CN109459372B

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Info

Publication number: CN109459372B
Application number: CN201811264421.XA
Authority: CN
Inventors: 刘旭; 蔡清华; 王丹凤; 林月; 朱子是; 魏学芹
Original assignee: Dirui Medical Technology Co Ltd
Current assignee: Dirui Medical Technology Co Ltd
Priority date: 2018-10-29
Filing date: 2018-10-29
Publication date: 2021-03-26
Anticipated expiration: 2038-10-29
Also published as: CN109459372A

Abstract

Nucleated red blood cell simulation particles and a preparation method and application thereof, belonging to the technical field of medical detection. Solves the problem of how to provide a preparation method of the nucleated erythrocyte simulated particles with simple process, high yield and good product stability. The preparation method comprises the following steps: separating and washing erythrocytes to obtain purified erythrocytes; diluting the purified red blood cells by using a diluent, adding a loading agent, and introducing the loading agent into the purified red blood cells under the action of a pulsed electromagnetic field; then adding cell fixing liquid into the cells with the added carrier, fixing at 18-28 ℃, washing the cells, and preserving by using cell preserving liquid to obtain the nucleated erythrocyte simulated particles. The preparation method has the advantages of simple steps, single reagent component, high yield of the prepared product, and suitability for mass production.

Description

Nucleated erythrocyte simulated particle and preparation method and application thereof

Technical Field

The invention belongs to the technical field of medical detection, and particularly relates to a nucleated red blood cell simulation particle and a preparation method and application thereof.

Background

Normal human mature erythrocytes do not contain cell nuclei, have the main components of protein and iron, have the capacity of transporting oxygen, and nucleated erythrocytes belong to the initial juvenile state in the erythrocyte development process and generally exist in bone marrow.

The clinical significance is as follows:

1. hyperplastic anemia: most commonly seen in various anemias, acute blood loss anemia, megaloblastic anemia, severe hypopigmented anemia; the appearance of late or intermediate erythroblasts is common; the appearance of nucleated red blood cells in peripheral blood indicates that erythroid hyperplasia in bone marrow is obviously active;

2. erythroleukemia, erythroleukemia: the abnormal proliferation of immature erythrocyte in marrow and releasing into blood, and the abnormal proliferation of primitive erythrocyte and early erythrocyte is common;

3. extramedullary hematopoiesis: when bone marrow fibrosis is carried out, tissues such as spleen, liver, lymph node and the like recover the hematopoietic function of embryonic period, and because the tissues lack the regulation and control capability on the release of blood cells, a large amount of immature blood cells enter peripheral blood; the erythroblasts at all developmental stages are visible, and the immature granulocytes and megakaryocytes can be seen;

4. and others: such as metastatic cancer of the bone marrow, severe hypoxia, etc.

Currently, more and more blood cell analyzers have a function of detecting nucleated red blood cells, and in order to ensure the accuracy of the measured value, quality control is required, that is, detection is performed using a quality control product containing nucleated red blood cell-simulating particles.

US patent US5559037(Kim et al) discloses a method for flow cytometric analysis of nucleated red blood cells and white blood cells. This method uses fluorescence, low angle light scattering and axial light loss measurements to distinguish NRBC from white blood samples. US5879900(Kim et al) further discloses a method of discriminating NRBC, leukocytes and damaged leukocytes, and a method of providing leukocyte differentiation in blood samples by flow cytometry, which has the disadvantage of poor accuracy.

US patents US7176031 and US 69662817 disclose methods of simulating human nucleated red blood cells using artificially synthesized particles. The method has the defects of complex production process and high cost of the synthesized particles. US patent US7354767 discloses a method for preparing nucleated red blood cell mimetic particles using normal mammalian red blood cells (without nuclei). The simulated particles prepared by the method are close to the nucleus size of human nucleated red blood cells, and are suitable for a blood cell analyzer for detecting the nucleated red blood cells by adopting an impedance method. However, such a mimic particle does not contain a nucleus and is not suitable for mimicking the fluorescent properties of human nucleated red blood cells stained with a specific fluorescent dye. The technical scheme disclosed in the U.S. Pat. No. 3,95919 is to link biomacromolecules (such as nucleic acid, skin chain, etc.) on the surface of erythrocytes to make them able to simulate the fluorescence characteristics of human nucleated erythrocytes. The method has the disadvantages of complex process and high cost of biomacromolecules as raw materials.

