KEGG pathway comparison of mild versus severe disease identified affected downstream systems: GAREM, IGF‐1, CALCRL, SMAD2&3, glutathione peroxidase, LDH, and NOS. This feasibility study demonstrates that miRNA and metabolomics are a source of potential plasma biomarkers for disease severity in AMN, providing both a disease signature and individual markers with strong clinical correlations. Network analyses of affected systems implicate differentially altered vascular, inflammatory, and oxidative stress pathways, suggesting disease‐severity‐specific mechanisms as a function of disease severity.Adrenomyeloneuropathy (AMN), the slow progressive phenotype of adrenoleukodystrophy (ALD), has no clinical plasma biomarker for disease progression. This feasibility study aimed to determine whether metabolomics and micro‐RNA in blood plasma provide a potential source of biomarkers for AMN disease severity. Metabolomics and RNA‐seq were performed on AMN and healthy human blood plasma. Biomarker discovery and pathway analyses were performed using clustering, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, and regression against patient's clinical Expanded Disability Status Score (EDSS). Fourteen AMN and six healthy control samples were analyzed. AMN showed strong disease‐severity‐specific metabolic and miRNA clustering signatures. Strong, significant clinical correlations were shown for 7‐alpha‐hydroxy‐3‐oxo‐4‐cholestenoate (7‐HOCA) ( Micro‐RNA and metabolomics markers in adrenomyeloneuropathy blood plasma provide a rich potential source of biomarkers, correlating cross‐sectionally with disease progression.1ABCD1 gene have been identified (https://adrenoleukodystrophy.info), which lead to peroxisomal reduced fatty acid beta‐oxidation and hallmark impaired degradation and accumulation of very long chain fatty acids (VLCFA) in all tissues.ABCD1 pathogenic variant can confirm X‐ALD diagnosis in a suspected male patient; however, it cannot predict the clinical course of the disease progression (AMN or cALD). In the absence of a direct biomarker or genetic correlate, many groups have looked toward environmental factors, modifier genes, transcriptomics, lipidomics, and microRNA (miRNA) profiling to understand the heterogeneity of ALD and AMN.X‐linked adrenoleukodystrophy  is an inborn error of metabolism, due to defects in the peroxisomal membrane transporter protein. Close to 900 pathogenic variants of the underlying Metabolomics and miRNA expression allows for the quantification of the entire spectrum of potential markers in biofluids, which captures the functional state of the organism at a given time point. Omics‐based markers may serve two functions: as a wide net for biomarker discovery, and to stratify AMN phenotypes through biochemical signatures, which may help cluster patients yielding insight into underlying mechanisms. MiRNAs are small, single‐stranded noncoding RNAs that play a role in regulating gene expression, or in post‐translational epigenetic mechanisms.In this feasibility study, we sought to determine whether metabolomics and miRNA‐seq‐based approaches could be used to characterize, cluster, and identify potential plasma biomarkers for disease severity in AMN.22.1All patients and controls were seen at the Kennedy Krieger Institute and had a confirmed biochemical diagnosis of ALD. Patient data and samples were collected during routine physician visits between 2015 and 2018 and stored at −80°C until use. Male patients over the age of 18, with a genetically confirmed diagnosis of ALD, and pure AMN phenotype were included. At the time of sampling, patients had AMN without cerebral involvement. Patients with a pure AMN phenotype without cerebral involvement have an EDSS of at least 1 and do not have evidence of cerebral demyelination on brain MRI at the time of assessment. AMN patients were categorized as mild (EDSS 1–3) or severe (EDSS 4–9). Controls were non‐ALD healthy controls with no functional neurological deficit.2.2p values from the global F‐test were converted to false discovery rate  q‐values and the threshold for selection set at FDR ≤0.05. Post hoc testing of two‐group comparisons was conducted for the selected metabolites to determine the differences (p ≤ 0.05).Global metabolomics profiling by ultraperformance liquid chromatography mass spectrometry  was performed on blood plasma samples (200 μl each) from healthy controls and AMN subjects. The per‐metabolite data were median‐scaled and minimum‐value imputed. Data were log2‐transformed, for interpretation as fold change relative to the median. Analysis of variance (ANOVA) per metabolite was used to screen for differences in mean intensity across diagnosis groups. 2.3https://cutadapt.readthedocs.io/en/stable/). Reads with less than 16 bp insert sequences or less than 10 bp unique molecular index (UMI) sequences were also removed. The insert sequence reads were then aligned to human GRCh38 reference databases  using Bowtie (http://bowtie-bio.sourceforge.net/index.shtml). Read and UMI counts for each RNA type were subsequently quantified from the mapping results. Assessment of differential expression used negative binomial modeling (DESeq2 package) for each of the two‐sample comparisons of the diagnosis groups, with FDR controlled at 5% (Benjamini–Hochberg). Normalized counts were exported for use in graphing.Plasma samples (200 μl) were processed for next‐generation sequencing at the USC Norris Cancer Center Molecular Genomics Core. Samples were extracted using QIAGEN's miRNeasy Kit following the manufacturer's protocol. Libraries were prepared from extracted total RNA enriched in miRNA using QIAseq miRNA Library Kit (QIAGEN). The libraries were sequenced on the Illumina Nextseq500 platform on 1x75 read length. Raw miRNA‐sequencing reads in FASTQ files were uploaded to the QIAGEN GeneGlobe Data Analysis Center for primary quantification. The 3′ adapter of sequencing reads and low‐quality bases were first trimmed using Cutadapt  was performed on metabolomics and miRNA separately.2.5Metabolites: Enrichment of changes in 80 metabolic pathways from the Kyoto Encyclopedia of Genes and Genomes  was assessed by Fisher's exact test. An additional measure of the level of impact the alterations have on a pathway was estimated using betweenness‐centrality and was performed in Metaboanalyst 4.0 (July 2019). The list of measured metabolites was imported into Ingenuity Pathway Analysis (IPA), mapped by HMDB number, and core analysis was performed on the differential lists of metabolites.miRNA: Enrichment of biofunctions and disease pathways was performed using IPA core analysis. The full set of miRNA was used as the reference. Terms meeting the threshold of FDR < 0.05 were retained.2.6r2). Second, biomarkers with any overlap with severe AMN, or over 20% overlap range between control and any AMN were excluded. Xenobiotics and metabolites with known exogenous or dietary sources were excluded. Significance testing was performed using omnibus F‐testing within analyses of variance. Adjusted R2 values are reported. Bonferroni adjusted p values for multiple comparisons are used to determine levels of significance. Regression analyses were performed in python (3.8.0), with the packages pandas (1.0.1), seaborn (0.10.0), statsmodels (0.11.0), matplotlib (3.1.3), scipy (1.4.1), and numpy (1.18.1).A biomarker discovery pipeline was designed: First, ordinary least‐squares regression and mixed‐linear modeling were conducted on all metabolites and miRNA against the individual EDSS score of AMN only , and biomarkers were ranked by coefficient of determination  and severe . Mean EDSS of the mild group was 2 (1–3) and severe group was 5.5 (4–7). All AMN patients had a diagnosis of adrenal insufficiency. Demographic data are shown in Table Fourteen AMN and six healthy control subjects were included in the study. The mean age for AMN subjects was 46.4 years  and 42.2 years for controls . AMN patients were stratified by disease severity into mild . Most identified metabolites were increased in the AMN groups, though the patterns were not exclusively monotonic with severity , metabolic phenotypes of plasma from control and AMN patients demonstrate that strong clustering by disease severity group can be achieved Figure . Analysiy Figure .Profiling for miRNA in control and AMN plasma was performed using high‐throughput RNA‐seq, with 2545 miRNAs identified for analysis in the plasma samples. PLS‐DA analysis of miRNA revealed a good separation between control and AMN plasma samples Figure . Two‐gro3.3r2 = 0.83, p = 0.0002), dehydroepiandrosterone sulfate (DHEA‐S) , hypoxanthine , and miRNA: 432‐5p . A selected set of metabolites and miRNAs identified in the primary categoric ANOVA group analysis (control vs. mild vs. moderate/severe AMN) were regressed against EDSS and are shown in Figure In a first step, linear regression between molecular intensity and EDSS score (range: 1–7) in AMN was performed. Then, to identify potential biomarkers of disease severity which only show abnormal levels in AMN, those molecules that had intensity (expression) level overlap of more than 20% between control and AMN cases were excluded. Exogenous‐ and dietary metabolites were also excluded. There were three metabolites and one miRNA retained with strong clinical correlations Figure : 7‐alphar2 = 0.63, p = 0.002), aspartate , cystein‐glutathione disulfide , glutamate , glutamine , glycine , sarcosine , threonine , miR‐134‐5p , miR‐186‐5p , miR‐409‐3p , and miR‐451‐a .These include asparagine  within AMN. There was a trend to a positive correlation , which was not the case when the patient was included in the model , shown in , in order to determine which pathways are affected as a function of disease severity. KEGG analysis identified the following systems significantly altered by highest impact, that is the changed metabolites sit in key junction points of the pathway  phenylalanine, tyrosine, and tryptophane biosynthesis; (4) vitamin B6; (5) alanine, aspartate, and glutamate; (6) glycine, serine, and threonine; and (7) beta‐alanine. To examine biological functions related to altered miRNA between mild and moderate/severe AMN, pathway enrichment was conducted using the ingenuity knowledgebase. Inflammatory disease, inflammatory response, and neurological disease toped the ranked list of affected functions  taus Figure .We then conducted integrated network analyses to generate a pathway map that combined both significantly changed miRNAs and metabolites between mild and moderate/severe AMN. Central to altered miRNA were signaling pathways SMAD 2/3, SMAD 6/7, CALCRL, IGF1‐R, and GAREM1. Central to the altered metabolites were inflammatory pathways NOS, LDH complex, and glutathione peroxide. ERK1/2 was identified as a pathway node joining the miRNA and metabolites networks Figure .4Recent advances in drug discovery have resulted in identification of several potential therapeutics for adult men with AMN; however, clinical trials have been hampered by the rarity of the disease, resulting in limited availability of natural history data and a highly variable rate of progression in AMN, with some subjects progressive over years and others remaining stable for decades. There is a great need to identify markers that correlate with disease severity in AMN, which could be used for stratification of patients for clinical trials but also may serve as predictors of outcome in future prospective studies.In this feasibility study, UPLC–MS‐based