Fluorescein-stained confocal laser endomicroscopy versus conventional frozen section for intraoperative histopathological assessment of intracranial tumors.

BACKGROUND: The aim of this clinical trial was to compare Fluorescein-stained intraoperative confocal laser endomicroscopy (CLE) of intracranial lesions and evaluation by a neuropathologist with routine intraoperative frozen section (FS) assessment by neuropathology. METHODS: In this phase II noninferiority, prospective, multicenter, nonrandomized, off-label clinical trial (EudraCT: 2019-004512-58), patients above the age of 18 years with any intracranial lesion scheduled for elective resection were included. The diagnostic accuracies of both CLE and FS referenced with the final histopathological diagnosis were statistically compared in a noninferiority analysis, representing the primary endpoint. Secondary endpoints included the safety of the technique and time expedited for CLE and FS. RESULTS: A total of 210 patients were included by 3 participating sites between November 2020 and June 2022. Most common entities were high-grade gliomas (37.9%), metastases (24.1%), and meningiomas (22.7%). A total of 6 serious adverse events in 4 (2%) patients were recorded. For the primary endpoint, the diagnostic accuracy for CLE was inferior with 0.87 versus 0.91 for FS, resulting in a difference of 0.04 (95% confidence interval -0.10; 0.02; P = .367). The median time expedited until intraoperative diagnosis was 3 minutes for CLE and 27 minutes for FS, with a mean difference of 27.5 minutes (standard deviation 14.5; P < .001). CONCLUSIONS: CLE allowed for a safe and time-effective intraoperative histological diagnosis with a diagnostic accuracy of 87% across all intracranial entities included. The technique achieved histological assessments in real time with a 10-fold reduction of processing time compared to FS, which may invariably impact surgical strategy on the fly.

[Application of Deep Learning Algorithm in the Grading Assessment of Corneal Fluorescein Staining].

Objective: To explore the application value of applying deep learning (DL) algorithm in the grading assessment of corneal fluorescein staining. Methods: A cross-sectional study was carried out, covering 600 corneal fluorescein staining photos acquired in the Contact Lens Clinic, West China Hospital, Sichuan University between 2020 and 2022. Out of the 600 photos, 500 were used to construct the algorithm and the remaining 100 were used for the validation of the algorithm and a comparative analysis of the difference in grading accuracy (ACC) and the length of diagnostic time between artificial intelligence (AI) and optometry students. One month after finishing the first grading analysis, assessment by AI and optometry students was conducted for a second time and results from the two rounds of assessment were compared to examine the intrarater agreement ( kappa value) of the two analyses. The grading analysis results of 3 experienced optometrists were used as the gold standard in the study. Results: Findings of the cross validation with the complete dataset, the training dataset, and the test dataset showed that ResNet34 had the highest predictive accuracy among four DL models. ResNet34 DL model achieved an accuracy of 93.0%, sensitivity of 89.5%, and specificity of 89.6% in the grading of corneal staining. In the comparison of the grading accuracy of AI and two optometry students, AI showed better accuracy, with the respective grading accuracy being 87.0%, 78.0%, and 52.0% for AI, student 1, and student 2 ( P ACC=0.001). In addition, the average diagnostic time of AI was shorter than that of optometry students ( t AI=1.00 s, t S1=11.86 s, t S2=13.25 s, P t =0.001). In the comparative analysis of the intrarater agreement between the two assessments, AI ( kappa AI=0.658, P AI=0.001) achieved better consistency than the two optometry students did （ kappa S1=0.575, P S1=0.001; kappa S2=0.609, P S2=0.001). Conclusion: Applying deep learning algorithms in the grading assessment of corneal fluorescein staining has considerable feasibility and clinical value. In the performance comparison between AI and optometry students, AI achieved higher accuracy and better consistency, which indicates that AI has potential application value for assisting optometrists to make clinical decisions with speed and accuracy.

Morphological and functional assessment of the tear film and meibomian glands in keratoconus.

