Mononuclear cell simulation composition, preparation method, quality control substance and quality control method

Abstract

The invention relates to a monocyte simulation composition, a preparation method, a quality control substance and a quality control method. The mononuclear cell simulation composition comprises mononuclear cell simulation particles stored in a preservation solution, the volume ratio of the mononuclear cell simulation particles in the composition is 50% or above, the mononuclear cell simulation particles are obtained by treating neutrophils or lymphocytes separated from blood of mammals, and the mononuclear cell simulation particles are used for simulating the mononuclear cells or the lymphocytes. When the mononuclear cell simulation particle is tested by a hematology analyzer, the mononuclear cell simulation particle has the same or similar light scattering characteristics as human mononuclear cells, and the light scattering characteristics at least reflect the complexity of cell contents. The mononuclear cell simulation particles are independently prepared, so that the particles have good consistency and stability, and the obtained composition is small in batch-to-batch difference when serving as a quality control substance, so that the quality control of a hematology analyzer is facilitated, and the reliability of clinical examination of the hematology analyzer is improved.

Description

Mononuclear cell mimicry composition, preparation method, quality control materials and quality control methods Technical Field

[0001] This disclosure relates to a method for preparing mononuclear cell mimicry particles for quality control in a blood cell analyzer, and the mononuclear cell mimicry particles, compositions, quality control materials, and quality control methods obtained therefrom. Background Technology

[0002] Currently, the detection parameters of a five-part differential hematology analyzer include the count and proportion of lymphocytes, monocytes, neutrophils, eosinophils, and basophils. The quality control or calibrator required for its quality control and calibration needs to contain simulated particles of these cells.

[0003] Currently available quality control materials or calibrators are typically prepared from mammalian blood, containing monocyte mimic particles, neutrophils, and lymphocytes, for use in three-part differential hematology analyzers. Alternatively, they may contain monocyte mimic particles within extracted mammalian (e.g., bovine) leukocytes as multiple cell mimic particles. Currently, there are no commercially available single-particle clusters of monocyte mimic particles prepared separately. Using currently available quality control materials or calibrators may result in significant variations in monocyte counts and proportions during quality control testing on hematology analyzers. Furthermore, methods for simultaneously preparing multiple cell mimic particles are susceptible to variations in individual animal characteristics and physical condition, leading to poor raw material stability. The requirement for multiple cell mimic particles to simultaneously meet requirements results in a low pass rate. Therefore, such preparation methods also suffer from high production costs and difficulty in scaling up production.

[0004] Therefore, there is a need to develop a mononuclear cell mimicry particle and its preparation method that can be well adapted to a blood cell analyzer for optical detection of five differential white blood cells. Summary of the Invention

[0005] In view of the above, this disclosure aims to provide a mononuclear cell mimicry composition comprising independently prepared mononuclear cell mimicry particles. These mononuclear cell mimicry particles are obtained by processing neutrophils or lymphocytes from mammalian blood and possess light scattering properties similar to or identical to those of human mononuclear cells, and can be used for quality control and / or calibration of blood cell analyzers.

[0006] A first aspect of this disclosure provides a mononuclear cell mimicry composition comprising mononuclear cell mimicry particles preserved in a preservation solution, wherein the mononuclear cell mimicry particles constitute more than 50% of the volume of the composition, the mononuclear cell mimicry particles being obtained by processing neutrophils or lymphocytes isolated from the blood of mammals, wherein the mononuclear cell mimicry particles, when tested with a hematology analyzer, have light scattering properties that are the same as or similar to those of human mononuclear cells, the light scattering properties at least reflecting the complexity of the cell contents.

[0007] In some embodiments, the mononuclear cell mimic particles constitute 50%-70% of the volume of the composition. In a further embodiment, the composition consists of mononuclear cell mimic particles preserved in a preservation solution.

[0008] A second aspect of this disclosure further provides a method for processing neutrophils or lymphocytes isolated from the blood of mammals to obtain the mononuclear cell mimic particles.

[0009] The method for preparing mononuclear cell mimic particles includes: processing neutrophils or lymphocytes isolated from mammalian blood to obtain the mononuclear cell mimic particles, wherein the mononuclear cell mimic particles, when tested with a blood cell analyzer, have the same or similar light scattering characteristics as human mononuclear cells, and the light scattering characteristics at least reflect the complexity of the cell contents.

[0010] In some embodiments, isolating the neutrophils or the lymphocytes includes the steps of lysing the red blood cells with a red blood cell lysis buffer and removing the lysed red blood cells.

[0011] In some embodiments, the step of treating neutrophils isolated from mammalian blood includes treating the neutrophils with a condensate of nitro alcohol and formaldehyde. In some embodiments, the neutrophils are treated with a condensate of nitro alcohol and formaldehyde at a concentration of 0.5 wt% to 5 wt% at 30°C for 24 to 36 hours. Further, the condensate of nitro alcohol and formaldehyde is 2-bromo-2-nitropropane-1,3-diol and / or 5-bromo-5-nitro-1,3-dioxane.

[0012] In some embodiments, the neutrophils treated with the condensate of nitro alcohol and formaldehyde are further fixed with an aldehyde compound. In a specific embodiment, the fixation is performed with the aldehyde compound at a concentration of 1%-10% (v / v), preferably 2%-5% (v / v), for 5 hours to 3 days, preferably 5 hours to 24 hours.

[0013] In some embodiments, the cells obtained in each step of preparing mononuclear cell mimic particles from neutrophils isolated from mammalian blood are washed and resuspended in a buffer solution with physiological osmotic pressure. The physiological osmotic pressure is 280 mOsm-340 mOsm, preferably 290 mOsm-300 mOsm, and more preferably 288 mOsm-292 mOsm.

[0014] In some embodiments, the mammalian blood or a suspension of the mammalian blood cells is subjected to density gradient centrifugation to separate crude lymphocytes.

[0015] In a further embodiment, the density gradient centrifugation includes performing the density gradient centrifugation with a cell separation medium whose density is between that of mammalian granulocytes and lymphocytes. According to some embodiments, the density of the cell separation medium is greater than 1.055 g / mL and less than 1.085 g / mL, preferably 1.065 g / mL to 1.080 g / mL. According to a specific embodiment, the cell separation medium is Ficoll cell separation medium or Percoll cell separation medium.

[0016] In some embodiments, the step of isolating the lymphocytes further includes centrifuging the crude lymphocyte product after removing lysed red blood cells at 150g-500g for 3-8 minutes, preferably at 200g-300g for 4-5 minutes, to remove platelets.

[0017] In some embodiments, processing lymphocytes isolated from mammalian blood includes treating the lymphocytes with a fluorescent dye capable of staining proteins in the cell membrane and inside the cell.

[0018] In some embodiments, the fluorescent dye is selected from compounds represented by the following general formula I:

[0019]

[0020] Wherein, the compound represented by general formula I is a protein fluorescent dye with a carboxyl group activated by N-hydroxysuccinimide, R and the carboxyl group substituted on R together constitute the protein fluorescent dye, and n is an integer from 1 to 4.

[0021] In some embodiments, the lymphocytes are treated with the fluorescent dye at a concentration of 0.5 mg / L-5 mg / L, preferably 0.7 mg / L-1.5 mg / L, for 3 min-60 min, preferably 3 min-15 min.

[0022] In some embodiments, lymphocytes treated with fluorescent dyes are fixed with aldehyde compounds. In a specific embodiment, the fixation is performed with the aldehyde compound at a concentration of 1%-10% (v / v), preferably 2%-5% (v / v), for 5 hours to 3 days, preferably 5 hours to 24 hours.

[0023] In some embodiments, the cells obtained in each step of preparing mononuclear cell mimic particles from lymphocytes isolated from mammalian blood are washed and resuspended in a buffer solution having the physiological osmotic pressure of mammalian lymphocytes. The physiological osmotic pressure of the mammalian lymphocytes is 295 mOsm-340 mOsm, preferably 300 mOsm-315 mOsm, and more preferably 305 mOsm-310 mOsm.

[0024] A third aspect of this disclosure provides a mononuclear granulocyte mimic particle prepared by the above method.

[0025] The fourth aspect of this disclosure provides a quality control or calibrator for a blood cell analyzer, comprising a mononuclear cell mimicry composition of any of the above embodiments or mononuclear cell mimicry particles prepared according to the methods of any of the above embodiments.

[0026] The fifth aspect of this disclosure provides a white blood cell five-part differential quality control material, comprising at least mononuclear cell mimic particles prepared according to any of the above embodiments.

