HK1032421A - Method and apparatus for separating particulate matter from a liquid specimen - Google Patents

Description

Method and apparatus for separating particulate matter from a liquid sample

Technical Field

The present invention relates to an apparatus and method for collecting a uniform monolayer of particulate matter. The invention relates in particular to a device for collecting a uniform monolayer of cells from a biological fluid and to a manually or semi-automatically operated method for preparing a monolayer of cells for cytological starting material.

Background of the invention

In various technical fields, the ability to separate substances, usually particulate substances, from a fluid and/or the ability of the device to check for the presence of substances in a fluid is a critical part. Often times, the sample preparation obscures the target cells so that the operation is not sufficiently reliable or is too expensive.

This is also the case in many other fields including detection and/or diagnosis, including environmental testing, radiation detection, malignancy screening, cytological examination, microbiological testing, and hazardous waste contamination, to name a few.

In all of these efforts, limiting factors in sample preparation primordia include proper separation of particulate matter from its fluid carrier (e.g., physiological, biological, and environmental fluids), and easy and efficient collection and aggregation of particulate matter into a form convenient for microscopic observation.

In the case of cytological examinations, a sample of cells is obtained from a patient. Typically, this is achieved by scraping or swabbing a site, as is the case with cervical sampling, or by collecting body fluids, such as from the chest cavity, bladder, or spinal canal by aspiration with a fine needle. In conventional manually operated cytological examinations, particulate matter, including cells and debris in a fluid, is smeared onto a glass slide and subsequently air dried. Smearing results in uneven density and uneven distribution of cells and debris, often obscuring the target cell. Air drying can cause cell distortion and thus prevent accurate detection.

Rapid urine handling to maintain freshness has been shown to ensure accurate quantitative culture results, urinalysis and microscopy. Fresh cells adhere better to the glass slide than cells in stored urine, resulting in a more even distribution of cells on the glass. Delays in handling, careless maintenance of adjustments to inpatients or outpatients, and lack of refrigeration can result in poor glass sheet preparation. One known solution to the problem of delays is the use of chemical preservatives in the urine. The presence of a liquid preservative in a urine sample, in any event, will raise the specific gravity of the sample to an immeasurable degree and limit the potential use of urine in various conventional types of quantitative analysis, such as slide microscopy.

Diagnostics microbiology and/or cytology, particularly in the field of clinical pathology, are based on microscopic examination and other microscopic analysis of cells. The accuracy of the diagnosis and the processing of the best interpretable sample typically rely on proper sample preparation. The preparation of the preparations required for new methodologies such as immunocytochemistry and image analysis is reproducible, rapid, biohazard-free and inexpensive. Conventional cell processing techniques cannot cope with the occurrence of uneven cell density, uneven cell distribution and air-dried formation.

Typically, body fluid samples for cytological examinations are collected from containers containing a preservative solution to protect the cytological specimen during transfer from the collection site to the cytological laboratory. Furthermore, cytological specimens collected from the body cavity by swabbing, smearing, rinsing or brushing are also stored in containers with fixatives (e.g., alcohol or acetone fixatives) prior to transfer to a slide or film for staining or examination.

It would be desirable to provide a container for urine or other biological fluid samples that allows a liquid biological sample to be tested without removing the lid of the urine or biological fluid container. In any event, the prior art fails to address the problem of transferring cells to a slide in a monolayer state for testing without having to repeatedly immerse portions of the device in the sample (thereby increasing the risk of contamination) to form a high quality monolayer on the microscope slide, and to process the sample so that the fluid from which the cells are removed is protected.

Several methods, devices and structures for dispersing cells in a fluid are known. For example, U.S. Pat. No. 5,143,627 discloses a sample container in which a dispersing unit is inserted into a liquid suspension and the dispersing unit is rotated for several minutes. In another example, sputum is processed with the so-called "Saccomanno method", which is a time consuming method and involves many processing steps.

Another limiting factor in optimally preparing particulate matter for microscopic examination includes the solution and/or solutions used to immobilize the particulate matter on a microscope slide or the like.

Cytological samples, which form the examination form of cytological material, may be prepared using well known smear or fluidics techniques. Since there is a considerable time before these samples are stained, covered, etc. for further processing, it is in any case important to apply a fixative on the cytological material as a means of preserving and fixing the cells.

The proper fixation (i.e., preservation) of cytological material such as cells, cell aggregates and minute tissue fragments derived from cytological collections of human or animal tissue is a prerequisite for the accurate diagnosis of disease, particularly malignant tumors. The cytology must be fixed as soon as possible after acquisition to prevent cell distortion.

Air-dried and four chromium dye-stained cytological specimens, although popular abroad, are not commonly used in the united states. In contrast, wet fixation is a well-known cell fixation method, in which a slide is immersed in an alcohol solution, or a fixative is sprayed or an alcohol solution is directly poured into a cytological material to saturate the slide. Cell fixation is a prerequisite for the use of interpretable slide of papanicolaou, hematoxylin, tetrabromofluorescein or other stained cytological specimens.

Generally, alcoholic solutions in the range from 50% to 95% (v/v: methanol, ethanol, isopropanol) with or without other additives such as polyethylene glycol are known as solutions for wet fixation. When an alcohol solution higher than 50% (v/v) is used for collecting and fixing the high protein fluid, in any case, a deposit of protein will be formed and will subsequently harden. Protein deposits make it difficult to transfer immobilized cytology material to a slide for examination, whether the transfer is direct application to the slide, or cell infiltration through a micro-well filter, or centrifugation of cells onto a glass slide coated with a coagulant such as chrome-aluminum gel.

For over a century, tissue fixative mixtures for preserving and preparing tissues for analytical evaluation have been based on formaldehyde. A standard cocktail for tissue preservation and preparation of microtomed tissue is formalin. Formalin is a 3 to 10 percent aqueous solution of formaldehyde, typically containing about 15 percent methanol. The alcohol improves the preservation properties of the solution. Despite a number of disadvantages, most notably the highly toxic and irritant nature, formalin has been selected as a fixative for routine laboratory use because of its rapid response to exposed tissue surfaces to maximize cell preservation. Methanol can be detrimental to the structure of the tissue, making it too fragile or, more commonly, too soft to cut in preparation for a slide. It may also produce color formations or impurities that affect coloration. Nevertheless, formalin containing methanol provides preserved tissue that meets the requirements for cutting and staining for microscopy.

Histologists have long worked to develop effective immunohistochemical and morphological fixatives. In addition, it is necessary to preserve morphological details and antibodies of the tissue in order to perform immunohistochemical detection and antibody determination in the tissue.

Such fixatives make the protein difficult to solubilize. For example, formaldehyde can act as a cross-linking agent that forms covalent bonds between acetaldehyde and specific amino acids to stabilize proteins and turn the cytoplasm of cells into a gel-like state, which can prevent movement of autolytic enzymes. On the other hand, alcohol can be used as a fixative to precipitate proteins by denaturation.

Preferably, the fixative should prevent autolysis and decay and preserve morphological details and antigenicity. Unfortunately, an effective morphological fixative is not necessarily an effective immunohistochemical fixative.

Contrary to the conventional art, the solid substance preparation technique of the present invention can cope with the generation of non-uniform substance density, uneven substance distribution, and sample loss and contamination due to excessive steps in the preparation of the sample. Thus, the preparation according to the invention results in a flat distribution of solids with good morphology, improved visibility, and ease of placement and allows for light absorbance analysis without further processing or handling of the sample.

Summary of the invention

The present invention relates to a device and method for collecting a substance for detection, analysis, quantification, and/or visual observation. The apparatus and method of the present invention are particularly suited for separating particulate matter from biological, physiological and environmental fluids and subjecting the particulate matter to cytological examination in an improved morphology.

