Lymphocyte simulation particle composition, preparation method, quality control substance and quality control method

Abstract

The invention relates to a lymphocyte simulation composition, a preparation method, a quality control substance and a quality control method. The lymphocyte simulation composition comprises lymphocyte simulation particles stored in a preservation solution, the volume ratio of the lymphocyte simulation particles in the composition is 50% or above, the lymphocyte simulation particles are obtained by treating lymphocytes separated from blood of mammals, and the lymphocyte simulation particles are used for simulating the lymphocytes. When the lymphocyte simulation particle is tested by a hematology analyzer, the lymphocyte simulation particle has the same or similar light scattering characteristics as human lymphocytes, and the light scattering characteristics at least reflect the complexity of cell contents. By independently preparing the lymphocyte simulation particles, the particles have good consistency and stability, and the obtained composition has small 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

Lymphocyte mimic particle composition, preparation method, quality control materials and quality control methods Technical Field

[0001] This disclosure relates to a method for preparing lymphocyte mimic particles for quality control in a hematology analyzer, and the lymphocyte mimic particles, quality control materials, and quality control methods obtained therefrom. Background Technology

[0002] Human blood cells include three types: red blood cells, white blood cells, and platelets. White blood cells mainly include five types: lymphocytes, monocytes, neutrophils, eosinophils, and basophils. Accurate detection of white blood cell counts and clusters has significant clinical value, which necessitates accurate quality control and calibration of blood cell analyzers.

[0003] Stable and reliable production of well-suited, cost-effective lymphocyte mimic particles can improve the stability and reliability of hematology analyzers when detecting lymphocytes. Currently available lymphocyte mimic particles are mostly developed for hematology analyzers that perform three-part differential white blood cell analysis (including monocytes, neutrophils, and lymphocytes), primarily considering impedance channel compatibility. However, in the cell mimic particles used for optical quality control of five-part white blood cell differential analysis in hematology analyzers, there is a mismatch between the detection principles. This results in significant batch-to-batch variations in the properties of lymphocyte mimic particles, and even within the same batch of quality control material, the coefficient of variation in cell count proportions obtained from multiple tests is substantial.

[0004] Therefore, there is a need to develop a lymphocyte mimic particle that can be well adapted to a blood cell analyzer that uses optical methods to detect five-part differential white blood cells. Summary of the Invention

[0005] In view of the above, this disclosure is intended to provide a lymphocyte mimicry composition comprising independently prepared lymphocyte mimicry particles obtained by processing lymphocytes from mammalian blood, having the same or similar optical properties as human lymphocytes, and for use in quality control and / or calibration of blood cell analyzers.

[0006] The first aspect of this disclosure provides a lymphocyte-mimicking composition comprising lymphocyte-mimicking particles preserved in a preservation solution, wherein the lymphocyte-mimicking particles constitute more than 50% of the volume of the composition, and wherein the lymphocyte-mimicking particles are obtained by isolating lymphocytes from mammalian blood and processing the lymphocytes, wherein the lymphocyte-mimicking particles, when tested with a hematology analyzer, have optical properties that are the same as or similar to those of human lymphocytes.

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

[0008] A second aspect of this disclosure further provides a method for obtaining lymphocyte mimic particles by isolating lymphocytes from mammalian blood and processing the lymphocytes.

[0009] The method for preparing lymphocyte mimic particles includes isolating lymphocytes from mammalian blood and processing the lymphocytes to obtain the lymphocyte mimic particles, wherein the lymphocyte mimic particles, when tested with a blood cell analyzer, have the same or similar optical properties as human lymphocytes.

[0010] In some embodiments, isolating lymphocytes from mammalian blood includes: performing density gradient centrifugation on the mammalian blood or a suspension of the mammalian blood cells to separate crude lymphocytes.

[0011] 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.

[0012] In some embodiments, the process of isolating lymphocytes from mammalian blood further includes: lysing the erythrocytes in the mammalian blood with a erythrocyte lysis buffer before or after performing the density gradient centrifugation, and removing the lysed erythrocytes.

[0013] In some embodiments, the process of isolating lymphocytes from mammalian blood 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.

[0014] In some embodiments, processing the lymphocytes isolated from mammalian blood includes fixing the lymphocytes with an aldehyde compound. According to a further embodiment, the fixation is performed for 5 hours to 3 days, preferably 5 hours to 24 hours, with the aldehyde compound at a concentration of 1%-10% (v / v), preferably 2%-5% (v / v).

[0015] In some embodiments, the cells obtained in each step of preparing lymphocyte 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.

[0016] A third aspect of this disclosure provides a lymphocyte mimic particle prepared by the above method.

[0017] The fourth aspect of this disclosure provides a quality control or calibrator for a blood cell analyzer, comprising a lymphocyte-simulating composition of any of the above embodiments or lymphocyte-simulating particles prepared according to the methods of any of the above embodiments.

[0018] The fifth aspect of this disclosure provides a white blood cell five-part differential quality control material, comprising at least lymphocyte mimic particles prepared according to any of the above embodiments.

