CN113125392A - 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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- CN113125392A CN113125392A CN201911415630.4A CN201911415630A CN113125392A CN 113125392 A CN113125392 A CN 113125392A CN 201911415630 A CN201911415630 A CN 201911415630A CN 113125392 A CN113125392 A CN 113125392A
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Images
Classifications
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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
- G01N21/62—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
- G01N21/64—Fluorescence; Phosphorescence
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- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- 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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- 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
- G01N21/17—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
- G01N21/51—Scattering, i.e. diffuse reflection within a body or fluid inside a container, e.g. in an ampoule
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- G—PHYSICS
- G01—MEASURING; TESTING
- 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
Abstract
Sample analyzers, methods, and computer-readable storage media for detecting cryptococcus in a sample are disclosed. The sample analyzer includes: a sampling device, a sample preparation device, an optical detection device 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 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 method can quickly and accurately detect whether the sample has cryptococcus infection or not by utilizing the characteristic that the pathogenic cryptococcus has thick capsule.
Description
Technical Field
The present application relates to the field of in vitro diagnostics, and in particular to a sample analyzer, method and computer readable storage medium for detecting cryptococcus in a sample.
Background
Cryptococcus is a common fungus that can cause invasive infections in humans. Cryptococcus (Cryptococcus) includes 17 and 18 varieties, and most of the Cryptococcus neoformans (Cryptococcus neoformans) causing diseases in humans. Cryptococcus neoformans is widely distributed in nature and can also be present in the body surface, oral cavity and intestinal tract of a human body.
Cryptococcus is typically an exogenous infection. It invades human body through respiratory tract and invades all viscera tissues when spreading from lung meridian blood channel, and the most common part is central nervous system. Cryptococcosis neonatorum disease is better in patients with low cellular immune function, such as AIDS, malignant tumor, diabetes, organ transplantation and patients with large dose of glucocorticoid. The incidence of cryptococcus has been increasing in recent 20 years, and has become one of the most common complications of AIDS in foreign countries, which is the leading cause of death from AIDS. The incidence of cryptococcus infections is about 5% to 10% in immunosuppressed patients, and as high as 30% in AIDS patients. According to statistics, about 100 million people worldwide infect cryptococcus annually, and the death rate can reach 63 percent. Cryptococcosis and viral hepatitis are classified as B infectious diseases in China, and the cryptococcosis and the viral hepatitis are serious mycoses faced by human beings.
The existing methods for detecting cryptococcus include ink negative staining method, culture identification method, antigen detection method, antibody detection method, nucleic acid detection method and the like.
The cerebrospinal fluid and ink negative staining method is the most common cryptococcus detection means in clinic at present. The pathogenic cryptococcus has a wide capsule outside the bacterium body and strong refractivity, and when the bacterium body is dyed negatively by ink, the capsule wraps the bacterium body so that the bacterium body is not dyed by the ink, so that a thicker blank band is wrapped outside the round transparent bacterium body, and normal cells, other pathogenic bacteria and nonpathogenic cryptococcus in cerebrospinal fluid are dyed black by the ink and cannot be observed under a microscope.
The culture identification method is to inoculate the sample on a Sabouraud's medium, and the pathogenic cryptococcus can grow at 25 ℃ and 37 ℃ while the non-pathogenic cryptococcus does not grow at 37 ℃. Observing the morphological characteristics of the bacterial colony after culturing for 2-5 days, and taking the bacterial colony for biochemical identification. Cryptococcus neoformans is positive in phenol oxidase test and positive in urease test, can assimilate glucose, galactose, sucrose, inositol and raffinose, but cannot ferment sugar and alcohol.
The antigen detection method is a method for detecting a cryptococcus capsular polysaccharide specific antigen in cerebrospinal fluid or plasma by an immunological method such as latex agglutination test, ELISA, polyclonal antibody and monoclonal antibody method, among which the Latex Agglutination Test (LAT) is most commonly used. LAT is an indirect agglutination test in which polystyrene latex particles are used as a carrier, and soluble antibodies are bound to the polystyrene latex particles to prepare sensitized latex particles, which are then crosslinked with an antigen in a sample to be detected, thereby causing aggregation of a plurality of latex particles.
Antibody assays radioimmunoassays and tube agglutination assays were used to detect cryptococcus antibodies in serum.
Nucleic acid detection methods Polymerase Chain Reaction (PCR) is used to detect nucleic acid sequences specific to pathogenic cryptococcus in a sample, typically cryptococcus capsular membrane associated protein gene (CAP 10).
The detection sensitivity of the ink negative dyeing method and the culture identification method is low, and the culture time of the culture identification method is long. Although the detection methods of antigens and antibodies and the detection methods of nucleic acids have high sensitivity, the false positive rate is high, the detection cost is high, the detection is not a routine detection item, and whether the detection is carried out 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.
Therefore, there is a need to develop more convenient and accurate methods for detecting cryptococcus.
Disclosure of Invention
The application aims to provide a sample analyzer and a method for detecting cryptococcus conveniently, quickly and accurately.
To this end, a first aspect of the present application provides a sample analyzer comprising:
a sampling device which is provided with a pipette nozzle and is provided with a driving device, wherein the driving device is used for driving the pipette to quantitatively suck 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 tested drawn by a sampling device and a reagent supply portion for supplying a hemolysis reagent and a fluorescence reagent to the at least one reaction cell, so that the sample to be tested drawn by the sampling device and the hemolysis reagent and the fluorescence reagent supplied by the reagent supply portion are mixed in the reaction cell to prepare a sample to be tested;
an optical detection device comprising a light source, a flow chamber, at least one scattering light detector and a fluorescence detector, wherein particles of the sample to be detected can pass through the flow chamber one by one, the particles in the flow chamber are irradiated by light emitted by the light source to generate optical information, the at least one scattering light detector is used for collecting at least one scattered light intensity information, and the fluorescence detector is used for collecting fluorescence intensity information, wherein the optical information comprises the at least one scattered light intensity information and the fluorescence 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 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.
A second aspect of the present application provides another sample analyzer, comprising:
a sampling device which is provided with a pipette nozzle and is provided with a driving device, wherein the driving device is used for driving the pipette to quantitatively suck 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 tested aspirated by a 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 tested aspirated 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 tested;
an optical detection device comprising a light source, a flow chamber through which particles of the sample to be detected can pass one by one, a first scattering light detector for collecting forward scattered light intensity information, and a second scattering light detector for collecting side scattered light intensity information, wherein 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 device 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 a cryptococcus bacterium in a sample, the method comprising the steps of:
processing a sample to be tested to obtain a sample to be tested, wherein the processing of the sample to be tested comprises mixing the sample to be tested with a hemolysis reagent;
enabling the particles in the sample to be detected to pass through an optical detector one by one to obtain optical information of each particle in the sample to be detected;
and distinguishing the cryptococcus particle swarm in the sample to be detected according to the optical information.