PURPOSE: To evaluate morphological and functional state of the meibomian glands (MG) in keratoconus patients. METHODS: One hundred eyes of 100 keratoconus patients and 100 eyes of 100 age-matched control subjects were included into this study. Ocular Surface Disease Index (OSDI) scores, non-invasive break up time (NIBUT), findings of meibography, staining with fluorescein of the ocular surface, tear film break-up time (TBUT), and Schirmer I test were documented in all patients' eyes and control eyes and were compared between the groups . RESULTS: The mean TBUT and NIBUT were significantly lower, corneal staining and OSDI scores were statistically greater in the keratoconus group (p < 0.05). The mean meiboscore, partial gland, gland dropout and gland thickening scores for upper/lower eyelids were significantly greater in keratoconus patients than controls (p < 0.05). The NIBUT measurements significantly correlated with MG loss in upper/lower eyelids (p < 0.05). The severity of keratoconus seemed to correlate with meiboscore, partial gland, gland thickening scores in upper/lower eyelids. CONCLUSION: Our data suggests that corneal ectasia in keratoconus is related with alterations in ocular surface, tear film function and MG morphology. Early screening and treatment of MG dysfunction may improve ocular surface quality and allow better disease management in keratoconus patients.

Non-invasive assessment of intestinal permeability in healthy volunteers using transcutaneous fluorescence spectroscopy.

The permeability of the intestinal barrier is altered in a multitude of gastrointestinal conditions such as Crohn's and coeliac disease. However, the clinical utility of gut permeability is currently limited due to a lack of reliable diagnostic tests. To address this issue, we report a novel technique for rapid, non-invasive measurement of gut permeability based on transcutaneous ('through-the-skin') fluorescence spectroscopy. In this approach, participants drink an oral dose of a fluorescent dye (fluorescein) and a fibre-optic fluorescence spectrometer is attached to the finger to detect permeation of the dye from the gut into the blood stream in a non-invasive manner. To validate this technique, clinical trial measurements were performed in 11 healthy participants. First, after 6 h of fasting, participants ingested 500 mg of fluorescein dissolved in 100 ml of water and fluorescence measurements were recorded at the fingertip over the following 3 h. All participants were invited back for a repeat study, this time ingesting the same solution but with 60 g of sugar added (known to transiently increase intestinal permeability). Results from the two study datasets (without and with sugar respectively) were analysed and compared using a number of analysis procedures. This included both manual and automated calculation of a series of parameters designed for assessment of gut permeability. Calculated values were compared using Student's T-tests, which demonstrated significant differences between the two datasets. Thus, transcutaneous fluorescence spectroscopy shows promise in non-invasively discriminating between two differing states of gut permeability, demonstrating potential for future clinical use.

Microvascular neural blood flow assessment for a chronic nerve compression neuropathy mouse model by fluorescein angiography.

BACKGROUND AND AIMS: Intraoperative fluorescence angiography with indocyanine green or fluorescein is used in ophthalmology and neurosurgery. However, there are few reports on the use of fluorescence angiography for peripheral neuropathy. This study aimed to assess the validity of fluorescein angiography (FAG) for peripheral nerve entrapment neuropathy. METHODS: We used an established model of chronic nerve compression (CNC) neuropathy in C57BL/6 mice by entrapping their left sciatic nerve with a silastic tube. Mice were assigned to the uninjured group (control), two-week CNC neuropathy group, four-week CNC neuropathy group, or six-week CNC neuropathy group. We then performed FAG to assess neural blood flow and quantified the peak of the luminance at the compression site with luminance analysis software. Following FAG, histological examinations using an anti-fluorescein antibody and Masson's trichrome staining were performed to evaluate the area of fluorescein distribution and epineural fibrosis. RESULTS: The luminance in the CNC neuropathy groups was significantly lower than that in the control group. Histological analysis revealed the fluorescein positive areas in the CNC neuropathy groups were significantly smaller than that in the control group, and the epineural fibrosis areas in the CNC neuropathy groups were significantly larger than that in the control group. CONCLUSION: We observed a significant decline of luminance in the CNC neuropathy groups, and the histological assessment was consistent with this result. FAG was found to be a valid method for assessing CNC neuropathy in mice.

