Activated Partial Thromboplastin Time: New Tricks for an Old Dogma

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1 Activated Partial Thromboplastin Time: New Tricks for an Old Dogma Giuseppe Lippi, M.D., 1 and Emmanuel J. Favaloro, Ph.D., M.A.I.M.S. 2 ABSTRACT The activated partial thromboplastin time (APTT) is the most common coagulation test procedure performed in routine laboratories, apart from the prothrombin time. The test is traditionally used for identifying quantitative and qualitative abnormalities in the intrinsic and common pathways of coagulation, monitoring anticoagulant therapy with unfractionated heparin, and detecting inhibitors of blood coagulation, the most common of which is the lupus anticoagulant. Whereas short APTT values have been mostly overlooked in the past, recent evidence suggests that these might be associated with hypercoagulability. Although clinical relevance is yet to be clearly defined, hypercoagulability detected by a shortened APTT appears to be significantly associated with a major risk of venous thromboembolism independently from other variables such as blood group, the presence of inherited thrombophilia, and factor VIII levels. This novel finding suggests that this traditional, simple, and inexpensive test might have renewed utility along with traditional thrombophilic tests in the evaluation of venous thromboembolic risk. In addition, APTT waveform analysis is also providing mounting evidence of added utility, in particular for identifying sepsis and disseminated intravascular coagulation in critically ill patients (particularly where this might worsen the prognosis), for monitoring therapy in patients with inhibitors, and as a diagnostic aid to identify patients with antiphospholipid antibodies. In total, such emerging evidence suggests that the APTT is either an old dogma displaying new tricks or else might describe a new dogma for an old laboratory trick. KEYWORDS: Activated partial thromboplastin time, clotting assay, factor V, hemophilia, therapy Damage to blood vessels can result in disastrous consequences should the resulting hemorrhage not arrest in a timely manner. Hemostasis, defined as the arrest of bleeding from an injured blood vessel, requires the combined activity of vascular, platelet, and plasma factors. 1 This multifaceted and complex process traditionally develops through three distinct phases: activation of primary hemostasis (i.e., formation of a primary platelet plug via platelet/von Willebrand factor/tissue interaction to initially stop bleeding); secondary hemostasis (i.e., coagulation to stabilize the platelet plug); and clot dissolution (i.e., fibrinolysis). Each of these processes are closely linked and precisely regulated to enable repair of vessel wounds, promote vascular healing, and Sezione di Chimica Clinica, Università di Verona, Verona, Italy; 2 Department of Haematology, Institute of Clinical Pathology and Medical Research (ICPMR), Westmead Hospital, Westmead, Australia. Address for correspondence and reprint requests: Prof. Giuseppe Lippi, M.D., Sezione di Chimica Clinica, Dipartimento di Scienze Morfologico-Biomediche, Università degli Studi di Verona, Ospedale Policlinico G.B. Rossi, Piazzale Scuro, Verona, Italy ( giuseppe.lippi@univr.it). Laboratory Diagnostics in Thrombosis and Hemostasis: The Past, the Present, and the Future; Guest Editors, Emmanuel J. Favaloro, Ph.D., M.A.I.M.S., Giuseppe Lippi, M.D., and Massimo Franchini, M.D. Semin Thromb Hemost 2008;34: Copyright # 2008 by Thieme Medical Publishers, Inc., 333 Seventh Avenue, New York, NY 10001, USA. Tel: +1(212) DOI /s ISSN

2 ACTIVATED PARTIAL THROMBOPLASTIN TIME/LIPPI, FAVALORO 605 maintain vessel patency. The intricate model of blood coagulation was originally developed by two independent groups in the early 1960s 2,3 and involves several coordinated and calcium-dependent conversions of proenzymes to their respective serine proteases, culminating in the conversion (activation) of prothrombin to thrombin (Fig. 1). According to the most recent evidence, the blood coagulation system initiates with activation of the traditional extrinsic pathway by exposure of tissue factor on specific tissue surfaces and further develops through preliminary thrombin generation, catalyzed by an enzymatically active complex between activated factor VII and tissue factor. There is a small amount of thrombin generated during the initial step, which is necessary for propagation and amplification of the subsequent enzymatic reactions (the thrombin burst ) and promotes the feedback activation of the traditional intrinsic pathway characterized by generation of numerous active enzymatic complexes, conversion of fibrinogen to fibrin, and fibrin polymerization and stabilization by coagulation factor XIII and thrombin-activatable fibrinolysis inhibitor. The attenuation and termination of the blood coagulation is sustained by natural inhibitors, which regulate the blood clot growing process. The final dissolution and elimination of the blood clot is facilitated by a highly regulated enzymatic cascade called fibrinolysis. 