In a fourth aspect, the present application provides another method of detecting a cryptococcus bacterium in a sample, the method comprising the steps of:
processing a first portion of a test sample to obtain a first test sample, said processing the first portion of the test sample comprising mixing the first portion of the sample with a first hemolysis reagent and a first fluorescent dye;
enabling the particles in the first sample to be detected to pass through an optical detector one by one, and obtaining first optical information of each particle in the first sample to be detected;
distinguishing the cryptococcus particle swarm in the first sample to be detected according to the first optical information and counting to obtain a first cryptococcus particle count value;
processing a second portion of the test sample to obtain a second test sample, said processing the second portion of the test sample comprising mixing the second portion of the sample with a second hemolysis reagent that is different from the first hemolysis reagent;
enabling the particles in the second sample to be detected to pass through the optical detector one by one to obtain second optical information of each particle in the second sample to be detected;
distinguishing the cryptococcus particle swarm in the second sample to be detected according to the second optical information and counting to obtain a second cryptococcus particle count value;
and judging whether cryptococcus infection exists in the sample to be detected according to the first cryptococcus particle counting value and the second cryptococcus particle counting value.
A fifth aspect of the application provides a computer-readable storage medium having a computer program stored thereon, characterized in that the computer program, when being executed by a processor, is adapted to carry out the steps of the method according to the third and fourth aspects of the application.
The invention utilizes the characteristic that the pathogenic cryptococcus has thick capsule, and utilizes instruments such as a full-automatic blood analyzer to quickly and accurately detect whether the cryptococcus infection exists in the sample.
Drawings
In the drawings, like reference numerals designate like or similar parts.
FIG. 1 is a schematic flow diagram of a method for detecting Cryptococcus according to the invention;
FIG. 2 is a schematic flow diagram of a method for detecting Cryptococcus according to one embodiment of the present invention;
FIG. 3 is a schematic diagram illustrating the principle of detecting Cryptococcus according to a first embodiment of the present invention;
FIG. 4 is a schematic flow chart of a method for detecting Cryptococcus according to a first embodiment of the present invention;
FIG. 5 is a schematic flow chart of a method for detecting Cryptococcus in a cerebrospinal fluid sample according to a first embodiment;
FIG. 6 is an optical scattergram of negative and positive cerebrospinal fluid samples of Cryptococcus according to the detection method shown in FIG. 5;
FIG. 7 is a schematic 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 according to a second embodiment of the present invention;
FIG. 9 is a schematic flow chart of a method for detecting Cryptococcus according to a second embodiment of the present invention;
FIG. 10 is a scatter plot of optical measurements of negative and positive cerebrospinal fluid samples of Cryptococcus according to the detection method shown in FIG. 9;
FIG. 11 is a schematic 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 according to the invention;
FIG. 13 is a schematic diagram of a sample testing apparatus according to the present invention; and
fig. 14 is a schematic structural diagram of an optical detection device according to the present invention.
Detailed Description
The technical solutions of the present invention will be described clearly and completely with reference to the following specific embodiments and the accompanying drawings, and it is to be understood that the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments, which can be obtained by a person skilled in the art without any inventive step based on the embodiments of the present invention, are within the scope of the present invention.
Throughout the specification, unless otherwise specifically noted, terms used herein should be understood as having meanings 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. If there is a conflict, the present specification will control.
Reference herein to 'Cryptococcus', unless otherwise indicated, refers to the most common pathogenic Cryptococcus neoformans (Cryptococcus neoformans) of the genus Cryptococcus (Cryptococcus).
Reference herein to a 'sample' is to a body fluid sample, such as cerebrospinal fluid, pleural fluid, ascites, myocardial fluid, synovial fluid, peritoneal dialysis fluid or fluid resulting from peritoneal washing, or a blood sample, unless otherwise indicated.
Reference herein to a 'haemolysing agent' is, unless otherwise indicated, to an agent which is capable of lysing red blood cells whilst maintaining white blood cells in a basic cellular morphology.
Reference herein to a 'fluorescent dye' is to a dye which is capable of specifically binding to a nucleic acid species (e.g. DNA, RNA) and emitting fluorescence of a particular wavelength upon excitation, unless otherwise indicated. The nucleic acid-specific dye used in the present invention is not particularly limited. Commercially available nucleic acid fluorescent dyes and nucleic acid specific fluorescent dyes already disclosed in some patent applications can be used in the present invention. Among them, commercially available nucleic acid fluorescent dyes include SYTO series nucleic acid dyes from Thermofeisher company. In addition, the fluorescent dye disclosed in chinese patent application CN201010022414.6, the cyanine dye disclosed in CN200910109215.6, the fluorescent dye disclosed in CN200810216864.1, and the like can be used in the present invention. The entire contents of the above patent documents are incorporated by reference into the present application.
The principle of sample detection as referred to herein refers to that after a body fluid or blood sample is drawn by a blood cell analyzer, the sample is first treated with a hemolytic agent and optionally a fluorescent dye, red blood cells are destroyed and lysed by the hemolytic agent, other particles, such as white blood cells, cannot be lysed, and the fluorescent dye can enter the nuclei of other particles, such as white blood cells, with the help of the hemolytic agent and bind to nucleic acid material 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, scattered light with various angles corresponding to the characteristics of the particles is generated, and the scattered light is received by the signal detector to obtain the related information of the particle structure and composition. Wherein, the quantity and volume of Forward Scattering (FS) reaction particles, the complexity of the internal structure (such as particle or nucleus in particle) of Side Scattering (SS) reaction particles, and the content of nucleic acid substances in Fluorescence signal (FL) reaction particles. Using these parameters, particles in the sample can be classified and counted.
The inventors have found that the action of a hemolytic agent on the thick capsular membrane of pathogenic cryptococcus can produce optical properties different from other particles in the sample, thereby enabling the detection of pathogenic cryptococcus in the sample using the hemolysis channel of a conventional hematology analyzer. The detection method of the invention does not influence the normal detection of the hemolysis channel, so that the detection method can also detect the index which can be detected by the hemolysis channel in a sample.
