A method for correcting variations in fluorescent dyes by measuring fluorescence in a free state.

Abstract

The present invention relates to a method for calculating a correction factor to correct for variations in the fluorescence emission intensity of a reagent containing a fluorescent dye, wherein the correction factor is intended to be applied in the process of measuring the fluorescence emission intensity in a bound state when cells are labeled with the fluorescent dye, and the method comprises at least the following steps: - A step of measuring the free-state fluorescence emission intensity emitted from the reagent containing the fluorescent dye when the cells are not labeled before measuring the fluorescence emission intensity in the bound state, - The present invention relates to a method that includes the step of calculating the correction coefficient from the measured free-state fluorescence emission intensity.

Description

[Technical Field]

[0001] This invention relates to monitoring the fluorescence emission intensity of reagents containing fluorescent dyes used in flow cytometry. Hereinafter, "fluorescence" refers to luminescence radiant intensity or fluorescence emission radiant intensity.

[0002] Flow cytometry is a technique for characterizing blood cells in vitro. Inside a flow cytometer, blood cells flow through a series of transducers, which measure various parameters of the blood cells.

[0003] The measured parameters may depend on fundamental, inherent characteristics of blood cells, such as cell size, membrane roughness, nucleolus complexity, or internal particle size.

[0004] The use of fluorescent markers is essential for the sophisticated characterization of blood cells. Such fluorescent markers can be selected to exhibit high specificity to the target blood cell characteristics, such as the nucleic acid content. Fluorescent probes re-emit fluorescence when excited, i.e., when irradiated at a specific wavelength, making it possible to confirm and quantify the characteristic properties of the target blood cells.

[0005] Fluorescent dyes have been used in hematology for many years because they are the most accurate means of characterizing blood cells.

[0006] In the early 1900s, the first use of fluorophores in biological research was for staining tissues, bacteria, and other pathogens. This later developed into fluorescence microscopy by Carl Zeiss and Carl Reichert. In the early 1940s, fluorescent labeling was realized by Ellinger and Hirt.

[0007] The use of fluorescent dyes (acridine orange) in hematology was reported by Vander in 1963 for the characterization and counting of reticulocytes using a visual microscope.

[0008] Patent Document 1 (Katmensky) discloses pioneering research on the characterization of leukocytes by fluorescence flow cytometry. Patent Document 2 (Kleinerman) similarly discloses fluorescence flow cytometry for characterizing and counting subgroups of leukocytes, lymphocytes, monocytes, neutrophils, eosinophils, and other blood cell populations such as erythrocytes and reticulocytes.

[0009] The use of fluorescence measurements in flow cytometry has been increasing since its early days. All current flow cytometers have at least multiple fluorescence emission paths, and multiple excitation sources are now extremely common.

[0010] Fluorescence measurement is a powerful tool for accurately distinguishing subgroups of all types of blood cells, but it also has drawbacks and is difficult to use.

[0011] Fluorescent dyes are known to be fragile molecules that are susceptible to degradation due to several environmental factors, due to their structure.

[0012] For example, fluorescent dyes are susceptible to electromagnetic radiation, particularly at the excitation wavelength, and are also susceptible to high-energy excitation wavelengths that cause photodegradation.

[0013] Furthermore, fluorescent dyes are easily oxidized by oxygen in the air or oxidizing agents (such as peroxides, some detergents, or free radicals in solutions).

[0014] Furthermore, some fluorescent dyes are known to be unstable in aqueous solutions depending on their structure, and their hydrolysis is accelerated by temperature, causing them to gradually decompose over time. Because the by-products absorb short wavelengths, they do not emit fluorescence even when irradiated by the device's light source (and do not bind to nucleic acids in any case). As a result, the fluorescence emission intensity decreases, which may lead to errors in the characterization of blood cells.

[0015] Therefore, hydrolysis significantly limits (or shortens) the shelf life of aqueous fluorescent reagents.

[0016] This is even more true for certain types of fluorescent dyes, such as heterocyclic polymethines, which are asymmetric cyanines. Asymmetric cyanines, which have been used in medical diagnostics since the late 1980s, still have unparalleled advantages over other more stable fluorescent probes such as acridines or symmetric cyanines. See, for example, Patent Document 3 by Lee et al.

[0017] Asymmetric cyanines emit a much lower fluorescence intensity than acridine (about 1 / 20th), but they hardly fluoresce in their free state. In other words, even when excited to the absorption limit, asymmetric cyanines hardly fluoresce when suspended in solution. Conversely, when asymmetric cyanines bind to a target (DNA or RNA), the amount of re-emitted fluorescence increases dramatically, and the ratio of fluorescence emitted in the bound state to the free state (bound state / free state) exceeds 1000. At the same time, the ratio for acridine is only slightly above 1, while for symmetric cyanines it is about 3.

