Interaction between Solvatochromic Dyes and Water Sampled from a Natural Source Treated with High Dilutions of Phosphorus.

BACKGROUND: Highly diluted and succussed solutions interact with solvatochromic dyes, indicating that changes in solvent and solute polarity could be related to their mechanism of action. It is not known, however, how the activity associated with succussed high dilutions is transferred to untreated water and what the limits of this process are. AIMS: The aims of the present study were to ascertain whether a succussed high dilution of phosphorus (1.5 × 1-59 M; Phos 30cH) seeded into a natural water source that fed a fjord and two connected lakes could propagate itself through the lake system (total volume 2200 m3) and, moreover, whether the process could be tracked using solvatochromic dyes. METHODS: Samples of water were collected before and after seeding, at different times and places throughout the lake system. Controls comprised water taken from an untreated and adjacent, but independent, lake (1385 m3). RESULTS: Water samples taken up to 72 hours after the source treatment produced significant increases (p ≤ 0.03) in the absorbance of the solvatochromic dye methylene violet (MV), while samples from the control lake produced no changes. CONCLUSIONS: The study indicates that activity associated with Phos 30c can propagate itself through large volumes of water, causing changes throughout a whole connected lake system, and that these changes can be tracked using the solvatochromic dye MV. This in turn means the use of homeopathic medicines in large volumes of drinking water, in farming and ecological contexts, now has the potential to be assessed with physico-chemical monitoring.

Quantitative analysis of cell proliferation by a dye dilution assay: Application to cell lines and cocultures.

Cell proliferation represents one of the most fundamental processes in biological systems, thus the quantitative analysis of cell proliferation is important in many biological applications such as drug screening, production of biologics, and assessment of cytotoxicity. Conventional proliferation assays mainly quantify cell number based on a calibration curve of a homogeneous cell population, and therefore are not applicable for the analysis of cocultured cells. Moreover, these assays measure cell proliferation indirectly, based on cellular metabolic activity or DNA content. To overcome these shortcomings, a dye dilution assay employing fluorescent cell tracking dyes that are retained within cells was applied and was diluted proportionally by subsequent cell divisions. Here, it was demonstrated that this assay could be implemented to quantitatively analyze the cell proliferation of different types of cell lines, and to concurrently analyze the proliferation of two types of cell lines in coculture by utilizing cell tracking dyes with different spectral characteristics. The mean division time estimated by the dye dilution assay is compared with the population doubling time obtained from conventional methods and values from literature. Additionally, dye transfer between cocultured cells was investigated and it was found that it is a characteristic of the cells rather than a characteristic of the dye. It was suggested that this method can be easily combined with other flow cytometric analyses of cellular properties, providing valuable information on cell status under diverse conditions. © 2017 International Society for Advancement of Cytometry.

Correlation of ultrasound estimated with dye-determined or directly measured amniotic fluid volume revisited.

BACKGROUND/AIMS: To examine the relationship of the amniotic fluid index (AFI) and single deepest pocket (SDP) with an AFV as modelled by Brace or Magann. METHODS: AFI and SDP were evaluated for their correlation with an actual AFV using the Spearman correlation coefficient. RESULTS: 482 AFI and 468 SDP pregnancies were evaluated. There was a significant association between the AFI and SDP and an actual AFV (p < 0.0001). The AFI range of 5.1-20 was better correlated than 5.1-24 for normal AFVs Brace (κ = 0.175) and Magann (κ = 0.356) versus 5.1-24 (κ = 0.150 and κ = 0.319), respectively. The agreement level t for the AFI (κ = 0.175) and SDP (κ = 0.126) using Brace was slight and for the AFI (κ = 0.356) and SDP (κ = 0.295) using Magann was fair. CONCLUSIONS: Both the AFI and SDP were correlated with actual AFV using both models. AFI of 5.1-20 better categorizes normal volumes. Although the Magann model correlates AFI/SDP and AFV better, the superiority is minimal.

Optimal back-extrapolation method for estimating plasma volume in humans using the indocyanine green dilution method.

