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Last updated 5 January 2006
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McCullough: Transfusion Medicine; November 2004 (2nd edition), 592 pages, $62 (list). Presents a practical approach to the diagnosis and management of the most common disorders of red blood cells, white blood cells, and hemostasis. Each disease state is discussed in terms of underlying pathophysiology, clinical features which suggest the diagnosis, the use of state-of-the-art lab tests in the diagnosis and differential diagnosis of the condition, and current management.
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Table of Contents-Coagulation
Primary references, hemostasis-general, normal hemostasis, intrinsic pathway, extrinsic pathway, common pathway, protein C/S, thrombomodulin, antithrombin, fibrinolysis pathway, contact system
Bleeding disorders: general, laboratory approach
Acquired bleeding disorders: acute phase reaction, acquired dysfibrinogenemia, acquired von Willebrand’s disease, amyloidosis, bovine coagulation factor inhibitors, DIC, factor V inhibitor, factor VIII inhibitor, factor IX inhibitor, liver dysfunction, lupus anticoagulants, proteinuria, Vitamin K deficiency/warfarin
Hereditary bleeding disorders: general, algorithm for workup, factor I (fibrinogen) deficiency, factor II (prothrombin) deficiency, factor V deficiency, factor VII deficiency, factor VIII deficiency (hemophilia A), factor IX deficiency (hemophilia B), factor X deficiency, factor XI deficiency, factor XII deficiency, factor XIII deficiency, high molecular weight kininogen deficiency, prekallikrein deficiency, von Willebrand’s disease
Therapy related coagulopathies: warfarin, danaparoid, heparin, heparin-low molecular weight, hirudin, thrombolytic therapy
Acquired thrombophilia / hypercoagulopathies: general, antiphospholipid antibodies, heparin induced thrombocytopenia
Hereditary thrombophilia / hypercoagulopathies: general, activated protein C resistance / factor V Leiden, antithrombin deficiency, dysfibrinogenemia, elevated coagulation factors, heparin cofactor II deficiency, hyperhomocysteinemia, protein C deficiency, protein S deficiency, prothrombin gene mutation (G20210A), sickle cell disease
Coagulation laboratory tests: general, quality assurance, abnormal PT and PTT, activated clotting time, activated protein C resistance, anticardiolipin antibodies, antiplasmin, antithrombin, bleeding time, clot retraction, cryoglobulin / cryofibrinogen, D-dimer, ecarin clotting time, factor assays, factor I (fibrinogen) assay, factor V Leiden, factor VII assay, factor VIII assay, factor VIII inhibitor, factor IX assay, factor Xa assay, factor XI assay, factor XIII assay, heparin induced thrombocytopenia, heparinase, high molecular weight kininogen, homocysteine, hypercoagulation panel, INR, International sensitivity index, low molecular weight heparin, lupus anticoagulant, mixing studies, plasminogen assay, plasminogen activator antigen-1, platelet aggregation studies, platelet antibodies, platelet hyperaggregation studies, prekallikrein assay, protein C assays, protein S assays, prothrombin gene 20210A, PT, PTT, reptilase time, thrombin time, tPA, vWF testing-general, vWF antigen analysis, vWF activity, vWF multimer analysis
Archives of Pathology and Laboratory Medicine (Archives), January 1976 to July 2005
CAP Today, January 2000 to June 2005
Journal of Clinical Pathology, January 2001 to July 2005
Henry: Clinical Diagnosis and Management by Laboratory Methods, 2001 (20th edition)
Massachusetts General Hospital coagulation handbook
Journal search terms: coagulation
Please refer to these primary references for more detailed discussions and photographs
Involves formation of blood clots to stop bleeding from damaged vessels, and activation of natural anticoagulation and fibrinolytic systems to limit clot formation to sites of injury
Bleeding disorders are due to defects in clot formation or overactive fibrinolytic systems
Hypercoagulability disorders are due to defects in anticoagulant system or underactive fibrinolytic systems
Initial step is formation of platelet plug to stop bleeding from damaged vessel
Then, platelet plug is reinforced by fibrin clot
Then, fibrin clot is stabilized by activated factor XIII, which cross-links fibrin strands
Fibrin clot may occur via either intrinsic or extrinsic pathway (or both), though in vivo it occurs via a hybrid model
Coagulation factors in intrinsic or extrinsic pathway assemble on surface of activated platelets, which are usually at site of vascular injury
Many coagulation reactions also require calcium as a cofactor
Note: “a” after factor number indicates “activated”
Factor I: fibrinogen
Factor II: prothrombin
Factor III: tissue thromboplastin (tissue factor and phospholipid)
Factor IV: ionized calcium
Factor V: occasionally called labile factor or proaccelerin
Factor VI: unassigned
Factor VII: occasionally called stable factor or proconvertin
Factor VIII: antihemophilic factor
Factor IX: plasma thromboplastin component, Christmas factor
Factor X: occasionally called Stuart-Prower factor
Factor XI: occasionally called plasma thromboplastin antecedent
Factor XII: Hageman factor
Factor XIII: fibrin-stabilizing factor
Involves factors VIII, IX, XI, XII (Hageman factor), prekallikrein, high molecular weight kininogen
Merges with extrinsic pathway into common pathway
Activated when factor XII binds to negatively charged “foreign” surface exposed to blood
Then sequentially activates factors XI, IX, X, then factor II (prothrombin to thrombin), which converts fibrinogen to fibrin (see common pathway, below)
Once extrinsic pathway is inhibited by TFPI-Xa complex (see extrinsic pathway), factor VIIIa / IXa complex becomes dominant generator of factor Xa, thrombin and fibrin
Factor XIIa also converts prekallikrein to kallikrein, which activates more factor XIIa; both require high molecular weight kininogen as cofactors
Kallikrein also releases bradykinin from high molecular weight kininogen, which causes vasoconstriction
Involves tissue factor (TF), originally considered “extrinsic” to blood since it is present on cell surfaces not normally in contact with (i.e. extrinsic to) the circulatory system
The primary mechanism of the coagulation pathway in vivo is tissue factor binding to activated factor VII (factor VIIa)
TF-Factor VIIa complex activates factors X and IX (though in vivo it appears to first involve factors VIII and V from the intrinsic pathway, which then activate factors X and IX)
Activated factor IX activates more factor X, with cofactors activated factor VIII, anionic phospholipids (from activated platelets) and calcium
Activated factor X converts prothrombin to thrombin, with activated factor V, anionic phospholipids (from activated platelets) and calcium as cofactors; prothrombin factor 1.2 is released (see common pathway, below)
After initial activation, pathway is inhibited by the binding of tissue factor pathway inhibitor (TFPI) to factor Xa, which inhibits TF-Factor VIIa complex, and further coagulation is dependent on the intrinsic pathway
Merges with extrinsic pathway into common pathway
Involves fibrinogen (factor I), factors II (prothrombin), V, X
Thrombin converts soluble fibrinogen to insoluble fibrin; releases fibrinopeptides A and B; remaining fibrin monomers polymerize to form fibrin; thrombin also binds to antithrombin, which inhibits thrombin to prevent excessive clotting
Thrombin may also activate factor XI (part of intrinsic pathway), factors V, VIII, XIII, XI and platelets
Factor XIII cross links fibrin to increase stability of fibrin clot
Charts: see normal hemostasis (above)
Protein C / Protein S anticoagulant pathway
Pathway is a physiologic anticoagulant system to limit blood clot formation (i.e. fibrinogen to fibrin conversion) to site of vessel injury
Major anticoagulant systems are protein C and protein S, antithrombin and tissue factor pathway inhibitor (TFPI, see Extrinsic pathway above)