US6723563 and US6653137 propose nucleated erythrocyte-simulating particles prepared from erythrocytes of birds (such as turkeys or chickens), reptiles (such as alligator sinensis) or fish (such as frogs). US patents 6406915, US6403377, US6399388, US6221668 and US6200500 disclose methods for preparing nucleated red blood cell mimetic particles using turkey red blood cells. US patent US6448085 discloses a method for preparing nucleated red blood cell mimetic particles using chicken red blood cells. US6187590 and US 58790 disclose methods for preparing nucleated red blood cell mimetic particles using red blood cells from turkey, chicken and frog crouch fish. US7285417, US7135341 and US7198953 disclose methods of preparing nucleated red blood cell mimetic particles using alligator cells. The common points of the technical schemes are as follows: animal erythrocytes with nuclei, including avian, reptilian and fish erythrocytes, are used to mimic human nucleated erythrocytes. The disadvantage of this method is that the majority of birds, reptiles and fish erythrocytes are elliptical in shape and differ significantly from the near-circular shape of human nucleated erythrocytes. Thus, in some cases, these animal nucleated red blood cells do not mimic well the characteristics of human nucleated red blood cells. For example, when cells are examined by flow cytometry, elliptical cells do not pass through the flow cell in the same direction, thus producing forward scattered light signals of widely varying sizes, whereas circular or near-circular cells do not.

Therefore, the methods for preparing the nucleated red blood cell simulated particles in the prior art all have certain defects, and a preparation method of the nucleated red blood cell simulated particles with simple process, high yield and good product stability is urgently needed.

Disclosure of Invention

In view of the above, the present invention provides a method for preparing a nucleated red blood cell simulated particle with simple process, high yield and good product stability, and provides an application of the nucleated red blood cell simulated particle prepared by the method as a quality control material of a cellular blood analyzer using a fluorescence-scattering light method as a detection principle.

The technical scheme adopted by the invention for realizing the aim is as follows.

The preparation method of the nucleated erythrocyte simulated particle comprises the following steps:

step one, separating and washing red blood cells to obtain purified red blood cells;

step two, diluting the purified red blood cells by using a diluent, adding a loading agent, and introducing the loading agent into the purified red blood cells under the action of a pulse electromagnetic field;

the loading agent is artificially synthesized DNA or RNA fragments, and the addition amount of the loading agent is more than 1.5 mu g/L and within 3.0 mu g/L;

and step three, adding cell fixing liquid into the cells into which the loading agent is introduced, fixing at 18-28 ℃, washing the cells, and preserving by using cell preservation liquid to obtain the nucleated red blood cell simulation particles.

Preferably, in the first step, the red blood cell is a human red blood cell or a mammalian red blood cell with MCV similar to human red blood cell.

Preferably, in the second step, the purified red blood cells are diluted to 10 degrees with a diluent^12-10¹⁴And (2) per liter.

Preferably, in the second step, the pH value of the diluent is7.1-7.5, prepared from ATP and MgCl₂、KH₂PO₄、NaHCO₃Glucose and water, ATP concentration of 0.1-1.0%, MgCl₂The concentration of the component (A) is 0.1% -0.5%, KH₂PO₄0.5% -3.0% of NaHCO₃The concentration is 0.05-0.3%, and the concentration of glucose is 0.01-0.1%.

Preferably, in the second step, the process of introducing the loading agent into the purified red blood cells under the action of the pulsed electromagnetic field comprises: the electroporator is stimulated at 90-120V for 4-8 seconds.

Preferably, in the first step and the third step, the washing is performed by using a detergent comprising a buffer, a preservative, an inorganic salt and water.

Preferably, in the third step, the cell fixing solution comprises a fixing agent, a buffer solution, disodium ethylene diamine tetraacetate (EDTA-2Na), a preservative and water; the fixing agent is one or more of formaldehyde, acetaldehyde, glutaraldehyde, paraformaldehyde, methanol and acetone; the fixed time is 3-5 h.

Preferably, in the third step, the cell preservation solution comprises a buffer solution, a nutrient component, a preservative and water.

The invention also provides the nucleated erythrocyte simulated particle prepared by the preparation method of the nucleated erythrocyte simulated particle.

The invention also provides the application of the nucleated red blood cell simulation particle as a quality control substance of a blood cell analyzer which takes nucleic acid fluorescent staining as a detection principle.

