Novel Approach to Improve the Identification of the Bleeding Phenotype in Noonan Syndrome and Related RASopathies.

OBJECTIVES: To characterize the bleeding phenotype in Noonan syndrome (NS), to test the utility of following national guidelines in detecting this phenotype, to evaluate thromboelastography (TEG) as a diagnostic tool, and to evaluate the cohort for genotype-phenotype correlations. STUDY DESIGN: Participants with a clinical diagnosis of NS or related RASopathies were enrolled in a cohort study. Study procedures included clinical bleeding assessment, coagulation testing per guidelines, and hematology consultation. TEG was completed in a subset, and genetic testing was conducted for those without a molecular diagnosis. International Society of Haemostasis and Thrombosis Bleeding Assessment Tool scores were calculated with hematology consultation. Bleeding phenotype was defined as abnormal bleeding score. RESULTS: Twenty participants were enrolled; 12 completed clinical and laboratory evaluation, and five of whom met the definition for bleeding phenotype. Four of the five participants with a bleeding phenotype had platelet aggregation defects and at least one additional coagulation defect. TEG was performed in nine participants, four with bleeding phenotype and five without, and results were normal in all cases. No genotype-phenotype correlation was found. CONCLUSION: Five of the 20 participants had a bleeding phenotype identified. Based on available data, we do not recommend incorporating TEG into clinical practice for patients with NS. Platelet aggregation defects were the most common abnormalities, which would not be detected on tier 1 testing of current guidelines; therefore, we propose a new algorithm.

A Novel Hypomorphic Apex1 Mouse Model Implicates Apurinic/Apyrimidinic Endonuclease 1 in Oxidative DNA Damage Repair in Gastric Epithelial Cells.

Aims: Though best known for its role in oxidative DNA damage repair, apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that regulates multiple host responses during oxidative stress, including the reductive activation of transcription factors. As knockout of the APE1-encoding gene, Apex1, is embryonically lethal, we sought to create a viable model with generalized inhibition of APE1 expression. Results: A hypomorphic (HM) mouse with decreased APE1 expression throughout the body was generated using a construct containing a neomycin resistance (NeoR) cassette knocked into the Apex1 site. Offspring were assessed for APE1 expression, breeding efficiency, and morphology with a focused examination of DNA damage in the stomach. Heterozygotic breeding pairs yielded 50% fewer HM mice than predicted by Mendelian genetics. APE1 expression was reduced up to 90% in the lungs, heart, stomach, and spleen. The HM offspring were typically smaller, and most had a malformed tail. Oxidative DNA damage was increased spontaneously in the stomachs of HM mice. Further, all changes were reversed when the NeoR cassette was removed. Primary gastric epithelial cells from HM mice differentiated more quickly and had more evidence of oxidative DNA damage after stimulation with Helicobacter pylori or a chemical carcinogen than control lines from wildtype mice. Innovation: A HM mouse with decreased APE1 expression throughout the body was generated and extensively characterized. Conclusion: The results suggest that HM mice enable studies of APE1's multiple functions throughout the body. The detailed characterization of the stomach showed that gastric epithelial cells from HM were more susceptible to DNA damage. Antioxid. Redox Signal. 38, 183-197.

Familiarity with, perceptions of and attitudes toward butterflies of urban park users in megacities across East and Southeast Asia.

Perceptions of, and attitudes toward, wildlife are influenced by exposure to, and direct experiences with, nature. Butterflies are a conspicuous and ubiquitous component of urban nature across megacities that are highly urbanized with little opportunity for human-nature interactions. We evaluated public familiarity with, perceptions of and attitudes toward butterflies across nine megacities in East and Southeast Asia through face-to-face interviews with 1774 urban park users. A total of 79% of respondents had seen butterflies in their cities mostly in urban parks, indicating widespread familiarity with butterflies. Those who had seen butterflies also had higher perceptions of butterflies, whereas greater than 50% of respondents had positive attitudes toward butterflies. Frequent visits to natural places in urban neighbourhoods was associated with (i) sightings of caterpillars, indicating increased familiarity with urban wildlife, and (ii) increased connectedness to nature. We found two significant positive relationships: (i) between connectedness to nature and attitudes toward butterflies and (ii) between connectedness to nature and perceptions of butterflies, firmly linking parks users' thoughts and feelings about butterflies with their view of nature. This suggests that butterflies in urban parks can play a key role in building connectedness to nature and consequently pro-environmental behaviours and support for wildlife conservation among urban residents.

