KR102303487B1 - Methods and apparatuses for predicting risk of prostate cancer and prostate gland volume - Google Patents

Abstract

The present invention provides a method and apparatus for predicting the risk of prostate cancer and/or prostate volume. More particularly, the present invention relates to methods and apparatus for providing and using models for predicting risk of prostate cancer and/or for predicting prostate volume. Methods and apparatus for predicting prostate cancer risk and/or prostate volume are provided, at least in part, using information from a panel of kallikrein markers.

Description

METHODS AND APPARATUSES FOR PREDICTING RISK OF PROSTATE CANCER AND PROSTATE GLAND VOLUME

The present invention relates to methods and devices for predicting prostate cancer risk and/or prostate volume. More particularly, the present invention relates to methods and apparatus for providing and using models for predicting risk of prostate cancer and/or for predicting prostate volume.

Most men who have elevated blood levels of total prostate specific antigen (PSA)—the most common trigger for a biopsy among American men—do not have prostate cancer. Consequently, it has been estimated that there are close to 750,000 unnecessary prostate biopsies each year in the United States. There is considerable evidence that measuring the isoforms individually, rather than combining isoforms of PSA together in a single measure of total PSA, can help predict the presence of prostate cancer. This data includes studies showing that cancer is predicted by free PSA, BPSA or -2proPSA. Indeed, free PSA is often measured individually, and urologists are provided with results regarding total PSA and free-to-total PSA ratios, with an estimated 10 million free PSAs being measured per year. In addition, hK2, a molecule that converts PSA from its pro-to-active form, has evidence to inform prostate risk. However, none of these markers on their own are good predictors of prostate biopsy results.

Several attempts to build predictive models for prostate cancer, inter alia "Prostate Cancer Prevention Trial Risk Calculator", "Sunnybrook" and the European Randomized trial of Screening for Prostate Cancer (ERSPC) There was a risk calculator. The problem with these models is that they require rather extensive clinical work-up, ie the patient must visit a urologist. For example, the ERSPC risk calculator requires data on prostate volume, obtained by inserting an ultrasound probe into the rectum. Accordingly, new methods and devices for predicting prostate cancer risk and/or prostate volume would be beneficial.

SUMMARY OF THE INVENTION It is an object of the present invention to solve the above-mentioned problem.

Methods and devices for predicting prostate cancer risk and/or prostate volume are provided. More particularly, the present invention relates to methods and apparatus for providing and using models for predicting risk of prostate cancer and/or for predicting prostate volume. In some embodiments, methods and devices for predicting prostate cancer risk and/or prostate volume are provided, at least in part, using information from a panel of kallikrein markers. The patent subject matter of this application, in some cases, includes interrelated methods, alternative solutions to a particular problem, and/or multiple different uses of systems and devices.

One object of the present invention is to provide a method for obtaining the probability of an event using a logistic regression model for predicting the risk of prostate cancer in men.

In one set of embodiments, a computer for determining a probability of an event associated with prostate cancer is provided. The computer comprises an input interface configured to receive information on a plurality of blood markers, wherein the information on the plurality of blood markers includes free prostate-specific antigen (fPSA) values and total PSA (tPSA) values. The computer also includes at least one processor programmed to evaluate a logistic regression model based at least in part on the received information to determine a probability of an event associated with prostate cancer in the human body. Evaluating the logistic regression model is determining cubic spline terms for tPSA, and determining cubic spline terms for tPSA is a first internal knot between 2 and 5. ) and determining cubic spline terms for tPSA, comprising determining cubic spline terms for tPSA based on a first cubic spline having a second inner knot between 5 and 8; determining cubic spline terms for fPSA, wherein determining the cubic spline terms for fPSA is based on a second cubic spline having a third inner knot between 0.25 and 1 and a fourth inner knot between 1.0 and 2.0. determining cubic spline terms for the fPSA, including determining cubic spline terms for the fPSA by using determining a value of 1, determining a second value for fPSA based at least in part on the received fPSA value and cubic spline terms for the determined fPSA, and based at least in part on the first value and the second value thereby determining the probability of an event associated with prostate cancer. The computer also includes an output interface configured to output an indication of a probability of an event associated with prostate cancer.

In one set of embodiments, a system for determining a probability of an event associated with prostate cancer is provided. The system comprises a detector configured to measure values for a plurality of blood markers, wherein the plurality of blood markers detect free prostate specific antigen (fPSA), total PSA (tPSA) and intact PSA (iPSA). include The system also includes at least one processor in electronic communication with the detector. wherein the at least one processor is programmed to evaluate a logistic regression model based at least in part on values measured for fPSA, tPSA and iPSA to determine a probability of an event associated with high risk prostate cancer in a human body. . Evaluating the logistic regression model is determining cubic spline terms for tPSA, wherein determining cubic spline terms for tPSA includes a first inner knot between 4 and 5 and a second inner knot between 6 and 8. determining cubic spline terms for tPSA, comprising determining cubic spline terms for tPSA based on a first cubic spline with Determining the cubic spline terms for fPSA includes determining cubic spline terms for fPSA based on a second cubic spline having a third inner knot between 0.25 and 1 and a fourth inner knot between 1.0 and 2.0. determining cubic spline terms for the fPSA, and determining a first value for the tPSA based at least in part on the received tPSA value and the determined cubic spline terms for the tPSA, the received fPSA value and determining a second value for the fPSA based at least in part on the cubic spline terms for the determined fPSA, and determining, based at least in part on the first value and the second value, a probability of an event associated with prostate cancer. and outputting an indication of the probability of an event associated with prostate cancer.

In one set of embodiments, a method of determining a probability of an event associated with prostate cancer is provided. The method includes receiving information on a plurality of blood markers through an input interface, wherein the information on the plurality of blood markers includes free prostate specific antigen (fPSA) values and total PSA (tPSA) values. including the steps of The method further comprises evaluating, using the at least one processor, a logistic regression model based at least in part on the received information to determine a probability of an event associated with prostate cancer in the human body. Evaluating the logistic regression model is determining cubic spline terms for tPSA, wherein determining cubic spline terms for tPSA includes a first inner knot between 2 and 5 and a first inner knot between 5 and 8. determining cubic spline terms for tPSA, comprising determining cubic spline terms for tPSA based on a first cubic spline having 2 inner knots; and cubic spline terms for fPSA and determining the cubic spline terms for the fPSA based on the second cubic spline having a third inner knot between 0.25 and 1 and a fourth inner knot between 1.0 and 2.0. determining cubic spline terms for the fPSA, comprising determining cubic spline terms for determining a value of 1 and determining a second value for fPSA based at least in part on the received fPSA value and cubic spline terms for the determined fPSA, wherein the first value and the second value are at least in part based, determining a probability of an event associated with prostate cancer. The method further comprises outputting an indication of the probability of an event associated with prostate cancer.

In one set of embodiments, a computer-readable storage medium is provided that is encoded with a plurality of instructions that, when executed by a computer, perform a method of determining a probability of an event associated with prostate cancer. The method includes receiving information on a plurality of blood markers, wherein the information on the plurality of blood markers includes free prostate specific antigen (fPSA) values and total PSA (tPSA) values; evaluating the logistic regression model based at least in part on the received information to determine a probability of an event associated with prostate cancer within. Evaluating the logistic regression model is determining cubic spline terms for tPSA, wherein determining cubic spline terms for tPSA includes a first inner knot between 2 and 5 and a first inner knot between 5 and 8. determining cubic spline terms for tPSA, comprising determining cubic spline terms for tPSA based on a first cubic spline having 2 inner knots; determining, wherein the determining the cubic spline terms for fPSA is based on a second cubic spline having a third inner knot between 0.25 and 1 and a fourth inner knot between 1.0 and 2.0. determining cubic spline terms for fPSA, comprising determining cubic spline terms, and a first value for tPSA based at least in part on the received tPSA value and the determined cubic spline terms for tPSA determining the second value for fPSA based at least in part on the received fPSA value and the determined cubic spline terms for the fPSA; , determining a probability of an event associated with prostate cancer. The method further comprises outputting an indication of the probability of an event associated with prostate cancer.

In one set of embodiments, a computer for determining a probability of an event associated with prostate cancer is provided. The computer comprises an input interface configured to receive information about a plurality of blood markers, wherein the information about the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a total PSA (tPSA) value, an intact PSA (iPSA) values and human kallikrein 2 (hK2) values. The computer also includes at least one processor programmed to evaluate the logistic regression model based at least in part on the received information to determine a probability of an event associated with prostate cancer in the human body. Evaluating the logistic regression model includes determining a probability of an event associated with prostate cancer based at least in part on the tPSA value, the iPSA value, the hK2 value, and the ratio of the fPSA value to the tPSA value. The computer also includes an output interface configured to output an indication of a probability of an event associated with prostate cancer.

In one set of embodiments, a method of determining a probability of an event associated with prostate cancer is provided. The method includes receiving information on a plurality of blood markers through an input interface, wherein the information on the plurality of blood markers includes a free prostate-specific antigen (fPSA) value, a total PSA (tPSA) value, an intact PSA ( iPSA) values and human kallikrein 2 (hK2) values, using at least one processor to determine a probability of an event associated with prostate cancer in the human body; evaluating the logistic regression model based, at least in part, on the logistic regression model. Evaluating the logistic regression model comprises determining a probability of an event associated with prostate cancer based at least in part on the tPSA value, the iPSA value, the hK2 value, and the ratio of the fPSA value to the tPSA values, and outputting an indication of the probability of the event.

In one set of embodiments, a computer-readable storage medium is provided that is encoded with a plurality of instructions that, when executed by a computer, perform a method of determining a probability of an event associated with prostate cancer. The method includes receiving, through an input interface, information on a plurality of blood markers, wherein the information on the plurality of blood markers includes a free prostate-specific antigen (fPSA) value, a total PSA (tPSA) value, and an intact PSA. using at least one processor to determine a probability of an event associated with prostate cancer in the human body and the receiving step, comprising: an (iPSA) value and a human kallikrein 2 (hK2) value; evaluating the logistic regression model based, at least in part, on the logistic regression model. Evaluating the logistic regression model comprises determining a probability of an event associated with prostate cancer based, at least in part, on the tPSA value, the iPSA value, the hK2 value, and the ratio of the fPSA value to the tPSA values, and outputting an indication of the probability of the event.

In one set of embodiments, a computer for determining a probability of an event associated with prostate cancer is provided. The computer comprises an input interface configured to receive information about a plurality of blood markers, wherein the information about the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a total PSA (tPSA) value, an intact PSA (iPSA) values and human kallikrein 2 (hK2) values. The computer also includes at least one processor programmed to evaluate a logistic regression model based at least in part on the received information to determine a probability of an event associated with prostate cancer in the human body. Evaluating the logistic regression model determines the nonlinear term for tPSA by squaring the tPSA value by a first exponent, determining the nonlinear term for fPSA by squaring the fPSA value by a second exponent, and determining the tPSA value, the fPSA value. , determining the probability of an event associated with prostate cancer based at least in part on the iPSA value, the hK2 value, the nonlinear term for tPSA, and the nonlinear term for fPSA. The computer further comprises an output interface configured to output an indication of a probability of an event associated with prostate cancer.

In one set of embodiments, a method of determining a probability of an event associated with prostate cancer is provided. The method includes receiving, through an input interface, information on a plurality of blood markers, wherein the information on the plurality of blood markers includes a free prostate-specific antigen (fPSA) value, a total PSA (tPSA) value, an intact PSA (iPSA) values and human kallikrein 2 (hK2) values. The method further comprises evaluating, using the at least one processor, the logistic regression model based at least in part on the received information to determine a probability of an event associated with prostate cancer in the human body. Evaluating the logistic regression model comprises determining a nonlinear term for tPSA by squaring the tPSA value by a first exponent, determining a nonlinear term for fPSA by squaring the fPSA value by a second exponent, and tPSA value; determining the probability of an event associated with prostate cancer based at least in part on the fPSA value, the iPSA value, the hK2 value, the nonlinear term for tPSA and the nonlinear term for fPSA. The method further comprises outputting an indication of the probability of an event associated with prostate cancer.

In one set of embodiments, a computer-readable storage medium is provided that is encoded with a plurality of instructions that, when executed by a computer, perform a method of determining a probability of an event associated with prostate cancer. The method comprises receiving information on a plurality of blood markers, wherein the information on the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a total PSA (tPSA) value, an intact PSA (iPSA) value. and human kallikrein 2 (hK2) values. The method further comprises evaluating the logistic regression model based at least in part on the received information to determine a probability of an event associated with prostate cancer in the human body. Evaluating the logistic regression model comprises determining a nonlinear term for tPSA by squaring the tPSA value by a first exponent, determining a nonlinear term for fPSA by squaring the fPSA value by a second exponent, and tPSA value; determining the probability of an event associated with prostate cancer based at least in part on the fPSA value, the iPSA value, the hK2 value, the nonlinear term for tPSA and the nonlinear term for fPSA. The method further comprises outputting an indication of the probability of an event associated with prostate cancer.

In one set of embodiments, a computer for determining a probability of an event associated with prostate cancer is provided. The computer comprises an input interface configured to receive information about a plurality of blood markers, wherein the information about the plurality of blood markers includes a free prostate specific antigen (fPSA) value and a total PSA (tPSA) value, an intact PSA (iPSA) values and human kallikrein 2 (hK2) values. The computer also includes at least one processor programmed to evaluate the logistic regression model based at least in part on the received information to determine a probability of an event associated with prostate cancer in the human body. Evaluating the logistic regression model determines the linear spline terms for tPSA, determines the linear spline terms for fPSA, and the second for tPSA based at least in part on the received tPSA value and the determined linear spline terms for tPSA. determine a value of 1, determine a second value for fPSA based at least in part on the received fPSA value and linear spline terms for the determined fPSA, and determine a second value for fPSA based at least in part on the first value and the second value. and determining a probability of an associated event. The computer also includes an output interface configured to output an indication of a probability of an event associated with prostate cancer.

In one set of embodiments, a method of determining a probability of an event associated with prostate cancer is provided. The method includes receiving, through an input interface, information on a plurality of blood markers, wherein the information on the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a total PSA (tPSA) value, and Intact PSA (iPSA) values and human kallikrein 2 (hK2) values are included. The method further comprises evaluating, using the at least one processor, the logistic regression model based at least in part on the received information to determine a probability of an event associated with prostate cancer in the human body. Evaluating the logistic regression model comprises determining linear spline terms for tPSA, determining linear spline terms for fPSA, tPSA based at least in part on the received tPSA value and the determined linear spline terms for tPSA. determining a first value for fPSA, determining a second value for fPSA based at least in part on the received fPSA value and the determined linear spline terms for fPSA, the first value and the second value at least in part determining a probability of an event associated with prostate cancer based on the step. The method further comprises outputting an indication of the probability of an event associated with prostate cancer.

In one set of embodiments, a computer-readable storage medium is provided that is encoded with a plurality of instructions that, when executed by a computer, perform a method of determining a probability of an event associated with prostate cancer. The method comprises receiving information about a plurality of blood markers, wherein the information about the plurality of blood markers includes a free prostate specific antigen (fPSA) value, a total PSA (tPSA) value, an intact PSA (iPSA) values and human kallikrein 2 (hK2) values. The method further comprises evaluating the logistic regression model based at least in part on the received information to determine a probability of an event associated with prostate cancer in the human body. Evaluating the logistic regression model comprises determining linear spline terms for tPSA, determining linear spline terms for fPSA, tPSA based at least in part on the received tPSA value and the determined linear spline terms for tPSA. determining a first value for fPSA, determining a second value for fPSA based at least in part on the received fPSA value and the determined linear spline terms for fPSA, the first value and the second value at least in part determining a probability of an event associated with prostate cancer based on the step. The method further comprises outputting an indication of the probability of an event associated with prostate cancer.

In one set of embodiments, a system for determining a risk of high risk cancer is provided. The system comprises an input interface configured to receive information about a plurality of blood markers, wherein the information about the plurality of blood markers includes a free prostate specific antigen (fPSA), a total PSA (tPSA) value, an intact PSA ( iPSA) values and hK2 values. The system also includes at least one processor, wherein the at least one processor enters the received values into a logistic regression model, wherein at least a tPSA value and tPSA values use both linear and non-linear terms to form a logistic regression model. and programmed to evaluate the logistic regression model to determine the risk of high risk cancer.

In one set of embodiments, a system for determining a probability of an event associated with prostate cancer in a human body is provided. The system includes a microfluidic sample analyzer, the 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 is within the housing and a component configured to interface with a mating component on the cassette to detect the cassette in the . The system also includes a pressure control system positioned within the housing, the pressure control system configured to pressurize the at least one microfluidic channel within the cassette to move the sample through the at least one microfluidic channel. The system further comprises an optical system positioned within the housing, the optical system comprising at least one light source and at least one detector spaced apart from the light source, wherein the light source transmits light through the cassette when the cassette is inserted into the sample analyzer. and the detector is positioned opposite the light source to detect the amount of light passing through the cassette. The system includes a processor in electrical communication with a microfluidic sample analyzer and a user interface associated with the housing for inputting at least the age of the person, the processor configured to determine a probability of an event associated with prostate cancer in the human body. to evaluate the logistic regression model based at least in part on information received from the at least one detector, wherein evaluating the logistic regression model comprises calculating each of the plurality of variables by different coefficients to yield scaled variables. and summing the values for the scaled variables that are scaled to a value and used to calculate a probability of an event associated with prostate cancer in the human body, wherein the plurality of variables depend on age and information received from the detector. It comprises at least two variables included and is selected from the group consisting of fPSA, iPSA and tPSA.

In one set of embodiments, a method of determining a probability of an event associated with prostate cancer in a human body is provided. The method includes providing a microfluidic sample analyzer, the microfluidic sample analyzer comprising: a housing, an opening in the housing configured to receive a cassette having at least one microfluidic channel, the housing configured to detect the cassette within the housing A pressure control system positioned within an opening and a housing comprising a component configured to interface with a mating component on the cassette, wherein the pressure control system comprises at least one in the cassette for moving a sample through the at least one microfluidic channel. and a pressure control system configured to pressurize the microfluidic channel. A microfluidic sample analyzer is also an optical system positioned within the housing, the optical system comprising at least one light source and at least one detector spaced apart from the light source, the light source directing light through the cassette when the cassette is inserted into the sample analyzer. and a user interface associated with the housing for inputting the optical system and at least the age of a person, configured to pass therethrough, wherein the detector is positioned opposite the light source to detect an amount of light passing through the cassette. The method includes determining information on a plurality of blood markers using a microfluidic sample analyzer, wherein the information on the plurality of blood markers includes a free prostate-specific antigen (fPSA) value, a total PSA (tPSA) value, and no a logistic regression model based, at least in part, on the information, using at least one processor to determine a probability of an event associated with prostate cancer in a human body, comprising: an impaired PSA (iPSA) value; and evaluating the logistic regression model, wherein the evaluating the logistic regression model comprises scaling each of the plurality of variables to different coefficient values to yield scaled variables and probabilities of events associated with prostate cancer in the human body. summing values for the scaled variables used to calculate selected from the group being

In one set of embodiments, a system is provided. The system comprises a device, wherein the device comprises a first assay region comprising a first binding partner and a second assay region comprising a second binding partner, wherein the first binding partner is free prostate-specific. and wherein the second binding partner is adapted to bind at least one of an antigen (fPSA), an intact prostate specific antigen (iPSA) and a total PSA (tPSA), and the second binding partner is adapted to bind at least another of fPSA, iPSA and tPSA. The system is configured to evaluate a logistic regression model based at least in part on a detector associated with the first and second regions of analysis and information received from the detector to determine a probability of an event associated with prostate cancer in the human body. Comprising a programmed processor, wherein evaluating the logistic regression model scales each of the plurality of variables to different coefficient values to yield scaled variables and calculates a probability of an event associated with prostate cancer in the human body. summing values for the scaled variables used to is chosen

In one set of embodiments, a method is provided. The method comprises introducing a sample into a device, wherein the device comprises a first assay region comprising a first binding partner and a second assay region comprising a second binding partner, wherein the first binding partner is free and is adapted to bind at least one of 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 another of fPSA, iPSA and tPSA. The method comprises the steps of allowing any PSA of fPSA, iPSA and/or tPSA from the sample to bind to first and/or second binding partners in first and second analysis regions, the first and second analysis regions determining a characteristic of fPSA, iPSA and/or tPSA using one or more detectors associated with inputting characteristics of the fPSA, iPSA and/or tPSA into a processor programmed to evaluate the logistic regression model based in part on the evaluation of the logistic regression model, wherein evaluating the logistic regression model comprises: scaling each to a different coefficient value and summing the values for the scaled variables used to calculate a probability of an event associated with prostate cancer in the human body, wherein the plurality of variables are derived from age and detector. and inputting at least two variables included in the received information and selected from the group consisting of fPSA, iPSA and tPSA, and determining a probability of an event associated with prostate cancer.

