Establishing Monoclonal Gammopathy of Undetermined Significance as an Independent Pre-Disease State of Multiple Myeloma Using Raman Spectroscopy, Dynamical Network Biomarker Theory, and Energy Landscape Analysis.

Multiple myeloma (MM) is a cancer of plasma cells. Normal (NL) cells are considered to pass through a precancerous state, such as monoclonal gammopathy of undetermined significance (MGUS), before transitioning to MM. In the present study, we acquired Raman spectra at three stages-834 NL, 711 MGUS, and 970 MM spectra-and applied the dynamical network biomarker (DNB) theory to these spectra. The DNB analysis identified MGUS as the unstable pre-disease state of MM and extracted Raman shifts at 1149 and 1527-1530 cm-1 as DNB variables. The distribution of DNB scores for each patient showed a significant difference between the mean values for MGUS and MM patients. Furthermore, an energy landscape (EL) analysis showed that the NL and MM stages were likely to become stable states. Raman spectroscopy, the DNB theory, and, complementarily, the EL analysis will be applicable to the identification of the pre-disease state in clinical samples.

Loop-mediated isothermal amplification assay for fluorescence analysis and lateral flow detection of male DNA.

Male DNA screening is important in forensic investigations, such as sexual assault cases. Although quantitative real-time PCR is a robust method for detection of male DNA, it is time-consuming and labor-intensive. We herein report the development of a male DNA-targeted loop-mediated isothermal amplification (LAMP) assay that can be used for both laboratory-based fluorescence analysis and on-site lateral flow detection. The two detection systems are independent, but we streamlined the reaction before the detection by introducing a fluorescence probe and biotin-labeled primer into a single reaction. This allowed the evaluation of fluorescence signal followed by lateral flow detection. Both the fluorescence and lateral flow analyses detected as low as 10 pg of male DNA. We also integrated an alkaline lysis method with our LAMP assay. The direct assay successfully detected male DNA from forensic samples without purification. The workflow requires only <40 min for fluorescence analysis and <45 min for lateral flow detection. Furthermore, when combined with a lateral flow strip, this workflow does not require any sophisticated instruments. These findings suggest that our assay is a promising strategy for on-site male DNA screening as well as laboratory-based testing.

Sperm mRNA screening by reverse transcription-recombinase polymerase amplification (RT-RPA) assay: Decision-making for differential extraction.

The presence of sperm cells is an indicator for differential extraction on sexual assault samples. In general, sperm cells are identified by microscopic analysis; however, this conventional method takes time and effort, even for trained personnel. Here, we present a reverse transcription-recombinase polymerase amplification (RT-RPA) assay targeting sperm mRNA marker (PRM1). The RT-RPA assay requires only 40 min for PRM1 detection and demonstrates a sensitivity of 0.1 µL of semen. Our results indicate that the RT-RPA assay may be a rapid, simple, and specific strategy for screening sperm cells in sexual assault samples.

The FADS mouse: A novel mouse model of atopic keratoconjunctivitis.

BACKGROUND: Atopic keratoconjunctivitis (AKC) is a chronic allergic conjunctival disease. However, a mouse model of AKC to investigate the underlying mechanism of the therapeutic agents and estimate their efficacy has not been established. We recently generated mice in which Ikk2 is specifically deleted in facial skin fibroblasts and found that these mice spontaneously develop atopic dermatitis (AD)-like facial skin inflammation and scratching behaviors; thus, we named them facial AD with scratching (FADS) mice. OBJECTIVE: We sought to evaluate whether the ocular lesions that FADS mice spontaneously develop are similar to those of patients with AKC and to estimate the efficacy of topical treatments with tacrolimus and betamethasone for FADS mice by using tear periostin, a novel biomarker for allergic conjunctival disease. METHODS: FADS mice, in which Ikk2 is deleted in dermal fibroblasts, were generated by crossing female Ikk2Flox/Flox mice to male Nestincre; Ikk2Flox/+ mice. We conducted histologic analysis of the ocular lesions in FADS mice. Furthermore, we measured periostin in the tears collected from FADS mice untreated or treated with tacrolimus or betamethasone. RESULTS: The FADS mice exhibited severe blepharitis and scratch behaviors for their faces. In these mice, corneal epithelium and stroma showed hyperplasia and infiltration of eosinophils, mast cells, and TH2/TC2 cells. Periostin was significantly expressed in the lesions and tear periostin was upregulated. Betamethasone showed more suppressive effects than did tacrolimus on severe corneal lesions and increased tear periostin level. CONCLUSIONS: The FADS mouse is a novel mouse model of AKC and is useful to examine the therapeutic effects of anti-AKC agents.

