Quality control in neutrophil granulocyte (PMN) concentrates by flow cytometry

2005 ◽  
Vol 43 (7) ◽  
Author(s):  
Uwe Schwanke ◽  
Laura Schrader ◽  
Rainer Moog
Keyword(s):  
Flow Cytometry ◽  
Quality Control ◽  
Oxidative Burst ◽  
Neutrophil Granulocyte ◽  
Polymorphonuclear Cells ◽  
Autologous Plasma ◽  
Function Tests ◽  
Healthy Donors ◽  
Migration Capacity

AbstractBackground: In peripheral blood, chemotaxis, phagocytosis, and oxidative burst of polymorphonuclear cells (PMNs) can be assessed by flow cytometry, whereas function tests, i.e., quality control in PMN concentrates designed for neutropenia therapy, are lacking.Methods: PMN concentrates (n=6) harvested from healthy donors who had been premedicated with granulocyte colony-stimulating factor (G-CSF) and dexamethasone were stored undiluted (control, C; n=6) and diluted 1:4 (D; n=6) with autologous plasma for 72h. Commercial flow cytometry function tests were performed to quantify changes in chemotaxis, phagocytosis, and oxidative burst of PMNs over time.Results: Median levels of phagocytosis and oxidative burst levelled at 86% (82–94) and 98% (83–100) in C on the day of apheresis, respectively, but deteriorated to 15% (0–24) and 0% within 72h; in D these parameters remained close to 90%. Median levels of chemotaxis were comparable in C (69%, 65–74) and D (74%, 70–84) at baseline. No migration was detected in C after 72h; however, D retained approximately 63% (13–76) migration capacity.Conclusion: Quality control in PMN concentrates is practical using flow cytometry and commercial test kits. While phagocytosis and oxidative burst may be maintained for 72h in vitro, chemotaxis of apheresed PMNs is already reduced on the day of apheresis.

Quality Control of Platelet Concentrates by the Thrombostat 4000

1995 ◽  
Vol 21 (S 02) ◽  
pp. 91-95 ◽  
Author(s):  
Markus Böck ◽  
Joachim Groh ◽  
Anle Glaser ◽  
Klaus Storck ◽  
Michael Kratzer ◽  
...  
Keyword(s):  
Quality Control ◽  
Platelet Function ◽  
Test System ◽  
Serotonin Release ◽  
Small Sample ◽  
Platelet Concentrates ◽  
Platelet Function Tests ◽  
Special Equipment ◽  
Function Tests

Quality control of platelet concentrates (PC) is an important prerequisite for good transfusion praxis. However, direct measurement of platelet function is complex, since available methods (e.g. aggregometry, serotonin release) are time consuming and require special equipment. Therefore a test system is needed, which is easy to handle, fast, and achieves reliable results. The present paper compares the results of conventional platelet function tests with those of a modified in-vitro bleeding test (IVBT) (Thrombostat 4000) in liquid-stored and cryopreserved PCs. A high correlation between aggregometry, serotonin release, GMP 140 expression upon stimulation, and IVBT was demonstrated. Therefore IVBT seems to be a good alternative to the conventional platelet function tests for quality control of PCs. In addition, a good correlation between the results of IVBT of patients’ blood after PC transfusion and IVBT of patients blood before transfusion supplemented with platelets of the respective PC could be found. Therefore IVBT seems to be able to predict PC transfusion success. However, since these data were obtained in a small sample undergoing bone marrow transplantation, further studies are needed to verify this hypothesis.

scholarly journals Sickling-induced binding of immunoglobulin to sickle erythrocytes

Blood ◽  
1988 ◽  
Vol 71 (3) ◽  
pp. 636-639 ◽  
Author(s):  
GA Green ◽  
VK Kalra
Keyword(s):  
Flow Cytometry ◽  
Gradient Centrifugation ◽  
Protein A ◽  
Fluorescein Isothiocyanate ◽  
Low Density ◽  
Autologous Plasma ◽  
Sickle Cells ◽  
Sickle Erythrocytes

