CN103698501B - A kind of Cyanide-free hemolytic agent - Google Patents
A kind of Cyanide-free hemolytic agent Download PDFInfo
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Abstract
The invention belongs to medical instruments field, disclose a kind of Cyanide-free hemolytic agent, Cyanide-free hemolytic agent of the present invention comprises at least one lytic agent, at least one cosolvent, the agent of at least one ligands stabilize, pH2 ~ 11 damping fluid and water.Lytic agent and cosolvent make erythrocytolysis and discharge haemoglobin, make leucocyte film dissolve simultaneously and retain leucocyte core, can classify to leucocyte while measurement haemoglobin hemolytic agent and count like this.Ligands stabilize agent can form more stable derivant with haemoglobin, and make measurement result more accurately and reliably, consistance is better.And damping fluid can regulate the pH value of hemolytic agent, make to identify one or more WBC sub-population.Cyanide-free hemolytic agent good stability of the present invention, the hemolytic agent of cyanide-free environmental protection, measurement result is accurately and reliably, applied widely, and cost is low, be applicable to vast hospital and R&D units, be specially adapted to the instrument will sought quickness to measuring speed.
Description
Technical field
The invention belongs to hemolytic agent technical field, particularly relate to a kind of Cyanide-free hemolytic agent.
Background technology
Haemocyte, also known as " blood cell ", is the cell be present in blood, can with the flowing of blood throughout whole body.Haemocyte accounts for 45% of volumetric blood, comprises red blood cell (RBC), leucocyte (WBC) and blood platelet.The main function of red blood cell transports oxygen.Ripe red blood cell does not have nucleus, yet acellular device, and its shape is the round pie of two sides central fovea, is full of haemoglobin (hemoglobin, HGB) in kytoplasm.Haemoglobin is the main protein of mature erythrocyte, accounts for 96% of cell gross weight, accounts for 35% of cell volume.Have and combine and transport O
2and CO
2function.Leucocyte is the colourless sphaerocyst having core, and volume ratio red blood cell is large, can do amoeboid movement, has defence and immunologic function, mainly plays the role of immunity.When germs have invaded the human body, leucocyte can pass capillary wall, focuses on germ invasion position, is surrounded by germ, engulf.Leucocyte is divided into granulocyte, monocyte and lymphocyte, and granulocyte is divided into again neutrocyte, eosinophil leucocyte, basophil.Blood platelet is the fritter that in marrow, megacaryocyte apocytosis gets off, therefore acellular core, there is complete cell membrane on surface.Play an important role in coagulation process.
Under normal physiological conditions, red blood cell, leucocyte and blood platelet have certain morphosis, and have metastable quantity.But some illness and physiological change, such as, infect, fever, Pregnancy time, the change of quantity to a certain degree or bulk & form will be there is.So, detect haemocyte and medical diagnosis on disease and treatment are had very important significance.
A kind of reagent the most frequently used in blood analysis process when hemolytic agent is medical test is the key reagents of Measuring hemoglobin and leukocytic counting or classification.Hemolytic agent is under the mating reaction of dilution, on the one hand red blood cell is dissolved rapidly, discharge haemoglobin and be combined with hemolytic agent and form stable haemoglobin dervative, for Measuring hemoglobin, make leucocyte bare nucleus or keep peculiar form on the other hand, for white blood cell count(WBC) and classification.And the measuring method of haemoglobin has a variety of, wherein cyanmethemoglobin (HiCN) method is the referential method of ICSH's recommendation.
