aPTT Tests Research Articles

Human Factor IX Expression and Hemophilia Phenotypic Correction in Mice: A Comparison of AAV Serotypes.

Recombinant adeno-associated virus (rAAV) has been extensively studied as a vector for hemophilia gene transfer. AAV serotype 2 targeting the liver and skeletal muscle has been used in clinical trials for hemophilia B patients. Previously we reported that the use of recombinant non-AAV2 serotype vectors generated persistent expression of supra-normal levels of canine factor IX (cFIX) in immunodeficient mice (Mol Ther ‘2000), and resulted in sustained and complete hemophilia B phenotype correction in immune competent hemophilia B mice (Mol Ther ‘2001). In those studies AAV serotypes 1–5 were tested by intramuscular injection. In this study we tested rAAV serotypes 1–8 for hepatic transduction and FIX production in both C57BL/6 and FIX knockout mice. Animals received intrapotal vein injection of 1x1011 virion particles of rAAV 1 thru 8 carrying a human FIX cDNA linked to a chicken beta actin enhancer-CMV promoter and bovine growth hormone polyA. Animals were followed for 8 months and assessed for plasma levels of human FIX by ELISA. A rapid, sustained and maximal rate of hFIX expression was observed within 1–2 weeks with all vectors except AAV2 where the peak expression occurred at 8–10 weeks. hFIX expression was sustained for all serotyped vectors (n=5 animals tested at each AAV serotype). Animals that received serotypes 7 and 8 maintained hFIX at physiological levels (100% hFIX ~ 5000 ng/ml). A differential hFIX expression pattern emerged with rAAV7 (6213 ng/ml) > 8 (5111 ng/ml) > 5 (2367 ng/ml) > 1 (1090 ng/ml) > 4 (377 ng/ml) > 2 (314 ng/ml) > 3 (232 ng/ml). rAAV7 and rAAV8 generated 20 times more hFIX per virion particle than the rAAV2 vector. We did not detect anti-human FIX antibody in any of the experimental mice. To assess why hFIX production differed between each serotype we performed immunohistochemical staining of the mouse liver using a fluorescent-tagged anti-human FIX antibody. Based on this assay, rAAV7 and AAV8 transduced 30% of hepatocytes while rAAV2 transduced less than 2% of the hepatocytes. We subsequently tested FIX production and effect on hemophilia phenotype of rAAV 7 and 8 (1x1011 virion particles) in hemophilia B mice; the data was similar to that observed using the C57Bl/6 mice. Human FIX levels (>5000 ng/ml) were again sustained during the 6–8 month observation period. No anti-human FIX antibody was detected in these hemophilia B mice. Results of clotting function by aPTT testing demonstrated a normalization of clotting time in all the rAA8/hFIX treated hemophilia mice. Survival of all treated mice by tail clip (lethal for all non-treated knockout animals) confirmed that phenotypic correction had been achieved. Our results again demonstrate that AAV serotypes have differential transduction rates and that new serotypes with greater mouse hepatocyte transduction rates are more efficient for hemophilia gene transfer.

Anticoagulation Monitoring

Optimal management of anticoagulant therapy requires an understanding of the laboratory tests often employed to guide therapy. The activated partial thromboplastin time (aPTT) can detect abnormalities in the intrinsic and common clotting pathways. Despite numerous limitations in the aPTT test, it remains the gold standard for monitoring unfractionated heparin and direct thrombin inhibitor therapy. The aPTT can be performed in the central laboratory or at the bedside (point of care [POC] testing). The activated clotting time (ACT) is a POC test that is routinely employed to monitor high-dose heparin during invasive and surgical procedures. The ACT therapeutic range will depend on the specific procedure or surgery being performed. Heparin levels are becoming more routinely available and are used to establish the aPTT therapeutic range for heparin therapy as well as for direct monitoring of heparin and low-molecular-weight heparin therapy. The international normalized ratio (INR) is the gold standard for monitoring warfarin patients. The target INR depends on the indication for anticoagulation. POC monitoring for warfarin is becoming increasingly used. Clinicians should have a thorough understanding of the benefits as well as the limitations of warfarin POC monitoring.