Abstract Previously we demonstrated that sickle erythrocytes sedimenting at high densities after gradient centrifugation contain higher levels of surface immunoglobulin bound in vivo in comparison to low-density erythrocytes from the same patient. The present study examines the possibility that binding of autologous IgG to sickle erythrocytes may be associated with the sickling phenomenon. In the present study we subjected low-density erythrocytes to prolonged sickling under nitrogen in the presence of platelet-poor autologous plasma with added glucose for 24 hours (37 degrees C). After reoxygenation IgG bound in vitro was quantified by a nonequilibrium 125iodinated protein A-binding assay and by flow cytometry. Results show that sickle erythrocytes incubated under nitrogen bound significantly (P less than .001) more IgG, 439 +/- 41, molecules of IgG per cell (mean +/- SD) compared with sickle cells incubated under oxygenation (227 +/- 12 molecules of IgG per red cell) or compared with 196 +/- 26 molecules IgG per cell for untreated sickle cells. In contrast, normal erythrocytes incubated in autologous plasma exhibited no detectable IgG binding in vitro under either oxygenation or deoxygenation. Flow cytometry shows that deoxygenation of sickle cells generated a two-to-sixfold increase in the subpopulation of brightly fluorescent IgG-positive cells in comparison to oxygenated sickle cells and a 13.5% +/- 3.1% (mean +/- SD) increase in median fluorescence intensity for fluorescein isothiocyanate-labeled deoxygenated sickled cells compared with labeled oxygenated sickle cells. Our studies demonstrate that prolonged sickling will induce in vitro binding of autologous IgG to sickle erythrocytes. These findings indicate that sickle erythrocytes may be unique when compared with erythrocytes from other nonimmunologic hemolytic anemias or senescent red cells in that the primary events producing surface antigens recognized by autoantibody may include the sickling process. These findings also suggest that sickling in vivo may generate membrane alterations in sickle erythrocytes that lead to cumulative binding of autoantibody in vivo.

scholarly journals Control of the Oxidative Burst of Human Neutrophils by Staphylococcal Leukotoxins

2003 ◽  
Vol 71 (7) ◽  
pp. 3724-3729 ◽  
Author(s):  
Didier A. Colin ◽  
Henri Monteil
Keyword(s):  
Flow Cytometry ◽  
Oxidative Burst ◽  
Pore Formation ◽  
Human Neutrophils ◽  
Polymorphonuclear Cells ◽  
Phosphatidylinositol 3 Kinase ◽  
Intracellular Release ◽  
Two Component ◽  
Phosphatidylinositol 3

ABSTRACT The ability of staphylococcal two-component leukotoxins to induce an oxidative burst and/or to prime human polymorphonuclear cells (PMNs) was studied by using spectrofluorometry or flow cytometry. At sublytic concentrations, the HlgA-HlgB, HlgA-LukF-PV, LukS-PV-LukF-PV, and HlgC-LukF-PV combinations of leukotoxins, but not the LukS-PV-HlgB and HlgC-HlgB combinations, were able to induce H2O2 production similar to the H2O2 production induced by 1 μM N-formyl-Met-Leu-Phe (fMLP). In addition, when added at sublytic concentrations, all of the leukotoxin combinations primed PMNs for H2O2 production induced by fMLP. Leukotoxin activation was dependent on the presence of Ca2+ and was inhibited by wortmannin, an inhibitor of phosphatidylinositol 3-kinase, but not by N-methyl-l-arginine, an inhibitor of NO generation, which eliminates the possibility that NO plays a role in the action of leukotoxins. At higher concentrations, all leukotoxins inhibited H2O2 production by PMNs activated by fMLP, phorbol 12-myristate 13-acetate (PMA), or the leukotoxins themselves. This inhibition was not related to the pore formation induced by leukotoxins. Intracellular release of H2O2 induced by fMLP and PMA was not primed by leukotoxins but was inhibited. It seems that leukotoxin inhibition of H2O2 release is independent of pore formation but secondary to an intracellular event, as yet unknown, triggered by leukotoxins.

scholarly journals Comparison of Polymorphonuclear Cells from Healthy Donors and Differentiated HL-60 Cells as Phagocytes in an Opsonophagocytic Assay Using Antigen-Coated Fluorescent Beads

