Agustín D Rizzo, Marianela Sanz, Georgina Roffe, Elisa O Sajaroff, Damian A Prado, Emma Prieto, Verónica Goris, Jorge G Rossi, Andrea R Bernasconi
X-linked inhibitor of apoptosis (XIAP) deficiency is an infrequent inborn error of immunity caused by mutations in XIAP gene. Most cases present with absence of XIAP protein which can be detected by flow cytometry (FC), representing a rapid diagnostic method. However, since some genetic defects may not preclude protein expression, it is important to include a complementary functional test in the laboratory workup of these patients. L-selectin (CD62-L) is a molecule that is cleaved from the surface membrane of leukocytes upon stimulation of different receptors such as toll like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), including NOD2. Considering that XIAP deficiency impairs NOD2 signaling, we decided to assess CD62-L down-regulation by FC post-stimulation of neutrophils and monocytes with L18-muramyl Di-Peptide (L18-MDP), a NOD2 specific agonist, in order to develop a novel assay for the functional evaluation of patients with suspicion of XIAP defects. Whole blood samples from 20 healthy controls (HC) and four patients with confirmed molecular diagnosis of XIAP deficiency were stimulated with 200 ng/mL of L18-MDP for 2 h. Stimulation with 100 ng/mL of lipopolysaccharide (LPS) was carried out in parallel as a positive control of CD62-L shedding. CD62-L expression was evaluated by FC using an anti CD62-L- antibody and down-regulation was assessed by calculating the difference in CD62-L expression before and after stimulation, both in terms of percentage of CD62-L expressing cells (Δ%CD62-L) and median fluorescence intensity (ΔMFI%). Neutrophils and monocytes from XIAP deficient patients displayed a significantly diminished response to L18-MDP stimulation compared with HC (p < 0.0001), indicating a severely altered mechanism of CD62-L down-regulation following activation of NOD2-XIAP axis. On the other hand, the response to LPS stimulation was comparable between patients and heathy controls, suggesting preserved CD62-L shedding with a different stimulus. FC detection of CD62-L down-regulation in monocytes and neutrophils after whole blood stimulation with L18-MDP results in an effective and rapid functional test for the identification of XIAP deficient patients.
{"title":"CD62-L down-regulation after L18-MDP stimulation as a complementary flow cytometry functional assay for the diagnosis of XIAP deficiency.","authors":"Agustín D Rizzo, Marianela Sanz, Georgina Roffe, Elisa O Sajaroff, Damian A Prado, Emma Prieto, Verónica Goris, Jorge G Rossi, Andrea R Bernasconi","doi":"10.1002/cyto.b.22181","DOIUrl":"https://doi.org/10.1002/cyto.b.22181","url":null,"abstract":"<p><p>X-linked inhibitor of apoptosis (XIAP) deficiency is an infrequent inborn error of immunity caused by mutations in XIAP gene. Most cases present with absence of XIAP protein which can be detected by flow cytometry (FC), representing a rapid diagnostic method. However, since some genetic defects may not preclude protein expression, it is important to include a complementary functional test in the laboratory workup of these patients. L-selectin (CD62-L) is a molecule that is cleaved from the surface membrane of leukocytes upon stimulation of different receptors such as toll like receptors (TLRs) and nucleotide-binding oligomerization domain-like receptors (NLRs), including NOD2. Considering that XIAP deficiency impairs NOD2 signaling, we decided to assess CD62-L down-regulation by FC post-stimulation of neutrophils and monocytes with L18-muramyl Di-Peptide (L18-MDP), a NOD2 specific agonist, in order to develop a novel assay for the functional evaluation of patients with suspicion of XIAP defects. Whole blood samples from 20 healthy controls (HC) and four patients with confirmed molecular diagnosis of XIAP deficiency were stimulated with 200 ng/mL of L18-MDP for 2 h. Stimulation with 100 ng/mL of lipopolysaccharide (LPS) was carried out in parallel as a positive control of CD62-L shedding. CD62-L expression was evaluated by FC using an anti CD62-L- antibody and down-regulation was assessed by calculating the difference in CD62-L expression before and after stimulation, both in terms of percentage of CD62-L expressing cells (Δ%CD62-L) and median fluorescence intensity (ΔMFI%). Neutrophils and monocytes from XIAP deficient patients displayed a significantly diminished response to L18-MDP stimulation compared with HC (p < 0.0001), indicating a severely altered mechanism of CD62-L down-regulation following activation of NOD2-XIAP axis. On the other hand, the response to LPS stimulation was comparable between patients and heathy controls, suggesting preserved CD62-L shedding with a different stimulus. FC detection of CD62-L down-regulation in monocytes and neutrophils after whole blood stimulation with L18-MDP results in an effective and rapid functional test for the identification of XIAP deficient patients.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-05-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"141070162","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Kimberly A Shumate, Samantha N Williams, Aashish B Khatri, Vijaya Knight
Peripheral blood lymphocyte phenotyping panels typically include CD45 for discrimination of the lymphocyte population, and fluorophore-conjugated monoclonal antibodies to identify T, B, and Natural Killer (NK) cells. While CD45 combined with side scatter is generally sufficient to clearly distinguish lymphocytes from monocytes in the majority of peripheral blood samples, it is challenging to accurately gate lymphocytes in samples from patients with monocytosis or significant lymphopenia, or from very young infants. Addition of a monocyte marker to lymphocyte phenotyping panels for monocyte exclusion has previously been evaluated for improved discrimination of lymphocytes, albeit largely in healthy donor adult samples. Here we evaluate the effect of the addition of CD14 to a standard lymphocyte phenotyping panel on total lymphocyte, T, B, and NK cell percentages in a predominantly pediatric population of patients under evaluation chiefly for immunodeficiency, immune-depletion, or immune reconstitution. Addition of CD14 to the standard lymphocyte phenotyping improved discrimination of lymphocytes from monocytes, resulted in decreased NK cell percentages, likely because CD16+ and/or CD56+ monocytes were included in the CD56+CD16+ NK cell gate with conventional gating, and although less significant, resulted in an increased percentage of B cells, since relatively larger B cells were likely gated out by more restrictive light scatter gating used with the conventional gating approach. The change in NK and B cell percentages were more pronounced in samples from patients below a year of age, and in patients who were relatively lymphopenic. These data suggest that addition of CD14 to conventional lymphocyte phenotyping panels that utilize CD45 versus side scatter gating results in significant improvement in the accuracy of lymphocyte gating, and accurate quantification of NK and B cells particularly in samples from infants and lymphopenic individuals.
