CN1188703C - Reagent and method for differential determination of leukocytes in blood - Google Patents
Reagent and method for differential determination of leukocytes in blood Download PDFInfo
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- CN1188703C CN1188703C CNB971127913A CN97112791A CN1188703C CN 1188703 C CN1188703 C CN 1188703C CN B971127913 A CNB971127913 A CN B971127913A CN 97112791 A CN97112791 A CN 97112791A CN 1188703 C CN1188703 C CN 1188703C
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Abstract
The present invention relates to a lytic agent and a method for automatically distinguishing the subgroups of leukocytes in blood, particularly to a new lytic agent which can dissolve erythrocytes and can act on acidophilic cells distinguishing at least one subgroup of leukocytes. The lytic agent as an acid contains a high water seepage solution of an alkali metal salt of alkyl sulfuric acid, a lytic agent for acidophilic cells, nonionic surfactant and a physiological salt. When the lytic agent is utilized with a second lytic agent together, DC measurement and RF measurement can be used for distinguishing at least five parts of leukocytes.
Description
Invention field
The present invention relates to a kind of lytic agent and a kind of method of distinguishing white blood cell subgroup in the blood sample by suitable electronic device means.
Background of invention
By blood sample analysis white blood cell colony is necessary and basic part in the numerous disease methods of diagnosis.The acidophic cell of measuring the white blood cell subgroup is important for some disease of diagnosis.For example, find that in Hodgkin's disease, parasite and anaphylactia, the quantity of acidophic cell increases.
The tradition blood sample analysis comprises blood sample is coated on the microscopical microslide, then this microslide of observation analysis.This method is very consuming time, and the explanation of microslide analysis is had individual difference.Therefore having developed utilizes mobile white blood cell count(WBC) to analyze leukocytic method automatically.Utilizing a steps necessary in the automatic blood instrumental analysis white blood cell method is lysed erythrocyte.Up to the present, some solubilising reagents that are used for whole blood sample have been developed.
United States Patent (USP) 4485175 (Ledis etc.) has been described a kind of reagent system and white blood cell has been divided into the method for three kinds of subgroups with the automated cell counting assembly.This reagent system contains hemodilution agent and solubilising reagent.This solubilising reagent contains the potpourri of quaternary surfactant.Yet this reagent system is limited to its purposes white blood cell is divided into three kinds of subgroups: lymphocyte, monocyte and granulocyte.This reagent system can not further be divided into granulocyte three kinds of other subgroups.
United States Patent (USP) 4751179 (Ledis etc.) discloses a kind of reagent system and has distinguished leukocytic method with the automated cell counting assembly.This reagent system contains: 1) solubilising reagent, and it contains the single dissolved dilution agent of saponin or contains two kinds of solution of order adding of being made up of pre-thinning agent and the solubilising reagent that contains saponin; With 2) a kind of active crosslinker, as glutaraldehyde as fixing agent.Heating blood sample solubilising reagent system's potpourri and by DC and turbidity it is analyzed after, can determine four kinds of white blood cell subgroups.These four kinds of subgroups are lymphocyte, monocyte, neutrophil(e) cell and acidophic cell.This method requirement by before the Instrument measuring, needs sample mixture 60-75 ℃ of heating down.
United States Patent (USP) 5155044 (Ledis etc.) discloses a kind of reagent system and quick the separation and analysing leucocyte and can use the automatic hematology analyzer that can measure conductivity (DC), radiate frequency (RF) and light scattering (LS) to divide into five kinds of subgroups automatically that is used for whole blood sample.This reagent system is by the water-based solubilising reagent and aqueous salt cancellation (quench) solution composition that contain acid or acid and saponin potpourri.This method is can distinguish 5 part white blood cells fast and in a pacing amount from whole blood sample.This single-measurement step is to distinguish white blood cell with the single blood aliquot of identical solubilising reagent systematic survey.Yet using this reagent and method that white blood cell is divided into five kinds of subgroups needs senior and expensive laser aid.Detect iff DC and RF with this reagent system, the granulocyte subgroup can not divided into basicyte, acidophic cell and neutrophil's subgroup fully.Can not distinguish granulocyte, this is that the DC of subgroup and RF signal are overlapped because under the described condition of this patent.
By DC and RF measure with white blood cell divide into four or the other method of five kind of subgroup be to use plurality of reagents and carry out multistep and measure.Each reagent is used to distinguish specific subgroup.Usually, use first kind of solubilising reagent (as described in United States Patent (USP) 5116539 (Hamaguchi etc.) and United States Patent (USP) 5389549 (Hamaguchi etc.)) to come white blood cell to be divided into three kinds of subgroups (that is, monocyte, lymphocyte and granulocyte) by measuring DC and RF.
