Circulating TNF receptor levels are associated with estimated glomerular filtration rate even in healthy individuals with normal kidney function.

The association between serum tumor necrosis factor receptor (TNFRs: TNFR1, TNFR2) levels and estimated glomerular filtration rate (eGFR) observed in patients with diabetes has not been comprehensively tested in healthy subjects with normal kidney function. It also remains unclear whether TNFR levels differ by age and sex, and between healthy subjects and diabetics. We measured serum TNFR levels in 413 healthy subjects and 292 patients with type 2 diabetes. In healthy subjects, TNFR levels did not differ between men and women. Additionally, TNFR2, but not TNFR1, levels increased with age. In multivariate analysis, TNFR1 was associated only with cystatin C-based eGFR (eGFR-CysC), whereas TNFR2 was associated with systolic blood pressure in addition to eGFR-CysC. Both TNFRs were associated with lower eGFR (eGFR-Cys < 90 mL/min/1.73 m2) even after adjustment for relevant clinical factors. Upon combining healthy subjects and patients with diabetes, the presence of diabetes and elevated glycated hemoglobin level were significant factors in determining TNFR levels. TNFR levels were associated with eGFR-CysC, but were not affected by age and sex in healthy subjects with normal kidney function. TNFR levels in patients with diabetes appeared to be higher than in healthy subjects.

Investigation of the individual genetic evolution of SARS-CoV-2 in a small cluster during the rapid spread of the BF.5 lineage in Tokyo, Japan.

There has been a decreasing trend in new severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) cases and fatalities worldwide. The virus has been evolving, indicating the potential emergence of new variants and uncertainties. These challenges necessitate continued efforts in disease control and mitigation strategies. We investigated a small cluster of SARS-CoV-2 Omicron variant infections containing a common set of genomic mutations, which provided a valuable model for investigating the transmission mechanism of genetic alterations. We conducted a study at a medical center in Japan during the Omicron surge (sub-lineage BA.5), sequencing the entire SARS-CoV-2 genomes from infected individuals and evaluating the phylogenetic tree and haplotype network among the variants. We compared the mutations present in each strain within the BA.5 strain, TKYnat2317, which was first identified in Tokyo, Japan. From June 29th to July 4th 2022, nine healthcare workers (HCWs) tested positive for SARS-CoV-2 by real-time PCR. During the same period, five patients also tested positive by real-time PCR. Whole genome sequencing revealed that the infected patients belonged to either the isolated BA.2 or BA.5 sub-lineage, while the healthcare worker infections were classified as BF.5. The phylogenetic tree and haplotype network clearly showed the specificity and similarity of the HCW cluster. We identified 12 common mutations in the cluster, including I110V in nonstructural protein 4 (nsp4), A1020S in the Spike protein, and H47Y in ORF7a, compared to the BA.5 reference. Additionally, one case had the extra nucleotide-deletion mutation I27* in ORF10, and low frequencies of genetic alterations were also found in certain instances. The results of genome sequencing showed that the nine HCWs shared a set of genetic mutations, indicating transmission within the cluster. Minor mutations observed in five HCW individuals suggested the emergence of new virus variants. Five amino acid substitutions occurred in nsp3, which could potentially affect virus replication or immune escape. Intra-host evolution also generated additional mutations. The cluster exhibited a mild disease course, with individuals in this case, recovering without requiring any medical treatments. Further investigation is needed to understand the relationship between the genetic evolution of the virus and the symptoms.

Novel O/W Emulsions by Utilizing a Vesicle/Disc Transformation of Polyether Modified Silicone.

