CN113125392B - Sample analyzer, method and computer-readable storage medium for detecting cryptococcus - Google Patents
Sample analyzer, method and computer-readable storage medium for detecting cryptococcus Download PDFInfo
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- CN113125392B CN113125392B CN201911415630.4A CN201911415630A CN113125392B CN 113125392 B CN113125392 B CN 113125392B CN 201911415630 A CN201911415630 A CN 201911415630A CN 113125392 B CN113125392 B CN 113125392B
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- G—PHYSICS
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- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
- G01N21/47—Scattering, i.e. diffuse reflection
- G01N21/49—Scattering, i.e. diffuse reflection within a body or fluid
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- G—PHYSICS
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
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Abstract
本申请公开了检测样本中的隐球菌的样本分析仪、方法以及计算机可读存储介质。所述样本分析仪包括：采样装置、样本制备装置、光学检测装置和控制装置，其与所述光学检测装置电连接并包括处理器和存储有计算机程序的存储介质，其中，所述控制配置为当所述计算机程序被所述处理器执行时，执行以下步骤：从所述光学检测装置获取所述光学信息中的至少两种光强度信息，并且根据所述至少两种光强度信息区分出所述待测样本中的隐球菌粒子群。本发明利用致病性隐球菌具有厚荚膜的特性可快速准确地对样本是否存在隐球菌感染进行检测。
The present application discloses a sample analyzer, method and computer-readable storage medium for detecting cryptococci in a sample. The sample analyzer includes: a sampling device, a sample preparation device, an optical detection device and a control device, which is electrically connected to the optical detection device and includes a processor and a storage medium storing a computer program, wherein the control is configured to perform the following steps when the computer program is executed by the processor: obtaining at least two types of light intensity information in the optical information from the optical detection device, and distinguishing the cryptococcal particle group in the sample to be tested based on the at least two types of light intensity information. The present invention utilizes the characteristic of pathogenic cryptococci having a thick capsule to quickly and accurately detect whether a sample has a cryptococcal infection.
Description
Technical Field
The present application relates to the field of in vitro diagnostics, and in particular to a sample analyzer, a method and a computer readable storage medium for detecting cryptococcus in a sample.
Background
Cryptococcus is a common fungus that causes invasive infections in humans. Cryptococcus (Cryptococcus) comprises 17 and 18 varieties, most notably Cryptococcus neoformans (Cryptococcus neoformans) pathogenic to humans. Cryptococcus neoformans are widely distributed in nature and can also exist on the body surface, mouth and intestinal tracts of the human body.
Cryptococcus is generally an exogenous infection. Invasion into the human body through the respiratory tract and spreading from the pulmonary tract can invade all organ tissues, the most common part is the central nervous system. Cryptococcosis neonatorum is well developed in patients with cellular immune dysfunction, such as AIDS, malignant tumor, diabetes, organ transplantation, and patients with large doses of glucocorticoid. In recent 20 years, the incidence of cryptococcus has been increasing, and it has become one of the most common complications of AIDS in foreign countries, the leading cause of death of AIDS. In immunosuppressed patients, the incidence of cryptococcus infection is about 5% to 10%, and in AIDS patients, the incidence of cryptococcus infection can be as high as 30%. It is counted that about 100 tens of thousands of people are infected with cryptococcus every year worldwide, and mortality can be as high as 63%. Cryptococcosis and viral hepatitis have been equally classified as infectious diseases of type B in China, which is a serious mycosis faced by humans.
The existing detection methods of cryptococcus include ink negative staining method, culture identification method, antigen detection method, antibody detection method, nucleic acid detection method, etc.
The cerebrospinal fluid ink negative staining method is the most commonly used cryptococcus detection means in clinic at present. The pathogenic cryptococcus has wide and thick capsules outside the thallus, and has strong refraction, when the thallus is negatively dyed by ink, the capsules wrap the thallus so that the thallus is not dyed by the ink, thus the round transparent thallus is wrapped by a thicker blank zone, and normal cells, other pathogenic bacteria and non-pathogenic cryptococcus in cerebrospinal fluid are dyed into black by the ink and cannot be observed under a microscope.
Culture identification method is to inoculate specimen on sand culture medium, and pathogenic cryptococcus can grow at 25 deg.C and 37 deg.C, but pathogenic cryptococcus does not grow at 37 deg.C. After 2-5 days of culture, observing the morphological characteristics of the colony, and taking the colony for biochemical identification. Cryptococcus neoformans has positive phenol oxidase test and urease test, can assimilate glucose, galactose, sucrose, inositol and raffinose, but cannot ferment sugar and alcohols.
The antigen detection method is a method for detecting cryptococcus capsular polysaccharide specific antigen in cerebrospinal fluid or blood plasma by using immunological methods such as latex agglutination test, ELISA, polyclonal antibody and monoclonal antibody method, wherein the latex agglutination test (latex agglutination test, LAT) is most commonly used. LAT is an indirect agglutination assay that uses polystyrene latex particles as a carrier, and combines soluble antibodies with the polystyrene latex particles to produce Cheng Zhimin latex particles, which then crosslink with antigens in the sample to be tested, and agglutination occurs in a plurality of latex particles.
Antibody detection methods use radioimmunoassay and tube agglutination assays to detect cryptococcus antibodies in serum.
Nucleic acid detection methods use the polymerase chain reaction (Polymerase Chain Reaction, PCR) to detect specific nucleic acid sequences of cryptococcus pathogenic, typically the cryptococcus capsular related protein gene (CAP 10), in a sample.
The detection sensitivity of the ink negative-dyeing method and the culture identification method is lower, and the culture time of the culture identification method is longer. Although the antigen, antibody detection method and nucleic acid detection method have higher sensitivity, the false positive rate is higher, the detection cost is higher, and the detection is not a conventional detection item, and whether the detection is performed or not needs to be judged according to the experience of a clinician, so that subjective factors are also one of reasons for restricting the development of the detection item.
Thus, there is a need to develop a more convenient and accurate method of detecting cryptococcus.
Disclosure of Invention
The application aims to provide a sample analyzer and a method for conveniently, rapidly and accurately detecting cryptococcus.
To this end, a first aspect of the present application provides a sample analyzer comprising:
A sampling device having a pipette with a pipette nozzle and having a driving device for driving the pipette to quantitatively aspirate a sample to be measured through the pipette nozzle;
A sample preparation device having at least one reaction cell for receiving a sample to be measured which is sucked by a sampling device and a reagent supply section for supplying a hemolysis reagent and a fluorescent reagent to the at least one reaction cell, so that the sample to be measured which is sucked by the sampling device is mixed with the hemolysis reagent and the fluorescent reagent supplied by the reagent supply section in the reaction cell to prepare a sample to be measured;
An optical detection device comprising a light source, a flow cell through which particles of the sample to be detected can pass one by one, at least one scatter detector for collecting at least one scattered light intensity information, and a fluorescence detector for collecting fluorescence intensity information, wherein the optical information comprises the at least one scattered light intensity information and the fluorescence intensity information, the light emitted by the light source illuminating the particles in the flow cell to generate optical information; and
A control device electrically connected to the optical detection device and comprising a processor and a storage medium storing a computer program, wherein the control is configured to perform the following steps when the computer program is executed by the processor: and acquiring at least two kinds of light intensity information in the optical information from the optical detection device, and distinguishing the cryptococcus particle swarm in the sample to be detected according to the at least two kinds of light intensity information.
A second aspect of the present application provides another sample analyzer, comprising:
A sampling device having a pipette with a pipette nozzle and having a driving device for driving the pipette to quantitatively aspirate a sample to be measured through the pipette nozzle;
A sample preparation device having at least one reaction cell for receiving a sample to be measured sucked by the sampling device and a reagent supply portion configured to supply a hemolysis reagent to the at least one reaction cell so that the sample to be measured sucked by the sampling device and the hemolysis reagent supplied by the reagent supply portion are mixed in the at least one reaction cell to prepare a sample to be measured;
An optical detection device comprising a light source, a flow cell, a first scattered light detector and a second scattered light detector, wherein particles of the sample to be detected can pass through the flow cell one by one, the particles in the flow cell are irradiated by light emitted by the light source to generate optical information, the first scattered light detector is used for collecting forward scattered light intensity information, the second scattered light detector is used for collecting side scattered light intensity information, and the optical information comprises the forward scattered light intensity information and the side scattered light intensity information; and
A control device electrically connected to the optical detection device and comprising a processor and a storage medium storing a computer program, wherein the control is configured to perform the following steps when the computer program is executed by the processor: and acquiring the optical information from the optical detection device, and distinguishing the cryptococcus particle swarm in the sample to be detected according to the optical information.
In a third aspect the present application provides a method of detecting cryptococcus in a sample, the method comprising the steps of:
Processing the sample to be tested to obtain a sample to be tested, the processing the sample to be tested comprising mixing the sample to be tested with a hemolysis reagent;
enabling particles in the to-be-tested sample to pass through an optical detector one by one, and obtaining optical information of each particle in the to-be-tested sample;
and distinguishing the cryptococcus particle swarm in the sample to be detected according to the optical information.