In United States Patent (USP) 5116539, use another kind of solubilising reagent to carry out another measurement and obtain the acidophic cell subgroup.This can divide into lymphocyte, monocyte, acidophic cell and remaining granulocyte four parts.This solubilising reagent is a kind ofly to be adjusted in the hypotonic aqueous solution that the buffering agent in the 3-11 scope is formed by the polyoxyethylene non-ionic surfactant with solution PH.This solubilising reagent except acidophic cell not only lyse red blood cells also dissolve white blood cell, like this can be according to the volume of its remaining cell nuclear to count for eosinophil.Yet,, use the method for this solubilising reagent under 40 ℃ of temperature, sample mixture to be cultivated for 50 seconds in order to separate acidophic cell.This rising temperature needs obviously more complicated instrument, because must control the temperature of reaction.In addition, prolong the work efficiency that the reaction time meeting directly reduces automatic analyzer.
In United States Patent (USP) 5389549, need two kinds of other solubilising reagents and need carry out other twice measurement, obtain acidophic cell and basicyte so that distinguish five parts.In the document, from total granulocyte group, deduct respectively the acidophic cell measured and basicyte and and obtain neutrophil's subgroup.
United States Patent (USP) 5196346 (Lefevre etc.) discloses a kind of solubilising reagent and has used this solubilising reagent to measure (comprising red blood cell and white blood cell) method of the basicyte in the whole blood sample automatically by dissolving all haemocytes except that basicyte.Solubilising reagent is by polyethenoxy ether class surfactant, phthalic acid/HCL potpourri, lauryl sodium sulfate (SDS) and butylated hydroxy-methylbenzene type antioxidant.SDS reduces the formation that precipitates in the surveying instrument as protein denaturant and finds to promote cytolysis.Disclosed method requires temperature of reaction is controlled at 30-40 ℃ and be confined to only write down the number of basicyte.
Unsettled U.S. Patent application 08/488630 (in application on June 8 nineteen ninety-five) discloses a kind of solubilising reagent system and used DC, RF and light scattering measurement device is the method for five kinds of subgroups with the white blood cell automatic distinguishing.This solubilising reagent system is made up of the water-soluble solubilising reagent of long-chain amine compound that contains ethoxylation and acid and the stabilizing agent that contains the alkaline reagent composition that height oozes.The solubilising reagent system of the disclosure uses DC, RF and light scattering measurement to distinguish leukocytic five parts through one-shot measurement.Yet,, only obtain four kinds of subgroups, i.e. monocyte, lymphocyte, basicyte and neutrophil(e) cell and acidophic cell sum if when on the blood analyser that DC and RF measurement mechanism only are housed, using this solubilising reagent.
In addition, to disclose solubilising reagent and used DC, RF and light scattering measurement instrument be the method for five kinds of subgroups with the white blood cell automatic distinguishing for unsettled U.S. Patent application 08/496469 (application on June 29 nineteen ninety-five).This solubilising reagent is made up of water-based solubilising reagent that contains polyoxyethylene surfactant and acid and the high alkaline stable reagent composition that oozes.Also to distinguish white blood cell be five parts a step when disclosed solubilising reagent system used DC, RF and light scattering measurement.Yet,, only obtain four kinds of subgroups, i.e. monocyte, lymphocyte, basicyte and neutrophil(e) cell and acidophic cell sum if when on the blood analyser that DC and RF measurement mechanism only are housed, using this solubilising reagent.
Summary of the invention
The present invention relates to new solubilising reagent composition.Described solubilising reagent composition is acid high infiltration solution, and it contains alkali metal salt, acidophic cell lytic agent (eosinolyticagent), non-ionic surfactant and the physiological salt of alkylsurfuric acid.The solubilising reagent composition solubilized red blood cell that this is new also optionally influences from other granulocyte subgroup by selecting the shrinkage acidophic cell and separates the acidophic cell subgroup.So at least, can distinguish a kind of white blood cell subgroup.
This solubilising reagent composition has the advantage of operation fully at room temperature.In order to separate the acidophic cell subgroup fully, prior art is carried out under the temperature (30 ℃ or higher) that raises.This rising temperature needs obviously more complicated instrument, because must control the temperature of reaction.The present invention has overcome the defective that needs the control temperature by optimum operation at room temperature.
In addition, the present invention relates to use the solubilising reagent composition to measure the method for white blood cell subgroup in the blood automatically.Method of the present invention comprises to be mixed blood sample, at room temperature the sample mixture cultivation was no more than for 25 seconds and uses automatic blood instrument analytic sample potpourri with the solubilising reagent composition.The effect that the synergy of surfactant produces fast selective to the acidophilia subgroup is told the acidophic cell subgroup fast by simple DC and RF measurement from other white blood cell subgroup.And this method has the advantage that does not need expensive laser aid to measure the acidophic cell subgroup.
In addition, the present invention relates to use new solubilising reagent composition and second kind of solubilising reagent to come only to measure to obtain leukocytic at least five part differentiations by DC and RF.
The accompanying drawing summary
Fig. 1 a and 1b show by the described method of example I by the acidophic cell of distinguishing in the normal whole blood sample.Fig. 1 a shows DC and the X-Y scheme of OP (turbidity, the function of RF and DC) and the figure that Fig. 1 b shows RF and OP respectively.The white blood cell subgroup thing that serves as a mark shows.
