E; use with the QAP as an internal competency test for staff once trained and qualified; and an capability to examine efficiency with peers running the same assay. Published studies have addressed the intra- and inter-assay precision of ICS in entire blood and peripheral blood mononuclear cells (PBMC) (Nomura et al., 2000; Horton et al., 2007; Maecker et al., 2008; Nomura et al., 2008). A recent study by our group on standardization and precision of ICS between laboratories (Maecker et al., 2005) revealed that ICS could possibly be performed by a number of laboratories using a typical protocol with good inter-laboratory precision (18?four ). This precision improves as the frequency of responding cells increases. In an work to standardize the assays across laboratories, in 2005, we designed a QAP for ICS assays. This program was created to assess the inter-laboratory variability when sharing a typical standardized protocol and reagents. Right here, we present the data from seven consecutive rounds of testing. A total of 16 laboratories from seven diverse countries participated in the study in which pre-tested PBMC, along with lyophilized antigens and antibodies, had been distributed. The laboratories had been requested to identify the percentage of cytokine+, CD4+ and CD8+ cells in every sample. The evaluation on the information generated within this program has allowed us to recognize variables accountable for ICS variability among laboratories that need to be taken into consideration when performing Good quality TCN238 custom synthesis Assurance of flow cytometry assays and reporting information for vaccine clinical trials.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptJ Immunol Techniques. Author manuscript; readily available in PMC 2012 January five.Jaimes et al.Page2. Components AND METHODS2.1. Participating institutionsNIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptIn the initial round of testing, ten laboratories worldwide participated. This number improved to 16 by Round five. A list on the participants is supplied in Table 1. All participants have agreed on the content of this publication. Of note, most of these laboratories have been involved in a previous study aimed at standardizing the protocol employed within this ICS QAP (Maecker et al., 2005). two.2. PBMC preparation and cryopreservation Concentrated leukocytes have been prepared by machine leukopheresis with anticoagulant ACDA by BRT Laboratory (Baltimore, MD). PBMC were isolated inside eight hours postcollection making use of a ficoll gradient. Briefly, an average of 11ml of leukocytes have been diluted with phosphate buffered saline (PBS) to 35ml and underlaid with 12 ml of ficoll. Following centrifugation for 30 minutes at 450g (at space temperature), the cell layer was collected; the cells had been washed 3 occasions with PBS and re-suspended in RPMI-1640 media, supplemented with 10 heat-inactivated fetal bovine serum (FBS) PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/20554190 (cRPMI-10). Cell concentration was determined employing the Guava ViaCount assay (Guava Technologies, Inc., Hayward, CA), and PBMC were frozen at 15 ?106 cells/mL in freezing media (22 FCS, 7.5 DMSO and 70.five RPMI). Pre-screening with the PBMC donors for CMV responses was initially done at SeraCare Life Sciences (Gaithersburg, MD) employing an IFN- and IL-2 ELISpot assay. Later, the central laboratory (BD Biosciences, San Jose, CA) performed an ICS assay and chosen the donors for each and every round. Two vials of your cryopreserved PBMC for every donor have been shipped to participant laboratories making use of a liquid nitrogen dry shipper. A advised thaw.
