Celite Activated Clotting Time Research Articles

Comparative Studies Of Purified Poloxamer 188 Using ClotBased and Viscoelastic Measurements Of Coagulation

Background Poloxamer 188 (P188; Mast Therapeutics, Inc.) is a surface-active, non-ionic block copolymer which binds to hydrophobic surfaces on damaged cells improving membrane hydration and lowering adhesion and viscosity. It is known to improve microvascular function in various pathologic states. Currently, this agent is under investigation in a phase 3 clinical trial for treatment of sickle cell disease patients experiencing acute vaso-occlusive crisis. The effect of P188 on blood coagulation and platelet function has been evaluated in several clinical trials where no clinically significant effect has been observed. In these trials, coagulation studies were based on standard clot based methods (e.g., PT and aPTT) and did not include viscoelastic measurements such as thromboelastography (TEG). Given P188 alters viscosity, we compared the effect of this agent using various clotbased, chromogenic and viscoelastic measurements of blood coagulation. Materials and Methods Whole blood activated clotting time studies were carried out in groups of healthy individuals (n=10) at a concentration range of 1.872-15.0mg/mL. TEG analysis on native and citratedwhole blood was carried out on TEG 5000 (Haemoscope Corp, Niles, IL) at concentrations of 0-0.45 mg/mL. The effect of P188 on normal plasma clotting parameters, such as PT and aPTT, was measured at a concentration range of 0-10 mg/mL. The effect of P188 on thrombin-induced clot formation was investigated using a fibrinokinetic method. The effect of P188 on thrombin generation was measured using the fluormetric method (Technoclone, Vienna, Austria). The anti-protease effects of P188 were studied using chromogenic substrate methods using isolated biochemical systems. Results At concentrations up to 10 mg/mL, P188 did not produce any modification of Celiteactivated clotting time (Celite-ACT). At all concentrations the Celite-ACT values remained comparable to saline (138-140 sec for P188 vs. 140 sec for saline). In the TEG analysis, P188 produced a concentrationdependent hypocoagulant effect in both native and citratedblood as evidenced by increased angle and shortening of maximum amplitude (MA). In standard PT and aPTT tests, P188 did not produce any effect on the clotting profile at concentrations up to 20 mg/mL. In fibrinokinetic studies, P188 produced an increase in the fibrin clot density and rate of fibrin polymerization. Discussion These studies demonstrate that even at very high concentrations, P188 does not produce an effect on whole blood clotting as measured by the Celite-ACT assay; this result was confirmed in other standard assays. Fibrinokinetic studies revealed an increase in the rate of fibrin formation and clot density. However, at relatively low concentrations, P188 exhibited a hypocoagulant profile in TEG analysis. The marked discordance between TEG and other coagulation tests suggest that P188’s effect on viscosity and adhesive interactions result in an artifact in TEG analysis and an incorrect indication of a hypocoagulant effect. This effect may be due to the viscoelastic endpoint in the TEG assay. Further studies are needed to confirm this hypothesis. Disclosures: Fareed: Mast Therapeutics: Research Funding. Emanuele:Mast Therapeutics: Employment.

Defibrotide Interactions with Newer Oral Anticoagulants and Antithrombotic Agents

Abstract 3411Defibrotide is a polydisperse mixture of porcine-derived single-stranded oligonucleotides which has been used for multiple clinical indications. Recent clinical trials of defibrotide indicate that this drug may provide benefits both for the treatment and prophylaxis of hepatic veno-occlusive disease (VOD) in hematopoietic stem cell therapy. In VOD it is believed that defibrotide exerts two distinct effects; 1. Endothelial cell protection and 2. Restoration of the thrombotic-fibrinolytic balance. Although antithrombotic in nature, defibrotide does not produce any systemic anticoagulant effects atthe selected dose of 25 mg/kg/day Conventional oral anticoagulants are routinely not used in veno-occlusive disease however heparin is often used in the management of these patients. More recently, newer oral anticoagulant drugs such as dabigatran, apixiban and rivaroxiban have been approved for the management of post orthopaedic surgical venous thrombosis and stroke prevention in atrial fibrillation. The current study was undertaken to investigate the effect of defibrotide on the anticoagulant and antiprotease effects of the newer oral anticoagulant drugs in the whole blood, plasma and platelet rich plasma based systems. Materials and MethodsNative whole blood was drawn from 25 normal healthy individuals and supplemented with 100 μg/mL defibrotide and 250 ng/mL of each of the individual oral anticoagulant drugs alone and with defibrotide. Activated clotting time (ACT) measurements were made on a Hemachron instrument using celite ACT tubes. For the plasma based anticoagulant studies, a fixed concentration of defibrotide of 100 μg/mL was supplemented with defibrotide to pool plasma alone and with each of the individual oral anticoagulantagents in a concentration range of 0–1000 ng/mL. To investigate the effect of defibrotide on agonist induced platelet aggregation platelet rich plasma was prepared with varying amounts of defibrotide (0–100ug/ml). Such agonsits as ADP, epinephrine, collagen and arachodonic acid were used. To test the effects of newer oral anticoagulants, each agent was supplemented at 250–500ng/ml to the defibrotide enriched PRP (100ug/ml) and agonist induced aggregation studies were carried out in comparison to saline and defibrotide control. To test the effect of defibrotide on the conventional oral anticoagulant agents, plasma samples from patients with INR range of 1.5–3.0 were supplemented with 100ug/ml and the PT/INR was re-determined using Innovin® reagent. ResultsIn the whole blood studies apixaban and rivaroxaban did not produce any prolongation of the whole blood ACT. However, dabigatran produced a modest increase in the ACT at 250 ng/mL. In the anticoagulant assays, supplementation of these agents did not result in any prolongation of the PT with defibrotide and newer oral anticoagulants. In the aPTT assay, the apixaban and rivaroxaban did not have any interaction, at a concentration greater than 500 ng/mL. Dabigatran shows a slight interaction in the aPTT assay. Defibrotide, did not produce any alterations of the effect on the agonist (ADP, epinephrine, collagen, arachidonic acid) induced aggregation of platelets in concentrations up to 100 μg/mL. Studies carried out where defibrotide at 100 μg/mL is combined with 250 ng/mL of each of the new anticoagulants does not show any modification of the aggregation responses. Defibrotide supplementation to anti-platelet therapy treated patient’s blood collected did not result in any augmentation of the observed inhibitory responses. Defibrotide did not produce any changes in the PT/INR values of plasma samples collected from warfarin treated patients at concentrations of up to 100 μg/ml. ConclusionsThese studies indicate that in a concentration range of 0–1000 ng/mL the new anticoagulants do not exhibit any significant interactions with defibrotide. Since dabigatran exhibits some anticoagulant effect of its own, minor interactions with defibrotide may be observed. Based on the indications for the new oral anticoagulant drugs, their circulating levels and rapid clearance, it is unlikely that defibrotide will produce any interactions with these drugs. Disclosures:No relevant conflicts of interest to declare.

