Ahmad Al-Attar, Kirthi R. Kumar, Dorian Untersee, Marci O'Driscoll, Miguel Francoise S. Ventura, Leo Lin
Automation in flow cytometry has recently advanced from the partial laboratory automation and robotics islets, to more fully integrated systems. This article reviews three manufacturers' newest sample preparation systems: the Beckman CellMek, the Sysmex PS-10, and the BD FACSDuet. These three instruments are capable of performing many of the manual steps in flow cytometry sample processing (pipetting, staining, lysing, washing, fixing). General description, capabilities, advantages, and disadvantages of each system are compared. Overall, these systems have the potential to become mainstay items in today's busy clinical flow cytometry laboratories, and save a significant amount of hands-on time for laboratory staff.
{"title":"Automation in flow cytometry: Guidelines and review of systems","authors":"Ahmad Al-Attar, Kirthi R. Kumar, Dorian Untersee, Marci O'Driscoll, Miguel Francoise S. Ventura, Leo Lin","doi":"10.1002/cyto.b.22125","DOIUrl":"10.1002/cyto.b.22125","url":null,"abstract":"<p>Automation in flow cytometry has recently advanced from the partial laboratory automation and robotics islets, to more fully integrated systems. This article reviews three manufacturers' newest sample preparation systems: the Beckman CellMek, the Sysmex PS-10, and the BD FACSDuet. These three instruments are capable of performing many of the manual steps in flow cytometry sample processing (pipetting, staining, lysing, washing, fixing). General description, capabilities, advantages, and disadvantages of each system are compared. Overall, these systems have the potential to become mainstay items in today's busy clinical flow cytometry laboratories, and save a significant amount of hands-on time for laboratory staff.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"106 4","pages":"308-320"},"PeriodicalIF":2.3,"publicationDate":"2023-05-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9453519","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>In the era of personalized medicine, the list of targeted therapeutic drugs has been greatly expanded, many of which are employed to treat hematolymphoid neoplasms. These include drugs specifically targeting immune checkpoint signaling epitopes such as programmed cell death 1 (PD1) and its ligand PDL1 to inhibit T-cell activation. Alternatively, these drugs might target the surface antigens expressed by tumor cells such as CD19 and CD22 in B-cell neoplasms. Of the latter, targeted immunotherapies include monoclonal antibodies with or without drug conjugates, bispecific T-cell engagers, or chimeric antigen receptor (CAR) T-cell therapy. The binding sites of these therapeutic antibodies are often identical or in proximity with the binding sites of the diagnostic antibodies. Therefore, use of these therapies pose great challenges for clinical cytometry labs in the assessment of post-treatment samples, especially in the detection of measurable/minimal residual disease (MRD). In this issue, Chen, Gao, et al. (<span>2023</span>) provided an overview of MRD detection in B-lymphoblastic leukemia/lymphoma in the era of immunotherapy, and Gao et al. (<span>2023</span>) focused their review on the impact of targeted therapy on mature B- and plasma cell neoplasms utilizing flow cytometry assessments. In both reviews, the authors illustrated the challenges, identified the problems, and provided a list of available options and solutions. For each of the above-mentioned disease categories, optimal gating and analysis strategies were illustrated with literature review and inputs from the authors' experience and insights. The utility and interpretation of additional B-cell markers other than CD19 and CD20 for mature or immature B-cell neoplasms such as CD22, CD24, and cCD79a (Mikhailova et al., <span>2022</span>) were studied as well as VS38, CD229, and CD319 (Pojero et al., <span>2016</span>; Soh et al., <span>2021</span>) for plasma cell neoplasms. The authors recommended that the flow cytometry assays in the era of targeted therapies must contain significant redundancy in the antibody panels allowing the detection of the cells of interest and additionally should be ready to utilize several gating strategies for accurate and consistent population identification.</p><p>Neoplastic mature B-cells often show restricted kappa or lambda light chain expression; however, light chain expression may be absent in around 5%–10% of mature B-cell lymphoma (Li et al., <span>2002</span>). It is suggested that mature B-cells lacking surface light chain expression can be used as a surrogate marker to diagnose mature B-cell lymphomas. Huang et al. (<span>2023</span>) reported a series of 89 cases of surface light chain negative B-cell lymphoma which consisted primarily of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Interestingly, the authors also reported no detectable light chain expression in normal/reactive mature B cells collected from body fluids an