Thus, the present application provides 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 detected to obtain a sample to be detected, wherein the processing of the sample to be detected comprises at least processing the sample by a hemolysis reagent;
s12, enabling the particles in the sample to be detected to pass through an optical detector one by one, and obtaining the optical information of each particle in the sample to be detected; and
and step S13, distinguishing the cryptococcus particle swarm in the sample to be detected according to the optical information.
The hemolytic reagent of the present invention may contain those hemolytic agents conventionally used for detection of blood cells. For example, the hemolytic agent usable in the present invention may be selected from surfactants (such as quaternary ammonium salt type cationic surfactants, nonionic surfactants, e.g., alkanol polyoxyethylene ether type surfactants, etc.), alkyl glycosides, triterpenoid saponins, steroid saponins, etc. The hemolysis reagent may further comprise buffer (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 size range of the cryptococcus is wide and is about 5-20 mu m, the forward scattered light intensity is reflected in the trend of extending from small to large, and part of the forward scattered light intensity is superposed with the forward scattered light intensity of the leucocytes. On the other hand, the complexity of the internal structure of cryptococcus is lower than that of leukocytes, so that the side scattered light intensity of cryptococcus is slightly lower than that of leukocytes.
Therefore, in an embodiment, after treating the sample to be tested with the hemolytic agent, the particle group of cryptococcus in the sample to be tested can be distinguished according to the forward scattering light intensity information and the side scattering light intensity information in the optical information.
Further, the population of cryptococcus senticosus particles in the test sample can be counted, and optionally, when the count value of the population of cryptococcus senticosus particles exceeds a predetermined value, the existence of cryptococcus senticosus infection in the test sample is prompted.
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 with respect to the kind of the fluorescent dye, and any of the fluorescent dyes as defined above may be used as necessary.
Components such as buffers, surfactants, preservatives, metal chelators, tonicity adjusting agents and the like may also be included in the fluorescent dye reagent and will not be described in detail herein.
In a specific embodiment, the hemolysis reagent and the fluorochrome reagent may be two separate reagents, which are mixed with the sample sequentially or simultaneously to prepare the sample to be tested; alternatively, the hemolysis reagent and the fluorochrome reagent may be combined into one reagent comprising both the hemolysis reagent and the fluorochrome.
Referring to fig. 2, a schematic flow diagram of the present embodiment is shown. As shown in fig. 2, in this embodiment, first, in step S110, a sample to be tested is mixed with a hemolysis reagent and a fluorochrome reagent (or a reagent containing a hemolysis reagent and a fluorochrome) and reacted to obtain a sample to be tested. In step S120, the particles in the sample are individually passed through the optical detection device, and the optical information of each particle in the sample is obtained. And in S130, the cryptococcus particle in the sample to be tested can be distinguished from other particles based on the detected fluorescence intensity information and at least one scattered light intensity information, such as forward scattered light intensity information and/or side scattered light intensity information.
In S130, preferably, the group of cryptococcus particles in the sample to be tested is 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 is to be understood that, in step S130, the group of cryptococcus particles in the sample to be tested can also be distinguished according to the forward scattered light intensity information and the side scattered light intensity information in the optical information.
Further, the population of cryptococcus particles in the test sample may be counted, and optionally, when the count value of the population of cryptococcus particles exceeds a predetermined value, it is suggested that cryptococcus infection exists in the test sample.
According to a particular 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 population of cryptococcus particles in the sample may be distinguished based on the intensity information of the forward and side scattered light. Alternatively, the sample of body fluid to be tested may be treated with a hemolysis reagent and a fluorescent dye reagent, and optically detected, and then the population of cryptococcus particles in the sample may be discriminated from at least one of scattered light intensity information (forward scattered light intensity information and/or side scattered light intensity information) and fluorescence intensity information.
In the case where the sample to be measured is a body fluid sample, at least one particle group selected from a white blood cell particle group, a nucleated cell particle group, a mononuclear cell particle group, and a plurality of nucleated cell particle groups may be further distinguished according to specific requirements from the side scattered light intensity information and the fluorescence intensity information in the optical information, preferably from the forward scattered light intensity information, the side scattered light intensity information, and the fluorescence intensity information in the optical information. Still further, at least one particle group selected from the group consisting of a white blood cell particle group, a nucleated cell particle group, a mononuclear cell particle group, and a plurality of nucleated cell particle groups may be counted.
According to another specific case, the sample to be tested may be a blood sample. Also in this case, a blood sample may be treated with a hemolysis reagent 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 the population of cryptococcus is discriminated by the obtained optical information. Furthermore, the leukocyte subpopulations including lymphocytes, monocytes, neutrophils and eosinophils in the sample can be distinguished from the obtained optical information, i.e., the forward scattered light intensity information and the side scattered light intensity information. And further counting the population of cryptococcus particles and said differentiated subpopulation of leukocytes, and optionally, when the count of said population of cryptococcus particles exceeds a predetermined value, suggesting the presence of cryptococcus infection in said sample to be tested.
According to another embodiment, when the sample to be tested is a blood sample, the blood sample can be treated with a hemolysis reagent and a fluorochrome reagent (or a reagent containing the hemolysis reagent and the fluorochrome) to obtain a test sample. In this embodiment, a sample to be tested is optically detected to obtain optical information including fluorescence intensity information and at least one scattered light intensity information. In some embodiments, the population of cryptococcus particles is distinguished on the one hand by the optical information obtained (e.g., using one or both of scattered light intensity information and fluorescence intensity information); on the other hand, leukocyte subsets including lymphocytes, monocytes, neutrophils and eosinophils and/or immature granulocyte populations may be distinguished simultaneously from the side scattered light intensity information and the fluorescence intensity information in the optical information, preferably from the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information, and the distinguished leukocyte subsets and/or immature granulocyte populations may be further selected for counting. In other embodiments, the population of cryptococcus particles is distinguished on the one hand by the optical information obtained; on the other hand, at least one of the nucleated red blood cell population and the basophil subpopulation in the sample to be tested 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 populations of nucleated red blood cells and basophil subpopulations.
Further, the distinguishing the cryptococcus particle swarm in the sample to be detected according to the optical information comprises:
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
identifying particles in the characteristic region of the cryptococcus particle as cryptococcus particles.
