Patent Description:
Malaria, caused by malaria parasites, is one of the most serious diseases endangering human health. Currently, malaria parasites are detected usually by means of microscopic examination of a blood smear, but this method relies heavily on experience of an operator, requires a high level of expertise for the operator, and is time-consuming.

With the development of blood cell analysis technology, a variety of methods, which can quickly detect erythrocytes infected with malaria parasites by using a hematology analyzer, are currently known.

European Patent Application <CIT> discloses a method for staining infected erythrocytes with a plurality of fluorescent dyes under a non-hemolytic condition, so as to better discriminate between reticulocytes and infected erythrocytes.

European Patent Application <CIT> discloses a method for detecting malaria parasites with a fluorescent dye under a hemolytic condition, which can implement the classification and counting of malaria parasites, but cannot implement the classification and counting of leukocytes at the same time.

Patent Application <CIT> discloses a reagent capable of partially lysing cell membranes of erythrocytes infected with malaria parasites, such that the malaria parasites are retained in the erythrocytes, and a fluorescent dye can pass through the cell membranes. However, erythrocytes infected with malaria parasites cannot be accurately detected when there are high values of reticulocytes in a sample.

Chinese Patent Application <CIT> discloses a method for detecting erythrocytes infected with malaria parasites. In this method, a sample to be tested is treated with a specific fluorescent dye of a specific concentration, allowing more accurate detection of erythrocyte infected with malaria parasites than the solution disclosed in U. Patent Application <CIT>.

Chinese Patent Application <CIT> discloses a blood analysis apparatus and a blood analysis method that can classify leukocytes in a test sample into <NUM> types and detect malaria-infected erythrocytes while reducing the burden on users caused by reagent development. However, in this method, although a same hemolytic agent is used, two blood samples need to be provided for different processing, and differential detection of leukocytes and detection of malaria-infected erythrocytes are performed separately in two tests, causing increased test time, blood volume, and costs of the hemolytic agent.

European Patent <CIT> discloses a reagent for staining malaria infected cells and a method for detecting malaria infected cells using the same, wherein the regent is a staining solution comprising at least one first dye of an Auramine analogue, and a detecting method for malaria infected cells using the reagent by which malaria infected cells, in particular malaria infected erythrocytes, can be stained rapidly and specifically. The method includes: treating a test sample with a staining solution which comprises at least one first dye of an Auramine analogue and at least one second dye of a condensed benzene derivative; and optically detecting stained malaria infected cells.

Patent <CIT> discloses a blood analyzer including: a specimen preparation unit configured to mix a blood sample with a hemolyzing agent which hemolyzes red blood cells and with a fluorescence-labeled antibody reagent which labels a predetermined surface antigen on blood cells, to prepare a first measurement specimen; a flow cell through which the first measurement specimen prepared by the specimen preparation unit is caused to flow; a light source unit configured to emit light to the first measurement specimen flowing in the flow cell; light receivers configured to respectively receive first scattered light, second scattered light, and first fluorescence which are obtained from blood cells in the first measurement specimen as a result of the emission of the light; and a processing unit configured to identify and count lymphocytes in the first measurement specimen by using first scattered light information based on the first scattered light and second scattered light information based on the second scattered light, and configured to identify and count blood cells having thereon the predetermined surface antigen in the first measurement specimen by using first fluorescence information based on the first fluorescence. The specimen preparation unit mixes the blood sample, a diluent, a hemolyzing agent, and a staining solution for staining plasmodium nucleic acid together, to prepare a second measurement specimen to be used in second measurement. The light receivers respectively receive third scattered light and second fluorescence, which are obtained from blood cells in the second measurement specimen as a result of the emission of the light. The processing unit uses the third scattered light information and the second fluorescence information to count malaria-infected red blood cells in the second measurement specimen.

