WO2020132906A1 - 血小板模拟粒子及其制备方法以及含该模拟粒子的质控物或校准物 - Google Patents

血小板模拟粒子及其制备方法以及含该模拟粒子的质控物或校准物 Download PDF

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WO2020132906A1
WO2020132906A1 PCT/CN2018/123638 CN2018123638W WO2020132906A1 WO 2020132906 A1 WO2020132906 A1 WO 2020132906A1 CN 2018123638 W CN2018123638 W CN 2018123638W WO 2020132906 A1 WO2020132906 A1 WO 2020132906A1
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red blood
blood cells
platelet
particles
animal red
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PCT/CN2018/123638
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English (en)
French (fr)
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孙思祥
谢键
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深圳迈瑞生物医疗电子股份有限公司
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Priority to CN201880097228.3A priority Critical patent/CN112639467B/zh
Priority to PCT/CN2018/123638 priority patent/WO2020132906A1/zh
Publication of WO2020132906A1 publication Critical patent/WO2020132906A1/zh

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    • 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
    • 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

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  • the invention relates to the field of blood quality control calibrators, in particular to platelet simulation particles and preparation methods thereof, and quality control substances or calibrators containing the platelet simulation particles.
  • the blood cell analyzer is used to detect and analyze different types of cells (such as platelets, white blood cells, red blood cells, and reticulocytes) present in the blood sample.
  • the detection and analysis function is based on the electrical and/or optical properties of each type of cell Achieved.
  • a blood quality control substance is a liquid containing single or multiple components of blood cells or blood cell simulation particles, and has the same detectable properties as blood.
  • the stability of platelets is enhanced by using polyethylene glycol (abbreviation PEG), but the disadvantages of this preparation method are: 1) When platelets are suspended in suspension and leave the blood vessels It will rupture, which will lead to the release of prothrombinogen kinase and lead to blood coagulation; 2) The platelet simulation particles prepared by this method are easy to activate and trigger the aggregation effect, resulting in the failure of quality control products; 3) The difficulty and low efficiency of human platelet extraction 4) Commercial sources of human-derived platelets are expensive.
  • PEG polyethylene glycol
  • animal red blood cells In the method of using animal red blood cells to prepare platelet simulated particles, if animal red blood cells can be combined with a fluorescent dye to generate a fluorescent signal, the animal red blood cells need to be processed to show that they are carried by nucleic acids due to the low content of animal red blood cells. Come on display in the direction of fluorescence.
  • the platelet-simulated particles prepared by this method can be well exhibited in the direction of fluorescence.
  • the platelet simulation particles obtained by this method have a large gap with real human platelet particles and can easily be mixed with side noise and red blood cell simulation particle fragments on a scattergram, which affects the counting accuracy and the stability of quality control substances.
  • the existing platelet simulation particles are difficult to be completely dissolved by the hemolytic agent of the white blood cell grouping, so that the signal generated in the white blood cell grouping channel is too large, resulting in a sharp increase in the number of counting particles generated by the white blood cell grouping channel. Larger, the sampling time is shortened, which in turn affects the accuracy of the white blood cell cluster measurement results.
  • the platelet-simulated particles prepared by this method have good stability and suitable fixing strength.
  • the present invention provides a new method for preparing platelet-simulating particles, platelet-simulating particles prepared by the method, and a quality control substance or calibrator containing the platelet-simulating particles.
  • the present invention provides a method for preparing platelet mimic particles, in which animal red blood cells are modified with trifluoromethyl compounds to make platelet mimic particles capable of binding fluorescent dyes.
  • the volume size of the animal red blood cells may preferably be 2fL to 25fL.
  • the present invention uses trifluoromethyl compounds to modify animal red blood cells, so that the modified animal red blood cells can be simply and effectively combined with the fluorescent dye molecules in the hematology analyzer. Therefore, trifluoromethyl compounds Platelet mimic particles made from modified animal red blood cells can produce a fluorescent signal in the reticulocyte red channel (also called RET channel) that is similar to real human platelets. Compared with the prior art, the trifluoromethyl compound used for the modification treatment is easy to obtain and low in cost, and the operation of using the trifluoromethyl compound for the modification treatment is simple.
  • the trifluoromethyl-based compound of the present invention is an aldehyde substituted with at least one trifluoromethyl group and/or an acid substituted with at least one trifluoromethyl group.
  • R 1 and R 2 are each H, C 1 -C 4 alkyl, C 6 -C 10 aryl, C 7 -C 10 phenylalkyl and C 7 -C 10 alkylphenyl.
  • the trifluoromethyl compound of the present invention may be selected from one or more of the group consisting of p-trifluoromethylbenzaldehyde, 2,4-ditrifluoromethylbenzaldehyde , P-trifluoromethylbenzoic acid, trifluoroacetic acid and trifluoroacetaldehyde.
  • the reaction concentration of the trifluoromethyl compound used in the present invention may be about 0.1 v/v% to 1 v/v%, and preferably may be about 0.2 v/v% to 0.6 v/v% For example, it may be about 0.25v/v% or 0.5v/v%.
  • the duration of the modification treatment using the trifluoromethyl compound can be about 5 min to 40 min, preferably about 15 min to 30 min.
  • the method for preparing platelet mimetic particles according to the first aspect of the present invention may further include: fixing the animal red blood cells using a fixing agent.
  • the order of the fixing process and the modification process is not particularly limited.
  • the animal red blood cells can be fixed first, and then the fixed animal red blood cells can be modified by using trifluoromethyl compounds; or, the animal red blood cells can be modified by using trifluoromethyl compounds.
  • the modified animal red blood cells are fixed.
  • fixative there is no particular limitation on the type of fixative, and any common fixative for fixing platelet-simulating particles can be used.
  • exemplary fixatives may be, for example, one or more fixatives selected from the group consisting of paraformaldehyde, formaldehyde, acrolein, malondialdehyde, glutaraldehyde, adipaldehyde, methanol, ethanol, and acetic acid.
  • an aldehyde fixative may be used to fix animal red blood cells, such as one or more of paraformaldehyde, formaldehyde, acrolein, malondialdehyde, glutaraldehyde, and adipic aldehyde.
  • the aldehyde fixative includes glutaraldehyde.
  • the concentration of the fixing agent there is no particular limitation on the concentration of the fixing agent, as long as a conventional fixing effect can be achieved.
  • the method for preparing platelet-simulating particles of the present invention may further include fixing animal cells using tannic acid (also called tannin).
  • tannic acid also called tannin
  • the platelet-mimetic particles prepared by this embodiment can hemolyze under the action of a stronger hemolytic reagent (DIFF channel hemolytic agent) and weaker hemolytic reagent (RET channel diluent) is stable under the action, and has good storage stability.
  • aldehydes and tannic acid in the fixing process is not particularly limited in the present invention.
  • tannic acid can be used to fix animal red blood cells first, and then aldehyde compounds can be used to fix animal red blood cells; or, after aldehydes are used to fix animal red blood cells, tannic acid can be used to fix animal red blood cells. Fixed processing.
  • the reaction concentration of the aldehydes of the present invention may be about 0.0075 v/v% to 0.025 v/v%, and preferably may be about 0.01 v/v% to 0.0125 v/v%.
  • the reaction concentration of the aldehydes used in the present invention may be about 0.0075v/v%, 0.01v/v%, 0.0125v/v%, 0.015v/v%, 0.0175v/v%, 0.02v/v% Or 0.0225v/v%.
  • the reaction concentration of the tannic acid of the present invention may be about 5 mg/L to 75 mg/L, preferably about 15 mg/L to 37.5 mg/L, and more preferably about 15 mg/L ⁇ 25mg/L.
  • the reaction concentration of tannic acid used in the present invention may also be about 20 mg/L, 30 mg/L, 40 mg/L, 50 mg/L, 60 mg/L, or 70 mg/L.
