BACKGROUND:Ethylenediamine tetraacetate/glycine acid (EGA) and chloroquine diphosphate (CDP) are used in transfusion testing to dissociate IgG antibodies from red blood cells (RBCs). However, the ability of these reagents to dissociate IgM antibodies sensitized to RBCs has not been comprehensively elucidated. We investigated whether EGA and CDP could dissociate cold-reactive antibodies from RBCs and their effect on RBCs after dissociation treatment. STUDY DESIGN AND METHODS:Cold-reactive antibody-sensitized RBC samples were prepared by mixing group A RBCs and group B plasma and treated with EGA, CDP, and dithiothreitol (DTT). Before and after the dissociation treatment, changes in the agglutination of these RBCs were assessed using the test tube method. Flow cytometric analysis was used to confirm the nature of antibodies bound to RBCs. Additionally, RBC morphology was evaluated using scanning electron microscopy. This study utilized off-label use of EGA and CDP. RESULTS:Flow cytometric analysis showed that antibodies sensitized to RBCs were mainly IgM antibodies. After antibody dissociation, agglutination disappeared in the EGA-treated samples to the same degree as in the DTT-treated samples. However, IgM antibodies remained in the CDP-treated samples. Regarding RBC morphology, RBC surface appeared coarser in both EGA- and CDP-treated samples, and RBC area was significantly smaller in the CDP-treated samples than in the EGA-treated samples. DISCUSSION:EGA could dissociate cold-reactive antibodies, whereas CDP had a higher residual antibody content. This difference in dissociation ability appears to correlate with the antibody pH of the dissociation reagent. EGA treatment may be useful in cases of sensitization by high-titer cold-reactive antibodies.

BACKGROUND:Activated partial thromboplastin time (APTT) is susceptible to reagent composition. This study aimed to investigate a large number of specimens and determine the cause of discrepancies. METHOD:This study included 18,994 subjects who underwent coagulation tests at our hospital from May 2020 to December 2020. Measuring reagents included HemosIL SynthASil APTT (APTT-SS, Instrumentation Laboratory) and Coagpia APTT-N (APTT-N, Sekisui Medical). RESULTS:A total of 451 patients demonstrated APTT-N of >39 seconds and an APTT-N/SS ratio of >1.3. A C-reactive protein (CRP) level of ≥1.4 mg/L demonstrated a significant positive correlation, with a higher APTT-N/SS indicating higher CRP levels. All 28 subjects receiving no anticoagulants and who had remaining specimens underwent a cross-mixing test (CMT). Of them, 17 were suspected for lupus anticoagulant (LA) by both the waveform shape and the index of circulating anticoagulant (ICA) value, 6 by the ICA value, and 5 were difficult to determine. CONCLUSION:This study revealed that the APTT-N prolongation correlated with CRP degree and the transient involvement of LA in CMT results due to CRP. This study indicated various reactivities depending on the assay reagents used. Further testing is warranted if LA is suspected, considering the patient's background.

Thioredoxin-interacting protein (TXNIP) plays an important role in glucose metabolism, and its expression is regulated by DNA methylation (DNAm). Although the association between TXNIP DNAm and type 2 diabetes mellitus has been demonstrated in studies with a cross-sectional design, prospective studies are needed. We therefore examined the association between TXNIP DNAm levels and longitudinal changes in glycemic traits by conducting a longitudinal study involving 169 subjects who underwent two health checkups in 2015 and 2019. We used a pyrosequencing assay to determine TXNIP DNAm levels in leukocytes (cg19693031). Logistic regression analyses were performed to assess the associations between dichotomized TXNIP DNAm levels and marked increases in glycemic traits. At four years, the TXNIP DNA hypomethylation group had a higher percentage of changes in fasting plasma glucose (FPG) and hemoglobin A1c (HbA1c) compared to those in the hypermethylation group. The adjusted odds ratios for FPG and HbA1c levels were significantly higher in the TXNIP DNA hypomethylation group than in the hypermethylation group. We found that TXNIP DNA hypomethylation at baseline was associated with a marked increase in glycemic traits. Leukocyte TXNIP DNAm status could potentially be used as an early biomarker for impaired glucose homeostasis.