Clinical Utility of Combined Whole-cell Antigen and Recombinant Hemolysis Co-regulated Protein 1-Enzyme-linked Immunosorbent Assays Reveals Underdiagnosed Cases of Melioidosis in Vietnam.

Melioidosis is a fatal infectious disease in the tropics and subtropics. Currently, bacterial culture is the gold standard for diagnosis of the disease, but its sensitivity is relatively low. In this study, we evaluated four ELISAs using sera collected from culture-confirmed cases of melioidosis (n = 63), cases with other bacterial infections (n = 62), and healthy donors (n = 60). Antigens used for ELISAs were the whole-cell (WC) antigens and recombinant proteins of hemolysis co-regulated protein 1 (Hcp1), GroEL1, and alkyl hydroperoxide reductase subunit C (AhpC). Using the cutoff values for optical density at 490 nm defined at a specificity of > 95%, the sensitivity of the WC, Hcp1, GroEL1, and AhpC ELISAs was 93.7%, 87.3%, 61.9%, and 57.1%, respectively. The combined WC/Hcp1 ELISA showed the greatest sensitivity and specificity of 98.4% and 95.1%, respectively. Of 511 and 500 sera collected from clinically suspected febrile patients admitted to the General Hospital of Ha Tinh Province and the Hue Central Hospital, respectively, combined WC/Hcp1 ELISAs showed 52 (10.2%) and 41 (8.2%) patients positive for melioidosis, respectively. The assay detected 14 of 14 (100%) and 21 of 23 (91.3%) culture-confirmed cases of melioidosis at Ha Tinh and Hue, respectively. A follow-up study of 38 patients positive for melioidosis by combined WC/Hcp1 ELISAs but negative for Burkholderia pseudomallei by culture method or not assigned to examine for bacterial culture resulted in 2 (5.3%) culture-reconfirmed patients with melioidosis, 9 (23.7%) deaths, 17 (44.7%) unhealthy patients, and 10 (26.3%) healthy persons. Combined WC/Hcp1 ELISA was a reliable serological method to detect underdiagnosed cases of melioidosis. Further investigations are needed to estimate the true sensitivity and specificity of the assay and the true number of cases of melioidosis.

Coagulation factor protein abundance in the pre-septic state predicts coagulopathic activities that arise during late-stage murine sepsis.

BACKGROUND: Although sepsis accounts for 1 in 5 deaths globally, few molecular therapies exist for this condition. The development of effective biomarkers and treatments for sepsis requires a more complete understanding of host responses and pathogenic mechanisms at early stages of disease to minimize host-driven pathology. METHODS: An alternative to the current symptom-based approach used to diagnose sepsis is a precise assessment of blood proteomic changes during the onset and progression of Salmonella Typhimurium (ST) murine sepsis. FINDINGS: A distinct pattern of coagulation factor protein abundance was identified in the pre-septic state- prior to overt disease symptoms or bacteremia- that was predictive of the dysregulation of fibrinolytic and anti-coagulant activities and resultant consumptive coagulopathy during ST murine sepsis. Moreover, the changes in protein abundance observed generally have the same directionality (increased or decreased abundance) reported for human sepsis. Significant overlap of ST coagulopathic activities was observed in Gram-negative Escherichia coli- but not in Gram-positive staphylococcal or pneumococcal murine sepsis models. Treatment with matrix metalloprotease inhibitors prevented aberrant inflammatory and coagulopathic activities post-ST infection and increased survival. Antibiotic treatment regimens initiated after specific changes arise in the plasma proteome post-ST infection were predictive of an increase in disease relapse and death after cessation of antibiotic treatment. INTERPRETATION: Altered blood proteomics provides a platform to develop rapid and easy-to-perform tests to predict sepsis for early intervention via biomarker incorporation into existing blood tests prompted by patient presentation with general malaise, and to stratify Gram-negative and Gram-positive infections for appropriate treatment. Antibiotics are less effective in microbial clearance when initiated after the onset of altered blood proteomics as evidenced by increased disease relapse and death after termination of antibiotic therapy. Treatment failure is potentially due to altered bacterial / host-responses and associated increased host-driven pathology, providing insight into why delays in antibiotic administration in human sepsis are associated with increased risk for death. Delayed treatment may thus require prolonged therapy for microbial clearance despite the prevailing notion of antibiotic de-escalation and shortened courses of antibiotics to improve drug stewardship. FUNDING: National Institutes of Health, U.S. Army.