Perioperative management of a patient with severe factor XII deficiency

EDITOR: Factor XII deficiency is a hereditary state that is usually discovered because of an unexpected prolongation of activated partial thrombin time (aPTT) during preoperative evaluation. We report the perioperative management of an orthopaedic patient with severe factor XII deficiency. An 18-yr-old male (height: 175 cm, weight: 72 kg) with a lateral malleolar fracture was transferred to our institution from a county hospital for operative stabilization. A pre-anaesthetic laboratory screen during his first admission revealed an extremely prolonged aPTT that did not return to normal after the transfusion of 3 units of fresh frozen plasma (see Table 1). He had no relevant past medical history and he did not report any symptoms of abnormal haemostasis after traumatic injuries. The physical examination showed no pathologic findings, except for painful ankle oedema. A detailed investigation of haemostasis was performed, including the assessment of protein C, protein S, antithrombin, lupus anticoagulant, prekallikrein and activated protein C resistance. The only pathologic finding was a significant reduction in factor XII activity (4.6%, normal range >80%). This explained the marked prolongation in aPTT. The patient's haemostasis was regarded as sufficiently effective [1]. Nevertheless, he was considered at increased risk for thromboembolic complications and prophylaxis was started with reviparin sodium, 3500 anti-Xa units subcutaneously once daily [2]. The following morning (12 h after reviparin administration), an epidural catheter was placed atraumatically in L4-L5 interspace and the patient underwent an uneventful operative stabilization under epidural anaesthesia with levobupivacaine 0.5% 15 mL plus morphine 3 mg. No abnormal bleeding was observed during the 40 min of the surgical procedure. Postoperative analgesia consisted of patient controlled epidural analgesia with morphine (2.5 mg morphine bolus, 30 min lockout) and parecoxib sodium 40 mg once daily intravenously or intramuscularly. Pain relief was successful, allowing early mobilization. The epidural catheter was removed on the third postoperative day. The postoperative period was uneventful with no signs of deep venous thrombosis or spinal cord compression. Prophylaxis with low-molecular-weight heparin was continued until the 5th postoperative day and the patient was discharged.Table 1: PT and aPTT.The factor XII protein is synthesized in the liver. It has a half-life of about 60 h and its concentration in normal plasma is 30 μg mL−1. Factor XII deficiency is inherited in an autosomal recessive pattern. Factor XII, prekallikrein and high-molecular-weight kininogen are the major factors involved in the so-called 'contact' phase of coagulation. Factor XII is activated by plasma kallikrein during endothelial injury. Activated factor XII is the first component of the intrinsic pathway of the coagulation cascade, while simultaneously, factor XII fragments activate plasminogen during the initiation of normal fibrinolysis. The overall prevalence of factor XII deficiency in the normal population is estimated as 1.5-3.0%, with the severe factor XII deficiency (activity <1%) in a minority. In these rare individuals, aPTT is seriously prolonged with values exceeding 120 s [3]. We believed that our patient had severe factor XII deficiency because of the extreme prolongation of the initial aPTT (238 s) and the significantly decreased factor XII activity (4.6%) 24 h after the transfusion of three units of fresh frozen plasma. The question arises as to why our patient did not report a bleeding tendency in spite of the very low factor XII levels. Although factor XII is necessary for in vitro coagulation during aPTT testing, its presence seems unimportant for in vivo haemostasis. The role of factor XII and other contact factors in coagulation is not so clear and the extrinsic pathway is sufficient for the initiation of coagulation in human beings [1]. Patients with even severe factor XII deficiency are free of haemostatic problems. Therefore, transfusion with fresh frozen plasma or cryoprecipitate for the correction of low factor XII plasma concentration in patients with isolated severe factor XII deficiency is not justified. Patients with severe factor XII deficiency do not have a bleeding tendency. On the contrary, they have a high rate (8-10%) of premature venous thromboembolic events [1]. Factor XII deficiency may lead to hypercoagulability through a defective activation of fibrinolysis. Surgical trauma and immobilization increase the risk of thromboembolic complications. Severe factor XII deficiency places the patient at a moderately increased thromboembolic risk level even during minor surgery and necessitates the application of effective preventing measures such as low-molecular-weight heparin administration, early mobilization and regional anaesthesia [2,4]. Epidural anaesthesia and analgesia have favourable effects on fibrinolysis and offer conditions for sufficient postoperative analgesia and early mobilization [5]. If the placement and removal of an epidural catheter are carefully timed with the low-molecular-weight heparin administration, epidural anaesthesia can be used safely in the perioperative anaesthetic management of these patients. C. Dragoumanis G. Vretzakis T. Vogiatzaki Department of Anesthesiology; Cardiac Anesthesia Unit; University Hospital of Alexandroupolis; Alexandroupolis, Greece

Heparin and low-molecular-weight heparin therapy for venous thromboembolism: will unfractionated heparin survive?