metabolomics and RNAseq‐based quantification of miRNA expression performed in blood plasma of a cohort of well phenotyped AMN patients demonstrate that these blood parameters have the potential to serve as biomarkers reflecting disease severity. Encouragingly, both metabolites and miRNA showed strong clustering within groups, as confirmed by PLS‐DA. This first step identified potential metabolic and miRNA signatures, not just separating AMN from healthy controls but differentiating mild from severe AMN, as shown in Figure r2 = 0.83, p < 0.0001). However, regressed against age, 7‐HOCA first showed a trend toward a weak inverse correlation . Upon closer examination of these data, we identified an outlier patient greatly reducing the strength of the correlation. The mild AMN outlier  demonstrated a much higher 7‐HOCA value than his later‐age counterparts, similar to those of young AMN patients. Interestingly, the outlier patient was the only patient in this cohort found to have developed cerebral disease, diagnosed in 2019, 4 years after the sample date in 2015. Repeat sensitivity analyses excluding the outlier showed a significant correlation with age  within AMN, without affecting the significant correlation to EDSS. In controls, there was no significant correlation between 7‐HOCA and age. Further studies are needed to determine whether 7‐HOCA has any predictive value in identifying individuals who are at risk of developing cALD later in life.7‐HOCA is the main metabolite of oxysterol 27‐hydroxycholesterol (27‐OHC) in the brain.r2 = 0.11, p = 0.16). However, DHEA‐S is transiently high in blood plasma following DHEA administration, and our patients may be on DHEA supplementation, which may be a confounder.DHEA‐S is an endogenous androstane steroid and regulator of peroxisomal function, shown to induce proliferation and stimulate peroxisomal enzyme activity involved in fatty acid metabolism.Hypoxanthine is oxidized by xanthine oxidase to uric acid, a process which generates radical oxygen species (ROS).We have previously shown alterations in glutathione peroxidase metabolism, previous attempts to discriminate between ALD phenotypes.Two previous studies, including one from our group, have reported miRNA changes in patient cells.MiR‐134‐5p is shown to contribute to synapto‐dendritic plasticity.MiR‐409‐3p is a reported biomarker in CSF for Parkinson's disease,High miR‐186‐5p is associated with improved neurological outcomes in the spinal cord ischemia–reperfusion injury model.We show decreased miR‐432‐5p in severe AMN. A reported downstream target is CXC Chemokine ligand 5 (CXCL5),Combined IPA and KEGG network comparative analyses between mild and moderate/severe disease patients identified downstream pathways, previously implicated in ALD pathology literature: IGF‐1 dysfunction has been observed in both neonatal and early childhood  ALD patient fibroblastsWhile network analyses comparing disease severity profiles implicate interesting vascular, inflammatory, and oxidative stress pathways, these differences must be explored in cellular models for mechanistic studies in the future. One limitation of the study are exogenous effects on the human metabolome. Dietary effects were not accounted for in these patients and should be considered in future studies, as well as menstrual cycle in female heterozygote AMN.In summary, our data adds to previous reports showing differences between AMN patients and healthy controls,Jaspreet Singh conceived the idea, and provided direction and funding for the project. Bela Rui Turk and Laila Marie Poisson designed and performed analyses and drafted the manuscript and shared the role of first authorship. Christina Linnea Nemeth collected and processed tissue samples and reviewed the manuscript. Jordan Goodman and Richard Owen Jones collected patient data and reviewed the manuscript. Ann B. Moser collected and processed tissue samples and reviewed the manuscript. Ali Fatemi and Jaspreet Singh designed the study and reviewed the manuscript. Jaspreet Singh serves as a guarantor for the article, accepts full responsibility for the work and the conduct of the study, had access to the data, and controlled the decision to publish.This study was supported by R21 NS104560 from the National Institutes of Health (Jaspreet Singh). National Institutes of Child Health and Human Development (NICHD) P50HD103538 (Ali Fatemi), Run for ALD and Brian's Hope Foundation .Potential competing interest for Ali Fatemi: Paid member of drug safety monitoring board – Bluebird Bio, Co‐inventor of Compositions and methods for treatment of peroxisomal disorders and leukodystrophies, Patent #US20170119899A1. Potential competing interest for Bela Rui Turk: Co‐inventor of Compositions and methods for treatment of peroxisomal disorders and leukodystrophies, Patent #US20170119899A1. Laila Marie Poisson, Christina Linnea Nemeth, Jordan Goodman, Richard Owen Jones, and Jordan Goodman have no relevant potential competing interests to report.The study was approved by the Johns Hopkins Medicine Institutional Review Boards  and Henry Ford Hospital IRB#12159.Informed consent was given by the patient or his guardian prior to inclusion.Figure S1. Select molecules identified as differential between mild and moderate/severe AMN groups (ANOVA) are plotted with the estimated linear relationship (solid black line) and 95% confidence bands (gray) for (A) metabolites and (B) MiRNA. The range of observed molecular intensity for controls is denoted with green‐dashed lines.Click here for additional data file.Figure S2. Ordinary least squares regression of AMN patient age versus global score for clinical severity EDSS .Click here for additional data file.Figure S3. KEGG analysis of mild versus moderate/severe AMN. (A) System impact and significance, highlighted systems above 0.4 impact. (B) Ranking of associated disease system.Click here for additional data file.Table S1. Baseline characteristics of patients and controls included in the study.Click here for additional data file.


These toxins were detected at average concentrations of 1800 and 966.7 ƞg/kg for AFs in chicken muscle and egg, respectively, and 1400 and 1933.3 ƞg/kg for OTA in muscle and egg, respectively. Based on the survey, their estimated daily intakes through these poultry products tended to be lower than the limits 1 and 100 ƞg/kg bw/day for AFB1 and OTA, respectively). The margins of exposure (MOE) of the different population age groups to AFB1 and OTA obtained suggest that the public health concern associated with the presence of mycotoxins in poultry products shall not be underestimated.Consumption of chicken and eggs contaminated by mycotoxins could lead to a public health concern. This study was conducted to evaluate the dietary exposure of populations to aflatoxins (AFs) and ochratoxin A (OTA) through these poultry products in the three most urbanized regions of Cameroon . A survey was firstly carried out to know about the consumption frequency by the different population age groups as well as their awareness about mycotoxins. Chicken feed, broiler, and eggs were collected from modern poultry farms. AFs and OTA were analysed using the enzyme-linked immunosorbent assay (ELISA), and dietary exposure was evaluated using a deterministic approach. From the 900 households questioned, a daily consumption frequency of chicken and eggs was the most reported , with populations having a very weak knowledge of mycotoxins and their associated health risk (18%). Mean concentrations of AFs, AFB Aspergillus, Penicillium, and Fusarium) which can produce more than 500 toxins. Amongst these mycotoxins, some like aflatoxins and ochratoxin A exhibit pathogenic characteristics [1, and ochratoxin A in chicken and eggs and the associated health risk for the different population age groups.Poultries such as chicken are important sources of protein and other nutrients for human nutrition . Chickeneristics , 9. Theyeristics , 12 and eristics , 14. HowA survey on chicken and eggs consumption and population awareness about mycotoxins was firstly carried out. This was a cross-sectional, multistage cluster study conducted in three regions of Cameroon . Fifteen samples of chicken feed, 48 live market-ready broilers, and 180 fresh layer eggs were randomly collected as described by Akinmusire et al.  for feedFeed samples were ground with a blender , weighed in several aliquots of 5 g using a scale , and stored in sterile plastic bags at −20 C until analysis (maximum 1 week). Chicken samples were slaughtered, and their carcasses were scalded using hot water at 100 ± 2 C. The skeletal muscle, as well as the skin and liver of each broiler, were removed with a knife. The most consumed parts such as the muscle and the liver were taken from each broiler to make a bulk sample of each part for a broiler farm. Each bulk sample was ground separately and thoroughly mixed to give representative samples. Ground bulk samples were weighed in several aliquots of 5 g and stored at −80 C until analysis. Eggs samples were washed with distilled water, broken, homogenized, weighed in several aliquots of 5 g, and also stored at −80 C until analysis.1 (AFB1), and ochratoxin A (OTA) concentrations in the samples were determined using quantitative ELISA (enzyme-linked immune sorbent assay) kits . Samples  were mixed with 25 mL of 70% methanol (HPLC grade Merck) in 50 mL falcon tubes for 10 min on a vortex, followed by centrifugation at 4,000 ×g for 10 min using the Rotofix 32 A, centrifuge (Germany). Then, 100 μL of the supernatant was collected and diluted with 700 μL of a mixture of methanol . The mixture was used for total AFs, AFB1, and OTA analyses following the kit manufacturer's instructions. Their concentrations were inversely proportional to the colour intensity established using an automated microplate reader  at 450 nm and were estimated based on a calibration curve plotted before. The analytical test was evaluated using the internal quality control (IQC) approach and validated before usage. Five different IQCs were chosen to monitor the analytical sequence: calibration, blanks, midrange standard, spiked standard solution, certified references material, and duplicates. When acceptance criteria were not met for a sample, results were discarded, and the sample was reanalysed. The limit of detection (LOD) of the analysed samples varied between 0.06–0.3 μg/kg for AFs and 0.3–0.6 μg/kg for OTA, while the limit of quantification (LOQ) was in the range 0.2–1 μg/kg for AFs and 1–2 μg/kg for OTA. Samples with values below the limit of quantification (LOQ) were recorded as containing not detectable.Total aflatoxins (AFs), aflatoxin B1, and OTA [1 is the most dangerous and is known with OTA to be heat stable at 160°C and 180°C, respectively [A deterministic approach was used to calculate dietary exposure to AFT, AFB and OTA . Amongst10) of the toxin and the estimated toxin intake divided by 1000 [10 represents the lowest dose with a 95% certainty of not leading to a 10% increase in cancer incidence in rodents and was fixed at 400 ƞg/kg bw/day and 17.86 ƞg/kg bw/day for AFB1 and OTA, respectively [1, and OTA obtained in the muscle and the liver.The risk was majored as we assumed that the mycotoxins are not destroyed during the cooking process. To estimate the daily dietary exposure, the average body weights of children, teenagers, adults, and elderly people were considered as well as the quantity of chicken and eggs daily consumed. The margins