Chemistry: analytical and immunological testing – Composition for standardization – calibration – simulation,... – Preservative – buffer – anticoagulant or diluent
Reexamination Certificate
2000-12-22
2003-03-11
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Composition for standardization, calibration, simulation,...
Preservative, buffer, anticoagulant or diluent
C436S008000, C252S408100, C252S380000, C252S397000, C435S040500, C435S040510, C435S040520
Reexamination Certificate
active
06531317
ABSTRACT:
TECHNICAL FIELD OF THE INVENTION
The present invention is directed to a fixative composition, the use of the fixative composition in cytological procedures, and a method of preparing particulate matter, such as cytology, hematology and microbiology specimens, for cytological or histological examination comprising collecting a particulate matter sample in a uniform layer, preferably a monolayer, and fixing the particles in a composition according to the present invention.
BACKGROUND OF THE INVENTION
In a wide variety of technologies, the ability and/or facility in separating matter, typically particulate matter, from a fluid is a critical component in the ability to test for the presence of substances in the fluid. Too often, interference associated with sample preparation obscures the target cells to such a degree that the process is not sufficiently reliable, or too costly. Such issues apply to many other fields which involve detection and/or diagnosis, including environmental testing, radiation research, cancer screening, cytological examination, microbiological testing, and hazardous waste contamination, to name just a few.
All that is required for a cytological examination of a sample is that a sample of cells be obtained from the patient, which can typically be performed by any one of the number of well known techniques including any of the following techniques: direct scrapings, scraping or swabbing an area (as in the case of cervical samples), brushings, collection and concentration of a fluid specimen with a standard preparatory centrifuge, fine needle aspiration or any other known method, or by collecting body fluids, such as those obtained from the chest cavity, bladder, or spinal canal. In a conventional manual cytological examination, the cells in the fluid are then transferred onto a glass slide for viewing. In a conventional automated cytological examination, a filter assembly is placed in the liquid suspension and the filter assembly both disperses the cells and captures the cell on the filter, and the filter is removed and placed in contact with a microscope slide. In all of these endeavors, a limiting factor in the sample preparation protocol is adequately separating solid matter from its fluid carrier (e.g., a variety of fluids, such as physiological, biological and environmental), and in easily and efficiently collecting and concentration the solid matter in a form readily accessible to microscopic examination.
Another limiting factor in optimally preparing the particulate matter for microscopic examination involves the solution and/or solutions for fixing the particulate matter to a microscope slide or the like.
A number of urine or other biological fluid specimen containers have been developed to allow liquid biological specimens to be tested without removing the lid of the urine or biological fluid container. None of the prior art solves the problem of transferring cells in a uniform layer to a slide for examination while at the same time preserving the fluid from which the cells were taken.
Currently, body fluid samples are collected for cytological examinations using special containers. These containers usually contain a preservative solution for preserving the cytology specimen during shipment from the collection site to the cytology laboratory. Furthermore, cytology specimens collected from the body cavities using a swab, smear, flush or brush are also preserved in special containers with fixatives (e.g., alcohol or acetone fixatives) prior to transferring cells onto the slide or membrane for staining or examination.
Diagnostic microbiology and/or cytology, particularly in the area of clinical pathology, bases diagnoses on a microscopic examination of cells and other microscopic analyses. The accuracy of the diagnosis and the preparation of optimally interpretable specimens typically depends upon adequate sample preparation. New methodologies such as immunocytochemistry and image analysis require preparations that are reproducible, fast, biohazard-free and inexpensive. Different cell preparation techniques of the present invention address the issues of non-uniform cell densities, uneven cell distribution and air drying artifact. These preparations have resulted in an even distribution of cells that have superior morphology, which has improved light microscopic visualization and has allowed for the use of image cytometry instruments.
The solid matter preparation techniques of the present invention address the issues of non-uniform matter densities, uneven matter distribution, and sample loss due to the number of steps involved in the sample preparation. The preparations of the present invention result in an even distribution of solids that have superior morphology, improved visualization, and are readily positioned and available for light absorbence analysis without the need to further manipulate or prepare the sample.
Properly fixing (i.e., preserving) cytologic material such as cells, cell aggregates and small tissue fragments derived from cytologic collections of human or animal tissue is a prerequisite to the accurate diagnosis of disease, especially cancer. Cytologic material must be fixed as soon as possible after obtaining the material to prevent cell distortion.
Cytologic specimens, which constitute the examinable form of the cytologic material, may be prepared by well-understood smear or fluid techniques. Because there may be a considerable lapse of time before these specimens are further processed by staining, applying a cover slip, and so forth, however, it is important to apply a fixative to the cytologic material as a means of preserving and fixing the cells.
Air-dried and tetrachrome-dye stained cytologic specimens, although popular abroad, are not generally used in the United States. Rather, wet fixation, either by the immersion of slides into an alcohol solution, by saturation of slides with a spray fixative or by directly discharging cytologic material into an alcohol solution, is a known method of cell fixation. Cell fixation is a prerequisite for interpretable Papanicolaou, Hematoxylin and Eosin or other stained cytologic specimen slides.
Generally, alcohol solutions, with or without other additives such as polyethylene glycol, ranging from 50% to 95% (v/v: methanol, ethanol, isopropanol) are known solutions for use in wet fixation. When alcohol solutions greater than 50% (v/v) are used for collecting and fixing fluids high in protein, however, a protein sediment forms which subsequently hardens. Protein sedimentation makes the fixed cytologic material difficult to transfer to glass slides for examination, regardless of whether the transfer is done by direct application to the glass slide, by cytofiltration through a small pore filter, or by cytocentrifugtion onto glass slides coated with an adhesive such as chrome aluminum gelatin.
For over a century, tissue fixative compositions used to preserve and prepare tissue for analytical evaluation have been based on formaldehyde. The standard composition employed for tissue preservation and the preparation of thin-cut tissue for microscopic examination is Formalin. Formalin is a 3 to 10 percent solution of formaldehyde in water, usually containing about 15 percent methyl alcohol. Alcohol improves the preservative properties of the solution. Despite numerous disadvantages, most notably high toxicity and irritant properties, Formalin remains the fixative of choice in typical laboratory applications owing to its rapid reaction with exposed tissue surfaces and consequent maximized cellular preservation. Methanol may adversely affect the texture of the tissue, rendering it too brittle or, more usually, too soft for ease in cutting for slide preparation. It also may produce pigmented artifacts or impurities which interfere with staining. Formalin containing methanol nevertheless provides preserved tissue which can be satisfactorily sectioned and stained for microscopic examination.
Histologists have long endeavored to develop effective immunohistochemical fixatives and morphologic fixatives. Mo
El-Amin Mariamena
Guirguis Raouf A.
Foley & Lardner
LaMina, Inc.
Wallenhorst Maureen M.
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