Increased cerebrospinal fluid adenosine 5'-triphosphate in patients with amyotrophic lateral sclerosis.

BACKGROUND: Extracellular adenosine 5'-triphosphate (ATP) has been suggested to cause neuroinflammation and motor neuron degeneration by activating microglia and astrocytes in amyotrophic lateral sclerosis (ALS). Since we have developed a highly sensitive ATP assay system, we examined cerebrospinal fluid (CSF) ATP levels in patients with ALS whether it can be a useful biomarker in ALS. METHODS: Forty-eight CSF samples from 44 patients with ALS were assayed for ATP with a newly established, highly sensitive assay system using luciferase luminous reaction. CSF samples from patients with idiopathic normal pressure hydrocephalus (iNPH) were assayed as a control. Patients were divided into two groups depending on their disease severity, as evaluated using the Medical Research Council (MRC) sum score. Correlations between the CSF ATP levels and other factors, including clinical data and serum creatinine levels, were evaluated. RESULTS: CSF ATP levels were significantly higher in patients with ALS than in the iNPH (716 ± 411 vs. 3635 ± 5465 pmol/L, p < 0.01). CSF ATP levels were significantly higher in the more severe group than in the iNPH group (6860 ± 8312 vs. 716 ± 411 pmol/L, p < 0.05) and mild group (6860 ± 8312 vs. 2676 ± 3959 pmol/L, p < 0.05) respectively. ALS functional rating scale-revised (ALSFRS-R) (37.9 ± 5.7 vs. 42.4 ± 2.8, p < 0.01) and serum creatinine levels (0.51 ± 0.13 vs. 0.68 ± 0.23 mg/dL, p < 0.05) were significantly lower in the severe group than in the mild group respectively. A negative correlation of CSF ATP levels with MRC sum score was demonstrated in the correlation analysis adjusted for age and sex (r = -0.3, p = 0.08). CONCLUSIONS: Extracellular ATP is particularly increased in the CSF of patients with advanced ALS. CSF ATP levels may be a useful biomarker for evaluating disease severity in patients with ALS.

Infected simple renal cyst due to Streptococcus pneumoniae rapidly diagnosed by the melting temperature mapping method: a case report.

