Multicolor Flow Cytometry-based Quantification of Mitochondria and Lysosomes in T Cells

10.3791/58844 ◽  
2019 ◽  
Author(s):  
Chin-Wen Wei ◽  
Tyng-An Zhou ◽  
Ivan L. Dzhagalov ◽  
Chia-Lin Hsu
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Multicolor Flow Cytometry

scholarly journals Screening Donor Samples Using Multicolor Flow Cytometry Prior to Allogeneic Hematopoietic Stem Cell Transplantation Can Improve Disease Monitoring Post-Transplantation

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 39-40
Author(s):  
Hui Wang ◽  
Aixian Wang ◽  
Meiwei Gong ◽  
Junyi Zhen ◽  
Xueying Wu ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Nk Cells ◽  
Incidence Rate ◽  
Multicolor Flow Cytometry ◽  
Hematopoietic Stem ◽  
Abnormal Cells

Introduction Multicolor flow cytometry (MFC) has been frequently adopted as a method for minimal residual disease (MRD) detection, and is also a promising technique to detect post-transplant lymphoproliferative disorder. Some abnormal donor origin cells might be found when detecting MRD following an allogeneic hematopoietic stem cell transplantation (allo-HSCT). To minimize the effects from donor cells, using MFC prior to allo-HSCT to screen donor peripheral blood (PB) or bone marrow (BM) might be feasible. Methods We performed 3395 allo-HSCTs between January 2013 and December 2019 at Lu Daopei Hospital in Langfang, China. MRD was detected in recipients' BMs according to a conventional two-tube 8 or 9-color MFC panel. Abnormal cells were observed in BMs from three patients in complete remission (CR) one to four months post allo-HSCT. Abnormal neutrophils lacking CD16 expression were found in a patient with secondary acute myeloid leukemia (AML) that developed from a myelodysplastic/ myeloproliferative neoplasm (MDS/MPN). After ruling out MDS and paroxysmal nocturnal hemoglobinuria (PNH), we hypothesized that an Fcγ receptor IIIB (FcγRIIIB) gene deletion was the most likely reason. Abnormal natural killer (NK) cells were detected in the BM from an allo-HSCT recipient with T-cell acute lymphoblastic leukemia (ALL), and monoclonal B lymphocytosis (MBL) in allo-HSCT recipient with B-cell ALL. These three patinets' PBs were detected using MFC after the new finding to decide the cell origin. Besides, 4.54%(in WBC) CD4+ and CD8+ double positive T- cells which were monoclonal cells of the TCRVβ repertoire were detected in a PB sample from a donor prior to allo-HSCT. To evaluate the incidence rate The immunophenotypings were studied in the BMs from 79 NK lymphoma patients. Results Identical phenotypes were recognized in PBs obtained from the three respective donors. The fourth donor did not donate her cells for allo-HSCT, yet. The incidence rate of abnormal cells in donor samples was 0.1% (4/3395 cases), but this rate might be underestimated because MFC screening was not a routine procedure for donors. Additionally, only abnormal immunophenotyping related to patient diagnosis might have been found using an MRD panel as this panel only included markers related to diagnosis. Among general population, the incidence rate of suspicious FcγRIIIB deletion was 0.2% (11/5256 cases), the incidence rate of NK cells without CD2+ and homogeneously expressed CD159c was 0.05% (1/2000 cases) and none among the 79 NK lymphoma samples. The rate of MBL was 0.75% (15/2000 cases) and 1.36% in older than 40 years old people and the rate of monoclonal CD4/CD8 DP T-cells was 0.05% (1/2000 cases). All of these abnormal cells or polymorphism could be analyzed using a two tube MFC panel-- ckappa/clambda/(CD34)/CD19/ CD5/CD20/CD38 /CD45/CD56 and CD16/(CD117)/CD3/ CD4/CD5/ CD8/CD56/CD45/CD2. Conclusion Donor original abnormal cells or phenotypic polymorphisms could have an effect on MFC-based MRD or PTLD detection of recipients following allo-HSCT. These patients might be mis-diagnosed as being MRD positive or having PTLD if the technician lacks experience. To avoid mis-diagnosis and minimize the risk of allo-HSCT, it might be promising to utilize a suitable MFC panel to screen donor PB or BM samples prior to transplantation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Sézary Syndrome with CD4/CD8 Double-Negative Neoplastic T Cells in Peripheral Blood

