Berne Ferry

Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay.

INTRODUCTION Circulating cell-derived microparticles (MPs) have been implicated in several disease processes and elevated levels are found in many pathological conditions. The detection and accurate measurement of MPs, although attracting widespread interest, is hampered by a lack of standardisation. The aim of this study was to establish a reliable flow cytometric assay to measure distinct subtypes of MPs in disease and to identify any significant causes of variability in MP quantification. MATERIALS AND METHODS Circulating MPs within plasma were identified by their phenotype (platelet, endothelial, leukocyte and annexin-V positivity (AnnV+). The influence of key variables (i.e. time between venepuncture and centrifugation, washing steps, the number of centrifugation steps, freezing/long-term storage and temperature of thawing) on MP measurement were investigated. RESULTS Increasing time between venepuncture and centrifugation leads to increased MP levels. Washing samples results in decreased AnnV+MPs (P=0.002) and platelet-derived MPs (PMPs) (P=0.002). Double centrifugation of MPs prior to freezing decreases numbers of AnnV+MPs (P=0.0004) and PMPs (P=0.0004). A single freeze thaw cycle of samples led to an increase in AnnV+MPs (P=0.0020) and PMPs (P=0.0039). Long-term storage of MP samples at -80° resulted in decreased MP levels. CONCLUSIONS This study found that minor protocol changes significantly affected MP levels. This is one of the first studies attempting to standardise a method for obtaining and measuring circulating MPs. Standardisation will be essential for successful development of MP technologies, allowing direct comparison of results between studies and leading to a greater understanding of MPs in disease.

Effects of continuous positive airway pressure on systemic inflammation in patients with moderate to severe obstructive sleep apnoea: a randomised controlled trial

Background: Obstructive sleep apnoea syndrome (OSAS) has been associated with cardiovascular disease in epidemiological and observational studies. Continuous positive airway pressure (CPAP) is the treatment of choice for OSAS, but the impact of this intervention on systemic inflammation involved in the atherosclerotic process remains unclear. Methods: 100 men with moderate–severe OSAS were randomised to therapeutic (n = 51) or subtherapeutic (n = 49) CPAP treatment for 4 weeks to investigate the effects of active treatment on inflammatory markers such as highly sensitive C reactive protein (hsCRP), interleukin (IL)6, interferon γ (IFNγ) and anti-inflammatory adiponectin. Results: 4 weeks of therapeutic CPAP did not significantly change blood levels of hsCRP compared with the subtherapeutic control group (difference between median changes −0.24 mg/l (95% CI −0.88 to +0.24); p = 0.30). Plasma levels of IL6 and IFNγ did not change significantly following therapeutic compared with subtherapeutic CPAP (difference between median changes +0.52 and −0.07 pg/ml (95% CI −0.72 to +1.94 and −0.81 to +0.44); p = 0.45 and p = 0.82, respectively). Furthermore, 4 weeks of therapeutic CPAP did not significantly change levels of adiponectin in plasma compared with the subtherapeutic control group (difference between median changes +0.05 pg/ml (95% CI −0.36 to +0.47); p = 0.84). If patients with hsCRP values above 8 mg/l at baseline were excluded, differences between the changes in hsCRP, IL6, IFNγ and adiponectin after 4 weeks of CPAP were smaller, and again not statistically different between groups. Conclusions: 4 weeks of CPAP treatment has no beneficial effect on blood markers of inflammation and adiponectin in patients with moderate–severe obstructive sleep apnoea.

Measurement of peripheral B cell subpopulations in common variable immunodeficiency (CVID) using a whole blood method

Recent reports have described reduced populations of CD27+ memory B cells and increased percentages of undifferentiated B cells in peripheral blood of patients with common variable immunodeficiency (CVID). This work has prompted two attempts to classify CVID based on rapid flow cytometric quantification of peripheral blood memory B cells and immature B cells. Evidence to support the hypothesis that such in vitro B cell classification systems correlate with clinical subtypes of CVID is being sought. For the classification to be useful in routine diagnosis, it is important that the flow cytometric method can be used without prior separation of peripheral blood mononuclear cells (PBMC). We have examined 23 CVID patients and 24 controls, using both PBMC and whole blood, and find an excellent correlation between these methods. The reproducibility of the method was excellent. We classified the CVID patients by all three of the existing classifications, including secretion of immunoglobulin by B cells in vitro as described by Bryant, as well as the more recent flow cytometric classification methods. Only one patient changed classification as a result of using whole blood.

