CN1206537C - Reagent red blood cell - Google Patents
Reagent red blood cell Download PDFInfo
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- CN1206537C CN1206537C CNB021489580A CN02148958A CN1206537C CN 1206537 C CN1206537 C CN 1206537C CN B021489580 A CNB021489580 A CN B021489580A CN 02148958 A CN02148958 A CN 02148958A CN 1206537 C CN1206537 C CN 1206537C
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Abstract
The present invention provides a reagent red blood cell capable of being stored for a long time, particularly a cell storing system which comprises a phosphoric acid-citrate buffer system, a nutrition system composed of saccharide and nucleotide and a double-antibiotic anticorrosion system. The osmotic pressure is effectively regulated through salt and macromolecule substances, and thus, a red blood cell membrane is stabilized. Before the reagent red blood cell of the present invention is regulated, red blood cells need to be sieved and treated, and thus, the blood group antigen strength on the red blood cell membrane is ensured. The effective period of the reagent red blood cell of the present invention at 2 to 8 DEG C is more than 2 months.
Description
Invention field
The present invention relates to a kind of composition that is used for the reagent red blood cell that blood group identifies and preparation method thereof, relate to the blood transfusion medical domain.
Background of invention
Blood group is a kind of sign of human blood type, and up to the present, human blood group system surpasses 20 kinds, wherein with the blood transfusion relation maximum be ABO blood group system.ABO blood group system is finalized the design with antibody in antigen on the human erythrocyte and the serum, contains A antigen on all red blood cells, and the blood group that contains anti-B antibody in the serum is called the A type; Contain B antigen on the red blood cell, and the blood group that contains anti-A antibody in the serum is called Type B; Contain A and B antigen on the red blood cell, and in the serum nonreactive A, anti-B antibody be called the AB type; Do not contain A, B antigen on the red blood cell, and the blood group that contains anti-A and anti-B antibody in the serum is called the O type.
Correct evaluation blood group is the first step of safe transfusion, and the principle that abo blood group is identified is based on antigen-antibody reaction and produces cell agglutination, thereby by positive definite form (cell qualification test) and reverse type (serum qualification test), accurately identifies abo blood group.Because the transfusion reaction that the abo blood group mistake causes happens occasionally, ministry of Health of China has just required as far back as 1994 the blood of blood donation personnel is identified that the accuracy of abo blood group must reach 100%.The known agent red blood cell is to identify the employed requisite reagent of antibody in the blood group serology, the reverse type that is used for accurately carrying out the abo blood group of human red blood cell identifies and irregular antibody is identified, and the use of reagent red blood cell does not still have the standardization of realization at home at present, the red blood cell that each Blood Center and blood sampling website use all is oneself preparation, both nonstandard while storage life is also short, generally can only use about three days, the red blood cell of some manufacturer production also is difficult at clinical application because of quality problems such as short and easy haemolysis of shelf-life.This present situation accurately detects the hidden danger that abo blood group has brought inconvenience and typing mistake to hospital blood bank and blood sampling website, more because the difficulty in reagent red blood cell source, cause some hospitals only to do the ABO positive definite form, do not do reverse type, cause the appearance of blood typing mistake phenomenon, even cause serious blood transfusion accident.
In hospital and blood station, red blood cell is generally preserved with physiological saline, all is to prepare at any time, the concentration that is cell on the one hand can not get guaranteeing that when preserving more than 3 days, antigen levels promptly obviously weakens, be easy to generate the typing mistake, cause serious transfusion reaction, therefore just can not use; At any time red blood cell is without screening in the process for preparation on the other hand, so antigen intensity can not get detecting and confirms that the response intensity of the red cell antigens for preparing is difficult to ensure; Once more, because preparation process lacks filter, make that the red blood cell that has formed agglutinating particle can not get driving away, thereby make typing be easy to generate false-positive result; Simultaneously, except that red blood cell, other cell components such as leucocyte, blood platelet mix wherein, because other cell components of sneaking into are very easy to cracking, gathering, thereby produce a lot of cell fragments, and influence typing result's judgement can cause erythrocytic breaking simultaneously.
