CN1130567C - Cyanide-free reagent and method for hemoglobin determination and leukocyte differentiation - Google Patents
Cyanide-free reagent and method for hemoglobin determination and leukocyte differentiation Download PDFInfo
- Publication number
- CN1130567C CN1130567C CN 97126470 CN97126470A CN1130567C CN 1130567 C CN1130567 C CN 1130567C CN 97126470 CN97126470 CN 97126470 CN 97126470 A CN97126470 A CN 97126470A CN 1130567 C CN1130567 C CN 1130567C
- Authority
- CN
- China
- Prior art keywords
- agent
- reagent composition
- haemoglobin
- same parents
- kinds
- 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 - Lifetime
Links
Images
Landscapes
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The present invention relates to a reagent composition containing no cyanide ion and a method used for measuring the hemoglobin concentration of a blood sample, and additionally, the reagent composition can be used for measuring haemoglobin concentration and differentiating at least two kinds of white cells. The reagent composition comprises at least one kind of lysis agent and an oxidation inhibitor, wherein the lysis agent is selected from quaternary ammonium salt, pyridine*salt and the mixture thereof, and the content is enough for dissolving erythrocytes and eluting haemoglobin; the effective content of the oxidation inhibitor can convert released haemoglobin into hemochromogen. The pH value of the reagent composition can be regulated with a pH value regulating agent, and therefore, a pH value ranging from 5 to 11.5 is provided.
Description
This invention is about a kind of reagent and method thereof that is used for measuring the total haemoglobin of blood, and wherein this reagent does not contain prussiate and ferricyanide ion.In addition, this reagent and method thereof can utilize suitable electronic equipment in a blood sample whole leucocytes to be counted and definite two kinds of leucocytes at least.
The measurement of leucocyte and haemoglobin is very important to the clinical diagnosis such as diseases such as leukaemia and anemias in the blood sample.Define following term for understanding and explaining for the purpose of this invention.
1, haemoglobin----is contained in the red blood cell, in blood as the carrier of oxygen.It is a kind of chromoprotein, is made up of four heme group: two α and two beta globin chains.
2, the ability that heme group----combines with oxygen depends on the existence of heme group.It also makes haemoglobin have special color.Protoheme is made up of an organic moiety and an iron atom that is referred to as protoporphyrin.Iron atom in the protoheme combines with four nitrogen-atoms at the center of protoporphyrin ring.Iron atom can be ferrous state, divalence or iron oxide state, trivalent.
3, methemoglobin (symbol is Hi)----iron atom is oxidized to ferric haemoglobin.Alternative terms is methamoglobin and high price Ferri-hemoglobin.
4, methemoglobin prussiate----iron atom be oxidized to ferric iron and with the cyanide ion methemoglobin of complexing mutually.Alternative terms is a cyanomethemoglobin, cyanmethemoglobin and methamoglobin prussiate.
5, all Ferri-hemoglobin derivants that in blood circulation, exist usually of haemoglobin type----.Comprise deoxyhemoglobin (Hb), oxyhemoglobin (HbO2), Hb-S and HbA2, carbonyl hemoglobin (HbCO), methemoglobin (Hi).
6, determine NCCLS (council of the clinical testing standard country) refer step (H15-A Vol.4 No.3) of the haemoglobin in the blood.One is applicable to that the method by the methemoglobin prussiate measures the producer's of all clinical testing personnel of hemoglobin concentration and instrument, reagent and material standard procedure.
7, hemoglobinometry----can be measured whole hemoglobin concentration in conjunction with the spectral characteristic of the derivant of the ability of oxygen, carbon monoxide, sulphur etc. and haemoglobin according to some chemical composition such as physical characteristicss such as proportion and refractive index, haemoglobin molecule of blood, haemoglobin.
8, stabilized hemoglobin agent----is used to keep the chemical compound of the stable physicochemical characteristics (chemical constitution, optical density (OD) etc.) of haemoglobin dervative or formed chromogen during hemoglobinometry.
Usually come the leucocyte in the blood sample is counted and distinguishes with automatic hematology analyzer, when utilizing automatic hematology analyzer to come leucocyte counted, at first whole blood sample is diluted with a kind of isotonic diluent agent, and add a kind of erythrocytolysis agent with the erythrocytolysis in the sample, obtain containing leukocytic sample.Then, sample is by a tiny passage or a meticulous aperture of analyser test section.The detection signal that the mononuclear leukocyte that is passed through according to response produces counts to leucocyte, and it is distinguished.Be included in United States Patent (USP) 3,962 Deng further specifying of thinning agent of infiltration, in 125,4,346,018 and 4,521,518.Further specifying of the molten born of the same parents' agent of red blood cell is included in United States Patent (USP) 3,874, in 852,4,528,274,3,874,852,4,346,018 and 4,485,175.
For example, United States Patent (USP) 4,346,018 lectures a kind of this molten born of the same parents' agent system of infiltration multipurpose blood diluent and utilization that waits distinguishes the leukocytic method of two classes.United States Patent (USP) 4,485,175 have lectured a kind of quaternary ammonium salt of using as thinning agent and COULTER
COUNTER Model S-plus automatic blood numeration instrument (registered trademark of Cauer spy (COULTER) company of Florida Miami) is to the method and the reagent system of lymphocyte, monocyte and the leukocytic differentiation of granulocyte three classes.United States Patent (USP) 4,485,175 also lecture, for forming a kind of suitable chromogen that is used to measure haemoglobin, also available a kind of alkali metal cyanide, for example potassium cyanide.
Carried out the measurement of hemoglobin concentration with the method for cyanmethemoglobin.According to this method, in blood sample, add a kind of erythrocytolysis agent that contains non-ionic surfactant, to reduce the turbidity that erythrocytic cell membrane causes.D/d haemoglobin is by the oxidation of a kind of effect of oxygenant institute, such as potassium ferricyanide oxygenant, to produce methemoglobin.Then, cyanide ion combines with methemoglobin and forms cyanmethemoglobin (HiCN), can produce a kind of stable hemoglobinometry sample.The absorption of measuring the cyanmethemoglobin sample with a predetermined wavelength.This method is widely adopted as the standard method of determining hemoglobin concentration.
