The direct barbituric acid assay for nicotine metabolites in urine: a simple colorimetric test for the routine assessment of smoking status and cigarette smoke intake

The qualitative direct barbituric acid (DBA) method of detecting urine nicotine metabolites was modified to make it quantitative. The performance of the quantitative DBA method was compared with the qualitative method and an established cotinine radio-immunoassay (RIA), using a panel of urines from 128 reported smokers and 383 reported non-smokers. The quantitative DBA method results were highly correlated with the cotinine RIA results, r = 0.85. The coefficients of variation for the two methods were 6% and 10%, respectively. Assuming that the reported smoking history was correct the qualitative DBA method gave a smoking detection rate of 91% and a false positive rate of 3%. At cut-off levels chosen to yield the same false positive rate the quantitative DBA method detected 93% of smokers, close to that of 98% detected with the cotinine RIA. The quantitative DBA method can be used to analyse over 170 samples per day compared to about 70 per day by RIA. It is therefore a fast and inexpensive alternative to cotinine assays for the assessment of smoking status and cigarette smoke intake.     

Assessment of the automated colorimetric and the high-performance liquid chromatographic methods for nicotine intake by urine samples of smokers' smoking low- and medium-yield cigarettes

We have compared the high-performance liquid chromatographic method with the direct barbituric acid test in an assessment of nicotine exposure. The effect of the endogenous colour in urine on the direct barbituric acid method was also studied. The applicability of methods was evaluated with urine samples from 15 smokers who smoked 5, 10 and 20 low- and medium-nicotine cigarettes per day. Assessments of nicotine intake with the methods were well correlated, although the high-performance liquid chromatographic method detects only cotinine and the direct barbituric method most of the nicotine metabolites. Before endogenous colour subtraction in the DBA method the correlation coefficient was 0.558 and after that 0.784. Both methods indicated similar changes in nicotine exposure with the change of brand and also a dose dependent relationship to the number of cigarettes smoked. The coefficients of variation for both of the methods were 3.4%. The endogenous colour determination lessened the capacity of direct barbituric acid method to one half being about 150 samples per day. With the high-performance liquid chromatographic method the capacity was about 50 samples per working day.     

An automated amidolytic assay of thrombin generation: an alternative for the prothrombin-time test.

An automated method is presented for the determination of thrombin generation in plasma during activation by thromboplastin. The thrombin generated is allowed to split the chromogenic substrate Tol-Gly-Pro-Arg-pNA yielding p-nitroaniline. The increase in absorbance at 410 nm is recorded. This assay may serve as a specific, precise and fast alternative for the conventional clotting tests: "Prothrombin time" and "Thrombotest".     

The efficacy and predictive value of the Heavy Smoking Index for smoking cessation among daily smokers in a public health center

Objective: The present study tested the effectiveness of the Heavy Smoking Index (HSI) for the screening of high nicotine dependence and the predictive value of HSI on smoking cessation within a community sample in a public health center. Methods: The Fagerström Test for Nicotine Dependence (FTND) scores from 1069 smokers who visited a public health center in Korea was analyzed. Receiver operating characteristic analyses were performed to calculate sensitivity and specificity values to compare the effectiveness of HSI to items 1 and 4 of FTND. In addition, HSI at baseline was found to predict smoking cessation after 4 weeks and after 6 months using logistic regression. We assessed whether HSI at baseline would predict smoking cessation after 4 weeks and after 6 months using logistical regression. Results: For the results, a score of 4 on HSI was considered optimal. Additionally, the predictive value for smoking cessation of both HSI and FTND were found to be statistically valid at baseline. Conclusions: Our results indicate that HSI is a useful brief screening tool to detect high nicotine dependence in a public health center.     

Pediatric reference values for urine particle quantification by using automated flow cytometer: Results of a multicenter study of Italian urinalysis group

Objectives

The purpose of this Italian multicenter study was to define pediatric upper reference values for urine particle quantification by using automated flow cytometry.

