Method for compensating fluorescent dye change by free fluorescence measurement

Abstract

The invention relates to a method for compensating for a change in the fluorescence radiation intensity of an agent containing a fluorescent dye by determining a compensation factor intended to be applied during a bound-state fluorescence radiation intensity measurement when a cell is labeled by a fluorescent dye, said method comprising at least the following steps:-before the bound-state fluorescence radiation intensity measurement, determining the fluorescence radiation intensity of the agent containing the fluorescent dye; measuring the intensity of free fluorescence radiation emitted by the fluorescent dye-containing reagent when the fluorescent dye does not label the cells,-determining the compensation factor from the measured intensity of free fluorescence radiation.

Description

[0001] This invention relates to the monitoring of fluorescence intensity of reagents containing fluorescent dyes used in flow cytometry. In the following text, "fluorescence" refers to luminescent radiation intensity or fluorescent emission radiation intensity.

[0002] Flow cytometry is a technique used to characterize blood cells in vitro. In a flow cytometer, blood cells flow sequentially through sensors that measure various parameters of the blood cells.

[0003] The parameters measured can be based on the basic inherent characteristics of blood cells, such as cell size, membrane roughness, nucleus complexity, or internal granularity.

[0004] Enhanced characterization of blood cells relies on the use of fluorescent markers. These fluorescent markers can be selected to be highly specific for target blood cell characteristics, such as the content of blood cell nucleic acids. When excited, i.e., when irradiated at a specific wavelength, the fluorescent probe re-emits fluorescence, allowing the presence and quantification of the target blood cell characteristics to be detected.

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

[0006] In the early 20th century, fluorophores were first used in biological research to stain tissues, bacteria, and other pathogens. Later, Carl Zeiss and Carl Reichert developed this into fluorescence microscopy techniques. By the early 1940s, Ellinger and Hirt had achieved fluorescent labeling.

[0007] In 1963, Vander reported the use of a fluorescent dye (acidine orange) in hematology for the characterization and counting of reticulocytes by visual microscopy.

[0008] US3,684,377 (Katmensky) discloses pioneering work on the characterization of leukocytes using fluorescence flow cytometry. US3,916,205 (Kleinerman) also discloses the use of fluorescence flow cytometry to characterize and count leukocyte subsets, lymphocytes, monocytes, neutrophils, eosinophils, and other blood cell populations, such as erythrocytes and reticulocytes.

[0009] Since these early studies, the application of fluorescence measurements in flow cytometry has continued to evolve. All modern flow cytometers offer at least multiple fluorescence emission channels, and multiple excitation sources are now quite common.

[0010] Although fluorescent dyes are powerful tools for accurately distinguishing different blood cell subsets, they also have drawbacks and are difficult to process.

[0011] Due to their structural characteristics, fluorescent dyes are known to be fragile molecules that are easily degraded by a variety of environmental factors.

[0012] For example, they are sensitive to electromagnetic radiation: particularly at their excitation wavelength, but also at higher energy wavelengths that would trigger their photodegradation.

[0013] They are also easily oxidized by oxygen in the air or by oxidizing agents (such as peroxides in solution, some detergents, or free radicals).

[0014] Furthermore, based on their structure, some fluorescent dyes are known to be unstable in aqueous solutions and undergo temperature-promoted hydrolysis, leading to their gradual degradation over time. The byproducts absorb lower wavelengths of light and therefore do not emit fluorescence when illuminated by an instrument light source (and cannot bind to nucleic acids), resulting in decreased fluorescence intensity, which may cause errors in blood cell characterization.

[0015] Therefore, hydrolysis greatly limits (or reduces) the shelf life of water-based fluorescent reagents.

[0016] This is especially true for certain types of fluorescent dyes, such as heterocyclic polymethystylene asymmetric cyanine dyes. Although the latter have been used in medical diagnostics since the late 1980s and still have unparalleled advantages over more stable fluorescent probes such as acridine or symmetric cyanine dyes, see, for example, US 4,957,870 by Lee et al.

[0017] Although asymmetric cyanine dyes emit significantly lower fluorescence intensity than acridine dyes (approximately 20 times lower), they exhibit almost no fluorescence in their free state. This means that even when excited at their absorption maxima, free asymmetric cyanine dyes in solution emit almost no fluorescence. Conversely, when they bind to their targets (DNA or RNA), the amount of re-emitted fluorescence increases dramatically, with the ratio of bound-state fluorescence to free-state fluorescence exceeding 1000, while this ratio for acridine dyes is only slightly above 1 and for symmetric cyanine dyes it is approximately 3.

