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

Abstract

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.

Immunomodulation by ionizing radiation—impact for design of radio‐immunotherapies and for treatment of inflammatory diseases

“…One could hypothesis that low‐dose radiation‐induced amelioration of inflammation before the start of high‐dose RT might increase anti‐tumor efficacy of RIT, because chronic inflammation that fosters tumor growth is curtailed. Irrespective of whether RIT or radiation therapies of inflammatory diseases should be enhanced, knowledge on the individual immune status before, during and after therapy is needed . Future work should strongly focus on the discrimination between responses to inflammation and/or specifically to radiation …”

“…In addition, the functional state of the immune cell (sub)set is evaluated and additionally a few non‐leukocytes that also have been associated with the outcome of cancer therapy. It must be stressed that all parameters are obtained with deep IPT by detection of surface antigens in cells of whole blood . Future analyses will shed light into whether and how the immune modulations observed in the periphery are associated with the local situation in the tumor …”

“…Irrespective of whether RIT or radiation therapies of inflammatory diseases should be enhanced, knowledge on the individual immune status before, during and after therapy is needed. 188 Future work should strongly focus on the discrimination between responses to inflammation and/or specifically to radiation. 189…”

“…It must be stressed that all parameters are obtained with deep IPT by detection of surface antigens in cells of whole blood. 188 Future analyses will shed light into whether and how the immune modulations observed in the periphery are associated with the local situation in the tumor. 196…”

The Main Parameters of Cellular Immunity in Patients With Triple-Negative Breast Cancer: Relationship With Efficiency of Chemotherapy

“…В настоящее время многочисленные клинические исследования направлены на определение прогностической значимости различных популяций иммунокомпетентных клеток, инфильтрирующих опухоль [6,19,26]. В то же время иммунофенотипирование клеток периферической крови также является высокоинформативным методом и может использоваться как для анализа исходного состояния иммунитета, так и для мониторинга иммунологических показателей у пациентов в процессе лечения и при прогрессировании заболевания [22].…”