Patent Description:
Lateral flow immunoassays, or lateral flow tests (LFT), are medical tests used to detect the presence or absence of an analyte of interest in a sample of biological fluid obtained from a patient. The lateral flow test comprise a cassette that has a window through which a test strip arranged inside the cassette is observed, the test strip having one or more test lines and one control line. The cassette has a well where the sample to be analyzed is deposited, after which the sample is diffused by capillarity along the test strip, and once a reaction time has elapsed (which can range between <NUM> and <NUM> minutes) the results in the test strip are obtained and can be viewed through the window of the cassette. When the sample from the patient contains the analyte of interest, the test line is colored in the test strip, with the test being positive, and if the sample does not contain the analyte, the test line is not colored, with the test being negative. The control line must always be colored in the test strip after depositing the sample, otherwise the test is not valid.

The results of a lateral flow test can be read with the naked eye, or they can be read by means of a reader that performs an automatic reading. The reader usually has optical means, such as a light emitter that illuminates the test strip and a photodiode that receives the light reflected by the test strip to generate a result, such as "positive" or "negative". Lateral flow tests can be used for various types of testing, such as pregnancy detection, detection of antigens indicating virus infection, detection of disease biomarkers, metabolites and other molecular targets, as well as detection of animal diseases, chemicals, toxins, water pollutants, among others.

In laboratories it is common to use readers that perform accurate automatic readings, such as the readers described in <CIT> or <CIT>. These readers have a motor for controlled introduction of the lateral flow test into a reader housing where an optical unit with a light emitter and a sensor is arranged to obtain an accurate reading of the test, and the result is reproduced on a digital display. Although these motorized readers allow accurate readings to be obtained, they are expensive equipment and the patient generally has to wait approximately <NUM> to <NUM> hours for the sample to be sent to the laboratory and processed for the result to be known.

The worldwide pandemic caused by the SARS-CoV-<NUM> virus has popularized the use of lateral flow tests for self-diagnosis and has generated the need for tests on the market that allow results to be obtained in the shortest possible time and with the highest possible accuracy rate. In this regard, <CIT> shows a reader for a lateral flow test that can be used directly by a patient and that does not require a motor to introduce the test, but rather the test is introduced manually.

Namely, <CIT> shows a reading method of a lateral flow test that uses a cassette and a reader that has a housing for receiving the cassette. The cassette has a window through which a test strip arranged inside the cassette is observed, the test strip having a reactive zone with test lines and one control line. The reader has an optical unit with a light emitter for illuminating the cassette and a photodiode for receiving the light reflected by the cassette. The cassette is manually moved in the housing of the reader and an output signal comprising luminous intensity values of the light reflected by the cassette during the time that the cassette moves is obtained with the sensor. After obtaining the output signal, the luminous intensity values of the output signal are normalized in a range defined between a lower value and a higher value, then peaks in the output signal corresponding with the test and control lines are identified, and lastly the luminous intensity of the peaks is quantified. The concentration of the analyte of interest is related to the light reflected in the test line, and the higher the concentration is, the greater the intensity (color) of the test line is.

The reader has a tray in which is arranged the cassette with the test strip, and the tray is introduced inside the housing of the reader in which the light emitter and the photodiode are located. The tray has a calibration pattern with several parallel lines the darkness of which gradually varies from the lower value to the higher value, for example, on a gray scale. Every time a reading is performed, first, the photodiode receives the light reflected by the lines of the calibration pattern, and then it receives the light reflected by the test strip. In this way, every time a test strip reading is performed, the luminous intensity values of the light reflected by the test strip are normalized with the luminous intensity values of the light reflected by the lines of the calibration pattern which is arranged on the tray. In this sense, the readings of the photodiode are corrected due to non-linearity in the optics or electronics of the reader. In other words, the coordinates of the Y axis of the signal obtained with the photodiode are normalized with the calibration pattern.

In addition, the reader has an optical position encoder in the tray which is used to relate the time readings obtained by the photodiode to linear positions of the tray. In this way, each luminous intensity value of the light reflected by the test strip corresponds to a known linear position of the test strip. Thus, the reader can recognize the linear positions of the photodiode signal where the peaks corresponding to the test and control lines are expected to be, and therefore, the photodiode readings are independent of the speed at which the user removes the tray from the reader. In other words, with the optical encoder the X axis coordinates of the photodiode output signal are normalized.

The object of the invention is to provide a reading method of a lateral flow test, as defined in the claims.

The invention relates to a reading method of a lateral flow test comprising:.

