By a News Reporter-Staff News Editor at Life Science Weekly — From Washington, D.C., NewsRx journalists report that a patent application by the inventor Berndt, Klaus W. (Cockeysville, MD), filed on October 16, 2013, was made available online on July 3, 2014 (see also Becton, Dickinson And Company).
The patent’s assignee is Becton, Dickinson And Company.
News editors obtained the following quote from the background information supplied by the inventors: “Gram staining is one of the most frequently performed procedures in modern microbiology laboratories. Such procedures are used to broadly classify bacteria as ‘Gram-positive’ or ‘Gram-negative.’ After affixing a bacteria-containing sample to a microscope slide, the sample is treated using staining reagents such as crystal violet in combination with Gram’s iodine. This first step stains all bacteria a deep blue or violet. The principal difference between ‘Gram-positive’ and ‘Gram-negative’ bacteria is that in ‘Gram-positive’ samples, the staining reagents are absorbed within the whole cellular structure, while in the ‘Gram-negative’ samples, staining occurs only superficially. Consequently, when the sample is subsequently treated with a decolorizing agent (such as acid alcohol), Gram-negative bacteria tend to lose their color, while Gram-positive bacteria remain stained blue or violet.
“Gram stains are conventionally prepared and analyzed manually. Manual Gram staining is labor-intensive, requires skilled personnel, and may fail to achieve optimum staining characteristics in the sample set. Non-optimum staining may result in false Gram-positives as well as false Gram-negatives.
“There have been different attempts to automate the Gram staining procedure. See, for example, U.S. Pat. No. 4,029,470 to Wilkins et al. (‘Wilkins’). Similarly, the Aerospray Slide Stainer, available commercially from Wescor, Inc. of Logan, Utah is a rudimentary automated staining apparatus. Other staining devices include the Midas III Slide Stainer, commercially available from Merck KGaA of Darmstadt, Germany; the Poly Stainer, which is commercially available from IUL Instruments GmbH of Koenigswinter, Germany; and the Automated Gram Stainer, commercially available from the GG&B Company of Wichita Falls, Tex. and described generally in U.S. Pat. No. 6,468,764 to Gibbs et al. None of these instruments or techniques, however, allow for the capability of applying staining and/or decolorizing agents before, during, and/or after examination and/or image processing of a sample slide.
“Consequently, there exists a need for an improved automated staining method and system.”
As a supplement to the background information on this patent application, NewsRx correspondents also obtained the inventor’s summary information for this patent application: “The present invention, in various embodiments, provides a method and system that overcomes many technical problems with regard to the optimization of staining procedures (such as Gram staining, for example) for biological samples. Specifically in one embodiment, the invention provides a method for staining a sample, comprising steps for operably engaging a sample with a sample stage of an imaging system, and recording images of the sample using the imaging system before and after an application of a staining reagent and a decolorization reagent to the sample. The method also comprises generating a difference image based at least in part on a comparison of the images of the sample recorded before and after the application of the staining reagent and the decolorization reagent to the sample, and correcting the application of the staining reagent and the decolorization reagent based at least in part on the difference image. The method further comprises reapplying at least one of the staining reagent and the decolorization reagent according to the correcting step and generating a final image of the sample so as to differentiate at least one target of interest in the sample that may be rendered discernible by the reapplying step.
“Some embodiments further provide a method for performing a staining procedure for a sample comprising steps for recording a first image of the sample and applying a staining agent to the sample so as to prepare a stained sample comprising a plurality of stained entities. Some method embodiments further comprise steps for washing the sample with a washing solution prior to recording the first image of the sample so as to ensure that each sample is washed to establish a ‘zero’ level of staining prior to recording the first image (which provides a basis for comparison with images recorded of the sample after the application of at least one of the staining agent and the decolorization agent).
“The plurality of stained entities may include at least one of a plurality of stained entities and a plurality of superficially stained entities. In some such embodiments, the staining agent may comprise a Gram stain such that the superficially stained entities, if present, comprise a plurality of Gram-negative entities, and such that the stained entities, if present, comprise a plurality of Gram-positive entities. The method may further comprise recording a second image of the stained sample and generating a first difference image by comparing the second image to the first image so as to determine a location of at least one of the stained entities on the surface.
“The method may further comprise applying a decolorizing agent to the stained sample so as to prepare a partially decolorized sample wherein at least a portion of the staining reagent is removed from the superficially stained entities, and recording a third image of the partially decolorized sample. The method may also comprise generating a second difference image by comparing the third image to the second image so as to determine a location of at least one of the superficially stained entities on the surface. Finally, the method may also comprise analyzing the second difference image to determine an exposure time during which the decolorizing agent could be applied to the partially decolorized sample to substantially decolorize the superficially stained entities without substantially decolorizing the stained entities.
