A fundamental challenge of clinical flow cytometry is to distinguish abnormal from normal cells on the basis of their immunophenotypic properties. Several articles in this issue of Cytometry Part B: Clinical Cytometry highlight the protean nature of normal and neoplastic cellular phenotypes, particularly in patients undergoing treatment. For instance, since bright CD38 expression typifies normal and neoplastic plasma cells, CD38 is nearly ubiquitous as a gating reagent in clinical assays (Scott et al., 2019; Soh et al., 2021; Wang & Lin, 2019). However, the expanding clinical use of daratumumab (Mateos et al., 2018), a monoclonal antibody directed against CD38, calls for the characterization of alternative gating reagents in patients with myeloma. The report by Soh and colleagues in this issue suggests that CD319 (SLAMF7) may represent one such alternative reagent, not only in daratumumab-treated patients, but also in patients treated with elotuzumab, which appears to target an epitope of CD319 distinct from those recognized by commercially-available antibody conjugates (Soh et al., 2020). In patients undergoing treatment for B-lymphoblastic leukemia, phenotypic differences between normal and neoplastic B-cell precursors underlie the ability to detect residual disease by flow cytometry (DiGiuseppe & Wood, 2019). Chatterjee and colleagues now demonstrate phenotypic differences between normal regenerating B-cell precursors in patients treated for B- or T-lymphoblastic leukemia and those in control samples obtained prior to any treatment (Chatterjee et al., 2021), highlighting the importance of a comprehensive understanding of potential variability in normal B-cell maturation for the evaluation of measurable residual disease (MRD). Normal phenotypes may also be affected by microenvironmental factors, unrelated to treatment; Gadgeel and colleagues suggest in this issue (Gadgeel et al., 2021) that phenotypic maturation patterns in ectopic intrathyroidal thymic tissue differ from those described in normal thymus and thymoma (DiGiuseppe & Wood, 2019). Along these lines, microenvironmental cues have also been suggested to explain the apparent association between extrathymic immature T-cell populations and Castleman disease (Fromm et al., 2020). One must also be alert for unexpected phenotypes in hematolymphoid neoplasms, such as the case of CD45-negative follicular lymphoma reported by Khanlari and colleagues in the current issue (Khanlari et al., 2021).

The consistent use of well-designed multicolor panels is one approach to managing the problem of phenotypic variability (and in mitigating the risk of unanticipated pitfalls (Cherian et al., 2019), such as steric hindrance (Matos, 2021); in this issue of the journal, several such multicolor combinations are evaluated. Goshaw and colleagues describe a 14-color, single-tube assay incorporating antigens recommended by the ERIC and ESCCA harmonization project (Rawstron et al., 2018), which enables characterization of CD5+ B-cell lymphoproliferative disorders and detection of MRD in patients treated for chronic lymphocytic leukemia (CLL) (Goshaw et al., 2021). The authors further demonstrate the ability of dimensionality reduction (viSNE) to facilitate MRD detection in CLL, as has been shown previously in B-lymphoblastic leukemia (DiGiuseppe et al., 2015). Gupta and colleagues, also in this issue (Gupta et al., 2021), illustrate the use of another multiparametric data visualization method, the radar plot (Jafari et al., 2018; Violidaki et al., 2020), to facilitate distinction between acute promyelocytic leukemia with PML-RARA and AML with NPM1 mutation, whose phenotypes may overlap (Zhou et al., 2019). Other multicolor antibody panels described and evaluated in this issue are a 10-color screening panel for B-cell neoplasia (Espasa et al., 2021) that includes CD200 (Sorigue et al., 2020), and a 10-color panel for MRD assessment in T-lymphoblastic leukemia (Tembhare et al., 2021). The former panel (Espasa et al., 2021), which makes use of dry reagents, has also been evaluated in a multicenter study recently reported in the journal (Hedley et al., 2021).

