JP6871729B2 - Detection method and blood analyzer for red blood cells infected with Plasmodium - Google Patents

Description

The present invention relates to a method for detecting erythrocytes infected with Plasmodium malaria. The present invention also relates to a blood analyzer for detecting erythrocytes infected with Plasmodium malaria.

Malaria is an infectious disease caused by Plasmodium malaria and is prevalent mainly in the tropics and subtropics. Plasmodium malaria infects humans through the Anopheles mosquito. Since Anopheles mosquitoes live in abundant water sources such as ponds and swamps, many malaria patients are found in remote areas where access to medical institutions is difficult.

Malaria kills many lives each year, most of them in children under the age of five. This is because children under the age of 5 have not acquired immunity to Plasmodium, which causes serious symptoms and rapidly worsens. On the other hand, adults in malaria endemic areas have acquired immunity to Plasmodium malaria and are asymptomatic patients who have no symptoms even if infected with malaria. This is one of the reasons why malaria is difficult to eradicate. First, Anopheles mosquitoes become hosts for Plasmodium malaria by sucking the blood of adult asymptomatic patients. Then, when this Anopheles mosquito sucks the blood of a child, Plasmodium malaria is transmitted from an adult asymptomatic patient to the child. Therefore, without screening and treating asymptomatic patients, it is not possible to prevent infection in children and eradicate malaria. However, since the number (concentration) of Plasmodium malaria is often low in the blood of asymptomatic patients due to immunity, it is difficult to identify the infection by microscopic examination, which is a general malaria test method. Therefore, screening of asymptomatic patients requires genetic testing, but there are few facilities that can perform such testing in malaria endemic areas.

Japanese Unexamined Patent Publication No. 2006-304774

Tamura T, Blood Count Specimen, Rinsho Byori, vol.63, p.1387-1396, 2015 Nguyen-Dinh P. et al., Comparative Studies on the Cryopreservation of Malaria Parasites, WHO Informal Consultation on Malaria Parasite Strain characterization, Cryopreservation and Banking of Isolates, Geneva, 8-11 December 1980.

As shown in Patent Document 1, it is known to detect erythrocytes infected with Plasmodium malaria by a flow cytometer (FCM). FCM enables quantitative measurement of erythrocytes infected with Plasmodium malaria and is less costly than genetic testing. Therefore, FCM testing is extremely useful for screening asymptomatic patients. On the other hand, in order to carry out an accurate malaria test by FCM, it is necessary to immediately use the blood sample for FCM measurement after obtaining the blood sample. In blood samples, nutrients become deficient over time and dead cells begin to increase. Since FCM measures cells one by one, it is not possible to perform accurate tests on blood samples that have deteriorated over time.

Here, cryopreservation is known as a means for preventing deterioration of biological samples. However, when the blood sample is frozen, the water inside and outside the cell crystallizes and the cell membrane is physically destroyed. Freezing especially damages red blood cells and causes hemolysis. Therefore, it is difficult to detect cells from a thawed blood sample. Non-Patent Document 1 also describes that cryopreservation of blood samples used for FCM for measuring blood cell count is generally prohibited.

On the other hand, Non-Patent Document 2 describes that a cryopreservation solution such as glycerol or dimethyl sulfoxide (DMSO) was added to a blood sample collected from a malaria-infected patient and cryopreserved. In addition, this document describes that a smear was prepared from a thawed blood sample and the number of protozoans was counted. The action of the cryopreservation solution prevents the destruction of cells in the blood sample due to freezing.

In this way, by adding a cryopreservation solution to a blood sample collected from a malaria-infected patient and freezing it, the cells in the blood sample can be stably preserved. Therefore, it is not necessary to perform measurement quickly in hospitals, inspection facilities, etc., and convenience is improved. However, since the step of adding the cryopreservation solution to the blood sample is added, the work becomes complicated when the number of blood samples is large as in screening.

Furthermore, since the blood sample is diluted by the addition of the cryopreservation solution, the cell concentration changes from the concentration that the original blood sample had. Therefore, when measuring a blood sample to which a cryopreservation solution has been added by FCM, it is necessary to collect cells after thawing and adjust the cell concentration in the measurement sample. Alternatively, when adding the cryopreservation solution to the blood sample, it is necessary to accurately weigh and mix the amounts of the blood sample and the cryopreservation solution, and correct the FCM measurement result based on the dilution ratio. Adjustment of cell concentration does not necessarily accurately reflect the original cell concentration. Cells may also be damaged by centrifugation and resuspension during preparation. In addition, it is extremely difficult and impractical to collect and dilute blood in an accurate amount in malaria endemic areas. Therefore, it is required to develop a means capable of easily preventing deterioration of blood samples and quantitatively detecting erythrocytes infected with Plasmodium malaria.

Contrary to common general knowledge, the present inventor has attempted to detect erythrocytes infected with Plasmodium malaria (hereinafter, also referred to as "malaria-infected erythrocytes") from blood samples frozen without adding a cryopreservation solution. Then, the present inventor has completed the present invention by finding that in a blood sample frozen without adding a cryopreservation solution, normal erythrocytes are hemolyzed, but malaria-infected erythrocytes are hardly hemolyzed. Therefore, the present invention is infected with Plasmodium including a step of thawing a frozen blood sample substantially free of a cryopreservation solution and a step of detecting erythrocytes infected with Plasmodium in the thawed blood sample. A method for detecting red blood cells is provided.

The present invention also comprises a step of thawing a substantially undiluted frozen blood sample and a step of detecting erythrocytes infected with Plasmodium in the thawed blood sample of erythrocytes infected with Plasmodium. Provide a detection method.

Further, the present invention relates to a step of thawing a frozen blood sample containing substantially no additive or a frozen blood sample containing substantially only an anticoagulant as an additive, and Plasmodium in the thawed blood sample. Provided is a method for detecting erythrocytes infected with Plasmodium, which comprises a step of detecting infected erythrocytes.

Furthermore, the present invention comprises a sample preparation unit that prepares a measurement sample from a thawed blood sample that is substantially free of a cryopreservation solution, a reagent containing a fluorescent dye for nucleic acid staining, and a reagent that partially damages the cell membrane of blood cells. , A flow cell for flowing the measurement sample prepared by the sample preparation unit, a light source unit for irradiating the measurement sample flowing in the flow cell with light, and scattered light obtained when the measurement sample is irradiated with light. Provided is a blood analyzer including an optical detection unit for acquiring information and fluorescence information, and an analysis unit for detecting red blood cells infected with malaria protozoa in the blood sample based on scattered light information and the fluorescence information.

The present invention also includes a sample preparation unit that prepares a measurement sample from a thawed blood sample that is not substantially diluted, a reagent containing a fluorescent dye for nucleic acid staining, and a reagent that partially damages the cell membrane of blood cells. A flow cell for flowing the measurement sample prepared by the sample preparation unit, a light source unit for irradiating the measurement sample flowing in the flow cell with light, scattered light information obtained when the measurement sample is irradiated with light, and scattered light information. Provided is a blood analyzer including an optical detection unit for acquiring fluorescence information and an analysis unit for detecting red blood cells infected with malaria protozoa in the blood sample based on scattered light information and the fluorescence information.

In addition, the present invention provides a frozen blood sample that contains substantially no additives or a thawed blood sample that contains substantially only an anticoagulant as an additive, a reagent containing a fluorescent dye for nucleic acid staining, and a cell membrane of blood cells. A sample preparation unit that prepares a measurement sample from a partially damaging reagent, a flow cell for flowing the measurement sample prepared by the sample preparation unit, and a light source unit for irradiating the measurement sample flowing in the flow cell with light. An optical detection unit that acquires scattered light information and fluorescence information obtained when the measurement sample is irradiated with light, and erythrocytes infected with malaria protozoa in the blood sample based on the scattered light information and the fluorescence information. Provided is a blood analyzer provided with an analysis unit for detecting.

According to the present invention, it is possible to detect malaria-infected erythrocytes from a frozen blood sample that is substantially free of cryopreservation solution. That is, the present invention makes it possible to cryopreserve a blood sample without adding a cryopreservation solution, and it is possible to easily prevent deterioration of the blood sample. Moreover, since the blood sample is not substantially diluted with the cryopreservation solution, the present invention can be used for quantitative detection of malaria-infected erythrocytes.

