CN1149397C - Reagent for determining granulophilocyte and determination method - Google Patents
Reagent for determining granulophilocyte and determination method Download PDFInfo
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- CN1149397C CN1149397C CNB971107270A CN97110727A CN1149397C CN 1149397 C CN1149397 C CN 1149397C CN B971107270 A CNB971107270 A CN B971107270A CN 97110727 A CN97110727 A CN 97110727A CN 1149397 C CN1149397 C CN 1149397C
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Abstract
To obtain a resin solution composition useful for a color filter, excellent in dispersibility and stability of pigment, improved in heat resistance, transparency and contrast ratio, especially excellent in chemical resistance, comprising a specific siloxane cross-linked polyamide precursor and pigment. This composition comprises (A) a siloxane cross-linked polyamide precursor and (B) pigment. The component A consists essentially of (i) a polyamic acid composed of a constituent unit of the formula (R<1> is a polyfunctional amine residue; R<2> is a polyfunctional amine residue; (n) is an integer of 0 to 4) as a main component and (ii) the reaction product of an aminoalkyl polyfunctional alkoxysilane or an aminoaryl polyfunctional alkoxysilane hydrolyzate or their condensate and a polyfunctional carboxylic acid dianhydride. The composition is obtained, for example, by a method comprising a process for adding the solution of the component A to a pigment dispersion mainly constituted of a pigment dispersed into a lactone as a main solvent.
Description
The present invention relates to reticulocyte determination with reagent and assay method, relate to aspect clinical examination the reagent and the assay method that adopt in order to measure granulophilocyte in the blood and granulophilocyte mature index in more detail.
Granulophilocyte is that the normoblast in the marrow takes off the immature erythrocyte that just has been released to behind the nuclear in the peripheral blood.Granulophilocyte has following feature as the traces in the cell mature process: promptly cell contains RNA or the inferior organ of cells such as ribosomes, mitochondria that does not contain in a spot of mature erythrocyte.
Aspect clinical examination, it is extremely important that granulophilocyte is carried out differential count hematopoiesis state aspect in grasping patient's marrow.Its granulophilocyte of marrow hemopoiesis normal healthy people accounts for 0.5～3.0% of RBCM, work as marrow hemopoiesis and be in abnormality for example under the repressed state of marrow hemopoiesis, granulophilocyte quantity reduces, and under the hyperfunction state of marrow hemopoiesis, granulophilocyte quantity increases.Specifically be exactly that granulophilocyte quantity reduces when hypoplastic anemia, malignant tumor chemotherapy, and when hemolytic anemia, increase.
Reticulocyte count method in the past is by blood sample is mixed with the dyeing liquor that contains new methylene blue (NMB) or brilliant cresyl blue alkaline pigments such as (BCB), the above-mentioned remaining material that contains in the granulophilocyte is separated out, dyeing, examine under a microscope each cell, distinguish ripe red blood cell and granulophilocyte.This is a manual manipulation method.
Yet the problem of method hand-manipulated is that sample making is loaded down with trivial details, and the error that can produce between the test technician on the statistics that reasons such as personal equation that cell distinguishes and cell count quantity are few cause increases.
In order to address the above problem, can be undertaken by following method (flow cytometer method), replace top alkaline pigment that granulophilocyte is carried out fluorescent dye with fluorescence alkalescence pigment, with the forward scattering light intensity and the fluorescence intensity of cells were tested by flow cytometry cell, mainly distinguish mature erythrocyte and granulophilocyte and carry out differential count with the difference of fluorescence intensity.The pigment that is used for this method has pigments such as acridine orange that people know, thiazole orange, gold (look) amine O.
This method is the fluorescence intensity according to granulophilocyte, press its maturity to the granulophilocyte differential count, might calculate the mature index of granulophilocyte, people know, the ratio of the granulophilocyte that fluorescence intensity is high, that is, the ratio of newborn granulophilocyte is useful as the recovery index of hemopoietic function of bone marrow, the wait in expectation utilization of this method of people.
But a problem is arranged in addition: the light source as the fluorescence excitation pigment needs the high and bulky argon laser of price, so price height of device own and volume are big.In addition, dyeing needs clock time more than 5 minutes to granulophilocyte except the above-mentioned pigment the spendable gold of flow cytometer method (look) amine O.Particularly thiazole orange needs the dyeing time more than 60 minutes, and this is at Lee.L.G.etal:Cytometry; 7:508-517 (1986) reported, so that the specimen preparation full-automation is difficult, see it is problematic from the labour-saving viewpoint.Because by manually carrying out specimen preparation, so because of its manual skill or the operator is different makes data that deviation be arranged, especially on the granulophilocyte mature index, exist this shortcoming of very big data deviation.