在个体化医疗时代，靶向治疗药物的清单得到了极大的扩展，其中许多药物被用于治疗血淋巴肿瘤。这些药物包括专门针对免疫检查点信号表位的药物，如程序性细胞死亡1 (PD1)及其配体PDL1，以抑制t细胞活化。或者，这些药物可能靶向肿瘤细胞表达的表面抗原，如b细胞肿瘤中的CD19和CD22。在后者中，靶向免疫治疗包括单克隆抗体(含或不含药物偶联物)、双特异性t细胞接合物或嵌合抗原受体(CAR) t细胞治疗。这些治疗性抗体的结合位点通常与诊断性抗体的结合位点相同或接近。因此，使用这些疗法对临床细胞实验室在评估治疗后样本时提出了巨大的挑战，特别是在检测可测量/微小残留疾病(MRD)时。在这一期中，Chen、Gao等人(2023)概述了免疫治疗时代B淋巴母细胞白血病/淋巴瘤的MRD检测，Gao等人(2023)利用流式细胞术评估了靶向治疗对成熟B细胞和浆细胞肿瘤的影响。在这两篇综述中，作者都阐述了挑战，确定了问题，并提供了可用选项和解决方案的列表。对于上述每种疾病类别，通过文献综述和作者的经验和见解，说明了最佳的门控和分析策略。研究了除CD19和CD20以外的其他b细胞标志物(如CD22、CD24和cCD79a)在成熟或未成熟b细胞肿瘤中的应用和解释(Mikhailova等人，2022)，以及VS38、CD229和CD319 (Pojero等人，2016;Soh et al.， 2021)对浆细胞肿瘤的影响。作者建议，在靶向治疗时代，流式细胞术检测必须在抗体组中包含重要的冗余，以便检测感兴趣的细胞，此外还应准备好利用几种门控策略来准确和一致地进行群体识别。肿瘤成熟b细胞常表现出kappa或lambda轻链表达受限;然而，在大约5%-10%的成熟b细胞淋巴瘤中可能没有轻链表达(Li et al.， 2002)。提示缺乏表面轻链表达的成熟b细胞可作为诊断成熟b细胞淋巴瘤的替代标志物。Huang等(2023)报道了89例表面轻链阴性b细胞淋巴瘤，主要包括慢性淋巴细胞白血病/小淋巴细胞淋巴瘤(CLL/SLL)。有趣的是，作者还报告了从14例无临床和病理证据的淋巴瘤/白血病患者的体液和囊性液中收集的正常/反应性成熟B细胞中未检测到轻链表达。体液通常是蛋白质;因此，通常需要在kappa/lambda染色之前进行额外的洗涤，以获得足够的轻链染色，并且在解释从体液中收集的b细胞中的轻链表达时需要谨慎。众所周知，除慢性髓性白血病(CML)外(Soma et al.， 2016)，其他慢性髓性肿瘤，如骨髓增生异常综合征(MDS) (Xie et al.， 2019)和费城阴性骨髓增生性肿瘤(MPN)，淋巴母细胞危象极为罕见。Chan等人(2023)报道，在1262例MDS或ph阴性MPN中，有9例检测到低水平的异常b淋巴细胞，范围为0.012%至3.6%。细胞分选结合下一代测序表明，髓系肿瘤中异常的B淋巴母细胞通常(但并非总是)与髓系室克隆相关，分子遗传学结果表明，具有不同谱系输出的突变多能祖细胞可能由特定突变和突变发生的特定细胞阶段决定。重要的是，与CML不同，MDS和ph阴性mpn中存在异常b淋巴细胞并不一定表明母细胞危象，密切监测潜在髓系肿瘤的治疗被认为是一种合理的方法。这期的最后一篇文章(Chen, Zhang, et al.， 2023)研究了再生障碍性贫血(AA)中的粘膜相关不变T细胞(MAIT) (Godfrey et al.， 2019)，再生障碍性贫血是一种与异常免疫反应直接相关的骨髓衰竭。MAIT细胞表现出效应记忆表型，表达多种NK受体，如NKG2D和CD161，并在自身免疫性疾病的发展中发挥重要作用。作者发现再生障碍性贫血患者中具有激活和效应功能相关的细胞表面标记谱的MAIT细胞的频率增加。 NKG2D在MAIT细胞上表达的特异性提示这些细胞可能在AA免疫发病机制中具有独特的参与机制。
{"title":"Issue highlights—May 2023","authors":"Sa Wang MD","doi":"10.1002/cyto.b.22122","DOIUrl":"10.1002/cyto.b.22122","url":null,"abstract":"<p>In the era of personalized medicine, the list of targeted therapeutic drugs has been greatly expanded, many of which are employed to treat hematolymphoid neoplasms. These include drugs specifically targeting immune checkpoint signaling epitopes such as programmed cell death 1 (PD1) and its ligand PDL1 to inhibit T-cell activation. Alternatively, these drugs might target the surface antigens expressed by tumor cells such as CD19 and CD22 in B-cell neoplasms. Of the latter, targeted immunotherapies include monoclonal antibodies with or without drug conjugates, bispecific T-cell engagers, or chimeric antigen receptor (CAR) T-cell therapy. The binding sites of these therapeutic antibodies are often identical or in proximity with the binding sites of the diagnostic antibodies. Therefore, use of these therapies pose great challenges for clinical cytometry labs in the assessment of post-treatment samples, especially in the detection of measurable/minimal residual disease (MRD). In this issue, Chen, Gao, et al. (<span>2023</span>) provided an overview of MRD detection in B-lymphoblastic leukemia/lymphoma in the era of immunotherapy, and Gao et al. (<span>2023</span>) focused their review on the impact of targeted therapy on mature B- and plasma cell neoplasms utilizing flow cytometry assessments. In both reviews, the authors illustrated the challenges, identified the problems, and provided a list of available options and solutions. For each of the above-mentioned disease categories, optimal gating and analysis strategies were illustrated with literature review and inputs from the authors' experience and insights. The utility and interpretation of additional B-cell markers other than CD19 and CD20 for mature or immature B-cell neoplasms such as CD22, CD24, and cCD79a (Mikhailova et al., <span>2022</span>) were studied as well as VS38, CD229, and CD319 (Pojero et al., <span>2016</span>; Soh et al., <span>2021</span>) for plasma cell neoplasms. The authors recommended that the flow cytometry assays in the era of targeted therapies must contain significant redundancy in the antibody panels allowing the detection of the cells of interest and additionally should be ready to utilize several gating strategies for accurate and consistent population identification.</p><p>Neoplastic mature B-cells often show restricted kappa or lambda light chain expression; however, light chain expression may be absent in around 5%–10% of mature B-cell lymphoma (Li et al., <span>2002</span>). It is suggested that mature B-cells lacking surface light chain expression can be used as a surrogate marker to diagnose mature B-cell lymphomas. Huang et al. (<span>2023</span>) reported a series of 89 cases of surface light chain negative B-cell lymphoma which consisted primarily of chronic lymphocytic leukemia/small lymphocytic lymphoma (CLL/SLL). Interestingly, the authors also reported no detectable light chain expression in normal/reactive mature B cells collected from body fluids an","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"104 3","pages":"203-204"},"PeriodicalIF":3.4,"publicationDate":"2023-05-08","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22122","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9431290","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}
