Comparison Evaluation of Automated Nucleated Red Blood Cell Enumeration by Sysmex XN 1000 in Comparison With Microscopic Reference in Children Under 1 Year.

BACKGROUND: In newborns, elevated nucleated red blood cell (NRBC) levels can be associated with enhanced erythropoietic stress and might be predictive for adverse outcome. Also, the presence of NRBC in peripheral blood might lead to erroneous enumeration results of white blood cells in automated hematology analyzers. We aimed to assess the comparability of the Sysmex XN 1000 to manual slide reviews and correlation of NRBC with inflammation markers. METHODS: Specimens of 3397 children under 1 year were compared by automated and microscopic NRBC enumeration. Additionally, potential correlations between NRBC and age and inflammation markers were examined. RESULTS: Overall, there was good correlation (r = 0.97) between automated (range: 0%-3883%) and microscopic enumeration (range: 0%-3694%) of NRBC with high comparability up to a NRBC value of 200% and an increase in the variation between the two methods with increasing NRBC numbers. When 94 samples with ≤ 200% NRBC and ≥ 30% divergence between methods were separately reanalyzed with respect to overlapping cell populations in their scattergrams, Sysmex would have generated unrecognized incorrect automated results in 47 samples, corresponding to 1.4% of total study samples. NRBC counts were negatively correlated to age, but not to inflammation markers. CONCLUSION: Sysmex XN 1000 is highly precise in the enumeration of NRBC in children under 1 year up to counts of 200% and might replace time-intense manual counting in routine diagnostics. In the setting of neonatal and intensive care diagnostics, microscopic control and supervision of scattergrams are highly recommended for any automated NRBC enumeration processes.

The von Willebrand factor-binding aptamer rondaptivon pegol as a treatment for severe and nonsevere hemophilia A.

Factor VIII (FVIII) circulates in a noncovalent complex with von Willebrand Factor (VWF), the latter determining FVIII half-life. The VWF-binding aptamer rondaptivon pegol (BT200) increases plasma levels of VWF/FVIII in healthy volunteers. This trial assessed its safety, pharmacokinetics, and pharmacodynamics in hemophilia A. Nineteen adult patients (ages 20-62 years, 4 women) with hemophilia A (8 mild, 2 moderate, and 9 severe) received subcutaneous injections of rondaptivon pegol. After an initial fixed dose of 3 mg on days 0 and 4, patients received weekly doses of 2 to 9 mg until day 28. Severe hemophilia A patients underwent sparse-sampling population pharmacokinetics individual profiling after the final dose of rondaptivon pegol. Adverse events, pharmacokinetics, and pharmacodynamics were assessed. FVIII activity and VWF levels were measured. All patients tolerated rondaptivon pegol well. The geometric mean half-life of rondaptivon pegol was 5.4 days and rondaptivon pegol significantly increased VWF levels. In severe hemophilia A, 6 doses of rondaptivon pegol increased the half-lives of 5 different FVIII products from a median of 10.4 hours to 31.1 hours (range, 20.8-56.0 hours). Median FVIII increased from 22% to 48% in mild hemophilia A and from 3% to 7.5% in moderate hemophilia A. Rondaptivon pegol is a first-in-class prohemostatic molecule that extended the half-life of substituted FVIII approximately 3-fold and increased endogenous FVIII levels approximately 2-fold in hemophilia patients. This trial was registered at www.clinicaltrials.gov as #NCT04677803.

The VWF binding aptamer rondoraptivon pegol increases platelet counts and VWF/FVIII in type 2B von Willebrand disease.