In the normalization, the lower value and the higher value are established between the light reflected, or emitted, by the cassette outside the window, and the light reflected, or emitted, by the test strip outside the reactive zone. The lower value corresponds with the light reflected, or emitted, by the cassette outside the window, and the higher value corresponds with the light reflected, or emitted, by the test strip outside the reactive zone. The cassette has a different color than the test strip which causes the luminous intensity of the light reflected, or emitted, by the cassette outside the window to be different from the luminous intensity of the light reflected, or emitted, by the test strip outside the reactive zone.

In this way, only information about the light reflected, or emitted, by the cassette that has the test strip is used to normalize the luminous intensity values of the output signal of the sensor. In other words, the method does not require using an additional calibration pattern to correct possible failures in the output signal of the sensor, for example, failures due to non-linearity in the optics or electronics of the reader. For example, <CIT> uses a tray to place the cassette that has the test strip, and said tray has a known calibration pattern with several parallel lines on a gray scale. Every time the test strip of a cassette is read, the calibration pattern of the tray must be read first to normalize the luminous intensity values of the test strip reading, establishing a relationship between the calibration pattern reading and the test strip reading. The method proposed by the invention does not require using a tray with a calibration pattern for normalizing, since it uses known information about the light reflected, or emitted, by the cassette itself inside and outside the window where the test strip is located. The cassette outside the window and the test strip outside the reactive zone are known, and therefore information about the light reflected in those zones is used to establish the upper and lower values with which the output signal of the sensor is normalized. All the luminous intensity values of the light reflected, or emitted, by the cassette with the test strip are established proportionally according to said upper and lower values.

These and other advantages and feature of the invention will become apparent in view of the figures and detailed description of the invention.

The invention relates to a reading method of a lateral flow test <NUM> which is used to detect the presence or the absence of an analyte in a liquid sample from a patient. The lateral flow test <NUM> is read automatically by means of a reader <NUM>.

The lateral flow test may be used in different biological testing scenarios; by way of nonlimiting example, they may be used for the detection of bacteria or viruses, pregnancy or fertility, heart infections, diabetes, the detection of cancer biomarkers, or therapeutic drug monitoring (TDM).

The lateral flow test <NUM> comprises a cassette <NUM> that has a window <NUM> through which a test strip <NUM> arranged inside the cassette <NUM> is observed. The test strip <NUM> has a reactive zone RZ with at least one test line T and one control line C. The cassette <NUM> has a well <NUM> in which the liquid sample from the patient is deposited.

The test strip <NUM> comprises membranes <NUM>, <NUM>, <NUM> and <NUM> that are superimposed on one another and adhered on a lower support <NUM>. The membranes allow the liquid sample from the patient to diffuse by capillarity. The membranes comprise a sample pad <NUM>, a conjugate pad <NUM>, a porous membrane <NUM>, and an absorbent pad <NUM>.

The liquid sample is applied in the well <NUM> which is directly communicated with the sample pad <NUM>, which ensures that the analyte present in the sample is capable of binding to the capture reagents for capturing the conjugates arranged in the porous membrane <NUM>. The treated sample migrates through the conjugate pad <NUM>, which contains antibodies specific to the target analyte and conjugate with colored or fluorescent particles, (commonly latex or colloidal gold microspheres). The sample, together with the conjugated antibody bound to the target analyte, migrates to the porous membrane <NUM> (normally made up of nitrocellulose) with specific biological components (mainly antibodies or antigens) immobilized on a test line T which is oriented perpendicular to the longitudinal axis of the test strip <NUM>. Their function is to react with the analyte bound to the conjugated antibody. Recognition of the analyte of the sample results in a coloring of the test line T, whereas a response in the control line C indicates that a suitable amount of liquid flow has passed through the entire test strip <NUM>. Finally, the absorbent pad <NUM> absorbs the excess reagents and prevents reflux of the liquid.

The reading, represented by the coloring of the test line T and control line C, which are colored with different intensities, can be evaluated with the naked eye or by using the reader <NUM>. In some cases an ultraviolet light is required to visualize the coloring of the lines. The reader <NUM> can correlate the color intensity of the test line T with the concentration of the analyte in the liquid sample from the patient.

The reader <NUM> has a housing <NUM> for receiving the cassette <NUM>. The reader <NUM> has an optical unit <NUM> with a light emitter <NUM> for illuminating the cassette <NUM> and a sensor <NUM> for receiving the light reflected, or emitted, by the cassette <NUM>.

The cassette <NUM> may fit snugly moved in the housing <NUM> of the reader <NUM>, and the housing <NUM> may be opaque so that the reading of the sensor <NUM> is not affected by outside light. The reader <NUM> comprises a base <NUM> that is closed in the upper part with a cover <NUM>, and the housing <NUM> for receiving the cassette <NUM> is defined between the base <NUM> and the cover <NUM>. An electronics board <NUM> is also arranged between the base <NUM> and the cover <NUM>.

