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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., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Isaioglou et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

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., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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., (Copyright © 2020 Elsevier Inc. All rights reserved.)

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., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier B.V. All rights reserved.)

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.

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

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., (© 2018 Wiley Periodicals, Inc.)

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.

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., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2019. No commercial re-use. See rights and permissions. Published by BMJ.)

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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.

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.

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 ( 15 N or 13 C). 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 ( 1 H N -Γ 2 -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.

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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., (Copyright © 2017 Elsevier B.V. All rights reserved.)

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.

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., (© 2016 Wiley Periodicals, Inc.)

Cytomegalovirus (CMV) causes clinically significant gastrointestinal (GI) injury. CMV inclusions can be identified on routine hematoxylin and eosin (H&E) stain, but immunohistochemistry (IHC) is also available for identifying CMV in tissue. The advent of accountable care organization models of care bring into question whether it is cost-effective for immunohistochemistry to be performed upfront at the request of clinicians and whether the quality of viral detection is compromised when the diagnosis of CMV is predicated on histologic review. In this study, a retrospective review of GI biopsies with CMV evaluations was performed. There were 449 cases with clinical requests to rule out CMV and 238 CMV analyses initiated by the pathologist without a clinical request. Among the cases that included a clinician's request, 37 had CMV detected. Immunostaining was performed on 26 cases, while a diagnosis based on readily identifiable viral inclusions on H&E-stained slides was made in 11. Among pathologist-initiated work-ups, 15 were CMV+, 3 of which had inclusions identified by H&E only. Among 38 CMV cases for which IHC had been performed, 27 had overt viral inclusions obvious on H&E. Seventy-two cases revealed uninflamed GI mucosa, and although a clinical concern about CMV infection was present, a CMV IHC work-up was not initially performed; all were negative for CMV by IHC and H&E. Clinical suspicion for CMV has a high yield for CMV detection, but "upfront" testing is likely unnecessary. Careful histopathologic review by a pathologist remains critical in the efficient and cost-effective detection of CMV., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© American Society for Clinical Pathology, 2017. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com)

Aims: To evaluate how reflexive versus selective H. pylori stains affect detection rates, turnaround time (TAT), and cost savings in a real life practice environment following an institutional policy change., Methods: The aforementioned parameters were evaluated in all cases in the year preceding and the year following an institutional policy change from reflexive to selective staining., Results: 1497 patients comprised the reflexive stain (RS) group of which 228 (15.2%) were H. pylori positive. 1629 patients comprised the selective stain (SS) group of which 237 (14.5%) were H. pylori positive. There was no significant difference in H. pylori detection rates between the RS and SS groups (OR=0.95, 95% CI=0.78-1.15, p=0.59). TATs were similarly equivalent with a mean of 52.4h for the RS cohort and 53.7h for the SS cohort (p=0.344), both of which included a resident preview day. We calculated an average laboratory cost savings of $11.68 per case, which saved our department over $15,000 (37%) in the year following the policy change., Conclusions: Our results support a policy of selective staining for H. pylori as opposed to reflexive staining and go on to show that laboratories that change their policy can expect to generate cost savings without compromising detection rates or TAT., (Copyright © 2016 Elsevier GmbH. All rights reserved.)

Despite the recommendation of expert gastrointestinal pathologists, private and academic centers (including our own) have continued to use ancillary stains for identification of Helicobacter pylori. For a 1-month period, gastric biopsies were prospectively evaluated for H pylori using routine hematoxylin and eosin (H&E) and a reflex Diff-Quik stain. During this time, 379 gastric biopsies were collected on 326 patients. H pylori organisms were prospectively identified in 23 (7%) patients, all of whom had superficial dense lymphoplasmacytic inflammation expanding the lamina propria. An additional 2 patients with neutrophilic inflammation were found to have H pylori by immunohistochemical staining. One patient diagnosed as having normal gastric mucosa was retrospectively found to have inflammation with rare H pylori organisms originally overlooked on both H&E and Diff-Quik but later identified on immunostain (0.5%). No patients with chemical gastritis (16%) or chronic inflammation (27%) were found to have H pylori. During the study month, 9 immunostains for H pylori were performed in addition to the 379 Diff-Quik. After discontinuation of reflex Diff-Quik, approximately 20 immunostains are performed for H pylori each month, which decreases technical time spent for processing gastric biopsies and reduces cost to the health care system. In our population with a low prevalence of H pylori, reflex staining for organisms is not cost-effective. The organisms can be seen on routine H&E; when suspicious superficial or active inflammation is present without visible organisms, immunohistochemical stains will confirm presence or absence within a day. Discontinuation of up-front ancillary studies is cost-effective without compromising patient care., (Copyright © 2016 Elsevier Inc. All rights reserved.)

