Plasma lysosphingolipids in GRN-related diseases: Monitoring lysosomal dysfunction to track disease progression.

GRN mutations are among the main genetic causes of frontotemporal dementia (FTD). Considering the progranulin involvement in lysosomal homeostasis, we aimed to evaluate if plasma lysosphingolipids (lysoSPL) are increased in GRN mutation carriers, and whether they might represent relevant fluid-based biomarkers in GRN-related diseases. We analyzed four lysoSPL levels in plasmas of 131 GRN carriers and 142 non-carriers, including healthy controls and patients with frontotemporal dementias (FTD) carrying a C9orf72 expansion or without any mutation. GRN carriers consisted of 102 heterozygous FTD patients (FTD-GRN), three homozygous patients with neuronal ceroid lipofuscinosis-11 (CLN-11) and 26 presymptomatic carriers (PS-GRN), the latter with longitudinal assessments. Glucosylsphingosin d18:1 (LGL1), lysosphingomyelins d18:1 and isoform 509 (LSM18:1, LSM509) and lysoglobotriaosylceramide (LGB3) were measured by electrospray ionization-tandem mass spectrometry coupled to ultraperformance liquid chromatography. Levels of LGL1, LSM18:1 and LSM509 were increased in GRN carriers compared to non-carriers (p < 0.0001). No lysoSPL increases were detected in FTD patients without GRN mutations. LGL1 and LSM18:1 progressively increased with age at sampling, and LGL1 with disease duration, in FTD-GRN. Among PS-GRN carriers, LSM18:1 and LGL1 significantly increased over 3.4-year follow-up. LGL1 levels were associated with increasing neurofilaments in presymptomatic carriers. This study evidences an age-dependent increase of ß-glucocerebrosidase and acid sphingomyelinase substrates in GRN patients, with progressive changes as early as the presymptomatic phase. Among FTD patients, plasma lysoSPL appear to be uniquely elevated in GRN carriers, and thus might serve as suitable non-invasive disease-tracking biomarkers of progression, specific to the pathophysiological process. Finally, this study might add lysoSPL to the portfolio of fluid-based biomarkers, and pave the way to disease-modifying approaches based on lysosomal function rescue in GRN diseases.

Plasma GM2 ganglioside potential biomarker for diagnosis, prognosis and disease monitoring of GM2-Gangliosidosis.

GM2-Gangliosidosis are a group of inherited lysosomal storage pathologies characterized by a large accumulation of GM2 ganglioside in the lysosome. They are caused by mutation in HEXA or HEXB causing reduced or absent activity of a lysosomal ß-hexosaminidase A, or mutation in GM2A causing defect in GM2 activator protein (GM2AP), an essential protein for the activity of the enzyme. Biochemical diagnosis relies on the measurement of ß-hexosaminidases A and B activities, which is able to detect lysosomal enzyme deficiency but fails to identify defects in GM2AP. We developed a rapid, specific and sensitive liquid chromatography-mass spectrometry-based method to measure simultaneously GM1, GM2, GM3 and GD3 molecular species. Gangliosides were analysed in plasma from 19 patients with GM2-Gangliosidosis: Tay-Sachs (n = 9), Sandhoff (n = 9) and AB variant of GM2-Gangliosidosis (n = 1) and compared to 20 age-matched controls. Among patients, 12 have a late adult-juvenile-onset and 7 have an infantile early-onset of the disease. Plasma GM2 molecular species were increased in all GM2-Gangliosidosis patients (19/19), including the patient with GM2A mutation, compared to control individuals and compared to patients with different other lysosomal storage diseases. GM234:1 and GM234:1/GM334:1 ratio discriminated patients from controls with 100% sensitivity and specificity. GM234:1 and GM234:1/GM334:1 were higher in patients with early-onset compared to those with late-onset of the disease, suggesting a relationship with severity. Longitudinal analysis in one adult with Tay-Sachs disease over 9 years showed a positive correlation of GM234:1 and GM234:1/GM334:1 ratio with age at sampling. We propose that plasma GM2 34:1 and its ratio to GM3 34:1 could be sensitive and specific biochemical diagnostic biomarkers for GM2-Gangliosidosis including AB variant and could be useful as a first line diagnostic test and potential biomarkers for monitoring upcoming therapeutic efficacy.

Burden of liver disease progression in hospitalized patients with type 2 diabetes mellitus.