A preferred embodiment of the present invention is directed to an apparatus and method for collecting a uniform layer of cells from a urine or other biological fluid sample in a cytological collection apparatus or assay module and transferring the uniform layer of particulate matter onto a slide.

The apparatus and method of the present invention may be configured as a manually operated system or structure, or as a partially automated system or structure.

Such a device according to the present invention overcomes the problems of conventional devices for collecting cells or other particles for cytology by providing a relatively simple mechanism and operation for separating particles from a liquid solution, collecting a near known number of monolayers of cells, and transferring the collected cells onto a microscope slide. In some embodiments of the invention, the components of the device are not placed in the sample of liquid, thereby preventing unwanted contamination of the sample. Additionally, in some embodiments of the invention, the container in which the sample is stored is not open during collection and transfer of the cells, thereby eliminating the possibility of sample contamination during the course of the assay.

In all embodiments of the invention, a monolayer of particulate matter, e.g., cells, in a sample is collected on a filter via two fluid flow branches through and around the filter. Such filters can be found in U.S. patent nos. 5,301,685 and 5,471,994.

A separate container may be sealed by the patient or medical personnel handling the collection. The collection of cells according to the present invention can obtain a uniform cell slide without contamination of the cells with a preservative, a worker or an external substance. Transfer from the collection container to the cytological collection apparatus may be accomplished without pouring or aspirating the collected sample.

The present invention relates to a cell collection and dispensing device that is detachable to allow face-to-face transfer of cells from the device to a slide for microscopic examination. The present invention provides an improved apparatus and method for collecting and transferring a monolayer of cells onto a microscope slide. The efficiency of transferring a monolayer of cells from a filter to a microscope slide has been demonstrated to be very high without local cell loss. Microscopic examination showed that the distribution of cells on the slide was the same as on the filter.

The apparatus of the present invention does not require a trained technician to properly prepare the sample substrate. Thus, time, expense, and expertise are eliminated or reduced as limiting factors in sample preparation of the original material.

The apparatus and methods of the present invention also have advantages in sample preparation because they are suitable for use with fresh, untreated cells, unmodified cells, and are specifically designed to provide a thin uniform layer (up to about 40 microns or slightly more) of solid material. The present invention is particularly useful for collecting cells for Pap smears.

The devices and methods of the present invention have many advantages over conventional microbiology and hematology. The collected cells are located at a predetermined location that is accessible by the radiation source and a wavelength absorbance meter. Since the cells are concentrated in a single layer, they are almost always in a focal plane to eliminate or reduce interference from other particles, and virtually no specialist time and expertise is required in determining the appropriate reading. The minimal overlap of material achieved by the present invention ensures that all material is conveniently observed, and there is little possibility that critical solids are obscured by overlapping solids or debris clumps. Certain embodiments of the apparatus of the present invention may be used in conjunction with other automated equipment for the detection and analysis of any given amount of solid matter. They can also be used to perform detailed analyses on chemical mixtures of substances.

The invention also includes improved apparatus and methods for treating liquids containing particulate matter. The apparatus and method include dispersing particulate matter in a sample, preferably by rotating the sample container about a fixed agitator or rotating an agitator in a fixed sample container. The present invention agitates the sample in the vessel to ensure breaking up of large particulate matter, e.g. mucin in the case of a foam-like sample, and even distribution of cells throughout the liquid. Agitation may occur as a result of relative movement between the sample vessel components, non-uniform movement of the sample vessel, and/or inertial reaction forces of the vessel on the sample.

According to a preferred embodiment of the present invention, structure and means are provided for rotating an agitator relative to a container and/or a sample in a container. As explained in detail below, a preferred embodiment according to the present invention may include a lid in one lid, wherein the agitator is secured to a freely rotating outer lid and the inner lid is secured to a stationary sample container. This relative movement moves the stirrer relative to the sample, thereby dispersing the particulate matter in the fluid.

Furthermore, providing a container lid with a rotatable portion can agitate or disperse the particulate material without inserting an agitation mechanism into the sample, thereby eliminating a source of spotting with commercially available equipment. In a preferred embodiment of the invention, the cap on the sample container may comprise a hollow tube with or without a rotatable dispersion member to facilitate removal of the sample from the container.

In a preferred embodiment of the invention, the lid comprises a first part which is in fixed engagement with the container and a second part which is rotatable relative to the container. As used herein, rotation relative to the container involves relative movement of the first and second portions; the first portion may be fixed and the second portion movable, or the first portion may be movable and the second portion fixed. In a most preferred embodiment, the second or inner portion of the cover is stationary and the first or outer portion is rotatable. In a preferred embodiment of the invention, the stirrer is attached or fixed to the second part of the cover.

An apparatus according to a preferred embodiment of the present invention may be configured to hold, couple, and rotate a portion of a collection container to mix a sample according to the present invention. An exemplary collection vessel includes a vessel or cup adapted to collect and store a sample, a cap having a first position in which the cap is non-rotatable relative to the vessel and a second position in which the cap is rotatable relative to the vessel, and a stirrer attached or secured to a portion of the lid and extending into the vessel. As used herein, different configurations of support, attachment, and rotation may be used to implement a particular function. For example, an apparatus according to the present invention may comprise a vessel support for supporting at least one sample vessel and rotating the vessel by itself, and a sleeve or holder for attaching and holding a portion of a cap, the cap being in communication with a stirring element. Alternatively, the support may hold the container in a fixed position, and a pulley, sleeve, or holder may engage and rotate a portion of the cap fixedly attached to an agitator. In a preferred embodiment of the invention, a sleeve is attached to the inner portion of the cap and constrains the inner portion of the cap in a stationary position relative to the outer portion of the cap.

The structure or structure that engages a portion of the cap or that engages the vessel generally includes any component that positions, secures and/or moves a portion of the cap or the vessel. Exemplary components include, but are not limited to, a sleeve, one or more belts, one or more pulleys, one or more elastic bands, or the like.

The present invention is also an apparatus for processing a fluid into one or more components, typically to remove particulate matter from the fluid. The present invention relates to devices and methods for collecting fluids, such as biological, physiological, or environmental fluids, and removing particulate matter from the fluids without centrifugation, and for diagnosing and testing the matter. In a preferred embodiment of the invention, the particulate matter is collected in a monolayer and in a predetermined spatial arrangement.

Although the cytological collection apparatus according to the invention may be used with any biological fluid, it is particularly useful for preparing test samples from urine and associated cells for pap smear. The type of substance to be treated does not limit the present invention. In a most preferred embodiment of the invention, the liquid is urine and the particulate matter is cells. The particulate matter processing apparatus of the present invention may also be used to isolate and collect fresh cells and/or microorganisms from a biological fluid for DNA interrogation and staining analysis while the cells are lysed by a suitable buffer.

In the case of cervical examinations of the uterus, a long handled brush or broom is used for cervical scraping. The handle is then shortened by a snapping or telescoping motion and the brush is placed into the collection container. Typically, the container must be open to allow the brush to be removed during testing. This treatment increases the potential for contamination because the lid of the container must be open, and the brush will normally retain the cells if the test is not performed immediately after the cells are collected, and the operator must come into contact with it.

According to a preferred embodiment of the invention, these problems are avoided by providing a system in which the brush not only remains in the container, but can also be used to disperse the collected cells while stirring. Furthermore, the device of the present invention is a closed system; once the device is closed, the device need not be opened to process any cells collected on the brush.

Furthermore, providing a container lid with a rotatable portion can agitate or disperse particulate matter without having to insert a stirring mechanism into the sample, thereby eliminating a source of contamination that can cause spotting in commercially available equipment.

The present invention also relates to cytological collection and testing assemblies that include a cytological collection apparatus, a disposable refill, and/or other components as described below, and a means for holding ingredients for use with the admixture. The cytological collection assembly may also include a refill filter, a disposable refill, and/or other components, ingredients, or solutions typically used in cytological examinations. The assembly may also include washing, fixing, and/or buffer solutions. An assembly for the neck may include a brush or broom, and a fluid suitable for storing the brush used before particulate matter on the brush is disposed of by the filter assembly.

