Urine flow cytometry as a primary screening method to exclude urinary tract infections.

PURPOSE: To exclude urinary tract infections, culture is the gold standard method, although it is time consuming and costly. Current strategies using dipstick analysis are unsatisfactory as screening methods, because of inadequate sensitivity/specificity. Urine flow cytometry is an attractive alternative. To exclude urinary tract infections, a cutoff value to screen for negative cultures was determined. METHODS: 281 outpatients (51 % male) of a general population visiting the urology department were included. Urine samples were measured by flow cytometry and compared with culture results and dipstick analysis. ROC analysis was performed to evaluate the screening performance of flow cytometry and dipstick analysis compared to culture. RESULTS: 18 % of cultures were positive, defined as >10(4) colony forming units/mL. Bacterial count by flow cytometry alone provides the best sensitivity and specificity to exclude a urinary tract infection. A cutoff value of 60 bacteria/µL urine leads to a sensitivity of 100 % and a specificity of 60 %. Retrospectively, with a cutoff value of 60 bacteria/µL urine, 49 % of the cultures would have been redundant. 20 % of patients receiving antibiotics possibly had received those unnecessarily. The calculated percentage of false negatives was 0 % (95 % confidence interval 0-3.3 %). CONCLUSIONS: Urine flow cytometry is a reliable screening method to exclude urinary tract infections. With a cutoff value of 60 bacteria/µL urine, negative predictive value is 100 % and the calculated percentage of false negatives is 0 % (95 % confidence interval 0-3.3 %). Using flow cytometry as a screening method could lead to a reduction in cultures and antibiotics.

Cut-off values to rule out urinary tract infection should be gender-specific.

The diagnosis of urinary tract infection (UTI) by urine culture is an expensive and time-consuming procedure. Using a screening method, to identify negative samples, would improve the procedure and reduce costs. In this study, urine flow cytometry, of over 7000 urine samples, was assessed by retrospective analysis. With a cut-off value of >200bacteria/µl, we obtained a sensitivity of 93.0%, a specificity of 63.5%, and a negative predictive value (NPV) of 96.2%. As a result the culturing of 49% of all samples could be avoided. In addition, the data was retrospectively analyzed to determine if the introduction of gender-specific cut-off values could improve screening results. The obtained receiver operator curves are indeed significantly different when gender specific cut-offs were used. When a NPV of 95% is considered acceptable the unisex cut-off value of >200bacteria/µl can be used for women (NPV 94.9%), but the cut-off value for men could be raised to >400bacteria/µl without diminishing the NPV (NPV 95.0%).

Urine flow cytometry can rule out urinary tract infection, but cannot identify bacterial morphologies correctly.

The diagnosis of urinary tract infection (UTI) by urine culture is a time-consuming and costly procedure. Usage of a screening method, to identify negative samples, would therefore affect time-to-diagnosis and laboratory cost positively. Urine flow cytometers are able to identify particles in urine. Together with the introduction of a cut-off value, which determines if a urine sample is subsequently cultured or not, the number of cultures can be reduced, while maintaining a low level of false negatives and a high negative predictive value. Recently, Sysmex developed additional software for their urine flow cytometers. Besides measuring the number of bacteria present in urine, information is given on bacterial morphology, which may guide the physician in the choice of antibiotic. In this study, we evaluated this software update. The UF1000i classifies bacteria into two categories: 'rods' and 'cocci/mixed'. Compared to the actual morphology of the bacterial pathogen found, the 'rods' category scores reasonably well with 91% chance of classifying rod-shaped bacteria correctly. The 'cocci/mixed' category underperforms, with only 29% of spherical-shaped bacteria (cocci) classified as such. In its current version, the bacterial morphology software does not classify bacteria, according to their morphology, well enough to be of clinical use in this study population.

It takes acid, rather than ice, to freeze glucose.

Plasma glucose levels provide the cornerstone of diabetes evaluation. Unfortunately, glucose levels drop in vitro due to glycolysis. Guidelines provide suitable conditions which minimize glycolysis, such as immediate centrifugation or the use of ice/water slurry storage containers. For obvious practical reasons, most laboratories use blood collection tubes containing glycolysis inhibitors. We describe the effect of a variety of commonly used blood collection tubes on in vitro stability of glucose. Furthermore, we looked at the validity of the assumption that glycolytic activity is minimal when blood is kept in an ice/water slurry. Sodium fluoride alone does not reduce in vitro glycolysis in the first 120 minutes after phlebotomy. Addition of citrate almost completely prevented in vitro glycolysis, but showed a positive bias (0.2 mmol/l) compared to control. This is partly due to a small drop in glucose level in control blood, drawn according to the current guidelines. This drop occurs within 15 minutes, in which glycolysis has been described to be minimal and acceptable. NaF-EDTA-citrate based test tubes provide the best pre-analytical condition available. Furthermore, glucose levels are not stable in heparinized blood placed in an ice/water slurry. We strongly advise the use of NaF-EDTA-citrate based test tubes in diabetes research.

Interaction between electrical stimulation, protein coating and matrix elasticity: a complex effect on muscle fibre maturation.

In skeletal muscle tissue engineering, it remains a challenge to produce mature, functional muscle tissue. Mimicking the in vivo niche in in vitro culture might overcome this problem. Niche components include, for example, extracellular matrix proteins, neighbouring cells, growth factors and physical factors such as the elasticity of the matrix. Previously, we showed the effects of matrix stiffness and protein coating on proliferation and differentiation of muscle progenitor cells in a two-dimensional (2D) situation. In the present study we have investigated the additional effect of electrical stimulation. More precisely, we investigated the effect of electrical stimulation on primary myoblast maturation when cultured on top of Matrigel™ - or laminin-coated substrates with varying elasticities. The effect of electrical stimulation on differentiation and maturation was found to be dependent on coating and stiffness. Although electrical stimulation enhanced myoblast maturation, the effect was mild. We therefore conclude that, with the current regimen, electrical stimulation is not essential to create functional, mature muscle tissue.

Essential environmental cues from the satellite cell niche: optimizing proliferation and differentiation.

The use of muscle progenitor cells (MPCs) for regenerative medicine has been severely compromised by their decreased proliferative and differentiative capacity after being cultured in vitro. We hypothesized the loss of pivotal niche factors to be the cause. Therefore, we investigated the proliferative and differentiative response of passage 0 murine MPCs to varying substrate elasticities and protein coatings and found that proliferation was influenced only by elasticity, whereas differentiation was influenced by both elasticity and protein coating. A stiffness of 21 kPa optimally increased the proliferation of MPCs. Regarding differentiation, we demonstrated that fusion of MPCs into myotubes takes place regardless of elasticity. However, ongoing maturation with cross-striations and contractions occurred only on elasticities higher than 3 kPa. Furthermore, maturation was fastest on poly-d-lysine and laminin coatings.