In a fourth aspect, the application provides a method of detecting cryptococcus in a sample, the method comprising the steps of:
processing a first portion of the sample to be tested to obtain a first sample to be tested, the processing the first portion of the sample to be tested comprising mixing the first portion of the sample with a first hemolysis reagent and a first fluorescent dye;
Enabling particles in the first to-be-tested sample to pass through an optical detector one by one, and obtaining first optical information of each particle in the first to-be-tested sample;
Distinguishing and counting cryptococcus particle groups in the first to-be-detected sample according to the first optical information so as to obtain a first cryptococcus particle count value;
Processing a second portion of the sample to be tested to obtain a second sample to be tested, the processing the second portion of the sample to be tested comprising mixing the second portion of the sample with a second hemolysis reagent different from the first hemolysis reagent;
Enabling particles in the second to-be-tested sample to pass through the optical detector one by one, and obtaining second optical information of each particle in the second to-be-tested sample;
distinguishing and counting cryptococcus particle groups in the second to-be-detected sample according to the second optical information so as to obtain a second cryptococcus particle count value;
judging whether the cryptococcus infection exists in the sample to be tested according to the first cryptococcus particle count value and the second cryptococcus particle count value.
A fifth aspect of the present application provides a computer-readable storage medium having a computer program stored thereon, characterized in that the computer program, when executed by a processor, implements the steps of the method according to the third and fourth aspects of the present application described above.
The invention utilizes the characteristic that the pathogenic cryptococcus has thick capsule, and can rapidly and accurately detect whether the cryptococcus infection exists in the sample by utilizing instruments such as a full-automatic blood analyzer.
Drawings
In the drawings, like reference numerals refer to the same or similar parts.
FIG. 1 is a schematic flow chart of a method for detecting cryptococcus according to the present invention;
FIG. 2 is a schematic flow chart of a method for detecting cryptococcus in accordance with one embodiment of the present invention;
FIG. 3 is a schematic diagram of the detection of cryptococcus in accordance with a first embodiment of the present invention;
FIG. 4 is a schematic flow chart of a method for detecting cryptococcus in accordance with a first embodiment of the present invention;
FIG. 5 is a flow chart of a method for detecting cryptococcus in a cerebrospinal fluid sample according to a first embodiment;
FIG. 6 is an optical scatter plot of cryptococcus negative and positive cerebrospinal fluid samples obtained according to the detection method of FIG. 5;
FIG. 7 is a flow chart of a method for detecting cryptococcus in a blood sample according to a first embodiment;
FIG. 8 is a schematic diagram of the detection of cryptococcus in accordance with a second embodiment of the present invention;
FIG. 9 is a schematic flow chart of a method for detecting cryptococcus in accordance with a second embodiment of the present invention;
FIG. 10 is a plot of optical detection scatter of cryptococcus negative and positive cerebrospinal fluid samples obtained according to the detection method of FIG. 9;
FIG. 11 is a flow chart of a method for detecting cryptococcus in a blood sample according to a second embodiment;
FIG. 12 is a schematic flow chart of another method for detecting cryptococcus in accordance with the invention;
FIG. 13 is a schematic diagram of a sample detector according to the present invention; and
Fig. 14 is a schematic structural view of an optical detection device according to the present invention.
Detailed Description
The technical solutions of the present invention will be clearly and completely described below with reference to specific examples of the present invention and the accompanying drawings, and it is apparent that the described examples are only some embodiments of the present invention, not all embodiments. All other embodiments, based on the embodiments of the invention, which are apparent to those of ordinary skill in the art without inventive faculty, are intended to be within the scope of the invention.
Throughout the specification, unless specifically indicated otherwise, the terms used herein should be understood as meaning as commonly used in the art. Accordingly, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In case of conflict, the present specification will control.
Reference herein to 'Cryptococcus' refers, unless otherwise indicated, to the most common pathogenic Cryptococcus neoformans (Cryptococcus neoformans) in the genus Cryptococcus (Cryptococcus).
Reference herein to a 'sample' is to a body fluid sample, such as cerebrospinal fluid, hydrothorax, ascites, myocardial fluid, synovial fluid, peritoneal dialysis fluid, or fluid obtained by peritoneal cleaning, or a blood sample, unless otherwise indicated.
Reference herein to a 'haemolytic agent' unless otherwise indicated, refers to an agent capable of lysing erythrocytes while maintaining the basic cellular morphology of leukocytes.
Reference herein to a 'fluorescent dye' refers, unless otherwise indicated, to a dye that is capable of specifically binding to nucleic acid species (e.g., DNA, RNA) and emitting fluorescence of a particular wavelength upon excitation. The nucleic acid-specific dye used in the present application is not particularly limited. Commercial nucleic acid fluorescent dyes and nucleic acid specific fluorescent dyes already disclosed in some of the patent applications can be used in the present application. Among them, commercially available nucleic acid fluorescent dyes include SYTO series nucleic acid dyes from Thermofisher. In addition, the fluorescent dye disclosed in chinese patent application CN201010022414.6, the anthocyanin-based dye disclosed in CN200910109215.6, the fluorescent dye disclosed in CN200810216864.1, and the like can be used in the present application. The entire contents of the above patent documents are incorporated by reference into the present application.
The sample detection principle referred to herein refers to that after a blood cell analyzer sucks a body fluid or blood sample, the sample is first treated with a hemolyzing agent and optionally with a fluorescent dye, the red blood cells are destroyed and lysed by the hemolyzing agent, other particles, such as white blood cells, cannot be lysed, and the fluorescent dye can enter other particles, such as the nuclei of white blood cells, with the help of the hemolyzing agent and bind to the nucleic acid substance in the nuclei. Then, the particles in the processed sample pass through the laser detection holes one by one, when the laser beam passes through the particles, the characteristics (such as volume, dyeing degree, cell content size and content, cell nucleus density and the like) of the particles can block or change the direction of the laser beam, and scattered light with various angles corresponding to the characteristics of the characteristics is generated, and the scattered light can obtain relevant information on the structure and composition of the particles after being received by the signal detector. Wherein Forward Scatter (FS) reflects the number and volume of particles, side scatter (SIDE SCATTER, SS) reflects the complexity of the internal structure of the particles (e.g., particle or nucleus within the particle), and Fluorescence (FL) reflects the amount of nucleic acid species in the particle. The particles in the sample can be classified and counted using these parameters.
The inventors have found that the application of a haemolytic agent to the thick capsule of cryptococcus pathogenicus can produce optical properties that are different from other particles in the sample, thereby enabling detection of cryptococcus pathogenicus in the sample using the haemolytic channel of a conventional blood analyser. The detection method of the invention does not influence the normal detection of the hemolysis channel, so that the detection can be carried out on the index which can be detected by the conventional hemolysis channel in the sample.
Thus, the present application proposes a method for detecting cryptococcus in a sample. Referring to fig. 1, a schematic flow diagram of the present method is shown. The method comprises the following steps:
S11, processing a sample to be tested to obtain a sample to be tested, wherein the processing of the sample to be tested comprises at least processing the sample with a hemolysis reagent;
S12, enabling particles in the sample to be tested to pass through an optical detector one by one, and obtaining optical information of each particle in the sample to be tested; and
S13, distinguishing the cryptococcus particle swarm in the sample to be detected according to the optical information.
The hemolysis reagent of the present invention may contain those conventionally used for detecting blood cells. For example, the hemolyzing agent usable in the present invention may be selected from surfactants (such as quaternary ammonium salt type cationic surfactants, nonionic surfactants such as alkanol polyoxyethylene ether type surfactants, etc.), alkyl glycosides, triterpene saponins, steroid saponins, etc. The hemolysis reagent may also contain buffering agent (such as phosphoric acid and its salt, citric acid and its salt, acetic acid and its salt, etc.), antiseptic (such as sodium azide, proClin series, etc.), metal chelating agent (such as sodium salt of EDTA), osmotic pressure regulator (such as sodium chloride, etc.), etc.
The cryptococcus has a wide cell size range of about 5 to 20 μm, reflecting the tendency of forward scattered light intensities to extend from small to large, with some overlapping with the forward scattered light intensities of leukocytes. On the other hand, the internal structure of cryptococcus is less complex than that of leukocytes, so that the side scatter light intensity of cryptococcus is slightly lower than that of leukocytes.
Thus, in an embodiment, after the sample to be tested is treated with the hemolytic agent, the cryptococcus particle population in the sample to be tested can be distinguished based on the forward scattered light intensity information and the side scattered light intensity information in the optical information.
Further counting the cryptococcus particle swarm in the sample to be tested, and optionally prompting the existence of cryptococcus infection in the sample to be tested when the count value of the cryptococcus particle swarm exceeds a preset value.
According to one embodiment, the sample to be tested may be further treated with a fluorescent dye reagent.
In this embodiment, the fluorescent dye reagent may contain a fluorescent dye capable of staining nucleic acids. The present invention is not particularly limited in the kind of the fluorescent dye, and any of the fluorescent dyes as defined above may be used as required.
