Laboratory Diagnostics in Thrombosis and Hemostasis: The Past, the Present, and the Future

Welcome to another issue of Seminars in Thrombosis and Hemostasis. This is actually the first of a series of issues that we have planned with a focus on the clinical-laboratory interface. Laboratory scientists working in the field of pathology tend to be focused on laboratory test results and the mechanistic processes involved in generating test “numbers.” Nevertheless, it must be recognized that laboratory scientists also have a duty of care both to patients, whose samples they are testing, and to the clinicians who request the tests and who are caring for these patients. This duty of care involves ensuring not only the precision of test results (which can substantially be guaranteed these days given modern laboratory instrumentation and by making sure that laboratories follow appropriate quality processes) but also that test results accurately reflect the status of the patient under investigation (a goal that is sometimes more elusive for reasons often outside the control of the scientists performing the laboratory tests). Laboratory scientists also have a role in assisting clinicians beyond that of test performance, and this relates to postanalytical guidance. Clinicians also have a duty of care to ensure that they best manage patients under their care. This is best ensured by a thorough understanding of the laboratory test process and of the strengths and limitations of pathology testing. We hope that our planned series of issues of Seminars in Thrombosis and Hemostasis achieves the intended goal, namely the bridging of the laboratory-clinical interface, and generates a better appreciation of laboratory practice among clinical practitioners within the field of hemostasis and thrombosis. The current issue of Seminars in Thrombosis and Hemostasis begins this process largely by taking us back to laboratory fundamentals.

The first article, by Tripodi, accordingly takes us on a chronological journey of the history of laboratory testing for disorders of hemostasis and thrombosis, starting from the most simple tests that were based on the visual inspection and recording of the time needed for native whole blood to clot. The onward path has at times been frustrating and seemingly slow but has led inexorably to the current point in time and to the more complex test procedures based on the addition to plasma of various exogenous substances, the use of sophisticated coagulometers or synthetic substrates, and computer software to record coagulation times, visualize coagulation tracings or analyze complex enzyme systems, or generate thrombin generation (TG) curves. Tripodi explains how the simple tests have evolved over the years and how such old and time-honored tests as TG and thromboelastography, devised more than 50 years ago and then neglected for many years, are now once again gaining momentum because of the progress in technology combined with a better understanding of the background coagulation mechanisms themselves. Nevertheless, Tripodi believes that current tests are still somewhat far from being adequate and standardized to fully investigate hemostasis and thrombosis, a sentiment that most workers in the field, the guest editors included, would share.

The main tests of routine coagulation are the prothrombin time (PT), usually reflected as an international normalized ratio (INR), and the activated partial thromboplastin time (APTT). The INR is most often used to monitor patients on vitamin K antagonist (VKA) therapy, using the so-called coumarin drugs (such as warfarin). The situation in terms of standardization of testing, and thus of monitoring therapy and managing patients, is much improved compared with that of the past, but there is still some room for improvement. The APTT has a multitude of uses, including monitoring of unfractionated heparin anticoagulant therapy, as a screening test for factor deficiencies including factor VIII and factor IX (which are deficient in classic hemophilia), and as a screening test for the acquired thrombophilia-related condition we call lupus anticoagulant (LA). More recently, the concept that short APTTs might further be of clinical utility has also emerged.[1] [2] The challenge in the future will be to further improve standardization and/or harmonization of current assays and to also devise newer tests that more closely mimic what occurs in vivo, and which would thus be better able to reflect true physiology and help explain pathobiological processes.

The second article, by Favaloro and Adcock, continues to focus on the PT and the INR. The PT assay is the most clinically ordered coagulation test. The INR is in essence the patient's PT “mathematically adjusted” to a standardized value by taking into account the peculiarities of the test system and by applying two “correction factors” defined by an international sensitivity index (ISI) and the mean normal prothrombin time (MNPT). Although some manufacturers provide assigned ISI values for specific PT reagents and instrumentation, it is still recommended practice that laboratories check (or validate) these ISI values, as well as estimate the MNPT. Where a manufacturer does not provide an ISI, the laboratory needs to estimate its own value, on all of the instruments being used. Current recommendations suggest the use of commercial reference-plasma calibration sets, but there is limited information to validate their performance in laboratory practice. Favaloro and Adcock report some personal experience with use of some of these materials, as well as reviewing alternate or supplementary procedures for estimation and/or validation of ISI and MNPT. It is evident from their appraisal that further verification checks are required prior to acceptance of ISI and MNPT estimates generated from such commercial reference-plasma calibration sets. In particular, use of different calibration sets may lead to generation of different ISI and/or MNPT estimates,[3] and the laboratory would still need to confirm the appropriateness of such estimates. Favaloro and Adcock also detail various strategies to ensure that laboratories are best placed to provide INR values that best reflect patients' true anticoagulant status and thus for this test process to better assist clinicians in the therapeutic management of patients under their care.

