Serum metabolic signatures for Alzheimer's Disease reveal alterations in amino acid composition: a validation study.

INTRODUCTION: Alzheimer's Disease (AD) is complex and novel approaches are urgently needed to aid in diagnosis. Blood is frequently used as a source for biomarkers; however, its complexity prevents proper detection. The analytical power of metabolomics, coupled with statistical tools, can assist in reducing this complexity. OBJECTIVES: Thus, we sought to validate a previously proposed panel of metabolic blood-based biomarkers for AD and expand our understanding of the pathological mechanisms involved in AD that are reflected in the blood. METHODS: In the validation cohort serum and plasma were collected from 25 AD patients and 25 healthy controls. Serum was analysed for metabolites using nuclear magnetic resonance (NMR) spectroscopy, while plasma was tested for markers of neuronal damage and AD hallmark proteins using single molecule array (SIMOA). RESULTS: The diagnostic performance of the metabolite biomarker panel was confirmed using sparse-partial least squares discriminant analysis (sPLS-DA) with an area under the curve (AUC) of 0.73 (95% confidence interval: 0.59-0.87). Pyruvic acid and valine were consistently reduced in the discovery and validation cohorts. Pathway analysis of significantly altered metabolites in the validation set revealed that they are involved in branched-chain amino acids (BCAAs) and energy metabolism (glycolysis and gluconeogenesis). Additionally, strong positive correlations were observed for valine and isoleucine between cerebrospinal fluid p-tau and t-tau. CONCLUSIONS: Our proposed panel of metabolites was successfully validated using a combined approach of NMR and sPLS-DA. It was discovered that cognitive-impairment-related metabolites belong to BCAAs and are involved in energy metabolism.

Sulfate and acid-base balance.

It has been acknowledged for years that compounds containing sulfur (S) are an important source of endogenous acid production. In the metabolism, S is oxidized to sulfate, and therefore the mEq sulfate excreted in the urine is counted as acid retained in the body. In this study we show that pH in fluids with constant [Na] and [HEPES] declines as sulfate ions are added, and we show that titratable acidity increases exactly with the equivalents of sulfate. Therefore, sulfate excretion in urine is also acid excretion per se. This is in accordance with the down-regulation of proximal sulfate reabsorption under acidosis and the observation that children with distal renal tubular acidosis may be sulfate depleted. These results are well explained using charge-balance modeling, which is based only on the three fundamental principles of electroneutrality, conservation of mass, and rules of dissociation as devised from physical chemistry. In contrast, the findings are in contrast to expectations from conventional narratives. These are unable to understand the decreasing pH as sulfate is added since no conventional acid is present. The results may undermine the traditional notion of endogenous acid production since in the case of sulfur balance, S oxidation and its excretion as sulfate exactly balance each other. Possible clinical correlates with these findings are discussed.

Serum NMR-Based Metabolomics Profiling Identifies Lipoprotein Subfraction Variables and Amino Acid Reshuffling in Myeloma Development and Progression.

Multiple myeloma (MM) is an incurable hematological cancer. It is preceded by monoclonal gammopathy of uncertain significance (MGUS)-an asymptomatic phase. It has been demonstrated that early detection increases the 5-year survival rate. However, blood-based biomarkers that enable early disease detection are lacking. Metabolomic and lipoprotein subfraction variable profiling is gaining traction to expand our understanding of disease states and, more specifically, for identifying diagnostic markers in patients with hematological cancers. This study aims to enhance our understanding of multiple myeloma (MM) and identify candidate metabolites, allowing for a more effective preventative treatment. Serum was collected from 25 healthy controls, 20 patients with MGUS, and 30 patients with MM. 1H-NMR (Nuclear Magnetic Resonance) spectroscopy was utilized to evaluate serum samples. The metabolite concentrations were examined using multivariate, univariate, and pathway analysis. Metabolic profiles of the MGUS patients revealed lower levels of alanine, lysine, leucine but higher levels of formic acid when compared to controls. However, metabolic profiling of MM patients, compared to controls, exhibited decreased levels of total Apolipoprotein-A1, HDL-4 Apolipoprotein-A1, HDL-4 Apolipoprotein-A2, HDL Free Cholesterol, HDL-3 Cholesterol and HDL-4 Cholesterol. Lastly, metabolic comparison between MGUS to MM patients primarily indicated alterations in lipoproteins levels: Total Cholesterol, HDL Cholesterol, HDL Free Cholesterol, Total Apolipoprotein-A1, HDL Apolipoprotein-A1, HDL-4 Apolipoprotein-A1 and HDL-4 Phospholipids. This study provides novel insights into the serum metabolic and lipoprotein subfraction changes in patients as they progress from a healthy state to MGUS to MM, which may allow for earlier clinical detection and treatment.

