Development and reliability of the histological THROMBEX-classification rule for thrombotic emboli of acute ischemic stroke patients.

BACKGROUND: Thrombus histology has become a potential diagnostic tool for the etiology assessment of patients with ischemic stroke caused by embolic proximal vessel occlusion. We validated a classification rule that differentiates between cardiac and arteriosclerotic emboli in individual stroke patients. We aim to describe in detail the development of this classification rule and disclose its reliability. METHODS: The classification rule is based on the hypothesis that cardiac emboli arise out of separation thrombi and arteriosclerotic emboli result from agglutinative thrombi. 125 emboli recovered by thrombectomy from stroke patients and 11 thrombi serving as references for cardiac (n = 5) and arteriosclerotic emboli (n = 6) were Hematoxylin and eosin, Elastica-van Gieson and CD61 stained and rated independently by two histopathologists blinded to the presumed etiology by several pre-defined criteria. Intra- and interobserver reliabilities of all criteria were determined. Out of the different criteria, three criteria with the most satisfactory reliability values were selected to compose the classification rule that was finally adjusted to the reference thrombi. Reliabilities of the classification rule were calculated by using the emboli of stroke patients. RESULTS: The classification rule reached intraobserver reliabilities for the two raters of 92.9% and 68.2%, respectively. Interobserver reliability was 69.9%. CONCLUSIONS: A new classification rule for emboli obtained from thrombectomy was established. Within the limitations of histological investigations, it is reliable and able to distinguish between cardioembolic and arteriosclerotic emboli.

First approach to distinguish between cardiac and arteriosclerotic emboli of individual stroke patients applying the histological THROMBEX-classification rule.

Endovascular treatment of strokes caused by large vessel occlusion enables the histopathological investigation of the retrieved embolus, possibly providing a novel opportunity to contribute to the diagnostic workup of etiology and to define secondary prevention measures in strokes with uncertain genesis. We aimed to develop a classification rule based on pathophysiological considerations and adjustment to reference thrombi for distinction between cardiac and arteriosclerotic emboli and to validate this classification rule on a patient cohort. From 125 patients with stroke due to large vessel occlusion and thrombectomy, 82 patients with known etiology (55 cardioembolic and 27 arterioembolic strokes) were included. The corresponding emboli were histologically evaluated by two raters blinded to the etiology of stroke by means of a novel classification rule. Presumed etiology and classification results were compared. Agreement concerning cardiac emboli was 72.2% (95% CI: 58.4-83.5) for rater I and 78.2% (95% CI: 65.0-88.2) for rater II. Agreement concerning arteriosclerotic emboli was 70.4% (95% CI: 49.8-86.3) for rater I and 74.1% (95% CI: 53.7-88.9) for rater II. Overall agreement reached 71.6% (95% CI: 60.5-81.1) for rater I and 76.8% (95% CI: 66.2-85.4) for rater II. Within the limits of generally restricted accuracy of histological evaluations, the classification rule differentiates between cardiac and arteriosclerotic emboli of acute ischemic stroke patients. Further improvement is needed to provide valuable complementary data for stroke etiology workup.

In Vitro Examination of the Thrombolytic Efficacy of Desmoteplase and Therapeutic Ultrasound Compared with rt-PA.

The aim of the study described here was to evaluate the thrombolytic efficacy of combined treatment with the fibrin-selective plasminogen activator desmoteplase (DSPA) and therapeutic ultrasound (sonothrombolysis [STL]) compared with conventional rt-PA (recombinant tissue plasminogen activator) treatment in vitro. Lysis rates were determined by the weight loss of platelet-rich plasma (PRP) clots treated with rt-PA (60 kU/mL) or DSPA (2 µg/mL) combined with pulsed wave ultrasound (2 MHz, 0.179 W/cm(2)). To reveal the individual effects of medication and ultrasound, lysis rates were also determined for DSPA monotherapy and for combined treatment with rt-PA and ultrasound. Clots solely placed in plasma served as the control group. Lysis increased significantly with rt-PA (26.5 ± 7.8%) and DSPA (30.5 ± 6%) compared with the control group (18.2 ± 5.9%) (each p < 0.001). DSPA lysis was more effective than rt-PA lysis (without STL: p = 0.015, with STL: p = 0.01). Combined treatment with DSPA and 2-MHz STL significantly exceeded rt-PA lysis (32.8% vs. 26.5%, p < 0.001).

Development of a new clot formation protocol for standardized in vitro investigations of sonothrombolysis.