Using activated clotting time to estimate intraoperative aprotinin concentration

The use of aprotinin during cardiopulmonary bypass may be associated with renal dysfunction due to renal excretion of excess drug. We hypothesized that the difference between standard celite activated clotting time (ACT), which is prolonged by aprotinin, and kaolin ACT could provide an estimate of aprotinin blood level. Fresh porcine blood was collected from six donor pigs and heparinized. Blood was stored at 4 degrees Celsius, rewarmed and aprotinin was added: 0, 100, 200, and 400 kallikrein inhibitor units/ml. Specimens were incubated at 37 degrees Celsius. Two pairs of ACT tubes (one celite and one kaolin) were measured at 37 degrees Celsius and 20 degrees Celsius using two Hemochron 401 machines. A generalized estimating equation (GEE) statistical approach was used to estimate actual aprotinin from differences in celite and kaolin ACT. There was a significant relationship of the form y = exp(a+bx) between aprotinin concentration and the difference between celite and kaolin ACT at both 37 degrees Celsius (R(2) = 0.858) and 20 degrees Celsius (R(2) = 0.743). The time difference between celite and kaolin ACT may be a simple and inexpensive method for measuring the blood level of aprotinin during cardiopulmonary bypass. This technique may improve patient-specific dosing of aprotinin and reduce the risk of postoperative renal complications.

Defibrotide Augments the Anticoagulant Actions of Heparin and Low Molecular Weight Heparins

Defibrotide represents a polydeoxyribonucleotide derived antithrombotic and antiischemic drug, which has been used in the management of vascular disorders and is currently being developed in other clinical indications. Defibrotide is a polyelectrolyte-based agent with target effects on endothelium, platelets, and blood cells. In addition, the aptameric consensus sequences in the nucleotides exhibit inhibitory effects towards thrombin and related proteases. In the anticoagulant assays defibrotide exhibits relatively weak effects (<5 USP U/mg). These studies were undertaken to study whether there is an interaction between defibrotide and unfractionated heparin (UFH) in various systems of anticoagulation. The interaction of defibrotide with commercially available low molecular weight heparins (LMWHs), enoxaparin and dalteparin, was also studied. For the first investigation, to evaluate the effect of defibrotide on the anticoagulant effects of UFH, native whole blood freshly drawn from human volunteers (n = 20) was supplemented with UFH at a fixed concentration of 5 μg/mL (0.8 U/mL), and graded amounts of defibrotide were added in a concentration range of 12.5 – 100 μg/mL. The whole blood celite Activated Clotting Time test (ACT) and the thrombin generation markers fibrinopeptide A (FPA), thrombin-antithrombin complex (TAT), and prothrombin fragment 1.2 (F1.2) were measured. Parallel controls with saline were included. While defibrotide did not produce a significant prolongation of the ACT compared to saline (128 ± 9 s vs 132 ± 7 s), it produced a concentration-dependent increase in the heparinized whole blood leading to an almost doubling of the anticoagulant action of UFH (248 ± 19 s vs 418 ± 21 s). Additional studies carried out by varying the concentrations of the two agents also revealed supraadditive to synergistic effects. Defibrotide also augmented the inhibitory effects of UFH on thrombin generation markers in a concentration-dependent fashion. Similar studies carried out with the two LMWHs did not reveal a similar interaction in the anticoagulant assays such as the ACT; however, significant interactions between defibrotide and the LMWHs were observed in the thrombin generation studies. For the second investigation, studies were carried out using plasma samples collected from heparinized patients (aPTT of 50 – 100 s). These studies also revealed that supplementation of defibrotide augmented the anticoagulant effects of UFH in a concentration-dependent fashion. While defibrotide at 12.5 μg/mL did not significantly increase the aPTT of normal plasma, when supplemented to heparinized plasmas (n = 50 with aPTT of 64.6 ± 14.0 s) it produced a strong prolongation of the clotting time (96.1 ± 20.6 s). In the third investigation, animal models of thrombosis including the rat jugular vein clamping model, demonstrated an augmentation of the antithrombotic effects of intravenously administered UFH by defibrotide. However, no augmentation of the hemorrhagic effect was observed in the rat tail bleeding model. These studies demonstrate that defibrotide exhibits a strong anticoagulant interaction with UFH and to a lesser degree LMWH. While the combination of defibrotide and UFH exhibits enhanced anticoagulant/antithrombotic activities, it does not exhibit any alteration of the hemorrhagic profile. These studies clearly suggest that defibrotide can be combined with UFH to achieve a superior anticoagulant approach with better safety/efficacy profile.

