CN113820286A - Chip for calibrating DNA ploidy analysis system and calibration method - Google Patents
Chip for calibrating DNA ploidy analysis system and calibration method Download PDFInfo
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- CN113820286A CN113820286A CN202111135749.3A CN202111135749A CN113820286A CN 113820286 A CN113820286 A CN 113820286A CN 202111135749 A CN202111135749 A CN 202111135749A CN 113820286 A CN113820286 A CN 113820286A
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Abstract
The invention discloses a chip for calibrating a DNA ploid analysis system and a calibration method. The chip comprises a glass slide, wherein a plurality of functional areas are divided on the glass slide, the functional areas comprise one or more of a dyeing reference area, a reagent reference area and an equipment calibration area according to application requirements, the dyeing reference area is provided with a display material for displaying the dyeing effect presented by a correct dyeing method, the reagent reference area is provided with a standard reagent for displaying the image effect presented by a qualified reagent, and the equipment calibration area is used for monitoring and calibrating the IOD value of the system. The invention can be used for displaying whether the dyeing process is correct or not and whether the used reagent is qualified or not, has high standardization degree and can carry out quality control on the DNA dyeing effect.
Description
Technical Field
The invention relates to the technical field of medical instruments, in particular to a chip for calibrating a DNA ploid analysis system and a calibration method.
Background
The cell DNA ploidy analysis system judges the physiological and pathological states of cells by quantitatively detecting the ploidy of genetic materials (DNA) in cell nuclei.
The working principle of the cell DNA ploidy analysis system is as follows: the cell slice, print or smear is fixed by fixing liquid and then stained by DNA specificity (Feulgen), the staining method can make DNA molecule in cell nucleus combine with dye molecule in linear proportion specificity, thereby staining the nucleus, and the cell nucleus image is classified after integrating optical density measurement by image analysis equipment.
In order to ensure that a DNA ploidy analysis system works normally, in general, calibration sheets (pork liver sheets and mouse liver sheets) are put into the DNA ploidy analysis system from the beginning of dyeing, and IOD (integrated optical density), CV (coefficient of variation) and linear relations shown by the results of system scanning are displayed, so that the performance of the DNA ploidy analysis system is evaluated. However, the evaluation of the reagent and the staining effect is often omitted due to the lack of the control sample, and thus the degree of standardization is low.
Therefore, it is necessary to provide a chip for calibrating a DNA ploidy analysis system to perform quality control on DNA staining effects and to improve the degree of standardization.
Disclosure of Invention
The invention aims to provide a chip for calibrating a DNA ploidy analysis system so as to control the quality of DNA staining effect and improve the standardization degree.
In order to achieve the purpose, the invention adopts the following technical scheme:
the chip for calibrating the DNA ploidy analysis system comprises a glass slide, wherein a plurality of functional areas are divided on the glass slide, the functional areas comprise a dyeing reference area and a reagent reference area, the dyeing reference area is provided with a display material to display the dyeing effect presented by a correct dyeing method, and the reagent reference area is provided with a standard reagent to display the image effect presented by a qualified reagent.
Preferably, the functional area further comprises a device calibration area for monitoring and calibrating the IOD value of the system.
Preferably, the device calibration area is a light transmittance graded area for detecting and calibrating integrated optical density measurement and calculation functions of the DNA ploidy analysis system device, the device calibration area is composed of a set of unequal gray scale maps, the optical density is from 0% to 100%, and the adjacent gray scale optical densities are increased in increments at the same intervals.
Preferably, the simulation area is provided with a plurality of rows of square visual fields, each visual field is provided with a plurality of circular targets, the circular targets in different visual fields have different transmittances, and the diameter of each circular target is 10 micrometers.
Preferably, the functional zone further comprises a positive control zone and a negative control zone, and the positive control zone and the negative control zone are both used for quality control of DNA staining effect, so as to avoid false negative and false positive respectively. Preferably, the display material of the staining reference area is rat liver cells or pig liver cells, and the control material of the positive control area is a hela cell line; the control material of the negative control area is human peripheral blood mononuclear cells.