[0018] US 5,821,127 or US 5,891,731 disclose reagents for automating reticulocyte measurements. US 6,004,816 discloses reagents, for example, for leukocyte measurements. The aforementioned document from Yasumasa et al. teaches that dyes can advantageously be dissolved in very high concentrations in water-soluble or miscible organic solvents to overcome the instability of dyes in aqueous solutions. Such solvents may include lower alcohols (ethanol, methanol), ethylene glycol, or dimethyl sulfoxide (DMSO). In fact, fluorescent dyes are stable in organic solutions in the absence of water, and when protected against photodegradation, the corresponding reagents exhibit long shelf lives (greater than one year), which is an undeniable commercial advantage.

[0019] This stable organic solution is further diluted and instantaneously mixed with another aqueous solution when used for staining blood samples in the instrument. The aqueous solution contains salts, osmotic compensators, and possibly other components such as preservatives, or cationic detergents used as staining accelerators, or used in larger doses to promote erythrocyte lysis during leukocyte measurements; see, for example, EP 2 175 340.

[0020] However, there are several drawbacks to using concentrated organic dye solutions.

[0021] First, most of these organic solvents are not harmless: they are either flammable or raw materials that are suspected (or proven) to be harmful or even toxic to humans (manufacturing workers, as well as end users) or aquatic life.

[0022] In addition, they may be incompatible with instrument or packaging components (plastics, pipes, valves).

[0023] Moreover, the use of such reagents increases the number of containers required on the instrument, which unnecessarily disrupts laboratory management in areas such as order processing, inventory management, expiration date monitoring, waste disposal, and especially when the reagents are classified.

[0024] Finally, the viscosity and density of concentrated organic dye solutions differ from those of water-based reagents. To ensure proper sample handling over time, the final working solution must be mixed and homogenized. This undesirably complicates instrument fluid lines and mixing methods, leading to increased costs and a higher risk of malfunction.

[0025] Another method to avoid fluorescent dye aging is to apply a numerical compensation factor that adjusts as fluorescence intensity decreases. The lower the fluorescence, the higher the numerical compensation factor.

[0026] The above compensation principle involves monitoring and quantifying the decrease in dye fluorescence over time.

[0027] Degradation compensation can extend the shelf life of reagents containing fluorescent dyes without reducing analytical performance.

[0028] US 9,448,175 discloses a method for correcting fluorescence levels by measuring the optical density of the fluorescent dye at a given wavelength. This measurement is based on optical density measurements using spectrophotometry. These methods are spectrophotometric in nature and complement specialized means for measuring the fluorescence of labeled particles. Therefore, US 9,448,175 requires two distinct measurement units: one for spectrophotometric measurement of the optical density of the fluorescent dye, and another for fluorescence measurement of the labeled particles. US 9,448,175 explicitly discloses using optical density as a relevant parameter for monitoring the decay of the fluorescent dye. No other parameters are disclosed for monitoring this decay.

[0029] Hulspas's "Flow Cytometry and the Stability of Phycoerythrin-TandemDye Conjugates," Cytometry Part A, volume 75A, 11 Nov 2009, pp. 966-972, discloses a method for compensating for the variability of emission spectra of tandem dyes bound to proteins. Hulspas discloses the variations in the spectral properties of the two components (donor and acceptor) of the tandem dyes. In particular, Hulspas investigated the effect of the instability of the tandem dyes on the compensation matrix values ​​over long periods (p. 969, Col. 2). Spectral compensation is only necessary when multiple fluorescent dyes are used simultaneously. In this case, multiple fluorescence emission spectra overlap (spillover), and the compensation matrix is ​​used to minimize the spillover effect. Hulspas discloses simulations achieved by determining the compensation matrix for each time point of the particle-bound tandem dye group. Hulspas uses a compensation matrix that handles spillover between the spectra of multiple tandem dyes.

[0030] The purpose of this invention is to provide a method for quantifying and / or compensating for changes in fluorescence intensity of reagents containing fluorescent dyes in their binding state (i.e., when the fluorescent dye binds to blood cells).

[0031] Another object of the present invention is a novel method for determining a compensation factor for changes in the fluorescence radiation intensity of a reagent.