Blood Center in more American-European advanced countries, because the automaticity that blood group is identified improves, the reagent red blood cell that is used for the strict standardization of instrument detecting has had special supplier to provide.So-called standardization reagent red blood cell, key is to propose one can generally claim A Shi liquid the America and Europe in the Precerving liquid system of external effective stored red blood cell, according to record, what it was common consists of:
Chloromycetin: 0.33 gram
Citric acid list water (C
6H
8O
7H
2O): 0.55 gram
Glucose (C
6H
12O
6): 20.5 grams
Inosine (inosine): 2.0 grams
Neomycinsulphate (Neomycin sulphate): 0.5 gram
Sodium chloride: 4.2 grams
Citric acid trisodium (C
6H
5Na
3O
72H
2O): 8.0 grams
Distilled water: to 1000mL
The pH of this preservation liquid is 6.8 ± 0.2, osmotic pressure is 340 ± 10mOsm/kg, reagent red blood cell with this A Shi liquid preparation can be preserved one month at 4 ℃, if from external order, remove haulage time, the general term of validity of just having only in end user's hand about 2 weeks that arrives is not also considered the transport for long-distance stability of cell membrane and the influence of cell viability, and the cost of detection is very high for a lot of users.Also have producer to use this A Shi liquid, but it is satisfactory to find that in actual applications its effect is difficult to, and is difficult in clinical expansion.
Therefore, we can say that reagent red blood cell source and quality do not ensure, directly influence and restricted the development of blood group authenticate technology and the use of pin-point accuracy instrument evaluation.
Summary of the invention
Present situation in view of present domestic blood transfusion field, the present invention proposes a kind of composition of alserver's solution, can preserve for a long time and stable red blood cell, thereby realize the standardized production of reagent red blood cell and the satisfied better requirement of accurately identifying abo blood group is provided.
The present invention also provides the preparation method of this standardization reagent red blood cell, reaches the stable purpose of guarantee reagent red blood cell character.
According to a first aspect of the invention, provide a kind of reagent red blood cell standard items that can preserve the long period, it comprises the component with following concentration:
Citric acid trisodium 23-25 millimolar concentration (mM)
Potassium dihydrogen phosphate (KH
2PO
4) 1.5mM
Sodium hydrogen phosphate (Na
2HPO
4) 1mM
Sodium chloride (NaCl) 68-72mM
Glucose (Glucose) 50-55mM
Sucrose (Sucrose) 19-22mM
Adenine (Adenine) 1.5-1.7mM
Inosine (Inosine) 4.5-4.8mM
Neomycinsulphate (Neomycin Sulfate) 0.033-0.040%
Chloromycetin (Chloramphenicol) 0.01-0.02%
Above component distilled water constant volume.
According to the reagent red blood cell with above-mentioned preservation liquid of the present invention, be to add through the red blood cell of accurately having identified abo blood group to allocate in the above-mentioned preservation liquid to form, red blood cell concentration is 1.7 ± 0.1%, and with this reagent red blood cell examination criteria typing serum, it is tired and all is not less than 128.Generally the term of validity was not less than 2 months under 2-8 ℃ condition, and this reagent red blood cell detects with the standard typing serum after diluting 8 times, and red cell agglutination intensity reaches 4+.
The preparation method of reagent red blood cell of the present invention comprises that preparing alserver's solution in proportion allocates with the adding red blood cell, and wherein, the red blood cell that is added has passed through in advance and filtered and washing, and the red blood cell and the protein ingredient of aggegation wherein taken place in removal.
The present invention proposes a kind of standardized alserver's solution system, its characteristics are:
Designed new buffer system, adopted phosphoric acid-citrate buffer system, made the pH value of system maintain 6.6-6.9.Compare with single citric acid buffer system on the books, surge capability is stronger, helps keeping whole system to be in the buffering range of requirement, has guaranteed the ionic strength of whole preservation liquid simultaneously.
Adopted more effective nutrition system, form as can be seen from it, preservation liquid of the present invention adopts carbohydrate and nucleotide as adjuvant, prove through effect, can guarantee erythrocytic freshness first, second can effectively control erythrocytic settling velocity, three can make red blood cell be easy to suspend, avoid erythrocyte aggregation bonding.
Preservation liquid of the present invention also has osmotic pressure regulating power preferably, regulate except utilizing salt, make in vivo consistent of its osmotic pressure and red blood cell, prevent erythrocyte hemolysis, according to preferred version of the present invention, comprise that also molecular weight is not higher than 40000 glucosan in the preservation liquid, its concentration is 0.02-0.04%, and the adding of this macromolecular complex more helps stablizing erythrocyte membrane.
