METHOD FOR PREPARING RETICULOCYTE SIMULATING PARTICLES AND PLATELET SIMULATING PARTICLES, AND REFERENCE CONTROL
20190352609 ยท 2019-11-21
Assignee
Inventors
Cpc classification
G01N1/30
PHYSICS
G01N1/28
PHYSICS
International classification
G01N1/30
PHYSICS
Abstract
Provided are a method for preparing reticulocyte simulating particles and platelet simulating particles, and a reference control. The method for preparing the reticulocyte simulating particles comprises: staining mammalian anucleated red blood cells having a volume of 60-120 fL with a protein fluorescent dye activated by N-hydroxysuccinimide, and fixing the anucleated red blood cells to prepare the reticulocyte simulating particles. The platelet simulating particles are prepared from mammalian anucleated red blood cells having a volume of 2-25 fL, and the steps of preparing the platelet simulating particles are the same as that for the reticulocyte simulating particles. The preparation method comprises: using an protein fluorescent dye activated by N-hydroxysuccinimide to stain mammalian anucleated red blood cells having different volumes, so as to respectively obtain reticulocyte simulating particles and platelet simulating particles. The fluorescence and volume direction thereof in the scatter diagram are similar to the scatter diagram distribution of reticulocytes, reticulated platelets and platelets in fresh blood. The prepared simulating particles have good stability and do not interfere with the counting and differentiation of other cell particles.
Claims
1. A method for preparing reticulocyte simulating particles, comprising: staining mammalian anucleated red blood cells with a protein fluorescent dye having a carboxyl group activated by N-hydroxysuccinimide, and fixing the mammalian anucleated red blood cells to prepare the reticulocyte simulating particles, wherein the fixing is performed before or after the staining, and a volume of each of the mammalian anucleated red blood cells is 60-120 fL.
2. A method for preparing platelet simulating particles, comprising: staining mammalian anucleated red blood cells with a protein fluorescent dye having a carboxyl group activated by N-hydroxysuccinimide, and fixing the mammalian anucleated red blood cells to prepare the platelet simulating particles, wherein the fixing is performed before or after the staining, and a volume of each of the mammalian anucleated red blood cells is 2-25 fL.
3. The preparation method according to claim 1, wherein the staining of mammalian anucleated red blood cells with a protein fluorescent dye having a carboxyl group activated by N-hydroxysuccinimide comprises the following steps: extracting the mammalian anucleated red blood cells; providing the protein fluorescent dye activated by N-hydroxysuccinimide, wherein the protein fluorescent dye has an activatable carboxyl group, and the carboxyl group is activated by N-hydroxysuccinimide; incubating the mammalian anucleated red blood cells with the protein fluorescent dye activated by N-hydroxysuccinimide for staining the mammalian anucleated red blood cells, thus obtaining stained anucleated red blood cells.
4. The preparation method according to claim 1, wherein the method further comprises washing the stained and fixed cells.
5. The preparation method according to claim 1, wherein the protein fluorescent dye having a carboxyl group activated by N-hydroxysuccinimide has a structure represented by general formula I, ##STR00011## in general formula I, R is a protein fluorescent dye, n is an integer of 1-4, and n depends on the number of carboxyl groups in the protein fluorescent dye.
6. The preparation method according to claim 1, wherein the protein fluorescent dye has at least one of structures represented by general formula II, general formula III, general formula IV and general formula V, ##STR00012## in general formula II to general formula V, m is 0, 1, 2 or 3, and R.sub.1 and R.sub.2 are each independently selected from sulfonic acid group, halogen atom, or hydrogen atom; R.sub.3 and R.sub.4 are each independently selected from alkyl or carboxyl-containing alkyl; at least one of R.sub.3 and R.sub.4 is carboxyl-containing alkyl; R.sub.5 and R.sub.6 are each independently selected from sulfonic acid group, halogen atom, alkyl, or hydrogen atom.
7. The preparation method according to claim 1, wherein the protein fluorescent dye comprises at least one of fluorescent dyes A-H, structural formula of the fluorescent dye A is ##STR00013## structural formula of the fluorescent dye B is ##STR00014## structural formula of the fluorescent dye C is ##STR00015## structural formula of the fluorescent dye D is ##STR00016## structural formula of the fluorescent dye E is ##STR00017## structural formula of the fluorescent dye F is ##STR00018## structural formula of the fluorescent dye G is ##STR00019## structural formula of the fluorescent dye H is ##STR00020##
8. The preparation method according to claim 1, wherein a fixative used in the fixing comprises at least one of heavy metal salt, formaldehyde, pyruvic aldehyde, glutaraldehyde, and polyformaldehyde.
9. The preparation method according to claim 1, wherein the method further comprises sphering the mammalian anucleated red blood cells.
10-11. (canceled)
12. A reference control or calibrator for a blood analyzer, wherein the reference control or calibrator comprises reticulocyte simulating particles prepared by a method comprising: staining mammalian anucleated red blood cells with a protein fluorescent dye having a carboxyl group activated by N-hydroxysuccinimide, and fixing the mammalian anucleated red blood cells to prepare the reticulocyte simulating particles, wherein the fixing is performed before or after the staining, and a volume of each of the mammalian anucleated red blood cells is 60-120 fL.
13. The reference control or calibrator according to claim 12, wherein the reference control or calibrator further comprises at least one of the following: white blood cell simulating particles, red blood cell simulating particles, and nucleated red blood cell simulating particles.
14. A cell simulating particle for a blood analyzer, wherein the cell simulating particle is a mammalian anucleated red blood cell with a surface thereof cross-linked with at least one protein fluorescent dye molecule.
