Clinical Cytometry is pleased to begin the New Year with a Special Issue focusing on Cytometry Advancing Next Generation Drug Development. This issue represents a second collaboration between the American Association of Pharmaceutical Scientists (AAPS) Flow Cytometry Action Program Committee (APC) and the International Clinical Cytometry Society. The first collaboration between these two groups yielded the Special Issue of Cytometry B focusing on Receptor Occupancy in 2016 (Green et al., 2016; Litwin et al., 2016; Stewart et al., 2016).

For over 20 years flow cytometry has been a mainstay in the drug development toolbox (Litwin & O'Gorman, 2011; O'Hara et al., 2011). The importance of cytometry is found in every stage of drug development process. This includes initial target identification, compound screening, and lead compound characterization. Cytometry also assists with defining mechanisms of action, proof of concept studies, toxicology assessment, dose selection and finally identifying biomarkers for clinical development.

During clinical development, cellular biomarkers are primarily implemented in clinical studies supporting immune oncology, immune disorders, cell therapy, gene therapy, and vaccine development. Cytometry might be used without a predefined hypothesis in order to monitor the overall cellular immune status of the patient by evaluating the balance of naïve and memory T and B cell subsets or the level of suppressor cell populations. In early clinical trials, single cell analysis can be used to identify differences in patient versus healthy populations as well to evaluate pharmacodynamic effects. In immune oncology studies, cytometry might be used to evaluate the expression levels of check‐point inhibitors on healthy or malignant immune cells, or to determine the level of measurable residual disease. Insights gained from over a decade of investigation have given rise to novel and efficacious drug targets and shed light on mechanisms of resistance and immune dysregulation of various cancer indications. Cytometry is the most sensitive tool for monitoring the efficacy of cell depleting therapies (Dass et al., 2008). When the drug is a cellular therapy, cytometry is used during the manufacturing and product release phases, as well as post‐infusion monitoring of the circulating cell product levels and phenotypic changes (Sarikonda et al., 2021a,b). For vaccine development, intracellular cytokine assays are used to characterize the scope of antigen specific immune responses, while extensive phenotypic characterization of responder cells helps to predict response duration (i.e., long‐lasting immunity). For protein‐based therapies which bind to cell surface antigens, receptor occupancy (RO) assays have become required measurement components both preclinically and during clinical studies (Green et al, 2016; Litwin et al., 2016; Hilt et al., 2021).

A critical concern when implementing cytometric methods used for drug development or in clinical testing is the level of analytical method validation required for the particular context of use (COU). Additional concerns surround how to actually conduct the validation experiments. The upcoming Clinical Laboratory and Standards Institute (CLSI) guideline, H62 the Validation of Methods Performed by Flow Cytometry addresses these topics (CLSI, 2021). In addition, this Special Issue of Clinical Cytometry includes four new recommendation papers, generated by the AAPS Flow Cytometry APC, which also address different challenges associated with the validation of cell‐based assays (Cabanski et al., 2021; Hilt et al., 2021; Sarikonda et al., 2021a; Sommers et al., 2021).

The first step in any method validation should be to determine the COU of the assay and the associated regulatory requirements. While this may seem obvious enough, this step is often overlooked during the development and validation of flow cytometric methods and as a result the final assays fail to meet performance specifications. To improve the likelihood of successful outcomes, a best practice is to implement the Design Control process for risk evaluation and implementation planning. Design Control is a familiar process in regulated settings; however, this concept is likely to be new to many cytometrists. The four recommendation papers presented herein are framed in the context of Design Control (Cabanski et al., 2021; Hilt et al., 2021; Sarikonda et al., 2021a; Sommers et al., 2021). The paper by Sommer, et al. focusing on flow cytometric assays for the detection of rare events, is unique as it is the first recommendation paper to describe in depth how to validate the lower limit of quantification of a flow cytometric assay (Sommers et al., 2021). The paper by Cabanski, et al. is the first publication to present strategies for successful method transfer validation for flow cytometric methods (Cabanski et al., 2021). In addition to best practice recommendations, this paper includes four case studies which illustrate the wide variety of situations which require assay transfer and how the COU influences the method transfer strategy. The paper by Hilt, et al. supplements existing recommendation papers from the AAPS Flow Cytometry APC addressing the optimization and validation of receptor occupancy assays (Hilt et al., 2021; Green et al., 2016; Stewart et al., 2016). The importance of this challenging type assay continues to increase as more and more biologics are being developed. Strategies for addressing the most challenging aspects of RO assays such as receptor modulation and low target‐antigen density are presented. In addition, the paper addresses the challenges of evaluating RO for bi‐specific therapeutic compounds and compounds which bind multiple targets.

