METHOD AND SYSTEM FOR IMPROVING ACCURACY OF BIOLOGICAL ASSAY

A method of conducting a biological assay, comprises obtaining data corelative to a temperature of a reagent, mixing the reagent with a sample to provide a mixture, receiving from the mixture a signal indicative of an amount of an analyte in the sample, and correcting the amount based on the obtained data and on a type of the reagent.

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to a biological assay and, more particularly, but not exclusively, to a method and system for improving accuracy of a biological assay, particularly a thermally biased biological assay.

Biological assays are procedures for determining the presence, amount, activity, and/or other properties or characteristics of an analyte in a sample. Immunoassays are procedures that identify and/or measures a specific antigen or antibody in a sample by observing the interaction of the specific antigen or antibody with an antibody or antigen contained in a reagent.

Known are systems that perform biological assays automatically. For example, U.S. Published Application No. 20200290037 discloses a system for analyzing a body liquid. The system comprises a cartridge holder, adapted for receiving a multi-well cartridge device, an internal analyzer system for analyzing the body liquid in an analysis chamber, and a robotic arm system carrying a pipette. The robotic arm system visits the wells of the cartridge device to aspirates their contents into the tip of the pipette, and then visits the analysis chamber at which the content of the tip is analyzed.

Many biological assays that are designed to provide quantitative output, particularly immunoassays, require that the used reagents, and sometimes also the sample, be at a recommended range of temperatures, otherwise the quantitative output in inaccurate.

SUMMARY OF THE INVENTION

According to an aspect of some embodiments of the present invention there is provided a method of conducting a biological assay. The method comprises obtaining data corelative to a temperature of a reagent, mixing the reagent with a sample to provide a mixture, receiving from the mixture a signal indicative of an amount of an analyte in the sample, and correcting the amount based on the obtained data and on a type of the reagent.

According to some embodiments of the invention the method comprises measuring a temperature of the reagent, thereby providing the data.

According to some embodiments of the invention the reagent is contained in a cartridge, and the method comprises measuring a temperature of the cartridge, thereby providing the data.

According to some embodiments of the invention the method comprises measuring thermal changes in an environment encompassing the reagent, thereby providing the data.

According to some embodiments of the invention the reagent is contained in a cartridge being in thermal communication with a heating system having a temperature sensor, and wherein the measuring the thermal changes comprises measuring changes in a signal generated by the sensor.

According to some embodiments of the invention the signal is a digital signal Sm, and wherein the correcting comprises applying to the digital signal a correction function specific to the reagent, to provide a corrected signal Sc.

According to an aspect of some embodiments of the present invention there is provided a system for conducting a biological assay. The system comprises: an analyzer system for receiving a mixture containing a reagent and a sample and generating a signal indicative of an amount of an analyte in the sample; and a data processor having a circuit configured to receive data corelative to a temperature of a reagent, and to correct the amount based on the received data and on a type of the reagent.

According to some embodiments of the invention the reagent is contained in a cartridge, and the system comprises a sensor for measuring thermal changes in an environment encompassing the cartridge, thereby providing the data.

According to some embodiments of the invention the system comprises a heating system having the sensor, wherein the cartridge is in thermal communication with the heating system.

According to some embodiments of the invention the signal is a digital signal Sm, and wherein the processor is configured for applying to the digital signal a correction function specific to the reagent, to provide a corrected signal Sc.

According to some embodiments of the invention the corrected signal Scis within 30% of Sm/f(T,R), wherein T is the data, R is a scaling factor specific to the reagent, and f is a predetermined function of T and R.

According to some embodiments of the invention the predetermined function comprises a linear function.

According to some embodiments of the invention the signal comprises an optical signal.

According to some embodiments of the invention the signal comprises a non-optical signal.

According to an aspect of some embodiments of the present invention there is provided control circuitry for a photomultiplier tube. The control circuitry comprises: a capacitor, connectable to an external power source, for maintaining amplification voltage between an anode and a cathode of the photomultiplier tube; a switching circuit having a gate connected to a voltage feeding circuit, and a discharging channel connected to the capacitor, wherein when the external power source is turned off, the feeding circuit momentarily activates the gate such that the capacitor is discharged via the discharging channel.

