [00134] FIGS. 9-11F illustrate various exemplified modalities-
[00135] [00135] Cassette 520 may also include top and bottom covers 710 and 712, which may, for example, be made of a transparent material. In some embodiments, a cap may be in the form of a biocompatible adhesive and may be made from a polymer (e.g., polyethylene (PE), a cyclic olefin copolymer (COC), polyvinyl chloride (PVC)) or an inorganic material. , for example. In some cases, one or more caps are in the form of an adhesive film (eg, an adhesive tape). For some applications, the material and dimensions of a lid are chosen such that the lid is substantially impermeable to water vapor. In other embodiments, the cap may be non-adhesive, but may thermally bond to the microfluidic substrate by the application of direct heat, laser energy, or ultrasonic energy. Any inlet and/or outlet of a cassette channel can be sealed (eg, by placing an adhesive over the inlet and/or outlet) using one or more caps. In some cases, the cap substantially seals one or more reagents stored in the cassette.
[00136] [00136] As illustrated, cassette body 704 may include one or more ports 714 coupled to channel 706 on cassette body
[00137] [00137] Cassette body 704 may optionally include a liquid containment region such as a waste area, including an absorbent material 717 (e.g., a waste pad). In some embodiments, the liquid containment region includes regions that capture one or more liquids flowing in the cassette, allowing gases or other fluids in the cassette to pass through the region. This may be accomplished, in some embodiments, by positioning one or more absorbent materials in the liquid containment region to absorb the liquids. This configuration can be useful for removing air bubbles from a fluid stream and/or for separating hydrophobic liquids from hydrophilic liquids. In certain embodiments, the liquid containment region prevents liquids from passing through the region. In some such cases, the liquid containment region can act as a waste area that captures
[00138] [00138] The schematic view of cassette 520 illustrated in FIG. 10 shows an embodiment in which cassette 520 includes a first channel 706 and a second channel 707 spaced from the first channel 706. In one embodiment, the channels 706, 707 range in major cross-sectional dimension from about 50 micrometers to about 50 micrometers. 500 micrometers, although other channel sizes and configurations can be used, as described in more detail below.
[00139] [00139] The first channel 706 may include one or more analysis regions 709 used to analyze the sample. For example, in an illustrative embodiment, channel 706 includes four analysis regions 709 (eg, connected in series or in parallel) that are used during sample analysis. As described herein, each of the analysis regions can be adapted to detect one or more of iPSA, fPSA, tPSA and/or hK2.
[00140] [00140] In certain embodiments, one or more analysis regions are in the form of snaking regions (eg regions surrounding snaking channels). A meandering region may, for example, be defined by an area of at least 0.25 mm2, at least 0.5 mm2, at least 0.75 mm2 or at least 1.0 mm2, wherein at least 25 %, 50% or 75% of the area of the meandering region comprises an optical detection path. A detector that allows the measurement of a single signal across more than one adjacent segment of the snaking region can be positioned adjacent to the snaking region. In some cases, channel 706 is fluidly connected to at least two serpentine regions connected in series.
[00141] [00141] As described herein, the first channel 706 and/or the second channel 707 can be used to store one or more reagents (e.g. capture antibodies to iPSA, fPSA, tP-SA and/or hK2) used to process and analyze the sample prior to the first use of the cassette. In some embodiments, dry reagents are stored in one channel or section of a cassette, and wet reagents are stored in a second channel or section of the cassette. Alternatively, two separate sections or channels of a cassette may contain dry reagents and/or wet reagents. Reagents can be stored and/or arranged, for example, as a liquid, a gas, a gel, a plurality of particles or a film. Reagents may be positioned in any suitable portion of a cassette, including, but not limited to, a channel, reservoir, a surface and/or a membrane, which may optionally be part of a reagent storage area. A reagent can be associated with a cassette (or components of a cassette) in any appropriate way. For example, reagents can be cross-linked (eg, covalently or ionically), adsorbed, or adsorbed (physisorbed) onto a surface within the cassette. In a particular embodiment, all or a portion of a channel (such as a fluid passage of a fluid connector or a cassette channel) is coated with an anticoagulant (eg, heparin). In some cases, a liquid is contained within a channel or reservoir of a cassette before first use and/or before introducing a sample into the cassette.
[00142] [00142] In some embodiments, stored reagents may include fluid plugs positioned in linear order so that, during use, as fluids flow into an analysis region, they are distributed in a predetermined sequence. A cassette designed to run an assay, for example, may include, in series, a rinse fluid, a labeled antibody fluid, a rinse fluid, and an amplification fluid, all stored in it. Although fluids are stored, they can be kept separate by means of substantially immiscible separating fluids (e.g., a gas such as air), so that fluid reactants that would normally react with each other when in contact can be stored in a common channel.
[00143] [00143] Reagents can be stored in a cassette for various periods of time. For example, a reagent can be stored for more than 1 hour, for more than 6 hours, for more than 12 hours, for more than 1 day, for more than 1 week, for more than 1 month, for more than 3 months, for more than 6 months, for more than 1 year or for more than 2 years. Optionally, the cassette can be treated in an appropriate manner in order to prolong storage. For example, cassettes that store the reagents contained therein can be vacuum sealed, stored in a dark environment, and/or stored at low temperatures (eg, below 0 degrees C). The duration of storage depends on one or more factors such as the particular reagents used, the form of the reagents stored (e.g. wet or dry), the dimensions and materials used to form the layer(s). ) of the substrate and lid, the method of adhesion of the layer(s) to the substrate and lid, and how the cassette is handled or stored as a whole. Storing a reagent (e.g. a liquid or a dry reagent) in a channel may involve sealing the inlet(s) and outlet(s) and
[00144] [00144] As illustrated in the exemplary embodiment shown in FIGS. 10 and 11A-11F, channels 706 and 707 may not be in fluid communication with each other until fluidic connector 720 is mated to cassette 520. In other words, the two channels, in some embodiments, are not in fluid communication. with each other before first use and/or before introducing a sample into the cassette. Particularly, as illustrated, the substantially U-shaped channel 722 of connector 720 can fluidly connect the first and second channels 706, 707 such that reactants in the second channel 707 can pass through the U-shaped channel. 522 and selectively move in the 709 analysis regions on the first channel
[00145] [00145] In some embodiments, a cassette described herein may include one or more microfluidic channels, although such cassettes are not limited to microfluidic systems and may relate to other types of fluidic systems. A cassette, device, apparatus or system that is microfluidic may include, for example, at least one fluid channel that has a maximum cross-sectional dimension of less than 1 mm and a length ratio of less than 1 mm. ment and the largest cross-sectional dimension of at least 3:1.
[00146] [00146] The cross-sectional dimension (eg a diameter) of the channel is measured perpendicular to the direction of fluid flow. Most of the fluid channels in the cassette components described herein have maximum cross-sectional dimensions of less than 2 mm and in some cases less than 1 mm. In one set of embodiments, all fluid channels in a cassette are microfluidic or have a cross-sectional dimension of less than 2 mm or 1 mm. In another set of embodiments, the maximum cross-sectional dimension of the channel is less than 500 microns, less than 200 microns, less than 100 microns, less than 50 microns, or less than 25 microns. In some cases, the dimensions of the channel may be chosen such that the fluid can flow freely through the article or substrate. The dimensions of the channel can also be chosen, for example, in order to allow a certain volumetric or linear flow rate of the fluid in the channel. Of course, the number of channels and the shape of the channels can be varied by any suitable method known to those skilled in the art. In some cases, more than one channel or capillary may be used.
[00147] [00147] A channel may include a feature on or in an article (eg, a cassette) that at least partially directs the flow of a fluid. The channel may be of any suitable cross-sectional shape (circular, oval, triangular, irregular, square or rectangular or similar) and may be covered or uncovered. In embodiments where it is completely covered, at least a portion of the channel may have a cross-section that is completely enveloped, or the entire channel may be completely enveloped along its entire length, with the exception of its inlet and outlet. A channel may also have an aspect ratio (between length and average cross-sectional dimension) of at least 2:1, more typically at least 3:1, 5:1, or 10:1 or more. .
[00148] [00148] The cassettes described herein may include channels or channel segments positioned on one or two sides of the cassette (or a substrate layer of the cassette). In some cases, channels are formed on one surface of the cassette. Channel segments can be connected by an intervention channel that passes through the cassette. In some embodiments, channel segments are used to store reagents on the device prior to first use by an end user. The specific geometry of the channel segments and the positions of the channel segments within the cassettes can allow fluid reagents to be stored for extended periods of time without mixing, even during routine handling of the cassettes, such as during transport. cassettes, and cassettes are subject to physical shock or vibration.
[00149] [00149] In certain embodiments, a cassette includes optical elements that are fabricated on one side of a cassette opposite a series of fluidic channels. An "optical element" is used to refer to a feature formed or positioned on or in an article or cassette that is provided for and is used to change direction (e.g., through refraction or reflection), focus, polarization and/or other property of incident electromagnetic radiation relative to light incident on the article or cassette in the absence of the element. For example, an optical element may comprise a lens (e.g. concave or convex), mirror, grating, groove or another feature formed or positioned on or in a cassette. A cassette without an original feature, however, would not constitute an optical element, even if one or more of the incident light properties could be altered upon interaction with the cassette. Optical elements can guide incident light passing through the cassette in such a way that most of the light is scattered away from specific areas of the cassette, such as the intervening portions between the s fluidic channels. By decreasing the amount of light incident on these intervening portions, the amount of noise in a detection signal can be decreased when using certain optical detection systems. In some embodiments, the optical elements
[00150] [00150] A cassette or portions thereof may be made of any material suitable for forming a channel or other component. Non-limiting examples of materials include polymers (e.g. polyethylene, polystyrene, polymethyl methacrylate, polycarbonate, poly(dimethylsiloxane), PVC, PTFE, PET and a cycloolefin copolymer), glass, quartz and silicon. The material that forms the cassette and all associated components (eg a lid) can be hard or flexible. Elements skilled in the art can readily select the appropriate material(s) based, for example, on their rigidity, inertia (e.g., freedom from degradation) to a fluid to be passed, their robustness. the temperature at which a particular device is to be used, its transparency/opacity to light (eg in the ultraviolet and visible regions) and/or the method used to manufacture the material's characteristics. For example, for extruded or injection molded articles, the material used may include a thermoplastic material (e.g. polypropylene, polycarbonate, acrylonitrile-butadiene-styrene, nylon 6), an elastomer (e.g. polyisoprene, isobutene-isoprene, nitrile, neoprene, ethylene-propylene, Hypalon, silicone), a heat-set material (eg epoxy, unsaturated polyesters, phenolics) or combinations thereof. As described in more detail below, cassettes that include two or more components or layers can be formed from
[00151] [00151] In some embodiments, the material and dimensions (e.g., thickness) of a cassette and/or lid are chosen such that they are substantially impermeable to water vapor. For example, a cassette designed to store one or more fluids therein prior to first use may include a lid comprising a material known to provide a high vapor barrier, such as sheet metal, certain polymers, certain ceramics, and combinations thereof. Examples of materials that have low water vapor permeability are provided below. In other cases, material is chosen based, at least in part, on cassette shape and/or configuration. For example, certain materials can be used to form planar devices, whereas other materials are more suitable for forming devices that are curved or irregularly shaped.
[00152] [00152] In some examples, a cassette is comprised of a combination of two or more materials, such as those listed above. For example, cassette channels can be formed from polystyrene or other polymers (eg, by injection molding) and biocompatible adhesive tape can be used to seal the channels. The biocompatible adhesive tape or flexible material may include a material known to enhance vapor barrier properties (e.g. sheet metal, polymers or other materials known to have high vapor barriers) and may optionally allow access to inlets and outlets when punching or removing adhesive tape. A variety of methods can be used to seal a microfluidic channel or portions of a channel or to bond multiple layers of a device, including, but not limited to, the use of adhesives, tape, glue, bonding, laminating. of materials or by mechanical methods (eg by means of clamping, pressure, etc.).
