scholarly journals Cell-mediated cytotoxicity evaluation using monoclonal antibody staining for target or effector cells with annexinV/propidium iodide colabeling by fluorosphere-adjusted counts on three-color flow cytometry

2003 ◽  
Vol 56A (1) ◽  
pp. 53-60 ◽  
Author(s):  
Öner Özdemir ◽  
Yaddanapudi Ravindranath ◽  
Süreyya Savaşan
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Propidium Iodide ◽  
Color Flow ◽  
Effector Cells ◽  
Antibody Staining

scholarly journals Data-Driven Compensation for Flow Cytometry of Solid Tissues

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Nickolaas Maria van Rodijnen ◽  
Math Pieters ◽  
Sjack Hoop ◽  
Marius Nap
Keyword(s):  
Flow Cytometry ◽  
Propidium Iodide ◽  
Dna Content ◽  
Flow Cytometer ◽  
Data Driven ◽  
Color Flow ◽  
Operator Experience ◽  
Solid Tissues

Propidium Iodide is a fluorochrome that is used to measure the DNA content of individual cells, taken from solid tissues, with a flow cytometer. Compensation for spectral cross-over of this fluorochrome still leads to compensation results that are depending on operator experience. We present a data-driven compensation (DDC) algorithm that is designed to automatically compensate combined DNA phenotype flow cytometry acquisitions. The generated compensation values of the DDC algorithm are validated by comparison with manually determined compensation values. The results show that (1) compensation of two-color flow cytometry leads to comparable results using either manual compensation or the DDC method; (2) DDC can calculate sample-specific compensation trace lines; (3) the effects of two different approaches to calculate compensation values can be visualized within one sample. We conclude that the DDC algorithm contributes to the standardization of compensation for spectral cross-over in flow cytometry of solid tissues.

scholarly journals FRI0009 IMMUNO-PHENOTYPIC ANALYSIS OF PERIPHERAL BLOOD MONONUCLEAR CELLS IN RHEUMATOID ARTHRITIS PATIENTS TREATED WITH E6011, A HUMANIZED ANTI-FRACTALKINE MONOCLONAL ANTIBODY

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 575.2-576
Author(s):  
T. Yamada ◽  
J. Kakuta ◽  
E. Fusaoka-Nishioka ◽  
J. I. Ito ◽  
N. Yasuda ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
T Cells ◽  
Nk Cells ◽  
Peripheral Blood ◽  
Initial Treatment ◽  
Inadequate Response ◽  
Color Flow ◽  
Research Institute

Background:Fractalkine (FKN) and its solo receptor CX3CR1 are deeply involved in the pathogenesis of rheumatoid arthritis (RA). FKN is expressed on vascular endothelium, while CX3CR1 is expressed on peripheral blood leukocytes such as monocytes, macrophages, NK cells, effector CD8+T cells and a minor fraction of CD4+T cells. E6011, a novel humanized anti-FKN monoclonal antibody (mAb), is under clinical development in RA.Objectives:In order to continuously assess the E6011 pharmacodynamics by monitoring the alteration of peripheral blood immune cells, including CX3CR1-expressed cell populations, a series of multi-color flow cytometry (FCM) was conducted before and during the course of the E6011 treatment of active RA patients in phase 2 clinical trial.Methods:Immuno-phenotypic changes were explored by FCM during the E6011 administration in 190 Japanese RA patients with inadequate response to MTX (NCT02960438). Patient’s peripheral blood were drawn into fixative tube (Cyto-Chex® BCT, Streck) at each clinics and thereafter transported to the FCM facility at KAN Research Institute, Inc. within 30 hours after the blood collection to operate the FCM analysis by standardized method. Immuno-phenotyping was carried out by multi colors flow cytometry (BD FACSCantoIITM, BD LSRFortessaTM, BD Biosciences).Results:Based on these determined conditions, CX3CR1 expression on monocytes, NK cells and a part of CD8+and CD4+T cells were confirmed in this method. Interestingly, during the E6011 treatment, the proportion of CD16+monocytes, which highly express CX3CR1 within whole monocytes, were significantly decreased at 2 week after initial treatment from the baseline (E6011:p< 0.001, placebo:p> 0.48) and sustained up to 24 week, while that of CD16-monocytes were increased. The reduction of the frequency of CX3CR1+cells in NK cells, CD4+and CD8+T cells were not observed, but in some certain populations like CX3CR1-expressed CD56+CD16+NK cells and terminal differentiated effector CD8+T cells, the frequency of CX3CR1+cells in these populations tended to increase from the baseline at 2 week and kept increasing up to 24 week by the E6011 treatment.Conclusion:E6011 significantly decreased the proportion of CD16+monocytes in whole monocytes. Our results indicated that the reduction of CD16+monocytes after initial treatment might be a sensitive marker of E6011 in peripheral blood, possibly reflecting mechanism of action of E6011, since the CD16+monocytes highly express CX3CR1.References:[1]Tanaka Y, et al., Mod Rheumatol (2018) 28, 58-65Disclosure of Interests:Tomohiro Yamada Employee of: KAN Research Institute, Inc,, Jungo Kakuta Employee of: KAN Research Institute, Inc., Eri Fusaoka-Nishioka Employee of: KAN Research Institute, Inc., Jun-ichi Ito Employee of: EISAI, Nobuyuki Yasuda Employee of: KAN Research Institute, Inc., Tetsu Kawano: None declared, Toshio Imai: None declared

