CN109142761A - Reticulocyte mimics and the preparation method and application thereof - Google Patents
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Abstract
The present invention relates to a kind of reticulocyte mimics and the preparation method and application thereof, belong to blood Quality Control object technical field.Solve how to provide it is a kind of it is easy to operate, yield is higher, better reticulocyte mimics of stability and the preparation method and application thereof.Preparation method of the invention, steps are as follows: Step 1: separating, washing red blood cell, obtains purification of erythropoietin；Step 2: load agent is added with diluted purification of erythropoietin, load agent is introduced into purification of erythropoietin under the action of pulse electromagnetic field；Step 3: fixer is added to being introduced into the cell after adding carrier, room temperature is fixed, and is washed cell, is saved with cell-preservation liquid, obtain reticulocyte mimics.The analogies preparation step is simple, is suitble to produce in enormous quantities, can be used as the quality control substance application of Automatic Blood Cell Analyzer detection granulophilocyte.
Description
Technical field
The invention belongs to blood Quality Control object technical fields, and in particular to a kind of reticulocyte mimics and preparation method thereof
With application.
Background technique
The blood quality-control product being made of single or multi-component haemocyte or haemocyte analogies, has as blood
Detectable characteristic, can be used for the daily monitoring of the accuracy and accuracy of blood analyser.
Granulophilocyte is the cell of the transition stage during erythrocyte maturation, is immature red blood cell, feature
It is just to have sloughed nucleus, still remained a small amount of RNA in cytoplasm.The sum or percentage of granulophilocyte are for clinically to anaemia
And its diagnosis of related disease has important clinical meaning.Reticulocyte count is the important finger for reflecting hemopoietic function of bone marrow
Mark, is mainly manifested in the following aspects.
1, evaluate bone marrow productivity, judge anaemia type: 1. net knit it is red increase, indicate hemopoietic function of bone marrow it is vigorous, often
Various hyperplastic anemias such as hypoferric anemia, megaloblastic (lacking folic acid, vitamin B12) anaemia, blood loss anemia is seen,
Especially the most significant to increase when hemolytic anemia, often > 10%.Radiation in jury phase visible RET is of short duration and increases rapidly, is marrow function
It can restore more sensitive index.2. net knits red reduction: it is common in aplastic anemia, reticulocyte count is often lower than 0.005,
Granulophilocyte absolute value is lower than 15 × 109/L。
2, it evaluates curative effect: 1. observing anaemia curative effect, RET is one of the project of Anemic patients' follow-up examination；2. bone-marrow transplantation
Monitoring marrow hemopoiesis restores afterwards.
3, the monitoring of radiation and chemotherapy: the dynamic observation of granulophilocyte can instruct clinical adjustment therapeutic scheme in due course, keep away
Exempt to cause serious bone marrow suppression.
In conclusion the indices of granulophilocyte have in terms of the assessment of the diagnostic and therapeutic effects of anemic disorders
Have important clinical meaning, in recent years the study found that Reticulocyte Parameters during detecting cirrhosis also have it is important
Clinical meaning.
The cellanalyzer of middle and high end is all using granulophilocyte as the index of analysis and detection at present, to blood sample
The analysis of middle granulophilocyte has become the important component of blood analysis, is carried out using Quality Control object to reticulocyte count
Quality control is to guarantee the premise of cellanalyzer measurement result accuracy, therefore develop and accurately can be used for full-automatic blood
The granulophilocyte Quality Control object of cytoanalyze is very necessary.
Preparation currently for granulophilocyte Quality Control object is mainly the following mode:
1, using the granulophilocyte of pig blood as raw material, granulophilocyte is isolated by the methods of centrifuge separation, prepares net
It knits red blood cell quality-control product (US5736402, US5858789, US5945340, US6444471), the place of this preparation method raw material
Reason amount is big, and yield is bad, and the separation of blood platelet may be not thorough, required higher cost.
2, after being handled the erythrocyte membrane of mammal, nucleic acid substances are injected, granulophilocyte is simulated, prepares net and knit
Red blood cell quality-control product (US6399388, US6406915, US5432089), product made from this method is red blood cell and net knit it is red
The mixture of cell, the yield of granulophilocyte depend on nucleic acid injection ratio how much, and operating process is cumbersome.
3, reticulocyte mimics are prepared by the substances such as nucleic acid and the covalently bound mode of Surface of Erythrocytes
(US7195919), the storage effect phase of the granulophilocyte quality-control product of this method preparation depends on what nucleic acid was connect with cell surface
Intensity is difficult to ensure the effect phase in long term storage, and cumbersome.