In one set of embodiments, a device is provided. The device comprises a microfluidic system, the microfluidic system comprising a first microfluidic channel comprising at least one inlet and one outlet, a first reagent stored in the first microfluidic channel, a first microfluidic a second microfluidic channel comprising a seal covering an inlet of the first microfluidic channel and a seal covering an outlet of the first microfluidic channel for storing a first reagent in the channel, at least one inlet and one outlet includes The device also comprises a first analysis region, a second analysis region and a third analysis region, each comprising one of an anti-iPSA specific capture antibody, an anti-fPSA specific capture antibody and an anti-tPSA specific capture antibody. and at least one of the first, second and third analysis regions is in fluid communication with the second microfluidic channel. The device also includes a fluidic connector connectable with the microfluidic system, the fluidic connector comprising a fluid path comprising a fluid path inlet and a fluid path outlet, wherein upon connection, the fluid path inlet is coupled to the fluid path and It is connected to the outlet of the first microfluidic channel to enable fluid communication between the first microfluidic channels, and the fluid path outlet is at the inlet of the second microfluidic channel to enable fluid communication between the fluid path and the second microfluidic channel. connected, and the first and second microfluidic channels are not in fluid communication with each other with the absence of a connection through the fluidic connector. The device also includes a source of metal colloid conjugated to an antibody that binds anti-PSA.

In one set of embodiments, a method is provided for obtaining a probability of an event using a logistic regression model for predicting the risk of prostate cancer in men. The method is:

a) providing a logistic regression model obtained by using multivariate logistic regression analysis of data of a plurality of men, wherein the data includes data on the prostate cancer status for each male of the plurality of men and the prostate cancer status preceded by the data of age; data comprising determinations of blood markers, total prostate specific antigen (tPSA), free PSA (fPSA), intact PSA (iPSA) and optionally human kallikrein 2 (hK2) from blood samples of the men , and the logistic regression model is

to obtain the logistic regression model, where π is the probability of the event and β _i is a variable over j variables including age, tPSA, fPSA, iPSA and optionally hK2, respectively. providing a coefficient for x _{i ;}

b) providing the male's age in years;

c) from the male blood sample, the blood markers

i) tPSA,

ii) fPSA;

iii) iPSA;

iv) optionally determining each hK2;

d) said probability of said event of said man

을 규정하고,i) Conjugation formula:

stipulate,

로 획득하는 것ii) the probability

to obtain with

utilizing the logistic regression model using the provided age of step b) and the determined blood markers of step c) to obtain by

A feature of this method is that the risk for cancer in the logistic regression model is based solely on tPSA when tPSA is > 15 ng/ml, preferably > 20 ng/ml and most preferably > 25 ng/ml.

Another object of the present invention is to provide a method for predicting prostate volume using a linear regression model.

Embodiments of the present invention provide a method for predicting prostate volume using a linear regression model, wherein the method comprises:

a) providing a linear regression model obtained by using a linear regression analysis of data of a plurality of men, wherein the data is for each male of the plurality of men,

i) data on prostate volume and

ii) preceded by data for prostate volume, age; Blood markers from blood samples of the men: data comprising determinations of total prostate specific antigen (tPSA), free PSA (fPSA), intact PSA (iPSA) and optionally human kallikrein 2 (hK2). wherein the linear regression model has the formula:

to obtain the linear regression model, where V is the prostate volume and β _{i is the variable x i} over j variables each comprising age, tPSA, fPSA, iPSA and optionally hK2. providing the coefficient for;

b) providing the male's age in years;

c) determining from the male blood sample the blood markers tPSA, fPSA, iPSA and optionally hK2, respectively;

d) utilizing said linear regression model using said provided age of step b) and said determined blood markers of step c) to obtain said predicted prostate volume of said male.

A feature of this method is that the risk for cancer in the linear regression model is based solely on tPSA when tPSA is > 15 ng/ml, preferably > 20 ng/ml and most preferably > 25 ng/ml.

Other advantages and novel features of the invention will become apparent from the following detailed description of various non-limiting embodiments of the invention when considered in conjunction with the accompanying drawings. To the extent this specification and documents incorporated by reference contain conflicting and/or contradictory material, this specification will control. If two or more documents incorporated by reference contain conflicting and/or contradictory content with respect to each other, the document with the more recent effective date will govern.

BRIEF DESCRIPTION OF THE DRAWINGS Non-limiting embodiments of the present invention will be described by way of example with reference to the accompanying drawings, which are schematic and are not intended to be drawn to scale. In the drawings, each identical or nearly identically depicted component is typically represented by a single number. In the interest of clarity, not all components are labeled in all drawings or all components of each embodiment of the invention are shown where explanation is not required to enable one of ordinary skill in the art to understand the present invention.
1 is a flow diagram of a process for determining the probability of a positive cancer biopsy in accordance with some embodiments of the present invention.
2 is a flow diagram of a process for conditionally selecting a logistic regression model in accordance with some embodiments of the present invention.
3 is a schematic diagram of a computer system in which some embodiments of the present invention may be implemented.
4 is a diagram illustrating an exemplary network environment in which some embodiments of the present invention may be used.
5 is a block diagram illustrating various components that may be part of a microfluidic system and a sample analyzer that may be used to determine one or more blood markers in accordance with some embodiments of the present invention.
6 is a perspective view of a sample analyzer and cassette that may be used to determine one or more blood markers in accordance with some embodiments of the present invention.
7 is a perspective view of a cassette including a fluidic connector that may be used to determine one or more blood markers in accordance with some embodiments of the present invention.
8 is an exploded view of a fluidic connector that may be used to determine one or more blood markers in accordance with some embodiments of the present invention.
9 is an exploded view of a cassette that may be used to determine one or more blood markers in accordance with some embodiments of the present invention.
10 is a schematic diagram of a cassette comprising a fluidic connector that may be used to determine one or more blood markers in accordance with some embodiments of the present invention.
11A is a schematic diagram of a cassette that may be used to determine one or more blood markers in accordance with some embodiments of the present invention.
11B-11F are schematic diagrams of cassettes formed from multiple components that may be used to determine one or more blood markers according to a set of embodiments.
12 is a schematic diagram of a portion of a sample analyzer that may be used to determine one or more blood markers in accordance with some embodiments of the invention.
13 is a block diagram illustrating a control system of a sample analyzer associated with various different components that may be used to determine one or more blood markers in accordance with some embodiments of the present invention.
14 is a schematic diagram illustrating a microfluidic system of a cassette that may be used to determine one or more blood markers in accordance with some embodiments of the present invention.
15 is a plot depicting a measurement of optical density as a function of time indicative of determination of one or more blood markers in accordance with some embodiments of the present invention.

As noted above, many conventional techniques for predicting the probability of prostate cancer and/or prostate volume are based, at least in part, on the patient's clinical examination (eg, digital rectal exam or DRE). Some embodiments described herein relate to methods and apparatus for determining a predicted probability of prostate cancer and/or prostate volume based, at least in part, on a panel of blood markers, without requiring clinical scrutiny. . As will be described in more detail below, the predictive probability of prostate cancer and/or prostate volume provided in a biopsy is a reliable metric that may be useful in assisting decisions related to a prostate biopsy.

Some embodiments relate to a computer system comprising at least one processor programmed to assess a risk of prostate cancer, wherein the risk of prostate cancer is determined based, at least in part, on values for a plurality of blood markers. In some embodiments, the computer system may be implemented as an integrated system (eg, on an analyzer and/or chip/cassette) having one or more detectors that determine a value for one or more of the blood markers described herein. can In other embodiments, the computer system may comprise a computer located remote from one or more detectors, and values for one or more of the blood markers described herein may be entered manually using a user interface and/ or the values may be received via a network interface that is communicatively coupled to a network (eg, the Internet). At least one processor in the computer system may be programmed to apply one or more models to the received inputs to assess the risk of prostate cancer on a biopsy, as will be described in more detail below.

Models used in accordance with some embodiments of the invention help to unify information about a plurality of input factors. For example, the input factors may be PSA, free-to-total PSA ratio, and/or digital rectal examination (DRE) status. Continuing these examples, a first patient may have a PSA of 3 ng/ml, a free-to-total PSA ratio of 15%, and a negative DRE, and a second patient may have a PSA of 9.5 ng/ml, 50% free. The patient may have a -to-total PSA ratio and negative DRA, and the third patient may have a PSA of 1.5 ng/ml, a free-to-total PSA ratio of 29% and a positive DRE. For patient 1, given that the PSA is adequate and the DRE is negative, the urologist wonders if a low (but not extremely low) free-to-total PSA ratio is sufficient to warrant biopsy can do. For a second patient, a high PSA value will usually justify an immediate biopsy, but a very high free-to-total PSA ratio may be a strong indication that the PSA elevation is benign. For a third patient, a positive DRE is usually a very worrisome sign, but considering the low PSA and normal free-to-total PSA ratio may be insufficient evidence that biopsy is necessary. As should be appreciated from the foregoing, when these factors are presented to the physician separately, it can be difficult to determine when a biopsy is needed. In addition, as the number of input factors increases, the determination of whether to perform a biopsy based on numerical information on various input factors becomes more and more complicated.

Both patients and clinicians differ in their propensity to choose biopsy, depending on differences regarding how they value early detection of cancer compared to the risks, harms and inconveniences of biopsy. do. Use strict decision rules (eg, biopsy if PSA > 4 ng/ml or free-to-total ratio < 15%) or risk scores (eg, prostate health index of 29) It is often impractical to incorporate such preferences using a prostate health index (PHI). For example, if a man dislikes medical procedures, it can be difficult to determine how high a PSA and/or PHI score will be "high enough" to justify a biopsy.

Instead of using strict decision rules, according to some embodiments, the at least one processor is programmed to use the at least one statistical model to process a plurality of inputs that guide decisions regarding the prostate biopsy. Inputs to the statistical models may include, but are not limited to, blood marker values, patient characteristics (eg, age) and other suitable information to determine the probability of finding a positive biopsy for prostate cancer. Such probabilities represent an interpretable scale that can be used to guide biopsy decisions taking into account patient and clinician preferences.

1 depicts a flow diagram of a process in accordance with some embodiments of the present invention. In act 110 , one or more values for blood markers are received by at least one processor processing using one or more of the techniques described herein. As described in more detail below, the blood marker value(s) may be received via a local input interface, such as a keyboard, touch screen, microphone or other input devices, or value(s) from a device located remote from the processor(s). either received from a network connection interface that receives the may be received in any suitable manner, including, but not limited to, being received.

In response to receiving the blood marker value(s), the process proceeds to act 120 , wherein at least one logistic regression model is evaluated to determine a probability of a positive biopsy for prostate cancer, wherein the probability is at least in part Based on the blood marker value(s) received as As will be described in more detail below, information other than blood marker values received (eg, age, cancer grade, etc.) is optionally used as factors in determining a particular model to use and/or an input to evaluate the selected model. factors can be used.

After determining the probability of a positive cancer biopsy, the process proceeds to act 130, where the probability is output to a user (eg, physician, patient) to guide the process of determining whether a biopsy is necessary. This probability may be output in any suitable manner. For example, in some embodiments, the probability may be output by displaying a numerical value representing the probability on a display screen of the device. In other embodiments, the probability may be output using one or more lights or other visual indicators on the device. In yet another embodiment, the probability may be provided using audio output, tactile output, or any combination of one or more of audio, tactile and visual output. In some embodiments, outputting the probability comprises transmitting information to the network connected device to inform the user of the determined probability. For example, the probability may be determined by one or more processors located at the remote site and the indication of the probability is, in response to determining the probability at the remote site, the user's (eg, physician) using the one or more networks. may be transmitted to an electronic device. Electronic devices that provide output to a user in accordance with the techniques described herein include, but are not limited to, laptops, desktop or tablet computers, smartphones, pagers, personal digital assistants, and electronic displays. It may be any suitable device that does not

As noted above, some embodiments relate to a method of obtaining a probability of an event using a logistic regression model to predict prostate cancer risk and/or prostate volume for men. In some embodiments, the method comprises information from one or more kallikrein markers: total prostate specific antigen (tPSA), free PSA (fPSA), intact PSA (iPSA) and human kallikrein 2 (hK2). entails doing Any suitable logistic regression model may be used, and the techniques described herein are not limited in this respect. In some embodiments, the probability of an event is reproduced below, in equation (I):

probability =

, where logit (L) is determined using any one of a plurality of logistic regression models. Non-limiting examples of nine different types of logistic regression models that may be used in accordance with the techniques described herein include:

1) Simple model (tPSA only)

(Age: age, the same in the following formulas)

2) 4 assay model using free/total ratio

In this model, the free PSA term is replaced by the ratio of free PSA to total PSA.

3. Four Assessment Analysis Models Using Logarithmic (tPSA) and Free/Total Ratio

In this model, the tPSA term is replaced with the logarithm of tPSA to account for the increasing contribution of this predictor.

4. Polynomial model

In this model, additional nonlinear terms for tPSA and fPSA are included. In the exemplary equations provided below, the square of tPSA is used to highlight the direct relationship between this term and prostate cancer, and the square root of the free/total PSA term is used to reflect the inverse association of this term with risk. However, it will be appreciated that higher order (eg, cubic) polynomial terms may also be included in some embodiments.

5. Linear splines for all 4 evaluation analyses

In this model, linear splines are added with a single knot at the median. The splines can be determined using the following equations:

sp1(x) = x, if x < knot

sp1(x) = knot, if x≥knot

sp2(x) = 0, if x < knot

sp2(x) = x - knot, if x ≥ knot

where the model is:

is expressed as

6. Linear splines for tPSA and fPSA

In this model, linear splines are included only for tPSA and fPSA to reduce the number of variables and simplify the model.

7. Cubic splines for all 4 evaluation analyses

In this model, cubic splines are included for each term. In the example provided below, a cubic spline with four knots is described. However, it should be appreciated that a cubic spline using any suitable number of knots, including, but not limited to, 5 knots, 6 knots, 7 knots, and 8 knots, may alternatively be used. The splines can be determined using the following equations:

Here, knot1 and knot4 are external knots for the tertiary spline, and knot2 and knot3 are internal knots for the tertiary spline. In some embodiments, the inner knots are between about 2 and about 5 and between about 5 and about 8 for tPSA, between about 0.25 and about 1 and between about 1.0 and about 2.0 for fPSA, between about 0.2 and about iPSA between 0.5 and between about 0.4 and about 0.8, between about 0.02 and 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 inner knots for tPSA, values of 0.81 and 1.19 are used for inner knots for fPSA, and values of 0.3 and 0.51 are used for iPSA. used for inner knots and values of 0.036 and 0.056 are used for inner knots of hK2.

In certain embodiments, the one or more inner knots for the tPSA are independently 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 6.5, 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 about 1 and about 5, between about 1 and about 4, and between about 1 and about 3. Other ranges are also possible.

In certain embodiments, the one or more inner knots for the fPSA are independently 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 0.7 and about 0.9, between about 1.1 and about 1.6, between about 1.1 and about 1.2, and between about 1.1 and about 2. Other ranges are also possible.

In certain embodiments, the one or more inner knots for the iPSA are independently 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 and 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 ranges are also possible.

In certain embodiments, the one or more inner knots for hK2 are independently between about 0.01 and about 0.03, between about 0.01 and about 0.04, between about 0.01 and 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 0.4 and about 0.07, between about 0.04 and about 1.0, between about 0.5 and about 1.0, and between about 0.6 and about 1.0. Other ranges are also possible.

As noted above, tertiary splines incorporating any suitable number of internal knots (eg, 3, 4, 5, 6 internal knots) may be used, and a tertiary containing two internal knots may be used. Examples of splines are provided for illustrative purposes only and without limitation. In embodiments involving more than two inner knots, the knots may be placed within one or more of the ranges described above or within some other suitable range. For example, in some embodiments, the knots may be specified such that the length of the segment of the spline between each pair of neighboring pairs of knots is essentially the same.

The model can be expressed as:

8. Tertiary Splines for tPSA and fPSA

In this model, cubic splines are included only for tPSA and fPSA to reduce the number of variables and simplify the model.

In certain embodiments, the inner knots for tPSA and fPSA are specified using one or more of the ranges described above for a cubic spline model for all 4 evaluation analyses. For example, the inner knots may be designated within a range of between about 2 and about 5 and between about 5 and about 8 for tPSA, between about 0.5 and about 1 and between about 1.0 and about 1.5 for fPSA. can For example, in one implementation, values of 3.89 and 5.54 are used for inner knots for tPSA and values of 0.81 and 1.19 are used for inner knots for fPSA. However, it should be appreciated that other values and/or ranges may alternatively be used. Moreover, it is recognized that any number of knots (eg, not 4 knots) may alternatively be used in some embodiments, as described above with respect to the cubic spline model for all 4 evaluation analyses. should be

The model can be expressed as:

9. Age stratified 3-D splines for tPSA and fPSA

In this model, cubic splines are divided into 2 parts in the dataset to produce different coefficients (β) used for patients with age less than/greater/same age than a particular age (eg 65 years old). is applied as Thus, in this model, the same representation (using different coefficient values) is used for both groups of patients. Examples of different coefficients that may be used by this model are provided in Table 1 below.

The model can be expressed as:

For age < 65:

For age ≥ 65:

Each of the logistic regression models described above includes blood marker values for one or more of age and total PSA (tPSA), free PSA (fPSA), intact PSA (iPSA), and human kallikrein 2 (hK2). It includes a plurality of input factors that In some cases, blood marker values are concentrations of blood markers in a patient sample. In some of the logistic regression models described above, cubic splines for linear or non-linear terms are determined. It should be appreciated that higher order splines may alternatively be used, as the techniques described herein are not limited in this respect.

For the logistic regression models described above, each of the terms is multiplied by the corresponding coefficient value β. These coefficients may be determined in any suitable manner. For example, each of the models may be applied to a dataset including patient information, serum assay results, and biopsy results. For information in each dataset of the models for predicting cancer, a best fit can be determined and coefficients corresponding to the best fit result can be used in accordance with the techniques described herein. An example table of coefficients determined for each of the models described above is shown in Table 1 below. In the case of these models, age is input as year and the result of each evaluation analysis is measured in ng/mL.

Example coefficients (β) for each of the nine linear regression models described above. Coefficients were determined based on the best fit of each model to a dataset containing information from 1420 individuals.

It should be appreciated that the specific coefficients used in implementation of the techniques described herein may differ from those described in Table 1 as the values in Table 1 are provided for illustrative purposes only. Additionally, in some embodiments, different coefficients may be used to determine probabilities of different outcomes and/or in different patient populations. For example, different coefficients may be used for patients in different age ranges, as described above for the age stratified cubic spline model. Different coefficients may also be used to determine the probabilities of a positive biopsy for different grades of cancer. For example, embodiments used to determine the probability of a positive biopsy for a high-risk cancer (eg, a Gleason score ≥ 7) include determining the probability of a positive biopsy for a low-grade cancer for one or more of the above models. It is possible to use a different coefficient than the embodiments used for this. Moreover, different coefficients may be used based, at least in part, on whether one or more of the blood marker values have been determined from serum or from plasma.

In some embodiments, a first logistic regression model may be used when a value for one or more of the markers exceeds a certain threshold, and a second logistic regression model may be used when the value is below the threshold. 2 depicts a process for selecting a logistic regression model based on a threshold in accordance with some embodiments of the present invention. At act 210 , a value for a blood marker total PSA (tPSA) is received. Although the exemplary process of Figure 2 uses tPSA as the blood marker value to determine which logistic regression model to use, any other blood marker value, combination of blood marker values, or any other suitable information may alternatively be used. this should be acknowledged Accordingly, in some embodiments, at least one processor may be programmed to implement and select from a plurality of models based at least in part on one or more input values.

After receiving the value for tPSA, the process proceeds to act 212, where a logistic regression model is selected based at least in part on the received tPSA value. For example, in one embodiment, if the value of tPSA is ≧15 ng/ml, preferably ≧20 ng/ml, most preferably ≧25 ng/ml, the logistic regression model may be based solely on tPSA ( For example, the "simple model (tPSA only)" model described above is used). For this implementation, when the tPSA value is less than a certain threshold (eg, less than 15 ng/ml), one or more of the other logistic regression models may be selected.

