Flow Cytometry Megakaryocyte Counting via Fluorescence and Scattering
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Solution Overview
Problem
Current methods for counting megakaryocytes are inconsistent and imprecise due to ambiguous recognition standards and the low ratio of megakaryocytes in bone marrow, leading to difficulties in distinguishing them from other cells like plasmacytes, especially in bone marrow aspirates.
Innovation Solution
A method and apparatus that involve lysing erythrocytes, staining nucleic acid with a fluorescent dye, and detecting forward scattered light, side scattered light, and fluorescence to identify and count megakaryocytes based on their unique properties, using a flow cytometer to create two-dimensional distribution diagrams for precise differentiation.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Ease of manufacture
If visual examination method is used to count megakaryocytes, then the measurement process is simple, but the measurement precision is low due to ambiguous recognition standards and low megakaryocyte ratio
Solution Approach 1:
The patent replaces the manual visual examination method with an automated flow cytometry system. The flow cytometer uses optical detection (light scattering and fluorescence) to automatically identify and count megakaryocytes, eliminating the need for manual microscope examination. This substitution of mechanical/manual operation with an automated instrumental system resolves the contradiction by providing both automation and high precision through multi-parameter detection.
Solution Approach 2:
The patent transforms the identification criteria from subjective visual assessment to objective multi-parameter measurement. By detecting forward scattered light (FSC), side scattered light (SSC), and fluorescence intensity simultaneously, the system creates a multi-dimensional parameter space for cell identification. This parameter expansion allows precise differentiation of megakaryocytes from other cells based on their unique optical properties, resolving the precision issue while maintaining ease of operation.
2Measurement precision
If flow cytometer method with two-color measuring is used to identify and count megakaryocytes, then the measurement precision is improved, but the device complexity and operation complexity increase due to mononuclear cell separation requirement
Solution Approach 1:
The patent extracts and eliminates the problematic mononuclear cell separation step from the measurement process. By using a fluorescent dye that specifically stains nucleic acid in all cell types present in bone marrow aspirate, the method directly measures megakaryocytes in the whole sample without requiring preliminary separation. This extraction of the separation step simplifies the device and operation while maintaining precision through specific fluorescent staining of megakaryocyte nucleic acid.
Solution Approach 2:
The patent employs a universal fluorescent staining approach that works for all nucleated cells in the sample, eliminating the need for type-specific separation procedures. The fluorescent dye binds to nucleic acid universally, and megakaryocytes are identified through their characteristic position in the multi-parameter plot (FSC vs SSC vs fluorescence), making the method applicable to whole bone marrow aspirate without complex separation requirements.
3Productivity
If automatic hemocyte counting apparatus is used to measure megakaryocytes, then the productivity is improved, but the measurement precision deteriorates due to inability to distinguish megakaryocytes from plasmacytes
Solution Approach 1:
The patent adds another dimension to the measurement by incorporating fluorescence detection alongside forward and side scattered light detection. This creates a three-dimensional parameter space (FSC, SSC, fluorescence) that enables clear separation of megakaryocytes from plasmacytes. The fluorescence dimension provides an additional discriminatory parameter that automatically differentiates cell types based on their nucleic acid content and staining characteristics, resolving the precision issue while maintaining automation.
Solution Approach 2:
The patent introduces fluorescent dye staining as an intermediary mechanism to enable precise cell differentiation. The fluorescent dye acts as a mediator that binds to nucleic acid and provides a detectable signal that, when combined with light scattering parameters, allows the automated system to accurately distinguish megakaryocytes from other cells. This intermediary staining step transforms the automated counting into a precise identification process.
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 allows for precise counting of megakaryocytes by distinguishing them from other cells, improving accuracy and reducing the time required for measurement, as demonstrated by a high correlation with visual examination results.
Implementation Method 1
staining nucleic acid in the megakaryocytes with a fluorescent dye; irradiating cells in the measurement sample with excited light; detecting forward scattered light, side scattered light and fluorescence which is emitted from the cells
Implementation Method 2
detecting forward scattered light, side scattered light and fluorescence which is emitted from the cells; identifying megakaryocytes on the basis of the detected forward scattered light, side scattered light and fluorescence
Data Source
AI summary
A method for counting megakaryocytes in a specimen is described. In the method, first, erythrocytes in the specimen are lysed and nucleic acid in the megakaryocytes is stained with a fluorescent dye, and thereby, a measurement sample is prepared. Next, the cells in the measurement sample are irradiated with excited light so that the forward scattered light, the side scattered light and the fluorescence, which are emitted from the cells, are detected. Megakaryocytes are identified on the basis of the detected forward scattered light, the fluorescence and the side scattered light. Then, the identified megakaryocytes are counted.