“In some embodiments, the method may further comprise applying the decolorizing agent to the partially decolorized sample for the determined exposure time so as to prepare a substantially decolorized sample wherein the superficially stained entities are substantially decolorized and wherein the stained entities are not substantially decolorized. In some such embodiments, the method may further comprise: (1) recording a fourth image of the substantially decolorized sample; (2) generating a third difference image by comparing the fourth image to the second image, wherein the third difference image depicts the location of at least one of the superficially stained entities on the surface; and (3) generating a fourth difference image by comparing the fourth image to the first image, wherein the fourth difference depicts a location of at least one of the stained entities on the surface. In method embodiments wherein the locations of the stained entities and the superficially stained entities are at least partially discernible in at least one of the third and fourth difference images, the method may further comprise analyzing a morphology of the stained entities and/or the superficially stained entities.
“Various embodiments of the present invention may also provide systems for performing an optimized staining procedure for a sample. In one embodiment, the system comprises a flow chamber defining channel in fluid communication with a supply of a staining agent and a supply of a decolorizing agent. Furthermore, in some such embodiments, the flow chamber may comprise a surface configured for operably engaging the sample therewith. The system may also comprise a fluidics system in fluid communication with the flow chamber, the supply of the staining agent, and/or the supply of the decolorizing agent.
“According to such system embodiments, the fluidics system (in cooperation with the flow chamber, for example) may be configured for applying the staining agent to the sample so as to prepare a stained sample comprising a plurality of stained entities, wherein the plurality of stained entities may include at least one of a plurality of stained entities and a plurality of superficially stained entities. As described above with respect to various method embodiments of the present invention, the staining agent may comprise a Gram stain such that the superficially stained entities, if present, comprise a plurality of Gram-negative entities, and such that the stained entities, if present, comprise a plurality of Gram-positive entities. According to various system embodiments, the fluidics system (in cooperation with the flow chamber, for example) may be further configured for applying the decolorizing agent to the stained sample so as to prepare a partially decolorized sample wherein at least a portion of the stain is removed from the superficially stained entities.
“In various system embodiments, the flow chamber may be adapted for receiving a slide defining the surface for operably engaging the sample therewith. According to such embodiments, the flow chamber may further comprise a flow channel housing defining a slide aperture configured for receiving the slide such that the sample is disposed substantially between the slide and the flow channel housing when the slide is disposed in the slide aperture. Furthermore, the flow channel housing may comprise a substantially translucent material such that the imaging system is capable of recording images of the sample while the sample is disposed between the slide and the flow channel housing.
“The system may also comprise an imaging system disposed adjacent to the flow chamber such that the sample is positioned within a field of view of the imaging system. In such system embodiments, the imaging system may be configured for monitoring an application of at least one of the staining agent and the decolorizing agent via the flow chamber. Furthermore, the imaging system may also be configured for generating a difference image by comparing at least one image of the sample obtained before a first application of the decolorizing agent and at least one image of the sample obtained after the first application of the decolorizing agent. The imaging system may also be configured to be capable of determining an exposure time for a second application of the decolorizing agent based at least in part on the difference image so as to substantially decolorize the superficially stained entities without substantially decolorizing the stained entities such that the stained entities may be more readily discerned from the superficially stained entities.
“The system may also comprise, in some embodiments, a controller device in communication with the imaging system and the fluidics system. The controller device may be further configured to control the application of at least one of the staining agent and the decolorizing agent based at least in part on the images and/or difference images generated by the imaging system.
“In some system embodiments, the imaging system may comprise a camera device configured to be capable of recording images of the sample while the sample is disposed in the flow chamber (such as between the slide and the flow channel housing, for example). According to some such embodiments, the imaging system may also comprise an actuator device operably engaged with the camera device and configured for adjusting a position of the camera device relative to the flow chamber. Furthermore, in some system embodiments, the imaging system may also comprise an image processing computer configured for generating the difference image. In some such embodiments, the image processing computer may be configured for generating the difference image using processes that may include, but are not limited to: image subtraction; image addition; image ratio calculation; and combinations of such processes.
