Photoactivatable metabolic warheads enable precise and safe ablation of target cells in vivo.

Photoactivatable molecules enable ablation of malignant cells under the control of light, yet current agents can be ineffective at early stages of disease when target cells are similar to healthy surrounding tissues. In this work, we describe a chemical platform based on amino-substituted benzoselenadiazoles to build photoactivatable probes that mimic native metabolites as indicators of disease onset and progression. Through a series of synthetic derivatives, we have identified the key chemical groups in the benzoselenadiazole scaffold responsible for its photodynamic activity, and subsequently designed photosensitive metabolic warheads to target cells associated with various diseases, including bacterial infections and cancer. We demonstrate that versatile benzoselenadiazole metabolites can selectively kill pathogenic cells - but not healthy cells - with high precision after exposure to non-toxic visible light, reducing any potential side effects in vivo. This chemical platform provides powerful tools to exploit cellular metabolic signatures for safer therapeutic and surgical approaches.

Py3-FITC: a new fluorescent probe for live cell imaging of collagen-rich tissues and ionocytes.

Polypyrrole-based polyamides are used as sequence-specific DNA probes. However, their cellular uptake and distribution are affected by several factors and have not been extensively studied in vivo. Here, we generated a series of fluorescence-conjugated polypyrrole compounds and examined their cellular distribution using live zebrafish and cultured human cells. Among the evaluated compounds, Py3-FITC was able to visualize collagen-rich tissues, such as the jaw cartilage, opercle and bulbus arteriosus, in early-stage living zebrafish embryos. Then, we stained cultured human cells with Py3-FITC and found that the staining became more intense as the amount of collagen was increased. In addition, Py3-FITC-stained HR cells, which represent a type of ionocyte on the body surface of living zebrafish embryos. Py3-FITC has low toxicity, and collagen-rich tissues and ionocytes can be visualized when soaked in Py3-FITC solution. Therefore, Py3-FITC may be a useful live imaging tool for detecting changes in collagen-rich tissue and ionocytes, including their mammalian analogues, during both normal development and disease progression.

Zebrafish heart regenerates after chemoptogenetic cardiomyocyte depletion.

BACKGROUND: Zebrafish can regenerate adult cardiac tissue following injuries from ventricular apex amputation, cryoinjury, and cardiomyocyte genetic ablation. Here, we characterize cardiac regeneration from cardiomyocyte chemoptogenetic ablation caused by localized near-infrared excited photosensitizer-mediated reactive oxygen species (ROS) generation. RESULTS: Exposure of transgenic adult zebrafish, Tg(myl7:fapdl5-cerulean), to di-iodinated derivative of the cell- permeable Malachite Green ester fluorogen (MG-2I) and whole-body illumination with 660 nm light resulted in cytotoxic damage to about 30% of cardiac tissue. After chemoptogenetic cardiomyocyte ablation, heart function was compromised, and macrophage infiltration was detected, but epicardial and endocardial activation response was much muted when compared to ventricular amputation. The spared cardiomyocytes underwent proliferation and restored the heart structure and function in 45-60 days after ablation. CONCLUSIONS: This cardiomyocyte ablation system did not appear to activate the epicardium and endocardium as is noted in other cardiac injury models. This approach represents a useful model to study specifically cardiomyocyte injury, proliferation and regeneration in the absence of whole organ activation. Moreover, this system can be adapted to ablate distinct cell populations in any organ system to study their function in regeneration.

Switchable cascade C-H annulation to polycyclic pyryliums and pyridiniums: discovering mitochondria-targeting fluorescent probes.

Disclosed herein is a counterion additive-switched rhodium-catalyzed cascade triple C-H annulation of 4-hydroxy-1-naphthaldehydes with alkynes, in which six chemical bonds are formed in one-pot. This reaction enables the rapid assembly of diverse polycyclic pyrylium and pyridinium fluorophores, which leads to two specific mitochondria-labeling reagents with low cytotoxicity and superior photostability.

Immunological Effects of Aggregation-Induced Emission Materials.

