Impedance-based Flow Cytometry Research Articles

Multiplex immunoassays using virus-tethered gold microspheres by DC impedance-based flow cytometry

Bead-based multiplex immunoassays for common use require enhanced sensitivity and effective prevention of non-specific adsorption, as well as miniaturization of the detection device. In this work, we have implemented virus-tethered gold microspheres for multiplex immunoassay applications, employing a DC impedance-based flow cytometer as a detection element. The advantages of virus-tethered gold microspheres, including excellent prevention of non-specific adsorption, are extended to signal enhancement arising from the large quantity of antibody loading on each virion, and to flexible movement of filamentous virus. Individual virus-tethered beads generate their own DC impedance and fluorescence signals, which are simultaneously detected by a chip-based microfluidic flow cytometer. This system successfully realized multiplex immunoassays involving four biomarkers: cardiac troponin I (cTnI), prostate specific antigen (PSA), creatine kinase MB (CK-MB), and myoglobin in undiluted human sera, elevating sensitivity by up to 5.7-fold compared to the beads without virus. Constructive integration between filamentous virus-tethered Au-layered microspheres and use of a microfluidic cytometer suggests a promising strategy for competitive multiplex immunoassay development based on suspension arrays.

Study on the kinetics and influence of feline platelet aggregation and deaggregation.

BackgroundFeline platelets are prone to clumping after blood collection, rendering the determination of accurate platelet counts difficult for clinical laboratories and resulting in a high incidence of pseudothrombocytopenia in feline haematology reports. No information is available about the kinetics of platelet aggregate formation in feline ethylenediaminetetraacetic acid blood and the course of platelet counts over a clinically relevant time period. The aim of the present study was to determine platelet counts in healthy cats over a time period of 24 h after blood collection at 9 time points; to assess potential effects of platelet aggregates, anaesthesia and bleeding conditions on feline platelets and white blood cell counts; and finally, to investigate if glucose concentration is associated with the presence of aggregates. From 30 clinically healthy cats, blood samples were analysed at 9 different time points using two different haematology instruments (using fluorescence and impedance-based flow cytometry) in the counting chamber and by blood smear evaluation.ResultsFourteen of the 30 samples were thrombocytopenic at one to 8 time points after collection as analysed on a fluorescence flow cytometry haematology analyser. At the 24-h timepoint, all thrombocytopenic samples had returned to normal platelet counts. Seventeen of the 30 samples showed platelet aggregates in the counting chamber. Significant differences in platelet counts were associated with the presence and size of aggregates and time since bleeding. No statistically significant differences in counts were found with regard to the quality of blood collection or the use of anaesthesia. Platelet aggregation and, therefore, pseudothrombocytopenia occurred in 57 % of the investigated samples at different time points.ConclusionFor the first time, deaggregation of feline platelet aggregates could be demonstrated as a reversible effect of platelet aggregation. For clinical laboratories or veterinarians, it may be helpful to rerun feline samples with pseudothrombocytopenia to obtain a more reliable platelet count. The quality of blood collection seems not to be causative for platelet aggregation. Blood smear evaluation is absolutely indicated in cases when haematology instruments give PLT counts below the reference interval.Electronic supplementary materialThe online version of this article (doi:10.1186/s12917-015-0590-7) contains supplementary material, which is available to authorized users.

