Dye Washout Research Articles

Mapping the Transport Kinetics of Molecules and Particles in Idealized Intracranial Side Aneurysms

Intracranial side aneurysms (IA) are pathological blood-filled bulges in cerebral blood vessels. Unlike healthy blood vessels where mass transport is dominated by convection, both diffusion and convection can play an active role in aneurysm sites. Here, we study via dye washout experiments and numerical simulations, the transport characteristics of particles (1 micron) and small molecules (300 Da) into simplified side aneurysms models following bolus injection. Time-lapse fluorescent microscopy imaging performed in our idealized aneurysm models showed that the parent artery geometry (located on the inner vs. outer curvature) as well as the aneurysm aspect ratio (AR) affect the washout kinetics while the pulsatile nature of the flow, maintained within the physiological range, carries only a minor effect. Importantly, in the absence of effective diffusion, particles that are located on slow streamlines linger within the aneurysm cavity, a phenomenon that could be of importance in deposition of cells and nano/micro-particles within aneurysms. Altogether, mass transport studies may provide valuable insights for better understanding of aneurysm pathophysiology as well as for the design of new diagnostic and theranostic nano-medicines.

(997) - Experimental and Numerical Analysis of Dye Washout in Ventricular Assist Devices

PurposeVentricular Assist Devices (VADs) are associated with complications due to the damage done to the blood. This damage relates to shear stress and exposure time. Designing VADs to minimise exposure time, in addition to reducing stress, should then reduce damage. The aim of this work was to use washout of a dye to investigate local and overall residence times in VADs.MethodsA flow rig was developed such that initially dyed glycerol circulated and a three way tap was used to switch to a transparent solution. A camera was focused on the transparent VAD outlet tube, and the colour change in the images calibrated to dye concentration. The concentration change at the outlet, ‘dye washout curve’, was used as an indicator of the residence times in the VAD. Dye washout curves were measured for VADs both in development and commercially available.Computational Fluid Dynamics (CFD) was used to calculate dye washout curves. An established flow field was first obtained with a transient simulation. A scalar variable was introduced to represent the dye, and a transport equation was used to model its convection. Simulated dye washout curves were compared with experiment, and simulations were used to understand fluid dynamic factors determining the different curves.ResultsDifferences in experimental washout were clearest at a flow rate of 1 l/min. The time taken for the concentration to drop from 100 to 20 %, T20, was an initial indicator of residence time. T20 was 2.1 s in the CentriMag (magnetic suspension) and just 0.9 s in the HeartMate II (axial & contact bearings). This difference may be explained by the larger volume of the CentriMag. Despite a similar internal volume to the HeartMate II, the Calon MiniVAD (centrifugal & magnetic suspension) had T20 of 1.4 s. The difference is likely due to the secondary flow in the MiniVAD which is required for suspension.ConclusionThe secondary flow path plays a critical role in determining blood residence time in the VAD. Ongoing work is focussed on investigating the importance of secondary flow path parameters.

Fluorescent Dextran Diffusion Assay to Study Cardiac T-Tubules

T-tubules are membrane invaginations of cardiac myocytes critical for effective excitation-contraction coupling and are significantly altered in various diseases. However, the mechanisms underlying t-tubule remodeling remain largely unknown, particularly due to a number of limitations of currently available technologies. Therefore, we have developed an assay that employs the diffusion of fluorescent 3 KD dextran out of the t-tubule network of live intact isolated ventricular cardiomyocytes. To significantly improve the quality of measurements, dextran is applied locally using a patch-clamp pipette (∼2-3 μm diameter) under constant flow of washing solution through 0.5 mm ID capillary glass, which limits dextran filling of t-tubules to a small area of the cardiomyocyte. With 40X objective and small pinholes in the light paths, excitation is limited to ∼9-10 μm diameter area and fluorescence emission is measured from ∼5-6 μm diameter region using a photomultiplier. Upon cessation of dye application, fluorescence decline displays 2 major components: a fast component (τ ∼5-50 ms) corresponding to dye washout from the solution surrounding the cell and a slow component corresponding to dye diffusion out of the t-tubule system. In healthy mouse ventricular myocytes the slow component had a time constant of 1304 ± 98 ms (n=24). In contrast, “detubulation” by acute hyposmotic stress significantly decreased the time constant to 848 ± 99 ms (n=27, p<0.01) presumably due to shorter remaining t-tubule segments. In HEK293 cells, which lack t-tubules, the slow component has time constant of 210 ± 51 ms (n=5), suggesting that dextran unbinding from cellular membrane is significantly faster than its diffusion out of t-tubules. This technique allows for rapid (within seconds) repetitive measures of the diffusional constraints imposed by the t-tubule network and provides a novel tool for studying dynamics of t-tubule remodeling in various experimental conditions.