The invention also discloses a calibration method for the DNA ploid analysis system, which is characterized in that a staining reference area, a reagent reference area and an equipment calibration area are arranged on a glass slide, and a display material is arranged in the staining reference area and displays the staining effect presented by a correct staining method so as to check whether the staining is abnormal; setting a standard reagent in the reagent reference area, wherein the standard reagent displays the image effect presented by the qualified reagent so as to check whether the reagent is abnormal; the equipment calibration area calibrates the IOD value of the analysis system by setting targets with different transmittances.
Preferably, the device calibration area is a device calibration area, the device calibration area is composed of a set of unequal gray scale maps, and each level of the grading area corresponds to a transmittance T measured by a spectrophotometeriThe calibration sheet is placed on a scanning device and the average gray value G of the image acquired at each level is recordediThen, there is,
in formula (II), G'iFor the calibrated gray value, T0Denotes the minimum transmittance, G0Representing the average gray value of the image corresponding to the minimum transmittance;
according to the tested grey value GiHebiaoGradation value G 'after determination'iAnd constructing a lookup table and calibrating the analysis system.
Preferably, the device calibration area is a simulation area, the simulation area is provided with a plurality of fields, each field is provided with a plurality of targets, and the transmittances of the targets in different fields are different, so that the device calibration area moves to each field when calibrating the OD value of the analysis system, images of the targets in each field are acquired, the acquired images are subjected to image segmentation, the average OD value of all the targets in each field is calculated, a list of the transmittances and the OD values is obtained, and the IOD value of the device is estimated according to the list.
Preferably, the slide glass is provided with a positive control area and a negative control area, the control material of the positive control area is a Hela cell line, the control material of the negative control area is a human peripheral blood mononuclear cell, and the quality control is performed on DNA staining through the positive control area and the negative control area to respectively eliminate false negative and false positive.
After adopting the technical scheme, compared with the background technology, the invention has the following advantages:
1. the utility model has high standardization degree, and can calibrate whether the dyeing process is correct, whether the reagent is qualified and the system IOD according to the application requirements;
2. the reagent reference area is treated by the anti-dropping sheet, so that the service cycle is long;
3. the invention is provided with the equipment calibration area, can be used for a long time, and simultaneously, the invention discloses the equipment calibration method which is efficient and accurate;
4. the equipment calibration area is made of acid and alkali resistant materials, and is not affected by subsequent DNA specificity (Feulgen) dyeing;
5. the invention is provided with a positive control area and a negative control area, and can be used for carrying out quality control on DNA staining and respectively eliminating false negative and false positive;
6. the invention is provided with a mark area so as to record chip information in a handwriting, bar code or two-dimensional code mode.
Drawings
FIG. 1 is a schematic structural view of the present invention;
FIG. 2 is a schematic view of another embodiment of the present invention;
FIG. 3 is a schematic view of another embodiment of the present invention;
FIG. 4 is a detail view of a simulation zone;
FIG. 5 is a detail view of a single field of view;
FIG. 6 is a scatter plot;
FIG. 7 is an example of a lookup table;
FIG. 8 is a graph showing the relationship between transmittance and OD value.
Description of reference numerals:
a marking region 10;
a functional zone 20, a staining reference zone 21, a reagent reference zone 22, a device calibration zone 23, a positive control zone 24, and a negative control zone 25.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
In the description of the present invention, it is to be understood that the terms "center", "length", "width", "thickness", "upper", "lower", "front", "rear", "left", "right", "vertical", "horizontal", "top", "bottom", "inner", "outer", and the like, indicate orientations and positional relationships based on those shown in the drawings, and are used only for convenience in describing the present invention and simplifying the description, but do not indicate or imply that the device or element referred to must have a particular orientation, be constructed and operated in a particular orientation, and thus, should not be construed as limiting the present invention.