1 The in vivo success of the coagulation cascade is modeled in vitro by two basic coagulation tests: the prothrombin time (PT) and the activated partial thromboplastin time (APTT). Both tests are variably sensitive to abnormalities of the common pathway, but the PT also explores the function of the extrinsic pathway, and the APTT reflects abnormalities of the intrinsic pathway. The APTT can hence be considered a global clotting test sensitive to several plasma inhibitors and acquired or inherited deficiencies of coagulation factors of the intrinsic and common pathway. 1 The APTT, developed in 1953 by Langdell, Wagner, and Brinkhous, 4 has defined an era in coagulation medicine. In 1961, Proctor and Rapaport modified the test using kaolin as the contact activator, which had led to its adaptation in coagulation laboratories throughout the world. 5 Subsequent to the development of an easy and reproducible APTT assay came the development of specific factor one-stage assays using factor-deficient plasmas, and then began the important process of purifying the various coagulation plasma factors, leading ultimately to their characterization, the identification of the sequence of reactions resulting in the cascade hypothesis, and the generation Figure 1 Physiology of blood coagulation and its relationship with the in vitro tests prothrombin time and activated partial thromboplastin time (TF, tissue factor; F, factor).

3 606 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER of therapeutic concentrates. 6 The specific name of the APTT, which includes the term partial, reflects the absence of tissue factor from the reaction mixture, which otherwise would characterize the PT. The specimen required for the APTT is a blood sample anticoagulated by buffered sodium citrate at a final concentration of to mol/l (also known as 3.2%), as currently recommended by the Clinical and Laboratory Standards Institute (CLSI). 7 A variety of APTT reagents are currently available, and these vary widely in composition. Many different preparations of phospholipids, from human, animal, and vegetable origin, are also used, and APTT reagents also differ in the surface activators (such as kaolin, celite, silica, and ellagic acid) as well as manufacturer recommended incubation times. 8 After separation of plasma by centrifugation at 1500 g for no less than 15 minutes at room temperature, phospholipids and activators are mixed with the plasma and act to shorten the clotting time by providing a catalytic, negative charge. The further addition of calcium initiates the coagulation reaction, which is thence monitored (either visually for manual methods or more typically by the instrument) until a clot develops (Fig. 2). For automated methods of clot detection, clot formation is usually deemed to have occurred when the optical density of the mixture has exceeded a certain threshold (clot formation makes the mixture more opaque and less light passes through). Less frequently, clot formation is detected by a mechanical method (e.g., reduction or cessation of movement by small metal balls, detected by magnets as the clot forms and restricts such movement). Results are reported as the clotting time end point in seconds. 9 Reference values for APTT vary widely among laboratories but generally range between 25 and 38 seconds. The APTT of a newborn will usually be prolonged and may be up to 55 seconds at birth, decreasing to the adult range by 6 months of age, but they are still lower than those of the adult population both in the premature and the full-term newborn. 10 Like most other tests of hemostasis, the normal reference range for the APTT is generally obtained as the mean 2 standard deviations of a large number of Figure 2 A schematic depicting the APTT test and its relationship to various clinical conditions. APTT reagents contain a source of phospholipids and contact activator. When added to plasma, contact activation occurs, and subsequent addition of CaCl 2 permits clot formation. The time to clot (the APTT) is identified by sensors within the coagulation analyzer. A prolonged APTT is classically deemed significant and may be associated with many important clinical events. A short APTT may also now be considered as potentially significant and worthy of investigation. It is important to distinguish genuine cases of prolonged and shortened APTTs from in vitro artifacts arising from collection issues and other preanalytical events.