The fluorochrome reagent may also contain components such as buffers, surfactants, preservatives, metal chelators, osmolality adjusting agents and the like, which are not described in detail herein.
In a specific embodiment, the hemolysis reagent and the fluorescent dye reagent may be two separate reagents, which are mixed with the sample sequentially or simultaneously to prepare the test sample; alternatively, the hemolysis reagent and the fluorescent dye reagent may be combined into one reagent containing both the hemolysis reagent and the fluorescent dye.
Referring to fig. 2, a schematic flow chart of the present embodiment is shown. As shown in fig. 2, in this embodiment, first, in step S110, a sample to be measured is mixed with a hemolysis reagent and a fluorescent dye reagent (or a reagent containing a hemolysis reagent and a fluorescent dye), and reacted to obtain a sample to be measured. In step S120, the particles in the sample to be measured are passed through the optical detection device one by one, and optical information of each particle in the sample is obtained. And in step S130 cryptococcus particles in the test sample may be distinguished from other particles based on the detected fluorescence intensity information and at least one scattered light intensity information, e.g., forward scattered light intensity information and/or side scattered light intensity information.
In step S130, the cryptococcus particle population in the sample to be measured is preferably distinguished according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information.
It can be appreciated that in step S130, the cryptococcus particle population in the sample to be tested may also be distinguished based on the forward scattered light intensity information and the side scattered light intensity information in the optical information.
Further, the cryptococcus particle population in the test sample may be counted, and optionally, when the count value of the cryptococcus particle population exceeds a predetermined value, the presence of cryptococcus infection in the test sample is indicated.
According to a specific case, the sample to be tested is a body fluid sample, in particular a cerebrospinal fluid sample. In this case, the body fluid sample to be tested may be treated with a hemolysis reagent, and after optical detection, the cryptococcus particle population in the sample may be distinguished based on the forward and side scattered light intensity information. Alternatively, the body fluid sample to be tested may be treated with a hemolysis reagent and a fluorescent dye reagent, and after optical detection, the cryptococcus particle population in the sample may be distinguished based on at least one of scattered light intensity information (forward scattered light intensity information and/or side scattered light intensity information) and fluorescent light intensity information.
In the case that the sample to be measured is a body fluid sample, according to specific needs, at least one particle group of the leukocyte particle group, the nucleated cell particle group, the mononuclear cell particle group and the plurality of the nucleated cell particle groups can be further distinguished according to the side scattering light intensity information and the fluorescence intensity information in the optical information, preferably according to the forward scattering light intensity information, the side scattering light intensity information and the fluorescence intensity information in the optical information. Further, at least one of the leukocyte population, the nucleated cell population, the mononuclear cell population, and the plurality of the nucleated cell population may be counted.
According to another specific case, the sample to be measured may be a blood sample. Also in this case, the blood sample may be treated with a hemolyzing agent to obtain a sample to be tested, and then the sample to be tested is optically detected to obtain optical information including forward scattered light intensity information and side scattered light intensity information, and then the cryptococcus particle group is discriminated by the obtained optical information. Furthermore, the leukocyte subpopulations including lymphocytes, monocytes, neutrophils and eosinophils in the sample can also be distinguished from the obtained optical information, i.e., from the forward scattered light intensity information and the side scattered light intensity information, at the same time. And further counting the cryptococcus particle population and the differentiated leukocyte subpopulation, and optionally, prompting the presence of cryptococcus infection in the sample to be tested when the count value of the cryptococcus particle population exceeds a predetermined value.
According to another embodiment, when the sample to be tested is a blood sample, the blood sample may be treated with a hemolysis reagent and a fluorescent dye reagent (or with a reagent comprising a hemolysis reagent and a fluorescent dye) to obtain the sample to be tested. In this embodiment, the sample to be tested is optically inspected to obtain optical information including fluorescence intensity information and at least one scattered light intensity information. In some embodiments, the cryptococcus particle population is distinguished on the one hand by the obtained optical information (e.g., using one or both of scattered light intensity information and fluorescence intensity information); on the other hand, the leukocyte subpopulations including lymphocytes, monocytes, neutrophils and eosinophils and/or the immature granulocyte population can be distinguished from the side scatter light intensity information and the fluorescence intensity information in the optical information at the same time, preferably from the forward scatter light intensity information, the side scatter light intensity information and the fluorescence intensity information in the optical information, and the distinguished leukocyte subpopulations and/or the immature granulocyte population can be further selected for counting. In other embodiments, the cryptococcus particle population is distinguished by the obtained optical information on the one hand; on the other hand, at least one particle population of the nucleated red blood cell particle population and the basophilic granulocyte particle population in the sample to be measured can be distinguished according to the forward scattered light intensity information and the fluorescence intensity information in the optical information, preferably according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information. And further optionally counting at least one of the differentiated nucleated red blood cell particle population and the basophil particle population.
Further, the distinguishing the cryptococcus particle group in the sample to be detected according to the optical information includes:
generating a two-dimensional or three-dimensional scatter diagram according to the optical information;
obtaining a cryptococcus particle characteristic region according to the two-dimensional or three-dimensional scatter diagram; and
Particles in the cryptococcus particle characterization region are identified as cryptococcus particles.
Based on the characteristic of the pathogenicity of the cryptococcus neoformans in the presence of a broad and thick capsule outside the thallus, the inventors further found that erythrocyte hemolytic agents with different pH values can have different effects on the capsule of cryptococcus. Under neutral conditions, such as pH 6.5-7.5, the hemolytic agent does not substantially damage the capsule of cryptococcus, i.e., the broad and thick capsule outside the cryptococcus cell can prevent the "perforating action" of the neutral hemolytic agent to a certain extent, and further prevent the fluorescent dye from entering the cryptococcus cell to a certain extent. Under the pH condition, the hemolytic agent can break the cell membrane of the red blood cells, the cell membrane of the white blood cells can be damaged to a certain extent, so that a 'punching effect' is caused, and the fluorescent dye enters the cells through the pore diameter on the cell membrane of the white blood cells and is combined with nucleic acid substances in the white blood cells. Under acidic conditions, such as pH 2-4, the hemolytic agent also damages the capsule of cryptococcus to some extent, and produces a "perforating effect" similar to the cell membrane of leucocytes, so that the fluorescent dye can enter cryptococcus thallus.
Thus according to a more specific embodiment, the hemolysis reagent may have a pH of about 2.0 to 4.0, preferably a pH of about 3.0; or the hemolysis reagent may have a pH of about 6.5 to 7.5, preferably a pH of about 7.0. The following describes specific embodiments at different pH values.
According to a first embodiment, the sample is treated with a hemolysis reagent and further with a fluorochrome reagent to obtain a test sample. In this embodiment, the hemolysis reagent may have a pH of about 6.5 to 7.5, preferably a pH of about 7.0. In the sample to be detected, red blood cells are broken under the action of a hemolytic agent; the cell membrane of the white blood cells is perforated, and the fluorescent dye enters the white blood cells through the holes on the cell membrane, so that nuclear substances in the white blood cells are dyed; while the capsule of cryptococcus remains substantially intact and the nuclear material in the cell is substantially not stained, as shown in figure 3.
As shown in fig. 4, in this embodiment, first, in step S111, a sample is mixed with a hemolysis reagent and a fluorescent staining reagent, and then reacted to obtain a sample to be measured. Similarly, when the sample is processed, the hemolysis reagent and the fluorescent dye reagent may be added sequentially or simultaneously. The hemolysis reagent and the fluorochrome reagent may be present in separate reagents or may be present in the same reagent. In addition, other necessary processing may be performed on the sample, for example, in some embodiments, the sample may be subjected to a dilution process.
In step S112, the particles in the sample to be measured are passed through the optical detection device one by one, and optical information of each particle in the sample is obtained.
In this embodiment, since the nuclear material in the cryptococcus cell is not substantially stained with the fluorescent dye, a very low fluorescence intensity signal is exhibited, whereas nucleated cells such as white blood cells are stained to exhibit a certain fluorescence intensity. On the other hand, the complexity of the internal structure of cryptococcus is lower than that of leucocytes, so that the side scatter light intensity of cryptococcus is slightly lower than that of leucocytes. In yet another aspect, the cryptococcus has a broad cell size range, about 5 to 20m, reflecting a trend in forward scattered light intensity toward a small to large extension, wherein a portion coincides with the forward scattered light intensity of the white blood cells and the maximum forward scattered light intensity of the cryptococcus particles is greater than the maximum forward scattered light intensity of the white blood cell particles. Thus, cryptococcus particles may be distinguished from other particles in the sample in step S113 based on the detected fluorescence intensity and forward scattered light intensity, in particular based on fluorescence intensity, forward scattered light intensity and side scattered light intensity.
Next, the classified cryptococcus particle groups are counted in step S114, and the counted value is compared with a predetermined value in step S115, so that it is known whether or not the sample to be tested has a cryptococcus infection.
