CN105928907A - Erythrocyte-osmotic-fragility measuring method based on scattering turbidimetric technology - Google Patents
Erythrocyte-osmotic-fragility measuring method based on scattering turbidimetric technology Download PDFInfo
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Abstract
The invention discloses an erythrocyte-osmotic-fragility measuring method based on the scattering turbidimetric technology. The erythrocyte-osmotic-fragility measuring method includes the following steps that a low-pressure osmotic buffering salt solution is added into a cuvette of a scattering turbidimetric analysis module; erythrocyte is added into the cuvette, the mixture is stirred and mixed to be even, and then the scattering value A0 of the mixed solution is measured; after hemolysis is generated in the mixed solution, before the hemolysis balance state, the scattering value A1 of the mixed solution is measured; the hemolysis rate is calculated according to the formula that the hemolysis rate (%)=(A0-A1)/A0*100%, and the result is output. According to the method, the scattering turbidimetric technology is adopted, the osmotic fragility of erythrocyte is measured in one step, operation is easy, measuring time is short, a sample does not need to be pretreated, centrifuging is not required in the testing process, and full-automatic detection can be achieved.
Description
Technical field
The present invention relates to hematopathy detection field, relate generally to a kind of osmotic fragility assay method based on scattering turbidimetry technology.
Background technology
, if being placed in hypisotonic solution by erythrocyte, there is haemolysis because or even intraor extracellular there is permeable pressure head, hydrone entrance erythrocyte so that it is generation swelling, erythrocyte fragmentation in osmotic fragility: normal erythrocyte is double intended circle dish type.Erythrocyte occur in hypotonic saline solution the feature of haemolysis be osmotic fragility (the reddest, Wang Yusan, Shen Ziyu, whole nation Clinical Laboratory rule of operation (the 4th edition) [M]. Beijing: People's Health Publisher, 2015.66).
Assay method to osmotic fragility mainly has following three kinds the most clinically:
1, erythrocyte osmotic fragility test: this law is traditional manual method, its measuring method is: compound concentration is respectively the NaCl solution of 2.0g/L, 2.4g/L, 2.8g/L, 3.2g/L, 3.6g/L, 4.0g/L, 4.4g/L, 4.8g/L, 5.2g/L, 5.6g/L, 6.0g/L, 6.4g/L, 6.8g/L, 7.2g/L.Then add 1 whole blood toward each in the NaCl solution of above-mentioned 14 concentration, after shaking up gently, stand 2 hours, start to observe the haemolysis of whole 14 pipe solution from high concentration.Result judges: supernatant begins to show light red, and still there is a large amount of non-haemolysis cell person bottom for starting haemolysis pipe；The peony that full pipe solution is the most transparent, bottom is complete hemolysis pipe without remaining erythrocyte person.The method to use 14 test tubes, joins the sodium chloride solution of 14 kinds of variable concentrations, will wait 2 hours.Making with the naked eye to judge, accuracy is low；
2, a tube method: the method is a kind of method of improvement on the basis of the manual method of traditional erythrocyte osmotic fragility test, its principle is to judge erythrocytic osmotic fragility by the erythrocytic hemolysis rate of mensuration.Concrete measuring method is as follows: the red blood cell suspension taking two parts of equivalent is respectively placed in hypotonic saline solution and the hemolytic agent of certain certain concentration, after mixing, after 37 DEG C of temperature bath 5min, measures absorbance A and the C of two parts of solution respectively.Hemolysis rate %=(1-A/C) × 100%.Two pipe solution are only used due to the method, the measurement time is 5min, it was greatly lowered relative to 2 hours of traditional-handwork method, whole blood is after pretreatment, automatic clinical chemistry analyzer can be used to be measured, precision is further improved, and this method has the most become most of hospital and accepted the main flow assay method of osmotic fragility；
3, the turbid dynamic-analysis method of transmittance: in having automatic constant-temperature and self registering spectrophotometric cuvette, add a certain amount of hypotonic medium, after solution in cuvette is constant in predetermined temperature, add a certain amount of blood sample, quickly after mixing, start to record the absorbance of per unit time under certain specific wavelength measures at once, no longer drop to reaction end with absorbance, thus obtain the dynamic changing value (A1, A2, A3...Az) of absorbance.The relative hemolysis rate thinking the second in i-th second is (Ai-Ax)/Ai × 100%.And then calculate in reaction previous measuring point in certain time period to the relative hemolysis rate of a rear measuring point.With the time as abscissa, absorbance is vertical coordinate, draw out the dynamic curve diagram that can reflect that haemolysis changes, numbered features according to figure can carry out correlation analysis (Pan Yuhua to the erythrocyte fragility of sample, a kind of assay method of osmotic fragility]: China, CN 1268928C [P] .2006-8-9).