Compared with the prior art, the invention has the beneficial effects that:

the preparation method of the nucleated erythrocyte simulated particle introduces the nucleic acid substance into the mature erythrocyte by using the mode of stimulating the cell by the electric pulse to prepare the nucleated erythrocyte simulated particle, the preparation steps of the simulated particle are simple, the used reagent components are single, the yield of the prepared product is high, and the preparation method is suitable for mass production.

Drawings

FIG. 1 shows the test picture of the whole blood quality control substance prepared by mixing the nucleated red blood cell mimic particles without the introduction of the nucleic acid substance into the cells stimulated by the electric pulse with the mature red blood cell mimic substance according to the mass ratio of 1:4 on the blood cell analyzer using the nucleic acid fluorescent staining as the detection principle (only the nucleated red blood cell channel is provided).

FIG. 2 shows a picture of a whole blood quality control substance prepared by mixing the nucleated red blood cell mimetic particles prepared in example 1 with a mature red blood cell mimetic at a mass ratio of 1:4 and testing the same on a blood cell analyzer using fluorescence staining of nucleic acid as a detection principle (only a nucleated red blood cell channel is provided).

FIG. 3 shows the picture of the whole blood quality control substance prepared by mixing the nucleated red blood cell mock particles prepared in example 2 with the mature red blood cell mock substance in a mass ratio of 1:4 and testing on the blood cell analyzer using the nucleic acid fluorescent staining as the detection principle (only the nucleated red blood cell channel is provided).

FIG. 4 shows the picture of the whole blood quality control substance prepared by mixing the nucleated red blood cell mock particles prepared in example 3 with the mature red blood cell mock substance in a mass ratio of 1:4 and testing on the blood cell analyzer using the nucleic acid fluorescent staining as the detection principle (only the nucleated red blood cell channel is provided).

Detailed Description

For a further understanding of the invention, reference will now be made to the preferred embodiments of the invention, but it is to be understood that the description is intended to illustrate further features and advantages of the invention, and not to limit the scope of the claims.

The preparation method of the nucleated erythrocyte simulated particle comprises the steps of separating and washing erythrocytes to obtain purified erythrocytes; and then adding the purified red blood cells into the diluent, adding a loading agent, introducing the loading agent into the purified red blood cells under the action of a pulse electric field, finally adding a cell fixing solution into the cells into which the loading agent is introduced, fixing at 18-28 ℃, washing the cells, and preserving by using a cell preserving solution to obtain the nucleated red blood cell simulated particles.

In the technical scheme, the red blood cells can be human red blood cells or mammalian red blood cells with MCV values close to that of human, and the effect of preparing the simulant in the invention by using fresh blood is better. The separation method is to remove the white blood cells and the platelets in the blood sample by means of centrifugation, washing and filtration, wherein the filtration can remove the white blood cells by using a white blood cell filter.

In the technical scheme, the pH value of the diluent is 7.1-7.5; diluting with ATP and MgCl₂、KH₂PO₄、NaHCO₃Glucose and water; the concentration of ATP is 0.1% -1.0%; MgCl₂The concentration of (A) is 0.1% -0.5%; KH (Perkin Elmer)₂PO₄The concentration is 0.5% -3.0%; NaHCO 2₃The concentration is 0.05% -0.3%; the concentration of glucose is 0.01-0.1%; diluting purified red blood cells to 10 deg.C with common diluent¹²-10¹⁴And (2) per liter.

In the technical scheme, the loading agent is an artificially synthesized RNA fragment, and the addition amount of the loading agent is more than 1.5 mu g/L and within 3.0 mu g/L. The sequence of the artificially synthesized DNA or RNA fragment has no special requirement and can be a 200-and 1000-bp fragment of any sequence; pET-15b was used as the expression vector to which the DNA fragment was ligated. The method specifically comprises the following steps: the DNA shown in the sequence 1 or the sequence 2 is artificially synthesized and then cloned into a pET-15b vector, the expression vector is extracted, separated and purified after amplification, and finally a large number of DNA fragments, namely the loading agent, are obtained after enzyme digestion and purification.

In the technical scheme, the process of introducing the loading agent into the purified red blood cells under the action of the pulsed electromagnetic field comprises the following steps: the electroporator is stimulated at 90-120V for 4-8 seconds.

In the technical scheme, the fixing time of the fixing liquid is 3-5 h.