In this embodiment, information about other particles in the sample may be further obtained.
For example, referring to fig. 5, there is shown a detection procedure for a body fluid sample, particularly a cerebrospinal fluid sample, using a neutral hemolytic agent. The steps S1111 and S1122 are described with reference to the steps S111 and S112 shown in fig. 4. After obtaining the optical information of each particle in the sample, on the one hand, in step S1113, the cryptococcus particle group may be distinguished based on the optical information (specifically, forward scattered light intensity information and fluorescence intensity information, or forward scattered light intensity information, side scattered light intensity information and fluorescence intensity information); and the cryptococcus particle population may be further counted in step S1114; further, the counting result is compared with a predetermined value in step S1115, and it may be determined that there is cryptococcus infection in the cerebrospinal fluid sample when the counting value exceeds the predetermined value. On the other hand, in step S1116, at least one particle group of a white blood cell particle group, a nucleated cell particle group, a mononuclear cell (mono-nucleated cells) particle group, and a plurality of nucleated cell (poly-nucleated cells) particle groups may be distinguished further according to need from side scattered light intensity information and fluorescence intensity information, particularly forward scattered light intensity information, side scattered light intensity information, and fluorescence intensity information, among the measured optical information; at least one particle population may be further counted in step S1117.
Referring further to FIG. 6, there is shown a scattergram of optical information formed after detection of two cerebrospinal fluid samples using a Shenzhen Mierei biomedical electronics Limited BC-6800 hemocytometer and its DIFF channel matched 68-LD hemolysis reagent and corresponding fluorescent dye, the matched 68-LD hemolysis reagent having a pH of about 7.0. Wherein, in the scatter diagram of the cerebrospinal fluid sample positive by the cryptococcus, the cryptococcus and other particles can be distinguished (the circled range is the optical information of the corresponding cryptococcus) in the three-dimensional scatter diagram and the two-dimensional scatter diagram of the fluorescence-forward scattered light intensity, and the optical information of the cryptococcus particle is not detected at the corresponding position in the scatter diagram of the cerebrospinal fluid sample negative by the cryptococcus.
Referring to fig. 7, a specific detection procedure for a blood sample is shown with the use of a neutral hemolytic agent. Steps S1121, S1122 in fig. 7 are substantially identical to steps S111, S112 in fig. 4 described above, and in the example shown in fig. 7, blood samples are processed and tested using a reagent containing a hemolysis agent and a fluorescent dye in a conventional blood analyzer (e.g., the DIFF channel kit of the BC-6800 blood cell analyzer of shenzhen michaeli biomedical electronics inc.). Steps S1123, S1124, S1125 are the same as steps S113 and S114, S115, respectively, in the example shown in fig. 4. In step S1126, a detection step of leukocytes in a conventional DIFF channel is performed, i.e., leukocyte particle populations can be distinguished from the obtained optical information, and further leukocyte subpopulations can be distinguished. Specifically, from the side scatter light intensity information and the fluorescence intensity information in the measured optical information, preferably from the forward scatter light intensity information, the side scatter light intensity information and the fluorescence intensity information in the optical information, a leukocyte subpopulation including lymphocytes, monocytes, neutrophils and eosinophils and/or a naive granulocyte population can be distinguished. Further, in step S1127, the above-described leukocyte subpopulations and/or the immature granulocyte population that are discriminated may be counted.
According to a second embodiment, the sample is treated with a hemolysis reagent to obtain a test sample. In this embodiment, the hemolysis reagent may have a pH of about 2.0 to 4.0, preferably a pH of about 3.0. In the sample to be detected, red blood cells are crushed under the action of an acidic hemolytic agent; the cell membrane of the leucocyte is perforated, and the capsule of cryptococcus is also damaged to some extent, so that the perforation phenomenon similar to the cell membrane of the leucocyte is formed on the cell membrane. If the sample is further stained with a fluorescent staining reagent, the dye enters the inside of the leucocyte through the hole in the cell membrane and also enters the inside of the cryptococcus cell, so that both the leucocyte and the nuclear material of the cryptococcus are stained, as shown in fig. 8.
In this embodiment, referring to fig. 9, erythrocytes in the sample are crushed by the acidic hemolytic agent in step S121, and leucococcus and cryptococcus are both subjected to the hemolytic agent and exhibit a significant difference in scattered light intensity due to the difference in volume and complexity of each, so that cryptococcus can be distinguished from other particles such as leucococcus. If the sample is further stained with a fluorescent staining reagent, both the leucocyte and cryptococcus nucleic acid species can be stained. In step S122, the particles in the sample are passed through the optical detection device one by one to obtain optical information of each particle in the sample to be measured. In step S123, the cryptococcus particle population is discriminated based on the optical information. Wherein, without the treatment of the fluorescent staining reagent, the cryptococcus particle population can be distinguished based on the forward scattered light intensity information and the side scattered light intensity information. When the fluorescent staining reagent is used for treatment, cryptococcus particles can be distinguished from other particles such as leukocytes based on the forward scattered light intensity information and the side scattered light intensity information, in particular, based on the forward scattered light intensity information, the side scattered light intensity information and the fluorescent light intensity information. In the steps S124 and S125, by counting the differentiated cryptococcus particle groups and comparing with a predetermined value, it is known whether or not the sample to be tested has cryptococcus infection.
In this embodiment, the sample may not be treated with a fluorescent dye, but only with a hemolysis reagent. Similarly, if the treatment with the hemolysis reagent and the fluorochrome reagent is desired, they may be added sequentially or simultaneously. And the sample may be further processed.
Referring further to FIG. 10, there is shown a scatter plot of two different cerebrospinal fluid samples tested using a Shenzhen Mierei biomedical electronics Co., ltd., a BC-6800 haemolytic reagent and corresponding fluorescent dye, the reagent having a pH of about 3.0, and a 68-LN haemolytic reagent matched to its WNB channel. In the scatter plot of the cerebrospinal fluid sample positive for cryptococcus, the three-dimensional scatter plot and the forward scattered light intensity-side scattered light intensity two-dimensional scatter plot can distinguish cryptococcus from other particles (circled range is the optical information of the corresponding cryptococcus). No optical information of cryptococcus particles is detected in the three-dimensional scatter plot of the cryptococcus negative cerebrospinal fluid sample and in the corresponding position in the two-dimensional scatter plot of forward scattered light intensity versus side scattered light intensity.
Referring to fig. 11, one embodiment of this embodiment is shown with blood as a sample. This embodiment utilizes a reagent such as a conventional blood analyzer (e.g., shenzhen Maire biomedical electronics Co., ltd., BC-6800 series blood cell analyzer and its WNB channel kit) for detection in the WNB channel. In step S1221, the blood sample is treated with a hemolysis reagent and a fluorescent dye reagent under acidic conditions to obtain a sample to be tested, and further in step S1222 the sample is passed through an optical detection device to obtain optical information of each particle in the sample, i.e., scattered light intensity information and fluorescence intensity information.
Next, in steps S1223 to S1225, the cryptococcus particle group is distinguished according to the forward scattered light intensity information and the side scattered light intensity information in the optical information, especially according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information, and the cryptococcus particle group may be further counted, and then the counted value of the cryptococcus particle group is compared with a predetermined value to determine whether the cryptococcus infection exists in the sample.
Further, it is also possible to distinguish the nucleated red blood cell particle group and/or the basophil particle group from the forward scattered light intensity information and the fluorescence intensity information in the optical information, particularly from the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information at the same time in steps S1226 to S1227, and further count the nucleated red blood cell particle group and/or the basophil particle group.
The invention also provides another method for detecting the cryptococcus in the sample, wherein the hemolytic agent which has different actions on the capsule of the cryptococcus is used for respectively treating two partial samples of the sample to be detected, a first count and a second count of the cryptococcus in the sample to be detected are obtained, and whether the cryptococcus infection exists in the sample to be detected is determined according to the first count and the second count.
Specifically, referring to the method flowchart of fig. 12, in step S1310, a sample to be measured (body fluid sample or blood sample) is divided into a first partial sample and a second partial sample. The first and second portion of samples are then treated with different hemolysis reagents, respectively.
In steps S1311 to S1314, the first portion of the sample is treated with a first hemolysis reagent and a first fluorochrome reagent to obtain a first test sample, and the first optical information of the first test sample is obtained by detection by an optical detection device. And then distinguishing the cryptococcus particle swarm in the first to-be-detected sample according to the first optical information, and counting the cryptococcus particle swarm in the first to-be-detected sample to obtain a first count value.
In some embodiments, the first optical information comprises forward scattered light intensity information and fluorescence scattered light intensity information, preferably comprising forward scattered light intensity information, side scattered light intensity information and fluorescence intensity information.
In some embodiments, the first hemolysis reagent has a pH of about 6.5 to 7.5, preferably a pH of about 7.0.
