Mitral regurgitation is associated with similar loss of von Willebrand factor large multimers but lower frequency of anemia compared with aortic stenosis.

Background: Various cardiovascular diseases cause acquired von Willebrand syndrome (AVWS), which is characterized by a decrease in high-molecular-weight (large) von Willebrand factor (VWF) multimers. Mitral regurgitation (MR) has been reported as a cause of AVWS. However, much remains unclear about AVWS associated with MR. Objectives: To evaluate VWF multimers in MR patients and examine their impact on clinical characteristics. Methods: Moderate or severe MR patients (n = 84) were enrolled. VWF parameters such as the VWF large multimer index (VWF-LMI), a quantitative value that represents the amount of VWF large multimers, and clinical data were prospectively analyzed. Results: At baseline, the mean hemoglobin level was 12.9 ± 1.9 g/dL and 58 patients (69.0%) showed loss of VWF large multimers defined as VWF-LMI < 80%. VWF-LMI in patients with degenerative MR was lower than in those with functional MR. VWF-LMI appeared to be restored the day after mitral valve intervention, and the improvement was maintained 1 month after the intervention. Seven patients (8.3%) had a history of bleeding, 6 (7.1%) of whom had gastrointestinal bleeding. Gastrointestinal endoscopy was performed in 23 patients (27.4%) to investigate overt gastrointestinal bleeding, anemia, etc. Angiodysplasia was detected in 2 of the 23 patients (8.7%). Conclusion: Moderate or severe MR is frequently associated with loss of VWF large multimers, and degenerative MR may cause more severe loss compared with functional MR. Mitral valve intervention corrects the loss of VWF large multimers. Gastrointestinal bleeding may be relatively less frequent and hemoglobin level remains stable in MR patients.

von Willebrand factor Ristocetin co-factor activity to von Willebrand factor antigen level ratio for diagnosis of acquired von Willebrand syndrome caused by aortic stenosis.

Background: Severe aortic stenosis (AS) causes acquired von Willebrand syndrome by the excessive shear stress-dependent cleavage of high molecular weight multimers of von Willebrand factor (VWF). While the current standard diagnostic method is so-called VWF multimer analysis that is western blotting under nonreducing conditions, it remains unclear whether a ratio of VWF Ristocetin co-factor activity (VWF:RCo) to VWF antigen levels (VWF:Ag) of <0.7, which can be measured with an automated coagulation analyzer in clinical laboratories and is used for the diagnosis of hereditary von Willebrand disease. Objectives: To evaluated whether the VWF:RCo/VWF:Ag is useful for the diagnosis of AS-induced acquired von Willebrand syndrome. Methods: VWF:RCo and VWF:Ag were evaluated with the VWF large multimer index as a reference, which represents the percentage of a patient's VWF high molecular weight multimer ratio to that of standard plasma in the VWF multimer analysis. Results: We analyzed 382 patients with AS having transaortic valve maximal pressure gradients of >30 mmHg, 27 patients with peripheral artery disease, and 46 control patients free of cardiovascular disease with osteoarthritis, diabetes, and so on. We assumed a large multimer index of <80% as loss of VWF large multimers since 59.0% of patients with severe AS had the indices of <80%, while no control patients or patients with peripheral artery disease, except for 2 patients, exhibited the indices of <80%. The VWF:RCo/VWF:Ag ratios, measured using an automated blood coagulation analyzer, were correlated with the indices (rs = 0.470, P < .001). When the ratio of <0.7 was used as a cut-off point, the sensitivity and specificity to VWF large multimer indices of <80% were 0.437 and 0.826, respectively. Conclusion: VWF:RCo/VWF:Ag ratios of <0.7 may indicate loss of VWF large multimers with high specificity, but low sensitivity. VWF:RCo/VWF:Ag ratios in patients with AS having a ratio of <0.7 may be useful for monitoring the loss of VWF large multimers during their clinical courses.

Impact of intraoperative use of venovenous extracorporeal membrane oxygenation on the status of von Willebrand factor large multimers during single lung transplantation.