Type 2B von Willebrand disease (VWD) is characterized by an increased binding affinity of von Willebrand factor (VWF) to platelet glycoprotein Ib. This can lead to clearance of high-molecular-weight (HMW) multimers and thrombocytopenia with a resulting moderate-severe bleeding phenotype. Rondoraptivon pegol (BT200) is a pegylated aptamer binding to the A1 domain of VWF with a novel mechanism of action: it enhances VWF/factor VIII (FVIII) levels by decreasing their clearance. To study the potential benefit of rondoraptivon pegol in patients with type 2B VWD, we conducted a prospective phase 2 trial. Patients with type 2B VWD received 3 mg rondoraptivon pegol subcutaneously on study days 1, 4, and 7, followed by 6 to 9 mg every week until day 28. Five patients (male:female ratio = 3:2) were included. Rondoraptivon pegol rapidly tripled platelet counts from a median of 60 to 179 × 10E9/L (P < .001). Circulating VWF antigen increased from a median of 64% to 143%, which doubled FVIII activity levels from 67% to 134%. In all thrombocytopenic patients, plasma levels of VWF:GPIbM normalized, VWF ristocetin cofactor and VWF collagen-binding activity increased, and HMW multimers appeared. These pronounced improvements reversed during washout of the drug, thus demonstrating causality. The A1 domain binding aptamer directly corrects the underlying defect of type 2B VWD, thus providing a novel potential option for prophylaxis and treatment of patients with this VWD type. These data provide the basis for a phase 2b/3 trial in such patients. This trial was registered at www.clinicaltrials.gov as #NCT04677803.

The von Willebrand factor A-1 domain binding aptamer BT200 elevates plasma levels of von Willebrand factor and factor VIII: a first-in-human trial.

Von Willebrand factor (VWF) and factor VIII (FVIII) circulate in a noncovalent complex in blood and promote primary hemostasis and clotting, respectively. A new VWF A1-domain binding aptamer, BT200, demonstrated good subcutaneous bioavailability and a long half-life in non-human primates. This first-in-human, randomized, placebo-controlled, doubleblind trial tested the hypothesis that BT200 is well tolerated and has favorable pharmacokinetic and pharmacodynamic effects in 112 volunteers. Participants received one of the following: a single ascending dose of BT200 (0.18-48 mg) subcutaneously, an intravenous dose, BT200 with concomitant desmopressin or multiple doses. Pharmacokinetics were characterized, and the pharmacodynamic effects were measured by VWF levels, FVIII clotting activity, ristocetin-induced aggregation, platelet function under high shear rates, and thrombin generation. The mean half-lives ranged from 7-12 days and subcutaneous bioavailability increased dose-dependently exceeding 55% for doses of 6-48 mg. By blocking free A1 domains, BT200 dose-dependently decreased ristocetin-induced aggregation, and prolonged collagen-adenosine diphosphate and shear-induced platelet plug formation times. However, BT200 also increased VWF antigen and FVIII levels 4-fold (P<0.001), without increasing VWF propeptide levels, indicating decreased VWF/FVIII clearance. This, in turn, increased thrombin generation and accelerated clotting. Desmopressin-induced VWF/FVIII release had additive effects on a background of BT200. Tolerability and safety were generally good, but exaggerated pharmacology was seen at saturating doses. This trial identified a novel mechanism of action for BT200: BT200 dose-dependently increases VWF/FVIII by prolonging half-life at doses well below those which inhibit VWF-mediated platelet function. This novel property can be exploited therapeutically to enhance hemostasis in congenital bleeding disorders.

Limitations of a calibrated, quantitative APC-R assay under routine conditions.

INTRODUCTION: The aim of this study was to evaluate the Hemoclot Quanti. V-L assay in various clinical conditions. METHODS: We compared the Hemoclot Quanti.V-L assay with DNA testing and with the Pefakit assay in 60 normal (no mutation) vs carriers of the factor V (FV) Leiden mutation (56 heterozygous and three homozygous). We further investigated the interference of lupus anticoagulant on test results in normal and heterozygous individuals and of direct oral anticoagulants (DOACs) at trough and peak levels. Additionally, DOAC-Remove was tested in samples containing DOACs at peak levels. We further evaluated the influence of FV deficiency on this quantitative assay. RESULTS: There was a 100% agreement between the Quant. V-L assay and DNA testing in 60 normal individuals. However, 1.85% of heterozygous and 33% of homozygous samples were falsely classified with the quantitative assay, and no misclassification was observed with the Pefakit assay. Lupus anticoagulant did not influence the test results of the quantitative assay. DOACs also interfered with test results in heterozygous patients, but this effect was prevented with the DOAC-Remove procedure. Even mild FV deficiency affected the test results of the quantitative assay in heterozygous patients leading either to misclassification or the need for subsequent PCR testing. CONCLUSION: The quantitative FV-L assay has several limitations, especially FV deficiency and the presence of DOACs have to be ruled out before running this quantitative assay.

