CN104705287B - Erythrocyte cryopreservation liquid and application thereof - Google Patents
Erythrocyte cryopreservation liquid and application thereof Download PDFInfo
- Publication number
- CN104705287B CN104705287B CN201510047839.5A CN201510047839A CN104705287B CN 104705287 B CN104705287 B CN 104705287B CN 201510047839 A CN201510047839 A CN 201510047839A CN 104705287 B CN104705287 B CN 104705287B
- Authority
- CN
- China
- Prior art keywords
- erythrocyte
- stored frozen
- liquid
- defrosting
- glycerol
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Fee Related
Links
Landscapes
- Agricultural Chemicals And Associated Chemicals (AREA)
- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses an erythrocyte cryopreservation liquid. The erythrocyte cryopreservation liquid comprises 0.8M of potassium citrate monohydrate, 0.4M of sodium dihydrogen phosphate dihydrate, 0.2M of disodium hydrogen phosphate dodecahydrate, and glycerin with the volume concentration of 20-60%. The erythrocyte cryopreservation liquid allows erythrocytes to be disposed at -20DEG C for a long term, and components added to the liquid can improve the water permeability of cell membranes, reduce the formation of intracellular ice crystals and effectively protect erythrocyte membranes; and a thawing protection liquid is added when erythrocytes are thawed at room temperature, so the internal and external physiologic balance of erythrocyte membranes is realized, the rupture of the erythrocyte membranes is avoided, and the purpose of high recovery rate of the erythrocytes is reached.
Description
Technical field
The invention belongs to erythrocyte Cryopreservation Technology field is and in particular to erythrocyte stored frozen liquid and its application.
Background technology
In clinical blood transfusion work, most importantly defeated use erythrocyte transfusion, its objective is to improve the oxygen supply shape of clinical patients
State.Clinical blood transfusion practice finds, if existing for the anti-of other side's red cell antigenses in the serum of donee or blood donor
Body, it is possible to cause hemolytic blood transfusion untoward reaction, shows the clinical symptoms of Ink vessel transfusing or extravascular hemolysis, threatens and is subject to blood
Person's life security.For guaranteeing clinic blood transfusion safety, need before blood transfusion donee and blood donor's blood group antigen and antibody are entered
Row detection, it is to avoid the generation of hemolytic blood transfusion untoward reaction.
The detection of erythrocyte blood type antibody is primarily referred to as the detection of irregular antibody in order to prevent irregular antibody
Cause the generation of hemolytic blood transfusion untoward reaction.Because the conventional sense of blood group is not that all blood group antigen are all examined
Survey, exist by the possibility of not other blood group antigen stimulating immune systems generation irregular antibodies after testing, so need to be to not advising
Then antibody is detected, and provides the erythrocyte of no corresponding antigens for donee, to guarantee transfusion safety.
Detect unknown antibody with known antigen, be the basic skills of immunology detection.Resisted according to erythrocyte blood type
The requirement of body detection range, for detecting that the blood group antigen species entrained by the erythrocyte of unknown antibody should be as comprehensive as possible, shows
So derive from single individual erythrocyte and be difficult to meet requirements above, single individual erythrocyte can not possibly express all of blood group
Antigen, nor conflicting blood group antigen may be expressed simultaneously, such as a certain antigen can not possibly be negative, be sun again
Property.For meeting Identification of the antibodies, erythrocyte should be covered as far as possible with the requirement of all blood group antigen, the method for solution can only be selected
Multiple individuality erythrocyte.Would commonly be used for detection antibody specificity, the multiple individual erythrocyte carrying multiple blood group antigen claim
For cell erythrocyte (panel cell of erythrocyte), also referred to as panel erythrocyte.For ensureing the safety of clinical blood transfusion,
Select to meet two conditions during panel erythrocyte：It is anti-that red cell antigenses species should cover that local crowd has as much as possible
Former, and comprise the significant antigen of clinical meaning, such as containing RhD, RhC, RhE, Rhc, Rhe, M, N, S, s, P1, Lea、Leb、K、k、
Fya、Fyb、Jka、JkbDeng antigen.