It is a figure for demonstrating the region where each particle group of normal erythrocytes, malaria-infected erythrocytes, leukocytes, and platelets appears on a scattergram. It is an example of a scattergram showing the analysis result of a frozen blood sample containing malaria-infected erythrocytes. It is a schematic diagram which shows the structure of the blood analyzer of this embodiment. It is a perspective view which shows the structure of a flow cell. It is a block diagram which shows the structure of an analysis part. It is a flowchart which shows the flow of operation of the blood analyzer of this embodiment. It is a flowchart which shows the procedure of the measurement sample preparation process. It is a flowchart which shows the procedure of the measurement data analysis processing. It is a scattergram which shows the analysis result of the blood sample which contained the blood of a healthy person or the cultured Malaria protozoa, which was frozen without adding a cryopreservative. The left panel is a photograph of blood derived from a healthy person who has not been frozen under a microscope, and the right panel is a photograph of blood derived from a healthy person who has been frozen without adding a cryopreservative agent under a microscope. It is a photograph of cultured Malaria protozoa frozen without adding a cryopreservative, observed under a microscope. It is a photograph which observed with a microscope the test sample 1 (a mixture of cultured Malaria protozoa and blood derived from a healthy person) frozen without adding a cryopreservative. It is a scattergram which shows the analysis result of the blood derived from the frozen or re-frozen healthy person. It is a scattergram which shows the analysis result of the cultured malaria protozoan which was frozen or re-frozen. It is a scattergram which shows the analysis result of the frozen or refrozen blood sample containing the cultured Malaria protozoa and the blood derived from a healthy person. It is a scattergram showing the analysis result of the blood sample frozen at various temperatures.

[1. How to detect erythrocytes infected with Plasmodium]
In the method for detecting erythrocytes infected with Plasmodium malaria according to the present embodiment (hereinafter, also simply referred to as “detection method”), first, a frozen blood sample that does not substantially contain a cryopreservation solution is thawed.

A "frozen blood sample that does not substantially contain a cryopreservation solution" is a blood sample that is in a frozen state and that does not substantially contain a cryopreservation solution. As used herein, the term "blood sample that does not substantially contain a cryopreservation solution" refers to a blood sample that does not contain a cryopreservation solution or blood that contains the cryopreservation solution in an amount that does not produce the effect of the cryopreservation solution. Refers to a sample. As used herein, the "cryopreservative solution" is a liquid reagent having the effect of preventing damage to the cell membrane during freezing of a sample and maintaining the shape of cells during thawing. In order to obtain the above effects of the cryopreservation solution, it is generally necessary to add the cryopreservation solution in an amount corresponding to 20 to 100% of the amount of the blood sample. The amount at which the effect of the cryopreservation solution cannot be obtained may be less than the amount at which the above effect of the cryopreservation solution can be obtained (that is, the amount normally used), depending on the type of the cryopreservation solution. As an example of the amount at which the effect of the cryopreservation solution cannot be obtained, there is an amount in which the increase in volume due to the addition of the cryopreservation solution to the blood sample can be ignored in the complete blood count (CBC). With such an amount, the effect of the cryopreservation solution cannot be obtained. On the other hand, when the cryopreservation solution is added to the blood sample in a normal amount, the cell concentration of the original blood sample changes significantly, so that the increase in volume cannot be ignored in CBC. In addition, CBC is preferably carried out by the sheath flow DC measurement method using FCM.

The type of cryopreservation solution is not particularly limited, and examples thereof include glycerol, DMSO, sorbitol solution, hydroxyethyl starch solution, serum albumin solution, dextran solution, polyphenol solution, and a combination thereof. Also included is a commercially available cryopreservation solution.

In the present embodiment, the frozen blood sample that does not substantially contain the cryopreservation solution may contain additives other than the cryopreservation solution. Such an additive is appropriately selected from the additives used in the field of blood testing, and is preferably an anticoagulant.

The blood sample is preferably whole blood collected from the subject. The subject is not particularly limited, but is preferably a malaria patient or a person suspected of being infected with Plasmodium malaria. Those suspected of being infected with Plasmodium malaria are, for example, those with suspected malaria symptoms and asymptomatic malaria patients.

The detection method of the present embodiment may be a method using a frozen blood sample that is substantially undiluted. Here, the "substantially undiluted frozen blood sample" is a substantially undiluted blood sample in a frozen state. As used herein, the term "substantially undiluted blood sample" refers to a sample consisting of whole blood collected from a subject, or an addition used in the field of whole blood collected from a subject and a blood test. A sample consisting of an agent and the increase in volume due to the addition of the additive to the whole blood is negligible in CBC. Anticoagulants are known as such additives. The additive may be a liquid or a powder. As long as the additive is used within the range of normal usage in the field of blood testing, the increase in volume due to the addition can be ignored in the CBC.

Anticoagulants are the most commonly used additives for the storage of collected blood. As used herein, adding an anticoagulant to a blood sample in a normal amount is not included in diluting the blood sample. That is, in the present specification, the blood sample composed of whole blood and an anticoagulant collected from a subject corresponds to a blood sample that is not substantially diluted. In the field of blood tests, powdered anticoagulants are often used, but the increase in volume due to the addition of such powders is insignificant.

Cryopreservation solution is a kind of additive used in the field of blood test. However, as described above, when the cryopreservation solution is added to the blood sample in a normal amount, the cell concentration of the original blood sample changes significantly, so that the increase in volume cannot be ignored in CBC. That is, adding the cryopreservation solution to the blood sample in a normal amount is included in diluting the blood sample.

The detection method of the present embodiment may be a method using a frozen blood sample substantially containing no additive or a frozen blood sample containing substantially only an anticoagulant as an additive. Here, the "frozen blood sample substantially free of additives" is a blood sample in a frozen state that is substantially free of additives. In the present specification, the "blood sample containing substantially no additive" refers to a blood sample containing no additive or a blood sample containing the additive in an amount in which the effect of the additive cannot be obtained. A blood sample containing no additives is, in other words, a sample consisting of whole blood collected from a subject. The amount at which the effect of the additive cannot be obtained may be an amount lower than the usual amount of the additive, depending on the type of the additive. In this embodiment, such a small amount of additive may be contained in whole blood collected from a subject.

A "frozen blood sample containing substantially only an anticoagulant as an additive" is a blood sample in a frozen state containing substantially only an anticoagulant as an additive. In the present specification, the "blood sample containing substantially only an anticoagulant as an additive" is a sample consisting of whole blood and an anticoagulant collected from a subject, or a whole sample collected from a subject. A sample consisting of blood, an anticoagulant, and an additive other than the anticoagulant, and the amount of the additive other than the anticoagulant is an amount at which the effect of the additive cannot be obtained. Additives other than the anticoagulant are not particularly limited, and examples thereof include a cryopreservation solution. The amount at which the effect of the additive cannot be obtained is as described above.

The anticoagulant is appropriately selected from calcium ion chelating agents and antithrombin agents in the field of blood tests. Specific examples of the chelating agent include ethylenediaminetetraacetic acid (EDTA) salt and sodium citrate, and examples of the antithrombin agent include heparin and its analogs, with particular preference given to the EDTA salt. Examples of the EDTA salt include EDTA · 2K, EDTA · 3K and EDTA · 2Na.

The frozen blood samples used in the detection method of the present embodiment described above are collectively referred to as "frozen samples" below. Regardless of which frozen sample is used, the subsequent operation for detecting malaria-infected erythrocytes is the same.

The frozen sample can be obtained by freezing the blood sample, which is substantially free of cryopreservation solution, within 4 hours after blood collection from the subject, preferably immediately. The means for freezing the blood sample is not particularly limited, and examples thereof include a freezer, a cold storage container containing a coolant such as dry ice, and liquid nitrogen. The freezing temperature may be −20 ° C. or lower, preferably lower than −20 ° C., and more preferably −80 ° C. or lower.

The frozen sample is preferably stored in a frozen state until it is used in the detection method of the present embodiment. However, transportation from malaria endemic areas to inspectable facilities takes a long time. Therefore, there may be situations where the frozen sample is thawed during transport and the sample must be re-frozen. As shown in Examples below, the present inventor has confirmed that freezing and thawing a blood sample to which no cryopreservation solution has been added at least twice does not affect the detection of malaria-infected erythrocytes. .. Therefore, in the present embodiment, the frozen sample may be a sample obtained by refreezing a blood sample that has been thawed once.

