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.

Desmin immunostaining is effective for improving interobserver variability in the depth assessment of the submucosal invasion of colorectal cancers.

Tumor depth evaluation is essential for pathological tumor staging because it affects clinical management as an independent risk factor for lymph node metastasis in colorectal cancers. However, poor interobserver variability of invasion depth has been reported. This study aimed to clarify the effectiveness of desmin immunostaining in the histological diagnosis of colorectal cancer. Overall, 63 sets of slides of colorectal cancer stained with hematoxylin and eosin (H&E) and desmin were prepared and independently reviewed by four examiners. After reviewing the desmin-stained slides, the interobserver variability of H&E slides alone was significantly improved for all examiners. For the assessment of Tis vs. T1, the sensitivity and accuracy were significantly improved for all examiners by combining H&E and desmin immunostaining. For the diagnosis of T1b vs. Tis or T1a, specificity and accuracy were significantly improved by adding desmin immunostaining. Ancillary desmin staining to assess submucosal invasion in colorectal cancers significantly improved interobserver agreement, led to efficient screening of T1 cancers, and reduced excessive T1b diagnoses. The combination of desmin immunostaining and H&E staining is highly recommended for diagnosing invasive colorectal cancer.

Doubly labelled water for determining total energy expenditure in adult critically ill and acute care hospitalized inpatients: A scoping review.

BACKGROUND & AIMS: Doubly labelled water (DLW) is considered the reference standard method of measuring total energy expenditure (TEE), but there is limited information on its use in the Intensive Care Unit (ICU) and acute care setting. This scoping review aims to systematically summarize the available literature on TEE measured using DLW in these contexts. METHODS: Four online databases (MEDLINE, Embase, Emcare and CINAHL) were searched up to Dec 12, 2020. Studies in English were included if they measured TEE using DLW in adults in the ICU and/or acute care setting. Key considerations, concerns and practical recommendations were identified and qualitatively synthesized. RESULTS: The search retrieved 7582 studies and nine studies were included; one in the ICU and eight in the acute care setting. TEE was measured over 7-15-days, in predominantly clinically stable patients. DLW measurements were not commenced until four days post admission or surgery in one study and following a 10-14-day stabilization period on parenteral nutrition (PN) in three studies. Variable dosages of isotopes were administered, and several equations used to calculate TEE. Four main considerations were identified with the use of DLW in these settings: variation in background isotopic abundance; excess isotopes leaving body water as carbon dioxide or water; fluctuations in rates of isotope elimination and costs. CONCLUSION: A stabilization period on intravenous fluid and PN regimens is recommended prior to DLW measurement. The DLW technique can be utilized in medically stable ICU and acute care patients, with careful considerations given to protocol design.

Development of a multiplex immuno-oncology biomarker and digital pathology workflow for assessment of urothelial carcinoma.

BACKGROUND: Immune checkpoint inhibitors (ICI) therapies have demonstrated significant benefit in the treatment of many tumors including high grade urothelial cancer (HGUC) of the bladder. However, variability in patients' clinical responses highlights the need for biomarkers to aid patient stratification. ICI relies on an intact host immune response. In this context, we hypothesize that key players in the antitumor immune response such as markers of activated cytotoxic T lymphocytes (CD8, granzyme-B) and immune suppression (FOXP3) may help to identify patients who will derive the greatest therapeutic benefit from ICI. A major obstacle for deployment of such a strategy is the limited quantities of tumor-derived biopsy material. Therefore, in this technical study, we develop a multiplex biomarker with digital workflow. We explored the (1) concordance of conventional single stain results using digital image analysis, and (2) agreement between digital scoring versus manual analysis. METHODS: (1) For concordance study of single and multiplex stains, triplicate core tissue microarrays of 207 muscle invasive, HGUC of bladder had sequential 4-micron sections cut and stained with CD8, FOXP3 and granzyme-B. An inhouse developed tri-chromogen multiplex immunohistochemistry (m-IHC) assay consisting of CD8 (green), granzyme B (brown), and FOXP3 (red) was used to stain the next sequential tissue section. (2) Agreement between manual and digital analysis was performed on 19 whole slide sections of HGUC cystectomy specimens. All slides were scanned using Aperio ScanScope AT Digital Scanner at 40X. Quantitative digital image analysis was performed using QuPath version 0.2.3 open-source software. Scores from triplicate cores were averaged for each HGUC specimen for each marker. Intraclass correlation coefficients were used to compare percent positive cells between the single- and multi-plex assays. Lin's concordance correlation coefficients were used for manual versus digital analysis. RESULTS AND CONCLUSIONS: m-IHC offers significant advantages in characterizing the host immune microenvironment particularly in limited biopsy tissue material. Utilizing a digital image workflow resulted in significant concordance between m-IHC and individual single stains (p < 0.001 for all assessments). Moderate to good agreements were achieved between manual and digital scoring. Our technical work demonstrated potential uses of multiplex marker in assessing the host immune status and could be used in conjunction with PD-L1 as a predictor of response to ICI therapy.

