A fast, easy, cost-free method to remove excess dye or drug from small extracellular vesicle solution.

Tracking small extracellular vesicles (sEVs), such as exosomes, requires staining them with dyes that penetrate their lipid bilayer, a process that leaves excess dye that needs to be mopped up to achieve high specificity. Current methods to remove superfluous dye have limitations, among them that they are time-intensive, carry the risk of losing sample and can require specialized equipment and materials. Here we present a fast, easy-to-use, and cost-free protocol for cleaning excess dye from stained sEV samples by adding their parental cells to the mixture to absorb the extra dye much like sponges do. Since sEVs are considered a next-generation drug delivery system, we further show the success of our approach at removing excess chemotherapeutic drug, daunorubicin, from the sEV solution.

A simple, economical and environmental-friendly method for staining protein gels using an extract from walnut-husk.

We wish to present a simple, rapid, cost-effective and environmentally safe method for staining proteins in polyacrylamide gels, using aqueous-based natural extracts from fresh green walnut (Juglans regia) hulls/husks. The technique takes not more than 10 min for staining and is comparable in sensitivity to the most commonly used Coomassie R-250 staining method when applied to different concentrations of Bovine Serum Albumin (BSA) and various amounts of E. coli extracts. The protein (BSA) band (~0.5 µg) and E. coli extract comprising ~25 µg total protein can be visualized on polyacrylamide gels. Compared to both Coomassie and Ponceau S staining, the current method displayed more intense bands when proteins are transferred to polyvinylidene fluoride (PVDF) membrane. Although the walnut-dye (WD) method does not require a time-consuming destaining step, excess background stain can simply be removed by washing in water. Extract from old dried black husks and extract from fresh green husks kept for a year was also effective. Using LC-MS, Myricetin and/or Kaempferol were found to be active compounds responsible for staining proteins. Compared to traditional Coomassie method, the inclusion of expensive and toxic solvents (methanol and acetic acid) is completely avoided resulting in positive health, environmental and economic benefits. In view of all these advantages, the WD method has immense potential to replace currently used protein staining techniques.

Fast and cost-effective preparation of plant cells for scanning electron microscopy (SEM) analysis.

The analysis of plant cell structure provides valuable information about its morphological, physiological, and biochemical characteristics. Nowadays, scanning electron microscope (SEM) is widely used to provide high-resolution images at the surface of biological samples. However, biological specimens require preparation, including dehydration and coating with conductive materials for imaging by SEM. There are several techniques for providing images with maximum maintenance of cell structure and minimum cellular damage, but each requires the use of expensive and hazardous materials, which can be damaging to the cell in many cases. Therefore, the provision of new and effective preparation methods based on maintaining cell structure for imaging can be very practical. In the present study, a fast and cost-effective protocol was first performed for chemical fixation and preparation of the plant cells for imaging by SEM. Taxus baccata and Zhumeria majdae cells were chemically fixed using glutaraldehyde and then successfully dried with different percentages of ethanol including 70, 80, 90, and 100%. In addition, SEM was performed for imaging the cell surface in different micro-scales. This protocol can be used by plant cell biologists and biotechnologists who are interested in studying structural and biochemical responses of treated or stressed plant cells by SEM.

A practical method for preparing fluorescent-labeled glycans with a 9-fluorenylmethyl derivative to simplify a fluorimetric HPLC-based analysis.