Continuing the process of Figure 2, after a model has been selected, the process proceeds to act 214, where the selected model is the entire model (eg, including all four kallikrein markers) or all markers within the kallikrein panel. It is determined whether the partial model contains fewer markers than . If it is determined that the selected model is not the full model, then the process proceeds to act 216, where the probability of cancer is determined solely based on the received tPSA value, as described above. If it is determined that the selected model is the global model, the process proceeds to act 218, where a probability of cancer is determined based on the selected model using a plurality of blood markers. Regardless of the particular model selected, after the probability of cancer is determined, the process proceeds to act 220, where the probability of cancer is output as described above with respect to FIG. 1 .

In some embodiments of the present invention, the event from which the probability is obtained is evidence of prostate cancer in a prostate biopsy taken from an analgesic male or male with lower urethral symptoms.

In some embodiments of the present invention, the event for which the probability is obtained is evidence of high risk prostate cancer, ie, a Gleason score of 7 or higher on a prostate biopsy taken from an analgesic male or a male with lower urethral symptoms. Typically, the progression of prostate cancer or the status of prostate cancer is defined as (i) a Gleason score of 7 or greater, (ii) a Gleason grade of 4+3 or greater, or (iii) a Gleason score of 8 or greater.

In many preferred embodiments the data of a plurality of men may include the reason for the biopsy, the year of the biopsy, the number of biopsy cores, the number of positive cores, the percentage of positives in each core and any other thereof. one or more biopsy data selected from the group consisting of possible combinations.

As noted above, in many preferred embodiments, blood markers are included in a logistic regression model using up to two non-linear terms for at least one blood marker. In certain embodiments, blood markers are included in a logistic regression model using up to three nonlinear terms for at least one blood marker. In certain embodiments, blood markers are included in a logistic regression model using up to four nonlinear terms for at least one blood marker. In certain embodiments, blood markers are included in a logistic regression model comprising up to five nonlinear terms for at least one blood marker.

In some embodiments, the logistic regression model calculates that the expected event rate in a target population sampled of men for which event probabilities can be obtained is calculated by equation (II):

According to , it can be recalibrated when data differs from the event rate of a number of men for which data was used to obtain the logistic regression model, where ρ is the event in the data of the number of men. rate, and P is the formula (III):

is the expected rate of events in the target population defined according to

where π is the original probability from the model, equation (IV):

According to the formula (V):

_{to obtain a recalibrated} probability according to , where π recalibrated is the probability of the event.

Some embodiments relate to methods and apparatus for predicting prostate volume using a linear regression model, wherein the method provides a linear regression model obtained by a) using a linear regression analysis of data from a plurality of men. wherein the data precedes, for each male in the plurality of males: (i) data on prostate volume, and (ii) data on prostate volume; data including determinations of blood markers including tPSA, fPSA, iPSA and optionally hK2. The linear regression model is represented by Equation (VI):

where V is the prostate volume and β _i is the variable x over j variables each comprising age, tPSA, fPSA, iPSA and optionally hK2 to obtain the linear regression model. _It is a coefficient for i . The method comprises b) providing the male's age in years, c) determining the blood markers tPSA, fPSA, iPSA and optionally hK2, respectively, from a blood sample of the male, d) the predicted prostate volume of the male using the linear regression model using the provided age of step b) and the determined blood markers of step c) to obtain In some embodiments, the statistical model of risk for cancer is solely based on tPSA when tPSA is ≥15 ng/ml, preferably ≥20 ng/ml, most preferably ≥25 ng/ml.

It should be appreciated that any suitable logistic regression model, including, but not limited to, the models described above for determining the probability of prostate cancer upon biopsy, may be used in conjunction with embodiments of the present invention to determine prostate volume. do.

In some embodiments, the data of steps a) (ii) of providing a logistic regression model or a linear regression model and a determination of blood markers in the male comprises human kallikrein 2.

In many preferred embodiments of the method of the present invention in which the prostate volume is predicted, the prostate volume is provided as defined by transrectal ultrasonography.

In many preferred embodiments of the methods of the present invention, the data providing a logistic regression model or a linear regression model for each male in said plurality of males further comprises the results of a digital rectal examination (DRE) and thus DRE is performed on males and the results obtained are used when using either a logistic regression model or a linear regression model to obtain the above probabilities. Preferably the results of the DRE are expressed with or without a second value for the estimated volume, i.e. small = 0, medium = 1 and large = 1, or binary values, i.e. normal = 0, nodularity present = 1 do.

In some preferred embodiments of the method of the present invention, the data of a plurality of men for obtaining the model comprises only data of men with elevated levels of tPSA, defined as or above the median by age, and thus Probabilities of event or predicted prostate volume are obtained only for men with the elevated levels of tPSA.

In preferred embodiments of the method of the present invention determinations of blood markers for obtaining a model for each male of a plurality of males and, accordingly, such blood markers determined to obtain a probability or predicted prostate volume are preferably fresh or or from blood samples of frozen anticoagulant serum or plasma. Preferably all samples are of the same type, ie serum or plasma, fresh or frozen.

In some preferred embodiments of the method of the present invention a logistic regression model or a linear regression model is provided using data from a large number of men between the ages of 40 and 75, such that the probability of an event or predicted prostate volume is It is obtained from a male between the ages of 40 and 75 years.

In some preferred embodiments of the method of the present invention, the logistic regression model or the linear regression model indicates that the number of tPSA in the blood is ≥ highest age quintile, ≥ highest age quartile, ≥ highest age quintile, or ≥ highest age decile. provided using data from men and thus the probability of an event or predicted prostate volume, indicates that tPSA in blood is ≥ highest age quintile, ≥ highest age quartile, ≥ highest age quintile, or ≥ highest age decile, respectively. is obtained from As an example, for men aged 60 years, the corresponding total PSA values are: ≥1.5 ng/ml for the highest age quartile, ≥1.9 ng/ml for the highest age quartile, ≥ for the highest age quintile. 2.1 mg/ml, and ≥ 3 ng/ml for the highest age decile.

Example computer system

An example implementation of a computer system 300 in which some or all of the techniques and/or user interactions described herein may be implemented is shown in FIG. 3 . Computer system 300 may include one or more processors 310 and one or more computer-readable non-transitory storage media (eg, memory 320 and one or more non-volatile storage media 330 ). Processor(s) 310 writes data to memory 320 and non-volatile storage device 330 and writes data to memory 320 and non-volatile storage device 330 in any manner, as aspects of the invention described herein are not limited in this respect. 320 and reading data from the non-volatile storage device 330 .

To perform any of the functions described herein, the processor(s) 310 is one or more computer readable storage media that may serve as a non-transitory computer readable storage medium storing instructions executed by the processor 310 . It may execute one or more instructions, such as program modules, stored in a capable storage medium (eg, memory 320 ). Generally, program modules include routines, programs, objects, components, data structures, etc. that perform particular tasks or implement particular abstract data types. Embodiments may 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 may be located in both local and remote computer storage media including memory storage devices.

Computer 300 may operate in a networked environment using logical connections to one or more remote computers. The one or more remote computers may include a personal computer, server, router, network PC, peer device, or other common network node, many of the above elements typically associated with computer 300 . or all. Logical connections between computer 300 and one or more remote computers may include, but are not limited to, a local area network (LAN) and a wide area network (WAN) as well as other networks. can Such networks may be based on any suitable technology and may operate according to any suitable protocol and may include wireless networks, wired networks or fiber optic networks. Such networking environments are quite common in offices, enterprise-wide computer networks, intranets, and the Internet.

When used in a LAN networking environment, computer 300 may be connected to a LAN through a network interface or adapter. When used in a WAN networking environment, computer 30 typically includes a modem or other means to establish communications over a WAN, such as the Internet. In a networked environment, program modules or portions thereof may be stored in a remote memory storage device.

The various inputs described herein for assessing risk of prostate cancer and/or determining prostate volume may include one or more remote storage data associated with the inputs via a network (eg, LAN, WAN, or some other network). may be received by computer 300 from the computers or devices of One or more of the remote computers/devices may perform analysis on the remotely stored data before transmitting the analysis results as input data to the computer 300 . Alternatively, the remotely stored data may be transmitted to the computer 300 as the data was stored remotely without any telemetry. Additionally, inputs may be entered by a user of computer 300 using any of a number of input interfaces (eg, input interface 340 ) that may be incorporated into the computer as components of computer 300 . can be received directly.

The various outputs described herein, including the output of the prostate volume and/or probability of prostate cancer risk, may be provided visually on an output device (eg, a display) that is directly connected to the computer 300 or output The (s) may be provided to a remotely located output device that is connected to the computer 300 via one or more wired or wireless networks, and such embodiments of the invention are not limited in this respect. The outputs described herein may be provided in addition or as an alternative to using a visual presentation. For example, computer 300 or a remote computer for which an output is provided may include one or more output interfaces including, but not limited to, speakers and vibration output interfaces to provide an indication of the output.

Although computer 300 is shown as a single device in FIG. 3 , in some embodiments, computer 300 may include a plurality of devices that are communicatively coupled to perform some or all of the functions described herein. It should be appreciated that computer 300 is only one example implementation of a computer that may be used in accordance with embodiments of the present invention. For example, in some embodiments, computer 300 may be integrated into and/or in electronic communication with the system shown in FIG. 5 .

As noted above, in some embodiments, computer 300 may be included in a networked environment, wherein information about one or more blood markers used to determine the probability of prostate cancer and/or prostate volume is provided herein. transmitted from an external source to the computer 300 for analysis using one or more of the techniques described in An exemplary networked environment 400 in accordance with some embodiments of the present invention is shown in FIG. 4 . In networked environment 400 , computer 300 is connected to detector 420 via network 410 . As noted above, network 410 may be any suitable type of wired or wireless network and may include one or more local area networks (LANs) or wide area networks (WANs), such as the Internet.

Detector 420 may be configured to determine values for one or more of the blood markers used to determine the probability of prostate cancer and/or prostate volume in accordance with one or more of the techniques described herein. Although detector 420 is shown as a single detector in FIG. 4 , detector 420 may be implemented with multiple detectors, each detector being one of the blood marker values used in accordance with one or more of the techniques described herein. It should be appreciated that it is constituted to determine more than one. Additional examples of detectors and detection systems are provided in greater detail below (eg, FIG. 12 ).

In some embodiments, information corresponding to values for blood markers determined from detector 420 may be stored prior to transmitting these values to computer 300 . In such embodiments, the information corresponding to the values may be stored locally in a local storage 430 communicatively coupled to the detector 420 and/or stored in a network-connected central storage 440 . have. Accordingly, when values corresponding to blood markers are received by computer 300 in accordance with one or more of the techniques described herein, at least some of the values are from detector 420 or one or more storage in which the values were stored. It should be appreciated that the values may be received directly from the device (eg, local storage 430 , central storage 440 ), and such embodiments are not limited based on where the values are received.

Other systems and components

As described herein, in some embodiments, the system is in electronic communication with an analyzer to determine a probability of an event associated with prostate cancer (eg, risk of prostate cancer and/or prostate volume) for logistic regression analysis. It may include a processor or computer programmed to evaluate the model. The analyzer may be adapted and arranged to determine one or more characteristics of blood markers for input into a logistic regression model. In some embodiments, the analyzer is a microfluidic sample analyzer; For example, the analyzer may be adapted and arranged to determine a sample to be processed in the microfluidic device/cassette. However, it should be appreciated that other types of analyzers may also be used (eg, analyzers for microwell ELISA-type assays) as the systems described herein are not limited in this respect.

One example of such a system includes in one set of embodiments a housing, a microfluidic sample analyzer comprising an opening in the housing configured to receive a cassette having at least one microfluidic channel, wherein the housing comprises: and a component configured to interface with a mating component on the cassette to detect the cassette within the housing. The analyzer also includes a pressure control system positioned within the housing, the pressure control system configured to pressurize the at least one microfluidic channel in the cassette to move the sample through the at least one microfluidic channel. An optical system is positioned within the housing, the optical system comprising at least one light source and at least one detector spaced apart from the light source, wherein the light source passes light through the cassette when the cassette is inserted into the sample analyzer. and the detector is positioned opposite the light source to detect the amount of light passing through the cassette. The system may also include a user interface associated with the housing for inputting at least a person's age and/or other information for input into the linear regression model.

In certain embodiments, the processor is in electronic communication with (or adapted to be in electronic communication with) the microfluidic sample analyzer. In some cases, the processor is within a housing of the analyzer. However, in other embodiments, the processor is not contained within the housing of the analyzer but can be accessed by electronic means as described herein. The processor may be programmed to evaluate the logistic regression model based at least in part on information received from the at least one detector to determine a probability of an event associated with prostate cancer in the human body, wherein Evaluating a value for the scaled variables used to scale each of the plurality of variables to a different coefficient value to yield scaled variables and to calculate a probability of an event associated with prostate cancer in the human body. wherein the plurality of variables include 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.

A method of determining a probability of an event associated with prostate cancer in a human body can include, for example, providing a microfluidic sample analyzer. The microfluidic sample analyzer may include a housing, an opening in the housing configured to receive a cassette having at least one microfluidic channel, the housing configured to interface with a mating component on the cassette to detect the cassette within the housing contains components. The analyzer may further include a pressure control system positioned within the housing, the pressure control system configured to pressurize the at least one microfluidic channel within the cassette to move the sample through the at least one microfluidic channel. An optical system is positioned within the housing, the optical system comprising at least one light source and at least one detector spaced apart 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 A detector is positioned opposite the light source to detect the amount of light passing through the cassette. The analyzer may also include a user interface associated with the housing for entering at least the age of the person. The method may include determining information about a plurality of blood markers using a microfluidic sample analyzer, wherein the information about the plurality of blood markers includes an fPSA value, an iPSA value, a tPSA value and optionally hK2 contains a value. The method may also include evaluating, using at least one processor, a logistic regression model, based at least in part on the information, to determine a probability of an event associated with prostate cancer in the human body. wherein the evaluating the logistic regression model comprises scaling each of the plurality of variables to different coefficient values to yield scaled variables, and calculating a probability of an event associated with prostate cancer in the human body. summing the values for the scaled variables used to is chosen

Another example of a system is, in one set of embodiments, a device (eg, a microfluidic cassette) comprising a first assay region comprising a first binding partner and a second assay region comprising a second binding partner includes The first binding partner is adapted to bind to at least one of fPSA, iPSA and tPSA and the second binding partner is adapted to bind to at least another one of fPSA, iPSA and tPSA. In some embodiments, the device comprises a third analysis region comprising a third binding partner adapted to bind to a third of fPSA, iPSA and tPSA. Optionally, the device may comprise a fourth analysis region comprising a fourth binding partner adapted to bind hK2. The system is configured to evaluate a logistic regression model based at least in part on a detector associated with the first and second analysis regions and information received from the detector to determine a probability of an event associated with prostate cancer in the human body. It contains a programmed processor. Evaluating the logistic regression model involves scaling each of a plurality of variables to a different coefficient value to yield scaled variables and the scaled variables used to calculate the probability of an event associated with prostate cancer in the human body. summing values for the variables, wherein the plurality of variables comprises age and at least two variables included in information received from the detector and is selected from the group consisting of fPSA, iPSA and tPSA.

A method of determining the probability of an event associated with prostate cancer in such a system may include, for example, a device comprising a first analysis region comprising a first binding partner and a second analysis region comprising a second binding partner (eg, for example, introducing a sample into a microfluidic cassette), wherein the first binding partner is adapted to bind to at least one of fPSA, iPSA and tPSA and wherein the second binding partner is one of fPSA, iPSA and tPSA. adapted to combine with at least one other. In some embodiments, the device comprises a third analysis region comprising a third binding partner adapted to bind to a third of fPSA, iPSA and tPSA. Optionally, the device may comprise a fourth analysis region comprising a fourth binding partner adapted to bind hK2. The method comprises the steps of allowing any PSA of fPSA, iPSA and/or tPSA from the sample to bind with at least first and/or second binding partners in the first and second analysis regions and the first and second assays determining a characteristic of the fPSA, iPSA and/or tPSA using one or more detectors associated with the regions. The method includes an fPSA, iPSA and/or 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 human body. inputting the characteristics of the tPSA, wherein evaluating the logistic regression model scales each of the plurality of variables to different coefficient values to yield scaled variables and is associated with prostate cancer in the human body. summing the values for the scaled variables used to calculate the probability of the event, wherein the plurality of variables include age and at least two variables included in information received from the detector and include fPSA, iPSA and is selected from the group consisting of tPSA. Accordingly, the probability of an event associated with prostate cancer can be determined.

In certain embodiments, a device for determining blood markers (eg, fPSA, iPSA, tPSA and/or hK2) is provided. In some cases, the device may be capable of simultaneously determining blood markers, eg, on a single cassette. The device comprises a first microfluidic channel comprising at least one inlet and one outlet, a first reagent stored in the first microfluidic channel, and a first microfluidic channel to store a first reagent in the first microfluidic channel. and a microfluidic system comprising a seal covering the inlet and a seal covering the outlet of the first microfluidic channel. The device may further include a second microfluidic channel comprising at least one inlet and one outlet, a first analysis region, a second analysis region and a third analysis region. Each of the analysis regions may comprise 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 connectable to a microfluidic system, wherein the fluidic connector comprises a fluid path comprising a fluid path inlet and a fluid path outlet, wherein upon connection the fluid path inlet comprises a fluid path and a first microfluidic system. It is connected to the outlet of the first microfluidic channel to enable fluid communication between the fluidic channels, and the fluidic path outlet is connected to the inlet of the second microfluidic channel to enable fluid communication between the fluidic path and the second microfluidic channel. The first and second microfluidic channels are not in fluid communication with each other with the absence of a connection through the fluidic connector. The device may comprise a source of colloidal metal conjugated to an antibody that optionally binds to anti-PSA.

In some embodiments comprising the 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 the third microfluidic channel. The second and third analysis regions (as well as the second and third microfluidic channels) may be formed on the same substrate layer or on different substrate layers, for example as described herein. Additionally, in some embodiments, the third analysis region is in fluid communication with the fourth microfluidic channel. The third and fourth analysis regions (as well as the third and fourth microfluidic channels) may be formed on the same substrate layer or on different substrate layers, for example, as described herein. In some cases, each of the first, second and third (and optionally fourth) analysis regions are formed in different substrate layers. In other embodiments, the fourth analysis region (eg, which may comprise an anti-hK2 specific capture antibody) is a different substrate than a substrate layer comprising at least one of the first, second and third analysis regions. formed in layers. In some such embodiments, the first, second and third analysis regions are formed in the same substrate layer. Irrespective of whether the assay regions are formed in different substrate layers or in the same substrate layer, in some embodiments, the reagents may be administered to a first, second and/or third (optionally) prior to, for example, the device being used. 4) can be stored in and sealed within the analysis areas. Reagents may 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 (eg, when connecting a fluidic connector to a microfluidic system), the first microfluidic channel is in fluid with one or more of the first, second and third (and optionally fourth) assay regions. It may be arranged to communicate. For example, the fluidic connector can connect to one or more inlets of the second, third and/or fourth microfluidic channel(s) when connected to the microfluidic system. Examples of device configurations are described below in more detail.

In certain devices described herein, the assay comprises the use of a detection antibody that recognizes two or more of iPSA, fPSA, tPSA and hK2. For example, a detection antibody can recognize both PSA and hK2 and then a blocker can be used to interfere with PSA such that only hK2 is detected. For example, in one particular embodiment, the analysis region is an anti-hK2 capture antibody (which is also capable of capturing, for example, 5-10% tPSA and is stored within the analysis region prior to use as described herein). ) as well as blocker antibodies that block tPSA. An anti-hK2 detector antibody (which can also detect tPSA) can be used to detect the amount of binding of hK2. The different analysis regions may include, for example, anti-tPSA capture antibodies that capture both fPSA and tPSA (which may be stored in the analysis region prior to use as described herein). Two different detector antibodies can be used for detection, 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. The different analysis regions may include, for example, an anti-fPSA capture antibody and optionally an anti-iPSA capture antibody. Two different detector antibodies can be used for detection, 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.

However, in other embodiments, specific capture antibodies may be used for the detection of species. Each of the specific capture antibodies may be located in different analysis regions, as described herein. Advantageously, using specific capture antibodies and/or positioning the capture antibodies in different analysis regions may make it possible to use 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 makes it possible to use simplified detectors and/or optical components for detection. For example, in some embodiments, detecting comprises accumulating opaque material in different regions of analysis, which can be determined at a particular wavelength, as described in more detail below.

