Tissue rinse liquid‐based cytology: a feasible tool for the intraoperative pathological evaluation of sentinel lymph nodes in breast cancer patients

K. Yamashiro, K. Taira, M. Nakajima, D. Okuyama, M. Azuma, H. Takeda, H. Suzuki, H. Jotoku, K. Watanabe, M. Takahashi, K. Taguchi and M. Tamura
Tissue rinse liquid‐based cytology: a feasible tool for the intraoperative pathological evaluation of sentinel lymph nodes in breast cancer patients Objectives: A unique diagnostic method was designed for the intraoperative pathological evaluation of sentinel lymph nodes (SLNs) in breast cancer patients, and the results were verified with 2 years of experience. Methods: Excised lymph nodes were cut into 2‐mm‐thick slices and rinsed thoroughly in CytoRich Red^®. The sliced tissues were embedded in a paraffin block. Three cytological glass slides of the cells exfoliated in CytoRich Red^® were prepared by the SurePath^® liquid‐based cytology (LBC) technique. Two slides were stained by the Papanicolaou method, and the remaining slide was immunostained with an anti‐keratin antibody. This process is called tissue rinse liquid‐based cytology (TRLBC). The results of TRLBC were compared with those of the final pathological diagnoses, including immunostaining with an anti‐keratin antibody on paraffin blocks (PB). Results: This study analysed 444 SLNs from 247 consecutive breast cancer patients. It required 35 minutes to complete the intraoperative diagnosis on a single node, and it took an additional 5 minutes per node if more than one node was submitted. When the results of PB were assumed to be the gold standard, the sensitivity and specificity of TRLBC were 81.9% and 96.1%, respectively. TRLBC detected all nodes with macrometastasis and 23 of 24 nodes with micrometastasis. Fifteen false‐negative TRLBC results were ‘isolated tumour cell clusters’ on PB, but there was one with micrometastasis histologically. Four of 14 false‐positive TRLBC results were proven to be true positive by supplementary examination using step sectioning of the paraffin blocks of the nodes. Conclusion: TRLBC is a feasible and promising intraoperative cytopathological tool showing a comparable efficacy to PB while still allowing the conventional postoperative histological examination.     

Environment‐specific spectral modeling: A new tool for the analysis of biological specimens

The recent discovery of fluorescent dyes for improving pathologic tissues identification has highlighted the need of robust methods for performance validation especially in the field of fluorescence‐guided surgery. Optical imaging of excised tissue samples is the reference tool to validate the association between dyes localization and the underlying histology in a controlled environment. Spectral unmixing may improve the validation process discriminating dye from endogenous signal. Here, an innovative spectral modeling approach that weights the spectral shifts associated with changes in chemical environment is described. The method is robust against spectral shift variations and its application leads to unbiased spectral weights estimates as demonstrated by numerical simulations. Finally, spectral shifts values computed pixel‐wise from spectral images are used to display additional information with potential diagnostic value.      

Approval policies for modifications to machine learning‐based software as a medical device: A study of bio‐creep

Successful deployment of machine learning algorithms in healthcare requires careful assessments of their performance and safety. To date, the FDA approves locked algorithms prior to marketing and requires future updates to undergo separate premarket reviews. However, this negates a key feature of machine learning—the ability to learn from a growing dataset and improve over time. This paper frames the design of an approval policy, which we refer to as an automatic algorithmic change protocol (aACP), as an online hypothesis testing problem. As this process has obvious analogy with noninferiority testing of new drugs, we investigate how repeated testing and adoption of modifications might lead to gradual deterioration in prediction accuracy, also known as “biocreep” in the drug development literature. We consider simple policies that one might consider but do not necessarily offer any error‐rate guarantees, as well as policies that do provide error‐rate control. For the latter, we define two online error‐rates appropriate for this context: bad approval count (BAC) and bad approval and benchmark ratios (BABR). We control these rates in the simple setting of a constant population and data source using policies aACP‐BAC and aACP‐BABR, which combine alpha‐investing, group‐sequential, and gate‐keeping methods. In simulation studies, bio‐creep regularly occurred when using policies with no error‐rate guarantees, whereas aACP‐BAC and aACP‐BABR controlled the rate of bio‐creep without substantially impacting our ability to approve beneficial modifications.     