Analysis of glycans in glycoproteins is often performed by liquid chromatography (LC) separation coupled with fluorescence detection and/or mass spectrometric detection. Enzymatically or chemically released glycans from glycoproteins are usually labeled by reductive amination with a fluorophore reagent. Although labeling techniques based on reductive amination have been well-established as sample preparation methods for fluorometric HPLC-based glycan analysis, they often include time-consuming and tedious purification steps. Here, we reported an alternative fluorescent labeling method based on the synthesis of hydrazone and its reduction using 9-fluorenylmethyl carbazate (Fmoc-hydrazine) as a fluorophore reagent. Using isomaltopentaose and N-glycans from human IgG, we optimized the Fmoc-labeling conditions and purification procedure of Fmoc-labeled N-glycans and applied the optimized method for the analysis of N-glycans released from four glycoproteins (bovine RNase B, human fibrinogen, human α1-acid glycoprotein, and bovine fetuin). The complete workflow for preparation of fluorescent-labeled N-glycans takes a total of 3.5 h and is simple to implement. The method presented here lowers the overall cost of a fluorescently labeled N-glycan and will be practically useful for the screening of disease-related glycans or routine analysis at an early stage of development of biopharmaceuticals.

Implementation of the FilmArray ME panel in laboratory routine using a simple sample selection strategy for diagnosis of meningitis and encephalitis.

BACKGROUND: Infectious meningitis is a serious disease and patient outcome relies on fast and reliable diagnostics. A syndromic panel testing approach like the FilmArray ME can accelerate diagnosis and therefore decrease the time to pathogen specific therapy. Yet, its clinical utility is controversial, mainly because of a remaining uncertainty in correct interpretation of results, limited data on its performance on clinical specimens and its relatively high costs. The aim of this study was to analyze clinical performance of the assay in a real life setting at a tertiary university hospital using a pragmatic and simple sample selection strategy to reduce the overall cost burden. METHODS: Over a period of 18 months we received 4623 CSF samples (2338 hospitalizations, 1601 individuals). FilmArray ME analysis was restricted to CSF-samples with a high pretest probability of infectious meningitis, e.g. positive Gram-stain, samples in which leukocytes and/or bacteria were evident or urgent suspicion of infection was communicated by clinicians. N = 171 samples matched to our risk criteria and were subjected to FilmArray ME analysis. Those samples were also analyzed by reference methods: culture only (n = 45), PCR only (n = 20) or both methods (n = 106). RESULTS: 56/171 (32.75%) were FilmArray ME positive. Bacterial pathogens were detected in 30/56 (53.57%), viral pathogens were detected in 27/56 (48.21%) and yeast DNA was detected in 1/56 (1.79%) of positive samples. Double detection occurred in 2/56 samples. In 52/56 (92.86%) FilmArray ME positive samples, results could be confirmed by the reference assays (sensitivity = 96.30%, specificity =96.58%). CONCLUSION: The FilmArray ME assay is a fast and reliable diagnostic tool for the management of infectious meningitis and can easily be implemented in routine diagnostic workflows. However, correlation of test results and underlying clinical symptoms requires experienced users and the awareness of potentially false negative or false positive results. Moreover, considering the need for antimicrobial susceptibility testing, the use of molecular tests as a stand-alone diagnostic cannot be recommended.

Methods for detection of brain injury after photothrombosis-induced ischemia in mice: Characteristics and new aspects of their application.

BACKGROUND: Photothrombosis is a minimally invasive method for induction of cortical ischemia. However, different ways of applying some methods to assess photothrombosis-induced damage need to be developed. NEW METHODS: We applied the tongue protrusion test and H&E staining of brain sections to detect ischemic damage after photothrombosis. Evaluation of the local status of the BBB using Evans blue dye was proposed. We also assessed the sensitivity of the grid-walk test. Moreover, we examined the interchangeability of MRI and TTC staining to measure lesion volume. RESULTS: We evaluated ischemic outcomes at 24 h after photothrombosis in mice. The tongue protrusion test did not reveal impairments of the neurological status whereas the grid-walk test showed the high sensitivity. Using histological techniques, we determined the reduction in the number of neurons with normal morphology in the penumbra. 3D reconstruction of the brain, which reflected Evans blue dye distribution in the nervous tissue, revealed BBB disruption in areas remote from the ischemic core. We also showed the strong correlation between damage volumes assessed by MRI and TTC staining. COMPARISON WITH EXISTING METHODS: The present work demonstrates the efficacy of the classical histological approach and TTC staining that are more affordable than MRI and immunohistochemical methods. Detection of 3D distribution of Evans blue dye in the brain in contrast to its total extraction reveals BBB damage in details. CONCLUSIONS: We proposed the simple methods for describing the severity of brain ischemia at the cellular and whole organism levels without significant labor and financial expenditures.