For example, in one set of embodiments, the anti-iPSA specific capture antibody, the anti-fPSA specific capture antibody and the anti-tPSA specific capture antibody (and optionally the hK2 specific capture antibody) are as described herein. , may be included in different analysis areas, optionally with negative and positive controls. Detection antibodies, such as gold labeled antibodies that are anti-PSA and anti-hK2, can be used to detect each of iPSA, fPSA, tPSA and/or hK2. However, in other embodiments, a mixture of gold labeled antibodies may be used for detection, such as a gold labeled anti-hK2 antibody, a gold labeled anti-PSA antibody and/or a gold labeled anti-iPSA antibody. In such a system, the same wavelength may be used to determine each of the species, which may enable using simplified detectors and/or optical components for detection.

Examples of specific systems, devices, and analyzers that may be used in combination with embodiments provided herein are now described.

5 shows a block diagram 510 of a microfluidic system and various components that may be included in accordance with a set of embodiments. The microfluidic system may include, for example, a fluid flow source 540 , such as a pump (eg, to introduce one or more fluids into the cassette and/or to control rates of fluid flow), optionally a positive pressure ) or a fluid flow source 540 , such as a pump or vacuum, that may be configured to apply either or both of a vacuum (eg, for moving/removing one or more fluids within/from a cassette and/or or to control the rate of fluid flow), a valve system 528 (eg, to actuate one or more valves), a detection system 534 (eg, to detect one or more fluids and/or processes) and/or a cassette 520 operatively associated with one or more components, such as a temperature control system 541 (eg, to heat and/or cool one or more regions of the cassette). can These components may be external or internal to the microfluidic device, and may optionally include one or more processors that control the component or system of components. In certain embodiments, one or more such components and/or processors are associated with a sample analyzer 547 configured to process and/or analyze a sample contained within the cassette. The processor may optionally be programmed to evaluate a linear regression model as described herein.

In general, as used herein, a component that is “operatively associated with” one or more other components means that such components are not directly connected to each other, connected to each other, or not attached to each other. Components that are not in direct physical contact with each other, or directly connected to each other or not in contact with each other, but are so associated mechanically or electrically (transmitted through space) to enable or enable them to perform their intended function. (including via electromagnetic signals) or fluidically interconnected (eg, via channels such as tubes).

5 as well as other optional components as described herein may be operatively associated with the control system 550 . In some embodiments, the control system may be used to control fluids and/or perform quality control by using feedback from one or more events occurring in the microfluidic system. For example, the control system receives input signals from one or more components, calculates and/or controls various parameters, compares one or more signals or patterns of signals to signals pre-programmed into the control system, and/or transmit signals to one or more components to regulate fluid flow and/or control operation of the microfluidic system. The control system may also optionally include a user interface 554 , an identification system 556 , an external communication unit 558 (eg, USB) and/or other, as described in more detail below. It may be associated with other components, such as components.

Cassette (eg, microfluidic device) 520 may have any suitable configuration of channels and/or components to perform a desired assay. In one set of embodiments, cassette 520 is used, for example, to perform a chemical and/or biological reaction (eg, an immunoassay), as described in more detail herein. Contains stored reagents that can be used.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, for example, be fluid in some embodiments. Liquids and gases, including immiscible reagents, such as reagent solutions and cleaning solutions, which are optionally separated by immiscible fluids (eg, as described in more detail herein). ) may include one or more channels and/or reservoirs that can be partially or fully filled in. The cassette also includes a fluidic connector that can be used to connect reagent storage area 564 to optional assay area 568; The same may include an optional sample or reagent loading area 566. An analysis region that may include one or more areas (eg, analysis regions) for detecting a component in the sample may include an optional waste product. It may be in fluid communication with the area 570 and coupled to the outlet 572. In some cases, such or other device features may be incorporated into different components or layers of the cassette, as described in more detail herein. It is therefore recognized that a cassette may comprise a single component or a plurality of components that are attached during use, such as the combination of an article and an attachment fluidic connector as described herein. In one set of embodiments, the fluid can flow in the direction of the arrows shown in the figure.Additional descriptions and examples of such and other components are provided herein.

In some embodiments, sections 571 and 577 of the cassette are not in fluid communication with each other prior to introducing the sample into the cassette. In some cases, sections 571 and 577 are not in fluid communication with each other prior to first use of the cassette, and upon first use, the sections are brought into fluid communication with each other. However, in other embodiments, sections 571 and 577 are in fluid communication with each other prior to first use and/or prior to introducing the sample into the cassette. Other cassette configurations are also possible.

As shown in the example embodiment illustrated in FIG. 5 , one or more fluid flow sources 540 or other pressure control systems, such as pumps and/or vacuums, valve system 528 , detection system 534 , temperature control system 541 and/or other components include a reagent inlet 562 , a reagent storage area 564 , a sample or reagent loading area 566 , a reaction area 568 , a waste area 570 , an outlet 572 and/or or operatively associated with one or more of the other regions of the cassette 520 . Detection of processes or events within one or more regions of the cassette may generate a signal or pattern of signals that may be transmitted to control system 550 . Based on the signal(s) received by the control system, this feedback may be applied to the microfluidic device, such as by controlling one or more of a pump, vacuum, valve system, detection system, temperature control system, and/or other components. It can be used to manipulate fluids within or between each of these regions.

Turning to FIG. 6 , one embodiment of a microfluidic sample analyzer 600 is shown. As shown in the exemplary embodiment of FIG. 6 , the analyzer includes a housing 601 that is configured to cover or hold components of the analyzer, discussed in greater detail below. An opening 620 in the housing is configured to receive a cassette 520 . As set forth in more detail below, the analyzer 600 may also include a user interface 650 positioned within the housing, the interface configured for a user to enter information into the sample analyzer. In this particular embodiment, user interface 650 includes a touch screen, although the user interface may be configured differently, as described below.

In some embodiments, the analyzer detects the cassette within the housing and a fluid flow source (eg, vacuum system) configured to pressurize the cassette, an identification reader configured to read information associated with the cassette, and the housing. and a mechanical subsystem comprising a component configured to interface with the cassette to As noted above, the opening in the housing is configured to receive a cassette. The opening 620 may be configured as an elongated slot. The opening may in this manner be configured to receive a cassette in the shape of a substantially card. It should be appreciated that in other embodiments, the opening may be differently shaped and configured, as the invention is not so limited.

As described above, the microfluidic sample analyzer 600 may be configured to receive various types of cassettes 520 (eg, microfluidic devices). 7-11F show various exemplary embodiments of a cassette 520 for use with an analyzer 600 . As shown, the cassette may be substantially card-shaped (ie resembling a card key) with a substantially rigid plate-like structure.

Cassette 520 is configured to include a fluid connector 720 that can snap into one end of the cassette. In certain embodiments, a fluidic connector may be used to introduce one or more fluids (eg, a sample or reagent) into a cassette.

In one set of embodiments, a fluid connector is used to fluidly connect two (or more) channels of a cassette during first use, which channels are not connected prior to first use. For example, a cassette may include two channels that were not in fluid communication prior to first use of the cassette. Unconnected channels may be useful in certain cases, such as for storing different reagents within each of the channels. For example, a first channel may be used to store dry reagents and a second channel may be used to store wet reagents. Physically separating the channels from each other may prevent the reagent(s) stored in each of the channels from being stored in each of the channels, for example, by keeping the reagent(s) stored in dry form protected from moisture that may be generated by the reagent(s) stored in wet form. It can enhance long-term stability. In first use, the channels may be connected via a fluidic connector to allow fluid communication between the channels of the cassette. For example, the fluid connector may puncture the seals covering the inlets and/or outlets of the cassette to enable insertion of the fluid connector into the cassette.

As used herein, "prior to first use of a cassette" means the time or times before a cassette is first used by an intended user after commercial sale. Initial use may include any step(s) requiring manipulation of the device by the user. For example, initial use may include puncturing a sealed inlet to introduce reagents into a cassette, connecting two or more channels to establish fluid communication between the channels, preparing the device prior to analysis of the sample (e.g., , loading reagents into the device), loading the sample onto the device, preparing the sample in an area of the device, performing a reaction with the sample, detecting the sample, and the like. In this situation, first use does not involve manufacturing or other preparation or quality control steps taken by the manufacturer of the cassette. A person skilled in the art will be fully aware of the meaning of first use in this situation and will be able to easily determine whether or not the cassette of the present invention has experienced first use. In one set of embodiments, a cassette of the present invention may be disposed of after first use (eg, after completion of the assay), which is particularly evident when such devices are first used because anyway This is because it is typically impractical to use devices after first use (eg, to perform a second assessment analysis).

As shown in the exemplary embodiment illustrated in FIG. 8 , the fluidic connector 720 may contain a fluid and/or reagent (eg, a fluid sample and/or one or more detection antibodies) prior to being connected to the cassette. may include a substantially U-shaped channel 722 or any other suitable shaped channel. Channel 722 may be housed between two shell components forming connector 720 . In some embodiments, the fluidic connector may be used to collect a sample from a patient before the fluidic connector is connected to the cassette. For example, a lancet or other suitable instrument may be used to obtain a finger stick blood sample and then allow the blood sample to be collected by the fluidic connector 720 and loaded into the channel 722 by capillary action. can In other embodiments, the fluidic connector 720 may be configured to puncture a patient's finger to collect a sample within the channel 722 . In certain embodiments, the fluidic connector 720 does not contain a sample (or reagent) prior to connection to the cassette, but merely enables fluid communication between two or more channels of the cassette upon connection. In one embodiment, the U-shaped channel is formed with a capillary tube. A fluidic connector may also include other channel configurations and, in some embodiments, may include one or more channels that may be fluidically connected or disconnected from each other.

9-11F illustrate various exemplary embodiments of cassette 520 in greater detail. As illustrated by way of illustration in the exploded view of FIG. 9 , the cassette 520 is configured to receive a sample or reagent and includes a cassette body 704 comprising at least one channel 706 through which the sample or reagent may flow. may include. Cassette body 704 may also include latches 708 positioned at one end that interlock with fluid connector alignment element 702 for a snap fit.

Cassette 520 may also include top and bottom covers 710 and 712 , which may be made of, for example, a transparent material. In some embodiments, the cover may be in the form of a biocompatible adhesive and may contain a polymer (eg, polyethylene (PE), cyclic olefin copolymer (COC), polyvinyl chloride (PVC)) or For example, it may be made of an inorganic material. In some cases, the one or more covers are in the form of an adhesive film (eg, tape). For some applications, the material and dimensions of the cover are selected such that the cover is substantially impermeable to water vapor. In other embodiments, the cover may be non-adhesive, but thermally adhered to the microfluidic substrate by direct application of heat, laser energy or ultrasonic energy. Any inlet(s) and/or outlet(s) of the channel of the cassette may be sealed (eg, by placing adhesive over the inlet(s) and/or outlet(s)) using one or more covers. . In some cases, the cover substantially seals one or more stored reagents within the cassette.

As shown, the cassette body 704 can include one or more ports 714 that engage a channel 706 within the cassette body 704 . These ports 714 are fluid when the fluid connector 720 is coupled to the cassette 520 to fluidly connect the channel 706 in the cassette body 704 with the channel 722 in the fluid connector 720 . It may be configured to align with a substantially U-shaped channel 722 in connector 720 . In certain embodiments, a substantially U-shaped channel 722 may also couple channels 706 and 707 by being fluidically connected to channel 707 . As shown, a cover 716 may be provided over the port 714 , the cover 716 being pierced or otherwise open to fluidly connect the two channels 706 and 722 . for example, by connector 720 or by other means). Additionally, a cover 718 may be provided to cover the port 719 (eg, a vacuum port) in the cassette body 704 . As will be described in more detail below, port 719 may be configured to fluidly connect fluid flow source 540 with channel 706 to move a sample through the cassette. Cover 718 over port 719 may be configured to pierce or otherwise open channel 706 to fluidly connect with fluid flow source 540 .

Cassette body 704 may optionally include a liquid receiving area, such as a waste area comprising absorbent material 717 (eg, a waste pad). In some embodiments, the liquid receiving region includes regions that capture one or more liquids flowing within the cassette while it is possible for gases or other fluids in the cassette to pass through the region. This may be achieved, in some embodiments, by placing one or more absorbent materials in the liquid receiving area to absorb liquids. This configuration may be useful for removing air bubbles from a stream of fluid and/or for separating hydrophobic liquids from hydrophilic liquids. In certain embodiments, the liquid receiving region prevents liquids from passing through the region. In some such cases, the liquid receiving region may trap substantially all of the liquids within the cassette and serve as a waste area, thereby preventing liquid from escaping the cassette (eg, while gases are at the outlet of the cassette). makes it possible to escape from it). For example, the waste area may be used to store sample and/or reagents within the cassette after they have passed through channel 706 during analysis of the sample. These and other arrangements may be useful when the cassette is used as a diagnostic tool, as the liquid receiving area may prevent the user from being exposed to potentially harmful fluids within the cassette.

The schematic diagram of the cassette 520 shown in FIG. 10 shows one embodiment in which the cassette 520 includes a first channel 706 and a second channel 707 spaced apart from the first channel 706 . . In one embodiment, channels 706 and 707 have a maximum cross-sectional dimension ranging from about 50 micrometers to about 500 micrometers, although other channel sizes and configurations may be used, as described in more detail below.

The first channel 706 may include one or more analysis regions 709 used to analyze the sample. For example, in one illustrative embodiment, the channel 706 includes four analysis regions 709 (eg, connected in series or parallel) used during sample analysis. As described herein, each of the analysis regions may be adapted to detect one or more of iPSA, fPSA, tPSA and/or hK2.

In certain embodiments, the one or more analysis regions are in the form of meandering regions (eg, regions comprising meandering channels). The meandering area may be defined, for example, 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 area is a light detection path includes A detector capable of measuring a single signal through one or more adjacent segments of the meandering area may be positioned adjacent to the meandering area. In some cases, the channel 706 is fluidically connected to at least two meander regions that are connected in series.

As described herein, the first channel 706 and/or the second channel 707 may contain one or more reagents (eg, iPSA, fPSA) used to process and analyze the sample prior to first use of the cassette. , capture antibodies to tPSA and/or hK2). In some embodiments, dry reagents are stored in one channel or section of the cassette and wet reagents are stored in a second channel or section of the cassette. Alternatively, two separate sections or channels of the cassette may both contain dry reagents and/or wet reagents. Reagents may be stored and/or disposed of, for example, as a liquid, gas, gel, plurality of particles or film. Reagents may be located in any suitable portion within the cassette including, but not limited to, channels, in reservoirs, on a face, and optionally in or on a membrane that may be part of a reagent storage area. A reagent may be associated with the cassette (or components of the cassette) in any suitable manner. For example, reagents can be crosslinked (eg, covalently or ionic), absorbed, or absorbed (physically absorbed) on a face within the cassette. In one particular embodiment, all or part of a channel (such as a fluid pathway in a fluid connector or a channel in a cassette) is coated with an anticoagulant (eg, heparin). In some cases, the liquid is contained in a channel or reservoir of the cassette prior to first use and/or prior to introducing samples into the cassette.

In some embodiments, the stored reagents may include fluid plugs positioned in a linear order such that, during use, the reagents are delivered in a predetermined sequence as they flow into the assay region. A cassette designed to perform an assay may include, for example, a rinse fluid, a labeled antibody fluid, and an amplification fluid, all in series, all stored therein. Although fluids are stored, the fluids may be kept separated by substantially immiscible separation fluids (eg, a gas such as air) such that fluid reagents that would normally react with each other when contacted may be stored in a common channel. .

Reagents can be stored in the cassette for various periods of time. For example, the reagent is longer than 1 hour, longer than 6 hours, longer than 12 hours, longer than one day, longer than one week, longer than one month, longer than three months. It can be stored for a longer time, longer than 6 months, longer than 1 year, or longer than 2 years. Optionally, the cassette may be treated in any suitable manner to extend storage. For example, cassettes having stored reagents contained therein may be vacuum sealed, stored in a dark environment, and/or stored at low temperatures (eg, less than 0 degrees Celsius). The length of storage depends on the specific reagents used, the type of reagents stored (eg, wet or dry), the dimensions and materials used to form the substrate and cover layer(s), the substrate and cover layer(s). ) depends on one or more factors such as how it is attached and how the cassette is handled or stored as a whole. Storing reagents (eg, liquid or dry reagents) within the channel may include sealing the inlet(s) and outlet(s) of the channel prior to first use or during packaging of the device.

As described in the example embodiment shown in FIGS. 10 and 11A-11F , channels 706 and 707 may not be in fluid communication with each other until fluid connector 720 is coupled to cassette 520 . . That is, the two channels, in some embodiments, are not in fluid communication with each other prior to first use and/or prior to introducing the sample into the cassette. In particular, as shown, the substantially U-shaped channel 722 of the connector 720 allows reagents in the second channel 707 to pass through the U-shaped channel 522 and the first channel 706 . The first and second channels 706 and 707 may be fluidly coupled to be selectively movable into the analysis region 709 within the . In other embodiments, the two channels 706 and 707 are in fluid communication with each other prior to first use and/or prior to introducing the sample into the cassette, although a fluid connector further connects the two channels upon first use (e.g., to form a closed loop system).

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 be associated with other types of fluidic systems. A microfluidic cassette, device, apparatus or system can include, for example, at least one fluidic channel having a maximum cross-sectional dimension of less than 1 mm and a ratio of length to maximum cross-sectional dimension of at least 3:1.

The cross-sectional dimension (eg, diameter) of the channel is measured perpendicular to the direction of fluid flow. Most fluid channels in the components of the cassettes 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 of the cassette are microfluidic or have a maximum cross-sectional dimension that does not exceed 2 mm or 1 mm. In another set of embodiments, the maximum cross-sectional dimension of the channel(s) 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 selected to allow fluid to flow freely through the article or substrate. The dimensions of the channel may also be selected to allow, for example, a specific volume or linear flowrate of fluid within the channel. Of course, the number of channels and the shape of the channels may 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.

A channel may include an article (eg, a feature on or in a cassette) that at least partially directs the flow of fluid. The channel may have any suitable cross-sectional shape (circle, oval, triangular, irregular, square or rectangular). etc.) and covered or uncovered In embodiments where the channel is fully covered, at least one portion of the channel may have a fully enclosed cross-section or the entire channel may have its inlet(s) and outlet ( . average cross-sectional dimensions).

Cassettes described herein may include channels or channel segments located on one or both sides of the cassette (or substrate layer of the cassette). In some cases, the channels are formed on the face of the cassette. Channel segments may be connected by intervening channels passing through the cassette. In some embodiments, channel segments are used to store reagents within 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 cassette allow the fluid reagents to extend unmixed, such as during transport of cassettes, even during routine handling of cassettes, and when the cassettes are subjected to physical shock or vibration. It may be possible to be stored for a period of time.

In certain embodiments, the cassette includes optical elements fabricated opposite a series of fluid channels on one side of the cassette. An “optical element” refers to a direction (eg, through refraction or reflection), a focus, of electromagnetic radiation relative to light formed or positioned on or in a provided article or cassette and incident on the article or cassette in the absence of said element. , used to refer to features used to change polarization and/or other properties. For example, the optical element may include a lens (eg, concave or convex), mirror, grating, groove, or other feature formed or positioned in or on the cassette. However, since the cassette itself has no intrinsic features, it will not constitute an optical element, even if one or more properties of the incident light change upon interaction with the cassette. The optical elements may guide incident light passing through the cassette such that a majority of the light is scattered away from certain areas of the cassette, such as portions intervening between the fluid channels. By reducing the amount of light incident on these intervening portions, the amount of noise in the detection signal can be reduced when using certain optical detection systems. In some embodiments, the optical elements include triangular shaped grooves formed on or in the face of the cassette. The draft angle of the triangular shaped grooves may be selected such that incident light normal to the face of the cassette is redirected at an angle according to the refractive index of the external medium (eg air) and the cassette material. In some embodiments, one or more optical elements are positioned between adjacent segments of the meandering region of the analysis region.

