DOI 10.1515/cclm-2012-0387 Clin Chem Lab Med 2013; 51(1): 91–97

Review

Giuseppe Lippi * and Emmanuel J. Favaloro Laboratory hemostasis: milestones in and Laboratory

A b s t r a c t conditions or to trigger clot formation to prevent excessive when the integrity of the blood vessels is jeopard- ized. A fine balance between these opposite forces is essen- Hemostasis is a delicate, dynamic and intricate system, in tial (Figure 1 ). An excessive prevalence of anticoagulant which pro- and anti-coagulant forces cooperate for either forces following blood vessel injury would lead to various maintaining blood fluidity under normal conditions, or degrees of hemorrhage that can reach even life-threaten- else will prompt blood clot generation to limit the bleeding ing severity, whereas an excessive activity of procoagulant when the integrity of blood vessels is jeopardized. Excessive forces under physiological conditions (e.g., in the lack prevalence of anticoagulant forces leads to hemorrhage, of blood vessel damage) would instead lead to extensive whereas excessive activation of procoagulant forces trig- (excessive) and consequent . gers excessive coagulation and thrombosis. The hemostasis The definition of hemorrhagic disorders entails a hetero- laboratory performs a variety of first, second and third line tests, and plays a pivotal role in diagnostic and monitoring geneous range of inherited and acquired conditions charac- of most hemostasis disturbances. Since the leading targets terized by abnormal and/or excessive bleeding. Basically, of Clinical Chemistry and Laboratory Medicine include pro- bleeding disorders are classified according to the phase of motion of progress in fundamental and applied research, hemostasis that is principally affected, so that they can be along with publication of guidelines and recommendations typically divided into disorders of primary or secondary in laboratory diagnostics, this journal is an ideal source of hemostasis. The former class comprehends most frequently information on current developments in the laboratory tech- as well as congenital or acquired disor- nology of hemostasis, and this article is aimed to celebrate ders of function, whereas the latter conditions are some of the most important and popular articles ever pub- mostly due to inherited or acquired abnormalities of clot- lished by the journal in the filed of laboratory hemostasis. ting factors, that can be also categorized as quantitative, qualitative or both. (VWD) is char- Keywords: bleeding; coagulation; hemostasis; laboratory acterized by qualitative or quantitative abnormalities of von testing; thrombosis. Willebrand factor (VWF), and characteristically expresses disturbances of primary and secondary hemostasis (Figure 2 ) [1, 2] . In terms of frequency, the most recent survey of the *Corresponding author: Prof. Giuseppe Lippi, U.O. Diagnostica World Federation of Hemophilia (WFH) reports that the Ematochimica, Dipartimento di Patologia e Medicina di Laboratorio, total number of people with bleeding disorders identified Azienda Ospedaliero-Universitaria di Parma, Via Gramsci 14, 43126 Parma, Italy, E-mail: [email protected] is 242,517, whereas those carrying hemophilia A and B are Emmanuel J. Favaloro: Institute of Clinical and Medical 153,251, those with VWD are 62,158 and those carrying other Research (ICPMR) , Department of Haematology, Westmead Hospital, bleeding disorders are 27,030 [3] . These figures, however, are Westmead , Australia likely to underestimate the true incidence of bleeding disor- ders, because of diagnostic challenges worldwide [2, 4] . Thrombosis is a highly complex and multifaceted phe- nomenon. Although blood clots can potentially develop in

Introduction on physiological almost each and every human vessel containing , and pathological hemostasis coagulant factors and prothrombotic substances [5] , lym- phatic thrombosis is extremely rare [6] so that the leading Physiological hemostasis is a complex and multifaceted clinical manifestations of thrombosis typically encom- system, whereby pro- and anti-coagulant forces actively passes venous [7] and arterial occlusion [5] (Figure 3 ). cooperate to either preserve blood fluidity under normal Although the final event is virtually identical between 92 Lippi and Favaloro: Hemostasis testing: milestones in Clinical Chemistry and Laboratory Medicine