[00153] [00153] In some examples, a cassette comprises a combination of two or more separate components (eg layers or cassettes) assembled together. Independent channel networks (such as sections 571 and 577 of FIG. 5), which may optionally include reagents stored therein prior to first use, may be included on or in different components of the cassette. The separate components may be assembled together or associated with each other by any suitable device, as by the methods described herein, for example, to form a single (composite) cassette. In some embodiments, two or more channel networks are positioned on different components or cassette layers and are not fluidly connected prior to first use, but are fluidly connected upon first use, for example by using a fluidic connector. In other embodiments, two or more channel networks are fluidly connected before first use.
[00154] [00154] Advantageously, each of the different components or layers that form a composite cassette can be individually adapted depending on the designed function(s) of that component or layer. For example, in one set of embodiments, a component of a composite cassette can be adapted to store wet reagents. In some such embodiments, this component may be formed from a material that has a relatively low vapor permeability. In addition or alternatively, for example, depending on the quantity of fluids to be stored, the storage region(s) of this cassette can be made with dimensions in cross-section larger than the cassettes.
[00155] [00155] FIGS. 11B-11E show a device that may include multiple components or layers 520B and 520C that are combined to form a single cassette. As shown in these illustrative embodiments, component 520B may include a first side 521A and a second side 521B. Component 520C may include a first side 522A and a second side 522B. Device components or parts described herein as channels or other entities may be formed on, on or on the first side of a component, on a second side of a component and/or through the component in some embodiments. For example, as illustratively shown in FIG. 11C, component 520C may include a channel 706 that has an inlet and an outlet and can be formed from a first material. Channel 706 can be of any suitable configuration, as described herein, and can include, for example, one or more reagent storage regions, analysis regions, liquid containment regions, mixing regions, and so on. In some embodiments, channel 706 is not formed through the entire thickness of component 520B. That is, the channel can be formed on or on one side of the component. Channel 706 may optionally be enclosed by a cap, as described herein, such as an adhesive tape (not shown), another cassette component or layer, or other appropriate component. In other embodiments, the channel 706 is formed through the entire thickness of the component 520B, and caps are required on both sides of the cassette to enclose the channel. As described herein, different layers or components may include different analysis regions for determining the species within a sample. For example, capture antibodies for iPSA, fPSA, tPSA and/or hK2 can be positioned in different analysis regions, optionally in different components or layers of a cassette, as shown.
[00156] [00156] Component 520B may include channel 707 which has an inlet and an outlet and may be formed from a second material, which may be the same as or different from the first material. Channel 707 also
[00157] [00157] As illustratively shown in FIGS. 11D and 11E, components 520B and 520C may be substantially planar and may lie on top of each other. However, generally, two or more components that form a cassette can be in any configuration suitable for each other. In some cases, the components are adjacent to each other (eg, side by side, one on top of the other). The first components may completely overlap, or only portions of the components may overlap each other. For example, as illustratively shown in FIGS. 11D and 11E, component 520C may extend beyond component 520B such that a portion of component 520C is not overlapped or covered by component 520B. In some cases, this configuration may be advantageous when the 520C component is substantially transparent and requires light to travel through a portion of the component (e.g., a reaction area, an analysis region, or a detection region) and when the component 520B is opaque or less transparent than component 520C.
[00158] [00158] In addition, the first and second components may include any appropriate format and/or configuration. For example, in some embodiments, the first component includes a feature complementary to a feature of the second component so as to form a non-fluidic connection between the first and second components. Complementary features can, for example, help align the first and second components during assembly.
[00159] [00159] The first and second components can be integrally connected to each other in some embodiments. As used herein, the term "integrally connected", when referring to two or more objects, means those objects that do not become separated from one another during normal use, for example, cannot be separated manually; Separation requires at least the use of tools and/or the action of causing damage to at least one of the components, for example, by breaking, removing or separating the components joined together through adhesives or by iron. rams. Integrally connected components can be irreversibly joined together during normal use. For example, components 520B and 520C can be integrally connected using an adhesive or other bonding methods. In other embodiments, two or more components of a cassette may be reversibly joined together.
[00160] [00160] As described herein, in some embodiments, at least a first component and a second component that form a composite cassette may be formed of different materials. The system may be designed such that the first component includes a first material that aids or enhances one or more of the features of the first component. For example, if the first component is designed to store a liquid reagent (e.g. in a component channel) prior to first use by a user (e.g. for at least one day, one week, one month or one year) , the first material may be chosen to have a relatively low vapor permeability to reduce the amount of evaporation of the stored liquid over time. It should be understood, however, that the same materials may be used for multiple components (eg layers) of a cassette in some embodiments. For example, the first and second components of a cassette can be formed from a material that has a low permeability to water vapor.
[00161] [00161] In certain embodiments, the first and second components of a cassette have different degrees of optical clarity. For example, a first component can be substantially opaque and a second component can be substantially transparent. The substantially transparent component may be suitable for optical detection of a sample or analyte contained within the component.
[00162] [00162] In one set of embodiments, a material used to form a component (e.g., a first or second component) of a cassette has an optical transmission greater than 90% between 400 and 800 nm wavelength. light (eg light in the visible range). Optical transmission can be measured through a material having a thickness of, for example, about 2 mm (or in other embodiments, about 1 mm or about 0.1 mm). In some examples, optical transmission is greater than 80%, greater than 85%, greater than 88%, greater than 92%, greater than 94%, or greater than 96% between 400 and 800 nm in length of light wave. Another component of the device may be formed in a material that has an optical transmission less than 96%, less than 94%, less than 92%, less than 90%, less than 85%, less than than 80%, less than 50%, less than 30% or less than 10% between 400 and 800 nm wavelength of light.
[00163] [00163] As described herein, in some embodiments, a channel of a first component of a cassette is not in fluid communication with a channel of a second component of a cassette prior to first use by a user. For example, even after coupling the two components, as shown illustratively in FIG. 11D, channels 706 and 707 are not in fluid communication with each other. However, the cassette may additionally include other parts or components, such as the fluidic connector alignment member 702 (FIG. 11E), which may join the first and/or second components 520B and 520C or to other portions of the cassette. cassette. As described herein, the fluidic connector alignment element may be configured to receive and mate with the fluidic connector 720, which may allow fluid communication between the channels 706 and 707 of the first and second components, respectively. For example, the fluidic connector may include a fluid passage which includes an inlet for the passage of fluid and an outlet for the passage of fluid, wherein the inlet for the fluid passage may be fluidly connected to the outlet of the channel 706. , and the outlet for the passage of fluid can be fluidly connected to the inlet of channel 707 (or vice versa). The fluidic connector fluid passage may be of any suitable length (eg, at least 1 cm, at least 2 cm, at least 3 cm, at least 5 cm) to connect the channels. The fluidic connector can be part of a kit along with a cassette and can be packaged in such a way that the fluidic connector does not fluidly connect channels 706 and 707.
[00164] [00164] A fluidic connector may have any suitable configuration with respect to a cassette or components of a cassette. As illustratively shown in FIG. 11E, when connecting the fluidic connector to the cassette, the fluidic connector may be positioned on one side of a component (eg, component 520B) opposite another component (eg, component 520C). In other embodiments, a fluidic connector may be positioned between two components of a cassette. For example, the fluidic connector may be a component or a layer positioned between (eg, compressed between) two cassette components. Other configurations are also possible.
[00165] [00165] While much of the description of the present invention refers to a cassette that has one or more components or layers including channel networks, in other embodiments, a cassette may include more than 2, more than 3, or more than 4 such components. or layers. For example, as illustratively shown in FIG. 11F, a cassette may include components 520B, 520C, 520D, and 520E, each including at least one channel or network of channels. In some examples, the channel(s) of one or more components (e.g. 2, 3, or all components) may be fluidly disconnected before first use, but may be fluidly connected when first used, for example by using a fluidic connector. In other embodiments, the channel(s) of one or more components (eg, 2, 3, or all components) are fluidly connected prior to first use.
[00166] [00166] As described herein, each of the components or layers of a cassette can be designed to have a specific function that is different from a function of another component of the cassette. In other embodiments, two or more components may have the same function. For example, as shown in the illustrative embodiment of FIG. 11F , each of the components 520C, 520D and 520E may have one or multiple analysis regions 709 connected in series. When connecting the fluidic connector 722 to the composite cassette, portions of a sample (or multiple samples) can be introduced into the channel network at each of the components 520C, 520D, and 520E to perform multiple analyses. For example, each of the analysis regions may include one or more binding partners to detect one or more of iPSA, fPSA, tPSA and/or hK2 (e.g. capture antibodies to iPSA, fPSA, tPSA and/or hK2). As described herein, in some embodiments, the use of capture antibodies and/or the separation of specific capture antibodies into different analysis regions may allow the use of the same detection antibody to detect each of the species. In some such embodiments, the same wavelength may be used to determine each of the species. This may allow the use of simplified detectors and/or optical components for detection. For example, in some embodiments, detection involves the accumulation of an opaque material in different analysis regions that can be determined at a particular wavelength.
[00167] [00167] In some embodiments, at least the first and second components of a cassette may be part of a device or kit used for the determination of a particular chemical or biological condition. The device or kit may include, for example, a first component, which comprises a first channel in a first material, which first channel includes an inlet, an outlet and, between the first inlet and outlet, at least a portion having a dimension in cross section greater than 200 microns. The device or kit may also include a second component comprising a second channel in a second material, wherein the second channel includes an inlet, an outlet and, between the second inlet and outlet, at least a portion that has a cross-sectional dimension of less than 200 microns. In some cases, the device or kit is packaged in such a way that the first and second components are connected together. For example, the first and second components can be integrally connected to each other. In other embodiments, the first and second components are reversibly joined together. The device or kit may additionally include a fluidic connector for fluidly connecting the first and second channels, which fluidic connector comprises a fluid passageway, including an inlet for the passage of fluid and an outlet for the passage of fluid, wherein the inlet for the fluid passage can be fluidly connected to the outlet of the first channel, and the outlet for the fluid passage can be fluidly connected to the inlet of the second channel. In some embodiments, the device or kit is packaged in such a way that the fluidic connector does not fluidly connect the first and second channels in the package. Upon first use of the device by an intended user, the fluidic connector can be utilized to bring the first and second channels into fluid communication with each other.
[00168] [00168] A cassette described herein may be of any volume suitable for performing an analysis such as a chemical and/or biological reaction or other process. The entire volume of a cassette includes, for example, all reagent storage areas, analysis regions, liquid containment regions, waste areas, as well as any fluid connectors and fluidic channels associated therewith. In some embodiments, small amounts of reagents and samples are used, and the entire volume of the fluidic device is, for example, less than 10 ml, 5 ml, 1 ml, 500 µl, 250 µl, 100 µl, 50 µl, 25 µl, 10 µl, 5 µl or 1 µl.
[00169] [00169] A cassette described herein may be portable and, in some embodiments, may be loaded manually. The length and/or width of the cassette can be, for example, less than or equal to 20 cm, 15 cm, 10 cm, 8 cm, 6 cm or 5 cm. The thickness of the cassette can be, for example, less than or equal to 5 cm, 3 cm, 2 cm, 1 cm, 8 mm, 5 mm, 3 mm, 2 mm or 1 mm. Advantageously, portable devices may be suitable for use in diagnostic testing facilities.
[00170] [00170] It is to be understood that the cassettes and their respective components described herein are exemplary and that other configurations and/or types of cassettes and components may be used with the systems and methods described herein.