The main points preparing panel erythrocyte are to collect the multiple erythrocyte carrying multiple antigens, constitute the one of logical judgment general layout
Group erythrocyte, needs longer time just can complete, its key problem in technology point is to preserve different discovery periods how for a long time
Can be used as composing the erythrocyte of cell composition.Frozen red cellses are the effective technologies preserving erythrocyte for a long time, the frost commonly used at present
Store method is -80 DEG C or -196 DEG C, and these methods, in addition to required special cell-preservation liquid, also suffer from the drawback that：1. this
Class method needs expensive equipment, and -80 DEG C of cryopreservation methods need special ultra cold storage freezer, and -196 DEG C of cryopreservation methods need liquid
The frozen equipment of nitrogen, longtime running is costly；2. the process of thawed red blood cell is very long, needs multiple Washed Red Blood Cells, erythrocyte
Broken many, the response rate is very low.
Content of the invention
The technical problem to be solved is for above-mentioned the deficiencies in the prior art, provides a kind of erythrocyte frost
Preserve liquid, erythrocyte can be made to be positioned over and preserve for a long time under -20 DEG C of temperature conditionss, the component of addition can improve cell membrane and water is led to
Permeability, reduces the formation of intracellular ice crystal, effective protection erythrocyte membrane；The erythrocyte of frost is thawed at room temperature, adds solution
Physiological equilibrium after freezing protection liquid, inside and outside achievable erythrocyte membrane, it is to avoid the rupture of erythrocyte membrane, reaches erythrocyte high-recovery
Target.
For solving above-mentioned technical problem, the technical solution used in the present invention is, a kind of erythrocyte stored frozen liquid, including
Following components：Citric Acid Monohydrate tripotassium 0.8M, sodium dihydrogen phosphate dihydrate 0.4M, disodium hydrogen phosphate 0.2M and 20%～
The glycerol of 60% (V/V).
The invention also discloses a kind of method of erythrocyte stored frozen liquid stored frozen erythrocyte, the method includes following
Step：
Erythrocyte and erythrocyte stored frozen liquid are sufficiently mixed, the volume ratio of described erythrocyte and stored frozen liquid is 1:
1；It is honored as a queen liquid-tight for the erythrocyte-stored frozen of the mix homogeneously obtaining, cryopreservation；
When needing using erythrocyte, the erythrocyte after stored frozen-stored frozen liquid is carried out reclaiming after defrosting process
Obtain erythrocyte；
Wherein, described erythrocyte stored frozen liquid includes following components：Citric Acid Monohydrate tripotassium 0.8M, dihydrogen phosphate dihydrate
The glycerol of sodium 0.4M, disodium hydrogen phosphate 0.2M and 20%～60% (V/V).
Further, the cryopreservation temperature of erythrocyte-stored frozen liquid is -20 DEG C.
Further, the protection liquid that thaws includes following components：5% (g/V) sodium citrate, 10%～30% (V/V's) is sweet
Oil.
Further, erythrocyte stored frozen liquid includes following components：Citric Acid Monohydrate tripotassium 0.8M, dihydrogen phosphate dihydrate
The glycerol of sodium 0.4M, disodium hydrogen phosphate 0.2M and 40% (V/V).
Further, the protection liquid that thaws includes following components：5% (g/V) sodium citrate, the glycerol of 10% (V/V).
Further, the detailed process of defrosting process is：The erythrocyte of stored frozen is thawed at room temperature, is subsequently adding
Thaw protection liquid mix homogeneously, adds centrifugal treating after normal saline, obtains red cell suspension.
Further, following process in triplicate：Add centrifugal treating after normal saline.
Erythrocyte stored frozen liquid of the present invention, it is possible to achieve long-term preservation erythrocyte under -20 DEG C of temperature conditionss；Frozen
During it is not necessary to the equipment of costliness；Course of defrosting is to thaw under room temperature, after adding the protection liquid that thaws, you can given birth to by adding
Reason salt water washing reclaims erythrocyte, takes short, and the erythrocyte response rate is high.The application of erythrocyte stored frozen liquid of the present invention, realizes
Small molecule carbohydrate is imported in erythrocyte, small molecule carbohydrate is the component in freezen protective liquid, it is to avoid
The damage to erythrocyte membrane during stored frozen of the factors such as ice crystal, in course of defrosting, adds the protection liquid that thaws, realizes red
Physiological equilibrium inside and outside cell membrane, it is to avoid the rupture of erythrocyte membrane, reaches the target of erythrocyte high-recovery.Frost is protected after testing
The form of the erythrocyte after depositing does not change, and blood group antigen is not decreased obviously.