外周血淋巴细胞表型检测板通常包括用于区分淋巴细胞群的 CD45 和用于识别 T、B 和自然杀伤(NK)细胞的荧光团结合单克隆抗体。虽然 CD45 与侧散射相结合通常足以清楚地区分大多数外周血样本中的淋巴细胞和单核细胞,但在单核细胞增多症或严重淋巴细胞减少症患者或年幼婴儿的样本中,要准确区分淋巴细胞却很困难。在淋巴细胞表型检测板中加入单核细胞标记物以排除单核细胞的方法曾被评估用于提高淋巴细胞的分辨能力,尽管主要是在健康的成人供体样本中。在这里,我们评估了在标准淋巴细胞表型分析中加入 CD14 对淋巴细胞总数、T、B 和 NK 细胞百分比的影响,这些患者主要是儿科患者,主要接受免疫缺陷、免疫耗竭或免疫重建评估。在标准淋巴细胞表型中加入 CD14 提高了淋巴细胞与单核细胞的区分度,降低了 NK 细胞的百分比,这可能是因为 CD16+ 和/或 CD56+ 单核细胞被纳入了传统分型方法的 CD56+CD16+ NK 细胞门中,虽然意义不大,但却提高了 B 细胞的百分比,因为在传统分型方法中,相对较大的 B 细胞可能被限制性更强的光散射分型方法分出。NK 和 B 细胞百分比的变化在一岁以下的患者样本和淋巴细胞相对较多的患者样本中更为明显。这些数据表明,在使用 CD45 与侧散射分型的传统淋巴细胞表型板中加入 CD14,可显著提高淋巴细胞分型的准确性,并能准确量化 NK 和 B 细胞,特别是在婴儿和淋巴变性者样本中。
{"title":"Addition of CD14 improves discrimination of lymphocytes in the TBNK phenotyping panel.","authors":"Kimberly A Shumate, Samantha N Williams, Aashish B Khatri, Vijaya Knight","doi":"10.1002/cyto.b.22180","DOIUrl":"https://doi.org/10.1002/cyto.b.22180","url":null,"abstract":"<p><p>Peripheral blood lymphocyte phenotyping panels typically include CD45 for discrimination of the lymphocyte population, and fluorophore-conjugated monoclonal antibodies to identify T, B, and Natural Killer (NK) cells. While CD45 combined with side scatter is generally sufficient to clearly distinguish lymphocytes from monocytes in the majority of peripheral blood samples, it is challenging to accurately gate lymphocytes in samples from patients with monocytosis or significant lymphopenia, or from very young infants. Addition of a monocyte marker to lymphocyte phenotyping panels for monocyte exclusion has previously been evaluated for improved discrimination of lymphocytes, albeit largely in healthy donor adult samples. Here we evaluate the effect of the addition of CD14 to a standard lymphocyte phenotyping panel on total lymphocyte, T, B, and NK cell percentages in a predominantly pediatric population of patients under evaluation chiefly for immunodeficiency, immune-depletion, or immune reconstitution. Addition of CD14 to the standard lymphocyte phenotyping improved discrimination of lymphocytes from monocytes, resulted in decreased NK cell percentages, likely because CD16+ and/or CD56+ monocytes were included in the CD56+CD16+ NK cell gate with conventional gating, and although less significant, resulted in an increased percentage of B cells, since relatively larger B cells were likely gated out by more restrictive light scatter gating used with the conventional gating approach. The change in NK and B cell percentages were more pronounced in samples from patients below a year of age, and in patients who were relatively lymphopenic. These data suggest that addition of CD14 to conventional lymphocyte phenotyping panels that utilize CD45 versus side scatter gating results in significant improvement in the accuracy of lymphocyte gating, and accurate quantification of NK and B cells particularly in samples from infants and lymphopenic individuals.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-05-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140956581","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Wikum Dinalankara, David P. Ng, Luigi Marchionni, Paul D. Simonson
Multiparameter flow cytometry data is visually inspected by expert personnel as part of standard clinical disease diagnosis practice. This is a demanding and costly process, and recent research has demonstrated that it is possible to utilize artificial intelligence (AI) algorithms to assist in the interpretive process. Here we report our examination of three previously published machine learning methods for classification of flow cytometry data and apply these to a B-cell neoplasm dataset to obtain predicted disease subtypes. Each of the examined methods classifies samples according to specific disease categories using ungated flow cytometry data. We compare and contrast the three algorithms with respect to their architectures, and we report the multiclass classification accuracies and relative required computation times. Despite different architectures, two of the methods, flowCat and EnsembleCNN, had similarly good accuracies with relatively fast computational times. We note a speed advantage for EnsembleCNN, particularly in the case of addition of training data and retraining of the classifier.