According to some embodiments of the invention the switching circuit is a MOSFET.

According to some embodiments of the invention the MOSFET is a silicon carbide MOSFET.

According to some embodiments of the invention the voltage feeding circuit comprises an additional capacitor connected such that when the external power source is turned on, the additional capacitor is charged, and when the external power source is turned off the additional capacitor is discharged, causing the momentary activation of the gate.

According to some embodiments of the invention the additional capacitor is discharged via a channel of a transistor, and wherein the external power source is connected to a gate of the transistor.

According to some embodiments of the invention the discharging is characterized by a time constant of less than 100 ms.

DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

The present invention, in some embodiments thereof, relates to a biological assay and, more particularly, but not exclusively, to a method and system for improving accuracy of a biological assay, particularly a thermally biased biological assay.

It is appreciated that results of quantitative assays, particularly immunoassays, are sensitive to the temperatures of the reagents that are used, and so typical assay kits are accompanied by a recommended temperature range at which the reagents are to be maintained until the assay is executed. For example, when the reagent includes Antibodies (e.g., TRAIL antibodies) and enzymes (e.g., Alkaline-phosphatase enzyme) in a solution (e.g., TRIS buffer), the recommended temperature range is from about 2° C. to about 8° C. or below (e.g., frozen), and when the reagent are dried (e.g. Lyophilized or the like), the recommended temperature range is Room Temperature (e.g. from about 18° C. to about 25° C.).

The inventors found that while it is possible to equip the analyzing system that performs the assay with a temperature control system so as to maintain the reagents at the recommended temperature range within the system, such a configuration is less than optimal because it requires the reagents to be loaded upfront to the analyzing system, thereby increasing the footprint of the analyzing system, and also makes it useful only for assays that use the loaded reagents.

The inventors also found that is inconvenient to maintain the reagent within a separate a temperature control system until immediately before the assay because it poses a limitation on the user.

In some cases, the reagent is stored at a temperature that is outside the recommended temperature range, and the user is requested to extract the reagent from the storage a certain time period before the assay, so as to bring the reagent to the desired temperature, e.g., by allowing it to reach room temperature, or by heating it artificially. The inventors found that this poses a limitation on the user and results in prolonged assay time.

The inventors found a solution to the above problem, and have devised a technique suitable for improving the accuracy of a biological assay even when the assay is thermally biased.

Referring now to the drawings,FIG.1is a flowchart diagram of the method according to various exemplary embodiments of the present invention. It is to be understood that, unless otherwise defined, the operations described hereinbelow can be executed either contemporaneously or sequentially in many combinations or orders of execution. Specifically, the ordering of the flowchart diagrams is not to be considered as limiting. For example, two or more operations, appearing in the following description or in the flowchart diagrams in a particular order, can be executed in a different order (e.g., a reverse order) or substantially contemporaneously. Additionally, several operations described below are optional and may not be executed.

The method can be executed using any system that performs biological assays automatically, such as, but not limited to, the system described in U.S. Published Application No. 20200290037 supra. The method can also be executed by systems that are not fully automatic, for example, systems in which a mixture containing a sample and a reagent is manually introduced to an analysis chamber.

Computation parts of the method can be implemented by computer programs which can commonly be distributed to users on a distribution medium or downloaded from the internet. The computer programs can be run by loading the computer programs into the execution memory of a computer, configuring the computer to act in accordance with the method of this invention. All these operations are well-known to those skilled in the art of computer systems.

Computation parts of the method can be embodied in many forms. For example, they can be embodied in on a tangible medium such as a computer for performing the method steps. It can be embodied on a computer readable medium, comprising computer readable instructions for carrying out the method steps. It can also be embodied in electronic device having digital computer capabilities arranged to run the computer program on the tangible medium or execute the instruction on a computer readable medium. It can be embodied in a computerized controller of a system that performs biological assays automatically, or in a computer readable medium that is accessible by such a computerized controller.

The method begins at10and optionally and preferably continues to11at which data corelative to a temperature of a reagent is obtained.

Antibodies for measuring CRP include monoclonal antibodies for measuring CRP and polyclonal antibodies for measuring CRP.