Because of the exceptional clinical efficacy of CAR‐T cells there are an increasing number of both industry and investigator driven clinical trials, but the procedures to develop and validate CAR‐T products by flow cytometry have not been adequately addressed (Maryamchik et al., 2020; Demaret et al., 2020). This Special Issue of Cytometry B features two articles that discuss the development and validation of flow cytometric CAR‐T cell protocols. Sarikonda, et al. discuss the analytical considerations critical for development of flow cytometric methods to enumerate CAR‐T levels, the operational challenges associated with clinical trial sampling and transportation, and the different cellular kinetics observed between flow cytometry and qPCR (Sarikonda et al., 2021b). In addition to covering proper assay design and validation topics, the authors consider the development and construction of CAR‐T detection reagents and approaches to monitoring these cells in patients, post‐infusion. A second manuscript addressing CAR‐T cells which was written by members of the AAPS Flow Cytometry APC, covers considerations regarding the appropriate approach regarding specimen type, regulatory requirements, quality assurance, and quality controls for a variety of COU (Sarikonda et al., 2021a). Both of these manuscripts will be of interest to anyone developing assays to either evaluate CAR‐T cell products or monitor the cellular products in vivo. Moreover, they will also likely guide the development of even more efficacious next generation therapies based on specific harmonized phenotypic and functional attributes of CAR‐T cells.

The first three papers in this Special Issue of Cytometry B are reviews which set the stage for the subsequent papers. The paper by Sanjabi and Lear focuses on the success of cancer immunotherapy (CIT) and the role of cellular biomarkers to advance the understanding of the immune landscape (Sanjabi and Lear, 2021). The manuscript considers how advances in high complexity cytometry, including spectral cytometry and algorithm‐driven analysis will contribute to the future understanding of immunotherapy and cancer immunity models. This paper also explores the emerging field of bioinformatic and data analysis tools for high‐dimension cytometry data. The review by Ng, et al. provides an overview of the diagnostic, disease, efficacy, and safety biomarkers used in the clinical development of antiviral therapies (Ng et al., 2021). This paper focuses on hepatitis B virus (HBV), human immunodeficiency virus (HIV), and hepatitis C virus (HCV), and novel coronaviruses such as severe acute respiratory syndrome coronavirus (SARS‐CoV). The critical role that biomarker strategy plays in the development of antiviral and vaccine therapies capable of generating durable remission or cure is discussed in depth. The third review by Saksena and Chattopadhyay highlights the contribution of immune profiling by flow cytometry in increasing the understanding of how the host immune response is coordinated to combat SARS‐CoV2 infection, and how that response becomes dysregulated in severe disease (Saksena and Chattopadhyay, 2021). For example, in severe cases the massive release of cytokines in response to the infection can result in a cytokine storm and sepsis‐like symptoms that trigger multi‐organ failure accounting for many COVID‐19 deaths (Zhang et al., 2020). The manuscript takes an in‐depth look at the identification of COVID‐19 immune signatures and their implications for treatment strategies. The critical need for risk stratification biomarkers is emphasized (Grifoni et al., 2020).

The issue concludes with two manuscripts describing validation and implementation case studies. The paper by Estevam, et al. describes the development and validation process for a mass cytometry assay, based on accepted industry practices for the validation of flow cytometric assays (Estevam et al., 2021). The biomarkers outlined in this paper used to support clinical trials were developed to measure gastrointestinal trafficking peripheral blood mononuclear cells in patients with Celiac Disease. This paper details some of the key challenges unique to mass cytometry including optimizing antibody/isotope panels, as well as challenges common to all cell‐based assays such as sample instability (pre‐ and post‐labeled). Quadrini, et al. (2021) describe the validation of a flow cytometric laboratory developed test (LDT) for monitoring sepsis using downregulation of HLA‐DR expression on monocytes (Sanjabi and Lear, 2021). This LDT was first used as an exploratory endpoint biomarker for Phase 1b and Phase 2a clinical studies and then revalidated for use as an enrollment biomarker. Some of the patients included in these clinical trials were from New York State (NY State) and thus the investigators were required to submit their analytical method validation to the NY State Department of Health (NYSDOH) for review and approval. This manuscript is one of the first reports to detail the validation approach used to satisfy the NYSDOH requirements for an enrollment biomarker. While validation standards have not yet been defined by the FDA, they are in development. Since the FDA does accept NY State approval for LDT assays the FDA requirements are likely to have many commonalities with those of the NYSDOH (Oldaker et al., 2018). Here the authors describe the approaches they took to evaluate and report sample adequacy, accuracy, analytical specificity and sensitivity, instrument and assay precision, reportable range, reference interval, and the development of multi‐level quality control material. Ultimately, the approaches taken by these authors were accepted by NYSDOH and provide excellent guidelines for the development and validation of flow cytometric LDT assays.

