Development and Evaluation of "The Delta Plus-Minus Even Distribution Check": A Novel Patient-Based Real-Time Quality Control Method for Laboratory Tests.

BACKGROUND: Laboratory testing of large sample numbers necessitates high-volume rapid processing, and these test results require immediate validation and a high level of quality assurance. Therefore, real-time quality control including delta checking is an important issue. Delta checking is a process of identifying errors in individual patient results by reviewing differences from previous results of the same patient (Δ value). Under stable analytical conditions, Δ values are equally positively and negatively distributed. METHODS: The previous 20 Δ values from 3 tests (cholesterol, albumin, and urea nitrogen) were analyzed by calculating the R-value: "the positive Δ value ratio minus 0.5." This method of monitoring optimized R-values is referred to as the even-check method (ECM) and was compared with quality control (QC) testing in terms of error detection. RESULTS: Bias was observed on 4 of the 120 days for the 3 analytes measured. When QC detected errors, the ECM captured the same systematic errors and more rapidly. In contrast, the ECM did not generate an alarm for the one random error that occurred in QC. While QC did not detect any errors, the percentage of R-values exceeding the acceptable range was under 2%, the number of days generating alarms was between 16 and 21 days, with short alarm periods, and a median number of samples per alarm period between 7 and 9 samples. CONCLUSIONS: The ECM is a practical real-time QC method, controlled by setting R-value conditions, that quickly detects bias values.

Novel knit fabric rehabilitation equipment for finger impairment.

[Purpose] Intensive training can at least partially improve finger movement dysfunction observed after stroke or any neurodegenerative disease. Wearable equipment can significantly improve patients' quality of life. However, long-term use of conventional training gloves containing metal can injure joints. In this study, we investigated the safety and efficacy of a novel, metal-free, wearable strength-building device. [Participants and Methods] We enrolled 20 healthy participants in whom we measured grip and pinch strength before and while the equipment was worn. Additionally, we investigated the adverse effects and discomfort experienced while participants wore the equipment. [Results] The grip strength was reduced by approximately 20% while participants wore the equipment. We did not observe any serious adverse events. [Conclusion] The knitting equipment described in this study resists movements associated with gripping the hand and acts on all fingers, and may be useful for rehabilitation to improve finger function during routine activities.

Measuring serum prothrombin in patients treated with warfarin or direct oral anticoagulants.

BACKGROUND: Warfarin therapy influences generation of γ-carboxyglutamyl (Gla) residues in prothrombin, causing reduced coagulation activity. It will leave such inactive prothrombin in serum after clot formation, resulting in serum prothrombin constituting total inactive prothrombin in these patients. METHODS: An ELISA was developed to measure biologically inactive prothrombin in serum, and applied to serum from warfarin therapy causing a decrease in Gla residues or direct oral anticoagulant (DOAC) therapy as its contrast. RESULTS: The concentrations of serum prothrombin in both the warfarin and DOAC groups were higher than those in the healthy group (p < 0.01 and p < 0.001, respectively). When serum in the previous three groups was treated with barium carbonate to exclude prothrombin, which lost several Gla residues, the prothrombin concentration in the DOAC group decreased to the same level as that in the healthy group, indicating that prothrombin was obtained at a high level only in the warfarin group (p < 0.01). CONCLUSIONS: Warfarin and DOAC led to increase in serum prothrombin concentration. The reason is that DOAC decreases prothrombin recruitment during fibrin clot formation, while warfarin leads to the accumulation of inactive prothrombin, which have a decreased number of Gla residues.

Reference intervals of red blood cell parameters and platelet count for healthy adults in Japan.

Although it is known that red blood cell (RBC) parameters and platelet count depend on ethnicity and sex, their reference intervals in healthy Asian populations are limited. The aim of this study was to establish reference intervals for RBC parameters and platelet count for healthy adults in Japan. A total of 750 healthy adults (447 women and 303 men; median age 40 years (18-67 years) at seven Japanese centers who participated in regular medical checkups entered this study. Their RBC parameters and platelet count were measured using automated hematocytometers. The reference intervals of the RBC parameters and platelet count according to sex in healthy adults were determined. There was an age-specific decrease in RBC counts and an age-specific increase in mean corpuscular volume in men. This study emphasizes the need to consider sex and age in the clinical use of reference intervals of RBC parameters.