Low-cost internal controls for detection of Giardia cysts in water samples.

Giardia cysts stained with hot carbolfuchsin were used as internal controls in a concentration method for surface water samples. The morphological integrity of stained cysts and the stain's stability and intensity were tested with each of the chemical reagents used in the aluminum sulfate flocculation method. No alterations in morphology or color were noted. The stained cyst preparation has a low cost, high stability, and suitability for both light and immunofluorescent microscopy, making it affordable to researchers in low- and middle-income countries.

A Rapid Protocol for Intraoperative Assessment of Peripheral Nerve Myelinated Axon Count and Its Application to Cross-Facial Nerve Grafting.

BACKGROUND: Donor nerve myelinated axon counts correlate with functional outcomes in reanimation procedures; however, there exists no reliable means for their intraoperative quantification. In this article, the authors report a novel protocol for rapid quantification of myelinated axons from frozen sections, and demonstrate its applicability to surgical practice. METHODS: The impact of various fixation and FluoroMyelin Red staining strategies on resolved myelin sheath morphology from cryosections of rat and rabbit femoral and sciatic nerves was assessed. A protocol comprising fresh cryosection and rapid staining was developed, and histomorphometric results were compared against conventional osmium-postfixed, resin-embedded, toluidine blue-stained sections of rat sciatic nerve. The rapid protocol was applied for intraoperative quantification of donor nerve myelinated axon count in a cross-facial nerve grafting procedure. RESULTS: Resolution of myelinated axon morphology suitable for counting was realized within 10 minutes of tissue harvest. Although mean myelinated axon diameter appeared larger using the rapid fresh-frozen as compared to conventional nerve processing techniques (mean ± SD; rapid, 9.25 ± 0.62 µm; conventional, 6.05 ± 0.71 µm; p < 0.001), no difference in axon counts was observed on high-power fields (rapid, 429.42 ± 49.32; conventional, 460.32 ± 69.96; p = 0.277). Whole nerve myelinated axon counts using the rapid protocol herein (8435.12 ± 1329.72) were similar to prior reports using conventional osmium processing of rat sciatic nerve. CONCLUSIONS: A rapid protocol for quantification of myelinated axon counts from peripheral nerves using widely available equipment and techniques has been described, rendering possible intraoperative assessment of donor nerve suitability for reanimation.

Microscopic examination of Gram-stained smears for anogenital gonorrhoea in men who have sex with men is cost-effective: evidence from a modelling study.

OBJECTIVE: To assess the cost-effectiveness of three testing strategies with or without light microscopic Gram-stained smear (GSS) evaluation for the detection of anogenital gonorrhoea among men who have sex with men (MSM) at the Amsterdam STI clinic using a healthcare payer perspective. METHODS: Three testing strategies for MSM were compared: (1) GSS in symptomatic MSM only (currently practised strategy), (2) no GSS and (3) GSS in symptomatic and asymptomatic MSM. The three testing protocols include testing with nucleic acid amplification test to verify the GSS results in (1) and (3), or as the only test in (2). A transmission model was employed to calculate the influence of the testing strategies on the prevalence of anogenital gonorrhoea over 10 years. An economic model combined cost data on medical consultations, tests and treatment and utility data to estimate the number of epididymitis cases and quality-adjusted life years (QALY) associated with gonorrhoea. Incremental cost-effectiveness ratios (ICERs) for the testing scenarios were estimated. Uncertainty and sensitivity analyses were performed. RESULTS: No GSS testing compared with GSS in symptomatic MSM only (current strategy) resulted in nine extra epididymitis cases (95% uncertainty interval (UI): 2-22), 72 QALYs lost (95% UI: 59-187) and €7300 additional costs (95% UI: -€185 000 (i.e.cost-saving) to €407 000) over 10 years. GSS testing in both symptomatic and asymptomatic MSM compared with GSS in symptomatic MSM only resulted in one prevented epididymitis case (95% UI: 0-2), 1.1 QALY gained (95% UI: 0.1-3.3), €148 000 additional costs (95% UI: €86 000 to-€217 000) and an ICER of €177 000 (95% UI: €67 000-to €705 000) per QALY gained over 10 years. The results were robust in sensitivity analyses. CONCLUSIONS: GSS for symptomatic MSM only is cost-effective compared with no GSS for MSM and with GSS for both symptomatic and asymptomatic MSM.