[0018] Patent Document 4 or Patent Document 5 is known for disclosing a reagent enabling automatic measurement of reticulocytes. Patent Document 6 discloses, for example, a reagent for measuring white blood cells. These documents by Yasumasa et al. teach that in order to overcome the instability of dyes in aqueous solutions, the dyes can be advantageously dissolved at very high concentrations in water-soluble or miscible organic solvents. Such solvents may include lower alcohols (ethanol, methanol), ethylene glycol, or dimethyl sulfoxide (DMSO). In fact, in the absence of water, fluorescent dyes are stable in organic solvents when protected from photodegradation, and the corresponding reagents have a long shelf life (more than one year), which is undoubtedly a commercial advantage.

[0019] When this stable organic solvent is used to stain a blood sample in the device, it is further diluted and immediately mixed with another aqueous solution, which contains salts, osmotic pressure compensators, and optionally other components, such as preservatives, or cationic detergents used as staining accelerators, or cationic detergents used in large amounts to promote erythrocyte lysis for white blood cell measurement. See, for example, Patent Document 7.

[0020] However, the use of organic concentrated staining solutions has many drawbacks.

[0021] First of all, most of these organic solvents are not harmless. They are flammable and are suspected (or proven) to be harmful to humans (not only manufacturing workers but also end-users) and aquatic organisms, and are even toxic raw materials.

[0022] Moreover, they may have poor compatibility with the device or packaging components (plastics, tubes, valves).

[0023] Furthermore, the use of such reagents increases the number of containers required for the device, causing unnecessary obstacles to laboratory management in terms of ordering, inventory management, monitoring of expiration dates, waste disposal (especially when the reagents are classified), etc.

[0024] Finally, the viscosity and density of the concentrated organic dye solution are different from those of the aqueous reagent. In order to properly process the sample over a long period of time, it is essential to mix and homogenize the final standard solution. This leads to an undesirable situation where the fluid lines and mixing means of the device become complex, resulting in increased costs and the risk of failure.

[0025] Another possibility to prevent the deterioration of the fluorescent dye over time is to apply a numerical correction factor according to the decrease in fluorescence. The lower the fluorescence, the higher the numerical correction factor.

[0026] The above correction principle is to monitor and quantify the decrease in the fluorescence of the dye over time.

[0027] By correcting the deterioration, it becomes possible to extend the storage period of the reagent containing the fluorescent dye without degrading the analysis performance.

[0028] Patent Document 8 discloses a means for correcting the fluorescence level of a fluorescent dye by measuring the optical density of the fluorescent dye at a specific wavelength. This measurement is based on the optical density by a spectrophotometer. Such a means is specific to the spectrophotometer and is additional to the means dedicated to the fluorescence measurement of labeled particles. Therefore, in Patent Document 8, two different measurement units are required. One is for measuring the optical density of the fluorescent dye using a spectrophotometer, and the other is for measuring the fluorescence of labeled particles. Patent Document 8 clearly discloses the optical density as a related parameter for monitoring the attenuation of the fluorescent dye. No other parameters for monitoring this attenuation are disclosed.

[0029] Non-patent document 1 discloses a means for correcting variations in the emission spectrum of tandem dyes bound to proteins. Hulspas discloses variations in the spectral properties of the two components (donor and acceptor) of a tandem dye. In particular, Hulspas investigated the effect of tandem dye instability on correction matrix values ​​over long periods (p. 969, Col. 2). Spectral correction is only necessary when multiple fluorescent dyes are used simultaneously. In this case, multiple fluorescence emission spectra overlap (spillover), and a correction matrix is ​​used to minimize the spillover effect. Hulspas discloses a simulation achieved by clarifying the correction matrix for each time point of a group of tandem dyes bound to particles. Hulspas uses a correction matrix that handles spillover between multiple tandem dye spectra. [Prior art documents] [Patent Documents]

[0030] [Patent Document 1] U.S. Patent No. 3,684,377 [Patent Document 2] U.S. Patent No. 3,916,205 [Patent Document 3] U.S. Patent No. 4,957,870 [Patent Document 4] U.S. Patent No. 5,821,127 [Patent Document 5] U.S. Patent No. 5,891,731 [Patent Document 6] U.S. Patent No. 6,004,816 [Patent Document 7] European Patent No. 2175340 [Patent Document 8] U.S. Patent No. 9,448,175 [Non-patent literature]

[0031] [Non-Patent Document 1] Hulspas, “Flow Cytometry and the Stability of Phycoerythrin-Tandem Dye Conjugates”, Cytometry Part A volume 75A, 11 nov 2009, p966-972 [Overview of the project] [Problems that the invention aims to solve]

[0032] The object of the present invention is to provide a method for quantifying and / or correcting (compensating for) fluctuations in fluorescence emission intensity when a reagent containing a fluorescent dye is bound (i.e., when the fluorescent dye is bound to blood cells).