[00171] [00171] The methods and systems described herein may involve a variety of different types of analysis and may be used for the determination of a variety of different samples. In some cases, an analysis involves a chemical and/or biological reaction. In some embodiments, a chemical and/or biological reaction involves binding. Different types of binding may occur in the cassettes described herein. Binding may involve interaction between a pair of corresponding molecules (eg, binding partners) that exhibit mutual affinity or binding ability, typically specific or non-specific binding or interaction, including biochemical, physiological, and/or biochemical interactions. pharmaceuticals. Biological binding defines a type of interaction that occurs between pairs of molecules (eg, binding partners) including proteins, nucleic acids, glycoproteins, carbohydrates, hormones, and more. Specific examples include antibody/antigen, antibody fragment/antigen, antibody, antibody/hapten, enzyme/cofactor antibody fragment/hapten, binding protein/substrate, carrier protein/substrate, lectin/carbohydrate, receptor/hormone, receptor/effector, nucleic acid complementary strands, protein/nucleic acid repressor/inducer, ligand/cell surface receptor, virus/ligand, etc. Binding can also occur between proteins or other components and cells. Furthermore, the devices described herein can be used for other fluid analysis (which may or may not involve binding and/or reactions) such as component detection, concentration etc.
[00172] [00172] In some cases, a heterogeneous reaction (or assay) may occur in a cassette; for example, a binding partner may be associated with a surface of a channel and the complementary binding partner may be present in the fluid phase. Other solid phase assays that involve the affinity reaction between proteins or other biomolecules (eg, DNA, RNA, carbohydrates) or molecules that do not occur naturally can also be performed. Non-limiting examples of typical reactions that can be performed on a cassette include chemical reactions, enzymatic reactions, immuno-based reactions (eg, antigen-antibody), and cell-based reactions.
[00173] [00173] Typical sample fluids include physiological fluids such as human or animal whole blood, blood serum, blood plasma, semen, tears, urine, sweat, saliva, cerebrospinal fluid, vaginal secretions; in vitro fluids used in research or environmental fluids such as aqueous fluids suspected of being contaminated by analyte.
[00174] [00174] In some embodiments, one or more reagents that can be used for the determination of an analyte from a sample (e.g., a binding partner of the analyte to be determined) are stored in a channel or chamber of a cassette before first use in order to perform a specific test or trial. In cases where an antigen is analyzed, an antibody or a corresponding aptamer may be the binding partner associated with a surface of a microfluidic channel. If an antibody is the analyte, then an appropriate antigen or aptamer may be the binding partner associated with the surface. When a disease condition is being determined, it may be preferable to place the antigen on the surface and test it for an antibody that is produced in the individual. It should be appreciated that while antibodies are mentioned herein, antibody fragments may be used in combination with or in place of antibodies.
[00175] [00175] In some embodiments, a cassette is adapted and arranged to perform an analysis that involves accumulating an opaque material in a region of a microfluidic channel, exposing the region to light, and determining the transmission. light through the opaque material. An opaque material may include a substance that interferes with the transmittance of light at one or more wavelengths. An opaque material does not merely refract light, but reduces the amount of transmission through the material, for example, through the absorption or reflection of light. Different opaque materials or different amounts of an opaque material may allow a transmittance of less than, for example, 90, 80, 70, 60, 50, 40, 30, 20, 10 or 1 percent of the light illuminating the opaque material. Examples of opaque materials include molecular layers of metal (eg, elemental metal), ceramic layers, polymeric layers, and layers of an opaque substance (eg, a paint). The opaque material may, in some cases, be a metal that can be deposited non-electrolytically. These metals may include, for example, silver, copper, nickel, cobalt, palladium and platinum.
[00176] [00176] An opaque material that forms in a channel may include a series of independent, discontinuous particles that together form an opaque layer, but in one embodiment, it is a continuous material that generally assumes a planar shape. The opaque material may have a dimension (e.g., a width of length), for example, greater than or equal to 1 micron, greater than or equal to 5 microns, greater than 10 microns, greater than or equal to 25 microns or greater than or equal to 50 microns. In some cases, the material
[00177] [00177] In one set of modalities, a cassette described herein is used to perform an immunoassay (e.g., for the determination of tPSA, iPSA, fPSA, and/or hK2) and optionally uses silver enhancement for the signal amplification. In such an immunoassay, upon delivery of a sample containing a blood marker to be detected in the analysis regions, binding between the blood marker and the corresponding binding partner can occur. One or more reagents, which may optionally be stored in a channel of the device prior to use, may then flow over this binding pair complex. One of the stored reagents may include a solution that contains one or more metal colloids that bind to the antigen to be detected. For example, a gold-labeled antibody that is both anti-PSA and anti-hK2 can be used to detect each of iPSA, fPSA, tPSA and/or hK2. In another example, a mixture of gold-labeled antibodies, such as a gold-labeled anti-hK2 antibody, gold-labeled anti-PSA antibody, and/or gold-labeled anti-iPSA antibody, can be used for detection. Such reagents can be stored in the cassette, for example, before use. The metal colloid can provide a catalytic surface for the deposition of an opaque material, such as a layer of metal (eg, silver), on a surface of one or more analysis regions. The metal layer can be formed using a two-component system: a metal precursor (e.g., a solution of silver salts) and a reducing agent (e.g., hydroquinone, chlorohydroquinone, pyrogallol, methyl, 4 -aminophenol and phenidone), which can optionally be stored in different channels before use.
[00178] [00178] As a positive or negative pressure differential is applied to the system, silver salts and reducing solutions can mix (e.g., can melt at a channel intersection) and then flow over the analysis region. Therefore, if binding occurs between antibody and antigen in the analysis region, flow of the metal precursor solution through the region may result in the formation of an opaque layer, such as a silver layer, due to the presence of the metal precursor. catalytic metal colloid associated with the antibody-antigen complex. The opaque layer may include a substance that interferes with the transmittance of light at one or more wavelengths. An opaque layer that is formed in the channel can be detected optically, for example, by measuring a reduction in light transmittance across a portion of the analysis region (e.g., a serpentine channel region) compared to a portion of an area that does not include the antibody or antigen. Alternatively, a signal can be obtained by measuring the change in light transmittance as a function of time as the film is being formed in an analysis region. The opaque layer can provide an increase in assay sensitivity when compared to techniques that do not form an opaque layer. In addition, various amplification chemistries that produce optical signals (eg, absorbance, fluorescence, glow or evaporation chemiluminescence, electrochemiluminescence), electrical signals (eg, resistance or conductivity of metal structures created by a non-electrolytic process) ) or magnetic signals (eg magnetic beads) can be used to allow detection of a signal by a detector.
[00179] [00179] Various types of fluids can be used with the cassettes described here. As described herein, fluids may be introduced into the cassette on first use and/or stored within the cassette prior to first use. Fluids include liquids such as solvents, solutions and suspensions. Fluids also include gases and gas mixtures. When multiple fluids are contained in a cassette, the fluids may be separated by another fluid which is preferably substantially immiscible in each of the first two fluids. For example, if a channel contains two different aqueous solutions, a third fluid separating buffer may be substantially immiscible in both aqueous solutions. When aqueous solutions must be kept separate, substantially immiscible fluids that can be used as separators can include gases such as air or nitrogen or hydrophobic fluids that are substantially immiscible with aqueous fluids. Fluids can also be chosen based on the fluid's reactivity with adjacent fluids. For example, an inert gas such as nitrogen can be used in some embodiments and can help preserve and/or stabilize all adjacent fluids. An example of a substantially immiscible liquid for separating aqueous solutions is perfluorodecalin. The choice of a separating fluid can also be made based on other factors, including any effect that the separating fluid may have on the surface tension of adjacent fluid plugs. You may prefer to maximize the surface tension within any fluid plug to promote retention of the fluid plug as a single continuous unit under varying environmental conditions, such as variations in vibration, shock, and temperature. Separator fluids may also be inert to an analysis region to which the fluids will be provided. For example, if an analysis region includes a biological binding partner, a separating fluid such as air or nitrogen may have almost no effect on the binding partner. The use of a gas (e.g., air) as a separating fluid can also provide room for expansion within a channel of a fluidic device should the liquids contained in the device expand or contract due to changes such as these. such as variations in temperature (including freezing) or pressure.
[00180] [00180] The microfluidic sample analyzer may include a fluid flow source (e.g. a pressure control system) that can be fluidly connected to channels 706, 707, 722 to pressurize the channels to move the sample and/or other reagents through the channels. In particular, the fluid flow source may be configured to initially move a sample and/or reagent from the substantially U-shaped channel 722 to the first channel 706. The fluid flow source may also be used to monitor see reagents in the second channel 707 through the substantially U-shaped channel 722 and to the first channel 706. After the sample and reagents have passed through the analysis regions 709 and are analyzed, the fluid flow source 540 may be configured to move fluids to the absorbent material 717 of the cassette. In one embodiment, the fluid flow source is a vacuum system. It should be understood, however, that other sources of fluid flow such as valves, pumps and/or other components may be used. As described herein, in some embodiments, a vacuum source may be used to direct fluid flow. A vacuum source may include a pump, such as a solenoid operated diaphragm pump. In other embodiments, fluid flow may be directed/controlled through the use of other types of pumps or fluid flow sources. For example, in one embodiment, a syringe pump may be used to create a vacuum by pulling the syringe driver in an outward direction. In other embodiments, positive pressure is applied to one or more cassette inlets to provide a source of fluid flow.
[00181] [00181] In some embodiments, fluid flow occurs by applying a substantially constant non-zero pressure drop (ie, ∆P) across an inlet and outlet of a cassette. In one set of embodiments, an entire analysis is performed by applying a substantially constant non-zero pressure drop (ie, ∆P) across an inlet and outlet of a cassette. A substantially constant non-zero pressure drop can be obtained, for example, by applying a positive pressure at the inlet or a reduced pressure (eg a vacuum) at the outlet. In some cases, a substantially constant non-zero pressure drop is obtained when fluid flow does not occur predominantly by capillary forces and/or without the use of actuation valves (e.g., without changing a cross-sectional area of a flow channel). one flow of fluid from the cassette). In some embodiments, during essentially the entire analysis conducted in the cassette, a substantially constant non-zero pressure drop may be present, for example, crossing an inlet to an analysis region (which may be connected to a fluidic connector) and a downstream outlet from the analysis region (eg, an outlet downstream from a liquid containment region), respectively.
[00182] [00182] In one embodiment, a vacuum source is configured to pressurize a channel to about -60kPa (about 2/3 atmosphere). In another embodiment, the vacuum source is configured to pressurize a channel to about -30kPa. In certain embodiments, the vacuum source is configured to pressurize a channel, for example,
[00183] [00183] Once the cassette is positioned inside the analyzer, the fluid flow source can be attached to the cassette to ensure a fluid resistant connection. As mentioned above, the cassette may include a port configured to couple channel 706 and channel 707, if fluidly connected to 706, with the fluid flow source. In one embodiment, seals or o-rings are positioned around the port and a linear solenoid may be positioned above the o-rings to seal the o-rings against the cassette body. For example, as shown in the exemplary embodiment illustrated in FIG. 11A, in addition to port 719, there may be two vent ports 715 and a mixing port
[00184] [00184] In one embodiment, when a fluid flow source is activated, the channel 706, 707 in the cassette may be pressurized (e.g., to about -30kPa), which will direct fluids into the channel (at fluid sample as well as reagents) towards the outlet. In an embodiment that includes vent ports 715 and mix port 713, a vent valve connected to port 713 through the manifold may be initially open, which may allow all reagents downstream of the mixture 713 to move towards the outlet, but will not cause movement of the reactants upstream of the mixing port 713. Once the vent valve is closed, the reactants upstream of the mixing port 713 can move towards a mixing port and then towards the outlet. For example, fluids can be stored in series in a channel upstream of the mixing port and, after closing a vent valve positioned along the channel, the fluids can flow sequentially towards the outlet of the channel. . In some cases, fluids can be stored in separate channels that intersect, and after a vent valve closes, fluids can flow together toward an intersection point. This set of modalities can be used, for example, to controllably mix fluids as they flow. The timing of the application and the volume of fluid applied can be controlled, for example, by the timing of actuation of the breather valve.