Flow cytometric techniques for isolating and analysing leucocytes

Flow cytometric analysis and flow cytometric cell sorting (FACS) have revolutionized the understanding of immune cell biology and proved an indispensible tool not just for immunologists but for those interested in inflammatory processes. Right through from the early days of impedance-based flow cytometry counter devices to the first fluorescence-based flow cytometry instruments where side and forward scatter parameters with one or two florescent color instruments to state-of-the-art multicolour flow cytometric and mass cytometry with over 38-antibody panels, we are discovering new cell types and multiple immune cell phenotypes that exhibit a diverse array of functions. Today most laboratories use flow cytometry as a powerful technique to support their fundamental biological and medical research. Our own work has used flow cytometry to successfully measure mammalian (e.g., human and mouse) leucocyte function (e.g., cell polarization, reactive oxygen species release, shedding of CD62L, upregulation of CD11b, etc) and apoptosis (shedding of CD16 from neutrophils and fluorescently labeled annexin V binding to apoptotic cell surfaces, DNA hyplodiploid peak assessment of permeabilised cells, macrophage efferocytosis (phagocytosis of apoptotic cells and to sort highly pure non-perturbed neutrophils from autofluorescent eosinophils [1-3]. Furthermore, flow cytometry has been crucial in the identification, characterization and phenotypic analysis of inflammatory leucocytes isolated from healthy volunteers and patients [4] and from cells isolated from various inflammatory sites in mouse models of inflammation [5]. Recent advances in flow cytometry (especially in imaging flow cytometry) will undoubtedly provide further insights and new information in the already complex field of leucocyte biology.

Impedance flow cytometry gauges proliferative capacity by detecting TRPC1 expression

When examined, the expansion of many stem cell classes has been shown to be facilitated by mechanically-regulated calcium entry from the extracellular space that also helps direct their developmental programs towards mechanosensitive tissues such as muscle, bone, and connective tissues. Cation channels of the transient receptor potential C class (TRPC) are the predominant conduit for calcium entry into proliferating myoblasts. Nonetheless, methods to non-invasively study this calcium-entry pathway are still in their infancy. Here we show that a microfluidic configuration of impedance-based flow cytometry (IFC) provides a method to detect TRP channel expression in cells at high throughput. Using this technology we discern changes in the IFC signal that correlates with the functional expression of TRPC1 channels and coincides with cell proliferation. Pharmacological agents, mechanical conditions or malignant states that alter the expression of TRPC1 channels are reflected in the IFC signal accordingly, whereas pharmacological agents that alter cation-permeation through TRPC1 channels, or ionophores that independently increase calcium entry across the membrane, have little effect. Our results suggest that IFC detects changes in whole-cell membrane organization associated with TRPC1 activation and surface expression, rather than cation permeation through the channel per se. IFC-based technologies thus have the potential to identify living stem cells in their earliest stages of expansion without staining or chemical fixation.

Rosuvastatin Lowers Circulating Tissue Factor Antigen-Bearing Microparticles In Metastatic Breast Cancer: A Phase II, Multi-Dose, Randomized Trial (MicroSTAT Trial)

BackgroundElevated numbers of circulating bloodborne tissue factor antigen-bearing microparticles represent a significant risk factor for the development of venous thromboembolic events in cancer patients. We recently demonstrated the efficacy of low molecular weight heparin to prevent thrombosis in cancer patients with high levels of circulating tissue factor bearing microparticles. Enthusiasm for prophylactic anticoagulation in cancer cohorts is tempered by the requirement for daily self-injection and potential risk for hemorrhage. Statins significantly reduce the incidence of venous thromboembolic events in healthy individuals by an uncertain mechanism. Statins reduce the generation of tissue factor bearing microparticles in vitro and in animal models. Whether statins affect tissue factor bearing microparticles in cancer patients has not been previously assessed. We performed a randomized phase II study to evaluate whether rosuvastatin reduces the number of circulating tissue factor bearing microparticles in women with metastatic breast cancer. MethodsEligibility requirements included women with metastatic breast cancer on stable endocrine therapy (no changes within previous 6 weeks) with preserved bone marrow function. Patients were excluded if actively receiving chemotherapy (within prior 5 weeks) or currently taking statins. Baseline laboratory testing was performed at days 0 and 28 following enrollment. Study subjects were randomized to rosuvastatin 20 mg or 40 mg to be initiated from days 28 to 56. Repeat laboratory testing was performed on days 42 and 56 to assess for a reduction of tissue factor bearing microparticles and D-dimer. The number of circulating tissue factor bearing microparticles was measured by impedance-based flow cytometry and tissue factor activity was measured by a chromogenic Xa assay. Two-sided Wilcoxon signed rank test was used to compare the median levels at baseline and post treatment of laboratory endpoints within group, and Fisher exact test and Wilcoxon rank sum test to compare baseline characteristics between groups. ResultsA total of 19 women have been enrolled. Nine women were randomized to rosuvastatin 20 mg and ten were randomized to 40 mg. There were no significant differences between the two groups in baseline characteristics (e.g. age, body mass index, performance status, blood counts, renal function or number of tissue factor bearing microparticles). For the group randomized to rosuvastatin 20 mg, we observed no change in the number of tissue factor bearing microparticles (1576/μl vs 1888/μl , P= 0.55), microparticle-associated tissue factor activity, D-dimer or C-reactive protein (CRP), (P>0.05). In the group of women who received 40 mg rosuvastatin, the median decrease in the number of circulating tissue factor bearing microparticles per patient was -28%, with a median baseline value of 1763/μl versus a post-treatment value of 1216/μl (P=0.047). The group receiving 40mg rosuvastatin also demonstrated a non-significant median change in D-dimer of -21% (P=0.44), microparticle-associated tissue factor activity of -2% (P=0.84), and CRP of -10% (P= 0.81). ConclusionsIn a randomized phase II study in women with metastatic breast cancer, we observed a statistically significant 28% decrease in circulating tissue factor bearing microparticles following initiation of rosuvastatin 40 mg. Furthermore, these results emphasize that assay of tissue factor antigen-bearing microparticles does not correlate with assay of tissue factor activity. Based on the association between tissue factor-antigen bearing microparticles and cancer-associated thrombosis, these data provide additional support for a large scale clinical trial to assess the efficacy of statins to prevent thrombosis in cancer patients. Disclosures:Off Label Use: rosuvastatin to lower tissue factor bearing microparticles.

