Selective Immune Cell Lysis for Rapid Microorganism Detection
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Solution Overview
Problem
Current methods for detecting viable microorganisms in samples with high concentrations of immune cells or their progenitor cells, such as those used in Advanced Therapy Medicinal Products (ATMPs), face challenges due to long response times and the inability to preserve microorganism viability, leading to false negatives and negatives.
Innovation Solution
A method involving a lysis composition with a non-ionic detergent and saponin, combined with an endonuclease and endopeptidase, is used to selectively lyse immune cells or their progenitor cells, followed by filtration through a 0.30 to 0.50 µm filter, preserving microorganism viability for rapid detection by solid-phase cytometry.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If traditional cell culture methods are used for sterility testing, then detection reliability is improved, but response time increases to 7-14 days
Solution Approach 1:
The detection process is segmented into two distinct phases: a rapid initial detection phase using solid-phase cytometry that provides results within hours, followed by a confirmatory cell culture phase if needed. This segmentation allows the system to deliver fast preliminary results while maintaining overall detection reliability through the secondary confirmation step.
Solution Approach 2:
The patent introduces an intermediary preparation step involving controlled lysis buffers and filtration that bridges the gap between rapid detection needs and reliable microorganism identification. This intermediary process removes immune cells while preserving microorganisms, enabling the rapid solid-phase cytometry detection to proceed with high reliability.
2Measurement precision
If sample preparation methods are used to reduce immune cell concentration, then detection precision is improved, but microorganism viability may be compromised
Solution Approach 1:
The lysis buffer composition is optimized to have local quality characteristics that selectively affect different cell types. The buffer contains specific concentrations of non-ionic detergents and saponin that are tailored to lyse immune cells while being gentle enough to preserve microorganism integrity and viability, thus achieving both detection precision and reliability.
Solution Approach 2:
The patent employs controlled changes in chemical parameters of the lysis buffer, specifically adjusting detergent concentration and composition, to achieve selective lysis. By precisely controlling these parameters, the method removes immune cells that would interfere with detection precision while maintaining conditions that preserve microorganism viability for reliable detection.
3Productivity
If automated reading cell culture methods are used, then productivity is improved, but response time remains 7-10 days
Solution Approach 1:
The patent replaces the traditional mechanical cell culture incubation process with a solid-phase cytometry system that uses optical detection methods. This substitution eliminates the need for lengthy incubation periods while maintaining automated processing capabilities, reducing the response time from days to hours without sacrificing productivity.
4Loss of time
If solid-phase cytometry is applied directly to high cell concentration samples, then response time is reduced, but measurement precision deteriorates
Solution Approach 1:
The patent implements a preliminary sample preparation action that removes immune cells through controlled lysis and filtration before the solid-phase cytometry detection. This preliminary step clears the sample matrix of interfering elements, allowing the rapid detection method to achieve both fast response time and high measurement precision by eliminating sources of detection interference.
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 efficiently reduces immune cell concentration, maintains microorganism viability, and allows for rapid detection of viable microorganisms within 4-6 hours, minimizing false positives and negatives.
Implementation Method 1
a lysis composition comprising a non-ionic detergent and saponin
Implementation Method 2
a lysis composition comprising a non-ionic detergent and saponin
Implementation Method 3
an endonuclease for digesting the nucleic acids released by the action of the lysis composition
Implementation Method 4
an endopeptidase for degrading proteins released by the action of the lysis composition
Implementation Method 5
filtration through a 0.30 to 0.50 µm filter
Data Source
AI summary
The present invention relates to a method for isolating viable microorganisms potentially present in a sample comprising 106 to 108 immune cells or progenitor cells thereof, comprising the following steps: - Contacting the sample with • a lysing composition comprising ∘ a lysis buffer comprising a non-ionic detergent at a concentration of between 0.004% and 0.050% ∘ and/or a lysis solution comprising a saponin at a concentration of between 0.03% and 4% • an endonuclease for digesting the nucleic acids released by the action of the lysing composition • an endopeptidase acting at a pH of between 7 and 8, - Filtering the viable microorganisms potentially present through a filter whose pore diameter is between 0.30 and 0.50 µm. The present invention also relates to the associated method of detection by solid phase cytometry.