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Published: September 1, 2000
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Hemostasis testing: Past, present, and future

By: David G. M. Carville and Kirk E. Guyer

Newly developed immunoassays hold promise for overcoming the limitations inherent in traditional coagulation tests performed for cardiac patients.

Under normal physiological conditions, hemostasis, the readiness of the blood to clot to prevent loss, is kept in homeostatic balance by feedback mechanisms. These mechanisms comprise an extremely complex series of steps on each side of the coagulation cascade—that is, clot formation and clot breakdown (see Figure 1). Should the homeostatic balance be upset, pathological clotting (vessel blockage) or bleeding (hemorrhage) can supplant normal hemostasis. Abnormalities of the hemostatic system can be acquired (via, for example, aspirin ingestion, immune dysfunction, or anemia) or congenital (relating to vWF disease, hemophilia, or Factor V Leiden, for instance). In such cases it is clinically essential to diagnose, monitor, and manage the patient in order to optimize therapeutic intervention. The postsurgical outpatient who is receiving oral anticoagulant therapy also must be treated in order to maintain the homeostatic balance of the hemostatic system.

Most coagulation testing involves end-point assays in which, after the blood sample is incubated with exogenous reagents, the time of clot formation is measured. These tests have traditionally been performed either in the central laboratory or, more recently, at the point of care.1–3 But now other test platforms are expected to play an important role in quantitative assessment of the coagulation system. These include cellular assays (platelet aggregation testing) and immunoassays that are being developed to measure peptides and factors of the coagulation cascade.4–7 This article describes some of these tests and notes their position in the hemostasis and thrombosis pathways.

Traditional Coagulation Tests

Traditional coagulation testing encompasses measurement of the activated clotting time (ACT), the plasma thrombin time, the activated partial thromboplastin time (APTT) and the prothrombin time (PT). The last of these measurements is also referred to as the tissue factor–induced coagulation time of blood or plasma.8 These tests are used to evaluate both the intrinsic and extrinsic coagulation systems (see Figure 1). The coagulation assays to monitor anticoagulation treatment are generally performed as stat tests either in the central coagulation laboratory or at the hospital bedside, or, in the case of PT tests, in the patient's home following discharge from the hospital.

The ACT assay is used primarily to monitor the efficacy of anticoagulation treatment in clinical procedures, such as percutaneous transluminal coronary angioplasty (PTCA) and cardiopulmonary bypass surgery, that involve the administration of high doses of heparin. Such monitoring is important, as there can be considerable variation in a patient's response to heparin therapy. Underdosing may result in pathological thrombus formation, whereas overdosing may lead to serious hemorrhagic complications.9

The thrombin time test measures the rate of clot formation when a standard amount of thrombin is added to a patient's plasma that has been depleted of platelets. This rate is compared to that of a normal plasma control. The test has been used as an aid in the diagnosis of disseminated intravascular coagulation and liver disease.

The APTT test is used to evaluate the intrinsic coagulation system (Factors I, II, V, VIII, IX, X, XI, and XII). In this test, the intrinsic system is activated by the addition of phospholipid, an activator (ellagic acid, kaolin, or micronized silica), and Ca2+ ions to plasma. Formation of the prothrombinase complex on the phospholipid surface allows conversion of prothrombin to thrombin with subsequent clot formation. The time in seconds required for this reaction is the APTT. The APTT test is used preoperatively to screen for bleeding tendencies.10

First described by A. J. Quick et al. in 1935, the PT test is employed as a simple screening procedure to evaluate the integrity of the extrinsic coagulation pathway. It is sensitive to coagulation Factors I, II, V, VII, and X.11 The test is performed by adding thromboplastin and Ca2+ to the patient sample and measuring the time to clot formation. If the time of clotting is prolonged, the interpretation is that the patient has a deficiency or an inhibitor of one or more of the coagulation factors just enumerated. This test is widely used to monitor oral anticoagulation therapy. Both the APTT and the PT test are used to assess overall coagulation competence.12

Figure 1. A representation of the intrinsic and extrinsic coagulation systems (after DA Triplett, Indiana University School of Medicine, 1992; used with permission).