A preferred embodiment of the present invention is also directed to a tissue fixative cocktail for histopathological procedures that can rapidly penetrate the tissue surface to maximize preservation of cells, leave minimal staining artifacts, and allow precise staining. The present invention is also directed to providing a cytological and histological fixative formulation that can fix and preserve cells, cell aggregates and minute tissue fragments in a liquid suspension.

The present invention also relates to the provision of a fixative composition which will hold the tissue sample in the event that the sample is taken with the histological material for further histological processing.

The present invention is also directed to providing a fixative formulation that allows a liquid suspension of cells, cell aggregates and tissue debris to be transported under conditions commonly encountered in postal delivery so that a cytologist, cytotechnologist, physician or other remote user, who is experienced in preparing cytology specimens, may fix the cytology specimen for later use, thereby technically satisfying the cytology specimen slides prepared therefrom.

In another aspect, the invention relates to a method of pre-processing tissue for cutting, staining and/or microscopic observation, wherein the sample tissue is preserved prior to dehydration using the storage-stable tissue cocktail solution of the invention.

A unique cytological and histological fixative formulation and methods of using the same are disclosed. The formulation can fix individual cells, clumps of cells and minute pieces of tissue in a liquid suspension; minimizing precipitation of protein in the suspension; optionally eliminating or reducing red blood cell contamination of the cytological material and cytological specimen slides; where the sample is collected with a histological material for further histological processing, the tissue sample may be retained; and allows the transport of cytological material under conditions normally encountered in postal delivery, so that users in remote areas who are not available with cytologists, cytotechnologists, doctors or other personnel experienced in preparing cytological specimens may have cytological specimen slides that technically meet the requirements.

According to another aspect of the invention, the material collection device may further include additional modules for handling fluids that may be disassembled or assembled. For example, the fluid may be processed with a material collection module, along with a debris removal module, a chromatography module, and assay module, or with a combination of these modules and other devices. These and other modules or processing schemes provide the features required to incorporate a sample preparation device according to the present invention.

The apparatus and method of the present invention have many advantages over conventional cytology. The location of the cells within a predetermined area creates an advantage in saving time for reviewing the slide. Problems such as cells being located outside the coverslip or at the end of the frosted glass are eliminated. Since the cells are in a monolayer, low magnification objectives are most often used to view the slide when a 10X objective is used, the cells always being in a single focal plane. Even with a 40X objective, most cells are in focus. This avoids frequent refocusing and saves time.

These and other objects, features and advantages of novelty are readily apparent from the accompanying drawings that illustrate embodiments of the invention.

Description of the drawings

Fig. 1 is a cross-sectional view of a first preferred embodiment of the present invention.

Fig. 2 is a cross-sectional view of a particulate matter separation chamber according to the present invention.

FIG. 3 is a cross-sectional view of a liquid flow path through a particulate matter separation chamber.

FIG. 4A is a top view of the base and recess arrangement forming the bottom of the particulate matter separation chamber.

Fig. 4B is a top view of the bottom of the particulate matter separation chamber showing the modified dial surface of the recessed portion.

FIG. 4C is a top view of the bottom of the particulate matter separation chamber showing the modified grid-like surface of the recessed portion.

FIG. 5 is a cross-sectional view of the base, hollow tube, and container, removed.

Fig. 6 is a bottom view of the top portion of the particulate matter separation chamber.

FIG. 7 is a cross-sectional view of a bottom portion of a particulate matter separation chamber showing an optional channel and an optional flap.

FIG. 8 is a cross-sectional view of a bottom portion of a particulate matter separation chamber showing an optional passage and an optional O-ring.

FIG. 9 is a cross-sectional view of a bottom portion of a particulate matter separation chamber showing an optional channel and an optional flap.

Fig. 10 is a cross-sectional view of a second preferred embodiment of the present invention.

Fig. 11 is a cross-sectional view of a third preferred embodiment of the present invention.

Fig. 12 is a combination of bottom and side views of a filter assembly in accordance with a preferred embodiment of the present invention.

FIG. 13 is a cross-sectional view of an apparatus for use in a semi-automated process according to a preferred embodiment of the present invention.

Fig. 14 is a schematic view of the device of fig. 13 in a first position.

Fig. 15 is a schematic view of the device of fig. 13 in a second position.

Detailed description of the invention

The present invention is a sample container that includes a particle-location separation chamber or module in fluid communication with the sample container.

The invention is also an apparatus for processing a liquid into one or more components, typically to remove particulate matter from the liquid.

The present invention also includes apparatus and methods for collecting fluids, such as biological, physiological, or environmental fluids, removing desirable particulate matter from the fluid without the use of centrifugation, and identifying and testing the particulate matter. In a preferred embodiment of the invention, the particulate matter is collected in a monolayer and in a predetermined spatial arrangement.

The invention also includes an improved apparatus and method for treating a liquid containing particulate matter. The apparatus and method include passing the liquid through a particulate matter separation chamber having a seat for a porous filter means, the seat including structure for aligning the collected particulate matter in a predetermined spatial arrangement, structure for enhancing the flow of the liquid through the particulate matter separation chamber, and/or structure for promoting and maintaining porosity and/or compressing the porous filter means located within the particulate matter separation chamber.

The present invention is also an improved apparatus for collecting a liquid, typically a biological liquid. The apparatus comprises a particulate matter separation chamber having one or more of the following: an acquisition section; a porous filter device comprising a membrane for separating particulate matter from a liquid and a porous support frit; the porous filtering means forming at least two liquid passages through the particulate matter separating chamber; a housing defining a predetermined spatial arrangement of the collected particulate matter; a particulate matter separation chamber having a concentric passageway; a channel having one or more resilient members; a housing having one or more resilient members; a housing or base with a post; a housing having one or more predetermined surface modifications; a housing having one or more components for creating a predetermined spatial arrangement of particulate matter at a collection site; and a structure to enhance the flow of liquid through the particulate matter separation chamber.

An apparatus according to the present invention may further comprise structure configured to agitate the sample collected in the sample container. Exemplary configurations include, but are not limited to, a sample container with a relatively rotatable cap, or a portion of a cap; a cap or cap portion movable relative to the sample container; and a tube or the like extending into the sample container. The tube may carry one or more components for agitating the sample. The cap may also include a portion that may be fluid-tightly engaged with a portion of the lid of the particulate matter separation chamber. The cap may also include a portion that engages a portion of the cap in a liquid-tight, but non-fluid-tight manner.

The device according to the invention may also comprise a pump or a syringe. The pump or syringe may optionally include one or more components configured to allow a predetermined amount of fluid to enter the pump or syringe.

The invention also includes preparing a sample for microscopy using an apparatus according to the invention for processing a liquid, and collecting particulate matter at a collection site within the apparatus.

The invention also includes a method comprising collecting fluid into a chamber for analysis of a substance, collecting particulate matter at a collection site, and transferring the particulate matter collected at the collection site onto a microscope slide or the like. Preferably, both acquisition steps can be performed indoors.

The device according to the invention may also comprise one or more separate parts. In a preferred embodiment of the invention, the apparatus comprises a separable particulate matter separation chamber. In a most preferred embodiment of the invention, the apparatus comprises a porous filtration device at least partially retained at the top of the chamber.

The invention also includes an assembly having an assay module with a particulate matter collection element according to the invention, a liquid sample container, and a pump for moving liquid from the sample container to the assay module.

In a preferred embodiment of the invention, the fluid sample in the container is in fluid communication with a particulate matter separation chamber or module for separating particulate matter from the fluid and collecting the separated particulate matter at the collection site. In a most preferred embodiment of the invention, the separated particulate matter is collected in the form of a monolayer at the collection site. A preferred embodiment of the present invention further comprises a hollow tube to provide fluid communication between the sample container and the particulate matter separation chamber. More preferably, the hollow tube includes means to agitate the sample and/or disperse particulate matter in the sample.