Brief description
Fig. 1 is erythrocytolysis situation after the stored frozen liquid stored frozen of different ratio, after defrosting for the 1ml erythrocyte
Figure；
Fig. 2 is response rate comparison diagram after stored frozen, after defrosting and without frozen erythrocyte for the 1ml erythrocyte；
Fig. 3 be erythrocyte after stored frozen, the aspect graph that detects under an atomic force microscope after defrosting；
Fig. 4 be erythrocyte after stored frozen, after defrosting detection Staphylococal Protein A and RhD antigen coagulation situation map；
Fig. 5 be erythrocyte after stored frozen, after defrosting detection Staphylococal Protein A and IgM anti-D monoclonal antibody coagulation situation
Figure.
Specific embodiment
Take healthy premenopausal volunteers venous blood 2ml, EDTA anticoagulant, with brine 3 times, remove plasma fraction, leukocyte
And platelet.By the erythrocyte of the enrichment after washing according to 1:The ratio of 1 volume ratio adds stored frozen liquid, is sufficiently mixed.Ice
Freeze preservation liquid composition and include Citric Acid Monohydrate tripotassium 0.8M, sodium dihydrogen phosphate dihydrate 0.4M, disodium hydrogen phosphate 0.2M, with
And the glycerol of 20%～60% (V/V)；Thaw and protect the composition of liquid to include 5% (g/V) sodium citrate, 10%～30% (V/V)
Glycerol.After thawing, observe the dissolving situation of erythrocyte.
As shown in figure 1, No. 0 test tube of in figure is the full rupture comparison adding water in erythrocyte, it is comparative example 1；No. 1 and No. 2
It is the normal control adding normal saline in erythrocyte in test tube, be comparative example 2 and comparative example 3；It is to add to implement in No. 3 test tubes
Erythrocyte state after the defrosting of the stored frozen liquid in example 1 and the protection liquid that thaws；It is to add ice in embodiment 2 in No. 4 test tubes
Freeze the erythrocyte state after preserving liquid and the defrosting of the protection liquid that thaws；It is using the stored frozen liquid in embodiment 3 in No. 5 test tubes
State with the protection liquid that thaws；It is the state using the stored frozen liquid in embodiment 4 and the protection liquid that thaws in No. 6 test tubes；No. 7
It is the state using the stored frozen liquid in embodiment 5 in test tube.It can be seen that using red in embodiment 1 embodiment 5
After the frozen erythrocyte of cell stored frozen liquid, hematoclasis is less, wherein minimum with the hematoclasis in embodiment 3, on
Clear liquid transparent clear.
Table 1 embodiment and comparative example parameter list (component of the protection liquid that wherein thaws is certain)
Erythrocyte is thawed using after erythrocyte stored frozen liquid stored frozen, adds the erythrocyte solution that the present invention provides
Freeze protection liquid.Using the component frozen 1ml erythrocyte of the erythrocyte stored frozen liquid in embodiment 3, during defrosting, add different groups
The defrosting protection liquid dividing, as shown in table 2, observes the yield of erythrocyte.As shown in Fig. 2 being using real in No. 1 test tube of in figure
Apply the state that liquid and stored frozen liquid gained erythrocyte are protected in the defrosting in example 6；It is using the solution in embodiment 7 in No. 2 test tubes
Freeze the state of protection liquid and stored frozen liquid gained erythrocyte；In No. 3 test tubes be using be in embodiment 8 defrosting protection liquid and
The state of stored frozen liquid gained erythrocyte；It is erythrocyte in No. 4 test tubes without frost and course of defrosting, only brine
The state of 3 times, it as a comparison case 4.In embodiment 6, substantially not poor in erythrocyte yield and comparative example 4 after defrosting
Different, and in embodiment 7 and embodiment 8, the erythrocyte response rate is relatively low.
Table 2 is embodiment parameter list (constituent content of wherein stored frozen liquid is certain)
Using the erythrocyte after processing in embodiment 3, with brine 3 times, with the PBS of pH 7.4 by blood
Liquid is configured to 1～2 ‰ concentration, takes 5 μ L diluent Deca on clean mica sheet, fixes 15min with 1% glutaraldehyde, uses
PBS rinses 3 times, air-dries standby.Sample is scanned be imaged with atomic force microscope, as shown in figure 3, can from figure
Know, the red cell morphology after defrosting is complete, smooth in appearance discoid in concave-concave, the stored frozen liquid energy that the present invention provides is described
Enough preservation erythrocyte well.
