[Measurement of Liver Fibrosis Marker Targeting Sugar Chain Marker].

The degree of liver fibrosis progression is an important factor in hepatocarcinogenesis, and monitoring liver fibrosis is important for predicting and preventing hepatocellular carcinoma. It is proportional to the appearance of a new hepatitis C therapy, or the expectation of liver fibrosis therapy, and liver fibrosis research is attracting attention. Although the Gold Standard for the diagnosis of liver fibrosis is liver biopsy, various problems, such as in the difficulty of invasive and frequent measurement, exist. The present non-invasive examination methods for the assessment of liver fibrosis also have a problem in the fields of organ specificity and diagnostic performance. Using a fully automated immunoassay system "HISCL", an assay system based on the lectin bound sugar reaction which is not an antigen-bound antibody reaction was developed. Measurements using the fully automated immunoassay system "HISCL" series and HISCL M2BPGi assay kit facilitated rapid assay (17 minutes) with a small sample volume (10 µL). Serum M2BPGi values can be used in various ways, such as for assessment of the risk and treatment associated with hepatocellular carcinoma, reflecting the liver fibrosis stage. Furthermore, many studies are currently in progress. The development of a new assay system for the detection of a cancer production sugar chain marker is expected in the future owing to the advent of a lectin-bound sugar chain reaction system.

Heme-related molecules induce rapid production of neutrophil extracellular traps.

BACKGROUND: Pulmonary endothelial cell damages caused by neutrophil overactivation could result in acute lung injuries including transfusion-related acute lung injury (TRALI). We previously reported that heme-related molecules derived from hemolysis induced the production of reactive oxygen species from neutrophils. Recently, neutrophil extracellular traps (NETs) have been demonstrated to associate with the onset of TRALI. STUDY DESIGN AND METHODS: In this study, neutrophils' morphologic changes induced by the heme-related molecule hemin were confirmed to be NETs via confocal laser scanning microscopy and electron microscopy (EM). Additionally, concentrations of hemin in red blood cell (RBC) components were measured via enzyme-linked immunosorbent assay and possible contribution of these molecules to the onset of TRALI was discussed. RESULTS: SYTOX green staining observation via confocal laser scanning microscopy revealed that neutrophil morphology changed rapidly upon addition of hemin. The nuclei began to be enlarged and become segmented after 5 minutes, and NET-like structures were released from neutrophils after 15 minutes. In EM observation, NET-like structures appeared after 10 minutes and the nucleoplasm was partially separated from the nuclear membrane, which were consistent with the features of NET formation. These structures stained positively for both myeloperoxidase and histone H3 antibodies. CONCLUSION: Thus, our results suggest that hemin induced NETs in 15 minutes, a quicker reaction than NET induction by phorbol myristate acetate requiring 3 hours. Moreover, since RBC components, especially those with long-term storage, contained sufficient hemin concentration to induce NETs, special attention to hemolysis of stored RBC components is important.

Non-activated T and B lymphocytes become morphologically distinguishable after detergent treatment.

T and B lymphocytes are difficult to distinguish morphologically even with electron microscopy, and antibodies are generally used to make the distinction. A specific reagent, consisting of nonionic and cationic detergents, is used for leukocyte differentiation using the Sysmex automated blood analyzer. This reagent increases cell membrane porosity and enables the introduction of fluorescent dye into leukocytes. In this study, we investigated the effect of this specific detergent on the morphology of T and B lymphocytes. T and B lymphocytes were obtained by density gradient centrifugation and magnetic cell sorting, with a minimum of 90% isolation efficiency. T and B lymphocytes were then treated with the specific detergent and fluorescent dye, and their distribution was analyzed based on side scatter and fluorescence intensity using general-purpose flow cytometry (FCM). Fluorescent images were observed using a confocal laser scanning microscope (CLSM), cellular inner structures using a transmission electron microscopy (TEM), and cell surfaces using a scanning electron microscope (SEM). The ratio of cholesterol to total lipid in cell membranes of B and T lymphocytes was measured using a fluorescent assay kit. The distribution of fluorescence intensity was different between T and B lymphocyte clusters, according to the FCM analysis. CLSM observations revealed that the fluorescent dye mainly stained cytoplasmic organelles. FCM, TEM, and SEM observations revealed that B lymphocytes are more likely to lose surface antigens and intracellular organelles than T lymphocytes, which allows the visual distinction between T and B lymphocytes. The ratio of cholesterol to total lipid in T lymphocyte membranes had tendency higher than that in B lymphocyte membranes. In this study, we demonstrate that cells with differences in cell membrane cholesterol amounts, such as B and T lymphocytes could be identified using an inexpensive detergent, as an alternative to costly antibodies.