Abstract 8: Oxidized LDL Induces Macrophage Production of Prothrombotic Microparticles

Background: Microparticles (MP) are submicron vesicles produced by activated or apoptotic vascular cells. Agonist proteins borne by MPs enables them to function as mediators of inflammation and thrombosis. Atherosclerotic plaques contain high levels of MPs, and plasma MP levels increase during acute coronary syndromes. The thrombotic consequences of plaque rupture may involve macrophage (MΦ) MPs. The activation pathways that promote MΦMP production remain poorly defined, and this study therefore tested the hypothesis that signals implicated in atherogenesis stimulates MΦMP production. Methods and Results: We stimulated human primary MΦs with pro-inflammatory cytokines and atherogenic lipids, and assessed MΦMP production using light- and impedance-based flow cytometry. Oxidized LDL (oxLDL, 6-17 moles MDA/mg protein), but not IFN-γ, IL-1β, LPS or TNF, induced MΦMP production in a concentration- and time-dependent manner (293% increase, 16h peak, p<0.001). Non-oxidized LDL and minimally oxidized LDL components (POV-PC and PG-PC) did not augment MΦMP generation. oxLDL stimulation increased MΦMP tissue factor (TF) content by 78% (p<0.05), and oxLDL-induced MΦMP production correlated with activation of PI-3K/Akt signaling pathways. Inhibition of Akt signaling with the Akt inhibitor, KP372-1, reduced MΦMP release by 29% (p<0.01). Because statins inhibit inflammatory signaling that promotes MP production, we also examined the ability of mevastatin to reduce ox-LDL-stimulated MΦMP generation. Mevastatin treatment (10μM) reduced oxLDL-stimulated MΦMP production by 60% (p<0.01), MΦMP TF content by 43% (p<0.05), and attenuated oxLDL-stimulated Akt activation. Conclusion: oxLDL induces Akt-dependent production of prothrombotic MPs, a process inhibited by pretreatment with mevastatin. These findings link atherogenic signaling pathways, inflammation, and plaque thrombogenicity, and identify a novel potential mechanism for the LDL-independent antithrombotic effects of statins.