 

Although performed routinely, all of these coagulation assays have inherent limitations that make them potentially unreliable as tools for monitoring coagulation. Moreover, there is not always a good correspondence between the results of coagulation tests and the prevention of postoperative hemorrhage or recurrent thrombosis. Most of the limitations relate to the fact that these are end-point tests that measure the time of clot formation in vitro and require the addition of exogenous reagents (thrombin or Ca2+ ions to replenish those bound by an anticoagulant), and thus do not necessarily reflect the patient's thrombotic potential. Indeed, it is now widely recognized that current ACT tests may also be suboptimal owing to the nature of the activators used. As originally developed, ACT testing involved glass tubes, particulate activators, heating, and continuous mixing.13,14 Unfortunately, when automated ACT platforms were being developed, some key test parameters were ignored. Most such platforms lack sensitivity to the onset of clotting and instead detect the end point of coagulation as a stable clot, resulting in extremely poor ACT test reproducibility.

But now, with the recent development of a new ACT test format approved by FDA, such limitations can be overcome. The MAX-ACT system, developed by Helena Point-of-Care (Beaumont, TX) with the goal of global standardization of contact activation, utilizes a cocktail of activators to ensure complete activation of multispecied Factor XII (see Figure 2). The cocktail brings the total patient population maximum Factor XII activation via both variety and volume of activator. This novel ACT test, in conjunction with the Actalyke platform also created by Helena Point-of-Care, can detect the earliest stages of clot formation with considerable accuracy, which is more clinically relevant in the heparinized patient than end-point determination.9

What makes the union of the MAX-ACT test and the Actalyke platform innovative is the ability of the MAX-ACT cocktail and the two-point Actalyke detection system to read highly heparinized samples that are beyond the capabilities of single-point clot-detection mechanisms (see Figure 3). The clots in such samples are extremely fragile; even though there is clot formation, single-point ACT tests may not trigger until a stable (hard) clot is formed. The clinical implications of this fact could be inaccurate ACT test results, poor reproducibility, or tests that "time out" owing to the nature of a single-point mechanism. The combination of MAX-ACT and Actalyke has reduced the error in ACT testing within the therapeutic range of heparin to less than 5%. (Comparative performance data for the Actalyke and Hemochron 8000 (International Technidyne Corp.; Edison, NJ) with MAX-ACT tubes and several other ACT tests are provided in Table I.

Figure 2. A comparison of probable Factor XII activation as measured by current methodology (a) and multispecied Factor XII activation as measured by MAX-ACT methodology (b).

 

Another limitation of the traditional end-point coagulation tests is that they do not account for thrombin previously bound to fibrinogen. This thrombin retains its enzymatic capability and may continue to induce fibrin formation. Therefore, the traditional tests measure levels of circulating heparin rather than providing a measurement of active thrombosis. Indeed, a recent study demonstrated that, of patients undergoing PTCA who were administered heparin to achieve an ACT of more than 300 seconds and were thus assumed to be adequately heparinized, 16% went on to develop thrombotic complications. Neither are these tests suitable for evaluating the antithrombotic or anticoagulant effects of newer agents such as hirudin, hirulog, or related thrombin inhibitors. They are also unsuitable for the measurement of hemostatic abnormalities associated with platelet dysfunction.12

Platelet Function Testing

Platelets perform an extremely important role as the first line of defense against any challenge to the hemostatic system. In response to an insult to the vasculature, platelets adhere to exposed collagen, then change shape and stimulate the release of adenosine diphosphate (commonly called ADP), a physiological agonist that activates additional platelets. The primary hemostatic plug is achieved by the cyclic stimulation of platelets as they aggregate, release other agonists, and interact with the proteins of the coagulation cascade, forming a consolidated clot. Although under normal physiological circumstances these processes are closely regulated by homeostatic feedback mechanisms, in the acute-care environment of interventional cardiology an upset in this homeostatic balance could occur which might lead to either abnormal hemorrhage (bleeding) or thrombosis (vascular clot formation).1,2