[0032] The present invention also aims to provide a simple, efficient and rapid method for compensating for changes in fluorescence radiation intensity.

[0033] At least one of the above objectives can be achieved by using a method that determines a compensation factor to compensate for changes in the fluorescence intensity of a reagent containing a fluorescent dye, said compensation factor being intended to be applied during measurements of the bound-state fluorescence intensity when cells are labeled with a fluorescent dye, said method comprising at least the following steps:

[0034] - Before measuring the bound state fluorescence intensity, measure the free state fluorescence intensity emitted by the reagent containing the fluorescent dye when the fluorescent dye is not bound to the cell.

[0035] - Determine the compensation factor based on the measured free-state fluorescence radiation intensity.

[0036] This invention can be advantageously used to compensate for changes in the fluorescence intensity of reagents containing fluorescent dyes due to aging of the fluorescent dye or batch-to-batch variations in composition. Composition variations can include chemical / process variations or variations due to storage / transport conditions.

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

[0038] Free-state fluorescence measurements are performed using a reagent containing a fluorescent dye; this reagent is then used to label cells.

[0039] The method according to the invention uses the fluorescence parameters of the dye in its unbound free state to monitor and compensate for changes in the fluorescence intensity of the dye when it is used in its bound state in subsequent measurements. Subsequent measurements may include, for example, cell characterization.

[0040] Fluorescent molecules (or dyes) used in flow cytometry have two main characteristics: their ability to bind to specific features of blood cells and their ability to re-emit light (fluorescence) when properly excited. Fluorescent dyes are known to degrade over time due to various environmental factors. That is, some molecules of the dye may break down into several parts over time due to environmental factors. If the molecular structure is disrupted, it will lose its ability to bind to specific features of blood cells and its ability to re-emit light (fluorescence) when properly excited.

[0041] The inventors of this invention have noted that fluorescence loss due to molecular decomposition exists simultaneously in both the free state of the dye (not bound to blood cell features) and the bound state of the dye (bound to specific blood cell features).

[0042] According to the present invention, the decrease in the fluorescence of the dye in its free state also reflects the decrease in its bound state fluorescence. Therefore, the decrease in the fluorescence of the dye in its bound state can be monitored by measuring the fluorescence of the dye in its free state.

[0043] In particular, a compensation algorithm for the change in fluorescence intensity in the bound state can be derived by monitoring and quantifying the evolution of its free-state fluorescence intensity.

[0044] The present invention can effectively quantify and / or compensate for the decrease in fluorescence intensity of reagents containing fluorescent dyes as the fluorescent dyes age.

[0045] In contrast, existing technologies such as document US9448175 monitor optical density; this is not free fluorescence.

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

[0047] The excitation wavelength and detection wavelength are the same. In this invention, we directly measure the free-state fluorescence of the dye used in its ionized (unbound) state, for example using the same optical system used to measure the re-emission of fluorescence from a fluorescent dye bound to a cell. This is not optical density (the light transmitted through the sample at a given wavelength) because when excited by an illumination source, the fluorescent light is re-emitted by the fluorescent dye at a wavelength greater than that of the illumination source. The excitation spectrum and emission spectrum are different.

[0048] The existing 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 the decrease in bound-state fluorescence do not require the use of any calibration materials (particles, proteins, nucleic acids, calibration materials, etc.).

[0049] In this invention, the same type of physical phenomenon (light emission) was measured to quantify the changes in the fluorescence of the dye in its free state and the final dye-bound state measurement parameters used for blood cell characterization.

[0050] The measurement of the optical density of fluorescent dyes disclosed in US 9,448,175 is susceptible to various measurement errors. Spectrophotometric measurements of a substance can be affected by many factors in the substance: absorption and / or scattering by other particles, impurities, bubbles, inhomogeneity, etc.

[0051] Unlike absorption, fluorescence emission is not at risk of being interfered with or impaired by impurities or inhomogeneities; it is a more robust parameter.

[0052] According to the embodiments, the present invention may include performing at least two measurements of the free state fluorescence intensity at different times before measuring the bound state fluorescence intensity; and determining a compensation factor based on the measured value of the free state fluorescence intensity.

[0053] This invention provides a method for performing multiple free-state fluorescence measurements to obtain multiple values, thereby accurately deriving the compensation factor for bound-state changes. Those skilled in the art, knowing the average rate of change of the fluorescent dye, can determine different appropriate times to perform the free-state fluorescence measurements.

