Capillary gel electrophoresis of very high molecular weight glycoproteins. Commercial and tailor-made gels for analysis of human monomeric and secretory immunoglobulin A.

Capillary gel electrophoresis (CGE) has been widely used for analysis of proteins according to their size. However, to our knowledge, this technique has not been optimized to immunoglobulin A (IgA) analysis, a protein of current and emerging high interest in several fields. IgA is the first barrier of human body against pathogens. This protein in human milk and colostrum is essential for immune protection of newborns and treatment of milk for storage in Human Milk Banks may alter IgA. The emerging use of IgA as therapeutic treatment also encourages the development of analysis methods for this class of immunoglobulins. IgA is far more heterogeneously glycosylated and complex than the well-studied IgG molecules. IgA in serum is mainly monomeric (mIgA) with about 160 kDa, while in secretions such as saliva, milk, colostrum, etc, secretory immunoglobulin A (sIgA) is the predominant form. This is a dimer where both monomers are linked by the J-chain and the secretory component accounting all together for a MW higher than 400 kDa including the glycans. This size is far from the 225 kDa MW for which commercial CGE kits are intended. The general rules governing CGE behavior of analytes cannot be directly applied to every protein. Addressing studies directed specifically to target proteins is specially needed for the large size and highly complex target analytes of this study. In this work the effect of several factors on CGE analysis of human serum and colostrum IgA is studied. The feasibility of performing analysis of both IgA classes using a commercial CGE kit is shown. In addition, this work introduces another novelty by preparing tailor-made reproducible gel buffers and to characterize them in terms of dynamic viscosity, conductivity, and electroosmotic flow mobility in bare fused silica capillaries. The possibility of analyzing mIgA and sIgA in less than 10 min using these tailor-made gels is demonstrated. Inter-day variation (RSD) for the main peak of sIgA is 0.25% for migration time (tm) and 0.27% for percentage corrected peak area (Acorr).

Simple, rapid, and reproducible capillary gel electrophoresis separation and laser-induced fluorescence detection of DNA topoisomers with unmodified fused silica separation capillaries.

The topology of DNA is a critical quality attribute for plasmid-based pharmaceuticals, making quantification of trace levels of plasmid topoisomers an important analytical priority. An automated and cost-effective method based on capillary gel electrophoresis laser-induced fluorescence detection is described. The method outlined in this report is significant because it is easily implemented by any laboratory for which routine analyses of plasmid topology are critical for the development of new plasmid-based therapies as well as for quality control of gene therapies utilizing supercoiled DNA. Detection of topoisomers was achieved by incorporating ethidium bromide in the separation medium. The detector response was improved by 3 orders of magnitude by utilizing a 605-nm optical filter with a 15-nm bandwidth. Separations of linear, open circle, supercoiled, and multimer DNA plasmids ranging from 4.2 to 10.5 kbp were accomplished in under 6 min using an unmodified fused silica capillary (50-µm internal diameter). The background electrolyte was comprised of 0.5% gel, which was hydroxypropylmethyl cellulose, 1 mM ethylenediaminetetraacetic acid, and 50 mM N-(2-acetamido)-2-aminoethanesulfonic acid (pH of 6.25). The separations, which balanced the bulk electroosmotic flow, the electrophoretic mobility of the DNA, and gel sieving were dependent upon the pH of the electrolyte and the gel concentration. Reproducibility was dependent upon the procedure used to prepare the gel as well as other factors including the ethidium bromide concentration and capillary conditioning. A single unmodified capillary operated for more than 150 runs had an across-day migration time precision of 1% relative standard deviation and percent area precision of 10% relative standard deviation.

Preservation of vapor samples on adsorbent alumina capillaries and implications for field sampling.

