I would like to start this article by hoping that all reading this are safe and healthy, and that your friends and loved ones are also doing well throughout the terrible pandemic that we all are enduring. Hopefully by the time you are reading this, the pandemic is beginning to ebb and we can start returning to our normal routines. Thankfully, people and systems have been in place to assure the publication of ongoing issues of this and other scientific journals, enabling us to keep abreast of the latest scientific developments while we social distance from one another.

J. Philip McCoy, Jr [Color figure can be viewed at wileyonlinelibrary.com]

In this issue there are 6 articles and a brief communication with the subjects of articles ranging from studies in minimal residual disease and myelodysplastic syndromes to the use of imaging flow cytometry in the assessment of erythrocytic disorders to various topics concerning T cells.

Rossi and colleagues (1) examine leukemia‐associated immunophenotypes (LAIP) in bone marrow specimens from both healthy donors as well as from the post‐treatment, recovering patients who had suffered various hematologic malignancies. It is now rather difficult to believe that 30 years ago minimal residual disease was defined microscopically by manually counting blasts under a slide and flow cytometric determinations of MRD were only just being proposed (2). Past studies have demonstrated that these LAIP, which consist of aberrant expressions of surface markers, are highly useful in the detection of minimal residual disease (MRD) (3) but not without possible pitfalls (4). In the current study, Rossi et al assign LAIP frequencies into “categories of specificity” ranging from strong to weak based on the number and combination of LAIPs studied and suggest that this approach may increase the sensitivity of monitoring MRD in specific patient populations.

The assessment of myelodysplastic syndromes (MDS) has always presented a challenge in the clinical laboratory. A recent review by Duetz and colleagues (5) nicely overviews developments and possible future directions of these studies. In this issue, Barreau et al. (6) validate a one tube, 10 antibody panel to study maturing granulocyte and monocyte populations in bone marrow samples from 251 patients. A number of panels and markers have been proposed to MDS evaluation, and as shown by Font et al (7), standardization among laboratories is accomplished by substantial effort. The one tube, 10 color panel with defined gating strategies proposed by Barreau et al could be amenable to interlab standardization. The authors propose using this panel to score abnormal expression patterns on monocytes and granulocytes, and in adding these to the Ogata score (8), establish a simple, reproducible, and sensitive method for MDS diagnosis.

Dimopoulos and coworkers (9) delve beyond MDS into the gray zone of idiopathic and clonal cytopenia of undetermined significance (ICUS/CCUS), asking the question if flow cytometric analyses would shed more light on these samples and provide enhancement of the prognostic picture of these patients. A recent review by Tanaka and Bejar discusses the complexity of MDS and MDS overlap disorders (10). In the current study Dimopoulos and colleagues use four eight antibody tubes from the Euroflow AML panel (11) as well as morphological reassessment and targeted DNA sequencing to study 79 patients with unexplained cytopenia. Among the many findings of this study was that a blinded morphological reevaluation of these samples by pathologists led to one or more of the reviewers finding more that 10% dysplasia in the marrows which would have led to a diagnosis of MDS. The flow cytometry was suggestive of dysplasia in 11 out of 12 of these samples; thus, initial immunophenotyping of these samples might have led to an initial reevaluation of these marrow. This and novel studies such as that by Raimbault et al. (12) promise to refine our knowledge of MDS and MDS‐like disorders and ultimately result in better patient care.

Switching gears a bit, More and colleagues bring us a study of erythrocyte membrane disorders using imaging flow cytometry (13). Imaging flow cytometry (IFC), a technology that became commercially available roughly a dozen years ago, has yet to gain substantial traction for use in the clinical flow cytometry laboratory. IFC is an extremely interesting and powerful methodology as it provides multicolor single cells analysis similar to more tradition flow cytometry while adding the ability to examine morphology and stain distribution of each cell acquired in a data file (14). Adding these features overcomes a longstanding limitation of flow cytometry – that of the lack of morphological information about a cell, while also overcoming constraints of microscopy by examining statistically rigorous numbers of cells in a non‐subjective manner. Further, the features acquired by IFC can be thoroughly studied using a long list of analytical tools to study various aspects of the cellular morphology and staining patterns. An example of how this technology can be applied to erythrocyte disorders was seen in a previous study which demonstrated the utility of IFC in the detection and characterization of sickled erythrocytes in clinical samples (15) as well as other erythrocyte disorders (16). More et al ask the question whether IFC can be used in the detection of hereditary spherocytosis (HS) and the answer appears to be a definitive “yes”. Using eosin‐5‐maleimide (EMA) to stain the surface of RBCs, the mean fluorescence intensity and other features were measured on samples from 50 control subjects and 165 patients with putative RBC disorders. HS samples showed a statistically significant decrease in the MFI of EMA compared to the control populations while other erythrocyte disorders showed no significant decrease. It would be of interest to determine of the recently published “new staining protocol” for fixation and permeabilization of erythrocytes would be adaptable to IFC for further studies of RBC disorders involving intracellular targets.