Simultaneously or sequentially, in the steps S1321 to S1324, the second portion of the sample is treated with a second hemolysis reagent different from the first hemolysis reagent to obtain a second sample to be tested, and the second optical information of the second sample to be tested is obtained by detection by the optical detection device. And distinguishing the cryptococcus particle group of the second to-be-detected sample according to the second optical information, and counting the cryptococcus particle group in the second to-be-detected sample to obtain a second count value.
In some embodiments, the second optical information includes forward scattered light intensity information and side scattered light intensity information.
In some embodiments, step S1321 includes: the second portion of the sample is mixed with a second hemolysis reagent different from the first hemolysis reagent and a second fluorescent reagent different from the first fluorescent reagent. Wherein the second optical information includes forward scattered light intensity information, side scattered light intensity information, and fluorescence intensity information.
In some embodiments, the second hemolysis reagent has a pH of about 2.0 to 4.0, preferably a pH of about 3.0.
In step 1330, it is determined whether there is a cryptococcus infection in the sample to be tested according to the first count value and the second count value. For example, when the first count value exceeds the first preset value, the second count value exceeds the second preset value, and the difference between the first count value and the second count value is within the preset range, it is determined that there is cryptococcus infection in the sample to be tested.
In a specific embodiment, for example, the first part sample and the second part sample of the sample to be tested are simultaneously detected through two detection channels of the sample analyzer (for example, a DIFF channel and a matched reagent thereof and a WNB channel and a matched reagent thereof of a Shenzhen micui biomedical electronics limited company BC-6800 series blood cell analyzer) to obtain a first cryptococcus count value and a second cryptococcus count value.
Accordingly, the present application further provides a sample analyzer that can detect cryptococcus.
Referring to fig. 13, the sample analyzer includes: first housing 100, second housing 200, sampling device 10, sample preparation device 30, optical detection device 50, control device 70, and output section 90. In practical applications, the output 90 may be a user interface. The optical detection device 50 and the data processing device 70 are disposed inside the second casing 200, and may be disposed on both sides of the second casing 200, for example. The sample preparation device 30 is disposed inside the first housing 100, for example, and the output section 90 is disposed on the outer surface of the first housing 100, for example, and is used to display the detection result of the sample analyzer, for example, the detection result of cryptococcus to the user.
The sampling device 10 has a pipette with a pipette nozzle and has a drive device for driving the pipette to quantitatively aspirate a sample to be measured through the pipette nozzle. The sampling device may deliver the collected blood sample to the sample preparation device 30. According to various embodiments, the sampling device may collect multiple blood samples, provide different chambers of the sample preparation device for different treatments, and then perform different tests. The sampling device may be used to aspirate a body fluid sample or a blood sample.
The sample preparation device 30 has at least one reaction cell for receiving the sample to be measured sucked by the sampling device and a reagent supply section for supplying a hemolysis reagent and a fluorescent reagent to the at least one reaction cell so that the sample to be measured sucked by the sampling device is mixed with the hemolysis reagent and the fluorescent reagent supplied by the reagent supply section in the reaction cell to prepare a sample to be measured.
The optical detection device 50 includes a light source, a flow cell in which particles of the sample to be detected can flow, at least one scattered light detector for collecting at least one scattered light intensity information, and a fluorescence detector for collecting fluorescence intensity information, wherein the optical information includes the at least one scattered light intensity information and the fluorescence intensity information, and the light emitted by the light source irradiates the particles in the flow cell to generate optical information.
As shown in fig. 14, one specific example of the optical detection device 50 is shown therein. The optical detection device has a light source 101, a beam shaping assembly 102, a flow cell 103 and a forward scatter detector 104 arranged in sequence in a straight line. On one side of the flow chamber 103, a dichroic mirror 106 is arranged at an angle of 45 ° to the straight line. A part of the lateral light emitted by the particles in the flow cell 103 is transmitted through the dichroic mirror 106, and is captured by the fluorescence detector 105 arranged behind the dichroic mirror 106 at an angle of 45 ° to the dichroic mirror 106; another portion of the side light is reflected by the dichroic mirror 106 and captured by a side scatter detector 107 arranged in front of the dichroic mirror 106 at an angle of 45 ° to the dichroic mirror 106.
The control device 70 is electrically connected to the optical detection device and comprises a processor and a storage medium storing a computer program, wherein the control device is configured to perform the following steps when the computer program is executed by the processor: and acquiring at least two kinds of light intensity information in the optical information from the optical detection device, and distinguishing the cryptococcus particle swarm in the sample to be detected according to the at least two kinds of light intensity information.
The output section 90 is configured to output a detection result corresponding to cryptococcus in the sample, and optionally other particles.
Wherein the sample, the hemolysis reagent and the fluorescent staining reagent are as described above and are not described in detail herein.
In some embodiments, the at least one scatter detector comprises a forward scatter detector for collecting forward scatter light intensity information, optionally a side scatter detector for collecting side scatter light intensity information. Wherein the control device 70 is configured to perform the following steps when the computer program is executed by the processor:
And distinguishing the cryptococcus particle swarm in the sample to be detected according to the forward scattering light intensity information and the fluorescence intensity information in the optical information, preferably according to the forward scattering light intensity information, the side scattering light intensity information and the fluorescence intensity information in the optical information.
In some embodiments, for the case where the sample to be tested is a cerebrospinal fluid sample, the control device 70 is configured to further perform the following steps when the computer program is executed by the processor:
And distinguishing at least one particle group of a white blood cell particle group, a nucleated cell particle group, a single nucleated cell particle group and a plurality of nucleated cell particle groups according to the side scattering light intensity information and the fluorescence intensity information in the optical information of the sample to be detected, preferably according to the forward scattering light intensity information, the side scattering light intensity information and the fluorescence intensity information in the optical information, and optionally counting the at least one particle group.
In some embodiments, for the case where the sample to be tested is a blood sample, the control device is configured to further perform the following steps when the computer program is executed by the processor:
differentiating leukocyte subpopulations including lymphocytes, monocytes, neutrophils and eosinophils and/or differentiating naive granulocyte populations in said test sample from side scatter light intensity information and fluorescence intensity information in said optical information or from forward scatter light intensity information and side scatter light intensity information in said optical information, preferably from forward scatter light intensity information, side scatter light intensity information and fluorescence intensity information in said optical information, and optionally counting differentiated leukocyte subpopulations and/or naive granulocyte populations, or
And distinguishing at least one particle group of nucleated red blood cell particle groups and basophilic particle groups in the sample to be detected according to the forward scattering light intensity information and the fluorescence intensity information in the optical information, preferably according to the forward scattering light intensity information, the side scattering light intensity information and the fluorescence intensity information in the optical information, and optionally counting the at least one particle group.
Further, the control device may be configured to perform the following steps when distinguishing the cryptococcus particle population in the sample to be tested according to the at least two light intensity information:
generating a two-dimensional or three-dimensional scatter diagram according to the at least two light intensity information;
obtaining a cryptococcus particle characteristic region according to the two-dimensional or three-dimensional scatter diagram; and
Particles in the cryptococcus particle characterization region are identified as cryptococcus particles.
The control device is configured to perform the following steps when the computer program is executed by the processor:
Counting cryptococcus particle swarms in the sample to be detected; and
Optionally, when the count value of the cryptococcus particle swarm exceeds a predetermined value, indicating that there is cryptococcus infection in the sample to be tested.
The application also provides another sample analyzer which has basically the same structure as the analyzer shown in fig. 13, and also comprises the first shell, the second shell, the sampling device, the sample preparation device, the optical detection device, the control device and the output part.
The sample preparation device of the sample analyzer is different in that the sample preparation device has at least one reaction cell for receiving a sample to be measured sucked by a sampling device and a reagent supply section for supplying a hemolyzing reagent to the at least one reaction cell so that the sample to be measured sucked by the sampling device and the hemolyzing reagent supplied by the reagent supply section are mixed in the reaction cell to prepare a sample to be measured.
Or alternatively, the reagent supply part of the sample preparation device may supply the hemolysis reagent and the fluorescent dye reagent to the at least one reaction cell, so that the sample to be measured sucked by the sampling device is mixed with the hemolysis reagent and the fluorescent dye reagent supplied by the reagent supply part in the reaction cell to prepare the sample to be measured.
In some embodiments, the optical detection device comprises a light source, a flow cell, a first scatter detector, and a second scatter detector, and preferably further comprises a fluorescence detector. Particles of the test sample may flow within the flow chamber, the light emitted by the light source irradiates the particles in the flow chamber to generate optical information, the first scatter detector is used to collect forward scatter light intensity information, the second scatter detector is used to collect side scatter light intensity information, wherein the optical information includes the forward scatter light intensity information and the side scatter light intensity information, and preferably, when a fluorescence detector is included, the fluorescence detector is used to collect fluorescence intensity information, and the optical information includes forward scatter light intensity information, side scatter intensity information, and fluorescence intensity information.
Likewise, a control device is electrically connected to the optical detection device and comprises a processor and a storage medium storing a computer program, wherein the control is configured to perform the following steps when the computer program is executed by the processor: and acquiring the optical information from the optical detection device, and distinguishing the cryptococcus particle swarm in the sample to be detected according to the optical information.
