WO2020132875A1 - 红细胞模拟粒子、其制备方法及含其的质控物或校准物 - Google Patents

红细胞模拟粒子、其制备方法及含其的质控物或校准物 Download PDF

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WO2020132875A1
WO2020132875A1 PCT/CN2018/123529 CN2018123529W WO2020132875A1 WO 2020132875 A1 WO2020132875 A1 WO 2020132875A1 CN 2018123529 W CN2018123529 W CN 2018123529W WO 2020132875 A1 WO2020132875 A1 WO 2020132875A1
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red blood
erythrocyte
blood cell
treatment solution
acid
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French (fr)
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宋瑞霞
谢键
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Shenzhen Mindray Bio Medical Electronics Co Ltd
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Shenzhen Mindray Bio Medical Electronics Co Ltd
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Priority to PCT/CN2018/123529 priority Critical patent/WO2020132875A1/zh
Priority to CN201880098394.5A priority patent/CN112805567B/zh
Publication of WO2020132875A1 publication Critical patent/WO2020132875A1/zh
Priority to US17/353,532 priority patent/US12332256B2/en
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    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N1/00—Preservation of bodies of humans or animals, or parts thereof
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/96—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood or serum control standard
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N1/00—Sampling; Preparing specimens for investigation
    • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
    • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
    • G01N2001/305—Fixative compositions
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483—Physical analysis of biological material
    • G01N33/487—Physical analysis of biological material of liquid biological material
    • G01N33/49—Blood

Definitions

  • the conventional method for preparing erythrocyte simulation particles adopts the method of aldehyde fixation, and there is a problem that the cell volume changes with time.
  • dichromate can cause environmental pollution problems.
  • the present invention provides a method for preparing red blood cell simulated particles, and thus provides a red blood cell simulated particle thus prepared and a quality control substance/calibrator containing the red blood cell simulated particle.
  • the method of the present invention and the red blood cell simulated particles and quality control/calibrators thus obtained solve at least one of the above problems.
  • a method for preparing erythrocyte simulated particles comprising:
  • the at least one oxidizing agent is perchlorate, especially sodium perchlorate and the concentration in the erythrocyte treatment solution is 8-20 g/L, preferably 8-10 g/L.
  • the at least one fixing agent is selected from the group consisting of formaldehyde, glutaraldehyde, glyoxal, pyruvaldehyde, p-trifluoromethylbenzaldehyde, paraformaldehyde, chromic acid, picric acid, tannic acid and acetic acid Composed of groups.
  • the erythrocyte treatment solution further includes a necessary buffer and an optional osmotic pressure regulator. Further, the erythrocyte treatment solution may further contain a preservative.
  • the erythrocyte treatment solution and the erythrocyte to be treated are mixed in a ratio of 1 to 2:1 to perform the treatment .
  • the erythrocytes treated with the first erythrocyte treatment solution and the second erythrocyte treatment solution are washed and suspended in the preservation solution.
  • the at least one oxidizing agent is selected from the group consisting of hypohalites, halites, halides, and perhalates of chlorine, bromine, and iodine.
  • the at least one oxidizing agent is selected from the group consisting of hypochlorous acid, chlorous acid, chloric acid, perchloric acid, bromic acid, iodic acid, and sodium and potassium salts of periodic acid.
  • the at least one oxidizing agent is selected from the group consisting of sodium perchlorate, potassium perchlorate, sodium bromate, potassium bromate, sodium chlorate and potassium chlorate.
  • the at least one oxidizing agent is perchlorate, especially sodium perchlorate and the concentration in the first erythrocyte treatment solution is 8-20 g/L, preferably 8-10 g /L.
  • the at least one oxidizing agent is bromate, especially sodium bromate and the concentration in the first erythrocyte treatment solution is 0.25-2 g/L, preferably 0.25-1 g/ L, more preferably 0.25 to 0.5 g/L.
  • the at least one fixing agent is selected from the group consisting of formaldehyde, glutaraldehyde, glyoxal, pyruvaldehyde, p-trifluoromethylbenzaldehyde, paraformaldehyde, chromic acid, picric acid, tannic acid and acetic acid .
  • the volume concentration of the at least one fixing agent in the second erythrocyte treatment solution is 0.01 to 0.5 vol%.
  • the oxidized red blood cells are washed with a buffer solution, and then the fixing treatment is performed.
  • first and second erythrocyte treatment fluids also contain necessary buffers and optional osmotic pressure regulators. Further, the first and/or second red blood cell treatment liquid may further include a preservative.
  • the first red blood cell treatment liquid and the red blood cells to be treated are mixed in a ratio of 1 to 2:1 to perform oxidation treatment.
  • the second erythrocyte treatment solution and the erythrocytes to be treated are mixed at a ratio of 1 to 2:1 to perform immobilization treatment.
  • the preservation solution has a pH of 5.0 to 9.0, an osmotic pressure of 300 to 800 sm/kg ⁇ H 2 O, and includes 0.01 to 10 g/L of preservative and 0.01 to 5g/L stabilizer.
  • the preservative is at least one selected from the group consisting of azide compounds, carcassone and aminoglycoside antibiotics, capable of inhibiting microbial contamination in red blood cell simulated particles and not affecting the stability of red blood cell simulated particles .
  • the stabilizer is at least one selected from imidazolidinyl ureas, which helps to maintain the stability of the erythrocyte simulated particles for a long time.
  • the preservatives of the present invention also include buffers and optional osmotic pressure regulators. Further, the preservative may further include glucose.
  • the method for preparing red blood cell simulation particles of the present invention further includes the step of spheroidizing the red blood cells before the red blood cells are treated with the (first) red blood cell treatment solution.
  • a red blood cell simulation particle is provided.
  • the erythrocyte simulation particle is prepared by any one of the methods of the first embodiment and the second embodiment in the above-mentioned first aspect.
  • red blood cell simulated particles prepared according to the method of the present invention in a storage solution containing a preservative and an imidazolidinyl urea stabilizer contributes to the long-term stability of the red blood cell simulated particles.
  • FIG. 2 is a graph showing the change of the average erythrocyte volume with time continuously measured within 18 weeks of the erythrocyte simulated particles prepared according to Example 1.
  • FIG. 2 is a graph showing the change of the average erythrocyte volume with time continuously measured within 18 weeks of the erythrocyte simulated particles prepared according to Example 1.
  • FIG. 3 is a graph showing the change in the average erythrocyte volume with time continuously measured within 18 weeks of the erythrocyte simulated particles prepared according to Example 2.
  • FIG. 3 is a graph showing the change in the average erythrocyte volume with time continuously measured within 18 weeks of the erythrocyte simulated particles prepared according to Example 2.
  • FIG. 4 is a graph showing the change of the average erythrocyte volume with time continuously measured within 18 weeks of the erythrocyte simulated particles prepared according to Example 3.
  • FIG. 4 is a graph showing the change of the average erythrocyte volume with time continuously measured within 18 weeks of the erythrocyte simulated particles prepared according to Example 3.
  • Example 6 is a graph showing the change in the average red blood cell volume measured with time continuously for the red blood cell simulated particles prepared according to Example 5 over 18 weeks.
  • FIG. 9 is a graph showing the change in the average red blood cell volume over time continuously measured over 18 weeks for the red blood cell simulated particles prepared according to Example 8.
  • FIG. 9 is a graph showing the change in the average red blood cell volume over time continuously measured over 18 weeks for the red blood cell simulated particles prepared according to Example 8.
  • FIG. 10 is a graph showing the change of the average erythrocyte volume with time continuously measured for the erythrocyte simulated particles prepared according to Example 9 within 18 weeks.
  • FIG. 12 is a graph showing the change in the average erythrocyte volume with time for the quality control prepared according to Example 11 continuously measured within 18 weeks.
  • a volume is stable, in order to obtain an erythrocyte simulated particle that can be produced in an environmentally friendly method.
  • oxidative treatment of naturally-derived red blood cells is carried out using halogenated oxo acid salts, and further immobilization treatment is carried out with an immobilizing agent, thereby obtaining stable red blood cell simulated particles.
  • the inventor believes that in addition to the spheroidization treatment, the denaturation treatment is required to change the conformation of the cell membrane skeleton protein and hemoglobin.
  • the commonly used protein denaturants are strong acids, strong bases, salts containing heavy metals, urea, acetone, etc.