Central nervous system (CNS) involvement in extranodal NK/T cell lymphoma (ENKTCL) is rare and confers a dismal prognosis. Though newer treatment modalities like L-asparaginase based chemotherapy, immunotherapy, and hematopoietic stem cell transplant (HSCT) have yielded encouraging results, treatment failure with relapse of primary disease continues to be a major a concern (Yamaguchi et al., 2018). Herein, we report a rare phenomenon of isolated CNS relapse in a patient with ENKTCL within 3 months of allogenic HSCT subsequent to SMILE (dexamethasone, methotrexate, ifosfamide, L-asparaginase, and etoposide) and nivolumab induced disease remission. Our patient, a 51-year-old male, was a known case of ENKTCL with simultaneous involvement of both nasal and right testes 1 year back. Initial diagnosis was made on histopathology and EBER in situ hybridization was positive on tissue sections. No signs or symptoms of CNS involvement was present at initial diagnosis and CSF was clear of any disease. He had undergone right sided orchiectomy and received six cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) with gemcitabine and prophylactic intrathecal methotrexate. He experienced a relapse 6 months into therapy with a left testicular hypoechoic mass lesion and required left sided orchiectomy. Subsequently, he was treated with low dose nivolumab and SMILE regimen for six cycles and achieved remission. This was followed by allo-HSCT after a 10/10 HLA match with his blood brother. The initial 2 months post-transplant time period was uneventful and chimerism was 100%. On day 62 of post allo-HSCT, he presented to our outpatient department with complaints of headache since last 4 days, blurring of vision in the right eye and difficulty in chewing solid food. Physical examination revealed diminished distant vision in the right eye, weakness in the left side of face, altered taste sensation and dysarthria in speech. MRI of brain and orbits showed borderline enhancement of bilateral optic nerves at optic disc level along with right facial and trigeminal nerve enhancement. Laboratory investigation showed normal complete blood counts with no atypical cells on peripheral smear. CSF cell count on was 15 cells/μL and biochemistry showed high protein (138 mg/dL) and low sugar (26 mg/dL) levels. Meningitis panel through multiplex PCR for viral, bacterial, and parasitic infection was negative; however, the panel did not include EBV target. Though CSF cytology was suspicious, a definite opinion could not be furnished due to scant number of cells and in the absence of obvious atypia. But, flow cytometry immunophenotyping (FCMI) through stain-no lyse-no wash technique on CSF revealed an abnormal NK cell population with CD45 bright, surfaceCD3 , CD7 , CD56 bright+, CD16 , CD2dim+, CD4 , CD8 , CD5 (Figure 1a–e) confirming CNS relapse of the primary disease. PET scan did not show any other lesion elsewhere in the body and bone marrow examination was unre
{"title":"Extranodal NK/T-cell lymphoma with isolated central nervous system relapse: A defiant disease and the role of flow cytometry in monitoring","authors":"Devasis Panda, Amardeep Pathak, Narender Tejwani, Anurag Mehta","doi":"10.1002/cyto.b.22124","DOIUrl":"10.1002/cyto.b.22124","url":null,"abstract":"Central nervous system (CNS) involvement in extranodal NK/T cell lymphoma (ENKTCL) is rare and confers a dismal prognosis. Though newer treatment modalities like L-asparaginase based chemotherapy, immunotherapy, and hematopoietic stem cell transplant (HSCT) have yielded encouraging results, treatment failure with relapse of primary disease continues to be a major a concern (Yamaguchi et al., 2018). Herein, we report a rare phenomenon of isolated CNS relapse in a patient with ENKTCL within 3 months of allogenic HSCT subsequent to SMILE (dexamethasone, methotrexate, ifosfamide, L-asparaginase, and etoposide) and nivolumab induced disease remission. Our patient, a 51-year-old male, was a known case of ENKTCL with simultaneous involvement of both nasal and right testes 1 year back. Initial diagnosis was made on histopathology and EBER in situ hybridization was positive on tissue sections. No signs or symptoms of CNS involvement was present at initial diagnosis and CSF was clear of any disease. He had undergone right sided orchiectomy and received six cycles of CHOP (cyclophosphamide, doxorubicin, vincristine, and prednisone) with gemcitabine and prophylactic intrathecal methotrexate. He