Flow Cytometry Analysis — Conventional & Spectral

Two analyses a QC/analytical flow scientist runs daily: (1) conventional CAR-T immunophenotyping and release gating, and (2) spectral panel quality — full-spectrum signatures, similarity, and unmixing risk.
Analysis demo by Kalif Shear
Flow Cytometry SME · CGT QC / Analytical
Gating: Cells → Singlets → Live (7-AAD⁻) → CD3⁺ → CAR⁺. CAR⁺ frequency and viable dose drive lot release.

Gating Hierarchy

% of parent at each gate

CD4 vs CD8 — T-cell subsets

Gated on live CD3⁺ lymphocytes

CD3 vs CAR — transduced population

CAR⁺ of CD3⁺ = product-defining frequency

Population Summary & Release Check

Fluorophore Emission Signatures

Normalized full-spectrum emission across the detector array. In spectral cytometry, each fluorophore is identified by its whole signature — not a single detector. Click a fluorophore to isolate it.

Spectral Similarity Matrix

Cosine similarity between signatures. High similarity (hot cells) = fluorophores that are hard to unmix and will spread spillover — the key panel-design risk to catch before running samples.

Unmixing Quality Check

Single-stain reference vs unmixed output for the selected pair — clean separation vs residual spread.