A multi-center evaluation of TECHNOSCREEN® ADAMTS-13 activity assay as a screening tool for detecting deficiency of ADAMTS-13.

BACKGROUND: Quantifying A disintegrin-like and metalloprotease with thrombospondin type 1 motif, member 13 (ADAMTS-13) activity enhances thrombotic thrombocytopenic purpura (TTP) diagnosis but most assays are time consuming, technically demanding, and mainly available in reference centers. OBJECTIVE: Evaluate a simple, semiquantitative ADAMTS-13 activity screening test for early identification/exclusion of TTP. PATIENTS/METHODS: Plasma from 220 patients with suspected thrombotic microangiopathy at three reference centers were tested with TECHNOSCREEN® ADAMTS13 activity screening test in comparison with TECHNOZYM® ADAMTS-13 activity ELISA at two centers, and in-house fluorescence resonance energy transfer assay at the third center. The screening test indicates if ADAMTS-13 activity is at one of four level-indicator points: 0, 0.1, 0.4, or 0.8 IU/mL. RESULTS: Screen results were interpreted as binary data in that ADAMTS-13 activity was above or below the 0.1 IU/mL TTP clinical threshold. Combining all sites' data, the screen exhibited 88.7% sensitivity, 90.4% specificity, 74.6% positive predictive value, and 96.2% negative predictive value, comparable to published data for quantitative assays. Five samples with quantitative results below the threshold gave screen readings of 0.1 IU/mL and seven marginally above the threshold gave screen readings of zero. All would warrant plasma exchange while the level is quantified. Nine samples with normal/near normal results gave screens of zero and confirmatory quantifications would prompt early treatment withdrawal, as is current practice. One sample generated screen/quantitative results of 0.4/0.00 IU/mL respectively and was the only clear false-negative. CONCLUSIONS: The screening test provides more rapid ADAMTS-13 level evaluation than most currently available assays. Its simple operation renders it suitable for adoption in routine or specialist laboratory environments.

Transition from fresh frozen plasma to solvent/detergent plasma in the Netherlands: comparing clinical use and transfusion reaction risks.

Plasma transfusion is indicated for replenishment of coagulative proteins to stop or prevent bleeding. In 2014, the Netherlands switched from using ~300mL fresh frozen plasma (FFP) units to using 200mL Omniplasma, a solvent/detergent treated pooled plasma (SD plasma), units. We evaluated the effect of the introduction of SD plasma on clinical plasma use, associated bleeding, and transfusion reaction incidences. Using diagnostic data from six Dutch hospitals, national blood bank data, and national hemovigilance data for 2011 to 2017, we compared the plasma/red blood cell (RBC) units ratio (f) and the mean number of plasma and RBC units transfused for FFP (~300mL) and SD plasma (200mL) for various patient groups, and calculated odds ratios comparing their associated transfusion reaction risks. Analyzing 13,910 transfusion episodes, the difference (Δf = fSD - fFFP) in mean plasma/RBC ratio (f) was negligible (Δfentire_cohort = 0.01 [95% confidence interval (CI): -0.02 - 0.05]; P=0.48). SD plasma was associated with fewer RBC units transfused per episode in gynecological (difference of mean number of units -1.66 [95% CI: -2.72, -0.61]) and aneurysm (-0.97 [-1.59, -0.35]) patients. SD plasma was further associated with fewer anaphylactic reactions than FFP (odds ratio 0.37 [0.18, 0.77; P<0.01]) while the differences for most transfusion reactions were not statistically significant. SD plasma units, despite being one third smaller in volume than FFP units, are not associated with a higher plasma/RBC ratio. SD plasma is associated with fewer anaphylactic reactions than FFP plasma/RBC units ratio.

Age of platelet concentrates and time to the next transfusion.