Though the compound that forms with the method for cyanmethemoglobin in case be a kind of material of stabilizer pole after forming, uses a kind of oxygenant will finish the oxidation of using Drabkins reagent with more than 10 minutes time to erythrocytic oxidation.In addition, the dissolving of the dilution of erythrocytic dissolving, haemoglobin and red blood cell and platelet membrane is desired slower than robot.
For satisfying the time requirement of automatic hematology analyzer, method in the past is to use the erythrocytolysis agent that contains cyanide ion, and it can form stable hemochromogen, and has an absorption spectrum that is similar to cyanmethemoglobin.The erythrocytolysis agent has also reduced the time needs of cytolysis, haemoglobin elution and cell membrane dissolving.In addition, must as sodium hypochlorite, carry out detoxification and processing to useless liquid with a kind of suitable method, this is the work of extremely requiring great effort.
For this reason, also utilize another known oxyhemoglobin method.In the oxyhemoglobin method, the non-ionic surface active material that red blood cell is used to lysed erythrocyte and discharges haemoglobin dissolves.Haemoglobin is released, and with the form (HbO of oxyhemoglobin
2) it is measured.Measure the absorption of oxyhemoglobin with predetermined wavelength.Because the oxyhemoglobin method is without prussiate, thus not dangerous when reagent treatment, do not need to carry out the processing operation of the waste liquid of trouble yet.
Yet traditional oxyhemoglobin method has not only lysed erythrocyte of a lytic agent, and the defective that makes leucocyte become very little.This is favourable for absorptiometry, because it has reduced the light scattering that leucocyte causes, but on the other hand, it makes with molten born of the same parents' agent to the very difficulty that becomes more than two kinds of leukocytic measurements.
For avoiding this problem, use the automatic blood sample analyser of oxyhemoglobin method, by a sample being delivered to two test sections is that red blood cell and determination of white cells are prepared sample separately, and one is used for erythrocytic measurement, and one is used for leukocytic measurement.Yet, owing to not only need two independent test sections, and need two cover streamlines to prepare measuring sample, so having, this method needs costliness and the shortcoming of complex apparatus.
In addition, use the oxyhemoglobin method, can not accurately measure the high blood sample of ferrihemoglobin content, this is not can be converted into oxyhemoglobin at an easy rate because of methemoglobin.When using contrast blood, ferrihemoglobin content is extremely important.Contrast blood is generally used for controlling the analysis precision of automatic hematology analyzer.Contrast blood leaves refrigerating state usually in, and has stable hemoglobin concentration for a long time.Yet be higher than between the storage life of room temperature, the haemoglobin in the blood is converted into methemoglobin at leisure.Therefore, when being higher than the contrast blood of 22 ℃ of storages with the measurement of oxyhemoglobin method, can't measure the haemoglobin part that has changed methemoglobin into, therefore after in a few days, the measured value of haemoglobin can become and slowly be lower than initial value.
The method that addresses this problem is to use the NaLS (SLS) of a kind of lauryl sodium sulfate or equivalent, a kind of anionic surfactant and Triton X-100----non-ionic surfactant reagent in neutral buffered solution (the pH value is 7.2) to measure haemoglobin.This method is by instruction in the 15th volume 83 (1982) of " clinical biochemistry " such as Oshiro.In the method, with erythrocytolysis, and the haemoglobin that elution goes out is converted to the SLS haemoglobin by the effect of SLS and Triton X-100.Therefore, can not be subjected to methemoglobin to influence the measurement of carrying out blood hemoglobin concentration under the situation, and because do not use prussiate, there is no need to carry out the special process that waste liquid is disposed.Yet, can not from the blood sample of same processing, carry out the measurement of leukocyte differentiation and hemoglobin concentration with this method.Change under the SLS concentration of SLS haemoglobin leucocyte at a haemoglobin that need go out elution dissolved, makes and can not carry out simultaneously with the measurement of haemoglobin.
United States Patent (USP) 5,250,437 and 5,242,832 have taught other solutions of these problems.These public publications are mentioned, in order to measure the concentration of haemoglobin, but with the main effect of suitable erythrocytolysis agent by the quaternary ammonium salt of the haemoglobin in elution red blood cell lysed erythrocyte selectively.Almost simultaneously, eluted haemoglobin sex change, promptly its space structure is changed, and the oxygen that the heme iron in the haemoglobin is dissolved in the reagent is oxidized to trivalent ion from divalent ion, thereby obtains methemoglobin.At United States Patent (USP) 5,250, in 437, an amount of kation, nonionic and amphoteric surfactant are added the degree of adjusting the haemoglobin sex change in the erythrocytolysis agent, to obtain stable haemoglobin.Though there is not the stable mechanism of clear and definite haemoglobin, by inference, may be multiple surfactant with different molecular structures to causing the fixing of denaturation degrees on the predetermined level of acting on of haemoglobin or being converted into the variation of the space structure of methemoglobin.United States Patent (USP) 5,242,832 have improved United States Patent (USP) 5,250 by adding a kind of stabilized hemoglobin agent, the reagent in 437.Though do not understand fully the mechanism of action of stabilized hemoglobin agent, but the single electron pair of nitrogen-atoms in the stabilized hemoglobin agent molecule structure or the oxygen atom in the phenolic hydroxyl may be combined into chelate with the heme iron in the methemoglobin by inference, thereby has obtained stable haemoglobin.
Other method comprises United States Patent (USP) 4,853,338 in addition, and its uses pH value to be at least 11.3 anionic surfactant and measures whole haemoglobins.Ionic surface active agent can be used as a kind of alkali, or contains another highly basic that is independent of surfactant give alkaline ferriheme and react desired pH value in composition.The surfactant that is fit to give desired pH value comprises the alkyl trimethyl ammonium hydroxide of long-chain.The surfactant that is fit to combine with the independent component that gives desired pH value comprises zwitterionic surfactant and cationic quaternary ammonium halogenide.Document is also mentioned, and can use anionic surfactant, for example the alkylsurfuric acid alkali metal salt.Yet because the pH value of alkaline ferriheme, this method is not optimal.
