CN101424614B - Basophilia granulocyte analytical reagent and measuring method - Google Patents
Basophilia granulocyte analytical reagent and measuring method Download PDFInfo
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- CN101424614B CN101424614B CN2007101243034A CN200710124303A CN101424614B CN 101424614 B CN101424614 B CN 101424614B CN 2007101243034 A CN2007101243034 A CN 2007101243034A CN 200710124303 A CN200710124303 A CN 200710124303A CN 101424614 B CN101424614 B CN 101424614B
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Abstract
The invention discloses an analytical reagent of basophils, which comprises at least one alkyl phenol polyoxyethylene ether, at least one alkyl alcohol polyoxyethylene ether and a buffering agent for adjusting a pH (pondus Hydrogenii) value. The invention also discloses a method of adopting the reagent to determine the basophils. The reagent of the invention can crack red blood cells and platelets, damage cell membranes of other white blood cells except the basophils, and keep the structure integrity of the basophils so as to sort and count the basophils through optical signals determining the sizes and the internal structures of the cells.
Description
Technical field
The present invention relates to a kind of cell analysis reagent and a kind of cell analysis method, particularly relate to a kind of reagent and a kind of cell analysis method that basophilic granulocyte is measured that is used for the basophilic granulocyte analysis.
Background technology
In the normal human, the basophilic granulocyte negligible amounts only accounts for the 0-1% of total white blood cells.But in diseases such as heavy metal poisoning, Hodgkin's disease, chronic myelocytic leukemia, the quantity of basophilic granulocyte can obviously raise, so the basophilic granulocyte counting is significant for the diagnosis of these diseases.
In the past, often use some chemical dyes to come basophilic granulocyte is carried out dyeing counting clinically, comprising Rui Shi-Jim Sa compound staining, Toluidine blue staining method and dimethyl diaminophenazine chloride decoration method etc.Though these methods are simple, directly perceived, effectively because the number percent counting of basophilic granulocyte only can be provided, exist simultaneously the running time long, be subject to shortcoming such as the subjective influence of operator, so and be not suitable for the detection of clinical extensive sample.
Harmet etc. (Blood, 1975,46:279-286) introduced a kind of method of using the fully automatic blood cell analysis apparatus that basophilic granulocyte is counted.This method uses dyestuff A Li Xinlan (alcian blue) that leucocyte is dyeed under the condition of low pH value, by visible light and the infrared light scattering signal of measuring cell basophilic granulocyte and other leucocytes is distinguished then.Yet A Li Xinlan has shortcomings such as being difficult for dissolving and easily separates out, and may become infringement to some parts of instrument.
US4,882,284 disclose a kind of method of leucocyte being carried out differential count.Use fluorescent dye that leucocyte is dyeed in the method, leucocyte can have been divided lymphoblast, monocyte, neutrophil leucocyte, eosinophil and five subgroups of basophilic granulocyte by measuring fluorescence, side scattered light and forward angle light scatter light signal then.But because the fluorescence of basophilic granulocyte and forward angle light scatter light signal and neutrophil leucocyte, monocytic fluorescence and forward-scattering signal overlap; when therefore using this method that basophilic granulocyte quantity sample normal or on the low side is measured, counting and classification results are inaccurate.
US5196346 discloses a kind of reagent and method that is used for the basophilic granulocyte counting.Contain polyethenoxy ether class (polyoxyethylene ether) surfactant, phthalic acid (pathalicacid)-hydrochloric acid mixture, lauryl sodium sulfate (SDS) and antioxidant BHT components such as (butylatedhydroxytoluene type anti-oxidizing agent) in the reagent.This reagent can dissolve red blood cell fully, and destroys other leucocyte beyond the basophilic granulocyte, keeps the structural integrity of basophilic granulocyte simultaneously, therefore basophilic granulocyte and other leukocyte differentiations can be come by the impedance signal of measuring cell.But this method only uses single parameter (impedance signal) that basophilic granulocyte is carried out differential count, compares with the sorting technique of multiparameter, and its accuracy is lower.
