Background technology
The information that blood cell analysis obtains contributes to diagnosis, the antidiastole disease relevant with hematological system; Contribute to analysing patient's condition, observe the curative effect, judging prognosis, for prevent disease provides foundation; And instruct clinical application and carry out clinic study.Therefore haemocyte check (being routine blood test) becomes first of three large routine inspections in clinical examination (routine blood test, routine urinalysis, just conventional), and its clinical practice is also extensive.After nineteen fifty-three Mr. Ku Erte invention electrical impedance method blood-counter system, various automatic blood cell analysers are come out one after another, and the blood cell analysis technology is developed rapidly.
Generally by commercially available cellanalyzer, through the specific people, carry out under given conditions blood cell analysis at present.And traditional cellanalyzer is bulky, expensive, complicated operation, need the special messenger to use and carry out periodic maintenance, and the measurement reagent price supporting with it is more expensive, therefore generally be applicable to more, the more concentrated hospital inspection section office of test samples, and it is usually less to be unsuitable for sample size, and the different medical units such as village's clinic that disperse very much on time dimension.
Therefore be badly in need of developing and be applicable to Site Detection, emergency analysis, domestic. applications and primary care, the cellanalyzer of portability, simple to operateization, the just-in-time of reporting the result, to overcome tradition check kind equipment shortcoming, and meet the demand of different medical unit to measuring means.
Early 1990s, the Manz of Switzerland and Widmer propose take micro electronmechanical process technology first as basic micro-full analytical system (miniaturized total analysis systems, μ TAS).Its objective is the function of assay laboratory is transferred in portable analytical equipment, even be integrated on the chip of heart size, to adapt to the demand for development of analytical instrument microminiaturization, integrated, portability, robotization, for advantage has been created in the realization of " individualizing ", " family oriented " assay laboratory.
Micro-fluidic chip (microfluidic chip) is most active field and frontier development in μ TAS, and has been widely used in the fields such as immunoassay, DNA analysis, analysis of protein, PCR, cell sorting and single cell analysis.Carry out cell research and more and more be subject to people's attention on micro-fluidic chip, be applied to the aspects such as the interactional research of constituent analysis in cell cultivation, cell count and category filter, born of the same parents, molion and cell and single cell analysis.The microscale effect of micro-fluidic chip and be easy to integrated, make it there are the characteristics such as the sample reagent consumption is little, reaction velocity is fast, be the ideal platform of quick, efficient, the low-cost medical diagnosis on disease technology of development and new equipment, and show application potential preferably aspect medical diagnosis on disease.
Summary of the invention
The invention provides a kind of blood cell analysis chip, analyser and analytical approach, be intended to solve that existing cellanalyzer is bulky, expensive, complicated operation, the technical matters that needs the special messenger to use and carry out periodic maintenance.
Technical scheme provided by the invention is: a kind of blood cell analysis chip comprises: leucocyte/hemoglobin analysis chip and red blood cell/blood platelet analysis chip; Described leucocyte/hemoglobin analysis chip comprises four hive off cell analysis chip and basophilic granulocyte analysis chips; The described four cell analysis chips that hive off utilize chemical dyeing method, electrical impedance technology and optical absorption method to carry out cell classification, and adopt colorimetric determination haemoglobin, described basophilic granulocyte analysis chip to adopt biological dissolution technology, impedance bioelectrical measurement method and threshold value boundary mark method to carry out white blood cell count(WBC) and basocyte detection; Described red blood cell/blood platelet analysis chip adopts electrical impedance technology, boundary mark technology for red blood cell and platelet count.
Technical scheme of the present invention also comprises: described four hive off on analysis chip is provided with liquid storage tank, waste liquid pool, detection zone, colorimetric pool and quantitative assembly, described liquid storage tank is for the storage of blood sample and detect reagent, described waste liquid pool is for storing the blood sample through detecting, described detection zone carries out the electrical impedance detection while for the leucocyte that makes blood sample, passing through detection zone and optical absorption method detects, by absorptance and cell size difference, the blood sample detected is classified, described colorimetric pool adopts the colorimetric determination content of hemoglobin, the detection mode of described detection content of hemoglobin is for colorimetric under the wavelength of 530-550nm and measure the absorbance of blood sample, described quantitative assembly is for making the interior corresponding fluids of each liquid storage tank quantitative.