Contribution of the late sodium current to intracellular sodium and calcium overload in rabbit ventricular myocytes treated by anemone toxin.

Pathological enhancement of late Na(+) current (INa) can potentially modify intracellular ion homeostasis and contribute to cardiac dysfunction. We tested the hypothesis that modulation of late INa can be a source of intracellular Na(+) ([Na(+)]i) overload. Late INa was enhanced by exposing rabbit ventricular myocytes to Anemonia sulcata toxin II (ATX-II) and measured using whole cell patch-clamp technique. [Na(+)]i was determined with fluorescent dye Asante NaTRIUM Green-2 AM. Pacing-induced changes in the dye fluorescence measured at 37°C were more pronounced in ATX-II-treated cells than in control (dye washout prevented calibration). At 22-24°C, resting [Na(+)]i was 6.6 ± 0.8 mM. Treatment with 5 nM ATX-II increased late INa 8.7-fold. [Na(+)]i measured after 2 min of electrical stimulation (1 Hz) was 10.8 ± 1.5 mM and 22.1 ± 1.6 mM (P < 0.001) in the absence and presence of 5 nM ATX-II, respectively. Inhibition of late INa with GS-967 (1 μM) prevented Na(+) i accumulation. A strong positive correlation was observed between the late INa and the pacing-induced increase of [Na(+)]i (R(2) = 0.88) and between the rise in [Na(+)]i and the increases in cytosolic Ca(2+) (R(2) = 0.96). ATX-II, tetrodotoxin, or GS-967 did not affect [Na(+)]i in quiescent myocytes suggesting that late INa was solely responsible for triggering the ATX-II effect on [Na(+)]i. Experiments with pinacidil and E4031 indicate that prolongation of the action potential contributes to as much as 50% of the [Na(+)]i overload associated with the increase in late INa caused by ATX-II. Enhancement of late INa can cause intracellular Na(+) overload in ventricular myocytes.

IN VIVO EVALUATION OF INFARCTED TISSUE DEGRADATION AFTER ISCHEMIA USING NIR SPECTROSCOPY IN PIG HEARTS

IN VIVO EVALUATION OF INFARCTED TISSUE DEGRADATION AFTER ISCHEMIA USING NIR SPECTROSCOPY IN PIG HEARTS

TCT-635 ACUTE AND MID-TERM CLINICAL OUTCOMES OF THE EVEROLIMUS-ELUTING BIORESORBABLE VASCULAR SCAFFOLDS IN AN ALL-COMER COHORT