Patent Application <CIT> relates to a microfluidic microscopy device. The absorbent structure may include two or more dyes configured to stain different components of a cell or different types of cells. For example, the at least one dry dye for a liquid includes a first fluorescing dye for staining DNA and/or RNA and a second fluorescing dye for staining a plasma membrane or cytoplasmic component. The spectrofluorometer can be used to measure and record fluorescence spectra emitted from a fluorescent dye or dyes associated with the liquid sample in the tapered internal chamber. For example, the optical properties of the cellular components of the liquid sample in the tapered internal chamber is used to differentiate white blood cells from red blood cells. For example, the optical properties of the cellular components of the liquid sample in the tapered internal chamber is used to differentiate Plasmodium-infected and -uninfected red blood cells.

<CIT> discloses a blood detection method for obtaining a platelet count in a blood sample, which is treated with a first reagent and a hemolytic agent for lysing red blood cells in the blood sample into fragments having light scattering characteristics significantly different from those of platelets.

One objective of the disclosure is to provide an improved solution for detecting malaria parasites, in which simultaneous detection of leukocyte parameters and infected erythrocyte parameters can be implemented in one single test, especially in the current leukocyte detection channel. This solution can obtain a variety of detection parameters in one single test, save blood volume for detection, and reduce detection costs compared with the prior art.

Another objective of the disclosure is to provide an improved solution for detecting malaria parasites, in which the detection of infected erythrocyte parameters using two fluorescent dyes under a hemolytic condition can be implemented.

The invention is defined by the method of claim <NUM> and a sample analyzer according to claim <NUM>. Further aspects of the invention are defined in the dependent claims.

The embodiments of the disclosure will be clearly and completely described below in conjunction with the accompanying drawings. Apparently, the described embodiments are merely some, rather than all, of the embodiments of the disclosure. Based on the embodiments of the disclosure, all the other embodiments that would have been obtained by those of ordinary skill in the art without any creative efforts shall fall within the scope of protection of the disclosure.

The serial numbers themselves for the components herein, for example, "first" and "second", are merely used to distinguish the described objects, and do not have any sequential or technical meaning. Moreover, as used in the disclosure, "connection" or "coupling", unless otherwise stated, includes both direct and indirect connections (couplings).

The hematology analyzer used in the disclosure implements classification and counting of particles in a blood sample through a flow cytometry technique using a laser scattering method and a fluorescence staining method in combination. The detection principle of the hematology analyzer is as follows: first, a blood sample is aspirated, and the sample is treated with a hemolytic agent and a fluorescent dye, wherein erythrocytes are destroyed and hemolyzed by the hemolytic agent, while leukocytes will not be hemolyzed, but the fluorescent dye can enter nucleus of the leukocytes with the help of the hemolytic agent and then is bound with nucleic acid substances of the nucleus; and then, particles in the sample are passed through a detection aperture irradiated by a laser beam one by one. When the laser beam irradiates the particles, properties (such as volume, staining degree, size and content of cell contents, density of cell nucleus, etc.) of the particles themselves may block or change a direction of the laser beam, thereby generating scattered light at various angles that corresponds to their properties, and the scattered light can be received by signal detectors to obtain relevant information about a structure and composition of the particles. Forward-scattered light (FS) reflects a number and a volume of particles, side-scattered light (SS) reflects a complexity of a cell internal structure (such as intracellular particles or nucleus), and fluorescence (FL) reflects a content of nucleic acid substances in a cell. The optical information can be used to implement classification and counting of the particles in the blood sample.

<FIG> is a schematic diagram of a hematology analyzer used in an embodiment of the disclosure. The hematology analyzer <NUM> includes a sampling apparatus <NUM>, a sample preparation apparatus <NUM>, an optical detection apparatus <NUM>, and a processor <NUM>. The hematology analyzer <NUM> has a liquid circuit system (not shown) for connecting the sampling apparatus <NUM>, the sample preparation apparatus <NUM> and the optical detection apparatus <NUM> for liquid transport between these apparatuses.

The sampling apparatus <NUM> has a pipette with a pipette nozzle and has a driving apparatus for driving the pipette to quantitatively aspirate a blood sample to be tested through the pipette nozzle. The sampling apparatus can transport the aspirated blood sample to be tested to the sample preparation apparatus <NUM>.

