Starting in the early 1980s, flow cytometry has had a rich history in the evaluation of blood for cutaneous T‐cell lymphoma (CTCL). Prior to that, Sézary cells had been identified on review of peripheral blood smears (Main, Goodall, & Swanson, 1959; Sezary & Bouvrain, 1938), and the origin of T‐helper cells had been determined by e‐rosette and functional studies (Lamberg & Bunn Jr., 1979). One‐color flow cytometry applied to blood specimens with abundant disease confirmed these observations through the demonstration of antigens subsequently classified as CD3 (Figure 1) and CD4 (Boumsell et al., 1981; Haynes et al., 1981; Waldmann et al., 1984) and documented the association between blood involvement and a high CD4:CD8 ratio (Willemze, van Vloten, Hermans, Damsteeg, & Meijer, 1983). In the 1990s, adoption of two‐ to three‐color flow cytometry facilitated recognition that the T‐cells of Sézary syndrome lacked expression of CD7 (Wood et al., 1990) and often demonstrated weak intensity staining for CD3 (Bogen et al., 1996; Kuchnio et al., 1994; Figure 1) In addition, CD7‐negative T‐cells were identified in patients with inflammatory skin lesions (Moll et al., 1994). During the early 2000s, two‐ to four‐color flow cytometry was used to demonstrate that Sézary cells also often lacked expression of CD26 (Bernengo et al., 2001) and that this finding was probably more reliable than lack of CD7 in the distinction between Sézary syndrome and inflammatory dermatoses (Kelemen, Guitart, Kuzel, Goolsby, & Peterson, 2008).

The dermatology community was quick to recognize the utility of flow cytometry in the assessment of blood for CTCL. In 2002, the International Society for Cutaneous Lymphomas (ISCL) made a recommendation to add a B2 rating for high‐burden blood disease to the classification scheme for mycosis fungoides and Sézary syndrome, identified by an absolute Sézary cell count >1,000 cells/mm³; or flow cytometry with a CD4:CD8 ratio > =10 due to increased CD3+ or CD4+ cells; or aberrant expression of pan‐T‐cell markers CD2, CD3, CD4, and CD5, and note that deficient CD7 expression on T‐cells represented a tentative criterion of Sézary syndrome (Vonderheid et al., 2002). The ISCL held a series of workshops from 2002 to 2006 to discuss the adoption of new data and testing modalities, which resulted in the 2007 ISCL/EORTC revisions to the staging and classification of mycosis fungoides and Sézary syndrome (Olsen et al., 2007). The limitations of manual differential Sézary counts were acknowledged and the value of flow cytometry in the identification and quantitation of neoplastic cells recognized. Although the resulting 2007 blood classification was still primarily based on morphology, it was noted that, if Sézary cells (by morphology) cannot be used to determine burden for B2, one of the following flow cytometry findings can be used: expanded CD4+ or CD3+ cells with CD4:CD8 ratio of 10 or more or expanded CD4+ cells with abnormal immunophenotype including loss of CD7 or CD26, with the suggested criteria of CD4+, CD7− ≥40%, or CD4+ CD26− ≥30% (Olsen et al., 2007). These recommendations were subsequently included in the 2008 WHO classification of disorders of the lymphoid and hematopoietic neoplasms and, with the declining availability of morphologic Sézary counts, were widely adopted. However, B2 determined by manual Sézary count has never been compared in a prospective study to flow cytometry results. In addition, it is important to note that, at this point, the focus was still very much on the identification of high‐burden disease, which had recently been identified as having prognostic significance regardless of the Tumor, Node, Metastasis (TNM) stage (Kim, Liu, Mraz‐Gernhard, Varghese, & Hoppe, 2003).