Background: von Willebrand factors (vWFs), hemostatic factors, are produced as large multimers and are shear stress-dependently cleaved to become the appropriate size. A reduction in vWF large multimers develops in various conditions including the use of extracorporeal life support, which can cause excessive-high shear stress in the blood flow and result in hemostatic disorders. The objective of this prospective study was to investigate the impact of venovenous extracorporeal membrane oxygenation (VV ECMO) use on the status of vWF large multimers and hemostatic disorders during single lung transplantation (SLT). Methods: We prospectively enrolled 12 patients who underwent SLT at our center. Among them, seven patients were supported by VV ECMO intraoperatively (ECMO group) and the remaining five patients underwent SLT without ECMO support (control group). The vWF large multimer index (%) was defined as the ratio of the large multimer proportion in total vWF (vWF large multimer ratio) derived from a patient's plasma to that from standard human plasma. Results: The vWF large multimer index at the end of the surgery was significantly lower in the ECMO group than in the control group (112.6% vs. 75.8%, respectively; P<0.05). The intraoperative blood loss and the amounts of intraoperative transfusion products in the ECMO group tended to be greater than those in the control group; however, the differences were not significant. Conclusions: During SLT, the use of VV ECMO caused a decrease in the vWF large multimer index. The short duration of time of VV ECMO use in our study did not significantly affect the intra- and postoperative outcomes including blood loss, blood transfusion, and re-exploration thoracotomy for bleeding. Nevertheless, to comprehensively evaluate the actual influence of this decrease in the vWF large multimer index on intra- and postoperative outcomes, a multicenter larger-scale study is warranted.

Blood Culture Result Profile in Patients With Central Line-Associated Bloodstream Infection (CLABSI): A Single-Center Experience.

Background Central line-associated bloodstream infection (CLABSI) is among the most common bloodstream infections in the university hospital and intensive care unit settings. This study evaluated the routine blood test findings and microbe profiles of bloodstream infection (BSI) by the presence and types of central vein (CV) access devices (CVADs). Methods A total of 878 inpatients at a university hospital who were clinically suspected for BSI and underwent blood culture (BC) testing between April 2020 and September 2020 were enrolled. Data regarding age at BC testing, sex, WBC count, serum C-reactive protein (CRP) level, BC test results, yielded microbes, and usage and types of CVADs were evaluated. Results The BC yields were detected in 173 patients (20%), suspected contaminating pathogens in 57 (6.5%), and 648 (74%) with a negative yield. The WBC count (p=0.0882) and CRP level (p=0.2753) did not significantly differ between the 173 patients with BSI and the 648 patients with negative BC yields. Among the 173 patients with BSI, 74 used CVADs and met the diagnosis of CLABSI; 48 had a CV catheter, 16 had CV access ports, and 10 had a peripherally inserted central catheter (PICC). Patients with CLABSI showed lower WBC counts (p=0.0082) and serum CRP levels (p=0.0024) compared to those with BSI who did not use CVADs. The most commonly yielded microbes in those with CV catheters, CV-ports, and PICC were Staphylococcus epidermidis (n=9; 19%), Staphylococcus aureus (n=6; 38%), and S. epidermidis (n=8; 80%), respectively. Among those with BSI who did not use CVADs, Escherichia coli (n=31; 31%) was the most common pathogen, followed by S. aureus (n=13; 13%). Conclusion Patients with CLABSI showed lower WBC counts and CRP levels than those with BSI who did not use CVADs. Staphylococcus epidermidis was among the most common microbes in CLABSI and accounted for the majority of yielded microbes in patients who used PICC.

Rapid Detection of SARS-CoV-2 RNA Using Reverse Transcription Recombinase Polymerase Amplification (RT-RPA) with Lateral Flow for N-Protein Gene and Variant-Specific Deletion-Insertion Mutation in S-Protein Gene.

