Felipe Castillo, Constanza Morales, Biserka Spralja, Joaquín Díaz-Schmidt, Mirentxu Iruretagoyena, Daniel Ernst
Background
The diagnosis of T-cell non-Hodgkin lymphomas (NHL) is challenging. The development of a monoclonal antibody specific for T-cell receptor β constant region 1 (TRBC1) provides an alternative to discriminate clonal T cells. The aim of this study was to evaluate the diagnostic potential of an anti-TRBC1 mAb for the identification of T-NHL.
Methods
We performed a cross-sectional diagnostic analytic study of samples tested for lymphoma. All samples sent for lymphoma screening were first evaluated using the standard Euroflow LST, to which a second additional custom-designed T-cell clonality assessment tube was added CD45/TRBC1/CD2/CD7/CD4/TCRγδ/CD3. Flow cytometry reports were compared with morphological and molecular tests.
Results
Fifty-nine patient samples were evaluated. Within the T-cell population, cut-off percentages in the CD4+ cells were from 29.4 to 54.6% and from 23.9 to 52.1% in CD8+ cells. Cut-off ratios in CD4+ T cells were from 0.33 to 1.1, and in CD8+ cells between 0.22 and 1.0. Using predefined normal cut-off values, 18 of 59 (30.5%) samples showed a restricted expression of TRBC1. A final diagnosis of a T-NHL was confirmed clinically and/or by histopathological studies in 15 of the 18 cases (83.3%). There were no cases of T-NHL by morphology/IHC with normal TRBC1 expression. Non-neoplastic patient samples behaved between predefined TRBC1 cut-off values.
Conclusions
Expression of TRBC1 provides a robust method for T-cell clonality assessment, with very high sensitivity and good correlation with complementary methods. TRBC1 can be integrated into routine lymphoma screening strategies via flow cytometry.
{"title":"Integration of T-cell clonality screening using TRBC-1 in lymphoma suspect samples by flow cytometry","authors":"Felipe Castillo, Constanza Morales, Biserka Spralja, Joaquín Díaz-Schmidt, Mirentxu Iruretagoyena, Daniel Ernst","doi":"10.1002/cyto.b.22147","DOIUrl":"10.1002/cyto.b.22147","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>The diagnosis of T-cell non-Hodgkin lymphomas (NHL) is challenging. The development of a monoclonal antibody specific for T-cell receptor β constant region 1 (TRBC1) provides an alternative to discriminate clonal T cells. The aim of this study was to evaluate the diagnostic potential of an anti-TRBC1 mAb for the identification of T-NHL.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>We performed a cross-sectional diagnostic analytic study of samples tested for lymphoma. All samples sent for lymphoma screening were first evaluated using the standard Euroflow LST, to which a second additional custom-designed T-cell clonality assessment tube was added CD45/TRBC1/CD2/CD7/CD4/TCRγδ/CD3. Flow cytometry reports were compared with morphological and molecular tests.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>Fifty-nine patient samples were evaluated. Within the T-cell population, cut-off percentages in the CD4+ cells were from 29.4 to 54.6% and from 23.9 to 52.1% in CD8+ cells. Cut-off ratios in CD4+ T cells were from 0.33 to 1.1, and in CD8+ cells between 0.22 and 1.0. Using predefined normal cut-off values, 18 of 59 (30.5%) samples showed a restricted expression of TRBC1. A final diagnosis of a T-NHL was confirmed clinically and/or by histopathological studies in 15 of the 18 cases (83.3%). There were no cases of T-NHL by morphology/IHC with normal TRBC1 expression. Non-neoplastic patient samples behaved between predefined TRBC1 cut-off values.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>Expression of TRBC1 provides a robust method for T-cell clonality assessment, with very high sensitivity and good correlation with complementary methods. TRBC1 can be integrated into routine lymphoma screening strategies via flow cytometry.</p>\u0000 </section>\u0000 </div>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"106 1","pages":"64-73"},"PeriodicalIF":3.4,"publicationDate":"2023-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138444237","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}
Deepak Kumar, F. N. U. Kiran, Amanda Wheeler, Rory Dellamano, Richard D. Hammer
Background
Surface median immunofluorescence intensity (MFI) of plasma cells antigens, particularly CD138, by flow cytometry underestimates plasma cell populations when compared with that estimated by morphological assessment on Wright's-stained slides. CD138 MFI using traditional sample preparation methods for flow cytometric analysis is often dim and difficult to interpret due to multiple factors. This becomes critical when diagnosing and accurately classifying plasma cell dyscrasias.
Methods
In this study, we analyzed 280 flow cytometric results collected from 2016 to 2022 for CD38 and CD138 MFI on bone marrow aspirates performed by two different methods of sample processing—traditional method of lyse-wash and the alternative method of lyse-no-wash.
Results
Visual examination of histograms showed a clear advantage to CD138 expression intensity with the no-wash method. Although no significant difference was observed in CD38 MFI between the two techniques (p = 0.3), considerable improvement was observed in CD138 MFI with the lyse-no-wash technique of sample processing compared with the conventional method (p = 0.003).