Based on the characteristic that the pathogenic cryptococcus neoformans has a wide and thick capsule outside the thallus, the inventor further discovers that erythrocyte hemolytic agents with different pH values can produce different effects on the capsule of the cryptococcus neoformans. Under the neutral condition, such as pH 6.5-7.5, the hemolytic agent basically cannot damage the capsule of the cryptococcus, namely the wide and thick capsule outside the cryptococcus can prevent the 'punching effect' of the neutral hemolytic agent to a certain extent, and further prevent the fluorescent dye from entering the cryptococcus thallus to a certain extent. Under the condition of the pH, the hemolytic agent can rupture 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 the 'punching effect' is caused, and the fluorescent dye enters the cells through the apertures on the cell membrane of the white blood cells and is combined with nucleic acid substances in the white blood cells. Under an acidic condition, such as pH 2-4, the hemolytic agent can also cause certain damage to capsules of cryptococcus, so that a 'punching effect' similar to a leukocyte cell membrane is generated, and fluorescent dye can enter cryptococcus thalli.
Thus according to a more specific embodiment, the hemolysis reagent may have a pH of about 2.0-4.0, 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 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 a sample to be detected, red blood cells are broken under the action of a hemolytic agent; perforating a cell membrane of the white blood cells, and enabling a fluorescent staining agent to enter the interior of the white blood cells through the pores on the cell membrane so as to stain nuclear substances in the white blood cells; while the capsule of cryptococcus was still substantially intact, the inner core material of the thallus was substantially unable to stain, as shown in fig. 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 reacted to obtain a sample to be tested. Similarly, the hemolysis reagent and the fluorochrome reagent may be added sequentially or simultaneously to the sample being processed. The hemolysis reagent and the fluorochrome reagent may be present in separate reagents or may be present in the same reagent. In addition, the sample may be subjected to other necessary processing, for example, in some embodiments, the sample may be subjected to dilution processing.
In step S112, the particles in the sample to be measured are passed through the optical detection device one by one, and the optical information of each particle in the sample is obtained.
In this embodiment, since the nuclear substance in the cryptococcus bacterium is substantially not stained with the fluorescent dye, a very low fluorescence intensity signal is exhibited, and the nucleated cells such as leukocytes 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 leukocytes, so the side scattered light intensity of cryptococcus is slightly lower than that of leukocytes. On the other hand, the size range of the cryptococcus is wide and is about 5-20 m, which reflects the trend that the forward scattered light intensity extends from small to large, wherein part of the forward scattered light intensity is superposed with the forward scattered light intensity of the leucocytes, and the maximum forward scattered light intensity of the cryptococcus particles is larger than that of the leucocytes. Therefore, the cryptococcus particle can be distinguished from other particles in the sample based on the detected fluorescence intensity and forward scattered light intensity in the step S113, particularly according to the fluorescence intensity, forward scattered light intensity, and side scattered light intensity.
Then, the groups of the distinguished cryptococcus particles are counted in step S114, and the counted value is compared with a predetermined value in step S115, so that whether the cryptococcus infection exists in the tested sample can be known.
In this embodiment, information about other particles in the sample may be further obtained.
See, for example, fig. 5, which shows a detection step for a body fluid sample, in particular a cerebrospinal fluid sample, using a neutral hemolytic agent. The S1111 step and the S1122 step refer to the description of the S111 step and the S112 step shown in fig. 4. After obtaining the optical information of each particle in the sample, on one hand, in step S1113, the group of cryptococcus particles can be discriminated from 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 may be possible); and may further count the population of cryptococcus particles in step S1114; further, the counting result is compared with a predetermined value in step S1115, and when the counting value exceeds the predetermined value, it is determined that cryptococcus infection exists in the cerebrospinal fluid sample. On the other hand, in step S1116, at least one of a group of white blood cells, a group of nucleated cells, a group of single-nuclear cells, and a group of multi-nuclear cells may be further distinguished according to the intensity information of side scattered light and the intensity information of fluorescence, particularly, the intensity information of forward scattered light, the intensity information of side scattered light, and the intensity information of fluorescence, among the measured optical information, as necessary; the at least one particle group may be further counted in step S1117.
With further reference to FIG. 6, there is shown a scattergram formed by optical information after two cerebrospinal fluid samples were tested using the BC-6800 hematology analyzer of Shenzhen Merrill biomedical electronics Limited, its DIFF channel matched 68-LD hemolysis reagent with pH value of about 7.0 and the corresponding fluorochromes. Wherein, in the scatter diagram of the cerebrospinal fluid sample with the cryptococcus being positive, the cryptococcus can be distinguished from other particles in the three-dimensional scatter diagram and the two-dimensional scatter diagram of the fluorescence-forward scattered light intensity (the circled range is the optical information of the corresponding cryptococcus), and the optical information of the cryptococcus particle is not detected at the corresponding position in the scatter diagram of the cerebrospinal fluid sample with the cryptococcus being negative.
Referring to fig. 7, a specific detection procedure for a blood sample is shown in the case of a neutral hemolytic agent. Steps S1121, S1122 in fig. 7 are substantially the same as steps S111, S112 in fig. 4, and in the example shown in fig. 7, the blood sample is processed and detected in a conventional blood analyzer (e.g., DIFF channel kit reagent of BC-6800 blood cell analyzer from shenzhen meirui biomedical electronics limited) with reagents containing a hemolytic agent and a fluorescent dye. The steps S1123, S1124, S1125 are the same as the steps S113 and S114, S115, respectively, in the example shown in fig. 4. In step S1126, a step of detecting leukocytes in the conventional DIFF channel is performed, i.e., a leukocyte particle group can be distinguished based on the obtained optical information, and further a leukocyte subpopulation can be distinguished. Specifically, a leukocyte subset including lymphocytes, monocytes, neutrophils and eosinophils and/or a naive granulocyte population may be distinguished based on the side scattered light intensity information and the fluorescence intensity information in the measured optical information, preferably based on the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information. Further, in step S1127, the differentiated leukocyte subpopulations and/or immature granulocyte populations 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 2.0-4.0, preferably a pH of about 3.0. In a sample to be detected, red blood cells are broken under the action of an acid hemolytic agent; the cell membrane of the leucocyte is perforated, and the capsule of the cryptococcus is also damaged to a certain extent, so that the perforation phenomenon of the cell membrane similar to the leucocyte is formed on the cell membrane. If the sample is further stained with a fluorescent staining reagent, the dye will enter the inside of leukocytes through pores in the cell membrane and also enter the inside of the cryptococcus thallus, so that both leukocytes and the nuclear material of cryptococcus are stained, as shown in fig. 8.