The cassette or portions of the cassette may be made of any material suitable for forming a channel or other component. Non-limiting examples of materials include polymers (eg, polyethylene, polystyrene, polymethylmethacrylate, polycarbonate, poly(dimethylsiloxane)), PVC, PTFE, PET, and cyclic olefins. copolymer), glass, quartz and silicone. The material forming the cassette and any associated components (eg, cover) may be rigid or flexible. One of ordinary skill in the art would know, for example, its rigidity, its inertness to the fluid through which it is passed (eg, its degree of freedom from heat induced by the fluid), and its temperature at which a particular device can be used. Select the appropriate material(s) based on its robustness, its transparency/opacity to its light (e.g., in the infrared and visible regions) and/or the method used to fabricate the features in the material. can be easily selected. For example, for injection molding or other extruded articles, the material used may be a thermoplastic (eg, polypropylene, polycarbonate, acrylonitrile-butadiene-styrene, nylon 6), an elastomer. (elastomer) (e.g., polyisoprene, isobutene-isoprene, nitrile, neoprene, ethylene-propylene, hypalon, silicone), thermosetting material (e.g., epoxy, unsaturated polyester, phenol) or combinations thereof can do. As will be described in greater detail below, cassettes comprising two or more components or layers may be configured to convert components to each major function(s) of the components, for example, based on the above factors described above and herein described. It can be formed of different materials to customize to fit.

In some embodiments, the material and dimensions (eg, thickness) of the cassette and/or cover are selected such that it is substantially impermeable to water vapor. For example, a cassette designed to store one or more fluids therein prior to first use has been shown to provide a high vapor barrier, such as metal foil, certain polymers, certain ceramics, and combinations thereof. It may include a cover comprising known materials. Examples of materials with low water vapor permeability are provided below. In other cases, the material is selected based at least in part on the shape and/or configuration of the cassette. For example, certain materials may be used to form flat devices while other materials are more suitable for forming curved or irregularly shaped devices.

In some examples, the cassette is constructed of a combination of two or more materials, such as those described above. For example, the channels of the cassette may be formed from polystyrene or other polymers (eg, by injection molding) and a biocompatible tape may be used to seal the channels. The biocompatible tape or flexible material may include a material known to improve vapor barrier properties (eg, metal foil, polymers or other materials known to have high vapor barrier layers) and optionally It may be possible to access the inlets and outlets by puncturing or peeling the tape with a To seal a microfluidic channel or portions of a channel, or to bond multiple layers of a device, various methods may be used, including adhesives, adhesive tapes, gluing, bonding, material including, but not limited to, using lamination of metals or by mechanical methods (eg, clamping, snapping mechanisms, etc.).

In some examples, a cassette comprises a combination of two or more separate components (eg, layers or cassettes) that are mounted together. Proprietary channel networks (such as sections 571 and 577 of FIG. 5 ), which may optionally contain reagents stored therein prior to first use, may be incorporated on or within different components of the cassette. The separate components may be mounted together or otherwise associated with each other by any suitable means, such as by the methods described herein, for example to form a single (composite) cassette. In some embodiments, two or more channel networks are located in different components or layers of the cassette and are not fluidically connected prior to first use, but are fluidically connected, for example, by using a fluidic connector upon first use. do. In other embodiments, two or more channel networks are fluidically connected prior to first use.

Advantageously, each of the different components or layers forming the composite cassette can be individually tailored according to the designed function(s) of said component or layer. For example, in one set of embodiments, one component of the composite cassette may be customized to store wet reagents. In some such embodiments, the component may be formed of a material having a relatively low vapor permeability. Additionally or alternatively, for example, depending on the amount of fluids to be stored, the storage region(s) of the cassette may be made larger in cross-sectional dimensions than the channels or regions of other components not used for storage of liquids. can The material used to form the cassette is compatible with fabrication techniques suitable for forming larger cross-sectional dimensions. In contrast, a second component that may be customized for detection of an analyte may, in some embodiments, include channel portions having smaller cross-sectional dimensions. Smaller cross-sectional dimensions are, for example, in certain embodiments, for a given volume of fluid, between the fluids flowing within the channel (eg, reagent solution or wash fluid) and analyte bound to the face of the channel. It can be useful to enable more contact time. Additionally or alternatively, the channel portion of the second component may have a lower surface roughness compared to the channel portion of the other component (eg, a signal to noise ratio during detection). ) to increase). The smaller cross-sectional dimensions or lower surface roughness of the channel portions of the second component may require, in certain embodiments, some fabrication technique or fabrication tool other than that used to form the different component of the cassette. . Moreover, in some specific embodiments, the material used for the second component may be sufficiently characterized for protein attachment and detection. As such, it may be useful to form different channel portions used for different purposes on different components of the cassette that may then be joined together before being used by the intended user. Other advantages, features and examples of the components are provided below.

11B-11E illustrate a device that may include multiple components or layers 520B and 520C that are joined to form a single cassette. As shown in this example 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. The device components or portions described herein, such as channels or other entities, may in some embodiments, in some embodiments, a first side of a component, a second side of a component, on or within, and/or Alternatively, it may be formed through the above components. For example, as illustrated by way of example in FIG. 11C , component 520C may include a channel 706 having an inlet and an outlet, and may be formed of a first material. Channel 706 may have any suitable configuration as described herein and may include, for example, one or more reagent storage regions, assay regions, liquid receiving regions, mixing regions, and the like. In some embodiments, channel 706 does not form through the entire thickness of component 520B. That is, the channel may be formed on or in one side of the component. Channel 706 may optionally be surrounded by a cover, such as tape (not shown), another component or layer of a cassette, or other suitable component, as described herein. In other embodiments, channel 706 is formed through the entire thickness of component 520B and covers are required to enclose the channel on both sides of the cassette. As described herein, different layers or components may include different analysis regions to determine species in a sample. For example, capture antibodies to iPSA, fPSA, tPSA and/or hK2 may be located in different analysis regions, optionally in different components or layers of the cassette as shown.

Component 520B may include a channel 707 having an inlet and an outlet, and may be formed of a second material, which may be the same as or different from the first material. Channel 707 may also have any suitable configuration as described herein, and may or may not be formed through the entire thickness of component 520C. Channel 707 may be surrounded by one or more covers. In some cases, the cover is not a component that includes one or more fluid channels, such as component 520C. For example, the cover may be a biocompatible tape or other surface positioned between components 520B and 520C. In other embodiments, channel 707 may be substantially surrounded by component 520C. That is, face 522A of component 520C may form part of channel 707 when components 520B and 520C are placed directly adjacent to each other.

11D and 11E , components 520B and 520C may be substantially flat and may be stacked on top of each other. In general, however, two or more components forming a cassette may be placed in any suitable configuration relative to each other. In some cases, components are placed adjacent to each other (eg, side by side, stacked on top of each other). The first components may completely overlap or only a portion of the components overlap each other. For example, as illustrated by way of illustration in FIGS. 11D and 11E , component 520C is longer than component 520B such that portions of component 520C are not overlapped or covered by component 520B. can be extended far. In some cases, this configuration is when component 520C is substantially transparent and allows light to travel through a portion of the component (eg, a reaction area, analysis area, or detection area), and It may be advantageous if 520B is opaque or less transparent than component 520C.

Moreover, the first and second components may comprise any suitable shape and/or configuration. For example, in some embodiments, the first component includes a feature that complements the feature of the second component to form a non-fluid connection between the first and second components. Complementary features may assist in alignment of the first and second components during assembly, for example.

The first and second components may be integrally connected to each other in some embodiments. As used herein, the term "integrally connected" when referring to two or more objects means objects that do not separate from each other during the course of normal use, eg, cannot be separated by hand. Separation requires the use of at least tools and/or by causing damage to at least one of the components, for example breaking, peeling or separating components fixed to each other via adhesives or tools. according to The integrally connected components may be irreversibly attached to each other during the course of normal use. For example, components 520B and 520C may be integrally connected by using an adhesive or by using other bonding methods. In other embodiments, two or more components of the cassette may be reversibly attached to each other.

At least the first component and the second component forming the composite cassette in some embodiments as described herein may be formed of different materials. The system may be designed such that the first component includes a first material that assists or enhances one or more functions of the first component. For example, if the first component is designed to store (eg, for at least a day, a week, a month or a year) a liquid reagent (eg, within a channel of the component) prior to first use by a user; The first material may be selected to have a relatively low vapor permeability to reduce the amount of evaporation of the stored liquid over time. However, it should be understood that the same materials may be used for multiple components (eg, layers) of a cassette in some embodiments. For example, both the first and second components of the cassette may be formed of a material having low water vapor permeability.

In certain embodiments, the first and second components of the cassette have different degrees of optical clarity. For example, the first component can be substantially opaque and the second component can be substantially transparent. A substantially transparent component may be suitable for optical detection of a sample or analyte contained within the component.

In one set of embodiments, the material used to form the component (eg, first or second component) of the cassette is 400-800 nm light (eg, light in the visible range). transmits greater than 90% of light between the wavelengths of Light transmission may 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, the light transmission is greater than 80%, greater than 85%, greater than 88%, greater than 92%, greater than 94% or greater than between wavelengths of light between 400 and 800 nm. or greater than 96%. other components of the device are less than 96%, less than 94%, less than 92%, less than 90%, less than 85%, between wavelengths of light between 400 and 800 nm; It may be formed of a material that transmits light less than 80%, less than 50%, less than 30%, or less than 10%.

As described herein, in some embodiments the channel of the first component of the cassette is not in fluid communication with the channel of the second component of the cassette prior to first use by a user. For example, even after mating of the two components, channels 706 and 707 are not in fluid communication with each other, as illustrated by way of example in FIG. 11D . However, the cassette may have other parts or components, such as fluid connector alignment element 702 ( FIG. 11E ) that may be attached to first and/or second components 520B and 520C or to other parts of the cassette. may include more. As described herein, a fluid connector alignment element receives and is coupled with a fluid connector 720 that may enable fluid communication between channels 706 and 707 of the first and second components, respectively. may be configured to mate. For example, a fluidic connector can include a fluid path comprising a fluid path inlet and a fluid path outlet, wherein the fluid path inlet can be fluidically connected to an outlet of the channel 706, and the fluid path outlet can include a channel ( 707) (and vice versa). The fluid path of the fluid connector can have any suitable length (eg, at least 1 cm, at least 2 cm, at least 3 cm, at least 5 cm) for connecting the channels. The fluidic connector may be part of a kit with the cassette and may be packaged such that the fluidic connector does not fluidically connect the channels 706 and 707 .

The fluid connector may have any suitable configuration with respect to the cassette or components of the cassette. As illustrated by way of illustration in FIG. 11E , during connection of the fluidic connector to the cassette, the fluidic connector is coupled to an opposing component (eg, component 520B) to another component (eg, component 520C). ))). In other embodiments, a fluid connector may be positioned between two components of the cassette. For example, a fluid connector may be a component or layer positioned between (eg, interposed between) two components of a cassette. Other configurations are also possible.

Although much of the description herein relates to a cassette having one or more components or layers comprising channel networks, in other embodiments, the cassette may contain more than two, more than three, or more than four; It may include the same components or layers. For example, as illustrated by way of example in FIG. 11F , a cassette may include components 520B, 520C, 520D and 520E, each component including at least one channel or network of channels. . In some examples, the channel(s) of one or more components (eg, 2, 3 or all components) may not be fluidically connected prior to first use, but upon first use, for example , can be fluidically connected by using a fluid connector. In other embodiments, the channel(s) of one or more components (eg, 2, 3 or all components) are fluidically connected prior to first use.

As described herein, each of the components or layers of a cassette may be designed to have a specific function that differs from that of other components 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 components 520C, 520D, and 520E may have one or multiple analysis regions 709 connected in series. When the fluidic connector 722 is connected to the composite cassette, a portion of the sample (or multiple samples) is introduced into the channel network at each of the components 520C, 520D, and 520E to perform multiple assays. can be For example, each of the analysis regions may have one or more binding partners (eg, capture antibodies to iPSA, fPSA, tPSA and/or hK2) to detect one or more of iPSA, fPSA, tPSA and/or hK2. may include. As described herein, in some embodiments using specific capture antibodies in different analysis regions and/or separating the capture antibodies may make it possible to use the same detection antibody for the detection of each of the species. have. In some such embodiments, the same wavelength may be used to determine each of the species. This makes it possible to use simplified detectors and/or optical components for detection. For example, in some embodiments, detecting includes accumulating opaque material in different analysis regions that can be determined at a particular wavelength.

In some embodiments, at least the first and second components of the cassette may be part of a device or kit used to determine a particular chemical or biological condition. A device or kit may include, for example, a first component comprising a first channel in a first material, wherein the first channel comprises an inlet, an outlet and is greater than at least 200 microns between the first inlet and outlet. at least one portion having a large cross-sectional dimension. The device or kit may also include a second component comprising a second channel in a second material, wherein the second channel comprises an inlet and an outlet and has a cross-sectional dimension of less than 200 microns between the second inlet and outlet. At least one portion having a In some cases, the device or kit is packaged such that the first and second components are connected to each other. For example, the first and second components may be integrally connected to each other. In other embodiments, the first and second components are reversibly attached to each other. The device or kit may further include a fluidic connector fluidically connecting the first and second channels, the fluidic connector comprising a fluid path comprising a fluid path inlet and a fluid path outlet, wherein the fluid path inlet comprises a first The fluid path outlet may be fluidically connected to the outlet of the first channel and the fluid path outlet may be fluidically connected to the inlet of the second channel. In some embodiments, the device or kit is packaged such that the fluidic connector does not fluidically connect the first and second channels within the package. When the device is first used by an intended user, the fluid connector may be used to bring the first and second channels into fluid communication with each other.

Cassettes described herein may have any suitable volume for performing analysis or other processes such as chemical and/or biological reactions. The overall volume of the cassette includes, for example, any reagent storage area, assay areas, liquid receiving areas, waste areas as well as any fluid connectors and associated fluid channels. In some embodiments, small amounts of reagents and samples are used and the total 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.

The cassettes described herein may be portable, and in some embodiments may be compact. The length and/or width of the cassette may 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 may 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 point-of-care settings.

It should 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.

The methods and systems described herein can include a variety of different types of analyzes and can be used to determine a variety of different types of samples. In some cases, the assay includes a chemical and/or biological reaction. In some embodiments, the chemical and/or biological reaction comprises binding. Different types of binding can occur in the cassettes described herein. Binding is the formation of a corresponding pair exhibiting mutual affinity or binding capacity, typically a specific or unspecified binding or interaction, including biochemical, physiological and/or pharmaceutical interactions. interactions between molecules (eg, binding partners). Biological binding defines the type of interaction that occurs between pairs of molecules (eg, binding partners), including proteins, nucleic acids, glycoproteins, carbohydrates, hormones, and the like. Specific examples are antibody/antigen, antibody fragment/antigen, antibody/hapten, antibody fragment/hapten, enzyme/substrate, enzyme/inhibitor, enzyme/cofactor, binding protein/substrate, carrier Proteins/substrates, lectins/carbohydrates, receptors/hormones, receptors/effectors, complementary strands of nucleic acids, protein/nucleic acid repressors/inducers, ligands ( ligand)/cell surface receptor, virus/ligand, and the like. Binding can also occur between proteins or other components and cells. Moreover, the devices described herein may be used for other fluid assays (which may or may not include binding and/or reactions), such as detection of components, concentrations, and the like.

In some cases, a heterogeneous response (or assay) may occur within the cassette. For example, a binding partner may be associated with the surface of a channel, and a complementary binding partner may be in a fluid phase. Other solid phase assays can also be performed, including affinity reactions between proteins or other biomolecules (eg, DNA, RNA, carbohydrates), or non-naturally occurring molecules. Non-limiting examples of typical reactions that can be performed in a cassette include chemical reactions, enzymatic reactions, immune-based reactions (eg, antigen-antibody) and cell-based reactions.

Typical sample fluids include human or whole animal blood, serum, plasma, semen, tears, urine, sweat, saliva, cerebro-spinal fluid, physiological fluids such as vaginal secretions, and extracorporeal fluids used in research. environmental fluids, such as aqueous liquids, suspected of being contaminated with ions or analytes.

In some embodiments, one or more reagents that may be used to determine an analyte (eg, a binding partner of an analyte to be determined) in a sample are administered to a chamber of a channel or cassette prior to first use to perform a particular test or assay. stored in (chamber). In cases where an antigen is assayed, the corresponding antibody or aptamer may be a binding partner associated with the face of the microfluidic channel. If the antibody is an analyte, an appropriate antigen or aptamer may be a binding partner associated with the surface. Once the disease state has been determined, it may be desirable to place the antigen on a surface and test the antibody that has been produced in the subject. Although antibodies are referred to herein, it should be appreciated that antibody fragments may be used in combination with or in place of antibodies.

In some embodiments, the cassette is adapted and arranged to perform an analysis comprising accumulating an opaque material on a region of the microfluidic channel, exposing the region to light and determining transmission of light through the opaque material. The opaque material may include a material that interferes with the transmission of light at one or more wavelengths. An opaque material does not only refract light, but reduces the amount of transmission through the material, for example by absorbing or reflecting light. Different opaque materials or different amounts of opaque material enable for example less than 90, 80, 70, 60, 50, 40, 30, 20, 10 or 1 percent of light illuminating the opaque material to be transmitted. can do it Examples of opaque materials include molecular layers of metal (eg, elemental metal), ceramic layers, polymeric layers, and layers of opaque material (eg, dye). The opaque material may, in some cases, be a metal that can be deposited electrolessly. These metals may include, for example, silver, copper, nickel, cobalt, palladium and platinum.

The opaque material formed within the channel may comprise a series of discrete, independent particles that come together to form an opaque layer, but in one embodiment is a continuous material that assumes a generally planar shape. The opaque material may have a dimension (e.g., a width of length) 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, for example. can In some cases, the opaque material extends across the width of the channel (eg, the analysis region) containing the opaque material. The opaque layer may have a thickness of, for example, less than or equal to 10 microns, less than or equal to 5 microns, less than or equal to 1 micron, less than or equal to 100 nanometers, or less than or equal to 10 nanometers. Even at these small thicknesses, a detectable change in transmission can be obtained. An opaque layer can provide an increase in assay sensitivity compared to techniques that do not form an opaque layer.

In one set of embodiments, the cassette described herein is used to perform an immunoassay (e.g., to determine tPSA, iPSA, fPSA and/or hK2), and optionally, Silver reinforcement is used for signal amplification. In such an immunological evaluation assay, after delivery of a sample containing a blood marker that is detected in the assay regions, binding between the blood marker and the corresponding binding partner may occur. One or more reagents, which may optionally be stored in a channel of the device prior to use, may then flow across this binding pair complex. One of the stored reagents may comprise a solution containing one or more metal colloids that bind to the antigen being detected. For example, gold labeled antibodies that are anti-PSA and anti-hK2 can be used to detect iPSA, fPSA, tPSA and hK2, respectively. In another example, a mixture of gold labeled antibodies, such as gold labeled hK2 antibody, gold labeled anti-PSA antibodies and/or gold labeled anti-iPSA antibodies, can be used for detection. Such reagents may be stored, for example, in a cassette prior to use. The metal colloid may provide a catalytic surface for deposition of an opaque material, such as a layer of metal (eg, silver), on the surface of one or more analysis regions. The metal layer contains a metal precursor (eg, a solution of silver salts) and a reducing agent (eg, hydroquinone, Chlorohydroquinone, Pyrogallol) which can be selectively stored in different channels prior to use. ), metol, 4-aminophenol and phenidone).

When a positive or negative pressure differential is applied to the system, the silver salt and reducing liquids mix (eg, coalesce at the channel intersection) and then flow across the analysis region. Therefore, when antibody-antigen binding occurs in an assay region, the flow of metal precursor solution through the region can result in the formation of an opaque layer such as a silver layer due to the presence of a catalytic metal colloid associated with the antibody-antigen complex. The opaque layer may include a material that interferes with transmission of light at one or more wavelengths. The opaque layer formed within the channel can be optically determined, for example, by measuring a decrease in light transmission through a portion of the analysis region (eg, a meandering channel region) compared to a portion of the area that does not contain the antibody or antigen. can be detected. Alternatively, the signal may be obtained by measuring the change in light transmittance as a function of time as the film is being formed within the analysis area. An opaque layer can provide an increase in assay sensitivity when compared to techniques that do not form an opaque layer. Additionally, optical signals (eg, absorbance, fluorescence, incandescent or scintillation chemiluminescence, electrochemiluminescence), electrical signals (eg, resistance or conductivity of metal structures produced by an electroless process) ) or various amplification chemistries that generate magnetic signals (eg, magnetic beads) may be used to enable detection of the signal by a detector.