Emulsification is an important technology in the field of cosmetics and household products. Emulsions are in non-equilibrium state; therefore, the products vary depending on the preparation process, and their state changes with time. Furthermore, it is known empirically that different types of oils have different emulsification properties (preparation and stability). For these reasons, the variables in emulsification research are numerous and complicated to analyze. As a result, many industrial applications have had to rely on empirical rules. In this study, emulsions with a lamellar liquid crystalline phase as an adsorption layer at the emulsion interface were investigated. The characteristics of O/W emulsions formed with the excess solvent phases (aqueous and oil phases) separated from the lamellar liquid crystalline phase were investigated based on the phase equilibrium of the ternary system.As a result, it was found that by agitating the aqueous phase containing dispersed vesicles of emulsifier (polyether modified silicone) together with the oil phase, an emulsion with a uniform interfacial membrane of lamellar liquid crystalline phase could be obtained. The emulsions prepared by this method were found to have good stability against coalescence. The process of transformation from vesicles to the uniform liquid crystal interfacial membrane during the emulsification process was clarified by a freeze-fracture transmission electron micrograph and the calculation of interfacial membrane thickness based on precise particle size analysis. Furthermore, the emulsification properties of polyether-modified silicones were clarified using polar oils and silicone oils, which are a combination of high/low and low/high compatibility with hydrophilic (polyethylene glycol) and lipophilic (polydimethylsiloxane) groups of polyether modified silicone, respectively. It is expected that this research will lead to the evolution of various functionalities in products in the fields of cosmetics, household products, food, pharmaceuticals, paint and others.

Anti-adsorption Mechanism of Photoresist by Pluronic Surfactants: An Insight into Their Adsorbed Structure.

Photoresist stripping is the final step in the photolithography process that forms fine patterns for electronic devices. Recently, a mixture of ethylene carbonate (EC) and propylene carbonate (PC) has attracted attention as a new stripper based on its eco-friendliness and anti-corrosiveness. However, the EC/PC mixture causes re-adsorption of the photoresist during a process of subsequent water rinsing. In this study, we characterized the adsorption/desorption of the photoresist and a triblock Pluronic surfactant [poly(ethylene oxide)-poly(propylene oxide)-poly(ethylene oxide)] as a blocking agent on an indium tin oxide (ITO) substrate. In addition, we evaluated the dispersion of photoresist particles. The photoresist polymer formed a thin and rigid adsorption layer on an ITO substrate in the EC/PC mixture. When water was injected into the EC/PC mixture and the photoresist solutions, the photoresist polymer aggregated and was then deposited on the substrate. In contrast, the addition of Pluronic surfactant F-68 (PEO79PPO30PEO79) into the EC/PC mixture remarkably decreased the residual amount of the photoresist on the ITO after water injection. This variation was attributed to the PEO blocks of F-68 extended to the solution phase, whereas the PPO blocks of F-68 functioned as anchors for adsorption onto the photoresist. Therefore, the F-68-adsorbed layer prevented interaction between the photoresist particles or the photoresist and the ITO surface, which provides potential for future applications as new stripping agents with high removal performance.

Evaluation of bone marrow aspirates using the automated hematology analyzer Sysmex XN-3000.

INTRODUCTION: This study evaluated the feasibility of the Sysmex XN-3000 automated hematology analyzer for the assessment of total nucleated cells (TNC) and bone marrow (BM) cell density in routine bone marrow aspiration (BMA) samples. METHODS: A total of 54 BMA samples from 39 hematological patients were evaluated. The number of megakaryocytes was calculated by a specific gating algorithm using the body fluid mode of the WBC differential (WDF) channel. Lipid contents were calculated through a newly developed algorithm utilizing the WDF channel. The ratio of lipid particles over TNCs by the WNR channel was compared with the BM cellularity assessed by the BM biopsy. The myeloid/erythroid (M/E) ratio was calculated by measuring the number of myeloid cells in the WDF channel and the number of nucleated red blood cells (NRBCs) in the WNR channel. RESULTS: XN-3000 counts and microscopic results showed a linear correlation in TNC (R2  = .98, p < .001), megakaryocytes (R2  = .59, p = .002), NRBC (R2  = .84, p < .001), and M/E ratio (R2  = .59, p < .001). There were significant differences in the lipid/TNC ratios of hypercellular, normocellular, and hypocellular BMs measured by XN-3000 (p < .001). Receiver-operating characteristic analysis detected cut-off values of the lipid/TNC ratio of >0.4054 for hypoplasia and <0.157 for hyperplasia. The sensitivity and specificity for hypoplasia were 100% and 88%, and for hyperplasia were 89% and 86%, respectively. CONCLUSION: XN-3000 provides a quantitative assessment of BM cellularity, supporting the qualitative assessment by myelogram and BM biopsy.