Circulating cell-derived microparticles in patients with minimally symptomatic obstructive sleep apnoea.

Moderate–severe obstructive sleep apnoea (OSA) has been associated with several pro-atherogenic mechanisms and increased cardiovascular risk, but it is not known if minimally symptomatic OSA has similar effects. Circulating cell-derived microparticles have been shown to have pro-inflammatory, pro-coagulant and endothelial function-impairing effects, as well as to predict subclinical atherosclerosis and cardiovascular risk. In 57 patients with minimally symptomatic OSA, and 15 closely matched control subjects without OSA, AnnexinV-positive, platelet-, leukocyte- and endothelial cell-derived microparticles were measured by flow cytometry. In patients with OSA, median (interquartile range) levels of AnnexinV-positive microparticles were significantly elevated compared with control subjects: 2,586 (1,566–3,964) μL−1 versus 1,206 (474–2,501) μL−1, respectively. Levels of platelet-derived and leukocyte-derived microparticles were also significantly higher in patients with OSA (2,267 (1,102–3,592) μL−1 and 20 (14–31) μL−1, respectively) compared with control subjects (925 (328–2,068) μL−1 and 15 (5–23) μL−1, respectively). Endothelial cell-derived microparticle levels were similar in patients with OSA compared with control subjects (13 (8–25) μL−1 versus 11 (6–17) μL−1). In patients with minimally symptomatic obstructive sleep apnoea, levels of AnnexinV-positive, platelet- and leukocyte-derived microparticles are elevated when compared with closely matched control subjects without obstructive sleep apnoea. These findings suggest that these patients may be at increased cardiovascular risk, despite being minimally symptomatic.

T cell phenotypes in patients with common variable immunodeficiency disorders: associations with clinical phenotypes in comparison with other groups with recurrent infections.

Common variable immunodeficiency disorders (CVID) are a group of heterogeneous conditions that have in common primary failure of B cell function, although numerous T cell abnormalities have been described, including reduced proliferative response and reduced regulatory T cells. This study compared the T cell phenotype of CVID patients subdivided into clinical phenotypes as well as patients with partial antibody deficiencies [immunoglobulin (Ig)G subclass deficiency and selective IgA deficiency], X‐linked agammaglobulinaemia (XLA) and healthy and disease controls. Absolute numbers of T cell subpopulations were measured by four‐colour flow cytometry: naive T cells, central and effector memory and terminally differentiated (TEM) T cells, using CD45RA and CCR7 expression. Early, intermediate and late differentiation status of T cells was measured by CD27/CD28 expression. Putative follicular T cells, recent thymic emigrants and regulatory T cells were also assessed. Significant reduction in naive CD4 T cells, with reduced total CD4 and recent thymic emigrant numbers, was observed in CVID patients, most pronounced in those with autoimmune cytopenias or polyclonal lymphoproliferation. These findings suggest a lack of replenishment by new thymically derived cells. CD8 naive T cells were reduced in CVID patients, most significantly in the autoimmune cytopenia subgroup. There was a reduction in early differentiated CD4 and CD8 T cells and increased CD8 TEM in the CVID patients, particularly autoimmune cytopenia and polyclonal lymphoproliferation subgroups, suggesting a more activated T cell phenotype, due perhaps to an antigen‐driven process. XLA patients had significantly reduced putative follicular T cells, which may depend on B cells for survival, while no significant alterations were observed in the T cells of those with IgG subclass deficiency or selective IgA deficiency.

Intracellular cytokine expression in whole blood preparations from normals and patients with atopic dermatitis.