Cyanomethemoglobin uses Triton X-100 as hemolytic agent, after red blood cell is dissolved, discharges haemoglobin, the ferrous ion (Fe in haemoglobin
2+) be oxidized to ferric ion (Fe by high-potassium ferricyanide
3+).Haemoglobin changes into methemoglobin, and methemoglobin combines with cryanide ion (CN-) again, generates stable cyanmethemoglobin (HiCN).Cyanmethemoglobin has a wider absorption peak at wavelength 540nm place, it is directly proportional to the concentration of solution in the absorbance of 540nm, by spectrophotometer or carry out colorimetric with HiCN with reference to liquid and can record hemoglobin concentration in blood.Although this method HGB measurement result is accurate, owing to employing potassium cyanide or the sodium cyanide of severe toxicity, when touching skin, the healthy of reviewer can be jeopardized.Prussiate also can discharge more hypertoxic hydrogen cyanide gas when running into acid medium.Meanwhile, the waste liquid containing prussiate also can pollute soil and water resources.
Due to the potassium cyanide containing severe toxicity in HiCN reagent, for this reason, some are not developed in succession containing the hemoglobin method that records of KCN.
The people such as Oshiro [Clinical Biochemistry, vol.15, Issue 1 (1982) .pp.83-88] once reported that a kind of cyanide-free measured the hemolytic agent of hemoglobin concentration, this hemolytic agent forms primarily of lauryl sodium sulfate (SLS), Triton X-100 and a kind of damping fluid (PH 7.2).But the method cannot be classified to leucocyte and count while measurement haemoglobin hemolytic agent.
US Patent No. 4853338 discloses a kind of hemolytic agent formula of cyanide-free, and this hemolytic agent is made up of a kind of ionic surface active agent and highly basic, but this hemolytic agent needs to use (PH 11.3) under stronger alkalescence.
US Patent No. 5242832 discloses a kind of hemolytic agent of cyanide-free, this hemolytic agent have selected catechol-3,5-sodium disulfonate, oxine, associating pyridine, 1,10-phenanthroline, phenol compound, bisphenol-A, pyrazole compound, N-phenylpyrazolone, the aromatics such as imidazoles are as the stabilizing agent of haemoglobin dervative.But these stabilizing agents have larger toxicity.
Chinese patent CN102662067 discloses a kind of cyanogens-free hemolytic agent, and this hemolytic agent formula is by cationic surfactant, and a kind of acidulant, a kind of sodium pyrithione is stabilizing agent composition.But pyrithione sodium water solution is to photo-labile.
Therefore, exploitation one can Measurement accuracy hemoglobin concentration, may be used for leukocytic classification and counting simultaneously, and stability is better, and the hemolytic agent of environmental protection is significant.
Summary of the invention
In view of this, the object of the invention is to the defect for prior art, proposing one can Measurement accuracy hemoglobin concentration, may be used for leukocytic classification and counting simultaneously, accuracy is good, the hemolytic agent of cyanide-free environmental protection, reduces the pollution to environment.
For realizing object of the present invention, the present invention adopts following technical scheme:
A kind of Cyanide-free hemolytic agent, contains in hemolytic agent described in every 1L:
Preferably, described lytic agent is compound shown in formula I or formula II,
Wherein, R
1be selected from C
8-20alkyl, C
8-20thiazolinyl or C
8-20alkynyl;
R
2, R
3and R
4independently selected from C
1-4alkyl, C
1-4thiazolinyl or C
1-4alkynyl;
X-is selected from chlorion, bromide ion, fluorine ion, acetate ion or sulfate ion;
N is 7,8,9,10,11,12,13,14,15,16,17,18 or 19.
Preferably, described lytic agent is one in trimethyl ammonium chloride, tetradecyltrimethylammonium amine bromide and hexadecyl trimethyl ammonium bromide or combination in any.
Preferably, described cosolvent is one in Tween 20, polyoxyethylene two Span 20, poloxalkol, AEO and phenol polyoxyethylene ether or combination in any.
Preferably, described ligands stabilize agent is amides compound or hydrazide kind compound.
Preferably, described amides compound is compound shown in formula IV or formula V,
Wherein, R
5~ R
12independently selected from H, C
1-4alkyl, C
2-4thiazolinyl, C
2-4alkynyl, C
1-4alkoxy, C
1-4carboxyl or C
2-6heterocyclic radical;
N1 is 0,1,2,3 or 4.