{"title":"Comparison of three machine learning algorithms for classification of B-cell neoplasms using clinical flow cytometry data","authors":"Wikum Dinalankara, David P. Ng, Luigi Marchionni, Paul D. Simonson","doi":"10.1002/cyto.b.22177","DOIUrl":"10.1002/cyto.b.22177","url":null,"abstract":"<p>Multiparameter flow cytometry data is visually inspected by expert personnel as part of standard clinical disease diagnosis practice. This is a demanding and costly process, and recent research has demonstrated that it is possible to utilize artificial intelligence (AI) algorithms to assist in the interpretive process. Here we report our examination of three previously published machine learning methods for classification of flow cytometry data and apply these to a B-cell neoplasm dataset to obtain predicted disease subtypes. Each of the examined methods classifies samples according to specific disease categories using ungated flow cytometry data. We compare and contrast the three algorithms with respect to their architectures, and we report the multiclass classification accuracies and relative required computation times. Despite different architectures, two of the methods, flowCat and EnsembleCNN, had similarly good accuracies with relatively fast computational times. We note a speed advantage for EnsembleCNN, particularly in the case of addition of training data and retraining of the classifier.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-05-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140897264","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Eya Anane, Fatma Ben Lakhal, Sarra Fekih Salem, Ons Ghali, Emna Feki, Yosr Ben Abdennebi, Marwa Bahri, Emna Azza, Lamia Aissaoui, Wijden El Borgi, Emna Gouider
Meningeal infiltration in children with B acute lymphoblastic leukemia is one of the most serious complications. Timely diagnosis not only significantly enhances treatment efficacy but also leads to improve patient outcome and reduce risk of relapse. This is particularly crucial in low to middle income countries facing health constraints, where optimizing resources is essential. Conventional cytology (CC) study of cerebrospinal fluid (CSF) is considered in different countries to be the Gold-standard despite its low sensitivity (< 50%). The study of CSF by multiparametric flow cytometry (MFC) appears to be an alternative. The aim of our study was to assess MFC analytical performance compared with CC. Our cross sectional study was conducted over a six-month period in the biological hematology department. CSF samples underwent analysis for the presence of blasts using both CC and MFC. Cytological slides of the CSF were prepared by cytocentrifugation in a Shandon Cytospin 4™. Flow cytometric analysis was performed on the BD FACSLyric™ flow cytometer. All statistical analyses were performed using SPSS version 21.0 (SPSS Inc.). Agreement between the two methods was made using the Kappa index and χ2 test. This study was approved by the local ethics committee. Sixty CSF samples from 39 children with B acute lymphoblastic leukemia were analyzed. Meningeal infiltration was detected respectively in 20% of cases by MFC and 5% of cases by CC, with a significant difference p = 0.006. Comparing the two methods, the Kappa coefficient was 0.35, indicating weak agreement between the two methods. Moreover, MFC positivity was higher even for hypocellular samples. Of the 51 hypocellular samples, eight were positive by MFC while they were negative by CC. MFC shows better sensitivity while retaining good specificity for the detection of meningeal involvement. MFC could therefore be a complementary method to CC for detecting blast cells in the central nervous system.
B 型急性淋巴细胞白血病患儿的脑膜浸润是最严重的并发症之一。及时诊断不仅能显著提高治疗效果,还能改善患者预后,降低复发风险。这对于面临医疗限制的中低收入国家尤为重要,因为这些国家必须优化资源。脑脊液(CSF)的常规细胞学(CC)研究在不同国家被视为黄金标准,尽管其敏感性较低(2 检验)。本研究获得了当地伦理委员会的批准。本研究分析了 39 名 B 型急性淋巴细胞白血病患儿的 60 份脑脊液样本。MFC和CC分别有20%和5%的病例检测到脑膜浸润,差异显著(P = 0.006)。比较两种方法,卡帕系数为 0.35,表明两种方法的一致性较弱。此外,即使是低细胞样本,MFC 阳性率也较高。在 51 份低细胞样本中,有 8 份样本的 MFC 检测结果呈阳性,而 CC 检测结果呈阴性。MFC 在检测脑膜受累方面显示出更高的灵敏度,同时保留了良好的特异性。因此,MFC 可作为 CC 的补充方法,用于检测中枢神经系统中的爆炸细胞。
{"title":"Flow cytometry for meningeal infiltration in B acute lymphoblastic leukemia in a low middle income country.","authors":"Eya Anane, Fatma Ben Lakhal, Sarra Fekih Salem, Ons Ghali, Emna Feki, Yosr Ben Abdennebi, Marwa Bahri, Emna Azza, Lamia Aissaoui, Wijden El Borgi, Emna Gouider","doi":"10.1002/cyto.b.22179","DOIUrl":"https://doi.org/10.1002/cyto.b.22179","url":null,"abstract":"<p><p>Meningeal infiltration in children with B acute lymphoblastic leukemia is one of the most serious complications. Timely diagnosis not only significantly enhances treatment efficacy but also leads to improve patient outcome and reduce risk of relapse. This is particularly crucial in low to middle income countries facing health constraints, where optimizing resources is essential. Conventional cytology (CC) study of cerebrospinal fluid (CSF) is considered in different countries to be the Gold-standard despite its low sensitivity (< 50%). The study of CSF by multiparametric flow cytometry (MFC) appears to be an alternative. The aim of our study was to assess MFC analytical performance compared with