Dynamic vapor microextraction (DVME) is a vapor preconcentration method that employs a capillary trap coated with an adsorbent, followed by solvent elution to recover the sample. DVME has been developed for applications in the laboratory, including highly precise vapor pressure measurements, and in the field. When vapor collection is conducted outside the laboratory, samples must almost always undergo some interval of storage representing the time between collection and analysis. This interval may be hours, days, or longer, depending on the situation. Regardless, in all situations there must be confidence that the integrity of the samples is maintained until processing and analysis. In this paper, we present results of two studies that tested the stability of a 50% weathered gasoline headspace sample on alumina PLOT (porous layer open tubular) capillaries stored at room temperature for periods from 24 h up to 20 wk. We used principal component analysis (PCA) to reduce the dimensionality of the chromatographic and mass spectral data and elucidate trends in stability with respect to the complex sample's range of hydrocarbon classes and molecular weights. Both analyses identified changes over storage periods of six weeks or more. The hydrocarbon class analysis, which used selected ion monitoring (SIM) data as input, proved more sensitive to changes over shorter storage periods. Sample integrity was preserved for at least 24 h, but losses, especially of high-volatility compounds, occurred by 168 h (7 d). Near total loss of sample occurred by 20 wk. These findings, which are specific to the sample, adsorbent, and storage conditions, will guide choices in experimental and instrumental design to ensure that data from future field studies is reliable.

Daptomycin: A Novel Macrocyclic Antibiotic as a Chiral Selector in an Organic Polymer Monolithic Capillary for the Enantioselective Analysis of a Set of Pharmaceuticals.

Daptomycin, a macrocyclic antibiotic, is here used as a new chiral selector in preparation of chiral stationary phase (CSP) in a recently prepared polymer monolithic capillary. The latter is prepared using the copolymerization of the monomers glycidyl methacrylate (GMA) and ethylene glycol dimethacrylate (EGDMA) in the presence of daptomycin in water. Under reversed phase conditions (RP), the prepared capillaries were tested for the enantioselective nanoliquid chromatographic separation of fifty of the racemic drugs of different pharmacological groups, such as adrenergic blockers, H1-blockers, NSAIDs, antifungal drugs, and others. Baseline separation was attained for many drugs under RP-HPLC. Daptomycin expands the horizon of chiral selectors in HPLC.

Determination of finerenone - a novel, selective, nonsteroidal mineralocorticoid receptor antagonist - in human plasma by high-performance liquid chromatography-tandem mass spectrometry and its application to a pharmacokinetic study in venous and capillary human plasma.

A straightforward and rapid high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) assay allowing the sensitive and selective quantitation of finerenone (BAY 94-8862) in lithium heparin human plasma is described. Finerenone is a novel, selective, nonsteroidal mineralocorticoid receptor antagonist that is in phase III clinical trials for the treatment of chronic kidney disease. Finerenone quantitation is performed after addition of its stable isotope-labelled internal standard (ISTD) by protein precipitation with acidified acetonitrile followed by HPLC-MS/MS separation and detection. The determination of finerenone concentrations was validated for a plasma volume of 0.100 mL and subsequently also for a lower plasma volume of 0.010 mL, collected e.g. in paediatric studies. The analytical range was from 0.100 µg/L (lower limit of quantification) to 200 µg/L (upper limit of quantification). Inter-day accuracy was 99.7-105.0% for the plasma volume of 0.100 mL and 101.1-104.5% for the plasma volume of 0.010 mL. Inter-day precision was ≤ 7.0%, independent of the extracted plasma volume. A moderate, concentration-independent matrix effect on ionisation was observed for both finerenone and its ISTD of 0.535-0.617, which is fully compensated by the ISTD (ISTD-normalised matrix factors were 0.98-1.03). The assay was successfully applied with both validated plasma volumes to a clinical phase I study in which the pharmacokinetics of 20 mg finerenone were compared in capillary plasma (0.010 mL) and venous plasma (0.100 mL) in a concentration range from the lower limit of quantification to 310 µg/L (capillary plasma) and 252 µg/L (venous plasma). The area under the plasma concentration versus time curve was similar in both matrices, while maximum concentrations were 37% higher in capillary plasma. In conclusion, capillary sampling should not bias pharmacokinetic exposure estimates compared with venous plasma values, if limited to sampling times in the distribution and elimination phases of finerenone.

Clear Cell Change in Reactive Angiogenesis: A Potential Diagnostic Pitfall.