No. 5360739 patent of the U.S. announced a kind of cells were tested by flow cytometry method, promptly adopts comparatively cheap He/Ne lasing light emitter ， oxazine 750 as pigment, by measuring scattered light intensity and absorbance or fluorescent strength determining granulophilocyte.But this patent does not relate to the unusual lymphocytic problem of saying below.
The applicant also dedicates oneself to study and is used for correct and the reagent and the assay method of rapid test granulophilocyte and granulophilocyte mature index, and this reagent and assay method can use in having disposed the flow cytometer method of cheap miniature light sources.But because in the sample that unusual lymphocytic particular patients ' occurs, the fluorescence intensity of lymphocyte fluorescence intensity and granulophilocyte is similar, so their resolution has become difficulty.Therefore, with regard to the difficult problem of distinguishing of leucocyte and granulophilocyte, also do not establish reagent and the assay method of correctly and promptly measuring at present.This situation also at present commercially available with the argon laser be the flow cytometer of light source with reticulocyte determination with kit (Retic-COUNT
TM) operation instructions in given prompting: promptly need be when measuring the unusual patient's who increases of leukocyte count sample by other method affirmation.
According to the present invention, the 1.th can provide the reticulocyte determination reagent of being made up of at least a pigment that can make the granulophilocyte specific staining and at least a pigment that can make the leucocyte specific staining, wherein, the pigment that makes the granulophilocyte specific staining is suc as formula shown in (I), its concentration is the 0.1-100mg/ liter
In the formula, R
1Be hydrogen atom or low alkyl group; R
2Or R
3Identical or different, expression hydrogen atom or low alkyl group, R
4Be hydrogen atom or acyl group; R
5Be hydrogen atom, hydroxyl low-grade alkyl; Z is sulphur atom or oxygen atom; N is 1 or 2, X
-Be negative ion; The pigment that makes the leucocyte specific staining is suc as formula shown in (II) or the formula (III), and its concentration is the 0.001-200mg/ liter,
In the formula, R
6And R
7Identical or different, expression hydrogen atom or low alkyl group; R
8～R
11Identical or different, expression hydrogen atom or low alkyl group; X
-Be negative ion;
In the formula, R
12And R
13Identical or different, expression hydrogen atom, low alkyl group or the amino that is replaced by low alkyl group; R
14And R
15Identical or different, expression hydrogen atom or low alkyl group; X
-It is negative ion.
The 2.th provides a kind of reticulocyte determination reagent of above-mentioned 1 record, wherein contains the multivalent anions that suppresses erythrocytic non-specific fluorescence.The 3.th provides the reticulocyte determination reagent of a kind of above-mentioned 1 or 2 records, wherein contains to make pH keep certain damping fluid.The 4.th provides the reticulocyte determination reagent of record in a kind of above-mentioned 1 or 2, wherein contains the osmotic pressure compensation liquid that makes osmotic pressure maintain physiology osmotic pressure.The 5.th provides the reticulocyte determination reagent of record in a kind of above-mentioned 3, wherein contains the osmotic pressure compensation liquid that makes osmotic pressure maintain physiology osmotic pressure.
The 6.th provides the reticulocyte determination reagent of a kind of above-mentioned 1 or 2 records, wherein contains short dye liquor, for suc as formula the cationic surfactant shown in (IV):
In the formula, R
16Alkyl for carbon number 8～12; R
17, R
18, R
19Identical or different, the expression low alkyl group; Y
-Be halide ion, the low alkyl group definition is the same.The 7.th provides a kind of reticulocyte determination reagent of above-mentioned 5 records, wherein contains short dye liquor, for suc as formula the cationic surfactant shown in (IV):
In the formula, R
16Alkyl for carbon number 8～12; R
17, R
18, R
19Identical or different, the expression low alkyl group; Y
-Be halide ion, the low alkyl group definition is the same.
The 8.th provides in a kind of above-mentioned 6 the reticulocyte determination reagent of record, and cationic surfactant wherein is following at least a: dodecyltrimethyl ammonium chloride, bromination decyl trimethyl ammonium, bromination octyl group trimethyl ammonium.
The 9.th provides in a kind of above-mentioned 7 the reticulocyte determination reagent of record, and cationic surfactant wherein is following at least a: dodecyltrimethyl ammonium chloride, bromination decyl trimethyl ammonium, bromination octyl group trimethyl ammonium.