Prospective comparison of cytological specimen adequacy assessment by different rapid staining techniques for rapid on-site evaluation in fine needle aspiration cytology and their cost-effectiveness.

OBJECTIVE: Rapid on-site evaluation (ROSE) is a technique beneficial in determining the adequacy of the samples, thereby increasing the diagnostic yield, useful in triage of specimens for ancillary studies and can also help determine a preliminary diagnosis in emergency cases. The different rapid stains for on-site evaluation described in the literature are diff quik, toluidine blue (TB), brilliant cresyl blue (BCB), ultra-fast Pap stains, and rapid hematoxylin and eosin (H&E). This study was undertaken as there is sparse literature regarding the best and the most cost-effective rapid stain. METHOD: Fine needle aspiration samples from 200 patients with palpable swellings in easily accessible regions were taken. Smears stained by rapid and routine stains were assessed based on four parameters, with provisional diagnosis on the rapid stained smears. A comparative analysis of the advantages and disadvantages of the rapid stains was carried out with appropriate statistical tests with the routinely stained smears as gold standard. RESULTS: There was adequate material in 100% of ROSE smears. rapid pap stained smears showed well preserved cytoplasmic details, nuclear details, and background details. The time taken was least with TB and BCB being 5 s each. The most cost-effective was found to be TB. CONCLUSIONS: We conclude that TB is the most cost-effective, quick, least labor-intensive, and reliable rapid stain for ROSE especially in resource-poor settings.

Cost-effectiveness of surgeon performed intraoperative specimen ink in breast conservation surgery.

BACKGROUND: Re-excision rates after breast conservation surgery are reported to be 20%-40%. Inaccuracies with specimen orientation may affect margin assessment. This study examined whether the addition of surgeon performed intraoperative inking of the lumpectomy specimen after adoption of margin guidelines would be cost-effective. METHODS: A retrospective review of a prospective surgical database was performed from 2009 to 2017. Patients with initial lumpectomy and a preoperative diagnosis of invasive breast carcinoma or ductal carcinoma in situ (DCIS) were included. Re-excision rates and the surgical costs per 100 initial lumpectomies were compared across three periods: before margin guideline publication, after guideline adoption, and after the addition of intraoperative surgeon performed specimen inking. RESULTS: Four hundred initial lumpectomies were evaluated. Overall re-excision rate was 21% (n = 84). There was a nonsignificant reduction in re-excision rates after margin guidelines from 24% (n = 36) to 20% (n = 23) and to 19% (n = 25) after addition of intraoperative specimen ink. Re-excision rates were significantly lower for invasive cancer than for DCIS across three periods (20%, 15%, and 12% versus 37%, 33%, and 31%) (odds ratio 3.31, P = 0.007). The estimated cost of re-excision per 100 initial lumpectomies decreased after guidelines by 25% ($128,270) for invasive breast cancer and by 11% ($102,616) for DCIS. The addition of intraoperative specimen inking after margin guideline adoption resulted in further 17% cost savings ($66,692) for invasive breast cancer and 5% ($41,308) for DCIS. CONCLUSIONS: Surgeon performed intraoperative inking of the lumpectomy specimen after adoption of margin guidelines is a cost-effective technique in breast conservation surgery.