experienced a relapse 6 months into therapy with a left testicular hypoechoic mass lesion and required left sided orchiectomy. Subsequently, he was treated with low dose nivolumab and SMILE regimen for six cycles and achieved remission. This was followed by allo-HSCT after a 10/10 HLA match with his blood brother. The initial 2 months post-transplant time period was uneventful and chimerism was 100%. On day 62 of post allo-HSCT, he presented to our outpatient department with complaints of headache since last 4 days, blurring of vision in the right eye and difficulty in chewing solid food. Physical examination revealed diminished distant vision in the right eye, weakness in the left side of face, altered taste sensation and dysarthria in speech. MRI of brain and orbits showed borderline enhancement of bilateral optic nerves at optic disc level along with right facial and trigeminal nerve enhancement. Laboratory investigation showed normal complete blood counts with no atypical cells on peripheral smear. CSF cell count on was 15 cells/μL and biochemistry showed high protein (138 mg/dL) and low sugar (26 mg/dL) levels. Meningitis panel through multiplex PCR for viral, bacterial, and parasitic infection was negative; however, the panel did not include EBV target. Though CSF cytology was suspicious, a definite opinion could not be furnished due to scant number of cells and in the absence of obvious atypia. But, flow cytometry immunophenotyping (FCMI) through stain-no lyse-no wash technique on CSF revealed an abnormal NK cell population with CD45 bright, surfaceCD3 , CD7 , CD56 bright+, CD16 , CD2dim+, CD4 , CD8 , CD5 (Figure 1a–e) confirming CNS relapse of the primary disease. PET scan did not show any other lesion elsewhere in the body and bone marrow examination was unre","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"104 5","pages":"397-399"},"PeriodicalIF":3.4,"publicationDate":"2023-05-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9405267","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, Teri Oldaker, Kalpesh Shah, Andrea Illingworth
In the clinical laboratory, flow cytometry assays are critical to providing diagnostic and prognostic information to the treating clinicians. A validation or verification provides confidence that the assay will yield reliable results that can be trusted to make critical medical decisions. The following performance specifications should be included in a validation for laboratory developed tests as needed: accuracy (or trueness), precision (reproducibility and repeatability), detection capability, selectivity, reference range, and sample and reagent stability. We define these terms and present our approach to validation of several common flow cytometry assays, including examples of a leukemia/lymphoma assay and a paroxysmal nocturnal hemoglobinuria (PNH) assay.
{"title":"Summary of validation considerations with real-life examples using both qualitative and semiquantitative flow cytometry assays","authors":"Katherine A. Devitt, Teri Oldaker, Kalpesh Shah, Andrea Illingworth","doi":"10.1002/cyto.b.22123","DOIUrl":"10.1002/cyto.b.22123","url":null,"abstract":"<p>In the clinical laboratory, flow cytometry assays are critical to providing diagnostic and prognostic information to the treating clinicians. A validation or verification provides confidence that the assay will yield reliable results that can be trusted to make critical medical decisions. The following performance specifications should be included in a validation for laboratory developed tests as needed: accuracy (or trueness), precision (reproducibility and repeatability), detection capability, selectivity, reference range, and sample and reagent stability. We define these terms and present our approach to validation of several common flow cytometry assays, including examples of a leukemia/lymphoma assay and a paroxysmal nocturnal hemoglobinuria (PNH) assay.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"104 5","pages":"374-391"},"PeriodicalIF":3.4,"publicationDate":"2023-05-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9832512","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}