CD19 Targeting of Lymphoid Malignancies by Novel Fc-Domain Engineered Monoclonal Antibody.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3725-3725
Author(s):  
Farrukh Awan ◽  
Rosa Lapalombella ◽  
Rossana Trotta ◽  
Jonathan P Butchar ◽  
Bo Yu ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
B Cells ◽  
B Cell ◽  
Nk Cells ◽  
Nk Cell ◽  
Wild Type ◽  
Color Flow ◽  
Mechanistic Insight ◽  
Insight Into

Abstract Abstract 3725 Poster Board III-661 CD19 is a lineage-specific B-cell antigen, expressed at a high density on CLL cells, that contributes to B-cell receptor signaling but to date has not been effectively targeted with therapeutic monoclonal antibodies. XmAb5574 is a novel engineered anti-CD19 monoclonal antibody with a modified Fc-domain designed to enhance binding of FcγRIIIa that is predominately expressed on Natural Killer (NK)-cells. Utilizing freshly isolated chronic lymphocytic leukemia (CLL) patient B-cells we demonstrate that XmAb5574 lacks significant internalization seen with other anti-CD19 antibodies [maximum internalization for XmAb5574 was only 27.9% at 30-minutes (95%CI 14.5%, 41.4%)], thereby enhancing its ability to induce potent antibody-dependent cellular cytotoxicity (ADCC). Annexin V/PI flow cytometry analysis revealed that XmAb5574 mediates modest direct cytotoxicity not significantly different from Rituximab (0.6% increase, 95%CI -10.5%, 11.7%, p=0.91), and no complement mediated cytotoxicity (CDC) against primary CLL B-cells. Multi-color flow cytometry and monocyte derived macrophages (MDM) were used to assess XmAb5574 antibody dependent cellular phagocytosis (ADCP) against CLL cells and revealed no significant impact of the Fc-domain modification on MDM induced ADCP against CLL cells as compared to the wild type parental anti-CD19 antibody (12.37% vs. 10.51%, p=0.58). Interestingly, utilizing NK-cells and CLL cells isolated from normal donors and CLL patients, and employing autologous and allogeneic effector-target (E:T) conditions, XmAb5574 was found to mediate significantly higher ADCC when compared to the control humanized anti-CD19 non-engineered antibody (26.9% higher at E:T 25:1, p=0.0004 for allogeneic conditions, and 23.6% higher, p=0.004 for autologous conditions). ADCC mediated by XmAb5574 was also significantly higher as compared to Rituximab (33.5% higher at E:T 25:1, p<0.0001 for allogeneic conditions and 27.1% higher, p=0.004 for autologous conditions), a therapeutic antibody widely utilized in the treatment of CLL, hence confirming the functional in vitro efficacy and utility of the Fc-domain modification. By using inhibitor studies we further provide mechanistic insight into the XmAb5574–dependent ADCC mediated by NK-cells through a Granzyme B dependent mechanism. XmAb5574 also enhanced NK-cell activation as exhibited by an increased phosphorylation of Erk1/2 downstream of Fcγ receptor. The enhancement of subsequent cytolytic and secretory function was shown by the measurement CD107a up regulation on the surface of NK-cells (19.4% increase, p=0.005, as compared to wild type anti-CD19 antibody), and interferon-gamma release as measured by ELISA assays (6.4 times higher, p=0.007, as compared to wild type anti-CD19 antibody). Notably, enhanced NK-cell mediated ADCC observed with XmAb5574 against primary CLL B-cells could be augmented further by treatment with Lenalidomide (17.9% higher, p=0.04). These findings provide strong pre-clinical evidence for further clinical development of XmAb5574, both as monotherapy and in combination with Lenalidomide, for the therapy of CLL and related CD19+ B-cell malignancies. We also provide mechanistic insight into the utility and feasibility of Fc-domain engineering of specific antibodies, which will enhance their efficacy through an increased ability to recruit the innate immune system to more effectively control tumor progression. Disclosures: Desjarlais: Xencor: Employment.

