Every Step Documented, Every Metric Tracked
Click any stage to expand its QC parameters, processing details, and quality benchmarks.
Peripheral Blood Draw & PBMC Isolation
Whole blood collected via standard phlebotomy, PBMCs isolated through density gradient centrifugation within the processing window.
Standardized collection protocols ensure consistency across all patient cohorts. Samples are processed at I3H's dedicated facility with tracked chain-of-custody from draw to cryopreservation. Temperature controlled at room temp during transit – no cold-chain artifacts that would alter cell surface marker expression.
- ✓ Ficoll-Paque density gradient centrifugation
- ✓ Automated cell counting via hemocytometer / Countess
- ✓ Red blood cell lysis QC (visual + scatter verification)
- ✓ Sample metadata captured at point of collection
Sample Viability & Cell Quality Assessment
Rigorous viability gating ensures only high-quality, representative samples enter the profiling pipeline.
Viability is the single most critical QC checkpoint. Dead cells bind antibodies non-specifically, corrupting downstream data. I3H maintains viability well above the 85% minimum threshold through rapid processing, controlled thawing, and cisplatin-based dead cell exclusion during CyTOF staining.
- ✓ Cisplatin (195Pt) live/dead discrimination on CyTOF
- ✓ Trypan blue exclusion for pre-stain counting
- ✓ Controlled thaw protocol for cryopreserved samples
- ✓ Viability gate applied before all downstream analysis
- ✓ Samples below threshold flagged and excluded with documentation
Maxpar Direct Immune Profiling Assay (MDIPA)
Standardized lyophilized antibody cocktail – 30+ metal-conjugated markers measuring surface and intracellular proteins at single-cell resolution.
The MDIPA kit is the backbone of I3H's standardized profiling. As a pre-validated, lyophilized antibody cocktail, it eliminates lot-to-lot variability – critical for comparing results across thousands of patient visits spanning years.
- ✓ No spectral overlap – mass cytometry uses metal isotope tags
- ✓ Batch-matched reagents across all cohorts
- ✓ Lineage, activation, and functional markers in single tube
- ✓ Intercalator (iridium) DNA stain for singlet discrimination
- ✓ EQ Four Element Calibration Beads for signal normalization
Mass Cytometry Data Acquisition
Cells are nebulized, ionized, and analyzed by time-of-flight mass spectrometry – 30+ parameters per cell at thousands of events per second.
CyTOF acquisition is destructive – each cell is vaporized into heavy-metal ions and analyzed by mass spectrometry. This eliminates spectral overlap entirely. EQ beads run continuously for real-time calibration.
- ✓ EQ bead normalization across acquisition runs
- ✓ Gaussian discrimination parameters for doublet removal
- ✓ Instrument tuning verified before each batch
- ✓ Signal stability monitored throughout acquisition
Bead Normalization, Gating & QC
Raw FCS files undergo bead normalization, debris removal, and standardized gating before entering the analysis pipeline.
I3H has developed a computational analysis approach that aligns with how immunologists think about the data. Validated against manual expert gating with strong correlation – scalable without sacrificing immunological rigor.
- ✓ EQ bead removal and signal normalization
- ✓ Live/dead gating (cisplatin-negative)
- ✓ Singlet gating (DNA intercalator)
- ✓ CD45+ immune cell gating
- ✓ Automated population identification across 50 subsets
- ✓ Batch effect monitoring across acquisition runs
Pennsieve Platform – Structured Data Management
Processed FCS files, annotations, and patient metadata organized in Pennsieve for discoverable, FAIR-compliant data management.
Pennsieve provides the data management backbone – each FCS file is linked to its clinical metadata, processing provenance, and QC annotations, creating a queryable, cohort-spanning dataset.
- ✓ FAIR data principles (Findable, Accessible, Interoperable, Reusable)
- ✓ Provenance tracking from blood draw through analysis
- ✓ Cross-cohort data discovery and comparison
- ✓ Integration with Penn EMR systems
- ✓ API access for computational workflows
Immune Fingerprinting & Informatics
Comprehensive immune fingerprints – quantifying 50 populations to establish baselines, detect perturbations, and track longitudinal trajectories.
The final layer transforms population frequencies into actionable immune health profiles. Standardized upstream means fingerprints are directly comparable across patients, time points, and disease contexts.
- ✓ Population frequency quantification across 50 immune subsets
- ✓ Activation state profiling (HLA-DR, CD38, Ki-67)
- ✓ Longitudinal trajectory tracking per patient
- ✓ Cohort-level distribution analysis
- ✓ Integration with clinical outcomes data
- ✓ Dashboard visualization for investigators