The renal angina index (RAI) is a validated scoring tool for predicting acute kidney injury (AKI). We investigated the efficacy of the RAI in 2436 heterogeneous patients (mean age, 70 years) treated in cardiac intensive care units (CICUs). The RAI was calculated from creatinine and patient condition scores. AKI was diagnosed by the Kidney Disease: Improving Global Outcome criteria. The primary and secondary endpoints were the development of severe AKI and all-cause mortality, respectively. Four hundred thirty-three patients developed AKI, 87 of them severe. In multivariate analyses, the RAI was a significant independent predictor of severe AKI. During the 12-month follow-up period, 210 patients suffered all-cause death. Elevated RAI was independently associated with all-cause mortality, as was NT-proBNP (p < 0.001). The RAI is a potent predictor not only of severe AKI but also of adverse outcomes and substantially improved the 12-month risk stratification of patients hospitalized in CICUs.

Preexisting cardiovascular disease (CVD) is a pivotal risk factor for severe coronavirus disease 2019 (COVID-19). We investigated the longitudinal (over 1 year and 9 months) humoral and cellular responses to primary series and booster doses of mRNA COVID-19 vaccines in patients with CVD. Twenty-six patients with CVD who received monovalent mRNA COVID-19 vaccines were enrolled in this study. Peripheral blood samples were serially drawn nine times from each patient. IgG against the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) spike receptor-binding domain (RBD) was measured using an enzyme-linked immunosorbent assay. The numbers of interferon-γ-releasing cells in response to SARS-CoV-2 peptides were measured using an enzyme-linked immunospot assay. The RBD-IgG titers increased 2 weeks after the primary series and booster vaccination and waned 6 months after vaccination. The S1-specific T cell responses in patients aged < 75 years were favorable before and after booster doses; however, the Omicron BA.1-specific T cell responses were poor. These results suggest that regular vaccination is useful to maintain long-term antibody levels and has implications for booster dose strategies in patients with CVD. Additional booster doses, including Omicron variant-adapted mRNA vaccines, may be recommended for patients with CVD, regardless of age.

The CHA2DS2 -VASc score is a vital clinical tool for evaluating thromboembolic risk in patients with atrial fibrillation (AF). This study investigated the efficacy of the CHA2DS2 -VASc score in a cohort of 737 heterogeneous patients (mean age: 63 years) receiving care in cardiac intensive care units (CICUs), with a creatinine-based estimated glomerular filtration rate (eGFR) of ≥60 mL/min/1.73 m2 upon admission and discharge. Incident chronic kidney disease (CKD) was defined as the emergence of a new-onset eGFR<60 mL/min/1.73 m2, accompanied by a decline of >5 mL/min/1.73 m2 compared to that at discharge. The primary endpoint was the incidence of CKD, and the secondary endpoints included all-cause mortality, cardiovascular events, and progression to end-stage kidney disease. In this cohort, 210 (28%) patients developed CKD. Multivariate analyses revealed that CHA2DS2 -VASc score was a significant independent predictor of incident CKD, regardless of the presence of AF. Integration of CHA2DS2 -VASc scores with eGFR enhanced the predictive accuracy of incident CKD, as evidenced by the improved C-index, net reclassification improvement, and integrated discrimination improvement values (all p<0.05). Over the 12-month follow-up period, a composite endpoint was observed in 61 patients (8.3%), with elevated CHA2DS2 -VASc scores being independently associated with this endpoint. In conclusion, CHA2DS2-VASc scores have emerged as robust predictors of both CKD incidence and adverse outcomes. Their inclusion substantially refined the 12-month risk stratification of patients with preserved renal function hospitalized in the CICUs.

Acute heart failure is an important cause of unplanned hospitalizations and poses a significant burden through increased mortality and frequent hospitalizations. Heart failure with preserved ejection fraction (HFpEF) presents as a diverse condition characterized by complex cardiovascular and non-cardiovascular pathology. This study aimed to identify distinct clinical phenotypes in acute decompensated HFpEF (ADHF) using cluster analysis and assess their prognostic significance. We applied a latent class analysis to 1,281 ADHF patients admitted to a single cardiac intensive care unit between 2008 and 2022 with a left ventricular ejection fraction ≥ 50%. We used 83 factors obtained at hospitalization. We evaluated the association between phenogroups and clinical outcomes using either Cox regression model or Fine-Gray competing risk model. We identified 4 phenogroups: Phenogroup 1 (n = 133, 10%) included younger patients with metabolic disorders and a low level of B-type natriuretic peptide (BNP); Phenogroup 2 (n = 346, 27%) had systemic congestion and high BNP levels; Phenogroup 3 (n = 514, 40%) had multiple comorbidities and vascular disorders; Phenogroup 4 (n = 288, 22%) included older patients with bradyarrhythmia and atrial fibrillation. After adjusting for age, sex, and Get with the Guidelines-Heart Failure risk score, Phenogroup 2 had the highest risk of all-cause death and cardiac death. In conclusion, we identified 4 clinically relevant phenogroups of ADHF patients, each associated with different adverse outcomes. Phenotyping may provide a better understanding of the underlying mechanisms involved in the heterogeneity of ADHF and decompensation. Furthermore, it may facilitate the search for phenotype-specific therapeutic strategies.

