Eosin staining techniques
Inventors
Blehm, Benjamin H. • Graves, Evan T. • Durrant, Edward E.
Assignees
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Publication Number
US-12332153-B2
Publication Date
2025-06-17
Expiration Date
Abstract
Presented herein are methods of improving the consistency of staining with a counterstain. In some embodiments, the method makes use of an automated specimen processing apparatus or other staining device. In some embodiments, the staining methods are applied manually. In some embodiments, the counterstain includes eosin.
Core Innovation
The invention relates to automated specimen processing for H&E staining in which predetermined amounts of staining and treatment solutions are dispensed and at least partially removed from a specimen on a substrate. The sequence includes dispensing a primary staining solution, dispensing an alkaline solution, dispensing at least one additional solution, dispensing a counter staining solution, and then dispensing an acid solution, with predetermined amounts and partial removals performed by an automated specimen processing apparatus.
A core aspect of the invention is repositioning and/or replacing fluid-dispensing steps around eosin to address improved consistency of eosin counterstaining. The embodiments describe options where acid is applied immediately after eosin with no acid between hematoxylin and eosin, and embodiments where acid is applied between hematoxylin and eosin and again immediately after eosin.
The invention further characterizes the transfer solution as neutral and includes automated systems and algorithms that dispense predetermined aliquot amounts and avoid acid prior to eosin in relevant embodiments. Example outcomes are described as improved eosin staining consistency, eosin intensity, and diagnostic clarity and preference across multiple tissues, with other attributes affected differently depending on whether wash dispenses are replaced with transfer steps or whether acid placement is repositioned.
Claims Coverage
The independent claims include three inventive categories with three inventive features: an automated staining method with a controlled sequence ending with acid dispensing, a specimen staining method with an order constraint prohibiting acid between primary and counter staining, and a non-transitory computer-readable storage medium controlling an automated histological staining system to dispense eosin and additional solutions where at least two are transfer solutions.
Automated sequential staining with controlled acid placement
A method of staining a specimen disposed on a substrate with an automated specimen processing apparatus comprising dispensing predetermined amounts of a primary staining solution and at least partially removing it, dispensing predetermined amounts of an alkaline solution and at least partially removing it, dispensing predetermined amounts of at least one additional solution and at least partially removing it, dispensing predetermined amounts of a counter staining solution and at least partially removing it, and then dispensing a predetermined amount of an acid solution to the specimen.
No acid between primary and counter staining
A method of staining a specimen comprising applying a predetermined amount of a primary staining solution, at least partially removing the primary staining solution, applying a predetermined amount of a counter staining solution, at least partially removing the counter staining solution, applying a predetermined amount of an acid solution, at least partially removing the acid solution, applying at least one additional solution, wherein no acid solution is applied between applying the primary staining solution to the specimen and the applying of the counter staining solution to the specimen.
Computer-readable control for eosin and predominantly transfer additional solutions
A non-transitory computer-readable storage medium encoded with instructions that, when executed, cause an automated histological staining system to dispense a predetermined amount of a counter staining solution to a specimen, at least partially remove the counter staining solution from the specimen, and sequentially dispense to the specimen and remove from the specimen four additional solutions selected from wash solutions and transfer solutions, wherein at least two of the four additional solutions are transfer solutions.
Together, the independent claims cover automated histological staining workflows using predetermined dispense and remove steps and additional solutions, with key limitations focused on how acid is ordered relative to counter staining and how wash versus transfer solutions are selected and sequenced for counterstaining.
Stated Advantages
Improves eosin staining consistency.
Improves eosin intensity.
Improves diagnostic clarity and diagnostic preference across multiple tissues.
Documented Applications
Automated histological staining (H&E staining) for eosin counterstaining as part of automated specimen processing.
Computer-controlled automated histological staining system dispensing sequences involving counter staining solution and additional wash/transfer solutions.
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