The Guest Editors wish to thank all of the authors for their contributions to this issue, the production team at Wiley and most of all Doris Regal, Managing Editor, who never fails to work her magic in making the issue concept a reality, and the Editor‐in‐Chief, Frederic Preffer, for his full support and unending enthusiasm for this project.

临床细胞计量学很高兴以一期特刊开始新的一年，重点关注细胞计量学促进下一代药物开发。本期是美国药学科学家协会 (AAPS) 流式细胞术行动计划委员会 (APC) 与国际临床细胞术学会之间的第二次合作。这两个小组之间的第一次合作产生了 2016 年专注于受体占用率的细胞学B 特刊（Green 等人，2016 年；Litwin 等人，2016 年；Stewart 等人，2016 年）。

20 多年来，流式细胞术一直是药物开发工具箱中的中流砥柱（Litwin & O'Gorman，2011 年；O'Hara 等人，2011 年）。在药物开发过程的每个阶段都发现了细胞计数的重要性。这包括初始目标识别、化合物筛选和先导化合物表征。细胞学还有助于定义作用机制、概念研究证明、毒理学评估、剂量选择，并最终确定临床开发的生物标志物。

在临床开发过程中，细胞生物标志物主要用于支持免疫肿瘤学、免疫疾病、细胞治疗、基因治疗和疫苗开发的临床研究。为了通过评估初始和记忆 T 和 B 细胞亚群的平衡或抑制细胞群的水平，可以在没有预定义假设的情况下使用细胞计数法来监测患者的整体细胞免疫状态。在早期临床试验中，单细胞分析可用于识别患者与健康人群的差异以及评估药效学效果。在免疫肿瘤学研究中，细胞计数可用于评估检查点抑制剂在健康或恶性免疫细胞上的表达水平，或确定可测量的残留疾病水平。从十多年的研究中获得的见解已经产生了新的和有效的药物靶点，并阐明了各种癌症适应症的耐药性和免疫失调机制。细胞计数法是监测细胞耗竭疗法疗效的最敏感工具（Dass 等人，2008 年）。当药物是细胞疗法时，在制造和产品发布阶段使用细胞计数，以及循环细胞产品水平和表型变化的输注后监测（Sarikonda 等，2021a，b）。对于疫苗开发，细胞内细胞因子测定用于表征抗原特异性免疫应答的范围，而应答细胞的广泛表型表征有助于预测应答持续时间（即持久免疫）。对于与细胞表面抗原结合的基于蛋白质的疗法，受体占用 (RO) 检测已成为临床前和临床研究期间所需的测量组件（Green 等，2016；Litwin 等，2016；Hilt 等，2021） .

在实施用于药物开发或临床测试的细胞计数方法时，一个关键问题是特定使用环境 (COU) 所需的分析方法验证水平。其他问题围绕如何实际进行验证实验。即将发布的临床实验室和标准协会 (CLSI) 指南 H62 流式细胞术方法验证解决了这些主题（CLSI，2021 年）。此外，本期临床细胞学特刊包括四篇由 AAPS 流式细胞术 APC 生成的新推荐论文，这些论文还解决了与基于细胞的检测验证相关的不同挑战（Cabanski 等人，2021 年；Hilt 等人， 2021；Sarikonda 等人，2021a；Sommers 等人，2021 年）。