The present invention can provide the method for using mentioned reagent to measure granulophilocyte simultaneously.
Reagent of the present invention and assay method for resemble above-mentioned patient's test sample, promptly also can correctly and promptly measure granulophilocyte for a lot of unusual lymphocytic patient's samples occurring.Reagent of the present invention is particularly suitable for using in the flow cytometer method.In other words, for a lot of unusual lymphocytic patient's samples occurring, the reagent of the application of the invention can not allow the fluorescence intensity of ripe red blood cell and granulophilocyte change, and leukocytic fluorescence intensity is increased, so can precision highland differential count granulophilocyte more.
Reagent of the present invention is made up of pigment that makes the granulophilocyte specific staining and the pigment that makes the leucocyte specific staining.What is called is meant with this pigment dyeing the pigment of granulophilocyte specific staining the difference of granulophilocyte and other haemocyte is come.For example: formula (I) compound
In the formula, R
1Be hydrogen atom or low alkyl group; R
2And R
3Can be identical or different, be hydrogen atom, low alkyl group or lower alkoxy; R
4Be hydrogen atom, acyl group or low alkyl group; R
5Be hydrogen atom, can substituted low alkyl group; Z is sulphur atom, oxygen atom or the carbon atom that replaced by low alkyl group; N is 1 or 2; X
-It is negative ion.
The R of formula (I)
1In low alkyl group be meant that carbon number is the alkyl of 1～6 straight or branched, as methyl, ethyl, propyl group, butyl, isobutyl, sec-butyl, the tert-butyl group, amyl group or hexyl.Wherein it is desirable to methyl and ethyl.
R
2And R
3In low alkyl group same as described above, and be meant that as lower alkoxy carbon number is 1～6 alkoxy, as methoxyl, ethoxy or propoxyl group, wherein it is desirable to methoxyl and ethoxy, and R
2And R
3Better when being hydrogen atom.
R
4In acyl group it is desirable to the acyl group of deriving from aliphatic carboxylic acid, for example acetyl group and propiono wherein it is desirable to acetyl group.And low alkyl group is with above-mentioned the same.
R
5In low alkyl group with above-mentioned the same, what is called can substituted low alkyl group be meant the low alkyl group that can be replaced by 1～3 hydroxyl or halogen atom (as fluorine, chlorine, bromine, sulphur) etc., wherein, it is desirable to methylol and hydroxyethyl.
Low alkyl group among the Z it is desirable to sulphur atom with above-mentioned the same as Z.
X
-In negative ion halide ion (fluorine, oxygen, bromine or iodine ion, halogenation boron ion (BF are arranged
4 -, BCl
4 -, BBr
4 -Deng), halogen atom or the substituent tetraphenyl boron compound of haloalkyl ion etc. are arranged on the phosphorus compound ion, oxyhalogen-acid ion, fluorosulfuric acid ion, methyl-sulfuric acid ion, phenyl ring, wherein, it is desirable to bromide ion and BF
4 -
Provided the particular compound of formula (I) below.
The compound of n=1 in the top formula (I) can be synthetic through following approach.Compound and the N that represents with following formula for example, N-two replaces the carbonamidines reaction,
Its product reacts with the quinoline of representing with following formula again, handles through sodium fluoborate then, can synthesize.
And the compound of n=2 in the formula (I) is to replace N with two (benzene imido) salt of MDA in above-mentioned reaction, and N-two replaces carbonamidine and reacts synthetic.
The example of the pigment that other may use, its structural formula is as follows:
With
[the B here is
Or
C ' is
X ' is O, S, Se or＞C (CH
3)
2
Y ' is Cl, Br or I,
R
1', R
2', R
3', R
4', R
5', R
6' or R
7' identical or inequality, expression low alkyl group, low-grade alkenyl or halogenated lower alkyl] for example:
(1) 1,1 ', 3,3,3 ', 3 '-hexamethyl indoles, two quinoline blue pigment iodide (HIDCI can buy from LAMBDA PHYSIK society)
(2) 1,1 '-diethyl-2,4 '-quinone quinoline blue pigment iodide (NK-138 can buy from Japanese photopigment research institute)
(3) pinacyanol chloride
(4) 1,3 '-diethyl-2,2 '-quinone thia quinoline blue pigment iodide.
The pigment that detects usefulness as granulophilocyte also can use Zhong known De oxazine 750.
For making granulophilocyte dyeing, it is sufficient that the above-mentioned pigment consumption in the reagent of the present invention is wanted, and can form according to kind, the reagent of pigment and suitably adjust.For example, be approximately 0.1～100mg/ liter, better amount approximately is 0.3～30mg/ liter, it would be desirable 0.3～3mg/ liter.