Nanotechnology is widely used in the fields of biology and medicine. Some special nanoparticles with good biocompatibility, hydrophilicity, and photostability can be used as ideal systems for biomedical imaging in early diagnosis and treatment of diseases. Among them, aggregation-induced emission materials are new antiaggregation-caused quenching nano-imaging materials, which have advantages in biocompatibility, imaging contrast, and light stability. Meanwhile, heterogeneity of nanoparticles may cause various adverse immune reactions. In response to the above problems, many researchers have modified nano-materials to be multifunctional nano-composites, aiming at combining diagnosis and treatment with simultaneous imaging and targeted therapy and additionally avoiding immune reactions, which is of great potential in imaging-guided therapy. This review discusses the application of aggregation-induced emission materials, and other nano-imaging materials are also mentioned. We hope to provide new ideas and methods for the imaging of nano-materials in diagnosis and treatment.

pHLIP ICG for delineation of tumors and blood flow during fluorescence-guided surgery.

Fluorescence imaging has seen enduring use in blood flow visualization and is now finding a new range of applications in image-guided surgery. In this paper, we report a translational study of a new fluorescent agent for use in surgery, pHLIP ICG, where ICG (indocyanine green) is a surgical fluorescent dye used widely for imaging blood flow. We studied pHLIP ICG interaction with the cell membrane lipid bilayer, the pharmacology and toxicology in vitro and in vivo (mice and dogs), and the biodistribution and clearance of pHLIP ICG in mice. The pHLIP ICG tumor targeting and imaging efficacy studies were carried out in several murine and human mouse tumor models. Blood vessels were imaged in mice and pigs. Clinical Stryker imaging instruments for endoscopy and open surgery were used in the study. Intravenously administered pHLIP ICG exhibits a multi-hour circulation half-life, offering protracted delineation of vasculature. As it clears from the blood, pHLIP ICG targets tumors and tumor stroma, marking them for surgical removal. pHLIP ICG is non-toxic, marks blood flow for hours after injection, and effectively delineates tumors for improved resection on the day after administration.

Fluorescein-guided surgery for spinal gliomas: Analysis of 220 consecutive cases.

OBJECTIVE: Sodium fluorescein (FL) is widely used as a fluorescent tracer for brain tumor resection. However, FL-guided resection of spinal gliomas has been reported only occasionally. To evaluate the safety, characteristics, and usefulness of FL-guided surgery in the resection of spinal glioma. METHODS: Between January 2015 and December 2018, 220 consecutive patients with 227 spinal gliomas underwent FL-guided resection using the Zeiss Pentero 900 surgical microscope with an integrated YELLOW 560 filter. FL evaluation and clinical outcomes were analyzed. RESULTS: No FL-related complications occurred in this series. Entire tumor fluorescence was observed in 161 (70.93%) gliomas, nodular fluorescence in 46 (20.26%) tumors, and no fluorescence in 20 (8.81%) tumors. The intraoperative fluorescence of 217 (95.59%) gliomas was highly correlated with preoperative contrast-enhancing magnetic resonance imaging, except in eight ependymomas, one pilocytic astrocytoma, and one diffuse midline glioma. Gross-total resection was achieved in 78.85% (179/227) of spinal gliomas, including 94.30% (149/158) ependymal tumors and 43.48% (30/69) astrocytic and oligodendroglial tumors. At the final clinical follow-up, the spinal function of 75 (33.04%) patients showed significant improvement, 105 (46.26%) showed stabilization, and 47 (20.70%) showed deterioration. CONCLUSION: FL is a safe and useful real-time tool that could enhance tumor borders or residual tumors and hence increase the gross-total resection rate in cases with contrast-enhanced tumors.

First-in-Human Assessment of cRGD-ZW800-1, a Zwitterionic, Integrin-Targeted, Near-Infrared Fluorescent Peptide in Colon Carcinoma.

PURPOSE: Incomplete oncologic resections and damage to vital structures during colorectal cancer surgery increases morbidity and mortality. Moreover, neoadjuvant chemoradiotherapy has become the standard treatment modality for locally advanced rectal cancer, where subsequent downstaging can make identification of the primary tumor more challenging during surgery. Near-infrared (NIR) fluorescence imaging can aid surgeons by providing real-time visualization of tumors and vital structures during surgery. EXPERIMENTAL DESIGN: We present the first-in-human clinical experience of a novel NIR fluorescent peptide, cRGD-ZW800-1, for the detection of colon cancer. cRGD-ZW800-1 was engineered to have an overall zwitterionic chemical structure and neutral charge to lower nonspecific uptake and thus background fluorescent signal. We performed a phase I study in 11 healthy volunteer as well as a phase II feasibility study in 12 patients undergoing an elective colon resection, assessing 0.005, 0.015, and 0.05 mg/kg cRGD-ZW800-1 for the intraoperative visualization of colon cancer. RESULTS: cRGD-ZW800-1 appears safe, and exhibited rapid elimination into urine after a single low intravenous dose. Minimal invasive intraoperative visualization of colon cancer through full-thickness bowel wall was possible after an intravenous bolus injection of 0.05 mg/kg at least 2 hours prior to surgery. Longer intervals between injection and imaging improved the tumor-to-background ratio. CONCLUSIONS: cRGD-ZW800-1 enabled fluorescence imaging of colon cancer in both open and minimal invasive surgeries. Further development of cRGD-ZW800-1 for widespread use in cancer surgery may be warranted given the ubiquitous overexpression of various integrins on different types of tumors and their vasculature.

