Erapies is the tissue source, with preference for easily accessible material, such as body fluids (blood and urine) rather than serial tumor biopsies, which are possible for cutaneous melanoma and hematologic malignancies, and more challenging for other tumor types where core biopsies (if anything) are more common. In this context, the capacity of the technology, the reproducibility of results, the assay stability and the ability to validate the results are essential considerations. Sample generation, isolation, and processing are important issues, since significant differences have been observed between different methods and consumables used for the purification of serum, plasma, and immune cells obtained from peripheral blood and from tumor tissues [18?0]. In addition, the sample holding timesStroncek et al. Journal for ImmunoTherapy of Cancer (2017) 5:Page 3 ofbefore processing (1 ?48 h.), the blood collection method, and immunoglobulin G (IgG) purification from these samples could PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28607003 affect analysis, leading to reproducibility problems [21].Serum and plasmaSerum and plasma samples prepared from peripheral blood are easily obtainable from patients and are often collected as a part of clinical studies and stored in biobanks. In particular, serum or plasma is collected for the evaluation of cytokines, chemokines, and growth factors, as well as other soluble molecules, including antibodies, matrix metalloproteinases (MMP), and adhesion molecules [22, 23]. In addition, the newly appreciated role of exosomes and extracellular vesicles (EV) as cancer biomarkers [24] and in immune surveillance [25], begs for the development of sample collection methods compatible with multiple downstream analyses, including that of exosomes/microvesicles. The considerations linked to the choice of the source (whole blood versus plasma or serum), and the method of purification have been discussed in a position paper from the International Society of Extracellular Vesicles [26], which concluded that plasma is the most physiological relevant milieu to study blood EV. If such studies are envisioned, then plasma should be collected. Serum samples can be collected using silica-coated serum separating tubes. Serum can then be incubated at a dilution of 1:50 in 0.5 casein-PBS (phosphate buffered saline) blocking buffer to suppress non-specific binding of sera proteins. For Luminex and ELISA, plasma samples can be collected in tubes containing one of three distinct anti-coagulants: (i) sodium heparin, (ii) sodium citrate dextrose and (iii) ethylenediaminetetraacetic acid (EDTA) [27]. To determine the serum peptidome profile, three different protocols can be used for mass spectrometric analyses of serum and plasma proteins. For the first, crude plasma samples can be directly subjected to tryptic cleavage. Otherwise, buffer components can be removed from the samples and samples can be concentrated using macro spin plates. For the third protocol, the process of depletion can be carried out using a proteoprep immunoaffinity albumin and IgG depletion kit, followed by trypsinization and peptide extraction on macro spin buy AZD-8835 centrifuge plates. The protein digestion can be performed using trypsin at 37 for three hours with stirring. The trypsinization is then terminated by adding trichloroacetic acid, the pH adjusted, the trypsinized plasma is dried, resolved in liquid chromatography solution, spiked with isotypically labeled peptide standard and then used for mass spectrometric.