of exposures (MOEs) to the studied mycotoxins were calculated as a ratio of the benchmark dose low limit 10% lower bound .Data obtained from survey and mycotoxin analysis were compiled in a database system using Microsoft Excel software. Descriptive statistics were performed to summarize data as the mean ± standard deviation (SD) for continuous variables and percentages for categorical variables. The Pearson correlation coefficient was used to evaluate the relationship between the tested variables using SPSS 20.0. Variables with p < 0.05). This survey globally revealed a daily consumption of chicken and eggs, the highest percentage being recorded in the Centre region (45%) for daily chicken consumption and in the West region (72%) for daily egg consumption. Data from this survey led to an estimation of the mean body weight of chicken and egg consumers of 25.2 ± 5.2 kg for children (4–12 years old), 56.3 ± 6.0 kg for teenagers (13–20 years old), 71.4 ± 10.2 kg for adults (21–59 years old), and 76.9 ± 7.7 kg for elderly people (≥60 years old)  . The est1, and OTA were detected in all chicken feed, chicken tissues, and eggs  [ƞg/kg; [ƞg/kg; [ƞg/kg; [1 was also found in all chicken feed samples in an average concentration of 1500–19300 ƞg/kg. However, just 2 samples over the 13 feed samples tested (2/13) showed concentrations above the regulatory limit which are 10 μg/kg (10000 ƞg/kg) [Aspergillus of which presence has already been reported [ƞg/kg) were also below the maximum tolerable limit of 5 μg/Kg (5000 ƞg/kg) [ƞg/kg [ƞg/kg [1, and OTA) were also found in bulk chicken muscle, liver and egg samples. For aflatoxins, the level of AFB1 in chicken samples (700–1200 ƞg/kg) and eggs (800–1100 ƞg/kg) was close to the total AFs levels observed . Hopefully, the concentrations detected respect the maximum tolerable level of 2 μg/kg (2000 ƞg/kg) for AFB1 in human food products set by the European Commission and many other countries [ƞg/kg for bulk muscle; 2266.7 ± 1000 ƞg/kg for bulk liver, and 1933.3 ± 208.2 ƞg/kg for bulk layer eggs. This higher presence of OTA in the liver than in the muscle was not observed with AFs but may be explained by the fact that liver is the organ where mycotoxins are metabolized [AFs, AFBand eggs . Concern0 ƞg/kg) . This is [ƞg/kg; ), Nigeri [ƞg/kg; ), and Ca [ƞg/kg; ). Aflato0 ƞg/kg) . This is0 ƞg/kg) ). Previo) [ƞg/kg ; 5400 ƞgg [ƞg/kg . The stuountries . Consideountries . Ochratoabolized . The preabolized . These rabolized  in 2017 1, and OTA contamination levels observed in chicken tissues and eggs as well as the consumption of these foods collected, the daily intake  of the toxins analysed were determined  which is the health-based reference value established for AFs by international scientific expert committees: the Joint Expert Committee on Food Additives (JECFA) and Scientific Committee for Food (SCF) [1 MOE, and their comparison to the related standard  [1 through chicken consumption. Mean MOE values were globally higher or around 600 with eggs, while with chicken it was also the case except for elderly people with an MOE value of 316 (risky). Indeed, AFB1 in food shall obey to the ALARA principle. Ananditya et al. [ƞg/kg bw/day but MOEs showing an important risk in Indonesia. This may be explained by the fact that limits of MOE were defined based on animal studies which can often be misleading as the animals may be more resistant to aflatoxins than humans [ƞg/kg bw/day and 0.5–1.6 ƞg/kg bw/day, respectively (ƞg/kg bw/day as recommended by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) and the European Food Safety Authority (EFSA), respectively [Based on the AFT, AFBtermined . The estod (SCF) , 37. Thie versa)  clearly a et al.  also repn humans . In the ectively . This suectively , 33. Howectively , 33, theectively , and the1, and OTA in these poultry products globally respects the limits, but a health risk should not be underestimated based on the populations' consumption frequency. A total diet study as well as the influence of local cooking processes' impact on mycotoxin content will be required before confirming this exposure.Chicken and egg consumers in urban cities of Cameroon are not aware of mycotoxins and their potential health risk. The presence of AFs, AFB


Necrobiotic xanthogranuloma (NXG) is a rare dermatosis that is often associated with monoclonal paraproteinemia and hematological malignancies. We report the rare case of an 84‐year‐old gentleman with extensive truncal NXG and IgG‐kappa paraproteinemia who achieved significant disease regression following six cycles of combination melphalan/prednisone therapy. Skin infiltration by necrobioc xanthogranuloma: Before treatment above in A, after treatment in B, with paraprotein response to therapy. Several case reports and small, retrospective case series have shown variable clinical responses to topical steroid applications, interferon‐α, corticosteroid therapy , infliximab, intravenous immunoglobulin, radiotherapy, surgery, and extracorporeal photopheresis.We present the case of an elderly Caucasian gentleman with extensive, upper truncal NXG lesions and an associated IgG kappa paraproteinemia who responded to combination melphalan/prednisone therapy. The therapeutic challenges of NXG are considered. The case highlights that a prolonged course of alkylating therapy combined with corticosteroid is capable of achieving disease regression in NXG. Further, clinical and biochemical correlates defining disease relapse are offered, suggesting the need to review published treatment and monitoring guidelines for NXG.2An 84‐year‐old Caucasian gentleman was referred to a tertiary hematology clinic in late June 2021 for assessment of a monoclonal paraproteinemia and worsening truncal skin lesions. He reported a four‐year history of progressive erythematous, scaly lesions across his bilateral shoulders and scapulae, which had started to increase in size in the preceding three months. He complained of associated pruritus, but no pain or ulceration. He denied any fevers, drenching night sweats, unintentional weight loss, constitutional symptoms, petechiae, bone pains, bleeding diatheses, recent travel outside of Australia or exposure to pets. There was no family history of any hematological diseases. He was a non‐smoker and did not consume alcohol. His past medical history included a previous myocardial infarction secondary to a total, mid left anterior descending artery occlusion for which he had a drug eluding stent inserted, hyperlipidaemia, type 2 diabetes mellitus, atrial fibrillation complicated by a previous ischaemic stroke and splenic infarct, left carotid artery stenosis which had been stented, complex partial seizures, a small, left supramarginal gyrus multinodular and vacuolating neuronal tumor, iron deficiency requiring parenteral iron support, shingles, a provoked right upper limb deep vein thrombosis, and, a previous inguinal hernia mesh repair. His chronic medications included apixaban 2.5 mg bd, aspirin 100 mg daily, atorvastatin 40 mg daily, bisoprolol 2.5 mg daily, metformin 1 g daily, and levetiracetam 1 g mane and 1.5 g nocte. He was generally fit and well and resided at home with his wife.He examined as an elderly gentleman with a normal BMI. Large, confluent red‐yellow, indurated patches and plaques without ulceration were noted across his bilateral scapulae, right anterior neck, left upper chest and shoulder Figure . There w9/L) and platelets (235 × 109/L). His renal and liver functions were generally unremarkable with a normal creatinine (79 μmol/L), eGFR 78 ml/min/1.73m2 and only mildly elevated globulins of 43 g/L (reference range: 25–42 g/L). His calcium level was normal. His erythrocyte sedimentation rate (ESR) was elevated , as was his C‐reactive protein  and fibrinogen . An autoimmune screen, including a rheumatoid factor, and anti‐CCP were both negative. His lipid profile was previously unremarkable except for a mildly suppressed HDL . His immunoglobulin G (IgG) level was raised  with normal IgA and IgM serum levels. Subsequent serum protein electrophoresis and immunofixation identified a monoclonal IgG kappa paraproteinemia (13 g/L). His serum free kappa was quantified at 21.9 mg/L (reference range: 3.3–19.4 mg/L) with a normal free kappa/lambda ratio of 1.60 (reference range: 0.26–1.65). His beta‐2‐microglobulin was marginally elevated at 3.1 mg/L (reference range: <2.9 mg/L). Computerized tomography imaging of his chest, abdomen, and pelvis did not reveal any lymphadenopathy, hepatosplenomegaly, or visceral organ involvement of his NXG. Recent endoscopic investigations had excluded a bowel malignancy. He proceeded to a bone marrow biopsy that showed a mildly hypercellular, reactive bone marrow with normal trilineage hematopoiesis and a small (2%) monoclonal plasma cell population. These features were consistent with a diagnosis of monoclonal gammopathy of uncertain significance (MGUS) in the context of his NXG.Concurrent dermatology review was obtained and, following biopsy of these skin lesions to exclude other typical lesions , features were determined to be consistent with NXG Figure . Given tDuring this period of investigation, his NXG lesions progressed with worsening pruritus, and he developed a new, periumbilical lesion. Given the ongoing and rapid progression of his NXG lesions across multiple skin areas, oral alkylating and corticosteroid therapy treatment was recommended. He commenced treatment three months later with melphalan 0.15 mg/kg (10 mg) daily and prednisone 25 mg daily for a total of five days out of every 28‐day cycle. He tolerated this treatment without any major adverse effects, except for some mild diarrhea with each cycle while taking melphalan. His skin lesions regressed significantly Figure  since hi3The clinical manifestations, treatment, and ongoing monitoring of patients with NXG are both heterogenous and complex. Treatment options are varied and depend on the coexistence of hematological neoplasms, location of dermal lesions, and consideration of visceral involvement.Several case reports have trialed alkylating therapies with and without corticosteroids in patients with NXG.The pathogenetic processes underpinning NXG are poorly understood. Two theories have been proposed; both of which are reliant on the patient having a concurrent monoclonal paraproteinemia.There is a paucity of literature determining treatment failure in NXG and the clinical and/or biochemical features constituting disease relapse. Up to 42% of patients with NXG may relapse within 6–12 months of initial treatment, with relapsed NXG often demonstrating more aggressive visceral infiltration.Necrobiotic xanthogranuloma is a complex disease with a heterogenous presentation. In elderly patients with extensive dermal NXG and an IgG‐kappa paraproteinemia, combination melphalan/prednisone therapy provided over six cycles every 28 days is both safe and tolerable. This treatment regimen appears to induce significant disease regression and reduce monoclonal paraproteinemia in patients with NXG.JAJM and JC cared for the patient and collected longitudinal data. JAJM and JC analyzed and synthesized all the data. JAJM undertook a comprehensive literature review. JAJM wrote the manuscript. All authors critically revised and approved the final manuscript.The authors declare that they have no conflicts of interest.Written informed consent for the publication of this case and the accompanying images was obtained from the patient.