  • the source of animal red blood cells is not particularly limited in the present invention, and any animal-derived red blood cells that can be used to prepare platelet-mimicking particles can be used in the present invention.
  • the animal red blood cells are red blood cells derived from mammals, especially one or more of goat red blood cells and sheep red blood cells.
  • the present invention provides a method for preparing platelet mimic particles, wherein tannic acid and aldehydes are used to fix animal red blood cells to make platelet mimic particles.
  • the volume size of the animal red blood cells may preferably be 2fL to 25fL.
  • platelet simulating particles are prepared by fixing animal erythrocytes with tannic acid and aldehydes. As demonstrated in the following examples, after this fixation treatment, the prepared platelet mimetic particles can hemolyze under the action of a stronger hemolytic reagent (DIFF channel hemolytic agent), while in a weaker hemolytic reagent (RET channel diluent) ) It is stable under the action and has good storage stability.
  • a stronger hemolytic reagent DIFF channel hemolytic agent
  • RET channel diluent a weaker hemolytic reagent
  • aldehydes and tannic acid during fixation treatment is not particularly limited in the present invention.
  • tannic acid may be used to fix animal red blood cells first, and then aldehyde compounds may be used to fix animal red blood cells; or After fixing animal red blood cells with aldehydes, fix animal red blood cells with tannic acid.
  • the animal red blood cells are fixed using tannic acid first, and then the animal red blood cells are fixed using aldehyde compounds.
  • the aldehyde fixative is selected from one or more of the following: paraformaldehyde, formaldehyde, acrolein, malondialdehyde, glutaraldehyde, and adipic aldehyde.
  • the aldehyde fixative includes glutaraldehyde.
  • the reaction concentration of the aldehydes of the present invention may be about 0.0075 v/v% to 0.025 v/v%, and preferably may be about 0.01 v/v% to 0.0125 v/v%.
  • the reaction concentration of the aldehydes of the present invention may be about 0.0075v/v%, 0.01v/v%, 0.0125v/v%, 0.015v/v%, 0.0175v/v%, 0.02v/v% or 0.0225 v/v%.
  • the reaction concentration of tannic acid used in the present invention may be about 5 mg/L to 75 mg/L, preferably about 15 mg/L to 37.5 mg/L, and more preferably about 15 mg /L ⁇ 25mg/L.
  • the reaction concentration of tannic acid used in the present invention may also be about 20 mg/L, 30 mg/L, 40 mg/L, 50 mg/L, 60 mg/L, or 70 mg/L.
  • reaction concentration refers to the final concentration of the treatment agent when it reacts with animal red blood cells during processing of animal red blood cells.
  • the method for preparing simulated particles according to the second aspect of the present invention further includes: modifying the animal red blood cells so that the animal red blood cells can generate a fluorescent signal in the platelet detection channel.
  • modification treatment may be performed first, and then erythrocytes are fixed using tannic acid and aldehydes; or modification treatment may be performed after erythrocytes are fixed using tannic acid and aldehydes.
  • the step of modifying the animal red blood cells so that the animal red blood cells can generate a fluorescent signal in the platelet detection channel includes: modifying the animal red blood cells using trifluoromethyl compounds to So that the animal red blood cells can be combined with a fluorescent dye; or the fluorescent red dye is used to stain the animal red blood cells.
  • the prepared platelet simulated particles exhibited better in the fluorescence direction.
  • the use of trifluoromethyl compounds for modification is simpler and less costly.
  • the animal red blood cells may be modified by using a substance that specifically binds to the dye, such as injecting nucleic acid into the animal red blood cells.
  • the trifluoromethyl compound is an aldehyde substituted with at least one trifluoromethyl group and/or an acid substituted with at least one trifluoromethyl group.
  • R 1 and R 2 are each H, C 1 -C 4 alkyl, C 6 -C 10 aryl, C 7 -C 10 phenylalkyl or C 7 -C 10 alkylphenyl.
  • the trifluoromethyl compound of the present invention may be selected from one or more of the group consisting of p-trifluoromethylbenzaldehyde, 2,4-ditrifluoromethylbenzaldehyde, P-trifluoromethylbenzoic acid, trifluoroacetic acid and trifluoroacetaldehyde.
  • the reaction concentration of the trifluoromethyl compound of the present invention may be about 0.1 v/v% to 1 v/v%, and preferably may be about 0.2 v/v% to 0.6 v/v%, for example It may be about 0.25v/v% or 0.5v/v%.
  • the duration of the modification treatment using the trifluoromethyl compound may be about 5 min to 40 min, and preferably may be about 15 min to 30 min.
  • a fluorescent dye when used to stain the erythrocytes, those skilled in the art can determine the dye used to stain animal red blood cells for the fluorescent dye used in the staining treatment.
  • An exemplary dye may be an N-hydroxysuccinimide activated protein fluorescent dye, but the present invention is not limited thereto.
  • Exemplary staining steps may include: using N-hydroxysuccinimide to activate the protein fluorescent dye to activate the carboxyl group of the protein fluorescent dye; incubating the activated protein fluorescent dye with animal red blood cells to stain the animal red blood cells to obtain staining Red blood cells.
  • the source of animal red blood cells is not particularly limited in the present invention, and any animal-derived red blood cells that can be used to prepare platelet-mimicking particles can be used in the present invention.
  • the animal red blood cells are red blood cells derived from mammals, especially one or more of goat red blood cells and sheep red blood cells.
  • the method according to the present invention further comprises washing the modified and fixed animal red blood cells.
  • the conventional buffer solution is used for washing, such as a mixed solution containing citric acid and sodium citrate, or a mixed solution of citric acid, sodium citrate and sodium chloride, which is not specifically limited herein.
  • the simulated platelet particles prepared by the present invention can be further spheroidized to meet different usage requirements.
  • the specific steps of the spheroidization process can be integrated into the preparation method of the present invention, and are not specifically limited herein.
  • the present invention provides a platelet mimic particle prepared by the method according to the first or second aspect of the present invention, wherein the platelet particle contains reticulated platelet mimic particles.
  • the present invention provides a quality control substance or calibrator for a hematology analyzer, wherein the quality control substance or calibrator contains the platelet simulation particles of the present invention.
  • quality control substance refers to a suspension synthesized to ensure the reliability of the blood cell analyzer, which has certain physical and chemical properties similar to blood, and includes the size and traits that are present in human blood Cells or granules that are very close to each other.
  • the quality control or calibrator of the present invention further includes at least one of white blood cell simulated particles, red blood cell simulated particles, and nucleated red blood cell simulated particles.
  • the concentration of components such as red blood cell simulated particles, platelet simulated particles, white blood cell simulated particles, and nucleated red blood cell simulated particles can be adjusted according to different usage requirements to prepare Low-value, medium-value, and high-value quality control substances or calibrators are not specifically limited here.