3D‐liquid chromatography as a complex mixture characterization tool for knowledge‐based downstream process development

Knowledge‐based development of chromatographic separation processes requires efficient techniques to determine the physicochemical properties of the product and the impurities to be removed. These characterization techniques are usually divided into approaches that determine molecular properties, such as charge, hydrophobicity and size, or molecular interactions with auxiliary materials, commonly in the form of adsorption isotherms. In this study we demonstrate the application of a three‐dimensional liquid chromatography approach to a clarified cell homogenate containing a therapeutic enzyme. Each separation dimension determines a molecular property relevant to the chromatographic behavior of each component. Matching of the peaks across the different separation dimensions and against a high‐resolution reference chromatogram allows to assign the determined parameters to pseudo‐components, allowing to determine the most promising technique for the removal of each impurity. More detailed process design using mechanistic models requires isotherm parameters. For this purpose, the second dimension consists of multiple linear gradient separations on columns in a high‐throughput screening compatible format, that allow regression of isotherm parameters with an average standard error of 8%. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:1283–1291, 2016     

Aggregation‐induced emission enhancement of anthracene‐derived Schiff base compounds and their application as a sensor for bovine serum albumin and optical cell imaging

Three anthracene‐based Schiff base complexes, R1–R3 ( R1 = (E)‐N´‐((anthracen‐10‐yl)methylene)benzohydrazide; R2 = (E)‐1‐((anthracen‐10‐yl)methylene)‐4‐phenylsemicarbazide; and R3 = (E)‐1‐((anthracen‐10‐yl)methylene)‐4‐phenylthiosemicarbazide) were synthesized from 9‐anthracenecarboxaldehyde, benzohydrazide, 4‐phenylsemicarbazide and 4‐phenylthiosemi‐carbazide respectively, and characterized by various spectral techniques. The absorption spectral characteristics of R1–R3 were bathochromically tuned to the visible region by extending the π conjugation. These target compounds were weakly fluorescent in tetrahydrofuran (THF) solution because of rapid isomerization of the C=N double bond in the excited state. However, the aqueous dispersion of R1–R3 in the THF/water mixture by the gradual addition of water up to 90% resulted in an increase in the fluorescence intensity mainly due to aggregation‐induced emission enhancement (AIEE) properties. The formation of nanoaggregates of R1–R3 were confirmed by scanning electron microscopy (SEM) and atomic force microscopy (AFM) techniques. The compounds R1–R3 are ideal probes for the fluorescence sensing of bovine serum albumin (BSA) and breast cancer cells by optical cell imaging.     

Three‐dimensional imaging technologies: a priority for the advancement of tissue engineering and a challenge for the imaging community

Tissue engineering/regenerative medicine (TERM) is an interdisciplinary field that applies the principle of engineering and life sciences to restore/replace damaged tissues/organs with in vitro artificially‐created ones. Research on TERM quickly moves forward. Today newest technologies and discoveries, such as 3D‐/bio‐printing, allow in vitro fabrication of ex‐novo made tissues/organs, opening the door to wide and probably never‐ending application possibilities, from organ transplant to drug discovery, high content screening and replacement of laboratory animals. Imaging techniques are fundamental tools for the characterization of tissue engineering (TE) products at any stage, from biomaterial/scaffold to construct/organ analysis. Indeed, tissue engineers need versatile imaging methods capable of monitoring not only morphological but also functional and molecular features, allowing three‐dimensional (3D) and time‐lapse in vivo analysis, in a non‐destructive, quantitative, multidimensional analysis of TE constructs, to analyze their pre‐implantation quality assessment and their fate after implantation. This review focuses on the newest developments in imaging technologies and applications in the context of requirements of the different steps of the TERM field, describing strengths and weaknesses of the current imaging approaches.