Consistent with past criticisms of using fight rather than fighter data to examine fighting success, which have argued that individual fights can be suddenly and unexpectedly determined and do not capture an individual's overall ability to succeed, Study 3 (N = 1367 fights) found no association between fWHR and singular victories. Studies 1–3 showed continual evidence that larger fWHRs were associated with grappling abilities, even after controlling for demographic and allometric factors. Strikingly, Study 3 discovered associations between all fWHR measures and grappling skill that remained robust before and after controlling for 17 different control variables. We discuss that grappling, or the act of taking down an opponent, involves a more aggressive, close‐combat approach than does striking. Combined, these results offer additional support for the argument that fWHR may have been shaped by sexual selection.Zilioli et al. (2014) were the first to show an association between male facial width‐to‐height ratio (fWHR) and physical aggression and fighting ability in professional mixed‐martial‐arts fighters. Here, we re‐examined this relationship by replicating  and extending (using 23 new variables related to fighting performance) Zilioli et al. (2014) in a statistically well‐powered sample of 520 fighters using automatic and manual measures of the fWHR involving both eyelid and eyebrow landmarks, used interchangeably in previous reports (Studies 1–2). Most importantly, we successfully replicated Zilioli et al.'s (2014) central finding that fighters' fWHR, when manually calculated using the eyebrow landmark, predicted their fighting success ( In a subsequent commentary, Třebický et al. (N = 146 combatants). Třebický et al.  fighters competing in the Ultimate Fighting Championships©  but could be associated with their underlying anatomical components that collectively contribute to force output. Thus, our study employed two methods for examining the relation between fWHR and potential force output: (1) by directly examining the association between fWHR and knockout wins (a proxy for force output) and (2) by examining the association between fWHR and morphological structures implicated in force output . Previous research has indeed interpreted links between physiological features  and bodily size as evidence for the physiological feature being an indicator of the individual's RHP . Further, de Kok . Study 3 extended Zilioli et al.  by exami44.14.1.1ufc.com or espn.com up to April 4th, 2020. In line with the original study, we excluded fighters without facial photographs (n = 19) and female fighters (n = 113). An independent research assistant then coded these facial photographs in accordance with Zilioli et al.'s (n = 5), hair/beards that covered the zygions (n = 15), head tilt (n = 17), and UFC retirement/termination (n = 45), leaving a final sample of 520 fighters. While our data analyses hereafter employs this sample of 520 fighters, we ran additional analyses with the retired/terminated fighters (see ESM). In line with Zilioli et al. (n = 353) or non‐Caucasian (n = 167). Zilioli et al. , total fights , weight , and height . To directly replicate the original study, which controlled for body mass index (BMI) rather than weight or height themselves, we calculated BMI  using the formula: weight (lbs)/(height[inches] × height[inches]) × 703 (with 703 being the number used to convert lbs/inches2 to kg/m2). In accord with the original study, we calculated fight success  using previously published methods  and eyelid  fWHR measurements. There is no variation between research assistants in this process because the fWHR code is simply entered into the terminal and the fWHR calculator consequently provides the resulting fWHR statistic. Both measurements were strongly associated (r = .85). The fWHR calculator extracts the bizygomatic and eyebrow/eyelid landmarks that have been placed by the face recognition package, and divides the individual's bizygomatic width by their facial height . Given that Zilioli et al. , arm reach , leg reach , and debut date . Zilioli et al.  whereas force output was measured via fighters' cumulative wins by KO/TKO.We collected all available data on Additionally, we collected data on 15 measures of striking  and grappling abilities , which allowed us to extend the original study by examining the relations between fWHR and acts of aggression.ufc.com and espn.com, including striking defense, takedown defense, wins and losses by submission, and wins and losses by decision. Definitions taken from James et al. (https://osf.io/scde7/).We also included multiple exploratory variables available from s et al.  and Kirks et al.  and desc4.24.2.1n = 265), middleweight , and heavyweight  fighters. There were no statistically significant relationships when restricting analyses to weight categories  and then among fighters of different ethnicities within each weight category Table . All ass4.2.2B = −0.04, SE = 0.03, t = −1.23, p = .22; total wins: standardised interaction B = −0.03, SE = 0.03, t = −1.03, p = .30; win percentage: standardised interaction B = 0.02, SE = 0.04, t = 0.45, p = .65; win percentage, controlling for total fights: standardised interaction B = −0.03, SE = 0.05, t = 0.16, p = .87).Thus far, we report limited evidence for associations between fWHR and formidability. However, fighters might adjust their strategies in response to their opponent. For example, fighters with large fWHRs with prior wins by knockout earlier in their career, subsequent opponents might strategically avoid stand‐up combat, potentially suppressing any associations between fWHR and fighting success. If the relationship between fWHR and fighting success is stronger among early than late‐career fighters, then fighters' debut date may moderate the relationship between fWHR and fighting success. Using Hayes'  SPSS PRO4.2.3Statistical associations between fWHR and the 23 measures of fighting abilities—as proxies for aggression, blunt force trauma resistance, and force output, as well as the exploratory variables—are reported in Table 4.3There were no significant relations between fWHR and fighting success. These results support other studies reporting a lack of association between fWHR and men's behavior  but not in automatic (p = .052) measures . The manual fWHRbrow measurement was also strongly correlated with the automatic fWHRbrow measurement , as was the manual fWHRlid measurement with the automatic fWHRlid measurement . For the direct replication aspect, fWHRlid associations are reported in the ESM because Zilioli et al. (https://osf.io/5v6mn/).In line with de Kok's  calculati et al.  did not 5.25.2.1Table We then restricted analyses to within ethnicities  and then among fighters of different ethnicities within each weight category Table . There w5.2.2B = −0.01, SE = 0.03, t = −0.26, p = .80; total wins: standardised interaction B = −0.002, SE = 0.03, t = −0.04, p = .97; win percentage: standardised interaction B = 0.03, SE = 0.04, t = 0.76, p = .45; win percentage, controlling for total fights: standardised interaction B = 0.03, SE = 0.04, t = 0.72, p = .47).As in Study 1, four moderation analyses were performed to examine the effect of debut date on the relationship between fWHRbrow and total fights, total wins, win percentage, win percentage controlling for total fights. Debut date did not significantly moderate the relationship between fWHRbrow and fight success . Our results suggest some error between automatic and manual measures, and research should ideally report both methods of measurement in fWHR research.When we used manual measurements—and, therefore, methodology identical to Zilioli et al. —we succeConsistent with the previous study, we failed to find support for the association between fWHR and blunt‐force trauma resistance. While we failed to find support for an association between fWHR and knockout power, there was a significant relationship between fWHR and body size. There was some support for a relationship between fWHR and aggression, with our strongest support being between fWHR and grappling ability  which remained robust after controlling for demographic and allometric variables.However, an additional limitation within Zilioli et al.  was the 66.1ufcstats.com. There were 4566 unique fights and 1674 unique fighters who had participated in at least one UFC fight  from March 21st, 2010, to February 6th, 2021. Fighters were assigned to be either blue or red fighters for a fight, but the fighter's color has been suggested to be associated with their abilities  and method of resolution  data, the latter of which was used to examine the relationship between fWHR and blunt‐force resistance and force output  and manual  fWHR measures, fighters' retirement/termination, debut date, leg reach, and total fights  to the respective focal and non‐focal fighter names in the individual fight dataset . Data merging was performed only for fighters who met the inclusion criteria for Studies 1 and 2, but now including retired fighters . As in Studies 1 and 2, only male fighters who did not have nonneutral facial expressions, head tilts, or hair/beards that covered the zygions were merged. This resulted in 1367 unique fights where both focal and nonfocal fighter possessed both the automatic and manual fWHR measures—making this the largest individual fight dataset in an empirical paper to date —one for each fWHR measurement —were conducted with a binomial error family to analyze the effect of focal fWHR on the focal outcome . We controlled for the focal and nonfocal fighters' height, reach, weight, age, retirement status, debut date, leg reach, and total fights, as well as the nonfocal fighters' respective fWHR measurement. It should nonetheless be noted that inclusion of these covariates for our fighting success analyses did not affect results; our results are the same regardless of the inclusion of covariates. Similar to Lane and Briffa's  method o6.1.2Twenty‐four LMMs—six for each fWHR measurement —with model specification via backwards elimination were conducted to examine the association between fWHR and an aggression‐related  in the ESM. In addition to our intercepts‐only models, we also included random slopes for both focal and nonfocal fighters' fWHR measurements; then, in another assortment of analyses, only focal fighters' fWHR measurements. Compared to the intercepts‐only analyses, equivalent intercepts‐and‐slopes models frequently resulted in a singular fit or a convergence error; nonetheless, intercepts‐and‐slopes models exhibited the same pattern of results as the intercepts‐only models. Yet, because singular fits are problematic for multilevel modelling, we reported the intercepts‐and‐slopes models in the ESM while the intercepts‐only models are reported in‐text. The R code and dataset for Study 3 are available on the OSF (https://osf.io/scde7/).All analyses were carried out in RStudio using the package lme4 , such that those with larger fWHRs were not significantly more likely to win the fight. There was also no significant interaction between fWHRbrow  and the method of resolution on the focal outcome , such that those with larger fWHRs were not significantly more likely to win or lose via a specific strategy .Results showed no significant association between fWHRbrow  and focal outcome , significant strikes attempted , striking accuracy , takedowns landed , or takedowns attempted . However, there was a significant association between fWHRbrow and grappling accuracy , such that those with larger fWHRs were more skilled grapplers. This latter effect was performed on the minimal adequate model (6 control variables) but was also significant in the most complex model with all 17 control variables  and also when there were no covariates . Strikingly, this relation between fWHR and grappling accuracy held across all three other fWHR measurements (ESM). These associations further held when random slopes were included (ESM).There were no significant associations between fWHRbrow  and significant strikes landed . Because grappling uses more close combat strategies, grappling could be argued as a more aggressive approach because: (a) there is a higher likelihood of subsequent punches being landed ; (b) there is a higher likelihood of now using elbows, fists, and knees to inflict damage; (c) landed strikes may be more damaging when in close quarters; (d) there is a reduced likelihood of escape for the one being struck; (e) there is a higher likelihood of using other methods to defeat their opponent other than strikes . There are several potential mediating mechanisms for this link between fWHR and grappling‐based aggression; we discuss the role of testosterone, allometric scaling, and opponent intimidation.7.17.1.1First, fWHR might be associated with aggressive outcomes due to its association with testosterone. However, links between fWHR and testosterone remain controversial  analyses are a statistical technique widely used in the biological sciences, validated in the 1980s and 1990s long before research began on fWHR . Indeed, morphological features can evolve through sexual selection by acting as a threat display  opponents (Briffa & Lane, 8While much research implicates fWHR in a suite of behavioral outcomes (Geniole et al., Supporting information.Click here for additional data file.