BACKGROUND: The indocyanine green dilution method is one of the methods available to estimate plasma volume, although some researchers have questioned the accuracy of this method. METHODS: We developed a new, physiologically based mathematical model of indocyanine green kinetics that more accurately represents indocyanine green kinetics during the first few minutes postinjection than what is assumed when using the traditional mono-exponential back-extrapolation method. The mathematical model is used to develop an optimal back-extrapolation method for estimating plasma volume based on simulated indocyanine green kinetics obtained from the physiological model. RESULTS: Results from a clinical study using the indocyanine green dilution method in 36 subjects with type 2 diabetes indicate that the estimated plasma volumes are considerably lower when using the traditional back-extrapolation method than when using the proposed back-extrapolation method (mean (standard deviation) plasma volume = 26.8 (5.4) mL/kg for the traditional method vs 35.1 (7.0) mL/kg for the proposed method). The results obtained using the proposed method are more consistent with previously reported plasma volume values. CONCLUSIONS: Based on the more physiological representation of indocyanine green kinetics and greater consistency with previously reported plasma volume values, the new back-extrapolation method is proposed for use when estimating plasma volume using the indocyanine green dilution method.

Amniotic fluid volume in normal singleton pregnancies: modeling with quantile regression.

PURPOSE: To develop uniform and reliable reference ranges for amniotic fluid volume (AFV) across gestation in normal singleton pregnancies using quantile regression (QR). METHODS: An analysis of true AFVs determined by dye-dilution techniques or by direct measurement at cesarean delivery in normal singleton pregnancies. AFV centiles were established by QR, a flexible semi-parametric approach of estimating rates of change across the entire distribution of AFV rather than just in the mean as is observed with standard linear regression. RESULTS: The study evaluated 379 women with normal singleton pregnancies between 16 and 41 weeks gestation. QR was used to determine the association between AFV and gestational age (GA). A second-order quantile regression model indicated a nonlinear relationship between AFV and gestational age at the upper centile range (≥80th percentile). CONCLUSION: This study defines normative centile charts for true AFVs between 16 and 41 weeks gestation in normal singleton pregnancies using QR. This statistical approach more appropriately reflects true AFV across gestation at each centile of interest (e.g. 5th, 50th, 95th, etc.) as compared to standard linear regression.

A method for evaluating the use of fluorescent dyes to track proliferation in cell lines by dye dilution.

Labeling nonquiescent cells with carboxyfluorescein succinimidyl ester (CFSE)-like dyes gives rise to a population width exceeding the threshold for resolving division peaks by flow cytometry. Width is a function of biological heterogeneity plus extrinsic and intrinsic error sources associated with the measurement process. Optimal cytometer performance minimizes extrinsic error, but reducing intrinsic error to the point of facilitating peak resolution requires careful fluorochrome selection and fluorescent cell sorting. In this study, we labeled the Jurkat and A549 cell lines with CFSE, CellTraceViolet (CTV), and eFluor 670 proliferation dye (EPD) to test if we could resolve division peaks in culture after reducing the labeled input widths by cell sorting. Reanalysis of the sorted populations to ascertain the level of reduction achieved always led to widths exceeding the gated limits due to the contribution of errors. Measuring detector-specific extrinsic error by sorting uniform fluorescent particles with similar spectral properties to the tracking dyes allowed us to determine the intrinsic error for each dye and cell type using a simple mathematical approach. We found that cell intrinsic error ultimately dictated whether we could resolve division peaks, and that as this increased, the required sort gate width to resolve any division peaks decreased to the point whereby issues with yield made A549 unsuitable for this approach. Finally, attempts to improve yields by setting two concurrent sort gates on the fluorescence distribution enriched for cells in different stages of the cell cycle that had nonequivalent proliferative properties in culture and thus should be practiced with caution.

[Extravascular lung water: from genes to telemedicine]. / Acqua extravascolare polmonare: dalla genetica alla telemedicina.