BACKGROUND: Storage time of platelet (PLT) concentrates has been negatively associated with clinical efficacy outcomes. The aim of this study was to quantify the association between storage time of PLT concentrates and interval to the next PLT transfusion for different types of PLT components, stored for up to 7 days and transfused to transfusion-dependent hematooncology patients with thrombocytopenia. STUDY DESIGN AND METHODS: From a cohort of patients from 10 major Dutch hospitals, patients were selected whose transfusion patterns were compatible with PLT transfusion dependency due to hematooncologic disease. Mean time to the next transfusion and mean differences in time to the next transfusion for different storage time categories (i.e., fresh, <4 days; intermediate, 4-5 days; and old, >5 days) were estimated, per component type, using multilevel mixed-effects linear models. RESULTS: Among a cohort of 29,761 patients who received 140,896 PLT transfusions we selected 4441 hematooncology patients who had received 12,724 PLT transfusions during periods of PLT transfusion dependency. Transfusion of fresh, compared to old, buffy coat-derived PLTs in plasma was associated with a delay to the next transfusion of 6.2 hours (95% confidence interval [CI], 4.5-8.0 hr). For buffy coat-derived PLTs in PAS-B and -C this difference was 7.7 hours (95% CI, 2.2-13.3 hr) and 3.9 hours (95% CI, -2.1 to 9.9 hr) while for apheresis PLTs in plasma it was only 1.8 hours (95% CI, -3.5 to 7.1 hr). CONCLUSION: Our results indicate that the time to the next transfusion shortens with increasing age of transfused buffy coat-derived PLT concentrates. This association was not observed for apheresis PLTs.

Validation of multisource electronic health record data: an application to blood transfusion data.

BACKGROUND: Although data from electronic health records (EHR) are often used for research purposes, systematic validation of these data prior to their use is not standard practice. Existing validation frameworks discuss validity concepts without translating these into practical implementation steps or addressing the potential influence of linking multiple sources. Therefore we developed a practical approach for validating routinely collected data from multiple sources and to apply it to a blood transfusion data warehouse to evaluate the usability in practice. METHODS: The approach consists of identifying existing validation frameworks for EHR data or linked data, selecting validity concepts from these frameworks and establishing quantifiable validity outcomes for each concept. The approach distinguishes external validation concepts (e.g. concordance with external reports, previous literature and expert feedback) and internal consistency concepts which use expected associations within the dataset itself (e.g. completeness, uniformity and plausibility). In an example case, the selected concepts were applied to a transfusion dataset and specified in more detail. RESULTS: Application of the approach to a transfusion dataset resulted in a structured overview of data validity aspects. This allowed improvement of these aspects through further processing of the data and in some cases adjustment of the data extraction. For example, the proportion of transfused products that could not be linked to the corresponding issued products initially was 2.2% but could be improved by adjusting data extraction criteria to 0.17%. CONCLUSIONS: This stepwise approach for validating linked multisource data provides a basis for evaluating data quality and enhancing interpretation. When the process of data validation is adopted more broadly, this contributes to increased transparency and greater reliability of research based on routinely collected electronic health records.

Storage time of platelet concentrates and all-cause bacteremia in hematologic patients.

BACKGROUND: Extension of storage time of platelet (PLT) concentrates may result in an increased risk of bacteremia, directly via transfusion of contaminated products or indirectly via transfusion-related immunomodulation. We aimed to quantify the association of storage time of PLT concentrates and all-cause bacteremia in hematologic patients. STUDY DESIGN AND METHODS: We established a cohort of hematologic patients who received a PLT transfusion between 2005 and 2015. Cases were defined as patients with a bacteremia the day after transfusion and matched to as many controls as possible. A conditional logistic regression was performed, stratified by storage medium. RESULTS: Among 3514 patients receiving 36,032 PLT concentrates stored in plasma, 613 cases of bacteremia were found. The relative risk of all-cause bacteremia the day after transfusion was 0.80 (95% confidence interval [CI], 0.58-1.12) for PLT concentrates stored 3 to 4 days and 0.67 (95% CI, 0.49-0.92) for at least 5 days, compared to no more than 2 days. Among 1527 patients receiving 11,822 PLT concentrates stored in PLT additive solution, 182 cases of bacteremia were found. The relative risk of all-cause bacteremia was 1.14 (95% CI, 0.70-1.84) for PLT concentrates stored for 3 to 4 days and 1.19 (95% CI, 0.70-2.01) for at least 5 days, compared to not more than 2 days. CONCLUSION: Storage time of PLT concentrates was not associated with increased occurrence of all-cause bacteremia the day after transfusion. If anything, fewer cases of bacteremia occurred with increasing storage time of PLT concentrates in plasma. These bacteremias are not directly caused by transfusion of a contaminated product and the underlying mechanism warrants further research.

