Red blood cell simulant particles, preparation method thereof, and quality control or calibration material containing the red blood cell simulant particles

Abstract

The present invention provides a red blood cell simulation particle, a preparation method thereof, and a quality control material or calibration material containing the red blood cell simulation particle. The preparation method can be used to process human red blood cells and animal red blood cells, and the stability of red blood cells can be increased and the storage time of red blood cells can be extended. The red blood cell simulation particles prepared by the preparation method can be used as simulation particles of quality control materials and calibration materials for human or animal blood cell analyzers. The preparation method uses two treatment reagents. Treatment reagent A is used to increase cell membrane fluidity, so that oxidants and preservatives can treat red blood cells more evenly and more deeply, and the oxidation and preservative effects on proteins in cells are more sufficient; treatment reagent B is used to reduce cell membrane fluidity, denature proteins on cell membranes, reduce cell activity, and slow down cell metabolic activities, thereby achieving the purpose of increasing the stability of red blood cells and extending the storage time of red blood cells.

Description

Technical Field

[0001] The present invention relates to the field of blood cell analysis, and in particular to a red blood cell simulant particle, a preparation method thereof, and a quality control material or a calibration material containing the red blood cell simulant particle. Background Art

[0002] Red blood cells are an important component of human blood. The number and morphology of red blood cells are important parameters for routine blood screening. Blood cell analyzers can obtain red blood cell parameter information by analyzing peripheral whole blood. In order to effectively monitor and calibrate the red blood cell-related output of the instrument, it is necessary to prepare red blood cell simulant particles that can effectively simulate the characteristics of red blood cells.

[0003] The existing red blood cell simulants have the problem of large changes in red blood cell volume during the shelf life, which is reflected in the large changes in the MCV parameters on the instrument; because the preservation solution contains nutrients required for cell metabolism, the antiseptic effect of the reagents is not ideal; the treatment reagents used in the currently published patents involve highly toxic or easily manufactured drugs under national control, which also brings certain difficulties to the procurement of reagents. Therefore, how to solve the above problems is one of the research directions of technicians in this field. Summary of the invention

[0004] In view of this, the present invention provides a method for preparing red blood cell mimetic particles.

[0005] The present invention also provides a red blood cell simulation particle prepared by the above preparation method.

[0006] The present invention also provides a blood cell analysis quality control material or calibrator containing the red blood cell simulant particles.

[0007] The present invention provides a method for preparing red blood cell simulant particles, comprising the following steps:

[0008] (1) removing white blood cells and platelets from a blood sample by centrifugation and standing, and washing the sample to obtain a purified red blood cell suspension;

[0009] (2) preparing a red blood cell treatment solution, wherein the red blood cell treatment solution comprises a treatment reagent A and a treatment reagent B, wherein the treatment reagent A comprises: an alkaloid, a heavy metal salt, a buffer system and a preservative, and the osmotic pressure of the treatment reagent A is controlled within a range of 220-1500 mOsm / L, and the pH value is controlled within a range of 6-10; the treatment reagent B comprises: a fluoride, an aldehyde and a buffer system, and the osmotic pressure of the treatment reagent B is controlled within a range of 220-1500 mOsm / L, and the pH value is controlled within a range of 6-10;

[0010] (3) taking an appropriate amount of the red blood cell suspension and mixing it with the treatment reagent A in a certain proportion, and then placing it in a 37° C. environment for treatment for 1 hour;

[0011] (4) washing the red blood cells treated in step (3), mixing with the treatment reagent B in a certain proportion, and leaving at room temperature for 0.5 h or 1 h;

[0012] (5) Washing and preserving the red blood cells treated in step (4) to obtain red blood cell mimetic particles.

[0013] In some embodiments, the method of allowing the blood sample to stand in an upright position for 3 days is as follows:

[0014] In some embodiments, the 37°C environment in step (3) is a 37°C water bath or a 37°C constant temperature incubator.

[0015] In some embodiments, the method of preserving red blood cells in step (5) is: diluting with a self-prepared preservation solution and storing at 2-8°C, wherein the preservation solution comprises a buffer system, sugars, a preservative and a dispersant.

[0016] In some embodiments, the treatment reagent A comprises: 3 g / L solanine, 1 g / L sodium nitrite, 0.2 g / L potassium dihydrogen phosphate, 2.9 g / L disodium hydrogen phosphate dodecahydrate, 8 g / L sodium chloride, 5 g / L PC300 and 1 L of pure water; the treatment reagent B comprises: 0.4 g / L sodium dihydrogen phosphate, 5.8 g / L disodium hydrogen phosphate, 5 g / L sodium chloride, 0.3 g / L formaldehyde, 2 g / L sodium fluoride and 1 L of pure water.