Reference intervals of white blood cell parameters for healthy adults in japan.

INTRODUCTION: While white blood cell (WBC) parameters have been suggested to depend on ethnicity and gender, reference intervals in healthy Asian populations are limited. The present study established reference intervals of WBC parameters for healthy adults in Japan. METHODS: A total of 750 healthy adults (447 women and 303 men; 18-67 years old, median 40 years old) at 7 Japanese centers who participated in regular medical checkups entered this study. The WBC parameters were measured using automated hematocytometers and blood film reviews by a manual microscopic examination. RESULTS: The reference intervals of the WBC parameters according to gender in healthy adults were determined. Age-specific decreases in WBC counts of both gender groups and in neutrophil counts of women were noted. Favorable correlations between the hematocytometer and microscopic methods were found in neutrophils, lymphocytes, and eosinophils but not in monocytes or basophils. CONCLUSION: This study suggests the need to consider gender and age in the clinical use of reference intervals of WBC parameters.

The Plot Thickens: Haploid and Triploid-Like Thalli, Hybridization, and Biased Mating Type Ratios in Letharia.

The study of the reproductive biology of lichen fungal symbionts has been traditionally challenging due to their complex lifestyles. Against the common belief of haploidy, a recent genomic study found a triploid-like signal in Letharia. Here, we infer the genome organization and reproduction in Letharia by analyzing genomic data from a pure culture and from thalli, and performing a PCR survey of the MAT locus in natural populations. We found that the read count variation in the four Letharia specimens, including the pure culture derived from a single sexual spore of L. lupina, is consistent with haploidy. By contrast, the L. lupina read counts from a thallus' metagenome are triploid-like. Characterization of the mating-type locus revealed a conserved heterothallic configuration across the genus, along with auxiliary genes that we identified. We found that the mating-type distributions are balanced in North America for L. vulpina and L. lupina, suggesting widespread sexual reproduction, but highly skewed in Europe for L. vulpina, consistent with predominant asexuality. Taken together, we propose that Letharia fungi are heterothallic and typically haploid, and provide evidence that triploid-like individuals are hybrids between L. lupina and an unknown Letharia lineage, reconciling classic systematic and genetic studies with recent genomic observations.

Influence of Warfarin Therapy on Prothrombin Production and Its Posttranslational Modifications.

BACKGROUND: Protein induced by vitamin K absence-II (PIVKA-II) is produced by the liver during hepatoma and upon warfarin administration. Those patients have disturbed protein synthesis and glycosylation in the liver. This decreases the number of γ-carboxyglutamyl (Gla) residues on prothrombin, converting prothrombin into PIVKA-II. The mechanism of this conversion, however, is not clearly understood. METHODS: Prothrombin was isolated from healthy and warfarin-treated individuals whose liver function of protein production was quantitatively normal. Glycan structures in the purified prothrombin containing PIVKA-II were qualitatively analyzed by high performance liquid chromatography after labeling the glycan with fluorophore 2-aminobenzamide. RESULTS: The concentration of PIVKA-II was significantly higher in the warfarin-treated individuals than in the healthy individuals (P< 0.001). Although protein production in the liver was normal in both groups, the concentration of prothrombin was lower in the warfarin-treated individuals than in the healthy individuals (P < 0.001). The main glycan was A2 in the healthy and warfarin-treated individuals (86.6 ± 4.4% and 85.6 ± 3.4%, respectively). Eight types of glycan were characterized in both groups, although generation of PIVKA-II in the warfarin-treated individuals did not lead to variation in glycosylation of prothrombin. CONCLUSIONS: Warfarin therapy leads to lower amounts of prothrombin and Gla residues within prothrombin without exerting qualitative and quantitative change in glycan profile and protein synthetic function in the liver.

[Successful treatment with enzyme replacement therapy for pelvic fragile fracture in an elderly case of type I Gaucher's disease].