[0027] In some embodiments, the white blood cell five-part differential quality control material further includes lymphocyte mimic particles, basophil mimic particles, neutrophils, and eosinophils. According to some embodiments, one or more of the lymphocyte mimic particles, basophil mimic particles, neutrophils, and eosinophils are prepared independently. Preferably, the lymphocyte mimic particles, basophil mimic particles, neutrophils, and eosinophils are all prepared independently.

[0028] The sixth aspect of this disclosure provides a whole blood quality control material, including the white blood cell five-part quality control material of any of the above embodiments, as well as red blood cell mimic particles, platelet mimic particles and reticulocyte mimic particles.

[0029] The seventh aspect of this disclosure provides a quality control method for a hematology analyzer, comprising: providing a quality control material containing at least five-part differential white blood cell particles, the quality control material comprising: mononuclear cell particles prepared according to any of the above embodiments, independently prepared lymphocyte particles, independently prepared basophil particles, independently prepared neutrophils, and independently prepared eosinophils; detecting the quality control material in quality control mode using the hematology analyzer to obtain a count of mononuclear cells in the quality control material; and comparing the count with a standard value of the quality control material.

[0030] In some embodiments, the deviation between the two counts of mononuclear cells in the quality control material obtained by performing two tests with the blood cell analyzer in quality control mode is less than 5%.

[0031] In some implementations, the coefficient of variation for each mononuclear cell count in all tests over one opening cycle of the quality control material is less than 10%.

[0032] In some embodiments, the test is performed on two batches of the quality control material separately, and the deviation between the two counts of monocytes in the two batches of the quality control material is less than 10%. The monocyte simulated particles in the monocyte simulated composition provided in this disclosure are prepared independently, resulting in small batch-to-batch differences and better consistency and stability of the simulated particle properties. This is beneficial for the quality control of the hematology analyzer and thus improves the reliability of the hematology analyzer for clinical testing. Attached Figure Description

[0033] Figure 1 shows the forward-scattered light intensity-side-scattered light intensity (FS-SS) scatter plot and the fluorescence intensity-side-scattered light intensity (FL-SSC) scatter plot obtained by testing the mononuclear cell simulated particles prepared in Example 1 and the blood samples from healthy subjects using two different blood cell analyzers.

[0034] Figure 2 shows the forward-scattered light intensity-side-scattered light intensity (FS-SS) scatter plot and fluorescence intensity-side-scattered light intensity (FL-SSC) scatter plot obtained by testing the mononuclear cell simulated particles prepared in Example 2 and blood samples from healthy subjects using two different blood cell analyzers.

[0035] Figure 3 shows a scatter plot of fluorescence intensity versus lateral scattering intensity of porcine blood lymphocytes isolated in Example 3 (Figure a) and mononuclear cell mimic particles prepared after further processing (Figure b) after testing with a hematology analyzer;

[0036] Figure 4 shows a scatter plot of fluorescence intensity versus side-scattered light intensity of bovine blood lymphocytes isolated in Example 4 (Figure a) and mononuclear cell mimic particles prepared after further processing (Figure b) after testing with a hematology analyzer;

[0037] Figure 5 shows the stability test results of the mononuclear cell mimic particles prepared in Examples 1-4 after 22 weeks of storage;

[0038] Figure 6 shows the forward scatter intensity-side scatter intensity (FS-SS) scatter plot and fluorescence intensity-side scatter intensity (FL-SSC) scatter plot (a) of the white blood cell five-part differential quality control material prepared in Example 6, obtained by testing with two different blood cell analyzers, and the test results (b) of the fluctuation in the proportion of mononuclear cell simulated particles to five-part white blood cells after 22 weeks of storage.

[0039] Figure 7 shows the test results of the fluctuation in the proportion of mononuclear cell simulated particles to white blood cells after 22 weeks of storage of whole blood quality control material prepared in Example 7;

[0040] Figure 8 shows a scatter plot of fluorescence intensity versus side-scatter light intensity of the whole blood control prepared in Example 7 (a), as well as the commercially available BC6D control (b) and BR60 control (c) on a hematology analyzer. Detailed Implementation

[0041] The technical solutions of this disclosure will be clearly and completely described below in conjunction with the implementation methods and embodiments. Obviously, the specific implementation methods described are only a part of the implementation methods, and not all of them. Based on these implementation methods, all other implementation methods obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0042] Throughout this specification, unless otherwise specified, the terminology used herein should be understood as having the meaning commonly used in the art. Therefore, unless otherwise defined, 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 pertains. In the event of any conflict, this specification shall prevail.

[0043] In this document, the terms “comprising,” “including,” or any other variations thereof are intended to cover non-exclusive inclusion, such that a method or product that comprises a list of elements includes not only the elements expressly stated, but also other elements not expressly listed, or elements inherent to carrying out the method or product.

[0044] Unless otherwise stated, the singular forms “a / kind” and “the / said” as used herein include the plural form of the noun they refer to.

[0045] The mammals mentioned in this disclosure refer to mammals other than humans. Specifically, the mammals may include cattle, pigs, etc., especially cattle.

[0046] The term "'single-particle cluster' cell-mimicking particle" as used in this disclosure refers to a single-species cell-mimicking particle containing only one cell type, particularly one leukocyte subtype. In this disclosure, unless otherwise specified, the qualifier "single-particle cluster" may be omitted, and the particle may simply be referred to as "cell-mimicking particle." For example, a mononuclear cell-mimicking particle refers to a mimicking particle of a single mononuclear cell.

[0047] The "independently prepared" cell mimic particle mentioned in this disclosure refers to a single type of cell mimic particle prepared using a relevant preparation method, that is, a single-particle cluster of a cell mimic particle prepared by that relevant preparation method. For example, independently prepared lymphocyte mimic particles refer to single-particle clusters of lymphocyte mimic particles prepared using a relevant preparation method.

[0048] The term "physiological osmotic pressure" as used in this disclosure refers to the osmotic pressure that enables blood cells (such as leukocytes) with cellular morphology to maintain an appropriate size in a solution (e.g., a treatment solution or preservation solution). Suitably, physiological osmotic pressure is in the range of 280 mOsm to 340 mOsm. The term "physiological osmotic pressure buffer" as used in this disclosure refers to a buffer solution having an appropriate physiological osmotic pressure.

[0049] The "physiological osmotic pressure of mammalian lymphocytes" mentioned in this disclosure refers to the osmotic pressure that enables mammalian lymphocytes to maintain an appropriate size in a solution (e.g., a treatment solution or a preservation solution). Suitablely, the physiological osmotic pressure is above 295 mOsm, particularly in the range of 300 mOsm to 315 mOsm.

[0050] The five-part differential white blood cell mimicry particles (including lymphocytes, monocytes, neutrophils, eosinophils, and basophils) commonly used in hematology analyzers are obtained by processing white blood cells from the blood of mammals such as cattle and sheep. There is currently no method to prepare monocyte mimicry particles independently. This is likely because the number of monocytes in blood is relatively small, and directly isolating monocytes is difficult, making the industrialization of preparing monocyte mimicry particles from mammalian monocytes highly improbable. Furthermore, other types of mammalian white blood cells differ significantly from human monocytes in their optical properties. For example, neutrophils from mammals, such as cattle, differ significantly from human monocytes in cell size, the quantity and structure of their contents, resulting in significant differences in their optical properties, particularly light scattering properties, when detected by hematology analyzers. Similarly, bovine lymphocytes differ significantly from human monocytes in their nucleic acid composition, leading to substantial differences in their fluorescence intensity characteristics.

[0051] Prior to this disclosure, no method had been developed for preparing single-particle clusters of mononuclear cell-mimicking particles from mammalian blood cells.

[0052] Therefore, this disclosure provides a method for preparing mononuclear cell mimic particles and mononuclear cell mimic particles of single particle clusters obtained therefrom, and thereby provides a mononuclear cell mimic composition.

[0053] Specifically, the method includes the step of processing neutrophils or lymphocytes isolated from mammalian blood to obtain monocyte-mimicking particles. When tested with a hematology analyzer, the monocyte-mimicking particles exhibit light scattering characteristics similar to or identical to those of human monocytes, which at least reflect the complexity of the cell contents. According to some embodiments, when tested with a hematology analyzer (particularly a five-part differential hematology analyzer), the monocyte-mimicking particles exhibit optical characteristics similar to or identical to those of human monocytes, including fluorescence (fluorescence FL after binding with nucleic acid dyes), forward scattering (FS), and side scattering (SS) characteristics, which at least reflect the cell's nucleic acid content (FL), size (FS), and complexity of intracellular contents (SS).

