Cellular Specimens Research Articles

A comprehensive study of LPBF parameter influence in 316L stainless steel mechanical properties, macrostructure, and printability

Additive manufacturing, particularly using Laser Powder Bed Fusion (LPBF) technologies with 316L stainless steel, has proven highly promising in biomedical engineering due to the material’s exceptional mechanical properties and biocompatibility. This study, which is a foundational step towards developing porous bone implants, focuses on assessing the influence of a broad range of parameters - spot size, scanning speed, laser power, and energy density - on the mechanical performance and structural integrity of 316L stainless steel parts across three distinct specimen geometries: fully dense parallelepipedic, fully dense cubic, and cellular (lattice) specimens. By analysing 17 different parameter sets and producing over 150 specimens, a thorough series of experimental tests were conducted, including flexural tests, compression tests, hardness tests, macroscopic evaluation of the surface of fully dense specimens, and macroscopic evaluation of the lattice structures.e in powder accumulation within the pores of cellular structures, with higher energy densities (100 J/mm3) leading to a significantly greater retention compared to lower energy densities (66 J/mm3). This underexplored phenomenon has significant implications for the selection of parameters in the fabrication of bone implants. Further research is needed to refine LPBF printing parameters and enhance the quality of porous bone implants.

An open-source membrane stretcher for simultaneous mechanical and structural characterizations of soft materials and biological tissues

The ability to simultaneously measure material mechanics and structure is central for understanding their nonlinear relationship that underlies the mechanical properties of materials, such as hysteresis, strain-stiffening and -softening, and plasticity. This experimental capability is also critical in biomechanics and mechanobiology research, as it enables direct characterizations of the intricate interplay between cellular responses and tissue mechanics. Stretching devices developed over the past few decades, however, do not often allow simultaneous measurements of the structural and mechanical responses of the sample. In this work, we introduce an open-source stretching system that can apply uniaxial strain at a submicron resolution, report the tensile force response of the sample, and be mounted on an inverted microscope for real-time imaging. Our system consists of a pair of stepper-based linear motors that stretch the sample symmetrically, a force transducer that records the sample tensile force, and an optically clear sample holder that allows for high-magnification microscopy. Using polymer samples and cellular specimens, we characterized the motion control accuracy, force measurement robustness, and microscopy compatibility of our stretching system. We envision that this uniaxial stretching system will be a valuable tool for characterizing soft and living materials.

Harvesting deformation modes for micromorphic homogenization from experiments on mechanical metamaterials

A micromorphic computational homogenization framework has recently been developed to deal with materials showing long-range correlated interactions, i.e. displaying patterning modes. Typical examples of such materials are elastomeric mechanical metamaterials, in which patterning emerges from local buckling of the underlying microstructure. Because pattern transformations significantly influence the resulting effective behaviour, it is vital to distinguish them from the overall deformation. To this end, the following kinematic decomposition into three parts was introduced in the micromorphic scheme: (i) a smooth mean displacement field, corresponding to the slowly varying deformation at the macro-scale, (ii) a long-range correlated fluctuation field, related to the buckling pattern at the meso-scale, and (iii) the remaining uncorrelated local microfluctuation field at the micro-scale. The micromorphic framework has proven to be capable of predicting relevant mechanical behaviour, including size effects and spatial as well as temporal mixing of patterns in elastomeric metamaterials, making it a powerful tool to design metamaterials for engineering applications. The long-range correlated fluctuation fields need to be, however, provided a priori as input parameters. The main goal of this study is experimental identification of the decomposed kinematics in cellular metamaterials based on the three-part ansatz. To this end, a full-field micromorphic Integrated Digital Image Correlation (IDIC) technique has been developed. The methodology is formulated for finite-size cellular elastomeric metamaterial specimens deformed in (i) virtually generated images and (ii) experimental images attained during in-situ compression of specimens with millimetre sized microstructure using optical microscopy. The proposed IDIC method identifies the different kinematic fields, both before and after the microstructural buckling, and without any prior knowledge determines correctly the relevant patterning modes required by the homogenization scheme. It is further argued that patterning modes are independent of the unit cell size, the hole diameter to cell size ratio, as well as local material properties, allowing for modelling and design of (finite- and infinite-size) metamaterials and specimens with graded microstructures in terms of geometry and/or material properties. It is shown that the proposed methodology is also applicable to cellular metamaterials and structures with different microstructural designs.

