Chitosan Film Sensor for Ammonia Detection in Microdiffusion Analytical Devices.

Chitosan films have attracted increased attention in the field of sensors because of chitosan's unique chemico-physical properties, including high adsorption capacity, filmability and transparency. A chitosan film sensor was developed through the dispersion of an ammonia specific reagent (Nessler's reagent) into a chitosan film matrix. The chitosan film sensor was characterized to assess the film's properties by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA), scanning electron microscopy (SEM) and differential scanning calorimetry (DSC). A gas diffusion device was prepared with the chitosan film sensor, enabling the collection and detection of ammonia vapor from biological samples. The chitosan film sensor color change was correlated with the ammonia concentration in samples of human serum and artificial urine. This method enabled facile ammonia detection and concentration measurement, making the sensor useful not only in clinical laboratories, but also for point-of-care devices and wherever there is limited access to modern laboratory facilities.

An origami microfluidic paper device for on-site assessment of urine tampering. First use of Nessler's reagent for the colorimetric determination of creatinine.

The relevance of the problem of urine tampering is well-known in forensic toxicology, with sample dilution being the most used method to cheat toxicological controls. Among the criteria to assess urine integrity, the quantification of creatinine probably represents the most popular method. The present paper presents a simple and low-cost analytical device for on-site creatinine determination as first-line screening for urine dilution. The proposed microfluidic devices were designed as a three-dimensional origami pattern. The device included three colorimetric reactions based on picric acid (PA-based reagent), 3,5-dinitrobenzoic acid (DNBA-based reagent), and Nessler's reagent. The last one, to the best of our knowledge, has never been used before for creatinine determination. In order to assure the highest ease and economy of operation, the color detection and data processing were performed using a built-in smartphone camera and the associated software. The optimized device showed a detection limit of 0.02 g/L. The proposed method was used for the qualitative screening for urine dilution of 48 samples, showing a diagnostic sensitivity and specificity for PA-based, DNBA-based and Nessler's reagent of 83.3%-80.0%, 72.2%-70.0%, and 100.0%-93.3% respectively, versus reference enzymatic method adopting a cut-off of 0.2 g/L. In conclusion, the present preliminary study shows that the proposed device could be a useful tool for on-site screening for urine tampering at the time of sample collection for toxicological testing.

Development of a new ultra-high-performance liquid chromatography-tandem mass spectrometry method for the determination of digoxin and digitoxin in plasma: Comparison with a clinical immunoassay.

Cardiac glycosides digoxin and digitoxin are used in therapy for the treatment of congestive heart failure. Moreover, these compounds can be responsible for intoxication cases caused by fortuitous ingestion of leaves of Digitalis. Due to the narrow therapeutic range of these drugs, therapeutic drug monitoring is recommended in the clinical practice. In this context, immunoassays-based methods are generally employed but digoxin- and digitoxin-like compounds can interfere with the analysis. The aim of this study was to develop and validate an original UPLC-MS/MS method for the determination of digoxin and digitoxin in plasma. The method shows adequate sensitivity and selectivity with acceptable matrix effects and very good linearity, accuracy, precision, and recovery. A simple liquid-liquid extraction procedure was used for sample clean-up. The method was applied for the analysis of n = 220 plasma samples collected in two different clinical chemistry laboratories and previously tested by the same immunoassay. The statistical comparison showed a relevant negative bias of the UPLC-MS/MS method versus the immunoassay. These results are consistent with an immunoassay overestimation of digoxin plasmatic levels due to cross-reaction events with endogenous digoxin-like substances.

Hypeprolactinemia: still an insidious diagnosis.

Hyperprolactinemia can have different causes: physiological, pharmacological, and pathological. When investigating the etiology of hyperprolactinemia, clinicians need to be aware of several conditions leading to misdiagnosis. The most popular pitfalls are: acute physical and psychological stress, macroprolactin, hook effect, even though antibodies interferences and biotine use have to be considered. A 52-year-old woman was referred to Endocrinology clinic for oligomenorrhoea and headache. She worked as a butcher. Hormonal evaluation showed very high PRL (305 ng/ml, reference interval: <24 ng/ml) measured with the ECLIA immunoassay analyzer Elecsys 170. The patient's pituitary MRI was normal and macroprolactin was normal. Hormonal workup showed LH: 71.5 mU/ml (2-10.9 mU/ml), FSH: 111.4 mU/ml (3.9-8.8 mU/ml), Estradiol: 110.7 pg/mL (27-122 pg/ml). Since an interference was suspected, the sample was sent to another laboratory using a different assay. After antibody blocking tubes treatment (Heterophilic Blocking Tube, Scantibodies) PRL was 28.8 ng/ml (reference interval < 29.2 ng/ml). Analytical interference should be suspected when assay results are not consistent with the clinical picture. Endogenous antibodies (EA) include heterophile, human anti-animal, autoimmune and other nonspecific antibodies, and rheumatoid factors, that have structural similarities and can cross-react with the antibodies employed by the immunoassay, causing hyperprolactinemia misdiagnosis. The patient's job (butcher), led us to suspect the presence of anti-animal antibodies. Clinicians should also carefully investigate the use of supplements. Biotin can falsely increase hormone concentration in competitive assays. Many clinicians are still not informed about these pitfalls that are not mentioned in some recent reviews on PRL measurement.