Incidence of New Onset Atrial Fibrillation After Cardiovascular Surgery in Vietnam: Results From A Novel Screening Strategy.

OBJECTIVE: To examine the incidence of atrial fibrillation (AF) newly developed after cardiovascular surgery in Vietnam, its associated risk factors, and postoperative complications. We also sought to evaluate the feasibility of a novel screening strategy for post-operative AF (POAF) using the combination of two portable devices. METHODS: Single-centre, prospective cohort study at the Cardiovascular Centre, E Hospital, Hanoi, Vietnam. All patients aged≥18 years, undergoing cardiovascular surgery and in sinus rhythm preoperatively were eligible. The primary outcome was occurrence of new-onset POAF detected by hand-held single-lead electrocardiography (ECG) or a sphygmomanometer with AF-detection algorithm. Multivariate logistic regression was used to identify risk factors of developing post-operative AF. Feasibility was evaluated by compliance to the protocol and semi-structured interviews. RESULTS: 112 patients were enrolled between 2018-2019: mean age 52.9±12.2 years; 50.9% female;92.0% (n=103) valve surgery; 9.8% (n=11)coronary surgery. New-onset POAF developed in 49patients (43.8%) with median time to onset 1.27days (IQR 0.96 -2.00 days). Age≥65 years was the only significant risk factor for the development of POAF(OR 3.78, 95% CI 1.16-12.34).The median thromboembolism risk scores (CHA2DS2-VASc score) were comparable among patients with and without POAF (1.0 vs. 1.0, p=0.104). The occurrence of POAF was associated with higher rates of postoperative complications (24.5% vs. 3.2%, p<0.001). Both doctors and nurses found this screening strategy feasible to be implemented long-term with the main difficulties being the instructions on both devices were in English, and an increase in workload. CONCLUSIONS: In this single-centre study, new-onset POAF occurred in 43.8% of patients who underwent cardiovascular surgery. This novel POAF screening strategy was feasible in a low resource setting, and its implementation could be improved by providing continuous training and translation to local language.

Endothelial Heparan Sulfate Mediates Hepatic Neutrophil Trafficking and Injury during Staphylococcus aureus Sepsis.

Hepatic failure is an important risk factor for poor outcome in septic patients. Using a chemical tagging workflow and high-resolution mass spectrometry, we demonstrate that rapid proteome remodeling of the vascular surfaces precedes hepatic damage in a murine model of Staphylococcus aureus sepsis. These early changes include vascular deposition of neutrophil-derived proteins, shedding of vascular receptors, and altered levels of heparin/heparan sulfate-binding factors. Modification of endothelial heparan sulfate, a major component of the vascular glycocalyx, diminishes neutrophil trafficking to the liver and reduces hepatic coagulopathy and organ damage during the systemic inflammatory response to infection. Modifying endothelial heparan sulfate likewise reduces neutrophil trafficking in sterile hepatic injury, reflecting a more general role of heparan sulfate contribution to the modulation of leukocyte behavior during inflammation. IMPORTANCE Vascular glycocalyx remodeling is critical to sepsis pathology, but the glycocalyx components that contribute to this process remain poorly characterized. This article shows that during Staphylococcus aureus sepsis, the liver vascular glycocalyx undergoes dramatic changes in protein composition associated with neutrophilic activity and heparin/heparan sulfate binding, all before organ damage is detectable by standard circulating liver damage markers or histology. Targeted manipulation of endothelial heparan sulfate modulates S. aureus sepsis-induced hepatotoxicity by controlling the magnitude of neutrophilic infiltration into the liver in both nonsterile and sterile injury. These data identify an important vascular glycocalyx component that impacts hepatic failure during nonsterile and sterile injury.

First record of the Cyrtodactylus brevipalmatus group (Squamata: Gekkonidae) from Vietnam with description of a new species.

A new species of the genus Cyrtodactylus is described from Dien Bien Province, northwestern Vietnam based on morphological and molecular data. Cyrtodactylus ngati sp. nov. can be distinguished from remaining congeners by the following combination of characters: maximum SVL 69.3 mm; dorsal pattern consisting of six dark irregular transverse bands between limb insertions; inter-supranasals one; dorsal tubercles present on occiput, body, hind limbs and on first half of tail; 1722 irregular dorsal tubercle rows at midbody; lateral folds clearly defined, with interspersed tubercles; 3238 ventral scales between ventrolateral folds; 13 precloacal pores separated by a diastema of 5/5 poreless scales from a series of 7/7 femoral pores in enlarged femoral scales; precloacal and femoral pores absent in females; 13 postcloacal tubercles on each side; transversely enlarged median subcaudal scales absent. In the molecular analyses, the new species is shown to be the sister taxon to C. interdigitalis from Thailand. This is the 47th species of the genus Cyrtodactylus and the first member of the C. brevipalmatus species group recorded from Vietnam.