Regulation of histamine and diamine oxidase in patients undergoing orthotopic liver transplantation.

Increased concentrations of the vasodilator histamine have been observed in patients undergoing abdominal surgery. The role of histamine during orthotopic liver transplantation (OLT) has only been studied in animals. The aim of this study was to measure plasma concentrations of histamine and its degrading enzyme diamine oxidase (DAO) in patients undergoing orthotopic liver transplantation, and assess whether histamine or DAO correlate with intraoperative noradrenaline requirements. Histamine and DAO concentrations were measured in 22 adults undergoing liver transplantation and 22 healthy adults. Furthermore, norepinephrine requirements during liver transplantation were recorded. Baseline concentrations of histamine and DAO were greater in patients, who underwent liver transplantation, than in healthy individuals (Histamine: 6.4 nM, IQR[2.9-11.7] versus 4.3 nM, IQR[3.7-7.1], p = 0.029; DAO: 2.0 ng/mL, IQR[1.5-4.1] versus <0,5 ng/mL, IQR[<0.5-1.1], p < 0.001). During liver transplantation, histamine concentrations decreased to 1.8 nM, IQR[0.5-4.9] in the anhepatic phase (p < 0.0001 versus baseline), and to 1.5 nM, IQR[0.5-2.9] after reperfusion (p < 0.0001 versus baseline). In contrast, DAO concentrations increased to 35.5 ng/ml, IQR[20-50] in the anhepatic phase (p = 0.001 versus baseline) and to 39.5 ng/ml, IQR[23-64] after reperfusion (p = 0.001 versus baseline), correlating inversely with histamine. Norepinephrine requirements during human liver transplantation correlated significantly with DAO concentrations in the anhepatic phase (r = 0.58, p = 0.011) and after reperfusion (r = 0.56; p = 0.022). In patients undergoing orthotopic liver transplantation, histamine concentrations decrease whereas DAO concentrations increase manifold. Diamine oxidase correlates with intraoperative norepinephrine requirements in patients undergoing OLT.

Interference in specialized coagulation assays affecting the protein C pathway: Effects of marked haemolysis, hyperbilirubinaemia and lipaemia on chromogenic and clotting tests on two coagulation platforms.

BACKGROUND: Haemolysis, lipaemia and hyperbilirubinaemia represent important challenges in the coagulation laboratory. Test results are influenced not only by the degree of the interfering substance but also by the detection system. METHODS: We investigated the interference of free haemoglobin, triglycerides and bilirubin on a "modified activated protein C (APC) resistance test," protein C activity and protein S (antigen and activity) with two coagulation analysers, the STA-R Evolution and the ACL TOP. RESULTS: Haemolysis interfered with all assays on the STA-R Evolution resulting in higher levels of protein C activity and lower levels of protein S and a decreased APC ratio compared with baseline levels. On the ACL TOP, haemolysis only diminished protein S antigen levels and the APC ratio. Lipaemia increased protein C activity and protein S activity levels on the STA-R Evolution, whereas APC-R decreased on the ACL TOP and protein S antigen could not be measured in any lipaemic samples. Hyperbilirubinaemia caused an increase in protein C activity and in protein S antigen and a decrease in APC-R on the STA-R Evolution, whereas a decline of protein C activity, of protein S antigen and of the APC-R could be observed in icteric samples on the ACL TOP. CONCLUSIONS: Our data show that the degree of interference associated with haemolysis, lipaemia and hyperbilirubinaemia is different in several assays. Some assay limitations were not reproduced, and limitations stated in kit inserts cannot be assumed to apply to all analysers.

Inhibition of complement C1s improves severe hemolytic anemia in cold agglutinin disease: a first-in-human trial.

