Manipulation Of Specimens Research Articles

A Raman topography imaging method toward assisting surgical tumor resection

AbstractAchieving complete tumor resection upon initial surgical intervention can lead to better patient outcomes by making adjuvant treatments more efficacious and reducing the strain of repeat surgeries. Complete tumor resection can be difficult to confirm intraoperatively. Methods like touch preparation (TP) have been inconsistent for detecting residual malignant cell populations, and fatty specimens like breast cancer lumpectomies are too fatty to process for rapid histology. We propose a novel workflow of immunostaining and topographic surface imaging of freshly excised tissue to ensure complete resection using highly sensitive and spectrally separable surface-enhanced Raman scattering nanoparticles (SERS NPs) as the targeted contrast agent. Biomarker-targeting SERS NPs are ideal contrast agents for this application because their sensitivity enables rapid detection, and their narrow bands enable extensive intra-pixel multiplexing. The adaptive focus capabilities of an advanced Raman instrument, combined with our rotational accessory device for exposing each surface of the stained specimen to the objective lens, enable topographic mapping of complete excised specimen surfaces. A USB-controlled accessory for a Raman microscope was designed and fabricated to enable programmatic and precise angular manipulation of specimens in concert with instrument stage motions during whole-surface imaging. Specimens are affixed to the accessory on an anti-slip, sterilizable rod, and the tissue surface exposed to the instrument is adjusted on demand using a programmed rotating stepper motor. We demonstrate this topographic imaging strategy on a variety of phantoms and preclinical tissue specimens. The results show detail and texture in specimen surface topography, orientation of findings and navigability across surfaces, and extensive SERS NP multiplexing and linear quantitation capabilities under this new Raman topography imaging method. We demonstrate successful surface mapping and recognition of all 26 of our distinct SERS NP types along with effective deconvolution and localization of randomly assigned NP mixtures. Increasing NP concentrations were also quantitatively assessed and showed a linear correlation with Raman signal with an R2 coefficient of determination of 0.97. Detailed surface renderings color-encoded by unmixed SERS NP abundances show a path forward for content-rich, interactive surgical margin assessment.

Repeat patient Testing-Quality control: An example of implementation in a network of commercial laboratories.

The theory and calculations underpinning Repeat Patient Testing-Quality Control (RPT-QC) have been presented in prior publications. This paper gives an example of the process used for implementing RPT-QC in a network of veterinary commercial reference laboratories and the stages associated with the transition to the sole use of RPT-QC. To employ RPT-QC in this commercial laboratory network, eight stages of implementation were identified: (1) education, (2) data collection, (3) calculations, (4) QC recording and documentation, (5) running RPT-QC in parallel with a commercially available quality control material (QCM), (6) development of a Standard Operating Procedure (SOP), (7) development of complementary aspects supporting RPT-QC, and (8) sole use of RPT-QC. Advantages of RPT-QC included cost savings for QCM and External Quality Assessment (EQA) participation and the ability to use commutable specimens with a veterinary matrix at a result level that is of clinical significance for the species. A disadvantage of RPT-QC using a single level of control was the inability to demonstrate stable performance over a range of results. Future avenues for investigation include ongoing refinement of control limits using a pooled standard deviation of the duplicates (SDdup), Sdup over time, investigation of blood samples from species other than the dog, and manipulation of specimens to produce "low abnormal" or "high abnormal" RPT-QC specimens.

Semi-Automated EXLO for Ambient and Cryogenic TEM Specimen Manipulation.

Semi-Automated EXLO for Ambient and Cryogenic TEM Specimen Manipulation.