The third article in this issue of Seminars in Thrombosis and Hemostasis, by Lippi and Favaloro, takes a longer look at the second most often performed routine coagulation test, namely the APTT. As briefly mentioned above, this 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 LA. Whereas short APTT values have been mostly overlooked in the past, recent evidence suggests that these might be associated with hypercoagulability.[1] [2] 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 either is an old dogma displaying new tricks or else might describe a new dogma for an old laboratory trick.

The next article, by Favaloro, Lippi, and Adcock, provides a detailed overview on extra-analytical issues in hemostasis testing. Thus, the advent of modern instrumentation, with associated improvements in test reliability, together with appropriate internal quality control (IQC) and external quality assurance (EQA) measures, has led to substantial reduction in analytical errors within hemostasis laboratories. This is mirrored by vastly improved precision in hemostasis testing. Unfortunately, the reporting of incorrect or inappropriate test results still occurs, perhaps even as frequently as in the past. Many of these cases will arise due to a variety of events largely outside the control of the laboratories and will primarily comprise preanalytical events related to patient collection and sample processing, as well as postanalytical events related to the reporting and interpretation of test results. This article provides an overview of these events, as well as some suggestions on how they can be minimized or prevented, thereby ensuring that the test results the clinician receives actually represents the true clinical status of the patient rather than just reflecting the status of the (potentially inappropriate) clinical sample received and tested. This article should be of interest to both laboratory scientists working in hemostasis and the clinicians that request hemostasis-based laboratory tests: the former professionals because laboratory scientists are ultimately responsible for the test results they provide to clinicians, and they are tasked to provide both accurate and precise results to enable clinicians to manage patients appropriately and to avoid the need to recollect and retest; the latter professionals because unless clinicians gain an appreciation of these issues they will not be in a position to best manage their patients, particularly when face with unexpected results.

The next article, by Banfi and Del Fabbro, is on the biological variability of coagulation testing. The two components of biological variability are interindividual variability, which is the variability due to the heterogeneity of physiologic influences among subjects, and intraindividual variability, which is due to the variability in the same individual over time. Analysis of biological variation is crucial for estimating the critical difference, which corresponds with a threshold suggestive of a statistically significant difference between two consecutive results of a laboratory parameter in the same subject, and which is therefore unlikely attributable to casual (random) oscillation of values. In other words, an appreciation of biological variation will allow clinicians to recognize whether different test results are reflective of a “true” difference or potentially due to simple “day-to-day” variation. Banfi and Del Fabbro advise us that studies on biological variation of tests of hemostasis are outdated and that currently published results require validation using modern automated methods. The above notwithstanding, biological variation for coagulation screening tests (PT [INR] and APTT) is low and comparable with the values obtained for hematologic parameters. However, the index of individuality (ratio between intraindividual and interindividual variability) suggests that the usual preoperative screening for coagulation disorders is greatly influenced by the between-subjects variability. On the other hand, the thrombin time (TT) is very constant within and between subjects. Proteins such as fibrinogen, clotting factors, and antithrombin (AT) show a low biological variability. In contrast, fibrinolytic parameters, such as plasminogen activator inhibitor 1 and fibrinopeptide A, show very high variability, and their interpretation in the clinical setting must take this into consideration.

The sixth article in this issue of Seminars in Thrombosis and Hemostasis, by Plebani and colleagues, discusses the issue of quality control (QC) in laboratory medicine. QC refers to all the procedures commonly used in clinical laboratories to monitor the routine performance of testing processes, to detect possible errors, and to correct any problems before test results are reported and relayed to the requesting clinicians. In particular, IQC and EQA programs are widely used to evaluate and improve quality in laboratory medicine. Although clinicians appreciate that laboratory testing is necessary for the diagnosis and treatment of patients with hemostatic disorders, they may not be aware of the controls in place within the laboratory to ensure that tests remain both accurate and precise over time. Nonetheless, the benefits of IQC and EQA in hemostasis have been demonstrated many times, although the inherent complexities in the specific tests performed within hemostasis leads to the conclusion that control is often more elusive than that we can achieve in other fields of laboratory medicine such as clinical chemistry. Plebani et al go on to say that analytical quality in coagulation testing is far from reaching a state of excellence but also highlights the need for more objective establishment of performances goals. New challenges to EQA schemes for coagulation testing derive from the introduction of innovative tests, genetic analysis, and the need to assess not only analytical procedures but also all the steps involved in the total testing process.