2021 ◽  
Vol 2021 ◽  
pp. 1-5
Author(s):  
Yuki Kageyama ◽  
Kenshiro Tsuda ◽  
Yuma Nato ◽  
Keiki Nagaharu ◽  
Kazutaka Suzuki ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
T Cell ◽  
Peripheral Blood ◽  
Tumor Burden ◽  
Sézary Syndrome ◽  
Sezary Syndrome ◽  
Double Negative ◽  
Multicolor Flow Cytometry ◽  
Sézary Cells

Sézary syndrome is a rare leukemic type of cutaneous T-cell lymphoma characterized by the presence of neoplastic T cells with cerebriform nuclei (Sézary cells) in the skin, lymph nodes, and peripheral blood. Typical Sézary cells have a CD3+CD4+CD8– phenotype; however, in cases of the aberrant loss of antigens on Sézary cells, especially the loss of critically important T-cell antigens such as CD4, there is a possibility of misdiagnosing the disease or underestimating the tumor burden of the disease. Here, we report a rare case of Sézary syndrome with CD4/CD8 double-negative Sézary cells in the peripheral blood. Most of the Sézary cells in the peripheral blood had lost CD4 expression, and we diagnosed the disease and evaluated the tumor burden by multicolor flow cytometry. Intriguingly, the Sézary cells showed a typical CD4+CD8–CD7– phenotype in the skin even though the cells in the peripheral blood lacked CD4. The patient responded well to treatment with bexarotene and narrow-band ultraviolet B therapy. Analysis by multicolor flow cytometry is essential to diagnose this rare type of Sézary syndrome and evaluate the tumor burden.

OMIP-020: Phenotypic characterization of human γδ T-cells by multicolor flow cytometry

2014 ◽  
Vol 85 (6) ◽  
pp. 522-524 ◽  
Author(s):  
Kilian Wistuba-Hamprecht ◽  
Graham Pawelec ◽  
Evelyna Derhovanessian
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Γδ T Cells ◽  
Phenotypic Characterization ◽  
Multicolor Flow Cytometry

scholarly journals Assessment of chilling injury in hypothermic stored boar spermatozoa by multicolor flow cytometry

2021 ◽  
Author(s):  
Helen Jäkel ◽  
Heiko Henning ◽  
Anne‐Marie Luther ◽  
Karl Rohn ◽  
Dagmar Waberski
Keyword(s):  
Flow Cytometry ◽  
Chilling Injury ◽  
Multicolor Flow Cytometry ◽  
Boar Spermatozoa

scholarly journals Culture, expansion, and flow-cytometry-based functional analysis of pteropid bat MR1-restricted unconventional T cells

2021 ◽  
Vol 2 (2) ◽  
pp. 100487
Author(s):  
Wan Rong Sia ◽  
Ying Ying Hey ◽  
Randy Foo ◽  
Lin-Fa Wang ◽  
Edwin Leeansyah
Keyword(s):  
Flow Cytometry ◽  
Functional Analysis ◽  
T Cells

scholarly journals Evaluation of the Expression of CCR5 and CX3CR1 Receptors and Correlation with the Functionality of T Cells in Women infected with ZIKV during Pregnancy

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 191
Author(s):  
Débora Familiar-Macedo ◽  
Iury Amancio Paiva ◽  
Jessica Badolato-Corrêa da Silva ◽  
Fabiana Rabe de Carvalho ◽  
Helver Gonçalves Dias ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Clinical Outcome ◽  
Cell Responses ◽  
Functional Assays ◽  
Asymptomatic Children ◽  
Cd107a Expression ◽  
Congenital Zika Syndrome ◽  
Ifn Γ

There have been reports of neurological abnormalities associated with the Zika virus (ZIKV), such as congenital Zika syndrome (CZS) in children born to mothers infected during pregnancy. We investigated how the immune response to ZIKV during pregnancy is primed and conduct a thorough evaluation of the inflammatory and cytotoxic profiles as well as the expression of CCR5 and CX3CR1. We compared the reactivity of T cells to ZIKV peptides in convalescent mothers infected during pregnancy. The child’s clinical outcome (i.e., born with or without CZS) was taken to be the variable. The cells were stimulated in vitro with ZIKV peptides and evaluated using the ELISPOT and flow cytometry assays. After in vitro stimulation with ZIKV peptides, we observed a tendency toward a higher Interferon gamma (IFN-γ)-producing T cell responses in mothers who had asymptomatic children and a higher CD107a expression in T cells in mothers who had children with CZS. We found a higher frequency of T cells expressing CD107a+ and co-expressing CX3CR1+CCR5+, which is much clearer in the T cells of mothers who had CZS children. We suggest that this differential profile influenced the clinical outcome of babies. These data need to be further investigated, including the evaluation of other ZIKV peptides and markers and functional assays.