(NIR) dye, Cy5.5-SE covalently binds to the extracellular proteins through their NH2 groups. Proteolysis and tissue degradation should result in dye washout and loss of its absorption, which may provide a measure of these processes. METHODS: In domestic pigs (w30 kg, n 1⁄4 14) following intercostal incision, prospective at risk and remote areas were labeled with Cy5.5-SE by 1-min touch of the epicardium with a 6-mm sponge soaked in 0.2 mM dye solution in 0.1 M NaPi buffer (pH 8.5). Ten min after staining, the 1st and 2nd diagonal branches of the left anterior descending coronary artery (LAD) were ligated. Immediately after epicardial staining and shortly after ligation, point spectra (400-1200 nm) were acquired using light guide attached to a spectrometer, which was followed by chest closure. Seven, 14 and 21 days later, the same procedure of spectra acquisition were repeated after the chest was opened again. Peaks of Cy5.5 (680 nm) as well as oxyand deoxy-(hemoglobin(Hb)+myoglobin(Mb)) (560-580 nm) were analyzed. Post-mortem analysis was achieved via both macroscopical staining of shortaxis heart slices with triphenyltetrazolium chloride (TTC) and microscopical H&E and trichrome collagen staining of fixed myocardial specimen. RESULTS: Ligation of the LAD branches resulted in immediate deoxygenation of the area at risk while subepicardial labeling of Cy5.5 did not affect this process. Infarction progression, as evidenced by TTC-negative area on short-axis slice, resulted in faster signal decay of Cy5.5 within the infracted subepicardium than that in the remote area (Table 1). After 7 days, signal of total-(Hb+Mb) decreased by 42% in the infarct relative to the remote area (0.23 0.11 vs. 0.40 0.08, p<0.05), which correlated with change of Cy5.5. Post-mortem microscopic examination revealed gradual cardiomyocyte and vasculature degradation and collagen accumulation, which was comparable with signal change of Cy5.5 and total-(Hb+Mb). CONCLUSION: The observations are consistent with activation of tissue degradation within the infracted myocardium due to inflammation and proteolysis. The method has potential for in vivo evaluating the efficacy of regenerative interventions on infracted myocardium with great accuracy.

In Vitro Study of Cerebrospinal Fluid Dynamics in a Shaken Basal Cistern after Experimental Subarachnoid Hemorrhage

BackgroundCerebral arterial vasospasm leads to delayed cerebral ischemia and constitutes the major delayed complication following aneurysmal subarachnoid hemorrhage. Cerebral vasospasm can be reduced by increased blood clearance from the subarachnoid space. Clinical pilot studies allow the hypothesis that the clearance of subarachnoid blood is facilitated by means of head shaking. A major obstacle for meaningful clinical studies is the lack of data on appropriate parameters of head shaking. Our in vitro study aims to provide these essential parameters.Methodology/Principal FindingsA model of the basal cerebral cistern was derived from human magnetic resonance imaging data. Subarachnoid hemorrhage was simulated by addition of dyed experimental blood to transparent experimental cerebrospinal fluid (CSF) filling the model of the basal cerebral cistern. Effects of various head positions and head motion settings (shaking angle amplitudes and shaking frequencies) on blood clearance were investigated using the quantitative dye washout method. Blood washout can be divided into two phases: Blood/CSF mixing and clearance. The major effect of shaking consists in better mixing of blood and CSF thereby increasing clearance rate. Without shaking, blood/CSF mixing and blood clearance in the basal cerebral cistern are hampered by differences in density and viscosity of blood and CSF. Blood clearance increases with decreased shaking frequency and with increased shaking angle amplitude. Head shaking facilitates clearance by varying the direction of gravitational force.Conclusions/SignificanceFrom this in vitro study can be inferred that patient or head shaking with large shaking angles at low frequency is a promising therapeutic strategy to increase blood clearance from the subarachnoid space.

Tri‐Leaflet Valve Design With a Purge Flow for Heart‐Assist Devices: An In Vitro Optimization Study

The objective of this study is to assess the effect of a purge flow on valves of pulsatile heart-assist devices. Clinical applications of these devices are still limited because of frequent thromboembolic complications. These complications often originate at the valves and the unavoidable flow separation regions that are found behind the leaflets. The flow separations cause a long residence time of blood that is considered particularly detrimental. To solve this problem, a valve with a purge flow is proposed. A purge flow is a jet, which is separated from the main blood flow and directed behind the leaflets into the sinus to flush it. Even though the purge flow does not prevent a flow separation, it shortens the residence time of the blood in the sinus. Thus, the purge flow improves the periodic washout of the blood in the region of flow separation. The complex purge flow was investigated in a tri-leaflet valve. The geometrical parameters of the valve were varied systematically. A statistical technique--the Taguchi method--was used to reduce the number of investigated models to 12. The flows through the resulting valve models were numerically simulated and analyzed. The evaluated models with the best results were subsequently investigated experimentally using different methods: hemodynamic tests in a valve tester and flow visualization using the dye washout method. It was shown that the purge flow can effectively wash out the sinus. Therefore, the purge flow valve reduces the potential of a thrombus formation normally associated with the valve.