Other method also comprises United States Patent (USP) 4,656,139 and 4,617,275, and they disclose a kind of haemoglobin that prevents and have been transformed into the oxyhemoglobin method of methemoglobin.In order to prevent that haemoglobin is transformed into methemoglobin, (2-pyridylthio-1-oxidation) sodium is added in the borate buffer solution as a kind of protective agent.In addition, in reagent system, use EDTA-2K as a kind of sequestrant.The pH value of reagent system is 6～8, and osmotic pressure is 240～310mOsm/kg.
In addition, additive method comprises United States Patent (USP) 3,874,852 disclosed usefulness are a kind of carries out the measurement of blood middle leukocytes and haemoglobin by containing fully lysed erythrocyte and platelet cell and haemoglobin being changed into the reagent that the aqueous solution of the no ferricyanide of the quaternary ammonium ion of the hemochromogen that is used to measure and cyanide ion forms.
Another method comprises United States Patent (USP) 4,185, the solubilising reagent of mentioning in 964 that is used for blood analyser, rapid damage leucocyte.Reagent and haemoglobin reaction or complexing form a kind of hemochromogen with enough stability, allow to carry out the spectral measurement of haemoglobin.
Another method also comprises United States Patent (USP) 4,800,167, it mentions a kind of reagent system that is used for measuring the content of hemoglobin in the whole blood, and whole blood comprises that molecular weight is approximately from 10,000～360,000 polyvinylpyrrolidone aqueous solution, the pH value is greater than 8, and the polyvinylpyrrolidone aqueous solution makes the haemoglobin sex change in the whole blood, form a kind of can be at the stable material of the wavelength measurement of about 575 nanometers.
Although prior art discussed above is arranged, but develop necessity that other have the reagent of one or more following characteristics in addition: it should be nontoxic, when using, the solution that oozes with a kind of grade of not deleterious effect stabilized hemoglobin produces a kind of stabilised blood chromogen, and compatible mutually with other blood measuring parameters, as white blood cell count(WBC) and leukocyte differentiation.
The present invention relates to a kind of reagent composition and a kind of method of measuring the hemoglobin concentration in the blood sample that does not contain prussiate or iron cyanogen compound ion.Reagent composition comprises fully lysed erythrocyte and discharge molten born of the same parents' agent and a kind of antioxidant that can enough the haemoglobin of release be converted to methemoglobin of haemoglobin of at least a that choose, content from quaternary ammonium salt, pyridiniujm and its potpourri.Usually, when molten born of the same parents' agent and antioxidant combination, reagent composition has about 6～7.5 pH value.Reagent composition can be effectively in 5～11.5 pH value scope abundant lysed erythrocyte and discharge haemoglobin, preferably from pH9-11,9.5-10.5 most preferably.In reagent composition, can add a kind of pH value correctives, big pH value in 5～11.5 scopes is provided.
The present invention also relates to a kind of method of the measurement hemoglobin concentration of forming by following steps: allow blood sample and a kind of reagent composition that does not contain prussiate or ferricyanide ion react, wherein said reagent composition comprises (i) thereby fully lysed erythrocyte and the molten born of the same parents' agent and the (ii) a kind of content that discharge haemoglobin can convert the haemoglobin that discharges to antioxidant that methemoglobin forms a kind of reaction product at least a that select from quaternary ammonium salt, pyridiniujm and its potpourri, content enough; And the hemoglobin concentration of described blood sample is determined in the absorption of measuring described reaction product.Usually, when molten born of the same parents' agent and antioxidant combination, reagent composition has about 6～7.5 pH value.Reagent composition 5～11.5, preferably 9～11 and best be can be effectively in the pH value scope 9.5～10.5 abundant lysed erythrocyte and discharge haemoglobin.In reagent composition, can add a kind of pH value correctives, big pH value in 5～11.5 scopes is provided.
In addition, the present invention relates to a kind of reagent composition and the method that can from the reaction product of same blood sample and reagent composition, carry out hemoglobin concentration measurement and leukocyte differentiation.Reagent composition comprise at least a be selected from quaternary ammonium salt, pyridiniujm and its potpourri, the abundant lysed erythrocyte and discharge molten born of the same parents' agent of haemoglobin and thereby a kind of content can convert the haemoglobin that discharges to antioxidant that methemoglobin forms reaction product effectively of content.Usually, when molten born of the same parents' agent and antioxidant combination, reagent composition has about 6～7.5 pH value.Reagent composition 5～11.5, preferably from 9～11 and preferably can be effectively in 9.5～10.5 the pH value scope abundant lysed erythrocyte and discharge haemoglobin.In reagent composition, can add a kind of pH value correctives, big pH value in 5～11.5 scopes is provided.
Measure in a kind of blood sample in hemoglobin concentration and the leukocytic method of differentiation at least two classes, innovative approach comprises the absorption of the reaction product hemochromogen of measuring blood sample and reagent composition of the present invention reaction gained and distinguishes at least two class leucocytes according to described reaction product.
Fig. 1 shows the COULTER COUNTER that the special company of the Cauer that uses Florida State Miami makes
Correlativity between hemoglobin concentration is measured in the different samples of the normal whole blood of Model S-plus IV diff instrument.The x axle represents to utilize commercially available prod LYSES
The molten born of the same parents' agent of III diff (registered trademark of the special company of the Cauer of Florida State Miami), special company produces by Cauer, and the y axle represents to use the reagent of embodiment 1, is expressed as LYSE S4 in the drawings.
Fig. 2 shows among Fig. 1 the correlativity between hemoglobin concentration is measured in the different samples of the abnormal whole blood of using COULTER COUNTER Model S-plus IVdiff instrument.The x axle represents to utilize the molten born of the same parents' agent of commercially available prod LYSES III diff, and the y axle represents to use the reagent of embodiment 1, is expressed as LYSE S4 in the drawings.
Fig. 3 shows leucocyte (WBC) in the normal whole blood sample of the method for utilizing embodiment 2 and the COULTER COUNTER ModelS-plus IV diff instrument correlativity between measuring.The x axle represents to utilize the molten born of the same parents' agent of commercially available prod LYSES III diff, and the y axle represents to use the reagent of embodiment 1, is expressed as LYSE S4 in the drawings.