Conclusions
We concluded that the method of lyse-no-wash is superior to traditional methods especially when it comes to handling bone marrow aspirate samples for plasma cell immunophenotyping. This alternate technique increases the sensitivity of flow cytometry to detect plasma cells resulting in bright and crisp signal intensity for surface CD138. This technique may be particularly advantageous when analyzing low tumor burden such as minimal residual disease.
{"title":"An alternative processing approach to increase CD138 intensity in flow cytometric analysis of plasma cells","authors":"Deepak Kumar, F. N. U. Kiran, Amanda Wheeler, Rory Dellamano, Richard D. Hammer","doi":"10.1002/cyto.b.22149","DOIUrl":"10.1002/cyto.b.22149","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>Surface median immunofluorescence intensity (MFI) of plasma cells antigens, particularly CD138, by flow cytometry underestimates plasma cell populations when compared with that estimated by morphological assessment on Wright's-stained slides. CD138 MFI using traditional sample preparation methods for flow cytometric analysis is often dim and difficult to interpret due to multiple factors. This becomes critical when diagnosing and accurately classifying plasma cell dyscrasias.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>In this study, we analyzed 280 flow cytometric results collected from 2016 to 2022 for CD38 and CD138 MFI on bone marrow aspirates performed by two different methods of sample processing—traditional method of lyse-wash and the alternative method of lyse-no-wash.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>Visual examination of histograms showed a clear advantage to CD138 expression intensity with the no-wash method. Although no significant difference was observed in CD38 MFI between the two techniques (<i>p</i> = 0.3), considerable improvement was observed in CD138 MFI with the lyse-no-wash technique of sample processing compared with the conventional method (<i>p</i> = 0.003).</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>We concluded that the method of lyse-no-wash is superior to traditional methods especially when it comes to handling bone marrow aspirate samples for plasma cell immunophenotyping. This alternate technique increases the sensitivity of flow cytometry to detect plasma cells resulting in bright and crisp signal intensity for surface CD138. This technique may be particularly advantageous when analyzing low tumor burden such as minimal residual disease.</p>\u0000 </section>\u0000 </div>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"106 2","pages":"113-116"},"PeriodicalIF":3.4,"publicationDate":"2023-11-27","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138444236","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}
Peter Slanina, Julie Stichova, Veronika Bosakova, Iva Staniczkova Zambo, Marcela Hortova Kohoutkova, Petra Laznickova, Zita Chovancova, Jiri Litzman, Terezie Plucarova, Jan Fric, Marcela Vlkova
Common variable immunodeficiency disorder (CVID) is the most common form of primary antibody immunodeficiency. Due to low antibody levels, CVID patients receive intravenous or subcutaneous immunoglobulin replacement therapy as treatment. CVID is associated with the chronic activation of granulocytes, including an increased percentage of low-density neutrophils (LDNs). In this study, we examined changes in the percentage of LDNs and the expression of their surface markers in 25 patients with CVID and 27 healthy donors (HD) after in vitro stimulation of whole blood using IVIg. An oxidative burst assay was used to assess the functionality of LDNs. CVID patients had increased both relative and absolute LDN counts with a higher proportion of mLDNs compared to iLDNs, distinguished based on the expression of CD10 and CD16. Immature LDNs in the CVID and HD groups had significantly reduced oxidative burst capacity compared to mature LDNs. Interestingly we observed reduced oxidative burst capacity, reduced expression of CD10 after stimulation of WB, and higher expression of PD-L1 in mature LDNs in CVID patients compared to HD cells. Our data indicate that that the functional characteristics of LDNs are closely linked to their developmental stage. The observed reduction in oxidative burst capacity in mLDNs in CVID patients could contribute to an increased susceptibility to recurrent bacterial infections among CVID patients.