[0033] Another object of the present invention is a novel method for calculating a compensation factor for variations in the fluorescence emission intensity of this reagent.

[0034] The present invention also intends to provide a simple, efficient, and rapid method for correcting fluctuations in fluorescence emission intensity. [Means for solving the problem]

[0035] At least one of the above objectives is achieved by a method for calculating a correction factor to compensate for variations in the fluorescence emission intensity of reagents containing fluorescent dyes, the correction factor being intended to be applied in the process of measuring the fluorescence emission intensity in the bound state when cells are labeled with the fluorescent dye, the method comprising at least the following steps: - A step to measure the free-state fluorescence emission intensity emitted from a reagent containing a fluorescent dye when the cells are not labeled before measuring the fluorescence emission intensity in the bound state. - A process for calculating a correction factor from the measured free-state fluorescence emission intensity. Includes.

[0036] The present invention can be advantageously used to compensate for variations in the fluorescence emission intensity of reagents containing fluorescent dyes, which are caused by aging degradation of the fluorescent dyes or variations in composition between batches of reagents. Variations in composition can include chemical variations and process variations resulting from storage and transportation conditions.

[0037] In free-state fluorescence measurements, the fluorescent dye is not labeled to the cells. Conversely, measuring the fluorescence of cells labeled with a fluorescent dye is called bound-state fluorescence measurement.

[0038] Fluorescence measurements in a free state are performed on a reagent containing a fluorescent dye, and then cells are labeled using this reagent.

[0039] In the method according to the present invention, the fluorescence parameters of the free state of the dye when the dye is not bound are used to monitor and correct for fluctuations in the fluorescence emission intensity of the dye in subsequent measurements using the bound dye. Subsequent measurements include, for example, cell characterization.

[0040] Fluorescent molecules (or dyes) used in flow cytometry primarily possess two properties: the ability to bind to specific sites on blood cells and the ability to re-emit light (fluorescence) when properly excited. Fluorescent dyes are known to degrade over time due to several environmental factors. In other words, parts of the dye molecule break down into multiple parts over time due to environmental factors. When the molecular structure is broken down, it loses both the ability to bind to specific sites on blood cells and the ability to re-emit light (fluorescence) when properly excited.

[0041] The inventors of this invention focused on the fact that the disappearance of fluorescence due to molecular degradation applies to both the free state of the dye (not bound to a specific site on the blood cell) and the bound state of the dye (bound to a specific site on the blood cell).

[0042] According to the present invention, the decrease in fluorescence of a dye in its free state also reflects the decrease in fluorescence in its bound state. Therefore, by measuring the fluorescence in the free state, it is possible to monitor the decrease in fluorescence in the bound state of the dye.

[0043] In particular, by monitoring and quantifying the change in fluorescence emission intensity in the free state, it becomes possible to derive a correction algorithm for fluctuations in fluorescence emission intensity in the bound state.

[0044] The present invention makes it possible to effectively quantify and / or correct the decrease in fluorescence emission intensity of reagents containing fluorescent dyes as the fluorescent dyes degrade over time.

[0045] On the other hand, prior art such as Patent Document 8 monitors a parameter called optical density, but this is not free-state fluorescence.

[0046] Optical density is defined as the logarithm of the ratio of the incident radiation intensity to the transmitted radiation intensity on a sample.

[0047] The excitation wavelength and the detection wavelength are the same. In this invention, the free-state fluorescence of a dye used in a free (unbound) state is directly measured using the same optical system used to measure the fluorescence re-emitted from a fluorescent dye bound to a cell, for example. As long as fluorescence is re-emitted from the fluorescent dye at a wavelength longer than the wavelength of the illumination light source when excited by the illumination light source, this is not optical density (the light transmittance of the sample at a given wavelength). The excitation spectrum and the emission spectrum are different.

[0048] Prior art methods require additional specific hardware to monitor the optical density of the fluorescent dye. The free-state fluorescence parameters of the dye, used to monitor fluorescence reduction in the bound state, do not require the use of any calibrators (particles, proteins, nucleic acids, etc.).

[0049] In this invention, the fluorescence fluctuations of the dye in its free state are quantified, and the same type of physical phenomenon (luminescence) is measured as a measurement parameter for the final dye-binding state used in characterizing blood cells.

[0050] The measurement of the optical density of fluorescent dyes disclosed in Patent Document 8 is susceptible to multiple determination errors. The measurement of a substance using a spectrophotometer may be hindered by many factors, including absorption and / or scattering due to other particles, impurities, bubbles, heterogeneity, etc. within the substance.

