Microfluidic manufacture of rt-PA -loaded echogenic liposomes.

Echogenic liposomes (ELIP), loaded with recombinant tissue-type plasminogen activator (rt-PA) and microbubbles that act as cavitation nuclei, are under development for ultrasound-mediated thrombolysis. Conventional manufacturing techniques produce a polydisperse rt-PA-loaded ELIP population with only a small percentage of particles containing microbubbles. Further, a polydisperse population of rt-PA-loaded ELIP has a broadband frequency response with complex bubble dynamics when exposed to pulsed ultrasound. In this work, a microfluidic flow-focusing device was used to generate monodisperse rt-PA-loaded ELIP (µtELIP) loaded with a perfluorocarbon gas. The rt-PA associated with the µtELIP was encapsulated within the lipid shell as well as intercalated within the lipid shell. The µtELIP had a mean diameter of 5 µm, a resonance frequency of 2.2 MHz, and were found to be stable for at least 30 min in 0.5 % bovine serum albumin. Additionally, 35 % of µtELIP particles were estimated to contain microbubbles, an order of magnitude higher than that reported previously for batch-produced rt-PA-loaded ELIP. These findings emphasize the advantages offered by microfluidic techniques for improving the encapsulation efficiency of both rt-PA and perflurocarbon microbubbles within echogenic liposomes.

Randomized Controlled Noninferiority Trial Comparing Daptomycin to Vancomycin for the Treatment of Complicated Skin and Skin Structure Infections in an Observation Unit.

BACKGROUND: Incidence of methicillin-resistant Staphylococcus aureus (MRSA) is increasing in complicated skin and skin structure infection (cSSSI) presenting to emergency departments (EDs). Treatment is heterogeneous and can require inpatient admission to an observation unit (OU). Vancomycin is commonly used in the OU for treatment, but increasing MRSA resistance to vancomycin suggests the need for alternatives. Daptomycin is an alternative but it is not known how it compares with vancomycin. OBJECTIVE: This study tested the hypothesis that daptomycin is noninferior to vancomycin for the treatment of cSSSI in an OU, using a relative risk (RR) of 1.3 as the noninferiority limit. METHODS: Subjects admitted to an ED-based OU with a diagnosis of cSSSI were eligible. Consenting subjects were randomized 1:1 to intravenous (i.v.) vancomycin at 15 mg/kg dosing every 12 h or i.v. daptomycin at 4 mg/kg once. Subjects were followed until they met objective criteria for discharge home or hospital admission. Discharged patients were prescribed 10-14 days of oral cephalexin and trimethoprim-sulfamethoxazole, or clindamycin if allergic to either of these medications. The primary endpoint was meeting objective discharge criteria with no change in antibiotic therapy or return to the ED for the same cellulitis within 30 days of OU discharge. RESULTS: There were 100 patients enrolled. RR for satisfying the endpoint was 1.07 (95% confidence interval 0.58-1.98) for daptomycin compared with vancomycin. Hospital admission rates were 36% and 32% for daptomycin and vancomycin treatment, respectively. CONCLUSION: Daptomycin was not inferior to vancomycin in the treatment of cSSSI in an OU.

Effect of low frequency ultrasound on combined rt-PA and eptifibatide thrombolysis in human clots.