ADN) and papaverine (Pd/PaPAP) in 326 patients (326 vessels). The overestimation of FFR was calculated as Pd/PaADN–Pd/PaPAP. The anatomical–functional mismatch was defined as diameter stenosis > 50% and Pd/PaADN > 0.80. Reclassification was indicated by Pd/PaADN > 0.80 and Pd/PaPAP ≤ 0.80. The mismatch (n = 72) had a greater overestimation of FFR than the non-mismatch (n = 99): median 0.02 (interquartile range 0.01–0.05) versus 0.01 (0.00–0.04), p = 0.014. Multivariable analysis identified the overestimation of FFR (p = 0.003), minimal luminal diameter (p = 0.001), and non-left anterior descending artery (LAD) location (p < 0.001) as determinants of the mismatch. Reclassification was indicated in 29% of the mismatch and was more frequent in the LAD than in the non-LAD . The overestimation of FFR is an independent determinant of anatomical−functional mismatch. Anatomical−functional mismatch, specifically in the LAD, may suggest a false-negative result.Adenosine occasionally results in overestimation of fractional flow reserve (FFR) values, compared with other hyperemic stimuli. We aimed to elucidate the association of overestimation of FFR by adenosine with anatomically significant but functionally non-significant lesions  and its influence on reclassification of functional significance. Distal-to-aortic pressure ratio (Pd/Pa) was measured using adenosine (Pd/Pa Coronary angiography alone, however, has limited ability to differentiate functionally significant lesions7. Pressure wire-derived fractional flow reserve (FFR) has emerged as the gold standard for assessing the functional impact of stenotic lesions, and a vessel with an FFR value ≤ 0.80 is considered to have functionally significant stenosis11. It is well known that the anatomical and functional significance of coronary lesions does not always match4. Previous studies have identified clinical and pathophysiological factors associated with anatomically significant but functionally non-significant lesions 12.Diameter stenosis (DS) of > 50% on invasive coronary angiography is deemed anatomically significant13. If maximal hyperemia is not induced, an FFR value becomes erroneously higher than the true FFR value. This overestimation of FFR underestimates the severity of ischemia and poses a risk of leaving an ischemic lesion untreated14. Intravenous adenosine, used most widely for hyperemia induction15, occasionally fails to induce maximal hyperemia when compared with other hyperemic stimuli, especially if the subject has consumed caffeine, a competitive antagonist of the target receptor of adenosine20. Although an overestimation of FFR by adenosine may cause an anatomical–functional mismatch (DS > 50% and FFR > 0.80), the association between the overestimation of FFR by adenosine and anatomical–functional mismatch has not been fully explored. Previous studies on anatomical–functional mismatch have used only adenosine to produce hyperemia, thereby precluding assessment of adenosine’s role in the mismatch12. If the anatomical–functional mismatch is associated with the overestimation of FFR by adenosine, vessels having the mismatch with adenosine may show functional significance when using another stimulus.The principle of FFR is based on the premise that coronary pressure is linearly related to myocardial blood flow under maximal hyperemia2a receptors21. This study compared distal-to-aortic pressure ratio (Pd/Pa) associated with adenosine (Pd/PaADN) and Pd/Pa associated with papaverine (Pd/PaPAP), as a reference standard. We sought to determine whether (1) the overestimation of FFR by adenosine is associated with the discordance between the anatomical and functional significance and (2) functional significance in such lesions is reclassified when papaverine is used as the indicator.Papaverine induces maximal hyperemia most reliably by directly relaxing the vascular smooth muscle, without involving the adenosine AThis study retrospectively analyzed the data of 365 consecutive patients with chronic coronary syndrome undergoing clinically indicated coronary angiography and an FFR assessment for coronary stenosis of 30–90% based on visual estimations during angiography. If FFR was measured in two or more vessels in a patient, only the first vessel was included in this study. All patients were asked to abstain from food and beverages for > 3 h before the catheterization. Periods for caffeine abstinence were left to the referring physicians’ discretion. This study excluded vessels with lesions on the coronary ostium, severe arrhythmia , a prior coronary artery bypass graft, significant valvular disease, or any contraindications for adenosine or papaverine, as well as patients taking theophylline-containing medications. Patients with insufficient pressure data quality, including inadequate waveform tracings and signal drift more than ± 0.03 after the pullback of the pressure wire, were also excluded from the analysis.The coronary physiology assessment was performed as part of the routine diagnostic coronary angiography procedures for clinical purposes. All methods were performed in accordance with the relevant guidelines and regulations. Written informed consent for the invasive physiology assessment was obtained from all of the patients before the procedure. The Institutional Review Board approved this retrospective study and waived the requirement to obtain patient approval and written informed consent for the review of patient data and medical records .13. After administration of intracoronary isosorbide dinitrate, distal coronary pressure and aortic pressure were simultaneously measured at baseline and during hyperemia. The guiding catheter was disengaged from the ostium during FFR measurements, and special care was taken not to alter the wire position.Diagnostic coronary angiography was performed in a standard manner. Thereafter, the FFR was measured using a 5- or 6-F guiding catheter without side holes and a commercially available FFR system (Philips Volcano or Abbott Vascular) in accordance with standardized procedures22. When Pd/Pa values were not stable during adenosine infusion, adenosine was continued for more than 180 s. Papaverine was used as the last agent to obtain a reliable pull-back curve, as it induces hyperemia with minimal variations in Pd/Pa23. After confirming that Pd/Pa values had returned to the baseline level, with an interval of ≥ 5 min, intracoronary papaverine (8–10 mg in the right coronary artery or 12–15 mg in the left coronary artery) was given through the coronary catheter, followed by 5 ml of saline13. Approximately 20 s after the papaverine injection, an FFR pullback recording was performed manually, and the presence of pressure-wire drift was checked.Adenosine was continuously given via a femoral vein or a large forearm vein at a dose of 140 μg/kg/min for more than 150 sADN was determined during the steady-state hyperemic plateau phase > 60 s after the initiation of adenosine and > 15 s after the transition to hyperemia24. The lowest Pd/Pa values on a beat-to-beat basis for adenosine and papaverine were regarded as Pd/PaADN and Pd/PaPAP, respectively25. The overestimation of FFR by adenosine was defined as Pd/PaADN – Pd/PaPAP19.Experienced observers blinded to patients’ coronary angiography results and clinical data manually reviewed the pressure recordings. Pd/Pa26. The anatomical–functional mismatch was defined as DS > 50% and Pd/PaADN > 0.80, and the reverse mismatch was defined as DS ≤ 50% and Pd/PaADN ≤ 0.80.Quantitative coronary angiography was performed with a commercially available system  by independent investigators unaware of patients’ FFR results or clinical data. Reference diameter, minimum lumen diameter, and lesion length were measured using the external diameter of the catheter as a scaling device, and DS was calculated. Proximal location was defined as Syntax segments 1, 5, 6, and 11ADN > 0.80 and FFRPAP ≤ 0.80 , and reverse reclassification as FFRADN ≤ 0.80 and FFRPAP > 0.80 .Reclassification of functional significance was indicated by FFR2 test or Fisher’s exact test for categorical variables, as appropriate. The correlation between DS and Pd/PaADN was assessed with Spearman’s rank correlation coefficient. Multivariable logistic regression analysis was used to determine factors associated with the anatomical–functional mismatch and the reverse mismatch. Clinical, angiographic, and hemodynamic parameters with a univariable association of p < 0.10 were entered into the multivariable model. Results were presented as odds ratios (ORs) and 95% confidence intervals (CIs). The numbers of the mismatch in vessels with DS > 50% and the reverse mismatch in vessels with DS ≤ 50% were compared between adenosine and papaverine using the McNemar test. Statistical analyses were performed using JMP® Pro, version 16.0.0. . A p value of < 0.05 was considered statistically