任何方法验证的第一步都应该是确定检测的 COU 和相关的监管要求。虽然这看起来很明显，但在流式细胞术方法的开发和验证过程中，这一步经常被忽略，因此最终的检测无法满足性能规范。为了提高成功结果的可能性，最佳实践是实施设计控制流程以进行风险评估和实施规划。设计控制是受监管环境中熟悉的过程；然而，这个概念对于许多细胞分析员来说可能是新的。本文提出的四篇推荐论文是在设计控制的背景下构建的（Cabanski 等人，2021 年；Hilt 等人，2021 年；Sarikonda 等人，2021a；Sommers 等人，2021 年）。Sommer 等人的论文。专注于检测罕见事件的流式细胞术检测是独一无二的，因为它是第一篇深入描述如何验证流式细胞术检测定量下限的推荐论文（Sommers 等，2021）。Cabanski 等人的论文。是第一本介绍成功的流式细胞术方法转移验证策略的出版物（Cabanski 等人，2021 年）。除了最佳实践建议外，本文还包括四个案例研究，它们说明了需要化验转移的各种情况以及 COU 如何影响方法转移策略。希尔特等人的论文。补充了 AAPS 流式细胞术 APC 中关于优化和验证受体占用测定的现有推荐论文（Hilt 等人，2021 年；Green 等人，是独一无二的，因为它是第一篇深入描述如何验证流式细胞术定量下限的推荐论文（Sommers 等，2021）。Cabanski 等人的论文。是第一本介绍成功的流式细胞术方法转移验证策略的出版物（Cabanski 等人，2021 年）。除了最佳实践建议外，本文还包括四个案例研究，它们说明了需要化验转移的各种情况以及 COU 如何影响方法转移策略。希尔特等人的论文。补充了 AAPS 流式细胞术 APC 中关于优化和验证受体占用测定的现有推荐论文（Hilt 等人，2021 年；Green 等人，是独一无二的，因为它是第一篇深入描述如何验证流式细胞术定量下限的推荐论文（Sommers 等，2021）。Cabanski 等人的论文。是第一本介绍成功的流式细胞术方法转移验证策略的出版物（Cabanski 等人，2021 年）。除了最佳实践建议外，本文还包括四个案例研究，它们说明了需要化验转移的各种情况以及 COU 如何影响方法转移策略。希尔特等人的论文。补充了 AAPS 流式细胞术 APC 中关于优化和验证受体占用测定的现有推荐论文（Hilt 等人，2021 年；Green 等人，是第一本介绍成功的流式细胞术方法转移验证策略的出版物（Cabanski 等人，2021 年）。除了最佳实践建议外，本文还包括四个案例研究，它们说明了需要化验转移的各种情况以及 COU 如何影响方法转移策略。希尔特等人的论文。补充了 AAPS 流式细胞术 APC 中关于优化和验证受体占用测定的现有推荐论文（Hilt 等人，2021 年；Green 等人，是第一本介绍成功的流式细胞术方法转移验证策略的出版物（Cabanski 等人，2021 年）。除了最佳实践建议外，本文还包括四个案例研究，它们说明了需要化验转移的各种情况以及 COU 如何影响方法转移策略。希尔特等人的论文。补充了 AAPS 流式细胞术 APC 中关于优化和验证受体占用测定的现有推荐论文（Hilt 等人，2021 年；Green 等人，本文包括四个案例研究，它们说明了需要化验转移的各种情况以及 COU 如何影响方法转移策略。希尔特等人的论文。补充了 AAPS 流式细胞术 APC 中关于优化和验证受体占用测定的现有推荐论文（Hilt 等人，2021 年；Green 等人，本文包括四个案例研究，它们说明了需要化验转移的各种情况以及 COU 如何影响方法转移策略。希尔特等人的论文。补充了 AAPS 流式细胞术 APC 中关于优化和验证受体占用测定的现有推荐论文（Hilt 等人，2021 年；Green 等人，2016 年；斯图尔特等人，2016 年）。随着越来越多的生物制剂被开发出来，这种具有挑战性的类型检测的重要性不断增加。提出了解决 RO 检测最具挑战性方面的策略，例如受体调节和低靶抗原密度。此外，该论文解决了评估双特异性治疗化合物和结合多个靶标的化合物的 RO 的挑战。