[0054] The blood cell analyzer mentioned in this article refers to a fully automated analyzer used for clinical testing of blood samples from subjects, capable of obtaining at least a complete blood count (CBC) analysis result. The blood cell analyzer possesses at least optical detection components capable of detecting the optical properties of particles in the sample, including the intensity of scattered light and fluorescence. The blood cell analyzer may also include detection components such as impedance detection components.

[0055] One embodiment of this disclosure provides a mononuclear cell mimicry composition comprising mononuclear cell mimicry particles preserved in a preservation solution, wherein the mononuclear cell mimicry particles constitute more than 50% of the volume of the composition, and the mononuclear cell mimicry particles are obtained by processing neutrophils or lymphocytes isolated from the blood of mammals, wherein the mononuclear cell mimicry particles, when tested with a hematology analyzer, have the same or similar light scattering characteristics as human mononuclear cells, and the light scattering characteristics at least reflect the complexity of the cell contents.

[0056] The method for obtaining the mononuclear cell-mimicking particles is described in detail below.

[0057] The method disclosed herein comprises two steps: first, neutrophils or lymphocytes are isolated from mammalian blood; then, the isolated neutrophils or lymphocytes are processed accordingly to adjust their size, content, and structure, so that their light scattering and fluorescence properties can mimic the optical properties of human monocytes when detected using a hematology analyzer. The methods for preparing monocyte-mimicking particles from mammalian neutrophils and lymphocytes are described in detail below.

[0058] Preparation of mononuclear cell mimic particles from mammalian neutrophils

[0059] Isolation of neutrophils

[0060] There are no particular restrictions on the method for isolating neutrophils from mammalian blood. First, the mammalian blood is centrifuged to separate the lower precipitate; then, the lower precipitate is treated with erythrocyte lysis buffer and centrifuged again to remove the upper layer, yielding neutrophils.

[0061] Mammalian blood is obtained from anticoagulated whole blood of mammals, such as cattle or pigs. After centrifugation, it separates into a plasma layer, a middle layer, and a lower sediment layer from top to bottom. The plasma and middle layers are removed, yielding the lower sediment layer containing erythrocytes and neutrophils. This lower sediment layer is mixed with erythrocyte lysis buffer, stirred thoroughly, and then allowed to stand at room temperature for 5 to 60 minutes, preferably 5 to 30 minutes, to lyse the erythrocytes. The mixture after lysis is centrifuged, then slowly poured off, removing the supernatant; the resulting precipitate is the neutrophil.

[0062] The erythrocyte lysis buffer mentioned herein is a lysis buffer that can lyse erythrocytes into fragments while allowing leukocytes to retain their cellular morphology. The erythrocyte lysis buffer is a solution containing a lysis agent. Commonly used lysis agents for erythrocyte lysis can be used, and this disclosure does not impose any particular limitation on this. For example, erythrocyte lysis buffers that can be used in this disclosure include, but are not limited to, Tris-ammonium chloride erythrocyte lysis buffer, Gey's erythrocyte lysis buffer, and ACK erythrocyte lysis buffer. The pH of the erythrocyte lysis buffer is 6.5-9.0 and can be adjusted using acid or base.

[0063] In some embodiments, the erythrocyte lysis buffer has the aforementioned physiological osmotic pressure to maintain appropriate leukocyte cell size. Specifically, the physiological osmotic pressure is 280 mOsm-340 mOsm. There are no particular limitations on the mixing ratio of mammalian whole blood and erythrocyte lysis buffer; they can be mixed in a ratio where the volume of erythrocyte lysis buffer is greater than the volume of mammalian whole blood, for example, a ratio of (1.1-5):1. The specific ratio can be determined based on the concentration of the erythrocyte lysis buffer and the actual cell lysis situation, as long as erythrocyte removal is achieved.

[0064] The isolated neutrophil pellet is further washed with a buffer solution and then resuspended therein for preservation. The buffer solution may be phosphate buffer, sodium citrate buffer, Tris buffer, PBS buffer, or HEPES buffer, but is not limited to these. In some embodiments, the buffer solution has the aforementioned physiological osmotic pressure, with a pH between 6.5 and 9.0. According to a preferred embodiment, the osmotic pressure of the buffer solution with physiological osmotic pressure is particularly between 280 mOsm and 300 mOsm, especially between 288 mOsm and 292 mOsm. Washing the obtained neutrophils at the above osmotic pressure allows the cells to be placed in a suitable osmotic environment, thereby adjusting the cells to have an appropriate cell volume.

[0065] Treatment of neutrophils

[0066] First, the obtained neutrophils are treated with a condensate of nitro alcohol and formaldehyde. Specifically, the neutrophils are treated with a condensate of nitro alcohol and formaldehyde at a concentration of 0.5%-5% at a temperature of 20°C-37°C for 12 hours to 7 days, preferably 24 to 36 hours.

[0067] The condensate of nitro alcohol and formaldehyde can cause partial loss of intracellular material in cells. When mammalian neutrophils isolated above are treated with the condensate of nitro alcohol and formaldehyde, at least some of the intracellular material in the neutrophils is lost, resulting in a decrease in side-scattered light intensity and an increase in forward-scattered light intensity when the treated neutrophils are detected by a hematology analyzer.

[0068] Therefore, after processing neutrophils isolated from mammals under the aforementioned suitable conditions, these neutrophils exhibit the same or similar light-scattering characteristics as human monocytes when detected using a blood cell analyzer.

[0069] Meanwhile, the inventors observed that there is a certain difference in fluorescence intensity between mammalian neutrophils and human monocytes after fluorescent staining. Neutrophils treated as described above exhibit enhanced binding to nucleic acid fluorescent dyes and reduced binding to protein fluorescent dyes, thus displaying the same or similar fluorescence characteristics, particularly fluorescence intensity, as human monocytes when detected using a hematology analyzer.

[0070] The fluorescence characteristics of monocytes mentioned in this disclosure refer to the fluorescence characteristics, especially the fluorescence intensity characteristics, exhibited by monocytes in a sample when detected using a blood cell analyzer.

[0071] Preferably, the neutrophils are treated with a condensate of nitro alcohol and formaldehyde at a concentration of 0.5-1% in physiological osmotic buffer at a temperature of 25-35°C for 24-36 hours.

[0072] The condensate of the nitro alcohol and formaldehyde may be 2-bromo-2-nitropropane-1,3-diol and / or 5-bromo-5-nitro-1,3-dioxane.

[0073] Next, the neutrophils treated with the condensate of nitro alcohol and formaldehyde are fixed to maintain stable physical properties of the cell particles over a longer period of time. Cell fixation can be performed using conventional methods, such as treatment with aldehydes to denature cell membrane proteins and thus stabilize the cell structure; this disclosure does not impose any particular limitation on this method. Specifically, for example, the treated neutrophils can be suspended in a fixation solution and left to stand at 20°C-37°C for 5 hours to 3 days; preferably, they can be left to stand at 25°C-30°C for 5 hours to 24 hours.

[0074] For example, the fixed treatment solution is a solution containing aldehydes. The concentration of the aldehydes is 1%-10% (v / v), preferably 2%-5% (v / v). The aldehydes are selected from one or more of formaldehyde, glutaraldehyde, glyoxal, acetone aldehyde, p-trifluoromethylbenzaldehyde, and paraformaldehyde.

[0075] The above-mentioned treatment steps for neutrophils can be carried out in a treatment environment with a buffer solution having physiological osmotic pressure.

[0076] Neutrophils after fixation are used as monocyte mimic particles. The prepared monocyte mimic particles are washed and then suspended in a preservation solution for storage.

[0077] The washing solution used in the washing steps following the above treatment, as well as the preservation solution for the finally obtained mononuclear cell mimic particles, can all be the aforementioned buffer solution with physiological osmotic pressure to provide an environment with suitable osmotic pressure. This disclosure thereby provides mononuclear cell mimic particles prepared by the above method. Preparation of mononuclear cell mimic particles from mammalian lymphocytes

[0078] Isolate lymphocytes

[0079] Methods for isolating lymphocytes from mammalian blood include the step of density gradient centrifugation of mammalian blood to obtain crude lymphocyte products.

[0080] Blood leukocytes mainly consist of granulocytes and lymphocytes. In mammals, the density of granulocytes is typically 1.085 g / mL–1.100 g / mL, while the density of lymphocytes is typically 1.055 g / mL–1.065 g / mL. Due to their similar densities, it is difficult to remove granulocytes from lymphocytes. Therefore, although the proportion of lymphocytes in blood is high, there are no reports of preparing cell-mimicking particles by isolating mammalian lymphocytes. Conventional centrifugation methods cannot separate lymphocytes with high purity, thus hindering industrial-scale production.