     

sandflyDST: a dynamic web‐based decision support tool for the morphological identification of sandflies present in Anatolia and mainland Europe,and user study

Species identification of sandflies is mainly performed according to morphological characters using classical written identification keys. This study introduces a new web‐based decision support tool (sandflyDST) for guiding the morphological identification of sandfly species present in Anatolia and mainland Europe and classified in the Phlebotomus and Sergentomyia genera (both: Diptera: Psychodidae). The current version of the tool consists of 111 questions and 36 drawings obtained from classical written keys, and 107 photographs for the quick and easy identification of 26 species of the genus Phlebotomus and four species of the genus Sergentomyia. The tool guides users through a decision tree using yes/no questions about the morphological characters of the specimen. The tool was applied by 30 individuals, who then completed study questionnaires. The results of subsequent analyses indicated that the usability () and users' level of appreciation (86.6%) of the tool were quite high; almost all of the participants considered recommending the tool to others. The tool may also be useful in training new entomologists and maintaining their level of expertise. This is a dynamic tool and can be improved or upgraded according to feedback. The tool is now available online at http://parasitology.ege.edu.tr/sandflyDST/index.php .     

Volumetric hand‐held optoacoustic angiography as a tool for real‐time screening of dense breast

Existing mammographic screening solutions are generally associated with several major drawbacks, such as exposure to ionizing radiation or insufficient sensitivity in younger populations with radiographically‐dense breast. Even when combined with ultrasound or magnetic resonance imaging, X‐Ray mammography may still attain unspecific or false positive results. Thus, development of new breast imaging tools represents a timely medical challenge. We report on a new approach to high‐resolution functional and anatomical breast angiography using volumetric hand‐held optoacoustic tomography, which employs light intensities safe for human use. Experiments in young healthy volunteers with fibroglandular‐dominated dense breasts revealed the feasibility of rendering three‐dimensional images representing vascular anatomy and functional blood oxygenation parameters at video rate. Sufficient contrast was achieved at depths beyond 2 cm within dense breasts without compromising the real‐time imaging performance. The suggested solution may thus find applicability as a standalone or supplemental screening tool for early detection and follow‐up of carcinomas in radiographically‐dense breasts.

Volumetric handheld optoacoustic tomography scanner uses safe pulses of near‐infrared light to render three‐dimensional images of deep vascular anatomy, blood oxygenation and breast parenchyma at video rate.     

A distance‐dependent atomic knowledge‐based potential and force for discrimination of native structures from decoys

The purpose of this article is to introduce a novel model for discriminating correctly folded proteins from well designed decoy structures using mechanical interatomic forces. In our model, we consider a protein as a collection of springs and the force imposed to each atom is calculated. A potential function is obtained from statistical contact preferences within known protein structures. Combining this function with the spring equation, the interatomic forces are calculated. Finally, we consider a structure and define a score function on the 3D structure of a protein. We compare the force imposed to each atom of a protein with the corresponding atom in the other structures. We then assign larger scores to those atoms with lower forces. The total score is the sum of partial scores of atoms. The optimal structure is assumed to be the one with the highest score in the data set. To evaluate the performance of our model, we apply it on several decoy sets. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Wide‐field color imaging of scatter‐based tissue contrast using both high spatial frequency illumination and cross‐polarization gating

