TITLE: QC Metrics for KPMP Data Collection by VU TIS
AUTHORS: Katerina V Djambazova, Lukasz Migas, Jamie Allen, Madeline E. Colley, Martin Dufrense, Allison Esselman, Léonore Tideman, Ellie Pringy, Angela R.S. Kruse, Melissa Farrow, Raf Van De Plas, Jeff Spraggins

[DESCRIPTION]
This protocol describes the Go/No-Go criteria, QC/QA steps, and metrics used in the "Multimodal Molecular Imaging" pipeline by the Vanderbilt University Tissue Interrogation Site (VU TIS) as part of the Kidney Precision Medicine Project (KPMP).

[STEPS]
SECTION: Biopsy Screening &amp; Go/No-Go Criteria
1. To accept KPMP samples for analysis, the following sample processing Go/No-Go criteria in Table 1 must be met:

Table 1. Sample Processing Go/No-Go Criteria
   AB1Go/No-Go Criteria  Reasoning  2Biopsy must be LN2 core  Other storage conditions can induce analyte suppression effect in MALDI IMS analysis  3Biopsy size must be > 3mm  Smaller tissue sections are difficult to handle, section, and mount  4Time to preservation must be < 10 minProlonged exposure prior to preservation can cause metabolite/lipid degradation  

Accepted biopsies will be cryo-sectioned to 10 µm thickness and visually assessed following the QC metrics described in Table 2 before data collection. 

Table 2. Tissue QC Metrics
   AB1Tissue QCReasoning / Data Flags2Are there any glomeruli in the biopsy section?Ensures proper coverage of biological features3Visible cracks/folds on the tissue surfaceThis can lower FTU segmentation performance, due to non-segmentable features4Visible freezing artifacts on the tissue surfaceFreezing artifacts can induce analyte delocalization, jeopardizing MALDI IMS spatial integrity

SECTION: Autofluorescence Microscopy &amp; FTU Segmentation Performance
2. Autofluorescence microscopy QC is performed prior to collecting autofluorescence data from KPMP biopsies. A detailed protocol for Autofluorescence Microscopy QC data collection is included here.

The mean fluorescence intensity (above the noise level) is calculated for each channel (eGFP, DAPI, DsRed) and monitored over time. To pass QC, intensity should be within ±15% of the mean for each channel.
SECTION: Autofluorescence Microscopy &amp; FTU Segmentation Performance
3. Autofluorescence-based FTU segmentation is performed on each KPMP biopsy and for one reference tissue, termed BlockQC. 

Percent coverage is calculated for each tissue section. A coverage >60% meets QC standards. When lower segmentation coverage is observed, the data is flagged for re-evaluation.

SECTION: Imaging Mass Spectrometry
4. MALDI IMS Data Collection
Mass calibration is performed prior to MALDI IMS data collection. The mass must be calibrated to <1 ppm error, corresponding to a 95% calibration score in the software. 
Mass calibration and subsequent MALDI IMS data are also collected on an external reference tissue section (BlockQC).
SECTION: Imaging Mass Spectrometry
5. MALDI IMS Data Analysis

Global Parameter Monitoring
Table 3 Summarizes the QC metrics for tracking the global performance of MALDI IMS. QC metrics for LC-MS/MS data analysis are also included in the table. 

Table 3. QC Metrics for MALDI IMS Global Parameters
     Parameter      Expected
  Performance      Notes        Mean
  intensity variability*      ± 15%      BlockQC section: Significant
  deviation from the mean intensity may suggest problems with the
  instrumentation. Instrument performance will be assessed.        Number
  of detected peaks*      > 600 (positive
  mode)  > 900 (negative
  mode)      Adaptive
  S/N thresholding is applied until the minimal acceptable peaks are detected.        Number
  of annotated peaks      ~ 270 (positive
  mode)  ~ 450 (negative
  mode)      Expected
  range, but real numbers may vary, especially in KPMP biopsies with CKD or AKI.        Common
  annotated peaks      > 150 (positive
  mode)  > 220 (negative
  mode)      Expected
  range, but real numbers may vary, especially in KPMP biopsies with CKD or AKI.        LC-MS/MS      > 300
  (positive mode)  > 300
  (negative mode)      Expected
  range, but real numbers may vary, especially in KPMP biopsies with CKD or AKI.   


Individual Ion Monitoring
For a set of diagnostic ions (in each polarity), we will monitor five parameters Listed in Table 4. 

Table 4. QC Metrics for MALDI IMS Individual Ion Parameters
     Parameter      Expected
  Performance      Notes        Peak
  Alignment      -      Visual
  interpretation and marked for pre-processing if not well aligned. Realignment
  may be performed.        Peak
  Width      ± 10%      Expected
  deviation, however real values may vary for lower intensity species.         Mass
  Error*      ± 5 ppm      BlockQC: If abnormally
  high ppm errors are detected for the set panel of individual ions, the data are
  flagged for pre-processing. Individual datasets can be recalibrated
  (internally) with different ions.        Mass
  Resolution      ± 10%      Significant
  deviations may be indicative of compromised instrument performance.
  Instrument performance will be assessed.        Peak
  Intensity      ± 15%      BlockQC: We allow for a
  wider peak intensity variability, as even within the same tissue block,
  analyte intensity is highly dependent on the sampled structures.   KPMP
  biopsies: CKD and AKI may significantly impact the peak intensity of the monitored
  panel of ions; therefore no meaningful boundary can be estimated here.   

Global parameters and individual ion monitoring is performed on both KPMP samples as well as for the external reference tissue.
SECTION: QC Data Collection &amp; Recording
6. The collected QC data 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.