In the present embodiment, the means for thawing the frozen sample is not particularly limited as long as the blood cells are not damaged. For example, the frozen sample may be heated at a temperature of 1 ° C. or higher and 35 ° C. or lower. Specifically, the frozen sample may be heated in a water bath or an incubator set to such a temperature. Alternatively, the frozen sample may be simply allowed to stand at room temperature. It is preferable that the thawed blood sample is promptly subjected to the detection step described later.

In the thawed blood sample, some of the normal red blood cells are hemolyzed. Specifically, the number of normal red blood cells in the thawed blood sample is reduced by about 20%, about 30%, about 40% or about 50% as compared with that before freezing.

Next, in the detection method of the present embodiment, erythrocytes infected with Plasmodium protozoan are detected in the thawed blood sample.

The malaria-infected erythrocytes themselves in the thawed blood sample can also be confirmed by microscopic observation. However, microscopic observation does not allow quantitative detection of malaria-infected erythrocytes. For the quantitative detection of malaria-infected erythrocytes by microscopic observation, it is necessary to calculate the ratio between the number of normal erythrocytes (non-infected erythrocytes) and the number of malaria-infected erythrocytes. However, since some normal erythrocytes are hemolyzed in the thawed blood sample, normal erythrocytes cannot be accurately counted by microscopic observation, and the above ratio cannot be calculated. Therefore, in this embodiment, it is preferable to detect malaria-infected erythrocytes in a blood sample using FCM. Since FCM measures all blood cells contained in a predetermined amount of blood sample, the number of malaria-infected red blood cells per predetermined amount can be obtained. Therefore, if the thawed blood sample is measured by FCM, malaria-infected erythrocytes can be quantitatively detected.

When FCM is used, it is also referred to as a thawed blood sample, a reagent that partially damages the cell membrane of blood cells (hereinafter, also referred to as "sample treatment solution"), and a reagent containing a fluorescent dye for nucleic acid staining (hereinafter, "staining solution"). It is preferable to prepare a measurement sample by mixing with (referred to as). In the measurement by FCM, the amount of the thawed blood sample may be about 20 μL to 500 μL.

The sample treatment solution contains a surfactant for damaging the cell membranes of normal erythrocytes, malaria-infected erythrocytes and leukocytes to the extent that the above-mentioned fluorescent dye can permeate. Such a surfactant can be appropriately selected from a cationic surfactant, an anionic surfactant, amphoteric surfactant and nonionic surfactant, but is preferably a cationic surfactant. Examples of cationic surfactants include lauryltrimethylammonium chloride (LTAC), stearyltrimethylammonium chloride (STAC), myristyltrimethylammonium chloride, myristyltrimethylammonium bromide, cetyltrimethylammonium chloride, octyltrimethylammonium bromide, and decyltrimethylammonium bromide. , Cetylpyridinium chloride and the like.

In the present embodiment, the sample treatment liquid preferably contains a nonionic surfactant in addition to the above cationic surfactant. Examples of such nonionic surfactants include polyoxyethylene alkyl ether, polyoxyethylene sterol, polyoxyethylene castor oil, polyoxyethylene sorbit fatty acid ester, polyoxyethylene alkyl amine, and polyoxyethylene polyoxypropylene alkyl. Examples include ether. Among them, polyoxyethylene (20) polyoxypropylene (8) cetyl ether (also called PBC-44) is particularly preferable.

In the present embodiment, it is preferable to use at least two kinds of surfactants having different dissolving powers for the cell membrane of erythrocytes in combination. Such combinations include, for example, a combination of LTAC and STAC, a combination of LTAC and STAC and PBC-44, a combination of myristyltrimethylammonium chloride and cetyltrimethylammonium chloride, and the like.

The concentration of the surfactant in the sample treatment liquid can be appropriately adjusted according to the type of the surfactant. Further, the concentration of the cationic surfactant in the sample treatment liquid is adjusted according to the final concentration of the cationic surfactant in the measurement sample described later.

The lower limit of the pH of the sample treatment liquid is usually 5.0, preferably 5.5. The upper limit of the pH of the sample treatment liquid is usually 7.0, preferably 6.5. More specifically, the pH of the sample treatment liquid is 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, It is 6.9 or 7.0.

The sample treatment solution preferably contains a buffer to maintain the pH in the above range. The buffer can be appropriately selected from, for example, citric acid, phosphoric acid, succinic acid and Good's buffer. Good buffers include, for example, ADA, PIPES, MES, tricine, tris, bicine, bis-tris, bis-tris-propane, ACES, MOPS, MOPSO, BES, TES, HEPES, HEPPS, TAPS, etc. .. An acid such as hydrochloric acid or a base such as sodium hydroxide may be added to the sample treatment solution to adjust the pH.

The lower limit of the osmotic pressure of the sample treatment liquid is usually 200 mOsm / kg · H ₂ O, preferably 220 mOs m / kg · H ₂ O. The upper limit of the osmotic pressure of the sample treatment liquid is usually 300 mOsm / kg · H ₂ O, preferably 280 mOs m / kg · H ₂ O. More specifically, the osmotic pressure of the sample treatment liquid is 200, 210, 220, 230, 240, 250, 260, 270, 280, 290 or 300 mOsm / kg · H ₂ O.

The sample treatment liquid preferably contains an osmotic pressure adjusting agent in order to adjust the osmotic pressure within the above range. Examples of the osmotic pressure adjusting agent include alkali metal salts such as sodium chloride, magnesium chloride and alkaline earth metal salts such as magnesium chloride, and sugars such as glucose and mannose.

The solvent of the sample treatment liquid is not particularly limited as long as it does not interfere with blood analysis by FCM, and can be appropriately selected from water, an organic solvent, and a mixture thereof. Water is preferred. Examples of the organic solvent include lower alcohols having 1 to 6 carbon atoms (particularly ethanol), ethylene glycol, diethylene glycol, polyethylene glycol, DMSO and the like.

The stain contains a fluorescent dye capable of selectively staining nucleic acids, especially DNA. As such a fluorescent dye, a bisbenzimide-based fluorescent dye is preferable, and a fluorescent dye represented by the following chemical formula (Hoechst 34580: Sigma-Aldrich) is particularly preferable.

Hoechst 34580 is a DNA-selective bisbenzimide fluorescent dye that stains DNA more strongly than RNA and is excited by light with a wavelength of 405 nm. In addition to Hoechst 34580, Hoechst 33258 and Hoechst 33342 may be used as DNA-selective bisbenzimide-based fluorescent dyes. Although these fluorescent dyes have different side chains from Hoechst 34580, they can all be excited by light with a wavelength of 405 nm.

The concentration of the fluorescent dye in the dyeing solution is usually 3 μM or more and 200 μM or less, preferably 10 μM or more and 180 μM or less. When the measurement sample is prepared by a fully automatic blood analyzer, the concentration of the fluorescent dye in the staining solution may be determined from the above range in consideration of the amount of liquid that can be sucked by the device.

The solvent of the staining solution is not particularly limited as long as it does not interfere with blood analysis by FCM, and can be appropriately selected from water, an organic solvent, and a mixture thereof. It is preferably an organic solvent. Examples of the organic solvent include lower alcohols having 1 to 6 carbon atoms (particularly ethanol), ethylene glycol, diethylene glycol, polyethylene glycol, DMSO and the like, and ethylene glycol is particularly preferable.

In the present embodiment, the final concentration of the fluorescent dye in the measurement sample is preferably 0.15 μM or more and 1.0 μM or less. By staining blood cells at such a final concentration, the ability of FCM to separate malaria-infected erythrocytes from normal erythrocytes is further improved. In the present embodiment, the final concentration of the cationic surfactant in the measurement sample is not particularly limited, but may be, for example, 1.72 mM or more and 5.97 mM or less.

In the present embodiment, the order in which the thawed blood sample, the sample treatment solution, and the staining solution are mixed is not particularly limited. These may be mixed sequentially or at the same time. Alternatively, a mixed solution of the sample treatment solution and the staining solution may be prepared in advance, and the mixed solution and the thawed blood sample may be mixed. When mixing these, it is preferable to heat them at a temperature of 30 to 45 ° C. for 5 to 60 seconds.