CC. Our cross sectional study was conducted over a six-month period in the biological hematology department. CSF samples underwent analysis for the presence of blasts using both CC and MFC. Cytological slides of the CSF were prepared by cytocentrifugation in a Shandon Cytospin 4™. Flow cytometric analysis was performed on the BD FACSLyric™ flow cytometer. All statistical analyses were performed using SPSS version 21.0 (SPSS Inc.). Agreement between the two methods was made using the Kappa index and χ<sup>2</sup> test. This study was approved by the local ethics committee. Sixty CSF samples from 39 children with B acute lymphoblastic leukemia were analyzed. Meningeal infiltration was detected respectively in 20% of cases by MFC and 5% of cases by CC, with a significant difference p = 0.006. Comparing the two methods, the Kappa coefficient was 0.35, indicating weak agreement between the two methods. Moreover, MFC positivity was higher even for hypocellular samples. Of the 51 hypocellular samples, eight were positive by MFC while they were negative by CC. MFC shows better sensitivity while retaining good specificity for the detection of meningeal involvement. MFC could therefore be a complementary method to CC for detecting blast cells in the central nervous system.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-05-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140862490","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Katherine A. Devitt, Wolfgang Kern, Weijie Li, Xuehai Wang, Allyson J. Wong, Felipe M. Furtado, Jean S. Oak, Andrea Illingworth
The assessment of T-cell clonality by flow cytometry has long been suboptimal, relying on aberrant marker expression and/or intensity. The introduction of TRBC1 shows much promise for improving the diagnosis of T-cell neoplasms in the clinical flow laboratory. Most laboratories considering this marker already have existing panels designed for T-cell workups and will be determining how best to incorporate TRBC1. We present this comprehensive summary of TRBC1 and supplemental case examples to familiarize the flow cytometry community with its potential for routine application, provide examples of how to incorporate it into T-cell panels, and signal caution in interpreting the results in certain diagnostic scenarios where appropriate.
长期以来,流式细胞术对 T 细胞克隆性的评估一直不够理想,主要依赖于异常标记物的表达和/或强度。TRBC1 的引入为改善临床流式细胞实验室对 T 细胞肿瘤的诊断带来了希望。大多数考虑采用该标记物的实验室已经有了为 T 细胞检查设计的现有检测板,并将确定如何以最佳方式纳入 TRBC1。我们提交这份关于 TRBC1 的全面总结和补充案例,旨在让流式细胞仪界熟悉其常规应用的潜力,提供如何将其纳入 T 细胞检测板的示例,并提示在某些诊断情况下酌情谨慎解释结果。
{"title":"TRBC1 in flow cytometry: Assay development, validation, and reporting considerations","authors":"Katherine A. Devitt, Wolfgang Kern, Weijie Li, Xuehai Wang, Allyson J. Wong, Felipe M. Furtado, Jean S. Oak, Andrea Illingworth","doi":"10.1002/cyto.b.22175","DOIUrl":"10.1002/cyto.b.22175","url":null,"abstract":"<p>The assessment of T-cell clonality by flow cytometry has long been suboptimal, relying on aberrant marker expression and/or intensity. The introduction of TRBC1 shows much promise for improving the diagnosis of T-cell neoplasms in the clinical flow laboratory. Most laboratories considering this marker already have existing panels designed for T-cell workups and will be determining how best to incorporate TRBC1. We present this comprehensive summary of TRBC1 and supplemental case examples to familiarize the flow cytometry community with its potential for routine application, provide examples of how to incorporate it into T-cell panels, and signal caution in interpreting the results in certain diagnostic scenarios where appropriate.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22175","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140841564","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Aryane Cruz Oliveira Pinho, Pedro Barbosa, Maria João Pereira, Artur Paiva, Eugenia Carvalho, Paula Laranjeira
CD20+ T cells constitute a small subset of T cells. These are found among CD4+, CD8+, CD4+CD8+, CD4−CD8− T, and TCRγδ+ T cells, and have been poorly characterized. The aim of this study was to characterize peripheral blood (PB) CD20+ T cells and compare them to their PB CD20− T cell counterparts. PB from 17 healthy individuals was collected. The distribution of CD20+ T cells among maturation-associated T cells compartments (naïve, central memory, transitional memory, effector memory, and effector T cells), their polarization, activation status, and expression of immune-regulatory proteins were evaluated by flow cytometry. Their function was also assessed, by measuring IFN-γ, TNF-α, and IL-17 production. Compared with CD20− T cells, CD20+ T cells represent a higher proportion of transitional memory cells. Furthermore, CD20+ T cells display a proinflammatory phenotype, characterized by the expansion of Th1, Th1/17, and Tc1 cell subsets , associated to a high expression of activation (CD25) and exhaustion (PD-1) markers. In addition, the simultaneous production of the proinflammatory cytokines IFN-γ, TNF-α, and IL-17 was also detected in CD4+CD20+ T cells. Our results show that CD20+ T cells are phenotypically and functionally different from CD20− T cells, suggesting that these cells are a distinct subset of T cells.