Morphological and flow-cytometric analysis of haemin-induced human neutrophil activation: implications for transfusion-related acute lung injury.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is associated with vascular endothelial cell injury following neutrophil activation. Recently, it has been suggested that haem-related molecules induce activation of neutrophils and that erythrocyte-derived substances contained in blood preparations are involved in TRALI. We observed the morphological effects and reactive oxygen species (ROS) production of haem-related molecules and investigated the effects of signal transduction inhibitors on haem-induced neutrophil activation. MATERIALS AND METHODS: The polymorphonuclear cell fraction was isolated and stimulated using a control stimulant, PMA or fMLP, or by haem-related molecules, haemin, ferric citrate, or protoporphyrin IX. After stimulation, neutrophil was analysed using electron microscopy, a flowcytometer (FCM) and confocal laser scanning microscope to determine the fluorescent intensity of aminophenyl fluorescein (to detect ROS). RESULTS: In FCM analysis, haemin and protoporphyrin IX, both of which have a porphyrin ring, induced ROS production in neutrophils. Ferric citrate, which has no porphyrin ring, did not induce neutrophil activation. Haemin alone induced ROS production at relatively high concentrations, whereas low-level fMLP acted as an agonist in the presence of low concentrations of haemin. Haem-related molecules induced ROS production in neutrophil granules through signal transduction in a way similar to PMA. However, in electron microscopy studies, haemin stimulated neutrophils showed minute process at their surface and did not show the vacuolation observable following stimulation with PMA or fMLP. DISCUSSION: We suggest that low concentrations of haem-related molecules with porphyrin rings in the presence of other stimulating agent are important for ROS production and possibly the onset of TRALI. The ROS production induced by these molecules is dependent on a signal transduction pathway in a way similar to PMA.

Rapid discrimination of Gram-positive and Gram-negative bacteria in liquid samples by using NaOH-sodium dodecyl sulfate solution and flow cytometry.

BACKGROUND: For precise diagnosis of urinary tract infections (UTI), and selection of the appropriate prescriptions for their treatment, we explored a simple and rapid method of discriminating gram-positive and gram-negative bacteria in liquid samples. METHODOLOGY/PRINCIPAL FINDINGS: We employed the NaOH-sodium dodecyl sulfate (SDS) solution conventionally used for plasmid extraction from Escherichia coli and the automated urine particle analyzer UF-1000i (Sysmex Corporation) for our novel method. The NaOH-SDS solution was used to determine differences in the cell wall structures between gram-positive and gram-negative bacteria, since the tolerance to such chemicals reflects the thickness and structural differences of bacterial cell walls. The UF-1000i instrument was used as a quantitative bacterial counter. We found that gram-negative bacteria, including E. coli, in liquid culture could easily be lysed by direct addition of equal volumes of NaOH-SDS solution. In contrast, Enterococcus faecalis, which is a gram-positive bacterium, could not be completely lysed by the solution. We then optimized the reaction time of the NaOH-SDS treatment at room temperature by using 3 gram-positive and 4 gram-negative bacterial strains and determined that the optimum reaction time was 5 min. Finally, in order to evaluate the generalizability of this method, we treated 8 gram-positive strains and 8 gram-negative strains, or 4 gram-positive and 4 gram-negative strains incubated in voluntary urine from healthy volunteers in the same way and demonstrated that all the gram-positive bacteria were discriminated quantitatively from gram negative bacteria using this method. CONCLUSIONS/SIGNIFICANCE: Using our new method, we could easily discriminate gram-positive and gram-negative bacteria in liquid culture media within 10 min. This simple and rapid method may be useful for determining the treatment course of patients with UTIs, especially for those without a prior history of UTIs. The method may be easily applied in order to obtain additional information for clinical prescriptions from bacteriuria.

[Scientific support for emerging countries--introduction to activities of a company].

Medical care cost per capita in developing countries is significantly low, only 1/100 of that of developed countries. In these countries, treatment comes first, without sufficient clinical testing. In emerging countries, on the other hand, economic growth increases medical care cost, enabling more clinical testing; however, in many cases, quality control is not sufficiently performed for economic and technical reasons. To promote evidence-based medical care, it is important to promote high-quality clinical testing in these countries. To do so, our company has provided 400 scientific documents in 17 languages. We have also held 49 scientific seminars in 10 countries, with more than 10,000 participants, to share the latest medical information. We especially focus on supporting the standardization and promotion of external quality control to improve the quality of clinical testing. To support the standardization of hematology testing in China, we set up an environment for the international standard method of blood cell counting, in 2002 for the organizer of an external quality assessment program, and in 2011 for an agency responsible for practical tests of medical equipments. We have also been supporting national external quality assessment programs since 1998, first in Thailand and now including the Philippines and Mongolia. For external quality assessment programs in these countries, we applied the approach shown by Japanese doctors and medical technologists in their effort for accuracy improvement. We believe the experience and knowledge of Japan can be utilized to support developing and emerging countries.