According to one embodiment, the light emitter <NUM> emits visible light on the cassette <NUM> (including the test strip <NUM>), and the sensor <NUM> receives the light directly reflected by the cassette <NUM>. In this way, the reader <NUM> may be used with a lateral flow test which works according to the principle of reflectance (colorimetry). When visible light is applied on the control and test lines, the light reflected is reduced due to the particles present in the lines that absorb light at a certain wavelength. The particles can be colloidal gold particles or particles of another type initially present in the conjugate pad <NUM>, such as latex particles, carbon nanoparticles, etc..

According to another embodiment, the light emitter <NUM> emits UV light which is absorbed by the cassette <NUM> (including the test strip <NUM>), and the sensor <NUM> receives visible light emitted by the cassette <NUM>, as a contrast to the UV light. In this way, the reader <NUM> may be used with a lateral flow test which works according to the principle of luminescence. The control line C and test line T of the test strip <NUM> contain fluorescent particles which, upon receiving UV light, absorb UV light and in turn emit a visible light which is captured by the sensor <NUM>.

The reading method of the lateral flow test <NUM> comprises using the cassette <NUM> that has the test strip <NUM> with the test line T and the control line C and using the reader <NUM> that has the optical unit <NUM> with the light emitter <NUM> for illuminating the cassette <NUM> and the sensor <NUM> for receiving the light reflected, or emitted, by the cassette <NUM>.

<FIG> shows a flow chart of the steps of the reading method. In a first step <NUM>, after obtaining the sample from the patient and depositing it in the well <NUM> of the cassette <NUM>, the cassette <NUM> is manually moved in the housing <NUM> of the reader <NUM>. The light emitter <NUM> of the optical unit <NUM> sends light which illuminates the cassette <NUM> and the test strip <NUM> during the movement of the cassette <NUM>, and the sensor <NUM> of the optical unit <NUM> receives the light reflected, or emitted, by the cassette <NUM> and the test strip <NUM>. The light reflected, or emitted, by the cassette <NUM> and the test strip <NUM> is related to the light sent by the light emitter <NUM>.

In a second step <NUM>, an output signal S comprising luminous intensity values of the light reflected by the cassette <NUM> during the time that the cassette <NUM> moves is obtained with the sensor <NUM>. The output of the sensor <NUM> represents raw data from the reading of the sensor <NUM>. The output signal S represents luminous intensity values "Iv" measured by the sensor <NUM> during the time "t" that the cassette <NUM> is manually moved. The Y axis of the signal shows luminous intensity values "Iv" and the X axis shows time "t".

The cassette <NUM> is manually moved between a first position in which the window <NUM> of the cassette <NUM> is inside the housing <NUM> of the reader <NUM> and a second position in which the window <NUM> of the cassette <NUM> is outside the housing <NUM> of the reader <NUM>. Preferably, the signal S is obtained during the movement of the cassette <NUM> between the first and the second position, i.e., during the removal of the cassette <NUM> from the housing. Alternatively, the signal S may be obtained during the movement of the cassette <NUM> between the second and the first position, i.e., during the introduction of the cassette <NUM> into the housing. Alternatively, two signals may be obtained, one during the introduction and the other during the removal, with one of the signals being used as a redundant signal.

In a third step <NUM>, the luminous intensity values of the output signal S are normalized in a range R defined between a lower value and a higher value. The raw readings of the sensor <NUM> are normalized in the range R defined between said lower value and higher value so that all the readings performed with the sensor <NUM> are represented on a common scale and are comparable with other readings. For example, normalization allows the correction of possible failures due to non-linearity in the optics or electronics of the reader. The Y axis of the signal shows adimensional values in the range R and the X axis shows time "t". The X coordinates of the output signal S may be discrete readings of the sensor captured every <NUM>.

In a fourth step <NUM>, peaks in the output signal S corresponding with the test line T and the control line C are identified. The identification of the peaks allows detection of the presence of an analyte in the test line T, and therefore detection of the test being 'positive', and also allows detection of the presence of the control line, and therefore detection of the test being 'valid'.

In an optional fifth step <NUM>, after identifying the peaks in the output signal S, the luminous intensity of the peak of the test line T is quantified, with the luminous intensity being related to the concentration of an analyte present in the test line T. Generally, the control line C usually exhibits a similar luminous intensity when the test is valid, but the test line T usually exhibits a different luminous intensity depending on the concentration of the analyte present in the test line T.