Due to the action of the sample treatment solution, the cell membranes of normal erythrocytes, malaria-infected erythrocytes and leukocytes in the blood sample are partially damaged, and the fluorescent dye can permeate. In this embodiment, normal erythrocytes may be contracted by the action of the sample treatment solution. Normal red blood cells do not have nuclei and are not stained with fluorescent dyes. Since platelets also do not have a nucleus, they are not stained with a fluorescent dye. In this embodiment, platelets may be aggregated. On the other hand, in malaria-infected erythrocytes, the nuclei of Plasmodium existing inside are stained with a fluorescent dye. Since leukocytes also have nuclei, they are stained with a fluorescent dye.

In the measurement by FCM, it is preferable to irradiate the prepared measurement sample with light to acquire scattered light information and fluorescence information. Specifically, first, the measurement sample is introduced into the FCM flow cell, and when each blood cell in the sample passes through the flow cell, the blood cell is irradiated with light. Then, the scattered light and fluorescence emitted from the blood cell are measured to obtain scattered light information and fluorescence information.

The scattered light information includes, for example, the pulse peak, pulse width, pulse area, scattered light intensity, etc. of the forward scattered light (for example, the light receiving angle is around 0 to 20 degrees) and the side scattered light (the light receiving angle is around 90 degrees). Can be mentioned. In this technique, it is known that laterally scattered light reflects internal information such as cell nuclei and granules, and forward scattered light reflects cell size. In the present embodiment, it is preferable to acquire the forward scattered light intensity as the scattered light information. Examples of the fluorescence information include fluorescence intensity, fluorescence pulse width, fluorescence pulse area, and the like, and fluorescence intensity is preferable. The wavelength of the excitation light to be irradiated can be appropriately selected according to the fluorescent dye.

The light source of the FCM is not particularly limited, and a light source having a wavelength suitable for exciting the fluorescent dye can be appropriately selected. As the light source, for example, a blue semiconductor laser, a red semiconductor laser, an argon laser, a He-Ne laser, a mercury arc lamp, or the like is used. When Hoechst 34580 is used, the light source is preferably a blue semiconductor laser.

Plasmodium grows in erythrocytes in the order of ring foam, trophozoite, and schizont. As it grows, the amount of Plasmodium DNA and the size of infected red blood cells increase. Therefore, in the present embodiment, the morphology of Plasmodium can be distinguished based on the scattered light information and the fluorescence information. Further, the ring foam includes a ring foam (single) in which one Plasmodium invades one erythrocyte and a ring foam (multi) in which a plurality of Plasmodium invades one erythrocyte. The amount of DNA in the ring foam (multi) is larger than that in the ring foam (single). Therefore, in the present embodiment, the ring foam (single) and the ring foam (multi) can be distinguished based on the fluorescence information.

In this embodiment, malaria-infected erythrocytes in a thawed blood sample are detected based on the above scattered light information and fluorescence information. Detection of malaria-infected erythrocytes based on scattered light information and fluorescence information is performed by creating a scattergram having two axes of scattered light information and fluorescence information and identifying the region where malaria-infected erythrocytes appear on the scattergram. It is preferable to do so.

The appearance region of each cell group on the scattergram with the fluorescence intensity on the horizontal axis and the forward scattered light intensity on the vertical axis will be described with reference to FIG. 1A. As shown in FIG. 1A, blood samples containing malaria-infected erythrocytes are classified by FCM into four groups: normal erythrocytes (non-infected erythrocytes), malaria-infected erythrocytes, leukocytes and platelets. The scattergram can be created and the cell group can be classified by using appropriate analysis software. Such analysis software is usually stored in the FCM itself or in the computer that controls the FCM.

With reference to FIG. 1A, the leukocyte-containing group appears in the region 10 where the forward scattered light intensity and the fluorescence intensity are high. The group containing platelets appears in the region 20 in which the forward scattered light intensity is similar to that of the group containing leukocytes and the fluorescence intensity is smaller than that of the group containing leukocytes. The platelet-containing group may contain aggregates of two or more platelets. As described above, normal erythrocytes are hardly stained with the staining solution, and contract due to the action of the sample treatment solution. Therefore, the group containing normal erythrocytes appears in the region 30 where the forward scattered light intensity and the fluorescence intensity are small. Malaria-infected erythrocytes contract due to the action of the sample treatment solution, but the nucleus of the Plasmodium inside is stained. Therefore, the group containing malaria-infected erythrocytes appears in the region 40 in which the forward scattered light intensity is smaller than the group containing leukocytes and the fluorescence intensity is larger than the group containing normal erythrocytes.

Actually, when a scattergram is prepared based on the measurement result of a blood sample containing malaria-infected erythrocytes, a scattergram as shown in FIG. 1B is obtained. By using the above sample treatment solution and staining solution, the blood cell component in the thawed blood sample can satisfactorily separate normal erythrocytes (non-infected erythrocytes) and malaria-infected erythrocytes as shown in FIG. 1B. In addition, in FIG. 1B, since the particles whose fluorescence intensity is equal to or less than a predetermined threshold value are excluded, most of the particles showing normal erythrocytes are excluded from the scattergram.

The analysis software can also count the number of dots (representing cells) that appear in any region on the scattergram. In this embodiment, malaria-infected erythrocytes appear in the region 40 of FIG. 1A. By counting the number of cells appearing in this region, the number of erythrocytes infected with Plasmodium can be obtained. Therefore, the detection method of the present embodiment may further include a step of counting red blood cells infected with Plasmodium.

As mentioned above, FCM measures all blood cells in a predetermined amount of blood sample introduced into the flow cell. Further, as shown in Examples described later, in a blood sample frozen and thawed without adding a cryopreservation solution, normal erythrocytes are hemolyzed, but malaria-infected erythrocytes are not. Therefore, the number of malaria-infected erythrocytes per predetermined amount (for example, 1 μL), that is, the concentration of malaria-infected erythrocytes in a blood sample can be obtained from the number of cells in the region where malaria-infected erythrocytes appear on the scattergram. Therefore, the detection method of the present embodiment may further include a step of obtaining the concentration of Plasmodium-infected erythrocytes in a blood sample based on the number of erythrocytes infected with Plasmodium-infected erythrocytes obtained in the counting step.

When CBC by the sheath flow DC measurement method is also performed in addition to the optical measurement by the flow cytometry method described above, a thawed blood sample and a diluted solution for DC measurement are mixed to prepare a second measurement sample. You may. Unlike the sample treatment solution, the diluted solution for DC measurement does not have the effect of damaging the cell membrane of blood cells. The diluted solution for DC measurement itself is known in the art, and examples thereof include Cellpack (Sysmex Corporation). The diluted solution for DC measurement can also be used as a sheath solution for optical measurement and DC measurement.

[2. Blood analyzer]
The scope of the present invention also includes a blood analyzer for carrying out the detection method of the present embodiment. That is, the blood analyzer of the present embodiment is a device for detecting malaria-infected erythrocytes in a thawed blood sample that does not substantially contain a cryopreservation solution. Here, the thawed blood sample that does not substantially contain the cryopreservation solution is a sample obtained by thawing the above-mentioned "frozen blood sample that does not substantially contain the cryopreservation solution". The means for thawing the frozen sample is the same as that described for the detection method of this embodiment.

In a further embodiment, the blood sample may be a thawed blood sample that is substantially undiluted. The thawed blood sample that is not substantially diluted is a sample obtained by thawing the above-mentioned "substantially undiluted frozen blood sample".

In a further embodiment, the blood sample may be a thawed blood sample that is substantially free of additives or a thawed blood sample that contains substantially only an anticoagulant as an additive. The thawed blood sample substantially free of additives is a sample obtained by thawing the above-mentioned "frozen blood sample substantially free of additives". The thawed blood sample containing substantially only an anticoagulant as an additive is a sample obtained by thawing the above-mentioned "frozen blood sample containing substantially only an anticoagulant as an additive".

The thawed blood samples used in the blood analyzer of the present embodiment described above are collectively referred to as "thawed samples" below. Regardless of which thawed sample is used, the configuration and operation of the blood analyzer are the same.