It has long been established that both platelet count and platelet function (the ability to aggregate) may be compromised by such procedures as cardiopulmonary bypass (CPB) surgery and cardiac catheterization with and without percutaneous coronary intervention (PCI). Hemodilution and hemorrhage (with subsequent hypovolemia) are common side reactions of CPB surgery, and adverse thrombosis attributed to overactivated or overaggregated platelets is a common occurrence resulting from PCI.15–18

To optimize patient outcomes in these clinical settings, some have suggested, treatment regimens should include combination therapies that consist of adequate anticoagulation, possible use of thrombolytics, and the administration of antiplatelet agents. Such combination regimens have demonstrated efficacy in significantly reducing the death rate associated with problematic cardiological procedures.19–23 The benefits of proper anticoagulation and thrombolytic therapy have been recognized for some time.

More recently, very effective antiplatelet agents have been introduced for use in alternative or combination therapies in both CPB and PCI. These include agents that target the platelet glycoprotein IIb/IIIa (GPIIb/IIIa) receptor, which is identified as the penultimate step in platelet aggregation. The agents have been approved for use in preventing adverse thrombotic complications during and subsequent to PCI procedures.21–24

Although anti-GPIIb/IIIa agents have demonstrated efficacy in reducing adverse platelet deposition at the site of interventional injury, they carry with them the major side effect of an increased risk of hemorrhage. This has led to the suggestion that the patient's hemostatic status be sequentially monitored throughout the cardiac procedure.25,26 Indeed, it has been suggested (and reinforced in package inserts) that it may be clinically important to quantitate the level of platelet function, thereby facilitating adequate dosing of the antiplatelet agent while at the same time minimizing hemorrhagic risk. Such monitoring must be conducted on a patient-to-patient basis because of individual variation.

Historically, the in vitro assessment of platelet function has relied on the measurement by optical methods of platelet aggregation in the presence of an agonist.27,28 Utilizing platelet-rich plasma (PRP), these methods require specialized equipment and specially skilled personnel, and are both laborious and indirect. Moreover, it has been suggested, these tests may not be an accurate reflection of the in vivo condition, as the preparation of PRP may exclude platelets of high density.29 This has necessitated the development of several whole-blood assays that use electrical impedance to quantitate platelet aggregation, and the introduction of a system, the PFA-100 from Dade Behring (Deerfield, IL), that monitors the inhibition of blood flow on a filter.30,31 Also, two platforms for monitoring platelet dysfunction have recently been approved by FDA for near-patient testing: the Ultegra system (Accumetrics, San Diego) and the Plateletworks/Ichor test platform (Helena Point-of-Care).32,33 PFA-100, Ultegra, and Plateletworks/ Ichor have all demonstrated clinical utility in monitoring platelet dysfunction periprocedurally. However, each system monitors the dysfunction differently.

The point-of-care PFA-100 is an instrument/test cartridge system that simulates in vitro the process of platelet adhesion and aggregation following a vascular injury, evaluating platelet function on small samples of anticoagulated whole blood according to historical principles. Results are provided as "closure time," that is, how long it takes the blood to plug an aperture on the cartridge. The PFA-100 has potential as a first-line screen for platelet dysfunction in clinical practice.

Figure 3. The two-point clot-detection system of the Actalyke platform brings sensitivity to the early stages of clot formation to ACT testing. The clot mass does not have to be stable to travel the minimal detection distance of 46° to shut off the instrument.