2000 ◽  
Vol 7 (2) ◽  
pp. 314-317 ◽  
Author(s):  
Bruno Guy ◽  
Catherine Testart ◽  
Sophie Gimenez ◽  
Violette Sanchez ◽  
Philippe Lheritier ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Streptococcus Pneumoniae ◽  
Polymorphonuclear Cells ◽  
Vaccine Candidates ◽  
Specific Antibodies ◽  
Fluorescent Beads ◽  
Healthy Donors ◽  
Latex Beads ◽  
The Mean

ABSTRACT Polymorphonuclear cells (PMNs) from healthy donors and differentiated HL-60 cells were compared in an opsonophagocytic assay using fluorescent latex beads coated with Streptococcus pneumoniae polysaccharide conjugates. Serum-specific phagocytosis was efficiently mediated by both sources of cells, as measured by flow cytometry, but the mean number of beads ingested per cell was three- to fivefold higher when PMNs were used than when HL-60 cells were used. Nevertheless, differentiated HL-60 cells could be a convenient and standardized source of cells to evaluate the functionality of specific antibodies to vaccine candidates as a coating on fluorescent beads.

scholarly journals Cross-Reactivity and Functionality of Approved Human Immune Checkpoint Blockers in Dogs

Cancers ◽  
2021 ◽  
Vol 13 (4) ◽  
pp. 785
Author(s):  
Stanislav Pantelyushin ◽  
Elisabeth Ranninger ◽  
Diego Guerrera ◽  
Gregor Hutter ◽  
Caroline Maake ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Mononuclear Cells ◽  
Cytokine Production ◽  
Disease Model ◽  
Cross Reactivity ◽  
Functional Responses ◽  
Peripheral Blood Mononuclear ◽  
Cancer Models ◽  
Healthy Donors

Background: Rodent cancer models have limitations in predicting efficacy, tolerability and accompanying biomarkers of ICIs in humans. Companion dogs suffering from neoplastic diseases have gained attention as a highly relevant translational disease model. Despite successful reports of PD-1/PD-L1 blockade in dogs, no compounds are available for veterinary medicine. Methods: Here, we assessed suitability of seven FDA-approved human ICIs to target CTLA-4 or PD-1/PD-L1 in dogs. Cross-reactivity and blocking potential was assessed using ELISA and flow cytometry. Functional responses were assessed on peripheral blood mononuclear cells (PBMCs) derived from healthy donors (n = 12) and cancer patient dogs (n = 27) as cytokine production after stimulation. Immune composition and target expression of healthy donors and cancer patients was assessed via flow cytometry. Results: Four candidates showed cross-reactivity and two blocked the interaction of canine PD-1 and PD-L1. Of those, only atezolizumab significantly increased cytokine production of healthy and patient derived PBMCs in vitro. Especially lymphoma patient PBMCs responded with increased cytokine production. In other types of cancer, response to atezolizumab appeared to correlate with a lower frequency of CD8 T cells. Conclusions: Cross-functionality of atezolizumab encourages reverse translational efforts using (combination) immunotherapies in companion dog tumor patients to benefit both veterinary and human medicine.

scholarly journals In Vitro Characterization of Peripheral Blood Progenitor Cell Differentiation and Platelet Function in Essential Thrombocythemia (ET)

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1877-1877
Author(s):  
Petros Papadopoulos ◽  
Carla AL Assaf ◽  
Sanne Smits ◽  
Laura Gutierrez ◽  
Els Lierman ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Peripheral Blood ◽  
Progenitor Cell ◽  
Triple Negative ◽  
Molecular Subtypes ◽  
Jak2 V617f ◽  
Aggregation Assay ◽  
Functional Differences ◽  
Healthy Donors