The sample preparation apparatus <NUM> has at least one reaction cell and a reagent supply portion, wherein the at least one reaction cell is configured to receive the blood sample to be tested that is aspirated by the sampling apparatus <NUM>, and the reagent supply portion is configured to supply a hemolytic agent and fluorescent dyes (including a first dye capable of staining leukocytes and a second dye capable of staining infected erythrocytes) to the at least one reaction cell, such that the blood sample to be tested that is aspirated by the sampling apparatus is mixed in the reaction cell with the hemolytic agent and the fluorescent dyes supplied by the reagent supply portion to prepare a test sample solution. The hemolytic agent may be any of existing hemolytic agents used for classification of leukocytes in an automated hematology analyzer. The hemolytic agent may be any one or a combination of a cationic surfactant, a non-ionic surfactant, an anionic surfactant, and an amphiphilic surfactant. Details of the first dye and the second dye will be further explained below.

The optical detection apparatus <NUM> includes a light source, a flow cell, at least one scattered light detector, and at least two fluorescence detectors, wherein the light source is configured to emit a light beam to irradiate the flow cell; the flow cell is connected with the reaction cell, and particles in the test sample solution are capable of passing through the flow cell one by one; the scattered light detector is configured to detect scattered light signals generated by the particles when passing through the flow cell after being irradiated with the light beam; and the fluorescence detectors are configured to detect fluorescence signals generated by the particles when passing through the flow cell after being irradiated by light.

In some embodiments, the optical detection apparatus <NUM> includes a forward-scattered light detector for detecting forward-scattered light or a side-scattered light detector for detecting side-scattered light. The optical detection apparatus <NUM> preferably includes both the forward-scattered light detector and the side-scattered light detector.

<FIG> shows a specific example of the optical detection apparatus <NUM>. The optical detection apparatus <NUM> includes a laser <NUM>, a front optical assembly <NUM>, a flow cell <NUM>, a forward-scattered light detector <NUM>, a first dichroscope <NUM>, a side-scattered light detector <NUM>, a second dichroscope <NUM>, a first fluorescence detector <NUM>, and a second fluorescence detector <NUM>. The first fluorescence detector <NUM> is configured to detect first fluorescence signals that correspond to the first dye and that are generated by the particles when passing through the flow cell <NUM> after being irradiated with the light beam, and the second fluorescence detector <NUM> is configured to detect second fluorescence signals that correspond to the second dye and that are generated by the particles when passing through the flow cell <NUM> after being irradiated with the light beam. Here, the laser <NUM>, the front optical assembly <NUM>, the flow cell <NUM>, and the forward-scattered light detector <NUM> are sequentially arranged on an optical axis in a direction of the optical axis, and the front optical assembly is configured such that excitation light emitted by the laser <NUM> converges in a detection region of the flow cell <NUM> in a flow direction of the particles, and the particles flowing through the detection region of the flow cell <NUM> can thus generate scattered light. On one side of the flow cell <NUM>, the first dichroscope <NUM> is arranged at an angle of <NUM>° to the optical axis. Part of side light generated by the particles when flowing through the detection region of the flow cell <NUM> is reflected by the first dichroscope <NUM> and is captured by the side-scattered light detector <NUM>, while the other part of the side light is transmitted through the first dichroscope <NUM> to the second dichroscope <NUM>, and the second dichroscope <NUM> is also arranged downstream of the first dichroscope <NUM> at an angle of <NUM>° to the optical axis. Part of the side light that is transmitted through the first dichroscope <NUM> is reflected by the second dichroscope <NUM> and is captured by the first fluorescence detector <NUM>, while the other part of the side light that is transmitted through the second dichroscope <NUM> is captured by the second fluorescence detector <NUM>.

In other embodiments, as shown in <FIG>, unlike the optical detection apparatus shown in <FIG>, the forward-scattered light detector <NUM> may also be arranged to be inclined to the optical axis. On the optical axis, a mirror <NUM> is arranged downstream of the flow cell in the direction of the optical axis. The mirror reflects the forward-scattered light of the particles into the forward-scattered light detector <NUM> arranged to be inclined to the optical axis.