Various types of fluids can be used with the cassettes described herein. As described herein, fluids may be introduced into the cassette upon 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 mixtures of gases. When multiple fluids are contained within the cassette, the fluids may be separated by another fluid, preferably substantially immiscible with each of the first two fluids. For example, if the channel contains two different aqueous solutions, the separation plug of the third fluid may be substantially immiscible in both of these aqueous solutions. 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, provided the aqueous solutions can be kept separate. Fluids may also be selected based on their reactivity with adjacent fluids. For example, in some embodiments an inert gas such as nitrogen may be used and this inert gas may help preserve and/or stabilize any adjacent fluids. An example of a substantially immiscible liquid for separating aqueous solutions is perfluorodecalin. The selection of the separator fluid may also be made based on other factors, including any effect that the separator fluid may have on the surface tension of adjacent fluid plugs. It may be desirable to maximize the surface tension within any fluid plug to facilitate maintenance of the fluid plug as a single continuous unit under changing environmental conditions such as vibration, shock and temperature changes. Separator fluids may also be inert to the assay region to which the fluid is to be supplied. For example, if the analysis region comprises a biological binding partner, a separator fluid such as air or nitrogen may have little or no effect on the binding partner. The use of a gas (eg, air) as a separator fluid also results in changes in temperature (including sub-zero) or pressure changes within a channel of a fluid device when the liquids contained within the device expand or contract. may provide room for expansion.

A microfluidic sample analyzer is a fluid flow source (eg, a pressure control system) that can be fluidly coupled to channels 706 , 707 , 722 to pressurize the channels to move sample and/or other reagents through the channels. may include. In particular, the fluid flow source may be configured to move the sample and/or reagent from the initially substantially U-shaped channel 722 to the first channel 706 . A fluid flow source may also be used to move reagents within the second channel 707 through the substantially U-shaped channel 722 and into the first channel 706 . After the sample and reagents have passed through the analysis region 709 and analyzed, the fluid flow source 540 may be configured to move the fluid into the absorbent material 717 of the cassette. In one embodiment, the fluid flow source is a vacuum system. However, it should be understood that other sources of fluid flow may be used, such as valves, pumps, and/or other components.

As described herein, in some embodiments a vacuum source may be used to drive a fluid flow. The vacuum source may include a pump such as a solenoid operated diaphragm pump. In other embodiments, fluid flow may be flowed/controlled through the use of other types of pumps or sources of fluid flow. For example, in one embodiment, a syringe pump may be used to create a vacuum by pulling a syringe plunger in an outward direction. In other embodiments, positive pressure is applied to one or more inlets of the cassette to provide a source of fluid flow.

In some embodiments, fluid flow occurs while applying a substantially constant non-zero pressure drop (ie, ΔP) across the inlet and outlet of the cassette. In one set of embodiments, the full analysis is performed while applying a substantially constant non-zero pressure drop (ie, ΔP) across the inlet and outlet of the cassette. A substantially constant non-zero pressure drop may be achieved, for example, by applying positive pressure to the inlet or reduced pressure (eg, vacuum) to the outlet. In some cases, a substantially constant non-zero pressure drop causes fluid flow to occur mostly by capillary forces and/or without the use of actuating valves (eg, without changing the cross-sectional area of a channel of the fluid path of the cassette). achieved while not doing so. In some embodiments, during the entire assay performed essentially in the cassette, a substantially constant non-zero pressure drop, for example, an inlet to the assay region (which may be connected to a fluid connector) and downstream of the assay region, respectively. may exist over an outlet downstream (eg, an outlet downstream of the liquid receiving region).

In one embodiment, the vacuum source is configured to pressurize the channel to approximately -60 kPa (approximately 2/3 atmospheric pressure). In another embodiment, the vacuum source is configured to pressurize the channel to approximately -30 kPa. In certain embodiments, the vacuum source is configured to pressurize the channel, for example between -100 kPa and -70 kPa, between -70 kPa and -50 kPa, between -50 kPa and -20 kPa, or between -20 kPa and -1 kPa.

Once the cassette is positioned within the analyzer, a fluid flow source may be coupled to the cassette to ensure a fluid-tight connection. As noted above, the cassette may include a port configured to couple a channel 707 and, when fluidically coupled to the channel 706 , couple the channel 706 with a fluid flow source. In one embodiment, seals or o-rings may be positioned around the port and a linear solenoid positioned over the o-rings to seal by pressing the o-rings against the cassette body. For example, as shown in the example embodiment illustrated in FIG. 11A , in addition to port 719 , there may be two vent ports 715 and one mixing port 713 . The interface between each port and the manifold may be independent (eg, there may not be any fluidic connections within the manifold).

In one embodiment, when the fluid flow source is activated, the channels 706, 707 in the cassette can be pressurized (eg, to about -30 kPa) and the fluids in the channel (fluid samples as well as reagents) all) will flow towards the outlet. In one embodiment that includes a vent port 715 and a mixing port 713 , a vent valve connected to the port 713 via a manifold may initially be open and this is the mixing port 713 . It may allow all of the reagents downstream of the to move towards the outlet, but will not cause the reagents upstream of the mixing port 713 to move. Once the vent valve is closed, reagents upstream of the mixing port 713 can travel towards the mixing port and then to the outlet. For example, fluids may continue to be stored in a channel upstream of the mixing port and after closing a vent valve located along the channel, the fluids may continuously flow toward the channel outlet. In some cases, the fluids may be stored in separate crossover channels and after closing the vent valve, the fluids will flow together towards the crossover point. This set of embodiments can be used, for example, to controllably mix fluids as they flow together. The timing of delivery and the volume of fluid delivered may be controlled, for example, by the timing of vent valve actuation.

Advantageously, vent valves can be operated without constricting the cross-section of the microfluidic channel in which they operate, as occurs with certain valves in the prior art. Such a mode of operation may be effective in preventing leakage across the valve. Moreover, because vent valves may be used, some of the systems and methods described herein have, for example, their high cost, complexity of manufacture, fragility, limited compatibility with mixed gas and liquid systems, and/or It does not require the use of specific internal valves, which can be problematic due to unreliability in microfluidic systems.

Although vent valves are described, it should be understood that other types of valve mechanisms may be used with the systems and methods described herein. Non-limiting examples of valve mechanisms that may be operatively associated with a valve include a diaphragm valve, a ball valve, a gate valve, a butterfly valve, a glove valve, a needle valve, a pinch valve. ), a poppet valve, or a pinch valve. The valve mechanism may be actuated by any suitable means including a solenoid, a motor, manually, by electrical actuation, or by hydraulic/pneumatic actuation.

As described above, all of the liquid (samples and reagents) in the cassette may be moved into a liquid containing area, which may contain absorbent material 717 . In one embodiment, the absorbent material absorbs only liquid so that gases can flow out of the cassette through the outlet.

Various determination (eg, measurement, quantitation, detection and validation) techniques may be used, for example, to analyze a sample component or other component or condition associated with a microfluidic system or cassette described herein. Crystallization techniques may include optical based techniques such as light transmission, light absorption, light scattering, light reflection, and visual techniques. Crystalline techniques may also include luminescent techniques such as photoluminescence (eg, fluorescence), chemiluminescence, bioluminescence, and/or electrochemiluminescence. In other embodiments, crystallization techniques may measure conductivity or resistance. Accordingly, the analyzer may be configured to include such and other suitable detection systems.

Different optical detection techniques provide a number of options for determining response (eg, assay) results. In some embodiments, measuring transmission or absorption means that light can be detected at the same wavelength emitted from the light source. Although the light source may be a narrow band source emitting at a single wavelength, the light source may be a broad spectrum source emitting over a variety of wavelengths, as many opaque materials can effectively block a wide range of wavelengths. In some embodiments, the system may be operated with a minimum of optical devices (eg, a simplified optical detector). For example, the crystal device may lack a photomultiplier, or may lack a wavelength selector such as a grating, prism or filter, or a device that directs or columns light, such as a columnator. may be absent, or magnifying glasses (eg, lenses) may be absent. Removal or reduction of these features may result in lower cost and stronger devices.

12 shows an example optical system 800 that may be positioned within the housing of the analyzer. As illustrated by way of example in this embodiment, the optical system includes at least one first light source 882 and a detector 884 spaced apart from the first light source. The first light source 882 may be configured to pass light through the first analysis region of the cassette when the cassette is inserted into the analyzer. A first detector 884 may be positioned opposite the first light source 882 to detect an amount of light passing through the first analysis region of the cassette 520 . It should be appreciated that in other embodiments, the number of light sources and detectors may vary as the invention is not so limited. As described 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 is aligned with a light source and a corresponding detector. In some embodiments, the light source includes an optical aperture that can help direct light from the light source to a specific area within the analysis area of the cassette.

In one embodiment, the light sources are light emitting diodes (LEDs) or laser diodes. For example, an InGaAlP red semiconductor laser diode emitting at 654 nm can be used. Other light sources may also be used. The light source may be located within a nest or housing. The nest or housing may include a narrow aperture or thin tube that may assist in collimating the light. The light sources may be positioned above where the cassette is inserted into the analyzer so that the light source shines downward onto the top surface of the cassette. Other suitable configurations of the light source relative to the cassette are also possible.

It should be appreciated that the wavelength of the light sources may vary as the invention is not so limited. 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 accommodates a different wavelength of light. However, in other embodiments, 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 the same and different wavelengths of light are also possible.

As noted, a detector 884 may be positioned below and spaced apart from the light source 882 to detect the amount of light passing through the cassette. In one embodiment, one or more of the detectors are photodetectors (eg, photodiodes). In certain embodiments, the photo detector may be any suitable device capable of detecting transmission of light emitted by a light source. One type of photodetector is an optical integrated circuit (IC) comprising a photodiode, an amplifier and a voltage regulator with maximum sensitivity at 700 nm. 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 analysis region 709 is measured at the detector 884 . If the light source is pulse modulated, the photodetector may include a filter to remove the effect of light not at a selected frequency. When multiple and neighboring signals are detected at the same time, the light source used for each analysis area (eg, detection area) may be modulated at a frequency sufficiently different from the frequency of the light source adjacent thereto. In this configuration, each detector can be configured (eg, using software) to be selected for its own attributed light source, thereby preventing interfering light from neighboring optical pairs.

Applicants claim that the amount of light passing through the analysis area of the cassette will be used to determine information about the samples as well as information about specific processes (eg, mixing of reagents, flow rate, etc.) occurring in the fluid system of the cassette. recognized that it could. In some cases, the measurement of light through an area can be used as feedback to control fluid flow within the system. In certain embodiments, quality control or anomalies in the operation of the cassette may be determined. For example, feedback from the analysis domain to a control system can be used to determine anomalies that have occurred in a microfluidic system, where the control system sends a signal to one or more components to cause all or parts of the system to be shut off. can do. Consequently, the quality of processes being performed in a microfluidic system can be controlled using the systems and methods described herein.

It should be appreciated that a clear liquid (such as water) may 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 in the analysis region 709 may cause less light to be transmitted through the analysis region 709 because more light may be scattered within the channel compared to when clear liquid is present. When the blood samples are within the analysis area 709 , a significantly lesser amount of light may pass to the detector 884 due to absorption as well as light scattering of the blood cells. In one embodiment, very little light is transmitted through the analysis region 709 when the silver is associated with a sample component that engages a facet within the analysis region and when the silver accumulates within the analysis region.

It is recognized that by measuring the amount of light detected by each detector 884 a user can determine which reagents are within a particular analysis area 709 at a particular point in time. It is also recognized that it is possible to measure the amount of silver deposited in each analysis region 709 by measuring the amount of light detected by each detector 884 . This amount may correspond to the amount of analyte captured during the reaction and thus it may provide a measure of the concentration of analyte in the sample.

As described above, Applicants have recognized that the optical system 880 can be used for various quality control. First, the time it takes a sample to arrive at the analysis region in which the optical system detects light passing through the analysis region can be used to determine if the sample is deficient or has an obstruction within the optical system. Also, if the sample is predicted to be of a particular volume, eg, about 10 microliters, there is an expected flow time that will be associated with the sample passing through the channels and analysis regions. If the sample is outside the range of the expected flow time, this may be an indication that there are not enough samples to perform the analysis and/or that the wrong type of sample has been loaded into the analyzer. Moreover, a range of expected results may be determined based on the type of sample (eg, serum, blood, urine, etc.) and if the sample is outside the expected range, this may be an indication of an error.

In one embodiment, the analyzer includes a temperature control system positioned within the housing, the temperature control system may be configured to regulate a temperature within the analyzer. For certain sample analysis, the sample may need to be maintained within a certain temperature range. For example, in one embodiment, it is desirable to maintain the temperature within the analyzer at about 37°C. Accordingly, in one embodiment, the temperature control system includes a heater configured to heat the cassette. In one embodiment, the heater is a resistive heater that may be located below where the cassette is placed within the analyzer. In one embodiment, the temperature control system may also include a thermistor to measure the temperature of the cassette and a control circuit may be provided to control the temperature.

In one embodiment, the passive flow of air within the analyzer may serve to cool the air within the analyzer as needed. A fan may optionally be provided within the analyzer to lower the temperature within the analyzer. In some embodiments, the temperature control system may include Peltier thermoelectric heaters and/or coolers within the analyzer.

In certain embodiments, an identification system comprising one or more identifiers is used and associated with one or more components or materials associated with the cassette and/or analyzer. “Identifiers” refer to “information and encoding” (i.e., radio frequency identification (RFID) tags or bar codes about the components they contain an identifier), as described in more detail below. may carry or contain information, such as using a device for carrying, storing, generating or transmitting information, such as as It may be associated with information (eg, information about a user and/or a sample to be analyzed) that may be included in a database on or on a computer readable medium. In the latter example, detection of such an identifier may trigger retrieval and use of the associated information from the database.

Identifiers "encoded" with information about a component are not necessarily encoded with the complete set of information about the component. For example, in certain embodiments, the identifier may only be encoded with enough information to uniquely identify the cassette (eg, regarding a serial number, part number, etc.), while additional information regarding the cassette Information (eg, type, use (eg, type of assessment), ownership, location, location, access, content, etc.) may be stored remotely and only associated with an identifier.

“Information about” or information associated with “a cassette, material or component, etc.” means the identity, positioning or location of the cassette, material or component or the identity, positioning or location of the content of the cassette, material or component. information about the cassette, and may further include information about the nature, state or composition of the cassette, material, component, or content. “Information about” or “information associated with” a cassette, material or component or content thereof that identifies a cassette, material, component, or content thereof and distinguishes the cassette, material, component, or content thereof from others may contain information. For example, “information about” or “information associated with” a cassette, material or component or content thereof may include the type or what the cassette, material or component or content thereof is, where or where it should be located; what it is or how it should be positioned; the function or purpose of the cassette, material or component or its contents; how the cassette, material or component or its contents should be connected with other components of the system; Information may refer to information indicating information, expiration date, destination, manufacturer or ownership of the cassette, material or component or its contents, the type of analysis/assessment analysis to be performed within the cassette, information about whether the cassette has been used/analyzed, etc. .

Non-limiting examples of identifiers that may be used in the context of the present invention are radio frequency identification (RFID) tags, barcodes, serial numbers, color tags, fluorescent or optical tags (eg, quantum (using quantum dots), chemical compounds, radio tags, magnetic tags.

In one embodiment, the identification reader is an RFID reader configured to read an RFID identifier associated with the cassette. For example, in one embodiment, the analyzer includes an antenna and an RFID module configured to read information from a cassette inserted into the analyzer. In another embodiment, the identification reader is a barcode reader configured to read a barcode associated with the cassette. Once the cassette is inserted into the analyzer, the identification reader can read information from the cassette. The identifier on the cassette may include one or more of the following types of information: cassette type, type of assay/assessment being performed, product number, information about whether the cassette was used/analyzed, and other information described herein. The reader may also be configured to read information such as, but not limited to, calibration information provided by a group of cassettes, an expiration date, and any additional information specific to the product, such as in a box of cassettes. The identified information may optionally be displayed to the user, eg, to confirm that the correct cassette and/or type of assay is being performed.

In some cases, the identification reader may be integrated with the control system via communication paths. Communication between the identification readers and the control system may occur along a hardwired network or may be transmitted wirelessly. In one embodiment, the control system provides a specific identifier (eg, of the cassette associated with information regarding the cassette type, manufacturer, assay to be performed, etc.) when indicating that a cassette is properly connected or inserted into an analyzer of a specific type. can be programmed to recognize

In one embodiment, the identifier of the cassette is predetermined or included in a database for use of the system or cassette for a particular purpose, user or product or with particular reaction conditions, sample types, reagents, users, etc. It may be associated with programmed information. If an incorrect match is detected or the identifier is deactivated, the process may be aborted or the system may be rendered inactive until notified to the user or with an acknowledgment by the user.

Information from or associated with the identifier may, in some embodiments, be stored, for example, in a computer memory or on a computer readable medium for future reference or archival purposes. For example, certain control systems may use identifiers from identifiers to identify which components (eg, cassettes) or type of cassettes were used in a particular analysis, date, time and duration of use, conditions of use, etc. Information associated with the identifiers or identifiers may be used. Such information may be used, for example, to determine if one or more components of the analyzer should be cleaned or replaced. Optionally, the control system or any other suitable system, including information encoded by or associated with identifiers, may be used to provide proof of conformance with regulatory standards or verification of quality control, including information collected. You can create a report from

Information encoded into or associated with the identifier may also be used, for example, to determine whether a component (eg, a cassette) associated with the identifier is genuine or fake. In some embodiments, determining that a fake component is present causes the system to lockout. In one example, the identifier may include a unique identity code. In this example, the process control software or analyzer will not allow the system to run (eg, the system may be disabled) if an external or mismatched identity code has been detected (or if no identity code has been detected).

In certain embodiments, information obtained from or associated with an identifier may be used to verify the identity of a customer for which a cassette and/or analyzer is to be sold or a biological, chemical or pharmaceutical process is to be performed. In some cases, information obtained from or associated with an identifier is used as part of a process of collecting data for troubleshooting a system. The identifier may also include or be associated with information such as batch histories, assembly process and instrumentation diagrams (P and IDs), troubleshooting histories, among others. Troubleshooting the system may, in some cases, be accomplished via remote access or include using diagnostic software.

In one embodiment, the analyzer includes a user interface positioned within the housing and configurable for a user to enter information into the sample analyzer. In one embodiment, the user interface is a touch screen.

The touch screen may guide the user through the operation of the analyzer providing textual and/or graphical instructions for use of the analyzer. A touch screen user interface may guide the user to insert a cassette into the analyzer, for example. This may then guide the user to enter the patient's name or other patient identification source/number (eg, age, results of a DRE test, etc.) into the analyzer. It should be appreciated that patient information such as name, date of birth and/or patient ID number may be entered into the touch screen user interface to identify the patient. The touch screen may display the amount of time remaining to complete the analysis of the sample. The touch screen user interface may then show the results of the sample analysis along with the patient's name or other identifying information.

In other embodiments, the user interface may be configured differently, such as an LCD display and single button scrolling through menus. In another embodiment, the user interface may simply include a start button that launches the analyzer. In another embodiment, a user interface from separate, standalone devices (such as a smartphone or mobile computer) may be used to interface with the analyzer.

The analyzer described above can be used in a variety of ways to process and analyze a sample disposed within the analyzer. In one particular embodiment, once the mechanical component configured to interface with the cassette indicates that the cassette has been properly loaded into the analyzer, the identification reader reads and identifies information associated with the cassette. The analyzer may be configured to compare this information with data stored in the control system to ensure that it has calibration information for this particular sample. If the analyzer does not have the appropriate calibration information, the analyzer may output a request to the user to upload the specific information required. The analyzer may also be configured to review expiration date information associated with the cassette and abort analysis if the expiration date has passed.

In one embodiment, once the analyzer has determined that the cassette can be analyzed, a fluid flow source, such as a vacuum manifold, can be configured in contact with the cassette to ensure a hermetic seal 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 light sources both activated and deactivated.

To initiate movement of the sample, a vacuum system may be activated, which may rapidly change the pressure in one or more channels (eg reduced to about -30 kPa). A decrease in pressure in this channel may cause the sample to flow into the channel and through each of the analysis regions 709A-709D (see FIG. 10 ). After the sample has reached the final analysis region 709D, the sample may continue to flow into the liquid receiving region 717 .

In one particular set of embodiments, a microfluidic sample analyzer is used to measure the level of iPSA, fPSA, tPSA and/or hK2 in a blood sample. In some embodiments, 3, 4, 5, 6 or more analysis regions (eg, analysis regions 709A-709D) may be used to analyze a sample. For example, in the first analysis region, the walls of the channel block proteins (bovine serum albumin) such that little or no proteins in the blood sample adhere to the walls of the analysis region (except possibly for some non-specific binding that can be washed away). (such as bovine serum albumin). The first analysis region may serve as a negative control.

