CN100577797C - Method for preparing human lymphoid leucocyte analogue - Google Patents
Method for preparing human lymphoid leucocyte analogue Download PDFInfo
- Publication number
- CN100577797C CN100577797C CN200510035800A CN200510035800A CN100577797C CN 100577797 C CN100577797 C CN 100577797C CN 200510035800 A CN200510035800 A CN 200510035800A CN 200510035800 A CN200510035800 A CN 200510035800A CN 100577797 C CN100577797 C CN 100577797C
- Authority
- CN
- China
- Prior art keywords
- osmotic pressure
- erythroblast
- analogue
- leucocyte
- volume
- 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
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a method to make human lymphocyte simulacra that includes the following steps: after getting animal erythrocyte, fixing the volume of the erythrocyte; making the volume of nucleated erythrocyte in the human lymphocyte normal distribution range by adjusting osmotic pressure; making the osmotic pressure over physiology osmotic pressure of the nucleated erythrocyte when adjusting the volume lower, making the osmotic pressure below physiology osmotic pressure of the nucleated erythrocyte when adjusting the volume higher. The advantages of the invention are that: the processed erythrocyte has good distribution, strong stability and satisfying quality controlling request; and it has abundant sample resources and no limits
Description
Technical field
The present invention relates to particularly relate to the technology that the erythrocyte that utilizes animal prepares human lymphoid leucocyte analogue from the medical compounding process of the erythrocyte of animal.
Background technology
People's peripheral leukocytes mainly is made up of lymph, monokaryon, 3 groups of cells of granulocyte, and they are the integral body of a continuous distribution on volume, and its test is mainly undertaken by blood cell analyzer, and cell in the distribution situation of instrument test as shown in Figure 1.In order to guarantee the accurate of testing tool, need manufacturing Quality Control steady in a long-term and caliberator carry out quality control and safeguard with calibrating to testing tool.In the Quality Control thing of multiparameter, contain the cell particle of certain size, be called people's cell analogue or class cell.These particles be generally the plastic cement particle or fixing after the animal erythrocyte particle.U.S. Pat 3541137, US4179398, US4264470, US4704364 etc. have described 3 white corpuscle and the thrombocyte methods of production of hiving off the blood cell analyzer Quality Control, and wherein the human granulocyte stand-in can obtain through fixing back from reptiles, fish red corpuscle; Person monocytic cell's stand-in use the fixing back of handling of turkey red corpuscle to obtain; Lymphoid leucocyte analogue is handled the back by people or other mammalian erythropoietins and is obtained, and platelet analogue uses goat red corpuscle processing back to obtain or the like.
Human lymphocyte distributes and has two characteristics: the one, and volume is little, and the 2nd, narrowly distributing.There is certain deficiency in existing human lymphoid leucocyte analogue: use plastic cement particle cost than higher, distribution range is not suitable for reducing the cost of clinical position than broad, and widespread use is restricted; And use zoogenous fixedly red corpuscle particle generally to be not easy to make it to be in human lymphocyte proper distribution scope, a wherein important factor is exactly because the lymphocyte volume is little, the white corpuscle that is in blood cell analyzer is measured near the thresholding, if its distribution is not good, can cause white corpuscle to be measured and occur than mistake, make the Quality Control failure greatly so be easy to generate data variation, or produce insignificant alarm message.In order to dwindle the lymphocyte count error as far as possible, a good method is exactly the cell quantity that increases the lymphocyte central distribution zone of blood cell analyzer mensuration, little positive rise step appears in the zone, the leftmost side (minimum lymphocyte zone) that will distribute at the lymphocyte of Instrument measuring like this, because this part cell proportion in whole white corpuscle distributes is very small, the instrument thresholding changes the white blood cell count(WBC) influence also just very little, thereby has further improved the stability of Quality Control.But the red corpuscle in end user's red corpuscle or other Mammalss source is not easy to reach above requirement, and the lymphocyte of Instrument measuring distributes and lacks little positive rise, occurs the cell debris alarm message easily, also is not easy to improve counting stability.And in mixing the white corpuscle particle, lymphocytic distribution is wide also can be covered monokaryon zone cell, because the quantity of monokaryon seldom, the lymphocyte distribution right side cell of Instrument measuring will obviously influence monocytic distribution curve for a long time.These change as shown in Figure 2: the I among Fig. 2-1
1Part curve display primary lymphedema particle is near the Instrument measuring thresholding I among Fig. 2-2
2The big lymph particle of part curve display influences the distribution curve of monokaryon district cell, and R2 is an alarm message among the figure.