Quantitative nuclear histomorphometry predicts oncotype DX risk categories for early stage ER+ breast cancer.

BACKGROUND: Gene-expression companion diagnostic tests, such as the Oncotype DX test, assess the risk of early stage Estrogen receptor (ER) positive (+) breast cancers, and guide clinicians in the decision of whether or not to use chemotherapy. However, these tests are typically expensive, time consuming, and tissue-destructive. METHODS: In this paper, we evaluate the ability of computer-extracted nuclear morphology features from routine hematoxylin and eosin (H&E) stained images of 178 early stage ER+ breast cancer patients to predict corresponding risk categories derived using the Oncotype DX test. A total of 216 features corresponding to the nuclear shape and architecture categories from each of the pathologic images were extracted and four feature selection schemes: Ranksum, Principal Component Analysis with Variable Importance on Projection (PCA-VIP), Maximum-Relevance, Minimum Redundancy Mutual Information Difference (MRMR MID), and Maximum-Relevance, Minimum Redundancy - Mutual Information Quotient (MRMR MIQ), were employed to identify the most discriminating features. These features were employed to train 4 machine learning classifiers: Random Forest, Neural Network, Support Vector Machine, and Linear Discriminant Analysis, via 3-fold cross validation. RESULTS: The four sets of risk categories, and the top Area Under the receiver operating characteristic Curve (AUC) machine classifier performances were: 1) Low ODx and Low mBR grade vs. High ODx and High mBR grade (Low-Low vs. High-High) (AUC = 0.83), 2) Low ODx vs. High ODx (AUC = 0.72), 3) Low ODx vs. Intermediate and High ODx (AUC = 0.58), and 4) Low and Intermediate ODx vs. High ODx (AUC = 0.65). Trained models were tested independent validation set of 53 cases which comprised of Low and High ODx risk, and demonstrated per-patient accuracies ranging from 75 to 86%. CONCLUSION: Our results suggest that computerized image analysis of digitized H&E pathology images of early stage ER+ breast cancer might be able predict the corresponding Oncotype DX risk categories.

A rapid and nondestructive protocol for whole-mount bone staining of small fish and Xenopus.

Here we propose a new protocol for whole-mount bone staining, which allows the rapid preparation of highly cleared and nondestructive specimens. It only takes 3 days to complete whole procedure for small vertebrates, such as medaka, zebrafish, and Xenopus frogs. In this procedure, we used a newly developed fixative containing formalin, Triton X-100, and potassium hydroxide, which allows the fixation, decolorization, and transparentization of specimens at the same time. A bone staining solution containing alizarin red S with ethylene glycol and a clearing solution containing Tween 20 and potassium hydroxide also contributed the specificity and swiftness of this new system. As expected, although details of the skeletal system could be observed in specimens with high transparency, it was noteworthy that high-resolution fluorescence images acquired using zoom microscopes clearly delineated the shape of each bone. This new procedure would be expected to be widely used as a standard procedure for bone staining in the testing the developmental toxicity of chemicals and in the screening test of knockout or mutant animals.

Simultaneous detection of intra- and inter-molecular paramagnetic relaxation enhancements in protein complexes.