Validation sentinel lymph node biopsy study in cN0 axilla using low-cost dual dye technique: potential solution for resource poor settings.

BACKGROUND: Sentinel lymph node biopsy (SLNB) using radio-pharmaceutical and a blue dye is gold standard for axillary staging in clinically node-negative breast cancer. High costs and limited availability of radio-pharmaceutical and/or gamma probe are major deterrents in performing SLNB in developing countries. In this study, we evaluated feasibility of SLN identification (SLN-IR) of fluorescein-guided (FG) SLNB in combination with methylene blue dye (MBD). METHODS: This was a prospective cross-sectional non-randomized validation study in patients with clinically node-negative axilla. Patients underwent validation SLNB using fluorescein (and blue LED light) and MBD. Axillary dissection was performed irrespective of SLNB histology. SLIN-IR and False Negative Rate (FNR) were assessed for both groups. RESULTS: The SLNs were identified in 29 (96.6%) pre-chemotherapy patients and 23 (82%) post Neoadjuvant Chemotherapy (NACT) patients. The median number of sentinel lymph nodes identified was 3 (range of 1-5) in pre-chemotherapy patients and 1 (range of 1-3) in post NACT patients. The SLN-IR using MBD was 90%, FD was 86.7% and combined MBD FD was 96.7% in pre-chemotherapy patients. The SLN-IR using MBD was 82%, FD was 71% and combined MBD FD was 82% in in post NACT patients. The false negative rate (FNR) in pre-chemotherapy group was 8.0% (MBD), 8.3% (FD) and 7.4% (MBD + FD). The FNR in post NACT group was 8.7% (MBD), 10% (FD) and 8.7% (MBD + FD). CONCLUSION: This prospective validation study showed adequate SLN-IR and FNR using low-cost dual dyes in early breast cancer patients and can be used in low resource settings. However, SLNB in post NACT axilla though viable along with a satisfactory FNR, is associated with low identification rate and needs further evaluation.

Cost-effectiveness of agents used for evaluation of ureteral patency during intraoperative cystoscopy in gynecologic and urogynecologic surgery.

BACKGROUND: Intraoperative evaluation of ureteral patency is often performed in gynecologic and urogynecologic surgery. Many agents are used to help assess the patency, each with its own associated cost, ease of use, and adverse reactions. Some agents, such as dextrose, are used as an instillation fluid to create a viscosity difference and aid the visualization of a ureteral jet. Others, such as oral phenazopyridine or the intravenous use of sodium fluorescein and indigo carmine, cause a color change of the urine to directly aid the visualization of ureteral jets. Recently, numerous studies have examined the efficacy and surgeon satisfaction of these agents. The studies have also emphasized certain options as associated with a lower cost. However, there have not been any cost studies comparing these agents. OBJECTIVE: To compare the cost-effectiveness of the following 4 agents that are commonly used in assessing ureteral patency intraoperatively: oral phenazopyridine, dextrose instillation, intravenous sodium fluorescein, and intravenous indigo carmine. STUDY DESIGN: We constructed a decision-analytic model to compare cystoscopy using oral phenazopyridine, dextrose instillation, intravenous sodium fluorescein, and intravenous indigo carmine. Failure to see efflux resulted in work-ups for ureteral obstruction. The probabilities were obtained from published studies, and the probability of successfully seeing efflux ranged from 0.92 with oral phenazopyridine to 0.99 with intravenous indigo carmine. The costs of the agents, adverse effects, and ureteral obstruction work-ups were obtained from the University of North Carolina at Chapel Hill Department of Pharmacy, the Healthcare Cost and Utilization Project 2016 database and the FAIR Health Consumer database. The cost of a ureteral obstruction work-up used in our model ranged from $9755 for intraoperative evaluation with retrograde pyelograms and stents to $29,034 for hospitalization. Our primary outcome was the incremental cost-effectiveness ratio per unnecessary work-up for ureteral obstruction avoided. Sensitivity analyses were performed to identify the key uncertainties. RESULTS: Oral phenazopyridine, followed by an intravenous agent if needed, had a mean cost of $110 per patient. Dextrose averaged $151 more per patient, with only a slight improvement in avoiding unnecessary ureteral obstruction work-ups and a higher cost associated with adverse reactions (incremental cost-effectiveness ratio, $62,000). Intravenous agents cost approximately $1000 more per patient and were less effective at preventing unnecessary work-ups. Sensitivity analyses did not identify any thresholds that would significantly change the outcomes. CONCLUSION: Our model suggests that oral phenazopyridine and dextrose instillation are the least expensive and the most effective agents to aid in the visualization of ureteral patency during intraoperative cystoscopy, although dextrose is associated with higher costs owing to a higher rate of adverse reactions (primarily urinary tract infections). Intravenous sodium fluorescein and indigo carmine are historically popular first-choice agents. However, they were found to be more expensive and less effective as primary agents in our model and should likely be reserved for use as secondary agents in the event that the visualization of ureteral jets is unclear with the initial use of phenazopyridine or dextrose.

