WBC Analysis via Fluorescence Triggering and Scatter
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Solution Overview
Problem
Current hematology assays face challenges in accurately analyzing white blood cells (WBCs) due to the low concentration of WBCs in blood samples and interference from unlysed red blood cells (RBCs) and their fragments, which can lead to incorrect WBC counting and classification, especially in samples with lysis-resistant RBCs or fragile lymphocytes.
Innovation Solution
The method involves fluorescence staining with a cell membrane-permeable fluorescent dye and a fluorescence triggering strategy, using a combination of axial light loss, intermediate angle scatter, 90° polarized side scatter, 90° depolarized side scatter, and fluorescence emission measurements to differentiate and count WBCs, effectively eliminating interference from unlysed RBCs and fragments, and developing milder WBC reagents for fragile lymphocytes.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If strong lysis reagents are used to completely lyse RBCs, then RBC lysis is improved, but WBC cell membranes are damaged and classification accuracy deteriorates
Solution Approach 1:
The patent changes the chemical parameters of the lysis reagent by using non-ionic surfactants (Tween-20, Tween-80, Triton X-100) instead of traditional strong ionic detergents. This parameter change allows complete RBC lysis while preserving WBC membrane integrity, resolving the contradiction between lysis completeness and WBC preservation
Solution Approach 2:
The patent introduces fluorescent dyes (acridine orange, propidium iodide, SYTO 13, SYBR Green) as intermediaries that selectively bind to nucleic acids in WBCs. These fluorescent markers serve as mediators to identify and count WBCs without requiring strong lysis, thereby preserving WBC integrity while enabling accurate detection
2Reliability
If lysis time is extended to ensure complete RBC lysis, then lysis completeness is improved, but WBC integrity deteriorates
Solution Approach 1:
The patent changes the temporal parameter by reducing lysis time to 2-5 minutes using mild non-ionic surfactants. This time optimization ensures complete RBC lysis while minimizing WBC exposure, preventing membrane damage and maintaining WBC integrity for accurate classification
3Quantity of substance
If traditional WBC reagents are used, then RBC lysis is achieved, but interference from unlysed RBCs and fragments persists
Solution Approach 1:
The patent introduces fluorescent dyes as intermediaries that selectively bind to WBC nucleic acids. This allows differentiation of WBCs from unlysed RBCs and fragments through fluorescence detection, eliminating interference in counting and classification
Solution Approach 2:
The patent changes the chemical composition of the reagent by using non-ionic surfactants that provide milder lysis. This parameter change achieves complete RBC lysis while minimizing fragment formation, reducing interference in WBC analysis
4Measurement precision
If conventional fluorescence dyes are used, then WBC detection is possible, but lysis-resistant RBCs still interfere with counting
Solution Approach 1:
The patent uses fluorescent dyes (acridine orange, propidium iodide, SYTO 13, SYBR Green) as intermediaries that selectively bind to nucleic acids. Since lysis-resistant RBCs lack nuclei, they do not bind these dyes and do not fluoresce, allowing clear differentiation from fluorescent WBCs and eliminating interference in counting
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
This approach ensures accurate counting and classification of WBCs and sub-populations by substantially eliminating interference from unlysed RBCs and fragments, providing reliable results even in challenging sample types, such as those with lysis-resistant RBCs or aged samples.
Implementation Method 1
staining a blood sample with an exclusive, cell membrane permeable, fluorescent dye, which corresponds in emission spectrum to an excitation source of a hematology instrument
Implementation Method 2
using a combination of measurements of (1) axial light loss, (2) intermediate angle scatter, (3) 90° polarized side scatter, (4) 90° depolarized side scatter
Data Source
Figure 1A~1C
Figure 1D~1E
Figure 2~3
AI summary
Systems and methods for analyzing blood samples, and more specifically for performing a white blood cell (WBC) differential analysis. The systems and methods screen WBCs by means of fluorescence staining and a fluorescence triggering strategy. As such, interference from unlysed red blood cells (RBCs) and fragments of lysed RBCs is substantially eliminated. The systems and methods also enable development of relatively milder WBC reagent(s), suitable for assays of samples containing fragile WBCs. In one embodiment, the systems and methods include: (a) staining a blood sample with an exclusive cell membrane permeable fluorescent dye, which corresponds in emission spectrum to an excitation source of a hematology instrument; (b) using a fluorescence trigger to screen the blood sample for WBCs; and (c) using measurements of (1) axial light loss, (2) intermediate angle scatter, (3) 90° polarized side scatter, (4) 90° depolarized side scatter, and (5) fluorescence emission to perform a differentiation analysis.