There are two major methodical approaches with which changes of status in stomatal pores are addressed: indirectly by measurement of leaf transpiration, and directly by measurement of stomatal apertures. Application of the former method requires special equipment, whereas microscopic images are utilized for the direct measurements. Due to obscure visualization of cell boundaries in intact leaves, a certain degree of invasive leaf manipulation is often required. Our aim was to develop a protocol based on the minimization of leaf manipulation and the reduction of analysis completion time, while still producing consistent results. We applied rhodamine 6G staining of Arabidopsis thaliana leaves for stomata visualization, which greatly simplifies the measurement of stomatal apertures. By using this staining protocol, we successfully conducted analyses of stomatal responses in Arabidopsis leaves to both closure and opening stimuli. We performed long-term monitoring of living stomata and were able to document the same leaf before and after treatment. Moreover, we developed a protocol for rapid-fixation of epidermal peels, which enables high throughput data analysis. The described method allows analysis of stomatal apertures with minimal leaf manipulation and usage of the same leaf for sequential measurements, and will facilitate the analysis of several lines in parallel., Competing Interests: The authors have declared that no competing interests exist.

Background: The diagnosis of superficial mycosis such as dermatophytosis is often done clinically. However, in difficult cases, a rapid test with high sensitivity and specificity helps in the immediate confirmation and administration of treatment., Methods: The efficacy, rapidity of detection, and cost-effectiveness of KOH preparation, calcofluor white (CW) stain, and Chicago sky blue (CSB) stain in the identification of fungal elements were assessed in patients with dermatophytoses attending the dermatology clinic of a tertiary care hospital. All three tests were performed on each sample collected from 73 patients according to standard procedure. The slides were examined after 5 and 30 minutes in × 10 and × 40 magnifications. The sensitivity and specificity of CW and CSB at 5 and 30 minutes were calculated using KOH preparation as the standard test., Results: CSB stain showed highest positivity (94.5%) within 5 minutes when compared to KOH (75.3%) and CW (83.5%). After 30 minutes, positivity increased in KOH (84.9%) and CW stains (89%), but it remained the same in CSB stain. Both CW and CSB stains when compared to 10% KOH are equally sensitive (100%), but CW was more specific (72.7%), particularly at 30 minutes. When cost of performing tests on 100 specimens is considered, KOH, CW, and CSB stains cost Rs 5, 100, and 15, respectively., Conclusion: CSB stain is a better stain for rapid diagnosis of dermatophytoses because of ease of performance, rapidity of detection, better appreciation of morphology of fungal elements, and cost effectiveness., (© 2016 The International Society of Dermatology.)

Active enzymes, such as proteases, often serve as valuable biomarkers for various disease pathologies. Therefore, methods to detect specific enzyme activities in biological samples can provide information to guide disease detection and diagnosis and to increase our understanding of the biological roles of specific enzyme targets. In this protocol, we outline methods for the topical application of fluorescently quenched activity-based probes (qABPs) to fresh-frozen tissue samples. This technique enables rapid imaging of enzyme activity at cellular resolution, and it can be combined with antibody labeling for immunodiagnosis. In this method, fresh-frozen tissue sections are fixed, incubated with the probe and imaged using fluorescence microscopy. This provides an advance over classical immunohistochemistry (IHC) in that it is rapid (4-8 h) and inexpensive, and it provides information on enzyme activity. Furthermore, it can be used with any of the growing number of fluorescent ABPs to provide data for more effective disease monitoring and diagnosis.