Technical scheme of the present invention also comprises: be provided with liquid storage tank, waste liquid pool, detection zone and quantitative assembly on described basophilic granulocyte analysis chip, described liquid storage tank is for the storage of blood sample and detect reagent, described waste liquid pool is for storing the blood sample through detecting, described detection zone adopts electrical impedance technology to be detected according to cell size for the cell of blood sample during through detection zone, calculate the Effective Numerical of leucocyte bare nucleus and basophilic granulocyte by electronic impulse size and number, described quantitative assembly is for making the interior corresponding fluids of each liquid storage tank quantitative.
Technical scheme of the present invention also comprises: on described red blood cell/blood platelet analysis chip, be provided with liquid storage tank, waste liquid pool, detection zone and quantitative assembly, described liquid storage tank detects reagent and blood sample for storing, described waste liquid pool is for storing the blood sample through detecting, when passing through detection zone, the cell of blood sample adopt electrical impedance technology to be detected according to cell size, according to the height of pulse, red blood cell and blood platelet are counted respectively, described quantitative assembly is for making the interior corresponding fluids of each liquid storage tank quantitative, wherein, described red blood cell and enumeration of thrombocytes mode are: the measurement by impulse magnitude determines cell volume, number by recording impulse obtains Cytometric result, difference setting threshold according to blood platelet and erythrocyte volume, to be defined as red blood cell higher than the pulse signal of threshold value, to be defined as blood platelet lower than the pulse signal of threshold value, and pass through the number of produced electronic impulse and big or smallly carry out red blood cell and enumeration of thrombocytes and measure analysis.
Technical scheme of the present invention also comprises: the material that described leucocyte/hemoglobin analysis chip and red blood cell/blood platelet analysis chip adopts comprises quartz, glass, monocrystalline silicon or macromolecule polymeric material; Described liquid storage tank sample introduction adopts Micropump, electrokinetic injection, positive pressure to drive sample introduction, Ngatively pressurized sampling or electric osmose sample introduction various ways.
Another technical scheme provided by the invention is: a kind of cellanalyzer, comprise blood cell analysis chip as claimed in claim 1, detecting unit and signal processing system, described blood cell analysis chip, detecting unit is connected successively with signal processing system, described blood cell analysis chip is for carrying haemocyte, described detecting unit is irradiated haemocyte for detection zone is applied to laser beam, described signal processing system applies steady current to the upper and lower two ends of detection zone, detect each cell and produce electronic impulse proportional to cell volume, according to the threshold value of setting, distinguish leucocyte bare nucleus and basophilic granulocyte, provide total white blood cells and basophilic granulocyte Effective Numerical accurately, or described signal processing system adopts absorptivity measurement cell to cross dye levels.
The another technical scheme that the present invention takes is: a kind of blood cell analysis method comprises:
Step a: utilize chemical staining, electrical impedance technology and optical absorption method to be classified to cell, utilize the colorimetric determination content of hemoglobin;
Step b: adopt biological dissolution technology, electrical impedance method and threshold value boundary mark method to detect basophilic granulocyte;
Step c: adopt electrical impedance technology, boundary mark technology for detection red blood cell and blood platelet.
Technical scheme of the present invention also comprises: described step a comprises: anticoagulation, coloring agent sample introduction respectively arrive corresponding liquid storage tank A1, A2, blood carries out the haemolysis processing to destroy the RBC/PLT/BASO cell, and the special particle to monocytic primary granule, eosinophil and neutrophil leucocyte carries out dyeing in various degree, simultaneously cell membrane is carried out in various degree painted; The sheath fluid sample introduction is to liquid storage tank B1, through the blood sample of chemical staining under the effect of sheath fluid stream, leucocyte is in line and, successively by detection zone S1 and colorimetric pool E, according to absorptance and cell size difference, the blood sample detected is classified, and the colorimetric determination content of hemoglobin.
Technical scheme of the present invention also comprises: described step b comprises: the anticoagulation sample introduction is to liquid storage tank A1, basophilic granulocyte dilution sample introduction is to liquid storage tank A3, quantitatively anticoagulation and dilution flow out and mix from liquid storage tank separately, the sheath fluid sample introduction is to liquid storage tank B2, treated blood sample is under the effect of sheath fluid stream, leucocyte is in line and passes through successively detection zone S2, the upper and lower two ends of detection zone S2 apply steady current, detect and flow into waste liquid pool W2 through electrical impedance, according to Threshold through the signal processing system Treatment Analysis, distinguish leucocyte bare nucleus and basophilic granulocyte, provide total white blood cells and basophilic granulocyte Effective Numerical accurately.