INTRODUCTION: Fibrinolytics such as recombinant tissue plasminogen activator (rt-PA) are used to treat thrombotic disease such as acute myocardial infarction (AMI) and ischemic stroke. Interest in increasing efficacy and reducing side effects has led to the study of adjuncts such as GP IIb-IIIa inhibitors and ultrasound (US) enhanced thrombolysis. Currently, GP IIb-IIIa inhibitor and fibrinolytic treatment are often used in AMI, and are under investigation for stroke treatment. However, little is known of the efficacy of combined GP IIb-IIIa inhibitor, fibrinolytic and ultrasound treatment. We measure the lytic efficacy of rt-PA, eptifibatide (Epf) and 120 kHz ultrasound treatment in an in-vitro human clot model. MATERIALS AND METHODS: Blood was drawn from 15 subjects after IRB approval. Clots were made in 20 microL pipettes, and placed in a water tank for microscopic visualization during lytic treatment. Clots were exposed to control, rt-PA (rt-PA), eptifibatide (Epf), or rt-PA+eptifibatide (rt-PA + Epf), with (+US) or without (-US) ultrasound for 30 minutes at 37 degrees C in human plasma. Clot lysis was measured over time, using a microscopic imaging technique. The fractional clot loss (FCL) and initial lytic rate (LR) were used to quantify lytic efficacy. RESULTS AND CONCLUSIONS: LR values for (- US) treated clots were 0.8+/-0.1(control), 1.8+/-0.3 (Epf), 1.5+/-0.2 (rt-PA), and 1.3+/-0.4 (rt-PA + Epf) (% clot width/minute) respectively. In comparison, the (+ US) group exhibited LR values of 1.6+/-0.2 (control), 4.3+/-0.4 (Epf), 6.3+/-0.4 (rt-PA), and 4.6+/-0.6 (rt-PA + Epf). For (- US) treated clots, FCL was 6.0+/-0.8 (control), 9.2+/-2.5 (Epf), 15.6+/-1.7 (rt-PA), and 28.0+/-2.2% (rt-PA + Epf) respectively. FCL for (+ US) clots was 13.5+/-2.4 (control), 20.7+/-6.4 (Epf), 44.4+/-3.6 (rt-PA) and 30.3+/-3.6% (rt-PA + Epf) respectively. Although the addition of eptifibatide enhances the in-vitro lytic efficacy of rt-PA in the absence of ultrasound, the efficacy of ultrasound and rt-PA is greater than that of combined ultrasound, rt-PA and eptifibatide exposure.

Ultrasound-enhanced tissue plasminogen activator thrombolysis in an in vitro porcine clot model.

INTRODUCTION: Thrombolytics such as recombinant tissue plasminogen activator (rt-PA) have advanced the treatment of ischemic stroke, myocardial infarction, deep vein thrombosis and pulmonary embolism. OBJECTIVE: To improve the efficacy of this thrombolytic therapy, the synergistic effect of rt-PA and 120 kHz or 1.0 MHz ultrasound was assessed in vitro using a porcine clot model. MATERIALS AND METHODS: Fully retracted whole blood clots prepared from fresh porcine blood were employed to compare rt-PA thrombolytic treatment with and without exposure to 120-kHz or 1-MHz ultrasound. For sham studies (without ultrasound), clot mass loss was measured as a function of rt-PA concentration from 0.003 to 0.107 mg/ml. For combined ultrasound and rt-PA treatments, peak-to-peak pressure amplitudes of 0.35, 0.70 or 1.0 MPa were employed. The range of duty cycles varied from 10% to 100% (continuous wave) and the pulse repetition frequency was fixed at 1.7 KHz. RESULTS: For rt-PA alone, the mass loss increased monotonically as a function of rt-PA concentration up to approximately 0.050 mg/ml. With ultrasound and rt-PA exposure, clot mass loss increased by as much as 104% over rt-PA alone. Ultrasound without the presence of rt-PA did not significantly enhance thrombolysis compared to control treatment. The ultrasound-mediated clot mass loss enhancement increased with the square root of the overall treatment duration. CONCLUSIONS: Both 120-kHz and 1-MHz pulsed and CW ultrasound enhanced rt-PA thrombolysis in a porcine whole blood clot model in vitro. No clear dependence of the observed thrombolytic enhancement on ultrasound duty cycle was evident. The lack of duty cycle dependence suggests a more complex mechanism that could not be sustained by merely increasing the pulse duration.

Serum cleaved tau does not predict postconcussion syndrome after mild traumatic brain injury.

OBJECTIVES: Our objective was to determine if the biomarker for axonal injury, serum cleaved tau (C-tau), predicts postconcussion syndrome (PCS) in adults after mild traumatic brain injury (mTBI). METHODS: C-tau was measured from blood obtained in the emergency department. Outcome was assessed at 3 months post injury using the Rivermead Postconcussion Symptoms Questionnaire and Acute Medical Outcomes SF-36v2 Health Survey (SF-36). RESULTS: Of 50 patients, there were 15 patients with detectable levels of C-tau, 10 patients with abnormal findings on initial head computed tomography (CT) and 22 patients with PCS. One-third of patients with detectable C-tau and 14.3% of patients without detectable C-tau had abnormal findings on head CT (P = .143). Serum C-tau was not detected more frequently in patients with PCS than those without, neither for all patients (P = .115) nor the subgroup with negative head CT (P = .253). CONCLUSIONS: C-tau is a poor predictor of PCS after mTBI regardless of head CT result.

Arrhenius temperature dependence of in vitro tissue plasminogen activator thrombolysis.