BACKGROUND & AIMS: There are uncertainties regarding the burden of liver disease in patients with type 2 diabetes (T2D). Thus, we aimed to quantify the burden of liver disease, identify risk factors, and estimate attributable risks in patients with T2D. METHODS: We measured adjusted hazard ratios of liver disease progression to hepatocellular carcinoma and/or decompensated cirrhosis in a 2010-2020 retrospective, bicentric, longitudinal, cohort of 52,066 hospitalized patients with T2D. RESULTS: Mean age was 64±14 years and 58% were men. Alcohol use disorders accounted for 57% of liver-related complications and were associated with all liver-related risk factors. Non-metabolic liver-related risk factors accounted for 37% of the liver burden. T2D control was not associated with liver disease progression. The incidence (95% CI) of liver-related complications and of competing mortality were 3.9 (3.5-4.3) and 27.8 (26.7-28.9) per 1,000 person-years at risk, respectively. The cumulative incidence of liver disease progression exceeded the cumulative incidence of competing mortality only in the presence of well-identified risk factors of liver disease progression, including alcohol use. The incidence of hepatocellular carcinoma was 0.3 (95% CI 0.1-0.5) per 1,000 person-years in patients with obesity and it increased with age. The adjusted hazard ratios of liver disease progression were 55.7 (40.5-76.6), 3.5 (2.3-5.2), 8.9 (6.9-11.5), and 1.5 (1.1-2.1), for alcohol-related liver disease, alcohol use disorders without alcohol-related liver disease, non-metabolic liver-related risk factors, and obesity, respectively. The attributable fractions of alcohol use disorders, non-metabolic liver-related risk factors, and obesity to the liver burden were 55%, 14%, and 7%, respectively. CONCLUSIONS: In this analysis of data from 2 hospital-based cohorts of patients with T2D, alcohol use disorders, rather than obesity, contributed to most of the liver burden. These results suggest that patients with T2D should be advised to drink minimal amounts of alcohol. LAY SUMMARY: There is uncertainty on the burden of liver-related complications in patients with type 2 diabetes. We studied the risks of liver cancer and complications of liver disease in over 50,000 patients with type 2 diabetes. We found that alcohol was the main factor associated with complications of liver disease. This finding has major implications on the alcohol advice given to patients with type 2 diabetes.

Serum neuron-specific enolase: a new tool for seizure risk monitoring after status epilepticus.

BACKGROUND AND PURPOSE: There is a need for accurate biomarkers to monitor electroencephalography (EEG) activity and assess seizure risk in patients with acute brain injury. Seizure recurrence may lead to cellular alterations and subsequent neurological sequelae. Whether neuron-specific enolase (NSE) and S100-beta (S100B), brain injury biomarkers, can reflect EEG activity and help to evaluate the seizure risk was investigated. METHODS: Eleven patients, admitted to an intensive care unit for refractory status epilepticus, who underwent a minimum of 3 days of continuous EEG concomitantly with daily serum NSE and S100B assays were included. At 103 days the relationships between serum NSE and S100B levels and two EEG scores able to monitor the seizure risk were investigated. Biochemical biomarker thresholds able to predict seizure recurrence were sought. RESULTS: Only NSE levels positively correlated with EEG scores. Similar temporal dynamics were observed for the time courses of EEG scores and NSE levels. NSE levels above 17 ng/ml were associated with seizure in 71% of patients. An increase of more than 15% of NSE levels was associated with seizure recurrence in 80% of patients. CONCLUSIONS: Our study highlights the potential of NSE as a biomarker of EEG activity and to assess the risk of seizure recurrence.

Absolute Versus Relative Changes in Cardiac Troponin T: Corresponding Cutoffs Based on Quantile Generalized Additive Models (QGAM).