Use of immuno-magnetic beads for direct capture of nanosized microparticles from plasma

Increased microparticle tissue factor (TF) activity is not only found in cancer patients, but also in patients with cardiovascular and inflammatory diseases. Methods such as flow cytometry and impedance-based flow cytometry allow the analysis of microparticle subsets but provide no insight on which microparticles carry active TF. Conversely, the microparticle-TF activity itself does not reveal the cellular origin of the microparticles carrying the active TF.For this reason, we developed an immuno-magnetic bead method to capture subsets of microparticles directly from plasma. The method was optimized for capture of platelet-derived microparticles (PMPs) from plasma. Only 100 μl platelet-poor plasma (PPP) was needed in combination with 135 μl (27 μg) of biotinylated antihuman CD41 monoclonal antibody (MoAb) and 200 μl of streptavidin beads to achieve complete separation of PMPs from plasma. As a control, biotinylated mouse IgG1 isotype control MoAb was used instead of the anti-CD41 MoAb. Using biotinylated anti-CD14 MoAb, CD14-positive microparticles were captured from normal plasma spiked with microparticles isolated from the supernatant of lipopolysaccharide-stimulated monocytes (MoMPs). TF activity was found both in the positive (selected) and negative (depleted) fractions indicating that both CD14-positive and negative MoMPs carry active TF. We propose that this method can be used in the future to investigate the source of microparticles carrying active TF in plasma of patients with cancer and other diseases.

State-of-the-Art and Current Controversies in the Detection and Quantitation of Tissue Factor-Bearing Microparticles

Abstract SCI-11 Microparticles are submicron vesicles that circulate in blood and are derived from a variety of vascular cells. Microparticles expressing tissue factor have been shown in animal thrombosis models to contribute to thrombus formation in vivo, which has led to the evaluation of tissue factor bearing microparticle populations in a variety of thrombotic disorders including malignancy, sepsis, and coronary thrombosis. Central to the evaluation of tissue factor-bearing microparticle populations in health and disease is the establishment of accurate means for qualitative or quantitative assessment of circulating microparticles. Whilst there is considerable interest in evaluating whether pathologic disturbances in tissue factor-bearing microparticles contribute to thrombosis, analytic techniques for microparticle measurement remain in the validation stage. The method most commonly employed for the detection of tissue factor-bearing microparticles is light-scatter flow cytometry, which relies on the sizing of particles based on light-scatter profile. Light scatter, however, is also influenced by variables independent of particle size, including the wavelength of incident light, particle shape, presence of surface absorptive material, and relative refractive indices of particles and suspension medium. A number of potentially more sensitive methodologies are currently under investigation such as impedance-based flow cytometry, quasi-elastic light scattering based on Brownian movement, and atomic force microscopy. Alternatively, assays have been developed that measure tissue factor antigen or activity following isolation of microparticles from plasma or whole blood but without determination of particle size. Absent a gold standard for the measurement of tissue factor-bearing microparticle populations by which these approaches can be validated, the development of accurate methodology will require inter-assay and intra-assay precision as well as validation of clinical relevance in human studies. Disclosures: No relevant conflicts of interest to declare.

Impedance-Based Flow Cytometry for the Measurement of Microparticles

Over the last several years, there has been considerable interest in evaluating the biological relevance of alterations in blood-borne microparticle populations. The most commonly employed technique for the characterization of microparticles is light scatter flow cytometry. However, the enumeration and sizing of submicron particles based on light scattering properties can be problematic. Impedance-based flow cytometry based on the Coulter principle offers a sensitive methodology to characterize microparticles. This review details the rationale for employing impedance-based flow cytometry in the measurement of blood-borne microparticles.

Tumor-Derived Tissue FactorBearing Microparticles Are Associated With Venous Thromboembolic Events in Malignancy