Stroke is a devastating disease and a leading cause of death and disability. Currently, the only FDA approved therapy for acute ischemic stroke is the intravenous administration of the thrombolytic medication, recombinant tissue plasminogen activator (tPA). However, this treatment has many contraindications and can have dangerous side effects such as intra-cerebral hemorrhage. These treatment limitations have led to much interest in potential adjunctive therapies, such as therapeutic hypothermia (T

Duty cycle dependence of ultrasound enhanced thrombolysis in a human clot model.

Combined ultrasound and tissue plasminogen activator (rt-PA) therapy, or ultrasound enhanced thrombolysis (UET), has been shown to improve recanalization in patients with acute ischemic stroke. We measured the effect of ultrasound duty cycle on the lytic efficacy of 120 kHz UET in an in vitro human clot model. The hypothesis was that an increase in duty cycle increases rt-PA lytic efficacy. Human whole blood clots were exposed to 120-kHz ultrasound and rt-PA for 30 min in human plasma. The duty cycle ranged from 0% to 80%, where 0% represents sham exposure. Clot lytic rate was measured by recording the clot width over time. The clot width after 30 min exposure to rt-PA and ultrasound decreases with increasing duty cycle. The initial lytic rate increased linearly with duty cycle.

Effect of mild hypothermia on the thrombolytic efficacy of 120 kHz ultrasound enhanced thrombolysis in an in-vitro human clot model.

INTRODUCTION: Ischemic stroke causes substantial death and disability. Currently, recombinant tissue plasminogen activator (rt-PA) is the only FDA approved therapy. However, there are dangerous side effects and therapy must start within 3 h of onset. Therefore, there is interest in adjunctive therapies such as ultrasound enhanced thrombolysis (UET) and hypothermia. Recently, transcranial ultrasound during rt-PA therapy was shown to improve vessel recanalization. Also hypothermia (32-34 degrees C) was shown to be safe and possibly improve outcome. This suggests combining UET and hypothermia to treat ischemic stroke. Little is known about the effects of hypothermia on UET, and in-vitro rt-PA efficacy is reduced for T<37 degrees C. Here, the effects of hypothermia on UET in in-vitro human clot are presented. It is hypothesized that UET efficacy at 33 degrees C is less than at 37 degrees C. MATERIALS AND METHODS: Whole blood was drawn from volunteers. Clots were made, incubated at 37 degrees C and aged for 2 days for maximal lytic resistance. Clots were exposed to human fresh-frozen plasma (control), hFFP and rt-PA ([rt-PA]=3.2 microg/ml), hFFP and 120 kHz ultrasound (US), and hFFP, rt-PA and ultrasound (UET) at 33 degrees C and 37 degrees C. Clot percent mass loss (Deltam) was measured to determine thrombolytic efficacy. Data were analyzed using mixed-model analysis of variance. RESULTS AND CONCLUSIONS: US and rt-PA independently increased Deltam (3.5+/-1.0% and 5.1+/-0.9% respectively; p<0.01) over control. UET increased Deltam an additional 8.1+/-1.3% (p=0.026) The effect of temperature on Deltam (-1.6+/-0.7%) was not significant (p=0.09). Hypothermia did not reduce UET efficacy in this in-vitro model.

A Novel Tool for Evaluation of Mild Traumatic Brain Injury Patients in the Emergency Department: Does Robotic Assessment of Neuromotor Performance Following Injury Predict the Presence of Postconcussion Symptoms at Follow-up?

OBJECTIVES: Postconcussion symptoms (PCS) are a common complication of mild traumatic brain injury (TBI). Currently, there is no validated clinically available method to reliably predict at the time of injury who will subsequently develop PCS. The purpose of this study was to determine if PCS following mild TBI can be predicted during the initial presentation to an emergency department (ED) using a novel robotic-assisted assessment of neurologic function. METHODS: All patients presenting to an urban ED with a chief complaint of head injury within the preceding 24 hours were screened for inclusion from March 2013 to April 2014. The enrollment criteria were as follows: 1) age of 18 years or greater, 2) ability and willingness to provide written informed consent, 3) blunt head trauma and clinical diagnosis of isolated mild TBI by the treating physician, and 4) blood alcohol level of <100 mg/dL. Eligible mild TBI patients were enrolled and their neuromotor function was assessed in the ED using a battery of five tests that cover a range of proprioceptive, visuomotor, visuospatial, and executive function performance metrics. At 3 weeks postinjury, participants were contacted via telephone to complete the Rivermead Post-Concussion Symptoms Questionnaire to assess the presence of significant PCS. RESULTS: A total of 66 mild TBI patients were enrolled in the study with 42 of them completing both the ED assessment and the follow-up; 40 patients were included in the analyses. The area under the receiver operating characteristic curve (AUC) for the entire test battery was 0.72 (95% confidence interval [CI] = 0.54 to 0.90). The AUC for tests that primarily measure visuomotor and proprioceptive performance were 0.80 (95% CI = 0.65 to 0.95) and 0.71 (95% CI = 0.53 to 0.89), respectively. CONCLUSIONS: The robotic-assisted test battery has the ability to discriminate between subjects who developed PCS and those who did not. Additionally, poor visuomotor and proprioceptive performance were most strongly associated with subsequent PCS.