[0051] Unlike absorption, fluorescence emission is a far more reliable parameter because it is not susceptible to interference or impairment by impurities or heterogeneity.

[0052] According to one embodiment, the present invention may include at least two measurements of free-state fluorescence intensity performed at separate times before measuring fluorescence intensity in the bound state, and the correction factor is calculated from the measured free-state fluorescence intensity values.

[0053] This invention provides a method for obtaining multiple values ​​by performing fluorescence measurements in the free state several times in order to accurately derive a coefficient for correcting fluctuations in the bound state. Those skilled in the art can determine various appropriate timings for performing fluorescence measurements in the free state by knowing the average rate of variation of the fluorescent dye.

[0054] In particular, one free-state fluorescence intensity measurement can be performed at time t1 after the preparation of the reagent containing the fluorescent dye, and another free-state fluorescence measurement can be performed at time t2, which is after t1 but before the bound-state fluorescence intensity measurement. The time from t1 to t2 is at least longer than the time from t2 to the bound-state fluorescence intensity measurement.

[0055] This invention proposes performing the first free-state fluorescence intensity measurement immediately after the preparation of a reagent containing a fluorescent dye, and the second free-state fluorescence measurement immediately before the fluorescence measurement of cells labeled with the fluorescent dye. By performing these two measurements at appropriate times, it becomes possible to accurately calculate the correction factor.

[0056] Patent Document 8 also requires two measurements, but these are based on the measurement of optical density. The correction coefficient in Patent Document 8 is based on the change, i.e., fluctuation, of optical density, so two measurements of different optical densities are essential. In contrast, the present invention proposes an alternative solution that can be completed with only one measurement.

[0057] According to a preferred embodiment of the present invention, the correction coefficient is a predetermined normalization function Φ N The following can be calculated from the measured free-state fluorescence emission intensity of a reagent containing a fluorescent dye, and at least one value of this intensity can be calculated using a predetermined normalization function Φ N It is at least one argument.

[0058] According to the present invention, the value obtained by measuring the fluorescence emission intensity of a reagent containing a fluorescent dye in the free state can be used as an argument to a normalization function used as a variation correction coefficient for subsequent measurements of fluorescence emission intensity in the bound state.

[0059] Advantageously, the fluorescence emission intensity of a reagent containing a fluorescent dye in the free state is measured only once and then normalized using a predetermined normalization function Φ N It can be a single argument.

[0060] In this invention, the fluorescence emission intensity of a reagent containing a fluorescent dye is measured only once in the free state and normalized by the function Φ N This is used as an argument to the function. This method does not rely on estimating the variation in fluorescence emission intensity in the free state, but rather on the fluorescence level in the bound state of the dye, using the normalization function Φ. N This method involves systematically normalizing the data using [a specific method / tool].

[0061] The normalization function according to the present invention requires only one measurement of the fluorescence emission intensity in a free state, which is one of the properties of the dye, in order to derive the fluorescence correction coefficient necessary for subsequent measurements when the dye is bound to blood cells.

[0062] This eliminates the need for initial measurement of the properties of the fluorescent dye immediately after manufacturing, significantly easing constraints in the manufacturing process.

[0063] Subsequent fluorescence measurements in the bound state, i.e., when the dye is bound to blood cells, are performed using a function Φ with a single argument (pre-measured fluorescence emission intensity in the free state). N It is normalized by [this method].

[0064] According to one embodiment of the present invention, a given normalization function Φ N This can be a polynomial function, a ratio of polynomial functions, an exponential function, a logarithmic function, a hyperbolic function, or a combination thereof. It can also be defined in parts; that is, different analytical definitions can be defined for different ranges of its single argument.

[0065] According to a preferred embodiment of the present invention, fluorescence emission intensity can be measured in a free state for a first reagent sample, and fluorescence emission intensity can be measured in a bound state for a second reagent sample, and the first and second reagent samples are from a single container containing a fluorescent dye.

[0066] In other words, the first reagent sample and the second reagent sample are from the same reagent.

[0067] Another object of the present invention is to correct for variations in fluorescence emission intensity due to the binding state of the fluorescent reagent between manufacturing batches, using the correction mechanism disclosed above based on fluorescence measurement in a free state.

[0068] In the manufacturing process of fluorescent reagents, slight variations in reagent composition (e.g., fluorescent dye concentration) may occur due to variability in the purity of raw materials, human factors, and environmental factors (accuracy of cumulative weighing, inaccuracy of filling measurements).