According to the invention, in the normalization of the third step <NUM>, the lower value and the higher value are established between the light reflected, or emitted, by the cassette <NUM> outside the window <NUM>, and the light reflected, or emitted, by the test strip <NUM> outside the reactive zone RZ. Preferably, in the normalization of the third step <NUM>, the lower value corresponds with the light reflected, or emitted, by the cassette <NUM> outside the window <NUM>, and the higher value corresponds with the light reflected, or emitted, by the test strip <NUM> outside the reactive zone RZ.

Thus, the luminous intensity values of the output signal S of the sensor <NUM> are normalized (scaled) in the range R defined between said lower value and said higher value. All the luminous intensity values of the light reflected, or emitted, by the cassette <NUM> and the test strip <NUM> are established proportionally according to said upper and lower values.

The cassette <NUM> is a known element, since the color and the type of material used for manufacturing same are known, so the luminous intensity value of the light reflected by the cassette <NUM> outside the window <NUM> is known. Moreover, the test strip <NUM> is also a known element, since its color (white) and the material used for manufacturing same are known, so the luminous intensity value of the light reflected by the test strip <NUM> is also known. In this way, the actual elements making up the cassette for carrying out the normalization of the values on a known common scale are utilized.

Preferably, the cassette <NUM> has a different color than the test strip <NUM> which causes the luminous intensity of the light reflected, or emitted, by the cassette <NUM> outside the window <NUM> to be different from the luminous intensity of the light reflected, or emitted, by the test strip <NUM> outside the reactive zone RZ. For example, the test strip <NUM> is made of nitrocellulose and has a white color different from the color of the cassette <NUM>, which causes the light reflected by the test strip <NUM> and the cassette <NUM> to be different and said information may be used to normalize the output signal S.

Even more preferably, the cassette <NUM> has a black color and the test strip <NUM> a white color. In this way, the difference between the light reflected, or emitted, by both elements is maximized. The white color of the test strip <NUM> absorbs virtually none of the light sent by the emitter <NUM>, whereas the black color of the cassette <NUM> absorbs virtually all the light. In other words, the white color of the test strip <NUM> reflects virtually all the light, whereas the black color of the cassette <NUM> reflects virtually no light.

In the fourth step <NUM>, luminous intensity variations in the output signal S of the sensor <NUM> are identified, and each of said variations may be a peak corresponding with a test line T or a control line C.

In the fifth step <NUM>, after identifying the peaks in the output signal S the luminous intensity of the peaks of the test line T may be quantified, with the luminous intensity being related to the concentration of an analyte which is present in the test line T. For example, data may be stored in a memory with previous tests measured with the reading method of the invention and in which the luminous intensity values measured with the sensor <NUM> correlate to the concentration of the analyte in the test line T.

The sensor <NUM> may be a photodiode. <FIG> shows raw data of the output signal S of a sensor in a single wavelength, which may have been obtained with a photodiode, and <FIG> shows said signal after normalization.

Preferably, the sensor <NUM> is a spectrometric sensor which obtains an output signal in channels, and each channel corresponds with the light reflected, or emitted, by the cassette in a certain wavelength. <FIG> shows raw data of the output signal S of a spectrometric sensor in several wavelengths and <FIG> shows the signal S of <FIG> after normalization. The spectrometric sensor has several channels, for example <NUM>, but for the sake of clarity only two channels S1 and Sn of the output signal S are represented.

In <FIG> and <FIG>, the Y axis is represented in luminous intensity values "Iv", and in <FIG> and <FIG>, the Y axis is adimensional, whereas the X axis is represented in all the figures in values of time "t".

As shown in <FIG> and <FIG>, in the normalization of the output signal S a lower curve B and an upper curve W are obtained, and the range R is defined between the lower curve B and the upper curve W.

<FIG> shows a correspondence between the raw data of the output signal S of the sensor and the cassette <NUM> that has the test strip <NUM>. At least four points b_s, w_s, b_e and w_e are identified in said output signal S. The lower curve B is defined with at least one first point b_s and one second point b_e. The first point b_s corresponds with the light reflected, or emitted, by the cassette <NUM> at the start of the window <NUM> and the second point b_e corresponds with the light reflected, or emitted, by the cassette <NUM> at the end of the window <NUM>. The upper curve W is defined with at least one third point w_s and one fourth point w_e. The third point w_s corresponds with the light reflected, or emitted, by the test strip <NUM> at the start of the window <NUM> and the fourth point w_e corresponds with the light reflected, or emitted, by the test strip at the end of the window <NUM>. The points are identified by solving an optimization problem. The start and the end of the window <NUM> are the transitions existing between the cassette <NUM> and the test strip <NUM>.