  • the principle of action of various denaturants is not the same, but heavy metal salts have a significant effect on increasing the stability of cell simulated particles, but heavy metals It has serious pollution to the environment. According to more and more stringent environmental protection requirements, more environmentally friendly reagents are needed to replace heavy metal salts.
  • the method for preparing red blood cell simulation particles of the present invention includes:
  • the red blood cell treatment solution with a pH of 5.0 to 9.0 and an osmotic pressure of 300 to 800 sm/kg ⁇ H 2 O is used to oxidize and fix the red blood cells, wherein the red blood cell treatment solution contains at least one selected from halogen oxyacids An oxidizing agent and at least one fixing agent selected from aldehydes or acids; and
  • the red blood cells treated with the red blood cell treatment solution are washed and suspended in the preservation solution.
  • the method for preparing red blood cell simulated particles of the present invention includes:
  • the red blood cell treatment solution is fixed with a second red blood cell treatment solution having a pH of 5.0 to 9.0 and an osmotic pressure of 300 to 800 sm/kg ⁇ H 2 O, wherein the second red blood cell treatment solution contains at least one selected from aldehyde or acid Fixative; and
  • the erythrocytes treated with the first erythrocyte treatment solution and the second erythrocyte treatment solution are washed and suspended in the preservation solution.
  • the oxidation treatment and the fixing treatment may be performed simultaneously in one solution, or the oxidation treatment may be performed before the fixing treatment
  • the halogen oxyacid salt may be hypohalite, halite, halide or perhalate of chlorine, bromine or iodine, especially an alkali metal salt.
  • the halogen oxyacid salt of the present invention may be selected from sodium hypochlorite, potassium hypochlorite, sodium chlorite, potassium chlorite, sodium chlorate, potassium chlorate, sodium perchlorate, potassium perchlorate, sodium bromate, potassium bromate, sodium iodate , Potassium iodate, sodium periodate and potassium periodate.
  • sodium perchlorate, potassium perchlorate, sodium bromate, potassium bromate, sodium chlorate and/or potassium chlorate are particularly preferred.
  • the concentration of the at least one oxidizing agent of the present invention in the erythrocyte treatment liquid is 0.01-20 g/L, preferably 0.025-10 g/L, more preferably 0.025-5 g/L, and even more preferably 0.025-2 g/L .
  • the oxidant may be, for example, perchlorate, especially sodium perchlorate, and its concentration in the erythrocyte treatment solution may be 8-20 g/L, preferably 8-10 g/L; or bromate , Especially sodium bromate, its concentration in the erythrocyte treatment solution is 0.25-2g/L, preferably 0.25-1g/L, more preferably 0.25-0.5g/L; or, chlorate, especially chloric acid
  • concentration of sodium in the erythrocyte treatment liquid is 0.5 to 2 g/L, preferably 0.5 to 1 g/L.
  • Bromate especially sodium bromate
  • the concentration of bromate is low, and with the following fixatives, more stable (especially volume-stabilized) red blood cell simulation particles can be obtained. According to the following specific examples, it can be seen that the simulated particles obtained by treating the red blood cell particles with different concentrations of bromate in the 18-week continuous measurement experiment have a volume change of only about 1 femtoliter.
  • the volume concentration of the fixative in the treatment solution (for the first solution is the red blood cell treatment solution and for the second solution is the second red blood cell treatment solution) can be 0.01 to 0.5 vol%. Within range.
  • the buffer is not particularly limited, and may be any buffer suitable for red blood cells. Examples include: sodium citrate, Tris, PBS, HEPES, etc., but are not limited thereto.
  • the osmotic pressure regulator is not particularly limited. Suitable osmotic pressure adjusting agents include: sodium chloride, sodium dihydrogen phosphate, potassium chloride, sodium citrate, etc., but are not limited thereto.
  • the concentration of the buffering agent and the osmotic pressure regulator is selected so that the osmotic pressure of the erythrocyte treatment solution of the present invention is in the range of 300 to 800 sm/kg ⁇ H 2 O, and the pH value is in the range of 5.0 to 9.0.
  • the erythrocyte treatment solution of the present invention may further contain preservatives, such as azides (such as sodium azide) and casons, such as proclin series (such as proclin300, proclin950), etc., but it is not limited thereto.
  • preservatives such as azides (such as sodium azide) and casons, such as proclin series (such as proclin300, proclin950), etc., but it is not limited thereto.
  • the amount of preservatives is usually 0.01 ⁇ 10g/L.
  • both the erythrocyte treatment solution and the erythrocytes to be treated can be mixed in a ratio of about 1 to 2:1 for corresponding treatment.
  • the treatment time is usually 1 to 3 hours.
  • the oxidized red blood cells are washed with a buffer solution, and then the fixing treatment is performed.
  • the pH of the storage solution is 5.0 to 9.0
  • the osmotic pressure is 300 to 800 sm/kg ⁇ H 2 O.
  • the preservative is at least one selected from the group consisting of azide, carson, and aminoglycoside antibiotics;
  • the stabilizer is at least one selected from imidazolidinyl ureas, preferably containing at least one carson Preservatives and at least one aminoglycoside antibiotic.
  • the combination of the above preservatives and stabilizers is more conducive to the long-term stability of the red blood cell simulated particles prepared by the present invention.
  • azides examples are sodium azide.
  • casin antibacterial agents examples include proclin series, such as proclin300, proclin950 and so on.
  • aminoglycoside antibiotics examples include streptomycin sulfate, kanamycin sulfate, neomycin sulfate, and the like.
  • imidazolidinyl ureas examples include imidazolidinyl urea, bisimidazolidinyl urea and the like.
  • the preservatives of the present invention also include buffers, and optional osmotic pressure regulators. Further, the preservative may further include glucose.
  • the buffering agent and osmotic pressure adjusting agent are as described above.
  • the preservative of the present invention contributes to the stable storage of blood cell simulation particles including red blood cell simulation particles.
  • the method of preparing red blood cell simulation particles of the present invention further includes the step of spheroidizing the red blood cells before processing the red blood cells with the (first) red blood cell treatment solution.
  • the step of spheroidizing treatment can be carried out using a suitable surfactant.
  • the type and concentration of the surfactant are not particularly limited, as long as the surfactant and the appropriate concentration thereof can spheroidize red blood cells without damaging the cells, and can be used in the present invention.
  • Particularly preferred are alkyl quaternary ammonium cationic surfactants or alkyl sulfonic acid anionic surfactants, but are not limited thereto.
  • the present invention can still obtain erythrocyte simulated particles with desired performance without performing spheroidization treatment.
  • the erythrocyte simulated particles obtained by the above method can be used as a quality control or calibrator in a blood cell analyzer.
  • the quality control substance or calibrator is used to simulate a human blood sample, and monitor and calibrate the red blood cell detection result of the analyzer.
  • the quality control or calibrator used in the blood cell analyzer usually also contains at least one of white blood cell simulation particles, platelet simulation particles, and nucleated red blood cell simulation particles.
  • step (3) Wash the red blood cells treated in step (2) three times with physiological saline, and then suspend the red blood cells using a storage solution with a pH of 8.0 and an osmotic pressure of 350 Osm/kg.H 2 O to obtain red blood cell simulated particles. Store at 8°C.
  • red blood cells treated in step (2) are washed three times with sodium citrate buffer, and then the red blood cells are suspended using the above storage solution to obtain red blood cell simulated particles, which are stored at 2-8°C.
  • Example 3 Treatment with about 0.25g/L sodium bromate
  • red blood cells treated in step (2) are washed three times with PBS buffer, and then the red blood cells are suspended using the above storage solution to obtain red blood cell simulated particles, which are stored at 2-8°C.
  • the red blood cell simulated particles in Example 3 were continuously tested for MCV on a blood cell analyzer for 18 weeks, and not less than twice a week, and the average value of MCV was taken.
  • the test results are shown in Figure 4.
  • the range (maximum change) of the MCV during the entire detection period was measured to be 1.1 fl, and the SD (standard deviation) was 0.32.
  • Example 4 Treatment with about 0.5g/L sodium bromate
  • red blood cell-containing blood sample and the above-mentioned red blood cell treatment solution A were mixed at a volume ratio of 1:1, mixed, and left at room temperature for 1 hour to oxidize the red blood cells.