Objective: Reflexive testing of bronchoalveolar lavage (BAL) specimens with Gomori methenamine silver (GMS) and acid-fast bacillus (AFB) stains is not routinely performed by most institutions. Instead, these stains are usually ordered to evaluate for the presence of fungal elements and/or acid-fast organisms if initial histopathologic assessment suggests the presence of these pathogens. Our institution, however, performs these stains on all BAL specimens. Thus, we sought to determine whether this practice was cost effective, considering the turnaround time and diagnostic efficacy of these tests., Methods: We retrospectively reviewed 488 BAL specimens performed at two military healthcare institutions over a 2-year period and performed a cost analysis with review of the impact on turnaround time., Results: Of the 488 cases, we identified only 3 (~0.6%) with infections by acid-fast or fungal organisms, at an estimated total cost of $12,151.20 and an average delay of 3.0 to 3.5 hours for slide preparation., Conclusion: Our results suggest that in a largely young and healthy population such as ours, it may be more feasible to perform these stains on BAL specimens on a case-by-case basis rather than automatically on every specimen, to control costs and enhance productivity., (Copyright© by the American Society for Clinical Pathology (ASCP).)

Eriochrome cyanine R (ECR) is a synthetic anionic dye that forms complexes with cations such as iron. We found that an iron-ECR (Fe-ECR) mixture provided either nuclear or myelin staining depending on the differentiator used. Selective nuclear staining was obtained by differentiation in an aqueous HCl solution, pH 0.95, followed by a wash in slightly alkaline tap water; the pH difference facilitated control of differentiation. When used with an eosin B counterstain, results were nearly indistinguishable from standard hematoxylin and eosin (H & E) staining. Nuclear staining with Fe-ECR provides tinctorial features similar to regressive aluminum-hemateins as well as resistance to acidic solutions such as those of iron hemateins. Fe-ECR also stained selectively intestinal cells of the diffuse neuroendocrine system (DNES). In addition to its use as an H & E substitute, acid differentiated Fe-ECR produced acid-resistant and selective nuclear counterstaining in combination with Alcian blue, and in the Papanicolaou and van Gieson techniques. With alkali differentiation, Fe-ECR produced selective myelin staining, which was compatible with neutral red counterstaining. Myelin sheaths were stained aqua blue. Fe-ECR could be used for both cytological and histological samples, and was suitable for use in automated tissue stainers. ECR also is less expensive than hematoxylin. Hematoxylin still may be preferred as a nuclear counterstain for some immunostaining methods for which Fe-ECR mixtures probably are too acidic.

Objectives: The practice of routine upfront use of special stains in biopsy specimens with minimal or no inflammation has not been evaluated. This study was conducted to determine the value of special stains for Helicobacter pylori in gastric biopsy specimens showing variable degrees of inflammation and to evaluate the practice of upfront ancillary staining of all mucosal biopsy specimens., Methods: Immunohistochemical or Diff-Quik stains were done on sections of 570 gastric biopsy specimens. The rates of positivity for H pylori were calculated in each category based on the degree of inflammation and classified as normal, minimal, mild, moderate, or severe., Results: H pylori was not detected in 386 (67.7%) biopsy specimens that were classified as normal (6.0%) or with minimal inflammation (28.2%) or mild inactive gastritis (33.5%). The organism was identified by Diff-Quik or immunohistochemical stain in 76 (89.4%) of 85 with moderate active gastritis and 30 (93.8%) of 32 biopsy specimens showing severe active gastritis., Conclusions: Upfront staining of gastric biopsy specimens is not cost-effective since a significant proportion of the gastric biopsy specimens were normal or showed minimal inflammation; in none of these biopsy specimens was the organism found. Special stains should be performed only in selected biopsy specimens that reveal any degree of chronic active gastritis or moderate inactive gastritis., (Copyright© by the American Society for Clinical Pathology.)

A novel fluorescence detection method for phosphoproteins in 1-D and 2-D SDS-PAGE by using purpurin is developed in this study. Phosphoproteins as low as 4-8 ng could be specifically detected by purpurin within 60 min, and the detection limit is similar to or better than that of Pro-Q Diamond staining. Only 2 steps (staining and destaining) are needed for purpurin staining without requiring excessive fixing and washing steps, and for single use, $0.8 is enough for purpurin staining. By comprehensively comparing with Pro-Q Diamond staining, it is concluded that purpurin staining is a simple, rapid and low-cost staining method for a broad application to the research of phosphoproteins.