In the description of the present invention, it should be noted that, unless otherwise explicitly specified or limited, the terms "mounted," "connected," and "connected" are to be construed broadly, e.g., as meaning either a fixed connection, a removable connection, or an integral connection; can be mechanically or electrically connected; either directly or indirectly through intervening media, either internally or in any other relationship. The specific meanings of the above terms in the present invention can be understood by those skilled in the art according to specific situations.
Examples
Referring to FIG. 1, the present invention discloses a chip for calibrating a DNA ploidy analysis system, which comprises a glass slide. The slide glass is divided into a marking area 10 and a functional area 20.
The marking area 10 is a frosted area, is arranged at one end of the glass slide, and records chip information in the forms of handwriting, one-dimensional codes, two-dimensional codes and the like.
The functional area 20 includes a staining reference area 21 and a reagent reference area 22, wherein the staining reference area 21 is provided with a display material for displaying the staining effect of the correct staining method, in this embodiment, the display material is rat liver cells or pig liver cells. The reagent reference area 22 is provided with a standard reagent to show an image effect exhibited by the acceptable reagent, and the reagent reference area 22 does not add the specimen and is subjected to the separation preventing treatment, processing a reagent such as APES or the like.
The functional region 20 further includes a device calibration region 23, the device calibration region 23 being used to detect and calibrate integrated optical density measurement and calculation functions of the DNA ploidy analysis system device.
Referring to fig. 1 and 2, in the present invention, the device calibration area 23 may be a light transmittance gradation area, which is composed of a set of unequal gray scale patterns with 50-256 density levels, the optical density is from 0% to 100%, and the optical density of adjacent gray scales increases at the same interval. Meanwhile, the equipment calibration area 23 is made of an acid and alkali resistant material (such as polysiloxane) and is arranged on the glass slide through photoetching or coating process to realize a gray scale image, and the influence of subsequent DNA specificity (Feulgen) dyeing can be avoided. Thus, the IOD value of the system can be quantitatively evaluated through the light transmittance graded region.
The equipment calibration area adopts the modes of photoetching, film coating and the like to place the acid-base resistant material on the glass slide, and the main photoetching process comprises the steps of glass slide cleaning and drying, bottom coating, photoresist spinning, soft drying, alignment exposure, post-drying, developing, hard drying, etching and the like; the main coating process includes cleaning glass slide, removing oil film, drying, and coating glass coating liquid.
Referring to fig. 3, the device calibration area 23 may be an analog area, and referring to fig. 4, the analog area has a plurality of sequences, wherein a sequence represents the optical density on a transparent background, B sequence represents the optical density on a black background, and C sequence represents the unpatterned optical density. Each sequence has several columns of fields in which several targets are located (9 targets in each field, each target being circular and 10 microns in diameter as shown in fig. 5), and the targets in different fields have different transmittances (e.g., increasing in units of 10 from 0-100 in column a). Thus, through the simulation region, a quantitative evaluation of the IOD value of the system can be performed.
For quality control of DNA staining, please refer to FIG. 2, the functional region 20 further comprises a positive control region 24 and a negative control region 25.
In this embodiment, the control material of the positive control area 24 is a hela cell line, which is placed on the slices with light transmittance graded area after anti-shedding treatment, and the slices can be made by sedimentation, membrane type, manual dropping, etc. Thus, when the specimen to be tested is negative and the positive control is positive, the possibility of false negative of the specimen to be tested can be eliminated.
In this embodiment, the control material of the negative control area 25 is normal human peripheral blood mononuclear cells separated by Ficoll density gradient centrifugation or the like, and the sheet preparation method may be sedimentation, membrane type, manual dropping, or the like, so that when the specimen to be tested is a positive structure and the negative control is a negative reaction, false positives due to non-specific staining can be excluded.