  • 1A is a fluorescent-forward scattered light scattergram obtained by detecting goat red blood cells without modification treatment with p-trifluoromethylbenzaldehyde in the RET channel of a blood cell analyzer in Example 1;
  • 1B is a fluorescence-forward scattered light scatter diagram obtained by detecting platelet simulated particles prepared according to the method steps of Example 1 in the RET channel of a blood cell analyzer;
  • 2A is a fluorescence-forward scattered light scattergram obtained by detecting goat red blood cells without modification treatment with p-trifluoromethylbenzaldehyde in the RET channel of a blood cell analyzer in Example 2;
  • 2B is a fluorescence-forward scattered light scattergram obtained by detecting platelet simulated particles prepared according to the method steps of Example 2 in the RET channel of the blood cell analyzer;
  • 3A is a fluorescence-forward scattered light scattergram obtained by detecting goat red blood cells without modification treatment with p-trifluoromethylbenzaldehyde in the RET channel of a blood cell analyzer in Example 3;
  • 3B is a fluorescence-forward scattered light scattergram obtained by detecting the platelet simulated particles prepared according to the method steps of Example 3 in the RET channel of the blood cell analyzer;
  • 4A is a fluorescent-forward scattered light scattergram obtained by detecting goat red blood cells not modified with 2,4-ditrifluoromethylbenzaldehyde in the RET channel of a blood cell analyzer in Example 4;
  • 4B is a fluorescence-forward scattered light scattergram obtained by detecting platelet simulated particles prepared according to the method steps of Example 4 in the RET channel of the blood cell analyzer;
  • 5A is a fluorescence-forward scattered light scattergram obtained by detecting goat red blood cells without modification treatment with p-trifluoromethylbenzoic acid in the RET channel of a blood cell analyzer in Example 5;
  • 5B is a fluorescence-forward scattered light scattergram obtained by detecting platelet simulated particles prepared according to the method steps of Example 5 in the RET channel of the blood cell analyzer;
  • FIG. 6A is a side scattered light-fluorescence scattergram obtained by detecting platelet simulated particles prepared after glutaraldehyde fixation with a reaction concentration of 0.0125v/v% in the DIFF channel of a blood cell analyzer;
  • 6B is a fluorescence-forward scattered light scatter diagram obtained by detecting the same platelet simulated particles in the RET channel of the blood cell analyzer as shown in FIG. 6A;
  • FIG. 6C is a side scattered light-fluorescence scatter diagram obtained by detecting platelet simulated particles prepared in a DIFF channel of a blood cell analyzer after being fixed with glutaraldehyde at a reaction concentration of 0.025v/v%;
  • FIG. 6D is a fluorescence-forward scattered light scattergram obtained by detecting the same platelet simulated particles in the RET channel of the blood cell analyzer as shown in FIG. 6C.
  • Fluorescent dye solution Dissolve 5 mg of N-hydroxysuccinimide activated protein fluorescent dye in 650 ⁇ L of dimethyl sulfoxide and prepare a fluorescent dye solution.
  • Sodium citrate buffer contains 10.0g/L trisodium citrate dihydrate, 0.4g/L citric acid monohydrate, 6.875g/L sodium chloride.
  • PBS buffer contains 5.9g/L disodium hydrogen phosphate dodecahydrate, 0.5g/L sodium dihydrogen phosphate dihydrate, 9.0g/L sodium chloride.
  • the preservative solution in each example is a commercially available preservative solution.
  • FIGS. 1A and 1B The Mindray BC-6800 blood cell analyzer was used to detect the platelet simulated particles before treatment and the platelet simulated particles after treatment in Example 1, and the results are shown in FIGS. 1A and 1B.
  • FIG. 1A shows the platelet simulated particles before treatment in this Fluorescence-forward scattered light scatter diagram detected in the RET channel of the hematology analyzer.
  • FIG. 1B is a fluorescence-forward scattered light scatter diagram of the treated platelet simulated particles detected in the RET channel of the hematology analyzer. .
  • platelet simulated particles modified with p-trifluoromethylbenzaldehyde can obtain significantly better fluorescence display in the RET channel.
  • FIG. 2A shows the platelet simulated particles before treatment. Fluorescence-forward scattering light scatter diagram detected in the RET channel of the hematology analyzer.
  • FIG. 2B is the fluorescence-forward scattering light scatter diagram of the treated platelet simulated particles detected in the RET channel of the hematology analyzer. .
  • platelet simulated particles that were fixed with glutaraldehyde and then modified with p-trifluoromethylbenzaldehyde can also obtain significantly better fluorescence display.
  • FIGS. 3A and 3B The Mindray BC-6800 blood cell analyzer was used to detect the platelet simulated particles before treatment and the platelet simulated particles after treatment in Example 3, and the results are shown in FIGS. 3A and 3B.
  • FIG. 3A shows the platelet simulated particles before treatment in this Fluorescence-forward scattering light scatter diagram detected in the RET channel of the hematology analyzer.
  • FIG. 3B is the fluorescence-forward scattering light scatter diagram of the treated platelet simulated particles detected in the RET channel of the hematology analyzer. .
  • platelet simulated particles modified with p-trifluoromethylbenzaldehyde first, and then fixed with tannic acid and glutaraldehyde can also obtain significantly better fluorescence. display.
  • FIGS. 4A and 4B The Mindray BC-6800 blood cell analyzer was used to detect the platelet simulated particles before treatment and the platelet simulated particles after treatment in Example 4, and the results are shown in FIGS. 4A and 4B.
  • FIG. 4A shows the platelet simulated particles before treatment in this Fluorescence-forward scattering light scatter diagram detected in the RET channel of the hematology analyzer.
  • FIG. 4B is the fluorescence-forward scattering light scatter diagram of the treated platelet simulated particles detected in the RET channel of the hematology analyzer. .
  • platelet simulated particles modified with 2,4-ditrifluoromethylbenzaldehyde can obtain significantly better fluorescence display in the RET channel .
  • FIGS. 5A and 5B The Mindray BC-6800 blood cell analyzer was used to detect the platelet simulated particles before treatment and the platelet simulated particles after treatment in Example 5, and the results are shown in FIGS. 5A and 5B.
  • FIG. 5A shows the platelet simulated particles before treatment in this Fluorescence-forward scattered light scatter diagram detected in the RET channel of the hematology analyzer.
  • FIG. 5B is a fluorescence-forward scattered light scatter diagram of the treated platelet simulated particles detected in the RET channel of the hematology analyzer. .
  • platelet simulated particles modified with p-trifluoromethylbenzoic acid exhibited significantly better fluorescence in the RET channel.
  • Scheme A uses tannic acid followed by glutaraldehyde fixation to prepare simulated platelet particles
  • Scheme B Use glutaraldehyde and tannic acid fixation in sequence to prepare simulated platelet particles
  • the processed samples are placed at 30°C, and each sample is periodically counted by a blood cell analyzer every day, and the statistics are counted separately. Changes in the number of RET channel particles for each sample on Day 1, Day 3, Day 4, and Day 7. Among them, the number of RET channel particles on the treatment day (reported as the first day) was recorded as 100%. The results are shown in Table 2.
  • Table 1 and Table 2 respectively show the percentage of the number of RET channel particles of the prepared platelet mimetic particles relative to day 1 after several days of fixation treatment. It can be seen that compared with the solution C using glutaraldehyde fixation alone, the solutions A and B using tannic acid and glutaraldehyde combined fixation treatment have achieved significantly better results in the stability of platelet simulated particles.
  • the preparation method of platelet simulated particles in Scheme A of Example 6 was used, and the reaction concentration of tannic acid was adjusted to investigate the effect of different reaction concentrations of tannic acid on the stability of prepared platelet simulated particles.
  • the processed samples were placed at 30°C, and each sample was periodically counted by a blood cell analyzer to observe changes in the number of particles in the RET channel of each sample. Among them, the number of RET channel particles on the day of treatment (reported as Day 1) is recorded as 100%.
  • the results are shown in Tables 3 and 4, wherein the sources of goat red blood cells in Tables 3 and 4 are different.
  • Table 3 shows the percentage of the number of RET channel particles of the prepared platelet mimetic particles relative to the first day on days 4, 6, and 8 after the treatment. It can be seen that when the reaction concentration of tannic acid is 25mg/L, 37.5mg/L, 50mg/L and 75mg/L, the prepared platelet simulated particles have good stability, of which 25mg/L and 37.5mg The stability effect is better at /L, and the best stability effect is obtained at 25mg/L.
  • Table 4 shows the percentage of the number of RET channel particles of the prepared platelet mimetic particles relative to day 1 on days 4 and 5 after the treatment. It can be seen that when the reaction concentration of tannic acid is 5 mg/L and 15 mg/L, the prepared platelet simulated particles have good stability, and the stability effect is better at 15 mg/L.