4, non-nucleated mammalian red blood cell is isolated and purified, prepares granulophilocyte by the way that cell treatment fluid and fixer is added
Quality-control product (CN CN101881778), the preparation method operating process is relatively easy, but protein is utilized in its preparation process
Denaturant makes intracellular molecular structure change to reach the suction-operated with dyestuff, the effect degree meeting of protein denaturant
Influence the yield of reticulocyte mimics.
Summary of the invention
In view of this, the object of the present invention is to provide it is a kind of it is easy to operate, yield is higher, the better net of stability knit it is red thin
Born of the same parents' analogies and the preparation method and application thereof.
The preparation method of reticulocyte mimics of the invention, steps are as follows:
Step 1: separating, washing red blood cell, obtains purification of erythropoietin；
Step 2: load agent is added, agent will be loaded under the action of pulse electromagnetic field with diluted purification of erythropoietin
It is introduced into purification of erythropoietin；
The dilution is made of volume ratio for the dilution I and dilution II of 1:(30-50)；Dilution I by ATP,
MgCl2It is formed with water, the concentration of ATP is 10%-40%, MgCl2Concentration be 5%-28%；Dilution II is by KH2PO4、
NaHCO3, glucose and water composition, the pH value of dilution II be 7.0-7.5, KH2PO4Concentration is 0.5%-2.0%, NaHCO3It is dense
Degree is 0.05%-0.2%, the concentration 0.01%-0.1% of glucose；
Step 3: fixer is added to being introduced into the cell after adding carrier, 18-28 DEG C of fixation is washed cell, is protected with cell
Liquid storage saves, and obtains reticulocyte mimics.
Preferably, the red blood cell is human red blood cells or the MCV mammalian erythropoietin similar with human red blood cells.
Preferably, with diluted purification of erythropoietin to 1012-1014A/L.
Preferably, the adding carrier is the DNA fragmentation or artificial synthesized RNA piece of artificial synthesized 100bp-2000bp
Section, the dosage of adding carrier are 1.0-1.5 μ g/L.
Preferably, load agent will be introduced into the process in purification of erythropoietin under the action of pulse electromagnetic field are as follows: electricity is worn
Hole instrument 90-120V voltage stimulates 4-8 seconds.
Preferably, the cell fixer is one of formaldehyde, glutaraldehyde, acetone, ethyl alcohol or a variety of, set time
It is 3-5 hours.
Preferably, washed in step 1 and step 3 the cleaning solution that uses for PBS buffer solution, D-HANKS buffer,
HEPES buffer solution, citrate buffer solution, sodium chloride buffer or borate buffer.
Preferably, the cell-preservation liquid includes buffer, nutritional ingredient and preservative.
The present invention also provides the reticulocyte mimics of the preparation method of above-mentioned reticulocyte mimics preparation.
The present invention also provides above-mentioned reticulocyte mimics as Automatic Blood Cell Analyzer detection granulophilocyte
Quality control substance application.
Compared with prior art, the invention has the benefit that
The preparation method of reticulocyte mimics of the invention, by nucleic acid substances by way of electric pulse stimulation cell
It introduces in mature erythrocyte and prepares reticulocyte mimics, the analogies preparation step is simple, is suitble to produce in enormous quantities.
Reticulocyte mimics of the invention are handled by pulse by people or the red blood cell of mammal and are introduced core
Processing is fixed to cell after acid or its analog, and then simulates the state of granulophilocyte, prepares granulophilocyte mould
Quasi- object.
Detailed description of the invention
Fig. 1 is reticulocyte mimics prepared by embodiment 1, using nucleic acid fluorescent dyeing as the blood of testing principle
Picture is tested on cytoanalyze (channel RET is only provided).
Fig. 2 is reticulocyte mimics prepared by embodiment 2, using nucleic acid fluorescent dyeing as the blood of testing principle
Picture (the only channel RET) is tested on cytoanalyze.
Fig. 3 is reticulocyte mimics prepared by embodiment 3, using nucleic acid fluorescent dyeing as the blood of testing principle
Picture (the only channel RET) is tested on cytoanalyze.
Fig. 4 is reticulocyte mimics prepared by embodiment 4, using nucleic acid fluorescent dyeing as the blood of testing principle
Picture (the only channel RET) is tested on cytoanalyze.
Specific embodiment
For a further understanding of the present invention, the preferred embodiment of the invention is described below, but it is to be understood that this
A little descriptions are only further explanation the features and advantages of the present invention, rather than limiting to the claimed invention.