Abstract Bleeding and thrombotic events are major clinical complications of ET, together with progression to myelofibrosis and leukemic transformation (AML). The JAK2 V617F mutation, the thrombopoietin receptor mutation MPL W515K/L and the most recently discovered calreticulin (CALR) mutations are mutually exclusive in ET, account for up to 80-90 % of ET cases and support a novel molecular categorization of ET. However, the link between these driver mutations and ET pathogenesis and complications remains elusive. We have studied megakaryocyte (MK) differentiation of peripheral blood progenitor cells in vitro as well as the platelet aggregation capacity in ET patients. Progenitors cells were grown under standard conditions in serum-free media supplemented with stem cell factor (SCF), FLT3 ligand, erythropoietin (Epo) and thrombopoietin (TPO), and gradually were switched to TPO only media after almost a week of expansion of the hematopoietic progenitors. ET MKs are larger in size, they are more granular and they display higher ploidy levels when compared to healthy MKs. They also co-express the megakaryocytic and erythroid markers, i.e. CD41 and CD71 at higher levels suggesting aberrant lineage specification from multipotent progenitors that can give rise to both the megakaryocytic and erythroid lineage. Remarkably, ET peripheral blood progenitors have a prolonged survival in vitro under the tested culture conditions when compared to healthy MKs. Megakaryocytic cultures from healthy donors last for 12-14 days in vitro while the ET MK cultures can be maintained for up to two extra weeks. Electron microscopy analysis of cultured MKs is consistent with the morphological observations mentioned above regarding size and granularity, which is higher in ET MKs, and hence support aberrant differentiation pathways. However, no differences were found between the molecular subtypes of ET. Therefore, we performed transcriptional analysis by RNA sequencing of megakaryocytic and erythroid cultures of progenitors from JAK2 V617F ET, CALR mutant ET or triple negative ET in order to identify the molecular cues that characterize abnormal megakaryopoiesis in ET subgroups. We have analyzed three biological replicates per genotype and we are currently confirming the results by RT-PCR and functional assays in primary cells and cell lines. In parallel, we analyzed the aggregation capacity of JAK2 V617F, CALR mutant and triple negative ET platelets by a novel flow cytometry-based aggregation assay (De Cuyper IM et al. Blood 2013) in order to dissect potential signaling defects due to single receptor malfunction. We were able to pinpoint a significant defect in the response to collagen mediated by integrin a2b1 in ET platelets, regardless of the driver mutation, in comparison with platelets from healthy donors. However, convulxin-stimulated aggregation mediated by GPVI was normal. We also explored the degranulation capacity of ET platelets upon stimulation with thrombin peptide PAR-1. Platelets from CALR mutant or triple negative ET exhibited a reduced response as measured by the change of surface expression of the alpha granule protein CD62P compared to WT (healthy donors) and ET JAK2 V617F platelets. This was correlated with increased basal CD62P expression, suggesting that platelets in CALRmutant or triple negative ET degranulate spontaneously. In summary, we have developed in vitro assays to explore peripheral blood progenitor cell differentiation and platelet function in ET. Our findings indicate functional differences between JAK2 V617F ET as compared with CALR mutant or triple negative ET. The correlation between these functional differences and the clinical behavior of the molecular subtypes of ET needs to be further investigated. References Cuyper IM, Meinders M, van de Vijver E, de Korte D, Porcelijn L, de Haas M, Eble JA, Seeger K, Rutella S, Pagliara D, Kuijpers TW, Verhoeven AJ, van den Berg TK, Gutiérrez L. A novel flow cytometry-based platelet aggregation assay. Blood 2013 Mar 7;121(10):e70-80 Disclosures No relevant conflicts of interest to declare.

scholarly journals Simultaneous Measurement of Changes in Neutrophil Granulocyte Membrane Potential, Intracellular pH, and Cell Size by Multiparametric Flow Cytometry

Biomedicines ◽  
2021 ◽  
Vol 9 (11) ◽  
pp. 1504
Author(s):  
Alexander Elias Paul Stratmann ◽  
Lisa Wohlgemuth ◽  
Maike Elisabeth Erber ◽  
Stefan Bernhard ◽  
Stefan Hug ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Membrane Potential ◽  
Intracellular Ph ◽  
Defense Mechanisms ◽  
Neutrophil Granulocyte ◽  
Multiparametric Flow Cytometry ◽  
Proinflammatory Mediators ◽  
Inflammatory Conditions ◽  
Typical Sequence