Protein C and S: vitamin K dependent anticoagulant proteins produced mainly in liver (“C” because was third peak to elute from a diethylaminoethyl affinity column)
Activation: endothelial cell protein C receptor binds thrombin-thrombomodulin complex, which activates protein C, which binds to free protein S on endothelial or platelet phospholipids surfaces; this protein C / protein S complex degrades factors Va and VIIIa, which reduces fibrin formation
Activated protein C also indirectly promotes fibrinolysis
60-70% of protein S is bound to and inactivated by C4b binding protein, an acute phase reactant
Clinical note: since C4b increases during pregnancy, the protein S level will routinely fall below the normal non-pregnant range
References: Archives 2002;126:1337
Intrinsic membrane glycoprotein on luminal surface of endothelial cells that binds thrombomodulin and facilitates the activation of protein C
C/T dimorphism at nucleotide 1418 is associated with premature myocardial infarction, but no definite association with venous thromboembolism
References: BMC Neurology 2004;4:21
Formerly called antithrombin III
Functions as anticoagulant by inhibiting activated factors II (thrombin), IX, X, XI, XII, kallikrein, plasmin and probably factor VII (all are serine proteases)
Activity is accelerated 1000x by interaction with heparin or heparan sulfate (located on endothelial cells)
Member of serine protease inhibitor (serpin) gene family on #1q23-25
References: Archives 2002;126:1326
Process of degrading the fibrin clot when it is no longer needed
Also prevents extension of clot beyond site of injury
Initiated by tPA (tissue plasminogen activator) or uPA (urokinase-like plasminogen activator), which convert plasminogen to plasmin in the presence of fibrin by cleaving the Arg561-Val562 peptide bond
Plasmin degrades the fibrin clot and intact fibrinogen to soluble fibrin/fibrinogen degradation products (FDP)
Plasmin also inactivates factors Va and VIIIa (as does Protein C and Protein S)
tPA is produced by endothelial cells; its activation of plasminogen is major mechanism for lysis of fibrin clots
Recombinant tPA is used to treat myocardial infarction, stroke and some cases of acute thrombosis
uPA is produced by urine and plasma; keeps renal tracts free of blood clots; also is important for other cell surfaces and initiating nonfibrinolytic activities of plasmin
Excessive fibrinolysis is prevented by plasmin inhibitor (antiplasmin, formerly called alpha2-antiplasmin) and plasminogen activator inhibitor 1 (PAI-1, inhibits tPA and uPA)
PAI-1 is synthesized by hepatocytes and endothelial cells, is present in platelets and plasma; can bind to fibrin and inhibit plasminogen activators tPA and uPA
PAI-1 is an acute phase reactant protein, and may increase 30-50 fold over baseline, possibly immediately inactivating systemically administered tPA
Homozygous deficiency of plasminogen is associated with ligneous conjunctivitis (rare form of chronic pseudomembranous conjunctivitis), and replacement therapy with plasminogen is therapeutic
Neither heterozygous plasminogen deficiency (0.5 to 2.0% of patients with thrombosis) nor tPA deficiency are associated with increased risk of thrombosis
References: Archives 2002;126:1376
Consists of coagulation factors unknown in the 1950’s
Includes factor XII (Hageman factor), prekallikrein (PK; Fletcher factor), high molecular weight kininogen (Williams, Flaujeac or Fitzgerald factor); some authors include factor XI
Made in the liver
Decreased activity is associated with liver disease, hepatic immaturity in newborns, antiphospholipid syndrome, Asian descent (for factor XII)
Homozygous deficiencies are rare, autosomal recessive; cause very long PTT but no bleeding disorders and no definite association with hypercoagulability
Recommended to not measure their activity in routine evaluation of patients with arterial or venous thromboembolism or acute coronary syndromes (Archives 2002;126:1382)
Laboratory testing: homozygous deficiencies cause prolonged PTT; heterozygous deficiencies have near normal PTT; the test for a particular contact factor is based on the ability of the patient’s plasma to correct a prolonged PTT in plasma that is deficient in the factor being tested
Even though the PTT may be decreased by deficiencies of contact factors, this does not necessarily correlate with increased bleeding risk
Bleeding disorders
Clinical history is important:
(a) single site (structural lesion) vs. multiple sites (coagulopathy)
(b) for coagulopathies - hereditary (family history of bleeding or bleeding since childhood) or acquired (no previous bleeding history)
(c) time from “hemostasis challenge” to bleeding symptoms - immediate suggests platelet disorder (inability to form normal platelet plug); late suggests coagulopathy (breakthrough bleeding occurs after platelet plug due to impaired fibrin formation)
(d) physical exam: petechiae (platelet disorders) vs. hematoma or hemarthrosis (coagulation defects) vs. mucous membrane bleeding or bruising (nonspecific)
Bleeding disorders are often classified as defects of primary hemostasis (platelets, vessels, etc.) or of secondary hemostasis (coagulation cascade and its regulation)
Bleeding disorders - laboratory approach
Laboratory tests should be ordered if (a) history or physical exam is suspicious for bleeding disorder or (b) for routine preoperative testing (PT, PTT, platelet count)
See algorithms for prolonged PT, prolonged PTT, abnormal platelet count (platelet chapter) or hereditary bleeding disorder (PT, PTT, and platelet count normal)
References: Crookston, K. P., and Spiess, B.D.; "Coagulation Support in the Perioperative Setting." in Simon: Rossi’s Principles of Transfusion Medicine; 2002 (3rd edition)
Acquired bleeding disorders
Many serum proteins become elevated due to illness, injury, inflammation or stress; also pregnancy
Elevated levels return to normal after condition resolves
Causes increase in fibrinogen, factor VIII, vWF (up to 3x normal levels) and PAI-1 (up to 50x); decrease in PTT, decrease in protein S (due to binding to increased C4b)
Usually caused by liver or biliary tract disease, acute phase reaction, hepatocellular carcinoma or renal cell carcinoma
Due to increased sialylation of fibrinogen’s carbohydrate side changes; this increases its net negative charge, which promotes charge repulsion between fibrin monomers and decreases the rate of fibrin polymerization
Tumor cells may secrete abnormal fibrinogen
Usually does not cause bleeding or thrombosis, but may in alcoholic liver disease
Acquired von Willebrand’s disease
Rare, either spontaneous or associated with hematologic neoplasms or autoimmune disorders
Primary amyloidosis may cause acquired factor X deficiency due to the binding of amyloid to factor X
Also inhibits fibrinogen conversion to fibrin, causing prolongation of thrombin time and reptilase time
Bovine coagulation factor inhibitors
After use of bovine “fibrin glue” to achieve hemostasis, 1.7% develop a clinically significant inhibitor
Some fibrin glue contains bovine thrombin and cryoprecipitate (containing human fibrinogen)
Antibodies may be formed against bovine thrombin, also against bovine factors V, VII, X (Archives 1998;122:887)
Disseminated intravascular coagulation (DIC)
Common, due to massive tissue injury, sepsis or other excessive activation of coagulation system; also pregnancy complications (abruptio placenta, amniotic fluid embolism, acute fatty liver of pregnancy, septic abortion), acute hemolytic transfusion reaction, snake bites, homozygous protein C deficiency, adult respiratory distress syndrome and hyaline membrane disease; chronic causes are cancer, liver disease, aortic aneurysm, retained dead fetus, giant hemangioma or head trauma
Anticoagulant and fibrinolytic systems are activated simultaneously and overwhelmed, leading to disseminated microthrombi and tissue ischemia, consumption of platelets, coagulation factors and natural anticoagulants, and variable bleeding