CD20+ T 细胞是 T 细胞的一个小亚群。这些细胞存在于 CD4+、CD8+、CD4+CD8+、CD4-CD8- T 和 TCRγδ+ T 细胞中,其特征还不十分明确。本研究旨在确定外周血(PB)CD20+ T 细胞的特征,并将其与 PB CD20- T 细胞进行比较。研究人员收集了 17 名健康人的外周血。通过流式细胞术评估了 CD20+ T 细胞在成熟相关的 T 细胞分区(幼稚、中枢记忆、过渡记忆、效应记忆和效应 T 细胞)中的分布、极化、活化状态以及免疫调节蛋白的表达。此外,还通过测量 IFN-γ、TNF-α 和 IL-17 的产生来评估它们的功能。与 CD20- T 细胞相比,CD20+ T 细胞代表了更高比例的过渡记忆细胞。此外,CD20+ T 细胞还表现出一种促炎表型,其特征是 Th1、Th1/17 和 Tc1 细胞亚群的扩增,与活化(CD25)和衰竭(PD-1)标志物的高表达有关。此外,在 CD4+CD20+ T 细胞中还检测到同时产生了促炎细胞因子 IFN-γ、TNF-α 和 IL-17。我们的研究结果表明,CD20+ T 细胞在表型和功能上都不同于 CD20- T 细胞,这表明这些细胞是 T 细胞的一个独特亚群。
{"title":"The role of CD20+ T cells: Insights in human peripheral blood","authors":"Aryane Cruz Oliveira Pinho, Pedro Barbosa, Maria João Pereira, Artur Paiva, Eugenia Carvalho, Paula Laranjeira","doi":"10.1002/cyto.b.22178","DOIUrl":"10.1002/cyto.b.22178","url":null,"abstract":"<p>CD20<sup>+</sup> T cells constitute a small subset of T cells. These are found among CD4<sup>+</sup>, CD8<sup>+</sup>, CD4<sup>+</sup>CD8<sup>+</sup>, CD4<sup>−</sup>CD8<sup>−</sup> T, and TCRγδ<sup>+</sup> T cells, and have been poorly characterized. The aim of this study was to characterize peripheral blood (PB) CD20<sup>+</sup> T cells and compare them to their PB CD20<sup>−</sup> T cell counterparts. PB from 17 healthy individuals was collected. The distribution of CD20<sup>+</sup> T cells among maturation-associated T cells compartments (naïve, central memory, transitional memory, effector memory, and effector T cells), their polarization, activation status, and expression of immune-regulatory proteins were evaluated by flow cytometry. Their function was also assessed, by measuring IFN-γ, TNF-α, and IL-17 production. Compared with CD20<sup>−</sup> T cells, CD20<sup>+</sup> T cells represent a higher proportion of transitional memory cells. Furthermore, CD20<sup>+</sup> T cells display a proinflammatory phenotype, characterized by the expansion of Th1, Th1/17, and Tc1 cell subsets , associated to a high expression of activation (CD25) and exhaustion (PD-1) markers. In addition, the simultaneous production of the proinflammatory cytokines IFN-γ, TNF-α, and IL-17 was also detected in CD4<sup>+</sup>CD20<sup>+</sup> T cells. Our results show that CD20<sup>+</sup> T cells are phenotypically and functionally different from CD20<sup>−</sup> T cells, suggesting that these cells are a distinct subset of T cells.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-05-02","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22178","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140841644","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Christopher J. Trindade, Xiaoping Sun, Dragan Maric, Sachein Sharma, Hirsh D. Komarow, Christopher S. Hourigan, Amy Klion, Irina Maric
BackgroundFlow cytometry has been widely used to study immunophenotypic patterns of maturation of most hematopoietic lineages in normal human bone marrow aspirates, thus allowing identification of changes in patterns in many myeloid malignancies. Eosinophils play an important role in a wide variety of disorders, including some myeloid neoplasms. However, changes in flow cytometric immunophenotypic patterns during normal and abnormal bone marrow eosinophilopoiesis have not been well studied.MethodsFresh bone marrow aspirates from 15 healthy donors, 19 patients with hypereosinophilic syndromes (HES), and 11 patients with systemic mastocytosis (SM) were analyzed for candidate markers that included EMR‐1, Siglec‐8, CCR3, CD9, CD11a, CD11b, CD11c, CD13, CD16, CD29, CD34, CD38, CD45, CD44, CD49d, CD49f, CD54, CD62L, CD69, CD117, CD125 (IL‐5Rα), HLA‐DR, using 10 parameter flow cytometry. Putative CD34‐negative immature and mature normal eosinophil populations were first identified based on changes in expression of the above markers in healthy donors, then confirmed using fluorescence‐based cell sorting and morphological evaluation of cytospin preparations. The normal immunophenotypic patterns were then compared to immunophenotypic patterns of eosinophilopoiesis in patients with HES and SM.ResultsThe eosinophilic lineage was first verified using the human eosinophil‐specific antibody EMR‐1 in combination with anti‐IL‐5Rα antibody. Then, a combination of Siglec‐8, CD9, CD11b, CCR3, CD49d, and CD49f antibodies was used to delineate normal eosinophilic maturational patterns. Early stages (eosinophilic promyelocytes/myelocytes) were identified as Siglec‐8 dim/CD11b dim to moderate/CD9 dim/CCR3 dim/CD49d bright/CD49f dim, intermediate stages (eosinophilic myelocytes/metamyelocytes) as Siglec‐8 moderate/CD11b moderate to bright/CD9 moderate/CCR3 moderate/CD49d moderate/CD49f moderate and mature bands/segmented eosinophils as Siglec‐8 bright/CD11b bright/CD9 bright/CCR3 bright/CD49d dim/CD49f bright. Overall maturational patterns were also similar in patients with HES and SM; however, the expression levels of several surface markers were altered compared to normal eosinophils.ConclusionA novel flow cytometric antibody panel was devised to detect alterations in immunophenotypic patterns of bone marrow eosinophil maturation and evaluated in normal, HES and SM samples. This approach will allow us to elucidate changes in immunophenotypic patterns of bone marrow eosinophilopoiesis in other hematological diseases.