FIP1L1::PDGFRA rearranged myeloid neoplasms encompass a broad range of malignancies including chronic eosinophilic leukemia, MDS, MPN, MDS/MPN, AML and myeloid sarcoma. A little data are available pertaining to their antigen expression pattern till now due to low disease incidence. We hereby, describe a post-transplant case of FIP1L1::PDGFRA rearranged AML that had a typical extramedullary relapse with an additional RUNX1::CBFA2T2 fusion within the first year of post-transplant period and presented with a peculiar CD45 negative immunophenotype. A 25-year-old male patient of FIP1L1::PDGFRA rearranged AML, on follow-up day 280 of post allogenic hematopoietic stem cell transplant (allo-HSCT) presented with palpable left cervical lymphadenopathy and multiple subcutaneous nodules over chest, abdomen and bilateral lower limbs averaging from 0.5 to 1 cm in maximum dimen-sion. High-resolution computed tomography of thorax revealed bilateral pleural effusion and a solitary right lung upper lobe nodule measuring 1.0 (cid:1) 0.9 cm and multiple discrete mediastinal lymph nodes. Complete blood counts showed hemoglobin 11.5 g/dL, total leukocyte count 5.61 (cid:1) 10 3 / μ L, platelet count 110 (cid:1) 10 3 / μ L and no blast on peripheral blood (PB) differential leukocyte count. Bone marrow (BM) morphology was unremarkable and multicolor flow cytometry (MFC) measurable residual disease was negative. Neither PB nor BM showed any increase in eosinophil count. Liver function test showed
{"title":"Acquired RUNX1::CBFA2T2 fusion at extramedullary relapse in a patient of PDGFRA rearranged acute myeloid leukemia post allogenic HSCT","authors":"Devasis Panda, Amardeep Pathak, Narender Tejwani, Pooja Pandey, Anurag Mehta","doi":"10.1002/cyto.b.22121","DOIUrl":"10.1002/cyto.b.22121","url":null,"abstract":"FIP1L1::PDGFRA rearranged myeloid neoplasms encompass a broad range of malignancies including chronic eosinophilic leukemia, MDS, MPN, MDS/MPN, AML and myeloid sarcoma. A little data are available pertaining to their antigen expression pattern till now due to low disease incidence. We hereby, describe a post-transplant case of FIP1L1::PDGFRA rearranged AML that had a typical extramedullary relapse with an additional RUNX1::CBFA2T2 fusion within the first year of post-transplant period and presented with a peculiar CD45 negative immunophenotype. A 25-year-old male patient of FIP1L1::PDGFRA rearranged AML, on follow-up day 280 of post allogenic hematopoietic stem cell transplant (allo-HSCT) presented with palpable left cervical lymphadenopathy and multiple subcutaneous nodules over chest, abdomen and bilateral lower limbs averaging from 0.5 to 1 cm in maximum dimen-sion. High-resolution computed tomography of thorax revealed bilateral pleural effusion and a solitary right lung upper lobe nodule measuring 1.0 (cid:1) 0.9 cm and multiple discrete mediastinal lymph nodes. Complete blood counts showed hemoglobin 11.5 g/dL, total leukocyte count 5.61 (cid:1) 10 3 / μ L, platelet count 110 (cid:1) 10 3 / μ L and no blast on peripheral blood (PB) differential leukocyte count. Bone marrow (BM) morphology was unremarkable and multicolor flow cytometry (MFC) measurable residual disease was negative. Neither PB nor BM showed any increase in eosinophil count. Liver function test showed","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"104 5","pages":"404-406"},"PeriodicalIF":3.4,"publicationDate":"2023-04-13","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9343339","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}
Fan-Chi Hsu, Chad Hudson, Elisabeth R. Wilson, Laura M. Pardo, Timothy P. Singleton, Dongbin Xu, Barbara K. Zehentner, Johann Hitzler, Jason Berman, Denise A. Wells, Michael R. Loken, Lisa Eidenschink Brodersen
Background
Detection of measurable residual disease detection (MRD) by flow cytometry after the first course of chemotherapy is a standard measure of early response in patients with acute myeloid leukemia (AML). Myeloid leukemia associated with Down Syndrome (ML-DS) is a distinct form of AML. Differences in steady-state and regenerating hematopoiesis between patients with or without DS are not well understood. This understanding is essential to accurately determine the presence of residual leukemia in patients with ML-DS.
Methods
A standardized antibody panel defined quantitative antigen expression in 115 follow-up bone marrow (BM) aspirates from 45 patients following chemotherapy for ML-DS or DS precursor B-cell acute lymphoblastic leukemia (B-ALL-DS) with the “difference from normal (ΔN)” technique. When possible, FISH and SNP/CGH microarray studies were performed on sorted cell fractions.
Results
93% of BM specimens submitted post chemotherapy had a clearly identifiable CD34+CD56+ population present between 0.06% and 2.6% of total non-erythroid cells. An overlapping CD34+HLA-DRheterogeneous population was observed among 92% of patients at a lower frequency (0.04%–0.8% of total non-erythroid cells). In B-ALL-DS patients, the same CD34+CD56+ HLA-DRheterogeneous expression was observed. FACS-FISH/Array studies demonstrated no residual genetic clones in the DS-specific myeloid progenitor cells.
Conclusions
Non-malignant myeloid progenitors in the regenerating BM of patients who have undergone chemotherapy for either ML-DS or B-ALL-DS express an immunophenotype that is different from normal BM of non-DS patients. Awareness of this DS-specific non-malignant myeloid progenitor is essential to the interpretation of MRD by flow cytometry in patients with ML-DS.
