Torres, Richard

AbstractIntroducing a sensitive and specific peripheral blood flow cytometric assay for Sézary syndrome and mycosis fungoides (SS/MF) requires careful selection of assay design characteristics, and translation into a laboratory developed assay through development/optimization, validation, and continual quality monitoring. As outlined in a previous article in this series, the recommended design characteristics of this assay include at a minimum, evaluation of CD7, CD3, CD4, CD8, CD26, and CD45, analyzed simultaneously, requiring at least a 6 color flow cytometry system, with both quantitative and qualitative components. This article provides guidance from an international group of cytometry specialists in implementing an assay to those design specifications, outlining specific considerations, and best practices. Key points presented in detail are: (a) Pre‐analytic components (reagents, specimen processing, and acquisition) must be optimized to: (i) identify and characterize an abnormal population of T‐cells (qualitative component) and (ii) quantitate the abnormal population (semi/quasi‐quantitative component). (b)Analytic components (instrument set‐up/acquisition/analysis strategy and interpretation) must be optimized for the identification of SS/MF populations, which can vary widely in phenotype. Comparison with expert laboratories is strongly encouraged in order to establish competency. (c) Assay performance must be validated and documented through a validation plan and report, which covers both qualitative and semi/quasi‐quantitative assay components (example template provided). (d) Ongoing assay‐specific quality monitoring should be performed to ensure consistency.

AbstractA peripheral blood flow cytometric assay for Sézary syndrome (SS) or circulating mycosis fungoides (MF) cells must be able to reliably identify, characterize, and enumerate T‐cells with an immunophenotype that differs from non‐neoplastic T‐cells. Although it is also important to distinguish SS and MF from other subtypes of T‐cell neoplasm, this usually requires information in addition to the immunophenotype, such as clinical and morphologic features. This article outlines the approach recommended by an international group with experience and expertise in this area. The following key points are discussed: (a) At a minimum, a flow cytometric assay for SS and MF should include the following six antibodies: CD3, CD4, CD7, CD8, CD26, and CD45. (b) An analysis template must reliably detect abnormal T‐cells, even when they lack staining for CD3 or CD45, or demonstrate a phenotype that is not characteristic of normal T‐cells. (c) Gating strategies to identify abnormal T‐cells should be based on the identification of subsets with distinctly homogenous immunophenotypic properties that are different from those expected for normal T‐cells. (d) The blood concentration of abnormal cells, based on any immunophenotypic abnormalities indicative of MF or SS, should be calculated by either direct enumeration or a dual‐platform method, and reported.