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Clinical Laboratory

Coagulation

Main Laboratory, Hospital- B Level

Phone: 501-686-8109

Medical Director: Ginell Post, M.D., Ph.D  501-526-6776

Manager: Mandy Duncan, MLS(AMT) 501-686-8852

Supervisor: Stephanie Wooldridge, MLS(ASCP) 501-686-6520

CP Resident on Rotation: Business Hours  501-686-6397

CP Resident On-call: Pager 501-688-2820

Hours of Operation

Specimen Collection

Most coagulation procedures require a blue top citrated tube (see special collection requirements for exceptions). When collection is made with a vacutainer needle, blood should be allowed to enter the tube until the vacuum is exhausted. A 4.5ml tube will be approximately two-thirds full. A proper ratio of blood to citrate is required for valid results.

The blue top tubes are calibrated to maintain the correct ratio of plasma to the sodium citrate anticoagulant based upon a hematocrit up to 55%. Therefore, patients with a hematocrit greater than 55% or less than 21% will not have correct ratio of plasma to citrate and the clotting studies may give false negative or false positive results. Specimens must be delivered to the laboratory labeled with the patient’s name, unit number, date and time of collection as soon as possible after collection.

Available Tests and Clinical Indications

PLATELET FUNCTION SCREEN (PFA): Used to detect platelet dysfunction induced by intrinsic platelet defects, von Willebrand disease or exposure to platelet inhibiting agents such as acetylsalicylic acid (ASA) or medications containing ASA. The closure time is prolonged by thrombocytopenia (platelet counts < 100,000) and anemia.

VERIFY NOW: 
The VerifyNow® System measures the antiplatelet effect of individual antiplatelet medications.
The VerifyNow PRU Test is designed to measure P2Y12 receptor blockade.    Results of the PRU Tests are reported as P2Y12 Reaction Units (PRU).  PRU measures the extent of platelet aggregation in the presence of a P2Y12 inhibitor.   Lower PRU levels are associated with expected antiplatelet effect.

The VerifyNow Aspirin Test is a qualitative test to aid in the detection of platelet dysfunction.  The test is reported in Aspirin Reaction Units (ARU).
350-549 ARU – therapeutic range for platelet function
550-700 ARU – non-therapeutic range for platelet function

PROTHROMBIN TIME (PT): The prothrombin time (PT) measures the integrity of the extrinsic and common pathways of coagulation (factors VII, X, V and II). The PT can be used to: 1) monitor warfarin therapy, 2) screen for inherited and acquired deficiencies of factors in the extrinsic (factor VII) and common pathway (factors V, X and II) and 3) screen for inhibitors to factors VII, V, X and II.

ACTIVATED  PARTIAL THROMBOPLASTIN TIME (APTT or PTT): The activated partial thromboplastin time (APTT) measures the integrity of the intrinsic and common pathways of coagulation (factors XII, XI, IX, VIII, X, V, and II). The APTT can be used to: 1) monitor heparin therapy, 2) screen for congenital and acquired deficiencies of factor VIII (hemophilia A), factor IX (hemophilia B), Von Willebrand disease and other congenital or acquired deficiencies of the intrinsic and common pathway factors, 4) screen for inhibitors to factors VIII, IX, XI, XII, X, and V, and II, and 5) screen for the presence of a lupus anticoagulant. Heparin contamination must be excluded.

MIXING STUDY (APTT or PT): Mixing studies are used to determine whether a prolonged PT or APTT is due to a factor deficiency or an inhibitor. Mixing studies are based on two principles: 1) the inhibitor is present in excess, and if present, it will inhibit normal and patient plasma, and 2) that 50% of any factor is enough to yield a normal test result. Correction into the normal range indicates a factor deficiency. Lack of correction suggests the presence of an inhibitor. Heparin contamination must be excluded. Mixing studies cannot be performed when the PT or APTT is within 2 or 5 seconds of normal range, respectively.

