Leukocyte Target Labeling Without Centrifugation or Wash Steps
Find Innovative SolutionsGenerate Solutions
Solution Overview
Problem
Existing methods for labeling intracellular and extracellular targets of leukocytes are time-consuming, labor-intensive, require centrifugation steps, and cannot be easily automated, leading to cell damage and loss, and necessitate separate use of binding agents.
Innovation Solution
A method involving a combination of solutions to cross-link, permeabilize, and fix leukocytes without centrifugation, allowing for the use of binding agent cocktails and enabling automation, comprising steps of cross-linking, permeabilization, and fixation with specific agent compositions.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If conventional staining methods with multiple washing and centrifugation steps are used, then extracellular and intracellular markers can be stained, but the procedure becomes time-consuming (2-3 hours) and labor-intensive
Solution Approach 1:
The patent combines multiple separate staining procedures (extracellular staining, washing, fixation, permeabilization, intracellular staining) into a single continuous workflow without intermediate centrifugation or washing steps. Binding agents for both extracellular and intracellular targets are added simultaneously to the cell suspension, and all processing occurs in one tube, reducing the procedure from 2-3 hours to approximately 45 minutes while maintaining staining quality.
Solution Approach 2:
The patent extracts and eliminates the centrifugation and washing steps from the conventional protocol. By using a specialized buffer system and controlled reagent addition, unbound binding agents are removed without mechanical centrifugation, thereby eliminating time-consuming separation steps while preserving cell integrity and staining effectiveness.
2Measurement precision
If centrifugation steps are performed for washing and separation, then unbound binding agents are removed, but cell structure is damaged and light scatter signals worsen
Solution Approach 1:
The patent replaces the mechanical centrifugation system with a chemical/biochemical approach using specialized buffers and controlled reagent interactions. The buffer system enables selective binding and removal of unbound agents through chemical means rather than mechanical force, thereby preserving cell structure and maintaining light scatter properties essential for flow cytometry analysis.
3Measurement precision
If multiple separate staining procedures are performed sequentially, then specific intracellular and extracellular targets are labeled, but automation is almost impossible due to mandatory centrifugation steps
Solution Approach 1:
The patent merges multiple discrete manual operations into a single automated-compatible workflow. By eliminating centrifugation steps and performing all staining operations in one tube with controlled reagent addition, the procedure becomes suitable for automation using liquid handling robots and automated flow cytometers, while maintaining the specificity required for labeling both intracellular and extracellular targets.
4Measurement precision
If conventional protocols with multiple washing steps are used, then unbound binding agents are removed, but cell loss and cell clumping occur
Solution Approach 1:
The patent substitutes mechanical centrifugation-based washing with a biochemical approach using specialized buffers that enable selective removal of unbound binding agents through chemical interactions rather than mechanical separation. This prevents cell loss and clumping while maintaining staining specificity, as cells remain in suspension throughout the procedure without subjected to mechanical stress.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
The method significantly reduces processing time, preserves cell structure, improves light scatter properties, and enables efficient staining of both intracellular and extracellular targets using binding agent cocktails, facilitating automation.
Implementation Method 1
a first solution comprising one or more agent(s) that are capable of cross-linking intracellular proteins, lipoproteins and nucleic acids of said leukocytes
Implementation Method 2
one or more agent(s) that are capable of permeabilizing said leukocytes
Implementation Method 3
lysing said red blood cells
Implementation Method 4
neutralizing the cross-linking activity of said one or more agent(s) in said first solution
Implementation Method 5
at least one binding agent which specifically binds to an extracellular target of leukocytes, and at least one binding agent which specifically binds to an intracellular target of leukocytes
Data Source
AI summary
The present invention relates to methods for labeling intracellular and extracellular targets of leukocytes, as well as to kits for performing said methods.