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In this work, the design of a microfluidic paper-based analytical device (μPAD) for the quantification of nitrate in urine samples was described. Nitrate monitoring is highly relevant due to its association to some diseases and health conditions. The nitrate determination was achieved by combining the selectivity of the nitrate reductase enzymatic reaction with the colorimetric detection of nitrite by the well-known Griess reagent. For the optimization of the nitrate determination μPAD, several variables associated with the design and construction of the device were studied. Furthermore, the interference of the urine matrix was evaluated, and stability studies were performed, under different conditions. The developed μPAD enabled us to obtain a limit of detection of 0.04 mM, a limit of quantification of 0.14 mM and a dynamic concentration range of 0.14–1.0 mM. The designed μPAD proved to be stable for 24 h when stored at room temperature in air or vacuum atmosphere, and 60 days when stored in vacuum at −20 °C. The accuracy of the nitrate μPAD measurements was confirmed by analyzing four certified samples (prepared in synthetic urine) and performing recovery studies using urine samples.

Leukocyte esterase (LE) is a useful marker that can be used in establishing a diagnosis of urinary tract infections (UTIs). The development of a UTI diagnostic method with quantitative determinations of biomarkers across all age groups is becoming more important. In this report, microfluidic resistance sensors based on silver ink (Ag ink) and silver ink mixed with ZnO nanoparticles (Ag-ZnO ink) were synthesized and coated on cellulose paper, namely LE-Ag-μPADs and LE-Ag-ZnO-μPADs, respectively, for the sensitive detection of LE. The microfluidic design increases the precision of data and further allows for quantitative determination and early detection of LE in human urine. The quantification of LE relies on the change in the resistance readout coating with Ag ink as well as Ag-ZnO ink in the detection zone. A mixture of 3-(N-tosyl-l-alaninyloxy)-5-phenylpyrrole (PE) and 1-diazo-2-naphthol-4-sulfonic acid (DAS) was deposited in the sample zone to selectively recognize LE, and the resulting nonconductive products, i.e., azo compounds, further reacted with the Ag ink and Ag-ZnO ink to increase resistance. The quantitative detectable LE concentrations between 2 to 32 (×5.2 U mL-1), i.e. ≈12 to 108 μg L-1, cover the commercial dipstick range of trace, +1 and +2. The minimum detectable concentration of LE in urine was 1 (×5.2 U mL-1). The lower concentrations of LE detectable by LE-Ag-μPADs (1-8 × 5.2 U mL-1) are below the value achieved with the ELISA LE kit. Urine samples from inpatients with indwelling urinary catheters were used, and the LE levels measured by the present device were highly correlated with those determined by a commercial urine analyser.

Bladder cancer is the fourth most common cancer in men, and it is becoming a prevalent malignancy. Most of the regular clinical examinations are prompt evaluations with cystoscopy, renal function testing, which require high-precision instrument, well-trained operators, and high cost. In this study, a microfluidic paper-based analytical device (μPAD) was fabricated to detect nuclear matrix protein 22 (NMP22) and bladder cancer antigen (BTA) from the urine samples. Urine samples were collected from 11 bladder cancer patients and 10 well-beings as experiment and control groups, respectively, to verify the working efficiency of μPAD. A remarkable checkout efficiency of up to 90.91% was found from the results. Meanwhile, this method is feasible for home-based self-detection from urine samples within 10 min for the total process, which provides a new way for quick, economical, and convenient tumor diagnosis, prognosis evaluation, and drug response.