Correlativity between the white blood cell count(WBC) that Fig. 4 shows the abnormal whole blood of the method for utilizing embodiment 2 and COULTER COUNTER ModelS-plus IV diff instrument is measured.The x axle represents to utilize the molten born of the same parents' agent of commercially available prod LYSES III diff, and the y axle represents to use the reagent of embodiment 1, is expressed as LYSE S4 in the drawings.
Fig. 5 shows the correlativity between leukocytic lymphocyte kind is measured in the sample of normal whole blood of the method for utilizing embodiment 2 and COULTER COUNTER ModelS-plus IV diff instrument.The x axle represents to utilize the molten born of the same parents' agent of commercially available prod LYSES III diff, and the y axle represents to use the reagent of embodiment 1, is expressed as LYSE S4 in the drawings.
Fig. 6 shows the correlativity between leukocytic monocyte kind is measured in the sample of normal whole blood of the method for utilizing embodiment 2 and COULTER COUNTER ModelS-plus IV diff instrument.The x axle represents to utilize the molten born of the same parents' agent of commercially available prod LYSES III diff, and the y axle represents to use the reagent of embodiment 1, is expressed as LYSE S4 in the drawings.
Fig. 7 shows leukocytic granulocyte kind in the normal whole blood sample of the method for utilizing embodiment 2 and the COULTER COUNTER ModelS-plus diff instrument correlativity between measuring.The x axle represents to utilize the molten born of the same parents' agent of commercially available prod LYSES III diff, and special company produces by Cauer, and the y axle represents to use the reagent of embodiment 1, is expressed as LYSE S4 in the drawings.
The present invention has provided a kind of novel agent composition and side of measuring blood hemoglobin concentration Method does not wherein contain cyanide or iron cyanide ion in the reagent composition. More preferably, should Reagent composition and method utilize the product of blood sample and reagent reacting can carry out hemoglobin Measurement and at least two kinds of leukocytic differentiations.
Reagent composition comprises the molten born of the same parents' agent of a kind of red blood cell and a kind of antioxidant, and does not contain poisonous Material, for example cyanide ion. The sample liquid that carries out this measurement comprises whole blood sample and is used for the school Blood calibration agent and the blood reference substance of the normal performance of accurate and definite blood analyser. Calibration agent Can be taken from the human or animal with reference substance.
Molten born of the same parents' agent contains at least a becoming of choosing from quaternary ammonium salt, pyridiniujm and its mixture Divide. Quaternary ammonium salt has following molecular formula:
Wherein R1 is C8～C20 alkyl, alkenyl or kiki alkenyl group; R2, R3 and R4 C1～C8 alkyl, alkenyl or kiki alkenyl group; And X-is the root that forms salt, as Cl, Br, I, PO4And CH3SO
4 Preferably, R1 represents a kind of at least 12 carbon that have The alkyl of atom, and R2, R3 and R4 represent to have the short alkyl of 1～6 carbon atom. Pyridiniujm has following form:
Wherein, n is the integer from 7～19, and X-is an anionic group.
Preferred molten born of the same parents' agent is the mixture that utilizes at least two kinds of quaternary ammonium compounds. More specifically, excellent Molten born of the same parents' agent of choosing is by a kind of quaternary ammonium compound with alkyl of 12 carbon atoms of the representative of R1 wherein Wherein the quarternary ammonium salt compound of a kind of alkyl with 14～16 carbon atoms of R1 representative is mixed Compound forms. Best, molten born of the same parents' agent comprises and contains TTAB and dodecyl Trimethyl ammonium chloride. Other quaternary ammonium salt that can provide effective result comprises and the myristyl front three The softex kw that ammonium chloride mixes mutually or cetyldimethylethylambromide bromide Ammonium.
The effective content that reagent composition contains can discharge molten born of the same parents' agent of haemoglobin from red blood cell.Preferably, reagent composition contains the molten born of the same parents' agent in 5～80 grams per liter scopes.The scope that preferably contains molten born of the same parents' dosage is at 15～35 grams per liters.
With molten born of the same parents' agent haemoglobin is discharged from red blood cell, and d/d haemoglobin and a kind of antioxidant react.The haemoglobin that antioxidant is used for discharging changes into hemochromogen.The amount of employed antioxidant can change into hemochromogen to d/d haemoglobin effectively.If the quantity not sufficient of the antioxidant that uses, compare with using the molten born of the same parents' agent of commercially available prod LYSE S IIIdiff so, haemoglobin just is not enough to accurately measure the concentration value of haemoglobin to the conversion of hemochromogen on COULTER COUNTER Model S-Plus IV diff instrument.Preferably, the content range of antioxidant, is more preferably from 1 grams per liter to 3 grams per liters to 10 grams per liters from 0.1 grams per liter.These scopes can change, because the ratio of the amount of antioxidant and the volume of blood sample depends on the amount that joins the thinning agent in the blood sample and joins the amount of the molten born of the same parents' agent in the blood sample.
Antioxidant comprises reductive agent, and for example ascorbic acid, phosphorous acid, sodium sulphite, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate and have the oxyacid alkali metal salt of reduction characteristic sulphur also comprise its potpourri, and wherein the oxidation value of sulphur is from+2～+ 4.Preferably, antioxidant comprises sodium sulphite, sodium pyrosulfite, sodium bisulfite and their potpourri.Best, antioxidant comprises sodium sulphite.
Usually, when molten born of the same parents' agent and antioxidant combination, reagent composition has about 6～7.5 pH value.The pH value of reagent composition can be in about 5～11.5 scope, and preferably 9～11, preferably 9.5～10.5.In order to obtain a pH value in the reagent composition pH value scope, should add a kind of pH regulator agent.
The example that is suitable for regulating the pH regulator agent of required pH value comprises strong acid, for example hydrochloric acid, and highly basic, for example alkali metal hydroxide.The example of gratifying alkali metal hydroxide comprises NaOH and potassium hydroxide.Less preferred example comprises tetraalkylammonium hydroxide, and alkyl can contain 1～4 carbon atom, for example tetrabutylammonium hydroxide.