A 76-year-old male with lower-limb weakness was admitted to our hospital where thrombocytopenia and anemia were noticed. CT showed massive splenomegaly and multiple nodules inside the spleen. Bone marrow examination showed an increase of macrophages with large cytoplasm. Suspected of splenic lymphoma, the patient underwent splenectomy. Spleen specimens were histologically analyzed and suggested the probability of Gaucher's disease (GD). Leukocyte glucocerebrosidase (GBA) enzyme activity had decreased to 1.25 nmol/mg, and mutation analysis of GBA revealed two missense variants, p.D448H (D409H), p.L483P (L444P), which confirmed the diagnosis of type I GD. Fourteen months after splenectomy, he developed right buttock pain, and pelvic magnetic resonance imaging showed a fragile right pubic and pelvic fracture. We initiated injection of imiglucerase as enzyme replacement therapy (ERT) and administered bisphosphonate. His symptoms gradually improved without surgical treatment. In addition, thrombocytopenia and anemia also improved, and angiotensin-converting enzyme levels decreased. Type I GD should be considered a differential diagnosis of giant splenomegaly and thrombocytopenia, even in the elderly. ERT or substrate reduction therapy should be administrated to GD patients, while paying attention to the development of bone lesions.

Effect of Cu on the fluorescence of the Cu-hyperaccumulator lichen Stereocaulon sorediiferum.

Stereocaulon sorediiferum is expected to be a Cu-hyperaccumulator lichen and has fluorescent substances. To clarify the relationship between the fluorescence (FL) of the lichen and its Cu concentration, we collected S. sorediiferum samples at Cu-contaminated and uncontaminated sites in Japan, determined the concentration of Cu, K, Mg, Al, Ca, Mn, Fe, Zn, chlorophyll a,b, and total carotenoids in them, analyzed lichen secondary metabolites and fluorescent substances extracted from them, and measured the FL of them and their extracts. We found that the FL intensity of S. sorediiferum samples is significantly negatively correlated with their Cu concentration. The application of its FL for Cu monitoring may allow a new nondestructive quantitative method for assessing Cu contamination. The spectroscopic and chromatographic analysis shows that the fluorescent substances negatively correlated with Cu concentration are not major lichen secondary metabolites (lobaric acid and atranorin) and remain after immersion in acetone. The correlation analysis and the comparison with the causal relationship between Cu concentration and the chlorophyll a/b ratio suggest that the reason for the decrease in FL intensity with increasing Cu concentration is a structural change of the fluorescent substances by accumulated Cu. These findings lead to a better understanding of the relationship between the FL of S. sorediiferum and its Cu concentration and provide new insights into fluorescent lichen substances.

[Hyper-IL-6 syndrome mimicking IgG4-related disease].

Distinguishing between IgG4-related disease (IgG4-RD) and hyper-interleukin (IL) -6 syndrome, such as immune mediated conditions, autoimmune diseases, and idiopathic multicentric Castleman disease (iMCD) is challenging. Here, we report the case of a 69-year-old man with cervical lymphadenopathy who was admitted to our hospital and histologically diagnosed with hyper-IL-6 syndrome mimicking IgG4-RD phenotypically. Laboratory data detected polyclonal hypergammaglobulinemia comprising IgG, including IgG4 (2,350 mg/dl). Computed tomography revealed presence of systemic lymphadenopathy, enlarged bilateral submandibular glands, and infiltrative shadow in the right lower lung. Magnetic resonance imaging revealed diffusely enlarged pancreas the size of a sausage and hypointense rim on T2, suggesting autoimmune pancreatitis as part of IgG4-RD. Biopsy of the cervical lymph node revealed proliferation of IL-6-positive mature plasma cells in the expanded interfollicular area with an elevated IgG4+/IgG+ cell ratio (approximately 70%). These histological findings were consistent with hyper-IL-6 syndrome rather than IgG4-RD; however, the serum IL-6 level was slightly elevated. Bone marrow aspiration detected both IgG4- and IL-6-positive mature plasma cells. Although this case cannot be diagnosed as IgG4-RD because it failed to meet its diagnostic criteria, administration of oral prednisolone (0.5 mg/kg) resulted in rapidly improved lymphadenopathy, enlarged pancreas, and serological findings. This report can be helpful for the diagnostic assessment of polyclonal hypergammaglobulinemia conditions.