Novel morphological features of developing white matter pericytes and rapid scavenging of reactive oxygen species in the neighbouring endothelia

Capillary endothelia and pericytes form a close morphological arrangement allowing pericytes to regulate capillary blood flow, in addition to contributing to vascular development and support. Vascular changes associated with oxidative stress are implicated in important pathologies in developing whiter matter, but little is known about the vascular unit in white matter of the appropriate age or how it responds to oxidative stress. We show that the ultrastructural arrangement of post-natal day 10 (P10) capillaries involves the apposition of pericyte somata to the capillary inner basement membrane and penetration of pericyte processes onto the abluminal surface where they form close connections with endothelial cells. Some pericytes have an unusual stellate morphology, extending processes radially from the vessel. Reactive oxygen species (ROS) were monitored with the ROS-sensitive dye 2',7'-dichlorofluorescin (DCF) in the endothelial cells. Exposure to exogenous ROS (100 μm H(2) O(2) or xanthine/xanthine oxidase), evoked an elevation in intracellular ROS that declined to baseline during the ongoing challenge. A second challenge failed to evoke an intracellular ROS rise unless the nerve was rested for > 4 h or exposed to very high levels of exogenous ROS. Exposure to a first ROS challenge prior to loading with DCF also prevented the intracellular ROS rise from a second challenge, proving that dye washout during exposure to ROS is not responsible for the loss of a second response. Perfusion with 30 μm extracellular Ca(2+) or the voltage-gated Ca(2+) antagonist diltiazem partially prevented this rapid scavenging of intracellular ROS, but blocking either catalase or glutathione peroxidase did not. The phenomenon was present over a range of post-natal ages and may contribute to the high ROS-tolerance of endothelial cells and act to limit the release of harmful ROS onto neighbouring pericytes.

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.

Numerical Dye Washout Method as a Tool for Characterizing the Heart Valve Flow: Study of Three Standard Mechanical Heart Valves

To date, no ideal heart valve prosthesis for the replacement of a diseased natural valve or for use in ventricular assist devices exists. Valves still cause thromboembolic complications originating from thrombus formations in the valve's stagnant and recirculation zones. Optimization of valve design requires detailed flow field investigations. Usually, the regions that are more prone to thrombus formation can be estimated using a dye washout experiment. This successful experimental method was simulated using numerical methods. The proposed method was applied to three standard mechanical heart valves--Björk-Shiley, St-Jude, and Starr-Edwards valve. The dye washout was characterized by a time course of the gray value averaged over a defined region of interest. Finally, these curves were quantified by a half dye time (HDT), which characterizes the blood residence time. The HDT in the best valve, the Starr-Edwards valve, was 0.0747 s. The HDT in the worst valve, the Björk-Shiley, was 0.0942 s. The analysis of the hemodynamic valve parameters (pressure drop, velocity magnitudes and turbulence) revealed that the best valve is the St-Jude valve. The Starr-Edwards valve displayed the worst hemodynamic parameters. This study shows that the proposed numerical method of dye washout visualization can be used as an additional tool for the flow characterization.