15-19. (canceled)
20. The preparation method according to claim 2, wherein the staining of mammalian anucleated red blood cells with a protein fluorescent dye having a carboxyl group activated by N-hydroxysuccinimide comprises the following steps: extracting the mammalian anucleated red blood cells; providing the protein fluorescent dye activated by N-hydroxysuccinimide, wherein the protein fluorescent dye has an activatable carboxyl group, and the carboxyl group is activated by N-hydroxysuccinimide; incubating the mammalian anucleated red blood cells with the protein fluorescent dye activated by N-hydroxysuccinimide for staining the mammalian anucleated red blood cells, thus obtaining stained anucleated red blood cells.
21. The preparation method according to claim 2, wherein the method further comprises washing the stained and fixed cells.
22. The preparation method according to claim 2, wherein the protein fluorescent dye having a carboxyl group activated by N-hydroxysuccinimide has a structure represented by general formula I, ##STR00021## in general formula I, R is a protein fluorescent dye, n is an integer of 1-4, and n depends on the number of carboxyl groups in the protein fluorescent dye.
23. The preparation method according to claim 2, wherein the protein fluorescent dye has at least one of structures represented by general formula II, general formula III, general formula IV and general formula V, ##STR00022## in general formula II to general formula V, m is 0, 1, 2 or 3, and R.sub.1 and R.sub.2 are each independently selected from sulfonic acid group, halogen atom, or hydrogen atom; R.sub.3 and R.sub.4 are each independently selected from alkyl or carboxyl-containing alkyl; at least one of R.sub.3 and R.sub.4 is carboxyl-containing alkyl; R.sub.5 and R.sub.6 are each independently selected from sulfonic acid group, halogen atom, alkyl, or hydrogen atom.
24. The preparation method according to claim 2, wherein the protein fluorescent dye comprises at least one of fluorescent dyes A-H, structural formula of the fluorescent dye A is ##STR00023## structural formula of the fluorescent dye B is ##STR00024## structural formula of the fluorescent dye C is ##STR00025## structural formula of the fluorescent dye D is ##STR00026## structural formula of the fluorescent dye E is ##STR00027## structural formula of the fluorescent dye F is ##STR00028## structural formula of the fluorescent dye G is ##STR00029## structural formula of the fluorescent dye H is ##STR00030##
25. The preparation method according to claim 2, wherein a fixative used in the fixing comprises at least one of heavy metal salt, formaldehyde, pyruvic aldehyde, glutaraldehyde, and polyformaldehyde.
26. The preparation method according to claim 2, wherein the method further comprises sphering the mammalian anucleated red blood cells.
27. The reference control or calibrator according to claim 12, wherein the reference control or calibrator further comprises platelet simulating particles prepared by a method comprising: staining mammalian anucleated red blood cells with a protein fluorescent dye having a carboxyl group activated by N-hydroxysuccinimide, and fixing the mammalian anucleated red blood cells to prepare the platelet simulating particles, wherein the fixing is performed before or after the staining, and a volume of each of the mammalian anucleated red blood cells is 2-25 fL.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0076] Protein fluorescent dyes used in the present application refer to fluorescent dyes used to stain proteins in the biotechnology field.
[0077] As found in the study on staining anucleated red blood cells with a protein fluorescent dye in the present application, after activating carboxyl group contained in the protein fluorescent dye by using N-hydroxysuccinimide and then incubating the anucleated red blood cells with the protein fluorescent dye, cell membranes of the anucleated red blood cells will not be destroyed, and the dye will be stably bound to the cell membranes, such that the anucleated red blood cells, which originally do not emit fluorescence signal, will emit fluorescence signal under laser irradiation, allowing the anucleated red blood cells to be used as raw materials for preparing some blood reference controls which need to generate fluorescence signal during detection. Moreover, mammalian anucleated red blood cells having different volumes can be used to respectively prepare reticulocyte simulating particles and platelet simulating particles containing reticulated platelets. Herein, the protein fluorescent dye activated by N-hydroxysuccinimide, which is applicable to the present application, is represented by general formula I. Preferably, the protein fluorescent dye has at least one of the structures represented by general formula II, general formula III, general formula IV and general formula V. More preferably, the protein fluorescent dye comprises at least one of fluorescent dyes A-H disclosed in the present application.
[0078] Compared with the method of using plastic particles or liposome cross-linked dyes to simulate cells, the present application uses anucleated red blood cells as raw materials to prepare simulating particles with membrane properties similar to that of reticulocytes and reticulated platelets, which can meet reference control requirements of hemolytic and non-hemolytic detection channels. The simulating particles prepared in the present application not only have good stability, but also do not influence counting and differentiation of other cell particles. In addition, in the preparation method of the present application as shown in
[0079] The present application will be further described below in detail through specific embodiments and drawings. The following embodiments are only used for further illustration of the present application and should not be construed as limitations to the present application.
[0080] Unless otherwise specified, the instruments, equipments and solutions used in the embodiments are conventional selections. In the embodiments, the blood cell analyzer is a Mindray BC-6 series blood cell analyzer.
Embodiment 1
[0081] In this embodiment, the fluorescent dye A is activated by N-hydroxysuccinimide and dissolved in an organic solvent to prepare a mother solution of the fluorescent dye A. In this embodiment, specific steps of synthesizing the activated dye comprise: adding the dye A, N-hydroxysuccinimide (NHS) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC) into dichloromethane and stirring them to react for 1 hour at normal temperature, thus obtaining the activated dye A. In other embodiments, other organic solvents, such as methanol, may also be used according to the type of dyes. Reaction conditions may be adjusted according to conventional technical means in the field.
[0082] The preservation solution used in this embodiment is a commercially available conventional preservation solution, and the preservation solution used in the following embodiment s is the same as that in this embodiment.
[0083] A method for preparing reticulocyte simulating particles comprises the following steps:
[0084] 1. Human red blood cells were taken as raw materials and filtered, and the filtered red blood cells (abbreviated as RBCs) were collected.