Cold agglutinin disease is a difficult-to-treat autoimmune hemolytic anemia in which immunoglobulin M antibodies bind to erythrocytes and fix complement, resulting in predominantly extravascular hemolysis. This trial tested the hypothesis that the anti-C1s antibody sutimlimab would ameliorate hemolytic anemia. Ten patients with cold agglutinin disease participated in the phase 1b component of a first-in-human trial. Patients received a test dose of 10-mg/kg sutimlimab followed by a full dose of 60 mg/kg 1 to 4 days later and 3 additional weekly doses of 60 mg/kg. All infusions were well tolerated without premedication. No drug-related serious adverse events were observed. Seven of 10 patients with cold agglutinin disease responded with a hemoglobin increase >2 g/dL. Sutimlimab rapidly increased hemoglobin levels by a median of 1.6 g/dL within the first week, and by a median of 3.9 g/dL (interquartile range, 1.3-4.5 g/dL; 95% confidence interval, 2.1-4.5) within 6 weeks (P = .005). Sutimlimab rapidly abrogated extravascular hemolysis, normalizing bilirubin levels within 24 hours in most patients and normalizing haptoglobin levels in 4 patients within 1 week. Hemolytic anemia recurred when drug levels were cleared from the circulation 3 to 4 weeks after the last dose of sutimlimab. Reexposure to sutimlimab in a named patient program recapitulated the control of hemolytic anemia. All 6 previously transfused patients became transfusion-free during treatment. Sutimlimab was safe, well tolerated, and rapidly stopped C1s complement-mediated hemolysis in patients with cold agglutinin disease, significantly increasing hemoglobin levels and precluding the need for transfusions. This trial was registered at www.clinicaltrials.gov as #NCT02502903.

Inhibition of Protease-Activated Receptor (PAR1) Reduces Activation of the Endothelium, Coagulation, Fibrinolysis and Inflammation during Human Endotoxemia.

The protease-activated receptor-1 (PAR-1) is critically involved in the co-activation of coagulation and inflammatory responses. Vorapaxar is a reversible, orally active, low molecular weight, competitive antagonist of PAR-1.We investigated the effects of PAR-1 inhibition by vorapaxar on the inflammatory response, the activation of coagulation, fibrinolysis and endothelium during experimental endotoxemia. In this randomized, double blind, crossover trial, 16 healthy volunteers received a bolus infusion of 2 ng/kg lipopolysaccharide (LPS) ± placebo/vorapaxar with a washout period of 8 weeks. Vorapaxar dosing was guided by thrombin receptor-activating peptide-6-induced whole blood aggregometry. Participants received 10 mg vorapaxar or placebo as an initial dose and, depending on the aggregometry, potentially an additional 10 mg. Goal was > 80% inhibition of aggregation compared with baseline. Vorapaxar significantly reduced the LPS-induced increase in pro-thrombin fragments F1 + 2 by a median of 27% (quartiles: 11-49%), thrombin-anti-thrombin concentrations by 22% (-3 to 46%) and plasmin-anti-plasmin levels by 38% (23-53%). PAR-1 inhibition dampened peak concentrations of tumour necrosis factor -α, interleukin-6 and consequently C-reactive protein by 66% (-11-71%), 50% (15-79%) and 23% (16-38%), respectively. Vorapaxar decreased maximum von Willebrand factor levels by 29% (26-51%) and soluble E-selectin concentrations by 30% (25-38%) after LPS infusion. PAR-1 inhibition did not affect thrombomodulin, soluble P-selectin and platelet factor-4 concentrations.PAR-1 inhibition significantly reduced the activation of coagulation, fibrinolysis, the inflammatory response and endothelial activation during experimental human endotoxemia.

Single, very low rituximab doses in healthy volunteers - a pilot and a randomized trial: implications for dosing and biosimilarity testing.

There are no  dose-finding trials available for rituximab that could guide dosing in non-malignant diseases. We hypothesized that currently used doses (≥375 mg/m2) exceed several hundred-fold the half-maximal effective dose, which is most sensitive for detecting putative differences between biosimilars and important for dose finding. In an open label, exploratory trial healthy volunteers received single infusions of rituximab at doses of 0.1, 0.3 or 1.0 mg/m2. Subsequently, in a double-blind, randomized trial healthy volunteers received single infusions of two rituximab products at doses of 0.1 and 0.3 mg/m2. In the exploratory trial rituximab transiently depleted CD20+ cells by a mean 68% (range: 57-95%), 74% (55-82%) and 97% (94-100%) immediately after the infusion of 0.1 (n = 4), 0.3 (n = 4) and 1 mg/m2 (n = 8), respectively. In the randomized trial CD20+ cells decreased by a mean 48% (25-84%) - 55% (26-85%) and 81 (67-89%) - 87% (77-96%) after infusion of 0.1 mg/m2 (n = 12) or 0.3 mg/m2 (n = 8 proposed biosimilar, n = 4 reference product) of the proposed biosimilar or the reference product, respectively. It is important to understand that in healthy volunteers <1% of the authorized rituximab doses depletes almost all circulating B lymphocytes. Thus, for non-malignant diseases alternative, more cost-effective dosing regimens seem plausible, but require clinical testing. (EudraCT-No. 2010-023781-45; EudraCT-No. 2013-001077-24).