[00185] [00185] Advantageously, vent valves can be operated without squeezing the cross section of the microfluidic channel in which they are operated, as can occur with certain valves in the prior art. Such a mode of operation can be effective in preventing leakage through the valve. Furthermore, since breather valves can be used, some systems and methods described here do not require the use of certain internal valves, which can be problematic due, for example, to high expense, manufacturing complexity, fragility , limited compatibility with mixed gas and liquid systems, and/or unreliability in microfluidic systems.
[00186] [00186] It should be understood that when breather valves are described, other types of valve mechanisms may be used with the systems and methods described herein. Non-limiting examples of a valve mechanism that can be operatively associated with a valve include a diaphragm valve, ball valve, gate valve, butterfly valve, globe valve, needle valve, pinch valve, or lathe valve. The valve mechanism can be actuated by any appropriate device, including a solenoid, motor, manually, electronically, or by hydraulic/pneumatic pressure.
[00187] [00187] As previously mentioned, all liquids in the cassette (sample and reagents) may move to the liquid containment area which may include a 717 absorbent material. In one embodiment, the absorbent material only absorbs liquids, so such that gases can flow out of the cassette through the outlet.
[00188] [00188] A variety of determination techniques (e.g. measurement, quantification, detection and qualification) can be used, for example, to analyze a sample component or other component or condition associated with a microfluidic system or cassette. described here. Determination techniques may include optically based techniques such as light transmission, light absorbance, light scattering, light reflection and visual techniques. Determination techniques may also include luminescence techniques such as photoluminescence (eg fluorescence), chemiluminescence, bioluminescence and/or electroluminescence. In other embodiments, determination techniques can measure conductivity or resistance. In this way, an analyzer can be configured to include such and other appropriate detection systems.
[00189] [00189] Different optical detection techniques offer a number of options for determining reaction results (eg assay). In some embodiments, measuring transmission or absorbance means that light can be detected at the same wavelength as it is sent from a light source. Although the light source may be a narrow-band source that is emitted at a single wavelength, it can also be a broad-spectrum source, which emits over a range of wavelengths, as many opaque materials can effectively obstruct a wide range of wavelengths. In some embodiments, a system can be operated with a minimum of optical devices (eg, a simplified optical detector). For example, the determining device may be free of a photomultiplier, it may be free of a wavelength selector such as an optical grating, a prism or a filter, it may be free of a device for directing or columnaring light such as a columnador, or it may be free from the magnifying optics (eg lenses). Eliminating or reducing these features can result in a less expensive, more robust device.
[00190] [00190] FIG. 12 illustrates an exemplary optical system 800 that can be positioned in the housing of an analyzer. As illustratively shown in this embodiment, the optical system includes at least a first light source 882 and a detector 884 spaced from the first light source. The first light source 882 may be configured to pass light through a first analysis region of the cassette when the cassette is introduced into the analyzer. First detector 884 may be positioned opposite first light source 882 to detect the amount of light that passes through the first analysis region of cassette 520. It should be appreciated that, in other embodiments, the number of light sources and detectors may vary, since the invention is not limited to that. As mentioned above, cassette 520 may include a plurality of analysis regions 709, and cassette 520 may be positioned within the analyzer such that each analysis region aligns with a corresponding light source and detector. . In some embodiments, the light source includes an optical aperture that can help direct light from the light source to a particular region within an analysis region of the cassette.
[00191] [00191] In one embodiment, the light sources are light-emitting diodes (LEDs) or laser diodes. For example, a red laser InGaAlP semiconductor diode that emits at 654 nm can be used. Other light sources can also be used. The light source can be positioned inside a nest or enclosure. The nest or enclosure may include a narrow opening or thin tube that can help to collide the light. The light sources can be positioned above where the cassette is inserted into the analyzer, such that the light source shines down onto the top surface of the cassette. Other appropriate light source configurations with respect to the cassette are also possible.
[00192] [00192] It should be appreciated that the wavelength of light sources varies, as the invention is not limited thereto. For example, in one embodiment, the wavelength of the light source is about 670 nm, and in another embodiment, the wavelength of the light source is about 650 nm. It should be appreciated that, in one embodiment, the wavelength of each light source may be different, such that each analysis region of the cassette receives a different wavelength of light. In other embodiments, however, the wavelength of each light source may be the same, such that each analysis region of the cassette receives the same wavelength of light. Combinations of wavelengths of the same or different light sources are also possible.
[00193] [00193] As mentioned, a detector 884 can be spaced and positioned below a light source 882 to detect the amount of light that passes through the cassette. In one embodiment, one or more of the detectors are photodetectors (e.g., photodiodes). In certain embodiments, the photodetector can be any suitable device that can detect the transmission of light that is emitted by the light source. One type of photodetector is an optical integrated circuit (IC) including a photodiode that has a peak sensitivity at 700 nm, an amplifier, and a voltage regulator. The detector may be positioned within a nest or housing which may include a narrow aperture or thin tube to ensure that only light from the center of the 709 analysis region is measured in the 884 detector. If the light source is pulse modulated , the photodetector may include a filter to remove the effect of light that is not at the selected frequency. When multiple neighboring signals are detected at the same time, the light source used for each analysis region (eg, the detection region) can be modulated at a sufficiently different frequency from that of its neighboring light source. In this configuration, each detector can be configured (eg using software) to select its assigned light source, thereby preventing interfering light from forming neighboring optical pairs.
[00194] [00194] The applicant for the filed patent recognized that the amount of light transmitted through an analysis region of the cassette can be used to determine information not only about the sample, but also information about the specific processes that occur in the fluidic system of the cassette (e.g. mixing of reagents, flow rate, etc.). In some cases, measuring light across a region can be used as feedback to control the flow of fluid in the system. In certain modes, quality control or cassette operation anomalies can be determined. For example, feedback from an analysis region to a control system can be used to determine anomalies that have occurred in the microfluidic system, and the control system can emit a signal to one or more components to cause the entire system or portions thereof are shut down. Consequently, the quality of the processes being performed in the microfluidic system can be controlled using the systems and methods described herein.
[00195] [00195] It should be recognized that a transparent liquid (such as water) can allow a large amount of light to be transmitted from the light source 882, through the analysis region 709 and to the detector 884. The air within the region probe 709 can lead to less light transmitted through the probe region 709 because more light can be scattered within the channel compared to when a clear liquid is present. When a blood sample is in an analysis region 709, significantly less light can pass through the 884 detector, due to light scattering outside the blood cells and also due to absorbance. In one embodiment, the silver associates with a component of the sample bound to a surface within the analysis region, and as silver accumulates within the analysis region, less and less light is transmitted through the analysis region 709.
[00196] [00196] It is recognized that measuring the amount of light that is detected at each detector 884 allows a user to determine which reagents are in a particular analysis region 709 at a particular point in time. It is also recognized that by measuring the amount of light that is detected with each 884 detector, it is possible to measure the amount of silver deposited in each analysis region.
[00197] [00197] As noted above, the patent applicant has acknowledged that the 880 optical system can be used for a variety of quality control reasons. First, the time it takes for a sample to reach an analysis region where the optical system detects light passing through the analysis region can be used to determine if there is a leak or blockage in the system. Also, when the sample is expected to have a certain volume, for example about 10 microliters, there is an associated predicted flow time for the sample to pass through the channels and analysis regions. If the sample is outside this predicted flow time, this could be an indication that there is not enough sample to conduct the analysis and/or that the wrong type of sample has been loaded into the analyzer. In addition, a predicted range of results can be determined based on the type of sample (e.g. serum, blood, urine etc.) and if the sample was outside the predicted range this could be an indication of an error.
[00198] [00198] In one embodiment, the analyzer includes a temperature regulation system positioned within the housing, which can be configured to regulate the temperature within the analyzer. For a given sample analysis, the sample may need to be kept within a certain temperature range. For example, in one embodiment, it is desirable to keep the temperature inside the analyzer at about 37°C. Accordingly, in one embodiment, the temperature regulation system includes a heater configured to heat the cassette. In one embodiment, the heater is a resistive heater that can be positioned on the underside of where the cassette is placed in the analyzer. In one embodiment, the temperature regulation system also includes a thermistor for measuring the temperature of the cassette, and a controller circuit may be provided to control the temperature.
[00199] [00199] In one embodiment, the passive flow of air within the analyzer can act to cool the air inside the analyzer if necessary. A fan can optionally be provided on the analyzer to lower the temperature inside the analyzer. In some embodiments, the temperature regulation system may include Peltier thermoelectric heaters and/or coolers within the analyzer.
[00200] [00200] In certain embodiments, an identification system including one or more identifiers is used and associated with one or more components or materials associated with a cassette and/or analyzer. "Identifiers", as described in more detail below, may be "encoded with" the information (i.e., may carry or contain the information, such as by using a device to load, store, generate, or transmit information such as a radio frequency identification (RFID) stripe or bar code) on the component including the identifier, or they may not be encoded with the information about the component, but instead may only be associated with the information that they may be contained, for example, in a database on a computer or in a computer-readable medium (eg information about a user and/or sample to be analyzed). In the last example, detection of such an identifier can trigger the retrieval and use of associated information from the database.
[00201] [00201] Identifiers "encoded with" information about a component do not necessarily need to be encoded with a complete set of information about the component. For example, in certain embodiments, an identifier may be encoded with just enough information to allow an original identification of the cassette (e.g. with respect to a serial number, part number, etc.), as additional information that relates to the cassette (eg type, usage [eg trial type], possession, position, position, connectivity, content, etc.) can be stored remotely and be associated only with the identifier.
[00202] [00202] "Information about" or "information associated with" a cassette, material or component, etc. is information concerning the identity, placement or location of the cassette, material or component or the identity, placement or location of the contents of a cassette, material or component, and may additionally include information concerning the nature ,
[00203] [00203] Non-limiting examples of identifiers that may be used in the context of the invention include radio frequency identification (RFID) stripes, bar codes, serial numbers, colored, fluorescent or optical stripes (e.g. using quantum dots), chemical compounds, radio stripes, magnetic stripes, among others.
[00204] [00204] In one embodiment, an ID reader is an RFID reader configured to read an RFID tag associated with the cassette. For example, in one embodiment, the analyzer includes an RFID module and antenna that is configured to read cassette information entered into the analyzer. In another embodiment, the ID reader is a barcode reader configured to read a barcode associated with the cassette. Once the cas-
[00205] [00205] In some cases, the ID reader can be integrated with a control system via the communication paths. Communication between ID readers and the control system can take place over a wired network or can be transmitted wirelessly. In one embodiment, the control system may be programmed to recognize a specific identifier (e.g., of a cassette associated with information relating to a cassette type, manufacturer, assay to be performed, etc.) indicating that the cassette is properly connected or inserted into a particular type of analyzer.
[00206] [00206] In one embodiment, a cassette identifier is associated with predetermined or programmed information contained in a database regarding the use of the system or cassette for a particular purpose, user or product, or with the particular reaction conditions, sample types, reagents, users and more. If an incorrect match is detected, or an identifier is disabled, the process may be interrupted or the system may become inoperable until the user is notified, or upon recognition by a user.