The final three articles in this issue address T lymphocytes, but in widely varying contexts. Petsiou and coworkers examine the various ways in which T regulatory (Tregs) can be identified and propose one method to best identify Tregs in both effector and naïve populations (17). The markers most commonly used to identify Tregs can be found in various combinations on a number of different T cell populations as demonstrated by Biancotto et al. (18). Using various combinations of Treg‐associated markers including CD25, FoxP3, andCD127 as well as CD45, CD14, and CD45RO the authors find that the use of a panel consisting of CD4/CD25/CD45RO/FoxP3 identifies 6 CD4+ T cell fractions including effector and naïve Tregs. A previous study has proposed a standardized protocol for monitoring Tregs in clinical trials using a panel consisting of CD3/CD4/CD8/CD25/CD127/FoxP3 (19). A modification of that panel with the addition of CD45RO as suggested currently might be of interest, although the issue of gating on CD127 low cells would still need to be resolved. Studies such as these could have direct clinical impact in a number of areas, such as blood stem cell harvests (20).

An examination of T cell subsets in the peripheral blood of patients suffering from HIV neuro‐tuberculosis coinfections is presented by Rao and colleagues (21). In this study the investigators looked at six populations of patients: those with HIV and neurotuberculosis (nTB); HIV and systemic TB (sTB); asymptomatic HIV TB negative; non HIV nTB; non HIV sTB; and healthy controls. A myriad of differences were observed in numerous T cell populations among these patient groups. Although the ultimate impact of these differences on the courses of disease in these patients remains to be delineated, there is little doubt that these findings will augment our understanding of the pathogenesis when these two deadly infectious diseases occur together.

Finally, we have a brief communication from Fromm and coworkers (22) describing 12 samples of incidental T lymphoblast populations from 10 patients. Despite the encyclopedic knowledge available about normal T cell maturation, there remains a need to more accurately distinguish between the occurrence of indolent T cell precursors and T lymphoblastic lymphomas/leukemias, as illustrated by a recent case report from Yuan and colleagues (23). In the current study lymph node suspensions were stained with either a nine color or ten color panel. Eight of the cases showed a T cell phenotype of CD4‐/CD8‐ with most of these also expressing CD34, three cases showed CD4+/CD8+ T cells, and one case showed low CD8 without CD4 coexpression. Further study of the deeper immunophenotypes revealed no noted abnormalities. These data suggest that indolent T cell proliferations can display immunophenotypes representing various stages of T cell maturation while no aberrant phenotypes are observed.

我想从本文开始，希望所有阅读的内容都是安全健康的，并且在我们大家都忍受的可怕大流行中，您的朋友和亲人也都做得很好。希望当您阅读本文时，这种流行病开始消退，我们可以开始恢复正常生活。值得庆幸的是，已经有了人员和系统来确保本科学期刊和其他科学期刊的不断出版，使我们能够在与彼此保持社交距离的同时紧跟最新的科学发展。

J. Philip McCoy，Jr [可在wileyonlinelibrary.com上查看彩色图形]

在本期中，有6篇文章以及与这些文章的主题的简短交流，涉及范围从最小残留疾病和骨髓增生异常综合征的研究到在评估红细胞疾病中使用成像流式细胞术到涉及T细胞的各种主题。

Rossi及其同事（1）检查了健康供体以及治疗后的骨髓标本中与白血病相关的免疫表型（LAIP），康复了患有各种血液系统恶性肿瘤的患者。现在很难相信，30年前通过在幻灯片下手动计数胚细胞的显微镜下定义了最小残留病，而仅提出了流式细胞仪测定MRD（2）。过去的研究表明，这些由表面标志物异常表达组成的LAIP在检测最小残留疾病（MRD）方面非常有用（3）但并非毫无缺陷（4））。在当前的研究中，Rossi等人根据所研究的LAIP的数量和组合，将LAIP频率分为从强到弱的“特异性类别”，并建议这种方法可能会提高在特定患者人群中监测MRD的敏感性。

骨髓增生异常综合症（MDS）的评估一直是临床实验室面临的挑战。Duetz及其同事（5）最近的评论很好地概述了这些研究的发展和可能的未来方向。在本期中，Barreau等人。（6）验证了一个单管10抗体小组，以研究251位患者的骨髓样本中成熟的粒细胞和单核细胞群体。已经提出了许多面板和标记物用于MDS评估，如Font等人（7），实验室之间的标准化是通过大量的努力来实现的。Barreau等人提出的具有定义门控策略的单管10色面板可能适合实验室间标准化。作者建议使用此面板对单核细胞和粒细胞的异常表达模式进行评分，并将其添加到绪方评分（8）中，以建立一种简单，可重复且敏感的MDS诊断方法。