In the above technical scheme, the cell fixing solution, the cell preservation solution, and the washing agent used for washing the red blood cells and the products are all common substances in the field, and are not particularly limited. Wherein, the detergent is a conventional isotonic solution, which comprises a buffer solution, a preservative, inorganic salt and water, the cell fixing solution comprises a fixing agent, the buffer solution, EDTA-2Na, the preservative and water, and can also comprise nutrient substances; wherein the fixing agent is one or more of formaldehyde, acetaldehyde, glutaraldehyde, paraformaldehyde, methanol and acetone, and the concentration of each fixing agent is 0.1-2.0 mL/L. The cell preservation solution comprises buffer solution, nutrient components, preservative and water. In the technical scheme, the preservative is one or two of Proclin-150, Proclin-200, Proclin-300 and Proclin-500; the buffer solution is one or more of PBS buffer solution, D-HANKS buffer solution, HEPES buffer solution, citric acid buffer solution, sodium chloride buffer solution and boric acid buffer solution; the nutrient substances include glucose, mannitol, adenine, etc. As usual, the detergent consisted of 20.0g/L disodium hydrogen phosphate, 3.0g/L sodium dihydrogen phosphate, 5.5g/L sodium chloride, 0.2g/L Proclin-200 and 1L purified water; the cell fixing solution consists of 0.3-2.0mL/L acetaldehyde, 0.1-0.2mL/L glutaraldehyde, 2.0g/L sodium dihydrogen phosphate, 0.15g/L EDTA-2Na, 8.0g/L glucose, 2.5g/L adenine, 0.2g/L LProclin-200 and 1L purified water; the cell preservation solution consists of 0.2-0.5/L citric acid, 1.5-2.0g/L sodium citrate, 1.0g/L sodium dihydrogen phosphate, 8.0-15.0g/L glucose, 12.0g/L mannitol, 2.5g/L adenine, 0.3g/L Proclin-200 and 1L purified water.

The present invention is further illustrated by the following examples, in which the reagents used are analytical grade and are commercially available.

Example 1

The preparation method of the nucleated erythrocyte simulated particle comprises the following steps: placing the anticoagulated human whole blood in a centrifuge, centrifuging at 3000rpm for 10min, removing supernatant, washing the precipitated cells with detergent, centrifuging at 3000rpm for 10min, removing supernatant, washing repeatedly for 2-3 times, and removing leukocytes with a leukocyte filter to obtain purified erythrocytes. Diluting purified red blood cells to 10 with diluent 1¹²The loading agent 1 was added at a concentration of 1.51. mu.g/L, the electroporator was applied with a voltage of 100V for 5 seconds, and the electrically stimulated purified erythrocytes were fixed at room temperature for 3 hours by adding the cell fixing solution 1. And repeatedly washing the reaction product for 3-4 times by using a detergent, and suspending the product by using the cell preservation solution 1 to obtain a quality control substance. The quality control substance was detected by a blood cell analyzer using fluorescence staining of nucleic acid as a detection principle, and the result is shown in FIG. 2 (only a nucleated red blood cell channel image is provided).

The loading agent 1: the DNA shown in the sequence 1 is artificially synthesized and then cloned into a pET-15b vector, the expression vector is extracted, separated and purified after amplification, and finally a large number of DNA fragments are obtained after enzyme digestion and purification, namely the loading agent 1.

A detergent: 20.0g/L disodium hydrogen phosphate, 3.0g/L sodium dihydrogen phosphate, 5.5g/L sodium chloride, 0.2g/L LProclin-200, and 1L purified water.

Diluent 1: 1.0g/L magnesium chloride, 12.0g/L potassium dihydrogen phosphate, 1.2g/L sodium bicarbonate, 5.0g/LATP, 0.4g/L glucose, 1L purified water; the pH value is 7.2.

Cell fixing solution 1: 2.0mL/L acetaldehyde, 0.1mL/L glutaraldehyde, 2.0g/L sodium dihydrogen phosphate, 0.15g/L EDTA-2Na, 8.0g/L glucose, 2.5g/L adenine, 0.2g/L Proclin-200, 1L purified water.

Cell preservation solution 1: 0.2g/L citric acid, 1.5g/L sodium citrate, 1.0g/L sodium dihydrogen phosphate, 8.0g/L glucose, 12.0g/L mannitol, 0.3g/L proclin-200 and 1L purified water.