[00207] [00207] Information from or associated with an identifier may, in some embodiments, be stored, for example, in computer memory or on a computer readable medium, for future reference and recording purposes. record. For example, certain control systems may employ information from or associated with identifiers to identify which components (e.g. cassettes) or cassette types were used in a particular analysis, the date, time and duration of use, the conditions of use etc. Such information can be used, for example, to determine whether one or more analyzer components should be cleaned or replaced. Optionally, a control system or any other appropriate system can generate a report of the information collected, including information encoded by or associated with identifiers, which can be used to provide evidence of compliance with regulatory standards or verification of control of quality.
[00208] [00208] Information encoded in or associated with an identifier may also be used, for example, to determine whether the component associated with the identifier (eg a cassette) is authentic or forged. In some embodiments, determining the presence of a forged component causes the system to shut down. In one example, the identifier may contain a unique identity code. In this example, the process control software or analyzer would not allow the system to start (e.g. the system could be shut down) if a strange or incompatible identity code (or no identity code at all) was detected. - ted
[00209] [00209] In certain embodiments, information obtained from or associated with an identifier may be used to verify the identity of a customer to whom the cassette and/or analyzer is sold or to whom a biological, chemical or pharmaceutical process is to be be executed. In some cases, information obtained from or associated with an identifier is used as part of a data collection process to troubleshoot a system. The identifier can also contain or be associated with information such as group histories, assembly process and instrumentation diagrams (P and IDs), problem resolution histories, among others. Troubleshooting a system can be performed, in some cases, through remote access or include the use of diagnostic software.
[00210] [00210] In one embodiment, the analyzer includes a user interface, which can be positioned inside the housing and is configured so that a user receives information on the sample analyzer. In one embodiment, the user interface is a touch screen.
[00211] [00211] The touch screen can guide a user through the operation of the analyzer, providing text and/or graphical instructions for using the analyzer. The touch screen user interface can, for example, guide the user to insert the cassette into the analyzer. It can then guide the user to receive the patient's name or other source/patient identification number on the analyzer (eg age, results of a DRE exam, etc.). It should be appreciated that patient information such as name, date of birth and/or patient ID number can be received on the touch screen user interface to identify the patient. The touch screen can indicate the amount of time remaining to finish the sample analysis. The touch screen user interface can then illustrate the results of the sample analysis along with the patient's name or other identifying information.
[00212] [00212] In another embodiment, the user interface can be configured differently, such as with an LCD screen and a single button scrolling menu. In another embodiment, the user interface may simply include a launch key to activate the analyzer. In other embodiments, the user interface of separate and independent devices (such as a smartphone or mobile computer) can be used to interface with the analyzer.
[00213] [00213] The analyzer described above can be used in a variety of ways to process and analyze a sample placed inside the analyzer. In a particular embodiment, once a mechanical component configured to interface with the cassette indicates that the cassette is correctly loaded into the analyzer, the identification reader reads and identifies the information associated with the cassette. The analyzer can be configured to compare the information with data stored in a control system to ensure it has the calibration information for this particular sample. If the analyzer does not have the proper calibration information, the analyzer may send a request to the user to load the specific information needed. The analyzer can also be configured to review the expiration date information associated with the cassette and cancel the analysis if the expiration date has passed.
[00214] [00214] In one embodiment, once the analyzer has determined that the cassette can be analyzed, a fluid flow source such as the vacuum manifold can be configured to contact the cassette to ensure an airtight seal at around the vacuum port and vent ports. In one embodiment, the optical system may take initial measurements to obtain reference readings. Such reference readings can be taken with the light sources on or off.
[00215] [00215] To initiate sample movement, the vacuum system can be activated, which can quickly change the pressure within one or more channels (eg, reduce to about -30kPa). This pressure reduction within the channel can drive the sample into a channel and through each of the analysis regions 709A-709D (see FIG. 10). After the sample reaches the final analysis region 709D, the sample can continue to flow into the liquid containment region 717.
[00216] [00216] In a particular set of modalities, the microfluidic sample analyzer is used to measure the level of iPSA, fPSA, tP-SA and/or hK2 in a blood sample. In one embodiment, three, four, five, six or more analysis regions (eg, analysis regions 709A-709D) can be used to analyze the sample. For example, in a first analysis region, the canal walls can be blocked with a blocking protein (such as bovine serum albumin) so that almost no protein in the blood sample attaches to the walls of the region of analysis (except perhaps for some non-specific binding that can be washed away). This first analysis region can act as a negative control.
[00217] [00217] In a second analysis region, the canal walls can be coated with a large predetermined amount of a prostate specific antigen (PSA) to act as an elevated or positive control. Once the blood sample passes through the second analysis region, almost no PSA proteins in the blood can bind to the channel walls. Detection antibodies conjugated to gold in the sample can be dissolved within the 722 fluidic connector tube or can flow from any other appropriate position. These antibodies may not yet bind to PSA in the sample and thus may bind to PSA in the channel walls to act as an elevated or positive control.
[00218] [00218] In a third analysis region, the channel walls can be coated with a capture antibody to iPSA (e.g. an anti-iPSA antibody), which can bind to a different epitope on the PSA protein from that of the gold-conjugated signal antibody. As the blood sample flows through the third analysis region, the iPSA proteins in the blood sample can bind to the anti-iPSA antibody in a manner proportional to the concentration of these proteins in the blood.
[00219] [00219] In a fourth analysis region, the channel walls can be coated with a capture antibody to fPSA (e.g. an anti-fPSA antibody), which can bind to a different epitope on the PSA protein from that of the gold-conjugated signal antibody. As the blood sample flows through the fourth analysis region, the fPSA proteins in the blood sample can bind to the anti-fPSA antibody in a manner proportional to the concentration of these proteins in the blood.
[00220] [00220] In a fifth analysis region, the channel walls can be coated with a capture antibody to tPSA (e.g. an anti-tPSA antibody), which can bind to a different epitope on the PSA protein from that of the gold-conjugated signal antibody. As the blood sample flows through the fifth analysis region, the tPSA proteins in the blood sample can bind to the anti-tPSA antibody in a manner proportional to the concentration of these proteins in the blood.
[00221] [00221] Optionally, in a sixth analysis region, the channel walls can be coated with a capture antibody to hK2 (e.g. an anti-hK2 antibody), which can bind to a different epitope on the protein from that of the gold-conjugated signal antibody. As the blood sample flows through the sixth analysis region, the hK2 proteins in the blood sample can bind to the anti-hK2 antibody in a manner proportional to the concentration of these proteins in the blood.
[00222] [00222] A detection antibody such as a gold-labeled antibody that is anti-PSA and anti-hK2 can be used to detect each of iPSA, fPSA, tPSA and/or hK2. In other embodiments, however, a mixture of gold-labeled antibodies, such as an anti-hK2 gold-labeled antibody, gold-labeled anti-PSA antibody, and/or gold-labeled anti-iPSA antibody, may be used. be used for detection. In some embodiments, the gold-conjugated detection antibodies in the sample may be dissolved within the fluidic connector tube 722 or may flow from any other appropriate position.
[00223] [00223] In some examples, measurements from an analyzing region can be used to determine not only the concentration of an analyte in a sample, but also as a control. For example, a threshold measurement can be established at an early stage of amplification. Measurements above this value (or below this value) may indicate that the analyte concentration is outside the desired range for the assay. This technique can be used to identify, for example, if a High Dose Hook Effect is occurring during analysis, ie when a very high concentration of analyte produces an artificially low reading.
[00224] [00224] In other embodiments, different numbers of analysis regions may be provided, and an analysis may optionally include more than one analysis region that actually tests the sample. Additional analysis regions can be used to measure additional analytes, so the system can run multi-part assays simultaneously with a single sample.
[00225] [00225] In one particular embodiment, it takes about eight minutes for a 10 microliter blood sample to flow through the four analysis regions. The start of this analysis can be calculated when the pressure inside the channel is about -30kPa. During this time, the optical system is measuring the light transmission for each analysis region and, in one embodiment, this data can be transmitted to a control system about every 0.1 second. Using reference values, these measurements can be converted using the following formulas: Transmission = (l-ld)/(lr-ld) (1) where: l = intensity of light transmitted through an analysis region at a given point in time; ld = intensity of light transmitted through an analysis region with the light source turned off; lr = reference intensity (that is, the intensity of light transmitted in an analysis region with the light source activated or before an analysis begins, when only air is in the channel and Optical Density = - log(Transmission) ( two)
[00226] [00226] In this way, using these formulas, the optical density in an analysis region can be calculated.
[00227] [00227] FIG. 13 is a block diagram 900 illustrating how a control system 550 (see FIG. 12) can be operatively associated with a variety of different components according to one embodiment. The control systems described herein can be implemented in numerous ways, such as with dedicated hardware or firmware, using a processor that is programmed using microcode or software to perform the aforementioned functions or any appropriate combination of the above. A control system can control one or more operations from a single analysis (eg for a biological, biochemical or chemical reaction) or from multiple analyzes (separate or interconnected). For example, the control system can be positioned within the analyzer housing and can be configured to communicate with an ID reader, user interface, fluid flow source, optical system, and/or flow regulation system. temperature to analyze a sample in the cassette.
[00228] [00228] In one embodiment, the control system includes at least two processors, including a real-time processor that controls and monitors all of the subsystems that interface directly with the cassette. In one embodiment, at a particular time interval (e.g., every 0.1 second), this processor communicates with a second, higher-level processor that communicates with the user through the interface with the server. user and/or communication subsystem (discussed below) and controls analyzer operation (eg, determines when to begin analysis of a sample and interprets the results). In one embodiment, communication between these two processors takes place over a serial communication bus. It should be appreciated that, in another embodiment, the analyzer may only include one processor or more than two processors, as the invention is not limited thereto.
[00229] [00229] In one embodiment, the analyzer can connect to external devices and can, for example, include ports for connection to one or more external communication units. External communication can be carried out, for example, via USB communication. For example, as shown in FIG. 13, the analyzer can send the results of a sample analysis to a USB printer 901 or a computer 902. In addition, the data stream produced by the processor in real time can be sent to a computer or USB memory stick 904. In some modes
[00230] [00230] The calculation methods, steps, simulations, algorithms, systems and system elements described here can be implemented using a computer-implemented control system, such as the various modalities of the implemented systems. by the computer described below. The methods, steps, systems, and system elements described here are not limited in their implementation to any specific computer system described here, as many other different machines can be used.
[00231] [00231] The computer-implemented control system may be part of or coupled in operative association with a sample analyzer, and, in some embodiments, is configured and/or programmed to control and adjust the analyzer's operational parameters sample, as well as analyze and calculate values, as described above. In some embodiments, the computer-implemented control system can send and receive reference signals to adjust and/or control operating parameters of the sample analyzer and, optionally, other devices in the system. In other embodiments, the computer-implemented system may be separate from and/or located remotely with respect to the sample analyzer, and may be configured to receive data from one or more remote sample analyzer devices through an indirect device and/or or portable, such as through portable electronic data storage devices, such as magnetic disks, or via
[00232] [00232] The computer-implemented control system may include several known components and circuits, including a processing unit (i.e., processor), a memory system, input and output devices, and interfaces (e.g., a interconnect), as well as other components, such as transport circuitry (e.g., one or more buses), a video and audio data input/output (I/O) subsystem, special-purpose hardware, as well as other components and circuits, as described in more detail below. In addition, the computer system may be a multiprocessor computer system, or it may include multiple computers connected through a computer network.