The conservation of polyol transporter proteins and their involvement in lichenized Ascomycota.

In lichen symbiosis, polyol transfer from green algae is important for acquiring the fungal carbon source. However, the existence of polyol transporter genes and their correlation with lichenization remain unclear. Here, we report candidate polyol transporter genes selected from the genome of the lichen-forming fungus (LFF) Ramalina conduplicans. A phylogenetic analysis using characterized polyol and monosaccharide transporter proteins and hypothetical polyol transporter proteins of R. conduplicans and various ascomycetous fungi suggested that the characterized yeast' polyol transporters form multiple clades with the polyol transporter-like proteins selected from the diverse ascomycetous taxa. Thus, polyol transporter genes are widely conserved among Ascomycota, regardless of lichen-forming status. In addition, the phylogenetic clusters suggested that LFFs belonging to Lecanoromycetes have duplicated proteins in each cluster. Consequently, the number of sequences similar to characterized yeast' polyol transporters were evaluated using the genomes of 472 species or strains of Ascomycota. Among these, LFFs belonging to Lecanoromycetes had greater numbers of deduced polyol transporter proteins. Thus, various polyol transporters are conserved in Ascomycota and polyol transporter genes appear to have expanded during the evolution of Lecanoromycetes.

Norlichexanthone produced by cultured endolichenic fungus induced from Pertusaria laeviganda and its antioxidant activity.

Endolichenic fungi, nonobligate microfungi that live in lichen, are promising as new bioresources of pharmacological compounds. We found that norlichexanthone isolated from the endolichenic fungus in Pertusaria laeviganda exhibited high antioxidant activity. Norlichexanthone produced by endolichenic fungus had the antioxidant activity with same level of ascorbic acid. This is the first report of high antioxidant activity of norlichexanthone. Abbreviations: AAPH: 2,2'-azobis (2-methylpropionamidine) dihydrochloride; DPPH: 2,2-diphenyl-1-picrylhydrazyl; FL: fluorescein sodium salt; HPLC-PDA: high-performance liquid chromatography with photodiode array; LC-ESI-MS: liquid chromatography with electrospray ionization mass spectrometry; ORAC: oxygen radical absorbance capacity; PB: phosphate buffer; ROS: reactive oxygen species; TLC: thin-layer chromatography.

Response of secondary metabolites to Cu in the Cu-hyperaccumulator lichen Stereocaulon japonicum.

Lichen secondary metabolites are known to be associated with heavy metal uptake and tolerance in lichens. Understanding the relationship between their secondary metabolites and heavy metals in them is important for clarifying the mechanisms of their heavy metal accumulation and tolerance. To determine the relationships between the concentrations of secondary metabolites and Cu in the Cu-hyperaccumulator lichen Stereocaulon japonicum and to clarify its response to Cu, we collected Cu-contaminated and uncontaminated samples of the lichen and determined relative concentrations of secondary metabolites and concentrations of Cu, K, glucose, and sugar alcohols in them. We found significant negative correlations between the relative concentrations of secondary metabolites-atranorin and stictic acid-and the concentration of Cu. These negative correlations can be interpreted in one of two ways: (a) S. japonicum itself reduced the relative concentrations of secondary metabolites in response to the increase of Cu concentration or (b) its carbon and energy metabolism was damaged by Cu stress, resulting in the reduction of the relative concentrations of secondary metabolites. The analysis of K, glucose, and sugar alcohols showed no effect of Cu on these concentrations, which means that the carbon and energy metabolism was not damaged by Cu stress. Therefore, the negative correlations can be interpreted that S. japonicum itself reduced the relative concentrations of secondary metabolites with the increase of Cu concentration. These findings provide a deeper understanding of the response of secondary metabolites to Cu in the lichen.

[Early relapse after autologous peripheral blood stem cell transplantation in an elderly patient with enteropathy-associated T-cell lymphoma].