  • the present invention preferably uses tannic acid with a reaction concentration of 15 mg/L to 25 mg/L to fix the animal red blood cells.
  • Figures 6A and 6C show the side scattered light-fluorescence scattergrams on the DIFF channel.
  • Figure 6B and Figure 6D show the scattergrams of the fluorescence-forward scattered light on the RET channel.
  • FIG. 6A shows that when glutaraldehyde with a reaction concentration of 0.0125v/v% is used for the fixation treatment, the prepared platelet simulation particles are basically completely hemolyzed in the DIFF channel, which will not affect the white blood cell clustering.
  • 6B shows that the platelet simulated particles are stable on the RET channel; and from FIGS. 6C and 6D, when glutaraldehyde with a reaction concentration of 0.025 v/v% is used for the fixation treatment, although the prepared platelet simulated particles are in the RET The channel is stable, but there is incomplete hemolysis in the DIFF channel, and a large number of blood shadows appear, which may lead to inaccurate detection results of white blood cell clusters.
  • the present invention preferably uses glutaraldehyde with a reaction concentration of less than 0.025 v/v% to fix the animal red blood cells.

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Abstract

一种制备血小板模拟粒子的方法，其中使用三氟甲基类化合物对动物红细胞进行修饰处理，以制成能结合荧光染料的血小板模拟粒子。该方法能使血小板模拟粒子与荧光染料分子简单、有效地结合。还涉及另一种制备血小板模拟粒子的方法，其中使用单宁酸和醛类对动物红细胞进行固定处理，以制成血小板模拟粒子。该方法能提高血小板模拟粒子的稳定性。此外还涉及按照该方法制备的血小板模拟粒子及含有该血小板模拟粒子的质控物或校准物。

Description

血小板模拟粒子及其制备方法以及含该模拟粒子的质控物或校准物 技术领域
本发明涉及血液质控校准物领域，尤其是涉及血小板模拟粒子及其制备方法以及包含该血小板模拟粒子的质控物或校准物。
背景技术
血细胞分析仪用于对血液样品中存在的不同类型细胞(诸如血小板、白细胞、红细胞和网织红细胞等)进行检测分析，其检测分析功能是依据每一种类型细胞的电学和/或光学性质来实现的。
为保证分析结果的准确性和精确性，需要定期用血液质控物对血细胞分析仪进行日常监控。血液质控物是一种含有单一组分或多组分血细胞或血细胞模拟粒子的液体，具备如同血液一样的可检测特性。
在血液质控物所包含的各类血细胞模拟粒子中，血小板模拟粒子的制备所面临的问题比较多。
在以人源血小板为原材料制备血小板模拟粒子的方法中，通过使用聚乙二醇(缩写PEG)增强血小板稳定性，但是该制备方法的缺点在于：1)当血小板悬浮于悬液并离开血管时会发生破裂，从而引起促凝血酶原激酶的释放并导致凝血；2)该方法所制备的血小板模拟粒子易活化进而引发聚集效应，导致质控品失效；3)人血小板提取的困难且低效；4)人源血小板商业来源价格昂贵。
此外，已经开发了采用动物红细胞来制备血小板模拟粒子的方法，例如，US4179398A和US5008201A中使用山羊红细胞来制备血小板模拟粒子，CN1891230A中使用鹰隼血液来制备血小板模拟粒子等。
在使用动物红细胞制备血小板模拟粒子的方法中，如果要使动物红细胞能够与荧光染料结合从而产生荧光信号，由于动物红细胞核酸含量很低而需要对动物红细胞进行处理以使其表现出由核酸所带来的荧光方向上的展出。目前已提出了以下方法：1)采用与荧光染料产生特异性结合的物质对动物红细胞进行处理，例如向动物红细胞内注入核酸，以使其表现出由核酸带来的荧光方向上的展出，但是该方法成本较高且实现难度大；2)预先使用特定的荧光染料、例如N-羟基琥珀酰亚胺活化的蛋白荧光染料对动物红细 胞进行染色处理，使经染色处理的动物红细胞在检测通道中产生荧光信号，但是该方法对该荧光染料有较高的要求，需要预先进行复杂的活化步骤。
因此，亟需一种操作简便的血小板模拟粒子制备方法，该方法所制备的血小板模拟粒子可以实现在荧光方向上的良好展出。
另一方面，在使用动物红细胞来制备血小板模拟粒子的方法中，通常使用甲醛与戊二醛混合溶液对模拟粒子进行固定处理。然而，该方法所获得血小板模拟粒子与真实的人血小板粒子差距较大且易与旁伴噪声及红细胞模拟粒子碎片在散点图上混合，对计数准确性及质控物稳定性产生影响。此外，现有的血小板模拟粒子由于上述固定方式强度过大而导致难以被白细胞分群溶血剂完全溶解，使得在白细胞分群通道中产生的信号过大，致使白细胞分群通道所产生的计数粒子数剧烈增大，采样时间缩短，进而影响了白细胞分群测量结果的准确性。
因此，亟需一种血小板模拟粒子的制备方法，该方法制备的血小板模拟粒子具有良好的稳定性及合适的固定强度。
发明内容
因此，为了解决上述问题，本发明提供了新的制备血小板模拟粒子的方法、由该方法制备的血小板模拟粒子以及包含该血小板模拟粒子的质控物或校准物。
为此，在第一方面，本发明提供了一种制备血小板模拟粒子的方法，其中，使用三氟甲基类化合物对动物红细胞进行修饰处理，以制成能结合荧光染料的血小板模拟粒子。所述动物红细胞的体积大小优选可以为2fL至25fL。
本发明通过使用三氟甲基类化合物对动物红细胞进行修饰处理，使得经修饰处理的动物红细胞能够与血细胞分析仪中的荧光染料分子简单、有效地结合，因此，由经三氟甲基类化合物修饰处理的动物红细胞所制成的血小板模拟粒子能够在网织红通道(也称为RET通道)中产生与真实的人血小板相近的荧光信号。与现有技术相比，用于修饰处理的三氟甲基类化合物容易获得、成本低廉，而且使用三氟甲基类化合物进行修饰处理的操作简单。
在一些的实施方式中，本发明的三氟甲基类化合物为被至少一个三氟甲基取代的醛和/或被至少一个三氟甲基取代的酸。
在具体的实施方式中，被至少一个三氟甲基取代的醛的结构如式I所示：
