CN109142761B - Reticulocyte mimics and preparation method and application thereof - Google Patents
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Abstract
The invention relates to a reticulocyte mimic and a preparation method and application thereof, belonging to the technical field of blood quality control substances. Solves the problem of how to provide the reticulocyte mimics which are simple to operate, higher in yield and better in stability, and the preparation method and the application thereof. The preparation method 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; and step three, adding a fixing solution into the cells introduced with the carriers, fixing at room temperature, washing the cells, and preserving with a cell preservation solution to obtain the reticulocyte mimics. The preparation steps of the simulant are simple, the simulant is suitable for mass production, and the simulant can be used as a quality control substance for detecting reticulocytes of a full-automatic hematology analyzer.
Description
Technical Field
The invention belongs to the technical field of blood quality control substances, and particularly relates to a reticulocyte mimic and a preparation method and application thereof.
Background
Blood quality control products, consisting of single or multicomponent blood cells or blood cell mimetics, possess the same detectable characteristics as blood and can be used for routine monitoring of the accuracy and precision of blood analyzers.
Reticulocytes are cells in the transition stage of the red blood cell maturation process, are immature red blood cells, and are characterized in that a small amount of RNA remains in the cell nucleus and cytoplasm immediately after removal. The total number or percentage of reticulocytes is clinically significant for the diagnosis of anemia and its related diseases. Reticulocyte count is an important index reflecting the hematopoietic function of bone marrow, and is mainly shown in the following aspects.
1. Evaluating the myeloproliferative capacity and judging the type of anemia: increased reticuloendothelial redness, which indicates vigorous bone marrow hemopoiesis, is commonIt is indicated for various hyperplastic anemia such as iron deficiency anemia, megaloblastic (folic acid deficiency, vitamin B12 deficiency) anemia, blood loss anemia, especially hemolytic anemia, with the most significant increase>10 percent. Transient and rapid elevation of RET during hematopoietic recovery is a more sensitive indicator of bone marrow functional recovery. ② the net weaving red is reduced: commonly seen in aplastic anemia, reticulocyte counts are often below 0.005, with absolute values below 15 × 109/L。
2. Evaluation of the efficacy: firstly, the anemia curative effect is observed, and RET is one of the follow-up examination items of anemia patients; ② monitoring the hematopoietic recovery of bone marrow after bone marrow transplantation.
3. Monitoring of radiotherapy and chemotherapy: the dynamic observation of reticulocytes can guide the clinic to adjust the treatment scheme timely, so that the serious bone marrow suppression is avoided.
In summary, the various indices of reticulocytes are of great clinical significance in diagnosing anemia and evaluating therapeutic effects, and recent studies show that reticulocyte parameters are also of great clinical significance in detecting liver cirrhosis.
At present, reticulocytes are used as indexes for analysis and detection in middle and high-end hematology analyzers, the analysis of the reticulocytes in blood samples becomes an important component of blood analysis, and the quality control of reticulocyte counting by using a quality control substance is a precondition for ensuring the accuracy of the measurement result of the hematology analyzers, so that the development of an accurate reticulocyte quality control substance which can be used for a full-automatic hematology analyzer is very necessary.
Currently, there are several methods for preparing reticulocyte quality control substances:
1. reticulocyte of pig blood is taken as a raw material, and the reticulocyte is separated by a centrifugal separation method and other methods to prepare a reticulocyte quality control product (US5736402, US5858789, US5945340 and US 6444471).
2. The method comprises treating red blood cell membrane of mammal, injecting nucleic acid substance, simulating reticulocyte, and preparing reticulocyte quality control product (US6399388, US6406915, US5432089), wherein the obtained product is mixture of red blood cell and reticulocyte, the yield of reticulocyte depends on the injection ratio of nucleic acid, and the operation process is complicated.
3. The reticulocyte mimics (US7195919) are prepared by covalently bonding nucleic acid and other substances with the surface of an erythrocyte membrane, the storage period of the reticulocyte quality control product prepared by the method depends on the strength of connection of the nucleic acid and the cell surface, the period is difficult to guarantee during long-term storage, and the operation is complicated.
4. Separating and purifying mammal anucleated red blood cells, and preparing reticulocyte quality control product (CN101881778) by adding cell treatment liquid and fixing liquid, the preparation method has relatively simple operation process, but the preparation process utilizes protein denaturant to change the molecular structure in cells so as to achieve the adsorption effect with dye, and the effect degree of the protein denaturant can influence the yield of reticulocyte mimics.
Disclosure of Invention
In view of the above, the present invention provides a reticulocyte mimic with simple operation, higher yield and better stability, and a preparation method and applications thereof.