significant.Continuous variables were presented as medians with interquartile ranges (IQRs). Categorical variables were expressed as frequencies with percentages. Between-group comparisons were made with unpaired-sample t-test or Mann–Whitney U test for quantitative variables and with the χOf the 365 patients, 39 were excluded due to sensor drift n = 13), insufficient waveform tracings (n = 10), side effects of adenosine (n = 8) or papaverine (n = 2), or difficulty in advancing the pressure wire far enough distal from the index lesion (n = 6), leaving 326 patients (326 vessels) in the analysis. Table 3, insuffADN was 0.79 (0.73–0.86). Figure ADN. The relationship between DS and Pd/PaADN was modest, with a large scatter . DS > 50% was observed in 171 vessels (52%), and DS ≤ 50% in 155 vessels (48%). Among the vessels with DS > 50%, 72 and 99 vessels demonstrated Pd/PaADN > 0.80 and Pd/PaADN ≤ 0.80, respectively. Of the vessels with DS ≤ 50%, 76 and 79 vessels showed Pd/PaADN ≤ 0.80 and Pd/PaADN > 0.80, respectively.The median DS was 50.6% (IQR 42.4–57.5%), and the median Pd/PaADN > 0.80) and non-mismatch (Pd/PaADN ≤ 0.80) groups. There was no significant difference in age, sex, body size, or coronary risk factors between groups. Concerning lesion characteristics, the mismatch group was associated with a higher frequency of non-LAD location , a greater reference diameter , a larger minimal luminal diameter , a smaller DS , and a shorter lesion length  than the non-mismatch group.Table ADN values were significantly higher than Pd/PaPAP values, regardless of the presence of anatomical–functional mismatch . Figure p = 0.014).Pd/Pa6 IQR 0.8–0.89 vs.p = 0.014), non-LAD location (p < 0.001), reference diameter (p = 0.004), and minimal luminal diameter (p = 0.001) in the multivariable model. As shown in Table p = 0.003), non-LAD location , and minimal luminal diameter  as independent factors associated with the anatomical–functional mismatch.Based on the results from the univariable analysis . In the mismatch group, the reclassification of functional significance by papaverine (Pd/PaADN > 0.80 and Pd/PaPAP ≤ 0.80) was observed in 29% (21/72). Of these, 12 vessels had an overestimation of FFR of ≥ 0.05, and 7 vessels had Pd/PaPAP values of ≤ 0.75. Reclassification was more frequently observed in LAD lesions than in non-LAD lesions . In the non-mismatch group, reverse reclassification (Pd/PaADN ≤ 0.80 and Pd/PaPAP > 0.80) was observed in only 4% (4/99). In all vessels, Pd/PaADN showed borderline values (0.78–0.80), with small differences from Pd/PaPAP (≤ 0.03).Figure ADN ≤ 0.80) and non-reverse mismatch (Pd/PaADN > 0.80) groups. The reverse mismatch group was associated with male sex , a higher frequency of non-LAD location , a smaller reference diameter (2.6 mm [IQR 2.3–3.1 mm]) vs. 2.9 mm [IQR 2.6–3.3 mm], p = 0.002), and a smaller minimal luminal diameter (1.5 mm [IQR 1.3–1.8 mm]) vs. 1.7 mm [IQR 1.6–2.1 mm], p < 0.001) compared to the non-reverse mismatch group. No significant difference in the overestimation of FFR was observed between the two groups (p = 0.224).Table p = 0.042), body mass index (p = 0.061), LAD location (p = 0.022), reference diameter (p = 0.001), and minimal luminal diameter (p < 0.001) were included in the multivariable model. The results of the multivariable logistic regression analysis revealed the following as independent factors associated with the reverse mismatch (Table p = 0.034), LAD location , and minimal luminal diameter  as independent factors associated with the reverse mismatch . In the non-reverse mismatch group, reclassification of functional significance was observed in 11 vessels (14%). In the reverse mismatch group, reverse reclassification was in observed only 2 vessels (3%).Compared to adenosine, papaverine significantly increased the number of vessels with the anatomical–functional reverse mismatch  to positive by papaverine (Pd/PaPAP ≤ 0.80); and 3) this reclassification was observed more frequently in LAD lesions (52%) than in non-LAD lesions (20%).Evaluating FFR values obtained with adenosine and papaverine in relation to DS on coronary angiography, we found that 1) the overestimation of FFR by adenosine was an independent determinant of the anatomical–functional mismatch; 2) in 30% of vessels with the anatomical–functional mismatch, functional significance was reclassified from negative by adenosine  were associated with a high rate of death or myocardial infarction when treated with medical therapy alone37. The reclassification, specifically due to a large overestimation of FFR, may lead to worse clinical outcomes.A large randomized trial confirmed that revascularization reduced adverse cardiac events in patients who had coronary stenosis with FFR ≤ 0.8012. This is a reasonable result from a physiological point of view. Stenosis in the non-LAD vessels, which subtends smaller myocardial masses than that in the LAD, produces a less hyperemic gradient across the lesion. Even with the anatomically significant stenosis of DS > 50%, the non-LAD is less likely to have FFR ≤ 0.80 than the LAD. Indeed, in the present study, the frequency of the anatomical–functional mismatch was higher in the non-LAD than in the LAD. Of note, however, the reclassification was observed in more than one-half of LAD vessels with the anatomical–functional mismatch. Physicians should keep in mind that when using adenosine, the anatomical–functional mismatch, specifically in the LAD, may suggest a false-negative result.The present and previous studies found non-LAD location to be an independent determinant of the anatomical–functional mismatch12, our analyses revealed that male sex, LAD location, and minimal lumen diameter are independent determinants of the anatomical–functional reverse mismatch. A greater pressure gradient is produced across a lesion located in the LAD, subtending larger myocardial masses than a lesion located in the non-LAD12. Likewise, males  are likely to have lower FFR values5.In agreement with previous studies38, patients with overestimations of FFR may miss the opportunity to receive the benefits of revascularization. The concept of FFR was established under the assumption of minimal microvascular resistance9. Our findings underscore the importance of inducing maximal hyperemia in FFR measurements to avoid misinterpretations of the FFR results. Significantly, more than half of LAD lesions with the anatomical–functional mismatch demonstrated false-negative results. Considering that stenotic lesions in the LAD carry a worse prognosis than in other locations39, LAD lesions with anatomical–functional mismatch are more clinically relevant.Given that revascularization reduces adverse cardiac events even in patients with gray zone FFR values compared with medical therapy alone19; however the serum caffeine level is unpredictable even after caffeine abstinence because it depends on various factors, including the inter-individual variation in caffeine’s half-life, patient compliance, and the recently ingested caffeine dose. In the context of a lack of a rapid test kit to detect serum caffeine, the anatomical–functional mismatch provides a clue to insufficient adenosine-induced hyperemia.FFR overestimations by adenosine (i.e. insufficient adenosine-induced hyperemia) will not be identified unless another hyperemic stimulus is used. Serum caffeine was reported to be a major determinantWe acknowledge several limitations to this study. First, we did not determine the clinical significance of the anatomical–functional mismatch and/or the reclassification due to the overestimation of FFR by adenosine. This was because some of the vessels with DS > 50% and negative FFR results by adenosine were revascularized based on positive FFR results by papaverine. Further research is warranted to address this topic. Second, microvascular function was not assessed. High microcirculatory resistance might affect the overestimation of FFR.However, in the presence of insufficient adenosine-induced hyperemia, microcirculatory resistance cannot be accurately assessed by adenosine. Multiple injections of papaverine to measure microcirculatory resistance, together with FFR, may carry an increased risk of side effects. Third, the patients' serum caffeine levels were not measured in this study. Lastly, the order of hyperemic agents was fixed (papaverine last) because papaverine was used to obtain a reliable pullback curve. Although papaverine was administered after confirming that Pd/Pa values had returned to the baseline level, adenosine’s carry-over effect cannot be excluded.The overestimation of FFR by adenosine compared to papaverine is an independent determinant of anatomical–fucntional mismatch.It should be kept in mind that anatomical–functional mismatch, specifically in the LAD and potentially due to caffeine, may suggest a false-negative result.Supplementary Information.