Preferably, described hydrazide kind compound is compound shown in formula VI or formula VII,
Wherein, R
13~ R
21independently selected from H, C
1-4alkyl, C
2-4thiazolinyl, C
2-4alkynyl, C
1-4alkoxy, C
1-4carbonyl or C
2-6heterocyclic radical;
N2 is 0,1,2,3 or 4.
Preferably, described amides compound is one in formamide, acetamide, propionamide, sweet amine amide, propylamine acid amides, prolineamide, asparagine and glutamine or combination in any.
Preferably, described pH 2 ~ 11 damping fluid is phosphate buffer, citrate buffer, veronal buffer, phthalate buffer, glycinate damping fluid, Tris damping fluid or borate buffer solution.
Preferably, described inorganic salts are alkali-metal halogenide or alkali-metal sulfate.
The invention provides a kind of Cyanide-free hemolytic agent, described Cyanide-free hemolytic agent comprises at least one lytic agent, at least one cosolvent, the agent of at least one ligands stabilize, pH 2 ~ 11 damping fluid and water.Lytic agent and cosolvent make erythrocytolysis and discharge haemoglobin, with Measuring hemoglobin concentration, make leucocyte membrane portions dissolve simultaneously, and retain leucocyte core, classify like this and count while measurement hemoglobin concentration to leucocyte.Ligands stabilize agent can form stable derivant with haemoglobin, rapid solution is erythrocytic while, part can replace the cryanide ion (CN-) in potassium cyanide, forms stable derivant with the haemoglobin discharged, make measurement result more accurately and reliably, consistance is better.And damping fluid can regulate the pH value of hemolytic agent, make it possible to identify one or more WBC sub-population.Cyanide-free hemolytic agent good stability of the present invention, the hemolytic agent of cyanide-free environmental protection, measurement result accurately and reliably, applied widely, and cost is low, be applicable to vast hospital and R&D units, be specially adapted to the instrument will sought quickness to measuring speed, as semi-automatic or automatic blood analyzer.
Accompanying drawing explanation
Figure 1 shows that and use hemolytic agent of the present invention graph of a relation of hemoglobin concentration measured value on hemoglobin concentration measured value and reference instrument XE-5000 on supporting machine;
Figure 2 shows that and use hemolytic agent of the present invention graph of a relation of leucocyte measured value on leucocyte measured value and reference instrument XE-5000 instrument on supporting machine.
Embodiment
The embodiment of the invention discloses a kind of Cyanide-free hemolytic agent.Those skilled in the art can use for reference present disclosure, and suitable improving technique parameter realizes.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in the present invention.Product of the present invention is described by preferred embodiment, related personnel obviously can not depart from content of the present invention, spirit and scope product as herein described is changed or suitably change with combination, realize and apply the technology of the present invention.
For realizing object of the present invention, the present invention adopts following technical scheme:
A kind of Cyanide-free hemolytic agent, contains in hemolytic agent described in every 1L:
Cyanide-free hemolytic agent of the present invention is a kind of aqueous solution, comprises at least one lytic agent, at least one cosolvent, the agent of at least one ligands stabilize, pH 2 ~ 11 damping fluid and water.Lytic agent energy lysed erythrocyte also discharges haemoglobin, cosolvent can promote that erythrocytolysis discharges haemoglobin further, ligands stabilize agent can form stable compound with haemoglobin dervative, rapid solution is erythrocytic while, part can replace the cryanide ion (CN-) in potassium cyanide, form stable compound with the haemoglobin dervative discharged, make measurement result more accurately and reliably, consistance is better.And damping fluid can regulate the pH value of hemolytic agent.
Cyanide-free hemolytic agent of the present invention also comprises inorganic salts in some embodiments, to regulate conductivity and the osmotic pressure of hemolytic agent.
In some embodiments, lytic agent described in Cyanide-free hemolytic agent of the present invention is quaternary ammonium compound or quaternized pyridinium salt compounds.