Design and methods

Four hospital-based clinical laboratories participated in this multicenter investigation, which included a total study population of 161 Italian children aged from 1 to 12 years. Two laboratories used Sysmex UF-100 and analyzed 86 children, whereas the other two used Sysmex UF-1000i and analyzed 75 subjects. Particle quantification included the analysis of white blood cells (WBC), red blood cells (RBC), squamous epithelial cells (EC), transitional epithelial cells (TC), casts (CAST) and bacteria (BACT).

Results

The upper reference values in subjects tested with the Sysmex UF-100 were 9.7 WBC/μL, 10.1 RBC/μL, 7.5 EC/μL, 2.5 TC/μL, 0.7 CAST/μL and 3090 BACT/μL, whereas the upper reference values in subjects tested with the Sysmex UF-1000i were 10.5 WBC/μL, 8.3 RBC/μL, 7.2 EC/μL, 2.9 TC/μL, 0.7 CAST/μL and 48 BACT/μL. No statistically significant differences between genders were found in the value distribution of any of the parameters tested. Similarly, no statistically significant differences were observed between the two urine analyzers, except for BACT.

Conclusions

Automated analysis of urine particles appears a suitable means to optimize the workflow of routine urinalysis of children specimens. The upper reference limits for pediatric subjects obtained in this study were comparable to those previously reported in the literature, with no significant differences between genders and analyzers.     

斑点免疫酶渗滤法检测尿液绒毛膜促性腺激素

为了提供一种简便、快速、可靠的检测尿液绒毛膜促性腺激素（ＨＣＧ）的方法，以自制的抗ＨＣＧβ－亚单位单克隆抗体标记辣根过氧化物酶，用抗ＨＣＧ多克隆抗体包被硝酸纤维素膜，建立了一种检测尿液ＨＣＧ的快速斑点免疫酶渗滤法，并制成试剂盒供应临床。该方法简便，整个检测过程只需２分钟。停经５～７天检测即可获阳性结果，斑点色泽鲜艳，结果易于判断。与一般一步酶联免疫吸附法（ＥＬＩＳＡ）比较，符合率为９８％，消除了一般一步法ＥＬＩＳＡ常见的前带现象。经临床验证１０００例，结果与诊断符合率为１００％。试剂盒置４℃和３７℃分别可保存半年和１０天以上。该试剂盒可作为诊断早早孕的常规试剂盒。     

Bidirectional (positive/negative) interference of spironolactone,canrenone, and potassium canrenoate on serum digoxin measurement: elimination of interference by measuring free digoxin or using a chemiluminescent assay for digoxin

Spironolactone and potassium canrenoate (aldosterone antagonist diuretics) are often used with digoxin in clinical practice. Spironolactone, potassium canrenoate, and their common metabolite canrenone cross-react with the fluorescence polarization immunoassay (FPIA) for digoxin, and can falsely elevate serum digoxin concentrations. Serum digoxin concentrations were falsely lowered when the microparticle enzyme immunoassay (MEIA) was used. Aliquots of drug-free serum were supplemented with therapeutic and above-therapeutic concentrations of spironolactone, canrenone, and potassium canrenoate, and apparent digoxin activities were measured. We observed digoxin-like activities in the FPIA, but observed no activity with the MEIA or the chemiluminescent assay (CLIA). However, when serum digoxin pools prepared from patients receiving digoxin were supplemented with these compounds, we observed suppression of total digoxin levels with the MEIA. In contrast, no interference was observed in the presence of these compounds when CLIA was used for digoxin measurement. These compounds are strongly protein-bound, and no apparent digoxin activity was observed in the protein-free ultrafiltrate when drug-free sera were spiked with high levels of these compounds. Taking advantage of strong protein binding of these compounds and weak protein binding of digoxin (25%), interference of spironolactone, canrenone, and potassium canrenoate in FPIA and MEIA digoxin assays can be mostly eliminated by monitoring free digoxin concentration. Another approach to avoid this interference is to use the CLIA digoxin assay.     

