The peripheral blood is easier and has yielded important information. The percent of T cells expressing CARs can be measured using flow cytometry. If the scFV region of the monoclonal antibody used in the CAR is of mouse origin, then goat antibodies directed to mouse F(ab)2 can be used to quantitate CAR T cells. To detect CD19 CAR T cells by flow cytometry, anti-Fab antibody staining and labeled CD19 protein have been used [216]. Flow cytometry using anti-idiotype monoclonal antibody has been used to detect CD19 CAR T cells derived from CD19 mouse monoclonal antibody clone FMC63, [206, 217, 218]. CAR T cell expansion can also be detected by quantitative qPCR [219, 220].The rapid expansion of adoptively transferred CD19 CAR T cells and the disappearance of leukemic cells is associated with clinical toxicity due to cytokine storm [221]. Cytokine release syndrome (CRS) is a non-antigen specific toxicity that occurs as a result of high levels of activation of lymphocytes or myeloid cells. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28607003 It is associated with elevated circulating levels of several cytokines including IL-6, IFN-, and TNF-. Clinically, patients with CRS may experience fever, tachycardia, and hypotension. It can result in cardiac dysfunction, adult respiratory distress syndrome, renal failure, hepatic failure, or neurotoxicity [221]. It is more likely to occur in patients with higher tumor burdens and greater T cell expansion [207, 221]. IL-6 appears to play an important role in the pathogenesis of CRS and the anti-IL-6 receptor antibody, tocilizumab, is often an effective therapy. The clinical use of tocilizumab has also been explored in patients with acute lymphoblastic leukemia who develop CRS after Alvocidib cancer blinatumomab immunotherapy [222]. CRP has been found to be an effective biomarker for CRS [221]. CRP is an acute phase reactant produced by the liver. Its production is largely dependent on IL-6. In patients with ALL treated with CAR T cells, CRP levels have been found to be associated with the IL-6 levels and CRS severity [206].Conclusions and recommendations The field of immune monitoring has helped advance immunotherapy for cancer. All clinical trials of immune therapies for cancer should include a structured plan for sample collection, biomarker analysis, and data analysis. Sample collection and analysis must be adopted for each study, but several points should be considered (Table 3).Table 3 Type of sample and high throughput assessmentsSample Type Serum/plasma Suggested High-Throughput Assessment ?Luminex ?Protein arrays ?SEREX ?PROTEOMEX/SERPA ?SomaScan ?Flow cytometry ?Phospho-flow cytometry ?MHC multiplexed multimers ?nCounter Analysis System (NanoString) ?qPCR ?Gene expression microarrays ?NGS ?miRNA expression analysis ?ImmunoSEQ ?Multiplexed IHC or immunofluorescence ?Flow cytometry ?qPCR ?Gene expression microarrays ?NGS ?miRNA expression analysisPBMCTissueStroncek et al. Journal for ImmunoTherapy of Cancer (2017) 5:Page 18 ofDue to the complexity and our current limitedunderstanding of the underlying biology of cancer immunotherapies, routine, direct evaluation of tumor samples, archival as well as fresh paired tumor samples and direct comparison to peripheral samples, should be considered as a high priority. In addition to analyzing plasma, serum, and peripheral blood leukocytes, consideration should be given to the analysis of tissue samples, the microbiome and, if appropriate, adoptively transferred immune cells. Mutiplexed, high throughput assessment allows fo.