Rapid molecular testing for severe acute respiratory coronavirus 2 (SARS-CoV-2) variants may contribute to the development of public health measures, particularly in resource-limited areas. Reverse transcription recombinase polymerase amplification using a lateral flow assay (RT-RPA-LF) allows rapid RNA detection without thermal cyclers. In this study, we developed two assays to detect SARS-CoV-2 nucleocapsid (N) gene and Omicron BA.1 spike (S) gene-specific deletion-insertion mutations (del211/ins214). Both tests had a detection limit of 10 copies/µL in vitro and the detection time was approximately 35 min from incubation to detection. The sensitivities of SARS-CoV-2 (N) RT-RPA-LF by viral load categories were 100% for clinical samples with high (>9015.7 copies/µL, cycle quantification (Cq): < 25) and moderate (385.5-9015.7 copies/µL, Cq: 25-29.9) viral load, 83.3% for low (16.5-385.5 copies/µL, Cq: 30-34.9), and 14.3% for very low (<16.5 copies/µL, Cq: 35-40). The sensitivities of the Omicron BA.1 (S) RT-RPA-LF were 94.9%, 78%, 23.8%, and 0%, respectively, and the specificity against non-BA.1 SARS-CoV-2-positive samples was 96%. The assays seemed more sensitive than rapid antigen detection in moderate viral load samples. Although implementation in resource-limited settings requires additional improvements, deletion-insertion mutations were successfully detected by the RT-RPA-LF technique.

Evaluation of the performance of GeneSoC®, a novel rapid real-time PCR system, to detect Staphylococcus aureus and methicillin resistance in blood cultures.

Staphylococcus aureus bacteremia results in substantial mortality. Rapid identification and the determination of methicillin susceptibility are crucial for immediate treatment with appropriate antibiotics. In the present study, we aimed to evaluate the basic assay performance of GeneSoC®, a novel rapid quantitative polymerase chain reaction (qPCR) method, for the detection of methicillin-susceptible (MS) or -resistant (MR) S. aureus in blood culture (BC) bottles. qPCR pimers and probes were desinged for femA and mecA genes to diagnose S. aureus and its methicilline-resistance status. GeneSoC® system can detect target genes within 12 min per sample using microfludic thermal cycling. A total of 100 BC-positive samples, showing clusters of gram-positive cocci using microscopy, were tested. The analytical sensitivity was demonstrated for the target sequence of femA and mecA genes at 10 copies/µL, respectively. The detection limit of the MRSA bacterial burden using this system was 104 and 103 CFU/mL for femA and mecA, respectively. Compared with culture-based identification and susceptibility testing, the sensitivity and specificity for the detection of femA (+)/mecA (+) MRSA using GeneSoC® were 90.9 and 98.9%, respectively, whereas the sensitivity and specificity for detection of femA (+)/mecA (-) MSSA were 96.2% and 97.3%, respectively. In conclusion, although this was a small sample and pilot study, the GeneSoC® system is beneficial for rapid, reliable, and highly sensitive real-time testing of MRSA and MSSA in BC bottles.

Response to COVID-19 vaccine is reduced in patients with inflammatory bowel disease, but improved with additional dose.

BACKGROUND: Coronavirus disease 2019 (COVID-19) vaccination is recommended for patients with inflammatory bowel disease (IBD); however, suppressed immune responses have been reported for fully vaccinated patients under immunosuppressive therapy, mainly from Western countries. We prospectively analyzed antibody titers of IBD patients in Asia induced by two-dose and additional dose of messengerRNA COVID-19 vaccine. METHODS: After measuring high-affinity antibody titers, factors associated with antibody titers were identified by multiple regression analyses using the following covariates: sex, age (≥60 or <60 years), disease type (Crohn's disease or ulcerative colitis), vaccine type (BNT162b2 or mRNA-1273), time from second/third vaccination, molecular-targeted agent (anti-tumor necrosis factor [TNF] agents, ustekinumab, vedolizumab, tofacitinib, or no molecular-targeted agents), thiopurine, steroid, and 5-aminosalicylic acid. RESULTS: Among 409 patients analyzed, mean titer was 1316.7 U/mL (SD, 1799.3); 403 (98.5%) were judged to be seropositive (≥0.8 U/mL), and 389 (95.1%) had neutralizing antibodies (≥15 U/mL). After the third vaccination, mean titer raised up to 21 123.8 U/mL (SD, 23 474.5); all 179 were seropositive, and 178 (99.4%) had neutralizing antibodies. In 248 patients with genetic data, there was no difference in mean titer after two/third doses between carriers and non-carriers of HLA-A24 associated with severe disease during COVID-19 infection. A multiple regression analyses using covariates revealed that older age, vaccine type (BNT162b2), time from second/third dose, anti-TNF agent, tofacitinib, and thiopurine were independently associated with lower antibody titers. CONCLUSIONS: Our findings further support the recommendation for COVID-19 vaccination in patients under immunosuppressive therapy, especially additional third dose for patients receiving anti-TNF agents and/or thiopurine or tofacitinib.

