Feb 25, 2025
QC Metrics for KPMP Data Collection by VU TIS
- Katerina V Djambazova1,
- Lukasz Migas2,
- Jamie Allen1,
- Madeline E. Colley1,
- Allison Esselman1,
- Léonore Tideman2,
- Ellie Pringy1,
- Angela R.S. Kruse1,
- Melissa Farrow1,
- Raf Van De Plas2,
- Jeff Spraggins1,
- Martin Dufrense1
- 1Vanderbilt University;
- 2Delft University of Technology
- VU Biomolecular Multimodal Imaging Center / Spraggins Research Group
- KPMP
Protocol Citation: Katerina V Djambazova, Lukasz Migas, Jamie Allen, Madeline E. Colley, Allison Esselman, Léonore Tideman, Ellie Pringy, Angela R.S. Kruse, Melissa Farrow, Raf Van De Plas, Jeff Spraggins, Martin Dufrense 2025. QC Metrics for KPMP Data Collection by VU TIS. protocols.io https://dx.doi.org/10.17504/protocols.io.14egn3wqyl5d/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 11, 2023
Last Modified: February 25, 2025
Protocol Integer ID: 89175
Abstract
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).
Biopsy Screening & Go/No-Go Criteria
Biopsy Screening & Go/No-Go Criteria
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
| A | B | |
| 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 > 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 |
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
| A | B | |
| Tissue QC | Reasoning / Data Flags | |
| Are there any glomeruli in the biopsy section? | Ensures proper coverage of biological features | |
| Are there visible cracks/folds on the tissue surface? | This can lower FTU segmentation performance, due to non-segmentable features | |
| Are there visible freezing artifacts on the tissue surface? | Freezing artifacts can induce analyte delocalization, jeopardizing MALDI IMS spatial integrity |
Autofluorescence Microscopy & FTU Segmentation Performance
Autofluorescence Microscopy & FTU Segmentation Performance
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.
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.
Imaging Mass Spectrometry
Imaging Mass Spectrometry
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).
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
| A | B | C | |
| Parameter | QC Threshold | 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 (negativemode) | 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. |
*Denotes critical data flags in the workflow that require re-processing
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
| A | B | C | |
| Parameter | QC Threshold | 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 Resolving Power | ± 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. | |
| 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. |
*Denotes critical data flags in the workflow that require re-processing
Global parameters and individual ion monitoring is performed on both KPMP samples as well as for the external reference tissue.
QC Data Collection & Recording
QC Data Collection & Recording
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.
Protocol references
Include workflow references when available.