Simultaneous Trapping and Manipulation of Micro-Specimens Using an All-Dielectric One-Dimensional Photonic Crystal

We demonstrate a platform where optical evanescent vortex has been realized by focusing a laser beam with orbital angular momentum (OAM) onto an all-dielectric one-dimensional photonic crystal (1DPC). This platform can simultaneously trap and manipulate various micro-scale specimens (i.e., polymer microsphere, inorganic microwire, yeast cells and Escherichia coli cells). The working principle lies in the low loss of the excited Bloch surface waves (BSWs) that propagate on the dielectric 1DPC and form a virtual probe for trapping the specimens. The OAM encoded by the BSWs will then simultaneously manipulate the specimens, such as inducing the rotation or orbital motion. Both experimental and simulation results verify that the capability of this new platform is due to the two kinds of optical force induced by the BSWs, which presents a different mechanism from that by the commonly optical electric-field vortex. Optical trapping or manipulation of micro & nano specimens play important roles in physics, life and materials sciences, thus, this new platform will find applications in microscale assembly, cell-cell interaction, bioprinting and biosensing research, and also expand our understanding of the physics interaction between vector beam and BSWs.

Epaxial and hypaxial co-contraction: a mechanism for modulating strike pressure and accuracy during suction feeding in channel catfish.

Most fish species use concentric epaxial and hypaxial contractions to suction feed, whereby both muscle groups produce cranial expansion and negative intraoral pressures. In contrast, channel catfish (Ictalurus punctatus) suction feed with little to no cranial elevation and epaxial shortening, generating suction power primarily with hypaxial shortening and pectoral girdle retraction. We hypothesized that channel catfish (1) actively anchor the head via isometric contraction of the epaxials and (2) vary feeding performance by modulating the absolute and relative outputs of the co-contracting muscles. We used a combination of electromyography, intraoral pressure recordings and specimen manipulation, and developed a new dual-lever model to explore this idea. We detected epaxial and hypaxial co-contraction prior to suction force development in all strikes. Our model revealed that the differential between the co-contracting muscles may be used to modulate suction pressure and strike accuracy.

Microsurgical training through laboratory experience: A step-by-step practical guideline

BackgroundMicrosurgical laboratory training remains the mainstay to acquire, improve, and perfect surgical abilities. Microvascular anastomotic techniques are of key importance for revascularization procedures, such as in giant cerebral aneurysms, skull base complex tumours, and symptomatic ischemic cerebrovascular diseases. MethodsWe show the techniques, materials, and experience performed in a microsurgical laboratory of a neurosurgical referral center in Mexico City. ResultsWe describe the materials, non-specimen suture practices, specimen manipulation, and finally all the individual procedures we are capable to perform on live specimens. ConclusionsThe microsurgical laboratory is an indispensable tool for the acquisition of the so needed microsurgical skills in neurosurgery, having a very special emphasis in vascular neurosurgery.

Impact of Viral Decontamination Method on Cytokine Profile of COVID-19 Patients.

COVID-19 related morbidity and mortality have been often attributed to an exaggerated immune response. The role of cytokines and chemokines in COVID-19 and their contributions to illness severity are known, and thus their profiling from patient bronchoalveolar lavage (BAL) samples would help in understanding the disease progression. To date, limited studies have been performed on COVID-19 BAL samples, as the manipulation of such specimens (potentially containing live viruses) requires several laboratorial precautions, such as personnel training and special equipment, a requirement that not all laboratories can fulfil. Here, we assessed two fast and easily applicable methods (ultrafiltration and ultraviolet–C irradiation) for their impact on viral load removal or inactivation, respectively and on cytokine profiles preservation. Eight samples of BAL fluids from SARS-CoV2 patients with high viral load were tested. For both methods, complete removal was confirmed by lack of viral replication in Vero E6 cells and by RT-qPCR. Although both methods showed to remove completely the active SARS-CoV2 viral load, only UVC treatment has little or no quantitative effect on total cytokines/chemokines measurements, however cytokines profile and relative ratios are preserved or minimally altered when compared data obtained by the two different decontamination methods. Sample preparation and manipulation can greatly affect the analytical results; therefore, understanding if changes occurred after sample processing is of outmost importance for reliable data and can be useful to improve clinical practice.

Relationship between lymphocyte subsets values and C-reactive protein in COVID-19 patients.

