EP2550536A1 - Automatisches verfahren und automatisierte vorrichtung für die vorbereitung und analyse mehrerer zellsuspensionen - Google Patents
Automatisches verfahren und automatisierte vorrichtung für die vorbereitung und analyse mehrerer zellsuspensionenInfo
- Publication number
- EP2550536A1 EP2550536A1 EP11715954A EP11715954A EP2550536A1 EP 2550536 A1 EP2550536 A1 EP 2550536A1 EP 11715954 A EP11715954 A EP 11715954A EP 11715954 A EP11715954 A EP 11715954A EP 2550536 A1 EP2550536 A1 EP 2550536A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- analysis
- sample
- cell
- cell suspension
- settling
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
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- 230000002380 cytological effect Effects 0.000 claims description 35
- 238000002360 preparation method Methods 0.000 claims description 29
- 238000005070 sampling Methods 0.000 claims description 26
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- 238000010908 decantation Methods 0.000 claims description 8
- 238000012360 testing method Methods 0.000 claims description 7
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 6
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
-
- 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
- G01N1/31—Apparatus therefor
- G01N1/312—Apparatus therefor for samples mounted on planar substrates
-
- 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
- G01N1/2813—Producing thin layers of samples on a substrate, e.g. smearing, spinning-on
- G01N2001/2846—Cytocentrifuge method
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N35/00—Automatic analysis not limited to methods or materials provided for in any single one of groups G01N1/00 - G01N33/00; Handling materials therefor
- G01N35/10—Devices for transferring samples or any liquids to, in, or from, the analysis apparatus, e.g. suction devices, injection devices
- G01N2035/1027—General features of the devices
- G01N2035/1048—General features of the devices using the transfer device for another function
- G01N2035/1058—General features of the devices using the transfer device for another function for mixing
- G01N2035/106—General features of the devices using the transfer device for another function for mixing by sucking and blowing
Definitions
- the present invention relates to a method for preparing and analyzing a plurality of cell suspensions, of the type comprising at least the following successive steps:
- step (c) taking a sample of a cell suspension from a vial and depositing that sample in an assay container; step (c) being repeated for each vial to be analyzed.
- the invention also relates to a preparation and analysis automaton implementing this method.
- Cytological diagnosis includes diagnostic techniques based on morphological examination of cells. It is very well suited for screening for cancer and precancerous lesions, including the cervix.
- automata do not always make it possible to prepare cellular deposits for performing a satisfactory analysis and to obtain a reliable diagnosis.
- Such automata use devices generating a certain number of artifacts including problems of filter pressure management, with changes in cellular morphology and problems of clogging of the filter mesh with debris.
- the manipulations are different depending on the type of sampling: for example, it may be necessary to remove the red blood cells or mucus before collecting the cells on the filter.
- One of the aims of the invention is to overcome these disadvantages by completely automating the preparation and analysis of a plurality of cytological suspensions reliably, reproductively and standardized at a high rate. Regardless of the type of cytology, the method of preparation is similar.
- the subject of the invention is a method of sampling and analyzing a plurality of cell suspensions such as the step (c) of taking a sample of a cell suspension in a bottle and depositing this sample in an analysis container comprises at least one step of breaking up the cell clusters by means of pipetting-dispensing means.
- step (c) of taking a sample of a cell suspension in a vial and depositing this sample in an analysis container comprises at least one step of mixing and filtering the cell suspension in the vial.
- step (c) of taking a sample of a cell suspension in a vial and depositing this sample in an analysis container further comprises at least the following steps:
- each analysis container comprises a settling well and an analysis plate placed opposite the settling well and the method comprises at least the following successive steps:
- each analysis container comprises a sampling or aliquoting tube.
- the invention also relates to an automated sampling and analysis of at least one cell suspension comprising:
- At least one settling well of the cell suspension the settling well being positioned and maintained accurately and fixed on the tray;
- an analysis system comprising at least one analysis slide intended to receive / contain a sample of the cell suspension, the sample being a volume portion of the cell suspension containing elements of interest to be analyzed and the analysis being positioned and maintained accurately and fixed on the plate below and facing the settling well;
- the automaton comprises pipetting means able to take the sample of the cell suspension in the flask and to pour this sample into the settling well on the analysis blade of the analysis system, these means being arranged to allow a rupture of the cell clusters.