{"title":"The impact of Down syndrome-specific non-malignant hematopoietic regeneration in the bone marrow on the detection of leukemic measurable residual disease","authors":"Fan-Chi Hsu, Chad Hudson, Elisabeth R. Wilson, Laura M. Pardo, Timothy P. Singleton, Dongbin Xu, Barbara K. Zehentner, Johann Hitzler, Jason Berman, Denise A. Wells, Michael R. Loken, Lisa Eidenschink Brodersen","doi":"10.1002/cyto.b.22118","DOIUrl":"10.1002/cyto.b.22118","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>Detection of measurable residual disease detection (MRD) by flow cytometry after the first course of chemotherapy is a standard measure of early response in patients with acute myeloid leukemia (AML). Myeloid leukemia associated with Down Syndrome (ML-DS) is a distinct form of AML. Differences in steady-state and regenerating hematopoiesis between patients with or without DS are not well understood. This understanding is essential to accurately determine the presence of residual leukemia in patients with ML-DS.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>A standardized antibody panel defined quantitative antigen expression in 115 follow-up bone marrow (BM) aspirates from 45 patients following chemotherapy for ML-DS or DS precursor B-cell acute lymphoblastic leukemia (B-ALL-DS) with the “difference from normal (Δ<i>N</i>)” technique. When possible, FISH and SNP/CGH microarray studies were performed on sorted cell fractions.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>93% of BM specimens submitted post chemotherapy had a clearly identifiable CD34<sup>+</sup>CD56<sup>+</sup> population present between 0.06% and 2.6% of total non-erythroid cells. An overlapping CD34<sup>+</sup>HLA-DR<sup>heterogeneous</sup> population was observed among 92% of patients at a lower frequency (0.04%–0.8% of total non-erythroid cells). In B-ALL-DS patients, the same CD34<sup>+</sup>CD56<sup>+</sup> HLA-DR<sup>heterogeneous</sup> expression was observed. FACS-FISH/Array studies demonstrated no residual genetic clones in the DS-specific myeloid progenitor cells.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>Non-malignant myeloid progenitors in the regenerating BM of patients who have undergone chemotherapy for either ML-DS or B-ALL-DS express an immunophenotype that is different from normal BM of non-DS patients. Awareness of this DS-specific non-malignant myeloid progenitor is essential to the interpretation of MRD by flow cytometry in patients with ML-DS.</p>\u0000 </section>\u0000 </div>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"104 4","pages":"311-318"},"PeriodicalIF":3.4,"publicationDate":"2023-04-04","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10033479","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}
Measurement of minimal/measurable residual disease (MRD) in B-lymphoblastic leukemia/lymphoma (B-ALL) has become a routine clinical evaluation tool and remains the strongest predictor of treatment outcome. In recent years, new targeted anti-CD19 and anti-CD22 antibody-based and cellular therapies have revolutionized the treatment of the high-risk B-ALL. The new treatments raise challenges for diagnostic flow cytometry, which relies on the presence of specific surface antigens to identify the population of interest. So far, reported flow cytometry-based assays are developed to either achieve a deeper MRD level or to accommodate the loss of surface antigens post-target therapies, but not both.
Methods
We developed a single tube flow cytometry assay (14-color-16-parameters). The method was validated using 94 clinical samples as well as spike-in and replicate experiments.
Results
The assay was well suited for monitoring response to targeted therapies and reached a sensitivity below 10−5 with acceptable precision (coefficient of variation < 20%), accuracy, and interobserver variability (κ = 1).
Conclusions
The assay allows for sensitive disease detection of B-ALL MRD independent of CD19 and CD22 expression and allows uniform analysis of samples regardless of anti-CD19 and CD22 therapy.
