Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving fixed or stabilized – nonliving microorganism,...
Reexamination Certificate
2000-03-28
2002-07-30
Leary, Louise N. (Department: 1623)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving fixed or stabilized, nonliving microorganism,...
C435S014000, C435S243000, C435S004000, C435S975000, C435S810000
Reexamination Certificate
active
06426195
ABSTRACT:
TECHNICAL FIELD
This invention relates to a novel method for silver staining a pathologic sample.
BACKGROUND OF THE INVENTION
Humans are subject to a variety of fungal infections, including histoplasmosis, coccidioidomycosis, blastomycosis, paracoccidioidomycosis, sporotrichosis, cryptococcosis, candidiasis, aspergillosis, mucormycosis, mycetoma, chromomycoses and phaeohyphomycosis. Pathogenic fungi are opportunistic pathogens, usually causing disease only in immunocomprised hosts such as the very old, the very young, patients taking immunosuppressive agents, or patients whose immune systems are compromised by another disease or infectious agent. Such patients often show both an increased susceptibility and an increased frequency of fungal infections.
Diagnosis of many fungal infections involves the use of histopathological techniques which permit identification of the fungal pathogen with a high degree of reliability. These techniques allow the identification of the fungal pathogen based on size, morphology and the tissue that it infects. The techniques can be performed on pathologic samples such as tissues obtained from biopsy, autopsy or necropsy, for example from bone marrow, liver biopsy, lymph nodes, oral ulcerations, or infected tissues, or on bodily fluids including sputum, urine, blood, lymph, pleural fluid, cerebrospinal fluid, and exudate from draining lesions. The reliability, accuracy, and reproducibility of the technique used is very important because the identification of the organism involved is critical for diagnosis and treatment. Staining methods in particular are useful both for making the fungal pathogens visible and as diagnostic tools, in that the ability to be visualized with a given stain is characteristic of particular species.
Most fungi are relatively large and their cell walls are rich in polysaccharide. These polysaccharides can be oxidized and the cells walls then visualized using silver staining methods. Grocott's methenamine-silver nitrate procedure (“Histotechnology: A Self-Instructional Text,” p. 194-196, F. L. Carson, ASCP Press, Chicago, Ill, 1997) is one silver staining technique used for demonstration of fungal organisms in pathologic samples. The procedure requires several steps of incubation at different temperatures from 56° C. to 60° C. Dilute ammoniacal silver staining techniques such as developed by Churukian et al. (Lab. Med. 17(2):87, 1986) have been used to stain Pneumocystis carinii and fungi using steps requiring a similar temperature range.
The use of caustic, toxic solutions at elevated temperatures complicates the technique and presents a number of safety concerns. Exposure of laboratory personal to hot alkali solutions containing toxic silver is undesirable, and requires additional safety precautions. Manually preparing and maintaining baths at precise temperatures adds to the difficulty of performing the technique and in its reproducibility. Additionally, the need for elevated temperatures at only a few steps of the method complicates automation of the procedures, requiring additional components for raising and monitoring the temperature. This increases the cost of the automated device as well as the space it requires in what are often-crowded laboratories. High temperature incubations can also produce increased background staining and decrease the useful life of staining solutions.
There is a need in the art for a silver staining method which does not require elevated temperatures. Ideally, such a method would be useful in both manual and automated screening procedures. Such a method could be used to detect the presence of fungi, encapsulated bacteria, and other pathogens such as
Pneumocystis carinii,
as well as in place of silver staining techniques used to visualize glycogen, basement membranes, mucins, urate crystals, melanin, or neural components.
SUMMARY OF THE INVENTION
A method is provided that permits silver-staining of a pathologic sample at ambient temperatures. The method involves contacting the sample with a silver diamine complex under basic conditions in the presence of a reducing agent carrier. The method permits staining of sample components which previously were visualized through methods requiring incubation steps at elevated temperatures. The ability to use lower temperatures in the method decreases the exposure of laboratory personnel to the risk of scalding and to fumes produced from hot, toxic solutions. If desired, however, the novel method can also be performed at elevated temperatures as used in previous methods, for example up to about 60° C.
The method of the invention permits intense staining of sample components with low staining background. Fungal spores and hyphae, for example, can be intensively stained with low background at room temperature.
The method therefore does not require preparation of water baths at precise temperatures or control of bath temperatures. Additionally, the method permits automation using devices lacking temperature controls. Devices prepared to perform the method can thus be simpler, with fewer components to maintain and fewer controls to adjust.
In one embodiment, the method involves contacting a pathologic sample with a solution of chromic acid in order to oxidize components of the sample. The sample is then contacted with a basic solution comprising a silver diamine complex and sucrose which results in the depositing of silver metal within the sample. The sample is then treated with a solution of gold chloride, which tones the sample and improves visualization of the stained components. The sample is then treated with a solution of sodium thiosulfate to halt development and to complex with and thereby remove any remaining silver or gold ions. Finally the sample is counterstained by contacting it with a solution of Light Green stain.
A kit comprising reagents useful for performing the method is also provided. In one embodiment the kit comprises an oxidizing agent, a silver diamine complex, a reducing agent carrier and a stopping agent, each of which is received within a housing. A toning agent and a stain can also be included in the kit.
DETAILED DESCRIPTION OF THE INVENTION
A method for silver-staining a pathologic sample which can be performed at ambient temperatures is disclosed. The method involves contacting the sample with a silver diamine complex under basic conditions in the presence of a reducing agent carrier. The reducing agent carrier slowly releases a reducing agent during the course of the reaction, allowing time for the silver diamine complex time to permeate and be localized within the sample prior to reduction of the silver from the complex by the reducing agent. The sample can then be treated with a toning agent, a stopping agent, and a stain to improve visualization. A kit comprising reagents useful for performing the method is also disclosed.
The method of the invention was derived in order to provide a room temperature silver staining technique that could replace Grocott's Methenamine-Silver Nitrate method for staining fungal pathogens, encapsulated bacteria,
Pneumocystis carinii,
glycogen and mucin. Examples of pathogens which can be stained by this method include
Actinomyces israelii, Actinomyces bovis, Aspergillus fumigatus, Blastomyces dermatitidis, Candida albicans, Coccidioides immitis, Cryptococcus neoformans, Histoplasma capsulatum, Nocardia asteroides, Pneumocystis carinii,
and
Sporothrix schenckii.
The invention can, however, be used in many other types of silver staining methods and can be performed at the elevated temperatures used in previous methods. These methods include, for example, silver impregnation methods (Argentaffin method, Argyrophil method, Churukian and Schenk's argyrophil method, Alcoholic silver nitrate method for argyrophil cells), methods for staining axons and neuronal processes in the central nervous system (Bielschowski's silver stain for axons in frozen and paraffin sections; Marsland, Glees & Erikson's method for axons in paraffin embedded tissues), techniques for staining a
Aggarwal Sunil Kumar
Chen Taiying
Su Sheng-Hui
Zhong Shude
Leary Louise N.
Thoeming Charles L.
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