In the second analysis region, the walls of the channel may be coated with a predetermined amount of prostate specific antigen (PSA) to serve as a high or positive control. As the blood sample passes through the second analysis region, little or no PSA proteins in the blood may bind to the walls of the channel. The gold conjugated detection antibodies in the sample may dissolve from the inside of the fluid connector tube 722 or may flow from any other suitable location. As these antibodies may not yet bind to PSA in the sample, these antibodies may bind to PSA on the walls of the channel to serve as a high or positive control.

In a third analysis region, the walls of the channel may be coated with a capture antibody to iPSA (eg, anti-iPSA antibody) capable of binding to an epitope on the PSA protein that is different from the gold conjugation signal antibody. As the blood sample passes through the third analysis region, iPSA proteins in the blood sample may bind the anti-iPSA antibody in a manner proportional to the concentration of these proteins in the blood.

In the fourth analysis region, the walls of the channel may be coated with a capture antibody to fPSA (eg, anti-fPSA antibody) capable of binding to an epitope on the PSA protein that is different from the gold conjugation signal antibody. As the blood sample passes through the fourth analysis region, the fPSA proteins in the blood sample may bind the anti-fPSA antibody in a manner proportional to the concentration of these proteins in the blood.

In the fifth analysis region, the walls of the channel may be coated with a capture antibody to tPSA (eg, anti-tPSA antibody) capable of binding to an epitope on the PSA protein that is different from the gold conjugation signal antibody. As the blood sample passes through the fifth analysis region, the tPSA proteins in the blood sample may bind the anti-tPSA antibody in a manner proportional to the concentration of these proteins in the blood.

Optionally, the wall of the channel in the sixth analysis region may be coated with a capture antibody to hK2 (eg anti-hK2 antibody) capable of binding to an epitope on a different protein than the gold junction signal antibody. As the blood sample passes through the sixth analysis region, the hK2 proteins in the blood sample may bind the anti-hK2 antibody in a manner proportional to the concentration of these proteins in the blood.

Detection antibodies, such as gold labeled antibodies that are anti-PSA and anti-hK2, can be used to detect each of iPSA, fPSA, tPSA and/or hK2. However, in other embodiments, a mixture of gold labeled antibodies, such as a gold labeled anti-hK2 antibody, a gold labeled anti-PSA antibody, and/or a gold labeled anti-iPSA antibody, may be used for detection. In some embodiments, the gold conjugated detection antibodies in the sample may dissolve from the inside of the fluid connector tube 722 or flow from any other suitable location.

In some examples, measurements from the region being analyzed can be used to determine as well as control the concentration of an analyte in a sample. For example, a threshold measure may be established at the stage of initial amplification. Measurements above (or below this value) may indicate that the analyte concentration is outside the desired range for the assay. This technique can be used, for example, to identify if a High Dose Hook effect occurs during an assay, ie when a very high concentration of analyte artificially gives a very low reading.

In other embodiments, different numbers of assay areas may be provided, and the assay may optionally include one or more assay areas that actually test the sample. Additional analysis areas can be used to measure additional analytes so that the system can perform multiple assays simultaneously on a single sample.

In one particular embodiment, it takes about 8 minutes for a 10 microliter blood sample to flow through the four analysis areas. The start of this analysis can be calculated when the pressure in the channel is about -30 kPa. During this time, the optical system measures the light transmittance for each analysis area, and in one embodiment, this data is sent to the control system approximately every 0.1 seconds. Using reference values, these measurements can be transformed using the following equation:

Transmittance = (l-ld)/(lr-ld) (1)

here:

l = intensity of light transmitted through the analysis area at a given point in time

ld = intensity of light transmitted through the analysis area with the light source turned off

lr = reference intensity (i.e. the intensity of light transmitted in the analysis area prior to the start of the analysis when the light source is on or when only air is in the channel)

and

Optical density = - log(transmittance) (2)

Therefore, using this equation, the optical density within the analysis area can be calculated.

13 is a block diagram 900 illustrating how control system 550 (see FIG. 12 ) may be operatively associated with various different components according to one embodiment. The control systems described herein can be implemented in many ways, such as by dedicated hardware or firmware, using a processor that is programmed using software or microcode to perform the functions enumerated above, or any suitable combination of the foregoing. can be implemented. The control system may control one or more operations of a single assay (eg, for a biological, biochemical or chemical reaction) or multiple (separate or interconnected) assays. For example, the control system may be located within the housing of the analyzer and configured to communicate with an identification reader, a user interface, a fluid flow source, an optical system, and/or a temperature control system to analyze a sample in the cassette.

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 certain time intervals (eg, every 0.1 seconds), the processor communicates with a second higher level processor that communicates with the user via a user interface and/or communication subsystem (described below), and , directs the operation of the analyzer (eg, determines when to start analyzing a sample and interprets the results). In one embodiment, communication between the two processors occurs over a serial communication bus. It should be appreciated that, in another embodiment, the analyzer may include only one processor or two or more processors, as the invention is not so limited.

In one embodiment, the analyzer may interface with external devices and may include ports for connection with, for example, one or more external communication units. External communication may be achieved, for example, via USB communication. For example, as shown in FIG. 13 , the analyzer may output the result of sample analysis to the USB printer 901 or to the computer 902 . Additionally, the data stream generated by the real-time processor may be output to a computer or USB memory stick 904 . In some embodiments, the computer may also be capable of controlling the analyzer directly via a USB connection. Moreover, other types of communication options are available as the present invention is not limited in this respect. For example, Ethernet, Bluetooth and/or WI-FI communication with the analyzer may be established via the processor.

The computational methods, steps, simulations, algorithms, systems, and system elements described herein may be implemented using a computer-implemented control system, such as the computer-implemented systems of the various embodiments described below. These methods, steps, systems, and system elements described herein are not limited to any particular computer system described herein in their implementation, as many other different machines may be used.

A computer implemented control system may be part of, or operatively associated with, the sample analyzer, configured to control and adjust operating parameters of the sample analyzer as described above, as well as analyze and calculate values, in some embodiments; / or can be programmed. In some embodiments, the computer implemented control system may transmit and receive reference signals to set and/or control operating parameters of the sample analyzer and optionally other system devices. In other embodiments, the computer implemented system may be separate from and/or located remotely relative to the sample analyzer and via portable electronic data storage devices such as magnetic disks or via a computer network such as the Internet or a local intranet. It may be configured to receive data from one or more remote sample analyzer devices via indirect and/or portable means, such as via communication.

The computer implemented control system includes a processing unit (ie, a processor), a memory system, input and output devices and interfaces (eg, interconnection mechanism), as well as transportation circuitry (eg, as well as various well-known components and circuitry including other components such as, for example, one or more buses), video and audio data input/output (I/O) subsystems, and special purpose hardware. It may include other components and circuitry. Moreover, the computer system may be a multi-processor computer system or may include multiple computers connected through a computer network.

The computer-implemented control system may include, for example, series x86, Celeron and Pentium processors available from Intel, similar devices from AMD and Cyrix, 680X0 series microprocessors available from Motorola, PowerPC microprocessors and ARM processors from IBM. A commercially available processor may be included. Many different processors are available and computer systems are not limited to a particular processor.

The processor typically executes a program called an operating system, examples of which are WindowsNT, Windows95 or 98, Windows 7, Windows 8, UNIX, Linux, DOS, VMS, MacOS and OSX and iOS, which operating system can be used on other computers It controls the execution of programs and provides scheduling, 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 on which application programs in high-level programming languages are written. The computer implemented control system is not limited to a particular computer platform.

A computer implemented control system can typically include a memory system including computer-readable and writable non-volatile recording media, examples of which are magnetic disks, optical disks, flash memory, and tape. Such recording media may be removable, eg floppy disks, read/write CDs or memory sticks, or they may be permanent, eg hard drives.

Such recording media typically store signals in binary form (ie, in a form interpreted as a sequence of ones and zeros). A disk (eg, magnetic or optical disk) has a number of tracks in which such signals can be stored, typically in binary form, ie in a form that is interpreted as a sequence of ones and zeros. Such signals may specify a software program executed by a microprocessor, eg, an application program, or information processed by the application program.

The memory system of a computer implemented control system may also include an integrated circuit memory element, which is typically volatile random access memory, such as dynamic random access memory (DRAM), or static memory (SRAM). Typically, in operation, the processor causes programs and data to be read from the non-volatile recording medium into an integrated circuit memory element, and the integrated circuit memory element typically causes the processor to program instructions faster than is possible on the non-volatile recording medium. It is possible to access fields and data.

A processor typically manipulates data in an integrated circuit memory element according to program instructions and then copies the manipulated data to a non-volatile recording medium after processing is complete. Various mechanisms are known for managing data movement between a non-volatile recording medium and an integrated circuit memory element, and a computer implemented control system implementing the methods, steps, systems and system elements described above with respect to FIG. 13 . is not limited thereto. The computer implemented control system is not limited to a particular memory system.

At least a portion of such a memory system described above may be used to store one or more data structures (eg, lookup tables) or expressions described above. For example, at least a portion of the non-volatile recording medium may store at least a portion of a database including one or more of such data structures. Such a database may be any of the various types of databases, eg, one or more flat-file data structures in which data is organized into data units separated by delimiters. It can be a file system comprising .

The computer implemented control system may include a video and audio data I/O subsystem. The audio portion of the subsystem may include an analog-to-digital (A/D) converter that receives analog audio information and converts it to digital information. Digital information may be compressed using known compression systems for storage on a hard disk for use at different times. A typical video portion of the I/O subsystem may include a video image compressor/restorer, many of which are known in the art. Such compressors/decompressors convert analog video information to compressed digital information and vice versa. The compressed digital information may be stored on a hard disk for later use.

The computer implemented control system may include one or more output devices. Exemplary output devices include cathode ray tube (CRT) displays, liquid crystal displays (LCDs) and other video output devices, communication devices such as printers, modems or network interfaces, storage devices such as disk or tape, and audio output such as speakers. including devices.

The computer implemented control system may also include one or more input devices. Exemplary input devices include sensors and data input devices such as keyboards, keypads, track balls, mice, pens and tablets, communication devices as described above, and audio and video capture devices. The computer implemented control system is not limited to the specific input or output devices described herein.

It should be appreciated that one or more of any type of computer implemented control system may be used to implement the various embodiments 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 implement one or more of the methods, steps, simulations, algorithms, systems and system elements described above as part of a computer implemented control system described above or as a standalone component. have.

The computer implemented control system and components thereof may be programmable using any of a variety of one or more suitable computer programming languages. Such languages are procedural programming languages such as C, Pascal, Fortran and BASIC, object oriented languages such as C++, Java and Eiffel and a scripting language or even assembly language. language) may include other languages.

Methods, steps, simulations, algorithms, systems and system elements include procedural programming languages, object-oriented programming languages, other languages and combinations thereof, executable by such a computer system. It may be implemented using any of a variety of suitable programming languages. Such methods, steps, simulations, algorithms, systems, and system elements may be implemented as separate modules of a computer program, or may be individually implemented as separate computer programs. Such modules and programs may be executed on separate computers.

Such methods, steps, simulations, algorithms, systems, and system elements, individually or in combination, may include a computer-readable medium, such as a non-volatile recording medium, an integrated circuit memory element, or a combination thereof. may be embodied as a computer program product tangibly embodied as computer readable signals in Per such method, step, simulation, algorithm, system, or system element, such computer program product may include computer readable signals tangibly embodied on a computer readable medium, which signals may contain instructions for example For example, as part of one or more programs, the instructions instructing the computer to perform a method, step, simulation, algorithm, system, or system element as a result of being executed by the computer.

It should be appreciated that various embodiments may be formed with one or more of the features described above. As the present invention is not limited in this respect, the above aspects and features may be used in any suitable combination. It should also be appreciated that the drawings are illustrative of various components and features that may be incorporated into various embodiments. For simplicity, some of the drawings may illustrate one or more optional features or components. However, the present invention is not limited to the specific embodiments disclosed in the drawings. It is understood that the present invention may include embodiments that may include only some of the components illustrated in any one drawing figure, and/or may include embodiments that combine components illustrated in a number of different drawing figures. should be recognized

other preferred embodiments

It will be appreciated that the methods of the present invention may be incorporated in the form of various embodiments, only some of which are disclosed herein. It will be apparent to those skilled in the art that other embodiments exist and do not depart from the spirit of the invention. Therefore, the described embodiments are illustrative and should not be construed as limiting.

examples

Example 1

studies

In total, 7 separate studies using statistical models were performed. The studies included a total of 7,647 men with elevated PSA and 2,270 cancers, and 5 studies were externally validated. Moreover, the studies were systematically designed to cover a wide range of clinical scenarios. Perhaps most importantly, one of the studies involved the natural history method. Because biopsy results are a surrogate endpoint—it doesn't matter whether a man has prostate cancer, but whether that man is at risk for prostate cancer that will affect his or her life—the ideal study is from patients. After blood is drawn, patients will be followed for many years without further screening to determine prostate cancer outcomes. We were fortunate enough to be able to conduct such a study [2011, Vickers, A.J et al. Cancer Epidemiol Biomarkers Prey, 20(2), p.255-61].

The Malmoe Diet and Cancer research cohort was part of a large population-based study to identify dietary risk factors for cancer death, living in the city of Malmö, Sweden and born between 1923 and 1945. 11,063 men who provided anti-coagulant blood samples between 1991 and 1996. Results were confirmed through the Swedish Cancer Registry. Marker values were obtained from archived blood samples that were analyzed in 2008 and this blood sample was previously validated by obtaining accurate kallikrein measurements from stored blood [Ulmert, D et al., Clin, 2006. Chem., 52(2): p.235-9]. Although almost all cases were clinically diagnosed, the rate of testing of PSA was very low. As such, the study follows the “natural history” of prostate cancer in men with elevated PSA. Of the 792 men with 3 ng/ml PSA at baseline, at a mean follow-up of 11 years, 474 were subsequently diagnosed with prostate cancer. The predictive discrimination of the 4 kallikrein panel statistical model was significantly higher than the PSA for predicting the amount of any cancer and the advanced cancers (stage T3 or T4 or metastasized) most likely to be precisely fatal. As previous studies have shown, about 50% of men have a risk of prostate cancer in less than 20% of the models. It was estimated that only 13 men per 1000 with elevated PSA would have a risk of < 20% from the model, but would be diagnosed with cancer within 5 years; Only one male would have had advanced cancer at the time of diagnosis.

The Malmö group showed several important features of the predictive model. First, it was externally justified. Second, it indicates that the model predicts clinically diagnosed cancers that are not overdiagnosed by definition. Third, the study suggests that cancers missed by the model are those considered overdiagnosed: data from biopsy studies suggest that the panel ranks about 60 per 1000 people with biopsy detectable cancers as low-risk. indicates to classify as; Malmö cohort data suggest that less than 1 in 4 of these will become clinically evident after 5 years of follow-up. Fourth, it predicts very strongly the types of aggressive cancers the model is most likely to shorten lifespan of men. Finally, the data show that only one in 1000 people will have a low risk of prostate cancer according to the model, but that when subsequently diagnosed with advanced cancer, clinical use of the model will not cause significant damage by delayed diagnoses. indicates that it is A summary of the studies is provided in Table 2.

In summary, the preliminary studies can be summarized as follows:

1. Various kallikrein forms in the blood - total PSA, free PSA, intact PSA and hK2 - can predict the outcome of prostate biopsy in men with elevated total PSA.

2. A statistical prediction model based on 4 kallikreins was built using a single training set.

3. It integrates information from novel markers with clinical testing to provide predictive probabilities of cancer.

4. Overall, the panel was applied to over 7,500 men diagnosed with close to 2250 cancers, where 5 separate studies were externally justified.

5. The model has a much higher AUC than the statistical model based on standard predictors alone (total PSA, age and digital rectal examination), making it highly discriminatory for prostate cancer.

6. Using the 4-kallikrein statistical predictive model to determine transition to prostate biopsy can reduce clinical outcomes compared to alternative strategies, such as performing biopsies on all men, according to decision analysis. will improve

7. The model was valuable within a range of different clinical settings; with and without prior screening; with and without prior biopsy; With and without clinical work-up prior to transfer to biopsy.

Summary of studies group technology sample size Increase in AUC: PSA vs. 4 Kallikrein Model Increase in AUC: 4 kallikrein panels plus DRE model vs. PSA + DRE Gothenburg Round 1 unscreened men 740 Any cancer:
0.832 vs 0.680
High Risk:
0.870 vs 0.816 Any cancer:
0.836 vs 0.724
High Risk:
0.903 vs 0.868 Gothenburg follow-up rounds Men who have had a PSA test before 1241 Any cancer:
0.674 vs 0.564
High Risk:
0.819 vs 0.658 Any cancer:
0.697 vs 0.622
High Risk:
0.828 vs 0.717 Rotterdam Round 1 unscreened men 2186 Any cancer:
0.764 vs 0.637
High Risk:
0.825 vs 0.776 Any cancer:
0.776 vs 0.695
High Risk:
0.837 vs 0.806 Rotterdam follow-up rounds Men who have had a PSA test before 1501 Any cancer:
0.713 vs 0.557
High Risk:
0.793 vs 0.699 Any cancer:
0.711 vs 0.585
High Risk:
0.798 vs 0.709 Before Rotterdam Negative Biopsy Consistently elevated PSA after negative biopsy 925 not rated Any cancer:
0.681 vs 0.584
High Risk:
0.873 vs 0.764 tarn Clinical work-up before biopsy 262 not rated Any cancer:
0.782 vs 0.628
High Risk:
0.870 vs 0.767 Malmo Longitudinal follow-up without biopsy or screening 792 Any cancer:
0.751 vs 0.654
Advanced Cancer ^* :
0.824 vs 0.716 not rated

* T3/T4 at the time of diagnosis or 8. Applying the model to the archived blood samples of men who were longitudinally followed without screening showed that men with elevated PSA, but low risk from the statistical model were unlikely to develop aggressive cancers over the subsequent 5 to 10 years. gave. Consequently, clinically diagnosed aggressive cancers were common in men at high risk from the model. Example model used in this example

Age: Enter age in years

tPSA: Enter total PSA in ng/ml

fPSA: Enter free PSA in ng/ml

iPSA: Enter intact PSA in ng/ml

hK2: Enter hK2 in ng/ml

Use if tPSA ≥ 25: L = 0.0733628 x tPSA - 1.377984

Prostate cancer risk = exp(L)/[1 + exp(L)]

If tPSA < 25, use one of the two equations below, one incorporating clinical information and the other not:

The cubic spline 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)/(0.84-0.25)x(fPSA-0.25)^3+max(fPSA-0.84,0)^3

If fPSA > 11.8, then Spline1_fPSA

= (11.8-0.84)x(0.84-0.25)x(11.8 + 0.84 + 0.25 - 3 x fPSA)

If fPSA < 11.8, then Spline2_fPSA

= -(11.8-0.84)/(11.8-0.25)x(fPSA-0.25)^3+max(fPSA-1.29,0)^3

If fPSA > 11.8, then Spline2_fPSA

= (11.8-1.29)x(1.29-0.25)x(11.8 + 1.29 + 0.25 - 3 x fPSA)

For lab models :

stipulates:

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 = 3.968052 x iPSA

x4 = 4.508231 x hK2

therefore:

L = -1.735529 + 0.0172287 x age + x1 + x2 + x3 + x4

Prostate cancer risk = exp(L)/[1 + exp(L)]

This presents a risk of prostate cancer without any clinical information. If this risk is high, it is assumed that the clinician will recommend that the patient undergo a clinical workup and a digital rectal examination. The following model is then run twice to provide risks depending on whether the DRE is normal or abnormal, respectively, where the DRE is coded as 0 or 1.

stipulates:

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

Therefore, the risk of a positive DRE is:

L = -1.373544 + 0.9661025 + 0.0070077 x age + x1 + x2 + x3 + x4

About DRE negatives:

L = -1.373544 + 0.0070077 x age + x1 + x2 + x3 + x4

Determining the risk as follows:

Risk of prostate cancer: exp(L)/[1 + exp(L)]

For recalibration :

Recalibration may be used for men whose previous biopsies were negative, but recalibration may be used in other situations where event rates differ significantly from the event rates observed in the Rotterdam population (not previously screened) (29%). can be used

stipulates:

odds_cancer = Pr(cancer)/(1-(Pr(cancer))

odds_prediction = predicted cancer risk/(1 - predicted cancer risk) Therefore:

bayes_factor = odds_cancer/odds_prediction

y_adj = y + log(Bayes factor)

Risk of recorrected prostate cancer = exp(y_adj)/[1 + exp(y_adj)]

Example 2 (prediction)

This is a predictive example describing the use of a cassette and analyzer to electrolessly deposit silver onto gold particles associated with the sample to perform an assay to detect iPSA, fPSA, tPSA and hK2 in the sample. 14 includes a schematic illustration of a microfluidic system 1500 of a cassette used in this example. The cassette had a shape similar to the cassette 520 shown in FIG. 7 .