[0019] In some embodiments, the white blood cell five-part differential control material further includes monocyte mimic particles, basophil mimic particles, neutrophil mimic particles, and eosinophil mimic particles. According to some embodiments, one or more of the monocyte mimic particles, basophil mimic particles, neutrophil mimic particles, and eosinophil mimic particles are prepared independently. Preferably, at least the monocyte mimic particles, basophil mimic particles, neutrophil mimic particles, and eosinophil mimic particles are all prepared independently.

[0020] 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.

[0021] A 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 mimicry particles, the quality control material comprising: lymphocyte mimicry particles prepared according to any of the above embodiments, independently prepared monocyte mimicry particles, independently prepared basophil mimicry particles, independently prepared neutrophil mimicry particles, and independently prepared eosinophil mimicry particles; detecting the quality control material in quality control mode using the hematology analyzer to obtain at least a lymphocyte count result in the quality control material; and comparing the count result with a standard value of the quality control material.

[0022] In some embodiments, the deviation between the two lymphocyte counts in the quality control material obtained by performing two tests with the blood cell analyzer in quality control mode is less than 5%.

[0023] In some implementations, the coefficient of variation for each lymphocyte count in all tests over one opening cycle of the quality control material is less than 10%.

[0024] In some embodiments, the test is performed on two batches of the quality control material separately, and the deviation between the two lymphocyte counts in the two batches of the quality control material is less than 10%.

[0025] The lymphocyte mimicry composition provided in this disclosure contains lymphocyte mimicry particles that are independently prepared single-particle clusters of cell mimicry particles. Therefore, the differences between batches are small, and the properties of the mimicry particles have better consistency and stability, which is beneficial to the quality control of the blood cell analyzer and thus improves the reliability of the blood cell analyzer for clinical testing. Attached Figure Description

[0026] Figure 1 shows the leukocytes obtained in the process of preparing lymphocyte mimic particles from pig blood in Example 1 (a), the upper layer cells after density gradient centrifugation (b), the lower layer cells after density gradient centrifugation (c), and the forward-lateral scattering intensity scatter plot and fluorescence intensity-lateral scattering intensity scatter plot (d) of the finally obtained lymphocyte mimic particles and human blood samples tested by a hematology analyzer.

[0027] Figure 2 shows the upper layer cells (a) after density gradient centrifugation, the lower layer cells (b) after density gradient centrifugation, and the final lymphocyte mimic particles and human blood samples after testing with a hematology analyzer using blood cell analyzer.

[0028] Figure 3 shows the bovine blood leukocytes (a) separated during the preparation of lymphocyte mimic particles from bovine blood in Example 3, the upper layer cells after density gradient centrifugation (b), the lower layer cells after density gradient centrifugation (c), and the scatter plots of forward and side scatter light intensity and fluorescence intensity-side scatter light intensity (d) of the finally obtained lymphocyte mimic particles and human blood samples after testing with a hematology analyzer.

[0029] Figure 4 shows a scatter plot of fluorescence intensity versus side-scatter light intensity of lymphocyte mimic particles prepared in Comparative Example 1 and human blood samples after testing with a hematology analyzer.

[0030] Figure 5 shows the stability test results of the lymphocyte mimic particles prepared in Examples 1-3 after 22 weeks of storage;

[0031] Figure 6 shows a scatter plot of fluorescence intensity-side scatter intensity of the leukocyte five-part differential quality control material prepared in Example 5 after testing with a hematology analyzer (a), and the test results of the stability of the proportion of lymphocyte mimic particles to five-part differential leukocytes after 22 weeks of storage.

[0032] Figure 7 shows the test results of the stability of the proportion of lymphocyte mimic particles to white blood cells in the whole blood quality control prepared in Example 6 after 22 weeks of storage;

[0033] Figure 8 shows a scatter plot of fluorescence intensity versus side-scatter light intensity of leukocytes tested on a Mindray BC-7500 hematology analyzer for whole blood control (a) prepared in Example 7, as well as commercially available BC6D control (b) and BR60 control (c). Detailed Implementation

[0034] 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.

[0035] 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.

[0036] 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.

[0037] Unless otherwise stated, the singular forms “a / kind” and “the / said” as used herein include the plural form of the noun they refer to.

[0038] The mammals mentioned in this disclosure refer to mammals other than humans. Specifically, the mammals may include cattle, pigs, etc., especially cattle.

[0039] The term "'single-cluster' cell mimic particle" as used in this disclosure refers to a single-species cell mimic particle containing only one cell type, particularly one leukocyte subtype. In this disclosure, unless otherwise specified, the qualifier "single-cluster" may be omitted, and the particle may simply be referred to as "cell mimic particle." For example, a lymphocyte mimic particle refers to a mimic particle of a single lymphocyte.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] Mammalian lymphocytes are similar to human lymphocytes in size and contents, and also constitute a higher proportion of the blood. However, there are no reports of preparing lymphocyte-mimicking particles from mammalian lymphocytes.