[0085] 2. The RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 2.0 g/L disodium hydrogen phosphate, 0.2 g/L sodium dihydrogen phosphate and 9.0 g/L sodium chloride.
[0086] 3. The cell pellet was suspended in the washing solution I, and the mother solution of the fluorescent dye A was added until a final concentration of the fluorescent dye A reached 100 mg/L. The fluorescent dye was then added to the surface of the cell pellet and left standing for 3 minutes, so the fluorescent dye was in full contact with the cells and then mixed with the cells. After reaction, centrifugation was performed to discard the supernatant, a 4 times volume of a washing solution II was added, and centrifugation was performed to discard the supernatant, wherein the washing solution II was a solution containing 35 g/L sodium citrate and 0.2 g/L citric acid.
[0087] 4. The cell pellet was suspended in the washing solution II, a fixative containing 37-40% formaldehyde was added until a final concentration thereof reached 0.1%. And the cells were treated at room temperature for 2 hours. After fixing, the washing solution II was added until an appropriate concentration of the RBCs was reached, and then centrifugation was performed to discard the supernatant, and washing was repeated twice.
[0088] 5. The supernatant was discarded, and a preservation solution was added for suspending the RBCs until an appropriate concentration of the RBCs was reached, so as to obtain reticulocyte simulating particles. The simulating particles were refrigerated at 2-8 C., the supernatant preservation solution was replaced once every 24 hours, and after 3 times' replacement, the prepared reticulocyte simulating particles could be preserved for a long time in the preservation solution.
[0089] A method for preparing platelet simulating particles comprises the following steps:
[0090] 1. Goat blood cells were taken as raw materials and filtered, and the filtered goat red blood cells (abbreviated as RBCs) were collected.
[0091] 2. The goat RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 1.44 g/L disodium hydrogen phosphate, 0.27 g/L potassium dihydrogen phosphate, 8 g/L sodium chloride and 0.2 g/L potassium chloride.
[0092] 3. The cell pellet was suspended in the washing solution I, the mother solution of the fluorescent dye A was added until a final concentration of the fluorescent dye A reached 50 mg/L, and mixing was quickly performed after adding the fluorescent dye A. The cells were labeled at room temperature for 1 hour, during which mixing was performed 3-4 times to make the fluorescent dye fully contact with the cells. After reaction, an equal volume of a washing solution II was added for dilution, centrifugation was performed to discard the supernatant, and washing with the washing solution II was repeated once, wherein the washing solution II was a solution containing 35 g/L sodium citrate and 0.2 g/L citric acid.
[0093] 4. The cell pellet was suspended in the washing solution II, a fixative containing 37-40% formaldehyde was added until a final concentration thereof reached 0.05%, and the cells were treated at room temperature for 2 hours.
[0094] 5. After fixing, an equal volume of the washing solution II containing bovine serum albumin (abbreviated as BSA) with a final concentration of 0.05% was added for dilution. Centrifugation was performed to discard the supernatant, and washing was repeated twice.
[0095] 6. A preservation solution was added for suspending the PLTs until an appropriate concentration of the PLTs was reached, and the PLTs were refrigerated at 2-8 C., thus the preparation of platelet simulating particles containing reticulated platelets was completed.
[0096] Formulation of Combined Reference Controls:
[0097] White blood cell simulating particles, red blood cell simulating particles and nucleated red blood cell simulating particles were added into the reticulocyte simulating particles and platelet simulating particles containing reticulated platelets prepared in this embodiment, and they were mixed in proportion to form a reference control. The concentration of each component was as follows:
[0098] low-value: 2.810.sup.9/L white blood cell simulating particles, 2.810.sup.12/L red blood cell simulating particles, 7510.sup.9/L platelet simulating particles, 0.0210.sup.12/L reticulocyte simulating particles, and 0.1110.sup.9/L nucleated red blood cell simulating particles;
[0099] mid-value: 8.510.sup.9/L white blood cell simulating particles, 4.510.sup.12/L red blood cell simulating particles, 22510.sup.9/L platelet simulating particles, 0.1110.sup.12/L reticulocyte simulating particles, and 0.3510.sup.9/L nucleated red blood cell simulating particles; and
[0100] high-value: 2010.sup.9/L white blood cell simulating particles, 5.210.sup.12/L red blood cell simulating particles, 49010.sup.9/L platelet simulating particles, 0.2610.sup.12/L reticulocyte simulating particles, and 0.810.sup.9/L nucleated red blood cell simulating particles.
[0101] The reticulocyte simulating particles, the platelet simulating particles and the reference controls prepared in this embodiment were respectively detected by using a blood cell analyzer based on fluorescence staining as detection principle. Scatter diagrams of the reticulocyte simulating particles and the platelet simulating particles are shown in
Embodiment 2
[0102] The fluorescent dye B was activated by N-hydroxysuccinimide and dissolved in an organic solvent to prepare a mother solution of the fluorescent dye B.
[0103] Preparation of Reticulocyte Simulating Particles:
[0104] 1. Human red blood cells were taken as raw materials and filtered, and the filtered RBCs were collected.
[0105] 2. The RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 1.41 g/L disodium hydrogen phosphate, 0.27 g/L potassium dihydrogen phosphate and 11.8 g/L potassium chloride.
[0106] 3. The cell pellet was suspended in a potassium dichromate solution with a final concentration of 0.1 g/L, and a fixative containing 50% glutaraldehyde was added until a final concentration thereof reached 0.005%. And the fixing was performed at room temperature for 2 hours. After the fixing, the RBCs were centrifuged and washed twice with the washing solution I.