CDT reference values for monitoring chronic alcohol abuse in pregnancy.

INTRODUCTION AND AIM: Carbohydrate Deficient Transferrin (CDT) is one of the most used biomarkers for monitoring alcohol use in pregnancy. However, its effective application in this context is hampered by the demonstrated physiological progressive increase during pregnancy (even in abstinent women) of CDT values, which in the third trimester can reach values close or exceeding the cut-offs usually adopted in clinical and forensic diagnostics. The present work was aimed at the re-assessment of CDT reference values in pregnancy. MATERIALS AND METHODS: The CDT analysis was performed by a validated HPLC-UV Vis method on 284 serum samples of women with a physiological pregnancy and on 370 sera of non-pregnant woman from the general population (control group). All the samples were tested also for GGT for excluding alcohol abuse. The statistical analysis was performed using the MedCalc® Statistical Software. RESULTS: The re-definition of the specific reference concentrations was carried out according to the Horn and Pesce Robust Method. The resulting CDT upper reference values were 1.45%, 2.01% and 2.05% in the first, second, and third trimester, respectively. CONCLUSIONS: In order to prevent the development of maternal and fetal prenatal alcohol exposure complications, the use of alcohol biomarkers, including CDT, has been proposed. However, this biomarker, in the monitoring of alcohol use in pregnancy, has so far been applied adopting the same cut-off used for general population without taking into consideration the progressive physiological increase of its value throughout the pregnancy. In the present study, a specific re-assessment of the CDT reference concentrations of each trimester is reported.

Critical Evaluation of the Association Between Elevated Mean Corpuscular Volume and Alcohol-Related Traffic Accidents: A Retrospective Study on 6244 Car Crash Cases.

BACKGROUND: Erythrocyte mean corpuscular volume (MCV) has been used for decades as a biomarker of chronic alcohol abuse and in the treatment of alcohol dependence. More recently, it has also been adopted to investigate the fitness of subjects to hold the driving license to prevent traffic accidents. So far, however, the studies on the association of MCV with an increased risk of alcohol-associated car accidents are extremely scarce, if not totally absent. To the best of our knowledge, the present work is the first specifically aimed at studying a plausible association between elevated MCV and crash accidents correlated with alcohol abuse. METHODS: A total of 6,244 drivers involved in traffic accidents underwent mandatory laboratory analyses including blood alcohol concentration (BAC) determination and MCV analysis. BAC and MCV determinations were performed by headspace gas chromatography and complete blood count, respectively. RESULTS: The chi-square test evaluating the proportions of subjects with elevated MCVs (>95 fl) yielded a highly significant result (χ2  = 68.0; p < 0.001) in the blood samples where the BAC was above the legal limit (i.e., >0.5 g/l). However, when considering only drivers showing BACs in the range of 0.51 to 1.5 g/l, the frequencies of elevated MCV values are fairly comparable (χ2  = 0.062, p = 0.80). In contrast, limiting the evaluation to BACs > 1.5 g/l, the frequency of elevated MCVs raised to 19.1% (χ2  = 58.9, p value < 0.001 vs. the group with BAC within the legal limits). CONCLUSIONS: The present observations show that MCV increases are typically associated with drivers involved in accidents only if driving under severe alcohol intoxication, leading to a preliminary conclusion that, in the context of the certification of the fitness to the driving license, MCV fails to reveal individuals at risk who tend to drive in a condition of low-to-moderate alcohol intoxication.

"Positive" urine testing for Cannabis is associated with increased risk of traffic crashes.