Extravascular lung water is a crucial parameter for the management of many different pathological conditions, especially heart failure. Many modalities can be employed for its evaluation, each with different advantages and limitations. In a near future, we hope that extravascular lung water assessment could be performed by non-invasive wearable devices, allowing remote continuous monitoring of pulmonary congestion. Recently, it has been proposed that some specific genetic variations may be responsible for the heterogeneous individual response to the development of extravascular lung water. These data could be relevant to titrate and personalize both treatments and preventive interventions.

[A dye densitometry analysis method for noninvasive measurement of cardiac output based on NIRS].

Currently, there exist technology problems in cardiac output (CO) parameter detection clinically, such as invasive and complex operation, as well as possibility of infection and death for patients. In order to solve these problems, a noninvasive and continuous method based on NIRS for CO detection was presented. In this way, the concentration changing of indocyanine green (ICG) dye in the patient's arterial blood was dynamically measured and analyzed, so that the CO could be noninvasively and continuously measured according to the characteristic parameters of dye densitometry curve. While the ICG dye was injected into the patient's body by the median cubital vein, block of photoelectric pulse dye densitometry measurement system as the lower machine acquired pulse wave data and uploaded the data to upper computer. In the scheme, two specialized light sources of LED at 940 and 805 nm were used to capture the signals of sufferer's fingertip pulse wave synchronously and successively. The CO value could then be successfully calculated through drawing complete ICG concentration variation of dye dilution and excretion process and computing mean transmission time (MTT) by upper computer. Compared with the "gold standard" method of thermodilution, the maximum relative error of this method was below 9. 76%, and the mean relative error was below 4. 39%. The result indicates that the method can be used as a kind of convenient operation, noninvasive and continuous solution for clinical CO measurement.

Amniotic fluid and the clinical relevance of the sonographically estimated amniotic fluid volume: oligohydramnios.

The amniotic fluid volume (AFV) is regulated by several systems, including the in-tramembranous pathway, fetal production (fetal urine and lung fluid) and uptake (fetal swallowing), and the balance of fluid movement via osmotic gradients. The normal AFV across gestation has not been clearly defined; consequently, abnormal volumes are also poorly defined. Actual AFVs can be measured by dye dilution techniques and directly measured at cesarean delivery; however, these techniques are time-consuming, are invasive, and require laboratory support, and direct measurement can only be done at cesarean delivery. As a result of these limitations, the AFV is estimated by the amniotic fluid index (AFI), the single deepest pocket, and subjective assessment of the AFV. Unfortunately, sonographic estimates of the AFV correlate poorly with dye-determined or directly measured amniotic fluid. The recent use of color Doppler sonography has not improved the diagnostic accuracy of sonographic estimates of the AFV but instead has led to overdiagnosis of oligohydramnios. The relationship between the fixed cutoffs of an AFI of 5 cm or less and a single deepest pocket of 2 cm or less for identifying adverse pregnancy outcomes is uncertain. The use of the single deepest pocket compared to the AFI to identify oligohydramnios in at-risk pregnancies seems to be a better choice because the use of the AFI leads to an increase in the diagnosis of oligohydramnios, resulting in more labor inductions and cesarean deliveries without any improvement in peripartum outcomes.

An improved technique for calculating relative response in cellular proliferation experiments.

Investigating the response of cells to specific agonists may involve the use of cell tracking dyes to assess the extent of stimulated proliferation, frequently reported as the proliferation index (PI). Calculation of PI uses a model for cell division that expects the cell number to double as cells proliferate through each successive generation. It is often useful to compare the PI of a stimulated control population with that of a population in the presence of some agent, whether chemical, pharmacologic, or cellular. For such comparison studies, the nature of the metric being used must be taken into account to accurately assess the extent of inhibition. Specifically, the metric used in ModFit LT (Verity Software House, Topsham, ME) and in FCS Express (De Novo Software, Los Angeles, CA) uses a metric with a lower limit of unity, whereas the metric used in FlowJo (Treestar, Ashland, OR) has a lower limit of zero. For studies involving cell proliferation comparisons using tracking dye dilution, a new equation is proposed as the appropriate calculation to use when determining the percent of relative response based on proliferation index values for a metric whose lower limit is unity.

