CN1115561C - Diluent and cleaning liquid for blood analysis counter - Google Patents
Diluent and cleaning liquid for blood analysis counter Download PDFInfo
- Publication number
- CN1115561C CN1115561C CN98102346A CN98102346A CN1115561C CN 1115561 C CN1115561 C CN 1115561C CN 98102346 A CN98102346 A CN 98102346A CN 98102346 A CN98102346 A CN 98102346A CN 1115561 C CN1115561 C CN 1115561C
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- dilution
- blood
- diluent
- cleaning fluid
- blood analysis
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Abstract
The present invention relates to a diluent for a calculating instrument for blood analysis. Per liter of diluent contains 9 to 12g of sodium sulfate, 3 to 6g of sodium chloride, 0.1 to 2g of EDTA sodium salt, 0.05 to 2g of Na2HPO4, 0.05 to 2g of KH2PO4, 0.1 to 1g of NaN3 and water as the rest, and the diluent has the pH value of 6.9 plus or minus 0.1. cleaning liquid can be obtained after tween is added to the diluent, per liter of cleaning liquid has the advantages of easy acquisition of raw materials, low cost, simple preparation process and favorable effect of blood sample maintenance, and the cost for analyzing blood can be greatly reduced by the diluent and the cleaning liquid.
Description
The present invention relates to a kind of reagent and compound method thereof.Specifically, provide dilution and the cleaning fluid that a kind of automatic blood count classifying instrument (CELL-DYN) uses in analyzing blood.
The haemocyte that comprises three kinds of principal ingredients in people's the blood, i.e. white blood cell, red blood cell and blood platelet.Ratio (concentration) to the shared cell of these three kinds of cells in the healthy human body round-robin blood has certain limit, but people for patient disease, because a lot of diseases at first influence hematological system, cause that blood changes, therefore, provide a kind of very valuable information by situation of change for traditional diagnostic techniques to the relevant blood constituent that analysis provides of blood of human body composition.Like this, blood analysis counter also just is widely used as a kind of important medical equipment.
CELL-DYN system blood counting instrument is a kind of Medical Instruments that is exclusively used in blood cell composition in the analyzing blood that company of U.S. Abbott Laboratories (ABBDTT) produces.Utilize this instrument can analyze the hematology result that counting comprises 16 parameters of white blood cell, red blood cell and blood platelet in the blood of human body, align and make a definite diagnosis disconnected disease important basis is provided.When using this instrument to carry out blood analysis,, it is highly important that the stability that to keep the blood sample of gathering in order to obtain correct blood analysis result.Yet keeping the constant control various factors that needs of total cellular score in the blood, mainly is conditions such as osmotic pressure, pH, conductivity, population of ions, ionic type, surface tension and temperature.For this reason, CELL-DYN series classification instrument of blood count uses a kind of dilution, the blood sample that collects is joined in proportion be mixed with test sample in the dilution.U.S. Pat 4,745, disclose a kind of dilution as above-mentioned blood analysis in No. 071, this dilution is contains sodium sulfate, sodium chloride, 1, the aqueous solution of 3-dimethyl urea, 1-pyridone-2-thioketones, N-(2-acetylaminohydroxyphenylarsonic acid 2-iminodiacetic acid) (ADA buffering agent) and NaOH.Wherein the pH value with NaOH adjustment dilution is 6.9 ± 0.1.
In addition, utilize CELL-DYN series blood counting instrument to carry out blood analysis except that needing thinning agent, for guaranteeing the accuracy of different analysis of blood, after analysing a sample, per minute needs the instrument relative section to be cleaned with clean-out system, to remove residual raw sample reagent, guarantee analysis result accurately and reliably.Above-mentioned U.S. Pat, also having reported this clean-out system in 4,745,071 is to be mixed with by add a certain amount of washing agent in above-mentioned dilution.So-called washing agent can be selected azo dyes Diazopon for use, or poly ethyl phenol or ester, as Fergitol, and Zougl, esters such as Fluorad.
But prepare in above-mentioned dilution and the cleaning fluid as the organism of stabilizing agent and antiseptic composition high price not only, and be difficult to obtain again, therefore improved the cost that carries out blood analysis.