US5518928 discloses a kind of method that is used for the basophilic granulocyte counting.Used the reagent of forming by polyxyethylated alcohol (polyoxyethylene glycol ether) non-ionic surfactant Brij35, phthalic acid and antioxidant in the method, can destroy other leucocyte except that basophilic granulocyte, only keep the integrality of basophilic granulocyte structure.By measuring the low angle and the high angle scatter light signal of cell, basophilic granulocyte can be carried out differential count then.But this method need just can be measured blood and reagent after mixed 50 seconds, has therefore limited its application on the automated blood analysis device.
US5538893 discloses a kind of basophilic granulocyte analyrical reagent and method of being used for.Principal ingredient in the reagent comprises polyxyethylated alcohol (polyoxyethylene glycolether), the cationic surfactant of nonionic surfactants and is used for the buffering agent of regulator solution pH value.This reagent can keep the structural integrity of basophilic granulocyte only with the leucocyte bare nucleusization except that basophilic granulocyte.Then by the impedance of mensuration cell or the signal of low angle scattered light acquisition cell size aspect, pass through to measure the side direction of cell or the signal that high angle scatter light obtains the cell interior configuration aspects again, at last two kinds of signals are made up and can carry out differential count to basophilic granulocyte.But cationic surfactant that uses in the reagent and polyoxyethylene non-ionic surfactant, the two effect is separate, does not have synergy.
US5677183 discloses the method for a kind of leukocyte differential count and counting.Used two kinds of reagent in the method, first reagent is mainly used in leukocytic four class cells and hives off, and second reagent is mainly used in the counting of basophilic granulocyte.The principal ingredient of second reagent comprises polyxyethylated alcohol (polyoxyethylene glycol ether), the cationic surfactant of nonionic surfactants and is used for the buffering agent of regulator solution pH value.It can be with other leucocyte bare nucleusization except that basophilic granulocyte, but the structure of basophilic granulocyte still is kept perfectly.By two kinds of signals measuring reflection cell size and reflection cell interior structure basophilic granulocyte is carried out differential count then.But cationic surfactant that uses in the reagent and polyoxyethylene non-ionic surfactant, the two effect is separate, does not have synergy.
US6214625B1 discloses a kind of reagent and method that basophilic granulocyte and eosinophilic granulocyte are carried out differential count.Reagent is made up of a kind of hemolytic agent and a kind of stabilizing agent, and wherein the principal ingredient of hemolytic agent comprises long-chain amine (ethoxylated long chain amine), the quaternary ammonium salts cationic surfactant of ethoxylation and organic acid or the mineral acid that is used to regulate the pH value.Behind hemolytic agent and the blood cell mixing certain hour, only there is the structure of basophilic granulocyte to be kept perfectly, adds stabilizing agent subsequently and stop the further destruction of hemolytic agent basophilic granulocyte.Finally, by impedance, rotation scattered light (the rotated light scatter) signal of measuring cell, perhaps radio frequency (radio frequency), log light scattering (log light scatter) can be carried out differential count to basophilic granulocyte.But the parameter measured of this method is more, needs a plurality of special determinators, the apparatus structure complexity, costs an arm and a leg.
Summary of the invention
The objective of the invention is at the deficiencies in the prior art, provide a kind of energy to be used for basophilic granulocyte is carried out the cell analysis reagent of differential count convenient and simplely.
Another object of the present invention is to provide the above-mentioned cell analysis reagent of a kind of employing that basophilic granulocyte is carried out method for measuring.
For achieving the above object, the present invention has adopted following technical scheme:
The invention discloses a kind of reagent that basophilic granulocyte is analyzed that is used for, described reagent comprises
I, at least a APES (Polyoxyethylene phenyl ether),
Ii, at least a alkylol polyoxyethylene ether (polyoxyethylene glycol ether),
Iii, be used to regulate the buffering agent of pH.
Described APES preferably has following structure:
R
1-C
6H
4-(C
2H
4O)
m-OH
Wherein, R
1The expression carbon number is 8～9 alkyl, and m is 8～10 positive integer;
And/or
Described alkylol polyoxyethylene ether has following structure:
R
2-(C
2H
4O)
n-OH
Wherein, R
2The expression carbon number is 8～18 alkyl, and n is 2～23 positive integer.
