CN108426886A - A kind of detection recognition method and system of circulating tumor cell - Google Patents
A kind of detection recognition method and system of circulating tumor cell Download PDFInfo
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- 208000005443 Circulating Neoplastic Cells Diseases 0.000 title claims abstract description 68
- 238000001514 detection method Methods 0.000 title claims abstract description 65
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- 230000008054 signal transmission Effects 0.000 claims abstract description 9
- 210000004881 tumor cell Anatomy 0.000 claims description 9
- 238000005070 sampling Methods 0.000 claims description 7
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- 230000035945 sensitivity Effects 0.000 abstract description 14
- 238000012360 testing method Methods 0.000 abstract description 7
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/84—Systems specially adapted for particular applications
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N1/00—Sampling; Preparing specimens for investigation
- G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
- G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/01—Arrangements or apparatus for facilitating the optical investigation
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2201/00—Features of devices classified in G01N21/00
- G01N2201/10—Scanning
- G01N2201/104—Mechano-optical scan, i.e. object and beam moving
- G01N2201/1045—Spiral scan
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Abstract
The present invention provides a kind of detection recognition method of circulating tumor cell and systems, and light source module is used for shoot laser, and laser is focused on sample to be tested；Sample to be tested is the blood smear by filtering and non fluorescent stain；Ellipsoidal mirror is used for the laser reflection that scatters sample to be tested to the light-receiving surface of photoelectric conversion module；Photoelectric conversion module is used to the optical signal of acquisition being converted to electric signal, and by electric signal transmission to described image identification module；Picture recognition module is used to generate the image of sample to be tested according to electric signal, and the circulating tumor cell in image is identified.Since sample to be tested is the blood smear by filtering and non fluorescent stain, biologic specificity enrichment is not carried out, therefore, can not only reduce testing cost, but also the sensitivity, accuracy and detection efficiency of detection can be improved；Also, the dyeing carried out using non-fluorescence dyestuff when due to blood smear, the sensitivity of detection can be further increased.
Description
Technical field
The present invention relates to circulating tumor cell identification technology fields, more specifically to a kind of circulating tumor cell
Detection recognition method and system.
Background technology
Circulating tumor cell (circulating tumor cell, CTC) is that all kinds of tumours that are present in peripheral blood are thin
The general designation of born of the same parents derives from entity tumor lesion.Most of CTC occurs apoptosis or is swallowed after entering peripheral blood, and minority can
It escapes and develops into transfer stove, increase the mortality risk of malignant tumor patient.CTC is in 2007 by American Society of Clinical Oncology
It is included in tumor markers；Multinomial clinical research shows patient CTC quantity and metastases, individualized treatment, curative effect monitoring and pre-
It is afterwards etc. closely related.
A kind of system of existing detection identification circulating tumor cell is Cell Search systems, is based on immune magnetic
A kind of detecting instrument for semi-automation that property separation principle grows up.Cell Search systems are according to tumor cell surface
Epithelial cell adhesion molecule (Epithelial cell adhesion molecule, EpCAM) is positive and blood cell not table
Up to the characteristic of EpCAM, EpCAM antibody is coated in magnetic bead surfaces, under antigen-antibody reaction, these are rich in the EpCAM of magnetic bead
Antibody can be combined with circulating tumor cell.Wherein, the side of circulating tumor cell detection identification is carried out using Cell Search systems
Method includes:It is to be enriched with to the cell for expressing EpCAM using the magnetic bead for being coated with EpCAM antibody to prepare sample, and cell is consolidated
It is fixed, DAPI fluorescent nuclear dyes, CD45 fluorescence antibodies and CK8, CK18 and CK19 fluorescence antibody label cell is used in combination；With semi-automatic
Analysis is identified to the sample after label in four color fluorescence microscopes, to obtain the quantity of circulating tumor cell in blood.
