Clinical Coagulation Laboratory

A division of Duke University Regional Referral Laboratory Services

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Biodata PAP4 Platelet Aggregometer
Storage of Plasma specimens for future testing
Computer generated aggregation tracings

COAGULATION TEST PANELS

Due to the complexities of the hemostatic system and confusion regarding the best test for establishing specific diagnosis, the laboratory has created test panels for several clinical scenarios. The utility of the panels for the diagnosis and management of patients will be briefly described.

CONTACT FACTOR PATHWAY PANEL

The Contact Factor Pathway Panel includes factors XII (FXII, Hageman factor), prekallikrein (PK, Fletcher factor), and high molecular weight kininogen (HMWK, Williams-Fitzgerald-Flaujeac factor). Like FXII, PK and HMWK factors are needed for in vitro hemostasis, but do not appear to be essential for in vivo hemostasis. Contact factor pathway deficiencies are characterized by a prolonged activated partial thromboplastin time (aPTT) in the presence of normal concentrations of all other clotting factors. The prothrombin time (PT) will be normal as well. Patients with a defect in this pathway are asymptomatic and without a history of hemostatic disorder. Assays to rule out other factor deficiencies of the intrinsic pathway (FVIII, FIX, and FXI) should be performed prior to testing for contact factor pathway deficiencies. This panel includes testing for FXII by traditional aPTT based factor assay. To test for deficiencies of either PK or HMWK, patient plasma is first tested by performing an aPTT with an extended incubation of 10 minutes at 37° C. Correction of the aPTT with the prolonged incubation suggests a PK deficiency; no correction suggests HMWK deficiency. Patient plasma is then mixed with either a known PK or HMWK deficient plasma and incubated at 37° C for 60 minutes. A plasma that is deficient in either PK or HMWK will not correct the aPTT follow- ing this incubation.
Related assays:


DIC SCREEN

The DIC screen is designed as a hemostatic screen to identify abnormalities in the major coagulation pathways. The screen is also useful to monitor patients with mixed coagulopathies. A hemostatic defect can involve the extrinsic system or initiator pathway (PT), intrinsic or propagator pathway (aPTT), fibrin formation (fibrinogen level) or fibrinolysis (Fragment D-dimer levels). The Fragment D-dimer assay is more sensitive and specific for fibrinolysis than the FSP (FDP) assay.
Related Assays:


EXTENDED DIC PROFILE

The consumptive thrombohemorrhagic syndromes, for brevity, will be grouped under the heading of Disseminated Intra vascular Coagulation (DIC) although this family of disorders may vary in their clinical presentations and pre cipitating events. The DIC syndrome occurs in response to a pathologic stimulus that continuously activates the clotting system resulting first in widespread thrombosis of the microcirculation followed by a consumptive coagulopathy and stimulation of the fibrinolytic system associated with hemorrhage. Bleeding and thrombotic problems may be life-threatening in these patients. The extended DIC profile includes a PT, aPTT, TCT, fibrinogen, titered Fragment D-dimer level, ATIII functional level and alpha-2-plasmin inhibitor as a single panel of tests. The profile is designed to be used for those patients in whom more specific testing for DIC is desired. The extended DIC profile offers a fragment D-dimer (ELISA), TCT, ATIII and alpha-2-plasmin inhibitor as more sensitive indicators of consumption and increased fibrinolysis since the PT, aPTT and fibrinogen levels usually change more slowly. Any part of the panel may be ordered separately. Frequent monitoring of the patient’s status is still best served by the DIC screen while the Extended DIC profile is intended to be used in a more limited fashion to make the initial diagnosis and to monitor significant changes in the screening tests or the patient’s clinical status.
Related Assays:


HEPARIN-INDUCED PLATELET AGGREGATIONS

Agonists:

The type of heparin to be tested [Unfractionated Heparin - porcine, Low Molecular Weight Heparin (Enoxaparin, Fragmin, etc.), or Heparinoid (Danaparoid Sodium, ORG 10172), must be specified on the test requisition by requesting physician.

Note: A sample of Heparin (porcine), Low Molecular Weight Heparin, or Heparinoid the patient is receiving (or will receive) should accompany the patient sample whenever possible (optional) to optimize the specificity of this assay.

Platelets normally do not aggregate when exposed to heparin. When platelets from a normal donor are incubated with patient’s plasma and heparin (100 units/mL, 1.0 units/mL and 0.1 units/mL) aggregation should not occur for up to 15 minutes. If the patient has heparin-induced thrombocytopenia (HIT), platelet aggregation may occur with the addition of heparin. The assay is not a highly sensitive test for HIT. It should be used only to support a clinical diagnosis and not as the sole means of deciding on the presence or absence of the syndrome.