BACKGROUND: The diagnosis of myocardial injury/infarction (MI) mainly relies on relative changes in cardiac troponin. However, absolute change cutoffs provide greater diagnostic sensitivity. We determined the absolute changes in high-sensitive cardiac troponin T concentrations (absΔhs-cTnT) corresponding to the main relative cutoffs (relΔhs-cTnT), using a quantile generalized additive model (qgam). METHODS: Plasma Δhs-cTnT from patients selected with a time variation of 1 to 6 hours were collected over a 6-year period. The absΔhs-cTnT-to-relΔhs-cTnT relationship was fitted using qgam, after ordered quantile-based normalization (OQN) to reduce the influence of extreme values. RESULTS: The qgam regression curve was nonlinear. Classifying patients (n = 9,753) above the recommended relΔhs-cTnT and predicted absΔhs-cTnT cutoffs as positive, the MI diagnosis rates were similar, and more reliable using the OQN-transformed data-based qgam, as compared to the untransformed data-based one. CONCLUSIONS: Through an optimized qgam-based approach accounting for heavy-tailed distributions, absolute Δhs-cTnT are provided for the corresponding relative Δhs-cTnT cutoffs.

Disturbances of brain cholesterol metabolism: A new excitotoxic process associated with status epilepticus.

The understanding of the excitotoxic processes associated with a severe status epilepticus (SE) is of major importance. Changes of brain cholesterol homeostasis is an emerging candidate for excitotoxicity. We conducted an overall analysis of the cholesterol homeostasis both (i) in fluids and tissues from patients with SE: blood (n = 63, n = 87 controls), CSF (n = 32, n = 60 controls), and post-mortem brain tissues (n = 8, n = 8 controls) and (ii) in a mouse model of SE induced by an intrahippocampal injection of kainic acid. 24-hydroxycholesterol levels were decreased in kainic acid mouse hippocampus and in human plasma and post-mortem brain tissues of patients with SE when compared with controls. The decrease of 24-hydroxycholesterol levels was followed by increased cholesterol levels and by an increase of the cholesterol synthesis. Desmosterol levels were higher in human CSF and in mice and human hippocampus after SE. Lanosterol and dihydrolanosterol levels were higher in plasma from SE patients. Our results suggest that a CYP46A1 inhibition could occur after SE and is followed by a brain cholesterol accumulation. The excess of cholesterol is known to be excitotoxic for neuronal cells and may participate to neurological sequelae observed after SE. This study highlights a new pathophysiological pathway involved in SE excitotoxicity.

Relationships between renal function variations and relative changes in cardiac troponin T concentrations based on quantile generalized additive models (qgam).

OBJECTIVES: The relationship between high-sensitive cardiac troponin T concentration (hs-cTnT) and renal markers levels is known. However, the extent to which their variations are associated remains to be explored. Objective: model the relationship between relative changes in hs-cTnT (Δhs-cTnT) and variations in creatinine (Δcre) or estimated glomerular filtration rate (ΔeGFR), using a quantile generalized additive model (qgam). METHODS: Concomitant plasma Δhs-cTnT and Δcre from patients aged 18-100 years, selected with a time variation (Δtime) of 3 h-7 days, were collected over a 5.8-year period. Relationships between Δhs-cTnT and covariates Δcre (A) or ΔeGFR (B), including age, Δtime, hour of blood sampling (HSB) and covariates interactions were fitted using qgam. RESULTS: On the whole (n=106567), Δhs-cTnT was mainly associated with Δcre, in a positive and nonlinear way (-21, -6, +5, +20, +55% for -50, -20, +20, +50, +100%, respectively), but to a lesser extent with age (min -9%, max +2%), Δtime (min -4%, max +8%), and HSB (min -5%, max +7%). Δhs-cTnT was negatively associated with ΔeGFR (+46, +7, -5, -11, -20% for -50, -20, +20, +50, +100%, respectively). Classifying Δhs-cTnT as consistent or not with myocardial injury based on recommendations, an interpretation of Δhs-cTnT adjusted for model A or B led to statistically significant but small diagnostic discrepancies (<2%), as compared to an interpretation based on Δhs-cTnT only. CONCLUSIONS: From a laboratory and statistical standpoint, considering renal function variations when interpreting relative changes in cardiac troponin T has a minor impact on the diagnosis rate of myocardial injury.

Thyroid hormone and folinic acid in young children with Down syndrome: the phase 3 ACTHYF trial.