Hemostatic balance Laborator y hemostasis

Laboratory diagnostics plays an essential role in the diag- nosis, follow-up and therapeutic monitoring of most – if not all – hemostasis disturbances. Laboratory hemo- stasis basically entails first, second and third line tests Procoagulant Anticoagulant [11] , which find their suitable collocation within well- established diagnostic algorithms. First line analyses, which are also conventionally called “ screening ” tests, include the activated partial thromboplastin time (APTT) Thrombosis Bleeding [12 – 14] , prothrombin time (PT)/International Normal- ized Ratio (INR) [15 – 17] , fibrinogen [18, 19] , D-dimer [20, 21], and platelet count [22] . Thrombin generation assays

Figure 1 The hemostatic balance: a delicate equilibrium between [23, 24] along with thromboelastography [25, 26] are addi- pro- and anti-thrombotic forces. tional useful “ global ” tests of hemostasis, which have not found, however, widespread application in clinical and these two blood districts, i.e., the generation of a throm- laboratory practice. Second line assays are typically those bus that may in part or completely obliterate the lumen designed to provide further insights into abnormalities of of the vessel (i.e., cause vascular occlusion), the patho- screening tests, or used to monitor more accurately some genesis, the risk factors, the composition of the antithrombotic , and thereby include clotting as well as the clinical are different factors assays [27] , ristocetin-induced platelet agglutina- between veins and arteries, with very modest overlap [8] . tion and VWF antigen tests [28] , anticardiolipin (aCL) IgG Regardless of its origin, the consequences of both types of and IgM, anti- β (2) glycoprotein I (anti-β (2) GPI) antibodies thrombosis, i.e., for venous throm- IgG and IgM and phospholipid-dependent coagulation bosis (VTE) and myocardial infraction or stroke for arterial assays [29, 30] , platelet function tests such as Platelet Func- thrombosis-are the leading causes of death worldwide. The tion Analyzer-100 (PFA-100) and aggregometry [31, 32], wide group of consequent to arterial throm- assays for -induced thrombocytopenia [33, 34], bosis (i.e., cardiovascular disease which encompasses additional tests for screening including acute coronary syndrome, cerebral ischemia and periph- resistance to activated protein C, antithrombin, proteins eral artery occlusive disease) are in fact the first cause of C and S, and genetic polymorphisms/mutations (e.g., death and morbidity in western countries, whereas VTE, and ) [35, 36] along which include both (DVT) and pul- with ecarin clotting time, chromogenic anti-factor Xa and monary embolism (PE), is the leading cause of death and dilute Russell viper venom time (dRVVT) for monitoring morbidity in hospitalized patients [9, 10] . novel anticoagulants [37, 38]. Both first and second line

Primary hemostasis • Thrombocytopenia • Qualitative platelet disorders

Bleeding • von Willebrand disease

Secondary hemostasis • A and B • Haemophilia C • Other rarer bleeding disorders

Figure 2 Classification of bleeding disorders. Lippi and Favaloro: Hemostasis testing: milestones in Clinical Chemistry and Laboratory Medicine 93

Blood vessel

Thrombosis

Venous Arterial

Deep vein thrombosis Myocardial infraction Pulmonary embolism stroke Peripheral occlusive disease