Although recent Cannabis use is widely reported to be associated with drug-related traffic accidents, the evidence that Cannabis users show an increased risk of being involved in road crashes is still not unequivocally proved. The purpose of the present work is to provide an objective assessment of this hypothesis, by comparing the frequency of occurrence of positive urine analyses in drivers involved in traffic accidents (n = 1406) with that observed in a control population undergoing mandatory urine drug testing (n = 1953). Urine analyses for drugs of abuse were performed by screening immunometric techniques followed by confirmation with UHPLC-QQQ MS, adopting a cut-off concentration for THC-COOH of 15 ng/mL. A case was classified as "positive" when a driver admitted to hospital for road traffic injuries showed urine concentrations of THC-COOH higher than the cut-off. All samples showing positive results for any other controlled drug in urine or blood alcohol concentrations >0.5 mg/mL were excluded from the study. Subjects positive to THC-COOH, and negative to all the other tested substances were 116 in Group 1 (8.2%) and 16 in Group 2 (0.8%). Subjects resulting negative to any tested substances were 1290 in Group 1 and 1937 in Group 2. The frequency of THC-COOH detection in the two groups was compared by using the "chi square" test, which resulted = 119.57, i.e. highly significant (P <<< 0.01). The Odds Ratio of the two groups was =10.88, showing a high degree of association between the presence of THC-COOH in urine and the occurrence of traffic accidents (P < 0.0001). The presented data, proving a high degree of association between Cannabis use and the occurrence of traffic accidents with injuries of the driver, support the use of urine testing for Cannabis in the procedures for the issuing of the driving licence, particularly in the case of subjects formerly or presently using Cannabis. This finding looks even more relevant in the present times, because of the increasing success of the policies of legalization of Cannabis for medical and non-medical purposes.

Prognostic role of serum concentrations of high-sensitivity C-reactive protein in patients with metastatic colorectal cancer: results from the ITACa trial.

Serum levels of C-reactive protein are (CRP) higher in patients with neoplastic conditions and numerous studies have been performed to clarify the etiologic and prognostic role of the high-sensitivity CRP (hs-CRP) in cancer. Our study was conducted on patients enrolled in the prospective randomized "Italian Trial in Advanced Colorectal Cancer (ITACa)" to assess hs-CRP levels and their impact on overall survival (OS) and progression-free survival (PFS). Serum samples from 132 ITACa patients were collected at baseline and 2 months after starting first-line chemotherapy. The supernatant was immediately transferred to cryovials and stored at -80°C. After thawing, hs-CRP was measured with the Cobas c501 analyzer. High levels of hs-CRP (≥ 13.1 mg/L) were associated with poorer median PFS (p < 0.0001) and OS (p < 0.0001) than low hs-CRP levels (< 13.1 mg/L). hs-CRP values in 107 patients were evaluated again after 2 months of therapy, revealing that patients with low hs-CRP levels in both baseline and second serum samples had the best median PFS and OS. Our study confirms the prognostic value of hs-CRP in patients with metastatic colorectal carcinoma.

Assessment of an expert system for the automated validation of electrophoretic profiles.

BACKGROUND: The Core-lab of the Greater Romagna Area Hub Laboratory carries out about 250,000 capillary electrophoresis assays/year. The huge workload demands the assessing of an Experimental Expert System (EES) capable to sort out the negative samples. METHODS: Capillarys 2 analyzer has been employed coupled with an EES (based on five simple rules) integrated with the electrophoretic test management software PhoresisCore for assessing the entire workload of a week (5,683 samples). The classification was compared with that of two expert laboratorians. RESULTS: The expert system automatically classified 2974 profiles as negative and no positive samples were erroneously classified as negative (negative predictive value: 100%). CONCLUSIONS: The EES sensitivity was 100% and the FTE required for the validation was reduced from 1.26 to 0.63. The EES could be easily implemented in routine activity embedded in a middleware or directly running in the analyzer improving the workflow.

Vascular access: an historical perspective from Sir William Harvey to the 1956 Nobel prize to André F. Cournand, Werner Forssmann, and Dickinson W. Richards.

Sir William Harvey (1578-1657), who had many precursors, discovered blood circulation in 1628 after a significant number of anatomic dissection of cadavers; his studies were continued by Sir Christopher Wren and Daniel Johann Major. The first central vein catheterization was performed on a horse by Stephen Hales, an English Vicar. In 1844, a century later, the French biologist Claude Bernard attempted the first carotid artery cannulation and repeated the procedure in the jugular vein, again on a horse. He was first to report the complications now well known to be associated with this maneuver. In 1929 Werner Forssmann tried cardiac catheterization on himself, but could not investigate the procedure further since his findings were rejected and ridiculed by colleagues. His work was continued by André Frédéric Cournand and Dickinson Woodruff Richards Jr in the United States. In 1956 the three physicians shared the Nobel Prize for Medicine for their studies on vascular and cardiac systems. The genius and the perseverance of the three physicians paved the way towards peripheral and central catheter vein placement, one of the most frequently performed maneuvers in hospitals. Its history still remains unknown to most and deserves a short description.