SARS-CoV-2 evolution among patients with immunosuppression in a nosocomial cluster of a Japanese medical center during the Delta (AY.29 sublineage) surge.

Background: Previous studies have shown that patients with immunosuppression tend to have longer-lasting SARS-CoV-2 infections and a number of mutations were observed during the infection period. However, these studies were, in general, conducted longitudinally. Mutation evolution among groups of patients with immunosuppression have not been well studied, especially among Asian populations. Methods: Our study targeted a nosocomial cluster of SARS-CoV-2 infection in a Japanese medical center during Delta surge (AY.29 sublineage), involving ward nurses and inpatients. Whole-genome sequencing analyses were performed to examine mutation changes. Haplotype and minor variant analyses were furtherly performed to detect the mutations on the viral genomes in detail. In addition, sequences of the first wild-type strain hCoV-19/Wuhan/WIV04/2019 and AY.29 wild-type strain hCoV-19/Japan/TKYK15779/2021 were used as references to assess the phylogenetical development of this cluster. Results: A total of 6 nurses and 14 inpatients were identified as a nosocomial cluster from September 14 through 28, 2021. All were Delta variant (AY.29 sublineage) positive. 92.9% of infected patients (13 out of 14) were either cancer patients and/or receiving immunosuppressive or steroid treatments. Compared to AY.29 wild type, a total of 12 mutations were found in the 20 cases. Haplotype analysis found one index group of eight cases with F274F (N) mutation and 10 other haplotypes with one to three additional mutations. Furthermore, we found that cases with more than three minor variants were all cancer patients under immunosuppressive treatments. The phylogenetical tree analysis, including 20 nosocomial cluster-associated viral genomes, the first wild-type strain and the AY.29 wild-type strain as references, indicated the mutation development of the AY.29 virus in this cluster. Conclusion: Our study of a nosocomial SARS-CoV-2 cluster highlights mutation acquisition during transmission. More importantly, it provided new evidence emphasizing the need to further improve infection control measures to prevent nosocomial infection among immunosuppressed patients.

Effect of polyols on membrane structures of liposomes: A study using small-angle X-ray scattering data and generalized indirect Fourier transformation.

This study aimed to evaluate the membrane structure of distearoylphosphatidylcholine (DSPC) liposomes dispersed in water containing various types of polyols with low molecular weight such as glycerin (Gly), 1,3-butandiol (BG), and propylene glycol (PG). To clarify the detailed membrane structure, generalized indirect Fourier transformation (GIFT) analysis, which provides information about the bilayer spacing, bilayer thickness, number of lamellar layers, and membrane flexibility, was applied to small-angle X-ray scattering (SAXS) data of the present system. The GIFT results showed that the bilayer thickness of the DSPC liposomes followed the order Gly>>BG>PG. In addition, the membrane flexibility estimated by the Caille parameter was in the order Gly>>BG>PG; this result was supported by the gel-liquid crystal phase transition temperature (Tc) obtained by differential scanning calorimetry (DSC). These results, together with the Raman spectra, suggest that BG and PG incorporated into the bilayers of DSPC liposomes result in the formation of an interdigitated lamellar structure.

Molecular characterization of SARS-CoV-2 detected in Tokyo, Japan during five waves: Identification of the amino acid substitutions associated with transmissibility and severity.