The Evaluation of Hematologic Screening and Perioperative Management in Patients with Noonan Syndrome: A Retrospective Chart Review.

OBJECTIVES: To assess the potential impact of using screening recommendations for bleeding disorders in patients with Noonan syndrome on perioperative bleeding complications. STUDY DESIGN: We performed a retrospective, single-site cohort study; patients were identified by query of the electronic medical record. All patients with a clinical diagnosis of Noonan syndrome over a 10-year period were included. Data on surgeries, hematologic evaluation, bleeding symptoms, and bleeding complications were extracted. Surgeries were graded as major or minor. RESULTS: We identified 101 patients with Noonan syndrome, 70 of whom required surgery for a total of 164 procedures. Nine patients (9/70; 12.8%) had bleeding complications, occurring in those without comprehensive testing or perioperative intervention and undergoing major or dental surgery. Based on these findings, the risk of a bleeding complication for patients with Noonan syndrome who did not have comprehensive testing or perioperative intervention was 6.2% (95% CI 2.3%-10.1%), indicating the number needed to treat or screen would be 16 to prevent 1 bleeding complication (95% CI 9.9-43.9). The majority of patients had either no or incomplete evaluation (59 of 101; 58.4%). CONCLUSIONS: With proper evaluation and management, the bleeding risk in patients with Noonan syndrome can be minimized. Efforts are needed to address the knowledge and implementation gap in this evaluation.

Two new and potentially highly threatened Megophrys Horned frogs (Amphibia: Megophryidae) from Indochina's highest mountains.

Megophrys are a group of morphologically conserved, primarily forest-dependent frogs known to harbour cryptic species diversity. In this study, we examined populations of small-sized Megophrys from mid- and high elevation locations in the Hoang Lien Range, northern Vietnam. On the basis of morphological, molecular and bioacoustic data, individuals of these populations differed from all species of Megophrys known from mainland Southeast Asia north of the Isthmus of Kra and from neighbouring provinces in China. Further, the newly collected specimens formed two distinct species-level groups. We herein describe two new species, Megophrys fansipanensis sp. nov. and Megophrys hoanglienensis sp. nov. Both new species are range restricted and likely to be highly threatened by habitat degradation. These discoveries highlight the importance of the Hoang Lien Range for Vietnam's amphibian diversity.

Excessive postextraction bleeding associated with Cordyceps sinensis: a case report and review of select traditional medicines used by Vietnamese people living in the United States.

Many older people who have emigrated from Vietnam to the United States continue to use the traditional medicines that they used in their country of origin. Clinicians trained in the West may not be familiar with these products. We reviewed 6 Asian traditional medicines that are popular among older Vietnamese people living in the United States. Each medicine has significant side effects that can lead to complications in patients undergoing surgery. Here, we present the case of a patient who used Cordyceps sinensis daily as a tonic and experienced prolonged bleeding after dental surgery.

A new species of Leptolalax (Anura: Megophryidae) from Son Tra Peninsula, central Vietnam.

Morphological, acoustic and molecular analyses result in the description of Leptolalax rowleyae sp. nov., a new species of frog in the Megophryidae, belonging to the L. applebyi Rowley Cao species group from central Vietnam. It differs from its congeners by a combination of the following morphological attributes: (1) adult SVL 23.4-25.4 mm in males and 27-27.8 mm in females; (2) presence of distinct dark/brown dorsolateral markings, including black spots on flanks; (3) pinkish milk-white to light brown chest and belly with numerous white speckles; (4) tympanum distinct; (5) absence of webbing or lateral dermal fringes on fingers and toes; (6) pectoral glands comparatively small (3.3-4.7% of SVL); (7) ventrolateral glands indistinct; and (8) iris bicolored with copper tint in upper half fading to golden in lower third of iris. The male advertisement call of the new species consists of 4-6 notes, lacking a distinct introductory note, with an average dominant frequency of 3.2-3.5 kHz. The description of the tadpole constitutes the first description of larval morphology for a member of the L. appleybi species group. Genetically, an uncorrected sequence divergence of 7.4% for 16S rRNA separates the new species from its two closest relatives, L. ardens and L. melicus in the L. applebyi species group.