A Possible Flow Cytometry-Based Viability and Vitality Assessment Protocol for Pathogenic Vibrio cholerae O1 and O139 Postexposure to Simulated Gastric Fluid.

During the intake of contaminated water, for diarrheal disease to occur, Vibrio cholerae must survive through the bactericidal digestive secretion of gastric fluid during passage through the stomach. Determining the viability of these bacteria is challenging, with the standard cultivation methods for viability being time-consuming and unable to culture cells that may still function accordingly. This study assessed the use of enzyme action and membrane integrity as alternatives for determining vitality and viability, respectively, in gastric acid-stressed pathogenic Vibrio cholerae O1 and O139, using fluorescent probes thiazole orange (TO) for viability based on membrane integrity, carboxyfluorescein diacetate (CFDA) with acetoxymethyl ester (AM) for vitality based on metabolic activity, and propidium iodide (PI) for cell death/damage due to loss of membrane integrity, with flow cytometry. Simulated gastric fluid-treated bacterial cells were labelled with blends of TO+PI and CFDA-AM+PI, and these stained cells were separated into heterologous populations based on their fluorescence signal. The gastric acid exposed cells presented with high green fluorescence signals after staining with the metabolic probe CFDA-AM, which indicated intact (live) cells due to being metabolically active, whereas when the same cells were stained with the DNA probe (TO), these appeared to be in a "stressed state" due to loss of membrane integrity. Damaged cells (dead cells) showed high red fluorescence levels after staining with PI probe. The use of flow cytometry with fluorescent probes is a favorable method for evaluating the vitality and viability of bacteria when cells are labelled with a combination of CFDA-AM+PI.

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.

Preservation and Processing of Intestinal Tissue for the Assessment of Histopathology.

The intestine is often examined histologically in connection with allergies and in search for pathological changes. To be able to examine the intestine histologically with a microscope, it must be sampled and processed correctly. For microscopic analysis, the samples have to be cut into thin sections, stained, and mounted on slides. Since it is not possible to cut fresh samples without damaging them, they must first be fixed. The most common method, which is described herein, is the fixation in formalin with subsequent embedding in paraffin and staining of the slides with hematoxylin and eosin (H&E). Hematoxylin solutions (in this case Mayer's hemalum solution) stain the acidic components of the cell, i.e., cell nuclei, blue. The staining with eosin gives a pink staining of cytoplasm. This chapter describes the method of processing intestinal tissue for paraffin-embedding, sectioning, and staining with H&E. Tissue processing can be done in tissue processing machines or manually. We describe the manual processing that is often used for smaller batches of samples.

Assessment of Lung Eosinophils In Situ Using Immunohistological Staining.

Eosinophils are rare white blood cells that are recruited from circulation to accumulate in the lung in mouse models of allergic respiratory inflammation. In hematoxylin-eosin (HE) stained lungs, eosinophils may be difficult to detect despite their bright eosin staining in the secondary granules. For this reason, antibody-mediated detection of eosinophils is preferable for specific and clearer identification of these cells. Moreover, eosinophils may degranulate, releasing their granule proteins into surrounding tissue, and remnants of cytolysed cells cannot be detected by HE staining. The methods here demonstrate the use of eosinophil-specific anti-mouse antibodies to detect eosinophil granule proteins in formalin-fixed cells both in situ in paraffin-embedded lungs, as well as in cytospin preparations from the lung. These antibody staining techniques enable either colorimetric or fluorescence imaging of eosinophils or their granule proteins with the potential for additional antibodies to be added for detection of multiple molecules.

