PhD Scientific Days 2026

Poster Session 1.A - Molecular Medicine

Optimization of Peripheral Blood Mononuclear Cell (PBMC) Isolation Protocol for Immunological Studies

Name of the presenter

Hegedűs, Károly

Institute/workplace of the presenter

HUN-REN Research Centre for Natural Sciences, Doctoral School of Semmelweis University, Toxi-Coop Toxicological Research Center Plc.

Authors

Károly Hegedűs¹, Sámuel Balázs², Ferenc Fekete³, Annamária Ónodi², Máté Déri³, Katalin Monostory³
1: HUN-REN Research Centre for Natural Sciences, Doctoral School of Semmelweis University, Toxi-Coop Toxicological Research Center Plc.
2: HUN-REN Research Centre for Natural Sciences, Doctoral School of Semmelweis University
3: HUN-REN Research Centre for Natural Sciences

Text of the abstract

Introduction: The isolation of functionally intact leukocyte populations is a fundamental requirement for immunological studies and clinical diagnostics. Standard procedures must ensure that cellular integrity is preserved during sample preparation to enable accurate molecular analyses.
Aims: We aimed to develop and validate an optimized isolation and cell analysis protocol for peripheral blood mononuclear cells (PBMCs) that preserves immunological responsiveness for sensitive gene expression.
Methods: We compared three approaches: 1) direct whole blood activation, 2) activation of PBMC isolated from blood using red blood cell (RBC) lysis, 3) and using Ficoll density gradient centrifugation. Functional integrity was verified by monitoring cytokine gene expression changes using qPCR following chemical immunoactivation. To address challenges in PBMC sample analysis, such as platelet and RBC contamination, various washing techniques and differential staining procedures were evaluated. Türk’s solution was used for cell concentration determination, while Trypan Blue staining (validated using a 60°C heat shock control) was applied to assess cell viability.
Result: Ficoll gradient centrifugation better preserved functional cells than RBC lysis. Although attempts to eliminate platelet and residual RBC contamination (e.g., applying extra lysis steps or buffy coat isolation) reduced PBMC functionality, these anuclear components do not interfere with qPCR accuracy due to their lack of a nuclei and relevant mRNA expression. Such contaminants affected only cell counting, which was improved by using Türk’s staining to distinguish nucleated cells from other blood components. The final protocol consistently yielded near 100% viability, and cells retained their immunostimulatory potential even after cryopreservation.
Conclusion: We established an optimized methodology combining Ficoll-based isolation, Türk’s staining, and safe cryopreservation. This protocol provides reliable gene-expression results by minimizing cell manipulation and preserving native cellular functions.
Funding: This work was funded by the Hungarian Ministry of Innovation and Technology through the National Research, Development and Innovation Fund (2025-2.1.2-EKÖP-KDP-2025-00007) and the SE 250+ Excellence PhD Scholarship Program.