THROMBIN TIME (TT): The thrombin time (TT) screens for defects in the conversion of fibrinogen to fibrin. This test is a useful screening test for abnormal fibrin formation, including: 1) the diagnosis of hereditary fibrinogen deficiencies and dysfibrinogenemia, 2) DIC, 3) liver disease and 4) fibrinolysis. The thrombin time is prolonged by abnormally high fibrin degradation products, low amounts of heparin, thrombolytic agents, and direct thrombin inhibitors.

EUGLOBULIN CLOT LYSIS TIME (Send out test): The euglobulin clot lysis time is a screening procedure for the measurement of fibrinolytic activity and can detect either primary or secondary fibrinolysis. A result less than 2 hours indicates an increase in fibrinolysis. Prolonged results indicate inadequate fibrinolysis.

DISSEMINATED INTRAVASCULAR COAGULATION SCREEN: The most useful panel to screen for disseminated intravascular coagulation (DIC) includes D-dimer, prothrombin time (PT), activated partial thromboplastin time (APTT), fibrinogen and platelet count. However, these tests are not specific for DIC.

FIBRINOGEN ACTIVITY (FACTOR I): This test measures the concentration of functional fibrinogen and can be used to evaluate 1) disseminated intravascular coagulation (DIC), 2) liver disease, 3) monitor individuals undergoing thrombotic therapy, and 4) diagnose inherited and acquired coagulopathies (afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia). Fibrinogen is an acute phase reactant and can be increased in inflammatory states, pregnancy and malignancies. High concentrations of heparin or fibrin degradation products can result in abnormal results.

D-DIMER QUANTIFICATION: Fibrinolysis is mediated by plasmin, which degrades fibrin clots into D-dimers and fibrin degradation products (FDP). Plasmin can also degrade intact fibrinogen, generating FDP that are detected in FDP assays. Elevated D-dimers may be seen in DIC, thromboembolism, systemic fibrinolytic states and liver disease due to decreased clearance. D-dimers can also be elevated postoperatively, with significant bleeding, hemodialysis, eclampsia, sickle cell crisis and other conditions. Cancer patients often have positive D-dimers and FDP, usually representing low-grade, chronic DIC. Serial D-dimers may be used to monitor resolution of a known thromboembolism.

HEPARIN-PF4 IgG ANTIBODIES (HIT): Immune-mediated heparin induced thrombocytopenia (HIT type II) is due to an IgG antibody that recognizes heparin bound to platelet factor 4 (PF4). A monoclonal antibody, that mimics human HIT (Heparin Induced Thrombocytopenia) antibodies, is coated onto latex particles. In the presence of PF4 from human platelets, complexed to polyvinyl sulfonate (PVS), and the patient sample, a competitive agglutination reaction occurs. The degree of agglutination is inversely proportional to the concentration of antibodies in the sample and is determined by measuring the decrease of transmitted light caused by the aggregates.  PF4/H antibody testing combined with an appropriate clinical assessment is useful as an aid in the management of HIT suspected patients (4T score).  HemosIL HIT-Ab (PF4-H) results equal to or higher than 1.0 U/mL may indicate the presence of HIT antibodies.  A weak positivity for PF4 antibodies may indicate that the antibodies are non-platelet activating, while a strong positivity may indicate a higher risk for HIT. In both cases, confirmation with a functional test is recommended (serotonin release assay). In patients with HIT, the most common clinical complication is thrombosis (30-40% incidence) despite thrombocytopenia, and non-heparin anticoagulation is typically required.

HEPARIN LEVEL- Xa INHIBITION FOR LMWH (chromogenic): The heparin anti-factor Xa assay is used to monitor heparin therapy. It can be used to monitor standard, unfractionated heparin when the APTT cannot be used, such as in patients with lupus anticoagulant. The anti-Xa assay is the only test that works for low molecular weight heparin such as Lovenox ® or Fragmin ®, or the pentasaccharide fondaparinux.