In the specific embodiment of the present invention, described APES preferred structure formula is C
8H
17-C
6H
4-(C
2H
4O)
x-OH, x=9 or 10 OPEO, as Triton X-100, perhaps the preferred structure formula is C
9H
19-C
6H
4-(C
2H
4O)
8The NPE of-OH is as Nonidet P 40 (NP 40).Described alkylol polyoxyethylene ether is preferably dodecyl alcohol polyoxyethylene ether, and as Brij35, its structural formula is C
12H
25-(C
2H
4O)
23-OH perhaps is preferably cetyl alcohol polyoxyethylene ether, and as Brij56, its structural formula is C
16H
33-(C
2H
4O)
10-OH.
In the described reagent, the concentration of APES is 100～8000mg/L, is preferably 400～4000mg/L, and the concentration of alkylol polyoxyethylene ether is 100～10000mg/L, is preferably 500～4000mg/L.
In the reagent of the present invention, the pH that described buffering agent is regulated reagent is 1.8～3.5, is preferably 2.0～3.0.
The invention also discloses a kind of assay method of basophilic granulocyte, described method comprises, with above-mentioned reagent and blood sample in 10～100: 1 ratio is mixed, and pair cell is analyzed after hatching 5～60 seconds under 15～42 ℃ of temperature.
Described pair cell analysis is meant, basophilic granulocyte is classified and counts by the light signal (as forward direction and lateral scattering light signal) of measuring reflection cell size and inner structure.
Owing to adopted above technical scheme, the beneficial effect that the present invention is possessed is:
Reagent of the present invention can splitting erythrocyte and blood platelet, and destruction other leukocytic after birth except that basophilic granulocyte, its endochylema outflow, eucaryotic cell structure are disappeared, but the structural integrity that keeps basophilic granulocyte, thereby light signal that can be by measuring reflection cell size and inner structure can be classified and counts basophilic granulocyte.Reagent composition dissolubility of the present invention is good, is difficult for separating out, and can the parts of instrument not caused damage.Reagent composition of the present invention is simple, and utilizes reagent of the present invention to carry out basophilic granulocyte when analyzing, and only need be used the simple analysis device and just can carry out differential count to basophilic granulocyte.In addition, the APES that uses in the reagent is not only a kind of hemolytic agent efficiently, but also be a kind of that be widely used, powerful solubilizer, cell lysis and eliminate the fragment that lysis produces fast, its use can reduce the consumption of other solubilizer (as the alkylol polyoxyethylene ether), reaches the purpose of saving reagent cost.
Description of drawings
Fig. 1 is that a kind of Basophilia granulocyte analytical reagent of the present invention carries out the scatter diagram that basophilic granulocyte mensuration is generated;
Fig. 2 utilizes Basophilia granulocyte analytical reagent of the present invention and the correlation analysis that carries out blood measuring with sysmex XE-2100 blood cell analyzer figure as a result;
Fig. 3 is the APES of using in the reference examples in the alternative reagent of the present invention of cationic surfactant (hexadecyltrimethylammonium chloride), carries out basophilic granulocyte and measures the scatter diagram that is generated;
Fig. 4 is that another kind of Basophilia granulocyte analytical reagent of the present invention carries out the scatter diagram that basophilic granulocyte mensuration is generated;
Fig. 5 is that another Basophilia granulocyte analytical reagent of the present invention carries out the scatter diagram that basophilic granulocyte mensuration is generated;
Fig. 6 is that another Basophilia granulocyte analytical reagent of the present invention carries out the scatter diagram that basophilic granulocyte mensuration is generated.
Embodiment
Reagent of the present invention can be used for the classification and the counting of basophilic granulocyte.This reagent can splitting erythrocyte, and other class leucocyte bare nucleusization that will be except that basophilic granulocyte, the structural integrity that only keeps basophilic granulocyte, the light scattering signal by measuring reflection cell size and inner structure can be classified and counts basophilic granulocyte again.
The component of cell analysis reagent of the present invention mainly comprises:
1) at least a APES (Polyoxyethylene phenyl ether), it has following structure
R
1-C
6H
4-(C
2H
4O)
m-OH
Wherein, R
1The expression carbon number is 8～9 alkyl, and m is 8～10 positive integer.Object lesson such as OPEO Triton X-100, its structural formula is C
8H
17-C
6H
4-(C
2H
4O) x-OH, x=9～10, and NPE Nonidet P 40 (NP 40), its structural formula is C
9H
19-C
6H
4-(C
2H
4O)
8-OH.