The purpose of this invention is to provide a kind of dilution and cleaning fluid that blood count classifying instrument (CELL-DYN) uses that be used for.The required chemical reagent of this dilution and the cleaning fluid tool that is easy to get is inexpensive, therefore can reduce the cost of prepared and diluted liquid and cleaning fluid significantly.Simultaneously, this dilution also can reach the effect of effective maintenance blood constituent.
Of the present invention to be used for the dilution that blood cell analysis uses be the aqueous solution that contains anti-coagulants, antiseptic, buffer solvent and inorganic salts (sodium sulphate and sodium chloride), it is characterized in that containing in the per 1 liter of dilution of consisting of of dilution:
Sodium sulphate: 9~12g;
Sodium chloride: 3~6g;
EDTA sodium salt: 0.1~2g;
Na
2HPO
4:0.05~2g;
KH
2PO
4: 0.05~2g;
NaN
3: 0.1~1g;
All the other are water, and its pH is 6.9 ± 0.1.
EDTA sodium salt (sodium ethylene diamine tetracetate) adds as anti-coagulants in the dilution of the present invention, and also available its sylvite of EDTA sodium salt replaces, but in the preparation otherwise influence the osmotic pressure to blood, ionic strength and the pH value of dilution.NaN
3Be effectively to keep the constant corruption of organism in the blood as antiseptic.Na
2HPO
4With HK
2PO
4Be damping fluid adding, keep solution in certain pH range as dilution.Damping fluid also can be selected K for use
2HPO
4With NaH
2PO
4Perhaps K
2HPO
4CaHPO
4With KH
2PO
4Ca (H
2PO
4)
2, but should make osmotic pressure, ionic strength and pH value in the dilution remain on suitable scope equally.
The compound method of dilution of the present invention is very simple, earlier dissolve each salt component with low amounts of water (deionized water or distilled water) after, add water to 1 liter, promptly obtain dilution of the present invention.Add the pH value that can keep dilution behind metaphosphoric acid as damping fluid, the phosphite in the dilution of the present invention and maintain in 6.9 ± 0.1 the scope, therefore do not need the pH value of hydro-oxidation sodium adjustment solution.This makes the Na in the dilution
+, K
+Ion concentration is more easy to control, makes dilution be more conducive to the preservation of blood.
Cleaning fluid of the present invention is to add a certain amount of washing agent in dilution, it is characterized in that washing agent selects tween type (polyoxyethylene sorbitan carboxylic ester) product for use, and its addition is that to contain tween in per 1 liter of cleaning fluid be 2~5ml.
A certain amount of washing agent of adding or saponifier all can obtain cleaning performance cleaning fluid preferably in dilution of the present invention.Easily introduce the trace impurity compound may disturb analysis to blood in instrument but add common washing agent or soap, washing agent, saponifier easily produce foam simultaneously, also will adverse effect (influencing the correct volume of solution) be arranged to analysis.Cleaning fluid of the present invention selects for use commodity polysorbate60,80 can effectively avoid above-mentioned unfavorable factor.Simultaneously, also the pH value of cleaning fluid can be adjusted into 10 ± 0.5, can increase cleaning performance.Adjusting the available dilute sodium hydroxide of pH value carries out.
The compound method of above-mentioned cleaning fluid is identical with the diluent preparing process.Compound as each component of cleaning fluid can be dissolved with low amounts of water (deionized water or distilled water), add entry again to setting.Also available a little dissolving detergent of the dilution for preparing adds dilution then to setting.
The using method of dilution of the present invention and cleaning fluid is identical with the method for existing CELL-DYN series classification instrument of blood count used diluent and cleaning fluid, can be undertaken by its identical operations method.
Below by example technology of the present invention is given to illustrate in further detail.
The preparation of example 1 dilution
Get sodium sulphate 10.8g, sodium chloride 3.6g, EDTANa
21g, Na
2HPO
41g, KH
2PO
40.6g, NaN
30.5g add in the recorder jar with distilled water 200g dissolving back, the continuation adding distil water makes volume reach 1 liter can obtain dilution of the present invention.
The preparation of example 2 dilutions
Get Tween 80 (Beijing chemical reagents corporation product) 1.7ml.After adding the dilution dissolving of 20ml example 1 preparation, add again and the pH value is adjusted into 10 dilution with NaOH (10%) and makes cleaning fluid of the present invention to 500ml.