HYPERCOAGULATION PANEL: This panel tests for the presence of a hereditary or acquired deficiency of the more common factors predisposing patients to thrombosis. It includes ATIII activity, fibrinogen activity, Protein C activity, Protein S activity, APC resistance ratio, Factor V Leiden (PCR) and prothrombin gene mutation (PCR). This panel requires 2 lavender tubes and 2 blue tubes. One lavender tube must be kept unspun and at room temperature. One lavender tube must be kept on ice. The blue top tubes must be spun until they are platelet free (<10,000) and the plasma frozen in appropriately labeled tubes.

  • PT and PTT
  • ANTI-THROMBIN III ACTIVITY
  • PROTEIN C ACTIVITY
  • PROTEIN S ACTIVITY
  • APC RESISTANCE RATIO
  • HOMOCYSTEINE
  •  DNA-PROTHROMBIN MUTATION
  •  FACTOR V LEIDEN
  •   FIBRINOGEN

ANTITHROMBIN III ACTIVITY: A deficiency of antithrombin III (AT III), a natural anticoagulant protein, leads to a hypercoagulable state with an increased risk for venous thrombosis. Acquired AT III deficiencies are more common than hereditary deficiencies. An acquired AT III deficiency has been described in DIC, septic shock, nephrotic syndrome, liver disease, in L-asparaginase treatments and with heparin therapy or contamination.

PROTEIN C ACTIVITY: Protein C is a natural anticoagulant protein that uses protein S as a cofactor to degrade factors V and VIII. Deficiency of protein C leads to a hypercoagulable state with an increased risk for venous thrombosis. Protein C levels are decreased by warfarin. The functional assay cannot be performed in patients on hirudin or argatroban anticoagulation.

PROTEIN S ACTIVITY: Protein S is a nonenzymatic cofactor required for the anticoagulant activity of protein C. Deficiency of protein S leads to a hypercoagulable state with an increased risk for venous thrombosis. Protein S levels are decreased by estrogen, pregnancy, inflammation, infection, acute phase reactions, nephrotic syndrome, and warfarin. The functional assay cannot be performed in patients on hirudin or argatroban anticoagulation.

ACTIVATED PROTEIN C (APC) RESISTANCE: The activated form of protein C (APC) degrades Factor Va and Factor VIIIa and provides physiologic anti-thrombotic activity. APC resistance is the inability if protein C to cleave factors V and/or factor VIII. Resistance to activated protein C is a condition which leads to a hypercoagulable state with an increased risk of venous thrombosis. The majority of cases (85-90%) are explained by a mutation in the Factor V gene (FVL).

HOMOCYSTEINE LEVELS (Test performed by Chemistry Department): Homocysteine is a thiol containing amino acid formed by demethylation of methionine. It is metabolized by remethylation to methionine or by transsulfuration to cysteine. Elevated homocysteine levels may occur as a result of inherited disorders which alter enzyme activity in the transsulfuration and remethylation pathways (eg, MTHFR gene mutation). Alternatively, nutritional deficiencies of essential cofactors of the enzymes including cobalamin (vitamin B12), folate or pyridoxine (vitamin B6), can results in blockade of homocysteine pathways. Plasma homocysteine may be used as a graded risk factor for cardiovascular disease (<11 mmol/L: desirable; 11-14 mmol/L: intermediate; 15-29 mmol/L: high and >29 mmol/L: very high).

DNA-PROTHROMBIN G20210A MUTATION (Test performed by Molecular Biology Department): Prothrombin G20210A mutation is a common hereditary predisposition to venous thrombosis. A polymerase chain reaction (PCR)-based assay is used to determine the presence of this specific mutation at nucleotide position 20210 in the prothrombin gene.

FACTOR V LEIDEN MUTATION (FVL) (Test performed by Molecular Biology Department) : Individuals with factor V Leiden have a mutation at one of the arginine cleavage sites in factor V, such that factor V resists degradation by activated protein C. This is the most prevalent hereditary predisposition to venous thrombosis. A polymerase chain reaction (PCR)-based assay is used to determine the presence of this specific mutation.