But when carrying out the detection identification of circulating tumor cell using Cell Search systems, in the preparation of samples stage
Operating procedure is more, can lead to the loss and damage of circulating tumor cell in this way, can seriously affect sensitivity, the accuracy of detection
And detection efficiency.Further, since above method requirement after the fluorescer and antigen or antibody coupling of label for forming stabilization
Therefore conjugate can cause the activity of labeled circulating tumor cell to change, and sample surfaces are easy residual and largely dissociate
Fluorescent molecular, influence detection sensitivity.
Invention content
In view of this, the present invention provides a kind of detection recognition method of circulating tumor cell and system, to improve cycle
Sensitivity, accuracy and the detection efficiency of tumour cell detection.
To achieve the above object, the present invention provides the following technical solutions：
A kind of detection identifying system of circulating tumor cell, including light source module, ellipsoidal mirror, photoelectric conversion module
And picture recognition module；
The light source module is used for shoot laser, and the laser is focused on the sample to be tested on sample stage；It is described
Sample to be tested is the blood smear by filtering and non fluorescent stain；Plane in the sample to be tested where circulating tumor cell
It is overlapped with the plane where a focus of the ellipsoidal mirror, and the laser focuses in the focus；
The ellipsoidal mirror is used for the laser reflection that scatters the sample to be tested to the photoelectric conversion module
Light-receiving surface；The photoelectric conversion module light-receiving surface is overlapped with the plane where the ellipsoidal mirror another focus；
The photoelectric conversion module is used to the optical signal of acquisition being converted to electric signal, and by the electric signal transmission to institute
State picture recognition module；
Described image identification module is used to generate the image of the sample to be tested according to the electric signal, and to described image
In circulating tumor cell be identified.
Preferably, the light source module includes light source and object lens；
The light source is used for shoot laser；
The object lens are used to focusing on the laser into the sample to be tested on sample stage.
Preferably, the light source module further include the polarizer group that is successively set between the light source and the object lens and
Beam expanding lens；
The polarizer group includes at least two polarizing films, the laser that the polarizer group is used to be emitted the light source
Light intensity is adjusted；
The beam expanding lens is for expanding the laser, so that laser beam fills up the entrance pupil of the object lens.
Preferably, the photoelectric conversion module is photomultiplier；
The detection identifying system further includes the aperture being arranged in the photomultiplier light-receiving surface；
The laser of ellipsoidal mirror reflection be irradiated to after the aperture photomultiplier by
Smooth surface.
Preferably, further include sample stage drive module；
The sample stage drive module is for driving the sample stage and sample to be tested with constant speed along a side
It is rotated on center shaft to movement, while with constant angular speed, so as to focus on the hot spot on the sample to be tested surface in institute
Sample to be tested surface is stated to move along spiral of Archimedes；
The photoelectric conversion module is according to preset timing node sequence acquisition optical signal, to obtain equal multiple in interval
Sampled point.
Preferably, the timing node sequence is according to formula
It arrives, s is the total length of the spiral of Archimedes, and v is the speed moved in one direction, and ω is angle speed
Degree.
A kind of detection recognition method of circulating tumor cell is applied to the inspection of any one of them circulating tumor cell as above
Identifying system is surveyed, including：
Light source module shoot laser, and the laser is focused on the sample to be tested on sample stage, the sample to be tested
For the blood smear by filtering and non fluorescent stain；
The laser reflection that ellipsoidal mirror scatters the sample to be tested to photomultiplier light-receiving surface；
The optical signal of acquisition is converted to electric signal by the photomultiplier, and by the electric signal transmission to image recognition
Module；
Described image identification module generates the image of the sample to be tested according to the electric signal, and in described image
Circulating tumor cell is identified.
Preferably, further include before light source module shoot laser：
Make blood smear.
Preferably, the making blood smear includes：
Blood sample is filtered, and retains the circulating tumor cell in the blood sample；
Blood smear is made using the filtered blood sample；
The blood smear is dyed using non-fluorescence dyestuff.