Evaluation of Potential Systemic Adverse Events Related to Fluorescein Angiography in Pediatric Patients.

PURPOSE: To evaluate adverse events of fluorescein angiography (FA) in pediatric patients. DESIGN: Single-institution retrospective chart review. PARTICIPANTS: Patients 0 to 18 years of age who underwent FA between January 2010 and December 2015 at a single institution in the United States. METHODS: Pediatric patients who underwent FA by 3 surgeons were included in the study. Patients with fewer than 24 hours of documented follow-up were excluded. Significant adverse events within 24 hours of FA were evaluated. Detailed intraoperative and perioperative physiological parameters, including heart rate, blood pressure, oxygen saturation, and ventilation parameters, in inpatients undergoing simultaneous examination under anesthesia were reviewed. Peri-injection effects of FA were evaluated by 2-tailed paired t test comparison of mean 5-minute preinjection and 5-minute postinjection physiological data. MAIN OUTCOME MEASURES: Significant adverse events associated with FA. RESULTS: One hundred fifteen patients with a total of 214 FA examinations were included. No significant adverse events were associated directly with FA. Comparison of mean 5-minute preinjection and postinjection physiologic parameters in 27 patients who underwent intravenous FA during EUA did not reveal significant changes associated with FA. A significant difference was found in average patient age between inpatient (2.5 years) and outpatient (10.7 years) FA (P < 0.00001). The youngest patients who underwent successful FA were 3.8 years old in the outpatient setting and 32 weeks' postmenstrual age in the inpatient setting. Patients younger than 3.8 years accounted for most (77.6%; n = 85) inpatient FA examinations. Excluding patients with a need or likely need for laser or surgery, the reasons for inpatient FA in patients older than 3.8 years included the lack of availability of outpatient ultra-widefield FA (UWFA) and more challenging situations in patients with developmental delay. CONCLUSIONS: Fluorescein angiography was not found to be associated directly with systemic adverse events in pediatric patients in this study. Younger patients more commonly were found to require an inpatient FA, whereas older patients older than 4 years underwent outpatient UWFA.

Detection of Fluorescent Powders and Their Effect on Survival and Recapture of Aedes aegypti (Diptera: Culicidae).

The use of insect markers, such as fluorescent powders, is a useful tool for studying ecological and epidemiological questions. Evaluating their effect on vectors of human disease agents, such as the invasive mosquito vector Aedes aegypti (Linnaeus), is crucial for their practical and reliable use, especially in parameters linked to the risk of disease transmission such as adult survival, dispersal, and host-seeking. Seven fluorescent powders (Hercules Radiant, DayGlo (DG), Risk Reactor (RR), and Angstrom Technologies), applied externally on cohorts of Ae. aegypti female mosquitoes, were tested to determine their impact on survival and recapture by baited mosquito traps, and their detectability after being exposed to controlled laboratory and semifield environments. There were no significant differences in survival among marked and unmarked females across all powders. Marked females were significantly less likely to be captured in baited traps relative to unmarked females, except for one of the DG powders. All females remained visibly marked on five parts of their body for 30 d (under both environments), except for one of the RR powders. The tested powders and application method are suitable for tracking mosquitoes throughout most of their lives under different environments, without significantly affecting their survival, but with potential impact on recapture by baited traps, possibly due to effects on senses or other physiological traits.

Fluorescent Cholangiography in Laparoscopic Cholecystectomy: An Updated Canadian Experience.