PRAME immunohistochemistry as an adjunct for diagnosis and histological margin assessment in lentigo maligna.

AIMS: Lentigo maligna (LM), the most common type of melanoma in situ, is a diagnostically challenging lesion for pathologists due to abundant background melanocytic hyperplasia in sun-damaged skin. Currently, no laboratory methods reliably distinguish benign from malignant melanocytes. However, preferentially expressed antigen in melanoma (PRAME) has shown promise in this regard, and could potentially be applied to diagnosis and margin assessment in difficult cases of LM. METHODS AND RESULTS: Ninety-six cases with a diagnosis of LM (n = 77) or no residual LM (n = 19) following initial biopsy were identified and stained with an antibody directed towards PRAME. Immunohistochemistry (IHC) was scored as positive or negative, and measurement of histological margins by PRAME was performed and compared to the measurement of histological margins using conventional methods [haematoxylin and eosin (H&E) and/or sex-determining region Y-box 10 (SOX10) and/or Melan-A]. Of cases with LM, 93.5% (72 of 77) were PRAME+ and 94.7% (18 of 19) of cases with no residual LM were PRAME- . Of the 35 cases with no margin involvement by PRAME or conventional assessment, 14 cases (40.0%) had no difference in measurement, 17 (48.6%) had a difference of 1 mm or less and four (11.4%) differed by between 1 and 3.5 mm. There was a high correlation between margin assessment methods (r = 0.97, P < 0.0001). CONCLUSIONS: PRAME IHC is a sensitive (93.5%) and specific (94.7%) method for diagnosing LM on biopsy and excision, and measurement of histological margins by PRAME shows a high correlation with conventional methods for margin assessment. Furthermore, the nuclear expression of PRAME makes it a good target for use in dual-colour IHC stains.

Backscattered Scanning Electron Microscopy Approach for Assessment of Microvessels under Conditions of Normal Microanatomy and Pathological Neovascularization.

We evaluated the efficiency of an original method for studying of the microvascular bed under conditions of normal microanatomy and pathological neovascularization. The blood vessels, tissues surrounding the stent in the pulmonary artery and subcutaneously implanted titanium nickelide plate, atherosclerotic plaque, and vascular stent with restenosis were examined. The specimens were fixed in formalin and stained in OsO4, embedded into fresh epoxy resin, grinded, polished, and counterstained with uranyl acetate and lead citrate. Numerous vasa vasorum were found in all native vessels. Around the pulmonary artery stent and metal plates, numerous newly formed vessels of small diameter were seen. The intensity of neovascularization in atherosclerosis and carotid stent restenosis differed significantly. Our technique can be successfully used for evaluation of the microvascular bed.

A novel CD2 staining-based flow cytometric assay for assessment of natural killer cell cytotoxicity.

BACKGROUND: Assessing cytotoxicity is fundamental to studying natural killer (NK) cell function. Various radioactive and non-radioactive cytotoxicity assays measuring target cell death have been developed. Among these methods, the most commonly used 51 Chromium-release assay (CRA) and flow cytometry-based cytotoxicity assays (FCCs) are the major representatives. Nonetheless, several drawbacks, including dye leakage and the potential effects of prior labeling on cells, curb the broad applicability of the FCCs. METHODS: Here, we report a rapid FCC for quantifying target cell death after co-incubation with NK cells. In this assay, after 4 hours of NK cell-target cell co-incubation, fluorochrome-conjugated CD2 antibody was used to identify NK cells, and SYTOX Green and Annexin V-FITC were further used to detect target cell death in CD2-negative population. In parallel, both CRA and FCC assay using CFSE/ 7-AAD were performed to validate the reproducibility and replicability. RESULTS: We observed that CD2 is exclusively positive on NK cells other than the most common hematological target tumor cells, such as K562, HL60, MOLM13, Raji, NCI-H929, rpmi8226, MM.1S, and KMS11. Assessment of target cell death using the CD2-based FCC shows a significantly higher percent specific lysis of the target cells compared to the standard CRA and the FCC assay using CFSE and 7-AAD. CONCLUSIONS: We demonstrated that this CD2-based FCC is a fast, simple, and reliable method for evaluating NK cell cytotoxicity.