Factor Xa Inhibitors Can Be Differentiated in Xa, IIa and Microparticle Generation and Other Whole Blood Based Assays.

Currently used oral anticoagulants such as Vitamin K antagonists have drawbacks, which reportedly limit their safety and efficacy. Oral Factor Xa and IIa inhibitors are claimed to overcome these limitations. Factor Xa inhibitors provide more complete suppression of thrombin generation than Factor IIa inhibitors. Four Factor Xa anticoagulant agents, both direct and indirect, namely A,B,C and D with Ki values 6–200 nM, were studied in various assays at equigravimetric final concentration of 10 ug/ml to determine their relative potencies. Apparent differences in their biochemical profiles were noted in thrombin generation, Factor Xa generation and microparticle generation inhibition assays. Furthermore, the anticoagulant potential of these Xa inhibitors was studied in celite activated clotting time (ACT) and modified celite ACT system using different concentrations of tissue factor. Microparticle generation was performed using agent- supplemented whole blood that was incubated for three minutes with varying concentrations of tissue factor. Following three minutes the reaction was stopped using EDTA stop solution. The functional assay for the determination of microparticle procoagulant activity in the plasma was performed using Zymuphen MP-Activity assay kit from Hyphen BioMed (Neuville-sur-Oise, France). While three of the Xa inhibitors studied showed microparticle generation inhibition levels of 31.37 nM, one agent provided a level of 17.87 nM compared to a saline control value of 39.28nM. Supplementation studies were also carried out in whole blood PT, APTT and Heptest assays in the concentration range of 0 to 10 ug/ml. The whole blood prothrombin time assay showed a maximum of 208.8, 61.3, 77.8 and 15.9 seconds at a final concentration of 10 ug/ml for the respective anticoagulant agents when compared to a normal value of 12.3 seconds. Similarly the whole blood APTT assay showed a maximum of 267.9, 161.8, >300 and 71.9 seconds respectively when compared to a control value of 46.8 seconds. The whole blood Heptest assay showed a varying maximum anticoagulant effect from >300, 42.7, >300 and >300 seconds respectively when compared to a control value of 13.8 seconds. In each of these assays Factor Xa inhibitors showed concentration-dependent effects and had varying potencies. In TF-mediated platelet activation assays the different Xa agents produced varying effects which were not proportionate to their Ki values. Differentiation of Factor Xa inhibitors have a clinical impact in dosage selection, dosage adjustment and their monitoring when given in large dosages. Synthetic factor Xa agents exhibit Ki values of 20–200 nM, while pentasaccharide -AT complex has a Ki value of 60 nM. However, these results do not translate into proportionate anticoagulant effects to their Ki values. Large-scale clinical studies are necessary to validate these preliminary results.

In-Vitro Evaluation of the Multiple Coagulation Test System (MCTS).

A device that would provide a rapid evaluation of hemostasis would greatly aid physicians in making scientific decisions regarding transfusion therapy; especially with perioperative bleeding. We performed a prospective in-vitro study with reconstituted, recalcified whole blood (n = 6) separated into samples containing specific deficiencies in platelets, plasma, lyophilized platelets and lyophilized plasma such that the concentrations of the specific platelet or plasma were 0%, 10%, 30%, 50%, and 100%. The whole blood was obtained from consenting healthy volunteers. Samples from the various concentrations of the reconstituted blood were subjected to the following point of care coagulation tests: activated clotting time (ACT), thromboelastogram (TEG), laboratory prothrombin time (PT), laboratory activated partial thromboplastin time (APTT) and Signature+ PT. ACT's were performed for each of four celite doses (1.5 mg, 3.0 mg, 6.0 mg and 12 mg celite). The four dose celite ACT's were performed in duplicate and the mean recorded. The TEG was performed in duplicate, and the mean was recorded. The signature+ PT was performed in duplicate, and the mean recorded. The laboratory PT, aPTT, Platelet Count and Fibrinogen were performed once each. ACT values were analyzed within each of the three preparations using analysis of variance. This assessed the strength of the relationship between blood constituent concentration and ACT, and determined whether this relationship differed across varying amounts of celite. Other coagulation parameters including TEG (mA), PT and APTT were analyzed using analysis of variance with a single term for blood constituent concentration. This model determined the strength of the relationship between these coagulation parameters and blood constituent concentration. In addition, the kaolin activated TEG and the signature+ PT was evaluated to determine the strength of the relationship between these tests and the variations in platelet and plasma concentrations. There was a significant correlation between the plasma, platelets and lyophilized plasma concentrations and ACT results (all p < 0.001). There was a significant correlation between plasma platelets and lyophilized plasma and the TEG MA and angle (all p < 0.028). There was a significant correlation between laboratory PT and APTT and plasma, platelets, and lyophilized plasma, lyophilized platelets (all p < 0.001). There was a significant correlation between the Signature+ PT and plasma, lyophilized platelets, and lypholized plasma and platelets (all p < 0.005), (figure 1). A better point of care coagulation device that is based on in-vitro additions of various therapies, and comparison of their effects on coagulation tests may provide better treatment of the bleeding patient. The Signature+ PT could provide the endpoint for such a device whereas the ACT was nonspecific and had a small change in results with large changes in plasma concentration. The additional benefit of lypholized plasma to behave similarly to human plasma may allow such a device to be built. [Display omitted]

Aprotinin does not prolong the Sonoclot aprotinin-insensitive activated clotting time

Study Objective To determine whether a new Sonoclot-based, aprotinin-insensitive activated clotting time (aiACT) assay yields stable results over a broad range of aprotinin concentrations. Design Prospective trial conducted on in vitro blood samples. Setting Tertiary-care teaching medical center. Participants 19 healthy adult volunteers. Interventions Whole blood samples were collected from volunteers. Heparin (2 U/mL) and escalating concentrations of aprotinin of 160 to 500 kallikrein inhibitory units (KIU)/mL were added in vitro. Measurements and Main Results Celite ACT, kaolin ACT, and aiACT assays were completed. The aiACT showed stable activated clotting time (ACT) results on heparinized, noncitrated blood with added aprotinin ( P = nonsignificant). In contrast, celite ACT and kaolin ACT were greatly prolonged when aprotinin was added to heparinized, noncitrated, and citrated blood ( P < 0.05). The aiACT had consistent results at all aprotinin concentrations ( P = nonsignificant). Conclusions Aprotinin (160, 320, and 500 KIU/mL) significantly prolongs the ACT value with celite and kaolin activators but not with the aprotinin-insensitive activator.

Thromboplastin C Supplemented Modified Activated Clotting Time (ACT) is Quite Ideal for Monitoring Direct Thrombin Inhibitors at High Concentrations

Introduction Direct thrombin inhibitors (DTIs) such as Argatroban (Novastan) and recombinant hirudin (Refludan) have been approved by the USFDA for prophylaxis of venous thromboembolism in patients with heparin-induced thrombocytopenia, and Angiomax (Bivalirudin) for use during percutaneous interventional procedures. Melagatran and Dabigatran are in advanced stages of drug development. For lack of a specific antagonist, a careful monitoring of the anticoagulant effects of these agents is crucial in the management of patients with high circulating concentrations. Materials and Methods: Using the Hemochron ACT system, DTIs were studied at a final concentration of 5 ug/ml and heparin (UFH) at 1.0 Unit/ml. A volume of 200 ul of thromboplastin C+ was placed in each celite ACT tube for the modified ACT, and 200 ul of saline for the regular ACT. The ACTs were immediately performed. Results The modified ACT was significantly shortened when compared to the regular celite ACT (see graph). These results suggest that while the regular ACTs are sensitive and reach a threshold at high concentrations, the modifed celite ACT system may be used instead. Conclusions Modified celite ACT system may be quite ideal in monitoring the anticoagulant effects of direct thrombin inhibitors and UFH at high dosages administered for cardiac bypass surgery or other interventional procedures.

Aprotinin and Hemostasis Monitoring Concerns During Cardiac Surgery

Aprotinin (Trasylol) is a serine protease inhibitor, isolated from bovine lung that initially was marketed for the treatment of pancreatitis. In the mid 1980s, reports of its ability to decrease hemorrhaging after cardiopulmonary bypass surgery introduced the drug to the realm of cardiac surgery. Unfortunately, its introduction into this arena was followed by the publication of multiple studies and case reports that blamed aprotinin for poor outcomes in the form of early graft closure. More than 17 years have passed since the initial article describing the use of aprotinin during cardiopulmonary bypass, and with time there has been a significant increase in scientific knowledge and clinical experience. Interestingly, modern literature does not support the dogma that aprotinin is a procoagulant. Aprotinin increases the activated partial thromboplastin time (aPTT), as well as the kaolin- and celite-activated clotting time (ACT), regardless of heparin. Aprotinin, because of its ability to inhibit kallikrein, has been found to decrease thrombin antithrombin III complexes, fibrin-split products, fibrinopeptide1+2, prothrombin fragments, and all markers of thrombin formation. Some authors have suggested that it may have a synergistic effect with heparin to ensure graft patency. Anticoagulation monitoring during the use of aprotinin also has been developed based on early studies. Aprotinin administration does influence the results of various ACT tests, and consequently different methods of testing anticoagulation have been developed. Researchers have demonstrated that the celite ACT is not “artificially” prolonged in the presence of heparin and aprotinin, rather the kaolin ACT is “artificially” shortened. This article will review the scientific literature with regard to aprotinin’s anticoagulatory effects and review the current recommendations for hemostasis monitoring during the use of aprotinin.

Synthetic Anti-Xa Drugs Can Be Used for Parenteral Anticoagulation but Not Fondaparinux.

Targeting at the level of factor Xa and/or prothrombinase complex by synthetic anti-Xa agents such as JTV-803 (Akros Pharma, Princeton, NJ), DX-9065a (Daiichi Pharmaceuticals, Tokyo, Japan), and Fondaparinux (Sanofi-Synthelabo, Toulouse, France) represents an important approach in anticoagulant therapy. Factor Xa is an essential component of the prothrombinase complex and leads to the generation of thrombin. Synthetic heparin pentasaccharide, Fondaparinux (Arixtra®) has been approved by the United States Food and Drug Administration for the prophylaxis of deep vein thrombosis in patients undergoing hip and knee replacement surgeries. There is a rapid stride in the development of newer synthetic inhibitors of factor Xa such as DX-9065a, and JTV-803 in various thrombotic indications. In order to evaluate the parenteral anticoagulant potential of these synthetic factor Xa inhibitors, we used the Hemochron (celite) activated clotting time (ACT) assay. DX-9065a at a final concentration of 5 and 10 μg/ml increased the ACTs to 250 and 300 seconds, respectively. Similar anticoagulant potential was noticed with JTV-803. These findings suggest that these agents may be useful as parenteral anticoagulants during interventional cardiologic procedures, surgical anticoagulation and for the prevention of vascular access occlusion. Fondaparinux requiring antithrombin (AT) for its anticoagulant effect does not increase the celite ACT to the extent as other synthetic factor Xa inhibitors. Fondaparinux even at a final concentration of 100 μg/ml increase the ACT to about 200 seconds and hence are not suitable to be used as parenteral anticoagulants. The concentrations of JTV-803, DX-9065a and fondaparinux required to increase the ACT to near 250 seconds are 6.2, 5 and 125 μg/ml, respectively. While fondaparinux is AT-dependent, the synthetic anti-Xa agents are AT-independent in their actions. The antithrombin sparing effect of direct anti-Xa agents may contribute to an additional anticoagulant effect as reflected by increased ACT levels. Furthermore, there are some other advantages of direct anti-Xa agents when compared to fondaparinux. While direct anti-Xa agents may be used for patients with AT deficiency, fondaparinux being AT-dependent, may not. The inhibition of the clot-bound and prothrombinase-bound factor Xa are additional advantages of direct Xa inhibitors when compared to fondaparinux. Fondaparinux being AT-dependent and upon complexation with AT is not capable of inhibiting the clot-bound factor Xa. Oral Xa inhibitors are being developed and when available patients may have an ideal transition from a parenteral anti-Xa agent to an oral Xa inhibitor. The results clearly suggest that synthetic factor Xa inhibitors except fondaparinux may be used as parenteral anticoagulants. Large-scale clinical studies are warranted to evaluate these findings.

Is the kaolin or celite activated clotting time affected by tranexamic acid?

Background. During cardiopulmonary bypass, the activated clotting time is frequently used for determination of anticoagulation, and either Celite or kaolin are used as activators. If aprotinin is administered concomitantly, the Celite activated clotting time (C-ACT) becomes significantly higher than the kaolin activated clotting time (K-ACT). Therefore, insufficient anticoagulation using C-ACT in the presence of aprotinin is a major concern. Whether the application of tranexamic acid (TA), a pharmacologic alternative to aprotinin, has similar effects has not been studied before. Methods. An in vitro study using the blood of healthy volunteers was performed. Both C-ACT and K-ACT were measured at baseline, after adding TA, and after adding TA and heparin. In addition, 30 patients undergoing primary cardiac operations had simultaneous measurements of C-ACT and K-ACT after skin-incision, 5 minutes after the application of heparin and TA, every 30 minutes during cardiopulmonary bypass, and 10 minutes after the application of protamine. Results. In vitro, C-ACT and K-ACT correlated significantly at each measurement. Tranexamic acid had no influence on the activated clotting time. In vivo, C-ACT and K-ACT did not differ significantly, but at each time C-ACT tended to be greater than K-ACT ( p = 0.086). The average difference between K-ACT and C-ACT was stable before and after the application of TA ( p = 0.85) but the variability of the differences significantly increased during cardiopulmonary bypass ( p < 0.001). Conclusions. Application of TA does not seem to differentially affect the mean C-ACT and K-ACT. No recommendation seems warranted to prefer one activator over the other in patients receiving TA.

Evaluation of CoaguChek Pro ACT in cardiac catheterization laboratory

The objective of this study was to assess the analytical performance of CoaguChek Pro ACT assay versus Hemochron Celite ACT assay concerning activated clotting time (ACT) values and the correlations versus heparin. Enrolled were 158 patients and 101 normal subjects from five cardiac catheterization laboratories (cathlabs). Two different CoaguChek Pro ACT lots were compared to different lots of Hemochron Celite ACT. All sites used arterial blood and one site also used venous blood. Determinations were carried out before and directly after heparinization, and 1–4 h later. Besides the ACT values, hematocrit, platelet counts and factor Xa levels were also determined. The correlations between the Hemochron Celite lots and the two different CoaguChek Pro lots for arterial and venous blood for all sites were good (r=0.88 and 0.84). The agreement between both CoaguChek Pro ACT lots was excellent (r=0.99). The correlations between heparin and CoaguChek Pro ACT were similar to those for the Hemochron Celite lots. There was no influence of the hematocrit and the platelets. The imprecision of the method was very good (CV<6%). This demonstrates that the CoaguChek Pro ACT assay is especially useful for monitoring heparin in cathlabs.

Activated Clotting Time (ACT) Testing: Analysis of Reproducibility

Activated Clotting Time (ACT) has been the standard for monitoring heparin anticoagulation in cardiac surgery for three decades. Although a 10% coefficient of variation (CV) is the referenced standard for the test, no recent reports of precision are available. The precision of Hemochron FTCA510 (celite) and KACT (kaolin) ACT test tubes was evaluated using a retrospective analysis of results from both laboratory studies and routine clinical usage. Laboratory studies of reproducibility included analysis of the CV from repetitive testing using multiple lots of ACTs. Substrates used included 40 consecutive lots of control plasma and freshly heparinized donor blood. Across the lots of control plasma, the celite ACT yielded an average CV of 5.4% for the normal control level and 4.0% in the abnormal control level (range 3.6–9.7% and 2.7–6.3%, respectively). The KACT showed similar performance for the normal (mean = 4.5%, range 2.2–7.8%) and abnormal (mean = 3.8%, range 2.0–10.0%). These values, significantly less than 10%, reflect the combined variability of both the ACT tests and the lyophilized, single use vial, control material. Fresh whole blood samples exhibited improved ACT precision when compared to this artificial substrate. CVs for the celite ACT ranged from 0.6–6.0% at one unit heparin/ml blood to 2.4–11.6% at 5 units/ml where clotting times exceed 650 sec. The KACT showed even lower CVs at all heparin levels, with values of 2.4–7.0%. Clinical evaluations included samples (N = 56) collected from cardiac surgery patients with celite ACT values ranging to 744 sec. Duplicate values differed by an average of 7.5 sec or 1.8%. There was only one clinically significant difference in paired values; a 376 sec paired with a 406 sec, 400 sec being the clinical target time. This retrospective data analysis demonstrates that Hemochron ACT variability is significantly less than 10%.

Anticoagulation and Anticoagulation Reversal With Cardiac Surgery Involving Cardiopulmonary Bypass: An Update

Accelerated thrombin generation is central to the development of hemostatic abnormalities during cardiopulmonary bypass (CPB) that are associated with both thromboembolic complications and serious, abnormal bleeding. Thrombin not only converts fibrinogen to fibrin, but also activates platelets and coagulation factors V, VIII, and XI and causes release of von Willebrand factor from vascular endothelium. Thrombin can also downregulate the hemostatic system by inducing formation of platelet inhibitory agents, such as nitric oxide and prostacyclin, and release of tissue plasminogen activator, facilitating activation of protein C, and releasing tissue factor pathway inhibitor. Excessive thrombin activity may also result in substantial consumption of platelets, fibrinogen, and labile coagulation factors and abnormal bleeding. Elevated tissue plasminogen activator levels secondary to activation of the contact system and surgery catalyze the formation of plasmin, which also consumes or internalizes platelet glycoprotein receptors and coagulation factors V, VIII, and fibrinogen. Heparin can reduce the generation of and mediate neutralization of excessive and CPB-associated thrombin activity. Heparin anticoagulation is commonly monitored with the activated clotting time (ACT). However, the ACT may be prolonged by factors other than heparin during CPB, such as hemodilution and hypothermia, and therefore may not accurately reflect the extent of anticoagulation by heparin. Aprotinin, a nonspecific serine protease inhibitor used with CPB, can also prolong celite-based ACT values, rendering it less reliable for monitoring heparin anticoagulation. Therefore, several alternative anticoagulation strategies have been recommended when aprotinin is used, such as a higher celite ACT trigger (>750 seconds), monitoring of whole blood heparin concentrations (eg, >2.7 U/mL), or administration of heparin based on a CPB duration-dependent, fixed-dose regimen. Administration of heparin doses higher than those generally recommended, as guided by predetermined, patient-specific whole blood heparin concentration measurements during bypass, can reduce excessive thrombin-mediated consumption of platelets and coagulation factors as well as post-CPB blood loss and blood component transfusions. New modalities of improving suppression of excess thrombin generation during CPB include use of heparin-bonded CPB circuits, heparin cofactor II or related analogs, supplemental antithrombin III, direct thrombin inhibitors (eg, hirudin, argatroban), and inhibitors of the contact and tissue factor pathways. The safety and efficacy of these approaches remains to be established by additional, appropriately powered, prospective studies.

Extracorporeal heparin adsorption following cardiopulmonary bypass with a heparin removal device--an alternative to protamine.

To evaluate the therapeutic efficacy and applicability of a heparin removal device (HRD) based on plasma separation and poly-L-lysine (PLL) affinity adsorption as an alternative to protamine in reversing systemic heparinization following cardiopulmonary bypass (CPB). A prospective study. University research laboratory. Adult female swine (n=7). Female Yorkshire swine (n=7, 67.3+/-3.5 [SEM] kg) were subjected to 60 mins of right atrium-to-aortic, hypothermic (28 degrees C) CPB. After weaning from CPB, the right atrium was recannulated with a two-stage, dual-lumen cannula which was connected to an HRD via extracorporeal circulation. Blood flow was drained at 1431.2+/-25.4 mL/min from the inferior vena cava, through the plasma separation chamber of the HRD (where heparin was bound to PLL), and reinfused into the right atrium. The HRD run time was determined by a previously established mathematical model of first-order exponential depletion. Heart rate, mean arterial pressure, pulmonary arterial pressure, central venous pressure, kaolin and celite activated clotting time (ACT), activated partial thromboplastin time (APTT), heparin concentration, and plasma free hemoglobin were obtained before, during, and after the use of the HRD. Pre-CPB ACT was 167+/-89 secs (kaolin) and 99+/-7 secs (celite), and APTT was 34+/-5 secs. The HRD run time averaged 27.4 +/-1.5 mins targeted to remove 90% total body heparin. Use of the HRD was not associated with any adverse hemodynamic reactions or increases in plasma free hemoglobin. The heparin concentration immediately following CPB was 4.85+/-0.24 units/mL, with ACT >1000 secs and APTT >150 secs in all animals. During heparin removal, total body heparin content followed first-order exponential depletion kinetics. At the end of the HRD run, heparin concentration decreased to 0.51+/-0.09 units/mL, with kaolin ACT returning to 177+/-22 secs, celite ACT returning to 179+/-17 secs, and APTT returning to 27+/-3 secs (p > .05 vs. pre-CPB baseline for all variables). The HRD is capable of reversal of anticoagulation following CPB without significant blood cell damage or changes in hemodynamics. The HRD, therefore, can serve as an alternative to achieve heparin clearance in clinical situations where use of protamine may be contraindicated.

Antithrombin III during cardiac surgery: effect on response of activated clotting time to heparin and relationship to markers of hemostatic activation.

This study was designed to determine if, and to what extent, antithrombin III (AT) levels affect the response of the activated clotting time (ACT) to heparin in concentrations used during cardiac surgery, and to characterize the relationship between AT levels and markers of activation of coagulation during cardiopulmonary bypass (CPB). After informed consent, blood specimens obtained from eight normal volunteers (Phase I) were used to measure the response of the kaolin and celite ACT to heparin after in vitro addition of AT (200 U/dL) and after dilution with AT-deficient plasma to yield AT concentrations of 20, 40, 60, 80, and 100 U/dL. In Phase II, blood specimens collected before the administration of heparin and prior to discontinuation of CPB, were used to measure the response of the kaolin ACT to heparin (preheparin only), AT concentration, and a battery of coagulation assays in 31 patients undergoing repeat or combined cardiac surgical procedures. In Phase I, strong linear relationships were observed between kaolin (slope = 1.04 AT - 2, r2 = 0.78) and celite (slope = 1.36 AT + 6, r2 = 0.77) ACT slopes and AT concentrations below 100 U/dL. In the pre-CPB period of Phase II, only factors V (partial r = -0.49) and VIII (partial r = -0.63) were independently associated with heparin-derived slope using multivariate analysis; an inverse relationship was observed between AT and fibrinopeptide A levels (r = -0.41) at the end of CPB. Our findings indicate that the responsiveness of whole blood (ACT) to heparin at the high concentrations used with CPB is progressively reduced when the AT concentration decreases below 80 U/dL. Because AT is variably, and sometimes extensively, reduced in many patients before and during CPB, AT supplementation in these patients might be useful in reducing excessive thrombin-mediated consumption of labile hemostatic blood components, excessive microvascular bleeding, and transfusion of blood products. Heparin, a drug with anticoagulant properties, is routinely given to patients undergoing cardiac surgery to prevent clot formation within the cardiopulmonary bypass circuit. However, when levels are reduced, heparin is not as effective. Findings within this study indicate that administration of antithrombin III may help to preserve the hemostatic system during cardiopulmonary bypass.

Intrinsic pathway–dependent activated clotting time is not reliable for monitoring anticoagulation during cardiopulmonary bypass in neonates

The conventional activated clotting time (ACT) stimulated by celite is often prolonged during neonatal or pediatric cardiopulmonary bypass operations. 1'2 This prolonged ACT may lead to insufficient use of heparin during bypass and result in serious consequences such as intravascular coagulation after the operation. 3 Dilution of the clotting factors because of high-degree hemodilution during bypass is often blamed for the prolongation of ACT, 2' 4 but it is still unknown whether the total clotting capacity in the circulating blood is similarly affected by the extreme hemodilution. Because the celite ACT is a clotting test dependent on the intrinsic pathway, we postulate that it is the intrinsic pathway (rather than the extrinsic and the common pathways) that fails to respond properly under highdegree hemodilution during neonatal bypass operations. As a first step, we studied the in vitro effect of dilution on celite ACT by using heparinized blood taken from 10 adults having coronary artery bypass. The samples were taken after a 300 IU/kg dose of bovine heparin was given before the start of bypass. These patients received no drugs known to affect clotting and had a normal clotting profile before the operation. Blood samples were diluted in the laboratory to 25%, 50%, and 75% with saline solution or were left undiluted as control samples. Three different types of stimuli were used to initiate clotting: celite (final concentration 3 mg/ml) stimulating clotting through the intrinsic pathway, rabbit brain thromboplast in (final concentration 4 mg/ml) through the extrinsic pathway, and high-dose thrombin (final concentration 9 U/ml) through the common pathway. All measurements were performed with a Hemochron 8000 machine (International Technidyne Co., Edison, N.J.). An ACT longer than 1000 seconds was stopped and recorded as 1000 seconds. In blood samples stimulated by celite, ACT was prolonged significantly when the blood was diluted to 75% compared with the ACT of control samples (p < 0.01), whereas in samples stimulated by thromboplast in no significant change was observed. Furthermore, in blood samples stimulated by thrombin a significant reduction of ACT was found in proport ion to the dilution of blood (p < 0.01, Table I). These results