Preferably, further include：
Sample stage drive module drives the sample stage and sample to be tested to be moved in one direction with constant speed, together
When rotated on center shaft with constant angular speed, so as to focus on the hot spot on the sample to be tested surface in the sample to be tested
It is moved along spiral of Archimedes on surface；
Photoelectric conversion module is according to preset timing node sequence acquisition optical signal, to obtain the equal multiple samplings in interval
Point.
Compared with prior art, technical solution provided by the present invention has the following advantages：
The detection recognition method and system of circulating tumor cell provided by the present invention, light source module shoot laser, and will
Laser focuses on the sample to be tested on sample stage.Due to the position of a focus of the position and ellipsoidal mirror of sample to be tested
Coincidence is set, the position of photomultiplier light-receiving surface is overlapped with the position of another focus of ellipsoidal mirror, therefore, ellipsoid
Speculum can be by the light-receiving surface of the laser reflection of sample to be tested scattering to photoelectric conversion module；Photoelectric conversion module adopts light-receiving surface
The optical signal of collection is converted to electric signal, and picture recognition module generates the image of sample to be tested according to electric signal, and in image
Circulating tumor cell is identified.
Since sample to be tested is the blood smear by filtering and non fluorescent stain, biologic specificity richness is not carried out
Collection, therefore, can not only reduce testing cost, but also compared with prior art, less the preparation of samples stage the step of, cycle
The loss and damage of tumour cell are all less, so as to improve the sensitivity, accuracy and detection efficiency of detection；Also, by
The dyeing carried out using non-fluorescence dyestuff when blood smear, therefore, sample surfaces will not remain largely free fluorescent molecular,
To further improve the sensitivity of detection.
Description of the drawings
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technology description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 is the structural schematic diagram of the detection identifying system of circulating tumor cell provided in an embodiment of the present invention；
Fig. 2 is the reflection schematic diagram of ellipsoidal mirror provided in an embodiment of the present invention；
Fig. 3 (a) is the schematic diagram of the sampled point of uneven distribution；
Fig. 3 (b) is the schematic diagram of equally distributed sampled point provided in an embodiment of the present invention；
Fig. 4 (a) is the schematic diagram of the scan image of sample to be tested provided in an embodiment of the present invention；
Fig. 4 (b) is the partial enlarged view of Fig. 4 (a)；
Fig. 5 is the flow chart of the detection recognition method of circulating tumor cell provided in an embodiment of the present invention.
Specific implementation mode
As described in background, the fluorescer and antigen or antibody due to Cell Search system requirements for label
Stable conjugate is formed after coupling, therefore, the activity of labeled circulating tumor cell can be caused to change, and sample surfaces
The fluorescent molecular that easy residual is largely dissociated, influences the sensitivity of detection.
Although also having researched and developed a variety of optical bio sensing technologies for exempting from fluorescent marker in the prior art, these
Sensing technology is mostly complicated, biological smear manufacture difficulty is big, detection efficiency is low, accordingly, it is difficult to promotion and application.
Based on this, the present invention provides a kind of detection identifying systems of circulating tumor cell, to overcome the prior art to exist
The above problem, including light source module, ellipsoidal mirror, photoelectric conversion module and picture recognition module；
The light source module is used for shoot laser, and the laser is focused on to the sample to be tested surface on sample stage；Institute
It is the blood smear by filtering and non fluorescent stain to state sample to be tested；The position of the sample to be tested is reflected with the ellipsoid
The position of one focus of mirror overlaps；The ellipsoidal mirror is used for the laser reflection that scatters the sample to be tested to described
The light-receiving surface of photoelectric conversion module；Another coke of the position of the photoelectric conversion module light-receiving surface and the ellipsoidal mirror
The position of point overlaps；The photoelectric conversion module is used to the optical signal of acquisition being converted to electric signal, and the electric signal is passed
Transport to described image identification module；Described image identification module is used to generate the figure of the sample to be tested according to the electric signal
Picture, and the circulating tumor cell in described image is identified.
