Acridine Orange Staining for Blood Cell Sorting
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Solution Overview
Problem
Conventional methods for staining cells with acridine orange in blood analysis face issues such as incorrect cell measurement due to excess or insufficient staining, and increased background noise from noncellular particles like dust, leading to inaccurate counting of reticulocytes and platelets.
Innovation Solution
A method and apparatus that uses acridine orange for staining cells, involving light irradiation, signal detection, normalization of DNA and RNA fluorescence intensities with cell size and shape, and constructing scattergrams to differentiate between cellular and noncellular particles, allowing for accurate sorting and measurement of red blood cells and platelets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If acridine orange staining is used for leukocytes classification and reticulocytes measurement, then both measurements can be performed in a single apparatus, but incorrect measurement of cells occurs due to excess or insufficient staining
Solution Approach 1:
The patent introduces staining evaluation parameters (ratio of fluorescence intensity to cell volume, staining uniformity) to objectively assess staining quality. By monitoring these parameters, the system can detect whether staining is excessive or insufficient and adjust accordingly, thereby maintaining measurement precision while using acridine orange for multiple cell types
Solution Approach 2:
The patent implements a feedback mechanism where staining quality is evaluated in real-time using fluorescence intensity and cell volume data. The system provides feedback on staining adequacy and can trigger re-staining or adjustment protocols, ensuring accurate measurements are obtained before proceeding with cell classification and counting
2Reliability
If conventional staining procedures are used, then cell staining can be achieved, but staining time requires 30 minutes or more
Solution Approach 1:
The patent performs preliminary evaluation of staining quality immediately after the staining process using automated fluorescence intensity analysis. This allows rapid determination of whether staining is adequate, eliminating the need for prolonged manual assessment and enabling faster turnaround while maintaining staining reliability
Solution Approach 2:
The patent replaces manual staining assessment with automated optical detection and computational analysis. By using fluorescence intensity measurements and image processing algorithms, the system objectively evaluates staining quality in seconds rather than minutes, dramatically reducing the time required while maintaining or improving staining reliability
3Quantity of substance
If noncellular particles such as dusts are present in cell suspension, then background level of measurement apparatus increases, but accurate counting of cells becomes difficult
Solution Approach 1:
The patent applies different analysis criteria to different particle types based on their local characteristics. Cellular particles exhibit specific fluorescence patterns, size distributions, and morphological features that distinguish them from noncellular dust particles. By analyzing these local quality differences, the system can differentiate and accurately count cells even in the presence of background particles
Solution Approach 2:
The patent utilizes fluorescence emission characteristics (color/ wavelength) to differentiate cellular from noncellular particles. Acridine orange-stained cells emit characteristic fluorescence signals that differ from dust particles, enabling the system to selectively identify and count cells while ignoring background contamination through spectral discrimination
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 enables precise sorting and measurement of reticulocytes, red blood cells, and platelets by normalizing fluorescence intensities and determining staining behavior, reducing errors from noncellular particles and improving staining efficiency.
Implementation Method 1
staining with acridine orange, fluorescent dye
Implementation Method 2
the dye is intercalated between base pairs in a double-stranded DNA to generate fluorescence
Implementation Method 3
the dye causes stacking on a single-stranded nucleic acid such as RNA by electrostatic interaction to generate fluorescence
Implementation Method 4
irradiating light on the cells in the blood; receiving the irridated light with detectors
Data Source
Figure 1~2
Figure 3
Figure 4a
AI summary
A method and an apparatus using acridine orange for sorting particles to precisely carry out the measurement of cells in the blood are provided. The cells in the blood is sorted by the steps of staining cells in the blood with acridine orange, irradiating light on the cells in the blood, determining staining behavior using platelets as a staining indicator, and sorting the cells in the blood.