{"title":"Phenotype and oxidative burst of low-density neutrophil subpopulations are altered in common variable immunodeficiency patients","authors":"Peter Slanina, Julie Stichova, Veronika Bosakova, Iva Staniczkova Zambo, Marcela Hortova Kohoutkova, Petra Laznickova, Zita Chovancova, Jiri Litzman, Terezie Plucarova, Jan Fric, Marcela Vlkova","doi":"10.1002/cyto.b.22150","DOIUrl":"10.1002/cyto.b.22150","url":null,"abstract":"<p>Common variable immunodeficiency disorder (CVID) is the most common form of primary antibody immunodeficiency. Due to low antibody levels, CVID patients receive intravenous or subcutaneous immunoglobulin replacement therapy as treatment. CVID is associated with the chronic activation of granulocytes, including an increased percentage of low-density neutrophils (LDNs). In this study, we examined changes in the percentage of LDNs and the expression of their surface markers in 25 patients with CVID and 27 healthy donors (HD) after in vitro stimulation of whole blood using IVIg. An oxidative burst assay was used to assess the functionality of LDNs. CVID patients had increased both relative and absolute LDN counts with a higher proportion of mLDNs compared to iLDNs, distinguished based on the expression of CD10 and CD16. Immature LDNs in the CVID and HD groups had significantly reduced oxidative burst capacity compared to mature LDNs. Interestingly we observed reduced oxidative burst capacity, reduced expression of CD10 after stimulation of WB, and higher expression of PD-L1 in mature LDNs in CVID patients compared to HD cells. Our data indicate that that the functional characteristics of LDNs are closely linked to their developmental stage. The observed reduction in oxidative burst capacity in mLDNs in CVID patients could contribute to an increased susceptibility to recurrent bacterial infections among CVID patients.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"106 2","pages":"99-112"},"PeriodicalIF":3.4,"publicationDate":"2023-11-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22150","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"138298613","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}
{"title":"BCR::ABL1 fusion gene positive de novo acute myeloid leukemia with coexistence of NRAS mutation and presented with a peculiar CD58 positive immunophenotype","authors":"Xueya Zhang, Xizhe Guo","doi":"10.1002/cyto.b.22152","DOIUrl":"10.1002/cyto.b.22152","url":null,"abstract":"","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"106 2","pages":"146-148"},"PeriodicalIF":3.4,"publicationDate":"2023-11-15","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"134648632","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}
The CD34+CD38− population in bone marrow includes hematopoietic stem/progenitor cells. Recently, in acute myeloid leukemia, the focus has shifted to flow cytometry analysis targeting CD34+CD38− leukemic cells due to their effectiveness in minimal/measurable residual disease detection and prognosis prediction. Nevertheless, the immunophenotype and cell frequency of these cells in the bone marrow, in the absence of leukemic cells, remains unknown. We aimed to evaluate detailed characteristics of CD34+CD38− cells in both normal and leukemic cells by flow cytometry.
Methods
We compared the cell frequency and immunophenotype of the CD34+CD38− fraction in the following groups: patients with idiopathic thrombocytopenic purpura and malignant lymphoma as controls (n = 17), post-treatment patients without abnormal blasts (n = 35), and patients with myeloid malignancies (n = 86). The comparison was based on the presence or absence of CD45RA expression, a marker commonly used to prospectively isolate lymphoid-primed cell populations within the CD34+CD38− fraction.
Results
The CD34+CD38−CD45RA+ cell population exhibited a significant expansion in bone marrow without leukemic cells 1 month after cord blood transplantation and in various type of myeloid malignancies, compared to the control group (p < 0.01). Continuous CD45RA expression and notable expansion of the CD34+CD38−CD45RA− population were exclusively observed in myelodysplastic syndrome-related diseases. The CD34+CD38−CD45RA+ population displayed frequent expression of various markers in both leukemic and non-leukemic cells, in contrast to the CD34+CD38−CD45RA− population.
Conclusions
The CD34+CD38− fraction should be carefully evaluated considering the nature of normal hematopoietic precursor cells, their cell frequency and immunophenotype, including CD45RA expression pattern, for improving the accuracy of myeloid malignancy diagnosis.