[0081] The density gradient centrifugation is performed using a cell separation medium with a density between that of granulocytes and lymphocytes of the mammal. Specifically, the density of the cell separation medium is greater than 1.055 g / mL and less than 1.085 g / mL, preferably 1.065 g / mL to 1.080 g / mL.

[0082] In a specific implementation, leukocytes are suspended in, for example, Ficoll or Percoll cell separation medium and subjected to density gradient centrifugation. Ficoll cell separation medium, whose main component is sucrose, has a density of 1.077 g / mL and can be used directly. Percoll cell separation medium, whose main component is siliconized polyvinylpyrrolidone particles, has a density of 1.131 g / mL and is typically adjusted to the desired cell density before use. For example, buffer solutions or physiological saline can be mixed with Percoll separation medium at a specific volume ratio to obtain the required density.

[0083] During the research, the inventors unexpectedly discovered that although lymphocytes can be separated by density gradient centrifugation, the method of separating human blood lymphocytes to separate animal blood lymphocytes has problems such as low yield, inability to extract in large quantities, and low cell purity, which cannot meet the production conditions of quality control materials.

[0084] Further research revealed that the optimal physiological osmotic pressure for separating mammalian lymphocytes is slightly higher than that for separating other blood cells. Typically, the physiological osmotic pressure of blood cells is between 280 mOsm and 340 mOsm. Under this osmotic pressure, all blood cells maintain their morphological integrity. However, at lower osmotic pressures, granulocytes absorb water and swell, leading to a decrease in cell density and making effective separation from lymphocytes more difficult. This results in a higher concentration of granulocytes within the lymphocytes, failing to meet production purity requirements. By adjusting the osmotic pressure of the solution during separation, and ensuring the osmotic pressure of the washing solution after centrifugation is above 295 mOsm, it is possible to better maintain the cell morphology and density of each cell during the separation process, thus improving the purity of the simulated particles.

[0085] According to one embodiment, the cell separation solution has the physiological osmotic pressure of the mammalian lymphocytes. The physiological osmotic pressure of the mammalian lymphocytes is 295 mOsm-340 mOsm, preferably 300 mOsm-315 mOsm, and more preferably 305 mOsm-310 mOsm. The osmotic pressure of the cell separation solution can be adjusted using a buffer solution with a suitable osmotic pressure.

[0086] In some embodiments, centrifugation is performed at 300g-700g for 15 to 45 minutes, preferably at 400g-500g for 25 to 35 minutes. Because the lymphocyte density is low, they remain on top of the cell separation medium after centrifugation. The layer containing the lymphocytes is then removed to obtain the crude lymphocyte product.

[0087] To better separate lymphocytes from granulocytes, the density gradient centrifugation described above can be repeated several times. For example, one to two density gradient centrifugations can be performed to remove as many granulocytes as possible.

[0088] In some embodiments, the collected crude lymphocyte product is washed with a buffer solution having the physiological osmotic pressure of the lymphocytes of the mammal.

[0089] The buffer solution may be phosphate buffer, sodium citrate buffer, Tris buffer, PBS buffer or HEPES buffer, etc., but is not limited to these.

[0090] Before or after separating lymphocytes from other white blood cells, i.e. before or after performing the density gradient centrifugation, the red blood cells are lysed with red blood cell lysis buffer and the lysed red blood cells are removed.

[0091] According to one embodiment, mammalian whole blood is first treated with erythrocyte lysis buffer, or the mammalian whole blood is first centrifuged to remove plasma before being treated with erythrocyte lysis buffer (this method reduces the amount of erythrocyte lysis buffer used), causing erythrocyte lysis. By centrifugation, leukocytes are separated from the lysed erythrocytes, and the precipitate is retained to obtain leukocytes. The leukocytes are then resuspended in the aforementioned cell separation buffer and subjected to density gradient centrifugation to obtain crude lymphocytes. In this embodiment, erythrocytes have been removed from the crude lymphocytes.

[0092] According to another embodiment, mammalian blood can be directly centrifuged using a density gradient to separate crude lymphocytes. The crude lymphocytes are then treated with erythrocyte lysis buffer to lyse the erythrocytes, and the lysed erythrocytes are removed by centrifugation. In this embodiment, the crude lymphocytes also contain erythrocytes.

[0093] The red blood cell lysis buffer is defined as described above and will not be repeated here.

[0094] According to one specific embodiment, the erythrocyte lysis buffer has the aforementioned physiological osmotic pressure, preferably having the physiological osmotic pressure of mammalian lymphocytes, in order to maintain appropriate cell morphology for various types of leukocytes.

[0095] Similarly, after removing lysed red blood cells, the product can be washed with the buffer solution described above, which has the physiological osmotic pressure of the lymphocytes of the mammals.

[0096] After removing red blood cells and granulocytes, the crude lymphocyte product still contains platelets. Although some platelets were removed during the aforementioned centrifugation processes, some platelets remained. Low-speed centrifugation can effectively separate lymphocytes and platelets. Platelets have a similar density to lymphocytes, but due to their relatively smaller size, they settle more slowly under lower centrifugal forces, thus allowing the two cell types to be separated.

[0097] Specifically, the crude lymphocyte product is suspended in physiological osmotic buffer solution and centrifuged at 150g-500g for approximately 3 to 8 minutes, preferably at 200g-300g for approximately 4 to 5 minutes. The supernatant, which mainly contains platelets, is discarded, and the separated precipitate is lymphocytes.

[0098] To better separate lymphocytes from platelets, the above low-speed centrifugation can be repeated several times. For example, 3-6 centrifugations can be performed to remove as many platelets as possible.

[0099] In some embodiments, the precipitate obtained by centrifugation is washed with a buffer solution having the physiological osmotic pressure of the lymphocytes of the said mammal.

[0100] The resulting lymphocyte pellet was further washed with a physiological osmotic buffer containing the lymphocytes of the aforementioned mammal and then suspended and stored therein. The physiological osmotic buffer containing the lymphocytes of the aforementioned mammal is the same as described above and will not be repeated here.

[0101] The method disclosed herein allows for the isolation of lymphocytes that are essentially free of other blood cells, making it possible to prepare lymphocyte mimic particles from mammalian lymphocytes. In a preferred embodiment, the purity (quantity percentage) of lymphocytes isolated by the method disclosed herein can reach over 95%, and even over 98%.

[0102] Treatment of lymphocytes

[0103] While the light scattering characteristics of mammalian lymphocytes (such as bovine lymphocytes) are similar to those of human monocytes, their fluorescence characteristics after staining with fluorescent dyes differ significantly. Therefore, lymphocytes need to be pre-treated with fluorescent dyes to ensure they exhibit the same or similar fluorescence characteristics as human monocytes when detected by a hematology analyzer. Simultaneously, the light scattering characteristics of the treated lymphocytes will also become closer to, or even identical to, those of human monocytes.

[0104] Specifically, lymphocytes are treated with a fluorescent dye capable of staining cell membranes and intracellular proteins to modulate their fluorescence properties. The fluorescent dye is added to a suspension containing lymphocytes at a concentration of 0.5-5 mg / L, preferably 0.7-1.5 mg / L. The suspension is then incubated at 20-30°C for 3 to 60 minutes, preferably 3 to 15 minutes.

[0105] Preferably, the fluorescent dye is one that can be excited by both red and blue light to produce fluorescence. This allows the obtained mononuclear cell mimic particles to be used in the optical detection platforms of a wider range of blood cell analyzers.

[0106] In some embodiments, the fluorescent dye is a carboxyl-containing protein fluorescent dye activated with N-hydroxysuccinimide. The fluorescent dye is selected from compounds represented by the following general formula I:

[0107]

[0108] In this formula, R and the substituted carboxyl groups on R together constitute the protein fluorescent dye. The substituted carboxyl groups on R react with the hydroxyl groups of N-hydroxysuccinimide to form compounds of formula I above. n is an integer from 1 to 4, where the value of n depends on the number of carboxyl groups in the protein fluorescent dye R. Compounds of formula I can be excited by both red and blue light.

[0109] More specifically, the protein fluorescent dye is selected from compounds represented by the following general formulas II to IX:

[0110]

[0111]

[0112] In general formulas II to V,

[0113] m can be 0, 1, 2, or 3.

[0114] R1 and R2 are each independently selected from sulfonic acid groups, halogens, and H.