[0107] 4. The cell pellet was suspended in a washing solution II and the mother solution of the fluorescent dye B was added until a final concentration of the fluorescent dye B reached 50 mg/L, and mixing was quickly performed after adding the fluorescent dye B. The cells were labeled at room temperature for 2 hours, during which mixing was performed 3-4 times to make the fluorescent dye fully contact with the cells. After reaction, a 4 times volume of the washing solution II of was added for dilution, centrifugation was performed to discard the supernatant, and washing with the washing solution II was repeated once, wherein the washing solution II was a solution containing 17 g/L sodium citrate, 0.1 g/L citric acid and 5 g/L sodium chloride.
[0108] 5. The supernatant was discarded, and a preservation solution was added for suspending the RBCs until an appropriate concentration of the RBCs was reached, so as to obtain reticulocyte simulating particles. The simulating particles were refrigerated at 2-8 C., the supernatant preservation solution was replaced once every 24 hours, and after 3 times' replacement, the prepared reticulocyte simulating particles could be preserved for a long time in the preservation solution.
[0109] Preparation of Platelet Simulating Particles:
[0110] 1. Goat blood cells were taken as raw materials and filtered, and the filtered goat RBCs were collected.
[0111] 2. The RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 2.0 g/L disodium hydrogen phosphate, 0.2 g/L sodium dihydrogen phosphate and 9.0 g/L sodium chloride.
[0112] 3. The cell pellet was suspended in the washing solution I, the mother solution of the fluorescent dye B was added until a final concentration of the fluorescent dye B reached 5 mg/L, and mixing was quickly performed after adding the fluorescent dye B. The cells were labeled at room temperature for 15 minutes, during which mixing was performed 3-4 times to make the fluorescent dye fully contact with the cells. After reaction, an equal volume of a washing solution II was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated twice, wherein the washing solution II was solution containing 17 g/L sodium citrate, 0.1 g/L citric acid and 5 g/L sodium chloride.
[0113] 4. The cell pellet was suspended in the washing solution II, a fixative containing 50% glutaraldehyde was added until a final concentration thereof reached 0.005%, and a fixative containing 37-40% formaldehyde was added until a final concentration thereof reached 0.1%, and the cells were treated at room temperature overnight.
[0114] 5. After fixing, an equal volume of the washing solution II containing BSA with a final concentration of 0.025% was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated twice.
[0115] 6. A preservation solution was added for suspending the PLTs until an appropriate concentration of the PLTs was reached, and the PLTs were refrigerated at 2-8 C., thus the preparation of platelet simulating particles containing reticulated platelets was completed.
[0116] Formulation of Combined Reference Controls:
[0117] White blood cell simulating particles, red blood cell simulating particles and nucleated red blood cell simulating particles were added into the reticulocyte simulating particles and platelet simulating particles prepared in this embodiment, and they were mixed in proportion to form a combined reference control, such that the concentration of each component was as follows:
[0118] mid-value: 8.510.sup.9/L white blood cell simulating particles, 4.510.sup.12/L red blood cell simulating particles, 22510.sup.9/L platelet simulating particles, 0.1110.sup.12/L reticulocyte simulating particles, and 0.3510.sup.9/L nucleated red blood cell simulating particles.
[0119] The reticulocyte simulating particles, the platelet simulating particles and the reference controls prepared in this embodiment were respectively detected by using a blood cell analyzer based on fluorescence staining as detection principle. Scatter diagrams of the reticulocyte simulating particles and the platelet simulating particles are shown in
Embodiment 3
[0120] The fluorescent dye C was activated by N-hydroxysuccinimide and dissolved in an organic solvent to prepare a mother solution of the fluorescent dye C.
[0121] Preparation of Reticulocyte Simulating Particles:
[0122] 1. Human red blood cells were taken as raw materials and filtered, and the filtered RBCs were collected.
[0123] 2. The RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 1.44 g/L disodium hydrogen phosphate, 0.27 g/L potassium dihydrogen phosphate, 8 g/L sodium chloride and 0.2 g/L potassium chloride.
[0124] 3. The cell pellet was suspended in the washing solution I, and the mother solution of the fluorescent dye C was added until a final concentration of the fluorescent dye C reached 100 mg/L, and mixing was quickly performed after adding the fluorescent dye C. The cells were labeled at room temperature for 20 minutes, during which mixing was performed 3-4 times to make the fluorescent dye fully contact with the cells. After reaction, a 4 times volume of a washing solution II was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated once, wherein the washing solution II was a solution containing 17 g/L sodium citrate, 0.1 g/L citric acid and 5 g/L sodium chloride.
[0125] 4. The cell pellet was suspended in the washing solution II, a fixative containing 50% glutaraldehyde was added until a final concentration thereof reached 0.05%, and the cells were treated at room temperature for 2 hours. After fixing, the washing solution II containing BSA with a final concentration of 1% was added for diluting the RBCs until an appropriate concentration of the RBCs was reached. Centrifugation was performed to discard supernatant, and washing was repeated twice.
[0126] 5. A preservation solution was added for suspending the RBCs until an appropriate concentration of the RBCs was reached, so as to obtain reticulocyte simulating particles in this embodiment.
[0127] Preparation of Platelet Simulating Particles:
[0128] 1. Goat blood cells were taken as raw materials and filtered, and the filtered goat RBCs were collected.
[0129] 2. The goat RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 2.0 g/L disodium hydrogen phosphate, 0.2 g/L sodium dihydrogen phosphate and 9.0 g/L sodium chloride.
[0130] 3. The cell pellet was suspended in the washing solution I, a certain volume of the mother solution of the fluorescent dye C was added until a final concentration of the fluorescent dye C reached 5 mg/L, and mixing was quickly performed after adding the fluorescent dye C. The cells were labeled at room temperature for 15 minutes, during which mixing was performed 3-4 times to make the fluorescent dye fully contact with the cells. After reaction, an equal volume of a washing solution II was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated once, wherein the washing solution II was solution containing 17 g/L sodium citrate, 0.1 g/L citric acid and 5 g/L sodium chloride.