The preparation method of the reticulocyte mimics 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 diluent consists of a diluent I and a diluent II with the volume ratio of 1 (30-50); the diluent I is prepared from ATP and MgCl2And water, ATP concentration of 10-40%, MgCl2The concentration of (A) is 5-28%; the diluent II is prepared from KH2PO4、NaHCO3Glucose and water, the pH value of the diluent II is 7.0-7.5, KH2PO4Concentration of 0.5% -2.0%, NaHCO3The concentration is 0.05-0.2%, and the concentration of glucose is 0.01-0.1%;
and step three, adding a fixing solution into the cells into which the loading agent is introduced, fixing at 18-28 ℃, washing the cells, and preserving with a cell preservation solution to obtain the reticulocyte mimics.
Preferably, the red blood cell is a human red blood cell or a mammalian red blood cell having an MCV similar to a human red blood cell.
Preferably, the purified red blood cells are diluted to 10 deg.C with a diluent12-1014And (2) per liter.
Preferably, the loading agent is an artificially synthesized DNA fragment of 100bp to 2000bp or an artificially synthesized RNA fragment, and the dosage of the loading agent is 1.0 to 1.5 mu g/L.
Preferably, 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, the cell fixing solution is one or more of formaldehyde, glutaraldehyde, acetone and ethanol, and the fixing time is 3-5 hours.
Preferably, the washing solution used in the washing in the first step and the third step is PBS buffer, D-HANKS buffer, HEPES buffer, citric acid buffer, sodium chloride buffer or boric acid buffer.
Preferably, the cell preservation solution includes a buffer, nutrients, and preservatives.
The invention also provides the reticulocyte mimics prepared by the preparation method of the reticulocyte mimics.
The invention also provides the application of the reticulocyte mimics as a quality control substance for detecting the reticulocytes of a full-automatic hematology analyzer.
Compared with the prior art, the invention has the beneficial effects that:
the preparation method of the reticulocyte mimics introduces the nucleic acid substances into mature red blood cells by using the mode of stimulating the cells by electric pulses to prepare the reticulocyte mimics, and the mimics have simple preparation steps and are suitable for mass production.
The reticulocyte mimics are prepared by introducing nucleic acid or analogues thereof into human or mammal erythrocytes through pulse treatment, and then fixing the erythrocytes to simulate the state of the reticulocytes.
Drawings
FIG. 1 is a photograph showing the reticulocyte mimics prepared in example 1, which were tested on a blood cell analyzer using fluorescence staining of nucleic acids as a detection principle (RET channel only is provided).
Fig. 2 is a photograph showing the reticulocyte mimics prepared in example 2, which were tested on a hematology analyzer using fluorescence staining of nucleic acids as a detection principle (RET channel only).
FIG. 3 is a photograph showing the reticulocyte mimics prepared in example 3, which were tested on a hematology analyzer using fluorescence staining of nucleic acids as a detection principle (RET channel only).
Fig. 4 is a photograph of the reticulocyte mimics prepared in example 4 tested on a hematology analyzer using nucleic acid fluorescence staining as the detection principle (RET channel only).
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 reticulocyte mimics comprises the following steps: separating and washing red blood cells to obtain purified red blood cells; 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 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 preservation solution to obtain the reticulocyte mimics.
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 diluent consists of a diluent I and a diluent II, and the volume ratio of the diluent I to the diluent II is 1 (30-50), preferably 1 (32-42), and most preferably 1: 40. Wherein the diluent I is prepared from ATP and MgCl2And water; the concentration of ATP is 10% -40%, preferably 15% -35%, more preferably 20% -30%; MgCl2Is 5% to 28%, preferably 8% to 20%, more preferably 10% to 15%. The diluent II is prepared from KH2PO4、NaHCO3Glucose and water; the pH value of the diluent II is 7.0 to 7.5, preferably 7.2 to 7.4; KH (Perkin Elmer)2PO4The concentration of (A) is 0.5% -2.0%, preferably 0.8% -1.8%, more preferably 1.0% -1.4%; NaHCO 23Is 0.05% -0.2%, preferably 0.08% -0.18%, more preferably 0.1% -0.14%; the concentration of glucose is 0.01% -0.1%, preferably 0.04% -0.08%. Diluting purified red blood cells to 10 deg.C with common diluent12-1014And (2) per liter.