The preparation method of reticulocyte mimics of the invention: first separating, washing red blood cell obtains purification of erythropoietin；So
Purification of erythropoietin is added in dilution afterwards, is added load agent, the effect through impulse electric field will load agent, and to imported into purifying red thin
It is intracellular, fixer finally is added to being introduced into the cell after adding carrier, 18-28 DEG C of fixation is washed cell, protected with cell-preservation liquid
It deposits, obtains reticulocyte mimics.
In above-mentioned technical proposal, red blood cell can be the red blood cell of people, be also possible to MCV value and the close mammal of people
Red blood cell, with new blood preparation the present invention in analogies better effect.Separation method is by centrifugation, washing, filtering
Mode remove leucocyte and blood platelet in blood sample, wherein filtering can use leukocyte depletion filter remove leucocyte.
In above-mentioned technical proposal, dilution is that dilution I and dilution II is formed, the volume of dilution I and dilution II
Than for 1:(30-50), preferably 1:(32-42), most preferably 1:40.Wherein, dilution I is by ATP, MgCl2It is formed with water；
The concentration of ATP is 10%-40%, preferably 15%-35%, more preferably 20%-30%；MgCl2Concentration be 5%-28%,
Preferably 8%-20%, more preferably 10%-15%.Dilution II is by KH2PO4、NaHCO3, glucose and water composition；Dilution
The pH value of II is 7.0-7.5, preferably 7.2-7.4；KH2PO4Concentration be 0.5%-2.0%, preferably 0.8%-1.8%, more
Preferably 1.0%-1.4%；NaHCO3Concentration be 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%.General diluted is pure
Change red blood cell to 1012-1014A/L.
In above-mentioned technical proposal, load agent be can with dye molecule combine substance, such as artificial synthesized DNA fragmentation,
But it is not limited only to DNA fragmentation, is also possible to artificial synthesized RNA segment, the dosage of adding carrier is 1.0-1.5 μ g/L.It is artificial to close
At DNA fragmentation be not particularly limited, can be the DNA of the 100bp-2000bp of arbitrary sequence, preferably 200-1500bp, it is optimal
Select 500-800bp.For DNA molecular through artificial synthesized into expression vector, expression vector can be pUC57 carrier, but be not limited only to
PUC57 carrier can be other carriers that can be expanded in expression in escherichia coli；The DNA molecular in carrier is cloned into through big
Enterobacteria amplification, available largely containing the product of DNA fragmentation, product purifies to obtain the DNA piece for loading agent through digestion
Section.
In above-mentioned technical proposal, load agent will be introduced into the process in purification of erythropoietin under the action of pulse electromagnetic field
Are as follows: electroporation apparatus 90-120V voltage stimulates 4-8 seconds.
In above-mentioned technical proposal, cell fixer is one of conventional fixative such as formaldehyde, glutaraldehyde, acetone, ethyl alcohol
Or a variety of mixing.Set time is 3-5 hours.
In above-mentioned technical proposal, when carrying out carrying out washing treatment to red blood cell and product, cleaning solution is conventional isotonic solution, example
Such as PBS buffer solution, D-HANKS buffer, HEPES buffer solution, citrate buffer solution, sodium chloride buffer, borate buffer
It can be used, there is no stringent restrictions.
In above-mentioned technical proposal, cell-preservation liquid is substance commonly used in the art, is not particularly limited, cell-preservation liquid includes
Buffer, nutritional ingredient and preservative composition.
The present invention is further illustrated with reference to embodiments, and chemical reagent employed in embodiment is that analysis is pure, can be led to
It crosses commercially available.Involved reagent includes following several in embodiment.
Cleaning solution: 5.5g containing NaCl, Na in every liter of distilled water2HPO420.3g NaH2PO43.1g.With HCl or NaOH
PH to 7.0 ± 0.1 is adjusted, infiltration is adjusted with NaCl and is depressed into 300 ± 10mOsm/kgH2O。
Diluent A: dilution I: 20g containing ATP, MgCl in every 100ml distilled water212g.Dilution II: every liter distills
Contain KH in water2PO412g, NaHCO31.2g, glucose 0.4g, pH value 7.2.Dilution I: the volume ratio of dilution II is 1:
40。
Dilution B: dilution I: 30g containing ATP, MgCl in every 100ml distilled water215g.Dilution II: every liter distills
Contain KH in water2PO410g, NaHCO31g, glucose 0.5g, pH value 7.3.Dilution I: dilution II volume ratio 1:30.
Dilution C: dilution I: 25g containing ATP, MgCl in every 100ml distilled water210g.Dilution II: every liter distills
Contain KH in water2PO414g, NaHCO31.4g, glucose 0.8g, pH value 7.4.Dilution I: dilution II volume ratio 1:50.
