Visceral Adiposity Is an Independent Determinant of Hypercoagulability as Measured by Thrombin Generation in Morbid Obesity.

Introduction Increased visceral adipose tissue (VAT) has been shown to be associated with the development of insulin resistance, type 2 diabetes, stroke, and ischemic heart disease. It remains unknown whether fat distribution impacts on coagulation markers and/or the risk of venous thrombosis. This study evaluates markers of hypercoagulability in class III obesity (body mass index [BMI] >40 kg/m 2 ) compared with nonobese controls. We further investigated whether hypercoagulability was influenced by VAT, metabolic syndrome, and metabolic markers, including adiponectin. Patients and Methods Ninety patients were recruited from the obesity clinic at King's College Hospital from November 2009 to December 2011. The inclusion criteria were class III obesity (BMI ≥40 kg/m 2 ) and age 18 to 65 years. A control group (healthy ambulatory participants, with a BMI < 30 kg/m 2 ) was recruited from volunteers responding to advertisement. Abdominal VAT and subcutaneous adipose tissue surface areas were determined by evaluation of a single-slice CT at spinal vertebra L4. Results Thrombin generation revealed a significantly increased peak and endogenous thrombin potential in patients compared with controls. Lag time and time to peak (ttP) were also significantly prolonged in patients. VAT was found to have the strongest association with thrombin generation parameters: lag time (ß = 0.378; p < 0.001), peak thrombin (0.378; p = 0.04), and ttP (ß = 0.373; p = 0.001). BMI was found to be a predictor for lag time only (ß = 0.313; p = 0.003). SAT was not associated with any of the thrombin generation parameters (data not shown). VAT was found to be an independent determinant of peak thrombin, lag time, and ttP. The study suggests not only fat mass but also fat distribution, particularly visceral adiposity, mediates hypercoagulability in obesity.

Microfabricated glucose biosensor for culture well operation.

A water-based carbon screen-printing ink formulation, containing the redox mediator cobalt phthalocyanine (CoPC) and the enzyme glucose oxidase (GOx), was investigated for its suitability to fabricate glucose microbiosensors in a 96-well microplate format: (1) the biosensor ink was dip-coated onto a platinum (Pt) wire electrode, leading to satisfactory amperometric performance; (2) the ink was deposited onto the surface of a series of Pt microelectrodes (10-500 µm diameter) fabricated on a silicon substrate using MEMS (microelectromechanical systems) microfabrication techniques: capillary deposition proved to be successful; a Pt microdisc electrode of ≥100 µm was required for optimum biosensor performance; (3) MEMS processing was used to fabricate suitably sized metal (Pt) tracks and pads onto a silicon 96 well format base chip, and the glucose biosensor ink was screen-printed onto these pads to create glucose microbiosensors. When formed into microwells, using a 340 µl volume of buffer, the microbiosensors produced steady-state amperometric responses which showed linearity up to 5 mM glucose (CV=6% for n=5 biosensors). When coated, using an optimised protocol, with collagen in order to aid cell adhesion, the biosensors continued to show satisfactory performance in culture medium (linear range to 2 mM, dynamic range to 7 mM, CV=5.7% for n=4 biosensors). Finally, the operation of these collagen-coated microbiosensors, in 5-well 96-well format microwells, was tested using a 5-channel multipotentiostat. A relationship between amperometric response due to glucose, and cell number in the microwells, was observed. These results indicate that microphotolithography and screen-printing techniques can be combined successfully to produce microbiosensors capable of monitoring glucose metabolism in 96 well format cell cultures. The potential application areas for these microbiosensors are discussed.

The use of coated paramagnetic particles as a physical label in a magneto-immunoassay.

An ideal label for use in an immunoassay would require no further chemical or electromagnetic stimulation prior to its detection and would be free from interference from the sample matrix. Micron sized paramagnetic particles are able to perturb magnetic fields. This perturbation can be directly detected using a suitable electronic device and is independent of the sample matrix. In this study coated paramagnetic particles were used as a physical label in a non-competitive solid phase "sandwich" assay for the detection of human transferrin. The transferrin acted as a "biological bridge" allowing a dose dependant immobilization of the paramagnetic particles to a polyethylene terephthalate solid phase. Quantitation of the paramagnetic label was achieved using an electronic detection system allowing a linear dose response with a femtomolar detection limit (260 fmol).