Honeycomb gold specimen supports enabling orthogonal focussed ion beam-milling of elongated cells for cryo-ET

Cryo-focussed ion beam (FIB)-milling is a powerful technique that opens up thick, cellular specimens to high-resolution structural analysis by electron cryotomography (cryo-ET). FIB-milled lamellae can be produced from cells on grids, or cut from thicker, high-pressure frozen specimens. However, these approaches can put geometrical constraints on the specimen that may be unhelpful, particularly when imaging structures within the cell that have a very defined orientation. For example, plunge frozen rod-shaped bacteria orient parallel to the plane of the grid, yet the Z-ring, a filamentous structure of the tubulin-like protein FtsZ and the key organiser of bacterial division, runs around the circumference of the cell such that it is perpendicular to the imaging plane. It is therefore difficult or impractical to image many complete rings with current technologies. To circumvent this problem, we have fabricated monolithic gold specimen supports with a regular array of cylindrical wells in a honeycomb geometry, which trap bacteria in a vertical orientation. These supports, which we call “honeycomb gold discs”, replace standard EM grids and when combined with FIB-milling enable the production of lamellae containing cross-sections through cells. The resulting lamellae are more stable and resistant to breakage and charging than conventional lamellae. The design of the honeycomb discs can be modified according to need and so will also enable cryo-ET and cryo-EM imaging of other specimens in otherwise difficult to obtain orientations.

Structural Behavior of a Fixed-End Arched Cellular Steel Beam without Lateral Support

The arched cellular beam has the advantages of both the solid-web arch and the straight beam with web opening, and has become increasingly admired by architects in recent years. In this paper, four arched cellular beam specimens are designed using an orthogonal test method (OTM) with a three-factor and two-level approach. Firstly, the static loading test is carried out to analyze the mechanical response of the arched cellular beam under concentrated load. Then, a numerical analysis based on ABAQUS finite element (FE) software is carried out. The results show that the simulation results agree well with the test results, which indicates the accuracy of the simulation analysis method. Finally, the buckling load of the arched cellular beam under three different loads is calculated using the variable parameter FE analysis. Combined with the range analysis in the OTM, the influence of the target factor on the buckling load of the arched cellular beam is determined. The results show that the order of the factors affecting the out-of-plane elastic buckling is rise–span ratio > web height–thickness ratio > diameter–depth ratio.

Longitudinal Noninvasive Surveillance & Fragmentomic Characterization of Follicular Lymphoma