And conduct makes the pigment of leucocyte specific staining that the pigment of formula (II) and formula (III) representative for example be arranged
R in the formula
6And R
7Can be identical also can be different, expression hydrogen atom, low alkyl group, lower alkoxy or phenyl; R
8～R
11Can be identical or different, expression hydrogen atom or low alkyl group; X
-It is negative ion.
R in the formula
12And R
13Can be identical also can be different, expression hydrogen atom, low alkyl group, lower alkoxy or the amino that replaces with low alkyl group, R
14And R
15Identical or different, expression hydrogen atom or low alkyl group; X
-It is negative ion.
And low alkyl group in above-mentioned formula (II) and the formula (III) and lower alkoxy are with noted earlier the same.
For making leucocyte dyeing, the concentration of pigment is wanted sufficient and this concentration does not hinder reticulocyte determination with the dyeing of pigment to granulophilocyte in the reagent of the present invention.Concentration can suitably be adjusted according to the kind and the reagent composition of pigment.For example be 0.001～200mg/ liter, better approximately is 0.003～3mg/ liter, it would be desirable 0.003～0.03mg/ liter.
In reagent of the present invention is formed, the pigment of granulophilocyte specific staining can promptly be permeated in red blood cell, with intracellular RNA dyeing.Therefore, even only make the pigment of granulophilocyte specific staining, it also is possible that granulophilocyte is carried out differential count.Yet under special situations such as the abnormal cell that makes lymphocytic series because of acute lymphatic leukemia etc. increases, the fluorescence intensity of lymphocyte and immature granulophilocyte becomes about equally, has just become difficulty so distinguish these two kinds of cells.
On the other hand, contain the pigment that can make the leucocyte specific staining in the reagent of the present invention, it is possible only making leucocyte dyeing with above-mentioned concentration.Make the pigment of leucocyte specific staining also different because of the kind coloured differently of pigment, probably can make not to be included in specific stainings such as particulate component in the granulophilocyte or nuclear in the leucocyte, we think can make leukocytic fluorescence intensity strengthen thus.Its result, granulophilocyte and leukocytic fluorescence intensity can produce tangible difference, promptly, the fluorescence intensity of leukocytic fluorescence intensity ratio granulophilocyte is strong, because the fluorescence intensity of granulophilocyte does not change, so even distinguish that under situation as described above these two kinds of cells also are possible.
Though exemplified the pigment that the light with red wavelength can excite here, but be not limited to these pigments among the present invention, also can even in this case, also can improve granulophilocyte and the leukocytic rate of distinguishing with being used in combination by the light activated pigment of other such wavelength of blue wavelength light.
Also can make in addition and contain multivalent anions, damping fluid, osmotic pressure compensation liquid and/or the short dye liquor that suppresses erythrocytic non-specific staining in the reagent of the present invention.
The inventor finds that when research reagent is formed the principal ingredient of the osmotic pressure compensation liquid in normally used dye liquor is that NaCl (mainly is Cl
-) time, erythrocytic non-specific fluorescence becomes big, and the specific fluorescence of granulophilocyte diminishes, and its result makes distinguishing of red blood cell and granulophilocyte become difficulty.Also find the Cl in the dyeing liquor in addition
-When changing multivalent anions into, the non-specific fluorescence of mature erythrocyte is suppressed significantly, and distinguishing also of red blood cell and granulophilocyte become easily.Therefore it is desirable to make in the reagent of the present invention and contain multivalent anions.As multivalent anions, sulfate ion, phosphate anion, carbanion and polyvalent carboxylic acid's radical ion are particularly suitable for.The compound of supplying with these ions for example has citric acid, sulfuric acid, phosphoric acid, EDTA and their alkali metal salt etc.The concentration of multivalent anions is that proportion it is desirable to account for more than 70% more than 50% in total ion component of reagent.
In addition, can contain in the reagent of the present invention and make pH keep certain damping fluid.Use damping fluid to make the coloration result of the granulophilocyte that pH keeps necessarily keeping stable thus, the concentration of use is about number mM～100mM.The kind of damping fluid, so long as normally used, be not particularly limited, for example suitably use metal carboxylate, phosphoric acid salt, Gourde(G) damping fluid, taurine, triethanolamine etc. by desirable pH.The suitable pH of reagent is different different because of used pigment among the present invention, but generally is in 6.0～11.0 scopes, and desirable pH is in 7.0～10.0 scopes, and better is in 8.0～9.5 scopes.If pH crosses when low than above-mentioned scope, since the easy embrittlement of red blood cell, the easy haemolysis that becomes, and institute is so that the correct mensuration of granulophilocyte has become difficult.If pH is too high, because the acidic functionality on the erythrocyte membrane dissociates, it is easily combined with the kation pigment, increase erythrocytic non-specific fluorescence.Owing to this reason has the tendency that makes distinguishing of mature erythrocyte and granulophilocyte become difficulty, this is undesirable.If above-mentioned multivalent anions has the surge capability of pH regulator in the suitable pH scope, also can be also used as buffering agent.