Tear film interferometry assessment after intense pulsed light in dry eye disease: A randomized, single masked, sham-controlled study.

BACKGROUND: Inadequate meibomian glands (MGs) secretion can lead to dry eye signs and symptoms. Tear film lipid layer (TFLL) secreted by MGs protects and prevents rapid evaporation of tear film. The purpose was to assess TFLL alteration and function in patients with evaporative dry eye (EDE) using tear interferometry after optimal pulse light technology (OPT) intense pulsed light (IPL). METHODS: This prospective randomized examiner-masked sham- controlled study included 86 participants (142 eyes) with DED. IPL or sham procedure was performed on day 0, 21, and 42. Ocular Surface Disease Index (OSDI), non-invasive breakup time (NITBUT), interferometric fringe pattern determined TFLL quality, fluorescein staining (FS), and meibum gland (MG) were assessed at day 0, 21, 42 and 3-month. RESULTS: At 3-month, TFLL, NITBUT, MG drop-out, MG quality, MG expressibility, FS and OSDI improved significantly (P < 0.05) in the IPL group, while the sham group had no significant improvements. Except for Meibo-score and FS, all parameters significantly correlated with the improvement in TFLL following IPL treatment. Additionally, artificial tears usage was significantly less in the IPL group from D-42 onwards. CONCLUSION: IPL treatment demonstrated the ability to improve TFLL quality and clinically reduced sign and symptoms of DED thereby reducing the frequency of artificial tears use.

Development and Assessment of an Inexpensive Smartphone-Based Respiratory Droplet Simulation Model.

Background. Droplet simulation often requires expensive and inaccessible equipment. Herein, we develop and assess a low-cost droplet simulation model using easily accessible materials, open-source software, and a smartphone-based cobalt blue light. Methods. The simulation model was developed using commercial-grade materials and fluorescein dye. A clear face shield was assessed ten times following a simulated cough using fluorescein dye. A conventional ultraviolet Woods lamp was compared to a smartphone-based cobalt blue light to detect fluorescein illumination. Results. The simulation platform and smartphone-based cobalt blue light cost $20.18. A Wilcoxon signed rank test revealed that the median droplet area of fluorescence under the UV Wood's lamp was not significantly different than that of the smartphone-based cobalt blue light (2.89 vs 2.94, P = .386). Conclusions. This simulation model is inexpensive and easily reproducible. The smartphone application may be a convenient alternative to standard ultraviolet lights. This model has great potential for use in financially restricted academic centers during the COVID-19 pandemic and beyond.

Assessment of iris vasculature abnormalities in dogs with diabetes mellitus.