- the pipetting means comprises a first movable arm on which the pipetting means are fixed, said arm moving above at least the receiving plate holding the bottles, the settling wells and the analysis blades; it comprises marking or staining solutions which can be mixed with the cytological solution by the pipetting means;
- each bottle and each analysis slide comprises a visual marking and the automaton comprises means for reading the visual markings
- the reading means comprise a camera and are carried by a second movable arm, said second arm moving over at least the receiving tray holding the bottles, the settling wells and the analysis blades;
- the bottle comprises filtering means disposed above a settling cone, the pipetting means being arranged to be able to take a portion of the cell suspension under the filtering means and reinjecting said part onto the decanting cone in order to mixing said cell suspension and passing at least a portion of said suspension through the filtering means to break cell clusters larger than the mesh size of the filter;
- the sample analysis system comprises means for analyzing the cell density of the cell deposits at the bottom of the settling well on the analysis slide;
- the means for analyzing the cell density of the cell deposits at the bottom of the settling well on the analysis slide comprise a camera
- the sample analysis system comprises a device for producing a virtual plate of the sample
- the device for producing a virtual plate of the sample comprises a camera
- a support comprising means for positioning at least the plate capable of holding the tray fixedly and accurately and a button adapted to validate the position of at least the plate;
- the cell suspension is a cytological suspension
- the cytological suspension comprises a fixing solution comprising
- FIG. 1 is a schematic sectional view of a sampling and analysis automaton according to the invention
- FIG. 2 is a schematic perspective view of the automaton of FIG. 1;
- FIG. 3 is a schematic view from above of a receiving plate of the preparation and analysis automaton receiving vials each containing a cell suspension;
- FIG. 4 is a sectional view of a bottle to be received in the automaton
- FIG. 5 is a sectional view of a settling well intended to be received in the automaton
- FIG. 6 is a schematic perspective view of the analysis means of the automaton of FIGS. 1 and 2;
- FIG. 7 is a sectional view of a vial during a mixing step of the cell suspension preparation and analysis method.
- controller 2 for preparing and analyzing a cell suspension for automating this preparation and this analysis.
- FIGS. 1 and 2 show an automaton 2 for preparing and analyzing at least one vial 4 containing a cell suspension 5 to be analyzed, such as, for example, a fixed cytological suspension.
- Such a cytological suspension may, for example, come from a cervical smear screening operation, for example with the aid of a sampling brush (not shown).
- This cytological suspension comprises in particular cells.
- the brush is then immersed in a bottle 4 containing a fixative and rubbed against the filter to extract and collect the cells and thus form the cell suspension 5.
- the fixative, or fixing solution is intended to preserve and preserve samples or cytological samples, including cells, for later analysis by a cytologist. Therefore the fixator must maintain the integrity of the nucleated cells and red blood cells, in particular their morphology, in the state they were in before they were removed.
- the fixing solution is intended to preserve in vitro a cytological sample comprising biological cells intended to be analyzed by a cytologist.
- the alcoholic fixing solution may also comprise Carbowax® or Polyethylene Glycol (PEG).
- formalin may be combined with decalcifying or anti-aggregating agents such as ethylene diamine tetracetic acid (EDTA) and its salts.
- EDTA ethylene diamine tetracetic acid
- a mucolytic agent such as dithiothreitol (DTT) or acetylcysteine can be added to the specimens containing mucus.
- Formalin makes it possible to preserve the red blood cells and the nucleated cells without lysing them, guaranteeing a reference in terms of size making it possible to make a more relevant diagnosis to the cytologist.
- the fixing solution according to the invention does not contain acetone or compounds of the ketone family, nor acetic acid, because these products lyse red blood cells which, by bursting, release the hemoglobin which binds to the cells.