BACKGROUND: Spontaneous infection of preexisting solitary renal cysts has been documented in adults but is extremely rare in children. To date, no cases of simple renal cysts infected with Streptococcus pneumoniae have been described. Recently, reports have described the diagnosis of bacterial infection using the 16 S rRNA gene as well as the accompanying antimicrobial stewardship for microorganisms that are difficult to culture and for culture-negative cases after preceding antibacterial administration. CASE PRESENTATION: A four-year-old Japanese girl who had a pleuroperitoneal shunt inserted to drain a right pleural effusion due to occlusion of the hepatic portion of the inferior vena cava at three years old visited our hospital due to fever and respiratory discomfort. She was incidentally found to have a right simple renal cyst 10 months before admission. The patient was suspected to have pneumonitis or catheter-related blood stream infection on chest X-ray, which showed right-side pleural effusion. She was diagnosed with invasive pneumococcal infection, as Streptococcus pneumoniae was detected from blood culture on admission. Transient improvements in her symptoms and decreases in the white blood cell count and C-reactive protein level were observed after effective antibiotic administration, but her respiratory condition deteriorated. Enhanced CT showed right renal cyst enlargement and enhancement and thickening of the surrounding wall. Using the melting temperature (Tm) mapping method, S. pneumoniae was rapidly detected directly from pus 4.5 hours after drainage. The specimen culture was negative, but the extracted 16 S rDNA sequence revealed 100 % identity for S. pneumoniae from the same specimen the subsequent day. We successfully performed optimal treatment and reduced medical cost based on the positive Tm mapping method result. CONCLUSIONS: We report the first case of a S. pneumoniae-infected simple renal cyst. The drainage culture was negative, but the Tm mapping method rapidly detected S. pneumoniae directly from the drainage. The Tm mapping method may have great impacts on rapid diagnosis and effective antimicrobial stewardship.

Pyogenic liver abscess due to hypervirulent Klebsiella pneumoniae in a 14-year-old boy.

A 14-year-old otherwise healthy boy presented with right-sided back pain following high fever. Abdominal computed tomography scan showed a large liver abscess. Klebsiella pneumoniae (KP) was rapidly identified from peripheral blood using the melting temperature mapping (Tm) method, which enables identification of pathogenic microorganisms within four hours after patient sample collection. He was diagnosed with pyogenic liver abscess (PLA) caused by KP on the day of admission. The KP was the hypervirulent (hv) clinical variant (string test positive, serotype K1, sequence type 23, rmpA and magA positive). After intravenous antibiotic therapy and drainage of the abscess, his condition resolved. The highlights of this case report are a healthy child with hypervirulent Klebsiella pneumoniae liver abscess in Japan and the new Tm mapping method for rapid and accurate identification of the pathogenic microorganism.

Differential proteome analysis in adolescent idiopathic scoliosis patients with thoracolumbar/lumbar curvatures.

BACKGROUND: Although the pathogenesis of adolescent idiopathic scoliosis (AIS) remains unclear, there are little evidences of the pathogenesis in patients with thoracolumbar/lumbar AIS. The purpose of this study was to identify proteins or proteomes that may be causally related to the pathogenesis of AIS with structured thoracolumbar/lumbar curvature using two-dimensional fluorescence difference gel electrophoresis (2D-DIGE). METHODS: A total of 20 control volunteers and 61 AIS in patients with thoracolumbar/lumbar curvature were included. First, the plasma samples of each five AIS with pure thoracolumbar/lumbar curvature and control samples were subjected to 2D-DIGE analysis. Protein spots that were expressed differently by the AIS and control groups were selected and identified by nanoscale liquid chromatography-tandem mass spectrometry (nanoLC-MS/MS) analysis. To characterize the differently-expressed proteins in AIS patients, we performed functional pathway analysis using the Protein ANalysis THrough Evolutionary Relationships (PANTHER) system. Additionally, the proteins were compared between control and AIS using western blotting. Lastly, prospectively collected 15 control and 41 AIS with thoracolumbar/lumbar curvature samples were compared to the differentially expressed proteins. RESULTS: A total of 3862 ± 137 spots were detected, of which 11 spots met the criteria when compared with controls. Nine proteins were identified by nanoLC-MS/MS. Functional analysis showed the association of the proteins in AIS patients with blood coagulation using the PANTHER system. Of the proteins, vitamin D binding protein (DBP) significantly correlated with Cobb angle in thoracolumbar/lumbar curvatures. DBP expression of the prospectively collected AIS samples were significantly higher than those of controls (P < 0.05). CONCLUSIONS: This study suggests that DBP and several coagulation-related proteins may play a role in the pathogenesis of AIS. DBP appears to be a marker of severity of AIS with thoracolumbar/lumbar curvature.

Spondylodiscitis due to Parvimonas micra diagnosed by the melting temperature mapping method: a case report.

