CN109142012A - A kind of FFPE sample fast dewaxing device and its application method - Google Patents
A kind of FFPE sample fast dewaxing device and its application method Download PDFInfo
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- CN109142012A CN109142012A CN201810940799.0A CN201810940799A CN109142012A CN 109142012 A CN109142012 A CN 109142012A CN 201810940799 A CN201810940799 A CN 201810940799A CN 109142012 A CN109142012 A CN 109142012A
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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
- G01N1/36—Embedding or analogous mounting of samples
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Abstract
The invention discloses a kind of FFPE sample fast dewaxing devices, are related to FFPE sample dewaxing field.FFPE sample fast dewaxing device includes sample incubation chamber and heating module, the sample incubation chamber is connected with heating module, the heating module is arranged to heat sample incubation chamber to assigned temperature, and the sample incubation chamber top or side setting can be closed opening.The present invention combines FFPE dewaxing cleavage method that is time-consuming shorter and only needing same temperature to be incubated for, realize that the FFPE sample automatic dewaxing equipment that can be integrated in automation equipment, the device can carry out closed dewaxing operation to FFPE sample using artificial or self-reacting device.This device is easy to operate and is easily integrated into self-reacting device, and the operation complexity for the current FFPE sample process of simplification provides convenient, quick integral type solution.
Description
Technical field
The present invention relates to the dewaxing field FFPE more particularly to a kind of FFPE sample fast dewaxing device and its application methods.
Background technique
Formalin fixes paraffin embedding techniques (Formalin-Fixed and Paraffin-Embedded, FFPE)
A kind of tissue preservation techniques.This technology is first fixed with formalin in order to maintain Nuclear extract structure and then uses tissue
Solid paraffin embedding uses for making histodiagnosis or research after slice.Since it can sufficiently keep original group of sample
It knits pattern, Clinicopathologic Diagnosis, pathomechanism research, cancer use can be now widely used for by the advantages such as preservation for a long time
The fields such as medicine guidance.
And such as FFPE is organized to carry out molecular biology research or application, it is necessary first to carry out the extraction of nucleic acid.But
Formalin will lead to the crosslinking of nucleic acid, albumen to the fixation of sample, this to be crosslinked the extraction that will affect nucleic acid, and can be right
The activity of archaeal dna polymerase has an impact, and causes PCR amplification effect undesirable.In addition, being wrapped in the paraffin of tissue periphery can hinder
Infiltration of the digestive juice to tissue influences the digestion and nucleic acid release of tissue to make digestive juice that can not contact with tissue.Therefore,
For the influence for eliminating formalin when FFPE sample is fixed, paraffin digests tissue samples, increase nucleic acid extraction efficiency, improves
PCR amplification effect needs to carry out tissue under the premise of not increasing PCR mortifier additionally, reducing nucleic acid damaging to the greatest extent thorough
Dewaxing.
And current process for dewaxing mainly have dimethylbenzene process for dewaxing and two step temperature process for dewaxing (patent publication No.:
CN104232616A), but its operating procedure is sufficiently complex, and dimethylbenzene dewaxing needs the addition of multistep reagent and centrifugally operated, real
It is cumbersome to test process, long flow path, and dimethylbenzene is toxic reagent, slightly misoperation all can be to experimenter during the experiment
Personal safety causes damages.Although two step temperature methods dewaxing will dewaxing with cleavage step merge processing, due to its
After 60 DEG C have been incubated for, need to take out sample tube, thermal modules to be added place into after being warming up to 90 DEG C, to avoid caused by temperature-rise period
Solution crosslinking time makes the excessive fragmentation of nucleic acid too long；And during taking out the water phase below oil reservoir, since temperature change is led
The problems such as upper oil phase solidification of cause, causing it to take, liquid is difficult and liquid-taken amount is inaccurate or can not take out sample solution at all
The problems such as.In addition, often one day FFPE sample size to be measured just can reach tens of examples, above method people in large and medium-sized hospital
Work operating time length, complex steps, automation difficulty are high, can such as simplify FFPE dewaxing cracking process, establish one kind more conveniently
Manually or automatically instrument carries out the dewaxing and cracking of FFPE sample, then can provide for current FFPE sample process greatly just
It is prompt.