The processor <NUM> is configured to process optical signals collected by the optical detection apparatus <NUM>, to obtain a required result, for example, may be configured to generate a two-dimensional scattergram or a three-dimensional scattergram based on the collected optical signals, and analyze particles using a gating method on the scattergram. The processor <NUM> may also be configured to perform visualization processing on an intermediate operation result or a final operation result, and then display same by a display apparatus <NUM>. In embodiments of the disclosure, the processor <NUM> is configured to implement the method which will be described in detail below. The processor <NUM> include, but is not limited to, a central processing unit (CPU), a micro controller unit (MCU), a field-programmable gate array (FPGA), a digital signal processor (DSP) and other apparatuses for interpreting computer instructions and processing data in computer software. For example, the processor <NUM> is configured to execute each computer application program in a computer-readable storage medium, so that the hematology analyzer <NUM> preforms a corresponding detection process and analyzes, in real time, optical signals detected by the optical detection apparatus <NUM>.

In addition, the hematology analyzer <NUM> further includes a first housing <NUM> and a second housing <NUM>. The display apparatus <NUM> may be, for example, a user interface. The optical detection apparatus <NUM> and the processor <NUM> are provided inside the second housing <NUM>. The sample preparation apparatus <NUM> is provided, for example, inside the first housing <NUM>, and the display apparatus <NUM> is provided, for example, on an outer surface of the first housing <NUM> and configured to display test results from the hematology analyzer. In other embodiments, a computer having a display may be remotely and communicatively connected to the hematology analyzer <NUM>. The computer is installed, for example, in a place far away from a laboratory where the hematology analyzer is located, such as in a doctor's consulting room.

Next, the detection method proposed in the disclosure is described in detail. The method proposed in the disclosure and various embodiments thereof are particularly applied to the above hematology analyzer <NUM>, and are particularly implemented by the processor <NUM> of the above hematology analyzer <NUM>.

In order to implement simultaneous detection of infected erythrocytes and leukocytes in one single test, the disclosure first proposes treating a same blood sample with at least two fluorescent dyes under a hemolytic condition and detecting the treated blood sample, and then identifying both leukocytes and infected erythrocytes based on optical signals obtained in the same test of the same treated blood sample. In the disclosure, one dye is capable of staining leukocytes, while the other dye is capable of staining infected erythrocytes.

<FIG> is a schematic flowchart of a sample analysis method <NUM> according to an embodiment of the disclosure. The sample analysis method <NUM> includes the following steps.

In step S210, optical signals generated by particles in one test sample solution after being irradiated by excitation light when the particles pass through an optical detection region of an optical detection apparatus one by one are obtained in one single test. In this step, the test sample solution is obtained by treating a blood sample with a hemolytic agent, a first dye and a second dye, the first dye being capable of staining leukocytes, and the second dye being capable of staining infected erythrocytes, wherein the optical signals include scattered light signals, first fluorescence signals corresponding to the first dye, and second fluorescence signals corresponding to the second dye.

Specifically, a blood sample of a subject is first provided, which is generally stored in a test tube, and the sampling apparatus <NUM> aspirates a portion of the blood sample in the test tube through a pipette and then delivers same to the sample preparation apparatus <NUM>. The portion of the blood sample is mixed with the hemolytic agent, the first dye, and the second dye in the reaction cell of the sample preparation apparatus <NUM> and incubated for a period of time, such as for <NUM> to <NUM>, to ensure that erythrocytes membranes are destroyed by the hemolytic agent and cells are stained, so as to form a test sample solution. The test sample solution is transported to the flow cell <NUM> of the optical detection apparatus <NUM> through a liquid circuit system, and particles in the test sample solution are passed through a detection aperture of the flow cell one by one. Then, the scattered light detectors <NUM> and <NUM>, the first fluorescence detector <NUM>, and the second fluorescence detector <NUM> respectively detect the scattered light signals, the first fluorescence signals, and the second fluorescence signals generated by the particles when passing through the flow cell after being irradiated by light.

In step S210, the hemolytic agent, the first dye, and the second dye may be added to the blood sample sequentially or simultaneously. It is also possible that the first dye and the second dye are mixed and then added to the blood sample.