After measured, compare, serious problems can occur when under pH value 5, obtaining accurate hemoglobin concentration value with using the molten born of the same parents' agent of commercially available prod LYSE S III diff.Under pH value 9, the stability of reagent composition reduces.
Reagent composition should have other characteristic, so that itself and its desired use fit.These characteristics comprise the osmotic pressure with 220～370 millitsmols and the conductance of about 3～11 Siemenss.
Have been found that when using a kind of having mixed when being incorporated in 70 ℃ of aqueous solution of having deposited several days molten born of the same parents' agent and antioxidant the concentration of measured haemoglobin is different from the value that the aqueous solution fresh mixture that uses molten born of the same parents' agent and antioxidant is obtained.Utilize the concentration of the haemoglobin that the aqueous solution fresh mixture of molten born of the same parents' agent and antioxidant measures to compare with the value of using the molten born of the same parents' agent of LYSE S IIIdiff to be obtained.More specifically, when depositing the potpourri of molten born of the same parents' agent and antioxidant at elevated temperatures, the concentration value of measured haemoglobin is starkly lower than the concentration when the measured haemoglobin of comparing with the molten born of the same parents' agent of use LYSE S III diff.For addressing this problem, can before hemoglobin concentration is measured, reagent be mixed rapidly.
Be preferably in and add a kind of anti-oxidation stabilizing agent in the reagent composition so that the stability of reagent to be provided.Effective quantity with stability that reagent composition can be provided adds anti-oxidation stabilizing agent.Reagent composition stability is to make reagent can provide accurate hemoglobin concentration to measure after long-term storage.More specifically, when reagent composition of the present invention has deposited at least 6 week, and be that the measurement result of hemoglobin concentration can be compared in the measurement result that stands under the same storage condition with the molten born of the same parents' agent of LYSE SIII diff under the situation that storing temperature raises.
Suitable anti-oxidation stabilizing agent is ethylenediamine tetraacetic acid (EDTA) derivant, citric acid, tartrate, gluconic acid, saccharic acid and their compound.Preferably, anti-oxidation stabilizing agent is selected from disodium ethylene diamine tetraacetate, ethylene glycol bis (the amino monoethyl ether of 3-)-N, N '-tetraacethyl (EGTA), gluconic acid, N-(2-acetylamino) imido oxalic acid (ADA) and its potpourri.Most preferred stabilizing agent is EDTA.Preferably, the scope of the anti-oxidation stabilizing agent that reagent composition contains more preferably is 1～5 grams per liter at 0.1～10 grams per liter, most preferably is 2 grams～4 grams per liters.
For the validity of anti-oxidation stabilizing agent is described, between reagent composition, compare.Reagent 1 is not add the reagent that ethylenediamine tetraacetic acid two is received among the embodiment 1.Reagent 2 is the reagent among the embodiment 1.Reagent composition was deposited 7 days under 70 ℃ of conditions.The reagent composition that following explanation of tables contains anti-oxidation stabilizing agent provide can with the comparable hemoglobinometry of traditional molten born of the same parents' agent of LYSE S III diff.
Table one
Hemoglobin concentration
Agent combination matter sample LYSE S III reagent 1 reagent 2
The molten born of the same parents' agent of diff (no EDTA) is new blood 1 15.5 14.4 15.3 new bloods 2 12.4 11.4 12.2 (0.25%EDTA)
The method of measuring hemoglobin concentration relates to the detection of the chromogen that reaction produced of blood sample and reagent composition of the present invention.The measurement hemoglobin concentration may further comprise the steps: allow blood sample and the reagent composition that does not contain cyanide ion react, described reagent composition comprises at least a molten born of the same parents' agent that is selected from quaternary ammonium salt, pyridiniujm and its potpourri, the content of molten born of the same parents' agent can be effectively abundant lysed erythrocyte, and discharge haemoglobin; Its content can be effectively change into antioxidant and a kind of 5～11.5 pH value aqueous slkali with the formation reaction product that provides of hemochromogen with the haemoglobin that discharges; And the concentration of the haemoglobin of described blood sample is determined in the absorption of measuring described reaction product.
The reaction product chromogen has in 500～600 nano measurements absorption spectrum repeatably.
Correlativity between the hemoglobin concentration that Fig. 1 shows 36 kinds of normal whole blood samples of difference that utilize COULTER COUNTER Model S-Plus IV diff instrument is measured.The x axle represents to utilize the molten cytosol of commercially available prod LYSE S III diff, and the y axle represents to use the reagent among the embodiment 1 that is expressed as LYSE S4 in the drawings.The blood sample number of being tested is 36.Normal blood sample is defined as the people's of no known disease blood sample.
Related coefficient is r=0.9940, and the tropic, y=1.0115x-0.1671 expresses acceptable correlativity and deviation.
Correlativity between the hemoglobin concentration that Fig. 2 shows 82 kinds of different abnormal whole blood samples that utilize COULTER COUNTER Model S-Plus IV diff instrument is measured.The x axle represents to utilize the molten cytosol of commercially available prod LYSE S III diff, and the y axle represents to use the reagent among the embodiment 1 that is expressed as LYSE S4 in the drawings.Unusual blood sample obtains from the people who suffers from one or more diseases, for example anemia, leukaemia and abnormal blood cell number disease.
Related coefficient is r=0.9994, and the tropic, y=1.0249x-0.256 expresses acceptable correlativity and deviation.
Embodiment 1
By each composition being mixed with following composition reagent being higher than under the condition of room temperature.
Quantity (grams per liter) DTAC and Tetradecyl Trimethyl Ammonium Bromide 32 antioxidant 2EDTA disodium 2.5Pluronic 25R8 prill 1pH values adjust to 10
In addition, have in addition can be from carrying out the measurement of hemoglobin concentration and the advantage of leukocyte differentiation with a kind of blood sample and reagent composition reaction product for reagent composition of the present invention.Preferably can carry out two kinds of leukocytic differentiations at least according to the measurement of haemoglobin.These two kinds of leucocytes are lymphocyte and granulocyte.Most preferably, can carry out at least three kinds of leukocytic differentiations.These three kinds of leucocytes are lymphocyte, monocyte and granulocyte.