[0054] Specifically, a 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 duration between t1 and t2 is at least greater than the duration between t2 and the bound-state fluorescence intensity measurement.

[0055] This invention provides a method for performing a first measurement of free-state fluorescence intensity immediately after the preparation of a reagent containing a fluorescent dye, and a second measurement of free-state fluorescence shortly before fluorescence measurement of cells labeled with the fluorescent dye. These two measurements, performed at appropriate times, allow for accurate determination of the compensation factor.

[0056] US 9,448,175 also requires two measurements, but it is based on optical density measurement. The compensation factor in US 9,448,175 is based on changes in optical density, i.e., variations, thus absolutely requiring two different optical density measurements. In contrast, the present invention proposes an alternative solution below that requires only a single measurement.

[0057] According to a preferred embodiment of the present invention, the compensation factor can be based on a predetermined normalization function Φ. N Determine; at least one measured value of the free-state fluorescence radiation intensity of a reagent containing a fluorescent dye is used as a predetermined normalization function Φ. N At least one independent variable.

[0058] According to the present invention, the measured value of the free-state fluorescence intensity of the reagent containing the fluorescent dye can be used as the independent variable of a normalization function, which serves as a compensation factor for the variation in the subsequent bound-state fluorescence intensity measurement.

[0059] Advantageously, the value of the free-state fluorescence radiation intensity of a single measured reagent containing a fluorescent dye can be used as a predetermined normalization function Φ. N A single independent variable.

[0060] This invention utilizes the measurement of a single free-state fluorescence intensity of a reagent containing a fluorescent dye as a normalization function Φ. N The scheme does not rely on estimating changes in free-state fluorescence intensity; it uses a normalized function Φ as the single independent variable. N Systematic normalization of the fluorescence level of the dye-bound state.

[0061] According to the normalization function of the present invention, only a single measurement of one characteristic of the dye, namely its free-state fluorescence intensity, is required to derive the fluorescence compensation factor needed for subsequent measurements of dye binding to blood cells.

[0062] This eliminates the need for initial measurements of fluorescent dye properties immediately after preparation, thus greatly relaxing restrictions on the preparation process.

[0063] Any subsequent binding state fluorescence measurement, i.e., when the dye binds to blood cells, is performed via the function Φ. N After normalization, the function has a single independent variable: the previously measured intensity of free-state fluorescence radiation.

[0064] According to an embodiment of the present invention, the predetermined normalization function Φ N It can be a polynomial function, the ratio of a polynomial function, an exponential function, a logarithmic function, a hyperbolic function, or a combination thereof. It can also be piecewise defined, meaning that different ranges of its single independent variable have different analytical definitions.

[0065] According to a preferred embodiment of the present invention, the free state fluorescence intensity of the first reagent sample can be measured; the bound state fluorescence intensity of the second reagent sample can be measured, wherein 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 came from the same reagent.

[0067] Another objective of this invention is to use the compensation mechanism based on free-state fluorescence measurement disclosed above to compensate for the changes in the intensity of bound-state fluorescence radiation of fluorescent reagents between preparation batches.

[0068] During the preparation of fluorescent reagents, slight changes in the reagent composition may occur due to variations in the purity of raw materials or human and environmental factors (accumulated weighing, inaccurate filling measurements), such as slight changes in the concentration of fluorescent dyes.

[0069] Although all prepared batches met the predetermined acceptance criteria for the defined fluorescent reagents, the free-state fluorescence intensity compensation of this technique can further smooth out slight variations in the intensity of bound-state fluorescence that may persist, and ensure better consistency of the final results.

[0070] The compensation factor, determined by measurements of free-state fluorescence radiation intensity, normalizes the results and yields a fluorescence scatter plot when using newly bottled reagents containing fluorescent dyes, regardless of storage time or slight variations in composition (especially the concentration of fluorescent dyes).

[0071] The composition of the first and second reagents can be the same or slightly different. For example, they can be prepared at different times, in different regions, or using different components.

[0072] The present invention may include multiple steps of measuring the intensity of free-state fluorescence radiation of a reagent containing a fluorescent dye in order to accurately monitor changes in fluorescence radiation intensity.