The summary of the invention the technical problem to be solved in the present invention is to avoid above-mentioned the deficiencies in the prior art part and proposes a kind of method for preparing human lymphoid leucocyte analogue, solve the volume size issue of lymphoid leucocyte analogue and the lymphocyte distribution problem of Instrument measuring, make the volume stable for extended periods of time of human lymphoid leucocyte analogue.
The present invention solve the technical problem can be by realizing by the following technical solutions:
A kind of method for preparing human lymphoid leucocyte analogue is proposed, obtain animal erythrocyte after, described red corpuscle carried out volume is fixing to be handled; Described red corpuscle is an erythroblast, and when this erythroblast was carried out the fixing processing of volume, the volume of regulating described erythroblast by osmotic pressure made it be in human lymphocyte proper distribution scope; Described osmotic pressure value was higher than the physiological osmotic pressure value of this erythroblast when the erythroblast volume was turned down, transferred described osmotic pressure value when big to be lower than the physiological osmotic pressure value of this erythroblast the erythroblast volume.
Through discovering, use contains nuclear erythrocytic distribution and has good preceding positive rise, and it is narrower than people or other Mammalss to distribute, can better simulate lymphocytic form, this class red corpuscle such as fowl, avian erythrocytes. but this class erythrocyte volume size and people's lymph obvious difference, so must consider cell volume is adjusted to the human lymphocyte scope.
Use osmotic pressure effectively to address this problem.Use Hyposmolality that cell volume is risen greatly, use high osmotic pressure that cell volume is dwindled, cooperate the fixating reagent effect again, cell pellets sub-volumes after handling like this meets the human lymphocyte volumetric region under the instrument reagent effect, and has kept the distribution characteristics of animal erythroblast.Compared with prior art, technique effect of the present invention is:
1, processed red cell distribution is good, and the small-particle proportion is few, and stability is strong, is not easy to produce abnormal alarm information, satisfies quality control requirement;
2, sample source is abundant and without limits, and is with low cost.
Description of drawings
Fig. 1 is normal people's peripheral blood leukocyte test distribution situation synoptic diagram;
Fig. 2 is that people's peripheral leukocytes is tested distribution situation because the lymphocyte volume change causes the synoptic diagram of variation in the prior art, and wherein: Fig. 2-1 illustrates that emphatically the left side that test distributes changes the I among the figure
1Part curve display primary lymphedema particle is near the Instrument measuring thresholding; Fig. 2-2 illustrates that emphatically the right side that test distributes changes the I among the figure
2The big lymph particle of part curve display influences the distribution curve of monokaryon district cell;
Fig. 3 is that the bird red corpuscle uses osmotic pressure to regulate fixed test distributional difference situation synoptic diagram, wherein Fig. 3-the 1st regulates directly the fixedly test distribution situation synoptic diagram of bird erythrocyte volume without osmotic pressure, and Fig. 3-the 2nd regulates the fixedly test distribution situation synoptic diagram of bird erythrocyte volume through osmotic pressure.
Among the figure, X-coordinate unit is a cell volume unit: ascend to heaven (f1), ordinate zou unit are cell quantity unit; R2 is an alarm message.
Embodiment
Be described in further detail below in conjunction with the most preferred embodiment shown in the accompanying drawing.
It is historical for many years to adopt the fixing back of zooblast anthropomorphic dummy's white corpuscle to have, but because animal erythrocyte is not easy to meet the proper distribution of people's cell, so the processing of pair cell stand-in and preservation have proposed very high request.Even also lack the cell analogue of highly stable constant red blood cell source like this, at present.
Human lymphocyte makes a group cell minimum in the peripheral blood, and its minicell colony measures and is near the Instrument measuring thresholding, if this part cell is a lot, will bring white blood cell count(WBC) result's bigger variation.So have higher requirement for instrument quality control product.To dwindle the quantity of this part lymphocyte in whole white corpuscle particle exactly as far as possible.In order to reduce distribution influence, must reduce the quantity of right side macrolymphocyte particle simultaneously, otherwise alarm message appears in monokaryon district cell distribution situation variation easily equally to monokaryon zone cell as far as possible.Above 2 ratios that require lymphoid leucocyte analogue must as far as possible increase centrocyte.Human lymphocyte distributes narrow, and end user or the processing of other mammalian erythropoietins are not easy to meet the demands.Found through experiments, the red cell distribution that contains nuclear animal erythrocyte such as bird or birds is just narrow, but the volume size often departs from the human lymphocyte volume range, use the volume range of such animal erythrocyte of osmotic pressure regulating and controlling this moment, just can make reasonable lymphoid leucocyte analogue in conjunction with fixed count.
