Mass transport of lipopolysaccharide induced H2O2 detected by an intracellular carbon nanoelectrode sensor.

Hydrogen peroxide is a key component of the innate immune response, regulating how a cell responds to a bacterial threat; however, being transient in nature makes it extremely difficult to detect. We show the development of an improved biosensor capable of the rapid detection of the hydrogen peroxide produced intracellularly in response to both smooth and rough lipopolysaccharides (LPS) structures. The arising signal and mass transport behaviour to the electrodes were characterised. This response was detected utilising a single walled carbon nanotube-based sensor that has been functionalised with an osmium complex for specificity and detecting the change in intracellular concentrations of hydrogen peroxide through chronoamperometry. This was conducted within murine macrophage (RAW264.7) cells and using ultra-pure LPS extracted from two different serotypes of bacteria (0111:B4 and Re495). This allowed the comparison of the immune response when infected with different structures of LPS. We demonstrate that the hydrogen peroxide signal can be electrochemically detected within 3 seconds post injection. Combining the nature of the mass transport of hydrogen peroxide and concentration characteristics, a bacterial 'fingerprint' was identified. The impact of this work will be demonstrated in allowing us to develop a rapid diagnostic for bacterial detection.

Real-time bacterial detection with an intracellular ROS sensing platform.

Reactive oxygen species are highly reactive molecules that as well as being ubiquitously expressed throughout the body, are also known to be involved in many diseases and disorders including bacterial infection. Current technology has limited success in the accurate detection and identification of specific reactive oxygen species. To combat this, we have developed an electrochemical biosensor that is constructed from single walled carbon nanotubes that have been immobilised on an indium tin oxide surface functionalised with osmium-based compound. This sensor was integrated within mouse macrophage cells (RAW 264.7) with multiple serotypes of bacteria used to initiate an immune response. Intracellular hydrogen peroxide was then measured in response to the interaction of the lipopolysaccharides, present on the outer wall of Gram-negative bacteria, with the Toll-like Receptor 4. Additional controls of n-acetylcysteine and sodium pyruvate were implemented to prove the specificity of the sensor towards hydrogen peroxide. The sensors were found to have a lower limit of detection of 368 nM hydrogen peroxide. An increase in intracellular hydrogen peroxide was detected within 3 seconds of interaction of the bacteria with the macrophage cells. This low limit of detection combined with the rapid response of the sensor resulted in the unprecedented detection of hydrogen peroxide on a temporal level not previously seen in response to a bacterial threat. From the three serotypes of Gram-negative bacteria that were tested, there were distinct differences in hydrogen peroxide production. This proves that the innate immune system has the ability to respond dynamically and rapidly, after infection prior to the activation of the adaptive immune system.

Electrochemical communication with the inside of cells using micro-patterned vertical carbon nanofibre electrodes.

With the rapidly increasing demands for ultrasensitive biodetection, the design and applications of new nano-scale materials for development of sensors based on optical and electrochemical transducers have attracted substantial interest. In particular, given the comparable sizes of nanomaterials and biomolecules, there exist plenty of opportunities to develop functional nanoprobes with biomolecules for highly sensitive and selective biosensing, shedding new light on cellular behaviour. Towards this aim, herein we interface cells with patterned nano-arrays of carbon nanofibers forming a nanosensor-cell construct. We show that such a construct is capable of electrochemically communicating with the intracellular environment.

A Case of Bilateral Cystic Partially Differentiated Nephroblastoma vs Cystic Wilms' Tumor: Highlighting a Diagnostic Dilemma.

Cystic partially differentiated nephroblastoma (CPDN) is a rare multicystic renal tumor along the spectrum of cystic nephroma and cystic Wilms' tumor. There have only been two previously reported cases of bilateral CPDN in the literature. We present here a case of bilateral CPDN vs cystic Wilms' tumor treated with neoadjuvant and adjuvant chemotherapy in addition to a bilateral partial nephrectomy. We also review the relevant literature regarding CPDN in an effort to aid in diagnosis and management of these rare cystic renal tumors.

High-altitude precipitation and exacerbation of protein-losing enteropathy after a Fontan operation.