4 ACTIVATED PARTIAL THROMBOPLASTIN TIME/LIPPI, FAVALORO 607 (preferably 100 or more) normal individuals. Because most tests of hemostasis show non-gaussian distributions, the data usually requires (e.g., logarithmic) transformation to enable derivation of data. In any case, it is important to recognize that, by definition, around 2.5% of normal individuals will have APTTs above the normal range, and around 2.5% of normal individuals will have APTTs below the normal range. 11 PROLONGED APTT: WHAT DOES IT TELL US? Occasionally, a prolonged APTT will just reflect a normal range variation effect, a specimen or analytical issue or some other preanalytical issue. 11 Alternatively, a prolonged APTT can reflect a significant clinical scenario. Regardless of the substantial heterogeneity in the composition of APTT reagents, there are some crucial requisites that should be fulfilled: they should not miss the mild or moderate factor deficiency (including inhibitors to these factors), they should be sensitive to the therapeutic dosage of heparin, and generally they should enable detection of inhibitors like lupus anticoagulants, in which case a low-phospholipid-containing APTT reagent or a lupus-sensitive APTT reagent might be preferred. 9,12,13 Sometimes laboratories select more than one type of APTT reagent for differential purposes. For example, a laboratory might specifically choose to use a lupus-insensitive APTT reagent for general use to avoid identification of asymptomatic lupus-positive cases, which will otherwise be a frequent finding if screening elderly populations for preoperative bleeding risk, and as this will lead to surgical delays and unnecessary and costly investigations. More recently, the combined differential use of lupus-sensitive versus lupus-insensitive APTT reagents provides another integrated means of differentially identifying lupus anticoagulants. 14 Ideally, the APTT is prolonged when levels of coagulation factor activity fall below the 95% confidence limit of the reference interval. However, several studies have shown considerable differences in the sensitivity of the various APTT reagents to mild and moderate factor deficiencies. Meaningful differences have been reported for the ability of different APTT reagents to identify mild deficiency of coagulation factor VIII and factor IX. Some reagents showed abnormal APTT results in mild cases of factor VIII and factor IX deficiency without producing a large number of falsely prolonged APTT with normal plasma. 13 Also, some reagents are oversensitive to factor XII deficiency, and low factor XII levels (i.e., < 50%) are a common finding in otherwise normal individuals. 15 A similarly variable sensitivity of the APTT to circulating lupus anticoagulants has been reported. Likewise, marked APTT variability in responsiveness to heparin has been observed among commercially available APTT reagents. 9 SHORT APTT: WHAT DOES IT TELL US? Looking at this story thus far, it might be concluded that APTT results have been taken as an index of loss-offunction and much less as an index of gain-of-function of the coagulation factors, in that until recently only abnormal prolongations of this test have been considered clinically meaningful. 16 However, recent clinical and biological evidence indicates that shortened APTT results also deserve scrutiny. A short APTT occurs in nearly 6 to 9% of routine APTTs, a proportion that might be strongly influenced by the sensitivity of the APTT reagent used, and thus rising up to 15%. 17,18 Although the clinical significance of prolonged APTTs is well established, the clinical significance of short APTTs has not been fully acknowledged to date. Like the prolonged APTT, a short APTT will occasionally just reflect a normal range variation effect, a specimen or analytical problem or some other preanalytical issues. 11 However, several studies have now indicated that a short APTT might be considered either as a marker or a risk factor for hypercoagulability. A historical prospective study found that a short APTT is related to a 10-fold increased incidence of thromboembolic events assessed by chart review on discharge for 100 medical and surgical inpatients. 19 Reddy et al reported that 25% of patients with short APTTs will develop thrombotic events within 1 year of detection, whereas in the control group the incidence was < 3%. 20 Korte et al reported that patients with short APTTs are at increased risk for thromboembolism, mainly venous thromboses, despite the fact that a short APTT can occur in the acute setting of bleeding. This finding was interesting and mostly unexpected, because 36% of patients with a short APTT and a bleeding episode during hospitalization also had thromboembolic events during the 18 months proximate to the blood collection. 