[0115] R3 and R4 are each independently selected from C1-C5 alkyl groups and C1-C5 alkyl groups substituted with carboxyl groups, and at least one of R3 and R4 is a C1-C5 alkyl group substituted with carboxyl groups.

[0116] R5 and R6 are each independently selected from sulfonic acid groups, halogens, C1-C5 alkyl groups, and H;

[0117] In general formulas VI to IX,

[0118] Me represents methyl, and Ar represents phenyl.

[0119] z is an integer between 0 and 8.

[0120] R7 to R 14 Each is independently selected from C1-C5 alkyl, C1-C5 alkyl substituted with a carboxyl group, sulfonic acid group, halogen and H, and each general formula has at least one C1-C5 alkyl substituted with a carboxyl group.

[0121] More specifically, the fluorescent dye may be selected from the following compounds:

[0122]

[0123]

[0124] Lymphocytes treated with fluorescent dye were washed with the aforementioned buffer solution. Preferably, the buffer solution was phosphate buffer, sodium citrate buffer, Tris buffer, PBS buffer, or HEPES buffer. The buffer solution may have the aforementioned physiological osmotic pressure.

[0125] When lymphocytes treated with fluorescent dyes are detected using a blood cell analyzer, they not only exhibit fluorescence intensity characteristics similar to or similar to those of human monocytes, but also scattered light intensity characteristics similar to or similar to those of human monocytes, thus simulating human monocytes.

[0126] The treated lymphocytes were then fixed to obtain monocyte-mimicking particles. The fixation process for lymphocytes was the same as that for neutrophils described above, using the same fixatives and methods, and will not be repeated here.

[0127] The prepared mononuclear cell mimic particles were washed and then suspended in a preservation solution for storage.

[0128] The washing solution used in the washing steps after the above treatment, as well as the preservation solution for the finally obtained mononuclear cell mimic particles, can all be the buffer solution with the physiological osmotic pressure of mammalian lymphocytes, so as to provide an environment with suitable osmotic pressure.

[0129] This disclosure thereby provides mononuclear cell mimic particles prepared by the above method.

[0130] Mononuclear cell mimicry composition

[0131] This disclosure also provides a mononuclear cell mimicry composition. The composition comprises mononuclear cell mimicry particles preserved in a preservation solution, the mononuclear cell mimicry particles comprising more than 50% of the volume in the composition. The mononuclear cell mimicry particles are obtained by processing neutrophils or lymphocytes isolated from mammalian blood, wherein, when tested with a hematology analyzer, the mononuclear cell mimicry particles exhibit light scattering properties similar to or similar to those of human mononuclear cells, the light scattering properties at least reflecting the complexity of the cell contents.

[0132] In some embodiments, the mononuclear cell mimic particles constitute 50%-70% of the volume of the composition.

[0133] According to some embodiments, the composition comprises mononuclear cell mimicry particles preserved in a preservation solution. That is, the cell mimicry particles in the composition are only mononuclear cell mimicry particles in single-particle clusters.

[0134] Furthermore, the light scattering properties also reflect the size of the cell.

[0135] In some embodiments, when the mononuclear cell mimic particles are tested using a blood cell analyzer, they have the same or similar fluorescence characteristics as human mononuclear cells, especially fluorescence intensity characteristics.

[0136] In this disclosure, the mononuclear cell mimic particles in the composition are prepared by the method described above.

[0137] The preservation solution can be a solution commonly used for simulating particle preservation, and this disclosure does not particularly limit it. Exemplarily, the preservation solution may include common buffers such as phosphate buffer, sodium citrate buffer, Tris buffer, PBS buffer, or various commercially available blood cell preservation solutions.

[0138] In the composition, the particle concentration of the mononuclear cell mimic particles is approximately (700-800) × 10⁻⁶. 9 per mL.

[0139] Quality control materials or calibrators for blood cell analyzers

[0140] This disclosure also provides a quality control material or calibrator for a blood cell analyzer, including the above-described mononuclear cell mimic particles or the above-described mononuclear cell mimic composition.

[0141] Quality control materials used in hematology analyzers are used to monitor and evaluate the precision of the hematology analyzer.

[0142] Calibrators used in hematology analyzers accurately simulate human blood when tested on the analyzer. They are used to establish metrological traceability of hematology analyzer measurement results.

[0143] In one embodiment, the quality control material is a five-part differential white blood cell control material. The five-part differential white blood cell control material includes at least the aforementioned mononuclear cell mimic particles.

[0144] Furthermore, the five-part differential white blood cell quality control material also includes lymphocyte mimic particles, basophil mimic particles, neutrophils, and eosinophils.

[0145] According to one embodiment, one or more of the lymphocyte mimic particles, basophil mimic particles, neutrophils, and eosinophils are prepared independently. According to a specific embodiment, the lymphocyte mimic particles, basophil mimic particles, neutrophils, and eosinophils are all prepared independently.

[0146] Independently prepared lymphocyte mimic particles can be bovine or porcine blood lymphocytes fixed with aldehydes. Lymphocytes can be isolated from mammalian blood using the method disclosed herein, and then directly fixed with aldehydes (such as formaldehyde, glutaraldehyde, glyoxal, acetone aldehyde, p-trifluoromethylbenzaldehyde, and paraformaldehyde) at a concentration of 1%-10% (v / v), preferably 2-5% (v / v) for 5 hours to 3 days, preferably 5-24 hours.

[0147] The independently prepared basophil mimic particles can be prepared using the method disclosed in CN116929866A, the full text of which is incorporated herein by reference.

[0148] The independently prepared neutrophils can be aldehyde-fixed bovine blood neutrophils. The method for preparing single-population leukocyte mimic particles disclosed in CN101311724A can be referred to. The difference is that mammalian neutrophils are first extracted and then processed in the same way as in the patent document, the full text of which is incorporated herein by reference.

[0149] Independently prepared eosinophils can be, for example, aldehyde-fixed bovine blood neutrophils, prepared using the method disclosed in CN101887059A, the full text of which is incorporated herein by reference.

[0150] The batch-to-batch variation of independently prepared cell-mimicking particles is small, and the cell properties have good consistency and stability. Therefore, when all subtypes of leukocytes are prepared independently, the overall consistency and stability of the leukocyte five-part differential quality control material are good.

[0151] In one embodiment, the quality control material is a whole blood quality control material. The whole blood quality control material includes at least the aforementioned white blood cell five-part differential quality control material. Further, the whole blood quality control material also includes red blood cell mimic particles, platelet mimic particles, and reticulocyte mimic particles.

[0152] The red blood cell mimic particles can be, for example, human red blood cells fixed with aldehydes, and can be produced using the method disclosed in CN105717312A, the full text of which is incorporated herein by reference.

[0153] Platelet-simulating particles can be, for example, sheep blood erythrocyte particles treated with fluorescent dyes and fixed with aldehydes, using the method disclosed in CN110140051A, the full text of which is incorporated herein by reference.

[0154] The reticulocyte mimic particles can be, for example, human red blood cells treated with fluorescent dyes and fixed with aldehydes, using the method disclosed in CN110140051A, the full text of which is incorporated herein by reference.

[0155] The quality control material also includes a preservation solution. The preservation solution has been described above and will not be repeated here.

[0156] The quality control material was stored at 2-8°C.

[0157] Quality control methods for blood cell analyzers

[0158] This disclosure also provides a quality control method for a blood cell analyzer. The method includes the following steps:

[0159] A quality control material containing at least five different white blood cell fractions mimics is provided, the quality control material comprising: the aforementioned monocyte mimics, independently prepared lymphocyte mimics, independently prepared basophil mimics, independently prepared neutrophils, and independently prepared eosinophils; the quality control material is tested using the blood cell analyzer in quality control mode to obtain at least the monocyte count result in the quality control material; and the count result is compared with the standard value of the quality control material.

[0160] Using the quality control material disclosed herein, the blood cell analyzer was used to perform two tests on the quality control material in quality control mode, and the deviation between the two counts of mononuclear cells in the quality control material was less than 5%.

[0161] Each of the quality control products can be tested multiple times during use. With this disclosed quality control, the deviation of individual mononuclear cell counts is less than 10% across all tests conducted in one opening cycle on the same device.

[0162] Furthermore, the quality control materials of this disclosure are of stable quality across batches. When the tests were performed on two batches of the quality control materials, the deviation between the two monocyte counts in the two batches was less than 10%.

[0163] The counting result is the count of monocyte-simulated particles, or the percentage of monocyte-simulated particles in white blood cells.

[0164] The advantages of this disclosure will be further illustrated by specific embodiments below.