The present invention also provides a kind of detection recognition methods of circulating tumor cell, which is characterized in that is applied to institute as above
The detection identifying system for the circulating tumor cell stated, including：
Light source module shoot laser, and the laser is focused on the sample to be tested on sample stage, the sample to be tested
For the blood smear by filtering and non fluorescent stain；The laser reflection that ellipsoidal mirror scatters the sample to be tested is to light
The light-receiving surface of electric multiplier tube；The optical signal of acquisition is converted to electric signal by the photomultiplier, and by the electric signal transmission
To picture recognition module；Described image identification module generates the image of the sample to be tested according to the electric signal, and to described
Circulating tumor cell in image is identified.
The detection recognition method and system of circulating tumor cell provided by the invention, since sample to be tested is only by filtering
With the blood smear of non fluorescent stain, biologic specificity enrichment is not carried out, therefore, can not only reduce testing cost, but also
Compared with prior art, less the preparation of samples stage the step of, the loss and damage of circulating tumor cell are all less, so as to
To improve sensitivity, accuracy and the detection efficiency of detection；Also, the dye carried out using non-fluorescence dyestuff when due to blood smear
Therefore color does not interfere with the activity of labeled circulating tumor cell, and sample surfaces will not remain largely free fluorescence
Molecule, to further improve the sensitivity of detection.
It is core of the invention thought above, to keep the above objects, features and advantages of the present invention more obvious easily
Understand, following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention is clearly and completely retouched
It states, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.Based on the present invention
In embodiment, every other implementation obtained by those of ordinary skill in the art without making creative efforts
Example, shall fall within the protection scope of the present invention.
An embodiment of the present invention provides a kind of detection identifying systems of circulating tumor cell, are mainly used in peripheral blood and follow
The detection of ring tumour cell identifies that the detection identifying system is that ellipsoid is copolymerized burnt details in a play not acted out on stage, but told through dialogues rotating scan imaging system, such as Fig. 1 institutes
Show, including light source module 1, ellipsoidal mirror 2, photoelectric conversion module 3 and picture recognition module (not shown).
Wherein, light source module 1 is used for shoot laser, and laser is focused on the sample to be tested 4 on sample stage.This is to be measured
Sample 4 is the blood smear by filtering and non fluorescent stain.Plane in the sample to be tested 4 where circulating tumor cell with it is ellipse
Plane where one focus of spherical reflector 2 overlaps, and laser focuses in the focus, the light-receiving surface of opto-electronic conversion mould 3
It is overlapped with the plane where ellipsoidal mirror 2 another focus.
Ellipsoidal mirror 2 is used to reflex to the light that sample to be tested 4 scatters the light-receiving surface of photoelectric conversion module 3；Light
Electric conversion module 3 is used to the optical signal of acquisition being converted to electric signal, and by electric signal transmission to picture recognition module 4；Image
Identification module 4 is used to generate the image of sample to be tested 4 according to electric signal, and the circulating tumor cell in image is identified.
As shown in Fig. 2, in rectangular coordinate system, ellipsoidal mirror 2 can be expressed as：(1), institute
With the spacing between 2 two conjugate focuses of ellipsoidal mirror can be expressed as：Root
According to geometric optical theory, a focus F1The light sent out can focus on another coke after the reflection of ellipsoidal mirror 2
Point F2On, therefore, sample to be tested 4 is placed on to a focus F of ellipsoidal mirror 21On, from F1The scattering light of reflection passes through again
It can be in another focus F after being reflected by ellipsoidal mirror 22It assembles, i.e., is assembled on 3 light-receiving surface of opto-electronic conversion mould.