Venous thromboembolism Cardiovascular disease

Figure 3 Pathogenesis of venous and arterial thrombosis. tests might be available to most clinical laboratories, preanalytical quality issues include order of collection whereas third line tests – which are intended to trouble- and appropriate filling of primary collection tubes [55] . shoot the most challenging conditions and encompass These two aspects are pivotal in order to prevent cross- analyses such as VWF collagen binding, VWF ristocetin contamination between sequential tubes and appropri- cofactor assay, VWF-FVIII binding assay, multimer and ate combination of blood and additive. Similar emphasis molecular analysis for the precise classification of VWD can be placed on sample processing in hemostasis [56] . [39, 40], coagulation factors inhibitors testing [41, 42], Although there is large evidence to support implementa- analyses of rare thrombophilic mutations [43] , rare plate- tion and monitoring of analytical quality specifications let functional disorders [44] , pharmacogenetics testing in clinical chemistry, less data has become available for [45, 46] – are occasionally used and typically available in coagulation testing and further efforts should be planned specialized laboratories. for improvement. The leading objectives of Clinical Quality in laboratory diagnostics is irrecusable, since Chemistry and Laboratory Medicine include promotion spurious results obtained on unsuitable specimens may of progress in fundamental and applied research, along negatively bias the clinical decision-making and jeopard- with publication of guidelines and recommendations in ize patient safety. Laboratory errors may develop from laboratory diagnostics. As such, since the journal is an any step throughout the testing process [47, 48], but are ideal source of information on current developments in characteristically prevalent in the manually intensive the medical and laboratory technology arena, this article activities of the preanalytical phase [49 – 51] . Although the is aimed to celebrate some of the most important and classical model of the “ brain-to-brain loop ” applies to all popular articles ever published by the journal in the filed fields of laboratory medicine, its significance is substan- of laboratory hemostasis. tial for hemostasis testing [48] . The vast armamentarium of laboratory hemostasis share some basic requisites and quality characteristics of traditional clinical chemistry, immunochemistry and diagnostics, but there Laboratory hemostasis throughout are also some specific preanalytical, analytical and posta- Clinical Chemistry and Laboratory nalytical requirements [52 – 54] . Basically, the sample matrix of clotting tests is peculiar since the clotting assays Medicine history require the use of buffered sodium citrate plasma, with the anticoagulant provided preferably at a final concen- The selection of the most representative hemostasis tration of 3.2% (i.e., 105 – 109 mM). Additional and specific articles published in Clinical Chemistry and Laboratory 94 Lippi and Favaloro: Hemostasis testing: milestones in Clinical Chemistry and Laboratory Medicine

Medicine since its founding in 1976 (formerly as Journal of In 1998 Heil et al. carried out an influential study that Clinical Biochemistry and Clinical Biochemistry, and then is still the basis for several guidelines and recommenda- European Journal of Clinical Biochemistry and Clinical Bio- tions about coagulation testing [59] . Basically, the authors chemistry) is obviously challenging and may be somehow determined the influence of storage time and temperature arbitrary when objective criteria are not applied. As such, on PT, APTT, thrombin time (TT), fibrinogen, factors V and we chose to base our initial selection according to one of VIII, antithrombin, protein C and S in plasma from healthy the most unbiased standards, that is the number of cita- subjects and patients receiving heparin . When tions retrieved from the Thomson (former ISI) database plasma stability was defined as the period during which (Table 1 ) (Thomson Reuters, Web of Science, available at: a change < 10% from the initial value could be recorded, http://thomsonreuters.com), using the keywords “ Clini- significant biases were reported after 8 h for APTT, 24 h for cal Chemistry and Laboratory Medicine” and/or “ Journal PT, 48 h for factor V and 7 days for TT, fibrinogen, protein of Clinical Biochemistry and Clinical Biochemistry” and/ C and antithrombin in healthy subjects when plasma or “ European Journal of Clinical Biochemistry and Clini- was held at 6° C. In volunteers untreated with heparin cal Biochemistry” and/or “ hemostasis ” and/or “ coagula- therapy, APTT was stable for 8 h, PT for 48 h, TT, fibrin- tion” . The articles will be briefly described, in chronologi- ogen and antithrombin for 7 days with plasma sample cal order. storage at room temperature. In patients under therapy Probably, the foremost article ever to appear in the with heparin, the stability in plasma stored at 6° C was journal and dealing with coagulation and hemostasis, was 8 h for TT, 24 h for PT and APTT and 7 days for fibrinogen published by van Wersch et al. in 1990 [57] . The authors and antithrombin. Factors V and VIII displayed a reduc- investigated 22 patients with exacerbation of inflamma- tion of 13% and 20 % after 8 h. Nevertheless, when plasma tory bowel disease and found that most of them exhibited was stored at room temperature, factor V was stable for abnormal platelet function (i.e., non-hyperaggregability 8 h, and PT for 24 h, whereas fibrinogen and antithrombin by ADP 2 μmol/L), as well as enhanced fibrinogen and remained unchanged for up to 7 days. The APTT showed D-dimer concentrations. It was hence shown that there an increase of 13% , TT a fall of 16 %, and factor VIII a was a high prevalence of prothrombotic factors in patients decrease of 18% after 8 h. Another milestone published with exacerbation of inflammatory bowel disease. One in the journal is the comprehensive overview by Ros é n year later, the same team of authors published another and Sturk, about the recently (at that time) discovered seminal article about blood coagulation and fibrinolysis resistance to activated protein C [60] . Another important during normal pregnancy and provided valuable clues review article was that published by Korte in 2000 and about activation of coagulation and fibrinolytic systems dealing with changes of the coagulation and fibrinoly- with ongoing pregnancy [58] . sis system in malignancy and their possible impact on