<Structure of blood analyzer>
An example of the blood analyzer according to the present embodiment will be described with reference to the drawings. However, this embodiment is not limited to this example. As shown in FIG. 2, the blood analyzer 1 includes a measuring unit 2 and an analysis unit 3. The measuring unit 2 takes in a blood sample, prepares a measurement sample from the blood sample, and optically measures the measurement sample. The analysis unit 3 processes the measurement data obtained by the measurement of the measurement unit 2 and outputs the analysis result of the blood sample.

The measurement unit 2 includes a suction unit 4, a sample preparation unit 5, an optical detection unit 6, a second detection unit 7, a signal processing circuit 81, a microcomputer 82, and a communication interface 83. The suction unit 4 has a suction tube (not shown). The suction unit 4 sucks the thawed sample contained in the test tube by the suction tube.

The sample preparation unit 5 has a reaction vessel 54 and is connected to the reagent containers 51, 52 and 53. The reagent container 52 contains the sample treatment liquid. The reagent container 53 contains the staining solution. The reagent container 51 contains a diluted solution for DC measurement (hereinafter, also simply referred to as “diluted solution”). The sample treatment liquid, the stain liquid, and the diluent are the same as those described for the detection method of the present embodiment. The suction unit 4 moves the suction tube above the reaction tank 54 and discharges the sucked thawed sample into the reaction tank 54. The thawed sample, the sample processing solution, and the staining solution are mixed in the reaction vessel 54 to prepare a measurement sample. One reagent which is a mixture of a sample processing solution and a staining solution may be mixed with a thawed sample to prepare a measurement sample. The measurement sample is used for optical measurement of blood cells by the flow cytometry method. The thawed sample sucked by the suction unit 4 and the diluent are mixed in the reaction tank 54 to prepare a second measurement sample. The second measurement sample is subjected to CBC by the sheath flow DC measurement method. In this example, the embodiment including one reaction tank has been described, but the blood analyzer of the present embodiment may include two or more reaction tanks.

The optical detection unit 6 is used for optical measurement of blood cells by the flow cytometry method. The optical detection unit 6 includes a flow cell 61, a light source unit 62, and detection units 63 and 64. The flow cell 61 is supplied with the diluent contained in the reagent container 51 and the measurement sample prepared by the sample preparation unit 5.

The flow cell 61 is made of a translucent material such as quartz, glass, or synthetic resin in a tubular shape. The inside of the flow cell 61 is a flow path through which the measurement sample and the sheath liquid, which is a diluted solution for DC measurement, flow. With reference to FIG. 3, the flow cell 61 is provided with an orifice 61a whose internal space is narrowed down more than other portions. Further, the vicinity of the inlet of the orifice 61a has a double tube structure, and the inner tube portion thereof is a sample nozzle 61b, through which the measurement sample prepared by the sample preparation unit 5 is supplied. The space outside the sample nozzle 61b is a flow path 61c through which the sheath liquid flows. The sheath liquid is introduced into the orifice 61a through the flow path 61c. In this way, the sheath liquid supplied to the flow cell 61 flows so as to wrap the measurement sample discharged from the sample nozzle 61b. Then, the flow of the measurement sample is narrowed down by the orifice 61a, and the blood cells in the measurement sample wrapped in the sheath liquid pass through the orifice 61a one by one.

The light source unit 62 is a semiconductor laser light source, and irradiates the orifice 61a of the flow cell 61 with a bluish-purple laser beam having a wavelength of 405 nm. The detection units 63 and 64 detect the light emitted from the individual cells in the measurement sample when the flow of the measurement sample in the flow cell 61 is irradiated with light. Avalanche photodiodes can be used for the detectors 63 and 64. In the following description, the direction connecting the light source unit 62 and the flow cell 61 is referred to as "X direction", and the direction orthogonal to the X direction is referred to as "Y direction". The detection unit 63 can detect the fluorescence emitted from each cell in the measurement sample through the mirror 65 arranged on the Y direction side from the flow cell 61. The detection unit 64 is arranged on the X direction side from the flow cell 61. Specifically, the detection unit 64 is arranged on the opposite side of the light source unit 62 with the flow cell 61 interposed therebetween. The detection unit 64 can detect the forward scattered light emitted from individual cells in the measurement sample. In addition, instead of the forward scattered light, other scattered light such as side scattered light and backscattered light may be detected.

In the present embodiment, an irradiation lens system composed of a plurality of lenses (not shown) may be arranged between the light source unit 62 and the flow cell 61. The irradiation lens system allows the parallel beam emitted from the semiconductor laser light source to be focused on the beam spot. Further, a beam stopper (not shown) may be arranged on the optical axis linearly extending from the light source unit 62 so as to face the light source unit 62 with the flow cell 61 interposed therebetween. The beam stopper blocks the direct light from the semiconductor laser light source, and the detection unit 64 can receive only the forward scattered light from the measurement sample.

Each of the detection units 63 and 64 photoelectrically converts the received fluorescence and the forward scattered light, and outputs an analog signal indicating the light reception intensity. Hereinafter, the analog signal output from the detection unit 63 is referred to as a “fluorescence signal”, and the analog signal output from the detection unit 64 is referred to as a “forward scattered light signal”.

The second detection unit 7 is used for CBC by the sheath flow DC measurement method. A second measurement sample is supplied from the sample preparation unit 5 to the second detection unit 7. The second detection unit 7 includes a sheath flow cell (not shown), and applies a voltage to the second measurement sample flowing in the sheath flow cell. When the blood cell passes through the sheath flow cell, the voltage changes due to the electrical resistance of the blood cell. The second detection unit 7 detects the electric resistance by capturing the change in the voltage, thereby detecting the blood cells. The second detection unit 7 outputs an analog signal indicating a voltage.

In the thawed sample, the numbers of normal red blood cells, white blood cells and platelets are changed as compared with those before freezing. In particular, since normal red blood cells are hemolyzed, the number of normal red blood cells obtained by CBC of the thawed sample does not accurately reflect the state of blood before freezing. Therefore, the second detection unit 7 is not essential for the blood analyzer of the present embodiment. However, the blood analyzer generally has a configuration for performing CBC by the sheath flow DC measurement method.

The signal processing circuit 81 performs signal processing on the analog signals output by the detection units 63 and 64 and the second detection unit 7. The signal processing circuit 81 extracts the peak value of the pulse included in the fluorescence signal and the forward scattered light signal as a feature parameter. Hereinafter, the peak value of the fluorescence signal is referred to as "fluorescence intensity", and the peak value of the forward scattered light signal is referred to as "forward scattered light intensity". The signal processing circuit 81 extracts the peak value of the output signal of the second detection unit 7 as blood cell detection data.

The microcomputer 82 controls the suction unit 4, the sample preparation unit 5, the optical detection unit 6, the second detection unit 7, the signal processing circuit 81, and the communication interface 83. The communication interface 83 is connected to the analysis unit 3 by a communication cable. The measurement unit 2 performs data communication with the analysis unit 3 by the communication interface 83. When the thawed sample is measured, the communication interface 83 transmits measurement data including each feature parameter to the analysis unit 3.

The configuration of the analysis unit 3 will be described with reference to FIG. The analysis unit 3 includes a main body 300, an input unit 309, and an output unit 310. The main body 300 includes a CPU (Central Processing Unit) 301, a ROM (Read Only Memory) 302, a RAM (Random Access Memory) 303, a hard disk 304, a reading device 305, an input / output interface 306, and an image output interface 307. And a communication interface 308. In the present embodiment, a display that displays an image is used as the output unit 310. As the output unit 310, a printer that outputs to paper or the like by printing may be used.

The CPU 301 executes a computer program stored in the ROM 302 and a computer program loaded in the RAM 303. The RAM 303 is used to read a computer program recorded on the ROM 302 and the hard disk 304. The RAM 303 is also used as a work area of the CPU 301 when executing a computer program. A computer program 320 for analyzing the measurement data given by the measurement unit 2 and outputting the analysis result is installed on the hard disk 304.

The reading device 305 is a flexible disk drive, a CD-ROM drive, a DVD-ROM drive, or the like, and can read a computer program or data recorded on the portable recording medium 321. Further, the portable recording medium 321 stores a computer program 320 for causing the computer to function as the analysis unit 3. The computer program 320 read from the portable recording medium 321 is installed on the hard disk 304.