Clinical Note: Heparin Induced Thrombocytopenia and Thrombosis:
Heparin induced thrombocytopenia (HIT) may develop in as many as 5% of patients receiving heparin anticoagulation. HIT is an immune disorder that is mediated by a heparin-dependent IgG antibody. It is manifested by the development of progressive thrombocytopenia, developing between four to eight days after the initiation of heparin. Thrombocyto penia may develop within a day in patients who have had prior exposure to heparin. Paradoxically, a small but significant proportion of patients with HIT will develop potentially life-threatening thromboembolic complications that can involve both the arterial as well as the venous circulation. Treatment requires the discontinuation of heparin, after which the platelet count should return to normal over several days.
Related Assays:
* Not Performed at DUMC Clinical Coagulation Laboratory

LUPUS ANTICOAGULANT PANEL

The lupus anticoagulant panel (LA panel) consists of a PT , aPTT, mixing studies, TCT, DRVVT, DRVVT mix, and DRVVT confirm test, combined as a single battery of tests. The mixes and the DRVVT confirm test are only performed if the screening tests (PT, aPTT or the DRVVT) are prolonged. This panel is an adequate screen for the identification of lupus anticoagulants (LA), but additional assays may be required to aid in the diagnosis. Heparin interferes with all clotting tests for the lupus anticoagulant and the TCT is on the panel as a sensitive screen for heparin. Figure 1 is an algorithm that outlines the approach to diagnosis of a lupus anticoagulant.
Figure 1
Related Assays:


PLATELET AGGREGATION STUDIES

Agonists:

Aggregating agents (agonists) are added to patient’s platelet-rich plasma to evaluate the ability of the patient’s platelets to respond to physiologic stimulus. The agonists used by this laboratory are ADP, collagen, arachidonic acid, and an analog of thromboxane A2 (TXA2). These agents have been chosen because they stimulate aggregation through different pathways. This combination of aggregating agonists help determine: 1) storage pool or platelet granule defects (abnormal ADP and collagen aggregation); 2) Glanzmann’s Thrombasthenia (no reaction to any aggregat ing agent due to lack of platelet receptor IIb/IIIa); and 3) an aspirin-like defect or defect of the prostaglandin pathway (no response to arachidonic acid, the substrate of the pathway but continued reaction to TXA2, the product of the pathway). Platelet aggregation will be abnormal if the patient has ingested aspirin within 7-10 days of testing. Numerous other drugs or clinical conditions may affect platelet aggregation assays. Platelet aggregations are helpful in defining inherited or acquired qualitative defects of platelets. However, platelet aggregation tests are not good global predictors of primary hemostasis.

Ristocetin, also used as a reagent, differs from the aggregating agents. It induces platelet agglutination in the presence of plasma von Willebrand factor and an intact platelet receptor glycoprotein Ib (GPIb). This reaction is distinct from platelet aggregation induced by the agonists listed above, all of which induce the binding of fibrinogen to the platelet receptor GPIIb-IIIa. Bernard-Soulier disease is characterized by normal aggregation of platelets to all aggregating agents but no reaction to ristocetin due to lack of the platelet vWF receptor, GPIb.

Related Assays:
* Not performed at DUMC Clinical Coagulation Laboratory

RISTOCETIN TITRATION PLATELET AGGREGATION STUDY

Agonists:

Ristocetin is added in multiple concentrations to patient’s platelet-rich plasma to evaluate the ability of the patient’s platelets to respond to physiologic stimulus. Ristocetin differs from the aggregating agents. It induces platelet agglutination in the presence of plasma von Willebrand factor and an intact platelet receptor GPIb. This reaction is distinct from platelet aggregation induced by the agonists listed under Platelet Aggregation Study, all of which induce the binding of fibrinogen to the platelet receptor GPIIb-IIIa. Bernard-Soulier disease is characterized by normal aggregation of platelets to all aggregating agents, but no reaction to ristocetin due to lack of the platelet vWF receptor, glycoprotein Ib. Patients with specific sub-types of von Willebrand’s disease display varying responses to different concentrations of Ristocetin.
Related Assays:
* Not performed at DUMC Clinical Coagulation Laboratory

THROMBOSIS PANEL

Recent advances in the biochemistry of blood coagulation have led to the discovery that specific deficiencies in anti thrombin III, protein C, protein S, or plasminogen may lead to a thrombotic tendency. All of these abnormalities produce similar clinical findings and should always be considered as a cause for recurrent thrombosis. The Thrombosis Panel is designed to evaluate those proteins which have been clearly identified as inherited or acquired thrombotic disorders and includes: functional antithrombin III, functional plasminogen, functional protein C, and functional protein S. Coumadin therapy interferes with protein C and S measurements. Heparin therapy may lower antithrombin III levels. For this reason, the thrombosis panel should be performed before anticoagulation therapy is initiated. However, consumption of clotting factors during the acute thrombotic period may also decrease the anticoagulant proteins, and therefore, interpretation of these tests taken too close to the thrombotic event may also complicate interpretation. An initially low protein level will need to be repeated and/or identification of the abnormality in family members may be necessary before the deficiency can be confirmed.
Related Assays


von WILLEBRAND PANEL

There is significant variability in the clinical and laboratory manifestations of this von Willebrand’s disease. Whenever this diagnosis is considered, a battery of tests may be required to confirm the diagnosis. This panel includes: Factor VIII coagulant assay, von Willebrand factor Antigen (vWF:Ag), and von Willebrand Factor: ristocetin cofactor (vWF activity). The von Willebrand Factor: multimer pattern should be ordered in patients found to have abnormal vWF levels or in the rare patient suspected of a dysfunctional vWF syndrome not associated with significant decreases in levels. Typically, a patient will only require a single determination of the multimer type as it is very unlikely to change, except in the rare case of a newly acquired defect or inhibitor. vWF is an acute phase reactant; it may be difficult to establish true baseline levels in some patients, especially if they are pregnant, acutely stressed or are on estrogen therapy (increases baseline levels). Several panels over time may be required to exclude the diagnosis in a patient whose clinical history is highly suggestive.
Related Assays:



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Clinical Coagulation Laboratory
Duke University Medical Center
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Box 3514
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Durham, North Carolina 27710
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Last Modified: September 13, 2003

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