Evidence for a systemic immune activation during depression: results of leukocyte enumeration by flow cytometry in conjunction with monoclonal antibody staining

1992 ◽  
Vol 22 (1) ◽  
pp. 45-53 ◽  
Author(s):  
M. Maes ◽  
J. Lambrechts ◽  
E. Bosmans ◽  
J. Jacobs ◽  
E. Suy ◽  
...  
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Immune Activation ◽  
Class Ii ◽  
Blast Transformation ◽  
T Helper ◽  
Systemic Immune Activation ◽  
Antibody Staining ◽  
Hla Dr ◽  
Class Ii Mhc

SYNOPSISSeveral studies have reported a suppressed immune function (e.g. blast transformation) during depression. In an attempt to define the cellular basis of the reported immune disorders, the present study investigates the leukocyte cell subset profile of minor, simple major, and melancholic depressives, versus normal controls. We have counted the number of white blood cells (WBC) lymphocytes, monocytes, and granulocytes, while the number of lymphocyte (sub)populations has been identified by phenotype, using monoclonal antibody staining in conjunction with flow cytometry. The following cell surface antigens were determined: CD3+ (pan T), CD19+ (pan B), CD4+ (T helper/inducer), CD8+ (T suppressor/cytotoxic), CD4+CD45RA (T-memory cells), CD4+CD45RA+ (T-virgin cells), surface Ig, class II MHC HLA-DR, and CD25+ (IL-2 receptor). By means of pattern recognition methods, we established distinct immunological changes in minor and simple major depressed and in melancholic patients, setting them apart from the reference population. Depression, per se, is characterized by a higher number of WBC, monocytes, class II MHC HLA-DR, and memory T cells. Minor and simple major depressives exhibited an increased T helper/suppressor ratio. Increased numbers of IL-2 receptor bearing cells are a hallmark for major depression. Melancholics showed an increased number of pan T, pan B and T suppressor/cytotoxic cells. It was concluded that the established immune cell profile of depressed patients may point towards the existence of a systemic immune activation during that illness.

POTENTIAL ROLE of 8-COLOR FLOW CYTOMETRY for CHARACTERIZATION of MDS with Del(5q) ABNORMALITIES

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 5020-5020
Author(s):  
Uta Oelschlaegel ◽  
Katja Sockel ◽  
Brigitte Mohr ◽  
Christian Thiede ◽  
Gerhard Ehninger ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Conflicts Of Interest ◽  
Residual Disease ◽  
Color Flow ◽  
Quantitative Expression ◽  
Antibody Staining ◽  
Minimal Residual Disease Monitoring ◽  
Healthy Donors ◽  
Lineage Infidelity ◽  
Diagnostic Panel