The performance of example 3 dilutions of the present invention and cleaning fluid
Utilize the dilution of example 1 and 2 described method preparations and dilution and the cleaning fluid that cleaning fluid is made CELL-DYN 1700 type classification instrument of blood count, measure following parameters in the blood: 1.WBC total white blood cells order; 2.LYM# lymphocyte number; 3.LYM% lymphocyte number percent; 4.MID# median size cell number; 5.MID% median size cell number percent; 6.GRAN# granulocyte number; 7.GRAN% granulocyte number percent; 8.RBC red blood cell sum; 9.HGB haemoglobin; 10.HCT packed cell volume capacity; 11.MCV MCV; 12.MCH average haemoglobin; 13, the concentration of the average haemoglobin of MCHC; 14.RDW RDW; 15.PLT total number of blood platelet; 16.MPV mean platelet volume; 17.PDW blood platelet distributed density; 18. thrombocytocrit capacity.Its blood analysis process is pressed the conventional method operation of CELL-DYN instrument, is divided into and analyses 12 blood samples.Wherein sample 1-5 and 11-12 analyze and to carry out next sample analysis again after the back is cleaned with above-mentioned cleaning fluid, and sample 6-10 cleans with the cleaning fluid of preparing by 4,745, No. 071 institute of U.S. Pat report method, its analysis the results are shown in table 1
The performance of comparative example dilution and cleaning fluid
By the prepared and diluted liquid and the cleaning fluid of 4,745, No. 071 report of U.S. Pat, and be used for blood analysis.Adopt the blood sample 1-12 of example 3 identical collections, with example 3 the same terms under carry out dilution and the comparative experiments of cleaning fluid performance.Cleaning process behind each sample analysis is also identical with example 3, and its analysis result is with listing in table 1.
Result by table 1 can see that dilution provided by the present invention and cleaning fluid have performance same as the prior art, but the compound method of dilution of the present invention and cleaning fluid is simpler than the method for prior art, and raw material is easy to get, and inexpensive.Therefore, use dilution of the present invention and cleaning fluid can reduce the cost of analyzing blood sample.
Table 1 analysis of blood result
Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 | |
Project | Comparative example of the present invention | Comparative example of the present invention | Comparative example of the present invention | Comparative example of the present invention | Comparative example of the present invention | Comparative example of the present invention |
1.WBC(G/L) 2.LYM 3.LYM(%L) 4.MLD 5.MLD(%M) 6.GRAN 7.GRAN(%G) 8.RBC(T/L) 9.HGB(G/L) 10.HCT(L/L) 11.MCV(F/L) 12.MCH(PG) 13.MCHC(G/L) 14.RDW(%) 15.PLT(G/L) 16.MPV(FL) 17.PCT(ML/L) 18.PDW 10(GSD) | 2.4 2.4 0.6 0.5 26.6 22.4 0.1 0.1 3.1 2.7 1.7 1.8 70.3 74.9 2.68 2.61 66 67 236 227 88 87 24.6 25.7 280 295 17.6 16.6 80 74 9.1 8.9 0.7 0.7 19.0 17.3 | 5.8 5.8 2.4 2.4 40.6 40.6 0.6 0.6 11.0 11.0 2.8 2.8 48.4 48.4 3.72 3.71 89 89 264 264 71 72 23.9 23.9 337 337 22.6 22.6 324 324 9.5 9.5 0.31 0.31 16.4 16.4 | 6.0 6.0 2.4 2.4 39.9 39.9 0.6 0.6 10.8 10.8 3.0 3.0 49.3 49.3 3.74 3.74 91 90 266 266 71 71 24.1 24.1 338 338 21.8 21.9 321 321 9.4 9.4 0.30 0.30 16.8 16.8 | 6.7 6.7 1.9 1.9 27.7 27.7 0.7 0.7 11.0 11.0 4.1 4.1 61.3 61.3 3.64 3.64 100 101 353 353 96 97 27.7 27.7 286 286 15.8 15.8 214 214 9.3 9.3 2.0 2.0 17.4 17.4 | 9.6 9.6 2.3 2.3 24.0 24.0 0.8 0.8 8.8 8.8 6.6 6.6 67.2 67.2 5.23 5.24 145 144 498 498 95 95 27.5 27.5 289 289 15.5 15.5 225 227 10.7 10.7 2.4 2.4 18.2 18.4 | 16.1 16.1 0.9 0.9 5.6 5.6 0.2 0.2 1.3 1.3 15.0 15.0 93.1 93.1 4.41 4.41 123 122 388 388 88 88 27.7 27.7 314 314 15.1 15.1 149 149 8.5 8.5 1.3 1.3 17.0 17.0 |