Reactive angiogenesis is commonplace, occurs in many circumstances, and is important in the repair of injured tissue. Histologically, it is characterized by newly formed capillaries arranged in a lobular architecture and lined by plump endothelial cells. We have encountered a form of reactive angiogenesis not well described; composed of large endothelial cells with abundant clear cytoplasm that causes diagnostic challenges. The cohort includes 10 patients, aged 4 to 61, mean 40 years; 7 males, 3 females. One case involved bone (ilium), and 9 involved soft tissue: fingers (n=2), toes (n=2), hip joint (n=1), shoulder (n=1), thigh (n=2), and anal mucosa (n=1). Clinically, the patients had chronic ulcers, osteomyelitis, or localized infection. All cases exhibited a lobular proliferation of capillaries lined by large polyhedral endothelial cells that obscured the vessel lumens and were admixed with acute and chronic inflammation. The endothelial nuclei were vesicular with small nucleoli and the cytoplasm was abundant and clear or palely eosinophilic. The endothelial cells were stained with CD31 and ERG (7/7 cases), CD34 (6/6), FLI1 (4/4), and were negative for keratin and CD68 (6/6). Periodic acid-Schiff stain and periodic acid-Schiff stain-diastase on 3 cases did not demonstrate glycogen. Using a polymerase chain reaction, no Bartonella henselae was found in all 6 cases tested. Reactive angiogenesis with clear cell change unassociated with Bartonella spp. has not been described. It causes diagnostic challenges and the differential diagnosis includes benign and malignant tumors, as well as unusual infections. It is important to distinguish between these possibilities because of the significant impact on treatment and prognosis.

Retina-specific targeting of pericytes reveals structural diversity and enables control of capillary blood flow.

Pericytes are a unique class of mural cells essential for angiogenesis, maintenance of the vasculature and are key players in microvascular pathology. However, their diversity and specific roles are poorly understood, limiting our insight into vascular physiology and the ability to develop effective therapies. Here, in the mouse retina, a tractable model of the CNS, we evaluated distinct classes of mural cells along the vascular tree for both structural characterization and physiological manipulation of blood flow. To accomplish this, we first tested three inducible mural cell-specific mouse lines using a sensitive Ai14 reporter and tamoxifen application either by a systemic injection, or by local administration in the form of eye drops. The specificity and pattern of cre activation varied significantly across the three lines, under either the PDGFRß or NG2 promoter (Pdgfrß-CreRha, Pdgfrß-CreCsln, and Cspg4-Cre). In particular, a mouse line with Cre under the NG2 promoter resulted in sparse TdTomato labeling of mural cells, allowing for an unambiguous characterization of anatomical features of individual sphincter cells and capillary pericytes. Furthermore, in one PDGFRß line, we found that focal eye drop application of tamoxifen led to an exclusive Cre-activation in pericytes, without affecting arterial mural cells. We then used this approach to boost capillary blood flow by selective expression of Halorhodopsin, a highly precise hyperpolarizing optogenetic actuator. The ability to exclusively target capillary pericytes may prove a precise and potentially powerful tool to treat microcirculation deficits, a common pathology in numerous diseases.

Two new reliable immunohistochemical methods for simultaneous identification of capillaries, the three types of fibers and basal lamina in human skeletal muscle.

Capillary network of skeletal muscle has a crucial role in oxygen supply and is strongly associated with the phenotype and metabolic profile of muscle fibers. Abundant literature has explored capillarization of skeletal muscle in different populations and in response to different interventions. Capillary and fiber type identification techniques have considerably evolved over the last decades, but to the best of our knowledge, no validated immunohistochemical method has yet been developed to simultaneously identify capillaries (using CD31), the three different muscle fiber types, and basal lamina. Nine human muscle biopsies of vastus lateralis were stained using 5 different methods to test: the reliability of different CD31 antibodies for capillary identification, the reliability between single-section or serial-section methods, and the intra-experimenter reproducibility in visual detection of capillaries. High reliability for the different antibodies directed against capillaries was observed for capillary contacts (CC) measurements (intra-class correlations (ICC) [ICC95%] of 0.89 [0.72; 0.96] for type I fibers, 0.93 [0.81; 0.97] for type IIA fibers, 0.88 [0.71; 0.96] for type IIX fibers, 0.95 [0.86; 0.98] for all fiber types) as well as a high level of similarity between single and serial sections methods. A strong similarity in capillary analysis between the different methods was obtained for each sample measurements. Analysis of Lin's concordance correlation coefficients and Bland and Altman's graphics showed a strong intra-experimenter reproducibility. This article proposes two time- and tissue-sparing immunohistochemical methods to accurately assess a complete fiber typing (type I, IIA, and IIX) along with muscle capillarization on a single muscle section.

