Sirak Petros

Effect of standardization and normalization on imprecision of calibrated automated thrombography: an international multicentre study

Calibrated automated thrombography (CAT) enables continuous measurement of thrombin generation (TG). Initial clinical studies using the CAT method showed large variability of normal values, indicating the necessity for a standardized CAT protocol. This international study assessed the intra‐ and inter‐assay imprecision of CAT as well as the inter‐centre variability of results in five European centres using locally available reagents and conditions (study 1) and a standardized protocol in which results were normalized (study 2). Samples with and without corn trypsin inhibitor from six healthy volunteers, two haemophilia patients and one protein C deficient patient were assayed. Study 1 confirmed that the use of different sources and concentrations of tissue factor (TF) and different phospholipid (PL) mixtures produced large variability in results. The second study demonstrated that, using the same source and concentration of TF, PL and the same test procedure, this variability could be significantly reduced. Normalization of results improved the inter‐centre variability. The benefit of contact factor inhibition prior to TG measurement was confirmed. These results demonstrated that standardization of CAT reduces the variability of results to acceptable limits. Standardization and normalization should be considered in future clinical studies which apply TG testing to clinical decision making.

Thrombin generation in severe haemophilia A and B: the endogenous thrombin potential in platelet-rich plasma

Thrombin generation was investigated in platelet-rich plasma (PRP) from 11 healthy controls, 17 patients with severe haemophilia A and 7 patients with severe haemophilia B. Mean endogenous thrombin potential (ETP) in arbitrary fluorescence units (FU) was 226.9 +/- 44.6, 186.4 +/- 22.5, 154.2 +/- 41.3 in controls, haemophilia A and B, respectively, all at a platelet count of 200 x 10(9)/l (p = 0.004 for controls vs. haemophilia A, p = 0.003 for controls vs. haemophilia B, no significant difference between haemophilia A and B). The contribution of FVIII to thrombin generation in haemophilia A was 1.31 +/- 0.16 FU/% of FVIII:C activity, while for FIX in haemophilia B this was 0.80 +/- 0.21 FU/% of FIX activity. There was an almost linear relationship between increasing platelet count and thrombin generation up to a mean platelet count of 100 x 10(9)/l. Further increase in platelet count has only a marginal influence on thrombin generation. Platelets increase ETP in haemophilia A by 0.184 +/- 0.022 FU/10(9) platelets/l and in haemophilia B by 0.319 +/- 0.085 FU/10(9) platelets/l, and this was significantly different between the two groups (p = 0.0002). This influence of plate-lets diminishes with increasing concentration of either FVIII or FIX. In conclusion, there is a difference in thrombin generation between haemophilia A and B, and this may be attributed to the role of platelets in the assembly of the tenase complex on their surface.

Hypocaloric vs Normocaloric Nutrition in Critically Ill Patients A Prospective Randomized Pilot Trial

BACKGROUND Optimal nutrition of critically ill patients is still a matter of debate. This pilot trial aimed to compare the impact of normocaloric vs hypocaloric feeding in critically ill patients in the first 7 days in the intensive care unit (ICU). The primary end point was the rate of nosocomial infections during the ICU stay. METHODS Critically ill patients requiring artificial nutrition for at least 72 hours were included within 24 hours of ICU admission and randomized into a normocaloric group (receiving 100% of their daily energy expenditure) and a hypocaloric group (receiving 50% of their daily energy expenditure). RESULTS One hundred patients were included (54 in the normocaloric group and 46 in the hypocaloric group). There were 66 male and 34 female patients with a mean age of 65.8 ± 11.6 years. The mean daily caloric supply was 19.7 ± 5.7 kcal/kg for the normocaloric group and 11.3 ± 3.1 kcal/kg for the hypocaloric group (P = .0001). Insulin demand was significantly higher and gastrointestinal intolerance more frequent in the normocaloric group than in the hypocaloric group. Nosocomial infections were detected more frequently in the hypocaloric group than in the normocaloric group (26.1% vs 11.1%, respectively). The ICU mortality rate was 22.2% in the normocaloric group and 21.7% in the hypocaloric group (not significant). The hospital mortality rate was 31.5% in the normocaloric group and 37.0% in the hypocaloric group (P = .67). CONCLUSION Hypocaloric feeding in the first 7 days in critically ill patients was associated with more nosocomial infections but less insulin demand and less gastrointestinal intolerance compared with normocaloric feeding. TRIAL REGISTRATION NUMBER DRKS00000104 (German Clinical Trials Register).

Adrenocortical hormones in survivors and nonsurvivors of severe sepsis: diverse time course of dehydroepiandrosterone, dehydroepiandrosterone-sulfate, and cortisol.