Recent improvements in clinical trials methodology and the use of accurate objective tests to detect venous thromboembolism (VTE) have made it possible to carry out a series of randomized trials to evaluate various treatments for VTE. The results of these trials have resolved many of the uncertainties a clinician confronts in selecting the appropriate course of anticoagulant therapy. These trials have shown that the intensity of both initial heparin treatment and long-term anticoagulant therapy must be sufficient to prevent unacceptable rates of recurrence of VTE. Patients with proximal deep vein thrombosis who receive inadequate anticoagulant therapy have a risk of clinically evident, objectively documented recurrent VTE that approaches 20% to 25%. The need for adequate therapy with heparin and the importance of monitoring blood levels of the effect of heparin have been established. The importance of achieving adequate heparinization was suggested by a nonrandomized trial in 1972 and randomized trials in the 1980s have confirmed this finding. Furthermore, randomized trials have demonstrated the importance of achieving adequate heparinization early in the course of therapy. Unfractionated heparin by continuous intravenous infusion has provided an effective therapy for more than half a century, but the need to monitor therapy and establish therapeutic levels is a fundamental problem. It is evident that validated heparin protocols are more successful in establishing adequate heparinization than intuitive ordering by the clinician. However, even with the best of care using a heparin protocol, some patients treated with intravenous heparin will receive subtherapeutic treatment. In this context, subtherapeutic treatment reflects a practical limitation of the use of unfractionated heparin, rather than a poor standard of care. Furthermore, it is recognized that the practical difficulties associated with heparin administration are compounded by the substantive practical difficulties of standardizing activated partial thromboplastin time (aPTT) testing and the therapeutic range. Our findings have emphasized the confounding effect that initial heparin treatment has on long-term outcome. In all trials of long-term treatment, it is imperative that the initial therapy is of adequate intensity and duration; failure to administer adequate initial treatment may lead to a poor outcome that is falsely attributed to the long-term therapy under evaluation. Treatment with low-molecular-weight heparin (LMWH), which does not require monitoring or dose finding, has largely replaced unfractionated heparin for the initial management of VTE. Efficacy in terms of recurrent VTE or extension of thrombus has been at least as good with LMWH as unfractionated heparin and there is evidence that the incidence of major bleeding, heparin-induced thrombocytopenia, and osteoporosis are less with LMWH as compared with unfractionated heparin. Although unfractionated heparin may survive as a treatment option for acute VTE, its use has been largely supplanted by LMWH.

Reducing unnecessary coagulation testing in hypertensive disorders of pregnancy

Objective: To estimate the magnitude of laboratory testing for hypertension in pregnancy and determine whether abnormalities in prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen can be predicted by results of common, less expensive tests. Materials and Methods: Laboratory records were searched and charts were reviewed to identify gravidas tested for hypertension and to exclude conditions producing coagulopathy. Contingency tables were constructed to assess the ability of the platelet count, lactate dehydrogenase, and transaminases to predict coagulation test results. Results: Preliminary data on 73 gravidas found that a platelet count plus a lactate dehydrogenase test best predicted coagulation abnormalities. Results on another 732 gravidas indicated that coagulation tests were obtained in about 30%. No patient had a PT greater than 18 seconds, two had an aPTT greater than 40 seconds, and three had fibrinogen levels less than 200 mg/dL. The combination of a normal platelet count plus a normal lactate dehydrogenase had a negative predictive value of 100% for clinically significant abnormalities of PT and aPTT, and 99% for significant abnormalities of fibrinogen. Conclusions: Substantial coagulation testing was done on gravidas evaluated for a hypertensive disorder even though the prevalence of clinically significant abnormalities was low. Laboratory evaluation of patients suspected of having preeclampsia need not include a PT, aPTT, or fibrinogen test when there is no evidence of bleeding or of a condition that could produce coagulopathy and when the platelet count and lactate dehydrogenase level are both normal.

Anticoagulant activity of immobilized heparin on the polypropylene nonwoven fabric surface depending upon the pH of processing environment.