Determination of piperaquine concentration in human plasma and the correlation of capillary versus venous plasma concentrations.

BACKGROUND: A considerable challenge in quantification of the antimalarial piperaquine in plasma is carryover of analyte signal between assays. Current intensive pharmacokinetic studies often rely on the merging of venous and capillary sampling. Drug levels in capillary plasma may be different from those in venous plasma, Thus, correlation between capillary and venous drug levels needs to be established. METHODS: Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) was used to develop the method. Piperaquine was measured in 205 pairs of capillary and venous plasma samples collected simultaneously at ≥24hr post dose in children, pregnant women and non-pregnant women receiving dihydroartemisinin-piperaquine as malaria chemoprevention. Standard three-dose regimen over three days applied to all participants with three 40mg dihydroartemisinin/320mg PQ tablets per dose for adults and weight-based dose for children. Correlation analysis was performed using the program Stata® SE12.1. Linear regression models were built using concentrations or logarithm transformed concentrations and the final models were selected based on maximal coefficient of determination (R2) and visual check. RESULTS: An LC-MS/MS method was developed and validated, utilizing methanol as a protein precipitation agent, a Gemini C18 column (50x2.0mm, 5µm) eluted with basic mobile phase solvents (ammonium hydroxide as the additive), and ESI+ as the ion source. This method had a calibration range of 10-1000 ng/mL and carryover was negligible. Correlation analysis revealed a linear relationship: Ccap = 1.04×Cven+4.20 (R2 = 0.832) without transformation of data, and lnCcap = 1.01×lnCven+0.0125, (R2 = 0.945) with natural logarithm transformation. The mean ratio (±SD) of Ccap/Cven was 1.13±0.42, and median (IQR) was 1.08 (0.917, 1.33). CONCLUSIONS: Capillary and venous plasma PQ measures are nearly identical overall, but not readily exchangeable due to large variation. Further correlation study accounting for disposition phases may be necessary.

The mouse olfactory peduncle 4: Development of synapses, perineuronal nets, and capillaries.

Olfaction is critical for survival in neonatal mammals. However, little is known about the neural substrate for this ability as few studies of synaptic development in several olfactory processing regions have been reported. Odor information detected in the nasal cavity is first processed by the olfactory bulb and then sent via the lateral olfactory tract to a series of olfactory cortical areas. The first of these, the anterior olfactory nucleus pars principalis (AONpP), is a simple, two layered cortex with an outer plexiform and inner cell zone (Layers 1 and 2, respectively). Five sets of studies examined age-related changes in the AONpP. First, immunocytochemistry for glutamatergic (VGlut1 and VGlut2) and GABAergic (VGAT) synapses demonstrated that overall synaptic patterns remained uniform with age. The second set quantified synaptic development with electron microscopy and found different developmental patterns between Layers 1 and 2. As many of the interhemispheric connections in the olfactory system arise from AONpP, the third set examined the development of crossed projections using anterograde tracers and electron microscopy to explore the maturation of this pathway. A fourth study examined ontogenetic changes in immunostaining for the proteoglycans aggrecan and brevican, markers of mesh-like extracellular structures known as perineuronal nets whose maturation is associated with the end of early critical periods of synaptogenesis. A final study found no age-related changes in the density of vasculature in the peduncle from P5 to P30. This work is among the first to examine early postnatal changes in this initial cortical region of the olfactory system.

The amyloid-ß degradation intermediate Aß34 is pericyte-associated and reduced in brain capillaries of patients with Alzheimer's disease.