In recent years, the importance of characterizing the role of cytokines in a wide range of clinical conditions has resulted in development of new methods to assess cytokine expression in clinical samples. The use of anti‐cytokine MoAbs and flow cytometry to detect cytokines intracellularly at the single‐cell level has the potential to quantify cytokine production in different diseases. For this technique to be useful in a clinical setting, rapid throughput of clinical samples and a cheap, reliable assay would be required, therefore the development of the above technique using unseparated whole blood samples would be advantageous. Using this technique, only one study to date (Maino et al., 1996) has used unseparated whole blood as the source of cells for detecting intracellular cytokines. In clinical practice, whole blood may be optimal, since this most closely approximates conditions in vivo: as no purification of blood mononuclear cells is required, very little blood is needed to detect a number of cytokines simultaneously in various lymphocyte subpopulations, and the assay can be applied to samples from infants and children. In this study we describe an intracellular cytokine assay using unseparated whole blood from normals. In activated CD8− T cells, IL‐2 and interferon‐gamma (IFN‐γ) were optimally induced after 10 h stimulation with phorbol 12‐myristate acetate (PMA)/ionomycin, and in CD8+ T cells IL‐2 was optimally induced after 10 h and IFN‐γ after 6 h. The levels of IL‐2 and IFN‐γ in CD8+ and CD8− T cells in four healthy individuals were consistent on four occasions over a 3‐month period. In a large group of 34 normal subjects, there was considerable heterogeneity in CD3/IL‐2+ (range 9.7–41.3) and CD3/IFN‐γ+ cells (10.1–44), expressed as a percentage of total lymphocytes. In patients with atopic dermatitis (n = 5) there was a significantly decreased percentage of CD3+/CD8+ peripheral blood T cells expressing IFN‐γ and an increased percentage of CD3+/CD8− T cells expressing IL‐4 compared with non‐atopic dermatitis controls (n = 5). Possible applications of this technique are discussed.

Dynamic Release and Clearance of Circulating Microparticles During Cardiac Stress

Rationale: Microparticles are cell-derived membrane vesicles, relevant to a range of biological responses and known to be elevated in cardiovascular disease. Objective: To investigate microparticle release during cardiac stress and how this response differs in those with vascular disease. Methods and Results: We measured a comprehensive panel of circulating cell-derived microparticles by a standardized flow cytometric protocol in 119 patients referred for stress echocardiography. Procoagulant, platelet, erythrocyte, and endothelial but not leukocyte, granulocyte, or monocyte-derived microparticles were elevated immediately after a standardized dobutamine stress echocardiogram and decreased after 1 hour. Twenty-five patients developed stress-induced wall motion abnormalities suggestive of myocardial ischemia. They had similar baseline microparticle levels to those who did not develop ischemia, but, interestingly, their microparticle levels did not change during stress. Furthermore, no stress-induced increase was observed in those without inducible ischemia but with a history of vascular disease. Fourteen patients subsequently underwent coronary angiography. A microparticle rise during stress echocardiography had occurred only in those with normal coronary arteries. Conclusions: Procoagulant, platelet, erythrocyte, and endothelial microparticles are released during cardiac stress and then clear from the circulation during the next hour. This stress-induced rise seems to be a normal physiological response that is diminished in those with vascular disease.

A rapid flow cytometric assay to detect CD4+ and CD8+ T‐helper (Th) 0, Th1 and Th2 cells in whole blood and its application to study cytokine levels in atopic dermatitis before and after cyclosporin therapy

Background  The immune response in atopic dermatitis (AD) is thought to be driven by T‐helper (Th) 2 cytokines. Using flow cytometry, higher frequencies of peripheral blood CD4+ and CD8+ T cells producing interleukin (IL)‐4 and correspondingly lower frequencies of CD4+ T cells producing interferon (IFN)‐γ have been found in patients with AD compared with healthy controls. It would be of interest to know whether other Th1 and Th2 cytokines such as IL‐5, IL‐13 and tumour necrosis factor (TNF)‐α are similarly skewed in patients with AD and whether this immune skewing, detected via a simple blood assay, can be correlated with other clinical measurements or treatments in AD.