While these classification schemes were found to be useful for patient management, it was recognized that clinical trials required better standardized end points and response criteria. Workshops held in 2004–2009 by ISCL, United States and the Cutaneous Lymphoma Consortium (USCLC) and Cutaneous Lymphoma Task Force of the European Organization for Research and Treatment of Cancer (EORTC) led to 2011 consensus recommendations, including use of the absolute number of abnormal lymphocytes determined by flow cytometric analysis to determine B2 status for clinical trials (Olsen et al., 2011). These recommendations added that the absolute number of CD4+ CD26− cells determined by flow cytometry was the most reasonable, quantifiable measure of potential blood involvement by mycosis fungoides and Sézary syndrome (Olsen et al., 2011) and suggested using a similar approach to define lower levels of blood involvement. They proposed that since “a normal value for CD4+ CD26‐ or CD4+ CD7‐ cells by flow cytometry is lower than 15%” and “Based on an upper limit of normal value of 1,600/μL for CD4 cells in the blood, an absolute count of lower than 250/μL CD4+CD26‐ or CD4+CD7‐ cells would appear to be a normal value” and “could be used to define the absence of or normalization of blood involvement (B0)” (Olsen et al., 2011). However, a subsequent publication applying these criteria concluded that “our current knowledge of the implication of ‘minor’ blood involvement (B0‐1) is rudimentary and should not be used as a primary indicator to change patients' treatments” (Scarisbrick et al., 2018). From a cytometrist's perspective, it is not surprising that these assumptions and calculations relating to lower levels of disease have limited utility. While the traditional flow cytometric evaluation of the overall CD4:CD8 ratio and separate assessment of the proportion of CD4+ CD26− and CD4+ CD7− T‐cells were sufficient to determine the presence of high‐burden disease, it is not effective for lower levels of disease where the contribution of admixed normal CD4 + CD26‐ and CD4 + CD7‐ T‐cells becomes more significant. Therefore, it is fortunate that clinical flow cytometric immunophenotyping has evolved and is ready to step up and meet dermatologists' need for a reliable, reproducible method for determining disease burden and monitoring disease over time and with treatment.

Multicolor flow cytometric immunophenotyping provides a more appropriate approach to the evaluation of Sézary syndrome and mycosis fungoides. Using this approach, multiple antigens are evaluated simultaneously to identify T‐cells that have a combination of findings that differ from the norm, such as lack of CD7 and CD26 and weak intensity staining for CD3 (Figure 1). This immunophenotyping approach can be applied to more confidently identify abnormal T‐cells, even when they represent a low proportion of all lymphoid cells (Horna et al., 2014; Scarisbrick et al., 2018; Washington, Huh, Powers, Duvic, & Jones, 2002), or in the unusual situation of CD8‐positive disease. In addition, evaluation of multiple antigens for abnormal expression helps to identify disease that does not express the typical CD4+ CD7− and CD4+ CD26− phenotype. The proportion of cells with an abnormal immunophenotype can be used to determine the absolute count of abnormal cells for use as a monitor of disease burden over time, even if the immunophenotype of the neoplastic cells varies (Horna et al., 2014; Novelli et al., 2015; Washington et al., 2002).

The time is right for the widespread adoption of this multicolor flow cytometry immunophenotyping approach for the evaluation of peripheral blood involvement mycosis fungoides and Sézary syndrome. Clinical flow cytometry laboratories have adopted flow cytometric systems that can evaluate more than four colors and can therefore perform simultaneous assessment for multiple antigens. The “difference from normal” approach is widely adopted for the identification of cells from a variety of hematolymphoid neoplasms, including the assessment for residual disease following therapy when neoplastic cells cannot be identified by morphologic evaluation, as outlined in the series of articles from the International Clinical Cytometry Society and the Society for Hematopathology (DiGiuseppe & Wood, 2019; Jevremovic & Olteanu, 2019). Finally, recently developed guidelines for the validation of flow cytometric assays (Clinical Laboratory Standards Institute H62) (Clinical Laboratory Standards Institute, n.d.) will help laboratories design and implement reproducible assays that are able to reliably meet the assay requirements. This issue of the Journal includes four articles developed by an international group of experts, which address the required performance characteristics for a multicolor flow cytometry immunophenotyping assay for mycosis fungoides and Sézary syndrome and include practical guidelines for adoption (Pulitzer, Horna, & Almeida, n.d.; Guitart, n.d.; Illingworth et al., n.d.; Horna et al., n.d.). The first article describes the clinicians' perspective (Guitart, n.d.). The second article provides an overview of Sézary syndrome and mycosis fungoides, including the role of immunophenotyping (Pulitzer et al., n.d.). The third and fourth articles describe flow cytometric assay design and validation (Horna et al., n.d.; Illingworth et al., n.d.). In 2020, flow cytometry remains essential for the evaluation of peripheral blood for Sézary syndrome and mycosis fungoides, but in contrast to the traditional approach, multicolor immunophenotyping can more reliably identify and quantitate neoplastic cells, which is essential for the identification of clinically relevant thresholds for disease burden and response to therapy.