PURPOSE: To determine whether folinic acid (FA) and thyroxine, in combination or alone, benefit psychomotor development in young patients with Down syndrome (DS). METHODS: The Assessment of Systematic Treatment With Folinic Acid and Thyroid Hormone on Psychomotor Development of Down Syndrome Young Children (ACTHYF) was a single-center, randomized, double-blind, placebo-controlled phase 3 trial in DS infants aged 6-18 months. Patients were randomly assigned to one of four treatments: placebo, folinic acid (FA), L-thyroxine, or FA+L-thyroxine, administered for 12 months. Randomization was done by age and sex. The primary endpoint was adjusted change from baseline in Griffiths Mental Development Scale global development quotient (GDQ) after 12 months. RESULTS: Of 175 patients randomized, 143 completed the study. The modified intention-to-treat (mITT) population included all randomized patients who did not prematurely discontinue due to elevated baseline thyroid stimulating hormone (TSH). Baseline characteristics in the mITT were well balanced between groups, with reliable developmental assessment outcomes. Adjusted mean change in GDQ in the mITT showed similar decreases in all groups (placebo: -5.10 [95% confidence interval (CI) -7.84 to -2.37]; FA: -4.69 [95% CI -7.73 to -1.64]; L-thyroxine: -3.89 [95% CI -6.94 to -0.83]; FA+L-thyroxine: -3.86 [95% CI -6.67 to -1.06]), with no significant difference for any active treatment group versus placebo. CONCLUSION: This trial does not support the hypotheses that thyroxine and/or folinic acid improve development of young children with DS or are synergistic. This trial is registered with ClinicalTrials.gov number, NCT01576705.

Circulating Receptor Activator of Nuclear Factor kB Ligand and triglycerides are associated with progression of lower limb arterial calcification in type 2 diabetes: a prospective, observational cohort study.

BACKGROUND: Lower limb arterial calcification is a frequent, underestimated but serious complication of diabetes. The DIACART study is a prospective cohort study designed to evaluate the determinants of the progression of lower limb arterial calcification in 198 patients with type 2 diabetes. METHODS: Lower limb arterial calcification scores were determined by computed tomography at baseline and after a mean follow up of 31.20 ± 3.86 months. Serum RANKL (Receptor Activator of Nuclear factor kB Ligand) and bone remodeling, inflammatory and metabolic parameters were measured at baseline. The predictive effect of these markers on calcification progression was analyzed by a multivariate linear regression model. RESULTS: At baseline, mean ± SD and median lower limb arterial calcification scores were, 2364 ± 5613 and 527 respectively and at the end of the study, 3739 ± 6886 and 1355 respectively. Using multivariate analysis, the progression of lower limb arterial log calcification score was found to be associated with (ß coefficient [slope], 95% CI, p-value) baseline log(calcification score) (1.02, 1.00-1.04, p < 0.001), triglycerides (0.11, 0.03-0.20, p = 0.007), log(RANKL) (0.07, 0.02-0.11, p = 0.016), previous ischemic cardiomyopathy (0.36, 0.15-0.57, p = 0.001), statin use (0.39, 0.06-0.72, p = 0.023) and duration of follow up (0.04, 0.01-0.06, p = 0.004). CONCLUSION: In patients with type 2 diabetes, lower limb arterial calcification is frequent and can progress rapidly. Circulating RANKL and triglycerides are independently associated with this progression. These results open new therapeutic perspectives in peripheral diabetic calcifying arteriopathy. Trial registration NCT02431234.

Cerebrospinal fluid and blood biomarkers of status epilepticus.

Status epilepticus is a condition resulting either from the failure of the mechanisms responsible for seizure termination or from the initiation of mechanisms that lead to abnormally prolonged seizures and require urgent administration of antiepileptic drugs. Refractory status epilepticus requires anesthetics drugs and may lead to brain injury with molecular and cellular alterations (eg, inflammation, and neuronal and astroglial injury) that could induce neurologic sequels and further development of epilepsy. Outcome scores based on demographic, clinical, and electroencephalography (EEG) condition are available, allowing prediction of the risk of mortality, but the severity of brain injury in survivors is poorly evaluated. New biomarkers are needed to predict with higher accuracy the outcome of patients admitted with status in an intensive care unit. Here, we summarize the findings of studies from patients and animal models of status epilepticus. Specific protein markers can be detected in the cerebrospinal fluid and the blood. One of the first described markers of neuronal death is the neuron-specific enolase. Gliosis resulting from inflammatory responses after status can be detected through the increase of S100-beta, or some cytokines, like the High Mobility Group Box 1. Other proteins, like progranulin may reflect the neuroprotective mechanisms resulting from the brain adaptation to excitotoxicity. These new biomarkers aim to prospectively identify the severity and development of disability, and subsequent epilepsy of patients with status. We discuss the advantages and disadvantages of each biomarker, by evaluating their brain specificity, stability in the fluids, and sensitivity to external interferences, such as hemolysis. Finally, we emphasize the need for further development and validation of such biomarkers in order to better assess patients with severe status epilepticus.