17 In a further study on 605 patients referred for thrombophilia testing after documented venous thromboembolism (VTE) and 1290 controls, median APTT ratio values were significantly shorter in patients (0.97) than in controls (1.00; p < 0.001). In patients who had an APTT ratio lower than the fifth percentile of the distribution in controls, the odds ratio for VTE was 2.4 and was independent of inherited thrombophilic abnormalities. 21 Measurement of APTT allows stratification of patients with VTE into high- and low-risk categories with regard to recurrence. Patients without recurrence have a greater APTT ratio than do those with recurrence (0.97 vs. 0.93). After 4 years, the probability of recurrent VTE is 8.5% among patients with a ratio 0.95 and 15.6% among patients with a lower ratio. Compared with patients with an APTT ratio < 0.95, the relative risk of recurrence among patients with a ratio 0.95 is 0.56 before and 0.58 after adjustment for sex, age, factor V Leiden, and factor II G20210A. 22 These findings were supported by the study of Legnani et al, who observed that the VTE recurrence

5 608 SEMINARS IN THROMBOSIS AND HEMOSTASIS/VOLUME 34, NUMBER Q1 rate was 17.5% and 7.5% in patients with APTT in the lower (ratio 0.90) and in the upper (ratio > 1.05) quartiles. The recurrence risk was more than 2-fold higher in patients with ratio 0.90 versus those of the reference category (relative risk: 2.38). 23 Regardless of the epidemiologic association with venous thromboembolism, shortened APTT on admission also predicts overall in-hospital mortality (odds ratio ¼ 2.6); nonsurvivors with short APTTs have significantly higher plasma D-dimer, C-reactive protein, and glucose levels compared with those of survivors. 24 Whereas the clinical relationship between a short APTT and the risk of venous thrombosis is adequately supported by the current scientific literature, less is known about the biological mechanisms that mediate this epidemiologic association. A shortening of the APTT may result from accumulation of circulating activated coagulation factors in plasma caused by enhanced coagulation activation in vivo. Interestingly, short APTTs have been described in a significant number of patients with low-grade disseminated intravascular coagulation, supporting the above comments. 25 Accordingly, Korte et al showed that a short APTT was indeed related to an increase in thrombin generation, as substantiated by the significantly increased prothrombin fragment F1 þ 2 Q1, a molecular marker reflecting activation of coagulation and fibrinolytic systems. 17 In this study, the presence of an abnormally short APTT associated with increased prothrombin fragment 1 þ 2 levels in patients with some type of bleeding during hospitalization was explained as a physiologic response of the coagulation system to an abnormal blood loss. Ten Boekel and Bartels showed that plasma levels of prothrombin fragment 1 þ 2, thrombin-antithrombin complex, D-dimer, and factor VIII are markedly higher in subjects with short APTTs compared with those displaying normal APTTs. The APTT values were also inversely related to prothrombin fragment 1 þ 2, thrombin-antithrombin complex, D-dimer, and factor VIII levels, though the prevalence of G1691A factor V and G20210A-prothrombin gene mutation did not influence APTT values. 26 In the study of Legnani et al, the increase in recurrence risk disappeared after adjustment for factor VIII, IX, and XI levels (relative risk, 1.74), but the risk was persistently increased in patients with a ratio 0.90 (relative risk, 2.07) after adjustment for age, gender, inherited thrombophilic alterations, and D-dimer level. 23 Mansvelt et al reported, however, that elevated factor VIII levels are associated with relatively shorter APTTs although they remain within the reference range of healthy subjects, thus concluding that factor VIII may not be the sole determinant of a short APTT. 27 This hypothesis was further confirmed by Tripodi et al, who demonstrated that adjustment for blood group, the presence of inherited thrombophilia, and factor VIII levels only reduces the odds ratio for venous thromboembolism associated with shortened APTT from 2.7 to 2.1, indicating that the risk might be partially mediated by high factor VIII levels. 