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.

Viability assessment of spermatozoa in large falcons (Falco spp.) using various staining protocols.

Viability assessment is an important part of semen analysis, and various live/dead staining protocols have been used in semen of avian species. Results of live/dead count differed between dyes, staining protocols and bird species, impeding comparability between studies and requiring species-specific comparisons of viability stains. In raptor semen, similar comparisons are absent. Thus, the aim of the present study was to compare eight conventional viability stains. Eosin blue 2% [EB], eosin blue 2% with the addition of 3% sodium citrate [EB2], eosin blue-nigrosin 5% [EBN5], eosin yellow-nigrosin 5% [EYN5], eosin yellow-nigrosin 10% [EYN10], eosin blue-aniline blue [EBA], eosin yellow-aniline blue [EYA] and bromophenol blue-nigrosin [BBN] were evaluated in comparison with the fluorescence stain SYBR® Green-propidium iodide [SYBR-PI] in spermatozoa of falcons. The comparison was performed using conventional light microscopy which is applicable in breeding centres, veterinary practices and field studies. Additionally, live/dead stains were correlated to motility values of the same samples to validate sperm viability. Light microscopy using EB and using SYBR-PI enabled an effective and clear differentiation between alive and dead spermatozoa of falcons. Motility values correlated significantly and strongly with EB only (r = .629; p < .001), but not with any other stain used in the study. Therefore, our results suggest EB as the most suitable stain for viability assessment in the semen of large falcons.

Automated assessment of Ki-67 in breast cancer: the utility of digital image analysis using virtual triple staining and whole slide imaging.

AIMS: Precise evaluation of proliferative activity is essential for the stratified treatment of luminal-type breast cancer (BC). Immunohistochemical staining of Ki-67 has been widely used to determine proliferative activity and is recognised to be a useful prognostic marker. However, there remains discussion concerning the methodology. We aimed to develop an automated and reliable Ki-67 assessment approach for invasive BC. MATERIALS AND RESULTS: A retrospective study was designed to include two cohorts consisting of 152 and 261 consecutive patients with luminal-type BC. Representative tissue blocks following surgery were collected, and three serial sections were stained automatically with Ki-67, pan-cytokeratin and p63. The whole slides were scanned digitally and aligned using VirtualTripleStaining - an extension to the VirtualDoubleStaining™ technique provided by Visiopharm software. The aligned files underwent automated invasive cancer detection, hot-spot identification and Ki-67 counting. The automated scores showed a significant positive correlation with the pathologists' scores (r = 0.82, P < 0.0001). Among selected patients with curative surgery and standard adjuvant therapies (n = 130), the digitally assessed low Ki-67 group (<20%) demonstrated a significantly better prognosis (breast cancer-specific survival, P = 0.030; hazard ratio = 0.038) than the high Ki-67 group. CONCLUSIONS: Digital image analysis yielded similar results to the scores determined by experienced pathologists. The prognostic utility was verified in our cohort, and an automated process is expected to have high reproducibility. Although some pitfalls were confirmed and thus need to be monitored by laboratory staff, the application could be utilised for the assessment of BC.

A technical note on the assessment of human sperm vitality using eosin-nigrosin staining.

