Method and composition for staining and sample processing
Inventors
Assignees
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Publication Number
US-9279750-B2
Publication Date
2016-03-08
Expiration Date
Abstract
The present disclosure relates to a staining methodology employing a particle contrast agent composition capable of rapidly staining cells in a single step. The particle contrast agent composition can be comprised of a combination of one or more particle contrast agents, one or more permeabilizing agents, and one or more fixing agents. The particle contrast agent composition can include Crystal Violet, New Methylene Blue, Saponin, and Gluteraldehyde.
Core Innovation
The invention provides a particle contrast agent composition for staining a blood fluid sample that is imaged in an automated particle analysis system. The composition includes at least one particle contrast agent selected from Crystal Violet, New Methylene Blue, Methyl Green, Eosin Y, and Safranin O, together with a permeabilizing agent including saponin and a fixing agent including gluteraldehyde. The amounts of saponin and gluteraldehyde are defined under staining conditions as producing saponin concentrations between about 50 mg/L and about 750 mg/L and gluteraldehyde concentrations at or below 0.1%.
The invention further specifies embodiments where the particle contrast agent composition supports differential staining of blood cells, including Wright-like staining, while maintaining membrane integrity of white blood cells. In examples, specific dye combinations emphasize Crystal Violet, New Methylene Blue, and Eosin-Y, with optional inclusion of Methyl Green and Safranin O. The document describes selecting Eosin-Y as a third dye in order to improve staining compared with alternatives.
A method aspect of the invention treats particles of a blood fluid sample to be imaged using an automated particle analysis system by combining the blood fluid sample with the particle contrast agent composition to obtain a sample mixture and incubating the mixture at a temperature between about 37°C and about 60°C for fewer than 90 seconds. The document ties this rapid incubation concept to preserving membrane integrity while enabling imaging, and it describes compatibility with automated flowcell imaging systems.
Claims Coverage
The patent includes two independent claims, one composition claim and one method claim. Across them, the core inventive coverage concentrates on combining a defined set of particle contrast dyes with a saponin permeabilizing agent and a gluteraldehyde fixing agent, and on performing a rapid incubation under specified temperature and time limits for automated particle imaging.
Particle contrast agent composition with saponin and gluteraldehyde under defined concentration ranges
A particle contrast agent composition for staining a blood fluid sample being imaged in an automated particle analysis system comprising at least one particle contrast agent selected from Crystal Violet, New Methylene Blue, Methyl Green, Eosin Y, and Safranin O; a permeabilizing agent including saponin present in amounts sufficient to result in concentrations between about 50 mg/L and about 750 mg/L under staining conditions; and a fixing agent including gluteraldehyde present in amounts sufficient to result in concentrations at or below 0.1% under staining conditions.
Rapid method for treating and incubating blood sample particles for automated particle imaging
A method of treating particles of a blood fluid sample which will be imaged using an automated particle analysis system comprising combining the blood fluid sample with a particle contrast agent composition to obtain a sample mixture; and incubating the sample mixture at a temperature between about 37°C and about 60°C for fewer than 90 seconds, wherein the particle contrast agent composition includes at least one particle contrast agent selected from Crystal Violet, New Methylene Blue, Methyl Green, Eosin Y, and Safranin O; a permeabilizing agent including saponin present in amounts sufficient to result in concentrations between about 50 mg/L and about 750 mg/L under staining conditions; and a fixing agent including gluteraldehyde present in amounts sufficient to result in concentrations at or below 0.1% under staining conditions.
Together, the independent claims cover the specific composition constraints for a dye/saponin/gluteraldehyde staining formulation for imaging in an automated particle analysis system and a rapid sample treatment method that uses the same defined dye, saponin, and gluteraldehyde concentration constraints.
Stated Advantages
Enables differential staining of white cell subtypes and subcellular/nuclear features while maintaining membrane integrity of white blood cells.
Supports rapid, one-step staining/sample processing suitable for automated imaging of blood cells.
Documented Applications
Staining and imaging of a blood fluid sample in an automated particle analysis system, including automated flowcell imaging systems.
Treating particles of a blood fluid sample for imaging in an automated particle analysis system using a rapid incubation process.
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