Dysfibrinogenemia-Potential Impact of Genotype on Thrombosis or Bleeding.

The congenital dysfibrinogenemias, most often associated with bleeding disorders, encompass mutations in the amino-terminal end of fibrinogen α-chain consisting of Gly17-Pro18-Arg19-Val20, known as knob A, which is a critical site for fibrin polymerization. Here we review the studies reporting dysfibrinogenemia due to mutations affecting fibrinogen knob A and identified 29 papers. The number of reports on dysfibrinogenemias related to residues Gly17, Pro18, Arg19, and Val20 is 5, 4, 18, and 2, respectively. Dysfibrinogenemias related to residues Gly17, Pro18, and Val20 are exclusively associated with bleeding tendency. However, the clinical picture associated with dysfibrinogenemia related to residue Arg19 varies, with most patients suffering from bleeding tendencies, but also transitory ischemic attacks and retinal thrombosis may occur. The reason for this variation is unclear. To elaborate the genotype-phenotype associations further, we studied a Danish family with knob A-related dysfibrinogenemia caused by the Aα Arg19Gly (p.Arg19Gly) mutation using whole-exome sequencing and fibrin structure analysis. Our family is the first reported carrying the p.Arg19Gly mutation combined with one or more single nucleotide polymorphisms (SNP)s in FGA, FGB, and/or FGG and increased fibrin fiber thickness and fibrin mass-to-length ratio suffering from pulmonary emboli, suggesting that compound genotypes may contribute to the thrombogenic phenotype of these patients. Our review, accordingly, focuses on significance of SNPs, compound genotypes, and fibrin structure measures affecting the genotype-phenotype associations in fibrinogen knob A mutations.

Circulating microvesicles and exosomes in small cell lung cancer by quantitative proteomics.

BACKGROUND: Early detection of small cell lung cancer (SCLC) crucially demands highly reliable markers. Growing evidence suggests that extracellular vesicles carry tumor cell-specific cargo suitable as protein markers in cancer. Quantitative proteomic profiling of circulating microvesicles and exosomes can be a high-throughput platform for discovery of novel molecular insights and putative markers. Hence, this study aimed to investigate proteome dynamics of plasma-derived microvesicles and exosomes in newly diagnosed SCLC patients to improve early detection. METHODS: Plasma-derived microvesicles and exosomes from 24 healthy controls and 24 SCLC patients were isolated from plasma by either high-speed- or ultracentrifugation. Proteins derived from these extracellular vesicles were quantified using label-free mass spectrometry and statistical analysis was carried out aiming at identifying significantly altered protein expressions between SCLC patients and healthy controls. Furthermore, significantly expressed proteins were subjected to functional enrichment analysis to identify biological pathways implicated in SCLC pathogenesis. RESULTS: Based on fold change (FC) ≥ 2 or ≤ 0.5 and AUC ≥ 0.70 (p < 0.05), we identified 10 common and 16 and 17 unique proteins for microvesicles and exosomes, respectively. Among these proteins, we found dysregulation of coagulation factor XIII A (Log2 FC = - 1.1, p = 0.0003, AUC = 0.82, 95% CI: 0.69-0.96) and complement factor H-related protein 4 (Log2 FC = 1.2, p = 0.0005, AUC = 0.82, 95% CI; 0.67-0.97) in SCLC patients compared to healthy individuals. Our data may indicate a novel tumor-suppressing role of blood coagulation and involvement of complement activation in SCLC pathogenesis. CONCLUSIONS: In comparing SCLC patients and healthy individuals, several differentially expressed proteins were identified. This is the first study showing that circulating extracellular vesicles may encompass specific proteins with potential diagnostic attributes for SCLC, thereby opening new opportunities as novel non-invasive markers.