The present invention prepares the method for human lymphoid leucocyte analogue, is after obtaining animal erythrocyte, and described red corpuscle is fixed processing; Described red corpuscle is an erythroblast, when this erythroblast is carried out the fixing processing of volume, makes the volume of described erythroblast be in human lymphocyte proper distribution scope by regulating osmotic pressure; Described osmotic pressure value was higher than the physiological osmotic pressure value of this erythroblast when the erythroblast volume was turned down, transferred described osmotic pressure value when big to be lower than the physiological osmotic pressure value of this erythroblast the erythroblast volume.By evidence, when the erythroblast volume was turned down, in general, osmotic pressure value must be higher than 350mOsm/kgH
2O, because cell can be not impaired under the high osmotic pressure condition, thereby the high osmotic pressure value does not have the upper limit in theory; And the erythroblast volume is transferred when big, in general, osmotic pressure value is lower than 250mOsm/kgH
2O, the lower limit of Hyposmolality value is higher than the critical osmotic pressure value of the obvious disruptive of cell and gets final product, for this critical osmotic pressure value difference of different zooblasts, but this critical osmotic pressure value can determine that whether breaking as observation of cell discharges content by simple test.When corresponding each concrete animal erythrocyte is selected osmotic pressure, at first, greatly still turn to determine to select Hyposmolality or high osmotic pressure down according to cell needs accent; Then, from the cell physiological osmotic pressure value that needs are regulated, reduce or the increasing osmotic pressure value, until obtaining cell volume being adjusted to the concrete osmotic pressure value that is in human lymphocyte proper distribution scope.
The method concrete steps that prepare human lymphoid leucocyte analogue with the osmotic pressure regulation and control are as follows:
1, reagent preparation.
Antithrombotics: physiological saline adds 1% disodium ethylene diamine tetraacetate (EDTA2 sodium salt);
Basis reagent: 1～5g polyoxyethylene glycol (PEG)
1～5g glucose
0.5～5g Trisodium Citrate
0.1～2g penicillin
0.1～2g Streptomycin sulphate
0.5～5g Thiomersalate
1～9g sodium-chlor
Above-mentioned composition is added distilled water be formulated as 1L, use sodium-chlor to regulate infiltration and be pressed onto 300mOsm/kgH
2(the normal osmotic pressure of people is 300mOsm/kgH to O
2O), can use hydrochloric acid and sodium hydroxide to regulate reagent ph scope.
Fixating reagent 1: in basic reagent, add sodium-chlor, osmotic pressure is adjusted to required osmotic pressure value; The different required osmotic pressure value differences of animal erythrocyte, the required osmotic pressure value of various zooblasts can draw by ordinary test.As the process evidence, for three emperor's chicken red blood cells, required osmotic pressure value is 600mOsm/kgH
2O is promptly at 600mOsm/kgH
2Under this numerical value of O, three emperor's chicken red blood cell volumes can narrow down to the human lymphocyte proper distribution scope that is in.
Fixating reagent 2: concentration can be selected 37% commercially available formaldehyde solution for use greater than 5% formaldehyde solution in the actually operating.
2, treating processes
(1) get blood from bird or birds, as three emperor chickens, and with the animal blood use antithrombotics anti-freezing that obtains;
(2) blood after using basic reagent to anti-freezing carries out centrifuge washing 1～3 time, removes the plasma protein composition in the supernatant.A plurality of samples can be mixed to increase and handle sample total, recentrifuge concentrates;
(3) remove supernatant liquor, use fixating reagent 1 to suspend through the red corpuscle after the centrifugal treating, fixating reagent 1 volume of adding is greater than through the erythrocyte volume after centrifugal;
(4) sample left standstill 10-30 minute;
(5) adding fixating reagent 2, is 2～5% until the formaldehyde final concentration, leaves standstill the time more than 30 minutes of fixing;
(6) use basic reagent or the abundant centrifuge washing of physiological saline to remove formaldehyde, staticly settle red corpuscle, obtain human lymphoid leucocyte analogue.