- Certain types of staining such as the staining of Papanicolaou, because of the complexity of coloring agents associating several nuclear dyes and hemoglobin, make the cytological analysis, and in particular nuclear, very difficult if not impossible.
- immuno-cytochemical studies are often hampered by hemoglobin deposition.
- the fixing solution described above thus allows excellent preservation of the integrity of nucleated cells and red blood cells for their analysis.
- the fixing solution described above comprising PEG and / or formaldehyde, and has a density different from conventional fixators used in cytology, which are essentially alcoholic.
- the Applicant has found that the solution of fixation thus allows selection of the cells of interest through a density gradient more efficiently than conventional fixatives used in cytology.
- the automaton 2 further comprises at least one receiving tray 6 carrying at least the vial 4 containing the cell suspension 5 to be analyzed and a support 8 on which the receiving tray 6 is placed.
- the receiving tray 6 is intended to support a plurality of bottles 4 each containing a cell suspension 5, in order to quickly and automatically produce a plurality of cell suspensions 5 to be analyzed, simultaneously or sequentially, of any or part of the suspensions.
- a receiving plate 4 has already been described in the patent application EP-211 110, filed in the name of the applicant, and allows to position and maintain accurately and fixed at least the bottle 4 on the tray 6. The skilled person can refer to this document and this plateau will not be described in more detail here. A schematic view of the plateau is shown in Figure 3.
- the support 8 comprises plate positioning means 6 able to maintain the plate fixedly and precisely in the automaton 2.
- These positioning means comprise receiving means 9 of an end portion of the plate 6, the plate being disposed against or in said means 9 when it is positioned in the automaton 2.
- the plate 6 comprises positioning means on the support 8.
- These positioning means comprise at least one orifice 10 dug in the thickness of the plate without passing through it and intended to precisely fix the plate on the support 8 of the automaton 2.
- the plate 6 comprises two identical orifices 10 arranged orthogonally to the longest side of the plate 4.
- the support 8 of the automaton 2 comprises positioning means complementary to those of the plate 6. These means comprise at least one lug 12 and preferably as many lugs as orifices 10 of the plate 6.
- the shape of the lug 12 is complementary to that of the orifice 10 of the tray and of slightly smaller size so that during the establishment of the plate 6 on the support 8, the lug 12 is located inside the orifice 10 corresponding.
- the plate 6 is held fixedly and accurately on the support 8.
- the support 8 of the controller 2 comprises a button 14 or press, intended to validate the proper horizontal placement of the plate 6 on the support 8. Indeed, when the plate 6 is well placed, that is that is to say when the entire lower face of the plate is in contact with the support 8, since the lugs 12 are received in the orifices 10, the button 14 is pressed into the support 8, in a space 16 provided for this purpose ensuring and the proper implementation of the tray 6 and allowing the controller 2 to operate.
- the controller 2 comprises pipetting-dispensing means 20, called pipetting or sampling means thereafter, that is to say, to collect and / or pour / fill a sample of the cell suspension 5.
- pipetting means extend over the receiving tray 6.
- the sample is a precise portion by volume of the cell suspension containing elements of interest to be analyzed, for example cells.
- the pipetting means are movable so as to pass over each vial 4 to perform sampling and / or filling as represented by the arrows of FIG. 1.
- the pipetting means are formed of at least one pipette 22 or needle, and preferably a plurality of pipettes, e.g. four or eight, arranged in parallel so as to be able to simultaneously take samples from a plurality of vials 4 and prepare simultaneously for analysis.
- the pipetting means 20 are fixed on a first mobile robotic arm on the automaton 2 above the plates 6.
- the device (orifices 10 / lugs 12 and button 14) ensuring the positioning of the plate accurately on the support makes it possible to avoid breaking the needles or pipettes
- the automaton 2 comprises a preparation and analysis system 30 comprising at least one analysis support intended to receive / contain the sample of the cell suspension 5, taken and poured into or on the analysis support. by the means
- the analysis supports of this preparation and analysis system may comprise spreading blades 32, sampling or aliquoting tubes 34, analysis or settling wells 36 according to the method of analysis. analysis chosen by the practitioner.