In vitro microscopic imaging of enhanced thrombolysis with 120-kHz ultrasound in a human clot model.

Substantial enhancement of recombinant tissue plasminogen activator (rt-PA) thrombolysis can be achieved with ultrasound, suggesting its use as an adjunctive treatment in thrombolytic therapy for stroke. A microscopic visualization method was used to measure the lysis of human whole-blood clots treated with human fresh frozen plasma (HFFP), rt-PA, and 120-kHz ultrasound for 30 min at T = 37 ° C. The clot-plasma interface was imaged using an inverted optical microscope and the thrombolytic front analyzed as a function of time. Ultrasound treatment significantly enhanced the mean lytic rate from 0.5 to 3.4 µm/min (a 580% change) compared with rt-PA treatment alone.

Plasmin-loaded echogenic liposomes for ultrasound-mediated thrombolysis.

Plasmin, a direct fibrinolytic, shows a significantly superior hemostatic safety profile compared to recombinant tissue plasminogen activator (rtPA), the only FDA-approved thrombolytic for the treatment of acute ischemic stroke. The improved safety of plasmin is attributed to the rapid inhibition of free plasmin by endogenous plasmin inhibitors present in very high concentrations (1 µM). However, this rapid inhibition prevents the intravenous (IV) administration of plasmin. In emergency situations, catheter-based local administration is not practical. There is a need for an alternative technique for IV administration of plasmin. A possible solution is the encapsulation of plasmin in echogenic liposomes (ELIP) for protection from inhibitors until ultrasound (US)-triggered release at the clot site. ELIP are bilayer phospholipid vesicles with encapsulated gas microbubbles. US induces oscillation and collapse of the gas bubbles, which facilitates ELIP rupture and delivery of the encapsulated contents. Plasmin-loaded ELIP (PELIP) were manufactured and characterized for size, gas and drug encapsulations, and in vitro thrombolytic efficacy using a human whole blood clot model. Clots were exposed to PELIP with and without exposure to US (center frequency 120 kHz, pulse repetition frequency 1667 Hz, peak-to-peak pressure of 0.35 MPa, 50 % duty cycle). Thrombolytic efficacy was calculated by measuring the change in clot width over a 30-min treatment period using an edge detection MATLAB program. The mean clot lysis obtained with PELIP in the presence of US exposure was 31 % higher than that obtained without US exposure and 15 % higher than that obtained with rtPA treatment (p < 0.05).The enhanced clot lysis is attributed to the US-mediated release of plasmin from the liposomes.

Integration of New Technology for Research in the Emergency Department: Feasibility of Deploying a Robotic Assessment Tool for Mild Traumatic Brain Injury Evaluation.

UNLABELLED: The objective of this paper is to demonstrate the effective deployment of a robotic assessment tool for the evaluation of mild traumatic brain injury (mTBI) patients in a busy, resource-constrained, urban emergency department (ED). METHODS: Functional integration of new robotic technology for research in the ED presented several obstacles that required a multidisciplinary approach, including participation from electrical and computer engineers, emergency medicine clinicians, and clinical operations staff of the hospital. Our team addressed many challenges in deployment of this advanced technology including: 1) adapting the investigational device for the unique clinical environment; 2) acquisition and maintenance of appropriate testing space for point-of-care assessment; and 3) dedicated technical support and upkeep of the device. Upon successful placement of the robotic device in the ED, the clinical study required screening of all patients presenting to the ED with complaints of head injury. Eligible patients were enrolled and tested using a robot-assisted test battery. Three weeks after the injury, patients were contacted to complete follow-up assessments. RESULTS: Adapting the existing technology to meet anticipated physical constraints of the ED was performed by engineering a mobile platform. Due to the large footprint of the device, it was frequently moved before ultimately being fully integrated into the ED. Over 14 months, 1423 patients were screened. Twenty-eight patients could not be enrolled because the device was unavailable due to operations limitations. Technical problems with the device resulted in failure to include 20 patients. A total of 66 mTBI patients were enrolled and 42 of them completed both robot-assisted testing and follow-up assessment. Successful completion of screening and enrollment demonstrated that the challenges associated with integration of investigational devices into the ED can be effectively addressed through a collaborative patient-oriented research model. CONCLUSION: Effective deployment and use of new robotic technology for research in an urban academic ED required significant planning, coordination, and collaboration with key personnel from multiple disciplines. Clinical Impact: A pilot clinical study on mTBI patients using the robotic device provided useful data without disrupting the ED workflow. Integration of this technology into the ED serves as an important step toward pursing active clinical research in an acute care setting.