Example 2

The preparation method of the nucleated erythrocyte simulated particle comprises the following steps: placing the anticoagulated human whole blood in a centrifuge, centrifuging at 3000rpm for 10min, removing supernatant, washing the precipitated cells with detergent, centrifuging at 3000rpm for 10min, removing supernatant, washing repeatedly for 2-3 times, and removing leukocytes with a leukocyte filter to obtain purified erythrocytes. Diluting purified red blood cells to 10 degree with diluent 2¹²The loading agent 2 was added at 2.0. mu.g/L, the electroporator was applied with a voltage of 100V for 8 seconds, and the electrically stimulated purified erythrocytes were fixed at room temperature for 4 hours by adding the cell fixing solution 2. And repeatedly washing the reaction product for 3-4 times by using a detergent, and suspending the product by using the cell preservation solution 2 to obtain a quality control substance. The quality control substance was detected by a blood cell analyzer using fluorescence staining of nucleic acid as a detection principle, and the result is shown in FIG. 3 (only a nucleated red blood cell channel image is provided).

And (3) a loading agent 2: and (3) artificially synthesizing the DNA shown in the sequence 2, cloning the DNA into a pET-15b vector, amplifying, extracting, separating and purifying the expression vector, and finally performing enzyme digestion and purification to obtain a large number of DNA fragments, namely the loading agent 2.

A detergent: 20.0g/L disodium hydrogen phosphate, 3.0g/L sodium dihydrogen phosphate, 5.5g/L sodium chloride, 0.2g/L LProclin-300, and 1L purified water.

Diluent 2: 5.0g/L magnesium chloride, 12.0g/L potassium dihydrogen phosphate, 1.2g/L sodium bicarbonate, 8.0g/LATP, 0.6g/L glucose, 1L purified water; the pH value is 7.4.

Cell fixing solution 2: 0.5mL/L formaldehyde, 0.1mL/L glutaraldehyde, 2.0g/L sodium dihydrogen phosphate, 0.15g/L EDTA-2Na, 0.2g/L LPCrine-500, 1L purified water.

Cell preservation solution 2: 0.5g/L citric acid, 2.0g/L sodium citrate, 1.0g/L sodium dihydrogen phosphate, 15.0g/L glucose, 2.5g/L adenine, 0.3g/L proclin-300 and 1L purified water.

Example 3

The preparation method of the nucleated erythrocyte simulated particle comprises the following steps: placing the selected human anticoagulated whole blood in a centrifuge, centrifuging at 3000rpm for 10min, removing supernatant, washing the precipitated cells with detergent, centrifuging at 3000rpm for 10min, removing supernatant, washing repeatedly for 2-3 times, and removing leukocytes with leukocyte filter to obtain purified erythrocytes. Diluting the purified red blood cells to 10 degree with diluent 3¹²The loading agent 1 was added at a concentration of 3.0. mu.g/L, the electroporator was applied with a voltage of 100V for 6 seconds, and the electrically stimulated purified erythrocytes were fixed at room temperature for 5 hours by adding the cell fixing solution 3. And repeatedly washing the reaction product for 3-4 times by using a detergent, and suspending the product by using a cell preservation solution 3 to obtain a quality control substance. The quality control substance was detected by a blood cell analyzer using fluorescence staining of nucleic acid as a detection principle, and the result is shown in FIG. 4 (only a nucleated red blood cell channel image is provided).

Diluent 3: 5.0g/L magnesium chloride, 11.5.0g/L potassium dihydrogen phosphate, 2.0g/L sodium bicarbonate, 10.0g/LATP, 1.0g/L glucose, 1L purified water; the pH value is 7.5.

Cell fixing solution 3: 0.3mL/L formaldehyde, 0.2mL/L glutaraldehyde, 2.0g/L sodium dihydrogen phosphate, 0.15g/L EDTA-2Na, 0.2g/L LPCrine-300, 1L purified water.

Cell preservation solution 3: 0.5g/L citric acid, 2.0g/L sodium citrate, 1.0g/L sodium dihydrogen phosphate, 15.0g/L glucose, 0.3g/L LProclin-300 and 1L purified water.

As can be seen from FIGS. 1 to 4, when the quality control substance of nucleated red blood cells prepared by the embodiments of the present invention is detected and analyzed in a blood analyzer, the simulated particles without nucleic acid substances cannot see the nucleated red blood cells on the scattergram, as shown in FIG. 1; after the electromagnetic pulse treatment, the nucleated red blood cell region having distinct boundaries with the mature red blood cell region and the white blood cell region can be seen on the scattergram, as shown in fig. 2 to 4. In conclusion, the preparation method has simple process and high yield, is suitable for mass production, and has good prospect when the prepared nucleated red blood cell simulation particles are used as the quality control material of a cell blood analyzer which takes a fluorescence-scattering light method as a detection principle.