Antibodies for measuring CRP also include antibodies that were developed to target epitopes from the list comprising of: Human plasma derived CRP, Human serum derived CRP, Mouse myeloma cell line NS0¬derived recombinant human C-Reactive Protein/CRP (Phe17-Pro224 Accession # P02741).

Antibodies for measuring IP-10 include monoclonal antibodies for measuring IP-10 and polyclonal antibodies for measuring IP-10.

Antibodies for measuring IP-10 also include antibodies that were developed to target epitopes from the list comprising of: Recombinant human CXCL10/IP-10, non-glycosylated proteins chain containing 77 amino acids (aa 22-98) and an N-terminal His tag Interferon gamma inducible protein 10 (125 aa long), IP-10 His Tag Human

Recombinant IP-10 produced inE. Colicontaining 77 amino acids fragment (22-98) and having a total molecular mass of 8.5 kDa with an amino-terminal hexahistidine tag, E. coli-derived Human IP-10 (Va122-Pro98) with an N-terminal Met, Human plasma derived IP-10, Human serum derived IP-10, recombinant human IP-10 where first amino acid is between position 1-24 and the last amino acid is at position 71-98.

The temperature of the reagent can be obtained in more than one way. In some embodiments of the present invention the temperature of the reagent is measured directly by a temperature sensor. This can be done, before loading the reagent into the system that performs the biological assays, or, alternatively, one or more temperature sensors can be a component of the system, in which case the temperature of the reagent is measured after the reagent is loaded to the system.

In some embodiments of the invention, the reagent is contained in a cartridge, and the temperature of cartridge is measured. Also contemplated, are embodiments in which the method measures thermal changes in an environment encompassing the reagent. These embodiments are useful when the system that performs the biological assays includes one or more temperature sensors, and the reagent is contained in a cartridge. In this case, the method can obtain signals from the sensor(s) before and after loading the cartridge to the system, thereby obtaining data that is correlative to the temperature of the reagent. For example, when the cartridge including the reagent is stored at a temperature that is below the ambient temperature, once the cartridge is loaded to the system it reduces the temperature of the environment encompassing the cartridge in a manner that is correlative to the temperature of the reagent in the cartridge.

In some embodiments, two or more of the above ways to obtain the data are employed. For example, the data can be obtained both by directly measuring the temperature of the cartridge and by indirectly measuring temperature changes in the environment.

The method continues to12at which the reagent is mixed with a sample to provide a mixture. The mixing can be done in any fluidic device, such as, but not limited to, within a mixing well or within a pipette tip. The mixing can be done automatically, for example, by a robotic arm carrying a pipette that releases the reagent and/or the sample into the same well, or aspirates the reagent and the sample into the same pipette tip.

The method continues to13at which a signal indicative of an amount of an analyte in the sample is received from the mixture. The signal can be any type of signal that is capable of identifying the analyte in the sample. Preferably, the signal is an optical signal (e.g., a chemiluminescent signal or a fluorescent signal) emitted by a substrate that directly or indirectly interacts with the analyte in the sample. Alternatively the signal can be an optical signal transmitted through the mixture, quantifying the absorbance properties of the analyte. The optical signal is indicative of the amount of the analyte in the sample, and it can be detected by an optical detector providing an electrical signal that is also indicative of this amount. Alternatively, the signal can be a non-optical signal, such as, but not limited to, an electrochemical signal, or the like.

The method proceeds to14at which the measured amount of analyte in the sample is corrected based on the obtained data and on the type of reagent. The correction can be applied directly or indirectly to the amount of analyte in the sample. When the correction is applied directly, the amount of analyte in the sample is extracted from the signal measured at13, and the correction is applied to the extracted amount. When the correction can be applied indirectly, the correction is applied to a digital form of the signal measured at13, and the amount of the analyte in the sample is extracted from the corrected signal.

The correction14is preferably applied by accessing a database including a correction function for each of a plurality of reagents or reagent combinations. Alternatively, the method can employ a predetermined correction function, and the database can include a specific set of parameters for the predetermined correction function for each of the plurality of reagents or reagent combinations. The database can be prepared in advance and be stored in a computer readable medium accessible to the method. Utilizing the database, the method can apply the correction function to a digital signal Smto provide a corrected signal Sc. Preferably, the correction is applied irrespectively of the type of signal obtained at13or the procedure employed to obtain the signal.