In some embodiments, the sample preparation device comprises a first reaction cell and a second reaction cell, the reagent supply part of the sample preparation device provides a first hemolysis reagent and a first fluorescent dye to the first reaction cell, so that the sample to be tested sucked by the sampling device is mixed with the first hemolysis reagent and the first fluorescent dye provided by the reagent supply part in the first reaction cell to prepare a first sample to be tested, and the reagent supply part also provides a second hemolysis reagent to the second reaction cell, so that the sample to be tested sucked by the sampling device is mixed with the second hemolysis reagent provided by the reagent supply part in the second reaction cell to prepare a second sample to be tested. That is, the control device is configured to perform the following steps when the computer program is executed by the processor:
controlling the sample preparation device to prepare a first sample to be tested by mixing a first portion of the sample to be tested with a first hemolysis reagent and a first fluorescent reagent to obtain the first sample to be tested;
Controlling the optical detection device to detect the first sample to be tested so as to acquire first optical information of each particle in the first sample to be tested;
Distinguishing and counting cryptococcus particle groups in the first to-be-detected sample according to at least two light intensity information in the first optical information to obtain a first cryptococcus particle count value;
Controlling the sample preparation device to prepare a second test sample by mixing a second portion of the sample of the test sample with a second hemolysis reagent different from the first hemolysis reagent to obtain the second test sample;
controlling the optical detection device to detect the second to-be-detected sample so as to acquire second optical information of each particle in the second to-be-detected sample;
Distinguishing and counting cryptococcus particle groups in the second to-be-detected sample according to at least two light intensity information in the second optical information to obtain a second cryptococcus particle count value;
judging whether the cryptococcus infection exists in the sample to be tested according to the first cryptococcus particle count value and the second cryptococcus particle count value.
In some embodiments, the first optical information comprises forward scattered light intensity information and fluorescence intensity information, preferably comprising forward scattered light intensity information, side scattered light intensity information and fluorescence intensity information.
In some embodiments, the second optical information includes forward scattered light intensity information and side scattered light intensity information.
In some embodiments, the control device is configured to, when controlling the sample preparation device to prepare the second test sample, perform the steps of:
Controlling the sample preparation device to prepare a second test sample by mixing a second portion of the sample of the test sample with a second hemolysis reagent different from the first hemolysis reagent and a second fluorescent reagent different from the first fluorescent reagent to obtain the second test sample;
wherein the second optical information includes forward scattered light intensity information, side scattered light intensity information, and fluorescence intensity information.
In some embodiments, the control device is configured to, when determining whether there is a cryptococcus infection in the sample to be tested, perform the steps of:
And when the difference value of the first cryptococcus particle count value and the second cryptococcus particle count value is within a preset range, judging that the cryptococcus infection exists in the sample to be detected.
The application also provides a computer-readable storage medium having stored thereon a computer program, characterized in that the computer program when executed by a processor realizes the steps of the aforementioned method.
The computer readable storage medium may be volatile memory or nonvolatile memory, and may include both volatile and nonvolatile memory. The nonvolatile 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, a compact disc, or a read-only compact disc; the magnetic surface memory may be a disk memory or a tape memory. The volatile memory may be a random access memory that acts as an external cache. By way of example, and not limitation, many forms of RAM are available, such as sram, dram, sdram, ddr, sdram with enhanced, sdram with synchronous connection, dram with direct memory bus, and RAM with direct memory bus. The memory described in embodiments of the present invention is intended to comprise these and any other suitable types of memory.
The above-mentioned features can be combined with each other arbitrarily as long as they are meaningful within the scope of the present invention. The advantages and features described for the various aspects of the method are applicable in a corresponding manner to the corresponding sample analyzer and the corresponding computer readable storage medium and vice versa.
The foregoing description is only of the preferred embodiments of the present invention, and is not intended to limit the scope of the invention, but rather, the equivalent structural changes made by the description of the present invention and the accompanying drawings or the direct/indirect application in other related technical fields are included in the scope of the invention.
Claims (61)
1. A sample analyzer, the sample analyzer comprising:
A sampling device having a pipette with a pipette nozzle and having a driving device for driving the pipette to quantitatively aspirate a sample to be measured through the pipette nozzle;
A sample preparation device having at least one reaction cell for receiving a sample to be measured which is sucked by a sampling device and a reagent supply section for supplying a hemolysis reagent and a fluorescent reagent to the at least one reaction cell, so that the sample to be measured which is sucked by the sampling device is mixed with the hemolysis reagent and the fluorescent reagent supplied by the reagent supply section in the reaction cell to prepare a sample to be measured;
An optical detection device comprising a light source, a flow cell through which particles of the sample to be detected can pass one by one, at least one scatter detector for collecting at least one scattered light intensity information, and a fluorescence detector for collecting fluorescence intensity information, wherein the optical information comprises the at least one scattered light intensity information and the fluorescence intensity information, the light emitted by the light source illuminating the particles in the flow cell to generate optical information; and
A control device electrically connected to the optical detection device and comprising a processor and a storage medium storing a computer program, wherein the control is configured to perform the following steps when the computer program is executed by the processor:
Acquiring at least two kinds of light intensity information in the optical information from the optical detection device; and
Generating a two-dimensional or three-dimensional scatter diagram from the at least two light intensity information, obtaining a cryptococcus particle characterization area from the two-dimensional or three-dimensional scatter diagram, and identifying particles in the cryptococcus particle characterization area as cryptococcus particles,
Wherein the at least two light intensity information comprises forward scattered light intensity information, an
Wherein the forward scattered light intensity of the cryptococcus particles in the two-dimensional or three-dimensional scatter plot tends to extend from small to large.
2. The sample analyzer according to claim 1, wherein, in the sample to be measured, a capsule of the cryptococcus is destroyed, a nuclear substance in a cell is stained with the fluorescent reagent,
Wherein the at least one light intensity information further comprises side scatter light intensity information and the control is configured to perform the following steps when the computer program is executed by the processor:
Generating a two-dimensional or three-dimensional scatter diagram from at least two light intensity information including the forward scattered light intensity information and the side scattered light intensity information, obtaining a cryptococcus particle characteristic region from the two-dimensional or three-dimensional scatter diagram, and identifying particles in the cryptococcus particle characteristic region as cryptococcus particles.
3. The sample analyzer according to claim 1, wherein in the test sample, the capsule of the cryptococcus remains substantially intact, the cell core material is substantially not stained by the fluorescent reagent,
Wherein the at least one light intensity information further comprises fluorescence intensity information, and the control is configured to perform the following steps when the computer program is executed by the processor:
generating a two-dimensional or three-dimensional scatter diagram from at least two light intensity information including the forward scattered light intensity information and the fluorescence intensity information, obtaining a cryptococcus particle characteristic region from the two-dimensional or three-dimensional scatter diagram, and identifying particles in the cryptococcus particle characteristic region as cryptococcus particles.
4. A sample analyzer according to any of claims 1-3 wherein the sampling device is configured for aspirating a body fluid sample or a blood sample.
5. The sample analyzer of claim 1 or 2, wherein the at least one scatter detector comprises a forward scatter detector for collecting forward scatter light intensity information;
Wherein the control device is configured to perform the following steps when the computer program is executed by the processor:
And distinguishing the cryptococcus particle swarm in the sample to be detected according to the forward scattered light intensity information and the fluorescence intensity information in the optical information.
6. The sample analyzer of any of claims 1-3, wherein the at least one scatter detector comprises a forward scatter detector for collecting forward scatter light intensity information and a side scatter detector for collecting side scatter light intensity information;
Wherein the control device is configured to perform the following steps when the computer program is executed by the processor:
And distinguishing the cryptococcus particle swarm in the sample to be detected according to the forward scattered light intensity information and the fluorescence intensity information in the optical information, or according to the forward scattered light intensity information and the side scattered light intensity information in the optical information, or according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information.
7. The sample analyzer of claim 6, wherein the sample to be measured is a body fluid sample;
Wherein the control device is configured to further perform the following steps when the computer program is executed by the processor:
And distinguishing at least one particle group of the white blood cell particle group, the nucleated cell particle group, the mononuclear cell particle group and the plurality of the nucleated cell particle groups according to the side scattering light intensity information and the fluorescence intensity information in the optical information.
8. The sample analyzer of claim 6, wherein the sample to be measured is a body fluid sample;
Wherein the control device is configured to further perform the following steps when the computer program is executed by the processor:
and distinguishing at least one particle group of white blood cell particle groups, nucleated cell particle groups, single nucleated cell particle groups and a plurality of nucleated cell particle groups according to the side scattering light intensity information and the fluorescence intensity information in the optical information, and counting the at least one particle group.
9. The sample analyzer of claim 6, wherein the sample to be measured is a body fluid sample;
Wherein the control device is configured to further perform the following steps when the computer program is executed by the processor:
And distinguishing at least one particle group of white blood cell particle groups, nucleated cell particle groups, single nucleated cell particle groups and a plurality of nucleated cell particle groups according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information.
