Process

Overview

HGMS profiling, both during test development and following commercial deployment, requires proprietary workflows and operating procedures be established for each test. In addition, HGMS profiling requires significant technological capabilities in order to attain efficient, high throughput operations. These workflows, procedures, and capabilities are specific to each step of the testing process:

arrow-small-green Specimen preparation
arrow-small-green Annotation
arrow-small-green Specimen analysis
arrow-small-green Data processing

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Specimen Preparation

Proper specimen preparation is critical for robust and reproducible HGMS profiling of the proteomic content within the histopathologically relevant areas of tissue sections.

  1. H&E Staining:  One tissue section is stained with hematoxylin and eosin (H&E) to enable our experienced veterinary pathologists to review and annotate the patient specimen.
  2. Antigen Retrieval:  A second tissue section undergoes a process that denatures the proteins within the patient specimen.
  3. Trypsin Digestion:  Trypsin enzyme is sprayed onto the second tissue section in very thin layers to promote reproducible protein digestion.
  4. Matrix Application:  MALDI matrix is sprayed onto the second tissue section in very thin layers using a robotic sprayer.

Annotation

Specimen annotation is performed by experienced veterinary pathologists using a portal that enables them to review and annotate high resolution digital images, targeting cells of interest down to a 50 µm diameter. The portal provides our veterinary pathologists the ability to target specific groups of cells, distinguish between various cell types, and reject specimens based on a test’s stated specimen requirements.

Annotated Image

Specimen Analysis

The Bruker rapifleX™ is a matrix assisted laser desorption/ionization (MALDI) time of flight (TOF) mass spectrometer. Slides containing adhered tissue are directly inserted into the instrument, and a laser is rastered over designated tissue regions causing bombarded molecules to be ablated from the surface and ionized. Charged ions are then accelerated through the flight tube within the mass spectrometer toward the ion detector. Ions with a smaller mass/charge (m/z) ratio move faster through the flight tube than those with a larger m/z value. Because the flight tube is a known length, the “time of flight” correlates directly to the m/z ratio of the ion, which can be calculated using calibration standards of known molecular weight. The resulting mass spectrum displays all observed m/z ions that were detected within a specified mass range.

View MALDI TOF Process

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Data Processing

Molecular profiling data are analyzed using SCiLS Lab software. During test development, this software enables us to perform computational analysis of large data sets pertaining to two or more phenotypes, harness machine learning to improve classification accuracy as specimen numbers are increased, and to optimize classification algorithms prior to validation. Once a classification algorithm has been validated for a specific test, we can lock the algorithm and transition it to our production environment.

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