Several reports in this issue address specific clinical applications of flow cytometry. In a multicenter study of flow cytometric assessment of CD30 expression in non-Hodgkin lymphomas (NHLs) (Debliquis et al., 2021), Debliquis and colleagues suggest that flow cytometry may permit the detection of dim CD30 expression in a subset of NHLs classified as CD30-negative by immunohistochemistry. This finding suggests that standardized flow cytometric assessment of CD30 expression in patients with NHL might potentially increase the number of patients eligible to receive brentuximab vedotin, an antibody-drug conjugate targeting CD30 (Debliquis et al., 2021). A similar application for flow cytometry in identifying patients with acute leukemia who might be candidates for CD123-directed therapy has also been described (Bras et al., 2019). One common application of flow cytometry is the evaluation of body fluids for involvement by neoplastic cells (Berg et al., 2021; Del Principe et al., 2021; Eidhof et al., 2021; Galán et al., 2019). Umino and colleagues illustrate the usefulness of flow cytometry in identifying neoplastic cells in pleural and cerebrospinal fluid specimens from a patient with monomorphic epitheliotropic intestinal T-cell lymphoma (Umino et al., 2021). Enumeration of CD34+ hematopoietic stem and progenitor cells has long been accomplished by flow cytometry (Saraiva et al., 2019); in the current issue, Lowes and colleagues demonstrate that a commercially-available lysing reagent may obviate the requirement for daily preparation of NH₄Cl (Lowes et al., 2021). Flow cytometry may also be used to monitor intracellular signaling in disease states, as exemplified by another study in this issue. Using B-cell receptor-induced phosphorylation of protein kinase B (Akt) and ribosomal S6 kinase protein (S6) to measure phosphoinositide 3-kinase (PI3K) signaling in B cells, Del Pino-Molina and colleagues demonstrate impaired PI3K signaling in B cells from patients with common variable immunodeficiency (CVID) (Del Pino-Molina et al., 2021). The authors further demonstrate the ability of the assay to monitor therapeutic inhibition of the mechanistic target of rapamycin (mTOR) in a patient with activated phosphoinositide 3-kinase delta syndrome (APDS) during treatment with the mTOR inhibitor, sirolimus (Del Pino-Molina et al., 2021).

Also in this issue, McGinnis and colleagues describe the clinical and laboratory correlates of myeloperoxidase (MPO) positivity by flow cytometry in a series of cases of B-lymphoblastic leukemia (McGinnis et al., 2021). Although MPO is typically regarded as a specific marker of myeloid lineage, and in conjunction with B-lineage antigens would raise the consideration of mixed phenotype acute leukemia (Porwit & Béné, 2019), MPO immunoreactivity should not automatically exclude a diagnosis of B-lymphoblastic leukemia in an otherwise-typical case (DiGiuseppe & Wood, 2019). Moreover, artifactual MPO positivity, which is well described in B-lymphoblastic leukemia (Savaşan et al., 2018), should be excluded. Among the authors' findings were a higher proportion of hyperdiploidy and a lower proportion of ETV6-RUNX1 among MPO-positive cases compared with MPO-negative cases (McGinnis et al., 2021). These findings complement other recently described immunophenotypic-genotypic correlations in B-lymphoblastic leukemia (Collins et al., 2021; Gudapati et al., 2020).

Finally, this issue brings new markers and new cells. Churchill and colleagues evaluate leukocyte immunoglobulin-like receptor B1 (LILRB1) and leukocyte immunoglobulin-like receptor B4 (LILRB4) as potential markers of monocytic differentiation in acute myeloid leukemia (AML), and conclude that these antigens are more sensitive and specific than other markers commonly evaluated (Churchill et al., 2021). Innate-like or NK-like CD8+ T cells are distinguished functionally by their rapid production of interferon gamma in response to stimulation with the cytokines IL-12 and IL-18, and phenotypically by their expression of KIR, NKG2A, CD49d, and the transcription factor, Eomesodermin (Eomes) (Barbarin et al., 2017). In the course of studying KIR+NKG2A+Eomes+CD8+ T cells, Kasakovski and colleagues identified an apparently novel subset of KIR+NKG2A+CD8+ T cells, which lack expression of Eomes (Kasakovski et al., 2021). Compared with Eomes+ NK-like CD8+ T cells, these Eomes- cells appeared to be less well differentiated, expressed lower levels of senescence markers, and declined with age (Kasakovski et al., 2021). In view of the increasingly high-dimensional nature of clinical immunophenotyping, the discovery of novel cell populations with potential pathogenic significance seems likely to continue (Bandyopadhyay et al., 2019; Behbehani et al., 2020).

