TITLE: VU TIS Multimodal Molecular Imaging Pipeline for KPMP Biopsy Interrogation 
AUTHORS: Katerina V Djambazova, Lukasz Migas, Jamie Allen, Martin Dufresne, Madeline E. Colley, N. Heath Patterson, Léonore Tideman, Allison Esselman, Ellie Pingry, Angela R.S. Kruse, Melissa Farrow, Raf Van De Plas, Jeff Spraggins

[DESCRIPTION]
This protocol provides an overview of the Multimodal Molecular Imaging pipeline used by the Vanderbilt University Tissue Interrogation Site (VU TIS) as part of the Kidney Precision Medicine Project (KPMP). Individual protocols are contextualized within our larger workflow.

[STEPS]
SECTION: Sample Interrogation Assays
3. Autofluorescence Microscopy (AF) is collected on all KPMP tissue sections prior to IMS. AF is also collected on an external reference tissue termed "BlockQC".
AF QC Data Collection Protocol: AFQC Protocol
AF protocol: AF Microscopy
 

SECTION: Sample Interrogation Assays
4. MALDI Imaging Mass Spectrometry (IMS)  is collected on all KPMP tissue sections and on a BlockQC tissue section.
Matrix Deposition Protocol (Sublimation): Tissue Washing and Matrix deposition Protocol
High Spatial Resolution MALDI IMS Data Acquisition: MALDI IMS Protocol
SECTION: Sample Interrogation Assays
5. LC-MS/MS Lipidomics is performed on a single serial KPMP biopsy and a BlockQC tissue section. 
Protocol: LC-MS/MS Protocol
SECTION: Sample Interrogation Assays
6. Post-IMS Autofluorescence Microscopy is collected on all KPMP tissue sections and a single BlockQC tissue section to aid in multimodal data registration.
Protocol: Post-IMS AF
SECTION: Sample Interrogation Assays
7. PAS Staining is performed on all tissue sections including BlockQC
Matrix removal and Staining: PAS Staining Protocol
SECTION: Data Pre-Processing and Analysis
9. Image registration - AF, PAS and IMS is performed on all tissue sections including BlockQC
Protocol: Image Registration
 

SECTION: Tissue Screening and Assessment
2. Asses Kidney Tissue Sections
To accept biopsies for multimodal molecular imaging analysis by VU TIS, the samples must meet the following Go/No-Go conditions (Table 1). 

Table 1. Sample Processing Go/No-Go Criteria 
     Sample Processing   Go/No-Go Criteria      Reasoning        Biopsy must be LN2 core      Other storage conditions can induce   analyte suppression effect in MALDI IMS analysis        Biopsy size must be ³ 3mm      Smaller tissue sections are difficult to   handle, section, and mount        Time to preservation must be < 10 min      Prolonged exposure prior to preservation   can cause metabolite/lipid degradation   

Sectioning
Accepted biopsies are sectioned and visually assessed following the QC metrics described in Table 2. Biopsies are cryo-sectioned to 10 µm thickness on indium-tin-oxide (ITO)-coated (for imaging mass spectrometry) or glass (for stained microscopy) slides. For workflow QC, a normal human kidney reference tissue section (termed BlockQC) is sectioned along with KPMP samples.  For cryo-sectioning,  samples are mounted to a chuck using a small amount of optimal cutting temperature (OCT) compound. Photos are taken before, during, and after sectioning (with and without mounting media) to document the process and ensure proper handling. After sections are obtained, the OCT is removed from the tissue block, and the samples are stored at -80oC. Tissue sections are visually assessed following the QC metrics described in Table 2 before data collection.

Table 2. On-Site Tissue Integrity Considerations
Tissue QCReasoning / Data FlagsAre there any glomeruli in the biopsy section?Ensures proper coverage of biological featuresVisible cracks/folds on the tissue surfaceThis can lower FTU segmentation performance, due to non-segmentable featuresVisible freezing artifacts on the tissue surfaceFreezing artifacts can induce analyte delocalization, jeopardizing MALDI IMS spatial integrity

 
SECTION: Data Pre-Processing and Analysis
11. Analyte Annotation
Tentative lipid annotations are curated from the data
Protocol: Tentative Lipid Annotation
SECTION: Data Pre-Processing and Analysis
10. Functional Tissue Unit (FTU) Segmentation is performed on all KPMP biopsies and BlockQC tissues.
Protocol: FTU Segmentation
 

SECTION: Data Pre-Processing and Analysis
8. Data Pre-Processing is performed on all MALDI IMS data.
Protocol: MALDI IMS Data Pre-Processing
SECTION: QC Data Collection and Recording
12. QC data collected during this work will be compiled in Levy-Jennings plots to longitudinally track the performance of the assays. QC data for both the KPMP samples and the reference tissue are summarized and reported. QC/QA protocols for the multimodal molecular imaging pipeline are summarized in the protocol QC Metrics for KPMP Data Collection by VU TIS.
SECTION: Sample Shipment, Storage, and Handling
1. KPMP Biopsy Shipment to VU TIS
KPMP biopsies are shipped on dry ice to VU TIS. Temperature is monitored using MadgeTech data loggers. Specimens and relevant information are recorded in Redcap, and samples are store at -80oC until sectioning. Prior to sectioning, samples are assessed according to Table 1, described below.