In step S220, optical information of leukocytes of the blood sample is obtained based on the first fluorescence signals and at least one type of the scattered light signals. Here, the optical information of leukocytes is optical information related to leukocytes.

For example, the optical information of leukocytes may be a first scattergram. In this step, a first scattergram of the blood sample is generated based on the first fluorescence signals and at least one type of the scattered light signals, and then leukocytes in the test sample solution are classified and/or counted based on the first scattergram. The first scattergram may be a two-dimensional scattergram generated based on forward-scattered light signals and the first fluorescence signals, or a two-dimensional scattergram generated based on side-scattered light signals and the first fluorescence signals, or preferably a three-dimensional scattergram generated based on the forward-scattered light signals, the side-scattered light signals, and the first fluorescence signals. It should be noted that, the scattergram herein is not limited to being presented graphically, and may also be presented in the form of data, such as in the form of digital tables or lists with the same or similar resolution as that of the scattergram, or in any other suitable manner known in the field.

In step S230, optical information of infected erythrocytes of the blood sample is obtained based on the second fluorescence signals and at least one type of the scattered light signals or at least based on the first fluorescence signals and the second fluorescence signals, that is, the optical information of infected erythrocytes is obtained based on the second fluorescence signals, and one type of other optical signals than the second fluorescence signals. Here, the optical information of infected erythrocytes is optical information related to infected erythrocytes.

Similarly, the optical information of infected erythrocytes may be a second scattergram. For example, the second scattergram may be a two-dimensional scattergram generated based on the forward-scattered light signals and the second fluorescence signals or based on the side-scattered light signals and the second fluorescence signals, or a two-dimensional scattergram generated based on the first fluorescence signals and the second fluorescence signals.

In some embodiments, the first dye is a non-nucleic acid-specific dye, and the second dye is a deoxyribonucleic acid (DNA)-specific fluorescent dye. The first fluorescence signals are fluorescence emitted after binding the non-nucleic acid-specific dye with leukocytes, and the second fluorescence signals are fluorescence emitted after binding the nucleic acid-specific dye with malaria-infected cells. The nucleic acid dye can specifically stain infected erythrocytes, and since there difference in nucleic acid content of infected erythrocytes of different types and/or at different development stages, the disclosure can also distinguish between infected erythrocytes of different types and/or at different development stages by staining degree of the second dye while counting infected erythrocytes.

Particularly advantageous, in the optical detection apparatus <NUM> of the disclosure, excitation light at a single wavelength is used to irradiate the test sample solution in the flow cell, that is, the optical signals are generated by the particles in the test sample solution after being irradiated by the excitation light at the single wavelength when the particles pass through the optical detection region of the optical detection apparatus one by one. In other words, the light source <NUM> of the optical detection apparatus <NUM> is configured as a laser that emits an excitation light at a single wavelength. In some embodiments, the light source <NUM> may be a laser that emits blue-green or red light, for example, may be a laser that emits light with a wavelength of <NUM> or <NUM> nanometers.

In some embodiments, as shown in <FIG>, the sample analysis method <NUM> may further include step <NUM>: classifying and/or counting leukocytes in the test sample solution based on the optical information of leukocytes.

For example, step S221 may include: classifying the leukocytes in the test sample solution into a neutrophil granulocyte population, a lymphocyte population, a monocyte population, and an eosinophil granulocyte population based on the optical information of leukocytes. Specifically, a first scattergram is generated based on the side-scattered light signals and the first fluorescence signals or based on the forward-scattered light signals, the side-scattered light signals and the first fluorescence signals, and on the first scattergram, the leukocytes in the test sample solution are classified into a neutrophil granulocyte population, a lymphocyte population, a monocyte population, and an eosinophil granulocyte population by using a gating technique, and the cell populations are then counted.

In an alternative embodiment, step S221 may include: identifying basophils in the test sample solution and counting the leukocytes in the test sample solution based on the optical information of leukocytes. Specifically, a first scattergram is generated based on the forward-scattered light signals and the first fluorescence signals, and basophils in the test sample solution are identified and the leukocytes in the test sample solution are counted based on the first scattergram. Further, in this embodiment, nucleated erythrocytes in the test sample solution can also be identified while identifying the basophils.