With malignancy, may get large vessel thrombosis
Activation of fibrinolytic system causes plasmin activation and formation of D-dimers and other fibrin degradation products
Schistocytes (fragmented red blood cells) are formed as red blood cells are severed flowing through fibrin strands
Laboratory: elevated D-dimers and other fibrin degradation products (FDPs), prolonged PT (70% of cases) and PTT (50%), decreasing platelets and fibrinogen (50%), schistocytes (50%); with chronic causes, fibrinogen and platelets may actually be elevated as acute phase reactants; all factors may be variably decreased due to factor activation and consumption
Baseline coagulation studies and serial followup are needed to follow the trends
Note: D-dimer may be falsely positive in HIV+ Castleman’s disease due to interference from monoclonal gammopathy (Archives 2004;128:328)
Treatment: treat underlying disease, transfuse fresh frozen plasma, platelets, cryoprecipitate if bleeding; keep fibrinogen levels above 100 mg/dL with cryoprecipitate or fresh frozen plasma; monitor PT, PTT, platelet count, fibrinogen and possibly antithrombin levels
Case reports: 30 year old woman with DIC due to amniotic fluid embolism (Archives 2002;126:869)
May behave like factor VIII inhibitor in mixing studies, with increasing PTT or PT after 1-2 hours
Most common clinically significant inhibitor; develops in 10-20% of patients with severe hemophilia A after infusion of factor VIII containing products, less often with mild/moderate disease
Rarely arises in patients without hereditary hemophilia, causing acquired hemophilia A (see below)
Causes prolonged PTT; in mixing study, PTT may initially be normal, then increases after 1-2 hours incubation
A nonlinear curve in a factor assay is often a clue to the presence of an inhibitor
Associated with normal PT
In patients with hemophilia A and factor VIII inhibitor, titer of inhibitor often increases after treatment with factor VIII containing products - this does not happen with autoimmune factor VIII inhibitor
Treatment with recombinant factor concentrates appears to lead to more inhibitors than plasma-derived concentrates
Each Bethesda unit of inhibitor indicates a decrease of factor VIII concentration in assay by 50% (1 unit: 100% to 50%; 2 units: 100% to 25%; 3 units: to 12.5%, etc.)
Treatment: porcine factor VIII (if no cross reactivity with inhibitor), prothrombin complex concentrates, FEIBA, recombinant factor VIIa (J Thromb Haemost 2004;2:899), immunosuppression for autoimmune based inhibitors
Nonhemophilic patients (autoimmune based inhibitors)
Incidence of 0.2 to 1 per million population per year
Causes bleeding
50% occur in patients with no known medical problems
Also associated with rheumatoid arthritis, SLE, post-partum; also solid tumors and hematologic malignancies
Laboratory: normal D-dimer and fibrinogen; prolonged PTT not corrected by mixing studies, nonlinear curve on assay for factor VIII
Treatment: recombinant factor VIIa, plasmapheresis (variable success), factor VIII, immunosuppression
References: Archives 2000;124:730
Develops in 2-12% of patients with severe hemophilia B after transfusion of factor IX containing products, less commonly with mild/moderate disease
Rarely arises in patients without hereditary hemophilia, causing acquired hemophilia B
Causes a prolonged PTT, not corrected by mixing studies, and a nonlinear curve on the factor assay
Can quantitate titer of inhibitor
Liver is site of production of most coagulation factors, but response of each factor to liver disease is variable due to differences in biologic half lives and acute phase reactions
PT usually prolonged first, then PTT
Factor VII: shortest biologic half life, often affected earliest with largest decrease in serum level
Clinical note: Factor VII also decreases earliest with warfarin treatment
Factor VIII: may be normal or elevated due to acute phase reaction
Factors XI and XII: long biologic half lives, may be normal until liver disease is advanced
Common
Antibodies against protein-phospholipid complexes
Causes prolonged PTT (not time dependent), increased or normal PT; may interfere with assays for factors VIII, IX, XI and XII without causing a true decrease in factor levels
May be mistaken for a factor VIII inhibitor if dilutions to abnormal factor assays are not done
Not associated with bleeding, except when accompanied by severe thrombocytopenia or acquired factor II deficiency (rare)
May cause factor II deficiency if lupus anticoagulant binds to factor II
May be associated with thrombosis
Case reports: due to phenytoin use (Archives 1987;111:719)
Patients with nephrotic syndrome may have decreased factors XI and XII
Vitamin K deficiency / warfarin use
Both have same molecular mechanism for their effects
Warfarin (coumadin): therapeutic anticoagulant to reduce risk of thromboembolism; impairs regeneration of active vitamin K
Vitamin K: cofactor in carboxylation of glutamic acid residues of factors II, VII, IX and X and protein S and C
Warfarin administration or Vitamin K deficiency cause prolonged PT; severe cases have prolonged PTT also
Vitamin K deficiency: due to fat malabsorption syndromes (vitamin K is a fat soluble vitamin), malnutrition, antibiotics (destroy bacteria producing vitamin K or interfere with vitamin K carboxylation), newborns
Treatment (warfarin overdose - INR > 5.0): fresh frozen plasma or vitamin K; PT should normalize within 12-24 hours
Clinical note: if a large dose of vitamin K is given, then it may be difficult to reach a therapeutic level of warfarin very quickly if the patient continues on warfarin therapy
Treatment (vitamin K deficiency): vitamin K once, then 12-24 hours later, then measure PT (should normalize)
Hereditary bleeding disorders
Hereditary bleeding disorders-general
Acquired factor deficiencies (due to liver disease, DIC, lupus anticoagulants, heparin, warfarin or other anticoagulants) are more common than hereditary factor deficiencies, and should be ruled out first
Heterozygous patients have 30-60% of normal values of affected factors, usually with no or minor bleeding disorder
However, factor I (hypofibrinogenemia or dysfibrinogenemia), X, XI or XIII deficient heterozygotes may have bleeding symptoms
Homozygous deficient patients have <30% of normal values of affected factors
In hemophilia A and B, small differences in factor levels (i.e. 1% vs. 3% vs. 10%) may markedly affect the clinical presentation and course
Hereditary disorders are confirmed by measuring factor levels in relatives
Combined factor deficiencies are very rare
Combined factor V and VIII: autosomal recessive, due to mutation in endoplasmic reticulum-Golgi gene ERGIC 53 on #18 that transports these factors
Combined factors II, VII, IX and X deficiency: due to mutation in gamma-glutamyl carboxylase gene, whose protein carboxylates glutamate residues in vitamin K-dependent coagulation factors
Very rare to have bleeding disorders due to deficiency in PAI-1 or antiplasmin
Symptoms: bleeding associated with surgery, trauma, dental extractions, postpartum, circumcision or umbilical stumps, GI bleeding, intracranial hemorrhage, hemarthrosis or soft tissue hematomas, easy bruising, epistaxis, menorrhagia
Algorithm for workup of hereditary bleeding disorders
Often there is a family history of bleeding disorder
PT, PTT, platelet count often normal
(a) Test for von Willebrand’s disease (most common hereditary bleeding disorder)
Testing for vWD includes Factor VIII activity, vWF antigen, vWF activity (often done by the “ristocetin cofactor” method); these results may lead to vWF multimer assays and blood type (type O patients have reduced vWF activity)
(b) Assays for fibrinogen, platelet aggregation or platelet function assay (e.g. PFA-100), factor XIII, dysfibrinogenemia (thrombin time or reptilase time)
(c) Also assays for factors VIII, IX or XI, even with normal PTT