Antiatherogenic properties of high-density lipoproteins from arterial plasma are attenuated as compared to their counterparts of venous origin.

BACKGROUND AND AIMS: High-density lipoprotein (HDL) particles play atheroprotective roles by their ability to efflux cholesterol from foam cells and to protect low-density lipoproteins (LDLs) from oxidative damage in the arterial intima. We hypothesized that antioxidative properties of HDLs can be attenuated in the oxygen-rich prooxidative arterial environment, contributing to the development of atherosclerosis. To evaluate this hypothesis, we compared antioxidative activity of HDLs from arterial and venous plasmas. METHODS AND RESULTS: Arterial and venous blood samples were simultaneously obtained from 16 patients (age 68 ± 10 years; 75% males) presenting with ischemic or valvular heart disease. Major HDL subfractions and total HDLs were isolated by density gradient ultracentrifugation and their chemical composition and the capacity to protect LDLs from in vitro oxidation were evaluated. HDL-cholesterol, triglycerides and apolipoprotein (apo) B-100 levels were slightly but significantly reduced by -4 to -8% (p < 0.01) in the arterial vs. venous samples. Total mass of HDL subpopulations was similar and HDL subpopulations did not reveal marked compositional differences between the arterial and venous circulation. Potent antioxidative activity of the small, dense HDL3c subpopulation was significantly reduced in the particles of arterial origin vs. their counterparts from venous plasma (increase of +21% in the propagation rate of LDL oxidation, p < 0.05). Interestingly, antioxidative properties of venous HDLs were enhanced in statin-treated patients relative to untreated subjects. CONCLUSION: Antioxidative properties of small, dense HDLs from arterial plasma are attenuated as compared to the particles of venous origin, consistent with the development of atherosclerosis in the arterial wall.

Non-linearity and gaps in results distribution over successive Roche Lipase method applications: improvement but persistence.

Non-linearity within the primary measurement range of a lipase assay (<300 U/L) has been shown on Cobas® Roche analyzers, causing gaps in results distribution between 300 and 400 U/L. Since, new lipase method applications (LMAs) have been used. The purpose is to retrospectively evaluate their impact on relative frequencies of lipase results (RFLs).Plasma lipase results from two hospital laboratories, assayed over 7.2 years, were collected. Over this period, three successive LMAs, characterized by automated repeat-on-dilution (1/11, 1/2, or 1/10), were applied for lipase results >300 U/L: LMA1 and LMA2 on the Modular®P800, Cobas®c501 and Cobas®C701 analyzers, and LMA3 on the Cobas®C701. RFLs were determined, linearity tests were performed, and inter-agreements between lipase results corrected and uncorrected for nonlinear biases were assessed, using 180 U/L as a decisional cut-off for acute pancreatitis.Overall, RFL gaps narrowed from LMA1 (300 to ∼380 U/L) to LMA3 (300 to ∼330 U/L). For a lipase activity fixed at 300 U/L, non-linearity biases were determined at -11.2% on the Modular®P800 (LMA1), -20.8% on the Cobas®c501 (LMA1), and -3.5% (LMA2) and -2.2% (LM3) on the Cobas®C701. Diagnostically, a maximum of 0.48% lipase results were misclassified as negative (LMA1 on the Cobas®c501), and a minimum of 0.01% misclassified as negative (LMA3 on the Cobas®C701). In conclusion, successive Roche lipase method applications improved linearity within the primary measurement range. While persisting, gaps in lipase results distribution narrowed with the evolution of the methods, with a minor impact in terms of diagnostic of acute pancreatitis.

Plasma cystatin C as a marker for estimated glomerular filtration rate assessment in HIV-1-infected patients treated with dolutegravir-based ART.