Proteinuria is a condition in which an excessive amount of protein is excreted in urine. It is, among others, an indicator of kidney disease or risk of cardiovascular disease. Rapid and reliable diagnosis and monitoring of proteinuria is of great importance for both patients and their physicians. For that reason, a paper-based sensor for proteinuria diagnosis was designed, optimized, and validated utilizing smartphone-assisted signal acquisition. In the first step, a few commonly employed protein assays were optimized and compared in terms of analytical performance on paper matrix. The tetrabromophenol blue method was selected as the one providing a sufficiently low limit of detection (39 mg·L−1) on the one hand and appropriate long-term stability (up to 3 months) on the other hand. The optimized assay was employed for protein-to-creatinine ratio (PCR) determination on a single paper-based sensor. For both analytes the linear ranges were within the clinically relevant range. The analytical usefulness of the developed sensors was demonstrated by a PCR recovery study in artificial urine. The obtained PCR recoveries were from ca. 80 to 150%.

In this study, a slug-flow microextraction (SFME) coupled with paper spray mass spectrometry (PS-MS) method was developed for rapid and on-site analysis of trace amphetamine-type illicit drugs including amphetamine (AM), methamphetamine (MA) and 3,4-methylenedioxy-N-methylamphetamine (MDMA) in complex biological samples such as whole blood and raw urine. The method involved the application of SFME for rapid extraction of trace amphetamine-type illicit drugs from whole blood and raw urine samples, followed by PS for direct MS analysis under ambient and open-air conditions. The experimental parameters including extraction solvent, extraction cycle, high voltage, and spray solvent, etc., were all investigated, and the optimized conditions showed an enhanced sensitivity of 1–2 order of magnitudes compared with PS-MS. The method showed good linearity, with correlation coefficient values (r) of no less than 0.9979 for analysis of AM, MA, and MDMA in human whole blood and raw urine without additional sample pretreatments. The limits of detection and quantification were 0.01–0.05 ng/mL and 0.05–0.2 ng/mL, respectively. Satisfactory recoveries were also obtained, with 74.2–94.9% for whole blood and 80.2–103.6% for raw urine, respectively. All of our experimental results demonstrated that SFME-PS-MS showed great potential for rapid, on-site, in situ, and high-throughput screening of amphetamine-type illicit drugs in various biological samples.

Made available in DSpace on 2018-12-11T17:34:37Z (GMT). No. of bitstreams: 0 Previous issue date: 2018-01-02 Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) In this paper, we describe a validated paper-based microfluidic analytical device for the simultaneous quantification of two important biomarkers of renal function in urine. This paper platform provides an inexpensive, simple, and easy to use colorimetric method for the quantification of creatinine (CRN) and uric acid (UA) in urine samples. The microfluidic paper-based analytical device (μPAD) consists of a main channel with three identical arms, each containing a circular testing zone and a circular uptake zone. Creatinine detection is based on the Jaffé reaction, in which CRN reacts with picrate to form an orange-red product. Uric acid quantification is based on the reduction of Fe3+ to Fe2+ by UA, which is detected in a colorimetric reaction using 1,10-phenanthroline. Under optimum conditions, obtained through chemometrics, the concentrations of the analytes showed good linear correlations with the effective intensities, and the method presented satisfactory repeatability. The limits of detection and the linear ranges, respectively, were 15.7 mg L−1 and 50–600 mg L−1 for CRN and 16.5 mg L−1 and 50–500 mg L−1 for UA. There were no statistically significant differences between the results obtained using the μPAD and a chromatographic comparative method (Student's t-test at 95% confidence level). Instituto de Química Universidade Estadual Paulista “Julio de Mesquita Filho”- UNESP, R. Prof. Francisco Degni 55, P.O. Box 355 Instituto de Química de São Carlos Universidade de São Paulo, Av. Trabalhador São-carlense, 400 Instituto de Química Universidade Estadual Paulista “Julio de Mesquita Filho”- UNESP, R. Prof. Francisco Degni 55, P.O. Box 355 CNPq: 132646/2014-5 CAPES: 2014/17749-7 FAPESP: 2015/21733-1 CNPq: 308788/2015-0