Neutrophils provide rapid and efficient defense mechanisms against invading pathogens. Upon stimulation with proinflammatory mediators, including complement factors and bacterial peptides, neutrophils respond with cellular changes in their membrane potential, intracellular pH, and cellular size. This study provides an approach to quantify these changes simultaneously using multiparametric flow cytometry, thereby revealing a typical sequence of neutrophil activation consisting of depolarization, alkalization, and increase in cellular size. Additionally, the time resolution of the flow cytometric measurement is improved in order to allow changes that occur within seconds to be monitored, and thus to enhance the kinetic analysis of the neutrophil response. The method is appropriate for the reliable semiquantitative detection of small variations with respect to an increase, no change, and decrease in those parameters as demonstrated by the screening of various proinflammatory mediators. As a translational outlook, the findings are put into context in inflammatory conditions in vitro as well as in a clinically relevant whole blood model of endotoxemia. Taken together, the multiparametric analysis of neutrophil responsiveness regarding depolarization, alkalization, and changes in cellular size may contribute to a better understanding of neutrophils in health and disease, thus potentially yielding innovative mechanistic insights and possible novel diagnostic and/or prognostic approaches.

The Activation of Fas Receptor by APO010, a Recombinant Form of Fas Ligand, Induces In Vitro and In Vivo Antimyeloma Activity.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1515-1515 ◽  
Author(s):  
Enrique M. Ocio ◽  
Patricia Maiso ◽  
Mercedes Garayoa ◽  
Marc Dupuis ◽  
Atanasio Pandiella ◽  
...  
Keyword(s):  
Flow Cytometry ◽  
Cell Lines ◽  
Fas Ligand ◽  
Annexin V ◽  
Recombinant Form ◽  
Caspase 8 ◽  
Fas Receptor ◽  
Healthy Donors

Abstract Background & Aims Fas receptor is expressed on the surface of many malignant cells and its activation represents a potentially relevant anticancer target. APO010 is a recombinant form of Fas Ligand with hexameric structure, which is currently being evaluated in Phase 1 clinical trials. In order to identify possible targeted indications, we tested the in vitro and in vivo anti-tumor efficacy of APO010 on multiple myeloma (MM) cells. Material & methods In vitro cytotoxicity was tested by MTT and Annexin V staining in 8 MM cell lines and PBMCs from 3 healthy donors. Other techniques used for mechanistic studies were propidium iodide uptake by flow cytometry, Western-blotting, BrdU uptake and gene expression profile analysis. The in vivo antimyeloma effect of APO010 was tested in a xenograft of human plasmocytoma in CB17-SCID mice. When tumors became palpable mice were randomized to receive APO010 15 μg/Kg ip × 5d/sem (n=7), APO010 5 μg/Kg ip × 5d/sem (n=8) or vehicle alone (n=8). Tumor volumes, clinical features and weight were monitored three times a week. Results Six of the 8 MM cell lines studied by MTT were highly sensitive to APO010 with IC-50 at 24h of 0.5–20 ng/ml (2.5–100 pM), whereas two were resistant (RPMI-8226 and OPM-1). This sensitivity was correlated with the expression of Fas receptor by flow cytometry. Activation of apoptosis was rapid (within two hours of incubation) with maximum effect at 10 hours, as determined by Annexin V staining. Interestingly, APO010 was not toxic against PBMCs (both resting and activated) from 3 healthy donors at doses effective against MM cell lines. The presence of the microenvironment, as simulated by the coculture of MM1S cells with IL-6, IGF-1 and BMSCs, was not able to abrogate the APO010 antimyeloma effect. The combination of APO010 with Doxorubicin and Bortezomib, and, to a less extent, with Melphalan and Lenalidomide, potentiated the efficacy of the drugs alone. Regarding the mechanism of action, APO010 antiproliferative activity is mediated through caspase dependent apoptosis (Annexin-V staining, and PARP, caspase-3, caspase-7, caspase-8 and caspase-9 cleavage) and is independent of variations on the cell cycle profile. In this sense, the presence of the pan-caspase or caspase-8 inhibitors (Z-VAD-FMK and Z-IETD-FMK respectively) were able to completely abrogate APO010-induced cell death. Treatment of MM1S cells with APO010 for just one hour induced changes in the expression of 52 genes, many of them implicated in regulation of transduction (n=16). Three of the 4 most upregulated genes were the 3 members of the nuclear receptor subfamily 4, group A (Nurr1, Nor1 and Nur77). Other upregulated transcription factors were members of the Fos/Jun family such as Jun, JunB or FosL2. Western-Blot studies revealed that APO010 also provoked cleavage of MCL-1 and BIM, a decrease of BID and an important downregulation of pAKT. In the in vivo studies, APO010 treatment inhibited tumor growth as compared with the control group (p=0.02) without differences among the two doses of APO010. No significant toxicity was observed regarding body weight loss or increase in liver enzymes. Conclusions These data show that Fas activation with APO010 induces in vitro and in vivo cytotoxicity in MM cell lines, mainly through transcriptional regulation. This study provides an initial rationale for the use of this compound for treatment of MM patients.