In some embodiments of the present invention the corrected signal Scis within 30% or within 20% or within 10% of the expression Sm/f(T,R), wherein T is the data obtained at11, R is a scaling factor parameter that is extracted from the database and that is specific to the reagent, and f is a predetermined function of T and R. Preferably, but not necessarily, f comprises a linear function of T and R, but may optionally also includes one or more terms that are non-linear in R and/or T.

For example, in experiments performed by the Inventors, it was found that adequate results are obtained when f(T,R) has the form:

where ΔT is the temperature change that is induced by the reagent on the environment, and ΔTrefis a predetermined reference temperature difference. ΔT can be expressed relative to a temperature measured immediately before using the reagent. For example, when the system that performs the biological assays includes a temperature sensor, ΔT can be the difference between the temperatures measured by the sensor before and after the reagent is loaded to the system. The reference temperature difference ΔTrefcan be global, or assay-specific or, more preferably, specific to the recommended temperature range at which the reagents are to be maintained until the assay is executed. For example, ΔTrefcan be measured in advance and be defined as the value of ΔT when the temperature of the reagent is within the recommended temperature range of the respective reagent (e.g., the central temperature within the recommended temperature range.

In experiments performed by the Inventors using TRAIL, IP10, and CRP antibodies, ΔTrefwas set to the value of 1.3° C. which was found to be the value of ΔT when the temperature of a cartridge containing the reagents was about 8° C. It is to be understood, however, that other value for ΔTref, e.g., from about 1° C. to about 1.6° C., or values that exceed to about 1.6° C. or are below 1.0° C., are also contemplated according to some embodiments of the present invention.

Representative examples of suitable values of the scaling factor parameter R for various reagent combinations are provided in the Examples section that follows. It is appreciated that the correction function can also use additional observables, other than the temperature change ΔT. For example, the correlation function can consider the nominal temperature of the cartridge that contains the reagent, the nominal environmental temperature, the temperature of other components of the system, the temperature of the sample, and the like.

When there is more than one analyte to be detected, each using a different reagent, the method optionally and preferably calculates the correction separately to the reagents using different correlation functions, or different parameters (e.g., different values for the scaling factor parameter R).

The method ends at15.

In some embodiments of the present invention the analyte in the sample is TRAIL, in some embodiments of the present invention the analyte in the sample is CRP, and some embodiments of the present invention the analyte in the sample is IP-10. Reagents for immunoassays capable for measuring amounts of these analytes are found, for example, in U.S. Published Application No. 20200290037, supra.

FIG.2is a schematic illustration of a system100for analyzing a liquid (e.g., body liquid), according to some embodiments of the present invention. System100can be used for executing the method described above with reference toFIG.1. In some embodiments of the present invention system100is a point-of-care (POC) system.

System100comprises a cartridge holder102, adapted for receiving a cartridge device101having a plurality of wells, and an internal analyzer system104, having an analysis chamber106and being configured for analyzing the liquid (e.g., body liquid) when enclosed in analysis chamber106. System100can also comprise a robotic arm system108carrying a pipette110having a disposable tip111. Pipette110can be a controllable air displacement pipette, as known in the art, and tip111can be detachable from pipette110. Preferably, device101can hold one or more disposable tips to be used by system100. System100further comprises a controller112configured for controlling robotic arm system108to establish a relative motion between device10and pipette110such that tip111of pipette110sequentially visits at least cartridge device101and analysis chamber106. Controller112optionally and preferably ensures that pipette110connects to, and picks up, one of the tips111from device110before visiting the wells and, and further ensures that pipette110releases tip111into back into device101, after visiting analysis chamber106. Controller112optionally also configured to control pipette110(e.g., by controlling piston motions within pipette110) to aspirate liquids into tip111and/or dispense liquid out of tip111. Controller112optionally and preferably receives signals from a data processor113. Preferably, but not necessarily, both controller112and data processor113are mounted on the same control board138.