ObjectiveActivation and suppression of immune responses are crucial events during sepsis. Based on substantial new data, a complex picture of differential immune-enhancing and immunosuppressive actions of adrenocortical steroids is emerging. The adrenal androgen dehydroepiandrosterone and its precursor, dehydroepiandrosterone-sulfate, show a considerable decrease with increasing age and serve as functional antagonists to endogenous glucocorticoids. Therefore, we examined time-dependent changes in dehydroepiandrosterone, dehydroepiandrosterone-sulfate, cortisol, adrenocorticotropin, and inflammatory variables in surviving and nonsurviving patients with severe sepsis. DesignProspective observational study in consecutive patients. SettingMedical and interdisciplinary intensive care units in two university hospitals and one city hospital. PatientsThirty nonsurgical patients (25 men and 5 women) with severe sepsis (American College of Chest Physicians/Society of Critical Care Medicine criteria); 15 survivors (mean age, 54 ± 14 yrs; Acute Physiology and Chronic Health Evaluation III score, 59 ± 35) and 15 nonsurvivors (mean age, 63 ± 15 yrs; Acute Physiology and Chronic Health Evaluation III score, 67 ± 24) were included. Hormones were compared individually and between survivors/nonsurvivors by sequential blood drawings from early sepsis till time of recovery/death. InterventionsNone. Measurements and Main ResultsDuring early sepsis, cortisol (nmol/L) was not significantly higher in survivors than nonsurvivors (750 ± 121 vs. 454 ± 92, p < .08) and decreased in survivors (p < .01) during late sepsis. During early sepsis, dehydroepiandrosterone-sulfate (percentage of age-matched normal levels) was higher in survivors than nonsurvivors (85 ± 19 vs. 22 ± 7, p < .01). Dehydroepiandrosterone-sulfate decreased in survivors (p = .0001) but remained low in nonsurvivors during late sepsis. Dehydroepiandrosterone (percentage of age-matched normal levels) was not significantly elevated in survivors compared to nonsurvivors during early sepsis (282 ± 42 vs. 214 ± 63, p < .08). Dehydroepiandrosterone decreased in survivors (p < .01) but not in nonsurvivors during late sepsis. Linear regression for dehydroepiandrosterone levels showed a reconstitution of age dependence only in survivors during recovery. Adrenocorticotropin levels did not change. The dehydroepiandrosterone-sulfate/cortisol ratio decreased significantly in both survivors and nonsurvivors, whereas dehydroepiandrosterone/cortisol ratio only decreased in survivors during course of sepsis. ConclusionsDuring sepsis, adrenal androgens and glucocorticoids show a diverse time-dependent course in survivors and nonsurvivors.

The endogenous thrombin potential and high levels of coagulation factor VIII, factor IX and factor XI

High plasma concentrations of factor VIII, factor IX and factor XI have been reported as thrombosis risk factors. Using the thrombin generation test in platelet-poor plasma, it was aimed to describe the mechanism for this increased thrombosis risk. Endogenous thrombin potential was measured in platelet-poor plasma in 180 patients with a history of thromboembolism, and results were compared with those of 180 age-matched and sex-matched controls. Subjects with major hereditary and acquired thrombophilia were excluded. Plasma concentrations of the clotting factor VIII, factor IX and factor XI were significantly elevated in patients compared with controls. The mean endogenous thrombin potential was significantly higher in patients than in controls: 191.3 ± 3.1 (95% confidence interval, 185.3–197.4) arbitrary units versus 180.8 ± 2.6 (95% confidence interval, 175.7–185.9) arbitrary units (P = 0.009). The endogenous thrombin potential was significantly higher in patients with elevated factor IX and factor XI, but elevated factor VIII was not associated with a significant increase in endogenous thrombin potential. In conclusion, the increased thrombosis risk associated with high plasma concentrations of factor IX and factor XI may be explained by the increase in endogenous thrombin potential. However, this did not help explain the association between elevated factor VIII and thrombosis risk.

Effect of biobanking conditions on short-term stability of biomarkers in human serum and plasma.

Abstract Background: Liquid biobanking is an important tool for laboratory diagnostics in routine settings and clinical studies. However, the current knowledge about adequate storage conditions for different classes of biomarkers is incomplete and, in part, contradictory. Here, we performed a comprehensive study on the effects of different storage conditions on the stability of various biomarkers in human serum and plasma. Methods: Serum and citrated plasma were aliquoted and stored at 4 °C, –20 °C, –80 °C, and <–130 °C for 0, 7, 30, and 90 days, respectively (5–10 pools/condition). Additionally, frozen aliquots were temporarily exposed to higher temperatures during storage to simulate removing individual samples. Stability was tested for 32 biomarkers from 10 different parameter classes (electrolytes, enzymes, metabolites, inert proteins, complement factors, ketone bodies, hormones, cytokines, coagulation factors, and sterols). Results: Biobanking at –80 °C and <–130 °C for up to 90 days did not lead to substantial changes (defined as >3 interassay coefficients of variation and p<0.01) of any biomarker concentration. In contrast, storage at 4 °C and –20 °C induced substantial changes in single biomarker concentrations in most classes. Such substantial changes were increases (<20%) in electrolytes, metabolites, and proteins, and decreases (<96%) in enzymes, ketone bodies, cytokines, and coagulation factors. Biomarker stability was minimally affected by occasional short-term thermal exposure. Conclusions: Based on these results, we provide recommendations for storage conditions of up to 90 days for several biomarkers. Generally, storage at ≤–80 °C for at least 90 days including occasional short-term thermal exposure is an excellent storage condition for most biomarkers.