Fixer: contain formalin 100ml, glutaraldehyde solution 20ml, disodium ethylene diamine tetraacetate in every liter of fixer
0.6g, NaH2PO4 0.15g。
Save liquid: 0.2g containing citric acid in every liter of distilled water, sodium citrate 1.5g, NaH2PO41g, glucose 8g, sweet dew
Alcohol 12g, glycine 7g, adenine 0.2g, 200,000 unit of gentamicin, after mixing with HCl or NaOH adjust pH value to 7.0 ±
0.1, infiltration, which is adjusted, with NaCl is depressed into 300 ± 10mOsm/kgH2O。
Load agent 1: the DNA as shown in sequence 1 in sequence table is through artificial synthesized rear clone to pUC57 carrier, through large intestine bar
Amplification, extraction separation and purification expression vector obtain a large amount of DNA fragmentations through digestion after purification in bacterium, obtain load agent 1.
Load agent 2: the DNA as shown in sequence 2 in sequence table through artificial synthesized rear clone to pcDNA3.1 carrier, through large intestine
Amplification, extraction separation and purification expression vector obtain a large amount of DNA fragmentations through digestion after purification in bacillus, obtain load agent 2.
Embodiment 1
Anticoagulated whole blood using EDTA-2K as anti-coagulants is placed in centrifuge, 3000rpm is centrifuged 5min, discards supernatant
Liquid cleans sedimentation cell with cleaning solution, and 3000rpm is centrifuged 5min, discards supernatant liquid, washes repeatedly 1-2 times, uses leukocyte depletion filter
Except leucocyte-removing, red blood cell is diluted to 10 with diluent A12Load agent 1, electroporation apparatus 100V is added by 1.5 μ g/L in a/L
Voltage stimulates 5 seconds, fixes 4 hours at room temperature for fixative is added in the sample after electro photoluminescence.It is anti-with cleaning solution repeated washing
It answers product 3-4 times, is suspended with the appropriate liquid that saves to product, be the blood cell point of testing principle to nucleic acid fluorescent dyeing
Analyzer is to above-mentioned Quality Control analyte detection, as a result referring to Fig. 1 (only providing RET channel image).
Embodiment 2
Anticoagulated whole blood using EDTA-2K as anti-coagulants is placed in centrifuge, 3000rpm is centrifuged 5min, discards supernatant
Liquid cleans sedimentation cell with cleaning solution, and 3000rpm is centrifuged 5min, discards supernatant liquid, washes repeatedly 1-2 times, uses leukocyte depletion filter
Except leucocyte-removing, red blood cell is diluted to 10 by dilution B13A/L loads agent 2, electroporation apparatus 90V by the addition of 1.2 μ g/L
Voltage stimulates 8 seconds, fixes 4 hours at room temperature for fixative is added in the sample after electro photoluminescence.It is anti-with cleaning solution repeated washing
It answers product 3-4 times, is suspended with the appropriate liquid that saves to product, be the blood cell point of testing principle to nucleic acid fluorescent dyeing
As a result analyzer (only provides RET channel image) referring to fig. 2 to above-mentioned Quality Control analyte detection.
Embodiment 3
Anticoagulated whole blood using EDTA-2K as anti-coagulants is placed in centrifuge, 3000rpm is centrifuged 5min, discards supernatant
Liquid cleans sedimentation cell with cleaning solution, and 3000rpm is centrifuged 5min, discards supernatant liquid, washes repeatedly 1-2 times, uses leukocyte depletion filter
Except leucocyte-removing, red blood cell is diluted to 10 by dilution B14A/L loads agent 2, electroporation apparatus 120V by the addition of 1.0 μ g/L
Voltage stimulates 4 seconds, fixes 3 hours at room temperature for fixative is added in the sample after electro photoluminescence.It is anti-with cleaning solution repeated washing
It answers product 3-4 times, is suspended with the appropriate liquid that saves to product, be the blood cell point of testing principle to nucleic acid fluorescent dyeing
Analyzer is to above-mentioned Quality Control analyte detection, as a result referring to Fig. 3 (only providing RET channel image).
Embodiment 4
Anticoagulated whole blood using EDTA-2K as anti-coagulants is placed in centrifuge, 3000rpm is centrifuged 5min, discards supernatant
Liquid cleans sedimentation cell with cleaning solution, and 3000rpm is centrifuged 5min, discards supernatant liquid, washes repeatedly 1-2 times, uses leukocyte depletion filter
Except leucocyte-removing, red blood cell is diluted to 10 by dilution B12A/L loads agent 2, electroporation apparatus 100V by the addition of 1.3 μ g/L
Voltage stimulates 5 seconds, fixes 5 hours at room temperature for fixative is added in the sample after electro photoluminescence.It is anti-with cleaning solution repeated washing
It answers product 3-4 times, is suspended with the appropriate liquid that saves to product, be the blood cell point of testing principle to nucleic acid fluorescent dyeing
As a result analyzer (only provides RET channel image) referring to fig. 4 to above-mentioned Quality Control analyte detection.