(d) Factor XIII assay if delayed bleeding is present (often done by “urea clot lysis” method)
(e) More esoteric assays include PAI-1 activity and antiplasmin
Factor I / fibrinogen deficiency or disorders
Rare
Autosomal inheritance; most mutations are in alpha-fibrinogen chain gene, sparing beta and gamma chains
Often associated with bruising, epistaxis, menorrhagia, GI/GU bleeding, umbilical stump bleeding, miscarriage, poor wound healing; also bleeding after surgery, trauma, dental procedures, pregnancy or circumcision
May prolong PT and PTT
Need 100 mg/dL of fibrinogen for surgical hemostasis; biologic half life is 72-120 hours; less is needed in non-surgical conditions, if there is no severe hemostatic challenge
Afibrinogenemia: homozygous form; causes severe quantitative deficiency of fibrinogen and increased risk of bleeding; associated with intracranial hemorrhages
Hypofibrinogenemia: heterozygous form; mild/moderate reductions in fibrinogen; little/no bleeding
Dysfibrinogenemia: qualitative fibrinogen deficiency with production of dysfunctional fibrinogen; usually heterozygous; usually either no symptoms or mild bleeding; may paradoxically be associated with thrombosis, with or without bleeding; often prolonged thrombin time and reptilase time, PT and PTT
Acquired causes: DIC, liver dysfunction - more common than hereditary deficiencies
Treatment: 1 unit of cryoprecipitate per 7 kg as needed to keep fibrinogen above 100 mg/dL, if symptomatic; one unit raises fibrinogen by 10 mg/dL (approximately); one unit of fresh frozen plasma contains at least twice as much fibrinogen as one unit of cryoprecipitate, but in a much larger volume
Factor II (prothrombin) deficiency
Rare
Autosomal inheritance
Need 10-40% for surgical hemostasis; biologic half life is 48-120 hours
Severe deficiencies associated with intracranial hemorrhage
Acquired hypoprothrombinemia can be associated with lupus-like inhibitors
Treatment: recombinant factor VIIa, alternatively 10-20 ml fresh frozen plasma/kg, then 3 ml/kg every 12-24 hours as necessary; prothrombin complex concentrates may be used for serious bleeding
Also called labile factor deficiency
Rare
Autosomal inheritance
Need 10-30% for surgical hemostasis, biologic half life is 12-36 hours
May be associated with bruising, epistaxis, menorrhagia, GI/GU bleeding, umbilical stump bleeding or bleeding after surgery, trauma, dental procedures, pregnancy or circumcision
Severe deficiencies associated with intracranial hemorrhage, although levels don’t always correlate with severity of symptoms
Treatment: 10-20 ml fresh frozen plasma/kg, then 3-6 ml/kg every 12 hours as necessary; commercial concentrates of factor V are not available
Also called autoprothrombin I deficiency
Rare
Autosomal inheritance
Need 10-25% for surgical hemostasis, biologic half life is 4-7 hours
May be associated with bruising, epistaxis, menorrhagia, GI/GU bleeding, umbilical stump bleeding or bleeding after surgery, trauma, dental procedures, pregnancy or circumcision
Severe deficiencies may resemble hemophilia A or B, and are associated with intracranial hemorrhage
Levels don’t always correlate with severity of symptoms
Treatment: 10-20 ml fresh frozen plasma/kg, then 3-6 ml/kg every 4 hours as necessary
Factor VIII deficiency (Hemophilia A)
Most common severe hereditary bleeding disorder
X linked recessive disorder; (gene is on X chromosome)
Need 80-100% for surgical hemostasis with major surgery or major bleeding, 30-50% postoperatively or to prevent minor bleeding
Biologic half life is 8-12 hours
Affects 1 per 5-10K males; female carriers are usually unaffected unless they have imbalanced inactivation (lyonization), Turner’s syndrome or other rare X chromosomal abnormalities; females with disease are rare (daughters of affected male and carrier female)
Clinical severity varies with factor levels: >5%: bleeding only with surgery or trauma; 1-5%; moderate bleeding; <1%: severe disease with spontaneous bleeding
30% of cases arise from new mutations, so there may be no family history
Symptoms: bleeding into muscle, soft tissue or joints (hemarthrosis), GI/GU tract; easy bruising, excessive bleeding after surgery, trauma, dental procedures or circumcision; epistaxis, poor wound healing, post-traumatic intracranial hemorrhage
Laboratory: prolonged PTT and normal PT in males with unexplained bleeding; measure factor VIII and IX levels (values of 20-30% of normal may cause prolonged PTT) and von Willebrand test panel (reduced factor VIII may be due to decrease in vWF; in female hemophilia A carriers, factor VIII/vWF ratio is 0.5 vs. 1.0 in normal females)
Notes: (a) factor VIII and vWF may be elevated during acute phase reactions, including pregnancy; must repeat tests when acute phase reaction has subsided; (b) factor VIII is labile at room temperature, and mild/moderate decreases may be due to improper processing and storage
Molecular: 40% in Caucasians are due to inversion of intron 22; also numerous other mutations; gene is large; RFLP analysis may be useful in families without the intron 22 mutation
Treatment: factor VIII concentrates (now treated to destroy viruses, but HIV+ in early 1980’s); 1 unit/kg raises levels in vivo by 2%
major surgery/bleeding - 40-50 units factor VIII concentrate/kg every 12 hours as necessary, usually for 7-10 days,
postoperatively - 15-25 units/kg every 12 hours as necessary, usually for 7-10 days
minor bleeding - 15-20 units/kg every 12-24 hours as necessary for minor bleeding
mild/moderate bleeding - DDAVP (if patients respond to DDAVP)
References: Mol Pathol 2002;55:127 (molecular aspects)
Factor IX deficiency (hemophilia B)
Also called Christmas disease, autoprothrombin II deficiency
Severe hereditary bleeding disorder
X lined recessive disorder (gene is on X chromosome)
Need 50-80% of normal levels for surgical hemostasis with major surgery or major bleeding, 40% postoperatively, 30-50% to prevent minor bleeding
Affects 1 per 25-30K males; female carriers are unaffected; females with disease are rare
Clinical severity varies with factor levels: >5%: bleeding only with surgery or trauma; 1-5%; moderate bleeding; <1%: severe disease with spontaneous bleeding
Factor half life is 18-24 hours
Symptoms: bleeding into muscle, soft tissue or joints (hemarthrosis), GI/GU tract; easy bruising, excessive bleeding after surgery, trauma, dental procedures or circumcision; epistaxis, poor wound healing, post-traumatic intracranial hemorrhage
Laboratory: prolonged PTT and normal PT in males with unexplained bleeding; measure factor VIII and IX levels (values of 20-30% of normal may cause prolonged PTT) and von Willebrand test panel
Molecular: numerous mutations; genetic testing for female carriers or prenatal detection uses RFLP analysis
Treatment: factor IX concentrates (now treated to destroy viruses, but HIV+ in early 1980’s); 1 unit/kg raises levels in vivo by 1%
major surgery/bleeding - 50-80 units factor IX concentrate/kg every 12-24 hours as necessary, usually for 7-10 days
postoperatively - 40 units/kg every 12-24 hours, usually for 7 days
minor bleeding - postoperatively; 30-40 units/kg q 12-24 hours as necessary
Rare
Autosomal inheritance
Need 10-40% for surgical hemostasis, biologic half life is 24-48 hours
May be associated with bruising, epistaxis, menorrhagia, GI/GU bleeding, umbilical stump bleeding or bleeding after surgery, trauma, dental procedures, pregnancy or circumcision
Severe deficiencies may resemble hemophilia A or B, and are associated with intracranial hemorrhage
Treatment: 10-20 ml fresh frozen plasma/kg, then 3-6 ml/kg every 12 hours as necessary; may use prothrombin complex concentrates for serious bleeding
Common among Ashkenazi Jews
Autosomal inheritance
Need 15-50% for surgical hemostasis, biologic half life is 40-84 hours
May be associated with bruising, epistaxis, menorrhagia, GI/GU bleeding, umbilical stump bleeding or bleeding after surgery, trauma, dental procedures, pregnancy or circumcision