临床流式细胞术的一个基本挑战是根据它们的免疫表型特性区分异常细胞和正常细胞。本期Cytometry Part B：Clinical Cytometry 中的几篇文章强调了正常和肿瘤细胞表型的多变性，特别是在接受治疗的患者中。例如，由于明亮的 CD38 表达代表正常和肿瘤性浆细胞，因此 CD38 作为门控试剂在临床试验中几乎无处不在（Scott 等人，  2019 年；Soh 等人，  2021 年；Wang & Lin，  2019 年）。然而，达雷妥尤单抗的临床应用不断扩大（Mateos 等，  2018)，一种针对 CD38 的单克隆抗体，呼吁对骨髓瘤患者的替代门控试剂进行表征。Soh 及其同事在本期中的报告表明，CD319 (SLAMF7) 可能代表一种这样的替代试剂，不仅适用于接受 daratumumab 治疗的患者，而且适用于接受 elotuzumab 治疗的患者，它似乎靶向 CD319 的表位，不同于公认的那些通过市售的抗体偶联物 (Soh et al.,  2020 )。在接受 B 淋巴细胞白血病治疗的患者中，正常和肿瘤 B 细胞前体之间的表型差异是通过流式细胞术检测残留疾病的能力的基础（DiGiuseppe & Wood，  2019）。Chatterjee 及其同事现在证明了在接受 B 或 T 淋巴细胞白血病治疗的患者中正常再生 B 细胞前体与在任何治疗前获得的对照样本中的表型差异（Chatterjee 等人，  2021 年），强调了全面的重要性了解正常 B 细胞成熟的潜在变异性以评估可测量残留病 (MRD)。正常表型也可能受到与治疗无关的微环境因素的影响；Gadgeel 及其同事在本期 (Gadgeel et al., 2021 ) 中指出，异位甲状腺 内胸腺组织中的表型成熟模式与正常胸腺和胸腺瘤中描述的不同 (DiGiuseppe & Wood,  2019）。沿着这些思路，还提出了微环境线索来解释胸腺外未成熟 T 细胞群与 Castleman 病之间的明显关联（Fromm 等人，  2020 年）。人们还必须警惕血液淋巴肿瘤中的意外表型，例如 Khanlari 及其同事在当前问题中报告的 CD45 阴性滤泡性淋巴瘤病例（Khanlari 等人，  2021 年）。

始终如一地使用精心设计的多色面板是管理表型变异性问题的一种方法（以及降低意外陷阱的风险（Chrian 等人，  2019 年），例如空间位阻（Matos，  2021 年）；在本期中Goshaw 及其同事描述了一种 14 色单管检测方法，该检测方法结合了 ERIC 和 ESCCA 协调项目（Rawstron 等人，2018 年）推荐的抗原 ，能够表征 CD5+ B-慢性淋巴细胞白血病 (CLL) 治疗患者的细胞淋巴增殖性疾病和 MRD 检测（Goshaw 等人，  2021 年））。作者进一步证明了降维 (viSNE) 促进 CLL 中 MRD 检测的能力，正如之前在 B 淋巴细胞白血病中所显示的那样 (DiGiuseppe et al.,  2015 )。Gupta 及其同事也在本期（Gupta 等人，  2021 年）中说明了如何使用另一种多参数数据可视化方法，即雷达图（Jafari 等人，  2018 年； Violidaki 等人，  2020 年），以促进区分急性早幼粒细胞白血病伴PML-RARA和伴有NPM1突变的AML ，其表型可能重叠（Zhou et al.,  2019）。本期中描述和评估的其他多色抗体组包括用于 B 细胞瘤形成的 10 色筛查组（Espasa 等人，  2021 年），其中包括 CD200（Sorigue 等人，  2020 年），以及用于 MRD 的 10 色组T 淋巴细胞白血病的评估（Tembhare 等人，  2021 年）。前一个小组（Espasa 等人，2021 年）使用干试剂，该小组 最近在该杂志（Hedley 等人，2021 年）上报道的一项多中心研究也进行了评估 。