[00233] [00233] The control system implemented by the computer may include a processor, for example a commercially available processor such as an x86 series, Celeron and Pentium processors available from Intel, similar devices from AMD and Cyrix, microprocessors from 680X0 series available from Motorola, IBM's PowerPC microprocessor and ARM processors. Many other processors are available and the computer system is not limited to a particular processor.
[00234] [00234] A processor typically executes a program called an operating system, and WindowsNT, Windows95 or 98, Windows 7, Windows 8, UNIX, Linux, DOS, VMS, MacOS and OSX and iOS are examples, which control the execution of other computer programs and provide programming, debugging, input/output control, accounting, compilation, storage allocation, data management and memory management, communication control, and related services. The processor and operating system together define a computer platform for application programs in high-level programming languages to be written. The computer-implemented control system is not limited to a particular computer platform.
[00235] [00235] The computer-implemented control system may include a memory system, which typically includes a non-volatile recording medium that can be read and written by a computer, including a magnetic disk, optical disk, flash memory, and tape are examples. Such recording medium may be removable, for example a floppy disk, read/write CD or memory card, or may be permanent, for example a hard drive.
[00236] [00236] Such a recording medium stores signals, typically in binary form (ie a form interpreted as a sequence of one and zero). A disk (eg magnetic or optical) has a number of tracks, on which such signals can be stored, typically in binary form, that is, a form interpreted as a sequence of one and zero. Such signals can define a software program, for example, an application program, to be executed by the microprocessor or, the information to be processed by the application program.
[00237] [00237] The memory system of the control system implemented by the computer may also include an integrated circuit memory element, which is typically a volatile, random access memory such as dynamic random access memory (DRAM) or memory. static (SRAM). Typically, in operation, the processor causes programs and data to be read from the nonvolatile recording medium to the integrated circuit memory element, which typically allows faster access to program instructions and data. by the processor than the non-volatile recording medium.
[00238] [00238] The processor generally manipulates the data within the integrated circuit memory element in accordance with the program instructions and then copies the manipulated data onto the non-volatile recording medium after processing is complete. A variety of mechanisms are known to manage the movement of data between the nonvolatile recording medium and the integrated circuit memory element, and the computer-implemented control system that executes the methods, steps, systems, and processes. system elements described above with respect to FIG. 13 is not limited to that. The control system implemented by the computer is not limited to a particular memory system.
[00239] [00239] At least part of such a memory system described above may be used to store one or more data structures (eg check tables) or equations described above. For example, at least part of the non-volatile recording medium may store at least part of a database that includes one or more such data structures. Such a database can be any of a variety of database types, for example, a file system that includes one or more one-dimensional data structures, where data is organized into data units. delimiter-separated databases, a relational database, where data is organized into data units stored in tables, an object-oriented database, where data is organized into data units stored as objects, another type of database or any combination thereof.
[00240] [00240] The computer-implemented control system may include an audio and video data I/O subsystem. A portion of the audio subsystem may include an analog-to-digital (A/D) converter, which receives analog audio information and converts it to digital information. Digital information can be compressed
[00241] [00241] The computer-implemented control system may include one or more output devices. Exemplifying output devices include a cathode ray tube (CRT) display, liquid crystal displays (LCD) and other video output devices, printers, communication devices such as a modem or a computer interface. network, storage devices such as disk or tape, and audio output devices such as a speaker.
[00242] [00242] The computer-implemented control system may also include one or more input devices. Exemplary input devices include a keyboard, keypad, trackball, mouse, pen and tablet, communication devices such as described above, and data input devices such as audio and video capture devices and sensors. The computer-implemented control system is not limited to the particular input or output devices described herein.
[00243] [00243] It should be appreciated that one or more of any type of computer-implemented control system may be used to perform various modalities described herein. Aspects of the invention may be implemented in software, hardware or firmware or any combination thereof. The computer-implemented control system may include specially programmed special-purpose hardware, for example, an application-specific integrated circuit (ASIC). Such special-purpose hardware may be configured to execute one or more of the methods, steps, simulations, algorithms, systems, and system elements described above as part of the computer-implemented control system described above. above or as an independent component.
[00244] [00244] The computer-implemented control system and components thereof may be programmable using any of a variety of one or more appropriate computer programming languages. Such languages may include procedural programming languages, for example, C, Pascal, Fortran, and BASIC, object-oriented languages, for example, C++, Java, and Eiffel, and other languages, such as a scripting language or a scripting language. assembly language.
[00245] [00245] Methods, steps, simulations, algorithms, systems, and system elements can be implemented using any of a variety of appropriate programming languages, including procedural programming languages, object-oriented, other languages and combinations thereof, which can be executed by such a computer system. Such methods, steps, simulations, algorithms, systems and system elements can be implemented as separate modules of a computer program or they can be implemented individually as separate computer programs. Such modules and programs can be run on separate computers.
[00246] [00246] Such methods, steps, simulations, algorithms, systems and system elements, individually or in combination, may be implemented as a product of the embedded computer program tangibly as computer-readable signals.
[00247] [00247] It should be appreciated that various embodiments may be formed with one or more of the characteristics described above. The above aspects and features may be employed in any suitable combination, as the present invention is not limited in this regard. It should also be appreciated that the drawings illustrate various components and features that may be incorporated in various embodiments. For simplicity, some of the drawings may depict more than one feature or optional component. However, the invention is not limited to the specific embodiments described in the drawings. It should be recognized that the invention encompasses embodiments which may include only a portion of the components illustrated in any one figure and/or may also encompass embodiments which combine the components illustrated in multiple different figures. Other preferred modalities
[00248] [00248] It will be appreciated that the methods of the present invention may be embodied in a variety of embodiments, only a few of which are described herein. It will be evident to the person skilled in the art that other embodiments exist and do not deviate from the character of the invention. Thus, the described modalities are illustrative and should not be interpreted as restrictive.
[00249] [00249] In total, seven separate studies using the statistical model were performed. The studies comprise a total of 7,647 men with elevated PSA and 2,270 cancers, with five of these studies constituting external validation. In addition, studies were systematically designed to cover a wide range of clinical scenarios. Perhaps, in a remarkable way, one of the studies includes a natural history approach. Since the biopsy result is a surrogate outcome—what matters is not whether a man has prostate cancer, but whether he is at risk of developing prostate cancer that affects his life—the ideal study would draw blood from patients, then follow these patients for several years in the absence of further screening to determine prostate cancer outcomes. Fortunately, this study was conducted [Vickers, A.J., et al., Cancer Epidemiol Biomarkers Prey, 2011. 20(2): p. 255-61].
[00250] [00250] The Malmö Diet and Cancer cohort is part of a large population-based study to identify dietary risk factors in cancer mortality. 11,063 men who lived in the city of Malmö, Sweden and who were born between 1923 and 1945, provided an EDTA anticoagulated blood sample 1991 - 1996. The result was confirmed by the Swedish Cancer Registry. Marker values were obtained from archived blood samples analyzed in 2008, which were previously validated as obtaining exact measurements of kallikrein from stored blood [Ulmert, D., et al., Clin. Chem., 2006. 52(2): p. 235-9]. The PSA test rate was very low, with almost all cases diagnosed clinically. In this way, the study follows the "natural history" of prostate cancer in men with elevated PSA. Of 792 men who had a PSA of 3 ng/ml at baseline, 474 were subsequently diagnosed with prostate cancer at a median follow-up of 11 years. The predictive discrimination of the four kallikrein panel statistical model was noticeably higher than PSA for predicting any cancer and advanced cancers (stage T3 or T4, or metastatic), precisely those cancers that are most likely to be fatal. As seen in previous studies, about 50% of men had a model prostate cancer risk of less than 20%. It was estimated that only 13 men per 1000 with elevated PSA would have a risk < 20% of the model, even if diagnosed with cancer within five years; only 1 man would have advanced cancer at diagnosis.
[00251] [00251] The Malmö cohort demonstrates several important features of our predictive model. First, it constitutes an external validation. Second, it shows that the model predicts clinically diagnosed cancers that, by definition, do not constitute overdiagnosis. Third, the study suggests that cancers not covered by the model are those considered to be overdiagnosed: data from our biopsy studies indicate that the panel rates about 60 men per 1000 who have biopsy-detectable cancers as low risk. ; Malmö cohort data suggest that less than 1 in 4 of these would become clinically evident after 5 years of follow-up. Fourth, it demonstrates that the model is very strongly predictive of the types of aggressive cancers that are most likely to shorten a man's life. Finally, the data indicate that the clinical use of the model would not lead to significant harm in terms of late diagnoses, since only
[00252] [00252] An illustrative template used in this example: Age: insert age in years tPSA: insert total PSA in ng/ml fPSA: insert free PSA in ng/ml iPSA: insert intact PSA in ng/ml hK2: insert a hK2 in ng/ml If tPSA ≥ 25, then use: L = 0.0733628 x tPSA – 1.377984 prostate cancer risk = exp(L)/[1 + exp(L)] If tPSA < 25 , then use one of the two equations below, one that incorporates clinical information and the other that does not:
[00253] [00253] The cubic slot variables are determined as follows: Spline1_tPSA = - (162 - 4.4503)/(162 - 3) x (tPSA - 3)^3 + max(tPSA – 4.4503, 0) ^3 Spline2_tPSA = - (162 – 6.4406)/(162 - 3) x (tPSA - 3)^3 + max(tPSA – 6.4406, 0)^3 If fPSA < 11.8, then Spline1_fPSA = - (11.8 – 0.84)/(11.8 – 0.25) x (fPSA – 0.25)^3 + max(fPSA – 0.84, 0)^3 If fPSA > 11.8, then Splinel_fPSA = (11.8 - 0.84) x (0.84 - 0.25) x (11.8 + 0.84 + 0.25 - 3 x fPSA)
[0233] [0233] Set the following: x1 = 0.0846726 x tPSA + -.0211959 x Spline1_tPSA + .0092731 x Spline2_tPSA x2 = -3.717517 x fPSA – 0.6000171 x Spline1_fPSA + 0.275367 x Spline2_fPSA x3 = 52.9680 x iPSA x4 = 4.508231 x hK2
[0234] [0234] So: L = -1.735529 + 0.0172287 x Age + x1 + x2 + x3 + x4 prostate cancer risk = exp(L)/[+ exp(L)]
[00254] [00254] This provides the risk of prostate cancer in the absence of any clinical information. We assume that if this risk is high, the clinician will ask the patient to present for a thorough clinical examination and digital rectal examination. The following model is then run twice, with the DRE coded as 0 or 1, to provide risks depending on whether the DRE is normal or abnormal, respectively. Set the following: x1 = 0.0637121 x tPSA – 0.0199247 x Spline1_PSA + 0.0087081 x Spline2_tPSA x2 = -3.460508 x fPSA – 0.4361686 x Spline1_fPSA + 0.1801519 x Spline2_fPSA x3 = 4.014925 x iPSA x4 = 3.523849 x hK2
[00255] [00255] Recalibration can be used for men with a previous negative biopsy, but recalibration can be used in other situations where event rates are markedly different from the observed (previously untracked) event rate in the Rot- cohort. terdam (29%). Set the following: odds_cancer = Pr(cancer)/(1-(Pr(cancer)) odds_prediction = predicted cancer risk/(1 – predicted cancer risk) Then: bayes_factor = odds_cancer/odds_prediction y_adj = y +log(bayesfactor ) recalibrated risk of prostate cancer = exp(y_adj)/[1 + exp(y_adj) Example 2 (Prophetic)
[00256] [00256] This is a prophetic example describing the use of a cassette and an analyzer to perform an assay to detect iPSA, fPSA, tPSA and hK2 in a sample through the non-electrolytic deposition of silver on gold particles that are associated with the sample. FIG. 14 includes a schematic illustration of a microfluidic system 1500 of a cassette used in this example. The cassette was similar in shape to cassette 520 shown in FIG. 7.
[00257] [00257] The microfluidic system included analysis regions 1510A-1510F, waste containment region 1512 and an outlet