  • red blood cells treated in step (4) are mixed with the above red blood cell treatment solution B in a volume ratio of 1:1, mixed, and left at room temperature for 2 hours to immobilize the red blood cells, and then Red blood cells are suspended using the above-mentioned storage solution to obtain red blood cell simulated particles, which are stored at a low temperature of 2-8°C.
  • the red blood cell simulated particles in Example 4 were continuously tested for MCV on a hematology analyzer for 18 weeks, and the test was conducted not less than twice a week, and the average value of MCV was taken. The test results are shown in Figure 5.
  • the range (maximum change) of MCV during the entire detection period was 1.0fl, and the SD (standard deviation) was 0.31
  • Example 5 Treatment with about 0.75g/L sodium bromate
  • red blood cells treated in step (3) are washed twice with Tris buffer, and then the red blood cells are suspended using the above-mentioned storage solution to obtain red blood cell simulated particles and stored at 2-8°C.
  • the red blood cell simulated particles in Example 5 were continuously tested for MCV on a blood cell analyzer for 18 weeks, and not less than twice a week, and the average value of MCV was taken.
  • the test results are shown in Figure 6.
  • the range (maximum change) of MCV during the entire detection period was measured to be 1.3fl, and the SD (standard deviation) was 0.33.
  • Example 6 Treatment with about 1g/L sodium bromate
  • red blood cells treated in step (3) are washed twice or more with HEPES buffer, and then the red blood cells are suspended using the above storage solution to obtain red blood cell simulated particles, which are stored at 2-8°C.
  • the red blood cell simulated particles in Example 6 were continuously tested for MCV on a hematology analyzer for 18 weeks, and not less than twice a week, and the average value of MCV was taken.
  • the test results are shown in Figure 7.
  • the range (maximum change) of the MCV during the entire detection period was measured to be 1.1fl, and the SD (standard deviation) was 0.31.
  • Example 7 treatment with about 0.5g/L sodium chlorate
  • red blood cells treated in step (3) are washed twice or more with HEPES buffer, and then the red blood cells are suspended using the above storage solution to obtain red blood cell simulated particles and stored at 2-8°C.
  • the red blood cell simulated particles in Example 7 were continuously tested for MCV on a hematology analyzer for 18 weeks, and not less than twice a week, and the average value of MCV was taken.
  • the test results are shown in Figure 8.
  • the range (maximum change) of the MCV during the entire detection period was measured to be 1.6fl, and the SD (standard deviation) was 0.45.
  • Example 8 Treatment with about 0.75g/L sodium chlorate
  • red blood cells treated in step (3) are washed twice or more with PBS buffer, and then the red blood cells are suspended using the above storage solution to obtain red blood cell simulated particles, which are stored at 2-8°C.
  • the red blood cell simulated particles in Example 8 were continuously tested for MCV on a hematology analyzer for 18 weeks, and not less than twice a week, and the average value of MCV was taken.
  • the test results are shown in Figure 9.
  • the range (maximum change) of the MCV during the entire detection period was measured to be 2.0fl, and the SD (standard deviation) was 0.58.
  • Example 9 Treatment with about 1g/L sodium chlorate
  • red blood cells treated in step (3) are washed twice or more with PBS buffer, and then the red blood cells are suspended using the above storage solution to obtain red blood cell simulated particles, which are stored at 2-8°C.
  • the red blood cell simulated particles in Example 9 were continuously tested for MCV on a hematology analyzer for 18 weeks, and not less than twice a week, and the average value of MCV was taken.
  • the test results are shown in Figure 10.
  • the range (maximum change) of the MCV during the entire detection period was measured to be 2.1fl, and the SD (standard deviation) was 0.66.
  • Example 10 Treatment with about 2g/L sodium chlorate
  • Example 7 (2) Prepare the preservation solution according to the formula of Example 7. (3) Take 20 ml of human anticoagulated blood, filter and centrifuge to remove white blood cells and platelets, wash twice with PBS buffer, discard the supernatant, and retain the packed blood cells to obtain blood samples containing red blood cells. The blood sample containing red blood cells and the above-mentioned red blood cell treatment solution were mixed at a volume ratio of 1:1, mixed, and left at room temperature for 3 hours to oxidize and immobilize the red blood cells.
  • red blood cells treated in step (3) are washed twice or more with HEPES buffer, and then the red blood cells are suspended using the above storage solution to obtain red blood cell simulated particles and stored at 2-8°C.
  • the red blood cell simulated particles in Example 10 were continuously tested for MCV on a hematology analyzer for 18 weeks, and the test was conducted not less than twice a week, and the average value of MCV was taken.
  • the test results are shown in Figure 11.
  • the range (maximum change) of MCV during the entire detection period was measured to be 3.3fl, and the SD (standard deviation) was 0.97.
  • the white blood cell simulated particles, platelet simulated particles and red blood cell simulated particles suspended in the storage solution prepared in Example 6 were mixed uniformly at a certain ratio to prepare a blood cell analysis quality control substance and stored at a low temperature of 2-8°C.
  • the leukocyte simulation particles and platelet simulation particles can be commercial products, or they can be prepared by conventional or known methods. Here, they can be Mindray BR60/B55/B30 and Mindray BR60/B55/
  • the platelet simulated particles of B30 are preferably replaced with the same storage solution of erythrocyte simulated particles by washing and centrifuging.
  • the above blood cell analysis quality control substance is continuously tested for MCV on the blood cell analyzer for 18 weeks, and the test is performed not less than twice a week, and the average value of MCV is taken.
  • the test results are shown in Figure 12.
  • the range (maximum change) of the MCV during the entire detection period was measured to be 1.3fL, and the SD was 0.32.
  • Fig. 13(a) is a two-dimensional fluorescence-scattered light scattergram (a1) and RBC histogram (a2) of WBC obtained from a fresh blood sample on a hematology analyzer;
  • FIG. 13(a) is a two-dimensional fluorescence-scattered light scattergram (a1) and RBC histogram (a2) of WBC obtained from a fresh blood sample on a hematology analyzer;
  • 13(b) is the blood of this embodiment
  • the two-dimensional fluorescence-scattered light scattergram of WBC on the blood cell analyzer of the cell analysis quality control materials including white blood cell simulated particles, platelet simulated particles and red blood cell simulated particles suspended in the storage solution prepared in Example 6) b1) and RBC histogram (b2).
  • results show that the simulated red blood cell particles prepared by this method have a volume similar to that of fresh red blood cells and hemolysis similar to that of fresh red blood cells, and do not affect the classification of white blood cells on the diff classification map.
  • quality control substance containing the erythrocyte simulated particles prepared by the method of the present application performs similarly to the fresh blood on the blood analyzer.