Background. Laparoscopic cholecystectomy (LC) is one of the most common general surgery procedures in Canada with approximately 100 000 cases performed per year. Bile duct injury remains a morbid complication with an incidence rate of 0.3% to 0.5%. Indocyanine green (ICG) fluorescent cholangiography is a noninvasive technology aiding in real-time identification of biliary structures for safe dissection within Calot's triangle. The objectives were to provide an update to our initial experience with ICG aiding in the identification of biliary structures and ensuring that no adverse patient reactions occurred with ICG administration. Methods. Prospective case series from 2016 to 2018 for elective LC with ICG technology performed at a single academic teaching institution. Patient demographics, indications for operation, biliary structures visualized, amount of ICG used, operative times, and complications were recorded. Results. One hundred eight cases were included for review. The cystic duct, common hepatic duct, and common bile duct were identified with ICG in 90%, 48%, and 84% of cases, respectively. ICG simultaneously visualized at least 2 of 3 biliary structures 83.4% of the time. Only 1 biliary structure was identified in 10% of cases. No biliary structures were identified in 6% of cases. Mean initial ICG dose given was 1.65 mL. No adverse patient reactions to ICG were noted. Conclusions. This updated series illustrates that administration of ICG enhances visualization of the biliary system during outpatient LC. ICG is safe and its application should be further studied in early LC for acute cholecystitis.

Extracapsular dissection in peripheral nerve schwannoma surgery using bright light and fluorescein sodium visualization: case series.

BACKGROUND: Schwannomas are the most frequent peripheral nerve sheath tumors and are treated by surgical resection when symptomatic. Tumor removal is performed by intraneural dissection and enucleation. In order to safely remove the tumor from the nerve, the use of sodium fluorescein has recently been proposed to distinguish the tumor from the adjacent normal nerve fibers, before incision of the tumor pseudocapsule and during intraneural tumor dissection. METHODS: We report a consecutive case series of 5 peripheral nerve schwannomas operated in 4 patients, in which we evaluate the usefulness of sodium fluorescein compared to usual visual landmarks, at each step of the surgical procedure. RESULTS: After exposition of the schwannoma, sodium fluorescein helped with the localization of intracapsular en passant nerve fascicles in only one case. Hence, the definition of a safe entry zone for capsular incision relied mainly on nerve monitoring and direct visualization of en passant nerve fascicles under microscope. During intraneural dissection, there was a sharp contrast between the fluorescent tumor and the non-fluorescent adjacent pseudocapsule in most cases but the colorimetric variation between tumor and normal tissue induced by fluorescence did not outperform the natural contrast between the yellow true capsule and the gray-red layers of the pseudocapsule. CONCLUSION: Based on these results, we consider that the limited additional value of sodium fluorescein in primary peripheral nerve schwannoma surgery does not warrant its use in daily clinical practice. Additional studies are needed to assess its usefulness during the surgery of recurrences and tumors which are intertwined with several fascicles of origin such as neurofibromas.

Impact of fluorescence and autofluorescence on surgical strategy in benign and malignant neck endocrine diseases.

Fluorescence and autofluorescence have been shown by several recent studies to be valuable adjuncts in identifying parathyroid glands during thyroidectomy and parathyroidectomy. The aim of this chapter is to review the impact of this new technology on surgical strategy concerning identification and preservation of parathyroid glands during thyroidectomy, identification of parathyroid glands in hyperparathyroidism, and the potential role in thyroid cancer surgery.

Tumor Targeting Strategies of Smart Fluorescent Nanoparticles and Their Applications in Cancer Diagnosis and Treatment.

Advantages such as strong signal strength, resistance to photobleaching, tunable fluorescence emissions, high sensitivity, and biocompatibility are the driving forces for the application of fluorescent nanoparticles (FNPs) in cancer diagnosis and therapy. In addition, the large surface area and easy modification of FNPs provide a platform for the design of multifunctional nanoparticles (MFNPs) for tumor targeting, diagnosis, and treatment. In order to obtain better targeting and therapeutic effects, it is necessary to understand the properties and targeting mechanisms of FNPs, which are the foundation and play a key role in the targeting design of nanoparticles (NPs). Widely accepted and applied targeting mechanisms such as enhanced permeability and retention (EPR) effect, active targeting, and tumor microenvironment (TME) targeting are summarized here. Additionally, a freshly discovered targeting mechanism is introduced, termed cell membrane permeability targeting (CMPT), which improves the tumor-targeting rate from less than 5% of the EPR effect to more than 50%. A new design strategy is also summarized, which is promising for future clinical targeting NPs/nanomedicines design. The targeting mechanism and design strategy will inspire new insights and thoughts on targeting design and will speed up precision medicine and contribute to cancer therapy and early diagnosis.