[0044] Through extensive and long-term experimental exploration, the inventors have developed a method for preparing lymphocyte-mimicking particles by isolating and purifying mammalian lymphocytes.

[0045] Therefore, this disclosure provides a method for preparing lymphocyte-mimicking particles with optical properties that are the same as or similar to those of human lymphocytes, and lymphocyte-mimicking particles of single particle clusters obtained therefrom.

[0046] Specifically, the method includes the steps of isolating lymphocytes from mammalian blood and processing the lymphocytes to obtain lymphocyte-mimicking particles. When tested with a blood cell analyzer, the lymphocyte-mimicking particles exhibit light scattering properties similar to or identical to those of human lymphocytes.

[0047] 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.

[0048] The method disclosed herein comprises two steps: first, lymphocytes are isolated from mammalian blood; then, the isolated lymphocytes are processed. Since mammalian lymphocytes are similar to human lymphocytes in size and contents, the method, after isolating the mammalian lymphocytes, only requires fixation to obtain lymphocyte-mimicking particles. The optical properties of the lymphocyte-mimicking particles obtained by this disclosure can simulate the optical properties of human lymphocytes when detected using a blood cell analyzer. The method for preparing lymphocyte-mimicking particles from mammalian lymphocytes is described in detail below.

[0049] Isolate lymphocytes

[0050] Methods for isolating lymphocytes from mammalian blood include the step of density gradient centrifugation of mammalian blood to obtain crude lymphocyte products.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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.

[0055] 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.

[0056] 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.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] The buffer solution may be phosphate buffer, sodium citrate buffer, Tris buffer, PBS buffer or HEPES buffer, etc., but is not limited to these.

[0061] 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.

[0062] 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.

[0063] 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.

[0064] The erythrocyte lysis buffer mentioned herein is a solution containing a lysis agent that lyses erythrocytes into fragments while allowing leukocytes to retain their cellular morphology. Commonly used lysis agents for erythrocyte lysis can be used, and this disclosure does not impose any particular limitation. Examples of 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.

[0065] According to one specific embodiment, the erythrocyte lysis buffer has the aforementioned physiological osmotic pressure, preferably the physiological osmotic pressure of mammalian lymphocytes, in order to maintain appropriate cell morphology for various types of leukocytes. There is no particular limitation 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.

[0066] 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.

[0067] 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.

[0068] 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.

[0069] 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.

[0070] 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.

[0071] 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.

[0072] 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%.

[0073] Treatment of lymphocytes

[0074] The size and contents of lymphocytes in mammals (such as cattle) are similar to those in humans. Therefore, lymphocyte-mimicking particles can be obtained simply by fixing the cells to maintain stable physical properties over a longer period of time.

[0075] The fixation of lymphocytes can be performed using conventional methods, such as treatment with aldehydes to denature cell membrane proteins and thus stabilize cell structure. This disclosure does not impose any particular limitation on this method. Specifically, for example, lymphocytes can be suspended in a fixation solution and left to stand at 20°C-37°C for 5 hours to 3 days, preferably at 25°C-30°C for 5 hours to 24 hours.

[0076] For example, the isolated lymphocytes can be suspended in the buffer solution, and a certain amount of aldehyde substance can be added. The concentration of the aldehyde substance is 1%-10% (v / v), preferably 2-5% (v / v). The aldehyde substance is selected from one or more of formaldehyde, glutaraldehyde, glyoxal, acetone aldehyde, p-trifluoromethylbenzaldehyde, and paraformaldehyde. The physiological osmotic pressure buffer solution is as described above. Preferably, the physiological osmotic pressure buffer solution is a buffer solution having the osmotic pressure of the lymphocytes of the mammal.

[0077] The fixed lymphocytes are then used as lymphocyte mimic particles. After washing, the prepared lymphocyte mimic particles are suspended in a preservation solution for storage. Both the washing solution and the preservation solution can be physiological osmotic buffers for the lymphocytes of the aforementioned mammals.

[0078] This disclosure thereby provides lymphocyte-mimicking particles prepared by the above method.

[0079] Lymphocyte mimicry composition

[0080] This disclosure also provides a lymphocyte-mimicking composition. The composition comprises lymphocyte-mimicking particles preserved in a preservation solution, the lymphocyte-mimicking particles comprising more than 50% by volume in the composition. The lymphocyte-mimicking particles are obtained by processing lymphocytes isolated from the blood of mammals, wherein the lymphocyte-mimicking particles, when tested with a hematology analyzer, possess optical properties identical or similar to those of human lymphocytes.

[0081] In some embodiments, the lymphocyte mimic particles constitute 50%-70% of the volume of the composition.

[0082] According to one specific embodiment, the composition comprises lymphocyte-mimicking particles preserved in a preservation solution. That is, the cell-mimicking particles in the composition are only single-particle clusters of lymphocyte-mimicking particles.

