Spatial Analysis of FFPE Samples Using Probe Hybridization
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Solution Overview
Problem
Existing methods for analyzing nucleic acid integrity in formalin-fixed paraffin-embedded (FFPE) samples are limited, especially for gene expression analysis, due to the crosslinks introduced during fixation, which affect the integrity of nucleic acids and make it challenging to determine their spatial distribution within tissue samples.
Innovation Solution
The development of methods and systems for assessing the integrity of nucleic acids from FFPE samples, including determining RNA integrity numbers (RIN) and spatial fragment distribution values (DV), and performing spatial analysis of analytes in these samples, even when stored for extended periods.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Shape
If formalin fixation is used to preserve tissue morphology, then tissue structure and cell morphology are maintained, but nucleic acid integrity is significantly degraded
Solution Approach 1:
The patent introduces an intermediary quality control assay that uses probe hybridization to detect and measure nucleic acid integrity in fixed tissue samples. This intermediary step allows assessment of whether nucleic acids are sufficiently intact for downstream spatial transcriptomics, bridging the gap between formalin fixation and gene expression analysis without requiring abandonment of either approach.
2Shape
If spatial analysis is performed on fixed biological samples, then tissue architecture is preserved, but measurement precision of analyte integrity is reduced
Solution Approach 1:
The patent segments the analysis into distinct components: first assessing nucleic acid integrity in specific spatial regions using probe hybridization, then determining whether to proceed with spatial transcriptomics on those regions. This segmentation allows independent evaluation of tissue architecture preservation versus analyte integrity, enabling selective analysis of regions that meet both criteria.
3Duration of action of stationary object
If long-term storage of FFPE samples is performed, then sample availability is maintained, but nucleic acid quality deteriorates over time
Solution Approach 1:
The patent implements preliminary action by performing quality control assessment of nucleic acid integrity before initiating spatial transcriptomics or other downstream analyses on stored FFPE samples. This preliminary step uses probe hybridization to predict whether the sample will yield usable data, preventing wasted resources on degraded samples and enabling informed decisions about sample suitability after long-term storage.
4Productivity
If gene expression analysis is performed on degraded nucleic acids, then analysis throughput is maintained, but data quality and interpretability are compromised
Solution Approach 1:
The patent implements feedback by using the results of probe hybridization-based quality control assessment to determine whether to proceed with spatial transcriptomics. This feedback loop provides information about nucleic acid integrity that guides downstream experimental decisions, allowing researchers to identify and analyze only those samples and regions likely to yield interpretable results, thereby optimizing both throughput and data quality.
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
These methods enable the evaluation of nucleic acid integrity and the performance of spatial analysis on FFPE samples, ensuring that sub-areas of the sample contain nucleic acids of sufficient quality for downstream analyses, including spatial transcriptomics, even after long-term storage.
Implementation Method 1
hybridizing a first probe and a second probe to the analyte of the fixed biological sample
Data Source
AI summary
Provided herein are methods of analyzing an analyte in a fixed biological sample on a first substrate that has been stored for a long period of time, the method comprising: (a) hybridizing a first probe and a second probe to the analyte of the fixed biological sample; (b) coupling the first probe and the second probe, thereby generating a connected probe; (c) aligning the first substrate with a second substrate comprising an array, wherein the array comprises a plurality of capture probes, wherein a capture probe of the plurality of capture probes comprises: (i) a spatial barcode and (ii) a capture domain; (d) when the biological sample is aligned with at least a portion of the array, (i) releasing the connected probe from the analyte and (ii) migrating the connected probe from the biological sample to the array; and (e) hybridizing the connected probe to the capture domain.