scholarly journals POS0776 CORRELATION OF PERIPHERAL CD4+GRANZB+CTLS WITH DISEASE SEVERITY IN PATIENTS WITH PRIMARY SJÖGREN’S SYNDROME

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 641.3-642
Author(s):  
Q. Wang ◽  
L. Gu ◽  
M. Zhang
Keyword(s):  
Flow Cytometry ◽  
T Cells ◽  
Sjögren’S Syndrome ◽  
Mhc Class Ii ◽  
Sjögren's Syndrome ◽  
Sjogren’S Syndrome ◽  
Sjogren's Syndrome ◽  
Healthy Controls ◽  
Extraglandular Manifestations ◽  
Sjögren‘S Syndrome

Background:Sjögren’s syndrome (SS) is a chronic autoimmune disorder. The major histopathologic lesion of it is a focal lymphocytic infiltrate around ductal and acinar epithelial cells, which include a majority of CD4+T. Several studies have shown that the epithelial cells in SS present diverse phenomena, such as MHC class II overexpression. CD4+T cells with cytotoxic activity (CD4 CTL) have been detected in various immune responses. They are characterized by their ability to secrete perforin and granzyme B to kill the target cells in an MHC class II-restricted fashion.Objectives:So this study was to investigate the correlation of peripheral CD4+GranzB+CTLs with disease severity and organ involvement in patients with primary Sjögren’s syndrome.Methods:We recruited 116 pSS patients and 46 healthy controls using flow cytometry to examine proportion of CD4+GranzB+CTLs in their peripheral blood, and immunofluorescence to test the expression of CD4+GranzB+CTLs in labial gland. The correlations of CD4+GranzB+CTLs and the relevant clinical data were analyzed.Results:We analyzed the percentage of CD4+GranzB+cytotoxic T cells in peripheral blood mononuclear cells (PBMCs) by flow cytometry. Frequency of peripheral CD4+GranzB+CTLs were measured in 116 patients with pSS and 46 healthy controls matched for age and sex. The percentage of CD4+GranzB+CTLs were significantly up-regulated in pSS patients than healthy controls (7.1%±4.9% vs 3.1%±1.9%, p <0.0001) and positive correlation with ESSDAI in pSS patients(r = 0.6332, p<0.001). The percentage of CD4+GranzB+CTLs were markedly higher in pSS patients with extraglandular manifestations. Moreover, CD4+GranzB+CTLs were observed in the lymphocytic foci and periductal areas of the LSGs and were elevated with increased foci index (FI). After excluding the other risk factors associated with pSS, CD4+GranzB+CTLs were still related to ESSDIA and extraglandular manifestations independently(p<0.05). ROC curve analysis indicated that the area under the curve (AUC) of CD4+GranzB+CTLs was 0.796 to predict the activity of pSS, and 0.851 to presume extraglandular manifestations. The best diagnostic cut-off point was 4.865 for pSS patients.Conclusion:In this study, We provide new evidence indicating involvement of CD4+GranzB+CTLs over activation in the disease pathophysiology of pSS, which may serve as a new biomarker to evaluate the activity and severity of pSS.References:[1]Takeuchi A, Saito T. Front Immunol. (2017) 23:194.[2]Brown DM, et al. Front Immunol. (2016) 9:93.[3]Polihronis M, et al. Clin Exp Immunol. (1998) 114:485-90.[4]Xanthou G, et al. Clin Exp Immunol. (1999) 118:154-63.[5]Maehara T, et al. Ann Rheum Dis. (2017) 76:377-385.[6]Goules AV, et al. Clin Immunol. (2017) 182:30-40.[7]Hashimoto K, et al. Proc Natl Acad Sci U S A. (2019) 116:24242-24251.[8]Croia C, et al. Arthritis Rheumatol. (2014) 66:2545-57.[9]Schmidt D,et al. J Clin Invest. (1996) 97:2027–37.[10]Pandya JM, et al. Arthritis Rheum. (2010) 62:3457–66.[11]Moosig F, et al. Clin Exp Immunol. (1998) 114:113–8.[12]Peeters LM, et al. Front Immunol. (2017) 20:1160.Table 1.Multivariate analysis of CD4+GranzB+CTLs influenced by pSS-related factorsregression coefficientstandard errort-statisticsp value95%CICD8+GranzB+CTLs(%)0.1440.0334.3346.9E-50.077, 0.211ESSDAI0.2560.1222.0950.0410.011, 0.502extraglandular manifestations2.6121.2682.0590.0450.065, 5.158Figure 1.Receiver operating characteristic (ROC) curve of the frequency of CD4+GranzB+CTLs to predict ESSDAI and extraglandular manifestations responseDisclosure of Interests:None declared

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