Technical scheme of the present invention also comprises: in described step c, the anticoagulation sample introduction is to liquid storage tank A4, the sheath fluid sample introduction is to liquid storage tank B3, anticoagulation is under the sheath fluid effect, haemocyte is in line and passes through successively detection zone S3, detect and flow into waste liquid pool W3 through electrical impedance, described red blood cell and enumeration of thrombocytes mode are: the measurement by impulse magnitude determines cell volume, and the number by recording impulse obtains Cytometric result; Difference setting threshold according to blood platelet and erythrocyte volume, to be defined as red blood cell higher than the pulse signal of threshold value, to be defined as blood platelet lower than the pulse signal of threshold value, and pass through the number of produced electronic impulse and big or smallly carry out red blood cell and enumeration of thrombocytes and measure analysis.
Technical scheme of the present invention has following advantage or beneficial effect: blood cell analysis chip, analyser and the analytical approach of the embodiment of the present invention are carried out somatotype to haemocyte in anticoagulation, have simple in structure, volume is little, cost is low, easy to operate, easy care, easily transportation, chip are the advantage such as discardable by mistake, the demand for development that meets analytical instrument microminiaturization, integrated and portability, be applicable to the uses such as hospital, clinic, community and individual family.
Embodiment
In order to make purpose of the present invention, technical scheme and advantage clearer, below in conjunction with drawings and Examples, the present invention is further elaborated.Should be appreciated that specific embodiment described herein, only in order to explain the present invention, is not intended to limit the present invention.
Referring to Fig. 1, is the structural representation of the blood cell analysis chip of the embodiment of the present invention.The blood cell analysis chip of the embodiment of the present invention comprises leucocyte/hemoglobin analysis chip and red blood cell/blood platelet analysis chip.
Described leucocyte/hemoglobin analysis chip comprises: four hive off cell analysis chip and basophilic granulocyte analysis chips.Refer to Fig. 2, Fig. 2 has disclosed four structural representations that hive off the cell analysis chip of the blood cell analysis chip of the embodiment of the present invention.Four hive off on the cell analysis chip is provided with liquid storage tank, waste liquid pool, detection zone, colorimetric pool and quantitative assembly.Described liquid storage tank is for the storage of blood sample and detect reagent; Described waste liquid pool is for storing the blood sample through detecting; Described detection zone carries out the electrical impedance detection while for the leucocyte that makes blood sample, passing through detection zone and optical absorption method detects, and by absorptance and cell size difference, the blood sample detected is classified; Described colorimetric pool is for detection of the concentration of haemoglobin; Described quantitative assembly is for making the interior corresponding fluids of each liquid storage tank quantitative.In embodiments of the present invention, liquid storage tank arranges at least one, when carrying out white blood cell count(WBC), anticoagulation (being the most frequently used in visiting work and one of most important anti-coagulants and reagent) sample introduction is to the A1 liquid storage tank, coloring agent (contains hemolytic agent and dyestuff, 100-150mOsm/kg or 800-1500mOsm/kg, the solution that pH is 2-8) sample introduction is to the A2 liquid storage tank, makes anticoagulation and coloring agent flow out and realize mixing from liquid storage tank separately.The effect of this process is: 1. blood carries out the haemolysis processing to destroy the RBC/PLT/BASO cell, and fragment is much smaller than leucocyte; 2. the special particle of monocytic primary granule, eosinophil and neutrophil leucocyte carried out to dyeing in various degree, the film (cell membrane, nuclear membrane, membrana granulosa) of cell is also carried out in various degree painted simultaneously; 3. the form of fixed cell, make it keep state of nature.The sheath fluid sample introduction is to the B1 liquid storage tank, and dyed quantitative blood sample is under quantitative sheath fluid effect, and leucocyte is in line and, one by one by the narrow structures (upper and lower two ends apply steady current) of detection zone S1 middle and lower reaches, detects and the inflow