R 1-CHO       (I)；
被至少一个三氟甲基取代的酸的结构如式II所示：
R 2-COOH     (II)；
其中，R 1和R 2各自为H、C 1-C 4烷基、C 6-C 10芳基、C 7-C 10苯烷基和C 7-C 10烷基苯基。
在一些实施方式中，本发明的三氟甲基类化合物可以选自以下项组成的组中一种或更多种：对三氟甲基苯甲醛、2,4-二三氟甲基苯甲醛、对三氟甲基苯甲酸、三氟乙酸和三氟乙醛。
在具体的实施方式中，本发明所使用的三氟甲基类化合物的反应浓度可以约为0.1v/v％～1v/v％、优选可以约为0.2v/v％～0.6v/v％，例如可以约为0.25v/v％或0.5v/v％。
在具体的实施方式中，使用三氟甲基类化合物进行修饰处理的时长可约为5min～40min、优选约为15min～30min。
进一步地，按照本发明的第一方面的制备血小板模拟粒子的方法可以进一步包括：使用固定剂对动物红细胞进行固定处理。
在此，对于所述固定处理与所述修饰处理的顺序没有特别的限制。例如，可以先对动物红细胞进行固定处理，再使用三氟甲基类化合物对经固定的动物红细胞进行修饰处理；或者，可以在使用三氟甲基类化合物对动物红细胞进行修饰处理之后，再对经修饰的动物红细胞进行固定处理。
根据本发明的第一方面，对于固定剂的种类没有特别的限制，任何用于固定血小板模拟粒子的常见固定剂都可以使用。示例性固定剂例如可以是选自以下项组成的组中一种或多种固定剂：多聚甲醛、甲醛、丙烯醛、丙二醛、戊二醛、己二醛、甲醇、乙醇和乙酸。
在有利的实施方式中，可以使用醛类固定剂对动物红细胞进行固定处理，例如多聚甲醛、甲醛、丙烯醛、丙二醛、戊二醛和己二醛中的一种或更多种。优选的，所述醛类固定剂包括戊二醛。
根据本发明的第一方面，对于固定剂的浓度没有特别的限制，只要能够实现常规的固定效果即可。
更进一步地，当使用醛类对动物红细胞进行固定处理时，本发明的制备血小板模拟粒子的方法还可以包括使用单宁酸(也称为单宁)对动物细胞进行固定处理。在此情况下，与单独使用醛类固定相比，通过该实施方式所制备的血小板模拟粒子能够在较强溶血能力试剂(DIFF通道的溶血剂)作用下溶血，而在较弱溶血能力试剂(RET通道稀 释液)作用下稳定，并且具有较好的保存稳定性。
对于醛类和单宁酸在固定处理时的使用顺序，本发明没有特别的限制。例如，可以先使用单宁酸对动物红细胞进行固定处理，再使用醛类化合物对动物红细胞进行固定处理；或者，在使用醛类对动物红细胞进行固定处理之后，再使用单宁酸对动物红细胞进行固定处理。
在本发明的有利实施方式中，本发明的醛类的反应浓度可以约为0.0075v/v％～0.025v/v％、优选可以约为0.01v/v％～0.0125v/v％。例如，本发明所使用的醛类的反应浓度可以约为0.0075v/v％、0.01v/v％、0.0125v/v％、0.015v/v％、0.0175v/v％、0.02v/v％或0.0225v/v％。
在本发明的有利实施方式中，本发明的单宁酸的反应浓度可以约为5mg/L～75mg/L、优选可以约为15mg/L～37.5mg/L、更优选可以约为15mg/L～25mg/L。例如，本发明所使用的单宁酸的反应浓度还可以约为20mg/L、30mg/L、40mg/L、50mg/L、60mg/L或70mg/L。
对于动物红细胞的来源，本发明没有特别的限制，任何可用于制备血小板模拟粒子的来源于动物的红细胞均可以用于本发明。
在优选的实施方式中，所述动物红细胞是来源于哺乳动物的红细胞，尤其是山羊红细胞和绵羊红细胞中的一种或更多种。
在第二方面，本发明提供了一种制备血小板模拟粒子的方法，其中，使用单宁酸和醛类对动物红细胞进行固定处理，以制成血小板模拟粒子。所述动物红细胞的体积大小优选可以为2fL至25fL。
本发明通过单宁酸和醛类对动物红细胞进行固定处理来制备血小板模拟粒子。如以下实施例中所证实的，经该固定处理后，所制备的血小板模拟粒子能够在较强溶血能力试剂(DIFF通道溶血剂)作用下溶血，而在较弱溶血能力试剂(RET通道稀释液)作用下稳定，并且具有较好的保存稳定性。
对于醛类和单宁酸在固定处理时的使用顺序，本发明没有特别的限制，例如，可以先使用单宁酸对动物红细胞进行固定处理，再使用醛类化合物对动物红细胞进行固定处理；或者，在使用醛类对动物红细胞进行固定处理之后，再使用单宁酸对动物红细胞进行固定处理。优选地，先使用单宁酸对动物红细胞进行固定处理，再使用醛类化合物对动物红细胞进行固定处理。
在具体的实施方式中，醛类固定剂选自以下项中的一种或多种：多聚甲醛、甲醛、 丙烯醛、丙二醛、戊二醛和己二醛。优选的，所述醛类固定剂包括戊二醛。
在本发明的有利实施方式中，本发明的醛类的反应浓度可以约为0.0075v/v％～0.025v/v％、优选可以约为0.01v/v％～0.0125v/v％。例如，本发明的醛类的反应浓度可以约为0.0075v/v％、0.01v/v％、0.0125v/v％、0.015v/v％、0.0175v/v％、0.02v/v％或0.0225v/v％。
在本发明的有利实施方式中，本发明所使用的单宁酸的反应浓度可以约为5mg/L～75mg/L、优选可以约为15mg/L～37.5mg/L、更优选可以约为15mg/L～25mg/L。例如，本发明所使用的单宁酸的反应浓度还可以约为20mg/L、30mg/L、40mg/L、50mg/L、60mg/L或70mg/L。
在此应该说明的是，本文所说的“反应浓度”是指处理剂在动物红细胞处理过程中与动物红细胞反应时的终浓度。
进一步地，按照本发明的第二方面的制备模拟粒子的方法还包括：对动物红细胞进行修饰处理，以使得所述动物红细胞能在血小板检测通道中产生荧光信号。
对于修饰处理与固定处理的顺序没有特别的限制。例如，可以先进行修饰处理，再使用单宁酸和醛类对动物红细胞进行固定处理；或者，可以在使用单宁酸和醛类对动物红细胞进行固定处理之后，再进行修饰处理。
在具体的实施方式中，所述对动物红细胞进行修饰处理，以使得所述动物红细胞能在血小板检测通道中产生荧光信号的步骤包括：使用三氟甲基类化合物对动物红细胞进行修饰处理，以使得所述动物红细胞能结合荧光染料；或者使用荧光染料对所述动物红细胞进行染色处理。在此情况下，与未经修饰处理相比，所制备的血小板模拟粒子在荧光方向上展出更好。与使用荧光染料进行染色处理相比，使用三氟甲基类化合物进行修饰处理的操作更加简单且成本更低。当然，在牺牲成本和操作性的情况下，也可以使用与染料特异性结合的物质对动物红细胞进行修饰处理，例如向动物红细胞内注入核酸。
在一些的实施方式中，所述三氟甲基类化合物为被至少一个三氟甲基取代的醛和/或被至少一个三氟甲基取代的酸。
在具体的实施方式中，被至少一个三氟甲基取代的醛的结构如式I所示：
R 1-CHO       (I)；
被至少一个三氟甲基取代的酸的结构如式II所示：
R 2-COOH     (II)；
其中，R 1和R 2各自为H、C 1-C 4烷基、C 6-C 10芳基、C 7-C 10苯烷基或C 7-C 10烷基苯基。
在一些实施方式中，本发明的三氟甲基类化合物可以选自以下项组成的组中一种或多种：对三氟甲基苯甲醛、2,4-二三氟甲基苯甲醛、对三氟甲基苯甲酸、三氟乙酸和三氟乙醛。
在具体的实施方式中，本发明的三氟甲基类化合物的反应浓度可以约为0.1v/v％～1v/v％、优选可以约为0.2v/v％～0.6v/v％，例如可以约为0.25v/v％或0.5v/v％。
在具体的实施方式中，使用三氟甲基类化合物进行修饰处理的时长可约为5min～40min、优选可以约为15min～30min。