A new species of Cyrtodactylus (Squamata: Gekkonidae) from Ninh Binh Province, Vietnam.

We describe a new species of the genus Cyrtodactylus on the basis of six specimens collected from the limestone forest of the Van Long Wetland Nature Reserve, Ninh Binh Province, Vietnam. Cyrtodactylus soni sp. nov. can be distinguished from its congeners by genetic distinction and morphological differences in number of femoral and precloacal pores, femoral scales, ventral scales, lamellae, subcaudals, and dorsal tubercle arrangement, as well as in size and color pattern. In the phylogenetic analyses, the new species is nested in a clade containing taxa from northwestern and northcentral Vietnam and northern Laos.

New provincial records of skinks (Squamata: Scincidae) from northwestern Vietnam.

We report six new records of skinks from northwestern Vietnam: Eutropis macularius, Scincelladevorator , S.monticola, S.ochracea, Sphenomorphuscryptotis and S.indicus. Our new findings increase the species number of skinks (Scincidae) to nine in Dien Bien Province and to 14 in Son La Province. We also provide additional natural history data of aforementioned species.

Small molecule antagonists of cell-surface heparan sulfate and heparin-protein interactions.

Surfen, bis-2-methyl-4-amino-quinolyl-6-carbamide, was previously reported as a small molecule antagonist of heparan sulfate (HS), a key cell-surface glycosaminoglycan found on all mammalian cells. To generate structure-activity relationships, a series of rationally designed surfen analogs was synthesized, where its dimeric structure, exocyclic amines, and urea linker region were modified to probe the role of each moiety in recognizing HS. An in vitro assay monitoring inhibition of fibroblast growth factor 2 binding to wild-type CHO cells was utilized to quantify interactions with cell surface HS. The dimeric molecular structure of surfen and its aminoquinoline ring systems was essential for its interaction with HS, and certain dimeric analogs displayed higher inhibitory potency than surfen and were also shown to block downstream FGF signaling in mouse embryonic fibroblast cells. These molecules were also able to antagonize other HS-protein interactions including the binding of soluble RAGE to HS. Importantly, selected molecules were shown to neutralize heparin and other heparinoids, including the synthetic pentasaccharide fondaparinux, in a factor Xa chromogenic assay and in vivo in mice. These results suggest that small molecule antagonists of heparan sulfate and heparin can be of therapeutic potential for the treatment of disorders involving glycosaminoglycan-protein interactions.

Inducing host protection in pneumococcal sepsis by preactivation of the Ashwell-Morell receptor.

The endocytic Ashwell-Morell receptor (AMR) of hepatocytes detects pathogen remodeling of host glycoproteins by neuraminidase in the bloodstream and mitigates the lethal coagulopathy of sepsis. We have investigated the mechanism of host protection by the AMR during the onset of sepsis and in response to the desialylation of blood glycoproteins by the NanA neuraminidase of Streptococcus pneumoniae. We find that the AMR selects among potential glycoprotein ligands unmasked by microbial neuraminidase activity in pneumococcal sepsis to eliminate from blood circulation host factors that contribute to coagulation and thrombosis. This protection is attributable in large part to the rapid induction of a moderate thrombocytopenia by the AMR. We further show that neuraminidase activity in the blood can be manipulated to induce the clearance of AMR ligands including platelets, thereby preactivating a protective response in pneumococcal sepsis that moderates the severity of disseminated intravascular coagulation and enables host survival.

A phenotype survey of 36 mutant mouse strains with gene-targeted defects in glycosyltransferases or glycan-binding proteins.