In some embodiments, the sample analysis method <NUM> may further include identifying immature leukocytes in the test sample solution based on the first fluorescence signals and at least one type of the scattered light signals.

In some embodiments, as shown in <FIG>, the sample analysis method <NUM> may further include steps <NUM> and <NUM>. In step <NUM>, infected erythrocytes are counted based on the optical information of infected erythrocytes, to obtain a count value. For example, a second scattergram is generated based on the forward-scattered light signals and the second fluorescence signals or based on the first fluorescence signals and the second fluorescence signals, and a region representing infected erythrocytes is obtained based on the second scattergram by using a gating technique, and scatters falling into the region are counted to obtain the count value of the infected erythrocytes. In step <NUM>, if the count value of the infected erythrocytes is greater than a predetermined threshold, an alarm prompt is outputted (determining that the blood sample is a malaria positive sample). Further, infected erythrocytes of different types and/or infected erythrocytes at different development stages can also be classified and counted based on the optical information of infected erythrocytes, for example, the infected erythrocytes are classified at least into rings, and for example, the infected erythrocytes can be classified into rings, trophozoites, and schizonts.

Preferably, in order to be able to more accurately distinguish between the leukocytes and the infected erythrocytes by two dyes under the hemolytic condition, particularly when the same excitation light source is used, the first dye and the second dye are selected such that an absolute value of a difference between wavelengths corresponding to peaks of emission spectra of the first dye and the second dye is greater than <NUM> nanometers and less than <NUM> nanometers. Alternatively or additionally, the first dye and the second dye are selected such that an overlap between emission spectra of the first dye and the second dye is not greater than <NUM>%. Through such selection of the first dye and the second dye, not only can interference between detecting the first fluorescence signals and detecting the second fluorescence signals be greatly reduced, that is, the degree of discrimination between the first fluorescence signals and the second fluorescence signals is greatly reduced, but the volume and complexity of the optical detection apparatus will not be increased.

<FIG> is a schematic diagram of emission spectra of the first dye and the second dye, in which a curve shown by a solid line is an emission spectrum <NUM> of the first dye, and a curve shown by a dotted line is an emission spectrum <NUM> of the second dye. A peak point of the emission spectrum <NUM> of the first dye is D, and a peak point of the emission spectrum <NUM> of the second dye is A. Here, a difference between respective abscissas of the peak point D and the peak point A (i.e., a difference between wavelengths corresponding to the peaks) is greater than <NUM> nanometers and less than <NUM> nanometers. In addition, an overlap between the emission spectrum <NUM> of the first dye and the emission spectrum <NUM> of the second dye may be a ratio of the area of a first polygon to the area of a second polygon, where the area of the first polygon is equal to the area of a curved polygon surrounded by three points, namely the point E, the point G, and the point C, and the area of the second polygon is equal to the area of a curved polygon surrounded by the emission spectrum <NUM> of the first dye (or the emission spectrum <NUM> of the second dye) and a reference line <NUM>. The reference line <NUM> is a dotted horizontal line parallel to a horizontal axis as shown in <FIG>, and the dotted horizontal line is at <NUM>% of a normalized peak of the emission spectrum <NUM> of the first dye and the emission spectrum <NUM> of the second dye. The point E and the point F are respectively a left intersection and a right intersection of the emission spectrum <NUM> of the first dye and the reference line <NUM>, and the point B and the point C are respectively a left intersection and a right intersection of the emission spectrum <NUM> of the second dye and the reference line <NUM>. Here, the overlap between the emission spectrum <NUM> of the first dye and the emission spectrum <NUM> of the second dye is not greater than <NUM>%.

Further, advantageously, especially when irradiated by a single light source, an absolute value of a difference between wavelengths corresponding to the peaks of the emission spectra of the first dye and the second dye is greater than <NUM> nanometers and less than <NUM> nanometers, preferably greater than <NUM> nanometers and less than <NUM> nanometers, more preferably greater than <NUM> nanometers and less than <NUM> nanometers. In this case, the interference between detecting the first fluorescence signals and detecting the second fluorescence signals can be further reduced without increasing the volume and complexity of the optical detection apparatus.