We describe the development and exacerbation of protein-losing enteropathy after relocating to an environment at an altitude of 3695 feet in El Paso, Texas, in a patient who had undergone a Fontan operation. This report should heighten awareness to the possibility of such patients developing protein-losing enteropathy at high-altitude, with hypoxemia-induced pulmonary vasoconstriction, and subsequent elevation of central venous pressure, the most likely underlying mechanism.

Recommendations and opinions for the use of point-of-care testing for hospitals and primary care: summary of a 1999 symposium.

As part of a symposium on laboratory medicine, a colloquium on point-of-care testing was held in June 1999 where four experts were invited to produce recommendations and opinions on the use of point-of-care testing under various clinical venues. Each commented on costs for providing POCT services. A total of eleven recommendations and four opinions were rendered and discussed in an open forum. While one expert concluded that some forms of POCT are less expensive than central laboratory testing if entire laboratory workstations are eliminated, another expert suggested that POCT offered little advantage if rapid transport systems are available. A recommendation was made that POCT be considered for analytes that have a required reporting turnaround time of <30 min, and that the goals for precision and accuracy should be dictated by the clinical need and not by analytical limitations. Recommendations for POCT in specific clinical situations include use of glycated hemoglobin and urine albumin testing with personal glucose monitoring at the time of consultation, use of glycated albumin for gestational diabetes, leukocyte esterase and nitrite testing in urine to screen for urinary tract infections, coagulation tests for monitoring patients on oral anticoagulant therapy and in the operating room, testing for H. pylori for patients with dyspepsia, and cardiac markers and urine drugs-of-abuse testing in the emergency department.

The cardiovascular responses of the red-eared slider (Trachemys scripta) acclimated to either 22 or 5 degrees C. II. Effects of anoxia on adrenergic and cholinergic control.

Cardiovascular control in cold-acclimated freshwater turtles during chronic anoxic exposure is not well understood. We tested the hypothesis that the observed bradycardia in Trachemys scripta results from increased cholinergic inhibitory tone and reduced sympathetic activity. Cardiovascular status was measured in vivo in turtles acclimated to either 22 degrees C or 5 degrees C and either acutely exposed (6 h) to anoxia at 22 degrees C or chronically exposed (22 days) to anoxia at 5 degrees C. In 22 degrees C-acclimated turtles, injection of the cholinergic antagonist atropine induced a significant tachycardia under both normoxic and anoxic conditions. However, in 5 degrees C-acclimated turtles, atropine injection had little effect on heart rate. Therefore, cholinergic control of heart rate was suppressed during cold acclimation; instead, temperature effects are more important in bringing about bradycardia, while the intrinsic effects of anoxia and acidosis are probably important during chronic anoxia. Injection of adrenaline caused a pressor response through increased systemic resistance at both acclimation temperatures. This response was blunted by acute and chronic anoxic exposure, suggesting that systemic vasomotor control was altered independently of acclimation temperature. This anoxic blunting may be related in part to the anoxia-induced increase in systemic resistance. Injection of nadolol after atropine decreased systemic cardiac output. The tonic beta-adrenergic cardiac stimulation was attenuated by acute and chronic anoxic exposure. Some of this attenuation of beta-adrenergic control could be attributed to the 39-40 % reduction in cell surface beta-adrenoreceptor density in the ventricles of these turtles that accompanied acute and chronic anoxic exposure. In conclusion and contrary to our original hypothesis, cholinergic and adrenergic control of the cardiovascular system in turtles was attenuated under cold anoxic conditions, perhaps assisting in the depressed physiological state of these animals.

The cardiovascular responses of the red-eared slider (Trachemys scripta) acclimated to either 22 or 5 degrees C. I. Effects of anoxic exposure on in vivo cardiac performance.