A zwitterionic near-infrared fluorophore for real-time ureter identification during laparoscopic abdominopelvic surgery.

Iatrogenic injury of the ureters is a feared complication of abdominal surgery. Zwitterionic near-infrared fluorophores are molecules with geometrically-balanced, electrically-neutral surface charge, which leads to renal-exclusive clearance and ultralow non-specific background binding. Such molecules could solve the ureter mapping problem by providing real-time anatomic and functional imaging, even through intact peritoneum. Here we present the first-in-human experience of this chemical class, as well as the efficacy study in patients undergoing laparoscopic abdominopelvic surgery. The zwitterionic near-infrared fluorophore ZW800-1 is safe, has pharmacokinetic properties consistent with an ideal blood pool agent, and rapid elimination into urine after a single low-dose intravenous injection. Visualization of structure and function of the ureters starts within minutes after ZW800-1 injection and lasts several hours. Zwitterionic near-infrared fluorophores add value during laparoscopic abdominopelvic surgeries and could potentially decrease iatrogenic urethral injury. Moreover, ZW800-1 is engineered for one-step covalent conjugatability, creating possibilities for developing novel targeted ligands.

QuatCy: A Heptamethine Cyanine Modification With Improved Characteristics.

A major restriction on optical imaging techniques is the range of available fluorophores that are compatible with aqueous media without aggregation, absorb light above 750 nm with high extinction coefficients, fluoresce with relatively high quantum yields, and resist photodecomposition. Indocyanine green (ICG or A in this paper) is an important example of a fluorophore that fits this description. Other dyes that are becoming increasingly prevalent are select heptamethine cyanine dyes (Cy7) which feature a cyclohexyl framework to rigidify the conjugated alkenes, and meso-chlorine substitution; MHI-148 (B) is one example. Methods: Research described here was initiated to uncover the consequences of a simple isoelectronic substitution to MHI-148 that replaces a cyclohexyl methylene with a dialkyl ammonium fragment. Solubility experiments were carried out in aqueous and cell culture media, photophysical properties including fluorescence quantum yields, brightness and stability were measured. Moreover, in vivo pharmacokinetics, distribution and tumor seeking properties were also explored. Results: Modification to incorporate dialkyl ammonium fragment leads to a brighter, more photostable fluorophore, with a decreased tendency to aggregation, complementary solubility characteristics, and a lower cytotoxicity. Conclusion: All the above-mentioned parameters are favorable for many anticipated applications of the new dye we now call quaternary cyanine-7 or QuatCy.

A Highly Efficient Tumor-Targeting Nanoprobe with a Novel Cell Membrane Permeability Mechanism.

Efficient tumor targeting has been a great challenge in the clinic for a very long time. The traditional targeting methods based on enhanced permeability and retention (EPR) effects show only an ≈5% targeting rate. To solve this problem, a new graphene-based tumor cell nuclear targeting fluorescent nanoprobe (GTTN), with a new tumor-targeting mechanism, is developed. GTTN is a graphene-like single-crystalline structure amphiphilic fluorescent probe with a periphery that is functionalized by sulfonic and hydroxyl groups. This probe has the characteristic of specific tumor cell targeting, as it can directly cross the cell membrane and specifically target to the tumor cell nucleus by the changed permeability of the tumor cell membranes in the tumor tissue. This new targeting mechanism is named the cell membrane permeability targeting (CMPT) mechanism, which is very different from the EPR effect. These probes can recognize tumor tissue at a very early stage and track the invasion and metastasis of tumor cells at the single cell level. The tumor-targeting rate is improved from less than 5% to more than 50%. This achievement in efficient and accurate tumor cell targeting will speed up the arrival of a new era of tumor diagnosis and treatment.

Safety and feasibility of low-dose fluorescein-guided resection of glioblastoma.