Objectives: Inhibition of the organic cation transporter-2 renal tubule transporter by dolutegravir leads to serum creatinine increase. Serum cystatin C is a non-organic cation transporter-2-dependent marker, possibly enabling glomerular filtration rate (GFR) estimation under dolutegravir. Our goal was to evaluate the changes in creatinine- and cystatin C-based estimated GFR values before and after dolutegravir initiation. Methods: Creatinine and cystatin measurements were carried out on frozen plasma samples from HIV-1-infected patients, before and after dolutegravir initiation, between October 2016 and March 2017 at Pitié-Salpêtrière Hospital. CKD-EPI equations were used to estimate mean GFR from creatinine and cystatin C values. Variations were analysed by paired t-test. Results: Forty-four patients were included [median age = 48 years (IQR 36-58) and median CD4 count = 592 cells/mm3 (IQR 388-728)], including 6 ART-naive patients and 38 on switch strategies [72% with viral load <50 copies/mL and median ART duration = 13 years (IQR 5-20)]. Before dolutegravir initiation (median time = 41 days), 19 patients (43%) had creatinine-based estimated GFR <90 mL/min/1.73 m2 and 11 (25%) had cystatin C-based estimated GFR <90 mL/min/1.73 m2. After dolutegravir initiation, serum creatinine values significantly increased (+8.6 µmol/L, 95% CI +5.8; +11.4, P < 0.001) and associated estimated GFR significantly decreased (-7.7 mL/min/1.73 m2, 95% CI -10.4; -5.1, P < 0.001). In contrast, there was no significant change in cystatin C value variation and associated estimated GFR. The same results were observed regardless of renal function at baseline. Conclusions: Creatinine values increased after dolutegravir initiation, whereas no change was observed for cystatin C values. Use of cystatin C may enable better understanding of plasma creatinine fluctuations after dolutegravir initiation, particularly in high-risk renal patients.

Determination of age- and sex-specific 99th percentiles for high-sensitive troponin T from patients: an analytical imprecision- and partitioning-based approach.

BACKGROUND: Detection of acute myocardial infarction (AMI) is mainly based on a rise of cardiac troponin with at least one value above the 99th percentile upper reference limit (99th URL). However, circulating high-sensitive cardiac troponin T (hs-cTnT) concentrations depend on age, sex and renal function. Using an analytical imprecision-based approach, we aimed to determine age- and sex-specific hs-cTnT 99th URLs for patients without chronic kidney disease (CKD). METHODS: A 3.8-year retrospective analysis of a hospital laboratory database allowed the selection of adult patients with concomitant plasma hs-cTnT (<300 ng/L) and creatinine concentrations, both assayed twice within 72 h with at least 3 h between measurements. Absence of AMI was assumed when the variation between serial hs-cTnT values was below the adjusted-analytical change limit calculated according to the inverse polynomial regression of analytical imprecision. Specific URLs were determined using Clinical and Laboratory Standards Institute (CLSI) methods, and partitioning was tested using the proportion method, after adjustment for unequal prevalences. RESULTS: After outlier removal (men: 8.7%; women: 6.6%), 1414 men and 1082 women with estimated glomerular filtration rate (eGFR) ≥60 mL/min/1.73 m2 were assumed as non-AMI. Partitioning into age groups of 18-50, 51-70 and 71-98 years, the hs-cTnT 99th URLs adjusted on French prevalence were 18, 33, 66 and 16, 30, 84 ng/L for men and women, respectively. Age-partitioning was clearly required. However, sex-partitioning was not justified for subjects aged 18-50 and 51-70 years for whom a common hs-cTnT 99th URLs of about 17 and 31 ng/L could be used. CONCLUSIONS: Based on a laboratory approach, this study supports the need for age-specific hs-cTnT 99th URLs.

Wide-range CRP versus high-sensitivity CRP on Roche analyzers: focus on low-grade inflammation ranges and high-sensitivity cardiac troponin T levels.