A novel measuring system for the determination of paramagnetic particle labels for use in magneto-immunoassays.

Coated micrometer-sized paramagnetic particles (PMPs) are readily available and widely used in immunoassays, mainly for separation and as a solid phase. We have described in a separate paper a model sandwich assay in which approximately 1 x 10(5) to 1 x 10(6) PMPs (2.8 microm diameter) are immobilised on a plastic strip at the end of the assay. In this paper, we describe the design of an instrument that is capable of determining the number of PMPs on the plastic strip. The paper also describes a method of making standard plastic strips with known numbers of PMPs on them. A strip, when placed in a coil of wire in parallel with a capacitor, causes the resonant frequency of the coil to decrease because of the presence of the PMPs. The decrease in frequency relates directly to the number of PMPs on the strip. A circuit based on a voltage-controlled oscillator and a phase-locked loop is used to accurately measure the resonant frequency of the coil. The instrument is capable of detecting at least 1 x 10(5) PMPs immobilised on a plastic strip and has a linear response (r=0.99) for up to at least 3.33 x 10(6) PMPs. In terms of the iron content of the PMPs, the detection limit is approximately equal to 1.2 microg Fe in the paramagnetic particles and the sensitivity is approximately equal to 3 Hz per microg of Fe. The instrument is small and compact and together with a suitable magneto-immunoassay will have many applications, including near-patient monitoring.

Towards the development of molecularly imprinted polymer based screen-printed sensors for metabolites of PAHs.

This paper describes the fabrication of a sensor for 1-hydroxypyrene (1-OHP) based on a screen-printed carbon electrode (SPCE) modified with a molecularly imprinted polymer (MIP); 1-OHP was chosen as a model metabolite of polyaromatic hydrocarbons (PAHs). It was shown that 1-OHP could be readily oxidised at a plain SPCE and the electrochemical mechanism was found to involve an ECE (electron transfer-chemical reaction-electron transfer) process. The MIP for 1-OHP was prepared using only divinylbenzene (DVB) and styrene as monomers and the binding was only based on hydrophobic interactions. Batch binding studies revealed that optimum uptake of 1-OHP by the MIP occurred from solutions containing 35% water in methanol. Selectivity of the binding sites in the MIP was examined by performing uptake studies in the same solution containing either phenol or 1-naphthol; the specific binding of 1-OHP was twenty times greater than the former and five times greater than the latter. Preliminary calibration studies were performed with the MIP-SPCE using a two-step approach; accumulation was carried out in 35% water in methanol followed by measurement in 50% methanol-0.025 mol dm(-3) phosphate buffer pH 12. This two-step non-competitive affinity assay gave encouraging results and indicated potential for use in pollution studies.

Studies towards a disposable screen-printed amperometric biosensor for progesterone.

A screen-printed carbon electrode (SPCE) has been investigated as the base transducer for a disposable amperometric progesterone biosensor. The biorecognition element was a monoclonal sheep anti-progesterone antibody (mAb). This was immobilized onto the transducer by interaction with a layer of rabbit IgG which had been previously coated onto the SPCE; optimum conditions for these loadings were deduced experimentally. The device was employed in a competitive assay using alkaline phosphatase-labelled progester-one. Three possible substrates for the enzyme were considered, namely, phenyl phosphate, phenolphthalein phosphate and 4-aminophenol phosphate. Cyclic voltammetry and amperometry were carried out on the corresponding aromatic phenols and phenol itself was found to give the best electrochemical characteristics; consequently, phenyl phosphate was employed as the substrate. Chronoamperometry was used to measure the phenol produced by the reaction of bound enzyme-labelled progesterone and substrate. The chronoamperometric response was dependent on unlabelled progesterone over at least three orders of magnitude with a detection limit of about 1 x 10(-9) mol/dm3. This suggests that the device may have applications for the analysis of biological fluids.

