Abstract:
A separation device for preparing biological samples for cytological, cytohistological and histological analysis includes a series of communicating chambers, means for moving a fluid through the chambers and one or more separating units for separating material contained in the fluid, wherein the separator units are arranged along the path of the fluid between the chambers.

Description:
FIELD OF THE INVENTION 
     The present invention refers to a device for preparing some biological samples to be used in cytological, cytohistological and histological analysis. 
     STATE OF THE ART 
     It is known that the diagnostic cytology is the branch of pathological anatomy which aims at observing, with different detection instruments and methods, cells and/or cellular aggregates, obtained in any manner, arranged on a microscope slide, suitably fixated with the aim of identifying the altered cells and/or cellular groups and thus formulate a diagnostic hypothesis useful to the doctor to identify the pathology in course. 
     The diagnostic cytology applies various techniques aimed at valorising the material obtained in any manner from tissues and/or organs of subjects and prepared on microscope slides adapted to allow suitable study. The preparations follow the collection of organic material generally conducted through exfoliative methods (spontaneous urine, phlegm, etc), abrasive methods (pap-test, brushings, fixation, scraping of tissues and/or mucosa, washing), invasive techniques (various organic liquids, needle aspiration or FNAB, etc.) methods. 
     Furthermore, technically, the preparations are usually divided into: 
     direct preparations on microscope slides of the material collected according to any of the abovementioned techniques and suitably fixated according to the diagnostic needs (fixation at the air, fixation with alcoholic solutions, etc); 
     preparation of material in liquid phase after enriching through centrifugation; 
     preparation of material considered having poor cellularity with resuspension of the bottom of the centrifugate and thus cytocentrifugation directly on the microscope slide; 
     preparation by cytoinclusion of material, generally microfrustolated, previously enriched and concentrated on the bottom of a test tube by trapping the same through various techniques (agar, celloidine, thromboplastin, etc); 
     preparations by cytoinclusion of semi-solid material; 
     specific preparations such as suspensions in Saccomano fluid, suspension of materials obtained by abrasion and “millipore” and/or “nucleopore” treatment, suspension in means suitable for differential separation (Sephadex®) and others. 
     All illustrated methods, usually used in cytology laboratories, provide for numerous technical steps such as centrifugation, resuspension and cytocentrifugation, and inclusion of the residue bottom in the test tubes after centrifugation. These steps are necessary for recovering the study material in the abovementioned form specifically required by the particular type of study to be conducted and the relative working diagnosis inherent with the indication under analysis. 
     In particular, when the technician is dealing with a given method to be conducted on a biological sample, the technician is necessarily required to process the sample so as to recover the material in a well determined form so as to provide the preparation to the utmost. The optimisation of such operation requires a separation of the material of interest from what constitutes the overall of the collected material. In other words, for some particular and/or special analysis, the material to be prepared and analysed should be separated from anything that can alter the preparation result and, especially, the ensuing study in any manner whatsoever. In order to obtain this, the technician is forced to conduct often sensitive and complex operations such as several centrifugation and resuspension operations with the ensuing waste of time, work materials and with the risk on one hand of not accurately preserving the study material and on the other hand wasting considerable amount of said material. Furthermore, the centrifugations must be provided under determined and precise conditions depending on the type of preparation and study to be carried out. 
     The biological material subject of study is always rich with information and the object of the various processing techniques is to extract from the latter as much data as possible, useful for diagnosis and/or follow-up of the patient and relative ensuing therapeutic choices. 
     In some cases, it is necessary to subject the sample to various treatment and/or separation steps. In case of starting from a liquid sample (blood plasma, urine, exudate or other biological fluid), the separation of one or more cellular components is often indispensible before conducting the required analysis on the fluid. Given analysis and determinations may instead be conducted on the cellular material separated from the fluid, always with the aim of obtaining the largest amount of information as possible from the sample. 
     It would thus be desirable to have a device capable of facilitating such operation, both in terms of speed of execution and in terms of efficiency and safety of use. 
     SUMMARY OF THE INVENTION 
     The technical problem on which the present invention is based is thus that of providing a device capable of allowing reducing the steps of preparing the biological material that should be prepared for different treatments as much as possible simultaneously considerably increasing the separation efficiency as well as the quality and quantity of the biological material available for the specific preparation. 
     Such problem is overcome by a separation device as described and claimed below. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       Further characteristics and advantages of the present invention shall be more apparent from the following description of some embodiments, provided purely by way of non-limiting example, with reference to the figures wherein; 
         FIG. 1  represents a schematic view of an embodiment of the device of the invention; 
         FIG. 2  represents a schematic view of a variant of the device of  FIG. 1 ; 
         FIG. 3  represents a schematic view of a further variant of the device of  FIG. 1 ; 
         FIG. 4  represents a schematic view of a different embodiment of the invention. 