Since sample to be tested 4 is transparent, therefore, it is necessary to eliminate the shot noise from 4 back side of sample to be tested.Due to right
For ellipsoidal mirror 2, the position of ideal image only has focus point a F1 and F2, therefore, passes through precision in the present invention
The height and hot spot for adjusting sample stage fall the position on 4 surface of sample to be tested so that the circulating tumor cell in sample to be tested 4
Place plane is overlapped with plane where focus F1, and the hot spot of laser is focused on focus F1, to be measured so as to avoid
The scattered signal at 4 back side of sample is focused, and then the purpose of Single Slice Mode may be implemented.
In the present embodiment, as shown in Figure 1, light source module 1 includes light source 10 and object lens 11, is successively set on light source 10 and object
Polarizer group 12 between mirror 11 and beam expanding lens 13.Light source 10 is used for shoot laser；Object lens 11 are used to laser focusing on sample
4 surface of sample to be tested on platform.Polarizer group 12 includes at least two polarizing films, what which was used to be emitted light source 10
The light intensity of laser is adjusted；Beam expanding lens 13 is for expanding laser, so that laser beam fills up the incident light of object lens 11
Pupil.
Preferably, the light source 10 in the present embodiment is laser diode, and the laser of outgoing is the light beam of 405nm wavelength.
Object lens 11 are the object lens of 3.47 centimetres of focal lengths, and the diameter for the hot spot for focusing on 4 surface of sample to be tested is about 20 microns.Polarization
Piece group 12 includes two polarizing films, and the laser for being incident on beam expanding lens 13 can be adjusted by adjusting the angle between two polarizing films
Light intensity.
Further, in this embodiment photoelectric conversion module 3 be photomultiplier.The detection identifying system further includes setting
Set the aperture 5 in photomultiplier light-receiving surface；The laser that ellipsoidal mirror 2 reflects is irradiated to after aperture 5
The light-receiving surface of photomultiplier.Preferably, the hole diameter of aperture 5 is 5 μm.
In the present embodiment, space filtering is carried out to light using aperture 5, can exclude particle in space scatter with
And the high-frequency noise that the other impurities scattering in light path introduces, so as to improve sampling efficiency and the scanning of detection identifying system
Resolution ratio.
In addition, the detection identifying system in the present embodiment further includes sample stage drive module (not shown).The sample
Platform drive module is for driving the sample to be tested 4 on sample stage and sample stage to be moved in one direction with constant speed v, together
When rotated on center shaft with constant angular velocity omega, so as to focus on the hot spot on 4 surface of sample to be tested on 4 surface of sample to be tested
It is moved along spiral of Archimedes, i.e., each sampled point on 4 surface of sample to be tested constitutes spiral of Archimedes.
When the laser that light source 10 is emitted is focused at a position of sample to be tested 4, what picture recognition module 4 obtained is
The image of 4 one sampled points of sample to be tested, when focusing on the hot spot on 4 surface of sample to be tested on 4 surface of sample to be tested along A Ji meter
When moral helix moves, picture recognition module 4 can obtain the image of multiple sampled points, and be obtained according to the image of multiple sampled points
It obtains entire sample to be tested 4 and is the image of blood smear, and then the circulating tumor cell in entire sample to be tested 4 can be known
Not, to obtain the quantity of the circulating tumor cell in entire sample to be tested 4.
It should be noted that if sample stage drive module only drive the sample to be tested 4 on sample stage and sample stage with
Constant speed v is moved in one direction, while being rotated on center shaft with constant angular velocity omega, then, with sample stage
Movement, as shown in Figure 3a, the interval between adjacent two sampled point a can be increasing, can cause detection zone sample
Inhomogeneities, to reduce the accuracy of testing result.
Assuming that indicating the time with t, then the distance between two adjacent sampled point a Δ s can be expressed as：The total length s for the spiral of Archimedes that multiple sampled point a are constituted can be expressed as：If being previously determined the overall length of spiral of Archimedes
S and constant time interval is spent, it is possible to obtain a sampling time sequence node according to formula (4).Photoelectricity turns
Mold changing block 3 is sampled according to this timing node sequence, so that it may which to obtain, interval as shown in Figure 3b is equal, is evenly distributed
Multiple sampled point a.