downstream through electrical impedance.The electrolytic solution oozed than grade due to the conductive properties of haemocyte is low, when haemocyte passes through narrow structures, narrow structures place voltage changes, the signal receiver of corresponding electrical impedance technology setting will receive a pulse signal, the size of this pulse signal depends on the blood cell volume size by narrow structures, the number of pulse signal depends on by the haemocyte of narrow structures how many, thereby how many and size obtains the cell volume measurement result according to pulse signal.Because lymphocyte, monocyte, neutrophil leucocyte and eosinophil are different to the coloring degree of coloring agent, the structure of every kind of specific karyomorphism of cell and particle causes the intensity of light scattering different, produced specific absorptance, carry out the absorptivity analysis at the narrow structures place with situations such as judgement cellular morphology and contents, raji cell assay Raji information shows in the relevant position of scatter diagram the most at last simultaneously.The characteristic showed on these two analytical parameters according to every class cell, the scatter diagram of formation Two dimensional Distribution.Can complete leucocyte medium size lymphocyte, monocyte, neutrophil leucocyte and acidophil group's classification in this detection zone.Simultaneously, RBC(Red Blood Cell, red blood cell count(RBC), refer to mean constant of red blood cell contained in unit volume blood) in destroyed, discharge haemoglobin, haemoglobin and hemolytic agent (haemoglobin detected components and erythrocyte hemolysis agent, hemolytic agent is hypotonic, acid, the solution that contains appropriate wetting agent, red blood cell in the rapid dissolved destruction blood of energy, discharge haemoglobin and form with it stable compound) reaction formation haemoglobin dervative, enter the haemoglobin test macro, be colorimetric pool E, and under the specific wavelength of 530-550nm colorimetric, in the variation of absorbance and liquid, the content of haemoglobin is proportional, can record the concentration of haemoglobin thus.
Refer to Fig. 2, Fig. 2 has disclosed the structural representation of basophilic granulocyte analysis chip of the blood cell analysis chip of the embodiment of the present invention.Be provided with liquid storage tank, waste liquid pool, detection zone and quantitative assembly on the basophilic granulocyte analysis chip of the embodiment of the present invention.Liquid storage tank is for the storage of blood sample and detect reagent, and waste liquid pool is for storing the blood sample through detecting, and detection zone is for calculating the Effective Numerical of leucocyte bare nucleus and basophilic granulocyte.Wherein, liquid storage tank quantity is at least one, is respectively used to store anticoagulation, basophilic granulocyte dilution and sheath fluid.In embodiment of the present invention, the detailed process that the basophilic granulocyte analysis chip is detected is: the anticoagulation sample introduction is to the A1 liquid storage tank, basophilic granulocyte dilution (hemolytic agent, 100-150mOsm/kg or 800-1500mOsm/kg, the solution that pH is 2-8) sample introduction is to the A3 liquid storage tank, quantitatively anticoagulation and quantitatively hemolytic agent flow out and realize mixing from liquid storage tank separately, lysed erythrocyte, because basophilic granulocyte has resistance to acid, can keep complete form, and other leucocyte endochylemas overflow, become the bare nucleus state.The sheath fluid sample introduction is to the B2 liquid storage tank, treated blood sample is under quantitative sheath fluid effect, leucocyte in blood is in line and the aperture by detection zone S2 middle and lower reaches (upper and lower two ends apply steady current) one by one, detect (being that each cell results from the proportional electronic impulse of cell volume) and flow into waste liquid pool W2 through electrical impedance, take the cell volume size as horizontal ordinate drafting WBC/BASO histogram.Through the signal processing system Treatment Analysis, distinguish leucocyte bare nucleus and basophilic granulocyte according to Threshold, provide total white blood cells and basophilic granulocyte Effective Numerical more accurately.In embodiment of the present invention, the basophilic granulocyte analysis chip adopts biological dissolution technology and electrical impedance technology to detect basophilic granulocyte, further improves and detects performance.
In Fig. 2, quantitatively assembly F1 to F8 is for making in each liquid storage tank corresponding fluids quantitative.
