Lymphocyte Enumeration via Fluorescent Antibody Markers
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Solution Overview
Problem
Current methods for identifying lymphocyte populations in blood samples require measurements of cellular properties like forward or side light scatter in addition to cell surface markers, making it challenging for low-cost point-of-care applications to analyze white blood cells solely based on surface markers.
Innovation Solution
A method using fluorescent dye-labeled binding compounds specific for CD2, CD3, and CD45RA markers, allowing for the enumeration of lymphocytes and determination of T-helper lymphocytes without the need for side scatter measurements, utilizing the same or distinct fluorescent dyes to distinguish lymphocyte subpopulations.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Measurement precision
If side scatter measurement is used to identify lymphocyte populations, then measurement precision is improved, but device complexity and cost increase
Solution Approach 1:
The patent extracts the lymphocyte identification function from the complex flow cytometry system by using a specific antibody combination (anti-CD45RA and anti-CD3 or anti-CD2) that can identify lymphocytes through fluorescence intensity alone, removing the requirement for side scatter measurement and simplifying the device
Solution Approach 2:
The patent introduces fluorescently labeled antibodies as intermediaries that mediate the identification process. These antibodies bind to specific cell surface markers (CD45RA and CD3/CD2) and provide a fluorescent signal that serves as a surrogate for the complex side scatter measurement, enabling lymphocyte identification through a simpler optical detection system
2Measurement precision
If multiple fluorescent dyes with distinct emission spectra are used to identify lymphocyte subsets, then measurement precision is improved, but ease of operation deteriorates due to spectral overlap complexity
Solution Approach 1:
The patent employs a simplified fluorescent labeling strategy using antibodies labeled with fluorophores that have sufficiently separated emission spectra, allowing for straightforward optical filtering and detection without requiring complex spectral unmixing algorithms or specialized equipment, making the assay more accessible for routine clinical use
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
Enables effective identification and enumeration of lymphocytes and T-helper lymphocytes in blood samples solely based on cell surface markers, suitable for low-cost point-of-care devices and image-based cell identification, improving accuracy and simplifying the analysis process.
Implementation Method 1
combining said sample with a fluorescent dye-labeled first binding compound specific for a T lymphocyte-specific marker that is CD2 or CD3 and a fluorescent dye-labeled second binding compound specific for CD45RA
Data Source
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AI summary
The invention provides methods and compositions for identifying and counting lymphocytes in a biological sample, such as whole blood, by means of a probe comprising at least one binding compound specific for a T lymphocyte-specific marker, e.g. a CD2 or CD3, and at least one binding compound specific for CD45RA. Lymphocytes within the sample combine with such a probe to form a distinguishable subpopulation based on the amount of probe that specifically binds to their surfaces, thereby permitting such lymphocytes to be detected and enumerated on the basis of the intensity of the signal generated by the probe, and without the need of a separate physical measurement, such as light scatter. With additional probes specific for additional blood cell markers, percentages of lymphocytes, monocytes and granulocytes in a sample may be determined.