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Abstract

一种红细胞模拟粒子、其制备方法及含有该红细胞模拟粒子的质控物或校准物。制备红细胞模拟粒子的方法包括采用pH为5.0～9.0、渗透压为300～800 Osm/kg•H 2O的红细胞处理液对红细胞进行氧化处理和固定处理，其中，该红细胞处理液含有选自卤素含氧酸盐的至少一种氧化剂和选自醛或酸的至少一种固定剂；和将经该红细胞处理液处理后的红细胞洗涤并悬浮在保存液中。该方法采用环境友好的卤素含氧酸盐作为氧化剂，获得了能够长期稳定保存的且在红细胞检测通道中与天然血液红细胞粒子性质类似的红细胞模拟粒子。

Description

红细胞模拟粒子、其制备方法及含其的质控物或校准物 技术领域
本发明涉及用于血液细胞分析仪的红细胞模拟粒子、其制备方法以及包含这样的红细胞模拟粒子的质控物或校准物。
背景技术
血液细胞分析仪是用于对血液样本进行包括红细胞参数在内的全自动生化分析设备。为了有效对分析仪的红细胞检测结果进行监控和校准，需要能够有效模拟红细胞特征的红细胞模拟粒子来制备质控物和标准物。
常规方法制备红细胞模拟粒子是采用醛固定的方法，存在细胞体积随时间产生变化的问题。本申请人提出了使用重铬酸盐与醛类联合对红细胞球进行固定以使其稳定的方法。然而，重铬酸盐的使用会导致环境污染问题。
基于对减轻环境污染问题越来越高的要求，有必要提供环保的红细胞模拟粒子的制备方法，同时还能够使所制备的红细胞模拟粒子在形态、体积等方面在有效期内保持稳定。
发明内容
针对现有技术中的问题，本发明提供了一种制备红细胞模拟粒子的方法，并由此提供一种由此制备的红细胞模拟粒子和包含该红细胞模拟粒子的质控物/校准物。本发明的方法及由此获得的红细胞模拟粒子和质控物/校准物解决了至少一个上述问题。
在本发明的第一方面中，根据第一实施方式，提供一种制备红细胞模拟粒子的方法，所述方法包括：
采用pH为5.0～9.0、渗透压为300～800sm/kg·H 2O的红细胞处理液对红细胞进行氧化处理和固定处理，其中，所述红细胞处理液含有选自卤素含氧酸盐的至少一种氧化剂和选自醛或酸的至少一种固定剂；和
将经所述红细胞处理液处理后的红细胞洗涤并悬浮在保存液中。
本发明的所述至少一种氧化剂选自由氯、溴和碘的次卤酸盐、亚卤酸盐、卤酸盐和高卤酸盐所组成的组。优选地，所述至少一种氧化剂选自由次氯酸、亚氯酸、氯酸、高氯酸、溴酸、碘酸和高碘酸的钠盐和钾盐所组成的组。进一步优选，所述至少一种氧化剂选自由高氯酸钠、高氯酸钾、溴酸钠、溴酸钾、氯酸钠和氯酸钾所组成的组。
本发明的所述至少一种氧化剂在所述红细胞处理液中的浓度为0.01～20g/L，优选为0.05～10g/L，更优选为0.05～5g/L。
根据一种具体实施方式，所述至少一种氧化剂为高氯酸盐、尤其是高氯酸钠并且在所述红细胞处理液中的浓度为8～20g/L，优选为8～10g/L。
根据另一种具体实施方式，所述至少一种氧化剂为溴酸盐、尤其是溴酸钠并且在所述红细胞处理液中的浓度为0.25～2g/L、优选为0.25～1g/L，更优选为0.25～0.5g/L。
根据再一种具体实施方式，所述至少一种氧化剂为氯酸盐、尤其是氯酸钠并且在所述红细胞处理液中的浓度为0.5～2g/L、优选为0.5～1g/L。
根据本发明，所述至少一种固定剂选自由甲醛、戊二醛、乙二醛、丙酮醛、对三氟甲基苯甲醛、多聚甲醛、铬酸、苦味酸、单宁酸和醋酸所组成的组。
本发明中，所述至少一种固定剂在所述红细胞处理液中的体积浓度为0.01～0.5vol％。
该实施方式中，所述红细胞处理液还包含必要的缓冲剂，和可选的渗透压调节剂。进一步地，所述红细胞处理液还可包含防腐剂。
根据一种具体实施方式，本发明的方法中，在用所述红细胞处理液进行处理的步骤，所述红细胞处理液与待处理的红细胞以1～2:1的比例混合，以进行所述处理。
根据第二实施方式，本发明还提供一种制备红细胞模拟粒子的方法，所述方法包括：
采用pH为5.0～9.0、渗透压为300～800sm/kg·H 2O的第一红细胞处理液对红细胞进行氧化处理，其中，所述第一红细胞处理液含有选自卤素含氧酸盐的至少一种氧化剂；
采用pH为5.0～9.0、渗透压为300～800sm/kg·H 2O的第二红细胞处理液对红细胞进行固定处理，其中，所述第二红细胞处理液含有选自醛或酸的至少一种固定剂；和
将经所述第一红细胞处理液处理和所述第二细胞处理液后的红细胞洗涤并悬浮在保存液中。
同样的，所述至少一种氧化剂选自由氯、溴和碘的次卤酸盐、亚卤酸盐、卤酸盐和高卤酸盐所组成的组。优选地，所述至少一种氧化剂选自由次氯酸、亚氯酸、氯酸、高氯酸、溴酸、碘酸和高碘酸的钠盐和钾盐所组成的组。进一步优选，所述至少一种氧化剂选自由高氯酸钠、高氯酸钾、溴酸钠、溴酸钾、氯酸钠和氯酸钾所组成的组。
在该实施方式中，所述至少一种氧化剂在所述第一红细胞处理液中的浓度也同样为0.01～20g/L，优选为0.05～10g/L，更优选为0.05～5g/L。
在一种具体实施方式中，所述至少一种氧化剂为高氯酸盐、尤其是高氯酸钠并且在所述第一红细胞处理液中的浓度为8～20g/L，优选为8～10g/L。
在另一种具体实施方式中，所述至少一种氧化剂为溴酸盐、尤其是溴酸钠并且在所述第一红细胞处理液中的浓度为0.25～2g/L、优选为0.25～1g/L，更优选0.25～0.5g/L。
在再一种具体实施方式中，所述至少一种氧化剂为氯酸盐、尤其是氯酸钠并且在所述第一红细胞处理液中的浓度为0.5～2g/L、优选为0.5～1g/L。
同样所述至少一种固定剂选自由甲醛、戊二醛、乙二醛、丙酮醛、对三氟甲基苯甲醛、多聚甲醛、铬酸、苦味酸、单宁酸和醋酸所组成的组。
所述至少一种固定剂在所述第二红细胞处理液中的体积浓度为0.01～0.5vol％。
在该实施方式中，进行所述氧化处理后，用缓冲液洗涤经氧化处理的红细胞，然后进行所述固定处理。
该实施方式中，所述第一和第二红细胞处理液还包含必要的缓冲剂，和可选的渗透压调节剂。进一步地，所述第一和/或第二红细胞处理液可进一步包含防腐剂。
根据一种具体的实施方式，在用所述第一红细胞处理液进行处理步骤中，用所述第一红细胞处理液与待处理的红细胞以1～2:1的比例混合以进行氧化处理。在用所述第二红细胞处理液进行处理步骤中，用所述第二红细胞处理液与所述待处理的红细胞以1～2:1的比例混合以进行固定化处理。
在本发明的第一和第二实施方式中，所述保存液的pH为5.0～9.0、渗透压为300～800sm/kg·H 2O，且包括0.01～10g/L的防腐剂和0.01～5g/L的稳定剂。
在一种具体实施方式中，所述防腐剂为选自叠氮化合物、卡松类和氨基糖苷类抗生素中的至少一种，能够抑制红细胞模拟粒子中的微生物污染且不影响红细胞模拟粒子稳定性。所述稳定剂为选自咪唑烷基脲类中的至少一种，有助于长期保持红细胞模拟粒子的稳定性。
本发明的保存剂还包括缓冲剂以及可选的渗透压调节剂。进一步地，所述保存剂还可包括葡萄糖。
在本发明的上述两种实施方式中，本发明制备红细胞模拟粒子的方法 进一步包括，在用所述(第一)红细胞处理液对红细胞进行处理之前，对所述红细胞进行球形化处理的步骤。
本发明制备红细胞模拟粒子的方法中所用的红细胞来自哺乳动物。优选来自人类或红细胞体积与人类接近的哺乳动物，例如猴或猪。
本发明的第二方面，提供一种红细胞模拟粒子。所述红细胞模拟粒子由上述第一方面中的第一实施方式和第二实施方式中的任何一种方法制备得到。
本发明的第三方面，提供一种用于血液细胞分析仪的质控物或校准物，其中包括本发明的红细胞模拟粒子。
具体地，所述质控物或校准物还含有白细胞模拟粒子、血小板模拟粒子和有核红细胞模拟粒子中的至少一种。
