Lensless imaging-based discrimination between tumour cells and blood cells towards circulating tumour cell cultivation.

Circulating tumour cells (CTCs) are recognized as important markers for cancer research. Nonetheless, the extreme rarity of CTCs in blood samples limits their availability for multiple characterization. The cultivation of CTCs is still technically challenging due to the lack of information of CTC proliferation, and it is difficult for conventional microscopy to monitor CTC cultivation owing to low throughput. In addition, for precise monitoring, CTCs need to be distinguished from the blood cells which co-exist with CTCs. Lensless imaging is an emerging technique to visualize micro-objects over a wide field of view, and has been applied for various cytometry analyses including blood tests. However, discrimination between tumour cells and blood cells was not well studied. In this study, we evaluated the potential of the lensless imaging system as a tool for monitoring CTC cultivation. Cell division of model tumour cells was examined using the lensless imaging system composed of a simple setup. Subsequently, we confirmed that tumour cells, JM cells (model lymphocytes), and erythrocytes exhibited cell line-specific patterns on the lensless images. After several discriminative parameters were extracted, discrimination between the tumour cells and other blood cells was demonstrated based on linear discriminant analysis. We also combined the highly efficient CTC recovery device, termed microcavity array, with the lensless-imaging to demonstrate recovery, monitoring and discrimination of the tumour cells spiked into whole blood samples. This study indicates that lensless imaging can be a powerful tool to investigate CTC proliferation and cultivation.

Colony Fingerprint-Based Discrimination of Staphylococcus species with Machine Learning Approaches.

Detection and discrimination of bacteria are crucial in a wide range of industries, including clinical testing, and food and beverage production. Staphylococcus species cause various diseases, and are frequently detected in clinical specimens and food products. In particular, S. aureus is well known to be the most pathogenic species. Conventional phenotypic and genotypic methods for discrimination of Staphylococcus spp. are time-consuming and labor-intensive. To address this issue, in the present study, we applied a novel discrimination methodology called colony fingerprinting. Colony fingerprinting discriminates bacterial species based on the multivariate analysis of the images of microcolonies (referred to as colony fingerprints) with a size of up to 250 µm in diameter. The colony fingerprints were obtained via a lens-less imaging system. Profiling of the colony fingerprints of five Staphylococcus spp. (S. aureus, S. epidermidis, S. haemolyticus, S. saprophyticus, and S. simulans) revealed that the central regions of the colony fingerprints showed species-specific patterns. We developed 14 discriminative parameters, some of which highlight the features of the central regions, and analyzed them by several machine learning approaches. As a result, artificial neural network (ANN), support vector machine (SVM), and random forest (RF) showed high performance for discrimination of theses bacteria. Bacterial discrimination by colony fingerprinting can be performed within 11 h, on average, and therefore can cut discrimination time in half compared to conventional methods. Moreover, we also successfully demonstrated discrimination of S. aureus in a mixed culture with Pseudomonas aeruginosa. These results suggest that colony fingerprinting is useful for discrimination of Staphylococcus spp.

Line image sensor-based colony fingerprinting system for rapid pathogenic bacteria identification.

The rapid identification of pathogenic bacteria is crucial across various industries, including food or beverage manufacturing. Bacterial microcolony image-based classification has emerged as a promising approach to expedite identification, automate inspections, and reduce costs. However, conventional imaging methods have significant practical limitations, namely low throughput caused by the limited imaging range and slow imaging speed. To address these challenges, we developed an imaging system based on a line image sensor for rapid and wide-field imaging compared to existing colony imaging methods. This system can image a standard Petri dish (92 mm in diameter) completely within 22 s, successfully acquiring bacterial microcolony images. This process yielded a set of discrimination parameters termed as colony fingerprints, which were employed for machine learning. We demonstrated the performance of our system by identifying Staphylococcus aureus in food products using a machine learning model trained on a colony fingerprint dataset of 15 species from 9 genera, including foodborne pathogens. While conventional mass spectrometry-based methods require 24 h of incubation, our colony fingerprinting approach achieved 96% accuracy in just 10 h of incubation. Line image sensor offer high imaging speeds and scalability, allowing for swift and straightforward microbiological testing, eliminating the need for specialized expertise and overcoming the limitations of conventional methods. This innovation marks a transformative shift in industrial applications.