{"title":"Flow cytometric analysis of CD34+CD38− cells; cell frequency and immunophenotype based on CD45RA expression pattern","authors":"Shumpei Mizuta, Makoto Iwasaki, Noriko Bandai, Saya Yoshida, Asami Watanabe, Hiroshi Takashima, Takeshi Ueshimo, Kazuhiro Bandai, Kensuke Fujiwara, Naoko Hiranuma, Yusuke Koba, Takahito Kawata, Akira Tamekane, Mitsumasa Watanabe","doi":"10.1002/cyto.b.22148","DOIUrl":"10.1002/cyto.b.22148","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Introduction</h3>\u0000 \u0000 <p>The CD34<sup>+</sup>CD38<sup>−</sup> population in bone marrow includes hematopoietic stem/progenitor cells. Recently, in acute myeloid leukemia, the focus has shifted to flow cytometry analysis targeting CD34<sup>+</sup>CD38<sup>−</sup> leukemic cells due to their effectiveness in minimal/measurable residual disease detection and prognosis prediction. Nevertheless, the immunophenotype and cell frequency of these cells in the bone marrow, in the absence of leukemic cells, remains unknown. We aimed to evaluate detailed characteristics of CD34<sup>+</sup>CD38<sup>−</sup> cells in both normal and leukemic cells by flow cytometry.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>We compared the cell frequency and immunophenotype of the CD34<sup>+</sup>CD38<sup>−</sup> fraction in the following groups: patients with idiopathic thrombocytopenic purpura and malignant lymphoma as controls (<i>n</i> = 17), post-treatment patients without abnormal blasts (<i>n</i> = 35), and patients with myeloid malignancies (<i>n</i> = 86). The comparison was based on the presence or absence of CD45RA expression, a marker commonly used to prospectively isolate lymphoid-primed cell populations within the CD34<sup>+</sup>CD38<sup>−</sup> fraction.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The CD34<sup>+</sup>CD38<sup>−</sup>CD45RA<sup>+</sup> cell population exhibited a significant expansion in bone marrow without leukemic cells 1 month after cord blood transplantation and in various type of myeloid malignancies, compared to the control group (<i>p</i> < 0.01). Continuous CD45RA expression and notable expansion of the CD34<sup>+</sup>CD38<sup>−</sup>CD45RA<sup>−</sup> population were exclusively observed in myelodysplastic syndrome-related diseases. The CD34<sup>+</sup>CD38<sup>−</sup>CD45RA<sup>+</sup> population displayed frequent expression of various markers in both leukemic and non-leukemic cells, in contrast to the CD34<sup>+</sup>CD38<sup>−</sup>CD45RA<sup>−</sup> population.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>The CD34<sup>+</sup>CD38<sup>−</sup> fraction should be carefully evaluated considering the nature of normal hematopoietic precursor cells, their cell frequency and immunophenotype, including CD45RA expression pattern, for improving the accuracy of myeloid malignancy diagnosis.</p>\u0000 </section>\u0000 </div>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"106 1","pages":"35-44"},"PeriodicalIF":3.4,"publicationDate":"2023-11-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"71479197","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}
<p>[Color figure can be viewed at wileyonlinelibrary.com]</p><p>Despite the reality that my entire career has been spent devoted to flow cytometry, it is undeniable to me that the gold standard remains an image of the cell, and when visualized within tissue, the architectural relationships between cells. Thus my keen interest in bringing the Amnis technology into my research laboratory years ago and my current enthusiasm for the cell-imaging capacities of more recent technologies such as those from Becton-Dickinson (Cell-View), ThermoFisher (Attune CytPix) and other imaging flow cytometers currently or soon available. In this issue I am therefore extremely interested in Cyclic Analysis of Single-Cell Subsets and Tissue Territories (CASSATT) which identifies scanned slide images through multiple staining rounds, segments nuclei and assesses marker expression on each detected cell (Brockman et al., <span>2023</span>). Cyclic immunochemistry (cycIHC) exploits multiple rounds of immunostaining and imaging for mapping and locating cells of interest with the speed and simplicity of brightfield microscopy, making the collection of entire tissue sections and slides possible. CASSATT employs registered scanned slide images across all rounds of staining, segments individual nuclei, and measures marker expression on each detected cell. It further explores the spatial relationships between cell populations and the odds of interaction frequencies between cell populations within tissue regions, helping to identify cells that interact or do not interact. In this report, a test dataset of six glioblastoma tissue sections were cyclically stained for eight biomarkers for a total of 48 scanned slide images. The authors describe an efficient workflow that provided answers to questions commonly encountered in discovery research in tissue specimens, such as the overall abundance of cells of interest within a tissue section as well as whether groups of cells were in close proximity. CASSATT gathered together and streamlined all steps necessary to produce single cell expression information from cycIHC datasets and did so utilizing an open source environment. In addition, CASSATT systematically analyzed the spatial relationships between cell populations and via unsupervised algorithms, identified clusters of cell niches.