In another embodiment of the invention, the device comprises a sample container and a particulate matter separation chamber as described above, and a pump, syringe or the like. In this embodiment of the invention, various structures provide a fluid flow path from the sample container through the particulate matter separation chamber and into the pump or syringe.

As used herein, the term "specimen" or "sample" refers to any fluid that is associated with a solid substance, such as a particulate substance, and requires the collection of particulate components from the specimen for the purpose of determining their identity or presence in the specimen. Typically, the fluid component of the sample is a liquid. In any case, the fluid may be air or gas. As an example, it is desirable to determine the presence of cancer cells or certain proteins in a biological fluid, such as urine. In another embodiment, it may be desirable to assess the nature of contamination, such as molecular contamination, in ultra-pure water used in the electronics industry. Other typical fluids include, but are not limited to, bodily fluids such as blood, spinal or amniotic fluids, bronchial washings, sputum, fine needle aspirates, groundwater, industrial process fluids, electronic or medical dialysis fluids, to name a few. It is to be noted here that the present invention is not limited by the type of fluid that can be treated.

As used herein, "particulate matter" refers to any matter in a fluid that can be collected and preferably evaluated by cytological examination. Typical particulate materials include, but are not limited to, cells and cell debris, proteins, molecules, polymers, rubbers, stabilizers, antioxidants, accelerators, silicones, alkyds, polysulfide rubbers, waxes, heat-fusible plastics, bacteria, pesticides, and herbicides. Specific exemplary polymeric materials include, but are not limited to, polyethylene, polypropylene, polyisobutylene, polyacrylonitrile, polyethylene glycol, polyvinyl chloride, polystyrene, polysulfide, plexiglass, polyethylene terephthalate, bisphenol a (a common environmental pollutant), ethyl cellulose, nitrocellulose, polyurethane, and nylon. Specific exemplary biological substances include cancer cells, including cancer cells that discriminate between metastatic and standard, proteins, nucleic acids, antibodies, or the like.

As used herein, the terms "adapted for communication," "intercommunicating," or similar terms refer to any device, structure, or method for establishing fluid flow through a system, as is well known to those of skill in the art. A typical structure is shown in the drawings. For example, a catheter may have a connector adapted to receive or connect with a mating connector or another catheter. As used herein, the term "connector" refers to any structure used to form an interface or to connect itself to another component. These connectors or fittings form fluid flow paths through the various components of the device, assembly, or system. Typical connectors include, but are not limited to, mating connectors, such as luer type, screw type, friction type, or bonded together connectors.

As used herein, "adapted to engage," "engage," or similar terms refer to complementary structures that may be aligned, engaged, coupled, or located adjacent, against, or within each other. Typical constructions include the connectors described above.

The apparatus 10 according to an exemplary embodiment of the present invention, shown in fig. 1, includes a sample container 20 storing a fluid sample 23, a particulate matter separation chamber 30 with a porous filter device, and a pump 40. Fig. 1 also shows a hollow tube 50 and includes a dispersion member.

Each of these components will now be described in detail. Collection container

According to the present invention, the sample container 20 comprises any container suitable for storing a fluid 23, preferably a biological fluid. The exemplary container includes a side wall 21 and a bottom wall 22 that together contain a sample 23. The sample container 20 also has an open end 24 for collecting, storing, or containing the fluid 23. Typical fluids include, but are not limited to, biological fluids, such as body fluids, waste fluids, or the like. Typical bodily fluids include urine or other biological fluids such as blood, cerebrospinal fluid (CSF), bronchial washings, sputum or fine needle aspirates.

The structure and materials used to make the container (and any components that make up the apparatus of the invention) can be any of a wide variety of materials, shapes, and sizes. For example, the cup may be made of any material compatible with the fluid to be treated. It will be appreciated that the assembly of the container and side walls to the base plate may be any conventional assembly. In a preferred embodiment of the invention, the base plate 22 is a conical member, as shown in FIG. 1. Optionally, floor 22 or sidewall 21 may include one or more ribs or the like (not shown) that extend into the interior of container 20. One embodiment of the invention, described in more detail below, shows that these fins are desirable where the sample in the container is agitated by rotating the container.

As shown in fig. 1 and 2, an apparatus according to the invention further comprises a cap 31. In a preferred embodiment of the present invention, the cap 31 is configured or adapted to receive a lower portion 32 of a particulate matter separation chamber 30. The cap 31 may have different configurations to perform the desired function. A preferred embodiment is shown in fig. 2. Cap 31 may include a downwardly extending member 51 to form an engagement with sidewall 21 of container 20. It is noted herein that cap 31 may be of any configuration or shape that closes or seals open end 24 of container 20.

The cap also includes a portion 52 with an opening 53 adapted to receive the lower portion 32 of the particulate matter separation chamber 30. Although the connection between the cap portion 52 and the lower portion 32 may be made in a variety of configurations, the lower portion 32 preferably includes a slot 53 adapted to receive a projection 54 of the cap portion 52. In a most preferred embodiment of the invention, the connection is a snap connection, and the engagement between lower part 32 and projection 54 allows lower part 32 to rotate relative to cap part 52. This structure is preferably liquid tight, in a most preferred embodiment of the invention the seal is liquid tight, but not gas (e.g. air) tight.

A preferred construction of the cap 31 will now be described with respect to fig. 1. The cap 31 may have a different structure to achieve the desired function. According to this embodiment of the invention, the cap 31 is provided with structure and means for moving the outer cap 71 relative to the inner cap 72. The outer cap 71 is preferably fixed to and/or in fluid communication with the tube 50. In a preferred embodiment of the invention, the outer cap 71 and the tube 50 are rotatable relative to the inner cap 72 while the inner cap 72 is secured to the container 23. This relative movement between the outer cap 71 and the inner cap 72 moves the sample within the container 23 relative to the agitator 58A (fig. 1), the brush 58B (fig. 10), or the brush 58C (fig. 11).

In embodiments of the invention with inner and outer caps, the inner and outer caps are preferably adapted to engage each other so that they do not rotate relative to the respective cap until the final closure of the container. It is to be noted here that at least initially, the corresponding cap corresponds to an integral cap. When the capping unit is secured in a predetermined position, whatever structure is required to secure the inner cap 72 in place relative to the outer cap 71 may be pulled apart or disengaged so that the inner and outer caps are free to rotate relative to each other. For example, the inner cap 72 may be used to seal the container and the outer cap 71 snap-fits onto the inner cap 72. In this embodiment of the invention, a tab or the like on the inside of the outer cap 71 prevents relative movement between the inner and outer caps when the respective cap is in the first position. When the outer cap 71 is moved to the second position, for example, by pulling the tab open, the outer cap 71 may be rotated relative to the inner cap 72. Alternatively, it is contemplated that a temporary spacer (not shown) may initially axially retain the inner and outer caps in the spaced apart position. In securing inner cap 72 to container 20, the septum is removed and outer cap 71 is slid axially over inner cap 72 to a position free to rotate relative to inner cap 72.

Another or additional structure in embodiments of the present invention includes a cover having a flexible wall 55, preferably circular or oval, connected to or supporting a portion 45 of the particle position separation chamber 30. In a most preferred embodiment of the invention, the wall 55 includes one or more spaced apart slots (not shown). The intention is to provide the walls with a degree of flexibility so that, if desired, the lower portion of the particle position separation chamber 30 can be disengaged from the cap 31 (see, e.g., fig. 5).

Fig. 5 also shows another embodiment of the invention, and refers to a cap 31 with an aperture through which either the stirrer 58A or the brush 58C can be placed into the container 20. In a preferred embodiment of the invention, the aperture or opening in cap 31 may be covered with a removable and/or penetrable cover to prevent contamination of the interior of container 20 prior to preparation of container 20 for use. For example, a brush 58B or the like may be used to collect a cervical specimen, and the cover may then be removed from the cap 31, placing the brush 58B in the container 20.

