Enumeration of TBNK cells: A case for a bioanalytical assay to support clinical studies for the development of modified risk tobacco products
Smoking has been reported to lessen the body’s immune response making it more susceptible to infections. Chronic cigarette smoke exposure causes imbalance in immune homeostasis affecting the circulating immune cells. Peripheral blood lymphocytes are heterogeneous in nature. Based on biological function and cell-surface antigen expression, lymphocytes are classified into three major populations: T lymphocytes, B lymphocytes and NK (Natural Killer) lymphocytes (TBNK). Measurement of these subsets can be valuable tool in understanding the impact of modified risk tobacco products (MRTP) in comparison with traditional combustible tobacco products.
Current clinical protocols for enumeration of specific cell populations within whole blood require that the sample be analysed within 24-48 hrs. Staining fresh whole blood is not practical in large clinical studies, and it is not possible to batch or re-run samples. Furthermore, it is very challenging if not impossible to ship samples from clinical study sites to a central analytical laboratory given these time constraints. Isolation and cryopreservation of PBMCs for downstream analysis by cell flow cytometry is an alternative, but is technically demanding, highly variable and labor intensive.
Here we report a flow cytometry-based TBNK assay, which overcomes the limitations of a clinical diagnostics assay mentioned as above. The assay utilizes stabilized whole blood simplifying the sample collection at the clinical sites. We have established stability in whole blood with stabilizer for more than 30 days post-freezing at -80 °C. This innovation allows the analysis of clinical samples in batches by subject minimizing the process related variability within subjects. The innovation allows the preparation and use of quality control samples in each batch for the monitoring of assay performance. During the method validation relative accuracy, precision (inter-operator and inter-instrument), post-staining stability, pre-process stability, post-staining stability and freeze-thaw stability were also tested. The method was found to be an attractive solution and alternative to highly variable clinical diagnostics for the enumeration of TBNK for the purposes of MRTP evaluation in a clinical setting.