Rapid discrimination of fungal species by the colony fingerprinting.

The contamination of foods and beverages by fungi is a severe health hazard. The rapid identification of fungi species in contaminated goods is important to avoid further contamination. To this end, we developed a fungal discrimination method based on the bioimage informatics approach of colony fingerprinting. This method involves imaging and visualizing microbial colonies (referred to as colony fingerprints) using a lens-less imaging system. Subsequently, the quantitative image features were extracted as discriminative parameters and subjected to analysis using machine learning approaches. Colony fingerprinting has been previously found to be a promising approach to discriminate bacteria. In the present proof-of-concept study, we tested whether this method is also useful for fungal discrimination. As a result, 5 fungi belonging to the Aspergillus, Penicilium, Eurotium, Alternaria, and Fusarium genera were successfully discriminated based on the extracted parameters, including the number of hyphae and their branches, and their intensity distributions on the images. The discrimination of 6 closely-related Aspergillus spp. was also demonstrated using additional parameters. The cultivation time required to generate the fungal colonies with a sufficient size for colony fingerprinting was less than 48 h, shorter than those for other discrimination methods, including MALDI-TOF-MS. In addition, colony fingerprinting did not require any cumbersome pre-treatment steps prior to discrimination. Colony fingerprinting is promising for the rapid and easy discrimination of fungi for use in the ensuring the safety of food manufacturing.

Electrochemical detection of HbA1c, a marker [correction of maker] for diabetes, using a flow immunoassay system.

An on-chip electrochemical flow immunoassay system for the detection of hemoglobin A1c (HbA1c) was developed using anti-human hemoglobin (Hb) IgG labeled with ferrocene monocarboxylic acid (Fc-COOH) and boronate-affinity chromatography. An on-chip column packed with boronate-activated agarose beads was used for the separation of HbA1c from both non-glycated Hb and free antibody. Anti-human Hb IgG conjugated to Fc-COOH (Fc-IgG) was used for the electrochemical detection of HbA1c. The assay procedure included immunoreactions with Fc-IgG and HbA1c, separation of immunocomplexes by boronate affinity, and electrochemical detection of Fc-IgG-HbA1c immunocomplexes. The immunoreaction mixtures were injected onto a boronate-affinity column. HbA1c-antibody complexes were then trapped onto the column by the affinity of HbA1c to boronic acid. Subsequently, elution buffer containing sorbitol was applied to elute HbA1c-antibody complexes and a current was detected by applying 600 mV versus Ag/AgCl. The elution signal was an estimation of the HbA1c amount. A linear correlation between the increase of current and HbA1c concentration was obtained up to an HbA1c concentration of 500 microg/ml. The HbA1c flow immunoassay was successfully achieved using hemolysates. This electrochemical flow immunoassay system enabled us to construct a novel point-of-care testing device for the monitoring of glycated proteins including HbA1c.

Clinicopathologic features and treatment outcomes in malignant lymphoma of pediatric and young adult patients in Korea: comparison of korean all-ages group and Western younger age group.

PURPOSE: The aim of this study is to define distinctive clinicopathologic features of malignant lymphoma in pediatric and young adult patients, particularly in Korea. PATIENTS AND METHODS: From May 1993 to November 2005, 294 pediatric and young adult patients (age range, 0-31 years) with malignant lymphoma were analyzed in this study at Samsung Medical Center. We also compared this group with the Korean all-ages group and Western younger age group using previously reported data. RESULTS: Hodgkin disease appears more common in the younger age group than in the all-ages group (15% vs. 5.3%; P = .001). Among patients with non-Hodgkin lymphoma (NHL), T/natural killer cell immunophenotype is more common in the present younger age group than the all-ages group (45.5% vs. 25%; P = .001) and Western younger age group (45.5% vs. 13.3%; P = .001). Lymphoblastic lymphoma and T-anaplastic large-cell lymphoma included relatively higher proportions in the younger age group. Overall survival for patients in the group aged 21-31 years was significantly inferior to that of the other younger age group (P = .014). CONCLUSION: The incidence of Hodgkin disease and T-cell NHL is relatively higher in pediatric and young-adult population group than the all-ages group. However, treatment outcome of the younger age group, excluding lymphoblastic lymphoma, seems to be similar to those in any age group.