Using the erythrocyte after processing in embodiment 6, the erythrocyte after thawing, brine 3 times use physiology salt
Water is made into the red cell suspension of 3% (V/V), is separately added into IgM anti-A monoclonal antibody, the Staphylococal Protein A of detection ABO blood group system
(sugar antigen)；Add IgM anti-D monoclonal antibody, detection RhD antigen (transmembrane protein class antigen), see Fig. 4 and Fig. 5.Fig. 4 is
The reaction result of A monoclonal antibody anti-with IgM, 1 is O-shaped erythrocyte (negative control), and 2 is red blood cells of type A (positive control),
3 is the red blood cells of type A after freezing and thawing.It can be seen that the red blood cells of type A after freezing and thawing, occur by force with anti-A monoclonal antibody
Coagulation, illustrates that its Staphylococal Protein A does not weaken.Fig. 5 is D monoclonal antibody reactive result anti-with IgM, and 1 is RhD positive red blood cell (sun
Property comparison), 2 and 3 be freeze and thaw after RhD positive red blood cell, 4 be RhD feminine gender erythrocyte (negative control).In figure is visible
RhD positive red blood cell after freezing and thawing, there is strong coagulation in D monoclonal antibody anti-with IgM, illustrate that its D antigen does not subtract
Weak.
Erythrocyte blood type sugar antigen and transmembrane protein class antigen easily come off from erythrocyte surface or lose, using the present invention
The method providing freeze and thaw after erythrocyte, this two classes antigen all no substantially weakens, and illustrate that the present invention is applied to and freezes for a long time
Deposit erythrocyte, can be used for preparing panel erythrocyte, can be used for the detection of blood group irregular antibody after thawing.
Claims (6)
1. a kind of method of erythrocyte stored frozen liquid stored frozen erythrocyte is it is characterised in that the method comprises the following steps：
Erythrocyte and erythrocyte stored frozen liquid are sufficiently mixed, the volume ratio of described erythrocyte and stored frozen liquid is 1:1；Will
Erythrocyte-the stored frozen of the mix homogeneously obtaining is liquid-tight to be honored as a queen, cryopreservation；The low temperature of described erythrocyte-stored frozen liquid
Storage temperature is -20 DEG C；
When needing using erythrocyte, the erythrocyte after stored frozen-stored frozen liquid is carried out recovery after defrosting process and obtains
Erythrocyte；
Wherein, described erythrocyte stored frozen liquid includes following components：Citric Acid Monohydrate tripotassium 0.8M, sodium dihydrogen phosphate dihydrate
The glycerol of 0.4M, disodium hydrogen phosphate 0.2M and 20%～60% (V/V).
2. according to a kind of stored frozen erythrocyte described in claim 1 method it is characterised in that described defrosting protection liquid bag
Include following components：5% (g/V) sodium citrate, the glycerol of 10%～30% (V/V).
3. according to a kind of stored frozen erythrocyte described in claim 1 or 2 method it is characterised in that described erythrocyte ice
Freeze preservation liquid and include following components：Citric Acid Monohydrate tripotassium 0.8M, sodium dihydrogen phosphate dihydrate 0.4M, disodium hydrogen phosphate
The glycerol of 0.2M and 40% (V/V).
4. according to a kind of stored frozen erythrocyte described in claim 3 method it is characterised in that described defrosting protection liquid bag
Include following components：5% (g/V) sodium citrate, the glycerol of 10% (V/V).
5. according to a kind of stored frozen erythrocyte described in claim 4 method it is characterised in that described defrosting process tool
Body process is：The erythrocyte of stored frozen is thawed at room temperature, is subsequently adding defrosting protection liquid mix homogeneously, adds physiology salt
Centrifugal treating after water, obtains red cell suspension.