[0165] Example

[0166] The following examples use conventional reagents:

[0167] Red blood cell lysis buffer: Tris-ammonium chloride red blood cell lysis buffer was used for all cases.

[0168] Phosphate buffer: pH = 7.00 ± 0.2. Examples 3 and 4 used a buffer osmotic pressure of 307 ± 5 mOsm, while the other examples used an osmotic pressure of 290 ± 5 mOsm.

[0169] Preservative solution: Phosphate buffer containing sugars and antibacterial agents, pH = 7.00 ± 0.2, osmotic pressure 305-310 mOsm.

[0170] The following tests were conducted using the following blood cell analyzers: Mindray BC-7500, with the following testing conditions: counting interface, micro-volume whole blood, CDR mode; and Mindray BC-5390CRP, with the following testing conditions: counting interface, micro-volume whole blood, CBC+Diff mode.

[0171] Example 1: Preparation of mononuclear cell mimic particles from bovine blood neutrophils

[0172] 1. Isolation of neutrophils

[0173] Centrifuge 500 mL of anticoagulated bovine blood at 3600 rpm for 8 min. Then, use a peristaltic pump to aspirate the plasma layer and the white cell layer at the boundary, leaving a uniformly colored bottom cell layer. Add erythrocyte lysis buffer (pH 7.0) at 1.5 times the volume of the bottom cell layer. After adding, mix thoroughly and incubate at room temperature for 10 min to allow the erythrocytes to lyse completely. Then, centrifuge at 2400 rpm for 5 min, and discard the supernatant after centrifugation.

[0174] Wash cells twice with phosphate-buffered saline (PBS): Resuspend cells in PBS in centrifuge tubes, centrifuge at 1800 rpm for 5 min, and remove the supernatant. Repeat the same procedure once to obtain neutrophils.

[0175] 2. Treatment of neutrophils

[0176] The neutrophils obtained above were resuspended in phosphate buffer containing 1 wt% bromonitroethanol (2-bromo-2-nitropropane-1,3-diol), thoroughly mixed, and incubated at 30°C for 24 h. After treatment, the cells were centrifuged at 1800 rpm for 5 min, and the supernatant was removed.

[0177] Resuspend the cells in phosphate buffer, add 5% (v / v) of the cell suspension volume of formaldehyde (which can be replaced with 5% (v / v) paraformaldehyde or 2% (v / v) glutaraldehyde), mix thoroughly, and fix at 30°C for 18 h.

[0178] After fixation, centrifuge at 1800 rpm for 5 min and remove the supernatant. Resuspend the cells in phosphate buffer, centrifuge at 1800 rpm for 5 min, remove the supernatant, and obtain mononuclear cell mimic particles.

[0179] Cells were resuspended in preservation solution at a concentration of 50 × 10⁶ cells / mL. 9 Quantity / mL, store at 2-8℃.

[0180] The storage solution of the mononuclear cell mimic particles prepared above and blood samples obtained from healthy subjects were tested on a hematology analyzer. The test results are shown in Figure 1. The left side shows a scatter plot of the forward and side scatter light intensity of the blood sample from the healthy subject and the mononuclear cell mimic particles prepared in Example 1 on a Mindray BC-5390CRP. The position of the mononuclear cell mimic particles in the figure is close to the position of the mononuclear cells tested in the blood sample from the healthy subject. The right side shows a scatter plot of the fluorescence intensity and side scatter light intensity of the sample on a Mindray BC-7500. The position of the mononuclear cell mimic particles in the figure has a slightly higher fluorescence intensity than the position of the mononuclear cells tested in the blood sample from the healthy subject. This is because fixation increases the fluorescence intensity of the cell particles, but the particles are still within the mononuclear cell recognition range and can be correctly identified as mononuclear cells.

[0181] Example 2: Preparation of mononuclear cell mimic particles from bovine blood neutrophils

[0182] 1. Isolation of neutrophils

[0183] Neutrophils were isolated from anticoagulated bovine blood using the same method as in Example 1.

[0184] 2. Treatment of neutrophils

[0185] Neutrophils obtained were resuspended in phosphate buffer containing 1 wt% bromonitidiaoxane (5-bromo-5-nitro-1,3-dioxane), thoroughly mixed, and incubated at 30°C for 24 h. After treatment, the cells were centrifuged at 1800 rpm for 5 min, and the supernatant was removed.

[0186] Resuspend the cells in phosphate buffer, add 5% formaldehyde (by volume of the cell suspension), mix thoroughly, and fix at 30°C for 18 hours.

[0187] After fixation, centrifuge at 1800 rpm for 5 min and remove the supernatant. Resuspend the cells in phosphate buffer and centrifuge at 1800 rpm for 5 min to obtain mononuclear cell mimic particles.

[0188] Cells were resuspended in preservation solution at a concentration of 50 × 10⁶ cells / mL. 9 Quantity / mL, store at 2-8℃.

[0189] The storage solution of the mononuclear cell mimic particles prepared above was tested on a blood cell analyzer, and the results are shown in Figure 2. Similarly, it can be seen that the positions of the mononuclear cell mimic particles prepared in Example 2 in the scatter plots obtained from the tests on the Mindray BC-5390CRP and BC-7500 blood cell analyzers are close to the positions of mononuclear cells in the blood samples of healthy subjects.

[0190] Example 3: Preparation of mononuclear cell mimic particles from porcine blood lymphocytes

[0191] 1. Isolation of lymphocytes

[0192] Centrifuge 500 mL of anticoagulated porcine blood at 3000 rpm for 5 min, then aspirate the plasma layer using a peristaltic pump. Add erythrocyte lysis buffer (pH 7.0) at 1.5 times the cell volume. After adding, mix thoroughly and incubate at room temperature for 10 min to allow the erythrocytes to lyse completely. Then centrifuge at 2400 rpm for 5 min. After centrifugation, discard the supernatant and resuspend the cells in phosphate buffer.

[0193] Add Ficoll cell separation medium to a separate centrifuge tube, then slowly add an equal volume of cell suspension. Centrifuge at 1400 rpm for 30 min. After centrifugation, the cells will separate into upper and lower fractions. Collect the upper fraction of cells into a clean centrifuge tube to obtain crude lymphocytes. Centrifuge the collected upper fraction of cells at 900 rpm (200 g) for 8 min (or alternatively, centrifuge at 1500 rpm (400 g) for 4 min), then remove the supernatant to obtain lymphocytes. Resuspend the cells in phosphate buffer, centrifuge under the same conditions, and remove the supernatant to wash the lymphocytes. Repeat the washing process three times, then resuspend the cells in phosphate buffer.

[0194] The test was performed on the Mindray BC-7500 blood cell analyzer, and the results are shown in Figure 3a. At this point, granulocytes have been removed, and only lymphocytes remain.

[0195] 2. Treatment of lymphocytes

[0196] Add a solution of N-hydroxysuccinimide-activated protein fluorescent dye A (7.7 g / L) dissolved in dimethyl sulfoxide at a volume ratio of approximately 0.01 to 0.02% of the cell resuspension, mix well, and let stand at room temperature for 10 min.

[0197]

[0198] Centrifuge at 1800 rpm for 5 min and remove the supernatant. Resuspend the cells in phosphate buffer, centrifuge at 1800 rpm for 5 min, and remove the supernatant.

[0199] Resuspend the cells again in phosphate buffer, add 5% (v / v) of the cell suspension volume of formaldehyde (which can be replaced with 5% (v / v) paraformaldehyde or 2% (v / v) glutaraldehyde), mix thoroughly, and fix at 30°C for 18 h.

[0200] After fixation, the cells were centrifuged at 1800 rpm for 5 min and the supernatant was removed. The cells were resuspended in phosphate buffer, centrifuged at 1800 rpm for 5 min, and the supernatant was removed to obtain mononuclear cell mimic particles.

[0201] Cells were resuspended in preservation solution at a concentration of 50 × 10⁶ cells / mL. 9 Cells / mL, store at 2-8℃.

[0202] The storage solution of the mononuclear cell mimic particles prepared above was tested on a Mindray BC-7500 hematology analyzer, and the results are shown in Figure 3b. Similarly, it can be seen from the fluorescence intensity-side scattering intensity scatter plot of the mononuclear cell mimic particles prepared in Example 3 that the position of the mimic particles is within the instrument's mononuclear cell recognition area, and they can be correctly identified as mononuclear cells.

[0203] Example 4: Preparation of mononuclear cell mimic particles from bovine blood lymphocytes

[0204] 1. Isolation of lymphocytes

[0205] Lymphocytes were isolated from 500 mL of anticoagulated bovine blood using the same method as in Example 3. The results, as shown in Figure 4a, were obtained by testing on a Mindray BC-7500 hematology analyzer. At this point, granulocytes had been removed, leaving only lymphocytes.

[0206] 2. Treatment of lymphocytes

[0207] The bovine lymphocytes obtained above were processed using the same method as in Example 3.

[0208] Cells were resuspended in preservation solution at a concentration of 50 × 10⁶ cells / mL. 9 Cells / mL, store at 2-8℃.

[0209] The storage solution of the mononuclear cell mimic particles prepared above was tested on a Mindray BC-7500 hematology analyzer, as shown in Figure 4b. Similarly, it can be seen from the fluorescence intensity-side scattering intensity scatter plot of the mononuclear cell mimic particles prepared in Example 4 that the position of the mimic particles is within the instrument's mononuclear cell recognition area, and they can be correctly identified as mononuclear cells.

[0210] Example 5: Stability of Mononuclear Cell-Mimetic Particles

[0211] The count of the mononuclear cell-simulated particles prepared in Examples 1-4 was adjusted to 9 × 10⁻⁶. 9 From 10 × 10⁶ cells / mL to 10 × 10⁻⁶ cells / mL 9 The samples were collected at a concentration of 1 / mL and stored in a refrigerator at 2-8℃. They were taken out and tested once a week for 22 weeks. The test results are shown in Figure 5.

[0212] During nearly five months of testing, the number of mononuclear cell mimic particles prepared in Examples 1-4 remained relatively stable, mostly within the range of (9.5-10.5) × 10⁻⁶. 9 The fluctuation within the range of cells / mL indicates that it has good stability and can meet the stability requirements for quality control materials.

[0213] Example 6: White blood cell five-part differential quality control material

[0214] 1. Prepare independently synthesized leukocyte mimic particles of various subtypes:

[0215] Mononuclear cell mimic particles: prepared using those described in Example 1.

[0216] Neutrophil mimic particles: Neutrophils were extracted from bovine blood and prepared by treating neutrophils with the leukocyte treatment solution disclosed in Example 1 of CN101311724A and fixing them with formaldehyde.

[0217] Lymphocyte mimic particles: Lymphocytes were isolated from pig blood using the same method as in Example 3 and fixed with 2 (v / v) glutaraldehyde for 18 h to prepare the particles.

[0218] Eosinophil mimic particles: prepared using glutaraldehyde-fixed bovine blood neutrophils according to the method disclosed in Example 1 of CN101887059A.

[0219] Basophil mimic particles: prepared using bovine blood neutrophils stained with neutral red and protein fluorescent dyes and fixed with glutaraldehyde, according to the method disclosed in Example 2 of CN116929866A.

[0220] The cell-simulated particles obtained above were suspended in preservation solution, and the cell count was adjusted to 50 × 10⁶.9 per mL.

[0221] The above-mentioned cell-simulated particle preservation solution was mixed in a volume ratio of neutrophil-simulated particles: lymphocyte-simulated particles: monocyte-simulated particles: eosinophil-simulated particles = 60:30:6:4 to prepare a five-part differential white blood cell quality control sample, which was then stored in a refrigerator at 2-8℃.

[0222] The above-mentioned five-part differential white blood cell quality control samples were tested on Mindray BC-5390CRP and Mindray BC-7500 hematology analyzers, respectively. The results showed that each cell cluster was clearly separated and could be correctly identified and grouped by the hematology analyzer. The test results and the results of the test results with the blood of healthy subjects are shown in Figure 6a.

[0223] The leukocyte five-part differential quality control sample was stored at 2-8°C, and samples were tested weekly to analyze the stability of the mononuclear cell mimic particle ratio. It was found that during the 22-week test, the proportion of mononuclear cell mimic particles to leukocyte five-part differential cell mimic particles remained stable, as shown in Figure 6b. This indicates that the mononuclear cell mimic particles described in this invention perform well in the leukocyte five-part differential quality control sample.

[0224] Example 7: Whole blood quality control material

[0225] The leukocyte five-part differential quality control material prepared in Example 6 was used as leukocyte mimic particles.

[0226] Red blood cell mimic particles: prepared using human red blood cells fixed with glutaraldehyde according to the method disclosed in Example 1 of CN105717312A.

[0227] Reticulocyte mimic particles: prepared using human red blood cells treated with fluorescent dyes and fixed with formaldehyde according to the method disclosed in Example 1 of CN110140051A.

[0228] Platelet-simulated particles: prepared using sheep blood erythrocyte particles treated with fluorescent dyes and fixed with formaldehyde, according to the method disclosed in Example 1 of CN110140051A.

[0229] The cell-simulated particles obtained above were suspended in preservation solutions. Based on normal human blood cell count levels, the suspended particles were mixed to prepare a median whole blood quality control; based on count levels below normal human blood cell count levels, the suspended particles were mixed to prepare a low-value whole blood quality control; and based on count levels above normal human blood cell count levels, the suspended particles were mixed to prepare a high-value whole blood quality control. All quality controls were stored at 2-8°C.

[0230] The whole blood quality control material needs to remain stable for at least 4 months, with no trend changes in cell population counts and proportions. All test data should show a monocyte proportion variation of less than 2% and a coefficient of variation of less than 10%. Samples were tested weekly, and the stability of the proportion of monocyte mimic particles to white blood cells in high, medium, and low-level whole blood quality control materials was statistically analyzed. It was found that the proportion of monocyte mimic particles remained stable throughout the 22-week test, as shown in Figure 7. The range of all test data was less than 1%, and the coefficient of variation was less than 4%. This indicates that the lymphocyte mimic particles described in this invention perform well in whole blood quality control materials.

[0231] Example 8: Comparison experiment between the quality control material of Example 7 and commercially available quality control materials

[0232] The whole blood control prepared in Example 7, the commercially available BC6D whole blood control, and the commercially available BR60 whole blood control were tested on a hematology analyzer to obtain scatter plots of leukocyte fluorescence intensity versus side-scatter light intensity, as shown in Figure 8. Compared with the leukocyte scatter plots of the commercially available BC6D control (Figure 8, Figure ...

[0233] Because all particles in the quality control materials described in this embodiment are individually manufactured, the proportion of each particle cluster can be freely adjusted according to actual needs, and the quality control materials exhibit better data stability between different batches. Three different batches of the three quality control materials were tested on a Mindray BC-7500 to obtain the percentage of each of the five differential white blood cell types based on the total white blood cell count, and the coefficient of variation of the percentage of monocytes between each batch was calculated, as shown in the table below.

[0234]

[0235] As can be seen from the table above, the deviation in the percentage of mononuclear cells measured between different batches of the quality control material prepared in Example 7 was significantly lower than that of the other two commercially available quality control materials.

[0236] The above descriptions are merely examples of some embodiments of the present invention and do not limit the patent scope of the present invention. All equivalent structural transformations made using the contents of the present invention's specification and drawings under the inventive concept of the present invention, or direct / indirect applications in other related technical fields, are included within the patent protection scope of the present invention.

Claims

1. A mononuclear cell mimicry composition comprising mononuclear cell mimicry particles preserved in a preservation solution, wherein the mononuclear cell mimicry particles constitute 50% or more of the volume in the composition, and wherein the mononuclear cell mimicry particles are obtained by treating neutrophils or lymphocytes isolated from mammalian blood, wherein... When the mononuclear cell-simulated particles are tested using a blood cell analyzer, they have the same or similar light scattering characteristics as human mononuclear cells, and the light scattering characteristics at least reflect the complexity of the cell contents.

2. The mononuclear cell mimicry composition according to claim 1, wherein, The mononuclear cell mimic particles constitute 50%-70% of the volume of the composition; preferably, the composition consists of mononuclear cell mimic particles preserved in a preservation solution.

3. The mononuclear cell mimicry composition according to claim 1 or 2, wherein, The process of isolating the neutrophils or the lymphocytes includes the steps of lysing the red blood cells with a red blood cell lysis buffer and removing the lysed red blood cells.

4. The mononuclear cell mimicry composition according to claim 3, wherein, The steps for separating the neutrophils include: performing the above-described steps of lysing red blood cells with the red blood cell lysis buffer and removing the lysed red blood cells on the mammalian blood; centrifuging the mammalian blood to separate the lower sediment; and performing the above-described steps of lysing red blood cells with the red blood cell lysis buffer and removing the lysed red blood cells on the lower sediment to obtain the neutrophils; preferably, the obtained neutrophils are washed with a buffer solution having physiological osmotic pressure.