In the reagent of the present invention, the working concentration of APES is generally 100～8000mg/L, is preferably 400～4000mg/L.Concentration is low excessively, may cause that then the leucocyte cracking beyond red blood cell, blood platelet even the basophilic granulocyte is incomplete; Excessive concentration then can be destroyed all leukocytic eucaryotic cell structures that comprise basophilic granulocyte.Therefore excessive concentration is hanged down the mistake that all can cause basophilic granulocyte classification and counting with crossing.
2) at least a alkylol polyoxyethylene ether (polyoxyethylene glycol ether), it has following structure
R
2-(C
2H
4O)
n-OH；
Wherein, R
2The expression carbon number is 8～18 alkyl, and n is 2～23 positive integer.Object lesson such as dodecyl alcohol polyoxyethylene ether Brij35, its structural formula C
12H
25-(C
2H
4O)
23-OH, and cetyl alcohol polyoxyethylene ether Brij 56, its structural formula C
16H
33-(C
2H
4O)
10-OH.
In reagent of the present invention, the working concentration of APES is generally 100～10000mg/L, is preferably 500～4000mg/L.Concentration is low excessively, may cause that then all leucocytes that comprise basophilic granulocyte are all cleaved, causes basophilic granulocyte correctly not distinguish with other leucocyte; Excessive concentration then may make the leucocyte cracking in addition of red blood cell, blood platelet and basophilic granulocyte incomplete, also can cause the mistake of basophilic granulocyte classification and counting.
3) be used to regulate the buffering agent of pH value
In the reagent of the present invention, the main effect of buffering agent is for the pH with reagent is stabilized between 1.8～3.5, preferably in 2.0～3.0 scopes.Reasonably the pH value helps basophilic granulocyte other leukocytic destruction in addition, optionally protects the integrality of basophilic granulocyte structure simultaneously.During pH low excessively (＜1.8), can cause all leucocytes that comprise basophilic granulocyte all destroyed; PH too high (＞3.5) then can cause the leucocyte cracking beyond the basophilic granulocyte incomplete.And both of these case all can influence the correct classification and the counting of basophilic granulocyte.
There is no particular limitation to can be used for this type of buffering agent of reagent of the present invention, can be organic acid (salt), mineral acid (salt) or multiple mixture of ingredients.Concrete example such as acetate, citric acid, phthalic acid, oxalic acid, glycocoll-hydrochloride buffer and Potassium Hydrogen Phthalate-hydrochloride buffer etc.The suitable concn of damping fluid is usually in 2～500mM scope.
When adopting method of the present invention that basophilic granulocyte is measured, use reagent provided by the invention to mix with a certain proportion of blood sample, both ratios are generally 10～100: 1, and incubation time was controlled at 5～60 seconds usually, and incubation temperature is controlled at 15～42 ℃ usually.Can suitably shorten incubation time when using higher incubation temperature, but and proper extension incubation time when using lower temperature.
After reagent and blood are hatched certain hour, pair cell is analyzed on the cell analysis apparatus that can detect forward direction and lateral scattering light signal, by the forward direction and the side scattered light two dimension scatter diagram of cell, basophilic granulocyte and other leucocyte can be distinguished then.Wherein, detecting the forward direction and the method for lateral scattering light signal and the method that pair cell is distinguished on two-dimentional scatter diagram is content well-known to those skilled in the art.
Below by specific embodiment the present invention is done further detailed description.
Embodiment 1
Press following one-tenth assignment system Basophilia granulocyte analytical reagent:
NP40 800mg/L
Brij35 2g/L
Citric acid 10mM
H
2O adds to 1L
Mentioned reagent pH value is about 2.8.Get above-mentioned Basophilia granulocyte analytical reagent 1mL and 20 μ L blood sample mixings, hatched 10 seconds for 40 ℃.Stepping forward direction and the lateral scattering light signal of measuring cell on the auspicious BC-5500 blood cell analyzer, the scatter diagram of generation such as Fig. 1 then.As shown in the figure, use forward direction and lateral scattering light signal basophilic granulocyte and other leucocyte can be distinguished.