Sepsis-associated changes of the arachidonic acid metabolism and their diagnostic potential in septic patients*

Objectives: Sepsis-associated changes of the arachidonic acid metabolism and the utility of arachidonic acid metabolites for the diagnosis of sepsis have been poorly investigated so far. Therefore, the primary objective of our study was to screen for differentially regulated arachidonic acid metabolites in septic patients using a lipopolysaccharide whole-blood model and to investigate their diagnostic potential. Design: Prospective, observational, single-center, clinical study. Setting: Intensive care unit at University Hospital Leipzig. Patients: Thirty-five patients (first cohort 25 patients, second cohort 10 patients) meeting the criteria for severe sepsis or septic shock were enrolled. Eighteen healthy volunteers (first cohort 15 subjects, second cohort 3 subjects) were enrolled as controls. Interventions: None. Measurements and Main Results: Arachidonic acid and its metabolites were investigated in supernatants of nonactivated (baseline) and lipopolysaccharide-activated heparinized whole blood of healthy subjects (n = 15) and septic patients (n = 25) by solid phase extraction and subsequent liquid chromatography-tandem mass spectrometry. Arachidonic acid, arachidonic acid analogues, and the cyclooxygenase-associated metabolites prostaglandin E2, 11-hydroxyeicosatetraenoic acid, and thromboxane B2 were identified as differentiating metabolites between septic patients and healthy subjects. Some of these compounds, including arachidonic acid, its analogues, and the cyclooxygenase metabolites prostaglandin E2 and thromboxane B2 differed at baseline. The inducibility of arachidonic acid and the cyclooxygenase metabolites 11-hydroxyeicosatetraenoic and prostaglandin E2 were reduced by 80% to 90% in septic patients. The degree of the inducibility was associated with severity of sepsis and clinical outcome. A reduced inducibility of COX-2 but preserved inducibility of mPGES-1 on gene expression level were confirmed in an independent cohort of septic patients (n = 10) by quantitative reverse-transcription polymerase chain reaction compared to healthy controls (n = 3). Conclusions: Arachidonic acid metabolism is markedly affected in patients with sepsis. Our data suggest that the analysis of arachidonic acid metabolites in an in vitro whole blood activation model may be a promising approach for risk estimation in septic patients that has to be further evaluated in subsequent large-scale clinical studies.

The effect of different anticoagulants on thrombin generation.

Decrease in thrombin generation is the key effect in anticoagulation. The aim of the present study was to investigate the effect of anticoagulants on thrombin generation and the relation to platelet count. Plasma samples from 10 healthy volunteers (mean age 43.0 ± 9 years) were incubated at preset platelet counts with different doses of the anticoagulants lepirudin, fondaparinux and low molecular weight heparins. Thrombin generation was measured in a tissue factor-mediated assay using a fluorometer and a slow-reacting fluorogenic substrate. The endogenous thrombin potential, the lag phase, the maximum reaction velocity (Vmax) and the concentration of a given anticoagulant required for 50% inhibition of thrombin generation (IC50) are presented. All three anticoagulants decreased endogenous thrombin potential and prolonged the lag phase in a dose-dependent manner. Fondaparinux and low molecular weight heparins, but not hirudin, decreased Vmax in a concentration-dependent manner. With increasing platelet count, the IC50 increased but the extent of this increase was not uniform for the three anticoagulants and the three variables investigated. The influence of anticoagulants on thrombin generation is variable, depending on their basic mechanism of action. In defining and comparing their effects, the endogenous thrombin potential, the lag phase and the maximum reaction velocity should be considered together. Platelets have a considerable influence on the magnitude of thrombin generation.

Serum Procalcitonin and Proinflammatory Cytokines in a Patient with Acute Severe Leptospirosis

Leptospirosis is a zoonosis, with clinical manifestations ranging from the imperceptible to severe, potentially fatal renal and liver failure accompanied by haemorrhage and jaundice. In this case report of a patient with severe leptospirosis, serum levels of procalcitonin decreased ahead of any obvious clinical improvement, and thus may be useful as a prognostic marker. Levels of soluble IL-2 receptor were very high and correlated well with the clinical course.Leptospirosis is a zoonosis, with clinical manifestations ranging from the imperceptible to severe, potentially fatal renal and liver failure accompanied by haemorrhage and jaundice. In this case report of a patient with severe leptospirosis, serum levels of procalcitonin decreased ahead of any obvious clinical improvement, and thus may be useful as a prognostic marker. Levels of soluble IL-2 receptor were very high and correlated well with the clinical course.