Serum oligoclonal IgG is a common and persistent finding in multiple sclerosis, and has a systemic source.

Synthesis of oligoclonal IgG within the central nervous system is a well established feature of multiple sclerosis. The occurrence of oligoclonal IgG in the serum of patients with multiple sclerosis has received little attention. We detected such a serum response in 20/45 consecutive patients (44%, 95% CI 30-59%) but in only 3/41 age- and sex-matched healthy controls (p < 0.01). We present qualitative and quantitative evidence that this oligoclonal IgG has a systemic origin. The plasma cell clones responsible for the serum response are often also represented in the intrathecal compartment. In a further study of the clinical significance of serum oligoclonal bands, in 80 patients, their presence was associated with elevated levels of intrathecal synthesis, increasing age, later disease onset and the presence of serum autoantibodies. These findings add to the evidence that there is a systemic immune disturbance in multiple sclerosis.

Affinity of antigen-specific IgG distinguishes multiple sclerosis from encephalitis.

The characteristics of antigen-specific IgG in patients with multiple sclerosis and patients with encephalitis have been compared. Both groups of patients showed antigen-specific oligoclonal bands locally synthesised in the CSF. When the affinity distribution of the antigen-specific IgG was measured there was a marked difference between the two groups. Encephalitis patients had high affinity antibody against the causative antigen. This was consistent with the antibody undergoing affinity maturation as a result of the immune system fighting a primary infection. Multiple sclerosis patients lacked high affinity response. This lack of high affinity antibody was also seen in those encephalitis patients when antigens other than the causative antigen were studied.

The JC virus antibody response in serum and cerebrospinal fluid in progressive multifocal leucoencephalopathy.

BACKGROUND: A clinical diagnosis of progressive multifocal leucoencephalopathy (PML) can be confirmed by histological or virological examination of brain material. Whilst a less invasive method is provided by the detection of JC DNA in cerebrospinal fluid (CSF), very few studies have been done to assess the value of JC virus (JCV) serology in PML diagnosis. OBJECTIVES: To study the JCV antibody response in the serum and CSF of PML patients. STUDY DESIGN: A retrospective study was done using haemagglutination inhibition (HI), M-antibody capture radioimmunoassay (MACRIA) and JC-specific oligoclonal IgG banding on one or more sera and/or CSFs from 28 confirmed PML patients. Seventy-one serum and CSF samples were tested from patients with memory loss or dementia as a control group. RESULTS: Twenty-seven PML patients (96%) had detectable JCV HI antibody in the serum, with titres ranging from 1 : 10 to > 1 : 20480, compared to 48 (68%) of the controls (P = <0.005). JCV IgM antibody was detected in the serum of 12/22 (55%) PML patients. JCV HI antibody was detected in the CSF in 12 of 18 (67%) PML patients, antibody index measurements being used to control for a possible breakdown of the blood-brain barrier. Intrathecal JCV antibody was not found in any control patient. Locally produced JCV-specific IgG bands were detected in the CSF of 7 PML patients tested, confirming the intrathecal origin and specificity of the HI antibody. CONCLUSIONS: The presence of intrathecal JCV antibody indicates active central nervous system infection with JC virus, and provides a useful diagnostic test for PML, with a sensitivity of 67% and a specificity of 100%. The absence of serum JCV antibody nearly always excludes a diagnosis of PML, but the titre of antibody, IgG or IgM, correlates with the underlying condition rather than the development of neurological symptoms.

Hairy leukaemic cell influx into the cerebrospinal fluid secondary to encephalomyelitis.

A case report of a 53 year old male with hairy cell leukaemia is presented in whom encephalomyelitis caused by toxoplasmosis resulted in an influx of hairy cells into the cerebrospinal fluid following disruption of the blood-brain barrier. These cells subsequently disappeared as the barrier reformed. It is suggested that the presence of hairy cells in the cerebrospinal fluid is a secondary self-limiting phenomenon.