Introduction While cell-free DNA (cfDNA) plays an increasingly defined role in aggressive lymphomas, its characteristics in indolent lymphomas are less established. Many follicular lymphoma (FL) patients experience durable remissions and late relapses ( Fig A). We therefore explored circulating tumor DNA (ctDNA) kinetics during long-term blood-based surveillance in patients with FL. In addition to noninvasive detection using somatic mutations, we also evaluated gene expression inference from cfDNA utilizing a novel fragmentomic method to detect histological transformation (tFL). Methods We studied 121 serial samples in a cohort of 17 FL patients (median 7 per patient) with long term clinical and blood-based MRD monitoring (median 5.6 years, max 10.8). We considered serial ctDNA status at 4 milestones: baseline/pre-treatment, post-treatment, in radiographic remission (MRD), and at relapse. We also profiled pre-treatment cfDNA for 9 tFL cases. Cases were genotyped to identify somatic mutations from diagnostic tumor or baseline plasma, with matched PBMCs used to censor germline variants and clonal hematopoiesis. ctDNA status in subsequent samples was evaluated via CAPP-Seq (SNVs) and PhasED-Seq (PVs). The cfDNA fragmentation profiles of pre-treatment FL and control samples were evaluated for inferred gene expression via EPIC-seq (Esfahani Nat Biotech 2022) using a novel 1676-gene panel focused on lymphoid neoplasms, including classic and transformed FL. Results Treatment & Relapse Kinetics Most patients had advanced disease (88% stage III-IV) and Low to Intermediate risk FLIPI (71% 0-2). At first cfDNA evaluation, 9/17 (53%) were previously untreated. Median pre-treatment ctDNA levels were 73.4 hGE/mL in FL, as compared to 236 hGE/mL in DLBCL (Kurtz JCO 2018). Post-treatment, median ctDNA levels dropped to 1.2 hGE/mL. Depth of response was similar between rituximab monotherapy and cytotoxic regimens, with median -2.2 log10 fold change from baseline. MRD Detection PVs were observed in all tumors, consistent with expected aberrant somatic hypermutation in FL. To evaluate MRD detection via PhasED-Seq, we considered plasma timepoints with undetectable ctDNA by CAPP-Seq (n=26) from patients (n=9) with subsequent clinical relapse. MRD was detected in 11 additional specimens (42%) by PhasED-Seq. Among FL patients tested for MRD while in radiographic remission, low-level ctDNA was detectable in 62% of plasma specimens (8/13) obtained within 1 month of negative PET/CT, suggesting potential additional utility for surveillance. Long-term Genomic Stability Sequential biopsies of FL tumors have shown genomic heterogeneity between diagnosis and relapse. We therefore evaluated ctDNA concordance across variant classes & regions. Relative AF was calculated pre-treatment and at relapse (median 2 years between samples, range 1-8 years), normalized by total ctDNA levels. The most stable lesions included missense mutations in CREBBP and TP53, nonsense KMT2D mutations, and BCL2 5'-UTR variants. BCL6 intronic mutations and chr2/chr14 SHM varied between diagnosis/relapse. Plasma versus Cellular Concentrations In contrast to DLBCL, circulating FL cells are frequently detectable at low levels in blood. To compare relative FL concentrations in the cellular versus plasma compartments, we profiled 10 paired specimens from patients with radiographic disease. While tumor-confirmed variants were detected in 7 of 10 cellular specimens, mean allelic frequency (AF) was 5.3x higher in plasma. Fragmentomic Assessment of Transformation We compared gene expression profiles inferred from cfDNA from pre-treatment lymphoma (FL=15, tFL=9) versus control (n=20) samples ( Fig B). Considering genes associated with FL (Huet Lancet Onc 2018), FL patients had significantly higher inferred expression than healthy adults (p=0.036). Likewise, patients with tFL showed higher inferred expression of genes associated with histologic transformation (Gentles Blood 2009, p=0.025). Conclusions Sensitive methods are required for effective MRD assessment in FL given lower ctDNA shedding as compared with DLBCL. Frequent detectable MRD in radiographic remission suggests that integrating molecular surveillance may augment serial imaging in the long-term management of FL. Simultaneous inferred gene expression from FL cfDNA appears to hold promise, including for noninvasive detection of transformation.

Low cycle fatigue behaviour of cellular materials: Experimental comparative study of strut-based and gyroid structures made of additively manufactured 316L steel

Cellular materials are an attractive option to improve mechanical properties in lightweight design. Given their complex geometry, cellular materials present small features at the meso-scale that make them highly susceptible to fatigue failures. Fatigue of these materials has been receiving adequate attention only in the last few years, nevertheless, studies of low cycle fatigue (LCF) behaviour are extremely scarce and fragmented. In this study, 316L steel strut-based (FBCCZ) and gyroid cellular specimens were successfully manufactured by laser-powder bed fusion and tested in the LCF regime, considering their post-manufacturing morphological characteristics. The cyclic elastoplastic response revealed a higher stiffness for the strut-based than the gyroid cellular structure. The latter, in contrast, exhibited higher fatigue strength in strain-control mode, thanks to the absence of severe stress and strain raisers when compared to the strut-based counterpart. Overall, strain-life curves of both types of cellular materials are shifted to lower number of cycles to failure with respect to the base material. Detailed fractographic analyses revealed complex and tri-dimensional fracture surfaces for the gyroid specimens, whereas the strut-based lattice displayed planar fracture surfaces.

Real-Time Evaluation of Thyroid Cytology Using New Digital Microscopy Allows for Sample Adequacy Assessment, Morphological Classification, and Supports Molecular Analysis.