In addition, advantageously, the overlap between the emission spectra of the first dye and the second dye is not greater than <NUM>%, preferably not greater than <NUM>%. In this case, the interference between detecting the first fluorescence signal and detecting the second fluorescence signal can also be further reduced.

In some embodiments, at least one of the first dye and the second dye, particularly the first dye, may be a dye with a large Stokes shift. Here, the dye with a large Stokes shift is a dye with a difference between wavelengths corresponding to respective peaks of an emission spectrum and an excitation spectrum being greater than a predetermined threshold.

<FIG> is a schematic diagram of a dye with a large Stokes shift, in which an excitation spectrum (also referred to as absorption spectrum) <NUM> of the dye with a large Stokes shift is shown by a dotted line, and an emission spectrum <NUM> of the dye with a large Stokes shift is shown by a solid line. A peak point of the excitation spectrum <NUM> is A1, and a peak point of the emission spectrum <NUM> is A2. A difference between respective abscissas of the peak point A2 and the peak point A1 (i.e., a difference between wavelengths corresponding to respective peaks of the emission spectrum and the excitation spectrum) is greater than a predetermined threshold. The predetermined threshold may be, for example, greater than <NUM> nanometers and less than <NUM> nanometers, preferably greater than <NUM> nanometers and less than <NUM> nanometers.

By using at least one dye with a large Stokes shift, interference between detecting the first fluorescence signals and detecting the second fluorescence signals can be reduced.

In some embodiments, a parent of the first dye may be a meso-amino-substituted cyanine dye, or a dye parent with a typical electronic push-pull system, such as carbazole and coumarin. For example, the first dye may have a parent structure of general formula I:
<CHM>
where R1, R2, and R3 are substituents, which can be any element, such as hydrogen element.

For more details of the first dye and the second dye of the disclosure, reference may be made to <CIT>.

In addition, the disclosure further provides a computer-readable storage medium having instructions stored thereon, wherein the instructions, when executed by a processor, cause the processor to implement the above sample analysis method <NUM> and one of the embodiments thereof.

The foregoing computer-readable storage medium may be a volatile memory or a non-volatile memory, or may include both a volatile memory and a non-volatile memory. The non-volatile memory may be a read-only memory, a programmable read-only memory, an erasable programmable read-only memory, an electrically erasable programmable read-only memory, a magnetic random access memory, a flash memory, a magnetic surface memory, an optical disc, or a compact disc read-only memory. The magnetic surface memory may be a disk memory or a magnetic tape memory. The volatile memory may be a random access memory, and is used as an external cache. In addition, many forms of RAMs can be applied to the disclosure, such as a static random access memory, a synchronous static random access memory, a dynamic random access memory, a synchronous dynamic random access memory, a double data rate synchronous dynamic random access memory, an enhanced synchronous dynamic random access memory, a synchlink dynamic random access memory, and a direct rambus dynamic random access memory.

Next, the specific embodiments of the disclosure and corresponding results are described by means of the following specific examples.

The first dye has the following general formula:
<CHM>
and the second dye has the following general formula:
<CHM>.

BC-<NUM> with 68LN hemolytic agent from Mindray Bio-medical Electronics Co. , Ltd was used.

Test method: <NUM> microliters of blood sample and <NUM> microliters of staining reagent were taken, simultaneously added to <NUM> of hemolytic agent, and incubated for <NUM> seconds, and after incubation was completed, a flow cytometer was used to detect the sample to be tested to collect forward-scattered light signals, first fluorescence signals, and second fluorescence signals. A first scattergram as shown in <FIG> was generated based on the forward-scattered light signals and the first fluorescence signals, and leukocytes were identified based on the first scattergram and were then counted, particularly basophils in the leukocytes could be identified. A second scattergram as shown in <FIG> was generated based on the forward-scattered light signals and the second fluorescence signals, and infected erythrocytes were identified based on the second scattergram, and were then classified into rings, trophozoites, and schizonts.