Radiographic lung density assessed by computed tomography is associated with extravascular lung water content.

BACKGROUND: We hypothesized that in acute lung injury (ALI), the volume of pulmonary tissue with aqueous density, as determined by spiral computed tomography (CT), is associated with extravascular lung water content. Our aim was to compare tissue volume index, as assessed by CT, before and after oleic acid-induced ALI, with extravascular lung water indexes (EVLWI), determined with single transpulmonary thermodilution (EVLWI(STD)), thermal-dye dilution (EVLWI(TDD)), and postmortem gravimetry (EVLWI(G)). METHODS: Seven instrumented sheep received an intravenous infusion of oleic acid 0.08 ml/kg (OA group) and four animals had vehicle only (Control group). The day before, and immediately after the experiment, sheep were anesthetized to undergo quantitative CT examinations during a short breath hold. Hemodynamics, oxygenation, EVLWI(STD), and EVLW(TDD) were registered. Linear regression analysis was used to assess the relationships between EVLWI(STD), EVLW(TDD), EVLWI(G), and lung tissue volume index (TVI(CT)) determined with CT. RESULTS: In the OA group, total lung volume increased compared with Controls. Poorly and non-aerated lung volumes increased a 3.6- and 4.9-fold, respectively, and TVI(CT) almost doubled. EVLWI(STD), EVLWI(TDD), and TVI(CT) were associated significantly with EVLWI(G) (r=0.85, 0.90, and 0.88, respectively; P<0.001). TVI(CT) deviated from the reference EVLWI(G) values to the greatest extent with a mean bias +/- 2SD of 4.0 +/- 6.0 ml/kg. CONCLUSIONS: In ovine oleic acid-induced ALI, lung tissue volume, as assessed by quantitative CT, is in close agreement with EVLWI, as determined by indicator dilution methods and postmortem gravimetry, but overestimates lung fluid content.

Is Sabin-Feldman dye test using T. gondii tachyzoites from animal inoculation still the best method for detecting Toxoplasma gondii antibodies?

Although the Sabin-Feldman dye test is the gold standard for detecting Toxoplasma antibodies in human, it is performed only in reference laboratories because live virulent T. gondii are used for the test. We collected 210 human serum samples and tested them by the dye test using in vivo tachyzoites (conventional method) then compared these results with three other methods: a dye test using cell culture-derived T. gondii tachyzoites and indirect immunofluorescent antibody tests (IFAT) using in vivo and in vitro tachyzoites. We found the conventional dye test detected the highest percent of cases (4.3%), followed by the IFAT using parasites from mice (3.8%), then the dye test and the IFAT using cell culture tachyzoites (both 2.8%). Agreement with the dye test when using mouse and cell culture derived tachyzoites was 96.7%. Using in vivo tachyzoites for the dye test and the IFAT gave 94.3% agreement, while using in vitro tachyzoites gave 94.8% agreement. When compared with the conventional dye test, the IFAT had 75% sensitivity and 100% specificity. The T. gondii tachyzoites obtained from cell culture had a lower virulence, as indicated by a three times longer survival period in the inoculated mice. We favor the conventional dye test as the gold standard for Toxoplasma antibody detection. In vitro tachyzoites can be used routinely in the dye test but false negative results may occur in some cases. The IFAT, using either in vivo or in vitro tachyzoites, are alternatives for laboratories where provision of live tachyzoites is limited.

Cardiac output and systemic transit time dispersion as determinants of circulatory mixing time: a simulation study.