- the analysis supports 32, 34, 36 are held in a precise and fixed manner on the plate 6.
- this bottle 4 has the same characteristics as those described in document EP-21111300 in order to be able to fix the bottle 4 on the receiving tray 6 and certain characteristics described in the document WO-2006/058989 in order to ability to prepare and analyze the cell suspension.
- the vial 4 containing the cytological suspension 5 comprises a body 40, for example substantially cylindrical. From this body 40 extends a lower edge 42 and locking means 44 disposed on either side of the body and projecting therefrom, for locking the bottle 4 on the receiving tray 6 as described in FIG. EP-2111,300 in longitudinal and elevation directions.
- the locking means 44 are intended to engage in impressions of the rails of the plate 6 so as to block the bottle 4 in a preferred direction.
- the skilled person can refer to this document to understand the operation of this bottle 4 with the receiving tray 6.
- the bottle 4 also comprises a lid 46, for example provided with a self-healing and pierceable membrane 48 allowing the passage of the pipetting means 10 of the automaton, for example a pipette.
- This portion 48 pierceable and self-healing allows in particular, not to remove the lid 46 of the bottle to perform a collection of cellular solution and therefore to maintain the integrity of the sample ensuring total security for it which after collection by the doctor or laboratory stays in a closed bottle throughout the preparation and analysis process. In addition, this allows a complete absence of risk of contamination or inhalation of the fixer for the technician in the laboratory.
- the lid 46 of each bottle 4 further comprises a visual marking 50 or identifier for identifying the cell suspension 5 contained in the bottle 4.
- this visual marking 50 is a bar code. This identifier 50 allows full traceability during preparation and analysis by computerized management of the sample numbers.
- the identification means are different, for example a tag carrying information or an electronic chip of the RFID chip type whose content can be read remotely by reading means.
- the bottle 4 is equipped with filter means 52 at least partially immersed in the suspension as described in document WO-2006. / 058989.
- These filtering means 52 are in the form of a web of filtering material, for example forming a basket, whose periphery is fixed on the body 40 of the bottle and whose center is connected to a tube 54, extending towards the opening of the bottle, associated with holding means in position in the bottle and adapted to allow the passage of the cell solution sampling means 20 of the automaton under the filtering means, in particular the suction pipette 22 of the suspension.
- the web of filtering material is braided nylon, allowing a mechanical action of detaching the cells when the brush is rubbed on the material without damaging the sample.
- the lower part of the bottle 4 has a settling cone 56.
- the bottle 4 is provided with an opening intended to receive a cytological sampling brush, detachably attached to a handling handle.
- the opening of the bottle comprises stop means for the brush, for locking it in the bottle and detach the handle.
- the bottle 4 is not equipped with a central tube or filtering means.
- the preparation and analysis system 30 preferably comprises a device 60 for depositing cells by decantation on an analysis slide.
- a device 60 for depositing cells by decantation on an analysis plate has already been described in document FR-2 917 165 and the person skilled in the art can refer to this document.
- This device 60 comprises at least one settling well 36 placed above an analysis blade 32, and an absorbent material 62.
- the analysis blade 32 has at one end an identification means 50, for example a visual marking of the barcode type.
- the suspension is poured, by the pipetting means 20, into the chamber 64 for receiving the settling well 36 placed above the analysis blade 32, and whose bottom is open and extends facing a cell deposition area of the analysis blade 32.
- the bottom of the chamber is in fluid communication with the absorbent material 62 of the preservative liquid or fixer to gradually absorb it and allow homogeneous deposition by settling of cells on the deposit area of cells of the analysis blade 32. This produces a uniform cell deposition in a reduced settling time.
- Absorbent material 62 is partially compressed by a settling press 66 between the bottom of the receiving chamber and the assay plate around the cell deposition area.
- the system 30 for preparing and analyzing comprises means 70 for measuring the cell density in the settling chamber, so as to produce an analysis blade 32.