Background: Golgi-Cox staining method is considered as one of the best neurohistological and fascinating staining techniques to reveal the cytoarchitecture of the brain. Requirement of longer time (more than a month), laborious section processing steps, requirement of sophisticated equipment's and costly ready to use kits limits extensive use of this technique., New Method: The need for a modified staining technique is to overcome some of these hurdles. Here we describe a modification of Golgi-Cox staining involving reduced impregnation time (7 days), omitting tissue dehydration steps, and alterations in section processing steps. Different impregnation duration (7 days, 14 days, 1 month, 6 months and 10 months) effects on optimized staining of dorsal hippocampus and basolateral amygdala were investigated., Results: Modified Golgi-Cox staining method was found to be effective in staining rat hippocampus and amygdala. Impregnation for 7 days, 14 days and 1 month resulted in giving good results and they were comparable. However, artifacts were slightly elevated with 6 months group but not extensively. Impregnation for 10 months negatively affected the staining process., Comparison With Existing Method(s): Compared to existing methods the current method was found to be cost effective, fast, reliable and can be executed in labs where infrastructure is limited., Conclusions: Current modification considerably benefitted in obtaining better results (good clarity and lesser artifact) in a short time. Longer impregnated brain sections were found to be unsuitable for morphometric evaluation due to more stain precipitation and artifact. The modified technique can be used to study cellular architecture in other brain regions., (Copyright © 2014 Elsevier B.V. All rights reserved.)

The increasing need for multiple-labeling of cells and whole organisms for fluorescence microscopy has led to the development of hundreds of fluorophores that either directly recognize target molecules or organelles, or are attached to antibodies or other molecular probes. DNA labeling is essential to study nuclear-chromosomal structure, as well as for gel staining, but also as a usual counterstain in immunofluorescence, FISH or cytometry. However, there are currently few reliable red to far-red-emitting DNA stains that can be used. We describe herein an extremely simple, inexpensive and robust method for DNA labeling of cells and electrophoretic gels using the very well-known histological stain methyl green (MG). MG used in very low concentrations at physiological pH proved to have relatively narrow excitation and emission spectra, with peaks at 633 and 677 nm, respectively, and a very high resistance to photobleaching. It can be used in combination with other common DNA stains or antibodies without any visible interference or bleed-through. In electrophoretic gels, MG also labeled DNA in a similar way to ethidium bromide, but, as expected, it did not label RNA. Moreover, we show here that MG fluorescence can be used as a stain for direct measuring of viability by both microscopy and flow cytometry, with full correlation to ethidium bromide staining. MG is thus a very convenient alternative to currently used red-emitting DNA stains.

Background: The aim of this paper was to develop a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, sensitive and inexpensive detection of astrovirus., Results: The detection limit of LAMP using in vitro RNA transcripts was 3.6 × 10 copies·μL⁻¹, which is as sensitive as the presently used PCR assays. However, the LAMP products could be identified as different colors with the naked eye following staining with hydroxynaphthol blue dye (HNB). No cross-reactivity with other gastroenteric viruses (rotavirus and norovirus) was observed, indicating the relatively high specificity of LAMP. The RT-LAMP method with HNB was used to effectively detect astrovirus in reclaimed water samples., Conclusions: The LAMP technique described in this study is a cheap, sensitive, specific and rapid method for the detection of astrovirus. The RT-LAMP method can be simply applied for the specific detection of astrovirus and has the potential to be utilized in the field as a screening test.