</p><p>The persistence of measurable residual disease (MRD) is a strong indicator for adverse outcomes in acute myeloid leukemia (AML) and has been shown to be a valid surrogate marker for disease-free survival and overall survival, irrespective of patients' age, AML subtype, sample type, time of MRD assessment and MRD detection method (Short et al., <span>2020</span>). Other hematopoietic malignancies such as B- and plasma cell lineages also benefit from MRD diagnostic assays (Chen et al., <span>2023</span>; Gao et al., <span>2023</span>; McMillan et al., <span>2023</span>; Zhou et al., <span>2023</span>). In a study by Wang et al. (<span>2023</span>) th
尽管我的整个职业生涯都致力于流式细胞术，但对我来说不可否认的是，黄金标准仍然是细胞的图像，当在组织中可视化时，细胞之间的结构关系。因此，多年前我对将Amnis技术引入我的研究实验室产生了浓厚的兴趣，并且我目前对最近技术的细胞成像能力充满热情，例如Becton-Dickinson (Cell-View)， ThermoFisher (tune CytPix)和其他目前或即将上市的成像流式细胞仪。因此，在这一期中，我对单细胞亚群和组织区域的循环分析(CASSATT)非常感兴趣，该分析通过多个染色轮、细胞核片段识别扫描的幻灯片图像，并评估每个检测细胞上的标记表达(Brockman等人，2023)。循环免疫化学(cycIHC)利用多轮免疫染色和成像来定位和定位感兴趣的细胞，具有明场显微镜的速度和简单性，使整个组织切片和载玻片的收集成为可能。CASSATT采用在所有染色轮中注册的扫描幻灯片图像，分割单个细胞核，并测量每个检测细胞上的标记表达。它进一步探讨了细胞群之间的空间关系和组织区域内细胞群之间相互作用频率的几率，有助于识别相互作用或不相互作用的细胞。在本报告中，对6个胶质母细胞瘤组织切片的测试数据集进行了8种生物标志物的循环染色，共扫描了48张幻灯片图像。作者描述了一个有效的工作流程，为组织标本发现研究中经常遇到的问题提供答案，例如组织切片中感兴趣的细胞的总体丰度以及细胞群是否接近。CASSATT收集并简化了从cycIHC数据集中产生单细胞表达信息所需的所有步骤，并利用开源环境完成了这一过程。此外，CASSATT系统地分析了细胞群体之间的空间关系，并通过无监督算法识别了细胞生态位集群。可测量残余疾病(MRD)的持续存在是急性髓性白血病(AML)不良结局的一个强有力的指标，已被证明是无病生存和总生存的有效替代标志物，与患者的年龄、AML亚型、样本类型、MRD评估时间和MRD检测方法无关(Short et al.， 2020)。其他造血恶性肿瘤，如B细胞和浆细胞谱系也受益于MRD诊断分析(Chen等人，2023;Gao et al.， 2023;McMillan et al.， 2023;Zhou et al.， 2023)。Wang等人(2023)在一项研究中分享了他们在CLSI HL62指南和FDA IDE(研究设备豁免)批准下验证12色AML MRD流式细胞术MRD检测的经验，包括面板设计，分析和解释的细节。在他们最近的细胞仪升级到12个标记容量允许更广泛的测试之前，这些研究人员使用了一个8个标记的AML MRD测试，设计用于区分正常和异常的骨髓单核细胞前体，使用结合检测白血病相关免疫表型(LAIP)和/或识别偏离正常(DfN)方法的分析方法。八色法与分子检测、临床结果交叉相关，并发表了多篇论文(Ouyang et al.， 2015,2016;Xu et al.， 2017)。在目前的研究中，通过检测已知的阳性和阴性样本，并与分子基因检测和后续骨髓检查的结果相关联，来评估检测的准确性。检测限(LOD)和定量限(LOQ)被验证为0.01%和0.1%之间的水平，取决于评估的细胞数量和与正常表型的偏差程度。检测线性度、精密度和结转研究均可接受。在61例患者中测试了该方法的临床有效性，以便通过与并发分子基因检测和/或随访骨髓检查结果的相关性来确定“真实性”;临床试验一致性93%，特异性98%，敏感性83%。最终，该试验最具挑战性的方面涉及识别白血病前细胞(持续克隆造血)或潜在骨髓增生异常克隆之间的差异，这些克隆来自AML MRD，具有免疫表型开关或亚克隆选择，强调需要进一步表征具有复发可能性的异常母细胞。 在临床流式细胞术实验室的一个染色管中处理尽可能多的单克隆抗体一直是我整个职业生涯的夙愿。与其说我是“心流极客”，不如说是我想要这种能力，而是我认为，这种做法显然为我们的病人提供了更好的医疗服务。在血液学恶性肿瘤的诊断中尤其如此，我认为肿瘤“不阅读与我们所阅读的谱系标记相同的书籍和期刊”，而是尽其所能地混淆它们的谱系，让我们对它们“不忠实的”抗原共表达感到困惑。我们对这类恶性肿瘤应用的标志物越多，以及内置的阳性和阴性对照(由混合的正常细胞提供)，我们就越能成为更好的诊断专家。这些并不是减少染色管数量的唯一优点，因为当样品是少细胞的时候，利用最少数量的管或孔处理细胞，更多的诊断信息也变得进一步可用。临床流式细胞术实验室可以利用的分析能力越强，我们可以提供的灵活性就越大，我们可以提出的问题也就越好，比如上面描述的与MRD以及许多其他领域相关的问题(Estevam等人，2021;Quirós-Caso等，2022;Shameli,罗山,2022;Sanjabi,李尔王,2021)。这些硬件、试剂和软件在我们的研究流式细胞仪实验室已经存在;我们都在等待这些技术完全进入临床领域，并看到这一领域的进步使我们更接近更好的实验室实践。在Hammerich等人(2023)提交的报告中，我们了解到三激光光谱流式细胞仪的应用，展示了31个标志物临床导向检测面板的分析。该小组使用的标准3激光极光分辨了405、488和640纳米激光激发的31个荧光色。研究中使用的所有荧光染料和滴定抗体都是市售的，简化了对面板的修改，并便于用其他可能更合适的标记替换感兴趣的标记。事实上，由于本研究中没有使用所有的检测器，因此可以假设，在未来，更多的偶联抗体可能会扩大该平台的分析能力。总之，我发现这是临床流诊断的一个伟大时刻，当然，除了我希望仪器中至少有一个，如果不是两到三个激光器，而不是在这篇文章中讨论的，这是我的荣幸和荣幸，为我们的杂志引入一种新的同行评审的提交，我们称之为“最佳实践”类别。虽然目前我们的期刊页面是新的，但在过去的几年里，这些以编号模块形式提供的信息片段已经提供给临床流动实验室社区，源于我们自己的ICCS质量和标准委员会。Devitt等人(2023)目前提交的报告强调了流式细胞术检测验证的重要性，它为临床护理人员在确定关键医疗决策时产生可靠的结果提供了信心。例如，作者为我们的读者提供了IVD(体外诊断)或LDT(实验室开发)测试急需的描述、解释和区别特征。IVD测试是由制造商开发的，他们优化、验证并将此类检测提交给监管机构，如FDA。监管机构批准验证并批准其临床使用，允许制造商将该分析方法出售给实验室用于检测患者样本。实验室必须遵循制造商描述的标准操作程序，并验证他们可以在实验室中复制制造商的性能规格。与IVD测试相比，Devitt等人(2023)侧重于LDT分析，这些分析是在使用特定实验室设备、试剂和工作人员的单个实验室开发、优化和验证的。在美国，目前不要求外部监管机构批准ldt，尽管已经建议通过联邦立法来改变这种模式(VALID法案)，而且许多受影响的医疗协会必须密切监测这种潜在的变化。实验室必须验证其内部开发的分析方法的独特性能规格。一旦验证，实验室就可以对患者样本进行分析;但是，检测不得出售给其他机构。对IVD分析的任何改变，如果偏离了制造商的说明，都可能使该测试成为LDT，需要验证。作者继续介绍ldt的其他方面，例如描述它们的报告结构(定量的、半定量的、定性的或混合的)，并提供每种结构的例子。 测试准确性，精度，