- Such measuring means 70 cell density are described in the document FR 2 922 019 to which the skilled person can refer.
- the means 70 for measuring the cell density comprise a camera 72 arranged to record images of the plate 6, that is to say the contents of the settling chamber 64 and the analysis blade 32.
- the means 70 for measuring the cell density comprise a sleeve 74 fixed around the camera so that, when the camera 72 is lowered and placed above the settling chamber 64 to acquire the images of the content of this chamber, that the sleeve 74 is in contact with the settling press 66 in order to achieve a dark chamber by obtaining a light-tightness in order to optimize the quality of image acquisition.
- the means 70 for measuring the cell density further include illumination means of the settling chamber to allow the camera 72 to perform the density measurement.
- illumination means comprise, for example, a light source 76 fixed to the camera 72.
- the light source 76 comes closest to the edge of the analysis blade 32, in order to have a light transmission. optimal light.
- the light source 76 then diffuses light in the analysis blade 32.
- the means 70 for measuring the cell density are fixed on a second mobile robotic arm above the bottles 4 of the trays 6.
- the analysis supports of this system 30 comprise at least one sampling or aliquoting tube 34 and a support 80 in which a plurality of tubes 34 can be inserted in order to maintain them in a fixed position.
- This support 80 of the aliquoting tubes 34 is fixed in a precise manner on the plate 6 so that the pipetting means 20 take the samples from the cytological solutions contained in the bottles 4 and pour them into the aliquoting tubes 34, the flasks being fixed on the same plate as the aliquoting tubes.
- the two corresponding possibilities for one of the settling well 36 placed above an analysis blade 32, and an absorbent material 62 and for the other to the sampling or aliquoting tube 34, can be associated on the same rack for a simultaneous or offset realization of complementary techniques in a systematic way or correlation with the results of the analysis of the density on non-colored slide allowing a better control of the pre-analytical step.
- the automaton 2 further comprises remote reading means 90 intended to identify the visual marking 32 of the bottles 4.
- These reading means 90 comprise a wide-field camera and extend above the reception plate 6.
- These means 90 remote reading are carried by a second mobile robot arm of the automaton, said second arm moving from each vial 4 fixed on the plate 6 to perform the reading of the visual marking 50 or identifier located on the cover 46.
- the camera is placed in such a way that it can read the identification information 50 from the analysis blade 32.
- This information is for example transmitted to a computer processing system that monitors the quality of a plurality of blades. analyzing and gathering information on the cell spread disposed on this slide. This information notably includes the origin of the cell spreading, the marking of the analysis slide being coupled to the labeling of the vials 4.
- the camera 72 of the cell density analysis means 70 makes it possible to read the visual markings 50.
- the cell density analysis means 70 and the remote reading means 90 are combined allowing a gain of space.
- the markings 50 are oriented in a precise and invariable direction when the lid is fixed on the body, which makes it easy to use the reading means 90 away from the markings.
- the invariable orientation can be obtained thanks to the blocking means 44 of the bottle 4 described in the document EP-2111300.
- These remote reading means 90 offer, because of the specific orientation of the bottles 4, a unitary or multiple visualization of these bottles in order to pair them unitarily or multiple to the envisaged analysis system (spreading blades 32, sampling or aliquoting tubes 34, analysis well 36 ...) itself having a fixed position. That is to say that the reading means 90 can read the marking 50 of a single bottle 4 or more markings at a time.
- This fixed positioning which corresponds, for example, to the analysis blades 32 positioned in the settling press 66, is in the extension of the rails, described in document EP-211 110, serving as a support for the bottles 4, so that one and the same plateau allows the proper positioning of the bottles 4 and blades 32 for remote reading means offering a unitary or multiple viewing.
- batch treatment of the bottles is possible, which allows a higher rate of analysis.
- the automaton further comprises a processor, not shown, for collecting the user-made parameter settings according to the nature of the cytological sample, then for controlling the robotic arms, the pipetting means 20, the reading means 90 , the means of analysis 70 of the cell density, the button 14 (displacements, volumes taken, acquisition by the camera, light source ).