10. The sample analyzer of claim 6, wherein the sample to be measured is a body fluid sample;
The control device is configured to further perform the following steps when the computer program is executed by the processor:
and distinguishing at least one particle group of white blood cell particles, nucleated cell particles, mononuclear cell particles and a plurality of nucleated cell particles according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information, and counting the at least one particle group.
11. The sample analyzer of claim 6, wherein the sample to be measured is a blood sample;
Wherein the control device is configured to further perform the following steps when the computer program is executed by the processor:
And distinguishing white blood cell subsets comprising lymphocytes, monocytes, neutrophils and eosinophils and/or distinguishing a naive granulocyte population in the sample to be detected according to the side scattered light intensity information and the fluorescence intensity information in the optical information or according to the forward scattered light intensity information and the side scattered light intensity information in the optical information.
12. The sample analyzer of claim 6, wherein the sample to be measured is a blood sample;
Wherein the control device is configured to further perform the following steps when the computer program is executed by the processor:
Distinguishing a leukocyte subset including lymphocytes, monocytes, neutrophils and eosinophils and/or distinguishing a naive granulocyte population in the sample to be tested according to the side scattered light intensity information and the fluorescence intensity information in the optical information or according to the forward scattered light intensity information and the side scattered light intensity information in the optical information, and counting the distinguished leukocyte subset and/or the naive granulocyte population.
13. The sample analyzer of claim 6, wherein the sample to be measured is a blood sample;
Wherein the control device is configured to further perform the following steps when the computer program is executed by the processor:
And distinguishing leukocyte subpopulations including lymphocytes, monocytes, neutrophils and eosinophils and/or distinguishing a naive granulocyte population in the sample to be detected according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information.
14. The sample analyzer of claim 6, wherein the sample to be measured is a blood sample;
Wherein the control device is configured to further perform the following steps when the computer program is executed by the processor:
And distinguishing the leukocyte subpopulations including lymphocytes, monocytes, neutrophils and eosinophils and/or distinguishing the immature granulocyte population in the sample to be detected according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information, and counting the distinguished leukocyte subpopulations and/or the immature granulocyte population.
15. The sample analyzer of claim 5, wherein the sample to be measured is a blood sample;
Wherein the control device is configured to further perform the following steps when the computer program is executed by the processor:
and distinguishing at least one particle group of nucleated red blood cell particle groups and basophilic granulocyte particle groups in the sample to be detected according to the forward scattered light intensity information and the fluorescence intensity information in the optical information.
16. The sample analyzer of claim 5, wherein the sample to be measured is a blood sample;
Wherein the control device is configured to further perform the following steps when the computer program is executed by the processor:
And distinguishing at least one particle group of nucleated red blood cell particle groups and basophilic granulocyte particle groups in the sample to be detected according to the forward scattered light intensity information and the fluorescence intensity information in the optical information, and counting the at least one particle group.
17. The sample analyzer of claim 6, wherein the sample to be measured is a blood sample;
Wherein the control device is configured to further perform the following steps when the computer program is executed by the processor:
And distinguishing at least one particle group of the nucleated red blood cell particle group and the basophilic granulocyte particle group in the sample to be detected according to the forward scattering light intensity information, the side scattering light intensity information and the fluorescence intensity information in the optical information.
18. The sample analyzer of claim 6, wherein the sample to be measured is a blood sample;
Wherein the control device is configured to further perform the following steps when the computer program is executed by the processor:
And distinguishing at least one particle group of nucleated red blood cell particle groups and basophilic particle groups in the sample to be detected according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information, and counting the at least one particle group.
19. The sample analyzer of claim 3, wherein the hemolysis reagent has a pH of 6.5 to 7.5.
20. The sample analyzer of claim 19, wherein the hemolysis reagent has a pH of 7.0.
21. The sample analyzer of claim 2, wherein the hemolysis reagent has a pH of 2.0-4.0.
22. The sample analyzer of claim 21, wherein the hemolysis reagent has a pH of 3.0.
23. The sample analyzer of claim 1, wherein the control device is configured to further perform the steps of, when the computer program is executed by the processor:
Counting the cryptococcus particle group in the sample to be tested.
24. The sample analyzer of claim 23, wherein the control device is configured to further perform the steps of, when the computer program is executed by the processor:
and when the count value of the cryptococcus particle swarm exceeds a preset value, prompting that the cryptococcus infection exists in the sample to be tested.
25. A sample analyzer, the sample analyzer comprising:
A sampling device having a pipette with a pipette nozzle and having a driving device for driving the pipette to quantitatively aspirate a sample to be measured through the pipette nozzle;
A sample preparation device having at least one reaction cell for receiving a sample to be measured sucked by the sampling device and a reagent supply portion configured to supply a hemolysis reagent to the at least one reaction cell so that the sample to be measured sucked by the sampling device and the hemolysis reagent supplied by the reagent supply portion are mixed in the at least one reaction cell to prepare a sample to be measured;
an optical detection device comprising a light source, a flow cell, a first scattered light detector and a second scattered light detector, wherein particles of the sample to be detected can pass through the flow cell one by one, the particles in the flow cell are irradiated by light emitted by the light source to generate optical information, the first scattered light detector is used for collecting forward scattered light intensity information, the second scattered light detector is used for collecting side scattered light intensity information, and the optical information comprises the forward scattered light intensity information and the side scattered light intensity information; and
A control device electrically connected to the optical detection device and comprising a processor and a storage medium storing a computer program, wherein the control is configured to perform the following steps when the computer program is executed by the processor:
acquiring the optical information from the optical detection device, and
Generating a two-dimensional or three-dimensional scatter diagram from the optical information, obtaining a cryptococcus particle characterization area from the two-dimensional or three-dimensional scatter diagram, and identifying particles in the cryptococcus particle characterization area as cryptococcus particles,
Wherein the forward scattered light intensity of the cryptococcus particles in the two-dimensional or three-dimensional scatter plot tends to extend from small to large.
26. The sample analyzer according to claim 25 wherein said sample to be measured is a blood sample, said control means being configured to further perform the steps of, when said computer program is executed by said processor:
From the optical information, leukocyte subpopulations including lymphocytes, monocytes, neutrophils and eosinophils are distinguished.
27. The sample analyzer according to claim 25 wherein said sample to be measured is a blood sample, said control means being configured to further perform the steps of, when said computer program is executed by said processor:
and differentiating leukocyte subpopulations including lymphocytes, monocytes, neutrophils and eosinophils based on the optical information, and counting the differentiated leukocyte subpopulations.
28. The sample analyzer according to claim 25, wherein the reagent supply section is configured to supply a hemolysis reagent and a fluorescence reagent to the at least one reaction cell, so that the sample to be measured sucked by the sampling device is mixed with the hemolysis reagent and the fluorescence reagent supplied by the reagent supply section in the reaction cell to prepare a sample to be measured, the optical detection device further comprising a fluorescence detector for collecting fluorescence intensity information, the optical information further comprising the fluorescence intensity information.
29. The sample analyzer of claim 28, wherein the control device is configured to perform the following steps when the computer program is executed by the processor:
controlling the sample preparation device to prepare a first sample to be tested by mixing a first portion of the sample to be tested with a first hemolysis reagent and a first fluorescent reagent to obtain the first sample to be tested;
Controlling the optical detection device to detect the first sample to be tested so as to acquire first optical information of each particle in the first sample to be tested;
Distinguishing and counting cryptococcus particle groups in the first to-be-detected sample according to the first optical information so as to obtain a first cryptococcus particle count value;
Controlling the sample preparation device to prepare a second test sample by mixing a second portion of the sample of the test sample with a second hemolysis reagent different from the first hemolysis reagent to obtain the second test sample;
controlling the optical detection device to detect the second to-be-detected sample so as to acquire second optical information of each particle in the second to-be-detected sample;
separating and counting cryptococcus particle groups in the second to-be-detected sample according to the second optical information to obtain a second cryptococcus particle count value; and
Judging whether the cryptococcus infection exists in the sample to be tested according to the first cryptococcus particle count value and the second cryptococcus particle count value.
30. The sample analyzer of claim 29 wherein the first optical information includes forward scattered light intensity information and fluorescence intensity information.
31. The sample analyzer of claim 29 wherein the first optical information includes forward scattered light intensity information, side scattered light intensity information, and fluorescence intensity information.
32. The sample analyzer of any one of claims 29 to 31, wherein the second optical information includes forward scattered light intensity information and side scattered light intensity information.
33. The sample analyzer of any one of claims 29 to 31, wherein the control device is configured to, when controlling the sample preparation device to prepare the second test sample, perform the steps of:
Controlling the sample preparation device to prepare a second test sample by mixing a second portion of the sample of the test sample with a second hemolysis reagent different from the first hemolysis reagent and a second fluorescent reagent different from the first fluorescent reagent to obtain the second test sample;
wherein the second optical information includes forward scattered light intensity information, side scattered light intensity information, and fluorescence intensity information.