We enrolled 33 patients with COVID‐19 (23 men and 10 women; age 59 ± 15; males, n = 23; females, n = 10) admitted to the Department of Infectious Diseases of Grande Ospedale Metropolitano “Bianchi‐Melacrino‐Morelli” of Reggio Calabria, Italy, between March and May 2020. Whole blood samples were collected before the start of therapeutic treatment using all virus spread containment measures. Sample preparation protocols were designed in order to minimize operators direct specimen's manipulation. On univariate analysis, circulating levels of CRP were strongly and inversely related to CD3+ (rho = −0.77, p < 0.001), CD3+4+ (rho = −0.74, p < 0.001), and CD3+8+ (rho = −0.66, p = 0.001) implying that the shared variances between absolute values T cells and CRP ranged from 44 to 59%. Of note, the strength of these associations was higher in patients with relatively lower (below the median value) white blood cells (WBC) as compared to those with WBC above the median value. CRP also correlated with NK bright (rho = −0.56, p = 0.005) but failed to be related with CD19+ (rho = −0.38, p = 0.07), CD4+/CD8+ ratio (rho = 0.03, p = 0.89), CD16+ CD56+ (rho = −0.18, p = 0.43), and NKdim (rho = −0.15, p = 0.49). Lymphocyte subsets alteration monitoring in COVID‐19 positive patients may be a valid aid to control treatment efficacy of therapy and to choose better clinical approach. In particular, the negative correlation between CD3+, CD3+CD4+, CD3+CD8+ T cells values and CRP could be a useful tool to predict patient's response to therapy, particularly in patients with relatively lower WBC.

P08 A Novel Technique for Large Specimen Manipulation and Extraction in the Thorax Using the Kiwi Complete Vacuum Delivery System

Removal of large specimens from the thoracic cavity can be challenging. Traditionally most surgeons extract large specimens using a sac or a bag. We present a case of a 68 year old female with a 15cmx13cmx20cm mediastinal mass extending into the right hemithorax who underwent a median sternotomy. We were not able to extract and manipulate the specimen by usual means—it was successfully taken out using a Kiwi Complete Vacuum Delivery System (Kiwi Omnicup).

A strategy to digitise natural history collections with limited resources

The present work is a contribution towards accelerating the digitisation process of natural history collections, usually a slow process. A two-stage process was developed at the herbarium of the University of Coimbra: (i) a new workflow was established to automatically create records in the herbarium master database with minimum information, while capturing digital images; (ii) these records are then used to populate a web-based crowdsourcing platform where citizens are involved in the transcription of specimen labels from the digital images. This approach simplifies and accelerates databasing, reduces specimen manipulation and promotes the involvement of citizens in the scientific goals of the herbarium. The novel features of this process are: (i) the validation method of the crowdsourcing contribution that ensures quality control, enabling the data to integrate the master database directly and (ii) the field-by-field integration in the master database enables immediate corrections to any record in the catalogue.

Herbaria Mundi: An image centric approach to manipulation of specimens from multiple collections

Addressing the challenges of the Climate Emergency and the Biodiversity Crisis requires us to understand how the world's vegetation is changing. This is not a trivial task,especially in highly diverse tropical areas. Frequently, the only way to characterise vegetation is to make voucher specimens of the plants present and identify them later in the lab. Fortunately there are extensive reference collections carefully curated in herbaria. Unfortunately specimens and expertise are dispersed over hundreds of different herbaria spread across many countries. It would be more efficient if scientists could see and manipulate specimens independently of where they are stored, especially as travel becomes more difficult due to both the need to reduce carbon emissions and virus spread. Herbaria Mundi is an application demonstrating how how this can be achieved using CETAF Specimen IDs and IIIF (International Image Interoperability Framework). The application mimics the way a botanist might work in a physical herbarium by enabling the gathering of specimens into groups and side-by-side comparison of specimens, but differs in that one can search for and manipulate specimens in multiple herbaria as if they were stored in a single collection. Voucher specimens are often not added to herbaria mainly because they do not add enough value to material already in the collections. Herbaria Mundi therefore includes the ability for researchers to upload specimen images to Zenodo and for them to appear in the user interface as if they were in an institutional herbarium. This proof of concept application is being developed as part of Task 4.3 of the Synthesys+ project funded by the European Commission. Two things need to occur to take this concept into production. More herbaria need to adopt the use of CETAF specimen identifiers and the IIIF image API. Botanists need to prioritise which features they would like developed first. More herbaria need to adopt the use of CETAF specimen identifiers and the IIIF image API. Botanists need to prioritise which features they would like developed first. The poster will be a catalyst to discuss how these things can be achieved.