The input unit 309 is connected to the input / output interface 306. The output unit 310 is connected to the image output interface 307. The communication interface 308 is connected to the communication interface 83 of the measuring unit 2.

<Operation of blood analyzer>
The operation of the blood analyzer 1 will be described with reference to FIG. First, the CPU 301 of the analysis unit 3 receives the measurement execution instruction from the user via the input unit 309 (step S101). Upon receiving the measurement execution instruction, the CPU 301 transmits instruction data instructing the measurement start to the measurement unit 2 (step S102), and the measurement unit 2 receives the instruction data (step S103). The microcomputer 82 executes the measurement sample preparation process (step S104), executes the first measurement process (step S105), and executes the second measurement process (step S106).

The measurement sample preparation process will be described with reference to FIG. The microcomputer 82 controls the suction unit 4 to supply a predetermined amount (for example, 17 μL) of thawed sample to the reaction vessel 54 (step S201). Next, the microcomputer 82 controls the sample preparation unit 5 to supply a predetermined amount (for example, 1 mL) of sample treatment liquid from the reagent container 52 to the reaction vessel 54, and a predetermined amount (for example, 1 mL) from the reagent vessel 53 to the reaction vessel 54. 20 μL) of the staining solution is supplied (step S202). The reaction tank 54 is heated to a predetermined temperature by a heater. In the heated state, the mixture in the reaction vessel 54 is stirred (step S203). By the operation of steps S201 to S203, the measurement sample is prepared in the reaction tank 54. The microcomputer 82 controls the sample preparation unit 5 to lead the measurement sample from the reaction tank 54 to the optical detection unit 6 (step S204).

Next, the microcomputer 82 controls the suction unit 4 to supply a predetermined amount (for example, 4 μL) of thawed sample to the reaction vessel 54 (step S205). Next, the microcomputer 82 controls the sample preparation unit 5 to supply a predetermined amount (for example, 2000 μL) of the diluted solution from the reagent container 51 to the reaction vessel 54 (step S206). The sample preparation unit 5 stirs the mixture in the reaction vessel 54 (step S207). By the operation of steps S205 to S207, the second measurement sample is prepared in the reaction tank 54. The microcomputer 82 controls the sample preparation unit 5 to lead the second measurement sample from the reaction tank 54 to the second detection unit 7 (step S208). As mentioned above, the preparation of the second measurement sample for CBC is not essential for the blood analyzer of this embodiment. However, blood analyzers are generally capable of performing CBC by sheath flow DC measurement.

When the process of step S208 is completed, the microcomputer 82 returns the process to the main routine.

With reference to FIG. 5, in the first measurement process, the measurement sample is measured by the optical detection unit 6. The sample preparation unit 5 supplies the measurement sample to the flow cell 61 together with the sheath liquid. When the measurement sample flows through the flow cell 61, blood cells pass through the orifice 61a of the flow cell 61 one by one. Here, blood cells may contain aggregates of two or more platelets. The light source unit 62 irradiates the measurement sample flowing in the flow cell 61 with light. More specifically, the light source unit 62 irradiates individual blood cells passing through the orifice 61a of the flow cell 61 with light. Each time a blood cell is irradiated with light, the blood cell emits fluorescence and forward scattered light.

The fluorescence emitted from the blood cells is detected by the detection unit 63. The forward scattered light emitted from the blood cells is detected by the detection unit 64. Each of the detection units 63 and 64 outputs an electric signal according to the light receiving level as a fluorescence signal and a forward scattered light signal. The signal processing circuit 81 extracts the fluorescence intensity from the fluorescence signal and extracts the forward scattered light intensity from the forward scattered light signal.

In the second measurement process, the second detection unit 7 measures the second measurement sample. The second detection unit 7 applies a voltage to each blood cell in the second measurement sample flowing in the sheath flow cell, and outputs an analog signal indicating the voltage to the signal processing circuit 81. Each time a blood cell passes through a sheath flow cell, the voltage changes according to its electrical resistance. The signal processing circuit 81 detects changes in electrical resistance by signal processing and detects blood cells. As a result, the signal processing circuit 81 converts the output signal of the second detection unit 7 into blood cell detection data. After the second measurement process, the microcomputer 82 transmits the measurement data including each feature parameter to the analysis unit 3 (step S107), and ends the process.

The analysis unit 3 receives the measurement data (step S108). After that, the CPU 301 executes the measurement data analysis process, generates the analysis result of the blood sample, and stores the analysis result in the hard disk 304 (step S109).

The measurement data analysis process will be described with reference to FIG. 7. When the measurement data analysis process is started, the CPU 301 first classifies normal erythrocytes, malaria-infected erythrocytes, leukocytes, and platelets based on the fluorescence intensity and forward scattered light intensity contained in the measurement data (step S301). The CPU 301 may create a catagram using the data of the forward scattered light intensity and the fluorescence intensity. Here, since normal erythrocytes do not have a nucleus, they are hardly stained with a fluorescent dye, and the normal erythrocytes in the measurement sample hardly emit fluorescence. Therefore, in the process of step S301, particles whose fluorescence intensity is equal to or less than a predetermined threshold value may be excluded. This excludes most normal erythrocytes from the scattergram and improves the separation of normal erythrocytes from malaria-infected erythrocytes.

In the process of step S301, the CPU 301 combines the particle size distribution of the forward scattered light intensity and the particle size distribution of the fluorescence intensity to produce a group containing leukocytes, a group containing platelet aggregation, a group containing normal erythrocytes, and malaria-infected erythrocytes. Identify the population that contains it. More specifically, the CPU 301 identifies a group of particles having high fluorescence intensity and forward scattered light intensity as a group containing leukocytes. The CPU 301 identifies a group containing platelets having a forward scattered light intensity similar to that containing white blood cells and a fluorescence intensity smaller than that containing white blood cells. The CPU 301 identifies a group containing normal erythrocytes, in which the forward scattered light intensity is smaller than the group containing leukocytes and the fluorescence intensity is smaller than the group containing platelets. The CPU 301 identifies a group containing malaria-infected erythrocytes, in which the forward scattered light intensity is smaller than the group containing leukocytes and the fluorescence intensity is higher than the group containing normal erythrocytes.

Next, the CPU 301 counts the number of cells in the group containing the malaria-infected erythrocytes classified in step S301 (step S302). Further, the CPU 301 calculates the concentration of malaria-infected erythrocytes in the thawed sample per predetermined amount (for example, 1 μL) based on the counted number of malaria-infected erythrocytes and the amount of the thawed sample used for preparing the measurement sample (step S303). ).

Then, the CPU 301 calculates the blood cell count using the blood cell detection data by the sheath flow DC measurement method included in the measurement data (step S304). Instead of the sheath flow DC measurement method, the blood cell count can be calculated using the forward scattered light intensity and the fluorescence intensity included in the measurement data. As described above, by treatment with the sample treatment solution and the staining solution, normal erythrocytes, malaria-infected erythrocytes, leukocytes and platelets each appear in a predetermined region on the scattergram. Therefore, the blood cell count can be calculated by counting the number of cells in each group classified in step S301. Instead of the forward scattered light intensity, the pulse width and pulse area of the forward scattered light may be used. Instead of the forward scattered light, the side scattered light may be detected and the peak value, pulse width, or pulse area of the pulse of the side scattered light may be used.

With the above, the CPU 301 ends the measurement data analysis process and returns the process to the main routine. When the measurement data analysis process as described above is completed with reference to FIG. 5, the CPU 301 outputs the analysis result to the output unit 310 (step S110), and ends the process.

Hereinafter, the present invention will be described in detail with reference to Examples, but the present invention is not limited to these Examples.

Example 1 Measurement of cryopreserved blood sample by FCM It was examined whether malaria-infected erythrocytes could be detected by measuring the cryopreserved blood sample by FCM without adding a cryopreservation solution.