The Effect of Rosuvastatin on Markers of Immune Activation in Treatment-Naive Human Immunodeficiency Virus-Patients.

Background. Immune activation has been implicated in the excess mortality in human immunodeficiency virus (HIV)-infected patients, due to cardiovascular diseases and malignancies. Statins may modulate this immune activation. We assessed the capacity of rosuvastatin to mitigate immune activation in treatment-naive HIV-infected patients. Methods. In a randomized double-blind placebo-controlled crossover study, we explored the effects of 8 weeks of rosuvastatin 20 mg in treatment-naive male HIV-infected patients (n = 28) on immune activation markers: neopterin, soluble Toll-like receptor (TLR)2, sTLR4, interleukin (IL)-6, IL-1Ra, IL-18, d-dimer, highly sensitive C-reactive protein, and CD38 and/or human leukocyte antigen-DR expression on T cells. Baseline data were compared with healthy male controls (n = 10). Furthermore, the effects of rosuvastatin on HIV-1 RNA, CD4/CD8 T-cell count, and low-density lipoprotein cholesterol were examined and side effects were registered. Results. T-cell activation levels were higher in patients than in controls. Patients had higher levels of circulating IL-18, sTLR2, and neopterin (all P < .01). Twenty patients completed the study. Rosuvastatin increased the CD4/CD8 T-cell ratio (P = .02). No effect on other markers was found. Conclusions. Patients infected with HIV had higher levels of circulating neopterin, IL-18, sTLR2, and T-cell activation markers. Rosuvastatin had a small but significant positive effect on CD4/CD8 T-cell ratio, but no influence on other markers of T-cell activation and innate immunity was identified (The Netherlands National Trial Register [NTR] NTR 2349, http://www.trialregister.nl/trialreg/index.asp).

Global coagulation tests: their applicability for measuring direct factor Xa- and thrombin inhibition and reversal of anticoagulation by prothrombin complex concentrate.

BACKGROUND: Specific mass spectrometry and direct activated factor X (Xa)- and thrombin inhibition assays do not allow determination of the reversal of anticoagulant effects of non-vitamin K direct oral anticoagulants (NOACs) by prothrombin complex concentrate (PCC). The objective of this study was the evaluation of the applicability of a variety of commercially available global coagulation assays in analyzing the reversal of NOAC anticoagulation by PCC. METHODS: Plasma and whole blood were spiked with apixaban or dabigatran and PCC was added to these samples. Prothrombin time (PT), modified PT (mPT), activated partial prothrombin time (APTT), thrombography (CAT method) and thromboelastography (ROTEM, TEG) were performed. RESULTS: Assays triggered by contact activation (APTT, INTEM) did not show inhibitor reversal by PCC. Assays triggered by tissue factor (TF) showed NOAC type and NOAC concentration dependent anticoagulation reversal effects of PCC ranging from partial normalization to overcorrection of the following parameters: clotting or reaction time (PT, mPT TEG-TF, EXTEM, FIBTEM); angle in thromboelastography (TEG-TF); thrombin generation (CAT) lag time, endogenous thrombin potential (ETP) and peak thrombin. Extent of reversal was assay reagent dependent. ETP (5 pM TF) was the only parameter showing complete reversal of anticoagulation by PCC for all NOACs ranging from 200 to 800 µg/L. CONCLUSIONS: ETP fits with the concept that reversal assessment of NOAC anticoagulation by PCC should be based on measurements on the clotting potential or thrombin generating potential of the plasma or whole blood patient sample. Low sensitivity of ETP for NOACs and its correlation with bleeding are issues that remain to be resolved.