BACKGROUND: It has been suggested that more than 100 bacterial species can be identified using only seven universal bacterial primer sets in the melting temperature (Tm) mapping method and that these findings can be obtained within 3 h of sterile site collection. CASE PRESENTATION: A 67-year-old Japanese man with type 2 diabetes visited our hospital complaining of progressive lower back pain for 2 months. The patient was suspected to have spondylodiscitis on magnetic resonance imaging of the spine. Blood culture and transcutaneous vertebral biopsy were subsequently performed. Using the Tm mapping method, Parvimonas micra was detected from a transcutaneous vertebral biopsy specimen in 3 h. Gram-positive cocci were also detected by Gram staining and P. micra was identified directly from the anaerobic blood culture by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Four days after admission, the biopsy specimen culture isolate was identified as P. micra. CONCLUSIONS: The Tm mapping method may be useful for the diagnosis of bacterial infections where diagnosis is challenging because of the difficulty of culturing.

The active zone protein CAST regulates synaptic vesicle recycling and quantal size in the mouse hippocampus.

Synaptic efficacy is determined by various factors, including the quantal size, which is dependent on the amount of neurotransmitters in synaptic vesicles at the presynaptic terminal. It is essential for stable synaptic transmission that the quantal size is kept within a constant range and that synaptic efficacy during and after repetitive synaptic activation is maintained by replenishing release sites with synaptic vesicles. However, the mechanisms for these fundamental properties have still been undetermined. We found that the active zone protein CAST (cytomatrix at the active zone structural protein) played pivotal roles in both presynaptic regulation of quantal size and recycling of endocytosed synaptic vesicles. In the CA1 region of hippocampal slices of the CAST knockout mice, miniature excitatory synaptic responses were increased in size, and synaptic depression after prolonged synaptic activation was larger, which was attributable to selective impairment of synaptic vesicle trafficking via the endosome in the presynaptic terminal likely mediated by Rab6. Therefore, CAST serves as a key molecule that regulates dynamics and neurotransmitter contents of synaptic vesicles in the excitatory presynaptic terminal in the central nervous system.

[Direct Oral Anticoagulants (DOACs): Pharmacological Characteristics and Laboratory Assessment].

Vitamin K antagonists such as warfarin, which inhibit vitamin K-dependent clotting factors II, VII, IX, and X, have been the only available oral anticoagulants for more than 50 years. Direct oral anticoagulants (DOACs), including direct anti-Xa and a thrombin inhibitor, have been introduced and provide advantages over warfarin. DOACs are rapidly-acting, target-specific anticoagulants that inhibit both free and bound activated serine proteases, unlike heparin that can inhibit only free proteases. The FXa inhibitors (Rivaroxaban, Apixaban, Edoxaban) bind directly to the catalytic site of FXa and inhibit both free and prothrombinase-bound FXa. The thrombin inhibitor (Dabigatran), designed to occupy and inactivate the serine proteolytic pocket of thrombin, prevents thrombin from acting on fibrinogen to produce fibrin. Although routine coagulation mon- itoring of DOACs is not required, it may be useful under some circumstances. Several reports have de- scribed the effect of DOACs on routine assays including the APTT and PT. Dabigatran will interfere with most APTT-based and some PT-based assays, which means that laboratory testing will lead to results that are not representative of the physiologic effects. Rivaroxaban and edoxaban prolong the PT to a greater extent than APTT, and these agents may interfere with PT-based factor activity assays (FVII, X, V, and II) causing factitiously low activities. Also discussed are the use of drug-specific assays and alternative meth- ods to determine the relative drug concentration, such as evaluating drug calibrators in APTT and PT assays. The actual drug concentration may be required for specific patients, such as those with deterioration of the renal function, before surgical interventions, bleeding or thrombotic episodes, and suspected overdoses, and to control adherence to therapy. The need to assess DOACs in emergent situations as well as their impact on routine and special coagulation assays has created challenges in most laboratories. [Review] '.