21 Because the majority (90%) of the patients with short APTTs will have normal values if measured several weeks or months later, it is hypothesized that short APTTs might reflect a temporary imbalance in the coagulation system associated with an increased thrombotic tendency. 26 The potential hypercoagulable state reflected (or detected) by a short APTT might also be extended beyond the epidemiologic association with VTE. Madi et al observed that a shortened APTT on presentation in patients with chest pain is associated with increased risk of acute myocardial infarction. Patients with a short/ normal APTT were significantly more likely to be diagnosed with myocardial infarction than were those with a prolonged APTT (relative risk, 2.83). 28 Although there is an acceptable agreement between the different commercial reagents for detection of short APTTs, a disparity between some of the reagents still persist. 18 In particular, shortened APTTs measured with Actin-FS reagent (Dade Behring, Marburg, Germany) are associated with enhanced concentrations of coagulation factors (factors VIII, IX, XI) accompanied with a moderate degree of coagulation activation, whereas shortened APTTs measured with other reagents such as HemosIL APTT-SP (Instrumentation Laboratory, Milano, Italy), Automated-APTT (biomerieux, Marcy l Etoile, France) and Platelin-LS (biomerieux) are associated with enhanced levels of coagulation factors but accompanied with a high degree of coagulation activation. 18 This finding led Ten Boekel et al Q2 to conclude that, due to the significant number of patients with high levels of coagulation factors missed when APTT is measured with some reagents, it is still unknown whether the association between short APTT and VTE pertain to specific APTT reagents or may be extrapolated to all those available on the market. The issue has been recently addressed in a case-control study investigating six widely used commercial APTT reagents for their ability to detect hypercoagulability in patients with a previous episode of deep vein thrombosis. Although the relative risk (expressed as odds ratio) estimated with different reagents was varied, all of them displayed odds ratio greater than one. The 95% coefficients of interval were relatively narrow with values encompassing the unity in only two of the six. These results suggest that the results obtained with one APTT may probably be applied to other commercial reagents. 16 WAVEFORM ANALYSIS: WHAT DOES IT TELL US? The value of the APTT has generally been well described in relation to its clot end-point determination. Q2

6 Q3 ACTIVATED PARTIAL THROMBOPLASTIN TIME/LIPPI, FAVALORO 609 However, the optical profile of clot formation in the APTT can be atypical in patients with hemostatic dysfunction. The so-called biphasic waveform was initially described when a decrease in plasma light transmittance before clot formation on the MDA-180 Q3 automated coagulation analyzer was shown to correlate with disseminated intravascular coagulation (DIC) in critically ill patients. 29,30 In contrast with the normal sigmoidal waveform pattern, which is characterized by an initial 100% light transmittance phase before clot formation, patients with a biphasic pattern had an immediate, progressive decrease in light transmittance that occurred even in the preclotting phase. The magnitude of this biphasic waveform often varies with sequential samples taken from individual patients and appears early in samples from patients who were later diagnosed with DIC by more conventional criteria. 31 The biphasic waveform depends on a substantially elevated level of C- reactive protein and levels of very-low-density lipoproteins or intermediate-density lipoproteins adequate to rapidly form a macroscopic precipitate on recalcification. This complex can also be induced by incubating acutephase serum containing elevated levels of C-reactive protein with serum from type III hyperlipidemic patients. 32 Whether the C-reactive protein and lipoproteins complex contributes to, or merely reflects, DIC is still a matter of debate. Mounting evidence, however, suggests that biphasic waveform might correlate with DIC and mortality. Matsumoto et al showed that the prevalence of the biphasic waveform is particularly high in patients with DIC and infection: 86.4% and 75.0% for DIC diagnosed by the International Society on Thrombosis and Haemostasis (ISTH), and the Japanese Ministry of Health and Welfare (JMHW) scores, respectively. Odds ratios are 29.9 and 19.0 for ISTH and JMWH scores, respectively. 33 Overall, Matsumoto et al concluded that biphasic waveform shows a moderate sensitivity (59.2% for the ISTH score; 47.9% for the JMWH score), but a high specificity (95.4% for both scores). 