## 4 The Importance of Biomarkers in Monitoring RT Toxicity
### Cell-Intrinsic Radiosensitivity Biomarkers
#### 4.1.2 DNA Damage Response
Last but not least, 53 BP1 is an early indicator of the starting activity of NHEJ/HR repair machinery. Hence, it is reasonable to investigate the behavior of 53 BP1 during DNA repair independently [76]. Immunofluorescence (IF) is commonly used to visualize \(\gamma\)H2 AX foci and 53 BPI, but this technique has some limitations related to a reduced level of quantification and subjectivity in interpretation. Another limitation of IF is that blood cells used for assessing the radiosensitivity of normal tissues are non-adherent cells that must be experimentally manipulated for attaching to slides so as to perform IF. Being dedicated to cells in suspension, flow cytometry appears to be more appropriate to analyze the response of blood leukocytes to IR. In standard flow cytometry, cells are labeled in suspension with specific functional and phenotypic fluorescent indicators and are analyzed one by one from the point of view of morphology (size and granularity) and surface/intracellular fluorescence intensity. The results are finally processed as a distribution of fluorescence intensity per cell, and the mean fluorescence and various other parameters of this distribution are finally calculated using dedicated software. Besides the analysis of cells in suspension, flow cytometry has several other advantages: (i) it allows gating and differential analysis of particular cell populations or sub-populations within a multi-cellular sample, (ii) it provides comprehensive multi-parametric information on single cells in suspension within a complex cellular sample, combining functional, phenotypic, and molecular parameters; for instance, in the radiosensitivity context, DNA damage evaluation (dsDNA breaks evaluated via flow cytometry with the fluorescent TUNEL and \(\gamma\)H2 AX foci or other specific assays) can be combined with investigations on leukocyte viability [77], apoptosis (annexin V-propidium iodide assay), senescence (\(\beta\)-galactosidase assay), leukocyte activation (MHCII, CD69), proliferation (CFDA-SE assay), and cell cycle (Vybrant orange stains) [78]. The currently available flow cytometers allow simultaneous measurement of more than eight parameters in the very same sample. Recently, the advent of high-dimensional time-of-flight mass cytometry (CyTOF) using antibodies labeled with metals and not with fluorophores enables broad dimensional and unbiased examination of blood cells [79], allowing simultaneous interrogation of more than 40 parameters in the same cellular sample. Clustering techniques were developed to reduce the dimensionality of the data in order to create a visual representation in a reduced number of dimensions. Altogether, this new investigational approach is highly important for deep biomarker screening in blood exposed to various challenges (such as IR), particularly when sample sizes are limited (i.e., immune cells in tumors). From another perspective, the new imaging flow cytometry platforms [80], which combine statistical power and high fluorescence sensitivity of standard flow cytometry with the spatial resolution and quantitative morphology of digital microscopy, provide a broader image of the IR impact on blood leukocytes.