(1) Blood sample Cultured malaria protozoa (obtained from the Institute of Microbial Diseases, Osaka University: hereinafter also referred to as "cultured malaria") is used as blood collected from healthy volunteers (hereinafter also referred to as "human whole blood") Addition was made to prepare test samples 1 and 2 mimicking malaria-infected blood. Anticoagulants are added to whole human blood in normal doses. Therefore, the test samples 1 and 2 also contain an anticoagulant. The cultured malaria protozoa is a culture obtained by infecting human blood from which leukocytes have been removed with the malaria protozoa, and is not the malaria protozoa itself. In the culture, Plasmodium is present in human erythrocytes. Test sample 1 is a sample assuming a patient with malaria symptoms, and contains malaria-infected erythrocytes at about 2,000 / μL. Test sample 2 is a sample for asymptomatic patients and contains malaria-infected erythrocytes at about 200 / μL. Whole human blood and cultured malaria were used as control blood samples, respectively.

_{(2) Reagent A solution (pH 6.1, 277 mOsm / kg · H 2} O) having the following composition was prepared as a reagent (sample treatment solution) that partially damages the cell membrane of blood cells. Purified water (1.0 L) was used as the solvent.
[Composition of sample treatment liquid]
LTAC (Cation Surfactant) 2.95 mM
STAC (Cation Surfactant) 1.11 mM
PBC-44 (nonionic surfactant) 2.90 mM
ADA (buffer) 20 mM
Appropriate amount of NaCl (amount at which the osmotic pressure becomes the above value)

A reagent (staining solution) containing a fluorescent dye for nucleic acid staining was prepared by dissolving 12.5 μg of Hoechst 34580 (Sigma-Aldrich) in ethylene glycol (1 mL). The concentration of Hoechst 34580 in the stain was about 22.3 μM.

(3) Freezing, thawing and measurement of blood samples
500 μL of each blood sample was dispensed into a 1.5 mL Eppendorf tube. These tubes were placed in a freezer and stored overnight (about 18 hours) at −20 ° C. Frozen blood samples were thawed at room temperature. After thawing, the treatment solution (1 mL) and the staining solution (20 μL) were immediately added to the blood sample (17 μL) and mixed at 41 ° C. for 20 seconds to prepare a measurement sample. The measurement sample was measured with a flow cytometer XN-30 (Sysmex Co., Ltd.) using a blue semiconductor laser with a wavelength of 405 nm as a light source. Scattergrams were prepared based on the forward scattered light intensity and fluorescence intensity obtained from each blood sample. For comparison, a part of each blood sample prepared in (1) above was taken and measured in the same manner as above without freezing to prepare a scattergram. Blood cell counts (CBC) for each blood sample were also performed.

(4) Results Scattergrams of each blood sample are shown in FIG. In addition, CBC data of whole human blood, cultured malaria, and test samples 1 and 2 are shown in Tables 1 to 4, respectively. The numerical value (/ μL) in FIG. 8 is the number of particles (number of cells) in the region where malaria-infected erythrocytes appear on the scattergram. In this example, this number of particles was counted as the number of malaria-infected red blood cells. In the table, WBC is leukocyte, RBC is erythrocyte, Hgb is hemoglobin, Hct is hematocrit, MCV is mean corpuscular volume, MCH is mean corpuscular hemoglobin amount, MCHC is mean erythrocyte hemoglobin concentration, PLT is platelet, MI-RBC # is malaria infection. Refers to red blood cells.

As can be seen from the scattergram of each blood sample before freezing shown in FIG. 8, the above FCM measurement was able to discriminate between normal erythrocytes, malaria-infected erythrocytes and leukocytes. The scattergram of each blood sample after freezing showed the same distribution as the scattergram of each blood sample before freezing. On the other hand, from Tables 1 to 4, the number of normal red blood cells (RBC) decreased in the frozen blood sample by about 20% to about 30% as compared with the blood sample before freezing. This indicates that the erythrocytes were hemolyzed by freezing. From this, it was expected that malaria-infected erythrocytes would also be hemolyzed by freezing. However, as shown in Tables 2-4, the number of malaria-infected erythrocytes after freezing was almost unchanged from the number before freezing. This can be seen from FIG. In the scattergrams of test samples 1 and 2 before freezing, the number of malaria-infected erythrocytes per μL was 2321 / μL and 220 / μL, respectively. Then, in the scattergrams of the test samples 1 and 2 after freezing, the number of malaria-infected erythrocytes per 1 μL was 2252 / μL and 252 / μL, respectively. These values are approximately the same as the concentration of malaria-infected erythrocytes assumed at the time of preparation of each test sample. Thus, FCM also confirmed that freezing blood samples showed almost no change in the number of malaria-infected erythrocytes.

If malaria-infected erythrocytes are hemolyzed, it is possible that Plasmodium malaria (merozoite) is released from the infected erythrocytes. However, in the scattergrams of the cultured malaria after freezing and test samples 1 and 2 shown in FIG. 8, no particles were observed in the region where merozoite appeared. From these facts, it was found that normal erythrocytes were hemolyzed by freezing the blood sample, but malaria-infected erythrocytes were hardly hemolyzed. Therefore, it was found that malaria-infected erythrocytes can be detected by measuring a blood sample cryopreserved without adding a cryopreservation solution by FCM.

In Example 1, since the blood sample is not diluted with the cryopreservation solution, the blood sample retains the original cell concentration. Therefore, FCM can count the number of malaria-infected red blood cells in a given amount of blood sample. From this, it was found that malaria-infected erythrocytes can be quantitatively detected from blood samples cryopreserved without adding a cryopreservation solution by FCM measurement.

Example 2 Microscopic observation of cryopreserved blood sample It was examined whether malaria-infected erythrocytes could be detected by observing the cryopreserved blood sample under a microscope without adding a cryopreservation solution.

(1) Preparation, Staining and Observation of Blood Samples Frozen human whole blood, cultured malaria and test sample 1 prepared in Example 1 were used as blood samples. These blood samples were thawed in the same manner as in Example 1 to prepare smears. These smears were Giemsa-stained according to a conventional method and observed under a microscope. For comparison, smears were prepared from whole human blood that had not been cryopreserved, stained with Giemsa, and observed under a microscope.

(2) Results Photographs of each stained blood sample are shown in FIGS. 9A to 9C. In the figure, arrows indicate malaria-infected erythrocytes. A large number of normal erythrocytes are present on the left panel of FIG. 9A (whole human blood), and the dented shape characteristic of erythrocytes can be clearly recognized. On the other hand, in the right panel of FIG. 9A, no normal erythrocytes were found, and it can be seen that hemolysis was caused by freezing. Similarly, in FIG. 9B (cultured malaria) and FIG. 9C (test sample 1), no normal erythrocytes (non-infected erythrocytes) were found, and the erythrocytes were hemolyzed. However, in FIGS. 9B and 9C, erythrocytes containing Plasmodium malaria of ring foam and trophozoite could be found. Therefore, it was found that the malaria-infected erythrocytes themselves can be detected even if the blood sample is frozen without being substantially diluted. However, since uninfected erythrocytes are hemolyzed, it is not possible to quantitatively detect malaria-infected erythrocytes. This is because it is necessary to count the number of uninfected erythrocytes for quantitative detection (calculation of infection rate) with a microscope.

Example 3 Effect of refreezing of blood sample on FCM measurement When the transportation of blood sample from the malaria endemic area to the laboratory takes a long time, the cold insulation material melts during the transportation and the frozen blood sample is thawed. It is possible to do. In addition, it is fully conceivable that the thawed blood sample may be re-frozen due to the additional addition of the cold insulation material. Therefore, the effects of refreezing and rethawing of blood samples on malaria-infected erythrocytes and their measurement were investigated.

(1) Preparation and measurement of blood sample As the blood sample, frozen human whole blood, cultured malaria and test sample 1 prepared in Example 1 were used. After thawing these blood samples at room temperature, they were placed in the freezer again and frozen at −20 ° C. The refrozen blood sample was thawed at room temperature. After thawing, a measurement sample was prepared in the same manner as in Example 1 and measured with a flow cytometer XN-30 (Sysmex Corporation). Scattergrams were prepared based on the forward scattered light intensity and fluorescence intensity obtained from each blood sample. The blood cell count of each refrozen blood sample was also calculated.

(2) Results The scattergrams of each refrozen blood sample are shown in FIGS. 10A to 10C together with the scattergram of Example 1. In addition, the CBC data of each refrozen blood sample is shown in Tables 5 to 7, together with the data of Example 1.