Many variants of SARS-CoV-2 have emerged around the world. It is therefore important to understand its global viral evolution and the corresponding mutations associated with transmissibility and severity. In this study, we analyzed 112 whole genome sequences of SARS-CoV-2 collected from patients at Juntendo University Hospital in Tokyo and the genome data from entire Japan deposited in Global Initiative on Sharing Avian Influenza Data (GISAID) to examine the relationship of amino acid changes with the transmissibility and the severity of each strain/lineage. We identified 12 lineages, including B.1.1.284, B.1.1.214, R.1, AY.29, and AY.29.1, which were prevalent specifically in Japan. B.1.1.284 was most frequently detected in the second wave, but B.1.1.214 became the predominant lineage in the third wave, indicating that B.1.1.214 has a higher transmissibility than B.1.1.284. The most prevalent lineage during the fourth and fifth wave was B.1.1.7 and AY.29, respectively. In regard to the severity of identified lineages, B.1.1.214 was significantly lower than the reference lineage, B.1.1.284. Analysis of the genome sequence and other traits of each lineage/strain revealed the mutations in S, N, and NSPs that increase the transmissibility and/or severity. These mutations include S: M153T, N: P151L, NSP3: S543P, NSP5: P108S, and NSP12: A423V in B.1.1.284; S: W152L and E484K in R.1; S: H69del, V70del, and N501Y in the Alpha strain; S: L452R, T478K, and P681R in the Delta strain. Furthermore, it is suggested that the transmissibility of B.1.1.214 could be enhanced by the mutations N: M234I, NSP14: P43L, and NSP16: R287I. To address the issue of the virus evolution, it is necessary to continuously monitor the genomes of SARS-CoV-2 and analyze the effects of mutations for developing vaccines and antiviral drugs effective against SARS-CoV-2 variants.

Meals and Room Temperature Storage do not Significantly Affect Feasibility of Direct RT-PCR Tests for SARS-CoV-2 Using Saliva.

BACKGROUND: Rapid detection of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) using saliva samples has emerged as a preferred technique since sample collection is easy and noninvasive. In addition, several commercial high-throughput PCR kits that do not require RNA extraction/purification have been developed and are now available for testing saliva samples. However, an optimal protocol for SARS-CoV-2 RT-PCR testing of saliva samples using the RNA extraction/purification-free kits has not yet been established. The aim of this study was to establish optimal preanalytical conditions, including saliva sample collection, storage, and dilution for RNA extraction/purification-free RT-PCR (direct RT-PCR). METHODS: Patients suspected with COVID-19 from March 02 to August 31, 2020, were enrolled in this study. A total of 248 samples, including 43 nasopharyngeal swabs and 205 saliva samples, were collected from 66 patients (37 outpatients and 29 inpatients) and tested using the 2019 Novel Coronavirus Detection Kit (nCoV-DK, Shimadzu Corporation, Kyoto, Japan). RESULTS: The detection results obtained using nasopharyngeal swabs and saliva samples matched 100%. The sampling time, i.e., either awakening time or post-breakfast, had no significant effect on the viral load of the saliva samples. Although saliva samples are routinely diluted to reduce viscosity, we observed that dilution negatively affected PCR sensitivity. Saliva samples could be stored at room temperature (25°C) for 24 hours or at 4°C for up to 48 hours. CONCLUSIONS: This study demonstrated the appropriate conditions of saliva sample collection, processing, and storage, and indicated that the nCoV-DK is applicable to saliva samples, making the diagnosis method simple and safe.

Preparation of Highly Stable Oil-in-Water Emulsions with High Ethanol Content Using Polyglycerol Monofatty Acid Esters as Emulsifiers.

Oil-in-water (O/W) emulsions containing ethanol have been used in food, cosmetics, paints, and other applications. However, O/W emulsions with long-term stability are difficult to produce at high ethanol concentrations because the adsorption of the emulsifier at the O/W interface is restricted by ethanol. In this study, to resolve this issue, we prepared ethanol-containing O/W emulsions with high dispersion stability using a series of polyglycerol monofatty acid esters (PGFEs) with different fatty acid chain lengths, which are bio-safe nonionic surfactants, as emulsifiers. First, aqueous PGFE solutions containing 0-50 wt% ethanol were prepared and then O/W emulsions were formed using limonene as the oil phase. When decaglycerol stearic acid ester (DGMS, C18) was used as the emulsifier, an O/W emulsion with fine droplets (~30 nm in size) was successfully obtained at an ethanol concentration of 35 wt%. This emulsion remained stable for more than four weeks, during which no phase separation occurred, indicating its high dispersion stability. Furthermore, aqueous DGMS solutions containing 30-40 wt% ethanol were viscous, and a lamellar liquid crystal phase was observed to be dispersed in these solutions. The formation of this lamellar liquid crystal phase at the O/W interface led to an interfacial film with superior viscoelastic properties. The results suggested that the stability of the emulsions was determined by the balance between the decrease in interfacial tension caused by the addition of ethanol and the density (rigidity) of the DGMS film formed at the O/W interface. Finally, to further improve the dispersion stability of the ethanol-containing O/W-type emulsions, O/W emulsions were prepared using a mixture of two PGFEs with different degrees of glycerol polymerization, that is, systems having different hydrophilic-lipophilic balance values.