- the automaton further comprises a support, not shown, fixed in a precise manner on the support 8 of the automaton 2 comprising bottles containing solutions generally used for staining or labeling of specific entities of the cells. such as nuclei, cytoplasm or other constituent elements of the cell.
- This support for staining or "marking” solutions makes it possible to carry out staining or marking steps on the analysis slides as commonly carried out by those skilled in the art.
- This staining or marking step is then carried out by means of pipetting means 20 which, after having been placed above the bottles of staining or marking solutions, take up the necessary volume and then move over the analysis slides where the pipetting means pour their contents.
- the analysis system 30 of the automaton 2 further comprises a device for producing a virtual plate of the sample as described in document FR-2 931 966 and comprising a camera. According to one embodiment, the same camera as that of the analysis means and / or reading means is used.
- the automaton then comprises a single camera forming the reading means 90, the analysis means 70 of the cell density and the device for producing a virtual plate, allowing a saving of space.
- the practitioner Beforehand, after having taken the cell samples 5 to be prepared and analyzed and transferred into vials 4, the practitioner permanently closes the vials 4 by means of their lids 46. Indeed, the method of preparation and analysis of these suspensions is implemented thereafter without subsequent opening of the vials 4.
- the practitioner can insert the cell sample by pricking his sampling needle through the healing membrane of the bottle, without having to open the lid at no time.
- the technician or user after a sufficient cell fixing time, for example at least two hours, load the vials 4 of cell suspensions 5 on the tray 6 of loading and as many containers of the analysis system 30, preferably analysis slides 32.
- each analysis blade (32) is disposed between the loading tray (6) and the absorption sheet (62).
- a plurality of decanting wells 36 are loaded into a press 66.
- the settling press 66 is installed above the plate 6 so that each settling well 36 is arranged above an analysis plate 32, such as described in document FR-2 917 165.
- the trays 6 are then loaded into the automaton 2 on the support 8 thereof, and fixed precisely thanks to the complementary positioning means 10, 12 of the trays 6 and the support 8 of the automaton 2 and the button 14. which validates this position if it is correct.
- the technician or user starts the software, selects the settings, very simple, according to the number and type of samples placed on the PLC, and starts the preparation session.
- the reading means 90 identify the visual markings 50 of the vials 4 and analysis blades 32 making it possible to establish a correlation between the vials 4 containing the solutions to be analyzed and the analysis slides 32 to be prepared.
- the sampling means 20 are moved above the vial 4 containing the cell solution 5 to be analyzed.
- the needle or pipette 22 of the sampling means 20 passes through the self-healing membrane 48 of the lid 46 of the vial 4 to the cell suspension.
- a sample of a cell suspension in a bottle 4 is then made by the pipette 22 of the sampling means 20.
- the cell suspension 5 is mixed in the analysis bottle 4 by means of the pipette 22 of the pipetting means 20, by aspirating / withdrawing a first volume of the cell solution and then exhaling / ejecting the first volume into the cell solution , as represented by the arrow F in FIG. 7.
- the first volume is substantially between 50 ⁇ ⁇ - and 2000 ⁇ ⁇ - of the cellular solution. This step aims to break / break the cell clusters of cytological sampling 5 to be analyzed on the wall of the settling cone 56 of the bottle 4.
- the cells reinjected into the bottle pass over the filtering means in order to break the cell clusters whose size is greater than the size of the mesh of the filter, this makes it possible to obtain a second filtration of the sample and a resuspension of the sample.
- a first settling of the cell solution is then carried out for a first time.
- the first duration is substantially between 5 minutes and 12 hours depending on the analysis mode chosen, for example equal to 15 minutes for the preparation of an analysis slide. This step makes it possible to establish a gradient between the cells of interest to be analyzed and the inflammatory or haemorrhagic cells, it is a differential settling.
- a suction step is performed for the solutions which will then be deposited in the analysis container.