33 In a separate investigation, an abnormal APTT waveform was seen in 32% of patients with a clinical suspicion of DIC and correlated well with the presence of disease (sensitivity 88%, specificity 97%). In 19% of patients, the APTT biphasic waveform-based diagnosis of DIC preceded the diagnosis based on the traditional ISTH scoring system. 34 Interestingly, biphasic waveform shows superior prediction over C-reactive protein alone, 35 and the combination with biphasic waveform also increases the specificity of procalcitonin testing. 36 Therefore, when the specific analyzer is available in intensive care units, APTT waveform analysis might be a promising, inexpensive, and rapid tool to provide additional information for the diagnosis of severe sepsis and the prognosis of septic patients Emerging evidence also suggests that the hemostatic response to factor VIII infusion and coagulationinhibitor bypassing agents in hemophilia A patients with high responding inhibitors may be monitored accurately and efficiently by clot waveform analysis. 40,41 However, biphasic waveform analysis is currently limited to select instrumentation and may thus be limited in general application. CONCLUSION The APTT is the most common coagulation procedure performed in routine laboratories, apart from the PT. The test is traditionally used for identifying quantitative and qualitative abnormalities in the intrinsic and common pathways of coagulation, monitoring anticoagulant therapy with unfractionated heparin, and detecting inhibitors of blood coagulation, the most common of which is the lupus anticoagulant. Recent evidence also suggests an emerging role for this test, in that (i) waveform analysis may enable identification of sepsis or disseminated intravascular coagulation in critically ill patients and (ii) shortened APTT values might reflect the presence of a hypercoagulable state, which is independently associated with VTE and, possibly, acute coronary syndrome. Taken together, these novel findings suggest that this traditional, simple, and inexpensive test might have several new indications including, for example, potential inclusion in the screening for thrombophilia and evaluating the risk of VTE. 21 Given the availability of specific instrumentation,mountingevidencealsosuggeststhatwaveform analysis of the APTT might be an additional and inexpensive aid in risk assessment of critical care patients, in whom development of DIC is known to worsen the prognosis. ABBREVIATIONS APTT activated partial thromboplastin time CLSI Clinical and Laboratory Standards Institute DIC disseminated intravascular coagulation ISTH International Society on Thrombosis and Haemostasis JMHW Japanese Ministry of Health and Welfare PT prothrombin time VTE venous thromboembolism REFERENCES 1. Lippi G, Franchini M, Guidi GC. Diagnostic approach to inherited bleeding disorders. Clin Chem Lab Med 2007;45: MacFarlane RG. 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8 ACTIVATED PARTIAL THROMBOPLASTIN TIME/LIPPI, FAVALORO 611 lipoprotein-complexed C reactive protein formation. Intensive Care Med 2003;29: Zakariah AN, Cozzi SM, Van Nuffelen M, Clausi CM, Pradier O, Vincent JL. Combination of biphasic transmittance waveform with blood procalcitonin levels for diagnosis of sepsis in acutely ill patients. Crit Care Med 2008;36: Chopin N, Floccard B, Sobas F, et al. Activated partial thromboplastin time waveform analysis: a new tool to detect infection? Crit Care Med 2006;34: Dempfle C-E, Borggrefe M. The hidden sepsis marker: aptt waveform analysis. Thromb Haemost 2008;100: Hussain N, Hodson D, Marcus R, Baglin T, Luddington R. The biphasic transmittance waveform: an early marker of sepsis in patients with neutropenia. Thromb Haemost 2008; 100: Kasuda S, Tanaka I, Shima M, et al. Effectiveness of factor VIII infusions in haemophilia A patients with high responding inhibitors. Haemophilia 2004;10: Shima M. Understanding the hemostatic effects of recombinant factor VIIa by clot wave form analysis. Semin Hematol 2004;41:

9 Author Query Form (STH/01450) Special Instructions: Author please write responses to queries directly on proofs and then return back. Q1: Au: Check term: fragment F1 þ 2 or rather fragment 1 þ 2 as elsewhere in text? Q2: Au: Cite here Ten Boekel et al reference by number. Q3: Au: Provide name and location of MDA-180 manufacturer. Q4: Please spell out NCCLS in reference 9 and check location. Q5: Editor: provide page range at page proof stage (in reference 11 "Favaloro, Lippi, Adcock, 2008"). Q6: EDITOR: Update Ref. 14 at page proof stage.