Fig. 2 shows the whole blood sample DC and rotation light scattering (RLS) figure that is undertaken by the described method of example I, and different with example I is to analyze by concentrating flow method (focused flow method) with the experimental blood analyser that DC, RF and light scattering device are housed.
The whole blood sample X-Y scheme that the solubilising reagent composition that Fig. 3 a and 3b demonstration use the present invention to enumerate by the method for example II is carried out.
Fig. 4 shows the example that uses the described second kind of solubilising reagent of EXAMPLE IV and the described stabilizing agent of example VI and the whole blood sample X-Y scheme that is undertaken by the method for example VII A.Every group of white blood cell subgroup of representing one or more marks.
The detailed description of invention
The present invention relates to water-based solubilising reagent composition and measure fast differentiation at least by DC and RF A kind of method of white blood cell subgroup. This solubilising reagent composition dissolves red blood cell and from other granulocyte Shrinkage acidophic cell subgroup optionally in the subgroup. More specifically, the present invention relates to this dissolving Reagent mixes rear cell volume different based on blood sample from the solubilising reagent composition with this solubilising reagent Distinguish and counting acidophic cell and without the method for laser measurement. In addition, providing a kind of only makes Distinguish the method for at least five kinds of white blood cell subgroups with DC and RF measurement.
In first embodiment of the present invention, water-based solubilising reagent composition contains the alkali of alkylsurfuric acid Slaine, acid, what promote that the acidophic cell subgroup selects shrinkage is selected from organophosphorus ester, organophosphor The alkali metal salt of acid esters, ethoxylated phosphate esters, ethoxylated mercaptans, ethoxylation lanonol, The acidophilia of ethoxylated alkyl phenols, ethoxylation alkanol, ethoxylation enol and composition thereof Cell lytic agent is protected the not lysed non-ionic surface active agent of neutrophil(e) cell and is enough to institute The osmolality of stating the solubilising reagent composition is adjusted between the 370-720mOsm The physiology salinity. These synergies with chemical substance of film activity can make solubilising reagent fast Instant solution red blood cell, simultaneously optionally shrinkage and from leukocytic other subgroup, distinguish acidophilia Cell.
The function of alkylsurfuric acid alkali metal salt is conclusive in the lytic agent of the present invention. It dissolves red blood Thereby distinguishing to analyze to white blood cell, ball provides chance. In addition, it also optionally affects acidophilia Cell subsets cell membrane and pyknotic cells volume fast-descending. The alkyl chain of alkylsurfuric acid alkali metal salt Long is 10-18 carbon atom, preferred 12-14 carbon atom. Alkylsurfuric acid alkali metal salt excellent Select example to comprise lauryl sodium sulfate and sodium tetradecyl sulfate. Most preferably use dodecyl sulphur Acid sodium. The concentration of alkylsurfuric acid alkali metal salt is approximately 0.02%-0.16%, preferably approximately 0.04%-0.12%.
The function of described acid helps to select lyse red blood cells and is selection shrinkage acidophic cell subgroup Sour environment is provided. Have been found that the selection shrinkage of acidophic cell subgroup is only in acid medium Take place.
The present invention can use various acid.Yet, preferably use PK to be lower than those acid of 3.This acid can be organic acid, mineral acid or its potpourri.The example of appropriate acid is acetate, citric acid, formic acid, propionic acid, hydrochloric acid, phosphoric acid, sulfuric acid and composition thereof in the lytic agent of the present invention.The amount of acid need be enough to quick lyse red blood cells, but can not cause the damage of other granulocyte subgroup simultaneously.The PH of lytic agent should remain within the scope of about 1.4-3.4, preferably approximately 1.8-2.6.
One group of surfactant plays the function of acidophic cell lytic agent.It is present in and helps the shrinkage of acidophic cell in the solubilising reagent composition and help the shrinkage acidophic cell to form to distinguish the group of analyzing of having any different.These acidophic cell lytic agents comprise phosphate, ethoxylated mercaptans, ethoxylation lanonol, ethoxylated alkyl phenols, ethoxylation alkanol, ethoxylation enol of alkali metal salt, the ethoxylation of organophosphate, organophosphate and composition thereof.
Because these surfactants are different chemical characteristics significantly, the mode of action of these surfactants under the condition of the present invention is unknown now.In a word, nonionic acidophic cell lytic agent has and is lower than about 15 hydrophilic-lipophilic balance value (HLB).Known these nonionic acidophic cell lytic agents are by strong globulolysis agent to gentleness under PH widely.Yet this can not explain that it distinguishes the ability of acidophic cell subgroup to the selection effect of acidophic cell cytoplasmic membrane and from other granulocyte subgroup.Effectiveness according to the concrete chemical substance of use determines the concentration range of acidophic cell lytic agent to be 0.05%-0.8%, can come by experiment to determine.
Another group surfactant plays the protectant function of white blood cell.The white blood cell protective agent has two functions.A function is to help optionally lyse red blood cells.Second function is that protection white blood cell, particularly neutrophil's subgroup are avoided damage during solubilizing reaction.Suitable example comprises that having HLB is 16 or the alkanol of bigger ethoxylation or enol of ethoxylation and composition thereof.