FIBRINOGEN (FACTOR I): This test measures the concentration of functional fibrinogen and can be used to evaluate 1) disseminated intravascular coagulation (DIC), 2) liver disease, 3) monitor individuals undergoing thrombotic therapy, and 4) diagnose inherited and acquired coagulopathies (afibrinogenemia, hypofibrinogenemia, and dysfibrinogenemia). Fibrinogen is an acute phase reactant and can be increased in inflammatory states, pregnancy and malignancies. High concentrations of heparin or fibrin degradation products can result in abnormal results.

PLASMINOGEN (Send out test): Plasminogen is the precursor of plasmin, which lysis fibrin clots. Hereditary plasmin deficiency is rare and it may predispose to venous thrombosis. The test is intended for the quantitative measure of plasminogen levels and may be considered in patients with familial venous thrombosis and no evidence for a more common hypercoagulable state or to monitor thrombolytic treatments.

LUPUS ANTICOAGULANT PANEL AND ANTI-PHOSPHOLIPID ANTIBODIES: Lupus anticoagulants (LAs) and antiphospholipid antibodies are a heterogeneous group of autoantibodies of the IgG, IgM or IgA subtypes that are directed against negatively charged phospholipids or complexes of phospholipids with proteins. The LAs prolong phospholipid-based in vitro clotting assays (PT and/or APTT) and are not corrected by mixing patient plasma with normal plasma. The lupus anticoagulant reflexive panel includes PT, APTT, APTT (or PT) mixing study, Russell’s Viper Venom Test and Silica Clot Time (SCT). The lupus anticoagulant panel requires 3 blue top tubes. The tubes must be spun down until they are platelet free (<10,000) and the plasma frozen in appropriately labeled tubes.

In contrast, antiphospholipid antibodies may not be associated with abnormalities in coagulation screening tests (PT or APTT) and include anticardiolipin antibodies (ACAs), anti beta 2-glycoprotein I antibodies (b2-GPI) and anti-phosphatidylserine antibodies. These autoantibodies are detected by ELISA. In-house tests require one full Gold top SST tube.  Send out tests require a second Gold top SST tube.

The presence of LA or antiphospholipid antibody is considered to be a significant risk factor in patients with otherwise unexplained thrombosis and are often present in women who have recurrent fetal loss.

LUPUS ANTICOAGULANT REFLEXIVE PANEL

  • PT
  • SILICA CLOT TIME (SCT)
  • DILUTE RUSSELL VIPER VENOM TEST (DRVVT)
  • THROMBIN TIME (Performed if PTT is prolonged)
  • Xa ASSAY (Performed if TT is prolonged)
  • 1:1 MIXING STUDIES (Performed if SCT or DVTT are prolonged)
  • CONFIRMATORY TESTS

ANTIPHOSPHOLIPID ANTIBODIES 

  • ANTI CARDIOLIPIN ANTIBODIES, IgG AND IgM (Multiplex-EIA)
  • ANTI- BETA 2-GLYCOPROTEIN I ANTIBODIES, IgG, IgM (Multiplex-EIA)
  • ANTI- BETA 2-GLYCOPROTEIN ANTIBODY IgA by Elisa (Send out test)
  • ANTI-PHOSPHATIDYLSERINE ANTIBODIES, IgG, IgM, IgA (Send out tests)

SCREENING TESTS: The SCT and DRRV are screening tests used to detect the presence of a lupus anticoagulant (LA). If these tests are negative, then no further testing is performed. If the tests are prolonged, thrombin time, anti-Xa assay, mixing studies and confirmatory assays are added.

THROMBIN TIME:  If the PTT is prolonged, the thrombin time is added. If the thrombin time is normal (no evidence of heparin), a PTT 1:1 mix will be added. If the thrombin time is prolonged, then anti-Xa assay for heparin levels is added.

MIXING STUDY (1:1): Mixing studies are used to determine whether a prolonged SCT or DRVVT is due to a factor deficiency or an inhibitor. Mixing studies are based on two principles: 1) the inhibitor is present in excess, and if present, it will inhibit normal and patient plasma, and 2) that 50% of any factor is enough to yield a normal test result. Correction into the normal range indicates a factor deficiency. Lack of correction suggests the presence of an inhibitor.