     
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     The idea on which the present invention is based is that of providing a device capable of facilitating the preliminary operations of preparing a biological sample for processing with the various methods of the diagnostic cytology so as to standardise them without being exposed to the risk of losing or altering the material of the sample. 
     A crucial step in any diagnostic cytology method is the separation of the material of interest from the sample collected from a patient. Such step usually comprises a series of compulsory steps, such as the ones described previously with reference to the conventional methods, in which the biological sample is treated under given conditions depending on the type of study to be carried out. As known, each type of treatment (centrifugation, resuspension, cytocentrifugation, etc) requires use of apparatus, supports for samples and solutions which require calibration from time to time. 
     In order to simplify the sample centrifugation and washing operations, a separation device which—through simple operations—allows preparing the sample for the various analytical operations in a quick manner and with as less human intervention as possible, was designed. 
     In given embodiments, the device of the invention allows subjecting the sample, as it is or partly or fully treated, to some analytical determinations in line with the separation device. 
     In given embodiments, the device of the invention is structured to allow an easy and safe interchangeability of the elements that compose it and the modularity of the device itself. 
     With reference to  FIG. 1 , the separation device of the invention, indicated in its entirety with number  1 , comprises a plurality of containers  2   a ,  2   b ,  2   c ,  2   d ,  2   e ,  2   f  connected in series in flow communication through suitable conduits  3   a ,  3   b ,  3   c ,  4   a ,  4   b . The number of containers and relative conduits of communication shall be variable depending on the type of use and treatment required. 
     A first container  2   a , for example a test tube or any other container that can be used for handling biological fluids, defines a decantation chamber  5  therein. Said first container  2   a  comprises a closure  6   a  having a through hole in which there is inserted the distal end of a tube  7 . The tube  7  is in flow communication, through the opposite end, with a reservoir  8 , typically a bag of the type used for medical purpose, containing the biological fluid F to be treated and analysed. 
     The closure  6   a  can be inserted on the container  2   a  by pressure or by screwing it. In such case, both the upper portion of the container  2   a  and the closure  6   a  shall be provided with complementary threads. 
     The tube  7  is inserted in the closure  6   a  in a sealed manner. For example, a sleeve gasket  10  may be provided for. 
     Within the first container  2   a  the biological fluid F is subjected to decantation so as to separate the coarse residue (part of the tissue, etc) which can be contained in the fluid on the bottom of the decantation chamber  5 . 
     A first touch racking conduit  3   a  transfers the decanted fluid F into a second container  2   b , within which there is defined a transfer chamber  9 . The conduit  3   a , which—in the embodiment of  FIG. 1  is obtained by means of a tube—develops substantially horizontal or inclined towards the container  2   b  downstream and connects the containers  2   a ,  2   b  through opposite openings in the lateral walls thereof, serving as the touching means for the decanted fluid F. For such purpose, the conduit  3   a  is arranged at a level such as to allow a suitable decantation of the fluid F, preferably at the upper portion at half the height of the chamber  5 . 
     The second container  2   b  comprises a closure  6   b  entirely analogous to the closure  6   a  of the first container  2   a , in a through hole in which a suction tube  11  which continues, outside the container  2   b , in a first transfer conduit  4   a , is inserted. Usually the suction tube  11  and the transfer conduit  4   a  are in a single piece. 
     The transfer conduit  4   a  connects said second container  2   b  with a third container  2   c  through openings on the respective closures  6   b ,  6   c  of said containers. The closure  6   c  of the third container  2   c  is entirely analogous to the closures  6   a ,  6   b  described previously. 
     The third container  2   c  defines a collection chamber  12  therein. The transfer conduit  4   a  terminates within said collection chamber  12  and comprises, typically at the open end thereof, first separation means  13 , adapted to separate a corpuscular component of the biological fluid F (for example, a cellular population, in single elements and/or in cellular aggregates and/or microfragments, or a protein or anything else depending on the mode of use) from the fluid. 
     In an embodiment, such separation means  13  are filtering means. A bag filter such as the one described in the patent application EP-A-2 052 231 in the name of Valenziano Susanna, whose description is incorporated herein for reference, may be advantageously used. Preferably, such filtering means have porosity P 1  capable of withholding corpuscles of dimensions greater 50 micro-meters. 