RESEARCH QUESTION: How many spermatozoa and slides need to be counted to make a reliable assessment of sperm vitality? Currently, various authorities recommend assessing human sperm vitality on counts of at least 200 cells, but on one or two slides. DESIGN: This was an observational study on duplicate eosin-nigrosin stained sperm vitality slides made from 58 ejaculates. Assessments were made using counts of 2 â€¯×  100 and 1 â€¯×  200 cells per slide, all performed by the same trained expert observer. RESULTS: Although assessments tend to show fewer and smaller outlier values when based on counts of 200 spermatozoa than 100, and on 2 â€¯×  200 than 1 â€¯×  200, counting 200 spermatozoa from one slide provides a result with sufficient accuracy for the clinical purpose of ascertaining whether the immotile spermatozoa in an ejaculate showing low sperm motility are alive or dead. CONCLUSIONS: While the increased accuracy of results derived from counts of 2 â€¯×  200 cells might be important in research studies where sperm vitality is the specific end-point of interest, counting at least 200 spermatozoa from one smear is sufficiently accurate for the clinical purpose of establishing whether the immotile spermatozoa seen in ejaculates with low sperm motility are alive or not. Consequently, the extra workload of performing replicate counts and the associated calculations does not increase the clinical value of the result, and hence is unnecessary in routine semen analysis. Careful laboratory technique as well as proper staff training and competence are essential. The conclusions might not be applicable to staining methods other than the recommended one-step eosin-nigrosin technique.

Mitochondrial morphology and function: two for the price of one!

Mitochondrial shape and function are known to be linked; therefore, there is a need to combine three-dimensional EM structural analysis with functional analysis. Cytochrome c oxidase labelling is one approach to examine mitochondrial function at the EM level. However, previous efforts to apply this method have had several issues including inconsistent results, disruption to mitochondrial ultrastructure, and a lack of optimisation for volume EM methods. We have used short fixation and microwave processing to address these issues. We show that our method gives consistent cytochrome c oxidase labelling and improves labelling penetration across tissue volume. We also quantify mitochondrial morphology metrics, including in volume EM, to show that ultrastructure is unaltered by the processing. This work represents a technical advance that allows the correlation of mitochondrial function and morphology with greater resolution and volume than has previously been feasible. LAY SUMMARY: Transmission electron microscopy (TEM) is a high-resolution technique used for the study of cells and their components, such as mitochondria. However, the two-dimensional nature of TEM means that quantification of these structures is difficult without making assumptions about their shape; a problem that was solved by the advent of three-dimensional EM approaches. Mitochondrial shape and function are known to be linked therefore there is a need to combine three-dimensional EM structural analysis with functional analysis. To do this we used electron microscopy to visualise a reaction that assesses the activity of cytochrome c oxidase in the mitochondrial respiratory chain. The reaction deposits a dark staining on mitochondrial cristae where cytochrome c oxidase is functioning and a lack of staining where it is not. We first optimised this technique for TEM, showing that the tissue was evenly stained and exhibited no effect on mitochondrial shape when compared to conventionally processed tissue. We then demonstrated that this was also true of a sample processed for three-dimensional EM imaging. This work presents an advance in three-dimensional EM imaging that allows us to look at both mitochondrial function and shape and to detect subtle changes in shape.

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.

Rapid determination of carbapenem resistance by low-cost colorimetric methods: Propidium Iodide and alamar blue staining.

Carbapenems are a class of ß-lactam antibiotics with a broad antimicrobial activity spectrum. Owing to their sturdy structures resistant to most ß-lactamases, they have been regarded as one of the last-resort antibiotics for combating multidrugresistant bacterial infections. However, the emergence of carbapenem resistance increases predominantly in nosocomial pathogens. To prevent spread of carbapenem resistance in early stages, it is imperative to develop rapid diagnostic tests that will substantially reduce the time and cost in determining carbapenem resistance. Thus, we devised a staining-based diagnostic method applicable to three different Gram-negative pathogens of Acinetobacter baumannii, Escherichia coli, and Klebsiella pneumoniae, all with the high potential to develop carbapenem resistance. Regardless of the resistance mechanisms presented by bacterial species and strains, double staining with propidium iodide (PI) and alamar blue (AB) identified resistant bacteria with an average sensitivity of 95.35%, 7 h after imipenem treatments in 343 clinical isolates. Among the three species tested, A. baumannii showed the highest diagnostic sensitivity of 98.46%. The PI and ABmediated staining method could be a promising diagnostic method with high-throughput efficacy and low cost.

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.