Paramagnetic relaxation enhancement (PRE) measurements constitute a powerful approach for detecting both permanent and transient protein-protein interactions. Typical PRE experiments require an intrinsic or engineered paramagnetic site on one of the two interacting partners; while a second, diamagnetic binding partner is labeled with stable isotopes (15N or 13C). Multiple paramagnetic labeled centers or reversed labeling schemes are often necessary to obtain sufficient distance restraints to model protein-protein complexes, making this approach time consuming and expensive. Here, we show a new strategy that combines a modified pulse sequence (1HN-Γ2-CCLS) with an asymmetric labeling scheme to enable the detection of both intra- and inter-molecular PREs simultaneously using only one sample preparation. We applied this strategy to the non-covalent dimer of ubiquitin. Our method confirmed the previously identified binding interface for the transient di-ubiquitin complex, and at the same time, unveiled the internal structural dynamics rearrangements of ubiquitin upon interaction. In addition to reducing the cost of sample preparation and speed up PRE measurements, by detecting the intra-molecular PRE this new strategy will make it possible to measure and calibrate inter-molecular distances more accurately for both symmetric and asymmetric protein-protein complexes.

Validation of simple and cost-effective stains to assess acrosomal status, DNA damage and mitochondrial activity in rooster spermatozoa.

Several methods have been developed to evaluate spermatozoa function in birds but many of these are sometimes complicated, costly and not applicable to field studies (i.e., performed within poultry breeding facilities). The objective was, therefore, to validate efficient, practical and inexpensive procedures to determine DNA fragmentation, acrosomal integrity, and mitochondrial activity in poultry spermatozoa. Initially, ejaculates were individually diluted and divided into control (4°C, 4h) and UV-irradiated aliquots (room temperature, 4h), and then samples containing different percentages of DNA-damaged spermatozoa (0%, 25%, 50%, 75% and 100%) were subjected to Toluidine Blue (TB) and Sperm Chromatin Dispersion assessments (SCD). Fast Green-Rose Bengal (FG-RB) and FITC-PSA staining protocols were subsequently used to assess acrosome status in aliquots comprising assorted amounts of acrosome-reacted spermatozoa. Furthermore, to validate 3,3'-diaminobenzidine (DAB) assay, ejaculates containing different gradients of spermatozoa with great amounts of mitochondrial activity were concurrently evaluated using DAB and JC-1 stains. The proportion of spermatozoa with abnormal DNA integrity when evaluated using the TB assessment correlated significantly with the expected percentages of UV-irradiated spermatozoa and with SCD results. A significant linear regression coefficient was also observed between expected amounts of acrosome-intact spermatozoa and FG-RB readings, and there was a significant correlation of the data when FG-RB and FITC-PSA were used. Likewise, the use of the DAB assay enabled for accurately ascertaining percentages of rooster spermatozoa with greater and lesser mitochondrial function, and results were highly correlated to results with staining with JC-1. Altogether, findings of the present study indicate acrosomal status, DNA integrity and mitochondrial activity in rooster spermatozoa can be easily and reliably determined using FG-RB, TB and DAB stains.

Sectioning Mammary Gland Whole Mounts for Lesion Identification.

Normal mammary gland development may be altered by exposure to environmental toxicants and pharmaceutical products, excessive exposure to hormones, and genetic alterations. Mammary gland whole mounts are an inexpensive method to capture the progression of morphological changes that may arise after exposure. However, in later life, when abnormalities are more prone to develop, sole reliance on this one method may not always provide enough information to make a proper diagnosis of the abnormality. Historically, in chemical test guideline studies, a single mammary gland is removed at necropsy and prepared as a hematoxylin and eosin (H&E)-stained section. The incorporation of contralateral mammary whole-mount collection and analysis decreases the likelihood of a false-negative assessment. Evaluation of the whole mount is limited by the presence of one or two entire mammary glands on a slide, and in some cases, the abnormalities observed in the whole mount are not uniformly represented in the H&E section.  The goal of this study was to develop a protocol for converting coverslipped mammary whole mounts to H&E-stained sections so that lesions that would otherwise have been missed or that are difficult to diagnose can be identified. Here, we detail a method to produce a high-quality, paraffin-embedded H&E section from a mammary gland that was initially prepared as a whole mount. In comparison to a tissue that was intentionally prepared for H&E sectioning, the whole mount requires additional preparation for tissue removal and processing. However, this method is considered inexpensive, as it requires common lab reagents and little additional time. As a result, this method can provide invaluable information on how chemical and environmental exposures alter normal mammary development, as well as display changes that occur because of genetic modifications.