In said method, traditional-handwork method, use 14 pipe hypotonic mediums to test, need manual sample-adding, complex operation, measure time length (2 hours), and need to be judged by naked eyes, easily affected by artificial subjective factors, the repeatability of result and poor accuracy.One tube method substantially employs two pipe solution, need manual whole blood is carried out pre-treatment can examination with computer, complex operation, the measurement time is more than 10 minutes, it addition, the result that this method records simply has reacted the absorbance of non-complete hemolysis and the ratio of complete hemolysis absorbance, and non-real hemolysis rate.The turbid dynamic-analysis method of transmittance needs reaction temperature 37 DEG C predetermined for pre-for hypotonic medium calorific value, record is needed to start all absorbances to reaction end from reaction, i.e. need to wait question response to terminate just can go out result, the measurement time is long, additionally need the dynamic curve diagram drawing haemolysis change, the erythrocyte fragility of sample is analyzed by the variation characteristic according to figure, analyzes method loaded down with trivial details.
Therefore, prior art has yet to be improved and developed.
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, it is an object of the invention to provide a kind of osmotic fragility assay method based on scattering turbidimetry technology, it is intended to the assay method solving existing osmotic fragility exists the problem that complex operation, time length or accuracy are low.
Technical scheme is as follows:
A kind of osmotic fragility assay method based on scattering turbidimetry technology, wherein, comprises the following steps:
In the cuvette of scattering turbidimetry analysis module, add the buffer salt solution of low-pressure permeability；
In above-mentioned cuvette, add erythrocyte, after stirring and evenly mixing, measure scattering value A0 of mixed liquor；
After mixed liquor generation haemolysis, before reaching haemolysis poised state, measure scattering value A1 of mixed liquor；
According to formula: hemolysis rate %=(A0-A1)/A0 × 100%, calculate hemolysis rate, export result.
Described osmotic fragility assay method based on scattering turbidimetry technology, wherein, the buffer salt solution of described Hyposmolality be electrical conductivity be 6.5～7.0mS/cm, pH value be NaCl or the buffer salt solution of other salt of 7.0-7.4.
Described osmotic fragility assay method based on scattering turbidimetry technology, wherein, hemocyte is 1:100～500 with the mixed volume ratio of Hyposmolality buffer salt solution.
Described osmotic fragility assay method based on scattering turbidimetry technology, wherein, the mode of stirring and evenly mixing is magnetic agitation blending manner or stirring rod blending manner.
Described osmotic fragility assay method based on scattering turbidimetry technology, wherein, the light source of scattering turbidimetry analysis module is laser, and the wavelength of laser is 600nm～800nm.
Described osmotic fragility assay method based on scattering turbidimetry technology, wherein, the step of assay method is as follows:
In the cuvette of scattering turbidimetry analysis module, add the buffer salt solution that 1.0mL contains 0.35%NaCl；
In above-mentioned cuvette, add 3uL erythrocyte, after stirring and evenly mixing, measure scattering value A0 of mixed liquor；
After waiting 30S, measure scattering value A1 of mixed liquor；
According to formula: hemolysis rate %=(A0-A1)/A0 × 100%, calculate hemolysis rate, export result.
Beneficial effect: the osmotic fragility assay method based on scattering turbidimetry technology of the present invention, has the advantage that a step realizes the mensuration of osmotic fragility, and the measurement time simple to operate is short；Reaction be certain a period of time during haemolysis in, the speed that mixed liquor scattering value declines, reaction is the hemolysis rate of real meaning；Sample is without carrying out pre-treatment, without centrifugal in test process, it is easy to accomplish full-automatic detection.