Wide-range C-reactive protein (wr-CRP) has been proposed as an economical alternative to high-sensitivity C-reactive protein (hs-CRP) for the evaluation of low-grade inflammation-associated cardiovascular risk (LGI-CVR). Concomitant values of serum hs-CRP and plasma wr-CRP ≤5 mg/L, and high-sensitivity cardiac troponin T (hs-cTnT), all assayed on Roche Diagnostics analyzers over a 1.8-year period, were extracted from a hospital laboratory database. Hs-CRP and wr-CRP values were compared (Bland-Altman method; Deming's correlation), then separately classified into low (<1 mg/L), moderate (1-3 mg/L) and high (>3 mg/L) LGI-CVR ranges for agreement test (κ), assessed before and after Deming's regression-based adjustment of wr-CRP (Adj-wr-CRP). Wr-CRP and hs-CRP values were strongly correlated, with linearity, whether below 5 mg/L (n = 744; τ = 0.933; p < .001) or below 1 mg/L (n = 283; τ = 0.823; p < .001). Overall, wr-CRP values were lower than hs-CRP (mean bias: -0.11 ± 0.17 mg/L). Agreement was good, with 8.1% of wr-CRP values misclassified compared to hs-CRP (κ: 0.874), and weakly improved after regression-based adjustment (7.7% reclassified values; κ: 0.881). Lowering the Adj-wr-CRP cutoff of the moderate LGI-CVR subrange from 1.0 to 0.9 mg/L resulted in an almost perfect agreement (3.2% reclassified data; κ: 0.950). Hs-cTnT concentration was positively associated with hs-CRP, wr-CRP, and Adj-wr-CRP (p < .001). Within each LGI-CVR subrange, hs-cTnT medians were similar regardless of the hs-CRP, wr-CRP or Adj-wr-CRP used for risk classification. Based on hs-cTnT, this study supports the use of wr-CRP as a low-cost alternative to hs-CRP for cardiovascular risk evaluation.

Use of HOMA-IR to diagnose non-alcoholic fatty liver disease: a population-based and inter-laboratory study.

AIMS/HYPOTHESIS: Recent European guidelines for non-alcoholic fatty liver disease (NAFLD) call for reference values for HOMA-IR. In this study, we aimed to determine: (1) the upper limit of normal HOMA-IR in two population-based cohorts; (2) the HOMA-IR corresponding to NAFLD; (3) the effect of sex and PNPLA3 genotype at rs738409 on HOMA-IR; and (4) inter-laboratory variations in HOMA-IR. METHODS: We identified healthy individuals in two population-based cohorts (FINRISK 2007 [n = 5024] and the Programme for Prevention of Type 2 Diabetes in Finland [FIN-D2D; n = 2849]) to define the upper 95th percentile of HOMA-IR. Non-obese individuals with normal fasting glucose levels, no excessive alcohol use, no known diseases and no use of any drugs were considered healthy. The optimal HOMA-IR cut-off for NAFLD (liver fat ≥5.56%, based on the Dallas Heart Study) was determined in 368 non-diabetic individuals (35% with NAFLD), whose liver fat was measured using proton magnetic resonance spectroscopy (1H-MRS). Samples from ten individuals were simultaneously analysed for HOMA-IR in seven European laboratories. RESULTS: The upper 95th percentiles of HOMA-IR were 1.9 and 2.0 in healthy individuals in the FINRISK (n = 1167) and FIN-D2D (n = 459) cohorts. Sex or PNPLA3 genotype did not influence these values. The optimal HOMA-IR cut-off for NAFLD was 1.9 (sensitivity 87%, specificity 79%). A HOMA-IR of 2.0 corresponded to normal liver fat (<5.56% on 1H-MRS) in linear regression analysis. The 2.0 HOMA-IR measured in Helsinki corresponded to 1.3, 1.6, 1.8, 1.8, 2.0 and 2.1 in six other laboratories. The inter-laboratory CV% of HOMA-IR was 25% due to inter-assay variation in insulin (25%) rather than glucose (5%) measurements. CONCLUSIONS/INTERPRETATION: The upper limit of HOMA-IR in population-based cohorts closely corresponds to that of normal liver fat. Standardisation of insulin assays would be the first step towards definition of normal values for HOMA-IR.

High-Sensitivity Cardiac Troponin T: a Preanalytical Evaluation.

BACKGROUND: Little is known about the effects of preanalytical conditions on high-sensitivity cardiac troponin T (hs-cTnT) concentrations. METHODS: Variations of hs-cTnT concentrations were evaluated under the following preanalytical conditions: 1) serum vs. lithium-heparin (Li-Hep) plasma, with or without separator gel; 2) centrifugation time (15-minutes vs. 10-minutes) and speed (1467 to 3756 g); 3) stability in Li-Hep plasma at room temperature and +4 degrees C for 4 days and at -80 degrees C for up to 12 months, for three concentrations; 4) four freeze-thaw cycles at -20 degrees C and -80 degrees C, for three concentrations. RESULTS: No significant changes were found regarding the type of blood collection tube, the centrifugation, and storage conditions. Minor decreases were observed after four freeze-thaw cycles at -20 degrees C (< 6.5%) and -80 degrees C (< 3.4%). CONCLUSIONS: High-sensitivity cardiac troponin T may be considered as not impacted by usual preanalytical conditions, thus strengthening its reliability in laboratory practice and clinical research.