BACKGROUND: Agreement about the most suitable clot formation protocol for sonothrombolysis investigations is lacking. Lysis rates vary strongly owing to different test conditions and, thus, cannot be compared. We aim to establish a simple but physiologically grounded protocol for in vitro coagulation to enable standardized sonothrombolysis investigations. METHOD: Clots were generated from platelet-rich plasma (PRP) obtained by centrifugation (10 min, 180 × g) of human venous blood (VB). PRP was mixed with the boundary layer formed between the supernatant and the erythrocyte layer. To achieve clots with different platelet counts, PRP was gradually substituted with platelet-free plasma (PFP), harvested from the supernatant of VB after centrifugation (10 min, 2570 × g). Clot types were examined for histological appearance, hydrodynamic resistance under physiological flows, and lysis rate measured by weight loss after a 2-h treatment with recombinant tissue plasminogen activator (rt-PA) (60 kU/ml). Lysis rates of the most suitable clot were measured after a 1-h treatment with rt-PA (60 kU/ml), and combined treatment with rt-PA and 2-MHz transcranial color-coded sonography (TCCS) (0.179 W/cm(2)) or 2-MHz transcranial Doppler (TCD) (0.457 W/cm(2)). RESULTS: With increased platelet count, the hydrodynamic resistance of the artificial clots increased, their histological appearance became more physiological, and lysis rates decreased. The most suitable clots consisted of 1.5-ml PRP, 2.0-ml PFP, and 0.5-ml boundary layer. Their lysis rates were 36.7 ± 7.8% (rt-PA), 40.8 ± 8.6% (rt-PA+TCCS), and 40.4 ± 8.3% (rt-PA+TCD). COMPARISON WITH EXISTING METHODS: These systemic investigations were conducted for the first time. CONCLUSION: This protocol should be used for standardized sonothrombolysis investigations.

Introduction of a new model for time-continuous and non-contact investigations of in-vitro thrombolysis under physiological flow conditions.

BACKGROUND: Thrombolysis is a dynamic and time-dependent process influenced by the haemodynamic conditions. Currently there is no model that allows for time-continuous, non-contact measurements under physiological flow conditions. The aim of this work was to introduce such a model. METHODS: The model is based on a computer-controlled pump providing variable constant or pulsatile flows in a tube system filled with blood substitute. Clots can be fixed in a custom-built clot carrier within the tube system. The pressure decline at the clot carrier is measured as a novel way to measure lysis of the clot. With different experiments the hydrodynamic properties and reliability of the model were analyzed. Finally, the lysis rate of clots generated from human platelet rich plasma (PRP) was measured during a one hour combined application of diagnostic ultrasound (2 MHz, 0.179 W/cm2) and a thrombolytic agent (rt-PA) as it is commonly used for clinical sonothrombolysis treatments. RESULTS: All hydrodynamic parameters can be adjusted and measured with high accuracy. First experiments with sonothrombolysis demonstrated the feasibility of the model despite low lysis rates. CONCLUSIONS: The model allows to adjust accurately all hydrodynamic parameters affecting thrombolysis under physiological flow conditions and for non-contact, time-continuous measurements. Low lysis rates of first sonothrombolysis experiments are primarily attributable to the high stability of the used PRP-clots.

The platelet-rich plasma clot: a standardized in-vitro clot formation protocol for investigations of sonothrombolysis under physiological flows.

No agreement exists about which protocol for in-vitro clot formation is suitable for sonothrombolysis investigations. Lysis rates vary considerably because of different clotting processes and cannot be compared. We aim to establish a new protocol for in-vitro coagulation to permit standardized sonothrombolysis investigations. The proposed procedure is based upon clots prepared from platelet-rich plasma (PRP). This clot material (group A) was compared with the two most commonly used procedures, namely, recalcification of citrate-anticoagulated whole venous blood (group B) and spontaneous clotting of nonanticoagulated venous blood (group C). Histological examinations were performed and clot stability was tested under physiological flow conditions in vitro for all groups (each n = 10). Lysis rates measured by mass loss were compared using buffered plasma and recombinant tissue plasminogen activator (60 kU/ml), or buffered plasma alone. PRP clots displayed a high degree of similarity to emboli specimens in histological examinations and remained stable under pulsatile flow conditions. B and C clots were mechanically unstable and did not resist physiological flow and pressure. Measuring the lysis rate by weighing seems to be inaccurate, with lowest variability in PRP clots. PRP clots appeared more resistant to lysis. PRP clots should be used for standardized sonothrombolysis investigations.