Levels don’t always correlate with severity of symptoms
Treatment: 10-20 ml fresh frozen plasma/kg, then 5-10 ml/kg every 24 hours as necessary
Note: factor XI concentrates may promote thromboembolic complications
Also called Hageman factor deficiency
Relatively common
Autosomal inheritance
Not needed in normal procoagulant pathways - deficiencies do not cause bleeding symptoms
May be implicated as cause of isolated, prolonged PTT after preanalytic variables and heparin contamination have been ruled out
Factor XII is activated by high molecular weight kininogen and prekallikrein
Activated factor XII converts prekallikrein to kallikrein, which activates more factor XII
Rare
Autosomal inheritance
Need 5-50% for surgical hemostasis, biologic half life is 9-12 days
Although fibrin clots form, they are weak and subsequently lyse
Normal PT and PTT
Patients commonly present with history of delayed bleeding; often associated with bruising, epistaxis, menorrhagia, GI/GU bleeding, umbilical stump bleeding, miscarriage, intracranial hemorrhage, poor wound healing; also bleeding after surgery, trauma, dental procedures, pregnancy or circumcision
Testing recommended if delayed bleeding, umbilical stump bleeding, or miscarriages (with normal PT and PTT)
Diagnosis: usually with “urea clot lysis” assay, although specific factor assay is available in specialized labs
50% of population has Val134Leu polymorphism, which may protect against deep venous thrombosis, but predispose to intracranial hemorrhage
Acquired causes of factor XIII deficiency: liver disease, DIC, Crohn’s disease, ulcerative colitis, Henoch-Schonlein purpura, leukemia, myelodysplasia, myeloproliferative disorders
Treatment: 500 ml plasma or 1 bag cryoprecipitate / 10 kg every 3 weeks
High molecular weight kininogen deficiency
Rare
Autosomal inheritance
Not needed in normal procoagulant pathways - deficiencies may cause marked prolongation of PTT, but do not cause bleeding symptoms
Rare
Autosomal inheritance
Prolonged PTT, but not associated with bleeding
Acquired cause are DIC or liver disease, rarely antibodies to prekallikrein
Most common hereditary bleeding disorder, affecting 1-2% of population, no gender preference
Often mild and undiagnosed; may be masked by acute phase reactions
Unmarked vWD is still a common underlying cause of hysterectomy; test women with menorrhagia during first few days of period
Due to quantitative or qualitative deficiencies of von Willebrand factor (vWF), found on #12
Symptoms are similar to a platelet function defect (epistaxis, easy bruising, bleeding, menorrhagia)
vWF is synthesized by (a) endothelial cells, stored in Weibel-Palade bodies, secreted into plasma and subendothelium and (b) megakaryocytes, present in platelets in alpha granules
vWF is large polypeptide that polymerizes to form multimers of up to 100 subunits
Plays a role in platelet plug and fibrin clot, both essential to hemostasis at site of endothelial injury, particularly in high flow vessels
vWF mediates platelet adhesion to endothelium (and formation of platelet plug) by serving as a bridge between them - binds to GPIb glycoprotein on platelet surface and to exposed subendothelium at site of endothelial injury
vWF supports coagulation (fibrin clot) by serving as protective carrier protein for factor VIII; without vWF, factor VIII has shorter half life and its plasma levels are lower
Note: type O patients have lower levels of vWF, type AB patients have highest levels; levels increase with age and with acute phase reactions
Treatment: DDAVP (desmopressin) temporarily increases vWF and factor VIII levels 2-3x; for the patients that don’t respond, give vWF-containing factor VIII concentrates
Testing: Factor VIIII activity, vWF antigen, vWF activity (often done by “ristocetin cofactor” assay); possible additional tests include vWF multimer size determination and blood type (vWF is significantly decreased in type O patients)
Repeat testing is often required because vWF and factor VIII become elevated during minor illnesses, injury, stress, pregnancy, estrogen use, other acute phase reactions or in newborns
Subtypes
Clinical note: As a general rule in coagulation, type I deficiencies refer to a decrease in the absolute amount of a normal factor and type II deficiencies indicate a defective protein that may be present in normal amounts
Type 1 (70-80%): most common, autosomal dominant, partial quantitative deficiency of vWF but normal function; causes mild/moderate bleeding disorder
Low factor VIII, vWF antigen and ristocetin cofactor; low/normal ristocetin induced platelet aggregation, normal or all sizes decreased in multimer analysis, mean ristocetin cofactor/vWF antigen ratio is 1.0; normal platelet count
Type 2 (15-20%): qualitative deficiency of vWF, variable quantitative deficiency, usually mild/moderate bleeding disorder, but may be severe
Type 2A: most common type 2 subtype; autosomal dominant; low/normal factor VIII activity and vWF; relative reduction of intermediate and high molecular weight multimers due to in vivo proteolytic degradation or defective multimer assembly and secretion; markedly reduced ristocetin cofactor, low ristocetin induced platelet aggregation; platelet vWF has similar abnormalities as plasma vWF; mean ristocetin cofactor/vWF antigen ratio is 0.3; normal platelet count
Type 2B: autosomal dominant, hemostatic defect due to intermittent thrombocytopenia and qualititatively abnormal vWF, with increased binding of vWF to GPIb (platelet vWF receptor), causing faster clearing of vWF coated platelets from the bloodstream; the platelet count drops further during pregnancy, surgery, DDAVP therapy; have low/normal factor VIII activity and vWF; reduction of high molecular weight multimers but increase in low molecular weight fragments; reduced ristocetin cofactor but increased ristocetin induced platelet aggregation; mean ristocetin cofactor/vWF antigen ratio is 0.6
Type 2C: autosomal recessive; reduction of high molecular weight multimers, increase in small multimers and qualititatively abnormal individual multimers; reduced ristocetin cofactor activity out of proportion to reductions in vWF
Type 2M: rare; autosomal dominant, decreased platelet directed function NOT due to a decrease of high molecular weight multimers, but otherwise similar to type 2A (may be due to mutation that impairs vWF and GPIb binding); low/normal factor VIII and vWF, normal multimer analysis, but very low ristocetin cofactor and low/normal ristocetin induced platelet aggregation; mean ristocetin cofactor/vWF antigen ratio is < 1.0
Type 2N: rare, autosomal recessive; markedly reduced affinity of vWF for factor VIII, causes reduction of factor VIII levels to 5% of reference range; other vWF lab tests are normal; often misdiagnosed as hemophilia A (which is X linked recessive), but males and females in type 2N are equally affected; assay that measures binding of factor VIII to vWF is available in specialized laboratories
Type 3: very rare; autosomal recessive, often associated with consanguinity; most severe clinical bleeding; homozygous patients have marked deficiencies of plasma vWF and factor VIII activity, no vWF in platelets and endothelial cells, no secondary transfusion response, no response to DDAVP; also undetectable ristocetin cofactor, low ristocetin induced platelet aggregation, all multimer sizes are absent
Platelet type or pseudo von Willebrand’s disease: rare disorder of mutation in GPIb (not vWF gene), causing increased binding of vWF to GPIb, with similar clinical findings as type 2B
Molecular basis of von Willebrand’s disease
Type 1: mutations throughout the gene, not well characterized
Type 2A: mutation in proteolysis site (most common type 2A mutation)
Type 2A: loss of propeptide, required for multimer formation from dimers
Type 2A: mutation in C-terminus, required for dimer formation from monomers
Type 2B: mutation in GPIb binding site, causing increased binding of vWF to GPIb