5. The mononuclear cell mimicry composition according to claim 4, wherein, The step of treating neutrophils isolated from mammalian blood includes: treating the neutrophils with a condensate of nitro alcohol and formaldehyde, preferably, treating the neutrophils with a condensate of nitro alcohol and formaldehyde at a concentration of 0.5wt%-5wt% at 30°C for 24-36 hours, more preferably, the condensate of nitro alcohol and formaldehyde is 2-bromo-2-nitropropane-1,3-diol and / or 5-bromo-5-nitro-1,3-dioxane; and even more preferably, washing the neutrophils treated with the condensate of nitro alcohol and formaldehyde with a buffer having physiological osmotic pressure.

6. The mononuclear cell mimicry composition according to claim 3, wherein, The step of separating the lymphocytes further includes: performing density gradient centrifugation on the mammalian blood or the mammalian blood cell suspension to separate crude lymphocytes; preferably, the crude lymphocytes obtained are washed with a buffer having the physiological osmotic pressure of mammalian lymphocytes.

7. The mononuclear cell mimicry composition according to claim 6, wherein, The density gradient centrifugation includes: performing the density gradient centrifugation with a cell separation solution whose density is between that of mammalian granulocytes and lymphocytes; preferably, the density of the cell separation solution is greater than 1.055 g / mL and less than 1.085 g / mL, more preferably 1.065 g / mL-1.080 g / mL; more preferably, the cell separation solution is Ficoll cell separation solution or Percoll cell separation solution, and even more preferably, the cell separation solution has the physiological osmotic pressure of mammalian lymphocytes.

8. The mononuclear cell mimicry composition according to claim 7, wherein, The density gradient centrifugation is performed at 300g-700g for 15min-45min; preferably, the density gradient centrifugation is performed at 400g-500g for 25min-35min; more preferably, the density gradient centrifugation is performed 1-2 times.

9. The mononuclear cell mimicry composition according to claim 6, wherein, The steps of lysing and removing lysed red blood cells in mammalian blood using red blood cell lysis buffer are performed before or after the density gradient centrifugation; preferably, the product after removing lysed red blood cells is washed with a buffer having the physiological osmotic pressure of mammalian lymphocytes.

10. The mononuclear cell mimicry composition according to claim 9, wherein the step of isolating the lymphocytes further comprises: The crude lymphocyte product after removing lysed red blood cells is centrifuged at 150g-500g for 3-8 minutes, preferably at 200g-300g for 4-5 minutes, to remove platelets. Preferably, the centrifugation is repeated 3-6 times. More preferably, the lymphocytes after removing platelets are washed with the buffer solution having the physiological osmotic pressure of mammalian lymphocytes, preferably 1-3 times.

11. The mononuclear cell mimicry composition according to any one of claims 1-10, wherein, When the mononuclear cell-simulated particles are tested using a blood cell analyzer, they further exhibit fluorescence properties that are the same as or similar to those of human mononuclear cells.

12. The mononuclear cell mimicry composition according to claim 11, wherein, The treatment of lymphocytes isolated from mammalian blood comprises: treating the lymphocytes with a fluorescent dye capable of staining proteins in the cell membrane and intracellular space; preferably, the fluorescent dye is selected from compounds represented by the following general formula I: Wherein, the compound represented by general formula I is a carboxyl-containing protein fluorescent dye activated by N-hydroxysuccinimide, wherein R and the substituted carboxyl groups on R together constitute the protein fluorescent dye, and n is an integer from 1 to 4; more preferably, the protein fluorescent dye is selected from the compounds represented by the following general formulas II to IX: In formulas II to V, m is 0, 1, 2, or 3; R1 and R2 are each independently selected from sulfonic acid groups, halogens, and H; R3 and R4 are each independently selected from C1-C5 alkyl groups and C1-C5 alkyl groups substituted with carboxyl groups, and at least one of R3 and R4 is a C1-C5 alkyl group substituted with carboxyl groups; R5 and R6 are each independently selected from sulfonic acid groups, halogens, C1-C5 alkyl groups, and H; in formulas VI to IX, Me represents methyl, Ar represents phenyl, z is an integer from 0 to 8, and R7 to R... 14 Each is independently selected from C1-C5 alkyl, C1-C5 alkyl substituted with a carboxyl group, sulfonic acid group, halogen and H, and each general formula has at least one C1-C5 alkyl substituted with a carboxyl group.

13. The mononuclear cell mimicry composition according to claim 12, wherein, The lymphocytes were treated with the fluorescent dye at a concentration of 0.5 mg / L-5 mg / L, preferably 0.7 mg / L-1.5 mg / L, for 3 min-60 min, preferably 3 min-15 min.

14. The mononuclear cell mimicry composition according to any one of claims 4 and 12-13, wherein, The treatment of neutrophils or lymphocytes isolated from mammalian blood further comprises: fixing neutrophils treated with a condensate of nitro alcohol and formaldehyde or lymphocytes treated with a fluorescent dye with an aldehyde compound, preferably with the aldehyde compound at a concentration of 1%-10% (v / v), more preferably 2%-5% (v / v), for 5 hours to 3 days, preferably 5 hours to 24 hours; more preferably, the aldehyde compound is selected from one or more of formaldehyde, glutaraldehyde, glyoxal, acetone aldehyde, p-trifluoromethylbenzaldehyde, and paraformaldehyde.

15. The mononuclear cell mimicry composition according to claim 4 or 5, wherein, The physiological osmotic pressure is 280mOsm-340mOsm, preferably 290mOsm-300mOsm, more preferably 288mOsm-292mOsm, and the pH value of the buffer solution with physiological osmotic pressure is 6.5-9.

0.

16. The mononuclear cell mimicry composition according to any one of claims 6-10, wherein, The physiological osmotic pressure of the mammalian lymphocytes is 295mOsm-340mOsm, preferably 300mOsm-315mOsm, and more preferably 305mOsm-310mOsm, and the pH value of the buffer solution having the physiological osmotic pressure of mammalian lymphocytes is 6.5-9.

0.

17. The mononuclear cell mimicry composition according to claim 1 or 2, wherein, The mammal in question is either a pig or a cow.

18. A method for preparing mononuclear cell mimic particles, comprising: The monocyte-like particles are obtained by processing neutrophils or lymphocytes isolated from mammalian blood, wherein the monocyte-like particles, when tested with a hematology analyzer, have the same or similar light scattering characteristics as human monocytes, and the light scattering characteristics at least reflect the complexity of the cell contents; preferably, the step of processing neutrophils or lymphocytes isolated from mammalian blood to obtain the monocyte-like particles is as defined in any one of claims 3-17.

19. A mononuclear granulocyte mimic particle, said mononuclear granulocyte mimic particle being prepared by the method of claim 18.

20. A quality control or calibrator for a hematology analyzer, comprising a mononuclear cell mimicry composition according to any one of claims 1-17 or a mononuclear cell mimicry particle according to claim 19.

21. A leukocyte five-part differential quality control material, comprising at least the mononuclear cell mimic particles according to claim 18.

22. The white blood cell five-part differential quality control material according to claim 21, further comprising lymphocyte mimic particles, basophil mimic particles, neutrophils, and eosinophils; preferably, one or more of the lymphocyte mimic particles, the basophil mimic particles, the neutrophils, and the eosinophils are prepared independently; more preferably, the lymphocyte mimic particles, the basophil mimic particles, the neutrophils, and the eosinophils are all prepared independently.

23. A whole blood quality control material, comprising the white blood cell five-part differential quality control material according to claim 21 or 22, and red blood cell mimic particles, platelet mimic particles and reticulocyte mimic particles.

24. A quality control method for a blood cell analyzer, comprising: A quality control material containing at least five different white blood cell fractions is provided, the quality control material comprising: monocyte fractions as described in claim 19, independently prepared lymphocyte fractions, independently prepared basophil fractions, independently prepared neutrophils, and independently prepared eosinophils; the quality control material is detected using the blood cell analyzer in quality control mode to obtain the monocyte count in the quality control material; and the count is compared with the standard value of the quality control material.

25. The quality control method according to claim 24, wherein the deviation between the two counts of monocytes in the quality control material obtained by performing two tests with the quality control material using the blood cell analyzer in quality control mode is less than 5%; preferably, the coefficient of variation of each count of monocytes in one batch of the quality control material is less than 10% in all tests during one opening cycle; and / or the deviation between the two counts of monocytes in the two batches of the quality control material obtained by performing the tests with two batches of the quality control material is less than 10%.

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