Bacterial meningitis secondary to a transethmoidal encephalocele presenting to the emergency department.

We present the case of a patient seen in the Emergency Department (ED) at the height of enteroviral meningitis season with the chief complaint of the worst headache of his life. He was subsequently found to have pneumococcal meningitis as the result of an encephalocele located within the left ethmoid sinus. The key features of the patient's past medical history, the steps to diagnosis, and a discussion of this exceedingly rare entity are detailed.

In vivo testing of a non-invasive prototype device for the continuous monitoring of intracerebral hemorrhage.

BACKGROUND: Intracerebral hemorrhage (ICH) is a stroke subtype with the highest mortality rate. Hematoma expansion and re-bleeding post-ICH are common and exacerbate the initial cerebral insult. There is a need for continuous monitoring of the neurologic status of patients with an ICH injury. NEW METHOD: A prototype device for non-invasive continuous monitoring of an ICH was developed and tested in vivo using a porcine ICH model. The device consists of receiving and transmitting antennae in the 400-1000 MHz frequency range, placed directly in line with the site of the ICH. The device exploits the differences in the dielectric properties and geometry of tissue media of a healthy brain and a brain with an ICH injury. The power received by the receiving antenna is measured and the percent change in power received immediately after infusion of blood and 30 min after the infusion, allowing for the blood to clot, is calculated. RESULTS: An increase in the received power in the presence of an ICH is observed at 400 MHz, consistent with previous in vitro studies. Frequency sweep experiments show a maximum percent change in received power in the 750-1000 MHz frequency range. COMPARISON WITH EXISTING METHODS: Currently, CT, MRI and catheter angiography (CA) are the main clinical neuroimaging modalities. However, these techniques require specialized equipment and personnel, substantial time, and patient-transportation to a radiology suite to obtain results. Moreover, CA is invasive and uses intra-venous dye or vascular catheters to accomplish the imaging. CONCLUSIONS: The device has the potential to significantly improve neurologic care in the critically ill brain-injured patient.

Quantitative assessment of post-concussion syndrome following mild traumatic brain injury using robotic technology.

Post-Concussion Syndrome (PCS) is a common sequelae of mild Traumatic Brain Injury (mTBI). Currently, there is no reliable test to determine which patients will develop PCS following an mTBI. As a result, clinicians are challenged to identify patients at high risk for subsequent PCS. Hence, there is a need to develop an objective test that can guide clinical risk stratification and predict the likelihood of PCS at the initial point of care in an Emergency Department (ED). This paper presents the results of robotic-assisted neurologic testing completed on mTBI patients in the ED and its ability to predict PCS at 3 weeks post-injury. Preliminary results show that abnormal proprioception, as measured using robotic testing is associated with higher risk of developing PCS following mTBI. In this pilot study, proprioceptive measures obtained through robotic testing had a 77% specificity (95CI: 46%-94%) and a 64% sensitivity (95CI: 41%-82%).

Individual lytic efficacy of recombinant tissue plasminogen activator in an in vitro human clot model: rate of "nonresponse".