Articles Citations van Wersch JW, Houben P, Rijken J. Platelet count, platelet function, coagulation activity and fibrinolysis in the acute phase 50 of inflammatory bowel disease. J Clin Chem Clin Biochem 1990;28:513 – 7. van Wersch JW, Ubachs JM. Blood coagulation and fibrinolysis during normal pregnancy. Eur J Clin Chem Clin Biochem 48 1991;29:45 – 50. Ros é n SB, Sturk A. Activated protein C resistance – A major risk factor for thrombosis. Eur J Clin Chem Clin Biochem 36 1997;35:501 – 16. Korte W. Changes of the coagulation and fibrinolysis system in malignancy: their possible impact on future diagnostic and 25 therapeutic procedures. Clin Chem Lab Med 2000;38:679 – 92. Lippi G, Franchini M, Guidi GC. Diagnostic approach to inherited bleeding disorders. Clin Chem Lab Med 2007;45:2 – 12. 24 Heil W, Grunewald R, Amend M, Heins M. Influence of time and temperature on coagulation analytes in stored plasma. Clin 22 Chem Lab Med 1998;36:459 – 62. Favaloro EJ, Lippi G, Franchini M. Contemporary platelet function testing. Clin Chem Lab Med 2010;48:579 – 98. 20 Samama MM, Guinet C. Laboratory assessment of new anticoagulants. Clin Chem Lab Med 2011;49:761 – 72. 12 Tripodi A, Di Iorio D, Lippi G, Testa S, Manotti C. Position paper on laboratory testing for patients taking new oral 0 anticoagulants. Consensus Document of FCSA, SIMeL, SIBioC and CISMEL. Clin Chem Lab Med, in press. Parenmark A, Landberg E. To mix or not to mix venous blood samples collected in vacuum tubes ? Clin Chem Lab Med 0 2011;49:2061–3.