Induction of Paraptosis by Cyclometalated Iridium Complex-Peptide Hybrids and CGP37157 via a Mitochondrial Ca2+ Overload Triggered by Membrane Fusion between Mitochondria and the Endoplasmic Reticulum.

We previously reported that a cyclometalated iridium (Ir) complex-peptide hybrid (IPH) 4 functionalized with a cationic KKKGG peptide unit on the 2-phenylpyridine ligand induces paraptosis, a relatively newly found programmed cell death, in cancer cells (Jurkat cells) via the direct transport of calcium (Ca2+) from the endoplasmic reticulum (ER) to mitochondria. Here, we describe that CGP37157, an inhibitor of a mitochondrial sodium (Na+)/Ca2+ exchanger, induces paraptosis in Jurkat cells via intracellular pathways similar to those induced by 4. The findings allow us to suggest that the induction of paraptosis by 4 and CGP37157 is associated with membrane fusion between mitochondria and the ER, subsequent Ca2+ influx from the ER to mitochondria, and a decrease in the mitochondrial membrane potential (ΔΨm). On the contrary, celastrol, a naturally occurring triterpenoid that had been reported as a paraptosis inducer in cancer cells, negligibly induces mitochondria-ER membrane fusion. Consequently, we conclude that the paraptosis induced by 4 and CGP37157 (termed paraptosis II herein) proceeds via a signaling pathway different from that of the previously known paraptosis induced by celastrol, a process that negligibly involves membrane fusion between mitochondria and the ER (termed paraptosis I herein).

Design, Synthesis, and Anticancer Activity of Triptycene-Peptide Hybrids that Induce Paraptotic Cell Death in Cancer Cells.

We report on the design and synthesis of triptycene-peptide hybrids (TPHs), 5, syn-6, and anti-6, which are conjugates of a triptycene core unit with two or three cationic KKKGG peptides (K: lysine and G: glycine) through a C8 alkyl chain. It was discovered that syn-6 and anti-6 induce paraptosis, a type of programmed cell death (PCD), in Jurkat cells (leukemia T-lymphocytes). Mechanistic studies indicate that these TPHs induce the transfer of Ca2+ from the endoplasmic reticulum (ER) to mitochondria, a loss of mitochondrial membrane potential (ΔΨm), tethering of the ER and mitochondria, and cytoplasmic vacuolization in the paraptosis processes.

Cyclometalated Iridium(III) Complex-Cationic Peptide Hybrids Trigger Paraptosis in Cancer Cells via an Intracellular Ca2+ Overload from the Endoplasmic Reticulum and a Decrease in Mitochondrial Membrane Potential.

In our previous paper, we reported that amphiphilic Ir complex-peptide hybrids (IPHs) containing basic peptides such as KK(K)GG (K: lysine, G: glycine) (e.g., ASb-2) exhibited potent anticancer activity against Jurkat cells, with the dead cells showing a strong green emission. Our initial mechanistic studies of this cell death suggest that IPHs would bind to the calcium (Ca2+)-calmodulin (CaM) complex and induce an overload of intracellular Ca2+, resulting in the induction of non-apoptotic programmed cell death. In this work, we conduct a detailed mechanistic study of cell death induced by ASb-2, a typical example of IPHs, and describe how ASb-2 induces paraptotic programmed cell death in a manner similar to that of celastrol, a naturally occurring triterpenoid that is known to function as a paraptosis inducer in cancer cells. It is suggested that ASb-2 (50 µM) induces ER stress and decreases the mitochondrial membrane potential (ΔΨm), thus triggering intracellular signaling pathways and resulting in cytoplasmic vacuolization in Jurkat cells (which is a typical phenomenon of paraptosis), while the change in ΔΨm values is negligibly induced by celastrol and curcumin. Other experimental data imply that both ASb-2 and celastrol induce paraptotic cell death in Jurkat cells, but this induction occurs via different signaling pathways.