“As described generally herein with respect to various method embodiments, the imaging system may be configured for recording a first image of the sample and subsequently recording a second image of the stained sample after applying the staining agent to the sample using the fluidics system in cooperation with the flow chamber. The imaging system may be further configured for generating a first difference image by comparing the second image to the first image so as to determine a location of at least one of the stained entities (i.e. the ‘Gram-positive’ entities, in some embodiments) on the surface. Furthermore, the imaging system may be further configured for recording a third image of the partially decolorized sample after applying the decolorizing agent using the flow chamber, and subsequently generating a second difference image by comparing the third image to the second image so as to determine a location of at least one of the superficially stained entities (i.e. the ‘Gram-negative’ entities, in some embodiments). The imaging system may be further configured for analyzing the second difference image to determine an exposure time during which the decolorizing agent should be applied to the partially decolorized sample to substantially decolorize the superficially stained entities without substantially decolorizing the stained entities. As described herein, in some such system embodiments, the flow chamber may be further configured for applying the decolorizing agent to the partially decolorized sample for the determined exposure time so as to prepare a substantially decolorized sample wherein the superficially stained entities, if present, are substantially decolorized and wherein the stained entities, if present, are not substantially decolorized.
“In some such system embodiments, the imaging system may be further configured for recording a fourth image of the substantially decolorized sample and subsequently generating a third difference image by comparing the fourth image to the second image. The third difference image may depict the location of at least one of the superficially stained entities. The imaging system may also be configured for generating a fourth difference image by comparing the fourth image to the first image, wherein the fourth difference depicts a location of at least one of the stained entities.
“Thus the methods and systems for performing staining procedures for biological materials, as described in the embodiments of the present invention, provide many advantages that may include, but are not limited to: providing methods and systems that are capable of monitoring discernible optical changes in a sample during a staining process so as to better control and/or optimize the staining and decolorization of the sample; providing methods and systems that may be especially capable of classifying and locating Gram-stained entities within a sample by monitoring optical changes that may be discernible between staining and/or decolorization steps and highlighting those optical changes that are unique to Gram-positive and/or Gram-negative entities within the sample; providing systems and methods that may be uniquely capable of minimizing ‘false positives’ and/or ‘false negatives’ when analyzing stained samples in search of entities of interest within the sample (such as disease-causing bacteria, for example); and providing an optimized staining method and system that may be monitored and/or controlled in real time by an optical imaging apparatus. Thus, various embodiments of the present invention may also provide an ‘all-in-one’ staining apparatus and sample analysis tool wherein finished (and optimized) stained sample slides may remain on the same imaging apparatus for downstream morphological investigations. This complete integration of the sample preparation (i.e. staining) steps and analysis steps may allow for more efficient laboratory throughput.
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
“Having thus described the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and wherein:
“FIGS. 1-10 show a series of ‘single-line’ images recorded of a sample containing both stained (i.e. Gram-positive, for example) and superficially stained (i.e. Gram-negative, for example) entities, according to one embodiment of the present invention;
“FIG. 1 shows a non-limiting depiction of a ‘single line’ image of an unstained sample, corresponding to a first image, produced according to one embodiment of the present invention;
“FIG. 2 shows a non-limiting schematic of a ‘single line’ of a sample, showing the locations of stained entities (i.e. Gram-positive entities) in the sample, according to one embodiment of the present invention;
“FIG. 3 shows a non-limiting schematic of a ‘single line’ of a sample, showing the locations of superficially stained entities (i.e. Gram-negative entities) in the sample, according to one embodiment of the present invention;
“FIG. 4 shows a non-limiting depiction of a ‘single line’ image of a stained sample, corresponding to a second image, produced according to one embodiment of the present invention;
“FIG. 5 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a first difference image, produced according to one embodiment of the present invention, highlighting the locations of stained and superficially stained entities in the sample;
“FIG. 6 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a third image, produced according to one embodiment of the present invention after a 35\% decolorization of the sample;
“FIG. 7 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a second difference image, produced according to one embodiment of the present invention, highlighting the effect of the 35\% decolorization on superficially stained entities in the sample;
“FIG. 8 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a fourth image, produced according to one embodiment of the present invention, after a 100\% decolorization of the sample;
“FIG. 9 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a third difference image, produced according to one embodiment of the present invention, highlighting the locations of superficially stained (i.e. Gram-negative, for example) entities in the sample;
“FIG. 10 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a fourth difference image, produced according to one embodiment of the present invention, highlighting the locations of stained entities (i.e. Gram-positive, for example) in the sample;