According to another preferred arrangement, the inner cap 71 may be provided with a collar (not shown) axially external to the tube 50 and extending partially into the container 20. The collar redirects the sample 23 back into the container 20 as it occurs when, for example, vortex agitation is performed. This is very advantageous because the ferrule has little or no resistance to relative rotation between the inner cap 71 and the outer cap 72. Further, it is contemplated that the outer cap 72 may have a connecting tube formed thereon for connection with the tube 50. A connecting tube may be formed on the outer cap 72 to coaxially project into the ferrule. Thus, a collapsible or frangible tube 50 can be used in its elongated shape to collect a sample and then changed to its compressed shape and attached to the hub. This arrangement according to this embodiment will also further reduce the likelihood of sample contamination by minimizing the storage time between sample collection and testing thereof. Particulate matter separation chamber

According to the invention, an apparatus according to the invention comprises a particulate matter separation chamber which may be of various shapes. One exemplary shape is shown in fig. 2. Any chamber 30 suitable for receiving a particulate matter collection assembly 33 may be used.

As shown in fig. 1 and 2, the particulate matter separation chamber 30 is preferably two chambers formed by a top portion 41 and a base portion 32. In a preferred embodiment of the invention, the top portion 41 and the base portion 32 are releasably connected; in any event, another structure or arrangement of chambers that can provide access to the porous filter arrangement 35 is suitable. In a preferred embodiment of the invention, the base portion 32 includes a generally annular sidewall 47, optionally including a serrated portion 63 (shown in FIG. 4A) that engages the sidewall 44 and base 42 of the top portion 41. It has been found that the optional serrated portion 63 of the lower portion 32 facilitates removal of the lower portion 32 from the top portion 41. Top portion 41 and base portion 32 may be connected or secured to one another using any connector or device capable of providing a liquid or fluid tight fit, such as a luer-type (threaded or unthreaded), a helical thread-type, a friction-type, a taper fit connection, or a snap connection (as described).

The base portion 32 includes a side wall and a floor adapted to receive a particulate matter filter assembly 33. The base portion 32 may also include a central aperture or orifice 34 in communication with the hollow tube 50. In a preferred embodiment of the present invention, the hollow tube 50 extends into the sample container 20. In a preferred embodiment of the invention, the base portion 32 may be a separate structure that is rotatable relative to the cap 31. To facilitate centrifugal rotation while maintaining a fluid-tight fit, base portion 32 may be matingly engaged with cap 31 by a tongue and groove arrangement (see FIG. 2).

In one embodiment according to the present invention, the base portion 32 of the particulate matter separation chamber 30 includes a floor or base 39. As shown in fig. 4A-4C, the base 39 may include one or more spaced ribs or protrusions 60. The configuration, size, and shape of the protrusion 60 is preferably sufficient to prevent flush contact of the porous means 35 with the base 39. In the embodiment shown in FIG. 4A, the protrusions 60 are concentric rings.

Another structure will be described in detail below. In a preferred embodiment of the invention, the protrusions 60 function in one or more of the following ways: the protrusions 60 may break the apparent tension between the porous filter arrangement 35 and the base 39 in use; as the porous filter device 35 is pulled out of the base 39, the first porous medium 36 is no longer in contact with the base 39; the protrusions 60 may even out the pressure distribution of the porous filter arrangement in the particulate matter separation chamber 30; the protrusions 60 may prevent or contain pressure of the porous filter device; and the projections 60 may be configured to cause any collected particulate matter to be distributed in a predetermined shape or space.

In accordance with the present invention, the surface of the base 39 may include one or more structures, shapes, or surface textures to enhance the ability of the porous filter device 35 to detach from the base 39, to facilitate a predetermined spatial distribution of particulate matter at the collection site, and/or to prevent or contain pressure from the porous filter device 35. A preferred embodiment of the present invention includes concentric projections such as projection 60 described above. Other structures include, but are not limited to, a lattice, a mesh, or the like, concentric squares or rectangles, or some continuous or intermittent structure, nubs, bumps, particles, or the like (see fig. 4B and 4C). It is to be noted that any element, structure, or chemistry that provides a texture on the surface of the base 39 to perform the above-described functions is suitable for use in the present invention.

In a preferred embodiment of the invention, the surface of the base is made in the form of a grid (see fig. 4C). In another preferred embodiment of the invention, the surface of the base is configured as a sundial or clock dial (see figure 4B). Both of these embodiments, as well as the other surface structures disclosed herein, facilitate rectangular collection of particulate matter at a collection site in a predetermined spatial arrangement. The configurations shown in fig. 4B and 4C are particularly desirable because the footprint of the base surface treatment can be transferred to a microscope slide and a coordinate system can be used to locate and identify specific particulate matter, such as cancer cell rectangles. It has been found that a substantial portion of the particulate matter is collected in the region at the collection site corresponding to the region 75 opposite the base. In contrast, the projections 76 correspond to regions on the collection surface where less particulate matter is collected. These areas will be imprinted on the microscope slide when the collection surface is in contact with the slide.

For example, it is noted that granular cells can be found at the 20 o' clock face angular position shown in FIG. 4B, and a technician reading a slide according to the present invention can thereby identify and locate the cells. Marking microscope slides in this manner advantageously speeds up the viewing of the slides and advantageously improves the ability of the technician to discover previously identified substances of interest. In connection with the present invention, the one or more structures on the surface of the base provide for reliable orientation of particulate matter as it is collected at a collection site and transferred to a microscope slide. For example, one suitable coordinate-identifying structure may be an arrow 71 as shown in FIG. 4B or the like.

According to another embodiment of the invention, the base 39 and/or lower portion 32 may optionally include a channel 70 or the like, examples of which are shown in FIGS. 4B, 4C and 7-9. In a preferred embodiment of the invention, the base 39 is slightly angled outwardly toward the channel 70. The slightly inclined base 39 and the channel 70 help to enhance the flow of fluid through the particulate matter separation chamber 30 and to reduce the surface tension of the base 39 on the filter device 35, both of which help to detach the porous filter device 35 from the lower portion 32 of the particulate matter separation chamber 30. This aspect of the invention is a structure that facilitates detachment of the porous device.

The additional structure shown in fig. 7-9 serves to or involves facilitating the flow of fluid through the particulate matter separation chamber 30 and also involves the disengagement of the porous filter arrangement 35 from the lower portion 32. Fig. 7 shows flap 72 extending from the end of base 39 down into channel 70. Fig. 8 shows an O-ring 73 or the like located within the channel 70, the upper surface of the O-ring 73 preferably being slightly above the plane of the base 39. This ensures that the O-ring 73 engages a portion of the porous filter arrangement 35 when placed in the lower section 32. Fig. 8 shows a flap 74 extending upwardly from the exterior of base 39, which ensures that flap 74 engages a portion of porous filter assembly 35 when it is placed in lower section 32. In a preferred embodiment of the present invention, flap 72, O-ring 73, and flap 74 are made of an elastomeric material, the preferred construction of which is shown in FIG. 9.

According to the present invention, the particulate matter separation chamber 30 may be configured to receive a porous device 35 having a particulate matter collection location 36 adapted to collect particulate matter as a fluid containing the particulate matter passes through the chamber 30.

A porous device 35 having a collection site 36 adapted to collect a substance may be placed across the fluid flow path, the collection site 36 and hollow tube 50 being in communication. The porous means 35 within the substance separation chamber is preferably adapted to define at least one fluid flow channel having first and second branches, a first branch 61 extending through the collection site 36 and a second branch 62 bypassing the collection site 36 (see, e.g., fig. 3).