[0083] The optical properties include light scattering properties and fluorescence properties. The light scattering properties include the characteristics of side-scattered and forward-scattered light, which reflect the complexity of the cell contents and the size of the cell.

[0084] In this disclosure, the lymphocyte-mimicking particles in the composition are prepared by the method described above.

[0085] The preservation solution can be a solution commonly used for simulating particle preservation, and this disclosure does not have any particular limitations. Exemplarily, the preservation solution can be common buffers such as phosphate buffer, sodium citrate buffer, Tris buffer, PBS buffer, or various commercially available blood cell preservation solutions.

[0086] In the composition, the concentration of lymphocyte-mimicking particles is approximately (700-800) × 10⁻⁶. 9 per mL.

[0087] Quality control materials or calibrators for blood cell analyzers

[0088] This disclosure also provides a quality control material or calibrator for a blood cell analyzer, including the above-described lymphocyte mimic particles or the above-described lymphocyte mimic composition.

[0089] Quality control materials used in hematology analyzers are used to monitor and evaluate the precision of hematology analyzers.

[0090] Calibrators used in blood cell analyzers are designed to accurately simulate human blood during testing on the analyzer, thereby establishing metrological traceability of the blood cell analyzer's measurement results.

[0091] 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 lymphocyte mimic particles.

[0092] Furthermore, the five-part differential white blood cell quality control material also includes monocyte mimic particles, basophil mimic particles, neutrophils, and eosinophils.

[0093] According to one embodiment, one or more of the monocyte mimic particles, basophil mimic particles, neutrophils, and eosinophils are prepared independently; preferably, at least the monocyte mimic particles are prepared independently. According to a specific embodiment, the monocyte mimic particles, basophil mimic particles, neutrophils, and eosinophils are all prepared independently, respectively.

[0094] All five categories of simulated particles were prepared independently, resulting in stable and highly distinguishable quality control materials. In particular, the distinguishability of lymphocyte and monocyte simulated particles was significantly improved, enabling consistent test results across multiple quality control tests conducted on the same batch of materials.

[0095] The independently prepared mononuclear cell mimic particles can be obtained using the following method. First, 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. The obtained neutrophils are treated with a condensate of nitro alcohol and formaldehyde (2-bromo-2-nitropropane-1,3-diol and / or 5-bromo-5-nitro-1,3-dioxane). Specifically, the neutrophils are treated with a condensate of nitro alcohol and formaldehyde at a concentration of 0.5%-5%, preferably 0.5-1%, at 25-35°C for 24-36 hours. The treated neutrophils are then suspended in a fixation solution containing 1%-10% (v / v), preferably 2%-5% (v / v) aldehydes and allowed to stand at 20°C-37°C for 5 hours to 3 days.

[0096] 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. The condensate of the nitro alcohol and formaldehyde is used to treat the neutrophils at a temperature of 20°C to 37°C for 12 hours to 7 days, preferably 24 to 36 hours.

[0097] 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.

[0098] 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.

[0099] 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.

[0100] The batch-to-batch variation of independently prepared cell-mimicking particles is small, and the cell properties exhibit good consistency and stability. Therefore, the overall consistency and stability of the five-part differential leukocyte quality control material obtained independently for all subtypes of leukocytes are good.

[0101] 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 and platelet mimic particles. Even further, the whole blood quality control material also includes reticulocyte mimic particles.

[0102] 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.

[0103] 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.

[0104] 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.

[0105] The quality control material was stored at 2-8°C.

[0106] The quality control material also includes a preservation solution. The preservation solution has been described above and will not be repeated here.

[0107] Quality control methods for blood cell analyzers

[0108] This disclosure also provides a quality control method for a blood cell analyzer. The method includes the following steps:

[0109] A quality control material containing at least five different white blood cell fractions mimics is provided, the quality control material comprising: the aforementioned lymphocyte mimics, independently prepared monocyte 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 lymphocyte count in the quality control material; and the count is compared with the standard value of the quality control material.

[0110] Using the quality control material disclosed herein, the quality control material was tested twice using the blood cell analyzer in quality control mode, and the deviation between the two lymphocyte count results in the quality control material was less than 5%.

[0111] Each vial of the quality control product can be tested multiple times during use. With this disclosed quality control, the deviation of individual lymphocyte counts in all tests within one open cycle on the same device is less than 10%.

[0112] Furthermore, the quality of each batch of the quality control material disclosed herein is stable. When the test was performed on two batches of the quality control material, the deviation between the two lymphocyte counts in the two batches of the quality control material was less than 10%.

[0113] The counting result is the count of lymphocyte mimic particles, or the percentage of lymphocyte mimic particles in white blood cells.

[0114] The advantages of this disclosure will be further illustrated by specific embodiments below.

[0115] Example

[0116] The following examples use conventional reagents:

[0117] Red blood cell lysis buffer: Tris-ammonium chloride red blood cell lysis buffer was used for all cases.

[0118] Phosphate buffer: pH = 7.00 ± 0.2, osmotic pressure 305-310 mOsm.