Thyroid cytological examination, a key tool in preoperative thyroid nodule evaluation, is specific and accurate; some drawbacks are due to inadequate or indeterminate cytological reports and there is a need for an innovative approach overcoming the limits of traditional cytological diagnostics. Fluorescence laser confocal microscopes (FCM) is a new optical technique for allowing immediate digital imaging of fresh unfixed tissues and real-time assessment of sample adequacy and diagnostic evaluation for small biopsies and cytological samples. Currently, there are no data about the use of FCMs in the field of thyroid nodular pathology. The aims of this study were to test FCM technology for evaluating the adequacy of FNA samples at the time of the procedure and to assess the level of concordance between FCM cytological evaluations, paired conventional cytology, and final surgical histology. The secondary aim was to define the integrity of nucleic acids after FCM evaluation through NGS molecular analysis. Sample adequacy was correctly stated. Comparing FCM evaluation with the final histology, all cases resulting in malignant or suspicious for malignancy at FCM, were confirmed to be carcinomas (PPV 100%). In conclusion, we describe a successful application of FCM in thyroid preoperative cytological evaluation, with advantages in immediate adequacy assessment and diagnostic information, while preserving cellular specimens for permanent morphology and molecular analysis, thus improving timely and accurate patient management.

Under the Hood: Understanding the Features of Mucin in Pseudomyxoma Peritonei.

Pseudomyxoma peritonei (PMP) is a rare malignant growth characterized by the production of mucin and the potential for peritoneal relapse. This study aimed to investigate the immunohistochemical and biological characteristics of mucin in patients with cellular and acellular PMP. We prospectively analyzed mucin specimens obtained from our patient cohort and described the composition and type of mucin present in each sample. A metagenomic analysis of the samples was performed to investigate the bacterial composition of the PMP microbiome. Secreted mucins 2 and 5AC and membrane-associated mucin-1 were the primary components of mucin in both cellular and acellular tumor specimens. The metagenomic study revealed a predominance of the phylum Proteobacteria and the genus Pseudomonas. Notably, Pseudomonas plecoglossicida, a species not previously reported in the human microbiome, was found to be the most abundant organism in the mucin of pseudomyxoma peritonei. Our findings suggest that the presence of MUC-2 and mucin colonization by Pseudomonas are characteristic features of both cellular and acellular disease. These results may have significant implications for the diagnosis and treatment of this rare entity.

Interruption of TRPC6-NFATC1 signaling inhibits NADPH oxidase 4 and VSMCs phenotypic switch in intracranial aneurysm

Intracranial aneurysm (IA) is a frequent cerebrovascular disorder with unclear pathogenesis. The vascular smooth muscle cells (VSMCs) phenotypic switch is essential for IA formation. It has been reported that Ca2+ overload and excessive reactive oxygen species (ROS) are involved in VSMCs phenotypic switch. The transient receptor potential canonical 6 (TRPC6) and NADPH oxidase 4 (NOX4) are the main pathway to participate in Ca2+ overload and ROS production in VSMCs. Ca2+ overload can activate calcineurin (CN), leading to nuclear factor of activated T cell (NFAT) dephosphorylation to regulate the target gene's transcription. We hypothesized that activation of TRPC6-NFATC1 signaling may upregulate NOX4 and involve in VSMCs phenotypic switch contributing to the progression of IA. Our results showed that the expressions of NOX4, p22phox, p47phox, TRPC6, CN and NFATC1 were significantly increased, and VSMCs underwent a significant phenotypic switch in IA tissue and cellular specimens. The VIVIT (NFATC1 inhibitor) and BI-749327 (TRPC6 inhibitor) treatment reduced the expressions of NOX4, p22phox and p47phox and the production of ROS, and significantly improved VSMCs phenotypic switch in IA rats and cells. Consistent results were obtained from IA Trpc6 knockout (Trpc6-/-) mice. Furthermore, the results also revealed that NFATC1 could regulate NOX4 transcription by binding to its promoter. Our findings reveal that interrupting the TRPC6-NFATC1 signaling inhibits NOX4 and improves VSMCs phenotypic switch in IA, and regulating Ca2+ homeostasis may be an important therapeutic strategy for IA.

Lossless Immunocytochemistry Based on Large-Scale Porous Hydrogel Pellicle for Accurate Rare Cell Analysis.