[Chairmen's Introductory Remarks].

At the 62nd Annual Meeting of the Japanese Society of Laboratory Medicine Scientific Sessions, a "promotion of globalization in clinical laboratory area" symposium of the Japanese Society of Laboratory Medicine (JSLM) and the Japanese Association of Medical Technologists (JAMT) was held. Studying abroad as well as the overseas dispatch of biomedical laboratory scientists have increased in re- cent years. However, there are still a number of problems. Both the JSLM and JAMT are focusing on in- ternational activities. For example, the JSLM has the International Committee, and the JAMT has set up a specially appointed Executive Director of International Affairs. Moreover, international standards, facility certification, and clinical trials are being established at the clinical laboratory. In other words, there is a growing need for the third-party evaluation of such international certification. The promotion of globaliza- tion in the clinical laboratory area is essential, and human resource development to nurture an international community in the future is important. However, the most important thing is that many biomedical laborato- ry scientists participate in international conferences. [Review].

Apixaban for the Treatment of Japanese Subjects With Acute Venous Thromboembolism (AMPLIFY-J Study).

BACKGROUND: Anticoagulation is recommended as standard of care for venous thromboembolism (VTE) (pulmonary embolism [PE]/deep vein thrombosis [DVT]), for which unfractionated heparin (UFH) and warfarin are used in Japan. In the multi-regional AMPLIFY study, a fixed-dose regimen of apixaban alone was non-inferior to conventional therapy for treatment of PE/DVT and was associated with significantly fewer bleeding events. METHODS AND RESULTS: Japan phase 3 study (AMPLIFY-J), randomized, active-controlled, open-label study in Japanese subjects with acute PE/DVT, was designed based on AMPLIFY. Key objectives were to investigate safety and efficacy of apixaban in symptomatic PE/DVT subjects during 24-week treatment. UFH/warfarin was used as control treatment. Apixaban was initiated at 10 mg twice daily for 7 days, followed by 5 mg twice daily for 23 weeks. All endpoints and imaging for thrombotic burden were assessed by an event adjudication committee. Eighty subjects were randomized, 33 subjects (41.3%) were aged <65 years. Proportion of major/clinically relevant non-major bleeding was lower in apixaban (7.5%) compared with well-controlled UFH/warfarin (28.2%; median TTR, 70.4%). [corrected]. Recurrent VTE occurred in no subjects in apixaban and in 1 subject in UFH/warfarin. Thrombotic burden results were similar in both groups. Proportions of subjects with adverse events was generally similar in both groups. CONCLUSIONS: Apixaban was well-tolerated and had a favorable safety profile. No clinically important efficacy difference compared with UFH/warfarin was observed.

N-terminal pro-brain natriuretic peptide as a predictor of reoperation in children with surgically corrected tetralogy of fallot.

BACKGROUND: Most patients with surgically corrected tetralogy of Fallot (TOF) are faced with multiple residua and sequelae such as pulmonary regurgitation (PR), resulting in reoperation for pulmonary valve replacement (PVR). Plasma brain natriuretic peptide (BNP) level and serum N-terminal pro-BNP (NT-pro-BNP) level are useful as diagnostic objective markers of chronic heart failure (CHF). The aim of the study was to examine whether these markers have predictive ability for reoperation in children with surgically corrected TOF. METHODS AND RESULTS: Fifty-eight patients (38 male, 20 female) aged 1-18 years (median, 7 years) were enrolled. Serum NT-pro-BNP in TOF patients was significantly higher than in age-matched hospital controls without CHF (359.5±449.7pg/ml vs. 86.1±45.1pg/ml, respectively; P<0.0001). BNP and NT-pro-BNP had a better correlation with CHF index, RVEDP, and LVEDV in TOF groups. Children with surgically corrected TOF who had indication for PVR had higher BNP and NT-pro-BNP and more severe PR than those without indication for PVR. On multivariate logistic regression analysis, NT-pro-BNP was the strongest predictor for reoperation in patients with surgically corrected TOF. Area under the curve of NT-pro-BNP for reoperation was 0.950 (P<0.001) with a sensitivity of 88.9% and specificity of 91.8%. CONCLUSIONS: NT-pro-BNP is a good biomarker for monitoring CHF, and is a good predictor of PVR in children with surgically repaired TOF.