As can be seen from Tables 5-7, refreezing the blood sample further reduced the number of normal erythrocytes, but little change in the number of malaria-infected erythrocytes. A similar tendency was observed in the scattergrams shown in FIGS. 10A-10C. As shown in FIGS. 10B and 10C, refreezing of the blood sample showed almost no change in the distribution of malaria-infected erythrocytes. From these facts, it was shown that even if the blood sample to which the cryopreservation solution was not added was repeatedly frozen and thawed at least twice, there was no effect on malaria-infected erythrocytes and it was detectable by FCM.

Example 4 Examination of freezing temperature For cryopreservation at -20 ° C, the temperature of a general freezer is assumed. During actual sample transportation, it is assumed that dry ice or liquid nitrogen will be used as the cold insulation material. Therefore, we investigated whether FCM could detect malaria-infected erythrocytes even when frozen at a lower temperature. For comparison, blood samples to which a cryopreservation solution was added were also examined.

(1) Blood sample Cultured Plasmodium was added to whole human blood to prepare test sample 3 containing malaria-infected erythrocytes at about 4,000 / μL. 300 μL of Test Sample 3 was dispensed into a 1.5 mL Eppendorf tube. Test sample 4 was prepared by adding 1.0 mL each of a cryopreservation solution having the following composition to a part of the tube containing the test sample 3.

[Cyropreservative solution]
Ultrapure water 72 mL
Sorbitol 4.2 g
Sodium chloride 0.9 g
Glycerol 28 mL
The obtained solution was filtered and sterilized with a 0.22 μm filter.

(2) Freezing, thawing and measurement of blood samples The tubes were stored at -20 ° C (freezer), -80 ° C (freezer) or -196 ° C (liquid nitrogen) for 2 days. Frozen blood samples were thawed at room temperature. For test sample 3, immediately after thawing, add the same treatment solution (1 mL) and stain solution (20 μL) as in Example 1 to test sample 3 (17 μL), mix at 41 ° C for 20 seconds, and mix the measurement sample. Prepared. The test sample 4 was immediately centrifuged after thawing, and 1 mL of the supernatant was removed (the remaining amount was about 300 μL). 17 μL was taken from the test sample 4 from which the supernatant was removed, and a measurement sample was prepared in the same manner as above. The prepared measurement sample was measured with a flow cytometer XN-30 (Sysmex Corporation) in the same manner as in Example 1. Scattergrams were prepared based on the forward scattered light intensity and fluorescence intensity obtained from each blood sample. As a control, a part of the test sample 3 prepared in (1) above was taken and measured in the same manner as above without freezing to prepare a scattergram.

(3) Results Scattergrams of each blood sample are shown in FIG. The numerical value (/ μL) in FIG. 11 is the number of particles (number of cells) in the region where malaria-infected erythrocytes appear on the scattergram. From the scattergram of test sample 3 (without cryopreservation solution), when the blood sample was cryopreserved at -80 ° C or lower, the number of infected erythrocytes was hardly reduced. On the other hand, when blood samples were cryopreserved at -20 ° C, the number of infected erythrocytes decreased slightly, suggesting that cell damage due to freezing was greater than storage at -80 ° C or lower. For test sample 4 (with cryopreservation solution), since the sample is diluted by the addition of the cryopreservation solution, an error may occur in the quantitative evaluation. Therefore, although it is not appropriate to quantitatively evaluate the test sample 4, the number of malaria-infected erythrocytes was significantly reduced by cryopreservation at −20 ° C.

1 Blood analyzer 2 Measurement unit 3 Analysis unit 5 Sample preparation unit 6 Optical detection unit 7 Second detection unit 61 Flow cell 62 Light source unit 63 Detection unit 64 Detection unit 301 CPU
310 Display 320 Computer program 321 Portable recording medium

Claims (15)

The process of thawing a frozen blood sample that contains virtually no cryopreservation solution,
The process of detecting erythrocytes infected with Plasmodium in thawed blood samples using a flow cytometer, and
Methods for detecting erythrocytes infected with Plasmodium, including. The method according to claim 1, wherein the frozen blood sample is a blood sample frozen at −20 ° C. or lower. The method according to claim 1, wherein the frozen blood sample is a blood sample frozen at −80 ° C. or lower. It further comprises the step of preparing a measurement sample from a thawed blood sample, a reagent containing a fluorescent dye for nucleic acid staining, and a reagent that partially damages the cell membrane of blood cells.
The method according to any one of claims 1 to 3 , wherein in the step of detecting erythrocytes infected with Plasmodium, the erythrocytes infected with Plasmodium in the prepared measurement sample are detected. In the step of detecting erythrocytes infected with Plasmodium,
The prepared measurement sample is irradiated with light to obtain scattered light information and fluorescence information.
The method according to any one of claims 1 to 4 , wherein erythrocytes infected with Plasmodium in a thawed blood sample are detected based on the acquired scattered light information and fluorescence information. The method according to any one of claims 1 to 5 , further comprising counting erythrocytes infected with Plasmodium in a blood sample based on the detected erythrocytes infected with Plasmodium. The method of claim 6 , further comprising the step of obtaining the concentration of Plasmodium-infected erythrocytes in a blood sample based on the counted number of Plasmodium-infected erythrocytes. The method according to any one of claims 1 to 7 , wherein a part of red blood cells is hemolyzed in the step of thawing the frozen blood sample. The process of thawing a frozen blood sample that is substantially undiluted,
The process of detecting erythrocytes infected with Plasmodium in thawed blood samples using a flow cytometer, and
Methods for detecting erythrocytes infected with Plasmodium, including. A step of thawing a frozen blood sample that contains substantially no additives or a frozen blood sample that contains substantially only an anticoagulant as an additive.
The process of detecting erythrocytes infected with Plasmodium in thawed blood samples using a flow cytometer, and
Methods for detecting erythrocytes infected with Plasmodium, including. It further comprises the step of preparing a measurement sample from a thawed blood sample, a reagent containing a fluorescent dye for nucleic acid staining, and a reagent that partially damages the cell membrane of blood cells.
In the step of detecting erythrocytes infected with Plasmodium,
The prepared measurement sample is irradiated with light to obtain scattered light information and fluorescence information.
Based on the obtained scattered light information and fluorescence information, it detects the infected erythrocyte malaria parasites in the blood sample thawing method according to claim 9 or 1 0. The method according to any one of claims 9 to 11, further comprising counting erythrocytes infected with Plasmodium in a blood sample based on the detected erythrocytes infected with Plasmodium. A sample preparation unit that prepares a measurement sample from a thawed blood sample that contains substantially no cryopreservation solution, a reagent containing a fluorescent dye for nucleic acid staining, and a reagent that partially damages the cell membrane of blood cells.
A flow cell for flowing the measurement sample prepared by the sample preparation unit, and
A light source unit for irradiating the measurement sample flowing in the flow cell with light,
An optical detection unit that acquires scattered light information and fluorescence information obtained when the measurement sample is irradiated with light, and
A blood analyzer comprising an analysis unit that detects red blood cells infected with Plasmodium in the blood sample based on the scattered light information and the fluorescence information. Measured samples from thawed blood samples that are substantially undiluted or thawed blood samples that contain only anticoagulants as additives, reagents that contain fluorescent dyes for nucleic acid staining, and reagents that partially damage the cell membrane of blood cells. Sample preparation department to prepare and
A flow cell for flowing the measurement sample prepared by the sample preparation unit, and
A light source unit for irradiating the measurement sample flowing in the flow cell with light,
An optical detection unit that acquires scattered light information and fluorescence information obtained when the measurement sample is irradiated with light, and
A blood analyzer comprising an analysis unit that detects red blood cells infected with Plasmodium in the blood sample based on the scattered light information and the fluorescence information. Frozen blood samples that are substantially free of additives or thawed blood samples that contain substantially only anticoagulants as additives, reagents containing fluorescent dyes for nucleic acid staining, and reagents that partially damage the cell membrane of blood cells. The sample preparation department that prepares the measurement sample from
A flow cell for flowing the measurement sample prepared by the sample preparation unit, and
A light source unit for irradiating the measurement sample flowing in the flow cell with light,
An optical detection unit that acquires scattered light information and fluorescence information obtained when the measurement sample is irradiated with light, and
A blood analyzer comprising an analysis unit that detects red blood cells infected with Plasmodium in the blood sample based on the scattered light information and the fluorescence information.

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