[0017] In some embodiments, in step (3), the red blood cell suspension is mixed with the treatment reagent A in a ratio of 1:1; in step (4), the washed sample solution is mixed with the treatment reagent B in a ratio of 1:2.

[0018] In some embodiments, the treatment reagent A comprises: 4.5 g / L of scopolamine, 2.5 g / L of mercuric chloride, 12 g / L of sodium citrate, 1 g / L of sodium thimerosal and 1 L of pure water; the treatment reagent B comprises: 10 g / L of sodium citrate, 0.6 g / L of paraformaldehyde, 0.5 g / L of cesium fluoride and 1 L of pure water.

[0019] In some embodiments, in step (3), the red blood cell suspension is mixed with the treatment reagent A in a ratio of 1:2; in step (4), the washed sample solution is mixed with the treatment reagent B in a ratio of 1:1.

[0020] In some embodiments, the treatment reagent A comprises: 8 g / L liensinine, 1.5 g / L copper sulfate, 0.8 g / L methylisothiazolinone and 1 L Tris buffer solution; the treatment reagent B comprises: 0.8 g / L beryllium fluoride, 0.08 g / L glutaraldehyde and 1 L Tris buffer solution.

[0021] In some embodiments, in step (3), the red blood cell suspension is mixed with the treatment reagent A in a ratio of 1:2; in step (4), the washed sample solution is mixed with the treatment reagent B in a ratio of 1:2.

[0022] The present invention also provides a red blood cell simulation particle, and the red blood cell simulation particle is prepared by the above preparation method.

[0023] The present invention also provides a blood cell analysis quality control material or calibrator, comprising red blood cell simulation particles prepared according to the above preparation method.

[0024] In summary, the present invention provides a red blood cell simulant particle, a preparation method thereof, and a quality control material or calibrant containing the red blood cell simulant particle. The preparation method can be used to process human red blood cells and animal red blood cells, thereby increasing the stability of red blood cells and extending the storage time of red blood cells. The red blood cell simulant particles prepared by the preparation method can be used as simulation particles of quality control materials and calibrants for human or animal blood cell analyzers.

[0025] This preparation method uses two treatment reagents. Treatment reagent A is used to increase the fluidity of the cell membrane, so that the oxidant and the preservative can treat the red blood cells more evenly and more deeply, and the oxidation and preservative effects on the proteins in the cells are more sufficient; treatment reagent B is used to reduce the fluidity of the cell membrane, denature the proteins on the cell membrane, reduce cell activity, and slow down the metabolic activity of the cells, thereby achieving the purpose of increasing the stability of the red blood cells and extending the storage time of the red blood cells. And the reagents involved in the present invention are all conventional reagents and are easy to purchase. The treated red blood cells are preserved using a self-prepared preservation solution and stored in an environment of 2-8°C to obtain a red blood cell simulant suspension, which can also be mixed with other white blood cell simulants and platelet simulants to make a blood quality control or calibration material. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 This is a monitoring data curve diagram of red blood cell simulant particles in Example 1 of the present invention.

[0027] Figure 2 This is a curve chart of monitoring data of red blood cell simulant particles in Example 2 of the present invention.

[0028] Figure 3 This is a curve chart of monitoring data of red blood cell simulant particles in Example 3 of the present invention. DETAILED DESCRIPTION

[0029] The following is a more detailed description of the present invention in conjunction with the following embodiments. It should be understood that the implementation of the present invention is not limited to the following embodiments, and any modifications and / or changes made to the present invention will fall within the scope of protection of the present invention.

[0030] The present invention provides a red blood cell simulant particle, a preparation method thereof, and a quality control material or a calibrator containing the red blood cell simulant particle. The preparation method of the red blood cell simulant particle comprises the following steps:

[0031] (1) removing white blood cells and platelets from a blood sample by centrifugation and standing, and washing the sample to obtain a purified red blood cell suspension;

[0032] (2) preparing a red blood cell treatment solution, wherein the red blood cell treatment solution comprises a treatment reagent A and a treatment reagent B, wherein the treatment reagent A comprises: an alkaloid, a heavy metal salt, a buffer system and a preservative, and the osmotic pressure of the treatment reagent A is controlled within a range of 220-1500 mOsm / L, and the pH value is controlled within a range of 6-10; the treatment reagent B comprises: a fluoride, an aldehyde and a buffer system, and the osmotic pressure of the treatment reagent B is controlled within a range of 220-1500 mOsm / L, and the pH value is controlled within a range of 6-10;