[0073] According to a preferred embodiment of the invention, the same optical sensor can be used to measure both the ionized fluorescence intensity and the bound fluorescence intensity. This ensures that there is no measurement bias between determining the change in ionized fluorescence intensity and measuring the fluorescence intensity of the fluorescent dye that ultimately binds to blood cells.

[0074] However, another dedicated sensor can also be used.

[0075] The spectrophotometric measurement of the optical density of fluorescent dyes disclosed in US 9,448,175 requires additional equipment. This necessitates at least one dedicated light source, photodetector, and acquisition chain for such measurements. With this invention, the same optical sensors can be used to derive the compensation factor and perform the final measurement. This results in a cheaper, more compact, and more robust instrument.

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

[0077] According to embodiments of the present invention, the same emission wavelength range can be used to measure the fluorescence intensity of the ionized state and the fluorescence intensity of the bound state.

[0078] According to an embodiment of the present invention, the concentrations of the fluorescent dyes used to measure the fluorescence intensity in the free state and the fluorescence intensity in the bound state can be the same or different.

[0079] This invention allows for the use of the same sensor to measure both the ionized fluorescence intensity and the final bound fluorescence intensity for cell characterization. Therefore, the ionized fluorescence intensity measurement and the final bound fluorescence intensity measurement can have the same excitation and emission spectral conditions. This improves the measurement consistency between monitoring the decay of the free fluorescence of the fluorescent dye and the final fluorescence measurement of the dye bound to blood cells.

[0080] According to an embodiment of the present invention, the step of measuring the intensity of free-state fluorescence radiation can be achieved using a reagent containing a single fluorescent dye.

[0081] In another embodiment, the step of measuring the intensity of free-state fluorescence radiation is performed using a reagent containing at least two fluorescent dyes. Depending on the excitation and emission spectra of the fluorescent dyes, additional illumination and / or detection modules may need to be added to the above embodiment.

[0082] If the fluorescent dyes contained in the reagent cannot be excited by the same light source due to incompatibility between the light source and the excitation spectrum of the fluorescent dyes, at least one illumination module needs to be added so that each fluorescent dye can be excited by an illumination module with a spectrum compatible with its excitation spectrum.

[0083] If the emission spectrum of the fluorescent dye contained in the reagent is incompatible with the wavelength range of a single fluorescence measurement module, at least one additional fluorescence measurement module is required so that the fluorescence emission of each fluorescent dye can be measured by a module with a bandwidth compatible with its emission spectrum.

[0084] The present invention also relates to an optical flow cytometer comprising a processing unit configured to:

[0085] -Measure the intensity of free-state fluorescence radiation of the fluorescent dye contained in the reagent.

[0086] -The compensation factor is determined based on the measured free-state fluorescence radiation intensity.

[0087] -Measure the intensity of bound-state fluorescence radiation in cells labeled with fluorescent dyes, and

[0088] - Apply the compensation factor to the measured fluorescence intensity of the bound state.

[0089] Further advantages and features of the invention will become apparent from a review of the detailed description and accompanying drawings of the non-limiting embodiments, in which:

[0090] Figure 1 This is a global view showing the components of an optical flow cytometer that can be used to implement the method according to the invention;

[0091] Figure 2 This is a flowchart illustrating a method for determining compensation factors according to the present invention;

[0092] Figure 3 This is a diagram illustrating the decay of free-state fluorescence;

[0093] Figure 4 This is a diagram illustrating the decay of fluorescence in the bound state;

[0094] Figure 5a and 5b Examples of uncompensated and compensated binding state fluorescence levels measured at t=0; and

[0095] Figure 6a and 6b Examples of uncompensated and compensated binding state fluorescence levels were measured at t = 10 months.

[0096] While the invention may have various modifications and alternatives, specific embodiments thereof are illustrated by way of example in the accompanying drawings and will be described in detail herein. It should be understood that the drawings and the detailed description thereof are not intended to limit the invention to the specific forms disclosed, but rather are intended to cover all modifications, equivalents, and alternatives falling within the scope of the invention as defined by the appended claims.

[0097] In the following, the invention will be described in detail by referring to the accompanying drawings and explaining exemplary embodiments thereof.

[0098] Figure 1 This is a global view showing the components of an optical flow cytometer that can be used to implement the method according to the invention.