Though thering is part patent of invention to refer to the extraction that can be used for FFPE at present, and the paraffin in unresolved pipetting processes is broken
The problem of piece and fragment of tissue blocking liquid-transfering device, and can not really realize the FFPE dewaxing of automation, therefore in the dewaxing of FFPE
Aspect has biggish room for promotion still to meet clinical application and basic research demand.Present invention incorporates one-step method dewaxings
Method realizes that lower layer's aqueous layer solution directly shifts using simple internal fluid path structure, while substantially shortening the dewaxing time
The performance requirement to heating module is reduced, and avoids influence of the paraffin to liquid relief of solidification, improves aqueous layer solution
Acquisition purity, reduce the operation difficulty of subsequent nucleic acid extraction.This device and instrument easy to operate can greatly simplify mesh
The Operating Complexity of preceding FFPE sample process provides convenient, quick FFPE sample dewaxing scheme.
Summary of the invention
In view of the above drawbacks of the prior art, the technical problem to be solved by the present invention is to current FFPE samples to dewax
Journey is complicated, larger workload and the automation for being difficult to realize dewaxing process.
To achieve the above object, the present invention provides a kind of FFPE sample fast dewaxing devices, which is characterized in that including sample
This incubation chamber and heating module, the sample incubation chamber are connected with heating module, and the heating module is arranged to heat
For sample incubation chamber to assigned temperature, the sample incubation chamber top or side setting can be closed opening.The heated mould
Block is preset with one, two or more temperature spots, and the temperature range of several temperature spots is respectively 50-60 DEG C and 75-95 DEG C.It should
Temperature spot dewaxes with a step and two step lost-wax processes match.
Further, the FFPE sample fast dewaxing device further includes valve and sample storage room, and the valve is located at
Sample incubation cavity bottom is simultaneously connected with sample storage room.Valve can be located at sample storage room, and valve and sample storage room are whole
Body is connected by modes such as screw thread, buckle or hinges with sample incubation chamber, and sample incubation cavity bottom is open at this time, opening
Size is suitable with valve size.Valve can also be located at sample incubation chamber, and sample storage room passes through screw thread, buckle or hinge
Etc. modes be connected with sample incubation chamber or valve.Preferably, the valve is solenoid valve or multiposition valve or pneumatic operated valve or machine
Any one in tool valve, valve can be closed by Mechanical course.
Further, further include filtering module at the FFPE valve, the filtering module can prevent uncracked sample into
Enter sample storage room.Filtering module filter opening size can be 60 mesh, 80 mesh, 100 mesh or 200 mesh and smaller.
Further, in order to guarantee that the aqueous phase liquid in sample incubation chamber completely flows into sample storage room as far as possible,
The sample storage building volume is equal to or less than aqueous phase liquid in default dewaxing lytic reagent.Preferably, sample stores chamber body
It is long-pending consistent with water phase volume in default dewaxing lytic reagent.
Further, it is after the sample storage room is connect with valve and the disconnected closed structure of atmosphere, sample storage
The materials such as deformable polypropylene can also be used in room, reduce composition of air therein, and subsequent samples is made to be incubated for the liquid in chamber
When inflow bubble will not be generated because of the air in discharge sample incubation chamber.Valve and sample storage room can be moved by machinery
Dynamic separation keeps final sample directly desirable.
Further, use and reduce for convenience the cross contamination between sample, the sample storage room or/or sample incubation
Chamber is disposable device, can preset dewaxing lysate in sample incubation chamber.Sample storage room can also be disposable
Use device, water phase close valve after flowing into sample storage room, can directly be taken by way of other Mechanical Movings such as rotation
Sample of nucleic acid to be extracted out, it is convenient and efficient.
Further, the FFPE sample fast dewaxing device further includes transfer tube, and the transfer tube is straight by aqueous phase liquid
Transport is connect to nucleic acid extraction position.The transfer tube can be syringe pump, air pump, diaphragm syringe pump method, wriggling syringe pump method, Electrowetting with
And other driving methods.