Table 1 is continuous
Sample 7 | Sample 8 | Sample 9 | Sample 10 | Sample 11 | Sample 12 | |
Project | Comparative example of the present invention | Comparative example of the present invention | Comparative example of the present invention | Comparative example of the present invention | Comparative example of the present invention | Comparative example of the present invention |
1.WBC(G/L) 2.LYM 3.LYM(%L) 4.MLD 5.MLD(%M) 6.GRAN 7.GRAN(%G) 8.RBC(T/L) 9.HGB(G/L) 10.HCT(L/L) 11.MCV(F/L) 12.MCH(PG) 13.MCHC(G/L) 14.RDW(%) 15.PLT(G/L) 16.MPV(FL) 17.PCT(ML/L) 18.PDW 10(GSD) | 9.6 9.6 2.5 2.5 26.1 26.1 1.3 1.3 13.7 13.7 5.8 5.8 60.2 60.2 5.23 5.23 143 143 518 518 98 99 27.3 27.3 276 276 16.7 16.7 255 257 9.6 9.7 2.6 2.6 21.5 21.5 | 6.3 6.3 2.1 2.1 33.7 33.7 0.4 0.4 6.9 6.9 3.7 3.7 59.4 59.4 5.32 5.33 144 143 469 469 81 88 26.8 26.8 305 305 13.5 13.5 177 178 6.6 6.6 1.2 1.2 18.1 18.1 | 22.6 22.6 2.4 2.4 10.5 10.5 0.9 0.9 3.8 3.8 19.4 19.4 85.7 85.7 3.48 3.48 96 95 341 341 97 98 27.3 27.3 279 279 16.4 16.4 234 234 6.2 6.2 1.6 1.5 18.2 18.2 | 6.2 6.2 3.0 3.0 48.9 48.9 0.6 0.6 8.9 8.9 2.6 2.6 42.2 42.2 4.74 4.75 109 109 394 394 84 83 22.9 229 277 277 18.1 18.1 296 296 9.0 9.0 2.7 2.7 19.4 19.4 | 6.0 6.0 2.2 2.3 37.4 38.3 0.7 0.6 11.0 10.2 3.1 3.1 51.6 51.5 3.82 3.78 90 89 267 265 70 70 23.8 23.5 341 336 20.9 22.3 305 304 8.9 8.9 0.27 0.27 15.6 15.6 | 16.7 16.7 1.1 1.1 6.6 6.6 0.8 0.8 4.5 4.5 14.8 14.8 88.9 88.9 3.82 3.82 95 96 359 359 94 94 25.1 25.1 26.7 26.7 22.2 22.2 110 111 12.3 12.3 1.4 1.4 18.8 18.8 |
Claims (4)
1, a kind of blood analysis counter dilution is the aqueous solution that contains anti-coagulants, antiseptic, buffer solvent, reaches inorganic salts sodium sulphate and sodium chloride, it is characterized in that containing in the per 1 liter of dilution of consisting of of dilution: sodium sulphate: 9~12g; Sodium chloride: 3~6g; EDTA sodium salt: 0.1~2g; Na
2HPO
4: 0.05~2g; KH
2PO
4: 0.05~2g; NaN
3: 0.1~1g; All the other are water, and its pH is 6.9 ± 0.1.
2,, it is characterized in that the EDTA sodium salt replaces with its sylvite by the described blood analysis counter dilution of claim 1.
3,, it is characterized in that damping fluid can select K for use by the described blood analysis counter dilution of claim 1
2HPO
4With NaH
2PO
4Perhaps K
2HPO
4CaHPO
4With KH
2PO
4Ca (H
2PO
4)
2
4, a kind of blood analysis counter cleaning fluid is characterized in that adding washing agent in dilution, consisting of in per 1 liter of dilution of described dilution contains: sodium sulphate: 9~12g; Sodium chloride: 3~6g; EDTA sodium salt: 0.1~2g; Na
2HPO
4: 0.05~2g; KH
2PO
4: 0.05~2g; NaN
3: 0.1~1g; All the other are water, and its pH is 6.9 ± 0.1; The rare sorbitan carboxylic esters of described detergent use polyoxy second, its addition are that to contain washing agent in per 1 liter of cleaning fluid be 2~5ml.