6. according to a kind of stored frozen erythrocyte described in claim 5 method it is characterised in that following mistake in triplicate
Journey：Add centrifugal treating after normal saline.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510047839.5A CN104705287B (en) | 2015-01-30 | 2015-01-30 | Erythrocyte cryopreservation liquid and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510047839.5A CN104705287B (en) | 2015-01-30 | 2015-01-30 | Erythrocyte cryopreservation liquid and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104705287A CN104705287A (en) | 2015-06-17 |
| CN104705287B true CN104705287B (en) | 2017-02-22 |
Family
ID=53405390
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510047839.5A Expired - Fee Related CN104705287B (en) | 2015-01-30 | 2015-01-30 | Erythrocyte cryopreservation liquid and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104705287B (en) |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101220346A (en) * | 2008-02-03 | 2008-07-16 | 西北农林科技大学 | Biological cell freezing antifreeze agent |
| CN102450248A (en) * | 2010-10-26 | 2012-05-16 | 陈浩杰 | Sperm freezing method and freezing liquid thereof |
-
2015
- 2015-01-30 CN CN201510047839.5A patent/CN104705287B/en not_active Expired - Fee Related
Also Published As
| Publication number | Publication date |
|---|---|
| CN104705287A (en) | 2015-06-17 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP0796040B1 (en) | Method and bag set for concentrating white cells | |
| AU2013328472B2 (en) | Method for preserving placental blood | |
| US20070179424A1 (en) | High concentration white cells, a method for agglomeration of the high concentration and a bag set for use in conjunction therewith | |
| JP2016508501A5 (en) | ||
| CN103702556A (en) | Improved platelet storage using a sialidase inhibitor | |
| CN108849854A (en) | A kind of peripheral blood mononuclear cells cryopreservation methods | |
| CN107410288A (en) | A kind of storing liquid of human umbilical cord mesenchymal stem cells | |
| CN104705287B (en) | Erythrocyte cryopreservation liquid and application thereof | |
| Taylor et al. | Selective destruction of leucocytes by freezing as a potential means of modulating tissue immunogenicity: membrane integrity of lymphocytes and macrophages | |
| Runnström et al. | Gamones from the sperm of sea urchin and salmon | |
| CN104090083B (en) | A kind of detection method of chemicals hemolysis in vitro | |
| Arnaud et al. | Use of hollow fiber membrane filtration for the removal of DMSO from platelet concentrates | |
| Huggins et al. | Advances in blood preservation | |
| CN103651332A (en) | Docosahexaenoic acid-glycine-glycerinum compound human seminal fluid cryoprotectant and preparation method thereof | |
| Choudhury et al. | Freeze preservation of platelets using hydroxyethyl starch (HES); a preliminary report | |
| Mohn et al. | A method for the preservation of human blood group erythrocyte antigens in liquid nitrogen for a test cell panel | |
| CN113170778B (en) | Cell cryopreservation solution and preparation method and application thereof | |
| Farrugia et al. | Cryopreservation of red blood cells: effect of freezing on red cell quality and residual lymphocyte immunogenicity. | |
| Tullis | Studies on permeability of the leucocyte | |
| MEGEL et al. | The relation of the adrenal to red blood cell fragility | |
| CN113261556B (en) | Erythrocyte preservation solution capable of reducing hemolysis rate and preparation method thereof | |
| CN207713738U (en) | Blood sampling separation bag | |
| Pegg et al. | The optimization of a mixture of two permeating cryoprotectants | |
| Klinger et al. | Storage lesion of human platelets as revealed by ultrathin sections and freeze-fracture replicas | |
| Wang et al. | Influence of unfrozen fraction to the survival of leucocytes during slow cooling |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| ASS | Succession or assignment of patent right |
Owner name: THE FIRST AFFILIATED HOSPITAL OF XI'AN JIAOTONG UN Free format text: FORMER OWNER: WANG BAOYAN Effective date: 20150723 |
|
| C41 | Transfer of patent application or patent right or utility model | ||
| TA01 | Transfer of patent application right |
Effective date of registration: 20150723 Address after: 710061 Yanta Road, Yanta District, Shaanxi, Xi'an, China, No. 277 Applicant after: The First Affiliated Hospital of Medical College of Xi'an Jiaotong University Address before: The First Affiliated Hospital of Yanta District Yanta West 710061 Shaanxi city of Xi'an province No. 277 Medical College of Xi'an Jiao Tong University Applicant before: Wang Baoyan |
|
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20170222 Termination date: 20190130 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |