Barbara M. Alving

Effect of ultrasound on tissue-type plasminogen activator-induced thrombolysis.

BackgroundThe efficacy of fibrinolytic therapy is limited by the small surface area of the clot that is available for the binding of the thrombolytic agent, such as tissue-type plasminogen activator (t-PA). We hypothesized that exposure of the clot to ultrasound during thrombolytic treatment could enhance lysis through perturbation of the thrombus, which would expose additional fibrin binding sites for t-PA Methods and ResultsWhole human blood clots containing radiolabeled fibrinogen were incubated in vitro for 200 minutes with Tris-albumin buffer containing t-PA at concentrations ranging from 3 to 3,000 IU/ml. In paired experiments, one of the clots also was exposed to intermittent ultrasound (1 MHz, 1.75 W/cm2) throughout the experiment. The ultrasound was delivered as a 2-second exposure followed by a 2-second rest interval. The overall difference in mean clot lysis between thrombi receiving ultrasound and those receiving no ultrasound was significant (p < 0.001) at all concentrations of t-PA. For clots incubated with t-PA at a concentration of 300 IU/ml, ultrasound increased the percent lysis at 200 minutes from 42±5% (mean±SEM) to 64±10%. In six paired experiments in a rabbit jugular vein thrombosis model, rabbits received 1 mg t-PA alone or t-PA and intermittent ultrasound (1 MHz, 1.75 W/cm2) for 200 minutes. For rabbits receiving ultrasound and t-PA, lysis was 55±11% at 100 minutes compared with 30±12% for rabbits receiving only t-PA. Lysis was 6±10% for rabbits (n = 4) receiving ultrasound alone. No evidence for tissue damage was noted in rabbits exposed to intermittent ultrasound ConclusionsExposure of whole blood clots in vitro to intermittent ultrasound combined with t-PA caused a significant enhancement of thrombolysis compared with t-PA alone. Intermittent ultrasound also showed a trend toward enhancement of t-PA-induced clot lysis in an animal thrombosis model. These data suggest that noninvasive intermittent ultrasound may be a useful adjunct to thrombolytic therapy.

Profound normovolemic hemodilution: hemostatic effects in patients and in a porcine model.

Previous systematic investigations of the hemostatic effects of normovolemic hemodilution (NHD) have not explored the influence of hematocrits less than 20% in humans or animals.However, clinical interest in maximizing the perioperative conservation of erythrocytes may involve profound NHD beyond traditionally accepted empiric end points. We report here on coagulation data in eight healthy adolescent patients undergoing profound NHD in concert with surgical correction of idiopathic scoliosis, and in 29 swine undergoing experimental stepwise NHD until death. Blood was replaced with 5% albumin in 0.9% saline in our patients, and with 5% albumin in lactated Ringers solution in our pigs. A 75% blood volume exchange in our patients yielded a platelet count (PLT) of 158 +/- 26 times 103/micro Liter, fibrinogen concentration (FIB), 50 +/- 7 mg/dL, prothrombin time (PT), 25.4 +/- 2.6 s, activated partial thromboplastin time (aPTT), 87 +/- 15 s, and a nadir hemoglobin of 2.8 +/- 0.2 g/dL; however, global oxygen delivery as assessed by body oxygen consumption remained adequate. Coagulation during the experimental porcine hemodilution was assessed by measuring PLT, FIB, PT, and aPTT, as well as by measurement of coagulation factor activities. In neither species did clinically significant thrombocytopenia (PLT < 100 times 103/micro Liter) become manifest prior to clinical or other laboratory evidence of coagulopathy. Rather, a combined deficiency of coagulation factors explains the coagulopathy developing during NHD in both patients and swine. Abnormal hemostasis develops prior to compromise of global tissue oxygenation, assessed by mixed venous oxygen saturation and total body oxygen consumption, during NHD in healthy patients anesthetized as described. Therefore, NHD may be more limited by preservation of normal coagulation than of global oxygen delivery and consumption. (Anesth Analg 1996;83:459-65)

Fibrin sealant: summary of a conference on characteristics and clinical uses

The 2‐day conference clearly outlined the formulations of products that are being developed or are commercially available in Europe. The major difference between products in the United States and those in Europe is that US manufacturers are preparing fibrin sealant that does not contain aprotinin, epsilon amino caproic acid, or any other type of antifibrinolytic agent, whereas antifibrinolytic agents are included in all such preparations used in Europe. The conference provided no clear consensus that such agents are essential to the efficacy of the product. Although many investigators believe in the clinical benefit of fibrin sealant, most of the studies to demonstrate efficacy have not been performed in a well‐controlled fashion. However, fibrin sealant, if found in a controlled trial to have clinical efficacy, could be approved by the FDA for a narrow indication. Opportunities remain for greater exploration of different forms of the product, not only as a hemostatic agent, but as an adjunct to wound healing and as a matrix for delivery of drugs and proteins with other biologic activities.

Maintaining iron balance in women blood donors of childbearing age: summary of a workshop

T he safety and availability of the national blood supply depends on individuals who can provide repeated blood donations. Frequent donations are generally not possible for women with borderline Hb values caused by iron deficiency. Although women donors have received iron replacement and have become successful repeat donors under well-defined research conditions, the potential value of this practice and the barriers to systemized implementation by organizations that collect volunteer donor blood have not been discussed in a national forum. The medical, economic, and operational feasibility of implementing a program to prevent iron deficiency in women blood donors of childbearing age was therefore explored in a workshop convened in Bethesda, MD, on June 8, 2001, which was cosponsored by the National Heart, Lung, and Blood Institute, the American Association of Blood Banks, America’s Blood Centers, and the American Red Cross. Speakers addressed the potential confounding issues of hemochromatosis and underlying disorders such as occult malignancy that might be masked by iron replacement. Potential protocol designs as well as possible means of implementation in blood centers were also reviewed. The following is a summary of the presentations and discussions of the meeting; the organizers are listed at the end of the report.

Hemostatic assessment of patients before tonsillectomy: A prospective study

The purpose of this prospective study in patients undergoing tonsillectomy was to determine whether perioperative bleeding could be predicted by use of a standardized questionnaire concerning bleeding risk combined with measurement of the activated partial thromboplastin time and prothrombin time. Of the 96 patients enrolled in the study, none had a history of a severe bleeding disorder, but 6 (6%) had histories suggestive of a mild bleeding disorder. Further evaluation showed possible von Willebrands disease in one of these patients. Of the 90 patients with negative questionnaires, 16% had prolongation of the activated partial thromboplastin time. One of these patients had possible von Willebrands disease. However, none of the patients with positive questionnaires or a prolonged activated partial thromboplastin time bled after surgery. Bleeding that resulted in additional hospital use occurred in 2% of patients, and blood-tinged sputum was described by 4% after discharge; all of these patients had negative questionnaires and normal screening studies. The data provide further evidence that routine measurement of the activated partial thromboplastin time and prothrombin time in asymptomatic patients is not useful for predicting postoperative bleeding. In addition, histories suggestive of a mild bleeding disorder are also not accurate predictors of postoperative bleeding. Excessive bleeding associated with tonsillectomy is usually not a result of an identifiable coagulation disorder.

A modified thromboelastographic method for monitoring c7E3 Fab in heparinized patients.

The monoclonal antibody, c7E3 Fab, binds to the platelet surface fibrinogen receptor (GPIIb/IIIa), inhibiting platelet aggregation and clot retraction.We performed an in vitro study to assess the ability of a modification of the thromboelastograph (MTEG) to detect inhibition of clot strength by c7E3 Fab and its reversal with platelet-rich plasma (PRP). In the modified assay (MTEG), thrombin was added to whole blood (WB) and platelet-poor plasma (PPP) and the resultant maximum amplitude (MA) was measured, MAWB and MAPPP, respectively. Anticoagulated blood samples from 17 patients scheduled for cardiac surgery were collected for a dose response (Part I; n = 5) and c7E3 Fab reversal (Part II; n = 12) study. Clot strength was reduced in a dose-dependent manner by c7E3 Fab. Ecteola cellulose effectively reversed the effect of heparin on the thrombin time and the addition of PRP significantly increased the MAWB (P < 0.0001) and MAWB-ppp (P < 0.0001). Subtracting the MAPPP from MAWB significantly magnified the response of MA to the addition of c7E3 Fab (P = 0.002) and its reversal with PRP (P = 0.005). This in vitro study indicates that the MTEG is a responsive assay demonstrating that inhibition by the antiplatelet c7E3 Fab is reversible with PRP. (Anesth Analg 1997;84:31-8)

Correlation between lupus anticoagulants and anticardiolipin antibodies in patients with prolonged activated partial thromboplastin times