Amphiphilic Cationic Triscyclometalated Iridium(III) Complex-Peptide Hybrids Induce Paraptosis-like Cell Death of Cancer Cells via an Intracellular Ca2+-Dependent Pathway.

We report on the design and synthesis of a green-emitting iridium complex-peptide hybrid (IPH) 4, which has an electron-donating hydroxyacetic acid (glycolic acid) moiety between the Ir core and the peptide part. It was found that 4 is selectively cytotoxic against cancer cells, and the dead cells showed a green emission. Mechanistic studies of cell death indicate that 4 induces a paraptosis-like cell death through the increase in mitochondrial Ca2+ concentrations via direct Ca2+ transfer from ER to mitochondria, the loss of mitochondrial membrane potential (ΔΨm), and the vacuolization of cytoplasm and intracellular organelle. Although typical paraptosis and/or autophagy markers were upregulated by 4 through the mitogen-activated protein kinase (MAPK) signaling pathway, as confirmed by Western blot analysis, autophagy is not the main pathway in 4-induced cell death. The degradation of actin, which consists of a cytoskeleton, is also induced by high concentrations of Ca2+, as evidenced by costaining experiments using a specific probe. These results will be presented and discussed.

Phase Behavior and Polymerization of the Ternary Polymerizable Cationic Gemini Surfactant/Fatty Alcohol/Water System.

We studied the phase behavior of a ternary polymerizable gemini surfactant (PC11-6-11)/1-undecanol/water system and stabilized these liquid crystalline structures through the polymerization of surfactants. The addition of 1-undecanol to a PC11-6-11/water system formed bicontinuous cubic (V1) and reversed hexagonal (H2) liquid crystal phases in addition to hexagonal (H1) and lamellar (Lα) phases, which were also formed using the binary system of PC11-6-11/water. These new phases were formed because the fatty alcohol penetrated the palisade layer of the PC11-6-11 micelles. The polymerization of PC11-6-11 with a thermal initiator successfully preserved the Lα and H2 phases. Layered or honeycomb structures of these liquid crystals were clearly observed by transmission electron microscopy. The nanomaterials have potential applications as membranes for nano- or microfiltration and catalyst support materials.

Accelerated Recombination of Lophyl Radicals with Self-Assembled Amphiphilic Lophine Dimer.

This report focuses on acceleration of the recombination of lophyl radicals with a lophine dimer derivative by forming molecular assemblies. A newly synthesized cationic amphiphilic lophine dimer formed molecular assemblies with a diameter of ~220 nm in an aqueous medium. When the molecular assemblies were formed, the rate of recombination of lophyl radicals, produced by ultraviolet light irradiation, was accelerated 50,000-fold compared to that in an organic solvent. The rate enhancement is likely derived from the short diffusion distance of the lophyl radicals in the molecular assemblies. These results revealed that accumulation of the lophine dimers via self-assembly remarkably accelerated the recombination of lophyl radicals. This novel photoisomerization system could rapidly control the interfacial properties or morphologies of molecular assemblies when used for applications, such as efficient delivery of drugs or active components.

A new highly sensitive real-time quantitative-PCR method for detection of BCR-ABL1 to monitor minimal residual disease in chronic myeloid leukemia after discontinuation of imatinib.