A 73-year-old male with melena was admitted to our hospital. Computed tomography (CT) scan revealed the thickening of the jejunal and ileal walls and swelling of the mesenteric lymph nodes. Type II enteropathy-associated T-cell lymphoma (EATL) was diagnosed based on the pathological analysis of the resected specimen. Positron emission tomography and CT scan showed complete remission (CR) after surgery, and he further received CHOP therapy. However, 2 months after the completion of the therapy, the patient's disease relapsed, and he presented with abdominal pain. Ifosfamide, dexamethasone, etoposide, and cytarabine therapy was administered, and the second CR was observed in the patient. Subsequently, the patient was administered high-dose chemotherapy (MCEC) with autologous peripheral blood stem cell transplantation (auto-PBSCT). The treatment was well tolerated. Engraftment was performed on day9, and he was discharged on day17 after auto-PBSCT. However, at 6 months after auto-PBSCT, the second relapse of the disease was observed in the patient. He received salvage therapy; however, the patient died because of disease progression. Because of the dismal prognosis of EATL treated with conventional chemotherapy, the feasibility and efficacy of auto-PBSCT have been investigated. To the best of our knowledge, there is no report on an elderly patient (age >70 years) with EATL who underwent auto-PBSCT. Thus, more data should be collected and analyzed to confirm that this therapy could be a promising treatment option for elderly patients with EATL.

Application of DNA markers for discrimination between Japanese and Australian Wagyu beef.

The objective of this study was to discriminate between original Japanese and Australian Wagyu beef, which is sold in the Singapore markets, using six previously developed DNA markers. To effectively evaluate the six markers for breed identification, the probability of identification as Australian Wagyu beef was calculated based on the estimated allele frequencies using 130 Australian Wagyu individuals. The combined use of six markers would allow the discrimination of Australian Wagyu beef with an estimated probability of 0.776. The probability to discriminate Australian Wagyu from Japanese Wagyu beef was sufficiently high. In addition, Australian Wagyu has maternal mitochondrial DNA of Bos indicus cattle with moderate high frequency of 0.377. The DNA marker system could also be used as a deterrent force against false sales, and contribute to the reduction and prevention of incorrect or falsified labeling of beef.

Primary adrenal diffuse large B-cell lymphoma positive for CD5 and CD10.

A 69-year-old man presented with back pain over the prior few months and was hospitalized because of bilateral adrenal masses and fracture of the left sixth rib. The mass on the right measured 6.5×3.6×7.0 cm, that on the left 8.1×4.8×6.9 cm, on CT. The final diagnosis was CD5- and CD10-positive primary adrenal diffuse large B-cell lymphoma (PA-DLBCL) with rib involvement. After EPOCH therapy accompanied with rituximab and intrathecal treatment, the tumors decreased dramatically. However, he died due to disease progression 8 months after the diagnosis. The prognosis of CD5- and CD10-positive PA-DLBCL may be very poor even with rituximab-containing chemotherapy.

Limitations of Species Delimitation Based on Phylogenetic Analyses: A Case Study in the Hypogymnia hypotrypa Group (Parmeliaceae, Ascomycota).

Delimiting species boundaries among closely related lineages often requires a range of independent data sets and analytical approaches. Similar to other organismal groups, robust species circumscriptions in fungi are increasingly investigated within an empirical framework. Here we attempt to delimit species boundaries in a closely related clade of lichen-forming fungi endemic to Asia, the Hypogymnia hypotrypa group (Parmeliaceae). In the current classification, the Hypogymnia hypotrypa group includes two species: H. hypotrypa and H. flavida, which are separated based on distinctive reproductive modes, the former producing soredia but absent in the latter. We reexamined the relationship between these two species using phenotypic characters and molecular sequence data (ITS, GPD, and MCM7 sequences) to address species boundaries in this group. In addition to morphological investigations, we used Bayesian clustering to identify potential genetic groups in the H. hypotrypa/H. flavida clade. We also used a variety of empirical, sequence-based species delimitation approaches, including: the "Automatic Barcode Gap Discovery" (ABGD), the Poisson tree process model (PTP), the General Mixed Yule Coalescent (GMYC), and the multispecies coalescent approach BPP. Different species delimitation scenarios were compared using Bayes factors delimitation analysis, in addition to comparisons of pairwise genetic distances, pairwise fixation indices (FST). The majority of the species delimitation analyses implemented in this study failed to support H. hypotrypa and H. flavida as distinct lineages, as did the Bayesian clustering analysis. However, strong support for the evolutionary independence of H. hypotrypa and H. flavida was inferred using BPP and further supported by Bayes factor delimitation. In spite of rigorous morphological comparisons and a wide range of sequence-based approaches to delimit species, species boundaries in the H. hypotrypa group remain uncertain. This study reveals the potential limitations of relying on distinct reproductive strategies as diagnostic taxonomic characters for Hypogymnia and also the challenges of using popular sequence-based species delimitation methods in groups with recent diversification histories.