根据本发明的制备红细胞模拟粒子的方法，采用环境友好的卤素含氧酸盐，使膜蛋白构象改变，从而加强了细胞膜骨架的稳定性，并可同时稳定细胞内的血红蛋白。另外，本发明使用醛类或酸类固定剂对细胞进行固定处理，进一步维持细胞的稳定性，从而获得在红细胞检测通道中与天然血液红细胞粒子性质类似的红细胞模拟粒子。本发明的方法不需对红细胞进行球形化处理就能获得较好的(例如在细胞形态和体积方面的)粒子稳定性，并避免使用不环保的重金属。此外，将根据本发明方法制得的红细胞模拟粒子保存在含有防腐剂和咪唑烷基脲类稳定剂的保存液中，有助于红细胞模拟粒子的长期稳定。
附图说明
图1是根据对比例1制备的红细胞模拟粒子在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图2是根据实施例1制备的红细胞模拟粒子在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图3是根据实施例2制备的红细胞模拟粒子在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图4是根据实施例3制备的红细胞模拟粒子在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图5是根据实施例4制备的红细胞模拟粒子在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图6是根据实施例5制备的红细胞模拟粒子在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图7是根据实施例6制备的红细胞模拟粒子在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图8是根据实施例7制备的红细胞模拟粒子在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图9是根据实施例8制备的红细胞模拟粒子在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图10是根据实施例9制备的红细胞模拟粒子在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图11是根据实施例10制备的红细胞模拟粒子在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图12是根据实施例11制备的质控物在18周内连续测定的平均红细胞体积随时间的变化曲线图。
图13是新鲜采集的人的抗凝血(a1和a2)及根据实施例11制备的质控物(b1和b2)在同一血液细胞分析仪上检测的白细胞(WBC)的散射光-荧光二维散点图(a1和b1)以及红细胞(RBC)的直方图(a2和b2)。
具体实施方式
下面将结合本发明具体实施方式和实施例，对本发明实施方式中的技 术方案进行清楚、完整地描述，显然，所描述的具有实施方式仅仅是本发明的一部分实施方式，而不是全部的实施方式。基于本发明中的实施方式，本领域普通技术人员在没有作出创造性劳动前提下所获得的所有其他实施方式，都属于本发明保护的范围。
在整个说明书中，除非另有特别说明，本文使用的术语应理解为如本领域中通常所使用的含义。因此，除非另有定义，本文使用的所有技术和科学术语具有与本发明所属领域技术人员的一般理解相同的含义。若存在矛盾，本说明书优先。
在文中，术语“包括”、“包含”或者其任何其他变体意在涵盖非排他性的包含，从而使得包括一系列要素的方法或者产品不仅包括所明确记载的要素，而且还包括没有明确列出的其他要素，或者还包括为实施所述方法或者产品所固有的要素。
除非另有说明，否则如在本文件所使用的单数形式“一个/种(a/an)”和“该/所述”包括所指名词的复数。
本发明为了获得一种能够以环保的方法制备出能够长期保持稳定的，尤其是体积稳定的红细胞模拟粒子，提供一种制备红细胞模拟粒子的方法。本发明的方法是采用卤素的含氧酸盐对天然来源的红细胞进行氧化处理，并进一步用固定化剂进行固定化处理，从而获得了稳定的红细胞模拟粒子。
因细胞中含有较大量的蛋白质，为制备更稳定的红细胞模拟粒子，本发明人认为，除了需要进行球形化处理外，还需要进行变性处理使细胞膜骨架蛋白和血红蛋白构象改变。而常用的蛋白质变性剂为强酸、强碱、含重金属的盐、尿素、丙酮等，各类变性剂其作用原理不尽相同，但重金属盐对于增加细胞模拟粒子的稳定性有着显著的效果，然而重金属对环境有严重的污染作用。根据越来越严格的环境保护要求，需要更为环境友好的试剂对重金属盐进行替换，本发明人在大量的实验中发现，卤素的含氧酸 盐通过对红细胞中膜蛋白(或者还包括血红蛋白)由变性作用，在同时进行固定处理的情况下，甚至不需要进行球形化处理，同样能够获得在血液细胞分析仪中行为与新鲜血液样本类似的、且稳定红细胞粒子，从而得到本发明。
与现有技术中使用的重金属含氧酸盐(诸如重铬酸盐或铬酸盐)相比，本发明的氧化剂避免使用对环境造成污染的重金属，是一种更为环境友好的试剂。而且经本发明的优选实施方式处理后的红细胞粒子甚至具有更为稳定的体积。
因此，本发明的制备红细胞模拟粒子的方法包括：
采用pH为5.0～9.0、渗透压为300～800sm/kg·H 2O的红细胞处理液对红细胞进行氧化处理和固定处理，其中，所述红细胞处理液含有选自卤素含氧酸盐的至少一种氧化剂和选自醛或酸的至少一种固定剂；和
将经所述红细胞处理液处理后的红细胞洗涤并悬浮在保存液中。
或者，作为替代方案，本发明的制备红细胞模拟粒子的方法包括：
采用pH为5.0～9.0、渗透压为300～800sm/kg·H 2O的第一红细胞处理液对红细胞进行氧化处理，其中，所述第一红细胞处理液含有选自卤素含氧酸盐的至少一种氧化剂；
采用pH为5.0～9.0、渗透压为300～800sm/kg·H 2O的第二红细胞处理液对红细胞进行固定处理，其中，所述第二红细胞处理液含有选自醛或酸的至少一种固定剂；和
将经所述第一红细胞处理液处理和所述第二细胞处理液后的红细胞洗涤并悬浮在保存液中。
即，根据本发明的方法，所述氧化处理和所述固定处理可以在一个溶液中同时进行，也可先进行氧化处理，再进行固定处理
本发明的上述两种方案中，卤素含氧酸盐可为氯、溴或碘的次卤酸盐、 亚卤酸盐、卤酸盐或高卤酸盐，特别是碱金属盐。
优选地，本发明的卤素含氧酸盐可选自次氯酸钠、次氯酸钾、亚氯酸钠、亚氯酸钾、氯酸钠、氯酸钾、高氯酸钠、高氯酸钾、溴酸钠、溴酸钾、碘酸钠、碘酸钾、高碘酸钠和高碘酸钾。进一步优选的是高氯酸钠、高氯酸钾、溴酸钠、溴酸钾、氯酸钠和/或氯酸钾。最优选的是溴酸盐，特别是溴酸钠。
本发明的所述至少一种氧化剂在所述红细胞处理液中的浓度为0.01～20g/L，优选为0.025～10g/L，更优选为0.025～5g/L，进一步优选为0.025～2g/L。
由于不同氧化剂的氧化能力有一些差异，因此为获得所需效果，本领域技术人员可以根据具体情况确定不同氧化剂的适宜浓度范围。
较佳地，氧化剂可为，例如，高氯酸盐、尤其是高氯酸钠，其在红细胞处理液中的浓度可为8～20g/L，优选为8～10g/L；或者溴酸盐、尤其是溴酸钠，其在红细胞处理液中的浓度为0.25～2g/L、优选为0.25～1g/L，更优选为0.25～0.5g/L；或者，氯酸盐、尤其是氯酸钠，其在红细胞处理液中的浓度为0.5～2g/L、优选为0.5～1g/L。
溴酸盐、尤其是溴酸钠是本发明优选的。因为采用溴酸盐的处理浓度低，且在与下述固定剂的配合下能够获得更为稳定的(特别是体积稳定)红细胞模拟粒子。根据以下具体实施例可知，采用不同浓度的溴酸盐对红细胞粒子进行处理得到的模拟粒子，在18周的连续测定实验中，体积变化仅为约1飞升。
本发明的上述两种方案中，固定剂可为醛或酸。例如，固定剂可选自由甲醛、戊二醛、乙二醛、丙酮醛、对三氟甲基苯甲醛、多聚甲醛、铬酸、苦味酸、单宁酸和醋酸所组成的组中的一种或多种。