从 1980 年代初开始，流式细胞术在评估皮肤 T 细胞淋巴瘤 (CTCL) 的血液方面有着丰富的历史。在此之前，Sézary 细胞已在外周血涂片检查中被鉴定出来（Main, Goodall, & Swanson, 1959 ; Sezary & Bouvrain, 1938），并且 T 辅助细胞的起源已通过 e-rosette 和功能研究确定(Lamberg & Bunn Jr.，1979 年)。应用于具有丰富疾病的血液样本的单色流式细胞术通过证明随后被分类为 CD3（图 1）和 CD4 的抗原（Boumsell 等人，1981 年；Haynes 等人，1981 年；Waldmann 等人，1984年) 并记录了血液受累与高 CD4:CD8 比率之间的关联（Willemze、van Vloten、Hermans、Damsteeg 和 Meijer，1983 年）。在 1990 年代，采用两到三色流式细胞术促进了人们认识到 Sézary 综合征的 T 细胞缺乏 CD7 表达（Wood 等人，1990 年）并且经常表现出对 CD3 的弱染色（Bogen 等人，1996 年；Kuchnio 等人，1994 年；图 1) 此外，在炎症性皮肤病变患者中发现了 CD7 阴性 T 细胞（Moll 等人，1994 年）。在 2000 年代初期，二到四色流式细胞术被用来证明 Sézary 细胞也经常缺乏 CD26 的表达（Berengo 等人，2001) 并且在区分 Sézary 综合征和炎症性皮肤病方面，这一发现可能比缺乏 CD7 更可靠（Kelemen、Guitart、Kuzel、Goolsby 和 Peterson，2008 年）。

皮肤病学界很快认识到流式细胞术在 CTCL 血液评估中的实用性。2002 年，国际皮肤淋巴瘤协会 (ISCL) 建议在蕈样真菌病和 Sézary 综合征的分类方案中增加 B2 等级的高负担血液病，通过绝对 Sézary 细胞计数 >1,000 个细胞/mm ³ ; 或由于 CD3+ 或 CD4+ 细胞增加，CD4:CD8 比率 > =10 的流式细胞术；或泛 T 细胞标志物 CD2、CD3、CD4 和 CD5 的异常表达，并注意 T 细胞上的 CD7 表达不足代表了 Sézary 综合征的暂定标准（Vonderheid 等，2002）。ISCL 于 2002 年至 2006 年举办了一系列研讨会，讨论采用新数据和测试模式，从而导致 2007 年 ISCL/EORTC 对蕈样真菌病和 Sézary 综合征的分期和分类进行了修订（Olsen 等，2007）。手动差异 Sézary 计数的局限性得到认可，流式细胞术在肿瘤细胞的识别和定量中的价值得到认可。尽管由此产生的 2007 年血液分类仍主要基于形态学，但有人指出，如果 Sézary 细胞（通过形态学）不能用于确定 B2 的负担，则可以使用以下流式细胞术发现之一：扩增的 CD4+ 或 CD3+ 细胞CD4:CD8 比率为 10 或更多或扩增的 CD4+ 细胞具有异常免疫表型，包括 CD7 或 CD26 缺失，建议标准为 CD4+、CD7- ≥40% 或 CD4+ CD26- ≥30%（Olsen 等，2007）。这些建议随后被纳入 2008 年 WHO 淋巴和造血系统肿瘤疾病分类中，随着形态学 Sézary 计数的可用性下降，这些建议被广泛采用。然而，在前瞻性研究中从未将手动 Sézary 计数确定的 B2 与流式细胞术结果进行比较。此外，需要注意的是，在这一点上，重点仍然是高负担疾病的识别，无论肿瘤、淋巴结、转移 (TNM) 分期如何，这些疾病最近都被确定为具有预后意义（Kim、Liu、Mraz-Gernhard、Varghese 和 Hoppe，2003 年）。