- This step comprises i) the aspiration by the pipetting means 20 of a volume of adhesive, adapted to adhere the cells to the analysis blade 32 of whatever type, or buffer, ii) optionally a volume of air as described in document FR 2 919 054, then iii) a volume of cellular solution at the bottom of the decantation cone 56, comprising relevant cells said relevantes, that is to say the cells located in the lower part of the solution following the first differential settling.
- the glue and cell solution volumes are equivalent and substantially between 50 ⁇ _ and 1000 ⁇ _, for example 250 ⁇ _.
- the glue volume is zero.
- this last step of taking a volume of cellular solution is itself done in two sub-steps: a first substep called "micro-mixing" and then a second sub-step suction volume desired cellular solution.
- the "micro-mixture” consists of the aspiration of a volume of the cytological solution just above the bottom of the decantation cone of the bottle, for example at 2 mm, the volume being between 20 ⁇ _ and 200 ⁇ _, for example 100 ⁇ _, then the pipetting means 20 reinject a portion of the volume taken, for example 50 ⁇ _, substantially at the same place, to avoid the "volume or dead pellet" 100, that is to say, to take off the cells from the bottom of the settling cone 56 through the injection force of the pipetting means 20 and create a very localized resuspension.
- This "micro-mixing" step is optional and can be replaced by a simple step of taking a volume of the cytological solution.
- the use of the adhesive adapted to adhere the cells to the analysis slides 32 makes it possible to use slides that have not undergone specific cell adhesion treatment and thus reduces the costs of preparation and analysis of the cells. cytological solutions to analyze.
- 250 ⁇ l of glue and 100 ⁇ l of the cytological solution containing the cells of interest are taken, then 50 ⁇ l of the sample of the cytological solution contained in the pipette is reinjected, and 200 ⁇ l of the cytological solution is taken in order to to have the same amount of glue or buffer and cells of interest.
- the pipette 22 is then automatically activated to homogeneously mix its contents, that is to say the glue or the buffer and the sample of the cytological solution, as described in the document FR 2 919 054.
- This step also makes it possible to dilute and resuspend homogeneous sampling in the adhesive or buffer according to a dilution method as described in document FR-2 919 054.
- the buffer solution may be supplemented with marking or staining elements.
- the pipetting means are then moved over a settling well 36 in order to pour / deposit the sample into the settling chamber 64 and to make an analysis slide 32 comprising a cell spread to be analyzed.
- Cell spreading results from the deposition of the sample in the settling well as described in document FR-2 917 165 and to which the skilled person can refer.
- a second settling takes place on the blade for a second time.
- the second duration is substantially between 5 and 60 minutes and preferably equal to 15 minutes for the preparation of an analysis slide.
- sampling and production steps of the slide 32 are repeated for each vial 4 to be analyzed by differentially selecting the pathological and relevant cells necessary for the diagnosis of the cytologist.
- a step of analyzing the deposition of the cells at the bottom of the settling wells 36 by measuring the cell density on the analysis slides 32, in order to propose a pre-analytical control for all the samples they are intended for cytological analysis or complementary techniques of biology, is then carried out, for example as described in document FR-2 922 019.
- This step makes it possible to determine whether the cell suspension contains enough cells to obtain a satisfactory cellular deposit that can lead to reliable diagnosis and thus automatically readjust the sample to obtain a suitable cell density for analysis of the blade 32.
- This step ensures quality monitoring of the analysis blade 32 prepared from the cell solution 5.
- this step of analyzing the deposition of the cells is followed by a staining or marking step in an automated manner.
- the pipetting means 20 comprise a plurality of needles, preferably four or eight, making it possible to prepare and analyze a plurality of cell suspensions in parallel and thus to increase the rate of preparation of the microparticles. 'analysis.
- the automaton according to the invention allows the automated production of smears and other cytological samples in a thin layer.