In some embodiments, described quaternary ammonium compound has structure shown in formula I,
Wherein, R
1be selected from C
8-20alkyl, C
8-20thiazolinyl or C
8-20alkynyl;
R
2, R
3and R
4independently selected from C
1-4alkyl, C
1-4thiazolinyl or C
1-4alkynyl;
X-is selected from chlorion, bromide ion, fluorine ion, acetate ion or sulfate ion.
In some embodiments, described quaternized pyridinium salt compounds has structure shown in formula II,
Wherein, X-is selected from chlorion, bromide ion, fluorine ion, acetate ion or sulfate ion;
N is 7,8,9,10,11,12,13,14,15,16,17,18 or 19.
Further, described lytic agent is one in dodecyl chlorination amine, myristyl amine bromide and cetyl amine bromide or combination in any in certain embodiments.
Lytic agent content described in Cyanide-free hemolytic agent of the present invention is 0.1-100g/L.In certain embodiments, described lytic agent content is 1-80g/L.Be 10g/L in certain embodiments.Be 20g/L in certain embodiments.Be 60g/L in certain embodiments.
In some embodiments, cosolvent described in Cyanide-free hemolytic agent of the present invention is polyoxyethylene type compounds.
Further, in some embodiments, described cosolvent is one in Tween 20, polyoxyethylene two Span 20, poloxalkol, AEO and phenol polyoxyethylene ether or combination in any.
In certain embodiments, described cosolvent is poloxamer, Tween 20, AEO or Octylphenol polyoxyethylene ether.
Cosolvent content described in Cyanide-free hemolytic agent of the present invention is 0.01-50g/L.In certain embodiments, described cosolvent content is 1-30g/L.Be 20g/L in certain embodiments.Be 10g/L in certain embodiments.Be 8g/L in certain embodiments.
In some embodiments, ligands stabilize agent described in Cyanide-free hemolytic agent of the present invention is amides compound or hydrazide kind compound.
In some embodiments, described amides compound has structure shown in formula IV or formula V,
Wherein, R
5~ R
12independently selected from H, C
1-4alkyl, C
2-4thiazolinyl, C
2-4alkynyl, C
1-4alkoxy, C
1-4carboxyl or C
2-6heterocyclic radical;
N1 is 0,1,2,3 or 4.
Further, described amides compound is formamide, acetamide, propionamide, sweet amine amide, propylamine acid amides, prolineamide, asparagine or glutamine in certain embodiments.
In some embodiments, described hydrazide kind compound has structure shown in formula VI or formula VII,
Wherein, R
13~ R
21independently selected from H, C
1-4alkyl, C
2-4thiazolinyl, C
2-4alkynyl, C
1-4alkoxy, C
1-4carbonyl or C
2-6heterocyclic radical;
N2 is 0,1,2,3 or 4.
Further, described hydrazide kind compound is acethydrazide, daminozide or maleic acid hydrazide in certain embodiments.
Ligands stabilize agent content described in Cyanide-free hemolytic agent of the present invention is 0.1-20g/L.In certain embodiments, described ligands stabilize agent content is 1-15g/L.Be 15g/L in certain embodiments.Be 8g/L in certain embodiments.Be 4g/L in certain embodiments.
In some embodiments, the damping fluid of pH 2 ~ 11 described in Cyanide-free hemolytic agent of the present invention is phosphate buffer, citrate buffer, veronal buffer, phthalate buffer, glycinate damping fluid, Tris damping fluid or borate buffer solution.Wherein, the buffer content of pH 2 ~ 11 described in Cyanide-free hemolytic agent of the present invention is 0.1mmol ~ 2mol.
In some embodiments, inorganic salts described in Cyanide-free hemolytic agent of the present invention are alkali-metal halogenide or alkali-metal sulfate.In certain embodiments, described inorganic salts are sodium sulphate, potassium sulfate, sodium chloride or potassium chloride.Wherein, inorganic salt content described in Cyanide-free hemolytic agent of the present invention is 0-30g/L.In certain embodiments, described inorganic salt content is the 1-10g/L of general assembly (TW).Be 2g/L in certain embodiments.Be 8g/L in certain embodiments.