Bisphenol A (BPA) is an endocrine disruptor frequently detected in human biofluids. Dermal absorption of BPA from thermal paper receipts occurs but BPA pharmacokinetics following dermal exposure is not understood. To compare the pharmacokinetics of dermal and dietary BPA exposure, six male participants handled simulated receipts containing relevant levels of BPA (isotope-labeled BPA-d16) for 5 min, followed by hand-washing 2 h later. Urine (0–48 h) and serum (0–7.5 h) were monitored for free and total BPA-d16. One week later, participants returned for a dietary administration with monitoring as above. One participant repeated the dermal administration with extended monitoring of urine (9 days) and serum (2 days). After dietary exposure, urine total BPA-d16 peaked within 5 h and quickly cleared within 24 h. After dermal exposure, cumulative excretion increased linearly for 2 days, and half the participants still had detectable urinary total BPA-d16 after 1 week. The participant repeating the dermal exposur...

A rapid and direct paper spray ionization/mass spectrometry (PSI/MS) method was developed for quantitative analysis of ephedrine, pseudoephedrine, norpseudoephedrine, and methylephedrine in human urine. This method involves the use of a triangular filter paper and high-resolution mass spectrometry, where the molecular ions of ephedrines were generated by applying high voltage after loading the spray solvent to the paper which urine sample was pre-loaded. Small amounts (2μL) of urine spiked with an internal standard were directly analyzed for ephedrines. The PSI/MS method was validated for linearity, within- and between-run precision, accuracy, and limit of detection. The results showed good linearity (R(2)≥0.9928) and acceptable precision and accuracy. Furthermore, the accuracy of the method was assessed by analyzing a blind urine sample from World Anti-Doping Agency and comparing the measured concentrations with the nominal concentrations. This test resulted in accuracies ranging from 96.4 to 106.1%, indicating that the PSI/MS method has the potential to be an alternative technique for the fast quantitation of ephedrines in doping control analysis.

Post-print RATIONALE: Drug overdose deaths due to fentanyls and other novel psychoactive substances (NPS) are on the rise. The higher potencies of fentanyl analogs compared with morphine require new technologies to identify and quantitate NPS. METHODS: Paper spray tandem mass spectrometry and high-resolution mass spectrometry were used to identify and measure fentanyl analogs as well as common drugs of abuse in urine samples from substance use disorder clinics. Ten-microliter urine samples were deposited directly on paper spray cartridges previously loaded with internal standards, dried, and analyzed with no other sample treatment. Quantitative results were obtained using tandem mass spectrometry (MS/MS). Individual drugs were identified using high resolution accurate mass spectrometry (HRAM), and confirmed by data dependent MS/MS. RESULTS: Calibration curves in urine were linear over a range of 0.5 - 50 ng/mL with R2 of 0.99 or better for eight representative fentanyl analogs. Cartridges preloaded with internal standards demonstrated satisfactory quantitative results compared with LC/MS. Direct identification and confirmation of fentanyl analogs and other common drugs of abuse in urine using high resolution accurate mass and MS/MS fragmentation were demonstrated at low picogram levels. CONCLUSIONS: Paper spray mass spectrometry can reliably identify and quantitate fentanyl analogs and other drugs of abuse in urine. Using paper spray cartridges as collection devices reduces exposure and transportation risks associated with biological fluids. Cartridges pre-loaded with labeled internal standards can be effective for targeted screening of fentanyl analogs and other drugs of abuse. Chris Gill would like to acknowledge the Natural Sciences and Engineering Research Council of Canada (NSERC) for funding under the Discovery Grants program (RGPIN-2016-05380), Vancouver Island University for their ongoing support of my research program, and an anonymous Vancouver Island University Foundation donor who funded the necessary travel to facilitate this timely collaboration. Post-print version This is the peer reviewed version of the following article: Kennedy, J.H., Palaty, J., Gill, C.G., & Wiseman, J.M. (2018). Rapid analysis of fentanyls and other novel psychoactive substances (NPS) in substance use disorder patient urine using paper spray mass spectrometry. Rapid Communications in Mass Spectrometry, 32(15), 1280-1286. DOI: 10.1002/rcm.8164, which has been published in final form at http://dx.doi.org/10.1002/rcm.8164. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Use of Self-Archived Versions. https://viurrspace.ca/bitstream/handle/10613/12782/Gill.RCM2018.pdf?sequence=4