本发明的上述两种方案中，固定剂在处理液(对于第一种方案为红细 胞处理液，对于第二种方案为第二红细胞处理液)中的体积浓度均可在0.01～0.5vol％的范围内。
用于本发明方法的两种方案的红细胞处理液还包含必要的缓冲剂，和可选的渗透压调节剂，以获得合适的pH值和渗透压。
本发明中，缓冲剂没有特别限制，可以是任何适于红细胞的缓冲剂。可以列举包括：柠檬酸钠、Tris、PBS、HEPES等，但不限于此。同样的，渗透压调节剂也没有特别限制。适宜的渗透压调节剂包括：氯化钠、磷酸二氢钠、氯化钾、柠檬酸钠等，但不限于此。
选择缓冲剂和渗透压调节剂的浓度，使本发明的红细胞处理液的渗透压在300～800sm/kg·H 2O的范围内，pH值在5.0～9.0的范围内。
进一步地，本发明的红细胞处理液还可包含防腐剂，例如叠氮化物(如叠氮化钠)和卡松类，如proclin系列(如proclin300，proclin950)等，但不限于此。防腐剂的用量通常为0.01～10g/L。
在本发明的上述两种方案中，在用本发明红细胞处理液(如果是分开进行氧化处理和固化处理，则是第一和第二红细胞处理液)进行处理的步骤，(第一/第二)红细胞处理液与待处理的红细胞均可以约1～2:1的比例混合，以进行相应的处理。处理时间通常为1～3小时。
根据本发明的第二方案的制备方法中，进行所述氧化处理后，用缓冲液洗涤经氧化处理的红细胞，然后进行所述固定处理。
此外，在第一方案中，在进行了氧化-固定处理后，或在第二方案中，在进行了固定处理后，均可用缓冲液洗涤经氧化-固定处理的红细胞，然后再将洗涤后的红细胞悬浮在保存液中。
用于洗涤的缓冲剂也没有特别限制，可以列举的有柠檬酸钠缓冲液、Tris缓冲液、PBS缓冲液或HEPES缓冲液。所述缓冲液也同样应具有5.0～9.0的pH值和300～800sm/kg·H 2O的渗透压。
在本发明的第一和第二方案中，同样的，保存液的pH为5.0～9.0、渗透压为300～800sm/kg·H 2O。
根据一种实例，保存液包括0.01～10g/L的防腐剂和0.01～5g/L的稳定剂。
具体地，防腐剂为选自叠氮化物、卡松类和氨基糖苷类抗生素中的至少一种；稳定剂为选自咪唑烷基脲类中的至少一种，优选包含至少一种卡松类防腐剂和至少一种氨基糖苷类抗生素。采用上述防腐剂和稳定剂的组合更加有利于本发明制备的红细胞模拟粒子的长期稳定。
叠氮化物的实例如叠氮化钠。
卡松类抑菌剂的实例有proclin系列，如proclin300，proclin950等。
氨基糖苷类抗生素的实例有硫酸链霉素、硫酸卡那霉素、硫酸新霉素等。
咪唑烷基脲类的实例有咪唑烷基脲、双咪唑烷基脲等。
本发明的保存剂还包括缓冲剂，以及可选的渗透压调节剂。进一步地，所述保存剂还可包括葡萄糖。
其中缓冲剂、渗透压调节剂如前文所述。
本发明的保存剂有助于包括红细胞模拟粒子的血细胞模拟粒子的稳定保存。
在本发明的上述两种方案中，本发明制备红细胞模拟粒子的方法进一步包括，在用所述(第一)红细胞处理液对红细胞进行处理之前，对所述红细胞进行球形化处理的步骤。
所述球形化处理的步骤可采用适宜的表面活性剂来进行。本发明对表面活性剂的种类及浓度没有特别限制，只要能够使红细胞球形化且不会破坏细胞的表面活性剂及其适宜浓度均可用于本发明。特别优选烷基季铵类阳离子表面活性剂或烷基磺酸类阴离子表面活性剂，但不限于此。
具体的表面活性剂及处理方法可采用CN105717312A中公开的那些表面活性剂及处理方法。CN105717312A全文通过引用合并于本文。
红细胞球形化进一步有助于在后续处理或长期放置中细胞形态和体积保持稳定。
当然，如前所述，本发明在不进行球形化处理的情况下，仍能得到所需性能的红细胞模拟粒子。
本发明制备红细胞模拟粒子的方法中所用的红细胞为来自哺乳动物的天然红细胞，优选来自人类或红细胞体积与人类接近的哺乳动物，例如猴或猪，其中人红细胞是更优选的。
由上述方法获得的红细胞模拟粒子可用于血液细胞分析仪的质控物或校准物中。所述质控物或校准物用于模拟人类的血液样本，对分析仪的红细胞检测结果进行监控和校准。用于血液细胞分析仪的质控物或校准物通常还含有白细胞模拟粒子、血小板模拟粒子和有核红细胞模拟粒子中的至少一种。
以下通过具体实施例来进一步说明本发明的优点。
对比例1：重金属离子处理
按以下步骤制备红细胞模拟粒子：
(1)按下表配制红细胞处理液。
Figure PCTCN2018123529-appb-000001
(2)取人抗凝血20ml使用生理盐水洗涤2次以上，离心至上清液中无过多悬浮细胞，弃去上清液，得到含有红细胞的血液样本。将该含有红 细胞的血液样本与上述红细胞处理液按照体积比为1:1的比例混合，混匀后放置于室温下静置1小时，对红细胞进行氧化和固定化处理。
(3)将经步骤(2)处理后的红细胞使用生理盐水洗涤3次，然后使用pH为8.0，渗透压为350Osm/kg.H 2O的保存液悬浮红细胞，得到红细胞模拟粒子，于2-8℃保存。
其中，保存液按下表配制。
序号 组分名称 组分量
1 葡萄糖 10g
2 Proclin 300 3ml
3 硫酸链霉素 0.2g
4 硫酸卡那霉素 0.2g
5 咪唑烷基脲 0.3g
6 HEPES缓冲液 1L
将对比例1中红细胞模拟粒子在血细胞分析仪上连续进行18周的平均红细胞体积(MCV)检测，每周检测不少于2次，取MCV的平均值。检测结果如图1所示。测得整个检测期间的MCV的极差(变化最大值)为1.6fl，SD(标准偏差)为0.42。
实施例1：用约8g/L高氯酸钠处理
按以下步骤制备红细胞模拟粒子：
(1)按下表配制红细胞处理液。
Figure PCTCN2018123529-appb-000002
(2)按下表配制保存液。
Figure PCTCN2018123529-appb-000003
Figure PCTCN2018123529-appb-000004
(3)取人抗凝血20ml进行过滤、离心去除白细胞和血小板，用HEPES缓冲液洗涤2次，弃去上清液，保留压积红细胞，得到含有红细胞的血液样本。将该含有红细胞的血液样本与上述红细胞处理液按照体积比为1:1的比例混合，混匀后放置于室温下静置3小时，对红细胞进行氧化和固定化处理。
(4)将经步骤(2)处理后的红细胞使用HEPES缓冲液洗涤3次，然后使用上述保存液悬浮红细胞，得到红细胞模拟粒子，于2-8℃保存。
将实施例1中红细胞模拟粒子在血细胞分析仪上连续进行18周的MCV检测，每周检测不少于2次，取MCV的平均值。检测结果如图2所示。测得整个检测期间的MCV的极差(变化最大值)为1.4fl，SD(标准偏差)为0.46。
实施例2：用约10g/L高氯酸钠处理
按以下步骤制备红细胞模拟粒子：
(1)按下表配制红细胞处理液。
Figure PCTCN2018123529-appb-000005
(2)按实施例1的配方配制保存液。
(3)取人抗凝血20ml进行过滤、离心去除白细胞和血小板，用柠檬酸钠缓冲液洗涤2次，弃去上清液，保留压积红细胞，得到含有红细胞的血液样本。将该含有红细胞的血液样本与上述红细胞处理液按照体积比为1:1的比例混合，混匀后放置于室温下静置2小时，对红细胞进行氧化和固定化处理。
(4)将经步骤(2)处理后的红细胞使用柠檬酸钠缓冲液洗涤3次，然后使用上述保存液悬浮红细胞，得到红细胞模拟粒子，于2-8℃保存。
将实施例2中红细胞模拟粒子在血细胞分析仪上连续进行18周的MCV检测，每周检测不少于2次，取MCV的平均值。检测结果如图3所示。测得整个检测期间的MCV的极差(变化最大值)为1.3fl，SD(标准偏差)为0.38。
实施例3：用约0.25g/L溴酸钠处理
按以下步骤制备红细胞模拟粒子：
(1)按下表配制红细胞处理液。