{"title":"Highly sensitive single tube B-lymphoblastic leukemia/lymphoma minimal/measurable residual disease test robust to surface antigen directed therapy","authors":"Qi Gao, Ying Liu, Umut Aypar, Jeeyeon Baik, Dory Londono, Xiaotian Sun, Jingping Zhang, Yanming Zhang, Mikhail Roshal","doi":"10.1002/cyto.b.22120","DOIUrl":"10.1002/cyto.b.22120","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>Measurement of minimal/measurable residual disease (MRD) in B-lymphoblastic leukemia/lymphoma (B-ALL) has become a routine clinical evaluation tool and remains the strongest predictor of treatment outcome. In recent years, new targeted anti-CD19 and anti-CD22 antibody-based and cellular therapies have revolutionized the treatment of the high-risk B-ALL. The new treatments raise challenges for diagnostic flow cytometry, which relies on the presence of specific surface antigens to identify the population of interest. So far, reported flow cytometry-based assays are developed to either achieve a deeper MRD level or to accommodate the loss of surface antigens post-target therapies, but not both.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>We developed a single tube flow cytometry assay (14-color-16-parameters). The method was validated using 94 clinical samples as well as spike-in and replicate experiments.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The assay was well suited for monitoring response to targeted therapies and reached a sensitivity below 10<sup>−5</sup> with acceptable precision (coefficient of variation < 20%), accuracy, and interobserver variability (<i>κ</i> = 1).</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusions</h3>\u0000 \u0000 <p>The assay allows for sensitive disease detection of B-ALL MRD independent of CD19 and CD22 expression and allows uniform analysis of samples regardless of anti-CD19 and CD22 therapy.</p>\u0000 </section>\u0000 </div>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"104 4","pages":"279-293"},"PeriodicalIF":3.4,"publicationDate":"2023-03-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22120","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"10392380","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}
Aren, M., Marce, S., Jurado, R., Tapia, G., Puigdefabregues, L., Raya, M., Cortes, M., Garcia-Caro, M., Junca, J., Mozas, P., Viñets, E., Cabezon, M., Plensa, E., Miljkovic, M., Sancho, J.-M., Navarro, J.-T., Zamora, L., & Sorigue, M. (2022). Flow cytometry to detect bone marrow involvement by follicular lymphoma. Cytometry Part B: Clinical Cytometry, 102(6), 427–439. https://doi.org/10.1002/cyto.b.22098
Coauthor Esther Plensa's affiliation was incorrectly given as Department of Hematology, ICO-Mataro, Badalona, Spain. The correct affiliation is Department of Hematology and Hemotherapy, Consorci Sanitari del Maresme, Hospital de Mataró, Institut Català d'Oncologia, Mataró, Spain.
{"title":"Correction to “Flow cytometry to detect bone marrow involvement by follicular lymphoma”","authors":"","doi":"10.1002/cyto.b.22111","DOIUrl":"10.1002/cyto.b.22111","url":null,"abstract":"<p>Aren, M., Marce, S., Jurado, R., Tapia, G., Puigdefabregues, L., Raya, M., Cortes, M., Garcia-Caro, M., Junca, J., Mozas, P., Viñets, E., Cabezon, M., Plensa, E., Miljkovic, M., Sancho, J.-M., Navarro, J.-T., Zamora, L., & Sorigue, M. (2022). Flow cytometry to detect bone marrow involvement by follicular lymphoma. <i>Cytometry Part B: Clinical Cytometry</i>, 102(6), 427–439. https://doi.org/10.1002/cyto.b.22098</p><p>Coauthor Esther Plensa's affiliation was incorrectly given as Department of Hematology, ICO-Mataro, Badalona, Spain. The correct affiliation is Department of Hematology and Hemotherapy, Consorci Sanitari del Maresme, Hospital de Mataró, Institut Català d'Oncologia, Mataró, Spain.</p><p>We apologize for this error.</p>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"104 2","pages":"195"},"PeriodicalIF":3.4,"publicationDate":"2023-03-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22111","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9193686","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}
Early T cell precursor-acute lymphoblastic leukemia (ETP-ALL) is a hematolymphoid malignancy where the blasts demonstrate T cell differentiation markers along with stem cell and myeloid antigen expression. The differential diagnosis of ETP-ALL from non-ETP ALL and mixed phenotype acute leukemia is often challenging due to its overlapping immunophenotypic picture with co-expression of myeloid antigens. In this study, we endeavored to describe the immune-phenotype profile of ETP-ALL in our patients and compared the utility of four different scoring systems for better discrimination of these entities.
Methods
This retrospective analysis included 31 ETP-ALL out of 860 acute leukemia cases consecutively diagnosed at the two tertiary care centers. Flowcytometry-based immunophenotype was reviewed for all the cases, and the utility of four flow-based objective scorings was assessed for the diagnosis of ETP-ALL. Receiver operating curves were drawn to compare the different flow-based scoring systems.
Results
The prevalence of ETP-ALL was 40% (n = 31/77 T-ALL) in our study group, comprised mainly of adults with a median age of 20 years. The five-marker scoring system had the maximum area under the curve, followed by the seven-marker scoring system. A cut-off of ≥2.5 was more specific (sensitivity: 91%; specificity: 100%), while a score of ≥1.5 was more sensitive but slightly less specific (sensitivity: 94%, specificity: 96%).
Conclusion
The WHO criteria for the diagnosis of ETP-ALL should be followed across all laboratories to avoid confusion and for better treatment stratification. Flow-based scoring systems can be objectively employed for better detection of cases.