The ARCHITECT galectin-3 assay: comparison with other automated and manual assays for the measurement of circulating galectin-3 levels in heart failure

Heart failure (HF) is a common disease and affects millions of patients worldwide. Diagnosis, risk assessment and treatment of HF are difficult and therefore there is a need for additional tools to improve clinical performance. Biomarkers may be helpful in this respect. Galectin-3 is a relatively new biomarker that has been shown to have strong associations with the development of HF. Galectin-3 plays a role in inflammation and fibrosis, which are key elements in the pathophysiology of HF. Circulating plasma or serum galectin-3 levels have strong associations with the severity of HF and may be used to prognosticate or risk-stratify HF patients. Currently, there are several commercially available assays that can measure circulating galectin-3. This article describes the role galectin-3 plays in HF and its prognostic consequences. We will summarize the technical specifications of various manual and automated galectin-3 assays, which may help in HF management.     

Comparison of the analytic sensitivities of a recombinant immunoblot assay and the radioimmune precipitation assay for the detection of antibodies to Trypanosoma cruzi in patients with Chagas disease

The diagnosis of chronic Chagas disease usually is made by detecting antibodies to Trypanosoma cruzi, the protozoan parasite that causes this illness. A highly sensitive and specific immunoblot assay developed by us showed a higher analytic sensitivity than the radioimmune precipitation assay, which is used widely as a confirmatory test.     

Nosocomial transmission of varicella to a healthcare provider positive for anti-varicella zoster virus antibodies: nonprotective positivity with an immune adherence hemagglutination assay

A 24-year-old male healthcare provider, having attended a varicella patient 2 weeks before, developed varicella himself. He had shown a positive result for anti-VZV antibodies measured with an immune adherence hemagglutination assay (1:4) 1 year before. The present case shows that a positive result with this assay does not necessarily indicate protection against VZV infection.     

Knowledge,attitudes, behavioral and organizational factors of health professions students for a competent smoking cessation practice: An instrument adaptation and psychometric validation study in Spanish and English samples

BackgroundTo improve smoking cessation, training of health professions students is essential. However, no specific instrument is available to assess factors that may affect students' learning about smoking cessation practice.AimTo adapt and validate the Knowledge, Attitudes, Behaviors and Organization questionnaire in the population of undergraduate health professions students.DesignMethodological research.MethodsThe researchers conducted this study with 511 Spanish and 186 English health professions students from four different universities. We used a four-step approach: 1) adaptation of the items to the target population and validation of the content by a panel of experts; 2) a pilot study to test face validity; 3) linguistic adaptation of the Spanish version to English; and 4) the psychometric assessment based on construct validity, criterion validity and internal consistency.ResultsExploratory factor analysis revealed four subscales for the Spanish version, namely ‘Individual knowledge and skills’, ‘Individual attitudes and beliefs’, ‘Organizational support’ and ‘Organizational resources’, which accounted for 85.1% of the variance. Confirmatory factor analysis in the holdout Spanish and English samples revealed adequate goodness-of-fit values, supporting the factor structure. Hypotheses testing demonstrated significant differences by capacitation in smoking cessation interventions and degree courses, providing further evidence regarding construct validity. All the subscales correlated positively with the criterion variables (5 A’s smoking cessation model), except for the ‘Organizational resources’ subscale, which was not significantly correlated with the 5 A's. The overall Cronbach’s alpha was.83 for the Spanish version and.88 for the English one.ConclusionsOur results provide empirical support for the use of the Knowledge, Attitudes, Behaviors and Organization questionnaire for Students as a reliable and valid instrument to assess knowledge, attitudes, behaviors and organization perceptions in health professions students, which is essential for competent smoking cessation practice. Interestingly, ‘Organizational resources’ subscale presented the lowest correlations among factors and did not correlate with any component of the 5 A's, suggesting the need of enhancing students' responsibility and involvement during their internships, as well as the interest of some organizations.     