Preferably, sample stage is persistently rotated in one direction with the frequency of constant 1000 hertz, to acquire
To a series of scattered signals changed over time.By high-speed rotating disk, static schema is converted to high-frequency signal, to effectively
Ground avoids the 1/f noise in static schema.For example, being scanned to blank sample, backscatter noise can be reduced to
0.0046v。
In the present embodiment, before the detection identification for carrying out circulating tumor cell using detection identifying system, need first to make
Make sample to be tested 4 i.e. blood smear.Make blood smear process be：First use the makrolon membrane filtration periphery in 8 μm of apertures
Blood sample is to screen circulating tumor cell.After human peripheral blood sample is filtered, smaller red blood cell and blood platelet are filtered, compared with
Big leucocyte and circulating tumor cell stays on polycarbonate membrane.Blood smear is made using filtered peripheral blood sample
Afterwards, blood smear is dyed, specifically, blood smear is contaminated using the method for silver staining and Rui Shi Giemsa stainings
Color.
As shown in Fig. 4 (a) and 4 (b), since tumour cell volume is 3 times or more of normal cell, nucleus is big, and core is abnormal
The strange shape of shape, the color after dyeing is deeper, and therefore, after blood smear is dyed and scanned, picture recognition module 4 can be from
Circulating tumor cell is identified in the image of blood smear, and the quantity of circulating tumor cell is counted, so that basis is followed
The quantity of ring tumour cell judges metastases situation.
The detection identifying system of circulating tumor cell provided in this embodiment, due to sample to be tested only by filtering and it is non-
The blood smear of fluorescent staining does not carry out biologic specificity enrichment, therefore, can not only reduce testing cost, and with it is existing
There is technology to compare, less the preparation of samples stage the step of, the loss and damage of circulating tumor cell are all less, so as to carry
Sensitivity, accuracy and the detection efficiency of high detection；Also, the dyeing carried out using non-fluorescence dyestuff when due to blood smear,
Therefore, the activity of labeled circulating tumor cell is not interfered with, and sample surfaces will not remain largely free fluorescence point
Son, to further improve the sensitivity of detection.
In the present embodiment, by using ellipsoidal mirror and aperture as the burnt details in a play not acted out on stage, but told through dialogues scanning imagery of the ellipsoid of core copolymerization
System carries out the detection identification of circulating tumor cell, and system structure is simple, and resolution ratio is higher, and the noise in detection process is relatively low,
Accuracy and the contrast for detecting signal are all higher；
In the present embodiment, sampled by the way of spatially uniform, rather than by the way of constant duration into
Row sampling, therefore, can make sampled point being more evenly distributed on sample to be tested, so as to improve sampling efficiency.
The embodiment of the present invention additionally provides a kind of detection recognition method of circulating tumor cell, applied to following as described above
The detection identifying system of ring tumour cell, as shown in figure 5, including：
S501：Light source module shoot laser, and the laser is focused on into the sample to be tested surface on sample stage, it is described to wait for
Sample is the blood smear by filtering and non fluorescent stain；
S502：The laser reflection that ellipsoidal mirror scatters the sample to be tested to photomultiplier light-receiving surface；
S503：The optical signal of acquisition is converted to electric signal by the photomultiplier, and the electric signal transmission is extremely schemed
As identification module；
S504：Described image identification module generates the image of the sample to be tested according to the electric signal, and to the figure
Circulating tumor cell as in is identified.
Wherein, further include before light source module outgoing laser beams：Make blood smear.
Further, the process for making blood smear includes：
Blood sample is filtered, and retains the circulating tumor cell in the blood sample；
Blood smear is made using the filtered blood sample；
The blood smear is dyed using non-fluorescence dyestuff.
