Method validation for automated isolation of viable peripheral blood mononuclear cells

Area of Science:

• Biotechnology
• Cell Biology
• Laboratory Medicine

Background:

• Formal method validation is crucial for biospecimen processing in accredited laboratories and biobanks.
• This study focuses on peripheral blood mononuclear cell (PBMC) isolation protocols.

Purpose of the Study:

• To optimize and validate both automated and manual protocols for isolating PBMCs from whole blood.
• To compare the performance and suitability of automated versus manual PBMC isolation methods.

Main Methods:

• Optimization of manual PBMC isolation involved centrifugation speed, gradient types (Ficoll, Leucosep, CPT), and freezing methods.
• Automated protocol optimization was performed on a Tecan liquid handler using a CPT gradient.
• Validation assessed cell yield, viability, recovery, white blood cell (WBC) subpopulation distribution, gene expression, and lymphoblastoid cell line (LCL) transformation.

Main Results:

• An initial centrifugation of whole blood at 2000 g was optimal for isolating plasma and PBMCs.
• Ficoll gradient showed advantages, and Mr. Frosty was selected as the optimal freezing method.
• Both manual and automated protocols demonstrated reproducibility across key quality and quantity metrics, with robustness acceptance rates higher for processing within 8 hours of blood collection.

Conclusions:

• The first fully automated method for viable PBMC isolation, including RNA analysis and LCL generation, has been validated.
• The study recommends processing blood samples within 8 hours of collection to ensure optimal results for both manual and automated PBMC isolation.