Rare cells, such as circulating tumor cells or circulating fetal cells, provide important information for the diagnosis and prognosis of cancer and prenatal diagnosis. Since undercounting only a few cells can lead to significant misdiagnosis and incorrect decisions in subsequent treatment, it is crucial to minimize cell loss, particularly for rare cells. Moreover, the morphological and genetic information on cells should be preserved as intact as possible for downstream analysis. The conventional immunocytochemistry (ICC), however, fails to meet these requirements, causing unexpected cell loss and deformation of the cell organelles which may mislead the classification of benign and malignant cells. In this study, a novel ICC technique for preparing lossless cellular specimens was developed to improve the diagnostic accuracy of rare cell analysis and analyze intact cellular morphology. To this end, a robust and reproducible porous hydrogel pellicle was developed. This hydrogel encapsulates cells to minimize cell loss from the repeated exchange of reagents and prevent cell deformation. The soft hydrogel pellicle allows stable and intact cell picking for further downstream analysis, which is difficult with conventional ICC methods that permanently immobilize cells. The lossless ICC platform will pave the way for robust and precise rare cell analysis toward clinical practice.

Single-virus tracking with quantum dots in live cells.

Single-virus tracking (SVT) offers the opportunity to monitor the journey of individual viruses in real time and to explore the interactions between viral and cellular structures in live cells, which can assist in characterizing the complex infection process and revealing the associated dynamic mechanisms. However, the low brightness and poor photostability of conventional fluorescent tags (e.g., organic dyes and fluorescent proteins) greatly limit the development of the SVT technique, and challenges remain in performing multicolor SVT over long periods of time. Owing to the outstanding photostability, high brightness and narrow emission with tunable color range of quantum dots (QDs), QD-based SVT (QSVT) enables us to follow the fate of individual viruses interacting with different cellular structures at the single-virus level for milliseconds to hours, providing more accurate and detailed information regarding viral infection in live cells. So far, the QSVT technique has yielded spectacular achievements in uncovering the mechanisms associated with virus entry, trafficking and egress. Here, we provide a detailed protocol for QSVT implementation using the viruses that we have previously studied systematically as an example. The specific procedures for performing QSVT experiments in live cells are described, including virus preparation, the QD labeling strategies, imaging approaches, image processing and data analysis. The protocol takes 1-2 weeks from the preparation of viruses and cellular specimens to image acquisition, and 1 d for image processing and data analysis.

An institutional experience with DICER1 mutated thyroid nodules-evaluating the cytomorphology and molecular phenotype.

DICER1 mutated thyroid nodules are commonly seen in pediatric populations often, as part of DICER1 syndrome. We seek to evaluate DICER1 mutated thyroid nodules in adult populations to assess whether there exists distinctive clinical, cytologic, histologic, and molecular characteristics that underline our institutional cohort. Retrospective analysis was performed on all fine-needle aspiration (FNA) specimens with a corresponding ThyroSeq panel, to select a cohort of cases with DICER1 mutations. Clinical, radiologic, and cytology materials were reviewed, and histology was reviewed for corresponding resection cases were available. ThyroSeq panel was further scrutinized for additional molecular alterations and variant allele frequency. DICER1 mutated thyroid nodules (n = 8), more commonly occurred in younger adults (P = 0.01) with larger (P = 0.01) nodules and only in female patients in our cohort. FNA commonly demonstrates cellular specimens with banal cytomorphologic cues including regular nuclei, inconspicuous nucleoli, smooth nuclear membranes, and abundant colloid. On retrospective review by 2 cytopathologists, the lesions were frequently diagnosed as Bethesda II (5 of 8) by both reviewers. Histology, when available, showed that all nodules were categorized as follicular adenomas (5 of 5), often demonstrating macrofollicles with papillary excrescences demonstrating bland nuclei (4 of 5). DICER1 mutational profile revealed a variant allele frequency of >40% in 25% of cases (2 of 8) and >30% in an additional 4 cases, highlighting a possible germline association. DICER1 mutated nodules may be under-reported due to banal cytomorphologic features and may be associated with an underlying germline alteration.

Intraductal papillary neoplasm of the bile duct: Cytomorphologic and molecular features.