进一步地，当使用荧光染料对动红物细胞进行染色处理时，对于染色处理所使用的荧光染料，本领域技术人员能够确定用于染色动物红细胞的染料。示例性的染料可以是N-羟基琥珀酰亚胺活化的蛋白荧光染料，但本发明不限于此。示例性染色步骤可以包括：使用N-羟基琥珀酰亚胺对蛋白荧光染料进行活化，使蛋白荧光染料的羧基活化；将经活化的蛋白荧光染料与动物红细胞孵育，对动物红细胞进行染色，获得染色的红细胞。
对于动物红细胞的来源，本发明没有特别的限制，任何可用于制备血小板模拟粒子的动物来源的红细胞均可以用于本发明。
在优选的实施方式中，所述动物红细胞是来源于哺乳动物的红细胞，尤其是山羊红细胞和绵羊红细胞中的一种或更多种。
优选地，按照本发明的方法还包括对经过修饰处理和固定处理的动物红细胞进行洗涤。可以理解，洗涤的目的是去除多余或者结合不稳定的修饰物质和固定剂，以免对后续步骤或者对融合质控物的其它组分造成影响。采用常规的缓冲液进行洗涤，如含柠檬酸和柠檬酸钠的混合溶液，或者柠檬酸、柠檬酸钠和氯化钠的混合溶液，在此不做具体限定。
此外可以理解，在本发明制备的血小板模拟粒子基础上，还可以进一步对其进行球形化处理，以满足不同的使用需求。当然，球形化处理的具体步骤可以整合到本发明的制备方法中，在此不做具体限定。
在第三方面，本发明提供了一种血小板模拟粒子，所述血小板模拟粒由本发明的第一方面或第二方面所涉及的方法制备得到，其中所述血小板粒子含有网织血小板模拟粒子。
在第四方面，本发明提供了一种用于血细胞分析仪的质控物或校准物，其中，所述质控物或校准物含有本发明的血小板模拟粒子。
在本发明中，“质控物”是指为确保血液细胞分析仪的可靠性所合成的悬浮液，其具 有与血液相似的某些物理和化学性质，且包含尺寸和性状与人血液中存在的不同类细胞十分接近的细胞或颗粒。
在一些实施方式中，本发明的质控物或校准物还包括白细胞模拟粒子、红细胞模拟粒子和有核红细胞模拟粒子中的至少一种。
需要说明的是，在本发明的质控物或校准物中，可以根据不同的使用需求调整红细胞模拟粒子、血小板模拟粒子、白细胞模拟粒子、有核红细胞模拟粒子等组份的浓度，以配制出低值、中值、高值质控物或校准物，在此不做具体限定。
附图说明
图1A是实施例1中的未经对三氟甲基苯甲醛修饰处理的山羊红细胞在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图；
图1B是按照实施例1的方法步骤制备得到的血小板模拟粒子在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图；
图2A是实施例2中的未经对三氟甲基苯甲醛修饰处理的山羊红细胞在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图；
图2B是按照实施例2的方法步骤制备得到的血小板模拟粒子在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图；
图3A是实施例3中的未经对三氟甲基苯甲醛修饰处理的山羊红细胞在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图；
图3B是按照实施例3的方法步骤制备得到的血小板模拟粒子在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图；
图4A是实施例4中的未经2,4-二三氟甲基苯甲醛修饰处理的山羊红细胞在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图；
图4B是按照实施例4的方法步骤制备得到的血小板模拟粒子在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图；
图5A是实施例5中的未经对三氟甲基苯甲酸修饰处理的山羊红细胞在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图；
图5B是按照实施例5的方法步骤制备得到的血小板模拟粒子在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图；
图6A是经过反应浓度为0.0125v/v％的戊二醛固定处理之后制备得到的血小板模拟 粒子在血液细胞分析仪的DIFF通道内检测得到的侧向散射光-荧光散点图；
图6B是与图6A相同的血小板模拟粒子在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图；
图6C是经反应浓度为0.025v/v％的戊二醛固定处理之后制备得到的血小板模拟粒子在血液细胞分析仪的DIFF通道内检测得到的侧向散射光-荧光散点图；
图6D是与图6C相同的血小板模拟粒子在血液细胞分析仪的RET通道内检测得到的荧光-前向散射光散点图。
具体实施方式
下面将结合本发明实施方式中的附图，对本发明实施方式中的技术方式进行清楚、完整地描述，显然，所描述的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。
细胞处理液的制备：
荧光染料溶液：将5mg的N-羟基琥珀酰亚胺活化的蛋白荧光染料溶于650μL二甲基亚砜，配置成荧光染料溶液。
柠檬酸钠缓冲液：包含10.0g/L二水合柠檬酸三钠、0.4g/L一水合柠檬酸、6.875g/L氯化钠。
PBS缓冲液：包含5.9g/L十二水合磷酸氢二钠、0.5g/L二水合磷酸二氢钠、9.0g/L氯化钠。
各实施例中的保存液为市售常规使用的保存液。
实施例1
血小板模拟粒子的制备：
1)取山羊红细胞，全血过滤，去除白细胞，收集滤后的红细胞(用作处理前的血小板模拟粒子)；
2)使用PBS缓冲液离心洗涤红细胞两次；
3)PBS缓冲液悬浮细胞，加入对三氟甲基苯甲醛至0.5％(v/v)，混匀保持30分钟使对三氟甲基苯甲醛与细胞反应充分，反应结束后，离心去上清，重复洗涤两次；
4)使用保存液悬浮粒子至适当计数，2～8℃冷藏，含有网织血小板的血小板模拟粒子制备完成(用作处理后的血小板模拟粒子)。
使用Mindray BC-6800血球分析仪分别对实施例1中的处理前的血小板模拟粒子和处理后的血小板模拟粒子进行检测，结果参见图1A和图1B，图1A是处理前的血小板模拟粒子在该血球分析仪的RET通道内检测得到的荧光-前向散射光散点图，图1B是处理后的血小板模拟粒子在该血球分析仪的RET通道内检测得到的荧光-前向散射光散点图。与用对三氟甲基苯甲醛修饰处理前相比，经过对三氟甲基苯甲醛修饰处理的血小板模拟粒子在RET通道内能够获得明显更好的荧光展出。
实施例2
血小板模拟粒子的制备：
1)取山羊红细胞，全血过滤，去除白细胞，收集滤后的红细胞(用作处理前的血小板模拟粒子)；
2)使用PBS缓冲液离心洗涤红细胞两次；
3)用PBS缓冲液悬浮细胞，加入50％戊二醛溶液，使其终浓度为0.0125％(v/v)，室温固定2小时，固定结束后，离心去上清，重复洗涤两次；
4)PBS缓冲液悬浮细胞，加入对三氟甲基苯甲醛至0.5％(v/v)，混匀保持15分钟使对三氟甲基苯甲醛与细胞反应充分。反应结束后，离心去上清，重复洗涤两次；
5)使用保存液悬浮粒子至适当计数，2～8℃冷藏，含有网织血小板的血小板模拟粒子制备完成(用作处理后的血小板模拟粒子)。