 

The Ultegra is a turbidimetric optical detection system that measures platelet-induced aggregation as an increase in light transmittance. It is a semiquantitative whole-blood platelet function assay used to measure GPIIb/IIIa receptor blockade in patients who are treated with abciximab. The system assesses platelet function on the basis of the ability of activated platelets to bind fibrinogen. Fibrinogen-coated microparticles agglutinate in anticoagulated whole blood in proportion to the number of unblocked GPIIb/IIIa receptors. Platelets with unblocked GPIIb/IIIa receptors are activated and cause microparticle agglutination. Results are provided in platelet aggregation units.

Assay Utility
Immunoassays for hemostasis  
Activated protein C–protein C inhibitor complex Index of protein C activation
Factor VIIa Index of activation of extrinsic pathway
Factor IXa–ATIII complex Index of Factor IX activation
Factor X activation peptide Indication of activation of intrinsic pathway
Factor XIIa–protein C complex Activation of intrinsic pathway
Protein C activation peptide Released from heavy chain of protein C
Prothrombin fragment (F1.2) Index of prothrombin (Factor II) activation
Thrombin-antithrombin complex Marker of thrombin formation
Immunoassays for thrombosis  
Activation fibrinopeptides (A and B) Indication of thrombin activity
Degradation products (D-dimer, fragment E) Result of fibrin degradation via plasmin
DesAA and desAABB–fibrin monomer Index of thrombin activity
Soluble fibrin (thrombus precursor protein) Marker of ongoing thrombosis

Table II. Immunochemical assays for the evaluation of coagulation factors (an index of thrombin generation) and thrombosis (an index of thrombin activity).
 

The Plateletworks/Ichor test platform is an in vitro diagnostic assay for the determination of percentage aggregation in fresh (nonanticoagulated) whole-blood samples taken during cardiac interventional procedures, as measured by a change in platelet count due to the activation of functional platelets. Providing exact numbers, it is a quantitative test for the measurement of platelet dysfunction. The system has demonstrated diagnostic clinical utility in the acute-care settings of cardiopulmonary bypass and cardiac catheterization; in addition to its ability to evaluate platelet aggregation with rapid turnaround, it provides an eight-parameter hematology profile that includes platelet count (see Figure 4). This is important given the increasing recognition of the risk of thrombocytopenia in patients treated with antiplatelet agents.18

The Potential of Immunochemical Assays

Alternatives to the traditional coagulation tests have been developed recently to provide quantitative evaluation of the activation of the coagulation cascade. Among them are immunochemical assays that can measure the concentration of peptides released following activation of the coagulation enzymes, the active enzymes themselves, or the molecular complexes that are formed upon activation or inhibition. These analytes should provide a direct correlation with the level of each particular activation factor. Other assays have been developed to measure fibrin monomer, soluble fibrin polymers, and degradation products (see Table II).

Hybridoma technology has enabled the generation of monoclonal antibodies for use in enzyme-linked immunoassays (EIA) to specifically and accurately determine the levels of individual coagulation factors (see Table II). These assays have demonstrated high sensitivities and specificities in the quantitative measurement of factors that have been activated but that do not recognize the inactive zymogen. For example, one such assay quantitatively measures Factor XIIa concentration but not Factor XII and, as such, has been proposed as a reliable indicator for activation of the contact system.5 Immunoassays have also been developed for Factors VIIa, IX, and X, prothrombin fragment (F1.2), and fibrinopeptide A (an activation product of fibrinogen).5–7 These assays provide an estimate of thrombin generation and activity.

Inhibition of the coagulation cascade is necessary for homeostatic regulation of hemostasis. Immunoassays have been developed that measure these inhibitors and the complexes they form. Activation of protein C by thrombin/thrombomodulin precedes inactivation of Factors VIIIa and Va. During activation a peptide is released from the heavy chain of protein C. Both the protein C activation peptide and activated protein C can be measured quantitatively by EIA.34 Also available are assays that measure the thrombin-antithrombin complex.35

Procedure Incidence (%)
  DVT PE
General surgery 23–25 1.6
Gynecology and obstetrics 7–45 1–5
Neurosurgery 9–50 1–3
Orthopedic surgery 45–70 ~20
Hip arthroplasty 45–57
Hip fracture 36–60
Knee arthroplasty 40–84
Urology ~25 1
Transvesical prostatectomy 40

Table III. Rates of development of intravascular thrombosis as a consequence of some common surgical procedures. DVT is deep vein thrombosis, and PE is its pathological sequela, pulmonary embolism.
 