Antenna coupling microwave plasma enables a highly oxidative treatment of the outmost surface of polypropylene (PP) nonwoven fabric within a short time period. Subsequently, grafting copolymerization with acrylic acid (AAc) makes the plasma-treated fabric durably hydrophilic and excellent in water absorbency. With high grafting density and strong water affinity, the pAAc-grafted support greatly becomes feasible as an intensive absorbent and as a support to promote heparin immobilization through amide bonds. For heparin immobilized in acidic condition, the carbonate groups of the molecule tend to dissolve and passive encapsulation of the molecule prevents its functional groups from bonding with the carboxylic acid of pAAc. This effect leads to inhibit the immobilization process and consequently reduces the quantity as well as the bioactivity of the immobilized heparin. In alkaline processing environment, the oxidized uronic acid residues in heparin-related glycans are presumably cleaved and the removal of some oxidized residuals before immobilization process is likely to reduce the chain length of heparin. In the latter case, anticoagulant Factors X and XII, but not thrombin, are unaffected. Anticoagulant activity test using activated partial thromboplastin time (aPTT) is more sensitive in assessing heparin-immobilized surfaces, since it corresponds to Factor X and initiates the inhibition of Factor XII and thrombin. Likewise, platelets adhesion on the surfaces decreases as the process shifted from acidic to alkaline condition, whereas the hydrophilic character of the grafted pAAc markedly contributes to extend physical insertion of platelets. The immobilized heparin has a great part of original bioactivity, depending on the pH of the processing environment and the immobilized quantity. Relative bioactivity based upon aPTT tests is partially held longer than 90 days for the sample prepared in the alkaline or neutral environment.

Comparison of Five Point-of-Care Prothrombin and Activated Partial Thromboplastin Time Devices Based on Age of Blood Sample

Delays in processing statium (STAT) blood samples have led to the production of an increasing number of point-of-care tests. Product inserts recommend measuring blood samples immediately after procurement, suggesting that delays may invalidate the test results. We studied the effect of the age of blood samples on point-of-care (POC) prothrombin time (PT) and an activated partial thromboplastin time (aPTT) result. Informed consent was obtained from 11 patients undergoing cardiopulmonary bypass (CPB). Blood samples (40 mL) were taken from each patient. Each blood sample was used to perform five PT tests and six aPTT tests on five POC devices (Gem PCL, Hemochron 801, Hemochron Jr. Signature, Hemochron Response, Rapidpoint Coag) at three different sample ages [&lt;60 s (fresh blood), 10 and 18 min after sample collection]. Blood samples were procured in a plastic syringe devoid of air bubbles, which was left undisturbed between tests but was gently agitated before initiating the 10- and 18-min tests. For tests requiring citrated whole blood, a fraction of each sample was anticoagulated (3.8% citrate) at each age. Statistical analysis was used for comparison of test results for fresh blood to aged samples (10 and 18 min). Test values were recorded as International Normalized Ratio (INR) and seconds for PT and aPTT, respectively. Two devices, the Hemochron 801 and Hemochron response showed statistically, although not clinically, significant variation in PT test results when the samples were aged to 10 and 18 minutes. As for aPTT results, Hemochron 801, Hemochron response, Hemochron Jr. signature, and Gem PCL showed statistically significant variation at 18 minutes. One device (Hemochron 801) reported results with 10-min aged blood that were statistically different from fresh blood. None of the aPTT tests results from any device produced results with aged blood that were clinically different from fresh blood. This study suggests that, in the tests evaluated, blood samples that have aged 10 or 18 min will produce clinically relevant aPTT and PT results, respectively.

An automated strategy for bedside aPTT determination and unfractionated heparin infusion adjustment in acute coronary syndromes: insights from PARAGON A.