Use the bird cell volume after the inventive method is handled to dwindle situation as shown in Figure 3, Fig. 3-the 1st regulates directly the fixedly test distribution situation synoptic diagram of bird erythrocyte volume without osmotic pressure, and Fig. 3-the 2nd regulates the fixedly test distribution situation synoptic diagram of bird erythrocyte volume through osmotic pressure.Comparison diagram 3-2, Fig. 3-1 and Fig. 1 obviously as can be seen, regulate the distribution of fixed bird erythrocyte volume through osmotic pressure and have been in human lymphocyte proper distribution scope in twos.And the processing cytotostatic phase after washing is preserved is long, does not contain fixating reagent, can not cooperated with other particles to form multiparameter Quality Control product by the surfactant dissolves in the reagent.
Claims (8)
1. a method for preparing human lymphoid leucocyte analogue is fixed processing with the animal erythrocyte after obtaining; It is characterized in that:
Described red corpuscle is an erythroblast, when this erythroblast is carried out the fixing processing of volume, makes the volume of described erythroblast be in human lymphocyte proper distribution scope by regulating osmotic pressure; Described osmotic pressure value was higher than the physiological osmotic pressure value of this erythroblast when the erythroblast volume was turned down, transferred described osmotic pressure value when big to be lower than the physiological osmotic pressure value of this erythroblast the erythroblast volume.
2. the method for preparing human lymphoid leucocyte analogue as claimed in claim 1 is characterized in that: the animal erythroblast after will obtaining prepares people's lymph stand-in with the osmotic pressure control technique and specifically may further comprise the steps:
Following basic reagent is meant: 1～5g polyoxyethylene glycol, 1～5g glucose, 0.5～5g Trisodium Citrate, 0.1～2g penicillin, 0.1～2g Streptomycin sulphate, 0.5～5g Thiomersalate and 1～9g sodium-chlor, use distilled water to be formulated as 1L, use sodium-chlor to regulate infiltration and be pressed onto the normal osmotic pressure level of people;
Following fixating reagent 1 is meant: add sodium-chlor in basic reagent, osmotic pressure is adjusted to required osmotic pressure value;
Following fixating reagent 2 is meant: formaldehyde solution;
1. the animal blood after will obtaining uses the antithrombotics anti-freezing; Blood after using basic reagent to anti-freezing carries out centrifuge washing, removes the plasma protein composition in the supernatant;
2. remove supernatant liquor, the red corpuscle that uses fixating reagent 1 suspended centrifugal to handle left standstill 10～30 minutes;
3. add fixating reagent 2, the formaldehyde ultimate density is 2～5%, leaves standstill above 30 minutes;
4. use the abundant centrifuge washing of basic reagent or physiological saline to remove formaldehyde, staticly settle, obtain human lymphoid leucocyte analogue.
3. the method for preparing human lymphoid leucocyte analogue as claimed in claim 2 is characterized in that: described antithrombotics is meant that physiological saline adds 1% disodium ethylene diamine tetraacetate.
4. the method for preparing human lymphoid leucocyte analogue as claimed in claim 2 is characterized in that: the commercially available concentration of described fixating reagent 2 employings is 37% formaldehyde solution.
5. the method for preparing human lymphoid leucocyte analogue as claimed in claim 1 or 2 is characterized in that: described erythroblast is the red corpuscle of bird or birds.
6. the method for preparing human lymphoid leucocyte analogue as claimed in claim 2 is characterized in that: described erythroblast is the red corpuscle of three emperor chickens.
7. the method for preparing human lymphoid leucocyte analogue as claimed in claim 6 is characterized in that: the osmotic pressure value of described fixating reagent 1 is adjusted to 600mOsm/kgH
2O.