[00258] [00258] Prior to first use, the microfluidic system was loaded with the liquid reagents that were stored in the cassette. A series of 7 buffers from wash 1523-1529 (water or buffer, about 2 microliters each) was loaded using a pipette into the secondary channels 1515 of channel 1516 using the through holes. Each of the wash buffers was separated by air plugs. Fluid 1528, containing a silver salt solution, was loaded into the branch channel through port 1519 using a pipette. Fluid 1530, containing a reducing solution, was charged to branch channel 1520 through port 1521. Each of the liquids shown was separated from the other liquids by air plugs. Ports 1514, 1519, 1521, 1536, 1539 and 1540 were sealed with an adhesive tape that could be easily removed or punctured. In this way, the liquids were stored in the microfluidic system before the first use.
[00259] [00259] When first used, ports 1514, 1519, 1521, 1536, 1539 and 1540 were opened by a user removing the tape covering the opening of the ports. A tube 1544 containing lyophilized anti-PSA and anti-hK2 antibodies labeled with colloidal gold and to which 10 microliters of sample blood (1522) was added, connected to ports 1539 and 1540. The tube was part of a fluid having a shape and configuration shown in FIG. 7. This created a fluidic connection between analysis region 1510 and channel 1516, which would otherwise be disconnected and not in fluid communication with each other prior to first use.
[00260] [00260] The cassette including the 1500 microfluidic system was inserted into an analyzer opening. The analyzer housing included an arm positioned within the housing that was configured to engage a raised surface on the cassette. The arm extended at least partially into the opening in the housing such that the cassette was inserted into the opening, and the arm was pushed away from the opening in a second position, allowing the cassette to enter the opening. Once the arm engaged the raised surface into the cassette, the cassette was positioned and retained within the analyzer housing, and the spring thrust prevented the cassette from sliding out of the analyzer. The analyzer detected the cassette insertion via a position sensor.
[00261] [00261] An identification reader (RFID reader) positioned inside the analyzer housing was used to read an RFID tag on the cassette, which includes the lot identification information. The analyzer used this identifier to combine the lot information (e.g. information about calibration, cassette expiration date, verification that the cassette is new, and about the type of analyzer).
[00262] [00262] The control system included the programmed instructions to perform the analysis. To start the analysis, a signal was sent to the electronics that control a vacuum system, which was part of the analyzer and was used to provide the fluid flow. A distributor with O-rings was pressed against the surface of the cassette by a solenoid. A port on the dispenser was sealed (by an O-ring) to port 1536 of the cassette's microfluidic system. This port on the distributor was connected by a tube to a single solenoid valve that was open to the atmosphere. A separate vacuum port on the manifold sealed (by the O-ring) to port 1514 of the cassette microfluidic system. A vacuum of about -30 kPa was applied to port 1514. Throughout the analysis, the channel including analysis region 1510 positioned between ports 1540 and 1514 had a substantially constant non-zero pressure drop of about -30 kPa. Sample 1522 flows in the direction of arrow 538 in each of analysis regions 1510A-1510H. As the fluid passed through the analysis regions, the PSA and hK2 proteins in sample 1522 were captured by the anti-PSA and anti-hK2 antibodies immobilized on the walls of the analysis region, as described in more detail below. . It took about 7-8 minutes for the sample to pass through the analysis regions, after which the remaining sample was captured in the waste containment region 1512.
[00263] [00263] The initiation of analysis also involved the control system that sends a signal to the optical detectors, which were positioned adjacent to each of the analysis regions 1510, to initiate detection. Each of the detectors associated with the analysis regions recorded the light transmission through the channels of the analysis regions. As the sample passed through each of the analysis regions, peaks were produced. The peaks (and troughs) measured by the detectors are signals (or are converted into signals) that are sent to the control system which compares the measured signals with the reference signals or pre-programmed values in the control system. . The control system included a pre-programmed set of instructions to provide feedback to the microfluidic system based, at least in part, on the comparison of signals/values.
[00264] [00264] In a first analysis region 1510-A of the device 1500 of FIG. 14 , the channel walls of this analysis region were blocked with a blocking protein (bovine serum albumin) before first use (eg, before sealing the device). Almost no protein in the blood sample has bound to the walls of the 1510-A analysis region (except, perhaps, for some nonspecific binding that can be washed away). This first analysis region acted as a negative control.
[00265] [00265] In a second analysis region 1510-B, the channel walls of this analysis region were coated with a large predetermined amount of a prostate specific antigen (PSA) before first use (e.g. before sealing the device) to act as an elevated or positive control. As the blood sample passed through the second 1510-B analysis region, almost no PSA proteins in the blood bound to the channel walls. The gold-conjugated signal antibodies in the sample cannot yet bind to PSA in the sample, and thus can bind to PSA in the channel walls to act as an elevated or positive control.
[00266] [00266] In a third analysis region 1510-C, the channel walls of this analysis region were coated with the capture antibody, an anti-iPSA antibody, which binds to a different epitope on the iPSA protein from that of the antibody of sign conjugated with gold. The walls were coated before first use (eg before sealing the device). As the blood sample flowed through the fourth analysis region during use, the iPSA proteins in the blood sample bound the anti-iPSA antibody in a manner proportional to the concentration of these proteins in the blood. Since the sample, which included iPSA, also included gold-labeled anti-iPSA antibodies coupled to the iPSA, the iPSA captured on the walls of the analysis region formed a sandwich-like immunocomplex.
[00267] [00267] In a fourth 1510-D analysis region, the channel walls of this analysis region were coated with the capture antibody, an anti-fPSA antibody, which binds to a different epitope on the fPSA protein than the antibody from sign conjugated with gold. The walls were coated before first use (eg before sealing the device). As the blood sample flowed through the fourth analysis region during use, the fPSA proteins in the blood sample bound to the anti-fPSA antibody in a manner proportional to the concentration of these proteins in the blood. Since the sample, which included the fPSA, also included the fPSA-coupled gold-tagged anti-fPSA antibodies, the fPSA captured on the walls of the analysis region formed a sandwich-like immunocomplex.
[00268] [00268] In a fifth 1510-E analysis region, the channel walls of this analysis region were coated with the capture antibody, an anti-tPSA antibody, which binds to a different epitope on the tPSA protein from that of the tPSA antibody. sign conjugated with gold.
[00269] [00269] While gold-labeled anti-iPSA, anti-fPSA, and anti-tPSA antibodies can be used, in other embodiments, gold-labeled anti-PSA antibodies that bind to any PSA protein can be used. used for detection.
[00270] [00270] The first, second, third, fourth and fifth analysis regions were formed in a single layer of the substrate. The sixth (1510-F), seventh (1510-G) and eighth analysis regions (1510-H) were formed in a separate layer of the substrate (1511).
[00271] [00271] In the sixth analysis region 1510-F, the channel walls of this analysis region were coated with the capture antibody, an anti-hK2 antibody, which binds to a different epitope on the hK2 protein than the conjugated signal antibody with gold. The walls were coated before first use (eg before sealing the device). As the blood sample flowed through the sixth analysis region during use, the hK2 proteins in the blood sample bound to the anti-hK2 antibody in a manner proportional to the concentration of these proteins in the blood. Since the sample, which included hK2, also included the gold-tagged anti-hK2 antibodies coupled to hK2, the hK2 captured on the walls of the analysis region formed a sandwich-type immunocomplex.
[00272] [00272] The seventh analysis region 1510-G can be used as a negative control as described above for the analysis region 1510-A. The eighth analysis region 1510-H can be used as an elevated or positive control as described above for the analysis region 1510-B.
[00273] [00273] Optionally, a ninth analysis region (not shown) can be used as a low control. In such an embodiment, the channel walls of this analysis region can be coated with a predetermined low amount of PSA before first use (eg, before sealing the device) to act as a low control. As the blood sample flows through this analysis region, almost no PSA proteins in the sample bind to the channel wall. The gold-conjugated signal antibodies in the sample can bind to PSA in the channel walls to act as a low control.
[00274] [00274] Wash fluids 1523-1529 followed the sample through analysis regions 1510 to waste containment region 1512 in the direction of arrow 1538. As the wash fluids passed through the regions analysis, the remaining unbound components were removed from the sample. Each wash buffer cleaned the channels in the analysis regions, providing progressively more thorough cleaning. The last wash fluid 1529 (water) removed salts that could react with the silver salts (eg chloride, phosphate, azide).
[00275] [00275] As shown in the batch illustrated in FIG. 15, while wash fluids are flowing through the analysis regions, each of the detectors associated with the analysis regions measure a 1620 pattern of peaks and troughs. The gutters correspond to the wash plugs (which are transparent liquids and thus provide maximum light transmission). The peaks between each plug represent the air between each plug of clear liquid. Since the assay included 7 wash buffers, 7 troughs and 7 peaks are present in batch 1600. The first trough 1622 is generally not as deep as the other troughs 1624, as the first wash buffer often captures the blood cells on the left in the channel and is thus not completely transparent.
[00276] [00276] The final peak of Air 1628 is much longer than the preceding peaks because there is no wash buffer following. Once a detector detects the length of this air spike, one or more signals are sent to the control system, which compares the time duration of this spike to a preset reference signal or input value that has a particular duration. If the measured peak time duration is long enough compared to the reference signal, the control system sends a signal to the electronics controlling the 1536 breather valve to actuate the valve and initiate mixing of the 1528 and 1530 fluids. (Note that the air peak signal 1628 may be combined with a signal that indicates: 1) the peak intensity; 2) where this peak is positioned as a function of time and/or 3) one or more signals indicating that a series of peaks 1620 of particular intensity has passed. In this way, the control system distinguishes the 1628 air peak from other long-duration peaks, such as the 1610 peak of the sample, for example, which uses a signal pattern.)
[00277] [00277] Again with reference to FIG. 14, to start mixing, the solenoid connected by the distributor to the breather port 1536 is closed. Since the vacuum remains and no air can enter through vent valve 1536, air enters the device through ports 1519 and 1521 (which are open). This forces the two fluids 1528 and 1530 in the two storage channels upstream of the vent valve 1536 to move substantially simultaneously towards the outlet 1514. These reagents mix at the intersection of the channels to form an amplification reagent (a solution of reactive silver) which has a viscosity of about 1x10-3 Pa-s. The ratio between the volumes of the 1528 and 1530 fluids was about 1:1. The amplification reagent continued through the downstream storage channel, through tube 1544, through analysis regions 1510 and then to residue containment region 1512. After a defined period of time (12 seconds), the analyzer reopens vent valve 1536 such that air flows through vent valve 1536 (instead of vent ports). This leaves some of the reagent behind in upstream storage channels 1518 and 1520 on the device. This also results in a single mixed amplification reagent buffer. The 12 seconds of breathing valve closure results in an amplification buffer of approximately 50 µl. (Instead of simple timing, another way to cause the vent valve to reopen would be to detect the amplification reagent as it enters the analysis regions.)
[00278] [00278] Since the Mixed Amplification Reagent is stable for only a few minutes (generally less than 10 minutes), mixing was run for less than one minute prior to use in the 1510 analysis region. The Amplification Reagent it is a transparent liquid, so when it enters the analysis regions, the optical density is at its lowest level. As the amplification reagent passes through the analysis regions, silver is deposited on the captured gold particles to increase the size of the colloids to amplify the signal. (As noted above, gold particles can be present in the low and high positive control analysis regions and, to the extent that PSA and hK2 were present in the sample, in the test analysis region.) Silver can be then deposited on top of the already deposited silver,
[00279] [00279] From the curves, the values (eg concentrations) of blood markers (eg iPSA, fPSA, tPSA and/or hK2) are determined using a computer (eg inside the analyzer) . The values are sent to a processor (which is in electronic communication with the analyzer) which is programmed to evaluate a logistic regression model (e.g. as described herein) based, at least in part, on the values received for the determination. a probability of the patient's prostate cancer risk, an indication of an estimated prostate gland volume, and/or an indication of a probability that a prostate cancer biopsy will be positive in the patient.
[00280] [00280] The test result is displayed on the analyzer screen and is communicated to a printer, computer or whatever output the user has selected. The user can remove the device from the analyzer and throw it away. The sample and all reagents used in the assay remain in the device. The analyzer is ready for another test.
[00281] [00281] This prophetic example shows that the analysis of a sample that contains iPSA, fPSA, tPSA and/or hK2 can be performed in a single microfluidic system using an analyzer that controls the fluid flow in the cassette and using feedback from one or more measured signals to modulate fluid flow. This prophetic example also shows that the results of such an analysis can be used for the determination of a probability of risk of prostate cancer in the patient, an indication of an estimated volume of the prostate gland and/or an indication of a probability that a prostate cancer biopsy will be positive in the patient.