Flow Cytometry-Based Photodynamic Diagnosis with 5-Aminolevulinic Acid for the Detection of Minimal Residual Disease in Multiple Myeloma.

Multiple myeloma is the cancer of plasma cells. Along with the development of new and effective therapies, improved outcomes in patients with multiple myeloma have increased the interest in minimal residual disease (MRD) monitoring. However, the considerable heterogeneity of immunophenotypic and molecular markers of myeloma cells has limited its clinical application. 5-Aminolevulinic acid (ALA) is a natural compound in the heme biosynthesis pathway. Following ALA treatment, tumor cells preferentially accumulate porphyrins because of the differential activities of aerobic glycolysis, known as Warburg effect. Among various porphyrins, protoporphyrine IX is a strong photosensitizer; thus, ALA-based photodynamic diagnosis has been widely used in various solid cancers. Here, the feasibility of flow cytometry-based photodynamic detection of MRD was tested in multiple myeloma. Among various human cell lines of hematological malignancies, including K562 erythroleukemia, Jurkat T-cell leukemia, Nalm6 pre-B cell leukemia, KG1a myeloid leukemia, and U937 monocytic leukemia, human myeloma cell line, KMS18, and OPM2 abundantly expressed ALA transporters, such as SLC36A1 and SLC15A2, and 1 mM ALA treatment for 24 h resulted in nearly 100% porphyrin fluorescence expression, which could be competitively inhibited by ALA transport with gamma-aminobutyric acid. Titration studies revealed that the lowest ALA concentration required to achieve nearly 100% porphyrin fluorescence in KMS18 cells was 0.25 mM, with an incubation period of 2 h. Under these conditions, incubation of primary peripheral blood mononuclear cells resulted in only 1.8 % of the cells exhibiting porphyrin fluorescence. Therefore, flow cytometry-based photodynamic diagnosis is a promising approach for detecting MRD in multiple myeloma.

[The Activity to Successfully Acquire ISO 15189 Accreditation].

Our facilities acquired ISO 15189 accreditation in April 2011. The lead time was a little less than 2 years. I reported on the lead time and activity after ISO 15189 acquisition. I reported how creating a quality manual and SOP was advanced as preparation work. I showed the examination method in detail and the process after examination. I think an increase in per- sonnel as internal auditors is important for quality management system maintenance.

Gene expression profiling identifies HOXB4 as a direct downstream target of GATA-2 in human CD34+ hematopoietic cells.

Aplastic anemia is characterized by a reduced hematopoietic stem cell number. Although GATA-2 expression was reported to be decreased in CD34-positive cells in aplastic anemia, many questions remain regarding the intrinsic characteristics of hematopoietic stem cells in this disease. In this study, we identified HOXB4 as a downstream target of GATA-2 based on expression profiling with human cord blood-derived CD34-positive cells infected with control or GATA-2 lentiviral shRNA. To confirm the functional link between GATA-2 and HOXB4, we conducted GATA-2 gain-of-function and loss-of-function experiments, and HOXB4 promoter analysis, including luciferase assay, in vitro DNA binding analysis and quantitative ChIP analysis, using K562 and CD34-positive cells. The analyses suggested that GATA-2 directly regulates HOXB4 expression through the GATA sequence in the promoter region. Furthermore, we assessed GATA-2 and HOXB4 expression in CD34-positive cells from patients with aplastic anemia (n = 10) and idiopathic thrombocytopenic purpura (n = 13), and demonstrated that the expression levels of HOXB4 and GATA-2 were correlated in these populations (r = 0.6573, p<0.01). Our results suggested that GATA-2 directly regulates HOXB4 expression in hematopoietic stem cells, which may play an important role in the development and/or progression of aplastic anemia.

