COAGULATION LABORATORY

Director:  Ginell Post, M.D., Ph.D (501-526-6776, Pager 688-2282)
Manager:   
Curtis Wade, BS, MLS(ASCP)CM (501-686-5226)

Supervisor: 
Elizabeth Guillory, MLS(ASCP) (501-686-8852)
CP Resident on Rotation:  Business Hours (501-686-6397)
CP Resident On-call:  Pager 501-688-2820

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.

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.

REPTILASE TIME (RT): The reptilase time is an alternative screening test for abnormal fibrin formation and can be used to: 1) identify heparin or a heparin-like inhibitor as a cause of a prolonged TT and 2) evaluation of patients with quantitative or qualitative fibrinogen disorders.  Unlike the thrombin time, the reptilase time is unaffected by heparin.

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 strong-positive ELISA result with positive confirmatory testing in a high probability patient is strongly suggestive of heparin induced thrombocytopenia (HIT).  However, it has been reported that a high incidence of anti-heparin PF4 antibodies is observed in the plasma of patients who have undergone cardiopulmonary bypass.  This does not necessarily mean HIT. Therefore, results should be used in conjunction with clinical findings, platelet counts and other laboratory tests.  In patients with HIT, the most common clinical complication is thrombosis (30-40% incidence) despite thrombocytopenia, and non-heparin anticoagulation is typically required.  Although the sensitivity of this testing is high (>90%), false negatives can occur. If there is a strong clinical suspicion of HIT and test results are negative, repeat testing within a few days may be useful since antibody titer can increase rapidly over time.

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
          HEPARIN NEUTRALIZATION (performed if APTT is prolonged)
          ANTI-THROMBIN III ACTIVITY
          PROTEIN C ACTIVITY
          PROTEIN S ACTIVITY
          APC RESISTANCE RATIO
          HOMOCYSTEINE
          DNA-PROTHROMBIN MUTATION
          FACTOR V LEIDEN
          FIBRINOGEN

HEPARIN NEUTRALIZATION: Heparin contamination of specimens can cause an unexpectedly prolonged APTT.  Heparinase (Hepzyme®) can be used to determine if the APTT prolongation is due to heparin (UFH and LMWH). In addition, heparinase can be used to remove heparin from samples to perform coagulation tests that demonstrate heparin interference.

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: 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: 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): 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 Staclot-LA. 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-glycoprotien I antibodies (b2-GPI) and anti-phosphatidylserine antibodies.  These autoantibodies are detected by ELISA. The test requires a red top tube and are send out tests.

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
PTT-LA
DILUTE RUSSELL VIPER ENOM TEST (DRVVT)
THROMBIN TIME (PERFORMED IF PTT IS PROLONGED
REPILASE TIME (PERFORMED IF TT IS PROLONGED)
PTT-HEPARIN NEUTRALIZED (PERFORMED IF PTT IS PROLONGED)
1:1 MIXING STUDIES (PERFORMED IF PTT OR DVVT ARE PROLONGED)
STACLOT LA

  ANTIPHOSPHOLIPID ANTIBODIES (SEND OUT TEST)
ANTI CARDIOLIPIN ANTIBODIES, IgG AND IgM (ELISA)
ANTI- BETA 2-GLYCOPROTIEN I ANTIBODIES, IgG, IgM AND IGgA (ELISA)

SCREENING TESTS: The PTT-LA 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, reptilase time, heparin neutralization, mixing studies and confirmatory assays are added.

THROMBIN TIME and HEPARIN NEUTRALIZATON: 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 reptilase time is added.  If the reptilase time is normal, PTT heparin neutralization is added. If heparin neutralization is prolonged, then a PTT 1:1 mix will be added.

MIXING STUDY (1:1): Mixing studies are used to determine whether a prolonged APTT 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.

LUPUS ANTICOAGULANT SCREEN (STACLOT LA): The Staclot LA test is used to detect lupus anticoagulants (LAs).  The patient’s plasma is incubated with and without hexagonal phase phosphatidylethanolamine (HPE).  An 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 HPE, 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.

BETA-2 GLYCOPROTEIN I ANTIBODIES (SEND OUT TEST):  Beta-2 Glycoprotein I is a protein cofactor that has a high binding affinity for negatively-charged phospholipids forming phospholipid-protein complexes.  Anti-beta-2 Glycoprotein I antibodies are more relevant for antiphospholipid antibodies with fewer false positive results than the anticardiolipin or antiphosphatidylserine assay. Anti-beta-2 glycoprotein I antibodies are detected by ELISA. 

ANTICARDIOLIPIN ANTIBODIES (SEND OUT TEST): Anticardiolipin antibodies are antibodies directed against phospholipid-binding proteins and are detected by ELISA.

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: 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: 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.