Despite the strong association between malignant disease and thromboembolic disorders, the molecular and cellular basis of this relationship remains uncertain. We evaluated the hypothesis that tumor-derived tissue factor-bearing microparticles in plasma contribute to cancer-associated thrombosis. We developed impedance-based flow cytometry to detect, quantitate, and size microparticles in platelet-poor plasma. We evaluated the number of tissue factor-bearing microparticles in a cohort of cancer patients of different histologies (N = 96) and conducted a case-control study of 30 cancer patients diagnosed with an acute venous thromboembolic event (VTE) compared with 60 cancer patients of similar age, stage, sex, and diagnosis without known VTE, as well as 22 patients with an idiopathic VTE. Tissue factor-bearing microparticles were detected in patients with advanced malignancy, including two thirds of patients with pancreatic carcinoma. Elevated levels of tissue factor-bearing microparticles were associated VTE in cancer patients (adjusted odds ratio, 3.72; 95% confidence interval, 1.18-11.76; P = 0.01). In cancer patients without VTE, a retrospective analysis revealed a 1-year cumulative incidence of VTE of 34.8% in patients with tissue factor-bearing microparticles versus 0% in those without detectable tissue factor-bearing microparticles (Gray test P = 0.002).The median number of tissue factor-bearing microparticles in the cancer VTE cohort (7.1 x 10(4) microparticles/microL) was significantly greater than both the idiopathic VTE and cancer-no VTE groups (P = 0.002 and P = 0.03, respectively). Pancreatectomy in three patients eliminated or nearly eliminated these microparticles which coexpressed the epithelial tumor antigen, MUC-1. We conclude that tumor-derived tissue factor-bearing microparticles are associated with VTE in cancer patients and may be central to the pathogenesis of cancer-associated thrombosis.

Eculizumab Therapy Results in Rapid and Sustained Decreases in Markers of Thrombin Generation and Inflammation in Patients with PNH

Paroxysmal Nocturnal Hemoglobinuria (PNH) is a clonal disorder of the bone marrow resulting from an acquired mutation in the PIG-A gene. The mutation decreases production of the glycosylphosphatidylinositol membrane anchor for a variety of membrane proteins. Loss of cell membrane CD59 and CD55 results in enhanced complement-mediated cell membrane injury. PNH is associated with an increased risk of venous (VTE) and arterial thrombosis. Eculizumab, a monoclonal antibody to complement C5, has received FDA approval for the treatment of PNH. Recent published data demonstrates a 92% reduction in thrombotic events with the use of eculizumab. However, the mechanism for this reduction is unclear. We have enrolled eight PNH patients (pts) in an ongoing IRB-approved study on the effect of eculizumab treatment on markers of thrombin generation and inflammation. Patients were treated by the FDA-approved treatment protocol with blood samples obtained prior to treatment day 1 and prior to each dose on days 8, 15, 22, 29, 43 and 90. Patients receiving anticoagulants and corticosteroids were continued on their baseline medications. Plasma samples were assayed for D-dimers (D-D), thrombin-antithrombin complex (TAT), interleukin 6 (Il-6) by ELISA and tissue factor microparticles (TFMP) by impedance-based flow cytometry. Mean age of pts was 40.8 years (26–70); 6 male pts and 2 females. One patient had a prior history of VTE; 4 pts were receiving anticoagulants (1 full dose low molecular weight heparin (LMWH), 2 prophylactic LMWH, 1 warfarin) and 2 pts were receiving prednisone at the initiation of eculizumab. The effect of eculizumab on markers of hemostatic activation and inflammation was evaluated using Wilcoxon signed-rank test and multilevel models.Results: Pretreatment levels of D-D were significantly elevated in all but two of the patients who were receiving anticoagulants. Pretreatment Il-6 levels were significantly elevated in all but two patients taking prednisone. With eculizumab treatment, there was a statistically significant decrease in LDH (p=0.0001), D-D (p=0.0057), TAT (0.0138) and Il-6 (p=0.0362) during the 4 week induction phase of treatment (days 1–29). TAT levels significantly decreased by day 8 (p=0.008), with little subsequent change to day29 and day 90. All decreases in D-D, TAT, Il-6 and LDH were sustained in the maintenance phase of treatment (days29–90). Plasma TFMP were detectable and significantly increased in all patients prior to treatment. There was a statistically significant decrease in TFMP by day 8 (p=0.0234) and TFMP levels remained below pretreatment levels for the duration of the study (p=0.030). However, there were wide individual variations in TFMP levels over the course of treatment. There were significant Spearman correlations between changes in D-D and TAT (0.521; p<0.0001), in D-D and IL-6 (0.4400; p=0.0007). Changes in LDH did not correlate with changes in D-D, TAT, TFMP or Il-6. Changes in TFMP did not correlate with changes in markers of thrombin generation (TAT or D-D).Conclusion: Eculizumab treatment of patients with PNH results in a rapid decrease in plasma tissue factor microparticles, thrombin generation and inflammation. These changes appear to be independent of eculizumab suppression of RBC hemolysis as characterized by decreases in serum LDH. A direct relationship between plasma TFMP levels and thrombin generation in PNH patients could not be confirmed in this study. Taken together, these data indicate the broader impact of eculizumab treatment to suppress inflammation and prothrombotic activity in patients with PNH.