Intravenous unfractionated heparin remains a cornerstone of anticoagulation therapy for patients with acute coronary syndromes, but regulation to a target aPTT is challenging. We assessed unfractionated heparin infusion regulation by bedside, whole-blood aPTT testing and computerized, algorithmic infusion adjustment, and further evaluated the relationship of achieving the target aPTT with clinical outcomes. We studied 1,275 patients randomized to unfractionated heparin in PARAGON-A, which tested lamifiban with or without unfractionated heparin versus unfractionated heparin. All patients had baseline and 6-hour blinded, bedside aPTTs, then aPTTs per algorithm. A central computer translated encrypted values to algorithmic dose-adjustment commands. We assessed the ability to achieve and maintain aPTTs of 50-70 seconds and associations of 6- and 12-hour aPTTs and time-to-target with 30-day outcomes.Overall, the median 6-hour aPTT was 50-70 seconds and remained so throughout infusion. Individually, only 33.6% of patients achieved 6-hour target-range aPTTs, and only 40% of all aPTTs were in-range. After achieving target, only 42% of subsequent measures were in-range. Thirty-day death or myocardial infarction (death/MI) increased non-significantly as time-to-target increased (p = 0.08). Thirty-day mortality was similar if target aPTT was reached, regardless of timing. Death/MI trended lower if target aPTT was reached by 8 hours (p = 0.10). The best clinical outcomes were associated with in-range aPTTs. This study represents the most systematic monitoring and regulation of unfractionated heparin anticoagulation to date. Although average anticoagulation achieved target range, wide inter- and intra-patient variability may have important implications for clinical outcomes.

Stability of prothrombin time and activated partial thromboplastin time tests under different storage conditions

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are common laboratory tests that are useful in the diagnosis of coagulation disorders and monitoring anticoagulant therapy. Recent expansions in the outreach laboratory services at our institution prompted us to investigate the shipping limitations for some tests, including PT and aPTT. Although we followed NCCLS guidelines for the collection of blood specimens, we observed falsely elevated PT and aPTT values due to the different storage conditions. The objective of this study is to determine the effect of conditions and duration of storage on PT and aPTT tests using plasma and whole blood samples, respectively. For this study, 36 plasma samples with normal and prolonged PT and aPTT were exposed to different storage conditions. Blood was centrifuged immediately and plasma was stored at room temperature (RT), refrigerated at 4°C, or frozen at −20°C. The samples were analyzed at 0 h and repeated at 6, 12 and 24 h under various conditions. Although statistically significant differences were observed for plasma samples for normal PT tests after 12 h at refrigerated and frozen storage conditions, the differences would not change the clinical interpretation of the results. On the other hand, samples stored refrigerated or at RT showed significant differences for aPTT at 24 h. These differences would change clinical interpretation, especially for samples with normal or near normal aPTT times. Interestingly, aPTT was significantly higher for samples stored frozen when compared to refrigerated and RT conditions at 6 h. Similar patterns were also observed on ten whole blood samples with normal PT and aPTT values. In conclusion, either plasma or whole blood samples can be accepted for PT testing up to 24 h and for aPTT testing up to 12 h only, when transported either at RT or at 4°C.

A survey of aPTT reporting in Canadian medical laboratories. The need for increased standardization. Thrombosis Interest Group of Canada.

A survey of all licensed medical laboratories performing activated partial thromboplastin time (aPTT) testing in Canada was undertaken; the response rate was 50.7%. Preanalytic phase of testing seemed generally satisfactory, although 46% of laboratories were still using 3.8% or a 129-mmol/L concentration of citrate, and only 59% of institutions routinely performed testing to verify the platelet-poor status of the plasma used for aPTT testing. There were also concerns relating to the speed and duration of centrifugation for specimen preparation. While more than 67% of institutions had established an individual therapeutic range for aPTT testing, only 47% of laboratories verified this range with heparinized samples. Approximately 67% of the institutions that had verified the range had done this by spiking heparin concentrations into pooled plasma rather than using ex vivo specimens from patients receiving heparin therapy. There seemed to be a need for increased education about circumstances under which the therapeutic range should be rechecked and current standards for screening for the lupus anticoagulant. More than 71% of Canadian institutions surveyed used low-molecular-weight heparin, which may obviate many of the issues surrounding aPTT testing. Overall performance as documented by survey results seemed similar to that reported for the United States and Australasia.

