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The Histology-Guided Mass Spectrometry (HGMS) profiling workflow, a targeted approach for analyzing discrete areas within a tissue section using histological staining as a guide. the importance of quality tissue sections, histological staining, digital imaging, image annotation, image merging, sample preparation, data collection, and data analysis. HGMS provides valuable biological insights not attainable by standard histology, such as predicting disease outcome, improving diagnosis, and predicting treatment response.
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Stained serial section annotated Tissue section on MALDI target Many tissues in one experiment Images of stained and unstained sections merged Trypsin and/or matrix applied Mass spectra collected
800 1200 1600 2000 0 5 10 15 20 25 Intensity m/z Benign Malignant
Histology-Guided Mass Spectrometry (HGMS) Profiling is a targeted approach in which only discrete areas within a tissue section are analyzed. Histological staining is used to guide the acquisition of spectra from the tissue section allowing each analyzed spot to be enriched for a single cell type. Due to the reduced data volume, this approach is highly conducive to statistical analysis and classification algorithm generation. HGMS can provide biological insight not attainable by standard histology; such as predicting disease outcome, improving diagnosis, predicting treatment response, etc.
Histological staining is used to allow for visualization of features and cell types of interest in a tissue section. Staining is typically carried out on a section immediately serial to the one to be analyzed by HGMS, but there are cases where staining can be used on the same section, if it will not interfere with data collection*. A variety of different stains can be used depending on the disease or types of cells to be targeted. These include: Hematoxylin and Eosin, Cresyl Violet, Congo Red, and Giemsa, among others, as well as immunohistochemical staining. *Chaurand P, Schawartz SA, Billheimer D, Xu BJ, Crecelius A, Caprioli RM. Anal Chem. 2004 , 76 , 1145-55.
Digital images are acquired of both the section for mass spectrometry and the stained serial section. Images of the mass spectrometry section are typically acquired with a flatbed document scanner with a resolution of 2400 dpi or higher. Images of the stained section are taken at microscopy resolution to allow for evaluation of histological features in the tissue. Histological images may be acquired with a stitching microscope using a 10X or higher objective or with a digital slide scanner.
Because the annotations have been placed on a serial stained section to the one being analyzed, the coordinates of those annotations must be transferred to the unstained MSI section. In order to accomplish this task, the images of the two sections must be digitally merged. This is accomplished using Photoshop or other image processing software. When necessary, the annotated image may be broken up into multiple pieces to account for slight differences between the two sections, including bends, folds, or tears. The merged digital image is then used to guide the data acquisition in the mass spectrometer.
Just as in traditional mass spectrometry imaging, appropriate sample preparation must be carried out for the class of molecules to be analyzed from the tissue section. This may include washing to remove biological salts and enhance signal, enzymatic digestion, and/or matrix application.
Depending on the goals of the study, the data can be subjected to a variety of statistical analyses. These include hypothesis testing, discriminant analysis (receiver operating characteristic curves), and principal component analysis, as well as the generation of a variety of machine learning algorithms for diagnostic or prognostic classification of samples.