A new approach to characterize the kinetics of intravascular mixing process is presented. The mixing time, defined as the time required for achieving 95% homogeneity, is calculated by numerical simulations using a circulatory model applied to the intravascular marker indocyanine green (ICG). The results suggest that the mixing time is determined by cardiac output and the relative dispersion of transit time distribution across the systemic circulation, whereby the rate of mixing increases with increasing cardiac output and decreasing transit time dispersion, and vice versa. The estimation of plasma volume from simulated ICG dilution data using the backextrapolation method shows that slow mixing is accompanied by an overestimation of blood volume. This error may be negligible for mixing times of less than approximately 3 min but high in disease states characterized by low cardiac output and/or high transit time dispersion. In view of the role of transit time dispersion as determinant of intravascular mixing, it would be interesting to know more about the effect of disease states on systemic transit time dispersion.

Motoneuron morphology and synaptic contacts: determination by intracellular dye injection.

The structure and dendritic connections of an identified crustacean motoneuron were analyzed by intracellular injection of dye. Some processes of the neuron end in the ganglionic neuropil, but most terminate on axons which pass through the ganglion in specific, identifiable tracts. The former processes are ipsilateral to the soma, while the latter, as well as their connections, display bilateral symmetry. Structural and functional evidence suggests that the demonstrated contacts are synaptic junctions, and that the approach can therefore be used to study patterns of synaptic organization in complex neural networks.

Cardiovascular monitoring by pulse dye densitometry or arterial indocyanine green dilution.

BACKGROUND: Noninvasive cardiac output (CO) monitoring is possible by indocyanine green (ICG) dilution measured by pulse dye densitometry (PDD). To validate the precision of this method, we compared hemodynamic variables derived from PDD (DDG-2001, Nihon Kohden, Japan) with those derived from simultaneously taken arterial blood ICG concentrations. METHODS: In 20 patients (6 M/14 F), ASA I or II, 36 sessions were performed (n = 24 with the PDD-finger probe, n = 10 with the PDD-nose probe). After IV administration of 10 mg ICG, 34 arterial blood samples were taken during each session, with 20 samples taken during the first 2 min. CO, central blood volume (CBV), and total blood volume (TBV) were calculated independently from ICG and PDD and the results compared between methods using Bland-Altman analysis. The results are reported as mean difference (bias) and limits of agreement (LOA = +/- 2 sd). RESULTS: PDD using the finger probe underestimated CO (LOA) by 5% (-56% and 47%); overestimated CBV by 21% (-54% and 96%) and underestimated TBV by -15% (-38% and 8%). PDD using the nose probe overestimated CO (LOA) by 30% (-67% and 127%); CBV by 48% (-98% and 193%) and underestimated TBV by -10% (-47% and 27%). CONCLUSION: Despite the permissible bias, the wide LOA of the PDD-derived hemodynamic variables CO and CBV, compared with those simultaneously obtained by invasive arterial ICG measurements, suggest that PDD is unsuitable for evaluation of cardiovascular variables in the individual patient. Hence, the reliability and clinical use of this method seem limited.

Multimodal flow visualization and optimization of pneumatic blood pump for sorbent hemodialysis system.

Renal Solutions Allient Sorbent Hemodialysis System utilizes a two-chambered pneumatic pump (Pulsar Blood Pump, Renal Solutions, Inc., Warrendale, PA, USA) to avoid limitations associated with peristaltic pumping systems. Single-needle access is enabled by counter-pulsing the two pump chambers, thereby obviating compliance chambers or blood reservoirs. Each chamber propels 20 cc per pulse of 3 s (dual access) or 6 s (single access) duration, corresponding to a peak Reynolds number of approximately 8000 (based on inlet velocity and chamber diameter). A multimodal series of flow visualization studies (tracer particle, dye washout, and dye erosion) was conducted on a sequence of pump designs with varying port locations and diaphragms to improve the geometry with respect to risk of thrombogenesis. Experiments were conducted in a simplified flow loop using occluders to simulate flow resistance induced by tubing and dialyzer. Tracer visualization revealed flow patterns and qualitatively indicated turbulence intensity. Dye washout identified dwell volume and areas of flow stagnation for each design. Dye erosion results indicated the effectiveness and homogeneity of surface washing. Compared to a centered inlet which resulted in a fluid jet that produced two counter-rotating vortices, a tangential inlet introduced a single vortex, and kept the flow laminar. It also provided better surface washing on the pump inner surface. However, a tangential outlet did not present as much benefit as expected. On the contrary, it created a sharp defection to the flow when transiting from filling to ejection.