[0099] Reference Figure 1 The optical flow cytometer 1 of the present invention includes an illumination module 2 for generating an illumination beam 3 toward a flow chamber 4 in which particles such as blood cells are flowing. The optical flow cytometer 1 includes motor and / or other means to drive and aggregate sample cells or blood cells into a flow that is encapsulated or not encapsulated by sheath fluid. Blood cells circulate through the flow chamber 4.

[0100] The illumination beam 3 is focused and passes through the cell stream, inducing fluorescence in the fluorescent sample particles or their labels. The fluorescence 7 produced by the labeled blood cells is collected by the fluorescence measurement module 5.

[0101] As blood cells pass through the optical detection zone, the illumination beam 3 also induces scattered light 8. The optical flow cytometer 1 includes a scattering measurement module 6, which collects the scattered light 8 from the flow chamber 4.

[0102] A processing unit 9 is provided to control the excitation module 2 used for the excitation signal. The processor unit 7 also controls the fluorescence measurement module 5 and the scattering measurement module 6 to detect direct and / or indirect scattering signals.

[0103] exist Figure 1 In one implementation, the scattering measurement module 6 is positioned opposite the illumination module 2, and the fluorescence measurement module 5 is positioned at 90° to the optical axis of the illumination beam. Other arrangements can be achieved by deflecting the light using lenses, mirrors, and / or beam splitters.

[0104] The present invention also relates to an epifluorescence configuration (not shown), wherein the excitation module 2 may include a fluorescence measurement module 5. In this configuration, the same focusing lens is used to focus the excitation beam onto the flow chamber and collect fluorescence from the flow chamber.

[0105] According to the present invention, the fluorescence measurement module 5 is preferably an optical sensor for measuring free fluorescence and bound fluorescence.

[0106] Processing unit 9, such as a microprocessor, is configured to implement the method according to the invention.

[0107] Figure 2 This is a flowchart illustrating a method for determining compensation factors according to the present invention.

[0108] like Figure 1 The device described herein can be used to implement the method according to the invention.

[0109] The first sequence according to the invention relates to the measurement of free-state fluorescence. In step 10, reagents containing fluorescent dyes in the device are considered.

[0110] In step 11, Figure 1The processing unit 9 drives different components of the device 1 to measure the free fluorescence of the fluorescent dye contained in the reagent.

[0111] Optical sensor 5 receives a fluorescence signal, which is processed to determine the value of fluorescence radiation intensity in step 12.

[0112] Multiple measurements can be performed between reagent preparation and the next binding state fluorescence measurement. As those skilled in the art will know, the function can be derived from multiple values, and a compensation factor can be identified each time a binding state fluorescence measurement is provided.

[0113] The compensation factor can be a fixed value determined shortly before the binding state fluorescence measurement. Alternatively, the compensation factor can be a function of time.

[0114] Alternatively, a single free-state fluorescence measurement can be performed and the fluorescence value calculated in step 11. In step 12, the fluorescence value is calculated according to a predetermined normalization function Φ. N Determine the compensation factor. The calculated fluorescence value is used as the predetermined normalization function Φ. N A single independent variable.

[0115] In step 13, the user prepares to use the optical flow cytometer 1, for example, to characterize different types of cells present in blood by fluorescence. This device can be designed to count the number of white blood cells contained in, for example, a blood sample, and determine their relative distribution in various subpopulations based on light scattering and fluorescence.

[0116] In step 13, the reagent is introduced into the bloodstream. This reagent contains a fluorescent dye for which a compensation factor has been determined based on multiple free-state fluorescence measurements or a single free-state fluorescence measurement.

[0117] In step 14, the compensation factor calculated during the free state fluorescence measurement is considered when performing the bound state fluorescence measurement.

[0118] For this purpose, illumination module 2 generates excitation light directed toward flow chamber 3. Fluorescence from blood cells is collected by optical sensor 5. Processing unit 9 calculates the fluorescence value, taking into account a compensation factor. This value is useful information for blood cell characterization in step 15.

[0119] Figure 3 This is a graph illustrating the decay of free-state fluorescence. In this example, multiple free-state fluorescence measurements were performed. Multiple fluorescence intensity values ​​(in arbitrary units) have been calculated. These values ​​can be plotted as a curve, i.e., a function of time. It can be seen that at t = 0 months, shortly after the preparation of the reagent containing the fluorescent dye, the level is approximately 900. At t = 6 months, the level drops to 500, and at t = 10 months, the level drops to 450.