Evaluation of between-, within- and day-to-day variation of coagulation measured by rotational thrombelastometry (ROTEM).

BACKGROUND AND AIMS: The aim of this study was to assess the circadian variation and the between- and within-subject variation in 10 healthy subjects over a period of 8 weeks by ROTEM®. We further evaluated the influence of elevated body mass index and the effect of low molecular weight heparin and antithrombin on clot formation. METHODS: Citrated blood samples were analysed in the NATEM® test system. The clotting time (CT), clot formation time (CFT), maximum clot firmness (MCF) and the maximum lysis (ML) were assessed. RESULTS: Duplicate measurements showed that 23% of the CT and 31% of the CFT measurements had a coefficient of variation (CV) greater than 10%. The within-subject CV was 16% for the CT and 30% for the CFT. The MCF was fairly constant (6%), whereas ML showed more variation (18%). The between-subject CV was 6% for the CT and 20% for the CFT. Analytical variability was improved by summing up CT and CFT. Compared to morning values, CT, CFT and the sum of CT + CFT were shortened in the afternoon. High body mass index was associated with faster clotting. High concentrations of antithrombin had similar effects on clot formation as 0.2 IU/ml of enoxaparin. CONCLUSIONS: To overcome the influence of diurnal variation, we recommend obtaining blood samples at specified times in the morning. The within-subject variation should be taken into account, when serial measurements of drug effects are required.

False-positive pregnancy test after transfusion of solvent/detergent-treated plasma.

BACKGROUND: The transmission of pathogens, antibodies, and proteins is a possible consequence of blood product transfusion. A female patient had an unexpected positive serum ß-human chorionic gonadotropin result, indicative of pregnancy, after she had received a transfusion with 1 unit of platelet concentrate, 4 units of red blood cells, and 4 units of pooled solvent/detergent-treated plasma (Octaplas). STUDY DESIGN AND METHODS: To investigate the possibility of passive transfusion of ß-human chorionic gonadotropin from the plasma transfusion, one additional unit from the same batch was thawed and analyzed. To validate the ß-human chorionic gonadotropin assay for use in solvent/detergent-treated plasma and to investigate any interference in the assay, dilution experiments were performed using the implicated plasma batch diluted with male and non-pregnant female sera. Also, plasma from a known pregnant woman was diluted with Octaplas (tested negative for ß-human chorionic gonadotropin) and with a male serum to validate the assay for use in solvent/detergent-treated plasma. RESULTS: The implicated solvent/detergent-treated plasma had a mean ß-human chorionic gonadotropin level of 91.5 mIU/mL. Results from the dilution experiments revealed an excellent correlation (r > 0.99) between ß-human chorionic gonadotropin measurement in solvent/detergent-treated plasma and male serum and no over or under recovery of the expected results. Further measurements of ß-human chorionic gonadotropin levels in the female recipient revealed an estimated half-life of 6 hours. CONCLUSION: This case demonstrates the importance of considering the possibility of passive transmission of analytes to a patient from the transfusion of blood products. Furthermore, the measurement of ß-human chorionic gonadotropin is valid in solvent/detergent-treated plasma using a Roche Cobas analyzer.

Colistin dampens fibrinolysis and endothelial activation during endotoxaemia. A randomised, double blind trial.