Other gets 155 parts of fresh clinical blood samples, use above-mentioned Basophilia granulocyte analytical reagent on auspicious BC-5500 blood cell analyzer advanced in years, basophilic granulocyte (%) to be measured as stated above, and the result is carried out correlation analysis with same blood preparation by the measured value of this area conventional method on sysmex XE-2100 blood cell analyzer.As shown in Figure 2, the correlativity of two groups of measured values is good, and its related coefficient reaches 0.84808.
Reference examples
Reference examples adopts the composition of the Basophilia granulocyte analytical reagent in the similar embodiment 1, and just NP40 is substituted by the hexadecyltrimethylammonium chloride of same concentrations, and it is composed as follows:
Hexadecyltrimethylammonium chloride 800mg/L
Brij35 2g/L
Citric acid 10mM
H
2O adds to 1L
Mentioned reagent pH value is about 2.8.Get mentioned reagent 1mL and 20 μ L blood sample mixings, hatched 10 seconds for 40 ℃.Stepping forward direction and the lateral scattering light signal of measuring cell on the auspicious BC-5500 blood cell analyzer, the scatter diagram of generation such as Fig. 3 then.As shown in the figure, when using the cationic surfactant (hexadecyltrimethylammonium chloride) with NP 40 same concentrations, there be basophilic granulocyte leucocyte and cell fragment dissolved destruction in addition incomplete, cause basophilic granulocyte and other leucocyte obviously not to distinguish.
Embodiment 2
Press following one-tenth assignment system Basophilia granulocyte analytical reagent:
NP40 800mg/L
Brij56 1g/L
Glycocoll 20mM
HCl transfers to pH 2.5 amount
H
2O adds to 1L
Use above-mentioned Basophilia granulocyte analytical reagent 1mL, add 20 μ L blood samples.40 ℃ hatch 40 seconds after, stepping forward direction and the lateral scattering light signal of measuring cell on the auspicious BC-5500 blood cell analyzer, generate scatter diagram as Fig. 4.As shown in the figure, can classify and count basophilic granulocyte by forward direction and lateral scattering light signal.
Embodiment 3
Press following one-tenth assignment system Basophilia granulocyte analytical reagent:
Triton?X-100?400mg/L
Brij35 500mg/L
Acetate 250mM
H
2O adds to 1L
Mentioned reagent pH value is about 2.8.Use above-mentioned Basophilia granulocyte analytical reagent 1mL,, hatched 10 seconds for 40 ℃ with 20 μ L blood sample mixings.Stepping forward direction and the lateral scattering light signal of measuring cell on the auspicious BC-5500 blood cell analyzer, its scatter diagram result such as Fig. 5 then.As shown in the figure, use forward direction and lateral scattering light signal basophilic granulocyte and other leukocyte differentiation can be come.
Press following one-tenth assignment system Basophilia granulocyte analytical reagent:
Triton?X-100 400mg/L
Brij56 1g/L
Potassium Hydrogen Phthalate 20mM
HCl transfers to pH 2.5 amount
H
2O adds to 1L
Use above-mentioned Basophilia granulocyte analytical reagent 1mL, add 20 μ L blood samples.40 ℃ hatch 40 seconds after, stepping forward direction and the lateral scattering light signal of measuring cell on the auspicious BC-5500 blood cell analyzer, generate scatter diagram as Fig. 6.As shown in the figure, can classify and count basophilic granulocyte by forward direction and lateral scattering light signal.
Above content be in conjunction with concrete preferred implementation to further describing that the present invention did, can not assert that concrete enforcement of the present invention is confined to these explanations.For the general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, can also make some simple deduction or replace, all should be considered as belonging to protection scope of the present invention.
Claims (10)
1. Basophilia granulocyte analytical reagent, it is characterized in that: described reagent is made up of water and following compositions
I, at least a APES,
Ii, at least a alkylol polyoxyethylene ether,
Iii, be used to regulate the buffering agent of pH.