[0069] While all manufactured batches meet the specified acceptance criteria for the fluorescent reagents, this technique can further smooth out slight variations in fluorescence intensity in the bound state by correcting the free-state fluorescence intensity, thereby maintaining and improving the consistency of the final results.

[0070] By calculating a correction factor from free-state fluorescence emission intensity measurements, the results and the resulting fluorescence dot plots can be normalized when preparing new bottles of fluorescent dye-containing reagents, regardless of the number of years the bottle has been used or slight changes in its composition, particularly the fluorescence dye concentration.

[0071] The compositions of the first and second reagents may be the same or slightly different, for example, because they may be manufactured from different components in different regions at different times.

[0072] The present invention may include a series of steps to measure the free-state fluorescence emission intensity of a reagent containing a fluorescent dye in order to accurately monitor fluctuations in fluorescence emission intensity.

[0073] According to a preferred embodiment of the present invention, the same optical transducer can be used for both measuring fluorescence emission intensity in the free state and in the bound state. This ensures that there is no bias in the measurement between the measurement of fluctuations in fluorescence emission intensity in the free state and the measurement of the final fluorescence emission intensity of the fluorescent dye bound to blood cells.

[0074] However, it is also possible to use a different, dedicated converter.

[0075] The spectrophotometer measurement of the optical density of fluorescent dyes disclosed in Patent Document 8 requires additional means. This measurement requires at least one dedicated light source, a photodetector, and an acquisition chain. In the present invention, the same photoconverter can be used for both the derivation of the correction coefficient and the final measurement. This results in a more inexpensive, compact, and robust device.

[0076] According to one embodiment of the present invention, the same excitation spectrum can be used to measure the fluorescence emission intensity in the free state and the fluorescence emission intensity in the bound state.

[0077] According to one embodiment of the present invention, the same emission wavelength range can be used to measure the fluorescence emission intensity in the free state and the fluorescence emission intensity in the coupled state.

[0078] According to one embodiment of the present invention, the concentrations of the fluorescent dye used to measure the fluorescence emission intensity in the free state and the fluorescence emission intensity in the bound state may be the same or different.

[0079] In this invention, the same transducer can be used for measuring fluorescence emission intensity in the free state and in the final bound state for cell characterization. Therefore, the excitation spectral conditions and emission spectral conditions can be the same for both free state and final bound state fluorescence emission intensity measurements. This improves the consistency between monitoring the fluorescence decay of the fluorescent dye in the free state and measuring the final fluorescence of the dye bound to blood cells.

[0080] According to one embodiment of the present invention, the process of measuring the fluorescence emission intensity in a free state can be performed using a reagent containing a single fluorescent dye.

[0081] In another embodiment, the step of measuring the fluorescence emission intensity in a free state is performed using a reagent containing at least two fluorescent dyes. Depending on the excitation and emission spectra of the fluorescent dyes, it may be necessary to add a new illumination module and / or detection module to the above embodiment.

[0082] If the excitation spectra of the light source and the fluorescent dyes do not match, and therefore the fluorescent dyes in the reagent cannot be excited by the same light source, then at least one additional illumination module is needed so that each fluorescent dye is excited by an illumination module with a spectrum that matches its excitation spectrum.

[0083] If the emission spectra of the fluorescent dyes contained in the reagent do not match the wavelength range of a single fluorescence measurement module, at least one additional fluorescence measurement module is required to measure the fluorescence emission of each fluorescent dye with a module that has a bandwidth that matches its emission spectrum.

[0084] The present invention - Measure the free-state fluorescence emission intensity of the fluorescent dye contained in the reagent. - A correction factor is calculated from the measured free-state fluorescence emission intensity, - Measure the fluorescence emission intensity in the bound state of cells labeled with a fluorescent dye, and - Apply the correction coefficient to the measured fluorescence emission intensity in the bound state. The present invention also relates to an optical flow cytometer that includes a processing unit configured in such a manner.

[0085] Further advantages and features of the present invention will become apparent upon closer examination of the detailed description of one non-limiting embodiment and the accompanying drawings. [Brief explanation of the drawing]

[0086] [Figure 1] This is an overall diagram showing the components of an optical flow cytometer that can be used to carry out the method according to the present invention. [Figure 2] This is a flowchart illustrating a method for calculating the correction coefficient according to the present invention. [Figure 3] This graph shows the fluorescence decay in a free state. [Figure 4] This graph shows the fluorescence decay in the bound state. [Figure 5]Figure 5a shows the uncorrected fluorescence level in the bound state measured at t=0. Figure 5b shows the corrected fluorescence level in the bound state measured at t=0. [Figure 6] Figure 6a shows the uncorrected fluorescence level in the bound state measured at t=10 months. Figure 6b shows the corrected fluorescence level in the bound state measured at t=10 months. [Modes for carrying out the invention]

[0087] While various modifications and alternative forms are possible with respect to the present invention, specific embodiments are illustrated in the drawings and described in detail herein. However, it should be understood that the drawings and their detailed description are not limited to the specific forms that disclose the invention, but rather are intended to encompass all modifications, equivalents, and alternative forms that fall within the scope of the invention as described in the appended claims.