The In Vitro Migration Capacity of Human Bone Marrow-Derived Mesenchymal Stem Cells in Response to Chemokines and Mesenchymal Growth Factors.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2315-2315
Author(s):  
Adriana Lopez ◽  
Emeline Marais ◽  
Nathalie Gallay ◽  
Olivier Herault ◽  
Bruno Delorme ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Flow Cytometry ◽  
Growth Factors ◽  
Positive Control ◽  
Negative Control ◽  
Differentiation Potential ◽  
Migratory Activity ◽  
Migration Capacity ◽  
High Level

Abstract Bone marrow (BM) mesenchymal stem cells (MSCs) are characterized by their wide differentiation potential into adipose, bone, cartilage, muscle, and neural tissues. This capacity makes MSCs potentially useful for cell therapy in case of tissue repair. The therapeutic efficacy of such cells depends on their capacity to migrate to damaged tissues in response to pro-migratory signals produced locally. In this study, we investigated the in vitro migration capacity of human BM MSCs (hMSCs) evaluating their response to several potential chemotactic factors, including chemokines and mesenchymal growth factors (MGF). BM cells obtained from patients undergoing orthopedic surgery, were cultured in alpha-MEM medium with 10% fetal calf serum (FCS). Non adherent cells were discarded after 2–3 days. Layers were passaged once or twice to deplete in hematopoietic cells. The three-lineal (adipogenic, osteoblastic, and chondrocytic) differentiation potential of CD45- cells was verified. hMSCs were then induced to migrate (37°C, 5% CO2 for 16 hours) through Transwells (8-μm pore filters), in response to purified chemotactic agents at optimal concentrations, as compared to that of medium with 30% FCS (positive control) and to that of medium alone (negative control). In parallel, the pattern of chemokine and growth factor receptor expression was assessed by flow cytometry and transcripts for cytokines, chemokines and receptors were quantified using Taqman Low Density Arrays® (RQ-PCR). Among MGF, the best pro-migratory activity was observed with PDGF-AB and IGF-1 (mean values of 64.9 and 37.9% of the positive control, respectively), while EGF and HGF activity was 20%–30% of the positive control and VEGF and FGF2 activity was comparable to that of the negative control (10%). Among chemokines, only MCP-1, Eotaxin-2 and MDC had significant activity (about 20 % of the control), while activity of RANTES and Eotaxin-1 was between 10 and 20% and that of SDF-1, Fractalkine, MIP-1alpha and GROalpha was below 10%. CCR1, CCR3, CCR4, CXCR4, PDGF-R, HGF-R, EGF-R and IGF1-R were expressed by hMSCs, as demonstrated by flow cytometry. RQ-PCR analysis indicated that some chemokines were expressed at high level (X-Y level of the GAPDH housekeeping gene), either constitutively (MCP-1, GROalpha), or after IL-1/TNF-alpha stimulation (RANTES, SDF-1), while others were expressed at intermediate (Fractalkine) or low (MIP-1alpha) level. Chemokine receptors were usually expressed at very low level (X-Y level of GAPDH), either constitutively (CCR3, CX3CR1) or only after stimulation (CCR1, CCR4, CXCR4 and CXCR5), while CCR2, CXCR-1 and CXCR-2 were not expressed at all. Transcripts for all MGF assayed in vitro and for their receptors were detected at intermediate/high level. In conclusion, mesenchymal growth factors appear to be better chemoattractants in vitro for BM hMSCs than chemokines. The reduced pro-migratory activity of exogenous chemokines might be related to the low level of their receptors, themselves downregulated by endogenous chemokines autocrinally produced.

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