[0131] 4. The cell pellet was suspended in the washing solution II, a fixative containing 37-40% formaldehyde was added until a final concentration thereof reached 0.25%, and the cells were treated at room temperature treatment for 4 hours.
[0132] 5. After fixing, an equal volume of the washing solution II containing BSA with a final concentration of 0.05% was added for dilution. Centrifugation was performed to discard the supernatant, and washing was repeated twice.
[0133] 6. A preservation solution was added for suspending the PLTs until an appropriate concentration of the PLTs was reached, and the PLTs were refrigerated at 2-8 C., thus the preparation of platelet simulating particles containing reticulated platelets was completed.
[0134] Formulation of Combined Reference Controls:
[0135] White blood cell simulating particles, red blood cell simulating particles and nucleated red blood cell simulating particles were added into the reticulocyte simulating particles and platelet simulating particles prepared in this example, and they were mixed in proportion to form a combined reference control material, such that the concentration of each component was as follows:
[0136] mid-value: 8.510.sup.9/L white blood cell simulating particles, 4.510.sup.12/L red blood cell simulating particles, 22510.sup.9/L platelet simulating particles, 0.1110.sup.12/L reticulocyte simulating particles, and 0.3510.sup.9/L nucleated red blood cell simulating particles.
[0137] The reticulocyte simulating particles, the platelet simulating particles and the reference controls prepared in this example were respectively detected by using a blood cell analyzer based on fluorescence staining as detection principle. Scatter diagrams of the reticulocyte simulating particles and the platelet simulating particles are shown in
Embodiment 4
[0138] The fluorescent dye D was activated by N-hydroxysuccinimide and dissolved in an organic solvent to prepare a mother solution of the fluorescent dye D.
[0139] Preparation of Reticulocyte Simulating Particles:
[0140] 1. Human red blood cells were taken as raw materials and filtered, and the filtered RBCs were collected.
[0141] 2. The RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 1.44 g/L disodium hydrogen phosphate, 0.27 g/L potassium dihydrogen phosphate, 8 g/L sodium chloride and 0.2 g/L potassium chloride.
[0142] 3. The cell pellet was suspended in the washing solution I, and the mother solution of the fluorescent dye D was added until a final concentration of the fluorescent dye D reached 40 mg/L, and mixing was quickly performed after adding the fluorescent dye D. The cells were labeled at 2-8 C. for 30 minutes, during which mixing was performed 1-2 times to make the fluorescent dye fully contact with the cells. After reaction, a 4 times volume of a washing solution II was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated once, wherein the washing solution II was solution containing 17 g/L sodium citrate, 0.1 g/L citric acid and 5 g/L sodium chloride.
[0143] 4. The cell pellet was suspended in the washing solution II, a fixative containing 50% glutaraldehyde was added until a final concentration thereof reached 0.05%, and the cells were treated at room temperature overnight. After fixing, the washing solution II containing BSA with a final concentration of 1% was added for diluting the RBCs until an appropriate concentration of the RBCs was reached, centrifugation was performed to discard supernatant, and washing was repeated twice.
[0144] 5. A preservation solution was added for suspending the RBCs until an appropriate concentration of the RBCs was reached, so as to obtain reticulocyte simulating particles.
[0145] Preparation of Platelet Simulating Particles:
[0146] 1. Goat blood cells were taken as raw materials and filtered, and the filtered goat RBCs were collected.
[0147] 2. The goat RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 1.44 g/L disodium hydrogen phosphate, 0.27 g/L potassium dihydrogen phosphate, 8 g/L sodium chloride and 0.2 g/L potassium chloride.
[0148] 3. The cell pellet was suspended in the washing solution I, a certain volume of the mother solution of the fluorescent dye D was added to until a final concentration of the fluorescent dye D reached 20 mg/L, and mixing was quickly performed after adding the fluorescent dye D. The cells were labeled at room temperature for 1 hour, during which mixing was performed 3-4 times to make the fluorescent dye fully contact with the cells. After reaction, an equal volume of a washing solution II was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated twice, wherein the washing solution II was a solution containing 35 g/L sodium citrate and 0.2 g/L citric acid;
[0149] 4. The cell pellet was suspended in the washing solution II, a fixative containing 50% glutaraldehyde was added until a final concentration thereof reached 0.03%, and a fixative containing 37-40% formaldehyde was added until a final concentration thereof reached 0.15%, and the cells were treated at room temperature treatment for 8 hours.
[0150] 5. After fixing, an equal volume of the washing solution II containing BSA with a final concentration of 0.15% was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated twice.
[0151] 6. A preservation solution was added for suspending the PLTs until an appropriate concentration of the PLTs was reached, and the PLTs were refrigerated at 2-8 C., thus the preparation of platelet simulating particles containing reticulated platelets was completed.
[0152] Formulation of Combined Reference Controls:
[0153] White blood cell simulating particles, red blood cell simulating particles and nucleated red blood cell simulating particles were added into the reticulocyte simulating particles and platelet simulating particles prepared in this embodiment, and they were mixed in proportion to form a combined reference control, such that the concentration of each component was as follows:
[0154] mid-value: 8.510.sup.9/L white blood cell simulating particles, 4.510.sup.12/L red blood cell simulating particles, 22510.sup.9/L platelet simulating particles, 0.1110.sup.12/L reticulocyte simulating particles, and 0.3510.sup.9/L nucleated red blood cell simulating particles.
[0155] The reticulocyte simulating particles, the platelet simulating particles and the reference controls prepared in this embodiment were respectively detected by using a blood cell analyzer based on fluorescence staining as detection principle. Scatter diagrams of the reticulocyte simulating particles and the platelet simulating particles are shown in
Embodiment 5
[0156] The fluorescent dye E was activated by N-hydroxysuccinimide and dissolved in an organic solvent to prepare a mother solution of the fluorescent dye E.