OBJECTIVE: To identify and characterize abnormalities of iris vasculature in dogs with diabetes mellitus, compared to clinically normal, age-matched control dogs, by means of anterior segment angiography. ANIMALS: 10 dogs with naturally occurring diabetes mellitus and 10 age-matched control dogs with no ocular or systemic disease. PROCEDURES: The day before iris vasculature abnormality (IVA) assessment, all dogs underwent complete physical and ophthalmic examinations and baseline clinicopathologic analyses. For diabetic dogs, serum fructosamine concentration and a 12-hour blood glucose concentration curve were generated. The next day, all dogs were sedated and anterior segment angiography (following IV injection of indocyanine green [1 mg/kg] and subsequently sodium fluorescein [20 mg/kg]) was performed with a full-spectrum camera and camera adapter system. Group findings were compared, and multiple linear regression analysis was performed to identify potential factor associations with IVAs. RESULTS: During anterior segment angiography, the arterial, capillary, and venous phases were identified in all dogs. Times to onset of all phases in diabetic dogs were significantly less than those in control dogs. Vascular disruptions within the peripupillary region (evident following sodium fluorescein administration) were common in diabetic dogs. Severity of dye leakage into the iris stroma and aqueous humor was significantly greater in diabetic dogs than in control dogs. Duration of disease, mean blood glucose concentration, and serum fructosamine concentration were significantly associated with IVAs. CONCLUSIONS AND CLINICAL RELEVANCE: In diabetic dogs, anterior segment angiography revealed IVAs that were not evident in control dogs. The severity of those changes appeared to be associated with disease duration and blood glucose regulation.

Fluorescence polarization immunoassay for rapid determination of dehydroepiandrosterone in human urine.

In this paper, five fluorescein-labeled dehydroepiandrosterone (DHEA) derivatives (tracers) with different chain lengths between the fluorescein and hapten were synthesized and featured so as to establish a fluorescence polarization immunoassay (FPIA) for DHEA detection in human urine samples with previously prepared polyclonal antibody against DHEA. The outcomes of the structure of tracer on FPIA sensitivity were investigated. Under the optimal condition, the fluorescence polarization value (FP) decreases linearly in DHEA concentration, ranging from 1.6 to 243.3 ng mL-1, with the limit of detection of 1.1 ng mL-1 and IC50 value of 25.1 ng mL-1. Moreover, the developed FPIA was time-saving as it could complete the detection within 3 min. FPIA and commercial enzyme-linked immunosorbent assay kit were both applied to analyze the spiked human urine samples with DHEA. Excellent recoveries (92.1-108.0%) and satisfactory correlation coefficient (R2 = 0.98) were acquired with the two methods, indicating that the developed FPIA was a fast and efficient screening immunoassay with accuracy and sensitivity for DHEA detection in human urine samples. Graphical abstract.

Aerosolised fluorescein can quantify FFP mask faceseal leakage: a cost-effective adaptation to the existing point of care fit test.

BACKGROUND: A qualitative fit test using bitter-tasting aerosols is the commonest way to determine filtering face-piece (FFP) mask leakage. This taste test is subjective and biased by placebo. We propose a cheap, quantitative modification of the taste test by measuring the amount of fluorescein stained filter paper behind the mask using image analysis. METHODS: A bitter-tasting fluorescein solution was aerosolised during mask fit tests, with filter paper placed on masks' inner surfaces. Participants reported whether they could taste bitterness to determine taste test 'pass' or 'fail' results. Filter paper photographs were digitally analysed to quantify total fluorescence (TF). RESULTS: Fifty-six healthcare professionals were fit tested; 32 (57%) 'passed' the taste test. TF between the taste test 'pass' and 'fail' groups was significantly different (p<0.001). A cut-off (TF = 5.0 × 106 units) was determined at precision (78%) and recall (84%), resulting in 5/56 participants (9%) reclassified from 'pass' to 'fail' by the fluorescein test. Seven out of 56 (12%) reclassified from 'fail' to 'pass'. CONCLUSION: Fluorescein is detectable and sensitive at identifying FFP mask leaks. These low-cost adaptations can enhance exiting fit testing to determine 'pass' and 'fail' groups, protecting those who 'passed' the taste test but have high fluorescein leak, and reassuring those who 'failed' the taste test despite having little fluorescein leak.