A Randomized Cross-over Study on the Effects of Levonorgestrel- and Desogestrel-containing Oral Contraceptives on the Anticoagulant Pathways

The use of oral contraceptives (OC) causes disturbances of the procoagulant, anticoagulant and fibrinolytic pathways of blood coagulation which may contribute to the increased risk of venous thrombosis associated with OC therapy. Here we report the results of a cycle-controlled randomized cross-over study, in which we determined the effects of so-called second and third generation OC's on a number of anticoagulant parameters. In this study, 28 non-OC using women were randomly prescribed either a second generation (150 microg levonorgestrel/30 microg ethinylestradiol) or a third generation OC (150 microg desogestrel/30 microg ethinylestradiol) and who switched to the other OC after a two month wash out period. The anticoagulant parameters determined were: antithrombin (AT), alpha2-macroglobulin (alpha2-M), alpha1-antitrypsin, protein C inhibitor (PCI), protein C, total and free protein S and activated protein C sensitivity ratios (APC-sr) measured with two functional APC resistance tests which quantify the effect of APC on either the activated partial thromboplastin time (aPTT) or on the endogenous thrombin potential (ETP). During the use of desogestrel-containing OC the plasma levels of alpha2-M, alpha1-antitrypsin, PCI and protein C significantly increased, whereas AT and protein S significantly decreased. Similar trends were observed with levonorgestrel-containing OC, although on this kind of OC the changes in AT, PCI and protein S (which was even slightly increased) did not reach significance. Compared with levonorgestrel, desogestrel-containing OC caused a significant decrease of total (p <0.005) as well as free protein S (p <0.0001) and more pronounced APC resistance in both the aPTT (p = 0.02) and ETP-based (p <0.0001) APC resistance tests. These observations indicate that the activity of the anticoagulant pathways in plasma from users of desogestrel-containing OC is more extensively impaired than in plasma from users of levonorgestrel-containing OC.

Reducing unnecessary coagulation testing in hypertensive disorders of pregnancy.

To estimate the magnitude of laboratory testing for hypertension in pregnancy and determine whether abnormalities in prothrombin time (PT), activated partial thromboplastin time (aPTT), and fibrinogen can be predicted by results of common, less expensive tests. Laboratory records were searched and charts were reviewed to identify gravidas tested for hypertension and to exclude conditions producing coagulopathy. Contingency tables were constructed to assess the ability of the platelet count, lactate dehydrogenase, and transaminases to predict coagulation test results. Preliminary data on 73 gravidas found that a platelet count plus a lactate dehydrogenase test best predicted coagulation abnormalities. Results on another 732 gravidas indicated that coagulation tests were obtained in about 30%. No patient had a PT greater than 18 seconds, two had an aPTT greater than 40 seconds, and three had fibrinogen levels less than 200 mg/dL. The combination of a normal platelet count plus a normal lactate dehydrogenase had a negative predictive value of 100% for clinically significant abnormalities of PT and aPTT, and 99% for significant abnormalities of fibrinogen. Substantial coagulation testing was done on gravidas evaluated for a hypertensive disorder even though the prevalence of clinically significant abnormalities was low. Laboratory evaluation of patients suspected of having preeclampsia need not include a PT, aPTT, or fibrinogen test when there is no evidence of bleeding or of a condition that could produce coagulopathy and when the platelet count and lactate dehydrogenase level are both normal.

Comparison of an Activated Partial Thromboplastin Time With a Russell Viper Venom Time Test in Screening for Factor VLeiden (FVR506Q)

Factor V(Leiden) is the most common abnormality detected in patients examined because of hereditary thrombophilia. The most widely used clot-based screening test is based on the activated partial thromboplastin (aPTT) time. This test has a low sensitivity. A comparison of the aPTT-based test with a Russell viper venom time test (RVVT) was performed in matched samples. All samples were analyzed by polymerase chain reaction (PCR) for the factor V(Leiden) defect. We studied 139 samples, of which 109 were PCR-negative; 30 were PCR-positive. Using the manufacturer's suggested threshold ratio of 2, the aPTT test showed a sensitivity of 0.43, a specificity of 0.86, and a positive predictive value (PPV) of 0.97. The RVVT test had a sensitivity of 1.0, a specificity of 0.95, and a PPV of 0.91. Segregation of a subpopulation of this study population into ABO group O vs non-group O showed an effect of ABO group on the aPTT test but not on the RVVT test, consistent with an influence of factor VIII clotting (factor VIII:C) on the aPTT test. The RVVT test seems superior to the unmodified aPTT test as a screening test for factor V(Leiden).