Specifically, first using the makrolon membrane filtration peripheral blood sample in 8 μm of apertures to screen circulating tumor cell.Externally
After all blood samples are filtered, smaller red blood cell and blood platelet are filtered, and larger leucocyte and circulating tumor cell stay in
On polycarbonate membrane.After blood smear is made using filtered peripheral blood sample, blood smear is dyed, specifically,
Blood smear is dyed using the method for silver staining and Rui Shi Giemsa stainings.
Then, with reference to figure 1, blood smear, that is, sample to be tested 4 is placed on sample stage, adjust sample stage height and
Hot spot falls the position on 4 surface of sample to be tested so that plane where the circulating tumor cell in sample to be tested 4 and the places focus F1
Plane overlaps, and the hot spot of laser is focused on focus F1, while ensureing that the light-receiving surface of opto-electronic conversion mould 3 and ellipsoid are anti-
Plane where penetrating another focus of mirror 2 F2 overlaps.
Later, 10 shoot laser of light source, laser are incident on object lens 11, object lens 11 after polarizer group 12 and beam expanding lens 13
Laser is focused on the sample to be tested 4 on sample stage, the light that sample to be tested 4 scatters is reflexed to light by ellipsoidal mirror 2
The light-receiving surface of electric conversion module 3；The optical signal of acquisition is converted to electric signal by photoelectric conversion module 3, and extremely by electric signal transmission
Picture recognition module 4；Picture recognition module 4 is used to generate the image of 4 one sampled points of sample to be tested according to electric signal.
During detecting identification, sample stage drive module can drive sample stage and wait for test sample 4 with constant speed v
It moves, while being rotated on center shaft with constant angular velocity omega in one direction, so as to focus on the hot spot on sample to be tested 4
It is moved along Archimedian screw on 4 surface of sample to be tested；Photoelectric conversion module 3 is believed according to preset timing node sequence acquisition light
Number, to obtain the equal multiple sampled points in interval.Picture recognition module is given birth to according to the image that the electric signal of multiple sampled points generates
It is identified at the image of sample to be tested 4, and to the circulating tumor cell in image, to obtain the cycle in entire sample to be tested
The quantity of tumour cell, to be judged metastases situation according to the quantity of circulating tumor cell.
Based on this, detection recognition method provided in this embodiment further includes：
Sample stage drive module drives the sample stage to be moved in one direction with constant speed, while with constant angle
Speed rotates on center shaft, so as to focus on the hot spot on the sample to be tested surface on the sample to be tested surface along A Ji meter
Moral spiral moves；
Photoelectric conversion module is according to preset timing node sequence acquisition optical signal, to obtain the equal multiple samplings in interval
Point.
The detection recognition method of circulating tumor cell provided in this embodiment, due to sample to be tested only by filtering and it is non-
The blood smear of fluorescent staining does not carry out biologic specificity enrichment, therefore, can not only reduce testing cost, and with it is existing
There is technology to compare, less the preparation of samples stage the step of, the loss and damage of circulating tumor cell are all less, so as to carry
Sensitivity, accuracy and the detection efficiency of high detection；Also, the dyeing carried out using non-fluorescence dyestuff when due to blood smear,
Therefore, the activity of labeled circulating tumor cell is not interfered with, and sample surfaces will not remain largely free fluorescence point
Son, to further improve the sensitivity of detection.
Each embodiment is described by the way of progressive in this specification, the highlights of each of the examples are with other
The difference of embodiment, just to refer each other for identical similar portion between each embodiment.For device disclosed in embodiment
For, since it is corresponded to the methods disclosed in the examples, so description is fairly simple, related place is said referring to method part
It is bright.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest range caused.