Twin antibiotic corrosion protection system of the present invention by a certain amount of antibiotic synergy, effectively prevents the procreation of bacterium, obtains the storage life of long period.
On the other hand, in order to guarantee the quality of reagent red blood cell of the present invention, in preparation process, also need red blood cell is screened and handles, promptly, at first to select suitable red blood cell, generally the condition that should satisfy is: be chosen in three days blood rbcs with interior collection normal person, require each inspection item all qualified, that is: HBsAg (-), HCV Ab (-), HIV Ab (-), syphilis (-), ALT is in range of normal value, red blood cell does not have haemolysis, do not contain under the prerequisite of any blood transfusion communicate illness factor in assurance, select the strong blood of antigen levels, be used to prepare reagent red blood cell as raw material.Raw material blood also should accurately be identified stand-by erythrocytic abo blood group with anti--A and anti--B antibody before the reagent preparation red blood cell.The raw material erythrocyte sample that screening is obtained need filter, available filter filters or filter paper filtering, purpose is to drive away the red blood cell coagula of leucocyte, blood platelet and aggegation in the raw material blood, prevents that agglutinating particle is mixed in the reagent red blood cell, produces false agglutinating reaction; Remove the plasma proteins of acellular composition then by washing, comprise the blood group antibody composition, further remove part leucocyte and blood platelet simultaneously, prevent that cell membrane and content that its back of breaking produces from producing harmful effect to reagent.Regulate erythrocytic concentration with Precerving liquid at last, make the normal concentration reagent red blood cell that is fit to the typing use.
The reagent red blood cell that obtains prepared according to the methods of the invention, its supernatant is white transparency liquid (little band opalescence), there is not any colony growth, and it is bright-colored behind the cell suspension, be always cerise in the storage life, can not become mulberry, the term of validity is not less than 2 months (reagent red blood cell storage life provided by the invention was 6 months).
In sum, why reagent red blood cell of the present invention can have long storage life, be the synergy that is the various compositions of whole system, the nutrition system guarantees that the energy consumption of red blood cell in the preservation process can obtain replenishing timely, keeps erythrocytic freshness and vigor; Buffer system is because surge capability strengthens, and the influence to system pH that red blood cell is produced because of the cell eubolism in the preservation process is eliminated, and keeps the needed pH of normal cell; Protective system then adopts twin antibiotic synergy, prevent cell in the preservation process because bacterial reproduction and inefficacy haemolysis; The sugared composition of preserving in the liquid adopts three kinds of carbohydrates, and glucose can provide metabolism necessary energy to red blood cell, and other two kinds of carbohydrates (sucrose and glucosan) can change the viscosity and the stabilizing cell membrane of system.Find out that thus the various compositions in the Precerving liquid all check and balance, synergy reaches the purpose that keeps the red blood cell vigor and prolong the cell storage life.
Red blood cell concentration of the present invention can be lower than the concentration of present routine operation.The general concentration that requires 2-4% that detects finds in practical measurement that still the false negative phenomenon that occurs often in identifying now is higher relevant with cell concentration; At present domestic mainly is to adopt test tube method and slide method to carry out blood group to identify, the present invention fixes on the red blood cell concentration standard about 1.7%, not only can satisfy the needs that conventional manual test tube method detects, along with a large amount of uses of China's robotization blood testing instrument, this concentration cell can also fully satisfy the requirement of robotization blood group instrument pair cell concentration simultaneously; Moreover the reduction of red blood cell concentration makes the total metabolism amount of cell in the preservation process reduce, and makes the nutrition of red blood cell in the preservation process etc. obtain better guarantee; And the present invention has effectively guaranteed erythrocytic vigor and purity for the use of erythrocytic prior filtration and carrying out washing treatment and specific preservation liquid, also provides guarantee for reducing red blood cell concentration.
In a word, enforcement of the present invention provides standardized reagent red blood cell product, and each hospital and blood station can directly obtain the reagent red blood cell of conformance to standard from the production supplier, is accurately to identify blood donor's abo blood group, guarantees that safe transfusion created condition.
Below describe enforcement of the present invention and the beneficial effect that had in detail by specific embodiment, be intended to help the reader to understand spirit of the present invention and essence better, can not constitute any qualification to practical range of the present invention.