[0120] The decay of the fluorescence intensity of a fluorescent dye in its free state reflects a decrease in its bound state fluorescence. Therefore, the decay of the bound state fluorescence of a fluorescent dye can be monitored by measuring its free state fluorescence.

[0121] Figure 4 yes Figure 3 The graph illustrates the fluorescence decay of the bound state of the fluorescent dye. Curve 16 shows the change in uncompensated fluorescence intensity, i.e., the change in the original data. (The last sentence appears to be incomplete and possibly contains errors.) Figure 3 Multiple bound-state fluorescence measurements were performed at the same time points for the free-state fluorescence. Values ​​of multiple fluorescence radiance intensities (in arbitrary units) were calculated. These values ​​were plotted as a function of time. It can be seen that at t = 0 months, shortly after the preparation of the reagent containing the fluorescent dye, the level was approximately 237. The level decreased to 175 at t = 6 months and to 150 at t = 10 months. The change in bound-state fluorescence without compensation corresponds to the change in free-state fluorescence. Specifically, a compensation algorithm for the decay of bound-state fluorescence can be derived by monitoring and quantifying the decay of free-state fluorescence.

[0122] The compensation factor was calculated after each measurement of the free state fluorescence intensity and the bound state fluorescence intensity at t = 0 months, 2 months, 4 months, 6 months, 8 months, 9 months and 10 months. Figure 4 Curve 17 in the figure illustrates the change in compensated fluorescence radiation intensity, i.e., the change in compensated data. It can be seen that curve 17 fluctuates around the initial value of 237 represented by curve 18.

[0123] According to the present invention, a predetermined normalization function Φ can be used. N This is used to determine the compensation factor. An example of such a normalization function is as follows:

[0124] Φ M (t, t0) = α*fluorescence 游离 (t0) / fluorescence 游离 (t)+β,

[0125] Where α and β are two coefficients that depend on the fluorescent dye used, fluorescence 游离 t is the intensity of free-state fluorescence radiation, and t is time.

[0126] The compensation can be applied using the following equation:

[0127] fluorescence 结合经补偿 (t) = fluorescence 结合原始 (t)*ΦN(t, t0),

[0128] Fluorescence 结合经补偿 (t) is the value of the compensated binding state fluorescence radiation intensity, fluorescence 结合原始t is the value of the uncompensated binding state fluorescence intensity, and t is time.

[0129] Figures 5 and 6 show graphs of fluorescence intensity measurements of different binding states of blood cells labeled with fluorescent dyes. The fluorescent dyes are included in reagents used in flow cytometry to characterize blood cell populations. Gy is the Y-value (i.e., the coordinate along the fluorescence axis) of the centroid of the {neutrophil + eosinophil} population.

[0130] Figure 5a This is a fluorescence scatter plot showing the fluorescence intensity of the bound state measured at t=0 months. No compensation was applied, and Gy=88. This measurement corresponds to the raw data at t=0.

[0131] Figure 5b This is a fluorescence scatter plot showing the fluorescence intensity of the bound state measured at t=0 months. In this case, compensation was applied, and Gy=87. The compensation factor is very close to 1, so it has no effect.

[0132] Figure 6a This is a fluorescence scatter plot showing the binding state fluorescence intensity measured at t = 10 months. No compensation was applied, and Gy = 50. The measurement results show a decrease along the Y-axis relative to the measurement at t = 0. To compensate for this decrease, at... Figure 6b A compensation factor was applied.

[0133] Figure 6b This is a fluorescence scatter plot showing the fluorescence intensity of the bound state measured at t = 10 months. In this case, with... Figure 5b The same applies; compensation was applied, and Gy = 87. The compensation factor is much greater than 1, thus correcting the descent on the Y-axis.

[0134] Once the foregoing disclosure is fully understood, those skilled in the art will recognize numerous variations and modifications. The following claims are intended to be construed as covering all such variations and modifications.

[0135] For example, this invention can be used to compensate for variations in fluorescence intensity between reagent batches. To this end, the intensity of ionized fluorescence can be automatically measured when reagent vials are changed (activated) on instruments such as cell analyzers.

