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BR-112020023832-B1 - SAMPLE FOR CALIBRATION OF MULTISPECTRAL ANALYSIS SYSTEMS AND METHOD FOR CALIBRATION OF MULTISPECTRAL ANALYSIS SYSTEMS

BR112020023832B1BR 112020023832 B1BR112020023832 B1BR 112020023832B1BR-112020023832-B1

Abstract

CALIBRATION OF MULTISPECTRAL ANALYSIS SYSTEMS. The present invention relates to methods for calibrating a multispectral analysis system (1000) that include calibrating the system to detect fluorescence emission from a first fluorescent entity in a biological sample (900) that includes the first fluorescent entity and a second fluorescent entity using a calibration sample, wherein the calibration sample features a first concentration of the first fluorescent entity and a second concentration of the second fluorescent entity, and wherein the first concentration is greater than the second concentration.

Inventors

  • Rongcong Wu
  • Daniel SCHOENER

Assignees

  • REVVITY HEALTH SCIENCES, INC

Dates

Publication Date
20260317
Application Date
20190610
Priority Date
20180608

Claims (12)

  1. 1. A method for calibrating a multispectral analysis system, characterized in that it comprises: calibrating the system to detect fluorescence emission from a first fluorescent entity in a biological sample comprising the first fluorescent entity and a second fluorescent entity, using a calibration sample, wherein the calibration sample comprises a first concentration of the first fluorescent entity and a second concentration of the second fluorescent entity; wherein the first concentration is greater than the second concentration; wherein a fluorescence emission spectrum of the first fluorescent entity at least partially overlaps with a fluorescence emission spectrum of the second fluorescent entity; wherein a fraction of the second fluorescent entity in the calibration sample relative to a total amount of the first and second fluorescent entities in the calibration sample is between 0.02 and 0.08; wherein the first and second fluorescent entities are each associated with spectral emission channels in the multispectral analysis system; and wherein the fluorescence emission from the first fluorescent entity is detected by the multispectral analysis system in the spectral emission channel associated with the second fluorescent entity; and in that the calibration composition calibrates the multispectral analysis system to compensate for fluorescence emission crosstalk by one or more of the fluorescent entities in the spectral emission channels corresponding to one or more of the other fluorescent entities; and wherein the calibration of the multispectral analysis system uses the flatness of the multicomponent amplification curve over 40 PCR amplification cycles as a metric for evaluating the degree of correction of the amplification curves produced by each sample.
  2. 2. Method according to claim 1, characterized in that the first fluorescent entity is Cy5 and the second fluorescent entity is Cy5.5.
  3. 3. Method according to claim 1, characterized in that the calibration sample is a calibration plate comprising a plurality of sample cavities.
  4. 4. Method according to claim 1, characterized in that the first and second fluorescent entities are each fluorescent dyes.
  5. 5. Method according to claim 1, characterized in that the first fluorescent entity is an endogenous fluorescent moiety, and the second fluorescent entity is a fluorescent dye.
  6. 6. Method according to claim 1, characterized in that the fraction of the second fluorescent entity in the calibration sample is between 0.03 and 0.07.
  7. 7. Method according to claim 1, characterized in that the fraction of the second fluorescent entity in the calibration sample is between 0.04 and 0.06.
  8. 8. Method according to claim 1, characterized in that it further comprises using the calibrated multispectral analysis system to identify one or more gene targets in the biological sample.
  9. 9. Calibration sample, for carrying out the method as defined in claim 1, characterized in that it comprises: a calibration plate comprising a plurality of sample cavities, wherein the calibration plate is sized to be received in a multispectral analysis system; and a calibration composition positioned in one or more of the cavities, the composition comprising a first fluorescent entity and a second fluorescent entity, wherein a fluorescence emission spectrum of the first fluorescent entity overlaps at least partially with a fluorescence emission spectrum of the second fluorescent entity, wherein a fraction of the second fluorescent entity in the composition relative to a total amount of the first and second fluorescent entities in the composition is between 0.02 and 0.08, wherein the calibration composition calibrates the multispectral analysis system to compensate for fluorescence emission crosstalk by one or more of the fluorescent entities in the spectral emission channels corresponding to one or more of the other fluorescent entities.
  10. 10. Calibration sample according to claim 9, characterized in that the fraction of the second fluorescent entity in the composition is between 0.03 and 0.07.
  11. 11. Calibration sample according to claim 9, characterized in that the fraction of the second fluorescent entity in the composition is between 0.04 and 0.06.
  12. 12. Calibration sample according to claim 9, characterized in that the first fluorescent entity is Cy5 and the second fluorescent entity is Cy5.5.

