Zebrafish: a tool to study hemostasis and thrombosis

PURPOSE OF REVIEW: In the past eight years our laboratory has developed the zebrafish model to study hemostasis and thrombosis. The purpose of this review is to explore current developments involving the zebrafish model in the study of hemostasis and thrombosis because the time is now ripe to apply this model to identify novel players that participate in hemostasis and thrombosis. RECENT FINDINGS: In the past twelve months, three papers appeared in the hemostasis and thrombosis area using the zebrafish model. The first one is a review article that summarizes establishment of the zebrafish model to study hemostasis and thrombosis. The second study is a methodological paper describing assays for measuring hemostasis and thrombosis by inducing vascular occlusion in zebrafish larvae. The third paper describes a knockdown of prothrombin in zebrafish, which recapitulates knockout studies in mouse, and marks the beginning of studies in the hemostasis and thrombosis area by this new knockdown technology. In addition to the above papers, there is one abstract that describes kinetics of thrombocyte and thrombocyte-microparticle recruitment in laser-induced arterial thrombus formation in zebrafish. SUMMARY: With the above advances, the zebrafish model has now matured to the point that it can address more important questions in the hemostasis and thrombosis area using genetic approaches. This review therefore summarizes the issues described in the above papers along with thoughts about future progress of the zebrafish model as a tool to study hemostasis and thrombosis.     

Identifying hypocoagulable states with a modified global assay of overall haemostasis potential in plasma.

To test the sensitivity of the global assay of overall haemostasis potential (OHP) in detecting hypocoagulation, the OHP was assayed in plasma containing exogenous thrombin (0.04 IU/ml), tissue-plasminogen activator (330 ng/ml), Ca and a platelet reagent. Commercial plasmas with factor II, V, VIII, IX, X, XI, XII or VII deficiency were mixed with normal plasma in different proportions to imitate different severities. Samples from patients with haemophilia and factor XII deficiency were also examined. No clot was found in the absence of factor II/factor X, indicating that the tiny dose of thrombin worked solely as a trigger for the intrinsic pathway activation. Changed levels of the investigated coagulants, apart from factor XII, influenced the outcome. OHPs were decreased in patients with haemophilia but were unchanged or even increased in those with factor XII deficiency. This modified OHP method may therefore be useful for estimating the bleeding tendency in haemophilic patients and to find suitable doses and intervals for prophylactic treatment. It may also be of use in investigations of the effect of antifibrinolytic drugs as well as for identifying a thrombotic tendency in patients with factor XII deficiency. For detection of other coagulation factor deficiencies, our investigations with the commercial plasmas suggest that the OHP assay is also valuable, especially when the intrinsic pathway of the coagulation cascade is impaired.     

Clinical hemostasis practice: the major impact of laboratory automation

This article has reviewed basic disease states of hemostasis and thrombosis with an emphasis on laboratory technologic advances comparing manual and semiautomated techniques with new fully automated technology that is now available for both aiding in a diagnosis and monitoring efficacy of therapy. As these new automated methods become generally available, more and more information will be accumulated to determine exactly how precise many of these modalities will be for diagnosis and monitoring. Many of these newer modalities have already gained general acceptance as highly sophisticated tools that certainly belong in any clinical laboratory having to deal with patients with thrombohemorrhagic disorders.     

The PFA-100: a potential rapid screening tool for the assessment of platelet dysfunction