Further, the cross contamination of sample room in order to prevent, the FFPE sample dewaxing device further includes cleaning module,
Good cleaning effect may be implemented in cleaning module between different samples.The cleaning module and sample incubation chamber and sample store up
It deposits room to be connected, effectively cleans the residual sample of sample incubation chamber and sample storage room.
Further, sample incubation chamber can be made of all-transparent or local transparent material, facilitate observation sample
Dewax the progress cracked.Sample incubation chamber is also possible to metal or other materials is made.
The present invention also provides a kind of FFPE sample fast dewaxing method, feature exists, the FFPE sample fast dewaxing
Method is dewaxed using FFPE sample fast dewaxing device above-mentioned.It is manually operable the object of the present invention is to provide a kind of
FFPE dewaxing cracker, which can also facilitate and instrument integration, realizes efficient FFPE dewaxing.The present invention can be used for a step
Lost-wax process, it can also be used to two step lost-wax processes；It can be used for manual lost-wax process, can also cooperate with other instruments and realize full-automatic take off
Wax method.
The present invention realizes that lower layer's aqueous layer solution directly shifts using simple internal fluid path structure, when substantially shortening dewaxing
Between while reduce performance requirement to heating module, and avoid influence of the paraffin to liquid relief of solidification, improve water
The acquisition purity of phase layer solution, reduces the operation difficulty of subsequent nucleic acid extraction.This device and instrument easy to operate can be big
The Operating Complexity for simplifying current FFPE sample process greatly provides convenient, quick FFPE sample dewaxing scheme.With common dewaxing
Device is compared, and the present invention has following beneficial technical effect:
1, using simple sample separation module, the separating treatment of FFPE sample can be conveniently and efficiently realized；
2, nucleic acid solution needed for directly obtaining below device, does not easily cause blocking of the paraffin fragment to sampler, can
Conveniently realize automation；
3, required reagent can be placed in advance in device, avoided uncapping and added the problems such as reagent causes Aerosol Pollution.
Detailed description of the invention
Fig. 1 is that the FFPE sample of the FFPE sample dewaxing device of a preferred embodiment of the invention does not dewax schematic diagram；
Fig. 2 is water phase separation of oil after the FFPE of the FFPE sample dewaxing device of a preferred embodiment of the invention dewaxes
Schematic diagram；
Fig. 3 is that the water phase of the FFPE sample dewaxing device of a preferred embodiment of the invention enters the signal of sample storage room
Figure；
Fig. 4 is that the FFPE sample of the FFPE sample dewaxing device of another preferred embodiment of the invention does not dewax signal
Figure；
Wherein, 101- sample incubation chamber, 102- heating module, 103-FFPE sample, 104- dewaxing lytic reagent, 105-
Valve, 106- sample storage room, 107- oil phase layer, 108- aqueous layer, 109- sample channel, 110- transfer tube.
Specific embodiment
Multiple preferred embodiments of the invention are introduced below with reference to Figure of description, keep its technology contents more clear and just
In understanding.The present invention can be emerged from by many various forms of embodiments, and protection scope of the present invention not only limits
The embodiment that Yu Wenzhong is mentioned.
In the accompanying drawings, the identical component of structure is indicated with same numbers label, everywhere the similar component of structure or function with
Like numeral label indicates.The size and thickness of each component shown in the drawings are to be arbitrarily shown, and there is no limit by the present invention
The size and thickness of each component.Apparent in order to make to illustrate, some places suitably exaggerate the thickness of component in attached drawing.
The object of the present invention is to provide a kind of FFPE dewaxing crackers being manually operable, which can also facilitate and instrument
Device is integrated, realizes efficient FFPE dewaxing.