Preferably, analysis chamber106is a dark chamber and internal analyzer system104is an optical analyzer configured for detecting chemiluminescent signals from the pipette tip111when the pipette tip is in dark chamber106. Internal analyzer system104can include an optical detector (not shown) such as, but not limited to, a photomultiplier tube (PMT) mounted on a side wall of chamber106. The optical detector provides a signal that is indicative of the amount of the analyte in the sample as further detailed hereinabove.

System100optionally and preferably comprises a display114for displaying information thereon. For example, display114can receive display instructions from internal analyzer system104to display the results of the analysis performed by internal analyzer system104. In some embodiments of the present invention, system100comprises a reader device136for reading information stored on device101.

In some embodiments of the present invention system100employs an analysis protocol based on the information read by reader device136, for example, by selecting a protocol from a list of protocols recorded on a computer readable medium accessible by data processor113. Alternatively, the list of protocols can be recorded on an external computer readable medium, in which case the information read by reader device136is optionally and preferably transferred over a network to an external computer (not shown), that selects the protocol from the list of protocols and transfers it to system100. The protocol to be run by system100may comprise instructions to controller112to perform the protocol, including but not limited to a particular assay to be run and a detection method to be performed.

In some embodiments of the present invention system100comprises a heating system124. Heating system124can be of any type. The heating system can be configured to heat the cartridge by conduction, radiation and/or convection. In some embodiments of the present invention the heating system heats the cartridge device by conduction. Alternatively, the heating system heats the cartridge device by radiation or convection but without conduction. Preferably, system100also comprises a temperature sensor126. For example, temperature sensor126, can be mounted on or be adjacent to the heating platform of heating system124which contacts device101once loaded to system100.

In use, cartridge device101, with wells filled with reagents and other substances for performing the assay, with sterile disposable tips placed within a dedicated compartment, and with a container containing a sample to be analyzed, is introduced by the operator to holder102. The robotic arm picks up one of tips from the dedicated compartment by way of driving the robotic arm into one of the tips in the dedicated compartment. Information stored on device101is read by reader136. Controller112establishes a relative motion between device101and pipette110such that pipette110such that pipette110aspirates into tip111the liquid to be analyzed and other reagents and substances for the assay. Controller112moves tip111of pipette110into chamber106for analysis by internal analyzer system104, which optionally and preferably uses processor113for the analysis. For example, processor113can receive signals from the optical detector of system104and determine the amount of the target substance in the liquid (e.g., body liquid) based on the intensity of the signals. Data processor113can receive from sensor126data corelative to the temperature of the reagent in device101, and also receive data pertaining to the type of reagents in device101from reader136. Data processor113can then correct the measured amount of the analyte in the sample based on the received data and on the type of reagent.

Once the analysis is completed, controller112establishes a relative motion between device101and pipette110until tip111of pipette110enters the dedicated compartment of device101. Controller112releases tip111of pipette110into the dedicated compartment.

Optionally and preferably, controller112causes robotic arm108to pick up another new pipette tip from the dedicated compartment and performs another assay by repeating the above operations protocol with another set of wells of the same cartridge device101. Processor113can instruct the display114to display the results obtained from one or more of the performed assays.

In embodiments in which the signal that is indicative of the amount of the analyte in the sample is an optical signal detected by a PMT, the PMT is preferably activated only after the pipette tip is fatedly introduced into the dark chamber such as to seal the dark chamber from ambient light. This is because the PMT is designed to operate at low light conditions, and so activating the PMT before the dark chamber is sealed may lead to deterioration in the performance (e.g. increased dark current with hours scale of recovery time), and even permanent damage. For the same reason, the PMT is deactivated before retreating the pipette tip off the dark chamber.

It was found by the Inventors, that convectional PMT remain biased for a prolonged period of time (tens of seconds) after they have been deactivated. The reason for this behavior is that the PMT employs a capacitor between its anode and cathode in order to maintain the necessary amplification voltage (about 1-2 kV) of the PMT. However, for a prolong time after a deactivation of the PMT, the capacitor remains charged, keeping the PMT biased. Such a behavior is disadvantageous because it requires the system to maintain the tip in the dark chamber after the measurement has been completed until the PMT is no longer biased.