Stanniocalcin-1 as a novel marker to detect minimal residual disease of human leukemia.

Stanniocalcin is a glycoprotein hormone that regulates the calcium level in fish. We found that mRNA of human stanniocalcin 1 (STC-1) is detectable in phytohemagglutinin-stimulated T cells and in most human leukemia cell lines, suggesting a role of STC-1 for cell proliferation. This finding prompts us to study the usefulness of STC-1 for monitoring acute leukemia. The levels of STC-1 transcripts increased in patients with acute leukemia at diagnosis and relapse, as judged by quantitative real-time RT-PCR. Levels of transcripts rapidly decreased to within the cut-off levels, when the blast numbers decreased with chemotherapy. Prolonged elevation of STC-1 levels after treatment was associated with a poor prognosis. All of 7 patients relapsed 1 to 4 months after they showed an elevated level of the transcripts in clinical remission. These results indicate that STC-1 is a novel marker for minimal residual disease of acute leukemia, and for an early diagnosis of relapse.

Detection of bone marrow infiltration of lymphoma cells by fluorescence in situ hybridization.

BACKGROUND: It is sometimes difficult to detect the bone marrow infiltration of lymphoma cells, because lymphoma cells are not distinguishable from normal lymphocytes due to the similarity of their phenotype. METHODS: Bone marrow involvement of 17 samples of 15 patients with follicular lymphoma, whose lymphoma cells were confirmed to harbor the translocation of chromosome14q32, were examined by microscopic analysis of bone marrow smear and biopsy, flow cytometorical analysis (FCM), chromosomal analysis of G-banding and fluorescence in situ hybridization (FISH). FISH was performed using a probe, which detects the split of IGH gene on 14q32. RESULTS: The positivity of FISH was highest among these methods and FISH was able to detect the bone marrow involvement in one case who was defined as negative by bone marrow biopsy. CONCLUSIONS: FISH can be used for detection of bone marrow involvement of malignant lymphoma that carries chromosomal rearrangement involving 14q32.

Quantitative analysis of a MDR1 transcript for prediction of drug resistance in acute leukemia.

BACKGROUND: Assessing the drug resistance of leukemic cells is important for treatment of leukemia. We developed a quantitative reverse transcription (RT)-PCR method for multidrug resistance 1 (MDR1) and multidrug resistance-related protein 1 (MRP1) transcripts to evaluate drug resistance, and applied it to clinical samples. METHODS: The cutoffs for copy numbers of MDR1 and MRP1 transcripts were defined based on copy numbers in healthy bone marrow mononuclear cells. To confirm that the cutoffs reflected biological resistance, we established vincristine (VCR)-resistant K562 sublines that showed various degrees of drug resistance and examined the correlation between the copy numbers of these transcripts and the biological resistance of these clones. In addition, we compared the sensitivity and specificity of quantitative RT-PCR to a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometric (FCM) analysis. RESULTS: The defined cutoff for copy numbers of MDR1 transcripts corresponded with the degree of biological resistance of VCR-resistant K562 sublines. Clinical study revealed that the concentrations of MDR1 mRNA in all relapsed patients with acute myelogenous leukemia (AML) were above the cutoff. Moreover, both AML and acute lymphoblastic leukemia patients with high MDR1 mRNA expression at diagnosis tended to show a low remission rate and short remission periods. No association was observed between the amounts of MRP1 transcripts and clinical outcomes. The specificity and sensitivity of quantitative RT-PCR for MDR1 were superior to the MTT assay and FCM analysis. CONCLUSION: These results suggest the efficacy of this quantitative analysis of MDR1 transcripts for the prediction of clinical drug resistance in acute leukemia.