The apparatus structure of a signal of the invention is as shown in Figure 1,101 be sample incubation chamber, for the cone of lower section aperture
Expects pipe is shaped, reagent system can be pre-placed in the pipe, or reagent is added simultaneously when FFPE sample is added, 102 be heated mould
Block, valve 105 is can be by the valve of machinery folding, as needed can be in sample incubation chamber 101 and 105 contact portion of valve
Increase filtering module to filter uncracked tissue block, 106 be sample storage room, and volume is preset as the water phase with reagent system
Liquid solution is consistent, sample storage room 106 be with the disconnected closed structure of atmosphere, the materials such as deformable polypropylene can also be used
Material, reduces composition of air therein, makes the liquid flow of subsequent samples incubation chamber 101 is fashionable will not be because of discharge sample storage room
Air in 106 and generate bubble in sample incubation chamber 101.Valve 105 and sample storage room 106 can pass through Mechanical Moving
Separation keeps final sample directly desirable, sample storage room 106 can also be changed to pump valve structure, directly by lower section aqueous phase liquid
It lashes into subsequent nucleic acid extraction chamber.
In actual mechanical process, valve 105 is closed state at this time, and FFPE sample is added in sample incubation chamber 101
103 and corresponding dewaxing lytic reagent 104.Heating module 102 heats sample incubation chamber 101, adds in one-step method dewaxing
In thermal process, in conjunction with the embodiments in lytic reagent, temperature is heated to 50-60 DEG C of incubation 30min-3h.After heating such as
Shown in Fig. 2, after incubation time to be heated, the paraffin in FFPE sample, since its is not soluble in water, can add from wherein dissolving out
Its in thermal process can enter top oil phase layer 107 and have the core of cracking in aqueous layer 108 at this time to separate with aqueous layer 108
Acid sample.
The lytic reagent 104 that dewaxes can use the reagent of one-step method process for dewaxing, it is also possible to the examination of existing two steps process for dewaxing
Corresponding water phase and oily phase reagent in agent box, can also be used in any existing FFPE deparaffinization reagents box or cell extraction kit
Tissue Lysis reagent adds the oily phase reagent in previous step process for dewaxing.
As shown in figure 3, keeping heating module 102 temperature-resistant, the valve 105 of 101 lower section of sample incubation chamber is opened, this
When aqueous layer 108 and oily phase layer 107 be liquid, directly lower layer's fixed volume aqueous phase solution can be transferred to down by connector
Square chamber, since the volume to sample storage room 106 is controlled in advance, only lower layer's aqueous layer 108 can pass through valve 105
Into sample storage room 106, the aqueous phase solution in sample storage room 106 is sample after dewaxing, and can be used for subsequent nucleic acid extraction
Operation.
Embodiment 1
Manually operated FFPE dewaxing device and process:
There is sealing device above sample incubation chamber 101, it is pre-packaged in sample incubation chamber 101 to have 200 μ L Tissue Lysis
Liquid, 20 μ L Proteinase Ks and 50 μ L oil phase reagents, such as mineral oil, paraffin oil, vegetable oil and silicone oil.
FFPE tissue samples are by being put into sample incubation chamber 101 after opening 101 upper seal device of sample incubation chamber
In, sealing state is maintained again by closeing the lid.
The volume of sample storage room 106 is 200 μ L, and sample storage room 106 can be removed from whole device, be protected after closeing the lid
It deposits.
Tissue lysates, Proteinase K and oily phase reagent are commercial product.
According to aforementioned dewaxing process (referring to schematic diagram 1-3), after sample storage room 106 is full of, valve 105 is closed, is taken
Lower sample storage room 106 is to get the tissue samples solution completed to dewaxing.If it is desired, 2 μ L can also be added in sample solution
RNase A is placed at room temperature for 5min to remove RNA.Resulting sample solution can directly carry out subsequent nucleic acid extraction step, be not necessarily to
Tissue Lysis is carried out again.
Embodiment 2
Connect the FFPE dewaxing device and process of subsequent automatic nucleic acid-extracting apparatus:
Schematic device used in embodiment 3 is shown in schematic diagram 4.
FFPE tissue samples are put into device by 101 top closing device (not expressing) of sample incubation chamber herein.
It include 500 μ L Tissue lysates, 10 μ L Proteinase Ks and 100 μ L oil phase reagents in sample incubation chamber 101.
109 structure one end of sample channel is connect with valve 105, and the other end can be connected to the sample chamber of subsequent nucleic acid-extracting apparatus
In.
Transfer tube 110 is the liquid driving device of signal, the liquid in sample incubation chamber 101 can be passed through sample channel
109 pumps are into subsequent samples chamber.