An Asymmetric Runaway Domain Swap Antithrombin Dimer as a Key Intermediate for Polymerization Revealed by Hydrogen/Deuterium-Exchange Mass Spectrometry.

Antithrombin deficiency is associated with increased risk of venous thrombosis. In certain families, this condition is caused by pathogenic polymerization of mutated antithrombin in the blood. To facilitate future development of pharmaceuticals against antithrombin polymerization, an improved understanding of the polymerogenic intermediates is crucial. However, X-ray crystallography of these intermediates is severely hampered by the difficulty in obtaining well-diffracting crystals of transient and heterogeneous noncovalent protein assemblies. Furthermore, their large size prohibits structural analysis by NMR spectroscopy. Here, we show how hydrogen/deuterium-exchange mass spectrometry (HDX-MS) provides detailed insight into the structural dynamics of each subunit in a polymerization-competent antithrombin dimer. Upon deuteration, this dimer surprisingly yields bimodal isotope distributions for the majority of peptides, demonstrating an asymmetric configuration of the two subunits. The data reveal that one subunit is very dynamic, potentially intrinsically disordered, whereas the other is considerably less dynamic. The local subunit-specific deuterium uptake of this polymerization-competent dimer strongly supports a ß4A-ß5A ß-hairpin runaway domain swap mechanism for antithrombin polymerization. HDX-MS thus holds exceptional promise as an enabling analytical technique in the efforts toward future pharmacological intervention with protein polymerization and associated diseases.

Preanalytical, analytical, and biological variation of blood plasma submicron particle levels measured with nanoparticle tracking analysis and tunable resistive pulse sensing.

BACKGROUND: Nanoparticle tracking analysis (NTA) and tunable resistive pulse sensing (TRPS) enable measurement of extracellular vesicles (EVs) in blood plasma but also measure other particles present in plasma. Complete isolation of EVs from similarly sized particles with full EV recovery is currently not possible due to limitations in existing isolation techniques. AIM: This study aimed to evaluate preanalytical, analytical, and biological variation of particle measurements with NTA and TRPS on blood plasma. METHODS: Blood from 20 healthy subjects was sampled in the fasting and postprandial state. Platelet free plasma (PFP) was analyzed immediately and after a freeze-thaw cycle. Additionally, the effect of prandial state and a freeze-thaw cycle on EV-enriched particle fractions obtained via size-exclusion chromatography (SEC) was examined. RESULTS: We observed analytical linearity in the range of 1.0-10.0 × 10(8) particles/mL for NTA and 1.0 × 10(8)-1.8 × 10(9) particles/mL for TRPS. The analytical variation was generally below 10%. A considerable intra- and inter-individual variation was demonstrated with estimated reference intervals of 1.4 × 10(11)-1.2 × 10(12) particles/mL for NTA and 1.8 × 10(8)-1.6 × 10(9) particles/mL for TRPS. Food intake and to a lesser extent a freeze-thaw cycle affected particle populations in PFP and, similarly, in EV-enriched fractions. CONCLUSION: In this study NTA and TRPS enabled acceptably precise concentration and size measurement of submicron particles in PFP. An appreciable intra- and inter-individual biological variation was observed. In studies on particle populations in PFP or EV-enriched fractions, we recommend analysis of fresh, fasting samples.

Amelioration of the severity of heparin-binding antithrombin mutations by posttranslational mosaicism.

The balance between actions of procoagulant and anticoagulant factors protects organisms from bleeding and thrombosis. Thus, antithrombin deficiency increases the risk of thrombosis, and complete quantitative deficiency results in intrauterine lethality. However, patients homozygous for L99F or R47C antithrombin mutations are viable. These mutations do not modify the folding or secretion of the protein, but abolish the glycosaminoglycan-induced activation of antithrombin by affecting the heparin-binding domain. We speculated that the natural ß-glycoform of antithrombin might compensate for the effect of heparin-binding mutations. We purified α- and ß-antithrombin glycoforms from plasma of 2 homozygous L99F patients. Heparin affinity chromatography and intrinsic fluorescence kinetic analyses demonstrated that the reduced heparin affinity of the α-L99F glycoform (K(D), 107.9 ± 3nM) was restored in the ß-L99F glycoform (K(D), 53.9 ± 5nM) to values close to the activity of α-wild type (K(D), 43.9 ± 0.4nM). Accordingly, the ß-L99F glycoform was fully activated by heparin. Similar results were observed for recombinant R47C and P41L, other heparin-binding antithrombin mutants. In conclusion, we identified a new type of mosaicism associated with mutations causing heparin-binding defects in antithrombin. The presence of a fully functional ß-glycoform together with the activity retained by these variants helps to explain the viability of homozygous and the milder thrombotic risk of heterozygous patients with these specific antithrombin mutations.