Takuo Aoyagi: Discovery of Pulse Oximetry

In the 1930s and 1940s, photo cells permitted German, English, and American physiologists to construct ear oximeters with red and infrared light, requiring calibration. In 1940 Squire recognized that changes of red and infrared light transmission caused by pneumatic tissue compression permitted saturation to be computed. In 1949 Wood used this idea to compute absolute saturation continuously from the ratios of optical density changes with pressure in an ear oximeter. In 1972 Takuo Aoyagi, an electrical engineer at Nihon Kohden company in Tokyo, was interested in measuring cardiac output noninvasively by the dye dilution method using a commercially available ear oximeter. He balanced the red and infrared signals to cancel the pulse noise which prevented measuring the dye washout accurately. He discovered that changes of oxygen saturation voided his pulse cancellation. He then realized that these pulsatile changes could be used to compute saturation from the ratio of ratios of pulse changes in the red and infrared. His ideas, equations and instrument were adapted, improved and successfully marketed by Minolta about 1978, stimulating other firms to further improve and market pulse oximeters worldwide in the mid 1980s. Dr. Aoyagi and associates provided a detailed history for this paper.

Methods for Detecting Internalized, FM 1-43 Stained Particles in Epithelial Cells and Monolayers

The membrane dye FM 1-43 has frequently been used to quantify exocytosis in neurons. In epithelia, intense lateral intracellular space staining and fluctuations in baseline labeling produced inconsistent results. Membrane retrieved in the presence of FM 1-43 retains the dye, however, and cells that undergo compensatory endocytosis during and following evoked exocytosis contain punctate, fluorescent particles after washout of external stain. As an alternative measure of trafficking, we quantified the fluorescent puncta retained after dye washout and tested our method on both coverslip-grown cell clusters and filter-grown intact monolayers. Images for analysis were acquired using serial sectioning with either epifluorescence or confocal microscopy. Tests with an intestinal goblet cell line that exhibits basal and ATP-stimulated granule trafficking confirmed that 1), the algorithm identified the same number of internalized particles with either epifluorescence or confocal microscopy acquired images; 2), low density clusters exhibited significantly more internalized particles per cell than either filter-grown monolayers or high density clusters; 3), ATP stimulation significantly increased the number of internalized particles in all preparations; and 4), the number of particles internalized was comparable to capacitance measurements of exocytosis. This method provides a single technique for quantifying membrane trafficking in both monolayers and unpolarized cells.

Characterization of an Artificial Valve Flow Using the Numerical Dye Washout Visualization Technique: Application to the Monoleaflet Valve With Purged Flow

Until today, no ideal heart valve prosthesis for the replacement of a diseased natural valve or for use in ventricular assist devices exists. Valves still cause thromboembolic complications originating from thrombus formations in the valve's stagnant zones. Optimization of valve design involves avoiding stagnation zones and zones of high shear stresses. This requires detailed flow field investigations. Usually, the regions which are more prone to thrombus formation can be estimated using a dye washout experiment. The method allows an assessment of regions with a high or low residence time that may in turn predict regions with a corresponding thrombus risk. This successful experimental method was simulated using numerical methods with a combination of the computational fluid dynamics program FLUENT (Fluent Inc., Lebanon, NH, USA) and of the visualization tool AMIRA (TGS Inc., San Diego, CA, USA). The numerical dye washout visualization was applied to four monoleaflet valves with varying valve housing geometries. The results show a significant difference in the washout processes of the examined valves. The dye washout was characterized by a time course of the gray value averaged over a defined region of interest. Finally, these curves were quantified by a half dye time. The half dye time in the best optimized valve was only 0.2753 s. The same time in the original valve was 0.6834 s. This study shows that the proposed numerical method of dye washout visualization can be used as an additional tool of the flow characterization in artificial organs.

SH3P7/mAbp1 deficiency leads to tissue and behavioral abnormalities and impaired vesicle transport