As observed in <FIG>, the range R is defined between the lower curve B and the upper curve W. The lower curve B and upper curve W are two straight lines. The value '<NUM>' may be assigned to the lower curve and the value '<NUM>' may be assigned to the upper curve W. In the normalization, the raw data of the output signal S of the sensor is established proportionally between said curves lower B and upper W.

As shown in <FIG>, when a spectrometric sensor is used the output signal S is represented in several channels S1 to Sn, and each channel corresponds with a wavelength. Said wavelengths may correspond with the wavelengths of the colors in the visible spectrum. For the sake of clarity, <FIG> and <FIG> show only two channels S1 and Sn.

A lower curve B1 and Bn and an upper curve W1 and Wn are obtained for each channel S1 and Sn. Each curve is obtained in the same way as described in <FIG>. The lower curve B1 is defined with at least one first point b_s1 and one second point b_e1. The lower curve Bn is defined with at least one first point b_sn and one second point b_en. The upper curve W1 is defined with at least one third point w_s1 and one fourth point w_e1. The upper curve Wn is defined with at least one third point w_sn and one fourth point w_en.

As shown in <FIG>, in the normalization, the lower curves B1 and Bn of the channels S1 and Sn are superimposed on a main lower curve BM, and the upper curves W1 and Wn of the channels S1 and Sn are superimposed on a main upper curve WM, with the range R being defined between the main lower curve BM and the main upper curve WM, and with the luminous intensity values of the light of each channel S1 and Sn being established proportionally between the main lower curve BM and the main upper curve WM.

As is also observed in <FIG>, the curves are straight lines. The value '<NUM>' may be assigned to the main lower curve BM and the value '<NUM>' may be assigned to the main upper curve WM. In the normalization, the raw data of all the channels S1 and Sn of the output signal S of the sensor <NUM> is established proportionally between said curves BM and WM.

Luminous intensity variations between the channels S1 and Sn are identified for each instant in time of the output signal S of the sensor <NUM>, and each of said variations is a peak corresponding with a test line T or a control line C. As observed in <FIG>, the channels S1 and Sn of the output signal S are superimposed at all the points except in the reactive zone RZ corresponding to the peaks T1, Tn, C1, and Cn of the test line T and control line C. It can be seen that the light reflected, or emitted, by the test line T and control line C show luminous intensity values different in each channel of the spectrometric sensor, whereas the light reflected, or emitted, by the test strip <NUM> outside the reactive zone RZ, or the light reflected, or emitted, by the cassette <NUM> outside the window, shows the same luminous intensity values in all the channels. In this way, luminous intensity variations that do not correspond to a test or control lines, such as defects in the test strip <NUM>, for example, can be discriminated.

Claim 1:
Reading method of a lateral flow test comprising:
- using a cassette (<NUM>) that has a window (<NUM>) through which a test strip (<NUM>) arranged in the cassette (<NUM>) is observed, the test strip (<NUM>) having a reactive zone (RZ) with at least one test line (T) and one control line (C),
- using a reader (<NUM>) that has a housing (<NUM>) for receiving the cassette (<NUM>), the reader (<NUM>) having an optical unit (<NUM>) with a light emitter (<NUM>) for illuminating the cassette (<NUM>) and a sensor (<NUM>) for receiving the light reflected, or emitted, by the cassette (<NUM>),
- manually moving the cassette (<NUM>) in the housing (<NUM>) of the reader (<NUM>),
- obtaining with the sensor (<NUM>) an output signal (S) comprising luminous intensity values of the light reflected, or emitted, by the cassette (<NUM>) during the time that the cassette (<NUM>) moves,
- normalizing the luminous intensity values of the output signal (S) of the sensor (<NUM>) in a range (R) defined between a lower value and a higher value, and
- identifying peaks in the output signal (S) of the sensor (<NUM>), said peaks corresponding with the test line (T) and the control line (C),
characterized in that the lower value and the higher value are established between the light reflected, or emitted, by the cassette (<NUM>) outside the window (<NUM>), and the light reflected, or emitted, by the test strip (<NUM>) outside the reactive zone (RZ), wherein the lower value corresponds with the light reflected, or emitted, by the cassette (<NUM>) outside the window (<NUM>), and the higher value corresponds with the light reflected, or emitted, by the test strip (<NUM>) outside the reactive zone (RZ), and wherein the cassette (<NUM>) has a different color than the test strip (<NUM>) which causes the luminous intensity of the light reflected, or emitted, by the cassette (<NUM>) outside the window (<NUM>) to be different from the luminous intensity of the light reflected, or emitted, by the test strip (<NUM>) outside the reactive zone (RZ).