Programmed cell death 1 (PD‐1)/programmed cell death ligand (PD‐L1) inhibitors plus chemotherapy (ICI + Chemo) is the standard treatment for advanced non‐small‐cell lung cancer (NSCLC). However, the impact of tumour burden on the efficacy of ICI + Chemo remains unknown.We retrospectively evaluated 92 patients with advanced NSCLC treated with ICI + Chemo. Tumour burden was assessed as the sum of the longest diameter of the target lesion (BSLD) and number of metastatic lesions (BNMLs). We categorised the patients into three groups based on the combined BSLD and BNML values.p = 0012). Twenty‐five patients (27%) in the low‐BNML group had a median PFS of 9.6 months, whereas patients in the high‐BNML group had a median PFS of 6.5 months . Low‐BSLD and low‐BNML were associated independently with improved PFS in multivariate analysis. Analysis of the tumour burden combined with BSLD and BNML revealed a trend towards improved PFS as the tumour burden decreased, with median PFS of 22.3, 8.7, and 3.9 months in the low‐ (N = 13), medium‐ (N = 42) and high‐burden (N = 37) groups respectively.Sixty‐eight patients (74%) had progressive disease or died. Forty‐four patients (48%) in the low‐BSLD group had a median progression‐free survival (PFS) of 9.5 months, whereas patients in the high‐BSLD group had a median PFS of 4.6 months  among patients with the low tumour burden, medium tumour burden and high tumour burden. The result of our study revealed a trend towards improved PFS as the tumour burden decreased. The following characteristics were analysed: age, sex, Eastern Cooperative Oncology Group performance status (ECOG‐PS), smoking history, histology, stage at diagnosis of lung cancer, PD‐L1 tumour proportion score (TPS) and metastatic sites. The study protocol was approved by the institutional ethical review board (registration number: J2020‐176‐2020‐1) and was performed in compliance with the principles of the Declaration of Helsinki. As this was a retrospective study, obtaining informed consent from the patients was not required. Immunohistochemical staining of PD‐L1 in tumour cells was performed using the pharmDx antibody .2.2All patients were assessed for all lesions in the chest, abdomen and intracranial region by computed tomography (CT) or magnetic resonance imaging (MRI) before the first administration of PD‐1/PD‐L1 inhibitors plus chemotherapy. In accordance with previous studies, BSLD was evaluated by measuring the target lesions based on the Response Evaluation Criteria for Solid Tumours (RECIST),The baseline number of metastatic lesions (BNML) was defined as any metastatic lesion identified on baseline CT or MRI scan, including both target and non‐target lesions. Metastatic thoracic lymph nodes (N1–N3) were collectively considered as a single metastatic lesion.To estimate the impact of tumour burden more accurately, we classified the patients into three groups by combining the BSLD and BNML data. Patients with low‐BSLD and low‐BNML were categorised into the ‘low tumour burden’ group, those with low‐BNML/high‐BSLD or high‐BNML/low‐BSLD into the ‘medium tumour burden’ group, and those with high‐BSLD and high‐BNML into the ‘high tumour burden’ group Figure .PD was identified by reviewing the follow‐up radiological imaging data, including the data of CT and MRI, after the initiation of PD‐1/PD‐L1 inhibitors plus chemotherapy. Tumour responses were classified according to RECIST version 1.1.2.3p < 0.2 in the univariate analysis, initial stage, and PD‐L1 TPS, as known predictors of PD‐1/PD‐L1 inhibitors, were included in the subsequent multivariate analysis. All categorical variables were analysed using Fisher's exact test and continuous variables were analysed using the t‐test. All analyses were performed using STATA software .The correlation between BSLD and BNML was evaluated based on Spearman's rank correlation. PFS was defined as the time from the start of PD‐1/PD‐L1 inhibitor plus chemotherapy to disease progression or death. Overall survival (OS) was defined as the time from the start of PD‐1/PD‐L1 inhibitor plus chemotherapy to death. PFS and OS were estimated using the Kaplan–Meier method and compared using the log‐rank test. Potential risk factors were assessed using univariate and multivariate analyses with a Cox proportional hazards model for PFS. Covariates in the univariate analysis included BSLD, BNML, PD‐L1 TPS, age, sex, smoking status, ECOG‐PS, stage (Stage IV vs. recurrence), and initial central nervous system metastasis. Factors with 33.1Of the 120 consecutive patients with advanced NSCLC who were treated with PD‐1/PD‐L1 inhibitors plus chemotherapy, we excluded 28 patients who participated in clinical trials. Thus, we included 92 patients in the study. The clinical characteristics of the entire cohort are summarised in Table 3.2p = 0.926).As shown in Figure p = 0.098). The ORR improved significantly in patients with low‐BNML than those with high‐BNML .The objective response rate (ORR) was higher in patients with low‐BSLD than in those with high‐BSLD . We observed 68 (74%) events of disease progression or death, and the median PFS was 7.6 months  for all patients. The median PFS was 9.5 months (95% CI = 7.5–12.3) in patients with low‐BSLD and 4.6 months (95% CI = 3.2–7.3) in those with high‐BSLD, that is patients in the low‐BSLD group had a significantly better PFS  for patients in the low‐BNML group and 6.5 months (95% CI = 3.9–8.6) for those in the high‐BNML group, that is the patients in the low‐BNML group had a significantly improved PFS  and low‐BNML  were favourable predictive factors for PFS. Additionally, a lack of central nervous system metastases tended to be a favourable predictive factor . In the multivariate analysis of PFS, low‐BSLD  and low‐BNML  were independent favourable predictive factors . Forty‐four patients (48%) had died at the cut‐off date. The median OS was 17.3 months (95% CI = 14.8–23.7) for all patients. The median OS was 20.7 months (95% CI: 15.8–NR) for patients with low‐BSLD and 15.0 months (95% CI: 9.0–NR) for those with high‐BSLD. Although there was no significant difference, patients with low‐BSLD had a more favourable OS than those with high‐BSLD  for patients with low‐BNML and 15.8 months (95% CI: 10.8–20.7) for those with high‐BNML, with patients in the low‐BNML group showing a significantly improved OS .To estimate the impact of tumour burden more accurately, we further classified the patients into low, medium, and high tumour burden groups based on the BSLD and BNML. Thirteen patients (14%) had a low tumour burden, 42 patients (46%) had a medium tumour burden and 37 patients (40%) had a high tumour burden. The ORR was significantly associated with the tumour burden according to this classification  and those with high tumour burden .As shown in Figure p = 0.019) and high tumour burden .As shown in Figure 4To the best of our knowledge, this is the first study to show the impact of tumour burden on the efficacy of PD‐1/PD‐L1 inhibitors plus chemotherapy in patients with advanced NSCLC. The results of our study revealed that a high tumour burden had a negative impact on the efficacy of PD‐1/PD‐L1 inhibitors plus chemotherapy, regardless of other clinical confounding factors. Our results also showed that there was no correlation between BSLD and BNML, and that these two indices complemented each other as indicators of the tumour burden. Furthermore, combined analysis of BNML and BSLD suggested that PD‐1/PD‐L1 inhibitors plus chemotherapy is the most effective when the tumour burden is the lowest. Therefore, simultaneous assessment of BSLD and BNML may reveal the actual tumour burden.As previously established, PD‐1/PD‐L1 inhibitors plus chemotherapy is not effective for all patients with advanced NSCLC; even in patients in whom this treatment is effective, a durable response is not necessarily achieved.A previous study on cytotoxic chemotherapy for advanced NSCLC has showed that BSLD is significantly associated with OS but not with PFS or the response rate.+ tumour‐infiltrating lymphocytes (TILs) are an essential component of the response to PD‐1/PD‐L1 inhibitor monotherapy.Additionally, several studies have indicated that elevated CD8A basic study revealed that tumour debulking improves the outcomes of PD‐1 inhibitor treatment.There were some limitations to our study. First, our analysis was limited because of the retrospective nature of our study and our inability to account for unknown confounders. The small number of patients in this study may have affected the statistical power. Furthermore, the study was conducted in a single cohort from a single institution and was not independently validated. Unlike the results of previous studies, high expression of PD‐L1 was not an independent predictive factor for the efficacy of PD‐1/PD‐L1 inhibitors plus chemotherapy in this study. Although patients with high PD‐L1 expression have been mainly treated with pembrolizumab monotherapy in our institution, those with a high tumour burden were treated with PD‐1/PD‐L1 inhibitors plus chemotherapy. This bias in treatment selection may have led to PD‐L1 expression in tumours not being a predictor of efficacy of PD‐1/PD‐L1 inhibitors plus chemotherapy in the present study. Furthermore, we could not directly evaluate tumour burden, as there is no common threshold for BNML or BSLD to estimate the tumour burden. However, this study and previous studies demonstrated the clinical relevance of the tumour burden in chemotherapy, PD‐1/PD‐L1 inhibitor use, and PD‐1/PD‐L1 inhibitor plus chemotherapy using a common threshold for BSLD.In conclusion, our findings suggest that a high tumour burden has a negative impact on the efficacy of PD‐1/PD‐L1 inhibitor plus chemotherapy in patients with advanced NSCLC. The addition of local ablative therapy for primary and metastatic sites to PD‐1/PD‐L1 inhibitors plus chemotherapy may improve survival outcomes by reducing the tumour burden, which should be further evaluated in prospective trials.Taichi Miyawaki: Conceptualisation, data curation, investigation, methodology, visualisation, writing—original draft. Hirotsugu Kenmotsu: Conceptualisation, Investigation, Methodology, Project administration, Supervision, Writing—review & editing. Kosei Doshita: Investigation. Hiroaki Kodama: Investigation. Naoya Nishioka: Investigation. Yuko Iida: Investigation. Eriko Miyawaki: Investigation. Nobuaki Mamesaya: Investigation. Haruki Kobayashi: Investigation. Shota Omori: Investigation. Ryo Ko: Investigation. Kazushige Wakuda: Investigation. Akira Ono: Investigation. Tateaki Naito: Conceptualisation, Investigation. Haruyasu Murakami: Investigation. Keita Mori: Methodology, Formal analysis, Supervision. Hideyuki Harada: Supervision. Masahiro Endo: Supervision. Kazuhisa Takahashi: Supervision. Toshiaki Takahashi: Project administration, Supervision, Writing—review & editing.This research did not receive any specific grant from funding agencies in the public, commercial, or not‐for‐profit sectors.Dr. Kenmotsu reports grants and personal fees from Chugai Pharmaceutical Co., Ltd., personal fees from Ono Pharmaceutical Co., Ltd., personal fees from Boehringer Ingelheim, personal fees from Eli Lilly K.K, personal fees from Kyowa Hakko Kirin Co., Ltd., personal fees from Bristol‐Myers Squibb, personal fees from MSD, grants and personal fees from Novartis Pharma K.K., grants and personal fees from Daiichi‐Sankyo Co., Ltd., grants and personal fees from