8. the method for preparing human lymphoid leucocyte analogue as claimed in claim 1 is characterized in that: described osmotic pressure value was higher than 350mOsm/kgH when the erythroblast volume was turned down
2O transfers the erythroblast volume that described osmotic pressure value is lower than 250mOsm/kgH when big
2O.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200510035800A CN100577797C (en) | 2005-07-04 | 2005-07-04 | Method for preparing human lymphoid leucocyte analogue |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200510035800A CN100577797C (en) | 2005-07-04 | 2005-07-04 | Method for preparing human lymphoid leucocyte analogue |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1891816A CN1891816A (en) | 2007-01-10 |
| CN100577797C true CN100577797C (en) | 2010-01-06 |
Family
ID=37597013
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200510035800A Expired - Fee Related CN100577797C (en) | 2005-07-04 | 2005-07-04 | Method for preparing human lymphoid leucocyte analogue |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100577797C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101561443B (en) | 2008-04-15 | 2013-08-21 | 深圳迈瑞生物医疗电子股份有限公司 | Five-classification leucocyte simulacrum particle, method for preparing same, and quality control substance and calibration substance containing same |
| CN101887059B (en) * | 2009-05-11 | 2014-03-19 | 深圳迈瑞生物医疗电子股份有限公司 | Eosinophil analogue, preparation method thereof and whole blood quality control substance |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4704364A (en) * | 1984-05-18 | 1987-11-03 | Coulter Electronics, Inc. | Hematology control compositions for three populations of leukocytes; and methods for their preparation and use in whole blood control systems |
| CN1263266A (en) * | 1999-02-08 | 2000-08-16 | 刘剑雄 | Development of whole blood quality control substance in cell three-classification of hematology |
| US6759246B1 (en) * | 2001-11-30 | 2004-07-06 | Research & Diagnostic Systems, Inc. | Hematology control composition including lymphocyte analogs and method for preparation and use |
-
2005
- 2005-07-04 CN CN200510035800A patent/CN100577797C/en not_active Expired - Fee Related
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4704364A (en) * | 1984-05-18 | 1987-11-03 | Coulter Electronics, Inc. | Hematology control compositions for three populations of leukocytes; and methods for their preparation and use in whole blood control systems |
| CN1263266A (en) * | 1999-02-08 | 2000-08-16 | 刘剑雄 | Development of whole blood quality control substance in cell three-classification of hematology |
| US6759246B1 (en) * | 2001-11-30 | 2004-07-06 | Research & Diagnostic Systems, Inc. | Hematology control composition including lymphocyte analogs and method for preparation and use |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1891816A (en) | 2007-01-10 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101311725B (en) | WBC differential count quality control matter and method for making same | |
| Levine et al. | The significance of megakaryocyte size | |
| JP4549440B2 (en) | Reference control blood and recipe | |
| Thompson et al. | The role of anticoagulation in the measurement of platelet volumes | |
| CN101285841B (en) | Three-classification whole blood quality control substance simulant and preparation method thereof | |
| JPH07504035A (en) | Suspension medium for blood compositions and methods of use thereof | |
| CN1030481A (en) | The method and the reagent system of the leukocytes in separation, evaluation and/or the analysis of whole blood sample | |
| CN107076766B (en) | Blood CRP Quality Control object and its quality control method | |
| Wilm et al. | Leukocyte adhesion as an indicator of oxygenator thrombosis during extracorporeal membrane oxygenation therapy? | |
| CN101672853A (en) | Blood cell analyzer calibrator and preparation process thereof | |
| de Baca et al. | Effects of storage of blood at room temperature on hematologic parameters measured on Sysmex XE-2100 | |
| Docan et al. | Effect of feeding with different dietary protein level on hematological indices of juvenile Siberian sturgeon, Acipenser baeri reared under recirculating systems condition | |
| Lew et al. | Distribution of plasma membrane Ca2+ pump activity in normal human red blood cells | |
| von Petersdorff-Campen et al. | Hemolysis testing in vitro: A review of challenges and potential improvements | |
| CN101246158B (en) | Hemolytic agent for measuring leukocyte in blood cell | |
| Li et al. | Comparison of hematologic and biochemical reference values in specific-pathogen-free 1-month-old Yorkshire pigs and Yorkshire‐Landrace crossbred pigs | |
| CN100577797C (en) | Method for preparing human lymphoid leucocyte analogue | |
| Kabata et al. | Hematologic values of New Zealand white rabbits determined by automated flow cytometry | |
| CN1891815B (en) | Method for preparing cell analogue | |
| Mahajan et al. | Haematological and haematopoietic responses to starvation in an air‐breathing fish Channapunctatus Bloch | |
| Lamirande et al. | Reference hematologic and plasma chemistry values of brown tree snakes (Boiga irregularis) | |
| US6146901A (en) | Composition for manipulating optical and electrical properties of particles to achieve target values for such properties and methods for using the composition | |
| CN110987553B (en) | Erythrocyte treatment reagent and application thereof | |
| CN1263266A (en) | Development of whole blood quality control substance in cell three-classification of hematology | |
| McShine et al. | Effect of EDTA on platelet count and other platelet parameters in blood and blood components collected with CPDA-1 |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| 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: 20100106 Termination date: 20160704 |
|
| CF01 | Termination of patent right due to non-payment of annual fee |