Development of a Modular Assay for Detailed Immunophenotyping of Peripheral Human Whole Blood Samples by Multicolor Flow Cytometry

The monitoring of immune cells gained great significance in prognosis and prediction of therapy responses. For analyzing blood samples, the multicolor flow cytometry has become the method of choice as it combines high specificity on single cell level with multiple parameters and high throughput. Here, we present a modular assay for the detailed immunophenotyping of blood (DIoB) that was optimized for an easy and direct application in whole blood samples. The DIoB assay characterizes 34 immune cell subsets that circulate the peripheral blood including all major immune cells such as T cells, B cells, natural killer (NK) cells, monocytes, dendritic cells (DCs), neutrophils, eosinophils, and basophils. In addition, it evaluates their functional state and a few non-leukocytes that also have been associated with the outcome of cancer therapy. This DIoB assay allows a longitudinal and close-meshed monitoring of a detailed immune status in patients requiring only 2.0 mL of peripheral blood and it is not restricted to peripheral blood mononuclear cells. It is currently applied for the immune monitoring of patients with glioblastoma multiforme (IMMO-GLIO-01 trial, NCT02022384), pancreatic cancer (CONKO-007 trial, NCT01827553), and head and neck cancer (DIREKHT trial, NCT02528955) and might pave the way for immune biomarker identification for prediction and prognosis of therapy outcome