[0119] Preservative solution: Phosphate buffer containing sugars and antibacterial agents, pH = 7.00 ± 0.2, osmotic pressure 305-310 mOsm.

[0120] 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.

[0121] Example 1: Preparation of Lymphocyte Mimic Particles from Porcine Blood Lymphocytes

[0122] 1. Isolation of lymphocytes

[0123] 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 500 mL of phosphate buffer.

[0124] The cell suspension was tested on a BC-7500 hematology analyzer, and the fluorescence intensity-side scatter light intensity scatter plot is shown in Figure 1a. At this time, the cell suspension contained various blood cells such as lymphocytes, neutrophils, and eosinophils.

[0125] Add Ficoll cell separation medium to a separate centrifuge tube, then slowly add an equal volume of the above cell suspension. Centrifuge at 400g for 30 minutes. After centrifugation, the cell suspension separates into upper and lower portions. Both portions are analyzed on a BC-7500 hematology analyzer. The upper portion mainly contains lymphocytes (Figure 1b), while the lower portion mainly contains neutrophils and eosinophils (Figure 1c). Collect the upper cells into a clean centrifuge tube to obtain crude lymphocytes.

[0126] The collected upper cells were centrifuged at 200g for 8 min (or 400g for 4 min), and the supernatant was removed. This process was repeated 3 times to obtain lymphocytes. The cells were resuspended in phosphate buffer, centrifuged under the same conditions, and the supernatant was removed to wash the lymphocytes. After washing 3 times, the cells were resuspended in phosphate buffer.

[0127] 2. Treatment of lymphocytes

[0128] Add 5% (v / v) of the cell suspension volume of formaldehyde (which can be replaced by 5% (v / v) paraformaldehyde or 2% (v / v) glutaraldehyde) to the cell suspension, mix thoroughly, and fix at 30°C for 18 h.

[0129] 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. The cells were then washed to obtain lymphocyte mimic particles.

[0130] The cells were resuspended in preservation solution to a cell concentration of approximately 50 × 10⁻⁶. 9 Cells / mL, store at 2-8℃.

[0131] The preservation solution containing lymphocyte mimic particles prepared above was tested on Mindray BC-5390CRP and Mindray BC-7500 hematology analyzers, respectively. Scatter plots of forward-scattered light intensity versus side-scattered light intensity and fluorescence-scattered light intensity versus side-scattered light intensity were obtained, as shown in Figure 1, d. It can be seen that the fluorescence, side-scattered light, and forward-scattered light properties of the cell mimic particles prepared in Example 1 are very close to those of lymphocytes tested in blood samples from healthy subjects under the same conditions. Furthermore, the content (purity) of the lymphocyte mimic particles prepared in this example was found to be above 95% on both devices.

[0132] Example 2: Preparation of Lymphocyte Simulation Particles from Porcine Blood Lymphocytes

[0133] 1. Isolation of lymphocytes

[0134] Prepare a phosphate buffer solution with a concentration 10 times that of the aforementioned phosphate buffer. Mix 9 parts of Percoll cell separation solution with 1 part of the 10-fold concentration of phosphate buffer solution at a volume ratio of 9:1 to obtain a solution with an osmotic pressure of (305-310 mOsm) and a density of 1.117 g / mL. Then dilute it with phosphate buffer solution (density 1.005 g / mL) to a density between 1.055-1.075 g / mL as the cell separation solution.

[0135] The prepared cell separation solution was added to a centrifuge bottle, followed by a slow addition of an equal volume of anticoagulated pig blood, and the mixture was thoroughly stirred. The mixture was centrifuged at 400g for 30 minutes. After centrifugation, the blood separated into upper and lower portions of the cell separation solution. Both portions were tested on a Mindray BC-7500 hematology analyzer. The fluorescence intensity-side scattering intensity scatter plot showed that the upper portion contained lymphocytes (Figure 2a), while the lower portion contained neutrophils and eosinophils (Figure 2b). The upper portion was collected into a clean centrifuge tube and centrifuged at 2400rpm for 5 minutes. The supernatant was then removed to obtain a crude lymphocyte product. This crude lymphocyte product contained lymphocytes, platelets, and erythrocytes.

[0136] Add erythrocyte lysis buffer at 10 times the cell volume, mix thoroughly, and incubate at room temperature for 10 minutes to allow the erythrocytes to lyse completely. Then centrifuge at 2400 rpm for 5 minutes. After centrifugation, discard the supernatant and resuspend the cells in phosphate buffer.

[0137] Centrifuge the cell suspension at 200g for 8 min (or 400g for 4 min), remove the supernatant, and repeat this process 3 times 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 3 times, and then resuspend the cells in phosphate buffer.

[0138] 2. Treatment of lymphocytes

[0139] Add 5% (v / v) of the cell suspension volume of formaldehyde (which can be replaced by 5% (v / v) paraformaldehyde or 2% (v / v) glutaraldehyde) to the cell suspension, mix thoroughly, and fix at 30°C for 18 h.