The PFA-100 is a device that simulates high shear dependent platelet function in vitro and thus is particularly useful for screening for von Willebrand's disease (VWD). The aim of this study was to assess the overall potential of the PFA-100 as a primary clinical screening tool using the wide spectrum of clinical samples assessed for platelet function within our institution. The PFA-100 test was performed using both collagen/ADP (CADP) and collagen/epinephrine (CEPI) cartridges on samples from 337 patients with a wide variety of haemostatic defects. One hundred and eighty-two patients were defined as having normal platelet function based on classical laboratory tests and von Willebrand factor levels. The overall clinical sensitivity of the PFA-100 for platelet abnormalities (including VWD) was 81% for CADP and 86% for CEPI. The overall specificity was found to be 82% for CADP and 80% for CEPI. When utilizing both cartridges in combination (with both results either higher or lower than the upper cutoff of the normal ranges), the overall false positive and false negative rates were 12% and 6%, respectively. The PFA-100 proved to be sensitive in detecting classical defects by giving prolonged closure times in samples from patients with major platelet function defects (e.g. von Willebrand's disease, Glanzmann's thrombasthenia and Bernard Soulier syndrome). However, there were a small number of false negative results (6%) obtained with various milder platelet defects (e.g. Hermansky Pudlak syndrome, storage pool and release defects, type I VWD and macrothrombocytopenia). The PFA-100 test provides a useful rapid screening tool and should increase the efficiency and reduce the cost of the routine diagnosis of platelet dysfunction.     

The laboratory as a tool to qualify tuberculosis diagnosis

OBJECTIVES: To evaluate the performance of laboratory diagnosis of tuberculosis, clinical samples underwent culture, species identification and drug susceptibility testing (DST). METHODS: A total of 554 samples from 269 patients were tested for smear microscopy using Kinyoun stain. Culture was performed in Ogawa-Kudoh medium and species identification was performed using the IS6110 amplified region. DST for rifampicin, isoniazid (INH) and streptomycin were carried out using the Resazurin assay. RESULTS: Cultures augmented the number of cases diagnosed by 22.1%, IS6110 amplification identified all Mycobacterium tuberculosis strains thus isolated and DST detected three strains resistant to INH and one multidrug-resistant strain. CONCLUSION: Simultaneous use of different techniques enhanced culture yield, species identification and detection of drug resistance even in a laboratory with limited facilities.     

血栓与止血检验进展

近年,随着基础理论和实验技术的发展,血栓与止血检验及其临床应用也有很大的进展,本文就此作一简述.     

Cell microencapsulation: a potential tool for the treatment of neuronopathic lysosomal storage diseases

Lysosomal storage disorders (LSD) are monogenic diseases caused by the deficiency of different lysosomal enzymes that degrade complex substrates such as glycosaminoglycans, sphingolipids, and others. As a consequence there is multisystemic storage of these substrates. Most treatments for these disorders are based in the fact that most of these enzymes are soluble and can be internalized by adjacent cells via mannose-6-phosphate receptor. In that sense, these disorders are good candidates to be treated by somatic gene therapy based on cell microencapsulation. Here, we review the existing data about this approach focused on the LSD treatments, the advantages and limitations faced by these studies.     

The single cell gel electrophoresis: a potential tool for DNA analysis of the patients with hematological malignancies

Single cell gel electrophoresis (SCGE) was used to evaluate the level of DNA damage in peripheral blood (PB), bone marrow (BM), and lymphatic node (LN) cells of patients with acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), chronic lymphocytic leukemia (CLL), chronic myeloid leukemia (CML) and non-Hodgkin's lymphoma (NHL). The level of DNA damage was compared with the level of basal DNA damage in control group, represented by healthy donors. Statistically significant increase of basal DNA damage was found in leukemia/lymphoma cells of patients suffered from AML, CML, ALL of T-cell subtype (T-ALL), and NHL, however, no difference in basal DNA damage was found in patients with ALL of early B-cell subtype (B-ALL) and CLL in comparison to control group. The mean basal DNA damage increased in the order CLL相似文献   

Wolbachia as a potential tool for suppressing filarial transmission

There is currently a great deal of interest in Wolbachia because of their wide distribution in arthropods and filarial nematodes and their striking effects on the biology of their hosts, including a possible role in speciation. They manipulate the reproduction of arthropod hosts through various effects on their hosts' biology, particularly cytoplasmic incompatibility (CI), to increase the proportion of infected individuals in the population, often to the point of fixation. This ability of Wolbachia to sweep through host populations indicates several potential applications of Wolbachia in the control of mosquito-borne disease. One uses Wolbachia-induced CI as a form of sterile-insect technique, to suppress mosquito populations. Another envisages the application of CI for population replacement, with the intention of preventing the transmission of human pathogens, by substituting desirable genotypes, including those carried in transgenes. A third possibility is to use Wolbachia to reduce the survival of mosquito populations and thereby reduce their ability to transmit the infection. This article provides an overview of the biological effects of Wolbachia on arthropod hosts, with discussion of the possible future exploitation of these effects in the control of filariasis.     