If neutrophil's subgroup is subjected to the influence of lytic agent during analyzing, their also shrinkages in size and disturb the classification of acidophic cell.Yet if use excessive white blood cell protective agent, the separation of acidophic cell subgroup only can be carried out under the prolongation reaction time greater than about 45 seconds, perhaps becomes and can not finish.So, need the white blood cell protective agent (can take by experiment to determine) of suitable concn.The concentration of the alkanol of ethoxylation or the enol of ethoxylation is approximately 0.5-3%, preferably is about 0.8-2%.
Use the physiology salinity to regulate the osmolality of solubilising reagent.The inventor has been found that the shrinkage of acidophic cell only at about 370-720mOsm, and preferably approximately the height of 400-570mOsm scope oozes under the environment and takes place.In preferred embodiments, use alkali metal chloride and sulfuric acid alkali metal salt for this purpose.Appropriate combination alkali metal chloride and sulfuric acid alkali metal salt, the shrinkage of acidophic cell can take place fast and the acidophic cell shrinkage after form distinguishing colony.
The example of suitable physiology salinity comprises sodium chloride or potassium chloride and sodium sulphate or glazier's salt.The concentration range of alkali metal chloride is about 0.1%-1.2%, preferably is about 0.3-1.0%.The concentration range of sulfuric acid alkali metal salt is about 0.4-2%, preferably is about 0.6-1.5%.
Randomly, can use polyoxyethylene and polyoxypropylene multipolymer to help the lyse red blood cells fragment and also reduce the noise that produces by fragment.In addition, have been found that multipolymer helps the acidophic cell subgroup to form distinct sets.The preferred scheme of the present invention is to use Pluronic multipolymer (being produced Parsippany, New Jersey by BASF AG), as Pluronicl OR5 and Pluronic L35.
Other dispensable adjuvant also can be included in the solubilising reagent composition, and its concentration is that [existing amount] is compatible with the key component of solubilising reagent composition.Wherein these adjuvants are the antistaling agent that has (to increase the period of storage of composition) of antioxygen characteristic and have antimicrobial property.Antiseptic with antioxygen characteristic includes but not limited to EDTA and butyl methyl phenol.Antiseptic with antimicrobial property includes but not limited to that dihydroxymethyl dimethyl hydantion and different sulphur shape kill phosphorus (isothiozolone) derivant.
In second embodiment of the present invention, provide a kind of method of dissolving red blood cell in the blood sample and analyzing last white blood cell subgroup.This method comprises: step 1) is exposed to the dissolved composition certain hour with blood sample; this time is enough to lyse red blood cells and tells at least a white blood cell subgroup in described blood sample; described lytic agent contains the alkali metal salt of alkylsurfuric acid; acid; promote the acidophic cell subgroup to select the organophosphate that is selected from of shrinkage; the alkali metal salt of organophosphate; ethoxylated phosphate esters; ethoxylation mercapto alcohol; the ethoxylation lanonol; ethoxylated alkyl phenols; the ethoxylation alkanol; the acidophic cell lytic agent of ethoxylation enol and composition thereof; the protection not lysed non-ionic surfactant of neutrophil's subgroup and be enough to osmolality with described solubilising reagent composition and be adjusted in physiology salinity between the 370-720mOsm; step 2) from acidophic cell; lymphocyte; distinguish out at least a white blood cell subgroup and step 3) among monocyte and the neutrophil(e) cell and report the result of described differentiation.
Blood sample is exposed to the dissolved composition certain hour, and this time is enough to lyse red blood cells and tells at least a white blood cell subgroup in described blood sample.Mix to make blood sample be exposed to lytic agent with blood sample by lytic agent, doubly with the about 45-70 of diluting blood sample.At room temperature cultivate the sample mixture of dilution.Preferably, temperature is greatly about 15-30 ℃.Incubation time is approximately 10-25 second, preferably is less than for 18 seconds.With automatic hematology analyzer analytic sample potpourri.This apparatus measures is also distinguished a kind of white blood cell subgroup at least, preferred acidophic cell subgroup or neutrophil's subgroup, and acidophic cell subgroup most preferably.
Use at least two kinds of metering systems that are selected from DC, RF and the light scattering measurement to distinguish.Preferably only use DC and RF measurement to distinguish.Any variations can take place because of the different of conductance between particle and the liquid medium during by narrow fluid passage in DC method test sample potpourri on electric current.In the DC method, the intensity of detection signal is basic and particle volume is proportional.In the RF method, sample mixture is by the narrow fluid passage between the tight adjacent electrode, because of the difference of electric medium constant between particle and the liquid medium so the variation that can record on electrical impedance between the electrode to be taken place.In the RF method, the signal that detects reflection information relates to the structure of particle and the character of their materials of composition.DC and RF detect the details of rule and technology and are assigning in CoulterElectronic, and the United States Patent (USP) 2656508 of Inc. and assigning in Coulter Electronic is instructed in the United States Patent (USP) 4298836 of Inc. to some extent.