When the concentration of measuring haemoglobin and leukocytic differentiation, the concentration of molten born of the same parents' agent is further adjusted an energy with effective content, make its abundant lysed erythrocyte, and deleterious effect leucocyte not, thereby can from same reaction product, carry out the measurement and the leukocytic differentiation of hemoglobin concentration.When using a kind of quarternary ammonium salt compound, the effective content of molten born of the same parents' agent is in the scope of 5～80 grams per liters.Preferably in the scope of 15～35 grams per liters.
Molten born of the same parents' agent lysed erythrocyte and deleterious effect leucocyte not.Yet, have in the blood sample that a certain amount of dissolved erythrocytic cell residue and leucocyte be retained in dissolving.Cell residue may produce ground unrest or cell flow blockage in leukocytic differentiation.Selectively, in reagent composition, can contain a kind of energy is eliminated the effective content that disturbs by dissolved cell particulate matter, protein residues surfactant.Surfactant concentrations scope from 0.1 to 2.5 grams per liter, and from 0.5 to 1.5 grams per liter more preferably.Suitable ionic surfactant pack is drawn together Pluronic (Pluronic) F, Pluronic L, Pluronic P, Pluronic R (the Pluronic surfactant is produced by BASF AG of the Parsippary of New Jersey) and polyxyethylated phenol.Preferred surfactants is Pluronic 25R8 Prill and Pluronic F127, and best surfactant is Pluronic 25R8 Prill.
When carrying out hemoglobin concentration measurement and leukocyte differentiation, the concentration of antioxidant is can effectively d/d haemoglobin be changed into the amount of hemochromogen.If use excessive antioxidant, the leukocytic value of recovery is lower.
Preferably, the content range of antioxidant at 0.1 grams per liter to 10 grams per liters, more preferably from 1 grams per liter to 3 grams per liters.These scopes can change, because the ratio of the amount of antioxidant and the volume of blood sample depends on the amount that joins the thinning agent in the blood sample and joins the amount of the molten born of the same parents' agent in the blood sample.
When carrying out the two the measurement of hemoglobin concentration and leukocyte differentiation, when molten born of the same parents' agent and antioxidant combination, reagent composition has about 6～7.5 pH value.The scope of reagent composition can be from 5～11.5, more preferably from 9～11, and preferably from 9.5～10.5.For obtaining the pH value of the reagent composition within suitable pH scope, can add a kind of pH regulator agent.When the pH value is higher than 11.5, when obtaining the hemoglobin concentration that molten born of the same parents' agent is obtained corresponding to use commercially available prod LISE SIII diff serious problems can appear.
When carrying out the two the measurement of hemoglobin concentration and leukocyte differentiation, the concentration of anti-oxidation stabilizing agent is adjusted to an amount that the stability of reagent composition can be provided effectively.More specifically, when reagent composition of the present invention has deposited at least 6 week, and be that the measurement result of leukocyte differentiation and hemoglobin concentration can be comparable in the measurement result that stands under the same storage condition with the molten born of the same parents' agent of LYSE S IIIdiff under the situation that temperature raises.Same anti-oxidation stabilizing agent can be used for the measurement and the leukocytic differentiation of hemoglobin concentration.Preferably, reagent composition contains the scope of anti-oxidation stabilizing agent at 0.1～10 grams per liter, and more preferably from 1～5 grams per liter, and preferably from 2～4 grams per liters.
Hemoglobin concentration and distinguishing at least two kinds of leukocytic methods in measuring blood sample, the improvement of the absorptiometry of the reaction product that is produced when the present invention relates to blood sample and according to the different leukocytic improvement of described reaction product differentiation at least two classes with reagent composition reaction of the present invention.
More specifically, method of the present invention comprises allows blood sample and the reagent composition that does not contain cyanide ion react, described reagent composition comprises at least a quaternary ammonium salt that is selected from, molten born of the same parents' agent of pyridiniujm and its composition, the content of molten born of the same parents' agent can be effectively abundant lysed erythrocyte and discharge haemoglobin, a kind of content can change into hemochromogen with the haemoglobin that discharges effectively, thereby form the antioxidant of reaction product, the concentration of the haemoglobin of described blood sample is determined in the absorption of measuring described reaction product, and distinguishes at least two kinds of leucocytes according to described reaction product.
Usually, when molten born of the same parents' agent and antioxidant were combined, the reagent composition of this method had 6～7.5 pH value.The scope of the pH value of reagent composition is approximately from 5～11.5, more preferably from 9～11, and preferably from 9.5～10.5.In order to obtain the pH value of reagent composition within pH value scope, should add a kind of pH regulator agent.
Person of skill in the art will appreciate that, can finish leukocytic differentiation by technology known in the art.These technology are for example understood in the instruction of people's such as people's such as United States Patent (USP) 3,874,852 Ledis of Hamill United States Patent (USP) 4,485,175 and Carter United States Patent (USP) 4,528,274.
Embodiment 2
A kind of whole blood sample oozes the dilution of balance thinning agent by a kind of grade of predetermined concentration, and thinning agent is adjusted to predetermined a pH value and an osmotic pressure.The such mode of variation with a kind of mononuclear blood cell volume of causing the leucocyte kind is within a certain period of time at least mixed resulting dilute blood mutually with reagent composition of the present invention, thereby can distinguish at least two class leucocytes.In addition, can determine to measure the leucocyte numeration.
Correlativity between the leucocyte (WBC) that Fig. 3 shows 36 normal whole blood samples of difference of the method for utilizing embodiment 2 and COULTER COUNTER ModelS-Plus IV diff instrument is measured.The x axle uses the molten born of the same parents' agent of commercially available prod LYSE S III diff, and the y axle uses the reagent among the embodiment 1 that is expressed as LYSE S4 in the drawings.Normal blood sample is defined as the people's of no known disease blood sample.Related coefficient is r=0.9989, and tropic y=0.9899x+0.0074 expresses acceptable correlativity and deviation.
Correlativity between the leucocyte (WBC) that Fig. 4 shows 82 kinds of different abnormal whole blood samples of the method for utilizing embodiment 2 and COULTER COUNTER ModelS-Plus IV diff instrument is measured.The x axle uses commercially available prod LYSE S III diff solution, and the y axle uses the reagent among the embodiment 1 that is expressed as LYSE S4 in the drawings.Related coefficient is r=0.9996, and tropic y=0.9962x-0.0251 expresses higher correlativity and very low deviation.