使用Mindray BC-6800血球分析仪分别对实施例2中的处理前的血小板模拟粒子和处理后的血小板模拟粒子进行检测，结果参见图2A和图2B，图2A是处理前的血小板模拟粒子在该血球分析仪的RET通道内检测得到的荧光-前向散射光散点图，图2B是处理后的血小板模拟粒子在该血球分析仪的RET通道内检测得到的荧光-前向散射光散点图。与用对三氟甲基苯甲醛修饰处理前相比，先经戊二醛固定处理后经对三氟甲基苯甲醛修饰处理的血小板模拟粒子同样能够获得明显更好的荧光展出。
实施例3
血小板模拟粒子的制备：
1)取山羊红细胞，全血过滤，去除白细胞，收集滤后的红细胞(用作处理前的血小板模拟粒子)；
2)使用柠檬酸钠缓冲液离心洗涤红细胞两次；
3)用柠檬酸钠缓冲液悬浮细胞，加入对三氟甲基苯甲醛至0.5％(v/v)，混匀保持15分钟使对三氟甲基苯甲醛与细胞反应充分。反应结束后，离心去上清，再加入柠檬酸钠缓冲液洗涤；
4)用柠檬酸钠缓冲液悬浮细胞并调节至适当浓度，加入单宁酸至15mg/L，室温固定2小时，固定结束后，离心去上清，重复洗涤两次；
5)PBS缓冲液悬浮细胞，加入50％戊二醛溶液，使其终浓度为0.0125％(v/v)，室温固定2小时，固定结束后，离心去上清，重复洗涤两次；
6)使用保存液悬浮粒子至适当计数，2～8℃冷藏，含有网织血小板的血小板模拟粒子制备完成(用作处理后的血小板模拟粒子)。
使用Mindray BC-6800血球分析仪分别对实施例3中的处理前的血小板模拟粒子和处理后的血小板模拟粒子进行检测，结果参见图3A和图3B，图3A是处理前的血小板模拟粒子在该血球分析仪的RET通道内检测得到的荧光-前向散射光散点图，图3B是处理后的血小板模拟粒子在该血球分析仪的RET通道内检测得到的荧光-前向散射光散点图。与用对三氟甲基苯甲醛修饰处理前相比，先经对三氟甲基苯甲醛修饰处理，后经单宁酸、戊二醛固定处理的血小板模拟粒子也能够获得明显更好的荧光展出。
实施例4
血小板模拟粒子的制备：
1)取山羊红细胞，全血过滤，去除白细胞，收集滤后红细胞(用作处理前的血小板模拟粒子)；
2)PBS缓冲液离心洗涤红细胞两次；
3)PBS缓冲液悬浮细胞，加入2,4-二三氟甲基苯甲醛至0.25％(v/v)，混匀保持30分钟使2,4-二三氟甲基苯甲醛与细胞反应充分。反应结束后，离心去上清，重复洗涤两次；
4)保存液悬浮粒子至适当计数，2～8℃冷藏，含有网织血小板的血小板模拟粒子制 备完成(用作处理后的血小板模拟粒子)。
使用Mindray BC-6800血球分析仪分别对实施例4中的处理前的血小板模拟粒子和处理后的血小板模拟粒子进行检测，结果参见图4A和图4B，图4A是处理前的血小板模拟粒子在该血球分析仪的RET通道内检测得到的荧光-前向散射光散点图，图4B是处理后的血小板模拟粒子在该血球分析仪的RET通道内检测得到的荧光-前向散射光散点图。与用2,4-二三氟甲基苯甲醛修饰处理前相比，经过2,4-二三氟甲基苯甲醛修饰处理的血小板模拟粒子在RET通道内能够获得明显更好的荧光展出。
实施例5
血小板模拟粒子的制备：
1)取山羊红细胞，全血过滤，去除白细胞，收集滤后红细胞(用作处理前的血小板模拟粒子)；
2)PBS缓冲液离心洗涤红细胞两次；
3)PBS缓冲液悬浮细胞，加入对三氟甲基苯甲酸至0.25％(v/v)，混匀保持30分钟使对三氟甲基苯甲酸与细胞反应充分。反应结束后，离心去上清，重复洗涤两次；
4)保存液悬浮粒子至适当计数，2～8℃冷藏，含有网织血小板的血小板模拟粒子制备完成(用作处理后的血小板模拟粒子)。
使用Mindray BC-6800血球分析仪分别对实施例5中的处理前的血小板模拟粒子和处理后的血小板模拟粒子进行检测，结果参见图5A和图5B，图5A是处理前的血小板模拟粒子在该血球分析仪的RET通道内检测得到的荧光-前向散射光散点图，图5B是处理后的血小板模拟粒子在该血球分析仪的RET通道内检测得到的荧光-前向散射光散点图。与用对三氟甲基苯甲酸修饰处理前相比，经过对三氟甲基苯甲酸修饰处理的血小板模拟粒子在RET通道内能够获得明显更好的荧光展出。
实施例6
方案A依次使用单宁酸、戊二醛固定来制备模拟血小板粒子
1)取山羊红细胞，全血过滤，去除白细胞，收集滤后的红细胞；
2)使用柠檬酸钠缓冲液离心洗涤红细胞两次；
3)柠檬酸钠缓冲液悬浮细胞，加入荧光染料溶液至0.1％(v/v)，混匀保持5分钟使荧光染料与细胞反应充分。反应结束后，离心去上清，再加入柠檬酸钠缓冲液洗涤；
4)柠檬酸钠缓冲液悬浮细胞并调节至适当浓度，加入单宁酸至15mg/L(v/v)，室温固定2小时，固定结束后，离心去上清，重复洗涤两次；
5)PBS缓冲液悬浮细胞，调整至适当计数后置于2-8℃冰箱冷藏一晚；
6)一晚后将细胞悬浊液复温，离心去上清并在此以PBS缓冲液悬浮细胞；
7)加入50％戊二醛溶液，使其终浓度为0.0125％(v/v)，室温处理2小时，固定结束后，离心去上清，重复洗涤两次；
8)保存液悬浮粒子至适当计数，2-8℃冷藏，含有网织血小板的血小板模拟粒子制备完成。
方案B依次使用戊二醛、单宁酸固定来制备模拟血小板粒子
1)取山羊红细胞，全血过滤，去除白细胞，收集滤后的红细胞；
2)使用柠檬酸钠缓冲液离心洗涤红细胞两次；
3)柠檬酸钠缓冲液悬浮细胞，加入0.1％荧光染料溶液，混匀保持5分钟使荧光染料与细胞反应充分。反应结束后，离心去上清，再加入柠檬酸钠缓冲液洗涤；
4)PBS缓冲液悬浮细胞，加入50％戊二醛溶液，使其终浓度为0.0125％(v/v)，室温处理2小时，固定结束后，离心去上清，重复洗涤两次；
5)柠檬酸钠缓冲液悬浮细胞并调节至适当浓度，加入单宁酸至15mg/L，室温固定2小时，固定结束后，离心去上清，重复洗涤两次；
6)保存液悬浮粒子至适当计数，2-8℃冷藏，含有网织血小板的血小板模拟粒子制备完成。
方案C单独使用戊二醛固定来制备模拟血小板粒子
1)取山羊红细胞，全血过滤，去除白细胞，收集滤后的红细胞；
2)使用柠檬酸钠缓冲液离心洗涤红细胞两次；
3)柠檬酸钠缓冲液悬浮细胞，加入0.1％荧光染料溶液，混匀保持5分钟使荧光染料与细胞反应充分。反应结束后，离心去上清，再加入柠檬酸钠缓冲液洗涤；
4)PBS缓冲液悬浮细胞，加入50％戊二醛溶液，使其终浓度为0.0125％(v/v)，室温固定2小时，固定结束后，离心去上清，重复洗涤两次；
5)保存液悬浮粒子至适当计数，2-8℃冷藏，含有网织血小板的血小板模拟粒子制备完成。
在分别按照上述方案A和C对同样来源的样本进行处理后，将处理后的样本置于在30℃的条件下，每天定期通过血液细胞分析仪对各个样本进行检测计数，分别统计第 1天、第2天、第3天和第4天的各样本的RET通道粒子数的变化。其中，处理当日(记为第1天)的RET通道粒子数记为100％，结果如表1所示。
表1联合固定与单独固定的稳定性比较
Figure PCTCN2018123638-appb-000001
此外，在分别按照上述方案A和B对同样来源的样本进行处理后，将处理后的样本置于在30℃的条件下，每天定期通过血液细胞分析仪对各个样本进行检测计数，分别统计第1天、第3天、第4天和第7天的各样本的RET通道粒子数的变化。其中，处理当日(记为第1天)的RET通道粒子数记为100％，结果如表2所示。
表2不同顺序的联合固定的稳定性比较
Figure PCTCN2018123638-appb-000002
表1和表2分别示出了固定处理后的若干天，所制备的血小板模拟粒子的RET通道粒子数相对于第1天的百分比。可见，与单独使用戊二醛固定的方案C相比，采用单宁酸、戊二醛联合进行固定处理的方案A和B在血小板模拟粒子的稳定性方面取得了明显更好的效果。
实施例7