In a preferred embodiment, the present invention comprises a porous filter device 35 having a first porous medium 37 adapted to prevent the passage of particulate matter, and a second porous medium 38 adapted to allow the passage of fluid. The second porous medium 38 may or may not be designed to remove particulate matter from the fluid 23, as selected by the needs of the particular apparatus. In a preferred embodiment, the first porous medium 37 is adapted to collect or collect particulate matter from the fluid 23, and more preferably, to collect or collect a uniform or monolayer of particulate matter. A preferred embodiment also includes a second porous medium adapted to support the first porous medium 37.

The nature of the substance used in the porous medium, the compatibility between the materials selected for the porous medium and the liquid to be treated are all factors that are considered in selecting a particular material for the porous medium for a given application.

The porous filter means 35 may comprise a unitary structure having a first porous medium 37 with a density and/or pore size suitable for preventing the passage of cells therethrough and a second porous medium 38 with a density and/or pore size suitable for the passage of cells therethrough.

In a preferred embodiment, the porous filter means 35 comprises a first porous medium 37 comprising a porous polycarbonate membrane, adapted to prevent the substance of interest from passing therethrough. Porous filter arrangement 37 may also include a second porous media 38 comprising a dense filter or filter plate. The thickening filter may be made of polypropylene or high-density polyethylene POREX  porous plastic. In a preferred embodiment of the present invention, the second porous media 38 may include a toothed or serrated downstream portion 64, an example of which is shown in FIG. 2. It is noted here that portion 64 is a structure and configuration that reduces or improves the pressure of the porous filter assembly 35 when placed in the particulate matter separation chamber 30.

It should be noted that various types of porous filter devices 35 and the present embodiment are used interchangeably. Although polycarbonate membrane 37 is particularly suitable for use in the cell collection device of the present invention, other porous membranes are also suitable. Typical porous membranes are well known in the art and are disclosed in U.S. patent nos. 5,471,994 and 5,301,685.

The pore size of the porous membrane 37 is preferably from about 0.22 microns to about 8 microns, more preferably from about 1 micron to about 6 microns, and most preferably about 2 microns, which allows for the retention of particulate matter, e.g., cells, having a size greater than 3 microns. The membrane is adapted to allow fluid to pass through while preventing particulate matter from passing through. The second porous medium 38 is adapted for fluid passage and may also remove particulate matter from the fluid 23. The pores of the second porous media 38 range in size from about 5 to about 60 microns, preferably from about 15 microns to about 45 microns, and most preferably about 35 microns.

Those skilled in the art will appreciate that the size of the pores of the porous membrane 37 and the porous depth filter 38 may be adjusted to collect particulate matter at the collection site depending on the type and/or size of the matter to be collected. In a preferred embodiment of the invention, the size of the pores is chosen such that the substance forms a homogeneous layer at the collection site, preferably a monolayer of the substance. For example, from about 3 microns to about 40 microns or slightly more have been shown to be effective, but it should be noted that the present invention is not limited by a certain range of pore sizes.

In a most preferred embodiment of the present invention, the first porous medium 37 is attached to the second porous medium with a liquid soluble binder. Such soluble binders include, but are not limited to, sugar mixtures, gels, and the like.

The first porous medium 37 and the second porous medium 38 may be positioned in any manner that is capable of performing the functions described herein. Those skilled in the art will appreciate that the porous filter means 35 can be constructed and arranged differently as required to achieve a particular purpose. For example, the first and second porous media may be separate, spaced apart media; the two media can be laminated together; the first media and the second porous media may be integrally or releasably engaged together; the acquisition component may comprise a high density region that mimics the function of said first porous medium and a low density region that mimics the function of said second porous medium. These different configurations are well within the skill of the art. The structure and various types of the constitution of the porous means will be described in detail below.

As shown in fig. 12, a porous support 38 with at least one through hole 73, preferably located near the periphery of the porous support, provides a direct suction path so that the filter membrane 37 remains on the open support 38 when the particulate matter separation chamber 30 is opened and the membrane 37 is exposed for further processing.

In another embodiment of the invention, lower portion 32, tube 50, and rib 58 comprise an integral unit and are separable from cap 31 to facilitate removal of the integral structure from container 20. A typical structure of this embodiment of the invention is shown in figure 5. Pump and method of operating the same

According to the invention, the sample container 10 comprises a pump 40. In a preferred embodiment of the present invention, the pump 40 is a syringe or the like for changing the pressure in the device so that fluid can be drawn from the sample container 20 through the particulate matter separation chamber 30.

According to the invention, the pump 40 may have different configurations. In a preferred embodiment of the present invention, the pump 40 includes an end portion that forms a cover portion 41 of the particulate matter separation chamber 30. The lid portion 41 includes a base 42 or the like to engage a downstream portion of the porous filter arrangement 35. In a preferred embodiment of the invention, the base 42 positions the porous filtering means 35 within the cover so that the porous filtering means 35 cannot move during use. In a most preferred embodiment of the present invention, the base 42 includes a plurality of protrusions or posts 43 sized, shaped, and numbered to position the porous filter arrangement 35 within the particulate matter separation chamber 30 to substantially evenly distribute pressure against the porous filter arrangement and reduce or prevent pressurization of the porous filter arrangement 35 that would affect the flow of fluid through the porous filter arrangement 35.

In a preferred embodiment of the present invention, lid portion 41 is releasably connected to bottom portion 32 to form particulate matter separation chamber 30. The lid portion 41 may be connected to the base portion 32 in any manner or configuration that enables the lid portion 41 to be removed from the base portion 32. In a preferred embodiment of the invention shown in fig. 2, the lid portion 41 includes a downwardly extending side wall 44 with a flange 45 or the like for releasable and/or resilient engagement with a projection 46 or the like on the base portion 32.

The flow of fluid through the collection device will be induced by maintaining a pressure differential between the source of the fluid and the destination of the fluid. A typical method of creating this pressure differential may be by applying pressure to any part of the system (e.g., for vessel 20) on the inlet side of particulate matter separation chamber 30; applying a vacuum to any part of the system (e.g., a syringe) at the outlet side of the chamber; or any form of pump, such as a vacuum glass fiber filter (manufactured by Genex corporation); a gravity height difference; or a flexible, collapsible container, such as a sample container, which can be squeezed to force fluid through the particulate matter separation chamber and into the syringe. In a preferred embodiment of the invention, a syringe draws fluid from the collection cup through the chamber. Hollow pipe

According to a preferred embodiment of the present invention, the sample container 20 includes a tube 50 or the like for drawing the fluid 23 into the particulate matter separation chamber 30. Typically, the tube 50 is hollow and open or openable at both ends. Tube 50 includes an open end 51 near the bottom of the collection chamber and may also include one or more apertures 52 into tube 50. The open end 51 and/or the apertures 52 allow for testing of the various fluid layers and deposits as the fluid is drawn into the particulate matter separation chamber 30.

According to another embodiment of the improved invention, the hollow tube 50 includes at least one protrusion or rib 58A or the like, as shown in FIG. 1. In a preferred embodiment of the invention, the hollow tube 50 is rotatable and the fins 58A agitate the liquid sample, and in a most preferred embodiment, disperse the cells and/or particulate matter, and/or disintegrate any large particulate matter such as a viscous liquid like object. In another preferred embodiment of the present invention, the hollow tube 50 and the lower section 32 are integrally formed, and the lower section 32, the tube 50, and the rib 58A are rotatable relative to the sample container 20. For example, if the container is rotatable, optional ribs on the sidewall and/or bottom of the container may create a concentric movement of the sample in the container, the presence of the ribs 58A will disrupt the movement. Alternatively, lower portion 32, tube 50, and rib 58A may rotate within a stationary container.