Another advantage of reagent composition of the present invention is that it can be used for as using traditional measurement method to carry out a kind of reagent of white blood cell count(WBC) in the blood sample.When being used to the measurement of white blood cell count(WBC), the agent prescription of reagent composition of the present invention is the same with the prescription of the reagent composition that is used for hemoglobin concentration measurement and leukocyte differentiation.This provides another advantage: can use with a kind of reagent composition and reaction is produced according to blood sample and reagent composition of the present invention allogeneic reaction product and carry out the measurement of hemoglobin concentration, leukocyte differentiation and white blood cell count(WBC).
Correlativity between the leukocytic lymphocyte that Fig. 5 shows 204 normal whole blood samples of difference of the method for utilizing embodiment 2 and COULTER COUNTER ModelS-Plus IV diff instrument is measured.The x axle uses the molten born of the same parents' agent of commercially available prod LYSE S III diff, and the y axle uses the reagent among the embodiment 1 that is expressed as LYSE S4 in the drawings.Related coefficient is r=0.9885, and tropic y=0.9761x-0.729 expresses acceptable correlativity and deviation.
Correlativity between the leukocytic monocyte that Fig. 6 shows 224 normal whole blood samples of difference of the method for utilizing embodiment 2 and COULTER COUNTER ModelS-Plus IV diff instrument is measured.The x axle uses commercially available prod LYSE S III diff solution, and the y axle uses the reagent among the embodiment 1 that is expressed as LYSE S4 in the drawings.Related coefficient is r=0.8528, and tropic y=0.9147x+1.3805 expresses preferably correlativity and acceptable deviation.
Correlativity between the leukocytic granulocyte that Fig. 7 shows 223 normal whole blood samples of difference of the method for utilizing embodiment 2 and COULTER COUNTER ModelS-Plus IV diff instrument is measured.The x axle uses commercially available prod LYSE S III diff solution, and the y axle uses the reagent among the embodiment 1 that is expressed as LYSE S4 in the drawings.Related coefficient is r=0.9851, and tropic y=0.976x+2.0711 indicates higher correlativity and very low deviation.
Detailed description of the present invention provides in instructions for the purpose of description.Do not break away from the spirit and scope of the present invention, those skilled in the art can do some variations to the details of being given.
Claims (14)
1, be used for measuring the reagent composition that does not contain cyanide ion of the hemoglobin concentration of blood sample, comprise:
A. comprise at least a molten born of the same parents' agent that is selected from quaternary ammonium salt, pyridiniujm and its composition, the content of molten born of the same parents' agent can be effectively abundant lysed erythrocyte and discharge haemoglobin; With
B. a content can change into the haemoglobin that discharges the antioxidant of hemochromogen effectively, wherein antioxidant is selected from phosphorous acid, sodium sulphite, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate and other has the oxyacid alkali metal salt of reduction characteristic sulphur, comprise its potpourri, wherein the oxidation value of sulphur is from+2～+ 4, and the pH scope of wherein said reagent composition is 5-11.5.
2, claim 1 is used for the reagent composition measuring the hemoglobin concentration of blood sample and distinguish the leucocyte kind, wherein have molten born of the same parents' agent of effective dose, it is measured enough lysed erythrocytes and discharges haemoglobin and influence leucocyte to determine two kinds of leukocytic kinds on volume.
3, claim 1 or 2 reagent composition, wherein molten born of the same parents' agent comprises two kinds of different quaternary ammonium salts that are selected from following formula:
Described quaternary ammonium salt has following molecular formula:
Wherein R1 is C8～C20 alkyl, alkenyl or kiki alkenyl group; R2, R3 and R4 are C1～C8 alkyl, alkenyl or kiki alkenyl group; And X-is the root that forms salt.
4, the reagent composition of claim 3, wherein R1 represents a kind of alkyl with at least 12 carbon atoms, and R2, R3 and R4 represent to have the short alkyl of 1～6 carbon atom, X-is Cl, Br, I, PO
4Or CH
3SO
4
5, the reagent composition of aforementioned arbitrary claim also contains a kind of pH regulator agent that 5～11.5pH value scope is provided, and described pH regulator agent is selected from strong acid and highly basic.
6, the reagent composition of aforementioned arbitrary claim, comprise that also a kind of its content can provide the anti-oxidation stabilizing agent of reagent composition stability effectively, and wherein stabilizing agent is selected from ethylenediamine tetraacetic acid (EDTA) derivant, citric acid, tartrate, gluconic acid, saccharic acid and its potpourri.
7, measure the method for hemoglobin concentration, comprise step:
A. blood sample and a kind of reagent composition that does not contain cyanide ion react, described reagent composition comprises (i) at least a quaternary ammonium salt that is selected from, pyridiniujm and its potpourri, molten born of the same parents' agent that it is measured enough lysed erythrocytes and discharges haemoglobin, (2) a kind of its amount haemoglobin that can be enough to discharge converts hemochromogen to, thereby form a kind of antioxidant of reaction product, wherein antioxidant is selected from ascorbic acid, phosphorous acid, sodium sulphite, sodium pyrosulfite, sodium bisulfite, sodium thiosulfate and other have the oxyacid alkali metal salt of reduction characteristic sulphur, comprise its potpourri, wherein the oxidation value of sulphur is from+2～+ 4, and the pH scope of wherein said reagent composition is 5-11.5.
B. the hemoglobin concentration of described blood sample is determined in the absorption of measuring described reaction product.
8, the method for hemoglobin concentration in the measurement blood sample of claim 1 and differentiation leucocyte kind, wherein the middle reagent composition of step (a) comprises the enough lysed erythrocytes of content and discharges haemoglobin and influence leucocyte by volume, so that determine molten born of the same parents' agent of at least two kinds of leucocyte kinds; This method also comprises distinguishes at least two kinds of leucocyte kinds that are contained in the reaction product.
