CytoFLEX mosaic Spectral Detection Module Enables Enhanced Spectral Unmixing of White Blood Cell Populations by Extracting Multiple Autofluorescence Signatures
Executive Summary
Autofluorescence (AF) arises from endogenous cellular fluorophores — including reduced pyridine nucleotides (NADH), oxidized flavin coenzymes (FMN and FAD), and aromatic amino acids — and poses a significant challenge in spectral flow cytometry. In complex, heterogeneous samples such as peripheral blood leukocytes, distinct AF signatures across cell types can lead to unmixing errors, aberrant (false‑positive and false‑negative) marker expression, and compromised data reliability.
This study evaluates how the CytoFLEX mosaic Spectral Detection Module addresses these challenges through multi‑channel AF extraction. The module can extract fluorescent signatures from up to 10 distinct AF populations; in this white blood cell study, extracting two AF signatures was sufficient to remove unmixing artifacts, restore true biological signal resolution, and support reliable immunophenotyping workflows.
Key Highlights & Findings
- Reduces False Positivity: Without AF extraction, fluorochromes emitting in the AF range (BUV496, BV510, BV570) produced aberrant false‑positive signals for CD16, CD4, and HLA‑DR across leukocytes; AF extraction reduced this false positivity while preserving true biological signals.
- Corrects Signal Distortions: AF interference can also reduce signal intensity — for example, artificial CD8 negativity (cFluor V547) in eosinophils — which appropriate AF extraction corrects.
- Targeted Multi‑AF Resolution: A single AF signature (neutrophils) removed autofluorescence from neutrophil, monocyte, and lymphocyte populations, while a second AF signature (eosinophils) was required to resolve the distinct granular AF profile of eosinophils.
- Optimal Balance: Two AF signatures provided the best balance of unmixing quality; adding further signatures (four total) did not improve marker resolution and increased spread (cumulative variance 0.337 → 0.386) in the classical monocyte population, underscoring the value of optimizing — not maximizing — the number of AF signatures.
Conclusion
Multi‑channel AF extraction using the CytoFLEX mosaic Spectral Detection Module improves the accuracy and resolution of spectral unmixing in complex biological samples. By extracting tailored AF signatures for highly autofluorescent populations such as eosinophils, researchers can achieve high‑resolution immunophenotyping while preserving true biological variation. The module’s capacity for up to 10 AF signatures offers additional flexibility for managing AF in more complex panels.
For Research Use Only. Not for use in diagnostic procedures.