Assessment of Blood-Brain Barrier Permeability Using Miniaturized Fluorescence Microscopy in Freely Moving Rats.

We report here the method of visualization of brain microcirculation and assessment of blood-brain barrier (BBB) permeability changes using the miniature integrated fluorescence microscope (i.e., miniscope) technology in awake, freely moving rats. The imaging cannula is implanted in the brain area of interest of anesthetized adult rats. After recovery and habituation, sodium fluorescein, a low-molecular-weight tracer, is injected i.v. Fluorescence intensity in the vicinity of microvessels, as an indicator of BBB permeability, is then recorded in vivo via the miniscope for extended periods of time. The method can be used to assess the changes in BBB permeability produced by pharmacologic agents; in this case, the drug of interest is administered after sodium fluorescein. An increase in the sodium fluorescein extravasation in brain microcirculation demonstrates an increase in BBB permeability. The method described here allows a high-resolution visualization of real-time changes in BBB permeability in awake, freely moving rats.

Silicon Photomultipliers as a Low-Cost Fluorescence Detector for Capillary Electrophoresis.

Capillary electrophoresis (CE) is a highly efficient separation method capable of handling small sample volumes (∼pL) and low (∼yoctomole) detection limits and, as such, is ideal for applications that require high sensitivity, such as single-cell analysis (Chen et al. Anal. Chem. 1996, 68 (4), 690-696; Cohen et al. Annu. Rev. Anal. Chem. 2008, 1 (1), 165-190; Vickerman et al. ACS Chem. Biol. 2018, 13 (7), 1741-1751). Low-cost CE instrumentation is quickly expanding, but low-cost, open-source fluorescence detectors with ultrasensitive detection limits are lacking (Vickerman et al. ACS Chem. Biol. 2018, 13 (7), 1741-1751; Fang et al. Electrophoresis 2016, 37 (17-18), 2376-2383; Casto et al. Anal. Chem. 2019, 40 (1), 65-78). Silicon photomultipliers (SiPM) are inexpensive, low-footprint detectors with the potential to fill the role as a detector when cost, size, and customization are important. In this work, we demonstrate the use of a SiPM in CE with zeptomolar detection limits and a dynamic range spanning 5 orders of magnitude, comparable to photomultiplier detectors. The performance of these detectors was measured using a continuous wave excitation laser in an epifluorescence detection configuration. We characterize the performance of the SiPM as a highly sensitive detector by measuring enzyme activity in single cells. This simple, small footprint, and low-cost (<$130) light detection circuit will be beneficial for open-source, portable, and budget-friendly instrumentation requiring high sensitivity.

Fluorescein dye as a novel cost-effective approach for staining raw specimens in ophthalmic pathology.

PURPOSE: To study the usefulness of sodium fluorescein dye for staining raw specimens in ophthalmic pathology. METHODS: Laboratory-based observational study. Eye specimens received in the ocular pathology department of a tertiary eye care center in northeast India were included in the study after obtaining the informed consent. The study period was from 2016 to 2019. Specimens received were a corneal button, lid, orbital tissues, enucleated eyeballs, eviscerated eye, explanted intraocular lens (IOLs), optic nerve and ocular parasites. Sections of the gross specimens were stained with sodium fluorescein (C20H12O5NA) dye. The average duration of tissue-stain contact time was 45 s. The sections were analyzed under the compound microscope. The intensity of illumination of the microscope was modulated to obtain high contrast digital photographs. RESULTS: 26 corneal buttons with or without limbal tissue specimens were analyzed with fluorescein staining procedure; limbus with its pigmented cells were seen in the enucleated eyeballs. 33 enucleated eyes (retinoblastoma [RB] (n = 24), phthisical eyes (n = 4), choroidal melanomas (n = 2), and others (n = 3) were included in the study cohort. In these 33 enucleated eyes, vitreous were also examined for the presence of hyalocytes and other pathological cells. Retinal pigment epithelial cells were also seen (n = 11). RB seeds were seen with fluorescein stain and documented in 14 specimens. The RB seeds were mostly in vitreous (n = 9) and subretinal space (n = 5). Fat cells (n = 8) from orbital tissues and sebaceous cells (n = 5) from frozen section specimens were also observed and documented. CONCLUSION: This study highlights a novel method of rapid staining of gross ophthalmic pathology specimens.