Utility of Acridine Orange staining for detection of bacteria from positive blood cultures.

The diagnostic performance of AO stain was evaluated for the detection of bacteria and or fungi from positive blood cultures. The sensitivity of Gram stain (GS) was 98.26% while Acridine Orange (AO) stain proved to be more sensitive (100%) with a Positive and Negative Predictive Value of 100% each. The specificity of both the stains was 100%. Overall agreement between the two stains was 98.23% (688/700). The organisms that were missed by GS and positive by AO were Candida species (Sutton, 2006) and Gram negative bacilli (GNB) (Sutton, 2006). Sensitivity of GS was 82.35% and AO was 100% among mixed cultures. Immediate reporting of the results of AO stain would have a significant impact on clinical management of patients with serious blood stream infections.

Tissue integrity, costs and time associated with different agents for histological bone preparation.

The selection of an appropriate demineralizing solution in pathology laboratories depends on several factors such as the preservation of cellularity, urgency of diagnostic and financial costs. The aim of this study was to test different decalcification bone procedures in order to establish the best value of these in formalin-fixed and paraffin-embedded samples. Femurs were removed from 13 adult male Wistar rats to obtain 130 bone disks randomly divided into five groups that were demineralized in different concentrations of nitric acid (Group I); formic acid (Group II); acetic acid (Group III); EDTA, pH7.4 (Group IV) and Morsés solution (Group V). Serial, 3-µm-thick sections were obtained and stained with hematoxylin-eosin to calculate the percentage of osteocyte-occupied lacunae. The sections were also stained with Masson's trichrome in conjunction with picrosirius red under polarized light followed by a semi-quantitative analysis to verify the adjacent muscle-to-bone integrity and preservation of collagen fibres. The highest percentage of osteocyte-occupied lacunae was found with 10% acetic acid solution (95.64 ± 0.95%) and Group I (nitric acid) demanded the shorter time (0.8-5.7days). Of all solutions, 5% nitric acid incurred the lowest cost to achieve complete demineralization compared with other solutions (p < .001). Group IV (EDTA) had the highest integrity of muscle and collagen type I and III (P < 0.01). Demineralization with 10% acetic acid was the most effective at preserving bone tissue, while 5% EDTA was the best at maintaining collagen and adjacent muscle to bone. In conclusion, nitric acid at 5% showed the most efficient result as it balanced both time and cost as a demineralizing solution.

Challenges to "Classic" Esophageal Candidiasis: Looks Are Usually Deceiving.

Objectives: We undertook the first case control study of histologically confirmed esophageal candidiasis (EC). Methods: A computer search from July 2012 through February 2015 identified 1,011 esophageal specimens, including 40 cases of EC and 20 controls. Results: The EC incidence was 5.2%; it was associated with immunosuppression and endoscopic white plaques and breaks. Smoking was a predisposing factor, and alcohol was protective. EC had no unique symptoms, and 54% of endoscopic reports did not suspect EC. Important histologic clues included superficial and detached fragments of desquamated and hyper-pink parakeratosis, acute inflammation, intraepithelial lymphocytosis, dead keratinocytes, and bacterial overgrowth. Thirty percent had no neutrophilic infiltrate. Pseudohyphae were seen on H&E in 92.5% (n = 37/40). "Upfront" periodic acid-Schiff with diastase (PAS/D) on all esophageal specimens would have generated $68,333.49 in patient charges. Our targeted PAS/D strategy resulted in $13,044.87 in patient charges (cost saving = 80.9%, $55,288.62). Conclusions: We describe the typical morphology of EC and recommend limiting PAS/D to cases where the organisms are not readily identifiable on H&E and with at least one of the following: (1) ulcer, (2) suspicious morphology, and/or (3) clinical impression of EC.