In this embodiment, referring to fig. 9, the erythrocytes in the sample are broken down by the action of the acidic hemolytic agent in step S121, and the leucocytes and cryptococcus are both subjected to the action of the hemolytic agent and exhibit a distinct difference in scattered light intensity due to the difference in volume and complexity of each, thereby being able to distinguish cryptococcus from other particles such as leucocytes. If the sample is further stained with a fluorescent staining reagent, both the leucocytes and the nucleic acid material of cryptococcus 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, groups of Cryptococcus are distinguished from the optical information. Wherein, in the case of not being treated with a fluorescent staining reagent, the group of Cryptococcus particles can be distinguished from the forward scattered light intensity information and the side scattered light intensity information. When the treatment is performed using a fluorescent staining reagent, cryptococcus particles can be distinguished from other particles such as leukocytes on the basis of the forward scattered light intensity information and the side scattered light intensity information, particularly the forward scattered light intensity information, the side scattered light intensity information, and the fluorescence intensity information. In the steps S124 and S125, the number of the distinguished cryptococcus particle groups is counted and compared with a preset value, so that whether cryptococcus infection exists in the tested sample can be known.
In this embodiment, the sample may be treated with no fluorochrome but only with a hemolysis reagent. Similarly, if it is desired to co-treat the hemolysis reagent and the fluorochrome reagent, they may be added sequentially or simultaneously. And the sample may be further processed.
With further reference to FIG. 10, there is shown a scattergram of two different cerebrospinal fluid samples after detection using the BC-6800 blood cell analyzer from Shenzhen Merrill biomedical electronics Limited and its WNB channel complement of 68-LN hemolysis reagent and corresponding fluorochromes, the complement of 68-LN hemolysis reagent having a pH of about 3.0. In the scattergram of the cerebrospinal fluid sample in which cryptococcus is positive, cryptococcus can be distinguished from other particles in the three-dimensional scattergram and the forward scattered light intensity-side scattered light intensity two-dimensional scattergram (the circled range is optical information corresponding to cryptococcus). No optical information of cryptococcus particles was detected in the three-dimensional scattergram of the cerebrospinal fluid sample where cryptococcus was negative and at the corresponding positions in the two-dimensional scattergram of forward scattered light intensity-side scattered light intensity.
Referring to fig. 11, a specific embodiment of the blood sample in this embodiment is shown. The embodiment utilizes a conventional blood analyzer (such as Shenzhen Merrill biomedical electronics Limited BC-6800 series blood cell analyzer and WNB channel matching reagents thereof) to detect in the WNB channel. In step S1221, the blood sample is treated with the hemolysis reagent and the fluorescent dye reagent under an acidic condition 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, that is, scattered light intensity information and fluorescence intensity information.
And in steps S1223 to S1225, distinguishing the cryptococcus particle swarm according to the forward scattering light intensity information and the side scattering light intensity information in the optical information, especially according to the forward scattering light intensity information, the side scattering light intensity information and the fluorescence intensity information, further counting the cryptococcus particle swarm, and then comparing the counted value of the cryptococcus particle swarm with a preset value to judge whether cryptococcus infection exists in the sample.
Further, in steps S1226 to S1227, the population of nucleated red blood cells and/or the subpopulation of basophils may be simultaneously distinguished from the forward scattered light intensity information and the fluorescence intensity information in the optical information, and in particular, from the forward scattered light intensity information, the side scattered light intensity information, and the fluorescence intensity information in the optical information, and the population of nucleated red blood cells and/or the subpopulation of basophils may be further counted.
The invention also provides another method for detecting cryptococcus in a sample, wherein two partial samples of the sample to be detected are respectively treated by hemolytic agents which have different effects on capsules of the cryptococcus, a first count and a second count of the cryptococcus in the sample to be detected are obtained, and whether 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 tested (a body fluid sample or a blood sample) is divided into a first partial sample and a second partial sample. The first and second portions of the sample are then processed with different hemolysis reagents, respectively.
In steps S1311 to S1314, the first sample portion is processed with the first hemolysis reagent and the first fluorochrome reagent to obtain a first test sample, and the first optical information of the first test sample is obtained by detecting with the optical detection device. And distinguishing the cryptococcus particle swarm in the first sample to be detected according to the first optical information, and counting the cryptococcus particle swarm in the first sample to be detected to obtain a first count value.
In some embodiments, the first optical information includes forward scattered light intensity information and fluorescence scattered light intensity information, and preferably includes 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 steps S1321 to S1324, a second portion of the sample is processed by a second hemolytic reagent different from the first hemolytic reagent to obtain a second test sample, and the second test sample is detected by the optical detection device to obtain second optical information of the second test sample. And distinguishing the cryptococcus particle swarm of the second sample to be detected according to the second optical information, and counting the cryptococcus particle swarm in the second sample to be detected 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 fluorescence reagent different from the first fluorescence 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 2.0-4.0, preferably a pH of about 3.0.
In step 1330, whether cryptococcus infection exists in the sample to be tested is determined according to the first count value and the second count value. For example, when the first count value exceeds a first preset value, the second count value exceeds a second preset value, and the difference between the first count value and the second count value is within a preset range, the cryptococcus infection in the sample to be tested is judged.
In a specific embodiment, for example, the first and second samples of the sample to be tested are simultaneously detected by two detection channels of the sample analyzer (for example, DIFF channel and its kit, WNB channel and its kit of shenzhen meirui biomedical electronics ltd.bc-6800 series blood cell analyzer) to obtain the first and second cryptococcus counts.
Accordingly, the present application further provides a sample analyzer that can detect cryptococcus.
Referring to fig. 13, the sample analyzer includes: a first housing 100, a second housing 200, a sampling device 10, a sample preparation device 30, an optical detection device 50, a control device 70, and an output unit 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 housing 200, and may be disposed on both sides of the second housing 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 aspirate a sample to be measured quantitatively by means of 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 processing, and then perform different tests. The sampling device may be used to draw a bodily fluid sample or a blood sample.
The sample preparation apparatus 30 has at least one reaction cell for receiving the sample to be tested drawn by the sampling apparatus and a reagent supply portion for supplying the hemolysis reagent and the fluorescence reagent to the at least one reaction cell, so that the sample to be tested drawn by the sampling apparatus and the hemolysis reagent and the fluorescence reagent supplied by the reagent supply portion are mixed in the reaction cell to prepare the sample to be tested.
The optical detection device 50 includes a light source, a flow chamber, at least one scattering light detector, and a fluorescence detector, wherein particles of the sample to be detected can flow in the flow chamber, light emitted by the light source irradiates the particles in the flow chamber to generate optical information, the at least one scattering light detector is used for collecting at least one scattered light intensity information, and the fluorescence detector is used for collecting fluorescence intensity information, wherein the optical information includes the at least one scattered light intensity information and the fluorescence intensity information.