The microfluidic system included analysis regions 1510A-1510F, a waste receiving region 1512 and an outlet 1514 . The assay regions contain microfluidic channels 50 microns deep and 120 microns wide with a total length of 175 mm. The microfluidic system also included a microfluidic channel 1516 and channel branches 1518 and 1520 (having inlets 1519 and 1521 respectively). Channel branches 1518 and 1520 were 350 microns deep and 500 microns wide. Channel 1516 is 350 microns deep and 500 microns wide and is formed of sub-channels 1515 located on alternating sides of the cassette, which sub-channels have through holes having a diameter of about 500 microns ( 1517). Although FIG. 14 shows reagents stored on a single side of the cassette, in other embodiments, reagents were stored on both sides of the cassette. Channel 1516 had a total length of 390 mm and branches 1518 and 1520 were each 360 mm long. Prior to sealing the channels, anti-PSA and anti-hK2 capture antibodies were attached to the surfaces of the microfluidic system in segments of analysis regions 1510 and 1511 , as described in more detail below.

Prior to first use, the microfluidic system was loaded with liquid reagents stored in the cassette. Seven wash plugs 1523-1529 in series (water or buffer, each about 2 microliters) were loaded using a pipette into sub-channels 1515 of channel 1516 using through holes. . Each of the cleaning plugs was separated by plugs of air. Fluid 1528 containing a solution of silver salt was loaded into branching channel 1520 through port 1519 using a pipette. Fluid 1530 containing reducing solution was loaded into branching channel 1520 through port 1521 . Each of the solutions shown was separated from the other liquids by plugs of air. Ports 1514, 1519, 1521, 1536, 1539 and 1540 were sealed with adhesive tape that could be easily removed or pierced. As such, the liquids were stored in the microfluidic system prior to first use.

Upon first use, ports 1514 , 1519 , 1521 , 1536 , 1538 and 1540 were unsealed by the user peeling off the tape covering the openings of the ports. A tube 1544 containing colloidal gold labeled lyophilized anti-PSA and anti-hK2 antibodies and to which 10 microliters of sample blood 1522 was added was connected to ports 1539 and 1540. The tube was part of a fluid connector having the shape and configuration shown in FIG. 7 . This tubing provided a fluid connection between the assay region 1510 and the channel 1516, which without the tube would cause the assay region 1510 and the channel 1516 to be disconnected and not in fluid communication with each other prior to first use.

The cassette containing the microfluidic system 1500 was inserted into the opening of the analyzer. The housing of the analyzer included an arm positioned within the housing configured to engage the cam-shaped face on the cassette. The arm extends at least partially into the opening in the housing such that when the cassette is inserted into the opening, the arm is pushed out of the opening into the second position so that the cassette enters the opening. Once the arm engaged the inwardly cammed face of the cassette, the cassette was positioned and held within the housing of the analyzer and the bias of the spring prevented the cassette from being pushed out of the analyzer. The analyzer detects the insertion of the cassette by means of a position sensor.

An identification reader (RFID reader) located within the housing of the analyzer was used to read an RFID tag on a cassette containing product identification information. The analyzer used this identifier to match product information stored in the analyzer (eg, calibration information, expiration date of the cassette, verification that the cassette is new, and the type of assay/assessment to be performed within the cassette). The user was prompted to enter information about the patient (from which the sample was obtained) into the analyzer using the touch screen. After the information on the cassette was verified by the user, the control system initiated the analysis.

The control system included programmed instructions to perform the analysis. To initiate the analysis, a signal was transmitted to electronics that were part of the analyzer and controlled the vacuum system used to provide fluid flow. The manifold with O-rings was pressed against the cassette face by a solenoid. One port on the manifold sealed (by an O-ring) port 1536 of the microfluidic system of the cassette. This port on the manifold was connected to a simple solenoid valve that was opened to atmosphere by a tube. A separate vacuum port on the manifold sealed (by an O-ring) port 1514 of the microfluidic system of the cassette. A vacuum of about -30 kPa was applied to port 1514 . Throughout the assay, there was a substantially constant non-zero pressure drop of about -30 kPa in the channel including the assay region 1510 positioned between ports 1540 and 1514 . Sample 1522 flowed into each of the analysis regions 1510A-1510H in the direction of arrow 538 . As the fluid passed through the analysis regions, the PSA and hK2 proteins in sample 1522 were captured by anti-PSA and anti-hK2 antibodies immobilized on the analysis region walls, as described in more detail below. The sample took about 7-8 minutes to pass through the analysis areas, after which the remaining sample was captured in the waste receiving area 1512 .

Initiation of analysis also included a control system that transmits a signal to adjacently located optical detectors in each of the analysis regions 1510 to initiate detection. Each of the detectors associated with the analysis regions recorded the transmittance of light through the channels of the analysis regions. As the sample passed through each of the analysis regions, peaks were generated. The peaks (and troughs) measured by the detectors are signals that have been transmitted to the control system (or are converted to signals), and the control system converts the measured signals into a reference signal pre-programmed into the control system. values or values were compared. The control system included a set of pre-programmed instructions that provide feedback to the microfluidic system based at least in part on the comparison of signals/values.

In a first assay region 1510 - A of device 1500 of FIG. 14 , the walls of the channel of this assay region are blocked with a blocking protein (bovine serum albumin) prior to first use (eg, prior to sealing the device). became Proteins in the blood sample have little or no attachment to the walls of analysis region 1510-A (except possibly for some non-specific binding that may be washed away). The first analysis area serves as a negative control.

In the second analysis region 1510-B, the walls of the channels of this analysis region are subjected to a predetermined mass of prostate-specific antigen (PSA) prior to first use (eg, prior to sealing the device) to serve as a high or positive control. ) was coated with As the blood sample passes through the second analysis region 1501-B, little or no PSA proteins in the blood bind to the walls of the channel. As the gold conjugated signal antibodies in the sample may not yet bind to PSA in the sample, they may bind to PSA on the walls of the channel to serve as a high or positive control.

In the third analysis region 1501-C, the walls of the channels of this analysis region were coated with a capture antibody, an anti-iPSA antibody that binds to an epitope on the iPSA protein that is different from the bear junction signal antibody. The walls were coated prior to first use (eg, prior to sealing the device). As the blood sample flowed through the fourth analysis region in use, the iPSA proteins in the blood sample bound to the anti-iPSA antibody in a manner proportional to the concentration of these proteins in the blood. As the sample containing the iPSA also contained gold labeled anti-iPSA antibodies that bound to the iPSA, the iPSA captured on the assay region walls formed a sandwiched immune complex.

In the fourth analysis region (1510-D), the walls of the channels of this analysis region were coated with a capture antibody, an anti-fPSA antibody that binds to a different epitope on the fPSA protein than the gold conjugated signal antibody. The walls were coated prior to first use (eg, prior to sealing the device). As blood flowed through the fourth assay 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. As the sample containing fPSA also contained gold labeled anti-fPSA that bound to fPSA, the fPSA captured on the analysis region walls formed a sandwich-type immune complex.

In the fifth analysis region (1510-E), the walls of the channels of this analysis region were coated with a capture antibody, an anti-tPSA antibody that binds to an epitope on the tPSA protein that is different from the gold conjugated signal antibody. The walls were coated prior to first use (eg, prior to sealing the device). As the blood sample flows through the fifth analysis region in use, the tPSA proteins in the blood sample bind to the anti-tPSA antibody in a manner proportional to the concentration of these proteins in the blood. As the sample containing tPSA also contained gold labeled anti-tPSA antibodies that bound tPSA, tPSA captured on the assay region walls formed a sandwiched immune complex.

Although gold labeled anti-iPSA, anti-fPSA and anti-tPSA antibodies may be used, in other embodiments gold labeled anti-PSA antibodies that bind to any PSA protein may be used for detection.

The first, second, third, fourth and fifth analysis regions were formed on a single substrate layer. Sixth (1510-F), seventh (1510-G) and eighth (1510-H) analysis regions were formed on separate substrate layers 1511 .

In the sixth analysis region (1510-F), the walls of the channels of this analysis region were coated with a capture antibody, an anti-hK2 antibody that binds to a different epitope on the hK2 protein than the gold conjugated signal antibody. The walls were coated prior to first use (eg, prior to sealing the device). As blood flowed through the sixth assay 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. As the sample containing hK2 also contained gold labeled anti-hK2 antibodies that bind to hK2, hK2 captured on the assay region walls forms a sandwiched immune complex.

The seventh analysis region 1510 -G may be used as a negative control as described above for the analysis region 1510 -A. The eighth analysis region 1510 -H may be used as a high or positive control as described above for the analysis region 1510 -B.

Optionally, a ninth analysis area (not shown) can be used as a low control. In such an embodiment, the walls of the channel of this assay region may be coated with a predetermined small amount of PSA prior to first use (eg, prior to sealing the device) to serve as a low control. As the sample flows through this analysis region, little or no PSA protein in the sample binds to the walls of the channel. Gold conjugated signal antibodies in the sample can bind PSA on the walls of the channel to serve as a low control.

Washing fluids 1523 - 1529 trail the sample in the direction of arrow 1538 through analysis regions 1510 to waste receiving region 1512 . As wash fluids were passed through the analysis regions, they washed away any remaining unbound sample components. Each wash plug purges the channels of the assay regions, providing progressively more complete cleaning. The final wash fluid 1529 (water) washes away salts that may react with silver salts (eg, chlorine, phosphate, azide).

As shown in the plot illustrated in FIG. 15 , each of the detectors associated with the analysis regions measures a pattern 1620 of peaks and troughs while cleaning fluids are flowing through the analysis regions. The troughs correspond to the cleaning plugs (which are clear liquids and therefore provide maximum light transmission). The peaks between each plug represent the air between each plug of clear liquid. Since the assay included 7 wash plugs, there are 7 troughs and 7 peaks in plot 1600 . The first trough 1622 is generally not as deep as the other troughs 1624 and is therefore not entirely clear as the first cleaning plug often catches the remaining blood cells in the channel.

The last peak of air 1628 is much longer than the previous peaks as there were no wash plugs to follow. When the detector detects the length of this air peak, one or more signals are sent to the control system that compares the length of time of this peak with a preset reference signal or input value having a specific length. If the length of time of the measured peak is long enough compared to the reference signal, the control system sends signals to the electronics controlling the vent valve 1536 to actuate the valve and initiate mixing of the fluids 1528 and 1530. transmit (signals at peak 1628 in air are 1) the intensity of the peak; Note that 2) where this peak is located as a function of time, and/or 3) a signal representing one or more signals indicating that a series of peaks 1620 of a particular intensity have already passed). In this way, the control system uses, for example, the pattern of signals to distinguish the peak 1628 in air from other peaks of long duration, such as the peak 1610 from the sample).

Referring back to FIG. 14 , the solenoid connected to the vent port 1536 by the manifold is closed to initiate mixing. Air enters the device through ports 1519 and 1521 (which are open) as the vacuum is still maintained and no air can enter through vent valve 1536 . This forces both 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 are mixed at the junction of the channels to form an amplification reagent (reacting silver solution) having a viscosity ^{of about 1x10 -3 Pa·s.} The ratio of the volumes of fluids 1528 and 1530 was about 1:1. Amplification reagent continued through the downstream storage channel, through tube 1544 , through assay regions 1510 and then into waste receiving region 1512 . After a set of times (12 seconds), the analyzer reopened the vent valve 1536 to allow air to flow through the vent valve 1536 (instead of the vent ports). This left some reagents in the upstream storage channels 1518 and 1520 on the device. The result is also a single plug of mixed amplification reagents. As a result of the vent valve closing in 12 seconds, the amplification plug is about 50 μL (instead of simple timing, another way to trigger the reopening of the vent valve would be to detect the amplification reagent when it first enters the assay regions). .

Because the mixed amplification reagents are stable for only a few minutes (typically less than 10 minutes), mixing was performed in less than 1 minute prior to use in the assay area 1510 . Since the amplification reagent is a clear liquid, the optical density is lowest when it enters the analysis areas. As the amplification reagent was passed through the assay regions, silver was deposited on the captured silver particles to increase the size of the colloids and thereby amplify the signal (as described above, gold particles were deposited in the low and high positive control assay regions). and PSA and hK2 may be present in the sample, to the extent that they were present in the test assay area). Silver can then be deposited over the already deposited silver, allowing more and more silver to be deposited in the analysis areas. As a result, the deposited silver reduces the transmittance of light through the analysis areas. This decrease in transmitted light is proportional to the amount of silver deposited and may be related to the amount of gold colloids trapped on the channel walls. In regions of analysis where silver is not deposited (eg, negative control or test areas when the sample contains no target protein), there will be no increase in optical density (or this increase will be minimal). In regions of the analysis where silver is predominantly deposited, the slope and ultimate level of the pattern of increasing optical density will be high. The analyzer monitors this pattern of optical density during amplification within the test area to determine the concentration of analyte in the sample. In one version of the test, the pattern is monitored within the first 3 minutes of amplification. The optical density of each of the regions of analysis as a function of time was recorded and shown as curves 1640-1647 in FIG. 14 . These curves corresponded to signals that occurred within the analysis regions. After 3 minutes of amplification, the analyzer stops testing. No more optical measurements are recorded and the manifold is disengaged from the device.

From the curves, values (eg, concentrations) of blood markers (eg, iPSA, tPSA and/or hK2) are determined using a computer (eg, in an analyzer). These values are based at least in part on a logistic regression model ( sent to a processor (in electronic communication with the analyzer) being programmed to evaluate (eg, as described above).

The test results are displayed on the analyzer screen and communicated to a printer, computer, or whatever output the user selects. The user can remove the device from the analyzer and put it away. The sample and all reagents used in the assay remain in the device. The analyzer is prepared for another test.

This predictive example shows that analysis of samples containing iPSA, fPSA, tPSA and hK2 is performed in a single microfluidic system using an analyzer to control fluid flow within the cassette, and from one or more measured signals to modulate fluid flow. Show that this can be done by using feedback. This predictive example also shows that results from such an analysis can be used to determine the probability that a patient has prostate cancer, an indication of an estimated prostate volume, and/or an indication of the likelihood that a prostate cancer biopsy will be positive in the patient.

Claims (16)

An assay region comprising one or more binding partners immobilized to a fixed phase portion therein, wherein the one or more binding partners are binding partners that bind total prostate specific antigen (tPSA), free prostate specific antigen (fPSA) the analysis region selected from the group consisting of a binding partner that binds to, a binding partner that binds an intact prostate specific antigen (iPSA), and a binding partner that binds human kallikrein 2 (hK2);
a detection device configured to detect the presence of at least one analyte in a sample from the analysis region, wherein the at least one analyte is selected from the group consisting of tPSA, fPSA, iPSA and hK2;
a user interface configured to allow a user to enter an age of a person associated with the sample; and
including computer;
the computer,
receive, from the user interface, first information comprising a value for an age of a person associated with the sample;
receive, from the detection device, second information indicative of the presence of at least one analyte in the sample, wherein the second information comprises a value for at least two of fPSA, iPSA, tPSA and hK2;
programmed to process the first information and the second information to determine a probability of an event associated with prostate cancer;
Processing the first information and the second information,
Scaling each of a plurality of values including a value for age and a value for at least two of fPSA, iPSA, tPSA, and hK2 with a different coefficient value to calculate a scaled value and logit evaluating the logistic regression model by summing the scaled values to determine
determining a probability of an event associated with cancer based on the logit;
outputting an indication of the probability of an event associated with prostate cancer;
evaluation analysis system. The method of claim 1,
The second information received from the detection device includes values for fPSA, iPSA, tPSA and hK2, and evaluating the logistic regression model includes values for age and fPSA, iPSA to calculate the scaled value. , comprising scaling the values for each of tPSA and hK2 to different coefficient values.
evaluation analysis system. The method of claim 1,
Outputting an indication of a probability of an event associated with prostate cancer comprises outputting an indication of a likelihood that a prostate cancer biopsy is positive.
evaluation analysis system. The method of claim 1,
The detection device is an optical or luminescent device selected from the group consisting of light transmission, light absorption, light scattering, light reflection, visual techniques, photoluminescence, fluorescence, chemiluminescence, bioluminescence and electrochemiluminescence. using detection technology
evaluation analysis system. The method of claim 1,
wherein the detection device detects chemiluminescence or fluorescence emission, wherein the chemiluminescence or fluorescence emission is indicative of an analyte that is bound to an analyte binding partner.
evaluation analysis system. The method of claim 1,
The detection device is configured to detect a change in the optical signal as a function of time.
evaluation analysis system. The method of claim 1,
The sample may be whole blood, serum or plasma.
evaluation analysis system. The method of claim 1,
wherein the assay region comprises a microfluidic channel having at least one inlet and one outlet.
evaluation analysis system. The method of claim 1,
wherein the at least one binding partner comprises a first binding partner and a second binding partner, wherein the first binding partner and the second binding partner are different
evaluation analysis system. 10. The method of claim 9,
wherein said first binding partner is particularly configured to bind to a first of fPSA, iPSA and tPSA
evaluation analysis system. 11. The method of claim 10,
wherein said second binding partner is particularly configured to bind to a second of fPSA, iPSA and tPSA
evaluation analysis system. 12. The method of claim 11,
wherein said at least one binding partner further comprises a third binding partner particularly configured to bind hK2
evaluation analysis system. The method of claim 1,
An indication of the probability of an event associated with prostate cancer comprising an interpretable scale used to guide a decision as to whether a person is obtaining a biopsy
evaluation analysis system. The method of claim 1,
An indication of the probability of an event associated with prostate cancer includes the determination of a positive biopsy for a high risk cancer or a low risk cancer.
evaluation analysis system. receiving, via the user interface, first information comprising a value for an age of a person associated with the sample;
detecting, by a detection device, second information indicative of the presence of at least one analyte in the sample from an assay region of an assay system comprising one or more binding partners immobilized to a stationary phase portion therein, wherein the second information is indicative of the presence of at least one analyte in the sample; The one or more binding partners include a binding partner that binds total prostate specific antigen (tPSA), a binding partner that binds free prostate specific antigen (fPSA), a binding partner that binds intact prostate specific antigen (iPSA), and human kallikrein 2 (hK2), wherein said at least one analyte is selected from the group consisting of tPSA, fPSA, iPSA, and hK2; and
processing the first information and the second information to determine a probability of an event associated with prostate cancer;
The processing of the first information and the second information comprises:
Scaling each of a plurality of values comprising a value for age and a value for at least two of fPSA, iPSA, tPSA, and hK2 to a different coefficient value to yield a scaled value and determining a logit; evaluating the logistic regression model by summing the scaled values;
determining a probability of an event associated with cancer based on the logit;
outputting an indication of the probability of an event associated with prostate cancer;
Way. A detection device configured to detect the presence of at least one analyte in a sample from an analysis region, said analysis region comprising one or more binding partners immobilized to a stationary phase portion therein, said one or more binding partners comprising total prostate specific antigen ( tPSA), a binding partner that binds free prostate-specific antigen (fPSA), a binding partner that binds intact prostate-specific antigen (iPSA), and a binding partner that binds human kallikrein 2 (hK2) wherein the at least one analyte is selected from the group consisting of tPSA, fPSA, iPSA and hK2;
a user interface configured to allow a user to enter an age of a person associated with the sample; and
A non-transitory computer-readable storage medium encoded with a plurality of instructions;
The plurality of instructions, when executed by a computer,
receiving, via the user interface, first information comprising a value for an age of a person associated with the sample;
receiving, from the detection device, second information indicative of the presence of at least one analyte in the sample, wherein the second information comprises a value for at least two of fPSA, iPSA, tPSA, and hK2; and
processing the first information and the second information to determine a probability of an event associated with prostate cancer;
do the method,
The processing of the first information and the second information comprises:
Scaling each of a plurality of values including a value for age and a value for at least two of fPSA, iPSA, tPSA, and hK2 with a different coefficient value to calculate a scaled value and logit evaluating the logistic regression model by summing the scaled values to determine
determining a probability of an event associated with cancer based on the logit;
outputting an indication of the probability of an event associated with prostate cancer;
evaluation analysis system.

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