     The third container  2   c  is in flow communication with a fourth container  2   d  through a second touch racking conduit  3   b . Such conduit  3   b  develops with a substantially horizontal development and connects the lateral walls of the two containers to a lower level with respect to the end of the separation means  13  or the transfer conduit  4   a , depending on which terminates lower. 
     The fourth container  2   d  is closed at the upper part by a closure  6   d , analogous to those described previously, and internally delimits a transfer chamber  14 . 
     A suction tube  15  is inserted into said transfer chamber  14 , traverses the closure  6   d  and continues into a transfer conduit  4   b , placing said fourth container  2   d  in flow communication with a fifth container  2   e.    
     The container  2   e  is provided with a closure  6   e , analogous to those described previously, which is traversed by said transfer conduit  4   b . Within the container  2   e  a second collection chamber  16  is defined. The transfer conduit  4   b  terminates within said collection chamber  16  and comprises, typically at the lower end thereof, separation means  13 ′. 
     In an embodiment, said separation means  13 ′ are filtering means, such as for example a bag filter, similar to those described regarding the separation means  13 , but having a porosity P 2  with lower diameter of the pores with respect to said porosity P 1 . Preferably, such porosity P 2  shall be such to allow the separation of corpuscular material of dimensions comprised between 50 micro-meters and 10 micro-meters. 
     The container  2   e  is in flow communication with at least one further container  2   f , through a touch racking conduit  3   c  which connects the lateral walls of the two containers to a level lower than the end of the separation means  13 ′ or the transfer conduit  4   b , depending on which terminates lower. 
     Also the container  2   f  comprises a chamber  31  and a closure  6   f  analogous to those described previously, through which a suction tube  17  is inserted. 
     In the embodiment shown in  FIG. 1 , the container  2   f  is the last of the series, hence the suction tube  17  serves as a conduit for evacuating the biological fluid F previously treated in the device of the invention. In such case, the suction tube  17  is operatively connected with means for moving said fluid which guarantee a flow of the biological fluid F through the device  1 . Such movement means, in the shown embodiment, comprise vacuum pumping means  18 . 
     The treated fluid F is then moved away through the tube  17  and then it is recovered downstream so as to be possibly subjected to further desired analysis. 
     It should be observed that the number of containers  2   a , . . .  2   f  may vary depending on the fluid F treatment needs, possibly being as few as four containers  2   a ,  2   b ,  2   c ,  2   d  and a single collection chamber  12 , should the fluid require a single separation stage. Vice versa, the number of separation or treatment stages may also be greater than the one shown in the drawing, thus requiring providing a greater number of chambers in series. 
     In particular, it may be required to provide at least one further separation stage, for example using filters capable of separating corpuscular material with grain size comprised between 2 and 10 micro-meters, downstream of the second separation means  13 ′ described above. Likewise, between the first separation means  13  and the second separation means  13 ′ there may be provided a stage for separating particles with intermediate grain size. 
     When all the fluid F has been treated, the separation means  13 ,  13 ′ shall be removed to recover the material withheld thereby, which may be possibly used for the desired clinical studies. 
     The means for moving the fluid may be arranged downstream of the device, as illustrated in the figures, or upstream. In this case they comprise pressurized pumping means. 
     The embodiment of the invention device shown in  FIG. 2  is similar to the one of  FIG. 1 , with the difference lying in that the stage of separating the corpuscular material from the fluid F between the first transfer chamber  9  and the first collection chamber  12  is actuated through separation means  113  arranged outside said first collection chamber  12  and comprising a separation system according to the MACCS® technology. Such technology, per se known and available in the market, provides for the advance marking of a cellular species intended to be separated through nanospheres made of super-paramagnetic material (for example, through suitable anti-bodies functionalised with such nanospheres) and the subsequent passage of the fluid containing the cellular species thus functionalised through a column  119  containing a suitable porous filling material and arranged between the poles of a permanent magnet  120 . The cellular species functionalised with the paramagnetic nanospheres is withheld in the column  119 , while the fluid containing the remaining components passes. At the end of the operation, the column  119  is removed and eluted so as to recover the withheld cellular species. The paramagnetic nanospheres are biodegradable, hence a step for removing the marking is not required. Thus, this allows removing a cellular species regardless of the dimensions thereof, but according to the capacity of being functionalised, for example through a suitable antibody. 
     In  FIG. 2 , the closure  6   b  of the second container  2   b  has a second hole for the introduction of an adding means  121  —for example a tube that can be connected to a syringe or provided with a loading hopper—which can be used for introducing the paramagnetic nanospheres for marking the desired cells of the fluid F or for introducing other reagents or markers. 