We report a paper-based assay platform for the detection of the kidney disease marker Trefoil Factor 3 (TFF3) in human urine. The sensor is based on a quantitative metalloimmunoassay that can determine TFF3 concentrations via electrochemical detection of environmentally stable silver nanoparticle (AgNP) labels attached to magnetic microbeads via a TFF3 immunosandwich. The paper electroanalytical device incorporates two preconcentration steps that make it possible to detect concentrations of TFF3 in human urine at the low end of the target TFF3 concentration range (0.03-7.0 μg mL(-1)). Importantly, the paper device provides a level of accuracy for TFF3 determination in human urine equivalent to that of a commercial kit. The paper sensor has a dynamic range of ∼2.5 orders of magnitude, only requires a simple, one-step incubation protocol, and is fast, requiring only 10 min to complete. The cost of the materials at the prototypic laboratory scale, excluding reagents, is just US$0.42.

Screening procedures using high resolution (HR)-mass spectrometry (MS) are getting more and more important, e.g., for drug testing or adherence monitoring. Approaches usually include time-consuming sample preparation and compound separation by liquid chromatography (LC). The paper spray ionization (PSI) technique coupled to MS might overcome these steps by direct analysis of complex mixtures without extraction and separation. In recent years, this technology proved its potential for quantification and/or qualitative screening in biofluids. However, so far, PSI-MS was only applied to procedures covering a limited number of targets. Therefore, a PSI-HR-MS/MS approach was developed and successfully validated for comprehensive urine screening. The procedure showed high matrix effects for most drugs but still acceptable limits of identification. Applicability was tested by analyses of three proficiency tests for systematic toxicological analysis and of 103 authentic human urine samples. Its screening power was compared to that of published LC-HR-MS/MS procedures after urine precipitation with conjugate cleavage (UglucP) or dried urine spot workup by conjugate cleavage and liquid extraction (DUSglucE). In the authentic samples, 73% of all 777 drug intakes were detectable with the new approach. The LC-HR-MS/MS screening approaches detected more drugs, particularly in samples with low analyte concentrations, with values of 88% after UglucP or 76% after DUSglucE. In conclusion, the new PSI-HR-MS/MS screening approach was suitable for comprehensive urine screening of different drug classes and might be a promising alternative to conventional procedures. However, limitations should be considered such as detection of drugs in low concentrations and risk of false positive or negative results caused by mixed spectra.

Background Mucopolysaccharidoses (MPSs) are a group of lysosomal storage diseases caused by the deficiency/absence of enzymes which catalyze the degradation of glycosaminoglycans (GAGs). The use of biological samples dried on filter paper has been increasing because it makes it easy to ship them to reference laboratories. Urinary GAGs are the main biomarkers of MPS and, thus, we studied the correlations of determinations to GAGs and creatinine, as well as compared the GAGs' profile on electrophoresis, between urine and dried urine in filter paper (DUFP) samples. We also assessed the GAG stability over time under different storage temperatures. Methods We quantified the GAG concentration in both sample types and compared the results by Pearson correlation. Results The results were very similar, with r = 0.97 for creatinine and with r = 0.94 and r = 0.98 for GAGs for controls and patients, respectively, with similar electrophoretic profiles. The GAG stability in DUFP was up to 30 days at − 20, 4, and 25 °C and up to 21 days at 37 °C. Conclusion Our proposal assessed urinary GAGs in DUFP and concluded that these samples can be used in the investigation of MPS, replacing urine samples in neonatal screening and monitoring of therapies, due to ease of transportation and storage.