The method of using solubilising reagent composition of the present invention to come blood sample is carried out the white blood cell compartment analysis is a kind of fast method that uses solubilising reagent.Solubilising reagent composition dissolves red blood cell also optionally influences the cytoplasmic membrane and the shrinkage acidophic cell cell subset volume of acidophic cell subgroup.Except that acidophic cell during solubilizing reaction, to be kept perfectly substantially.
Example I is described should have disclosed solubilising reagent composition to distinguish and write down the preferred embodiment of leukocytic number in the whole blood sample.Fig. 1 shows the method compartment analysis whole blood sample that uses DC and RF measurement to describe according to example I.Stream of cells is crossed a hole, and there is the impedance measurement device of a record cell to radiation frequency and direct current reaction at this place, hole.Handle electric signal and draw the X-Y scheme of Fig. 1 a and the described DC of 1b and turbidity or RF and turbidity respectively.As shown in the figure, blood sample is with after solubilising reagent composition of the present invention mixes, and the acidophic cell subgroup is separated from other white blood cell clusters fully.Although embodiment carries out with the non-instrument that flows of concentrating, use as shown in Figure 2 and concentrate flow system can obtain identical result.
Notice, reduced hugely that its RF signal is not discovered and is affected although mix the size of back acidophic cell with solubilising reagent.Using DC and RF detection technique difference neutrophil(e) cell and acidophic cell mainly is to finish by the significant difference of their sizes and turbidity.As described in Fig. 1 a and 1b, the turbidity of acidophic cell subgroup moves to right and shifts out from neutrophil(e) cell and other white blood cell subgroup fully.
Fig. 2 demonstration uses identical blood sample by DC with rotation light scattering diagram to prove conclusively of the present invention acidophic cell group by concentrated flow method by the method for example I on the experimental blood analyser that DC, RF and light scattering device are housed.In DC and light scattering X-Y scheme, from the neutrophil(e) cell, tell acidophic cell by the difference of its size and light scattering.The number of the acidophic cell of telling by the inventive method is consistent with the number of acidophic cell by conventional manual method record.
In the 3rd embodiment of the present invention, provide a kind of method of dissolving red blood cell in the blood sample and remaining white blood cell subgroup being divided at least five kinds of white blood cell subgroups.This method comprises: step 1) is handled the enough time of first blood aliquot with first kind of dissolving agent composition, so that globulolysis, and from described blood sample, tell at least a white blood cell subgroup, step 2) with second aliquot of second kind of described blood of lytic agent system handles, so that globulolysis, and from described blood sample, tell four kinds of white blood cell subgroups, step 3) is used first aliquot of DC and the described processing blood of RF Measurement and analysis, step 4) is used second aliquot of DC and the described processing blood of RF Measurement and analysis, at least five kinds of white blood cell subgroups of step 5) report.
The step 1 of handling first blood aliquot is to use the dissolving agent composition described in first embodiment of the present invention to finish.The step 3 of directly analyzing first blood aliquot is to finish by the method described in second embodiment of the present invention.
Step 2 is by second aliquot with second kind of lytic agent system handles blood sample, is enough to make globulolysis, and tells from described blood sample that four kinds of white blood cell subgroups finish.Second lytic agent system is divided into lymphocyte, monocyte, basicyte and the granulocyte except that basicyte with white blood cell.The incorporation time of blood sample and second kind of lytic agent was less than for 10 seconds.
Second kind of lytic agent will comprise solubilising reagent and stabiliser compositions usually.An example of second kind of lytic agent system is to comprise solubilising reagent, and it contains the long-chain amine compound of the ethoxylation shown in the following formula:
Wherein R is alkyl, alkenyl or the alkynyl with 12-22 carbon atom, m and n respectively be 1 or bigger and m+n between 20 and 40; Be adjusted in acid within the 2.0-3.6 scope with PH with solubilising reagent; Ooze the alkaline stabiliser composition with height.Preferred R is the alkyl with 14-20 carbon atom.In addition, preferably, the acid that is used to regulate PH contains organic acid.The acid of more preferably regulating PH contains formic acid and the effective potpourri that is selected from the acid of acetate, citric acid, oxalic acid, glycollic acid, propionic acid, hydrochloric acid, sulfuric acid and phosphoric acid and composition thereof.
Another example of second kind of lytic agent system is to contain solubilising reagent, and it contains the polyoxyethylene surfactant shown in the following formula:
R
1-R
2-(CH
2CH
2O)
n-H
R wherein
1For having the alkyl of 10-22 carbon atom, alkenyl or alkynyl, R
2For-O-or-COO-, and n is between 20-35; Be adjusted in acid within the 2.0-4.0 scope with PH with solubilising reagent; Ooze the alkaline stabiliser composition with height.Preferred R is the alkyl with 14-20 carbon atom.In addition, preferably, the acid that is used to regulate PH contains organic acid.The preferred acid of regulating PH contains formic acid and is selected from effective potpourri of the acid of acetate, citric acid, oxalic acid, glycollic acid, propionic acid, hydrochloric acid, sulfuric acid and phosphoric acid and composition thereof.