AstraZeneca K.K., personal fees from Pfizer, personal fees from Taiho Pharma, outside the submitted work.Dr. Mamesaya reports personal fees from AstraZeneca K.K., Pfizer Japan, Inc., personal fees from Chugai Pharmaceutical Co., Ltd., grants and personal fees from Boehringer Ingelheim, personal fees from MSD K.K., personal fees from Taiho Pharmaceutical Co., Ltd., personal fees from Oon Pharmaceutical Co., Ltd., outside the submitted work.Dr. Kobayashi reports personal fees from Eli Lilly K.K, personal fees from Taiho Pharmaceutical, personal fees from AstraZeneca, outside the submitted work.Dr. Omori reports personal fees from Chugai Pharmaceutical Co., Ltd., Ono Pharmaceutical, AstraZeneca K.K., Boehringer Ingelheim, Taiho Pharmaceutical, and MSD, which are unrelated to the submitted work.Dr. Ko reports grants and personal fees from Boehringer Ingelheim, grants and personal fees from AstraZeneca, personal fees from Taiho Pharmaceutical, personal fees from Chugai Pharmaceutical, personal fees from Ono Pharmaceutical, personal fees from Pfizer, personal fees from Lilly, outside the submitted work.Dr. Wakuda reports grants and personal fees from Chugai Pharmaceutical Co., Ltd., personal fees from Taiho Pharmaceutical, personal fees from Boehringer Ingelheim, personal fees from Eli Lilly K.K., personal fees from Ono Pharmaceutical, personal fees from MSD, grants and personal fees from AstraZeneca, grants from Novartis, grants from Abbvie, outside the submitted work.Dr. Ono reports grants from Taiho Pharmaceutical, grants from Ono Pharmaceutical, grants from Chugai Pharmaceutical Co., Ltd., grants from Novartis Pharma K.K., outside the submitted work.Dr. Murakami reports personal fees from AstraZeneca K.K., Ono Pharmaceutical, Bristol‐Myers Squibb Japan, Chugai Pharmaceutical Co., Ltd., Pfizer Inc., Novartis Pharma K.K., Boehringer Ingelheim, Taiho Pharmaceutical, Eli Lilly K.K., and MSD, which are unrelated to the submitted work.Dr. Harada reports personal fees from Daiichi‐Sankyo Pharmaceutical Co. during the conduct of the study as well as personal fees from Daiichi‐Sankyo Pharmaceutical Co., AstraZeneca K.K., Brain Labo Co., and Chugai Pharmaceutical Co. and grants from the Japan Agency for Medical Research and Development and the National Cancer Center Research and Development Fund, which are unrelated to the submitted work.Dr. Endo reports personal fees from Ono Pharmaceutical, personal fees from AstraZeneca, personal fees from Takeda Pharmaceutical Co., Ltd., personal fees from Daiichi‐Sankyo Co., Ltd., outside the submitted work.Dr. Kazuhisa Takahashi reports grants and personal fees from AstraZeneca K.K., Pfizer Japan, Inc., Eli Lilly K.K., MSD, and Boehringer Ingelheim as well as grants from Takeda Pharmaceutical Co., Ltd., Chugai Pharmaceutical Co., Ltd., Taiho Pharmaceutical Co., Ltd., KYORIN Pharmaceutical Co., Ltd., Ono Pharmaceutical Co., Ltd., GlaxoSmithKline Consumer Healthcare Japan K.K., Shionogi & Co., Ltd., and Novartis Pharma K.K., which are not related to the submitted work.Dr. Toshiaki Takahashi reports grants and personal fees from AstraZeneca K.K., Pfizer Japan, Inc., grants and personal fees from Eli Lilly Japan K.K., grants and personal fees from Chugai Pharmaceutical Co., Ltd., grants and personal fees from Ono Pharmaceutical Co., Ltd., grants and personal fees from MSD K.K., grants and personal fees from Boehringer Ingelheim Japan, Inc., grants and personal fees from Pfizer Japan, Inc., personal fees from Roche Diagnostics K.K., outside the submitted work.The remaining authors declare no conflict of interest.The study protocol was approved by the institutional ethical review board (registration number: J2020‐176‐2020‐1) and was performed in compliance with the principles of the Declaration of Helsinki.As this was a retrospective study, obtaining informed consent from the patients was not required.


Single‐use electrocardiography (ECG) leads have been developed to reduce healthcare‐associated infection. This study compared the validity and reliability of short‐term heart rate variability (HRV) obtained from single‐use disposable ECG leads.Thirty healthy subjects  underwent 5‐min resting HRV assessments using disposable (single use) ECG cable and wire system  and a standard, reusable ECG leads .2); 0.89 , HF power (ms2); 0.90 ] variables. The mean difference and upper and lower limits of agreement between disposable and reusable leads for time‐ and frequency‐domain variables were acceptable. Analysis of repeated measures using disposable leads demonstrated excellent reproducibility (ICC 95% CI) for R‐R interval (ms); 0.93 , RMSSD (ms); 0.93 , SDNN (ms); 0.88 , LF power (ms2); 0.87 , and HF power (ms2); 0.88  with coefficient of variation ranging from 2.2% to 5% .Intraclass correlation coefficient (ICC) with 95% confidence interval (CI) between disposable and reusable ECG leads was for the time domain , the root mean square of successive normal R‐R interval differences (RMSSD) (ms); 0.91 , the SD of normal‐to‐normal R‐R intervals (SDNN) (ms); 0.91  and frequency domain [low‐frequency (LF) normalized units (nu); 0.90 , high frequency (HF) nu; 0.91 , LF power (msSingle‐use Kendall DL™ ECG leads demonstrate a valid and reproducible tool for the assessment of HRV. For example, the standard deviation of normal‐to‐normal R‐R intervals (SDNN); a representation of beat‐to‐beat intervals without artefacts, provides estimates of overall HRV, and the root mean square of successive beat‐to‐beat interval differences (RMSSD), gives an indication of variations in parasympathetic activity.Although microbial contamination of the fomite electrocardiographic telemetry systems is significantly reduced after cleaning,22.1This was a single‐center, prospective, observational validity, and reliability study approved by the Coventry University Institute of Health and Wellbeing Research Ethics Committee (P109193). The study protocol and procedures were in accordance with the declaration of Helsinki, and participants provided written informed consent before taking part in the study. To ensure a high level of consistency in the physiological status of participants, individuals without a history of cardiovascular, respiratory, or other chronic or acute illnesses participated in the study. Participants attended the Cardiovascular Research Laboratory, for two visits lasting approximately 1.5 h each. All participants were asked to refrain from alcohol and caffeine on the study day and to avoid vigorous exercise 24 h before the study visit as these could alter HRV. While supine, participants’ blood pressure and heart rate were measured, and participants’ skin was prepared for the attachment of the ECG electrodes. The electrodes were placed on the participant's thorax by trained technicians using the standard 12‐lead placementMeasurements were repeated 1 week apart using the same approach, to assess the reliability of disposable leads in assessing HRV. Signal processing and generation of R‐R interval variability data were made automatically by the CardioExpress software. HRV measurements included the following variables: heart rate, R‐R interval, SDNN, RMSSD, HF power , and LF power .2.2Descriptive statistics were calculated for all variables. Data are presented as mean ± SD. Normal distribution was assessed via visual inspection of frequency histogram and with either a Kolmogorov–Smirnov or Shapiro–Wilk test. Heart rate, R‐R intervals, LF power, and HF power in normalized units were analyzed using raw data, whereas RMSSD, SDNN, LF power, and HF power were analyzed after log transformation due to non‐normal distribution. The agreement between disposable and reusable leads was performed using Bland–Altman analysis , intraclass correlation coefficient (ICC), and Pearson's coefficient of correlation. An ICC and Pearson's coefficient of correlation value above 0.9 was considered excellent correlation, while value ≥ 0.7 < 0.9 was considered very good. Test–retest reliability of the disposable leads was performed using ICC and coefficient of variation (CoV). CoV (%) was used to measure the degree of dispersion around the mean of repeated measurements and was calculated as the standard deviation of repeated measurements expressed as a percentage of the mean. Data were analyzed using SPSS (Version 27).3p > 0.05).Thirty healthy participants were recruited for the study. The physical characteristics of participants are presented in Table 2); 0.89 , HF power (ms2); 0.90 ] variables (Table 2); −0.18 , HF power (ms2); −0.16 ] variables  between disposable and reusable ECG leads was excellent for the time domain  and very good to excellent for frequency domain  (Table Repeated measures of disposable leads demonstrated excellent reproducibility (ICC 95% CI) for time‐domain variables  with CoV <5%. Frequency domain variables also showed very good to excellent reproducibility [LF nu; 0.77 0.52, 0.89), HF nu; 0.74 , LF power  Excellent agreement between disposable and reusable ECG leads in the assessment of time and frequency domain HRV measures and (ii) disposable ECG leads demonstrated an excellent degree of reliability for time domain HRV measures, and very good to excellent reliability for frequency domain HRV measures.Over the past two decades, several investigations have scrutinized the validity and reliability of commercially available technologies for the assessment of HRV.Evidence from available literature assessing multiple technologies suggests large discrepancies in the design and analytical methods used to analyze time and frequency domain variables leading to conflicting results and difficulty in making comparisons among studies. For example, some studiesAlthough there is no direct comparison with previous studies due to the novelty, all HRV variables assessed in this study fall within the normative range reported by previous studies.The present study is not without limitations. The reliability of the disposable ECG leads was assessed by comparing the original HRV values to values obtained 1‐week later. Although our results showed excellent reliability, any inherent error may have been masked by the natural variation exhibited by participants during the second measurement.In conclusion, the present study has shown that disposable ECG leads are a valid tool for evaluating HRV. The present study's authors agree with other studies suggesting that HRV variables should always be measured using the same device during HRV assessments.Nduka C. Okwose: Formal analysis; investigation; supervision; writing – original draft; writing – review and editing. Sophie L. Russell: Formal analysis; investigation; writing – review and editing. Mushidur Rahman: Formal analysis; investigation. Charles J. Steward: Investigation. Amy E. Harwood: Investigation; writing – review and editing. Gordon McGregor: Writing – review and editing. Srdjan Ninkovic: Writing – review and editing. Helen Maddock: Writing – review and editing. Prithwish Banerjee: Writing – review and editing. Djordje G. Jakovljevic: Conceptualization; funding acquisition; investigation; methodology; project administration; writing – review and editing.The authors declare no conflict of interest.The lead author Djordje G. Jakovljevic affirms that this manuscript is an honest, accurate, and transparent account of the study being reported; that no important aspects of the study have been omitted; and that any discrepancies from the study as planned  have been explained.