As shown in fig. 14, a specific example of the optical detection device 50 is shown. 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 series. On one side of the flow cell 103, a dichroic mirror 106 is arranged at an angle of 45 ° to the straight line. The lateral light emitted by the particles in flow cell 103, a portion of which is transmitted through dichroic mirror 106, is captured by fluorescence detector 105 arranged behind dichroic mirror 106 at an angle of 45 ° to dichroic mirror 106; another portion of the side light is reflected by dichroic mirror 106 and captured by side scatter detector 107, which is arranged at a 45 angle to dichroic mirror 106 in front of dichroic mirror 106.
A 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 90 is configured to output a detection result corresponding to the cryptococcus in the sample, and optionally a detection result of other particles.
Wherein the sample, the hemolysis reagent, and the fluorescence staining reagent are as described above and will not be described herein.
In some embodiments, the at least one scattered light detector comprises a forward scattered light detector for collecting forward scattered light intensity information, optionally a side scattered light detector for collecting side scattered 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, when the computer program is executed by the processor, further perform the following steps:
distinguishing at least one particle swarm of a leukocyte particle swarm, a nucleated cell particle swarm, a mononuclear cell particle swarm and a plurality of nuclear cell particle swarms according to the side scattered light intensity information and the fluorescence intensity information in the optical information of the sample to be detected, 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 optionally counting the at least one particle swarm.
In some embodiments, for the case where the sample to be tested is a blood sample, the control device is configured to, when the computer program is executed by the processor, further perform the steps of:
distinguishing leukocyte subsets including lymphocytes, monocytes, neutrophils and eosinophils and/or distinguishing immature granulocyte populations 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, 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 optionally counting the distinguished leukocyte subsets and/or immature granulocyte populations
Distinguishing at least one of a nucleated red blood cell population and a basophil subpopulation in the sample to be tested from the forward scattered light intensity information and the fluorescence intensity information in the optical information, preferably from the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information, and optionally counting the at least one particle population.
Further, the control device may be configured to perform the following steps when distinguishing the cryptococcus particle group in the sample to be tested according to the at least two kinds of light intensity information:
generating a two-dimensional or three-dimensional scatter diagram according to the at least two kinds of light intensity information;
obtaining a cryptococcus particle characteristic region according to the two-dimensional or three-dimensional scatter diagram; and
identifying particles in the characteristic region of the cryptococcus particle as cryptococcus particles.
The control apparatus is configured such that when the computer program is executed by the processor, it can further perform the steps of:
counting the particle swarm of the cryptococcus in the sample to be detected; and
optionally, when the count value of the population of cryptococcus is over a predetermined value, it is suggested that cryptococcus infection exists in the sample to be tested.
The present application also provides another sample analyzer, which has substantially the same structure as the analyzer shown in fig. 13, and also includes the aforementioned first housing, second housing, sampling device, sample preparation device, optical detection device, control device, and output unit.
The sample preparation device of the sample analyzer has at least one reaction cell for receiving a sample to be tested drawn by a sampling device, and a reagent supply portion for supplying a hemolysis reagent to the at least one reaction cell, so that the sample to be tested drawn by the sampling device and the hemolysis reagent supplied by the reagent supply portion are mixed in the reaction cell to prepare a sample to be tested.
Or, alternatively, the reagent supply part of the sample preparation apparatus may supply the hemolysis reagent and the fluorochrome reagent to the at least one reaction cell, so that the sample to be tested, which is drawn up by the sampling apparatus, is mixed with the hemolysis reagent and the fluorochrome reagent supplied from the reagent supply part in the reaction cell to prepare the sample to be tested.
In some embodiments, the optical detection device comprises a light source, a flow cell, a first scattered light detector and a second scattered light detector, and preferably also a fluorescence detector. The particles of the sample to be tested may flow in the flow cell, the light emitted by the light source irradiates the particles in the flow cell to generate optical information, the first scattering light detector is configured to collect forward scattered light intensity information, the second scattering light detector is configured to collect side scattered light intensity information, wherein the optical information includes the forward scattered light intensity information and the side scattered light intensity information, and preferably, when a fluorescence detector is included, the fluorescence detector is configured to collect fluorescence intensity information, and the optical information includes forward scattered light intensity information, side scattered intensity information, and fluorescence intensity information.
Also, 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 device 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, and the reagent supply portion of the sample preparation device supplies a first hemolysis reagent and a first fluorochrome to the first reaction cell, on one hand, so that the sample to be tested drawn by the sampling device is mixed with the first hemolysis reagent and the first fluorochrome supplied by the reagent supply portion in the first reaction cell to prepare a first sample to be tested, and also supplies a second hemolysis reagent to the second reaction cell, on the other hand, so that the sample to be tested drawn by the sampling device is mixed with the second hemolysis reagent supplied by the reagent supply portion in the second reaction cell to prepare a second sample to be tested. That is, the control apparatus is configured to, when the computer program is executed by the processor, perform the steps of:
controlling the sample preparation device to prepare a first test sample by mixing a first portion of the sample to be tested with a first hemolysis reagent and a first fluorescence reagent to obtain the first test sample;
controlling the optical detection device to detect the first sample to be tested so as to obtain first optical information of each particle in the first sample to be tested;
distinguishing the cryptococcus particle swarm in the first sample to be detected according to at least two kinds of light intensity information in the first optical information and counting 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 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 sample to be detected so as to obtain second optical information of each particle in the second sample to be detected;
distinguishing the cryptococcus particle swarm in the second sample to be detected according to at least two kinds of light intensity information in the second optical information and counting to obtain a second cryptococcus particle count value;
and judging whether cryptococcus infection exists in the sample to be detected according to the first cryptococcus particle counting value and the second cryptococcus particle counting value.
In some embodiments, the first optical information includes forward scattered light intensity information and fluorescence intensity information, and preferably includes 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 perform the following steps when controlling the sample preparation device to prepare the second test sample:
controlling the sample preparation device to prepare a second test sample by mixing a second portion of the sample with a second hemolysis reagent different from the first hemolysis reagent and a second fluorescence reagent different from the first fluorescence 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 perform the following steps in determining whether a cryptococcus infection is present in the sample to be tested:
and when the difference value of the first cryptococcus particle counting value and the second cryptococcus particle counting value is within a preset range, judging that cryptococcus infection exists in the sample to be detected.
The present application also provides a computer-readable storage medium having a computer program stored thereon, wherein the computer program, when executed by a processor, performs the steps of the aforementioned method.