Utilize Cyanide-free hemolytic agent of the present invention to measure the method for haemoglobin to be specially and to get fresh anticoagulation and mix with dilution, dilution ratio is from 125:1 to 500:1, then mix with Cyanide-free hemolytic agent of the present invention, ultraviolet-visible spectrophotometer is used to carry out measuring the absorbance of sample, thus measure the hemoglobin concentration of sample, also the blood cell analyzer simultaneously with electrical impedance and photometer device can be utilized, the Simultaneously test hemoglobin concentration value of white blood cell count(WBC) in sample.
Wherein said dilution can be the dilution of any one conventional hemolytic agent.In certain embodiments, described dilution is the supporting dilution of SC-8000A blood analyser.Dilution mixture in the proper ratio.According to appliance requires, fresh anticoagulation can select craft or machine automatically to mix with the potpourri of dilution.
In order to understand the present invention further, be described in detail to the invention provides below in conjunction with embodiment.
Embodiment 1: Cyanide-free hemolytic agent of the present invention
Embodiment 2: Cyanide-free hemolytic agent of the present invention
Embodiment 3: Cyanide-free hemolytic agent of the present invention
Embodiment 4: accuracy and correlation detection
In order to prove the accuracy of Cyanide-free hemolytic agent of the present invention, have selected 80 parts of fresh anticoagulation samples, wherein 50 parts of disease samples, 30 parts of normal samples, Cyanide-free hemolytic agent of the present invention is used to open in SC-8000A blood cell analyzer the wbc value and hemoglobin concentration value of measuring 80 increments bases, sample is measured (using the matched reagent of this type) on Sysmex XE 5,000 five classifies blood cell analyzer simultaneously, both contrast measurement result, the results are shown in Figure 1 and Fig. 2.
By the visible Cyanide-free hemolytic agent of the present invention of Fig. 1 and Fig. 2 result in the high correlation of the measurement result of SC-8000A blood cell analyzer and the measurement result of Sysmex XE 5000 analyser and accuracy.
The explanation of above embodiment is just for helping to understand product of the present invention and method and core concept thereof.It should be pointed out that for those skilled in the art, under the premise without departing from the principles of the invention, can also carry out some improvement and modification to the present invention, these improve and modify and also fall in the protection domain of the claims in the present invention.
Claims (5)
1. a Cyanide-free hemolytic agent, is characterized in that, contains in hemolytic agent described in every 1L:
Described ligands stabilize agent is amides compound or hydrazide kind compound;
Described lytic agent is compound shown in formula I or formula II,
Wherein, R
1be selected from C
8-20alkyl, C
8-20thiazolinyl or C
8-20alkynyl; R
2, R
3, R
4independently selected from C
1-4alkyl, C
1-4thiazolinyl or C
1-4alkynyl; X-is selected from chlorion, bromide ion, fluorine ion, acetate ion or sulfate ion; N is 7,8,9,10,11,12,13,14,15,16,17,18 or 19;
Described cosolvent is one in Tween 20, polyoxyethylene two Span 20, poloxalkol, AEO and phenol polyoxyethylene ether or combination in any;
Described amides compound is compound shown in formula IV or formula V,
Wherein, R
5~ R
12independently selected from H, C
1-4alkyl, C
2-4thiazolinyl, C
2-4alkynyl, C
1-4alkoxy, C
1-4carboxyl or C
2-6heterocyclic radical; N1 is 0,1,2,3 or 4;
Described hydrazide kind compound is compound shown in formula VI or formula VII,
Wherein, R
13~ R
21independently selected from H, C
1-4alkyl, C
2-4thiazolinyl, C
2-4alkynyl, C
1-4alkoxy, C
1-4carbonyl or C
2-6heterocyclic radical; N2 is 0,1,2,3 or 4.