{"title":"Issue highlights—September 2023","authors":"Professor Frederic I. Preffer","doi":"10.1002/cyto.b.22145","DOIUrl":"10.1002/cyto.b.22145","url":null,"abstract":"<p>[Color figure can be viewed at wileyonlinelibrary.com]</p><p>Despite the reality that my entire career has been spent devoted to flow cytometry, it is undeniable to me that the gold standard remains an image of the cell, and when visualized within tissue, the architectural relationships between cells. Thus my keen interest in bringing the Amnis technology into my research laboratory years ago and my current enthusiasm for the cell-imaging capacities of more recent technologies such as those from Becton-Dickinson (Cell-View), ThermoFisher (Attune CytPix) and other imaging flow cytometers currently or soon available. In this issue I am therefore extremely interested in Cyclic Analysis of Single-Cell Subsets and Tissue Territories (CASSATT) which identifies scanned slide images through multiple staining rounds, segments nuclei and assesses marker expression on each detected cell (Brockman et al., <span>2023</span>). Cyclic immunochemistry (cycIHC) exploits multiple rounds of immunostaining and imaging for mapping and locating cells of interest with the speed and simplicity of brightfield microscopy, making the collection of entire tissue sections and slides possible. CASSATT employs registered scanned slide images across all rounds of staining, segments individual nuclei, and measures marker expression on each detected cell. It further explores the spatial relationships between cell populations and the odds of interaction frequencies between cell populations within tissue regions, helping to identify cells that interact or do not interact. In this report, a test dataset of six glioblastoma tissue sections were cyclically stained for eight biomarkers for a total of 48 scanned slide images. The authors describe an efficient workflow that provided answers to questions commonly encountered in discovery research in tissue specimens, such as the overall abundance of cells of interest within a tissue section as well as whether groups of cells were in close proximity. CASSATT gathered together and streamlined all steps necessary to produce single cell expression information from cycIHC datasets and did so utilizing an open source environment. In addition, CASSATT systematically analyzed the spatial relationships between cell populations and via unsupervised algorithms, identified clusters of cell niches.</p><p>The persistence of measurable residual disease (MRD) is a strong indicator for adverse outcomes in acute myeloid leukemia (AML) and has been shown to be a valid surrogate marker for disease-free survival and overall survival, irrespective of patients' age, AML subtype, sample type, time of MRD assessment and MRD detection method (Short et al., <span>2020</span>). Other hematopoietic malignancies such as B- and plasma cell lineages also benefit from MRD diagnostic assays (Chen et al., <span>2023</span>; Gao et al., <span>2023</span>; McMillan et al., <span>2023</span>; Zhou et al., <span>2023</span>). In a study by Wang et al. (<span>2023</span>) th","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"104 5","pages":"341-343"},"PeriodicalIF":3.4,"publicationDate":"2023-10-10","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22145","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41182275","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}
Jesse M. Tettero, Naveen Dakappagari, Maaike E. Heidinga, Yvonne Oussoren-Brockhoff, Diana Hanekamp, Anil Pahuja, Kerri Burns, Pavinder Kaur, Zeni Alfonso, Vincent H. J. van der Velden, Jeroen G. te Marvelde, Willemijn Hobo, Jennichjen Slomp, Costa Bachas, Angele Kelder, Kevin Nguyen, Jacqueline Cloos
Background
Measurable residual disease (MRD) assessed by multiparametric flow cytometry (MFC) has gained importance in clinical decision-making for acute myeloid leukemia (AML) patients. However, complying with the recent In Vitro Diagnostic Regulations (IVDR) in Europe and Food and Drug Administration (FDA) guidance in the United States requires rigorous validation prior to their use in investigational clinical trials and diagnostics. Validating AML MRD-MFC assays poses challenges due to the unique underlying disease biology and paucity of patient specimens. In this study, we describe an experimental framework for validation that meets regulatory expectations.
Methods
Our validation efforts focused on evaluating assay accuracy, analytical specificity, analytical and functional sensitivity (limit of blank (LoB), detection (LLoD) and quantitation (LLoQ)), precision, linearity, sample/reagent stability and establishing the assay background frequencies.
Results
Correlation between different MFC methods was highly significant (r = 0.99 for %blasts and r = 0.93 for %LAIPs). The analysis of LAIP specificity accurately discriminated from negative control cells. The assay demonstrated a LoB of 0.03, LLoD of 0.04, and LLoQ of 0.1%. Precision experiments yielded highly reproducible results (Coefficient of Variation <20%). Stability experiments demonstrated reliable measurement of samples up to 96 h from collection. Furthermore, the reference range of LAIP frequencies in non-AML patients was below 0.1%, ranging from 0.0% to 0.04%.