[0140] 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. The cells were then washed to obtain lymphocyte mimic particles.

[0141] The cells were resuspended in preservation solution to a cell concentration of approximately 50 × 10⁻⁶. 9Cells / mL, store at 2-8℃.

[0142] The preservation solution containing lymphocyte mimic particles prepared above was tested on Mindray BC-5390CRP and Mindray BC-7500 hematology analyzers, respectively. Scatter plots of forward scatter intensity versus side scatter intensity and fluorescence scatter intensity versus side scatter intensity were obtained, as shown in Figure 2c. It can be seen that the fluorescence, side scatter, and forward scatter properties of the lymphocyte mimic particles prepared in Example 2 are very close to those of lymphocytes tested in blood samples from healthy subjects under the same conditions. Furthermore, the content (purity) of the lymphocyte mimic particles prepared in this example was found to be above 95% on both devices.

[0143] Example 3: Preparation of Lymphocyte Simulation Particles from Bovine Blood Lymphocytes

[0144] 1. Isolation of lymphocytes

[0145] Centrifuge 500 mL of anticoagulated bovine 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.

[0146] The cell suspension was tested on a Mindray BC-7500 hematology analyzer, and the fluorescence intensity-side scatter light intensity scatter plot is shown in Figure 3a. At this time, the cell suspension contained a large number of granulocytes in addition to lymphocytes.

[0147] Add Ficoll cell separation medium to a separate centrifuge tube, then slowly add an equal volume of the above cell suspension. Centrifuge at 400g for 30 minutes. After centrifugation, the cell suspension separates into upper and lower portions. Both portions are analyzed on a Mindray BC-7500 hematology analyzer. The fluorescence intensity-side scatter plot shows that the upper portion mainly contains lymphocytes (Figure 3b), while the lower portion mainly contains granulocytes (Figure 3c). Collect the cells from the middle portion into a clean centrifuge tube to obtain crude lymphocytes.

[0148] The collected mid-cells were centrifuged at 200g for 8 min (or 400g for 4 min), and the supernatant was removed. This process was repeated 3 times to obtain lymphocytes. The cells were resuspended in phosphate buffer, centrifuged under the same conditions, and the supernatant was removed to wash the lymphocytes. After washing 3 times, the cells were resuspended in phosphate buffer.

[0149] 2. Treatment of lymphocytes

[0150] Add 5% (v / v) of the cell suspension volume of formaldehyde (which can be replaced by 5% (v / v) paraformaldehyde or 2% (v / v) glutaraldehyde) to the cell suspension, mix thoroughly, and fix at 30°C for 18 h.

[0151] After fixation, the cells were centrifuged at 1800 rpm for 5 min and the supernatant was removed. The cells were then resuspended in phosphate buffer and centrifuged at 1800 rpm for 5 min to remove the supernatant and wash the cells, thereby obtaining lymphocyte mimic particles.

[0152] Cells were resuspended in preservation solution at a concentration of 50 × 10⁶ cells / mL. 9 Cells / mL, store at 2-8℃.

[0153] The preservation solution containing lymphocyte mimic particles prepared above was tested on Mindray BC-5390CRP and Mindray BC-7500 hematology analyzers, respectively, and scatter plots of forward-scattered light intensity versus side-scattered light intensity and fluorescence-scattered light intensity versus side-scattered light intensity were obtained, as shown in Figure 3d. It can be seen that the fluorescence, side-scattered light, and forward-scattered light properties of the lymphocyte mimic particles prepared in Example 3 are very close to those of lymphocytes tested in blood samples from healthy subjects under the same conditions. Furthermore, the content (purity) of the lymphocyte mimic particles prepared in this example was found to be above 95% on both devices.

[0154] Comparative Example 1: Preparation of Lymphocyte Mimic Particles from Porcine Blood Lymphocytes under Conventional Osmotic Pressure

[0155] 1. Isolation of lymphocytes

[0156] 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 500 mL of commercially available physiological saline (osmolarity 288-293 mOsm).

[0157] Add Ficoll cell separation medium to a separate centrifuge tube, then slowly add an equal volume of the above cell suspension. Centrifuge at 400g for 30 minutes. After centrifugation, the cell suspension separates into upper and lower portions. Collect the middle portion of cells into a clean centrifuge tube to obtain crude lymphocytes.

[0158] The collected supernatant was centrifuged at 200g for 8 minutes, and the supernatant was removed. This process was repeated 3 times to obtain lymphocytes. The cells were resuspended in the same physiological saline, centrifuged under the same conditions, and the supernatant was removed to wash the lymphocytes. After washing 3 times, the cells were resuspended in the same physiological saline.

[0159] 2. Treatment of lymphocytes

[0160] Add 5% (v / v) of the cell suspension volume of formaldehyde (which can be replaced by 5% (v / v) paraformaldehyde or 2% (v / v) glutaraldehyde) to the cell suspension, mix thoroughly, and fix at 30°C for 18 h.