Also contain osmotic pressure compensation liquid in the reagent of the present invention,, wish osmotic pressure is adjusted to physiological osmotic pressure in order to prevent erythrocytic hypotonic haemolysis.Usually adjust in the scope of 150～600mOsm/kg.Osmotic pressure compensation liquid is not specially limited.For example, carbohydrates such as the suitable alkali metal salt that acid such as propionic acid arranged, glucose, mannose.If do not reach 50% of total anionic ratio in the shared reagent, also can use alkali halide resemble NaCl, alkaline-earth halide etc.If in the time of osmotic pressure can being adjusted in the suitable scope with above-mentioned multivalent anions and damping fluid, just need not contain osmotic pressure compensation liquid.
Also can contain cationic surfactant in the reagent of the present invention with formula (IV) expression as short dye liquor.
In the formula, R
16For carbon number is the alkyl of 8-12; R
17, R
18, R
19Identical or different, the expression low alkyl group; Y
-Be halide ion.
Wherein, the alkyl of carbon number 8-12 comprises the alkyl of straight or branched, and for example octyl group, decyl or dodecyl it is desirable to octyl group and decyl in the base.
And low alkyl group is with above-mentioned identical.
Object lesson as cationic surfactant has bromination octyl group trimethyl ammonium (OTAB), bromination decyl trimethyl ammonium (DTAB) or chlorination dodecyl trimethyl ammonium (LTAC).
As the concentration of the cationic surfactant of urging dye liquor, total carbon number more increases, and gets final product effectively on a small quantity.For example: OTAB is 300～20000mg/ liter, and DTAB is 500～300mg/ liter, and LTAC to be 50～250mg/ rise gets final product.Though the mechanism of action of cationic surfactant is indeterminate,, it is believed that it can promote the permeability of pigment because it can cause and some of erythrocyte membrane interacts.Therefore will injure red blood cell if short dye liquor amount is many in the sample, cause haemolysis sometimes, this is undesirable.
Because cationic surfactant has the effect that makes the red blood cell spheroidizing, institute is so that the forward scattering light intensity distributions pack that red blood cell is rolled into a ball in addition.Its result has become easily blood platelet and distinguishing of erythron cell.Also have in the bead erythropathy, for example in the treatment of diseases such as hypoferric anemia, microspherocyte that produces because of disease in the blood and positive spherocyte mix, but if distinguish that by the forward scattering light intensity these two kinds of cells have also become possibility with reagent of the present invention.Also can distinguish simultaneously the cameloid cell of a small amount of appearance equally.So utilize the red blood cell spheroidization of cationic surfactant to act on when measuring granulophilocyte and also can detect erythrocytic abnormal morphologies such as microspherocyte and cameloid cell.
Except that containing above-mentioned constituent, also can contain for example antiseptic such as 2-pyridylthio-1-sodium oxide molybdena or bata-phenethyl alcohol in the reagent of the present invention.
The pigment that uses in reagent of the present invention can be dissolved in pigment when unstable in aqueous solution in the suitable non-aqueous solvent that can dissolve each other with water and preserve in addition, as ethanol, dimethyl sulfoxide (DMSO), ethylene glycol etc.Just can use during use with after the aqueous solution that contains other composition.It also is possible in the same non-aqueous solvent that each pigment is dissolved in, and also may be dissolved in other non-aqueous solvent.
Reagent of the present invention is effective especially when using the cells were tested by flow cytometry granulophilocyte.
Operation (1) is with formation determination sample after reticulocyte determination reagent of the present invention and the hematology sample mix.So-called hematology sample is meant any sample that contains the blood constituent that becomes determination object.For example, carried out peripheral blood or the bone marrow fluid that anti-coagulants is handled.The preparation of working sample it is desirable to reticulocyte determination with reagent with make its reaction after the hematology sample mixes in 100: 1～1000: 1 ratio, the temperature of reaction of this moment is suitable in 25～50 ℃ of scopes, more preferably at 35～45 ℃.And the suitable reaction time is different because of contained pigment in the reagent, but it is desirable to 10 seconds～about 5 minutes, better is about 20 seconds～2 minutes, it would be desirable about 20～60 seconds.