Test method: <NUM> microliters of blood sample and <NUM> microliters of staining reagent were taken, simultaneously added to <NUM> of hemolytic agent, and incubated for <NUM> seconds, and after incubation was completed, a flow cytometer was used to detect the sample to be tested to collect forward-scattered light signals, first fluorescence signals, and second fluorescence signals. A first scattergram as shown in <FIG> was generated based on the forward-scattered light signals and the first fluorescence signals, and leukocytes were identified based on the first scattergram and were then counted, particularly nucleated erythrocytes and basophils were identified based on the first scattergram. A second scattergram as shown in <FIG> was generated based on the forward-scattered light signals and the second fluorescence signals, and infected erythrocytes were identified based on the second scattergram, and were then classified into rings, trophozoites, and schizonts.

Test method: <NUM> microliters of blood sample and <NUM> microliters of staining reagent were taken, simultaneously added to <NUM> of hemolytic agent, and incubated for <NUM> seconds, and after incubation was completed, a flow cytometer was used to detect the sample to be tested to collect forward-scattered light signals, first fluorescence signals, and second fluorescence signals. A first scattergram as shown in <FIG> was generated based on the forward-scattered light signals and the first fluorescence signals, and leukocytes were identified based on the first scattergram and were then counted. A second scattergram as shown in <FIG> was generated based on the forward-scattered light signals and the second fluorescence signals, and infected erythrocytes were identified based on the second scattergram, and were then classified into rings, trophozoites, and schizonts.

Test method: <NUM> microliters of blood sample and <NUM> microliters of staining reagent were taken, simultaneously added to <NUM> of hemolytic agent, and incubated for <NUM> seconds, and after incubation was completed, a flow cytometer was used to detect a sample to be tested to collect forward-scattered light signals, first fluorescence signals, and second fluorescence signals. A first scattergram as shown in <FIG> was generated based on the forward-scattered light signals and the first fluorescence signals, and leukocytes were classified into a neutrophil granulocyte population, a lymphocyte population, a monocyte population, and an eosinophil granulocyte population based on the first scattergram. A second scattergram as shown in <FIG> was generated based on the forward-scattered light signals and the second fluorescence signals, and infected erythrocytes were identified based on the second scattergram.

BC-<NUM> with 68LD hemolytic agent from Mindray Bio-medical Electronics Co. , Ltd was used.

Test method: <NUM> microliters of blood sample and <NUM> microliters of staining reagent were taken, simultaneously added to <NUM> of hemolytic agent, and incubated for <NUM> seconds, and after incubation was completed, a flow cytometer was used to detect a sample to be tested to collect forward-scattered light signals, side-scattered light signals, first fluorescence signals, and second fluorescence signals. A first scattergram as shown in <FIG> was generated based on the side-scattered light signals and the first fluorescence signals, and leukocytes were classified into a neutrophil granulocyte population, a lymphocyte population, a monocyte population, and an eosinophil granulocyte population based on the first scattergram. A second scattergram as shown in <FIG> was generated based on the forward-scattered light signals and the second fluorescence signals, and infected erythrocytes were identified based on the second scattergram.

Claim 1:
A sample analysis method for analyzing a blood sample, comprising:
obtaining (<NUM>), in one single test, optical signals generated by particles in a test sample solution after being irradiated by excitation light when the particles pass through an optical detection region of an optical detection apparatus one by one, wherein the test sample solution is obtained by treating the blood sample with a hemolytic agent, a first dye and a second dye, the first dye being capable of staining leukocytes, and the second dye being capable of staining infected erythrocytes, and wherein the optical signals comprise scattered light signals, first fluorescence signals corresponding to the first dye and second fluorescence signals corresponding to the second dye, wherein the first fluorescence signals and the second fluorescence signals are respectively detected by different fluorescence detectors;
obtaining (<NUM>) optical information of leukocytes of the blood sample based on the first fluorescence signals and at least one type of the scattered light signals; and
obtaining (<NUM>) optical information of infected erythrocytes of the blood sample based on the second fluorescence signals and at least one type of the scattered light signals.