Quantitative proteome analyses suggest that the well-established stain colloidal Coomassie Blue, when used as an infrared dye, may provide sensitive, post-electrophoretic in-gel protein detection that can rival even Sypro Ruby. Considering the central role of two-dimensional gel electrophoresis in top-down proteomic analyses, a more cost effective alternative such as Coomassie Blue could prove an important tool in ongoing refinements of this important analytical technique. To date, no systematic characterization of Coomassie Blue infrared fluorescence detection relative to detection with SR has been reported. Here, seven commercial Coomassie stain reagents and seven stain formulations described in the literature were systematically compared. The selectivity, threshold sensitivity, inter-protein variability, and linear-dynamic range of Coomassie Blue infrared fluorescence detection were assessed in parallel with Sypro Ruby. Notably, several of the Coomassie stain formulations provided infrared fluorescence detection sensitivity to <1 ng of protein in-gel, slightly exceeding the performance of Sypro Ruby. The linear dynamic range of Coomassie Blue infrared fluorescence detection was found to significantly exceed that of Sypro Ruby. However, in two-dimensional gel analyses, because of a blunted fluorescence response, Sypro Ruby was able to detect a few additional protein spots, amounting to 0.6% of the detected proteome. Thus, although both detection methods have their advantages and disadvantages, differences between the two appear to be small. Coomassie Blue infrared fluorescence detection is thus a viable alternative for gel-based proteomics, offering detection comparable to Sypro Ruby, and more reliable quantitative assessments, but at a fraction of the cost.

SYBR Green coupled to melting curve analysis has been suggested to detect RNA viruses showing high genomic variability. Here, a SYBR Green-based real-time RT-PCR assay was developed for simultaneous detection and differentiation of highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV) and classical type 2 PRRSV (C-PRRSV). The different strains were identified by their distinctive melting temperatures: 82.98 ± 0.25 °C and 85.95 ± 0.24 °C for HP-PRRSVs or 82.74 ± 0.26 °C for C-PRRSVs. Specificity was tested using nine other viral and bacterial pathogens of swine. The detection limit was 1 TCID(50) for HP- or C-PRRSV. Furthermore, the detection results for samples from an animal trial with HP- or C-PRRSV infections showed that the SYBR Green-based real-time RT-PCR was more sensitive than the conventional RT-PCR. Additionally, an analysis of 319 field samples from North China, Central China and Northeast China showed that HP- and C-PRRSVs co-circulated in pig herds. Thus, the SYBR Green-based real-time RT-PCR, which can be performed within one hour, is a rapid, sensitive and low-cost diagnostic tool for rapid differential detection and routine surveillance of HP- and classical type 2 PRRSVs in China.

Transbronchial needle aspiration (TBNA) via flexible bronchoscopy is a well-established sampling modality for lung masses. The procedure is useful in the diagnosis of neoplastic and non-neoplastic lesions as well as for staging of bronchogenic carcinoma. Rapid on-site evaluation (ROSE) adds value as it has the advantage of triaging material during the procedure so avoiding a battery of investigations. Frequently used rapid stains are the modified Wright-Giemsa water-based stain (WG-ROSE) and the alcohol-based modified Papanicolaou stain (Pap-ROSE). Final review of laboratory-based Giemsa and Pap stains supplemented by ancillary investigations is essential for quality assurance. To investigate whether and how ROSE influenced the quantity and quality of the material submitted to the laboratory we randomized 126 patients to WG-ROSE, requiring only one pathologist on-site, or combined WG- and Pap-ROSE, requiring an additional person on-site to assist with staining. In those patients with positive TBNA we graded the laboratory-based slides of the first pass containing diagnostic material into insufficient, suspicious, adequate and excellent. The first diagnostic pass was found after 3.06 ± 1.94 (SD) passes and 3.13 ± 2.16 passes with WG-ROSE and combined ROSE (P = 0.87), respectively. Following WG-ROSE and combined ROSE 69% and 71.1% (P = 0.509) of slides were diagnostic (adequate or excellent) on laboratory-based Giemsa stains, and 93.3% and 100% (P = 0.134) were scored adequate or excellent on laboratory-based Pap stains. We concluded that the less costly and labour intensive WG-ROSE procedure is adequate for TBNA. This has cost implications especially in resource poor settings., (Copyright © 2011 Wiley Periodicals, Inc.)

Here we introduce a modified antibody staining method that uses up to 80% less antibody for flow cytometry. We demonstrate this method for the detection of antigens expressed at high, moderate, or low levels in mouse and rat lymphocytes as well as mouse mammary epithelial cells. We obtained reproducibly accurate results for the detection of up to seven parameters for activation induced-proliferation, cell cycle analysis, and phenotyping of cell-surface and intracellular antigens.