Dimopoulos及其同事（9）深入研究MDS以外的灰色区域，发现其原发性和克隆性血细胞减少症的重要性尚未确定（ICUS / CCUS），并提出了一个问题，即流式细胞术分析是否可以为这些样本提供更多的信息，并增强这些样本的预后情况耐心。Tanaka和Bejar最近的评论讨论了MDS和MDS重叠障碍的复杂性（10）。在当前的研究中，Dimopoulos及其同事使用了Euroflow AML小组的四个八支抗体管（11）以及形态学重新评估和靶向DNA测序，以研究79例原因不明的血细胞减少症患者。在这项研究的许多发现中，病理学家对这些样品进行了盲目形态重新评估，导致一名或多名审阅者发现骨髓中有10％以上的异型增生，这将导致MDS的诊断。流式细胞仪提示其中有12个样本中有11个发育异常。因此，这些样品的最初免疫表型可能导致了这些骨髓的最初重新评估。这种和新颖的研究，例如Raimbault等人的研究。（12）承诺完善我们对MDS和MDS样疾病的了解，并最终带来更好的患者护理。

稍微切换一下，More和同事通过成像流式细胞术为我们带来了红细胞膜疾病的研究（13）。成像流式细胞术（IFC）是大约十二年前市售的一项技术，尚未在临床流式细胞术实验室中获得广泛的应用。IFC是一种非常有趣且功能强大的方法，它提供了多色单细胞分析，类似于更传统的流式细胞术，同时还增加了检查数据文件中每个细胞的形态和染色分布的功能（14）。添加这些功能克服了流式细胞术长期以来的局限性–缺乏有关细胞形态信息的局限性，同时还通过以非主观的方式检查统计上严格的细胞数量来克服显微镜的限制。此外，可以使用一长串分析工具来全面研究IFC所获得的特征，以研究细胞形态和染色模式的各个方面。在先前的研究中可以看到该技术如何应用​​于红细胞疾病的一个例子，该研究证明了IFC在检测和表征临床样本中镰刀状红细胞（15）以及其他红细胞疾病（16）中的实用性。）。More等人提出了一个问题，即是否可以将IFC用于检测遗传性球菌增多症（HS），答案似乎是肯定的“是”。使用曙红5-马来酰亚胺（EMA）对RBC的表面进行染色，从50名对照受试者和165名假定的RBC疾病患者的样品中测量了平均荧光强度和其他特征。与对照人群相比，HS样品显示EMA的MFI有统计学意义的下降，而其他红细胞疾病则没有显着下降。确定最近发表的用于固定和透化红细胞的“新染色方案”将适合于IFC，以进一步研究涉及细胞内靶标的RBC疾病。

本期的最后三篇文章讨论了T淋巴细胞，但背景变化很大。Petsiou及其同事研究了可识别T调节剂（Tregs）的各种方法，并提出了一种在效应和未加工人群中最佳识别Treg的方法（17）。如Biancotto等人所证实的，可以在许多不同的T细胞群体上以各种组合找到最常用于鉴定Treg的标记。（18）。作者使用多种Treg相关标记的组合，包括CD25，FoxP3和CD127以及CD45，CD14和CD45RO，发现使用由CD4 / CD25 / CD45RO / FoxP3组成的面板可识别6个CD4 + T细胞组分，包括效应子和天真的Tregs。先前的研究提出了使用由CD3 / CD4 / CD8 / CD25 / CD127 / FoxP3组成的小组在临床试验中监测Treg的标准化方案（19）。尽管仍需要解决门控CD127低细胞的问题，但目前建议的对CD面板进行修改的建议是添加CD45RO。这些研究可能会在许多领域产生直接的临床影响，例如血液干细胞收获（20）。

Rao和同事提出了对患有HIV神经结核合并感染的患者外周血T细胞亚群的检查（21）。在这项研究中，研究人员研究了六类患者：艾滋病毒和神经结核患者；艾滋病毒和系统性结核病；无症状的艾滋病毒结核阴性；非艾滋病毒nTB；非艾滋病毒结核病；和健康的控制。在这些患者组的众多T细胞群体中观察到无数差异。尽管这些差异对这些患者病程的最终影响尚待确定，但毫无疑问的是，当这两种致命的传染病同时发生时，这些发现将增强我们对发病机理的理解。

最后，我们与Fromm及其同事（22）进行了简短的交流，描述了来自10位患者的12个偶然T淋巴母细胞群体的样本。尽管有关于正常T细胞成熟的百科全书知识，但仍然需要更准确地区分惰性T细胞前体的发生与T淋巴细胞母细胞性淋巴瘤/白血病的发生，如Yuan和他的同事最近的病例报告所说明的那样（23）。在本研究中，淋巴结悬液用九色或十色板染色。其中8例显示CD4- / CD8-的T细胞表型，其中大多数还表达CD34，3例显示CD4 + / CD8 + T细胞，而1例显示低CD8而无CD4共表达。对更深层次的免疫表型的进一步研究表明没有发现异常。这些数据表明，惰性T细胞增殖可以显示代表T细胞成熟各个阶段的免疫表型，而没有观察到异常表型。