[0136] If reagent bottle A is already installed on the instrument, at least one measurement of the ionized radiation intensity can be performed during its installation to determine the specific compensation factor for that batch at that storage time. If necessary (in cases where the reagent bottle is retained on the instrument for an extended period), other measurements can be performed to correct the compensation factor…

[0137] When changing from reagent bottle A to reagent bottle B (regardless of whether they are from the same or different batches, and regardless of their condition), a new measurement of the free-state fluorescence intensity is performed; then, if necessary, the fluorescence compensation factor on the instrument is adjusted. This new measurement eliminates:

[0138] - Chemical changes (changes in the purity of the fluorescent dye used, changes in the weight of reagent compounds, and changes in the liquid level in the storage tank during the production process),

[0139] - Changes in "time" (relative aging of the two reagent bottles),

[0140] - Changes in the "environment" (temperature and storage conditions of the reagent bottle).

Claims

1. A method for compensating for variations in fluorescence intensity of a reagent containing a fluorescent dye by determining a compensation factor, said compensation factor being intended to be applied during measurements of bound-state fluorescence intensity when cells are labeled with a fluorescent dye, said method comprising at least the following steps: - Before measuring the bound state fluorescence intensity, measure the free state fluorescence intensity emitted by the reagent containing the fluorescent dye when the fluorescent dye is not labeled in the cells. - Determine the compensation factor based on the measured free-state fluorescence radiation intensity.

2. The method according to claim 1, characterized in that... This includes performing at least two measurements of the free state fluorescence intensity at different times before measuring the bound state fluorescence intensity; and determining a compensation factor based on the measured values ​​of the free state fluorescence intensity.

3. The method according to claim 2, characterized in that... A free-state fluorescence intensity measurement is performed at time t1 after the preparation 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. The duration between t1 and t2 is at least greater than the duration between t2 and the bound-state fluorescence intensity measurement.

4. The method according to claim 1, characterized in that... According to the predetermined normalization function Φ N Determine the compensation factor; at least one value of the measured free-state fluorescence radiation intensity of the reagent containing the fluorescent dye is used as a predetermined normalization function Φ. N At least one independent variable.

5. The method according to claim 4, characterized in that... The reagent containing the fluorescent dye uses only a single measured value of the free-state fluorescence intensity as a predetermined normalization function Φ. N A single independent variable.

6. The method according to claim 4 or 5, characterized in that... The predetermined normalization function Φ N This includes polynomial functions, ratios of polynomial functions, exponential functions, logarithmic functions, hyperbolic functions, or combinations thereof.

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

8. The method according to any one of the preceding claims, characterized in that... The free-state fluorescence intensity of the first reagent sample was measured; the bound-state fluorescence intensity of the second reagent sample was measured. The first and second reagent samples were from a single container containing a fluorescent dye.

9. The method according to any one of the preceding claims, characterized in that... The procedure involves repeatedly measuring the intensity of free-state fluorescence to monitor the decrease in fluorescence intensity.

10. The method according to any one of the preceding claims, characterized in that... The same optical sensor was used to measure the fluorescence intensity in both the free and bound states.

11. The method according to any one of the preceding claims, characterized in that... The same excitation spectrum was used to measure the fluorescence intensity of the free state and the fluorescence intensity of the bound state.

12. The method according to any one of the preceding claims, characterized in that... The fluorescence intensity of the ionized state and the fluorescence intensity of the bound state were measured using the same emission wavelength range.

13. The method according to any one of the preceding claims, characterized in that... The concentrations of the fluorescent dyes used to measure the fluorescence intensity in the free state and the fluorescence intensity in the bound state may be the same or different.

14. The method according to any one of the preceding claims, characterized in that... The procedure involves measuring the intensity of free-state fluorescence radiation using a reagent containing a single fluorescent dye.

15. The method according to any one of claims 1 to 13, characterized in that... The step involves measuring the intensity of free-state fluorescence radiation using a reagent containing at least two fluorescent dyes; and wherein: Use at least one illumination module such that each fluorescent dye is excited by an illumination module having a spectrum compatible with its excitation spectrum, and / or Use at least one fluorescence measurement module so that the fluorescence emission of each fluorescent dye is measured by a module having a bandwidth compatible with its emission spectrum.

16. An optical flow cytometer comprising a processing unit configured to: -Measure the intensity of free-state fluorescence radiation of the fluorescent dye contained in the reagent. -The compensation factor is determined based on the measured free-state fluorescence radiation intensity. -Measure the intensity of bound-state fluorescence radiation in cells labeled with fluorescent dyes, and - Apply the compensation factor to the measured fluorescence intensity of the bound state.

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