Colony fingerprint for discrimination of microbial species based on lensless imaging of microcolonies.

Detection and identification of microbial species are crucial in a wide range of industries, including production of beverages, foods, cosmetics, and pharmaceuticals. Traditionally, colony formation and its morphological analysis (e.g., size, shape, and color) with a naked eye have been employed for this purpose. However, such a conventional method is time consuming, labor intensive, and not very reproducible. To overcome these problems, we propose a novel method that detects microcolonies (diameter 10-500 µm) using a lensless imaging system. When comparing colony images of five microorganisms from different genera (Escherichia coli, Salmonella enterica, Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans), the images showed obvious different features. Being closely related species, St. aureus and St. epidermidis resembled each other, but the imaging analysis could extract substantial information (colony fingerprints) including the morphological and physiological features, and linear discriminant analysis of the colony fingerprints distinguished these two species with 100% of accuracy. Because this system may offer many advantages such as high-throughput testing, lower costs, more compact equipment, and ease of automation, it holds promise for microbial detection and identification in various academic and industrial areas.

Automated DNA extraction from genetically modified maize using aminosilane-modified bacterial magnetic particles.

A novel, automated system, PNE-1080, equipped with eight automated pestle units and a spectrophotometer was developed for genomic DNA extraction from maize using aminosilane-modified bacterial magnetic particles (BMPs). The use of aminosilane-modified BMPs allowed highly accurate DNA recovery. The (A(260)-A(320)):(A(280)-A(320)) ratio of the extracted DNA was 1.9+/-0.1. The DNA quality was sufficiently pure for PCR analysis. The PNE-1080 offered rapid assay completion (30 min) with high accuracy. Furthermore, the results of real-time PCR confirmed that our proposed method permitted the accurate determination of genetically modified DNA composition and correlated well with results obtained by conventional cetyltrimethylammonium bromide (CTAB)-based methods.

Extranodal Marginal Zone B-Cell Lymphoma of Mucosa-Associated Tissue Type Involving the Dura.

Primary central nervous system marginal zone B-cell lymphoma (MZBCL) is very rare, with only a few reported cases worldwide. It has an indolent disease course with high cure potential. We experienced a rare case of dural MZBCL of mucosa-associated lymphoid tissue (MALT) in a 69-year-old man who presented with headache. A magnetic resonance imaging scan of brain showed a 1.9×3.6-cm-sized extra-axial mass with a broad based dural attachment to the anterosuperior aspect of the falx cerebri, radiographically consistent with meningioma. Surgical resection yielded a MZBCL of the MALT type. Histopathology revealed a lymphoplasmacytic infiltration of the dura, and immunohistochemical study showed a B-cell phenotype with CD20, bcl-2, MUM-1, Ki-67 positive. He was treated with chemotherapy after complete surgical resection and remained free of disease at 30 months after chemotherapy. MALT lymphoma must be considered in the differential diagnosis in patients presenting radiographically with meningioma.

Simple and rapid CD4 testing based on large-field imaging system composed of microcavity array and two-dimensional photosensor.

This study presents a novel method for CD4 testing based on one-shot large-field imaging. The large-field imaging system was fabricated by a microcavity array and a two-dimensional (2D) photosensor within the desk-top-sized instrument. The microcavity array was employed to separate leukocytes from whole blood based on differences in the size of leukocytes and other blood cells. The large-field imaging system with lower side irradiation enabled acquisition of cell signatures with high signal-to-noise ratio, because the metallic substrate of the microcavity array obstructed excessive excitation light. In this setting, dual-color imaging of CD4(+) and CD8(+) T cells was achieved within the entire image area (64 mm(2)) in 2s. The practical performance of the large-field imaging system was demonstrated by determining the CD4/CD8 ratio in a few microliter of control whole blood as small as those obtained by a finger prick. The CD4/CD8 ratios measured using the large-field imaging system correlated well with those measured by microscopic analysis. These results indicate that our proposed system provides a simple and rapid CD4 testing for the application of HIV/AIDS treatment.