Conclusion
In this manuscript, we present the validation of an AML MFC-MRD assay using BM/PB patient specimens, adhering to best practices. Our approach is expected to assist other laboratories in expediting their validation activities to fulfill recent health authority guidelines.
{"title":"Analytical assay validation for acute myeloid leukemia measurable residual disease assessment by multiparametric flow cytometry","authors":"Jesse M. Tettero, Naveen Dakappagari, Maaike E. Heidinga, Yvonne Oussoren-Brockhoff, Diana Hanekamp, Anil Pahuja, Kerri Burns, Pavinder Kaur, Zeni Alfonso, Vincent H. J. van der Velden, Jeroen G. te Marvelde, Willemijn Hobo, Jennichjen Slomp, Costa Bachas, Angele Kelder, Kevin Nguyen, Jacqueline Cloos","doi":"10.1002/cyto.b.22144","DOIUrl":"10.1002/cyto.b.22144","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>Measurable residual disease (MRD) assessed by multiparametric flow cytometry (MFC) has gained importance in clinical decision-making for acute myeloid leukemia (AML) patients. However, complying with the recent In Vitro Diagnostic Regulations (IVDR) in Europe and Food and Drug Administration (FDA) guidance in the United States requires rigorous validation prior to their use in investigational clinical trials and diagnostics. Validating AML MRD-MFC assays poses challenges due to the unique underlying disease biology and paucity of patient specimens. In this study, we describe an experimental framework for validation that meets regulatory expectations.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>Our validation efforts focused on evaluating assay accuracy, analytical specificity, analytical and functional sensitivity (limit of blank (LoB), detection (LLoD) and quantitation (LLoQ)), precision, linearity, sample/reagent stability and establishing the assay background frequencies.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>Correlation between different MFC methods was highly significant (<i>r</i> = 0.99 for %blasts and <i>r</i> = 0.93 for %LAIPs). The analysis of LAIP specificity accurately discriminated from negative control cells. The assay demonstrated a LoB of 0.03, LLoD of 0.04, and LLoQ of 0.1%. Precision experiments yielded highly reproducible results (Coefficient of Variation <20%). Stability experiments demonstrated reliable measurement of samples up to 96 h from collection. Furthermore, the reference range of LAIP frequencies in non-AML patients was below 0.1%, ranging from 0.0% to 0.04%.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusion</h3>\u0000 \u0000 <p>In this manuscript, we present the validation of an AML MFC-MRD assay using BM/PB patient specimens, adhering to best practices. Our approach is expected to assist other laboratories in expediting their validation activities to fulfill recent health authority guidelines.</p>\u0000 </section>\u0000 </div>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"104 6","pages":"426-439"},"PeriodicalIF":3.4,"publicationDate":"2023-09-28","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22144","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41113543","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}
Martijn W. C. Verbeek, Beatriz Soriano Rodríguez, Lukasz Sedek, Anna Laqua, Chiara Buracchi, Malicorne Buysse, Michaela Reiterová, Elen Oliveira, Daniela Morf, Sjoerd R. Oude Alink, Susana Barrena, Saskia Kohlscheen, Stefan Nierkens, Mattias Hofmans, Paula Fernandez, Elaine Sobral de Costa, Ester Mejstrikova, Tomasz Szczepanski, Lukasz Slota, Monika Brüggemann, Giuseppe Gaipa, Georgiana Grigore, Jacques J. M. van Dongen, Alberto Orfao, Vincent H. J. van der Velden
Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating & Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data.