“FIGS. 11-19 show a series of ‘single-line’ images recorded of a sample containing only stained (i.e. Gram-positive, for example) entities, according to one embodiment of the present invention;
“FIG. 11 shows a non-limiting depiction of a ‘single line’ image of an unstained sample, corresponding to a first image, produced according to one embodiment of the present invention;
“FIG. 12 shows a non-limiting schematic of a ‘single line’ of a sample, showing the locations of stained entities (i.e. Gram-positive entities) in the sample, according to one embodiment of the present invention;
“FIG. 13 shows a non-limiting schematic of a ‘single line’ of a sample, showing the lack of superficially stained entities (i.e. Gram-negative entities) in the sample, according to one embodiment of the present invention;
“FIG. 14 shows a non-limiting depiction of a ‘single line’ image of a stained sample, corresponding to a second image, produced according to one embodiment of the present invention;
“FIG. 15 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a first difference image, produced according to one embodiment of the present invention, highlighting the locations of stained entities in the sample;
“FIG. 16 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a third image, produced according to one embodiment of the present invention after a 35\% decolorization of the sample;
“FIG. 17 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a second difference image, produced according to one embodiment of the present invention, further depicting the lack of superficially stained entities in the sample;
“FIG. 18 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a third difference image, produced according to one embodiment of the present invention, highlighting the lack of superficially stained (i.e. Gram-negative, for example) entities in the sample;
“FIG. 19 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a fourth difference image, produced according to one embodiment of the present invention, highlighting the locations of stained entities (i.e. Gram-positive, for example) in the sample;
“FIGS. 20-29 show a series of ‘single-line’ images recorded of a sample containing only superficially-stained (i.e. Gram-negative, for example) entities, according to one embodiment of the present invention;
“FIG. 20 shows a non-limiting depiction of a ‘single line’ image of an unstained sample, corresponding to a first image, produced according to one embodiment of the present invention;
“FIG. 21 shows a non-limiting schematic of a ‘single line’ of a sample, showing the lack of stained entities (i.e. Gram-positive entities) in the sample, according to one embodiment of the present invention;
“FIG. 22 shows a non-limiting schematic of a ‘single line’ of a sample, showing the locations of superficially stained entities (i.e. Gram-negative entities) in the sample, according to one embodiment of the present invention;
“FIG. 23 shows a non-limiting depiction of a ‘single line’ image of a stained sample, corresponding to a second image, produced according to one embodiment of the present invention;
“FIG. 24 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a first difference image, produced according to one embodiment of the present invention, highlighting the locations of superficially stained entities in the sample;
“FIG. 25 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a third image, produced according to one embodiment of the present invention after a 35\% decolorization of the sample;
“FIG. 26 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a second difference image, produced according to one embodiment of the present invention, highlighting the effect of the 35\% decolorization on superficially stained entities in the sample;
“FIG. 27 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a fourth image, produced according to one embodiment of the present invention, after a 100\% decolorization of the sample;
“FIG. 28 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a third difference image, produced according to one embodiment of the present invention, highlighting the locations of superficially stained (i.e. Gram-negative, for example) entities in the sample;
“FIG. 29 shows a non-limiting depiction of a ‘single line’ image of a sample, corresponding to a fourth difference image, produced according to one embodiment of the present invention, highlighting the lack of stained entities (i.e. Gram-positive, for example) in the sample;
“FIG. 30 shows a non-limiting flow chart summarizing method steps for performing a staining procedure for a sample, according to one embodiment of the present invention;
“FIG. 31 is a non-limiting schematic depiction of a system according to one embodiment of the present invention, showing an imaging system disposed adjacent to a flow chamber configured for applying a staining agent and/or a decolorizing agent to a sample;
“FIG. 32 is a non-limiting schematic depiction of a system according to one embodiment of the present invention, including a flow channel and an imaging system including a camera device, an actuator device, an image processing computer, and a systems controller;
“FIG. 33 shows several non-limiting views of a flow chamber, according to one embodiment of the present invention, the flow chamber including a slide defining the surface for operably engaging the sample therewith and a flow channel housing defining a slide aperture configured for receiving the slide;
“FIG. 34 shows several non-limiting views of a flow chamber, according to one embodiment of the present invention, showing steps for operably engaging a slide with a flow channel housing of the flow chamber;
“FIG. 35 shows a non-limiting schematic of a flow chamber and an imaging system disposed adjacent to the flow chamber, according to one embodiment of the present invention; and
“FIG. 36 shows a non-limiting flow chart summarizing method steps for a method for optimally staining a sample, according to one embodiment of the present invention.”
For additional information on this patent application, see: Berndt, Klaus W. Method and Apparatus for Automated Staining of Biological Materials. Filed October 16, 2013 and posted July 3, 2014. Patent URL: http://appft.uspto.gov/netacgi/nph-Parser?Sect1=PTO2&Sect2=HITOFF&u=\%2Fnetahtml\%2FPTO\%2Fsearch-adv.html&r=3110&p=63&f=G&l=50&d=PG01&S1=20140626.PD.&OS=PD/20140626&RS=PD/20140626
Keywords for this news article include: Gram-Negative Bacteria, Becton Dickinson And Company.
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