The first step in curing a disease is being able to detect the disease effectively. Paper-based microfluidic devices are biodegradable and can make diagnosing diseases cost-effective and easy in almost all environments. We created a three-dimesnional (3D) paper device using wax printing fabrication technique and basic principles of origami. This design allows for a versatile fabrication technique over previously reported patterning of SU-8 photoresist on chromatography paper by employing a readily available wax printer. The design also utilizes multiple colorimetric assays that can accommodate one or more analytes including urine, blood, and saliva. In this case to demonstrate the functionality of the 3D paper-based microfluidic system, a urinalysis of protein and glucose assays is conducted. The amounts of glucose and protein introduced to the device are found to be proportional to the color change of each assay. This color change was quantified by use of Adobe Photoshop. Urine samples from participants with no pre-existing health conditions and one person with diabetes were collected and compared against synthetic urine samples with predetermined glucose and protein levels. Utilizing this method, we were able to confirm that both protein and glucose levels were in fact within healthy ranges for healthy participants. For the participant with diabetes, glucose was found to be above the healthy range while the protein level was in the healthy range.

Iodine deficiency is a world-wide health problem. A simple, convenient, and inexpensive method to monitor urine iodine levels would have enormous benefit in determining an individual's recent iodine intake or in identifying populations at risk for iodine deficiency or excess. Current methods used to monitor iodine levels require collection of a large volume of urine and its transport to a testing laboratory, both of which are inconvenient and impractical in parts of the world lacking refrigerated storage and transportation. To circumvent these limitations we developed and validated methods to collect and measure iodine and creatinine in urine dried on filter paper strips. We tested liquid urine and liquid-extracted dried urine for iodine and creatinine in a 96-well format using Sandell–Kolthoff and Jaffe reactions, respectively. Our modified dried urine iodine and creatinine assays correlated well with established liquid urine methods (iodine: R2=0.9483; creatinine: R2=0.9782). Results demonstrate that the dried urine iodine and creatinine assays are ideal for testing the iodine status of individuals and for wide scale application in iodine screening programs.

Dried urine spots (DUS) have been reported to provide a simple screening tool for congenital cytomegalovirus (CMV) infection. We developed a standardized method for CMV quantification from DUS. Two applications of 20 μL urine remained within the rim of the filter paper disc and were used to determine the analytical performance of Towne CMV spiked into urine and applied on the discs. The measurable range spanned 3.7 to ≥8.0 log(10) copies/mL. The detection limit was 22 DNA copies/disc. Urine samples from congenitally infected newborns and negative controls were either diluted 1:10 or applied on filter paper at the same volume. DNA copy number from DUS correlated well with copy number from 1:10 diluted urine, although there was a trend for lower levels from DUS (0.3 log(10) difference). Our standardized method for CMV detection and quantification may facilitate CMV studies in resource-limited areas and allow for longitudinal monitoring of viral loads in treated infants.

Mucopolysaccharidoses are complex lysosomal storage disorders caused by any of eleven different enzyme deficiencies resulting in the accumulation of substrates, mainly glycosaminoglycans (GAGs), in various tissues and biological fluids.We developed and validated a urine filter paper methodology for the analysis of GAGs using liquid chromatography-tandem mass spectrometry (LC-MS/MS) for mucopolysaccharidoses type I, type II and type VI patients. We focused on 2 objectives: first, its applicability to high-risk screening, and secondly, to facilitate the collection and shipping of samples to reference centers as part of diagnostic investigation, as well as from treated patients needing to be monitored for assessment of the efficacy of treatment. GAGs in urine dried onto filter paper were extracted and subjected to methanolysis to obtain the repeating disaccharides of the molecules. We devised a multiple reaction monitoring method in positive electrospray ionization mode.The use of deuterated internal standards for dermatan sulfate (DS) and heparan sulfate (HS) reduced a troubling matrix effect. The resulting CVs were14%. Linearity assessment showed Pearson correlation coefficients of 0.999 and 0.997, for DS and HS, respectively. The stability on filter paper was good for DS and HS for up to 6 weeks at various temperatures.We devised a robust and efficient LC-MS/MS methodology for GAGS quantification in urine dried on filter paper and subjected to environmental conditions likely to be encountered during collection, storage and shipping of specimens from referring physicians to medical centers.