An impairment of amyloid ß-peptide (Aß) clearance is suggested to play a key role in the pathogenesis of sporadic Alzheimer's disease (AD). Amyloid degradation is mediated by various mechanisms including fragmentation by enzymes like neprilysin, matrix metalloproteinases (MMPs) and a recently identified amyloidolytic activity of ß-site amyloid precursor protein cleaving enzyme 1 (BACE1). BACE1 cleavage of Aß40 and Aß42 results in the formation of a common Aß34 intermediate which was found elevated in cerebrospinal fluid levels of patients at the earliest disease stages. To further investigate the role of Aß34 as a marker for amyloid clearance in AD, we performed a systematic and comprehensive analysis of Aß34 immunoreactivity in hippocampal and cortical post-mortem brain tissue from AD patients and non-demented elderly individuals. In early Braak stages, Aß34 was predominantly detectable in a subset of brain capillaries associated with pericytes, while in later disease stages, in clinically diagnosed AD, this pericyte-associated Aß34 immunoreactivity was largely lost. Aß34 was also detected in isolated human cortical microvessels associated with brain pericytes and its levels correlated with Aß40, but not with Aß42 levels. Moreover, a significantly decreased Aß34/Aß40 ratio was observed in microvessels from AD patients in comparison to non-demented controls suggesting a reduced proteolytic degradation of Aß40 to Aß34 in AD. In line with the hypothesis that pericytes at the neurovascular unit are major producers of Aß34, biochemical studies in cultured human primary pericytes revealed a time and dose dependent increase of Aß34 levels upon treatment with recombinant Aß40 peptides while Aß34 production was impaired when Aß40 uptake was reduced or BACE1 activity was inhibited. Collectively, our findings indicate that Aß34 is generated by a novel BACE1-mediated Aß clearance pathway in pericytes of brain capillaries. As amyloid clearance is significantly reduced in AD, impairment of this pathway might be a major driver of the pathogenesis in sporadic AD.

3D analysis of capillary network in skeletal muscle of obese insulin-resistant mice.

In obesity, the skeletal muscle capillary network regresses and the insulin-mediated capillary recruitment is impaired. However, it has been shown that in the early stage of advanced obesity, an increased functional vascular response can partially compensate for other mechanisms of insulin resistance. The present study aimed to investigate the changes in the capillary network around individual muscle fibres during the early stage of obesity and insulin resistance in mice using 3D analysis. Capillaries and muscle fibres of the gluteus maximus muscles of seven high-fat-diet-induced obese and insulin-resistant mice and seven age-matched lean healthy mice were immunofluorescently labelled in thick transverse muscle sections. Stacks of images were acquired using confocal microscope. Capillary network characteristics were estimated by methods of quantitative image analysis. Muscle fibre typing was performed by histochemical analysis of myosin heavy chain isoforms on thin serial sections of skeletal muscle. Capillary length per muscle fibre length and capillary length per muscle fibre surface were increased by 27% and 23%, respectively, around small muscle fibres in obese mice, while there were no significant comparative differences around large fibres of obese and lean mice. Furthermore, the capillarization was larger around small compared to large fibres and there was a shift toward fast type myosin heavy chain isoforms, with no significant changes in muscle fibre diameters, tortuosity and anisotropy in obese mice. Overall, the results show that obese insulin-resistant mice have selective increase in capillarization around small predominantly intermediate muscle fibres, which is most likely related to the impaired glucose metabolism characteristic of type 2 diabetes.

Liquid-crystal organization of liver tissue.

Functional tissue architecture originates by self-assembly of distinct cell types, following tissue-specific rules of cell-cell interactions. In the liver, a structural model of the lobule was pioneered by Elias in 1949. This model, however, is in contrast with the apparent random 3D arrangement of hepatocytes. Since then, no significant progress has been made to derive the organizing principles of liver tissue. To solve this outstanding problem, we computationally reconstructed 3D tissue geometry from microscopy images of mouse liver tissue and analyzed it applying soft-condensed-matter-physics concepts. Surprisingly, analysis of the spatial organization of cell polarity revealed that hepatocytes are not randomly oriented but follow a long-range liquid-crystal order. This does not depend exclusively on hepatocytes receiving instructive signals by endothelial cells, since silencing Integrin-ß1 disrupted both liquid-crystal order and organization of the sinusoidal network. Our results suggest that bi-directional communication between hepatocytes and sinusoids underlies the self-organization of liver tissue.

Analysis of capillary microsamples obtained from a skin-prick to measure vancomycin concentrations as a valid alternative to conventional sampling: A bridging study.