As attached drawing 1-4 as it can be seen that blood Quality Control object made from various embodiments of the present invention tests and analyzes in blood analyser, obtain
To the granulophilocyte area with mature erythrocyte area with obvious boundary.Therefore, the granulophilocyte mould that the method for the present invention obtains
Quasi- object can be realized to be controlled by the quality of the granulophilocyte of principle of nucleic acid fluorescent dyeing.The obtained net of the present invention is knitted red thin
Born of the same parents' analogies can be used alone, and completes the quality detected to granulophilocyte and controls, while can also be with other blood Quality Controls
Object, leucocyte simulation particle, blood platelet simulation particle are prepared into whole blood quality control materials in proportion, complete the matter to cellanalyzer
Amount control.The granulophilocyte simulation particle being related in the present invention includes red blood cell simulation particle, simulates grain with red blood cell
The ratio of son can be adjusted effectively.Designed reticulocyte mimics preparation process is simple in the present invention, used
Agent formulations are more single, and products collection efficiency obtained is higher, are suitble to a large amount of production.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Sequence table
<110>Di Rui medical science and technology limited liability company
<120>reticulocyte mimics and the preparation method and application thereof
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 600
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
aggtgaaggt cggagtcaac ggatttggtc gtattgggcg cctggtcacc agggctgctt 60
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
<211> 480
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
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 (10)
1. the preparation method of reticulocyte mimics, which is characterized in that steps are as follows:
Step 1: separating, washing red blood cell, obtains purification of erythropoietin；
Step 2: load agent is added with diluted purification of erythropoietin, load agent is introduced under the action of pulse electromagnetic field
Into purification of erythropoietin；
The dilution is made of volume ratio for the dilution I and dilution II of 1:(30-50)；Dilution I is by ATP, MgCl2With
Water composition, the concentration of ATP are 10%-40%, MgCl2Concentration be 5%-28%；Dilution II is by KH2PO4、NaHCO3, grape
Sugar and water composition, the pH value of dilution II are 7.0-7.5, KH2PO4Concentration be 0.5%-2.0%, NaHCO3Concentration be
0.05%-0.2%, the concentration 0.01%-0.1% of glucose；
Step 3: fixer is added to being introduced into the cell after adding carrier, 18-28 DEG C of fixation washs cell, uses cell-preservation liquid
It saves, obtains reticulocyte mimics.
2. the preparation method of reticulocyte mimics according to claim 1, which is characterized in that the red blood cell is behaved
Red blood cell or the MCV mammalian erythropoietin similar with human red blood cells.
3. the preparation method of reticulocyte mimics according to claim 1, which is characterized in that pure with diluted
Change red blood cell to 1012-1014A/L.
4. the preparation method of reticulocyte mimics according to claim 1, which is characterized in that the adding carrier is behaved
Work synthesizes the DNA fragmentation of 100bp-2000bp or artificial synthesized RNA segment, the dosage of adding carrier are 1.0-1.5 μ g/L.
5. the preparation method of reticulocyte mimics according to claim 1, which is characterized in that by pulse electromagnetic field
Load agent is introduced into the process in purification of erythropoietin under effect are as follows: electroporation apparatus 90-100V voltage stimulates 4-8 seconds.
6. the preparation method of reticulocyte mimics according to claim 1, which is characterized in that the cell fixer
For one of formaldehyde, glutaraldehyde, acetone, ethyl alcohol or a variety of, the set time is 3-5 hours.
7. the preparation method of reticulocyte mimics according to claim 1, which is characterized in that step 1 and step 3
The middle cleaning solution used that washs delays for PBS buffer solution, D-HANKS buffer, HEPES buffer solution, citrate buffer solution, sodium chloride
Fliud flushing or borate buffer.
8. the preparation method of reticulocyte mimics according to claim 1, which is characterized in that the cell-preservation liquid
Including buffer, nutritional ingredient and preservative.
9. granulophilocyte simulation prepared by the preparation method of reticulocyte mimics described in claim 1-8 any one
Object.
10. reticulocyte mimics as claimed in claim 9 are as Automatic Blood Cell Analyzer detection granulophilocyte
The application of quality control substance.
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| 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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