Comparison of assays measuring extracellular vesicle-tissue factor in plasma samples: Communication from the ISTH SSC Subcommittee on Vascular Biology.

BACKGROUND: Scientific and clinical interest in extracellular vesicles (EVs) is growing. EVs that expose tissue factor (TF) bind factor VII/VIIa and can trigger coagulation. Highly procoagulant TF-exposing EVs are detectable in the circulation in various diseases, such as sepsis, COVID-19 or cancer. Many in-house and commercially available assays have been developed to measure EV-TF activity and antigen but only a few studies have compared some of these assays. The ISTH SSC Subcommittee on Vascular Biology initiated a multicenter study to compare the sensitivity, specificity and reproducibility of these assays. MATERIALS AND METHODS: Platelet-depleted plasma samples were prepared from blood of healthy donors. The plasma samples were spiked either with EVs from human milk, or EVs from TF-positive and TF-negative cell lines. Plasma was also prepared from whole human blood with or without LPS stimulation. Twenty-one laboratories measured EV-TF activity and antigen in the prepared samples using their own assays representing 18 functional and 9 antigenic assays. RESULTS: There was a large variability in the absolute values for the different EV-TF activity and antigen assays. Activity assays had higher specificity and sensitivity compared to antigen assays. In addition, there was a large intra-assay and inter-assay variability. Functional assays that used a blocking anti-TF antibody or immunocapture were the most specific and sensitive. Activity assays that used immunocapture had a lower coefficient of variation compared to assays that isolated EVs by high-speed centrifugation. CONCLUSION: Based on this multicenter study, we recommend measuring EV-TF using a functional assay in the presence of an anti-TF antibody.

A sensitive tissue factor activity assay determined by an optimized thrombin generation method.

BACKGROUND: Tissue factor (TF) is the principal activator of the coagulation system, but an increased concentration in the blood in cancer and inflammatory diseases has been suggested to play a role increasing the risk of venous thromboembolism. However, measurement of the TF concentration is difficult, and quantitation of activity is the most valid estimation. The objective of this study was to establish a sensitive method to measure TF activity based on thrombin generation. METHODS: The assay is based on thrombin generation (TG) measured on the Calibrated Automated Thrombogram (CAT). Various low concentrations of TF were prepared from reagents containing 1 pM TF and 4 µM phospholipid (PPL), and no TF and 4 µM PPL, and a calibration curve was produced from Lagtime vs TF concentration. TF in blood samples was measured after isolation and resuspension of extracellular vesicles (EVs) in a standard plasma from which EVs had been removed. The same standard plasma was used for the calibrators. RESULTS: Contact activation of the coagulation system was avoided using CTI plasma samples in Monovette tubes. EVs contain procoagulant phospholipids but addition of PPL only reduced lagtime slightly at very low concentrations of TF resulting in overestimation to a lesser extent at 10 fM but no interference at 30 fM or higher. Addition of EVs to the TG analysis induced a small unspecific TF-independent activity (i.e., an activity not inhibited by antibodies against TF) which also may result in a smaller error in estimation of TF activity at very low levels but the effect was negligible at higher concentrations. It was possible to measure TF activity in healthy controls which was found to be 1-6 fM (EVs were concentrated, i.e. solubilized in a lower volume than the original volume plasma). Coefficient of variation (CV) was below 20% at the low level, and below 10% at a level around 100 fM TF. However, the step with isolation of EVs have a higher inherent CV. CONCLUSION: A sensitive and rather precise one-stage TG-based method to measure TF activity has been established.

Preliminary investigation of elevated collagen and blood-clotting markers as potential noninvasive biomarkers for small cell lung cancer.