[0033] (3) taking an appropriate amount of the red blood cell suspension and mixing it with the treatment reagent A in a certain proportion, and then placing it in a 37° C. environment for treatment for 1 hour;

[0034] (4) washing the red blood cells treated in step (3), mixing with the treatment reagent B in a certain proportion, and leaving at room temperature for 0.5 h or 1 h;

[0035] (5) Washing and preserving the red blood cells treated in step (4) to obtain red blood cell mimetic particles.

[0036] Preferably, the method of allowing the blood sample to stand in an upright position for 3 days in the step (1).

[0037] Preferably, the 37°C environment in step (3) is a 37°C water bath or a 37°C constant temperature incubator.

[0038] Preferably, the method of preserving red blood cells in step (5) is: diluting with a self-prepared preservation solution and storing at 2-8°C, wherein the preservation solution comprises a buffer system, sugars, a preservative and a dispersant.

[0039] The present invention is described in detail below with reference to specific embodiments.

[0040] Embodiment 1

[0041] 1. Place the blood sample upright for 3 days until the red blood cells and plasma are completely separated, remove the upper platelets and the middle layer of white blood cells, and wash with normal saline 3 times to obtain a purified red blood cell suspension;

[0042] 2. Prepare treatment reagent A and treatment reagent B:

[0043] Treatment Reagent A:

[0044] Reagent name concentration Solanine 3g / L Sodium Nitrite 1g / L Potassium dihydrogen phosphate 0.2g / L Disodium hydrogen phosphate dodecahydrate 2.9g / L Sodium chloride 8g / L PC300 5g / L Pure water 1L

[0045] Treatment Reagent B:

[0046] Reagent name concentration Sodium dihydrogen phosphate 0.4g / L Disodium hydrogen phosphate 5.8g / L Sodium chloride 5g / L formaldehyde 0.3g / L Sodium fluoride 2g / L Pure water 1L

[0047] 3. Take an appropriate amount of the red blood cell suspension prepared in step 1 and mix it with the above-mentioned treatment reagent A in a ratio of 1:1. After mixing, put it into a 37°C water bath for 1 hour to increase the fluidity of the cell membrane, thereby allowing the internal protein and environment of the cell to be treated more fully;

[0048] 4. The red blood cells treated in step 3 were washed three times with normal saline, mixed with treatment reagent B at a ratio of 1:2, and placed at room temperature for 1 hour to reduce the fluidity of the cell membrane, slow down the metabolic activity of the red blood cells, and increase the stability of the cells;

[0049] 5. The red blood cells treated in step 4 are washed three times with physiological saline, diluted with preservation solution and stored to obtain red blood cell mimetic particles.

[0050] The red blood cell simulant particles prepared in Example 1 were monitored on a blood cell analyzer for 20 weeks, with one test per week. The average of three repeated test results was taken each time to observe the changes in the red blood cell MCV parameter. The final result showed that the extreme value of the average red blood cell volume change was 1.6 fl. Please refer to Figure 1 .

[0051] Embodiment 2

[0052] 1. Place the blood sample upright for 3 days until the red blood cells and plasma are completely separated, remove the upper platelets and the middle layer of white blood cells, and wash with normal saline 3 times to obtain a purified red blood cell suspension;

[0053] 2. Prepare treatment reagent A and treatment reagent B:

[0054] Treatment Reagent A:

[0055] Reagent name concentration Anisodamine 4.5g / L Mercuric chloride 2.5g / L Sodium Citrate 12g / L Sodium Thimerosal 1g / L Pure water 1L

[0056] Treatment Reagent B:

[0057] Reagent name concentration Sodium Citrate 10g / L Paraformaldehyde 0.6g / L Cesium fluoride 0.5g / L Pure water 1L

[0058] 3. Take an appropriate amount of the red blood cell suspension prepared in step 1 and mix it with the above-mentioned treatment reagent A in a ratio of 1:2. After mixing, put it in a 37°C constant temperature incubator for 1 hour to increase the fluidity of the cell membrane, thereby allowing the internal protein and environment of the cell to be treated more fully;

[0059] 4. The red blood cells treated in step 3 were washed three times with normal saline, mixed with treatment reagent B at a ratio of 1:1, and placed at room temperature for 1 hour to reduce the fluidity of the cell membrane, slow down the metabolic activity of the red blood cells, and increase the stability of the cells;

[0060] 5. The red blood cells treated in step 4 are washed three times with physiological saline, diluted with preservation solution and stored to obtain red blood cell mimetic particles.