Colistin electrostatically interacts with lipopolysaccharides (LPS). Pre-clinical studies demonstrated beneficial effects of colistin on LPS-induced coagulation and fibrinolysis. The objective of this trial was to investigate the effects of colistin during experimental endotoxaemia. In this randomised, double-blind, placebo-controlled, crossover trial 16 healthy volunteers received a 2 ng/kg LPS bolus after infusion of 2.5 million IU colistin or placebo. Plasma levels of F1+2 prothrombin fragments, thrombin-antithrombin complexes (TAT), von Willebrand factor antigen levels (vWF), E-selectin, plasmin-antiplasmin complexes (PAP), tissue-type plasminogen activator (t-PA) antigen and activity, plasminogen activator inhibitor-1 (PAI-1) were measured. Infusion of colistin significantly reduced peak concentrations of PAP complexes by 70 %, t-PA antigen levels by 63 % and t-PA activity by 48 %, while PAI-1 levels decreased numerically by 63 %. Two hours after the LPS bolus F1+2 levels and TAT complexes were slightly reduced in the colistin period, but peak concentrations were similar in both periods. Colistin blunted the LPS induced four-fold increase in soluble E-Selectin levels by ~50 % and the two-fold increase in vWF antigen levels by ~70 %. The LPS-scavenging actions of colistin significantly reduce endothelial activation and fibrinolytic response in the human endotoxaemia model, while the activation of the coagulation system remains largely unaffected.

Upregulation of cytokine mRNA in circulating leukocytes during human endotoxemia.

Endotoxemia induces pronounced changes in leukocyte count and enhances the release of many cytokines. However, the molecular regulation of this cytokine release is poorly characterized in humans. The time course of mRNA expression of 24 cytokines in circulating leukocytes was studied in a well-standardized model of human endotoxemia (2 ng/kg). Real-time polymerase chain reaction (RT-PCR) was used to quantify the lipopolysaccharide (LPS)-inducible mRNA levels of leukocytes from 16 healthy volunteers in a randomized, placebo-controlled trial. Baseline mRNA levels of interleukins including IL-1α, IL-3, IL-5, IL-6, IL-12p40, IL-13, IL-15, IL-17, granulocyte colony-stimulating factor (G-CSF) and granulocyte monocyte CSF (GM-CSF) were below detectable levels in normal blood of the healthy participants. After 2 h, LPS infusion increased median mRNA levels of IL-1α by >1100-fold and IL-1ß and IL-8 by 33-fold and 46-fold, respectively. In contrast, levels of tumor necrosis factor (TNF-α) and IL-10 mRNA increased by only 7-fold, whereas changes in mRNA expression of other cytokines showed either a more than two fold increase or were undetectable. In vitro incubation of whole blood with 50 pg/mL LPS for 2 h enhanced transcription levels of IL-1α mRNA by >10,000-fold, IL-6 and IL-12p40 by >1000-fold, IL-1ß by 400-fold, TNF-α by 100-fold, IL-8, IL-18, interferon γ (IFN-γ) and G-CSF by >10-25-fold, and IL-10, IL-12p35, TNF-ß, and IL-13 by 10-25-fold. Only half of the 24 evaluated cytokines were expressed at the mRNA level in circulating leukocytes under basal conditions and after an LPS challenge. Only IL-1α, IL-1ß, IL-10, IL-8, and TNF-α were upregulated in the circulating leukocytes, whereas several other cytokines (including IL-6 and G-CSF), were expressed on the mRNA level following in vitro incubation of blood with LPS. In addition, IL-1α and IL-1ß might be potential diagnostic targets in inflammatory diseases.

The use of frozen plasma samples in thromboelastometry.

Thromboelastometry is increasingly used in the clinical and scientific setting. The use of frozen plasma samples may be useful in overcoming certain limitations such as local and timely availability. Whole blood (WB) samples of 20 healthy volunteers were obtained, and plasma was generated. NATEM (n = 20), EXTEM (n = 20) and INTEM (n = 8) analyses were performed in WB, fresh plasma and frozen and thawed plasma. Dabigatran (500, 1000 ng/ml), rivaroxaban (100, 200 ng/ml) or alteplase (333 ng/ml) were added ex vivo to WB, and thromboelastometry was performed in WB and in frozen and thawed plasma samples. Clot formation time, mean clot firmness and the area under the curve were significantly altered in plasma compared to WB. In INTEM and EXTEM analysis, clotting time (CT) was comparable between WB (100%) and fresh (INTEM 114% and EXTEM 93%, ratio of the means) and frozen plasma samples (85 and 99%), whereas in NATEM analysis, the CT increased in fresh (193%) and frozen plasma samples (130%). Dabigatran dose-dependently increased the CT approximately 5- and 9-fold in WB and even more pronounced 10- and 26-fold in plasma. Accordingly, rivaroxaban dose-dependently increased the CT 2- and 2.7-fold in WB, and 3.5- and 4-fold in plasma samples. Hyperfibrinolysis was achieved by addition of alteplase in all WB samples and was reproducible in plasma samples. In conclusion, thromboelastometry, especially INTEM and EXTEM analyses, is possible using frozen and stored plasma samples with comparable results to the corresponding whole blood samples.