[0157] Preparation of Reticulocyte Simulating Particles:
[0158] 1. Human red blood cells were taken as raw materials and filtered, and the filtered RBCs were collected.
[0159] 2. The RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 1.44 g/L disodium hydrogen phosphate, 0.27 g/L potassium dihydrogen phosphate, 8 g/L sodium chloride and 0.2 g/L potassium chloride.
[0160] 3. The cell pellet was suspended in the washing solution I, and the mother solution of the fluorescent dye E was added until a final concentration of the fluorescent dye E reached 15 mg/L, and mixing was quickly performed after adding the fluorescent dye E. The cells were labeled at room temperature for 30 minutes, during which mixing was performed 3-4 times to make the fluorescent dye fully contact with the cells. After reaction, a 4 times volume of a washing solution I was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated once.
[0161] 4. The cell pellet was suspended in the washing solution II, a 37-40% formaldehyde was added until a final concentration thereof reached 0.1%, and the cells were treated at room temperature for 1 hour. After mixing, the washing solution II containing BSA with a final concentration of 0.5% was added for diluting the RBCs until an appropriate concentration of the RBCs was reached. Centrifugation was then performed to discard supernatant, and washing was repeated twice, wherein the washing solution II was a solution containing 17 g/L sodium citrate, 0.1 g/L citric acid and 5 g/L sodium chloride.
[0162] 5. A preservation solution was added for suspending the RBCs until an appropriate concentration of the RBCs was reached, so as to obtain reticulocyte simulating particles.
[0163] Preparation of Platelet Simulating Particles:
[0164] 1. Goat blood cells were taken as raw materials and filtered, and the filtered goat RBCs were collected.
[0165] 2. The goat RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 2.0 g/L disodium hydrogen phosphate, 0.2 g/L sodium dihydrogen phosphate and 9.0 g/L sodium chloride.
[0166] 3. The cell pellet was suspended in the washing solution I, a certain volume of the mother solution of the fluorescent dye E was added until a final concentration of the fluorescent dye E reached 10 mg/L, and mixing was quickly performed after adding the fluorescent dye E. The cells were labeled at room temperature for 1 hour, during which mixing was performed 3-4 times to make the fluorescent dye fully contact with the cells. After reaction, an equal volume of a washing solution II was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated once, wherein the washing solution II was a solution containing 17 g/L sodium citrate, 0.1 g/L citric acid and 5 g/L sodium chloride.
[0167] 4. The cell pellet was suspended in the washing solution II, a fixative containing 37-40% formaldehyde was added until a final concentration thereof reached 0.8%, and the cells were treated at room temperature treatment overnight.
[0168] 5. After fixing, the washing solution II containing BSA with a final concentration of 0.05% was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated twice.
[0169] 6. A preservation solution was added for suspending the PLTs until an appropriate concentration of the PLTs was reached, and the PLTs were refrigerated at 2-8 C., thus the preparation of platelet simulating particles containing reticulated platelets was completed.
[0170] Formulation of Combined Reference Controls:
[0171] White blood cell simulating particles, red blood cell simulating particles and nucleated red blood cell simulating particles were added into the reticulocyte simulating particles and platelet simulating particles prepared in this embodiment, and they were mixed in proportion to form a combined reference control, such that the concentration of each component was as follows:
[0172] mid-value: 8.510.sup.9/L white blood cell simulating particles, 4.510.sup.12/L red blood cell simulating particles, 22510.sup.9/L platelet simulating particles, 0.1110.sup.12/L reticulocyte simulating particles, and 0.3510.sup.9/L nucleated red blood cell simulating particles.
[0173] The reticulocyte simulating particles, the platelet simulating particles and the reference controls prepared in this embodiment were respectively detected by using a blood cell analyzer based on fluorescence staining as detection principle. Scatter diagrams of the reticulocyte simulating particles and the platelet simulating particles are shown in
Embodiment 6
[0174] The fluorescent dye F was activated by N-hydroxysuccinimide and dissolved in an organic solvent to prepare a mother solution of the fluorescent dye F.
[0175] Preparation of reticulocyte simulating particles:
[0176] 1. Human red blood cells were taken as raw materials and filtered, and the filtered RBCs were collected.
[0177] 2. The RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 1.44 g/L disodium hydrogen phosphate, 0.27 g/L potassium dihydrogen phosphate, 8 g/L sodium chloride and 0.2 g/L potassium chloride.
[0178] 3. The cell pellet was suspended in the washing solution I, the mother solution of the fluorescent dye F was added until a final concentration of the fluorescent dye F reached 55 mg/L, and the fluorescent dye was slowly added to the surface of the cells and left standing for 5 minutes, then mixing was performed. The cells were labeled at 2-8 C. for 30 minutes, so the fluorescent dye was in full contact with the cells. After reaction, a 4 times volume of a washing solution II was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated once, wherein the washing solution II was a solution containing 17 g/L sodium citrate, 0.1 g/L citric acid and 5 g/L sodium chloride.
[0179] 4. The cell pellet was suspended in the washing solution II, a fixative containing 50% glutaraldehyde was added until a final concentration thereof reached 0.05%, and the cells were treated at room temperature for 3 hours. After fixing, the washing solution II containing BSA with a final concentration of 0.6% was added for diluting the RBCs until an appropriate concentration of the RBCs was reached. Centrifugation was performed to discard supernatant, and washing was repeated twice.
[0180] 5. A preservation solution was added for suspending the RBCs until an appropriate concentration of the RBCs was reached, so as to obtain reticulocyte simulating particles.
[0181] Preparation of Platelet Simulating Particles:
[0182] 1. Goat blood cells were taken as raw materials and filtered, and the filtered goat RBCs were collected.