权利要求:
Claims (17)
[1]
1. System for determining a probability of an event associated with prostate cancer, characterized in that it comprises: an input interface configured to receive information from a plurality of blood markers, in which the information for the a plurality of blood markers include a free prostate specific antigen (fPSA) value and a total PSA (tPSA) value; at least one processor programmed to evaluate a logistic regression model based, at least in part, on information received for determining a probability of an event associated with prostate cancer in a person, wherein the regression model evaluation The logistics comprises: determining the cubic slot terms for tPSA, wherein determining the cubic slot terms for tPSA comprises determining the cubic slot terms for tPSA based on a first cubic slot that has a first internal node between 2 and 5 and a second internal node between 5 and 8; determining the cubic slot terms for fPSA, where determining the cubic slot terms for fPSA comprises determining the cubic slot terms for fPSA based on a second cubic slot that has a third internal node between 0.25 and 1 and a fourth internal node between 1.0 and 2.0; determining a first value for tPSA based, at least in part, on the received tPSA value and the cubic groove terms determined for tPSA; determining a second value for fPSA based, at least in part, on the received fPSA value and the cubic groove terms determined for fPSA; and determining the probability of the event associated with prostate cancer based, at least in part, on the first value and the second value; and an output interface configured to output an indication of the probability of the event associated with prostate cancer.
[2]
2. System according to claim 1, characterized in that the cubic slot terms for tPSA include a first cubic slot term and a second cubic slot term, wherein the cubic slot terms for fPSA include a third slot cubic term and a fourth cubic slot term; wherein determining the first value comprises scaling the received tPSA value by a value of the first coefficient, scaling the first cubic slot term by a second coefficient value, and scaling the second cubic slot term by a third coefficient value to produce scaled tPSA values and the sum of the scaled tPSA values; and wherein determining the second value comprises scaling the received fPSA value by a value of the fourth coefficient, scaling the third cubic slot term by a fifth value of the coefficient, and scaling the fourth slot term cubic by a sixth coefficient value to produce scaled fPSA values, and the sum of the scaled fPSA values.
[3]
3. System according to claim 1, characterized in that the information for the plurality of blood markers also includes an intact PSA (iPSA) value and a human kallikrein 2 (hK2) value, and in which the determination of the probability of the event associated with prostate cancer is also based, at least in part, on the iPSA value and the hK2 value.
[4]
4. System according to claim 1, characterized by the fact that the first internal node is specified as 3.89, the second
The second inner node is specified as 5.54, the third inner node is specified as 0.81, and the fourth inner node is specified as 1.19.
[5]
5. System according to claim 1, characterized in that the input interface comprises a network interface, and in which the network interface is configured to receive information for the plurality of blood markers through at least one network.
[6]
6. System, according to claim 1, characterized in that the input interface is also configured to receive patient information, in which the patient information includes age information, and in which the determination of event probability associated with prostate cancer is also based, at least in part, on age information through: comparing age information to a threshold value; selecting a first set of coefficients when the age information is above the threshold value and selecting a second set of coefficients when the age information is below the threshold value; and where the determination of the probability of the event associated with prostate cancer is also based, at least in part, on the first set of coefficients or second set of coefficients selected.
[7]
7. System according to claim 1, characterized in that it additionally comprises: a detection module configured to measure information for at least one first blood marker of the plurality of blood markers, wherein the detection module is configured to provide the information for at least one first blood marker to the at least one processor through the input interface after having measured the information for at least one first blood marker.
[8]
8. System according to claim 7, characterized in that the input interface is configured to receive at least a second blood marker from the plurality of blood markers from a source connected in a network through a network.
[9]
9. System according to claim 1, characterized in that the output interface comprises a display interface, a speaker interface, or at least one light source, and wherein the emission of the indication comprises the provision of a numerical indication of the probability on the display interface, providing an audio indication of the probability through the speaker interface, or by activating at least one light source.
[10]
10. System according to claim 1, characterized in that the emission of an indication of the probability of the event associated with prostate cancer comprises emission of an indication of an estimated volume of the prostate gland or the emission of an indication of a probability that a prostate cancer biopsy will be positive.
[11]
11. Method for determining a probability of an event associated with prostate cancer, wherein the method is characterized by the fact that it comprises: the reception, through an input interface, of information for a plurality of markers of the blood, wherein the information for the plurality of blood markers includes a free prostate specific antigen (fPSA) value and a total PSA (tPSA) value; the evaluation, using at least one processor, of a logistic regression model based, at least in part, on information received for determining a probability of an event associated with prostate cancer in a person, wherein the evaluation of the logistic regression model comprises: the determination of the cubic slot terms for tPSA, wherein the determination of the cubic slot terms for tPSA comprises the determination of the cubic slot terms for tPSA based on a first cubic slot that has a first inner node between 2 and 5 and a second inner node between 5 and 8; determining the cubic slot terms for fPSA, where determining the cubic slot terms for fPSA comprises determining the cubic slot terms for fPSA based on a second cubic slot that has a third internal node between 0.25 and 1 and a fourth internal node between 1.0 and 2.0; determining a first value for tPSA based, at least in part, on the received tPSA value and the cubic groove terms determined for tPSA; determining a second value for fPSA based, at least in part, on the received fPSA value and the cubic groove terms determined for fPSA; and determining the probability of the event associated with prostate cancer based, at least in part, on the first value and the second value; and issuing an indication of the probability of the event associated with prostate cancer.
[12]
12. System, according to claim 1, characterized in that it also comprises: a microfluidic sample analyzer in electronic communication with the emission interface, the microfluidic sample analyzer, which comprises: a housing; an opening in the housing configured to receive a cassette having at least one microfluidic channel, wherein the housing
cro includes a component configured to interface with a coupling component in the cassette to detect the cassette within the housing; a pressure control system positioned within the housing, wherein the pressure control system is configured to pressurize at least one microfluidic channel in the cassette to move the sample through the at least one microfluidic channel; and an optical system positioned within the housing, wherein the optical system includes at least one light source and at least one detector spaced from the light source, wherein the light source is configured to pass through the light of the cassette when the cassette is inserted into the sample analyzer and wherein the detector is positioned opposite the light source to detect the amount of light passing through the cassette; and a user interface associated with the wrapper to enter at least a person's age.
[13]
13. Method for determining a probability of an event associated with prostate cancer in a person, characterized in that it comprises: the provision of a microfluidic sample analyzer, which comprises: a housing; an opening in the housing configured to receive a cassette having at least one microfluidic channel, wherein the housing includes a component configured to interface with a coupling component in the cassette to detect the cassette within the housing; a pressure control system positioned within the housing, wherein the pressure control system is configured to pressurize at least one microfluidic channel in the cassette to move the sample through the at least one microfluidic channel; an optical system positioned within the housing, wherein the optical system includes at least one light source and at least one detector spaced from the light source, wherein the light source is configured to pass light through the cassette when the cassette is inserted into the sample analyzer and wherein the detector is positioned opposite the light source to detect the amount of light passing through the cassette; and a user interface associated with the wrapper for entering at least a person's age; determining the information for a plurality of blood markers using the microfluidic sample analyzer, wherein the information for the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a total PSA (tPSA) value ), and an intact PSA (iPSA) value; and the evaluation, using at least one processor, of a logistic regression model based, at least in part, on information for determining a probability of an event associated with prostate cancer in a person, which evaluates that person - tion of the logistic regression model comprises grading each variable of a plurality of variables by a different coefficient value to produce graded variables and the sum of values for graded variables used to produce the probability of the event associated with prostate cancer in a person, where the plurality of variables includes age and at least two variables included in the information received from the detector and is selected from the group consisting of fPSA, iPSA, and tPSA.
[14]
14. System, according to claim 1, characterized in that it also comprises: a device, which comprises:
a first analysis region comprising a first binding partner; and a second analysis region comprising a second binding partner, wherein the first binding partner is adapted to bind with at least one of free prostate-specific antigen (fPSA), intact prostate-specific antigen (iPSA). ) and total PSA (tPSA), and wherein the second binding partner is adapted to bind with at least one other of fPSA, iPSA, and tPSA, and a detector associated with the first and second analysis regions to determine the information about the plurality of blood markers, wherein the device is configured to provide the information about the plurality of blood markers to at least one processor.
[15]
15. Method, characterized in that it comprises: introducing a sample into a device comprising: a first analysis region comprising a first binding partner; and a second analysis region comprising a second binding partner, wherein the first binding partner is adapted to bind with at least one of free prostate-specific antigen (fPSA), intact prostate-specific antigen (iPSA). ) and total PSA (tPSA), and wherein the second binding partner is adapted to bind with at least one other of fPSA, iPSA, and tPSA, and provision for any one of fPSA, iPSA, and /or sample tPSA binds with the first and/or second binding partners in the first and second analysis regions; determining an fPSA, iPSA and/or tPSA characteristic using one or more detectors associated with the first and second analysis regions; the input of fPSA, iPSA, and/or tPSA characteristics into a processor programmed to evaluate a logistic regression model based, at least in part, on information received from at least one detector to determine a probability of an event associated with prostate cancer in a person, where the evaluation of the logistic regression model comprises grading each variable from a plurality of variables by a different coefficient value to produce graded variables and the sum of values for the graded variables used to produce the probability of the event associated with prostate cancer in a person, where the plurality of variables includes age and at least two variables included in the information received from the detector and are selected from the group consisting of fPSA, iPSA and tPSA; and determining the probability of the event associated with prostate cancer.
[16]
16. System, according to claim 1, characterized in that it also comprises: a microfluidic system in electronic communication with the emission interface to provide information on the plurality of blood markers to the emission interface, the system microfluidic, which comprises: a first microfluidic channel that includes at least one inlet and one outlet; a first reagent stored in the first microfluidic channel; a seal that covers the entrance of the first micro-channel
fluidic and a seal covering the outlet of the first microfluidic channel to store the first reagent in the first microfluidic channel; a second microfluidic channel that includes at least one inlet and one outlet; a first analysis region, a second analysis region, and a third analysis region, wherein each of the analysis regions includes one of an anti-iPSA specific capture antibody, an anti-fPSA specific capture antibody , and an anti-tPSA specific capture antibody; wherein one or more of the first, second and third analysis regions are in fluid communication with the second microfluidic channel; a fluidic connector that can be connected to the microfluidic system, wherein the fluidic connector comprises a fluid passageway that includes a fluid passageway inlet and a fluid passageway outlet, wherein, with the connection, the passageway inlet of fluid connects with the outlet of the first microfluidic channel to allow fluid communication between the fluid passage and the first microfluidic channel, and the outlet of the fluid passage connects with the inlet of the second microfluidic channel to allow fluid communication between the fluid passage and the second microfluidic channel, wherein the first and second microfluidic channels are not in fluid communication with each other without a connection through the fluidic connector; and a source of a metal colloid conjugated to an antibody that binds anti-PSA.
[17]
17. Invention, in any form of its embodiment or in any applicable category of claim, for example,
product, apparatus, process or use, or any other type of claim encompassed by the matter initially described, disclosed or illustrated in the patent application.

类似技术:

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公开号 | 公开日 | 专利标题

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AU2020203006B2|2021-04-22|Methods and apparatuses for predicting risk of prostate cancer and prostate gland volume

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JP2018200323A|2018-12-20|Systems and devices for analysis of samples

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JP2020073885A|2020-05-14|Method and device for predicting risk of prostate cancer and prostate volume

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US20170168060A1|2017-06-15|

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CN104364788A|2015-02-18|

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TWI638277B|2018-10-11|

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2018-03-27| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2019-10-01| B06U| Preliminary requirement: requests with searches performed by other patent offices: procedure suspended [chapter 6.21 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201261606554P| true| 2012-03-05|2012-03-05|

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FI20125238|2012-03-05|

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FI20125238|2012-03-05|

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US61/606,554|2012-03-05|

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PCT/US2013/028978|WO2013134179A2|2012-03-05|2013-03-05|Methods and apparatuses for predicting risk of prostate cancer and prostate gland volume|

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