Swimming and central venous catheter-related infections in the child with cancer

Hypercoagulability due to high coagulation factor levels resulting from host inflammatory response to cancer contributes to an increased risk of venous thromboembolism (VTE) in cancer patients. Central venous catheters (CVCs) further heighten this risk. Activated partial thromboplastin time (aPTT) can be used to broadly screen for elevated levels of relevant coagulation factors. Our objective was to determine if a shortened aPTT ratio (coagulation time of test- to- reference plasma) was a predictor of CVC-associated VTE in cancer patients.We performed a retrospective case–control study on cancer patients undergoing tunneled CVC insertion at our center from 1999 to 2006 and identified 40 patients who had CVC-associated VTE. VTE was confirmed with color duplex ultrasonography or computed tomography scan. For each case, we obtained 5 controls that had the same cancer diagnosis and were matched on the following factors: age, chemotherapy, hormone therapy (if applicable), tobacco use, TNM staging and year of diagnosis. All patients had aPTT testing within 30 days prior to surgery. We compared aPTT and aPTT ratio between cases and controls using Wilcoxon two sample test.aPTT ratio was significantly shorter in patients with CVC-related VTE as compared to controls [0.86 (95% confidence interval (CI) 0.78, 0.94) vs. 0.98 (0.94, 1.01), p = 0.0003]. Mean aPTT was also significantly shorter. [25.6 seconds (95% CI 23.2, 27.9) vs. 28.1 (26.9, 29.3), p = 0.001] aPTT ratios of the controls tended to spread across larger aPTT ratio values whereas those of cases tended to clustered around the mean.Cancer patients undergoing catheter placement who develop CVC-associated VTE have a shorter aPTT and aPTT ratio than those who do not develop VTE. aPTT, a simple and inexpensive test might be useful as a predictor of CVC-associated VTE risk in cancer patients.

Pharmacokinetics and Tolerability of a New Low Molecular Mass Heparin (RO-11) in Healthy Volunteers – A Dose-Finding Study within the Therapeutical Range

This paper reports on the results of a Phase I, dose-finding study with a new low molecular mass heparin (LMMH) called RO-11. The study focused on pharmacokinetics, dose-effect relationship and on tolerability of three single subcutaneous (s.c.) doses within the therapeutical range. After the injection of 7,500, 9,000 and 12,500 anti-FXa i.u., the anti-FXa effect peaked between 3-6 h and showed a dose-dependent response. The absorption and elimination were first-order processes and the long half-life (> 5 h) kept constant after increasing doses. The compound was tolerated very well and no clinically relevant prolongation of APTT, prothrombin and thrombin clotting tests was observed. At the dose of 7,500 i.u., which corresponded to 110 anti-FXa i.u./Kg, RO-11 exerted anti-FXa effect for at least 18-20 h. We recommend using this dose in a single s.c. injection, to evaluate the efficacy and safety of RO-11 in the initial treatment of DVT or PE.

Critical aspects in routine coagulation testing

Abstract

Anticardiolipin antibodies in systemic lupus erythematosus: Clinical and laboratory correlations

To examine the link between anticardiolipin antibodies and the features of antiphospholipid syndrome in patients with systemic lupus erythematosus (SLE). In this prospective cohort study conducted in a single center, 390 SLE patients were followed up between June 1991 and 1994. At each assessment, a complete history, physical examination, and laboratory evaluation (including measurement of anticardiolipin antibodies) were performed according to a standard protocol. Forty-seven percent of the patients had an elevated level of anticardiolipin antibodies. In the univariate analysis, elevated anticardiolipin antibody levels were found to correlate with thrombocytopenia (P = 0.006), prolonged activated partial thromboplastin time (aPTT) (P = 0.003), and positive direct and indirect Coombs' test result's (P < 0.001). No correlation was identified with any of the clinical features of antiphospholipid syndrome. In the multivariate analysis, anticardiolipin antibodies remained highly associated with thrombocytopenia (odds ratio [OR] 4.05, P = 0.02), positive direct Coombs' test (OR 2.31, P < 0.001), and prolonged aPTT (OR 1.73, P = 0.03). In the multivariate model using venous/arterial thrombosis as the outcome variable, only prolonged aPTT was associated with venous/arterial thrombosis (OR 7.9, P < 0.001). None of the laboratory variables were found to correlate with fetal loss. The presence of anticardiolipin antibodies in patients with SLE is associated with prolonged aPTT, thrombocytopenia, and positive Coombs' test result, but not with antiphospholipid syndrome. Prolonged aPTT is strongly associated with venous/arterial thrombosis.