Intraductal papillary neoplasm of the bile duct (IPNB) is a rare premalignant neoplasm that can progress to invasive adenocarcinoma. In this retrospective study, cases of IPNB were reviewed to examine cytomorphologic and molecular features. IPNB cytology cases with histopathologic confirmation were retrieved from the pathology archives. The cytomorphologic features such as cellularity, architecture, cell type, and cellular details were analyzed. The cohort included 13 cases (six brushings, six fine-needle aspirations [FNA], and one combined brushing and FNA). The lesions involved common bile duct in nine cases (69%) and hepatic duct in four cases (31%). Original cytological diagnoses included adenocarcinoma (five, 38%), suspicious for adenocarcinoma (one, 8%), neoplasm (three, 23%), atypical (three, 23%), and reactive (one, 8%). The cytomorphologic features included moderate/high cellularity (12, 92%), papillary and/or complex papillary architecture (10, 77%), columnar cells (11, 85%), vacuolated cytoplasm (12, 92%), enlarged nuclei (13, 100%), and fine granular chromatin (12, 92%). Background mucin, necrosis and acute inflammation were seen in four (31%), four (31%), and two (15%) cases, respectively. KRAS testing was performed in nine cases with mutant KRAS found in five (56%). Our study demonstrated that IPNB cytology specimens were relatively cellular with a wide spectrum of cytomorphology; however, most cases harbored adenocarcinoma or high-grade dysplasia. The characteristic cytomorphologic features included papillary/complex papillary clusters of columnar cells with vacuolated cytoplasm, enlarged nuclei, and fine granular chromatin in relatively cellular specimens. KRAS mutations identified may have potential diagnostic and therapeutic implications.

Crashworthiness design of graded cellular materials: Experimental verification of the backward design strategy

Graded cellular materials with a suitable design may improve the crashworthiness of protective structures. A backward design strategy considering the loading-rate sensitivity of cellular material has been proposed in the companion paper to guide the crashworthiness design of graded cellular material for protecting a moving object. Detailed experimental results are presented in this study to verify the feasibility and estimate the accuracy of this design strategy. A rate-dependent, rigid–plastic hardening (D-R–PH) idealization was employed to model cellular materials. A two-specimen test method was developed to determine the dynamic material parameters and their relationship with the relative density for the crashworthiness design. A power-law relationship between the dynamic material parameters and the relative density for the closed-cell foam with acrylonitrile butadiene styrene (ABS) plastic as the matrix material was obtained and employed in the crashworthiness design. Additive manufacturing was employed to fabricate the designed graded cellular specimens with ABS plastic, and the Taylor–Hopkinson pressure bar experimental technique together with high-speed photography was applied to perform impact tests. Graded ABS closed-cell foam specimens for different design conditions, e.g., the initial impact velocity, impact mass, and design target of impact force, were designed by the D-R–PH backward design strategy and prepared, and the corresponding impact tests were conducted. The impact force applied on the mass remains relatively stable and meets the crashworthiness requirement in all test cases. Therefore, the feasibility and reliability of the crashworthiness backward design strategy are verified, and this design strategy can be used to guide the crashworthiness design in engineering practice.

Prognostic value of minimal residual disease among patients with classical Hodgkin lymphoma undergoing autologous stem cell transplantation

Improved biomarkers are needed to guide patient selection for autologous stem cell transplantation (ASCT) and post-ASCT maintenance therapies in relapsed/refractory classical Hodgkin lymphoma (cHL). To assess the prognostic value of minimal residual disease (MRD) using immunoglobulin-based high-throughput sequencing (Ig-HTS), we analyzed pre- and post-ASCT peripheral blood and pre-ASCT apheresis stem cell (ASC) samples in 36 cHL patients. A tumor clonotype was detected in only 12 patients (33%). Among these patients, MRD within plasma samples was closely associated with impending relapse. All patients (n = 3) with detectable MRD in any post-ASCT plasma sample relapsed (100% specificity), and MRD was not detected in any patients in remission. MRD testing from cellular specimens (peripheral blood mononuclear cell or ASC samples) was not associated with relapse. In this small cohort, plasma-based MRD testing appeared to be a promising biomarker in cHL, but given low clonotype detection rates with Ig-HTS, alternative MRD approaches should be investigated.

Hydrogel Stamping for Rapid, Multiplexed, Point-of-Care Immunostaining of Cells and Tissues.

In the era of precision oncology, multicolor fluorescence imaging has become a core technology for multiplexed molecular analysis of cellular and tissue specimens. However, conventional solution-based staining is labor-intensive and time-consuming and requires considerable expertise to yield optimal results, which creates difficulties for employing this technology in resource-limited settings. Here, we report a new immunostaining method based on hydrogel stamping, which is simple, fast, easy to use, and reproducible. We showed that a hydrophilic hydrogel stamp could effectively transfer fluorescent antibodies to targets and withdraw an excess solution when the reaction is completed, obviating the need for extra washing. This unique property allows for quality immunostaining in 5 min for cells using one-eighth of antibody consumption compared to the conventional solution-based method. Furthermore, we implemented fluorescence quenching and immunocycling with hydrogel staining for multiplexed analysis of 9 protein markers at a single cell level. Finally, we applied the immunocycling method to human breast cancer tissue samples and showed quality immunostaining over a large area (∼2 cm2) in 30 min for molecular subtyping of breast cancer. The hydrogel immunostaining could open new opportunities for rapid, automated, and multiplexed profiling in compact point-of-care systems for molecular cancer diagnosis.