PURPOSEnAn increased incidence of thrombosis has been reported in patients with a prolonged activated partial thromboplastin time (APTT) due to a lupus anticoagulant (LA), which is an antibody to negatively charged phospholipids. The antiphospholipid antibodies can be quantitated in an enzyme-linked immunoabsorbent assay (ELISA) that utilizes cardiolipin as the antigen. With the development of the ELISA, two major areas of controversy have arisen. First, the correlation between assay results for LA and for the ELISA has varied widely among laboratories. Second, some investigators have described a correlation between high levels of anticardiolipin antibodies (ACA) and thrombotic disorders, whereas others have found no association between ACA levels and thrombosis in a general population of medical patients. To explore these issues further, the present study determined the sensitivity and specificity of an LA assay for detecting ACA in medical patients with a prolonged APTT. The association between the isotype and titer of ACA and thrombosis was examined in those patients positive for LA.nnnPATIENTS AND METHODSnPlasma samples from 70 medical patients with a prolonged APTT by routine screening studies were tested for the presence of LA by dilution of phospholipid in an APTT system and for IgM and IgG ACA according to a standardized ELISA. Clinical records were reviewed for a history of thrombotic events by an investigator who had no knowledge of the laboratory results.nnnRESULTSnThe ACA assay gave positive results in 47 patients, 44 of whom also tested positive for LA. Thus, the sensitivity for the LA assay for detecting ACA was 94% (confidence interval, 82% to 99%). The result of the LA assay was negative in 20 of 23 patients who were ACA-negative. The specificity of the LA assay was 87% (confidence interval, 67% to 98%). Twelve of the 47 patients (26%) had a history of venous or arterial thrombosis. Of these patients, 75% tested in the high-positive range for IgG or IgM ACA, or both. Of the 35 patients without thrombosis, only 14% were in this range. Patients with thrombosis had either underlying systemic lupus erythematosus, lymphoma, or no apparent etiology for LA. There was no history of thrombosis in patients with LA associated with infection or medication.nnnCONCLUSIONnA test for LA in medical patients with a prolonged APTT can be sensitive and specific for ACA. Determination of ACA levels in patients who have LA that is not induced by medication or infection may define those patients at increased risk for thrombosis.

Heparin-associated Thrombocytopenia in the Neurosurgical Patient

Although thrombocytopenia occurs in approximately 10% of medical patients who receive heparin, this complication has not been described in neurosurgical patients. We report the clinical course of two patients who in the immediate postoperative period developed heparin-associated thrombocytopenia that resulted in significant morbidity. In these two cases, the origin of the heparin was in flush solutions used to maintain the patency of indwelling vascular catheters and was infused at a dose of 250 to 500 units/day. The minimal daily dose previously reported to result in thrombocytopenia is 9000 units/day administered in divided doses subcutaneously. The case reports indicate that heparin in flush solutions should be considered as a cause for unexpected thrombocytopenia and that platelet counts should be monitored in patients receiving heparin in any amount.

INCREASED ANTICARDIOLIPIN ANTIBODIES ASSOCIATED WITH THE DEVELOPMENT OF ANETODERMA IN HIV‐1 DISEASE

Background and Objective. Anetoderma has been reported in patients with HIV‐1 disease. In patients with autoimmune disease, anetoderma has been associated with increased levels of antiphospholipid antibodies (APL) that include anticardiolipin antibodies (ACA) and lupus anticoagulant (LA). This has led to speculation that the autoimmune phenomena seen in HIV‐1 disease and the immune dysregulation induced by HIV‐1 disease may play a role in the development of these lesions. We have seen both primary and secondary lesions of anetoderma in patients followed for HIV‐1 disease. In this study, we wanted to determine whether there was an association in the development of anetoderma and elevated anticardiolipin antibodies (ACA) in HIV‐1 patients.

Inhibitory Effects of Lysine Analogues on t-PA Induced Whole Blood Clot Lysis

The lysine analogues epsilon-aminocaproic acid (EACA) and trans-4-amino-methyl cyclohexane carboxylic acid (AMCA) are used to prevent excessive bleeding in patients with coagulopathies, such as hemophilia and thrombocytopenia, or in those who have received tissue plasminogen activator (t-PA). However, their relative efficacy in inhibiting lysis of clots that have been formed in the presence of exogenous t-PA or that have been formed and then exposed to exogenous t-PA has not been well characterized. The present study utilized blood from normal volunteers and 125I-fibrinogen in a dilute whole blood clot assay to determine the relative concentrations of lysine analogues required for inhibition of clot lysis induced by exogenous t-PA. AMCA (0.06 mM) and EACA (0.6 mM) were effective in prolonging clot lysis if (1) whole blood clots were formed and then exposed to a lysine analogue and exogenous t-PA or if (2) whole blood clots were formed in the presence of exogenous t-PA and a lysine analogue. However, their inhibitory effect was markedly reduced if clots were formed in the presence of t-PA and then exposed to either of the lysine analogues. The analogues did not inhibit the initial binding of t-PA to fibrin. They did inhibit binding of plasminogen to fibrin as well as the activation of plasminogen by t-PA in the absence of fibrin. The data suggest that lysine analogues, even at low concentrations, reduce the rate of t-PA induced whole blood clot lysis by several mechanisms.