The computer readable storage medium may be either volatile memory or nonvolatile memory, and may include both volatile and nonvolatile memory. The nonvolatile memory can be a read-only memory, a programmable read-only memory, an erasable programmable read-only memory, an electrically erasable programmable read-only memory, a magnetic random access memory, a flash memory, a magnetic surface memory, an optical disc, or a read-only optical disc; the magnetic surface storage may be disk storage or tape storage. Volatile memory may be random access memory, which acts as external cache memory. By way of illustration and not limitation, many forms of RAM are available, such as SRAM, SDRAM, DRAM, SDRAM, DDR SDRAM, SSRAM, SDRAM, and DMA bus RAM. The memory described in connection with the embodiments of the invention is intended to comprise these and any other suitable types of memory.
The features mentioned above can be combined with one another as desired, insofar as they are within the scope of the invention. The advantages and features explained for the various aspects of the method apply in a corresponding manner to the corresponding sample analyzer and to the corresponding computer-readable storage medium, and vice versa.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention, and all modifications and equivalents made by the contents of the present specification and drawings, or directly/indirectly applied to other related technical fields, are included in the scope of the present invention.
Claims (35)
1. A sample analyzer, comprising:
a sampling device which is provided with a pipette nozzle and is provided with a driving device, wherein the driving device is used for driving the pipette to quantitatively suck 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 tested drawn by a sampling device and a reagent supply portion for supplying a hemolysis reagent and a fluorescence reagent to the at least one reaction cell, so that the sample to be tested drawn by the sampling device and the hemolysis reagent and the fluorescence reagent supplied by the reagent supply portion are mixed in the reaction cell to prepare a sample to be tested;
an optical detection device comprising a light source, a flow chamber, at least one scattering light detector and a fluorescence detector, wherein particles of the sample to be detected can pass through the flow chamber one by one, the particles in the flow chamber are irradiated by light emitted by the light source to generate optical information, the at least one scattering light detector is used for collecting at least one scattered light intensity information, and the fluorescence detector is used for collecting fluorescence intensity information, wherein the optical information comprises the at least one scattered light intensity information and the fluorescence 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 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.
2. The sample analyzer of claim 1, wherein the sampling device is configured to draw a bodily fluid sample or a blood sample.
3. The sample analyzer of claim 1 or 2, wherein the at least one scattered light detector comprises a forward scattered light detector for collecting forward scattered light intensity information, optionally a side scattered light detector for collecting side scattered light intensity information;
wherein the control apparatus 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 or according to the forward scattering light intensity information and the side scattering light 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.
4. The sample analyzer of claim 3, wherein the sample to be tested is a body fluid sample, in particular a cerebrospinal fluid sample;
wherein the control apparatus is configured to, when the computer program is executed by the processor, further perform the steps of:
distinguishing at least one particle group of a leukocyte particle group, a nucleated cell particle group, a mononuclear cell particle group, and a plurality of nucleated cell particle groups according to the side 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 optionally counting the at least one particle group.
5. The sample analyzer of claim 3, wherein the sample to be tested is a blood sample;
wherein the control apparatus is configured to, when the computer program is executed by the processor, further perform the steps of:
and distinguishing leukocyte subsets 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, 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 optionally counting the distinguished leukocyte subsets and/or naive granulocyte population.
6. The sample analyzer of claim 3, wherein the sample to be tested is a blood sample;
wherein the control apparatus is configured to, when the computer program is executed by the processor, further perform the steps of:
distinguishing at least one of a nucleated red blood cell population and a basophil subpopulation in the sample to be tested from the forward scattered light intensity information and the fluorescence intensity information in the optical information, preferably from the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information, and optionally counting the at least one particle population.
7. The sample analyser of any one of claims 1 to 6 wherein the hemolysis reagent has a pH of 6.5-7.5, preferably has a pH of 7.0.
8. The sample analyser of any one of claims 1 to 6 wherein the hemolysis reagent has a pH of 2.0-4.0, preferably has a pH of 3.0.
9. The method according to any one of claims 1 to 8, wherein the control device is configured to perform the following steps when distinguishing the group of cryptococcus particles in the sample to be tested from the at least two light intensity information:
generating a two-dimensional or three-dimensional scatter diagram according to the at least two kinds of light intensity information;
obtaining a cryptococcus particle characteristic region according to the two-dimensional or three-dimensional scatter diagram; and
identifying particles in the characteristic region of the cryptococcus particle as cryptococcus particles.
10. The sample analyzer of any one of claims 1 to 9 wherein the control device is configured to, when the computer program is executed by the processor, further perform the steps of:
counting the particle swarm of the cryptococcus in the sample to be detected; and
optionally, when the count value of the population of cryptococcus is over a predetermined value, it is suggested that cryptococcus infection exists in the sample to be tested.
11. A sample analyzer, comprising:
a sampling device which is provided with a pipette nozzle and is provided with a driving device, wherein the driving device is used for driving the pipette to quantitatively suck 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 tested aspirated by a 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 tested aspirated 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 tested;
an optical detection device comprising a light source, a flow chamber through which particles of the sample to be detected can pass one by one, a first scattering light detector for collecting forward scattered light intensity information, and a second scattering light detector for collecting side scattered light intensity information, wherein 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 device 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.
12. The sample analyzer of claim 11, wherein the sample to be tested is a blood sample, the control device being configured to perform the following further steps when the computer program is executed by the processor:
differentiating leukocyte subpopulations comprising lymphocytes, monocytes, neutrophils and eosinophils based on the optical information and optionally counting the differentiated leukocyte subpopulations.
13. The sample analyzer of claim 11 or 12, wherein the reagent supplying portion is configured to supply a hemolysis reagent and a fluorescence reagent to the at least one reaction cell, so that the sample to be tested aspirated by the sampling device is mixed with the hemolysis reagent and the fluorescence reagent supplied by the reagent supplying portion in the reaction cell to prepare a sample to be tested, and the optical detecting device further comprises a fluorescence detector for collecting fluorescence intensity information, the optical information comprising the fluorescence intensity information.
14. The sample analyzer of claim 13 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 test sample by mixing a first portion of the sample to be tested with a first hemolysis reagent and a first fluorescence reagent to obtain the first test sample;
controlling the optical detection device to detect the first sample to be tested so as to obtain first optical information of each particle in the first sample to be tested;
distinguishing the cryptococcus particle swarm in the first sample to be detected according to the first optical information and counting 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 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 sample to be detected so as to obtain second optical information of each particle in the second sample to be detected;
according to the second optical information, the cryptococcus particle swarm in the second sample to be detected is separated and counted to obtain a second cryptococcus particle count value; and
and judging whether cryptococcus infection exists in the sample to be detected according to the first cryptococcus particle counting value and the second cryptococcus particle counting value.