Haemolytic anaemia after nitrofurantoin treatment in a pregnant woman with G6PD deficiency.

We present a normotensive, pregnant woman with severe haemolytic anaemia in the third trimester of pregnancy. Owing to normal platelet count diagnoses other than HELLP syndrome were considered and investigated. The patient was treated with nitrofurantoin 3 weeks before presentation and she turned out to have a deficiency of glucose-6-phosphate dehydrogenase. After treatment with blood transfusion, vitamin B12 and folic acid the patient recovered completely. Caesarean delivery was performed because of maternal hypertension and fetal distress at 33 weeks' gestation.

Clinical performance evaluation of the CellaVision Image Capture System in the white blood cell differential on peripheral blood smears.

INTRODUCTION: Differential counting and morphological analysis of peripheral blood smears is of great diagnostic importance to the clinician. For economic and time-saving reasons, automated imaging processes have been successfully introduced over the years. The aim of this study was to investigate whether a new morphology system, the CellaVision Image Capture System (CICS), can be used to perform a white blood cell (WBC) differential on peripheral blood smears. METHODS: WBCs in 200 peripheral blood smears were analysed with the CICS method and compared with the DM96 method to establish accuracy, short-term imprecision and clinical sensitivity and specificity for morphology. For establishing long-term imprecision, two blood smears were analysed for 20 days with the CICS method. RESULTS: Evaluation of accuracy in 199 samples demonstrated a good correlation for the CICS when compared with the postclassification on the DM96. Regression coefficients ranged from 0.97 for monocyte counts to 0.99 for neutrophil counts. All regression lines showed slopes with 1 and intercepts with 0 within the 95% CI. Long-term imprecision was less than 5% for neutrophils, eosinophils, basophils, lymphocytes and monocytes. Comparison of the short-term imprecision demonstrated that the SD did not differ by more than 1.1% between the DM96 method and the CICS method. Clinical sensitivity of the CICS was 93.5% and specificity was 97.8%. Specificity and sensitivity for blasts was 100%. CONCLUSIONS: The CICS has proven to be a reliable and accurate tool in the differential WBC count on peripheral blood smears and provides small laboratories with a 24 h available real-time digital differential WBC count on peripheral blood smears and consultation for patients in remote locations.

Measuring Rivaroxaban in a clinical laboratory setting, using common coagulation assays, Xa inhibition and thrombin generation.

BACKGROUND: Rivaroxaban, a direct Xa inhibitor, is one of the new oral antithrombotic agents for which laboratory monitoring is thought to be unnecessary in most cases due to predictable pharmacokinetics. Circumstances are conceivable, however, in which reliable laboratory testing of Rivaroxaban is desirable. The aim of the present in vitro study was to investigate and compare the analytical and practical use of Rivaroxaban monitoring with routine screening assays, thrombin generation and anti-Xa activity, in a clinical laboratory setting. METHODS: Rivaroxaban was added to nine normal donor plasmas and to a normal pooled plasma in concentrations up to 1000 µg/L. Prothrombin time (PT), activated partial thromboplastin time (APTT), endogenous thrombin potential (ETP) and anti-Xa activity were measured in all donor samples. Responsiveness to Rivaroxaban and imprecision of Rivaroxaban recovery were assessed. RESULTS: Low intra-, but high inter-individual imprecision was found for PT displaying a linear dose-response relationship. Imprecision was much lower when directly measuring anti-Xa activity. Responsiveness of ETP lag-time was high, but of total thrombin generation was low, illustrating that the main effect of Rivaroxaban Xa inhibition lies in delaying thrombin formation rather than in preventing it. CONCLUSIONS: Despite a high inter-individual imprecision of the PT, this relatively fast and cost-friendly assay is sensitive to Rivaroxaban and integrates its effects on the global coagulant state of patients. Anti-Xa activity assays can be run to assess the actual Rivaroxaban concentration and in the future ETP could serve as a fine-tuned hemostatic balance indicator for patients using Rivaroxaban.

Comparative study of blood collection tubes and thromboplastin reagents for correction of INR discrepancies: a proposal for maximum allowable magnesium contamination in sodium citrate anticoagulant solutions.