{"title":"Flow cytometric immunophenotypic differentiation patterns of bone marrow eosinophilopoiesis","authors":"Christopher J. Trindade, Xiaoping Sun, Dragan Maric, Sachein Sharma, Hirsh D. Komarow, Christopher S. Hourigan, Amy Klion, Irina Maric","doi":"10.1002/cyto.b.22174","DOIUrl":"https://doi.org/10.1002/cyto.b.22174","url":null,"abstract":"BackgroundFlow cytometry has been widely used to study immunophenotypic patterns of maturation of most hematopoietic lineages in normal human bone marrow aspirates, thus allowing identification of changes in patterns in many myeloid malignancies. Eosinophils play an important role in a wide variety of disorders, including some myeloid neoplasms. However, changes in flow cytometric immunophenotypic patterns during normal and abnormal bone marrow eosinophilopoiesis have not been well studied.MethodsFresh bone marrow aspirates from 15 healthy donors, 19 patients with hypereosinophilic syndromes (HES), and 11 patients with systemic mastocytosis (SM) were analyzed for candidate markers that included EMR‐1, Siglec‐8, CCR3, CD9, CD11a, CD11b, CD11c, CD13, CD16, CD29, CD34, CD38, CD45, CD44, CD49d, CD49f, CD54, CD62L, CD69, CD117, CD125 (IL‐5Rα), HLA‐DR, using 10 parameter flow cytometry. Putative CD34‐negative immature and mature normal eosinophil populations were first identified based on changes in expression of the above markers in healthy donors, then confirmed using fluorescence‐based cell sorting and morphological evaluation of cytospin preparations. The normal immunophenotypic patterns were then compared to immunophenotypic patterns of eosinophilopoiesis in patients with HES and SM.ResultsThe eosinophilic lineage was first verified using the human eosinophil‐specific antibody EMR‐1 in combination with anti‐IL‐5Rα antibody. Then, a combination of Siglec‐8, CD9, CD11b, CCR3, CD49d, and CD49f antibodies was used to delineate normal eosinophilic maturational patterns. Early stages (eosinophilic promyelocytes/myelocytes) were identified as Siglec‐8 dim/CD11b dim to moderate/CD9 dim/CCR3 dim/CD49d bright/CD49f dim, intermediate stages (eosinophilic myelocytes/metamyelocytes) as Siglec‐8 moderate/CD11b moderate to bright/CD9 moderate/CCR3 moderate/CD49d moderate/CD49f moderate and mature bands/segmented eosinophils as Siglec‐8 bright/CD11b bright/CD9 bright/CCR3 bright/CD49d dim/CD49f bright. Overall maturational patterns were also similar in patients with HES and SM; however, the expression levels of several surface markers were altered compared to normal eosinophils.ConclusionA novel flow cytometric antibody panel was devised to detect alterations in immunophenotypic patterns of bone marrow eosinophil maturation and evaluated in normal, HES and SM samples. This approach will allow us to elucidate changes in immunophenotypic patterns of bone marrow eosinophilopoiesis in other hematological diseases.","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-04-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140805354","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Sihem Tarfi, Wolfgang Kern, Elodie Goulas, Dorothée Selimoglu-Buet, Orianne Wagner-Ballon, the CytHem-LMMC
The monocyte subset partitioning by flow cytometry, known as “monocyte assay,” is now integrated into the new classifications as a supporting criterion for CMML diagnosis, if a relative accumulation of classical monocytes above 94% of total circulating monocytes is observed. Here we provide clinical flow cytometry laboratories with technical support adapted for the most commonly used cytometers. Step-by-step explanations of the gating strategy developed on whole peripheral blood are presented while underlining the most common difficulties. In a second part, interpretation recommendations of circulating monocyte partitioning from the dedicated French working group “CytHem-LMMC” are shared as well as the main pitfalls, including false positive and false negative cases. The particular flow-defined inflammatory profile is described and the usefulness of the nonclassical monocyte specific marker, namely slan, highlighted. Examples of reporting to the physician with frequent situations encountered when using the monocyte assay are also presented.
