Analysis [28]. Sample collection and preparation are critical steps to obtain useful information in clinical proteomics analyses. Tocircumvent undesirable degradation of proteins and peptides, serum samples should be collected under specific standard operating procedures (SOP). However, the current protocols and guidelines for human bodily fluid collection and storage prior to proteomic analysis, in particular regarding blood plasma and serum, still need to be optimized. The influence of pre-analytical factors on the serum peptidome profile is significant, especially the type of blood collection tube, variations in clotting time and temperature, storage conditions, and the number of freeze and thaw cycles [29?2]. Briefly, all venous blood specimens should be collected with vacuum blood collection tubes. After standing upright at room temperature for 60 min, the serum fraction is separated by centrifugation at 1500 x g for 15 min (4 ) and immediately stored at -80 . Only one freeze PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28607003 and thaw procedure can be permitted for any serum sample used for mass spectrometric analysis (this is also critical for other assessments by approaches, such as Luminex because analytes are differentially sensitive to freeze/thaw cycles). The selection of the T0901317 biological activity preservatives and additives used in the collection of blood is important in determining future applicability of the samples. For example, the collection of whole blood in tubes containing any type of anti-coagulant may induce cytokine production in vitro and thus results in artificial measures. Some coagulants are recommended or even required for particular analytical purposes, while others might be contraindicated [33]. The samples should be collected prior to treatment (baseline) and at various time points (e.g., early, middle, and late depending on the treatment interval) during therapy as well as after therapy (early, middle, and late time points). The samples should be aliquoted prior to freezing.LeukocytesComplex immunoregulatory circuits, including the low frequency and activity of effector cells and high frequency of suppressive cells, have the potential to dampen the efficacy of immune interventions, thus cellular immune assessments should be considered an essential component of monitoring efforts in cancer immunotherapy clinical trials. Immune monitoring of peripheral blood and tumor immune cell infiltration offers insights into the mechanism(s) of action of immunotherapeutic strategies and may be prognostic of outcome. However, the selection of the methods and components analyzed during cellular monitoring of clinical trials clearly depends on the individual therapeutic modality and disease being investigated. For these analyses, PBMC obtained from fresh anticoagulated whole blood are isolated by gradient centrifugation using ficoll or Histopaque? Platelets are removed and any remaining contaminating red cells can beStroncek et al. Journal for ImmunoTherapy of Cancer (2017) 5:Page 4 ofeliminated with ammonium chloride potassium (ACK) lysing buffer prior to the use of the cells for downstream analyses, e.g., flow cytometry, transcriptomics, and proteomics. It is noteworthy that hemolysis during sample preparation could significantly affect the biomarker content of e.g. cytokines, microRNA (miRNA) [34].Leukocyte countsRecently, studies have indicated that early changes in immunological markers may be associated with improved survival. To date, many of these signals have come from single analy.