International normalized ratio (INR) discrepancies were noted between clinical laboratories using various prothrombin time (PT) systems. We studied the influence of different commercial blood collection tubes and different PT systems on INR measurements. INRs of fresh patient samples were determined by 3 laboratories, each using different PT systems. In the first part of the study, samples were drawn with Vacutainer tubes and in the second part with Monovette tubes. In the first part of the study, the maximum bias for all patients amounted to 0.46 INR (14%), and in the second part, to 0.14 INR (4.9%). The maximum bias for all patients could be reduced further by local system calibration using frozen pooled plasma specimens. The sodium citrate solutions in the blood collection tubes were contaminated with magnesium ions (approximately 2.7 mmol/L and 0.3 mmol/L in the Vacutainer and Monovette, respectively). INR discrepancies could be explained largely by this influence of blood collection tubes. The maximum allowable magnesium contamination in sodium citrate anticoagulant solutions should be less than 1 mmol/L.

Chronic renal failure is accompanied by endothelial activation and a large increase in microparticle numbers with reduced procoagulant capacity.

BACKGROUND: In patients with chronic renal failure (CRF), cardiovascular disease is the leading cause of increased morbidity and mortality. We hypothesized a role for endothelial activation and microparticle (MP) numbers and procoagulant activity in the pre-thrombotic state of these patients. METHODS: We analysed blood samples of 27 patients with CRF [8 chronic kidney disease Stage 4 (CKD4), 9 peritoneal dialysis (PD) and 10 haemodialysis (HD), samples taken before and after HD] and 10 controls. Degree and nature of endothelial activation were assessed by measuring mature von Willebrand factor (vWF) and vWF propeptide levels. Cellular MPs were characterized by flow cytometry and MP-specific thrombin generation (TG) measurements. RESULTS: CRF was accompanied by chronic (CKD4 and PD) or acute (HD) endothelial activation. Patients with CRF had substantially higher MP numbers than controls (median 9400 versus 4350×10(6)/L, P=0.001), without significant differences between the treatment subgroups or between pre- and post-HD. The vast majority of MPs were platelet derived. Of the minor populations, endothelial MPs and tissue factor-bearing MPs were more abundant in CRF. MPs were procoagulant, but the increase in numbers was not reflected in a proportional increase in MP-specific TG. CONCLUSION: Renal failure is accompanied by endothelial activation of a different nature in CKD4 and PD patients compared to HD patients, and results in all subgroups in an increase of mainly platelet-derived MPs that appear to be less procoagulant than in other disease states, possibly because of the uraemic functional defect of their cellular source.

Pre-acquisition system assessment of the Sysmex(®) Coagulation System CS-2100i and comparison with end-user verification; a model for the regional introduction of new analysers and methods.

BACKGROUND: Pre-acquisition system assessment of clinical laboratory analysers and/or methods are generally repeated independently in each individual organisation planning their introduction. In the course of replacing our 10-year-old Sysmex(®) CA-1500 for Sysmex(®) CS-2100i coagulometers, we designed and tested a model based on CLSI protocols in which one laboratory performs an extensive validation, allowing others to rely on concise verification. METHODS: Validation of the Sysmex(®) CS-2100i was performed largely according to CLSI Guideline H57-A and included EP-5, 7, 9 and 10 in the evaluation of 10 assays encompassing all measurement principles available. EP-15 was used for end-user verification. Practicability and results of validation and verification were compared. RESULTS: Analytical performance of the CS-2100i was as claimed by the manufacturer and complied with our own criteria. System verification results were compatible with those of the validation. Verification was time- and cost-effective. CONCLUSIONS: We have approved the Sysmex(®) CS-2100i analyser for introduction in our laboratory. For colleague laboratories in our region introducing this analyser, a system verification is proposed to be sufficient when referring to our data. It is our intention to use the validation vs. end-user verification model for future method introduction, and when harmonising between our different laboratory locations.

Elevated numbers and altered subsets of procoagulant microparticles in breast cancer patients using endocrine therapy.