CelLock TM: an innovative standardized cell-block preparation procedure

ABSTRACT The CelLock™ procedure kit is used to collect and prepare cellular specimens such as fine needle aspirates (FNA), cytology specimens, cultured cells, small tissue biopsies, and samples with scant tissue fragments or cells into a paraffin cell-block. This cell-block can be used for subsequent microtomy and staining using hematoxylin and eosin (H&E), special stains, immunohistochemistry (IHC), and applicable molecular techniques such as in situ hybridization (ISH). CelLock is a standardized method that provides optimal receipt, preservation, preparation, and processing of cell-blocks which, contain virtually all of the submitted specimens and are able to be embedded and sectioned in a reproducible fashion. The specimen contained within the cell-block is preserved such that all the cellular protein and genetic information is available for histological and ancillary testing.

The Effect of Different Modifying Methods on Physical, Mechanical and Thermal Performance of Cellular Geopolymers as Thermal Insulation Materials for Building Structures

Geopolymers represent a new class of inorganic materials that have great potential for practical application due to the properties of used raw materials, as well as the peculiarities of the cementitious matrix structure formed during the geopolymerization process. Cellular geopolymer specimens were produced in this study using class F fly ash product, which is characterized by low reactivity during geopolymerization. Several standard methods, as well as microstructural studies were applied to evaluate the effect of the following factors on the physical-mechanical and thermophysical characteristics of cellular geopolymers: the use of various mineral modifying components for synthesis of geopolymer systems; high-temperature treatment; the introduction method of alkaline activator. It was observed that “ageing” an aqueous alkali solution for 24 h before mixing with fly ash and foam agent was able to provide a boost of compressive strength of cellular geopolymer specimens up to about 2.5 times, while decreasing the average density by about 28% for all experimental mixes, except for PC-modified mixes. Additionally, high-temperature treatment at 600 °C enables an enhanced strengthening effect of pore structure in cellular geopolymer matrix up to 1.5 times. This phenomenon is especially pronounced for the mixes with 24 h “aged” alkaline solution with exception for PC-modified mixes; for those, high-temperature treatment at 600 °C leads to strength decrease up to 40%. The introduction method of alkaline activator and high-temperature treatment showed a controversial effect on thermal conductivity coefficient depending on the mineral modifying component used for the synthesis of cellular geopolymers. The proposed method for calculation of total porosity of cellular structure of geopolymers as a polycomponent material demonstrated a high degree of correlation with the R2 value of at least 0.96 between the average density and the calculated total porosity. However, a low degree of correlation with R2 not exceeding 0.29 was observed for the measured nanoporosity, regardless of the introduction method of alkaline activator and high-temperature treatment.

A quantitative analysis of thyroid fine needle aspiration (FNA) using needles with different gauges.

The aim of this study is to compare the results of three gauge (G) needles (22G, 23G and 25G) in terms of cell amount in thyroid fine needle aspiration (FNA). In the retrospective study, a total of 443 patients undergoing FNA for the first time between 2017 and 2018 were included in the study, and assigned to 3 groups with 22-gauge, 23-gauge and 25-gauge needles, respectively. The cell amount of a suspicion for the four diagnosis groups, including malignancy and malignant, benign nodules, follicular of undetermined significance (FLUS), and follicular neoplasia was mainly in the range of 0-10000, 0-300, 0-150, and 500-2500, respectively. The cut-off values of 22G needle 20000, 300, 1000, and 2500, while the cut-off values of 23G and 25G were 10000, 400, 1000, and 2500; 5000, 400, 1500, and 2000, respectively for the four diagnosis groups. Large-gauge needles resulted in more cellular specimens than small-gauge needles only in the cases of malignant tumors. Small-gauge needles resulted in a higher comfort level of the patients, and had no difference in cell number in nodules with abundant blood supply, compared with large-gauge needles.