Priority Applications (1)
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CN98102346A CN1115561C (en) | 1998-06-04 | 1998-06-04 | Diluent and cleaning liquid for blood analysis counter |
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---|---|---|---|
CN98102346A CN1115561C (en) | 1998-06-04 | 1998-06-04 | Diluent and cleaning liquid for blood analysis counter |
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CN1238455A CN1238455A (en) | 1999-12-15 |
CN1115561C true CN1115561C (en) | 2003-07-23 |
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CN98102346A Expired - Fee Related CN1115561C (en) | 1998-06-04 | 1998-06-04 | Diluent and cleaning liquid for blood analysis counter |
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CN101021456B (en) * | 2007-02-12 | 2010-11-10 | 南昌大学 | Method for producing tripartite group blood cell analyser diluent |
CN101226117B (en) * | 2007-12-05 | 2010-08-11 | 成都瑞琦科技实业有限责任公司 | Agent for preventing EDTA-K2 agent from crystallizing in vitro |
CN101281193B (en) * | 2008-06-04 | 2010-11-10 | 南昌百特生物高新技术股份有限公司 | Blood cell analyzer dilution |
CN101281194B (en) * | 2008-06-04 | 2010-11-10 | 南昌百特生物高新技术股份有限公司 | Blood cell analyzer cleaning fluid |
JP5288970B2 (en) * | 2008-09-26 | 2013-09-11 | シスメックス株式会社 | Method for measuring mean red blood cell volume using a reagent for diluting blood samples |
CN101559244B (en) * | 2009-05-21 | 2012-07-11 | 安徽信灵检验医学科技有限公司 | Ethylene diamine tetraacetic acid sylvite metacrystalline excipient |
CN101975850A (en) * | 2010-09-13 | 2011-02-16 | 南京卡博生物科技有限公司 | Diluent for blood cell analyzer |
CN103323308B (en) * | 2013-05-23 | 2016-01-06 | 北京博晖创新光电技术股份有限公司 | A kind of reagent and application thereof processing micro whole blood |
CN104698157B (en) * | 2015-02-13 | 2017-05-17 | 中山市创艺生化工程有限公司 | Agent for blood cell analyzer |
CN106168554B (en) * | 2016-02-29 | 2019-10-18 | 迈克生物股份有限公司 | A kind of reagent for hemocyte analyzers |
CN106525553A (en) * | 2016-11-30 | 2017-03-22 | 刘相东 | Diluent applied to blood analyzer and preparation method thereof |
CN107014664A (en) * | 2017-03-22 | 2017-08-04 | 安徽民泰医药科技有限公司 | Blood cell analysis dilution |
CN111413176B (en) * | 2020-05-09 | 2023-05-16 | 河北艾驰生物科技有限公司 | Erythrocyte and leucocyte quality control substance for urine component analysis and preparation method thereof |
CN112985966A (en) * | 2021-02-08 | 2021-06-18 | 桂林优利特医疗电子有限公司 | Diluent for analyzing urine visible components and preparation method thereof |
CN113267395A (en) * | 2021-07-05 | 2021-08-17 | 江苏荣盛嘉美生物试剂有限公司 | Reagent for urine visible component analyzer and preparation method thereof |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4745071A (en) * | 1985-09-05 | 1988-05-17 | Sequoia-Turner Corporation | Method for the volumetric differentiation of blood cells types |
CN1037776A (en) * | 1988-05-18 | 1989-12-06 | 中国人民解放军第二三○医院 | PC-703 type electronic blood cell counter reagent process |
CN1163401A (en) * | 1996-02-13 | 1997-10-29 | 李占元 | Fully automatic hemacytometer reagent and preparing method |
-
1998
- 1998-06-04 CN CN98102346A patent/CN1115561C/en not_active Expired - Fee Related
Patent Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US4745071A (en) * | 1985-09-05 | 1988-05-17 | Sequoia-Turner Corporation | Method for the volumetric differentiation of blood cells types |
CN1037776A (en) * | 1988-05-18 | 1989-12-06 | 中国人民解放军第二三○医院 | PC-703 type electronic blood cell counter reagent process |
CN1163401A (en) * | 1996-02-13 | 1997-10-29 | 李占元 | Fully automatic hemacytometer reagent and preparing method |
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CN1238455A (en) | 1999-12-15 |
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