PD-L1 lineage-specific quantification in malignant pleural effusions of lung adenocarcinoma by flow cytometry

ObjectivesPathologists encounter several challenges with programmed death ligand-1 (PD-L1) immunohistochemistry (IHC) tests in malignant effusions, including lineage specification (distinction between carcinoma vs. immune and mesothelial cells), background staining, sample fixation issues and inter-observer variability. We explored flow cytometric (FC) quantification of PD-L1 expression in malignant pleural effusions of lung adenocarcinoma patients as an alternative, automated, and objective quantification method compared to PD-L1 IHC. Materials and methodsWe examined 23 malignant pleural effusions of TTF-1-positive adenocarcinoma were subjected to FC with a panel of antibodies against CD45, CD3, CD200, EpCAM, D2-40 (podoplanin), and PD-L1 (clone MIH1). The PD-L1 gate was established using fluorescence-minus-one (FMO) isotype controls. Lineage-specific PD-L1 surface expression was quantified and the FC tumor proportion score (TPS) was assessed. PD-L1 IHC was performed on cell block sections using Dako PD-L1 IHC 22C3 pharmDx assay and assessed by two cytopathologists blinded to the FC PD-L1 TPS. ResultsFC analysis allowed for the distinction between carcinoma cells (CD45−/EpCAM+/D2-40-), leukocytes (CD45+/EpCAM-/D2-40-) and mesothelial cells (CD45-/EpCAM-/D2-40+). FC PD-L1 TPS ranged from 0% to 77 %, while the 22C3 IHC PD-L1 TPS ranged from 0% to 97 %. The FC and IHC TPS values correlated positively (R = 0.8). Best concordance was observed when FC was performed and cell blocks were generated in parallel (R = 0.99). FC also allowed for simultaneous PD-L1 quantification in mesothelial and T-cells. PD-L1 expression on mesothelial cells ranged from 0% to 90.9 %, which also correlated positively with IHC TPS (R = 0.54). PD-L1 expression on T-cells was limited (0.1–2.9 %). ConclusionFC permits rapid, objective and lineage-specific PD-L1 surface expression quantification with limited specimen manipulation. The FC and IHC concordance was impacted by different antibody clones being used, but the positive correlation suggests potential clinical utility, especially in malignant effusion specimens.

Lymph nodes detection and the mesorectal excision quality evaluation

Detection and examination of proper number of lymph nodes in patients after rectal resection is important for next treatment and management of patients with rectal carcinoma. There are no clear guideliness for minimal count of lymph nodes, variant recommendations agree on the number of 12 (1014) nodes. There are situations, when is not easy to reach this count, mainly in older age groups and in patients after neoadjuvant, especially radiation therapy. As amodality for improvement of lymph nodes harvesting seems to be establishing of defined protocols originally designed for mesorectal excision quality evaluation. The investigation group was formed by patients examined in 2 three-years intervals before and after implementation of the protocol. Elevation in count of harvested lymph nodes was rated generaly and in relation to age groups and gender. The average count of lymph nodes increased from 10 to 15 nodes, in subset of patients whose received neoadjuvant therapy from 7 to almost 14 nodes. The recommended number of lymph nodes was obtained in all investigated age groups. By the increased number of lymph nodes, rises also possibility of positive nodes found, that can lead to upstaging of the disease, in subset of patients whose received neoadjuvant therapy it is more than 4%. Our conclusions show, that forming of multidisciplinary cooperative groups (chiefly surgeon-pathologist), implementation of defined protocol of surgery, specimen manipulation and investigation by detached specialists lead to benefit consequences for further management and treatment of the patients with colorectal cancer.

Diagnostic Efficacy of Liquid-Based Cytology in Endoscopic Ultrasound-Guided Fine Needle Aspiration for Pancreatic Mass Lesions During the Learning Curve: A Retrospective Study.