[0088] The present invention will be described in detail below with reference to the attached drawings, illustrating exemplary embodiments of the present invention.

[0089] Figure 1 is an overall diagram showing the components of an optical flow cytometer that can be used to carry out the method according to the present invention.

[0090] Referring to Figure 1, the optical flow cytometer 1 of the present invention includes an illumination module 2 that generates an illumination beam 3 toward a flow cell 4 toward which particles such as blood cells are flowing. The optical flow cytometer 1 has motorized and / or other means for moving and focusing the sample cells or blood cells (which may or may not be surrounded by sheath fluid) into the flow. The blood cells circulate through the flow cell 4.

[0091] The illumination beam 3 is focused and directed perpendicularly across the flow of cells to induce fluorescence of fluorescent sample particles or their markers. The fluorescence 7 generated by the labeled blood cells is collected by the fluorescence measurement module 5.

[0092] The illumination beam 3 also induces scattered light 8 as blood cells pass through the photometric area. The optical flow cytometer 1 has a scattering measurement module 6 provided for collecting scattered light 8 coming from the flow cell 4.

[0093] A processing unit 9 is provided to control the excitation module 2 for the excitation signal. The processing unit 7 also controls the fluorescence measurement module 5 and the scattering measurement module 6 to detect direct and / or indirect scattering signals.

[0094] In the embodiment shown in Figure 1, the scattering measurement module 6 is positioned on the opposite side of the illumination module 2, and the fluorescence measurement module 5 is positioned at 90° from the optical axis of the illumination beam. Other arrangements are possible by deflecting the light with mirrors, lenses, and / or beam splitters.

[0095] The present invention also relates to epifluorescence configurations not shown, in which the excitation module 2 may include a fluorescence measurement module 5. In such a configuration, the same focusing lens is used to focus the excitation beam onto the flow cell, and fluorescence is collected from the flow cell.

[0096] According to the present invention, the fluorescence measurement module 5 is preferably a photoconverter used to measure free-state fluorescence and bound-state fluorescence.

[0097] A processing unit 9, such as a microprocessor, is configured to perform the method according to the present invention.

[0098] Figure 2 is a flowchart illustrating a method for calculating the correction coefficient according to the present invention.

[0099] The method according to the present invention can be carried out using the apparatus described with respect to Figure 1.

[0100] The first set of steps according to the present invention relates to the measurement of fluorescence in a free state. In step 10, it is assumed that a reagent containing a fluorescent dye is present in the apparatus.

[0101] In step 11, the processing unit 9 in FIG. 1 drives various components of the apparatus 1 to measure the fluorescence of the free fluorescent dye contained in the reagent. N 79> The optical converter 5 receives a fluorescence signal that is processed to calculate the value of the fluorescence emission intensity in step 12.

[0103] During the period from the production of the reagent to the measurement of the fluorescence in the next bound state, the measurement can be performed multiple times. As is well known to those skilled in the art, a function can be derived from multiple values, and a correction factor can be specified each time the fluorescence measurement in the bound state is performed.

[0104] The correction factor can be a fixed value calculated immediately before performing the fluorescence measurement in the bound state. The correction factor can also be a function that changes with time.

[0105] In step 11, the fluorescence measurement in the free state can also be performed once, and the fluorescence value can be calculated. In step 12, the correction factor is calculated from a predetermined normalization function Φ N The calculated fluorescence value is used as a single argument of a predetermined normalization function Φ N

[0106] In step 13, the user prepares to characteristically evaluate various types of cells present in, for example, blood by fluorescence using the optical flow cytometer 1. Such an apparatus can be designed, for example, to count the number of white blood cells contained in a blood sample and to clarify the relative distribution of white blood cells within various sub-populations based on light scattering and fluorescence.

[0107] In step 13, the reagent is injected into the blood. This reagent contains a fluorescent dye for which a correction factor has been calculated based on multiple free-state fluorescence measurements or one free-state fluorescence measurement.

[0108] In step 14, the fluorescence measurement in the bound state is performed taking into account the correction factor calculated from the fluorescence measurement in the free state.

[0109] ​To do this, the illumination module 2 generates excitation light directed toward the flow cell 3. Fluorescence from the blood cells is collected by the phototransistor 5. The processing unit 9 calculates the fluorescence value, taking into account a correction factor. This value is useful information for characterizing the blood cells in step 15.