A bridging study is presented to investigate the applicability of measuring vancomycin concentrations obtained by finger-prick. A total of 25 paired plasma samples, collected from finger prick as capillary microsampling and arterial plasma samples collected from an indwelling cannula as conventional sampling, were obtained from critically ill patients receiving vancomycin. The maximum concentration (Cmax) and the minimum concentration (Cmin) measured were 66.2 mg/L and 29.7 mg/L for capillary microsampling and 78.9 mg/L, 25.6 mg/L for conventional sampling, respectively. The area under the concentration-time curve from 0 to 6 h (AUC0-6h) ranged between 94.8 and 269 mg/L.h for capillary microsampling and from 106 and 303 mg/L.h for conventional sampling. The comparative study conducted was assessed using three different statistical approaches: Bland-Altman and Passing-Bablok regression analyses and the USFDA criterion for the incurred sample reanalysis. The results of this analysis revealed no significant bias and a strong correlation between both sampling methods, with 95% of the calculated concentrations from the paired plasma samples laying within 20% of difference of the mean. This bridging study verifies that capillary microsampling may serve as an alternative to conventional sampling techniques to support clinical applications for measuring vancomycin concentrations in plasma.

Capillary and Venous Blood Glucose Accuracy in Blood Glucose Meters Versus Reference Standards: The Impact of Study Design on Accuracy Evaluations.

BACKGROUND: Anecdotal blood glucose assessments conducted by health care professionals (HCPs) in the field have highlighted differences in results when methodology used is not according to best practices for measuring blood glucose. This study assessed the impact on accuracy of blood glucose measurements when methodology deviates from the recommended study design and recommended reference instrument. METHODS: Adults with type 1 or type 2 diabetes provided capillary and venous blood samples for accuracy assessments using OneTouch® Verio® (Verio) and OneTouch® Ultra 2® (Ultra) blood glucose meters (BGM) and two different reference instruments. RESULTS: Increases in mean bias were observed when comparing capillary to venous samples tested on the BGMs and the recommended reference instrument. Mean bias was even greater when a hospital blood glucose analyzer was used to measure venous plasma glucose. Increases in mean bias observed for Ultra BGM when testing venous blood on the meter compared to the recommended reference instrument was likely due to the interfering effects of low oxygen levels in the venous blood sample. Conversely, Verio meters, which are insensitive to low oxygen levels, showed little difference from baseline when testing venous blood on the meter compared to results from the same venous sample measured on a reference instrument. CONCLUSIONS: Deviations from the best practice study design of comparing capillary blood glucose results tested on the blood glucose meter with the manufacturer's stated reference instrument will affect accuracy of blood glucose measurements.

[Evaluation of the capillary assay of C-reactive protein (CRP) through the lenght of consultation in pediatric emergencies and its economic impact]. / Évaluation du dosage capillaire de la protéine C-réactive (CRP) sur la durée de consultation aux urgences pédiatriques et impact économique.

Fever is a frequent reason for consultation in pediatric emergency departments and raises the question of biological and radiological examinations. Rapidly obtaining the result of C-reactive protein (CRP) level is essential in front of the steady increase of the number of visits. We carried out a prospective study within the pediatric emergency department of the University Hospital of Clermont-Ferrand from January to April 2017, in order to evaluate the interest of the capillary CRP in point of care (POCT). In two periods, 68 patients (28 controls without and 40 cases with capillary CRP assayed on a Afinion® AS100) with naked fever greater than 48 hours were included. After a study of the analytical performances of Afinion® and a verification of the homogeneity and the comparability of the two groups on clinical criteria (age, sex, duration of the fever, antibiotics treatment) and biological (values of CRP), the interest of the CRP in POCT was evaluated. In the POCT group, a significant drop in the median of the emergency room consultation time (60 (IQR 33-125) versus 180 (IQR 158-208) minutes), the number of biological acts by patient (1 (IQR 1-3) versus 7 (IQR 3-8)), the global cost of biological and radiological examinations per patient (5.4 (IQR 5.4-32.6) versus 153.8 (IQR 46.9-180.4) euros), and the cost of reagents spent by the laboratory per patient (5.2 (IQR 5.2-6.4) versus 33.2 (IQR 2.3-34.2) euros). Thus, in the context of a clinical-biological partnership, the use of CRP in POCT present a practical and an economic interest.

Histomorphometric evaluation of the Ki-67 proliferation rate and CD34 microvascular and D2-40 lymphovascular densities drives the pulmonary typical carcinoid outcome.