INTRODUCTION: Microparticles (MP) can be elevated in cancer and thromboembolic disease. We hypothesized a role for MP in the hypercoagulable state in breast cancer patients using endocrine therapy, in whom both cancer and the use of endocrine therapy are independent risk factors for the development of thrombosis. DESIGN AND METHODS: Plasma samples were collected from 40 breast cancer patients using endocrine therapy (20 patients without metastases receiving adjuvant therapy and 20 patients with metastatic disease treated in a palliative setting) and from 20 female healthy controls. The endocrine therapy used was either an anti-estrogen or an aromatase inhibitor. Numbers and cellular origin of MP subsets were analyzed by flowcytometry. MP-associated procoagulant activity was measured using a thrombin generation assay using conditions that allow analysis of MP induced thrombin generation. RESULTS: Breast cancer patients using endocrine therapy had higher levels of MP positive for Annexin V (median 10000 vs 6500×10E6/l), P-selectin (330 vs 200×10E6/l), tissue factor (33 vs 15×10E6/l), and of MP derived from platelets (CD41) and leukocytes (CD45). Thrombin generation in plasma was dependent on the presence of MP and thrombin generation performed after addition of isolated MP to normal plasma showed a higher endogenous thrombin potential (1105 vs 1029 nM.min) in breast cancer patients. No differences were observed in MP levels and thrombin generation parameters between the metastatic and adjuvant group. CONCLUSION: Breast cancer patients using endocrine therapy have an increased MP number and a higher MP-dependent thrombin generation, irrespective of the presence of metastatic disease. Altered MP subset characteristics in these patients, especially the higher number of (activated) platelet derived MP and leukocyte derived MP, may in part explain a heightened procoagulant state in breast cancer patients using endocrine therapy.

Hemostasis during low molecular weight heparin anticoagulation for continuous venovenous hemofiltration: a randomized cross-over trial comparing two hemofiltration rates.

INTRODUCTION: Renal insufficiency increases the half-life of low molecular weight heparins (LMWHs). Whether continuous venovenous hemofiltration (CVVH) removes LMWHs is unsettled. We studied hemostasis during nadroparin anticoagulation for CVVH, and explored the implication of the endogenous thrombin potential (ETP). METHODS: This cross-over study, performed in a 20-bed teaching hospital ICU, randomized non-surgical patients with acute kidney injury requiring nadroparin for CVVH to compare hemostasis between two doses of CVVH: filtrate flow was initiated at 4 L/h and converted to 2 L/h after 60 min in group 1, and vice versa in group 2. Patients received nadroparin 2850 IU i.v., followed by 380 IU/h continuously in the extracorporeal circuit. After baseline sampling, ultrafiltrate, arterial (art) and postfilter (PF) blood was taken for hemostatic markers after 1 h, and 15 min, 6 h, 12 h and 24 h after converting filtrate flow. We compared randomized groups, and 'early circuit clotting' to 'normal circuit life' groups. RESULTS: Fourteen patients were randomized, seven to each group. Despite randomization, group 1 had higher SOFA scores (median 14 (IQR 11-15) versus 9 (IQR 5-9), p = 0.004). Anti-Xa art activity peaked upon nadroparin bolus and declined thereafter (p = 0.05). Anti-Xa PF did not change in time. Anti-Xa activity was not detected in ultrafiltrate. Medians of all anti-Xa samples were lower in group 1 (anti-Xa art 0.19 (0.12-0.37) vs. 0.31 (0.23-0.52), p = 0.02; anti-Xa PF 0.34 (0.25-0.44) vs. 0.51 (0.41-0.76), p = 0.005). After a steep decline, arterial ETPAUC tended to increase (p = 0.06), opposite to anti-Xa, while postfilter ETPAUC increased (p = 0.001). Median circuit life was 24.5 h (IQR 12-37 h). Patients with 'short circuit life' had longer baseline prothrombin time (PTT), activated thromboplastin time (aPTT), lower ETP, higher thrombin-antithrombin complexes (TAT) and higher SOFA scores; during CVVH, anti-Xa, and platelets were lower; PTT, aPTT, TAT and D-dimers were longer/higher and ETP was slower and depressed. CONCLUSIONS: We found no accumulation and no removal of LMWH activity during CVVH. However, we found that early circuit clotting was associated with more severe organ failure, prior systemic thrombin generation with consumptive coagulopathy, heparin resistance and elevated extracorporeal thrombin generation. ETP integrates these complex effects on the capacity to form thrombin. TRIAL REGISTRATION: Clinicaltrials.gov ID NCT00965328.