Type 2M: mutation in GPIb binding site, causing decreased binding of vWF to GPIb
Type 2N: mutation in N-terminis (factor VIII binding site), leading to decreased binding of vWF to factor VIII
Type 3: mutations throughout the gene, not well characterized
References: more information, OMIM 193400
Therapy related coagulopathies
Therapeutic anticoagulant to prevent thromboembolism by impairing regeneration of active vitamin K (warfarin is sodium salt of coumarin)
Vitamin K is a cofactor in reactions that carboxylate glutamic acid residues in factors II, VII, IX, X, protein C, protein S
Therapeutic warfarin or Vitamin K deficiency cause decreased activity of these proteins, which prolongs the PT
PTT may be normal if low warfarin levels
Takes 4-5 days for complete therapeutic effect due to long half-life of factors II and X
Therapeutic effect is measured by INR - goal is often INR between 2 and 3
INR may be elevated by lupus anticoagulants or use of hirudin with warfarin
Make be supplemented with heparin until INR is in therapeutic range for 2 consecutive days
Treatment of bleeding/overdose: vitamin K or fresh frozen plasma
Should not be used alone for acute heparin induced thrombocytopenia, because it causes paradoxical thrombosis - must add a rapid acting anticoagulant also (hirudin, danaparoid, argatroban) until INR is therapeutic
References: J Clin Path 2004;57:1132 (reversal of warfarin)
Approved to prevent deep venous thromboses; also an alternative to heparin for patients with heparin induced thrombocytopenia; use is decreasing due to newer anticoagulants
Composed of low molecular weight glycosaminoglycans that primarily inhibit factor Xa, factor IIa to a much lesser extent
Similar to low molecular weight heparin in having a more predictable anticoagulant effect, with less need for laboratory monitoring
Monitor, if desired, by measuring inhibitor of factor Xa using standard curve; draw 6 hours after subcutaneous injection; PT and PTT are unaffected
Therapeutic levels to treat DVT are 0.5-0.8 anti-factor Xa units/ml, lower for DVT prophylaxis
Long half-life, is prolonged with renal failure
No reversal agent is known
Also called unfractionated heparin
Short acting anticoagulant with half life of approximately 90 minutes
Used as initial anticoagulant therapy, to treat deep venous thrombosis, post-operatively and for other short-term indications
Decreases morbidity and mortality from acute thrombotic disease
Works by markedly enhancing activity of antithrombin, which inhibits activated factors II, IX, X, XI, XII, kallikrein and probably VII, but doesn’t cause a true decrease in factor levels
Derived from porcine intestinal mucosa or bovine lung, which contain heparin-rich mast cells
Most heparin preparations are heterogeneous, with a molecular weight between 7-25K daltons.
Anticoagulant activity is variable, since only 1/3 of heparin molecules have the pentasaccharide sequence necessary for antithrombin mediated anticoagulant activity
Complications include hemorrhage (overcoagulation) and heparin-induced thrombocytopenia (up to 3% of patients)
Recommended to monitor with PTT assay (that has been standardized using determination of heparin levels), activated clotting time (if high heparin levels present, as during cardiopulmonary bypass surgery, since no clotting occurs at these levels with PTT) or heparin levels assayed by measuring activity against factor Xa (therapeutic range is 0.3 to 0.7 anti-Xa units/ml) within 12 hours; also monitor platelet count within 72 hours, with platelet monitoring to continue for 20 days; PT is usually normal
Recommended that 90% of patients should achieve therapeutic anticoagulation within 24 hours
One study disputes recommendation to reevaluate PTT therapeutic range with each new heparin lot (Archives 2001;125:1458)
Note: often the cause of prolonged PTT is heparin in sample collected through indwelling line; identify by treating with heparinase
Treatment: protamine (for emergency reversal of heparin)
Heparin - low molecular weight
Can be used instead of standard heparin for many patients, with similar efficacy and safety
Produced by breaking heparin into shorter polysaccharide chains; molecular weight is approximately 5,000 daltons
Less likely to bind to acute phase reactant proteins, platelets, platelet factor 4, macrophages and other sites, due to its shorter length
Has more predictable anticoagulant effect than standard heparin, less need for laboratory monitoring, lower incident of heparin induced thrombocytopenia, greater bioavailability
Longer half life than standard heparin (4 vs. 1.5 hours), which is prolonged in renal failure
Inhibits factor Xa by 2 to 4x more than factor IIa, so does not substantially prolong PT and PTT
Unlike regular heparin, does not inhibit thrombin or factor IXa
May monitor (using tests measuring anti-factor Xa activity, drawn 4 hours after injection) in pregnancy, renal failure, obesity, prolonged use, infants and children, or high risk bleeding / thrombosis patients, although often don’t monitor except for periodic platelet counts
Therapeutic range is often 0.4 to 1.1 U/ml for twice a day dosing, higher for once a day dosing, 0.1 to 0.4 U/ml for prophylactic dosing
Effects are reversed with protamine
Derivates include lepirudin, refludan
Approved by FDA to treat thrombosis in patients with heparin induced thrombocytopenia
Recombinant protein, cloned from a leech, which directly inhibits factor IIa (thrombin)
Has more predictable anticoagulant effect than standard heparin, less need for laboratory monitoring, although close monitoring is still advised with PTT, though this assay has limitations (ecarin clotting time is preferable)
Prolongs PT, PTT, thrombin time, ACT, and interferes with most clotting based assays
Therapeutic range to treat DVT is PTT that is 1.5-2.5 x normal
Does not cause a heparin induced thrombocytopenia-type syndrome
Half life is approximately one hour, may be prolonged if antibodies develop, dramatically prolonged in renal failure
No reversal agent
Used to treat myocardial infarction, pulmonary embolism, arterial or venous thrombosis, thrombotic stroke
Thrombolytic agents include recombinant t-PA, urokinase, streptokinase
Laboratory: presence of fibrin degradation products and D-dimers, decreased fibrinogen and plasminogen, prolonged thrombin time, PT and PTT
Acquired thrombophilia / hypercoagulable states
Acquired thrombophilia-general
Thrombophilia: any disorder associated with increased risk of venous thromboembolic disease; more appropriately called “hypercoagulable state” if not genetic, although terms often used interchangeably
Common risk factors are post-operative state, trauma, pregnancy, oral contraceptives, obesity, immobility, chronic DIC, malignancy, nephrotic syndrome, paroxysmal nocturnal hemoglobinuria, systemic lupus erythematosus, polycythemia vera, essential thrombocythemia, hyperhomocysteinemia, antiphospholipid antibodies, prior thromboembolism, heparin induced thrombocytopenia, increasing age
The presence of more than one risk factor results in a further increased risk (Archives 2002;126:295)
Case reports: portal vein thrombosis associated with myeloproliferative disorder (Archives 2003;127:e385), fatal pulmonary emboli associated with hypereosinophilia (J Clin Path 2004;57:541)
Micro images: above case report with portal vein thrombosis - figures 1/2: old, recanalized thrombi in portal veins; 3: recanalization of portal vein webs with intimal tags; 4: bone marrow is hypercellular with large, dysplastic, abnormally clustered megakaryocytes
Acquired antibodies against phospholipid-protein complexes
Occurs in 3-5% of general population; most common cause of acquired thrombophilia
Rate of thrombosis per year is 1% if no history of thrombosis, 4% in systemic lupus erythematosus (SLE) patients, 5.5% in patients with a history of thrombosis, 6% if high titer of IgG anticardiolipin
Includes lupus anticoagulant (most patients don’t have SLE) and anticardiolipin antibody