Stability of Routine Biochemical Analytes in Whole Blood and Plasma From Lithium Heparin Gel Tubes During 6-hr Storage.

BACKGROUND: The stability of biochemical analytes has already been investigated, but results strongly differ depending on parameters, methodologies, and sample storage times. We investigated the stability for many biochemical parameters after different storage times of both whole blood and plasma, in order to define acceptable pre- and postcentrifugation delays in hospital laboratories. METHODS: Twenty-four analytes were measured (Modular® Roche analyzer) in plasma obtained from blood collected into lithium heparin gel tubes, after 2-6 hr of storage at room temperature either before (n = 28: stability in whole blood) or after (n = 21: stability in plasma) centrifugation. Variations in concentrations were expressed as mean bias from baseline, using the analytical change limit (ACL%) or the reference change value (RCV%) as acceptance limit. RESULTS: In tubes stored before centrifugation, mean plasma concentrations significantly decreased after 3 hr for phosphorus (-6.1% [95% CI: -7.4 to -4.7%]; ACL 4.62%) and lactate dehydrogenase (LDH; -5.7% [95% CI: -7.4 to -4.1%]; ACL 5.17%), and slightly decreased after 6 hr for potassium (-2.9% [95% CI: -5.3 to -0.5%]; ACL 4.13%). In plasma stored after centrifugation, mean concentrations decreased after 6 hr for bicarbonates (-19.7% [95% CI: -22.9 to -16.5%]; ACL 15.4%), and moderately increased after 4 hr for LDH (+6.0% [95% CI: +4.3 to +7.6%]; ACL 5.17%). Based on RCV, all the analytes can be considered stable up to 6 hr, whether before or after centrifugation. CONCLUSION: This study proposes acceptable delays for most biochemical tests on lithium heparin gel tubes arriving at the laboratory or needing to be reanalyzed.

Modification of the ß-Adrenoceptor Stimulation Pathway in Zucker Obese and Obese Diabetic Rat Myocardium.

OBJECTIVES: Although metabolic syndrome is associated with increased sympathetic activity that chronically stimulates ß-adrenoceptors, the ß-adrenoceptor signaling pathway has been poorly studied in this situation. We studied the ß-adrenoceptor signaling pathway in Zucker lean, obese, and obese diabetic rats. DESIGN: Experimental, prospective study. SETTING: University medical research laboratory. SUBJECTS: Adult male Zucker lean (control), obese, and obese diabetic rats. INTERVENTIONS: The effects of ß-adrenoceptor stimulation were investigated in vitro in isolated left ventricular papillary muscles in control, obese, and obese diabetic rats. ß1-, ß2-, and ß3-adrenoceptors and multidrug resistance-associated protein 4 were quantified by Western Blotting. Triglyceride, cholesterol, leptin, adiponectin, and C-peptide plasma concentrations were measured. Data are mean ± SD. MEASUREMENTS AND MAIN RESULTS: Hyperlipidemia, high leptin, and C-peptide concentrations were observed in obese and obese diabetic strains, whereas hyperglycemia occurred only in the diabetic strain. The positive inotropic effect of isoproterenol was slightly reduced in obese rats (183% ± 11% of baseline; p = 0.003; n = 7) and markedly reduced in obese diabetic rats (137% ± 18% of baseline; p < 0.001; n = 10) when compared with control rats (210% ± 17% of baseline; n = 9). ß1-adrenoceptors were down-regulated in obese (-41%; p = 0.02) and diabetic (-54%; p = 0.003) when compared with control rats, whereas ß3-adrenoceptors and multidrug resistance-associated protein expression remained unchanged. Direct stimulation of adenylate cyclase with forskolin or administration of 3',5'-cyclic adenosine monophosphate suggests that subtle impairments also occurred beside the down-regulation of ß1-adrenoceptor. CONCLUSIONS: The positive inotropic effect of ß-adrenoceptor stimulation is slightly decreased in Zucker obese rats and was more markedly decreased in Zucker diabetic rats. These decreases are mainly related to ß1-adrenoceptor down-regulation.