Quantitative High-speed Assessment of Droplet and Aerosol From an Eye After Impact With an Air-puff Amid COVID-19 Scenario.

PURPOSE: To quantify aerosol and droplets generated during noncontact tonometry (NCT) and assess the spread distance of the same. METHODOLOGY: This was an experimental study on healthy human volunteers (n=8 eyes). In an experimental setup, NCT was performed on eyes (n=8) of human volunteers under normal settings, with a single and 2 drops of lubricant. High-speed shadowgraphy, frontal lighting technique, and fluorescein analysis were used to detect the possible generation of any droplets and aerosols. Mathematical computation of the spread of the droplets was then performed. RESULTS: In a natural setting, there was no droplet or aerosol production. Minimal splatter along with droplet ejection was observed when 1 drop of lubricant was used before NCT. When 2 drops of lubricant were instilled, a significant amount of fluid ejection in the form of a sheet that broke up into multiple droplets was observed. Some of these droplets traversed back to the tonometer. Droplets ranging from 100 to 500 µm in diameter were measured. CONCLUSIONS: There was no droplet generation during NCT performed in a natural setting. However, NCT should be avoided in conditions with high-tear volume (natural or artificial) as it would lead to droplet spread and tactile contamination.

Low-cost Fluorescein as an Alternative to Radio-colloid for Sentinel Lymph Node Biopsy-a Prospective Validation Study in Early Breast Cancer.

BACKGROUND: Sentinel lymph node biopsy (SLNB) using radio-pharmaceutical (RP) and a blue dye is gold standard for axillary staging in clinically node-negative early breast cancer. High costs and limited availability of RP and/or gamma probe are major deterrents in performing SLNB in developing countries. Fluorescence-guided SLNB can obviate the need for RP and gamma probe. Fluorescein is an inexpensive fluorescent lymphatic tracer. In this study, we compared SLN identification rate (SLN-IR) and false negative rates (FNR) of fluorescein-guided SLNB and radio-guided SLNB using 99mTc-Sulfur-colloid, in isolation, or in combination with methylene blue dye (MBD). METHODS: Sixty-five cN0 early and large operable breast cancer patients underwent validation SLNB using fluorescein (and blue LED light), 99mTc-Sulfur-colloid (and gamma probe) and MBD. Inj Fluorescein 4% was injected, 1 ml each peri-tumoral and sub-areolar five minutes before axillary incision. Axillary dissection was performed irrespective of SLNB histology. The SLN-IR and FNR with various tracers and their combinations were compared. RESULTS: The mean number of SLNs identified was 3.5 ± 1.8 (range 1-6). The SLN-IR using RP alone was 94%, fluorescein alone was 92%, and MBD alone was 82%. The SLN-IR using fluorescein plus MBD combination was 95.4%, compared to 97% using MBD plus RP combination. FNR was 6.3% using fluorescein plus MBD, as well as RP plus MBD combinations. CONCLUSIONS: SLN-IR of > 90% and SLN-FNR of < 10% using fluorescein plus MBD combination are in acceptable range, and are comparable to that of RP plus MBD combination. Fluorescein can replace RP for performing SLNB, in combination with MBD.