The intracellular adaptor protein SH3P7 is the mammalian ortholog of yeast actin-binding protein 1 and thus alternatively named as mAbp1 (or HIP55). Structural properties, biochemical analysis of its interaction partners and siRNA studies implicated mAbp1 as an accessory protein in clathrin-mediated endocytosis (CME). Here, we describe the generation and characterization of mice deficient for SH3P7/mAbp1 owing to targeted gene disruption in embryonic stem cells. Mutant animals are viable and fertile without obvious deficits during the first weeks of life. Abnormal structure and function of organs including the spleen, heart, and lung is observed at about 3 months of age in both heterozygous and homozygous mouse mutants. A moderate reduction of both receptor-mediated and synaptic endocytosis is observed in embryonic fibroblasts and in synapses of hippocampal neurons, respectively. Recycling of synaptic vesicles in hippocampal boutons is severely impaired and delayed four-fold. The presynaptic defect of SH3P7/mAbp1 mouse mutants is associated with their constricted physical capabilities and disturbed neuromotoric behaviour. Our data reveal a nonredundant role of SH3P7/mAbp1 in CME and places its function downstream of vesicle fission.

NUMERICALL DYE WASHOUT METHOD AS A TOOL TO CHARACTERIZE THE HEART VALVE FLOW

CFD methods are widely used for the development of artificial valves. However, choosing the proper flow parameters to characterize the flow to predict the valve function in vivo, is still a big challenge. There is a set of parameters, which are used for flow characterization: pressure drop, maximal velocity magnitude, calculation of the residence time along path line and maximal values or histogramms of wall shear stress, shear stress and turbulence intencity. We propose to use the numerical dye washout method for flow characterization. This allows the assessment of regions with a long or short residence time and therefore predicts the regions of high or low thrombus risk. This successful experimental method is modeled now numerically. This allows a 3D-resolved flow characterization and a possibility to validate the results experimentally. In order to simulate the dye washout, an unsteady flow simulation is performed with the flow solver FLUENT. Furthermore, a species transport model simulating the convective diffusion mixing of species (entering/outflowing blood) is applied. The results are distributions of the fluid fraction for different time steps. These data were used for the dye washout visualization, which was realized using the software AMIRA. Results are images, which represent the projection of the absorbed light intensity. The analysis of the washout process was done by quantifying the time course of the gray value. The method was applied in 5 different mechanical valves. The results show that dye washout flow characterization seems to be a very useful parameter.

Phorbol Esters Target the Activity-Dependent Recycling Pool and Spare Spontaneous Vesicle Recycling

Using electrophysiology and styryl dye imaging, we studied the effect of phorbol 12-myristate 13-acetate (PMA) on activity-dependent and spontaneous vesicle recycling. In electrophysiological experiments, we found that the PMA effect depended on the maturational state of the synapses. Spontaneous neurotransmitter release from nascent synapses without a functional readily releasable pool (RRP) was unresponsive to PMA application. In contrast, mature synapses responded robustly to PMA application, consistent with previous studies. Using styryl dye imaging, we found that there was a PMA-dependent increase in the size of the RRP when PMA was present before, during, or after activity-dependent dye uptake, suggesting that this effect involves an increase in the population of the RRP by vesicles recruited from the reserve pool. Additionally, we found that when PMA was present during spontaneous dye uptake, there was an increase in dye labeling, and these additional dye-loaded vesicles showed rapid destaining in response to strong stimulation and were also releasable by hypertonic sucrose. In contrast, these observations were not reproducible when PMA treatment was performed after spontaneous dye uptake and extracellular dye washout. Together, these findings suggest that the increased spontaneous neurotransmission in the presence of PMA was attributable to release of vesicles from the RRP rather than an effect of PMA on the spontaneously recycling pool. Thus, the phorbol esters selectively regulate the activity-dependent pool of vesicles, indicating that priming mechanisms that prepare vesicles for fusion, which are targeted by phorbol esters, are different for the spontaneous and evoked forms of fusion.

Experimental and numerical dye washout flow visualization

Flow visualization in realistic models is very important for the study of pathological vessel enlargements (aneurysms). Furthermore, flow visualization may help in treatment decisions. However, the most interesting parameter, the wall shear stress, is difficult to measure in vivo. This parameter can be provided by computational fluid dynamics. However, the numerical methods don't visualize the results as does of the dye washout method - a method often used in flow studies. This experimental method simulates the cine angiograms acquired during contrast agent injection used in medicine. In this paper we present the dye washout visualization of CFD results and compare these results with the conventional dye washout experiments in the same aneurysm model under steady flow conditions.