{"title":"Minimal residual disease assessment in B-cell precursor acute lymphoblastic leukemia by semi-automated identification of normal hematopoietic cells: A EuroFlow study","authors":"Martijn W. C. Verbeek, Beatriz Soriano Rodríguez, Lukasz Sedek, Anna Laqua, Chiara Buracchi, Malicorne Buysse, Michaela Reiterová, Elen Oliveira, Daniela Morf, Sjoerd R. Oude Alink, Susana Barrena, Saskia Kohlscheen, Stefan Nierkens, Mattias Hofmans, Paula Fernandez, Elaine Sobral de Costa, Ester Mejstrikova, Tomasz Szczepanski, Lukasz Slota, Monika Brüggemann, Giuseppe Gaipa, Georgiana Grigore, Jacques J. M. van Dongen, Alberto Orfao, Vincent H. J. van der Velden","doi":"10.1002/cyto.b.22143","DOIUrl":"10.1002/cyto.b.22143","url":null,"abstract":"<p>Presence of minimal residual disease (MRD), detected by flow cytometry, is an important prognostic biomarker in the management of B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, data-analysis remains mainly expert-dependent. In this study, we designed and validated an Automated Gating & Identification (AGI) tool for MRD analysis in BCP-ALL patients using the two tubes of the EuroFlow 8-color MRD panel. The accuracy, repeatability, and reproducibility of the AGI tool was validated in a multicenter study using bone marrow follow-up samples from 174 BCP-ALL patients, stained with the EuroFlow BCP-ALL MRD panel. In these patients, MRD was assessed both by manual analysis and by AGI tool supported analysis. Comparison of MRD levels obtained between both approaches showed a concordance rate of 83%, with comparable concordances between MRD tubes (tube 1, 2 or both), treatment received (chemotherapy versus targeted therapy) and flow cytometers (FACSCanto versus FACSLyric). After review of discordant cases by additional experts, the concordance increased to 97%. Furthermore, the AGI tool showed excellent intra-expert concordance (100%) and good inter-expert concordance (90%). In addition to MRD levels, also percentages of normal cell populations showed excellent concordance between manual and AGI tool analysis. We conclude that the AGI tool may facilitate MRD analysis using the EuroFlow BCP-ALL MRD protocol and will contribute to a more standardized and objective MRD assessment. However, appropriate training is required for the correct analysis of MRD data.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"106 4","pages":"252-263"},"PeriodicalIF":2.3,"publicationDate":"2023-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22143","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"41118047","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}
Monocyte (m)HLA-DR expression appears to be a potent marker of immunosuppression in critically ill patients. The persistence of low mHLA-DR expression is associated with an increased risk of nosocomial infections and mortality. To adapt this measurement to pediatric requirements and provide extensive 24/7 access, we have developed a whole blood no-lyse no-wash micromethod (MM) and compared it with the standardized method (SM).
Methods
mHLA-DR was quantified by flow cytometry using Quantibrite™ Anti-HLA-DR PE/Monocyte PerCP-Cy™5.5 with either the SM performed in a diagnostic hematology laboratory using manufacturer protocol, or a whole blood no-lyse no-wash MM using an Attune flow cytometer located in the pediatric ICU. Median fluorescence intensity was measured in both techniques and converted to antibodies per cell (AB/C) calibrated with BD Quantibrite™ PE beads. Blood and Quantibrite™ reagent volume used with the MM was reduced by 5-fold compared to SM. In addition to Quantibrite™ Anti-Human HLA-DR PE/Monocyte PerCP-Cy™5.5, MM required anti-CD45 and anti-CD19 labeling.
Results
We determined the expression of mHLA-DR in 34 patients, 20 adults, and 14 children admitted to ICU. Correlation between MM and SM was excellent (Pearson's correlation: y = 0.8192x + 678.7, r = 0.9270, p < 0.0001). The estimated bias was 2467 ± 1.96 × 3307 AB/C; CI 95% [−4016; +8949].
Conclusions
The no-lyse no-wash whole blood microvolume method for measuring mHLA-DR expression allows for simplified sample preparation without compromising accuracy of the data. This method may simplify immune monitoring of critically ill patient by the deployment of a point of care method.
{"title":"Whole blood no-lyse no-wash micromethod for the quantitative measurement of monocyte HLA-DR","authors":"Jordi Miatello, Valérie Faivre, Clémence Marais, Mégane Raineau, Didier Payen, Pierre Tissieres","doi":"10.1002/cyto.b.22142","DOIUrl":"10.1002/cyto.b.22142","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>Monocyte (m)HLA-DR expression appears to be a potent marker of immunosuppression in critically ill patients. The persistence of low mHLA-DR expression is associated with an increased risk of nosocomial infections and mortality. To adapt this measurement to pediatric requirements and provide extensive 24/7 access, we have developed a whole blood no-lyse no-wash micromethod (MM) and compared it with the standardized method (SM).</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>mHLA-DR was quantified by flow cytometry using Quantibrite™ Anti-HLA-DR PE/Monocyte PerCP-Cy™5.5 with either the SM performed in a diagnostic hematology laboratory using manufacturer protocol, or a whole blood no-lyse no-wash MM using an Attune flow cytometer located in the pediatric ICU. Median fluorescence intensity was measured in both techniques and converted to antibodies per cell (AB/C) calibrated with BD Quantibrite™ PE beads. Blood and Quantibrite™ reagent volume used with the MM was reduced by 5-fold compared to SM. In addition to Quantibrite™ Anti-Human HLA-DR PE/Monocyte PerCP-Cy™5.5, MM required anti-CD45 and anti-CD19 labeling.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>We determined the expression of mHLA-DR in 34 patients, 20 adults, and 14 children admitted to ICU. Correlation between MM and SM was excellent (Pearson's correlation: <i>y</i> = 0.8192<i>x</i> + 678.7, <i>r</i> = 0.9270, <i>p</i> < 0.0001). The estimated bias was 2467 ± 1.96 × 3307 AB/C; CI 95% [−4016; +8949].</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>The no-lyse no-wash whole blood microvolume method for measuring mHLA-DR expression allows for simplified sample preparation without compromising accuracy of the data. This method may simplify immune monitoring of critically ill patient by the deployment of a point of care method.</p>\u0000 </section>\u0000 </div>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"106 1","pages":"58-63"},"PeriodicalIF":3.4,"publicationDate":"2023-09-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22142","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10227410","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}
扫码关注我们
求助内容：
应助结果提醒方式：
京公网安备 11010802042870号