Flow Cytometry in Transplantation.

Flow Cytometry in Transplantation James A. Hutchinson, MBBChir, PhD Links [A] www.miltenyi. tv/vsc_2318_1848_1_vid_720343/ macs-academy-rare-cell-analysis-in-flowcytometry.html [B] www.fda. gov/ScienceResearch/FieldScience/ ucm171818.htm [C] flowcyt.sourceforge. net/ [D] icsh.org/published-standards/ [E] www.beckmancoulter. com/wsrportal/wsr/ research-and-discovery/products-and-services/ flow-cytometry/practical-flow-cytometry/index.htm [F] www.miltenyi. tv/vsc_2318_1848_1_vid_559890/ MACS-Academy-Immunophenotyping-by-flowcytometry.html [G] fccf.epfl. ch/files/content/sites/facs/files/ shared/titration%20of%20antibodies.pdf [H] www.hcdm. org/ [I] www.immunetolerance. org/researchers/clinicaltrials/transplantation [J] www.ctotstudies. org/index.htm [K] www.kcl. ac.uk/lsm/research/divisions/timb/ research/tolerance/index.aspx [L] cordis.europa. eu/result/rcn/52919_en. html [M] www.onestudy. org [N] www.duraclone. com/im/ F low cytometry has emerged as an important method of assessing human immune responses, both as a diagnostic and clinical research tool. Notably, flow cytometry has been applied in detection of donor-directed antibodies, identifying HLA-specific B cells, measuring responses to viral infections, quantifying efficacy of immunosuppressive therapy, and predicting allograft rejection or acceptance. As such techniques become more widely adopted, learning how to judge the quality of flow cytometry studies will benefit researchers and transplant clinicians alike. Modern cytometers produce extremely rich data, but the reliability and comprehensibility of this information depend on many factors apart from an instrument's technical capabilities. Clinical flow cytometry studies present various challenges that are not commonly encountered in basic research, including the need to maintain stable assays over long periods of time and difficulties with ensuring sample quality before analysis. In transplant recipients, immunologically relevant changes in leucocyte subsets may be small, both with respect to biological and technical variations. Multiparameter flow cytometry allows for detailed classification of leucocytes and enumeration of highly specified cell types; however, measurements of rare cells are inherently less precise [A] and the ability to define many subsets often leads to statistical pitfalls with uncorrected multiple comparisons. Hence, proper interpretation of flow cytometry results requires an understanding of assay design and performance, as well as appreciating their clinical context. This article collates online resources with relevance to the readership of Transplantation that provide guidance in design, performance, validation and interlaboratory harmonization of clinical flow cytometry assays. Although flow cytometry assays in accredited diagnostic laboratories are expected to conformwith quality management guidelines, there are no universal standards for researchonly methods. How then should the quality of a researchonly flow cytometry assay be judged? Development of a new flow cytometry assay is a 2-step process: In the design or “prevalidation” step, a method is defined and its specificity, feasibility, and robustness are assessed; in the