Two examples of second kind of lytic agent are provided in EXAMPLE IV and V.The example of the stabilizing agent that uses in second kind of lytic agent of EXAMPLE IV and V is provided in the example VI.
Step 4 is to finish by second aliquot using DC and RF to analyze lysed blood.Fig. 4 describes four kinds of white blood cell subgroups, and it is by using the stabiliser compositions described in second kind of lytic agent described in the EXAMPLE IV and the example VI to obtain by the described methods analyst blood sample of example VII A.As described in Figure 4, use DC and RF to measure and clearly isolate lymphocyte, monocyte, basicyte and the granulocytic four kinds of cell subsets except that basicyte.
At least five kinds of white blood cell subgroups of step 5 report.As implied above, directly obtain lymphocyte, monocyte, basicyte in the step 4.Obtain neutrophil's subgroup by deducting by the acidophic cell subgroup that obtains in the step 3 by the granulocyte except that basicyte that obtains in the step 4.
Dissolving agent composition, second kind of solubilising reagent system or its combination all can kit form sell, wherein solubilising reagent can be packaged in the container (as plastic containers).Preferably will how use the instructions of these components to be included in the container or on the container according to the present invention.
Further describe the present invention by reference the following example, these embodiment are illustrative and do not limit scope of the present invention.
Example I
Prepare dissolving agent composition with following component:
1, T-mulz (Harcros Chemical, Inc., the sylvite of 50% organophosphate) 5.0g
2, lauryl sodium sulfate 1.0g
3, Hetoxol STA30 (Heterene, the octadecanol of ethoxylation) 10.0g
4、Pluronic?10R5(BASF) 15.0g
5, formic acid 0.2ml
6, sodium sulphate 9.0g
7, potassium chloride 6.0g
8, the BHT 0.05g of predissolve in ethanol
9、proclin300(Rohm?&?Haas?Co.) 0.5ml
10, PH (regulating) 2.2 by 4M HCl
11, deionized water is adjusted to 1L
In the normal whole blood sample of 28ml EDTA anti-freezing, the dissolving agent composition that adds 1800ml, and under room temperature (about 212 ℃), potpourri was mixed for 9 seconds gently, prepare to carry out compartment analysis during about 13 seconds after adding dissolving agent composition by vibration.Blood mixture fluid remains under the condition of acidity (PH is about 2.2) and osmolality 484mOsm.On the experimental blood analyser that DC and RF device are housed, use non-concentrated flow technique to carry out compartment analysis.The X-Y scheme of gained has been described among Fig. 1 a and the 1b.The vertical pivot of Fig. 1 a and transverse axis are respectively DC and turbidity, and the vertical pivot of Fig. 1 b and transverse axis are respectively RF and turbidity.The acidophic cell group is distinguished and is counted, and reports the result by absolute value or with respect to the percentage that total white blood cell count calculates.
Example II
With following component preparation dissolving agent composition, and be used for white blood cell subgroup compartment analysis.In the normal whole blood sample of 28 μ lEDTA anti-freezings, the dissolving agent composition that adds 1600 μ l, and under room temperature (about 212 ℃), potpourri was mixed for 8 seconds gently, prepare to carry out compartment analysis during about 12 seconds after adding dissolving agent composition by vibration.Fig. 3 a and 3b show the figure of two DC and turbidity.Fig. 3 a is corresponding with normal blood sample, and Fig. 3 b is corresponding to the blood sample of acidophic cell rising (manual difference counting is 12%).
1, Burco TME (Burlington Chemical Co.Inc., the lauryl mercaptan of 1.5g ethoxylation, HLB=13.0)
2, lauryl sodium sulfate 1.0g
3, Hetoxol STA30 (Heterene, the octadecane 8.0g alcohol of ethoxylation)
4、Pluronic?L35(BASF) 10.0g
5, citric acid 17.0ml
6, sodium sulphate 9.0g
7, potassium chloride 6.0g
8, deionized water is adjusted to 1L
9、PH 2.2
EXAMPLE III
With following component preparation dissolving agent composition, and be used for white blood cell subgroup compartment analysis.In the normal whole blood sample of 28 μ lEDTA anti-freezings, the dissolving agent composition that adds about 1880 μ l, and under room temperature (about 212 ℃), potpourri was mixed for 11 seconds gently, prepare to carry out compartment analysis during about 15 seconds after adding dissolving agent composition by vibration.Analytic sample on experimental blood analyser then.
1, Tergitol NP-13 (Union Carbide, the nonyl phenol of ethoxylation) 1.0g
2, sodium tetradecyl sulfate 1.0g
3、Hetoxol?STA30(Heterene) 10.0g
4, formic acid 0.2g
5, sodium sulphate 9.0ml
6, potassium chloride 5.0g
7、Pluronic?10R5 15.0g
8, deionized water is adjusted to 1L
9、PH 2.2
EXAMPLE IV:
First example of second kind of lytic agent
The dissolving agent composition that comprises the long-chain amine compound of ethoxylation with following component preparation:
Long-chain amine compound with the cationic ethoxyization of following formula:
Wherein the value of m+n is 30, is dissolved in deionized water, and concentration is 20g/L.With formic acid PH is transferred to 3.2.Add following antistaling agent in addition: 0.2g/L EDTA, 0.5g/L Proclin300 (Rohm ﹠amp; Haas Co.) and 0.05g/L 2,6-two-tert-butyl group-4-methylphenol (being dissolved in ethanol in advance).