Unfortunately, abnormalities in the regulation of hemostasis are not necessarily coincident with the development or the extent of thrombosis. Consequently, recent research has focused on the high-molecular-weight proteins that ultimately lead to cross-linked fibrin formation. These include fibrin monomer and multimeric soluble fibrin polymers. Other researchers have focused on the "other side" of thrombosis, that is, the degradation proteins generated by the action of plasmin on fibrin (D-dimer and fibrin fragment E).36

Invasive surgical procedures carry a risk of developing thrombosis (see Table III). The final stage of thrombus formation is the conversion of fibrinogen to cross-linked fibrin, the protein component and structural support of blood clots (see Figure 1). Measurement of the penultimate protein moieties of hemostasis may provide a more accurate assessment of the degree to which a patient is at risk for thrombosis.4

Fibrin monomers (FM) are formed when fibrinopeptides are cleaved from fibrinogen by thrombin. Release of fibrinopeptides A and B produces desAA-FM and desAABB-FM, respectively. The desAABB-FM then associate in multimers, forming polymeric soluble fibrin, which is followed by lateral association and covalent cross-linking (influenced by Factor XIIIa) to form the stable clot. Immunochemical assays have been developed for fibrinopeptide A, FM, and soluble fibrin polymeric species.4–5 They have demonstrated utility in the diagnosis of intravascular thrombosis in a number of clinical conditions.

FM assays present the disadvantage that they tend to cross-react with other fibrin-like proteins (fibrinogen, fibrin, and their degradation products), thereby affecting their specificity. By measuring the immediate precursor proteins to the thrombus, soluble polymeric fibrin assays offer potential for use in the risk-stratification of patients likely to develop thrombosis. One such assay, developed by American Biogenetic Sciences (Copiague, NY), designated as Thrombus precursor Protein (TpP), and approved by FDA for diagnostic use, has demonstrated utility in monitoring intravascular clot formation in multiple disease states, including postsurgical risk of clot formation. Because these assays are currently in EIA formats, they are unfortunately not suitable for acute clinical settings such as the emergency room or as on-line assays in surgical suites. Ongoing research seeks to make the TpP assay available in near-patient settings.

Plasminogen activation by tPA, streptokinase, or urokinase results in the serine protease plasmin. The latter cleaves fibrin, leading to the formation of degradation products. One such fibrinolysis product is D-dimer, which is composed of two fibrin monomers covalently linked during fibrin formation. Immunoassays that quantitate the level of D-dimer may provide an indirect, yet reliable, indication of the extent of fibrin formation.36

Again, these assays have inherent drawbacks. By measuring fibrin breakdown, D-dimer assays measure clot formation but do not measure active thrombosis. And high levels of D-dimer have been measured in patients with nonthrombotic conditions.

Conclusion

Recently developed immunochemical assays for the measurement of markers of coagulation activation are more useful for understanding the regulation mechanisms of hemostasis than traditional clotting assays. They have made possible accurate quantitation of the proteins, peptides, and complexes of the coagulation system, and the additional information they provide points to appropriate intervention. They should not, however, be considered indices of the risk of developing thrombotic complications. Alternative assays for the measurement of thrombosis look promising, but they require clinical studies to more clearly define their usefulness. Such studies are needed to provide conclusive evidence that immunochemical assays that measure markers of coagulation activation and thrombosis are more suitable for monitoring anticoagulant and antithrombotic intervention than current tests. The advances represented by the new assays, along with the parallel development of near-patient test instrument platforms with multiplexed assays, make predictable the attainment of enhanced prophylactic intervention with a subsequent reduction in postdischarge morbidity and mortality and lower patient costs.