Comparison of the MGIT TBc immunochromatographic assay with the Accuprobe Gen-Probe TB assay for identification of Mycobacterium tuberculosis complex: results from a low-burden tuberculosis setting

We compared the BD MPT64 TBc Antigen assay with the Gen-Probe TB assay for identifying Mycobacterium tuberculosis (MTB) from liquid culture vials. The BD TBc Antigen assay was more sensitive than and as specific as the Gen-Probe TB assay, making it a useful alternative for the rapid detection of MTB.     

Development of a PCR-based method coupled with a microplate colorimetric assay for the detection of Porcine Parvovirus and application to diagnosis in piglet tissues and human plasma

A new method for Porcine Parvovirus (PPV) diagnosis was developed. The method is based on polymerase chain reaction (PCR) amplification followed by hybridization and colorimetric detection of PCR products in microwell plates. A highly specific and sensitive amplification step was ensured by primers carefully selected in the VP2 structural gene and optimized PCR conditions. Uracyl-DNA-Glycosylase (UDG) in combination with dUTP was used to avoid false-positive results, and 100 copies of internal control (IC) were added to each PCR reaction to reveal any false-negative samples. Biotinylated amplified fragments were hybridized on specific capture probes covalently linked to microwell plates. Finally, the detection of hybridized PCR products was performed by means of a colorimetric reaction, which was automated. The method permitted the detection of 10³copies (6 fg) of replicative form DNA (RF-DNA) in 20 mg of lung sample, and 500 copies (3 fg) in 100μl of plasma. It was used to analyse 24 field piglet tissue samples, and 35 human plasma or serum specimens collected from patients treated with porcine Factor VIII concentrates.     

Problem solved: a modified enzyme-linked immunosorbent assay for detection of human antibodies to pertussis toxin eliminates false-positive results occurring at analysis of heat-treated sera

Enzyme-linked immunosorbent assay (ELISA) for measurement of antibodies to pertussis toxin (PT) is a widely used method for diagnosis of whooping cough and is also frequently used for seroprevalence studies and for assessment of antibodies after vaccinations against whooping cough. The recommended ELISA procedure for assessment of PT antibodies in human serum does, however, have a serious problem with false-positive results when heat-treated sera are analyzed. Historic sera might have been exposed to routine heat treatment, and diagnostic sera from warm geographic regions are at risk of unintentional exposure to heat. A modified version of the PT ELISA, incorporating a blocking step with 1% milk and addition of 0.1% milk to the dilution buffer, eliminates the false-positive phenomenon occurring with heat-treated sera. Results for serum antibody concentrations correlate well with the currently recommended method. This ELISA modification is straightforward and cheap, and it should be recommended at all analyses incorporating sera with unknown history of heat exposure.     

Application of a six sigma model to the evaluation of the analytical performance of serum enzyme assays and the design of a quality control strategy for these assays: A multicentre study

BackgroundSix medical testing laboratories at six different sites in China participated in this study. We applied a six sigma model for (a) the evaluation of the analytical performance of serum enzyme assays at each of the laboratories, (b) the design of individualized quality control programs and (c) the development of improvement measures for each of the assays, as appropriate.MethodsInternal quality control (IQC) and external quality assessment (EQA) data for selected serum enzyme assays were collected from each of the laboratories. Sigma values for these assays were calculated using coefficients of variation, bias, and total allowable error (TEa). Normalized sigma method decision charts were generated using these parameters. IQC design and improvement measures were defined using the Westgard sigma rules. The quality goal index (QGI) was used to assist with identification of deficiencies (bias problems, precision problems, or their combination) affecting the analytical performance of assays with sigma values <6.ResultsSigma values for the selected serum enzyme assays were significantly different at different levels of enzyme activity. Differences in assay quality in different laboratories were also seen, despite the use of identical testing instruments and reagents. Based on the six sigma data, individualized quality control programs were outlined for each assay with sigma <6 at each laboratory.ConclusionsIn multi-location laboratory systems, a six sigma model can evaluate the quality of the assays being performed, allowing management to design individualized IQC programs and strategies for continuous improvement as appropriate for each laboratory. This will improve patient care, especially for patients transferred between sites within multi-hospital systems.