Antibodies are against phospholipids (usually transient, secondary to infection) or various plasma protein antigenic targets (beta2-glycoprotein I, protein C, protein S, annexin V, high and low molecular weight kininogens, thrombomodulin, prothrombin, factors XI and XII, complement factor H)
First described by Wassermann in 1906 (Wasserman test was complement fixation procedure using saline liver extracts from fetuses with congenital syphilis)
Associated with an increased risk of arterial or venous thrombosis, thrombocytopenia, recurrent miscarriages; causes 1/3 of strokes in patients younger than age 50 years (often due to mitral or aortic valve emboli), 15% of deep venous thromboses, 5-15% of recurrent spontaneous abortions, eclampsia, maternal DVT’s; also multi-infarct dementia, chorea, migraine, livedo reticularis in skin
Catastrophic antiphospholipid syndrome resembles TTP-HUS
Antiphospholipid antibody syndrome: diagnosis requires a positive lupus anticoagulant or anticardiolipin antibody on two separate occasions, at least 6-12 weeks apart, AND either venous or arterial thrombosis, thrombocytopenia or recurrent fetal loss
References: Archives 2002;126:1424
Heparin induced thrombocytopenia (HIT)
Common complication of heparin therapy, may cause life threatening venous or arterial thrombosis
Prevent by monitoring platelet count for at least 20 days after initiation of heparin therapy
Clinical note: platelet counts often fall slightly during the first 24 hours of heparin treatment, unrelated to heparin induced thrombocytopenia
Treatment: permanently discontinue heparin, avoid platelet transfusions; recommended to not use low molecular weight heparin (may cross react) or warfarin (may cause venous limb gangrene); instead use danaparoid, hirudin or argatroban
Hereditary thrombophilia / hypercoagulopathies
Hereditary thrombophilia-general
Thrombotic disorders due in part to deficiencies in natural anticoagulant or fibrinolytic systems
Acquired risk factors, such as oral contraceptive use, may synergistically increase the risk for thrombosis
Disorders often occur at a young age; usually affects venous system
Workup includes documenting all acquired risk factors for thrombosis
Activated protein C resistance
Most common hereditary predisposition to venous thrombosis (20% of first episodes of thrombosis, 50% of familial thrombosis)
Normally, activated protein C degrades activated factors V and VIII by cleaving specific arginine residues
Almost all patients with activated protein C resistance have Factor V Leiden mutation that causes resistance to degradation by activated protein C
Does not appear to reduce life expectancy
Factor V Leiden
95% with activated protein C resistance have point mutation at an arginine cleavage site (Arg506Gln, 1691 G to A) called R506Q or Factor V Leiden
Mutation causes delayed inactivation by activated protein C, prolonging its life span and procoagulant activity
3-5% frequency in heterozygous form in general white population; rare in African blacks and Asians
Heterozygotes have 3-10x increased risk for venous thrombosis
Homozygotes have 80x increased risk for venous thrombosis; risk occurs later in life
Homozygosity or heterozygosity without symptoms may not require treatment
Presence of second risk factor (genetic or acquired) is often necessary to produce thrombosis; acquired risk factors are malignancy, trauma, surgery, oral contraceptive use, estrogen replacement therapy, antiphospholipid antibody, heterozygosity for prothrombin G20210A, elevated serum homocysteine
Other low frequency factor V mutations (besides Factor V Leiden) are factor V Cambridge (Arg306Thr) and factor V Hong Kong (Arg306Gly); also HR2 haplotype with mutation 4070A to G (His199Arg) in exon 13 of factor V gene, which is associated with other polymorphisms; all have unclear association with venous thrombosis
Testing recommended if venous thromboemboli occur with these features: recurrent, before age 50 years, unprovoked at any age, at unusual anatomic sites (cerebral, mesenteric, portal or hepatic veins), in patient with first degree relative with venous thromboemboli before age 50 years; related to pregnancy or estrogen use or unexplained pregnancy loss in second or third trimesters; may be recommended in family members (with family history), female family members who are pregnant or considering oral contraceptives
Testing not recommended: general population screen, routine test during pregnancy, routine test before or during oral contraceptive use or hormone replacement therapy in patients without a family history of thrombosis; as newborn initial test, as initial test in patients with arterial thrombotic events
Treatment: treat venous thromboemboli similarly regardless of the presence of factor V Leiden
Case reports: 51 year old woman with heterozygous factor V Leiden and dural sinus thrombosis (Archives 2003;127:1359)
References: Archives 2002;126:577
Hereditary deficiencies occur in 0.07 to 0.17% of general population; many mutations exist (qualitative or quantitative); usually autosomal dominant
Present in 1-9% of patients with venous thrombosis
Higher risk for venous and arterial thrombosis than protein C or S deficiency or activated protein C resistance; often occurs with other genetic or acquired risk factors
Heterozygotes have levels 35-75% of normal; first thrombotic event occurs between ages 10-50 years; overall 50% have thrombosis
Homozygosity is very rare, usually incompatible with life due to neonatal thrombosis, except for those with a heparin-binding mutation subtype, who have severe thrombosis but may survive
Type I mutations: quantitative deficiency with 50% of normal levels; due to any of 80 point mutations
Type II mutations: dysfunctional protein; often asymptomatic
IIa: mutations affect reactive site of target protease
IIb: mutations affect heparin binding site
IIc: recurrent missense mutations;
Treatment: heparin (unfractionated or low molecular weight), followed by warfarin; may need increased doses of heparin or antithrombin concentrates/fresh frozen plasma if resistant to heparin; should monitor antithrombin levels (should be 80-120%)
Acquired causes: liver disease, malnutrition, inflammatory bowel disease, extensive burns, recent or active thrombosis (including DIC), heparin therapy, acute hemolytic transfusion reaction, malignancy, L-asparaginase therapy, acute thrombotic episodes, heparin therapy, nephrotic syndrome
References: Archives 2002;126:1326, more information #1, #2
Disorders of fibrinogen structure; have variable effects on function (25% associated with bleeding, 20% associated with thrombosis, 55% have no symptoms or prolonged thrombin time)
Bleeding due to defective fibrin clot formation (impaired release of fibrinopeptides A or B and impaired fibrin monomer polymerization)
Thrombosis due to (a) defective thrombin binding to fibrin, causing increased thrombin in circulation and more thrombosis; (b) defective binding of tPA or plasminogen to fibrin or fibrin resistance to plasmin; includes Dusart (Paris V) and Chappel Hill III dysfibrinogens that are resistant to degradation by plasmin
Congenital (hereditary) dysfibrinogenemia is a rare cause of hypercoagulability (350 reported cases, 0.8% of patients with venous thrombosis); usually due to single amino acid substitutions in fibrinogen Aalpha, Bbeta or gamma genes; recommended to use only as a second-line test in patients with thrombosis since dysfibrinogenemia is so rare
Autosomal dominant inheritance, but higher incidence in women due to pregnancy related thrombosis, particularly post-partum and in venous lower extremities, at mean age 27 years; also associated with spontaneous abortions