Accompanying drawing explanation
Fig. 1 is the scattering value change curve of mixed liquor during erythrocyte hemolysis.
Detailed description of the invention
The present invention provides a kind of osmotic fragility assay method based on scattering turbidimetry technology, and for making the purpose of the present invention, technical scheme and effect clearer, clear and definite, the present invention is described in more detail below.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Scattering turbidimetry technology refer to light along trunnion axis irradiate time, encounter short grained immune complex and may result in light scattering, the intensity of scattered light is directly proportional to the content of complex.Diagnostic field in vitro, scattered light urbidmetry is only applied to immunoassay at present, that is: by antibody absorption size to fit (100-200nm), uniformity latex microsphere granule on, when running into corresponding antigen, then make latex particle generation coagulation, single latex particle does not hinder light to pass through within incident light wave, scattered light is fewer, scattered light is then made to increase during two or more latex particle agglutination, the degree that scattered light increases is directly proportional to the degree of latex particle agglutination, namely proportional with determined antigen amount.(Tang Qiuyan, Wang Yunlong, Chen Xingye immune diagnostic reagent practical technique [M]. Beijing: Maritime Press, 2009.226).
In osmotic fragility assay method based on scattering turbidimetry technology provided by the present invention, described scattering turbidimetry technology refers to: after being mixed by the saline solution of whole blood sample with specific Hyposmolality, owing to intra-erythrocyte exists permeable pressure head, hydrone enters inside erythrocyte, bring it about expansion, scattered light first raises, and can reach maximum；When expanding into a certain degree, erythrocyte membrane can rupture and haemolysis, and scattered light declines along with erythrocytic level of breakage.The speed that mixed liquor scattered light declines is directly proportional to osmotic fragility.Utilize this principle, osmotic fragility can be measured.
Specifically, osmotic fragility assay method based on scattering turbidimetry technology provided by the present invention, comprise the following steps:
1, in the cuvette of scattering turbidimetry analysis module, the buffer salt solution of low-pressure permeability is added；
2, in above-mentioned cuvette, add erythrocyte, after stirring and evenly mixing, measure scattering value A0 of mixed liquor；
3, after mixed liquor generation haemolysis, before reaching haemolysis poised state, scattering value A1 of mixed liquor is measured；
4, according to formula: hemolysis rate %=(A0-A1)/A0 × 100%, calculate hemolysis rate, export result.
Wherein, the buffer salt solution of described Hyposmolality be electrical conductivity be 6.5～7.0mS/cm, pH value be NaCl or the buffer salt solution of other salt of 7.0-7.4.Described hemocyte is 1:100～500 with the mixed volume ratio of Hyposmolality buffer salt solution.The mode of described stirring and evenly mixing can be magnetic agitation blending manner or stirring rod blending manner.The light source of described scattering turbidimetry analysis module is laser, and the wavelength of described laser is 600nm～800nm.
The principle of this assay method is, scattering turbidimetry analytical technology is used to measure erythrocyte fragility: after being mixed by the saline solution of whole blood sample with specific Hyposmolality, owing to intra-erythrocyte exists permeable pressure head, hydrone enters inside erythrocyte, bring it about expansion, scattered light first raises, and can reach maximum；When expanding into a certain degree, erythrocyte membrane can rupture and haemolysis occurs, and scattered light slowly declines along with erythrocytic level of breakage.The speed that mixed liquor scattered light declines is directly proportional to osmotic fragility.Utilize this principle, osmotic fragility can be measured.When erythrocyte has just added in the buffer salt solution of Hyposmolality, the most there is not haemolysis in erythrocyte, now the scattering value of mixed liquor is the highest, growth over time, erythrocyte first expands, and haemolysis the most slowly occurs, and the scattering value of mixed liquor is gradually lowered, may eventually reach a stable state, the speed that mixed liquor scattered light declines is directly proportional to osmotic fragility.During erythrocyte hemolysis, the scattering value change curve of mixed liquor refers to Fig. 1.Therefore, erythrocyte is added in the buffer salt solution of Hyposmolality, scattering value A0 of mixed once liquid is first surveyed after mixing, after haemolysis a period of time (not up to terminal), survey scattering value A1 of mixed once liquid again, utilize formula: hemolysis rate %=(A0-A1)/A0 × 100%, hemolysis rate can be calculated.