Kitchen and colleagues take the concept of IQC and EQA further in the next article, this time specifically related to so-called point-of-care (POC) instruments, which continue to expand rapidly in the field of hemostasis within many countries. POC processes include use of global tests of hemostasis in operating theaters, but especially comprise use of POC monitors for determination of the INR for monitoring VKA therapy. One of the great challenges for POC testing remains training and education, given that these instruments are used by a wide variety of health care professionals, including laboratory scientists, nurses, clinicians, and pharmacists, but increasingly also the patients themselves. This article is largely focused on issues related to IQC and EQA for these devices. Accordingly, data from various EQA exercises involving users of several different POC-INR devices is described, and use of split samples where a patient sample is analyzed by both a POC device and by a conventional laboratory method is also discussed.

The next article, by Pereira and colleagues, defines a slight change of focus in this issue of Seminars in Thrombosis and Hemostasis and looks at the process of investigating mucocutaneous bleeding (MCB) in affected individuals and simply concludes that a definitive diagnosis is not always possible, a conclusion that this group have previously proposed.[4] [5] Patients with inherited MCB pose frequent and significant diagnostic challenges. Bleeding symptoms are frequent among the otherwise healthy population, and the clinical distinction between normal subjects and patients with genuine bleeding disorders is often complex. Screening or global laboratory assays are nonspecific and have low sensitivity to detect mild bleeding disorders (MBDs). Moreover, there are inherent difficulties in diagnosing von Willebrand disease and platelet function defects (PFDs), the best-characterized and most frequent disorders of primary hemostasis. Furthermore, some patients with moderate to severe clotting factor(s) deficiencies and those with increased fibrinolysis often present with MCB. Finally, in a significant proportion of patients with clinically defined MBDs, a specific and definitive diagnosis is not always possible, even after an extensive laboratory workup. Pereira and colleagues review the clinical and laboratory approach to the diagnosis of patients presenting with MCB, the limitations of the available methodologies to evaluate the clinical significance of bleeding, and the diagnostic yield of global and specific hemostasis tests used to investigate these patients.

The next article, by Franchini and Mannucci, defines another slight change of focus in this issue of Seminars in Thrombosis and Hemostasis, and looks at the concept of thrombotic risk associated with elevated plasma von Willebrand factor (VWF). VWF is a multimeric plasma protein that mediates platelet adhesion and aggregation at sites of vascular injury and also acts as a protective carrier of factor VIII. Although the acquired or inherited deficiency of VWF is associated with a bleeding tendency, there is increasing evidence that VWF also plays a pivotal role in thrombosis. The current article reviews the literature and provides evidence for the two-faced character that VWF therefore represents. Well recognized is that the presence in plasma of unusually large VWF multimers, due to the congenital or acquired deficiency of the VWF-cleaving metalloproteinase ADAMTS13, is implicated in the pathogenesis of thrombotic thrombocytopenic purpura (TTP). In addition, high plasma levels of VWF have been associated with an increased risk of atherothrombosis. The main question of interest is whether such high VWF levels are the cause of clinical atherothrombotic conditions or simply a marker of disease (i.e., cause or effect). In synopsis of the current literature, high VWF levels seem to be associated with a weakly increased risk of coronary artery disease, but little information is available on VWF and the risk of ischemic stroke.

Perhaps it is pertinent, however, to raise some additional thoughts here in relation to the limitations of current research. To date, all studies have used testing of plasma VWF by means of a VWF:Ag assay. These assays are not selective for the most functional or high-molecular-weight (HMW) forms of VWF. Studies using functional assays for VWF, including those that are selective for HMW forms of VWF, such as a validated or optimized VWF:CB,[6] are simply lacking. In the case of the bleeding disorder we call von Willebrand disease (VWD), bleeding risk is not just evidenced by a loss of VWF per se. Although this is so in the quantitative VWD types (i.e., 1 and 3), in the qualitative VWD types 2A and 2B, VWF:Ag levels may actually be normal, but bleeding risk is still evident and related to the relative loss of HMW VWF, which can be characterized by laboratory testing as a relatively lowered VWF:CB/VWF:Ag ratio.[7] Moreover, the thrombotic risk in TTP is thought not to result from the elevated VWF per se, but from the presentation of elevated ultralarge VWF. This would be reflected within laboratory testing by a relatively elevated VWF:CB/VWF:Ag ratio. Could the question therefore be raised that the failure of current studies to conclusively determine the thrombotic risk of elevated VWF is due to limited usage of the VWF:Ag assay in previous studies? Could it also be possible that past studies have additionally failed to fully optimize assays to the detection of very high levels of VWF, as normal laboratory practice is to optimize VWF assays to the preferential detection of low levels of VWF to identify VWD? Finally, thrombosis is now often thought to result from a double- or triple-hit process. Elevated VWF per se may not elevate the risk of thrombosis, but like a bullet waiting for the right gun, elevated VWF may promote a prothrombotic milieu that will facilitate prothrombotic mechanisms under the right (or wrong) conditions.