Recombinant soluble human thrombomodulin (thrombomodulin alfa) in the treatment of neonatal disseminated intravascular coagulation.

UNLABELLED: Recombinant soluble human thrombomodulin (TM-α) has been shown to be useful in the treatment of disseminated intravascular coagulation (DIC) in a heparin-controlled study and has been available for clinical use in Japan since 2008. However, data on its use for neonatal DIC have not been reported from any clinical studies, so efficacy and safety were analyzed in 60 neonatal DIC patients identified in post-marketing surveillance. The DIC resolution rate as of the day after last administration of TM-α was 47.1 %, and the survival rate at 28 days after last administration was 76.7 %. Hemostatic test result profiles revealed decreased levels of fibrin/fibrinogen degradation products and increased platelet counts and antithrombin activity. Incidences of adverse drug reactions, bleeding-related adverse drug reactions, and bleeding-related adverse events were 6.7, 6.7, and 16.7 %, respectively, with no significant differences between neonatal, pediatric (excluding neonates), and adult DIC patients. CONCLUSION: This surveillance provided real-world data on the safety and effectiveness of TM-alpha in the treatment of neonatal DIC in general practice settings.

[Points of revision of the guideline JSLM 2012].

The revised version of the guideline JSLM 2012 was published in December 2012 by the Japanese Society of Laboratory Medicine. This version included new sections, such as blood gas analysis, sleep apnea syndrome, interstitial lung disease, liver and pancreas cancer, chronic kidney disease, acute kidney disease, gout and hyperuricemia, bone metabolism abnormality, malignant lymphoma, and rheumatoid arthritis. The guideline committee was composed of specialists in each field of internal medicine, who were responsible for selecting and requesting the authors and also in promoting and proofreading the manuscripts. In special program I at the 60th annual meeting, each specialist gave lectures concerning the points of the revision in their fields. The questionnaire surveys were performed using FAX or the Internet. Analysis of eighty-seven (2.5%) responses from 3,500 individuals/facilities, which were sent the guideline, revealed that this guideline was graded as excellent by 38 readers, fair by 42, and average by in 6. Significant opinions on the guideline were obtained from the readers, and they will be the bases for the next revision. The main subjects of this guideline were confirmed to be the residents and general physicians, by whom it is hoped the routine laboratory tests will be properly utilized. Therefore, based on the section of 'approach of the laboratory test results', which is a representative characteristic of this guideline, the sections of 'symptoms' will be fulfilled for the next version. This guideline needs to be periodically revised with advances in medicine.

Melting temperature mapping method using imperfect-match linear long probes.

Identifying pathogenic microorganisms as early as possible is critical for selecting the appropriate antimicrobial therapy in infected patients. We previously reported the development of the Tm mapping method for identifying a broad range of pathogenic bacteria within 3 h of blood collection. However, the Tm mapping identification requires an analytical instrument with a tube-to-tube variation of no more than 0.1 °C, so we can only use a few instruments that have such high thermal accuracy. To address the problem, we developed the improved Tm mapping method using imperfect-match linear long quenching probes (IMLL Q-probes). Using IMLL Q-probes, almost all commercially available analytical instruments can be used for the Tm mapping method. Some bacterial species cannot be narrowed down to one species, but they can at least be narrowed down to the genus level. The Tm mapping method using IMLL Q-probes is useful for deciding on antimicrobial therapy in infected patients.