The current gold standard for diagnosing and assessing treatment response is tumor biopsy; however, biopsies are not always feasible, safe or easily repeated during treatment. Utilization of peripheral blood mononuclear cells (PBMCs) as a surrogate for tumor biopsy allows for longitudinal sampling and is a safer, more readily available option. However, collection conditions, sample transfer time across multiple clinical sites, and PBMC processing conditions are external pre-analytical factors that must be understood and controlled to mitigate bias in downstream functional analyses. This study aims to systematically evaluate the pre-analytical variables affecting PBMC integrity and functional immune readouts as a prerequisite for downstream translational biomarker applications. Peripheral blood samples were collected from 80 treatment-naive patients with a diagnosis of head and neck squamous cell carcinoma. Blood was collected in cell preparation tubes (CPT), potassium ethylenediaminetetraacetic acid (EDTA), or sodium heparin (SH) tubes and diluted 1:1 with sterile PBS or remained undiluted. PBMCs were processed and cryopreserved immediately or held for 8- and 24-hours before processing. PBMC viability was measured at cryopreservation and upon thawing. CD8+ T cells or natural killer (NK) cells derived from PBMCs were subjected to cytotoxicity assays using flow cytometry. CPT tubes provided lower cell viability and yield at cryopreservation and upon thaw compared to EDTA and SH tubes while dilution had no effect on viability. NK cell cytotoxicity was similar between EDTA and SH tubes irrespective of dilution. However, diluted EDTA tubes resulted in lower T cell cytotoxicity after 24-hour hold. Viability and NK and T cell cytotoxicity were equivalent between cryopreserved PBMCs that were processed immediately or processed after 8- or 24-hour hold. Here we report cryopreservation methods for reproducibility of viable cells that maintain functional immunological capacity even after significant delay in processing allowing flexibility and feasibility for collection from multiple clinical sites for deferred processing.

The authors have declared no competing interest.

This work was supported by the National Cancer Institute (U01 CA267985-01). This publication contents are the sole responsibility of the authors and do not necessarily represent the official view of the NIH.

I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

Blood procurement involving human subjects was approved by the Institutional Review Board (IRB) of the University of Cincinnati (UCCI-UMB-14-01, IRB# 2015-2344 and General Specimen Protocol IRB# 2017-2137) and conducted in accordance with Good Clinical Practice guidelines and the Declaration of Helsinki. Written and informed consent was collected from enrolled patients with HNSCC prior to sample collection.

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Patient-level deidentified datasets used during the current study are provided as supplemental tables. Additional information can be provided upon reasonable request to the corresponding author.