As another embodiment of the present invention, as shown in FIGS. 10 and 11, the agitator 58 may comprise a fiber, brush, swab, or brush or the like. Preferably, the fibers or brushes are adapted to disperse the particulate matter within the container when the sample is swirled relative to the agitator, brush, or broom. In a most preferred embodiment of the invention, the brush or swab is also adapted for collecting particulate matter from the patient, for example, a brush or swab for the neck or the like. It is noted here that the brush may be fixed to a portion of the cap 31, or the cap 31 may include an aperture, collar or the like for mating engagement with a portion of the handle at the opposite end of the brush. Mixer with a rotating shaft

Fig. 13-15 illustrate an apparatus for a semi-automated method according to a preferred embodiment of the present invention. In particular, FIGS. 13-15 illustrate a most preferred embodiment which includes a support sleeve A for positioning and rotating the container and inner cap 72. In the most preferred embodiment of the present invention, outer cap 71 is attached with one or more elastic bands B which are not attached to outer cap 71 in its relaxed or first position (FIG. 4) and which are attached to and support outer cap 71 in its secured or second position (FIG. 15) while inner cap 72 and container 20 are rotating. In another embodiment, the strap B may be a drive belt that rotates the outer cap 71, tube 50 and stirrer 58 as a unit relative to the container 20 and inner cap 72. Assembly

The present invention also relates to a particulate matter collection and testing assembly comprising collection device 10 as an integral unit. The assembly may include at least one sample container 20, at least one particulate matter separation chamber 30, at least one pump 40, and at least one opening filter device 35. An assembly according to the present invention may also include a replacement filter, a replacement disposable container, and/or other components or solutions typically used in particulate matter testing or inspection, e.g., cell testing. Fixing agent

The mixture according to the invention comprises one or more vehicles, preferably alcohols, in a volume of between about 35% and about 45%, ketones, in a volume of between about 2% and 3%, diluents, preferably diols or triols, in a volume of from about 1% to 3%; from about 0.4% to 3% by volume of a cross-linking agent, preferably acetaldehyde; from about 0.5% to about 2% by volume of glycerin; one or more detergents and/or dispersants, preferably nonionic, from about 0.01% to about 0.05% by volume; and from about 45 to about 65% by volume of a buffer. In a preferred embodiment of the invention, the pH of the mixture is between about 4 and about 7.

The invention also comprises a method for preparing particulate material, such as cells and the like, for cytological or histological examinations, comprising collecting the particulate material in a homogeneous layer, preferably a monomolecular layer, and immobilizing the cells in a mixture according to the invention.

Table 1 summarizes the ranges and preferred concentrations of the ingredients of a mix formulation according to the present invention.

TABLE 1

Composition (I)	Ranges (by volume,%)	Preferred (by volume,%)	Examples of the invention
				Solvent(s)	35-45	37-42	Alkanols
Ketone	2-3	2.1-2.4	Acetone (II)
				Diluent	1-3	1.6-1.9	Diols, triols
Glycerol	0.5-2	0.8-1.2	Glycerol
				Crosslinking agent	0.4-3	0.6-0.8	Acetaldehyde
Cleaning agent	0.01-0.05	0.02	Nonidet P40
				Buffering agent	45-65	50-55	Triphosphoric acid ester

One mixture according to the present invention includes one or more solvents to penetrate the tissue of the cells, dehydrate the cells, and/or inhibit bacterial and life viability. In a preferred embodiment of the invention, the solvent is a relatively slow penetrating alkanol that rapidly fixes the sample when mixed with other reagents. It denatures proteins by precipitating, depositing glycogen, and breaking down fats and lipids. The alkanol can be any well known alcohol having one to four carbons, for example, methanol, ethanol, n-propanol, isopropanol, n-butanol, and various branched butanols. The most preferred solvent is a mixture of methanol and isopropanol, typically about 30% and about 10% by volume, respectively.

Ketone is a fixative with similar action to alcohol, only glycogen is not easy to preserve. The ketone acts as a fixative and also allows all the cocktail to penetrate the cell. The preferred ketone is acetone.

The diluent forms a coating on the sample and helps prevent drying. Preferred diluents are diols and triols, most preferably polyethylene glycol (PEG), e.g., PEG-1500 (average molecular weight about 1450), also known as polyethylene glycol.

Glycerol prevents drying of the cells during sample processing. Cells that have been stored in the fixative solution for a considerable time become hardened due to the fixation process and are not easily scattered on the slide. Glycerol helps to flatten the cells on the slide.

The cross-linking agent and protein end groups act to cross-link the molecules and form insoluble products. The protein family includes amino, imino, amido, peptide, hydroxyl, carboxyl and sulfenyl. Methylene bridges are also commonly formed between similar groups such as NH2 and NH, but are believed to be reversible by water washing. Certain crosslinking agents, such as formaldehyde, are preservatives.

The preferred crosslinking agent is acetaldehyde, most preferably formaldehyde.

The detergent is a non-ionic detergent and dispersant to solubilize the protein and membrane components to reduce cell aggregation. Preferred detergents are Nonidet P40 or TritonX-100, both of which are well known detergents.

The buffer maintains the pH of the solution between about 4 and about 7 and provides a medium for delivery. The preferred buffer is Tris, a well known buffer. According to the invention, the buffer may also include a fixative to deposit the nuclear protein and one or more permeability-maintaining agents. The preferred nuclear protein precipitating agent is glacial acetic acid, typically ranging from about 0.2% to about 0.3% by weight, and helps maintain the buffer pH between about 7.4 and about 7.8. A preferred osmotic retention agent is dextrose, which typically ranges from about 0.7% to about 0.8% by weight.

In a preferred embodiment, the ingredients of the active fixative may be dissolved in a suitable solvent such as distilled water, which solution may then be used as a fixative in a variety of ways, as will be apparent to those skilled in the art. For example, the fixative solution may be used to preserve tissue samples that are to be shipped or transported to a testing site. In this process, a vial or jar having a liquid seal is filled with the reagent of the present invention, and the tissue sample is placed in the reagent-containing vial until it reaches the site where it will be further processed. Any suitable diluent does not alter the important chemical and physical characteristics of the formulation that may be used.

The tissue to be examined using the fixative of the present invention may be prepared for histological studies in any well-known conventional manner, such as by using paraffin, cutting devices, staining, fixing on a glass slide, or other procedures commonly used prior to performing a microscope or other examination. The present invention thus provides a safe, convenient and effective fixative solution that can be used in many known histological procedures employing such solutions. Method of producing a composite material

The invention also includes methods for removing particulate matter from a fluid and methods for transferring particulate matter, such as cells, onto a microscope slide. In contrast to currently available methods, the use of membrane filtration provides a means for placing cells on microscope slides with minimal overlap. This will make the observation clear and the identification with good accuracy.

The method includes collecting a fluid sample containing particulate matter from collection container 20. The container 20 is then capped with a device comprising one or more of the following ingredients: a lid 31, a particulate matter separation chamber 30, and a pump 40. The pump 40 is then activated, drawing fluid from the container 20 through the particulate matter separation chamber 30 into the pump 40, for example, by drawing a plunger in a syringe.

As fluid is drawn from the container 20 into the pump 40, the fluid will flow through the porous filter device 35, as shown in fig. 3, thereby forming a monolayer of particulate matter at the collection site 37. Once a monolayer of cells is formed, the fluid flow at the center of the porous filter device 35 will decrease and increase toward the edges of the porous filter device 35. This may be due to the blocking of the flow of fluid as the collected cells form a monolayer on the surface of the porous filter device 35. When the monolayer has covered a substantial portion of the porous filter device surface 37, the flow of fluid will bypass the first porous medium 37 and pass through the region of the side edge from which the second porous medium 38 protrudes. Thus, the second porous medium 38 extending beyond an end wall or side edge of the top portion acts as an outlet (with low flow resistance) preventing stacking or collection of cells in more than one monolayer. The fluid may pass through the porous device as many times back and forth as desired.