BACKGROUND: Small cell lung cancer (SCLC) is highly aggressive with limited therapeutic options and a poor prognosis. Moreover, noninvasive biomarker tools for detecting disease and monitoring treatment response are lacking. To address this, we evaluated serum biomarkers of extracellular matrix proteins not previously explored in SCLC. METHODS: We measured biomarkers in the serum of 16 patients with SCLC before and after chemotherapy as well as in the serum of 11 healthy individuals. RESULTS: Our findings demonstrated that SCLC serum had higher levels of collagen type I degradation, collagen type III formation, and collagen type XI formation than healthy controls. In addition, we observed higher levels of type XIX and XXII collagens, fibrinogen, and von Willebrand factor A formation in SCLC serum. The formation of type I collagen did not exhibit any discernible variation. However, we observed a decrease in the degradation of type I collagen following chemotherapy. CONCLUSION: Overall, our findings revealed elevated levels of collagen and blood-clotting markers in the serum of SCLC patients, indicating the potential of ECM proteins as noninvasive biomarkers for SCLC.

Combined oral contraceptives may activate the contact system in healthy women.

Background: The contact system (CAS) is part of the coagulation system, consisting of a group of plasma proteins stimulating inflammation, coagulation, and fibrinolysis when activated. CAS can be triggered by several activating surfaces, and CAS may play a potential role in thrombus formation. Combined oral contraceptives (COCs) are known to increase the risk of venous thromboembolism, and COCs induce various prothrombotic changes in the coagulation system, whereas the effect of COC on CAS has not been thoroughly investigated. Objectives: To investigate CAS in COC users compared with nonusers. Methods: Blood samples from 62 study subjects, 30 COC users, and 32 nonusers, were analyzed. Coagulation factor XII (FXII), prekallikrein (PK), H-Kininogen (HK), cleaved HK (cHK), C1-esterase inhibitor (C1-inh), and the endogenous kallikrein potential (EKP) were measured. Results: COC users had significantly higher FXII (median, 38.4 vs 28.9 mg/L) and lower C1-inh levels (0.20 vs 0.23 g/L) than nonusers. The levels of PK and HK were not significantly different. Measurement of EKP indicated an increased capacity of CAS in COC users (1860 vs 1500 nmol/L × min), and increased plasma levels of cHK (2.02 vs 1.07 µg/L) indicated an increased activity in vivo. Conclusion: This study demonstrates an increased CAS capacity in women using COC compared with nonusers and also an increased activity in vivo. The results indicate that increased contact activation may contribute to the increased thrombotic risk caused by COC.

Thrombin generation measured on ST Genesia, a new platform in the coagulation routine lab: Assessment of analytical and between-subject variation.

BACKGROUND: The thrombin generation (TG) assay, which measures global coagulation, has mainly been used as a research tool to investigate thrombotic and bleeding disorders. Recently, Diagnostica Stago launched the ST Genesia, a fully automated system to perform "routine version" of this assay. The objectives of this study were to evaluate the imprecision compared with the previous method, Thrombinoscope CAT, and to establish reference intervals. METHODS: Thrombin generation was measured in platelet-poor citrated plasma from 20 normal controls (fresh and after freezing at -80°C up to 12-13 weeks) on CAT and ST Genesia in duplicate to estimate the total variation, and within and between variations. The reference intervals were estimated nonparametrically in 30 men, 30 women taking combined oral contraceptives (COCs), and 30 women not taking COCs. These were sampled in both Vacutainer and Monovette tubes (i.e., tubes with a high and minimal contact activation, respectively). RESULTS: Freezing had minimal effects. Imprecision was comparable between the ST Genesia and CAT, with a strong correlation between the two methods. TG was higher when sampled in Vacutainer than in Monovette. We observed a distinct difference between women taking and not taking COCs, whereas men and women not taking COC were quite similar. CONCLUSIONS: Thrombin generation on ST Genesia showed an analytical variation similar to that of CAT. The results depended on the type of sample tubes; thus, reference intervals must be established for the collection tubes used in each laboratory. Furthermore, a considerable difference was observed between women using and not using COCs.

Validation of Postsurgical Venous Thromboembolism Diagnoses of Patients Undergoing Lower Limb Orthopedic Surgery in the Danish National Patient Registry.