David G. M. Carville, PhD, is president and chief scientific officer of Causeway Scientific (Mishiwaka, IN), and Kirk E. Guyer is president of Cascade Technologies (South Bend, IN). Both are members of the associate faculty at Indiana University, South Bend.

References

1. LA Harker and KG Mann, "Thrombosis and Fibrinolysis," in Thrombosis in Cardiovascular Disorders, ed. V Fuster and M Verstraete (Philadelphia: W. B. Saunders, 1992), 1–16.

2. L Badimon, JJ Badimon, and V Fuster, "Pathogenesis of Thrombosis," in Thrombosis in Cardiovascular Disorders, ed. V Fuster and M Verstraete (Philadelphia: W. B. Saunders, 1992), 17–39.

3. DGM Carville and KE Guyer, "Coagulation Testing, Part 1: Current Methods and Challenges," IVD Technology 4, no. 4 (1998): 59–66.

4. DGM Carville et al., "Thrombus Precursor Protein (TpP): A Marker of Thrombosis Early in the Pathogenesis of Myocardial Infarction," Clinical Chemistry 42, no. 9 (1996): 1537–1541.

5. PM Mannuci and A Tripodi, "Mechanisms, Markers, and Management of Hypercoagulable States," Haemostasis 26, no. 4 (1996): 1–8.

6. KA Bauer, "New Markers for In Vivo Coagulation," Current Opinion in Haematology 1 (1994): 341–346.

7. H Pelzer, A Schwartz, and W Stuber, "Determination of Human Pro-Thrombin Activation Fragment 1+2 in Plasma with an Antibody against a Synthetic Peptide," Thrombosis and Haemostasis 65 (1991): 153–159.

8. RHM Peters, AMPH Van den Besselar, and FMFG Olthuis, "A Multi-Center Study to Evaluate Method Dependency of the International Sensitivity Index of Bovine Thromboplastin," Thrombosis and Haemostasis 66 (1991): 442–445.

9. RA Esposito et al., "The Role of the Activated Clotting Time in Heparin Administration and Neutralization for Cardiopulmonary Bypass," Journal of Thoracic and Cardiovascular Surgery 85 (1983): 174–185.

10. The Illustrated Guide to Diagnostic Tests (Springhouse, PA: Springhouse, 1993), 52–65.

11. AJ Quick, M Stanley-Brown, and FW Bancroft, "A Study of the Coagulation Defects in Hemophilia and in Jaundice," American Journal of the Medical Sciences 190 (1935): 501.

12. M Verstraete and S Wessler, "Heparins and Oral Anticoagulants," in Thrombosis in Cardiovascular Disorders, ed. V Fuster and M Verstraete (Philadelphia: W. B. Saunders, 1992), 121–140.

13. P Hattersley, "Activated Coagulation Time of Whole Blood," Journal of the American Medical Association 196 (1966): 150–154.

14. P Hattersley, "Progress Report: The Activated Coagulation Time of Whole Blood (ACT)," American Journal of Clinical Pathology 66 (1976): 899–904.

15. GJ Despotis, VL Levine, and LT Goodnough, "Relationship between Leukocyte Count and Patient Risk for Excessive Blood Loss after Cardiac Surgery," Critical Care Medicine 25 (1997): 1338–1346.

16. M Sughayer and CF Arkin, "Monitoring Coagulation during and after Cardiopulmonary Bypass Surgery," American Society of Clinical Pathology, Thrombosis and Hemostasis 12, no. 4 (1990): 1–7.

17. RC Woodman and LA Harker, "Bleeding Complications Associated with Cardiopulmonary Bypass [review]," Blood 76 (1990): 1680–1697.

18. SD Berkowitz et al., "Acute Profound Thrombocytopenia after c7E3 Fab (Abciximab) Therapy," Circulation 95 (1997): 809–813.


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