     The embodiment of  FIG. 2  also comprises separation means  13 ′ comprising conventional filtering means, but such filtering means can also be replaced by the above-mentioned separation means  113  or that the first transfer conduit  4   a  comprises the separation means  13  for conventional filtration, while the separation means  113  described above are associated only to the second conduit  4   b , or even that the device  1  comprises further separation means  13  or  113  downstream. 
     In  FIG. 3  there is shown a further embodiment of the device of the invention, similar to the embodiment of  FIG. 1 , but with the difference that the transfer conduit  4   a  is intercepted by an analysis device  200  for the in line analysis of one or more components of the biological fluid F. Various types of analysis, such as for example spectroscopic analysis (for example, UV spectroscopy for determining DNA or RNA in solution), cells count (“Coulter counter)” or flow cytofluorometer (for determining the dimensions and/or cellular form) can be provided for. 
     For example, the flow cytofluorometer provides for the use of light beam, typically laser light from Argon source, and the determination of the light diffused in the same direction of the incident light (Forward Scatter) or according to an angle of 90° (Side Scatter) through a suitable device for detecting and analyzing results. In the first case there is obtained information on the dimensions of the analysed cells, while in the second case there can be determined the roughness characteristics of the cellular surface and the number of organelles present in the cell. Possibly, the analysed sample can be treated with various fluorochromes free or conjugated with suitable monoclonal anti-bodies, so as to highlight the presence of specific cellular components such as proteins (fluorescein isothiocyanate) nucleic acids (propidium iodide) and lipids (Nile Red) or the enzymatic activity of esterase, peroxidase and peptidase. The fluorochromes can be conjugated to nucleotide probes for identifying DNA or RNA sequences. 
     The cellular components of the biological fluid F can be analysed as they are or in lysate form. In this case, the fluid F may be pre-treated with a suitable lysing agent, typically a lysing buffer. 
     For such purpose, the device of the invention may provide for means  221  for adding reagents such as lysing agents, fluorochromes or other marking agents. The adding means  221  are typically constituted by a tube which places one of the containers  2   a ,  2   b  in flow communication upstream of the analysis device  220  with the external or directly with a source of said reagents. 
     In  FIG. 4  a further embodiment of the invention is shown, in which the device  301  comprises a plurality of modular elements  302   a ,  302   b ,  302   c ,  302   d ,  302   e ,  302   f  connectable in series through suitable coupling means  330 . Each of said modular elements  302   a , . . .  302   f  comprises a chamber  305 ,  309 ,  312 ,  314 ,  316 ,  331  for the decantation, the transfer or the collection of a biological fluid F, depending on the case, and respective closure plates  306   a ,  306   b ,  306   c ,  306   d ,  306   e ,  306   f.    
     The first modular element  302   a  comprises a decantation chamber  305  and communicates with the exterior through at least one channel  304   a  which traverses the thickness of the respective closure plate  306   a , so as to be connected with a bag or another container of a biological fluid F to be treated. One or more further channels  304   a  may be provided for supplying reagents, markers or another material, according to needs. 
     From the lateral wall of the decantation chamber  305  there originates a touch racking channel  303   a  which continues in a channel  332   a  which traverses the lateral wall of the second modular element  302   b  so as to transfer the decanted fluid F in the transfer chamber  309  of said second modular element  302   b . The channel  303   a ,  332   a  is arranged at a level such as to allow a suitable decantation of the fluid F, preferably at the upper portion at half the height of the chamber  305 . 
     The second modular element  302   b  comprises a suction tube  11  which continues, inside the corresponding closure plate  306   b , in a transfer channel  333  which forms a substantially right-angled elbow. The transfer channel  333  continues, inside the closure plate  306   c  of the third modular element  302   c , in a transfer channel  333 ′ having a substantially mirror-like development, which connects said second modular element  302   b  with a third modular element  302   c.    
     The closure plate  306   b  of the modular element  302   b  may comprise a channel  304   b  which places the chamber  309  in communication with the exterior, for supplying reagents, markers or other material. 
     Both the channel  304   b  and the channel  304   a  serve as means for adding said reagents, markers or other material and, if not used, they can be closed by suitable caps  350 . 
     The third modular element  302   c  defines a collection chamber  312  therein. The transfer channel  333 ′ terminates within said collection chamber  312  with a tube  334  which comprises first separation means  13 , adapted to separate a corpuscular component of the biological fluid F (for example, a cellular population, in single elements and/or in cellular aggregates and/or microfragments, or a protein or anything else depending on the mode of use) from the fluid. 