34. The sample analyzer of any one of claims 29 to 31, wherein the first hemolysis reagent has a pH of 6.5 to 7.5; and/or the second hemolysis reagent has a pH value of 2.0-4.0.
35. The sample analyzer of claim 34, wherein the first hemolysis reagent has a pH of 7.0; and/or the second hemolysis reagent has a pH value of 3.0.
36. The sample analyzer of any one of claims 29 to 31, wherein the control device is configured to, in determining whether there is a cryptococcus infection in the sample to be tested, perform the steps of:
And when the difference value of the first cryptococcus particle count value and the second cryptococcus particle count value is within a preset range, judging that the cryptococcus infection exists in the sample to be detected.
37. A method of detecting cryptococcus in a sample, the method comprising the steps of:
Processing the sample to be tested to obtain a sample to be tested, the processing the sample to be tested comprising mixing the sample to be tested with a hemolysis reagent;
enabling particles in the to-be-tested sample to pass through an optical detector one by one to obtain optical information of each particle in the to-be-tested sample, wherein the optical information at least comprises forward scattered light intensity information;
Distinguishing the cryptococcus particle swarm in the sample to be detected according to the optical information, comprising the following steps: generating a two-dimensional or three-dimensional scatter diagram from the optical information, obtaining a cryptococcus particle characterization area from the two-dimensional or three-dimensional scatter diagram, and identifying particles in the cryptococcus particle characterization area as cryptococcus particles,
Wherein the forward scattered light intensity of the cryptococcus particles in the two-dimensional or three-dimensional scatter plot tends to extend from small to large.
38. The method of claim 37, wherein the optical information further comprises side scatter light intensity information.
39. The method of claim 37, wherein the processing the sample to be tested further comprises: the sample to be measured is further mixed with a fluorescent reagent,
Wherein in the sample to be tested, the capsule of the cryptococcus is destroyed, the nucleus substance in the bacteria body is dyed by the fluorescent reagent, and the optical information further comprises side scattering light intensity information or further comprises side scattering light intensity information and fluorescent light intensity information; or alternatively
Wherein in the test sample, the capsule of the cryptococcus remains substantially intact, the cell nucleus substance cannot be substantially stained by the fluorescent agent, and the optical information further includes fluorescence intensity information or further includes fluorescence intensity information and side scatter light intensity information.
40. The method of claim 39, wherein the sample to be tested is a body fluid sample, the method further comprising:
And distinguishing at least one particle group of the white blood cell particle group, the nucleated cell particle group, the mononuclear cell particle group and the plurality of the nucleated cell particle groups according to the side scattering light intensity information and the fluorescence intensity information in the optical information.
41. The method of claim 39, wherein the sample to be tested is a body fluid sample, the method further comprising:
And distinguishing at least one particle group of white blood cell particle groups, nucleated cell particle groups, single nucleated cell particle groups and a plurality of nucleated cell particle groups according to the side scattering light intensity information and the fluorescence intensity information in the optical information, and counting the at least one particle group.
42. The method of claim 39, wherein the sample to be tested is a body fluid sample, the method further comprising:
And distinguishing at least one particle group of white blood cell particle groups, nucleated cell particle groups, single nucleated cell particle groups and a plurality of nucleated cell particle groups according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information.
43. The method of claim 39, wherein the sample to be tested is a body fluid sample, the method further comprising:
And distinguishing at least one particle group of white blood cell particle groups, nucleated cell particle groups, single nucleated cell particle groups and a plurality of nucleated cell particle groups according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information, and counting the at least one particle group.
44. The method of any one of claims 40 to 43, wherein the sample to be tested is a cerebrospinal fluid sample.
45. The method of claim 37 or 38, wherein the sample to be tested is a blood sample, the method further comprising:
from the forward scattered light intensity information and the side scattered light intensity information in the optical information, a leukocyte subpopulation including lymphocytes, monocytes, neutrophils and eosinophils is distinguished.
46. The method of claim 37 or 38, wherein the sample to be tested is a blood sample, the method further comprising:
And distinguishing leukocyte subpopulations including lymphocytes, monocytes, neutrophils and eosinophils according to the forward scattered light intensity information and the side scattered light intensity information in the optical information, and counting the distinguished leukocyte subpopulations.
47. The method of claim 39, wherein the sample to be tested is a blood sample, the method further comprising:
distinguishing a leukocyte subset including lymphocytes, monocytes, neutrophils and eosinophils and/or distinguishing a naive granulocyte population or counting the distinguished leukocyte subset and/or naive granulocyte population according to the side scatter light intensity information and the fluorescence intensity information in the optical information; or alternatively
And distinguishing at least one particle group of nucleated red blood cell particle groups and basophilic granulocyte particle groups in the sample to be detected according to the forward scattered light intensity information and the fluorescence intensity information in the optical information, or counting the at least one particle group.
48. The method of claim 39, wherein the sample to be tested is a blood sample, the method further comprising:
Distinguishing a leukocyte subset including lymphocytes, monocytes, neutrophils and eosinophils and/or distinguishing a naive granulocyte population or counting the distinguished leukocyte subset and/or naive granulocyte population based on the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information; or alternatively
And distinguishing at least one particle group of nucleated red blood cell particle groups and basophilic granulocyte particle groups in the sample to be detected according to the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information, or counting the at least one particle group.
49. The method of any one of claims 37 to 39, wherein the method further comprises:
Counting the cryptococcus particle group in the sample to be tested.
50. The method of claim 49, wherein the method further comprises:
and when the count value of the cryptococcus particle swarm exceeds a preset value, prompting that the cryptococcus infection exists in the sample to be tested.
51. The method of any one of claims 37 to 39, wherein the hemolysis reagent has a pH of 2.0 to 4.0; or the hemolysis reagent has a pH value of 6.5-7.5.
52. The method of claim 51, wherein the hemolysis reagent has a pH of 3.0; or the hemolysis reagent has a pH of 7.0.
53. A method of detecting cryptococcus in a sample, the method comprising the steps of:
processing a first portion of the sample to be tested to obtain a first sample to be tested, the processing the first portion of the sample to be tested comprising mixing the first portion of the sample with a first hemolysis reagent and a first fluorescent reagent;
Enabling particles in the first to-be-tested sample to pass through an optical detector one by one, and obtaining first optical information of each particle in the first to-be-tested sample;
Distinguishing and counting cryptococcus particle groups in the first to-be-detected sample according to the first optical information so as to obtain a first cryptococcus particle count value;
Processing a second portion of the sample to be tested to obtain a second sample to be tested, the processing the second portion of the sample to be tested comprising mixing the second portion of the sample with a second hemolysis reagent different from the first hemolysis reagent;
Enabling particles in the second to-be-tested sample to pass through the optical detector one by one, and obtaining second optical information of each particle in the second to-be-tested sample;
distinguishing and counting cryptococcus particle groups in the second to-be-detected sample according to the second optical information so as to obtain a second cryptococcus particle count value;
Judging whether the cryptococcus infection exists in the sample to be tested according to the first cryptococcus particle count value and the second cryptococcus particle count value,
Wherein the first optical information and the second optical information respectively include forward scattered light intensity information, wherein the forward scattered light intensity of the cryptococcus particles in a two-dimensional or three-dimensional scatter diagram generated from the first optical information and the second optical information respectively is in a trend of extending from small to large.
54. The method of claim 53, wherein the first optical information further comprises fluorescence scattered light intensity information.
55. The method of claim 54, wherein the first optical information further comprises side scatter light intensity information and fluorescence intensity information.
56. The method of any of claims 53 to 55, wherein the second optical information further comprises side scatter light intensity information.
57. The method of any one of claims 53 to 55, wherein the processing a second portion of the sample to be tested comprises mixing the second portion of the sample with a second hemolysis reagent different from the first hemolysis reagent and a second fluorescent reagent different from the first fluorescent reagent;
Wherein the second optical information further includes side scatter light intensity information and fluorescence intensity information.
58. The method of any one of claims 53 to 55, wherein the first hemolysis reagent has a pH of 6.5 to 7.5; and/or the second hemolysis reagent has a pH value of 2.0-4.0.
59. The method of claim 58, wherein said first hemolysis reagent has a pH of 7.0; and/or the second hemolysis reagent has a pH value of 3.0.
60. The method of any one of claims 53 to 55, wherein the determining whether there is a cryptococcus infection in the test sample based on the first cryptococcus particle count value and the second cryptococcus particle count value comprises:
And when the difference value of the first cryptococcus particle count value and the second cryptococcus particle count value is within a preset range, judging that the cryptococcus infection exists in the sample to be detected.
61. A computer readable storage medium having stored thereon a computer program, which when executed by a processor performs the steps of the method of any of claims 37 to 60.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911415630.4A CN113125392B (en) | 2019-12-31 | 2019-12-31 | Sample analyzer, method and computer-readable storage medium for detecting cryptococcus |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201911415630.4A CN113125392B (en) | 2019-12-31 | 2019-12-31 | Sample analyzer, method and computer-readable storage medium for detecting cryptococcus |
Publications (2)
| Publication Number | Publication Date |
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