Dairy cattle barns are a major source of NH3 emissions to the atmosphere. Previous studies have shown that the bedding material used in the barn can influence the magnitude of NH3 emissions, but little is known about which bedding characteristics are important in this respect. The aims of this study were to assess, at a laboratory scale, the relative importance of the chemical [pH, cation exchange capacity (CEC), C:N] and physical (urine absorbance capacity, bulk density) characteristics of 5 bedding materials (chopped wheat straw, sand, pine shavings, chopped newspaper, chopped corn stalks, and recycled manure solids) on NH3 emissions from dairy cattle urine. Recycled manure solids were the most absorbent of the bedding types (4.2 g of urine/g of bedding), and sand was the least (0.3 g of urine/g of bedding). When beddings were soaked in urine to their absorbance capacities, NH3 emissions over 48 h (expressed as a proportion of the urine N absorbed) were not significantly different among bedding types, despite differences in initial bedding pH, CEC, and C:N. When equal volumes of urine were applied to equal depths of dry bedding, NH3 emissions over 48 h were significantly less from sand and pine shavings (23 and 42% of applied urine N, respectively) than from chopped newspaper, chopped corn stalks, and recycled manure solids (62, 68, and 65% of applied urine N, respectively), whereas emissions from chopped wheat straw (55% applied urine N) only differed significantly from that from sand. Differences in the chemical characteristics of the beddings did not explain differences in emission; NH3 emissions increased linearly with CEC contrary to expectations, and there was no significant relationship with initial bedding pH. The physical characteristics of bedding materials were of more importance, as NH3 emissions increased linearly with absorbance capacity and decreased as the bulk density of the packed beddings increased.

Hormonal analysis of urine from free-ranging primates has been limited due to the difficulty of preserving samples under field conditions. Drying urine on filter paper is an alternative for field preservation. This study describes a new laboratory method for eluting steroids from filter paper with methanol, along with a series of experiments used to develop and validate this method. The overall elution recovery of estrone sulfate (ES) from filter paper was 86.4%. Estrone conjugate (E1C) levels in humans and captive orangutans were analyzed by radioimmunoassay (RIA). Values from samples dried on filter paper were significantly correlated with values from matched frozen samples, with elution efficiencies ranging between 97.1% and 102.4%. Creatinine (Cr) measurements from frozen urine compared to urine dried on filter paper were also significantly correlated (r=0.96) with an elution efficiency of 101.7%. After the samples were stored for 2 years, the absolute values of E1C and Cr were significantly lower but were still significantly correlated with frozen urine values. These data demonstrate the effectiveness of filter paper as a medium for preserving urinary steroid samples, and the efficiency of methanol as a solvent for eluting E1C and Cr. This method thus provides a viable alternative to the traditional procedure of freezing urine for field studies, where freezers are not available.

Background The mucopolysaccharidoses (MPS) are inherited metabolic disorders with bone, joint, and visceral abnormalities, leading to multi-organ dysfunction and, sometimes, neurological manifestations. These diseases are caused by storage of glycosaminoglycans (GAGs) and other complex molecules in tissues, among other pathogenic mechanisms. Definitive diagnosis of the affected individual is mainly based on the identification of the specific enzyme deficiency. New therapies are available or are in development for these pathologies, and early diagnosis seems to be important for the therapy outcomes. Almost all MPS patients have increased levels of GAGs in urine being their evaluation usually the first step in the screening of these conditions. Test on urine may be challenging as transportation of liquid urine samples in appropriate conditions for long distances, especially across international borders, could be difficult. Methods With the aim of overcoming the difficulties related to the use of liquid samples, we extended and validated previous studies about colorimetric determination of GAGs in dried-urine filter paper (DUFP) samples. Results In the conditions we described, there are no differences in the concentration of GAGs between urine and DUFP samples. Untreated patients with MPS and normal controls were well discriminated using any of the samples. Conclusions Dried-urine filter paper is a suitable sample for the colorimetric quantitation of GAGs, and that its incorporation as an additional tool for screening of MPS should be considered by reference laboratories.