This study characterizes the scatter‐specific tissue contrast that can be obtained by high spatial frequency (HSF) domain imaging and cross‐polarization (CP) imaging, using a standard color imaging system, and how combining them may be beneficial. Both HSF and CP approaches are known to modulate the sensitivity of epi‐illumination reflectance images between diffuse multiply scattered and superficially backscattered photons, providing enhanced contrast from microstructure and composition than what is achieved by standard wide‐field imaging. Measurements in tissue‐simulating optical phantoms show that CP imaging returns localized assessments of both scattering and absorption effects, while HSF has uniquely specific sensitivity to scatter‐only contrast, with a strong suppression of visible contrast from blood. The combination of CP and HSF imaging provided an expanded sensitivity to scatter compared with CP imaging, while rejecting specular reflections detected by HSF imaging. ex vivo imaging of an atlas of dissected rodent organs/tissues demonstrated the scatter‐based contrast achieved with HSF, CP and HSF‐CP imaging, with the white light spectral signal returned by each approach translated to a color image for intuitive encoding of scatter‐based contrast within images of tissue. The results suggest that visible CP‐HSF imaging could have the potential to aid diagnostic imaging of lesions in skin or mucosal tissues and organs, where just CP is currently the standard practice imaging modality.      

Genome‐Wide Analysis of circular RNAs and validation of hsa_circ_0006719 as a potential novel diagnostic biomarker in congenital scoliosis patients

Congenital scoliosis (CS) is a form of spinal curvature resulting from anomalous development of vertebrae. Recent studies demonstrated that circRNAs could serve as potential biomarkers of disease diagnosis. Genome‐wide circRNAs expression in seven CS patients and three healthy controls was initially detected. Bioinformatics analysis was conducted to explore the potential pathological pathway of CS. Quantitative PCR (qPCR) was performed to validate the selected circRNAs in the replication cohort with 32 CS patients and 30 healthy controls. Logistic regression controlling for gender was conducted to compare the expression difference. Receiver operating characteristic (ROC) curve analysis was performed to evaluate the diagnostic value. Twenty‐two differentially expressed circRNAs were filtered from genome‐wide circRNA sequencing. Seven circRNAs were validated by qPCR. Only hsa_circ_0006719 was confirmed to have a higher expression level in the CS group than the healthy control group (P = 0.036). Receiver operating characteristic curve also suggested that hsa_circ_0006719 had significant diagnostic value for CS (AUC = 0.739, P = 0.001). We described the first study of circRNAs in CS and validated hsa_circ_0006719 as a potential novel diagnostic biomarker of CS.     

Opisthorchis viverrini: identification of a glycine-tyrosine rich eggshell protein and its potential as a diagnostic tool for human opisthorchiasis

A cDNA encoding a novel eggshell protein (OvESP) with high-glycine (49.2%) and -tyrosine (27.8%) content was cloned from the human liver fluke Opisthorchis viverrini. In the adult parasite, the RNA products of the OvESP gene are limited to the vitelline follicles. They have a size of 800 nucleotides and are already present in 2-week-old juveniles. Immune sera of hamsters, experimentally infected, and humans, naturally infected with O. viverrini, detect bacterially expressed recombinant OvESP (rOvESP). A rabbit anti-rOvESP antiserum only reacts with the shells of intrauterine eggs in tissue sections of the parasite. Comparison of rOvESP with the parasite's excretion/secretion products as diagnostic tools for human opisthorchiasis shows a higher sensitivity (0.82-0.48) and specificity (0.97-0.91) of the recombinant protein in the ELISA technique. But the observed weak cross-reactivity of immune sera from mice infected with Schistosoma mansoni, Schistosoma mekongi, and Fasciola gigantica in Western blots of rOvESP indicates that the diagnostic quality of this protein might be compromised if infections by other trematodes are present.     

Diagnosis of early gastric cancer based on fluorescence hyperspectral imaging technology combined with partial‐least‐square discriminant analysis and support vector machine