Development and assessment of a novel ex vivo corneal culture technique involving an agarose-based dome scaffold for use as a model of in vivo corneal wound healing in dogs and rabbits.

OBJECTIVE: To develop and assess a novel ex vivo corneal culture technique involving an agarose-based dome scaffold (ABDS) for use as a model of in vivo corneal wound healing in dogs and rabbits. SAMPLE: Corneas from clinically normal dogs (paired corneas from 8 dogs and 8 single corneas) and rabbits (21 single corneas). PROCEDURES: 8 single dog corneas (DCs), 1 DC from each pair, and 10 rabbit corneas (RCs) were wounded with an excimer laser; 1 DC from each pair and 11 RCs remained unwounded. Corneas were cultured for 21 days on ABDSs (8 pairs of DCs and all RCs) or on flat-topped scaffolds (8 single DCs). The surface area of corneal fluorescein retention was measured every 6 (DCs) or 12 (RCs) hours until full corneal epithelialization was detected. Changes in corneal clarity were evaluated at 0, 7, 14, and 21 days. RESULTS: Median time to full epithelialization for wounded dog and rabbit corneas was 48 and 60 hours, respectively; among wounded DCs, time to full epithelization did not differ by scaffold type. After 21 days of culture on ABDSs, all DCs and RCs that epithelialized developed a circular, diffuse, cloud-like pattern of optical haze, whereas DCs cultured on flat-topped scaffolds developed a focal, crater-like region of optical haze. All corneas on the ABDSs maintained convex curvature throughout the study. CONCLUSIONS AND CLINICAL RELEVANCE: Wounded ex vivo DCs and RCs cultured on ABDSs reliably epithelialized, formed optical haze (consistent with in vivo wound healing), and maintained convex curvature. This culture technique may be adaptable to other species.

Protein Characterization in 3D: Size, Folding, and Functional Assessment in a Unified Approach.

Assessment of protein stability and function is key to the understanding of biological systems and plays an important role in the development of protein-based drugs. In this work, we introduce an integrated approach based on Taylor dispersion analysis (TDA), flow induced dispersion analysis (FIDA), and in-line intrinsic fluorescence which enables rapid and detailed assessment of protein stability and unfolding. We demonstrate that the new platform is able to efficiently characterize chemically induced protein unfolding of human serum albumin (HSA) in great detail. The combined platform enables local structural changes to be probed by monitoring changes in intrinsic fluorescence and loss of binding of a low-molecular weight ligand. Simultaneously, the size of the unfolding HSA is obtained by TDA on the same samples. The integration of the methodologies enables a fully automated characterization of HSA using only a few hundred nanoliters of sample. We envision that the presented methodology will find applications in fundamental biophysics and biology as well as in stability screens of protein-based drug candidates.

Assessment of Tear Functions in Patients with Acne Rosacea without Meibomian Gland Dysfunction.

Purpose: To evaluate the results of tear functions in acne rosacea. Methods: This prospective study includes 64 eyes of 32 acne rosacea patients without blepharitis and meibomian gland dysfunction and 90 eyes of 45 patients as control group. Tear functions of all were evaluated with ocular surface disease index (OSDI) questionnaire, and measurements of tear osmolarity were performed by using TearLab, Schirmer I tests without anesthesia and fluorescein tear break-up time (TBUT). Results: The mean Schirmer test result was 12.53 ± 6.54 in study group and 16.21 + 7.52 mm/5 min in control group (p = 0.28). The mean TBUT in study group was 8.21 ± 4.01 and in control group was 18.03 ± 6.45 s (p = 0.02). Mean tear osmolarity in study group was 304.77 ± 15.59and in control group was 275.23 + 28.52 mOsms/L (p = 0.03). Mean OSDI score in study group was 27.51 ± 16.73 and was 18.15 ± 7.05 in control group (p = 0.38). Conclusions: Our study demonstrated lower dry eye tests before the appearance of clinical signs of meibomian gland disease in acne rosacea.