The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Sequence listing

<110> Dirui medical science and technology Co., Ltd

<120> nucleated red blood cell simulation particle, and preparation method and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

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<211> 427

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

cttggaagag aggcgggtca aagaagtagt gaagaagcac tctcagttca taggctatcc 60

catcaccctt tatttgaaga aggagcgaga gaaggaaatt agtggtgatg aggcagagga 120

agagaaaggt gagaaagagg aggaagataa agatgatgaa gaaaagccca agatcgaaga 180

tgtgggttca gatgaggagg atgacagtgg taaggataag aagaagaaaa ctaagaagat 240

caaagagaaa tacattgatc aggaagagct aaacaagacc aagcctattt ggaccagaaa 300

ccctgatgac atcacccaag aggagtatgg agaattctat aagagcctca ccaatgactg 360

ggaagaccac ttggcagtca agcacttttc tgtagaaggt cagttggaat tcagggcatt 420

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atcgatagca ccctccagaa aatggtgctg acttccggct gtcaaatttt tctttctttc 60

ccagccacga cgaggtcgac agcatgagtt ctctaaagct ccagaagagg ctcgcagcct 120

ccgtgctgcg atgcggcaag aagaaggtct ggttggatcc caatgaaatc aacgagatcg 180

ccaacacaaa ctcgcgtcag aacatccgca agctcatcaa ggatggtctg atcatcaaga 240

agcccgtcgt ggtccactcc cgctaccgtg tgcgcaagaa caccgaggcc cgccgcaagg 300

gtcgtcactg cggattcgga aagcgcaagg gtactgcaaa cgcccgtatg cccaccaagc 360

tggtgtggat gcagcgccag cgcgttctgc gtcgcctgtt gaagaagtac cgcgacagca 420

agaagattga caggcacctg taccacgacc tgtacatgaa gtgcaagggt aacgtgttca 480

agaacaagcg cgtcctgatg gagtacatcc acaagaagaa ggctgagaag cagcgcagca 540

agatgctggc tgaccaagcc gaggctcgcc gacagaaggt gcgcgaggcc cgcaagcgcc 600

gcgaggagcg tatcgccacc aagaagcagg agctcattgc tctgcacgcc aaggaggacg 660

agatcgctgc caaggccgcc accgcgggtc actaagcagt ccggcctcgc tagtcgaggt 720

accccatatt gatagtactg tttatcatct gaataaaaca cctttggttt ggccgaaatg 780

attttgtaca aacgtgaaac 800

Claims

1. The preparation method of the nucleated erythrocyte simulated particle comprises the following steps:

the red blood cells are human red blood cells;

2. The method of claim 1, wherein in step two, the purified erythrocytes are diluted to 10 degrees with a diluent¹²-10¹⁴And (2) per liter.

3. The method for preparing nucleated red blood cell mimetic particles according to claim 1, wherein in said second step, said diluent has a pH of 7.1 to 7.5 and is composed of ATP and MgCl₂、KH₂PO₄、NaHCO₃Glucose and water, ATP concentration of 0.1-1.0%, MgCl₂The concentration of the component (A) is 0.1% -0.5%, KH₂PO₄0.5% -3.0% of NaHCO₃The concentration is 0.05-0.3%, and the concentration of glucose is 0.01-0.1%.

4. The method of claim 1, wherein the step two of introducing the loading agent into the purified red blood cells under the action of the pulsed electromagnetic field comprises: the electroporator is stimulated at 90-120V for 4-8 seconds.

5. The method of claim 1, wherein in step one and step three, the washing agent used in the washing step comprises buffer, preservative, inorganic salt and water.

6. The method of claim 1, wherein in step three, the cell fixative solution comprises fixative, buffer solution, EDTA-2Na, preservative and water; the fixing agent is one or more of formaldehyde, acetaldehyde, glutaraldehyde, paraformaldehyde, methanol and acetone; the fixed time is 3-5 h.

7. The method of claim 1, wherein in step three, the cell preservation solution comprises a buffer, nutrients, preservatives and water.

8. A nucleated red blood cell mimetic particle produced by the method for producing a nucleated red blood cell mimetic particle according to any one of claims 1 to 7.

9. Use of the nucleated red blood cell pseudoparticle according to claim 8 as a quality control material for a blood cell analyzer based on the detection principle of fluorescent staining of nucleic acids.