We investigated the prognostic value of cardiac myosin-binding protein C (cMyC), a novel cardiospecific marker, both independently and in combination with N-terminal pro-B-type natriuretic peptide (NT-proBNP), for predicting 6-month all-cause mortality in patients without acute coronary syndrome (ACS) treated at medical (nonsurgical) cardiac intensive care units (CICUs). Admission levels of cMyC, high-sensitivity cardiac troponin T (hs-cTnT), and NT-proBNP were measured in 1032 consecutive patients (mean age; 70 years) without ACS hospitalized acutely in medical CICUs for the treatment of cardiovascular disease. Serum cMyC was closely correlated with hs-cTnT and moderately with NT-proBNP (r = 0.92 and r = 0.49, respectively, p < 0.0001). During the 6-month follow-up period after admission, there were 109 (10.6

Background: Previous studies have proposed different formulas of estimating glomerular filtration rate (eGFR) among clinical patients. The comprehensive comparison of eGFR formulas is not well established in a Japanese population. We compared eGFR values and chronic kidney disease (CKD) classification of nine different eGFR in a Japanese general population sample.Methods: We analyzed 469 Japanese community-dwelling adults (184 men) without any self-reported kidney disease. GFR estimated using the 4- and 6-parameter Modification of Diet in Renal Disease (MDRD) formulas (MDRD4 and MDRD6); the CKD-EPI formulas based on creatinine with (CKD-EPI-2009) and without race coefficient (CKD-EPI-2021), on cystatin C (CKD-EPI-Cys), on both (CKD-EPI-CreCys); the Japanese creatinine-based formula (JPN-Cre), cystatin C-based formula (JPN-Cys), and modified CKD-EPI formula (JPN-CKD-EPI). CKD stages were defined by KDIGO guidelines (eGFR < 60 ml/min/1.73 m2).Results: eGFRJPN-Cre (mean = 71.2; SD = 14.3) were much lower than eGFRCKD-EPI-2021 (mean = 94.2; SD =12.7), while eGFRJPN-Cys (mean = 102.8; SD = 24.2) was comparable to the MDRD and CKD-EPI formulas. The difference between eGFRCKD-EPI-2021 and eGFRJPN-Cre showed a V-shaped distribution across eGFR levels, indicating complex errors between these formulas. We observed very low agreement in CKD classification between eGFRJPN-Cre and the eGFRCKD-EPI-2021 (kappa = 0.13; 95% confidence interval: 0.06, 0.23).Conclusions: JPN-Cre was substantially different from the CKD-EPI formula without race term (CKD-EPI-2021), which means that it is impossible to recalibrate those with a simple coefficient. Although a comparison with measured GFR should be necessary, choice of the estimation method needs caution in clinical decision-making and academic research.

BACKGROUND: Hemoglobin A1c (HbA1c), which is one of the important parameters considered when judging the degree of glucose concentrations during the preceding 8–12 weeks, is used as the standard assessment of glycemic control in patients with diabetes. However, some measurement principles may cause errors in HbA1c measurement. CASE REPORT: A 49-year-old male patient with hyperglycemic hyperosmotic syndrome had an extreme high blood glucose level of 2,161 mg/dL and an HbA1c level that was not measurable due to a large amount of labile HbA1c. Many institutions measure HbA1c by high-performance liquid chromatography (HPLC). We measure HbA1c using ARKRAY HA-8190V (ion-exchange HPLC) and report National Glycohemoglobin Standardization Program (NGSP) (%). HbA1c was not measurable in this case, so we requested glycated albumin measurement as a substitute which revealed an extreme abnormal value of 64.5%. HPLC analysis revealed a large #C peak, which appeared to be labile HbA1c, before the HbA1c peak. The patient was treated with insulin therapy, and the HbA1c was measurable and was 14.1% a day after the glucose level was controlled. CONCLUSION: The report emphasiz of HbA1c measurements in situations of extreme hyperglycemia.