Tyrosine kinase inhibitors (TKIs) targeting the BCR-ABL1 fusion protein, encoded by the Philadelphia chromosome, have drastically improved the outcomes for patients with chronic myeloid leukemia (CML). Although several real-time quantitative polymerase chain reaction (RQ-PCR) kits for the detection of BCR-ABL1 transcripts are commercially available, their accuracy and efficiency in laboratory practice require reevaluation. We have developed a new in-house RQ-PCR method to detect minimal residual disease (MRD) in CML cases. MRD was analyzed in 102 patients with CML from the DOMEST study, a clinical trial to study the rationale for imatinib mesylate discontinuation in Japan. The BCR-ABL1/ABL1 ratio was evaluated using the international standard (IS) ratio, where IS < 0.1% was defined as a major molecular response. At enrollment, BCR-ABL1 transcripts were undetectable in all samples using a widely-applied RQ-PCR method performed in the commercial laboratory, BML (BML Inc., Tokyo, Japan); however, the in-house method detected the BCR-ABL1 transcripts in five samples (5%) (mean IS ratio: 0.0062 ± 0.0010%). After discontinuation of imatinib, BCR-ABL1 transcripts were detected using the in-house RQ-PCR in 21 patients (21%) that were not positive using the BML method. Nineteen samples were also tested using a commercially available RQ-PCR assay kit with a detection limit of IS ratio, 0.0032 (ODK-1201, Otsuka Pharmaceutical Co., Tokyo, Japan). This method detected low levels of BCR-ABL1 transcripts in 14 samples (74%), but scored negative for five samples (26%) that were positive using the in-house method. From the perspective of the in-house RQ-PCR method, number of patients confirmed loss of MMR was 4. These data suggest that our new in-house RQ-PCR method is effective for monitoring MRD in CML.

Eprobe mediated RT-qPCR for the detection of leukemia-associated fusion genes.

The detection and quantification of leukemia-associated fusion gene transcripts play important roles in the diagnosis and follow-up of leukemias. To establish a standardized method without interlaboratory discrepancies, we developed a novel one-step reverse transcription quantitative PCR (RT-qPCR) assay, called "the Eprobe leukemia assay," for major and minor BCR-ABL1, RUNX1-RUNX1T1, and various isoforms of PML-RARA. This assay is comprised of Eprobes that are exciton-controlled hybridization-sensitive fluorescent oligonucleotides. Melting curve analyses were performed on synthetic quantitative standard RNAs with strict quality control. Quantification capacity was evaluated by comparison with TaqMan RT-qPCR using 67 primary leukemia patient samples. The lower limit of detection and the limit of quantification of this assay were less than 31.3 copies/reaction and 62.5 copies/reaction, respectively. This assay correctly detected the fusion genes in samples with 100% sensitivity and specificity. The specificity of the reactions was confirmed by melting curve analyses. The assay detected low-level expression of minor BCR-ABL1 co-expressed with major BCR-ABL1. These results illustrate the feasibility and high accuracy of the Eprobe leukemia assay, even for minimal residual disease monitoring.

Evaluation of cell count and classification capabilities in body fluids using a fully automated Sysmex XN equipped with high-sensitive Analysis (hsA) mode and DI-60 hematology analyzer system.

The XN series automated hematology analyzer has been equipped with a body fluid (BF) mode to count and differentiate leukocytes in BF samples including cerebrospinal fluid (CSF). However, its diagnostic accuracy is not reliable for CSF samples with low cell concentration at the border between normal and pathologic level. To overcome this limitation, a new flow cytometry-based technology, termed "high sensitive analysis (hsA) mode," has been developed. In addition, the XN series analyzer has been equipped with the automated digital cell imaging analyzer DI-60 to classify cell morphology including normal leukocytes differential and abnormal malignant cells detection. Using various BF samples, we evaluated the performance of the XN-hsA mode and DI-60 compared to manual microscopic examination. The reproducibility of the XN-hsA mode showed good results in samples with low cell densities (coefficient of variation; % CV: 7.8% for 6 cells/µL). The linearity of the XN-hsA mode was established up to 938 cells/µL. The cell number obtained using the XN-hsA mode correlated highly with the corresponding microscopic examination. Good correlation was also observed between the DI-60 analyses and manual microscopic classification for all leukocyte types, except monocytes. In conclusion, the combined use of cell counting with the XN-hsA mode and automated morphological analyses using the DI-60 mode is potentially useful for the automated analysis of BF cells.