[0183] 2. The goat RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 2.0 g/L disodium hydrogen phosphate, 0.2 g/L sodium dihydrogen phosphate and 9.0 g/L sodium chloride;
[0184] 3. The cell pellet was suspended in the washing solution I, and the mother solution of the fluorescent dye F was added until a final concentration of the fluorescent dye reached 15 mg/L, and the fluorescent dye was slowly added to the surface of the cells and left standing for 8 minutes, then mixing was performed. The cells were labeled at room temperature for 15 minutes, during which mixing was performed 3-4 times to make the fluorescent dye fully contact with the cells. After reaction, a 4 times volume of a washing solution II was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated once, wherein the washing solution II was a solution containing 17 g/L sodium citrate, 0.1 g/L citric acid and 5 g/L sodium chloride.
[0185] 4. The cell pellet was suspended in the washing solution II, a fixative containing glutaraldehyde was added until a final concentration thereof reached 0.015%, and the cells were treated at room temperature overnight.
[0186] 5. After fixing, an equal volume of the washing solution II containing BSA with a final concentration of 0.025% was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated twice.
[0187] 6. A preservation solution was added for suspending the PLTs until an appropriate concentration of the PLTs was reached, and the PLTs were refrigerated at 2-8 C., thus the preparation of platelet simulating particles containing reticulated platelets was completed.
[0188] Formulation of Combined Reference Controls:
[0189] White blood cell simulating particles, red blood cell simulating particles and nucleated red blood cell simulating particles were added into the reticulocyte simulating particles and platelet simulating particles prepared in this embodiment, and they were mixed in proportion to form a combined reference control, such that the concentration of each component was as follows:
[0190] mid-value: 8.510.sup.9/L white blood cell simulating particles, 4.510.sup.12/L red blood cell simulating particles, 22510.sup.9/L platelet simulating particles, 0.1110.sup.12/L reticulocyte simulating particles, and 0.3510.sup.9/L nucleated red blood cell simulating particles.
[0191] The reticulocyte simulating particles, the platelet simulating particles and the reference controls prepared in this embodiment were respectively detected by using a blood cell analyzer based on fluorescence staining as detection principle. Scatter diagrams of the reticulocyte simulating particles and the platelet simulating particles are shown in
Embodiment 7
[0192] The fluorescent dye G was activated by N-hydroxysuccinimide and dissolved in an organic solvent to prepare a mother solution of the fluorescent dye G.
[0193] Preparation of Reticulocyte Simulating Particles:
[0194] 1. Human red blood cells were taken as raw materials and filtered, and the filtered RBCs were collected.
[0195] 2. The RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 1.44 g/L disodium hydrogen phosphate, 0.27 g/L potassium dihydrogen phosphate, 8 g/L sodium chloride and 0.2 g/L potassium chloride.
[0196] 3. The cell pellet was suspended in the washing solution I, a certain volume of the mother solution of the fluorescent dye G was added until a final concentration of the fluorescent dye G reached 85 mg/L, and mixing was quickly performed after adding the fluorescent dye G. The cells were labeled at 2-8 C. for 45 minutes, during which mixing was performed 1-2 times to make the fluorescent dye fully contact with the cells. A 4 times volume of a washing solution II was added after reaction, centrifugation was performed to discard the supernatant, and washing was repeated once, wherein the washing solution II was solution containing 17 g/L sodium citrate, 0.1 g/L citric acid and 5 g/L sodium chloride.
[0197] 4. The cell pellet was suspended in the washing solution II, a fixative containing 50% glutaraldehyde was added until a final concentration thereof reached 0.1%, and the cells were treated at room temperature for 1 hour. After fixing, the washing solution II containing BSA with a final concentration of 1% was added for diluting the RBCs until an appropriate concentration of the RBCs was reached. Centrifugation was then performed to discard the supernatant, and washing was repeated twice.
[0198] 5. A preservation solution was added for suspending the RBCs until an appropriate concentration of the RBCs was reached, so as to obtain reticulocyte simulating particles.
[0199] Preparation of Platelet Simulating Particles:
[0200] 1. Goat blood cells were taken as raw materials and filtered, and the filtered goat RBCs were collected.
[0201] 2. The goat RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 1.44 g/L disodium hydrogen phosphate, 0.27 g/L potassium dihydrogen phosphate, 8 g/L sodium chloride and 0.2 g/L potassium chloride.
[0202] 3. The cell pellet was suspended in the washing solution I, and the mother solution of the fluorescent dye G was added until a final concentration of the fluorescent dye G reached 35 mg/L, and mixing was slowly performed after adding the fluorescent dye G. The cells were labeled at room temperature for 30 minutes, during which mixing was performed 3-4 times to make the fluorescent dye fully contact with the cells. A 4 times volume of a washing solution II was added for dilution after reaction, centrifugation was performed to discard the supernatant, and washing was repeated once, wherein the washing solution II was a solution containing 17 g/L sodium citrate, 0.1 g/L citric acid and 5 g/L sodium chloride.
[0203] 4. The cell pellet was suspended in the washing solution II, a fixative containing 37-40% formaldehyde was added until a final concentration thereof reached 0.3%, and the cells were treated at room temperature for 4 hours.
[0204] 5. After the fixing, an equal volume of a washing solution II containing BSA with a final concentration of 0.05% was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated twice.
[0205] 6. A preservation solution was added for suspending the PLTs until an appropriate concentration of the PLTs was reached, and the PLTs were refrigerated at 2-8 C., and the preparation of platelet simulating particles containing reticulated platelets was completed.