Operation (2), the test sample that will obtain in operation (1) imports in the flow system of the flow cytometer that the red wavelength light source is housed.As the red wavelength light source, so long as can produce near the light of excitation wavelength that uses pigment (fluorchrome), for example the light source of the light of the wavelength about 600～680nm gets final product, and is not specially limited.The light sources such as semiconductor laser of He/Ne laser instrument, red wavelength range for example.Fig. 1 has provided the opticator of the flow cytometer that contains the red wavelength light source that uses.A flow cell 3 has been adorned by convergent lens group 2 in the place ahead at semiconductor laser 1, has adorned forward scattering light sensitive lens 5 and photodiode 6 in the place ahead of flow cell by beam limit device 4.And the sensitive lens of using by side fluorescence in the side of flow cell 37 has been adorned bandpass filter 8 and photomultiplier 9.And the part that flows, data processing division other member that grades will can use after the improvement such as well-known structure.
Operation (3) flows through cell in the sheath stream (Sheath-flow) with the rayed of above-mentioned red wavelength.
Operation (4) is to measure scattered light and the fluorescence that is sent by cell.No matter the scattered light of this moment is that forward scattering light or side-scattered light can.No matter as forward scattering light is that angle scattered light (1～5 °) is hanged down in the place ahead or the place ahead high angle scatter light (6～20 °) can.Scattered light is the parameter of reflection cell size information, and other method of measuring the cell size information also has electric-resistivity method etc.The according to circumstances different electric-resistivity methods that can utilize replace the mensuration of scattered light to measure resistance signal.
Operation (5) is distinguished blood platelet and other cell according to the scatter diagram of scattered light intensity or scattered light intensity and fluorescence intensity.
Operation (6) is distinguished red blood cell, granulophilocyte and leucocyte according to the scatter diagram of fluorescence intensity or fluorescence intensity and scattered light intensity.
Operation (7) to red blood cell and granulophilocyte differential count, is calculated their cell number and cells ratio.
With the difference differential count of granulophilocyte by its maturity, specifically be to carry out according to the difference of fluorescence intensity, can calculate the ratio that they account for total granulophilocyte.
Embodiment 1: at first by following composition preparation reticulocyte determination reagent.
Make the pigment A 3mg of red blood cell specific staining
Make the pigment B 0.03mg of leucocyte specific staining
Triein (buffering agent) 1.79g
LTAC (cationic surfactant, accelerant) 150mg
1 liter of Purified Water
(pH being transferred to 9.0) with NaOH
To be added in the reagent of 2ml through the healthy human blood 10 μ l that anti-coagulants is handled, 40 ℃ of incubations were measured forward scattering light intensity and fluorescence intensity with the test sample that obtains with opticator shown in Figure 1 after 120 seconds.In scatter diagram shown in Figure 2, the granulophilocyte ratio is 2.0%.And method measures with East Asia medical electric system R-2000 in contrast also is 2.0%.
The same scatter diagram that obtains Fig. 3 when measuring the blood that lymphoid abnormal cell occurs.Can clearly distinguish granulophilocyte group and leucocyte group according to this scatter diagram.
Embodiment 2: at first by following composition preparation reticulocyte determination reagent.
Make the pigment A 3mg of granulophilocyte specific staining
Make the pigment C 30mg of leucocyte specific staining
Triein 1.79g
LTAC 150mg
1 liter of Purified Water
(pH being transferred to 9.0) with NaOH
The healthy human blood that 10 μ l are handled through anti-coagulants is added in the reagent of 2ml, and 40 ℃ of incubations were measured forward scattering light intensity and fluorescence intensity with the test sample that obtains with opticator shown in Figure 1 after 120 seconds.The granulophilocyte ratio is 1.8% in the scatter diagram that Fig. 4 provides, and is 1.7% when method is measured with East Asia medical electric system R-2000 in contrast.
Comparative example 1: at first by following composition preparation reticulocyte determination reagent.
Make the pigment A 3mg of granulophilocyte specific staining
Triein 1.79g
LTAC 150mg
1 liter of Purified Water
(pH being transferred to 9.0) with NaOH
The healthy human blood that 10 μ l was carried out the anti-coagulants processing is added in the 2ml reagent, and 40 ℃ of incubations were measured forward scattering light intensity and fluorescence intensity with the test sample that obtains with opticator shown in Figure 1 after 120 seconds.The ratio of granulophilocyte is 2.1% in the scatter diagram that Fig. 5 provides, and is 2.0% when method is measured with East Asia medical electric system R-2000 in contrast.