Referring to Fig. 3, is the structural representation of the red blood cell of the blood cell analysis chip of the embodiment of the present invention/blood platelet analysis chip.Be provided with liquid storage tank, waste liquid pool, detection zone and quantitative assembly on red blood cell/blood platelet analysis chip.Liquid storage tank is for the storage of blood sample and detect reagent; Waste liquid pool is for storing the blood sample through detecting; Detection zone adopts electrical impedance technology to be detected according to cell size for the cell of blood sample during through detection zone, according to the height of pulse, red blood cell and blood platelet is counted respectively; Quantitatively assembly is for making the interior corresponding fluids of each liquid storage tank quantitative.A kind of concrete embodiment of the present invention is: under the quantitative effect of quantitative assembly F9 and F10, the anticoagulation sample introduction is to liquid storage tank A4, sheath fluid (osmotic pressure 250-300mOsm/kg, the isotonic solution of pH7-7.4) sample introduction is to liquid storage tank B3, quantitatively anticoagulation is under quantitative sheath fluid effect, unicellular in blood is in line and one by one by detection zone S3, through electrical impedance, detects and flows into waste liquid pool W3.Because blood platelet and erythrocyte volume have obvious difference, setting threshold, according to the height of pulse, RBC and PLT are counted respectively, to be defined as red blood cell higher than the pulse signal of threshold value, otherwise be blood platelet, thereby according to pulse signal, how many and size is carried out red blood cell/platelet count and measure analysis, and wherein the quantity of leucocyte impact can be ignored.
Sheath stream technology is specially: for fear of cell in testing process, occur side by side or lateral flow district after testing, and the phenomenons such as cell backflow, turbulent flow or eddy current are brought the detection error, adopt sheath stream technology, be that the cell suspension sample is under the effect of swiftly flowing sheath fluid lateral compression effect, form the similar compression flow form that enters the sheath shape, guarantee the coated lower stream of cells that form single arrangement of sample cell at sheath fluid, pass through successively detection zone; Sheath stream technology can be applicable to two kinds of cell count principles: one is theory of electrical impedance, and cell count is carried out in the sensitizing range of sheath circulation small holes; Another kind is the laser counting principle, and cell liquid stream chamber is longer, with laser vertical, intersects, and laser beam produces light scattering after to each cell irradiation of flowing through, and utilizes this principle to carry out cell count; The boundary mark technology of floating is: because various intercellular boundaries can be with the cell actual size left or move right, therefore be called the boundary mark technology of floating; In normal specimen, red blood cell and volume of platelets differ larger, generally by red blood cell and hematoblastic boundary due to 35fl, large is red blood cell, little is blood platelet, also has to take that 30fl or 20fl be boundary; But under some pathologic condition, may there is large blood platelet to surpass the 35fl boundary, cause blood platelet to leak meter and make Lower result; Otherwise, if erythrocyte volume (as hypoferric anemia or Mediterranean property anaemia) less than normal may be blood platelet by part microcyte error count, make platelet count higher; For result accurate, calculating instrument utilizes computing machine to find the histogram minimum point between 5-35fl, be decided to be red blood cell and hematoblastic boundary with this, histogrammic boundary mark can be made corresponding adjustment automatically according to the variation of cell mass, can make thus counted numerical value tally with the actual situation.
In embodiments of the present invention, the liquid storage tank sample introduction adopts Micropump, electrokinetic injection, positive pressure to drive the various ways such as sample introduction, Ngatively pressurized sampling or electric osmose sample introduction, the blood cell analysis chip can be made by materials such as quartz, glass, monocrystalline silicon, high molecular polymerizations, such as polymetylmethacrylate, polydimethylsiloxane or polycarbonate etc.; Can be by adding the suitable modes such as adjuvant to alleviate or to avoid the chip microchannel surface to adsorb haemocyte in chip microchannel modifying inner surface, chip material modification or solution; Simultaneously, the present invention, except being applied to medical industry, is equally applicable to relate in the diameter of measurement of species particle or liquid that atomic quantity is carried out quantitatively and the industry such as analysis qualitatively, for example, in physico-chemical analysis in pure water, measure the content of its impurity and bacterium; Or the measurement of the degree of purity of various industrial high purity liquid or demarcation etc.