Figure PCTCN2018123529-appb-000006
(2)按下表配制保存液。
Figure PCTCN2018123529-appb-000007
Figure PCTCN2018123529-appb-000008
(3)取人抗凝血20ml进行过滤、离心去除白细胞和血小板，用PBS缓冲液洗涤2次，弃去上清液，保留压积红细胞，得到含有红细胞的血液样本。将该含有红细胞的血液样本与上述红细胞处理液按照体积比为1:1的比例混合，混匀后放置于室温下静置1小时，对红细胞进行氧化和固定化处理。
(4)将经步骤(2)处理后的红细胞使用PBS缓冲液洗涤3次，然后使用上述保存液悬浮红细胞，得到红细胞模拟粒子，于2-8℃保存。
将实施例3中红细胞模拟粒子在血细胞分析仪上连续进行18周的MCV检测，每周检测不少于2次，取MCV的平均值。检测结果如图4所示。测得整个检测期间的MCV的极差(变化最大值)为1.1fl，SD(标准偏差)为0.32。
实施例4：用约0.5g/L溴酸钠处理
按以下步骤制备红细胞模拟粒子：
(1)按下表配制红细胞处理液A和B。
Figure PCTCN2018123529-appb-000009
(2)按实施例3的配方配制保存液。
(3)取人抗凝血20ml进行过滤、离心去除白细胞和血小板，用Tris缓冲液洗涤2次，弃去上清液，保留压积红细胞，得到含有红细胞的血液样本。将该含有红细胞的血液样本与上述红细胞处理液A按照体积比为1:1的比例混合，混匀后放置于室温下静置1小时，对红细胞进行氧化处理。
(4)将经步骤(3)处理后的红细胞使用Tris缓冲液洗涤2次以上，然后再用Tris缓冲液悬浮。
(5)将经步骤(4)处理后的红细胞与上述红细胞处理液B按照体积比为1:1的比例混合，混匀后放置于室温下静置2小时，对红细胞进固定化处理，然后使用上述保存液悬浮红细胞，，得到红细胞模拟粒子，于2-8℃低温保存。
将实施例4中红细胞模拟粒子在血细胞分析仪上连续进行18周的MCV检测，每周检测不少于2次，取MCV的平均值。检测结果如图5所示。测得整个检测期间的MCV的极差(变化最大值)为1.0fl，SD(标准偏差)为0.31
实施例5：用约0.75g/L溴酸钠处理
按以下步骤制备红细胞模拟粒子：
(1)按下表配制红细胞处理液。
Figure PCTCN2018123529-appb-000010
(2)按实施例3的配方配制保存液。
(3)取人抗凝血20ml进行过滤、离心去除白细胞和血小板，用Tris缓冲液洗涤2次，弃去上清液，保留压积红细胞，得到含有红细胞的血液样本。将该含有红细胞的血液样本与上述红细胞处理液按照体积比为1:1的比例混合，混匀后放置于室温下静置2小时，对红细胞进行氧化和固定化处理。
(4)将经步骤(3)处理后的红细胞使用Tris缓冲液洗涤2次以上，然后使用上述保存液悬浮红细胞，得到红细胞模拟粒子，于2-8℃保存。
将实施例5中红细胞模拟粒子在血细胞分析仪上连续进行18周的MCV检测，每周检测不少于2次，取MCV的平均值。检测结果如图6所示。测得整个检测期间的MCV的极差(变化最大值)为1.3fl，SD(标准偏差)为0.33。
实施例6：用约1g/L溴酸钠处理
按以下步骤制备红细胞模拟粒子：
(1)按下表配制红细胞处理液。
Figure PCTCN2018123529-appb-000011
(2)按实施例3的配方配制保存液。
(3)取人抗凝血20ml进行过滤、离心去除白细胞和血小板，用HEPES缓冲液洗涤2次，弃去上清液，保留压积红细胞，得到含有红细胞的血液样本。将该含有红细胞的血液样本与上述红细胞处理液按照体积比为1:1的比例混合，混匀后放置于室温下静置2小时，对红细胞进行氧化和固定化处理。
(4)将经步骤(3)处理后的红细胞使用HEPES缓冲液洗涤2次以上，然后使用上述保存液悬浮红细胞，，得到红细胞模拟粒子，于2-8℃保存。
将实施例6中红细胞模拟粒子在血细胞分析仪上连续进行18周的MCV检测，每周检测不少于2次，取MCV的平均值。检测结果如图7所示。测得整个检测期间的MCV的极差(变化最大值)为1.1fl，SD(标准偏差)为0.31。
实施例7：用约0.5g/L氯酸钠处理
按以下步骤制备红细胞模拟粒子：
(1)按下表配制红细胞处理液。
Figure PCTCN2018123529-appb-000012
(2)按下表配制保存液。
Figure PCTCN2018123529-appb-000013
(3)取人抗凝血20ml进行过滤、离心去除白细胞和血小板，用HEPES缓冲液洗涤2次，弃去上清液，保留压积红细胞，得到含有红细胞的血液样本。将该含有红细胞的血液样本与上述红细胞处理液按照体积比为1:1 的比例混合，混匀后放置于室温下静置3小时，对红细胞进行氧化和固定化处理。
(4)将经步骤(3)处理后的红细胞使用HEPES缓冲液洗涤2次以上，然后使用上述保存液悬浮红细胞，得到红细胞模拟粒子，于2-8℃保存。
将实施例7中红细胞模拟粒子在血细胞分析仪上连续进行18周的MCV检测，每周检测不少于2次，取MCV的平均值。检测结果如图8所示。测得整个检测期间的MCV的极差(变化最大值)为1.6fl，SD(标准偏差)为0.45。
实施例8：用约0.75g/L氯酸钠处理
按以下步骤制备红细胞模拟粒子：
(1)按下表配制红细胞处理液。
Figure PCTCN2018123529-appb-000014
(2)按实施例7的配方配制保存液。
(3)取人抗凝血20ml进行过滤、离心去除白细胞和血小板，用PBS缓冲液洗涤2次，弃去上清液，保留压积红细胞，得到含有红细胞的血液样本。将该含有红细胞的血液样本与上述红细胞处理液按照体积比为1:1的比例混合，混匀后放置于室温下静置3小时，对红细胞进行氧化和固定化处理。
(4)将经步骤(3)处理后的红细胞使用PBS缓冲液洗涤2次以上，然后使用上述保存液悬浮红细胞，得到红细胞模拟粒子，于2-8℃保存。
将实施例8中红细胞模拟粒子在血细胞分析仪上连续进行18周的MCV检测，每周检测不少于2次，取MCV的平均值。检测结果如图9所示。测得整个检测期间的MCV的极差(变化最大值)为2.0fl，SD(标准偏差)为0.58。
实施例9：用约1g/L氯酸钠处理
按以下步骤制备红细胞模拟粒子：
(1)按下表配制红细胞处理液。
Figure PCTCN2018123529-appb-000015
(2)按实施例7的配方配制保存液。
(3)取人抗凝血20ml进行过滤、离心去除白细胞和血小板，用PBS缓冲液洗涤2次，弃去上清液，保留压积红细胞，得到含有红细胞的血液样本。将该含有红细胞的血液样本与上述红细胞处理液按照体积比为1:1的比例混合，混匀后放置于室温下静置3小时，对红细胞进行氧化固定化处理。
(4)将经步骤(3)处理后的红细胞使用PBS缓冲液洗涤2次以上，然后使用上述保存液悬浮红细胞，得到红细胞模拟粒子，于2-8℃保存。
将实施例9中红细胞模拟粒子在血细胞分析仪上连续进行18周的MCV检测，每周检测不少于2次，取MCV的平均值。检测结果如图10所示。测得整个检测期间的MCV的极差(变化最大值)为2.1fl，SD(标准偏差)为0.66。
实施例10：用约2g/L氯酸钠处理
按以下步骤制备红细胞模拟粒子：
(1)按下表配制红细胞处理液。
Figure PCTCN2018123529-appb-000016
(2)按实施例7的配方配制保存液。(3)取人抗凝血20ml进行过滤、离心去除白细胞和血小板，用PBS缓冲液洗涤2次，弃去上清液，保留压积红细胞，得到含有红细胞的血液样本。将该含有红细胞的血液样本与上述红细胞处理液按照体积比为1:1的比例混合，混匀后放置于室温下静置3小时，对红细胞进行氧化和固定化处理。
(4)将经步骤(3)处理后的红细胞使用HEPES缓冲液洗涤2次以上，然后使用上述保存液悬浮红细胞，得到红细胞模拟粒子，于2-8℃保存。