[0061] The red blood cell simulant particles prepared in Example 2 were monitored on a blood cell analyzer for 20 weeks, with one test per week. The average of three repeated test results was taken each time to observe the changes in the red blood cell MCV parameter. The final result showed that the extreme value of the average red blood cell volume change was 2.3 fl. Please refer to Figure 2 .

[0062] Embodiment 3

[0063] 1. Place the blood sample upright for 3 days until the red blood cells and plasma are completely separated, remove the upper platelets and the middle layer of white blood cells, and wash with normal saline 3 times to obtain a purified red blood cell suspension;

[0064] 2. Prepare treatment reagent A and treatment reagent B:

[0065] Treatment Reagent A:

[0066] Reagent name concentration Liensinine 8g / L Copper sulfate 1.5g / L Methylisothiazolinone 0.8g / L Tris buffer solution 1L

[0067] Treatment Reagent B:

[0068] Reagent name concentration Beryllium fluoride 0.8g / L Glutaraldehyde 0.08g / L Tris buffer solution 1L

[0069] 3. Take an appropriate amount of the red blood cell suspension prepared in step 1 and mix it with the above-mentioned treatment reagent A in a ratio of 1:2. After mixing, put it into a 37°C water bath for 1 hour to increase the fluidity of the cell membrane, thereby allowing the internal protein and environment of the cell to be treated more fully;

[0070] 4. The red blood cells treated in step 3 were washed three times with normal saline, mixed with treatment reagent B at a ratio of 1:2, and placed at room temperature for 0.5 h to reduce the fluidity of the cell membrane, slow down the metabolic activity of the red blood cells, and increase the stability of the cells;

[0071] 5. The red blood cells treated in step 4 are washed three times with physiological saline, diluted with preservation solution and stored to obtain red blood cell mimetic particles.

[0072] The red blood cell simulant particles prepared in Example 3 were monitored on a blood cell analyzer for 20 weeks, with one test per week. The average of three repeated test results was taken each time to observe the changes in the red blood cell MCV parameter. The final result showed that the extreme value of the average red blood cell volume change was 1.8 fl. Please refer to Figure 3 .

[0073] Examples 1 to 3 illustrate that the red blood cell simulant particles prepared by the preparation method of the present invention have good volume stability. The red blood cell simulant particles prepared by the present invention have a small change in MCV (mean corpuscular volume) within a certain period of time, a stable volume, can effectively simulate the characteristics of red blood cells, and can be used in blood cell analysis quality control and calibration materials to accurately control the quality of red blood cell analysis and determination.

[0074] In summary, the present invention provides a red blood cell simulant particle, a preparation method thereof, and a quality control material or calibrant containing the red blood cell simulant particle. The preparation method can be used to process human red blood cells and animal red blood cells, thereby increasing the stability of red blood cells and extending the storage time of red blood cells. The red blood cell simulant particles prepared by the preparation method can be used as simulation particles of quality control materials and calibrants for human or animal blood cell analyzers.

[0075] This preparation method uses two treatment reagents. Treatment reagent A is used to increase the fluidity of the cell membrane, so that the oxidant and the preservative can treat the red blood cells more evenly and more deeply, and the oxidation and preservative effects on the proteins in the cells are more sufficient; treatment reagent B is used to reduce the fluidity of the cell membrane, denature the proteins on the cell membrane, reduce cell activity, and slow down the metabolic activity of the cells, thereby achieving the purpose of increasing the stability of the red blood cells and extending the storage time of the red blood cells. And the reagents involved in the present invention are all conventional reagents and are easy to purchase. The treated red blood cells are preserved using a self-prepared preservation solution and stored in an environment of 2-8°C to obtain a red blood cell simulant suspension, which can also be mixed with other white blood cell simulants and platelet simulants to make a blood quality control or calibration material.

[0076] The concepts described herein may be implemented in other forms without departing from their spirit and characteristics. The specific embodiments disclosed should be considered illustrative rather than restrictive. Therefore, the scope of the present invention is determined by the appended claims rather than by the preceding description. Any changes within the literal meaning and equivalent scope of the claims should fall within the scope of these claims.