由于 CAR-T 细胞的卓越临床功效，越来越多的行业和研究人员驱动的临床试验，但尚未充分解决通过流式细胞术开发和验证 CAR-T 产品的程序（Maryamchik 等，2020 年；Demaret 等人，2020 年）。本期 Cytometry B 特刊刊登了两篇讨论流式细胞术 CAR-T 细胞方案的开发和验证的文章。萨里孔达等。讨论对于开发流式细胞术方法来枚举 CAR-T 水平至关重要的分析考虑、与临床试验采样和运输相关的操作挑战，以及流式细胞术和 qPCR 之间观察到的不同细胞动力学（Sarikonda 等，2021b）。除了涵盖正确的检测设计和验证主题外，作者还考虑了 CAR-T 检测试剂的开发和构建以及在输注后监测患者体内这些细胞的方法。AAPS 流式细胞术 APC 成员撰写的关于 CAR-T 细胞的第二份手稿，涵盖有关标本类型、监管要求、质量保证和各种 COU 质量控制的适当方法的考虑（Sarikonda 等人，2021a）。任何开发评估 CAR-T 细胞产品或监测细胞产品的检测方法的人都会对这两份手稿感兴趣体内。此外，它们还可能指导基于 CAR-T 细胞特定协调表型和功能属性的更有效的下一代疗法的开发。

本期 Cytometry B 特刊中的前三篇论文是评论，为后续论文奠定了基础。Sanjabi 和 Lear 的论文侧重于癌症免疫疗法 (CIT) 的成功以及细胞生物标志物在促进对免疫景观的理解方面的作用（Sanjabi 和 Lear，2021 年）。手稿考虑了高复杂性细胞术的进步，包括光谱细胞术和算法驱动的分析将如何有助于未来对免疫疗法和癌症免疫模型的理解。本文还探讨了用于高维细胞计数数据的生物信息学和数据分析工具的新兴领域。Ng 等人的评论。概述了抗病毒疗法临床开发中使用的诊断、疾病、功效和安全性生物标志物（Ng 等，2021）。本文重点介绍乙型肝炎病毒（HBV）、人类免疫缺陷病毒（HIV）和丙型肝炎病毒（HCV），以及新型冠状病毒，如严重急性呼吸系统综合症冠状病毒（SARS-CoV）。深入讨论了生物标志物策略在开发能够产生持久缓解或治愈的抗病毒和疫苗疗法中发挥的关键作用。Saksena 和 Chattopadhyay 的第三篇评论强调了通过流式细胞术进行免疫分析在增加对宿主免疫反应如何协调以对抗 SARS-CoV2 感染以及该反应如何在严重疾病中失调的理解方面的贡献（Saksena 和 Chattopadhyay，2021 年） ）。例如，2020 年）。该手稿深入研究了 COVID-19 免疫特征的识别及其对治疗策略的影响。强调了对风险分层生物标志物的迫切需求（Grifoni 等人，2020 年）。

本期以两份描述验证和实施案例研究的手稿结束。Estevam 等人的论文。描述了基于流式细胞分析验证的公认行业实践的质谱流式细胞分析的开发和验证过程（Estevam 等，2021）。本文概述的用于支持临床试验的生物标志物被开发用于测量腹腔疾病患者的胃肠道运输外周血单核细胞。本文详细介绍了质谱流式细胞术特有的一些关键挑战，包括优化抗体/同位素组合，以及所有基于细胞的检测所面临的常见挑战，例如样品不稳定性（标记前和标记后）。Quadrini 等。(2021) 描述了流式细胞仪实验室开发的测试 (LDT) 的验证，用于使用单核细胞上 HLA-DR 表达的下调来监测败血症（Sanjabi 和 Lear，2021）。该 LDT 首先用作 1b 期和 2a 期临床研究的探索性终点生物标志物，然后重新验证用作注册生物标志物。这些临床试验中包括的一些患者来自纽约州 (NY State)，因此研究人员需要将他们的分析方法验证提交给纽约州卫生部 (NYSDOH) 进行审查和批准。这份手稿是首批详细介绍用于满足 NYSDOH 对注册生物标志物要求的验证方法的报告之一。虽然 FDA 尚未定义验证标准，但它们正在开发中。2018 年）。在这里，作者描述了他们用来评估和报告样品充分性、准确性、分析特异性和灵敏度、仪器和测定精度、可报告范围、参考区间以及多级质量控制材料的开发的方法。最终，这些作者采用的方法被 NYSDOH 接受，并为流式细胞术 LDT 检测的开发和验证提供了极好的指导。

客座编辑要感谢对本期做出的贡献的所有作者、Wiley 的制作团队以及最重要的执行编辑 Doris Regal，他一直在努力使本期概念成为现实，以及编辑- 首席弗雷德里克·普雷弗 (Frederic Preffer)，感谢他对这个项目的全力支持和无尽的热情。