Prevalence and clinical characteristics of serum neuronal cell surface antibodies in first-episode psychosis: a case-control study

BACKGROUND Psychosis is a common presenting feature in antibody-mediated encephalitis, for which prompt recognition and treatment usually leads to remission. We aimed to investigate whether people with circumscribed schizophrenia-like illnesses have such antibodies-especially antibodies against the N-methyl-D-aspartate receptor (NMDAR)-more commonly than do healthy controls. METHODS We recruited patients aged 14-35 years presenting to any of 35 mental health services sites across England with first-episode psychosis, less than 6 weeks of treatment with antipsychotic medication, and a score of 4 or more on at least one selected Positive and Negative Syndrome Scale (PANSS) item. Patients and controls provided venous blood samples. We completed standardised symptom rating scales (PANSS, ACE-III, GAF) at baseline, and tested serum samples for antibodies against NMDAR, LGI1, CASPR2, the GABAA receptor, and the AMPA receptor using live cell-based assays. Treating clinicians assessed outcomes of ICD diagnosis and functioning (GAF) at 6 months. We included healthy controls from the general population, recruited as part of another study in Cambridge, UK. FINDINGS Between Feb 1, 2013, and Aug 31, 2014, we enrolled 228 patients with first-episode psychosis and 105 healthy controls. 20 (9%) of 228 patients had serum antibodies against one or more of the neuronal cell surface antibodies compared with four (4%) of 105 controls (unadjusted odds ratio 2·4, 95% CI 0·8-7·3). These associations remained non-significant when adjusted for current cigarette smoking, alcohol consumption, and illicit drug use. Seven (3%) patients had NMDAR antibodies compared with no controls (p=0·0204). The other antibodies did not differ between groups. Antibody-positive patients had lower PANSS positive, PANSS total, and catatonia scores than did antibody-negative patients. Patients had comparable scores on other PANSS items, ACE-III, and GAF at baseline, with no difference in outcomes at 6 months. INTERPRETATION Some patients with first-episode psychosis had antibodies against NMDAR that might be relevant to their illness, but did not differ from patients without NMDAR antibodies in clinical characteristics. Our study suggests that the only way to detect patients with these potentially pathogenic antibodies is to screen all patients with first-episode psychosis at first presentation. FUNDING Medical Research Council.

Inhibition of the transendothelial migration of human lymphocytes but not monocytes by phosphodiesterase inhibitors

This study describes an in vitro model of peripheral blood mononuclear cell (PBMC) migration through human endothelial cells, held on polycarbonate inserts, which allows automatic differential counting of migrated cells as lymphocytes and monocytes. Using this system it was found that treatment of PBMC with the phosphodiesterase (PDE) inhibitors theophylline (at 1 and 10 μg/ml) and RO‐20‐1724 (at 1 μm) inhibited the migration of the lymphocyte component to 64.2 ± 16.4%, 48.9 ± 3.0% and 47.5 ± 5.8% of the control values, respectively, while the migration of the monocytes component was largely unaffected. The PDE inhibitors needed to be present during the assay to inhibit migration, whereas pre‐treatment of either the endothelium or the PBMC did not consistently effect lymphocyte migration. The drugs also inhibited the migration of lymphocytes through control inserts, either uncoated or coated with fibronectin, suggesting that some of the inhibition is an effect on lymphocyte motility rather than lymphocyte–endothelial interactions. Lymphocyte migration through fibronectin‐coated filters was significantly enhanced compared with uncoated filters. Activation of the PBMC by anti‐CD3 MoAb increased motility and migration by up to 300%. This migration appeared to be greatly inhibited by the PDE inhibitors, although the effect was complicated by problems of lymphocyte aggregation. This study provides a novel method of measuring mononuclear cell transendothelial migration, and suggests a possible role of PDE inhibitors in reducing this process.