Development of Venous Thromboembolic Events in Cancer Patients with Elevated Levels of Tissue Factor-Bearing Microparticles

Cancer cells shed procoagulant vesicles containing tissue factor, and these tissue factor-bearing microparticles (TFMP) may play a role in thrombus formation in vivo. Using impedance-based flow cytometry to quantify microparticles and a high affinity monoclonal antibody specific for tissue factor, we previously demonstrated the presence of tissue factor-bearing microparticles in platelet-poor plasma in cancer patients. In this case control study, tissue factor-bearing microparticles represented a 4-fold risk factor for venous thromboembolic events (VTE) in cancer patients with acute VTE compared to age, stage, sex, diagnosis-matched controls with cancer but without acute VTE. To further assess the relationship between tissue factor-bearing microparticles and VTE, we performed a retrospective analysis of deep venous thrombosis or pulmonary emboli diagnosed in cancer patients initially enrolled without evidence of VTE. All radiographic reports for the cancer-no VTE group in the 2 years following enrollment were analyzed by a reviewer blinded to microparticle status. Only documented evidence of a new proximal extremity deep vein thrombosis or pulmonary embolism was included in the analysis. The TFMP and no-TFMP groups did not differ significantly for age, sex, active cancer treatment, smoking status, diabetes, or the presence of metastatic disease at time of enrollment. Sixteen of the 60 patients in this group had measurable tissue factor-bearing microparticles, 4 (4/16; 25%) of which subsequently developed radiographic evidence of VTE within 12 months of enrollment. No thrombotic events were recorded among the 44 patients without detectable tissue factor-bearing microparticles within the initial 12 months; however, one patient developed a pulmonary embolism 17 months following enrollment. Identifying death without VTE as a competing risk, the one-year estimate of the rate of VTE in cancer patients with detectable tissue factor-bearing microparticles was 34.8%; among the same group without detectable tissue factor-bearing microparticles, the 1-year rate was 0% (Log Rank p-value=0.002). The presence of tissue factor-bearing microparticles in cancer patients initially thrombosis-free predicted a 7-fold increased risk of thrombosis over cancer patients who were negative for tissue factor-bearing microparticles (OR 7.00, 95% CI 0.85–82.74, P=0.02). These tissue factor-bearing microparticles appear to be derived from the underlying malignancy since samples analyzed from patients with pancreatic cancer demonstrated co-expression of both tissue factor and MUC-1, a transmembrane glycoprotein overexpressed in epithelial malignancies. These data further support the role of tissue factor-bearing microparticles in the pathogenesis of cancer-associated thrombosis and as a biomarker for the prediction of cancer patients at risk of thrombosis. A prospective clinical study, currently being initiated, is required to evaluate this biomarker for the prediction of VTE risk in cancer patients and the utility of thromboprophylaxis in patients with elevated numbers of tissue factor-bearing microparticles.

Differential leukocyte count: manual or automated, what should it be?

Today's automated hematology analyzers capable of performing a full CBC and a differential leukocyte count (DLC) on whole blood, particularly in a closed tube system, using cytochemistry or impedance-based flow cytometry technology coupled with laser light scattering, conductivity and/or differential cell lysis, are here to stay. Their need and popularity among at least the large, cost and quality-conscious clinical laboratories have been growing for the past few years and will continue to do so in the years ahead. The efficiency and reliability of several of these analyzers in performing complete CBCD (CBC and DLC) and in flagging significant abnormalities have been tested and found acceptable with the need to review a stained blood smear or perform a manual DLC to confirm or obtain additional information on selected cases.