[0110] Figure 3 is a graph showing fluorescence decay in the free state. In this example, fluorescence measurements in the free state were performed several times. Several values ​​of fluorescence emission intensity (in arbitrary units) were calculated. Using these values, a curve, which is a function that changes over time, can be drawn. It can be seen that at t=0 months, i.e., immediately after the manufacture of the reagent containing the fluorescent dye, the level is approximately 900. The level decreases to 500 at t=6 months and to 450 at t=10 months.

[0111] The decay of fluorescence emission intensity in the free state of a fluorescent dye reflects a decrease in fluorescence in its bound state. Therefore, monitoring the fluorescence decay of a fluorescent dye in its bound state is possible by measuring the fluorescence in its free state.

[0112] Figure 4 is a graph showing the fluorescence decay of the bound fluorescent dye shown in Figure 3. Curve 16 shows the change in fluorescence emission intensity without correction, i.e., the raw data. Several measurements of the bound state fluorescence were performed simultaneously with the free state fluorescence shown in Figure 3. Several values ​​(in arbitrary units) of fluorescence emission intensity were calculated. Using these values, a curve, which is a function of change over time, can be drawn. At t=0 months, i.e., immediately after the manufacture of the reagent containing the fluorescent dye, the level is approximately 237. The level decreases to 175 at t=6 months and to 150 at t=10 months. The change in bound state fluorescence without correction is consistent with the change in free state fluorescence. In particular, by monitoring and quantifying the fluorescence decay in the free state, it is possible to derive a correction algorithm for the fluorescence decay in the bound state.

[0113] Correction factors were calculated each time the fluorescence emission intensity in the free state and the bound state was measured at t=0, 2, 4, 6, 8, 9, and 10 months. Curve 17 in Figure 4 shows the change in corrected fluorescence emission intensity, i.e., the corrected data. It can be seen that curve 17 changes around the initial value of 237 represented by curve 18.

[0114] According to the present invention, a predetermined normalization function Φ is used to calculate the correction coefficient. N You can use this. An example of such a normalization function is, Φ N (t,t0) = α × Fluo free (t0) / Fluo free (t)+β And α and β are two coefficients depending on the fluorescent dye used, Fluo free t is the fluorescence emission intensity in the free state, and t is time.

[0115] The correction can be applied using the following formula: Fluo Bound Compensated (t)=Fluo Bound Raw (t) × Φ N (t,t0) During the ceremony, Fluo Bound Compensated (t) is the corrected value of the fluorescence emission intensity in the bound state, Fluo Bound Raw is the uncorrected value of the fluorescence emission intensity in the bound state, and t is time.

[0116] Figures 5 and 6 show images of the fluorescence emission intensity measured for various binding states of blood cells labeled with a fluorescent dye. This fluorescent dye is included in the reagents used in flow cytometry to characterize blood cell populations. Gy is the Y-value (i.e., coordinate along the fluorescence axis) of the centroid of the {neutrophil + eosinophil} population.

[0117] Figure 5a is an image of the fluorescence spots showing the fluorescence emission intensity of the binding state measured at t=0 months. There is no correction, and Gy=88. This measurement corresponds to the raw data at t=0.

[0118] Figure 5b is an image of the fluorescence spots showing the fluorescence emission intensity of the binding state measured at t=0 months. In this case, a correction is applied, and Gy=87. The correction factor is very close to 1, so it has no effect.

[0119] Figure 6a is an image of the fluorescence spot showing the fluorescence emission intensity of the bound state measured at t=10 months. There is no correction, and Gy=50. This measurement shows the decay along the Y axis compared to the measurement at t=0. To correct for this decay, a correction factor is applied in Figure 6b.

[0120] Figure 6b is an image of the fluorescence spots showing the fluorescence emission intensity of the bound state measured at t=10 months. In this case, a correction is applied, with Gy=87, the same as in Figure 5b. Since the correction factor is considerably larger than 1, the attenuation along the Y axis is corrected.

[0121] A full understanding of the above disclosure will reveal numerous variations and modifications to those skilled in the art. The following claims are intended to encompass all such variations and modifications.

[0122] For example, the present invention can be used to compensate for variations in fluorescence emission intensity between reagent batches. To do this, the fluorescence emission intensity in a free state can be automatically measured when changing (preparing) bottles in a device such as a hemocytometer.

[0123] If bottle A is already installed in the apparatus, at least one measurement of its free-state radiation intensity can be taken while it is installed to calculate a specific correction factor for that batch over its lifespan. Additionally, other measures may be taken to adjust the correction factor as needed (if the bottle has remained in the apparatus for an extended period).