Claims (10)
1. a kind of detection identifying system of circulating tumor cell, which is characterized in that including light source module, ellipsoidal mirror, light
Electric conversion module and picture recognition module；
The light source module is used for shoot laser, and the laser is focused on the sample to be tested on sample stage；It is described to be measured
Sample is the blood smear by filtering and non fluorescent stain；Plane in the sample to be tested where circulating tumor cell and institute
Plane where stating a focus of ellipsoidal mirror overlaps, and the laser focuses in the focus；
The ellipsoidal mirror is used for the laser reflection that scatters the sample to be tested to the light of the photoelectric conversion module
Face；The photoelectric conversion module light-receiving surface is overlapped with the plane where the ellipsoidal mirror another focus；
The photoelectric conversion module is used to the optical signal of acquisition being converted to electric signal, and by the electric signal transmission to the figure
As identification module；
Described image identification module is used to generate the image of the sample to be tested according to the electric signal, and in described image
Circulating tumor cell is identified.
2. detection identifying system according to claim 1, which is characterized in that the light source module includes light source and object lens；
The light source is used for shoot laser；
The object lens are used to focusing on the laser into the sample to be tested on sample stage.
3. detection identifying system according to claim 2, which is characterized in that the light source module further includes being successively set on
Polarizer group between the light source and the object lens and beam expanding lens；
The polarizer group includes at least two polarizing films, the light intensity for the laser that the polarizer group is used to be emitted the light source
It is adjusted；
The beam expanding lens is for expanding the laser, so that laser beam fills up the entrance pupil of the object lens.
4. detection identifying system according to claim 1, which is characterized in that the photoelectric conversion module is photomultiplier transit
Pipe；
The detection identifying system further includes the aperture being arranged in the photomultiplier light-receiving surface；
The laser of the ellipsoidal mirror reflection is irradiated to the light-receiving surface of the photomultiplier after the aperture.
5. detection identifying system according to claim 1, which is characterized in that further include sample stage drive module；
The sample stage drive module is for driving the sample stage and sample to be tested to be moved in one direction with constant speed
It is dynamic, while being rotated on center shaft with constant angular speed, so that the hot spot for focusing on the sample to be tested surface is waited for described
It is moved along spiral of Archimedes on sample surface；
The photoelectric conversion module is according to preset timing node sequence acquisition optical signal, to obtain the equal multiple samplings in interval
Point.
6. detection identifying system according to claim 5, which is characterized in that the timing node sequence is according to formulaIt obtains, s is the total length of the spiral of Archimedes, and v is institute
The speed moved in one direction is stated, ω is the angular speed.
7. a kind of detection recognition method of circulating tumor cell, which is characterized in that be applied to described in any one of claim 1~6
Circulating tumor cell detection identifying system, including：
Light source module shoot laser, and the laser is focused on the sample to be tested on sample stage, the sample to be tested is warp
The blood smear of filtering and non fluorescent stain；
The laser reflection that ellipsoidal mirror scatters the sample to be tested to photomultiplier light-receiving surface；
The optical signal of acquisition is converted to electric signal by the photomultiplier, and by the electric signal transmission to image recognition mould
Block；
Described image identification module generates the image of the sample to be tested according to the electric signal, and to the cycle in described image
Tumour cell is identified.
8. the method according to the description of claim 7 is characterized in that further including before light source module shoot laser：
Make blood smear.
9. according to the method described in claim 8, it is characterized in that, the making blood smear includes：
Blood sample is filtered, and retains the circulating tumor cell in the blood sample；
Blood smear is made using the filtered blood sample；
The blood smear is dyed using non-fluorescence dyestuff.
10. the method according to the description of claim 7 is characterized in that further including：
Sample stage drive module drives the sample stage and sample to be tested to be moved in one direction with constant speed, while with
Constant angular speed rotates on center shaft, so as to focus on the hot spot on the sample to be tested surface on the sample to be tested surface
It is moved along spiral of Archimedes；
Photoelectric conversion module is according to preset timing node sequence acquisition optical signal, to obtain the equal multiple sampled points in interval.
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