Referring to Fig. 4, is the structural representation of the cellanalyzer of the embodiment of the present invention.The cellanalyzer of the embodiment of the present invention comprises blood cell analysis chip as described in Figure 1, detecting unit and signal processing system, described blood cell analysis chip, detecting unit is connected successively with signal processing system, described blood cell analysis chip is for carrying haemocyte, described detecting unit is irradiated haemocyte for detection zone is applied to laser beam, described signal processing system applies steady current to the upper and lower two ends of detection zone, detect each cell and produce electronic impulse proportional to cell volume, according to the threshold value of setting, distinguish leucocyte bare nucleus and basophilic granulocyte, provide total white blood cells and basophilic granulocyte Effective Numerical accurately, or described signal processing system adopts absorptivity measurement cell to cross dye levels.The cellanalyzer of the embodiment of the present invention is except being applied to medical industry, in every diameter that relates to requirement measurement of species particle and liquid, atomic quantity carries out quantitatively and qualitatively analyzing, can measure with this instrument, be that this instrument can develop into laser particle size analyzer, for example, in physico-chemical analysis in pure water, measure the content of its impurity and bacterium; The measurement of the degree of purity of various industrial high purity liquid, demarcate etc., to microelectronic component, and the production of integrated circuit, quality control, pharmaceutical industry and chemical industry, Food Hygiene Surveillances etc. have realistic meaning.
Referring to Fig. 5, is the process flow diagram of the blood cell analysis method of the embodiment of the present invention.The blood cell analysis method of the embodiment of the present invention comprises:
Step 100: utilize chemical staining, electrical impedance technology and optical absorption method to be classified to cell, utilize the colorimetric determination content of hemoglobin;
In step 100, referring to Fig. 2, anticoagulation, coloring agent difference sample introduction are to liquid storage tank A1, A2, blood carries out the haemolysis processing to destroy the RBC/PLT/BASO cell, and the special particle to monocytic primary granule, eosinophil and neutrophil leucocyte carries out dyeing in various degree, simultaneously cell membrane is carried out in various degree painted; The sheath fluid sample introduction is to liquid storage tank B1, through the blood sample of chemical staining under the effect of sheath fluid stream, leucocyte is in line and, successively by detection zone S1 and colorimetric pool E, according to absorptance and cell size difference, the blood sample detected is classified, and the colorimetric determination content of hemoglobin.
Step 200: adopt biological dissolution technology, electrical impedance method and threshold value boundary mark method to detect basophilic granulocyte;
In step 200, referring to Fig. 2, the anticoagulation sample introduction is to liquid storage tank A1, basophilic granulocyte dilution sample introduction is to liquid storage tank A3, quantitatively anticoagulation and dilution flow out and mix from liquid storage tank separately, the sheath fluid sample introduction is to liquid storage tank B2, treated blood sample is under the effect of sheath fluid stream, leucocyte is in line and passes through successively count detection district S2, the upper and lower two ends of detection zone S2 apply steady current, detect (being that each cell produces electronic impulse proportional to cell volume) through electrical impedance and flow into waste liquid pool W2, according to Threshold through the signal processing system Treatment Analysis, distinguish leucocyte bare nucleus and basophilic granulocyte, provide total white blood cells and basophilic granulocyte Effective Numerical accurately.
Step 300: adopt electrical impedance technology, boundary mark technology for detection red blood cell and blood platelet.
In step 300, referring to Fig. 3, the anticoagulation sample introduction is to liquid storage tank A4, the sheath fluid sample introduction is to liquid storage tank B3, anticoagulation is under the sheath fluid effect, and haemocyte is in line and, successively by count detection district S3, detects and inflow waste liquid pool W3 through electrical impedance, described red blood cell and enumeration of thrombocytes mode are: the measurement by impulse magnitude determines cell volume, and the number by recording impulse obtains Cytometric result; Difference setting threshold according to blood platelet and erythrocyte volume, to be defined as red blood cell higher than the pulse signal of threshold value, to be defined as blood platelet lower than the pulse signal of threshold value, and pass through the number of produced electronic impulse and big or smallly carry out red blood cell and enumeration of thrombocytes and measure analysis.
Blood cell analysis chip, analyser and the analytical approach of the embodiment of the present invention are carried out somatotype to haemocyte in anticoagulation, have simple in structure, volume is little, cost is low, easy to operate, easy care, easily transportation, chip are the advantage such as discardable by mistake, the demand for development that meets analytical instrument microminiaturization, integrated and portability, be applicable to the uses such as hospital, clinic, community and individual family.
The foregoing is only preferred embodiment of the present invention, not in order to limit the present invention, all any modifications of doing within the spirit and principles in the present invention, be equal to and replace and improvement etc., within all should being included in protection scope of the present invention.