This study investigated the feasibility of using fluorescence hyperspectral imaging technology to diagnose of early‐stage gastric cancer. Fluorescence spectral images of 76 patients who were pathologically diagnosed as non‐atrophic gastritis, premalignant lesions and gastric cancer were collected. Fluorescence spectra at 100‐pixel points were randomly extracted after binarization. Diagnostic models of non‐atrophic gastritis, premalignant lesions and gastric cancer were constructed through partial‐least‐square discriminant analysis (PLS‐DA) and support vector machine (SVM) algorithms. The prediction effects of PLS‐DA and SVM models were compared. Results showed that the average spectra of normal, precancerous and gastric cancer tissues significantly differed at 496, 546, 640 and 670 nm, and regular changes in fluorescence intensity at 546 nm were in the following order: normal > precancerous lesions > gastric cancer. Additionally, the effect of the diagnostic model established by SVM is significantly better than PLS‐DA which accuracy, specificity and sensitivity are above 94%. Experimental results revealed that the fast diagnostic model of early gastric cancer by combining fluorescence hyperspectral imaging technology and improved SVM was effective and feasible, thereby providing an accurate and rapid method for diagnosing early‐stage gastric cancer.      

Trifluoroethanol‐containing RP‐HPLC mobile phases for the separation of transmembrane peptides human glycophorin‐A,integrin alpha‐1, and p24: analysis and prevention of potential side reactions due to formic acid

Reversed‐phase high‐pressure liquid chromatography analysis and purification of three hydrophobic, aggregation‐prone peptides, composed mainly of the transmembrane (TM) sequence, were performed using elution systems containing 2,2,2‐trifluoroethanol (TFE). The addition of 10–16% TFE to a common mobile phase, such as a water/acetonitrile/propanol (PrOH) or a water/PrOH/formic acid system, markedly improved the chromatographic separation of these peptides. The superior performance of TFE‐containing systems in separating peptides over water/PrOH/formic acid systems [Bollhagen R. et al., J. Chromatogr. A, 1995; 711 : 181–186.] clearly demonstrated that adding TFE to the mobile phase is one of best methods for TM‐peptide purification. Characterization of the potential side reactions using MALDI and ESI‐LIT/Orbitrap mass spectrometry indicated that prolonged incubation of peptides in a mixture of TFE–formic acid possibly induces O‐formylation of the Ser residue and N‐formylation of the N‐terminus of peptides. The conditions for selective removal of the formyl groups from TM peptides were also screened. We believe that these results will expand our ability to analyze and prepare hydrophobic, aggregation‐prone TM peptides and proteins. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Engineering a chemical implementation device and an imaging device for detecting chemiluminescence with a Polaroid™ high‐speed detector film: application to influenza diagnostics with the ZstatFlu®‐II test

We describe the engineering and product development of the chemiluminescent ZstatFlu®‐II Test kit for influenza diagnostics. The reaction vessel is a chemical implementation device with a polystyrene bottom chamber and a polypropylene top chamber that screw together. The patient's specimen is dispersed in a proprietary diluent and mixed inside the bottom chamber with the influenza viral neuraminidase‐specific substrate, 1,2‐dioxetane‐4,7‐dimethoxy‐Neu5Ac. Neuraminidase catalysis releases the dioxetane. The top chamber contains 40% NaOH and is sealed at the top with an ABS plastic plug‐crush pin assembly. The top chamber floor is 85% thinner at the centre, forming a frangible flap. An automated imaging device serves as an incubator for the chemical implementation devices and also facilitates the piercing of the flap by the crush pin. This action results in NaOH flushing into the bottom chamber, initiating chemiluminescence. The imaging device also exposes the Polaroid? high‐speed detector film to chemiluminescence. At the end of exposure, the film is automatically processed and ejected. Chemiluminescence from an influenza virus‐positive specimen produces a ‘+’‐shaped white image, archiving the diagnostic outcome. The modular ZstatFlu®‐II test kit components are easily adaptable for the chemiluminescent detection of a wide range of analytes. Copyright © 2003 John Wiley & Sons, Ltd.     