Lymphocytic meningitis following insertion of a porcine dermis dural graft.

We describe a case of lymphocytic meningitis following insertion of a porcine dermis implant to repair an operative dural defect. Histology of the excised implant revealed local abscess formation with a granulomatous reaction. Oligoclonal Immunoglobulin G, part of which could be removed by absorbtion with the porcine dermis, was present in the patient's cerebrospinal fluid, and, to a less marked degree, in his serum. The cerebrospinal fluid glucose was markedly depressed. An unusual hypersensitivity reaction to the porcine implant was considered the most likely explanation for this meningitic illness. The patient went on to make a full recovery following excision of the implant.

The significance of serum oligoclonal bands in neurological diseases.

The presence of oligoclonal bands (OCBs) of immunoglobulin G (IgG) in CSF provides evidence for the occurrence of a humoral immune response, but it is not always appreciated that the oligoclonal IgG may have originated in the serum. To determine the diagnostic significance of serum OCBs 146 patients with serum OCBs were identified among 1874 patients with suspected neurological disorders (7.6%). Clear diagnoses had been made in 112 of these patients: in 56 identical CSF and serum bands were present, revealing a systemic immune response, while in 46 additional unique CSF bands indicated that intrathecal IgG synthesis was also occurring. In the first group neoplasia and peripheral neuropathies accounted for over 50% of the diagnoses, infections and systemic inflammatory disorders for 32%, and multiple sclerosis was diagnosed in only one case. These figures contrast considerably with those reported for patients with CSF OCBs alone. Diagnoses in the second group of patients, with unique CSF OCBs in addition to serum OCBs, resembled those among patients with CSF OCBs alone. Examining CSF and serum in parallel for OCBs of IgG provides more diagnostic information than examining CSF alone, and the latter is potentially misleading.

IgG paraproteins in neurological diseases: lack of association with neurotropic viral/bacterial antigens.

In this study we tested the hypothesis that IgG paraproteins in neurological diseases might be regarded as an anti-idiotypic response raised by a nervous system antigen associated with certain neurotropic agents. After initial ELISA screen, positive samples from 34 neurological patients who had paraproteins in their CSF and serum on routine IEF investigation, were tested by immunoblot technique for the presence of specific monoclonal immunoglobulin G (IgG) against eight different neurotropic antigens. Only one patient had specific monoclonal IgG against herpes simplex virus. The results of this study did not confirm our hypothesis. Further study of IgM/IgA paraproteins is indicated.

Oligoclonal immunoglobulin D in the cerebrospinal fluid of neurologic patients.

Oligoclonal immunoglobulin D (IgD) bands were observed in 20 of 110 CSF samples investigated by our method for isoelectric focusing (IEF) of IgD in unconcentrated CSF. These were mostly from demyelinating disorders, viral CNS infections, and CNS tumors. The patterns of expression of IgD were similar to that of IgG, except in cases with tumors where oligoclonal IgD may occur independently of IgG. The study shows for the first time the presence of oligoclonal IgD in CSF from neurologic patients.

A new qualitative method for detecting IgD in unconcentrated cerebrospinal fluid.

We report a method which is capable of demonstrating the isoelectric focusing (IEF) pattern of immunoglobulin D in unconcentrated cerebrospinal fluid (CSF) samples containing as little as 0.1-0.5 ng of total IgD. The method used was an immuno-sandwich technique, with alkaline phosphatase enzyme amplification. Oligoclonal and polyclonal IgD patterns were seen in CSF samples. No cross-reactivity with other immunoglobulins (IgG, IgA and IgM) was detected.

A study of immunoglobulin G in the cerebrospinal fluid of 1007 patients with suspected neurological disease using isoelectric focusing and the Log IgG-Index. A comparison and diagnostic applications.