OBJECTIVES: The extent of resection is an independent predictor of prognosis in patients with glioblastomas. Although fluorescein sodium may enhance intraoperative visualization of tumor margin and increase the extent of glioblastoma, the dose related anaphylactic reaction is still a major concern. In the present study, we used allergy skin testing to exclude the patients susceptible to anaphylaxis preoperatively, and then investigated the feasibility of low-dose fluorescein sodium to guide glioblastoma resection intraoperatively, thereby to improve the safety of fluorescein-guided glioma resection. PATIENTS AND METHODS: Patients with suspected glioblastoma based on brain MRI were subjected to allergy skin intradermal tests for fluorescein sodium preoperatively. Only those with negative allergy skin tests received intravenous injection of low dose fluorescein sodium (1-2 mg/kg) during microsurgical tumor resection under dedicated Yellow 560 filter. The degree of fluorescent staining was documented and the extent of resection was evaluated by MRI scan. RESULTS: One patient with positive allergy skin test was excluded from fluorescein sodium administration and no anaphylactic reaction was found during fluorescein sodium guided surgery in the patients who were negative for allergy skin tests. The low dose fluorescein sodium (1-2 mg/kg) could provide enough visualization of tumors with sufficient discrimination from surrounding normal brain tissue and improve the resection extent of glioblastoma. CONCLUSION: Preoperative allergy skin test is a useful method to exclude the patients susceptible to anaphylaxis, together with intraoperative low dose fluorescein sodium administration, may facilitate glioblastoma resection by fluorescence guidance while avoid safety concern of dose-related anaphylaxis.

Dynasore protects the ocular surface against damaging oxidative stress.

Water soluble "vital" dyes are commonly used clinically to evaluate health of the ocular surface; however, staining mechanisms remain poorly understood. Recent evidence suggests that sublethal damage stimulates vital dye uptake by individual living cells. Since cell damage can also stimulate reparative plasma membrane remodeling, we hypothesized that dye uptake occurs via endocytic vesicles. In support of this idea, we show here that application of oxidative stress to relatively undifferentiated monolayer cultures of human corneal epithelial cells stimulates both dye uptake and endocytosis, and that dye uptake is blocked by co-treatment with three different endocytosis inhibitors. Stress application to stratified and differentiated corneal epithelial cell cultures, which are a better model of the ocular surface, also stimulated dye uptake; however, endocytosis was not stimulated, and two of the endocytosis inhibitors did not block dye uptake. The exception was Dynasore and its more potent analogue Dyngo-4a, both small molecules developed to target dynamin family GTPases, but also having off-target effects on the plasma membrane. Significantly, while Dynasore blocked stress-stimulated dye uptake at the ocular surface of ex vivo mouse eyes when treatment was performed at the same time as eyes were stressed, it had no effect when used after stress was applied and the ocular surface was already damaged. Thus, Dynasore could not be working by inhibiting endocytosis. Employing cytotoxicity and western blotting assays, we went on to demonstrate an alternative mechanism. We show that Dynasore is remarkably protective of cells and their surface glycocalyx, preventing damage due to stress, and thus precluding dye entry. These unexpected and novel findings provide greater insight into the mechanisms of vital dye uptake and point the direction for future study. Significantly, they also suggest that Dynasore and its analogues might be used therapeutically to protect the ocular surface and to treat ocular surface disease.

Effect of fluorescein angiography on renal functions in type 2 diabetes patients: A pilot study.

Fluorescein angiography (FA) is an important tool for the diagnosis and management of diabetic retinopathy. However, the safety of fluorescein sodium on renal functions is not fully understood. One hundred type 2 diabetes patients, within the Ophthalmology Outpatient Clinic at Alexandria Main University Hospital, Egypt, were enrolled in this prospective observational study to determine the safety of FA on renal function. Serum creatinine and cystatin C were measured pre- and 2 days post-FA. Urinary neutrophil gelatinase-associated lipocalin (uNGAL) was measured pre- and 4 hours post-FA. Renal injury was defined as a 25% increase in serum creatinine, cystatin C, or uNGAL. The study included 71 females and 29 males, with a mean age of 55.73 ± 7.29 years. Baseline serum cystatin C and uNGAL were 0.89 ± 0.34 mg/L and 21.7 ± 2.39 ng/mL, respectively. Serum cystatin C and uNGAL significantly increased after FA to 0.95 ± 0.36 and 27 ± 2.81, respectively (P <0.001). Eleven patients (11%) experienced more than a 25% rise in serum cystatin C from baseline, whereas 40 patients (40%) experienced more than a 25% increase in uNGAL levels after FA. However, the mean serum creatinine level did not change significantly after FA (P = 0.061). Only one patient experienced more than a 25% rise in serum creatinine from baseline. FA showed a significant increase in early sensitive acute kidney injury biomarkers (as serum cystatin C and uNGAL) in substantial number of patients, suggesting but still not proving, a potential harmful effect of FA on kidney functions. These findings were not demonstrated using ordinary serum creatinine.