EXAMPLE V
Second example of second kind of lytic agent
The dissolving agent composition that comprises polyoxyethylene surfactant with following component preparation:
Polyoxyethylene surfactant with following formula:
C
18H
37O(CH
2CH
2O)
nH
Wherein the value of n is 30, is dissolved in deionized water, and concentration is 20g/L.The lauryl sodium sulfate of adding 0.8g/L (SDS, Aldrich).With formic acid PH is transferred to 2.8.
Use second kind of lytic agent and stabilizing agent to prepare white blood cell is separated into lymphocyte, monocyte, basicyte and remove the granulocytic lytic agent system of basicyte subgroup.Example VI has been described an example of stabilizing agent.
Example VI
The stabilizing agent of second kind of lytic agent
The stabilizing agent of preparing as indicated above, it comprises 14g/L NaCl, 32g/L Na
2SO
4And 6.6g/LNa
2CO
3Buffering agent, PH is transferred to 11.0.The osmolality of this reagent is 1080mOsm.
Example VII A
Distinguish the white blood cell clusters of blood sample
In the normal whole blood sample of 28ml EDTA anti-freezing, add the dissolving agent composition of 488ml EXAMPLE IV, and under room temperature (about 212 ℃), potpourri was mixed for 4 seconds gently by vibration.Stop solubilizing reaction by the stabilizing agent 209 μ l that add example VI.Prepare to carry out compartment analysis gently when mixed blood sample potpourri and about 15 seconds after adding stabilizing agent.Final blood mixture fluid remains on the height that neutral PH (being about 7) and osmolality be about 445mOsm and oozes under the condition.Fig. 4 shows the compartment analysis that carries out with DC and RF.
Although show some representational embodiment and details for the purpose of illustrating the invention, under the scope that does not break away from the definition of the present invention such as appended claim, can carry out various changes and modifications.
Claims (29)
1, a kind of water-based dissolving agent composition, contain:
A) alkali metal salt of alkylsurfuric acid, helping lysed erythrocyte and the concentration of the 0.02%-0.16%w/w of acidophic cell subgroup shrinkage is existed,
B) acid is that the effective dose of the sour environment of 1.4-3.4 exists to help lysed erythrocyte and being provided for to make the pH value of acidophic cell subgroup shrinkage,
C) promote the acidophic cell subgroup to select having of the alkali metal salt that is selected from organophosphate, organophosphate of shrinkage, ethoxylated phosphate esters, ethoxylated mercaptans, ethoxylation lanonol, ethoxylated alkyl phenols, ethoxylation alkanol, ethoxylation enol and composition thereof to be lower than the acidophic cell lytic agent of 15 hydrophilic-lipophilic balance value
D) the leukocytic neutrophil's subgroup of protection not lysed have greater than the non-ionic surfactant of 16 hydrophilic-lipophilic balance value and
E) be enough to osmolality with described dissolving agent composition and be adjusted in physiology salinity between the 370-720mOsm.
2, the dissolving agent composition of claim 1, wherein the alkyl of the alkali metal salt of alkylsurfuric acid has 10-18 carbon atom.
3, the dissolving agent composition of claim 1, the acid that wherein is used to regulate PH is selected from organic acid, mineral acid and composition thereof.
4, the dissolving agent composition of claim 3, wherein acid is selected from acetate, citric acid, formic acid, propionic acid, hydrochloric acid, phosphoric acid, sulfuric acid and composition thereof.
5, the dissolving agent composition of claim 1, the concentration range of wherein said acidophic cell lytic agent are 0.05-0.8%w/v.
6, the dissolving agent composition of claim 1; the not lysed ionic surfactant pack of wherein said protection neutrophil's subgroup is drawn together to be had the hydrophilic-lipophilic balance value and is at least 16 ethoxylation alkanol, or has the hydrophilic-lipophilic balance value and be at least 16 ethoxylation enol or its potpourri.
7, the dissolving agent composition of claim 6, wherein the concentration range of non-ionic surfactant is 0.5-3.0%w/v.
8, the dissolving agent composition of claim 1 wherein also comprises the polyoxyethylene and the polyoxypropylene multipolymer that are enough to promote red blood cell fragment meltage.
9, the dissolving agent composition of claim 1, wherein said physiology salinity comprises alkali-metal chloride, or the alkali metal salt of sulfuric acid or its potpourri.
10, the dissolving agent composition of claim 9, wherein alkali-metal muriatic concentration is 0.2%-1.3%w/v.
11, the dissolving agent composition of claim 9, wherein the concentration of the alkali metal salt of sulfuric acid is 0.4%-2.0%w/v.