The PFA‐100®: a potential rapid screening tool for the assessment of platelet dysfunction

The PFA‐100^® is a device that simulates high shear dependent platelet function in vitro and thus is particularly useful for screening for von Willebrand's disease (VWD). The aim of this study was to assess the overall potential of the PFA‐100^® as a primary clinical screening tool using the wide spectrum of clinical samples assessed for platelet function within our institution. The PFA‐100^® test was performed using both collagen/ADP (CADP) and collagen/epinephrine (CEPI) cartridges on samples from 337 patients with a wide variety of haemostatic defects. One hundred and eighty‐two patients were defined as having normal platelet function based on classical laboratory tests and von Willebrand factor levels. The overall clinical sensitivity of the PFA‐100^® for platelet abnormalities (including VWD) was 81% for CADP and 86% for CEPI. The overall specificity was found to be 82% for CADP and 80% for CEPI. When utilizing both cartridges in combination (with both results either higher or lower than the upper cutoff of the normal ranges), the overall false positive and false negative rates were 12% and 6%, respectively. The PFA‐100^® proved to be sensitive in detecting classical defects by giving prolonged closure times in samples from patients with major platelet function defects (e.g. von Willebrand's disease, Glanzmann's thrombasthenia and Bernard Soulier syndrome). However, there were a small number of false negative results (6%) obtained with various milder platelet defects (e.g. Hermansky Pudlak syndrome, storage pool and release defects, type I VWD and macrothrombocytopenia). The PFA‐100^® test provides a useful rapid screening tool and should increase the efficiency and reduce the cost of the routine diagnosis of platelet dysfunction.     

The VA relationship after differential atrial overdrive pacing: a novel tool for the diagnosis of atrial tachycardia in the electrophysiologic laboratory

Introduction: Despite recent advances in clinical electrophysiology, diagnosis of atrial tachycardia (AT) originating near Koch's triangle remains challenging. We sought a novel technique for rapid and accurate diagnosis of AT in the electrophysiologic laboratory.
Methods: Sixty-two supraventricular tachycardias including 18 ATs (10 ATs arising from near Koch's triangle), 32 atrioventricular nodal reentrant tachycardias (AVNRTs), and 12 orthodromic reciprocating tachycardias (ORTs) were studied. Overdrive pacing during the tachycardia from different atrial sites was performed, and the maximal difference in the postpacing VA intervals (last captured ventricular electrogram to the earliest atrial electrogram of the initial beat after pacing) among the different pacing sites was calculated (delta-VA interval).
Results: The delta-VA intervals were >14 ms in all AT patients and <14 ms in all AVNRT/ORT patients, and thus, the delta-VA interval was diagnostic for AT with the sensitivity, specificity, and positive and negative predictive values all being 100%. When the diagnostic value of the delta-VA interval and conventional maneuvers were compared for differentiating AT from atypical AVNRT, both a delta-VA interval >14 ms and "atrial-atrial-ventricular" response after overdrive ventricular pacing during the tachycardia were diagnostic. However, the "atrial-atrial-ventricular" response criterion was available in only 52% of the patients because of poor ventriculoatrial conduction.
Conclusions: The delta-VA interval was useful for diagnosing AT irrespective of patient conditions such as ventriculoatrial conduction.     