[0161] After fixation, the cells were centrifuged at 1800 rpm for 5 min and the supernatant was removed. The cells were then resuspended in the physiological saline, centrifuged at 1800 rpm for 5 min, and the supernatant was removed. The cells were then washed to obtain lymphocyte mimic particles.

[0162] The cells were resuspended in preservation solution to a cell concentration of approximately 50 × 10⁻⁶. 9 Cells / mL, store at 2-8℃.

[0163] The preservation solution containing lymphocyte-simulated particles prepared above was tested on a hematology analyzer, and the results are shown in Figure 4. Comparing the results with the cell-simulated particles prepared in Example 1, it can be found that the simulated particles prepared in this comparative example contain many granulocytes, and the lymphocyte purity is only about 70%. At this level, the cell particles cannot be used to prepare quality control materials; otherwise, the contaminating cells will affect the accurate grouping of the quality control materials.

[0164] Example 4: Stability of Lymphocyte-mimicking particles

[0165] The count of the lymphocyte-mimicking particles prepared in Examples 1-3 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.

[0166] The simulated particles used to prepare quality control materials need to remain stable for at least 4 months, with no trend in cell counts, and all test data having a relative range and coefficient of variation of less than 10%. In this example, during approximately 5 months of testing, the number of lymphocyte simulated particles prepared in Examples 1-3 remained essentially stable, with all test values ​​having a relative range of less than 8% and a coefficient of variation of less than 4%, indicating good stability and meeting the stability requirements for quality control materials.

[0167] Example 5: White blood cell five-part differential quality control material

[0168] Prepare independently synthesized leukocyte mimic particles of various subtypes:

[0169] Lymphocyte mimic particles: prepared in Example 1.

[0170] Mononuclear cell mimic particles were prepared using the following method:

[0171] 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.

[0172] 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.

[0173] 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.

[0174] 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.

[0175] 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.

[0176] Resuspend the cells in preservation solution and store at 2-8°C.

[0177] 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.

[0178] Eosinophil mimic particles: prepared using glutaraldehyde-fixed bovine blood neutrophils according to the method disclosed in Example 1 of CN101887059A.

[0179] 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.

[0180] The cell-simulated particles obtained above were suspended in preservation solution, and the cell count was adjusted to approximately 50 × 10⁻⁶. 9 per mL.

[0181] 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℃.

[0182] The above-mentioned five-part differential white blood cell quality control was tested on a blood cell analyzer. The results showed that each cell cluster was clearly divided and could be correctly identified and grouped by the blood cell analyzer (the test was conducted using Mindray BC-7500). The test results and the results of the test on the blood of healthy subjects are shown in Figure 6a.

[0183] 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 lymphocyte mimic particle ratio. It was found that during the 22-week test, the proportion of lymphocyte mimic particles to leukocyte five-part differential quality control particles remained stable, as shown in Figure 6b. This indicates that the lymphocyte mimic particles described in this invention perform well in the application of leukocyte five-part differential quality control samples.

[0184] Example 6: Whole blood quality control material

[0185] The leukocyte five-part differential quality control material prepared in Example 5 was used as leukocyte mimic particles.

[0186] Red blood cell mimic particles: prepared using human red blood cells fixed with glutaraldehyde according to the method disclosed in Example 1 of CN105717312A.

[0187] 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.

[0188] 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.

[0189] 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.

[0190] 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. The relative range and coefficient of variation for all test data should be less than 10%. Samples were tested weekly, and the stability of the proportion of lymphocyte mimic particles to white blood cells in high, medium, and low-level whole blood quality controls was statistically analyzed. It was found that the proportion of lymphocyte mimic particles remained stable throughout the 22-week test, as shown in Figure 7. The relative range for all test data was 5.2%, and the coefficient of variation was 1.4%. This indicates that the lymphocyte mimic particles described in this invention perform well in whole blood quality control materials.

[0191] Example 7: Comparison experiment between the quality control material of Example 6 and commercially available quality control materials

[0192] The whole blood control prepared in Example 6, the commercially available BC6D whole blood control, and the commercially available BR60 whole blood control were tested on a hematology analyzer to obtain a scatter plot of fluorescence intensity versus side scatter intensity for the five-part differential white blood cell count, as shown in Figure 8. Compared with the scatter plots of the commercially available BC6D control (Figure 8, Figure ...

[0193] Because all particles in the quality control materials in Example 6 were prepared individually, the proportion of each particle cluster could be freely adjusted according to actual needs, and the quality control materials showed 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 type of white blood cell based on the total white blood cell count, and the coefficient of variation of the percentage of lymphocytes between batches was calculated, as shown in the table below.

[0194]

[0195] As can be seen from the table above, the deviation in the percentage of lymphocytes measured between different batches of the quality control material prepared in Example 6 was significantly lower than that of the other two commercially available quality control materials.