Lupus-anticoagulant testing at NOAC trough levels.

Non-vitamin K antagonist oral anticoagulants (NOAC), including rivaroxaban, apixaban or dabigatran, regularly show relevant effects on coagulation tests, making the interpretation of results difficult. The aim of this study was to evaluate possible interferences of NOACs in trough level concentrations in lupus anticoagulant (LA) testing. Citrate plasma specimens of 30 healthy volunteers were spiked with rivaroxaban, apixaban or dabigatran in four plasma concentration levels at or below trough NOAC levels. The NOAC concentration was measured using dedicated surrogate concentration tests and a stepwise diagnostic procedure for LA-testing was applied using screening, mixing and confirmatory testing. Results were compared to NOAC-free specimens. Starting with a plasma concentration of 12.5 ng/ml, dabigatran-spiked specimens showed significant prolongations in the lupus anticoagulant-sensitive activated partial thromboplastin time (aPTT-LA) as well as in the Dilute Russell viper venom time (dRVVT), leading to 43.3 % false positives in confirmatory testing in the dRVVT. In contrast, rivaroxaban, beginning with 7.5 ng/ml, exclusively affected dRVVT-based tests. In confirmatory tests, 30.0 % of rivaroxaban-spiked specimens showed false positive results. Starting with 18.75 ng/ml apixaban, a significant prolongation of the dRVVT and up to 20.7 % false positives in confirmatory tests were found. In contrast to other NOACs tested, apixaban did not present with a dose-dependent increase of the dRVVT ratio. In conclusion, the rate of false positive results in LA-testing is unacceptably high at expected trough levels of NOACs. Even at plasma concentrations below the LLOQ of commercially available surrogate tests, LA testing is best avoided in patients with NOAC therapy.

Effect of preanalytical time-delay on platelet function as measured by multiplate, PFA-100 and VerifyNow.

BACKGROUND AND AIMS: Platelet function testing may help to identify poor responders to antiplatelet drugs. The aim of this study was to compare three commonly used platelet function tests with special focus on the pre-analytical influence of time-delay on the tested parameters. METHODS: We assessed ADP-induced platelet function by the Multiplate, Platelet Function Analyzer-100 (PFA-100) and VerifyNow in nine healthy volunteers and 36 patients receiving clopidogrel or prasugrel 1 and 3 hours after sampling. RESULTS: The PFA-100 demonstrated non-closure time in 23 patients. A more graded response could be detected with the two other devices. Aggregation in whole blood (Multiplate) decreased after 3 hours compared to 1 hour in all subjects (p < 0.05). Furthermore, aggregation levels obtained by the VerifyNow showed a decrease in patients taking P2Y12 inhibitors after 3 hours (p < 0.05), except in three patients, in whom an increase was observed. CONCLUSION: Responses to ADP are time-dependent after blood sampling for the Multiplate in all subjects and for the VerifyNow in patients on antiplatelet drugs. For both devices, platelet aggregation was reduced 3 hours after sampling which may affect data interpretation and clinical consequences.

Acquired von Willebrand factor deficiency caused by LVAD is ADAMTS-13 and platelet dependent.

