Classifications

• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
  • G01N21/39—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using tunable lasers
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N1/00—Sampling; Preparing specimens for investigation
  • G01N1/28—Preparing specimens for investigation including physical details of (bio-)chemical methods covered elsewhere, e.g. G01N33/50, C12Q
  • G01N1/30—Staining; Impregnating ; Fixation; Dehydration; Multistep processes for preparing samples of tissue, cell or nucleic acid material and the like for analysis
  • G01N2001/305—Fixative compositions
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/17—Systems in which incident light is modified in accordance with the properties of the material investigated
  • G01N21/25—Colour; Spectral properties, i.e. comparison of effect of material on the light at two or more different wavelengths or wavelength bands
  • G01N21/31—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry
  • G01N21/39—Investigating relative effect of material at wavelengths characteristic of specific elements or molecules, e.g. atomic absorption spectrometry using tunable lasers
  • G01N2021/396—Type of laser source
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
  • G01N21/75—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated
  • G01N2021/757—Systems in which material is subjected to a chemical reaction, the progress or the result of the reaction being investigated using immobilised reagents
• • G—PHYSICS
  • G01—MEASURING; TESTING
  • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
  • G01N2201/00—Features of devices classified in G01N21/00
  • G01N2201/12—Circuits of general importance; Signal processing
  • G01N2201/129—Using chemometrical methods

Definitions

• Tissue thin sections are used in histology in order to obtain representative infonnation about a tissue sample.
• the quality of the thin section should meet a number of characteristics in order to be properly representative of the overall tissue region where excision of the sample was performed.
• the size of the thin section generally should not be less than 2 ⁇ .
• tissue sections are prepared in the range between 2 and 5 ⁇ and should not vary in thickness by more than 50% over the lateral extent of the thin section in order to allow for appropriate further processing. Further factors that affect tissue section quality may include proper sample moisture and the temperature maintained during the sectioning process.
• the first principal component (PCI) (which carries the largest fraction of the overall variance) may be used alone or together with further principal components. Further uni- or multivariate analysis or combination of analysis schemes may be used. This information may then be used to determine whether or not to perform a subsequent analysis on the tissue sample, or whether a remedial tissue process (such as further fixation) should be performed. Molecular or tissue diagnostic tests can thus be reserved for tissue samples that are most likely to give
• Fig. 8B is an image of the average slope in the range between 1050-1080cm -1 for a fixed sample.
• fixation protocols use a combination of formaldehyde and glutaraldehyde. Glyoxal and acrolein are less commonly used aldehydes.
• Denaturation fixatives typically alcohols or acetone - act by displacing water in the cellular sample, which destabilizes hydrophobic and hydrogen bonding within proteins. This causes otherwise water-soluble proteins to become water insoluble and precipitate, which is largely irreversible.
• over-fixed refers to a tissue sample in which the fixation process obscures or inappropriately alters the morphological and/or molecular details of the sample.
• over-fixation involves an antibody being rendered incapable of binding to its target.
• a spectral analysis system 100 comprising a memory coupled to a processor, the memory to store computer-executable instructions that, when executed by the processor, cause the processor to perform operations.
• processor encompasses all kinds of apparatus, devices, and machines for processing data, including by way of example a programmable microprocessor, a computer, a system on a chip, or multiple ones, or combinations, of the foregoing.
• the apparatus can include special purpose logic circuitry, e.g., an FPGA (field programmable gate array) or an ASIC (application-specific integrated circuit).
• data associated with the acquired test spectrum is communicated to the spectral analysis system 100 from the SA device 101 , 111b or the storage medium 102, 1 11c.
• the spectral analysis system 100 evaluates the data to identify quality signatures within the test spectrum and to classify the test spectrum on the basis of this analysis. This process is illustrated at Fig. 2.
• Data associated with the test spectrum 220 is input into the spectral analysis system.
• a processor of the spectral analysis system 200 then implements a feature extraction (FE) module 210 to extract features of the test spectrum relevant to the quality signature being evaluated 230.
• FE feature extraction
• an unsupervised classification algorithm is used by the classifier.
• the concept of unsupervised classification e.g. cluster analysis, principal component analysis, k-nearest neighbour, etc.
• the algorithm is first trained on a plurality of spectra to generate a plurality of clusters spectra having similar features. Each cluster is then evaluated to determine whether the cluster correlates with a particular quality state. The trained algorithm is then applied to test spectrum, and the algorithm assigns the test spectrum to one of the clusters.
• samples sliced to 4mm thick were placed into 10% Neutral buffered formalin at room temperature (21 degrees Celsius) for 24 hours. These samples were then dehydrated, cleared and impregnated with wax in a standard overnight cycle on an automated tissue processor.
• FT-IR niicrospectroscopy was performed using a Bruker Hyperion 1000 (Bruker Optics, Ettlingen, Germany) together with a Tensor 27 in the wavenumber range 60()-60()0cm- 1 , corresponding to 16.7 ⁇ m. ... 1.67 ⁇ .
• a liquid-nitrogen cooled MCT detector (InfraRcd D326-025-M) was used. The spectral resolution was 4 cm-1.
• Tissue sections were mapped over an area of 60 x 60 steps using a 36x Cassegrain objective (NA: 0.5). A 3.75 ⁇ aperture was introduced into the microscope. The step width was 50 ⁇ .
• 25 forward/backward interferometer scans were collected.
• Blackman-Harris 3 -term apodization was performed prior to background correction and vector normalization. Second derivatives were calculated using Savitzky-Golay filtering. The total acquisition time per thin section amounted to 18 hours.
• the second derivative spectrum of samples fixed for 0, 4, and 24 hours are shown in Fig. 3.
• spectral differences are also observable in the spectral ranges of the QCLs used for illustration in this example (Fig 7):
• One simple example for such spectral differences is the average slope in the 1050 cm -1 - 1080 cm -1 spectral range. If simply this average slope is taken, as a measure of the state of fixation clear differences between the unfixed and fixed sample are illustrated (Fig.8A and Fig 8B, respectively). They may be former evaluated e.g.

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• Engineering & Computer Science (AREA)
• Investigating Or Analysing Materials By Optical Means (AREA)
• Investigating Or Analysing Biological Materials (AREA)

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• 2016
  • 2016-10-28 EP EP16790939.9A patent/EP3368885B1/en active Active
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