将实施例10中红细胞模拟粒子在血细胞分析仪上连续进行18周的MCV检测，每周检测不少于2次，取MCV的平均值。检测结果如图11所示。测得整个检测期间的MCV的极差(变化最大值)为3.3fl，SD(标准偏差)为0.97。
实施例11：血液细胞分析质控物
按以下方法制备血液细胞分析质控物：
将白细胞模拟粒子、血小板模拟粒子及实施例6制得的悬浮在保存液中的红细胞模拟粒子按一定比例混合均匀，制成血液细胞分析质控物，在2～8℃低温保存。这里的白细胞模拟粒子和血小板模拟粒子可以用商品化的产品，也可以是用常规或已知方法制备的模拟物，在此可以为迈瑞 BR60/B55/B30的白细胞模拟粒子和迈瑞BR60/B55/B30的血小板模拟粒子，优选通过洗涤离心方式置换为红细胞模拟粒子相同的保存液。
将上述血液细胞分析质控物在血液细胞分析仪上的连续进行18周的MCV检测，每周检测不少于2次，取MCV的平均值。检测结果如图12所示。测得整个检测期间的MCV的极差(变化最大值)为1.3fL，SD为0.32。
进一步将本实施例的质控品与人新鲜抗凝血作为分析样本，分别注入血液细胞分析仪(迈瑞BC6系列血液分析仪)，进行检测，结果如图13所示。图13(a)为新鲜血液样本在血细胞分析仪上获得的WBC的荧光-散射光二维散点图(a1)和RBC的直方图(a2)；图13(b)为本实施例的血液细胞分析质控物(包括白细胞模拟粒子、血小板模拟粒子及实施例6制得的悬浮在保存液中的红细胞模拟粒子)在血液细胞分析仪上的WBC的荧光-散射光二维散点图(b1)和RBC的直方图(b2)。
结果表明，本方法制备的红细胞模拟粒子具有和新鲜血红细胞类似的体积，也具有和新鲜血红细胞类似的溶血性，在diff分类图上不影响白细胞的分类。这说明含本申请方法制备的红细胞模拟粒子的质控物与新鲜血相比，在血液分析仪上表现类似。
以上所述仅为本发明的部分实施方式的实例，并非因此限制本发明的专利范围，凡是在本发明的发明构思下，利用本发明说明书及附图内容所作的等效结构变换，或直接/间接运用在其他相关的技术领域均包括在本发明的专利保护范围内。

Claims (20)

  1. 一种制备红细胞模拟粒子的方法，所述方法包括：
    采用pH为5.0～9.0、渗透压为300～800sm/kg·H 2O的红细胞处理液对红细胞进行氧化处理和固定处理，其中，所述红细胞处理液含有选自卤素含氧酸盐的至少一种氧化剂和选自醛或酸的至少一种固定剂；和
    将经所述红细胞处理液处理后的红细胞洗涤并悬浮在保存液中。
  2. 根据权利要求1所述的方法，其中所述至少一种氧化剂选自由氯、溴和碘的次卤酸盐、亚卤酸盐、卤酸盐和高卤酸盐所组成的组；优选地，所述至少一种氧化剂选自由次氯酸、亚氯酸、氯酸、高氯酸、溴酸、碘酸和高碘酸的钠盐和钾盐所组成的组；进一步优选，所述至少一种氧化剂选自由高氯酸钠、高氯酸钾、溴酸钠、溴酸钾、氯酸钠和氯酸钾所组成的组。
  3. 根据权利要求1或2所述的方法，其中所述至少一种氧化剂在所述红细胞处理液中的浓度为0.01～20g/L，优选为0.05～10g/L，更优选为0.05～5g/L。
  4. 根据权利要求3所述的方法，其中所述至少一种氧化剂为高氯酸盐、尤其是高氯酸钠并且在所述红细胞处理液中的浓度为8～20g/L，优选为8～10g/L；或者所述至少一种氧化剂为溴酸盐、尤其是溴酸钠并且在所述红细胞处理液中的浓度为0.25～2g/L、优选为0.25～1g/L，更优选为0.25～0.5g/L；或者所述至少一种氧化剂为氯酸盐、尤其是氯酸钠并且在所述红细胞处理液中的浓度为0.5～2g/L、优选为0.5～1g/L。
  5. 根据权利要求1～4中任一项所述的方法，其中所述至少一种固定剂选自由甲醛、戊二醛、乙二醛、丙酮醛、对三氟甲基苯甲醛、多聚甲醛、铬酸、苦味酸、单宁酸和醋酸所组成的组。
  6. 根据权利要求1～5中任一项所述的方法，其中所述至少一种固定剂在所述红细胞处理液中的体积浓度为0.01～0.5vol％。
  7. 根据权利要求1～6中任一项所述的方法，其中所述方法进一步包括：在用所述红细胞处理液对红细胞进行氧化处理和固定处理之前，对所述红细胞进行球形化处理。
  8. 一种制备红细胞模拟粒子的方法，所述方法包括：
    采用pH为5.0～9.0、渗透压为300～800sm/kg·H 2O的第一红细胞处理液对红细胞进行氧化处理，其中，所述第一红细胞处理液含有选自卤素含氧酸盐的至少一种氧化剂；
    采用pH为5.0～9.0、渗透压为300～800sm/kg·H 2O的第二红细胞处理液对红细胞进行固定处理，其中，所述第二红细胞处理液含有选自醛或酸的至少一种固定剂；和
    将经所述第一红细胞处理液处理和所述第二细胞处理液后的红细胞洗涤并悬浮在保存液中。
  9. 根据权利要求8所述的方法，其中所述至少一种氧化剂选自由氯、溴和碘的次卤酸盐、亚卤酸盐、卤酸盐和高卤酸盐所组成的组；优选地，所述至少一种氧化剂选自由次氯酸、亚氯酸、氯酸、高氯酸、溴酸、碘酸和高碘酸的钠盐和钾盐所组成的组；进一步优选，所述至少一种氧化剂选自由高氯酸钠、高氯酸钾、溴酸钠、溴酸钾、氯酸钠和氯酸钾所组成的组。
  10. 根据权利要求8或9所述的方法，其中所述至少一种氧化剂在所述第一红细胞处理液中的浓度为0.01～20g/L，更优选为0.05～10g/L，优选为0.05～5g/L。
  11. 根据权利要求10所述的方法，其中所述至少一种氧化剂为高氯酸盐、尤其是高氯酸钠并且在所述第一红细胞处理液中的浓度为8～20g/L，优选为8～10g/L；或者所述至少一种氧化剂为溴酸盐、尤其是溴酸钠并且在所述第一红细胞处理液中的浓度为0.25～2g/L、优选为0.25～1g/L，更优选0.25～0.5g/L；或者所述至少一种氧化剂为氯酸盐、尤其是氯酸钠并且在所 述第一红细胞处理液中的浓度为0.5～2g/L、优选为0.5～1g/L。
  12. 根据权利要求8～11中任一项所述的方法，其中所述至少一种固定剂选自由甲醛、戊二醛、乙二醛、丙酮醛、对三氟甲基苯甲醛、多聚甲醛、铬酸、苦味酸、单宁酸和醋酸所组成的组。
  13. 根据权利要求8～12中任一项所述的方法，其中所述至少一种固定剂在所述第二红细胞处理液中的体积浓度为0.01～0.5vol％。
  14. 根据权利要求8～13中任一项所述的方法，其中进行所述氧化处理后，用缓冲液洗涤经氧化处理的红细胞，然后进行所述固定处理。
  15. 根据权利要求8～14中任一项所述的方法，其中所述方法进一步包括：在用所述第一红细胞处理液对红细胞进行氧化处理之前，对所述红细胞进行球形化处理。
  16. 根据权利要求1至15所述的方法，其中所述保存液的pH为5.0～9.0、渗透压为300～8000sm/kg·H 2O且包括0.01～10g/L的防腐剂和0.01～5g/L的稳定剂，
    优选地，所述防腐剂为选自卡松类和氨基糖苷类抗生素中的至少一种，所述稳定剂为选自咪唑烷基脲类中的至少一种；
    更优选，所述防腐剂为至少一种卡松类防腐剂和至少一种氨基糖苷类抗生素。
  17. 根据权利要求1～16中任一项所述的方法，其中所述红细胞来自哺乳动物，优选来自人类或红细胞体积与人类接近的哺乳动物，例如猴或猪。
  18. 一种红细胞模拟粒子，由根据权利要求1～17中任一项所述的方法制备得到。
  19. 一种用于血液细胞分析仪的质控物或校准物，包括根据权利要求18所述的红细胞模拟粒子。
  20. 根据权利要求19所述的质控物或校准物，其中，所述质控物或校 准物还含有白细胞模拟粒子、血小板模拟粒子和有核红细胞模拟粒子中的至少一种。
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