INTRODUCTION: The high shear rates induced by left ventricular assist devices cause acquired von Willebrand disease (aVWD). We hypothesised that an ex vivo model could be established to study whether mechanical shear stress alone causes aVWD or whether this process depends also on the VWF cleavage protein ADAMTS-13 and on platelets. MATERIALS AND METHODS: Healthy volunteers and two patients with congenital ADAMTS-13 deficiency donated blood. In vitro closed extracorporeal circuits were established using medically approved left ventricular assist devices (LVAD). VWF multimers were quantified by gel electrophoresis; VWF antigen, ristocetin cofactor activity (VWF:RCo), ADAMTS-13 levels and platelet function were assessed. RESULTS: The high shear stress in the extracorporeal circulation rapidly decreased VWF:RCo and thereby the VWF:RCo/VWF:Ag ratio by 47% (p<0.01) to pathologically low values. Concomitantly, high molecular weight multimers (HMWM) decreased: up to 14-15 mers were visible on the gels at baseline, which were reduced by a maximum of 6-7 mers, corresponding to an average 68% lower densitometry signal of HMWM (p<0.001). This was accompanied by marked reduction of aggregation by various agonists (p<0.005). In contrast, the two patients with congenital thrombocytopenic purpura with virtually complete deficiency of ADAMTS-13 activity had only a minimal or no decrease in multimers (p<0.005 vs. healthy controls). Similarly, no or minimal depletion of large multimers occurred, when normal plasma circulated without platelets. CONCLUSION: An in vitro model for LVAD associated aVWD demonstrated that ADAMTS-13 and platelets contribute to the depletion of HMWM of VWF.

D-dimer and histamine in early stage bacteremia: A prospective controlled cohort study.

INTRODUCTION: Plasma histamine levels and D-dimer predict disease severity and mortality in advanced septic shock. We hypothesized that increased plasma histamine levels parallel coagulation activation and yield prognostic significance already at a very early stage of bacteremia. PATIENTS AND METHODS: This prospective controlled cohort study enrolled 72 consecutive non-surgical non-ICU-ward inpatients with newly culture-diagnosed bacteremia and a Pitt Bacteremia score ≤2 to determine the extent of histamine and D-dimer release and their predictive role on outcome at the earliest stage of blood stream infection. Age-matched healthy adults served as internal controls (n=36). A binominal logistic regression and a Cox proportional hazards regression analysis were performed to ascertain the effects of D-dimer and histamine on in-hospital mortality. RESULTS: In contrast to plasma histamine, D-dimer levels were significantly higher within hours of culture-proven bacteremia. In-hospital mortality occurred in 17%. Histamine levels were neither associated with D-dimer level (r=0.04; p>0.05) nor with ICU admissions (r=0.06; p>0.05) and outcome (crude OR 0.8, 95% CI 0.3-1.9; p=0.6). In contrast, early-elevated D-dimer levels predicted mortality: the odds to die increased with the D-dimer level, and was 12.6 (crude OR, 95% CI 3-52; p=0.001) in patients with a D-dimer ≥4µg/mL (n=13). CONCLUSION: Histamine levels are elevated in only few patients (4%) with newly diagnosed bacteremia. Our findings suggest that D-dimer, but not plasma histamine, could be a promising marker of lethality already at a very early stage of blood stream infection.

A model comparing how rapidly transfusion of solvent detergent plasma restores clotting factors versus infusion of albumin-saline.

BACKGROUND: A recent randomized controlled trial demonstrated the bioequivalence between universally applicable and AB0 compatible transfusion plasma in healthy volunteers. There was a limited change in coagulation factor levels and inhibitors before and after plasmapheresis and subsequent plasma transfusion. The aim of this extension trial was to investigate the true capacity of these plasma products to restore baseline levels of coagulation factors and inhibitors after plasma depletion in comparison to haemodilution induced by infusion of albumin solution. MATERIALS AND METHODS: Fourteen healthy subjects, who completed both plasma transfusion periods, underwent an additional plasmapheresis (600 mL) followed by an infusion of 1200 mL albumin (3.125%) in a third period. RESULTS: The fibrinogen levels, as well as other clotting factors (FII, FV, FVII and FXI), decreased by 10% after plasmapheresis, and subsequent infusion of albumin solution further aggravated this drop in clotting factors to approximately 20-25%. The clotting factors with a long half-life were not even restored 24 hours after infusion of albumin solution, whereas those with a short half-life were replenished by endogenous synthesis within 24 hours. In contrast, transfusion of either plasma product rapidly restored all clotting parameters and inhibitors (protein S and plasmin inhibitor) immediately after transfusion. CONCLUSION: This study demonstrates that albumin solution induces an enhanced dilution of clotting factors and inhibitors, whereas both plasma products quickly compensated for the experimental loss of these plasma proteins.