Multianalyte Conventional Reference Material (MacRM): A Useful Tool for Nationwide Standardization of Laboratory Measurements for Medical Care-A Model Study in Japan.

BACKGROUND: The Japanese Committee for Clinical Laboratory Standards (JCCLS) has developed a multianalyte conventional reference material (MacRM) for nationwide standardization of laboratory measurements. METHODS: To prepare the MacRM, pooled sera were obtained from healthy Japanese individuals. Target values of the pooled sera for 30 analytes were assigned on the basis of the measurement results of 45 certified clinical laboratories whose calibration was verified by measuring certified reference materials (CRMs) provided by the National Institute of Standards and Technology, the Institute for Reference Materials and Measurements, and JCCLS. Commutability of MacRM was assessed by comparison with results for 150 individual inpatients at Fukuoka University Chikushi Hospital. Survey samples were prepared by essentially the same method for MacRM but without target values. The survey samples were used to assess agreement among 165 laboratories that used various assay kits and platforms calibrated with the MacRM. RESULTS: The commutability of MacRM was confirmed for 30 analytes with sera from 150 individual patients. The imprecision (CV) of measurements of survey samples (high and low concentrations) among the 165 laboratories was 0.4%-10.0%. Twenty-six of 30 analytes were within the goals for interinstitutional allowable bias. An aliquot of MacRM stored frozen at -80 °C remained stable for ≥4 years. CONCLUSIONS: The MacRM was successfully applied as a calibrator to achieve nationwide standardization for 30 analytes measured by 165 laboratories that used various methods from different manufacturers.

[Successful treatment with rituximab of a patient with coincident acquired hemophilia A and thrombotic thrombocytopenic purpura].

A 66-year-old man was admitted for oral hemorrhage, purpura, and APTT prolongation. Factor VIII (FVIII) activity was decreased, due to the presence of FVIII inhibitor. He was diagnosed with acquired hemophilia A (AHA) and treated with prednisolone. Eight months later, the FVIII inhibitor titer again increased. Upon readmission, thrombocytopenia and autoimmune hemolytic anemia were found. We suspected Evans syndrome accompanied by AHA, and we treated the patient with IVIG. However, his platelet count did not increase. Speech disturbance and delirium were observed from the 12th day of hospitalization. He was subsequently diagnosed with thrombotic thrombocytopenic purpura (TTP) because ADAMTS13 inhibitor was detected, causing a decrease in ADAMTS13 activity. We initiated plasma exchange (PE) and steroid-pulse therapy. After PE for 3 days, laboratory test results and psychiatric symptoms showed dramatic improvement. However, after a 2-day period without PE, the patient's platelet count decreased markedly. Therefore, we administered rituximab to eliminate these inhibitors. His platelet count recovered rapidly, and we were able to gradually wean the patient from PE. After two additional administrations of rituximab, neither inhibitor was detected. To date, the patient has remained in complete remission for approximately 3 years.

Biosynthesis of Panaefluoroline B from the Cultured Mycobiont of Amygdalaria panaeola.

Panaefluoroline B (2) is a fluorescent yellowish-green pigment produced by the cultured mycobiont of a lichen, Amygdalaria panaeola. Panaefluoroline B (2) has an isoquinoline skeleton, a C5 unit, and an amino acid, glycine, in its structure. The biosynthetic pathway of 2 was revealed by feeding experiments using [1-(13)C]-sodium acetate and [1,2-(13)C2][(15)N]-glycine. The analysis of labeling patterns of 2 and its mass spectrum suggested the isoquinoline part is biosynthesized via the acetate-malonate pathway with glycine as the nitrogen source and that the C5 unit originates from the mevalonate pathway.