Objectives We evaluated the applicability of a machine learning–based low-density lipoprotein-cholesterol (LDL-C) estimation method and the influence of the characteristics of the training datasets. Methods Three training datasets were chosen from training datasets: health check-up participants at the Resource Center for Health Science ( N = 2664), clinical patients at Gifu University Hospital ( N = 7409), and clinical patients at Fujita Health University Hospital ( N = 14,842). Nine different machine learning models were constructed through hyperparameter tuning and 10-fold cross-validation. Another test dataset of another 3711 clinical patients at Fujita Health University Hospital was selected as the test set used for comparing and validating the model against the Friedewald formula and the Martin method. Results The coefficients of determination of the models trained on the health check-up dataset produced coefficients of determination that were equal to or inferior to those of the Martin method. In contrast, the coefficients of determination of several models trained on clinical patients exceeded those of the Martin method. The means of the differences and the convergences to the direct method were higher for the models trained on the clinical patients' dataset than for those trained on the health check-up participants' dataset. The models trained on the latter dataset tended to overestimate the 2019 ESC/EAS Guideline for LDL-cholesterol classification. Conclusion Although machine learning models provide valuable method for LDL-C estimates, they should be trained on datasets with matched characteristics. The versatility of machine learning methods is another important consideration.

Background: The increasing prevalence of non-alcoholic fatty liver disease (NAFLD) has become a global health problem. NAFLD has few initial symptoms and may be difficult to detect early, so there is need for a minimally invasive early detection marker. We hypothesized that miR-122 and miR-20a levels combined, as the miR-122/miR-20a ratio might detect NAFLD more sensitively. Methods: This study involved 167 participants with low alcohol intake. Those who had an increase in echogenicity of the liver parenchyma and hepato-renal contrast on ultrasonography were classified as the NAFLD group (n = 44), which was further classified into mild (n = 26) and severe (n = 18) groups based on echogenic intensity and hepatic vessel and diaphragm visualization. Participants without fatty liver were included in the normal group, except for those with an abnormal body mass index, glycated hemoglobin, and systolic blood pressure (n = 123) values. Serum miR-122 and miR-20a expression levels in participants were measured by real-time polymerase chain reaction, and the miR-122/miR-20a was calculated. Results: In the NAFLD group, miR-122 expression was significantly higher and the miR-20a was significantly lower than in the normal group, in agreement with previous studies. miR-122/miR-20a was also significantly higher in the NAFLD group. Receiver operating characteristic curve analysis was performed with miR-122/miR-20a as an NAFLD detection marker, and the area under the curve of miR-122/miR-20a was significantly larger than that of miR-122 or miR-20a alone. Conclusions: The miR-122/miR-20a ratio, combined with miR-122 and miR-20a levels, is a useful biomarker to detect NAFLD with high sensitivity.

Objectives High concentrations of low-density lipoprotein cholesterol (LDL-C) are a risk factor for cardiovascular disease. We validated the efficacy of the Martin method is useful in the estimation of LDL-C concentrations was validated in Japanese populations and derived a modified Martin method for easy laboratory information system applications. Methods We created 3 subject groups, including 2664 health check-up participants registered with the Resource Center for Health Science, 29,806 clinical patients (A) in the Gifu University Hospital, and 113,716 clinical patients (B) in the Fujita Health University Hospital. Each method to estimate serum LDL-C concentrations (Friedewald formula, Martin method and modified Martin method) was validated by correlation analysis with serum LDL-C concentrations measured using a direct method Results The correlation coefficients with the direct method in terms of the Friedewald formula, Martin method, and modified Martin method were 0.963, 0.972 and 0.970 in the health check-up participants; 0.946, 0.962 and 0.961 in clinical patients A; and 0.961, 0.979 and 0.978 in clinical patients B, respectively. Concordance ratios with using the direct method in the Friedewald formula, Martin method and modified Martin method were 82.8%, 85.5% and 85.3% in the health check-up participants; 76.4%, 80.5% and 80.2% in clinical patients A; and 76.1%, 82.6% and 82.6% in clinical patients B, respectively. Conclusion Our results show that the Martin and modified Martin methods display good performance in terms of the estimation of LDL-C concentrations among triglyceride concentrations of a wide range, and they may thus be useful for estimating LDL-C concentrations.