The diagnostic yield of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) cytology widely varies depending on the treatment method used. Liquid-based cytology (LBC) has gained popularity in the gynecological field because of its efficacy in collection of target cells and simplicity in the manipulation of specimens. Since the introduction of EUS-FNA at our institution, we have used LBC for the diagnosis of pancreatic mass lesions. This study aims to investigate the diagnostic efficacy of EUS-FNA with LBC in patients with pancreatic mass lesions during the learning curve for EUS-FNA. In this study, we retrospectively enrolled 222 patients with pancreatic mass lesions who were diagnosed using EUS-FNA with LBC between 2011 and 2016. The diagnostic yields for EUS-FNA with LBC for pancreatic mass lesions were evaluated. The diagnostic sensitivity, specificity, and accuracy for malignancy were found to be 93.9%, 95.1%, and 94.1%, respectively. This study suggests that EUS-FNA with LBC for specimens provides good diagnostic efficacy in patients with pancreatic mass lesions even during the learning curve for EUS-FNA.

Expedited large-volume 3-D SEM workflows for comparative microanatomical imaging.

To-date, different electron microscopy (EM) approaches are available (e.g., TEM, SEM, STEM, CLEM, etc.) to collect three-dimensional (3-D) information in tissues and cells from the microscale up to the nanometer scale. However, an abundant amount of possibilities and methodologies exist to reconstruct volumes of biological matter. In this topical paper we outline two specimen manipulation workflows for the generation of 3-D scanning electron microscopy (3-D SEM) data by means of serial-block face/focussed ion beam SEM and array tomography (AT). We applied these commonly used workflows to rodent and zebrafish liver as model experimental systems. In doing so, we outline the specific steps of each procedure and discuss in-depth the strengths vs. limitations for each of the respective 3-D SEM specimen manipulation workflows.

Biaxial flexure testing of free-standing thin film membrane with nanoindentation system

The advent of microelectromechanical devices has increased the demand for biaxial flexure testing at the micro- and nanoscale. However, testing at these scales is challenging owing to difficulties in manipulating very small samples and applying highly symmetric biaxial loads to them. In this study, we developed a facile technique for on-chip biaxial flexure testing. The principle of the technique was inspired by ball-on-ring biaxial testing commonly employed for macroscopic flexure analysis of ceramics. In our technique, the specimen is tested as a circular membrane fixed at its edges to a substrate. The center of the membrane is pushed from above by a rounded conical nanoindenter (NI) until the membrane fractures. Since this method does not require microscale specimen manipulation involving the securing of microscale components and/or samples by external fixtures, the test procedure is relatively easy to perform. In addition, the load and displacement curves obtained using the NI are high resolution, enabling precise strength evaluation and application to very weak structures such as nanoscale membranes. To demonstrate the test system, we designed and fabricated polysilicon membranes with diameters of 20 μm and thicknesses of 80 nm. The most likely source of error in the system is misalignment between the indenter and the membrane center. Accordingly, its effect was numerically analyzed. The results showed that a misalignment of less than 3.0 μm causes a 0.32% error in the first principal stress. Since the NI tester can easily attain this alignment accuracy, highly accurate stress estimates can be achieved. The measured fracture strengths of the polysilicon membranes were fitted to the Weibull distribution, revealing an average strength of 8.11 ± 0.31 GPa and a shape parameter of 13.9 ± 5.4, both of which agree well with the results from previous research. The fracture origins were observed near the center of the membranes. These results confirm the viability of our concept.

Use of High-Resolution Ultrasound (HRU) in the Assessment of Deep Injections of CHAP-Hyaluronic Acid (CHAP-HA) Fillers for Midface Lift

High-resolution ultrasound (HRU) imaging is a useful tool to study hyaluronic acid (HA) filler injection in the face. It is noninvasive, quick, well-tolerated, and can provide in vivo and dynamic information. The formations of pools or pearls in HA fillers could be observed real time during injection. The plane of injection could be determined accurately, and there were no specimen manipulation artifacts. It was observed that HA gel fillers with differing production technologies showed distinct spread and distribution patterns in the periocular tissues on HRU examination. The authors used HRU to assess deep injections of CHAP-Hyaluronic Acid (CHAP-HA) fillers for midface lift. 10 patients who underwent bilateral midface deep injections using CHAP-HA filler were examined with HRU before and immediately after treatment, and in 2 weeks and one month later. The CHAP-HA appeared as hypoechoic densities within the preperiosteal plane in HRU. CHAP-HA adopted variable morphology within the tissue depending on individual tissue densities and the compliance of the tissues in the plane of injection. CHAP-HA was unidentifiable with surrounding tissue after one month in 13 of the 20 injection sites. HRU allows in vivo study of CHAP-HA injection behavior and could be a tool for further studies of HA-tissue reactions.