[0206] Formulation of Combined Reference Controls:
[0207] White blood cell simulating particles, red blood cell simulating particles and nucleated red blood cell simulating particles were added into the reticulocyte simulating particles and platelet simulating particles prepared in this embodiment, and they were mixed in proportion to form a combined reference control, such that the concentration of each component was as follows:
[0208] mid-value: 8.510.sup.9/L white blood cell simulating particles, 4.510.sup.12/L red blood cell simulating particles, 22510.sup.9/L platelet simulating particles, 0.1110.sup.12/L reticulocyte simulating particles, and 0.3510.sup.9/L nucleated red blood cell simulating particles.
[0209] The reticulocyte simulating particles, the platelet simulating particles and the reference controls prepared in this embodiment were respectively detected by using a blood cell analyzer based on fluorescence staining as detection principle. Scatter diagrams of the reticulocyte simulating particles and the platelet simulating particles are shown in
Embodiment 8
[0210] The fluorescent dye H was activated by N-hydroxysuccinimide and dissolved in an organic solvent to prepare a mother solution of the fluorescent dye H.
[0211] Preparation of reticulocyte simulating particles:
[0212] 1. Human red blood cells were taken as raw materials and filtered, and the filtered RBCs were collected.
[0213] 2. The RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 1.44 g/L disodium hydrogen phosphate, 0.27 g/L potassium dihydrogen phosphate, 8 g/L sodium chloride and 0.2 g/L potassium chloride.
[0214] 3. The cell pellet was suspended in the washing solution I, and a certain volume of the mother solution of the fluorescent dye H was added until a final concentration of the fluorescent dye H reached 25 mg/L, and the fluorescent dye was slowly added to the surface of the cells and left standing for 4 minutes, mixing was then performed. The cells were labeled at room temperature for 20 minutes, during which mixing was performed 1-2 times to make the fluorescent dye fully contact with the cells. An equal volume of the washing solution I was added after reaction, centrifugation was performed to discard the supernatant, and washing was repeated twice.
[0215] 4. The cell pellet was suspended in a washing solution II, a fixative containing 37-40% formaldehyde was added until a final concentration thereof reached 0.5%, and the cells were treated at room temperature for 2 hours. After fixing, the washing solution II containing BSA with a final concentration of 1% was added for diluting the RBCs until an appropriate concentration of the RBCs was reached. Centrifugation was then performed to discard the supernatant, and washing was repeated twice, wherein the washing solution II was solution containing 35 g/L sodium citrate and 0.2 g/L citric acid.
[0216] 5. A preservation solution was added for suspending the RBCs until an appropriate concentration of the RBCs was reached, so as to obtain reticulocyte simulating particles.
[0217] Preparation of Platelets:
[0218] 1. Goat blood cells were taken as raw materials and filtered, and the filtered goat RBCs were collected.
[0219] 2. The RBCs were centrifuged and washed twice with a washing solution I, wherein the washing solution I was a solution containing 2.0 g/L disodium hydrogen phosphate, 0.2 g/L sodium dihydrogen phosphate and 9.0 g/L sodium chloride.
[0220] 3. The cell pellet was suspended in the washing solution I, and a certain volume of the mother solution of the fluorescent dye H was added until a final concentration of the fluorescent dye H reached 5 mg/L, and the fluorescent dye was slowly added to the surface of the cells and left standing for 10 minutes, mixing was then performed. The cells were labeled at room temperature for 15 minutes, during which mixing was performed 1-2 times to make the fluorescent dye fully contact with the cells. An equal volume of a washing solution II was added for dilution after reaction, centrifugation was performed to discard the supernatant, and washing was repeated once, wherein the washing solution II was a solution containing 35 g/L sodium citrate and 0.2 g/L citric acid.
[0221] 4. The cell pellet was suspended in the washing solution II, a fixative containing 50% glutaraldehyde was added until a final concentration thereof reached 0.05%, and the cells were treated at room temperature for 4 hours.
[0222] 5. After fixing, an equal volume of the washing solution II containing BSA with a final concentration of 0.04% was added for dilution, centrifugation was performed to discard the supernatant, and washing was repeated twice.
[0223] 6. A preservation solution was added for suspending the PLTs until an appropriate concentration of the PLTs was reached, and the PLTs were refrigerated at 2-8 C., thus the preparation of platelet simulating particles containing reticulated platelets was completed.
[0224] Formulation of Combined Reference Controls:
[0225] White blood cell simulating particles, red blood cell simulating particles and nucleated red blood cell simulating particles were added into the reticulocyte simulating particles and platelet simulating particles prepared in this embodiment, and they were mixed in proportion to form a combined reference control material, such that the concentration of each component was as follows:
[0226] mid-value: 8.510.sup.9/L white blood cell simulating particles, 4.510.sup.12/L red blood cell simulating particles, 22510.sup.9/L platelet simulating particles, 0.1110.sup.12/L reticulocyte simulating particles, and 0.3510.sup.9/L nucleated red blood cell simulating particles.
[0227] The reticulocyte simulating particles, the platelet simulating particles and the reference controls prepared in this embodiment were respectively detected by using a blood cell analyzer based on fluorescence staining as detection principle. Scatter diagrams of the reticulocyte simulating particles and the platelet simulating particles are shown in
[0228] On the basis of the above embodiments, the combined reference controls prepared in the eight embodiments of the present application were refrigerated at 2-8 C. for three months and were periodically detected by using a blood cell analyzer every week. The results show that, the scatter diagrams of the reference controls prepared in the present application in each channel did not change significantly, wherein the scatter diagrams of the low-value, mid-value and high-value combined reference controls prepared in embodiment 1 measured after three months are shown in
[0229] While certain embodiments have been described, these embodiments have been presented by way of example only, and are not intended to limit the scope of the inventions. Indeed, the novel embodiments described herein may be embodied in a variety of other forms; furthermore, various omissions, substitutions and changes in the form of the embodiments described herein may be made without departing from the spirit of the inventions. The accompanying claims and their equivalents are intended to cover such forms or modifications as would fall within the scope and spirit of the inventions.