Digital cell counting device integrated with a single-cell array.

In this paper, we present a novel cell counting method accomplished using a single-cell array fabricated on an image sensor, complementary metal oxide semiconductor sensor. The single-cell array was constructed using a microcavity array, which can trap up to 7,500 single cells on microcavities periodically arranged on a plane metallic substrate via the application of a negative pressure. The proposed method for cell counting is based on shadow imaging, which uses a light diffraction pattern generated by the microcavity array and trapped cells. Under illumination, the cell-occupied microcavities are visualized as shadow patterns in an image recorded by the complementary metal oxide semiconductor sensor due to light attenuation. The cell count is determined by enumerating the uniform shadow patterns created from one-on-one relationships with single cells trapped on the microcavities in digital format. In the experiment, all cell counting processes including entrapment of non-labeled HeLa cells from suspensions on the array and image acquisition of a wide-field-of-view of 30 mm(2) in 1/60 seconds were implemented in a single integrated device. As a result, the results from the digital cell counting had a linear relationship with those obtained from microscopic observation (r(2)  = 0.99). This platform could be used at extremely low cell concentrations, i.e., 25-15,000 cells/mL. Our proposed system provides a simple and rapid miniaturized cell counting device for routine laboratory use.

On-chip type cation-exchange chromatography with ferrocene-labeled anti-hemoglobin antibody and electrochemical detector for determination of hemoglobin A1c level.

An on-chip type cation-exchange chromatography system with electrochemical detection of HbA(1c), which is one of the most important diabetes marker protein, was developed using ferrocene-conjugated anti-human hemoglobin (Hb) monoclonal antibody (FcAb). The FcAb was used as an electrochemical probe for the detection of each Hb. The system contains syringe pump, on-chip type cation-exchange column consisted of PDMS and cation-exchange resin beads, and a three-electrode flow-cell system. The separation conditions of HbA(1c) in blood calibrator samples from other Hbs, e.g. HbA(0), HbA(1a) or HbA(1b), were optimized using the on-chip type system. The electrochemical oxidation current from FcAb reacting with each Hb was measured at 350 mV (vs. Ag/AgCl). Hbs including HbA(1a) and HbA(1b), HbA(1c) and HbA(0) fractions were eluted in this order. A linear relationship between HbA(1c) levels and electrochemical oxidation currents was obtained in the range from 4.0% to 12.6% HbA(1c). All procedure including antigen-antibody reaction was completed in 15 min. Furthermore, a good correlation was obtained between KO500 method (HPLC) and our proposed method. These results indicate that the on-chip type system with electrochemical detection can be applied to a novel POCT device for rapid and precise detection of HbA(1c).

Changes in serologic markers of hepatitis B following autologous hematopoietic stem cell transplantation.

Korea is an endemic area for hepatitis B virus (HBV) infection. Reactivation of HBV is a well-recognized complication in patients with chronic HBV infection undergoing cytotoxic or immunosuppressive therapy, and there are some reports of hepatitis B reverse seroconversion after HSCT. This study evaluated changes in HBV serology after HSCT. We reviewed the medical records of 141 patients who had available HBV serologic data after autologous HSCT. Patient information was retrospectively collected from the BMT database. Before transplantation, 12 patients were positive for hepatitis B surface antigen (HBsAg) and received lamivudine prophylaxis. There was 1 case of reactivation of HBV among these patients. One hundred twenty-nine patients were negative for HBsAg before HSCT, of whom 110 were positive and 19 were negative for hepatitis B surface antibody (anti-HBs). Sixty-two of the 110 patients who were positive for anti-HBs were also positive for hepatitis B core antibody (anti-HBc). Eight patients were negative for anti-HBs and anti-HBc. Seven patients who were initially negative for HBsAg were identified as positive after HSCT, and 5 of those 7 patients developed acute hepatitis, thus indicating reverse seroconversion. Univariate analysis showed that reverse seroconversions were observed more frequently with multiple myeloma than another disease (P = .005; relative risk, 11.854; 95% confidence interval, 1.381-101.770). Other factors, such as age, sex, and presence of HBcAb before HSCT, had no statistically significant affect on reverse seroconversion. In conclusion, reverse seroconversion of HBV is not a rare complication of autologous HSCT, and the risk of reverse seroconversion after treatment is a serious concern due to possible complications arising from patients' suppressed immune systems.