The use method of the invention comprises the following steps:
step 1: placing the murine or porcine hepatocytes into a staining reference zone 21; placing hela cells in the positive control zone 24; placing human peripheral blood mononuclear cells in the negative control zone 25;
step 2: the sections were stained with DNA specificity (Feulgen) along with other specimens to be tested.
The chip equipment calibration area 23 is used for equipment calibration after dyeing, and equipment performance is detected; the dyeing reference area 21 is used for displaying the dyeing effect presented by the correct dyeing method; the reagent reference area 22 is used to display the image effect exhibited by the use of the acceptable reagent; the positive control area 24 and the negative control area 25 are used to screen for false negatives and false positives, respectively.
The invention also discloses a calibration method for the DNA ploid analysis system, which is characterized in that a staining reference area, a reagent reference area and an equipment calibration area are arranged on the glass slide, and a display material is arranged in the staining reference area and displays the staining effect presented by a correct staining method so as to check whether the staining is abnormal; setting a standard reagent in the reagent reference area, wherein the standard reagent displays the image effect presented by the qualified reagent so as to check whether the reagent is abnormal; the equipment calibration area calibrates the IOD value of the analysis system by setting targets with different transmittances.
Wherein, when the equipment calibration area is a light transmittance grading area, the light transmittance grading area is composed of a group of unequal gray scale images, each grade of the grading area corresponds to a transmittance T measured by a spectrophotometeriTherefore, a total of N (50-255) sets of transmittance data; the calibration sheet is placed on a scanning device and the average gray value G of the image acquired under each grade is recordediThen, there is,
in formula (II), G'iFor the calibrated gray value, T0Denotes the minimum transmittance, G0Representing the average gray value of the image corresponding to the minimum transmittance;
according to the tested gray value Gi and the calibrated gray value G'iAnd a scatter diagram is drawn, as shown in fig. 6. Wherein the horizontal axis represents the gray value G of the testiThe ordinate represents the calibrated gray value G'i. The IOD height of the equipment can be qualitatively known according to the scatter diagram, if G'iIndicating IOD lower below the reference grey RefG, if G'iIndicating that IOD is high above a reference gray level RefG, which is the value for each point on a linear function of y ═ x.
According to the tested grey value GiAnd the calibrated gray value G'iAnd constructing a lookup table to calibrate the analysis system so as to improve the consistency of the equipment.
Look-up table according to GiAnd G'iThe formed data pairs are interpolated to form a look-up table, including but not limited to piecewise linear interpolation. FIG. 7 is example data for a look-up table. Wherein the horizontal axis represents the input of the lookup table, i.e. the original gray value of the image data, and the vertical axis represents the output of the lookup table, i.e. the corresponding gray value of the image data after applying the lookup table. The device imaging apparatus applies the look-up table after the image is acquired and subsequent calculations are calculated using the calibrated data.
When the device calibration area is the simulation area, as described above, the simulation area has several rows of views, each view has several targets, and the targets in different views have different transmittances, the device moves to each view when calibrating the OD value of the analysis system, acquires the image of each target in each view, performs image segmentation on the acquired image, calculates the average OD value of all targets in each view, obtains a list of transmittances and OD values (see fig. 8), and estimates the IOD value of the device according to the list. In fig. 8, the horizontal axis represents the transmittance (Transmission) of each circle, and the vertical axis represents the Optical Density (Optical Density) of the actually tested corresponding circle.
In summary, the quality control chip is placed in the invention from the beginning of staining, and DNA specificity (Feulgen) staining is carried out together with other slices to be tested, therefore, the invention can be used for displaying whether the staining process is correct and whether the used reagent is qualified or not and screening out false positive and false negative while calibrating the DNA ploidy analysis system.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present description refers to embodiments, not every embodiment may contain only a single embodiment, and such description is for clarity only, and those skilled in the art should integrate the description, and the embodiments may be combined as appropriate to form other embodiments understood by those skilled in the art.