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11 Thieme Medical Publishers, Inc. (the Publisher ) will be pleased to publish your article (the Work ) entitled in the Seminars in Thrombosis and Hemostasis, Volume 34, Number 7, The undersigned Author(s) hereby assigns to the Publisher all rights to the Work of any kind, including those rights protected by the United States Copyright laws. The Author(s) will be given permission by the Publisher, upon written request, to use all or part of the Work for scholarly or academic purposes, provided lawful copyright notice is given. If the Work, subsequent to publication, cannot be reproduced and delivered to the Author(s) by the publisher within 60 days of a written request, the Author(s) is given permission to reprint the Work without further request. The Publisher may grant third parties permission to reproduce all or part of the Work. The Author(s) will be notified as a matter of courtesy, not as a matter of contract. Lawful notice of copyright always will be given. Check appropriate box below and affix signature. [ ] I Sign for and accept responsibility for transferring copyright of this article to Thieme Medical Publishers, Inc. on behalf of any and all authors. Author s full name, degrees, professional title, affiliation, and complete address: Author s printed name, degrees Professional title Complete professional address Author s signature Date [ ] I prepared this article as part of my official duties as an employee of the United States Federal Government. Therefore, I am unable to transfer rights to Thieme Medical Publishers, Inc. Author s signature Date

12 Order Form for Offprints and additional copies of the Seminars in Thrombosis and Hemostasis (Effective October 2005) Please circle the cost of the quantity/page count you require (orders must be in increments of 100) Pages in Article / Cost Quantity 1 to 4 5 to 8 9 to to to $198 $317 $440 $578 $ $277 $444 $615 $809 $ $356 $570 $791 $1,041 $1, $396 $634 $879 $1,156 $1, $446 $713 $989 $1,301 $1, $792 $1,267 $1,758 $2,313 $2,772 Volume/Issue #: Page Range (of your article): Article Title: MC/Visa/AmEx No: Exp. Date: Signature: Name: Address: City/State/Zip/Country: Corresponding author will receive one complimentary copy of the issue in which the manuscript is published. Number of additional copies of the journal, at the discounted rate of $20.00 each: Notes 1. The above costs are valid only for orders received before publication of the issue. Please return the completed form, even if your institution intends to send a Purchase Order (the P.O. may sometimes be supplied after the issue has been printed). 2. Orders from outside the U.S. must be accompanied by payment. 3. A shipping charge will be added to the above costs. 4. Reprints are printed on the same coated paper as the journal and side-stapled. 5. For larger quantities or late orders, please contact reprints dept. Phone: +1(212) Fax: +1(212) reprints@thieme.com As an added benefit to all contributing authors, a discount is offered on all Thieme books. See below for details or go to

13 As a Thieme author you are entitled to a 25% discount for new books and a 35% discount for forthcoming books. We selected two books that might be of interest for you: new! 25% forthcoming! 35% Thurlbeck's Pathology of the Lung 3 rd Edition Andrew M. Churg, M.D. Ph. D. Professor of Pathology, University of British Columbia; Pathologist, Vancouver Hospital & Health Sciences Center, Vancouver, BC, Canada Thurlbeck's cornerstone textbook and reference on pulmonary pathology returns in a brand new edition! Updated with the latest advances in the field, you will save time with all-inclusive coverage of neoplastic, non-neoplastic, infectious, occupational/environmental, and developmental pathologies in one book, learn how molecular biology provides a greater understanding of lung development, gain new insights into the diagnosis of neoplastic and non-neoplastic lung disease, find pertinent information on clinical features, epidemiology, and pathogenetic mechanisms of lung disease and much more! Comprehensive in its scope and authoritative in its scholarship, Thurlbeck's Pathology of the Lung is a virtual one-volume encyclopedia written by a ''who's who'' list of specialists. It is the one text that no pathologist, pulmonologist, or resident in either specialty can afford to be without. Vascular Diagnosis with Ultrasound Cerebral and Peripheral Vessels Michael Hennerici, M.D. Professor and Chairman, Department of Neurology, University of Heidelberg, Mannheim, Germany Covering the entire venous and body circulation as examined by vascular ultrasound, this unique text/atlas is invaluable for diagnosing arterial and venous disease. It includes comprehensive chapters on vascular ultrasonography in the arteries and veins of the cerebral circulation and the peripheral upper and lower limb circulation, systematic coverage of all available ultrasound technologies, including continuous and pulsed-wave Doppler mode, b-mode, and conventional and color-coded duplex analysis in frequency and amplitude power modes, anatomy and physiology, normal and abnormal findings, test accuracy and sensitivity, pitfalls, and comparison with other diagnostic tests in each vascular region and special, difficult-to-interpret cases discussed in a separate section 2005, app.1032 pp., app.1064 illus., hardcover $ $ , 336 pp., 530 illus., hardcover, $ $97.47 ISBN ISBN If you want to view more Thieme books, fell free to visit Thieme Books Thieme Author order form For faster service, call TOLL-FREE or fax this order form to Quantity ISBN (last 4-digits only) Author/Title Price Subtotal: Shipping & Handling (Add $7.50 for the first book and $1.00 for each additional book): NY and PA residents add applicable sales tax: TOTAL: Enclosed is my check for $ Charge my: AMEX MasterCard VISA Discover Card# Exp. First Name MI Last Name Address City/State/Zip Telephone FAX Signature EM1-05