     In an embodiment, such separation means  13  are filtering means. A bag filter such as the one described in the patent application EP-A-2 052 231 in the name of Valenziano Susanna, whose description is incorporated herein for reference, may be advantageously used. Preferably, such filtering means have porosity P 1  capable of withholding corpuscles of dimensions greater 50 micro-meters. 
     The third modular element  302   c  is in flow communication with a fourth modular element  302   d  through a touch racking channel  303   b  which continues in a channel  332   b  which traverses the lateral wall of the fourth modular element  302   d  so as to transfer the fluid F in the transfer chamber  314  of said fourth modular element  302   d . Such conduit  303   b ,  332   b  develops with a substantially horizontal development and connects the lateral walls of the two modular elements to a lower level with respect to the end of the separation means  13  or of the tube  334 , depending on which terminates lower. 
     The fourth modular element  302   d  internally delimits a transfer chamber  314 . A suction tube  15  is inserted into said transfer chamber  314  and continues in a transfer channel  335  obtained in the body of the closure plate  306   d  and which forms a substantially right-angled elbow. The transfer channel  335  continues, within the closure plate  306   e  of the fifth modular element  302   e , in a transfer channel  335 ′ with a substantially mirror-like development, which connects said fourth modular element  302   d  with said fifth modular element  302   e.    
     The modular element  302   e  defines a collection chamber  316  therein. The transfer channel  335 ′ terminates within said collection chamber  316  with a tube  336  and comprises separation means  13 ′. 
     In an embodiment, said separation means  13 ′ are filtering means, such as for example a bag filter, similar to those described regarding the separation means  13 , but having porosity P 2  with smaller diameter of the pores with respect to said porosity P 1 . Preferably, such porosity P 2  shall be such as to allow the separation of corpuscular material having dimension comprised between 50 micro-meters and 10 micro-meters. 
     The modular element  302   e  is in flow communication with at least one further modular element  302   f , through a touch racking conduit  303   c  which continues in a channel  332   c  and connects the lateral walls of the two modular elements to a lower level with respect to the end of the separation means  13 ′ or the tube  336 , depending on which terminates lower. 
     Also the container  302   f  comprises a suction tube  17 . In the embodiment shown in  FIG. 4 , the modular element  302   f  is the last of the series, hence the suction tube  17  serves as a conduit for evacuating the biological fluid F that has been treated in the device of the invention and collected in the chamber  331  of such modular element  302   f . In such case, the suction tube  17  is operatively connected with vacuum pumping means  18  which guarantee a flow of the biological fluid F through the device  301 . 
     The treated fluid F is then moved away through the tube  17  and it is then recovered downstream so as to be possibly subjected to the desired analysis. 
     It should be observed that the number of containers  302   a , . . .  302   f  may vary depending on the fluid F treatment needs, possibly being as few as four containers  302   a ,  302   b ,  302   c ,  302   d  and a single collection chamber  312 , should the fluid require a single separation stage. Vice versa, the number of separation or treatment stages may also be greater than the one shown in the drawing, thus requiring providing a greater number of chambers in series. 
     The device  1 ,  301  according to the invention may be made of any material suitable for the described applications, such as plastic, metal or glass. The dimensions of the containers  2   a , . . .  2   f  or of the modular elements  302   a , . . .  302   f  shall depend on the amount of fluid to be treated, but it shall be generally such to allow the use in an analysis laboratory. 
     From the description above, it can be observed that the handling of the filtered/washed material is advantageously simple and applicable to any type of preparation provided for by the diagnostic cytology, as indicated in the introductory part of the present description and with a possible use for histological material (e.g. VABRA, TURV, etc) using a bag filter suitable in terms of porosity according to the dimension of the tissue fractions to be withheld. 
     Furthermore, the separation device of the invention allows obtaining various types of separation in a single passage, both by filtration and according to various methods, as well as an in-line analysis which may provide some indications to the pathologist. 
     Moreover, the inventive device can be adapted to standardized methods, thus satisfying a long felt need in the state of the art. 
     Obviously, the described device may be subjected—by a man skilled in the art—to various modifications all falling within the scope of protection of the claims that follow. 
     It may be observed that the filtration/separation device described previously may also advantageously be applied to other fields of the art such as botany for the separation for example of cells from culture media, studies in the mineralogy sector for separating powders or other elements, in the wine industry for separating sediments or impurities and similar applications. 
     Furthermore, it may also be provided for sterile sets, previously prepared, that may be directly positioned in line with respect to drainage pipes exiting from the body of the patient, also bedridden, and instantaneously perform some evaluations through detection systems of the calorimetric type for each step, to be defined from time to time depending on the clinical data and the ensuing working diagnoses.