Flow Cytometry in Transplantation.

Abstract

Flow cytometry has emerged as an important method of assessing human immune responses, both as a diagnostic and clinical research tool.1 Notably, flow cytometry has been applied in detection of donor-directed antibodies, identifying HLA-specific B cells,2 measuring responses to viral infections,3 quantifying efficacy of immunosuppressive therapy,4 and predicting allograft rejection5 or acceptance.6,7 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 conform with quality management guidelines,8 there are no universal standards for research-only methods. How then should the quality of a research-only 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 “validation” step, accuracy, sensitivity, imprecision, and stability of the assay are characterized. Only an assay has successfully passed through both steps of development can be regarded as fit-for-purpose [B and C]. Detailed guidance concerning the validation process for flow cytometry assays is available in a 5-part set of recommendations issued by the International Council for Standardization of Haematology (ICSH) and the International Clinical Cytometry Society, which can be downloaded from the ICSH website [D]. Links [A] www.miltenyi.tv/vsc_2318_1848_1_vid_720343/macs-academy-rare-cell-analysis-in-flow-cytometry.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-flow-cytometry.html [G] fccf.epfl.ch/files/content/sites/facs/files/shared/titration%20of%20antibodies.pdf [H] www.hcdm.org/ [I] www.immunetolerance.org/researchers/clinical-trials/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/ During the prevalidation of a flow cytometry assay, developers should address the following questions: What is the purpose of the assay? What performance criteria are expected of the assay? What level of confidence is required in the results obtained from the assay? How will consistency in assay quality be assured? Answers to these questions will influence the choice of reagents and decisions about appropriate controls.9 Selection of reagents is critical to the performance of a flow cytometry assay. The principles of multicolor panel design are clearly explained in Practical Flow Cytometry: 4th Edition, which can be freely downloaded from the Beckman Coulter website [E]. A webinar covering the basics of immunophenotyping by flow cytometry, including a helpful discussion of reagent selection, is presented on the Miltenyi website [F]. After selecting a panel of antibodies, it is essential to determine their optimal concentration for the particular assay in titration experiments [G]. Proving that a selected combination of antibodies allows specific identification of a cell subset is difficult; indeed, it may not be possible to show more than specificity of each antibody for its antigen [H] and a plausible gating strategy based on the chosen markers. In this case, the lack of universally accepted marker combinations that define certain leucocyte subsets (e.g., human regulatory T cells) can be problematic. Standardization of marker panels and methodologies is an alternative to development of “home-brew” immune phenotyping assays at different sites and has many obvious scientific advantages.10 Establishing standardized flow cytometry methods for use in solid organ transplantation has been a focus of many large research consortia, including the Immune Tolerance Network [I], the Clinical Trials in Organ Transplantation project [J], the Indices of Tolerance consortium [K], the Reprogramming the Immune System for the Establishment of Tolerance consortium [L] and The ONE Study [M]. Several antibody vendors now offer predesigned general immune phenotyping panels. Of particular relevance to solid organ transplantation is a reagent set produced by Beckman Coulter, which was designed by Sawitzki and colleagues for use in The ONE Study clinical trials [N]. Critically, adopting standardized methodology avoids the need for laboratories to perform time-consuming and expensive prevalidation processes, which is a particular benefit for centers wishing to set up new immune monitoring programs.FigureHaving successfully completed prevalidation, a flow cytometry assay must then undergo formal validation, including assessment of its accuracy, imprecision, sensitivity, and stability.9 Analytical accuracy, which expresses the closeness of a measured result to the true value, is a product of the specificity of the reagents and the appropriateness of the data analysis. Clinical accuracy is evaluated by correlating results from flow cytometry assays with other relevant clinical data to demonstrate that an assay has predictive value. ICSH/International Clinical Cytometry Society guidelines recommend that intra-assay imprecision of a flow cytometry assay is estimated by analyzing 3 or more replicate samples from 5 or more samples; the result is then expressed as mean % coefficients of variation. Likewise, the guidelines advise that interassay imprecision should be estimated using 3 or more replicate samples in 3 to 5 independent analytical runs. The “functional sensitivity” of a flow cytometry assay is defined by its lower limit of quantification (LLoQ). This is a critical property because changes in cell subset frequencies smaller than the LLoQ cannot be interpreted. A useful method for calculating LLoQ from coefficients of variation of replicate samples was described by Streitz et al11 and applied to The ONE Study reagent panel. The final steps in the validation of a flow cytometry assay are to characterize unprocessed and processed specimen stability, reagent stability, and to generate appropriate reference ranges. In principle, it is necessary for every laboratory performing a flow cytometry assay to independently validate the method. Recently, the concept of assay standardization in transplantation has been universalized after a proposal for the establishment of a Global Virtual Laboratory (GVL).12 In a recent position paper, the stated objectives of the Global Virtual Laboratory project include development of assays with a “high degree of validation and robust standard operating procedures” that can be made “widely available to the transplant community.” Furthermore, “this standardization will allow tests to be used reliably over time and between laboratories, thus providing a solid basis for comparison between trials.” Whether or not such a level of standardization can be achieved, and whether this would really permit statistically meaningful comparisons between transplantation trials, are an open questions; however, the many other virtues of standardized analytical methods are clear. In summary, flow cytometry is becomingly an increasingly important technique for monitoring immunological responses in transplant recipients; therefore, it is important for transplant clinicians and scientists to appreciate the power and limitations of the method in clinical settings. In addition to specialist literature about the quality of flow cytometry assays, there are now many excellent online sources of information, tools to aid assay design, and opportunities for collaborative research.