- One of the advantages of the automaton 2, according to the invention, is that it is entirely closed from the vial 4 to the analysis blade 32 thus ensuring the integrity of all the cytological solutions and samples while completely avoiding the risks. contamination and ensuring greater safety for the practitioner (no inhalation of the fixative). This advantage also results from the use of a vial combining both filtration means and decanting means for performing thin-layer cytological spreading.
- the bottles 4 are fixed on the support of the controller 2, it is the other systems such as the pipetting means 20, the reading means 70 and the density analysis means 90 which are movable above the flasks 4 ensuring greater safety of handling in the laboratory.
- the cell density adjustment method implemented by the automaton for producing the analysis plates makes it possible to increase, if necessary, the relative density in pathological cells, while keeping a context of clean but informative analysis, and improving the quality of the spreading and preservation of the selected elements. This allows a safer interpretation compared to traditional technique or semi-automated thin-layer cytology techniques.
- the automation allows the establishment of a real quality assurance system and a standardization of thin layer deposition improving the reproducibility of spreading, ensuring respect for cell integrity, and easy reading. It also allows a net decrease in technical and reading costs.
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- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Immunology (AREA)
- Pathology (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Physics & Mathematics (AREA)
- Chemical & Material Sciences (AREA)
- Investigating Or Analysing Biological Materials (AREA)
- Sampling And Sample Adjustment (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Apparatus Associated With Microorganisms And Enzymes (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR1052049A FR2957672B1 (fr) | 2010-03-22 | 2010-03-22 | Procede automatique et automate de preparation et d'analyse d'une pluralite de suspensions cellulaires |
PCT/FR2011/050575 WO2011117523A1 (fr) | 2010-03-22 | 2011-03-21 | Procédé automatique et automate de préparation et d'analyse d'une pluralité de suspensions cellulaires |
Publications (1)
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EP2550536A1 true EP2550536A1 (de) | 2013-01-30 |
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EP11715954A Withdrawn EP2550536A1 (de) | 2010-03-22 | 2011-03-21 | Automatisches verfahren und automatisierte vorrichtung für die vorbereitung und analyse mehrerer zellsuspensionen |
Country Status (9)
Country | Link |
---|---|
US (1) | US9983222B2 (de) |
EP (1) | EP2550536A1 (de) |
JP (1) | JP5894142B2 (de) |
KR (1) | KR101858056B1 (de) |
CN (1) | CN102812365B (de) |
BR (1) | BR112012023615A2 (de) |
FR (1) | FR2957672B1 (de) |
RU (1) | RU2556994C2 (de) |
WO (1) | WO2011117523A1 (de) |
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- 2011-03-21 JP JP2013500558A patent/JP5894142B2/ja not_active Expired - Fee Related
- 2011-03-21 RU RU2012144711/04A patent/RU2556994C2/ru active IP Right Revival
- 2011-03-21 US US13/636,665 patent/US9983222B2/en active Active
- 2011-03-21 CN CN201180015106.3A patent/CN102812365B/zh not_active Expired - Fee Related
- 2011-03-21 EP EP11715954A patent/EP2550536A1/de not_active Withdrawn
- 2011-03-21 BR BR112012023615A patent/BR112012023615A2/pt not_active Application Discontinuation
- 2011-03-21 KR KR1020127027348A patent/KR101858056B1/ko active IP Right Grant
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Also Published As
Publication number | Publication date |
---|---|
FR2957672A1 (fr) | 2011-09-23 |
CN102812365B (zh) | 2015-11-25 |
BR112012023615A2 (pt) | 2016-08-02 |
RU2012144711A (ru) | 2014-04-27 |
US9983222B2 (en) | 2018-05-29 |
KR20130064048A (ko) | 2013-06-17 |
US20130034874A1 (en) | 2013-02-07 |
KR101858056B1 (ko) | 2018-05-15 |
WO2011117523A1 (fr) | 2011-09-29 |
FR2957672B1 (fr) | 2013-03-15 |
JP2013522641A (ja) | 2013-06-13 |
JP5894142B2 (ja) | 2016-03-23 |
CN102812365A (zh) | 2012-12-05 |
RU2556994C2 (ru) | 2015-07-20 |
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