Staining of Intraocular Lenses with Various Dyes: A Study of Digital Image Analysis

Purpose: To evaluate the interaction of various intraocular lens (IOL) materials with commonly used dyes. Materials and Methods: One IOL of each of the five groups (polymethylmethacrylate, silicone, three-piece hydrophobic acrylic, single-piece hydrophobic acrylic and single-piece hydrophilic acrylic) was bathed in trypan blue 0.1%, fluorescein sodium 2% and indocyanine green (ICG) 0.5% for 15 min. Digital photographs of the IOLs were obtained prior to bathing and after the 15-min bath in each dye. The same IOLs were immersed in the same dye for another 15 min and digital images were reobtained to depict the 30-min dye uptake. New IOLs from the group that exhibited visible colour change after the 15-min bath were then bathed in twofold dilution and if there was still visible dye uptake, fourfold dilution was performed, repeating the 15-min bath and digital photography. The images were then processed using Adobe Photoshop 5.1 to get mean luminosity and red-green-blue values. These values were compared between the groups and the undyed control. Also, dye washout was observed in serum-containing vials. Results: None of the polymethylmethacrylate, silicone and hydrophobic acrylic IOLs were stained with the dyes used. The only IOL material that changed colour was hydrophilic acrylic and did so with all dyes. The most marked colour change was with fluorescein sodium 2%, the least was with trypan blue 0.05%. Twofold dilution of trypan blue and fourfold dilutions of fluorescein sodium and ICG still stained the hydrophilic acrylic IOL; however, fourfold dilution of trypan blue did not cause a significant colour change. Trypan blue washed out within 6 h, while the IOL stained with ICG remained stained for longer than 24 h. Comment: Hydrophilic acrylic IOLs should be used with caution together with dyes since this material demonstrates marked dye uptake and washout may also take some time.

Incidence of nonlocalization of sentinel lymph nodes using preoperative lymphoscintigraphy in 74 consecutive head and neck melanoma and Merkel cell carcinoma patients.

Lymphatic drainage pathways in the head and neck region are more variable than in any other location of the body. Occasionally, head and neck lymphoscintograms fail to identify a definitive lymphatic drainage pattern, making preoperative and intraoperative identification of sentinel nodes very difficult. The purpose of this study was to determine the incidence of nonlocalization on lymphoscintigraphy of sentinel nodes in patients with head and neck cutaneous malignancies. A retrospective chart review was conducted of a single surgeon's (WKS) 135 consecutive head and neck melanoma and Merkel cell cancer patients from August 1997 to August 2002. In all cases. technetium-99m sulfur colloid was the radioactive tracer used by the nuclear medicine department to perform the lymphoscintograms. Of the 135 patients, 74 underwent preoperative lymphoscintigraphy in preparation for performing a sentinel lymph node biopsy. Of these 74 patients, 5 (6.8%) were found to have nonlocalization of a sentinel node(s). Of the 5 patients who failed to localize, 3 had primary lesions near the midline scalp, while 2 had primary lesions located on the cheek. Two of the 5 patients underwent reinjection of the radioisotope by the treating surgeon (WKS) but failed to further localize the radiotracer. All 5 patients went on to have wide local excision of the primary cancer on the day of the lymphoscintogram, as well as undergoing intraoperative examination of all head and neck nodal basins with a handheld gamma detector. No focal areas of radiation were identified and no lymph nodes were biopsied. To date, 1 patient has developed distant metastases and has succumbed to her disease. The remaining 4 patients are free of disease. In a series of 135 consecutive patients with head and neck cutaneous malignancies, 74 of whom were treated with preoperative lymphoscintograms, a nonlocalization rate of 6.8% was found. This is a significant rate of nonlocalization and reflects either the inherent difficulty in imaging the head and neck region and/or the possible rapid rate of dye washout via multiple lymphatic drainage pathways that exist in this location.