Cerebrospinal fluid and serum immunoglobulin G from 1007 patients with suspected neurological disease were analysed by 2 methods: isoelectric focusing for the detection of oligoclonal banding, and quantitative measurement of IgG and albumin for the formulation of a Log IgG-Index. A comparison of the 2 methods in the detection of local synthesis of IgG showed that isoelectric focusing not only gave a much higher yield overall, with 282 patients showing local synthesis versus 225 for the Log IgG-Index, but also a higher specificity, with a false positive rate of 0% versus 3.5%. In addition, of the 282 patients positive by isoelectric focusing only 163 (58%) were positive by the Log IgG-Index. Of the 1007 patients studied, 206 had multiple sclerosis (MS), and isoelectric focusing showed local synthesis in 95% of clinically definite cases, with a 90% detection rate overall. The Log IgG-Index was positive in only 67% of clinically definite cases, with an overall 59% detection rate. Thus with the exceptions noted above, local synthesis of IgG as defined by isoelectric focusing is confined to demyelinating, inflammatory, infectious and postinfectious disorders. Our results compare very favourably with the published sensitivities of magnetic resonance imaging in the detection of abnormalities in multiple sclerosis, and better than those for evoked potentials. Where both these investigations are readily available isoelectric focusing provides a useful adjunct. For the majority of physicians and neurologists who do not have ready access to magnetic resonance imaging, isoelectric focusing is an excellent alternative. We would also recommend that it become the standard for the measurement of IgG abnormalities in the cerebrospinal fluid and that the use of quantitative data be abandoned for routine purposes.

Isoelectric focusing of cerebrospinal fluid immunoglobulin G: an annotated update.

A revised agarose isoelectric focusing method for detecting oligoclonal IgG in unconcentrated cerebrospinal fluid is presented. The technique is shown to be robust and reproducible and suitable for the detection of intrathecal IgG synthesis.

Affinity distributions of antigen-specific IgG in patients with multiple sclerosis and in patients with viral encephalitis.

We report a method for displaying the affinity distribution of a polyclonal antibody population using sodium thiocyanate elution followed by an ELISA detection technique. We have used this method to study the affinity distribution of antibodies in samples of CSF and serum from patients with MS, and compared the results to those obtained from patients with viral encephalitis. Patients with MS had predominantly low affinity antibody against a particular antigen whilst patients with a primary viral infection had predominantly high affinity antibody against the causative organism.

The occurrence of oligoclonal IgG in tears from patients with MS and systemic immune disorders.

Using isoelectric focusing (IEF) and immunoperoxidase staining of proteins transferred to nitrocellulose membranes, we have examined the IgG band pattern in tears and matched serum and CSF specimens of 28 patients with MS, 4 patients with optic neuritis (ON), 30 individuals with systemic, inflammatory, or other neurologic diseases, and 5 patients with tension headache. We found no evidence of positive oligoclonal IgG in tears in any MS or ON patients, while 10 out of 16 cases with systemic immune disorders or infections of the CNS had positive tear oligoclonal bands. We are thus not able to support the hypothesis that tears from MS patients reveal abnormalities in their humoral immune response.

Isoelectric focusing versus quantitative measurements in the detection of intrathecal local synthesis of IgG.

Specimens from 1007 patients with suspected neurological disturbances had quantitative and qualitative measurements made of cerebrospinal fluid and serum to investigate the presence of locally synthesised IgG. Qualitative measurement was recorded as the presence or absence of oligoclonal banding, and the quantitative measurement was derived by the use of the IgG index, the log index and the Reiber, Schuller and Tourtellotte formulae. The patients were divided into two categories, on the basis of banding: those with local synthesis and those without. The sensitivity, specificity and efficiency for each of the quantitative measurements were then calculated. Receiver-operator curves were also constructed for each of the quantitative measurements. 282 samples showed local synthesis of IgG by isoelectric focusing, whereas the best quantitative assay (log index) could only detect 198. Therefore, we conclude that oligoclonal banding should be adopted as the standard laboratory measurement of local synthesis of IgG in the diagnosis of neurological disorders, and that the diagnostic use of quantitative measurements should be abandoned for routine purposes. Furthermore, we suggest that quantitative analysis, at its current level, is misleading and has little value in the understanding of neurological disorders, but may be of use in serially monitoring individual patients as part of their therapeutic trials.