Claims (9)
1. The chip for calibrating the DNA ploidy analysis system comprises a glass slide, wherein functional areas are divided on the glass slide, and is characterized in that the functional areas comprise one or more of a staining reference area, a reagent reference area and an equipment calibration area according to application requirements, the staining reference area is provided with a display material to display staining effects presented by a correct staining method, the reagent reference area is provided with a standard reagent to display image effects presented by a qualified reagent, and the equipment calibration area is used for monitoring and calibrating the IOD value of the system.
2. The chip of claim 1 for calibration of a DNA ploid analysis system, wherein: the device calibration area is a light transmittance grading area which is composed of a group of unequal gray scale graphs, the light density is from 0% to 100%, and the optical density of adjacent gray scales is increased progressively at the same interval.
3. The chip of claim 1 for calibration of a DNA ploid analysis system, wherein: the equipment calibration area is a simulation area, a plurality of rows of visual fields are arranged on the simulation area, a plurality of targets are arranged in each visual field, and the transmittance of the targets in different visual fields is different.
4. The chip of claim 1 for calibration of a DNA ploid analysis system, wherein: the functional area also comprises a positive control area and a negative control area, and the positive control area and the negative control area are used for quality control of DNA staining effect and respectively avoid false negative and false positive.
5. The chip of claim 4 for calibration of a DNA ploid analysis system, wherein: the display material of the staining reference area is rat liver cells or pig liver cells, and the control material of the positive control area is a Hela cell line; the control material of the negative control area is human peripheral blood mononuclear cells.
6. The calibration method for the DNA ploidy analysis system is characterized by comprising the following steps: setting one or more of a staining reference area, a reagent reference area and an equipment calibration area on the glass slide according to application requirements; the dyeing reference area is provided with a display material which displays the dyeing effect presented by a correct dyeing method so as to check whether the dyeing is abnormal or not; a standard reagent is arranged in the reagent reference area and displays the image effect presented by the qualified reagent so as to check whether the reagent is abnormal or not; the equipment calibration area calibrates the IOD value of the analysis system by setting targets with different transmittances.
7. The method of claim 6 for calibrating a DNA ploidy analysis system, wherein: the device calibration area is a light transmittance grading area which is composed of a group of unequal gray scale images, and each grade of the grading area corresponds to a transmittance T measured by a spectrophotometeriThe calibration sheet is placed on a scanning device and the average gray value G of the image acquired at each level is recordediThen, there is,
in formula (II), G'iFor the calibrated gray value, T0Denotes the minimum transmittance, G0Representing the average gray value of the image corresponding to the minimum transmittance;
according to the tested grey value GiAnd the calibrated gray value G'iAnd constructing a lookup table and calibrating the analysis system.
8. The method of claim 7 for calibrating a DNA ploidy analysis system, wherein: the device calibration area is a simulation area, the simulation area is provided with a plurality of visual fields, a plurality of targets are arranged in each visual field, the transmittances of the targets in different visual fields are different, the device calibration area moves to each visual field when calibrating the OD value of the analysis system, images of the targets in each visual field are obtained, the obtained images are subjected to image segmentation, the average OD value of all the targets in each visual field is calculated, a list of the transmittances and the OD values is obtained, and the IOD value of the device is estimated according to the list.
9. The method of calibrating a DNA ploidy analysis system of claim 7, wherein: a positive control area and a negative control area are arranged on the glass slide, the control material of the positive control area is a Hela cell line, the control material of the negative control area is human peripheral blood mononuclear cells, and the quality control is carried out on DNA staining through the positive control area and the negative control area to respectively eliminate false negative and false positive.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114295589A (en) * | 2021-12-31 | 2022-04-08 | 信利光电仁寿有限公司 | Method for measuring optical density of hot melt adhesive |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN114295589A (en) * | 2021-12-31 | 2022-04-08 | 信利光电仁寿有限公司 | Method for measuring optical density of hot melt adhesive |
| CN114295589B (en) * | 2021-12-31 | 2024-05-07 | 信利光电仁寿有限公司 | Method for measuring optical density of hot melt adhesive |
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