The present invention also provides for one more preferred embodiment scheme, comprises the following steps:
1, in the cuvette of scattering turbidimetry analysis module, the buffer salt solution that 1.0mL contains 0.35%NaCl is added；
2, in above-mentioned cuvette, add 3uL erythrocyte, after stirring and evenly mixing, measure scattering value A0 of mixed liquor immediately；
3, after waiting 30S, scattering value A1 of mixed liquor is measured.
4, according to formula: hemolysis rate %=(A0-A1)/A0 × 100%, calculate hemolysis rate, export result.
Osmotic fragility assay method based on scattering turbidimetry technology provided by the present invention, has the advantage that
1, shortcoming that is loaded down with trivial details for prior art operation and that measure time length: using scattering turbidimetry technology, a step realizes the mensuration of osmotic fragility.Only need to take 3uL erythrocyte, be placed in equipped with in the reaction cup of hypisotonic solution, use scattering turbidimetry analysis-e/or determining, within 30 seconds, result can be gone out.
2, for the shortcoming of prior art algorithm: this method first surveys scattering value A0 of mixed liquor before haemolysis, after haemolysis a period of time (not up to terminal), then scattering value A1 of the most same pipe mixed liquor is surveyed, hemolysis rate %=(A0-A1)/A0 × 100%.Reaction be certain a period of time during haemolysis in, mixed liquor scattering value decline speed, be the hemolysis rate of real meaning.
3, the shortcoming realized for prior art full-automation detection difficulty: the sample of this law is without carrying out pre-treatment, without centrifugal in test process, it is easy to accomplish full-automatic detection.
It should be appreciated that the application of the present invention is not limited to above-mentioned citing, for those of ordinary skills, can be improved according to the above description or convert, all these modifications and variations all should belong to the protection domain of claims of the present invention.
Claims (6)
1. an osmotic fragility assay method based on scattering turbidimetry technology, it is characterised in that comprise the following steps:
(1) in the cuvette of scattering turbidimetry analysis module, the buffer salt solution of low-pressure permeability is added；
(2) in above-mentioned cuvette, add erythrocyte, after stirring and evenly mixing, measure scattering value A0 of mixed liquor；
(3), after mixed liquor generation haemolysis, before reaching haemolysis poised state, scattering value A1 of mixed liquor is measured；
(4) according to formula: hemolysis rate %=(A0-A1)/A0 × 100%, calculate hemolysis rate, export result.
Osmotic fragility assay method based on scattering turbidimetry technology the most according to claim 1, it is characterized in that, the buffer salt solution of described Hyposmolality be electrical conductivity be 6.5～7.0mS/cm, pH value be NaCl or the buffer salt solution of other salt of 7.0-7.4.
Osmotic fragility assay method based on scattering turbidimetry technology the most according to claim 1, it is characterised in that hemocyte is 1:100～500 with the mixed volume ratio of Hyposmolality buffer salt solution.
Osmotic fragility assay method based on scattering turbidimetry technology the most according to claim 1, it is characterised in that the mode of stirring and evenly mixing is magnetic agitation blending manner or stirring rod blending manner.
Osmotic fragility assay method based on scattering turbidimetry technology the most according to claim 1, it is characterised in that the light source of scattering turbidimetry analysis module is laser, and the wavelength of laser is 600nm～800nm.
Osmotic fragility assay method based on scattering turbidimetry technology the most according to claim 1, it is characterised in that the step of assay method is as follows:
(1) in the cuvette of scattering turbidimetry analysis module, the buffer salt solution that 1.0mL contains 0.35%NaCl is added；
(2) in above-mentioned cuvette, add 3uL erythrocyte, after stirring and evenly mixing, measure scattering value A0 of mixed liquor；
(3), after waiting 30S, scattering value A1 of mixed liquor is measured；
(4) according to formula: hemolysis rate %=(A0-A1)/A0 × 100%, calculate hemolysis rate, export result.
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| CN110530824A (en) * | 2019-09-04 | 2019-12-03 | 深圳褀氏生物科技有限公司 | A kind of osmotic fragility measuring method and its kit |
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