The pump 40 can then be removed from the base 31 to expose the porous filter arrangement 35. The first porous media 37 is readily accessible by the porous filter arrangement 35 once removed from the lower section 32. Alternatively, the top portion 41 of the pump 40 can be removed from the lower portion 32 and the porous means 35 can also be removed from the recessed portion 32.

The first porous medium 37 may then be pressed against a microscope slide to cause the collected particulate matter at the collection site, if collected, to be transferred to the slide. This would allow the practitioner to conduct cytological examinations without the intervention of a thin film well or delay due to processing requirements.

Since the clarity of the cells depends on the fixation, the fixation should preferably be performed immediately after the cells are placed on the slide. Too long a delay between preparation and fixation may allow drying due to cell exposure, which would be detrimental to the cell structure. Furthermore, the air-dried product may be detrimental to subsequent coloration results. One exception is that air drying is used as a fixation step when cells are stained by Wright-Giemsa.

In another embodiment of the invention, a monolayer of cells may be immobilized directly at the collection site. This may be accomplished by first placing a monolayer of cells on the collection site of the cytological collection apparatus and then flowing a solution containing a fixative, such as alcohol or acetone, through the cytological collection apparatus, as described above. Of course, in the most preferred embodiment of the invention, the above-described fixative will be used. Alternative constructions

The material collection device or module described above may be used in conjunction with other suitable filters or processing equipment. The devices typically include other debris and/or assay devices or modules that may be attached to the chamber 10. Typically, these additional modules include a chamber having an inlet and an outlet, and will also include a filtration, assay, or detection unit spanning the fluid flow path in the chamber. For example, the device may comprise a chamber comprising inlet and outlet portions defining a flow path between the inlet and outlet; a filter positioned across the flow path; and a freely movable chromatography and/or assay element, such as a substrate bead, placed on the outlet side of the filter. The chromatography and/or assay element is free to mix with substances in the fluid, capture the substances, and then be able to assay for the presence of the substances. Suitable devices are described in us patent 4,953,561; 5,224,489, respectively; 5,016,644, respectively; 5,139,031, respectively; 5,301,685, respectively; 5,042,502, respectively; and 5,137,031.

It is within the scope of the present invention to make a single slide from a patient's specimen, multiple slides from a single patient's specimen, or multiple slides from multiple patient's specimens. It is noted that a patient's sample may be administered individually, in a batch operation, or processed in a continuous manner. Additional slides for other staining uses are readily prepared. Human papillomavirus assays can be performed, for example, on additional slides using new methods such as immunocytochemistry or in situ hybridization. More slides are required for the development of oncogene products or other immunocytochemistry assays. The different fixatives required for these tests can be easily integrated with the test procedure, since this preparation does not require fixing the slide in only one way.

The staining most widely used in cytology to visualize cellular changes is the papanicolaou staining method. The colorant, which can be used in both gynecological and non-gynecological conditions, consists essentially of a counterstain of the blue nucleus with orange, red and green cytoplasm. Nuclear staining indicates a color pattern involving normal and abnormal cells, while cytoplasmic staining helps to illustrate the origin of the cells. The success of this approach can be attributed to the ability to observe a range of factors, including the definition of nuclear details and the differentiation of cells. This coloring method also results in color preparation that is very suitable for viewing and may reduce the burden on the eyes. This same slide preparation procedure can be used for virtually all forms of cytology.

Again, the use of disposable components, including all disposable components, is focused on the biological risks involved. Ultimately, enhanced cell display, leading to improved cytological interpretation, augments the cytological effects by providing more context and reliable diagnosis for the patient.

Also, the captured microorganisms may be cultured in a culture medium. After a monolayer of cells is collected in the cytological collection apparatus, the collection site may be backwashed with a fluid to remove any microorganisms collected from the collection site.

In bacterial testing, a first porous medium may be incubated with a Qualture apparatus (not shown) to determine the presence of specific bacterial colonies. The Qualture device is a plastic capsule containing a filter membrane and four dehydrated, selective media nutrient pads.

The Qualture technique is more sensitive than the agar plate method and allows for a faster determination of the desired diagnosis. The screen of the apparatus often completes the precipitation and the determination of the isolates caused by the bacteria in one step within 4 to 6 hours. Experiments have shown that separation with 50 ml of fluid is good and sensitive.

While the invention has been described with specific preferred embodiments, it is not to be limited to those embodiments. Those skilled in the art may devise additional embodiments, examples, and modifications that are encompassed within the invention.

Claims (25)

1. An apparatus for separating particulate matter from a fluid, comprising:

a container having a cap, said cap comprising a rotatable element;

a porous device in fluid communication with the reservoir and adapted to remove particulate matter from the liquid, said porous device being disposed within a chamber; and

a pump in fluid communication with the chamber.

2. An apparatus for treating a liquid containing particulate matter, comprising:

a container having a cap;

a porous device located within the chamber, said chamber including a first portion adapted to engage a portion of said cap; and

a pump, said pump including a portion adapted to engage a second portion of said chamber;

said chamber being adapted to be divided between said first portion and said second portion, and said second portion being adapted to hold said porous means.

3. The apparatus of claim 1, wherein: further comprising a chamber having a lower portion adapted to engage said porous means, said lower portion having a base with a predetermined surface structure.

4. The device of claim 3 wherein the surface structure comprises protrusions in a predetermined configuration.

5. The device of claim 4, wherein the configuration is a lattice.

6. The device of claim 4, wherein the configuration is a clock dial.

7. The device of claim 1 or 2, wherein said cap comprises a tube extending into said container.

8. The apparatus of claim 7 wherein said tube includes at least one fin located at the end of the tube extending into said container.

9. The device of claim 2 wherein said first portion is adapted to be in fluid tight engagement with said cap.

10. The device of claim 9 wherein said liquid seal is not a fluid seal.

11. The apparatus of claim 3, wherein: also included is a base with a slope extending radially from a center.

12. The apparatus of claim 1, wherein: further comprising a chamber having a lower portion adapted to engage said porous means, said lower portion having a channel in a side wall adjacent said lower portion.

13. The apparatus of claim 12 wherein said passage further comprises a resilient member positioned within said passage.

14. The apparatus of claim 13 wherein said resilient member is an O-ring or a flap.

15. The device of claim 3, further characterized in that said base further comprises a flap adapted to engage the porous means.

16. A device as claimed in claim 1 or 2, wherein the pump includes one or more stoppers for drawing a predetermined amount of liquid into the pump.

17. The device of claim 2 wherein said second portion comprises at least one nub adapted to engage the porous means.

18. The device of claim 1, further comprising a chamber having a lower portion with serrated sidewalls.

19. The apparatus of claim 1, further comprising a chamber having a base with one or more structures to reduce surface tension of the base.

20. The device of claim 1, further comprising a chamber having one or more structures that reduce the ability to retain the porous device in the lower portion of the chamber.

21. The device of claim 1, further comprising a chamber having one or more structures that increase retention characteristics for an upper portion of the chamber.

22. An apparatus for treating a liquid containing particulate matter, comprising:

a container having a cap;

a porous means located within the chamber, said chamber comprising a first portion adapted to engage a portion of said cap, said first portion comprising a tube extending into said container, said first portion being rotatable when engaged with said cap, said first portion being configured to release said porous means; and

a pump, said pump including a portion adapted to engage with said second portion of said chamber;

said chamber being adapted to be divided between said first portion and said second portion, and said second portion being adapted to retain said porous means.

23. The apparatus of any of claims 1-22, further comprising:

a motor for agitating the liquid by relative rotation between said container and said chamber.

24. A method for separating particulate matter from a liquid, comprising:

collecting the liquid into a container;

passing the liquid through a chamber having porous means adapted to remove particulate matter from the liquid;

separating a portion of said chamber, wherein said separating step comprises retaining the porous means in a portion of the chamber.

25. The method of claim 24, further comprising:

agitating the liquid in said container.