Abstract Abstract 5020 Four-colour based flow cytometry (FCM) of bone marrow (BM) samples has become a valuable tool in the diagnostics of patients (pts) with myelodysplastic syndrome (MDS). It is still unknown, whether aberrations detected by FCM differ between certain cytogenetic subgroups. By using an 8-color (5-tube) FCM diagnostic panel we aimed to investigate the antigen patterns of MDS pts with del(5q) abnormalities and compared these to MDS pts with non-del(5q) MDS. BM samples of healthy donors (n=19) and 57 MDS pts including 28 pts (15 RCMD; 3 RAEB-1; 8 RAEB-2; 1 RAEB-t; 1 CMMoL-1) with del(5q) MDS (single n=15) and 29 pts (1 RCUD; 2 RA±RS; 17 RCMD; 2 RAEB-2; 7 sAML including 1 RAEB-t) with non-del(5q) MDS were investigated. FCM procedure was performed at a FACS Canto II (200,000 events) and standardized including the analysis of all samples after overnight storage at room temperature and erythrocytes lysis prior to antibody staining. Thresholds were set according to median ± 2SD and/or ½ log differences compared to healthy BM. Aberrations were scored according to the FCSS by Wells et al. 2003 and modified by Van de Loosdrecht et al. 2008. In del(5q) MDS several phenotypic characteristics differed to non-del(5q) pts. Of note, lineage infidelity (CD2, CD5, CD7, CD19, CD56) was detectable in significantly fewer del(5q) pts compared to non-del(5q) MDS (in granulocytes: 8% vs. 33%, p=0.040 and in monocytes: 24% vs. 56%, p=0.027). Thus, especially aberrant CD56 expression contributed to these results with only small subpopulations being positive in del(5q) vs. often more than 50% of aberrant granulocytes/monocytes in non-del(5q) MDS pts. Additionally, the lymphoid-to-granulocytes/monocytes-ratios were higher in del(5q) (p=0.029 and 0.025) and hypogranularity was more pronounced in those pts (p=0.05). Intensity of expression of CD14 on monocytes as well as CD71 on granulocytes showed significant differences (2478 vs. 4402, p=0.035 and 2098 vs. 1552, p=0.007). Most of the above mentioned differences could also be confirmed if only RCMD pts of both groups were considered (e.g. lineage infidelity in granulocytes: 0% vs. 36%, p=0.041). Furthermore, trends were seen for a lower CD45-lymphocytes-to-blasts-ratio (4.8 vs. 5.4; p=0.09), higher CD33 intensity on blasts (2416 vs. 1673, p=0.08), and higher qualitative CD36 expression on granulocytes (3.2% vs. 2.5%, p=0.0619) as well as quantitative expression on monocytes (1940 vs. 663, p=0.06). Finally, monitoring of aberrations by FCM allowed for minimal residual disease monitoring in del(5q) MDS pts. Interestingly, the decreased CD19 expression on blasts seemed to be stable independent from the extent of treatment response. Eight-colour based FCM is a valuable tool in the diagnostics of MDS. It might also be able to distinguish del(5q) MDS from other MDS subtypes. Disclosures: No relevant conflicts of interest to declare.

Novel Methodology for the Detection of Cryptosporidium parvum: A Comparison of Cooled Charge Couple Devices (CCD) and Flow Cytometry

1993 ◽  
Vol 27 (3-4) ◽  
pp. 89-92 ◽  
Author(s):  
Andrew Campbell ◽  
Lucy Robertson ◽  
Huw Smith
Keyword(s):  
Monoclonal Antibody ◽  
Flow Cytometry ◽  
Cryptosporidium Parvum ◽  
Propidium Iodide ◽  
Three Dimensional ◽  
Flow Cytometer ◽  
Cryptosporidium Oocysts ◽  
Exact Size

Flow cytometry and CCD were assessed for their usefialness in the detection of oocysts of Cryptosporidium. Oocysts were labelled with FITC-monoclonal antibody and with the nuclear stains 4’6-diamidino-2-phenyl indole (DAPI) and propidium iodide (PI) before analysis by flow cytometer and CCD. Although the flow cytometer tested was able to concentrate particles and place them on a slide for subsequent viewing, readily sorting oocysts from contaminating debris, confirmation by fluoresence microscopy was still essential, even when additional parameters such as the inclusion of DAPI is used. Initial observations from the use of CCD, however, suggested that screening samples for oocysts was a possibility. Three dimensional visualisation of individual oocysts can map precisiely both the detailed morphology and the exact size of oocysts, thereby making confirmation by fluoresence microscopy unneccesary.

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