Influence of High-dose Aprotinin on Anticoagulation, Heparin Requirement, and Celite- and Kaolin-Activated Clotting Time in Heparin-pretreated Patients Undergoing Open-Heart Surgery

Aprotinin causes a prolongation of the celite-activated clotting time (CACT), but not of the kaolin-activated clotting time (KACT). Therefore, concern has been raised regarding the reliability of CACT to monitor anticoagulation in the presence of aprotinin. The current study was designed to test the efficacy of aprotinin to improve anticoagulation, and to investigate whether the prolongation of CACT reflects true anticoagulation or is an in vitro artifact. To elucidate this antithrombotic effect of aprotinin, this study was done in patients prone to reduced intraoperative heparin sensitivity. In a prospective, randomized, double-blind clinical trial, 30 male patients scheduled for elective primary coronary revascularization and treated with heparin for at least 10 days preoperatively, received either high-dose aprotinin (group A) or placebo (group C). The CACT and KACT were determined, but only CACT was used to control anticoagulation with heparin. Parameters of coagulation that are indicators of thrombin generation and activity (F1+2 prothrombin fragments, thrombin-antithrombin III complex, and fibrin monomers), parameters of fibrinolysis (D-dimers), aprotinin, and heparin plasma concentrations were measured. Postoperative blood loss and allogeneic blood transfused were recorded. Total heparin administered was 36,200 units (95% confidence interval: 31,400-41,000; group C) compared with 27,700 (25,500-29,800) units (group A; P < 0.05). Hemostatic activation during cardiopulmonary bypass (CPB) was significantly reduced in group A compared with group C. After 60 min of CPB, all parameters were significantly different (P < 0.05) between the groups (group C vs. group A): F1+2 prothrombin fragments, 9.7 (8.9-11.7) ng/ml versus 7.5 (6.2-8.6) ng/ml; thrombin-anti-thrombin III complex (TAT), 53 (42-68) ng/ml versus 29 (23-38) ng/ml; and fibrin monomers, 23 (12-43) ng/ml versus 8 (3-17) ng/ml. Fibrinolysis was also attenuated; D-dimers at the end of operation were 656 (396-1,089) and 2,710 (1,811-4,055) ng/ml for groups A and C, respectively (P < 0.05). The CACT 5 min after the onset of CPB was 552 (485-627) versus 869 (793-955) s for groups C and A, respectively (P < 0.05), whereas the KACT showed no differences between the groups (569 [481-675] vs. 614 [541-697] s for groups C and A, respectively; P = NS). The 24-h blood loss was 1,496 (1,125-1,995) versus 597 (448-794) ml for groups C and A, respectively (P < 0.05). Aprotinin treatment in combination with heparin leads to less thrombin generation during CPB. Aprotinin has anticoagulant properties. Celite-activated ACT is reliable for monitoring anticoagulation in the presence of aprotinin, because the prolonged CACT in the aprotinin group reflects improved anticoagulation. Kaolin-activated ACT does not reflect this effect of aprotinin.

Celite and kaolin produce differing activated clotting times during cardiopulmonary bypass under aprotinin therapy.

Since the introduction of the proteinase inhibitor aprotinin in cardiac surgery, a strong increase of the activated clotting time (ACT) during the extracorporeal circulation phase (ECC) was reported in many clinical studies, but with a lack of correlation between ACT and heparin concentration. In searching for a cause of this inconsistency we investigated different surface activators of the ACT in a clinical study. During ECC ACT was measured in parallel, using a Hemochron device and corresponding tubes (nominally 12 mg celite activator) for celite ACT, and a HemoTec device with corresponding double tubes (nominally 0.1 ml kaolin activator) for kaolin ACT. Under the conditions of ECC, the kaolin ACT values (482 +/- 145 sec) were significantly lower than the celite ACT values (985 +/- 267 sec). These results were confirmed in ex-vivo experiments using an activated partial thromboplastin time (aPTT) model. With heparin alone, aPTT activated with celite and kaolin were similar. Including aprotinin in this model, the celite aPTT showed no correlation to the heparin concentration, whereas the kaolin aPTT remained well correlated to the heparin concentration and similar to the values without aprotinin. With aprotinin alone there were no changes of the aPTT times, whereas the celite ACT times were without any correlation. Our results indicate that using kaolin instead of celite the ACT measurements under aprotinin therapy stay in the same ranges as without application of aprotinin: aprotinin has no detectable influence on kaolin-activated ACT. In our opinion, kaolin should be used as the surface activator for ACT measurements under the conditions of ECC, heparinization, and aprotinin therapy.(ABSTRACT TRUNCATED AT 250 WORDS)

The use of the Hemochron in assessment of heparin reversal after cardiopulmonary bypass

The use of the Hemochron activated clotting time (ACT) for determining adequacy of reversal of heparin following cardiopulmonary bypass was evaluated in 20 patients and compared with standard laboratory tests of coagulation. The commonly used Celite ACT method proved too insensitive to have any useful predictive value in detecting or quantifying residual heparin and removal of the Celite activation rendered the test even less sensitive. In contrast, the partial thromboplastin time with kaolin (PTTK) proved to correlate well with residual heparin ( r = 0.79). It is, therefore, recommended that the use of the Hemochron following protamine administration be abandoned in favour of the PTTK.