Claims

1. A method for preparing red blood cell mimetic particles, characterized in that: The following steps are involved: (1) removing white blood cells and platelets from a blood sample by centrifugation and standing, and washing the sample to obtain a purified red blood cell suspension; (2) preparing a red blood cell treatment solution, wherein the red blood cell treatment solution comprises a treatment reagent A and a treatment reagent B, wherein the treatment reagent A comprises: an alkaloid, a heavy metal salt, a buffer system and a preservative, and the osmotic pressure of the treatment reagent A is controlled within a range of 220-1500 mOsm / L, and the pH value is controlled within a range of 6-10; the treatment reagent B comprises: a fluoride, an aldehyde and a buffer system, and the osmotic pressure of the treatment reagent B is controlled within a range of 220-1500 mOsm / L, and the pH value is controlled within a range of 6-10; (3) taking an appropriate amount of the red blood cell suspension and mixing it with the treatment reagent A in a certain proportion, and then placing it in a 37° C. environment for treatment for 1 hour; (4) washing the red blood cells treated in step (3), mixing with the treatment reagent B in a certain proportion, and leaving at room temperature for 0.5 h or 1 h; (5) Washing and preserving the red blood cells treated in step (4) to obtain red blood cell mimetic particles.

2. The method for preparing red blood cell mimetic particles according to claim 1, characterized in that: The method of allowing the blood sample to stand in an upright position for 3 days is as follows:

3. The method for preparing red blood cell mimetic particles according to claim 1, characterized in that: The 37°C environment in step (3) is a 37°C water bath or a 37°C constant temperature incubator.

4. The method for preparing red blood cell mimetic particles according to claim 1, characterized in that: The method of preserving red blood cells in step (5) is: diluting with a self-prepared preservation solution and storing at 2-8°C, wherein the preservation solution comprises a buffer system, sugars, a preservative and a dispersant.

5. The method for preparing red blood cell mimetic particles according to claim 1, characterized in that: The treatment reagent A comprises: 3 g / L solanine, 1 g / L sodium nitrite, 0.2 g / L potassium dihydrogen phosphate, 2.9 g / L disodium hydrogen phosphate dodecahydrate, 8 g / L sodium chloride, 5 g / L PC300 and 1 L pure water; the treatment reagent B comprises: 0.4 g / L sodium dihydrogen phosphate, 5.8 g / L disodium hydrogen phosphate, 5 g / L sodium chloride, 0.3 g / L formaldehyde, 2 g / L sodium fluoride and 1 L pure water.

6. The method for preparing red blood cell mimetic particles according to claim 5, characterized in that: In the step (3), the red blood cell suspension is mixed with the treatment reagent A in a ratio of 1:1; in the step (4), the washed sample solution is mixed with the treatment reagent B in a ratio of 1:

2.

7. The method for preparing red blood cell mimetic particles according to claim 1, characterized in that: The treatment reagent A comprises: 4.5 g / L of scopolamine, 2.5 g / L of mercuric chloride, 12 g / L of sodium citrate, 1 g / L of sodium thimerosal and 1 L of pure water; the treatment reagent B comprises: 10 g / L of sodium citrate, 0.6 g / L of paraformaldehyde, 0.5 g / L of cesium fluoride and 1 L of pure water.

8. The method for preparing red blood cell mimetic particles according to claim 7, characterized in that: In the step (3), the red blood cell suspension is mixed with the treatment reagent A in a ratio of 1:2; in the step (4), the washed sample solution is mixed with the treatment reagent B in a ratio of 1:

1.

9. The method for preparing red blood cell mimetic particles according to claim 1, characterized in that: The treatment reagent A comprises: 8 g / L liensinine, 1.5 g / L copper sulfate, 0.8 g / L methylisothiazolinone and 1 L Tris buffer solution; the treatment reagent B comprises: 0.8 g / L beryllium fluoride, 0.08 g / L glutaraldehyde and 1 L Tris buffer solution.

10. The method for preparing red blood cell mimetic particles according to claim 9, characterized in that: In the step (3), the red blood cell suspension is mixed with the treatment reagent A in a ratio of 1:2; in the step (4), the washed sample solution is mixed with the treatment reagent B in a ratio of 1:

2.

11. A red blood cell mimetic particle, characterized in that: The red blood cell mimetic particles are prepared by the preparation method according to any one of claims 1-10.

12. A blood cell analysis quality control material or calibrator, characterized in that: The invention comprises red blood cell mimetic particles prepared according to the preparation method according to any one of claims 1 to 10.

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