2. Cyanide-free hemolytic agent according to claim 1, is characterized in that, described lytic agent is one in trimethyl ammonium chloride, tetradecyltrimethylammonium amine bromide and hexadecyl trimethyl ammonium bromide or combination in any.
3. Cyanide-free hemolytic agent according to claim 1, is characterized in that, described amides compound is one in formamide, acetamide, propionamide, sweet amine amide, propylamine acid amides, prolineamide, asparagine and glutamine or combination in any.
4. the Cyanide-free hemolytic agent according to claim 1-3 any one, it is characterized in that, described pH 2 ~ 11 damping fluid is phosphate buffer, citrate buffer, veronal buffer, phthalate buffer, glycinate damping fluid, Tris damping fluid or borate buffer solution.
5. the Cyanide-free hemolytic agent according to claim 1-4 any one, is characterized in that, described inorganic salts are alkali-metal halogenide or alkali-metal sulfate.
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| CN104297134A (en) * | 2014-11-05 | 2015-01-21 | 深圳市开立科技有限公司 | Hemolytic agent and application thereof as well as classifying and counting method for white blood cells |
| CN104634953A (en) * | 2015-02-28 | 2015-05-20 | 韩冰 | Environment-friendly and non-toxic hemolytic agent |
| CN106932350A (en) * | 2017-03-22 | 2017-07-07 | 安徽民泰医药科技有限公司 | Blood cell analysis hemolytic agent |
| CN108132343B (en) * | 2017-12-12 | 2020-04-07 | 山东兰桥医学科技有限公司 | Hemolytic agent for four-cluster analysis of white blood cells |
| CN110132899A (en) * | 2018-02-02 | 2019-08-16 | 深圳市帝迈生物技术有限公司 | A kind of hemolytic agent for leukocyte differential count |
| CN110231469A (en) * | 2018-03-06 | 2019-09-13 | 深圳市帝迈生物技术有限公司 | A kind of classification of leucocyte three hemolytic agent |
| CN109238801B (en) * | 2018-10-19 | 2021-08-13 | 武汉百合龙腾生物科技有限责任公司 | Hemolytic agent for blood cell analysis |
| CN110702892A (en) * | 2019-09-09 | 2020-01-17 | 金华市鑫科医疗器械有限公司 | Special reagent for measuring hemoglobin concentration for blood cell analyzer |
| CN110579595B (en) * | 2019-09-10 | 2022-02-18 | 核工业总医院 | Isohemolysin, preparation method thereof, biological sample treatment method and leukocyte membrane antigen detection method |
| CN110954489A (en) * | 2019-11-15 | 2020-04-03 | 中山市创艺生化工程有限公司 | Cyanide-free hemolytic agent and application thereof |
| CN111006990A (en) * | 2019-12-31 | 2020-04-14 | 山东博科生物产业有限公司 | Reagent for analyzing blood cells and use thereof |
| CN111157431A (en) * | 2020-01-14 | 2020-05-15 | 迪瑞医疗科技股份有限公司 | Reagent for classifying nucleated erythrocytes and basophils |
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| JP3320869B2 (en) * | 1993-12-22 | 2002-09-03 | シスメックス株式会社 | Leukocyte analysis reagent |
| DE69527947T2 (en) * | 1994-03-11 | 2003-05-28 | Abbott Laboratories, Abbott Park | CYANIDE-FREE REAGENT AND METHOD FOR DETERMINING HEMOGLOBIN |
| CN1277125C (en) * | 2003-08-20 | 2006-09-27 | 深圳迈瑞生物医疗电子股份有限公司 | Cyanideless hemolysin and its use |
| CN102662067B (en) * | 2012-05-20 | 2014-07-02 | 烟台卓越生物技术有限责任公司 | Cyanide-free hemolysin for determining hemoglobin concentration and carrying out white blood cell count by groups |
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