Description

CROSS-REFERENCE TO RELATED REQUESTS [001] This application claims priority to U.S. Provisional Patent Application No. 62/682,819, filed June 8, 2018, the entire contents of which are incorporated by reference herein. BACKGROUND [002] Multispectral analysis systems can be used in a variety of assays to determine information about fluorophore and chromophore binding and expression in biological samples. Typically, where multiple spectral contributors in a sample emit or absorb radiation, contributions from each of the spectral contributors are separated to individually assess attributes such as spatial location and concentration of each contributor. Such assessments can provide important information about a sample, including disease status, immune response, protein expression, and the effectiveness of pharmaceutical treatment. [003] Multispectral analysis systems are typically calibrated before sample evaluation. Calibration usually involves several steps to ensure that such systems can resolve individual contributions from different spectral contributors. SUMMARY [004] The methods and devices described herein use calibration dye mixtures in single calibration plate cavities to calibrate multispectral imaging systems. In particular, calibration dye mixtures are used to reduce or eliminate crosstalk between spectral channels that are relatively closely spaced. Crosstalk impairs the retrieval of accurate quantitative information from a variety of assays by allowing contributions from one spectral contributor to contaminate or obscure contributions from another spectral contributor. [005] In the methods and devices described herein, by calibration with mixtures of calibration dyes, the “antecedent” contributions of an interfering spectral contributor to measured emission or absorption of a spectral contributor of interest can be reduced or eliminated. [006] As a result, spectral crosstalk is reduced and spectral multiplexation is enhanced. In other words, by using calibration dye mixtures at particular wavelengths or calibration bands, the number of different fluorophores in a sample that can be analyzed can be increased, and contributions from fluorophores with emission spectra that at least partially overlap can be distinguished and quantitatively analyzed with accuracy and precision. [007] In general, in a first aspect, the description characterizes methods for calibrating a multispectral analysis system that includes calibrating the system to detect fluorescence emission from a first fluorescent entity in a biological sample that includes the first fluorescent entity and a second fluorescent entity using a calibration sample, where the calibration sample characterizes a first concentration of the first fluorescent entity and a second concentration of the second fluorescent entity, and where the first concentration is greater than the second concentration. [008] Implementations of the methods may include any one or more of the following characteristics. [009] The first fluorescent entity may be Cy5 and the second fluorescent entity may be Cy5.5. The calibration sample may be a calibration plate that includes a plurality of sample wells. [0010] A fluorescence emission spectrum of the first fluorescent entity may at least partially overlap with a fluorescence emission spectrum of the second fluorescent entity. The first and second fluorescent entities may each be associated with spectral emission channels in the multispectral analysis system, and fluorescence emission from the first fluorescent entity may be detected by the multispectral analysis system in the spectral emission channel associated with the second fluorescent entity. [0011] The first and second fluorescent entities may be fluorescent dyes. The first fluorescent entity may be an endogenous fluorescent moiety, and the second fluorescent entity may be a fluorescent dye. A fraction of the second fluorescent entity in the calibration sample relative to a total amount of the first and second fluorescent entities in the calibration sample may be between 0.02 and 0.08 (e.g., between 0.03 and 0.07, between 0.04 and 0.06). [0012] The methods may include using the calibrated multi-spectral analysis system to identify one or more gene targets in the biological sample. [0013] Embodiments of the methods may also include any of the other features described herein, including any combinations of individual features described together with different embodiments, except as expressly stated otherwise. [0014] In another aspect, the description characterizes sample calibrations that include a calibration plate that features a plurality of sample cavities, where the calibration plate is sized to be received in a multispectral analysis system, and a calibration composition positioned in one or more of the cavities, the composition including a first fluorescent entity and a second fluorescent entity, where a fluorescence emission spectrum of the first fluorescent entity