In the above technical solution, the loading agent is a substance capable of binding with the dye molecule, such as an artificially synthesized DNA fragment, but not limited to the DNA fragment, and may also be an artificially synthesized RNA fragment, and the amount of the loading agent is 1.0-1.5 μ g/L. The artificially synthesized DNA fragment is not particularly limited, and may be a DNA of 100bp-2000bp, preferably 200-1500bp, and most preferably 500-800bp of any sequence. The DNA molecule is artificially synthesized into an expression vector, wherein the expression vector can be a pUC57 vector, but is not limited to a pUC57 vector, and can be other vectors capable of expressing and amplifying in escherichia coli; the DNA molecules cloned into the vector are amplified by escherichia coli to obtain a large amount of products containing DNA fragments, and the products are subjected to enzyme digestion and purification to obtain the DNA fragments for the loading agent.
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 cell fixing solution is one or a mixture of more of formaldehyde, glutaraldehyde, acetone, ethanol and other conventional fixing solutions. The fixing time is 3-5 hours.
In the above-mentioned embodiment, when the washing treatment is performed on erythrocytes and products, the washing solution is a conventional isotonic solution, and may be any of, for example, a PBS buffer, a D-HANKS buffer, a HEPES buffer, a citrate buffer, a sodium chloride buffer, a borate buffer, and the like, without being limited thereto.
In the above technical scheme, the cell preservation solution is a substance commonly used in the art, and is not particularly limited, and the cell preservation solution includes a buffer solution, a nutrient component, and a preservative.
The present invention is further illustrated by the following examples, in which the reagents used are analytical grade and are commercially available. The reagents referred to in the examples include the following.
Washing liquid: each liter of distilled water contains NaCl 5.5g, Na2HPO4 20.3g，NaH2PO43.1 g. Adjusting pH to 7.0 + -0.1 with HCl or NaOH, and adjusting osmotic pressure to 300 + -10 mOsm/kgH with NaCl2O。
Diluent A: diluent I: 20g of ATP, MgCl per 100ml of distilled water212 g. And (3) diluting liquid II: KH in distilled water per liter2PO4 12g，NaHCO31.2g, glucose 0.4g, pH 7.2. The volume ratio of the diluent I to the diluent II is 1: 40.
And (3) diluting liquid B: diluent I: 30g of ATP and MgCl are contained in each 100ml of distilled water215 g. And (3) diluting liquid II: KH in distilled water per liter2PO4 10g，NaHCO31g, glucose 0.5g, pH 7.3. The volume ratio of the diluent I to the diluent II is 1: 30.
And (3) diluent C: diluent I: 25g of ATP, MgCl per 100ml of distilled water210 g. And (3) diluting liquid II: KH in distilled water per liter2PO4 14g，NaHCO31.4g, glucose 0.8g, pH 7.4. The volume ratio of the diluent I to the diluent II is 1: 50.
Fixing liquid: every liter of fixing solution contains 100ml of formaldehyde solution, 20ml of glutaraldehyde solution, 0.6g of disodium ethylenediamine tetraacetic acid and NaH2PO4 0.15g。
Preservation solution: each liter of distilled water contains 0.2g of citric acid, 1.5g of sodium citrate and NaH2PO4 1g, 8g of glucose, 12g of mannitol, 7g of glycine, 0.2g of adenine and 20 ten thousand units of gentamicin, uniformly mixing, adjusting the pH value to 7.0 +/-0.1 by using HCl or NaOH, and adjusting the osmotic pressure to 300 +/-10 mOsm/kgH by using NaCl2O。
The loading agent 1: the DNA shown as the sequence 1 in the sequence table is artificially synthesized and then cloned into a pUC57 vector, amplified in escherichia coli, extracted, separated and purified to express the vector, and subjected to enzyme digestion and purification to obtain a large number of DNA fragments, so that the loading agent 1 is obtained.
And (3) a loading agent 2: the DNA shown as the sequence 2 in the sequence table is artificially synthesized and then cloned into a pcDNA3.1 vector, amplified in escherichia coli, extracted, separated and purified to express the vector, and subjected to enzyme digestion and purification to obtain a large number of DNA fragments and obtain the loading agent 2.
Example 1
Placing anticoagulant whole blood with EDTA-2K as anticoagulant in centrifuge, centrifuging at 3000rpm for 5min, discarding supernatant, washing precipitated cells with washing solution, centrifuging at 3000rpm for 5min, discarding supernatant, repeatedly washing for 1-2 times, removing leukocyte with leukocyte filter, diluting erythrocyte to 10 with diluent A12Add vehicle 1 at 1.5. mu.g/L, stimulate the electroporator at 100V for 5 seconds, add the electrically stimulated sample to the fixative and fix it for 4 hours at room temperature. The reaction product was washed with washing solution 3-4 times, suspended with a proper amount of preservation solution, and the above-mentioned quality control substance was detected with a blood cell analyzer using fluorescence staining of nucleic acid as a detection principle, and the results are shown in FIG. 1 (only RET channel image is provided).