Disposable Autonomous Device for Swab-to-Result Diagnosis of Influenza.

A prototype of a self-contained, automated, disposable device for chemically amplified protein-based detection of influenza virus from nasal swab specimens was developed and evaluated in a clinical setting. The device required only simple specimen manipulation without any dedicated instrumentation or specialized training by the operator for interpretation. The device was based on a sandwich immunoassay for influenza virus nucleoprotein; it used an enzyme-labeled antibody and a chromogenic substrate to provide an amplified visible signal, in a two-dimensional paper network format. All reagents were stored within the device. Device performance was assessed at Seattle Children's Hospital; clinical staff collected nasal swab samples from 25 patients and then operated test devices on site to detect influenza A and B in those specimens. The total test time from device initiation to result was approximately 35 min. Device performance for influenza A detection was ∼70% accurate using in-house qRT-PCR influenza A as a gold-standard comparison. The ratio of valid to total completed device runs yielded a success rate of 92%, and the negative predictive value for both the influenza A and B assay was 81%. The ability to diagnose respiratory infections rapidly and close to the patient was well received by hospital staff, inspiring further optimization of device function.

Grating-flanked plasmonic coaxial apertures for efficient fiber optical tweezers

Subwavelength plasmonic apertures have been foundational for direct optical manipulation of nanoscale specimens including sub-100 nm polymeric beads, metallic nanoparticles and proteins. While most plasmonic traps result in two-dimensional localization, three-dimensional manipulation has been demonstrated by integrating a plasmonic aperture on an optical fiber tip. However, such 3D traps are usually inefficient since the optical mode of the fiber and the subwavelength aperture only weakly couple. In this paper we design more efficient optical-fiber-based plasmonic tweezers combining a coaxial plasmonic aperture with a plasmonic grating coupler at the fiber tip facet. Using full-field finite difference time domain analysis, we optimize the grating design for both gold and silver fiber-based coaxial tweezers such that the optical transmission through the apertures is maximized. With the optimized grating, we show that the maximum transmission efficiency increases from 2.5% to 19.6% and from 1.48% to 16.7% for the gold and silver structures respectively. To evaluate their performance as optical tweezers, we calculate the optical forces and the corresponding trapping potential on dielectric particles interacting with the apertures. We demonstrate that the enahncement in the transmission translates into an equivalent increase in the optical forces. Consequently, the optical power required to achieve stable optical trapping is significantly reduced allowing for efficient localization and 3D manipulation of sub-30 nm dielectric particles.

Combined Multidimensional Microscopy as a Histopathology Imaging Tool.

Herein, we present a highly versatile bioimaging workflow for the multidimensional imaging of biological structures across vastly different length scales. Such an approach allows for the optimised preparation of samples in one go for consecutive X-ray micro-computed tomography, bright-field light microscopy and backscattered scanning electron microscopy, thus, facilitating the disclosure of combined structural information ranging from the gross tissue or cellular level, down to the nanometre scale. In this current study, we characterize various aspects of the hepatic vasculature, ranging from such large vessels as branches of the hepatic portal vein and hepatic artery, down to the smallest sinusoidal capillaries. By employing high-resolution backscattered scanning electron microscopy, we were able to further characterize the subcellular features of a range of hepatic sinusoidal cells including, liver sinusoidal endothelial cells, pit cells and Kupffer cells. Above all, we demonstrate the capabilities of a specimen manipulation workflow that can be applied and adapted to a plethora of functional and structural investigations and experimental models. Such an approach harnesses the fundamental advantages inherent to the various imaging modalities presented herein, and when combined, offers information not currently available by any single imaging platform. J. Cell. Physiol. 232: 249-256, 2017. © 2016 Wiley Periodicals, Inc.