Thrombopénie à l'héparine sous hémodialyse en réanimation : utilisation d'un héparinoïde de bas poids moléculaire, l'ORG 10 172 (Orgaran ®)

A 48-yr-old patient was admitted to the ICU for cardiogenic shock and acute renal failure after coronary artery bypass graft surgery. A heparin-induced thrombocytopenia (HIT) occurred during haemodialysis with unfractioned heparin (UFH) as the anticoagulant. The dialysers, the circuits and the catheters were recurrently thrombosing and the platelet count decreased to 9 G · L −1 on postoperative day 7. UFH was discontinued. Attempts to substitute UFH with a low molecular weight heparin (LMWH) failed, due to the presence of a high cross-reactivity rate of LMWH with the heparin-dependent antibody. Intermittent haemodialysis without anti-coagulation using a predilution of the dialysers failed also and resulted in recurrent clotting. After informed consent of the patient, a new natural heparinoid Orgaran ® (Org 10172, Organon, Oss Holland) was administered. This agent is a mixture of several non heparin low molecular weight glycosaminoglycans, with proven anti-coagulant efficacy, low cross-reactivity with the HIT antibody, and a half-time prolonged over 18–25 hours. The treatment regimen consisted in a i.v. bolus of 40–45 IU · kg −1 prior to each dialysis procedure, performed every two days. The platelet count increased to 200 G · L −1, seven days after discontinuing heparin injection, and remained stable during the administration of Orgaran ®. No other thrombosis occurred again. Each procedure of four hours duration was monitored with the plasma anti-Xa activity and APTT test. The mean anti-Xa plasma concentrations (0.44 ± 0.55 IU · mL −1, 30 min after injection of Orgaran ®) were well correlated with APTT test (r = 0.73, p < 0.01). As the drug tends to accumulate in plasma in case of renal failure, the dose should be titrated according to the pre-dialysis plasma anti-Xa activity.

Bedside coagulation monitoring in heparin-treated patients with active thromboembolic disease: A coronary care unit experience

Patients with active venous and arterial thromboembolic disorders are known to benefit from systemic anticoagulation with heparin. Clinical studies have shown, however, that therapeutic anticoagulation is rarely achieved rapidly and often is not maintained over time. Prolonged laboratory turnaround time of the activated partial thromboplastin time (aPTT) may contribute directly to these common problems. A total of 272 aPTT determinations were performed on 120 heparin-treated patients admitted to the coronary care unit. The time from sample collection to data availability was 126 ± 84 minutes with standard laboratory aPTT testing. In contrast, a bedside coagulation device provided an aPTT within 3 minutes ( p < 0.001). Subtherapeutic aPTT values (<65 seconds) were documented in 21% of all patients; in each, the heparin dose was changed and a repeat aPTT was required. In a separate study of 33 heparinized patients randomized to either bedside or central laboratory aPTT testing (264 aPTT determinations), the time to achieve a therapeutic state of systemic anticoagulation was 8.2 hours and 18.1 hours, respectively ( p < 0.005). The time from aPTT determination to a decision regarding heparin titration adjustments was 14.5 minutes and 3 hours with bedside and laboratory testing, respectively ( p < 0.001). Thus bedside coagulation monitoring provides a convenient, rapid, and accurate assessment of systemic anticoagulation among heparin-treated patients with active thromboembolic disease in the coronary care unit. This technology warrants further clinical investigation.

Some recent developments with lupus anticoagulants

Lupus anticoagulants (LA) have been defined as phospholipid-interfering antibodies. Testing for them has become a frequently requested procedure in coagulation laboratories and new methods have recently become available. Activated partial thromboplastin time (aPTT) reagents with reduced levels or different types of phospholipid provide high sensitivity. Correction procedures resistant to heparin and based on aPTT and dilute Russell's viper venom time (DRVVT) tests with added hexagonal phase phospholipids have improved the specificity of testing. Simplified tests based on venom activators of factor X and prothrombin improve the reliability of LA testing and may facilitate the further categorization of circulating anticoagulants. Recent studies on the mechanism of LA derived from various patients have confirmed their heterogeneity, principally in the protein cofactors involved in their interactions with phospholipids. Perhaps one-third of LA require beta 2-glycoprotein 1 to exert an anticoagulant effect. The remainder may require human prothrombin as suggested from studies with reconstituted clotting factor systems.