Directly labelling locus-specific primers for microsatellite analysis is expensive and a common limitation to small-budget molecular ecology projects. More cost-effective end-labelling of PCR products can be achieved through a three primer PCR approach, involving a fluorescently labelled universal primer in combination with modified locus-specific primers with 5' universal primer sequence tails. This technique has been widely used but has been limited largely due to a lack of available universal primers suitable for co-amplifying large numbers of size overlapping loci and without requiring locus-specific PCR conditions to be modified. In this study, we report a suite of four high-performance universal primers that can be employed in a three primer PCR approach for efficient and cost-effective fluorescent end-labelling of PCR fragments. Amplification efficiency is maximized owing to high universal primer Tm values (approximately 60+ °C) that enhance primer versatility and enable higher annealing temperatures to be employed compared with commonly used universal primers such as M13. We demonstrate that these universal primers can be combined with multiple fluorophores to co-amplify multiple loci efficiently via multiplex PCR. This method provides a level of multiplexing and PCR efficiency similar to microsatellite fluorescent detection assays using directly labelled primers while dramatically reducing project costs. Primer performance is tested using several alternative PCR strategies that involve both single and multiple fluorophores in single and multiplex PCR across a wide range of taxa., (© 2012 Blackwell Publishing Ltd.)

The constantly growing number of genetic tools rapidly increases possibilities for various screens in different model organisms and calls for new methods facilitating screen performance. In particular, screening procedures involving fixation and staining of samples are difficult to perform at a genome-wide scale. The time-consuming task to generate these samples makes such screens less attractive. Here, we describe the use of multi-well filter plates for high throughput labellings of different Drosophila organs and zebrafish embryos. Our inexpensive vacuum-assisted staining protocol minimises the risk of sample loss, reduces the amount of staining reagents and drastically decreases labour and repetitive work. The simple handling of the system and the commercial availability of its components makes this method easily applicable to every laboratory.

Background: Yellow Fever virus (YFV) is an important arboviral pathogen in much of sub-Saharan Africa and the tropical Americas. It is the prototype member of the genus Flavivirus and is transmitted primarily by Aedes (Stegomyia) mosquitoes. The incidence of human infections in endemic areas has risen in recent years. Prompt and dependable identification of YFV is a critical component of response to suspect cases., Results: We developed a one-step SYBR Green I-based real-time quantitative RT-PCR (qRT-PCR) assay targeting the 5'NTR and capsid-gene junction--for rapid detection and quantification of YFV. The detection limit was 1 PFU/mL, 10-fold more sensitive than conventional RT-PCR, and there was no cross-reactivity with closely related flaviviruses or with alphaviruses. Viral load in samples was determined by standard curve plotted from cycle threshold (Ct) values and virus concentration. The efficacy of the assay in mosquitoes was assessed with spiked samples. The utility of the assay for screening of pooled mosquitoes was also confirmed. Replication of a Cameroon isolate of YFV in Ae. aegypti revealed a marked variation in susceptibility among different colonies at different days post infection (pi)., Conclusions: The SYBR Green-1 based qRT-PCR assay is a faster, simpler, more sensitive and less expensive procedure for detection and quantification of YFV than other currently used methods.

X-gal staining is a rapid and convenient histochemical technique used to detect reporter gene expression. A prerequisite is the creation or acquisition of transgenic reporter mouse lines, in which the bacterial LacZ gene has been knocked into the gene of interest or placed under the control of regulatory elements corresponding to the gene of interest. Expression is marked by a dark blue stain and can be detected at the single cell level, providing a robust visual readout of gene expression in the developing kidney. Here, we describe the methodology, applications, and limitations of this technique.

A recent prospective study published in the American Journal of Forensic Medicine and Pathology concluded that routine histopathologic examination lacked value. We disagreed with this assertion as we have found routine microscopic examination to be fruitful by documenting gross findings and by revealing interesting and unexpected findings.We designed a retrospective study to determine the benefit and cost of routine histopathologic examination at our institution. Forensic autopsy cases from January 2004 through June 2007 with lethal gross findings were reviewed to determine the number of cases in which microscopic examination provided the definitive cause of death. Cost was based on the average number of hematoxylin-eosin-stained slides per autopsy.One case was found in which the microscopic findings determined the correct cause of death despite compelling history and the initial impression from the autopsy findings. The cost of routine histopathologic examination during this period was approximately $39,000.We conclude that routine histopathologic examination has value. Despite having a low yield, the information it provides is nonetheless important, and its cost is not prohibitive. Furthermore, there are benefits gained from routine microscopic examination as exemplified in the 2 case reports presented in this article.