Example 2
Placing anticoagulant whole blood with EDTA-2K as anticoagulant in centrifuge, centrifuging at 3000rpm for 5min, discarding supernatant, washing precipitated cells with washing solution, centrifuging at 3000rpm for 5min, discarding supernatant, repeatedly washing for 1-2 times, removing leukocyte with leukocyte filter, diluting erythrocyte with diluent B to 1013At each liter, 1.2. mu.g/L of the loading agent 2 was added, the electroporator was applied with a voltage of 90V for 8 seconds, and the electrically stimulated sample was fixed in a fixed solution at room temperature for 4 hours. Washing the reaction product with washing solution for 3-4 times, suspending the product with appropriate amount of preservation solution, and detecting by nucleic acid fluorescent stainingThe blood cell analyzer detects the above quality control substances, and the result is shown in FIG. 2 (only RET channel image is provided).
Example 3
Placing anticoagulant whole blood with EDTA-2K as anticoagulant in centrifuge, centrifuging at 3000rpm for 5min, discarding supernatant, washing precipitated cells with washing solution, centrifuging at 3000rpm for 5min, discarding supernatant, repeatedly washing for 1-2 times, removing leukocyte with leukocyte filter, diluting erythrocyte with diluent B to 1014At each liter, 1.0. mu.g/L of the loading agent 2 was added, the electroporator was applied with a voltage of 120V for 4 seconds, and the electrically stimulated sample was added to a fixed solution and fixed at room temperature for 3 hours. The reaction product was washed with a washing solution 3 to 4 times, suspended with a proper amount of a preservation solution, and the above-mentioned quality control substance was detected with a blood cell analyzer using fluorescence staining of nucleic acid as a detection principle, and the result is shown in FIG. 3 (only an image of RET channel is provided).
Example 4
Placing anticoagulant whole blood with EDTA-2K as anticoagulant in centrifuge, centrifuging at 3000rpm for 5min, discarding supernatant, washing precipitated cells with washing solution, centrifuging at 3000rpm for 5min, discarding supernatant, repeatedly washing for 1-2 times, removing leukocyte with leukocyte filter, diluting erythrocyte with diluent B to 1012Each sample was fixed at room temperature for 5 hours by adding 1.3. mu.g/L of the carrier 2, stimulating with an electroporator at 100V for 5 seconds, and adding the electrically stimulated sample to a fixed solution. The reaction product was washed with washing solution 3-4 times, suspended with a proper amount of preservation solution, and the above-mentioned quality control substance was detected with a blood cell analyzer using fluorescence staining of nucleic acid as a detection principle, and the result is shown in FIG. 4 (only an image of RET channel is provided).
As can be seen from FIGS. 1-4, the blood quality control prepared according to the embodiments of the present invention was examined and analyzed in a blood analyzer to obtain a reticulocyte region having a distinct boundary with the mature erythrocyte region. Therefore, the reticulocyte mimics obtained by the method can realize the quality control of the reticulocytes based on the nucleic acid fluorescent staining. The reticulocyte simulacrum obtained by the invention can be used independently to complete the quality control of reticulocyte detection, and can also be prepared into a whole blood quality control substance in proportion with other blood quality control substances, leukocyte simulacrum particles and platelet simulacrum particles to complete the quality control of a blood cell analyzer. The reticulocyte mimic particle according to the present invention includes a red blood cell mimic particle, and the ratio of the red blood cell mimic particle to the reticulocyte mimic particle can be effectively adjusted. The reticulocyte mimics designed by the invention are simple in preparation process, the used reagent components are single, and the prepared product is high in yield and suitable for mass production.