{"title":"Technical, gating and interpretation recommendations for the partitioning of circulating monocyte subsets assessed by flow cytometry","authors":"Sihem Tarfi, Wolfgang Kern, Elodie Goulas, Dorothée Selimoglu-Buet, Orianne Wagner-Ballon, the CytHem-LMMC","doi":"10.1002/cyto.b.22176","DOIUrl":"10.1002/cyto.b.22176","url":null,"abstract":"<p>The monocyte subset partitioning by flow cytometry, known as “monocyte assay,” is now integrated into the new classifications as a supporting criterion for CMML diagnosis, if a relative accumulation of classical monocytes above 94% of total circulating monocytes is observed. Here we provide clinical flow cytometry laboratories with technical support adapted for the most commonly used cytometers. Step-by-step explanations of the gating strategy developed on whole peripheral blood are presented while underlining the most common difficulties. In a second part, interpretation recommendations of circulating monocyte partitioning from the dedicated French working group “CytHem-LMMC” are shared as well as the main pitfalls, including false positive and false negative cases. The particular flow-defined inflammatory profile is described and the usefulness of the nonclassical monocyte specific marker, namely slan, highlighted. Examples of reporting to the physician with frequent situations encountered when using the monocyte assay are also presented.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-04-24","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22176","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140859692","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Feng Zhang, Ya-Zhe Wang, Yan Chang, Xiao-Ying Yuan, Wei-Hua Shi, Hong-Xia Shi, Jian-Zhen Shen, Yan-Rong Liu
Thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF), prefibrotic/early (pre-PMF), and overt fibrotic PMF (overt PMF) are classical Philadelphia-Negative (Ph-negative) myeloproliferative neoplasms (MPNs). Differentiating between these types based on morphology and molecular markers is challenging. This study aims to clarify the application of flow cytometry in the diagnosis and differential diagnosis of classical MPNs. This study retrospectively analyzed the immunophenotypes, clinical characteristics, and laboratory findings of 211 Ph-negative MPN patients, including ET, PV, pre-PMF, overt PMF, and 47 controls. Compared to ET and PV, PMF differed in white blood cells, hemoglobin, blast cells in the peripheral blood, abnormal karyotype, and WT1 gene expression. PMF also differed from controls in CD34+ cells, granulocyte phenotype, monocyte phenotype, percentage of plasma cells, and dendritic cells. Notably, the PMF group had a significantly lower plasma cell percentage compared with other groups. A lasso and random forest model select five variables (CD34+CD19+cells and CD34+CD38− cells on CD34+cells, CD13dim+CD11b− cells in granulocytes, CD38str+CD19+/−plasma, and CD123+HLA-DR−basophils), which identify PMF with a sensitivity and specificity of 90%. Simultaneously, a classification and regression tree model was constructed using the percentage of CD34+CD38− on CD34+ cells and platelet counts to distinguish between ET and pre-PMF, with accuracies of 94.3% and 83.9%, respectively. Flow immunophenotyping aids in diagnosing PMF and differentiating between ET and PV. It also helps distinguish pre-PMF from ET and guides treatment decisions.
{"title":"A lasso and random forest model using flow cytometry data identifies primary myelofibrosis","authors":"Feng Zhang, Ya-Zhe Wang, Yan Chang, Xiao-Ying Yuan, Wei-Hua Shi, Hong-Xia Shi, Jian-Zhen Shen, Yan-Rong Liu","doi":"10.1002/cyto.b.22173","DOIUrl":"10.1002/cyto.b.22173","url":null,"abstract":"<p>Thrombocythemia (ET), polycythemia vera (PV), primary myelofibrosis (PMF), prefibrotic/early (pre-PMF), and overt fibrotic PMF (overt PMF) are classical Philadelphia-Negative (<i>Ph-negative</i>) myeloproliferative neoplasms (MPNs). Differentiating between these types based on morphology and molecular markers is challenging. This study aims to clarify the application of flow cytometry in the diagnosis and differential diagnosis of classical MPNs. This study retrospectively analyzed the immunophenotypes, clinical characteristics, and laboratory findings of 211 <i>Ph-negative</i> MPN patients, including ET, PV, pre-PMF, overt PMF, and 47 controls. Compared to ET and PV, PMF differed in white blood cells, hemoglobin, blast cells in the peripheral blood, abnormal karyotype, and WT1 gene expression. PMF also differed from controls in CD34<sup>+</sup> cells, granulocyte phenotype, monocyte phenotype, percentage of plasma cells, and dendritic cells. Notably, the PMF group had a significantly lower plasma cell percentage compared with other groups. A lasso and random forest model select five variables (CD34<sup>+</sup>CD19<sup>+</sup>cells and CD34<sup>+</sup>CD38<sup>−</sup> cells on CD34<sup>+</sup>cells, CD13<sup>dim+</sup>CD11b<sup>−</sup> cells in granulocytes, CD38<sup>str+</sup>CD19<sup>+/−</sup>plasma, and CD123<sup>+</sup>HLA-DR<sup>−</sup>basophils), which identify PMF with a sensitivity and specificity of 90%. Simultaneously, a classification and regression tree model was constructed using the percentage of CD34<sup>+</sup>CD38<sup>−</sup> on CD34<sup>+</sup> cells and platelet counts to distinguish between ET and pre-PMF, with accuracies of 94.3% and 83.9%, respectively. Flow immunophenotyping aids in diagnosing PMF and differentiating between ET and PV. It also helps distinguish pre-PMF from ET and guides treatment decisions.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":null,"pages":null},"PeriodicalIF":2.3,"publicationDate":"2024-04-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140805114","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}