A sensitive reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was developed for rapid visual detection of human enterovirus 71 subgenotype C4 (EV71-C4) and Coxsackievirus A16 (CVA16) infection, respectively. The reaction was performed in one step in a single tube at 65°C for 60 min with the addition of the hydroxynaphthol blue (HNB) dye prior to amplification. The detection limits of the RT-LAMP assay were 0.33 and 1.58 of a 50% tissue culture infective dose (TCID(50)) per reaction based on 10-fold dilutions of a titrated EV71 or CVA16 strain, respectively. No cross-reaction was observed with Coxsackievirus A (CVA) viruses (CVA2, 4, 5, 7, 9, 10, 14, and 24), Coxsackievirus B (CVB) viruses (CVB1,2,3,4, and 5) or ECHO viruses (ECHO3, 6, 11, and 19). The assay was further evaluated with 47 clinical stool specimens diagnosed previously with EV71, CVA16 or other human enterovirus infections. Virus isolates from stool samples were confirmed by virus neutralization testing and sequencing. RT-LAMP with HNB dye was demonstrated to be a sensitive and cost-effective assay for rapid visual detection of human EV71-C4 and CVA16., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Background: Loop-mediated isothermal amplification (LAMP) is a high performance method for detecting DNA and holds promise for use in the molecular detection of infectious pathogens, including Plasmodium spp. However, in most malaria-endemic areas, which are often resource-limited, current LAMP methods are not feasible for diagnosis due to difficulties in accurately interpreting results with problems of sensitive visualization of amplified products, and the risk of contamination resulting from the high quantity of amplified DNA produced. In this study, we establish a novel visualized LAMP method in a closed-tube system, and validate it for the diagnosis of malaria under simulated field conditions., Methods: A visualized LAMP method was established by the addition of a microcrystalline wax-dye capsule containing the highly sensitive DNA fluorescence dye SYBR Green I to a normal LAMP reaction prior to the initiation of the reaction. A total of 89 blood samples were collected on filter paper and processed using a simple boiling method for DNA extraction, and then tested by the visualized LAMP method for Plasmodium vivax infection., Results: The wax capsule remained intact during isothermal amplification, and released the DNA dye to the reaction mixture only when the temperature was raised to the melting point following amplification. Soon after cooling down, the solidified wax sealed the reaction mix at the bottom of the tube, thus minimizing the risk of aerosol contamination. Compared to microscopy, the sensitivity and specificity of LAMP were 98.3% (95% confidence interval (CI): 91.1-99.7%) and 100% (95% CI: 88.3-100%), and were in close agreement with a nested polymerase chain reaction method., Conclusions: This novel, cheap and quick visualized LAMP method is feasible for malaria diagnosis in resource-limited field settings.

The amplified fragment length polymorphism (AFLP) technique is a widely used multi-purpose DNA fingerprinting tool. The ability to size-separate fluorescently labelled AFLP fragments on a capillary electrophoresis instrument has provided a means for high-throughput genome screening, an approach particularly useful in studying the molecular ecology of nonmodel organisms. While the 'per-marker-generated' costs for AFLP are low, fluorescently labelled oligonucleotides remain costly. We present a cost-effective method for fluorescently end-labelling AFLPs that should make this tool more readily accessible for laboratories with limited budgets. Both standard fluorescent AFLPs and the end-labelled alternatives presented here are repeatable and produce similar numbers of fragments when scored using both manual and automated scoring methods. While it is not recommended to combine data using the two approaches, the results of the methods are qualitatively comparable, indicating that AFLP end-labelling is a robust alternative to standard methods of AFLP genotyping. For researchers commencing a new AFLP project, the AFLP end-labelling method outlined here is easily implemented, as it does not require major changes to PCR protocols and can significantly reduce the costs of AFLP studies., (© 2010 Blackwell Publishing Ltd.)