9, claim 7 or 8 method, wherein molten born of the same parents' agent comprises two kinds of different quaternary ammonium salts that are selected from following formula:
Described quaternary ammonium salt has following molecular formula:
Wherein R1 is C8～C20 alkyl, alkenyl or kiki alkenyl group; R2, R3 and R4 are C1～C8 alkyl, alkenyl or kiki alkenyl group; And X-is the root that forms salt.
10, the method for claim 9, wherein R1 represents a kind of alkyl with at least 12 carbon atoms, and R2, R3 and R4 represent to have the short alkyl of 1～6 carbon atom, X-is Cl, Br, I, PO
4Or CH
3SO
4
11, arbitrary method among the claim 7-10, wherein reagent composition also contains a kind of pH regulator agent that 5～11.5pH value scope is provided, and described correctives is selected from strong acid and highly basic.
12, arbitrary method among the claim 7-10, wherein reagent composition comprises that also a kind of its content can provide the anti-oxidation stabilizing agent of reagent composition stability effectively, and wherein stabilizing agent is selected from ethylenediamine tetraacetic acid (EDTA) derivant, citric acid, tartrate, gluconic acid, saccharic acid and its potpourri.
13, the method in the claim 8, the absorptiometry of wherein said reaction mixture carries out between 500～600 nanometers.
14, the method in the claim 13 is wherein distinguished at least three kinds of leucocyte kinds that are contained in the reaction product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 97126470 CN1130567C (en) | 1997-10-17 | 1997-10-17 | Cyanide-free reagent and method for hemoglobin determination and leukocyte differentiation |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN 97126470 CN1130567C (en) | 1997-10-17 | 1997-10-17 | Cyanide-free reagent and method for hemoglobin determination and leukocyte differentiation |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1215168A CN1215168A (en) | 1999-04-28 |
| CN1130567C true CN1130567C (en) | 2003-12-10 |
Family
ID=5177671
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN 97126470 Expired - Lifetime CN1130567C (en) | 1997-10-17 | 1997-10-17 | Cyanide-free reagent and method for hemoglobin determination and leukocyte differentiation |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1130567C (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102662067A (en) * | 2012-05-20 | 2012-09-12 | 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 |
Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101246113B (en) * | 2007-02-14 | 2012-08-15 | 深圳迈瑞生物医疗电子股份有限公司 | Monitoring method for hemoglobin test result |
| AU2010216932B2 (en) * | 2009-02-23 | 2012-12-06 | Prima Meat Packers, Ltd. | Allergen detection method using immunochromatography |
| CN110520728B (en) | 2017-03-03 | 2022-12-06 | 聚合物技术系统公司 | Systems and methods for enzymatic A1C detection and quantification |
| US11703513B2 (en) | 2017-08-07 | 2023-07-18 | Polymer Technology Systems, Inc. | Systems and methods for enzymatic A1C detection and quantification |
| MX2019010428A (en) * | 2017-12-12 | 2020-01-09 | Polymer Technology Systems Inc | Systems and methods for sample preparation for enzymatic a1c detection and quantification. |
| CN110687306B (en) * | 2019-10-30 | 2023-04-25 | 深圳上泰生物工程有限公司 | Double-reagent glycosylated hemoglobin detection kit for direct on-machine hemolysis enzyme method |
| CN113238060B (en) * | 2021-05-08 | 2022-10-11 | 迈克生物股份有限公司 | Kit for predicting or diagnosing myocarditis |
-
1997
- 1997-10-17 CN CN 97126470 patent/CN1130567C/en not_active Expired - Lifetime
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102662067A (en) * | 2012-05-20 | 2012-09-12 | 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 |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1215168A (en) | 1999-04-28 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP2836865B2 (en) | Reagents for measuring leukocytes and hemoglobin in blood | |
| US5834315A (en) | Cyanide-free reagent and method for hemoglobin determination and leukocyte differentitation | |
| US5882934A (en) | Composition and method for hemoglobin and cell analysis | |
| JP4263247B2 (en) | Cyanide-free lysis reagent composition and method for hemoglobin and cell analysis | |
| JP4889730B2 (en) | Cyanide-free lysis reagent composition; and method of use for the determination of hemoglobin and leukocytes | |
| CN1119657C (en) | Concentration determining reagent for white blood cell and haemoglobin | |
| JP2000162209A (en) | Reagent and method for sorting and counting of erythroblasts | |
| SE453693B (en) | SET FOR DIFFERENCE OF LYMPHOID AND MYELOID LEUKOCYTES, AND A BLOOD CELL STABILIZING ISOTONIC DILUTOR | |
| JP5600726B2 (en) | Sample analysis method | |
| EP0486645A1 (en) | Blood diluent and lysing agent for differential determination of white blood cells | |
| CN1130567C (en) | Cyanide-free reagent and method for hemoglobin determination and leukocyte differentiation | |
| EP0794435B1 (en) | Cyanide-free reagent and method for hemoglobin determination and leukocyte differentiation | |
| CN1277125C (en) | Cyanideless hemolysin and its use | |
| JP4338206B2 (en) | Classification and counting method of erythroblasts | |
| US6890756B2 (en) | Method of using cyanide-free lyse solution to emulate a cyanide-containing lyse solution in the measurement of hemoglobin | |
| US8614066B2 (en) | Method of using ligand-free lysing agent in hemoglobin analysis | |
| JP2891302B2 (en) | Reagents for measuring leukocytes and hemoglobin in blood | |
| JP3143064B2 (en) | Cyanide-free reagent, hemoglobin quantification method and leukocyte classification method | |
| CN112166322A (en) | Green concentrated reagent for hematology system | |
| JP2000329762A (en) | Method for preparing white blood cell measuring reagent and white blood cell measuring sample | |
| CN110954489A (en) | Cyanide-free hemolytic agent and application thereof | |
| CN1308235A (en) | Non-toxic hemolytic agent for blood analysis | |
| JP2006220665A (en) | Measuring method of hemoglobin concentration in blood | |
| MXPA99003779A (en) | Method and elaboration of a hemolizant without cyanides, for obtaining the leukocytes, differentiation in the hemaglobin concentration in automated apparatus |
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 | ||
| CX01 | Expiry of patent term |
Granted publication date: 20031210 |
|
| CX01 | Expiry of patent term |