12, a kind of dissolving blood sample red blood cell and the leukocytic method of analysis residue comprise:
A) blood sample is exposed to the dissolving agent composition certain hour, this time is enough to lyse red blood cells, and tells at least a white blood cell subgroup in described blood sample, and described dissolving agent composition contains:
1) alkali metal salt of alkylsurfuric acid, its concentration are 0.02%-0.16%w/v;
2) acid is enough to the pH value of described dissolving agent composition is remained in the 1.4-3.4 scope;
3) promote the acidophic cell subgroup to select the acidophic cell lytic agent of the alkali metal salt that is selected from organophosphate, organophosphate of shrinkage, ethoxylated phosphate esters, ethoxylated mercaptans, ethoxylation lanonol, ethoxylated alkyl phenols, ethoxylation alkanol, ethoxylation enol and composition thereof
4) protection the not lysed non-ionic surfactant of neutrophil(e) cell and
5) be enough to osmolality with described dissolving agent composition be adjusted between the 370-720mOsm the physiology salinity and
B) from acidophic cell, lymphocyte, monocyte and neutrophil(e) cell, distinguish out at least a white blood cell subgroup,
C) result of the described differentiation of report.
13, the method for claim 12, the time that wherein is enough to lyse red blood cells and tells at least a white blood cell subgroup in described blood sample is 10-25 second.
14, the method for claim 12, wherein lyse red blood cells and in described blood sample, tell at least a white blood cell subgroup at room temperature.
15, the method for claim 14, wherein room temperature is 15-30 ℃.
16, the method for claim 12, wherein said white blood cell subgroup is the acidophic cell subgroup.
17, the method for claim 12, wherein said white blood cell subgroup is neutrophil's subgroup.
18, the method for claim 12, described differentiation are by using at least two types of metering systems that are selected from conductivity, radiation frequency and light scattering to carry out.
19, the method for claim 18, wherein said differentiation is undertaken by using conductivity and radiation frequency measurement mode.
20, a kind of method of dissolving the blood sample red blood cell and analyzing residue white blood cell subgroup, this method comprises:
A) handle the enough time of first blood aliquot with each dissolving agent composition of claim 1-11, so that globulolysis, and from described blood sample, tell at least a white blood cell subgroup,
B) with second aliquot of second kind of described blood of lytic agent system handles, be enough to make globulolysis, and from described blood sample, tell four kinds of white blood cell subgroups, comprise lymphocyte, monocyte, basophil and basophil granulocyte in addition
C) use conductivity and radiation frequency measurement mode to analyze first aliquot of described processing blood, obtaining the detection of at least a leucocyte subgroup,
D) use conductivity and radiation frequency measurement mode to analyze second aliquot of described processing blood, with the detection that obtains to comprise other the granulocytic four kinds of leucocyte subgroups beyond lymphocyte, monocyte, basophil and the basophil and
E) utilize the result of step c and d to report at least five kinds of white blood cell subgroups.
21, the method for claim 20 wherein is enough to make globulolysis, and the time of telling at least a white blood cell subgroup from described blood sample is 10-25 second.
22, the method for claim 21 wherein is enough to make globulolysis, and the time of telling four kinds of white blood cell subgroups from described blood sample was 10 seconds.
23, the method for claim 20 wherein at room temperature makes globulolysis, and tells the white blood cell subgroup from first and second aliquots of described blood sample.
24, the method for claim 23, wherein room temperature is 15-30 ℃.
25, the method for claim 20, first aliquot that described processing blood is analyzed in wherein said use conductivity and radiation frequency measurement is to distinguish the acidophic cell subgroup.
26, the method for claim 20, second aliquot that described processing blood is analyzed in wherein said use conductivity and radiation frequency measurement are white blood cell to be divided into comprise lymphocyte, monocyte, basicyte and except that basicyte granulocytic four types.
27, the method for claim 20, wherein second kind of lytic agent system contains:
A) comprise the long-chain amine compound of the ethoxylation shown in the following formula and the PH of solubilising reagent is adjusted in the solubilising reagent of the acid within the 2.0-3.6 scope:
Wherein R is alkyl, alkenyl or the alkynyl with 12-22 carbon atom, m and n respectively be 1 or bigger and m+n between 20 and 40; With
B) height oozes the alkaline stabiliser composition.
28, the method for claim 20, wherein second kind of lytic agent system comprises:
A) solubilising reagent, it contains the polyoxyethylene surfactant shown in the following formula:
R
1-R
2-(CH
2CH
2O)
n-H is R wherein
1For having the alkyl of 10-22 carbon atom, alkenyl or alkynyl, R
2For-O-or-COO-, and n is between 20-35; Be adjusted in acid within the 2.0-4.0 scope with PH with solubilising reagent; With
B) height oozes the alkaline stabiliser composition.
29, the method for claim 28, wherein second kind of lytic agent system also contains the lauryl sodium sulfate of adding.
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| CNB971127913A CN1188703C (en) | 1997-06-17 | 1997-06-17 | Reagent and method for differential determination of leukocytes in blood |
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| CN1188703C true CN1188703C (en) | 2005-02-09 |
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