Embodiment
1, preserve the preparation of liquid:
Citric acid trisodium 2H
2O 25mM
Potassium dihydrogen phosphate (KH
2PO
4) 1.5mM
Sodium hydrogen phosphate (Na
2HPO
4) 1mM
Sodium chloride (NaCl) 70mM
Glucose (Glucose) 50mM
Sucrose (Sucrose) 20mM
Adenine (Adenine) 1.5mM
Inosine (Inosine) 4.5mM
Neomycinsulphate (Neomycin Sulfate) 0.038%
Chloromycetin (Chloramphenicol) 0.015%
Glucosan (molecular weight≤40000) (Dextran) 0.030%
Said components that will be except that microbiotic with the dissolved in distilled water of about 500-600mL, adds the microbiotic mixing earlier then, adds distilled water to 1000mL, must preserve liquid, and pH is at 6.6-6.9 for mensuration, and osmotic pressure is 340 ± 10mOsm/kg.
2, erythrocytic selection
Use the normal person's who gathers in three days raw material blood, detect every index, require: HBsAg (-), HCVAb (-), HIV Ab (-), syphilis (-), ALT is in range of normal value, and red blood cell does not have haemolysis.
Accurately identify stand-by erythrocytic abo blood group with anti--A and anti--B antibody, select the red blood cell sample of suitable accurate blood group, remove by filter red blood cell and other albumen agglutinating particle of leucocyte, aggegation with leukocyte depletion filter, then with physiological saline washing 5-6 time, remove plasma proteins and cell fragment, collect the haemocyte that obtains being suitable for.
3, the allotment of reagent red blood cell and check
Above-mentioned red blood cell is added preservation liquid, utilize spectrophotometer accurately to regulate cell concentration and reach 1.7% ± 0.1%, be that 128 standard typing serum (American I mmucol standard typing serum) is checked with tiring, guarantee that at first erythrocyte blood type is correct, be 128 standard typing serum (" moral is won in Changchun " standard typing serum) with known the tiring of this cytoscopy then, guarantee that it checks that tiring of gained is not less than 128.
4, aggegation intensity
With known the tiring of this cytoscopy is 128 standard typing serum, standard is finalized the design serum (" moral is won in Changchun " standard typing serum) according to 1: 2 doubling dilution totally 8 pipes, require red blood cell to being less than the strong positive agglutinating reaction that preceding 3 pipes (promptly 1: 2,1: 4 and dilution in 1: 8) all produce 4+ (being the whole aggegations generation of red blood cell one big grumeleuse).
5, preserve test
1) the mentioned reagent red blood cell was preserved more than 2 months, and in this storage life, this reagent red blood cell has following testing result:
-outward appearance: no haemolysis takes place, and the interface between the liquid level of red blood cell and preservation liquid is clear, and the red blood cell color is scarlet, does not have any bacteria breed sign, no red cell agglutination piece.
-all red blood cells all suspend with preserving liquid easily, do not have the phenomenon that do not suspend at the bottom of the red blood cell cake.
-be the response intensity that 128 standard typing serum is checked red cell antigens with known tiring, standard is finalized the design serum according to 1: 2 doubling dilution totally 8 pipes, red blood cell is managed the strong positive agglutinating reaction that (promptly 1: 2,1: 4 and dilution in 1: 8) all produces 4+ (being that the whole aggegations of red blood cell produce a big grumeleuse) to being less than preceding 3, and tiring of the standard serum that records is not less than 128.And antigen-reactive intensity with preserve before compare and do not change.
In-the storage life, the content of hemoglobin that supernatant is preserved in the liquid is not higher than 1.2g/L.
2) preserve under similarity condition with above-mentioned A Shi liquid, the term of validity is 1 month, after 1 month, the cell color becomes dark brown gradually, and the interface that cell and supernatant are preserved liquid thickens, and cell is difficult for suspending, and the free hemoglobin content that supernatant is preserved in the liquid obviously increases, when preserving 2 months, reach more than the 4g/L.