15. The sample analyzer of claim 14, wherein the first optical information comprises forward scattered light intensity information and fluorescence intensity information, preferably forward scattered light intensity information, side scattered light intensity information, and fluorescence intensity information.
16. The sample analyzer of claim 14 or 15, wherein the second optical information includes forward scattered light intensity information and side scattered light intensity information.
17. The sample analyzer of any one of claims 14 to 16, wherein the control device is configured to perform the following steps when controlling the sample preparation device to prepare the second test sample:
controlling the sample preparation device to prepare a second test sample by mixing a second portion of the sample with a second hemolysis reagent different from the first hemolysis reagent and a second fluorescence reagent different from the first fluorescence 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.
18. The sample analyser of any one of claims 14 to 17, wherein the first hemolysis reagent has a pH of 6.5-7.5, preferably has a pH of 7.0; and/or the second hemolysis reagent has a pH of 2.0-4.0, preferably a pH of 3.0.
19. The sample analyzer of any one of claims 14 to 18, wherein the control device is configured to perform the following steps in determining whether a cryptococcus infection is present in the sample to be tested:
and when the difference value of the first cryptococcus particle counting value and the second cryptococcus particle counting value is within a preset range, judging that cryptococcus infection exists in the sample to be detected.
20. A method of detecting cryptococcus in a sample, the method comprising the steps of:
processing a sample to be tested to obtain a sample to be tested, wherein the processing of the sample to be tested comprises mixing the sample to be tested with a hemolysis reagent;
enabling the particles in the sample to be detected to pass through an optical detector one by one to obtain optical information of each particle in the sample to be detected;
and distinguishing the cryptococcus particle swarm in the sample to be detected according to the optical information.
21. The method of claim 20, wherein said distinguishing the population of cryptococcus particles in the sample to be tested from the optical information comprises:
and distinguishing the cryptococcus particle swarm in the sample to be detected according to the forward scattering light intensity information and the side scattering light intensity information in the optical information.
22. The method of claim 20, wherein the processing a sample to be tested further comprises:
mixing the sample to be tested with a fluorescent dye reagent;
wherein, said differentiating the cryptococcus particle swarm in the sample to be tested according to the optical information comprises:
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 or according to the forward scattering light intensity information and the side scattering light 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.
23. The method according to claim 22, wherein the test sample is a body fluid sample, in particular a cerebrospinal fluid sample, the method further comprising:
distinguishing at least one particle group of a leukocyte particle group, a nucleated cell particle group, a mononuclear cell particle group, and a plurality of nucleated cell particle groups according to the side 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 optionally counting the at least one particle group.
24. The method of claim 20 or 21, wherein the test sample is a blood sample, the method further comprising:
differentiating leukocyte subsets including lymphocytes, monocytes, neutrophils and eosinophils based on the forward scattered light intensity information and the side scattered light intensity information of the optical information, and optionally counting the differentiated leukocyte subsets.
25. The method of claim 22, wherein the test sample is a blood sample, the method further comprising:
distinguishing leukocyte subsets including lymphocytes, monocytes, neutrophils and eosinophils and/or distinguishing a naive granulocyte population according to the side 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 optionally counting the distinguished leukocyte subsets and/or naive granulocyte population; or
Distinguishing at least one of a nucleated red blood cell population and a basophil subpopulation in the sample to be tested from the forward scattered light intensity information and the fluorescence intensity information in the optical information, preferably from the forward scattered light intensity information, the side scattered light intensity information and the fluorescence intensity information in the optical information, and optionally counting the at least one particle population.
26. The method of any one of claims 20 to 25, wherein said distinguishing a population of cryptococcus particles in the sample to be tested from said optical information comprises:
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;
identifying particles in the characteristic region of the cryptococcus particle as cryptococcus particles.
27. The method of any of claims 20 to 26, wherein the method further comprises:
counting the particle swarm of the cryptococcus in the sample to be detected; and
optionally, when the count value of the population of cryptococcus is over a predetermined value, it is suggested that cryptococcus infection exists in the sample to be tested.
28. The method according to any one of claims 20 to 27, wherein the first agent has a pH of 2.0-4.0, preferably a pH of 3.0; or the first reagent has a pH value of 6.5-7.5, preferably a pH value of 7.0.
29. A method of detecting cryptococcus in a sample, the method comprising the steps of:
processing a first portion of a test sample to obtain a first test sample, said processing the first portion of the test sample comprising mixing the first portion of the sample with a first hemolysis reagent and a first fluorescent dye;
enabling the particles in the first sample to be detected to pass through an optical detector one by one, and obtaining first optical information of each particle in the first sample to be detected;
distinguishing the cryptococcus particle swarm in the first sample to be detected according to the first optical information and counting to obtain a first cryptococcus particle count value;
processing a second portion of the test sample to obtain a second test sample, said processing the second portion of the test sample comprising mixing the second portion of the sample with a second hemolysis reagent that is different from the first hemolysis reagent;
enabling the particles in the second sample to be detected to pass through the optical detector one by one to obtain second optical information of each particle in the second sample to be detected;
distinguishing the cryptococcus particle swarm in the second sample to be detected according to the second optical information and counting to obtain a second cryptococcus particle count value;
and judging whether cryptococcus infection exists in the sample to be detected according to the first cryptococcus particle counting value and the second cryptococcus particle counting value.
30. The method of claim 29, wherein the first optical information comprises forward scattered light intensity information and fluorescence scattered light intensity information, preferably forward scattered light intensity information, side scattered light intensity information and fluorescence intensity information.
31. The method of claim 29 or 30, wherein the second optical information comprises forward scattered light intensity information and side scattered light intensity information.
32. The method of any one of claims 29 to 31, 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 fluorescence reagent different from the first fluorescence reagent;
wherein the second optical information includes forward scattered light intensity information, side scattered light intensity information, and fluorescence intensity information.
33. A process according to any one of claims 29 to 32, wherein the first hemolysis reagent has a pH of 6.5-7.5, preferably has a pH of 7.0; and/or the second hemolysis reagent has a pH of 2.0-4.0, preferably a pH of 3.0.
34. The method of any one of claims 29 to 33, wherein said determining whether a cryptococcus infection is present 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 counting value and the second cryptococcus particle counting value is within a preset range, judging that cryptococcus infection exists in the sample to be detected.
35. A computer-readable storage medium, on which a computer program is stored which, when being executed by a processor, carries out the steps of the method of any one of claims 20 to 34.
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