The consortium for functional glycomics (CFG) was a large research initiative providing networking and resources for investigators studying the role of glycans and glycan-binding proteins in health and disease. Starting in 2001, six scientific cores were established to generate data, materials and new technologies. By the end of funding in 2011, the mouse phenotype core (MPC) submitted data to a website from the phenotype screen of 36 mutant mouse strains deficient in a gene for either a glycan-binding protein (GBP) or glycosyltransferase (GT). Each mutant strain was allotted three months for analysis and screened by standard phenotype assays used in the fields of immunology, histology, hematology, coagulation, serum chemistry, metabolism and behavior. Twenty of the deficient mouse strains had been studied in other laboratories, and additional tests were performed on these strains to confirm previous observations and discover new data. The CFG constructed 16 new homozygous mutant mouse strains and completed the initial phenotype screen of the majority of these new mutant strains. In total, >300 phenotype changes were observed, but considering the over 100 assays performed on each strain, most of the phenotypes were unchanged. Phenotype differences include abnormal testis morphology in GlcNAcT9- and Siglec-H-deficient mice and lethality in Pomgnt1-deficient mice. The numerous altered phenotypes discovered, along with the consideration of the significant findings of normality, will provide a platform for future characterization to understand the important roles of glycans and GBPs in the mechanisms of health and disease.

Systemic mastocytosis in association with chronic lymphocytic leukemia and plasma cell myeloma.

Systemic mastocytosis with associated clonal haematological non-mast cell lineage disease (SM-AHNMD) is a heterogeneous group of mast cell disorders with different clinical, pathologic and underlying molecular characteristics. While myelomonocytic/myeloid neoplasia overwhelmingly predominates the AHNMD component, lymphoproliferative disorders rarely occur as an AHNMD component of SM-AHNMD. Here we report two cases of SM-AHNMD, in which the AHNMD component is chronic lymphocytic leukemia in one case, and concurrent chronic lymphocytic leukemia as well as plasma cell myeloma in another case. To the best of our knowledge, this is the first case report of SM-AHNMD with chronic lymphocytic leukemia and plasma cell dyscrasia simultaneously.

Physiological and glycomic characterization of N-acetylglucosaminyltransferase-IVa and -IVb double deficient mice.

N-Acetylglucosaminyltransferase-IV (GnT-IV) has two isoenzymes, GnT-IVa and GnT-IVb, which initiate the GlcNAcbeta1-4 branch synthesis on the Manalpha1-3 arm of the N-glycan core thereby increasing N-glycan branch complexity and conferring endogenous lectin binding epitopes. To elucidate the physiological significance of GnT-IV, we engineered and characterized GnT-IVb-deficient mice and further generated GnT-IVa/-IVb double deficient mice. In wild-type mice, GnT-IVa expression is restricted to gastrointestinal tissues, whereas GnT-IVb is broadly expressed among organs. GnT-IVb deficiency induced aberrant GnT-IVa expression corresponding to the GnT-IVb distribution pattern that might be attributed to increased Ets-1, which conceivably activates the Mgat4a promoter, and thereafter preserved apparent GnT-IV activity. The compensative GnT-IVa expression might contribute to amelioration of the GnT-IVb-deficient phenotype. GnT-IVb deficiency showed mild phenotypic alterations in hematopoietic cell populations and hemostasis. GnT-IVa/-IVb double deficiency completely abolished GnT-IV activity that resulted in the disappearance of the GlcNAcbeta1-4 branch on the Manalpha1-3 arm that was confirmed by MALDI-TOF MS and GC-MS linkage analyses. Comprehensive glycomic analyses revealed that the abundance of terminal moieties was preserved in GnT-IVa/-IVb double deficiency that was due to the elevated expression of glycosyltransferases regarding synthesis of terminal moieties. Thereby, this may maintain the expression of glycan ligands for endogenous lectins and prevent cellular dysfunctions. The fact that the phenotype of GnT-IVa/-IVb double deficiency largely overlapped that of GnT-IVa single deficiency can be attributed to the induced glycomic compensation. This is the first report that mammalian organs have highly organized glycomic compensation systems to preserve N-glycan branch complexity.

In vitro sonothrombolysis with duplex ultrasound: first results using a simplified model.

BACKGROUND: The main aim was to study the effects of ultrasound (US) alone, in combination with an US contrast agent (UCA), tissue plasminogen activator (tPA), or the combination of both upon blood clots. METHODS: In order to learn about sonothrombolysis with diagnostic duplex US, a simplified in vitro test model, using human whole blood clots in Petri dishes, was established. RESULTS: A total of 286 blood clots were analyzed. Improved sonothrombolysis due to insonation with diagnostic duplex US could be achieved, whether it was used alone or in combination with tPA. Although already described, a beneficial effect of UCA microbubbles on sonothrombolysis could not be confirmed due to the study design. CONCLUSION: Diagnostic duplex US improves thrombolysis significantly, even when it is used without tPA. To study the effect of UCA microbubbles on sonothrombolysis appropriately, any experimental design should provide continuous replenishment of microbubbles at the target site.