OBJECTIVES: Recombinant tissue plasminogen activator (rt-PA) is a lytic medication widely used in the emergency department to treat acute thrombotic disorders such as ischemic stroke and myocardial infarction. It is known in the clinical use of this drug that it can be less effective in approximately 25% of individuals receiving such treatment. However, there are no data on the variation of lytic efficacy of rt-PA in decreasing individuals' clot size over time. In this study, in vitro lytic efficacy was determined by measuring the decrease in clot diameter after 30 minutes of drug exposure. The authors sought to explore whether there are individuals who do not respond to this lytic therapy and to estimate the rate of nonresponse. METHODS: Human whole blood clots were made from blood drawn from 22 adult volunteers. The only exclusion criterion was the use of aspirin within 72 hours of the blood draw. Blood clots were allowed to spontaneously form at room temperature and were then incubated at 37°C for 3 hours to ensure complete clot retraction. Sample clots from the same individuals were then exposed to human fresh-frozen plasma (hFFP) control or rt-PA in hFFP (rt-PA) at a concentration of 3.15 µg/mL. All clots were exposed at 37°C for 30 minutes, and clot diameter was measured as a function of time, using a microscopic imaging technique. The fractional clot loss (FCL), which is the percentage decrease in clot diameter at 30 minutes, was used as a measure of lytic efficacy. RESULTS: Means with standard deviation (SD) FCL values were 8.6% (±3.0%) for control and 20.6% (±9.3%) for rt-PA-treated clots. The mean (±SD) difference in FCL values was 12.0% (±8.8%) and was significant (p < 0.05, paired t-test). Five of the 22 subjects (23%) were "rt-PA nonresponders," in that their FCL (rt-PA) values fell within that of the FCL control values. CONCLUSIONS: Overall, rt-PA does not produce clot lysis in vitro in clots from a substantial minority of the population, likely due to individual variations in clot composition and structure.

A prototype device for non-invasive continuous monitoring of intracerebral hemorrhage.

A prototype for a non-invasive, real-time, monitoring device was developed to detect changes in the brain secondary to disease or injury such as intracerebral hemorrhage (ICH). The eventual goal is a non-invasive, real time sensor that can alert the clinician to alterations in the comatose patient's brain resulting from hemorrhage, seizure or stroke. In this work, a 400 MHz electromagnetic (EM) signal was transmitted with an antenna (T), incident on a 'brain gel' in vitro ICH model, and received by a receiving (R) antenna. Changes in the received signal were found to be induced by the presence of blood. The received power (P(R)) was found to be a linear function of the cross sectional area of blood, as measured normal to the incident wave. In addition, the sensor was able to detect as little as 1 mL of blood in this 1000 mL in vitro model.

Comparison of electrical conductivities of various brain phantom gels: Developing a 'Brain Gel Model'

The use of conducting gels to mimic brain and other tissues is of increasing interest in the development of new medical devices. Currently, there are few such models that can be utilized at physiologic temperatures. In this work, the conductivities of agar, agarose and gelatin gels were manipulated by varying NaCl concentration from 0-1 mg/ml. The AC conductivity was measured at room and physiological temperatures (37°C) in the 100-500 Hz frequency range. Conductivity (σ) was nearly independent of frequency but increased linearly with NaCl concentration and was higher at physiological temperatures in these gels. A formula for predicting conductivity as a function of NaCl concentration was derived for each gel type. The overall goal is to develop a 'brain gel model', for studying low frequency electrical properties of the brain and other tissues at physiological temperatures.

Temperature affects thrombolytic efficacy using rt-PA and eptifibatide, an in vitro study.

The potential for hypothermia as a neuroprotectant during stroke has led to its increase in clinical use. At the same time, combination pharmaceutical therapies for ischemic stroke using recombinant tissue plasminogen activator (rt-PA), and GP IIb-IIIa inhibitors, such as Eptifibatide (Epf ), are under study. However, there is little data on how the reactions triggered by these agents are impacted by temperature. Here, clot lysis during exposure to the combination of rt-PA and Epf is measured in an in vitro human clot model at hypothermic temperatures. The hypothesis is that lytic efficacy of rt-PA and Epf decreases with decreasing temperature. Whole blood clots from 31 volunteers were exposed to rt-PA (0.5 µg/mL) and Epf (0.63 µg/mL) in human fresh-frozen plasma (rt-PA+Epf ), rt-PA alone in plasma (rt-PA Alone), or to plasma alone (Control), at temperatures from 30°C to 37°C, for 30 minutes. Clot lysis was measured using a microscopic imaging technique; the mean fractional clot loss (FCL) at 30 minutes was used to determine lytic efficacy. Temperature had a significant impact on FCL in clots exposed to rt-PA+Epf, with the FCL being lower at 30°C to 36°C than at 37°C. The FCL remained significantly higher for rt-PA+Epf­treated clots than Controls regardless of temperature, with the exception of measurements made at 30°C when no significant differences in the FCL were observed between groups. The use of hypothermia as a neuroprotectant may negatively impact the therapeutic benefit of thrombolytic agents.