The extreme anoxia-tolerance of freshwater turtles under cold conditions is well documented, but little is known about their cardiac performance in such situations. Using chronic catheterization techniques, we measured systemic cardiac power output (PO(sys)), systemic cardiac output (Q(.)(sys)), heart rate (fh), systemic stroke volume (V(s,sys)), systemic resistance (R(sys)) and mean arterial pressure (P(sys)) in red-eared sliders (Trachemys scripta). The effects of cold acclimation and anoxic exposure were studied. Turtles were acclimated to either 22 degrees C or 5 degrees C, and the anoxic exposure was either acute (6 h) at 22 degrees C or chronic (3 weeks) at 5 degrees C. Cold acclimation alone decreased PO(sys) by 15-fold, representing a Q(10) of 8.8. In addition, fh and V(s,sys) decreased significantly, while R(sys) increased and moderated the arterial hypotension. Acute and chronic anoxic exposures significantly decreased PO(sys), V(s,sys), fh and P(sys) and increased R(sys). But the changes were qualitatively much larger with chronic anoxia. For example, acute anoxia in 22 degrees C-acclimated turtles decreased PO(sys) by 6.6-fold, whereas chronic anoxia in 5 degrees C-acclimated turtles decreased PO(sys) by 20-fold. The remarkable cardiovascular down-regulation that accompanies long periods of cold anoxia in these turtles was characterized by comparing cardiovascular status during chronic anoxia at 5 degrees C with that during normoxia at 22 degrees C. Cardiac PO(sys) was reduced 330-fold, through decreases in Q(.)(sys) (120-fold), fh (24.2-fold), V(s,sys) (5.7-fold) and P(sys) (2. 2-fold), while R(sys) was increased 64.6-fold. We also compared cardiac glycolytic rates by assuming that PO(sys) was proportional to ATP supply and that glycolysis yielded 18 times less ATP per mole of glucose than oxidative metabolism. At 22 degrees C, the 6.6-fold decrease in PO(sys) with anoxia suggests that a Pasteur effect was needed in cardiac tissues during acute anoxia. However, this would not be so with chronic anoxia at 5 degrees C because of the 22-fold decrease in PO(sys). We propose that the suppression of the Pasteur effect and the large Q(10) values for cold acclimation would conserve glucose stores and enable turtles to withstand anoxia much longer under cold than under warm conditions.

Evaluation of i-STAT portable clinical analyzer in a neonatal and pediatric intensive care unit.

OBJECTIVE: To evaluate the Point-of-Care (POC) i-STAT system for measuring blood gases (pH, pCO2, pO2) and whole blood electrolytes (sodium, Na+, potassium, K+ and ionized calcium, iCa2+) in the neonatal and pediatric intensive care units. DESIGN AND METHODS: The i-STAT system was evaluated for imprecision, necessity of running quality control and accuracy. Comparison of patients' samples analyzed by the i-STAT system and the Ciba Coming 288 blood gas analyzer were performed. The reliability of the i-STAT system when performed by non-laboratory personnel was assessed. RESULTS: The system was evaluated for imprecision and linearity using three concentrations of aqueous standards. Except for pO2, the %CVs were < 3.0 for all the analytes (pH, pCO2, Na+, K+ and iCa2+) at all the three concentrations. Using whole blood studies the precision data gave %CVs that were < 3.5 for all the analytes. Linearity studies showed good linearity over the five different concentrations tested. Comparison of the i-STAT and the Ciba Coming 288 blood gas analyzer was assessed by split sample measurement. Patients' results from the i-STAT correlated well with the Ciba Coming 288 blood gas analyzer (r = 0.99 for pH, pCO2 and pO2 and 0.95 to 0.99 for Na+ and K+) with the exception of iCa2+ (r = 0.73). There was no significant difference in the results when operated by PICU/NICU nurses or laboratory personnel. A further study was made to assess whether routine quality control (QC) samples are necessary when using the i-STAT system. The regression analysis (slope and correlation coefficient) of the results from instruments run with and without QC samples gave results close to 1, indicating that there is no need to run additional quality control (QC). CONCLUSION: The POC testing analyzer i-STAT is a reliable alternative to the traditional blood gas analyzers and provides marginal improvement in turnaround time when compared with the service received from the PICU/NICU laboratory. Costs need to be carefully controlled.

Failure of current guidelines for cholesterol screening in urban African-American adolescents.