Multispectral imaging for quality control of laboratory‐reared Anastrepha fraterculus (Diptera: Tephritidae) pupae

The sterile insect technique (SIT) has been widely used to suppress several fruit fly species. In southern Brazil, millions of sterile flies of the South American fruit fly, Anastrepha fraterculus Wiedemann (Dipetra: Tephritidae), will be produced in a mass‐rearing facility called MOSCASUL to suppress wild populations from commercial apple orchards. In spite of standard rearing conditions, the quality of pupal batches can be inconsistent due to various factors. The quantification of poor quality material (e.g. empty pupae, dead pupae or larvae) is necessary to track down rearing issues, and pupal samples must be taken randomly and evaluated individually. To speed up the inspection of pupal samples by replacing the manual testing with the mechanized one, this study assessed a multispectral imaging (MSI) system to distinguish the variations in quality of A. fraterculus pupae and to quantify the variations based on reflectance patterns. Image acquisition and analyses were performed by the VideometerLab4 system on 7‐d‐old pupae by using 19 wavelengths ranging from 375 to 970 nm. The image representing the near infrared wavelength of 880 nm clearly distinguished among high‐quality pupae and the other four classes (i.e. low‐quality pupae, empty pupae, dead pupae and larvae). The blind validation test indicated that the MSI system can classify the fruit fly pupae with high accuracy. Therefore, MSI‐based classification of A. fraterculus pupae can be used for future pupal quality assessments of fruit flies in mass‐rearing facilities.     

Noninvasive molecular sexing: An evaluation and validation of the SRY‐ and amelogenin‐based method in three new lemur species

Many lemur species are arboreal, elusive, and/or nocturnal and are consequently difficult to approach, observe and catch. In addition, most of them are endangered. For these reasons, non‐invasive sampling is especially useful in primates including lemurs. A key issue in conservation and ecological studies is to identify the sex of the sampled individuals to investigate sex‐biased dispersal, parentage, social organization and population sex ratio. Several molecular tests of sex are available in apes and monkeys, but only a handful of them work in the lemuriform clade. Among these tests, the coamplification of the SRY gene with the amelogenin X gene using strepsirhine‐specific X primers seems particularly promising, but the reliability and validity of this sexing test have not been properly assessed yet. In this study, we (i) show that this molecular sexing test works on three additional lemur species (Microcebus tavaratra, Propithecus coronatus and P. verreauxi) from two previously untested genera and one previously untested family, suggesting that these markers are likely to be universal among lemurs and other strepsirrhines; (ii) provide the first evidence that this PCR‐based sexing test works on degraded DNA obtained from noninvasive samples; (iii) validate the approach using a large number of known‐sex individuals and a multiple‐tubes approach, and show that mismatches between the field sex and the final molecular consensus sex occur in less than 10% of all the samples and that most of these mismatches were likely linked to incorrect sex determinations in the field rather than genotyping errors. Am J Phys Anthropol, 2013. © 2013 Wiley Periodicals, Inc.     

Consistent refinement of submitted models at CASP using a knowledge‐based potential

Protein structure refinement is an important but unsolved problem; it must be solved if we are to predict biological function that is very sensitive to structural details. Specifically, critical assessment of techniques for protein structure prediction (CASP) shows that the accuracy of predictions in the comparative modeling category is often worse than that of the template on which the homology model is based. Here we describe a refinement protocol that is able to consistently refine submitted predictions for all categories at CASP7. The protocol uses direct energy minimization of the knowledge‐based potential of mean force that is based on the interaction statistics of 167 atom types (Summa and Levitt, Proc Natl Acad Sci USA 2007; 104:3177–3182). Our protocol is thus computationally very efficient; it only takes a few minutes of CPU time to run typical protein models (300 residues). We observe an average structural improvement of 1% in GDT_TS, for predictions that have low and medium homology to known PDB structures (Global Distance Test score or GDT_TS between 50 and 80%). We also observe a marked improvement in the stereochemistry of the models. The level of improvement varies amongst the various participants at CASP, but we see large improvements (>10% increase in GDT_TS) even for models predicted by the best performing groups at CASP7. In addition, our protocol consistently improved the best predicted models in the refinement category at CASP7 and CASP8. These improvements in structure and stereochemistry prove the usefulness of our computationally inexpensive, powerful and automatic refinement protocol. Proteins 2010. © 2010 Wiley‐Liss, Inc.