2. a kind of Basophilia granulocyte analytical reagent according to claim 1 is characterized in that: described APES has following structure:
R
1-C
6H
4-(O?C
2H
4)
m-OH
Wherein, R
1The expression carbon number is 8～9 alkyl, and m is 8～10 positive integer;
And/or
Described alkylol polyoxyethylene ether has following structure:
R
2-(O?C
2H
4)
n-OH
Wherein, R
2The expression carbon number is 8～18 alkyl, and n is 2～23 positive integer.
3. a kind of Basophilia granulocyte analytical reagent according to claim 2 is characterized in that: described APES is selected from:
Structural formula is C
8H
17-C
6H
4-(O C
2H
4)
x-OH, wherein x=9 or 10 OPEO, or,
Structural formula is C
9H
19-C
6H
4-(O C
2H
4)
8The NPE of-OH.
4. a kind of Basophilia granulocyte analytical reagent according to claim 2 is characterized in that: described alkylol polyoxyethylene ether is dodecyl alcohol polyoxyethylene ether or cetyl alcohol polyoxyethylene ether.
5. according to any described a kind of Basophilia granulocyte analytical reagent of claim 1～4, it is characterized in that: in the described reagent, the concentration of APES is 100～8000mg/L, and the concentration of alkylol polyoxyethylene ether is 100～10000mg/L.
6. a kind of Basophilia granulocyte analytical reagent according to claim 5 is characterized in that: in the described reagent, the concentration of APES is 400～4000mg/L, and the concentration of alkylol polyoxyethylene ether is 500～4000mg/L.
7. according to any described a kind of Basophilia granulocyte analytical reagent of claim 1～4, it is characterized in that: the pH that described buffering agent is regulated reagent is 1.8～3.5.
8. a kind of Basophilia granulocyte analytical reagent according to claim 7 is characterized in that: the pH that described buffering agent is regulated reagent is 2.0～3.0.
9. the assay method of a basophilic granulocyte, described method comprise, with any described reagent in the claim 1～8 and blood sample in 10～100: 1 ratio is mixed, and pair cell is analyzed after hatching 5～60 seconds under 15～42 ℃ of temperature.
10. the assay method of a kind of basophilic granulocyte according to claim 9 is characterized in that: described pair cell analysis is meant, basophilic granulocyte is classified and counts by the light signal of measuring reflection cell size and inner structure.
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Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5196346A (en) * | 1990-02-13 | 1993-03-23 | Abx | Reagent and method of using same for automatically counting basophilic leukocytes in the blood in resistivity variation measuring apparatus |
| CN1126836A (en) * | 1994-08-03 | 1996-07-17 | 东亚医用电子株式会社 | Method for sorting leucocyte |
| US5821128A (en) * | 1995-06-13 | 1998-10-13 | Hycel Diagnostics | Method for differentiating and isolating sub-populations of leucocytes in blood samples by treatment with polyxoxyethylene 9-lauryl ether, and reagent for its implementation |
| CN1084474C (en) * | 1994-04-21 | 2002-05-08 | 希森美康株式会社 | Agent for analysing leucocyte and classification of leucocyte |
| CN101046439A (en) * | 2006-03-29 | 2007-10-03 | 希森美康株式会社 | Method and apparatus for measuring hematological sample |
-
2007
- 2007-10-29 CN CN2007101243034A patent/CN101424614B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5196346A (en) * | 1990-02-13 | 1993-03-23 | Abx | Reagent and method of using same for automatically counting basophilic leukocytes in the blood in resistivity variation measuring apparatus |
| CN1084474C (en) * | 1994-04-21 | 2002-05-08 | 希森美康株式会社 | Agent for analysing leucocyte and classification of leucocyte |
| CN1126836A (en) * | 1994-08-03 | 1996-07-17 | 东亚医用电子株式会社 | Method for sorting leucocyte |
| US5821128A (en) * | 1995-06-13 | 1998-10-13 | Hycel Diagnostics | Method for differentiating and isolating sub-populations of leucocytes in blood samples by treatment with polyxoxyethylene 9-lauryl ether, and reagent for its implementation |
| CN101046439A (en) * | 2006-03-29 | 2007-10-03 | 希森美康株式会社 | Method and apparatus for measuring hematological sample |
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