The same scatter diagram that obtains Fig. 6 when lymphoid unusual lymphocytic blood occurring of measuring.Can not clearly distinguish granulophilocyte group and leucocyte group according to this scatter diagram.
Adopt reagent of the present invention and assay method, use the flow cytometer method that has disposed cheap, small-sized light source, both made in the special detection sample that unusual lymphocyte occurs and also can correctly, promptly measure granulophilocyte.Promptly, utilize reagent of the present invention and assay method for unusual lymphocytic patient's sample occurring, the fluorescence intensity of mature erythrocyte and granulophilocyte is changed, and only make leukocytic fluorescence intensity become big, therefore can be more accurately to the granulophilocyte differential count.
The simple declaration of accompanying drawing:
Fig. 1: the sketch of the opticator of the flow cytometer that has the red wavelength light source that in assay method of the present invention, uses.
Fig. 2: use and contain the reticulocyte determination reagent that makes the pigment A of granulophilocyte specific staining and make the pigment B of leucocyte specific staining of the present invention, the scatter diagram that obtains when the forward scattering light intensity of mensuration healthy human peripheral blood liquid sample and fluorescence intensity.
Fig. 3: use and contain the reticulocyte determination reagent that makes the pigment A of granulophilocyte specific staining and make the pigment B of leucocyte specific staining of the present invention, the scatter diagram that obtains when the forward scattering light intensity of the unusual lymphocytic peripheral blood of patients liquid sample of mensuration appearance and fluorescence intensity.
Fig. 4: use and to contain the reticulocyte determination reagent that makes the pigment A of granulophilocyte specific staining and make the pigment C of leucocyte specific staining of the present invention, the scatter diagram that obtains when measuring healthy people's the forward scattering light intensity of peripheral blood sample and fluorescence intensity.
Fig. 5: use of the present invention reticulocyte determination reagent that contains the pigment A that makes the granulophilocyte specific staining, the scatter diagram that obtains when the forward scattering light intensity of mensuration healthy human peripheral blood liquid sample and fluorescence intensity.
Fig. 6: use the reticulocyte determination reagent only contain the pigment A that makes the granulophilocyte specific staining, the scatter diagram that obtains when measuring the forward scattering light intensity of the peripheral blood of patients liquid sample that unusual lymphocyte appearance is arranged and fluorescence intensity.
Symbol description:
1. semiconductor laser
2. convergent lens group
3. flow cell
4. beam limit device
5. forward scattering light sensitive lens
6. photodiode
7. side fluorescence sensitive lens
8. band pass filter
9. photomultiplier
Claims (9)
1. reticulocyte determination reagent is made up of at least a pigment of granulophilocyte specific staining and at least a pigment of leucocyte specific staining that can make of making;
Wherein, the pigment that makes the granulophilocyte specific staining is suc as formula shown in (I), and its concentration is the 0.1-100mg/ liter,
In the formula, R
1Be hydrogen atom or low alkyl group; R
2Or R
3Identical or different, expression hydrogen atom or low alkyl group, R
4Be hydrogen atom or acyl group; R
5Be hydrogen atom, hydroxyl low-grade alkyl; Z is sulphur atom or oxygen atom; N is 1 or 2, X
-Be negative ion;
The pigment that makes the leucocyte specific staining is suc as formula shown in (II) or the formula (III), and its concentration is the 0.001-200mg/ liter,
In the formula, R
6And R
7Identical or different, expression hydrogen atom or low alkyl group; R
8～R
11Identical or different, expression hydrogen atom or low alkyl group; X
-Be negative ion;
In the formula, R
12And R
13Identical or different, expression hydrogen atom, low alkyl group or the amino that is replaced by low alkyl group; R
14And R
15Identical or different, expression hydrogen atom or low alkyl group; X
-It is negative ion;
Above-mentioned low alkyl group is the straight or branched alkyl of carbon number 1-6, and negative ion is halogen ion or halogenation boron ion.
2. the reticulocyte determination reagent of claim 1 record wherein contains the multivalent anions that suppresses erythrocytic non-specific fluorescence.
3. the reticulocyte determination reagent of claim 1 or 2 records wherein contain the damping fluid that makes pH maintenance certain.
4. the reticulocyte determination reagent of record in the claim 1 or 2 wherein contains the osmotic pressure compensation liquid that makes osmotic pressure maintain physiology osmotic pressure.