Contain filter membrane (not expressing herein) at valve 105, may filter that uncracked fragment of tissue and oily phase in aqueous layer 108
Liquid.
Tissue lysates, Proteinase K and oily phase reagent are commercial product.
According to aforementioned dewaxing process, 102 temperature of heating module is down to room temperature, valve 105 is opened, makes lower layer's aqueous layer
108 enter sample channel 109 by the filter membrane of 105 top of valve, are pumped aqueous phase solution to subsequent nucleic acid using transfer tube 110 and mentioned
It takes in device.
Embodiment 3
The present embodiment operating process is substantially the same manner as Example 1, the difference is that sample incubation chamber is disposable, chamber
Pre-add dewaxing lysate in room.In use, sample incubation chamber can pass through screw thread rotation or other methods and heating module and valve
Door is connected, and sample incubation chamber upper and lower, which is provided with, can be closed opening.FFPE sample is added from top when use, after to be heated
Aqueous phase separation is opened by valve can be closed opening under chamber, into sample storage room.Sample storeroom can also be disposable
Stage property.This embodiment can largely avoid the cross contamination of sample room.
Embodiment 4
The FFPE sample dewaxing device of the present embodiment further includes cleaning module.Dewaxing process is identical as embodiment 1,2.Cleaning
Module can clean automatically the inner cavity chamber of dewaxing device after the dewaxing of each sample, avoid the cross contamination of sample room.
The preferred embodiment of the present invention has been described in detail above.It should be appreciated that the ordinary skill of this field is without wound
The property made labour, which according to the present invention can conceive, makes many modifications and variations.Therefore, all technician in the art
Pass through the available technology of logical analysis, reasoning, or a limited experiment on the basis of existing technology under this invention's idea
Scheme, all should be within the scope of protection determined by the claims.
Claims (10)
1. a kind of FFPE sample fast dewaxing device, which is characterized in that including sample incubation chamber and heating module, the sample
It is incubated for chamber to be connected with heating module, the heating module is arranged to heat sample incubation chamber to assigned temperature, the sample
The setting of above this incubation chamber or side can be closed opening.
2. FFPE sample fast dewaxing device as described in claim 1, which is characterized in that the FFPE sample fast dewaxing dress
Setting further includes valve and sample storage room, and the valve is located at sample incubation cavity bottom and is connected with sample storage room.
3. FFPE sample fast dewaxing device as claimed in claim 2, which is characterized in that the valve is solenoid valve or multidigit
Any one in valve or pneumatic operated valve or mechanical valve.
4. FFPE sample fast dewaxing device as claimed in claim 2, which is characterized in that further included at the FFPE valve
Module is filtered, the filtering module prevents uncracked sample from entering sample storage room.
5. FFPE sample fast dewaxing device as claimed in claim 2, which is characterized in that the sample storage building volume is equal to
Or it is less than aqueous phase liquid in default dewaxing lytic reagent.
6. FFPE sample fast dewaxing device as claimed in claim 2, which is characterized in that the sample storage room and valve connect
It is and the disconnected closed structure of atmosphere after connecing.
7. FFPE sample fast dewaxing device as claimed in claim 2, which is characterized in that the sample storage room or/and sample
This incubation chamber is disposable device.
8. FFPE sample fast dewaxing device as claimed in claim 2, which is characterized in that the FFPE sample fast dewaxing dress
Setting further includes transfer tube, and the transfer tube directly transports aqueous phase liquid to nucleic acid extraction position.
9. FFPE sample fast dewaxing device as claimed in claim 2, which is characterized in that the FFPE sample dewaxing device is also
Including cleaning module.
10. a kind of FFPE sample fast dewaxing method, feature exist, the FFPE sample fast dewaxing method is wanted by such as right
FFPE sample fast dewaxing device described in 1-9 is asked to carry out the dewaxing of FFPE sample.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN113234589A (en) * | 2021-05-10 | 2021-08-10 | 宁波康程德诺生物医药有限公司 | Quadruple tube device, kit and extraction method for quickly extracting nucleic acid |
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Application publication date: 20190104 |