Current laboratory practices in the diagnosis and management of haemophilia: a global assessment

Haemophilia management is complicated by the extreme variability in laboratory practices. Lack of consistency or comparability in testing makes it difficult to establish diagnostic criteria or disease severity, and complicates response assessment. A global survey was conducted to document current practices. A 35‐min survey was completed by 30 laboratory scientists in each of seven countries (France, Germany, Italy, Japan, Spain, UK, USA; 210 in total); results were weighted by average country testing volume in haemophilia. Eighty‐three per cent of participants reported participation in a Quality Assurance scheme. Ninety per cent reported using clotting tests in haemophilia A and 88% in haemophilia B (55% and 53% frequent use respectively). Sixty‐eight per cent reported chromogenic assays were used in haemophilia A, with only 23% reporting frequent use, compared to only 11% reporting any use in haemophilia B. Twenty‐nine separate activated partial thromboplastin time (aPTT) reagents were reported for haemophilia A and 27 aPTT reagents were reported for haemophilia B, with one‐quarter or less obtaining reagents or kits from any single manufacturer. Fifty‐four per cent run a calibration curve with every factor VIII (FVIII) assay. The mean number of plasma dilutions varied from 2 to 4 for FVIII assays and from 1 to 3 for FIX assays. Results indicate very low consistency in materials and practices used to test for factor activity in haemophilia. A number of responses suggest that some laboratory scientists' understanding of best practices or guidelines in haemophilia could be improved. More education and broader understanding is recommended regarding assay types, assay components, test material and instrument features and capabilities.     

The global potential for increased storage of carbon on land

Constraining the climate crisis requires urgent action to reduce anthropogenic emissions while simultaneously removing carbon dioxide from the atmosphere. Improved information about the maximum magnitude and spatial distribution of opportunities for additional land-based removals of CO₂ is needed to guide on-the-ground decision-making about where to implement climate change mitigation strategies. Here, we present a globally consistent spatial dataset (approximately 500-m resolution) of current, potential, and unrealized potential carbon storage in woody plant biomass and soil organic matter. We also provide a framework for prioritizing actions related to the restoration, management, and maintenance of woody carbon stocks and associated soils. By comparing current to potential carbon storage, while excluding areas critical to food production and human habitation, we find 287 petagrams (PgC) of unrealized potential storage opportunity, of which 78% (224 PgC) is in biomass and 22% (63 PgC) is in soil. Improved management of existing forests may offer nearly three-fourths (206 PgC) of the total unrealized potential, with the majority (71%) concentrated in tropical ecosystems. However, climate change is a source of considerable uncertainty. While additional research is needed to understand the impact of natural disturbances and biophysical feedbacks, we project that the potential for additional carbon storage in woody biomass will increase (+17%) by 2050 despite projected decreases (−12%) in the tropics. Our results establish an absolute reference point and conceptual framework for national and jurisdictional prioritization of locations and actions to increase land-based carbon storage.