Claims (6)
1, a kind of reagent red blood cell is made up of alserver's solution and the red blood cell sample wherein allocated, and wherein alserver's solution comprises the component with following concentration:
Citric acid trisodium 23-25 millimolar concentration
Potassium dihydrogen phosphate 1.5 millimolar concentrations
Sodium hydrogen phosphate 1 millimolar concentration
Sodium chloride 68-72 millimolar concentration
Glucose 50-55 millimolar concentration
Sucrose 19-22 millimolar concentration
Adenine 1.5-1.7 millimolar concentration
Inosine 4.5-4.8 millimolar concentration
Neomycinsulphate 0.033-0.040%
Chloromycetin 0.01-0.02%
Above component distilled water constant volume.
2, the described reagent red blood cell of claim 1, wherein, described red blood cell sample is from having passed through the raw material blood that filters and wash.
3, claim 1 or 2 described reagent red blood cells, wherein, the term of validity of this reagent red blood cell was not less than 2 months.
4, claim 1 or 2 described reagent red blood cells, wherein, red blood cell concentration is 1.7 ± 0.1%.
5, claim 1 or 2 described reagent red blood cells, it preserves the glucosan that also contains molecular weight≤40000 in the liquid composition, and its concentration is 0.02-0.04%.
6, the preparation method of each described reagent red blood cell of claim 1-5, comprise and prepare alserver's solution in proportion and add the red blood cell allotment, wherein, the red blood cell that is added has passed through in advance and has filtered and washing, removes red blood cell and protein ingredient that aggegation has wherein taken place.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021489580A CN1206537C (en) | 2002-11-14 | 2002-11-14 | Reagent red blood cell |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB021489580A CN1206537C (en) | 2002-11-14 | 2002-11-14 | Reagent red blood cell |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1501084A CN1501084A (en) | 2004-06-02 |
| CN1206537C true CN1206537C (en) | 2005-06-15 |
Family
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB021489580A Expired - Lifetime CN1206537C (en) | 2002-11-14 | 2002-11-14 | Reagent red blood cell |
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| Country | Link |
|---|---|
| CN (1) | CN1206537C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104304235A (en) * | 2014-09-27 | 2015-01-28 | 何永勋 | A reagent erythrocyte preserving fluid and a preparing method thereof |
Families Citing this family (11)
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| CN100391473C (en) * | 2006-03-28 | 2008-06-04 | 中国人民解放军军事医学科学院野战输血研究所 | Pretreating liquid and freeze protecting liquid for cryopreservation of erythrocyte and their application |
| CN102338800A (en) * | 2011-05-25 | 2012-02-01 | 上海血液生物医药有限责任公司 | Antibody screening erythrocyte kit |
| CN103091306B (en) * | 2013-01-23 | 2015-07-01 | 苏州浩欧博生物医药有限公司 | Solution for sealing and preserving magnetic nanoparticles |
| CN105028389B (en) * | 2015-07-08 | 2017-09-22 | 苏州苏大赛尔免疫生物技术有限公司 | A kind of alserver's solution |
| CN105928753B (en) * | 2016-04-18 | 2019-01-15 | 中国科学院苏州生物医学工程技术研究所 | The flow cytometer calibration preparation method of chicken red blood cell |
| CN106561634A (en) * | 2016-11-14 | 2017-04-19 | 北京乐普医疗科技有限责任公司 | Red cell storage solution and preparation method thereof |
| CN107037220A (en) * | 2017-04-01 | 2017-08-11 | 合肥迪安医学检验所有限公司 | The compound method of ABO reverse type red cell suspensions |
| CA3117893A1 (en) * | 2018-11-19 | 2020-05-28 | Hemanext Inc. | Methods for the preservation of reagent red blood cells using carbon monoxide |
| CN109566602A (en) * | 2019-01-04 | 2019-04-05 | 宁波艾捷康宁生物科技有限公司 | A kind of alserver's solution and save set |
| CN111713487B (en) * | 2020-06-30 | 2021-10-08 | 上海市血液中心 | A kind of reagent red blood cell preservation system and preparation method thereof |
| CN112684191A (en) * | 2020-12-04 | 2021-04-20 | 上海润普生物技术有限公司 | ABO blood type positive and negative shaping and Rh blood type detection card and preparation method thereof |
-
2002
- 2002-11-14 CN CNB021489580A patent/CN1206537C/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104304235A (en) * | 2014-09-27 | 2015-01-28 | 何永勋 | A reagent erythrocyte preserving fluid and a preparing method thereof |
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| Publication number | Publication date |
|---|---|
| CN1501084A (en) | 2004-06-02 |
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