Table 1 Most popular articles published by Clinical Chemistry and Laboratory Medicine in the field of hemostasis and coagulation, according to the Thomson (former ISI) database, updated July 2012. Lippi and Favaloro: Hemostasis testing: milestones in Clinical Chemistry and Laboratory Medicine 95 future diagnostic and therapeutic procedures [61] . Seven cell-free hemoglobin. This is truly important information, years later, in 2007, the journal published another highly since it may pave the way to revising the current best prac- cited article by Lippi et al., who provided a comprehen- tice guidelines for collection and handling of blood speci- sive overview on the physiopathology of hemostasis along mens for coagulation testing [68]. with clear indications about the diagnostic approach to the inherited bleeding disorders [62] . In 2010, Favaloro et al. published an opinion paper that might be consid- Conclusions ered the ideal continuum of the previous article of Lippi et al., outlining the sequential process of platelet func- The abnormalities of primary and secondary hemosta- tion investigations, and discussing each of the essential sis, either thrombotic or hemorrhagic, represent serious components in some detail [31] . In more recent times, challenges for clinicians, while the contribution of lab- although the number of citations is understandably oratory diagnostics is indeed essential for clinical and lower, three articles will probably provide some predict- therapeutic decision-making. Several preanalytical, ana- able breakthrough into laboratory hemostasis. In 2011, lytical and postanalytical issues wait to be clarified in Samama et al. published a seminal guide in the intricate coagulation testing. All the previous articles noted above field of laboratory assessment of new anticoagulants (i.e., have produced breakthroughs in the field of laboratory fondaparinux, dabigatran, rivaroxaban or apixaban) [63] , hemostasis; some have also substantially influenced which has since been followed by a position paper issued clinical and laboratory practice, many others will prob- by the Italian Society of Clinical Biochemistry (SIBioC), ably do so as well. With this expectation, we encourage the Italian Society of Laboratory Medicine (SIMeL) and all readers of Clinical Chemistry and Laboratory Medicine the Italian Federation of Centers for Surveillance of Anti- to submit further high quality articles about this topic to coagulated patients (FCSA), which analogously discusses the journal. guidelines and recommendations on the same topic [64] . Another influential article to have recently appeared in the journal is that of Parenmark and Landberg [65] , who raised several doubts as to whether mixing of tubes with Conflict of interest statement liquid-based citrate buffer for coagulation testing may be really necessary. Practically, avoidance of mixing the Authors’ conflict of interest disclosure: The authors stated that there tubes seems to produce clinically negligible bias in results are no conflicts of interest regarding the publication of this article. Research funding: None declared. of screening coagulation tests, whereas instant and too Employment or leadership: None declared. vigorous mixing of blood samples after venipuncture Honorarium: None declared. may produce various degree of spurious hemolysis, and thereby jeopardize the reliability of several clotting [66] Received June 19, 2012; accepted June 20, 2012; previously published and platelet function tests [67] , due to the presence of online July 15, 2012

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Prof. Giuseppe Lippi is the director of the clinical chemistry and hematology laboratory of the Academic Hospital of Parma and Dr. Emmanuel J. Favaloro is a Principle Hospital Scientist, and Associate Professor of Clinical Biochemistry and Molecular Biology. currently the specialist scientist in charge of the Hemostasis He has a degree in Medicine and a specialization in Clinical Laboratories at the Institute of and Medical Biochemistry and Laboratory Medicine. He currently serves as Research (ICPMR), a part of the Pathology West Network, and based Clinical Chemistry and Laboratory Medicine associate editor of and at Westmead Hospital, one of the largest tertiary level academic Seminars in Thrombosis of Hemostasis. He is also chairman of the public teaching hospitals in Australia. He has co-authored over 300 scientific division of the Italian Society of Clinical Biochemistry and papers, is the current Editor-in-Chief of Seminars in Thrombosis and Molecular Biology (SIBioC), and member of the board of the Hemostasis, and also on the editorial board of many international European Association of Clinical Chemistry (EFCC). Prof. Lippi is journals, including CCLM. He is also a current or past member of author of more than 750 publications on international journal several International Society on Thrombosis and Hemostasis (ISTH) indexed in PubMed and Thomson, in more than 90% of them as main Scientific Standardisation Committees (SSC) or working parties, or senior author. The main areas of research include preanalytical a member of the CLSI Area Committee on Hematology and several variability, analytical and clinical validation of novel biomarkers, guideline working parties, and an advisory member of the Royal metabolism of lipoproteins and relevant assay methods, diagnosis College of Pathologists of Australasia (RCPA) Hematology Qual- and management of disorders of hemostasis. ity Assurance Program (QAP) hemostasis committee. His main interests are the diagnosis of bleeding and thrombotic disorders, and the development and evaluation of new or improved diagnostic processes.