Ki-67 has shown promise as a prognostic factor in pulmonary carcinoids. In this study, we sought to validate the importance of Ki-67 and study the relationships between Ki-67 and other stromal biomarkers of vascular density. We examined Ki-67, CD34, and D2-40 in tumor tissues from 128 patients with surgically excised typical carcinoid of the lung. We used immunohistochemistry and morphometry to evaluate the amount of tumor staining for cellular proliferation (Ki-67), microvascular density (CD34-MVD), and D2-40 lymphovascular density. The main outcome was overall survival, considered as life expectancy until death from metastasis. Specimens from patients with central tumors showed high CD34-MVD (P = .01), which was also significantly associated with a compromised surgical margin, lymph node metastasis, and clinical stage Ib. Equally significant was high D2-40 lymphovascular density in central specimens with a compromised surgical margin and lymph node metastasis. A high Ki-67 proliferation rate was significantly associated with tumors from patients with clinical stage IIb, IIIa, and IV disease. Multivariate Cox model analysis demonstrated that tumor location and stage, surgical margin, tumor size, and N stage were significantly related to survival time (P < .05). Quantitative staining of the tumor for Ki-67 and CD34-MVD served as prognostic factors (P < .05), which were more relevant than the surgical and pathological stage. Ki-67 greater than 5% and CD34-MVD greater than 7% staining comprise a subset of patients with higher death hazard; this outcome may harbor evidence for further prospective studies of target therapy after surgical resection.

Diabetic Ketoacidosis in Adolescents and Children: A Prospective Study of Blood versus Urine Ketones in Monitoring Therapeutic Response.

BACKGROUND: diabetic ketoacidosis (DKA) is a potentially lethal complication of diabetes mellitus (DM). There is no study in Indonesia that compares the much-preferred capillary beta hydroxybutirate (ß-OHB) measurement to urine acetoacetate in monitoring therapeutic response of DKA in adolescents. METHODS: a prospective study of 37 adolescents and children with DKA in Cipto Mangunkusumo Hospital was done between June 2006 and March 2011. The patients were followed until the time of DKA resolution. Hourly measurement of random blood glucose, capillary ß-OHB concentration, and urine ketones were done, while blood gas analysis and electrolyte were measured every four hours. RESULTS: median time to resolution was 21 (9-52) hours. Compared to urine ketones, capillary ß-OHB concentration showed stronger correlation with pH (r= -0,52, p= 0,003 vs r= -0,49, p= 0,005) and bicarbonate level (r=-0,60, p=0.000 vs r= -0.48, p=0.007) during the median time of DKA resolution. All capillary ß-OHB measurement yielded negative results at median time of DKA resolution, while urine ketones were still detected up to 9 hours after resolution. CONCLUSION: blood ketone concentration showed better correlation with pH and bicarbonate level, as a tool to monitor therapeutic response in DKA in adolescent, compared to traditional urine ketones test in adolescents.

Visual Detection of Multiple Nucleic Acids in a Capillary Array.

Multi-target, short time, and resource-affordable methodologies for the detection of multiple nucleic acids in a single, easy to operate test are urgently needed in disease diagnosis, microbial monitoring, genetically modified organism (GMO) detection, and forensic analysis. We have previously described the platform called CALM (Capillary Array-based Loop-mediated isothermal amplification for Multiplex visual detection of nucleic acids). Herein, we describe improved fabrication and performance processes for this platform. Here, we apply a small, ready-to-use cassette assembled by capillary array for multiplex visual detection of nucleic acids. The capillary array is pre-treated into a hydrophobic and hydrophilic pattern before fixing loop-mediated isothermal amplification (LAMP) primer sets in capillaries. After assembly of the loading adaptor, LAMP reaction mixture is loaded and isolated into each capillary, due to capillary force by a single pipetting step. The LAMP reactions are performed in parallel in the capillaries. The results are visually read out by illumination with a hand-held UV flashlight. Using this platform, we demonstrate monitoring of 8 frequently appearing elements and genes in GMO samples with high specificity and sensitivity. In summary, the platform described herein is intended to facilitate the detection of multiple nucleic acids. We believe it will be widely applicable in fields where high-throughput nucleic acid analysis is required.