BACKGROUND: Healthcare databases can be a valuable source of epidemiological research regarding postoperative venous thromboembolism (VTE), ie, deep vein thrombosis (DVT) and pulmonary embolism (PE), following orthopedic procedures, but only if the diagnoses are valid. We examined the validity of VTE diagnosis codes in the Danish National Patient Registry (DNPR) by calculating their positive predictive value (PPV) and negative predictive value (NPV) versus actual medical records. METHODS: We identified patients who had undergone lower limb surgery during the period 2009-2019 at a hospital in the North Denmark Region. Of these, 420 patients had at least one VTE diagnosis registered in the DNPR within 180 days after lower limb surgery. Each patient with a VTE diagnosis was matched with two patients on age and sex, as well as type, location and period of surgery. The entire medical record and diagnostic imaging were reviewed to confirm VTE diagnosis. RESULTS: The overall PPVs was 85.2% (95% CI: 81.5-88.5%) for first time VTE diagnosis following lower limb surgery, 82.6% (95% CI: 77.5-82.8%) for DVT, and 90.3% (95% CI: 84.3-94.6%) for PE. We found improvement in PPV during the study period when stratifying for three periods of the whole period. There were no significant differences when stratifying for sex, age, or surgery site. All negative predictive values were higher than 99%. A total of 113 additional VTE diagnoses were registered among 88 VTE patients during follow-up. Only four of the suspected recurrent VTEs were confirmed to be true recurrent VTEs. CONCLUSION: The VTE diagnosis codes in the DNPR after lower limb orthopedic surgery were highly valid against the actual medical records, and we observed better PPV over recent years.

The Physiological and Cardiologic Effects of Long Video Gaming Sessions in Adult Males.

The effect of long gaming sessions on energy intake, caffeine intake, blood pressure, heart rate, heart rate variability, and biochemical cardiac injury markers is unknown. The objective of this exploratory study was to investigate the changes in healthy male adults during two consecutive 18-hour sedentary video gaming sessions. Nine participants were enrolled in the study. Energy intake was noted in food diaries. Heart rate variability was monitored continuously; blood pressure and cardiac injury markers were measured every three to six hours. During the 42-hour study, the participants had an energy and caffeine intake of 8004.9 kcal and 1354.4 mg, respectively. The participants had a significant decrease in energy intake in the second session (p=0.01). A strong, negative correlation was found between body mass index and total energy intake (R=-0.84, p=0.005) and waist circumference and total energy intake (R=-0.70, p=0.036) in the first session. No nightly dip in blood pressure or heart rate was observed. Based on this study, long-term adverse effects of gaming cannot be ruled out. The non-dip of HR and BP suggests that long gaming sessions could be detrimental to cardiovascular health long term.

Reference intervals and age and gender dependency for arterial blood gases and electrolytes in adults.

BACKGROUND: Although results from blood gas analyzers are frequently used in clinical work surprisingly few and small studies have examined reference intervals for arterial blood gases and acid-base status. We have established reference values based on a large group of healthy people with a wide age distribution. METHODS: A group of medical students (n=182) aged 20-32 years old and a group of health professionals aged 21-76 years were used in this study. Arterial samples were analyzed on the blood gas analyzer ABL from Radiometer(TM). Age and gender dependency was examined for all analytes and reference intervals were calculated non-parametrically. RESULTS: Females had significantly higher pH and lower PaCO(2) (partial pressure of carbon dioxide in an arterial sample), base excess (BE, standard, extra cellular fluid), plasma standard and actual HCO(3), when compared to males (p<0.01). However, the differences were minor and common reference intervals were therefore also determined, generally at the same level as previously published. The lactate values were similar among the genders but with a high upper limit of 2.5 mmol/L. The non-smoker group of females and males had similar PaO(2) values (partial pressure of oxygen in an arterial sample). However, an age dependent effect was found and PaO(2) decreased by 0.29 kPa per decade (confidence interval of slope -0.11 to -0.47 kPa). Electrolytes and anion gap results depicted smaller differences from previous published reference intervals for sodium (136-141 mmol/L) and anion gap (10-16 mmol/L, with potassium included or 6-12 mmol/L without potassium). CONCLUSIONS: Reference intervals for analytes on modern blood gas analyzers were established on a large group of healthy people. Gender and age dependency is generally without clinical importance, except for a lower PaCO(2) in women and a decreasing PaO(2) with higher age.

Platelet response to aspirin and clopidogrel in patients with peripheral atherosclerosis.