The Inventors devised a solution to this problem by providing control circuitry for a PMT.FIG.3is a schematic illustration of control circuitry30for a PMT32. Control circuitry30can be implemented in system100in embodiments in which the internal analyzer system104comprises a PMT.

Circuitry30comprises a capacitor34for maintaining amplification voltage between the anode36and the cathode38of PMT32. Capacitor34is connectable to an external power source (not shown). Circuitry30also comprises a switching circuit42having a gate44connected to a voltage feeding circuit40, and a discharging channel46connected to capacitor34.

Switching circuit42is preferably a transistor, such as, but not limited to, a MOSFET, more preferably a silicon carbide MOSFET. Transistor42is illustrated as an n-channel MOSFET but can also be a p-channel MOSFET. Transistor42is optionally and preferably able to withhold voltage of at least 1 kV more preferably at least 2 kV, sufficiently low leakage current (IDSS), so that it does not load the PMT32. The transistor42is optionally and preferably turned on upon arrival of a pulse of from about 4V to about 5V to its gate44. Representative examples of types of silicon carbide MOSFET suitable for the present embodiments including, without limitation, IXYS-IXTA3N150HT, LSIC1MO170E1000, ROHM-SCT2H12NY, Infineon-IPW90R120C3, ST-STW21N150K5, CREE-C2M0045170P, UnitedSic. In experiments performed by the inventors, Genesic-G3R350MT12J was tested. The leakage current was measured to be less than 0.1 μA at PMT voltage of about 1 kV.

Voltage feeding circuit40is designed and configured such that when the external power source is turned off, feeding circuit40momentarily activates gate44such that capacitor34is discharged via discharging channel46. The discharging of capacitor34is preferably characterized by a time constant of less than 200 ms, more preferably less than 100 ms, more preferably less than 50 ms, less than 30 ms. Circuitry30is therefore advantageous since it provides a fast discharge of the capacitor34and so the PMT becomes unbiased a short time after it is powered off, allowing the system to retreat the pipette tip off the dark chamber shortly after the measurement.

Circuitry30can also comprise a resistor R3which, together with the capacitor34provides the time constant for the discharge. In experiments performed by the inventors, a capacitor34of about 14 nF, and a 200 kΩ resistor R3were used. The time scale for complete discharge was about 3 RC<10 ms. The resistor R3is optionally and preferably sufficiently large in case of a failure (shorted) of the transistor42. A 200 kΩ can create a maximum of 5 mA current and power dissipation of 5 W. The resistor can be rated accordingly.

In a representative implementation voltage feeding circuit40comprises an additional capacitor C1, connected such that when the external power source is turned on, additional capacitor C1is charged, and when the external power source is turned off additional capacitor C1is discharged, causing the momentary activation of gate44. Typically, additional capacitor C1is discharged via a channel of an additional transistor48, that serves as a pulse generator for switching transistor42to its on state. Alternatively, a relay can be used as the pulse generator.

The external power source is preferably connected to the gate50of additional transistor48. In some embodiments of the present invention additional transistor48is a p-channel MOSFET, but other types of pulse generators are also contemplated. As a representative example, when the external power source supplies voltage of Vin(say, 5 V) both the gate and the source voltages of transistor48also equal Vin, and so transistor48also is in cut-off mode and is not conducting. When the external power source is disconnected, the gate voltage of transistor48drops to 0 instantaneously, while the source voltage of transistor48drops remains at Vin, since capacitor C1is still charged. The transistor48is thus turned on and C1is discharged, for example, through another resistor R2. This creates a positive voltage on R2hence also on the gate44of transistor42, thus discharging the capacitor34.

The duration of the pulse that is applied to gate44is determined by the time scale R2·C1. It is optionally and preferably sufficiently long to keep the transistor42conducting for the entire discharge time of the PMT. The value of R2is optionally and preferably selected sufficiently small so as to prevent gating of transistor42by leakage current through transistor48.