{"title":"Comparison of flowcytometry-based scoring system for the diagnosis of early T precursor-acute lymphoblastic leukemia","authors":"Deepak Marballi Basavaraju, Shruti Mishra, Gaurav Chhabra, Sudarshan Chougule","doi":"10.1002/cyto.b.22119","DOIUrl":"10.1002/cyto.b.22119","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Background</h3>\u0000 \u0000 <p>Early T cell precursor-acute lymphoblastic leukemia (ETP-ALL) is a hematolymphoid malignancy where the blasts demonstrate T cell differentiation markers along with stem cell and myeloid antigen expression. The differential diagnosis of ETP-ALL from non-ETP ALL and mixed phenotype acute leukemia is often challenging due to its overlapping immunophenotypic picture with co-expression of myeloid antigens. In this study, we endeavored to describe the immune-phenotype profile of ETP-ALL in our patients and compared the utility of four different scoring systems for better discrimination of these entities.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>This retrospective analysis included 31 ETP-ALL out of 860 acute leukemia cases consecutively diagnosed at the two tertiary care centers. Flowcytometry-based immunophenotype was reviewed for all the cases, and the utility of four flow-based objective scorings was assessed for the diagnosis of ETP-ALL. Receiver operating curves were drawn to compare the different flow-based scoring systems.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The prevalence of ETP-ALL was 40% (<i>n</i> = 31/77 T-ALL) in our study group, comprised mainly of adults with a median age of 20 years. The five-marker scoring system had the maximum area under the curve, followed by the seven-marker scoring system. A cut-off of ≥2.5 was more specific (sensitivity: 91%; specificity: 100%), while a score of ≥1.5 was more sensitive but slightly less specific (sensitivity: 94%, specificity: 96%).</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Conclusion</h3>\u0000 \u0000 <p>The WHO criteria for the diagnosis of ETP-ALL should be followed across all laboratories to avoid confusion and for better treatment stratification. Flow-based scoring systems can be objectively employed for better detection of cases.</p>\u0000 </section>\u0000 </div>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"104 6","pages":"453-459"},"PeriodicalIF":3.4,"publicationDate":"2023-03-09","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9076006","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}
Paul Freund, Christopher M. Skopnik, Diana Metzke, Nina Goerlich, Jan Klocke, Emil Grothgar, Luka Prskalo, Falk Hiepe, Philipp Enghard
Introduction
Kidney diseases are a major health concern worldwide. Currently there is a large unmet need for novel biomarkers to non-invasively diagnose and monitor kidney diseases. Urinary cells are promising biomarkers and their analysis by flow cytometry has demonstrated its utility in diverse clinical settings. However, up to date this methodology depends on fresh samples, as cellular event counts and the signal-to-noise-ratio deter over time. Here we developed an easy-to-use two-step preservation method for conservation of urine samples for subsequent flow cytometry.
Methods
The protocol utilizes a combination of the formaldehyde releasing agent imidazolidinyl urea (IU) and MOPS buffer, leading to gentle fixation of urinary cells.
Results
The preservation method increases acceptable storing time of urine samples from several hours to up to 6 days. Cellular event counts and staining properties of cells remain comparable to fresh untreated samples.
Outlook
The hereby presented preservation method facilitates future investigations on flow cytometry of urinary cells as potential biomarkers and may enable broad implementation in clinical practice.
{"title":"Addition of formaldehyde releaser imidazolidinyl urea and MOPS buffer to urine samples enables delayed processing for flow cytometric analysis of urinary cells: A simple, two step conservation method of urinary cells for flow cytometry","authors":"Paul Freund, Christopher M. Skopnik, Diana Metzke, Nina Goerlich, Jan Klocke, Emil Grothgar, Luka Prskalo, Falk Hiepe, Philipp Enghard","doi":"10.1002/cyto.b.22117","DOIUrl":"10.1002/cyto.b.22117","url":null,"abstract":"<div>\u0000 \u0000 \u0000 <section>\u0000 \u0000 <h3> Introduction</h3>\u0000 \u0000 <p>Kidney diseases are a major health concern worldwide. Currently there is a large unmet need for novel biomarkers to non-invasively diagnose and monitor kidney diseases. Urinary cells are promising biomarkers and their analysis by flow cytometry has demonstrated its utility in diverse clinical settings. However, up to date this methodology depends on fresh samples, as cellular event counts and the signal-to-noise-ratio deter over time. Here we developed an easy-to-use two-step preservation method for conservation of urine samples for subsequent flow cytometry.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Methods</h3>\u0000 \u0000 <p>The protocol utilizes a combination of the formaldehyde releasing agent imidazolidinyl urea (IU) and MOPS buffer, leading to gentle fixation of urinary cells.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Results</h3>\u0000 \u0000 <p>The preservation method increases acceptable storing time of urine samples from several hours to up to 6 days. Cellular event counts and staining properties of cells remain comparable to fresh untreated samples.</p>\u0000 </section>\u0000 \u0000 <section>\u0000 \u0000 <h3> Outlook</h3>\u0000 \u0000 <p>The hereby presented preservation method facilitates future investigations on flow cytometry of urinary cells as potential biomarkers and may enable broad implementation in clinical practice.</p>\u0000 </section>\u0000 </div>","PeriodicalId":10883,"journal":{"name":"Cytometry Part B: Clinical Cytometry","volume":"104 6","pages":"417-425"},"PeriodicalIF":3.4,"publicationDate":"2023-03-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://onlinelibrary.wiley.com/doi/epdf/10.1002/cyto.b.22117","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"9413362","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}
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