采用实施例6方案A中血小板模拟粒子制备方法，并调整其中的单宁酸的反应浓度，以考察单宁酸的不同反应浓度对所制备的血小板模拟粒子稳定性的影响。在对同样来源的样本进行处理后，将处理后的样本置于在30℃的条件下，定期通过血液细胞分析仪对各个样本进行检测计数，观察各样本的RET通道粒子数的变化。其中，处理当日(记为第1天)的RET通道粒子数记为100％，结果如表3和表4所示，其中表3和表4的山羊红细胞的来源不同。
表3
Figure PCTCN2018123638-appb-000003
Figure PCTCN2018123638-appb-000004
表3示出了处理后第4、6和8天，所制备的血小板模拟粒子的RET通道粒子数相对于第1天的百分比。可见，单宁酸的反应浓度为25mg/L、37.5mg/L、50mg/L和75mg/L的情况下，所制备的血小板模拟粒子均有较好的稳定性，其中25mg/L和37.5mg/L时稳定效果更佳，25mg/L时取得最佳的稳定效果。
表4
Figure PCTCN2018123638-appb-000005
表4示出了处理后第4和5天，所制备的血小板模拟粒子的RET通道粒子数相对于第1天的百分比。可见，单宁酸的反应浓度为5mg/L和15mg/L的情况下，所制备的血小板模拟粒子均有较好的稳定性，其中15mg/L时稳定效果更佳。
因此，本发明优选采用反应浓度为15mg/L至25mg/L的单宁酸对动物红细胞进行固定处理。
实施例8
采用实施例6方案A中血小板模拟粒子制备方法，其中戊二醛的反应浓度调整为0.0125v/v％和0.025v/v％，以考察戊二醛的不同反应浓度对所制备的血小板模拟粒子溶血情况的影响。
使用血液细胞分析仪分别对使用上述不同浓度戊二醛所制备的含网织红细胞的血小板模拟粒子进行检测，其DIFF通道上的侧向散射光-荧光散点图分别如图6A和图6C所示，其RET通道上的荧光-前向散射光散点图分别如图6B和图6D所示
由图6A可知，当使用反应浓度为0.0125v/v％的戊二醛进行固定处理时，所制备的血小板模拟粒子在DIFF通道中基本上完全溶血，不会对白细胞分群造成影响，而且由图6B可知该血小板模拟粒子在RET通道上是稳定的；而由图6C和6D可知，当使用反应浓度为0.025v/v％的戊二醛进行固定处理时，虽然所制备的血小板模拟粒子在RET通道上是稳定的，但在DIFF通道中出现了未完全溶血情况，出现了大量血影，可能会 导致白细胞分群的检测结果不准确。
因此，本发明优选采用反应浓度小于0.025v/v％的戊二醛对动物红细胞进行固定处理。

Claims (20)

  1. 一种制备血小板模拟粒子的方法，其中，使用三氟甲基类化合物对动物红细胞进行修饰处理，以制成能结合荧光染料的血小板模拟粒子。
  2. 根据权利要求1所述的方法，其中，所述三氟甲基类化合物为被至少一个三氟甲基取代的醛和/或被至少一个三氟甲基取代的酸。
  3. 根据权利要求2所述的方法，其中，被至少一个三氟甲基取代的醛的结构如式I所示：
    R 1-CHO  (I)；
    被至少一个三氟甲基取代的酸的结构如式II所示：
    R 2-COOH  (II)；
    其中，R 1和R 2各自为H、C 1-C 4烷基、C 6-C 10芳基、C 7-C 10苯烷基和C 7-C 10烷基苯基。
  4. 根据权利要求1～3中任一项所述的方法，其中，所述三氟甲基类化合物选自对三氟甲基苯甲醛、2,4-二三氟甲基苯甲醛、对三氟甲基苯甲酸、三氟乙酸和三氟乙醛所组成的组。
  5. 根据权利要求1～4中任一项所述的方法，其中，在使用三氟甲基类化合物对动物红细胞进行修饰处理之前或之后，使用固定剂对所述动物红细胞进行固定处理。
  6. 根据权利要求5所述的方法，其中，所述使用固定剂对所述动物红细胞进行固定处理的步骤包括：使用醛类对所述动物红细胞进行固定处理；
    优选地，所述醛类选自由以下项所组成的组中的一种或多种：多聚甲醛、甲醛、丙烯醛、丙二醛、戊二醛和己二醛，所述醛类尤其包括戊二醛；
    优选地，所述醛类的反应浓度为0.0075v/v％～0.025v/v％、优选为0.01v/v％～0.0125v/v％。
  7. 根据权利要求6所述的方法，其中，所述使用固定剂对所述动物红细胞进行固定处理的步骤还包括：使用单宁酸对所述动物红细胞进行固定处理；
    优选地，所述单宁酸的反应浓度为5mg/L～75mg/L、优选为15mg/L～37.5mg/L、更优选为15mg/L～25mg/L。
  8. 一种制备血小板模拟粒子的方法，其中，使用单宁酸和醛类对动物红细胞进行固定处理，以制成血小板模拟粒子。
  9. 根据权利要求8所述的方法，其中，所述醛类选自由以下项所组成的组中的一种或多种：多聚甲醛、甲醛、丙烯醛、丙二醛、戊二醛和己二醛，所述醛类尤其是包括戊二醛；
    优选地，所述醛类的反应浓度为0.0075v/v％～0.025v/v％、优选为0.01v/v％～0.0125v/v％。
  10. 根据权利要求8或9所述的方法，其中，所述单宁酸的反应浓度为5mg/L～75mg/L、优选为15mg/L～37.5mg/L、更优选为15mg/L～25mg/L。
  11. 根据权利要求8至10中任一项所述的方法，其中，所述使用单宁酸和醛类对所述动物红细胞进行固定处理的步骤包括：
    先使用单宁酸对所述动物红细胞进行固定处理，然后使用醛类对所述动物红细胞进行固定处理；或者
    先使用醛类对所述动物红细胞进行固定处理，随后使用单宁酸对所述动物红细胞进行固定处理。
  12. 根据权利要求8至11中任一项所述的方法，其中，所述方法还包括：
    在所述固定处理之前或之后，对所述动物红细胞进行修饰处理，以使得所述动物红细胞能在血小板检测通道中产生荧光信号。
  13. 根据权利要求12所述的方法，其中，所述对动物红细胞进行修饰处理，以使得所述动物红细胞能在血小板检测通道中产生荧光信号的步骤包括：
    使用三氟甲基类化合物对所述动物红细胞进行修饰处理，以使得所述动物红细胞能结合荧光染料，或者
    使用荧光染料对所述动物红细胞进行染色处理。
  14. 根据权利要求13所述的方法，其中，所述三氟甲基类化合物为被至少一个三氟甲基取代的醛和/或被至少一个三氟甲基取代的酸。
  15. 根据权利要求15所述的方法，其中，被至少一个三氟甲基取代的醛的结构如式I所示：
    R 1-CHO  (I)；
    被至少一个三氟甲基取代的酸的结构如式II所示：
    R 2-COOH  (II)；
    其中，R 1和R 2各自为H、C 1-C 4烷基、C 6-C 10芳基、C 7-C 10苯烷基和C 7-C 10烷基苯基。
  16. 根据权利要求15所述的方法，其中，所述三氟甲基类化合物选自对三氟甲基苯甲醛、2,4-二三氟甲基苯甲醛、对三氟甲基苯甲酸、三氟乙酸和三氟乙醛所组成的组。
  17. 根据权利要求1至16中任一项所述的方法，其中，所述动物红细胞源自哺乳动物，优选源自山羊或绵羊。
  18. 一种血小板模拟粒子，所述血小板模拟粒子根据权利要求1至17中任一项所述的方法所制备。
  19. 一种用于血液分析仪的质控物或校准物，其中，所述质控物或校准物含有根据权利要求18所述的血小板模拟粒子。
  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、红细胞模拟粒子和有核红细胞模拟粒子中的至少一种。
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