Elevated procoagulant microparticles expressing endothelial and platelet markers in essential thrombocythemia.

BACKGROUND: Most cell types, including blood--and vascular cells, produce microparticles upon activation. Since cellular microparticles are known to be elevated in thromboembolic diseases, we hypothesized a role for microparticles in the pathogenesis of thrombosis in essential thrombocythemia. DESIGN AND METHODS: In plasma samples from 21 patients with essential thrombocythemia and ten healthy subjects, the levels and the cellular origin of microparticles were determined by flowcytometric analysis, while the microparticle-associated procoagulant activity was measured using a thrombin generation assay. RESULTS: Patients with essential thrombocythemia had significantly higher numbers of circulating annexin V-positive microparticles than controls (median 4500 vs. 2500x10(6) events/L; p=0.039), including significantly higher numbers of microparticles positive for the platelet marker CD61 (p=0.043), the endothelial markers CD62E (p=0.009) and CD144 (p=0.021), and for tissue factor (p=0.036). CD62E was co-expressed with the platelet marker CD41 on microparticles, suggesting a bilineage origin of such microparticles, which were observed only in patients with risk factors for thrombosis. Patients with essential thrombocythemia had higher plasma levels of mature von Willebrand factor (p=0.045) but similar propeptide levels compared to controls. In thrombin generation analyses, microparticle-rich plasma from patients with essential thrombocythemia had a shorter lag time (p=0.001) and higher peak height (p=0.038) than plasma from controls. Peak height correlated significantly with the total number of microparticles (R=0.634, p<0.001). CONCLUSIONS: Patients with essential thrombocythemia had higher number of circulating microparticles with platelet and endothelial markers, suggesting ongoing platelet and endothelial activation. This was confirmed by an increased level of mature von Willebrand factor, an abnormal mature von Willebrand factor/propeptide ratio, and a hypercoagulable state reflected in thrombin generation. These findings suggest a role for microparticles in thrombosis in essential thrombocythemia.

Circulating white blood cells and platelets amplify oxidative stress in heart failure.

BACKGROUND: Mitochondria of circulating white blood cells (WBC) and platelets sense oxidative stress during capillary passage and react by producing reactive oxygen species (ROS). Although evidence indicates that congestive heart failure (CHF) is associated with oxidative stress, the role of WBC and platelets as mediators in CHF has not been investigated. METHODS: Patients with CHF (n = 15) and healthy volunteers (n = 9) were enrolled between 2006 and 2007 into this observational study. Arterial and venous blood samples from participants were incubated with probes to detect cytosolic and mitochondrial ROS. Fluorescence-activated cell sorting was used to measure the degree of fluorescence in WBC and platelets. RESULTS: Patients with CHF had a higher proportion of ROS-positive arterial WBC and platelets than did controls (67% +/- 47% versus 16% +/- 9%; P <0.005), as well as venous WBC and platelets (77% +/- 43% versus 38% +/- 13%; P <0.01). In the control group, the proportion of cytosolic ROS-positive arterial WBC and platelets was lower than that for ROS-positive venous WBC and platelets (16% +/- 9% versus 38% +/- 13%; P <0.005). CHF patients had a higher proportion of mitochondrial ROS-positive arterial and venous WBC and platelets than did controls. CONCLUSION: In CHF, the proportion of WBC and platelets that are ROS-positive is raised, possibly because cytosolic ROS-positive WBC and platelets are normally cleared in the lungs; this function is deficient in CHF while mitochondrial ROS production is increased. The raised numbers of circulating ROS-positive WBC and platelets amplify oxidative stress in CHF.