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> reticulocyte mimics and preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
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ttaactctgg taaagtggat attgttgcca tcaatgaccc cttcattgac ctcaactaca 120
tggtttacat gttccaatat gattccaccc atggcaaatt ccatggcacc gtcaaggctg 180
agaacgggaa gcttgtcatc aatggaaatc ccatcaccat cttccaggag cgagatccct 240
ccaaaatcaa gtggggcgat gctggcgctg agtacgtcgt ggagtccact ggcgtcttca 300
ccaccatgga gaaggctggg gctcatttgc aggggggagc caaaagggtc atcatctctg 360
ccccctctgc tgatgccccc atgttcgtca tgggtgtgaa ccatgagaag tatgacaaca 420
gcctcaagat catcagcaat gcctcctgca ccaccaactg cttagcaccc ctggccaagg 480
tcatccatga caactttggt atcgtggaag gactcatgac cacagtccat gccatcactg 540
ccacccagaa gactgtggat ggcccctccg ggaaactgtg gcgtgatggc cgcggggctc 600
<210> 2
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<213> Artificial Sequence (Artificial Sequence)
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tcatccatga caactttggt atcgtggaag gactcatgac cacagtccat gccatcactg 60
ccacccagaa gactgtggat ggcccctccg ggaaactgtg gcgtgatggc cgcggggctc 120
tccagaacat catccctgcc tctactggcg ctgccaaggc tgtgggcaag gtcatccctg 180
agctgaacgg gaagctcact ggcatggcct tccgtgtccc cactgccaac gtgtcagtgg 240
tggacctgac ctgccgtcta gaaaaacctg ccaaatatga tgacatcaag aaggtggtga 300
agcaggcgtc ggagggcccc ctcaagggca tcctgggcta cactgagcac caggtggtct 360
cctctgactt caacagcgac acccactcct ccacctttga cgctggggct ggcattgccc 420
tcaacgacca ctttgtcaag ctcatttcct ggtatgacaa cgaatttggc tacagcaaca 480
Claims (7)
1. The preparation method of the reticulocyte mimics is characterized by comprising 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 diluentConsists of a diluent I and a diluent II with the volume ratio of 1 (30-50); the diluent I is prepared from ATP and MgCl2And water, ATP concentration of 10-40%, MgCl2The concentration of (A) is 5-28%; the diluent II is prepared from KH2PO4、NaHCO3Glucose and water, the pH value of the diluent II is 7.0-7.5, KH2PO4The concentration of the active component is 0.5 to 2.0 percent, and NaHCO is3The concentration of the glucose is 0.05-0.2%, and the concentration of the glucose is 0.01-0.1%;
step three, adding a fixing solution into the cells into which the loading agent is introduced, fixing at 18-28 ℃, washing the cells, and preserving with a cell preservation solution to obtain the reticulocyte mimics;
the red blood cell is a human red blood cell or a mammal red blood cell with MCV similar to the human red blood cell;
the loading agent is an artificially synthesized DNA fragment of 100bp-2000bp or an artificially synthesized RNA fragment, and the dosage of the loading agent is 1.0-1.5 mu g/L;
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 90-100V for 4-8 seconds.
2. The method of claim 1, wherein the purified erythrocytes are diluted to 10 degrees with a diluent12-1014And (2) per liter.
3. The method of claim 1, wherein the cell fixative is one or more of formaldehyde, glutaraldehyde, acetone, and ethanol, and the fixation time is 3-5 hours.
4. The method of claim 1, wherein the washing solution used in the first step and the third step is PBS buffer, D-HANKS buffer, HEPES buffer, citrate buffer, sodium chloride buffer, or borate buffer.
5. The method of preparing a reticulocyte mimic according to claim 1, wherein the cell preservation solution includes a buffer, nutrients, and preservatives.
6. A reticulocyte mimic prepared by the method of preparing a reticulocyte mimic according to any one of claims 1 to 5.
7. Use of the reticulocyte mimic of claim 6 as a quality controller for detecting reticulocytes in a fully automated blood cell analyzer.
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| CN101400996A (en) * | 2006-03-16 | 2009-04-01 | 贝克曼考尔特公司 | Reference control composition containing a nucleated red blood cell component made of non-nucleated blood cells |
| CN101881778A (en) * | 2009-05-06 | 2010-11-10 | 深圳迈瑞生物医疗电子股份有限公司 | Reticulocyte mimics and preparation method thereof |
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| US20020094515A1 (en) * | 2000-11-30 | 2002-07-18 | Erlach Julian Van | Method and apparatus for attaching a microdevice or a nanodevice to a biological member |
| JP4993603B2 (en) * | 2004-04-07 | 2012-08-08 | ベックマン コールター， インコーポレイテッド | Reference control containing nucleated red blood cell components |
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| US5432089A (en) * | 1992-03-02 | 1995-07-11 | Streck Laboratories, Inc. | Reference control for use with manual and flow cytometric reticulocyte counting devices |
| CN101400996A (en) * | 2006-03-16 | 2009-04-01 | 贝克曼考尔特公司 | Reference control composition containing a nucleated red blood cell component made of non-nucleated blood cells |
| CN101881778A (en) * | 2009-05-06 | 2010-11-10 | 深圳迈瑞生物医疗电子股份有限公司 | Reticulocyte mimics and preparation method thereof |
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