DILUTE RUSSELL VIPER VENOM TEST: The dilute Russell Viper Venom Confirm is intended to confirm the presence of a lupus anticoagulant (LA) in citrated plasma. Russell viper venom directly activates factor X and is not affected by contact factor abnormalities or factor VIII deficiencies/antibodies. Persistence of the positive test must be demonstrated on a separate blood sample collected at least 12 weeks apart. For negative results, consider repeat testing if clinical suspicion remains high or test for anticardiolipin or beta-2 glycoprotein 1 antibodies is recommended.

SILICA CLOT TIME: The Silica Clot Time test is used to detect lupus anticoagulants (LAs). The patient’s plasma is incubated with and without excess phospholipid.   An modified APTT is performed on both tubes using a lupus anticoagulant sensitive reagent. If LAs are present in the test plasma, it would be neutralized by excess phospholipid, and this would result in the shortening of the clotting time. Heparin levels greater than 1 IU/ml and thrombin inhibitors may lead to falsely positive results. Persistence of the positive test must be demonstrated on a separate blood sample collected at least 12 weeks apart. For negative results, consider repeat testing if clinical suspicion remains high or test for anticardiolipin or beta-2 glycoprotein 1 antibodies is recommended.

FACTOR ACTIVITY: The factor activity tests are used to determine the functional activity of factors in the extrinsic, intrinsic and common pathways of coagulation.

PROTHROMBIN (FACTOR II) ACTIVITY (SEND OUT)

FACTOR V ACTIVITY (SEND OUT)

FACTOR VII ACTIVITY (SEND OUT)

FACTOR VIII ACTIVITY

FACTOR IX ACTIVITY

FACTOR X ACTIVITY (SEND OUT)

FACTOR XI ACTIVITY (SEND OUT)

FACTOR XII ACTIVITY (SEND OUT)

FACTOR XIII SCREEN (SEND OUT)

The clot solubility test is used to detect deficiencies or inhibitors to Factor XIII. Clots are suspended in 5 M urea which will disrupt hydrogen bonds. If Factor XIII level is <5%, then the clot will dissolve in 24 hours.

FACTOR INHIBITORS: The inhibitor assays are used to determine the amount of immunoglobulin in a patient. Inhibitors occur in four clinical situations: hemophilia, post partum, immunologic disorders (e.g. RA, SLE, UC, eg) and old age without underlying disease. Factor inhibitor assays cannot be performed in patients with normal factor activity levels. This panel requires 2 blue tubes. They must be spun down until they are platelet free (<10,000) and the plasma frozen.

FACTOR VIII AND FACTOR IX INHIBITORS: The amount of inhibitor present can be calculated in Bethesda units. One Bethesda unit is defined as the amount that will inactivate 50% of the FVIII present.

VON WILLEBRAND PANEL (Send out test): This panel screens for von Willebrand’s Disease. It includes Factor VIII activity, von Willebrand factor (vWF) activity (ristocetin cofactor assay) and von Willebrand antigen. This panel requires 2 blue tubes. They must be spun down until they are platelet free (<10,000) and the plasma frozen.

FACTOR VIII ACTIVITY:  measures the activity of FVIII which is decreased in some forms of VWD.

VON WILLEBRAND ANTIGEN (Send out tests): measures the quantity of von Willebrand factor (vWF) in patient plasma.

RISTOCETIN COFACTOR ASSAY (Send out test):  measures the functional level of vWF protein in the plasma. The vWF activity is reduced in type 1 and 2 von Willebrand Disease and absent in type 3 vWD.

VON WILLEBRAND MULTIMER ANALYSIS (Send out test): Subclassification of von Willebrand Disease is based on the distribution of vWF multimers. vWF multimers are detected using SDS-agarose gel electrophoresis with radiolabeled antibody detection or transblotting and enzyme-linked antibody detection. Von Willebrand factor levels are influenced by ABO blood type. The following conditions may elevate vWF and mask the diagnosis of von Willebrand Disease: pregnancy, oral contraceptives, liver disease, inflammation, exercise and stress.