Emissions of carbon to the atmosphere must remain below ∼250 petagrams (PgC) (918 PgCO₂) from 2021 onward to achieve the Paris Agreement’s goal of limiting global temperature rise to well below 2 °C (1–3). At present rates, that amount of carbon will be emitted by 2045. It follows that even necessary and drastic cuts in emissions (i.e., a rapid transition from fossil fuels to renewable energy sources) must be accompanied by carbon dioxide removal (CDR) or negative emissions strategies (4). Promising options for large-scale CDR include improved land stewardship (5), commonly referred to as natural climate solutions (NCS) (6–8). In particular, increasing carbon storage in woody biomass (e.g., forest ecosystems) is widely recognized as having high climate mitigation potential while also affording an array of environmental and socio-economic cobenefits (6–9). While a growing body of research has estimated the near-term potential for land-based climate mitigation (6, 8, 10), these studies emphasize the climate benefit over short, 10- to 30-y planning horizons. They do not include estimates of the upper limit for additional land-based carbon storage or its spatial distribution. This information is essential for landscape-level planning and targeted implementation of NCS, given that the potential for additional carbon storage is necessarily defined by both the rate at which carbon can be sequestered and the magnitude of the available reservoir. Therefore, we provide 500-m-resolution global maps to quantify the maximum potential for additional carbon storage in ecosystems dominated by woody vegetation (i.e., trees and shrubs), under baseline (1960 to 1990) and future (representative concentration pathway scenario 8.5 [RCP8.5]) climate conditions. This information can be used to help direct NCS toward areas with the greatest maximum opportunity, inform when NCS will saturate, and identify the types of NCS actions that are best suited to a given location.One approach to estimating maximum additional carbon storage—or the difference between current and potential carbon, which we term “unrealized potential” carbon—is a bookkeeping approach that tracks carbon fluxes through time. Under this approach, net land-based emissions since 1850 are estimated to have been 108 to 188 PgC, including both biomass (above and below ground) and soil organic matter (13–17). Estimates that account for preindustrial (i.e., pre-1850) land use are more varied and increase post-1850 estimates by as much as 325 to 357 PgC (18) or as little as 48 to 153 PgC (11–13, 15). This high uncertainty limits the practical utility of this approach.Other investigators have sought instead to quantify unrealized potential by comparing estimates of current and potential land carbon storage. Sanderman et al. (6), considering only soil organic carbon (SOC), estimated net losses in the upper 2 m of soil from agricultural land use to be 116 PgC since 10,000 BC. Erb et al. (19), focusing on changes in vegetation biomass, found losses in carbon due to human land use to be significantly larger (447 PgC) than the studies cited above that consider only the postindustrial period, but generally consistent with some of those that account for preindustrial human disturbance (18). Bastin et al. (20), in a study focused on the restoration of global tree cover, identified an additional reservoir of 206 PgC when considering all carbon pools (aboveground and belowground biomass, soil, litter, and dead wood) after excluding cropland and urban areas.However, all of these global analyses fall short in delivering the robust spatially explicit information needed for targeted planning and implementation of landscape-level NCS. While the global dataset produced by Bastin et al. (20) has a reasonably high spatial resolution (30 arc seconds; approximately 900 m) and considers all land carbon pools, the product is limited to the storage potential afforded by the expansion of tree cover. Moreover, the result is subject to the uncertainty inherent in indirect estimates of carbon stock from area-based metrics of tree/forest cover (21). In comparison, the data product created by Erb et al. (19), which is based on several disparate yet direct estimates of terrestrial carbon storage, is limited by its treatment of only the biomass carbon pool and coarse spatial resolution (5 arc min; approximately 9.3 km). The authors themselves remark that “the uncertainty range could be narrowed if a single robust, validated method would be applied continuously in the stocktaking efforts” (19).Here, we apply a consistent suite of methods to generate spatially explicit global estimates of current (ca. 2016) and climate-constrained potential land carbon storage in aboveground woody biomass (AGB), belowground woody biomass (BGB), and SOC pools at a spatial resolution of approximately 500 m. The difference between current and potential land carbon storage represents the unrealized potential for additional carbon accumulation in global woody biomass and soils. We then disaggregate this global estimate of unrealized potential carbon storage using a conceptual framework we term the NCS opportunity space: seven discrete, internally consistent, and spatially explicit categories of broad NCS action (Fig. 1). Categories are defined quantitatively in terms of woody carbon density, thereby avoiding the uncertainty associated with derivative approximations of potential carbon storage based on forest area or canopy cover. After applying safeguards to lands currently utilized for food production, human habitation (e.g., urban areas), and sensitive biodiversity (nonwoody grasslands), we demonstrate the utility of the opportunity space framework for landscape-level NCS planning by analyzing the global, regional, and national potential for additional land carbon storage attributable to restoration (e.g., reforestation), management (e.g., improved natural forest stewardship), and maintenance (i.e., the sequestration benefit accrued through avoided forest conversion) of woody carbon stocks and associated soils. Finally, we evaluate the uncertainty that climate change poses to the magnitude and spatial distribution of the unrealized potential for additional carbon storage through 2050.Open in a separate windowFig. 1.The NCS opportunity space, consisting of seven categories defined by the ratio of current (x axis) to potential (y axis) carbon storage as well as carbon-based thresholds delineating NCS-relevant systems. Categories include: Restore/High suitability for forestry-based NCS (R/H; red), Maintain and manage/High suitability for forestry-based NCS (MM/H; dark green), Maintain/High suitability for forestry-based NCS (M/H; dark blue), Restore/Low suitability for forestry-based NCS (R/L; orange), Maintain and manage/Low suitability for forestry-based NCS (MM/L; light green), Maintain/Low suitability for forestry-based NCS (M/L; light blue), and Nonwoody (yellow). † denotes associated grassland/savanna biodiversity considerations.     

