Patent Application
20230296591
The disclosure relates to the field of blood testing, and in particular, to a sample analysis method, a sample analyzer, and a computer-readable storage medium.
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 EP 0613003 B1 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 EP 1406088 A2 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.
U.S. Patent Application US 2006/0223137 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 CN 106483278 B 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.S. Patent Application US 2006/0223137.
Chinese Patent Application CN 102016573 B discloses a blood analysis apparatus and a blood analysis method that can classify leukocytes in a test sample into 4 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.
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.
In order to achieve an objective of the disclosure, a first aspect of the disclosure relates to a sample analysis method for analyzing a blood sample, including:
A second aspect of the disclosure relates to a computer-readable storage medium having instructions stored thereon, wherein the instructions, when executed by a processor, cause the processor to implement the sample analysis method according to the first aspect of the disclosure.
A third aspect of the disclosure relates to a sample analyzer, including:
The processor of the sample analyzer according to the third aspect of the disclosure is particularly configured to implement the sample analysis method according to the first aspect of the disclosure.
In order to achieve another objective of the disclosure, a fourth aspect of the disclosure relates to another sample analysis method for analyzing a blood sample, including:
A fifth aspect of the disclosure relates to a computer-readable storage medium having instructions stored thereon, wherein the instructions, when executed by a processor, cause the processor to implement the sample analysis method according to the fourth aspect of the disclosure.
A sixth aspect of the disclosure relates to a sample analyzer, including:
In the technical solution provided in various aspects of the disclosure, a hemolytic agent, a first dye capable of staining leukocytes, and a second dye capable of staining infected erythrocytes are used to treat a same blood sample in one single test, particularly in a leukocyte test, to obtain a test sample solution, and then, an optical detection apparatus is used to detect scattered light signals, first fluorescence signals, and first fluorescence signals generated by particles in the test sample solution after being irradiated by excitation light, particularly excitation light at a single wavelength, such that optical information of leukocytes is obtained based on the first fluorescence signals and at least one type of the scattered light signals, and optical information of infected erythrocytes is obtained based on the second fluorescence signals and at least one type of the scattered light signals or based on the first fluorescence signals and the second fluorescence signals. Therefore, the optical information of leukocytes and the optical information of infected erythrocytes can be obtained simultaneously without increasing blood volume for use, thereby greatly reducing detection costs.
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.
The sampling apparatus 110 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 120.
The sample preparation apparatus 120 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 110, 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 130 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 130 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 130 preferably includes both the forward-scattered light detector and the side-scattered light detector.
In other embodiments, as shown in
The processor 140 is configured to process optical signals collected by the optical detection apparatus 130, 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 140 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 150. In embodiments of the disclosure, the processor 140 is configured to implement the method which will be described in detail below. The processor 140 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 140 is configured to execute each computer application program in a computer-readable storage medium, so that the hematology analyzer 100 preforms a corresponding detection process and analyzes, in real time, optical signals detected by the optical detection apparatus 130.
In addition, the hematology analyzer 100 further includes a first housing 160 and a second housing 170. The display apparatus 150 may be, for example, a user interface. The optical detection apparatus 130 and the processor 140 are provided inside the second housing 170. The sample preparation apparatus 120 is provided, for example, inside the first housing 160, and the display apparatus 150 is provided, for example, on an outer surface of the first housing 160 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 100. 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 100, and are particularly implemented by the processor 140 of the above hematology analyzer 100.
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.
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 110 aspirates a portion of the blood sample in the test tube through a pipette and then delivers same to the sample preparation apparatus 120. 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 120 and incubated for a period of time, such as for 10 to 30s, 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 133 of the optical detection apparatus 130 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 134 and 136, the first fluorescence detector 138, and the second fluorescence detector 139 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 130 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 131 of the optical detection apparatus 130 is configured as a laser that emits an excitation light at a single wavelength. In some embodiments, the light source 131 may be a laser that emits blue-green or red light, for example, may be a laser that emits light with a wavelength of 488 or 520 nanometers.
In some embodiments, as shown in
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 200 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
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 30 nanometers and less than 80 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 50%. 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.
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 40 nanometers and less than 80 nanometers, preferably greater than 50 nanometers and less than 80 nanometers, more preferably greater than 50 nanometers and less than 70 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 35%, preferably not greater than 15%. 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.
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:
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 Chinese patent application no. 202011008754.3, which is incorporated herein by reference in its entirety.
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 200 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.
Formula of staining reagent:
The first dye has the following general formula:
and the second dye has the following general formula:
BC-6800 with 68LN hemolytic agent from Mindray Bio-medical Electronics Co., Ltd was used.
Test method: 20 microliters of blood sample and 20 microliters of staining reagent were taken, simultaneously added to 1 ml of hemolytic agent, and incubated for 30 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
Formula of staining reagent:
The first dye has the following general formula:
and the second dye has the following general formula:
BC-6800 with 68LN hemolytic agent from Mindray Bio-medical Electronics Co., Ltd was used.
Test method: 20 microliters of blood sample and 20 microliters of staining reagent were taken, simultaneously added to 1 ml of hemolytic agent, and incubated for 30 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
Formula of staining reagent:
The first dye has the following general formula:
and the second dye has the following general formula:
BC-6800 with 68LN hemolytic agent from Mindray Bio-medical Electronics Co., Ltd was used.
Test method: 20 microliters of blood sample and 20 microliters of staining reagent were taken, simultaneously added to 1 ml of hemolytic agent, and incubated for 30 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
Formula of staining reagent:
The first dye has the following general formula:
and the second dye has the following general formula:
BC-6800 with 68LN hemolytic agent from Mindray Bio-medical Electronics Co., Ltd was used.
Test method: 20 microliters of blood sample and 20 microliters of staining reagent were taken, simultaneously added to 1 ml of hemolytic agent, and incubated for 30 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
Formula of staining reagent:
The first dye has the following general formula:
and the second dye has the following general formula:
BC-6800 with 68LD hemolytic agent from Mindray Bio-medical Electronics Co., Ltd was used.
Test method: 20 microliters of blood sample and 20 microliters of staining reagent were taken, simultaneously added to 1 ml of hemolytic agent, and incubated for 30 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
Formula of staining reagent:
The first dye has the following general formula:
and the second dye has the following general formula:
BC-6800 with 68LN hemolytic agent from Mindray Bio-medical Electronics Co., Ltd was used.
Test method: 20 microliters of blood sample and 20 microliters of staining reagent were taken, simultaneously added to 1 ml of hemolytic agent, and incubated for 30 seconds, and after incubation was completed, a flow cytometer was used to detect a sample to be tested to collect first fluorescence signals and second fluorescence signals. A second scattergram as shown in
The features or combinations thereof mentioned above in the description, accompanying drawings, and claims can be combined with each other arbitrarily or used separately as long as they are meaningful within the scope of the disclosure and do not contradict each other. The advantages and features described with reference to the sample analysis method provided in the disclosure are applicable in a corresponding manner to the sample analyzer and the computer-readable storage medium provided in the disclosure, and vice versa.
The foregoing description merely relates to the preferred embodiments of the disclosure, and is not intended to limit the scope of patent protection of the disclosure. All equivalent variations made by using the content of the specification and the accompanying drawings of the disclosure from the concept of the disclosure, or the direct/indirect applications of the contents in other related technical fields all fall within the scope of patent protection of the disclosure.
This application is a continuation of International Application No. PCT/CN2020/133039, filed Dec. 1, 2020, the contents of which are incorporated herein by reference.
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/CN2020/133039 | Dec 2020 | US |
| Child | 18203532 | US |