Self-poisoning in the acute care medicine 2005–2012

ObjectiveTo describe the trend of acute self-poisoning in the emergency and intensive care.MethodsElectronic charts of adults who presented to the emergency department of the University Hospital Leipzig with self-poisoning following a suicide attempt (suicide group), intoxication (intoxication group), drug overdose for relief of pain or discomfort (drug overdose group) between 2005 and 2012 were analyzed.Results3533 adults (62.6 % males) were identified, with the yearly admissions increasing from 305 in 2005 to 624 in 2012. The admission rate in relation to the total emergency department admissions also increased, from 1.2 % in 2005 to 1.9 % in 2012. 31.7 % of the patients were younger than 25 years. The reasons for self-poisoning were suicide attempt (18.1 %), intoxication (76.8 %) and drug overdose (2.9 %). The reason could not be clearly classified in 80 patients. Psychotropic drugs were used in 71.6 % of suicide attempts, while alcohol was the sole cause of intoxication in 80.1 % of cases in the intoxication group. Self-poisoning using at least two substances was observed in 52.0 % of the suicide attempts, 10.3 % of those with intoxication and 29.7 % of those with drug overdose. While alcohol remains the most common cause of intoxication, there was a drastic increase in the consumption of cannabinoids, Crystal Meth and gamma-hydroxybutyrate in the years 2011 and 2012. ICU admission was necessary in 16.6 % of the cases. There were 22 deaths (0.6 % of the study population), of whom 15 were in the suicide group (2.3 %), four (0.15 %) in the intoxication group, and three in the not clearly classified group (3.8 %).ConclusionAcute self-poisoning is an increasing medical issue. Psychotropic drugs remain the most common means of suicide attempt. Although alcohol intoxication is very frequent, intake of illicit drugs as the cause of emergency admission is increasing.ZusammenfassungZielBeschreibung des Trends der akuten Selbstvergiftung in der Notfall- und Intensivmedizin.MethodeElektronische Akten erwachsener Patienten, die zwischen 2005 und 2012 wegen einer akuten Selbstvergiftung infolge eines Selbstmordversuches (Selbstmordgruppe), einer Berauschung (Intoxikationsgruppe) oder Medikamentenüberdosierung zwecks Schmerzlinderung (Überdosierungsgruppe) in der Notaufnahme des Universitätsklinikums Leipzig eingewiesen worden waren, wurden ausgewertet.Ergebnisse3533 Patienten (62,6 % männlich) wurden identifiziert, mit einem Anstieg von 305 Fällen im Jahre 2005 auf 624 in 2012. Die Aufnahmerate bezogen auf die Gesamtaufnahmen stieg von 1,2 % im Jahre 2005 auf 1,9 % im Jahre 2012 an. 31,7 % der Patienten waren < 25 Jahre alt. Die Ursachen der Selbstvergiftung waren Suizidversuch (18,1 %), Intoxikation (76,8 %) und Medikamentenüberdosierung (2,9 %). Bei 80 Fällen war eine genaue Gruppenzuordnung nicht möglich. Während psychotrope Medikamente bei 71,6 % der Suizidversuche angewandt wurden, war Alkoholintoxikation die Ursache bei 80,1 % der Fälle in der Intoxikationsgruppe. Selbstvergiftung mit mindestens zwei Substanzen lag bei 52,0 % der Suizidversuche, 10,3 % der Intoxikationsgruppe und 29,7 % der Gruppe mit Medikamentenüberdosierung vor. Während die Alkoholintoxikation nach wie vor am häufigsten vorkam, gab es eine drastische Zunahme der Intoxikationen mit Cannabinoiden, Crystal Meth und Gamma-Hydroxybuttersäure in den Jahren 2011 und 2012. Eine Aufnahme auf die Intensivstation war bei 16,6 % der Fälle erforderlich. Es gab 22 Todesfälle (0,6 % der gesamten Studienpopulation), von denen 15 aus der Suizidgruppe (2,3 %), vier aus der Intoxikationsgruppe (0,15 %), und drei aus der nicht eindeutig zugeordneten Gruppe (3.8 %) waren.ZusammenfassungAkute Selbstvergiftung ist ein zunehmendes medizinisches Problem. Psychotrope Medikamente stellen nach wie vor die häufigsten Suizidversuchsmittel dar. Obwohl die Alkoholintoxikation weiterhin am häufigsten vorkommt, nehmen illegale Drogen als notfallmedizinischer Einweisungsgrund zu.