New laboratory perspectives for evaluation of vivax malaria infected patients: a useful tool for infection monitoring

In recent years, the number of cases with severe Plasmodium vivax malaria has shown an increasing trend. It is, therefore, important to identify routine laboratory markers that best characterize the acute disease phase and can serve as a tool for clinical follow-up of patients. In a cohort study, we followed 87 patients with acute P. vivax monoinfection acquired in an endemic region of the Brazilian Amazon. Forty-two different biochemical and hematological parameters frequently tested in clinical routine were evaluated at the acute phase and the convalescent phase. A total of 42 laboratory tests were performed: biochemical parameters measured were serum lipids levels, aminotransferases, bilirubin, amylase, glucose, urea, creatinine, albumin, globulin, uric acid, C-reactive protein, and alpha-1-acid glycoprotein. Hematological parameters included total and differential white blood cell and platelet counts, hemoglobin concentration, mean platelet volume, platelet width distribution, and plateletcrit. Our results show that several biochemical and hematological parameters were associated with acute phase P. vivax malaria and these parameters reverted to normal values in the convalescent phase. The use of these parameters during diagnosis and follow-up of the infection is a useful clinical tool to evaluate the clinical course and therapeutic response of patients with uncomplicated vivax malaria.     

Subjective global assessment: a simple and reliable screening tool for malnutrition among Indians.

BACKGROUND AND AIMS: Subjective global assessment (SGA) is a simple and reliable malnutrition-screening tool. The SGA has not been evaluated in India or in populations where chronic energy deficiency (CED) is rampant. We evaluated the value of preoperative nutrition, determined using the SGA, in predicting postoperative adverse outcomes in cancer patients. METHODS: Two hundred and ninety-four cancer patients undergoing elective surgery were screened for malnutrition using a modified version of the SGA, and 266 patients (aged 14-73 years [median 50]; 165 male) were eligible. All patients were followed up till discharge, and number of days on antibiotics, length of postoperative stay, occurrence of major adverse events, and death within 30 days were recorded. The association of preoperative SGA scores (A, B, or C) and BMI groups (< 18.5, 18.5-20 or> 20 Kg/m2) and four outcome variables were tested for statistical significance. RESULTS: The cancer sites included head and neck region in 112, gastrointestinal tract in 53, thoracic organs in 28, and other sites in 73 patients. The SGA scores were A in 152, B in 98, and C in 16 patients. The BMI was < 18.5 in 110 (41.8%) patients. The length of postoperative stay and the number of antibiotic days revealed a significant trend from SGA-A to SGA-C (p=0.000). Pre-defined adverse events occurred in 7.9%, 17.3% and 25% of SGA groups A, B, and C, respectively (p=0.025). The risk for adverse events was significantly higher in SGA-C group (OR 5.27, 95% CI 1.35-20.51, p< 0.016) compared to SGA-A group. Three patients in SGA-B group and one in SGA-C group died within 30 days (p=0.04). No significant association was detected between the three BMI groups and duration of antibiotic use, length of postoperative stay, adverse events or mortality. CONCLUSION: SGA is a simple and inexpensive way to identify clinically relevant malnutrition in Indian patients undergoing cancer surgery. Low BMI was not associated with postoperative adverse outcomes, and its use for nutritional screening is likely to overestimate severe malnutrition in Indian patients.