OBJECTIVE: The National Cholesterol Education Program (NCEP) and the American Academy of Pediatrics recommend selective screening to detect children and adolescents with hypercholesterolemia. We compared the effectiveness of these guidelines with other potential screening strategies in urban African-American adolescents. SUBJECTS AND METHODS: Two hundred sixty African-American adolescents and young adults (192 females and 68 males; age range, 12 to 20 years) who were free from illnesses or medications that disrupt lipid metabolism were included in the study. Participants completed a questionnaire regarding their smoking habits, blood pressure, contraceptive pill use, and family history of early heart disease and high cholesterol and had their blood lipids, lipoproteins, and apolipoproteins measured (152 fasting and 108 nonfasting). RESULTS: Nineteen percent of participants reported family histories of hypercholesterolemia; 26% reported family histories of premature heart disease; and 8% had family histories of both hypercholesterolemia and premature heart disease. Therefore, 37% of these participants would have been targeted for cholesterol screening, compared with the 25% predicted by the NCEP. Less than 50% of the participants with low-density lipoprotein cholesterol (LDL-C) levels greater than 110 or 130 mg/dL would have been detected by selective screening. Total cholesterol was superior as a screening test to apolipoprotein B in predicting LDL-C levels greater than 110 mg/dL (sensitivity, 92% vs 59%). However, total cholesterol minus high-density lipoprotein cholesterol showed better positive predictive value (100%) at LDL-C levels greater than 110 mg/dL than total cholesterol in the fasting (80%) and total groups (90%). CONCLUSIONS: In this population, selective screening with total cholesterol, as recommended by the NCEP, has such poor sensitivity and positive predictive value that other options may be superior. As an alternative, we recommend the measurement of high-density lipoprotein cholesterol together with total cholesterol at the initial screening step in adolescents and universal screening for those older than 16 years to capture a greater proportion of young adults with increased LDL-C. Furthermore, we recommend using the less stringent treatment guidelines established by the Adult Treatment Panel II for premenopausal women and men younger than 35 years for older adolescents and young adults.

Stability of twenty-five analytes in human serum at 22 degrees C, 4 degrees C, and -20 degrees C.

The stability of 25 analytes from serum of healthy donors was determined at room temperature, 4 degrees C, and -20 degrees C over 48 h, 14 days, and 4 months, respectively. Glucose, blood urea nitrogen, sodium, potassium, chloride, creatinine, calcium, phosphorus, uric acid, total protein, albumin, triglycerides, lipase, total creatine kinase, gamma-glutamyltransferase, iron, magnesium, and cholesterol were stable at all three temperatures for the specified times. Carbon dioxide, aspartate and alanine aminotransferases, lactate dehydrogenase, amylase, alkaline phosphatase, and high-density lipoprotein cholesterol demonstrated some loss over time. Proper storage temperatures and times must be considered for these analytes if measurement is not to take place immediately after specimen collection.

Is heparinized plasma suitable for use in routine biochemistry?

Using lithium heparin plasma and serum, we compared the values of 27 biochemical analyses performed with a Kodak Ektachem 500 analyzer. For 16 of the 27 tests, we observed statistical differences between the mean results obtained from the analysis of heparinized plasma and those obtained from the analysis of serum (Student t-test; P < .001). However, none of these differences were medically significant. When the concentration of lithium heparin was increased to three and five times normal (reflecting incomplete Vacutainer tube filling), alanine aminotransferase, amylase, aspartate aminotransferase, lipase, and potassium all exhibited some changes compared with serum. Therefore, heparin may be used to shorten the turnaround time in the routine biochemistry laboratory, but the anticoagulant tubes should be completely filled to prevent spurious intraindividual variations in serial specimen analysis.

Evaluation of the Technicon Immuno I random access immunoassay analyzer and calculation of pediatric reference ranges for endocrine tests, T-uptake, and ferritin.

OBJECTIVES: Evaluation of the precision, accuracy, and user-friendliness of the Technician Immuno I. Calculation of pediatric reference ranges for ferritin and endocrine tests run on Immuno I. METHODS: Precision and accuracy were measured using controls and method comparison studies. Pediatric reference ranges were calculated by comparing the Immuno I results for 100 patients with those of the Abbott IMx and TDx. The regression equation obtained was then used to convert the IMx and TDx reference ranges to reference ranges for the Immuno I. RESULTS: The Immuno I provided both accurate and precise measurement of drugs and endocrine hormones. Pediatric reference ranges were obtained for ferritin and all endocrine tests. CONCLUSION: The Immuno I is user-friendly and provides reliable measurement of both the drugs tested and endocrine tests on a micro-sample. Reagent and curve stability are excellent.