Cardiac output measurement during exercise in COPD: A comparison of dye dilution and impedance cardiography.

INTRODUCTION: Impedance cardiography (IC) derived from morphological analysis of the thoracic impedance signal is now commonly used for noninvasive assessment of cardiac output (CO) at rest and during exercise. However, in Chronic Obstructive Pulmonary Disease (COPD), conflicting findings put its accuracy into question. OBJECTIVES: We therefore compared concurrent CO measurements captured by IC (PhysioFlow: COIC ) and by the indocyanine green dye dilution method (CODD ) in patients with COPD. METHODS: Fifty paired CO measurements were concurrently obtained using the two methods from 10 patients (FEV1 : 50.5 ± 17.5% predicted) at rest and during cycling at 25%, 50%, 75% and 100% peak work rate. RESULTS: From rest to peak exercise COIC and CODD were strongly correlated (r = 0.986, P < 0.001). The mean absolute and percentage differences between COIC and CODD were 1.08 L/min (limits of agreement (LoA): 0.05-2.11 L/min) and 18 ± 2%, respectively, with IC yielding systematically higher values. Bland-Altman analysis indicated that during exercise only 7 of the 50 paired measurements differed by more than 20%. When data were expressed as changes from rest, correlations and agreement between the two methods remained strong over the entire exercise range (r = 0.974, P < 0.001, with no significant difference: 0.19 L/min; LoA: -0.76 to 1.15 L/min). Oxygen uptake (VO2 ) and CODD were linearly related: r = 0.893 (P < 0.001), CODD = 5.94 × VO2 + 2.27 L/min. Similar results were obtained for VO2 and COIC (r = 0.885, P < 0.001, COIC = 6.00 × VO2 + 3.30 L/min). CONCLUSIONS: These findings suggest that IC provides an acceptable CO measurement from rest to peak cycling exercise in patients with COPD.

Mat Seroprevalence Infers Low Rates of Toxoplasma gondii in Domestic Pigs from Yucatan, Mexico.

Toxoplasma gondii is a zoonotic parasite of worldwide distribution. The consumption of infected pork meat has been suggested to be an important source for human infection in the tropical area of Yucatan, Mexico. We performed a cross-sectional study of 12 farms across the state to investigate the seroprevalence of Toxoplasma gondii infection in domestic pigs. In total, 632 samples were obtained from 2 different environmental zones (tropical deciduous low forest and tropical sub-deciduous medium forest) and 2 abattoirs. The modified agglutination test (MAT) was used to assess the seroprevalence of T. gondii in pigs and to evaluate 2 globally used serological tests, the Dye test (DT) and ID Screen® ELISA multi-species, and a commercial ELISA kit (Human Toxo IgG, Human-diagnostics), which is widely used locally in this geographical area. The overall prevalence obtained with the MAT (cut-off ≥1:25) among the 632 pigs was 1.4% (95% CI, 0.6-2.7%). The seroprevalence obtained for the different age groups was 0.6%, 0.7%, 1.8%, and 6.8% among 2-3, 3-4, 4-5, and ≥5-mo-old pigs. This increase in the seroprevalence was statistically significant for the 2 older groups (odds ratio [OR] 3.9-7.1, P < 0.05) in comparison with younger groups. DT at >4 IU dilution had a perfect agreement and 100% of sensitivity and specificity when compared with the MAT. Although ID Screen® had only a fair agreement (κ = 0.389) with the MAT, the McNemar test showed that the results of these tests were comparable (P = 0.29). The Human Toxo ELISA showed no agreement with MAT, ID Screen®, and DT (κ = 0.000-0.023, McNemar P < 0.05). This ELISA was lacking in specificity, accuracy, and precision; hence, we do not recommend its use for T. gondii diagnosis in pig serum.