5. the reticulocyte determination reagent of record in the claim 3 wherein closes the osmotic pressure compensation liquid that makes osmotic pressure maintain physiology osmotic pressure.
6. the reticulocyte determination reagent of claim 1 or 2 records wherein contain short dye liquor, for suc as formula the cationic surfactant shown in (IV):
In the formula, R
16Alkyl for carbon number 8～12; R
17, R
18, R
19Identical or different, the expression low alkyl group; Y
-Be halide ion, low alkyl group defines with claim 1.
7. the reticulocyte determination reagent of claim 5 record wherein contains short dye liquor, for suc as formula the cationic surfactant shown in (IV):
In the formula, R
16Alkyl for carbon number 8～12; R
17, R
18, R
19Identical or different, the expression low alkyl group; Y
-Be halide ion, low alkyl group defines with claim 1.
8. the reticulocyte determination reagent of record in the claim 6, cationic surfactant wherein is following at least a: dodecyltrimethyl ammonium chloride, bromination decyl trimethyl ammonium, bromination octyl group trimethyl ammonium.
9. the reticulocyte determination reagent of record in the claim 7, cationic surfactant wherein is following at least a: dodecyltrimethyl ammonium chloride, bromination decyl trimethyl ammonium, bromination octyl group trimethyl ammonium.
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP9133596 | 1996-04-12 | ||
| JP091335/96 | 1996-04-12 | ||
| JP091335/1996 | 1996-04-12 |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1172953A CN1172953A (en) | 1998-02-11 |
| CN1149397C true CN1149397C (en) | 2004-05-12 |
Family
ID=14023575
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB971107270A Expired - Lifetime CN1149397C (en) | 1996-04-12 | 1997-04-11 | Reagent for determining granulophilocyte and determination method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1149397C (en) |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7598385B2 (en) | 2007-07-12 | 2009-10-06 | Shenzhen Mindray Biomedical Electronics Co., Ltd. | Asymmetric cyanine fluorescent dyes |
| US7709653B2 (en) | 2008-06-10 | 2010-05-04 | Shenzhen Mindray Bio-Medical Electronics Co. Ltd. | Asymmetric cyanine compounds, their preparation methods and their uses |
| US8067602B2 (en) | 2008-01-04 | 2011-11-29 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Asymmetric cyanine fluorescent dyes, compositions and their use in staining biological samples |
| US8367358B2 (en) | 2008-12-17 | 2013-02-05 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Reagent, kit and method for differentiating and counting leukocytes |
| US8685661B2 (en) | 2009-07-31 | 2014-04-01 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Reagent and kit for classifying and counting leukocytes, the preparation thereof, and process for classifying and counting leukocytes |
| US8940499B2 (en) | 2008-10-17 | 2015-01-27 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Reagent for blood analysis and method of use thereof |
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| CN101231243B (en) * | 2007-01-24 | 2011-11-16 | 深圳迈瑞生物医疗电子股份有限公司 | Agent and method for testing reticulocyte |
| CN101236158B (en) * | 2007-01-29 | 2011-11-16 | 深圳迈瑞生物医疗电子股份有限公司 | Reticular cell detection method and detection device |
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| CN113654955A (en) * | 2021-09-18 | 2021-11-16 | 迪瑞医疗科技股份有限公司 | Kit for reticulocyte counting and classifying and optical platelet counting |
-
1997
- 1997-04-11 CN CNB971107270A patent/CN1149397C/en not_active Expired - Lifetime
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US7598385B2 (en) | 2007-07-12 | 2009-10-06 | Shenzhen Mindray Biomedical Electronics Co., Ltd. | Asymmetric cyanine fluorescent dyes |
| US8067602B2 (en) | 2008-01-04 | 2011-11-29 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Asymmetric cyanine fluorescent dyes, compositions and their use in staining biological samples |
| US7709653B2 (en) | 2008-06-10 | 2010-05-04 | Shenzhen Mindray Bio-Medical Electronics Co. Ltd. | Asymmetric cyanine compounds, their preparation methods and their uses |
| US8940499B2 (en) | 2008-10-17 | 2015-01-27 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Reagent for blood analysis and method of use thereof |
| US8367358B2 (en) | 2008-12-17 | 2013-02-05 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Reagent, kit and method for differentiating and counting leukocytes |
| US8685661B2 (en) | 2009-07-31 | 2014-04-01 | Shenzhen Mindray Bio-Medical Electronics Co., Ltd. | Reagent and kit for classifying and counting leukocytes, the preparation thereof, and process for classifying and counting leukocytes |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1172953A (en) | 1998-02-11 |
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