Berntorp and Salvagno then take us on another journey, this time related to a test we have come to know as thrombin generation (TG). As mentioned at the start of this article, TG is actually an old test that has seen resurgence of late because of both evolved methodology and improved understanding of the basic hemostatic mechanisms. TG is a key process within hemostasis and ultimately determines the extent of a hemostatic plug or a thrombotic process. The ensuing thrombin burst is crucial for the formation of a stable fibrin clot. During its active life, thrombin exerts a multitude of highly regulated actions on the blood and the vessel wall, including the clotting of fibrinogen. The inappropriate generation of thrombin may lead to pathologic processes, foremost of which are hemorrhagic or thrombotic diseases. Berntorp and Salvagno reflect that the coagulation system is usually investigated by means of two classic in vitro clotting tests, PT and APTT, but that these assess only time to initiation of clot formation and do not entirely reflect the global hemostatic balance. The PT and APTT permit identification of connectivity between the component activities identified as required for plasma coagulation and define the concept of intrinsic and extrinsic coagulation pathways, which converge at the point of formation of the prothrombinase complex. However, the mechanisms established by in vitro tests are not truly mirrored in the human pathologies associated with bleeding or thrombosis. The recent development of newer tests based on the continuous registration of TG under in vitro conditions that mimic more closely what occurs in vivo will prompt a reinvestigation of the balance between procoagulants and anticoagulants in patients with various hemostatic disorders. Thrombin-generation assays (TGAs) not only provide an overall assessment of hemostasis but also target potential extrahemostatic effects of the generated thrombin, a potent agonist of a multitude of cellular activation pathways, cancer growth and spread, among others. Moreover, estimation of an individual's TG potential may correlate more closely with a hypercoagulable or hypocoagulable phenotype when compared with traditional coagulation tests. Berntorp and Salvagno discuss to what extent TG can be expected to reflect the clotting function of the blood, the development and use of different TGA systems suitable for detecting changes in the kinetics of TG, and the clinical utility of this test.

The final article in this issue of Seminars in Thrombosis and Hemostasis. by Enjeti et al, follows on from an earlier article in this journal[8] and discusses the potential role of microparticles (MPs) in health and disease. MPs are small fragments of membrane-bound cytoplasm that are shed from the surface of an activated or apoptotic cell. Recently, their function as vectors of transcellular exchange of biologic information, in addition to better-described forms of intercellular communication such as growth factors, cytokines, and chemokines, has become increasingly recognized. Circulating levels of MPs seem to reflect a balance between cell stimulation, proliferation, and death. The production of MPs is thought to predominately occur by vesiculation or blebbing of the cell membrane. The mechanisms governing the remodeling of the plasma membrane are complex, involving cytoskeletal changes and a shift from normal phospholipid asymmetry. Increased intracellular calcium subsequent to cell activation leads to intracellular increases in several proteins, as well as the activity of several enzymes, all playing important roles in the homeostasis of the cell membrane. The membrane vesiculation and phospholipids asymmetry that sustains the production of MPs occurs by the complex interplay of the proteins involved. There are several clinical conditions associated with elevated MPs, and most are also associated with an increased risk of thrombosis. Apart from cardiovascular disease and venous thromboembolism, MPs are also elevated in solid tumors with metastatic disease. The measurement of MPs is being regarded as a potential biomarker, given the range of conditions in which it is elevated and the association with prothrombotic states. The utility of measuring MPs as a diagnostic and prognostic marker is currently a subject of intense investigation. The further development of the various methods for the detection and measurement of MPs, along with the availability of prospective clinical trials establishing the utility of such tests, will be critical prior to any introduction of routine measurement of MPs in the diagnostic laboratory.

The guest editors of this issue of Seminars in Thrombosis and Hemostasis would like to sincerely thank all the authors for their interesting and timely contributions. We hope that you as readers enjoy the collation of articles—the first of this series of issues related to the laboratory-clinical interface.

REFERENCES

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