IMPORTANCE The associations of levels of diverse serum carotenoids ascertained via repeated measurements with all-cause, cancer, and cardiovascular disease (CVD) mortality risk have not been considered in previous prospective studies. OBJECTIVE To investigate the association between repeated measurement of serum carotenoid levels and all-cause and cause-specific mortality risk. DESIGN, SETTING, AND PARTICIPANTS This cohort study's baseline data were collected using information from physical examinations from 1990 to 1999. Eligible participants were followed up until December 2017, with a median (interquartile range) follow-up period of 22.3 (15.5-25.3) years. Included individuals were age 40 years or older at the baseline data collection, were residents of the study site in the town of Yakumo, Hokkaido, Japan, and participated in a physical examination at least once from 1990 to 1999. Among eligible participants, after excluding 332 individuals, 3116 individuals were included in the analysis. Data analysis was conducted in April 2020. EXPOSURES Repeated measurements of 6 serum carotenoid levels and 4 associated indices. MAIN OUTCOMES AND MEASURES All-cause, cancer, and CVD mortality, categorized by International Classification of Diseases, Ninth Revision and International Statistical Classification of Diseases and Related Health Problems, Tenth Revision codes, were recorded. A time-dependent Cox regression model was performed to examine associations between time-varying serum carotenoid levels and mortality. RESULTS Among 3116 individuals who received physical examinations, the mean (SD) age was 54.7 (10.6) years and 1883 (60.4%) were women. During the follow-up period, 762 deaths from all causes, 253 deaths from cancer, and 210 deaths from CVD were ascertained. In a time-dependent Cox regression analysis, for every 25% higher serum levels of total carotenoids, risks were statistically significantly lower for all-cause mortality (hazard ratio [HR], 0.85; 95% CI, 0.82-0.87; P < .001), cancer mortality (HR, 0.82; 95% CI, 0.78-0.87; P < .001), and CVD mortality (HR, 0.86; 95% CI, 0.81-0.91; P < .001). Using only baseline measures, for every 25% higher serum levels of total carotenoids, risks were also statistically significantly lower for all-cause mortality (HR, 0.92; 95% CI, 0.89-0.95; P < .001), cancer mortality (HR, 0.87; 95% CI, 0.83-0.93; P < .001), and CVD mortality (HR, 0.93; 95% CI, 0.88-0.99; P = .03) but with larger HRs than those associated with repeated measurements. CONCLUSIONS AND RELEVANCE This study found that higher levels of serum carotenoids in analysis using repeated measurements were associated with significantly lower all-cause and cause-specific mortality over a follow-up period of 25 years.

Abstract Background Although dementia is a huge problem in public health, no fundamental biomarker has been established to detect cognitive decline at the early stage. MicroRNAs (miRNAs) regulate gene expression, and are associated with the development of various diseases. Methods The subjects of this prospective study were 162 (75 men, 87 women) residents who attended a health examination in Yakumo town in Hokkaido, in 2012 and re-attended at least once while 2013 to 2015. Serum samples were collected in 2012 and serum miRNA were measured by qRT-PCR. We used a short version of the Mini-Mental State Examination (SMMSE) to screen cognitive function, and calculated the change in SMMSE score per year. We calculated odds ratios (ORs) and 95% confidence intervals (CIs) of serum miRNA levels for cognitive decline (decrease of greater than 0.5 points per year) using the lowest tertile group of miRNAs as the reference by logistic regression analysis. Results The mean age and change in SMMSE score of the subjects was 63.9±9.6 years and -0.03±1.19 points. Odds ratios (ORs) for cognitive decline were significantly higher in the highest tertile of serum miR-221 (OR = 3.24, 95%CI=1.20-8.72) and miR-222 (OR = 4.01, 95%CI=1.36-11.80) even if confounding factors were adjusted. Conclusions High serum levels of miR-221 and -222 were significantly associated with cognitive decline. Key messages High serum levels of miR-221 and -222 may be potential candidate biomarkers for prediction of cognitive decline.

BACKGROUND:Although a number of microRNAs (miRNA) reflecting kidney function has been identified, prospective studies are now urgently needed to determine a clinical utility of these miRNAs among general populations. The purpose of this study was to examine the associations between serum miRNAs and kidney function in a population-based study. METHODS:We conducted a five-year prospective study (2012-2017) of 169 individuals without chronic kidney disease (CKD) at the baseline survey (mean age, 62.5; 96 women). The real-time qPCR was used to measure serum levels of five previously reported miRNAs. Participants with eGFR < 60 mL/min/1.73 m2 were defined as having CKD. Changes in eGFR were defined as eGFR2017 - eGFR2012. RESULTS:After adjusting for covariates including baseline eGFR, lower serum levels (1st tertile) of miR-126 were associated with a greater decline of eGFR (β [SE] = -3.18 [1.50]) and a higher odds ratio (OR) of CKD onset over five years (OR [95% CI] = 3.85 [1.01-16.8]), compared with the 3rd tertile. CONCLUSIONS:We found baseline serum miR-126 levels were associated with changes in eGFR and new CKD cases in a five-year prospective study. This result suggests that miR-126 may be a potential biomarker of CKD even among general populations.