[0124] When replacing bottle A with bottle B (regardless of whether it's from the same batch or a different batch, or whether it's new or old), the free-state fluorescence intensity is measured anew. Then, the fluorescence correction coefficient of the instrument is adjusted as needed. This new measure allows for: - "Chemical" variations (purity of the fluorescent dye used, variability in the weight of the reagent compounds, and variability in the water level in the tank during the manufacturing process) - "Temporal" fluctuations (relative aging of the two bottles), - "Environmental" fluctuations (bottle temperature and storage conditions) It will disappear.

Claims

1. A method for calculating a correction factor to compensate for variations in the fluorescence emission intensity of a reagent containing a fluorescent dye, wherein the correction factor is intended to be applied in the process of measuring the fluorescence emission intensity in a bound state when cells are labeled with the fluorescent dye, and the method comprises at least the following steps: - A step of measuring the free-state fluorescence emission intensity emitted from the reagent containing the fluorescent dye when the cells are not labeled before measuring the fluorescence emission intensity in the bound state. - A step of calculating the correction coefficient from the measured free-state fluorescence emission intensity. Methods that include...

2. The method according to claim 1, characterized in that, prior to measuring the fluorescence emission intensity in the bound state, at least two measurements of the fluorescence emission intensity in the free state are performed at separate times, and the correction coefficient is calculated from the measured free state fluorescence emission intensity values.

3. The method according to claim 2, characterized in that a free-state fluorescence intensity measurement is performed once at time t1 after the production of the reagent containing the fluorescent dye, and another free-state fluorescence measurement is performed at time t2, which is after t1 but before the bound-state fluorescence intensity measurement, and the time from t1 to t2 is at least longer than the time from t2 to the bound-state fluorescence intensity measurement.

4. The correction coefficient is a predetermined normalization function Φ Ｎ The predetermined normalization function Φ is used to calculate at least one value of the measured free-state fluorescence emission intensity of the reagent containing the fluorescent dye. Ｎ The method according to claim 1, characterized in that it is at least one argument of

5. The fluorescence emission intensity of the reagent containing the fluorescent dye in a free state, measured only once, is obtained by the predetermined normalization function Φ Ｎ The method according to claim 4, characterized in that it is a single argument.

6. The predetermined normalization function Φ Ｎ The method according to claim 4 or 5, characterized in that it consists of a polynomial function, a ratio of polynomial functions, an exponential function, a logarithmic function, a hyperbolic function, or a combination thereof.

7. The predetermined normalization function Φ Ｎ The method according to any one of claims 4 to 6, characterized in that it is defined in parts.

8. The method according to any one of claims 1 to 7, characterized in that the fluorescence emission intensity is measured in a free state for a first reagent sample, and the fluorescence emission intensity is measured in a bound state for a second reagent sample, and the first reagent sample and the second reagent sample are from a single container containing the fluorescent dye.

9. The method according to any one of claims 1 to 8, characterized in that a plurality of steps for measuring the fluorescence emission intensity in a free state are performed to monitor the decrease in the fluorescence emission intensity.

10. The method according to any one of claims 1 to 9, characterized in that the same photoconverter is used for both measuring the fluorescence emission intensity in the free state and measuring the fluorescence emission intensity in the coupled state.

11. The method according to any one of claims 1 to 10, characterized in that the same excitation spectrum is used for measuring the fluorescence emission intensity in the free state and for measuring the fluorescence emission intensity in the bound state.

12. The method according to any one of claims 1 to 11, characterized in that the same emission wavelength range is used for measuring the fluorescence emission intensity in the free state and for measuring the fluorescence emission intensity in the coupled state.

13. The method according to any one of claims 1 to 12, characterized in that the concentrations of the fluorescent dye used for measuring the fluorescence emission intensity in the free state and the fluorescence emission intensity in the bound state are the same or different.

14. The method according to any one of claims 1 to 13, characterized in that the step of measuring the fluorescence emission intensity in a free state is carried out using a reagent containing a single fluorescent dye.

15. The step of measuring the fluorescence emission intensity in the free state is carried out using a reagent containing at least two types of fluorescent dyes. Use at least one illumination module so that each fluorescent dye is excited by an illumination module having a spectrum that matches its excitation spectrum, and / or Use at least one fluorescence measurement module to measure the fluorescence emission of each fluorescent dye with a module having a bandwidth that matches its emission spectrum. A method according to any one of claims 1 to 13, characterized by the above.

16. - Measure the free-state fluorescence emission intensity of the fluorescent dye contained in the reagent. - A correction factor is calculated from the fluorescence emission intensity measured in the free state, - Measure the fluorescence emission intensity of the cells labeled with the aforementioned fluorescent dye in their bound state. - Apply the correction coefficient to the fluorescence emission intensity in the measured binding state. An optical flow cytometer including a processing unit configured as follows.

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