Background: quantitative bioassays for determination of urinary creatinine concentrations using liquid urine and dried urine on paper disks (DUPD) specimens were developed and validated. The bioassays were cross-validated by means of measurement of the urinary creatinine concentrations applying DUPD (3 and 5 mm) and liquid urine specimens. Results: LLOQ and LOD for all bioassays were 0.88 and 0.27 mmol/l creatinine, respectively. All bioassays were cross-validated by the analysis of urine samples from healthy voluntary individuals and bioassays performed were statistically not significantly different. Mean observed urinary creatinine concentrations ranged between 0.44 and 25.6, 0.24 and 23.9, 0.13 and 31.26 mmol/l for liquid, DUPD-5mm and DUPD-3mm bioassays, respectively. Conclusion: the advantage of the DUPD bioassays was the relatively small amounts of urine necessary, making these microsampling techniques attractive when limited amounts of urine specimens of, for example, rodents from drug development studies are available.

Bisphenol A (BPA) has one of the highest production volumes of all chemicals worldwide. It has been widely studied because of its endocrine modulating activity. In addition to dietary intake, absorption of BPA via the skin from handling thermal papers is believed to be a relevant route of exposure. We studied BPA exposure via thermal paper receipts in simulation experiments performed by three volunteers, and examined urinary excretion of BPA. We also evaluated background BPA excretion among the Finnish working-age population. The geometric mean BPA excretion among non-occupationally exposed working-age Finns (n=121) was 2.6 μg/l, the range being 0.8-18.9 μg/l. The 95th percentile of the non-occupationally exposed people was 8 μg/l, and this was set as the reference limit for the non-occupationally exposed population. In the first simulation experiment, which was conducted under conditions representing the most likely exposure, i.e., the work of a cashier in a supermarket, BPA excretion remained below the reference limit in all participants. In the second simulation experiment, with more intensive, short-time handling of thermal paper (three times 5 min), urinary excretion also remained at or below background levels (highest value being 10.3 μg/l). The calculated maximum BPA excretion per day after handling thermal paper was less than 0.2 μg/kg of body weight, suggesting a total daily intake over 25 times lower than the European Food Safety Authority's (EFSA's) proposal for a temporary tolerable daily intake (temporary TDI) (5 μg/kg/day).

Summary 250 urine specimens were classified into 7 categories on the basis of viable counts and the bacteria found. The results of 7 bacteriuria screening tests performed on the same specimens are presented in relation to this classification. Four rapid culture tests and a triphenyltetrazolium chloride reduction test proved capable of detecting specimens classed as ‘significant’ or ‘not significant’ bacteriuria with accuracies between 95 and 85%. For more accurate and subtle categorization of specimens, a calibrated drop viable count procedure is recommended.

Five different anti-D antisera, each demonstrating both a prozone and specific blocking of D-anti-D agglutination, were fractionated by a variety of methods including DEAE-cellulose chromatography, preparative electrophoresis, and Sephadex gel filtration. In fractions with blocking activity, IgG was either the one immunoglobulin consistently present or the only immunoglobulin detectable. The most potent blocking activity was observed in fractions in which IgG had the most cathodic electrophoretic migration. The possibility that blocking reactions might be associated with either low-molecular-weight microglobulins or IgM monomers was considered, but no supporting evidence was found. Specific agglutination inhibition of D-anti-D was observed also with urine concentrates from donors undergoing active immunization, and IgG was the most prominent immunoglobulin in these preparations.