Disinfection of microorganisms by use of electrochemically regenerated periodate.

A new method for disinfection of microorganisms by electrochemically regenerated periodate was developed. Oxidation of iodate to periodate was observed at 1.25 V versus a silver/silver chloride electrode in a cyclic voltammogram of potassium iodate. When 1.25 V was applied in 1.0 mM potassium iodate, approximately 4-log inactivation of Escherichia coli was observed in 30 min.

Microfabricated on-chip-type electrochemical flow immunoassay system for the detection of histamine released in whole blood samples.

This paper describes an on-chip-type electrochemical flow immunoassay system with a multichanneled matrix column. The multichanneled matrix column was functionally coated with cation-exchange resin and used for separation of proteins. Antihistamine immunoglobulin G (IgG) antibody conjugated with ferrocenemonocarboxylic acid (Fc) was also prepared and used as a novel analytical reagent. Antibody-antigen complexes were separated from free Fc-conjugated IgG antibody (Fc-IgG) on the basis of differences in isoelectric point (pI) using the multichanneled matrix column coated with cation-exchange resin. The assay yields a good relationship between current and histamine concentration in the range of 200-2000 ng/mL. This simple technique enables the assay of histamine released in whole blood within 2 min. Furthermore, a good correlation was found between the response of the electrochemical immunoassay described in this paper and the conventional RIA (radioimmunoassay). This on-chip-type electrochemical flow immunoassay requires only minute quantities of whole blood samples and generates highly reproducible results.

Miniaturized amperometric flow immunoassay system using a glass fiber membrane modified with anion.

This paper describes a miniaturized amperometric flow immunoassay system using a glass fiber membrane modified with anion. The glass fiber membrane was functionally modified with gamma-glycidoxypropyltrimethoxysilane and sodium thiosulfate and was used for separation of protein. Anti-human chorionic gonadotrophin (HCG) immunoglobulin G (IgG) antibody conjugated with ferrocenemonocarboxylic acid (Fc), namely, Fc-conjugated IgG (Fc-IgG), was used as a novel analytical reagent. HCG and Fc-IgG complexes were separated from free Fc-IgG based on differences in isoelectric point (pI) using the glass fiber membrane modified with a thiosulfonyl acid functional group. The assay yields a linear relationship between current and HCG concentration in the range of 0-2000 mIU/mL. This simple technique enables the assay of HCG within 2 min. The modified glass fiber membrane was regenerated by occasional elution with malonate buffer (pH 6.0) containing 0.5 M NaCl, to remove free Fc-IgG. Free Fc-IgG recovered in this manner could be reused up to eight times without significant decreases in sensitivity. This miniaturized amperometric flow immunoassay requires only minute quantities of serum and generates highly reproducible results.

Preparation of a colored conductive paint electrode for electrochemical inactivation of bacteria.

In this study we describe the preparation of a colored conductive paint electrode containing In(2)O(3), SnO(2), or TiO(2) for the electrochemical inactivation of marine bacteria. When each colored conductive paint electrode was immersed in seawater containing 10(6) cells/mL for 90 min, marine microbe attachment to the TiO(2)/SnO(2)/Sb electrode surface was minimal. Preparation of electrodes coated with 40% particles is shown to be more cost-effective, and because of their more translucent coatings they can be painted over with bright colors. When a potential of 1.0 V was applied for 30 min to the colored conductive paint electrode (40 wt% TiO(2)/SnO(2)/Sb) in sterile seawater, the survival ratio decreased to 55%. When 1.5 V vs. saturated calomel electrode (SCE) was applied, all attached cells were inactivated. Chlorine was not detected below an applied potential of 1.5 V. A change in pH was not observed in the range of 0 to 1.5 V. This method might be effective for preventing bacterial cell accumulation and the formation of biofilms.