Pénélope Bourgoin, Thomas Dupont, Chantal Agabriel, Ania Carsin, Aurélie Verles, Maciej Cabanski, Alessandra Vitaliti, Jean‐Marc Busnel
The Basophil Activation Test (BAT) enables flow cytometry characterization of basophil reactivity against specific allergenic molecules. The focus now revolves around democratizing this tool, but, as blood sample stability could be challenging, after having developed a simplified approach, herein, we aimed to characterize two strategies for implementing BAT in multicentric studies: store and ship blood before or after sample processing. Fresh heparin‐ and EDTA‐anticoagulated whole blood samples followed both BAT workflows: “collect, store, process & analyze” or “collect, process, store & analyze”. Storage temperatures of 18–25 °C or 2–8 °C and preservation times from 0 to 7 days were considered. Interleukin‐3 was also evaluated. With the “collect, store, process & analyze” workflow, heparin‐anticoagulated blood and 18–25 °C storage were better than other conditions. While remaining possible, basophil activation exhibited a possible reactivity decay after 24 h. Under the conditions tested, interleukin‐3 had no role in enhancing basophil reactivity after storage. Conversely, the “collect, process, store & analyze” workflow demonstrated that either heparin‐ or EDTA‐anticoagulated blood can be processed and kept up to 7 days at 18–25 °C or 2–8 °C before being analyzed. Various strategies can be implemented to integrate BAT in multicentric studies. The “collect, store, process & analyze” workflow remains a simplified logistical approach, but depending on time required to ship from the clinical centers to the reference laboratories, it might not be applicable, or should be used with caution. The “collect, process, store & analyze” workflow may constitute a workflow improvement to provide significant flexibility without impact on basophil reactivity.
嗜碱性粒细胞活化测试(BAT)可通过流式细胞术鉴定嗜碱性粒细胞对特定过敏原分子的反应性。目前的重点是使这一工具平民化,但由于血液样本的稳定性可能具有挑战性,在开发出简化方法后,我们在本文中旨在描述在多中心研究中实施嗜碱性粒细胞活化测试的两种策略:在样本处理之前或之后储存和运输血液。新鲜肝素和 EDTA 抗凝全血样本均遵循两种 BAT 工作流程:"采集、储存、处理和分析 "或 "采集、处理、储存和分析"。储存温度为 18-25 °C 或 2-8 °C,保存时间为 0-7 天。还对白细胞介素-3 进行了评估。在 "收集、储存、处理和分析 "的工作流程中,肝素抗凝血液和 18-25 ° C 的储存条件优于其他条件。在所测试的条件下,白细胞介素-3 在储存后不会增强嗜碱性粒细胞的反应性。相反,"收集、处理、储存& 分析 "的工作流程表明,肝素或 EDTA 抗凝血都可以处理,并在分析前在 18-25 °C 或 2-8 °C 下保存长达 7 天。在多中心研究中,可以采用多种策略整合 BAT。收集、储存、处理和分析 "工作流程仍然是一种简化的后勤方法,但根据从临床中心运送到参考实验室所需的时间,这种方法可能并不适用,或应谨慎使用。收集、处理、储存和分析 "工作流程可能是对工作流程的一种改进,可在不影响嗜碱性粒细胞反应性的情况下提供极大的灵活性。
{"title":"Possible alternative strategies to implement basophil activation testing in multicentric studies","authors":"Pénélope Bourgoin, Thomas Dupont, Chantal Agabriel, Ania Carsin, Aurélie Verles, Maciej Cabanski, Alessandra Vitaliti, Jean‐Marc Busnel","doi":"10.1002/cyto.b.22172","DOIUrl":"https://doi.org/10.1002/cyto.b.22172","url":null,"abstract":"The Basophil Activation Test (BAT) enables flow cytometry characterization of basophil reactivity against specific allergenic molecules. The focus now revolves around democratizing this tool, but, as blood sample stability could be challenging, after having developed a simplified approach, herein, we aimed to characterize two strategies for implementing BAT in multicentric studies: store and ship blood before or after sample processing. Fresh heparin‐ and EDTA‐anticoagulated whole blood samples followed both BAT workflows: “collect, store, process & analyze” or “collect, process, store & analyze”. Storage temperatures of 18–25 °C or 2–8 °C and preservation times from 0 to 7 days were considered. Interleukin‐3 was also evaluated. With the “collect, store, process & analyze” workflow, heparin‐anticoagulated blood and 18–25 °C storage were better than other conditions. While remaining possible, basophil activation exhibited a possible reactivity decay after 24 h. Under the conditions tested, interleukin‐3 had no role in enhancing basophil reactivity after storage. Conversely, the “collect, process, store & analyze” workflow demonstrated that either heparin‐ or EDTA‐anticoagulated blood can be processed and kept up to 7 days at 18–25 °C or 2–8 °C before being analyzed. Various strategies can be implemented to integrate BAT in multicentric studies. The “collect, store, process & analyze” workflow remains a simplified logistical approach, but depending on time required to ship from the clinical centers to the reference laboratories, it might not be applicable, or should be used with caution. The “collect, process, store & analyze” workflow may constitute a workflow improvement to provide significant flexibility without impact on basophil reactivity.","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":null,"pages":null},"PeriodicalIF":3.4,"publicationDate":"2024-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"140561487","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":3,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}