In some embodiments of the present invention capacitor C1of circuit40is charged through a diode D2. The forward voltage on the diode D2is optionally and preferably as small as possible for the charging to be as close as possible to the power supply (5V, in the present example). The advantage of diode D2is that it prevents C1from discharging back through the power supply. Alternatively, or additionally, capacitor C1of circuit40is charged through a resistor of sufficiently high resistance. This resistor is optionally and preferably larger than R2(e.g., at least 5 times or 10 times larger) so the discharge of C1is determined by R2and not by this resistor. In some embodiments of the present invention the charging time is less than a predetermined value, which can depend on the application.

It is appreciated that whileFIG.3illustrates an embodiment in which a positive voltage is applied to the anode32, the skilled person, provided by the details presented herein, would be able to modify the circuit to the case in which a negative voltage is applied to the cathode38.

As used herein the term “about” refers to ±10%

The term “consisting of” means “including and limited to”.

EXAMPLES

Thermally Biased Biological Assay

FIG.4shows an example of a thermal bias of biological assay. The biological assay was conducted using MeMed Key® analyzer, marketed by MeMed, Israel. Cartridges containing the reagents were kept at controlled temperature of 2-8° C. The assays were conducted 1.5 min, 5 min, 10 min and 20 min after the cartridges were taken out of the controlled temperature. As shown, longer delays of the assay (corresponding to higher cartridge temperature) result in higher relative light unit outputs, which are converted to a higher assessed concentration.

FIG.5shows the measured output of the temperature sensor126of the heater block124(seeFIG.2), as a function of time since cartridge loading, in seconds. Since the cartridge contacts the heater block124there is a change upon loading of the cartridge to the system. The difference ΔT between the maximal measured temperature and the minimal measured temperature (represented as solid lines) was calculated and used for constructing the correction function.

FIG.6shows a correlation obtained between the change in the heater block temperature sensor and the cartridge temperature. A linear correlation is observed. The data demonstrates a correlation relation between ΔT and the cartridge temperature. The reference temperature difference ΔTrefwas selected to be the value of ΔT when the cartridge's temperature was about 8° C. As shown the obtained value of ΔTrefwas about 1.3° C.

FIG.7shows relation between output of the PMT in Relative Light Units (RLU) and ΔT (seeFIG.5). The dots represent RLU measured for CRP with a specific cartridge lot and a specific sample. That is to say, the dots represent CRP RLUs measured with the same type of samples and the same cartridge lot, and therefore the differences in RLUs are a result of the cartridge temperature change.

FIG.8shows the results of about 140 measurements of CRP output signal with two types of samples (named CAL1 and CAL3). Solid bar represents the precision obtained across two different cartridge lots when a correction for cartridge temperature is not employed. Hatched bar represents the precision obtained across two different cartridge lots when a correction for cartridge temperature was employed. A significant reduction (of roughly 50%) in the assay precision was obtained.

Table 1, below lists representative examples for values of the scaling factor parameter R, for assays performed serially using the cartridges. The first row of Table 1 corresponds to a quality control assay performed using an IP-10 substrate immediately after loading the cartridge, the second row of Table 1 corresponds to a quality control assay performed using a TRAIL substrate after completing the assay of the first row, the third row of Table 1 corresponds to an assay performed after completing the assay of the second row using a TRAIL substrate once conjugated to detect TRAIL levels, the fourth row of Table 1 corresponds to an assay performed after completing the assay of the third row using a CRP substrate once conjugated to detect CRP levels. The fifth row of Table 1 corresponds to an internal control assay performed using a TRAIL substrate.

Fast Discharge of a Photomultiplier Tube

Computer simulations of the time dependence of the PMT voltage provided using the circuitry30ofFIG.3, are shown inFIG.9. Shown are three waveforms representing the input Vin, the voltage at gate44, and the voltage on PMT32. The solid waveform starting from 1 kV in time t=0 s is the voltage on PMT32. The dash-dot waveform starting from 5V at time t=0 s is the input voltage Vin (multiplied by 100). The dashed waveform starting from V=0V is the voltage at gate44(multiplied by 100).

At time t=1 s the dash-dot waveform goes from 5V to 0V resembling the input low voltage turn-off time. At the same time, the dashed waveform goes from 0V to 5V switching the transistor46. At the same time, the solid waveform goes from 1 kV to about 150V on behalf of discharging the capacitor34.