虽然发现这些分类方案对患者管理有用，但人们认识到临床试验需要更好的标准化终点和反应标准。2004-2009 年由美国 ISCL 和皮肤淋巴瘤联盟 (USCLC) 和欧洲癌症研究和治疗组织 (EORTC) 皮肤淋巴瘤工作组举办的研讨会导致了 2011 年的共识建议，包括使用绝对数量的通过流式细胞术分析确定异常淋巴细胞以确定临床试验的 B2 状态（Olsen 等，2011）。这些建议补充说，通过流式细胞术确定的 CD4+ CD26− 细胞的绝对数量是蕈样真菌病和 Sézary 综合征（Olsen 等人，2011 年）潜在血液受累的最合理、可量化的衡量标准，并建议使用类似的方法来定义较低的血液受累程度。他们提出，由于“流式细胞术检测的 CD4+ CD26- 或 CD4+ CD7- 细胞的正常值低于 15%”和“基于血液中 CD4 细胞正常值 1,600/μL 的上限，绝对计数低于 250/μL CD4+CD26- 或 CD4+CD7- 细胞似乎是正常值”并且“可用于定义血液受累的缺失或正常化 (B0)”（Olsen 等人，2011 年））。然而，随后发表的应用这些标准的出版物得出结论：“我们目前对‘轻微’血液受累 (B0-1) 的影响的了解是初步的，不应用作改变患者治疗的主要指标”（Scarisbrick 等人，2017 年）。 , 2018）。从细胞计数器的角度来看，这些与较低疾病水平相关的假设和计算的效用有限，这并不奇怪。虽然传统的流式细胞术评估整体 CD4:CD8 比率和单独评估 CD4+ CD26- 和 CD4+ CD7- T 细胞的比例足以确定高负担疾病的存在，但它对较低水平的 T 细胞无效在这种疾病中，混合的正常 CD4 + CD26- 和 CD4 + CD7- T 细胞的贡献变得更加显着。因此，幸运的是临床流式细胞术免疫表型已经发展并准备加强并满足皮肤科医生对可靠、可重复的方法的需求，用于确定疾病负担和随时间和治疗监测疾病。

多色流式细胞术免疫表型分析为评估 Sézary 综合征和蕈样真菌病提供了更合适的方法。使用这种方法，可以同时评估多种抗原，以识别具有与正常情况不同的结果组合的 T 细胞，例如缺乏 CD7 和 CD26 以及 CD3 的弱强度染色（图 1）。这种免疫表型方法可用于更自信地识别异常 T 细胞，即使它们在所有淋巴细胞中所占比例很低（Horna 等人，2014 年；Scarisbrick 等人，2018 年；Washington, Huh, Powers, Duvic, &琼斯，2002)，或在 CD8 阳性疾病的异常情况下。此外，评估多种抗原的异常表达有助于识别不表达典型 CD4+ CD7- 和 CD4+ CD26- 表型的疾病。具有异常免疫表型的细胞比例可用于确定异常细胞的绝对计数，以用作随时间推移的疾病负担监测器，即使肿瘤细胞的免疫表型发生变化（Horna 等人，2014 年；Novelli 等人.，2015 年；华盛顿等人，2002 年）。

现在是广泛采用这种多色流式细胞术免疫表型方法来评估外周血受累蕈样真菌病和 Sézary 综合征的时候了。临床流式细胞仪实验室已采用流式细胞仪系统，可以评估四种以上的颜色，因此可以同时评估多种抗原。“与正常不同”的方法被广泛用于识别来自各种血淋巴肿瘤的细胞，包括当肿瘤细胞无法通过形态学评估识别时，对治疗后残留疾病的评估，如国际的系列文章所述。临床细胞学学会和血液病理学会 (DiGiuseppe & Wood, 2019; Jevremovic 和 Olteanu，2019 年）。最后，最近制定的流式细胞术检测验证指南（临床实验室标准协会 H62）（临床实验室标准协会，nd）将帮助实验室设计和实施能够可靠地满足检测要求的可重复检测。本期期刊包括由国际专家组撰写的四篇文章，这些文章讨论了真菌病和 Sézary 综合征的多色流式细胞术免疫表型分析所需的性能特征，并包括采用的实用指南（Pulitzer、Horna 和 Almeida，nd ; Guitart, nd ; Illingworth 等人, nd ; Horna 等人,nd )。第一篇文章描述了临床医生的观点（Guitart，nd）。第二篇文章概述了 Sézary 综合征和蕈样真菌病，包括免疫表型分析的作用（Pulitzer 等，nd）。第三和第四篇文章描述了流式细胞分析设计和验证（Horna 等人，nd；Illingworth 等人，nd）。2020 年，流式细胞术对于 Sézary 综合征和蕈样真菌病的外周血评估仍然必不可少，但与传统方法相比，多色免疫表型可以更可靠地识别和定量肿瘤细胞，这对于识别临床相关阈值至关重要疾病负担和对治疗的反应。