本期的几篇报告涉及流式细胞术的特定临床应用。在一项对非霍奇金淋巴瘤 (NHL) 中 CD30 表达进行流式细胞术评估的多中心研究中（Debliquis 等人，  2021 年），Debliquis 及其同事建议，流式细胞术可能允许检测分类为的 NHL 子集中的暗 CD30 表达CD30 免疫组化阴性。这一发现表明，对 NHL 患者 CD30 表达进行标准化流式细胞术评估可能会增加有资格接受 brentuximab vedotin 的患者数量，brentuximab vedotin 是一种靶向 CD30 的抗体-药物偶联物（Debliquis 等，  2021）。还描述了流式细胞术在识别可能适合 CD123 定向治疗的急性白血病患者中的类似应用（Bras 等人，  2019 年）。流式细胞术的一种常见应用是评估体液中是否有肿瘤细胞参与（Berg 等人，  2021 年；Del Principe 等人，  2021 年；Eidhof 等人，  2021 年；Galán 等人，  2019 年）。Umino 及其同事说明了流式细胞术在识别单形性上皮性肠 T 细胞淋巴瘤患者胸膜和脑脊液标本中的肿瘤细胞方面的有用性（Umino 等，  2021）。CD34+ 造血干细胞和祖细胞的计数早已通过流式细胞术完成（Saraiva 等人，  2019 年）；在本期杂志中，Lowes 及其同事证明，市售的裂解试剂可以免除每日制备 NH ₄ Cl的要求（Lowes 等人，  2021）。流式细胞术也可用于监测疾病状态下的细胞内信号传导，如本期另一项研究所示。Del Pino-Molina 及其同事使用 B 细胞受体诱导的蛋白激酶 B (Akt) 和核糖体 S6 激酶蛋白 (S6) 磷酸化来测量 B 细胞中的磷酸肌醇 3-激酶 (PI3K) 信号传导，证明 B 细胞中的 PI3K 信号传导受损来自患有常见变异型免疫缺陷 (CVID) 的患者（Del Pino-Molina 等人，  2021 年）。作者进一步证明了该测定法能够监测在使用 mTOR 抑制剂西罗莫司治疗期间，在患有活化磷酸肌醇 3-激酶 δ 综合征 (APDS) 的患者中，雷帕霉素的机械靶点 (mTOR) 的治疗抑制作用 (Del Pino-Molina 等人)等人，  2021 年）。

同样在本期杂志中，McGinnis 及其同事通过流式细胞术描述了一系列 B 淋巴细胞白血病病例中髓过氧化物酶 (MPO) 阳性的临床和实验室相关性（McGinnis 等人，  2021 年）。虽然 MPO 通常被认为是髓系的特异性标志物，并且与 B 系抗原结合会引起对混合表型急性白血病的考虑（Porwit & Béné，  2019 年），但 MPO 免疫反应性不应自动排除 B 淋巴细胞的诊断其他典型病例中的白血病（DiGiuseppe & Wood，  2019 年）。此外，人为的 MPO 阳性，这在 B 淋巴细胞白血病中得到了很好的描述（Savaşan 等人，  2018)，应排除。在作者的发现中，与 MPO 阴性病例相比，MPO 阳性病例中超二倍体的比例更高，而ETV6-RUNX1 的比例更低（McGinnis 等人，  2021 年）。这些发现补充了最近描述的 B 淋巴细胞白血病中的其他免疫表型-基因型相关性（Collins 等人，  2021 年；Gudapati 等人，  2020 年）。

最后，这个问题带来了新的标记和新的细胞。 Churchill 及其同事评估白细胞免疫球蛋白样受体 B1 (LILRB1) 和白细胞免疫球蛋白样受体 B4 (LILRB4) 作为急性髓性白血病 (AML) 中单核细胞分化的潜在标志物，并得出结论认为这些抗原比其他标志物更敏感和特异性普遍评估（Churchill 等人，  2021 年）。先天样或 NK 样 CD8+ T 细胞在功能上的区别在于它们响应细胞因子 IL-12 和 IL-18 的刺激而快速产生干扰素 γ，而表型上则在于它们的 KIR、NKG2A、CD49d 和转录因子，Eomesodermin (Eomes) (Barbarin et al.,  2017）。在研究 KIR+NKG2A+Eomes+CD8+ T 细胞的过程中，Kasakovski 及其同事发现了一个明显新的 KIR+NKG2A+CD8+ T 细胞亚群，它缺乏 Eomes 的表达（Kasakovski 等人，  2021 年）。与 Eomes+ NK 样 CD8+ T 细胞相比，这些 Eomes- 细胞似乎分化较差，表达的衰老标志物水平较低，并且随着年龄的增长而下降 (Kasakovski et al.,  2021 )。鉴于临床免疫表型越来越高维的性质，具有潜在致病意义的新细胞群的发现似乎可能会继续（Bandyopadhyay 等人，  2019 年；Behbehani 等人，  2020 年）。