[0196] 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 lymphocyte-mimicking composition comprising lymphocyte-mimicking particles preserved in a preservation solution, wherein the lymphocyte-mimicking particles constitute 50% or more of the volume in the composition, and wherein the lymphocyte-mimicking particles are obtained by isolating lymphocytes from mammalian blood and processing the lymphocytes, wherein... When the lymphocyte-simulating particles are tested using a blood cell analyzer, they have optical properties that are the same as or similar to those of human lymphocytes.

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

3. The lymphocyte mimicry composition according to claim 1 or 2, wherein, Isolating lymphocytes from mammalian blood includes: performing density gradient centrifugation on the mammalian blood or a suspension of mammalian blood cells to separate crude lymphocytes; preferably, washing the obtained crude lymphocytes with a buffer having the physiological osmotic pressure of mammalian lymphocytes.

4. The lymphocyte-mimicking composition according to claim 3, wherein, The density gradient centrifugation includes: performing the density gradient centrifugation with a cell separation solution with a density 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.

5. The lymphocyte-mimicking composition according to claim 4, 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.

6. The lymphocyte-mimicking composition according to claim 3, wherein, The process of isolating lymphocytes from mammalian blood further includes: lysing the erythrocytes in the mammalian blood with a erythrocyte lysis buffer before or after the density gradient centrifugation, and removing the lysed erythrocytes; preferably, washing the product after removing the lysed erythrocytes with the buffer having the physiological osmotic pressure of mammalian lymphocytes.

7. The lymphocyte-mimicking composition according to claim 6, wherein, The erythrocyte lysis buffer was a tris-ammonium chloride solution.

8. The lymphocyte-mimicking composition according to claim 6, wherein, The process of isolating lymphocytes from mammalian blood 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; preferably, repeating the centrifugation 3-6 times; more preferably, washing the lymphocytes after removing platelets with the buffer solution having the physiological osmotic pressure of mammalian lymphocytes, preferably 1-3 times.

9. The lymphocyte-mimicking composition according to claim 1, wherein, The treatment of the lymphocytes isolated from the blood of mammals includes fixing the lymphocytes with an aldehyde compound; preferably, the fixation is performed 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.

10. The lymphocyte-mimicking composition according to claim 3, 6, or 8, wherein, The physiological osmotic pressure of the mammalian lymphocytes is 295mOsm-340mOsm, preferably 300mOsm-315mOsm, and more preferably 305mOsm-310mOsm; preferably, the pH value of the buffer solution is 6.5-9.

11. The lymphocyte-mimicking composition according to claim 1, wherein, The optical properties include light scattering properties and fluorescence properties.

12. The lymphocyte-mimicking composition according to claim 1, wherein, The mammal in question is either a pig or a cow.

13. A method for preparing lymphocyte mimic particles, comprising: Lymphocytes are isolated from mammalian blood and processed to obtain lymphocyte mimic particles, wherein the lymphocyte mimic particles, when tested with a blood cell analyzer, have the same or similar optical properties as human lymphocytes.

14. The method according to claim 13, wherein, The steps of isolating lymphocytes from mammalian blood and processing the lymphocytes are as defined in any one of claims 3-12.

15. A lymphocyte mimic particle, said lymphocyte mimic particle being prepared by the method of claim 13 or 14.

16. A quality control or calibrator for a hematology analyzer, comprising a lymphocyte-mimicking composition according to any one of claims 1-12 or lymphocyte-mimicking particles according to claim 15.

17. A leukocyte five-part differential quality control material, comprising at least the lymphocyte mimic particles according to claim 15.

18. The white blood cell five-part differential quality control material according to claim 17, further comprising monocyte mimic particles, basophil mimic particles, neutrophil mimic particles, and eosinophil mimic particles; preferably, one or more of the monocyte mimic particles, basophil mimic particles, neutrophil mimic particles, and eosinophil mimic particles are prepared independently; more preferably, at least the monocyte mimic particles, basophil mimic particles, neutrophil mimic particles, and eosinophil mimic particles are all prepared independently.

19. A whole blood quality control material, comprising the white blood cell five-part differential quality control material according to claim 17 or 18, and red blood cell mimic particles, platelet mimic particles and reticulocyte mimic particles.

20. A quality control method for a blood cell analyzer, comprising: Provide a quality control material containing at least five different white blood cell fractions mimic particles, the quality control material comprising: lymphocyte mimic particles as described in claim 15, independently prepared monocyte mimic particles, independently prepared basophil mimic particles, independently prepared neutrophil mimic particles, and independently prepared eosinophil mimic particles; detect the quality control material using the blood cell analyzer in quality control mode to obtain at least the lymphocyte count result in the quality control material; and compare the count result with the standard value of the quality control material.

21. The quality control method according to claim 20, wherein, When the blood cell analyzer is used in quality control mode, two tests are performed on the quality control material, and the deviation between the two lymphocyte count results in the quality control material is less than 5%. Preferably, in all tests of one quality control material in one opening cycle, the coefficient of variation of each lymphocyte count result is less than 10%. When the tests are performed on two batches of the quality control material, the deviation between the two lymphocyte count results in the two batches of the quality control material is less than 10%.

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