Aspirin and clopidogrel are important drugs in the secondary prevention of ischemic events. A considerable individual variation in platelet response to these drugs has, however, been reported, and high residual platelet reactivity despite treatment may be an independent risk factor for ischemic events. Most studies have been undertaken in patients with coronary heart disease, but patients with peripheral artery disease (PAD) may exhibit greater residual platelet reactivity, possibly because of platelet activation by a larger area of diseased endothelium. It is yet unsettled which method that best measures platelet reactivity and an eventual lack of response to aspirin. Several instruments are promoted to measure platelet response and low-response to platelet inhibitors, but it is questionable if they measure this in comparable ways. We studied the comparability of three tests of platelet reactivity for the assessment of low-response to aspirin and clopidogrel in patients with PAD. In 263 patients, platelet function was assessed twice, 3 months apart, by the Platelet Function Analyzer-100 (PFA), light transmission aggregometry (LTA), and whole blood impedance aggregometry (IA). In a subgroup of 43 patients, we studied the effect of a single dose of 600 mg clopidogrel on platelet function. Low-response to aspirin assessed by analyses targeting cyclooxygenase-1 activity (LTA, IA) was rare (≤ 8.1%). With the PFA, we found 17% with low response at both visits, and 60% who were consistently responsive, whereas 23% were categorized differently at the two visits. Low response to clopidogrel, occurred in 0-23%, depending on the method and the criteria used. A low-response to aspirin, defined by lack of COX-1 inhibition, is a rare phenomenon whereas high residual platelet reactivity as determined by PFA may be a rather frequent finding but is not consistent over time in all patients. A low-response to clopidogrel depends very much on the method and definition used.

A third troponin T blood sample is not cost-effective in patients with suspected non-ST segment elevation acute coronary syndrome.

BACKGROUND: The diagnosis of acute myocardial infarction requires troponin assessment in at least two blood samples 6-9 hours apart, with an optional third sample 12-24 hours after admission if suspicion is high. Yet, in many institutions, this third sample is routinely drawn. This study aimed to evaluate cost-effectiveness of this third sample of troponin. METHODS: A total of 534 patients with possible Non ST-Elevation Acute Coronary Syndrome (NSTE-ACS) were included. Blood samples for cardiac TroponinT (cTnT) were obtained on arrival, after 6-9 hours, and 12-24 hours after admission. The costs of cTnT analysis, and hospital stay were calculated. RESULTS: Of the 534 patients, 124 had at least one elevated cTnT value. Among these, four patients (3.2%) had cTnT values increased only in the third sample. Based on their risk profile and/or ECG changes, these four patients were eligible for referral to coronary angiography even before the result of the third sample became available. The number of patients whose length of stay was extended solely because of the third sample was 275. Incremental cost of the third blood sample: [534 patients × Euro (EUR) 12 per cTnT analysis] + [275 patients × 0.5 day × EUR 1,550] = EUR 219,533. Approximately 1400 patients with suspected NSTE-ACS are admitted to our department each year. Thus, the total cost per year is: (1,400/534) × EUR 219,533 = EUR 575,555. CONCLUSION: A third troponin sample adds no vital information regarding patients' treatment or investigations plan. On the contrary, it may lead to an unnecessary extension of the admission period and increased costs.

Elevated thyroid-stimulating hormone level in a euthyroid neonate caused by macro thyrotropin-IgG complex.

UNLABELLED: Elevated thyroid-stimulating hormone (TSH) was discovered by routine neonatal screening in a newborn with no clinical symptoms. Thyroid function tests were repeated and confirmed a high TSH value but normal total thyroxine (T4) and triiodothyronine (T3). However, the mother also had elevated serum TSH with normal levels of T4 and T3. The results suggested a transmitted maternal interfering factor, and no treatment was started while further investigation was performed. Gel filtration chromatography of serum from both the infant and the mother showed a peak TSH with molecular mass consistent with a TSH-IgG complex (macro-TSH). TSH in the infant decreased to a normal level within 8 months in accordance with a normal rate of elimination of maternal IgG, whereas the TSH level of the mother remained high. CONCLUSION: This case suggests that interfering macro-TSH should be considered in a euthyroid neonate with elevated serum TSH and normal T4 and T3 levels to avoid unnecessary treatment.