Coomassie Brilliant Blue (CBB) protein stains are inexpensive but detect proteins at only at microgram levels. Because of acetic acid and methanol, they cause skin irritation and reduce work motivation by malodor. Recent mass spectrometric (MS) analyses demonstrated that nanogram-sensitive colloidal CBB staining resulted in in vitro methylations of proteins. We propose a rapid, inexpensive, sensitive, odorless, less harsh, and in vitro methylation-free CBB stain. CGP uses three components: citric acid, CBB G-250, and polyvinylpyrrolidone. CGP detects proteins at 12ng within 45min, and because it is nonalcohol, in principle in vitro methylation would be eliminated. Indeed, MS analysis of CGP-stained bands confirmed a lack of methylation., (2010 Elsevier Inc. All rights reserved.)

Objective: To devise a simple, cost-effective protocol for Papanicolaou (Pap) staining of cervicovaginal smears., Methods: Five hundred coded paired cervical smears were collected from women as part of routine cervical cancer screening. One set of smears was stained by conventional Pap staining protocol (CP) and the other by a modified protocol (MP) in which alcohol was replaced by 1% acetic acid in all the steps except during fixation and prior to mounting; in addition, one alcohol-based counterstain, OG, was omitted. The smears were examined blindly by the pathologists and then decoded. Each pair of smears was compared and the two protocols were analysed for staining quality and diagnoses by McNemar and chi-square tests., Results: The staining quality in the MP was satisfactory. The nuclear and cytoplasmic features were comparable to the CP. Cytoplasmic transparency was maintained in the MP and the differential staining of blue/green and pink was acceptable to the pathologists and technicians. The diagnoses agreed in all cases and there was no compromise in interpreting the smears. With MP it took only 3-4 minutes to stain a batch of 50 slides. in contrast to the 20 minutes taken by CP. The MP used almost one-seventh of the amount of alcohol compared with CP, which translated into a significant cost reduction per smear., Conclusions: The improvised Pap staining protocol with minimum alcohol use is a simple, cost-effective and technician-friendly procedure that can be easily adopted in high-volume, resource-limited laboratories for mass cervical cancer screening.

Context: Seminal vesicle invasion by prostatic carcinoma is directly associated with tumor staging; verification is challenging when the tumor demonstrates cribriform or papillary growth patterns or there are back-to-back small-gland proliferations. P504S is overexpressed in prostatic carcinoma and high-grade prostatic intraepithelial neoplasia with cytoplasmic immunoreactivity. p63 has positive immunoreactivity in basal cell nuclei of benign prostatic glands. Many researchers use a combination of these antibodies and their different colors., Objective: To evaluate the usefulness of a single-color P504S/p63 cocktail immunostain in verifying prostatic carcinoma within the seminal vesicle., Design: Sections from 57 radical prostatectomy specimens of pathologic stage pT3b that contain seminal vesicle with prostatic carcinoma involvement were immunostained with primary antibodies against prostate-specific antigen (PSA) and prostatic acid phosphatase (PAP) and a cocktail of antibodies against P504S and p63., Results: Prostatic carcinoma cells from all 57 cases were diffusely positive for P504S, PSA, and PAP with cytoplasmic staining and no p63 nuclear staining. Seminal vesicle epithelium from all 57 cases was negative for all 3 markers with distinct p63 nuclear staining of the basal cells. Benign prostatic tissue was positive for PSA and PAP, as well as for p63, but negative for P504S., Conclusions: The P504S/p63 one-color cocktail is a practical and cost-effective stain to differentiate prostatic carcinoma that involves the seminal vesicle from seminal vesicle epithelium. It is superior to PSA or PAP when sections contain both seminal vesicle and benign glands because PSA and PAP cannot distinguish benign from malignant glands.

We describe a visible dye-based staining method for DNA in polyacrylamide gels using ethyl violet (EV). The novel method is a background-free, sensitive, economical, and simple procedure involving only staining and washing steps that can be completed within 30 min. As little as 0.8-1.6 ng of phiX174 DNA/HaeIII can be detected by EV, which is about eightfold more sensitive than Nile blue (NB) stain and twofold less sensitive than ethidium bromide (EB) stain., (2010 Elsevier Inc. All rights reserved.)