Dried Blood Spot Metabolite Extraction for ADA-SCID Diagnosis
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Solution Overview
Problem
Current methods for diagnosing severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) or purine-nucleoside phosphorilase (PNP) defects are inadequate as they often require onset of infections for detection, leading to delayed diagnosis and treatment, and existing mass spectrometry methods are ineffective for detecting these metabolites in dried blood spots.
Innovation Solution
A method using a mixture of water and C1-3 linear or branched chain monoalcohol, preferably methanol, to extract Adenosine and Deoxyadenosine from dried blood samples, allowing for concurrent extraction of other metabolites, enabling sensitive and specific detection using mass spectrometry, particularly on Guthrie cards, facilitating early diagnosis.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If conventional mass spectrometry methods are used on dried blood spots, then other metabolites can be detected, but ADA and PNP deficiency metabolites (Adenosine and Deoxyadenosine) cannot be detected
Solution Approach 1:
The patent changes the extraction parameters by using a water-containing alcohol solution (30-70% v/v water) instead of conventional organic solvents. This parameter change enables the extraction of Adenosine and Deoxyadenosine from dried blood spots while maintaining compatibility with mass spectrometry detection, thereby expanding metabolite detection coverage without compromising reliability
Solution Approach 2:
The patent introduces a specific extraction solution (water-containing alcohol) as an intermediary substance that facilitates the extraction of previously undetectable metabolites. This intermediary enables the transfer of Adenosine and Deoxyadenosine from the dried blood spot matrix to the mass spectrometry detection system, resolving the detection limitation
2Measurement precision
If diagnosis is delayed until infection onset, then clinical suspicion can be formulated, but life-saving treatment opportunity is lost
Solution Approach 1:
The patent enables preliminary diagnosis by detecting Adenosine and Deoxyadenosine metabolites in dried blood spots collected at birth or early infancy, before clinical symptoms or infections occur. This preliminary detection allows treatment to be initiated in the preinfection period, maximizing therapeutic effectiveness and preventing severe complications
Solution Approach 2:
The patent utilizes routinely collected dried blood spots (Guthrie cards) already present in the healthcare system, transforming an existing sample collection practice into a diagnostic tool. This self-service approach enables early detection without requiring additional blood draws or specialized sampling procedures, thus eliminating time loss while maintaining diagnostic accuracy
3Ease of operation
If ADA activity evaluation is performed, then enzyme function can be assessed, but misleading results occur due to variable residual activity in non-immune cells
Solution Approach 1:
The patent extracts and measures the end metabolites (Adenosine and Deoxyadenosine) of the enzymatic pathway rather than measuring the enzyme activity itself. This extraction approach bypasses the complexity of ADA activity evaluation and the confounding effect of variable residual activity in different cell types, providing a direct and reliable measure of metabolic dysfunction
Solution Approach 2:
The patent replaces the biochemical assay system (ADA activity measurement) with a mass spectrometry-based metabolite detection system. This substitution eliminates the limitations of enzyme activity measurement, including the influence of residual enzyme activity in non-immune cells, and provides a more accurate reflection of the metabolic defect
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 reliable and reproducible early diagnosis of ADA-SCID at birth, allowing timely treatment and preventing severe complications, with high sensitivity and specificity, and the method can be used during enzyme replacement therapy to monitor treatment efficacy.
Implementation Method 1
A method using a mixture of water and C1-3 linear or branched chain monoalcohol, preferably methanol, to extract Adenosine and Deoxyadenosine from dried blood samples
Data Source
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AI summary
Is described a method for individuating with high sensitivity and specificity ADA metabolites from dried blood spot. The method described herein can be used to extract Adenosine and Deoxyadenosine from a sample under conditions that permit concurrently extracting other metabolites, such as amino acids, free carnitine, or acylcarnitines. For example, harsh extraction conditions (such as extreme acidity and high temperature) can be avoided. The method can be used, along with other neonatal screenings, on blood samples and preferably on dried blood spots (Guthrie cards) and more preferably on Guthrie cards obtained in the II-IV day of life. The method is reliable and reproducible, easy to perform and gives a definitive response within a short time (1 -2 days). One or more kit for use in the method if the invention are also described.