Laboratory testing: primary screening test is thrombin time (prolonged except for fibrinogens Oslo I and Valhalla - shortened); prolongation may also be due to heparin, heparin-like inhibitors, fibrin degradation products, hypofibrinogenemia, excess fibrinogen, paraproteins, excess protamine, anti-fibrinogen antibodies, anti-bovine thrombin antibodies, systemic amyloidosis, acquired dysfibrinogenemia; the sensitivity of thrombin time assays varies for dysfibrinogenemia because many assays are designed primarily to detect heparin contamination; some labs use the reptilase time, which is not affected by heparin
Confirmatory test (if thrombin time or reptilase time is prolonged): fibrinogen activity-antigen ratio below reference range; activity measured by Clauss method (rate of clot formation after adding high concentration of thrombin to citrated plasma; use standard curve relating clotting time to plasma of known fibrinogen activity); antigen concentration determined by ELISA, radial immunodiffusion, precipitation or thrombin clotting methods; perform both tests on same sample in same laboratory and using method-specific reference ranges
Diagnosis: similar laboratory test abnormalities in family members; if necessary, demonstrate abnormal structure or function of fibrinogen
Diagnosis of acquired dysfibrinogenemia: abnormal liver function tests, no dysfibrinogenemia in family members
References: Archives 2002;126:1387, Archives 2002;126:499, http://www.geht.org/databaseang/fibrinogen (online database of fibrinogen mutations)
Elevated coagulation factor levels in plasma
May predispose to thrombosis
Factor I (fibrinogen): high levels associated with increased risk for myocardial infarction and arterial thrombosis
Often elevated in hospitalized patients since it is an acute phase reactant
Not recommended to measure to assess thrombotic risk, since assay has not been standardized, independent effect appears to be modest, insufficient clinical data to demonstrate that lowering fibrinogen will present ischemic heart disease
Factor II (prothrombin): high levels associated with increased risk of venous thrombosis; see also G20210A mutation (below)
Standard activity assays are not useful in assessing individual patients for hyperprothrombinemia - use the genetic assay
Factor V: high levels associated with increased risk of arterial thrombosis
Not recommended to measure to assess thrombotic risk, since not associated with venous thrombosis, only one study relates factor V activity and ischemic heart disease, methodology is inadequate to assess individual levels, there are no studies showing that reduction of factor V activity will reduce risk for ischemic heart disease or stroke
Factor VII: high levels or certain genetic polymorphisms are associated with increased risk for myocardial infarction, but not an independent risk factor; levels are associated with triglyceride and cholesterol levels
Factor VIII: high levels associated with increased risk of venous thrombosis and arterial thrombosis; however normal range varies about 3 fold, levels vary with ABO blood type (15% lower with type 0) and is acute phase reactant; if no acute stress reaction, no aerobic exercise in past 24 hours, no estrogenic effects, then baseline value >150% is a risk factor
Not recommended to assess individual risk because no established standard for elevated levels, tremendous interlaboratory variation
Factor IX: high levels associated with increased risk of venous thrombosis; not recommended to assess individual venous thrombosis risk due to limited amount of clinical data and limited availability of factor IX ELISA
Factor XI: high levels (>121%) associated with increased risk of venous thrombosis; not recommended to assess individual venous thrombosis risk because (1) factor XI assays are not generally available; (2) one step clotting assay and PTT based assays may be too variable; (3) may be affected by other variables
Factor XIII: polymorphism Val34Leu may protect against venous thrombosis (Archives 2002;126:1391)
von Willebrand factor: high levels associated with increased risk of arterial thrombosis; not recommended to assess individual thrombotic risk because it is not an independent risk factor, and no studies show that reduction of vWF levels reduces risk of ischemic heart disease, stroke or peripheral arterial disease
References: Archives 2002;126:1405
Heparin cofactor II deficiency
Very rare hypercoagulable condition, either hereditary (autosomal dominant, 15 families documented through 2002) or acquired (liver disease)
Associated with thrombosis, but not a strong risk factor by itself
Testing patients with thromboembolic disease for heparin cofactor II deficiency is not recommended as a first or second line test (Archives 2002;126:1394)
Homocysteine is an amino acid, derived from methionine, may be converted to cysteine
Its metabolic pathways require vitamins B12, B6 and folate; elevated levels may be hereditary (due to mutations in these pathways) or acquired (due to deficiencies of vitamins B12, B6 or folate, renal failure, carcinoma, hypothyroidism or medications)
Elevations in homocysteine are associated with increased risk of arterial and venous thrombosis and atherosclerosis, based on retrospective case control studies; prospective studies show a weak positive association with arterial thrombosis, and no definite association for venous thrombosis
Homozygosity or heterozygosity for the C677T mutation in MTHFR gene (methylene tetrahydrofolate reductase), which is involved in homocysteine metabolic pathway, does not appear to be a risk factor for thrombosis; may be significant in folate-deficient patients; although MTHFR was previously thought to be associated with thrombosis, newer data suggests this test is not useful in the first-line evaluation of thrombosis
Consider testing patients with documented coronary artery disease, cerebrovascular disease or peripheral vascular disease for homocysteine; high levels can be treated with vitamins B6, B12 and folic acid, although they may not reduce the risk of future cardiovascular events
Homocysteinemia is usually associated with a moderately elevated plasma homocysteine, while homocysteinuria is a specific genetic entity with very high plasma homocysteine levels
References: Archives 2002;126:1367
Hereditary deficiencies occur in 0.14 - 0.5% of general population (the clinically significant incidence is much lower); >160 mutations exist, either type I (76%, usually quantitative) or type II (dysfunctional protein, normal protein levels)
Causes 1-11% of cases of venous thrombosis; these patients are also at risk for warfarin-induced skin necrosis if treated with warfarin and no heparin until warfarin levels are therapeutic; this paradoxical clotting is due to a faster fall in natural anticoagulant proteins than procoagulant proteins in these patients
Heterozygotes have levels 35-65% of normal; first thrombotic event occurs between ages 10-50 years; only 30% have thromboembolism, increasing to 75% if coexisting factor V Leiden
Homozygotes (1 per 500-750K births) with severely decreased levels present as newborns with DIC and purpura fulminans neonatorum, leading to death unless anticoagulation and replacement therapy with fresh frozen plasma is started
Must exclude acquired causes of protein C deficiency
Acquired causes of low protein C levels: clot formation, surgery, liver disease, warfarin (should be discontinued at least 10 days prior to testing) or Vitamin K antagonist therapy, DIC, vitamin K deficiency, L-asparaginase therapy
Acquired causes of increased protein C (may mask protein C deficiency): ischemic heart disease, pregnancy, postmenopausal women, hormone replacement therapy, oral contraceptives
References: Archives 2002;126:1337
Hereditary deficiencies occur in 0.7% of general population; many mutations exist (qualitative or quantitative); much lower prevalence of thrombophilia with clustering in families; variable penetrance may be due to coexisting risk factors, such as factor V Leiden
Causes 1-9% of cases of venous thrombosis; these patients also at risk for warfarin-induced skin necrosis if started on warfarin without the addition of heparin until warfarin levels are therapeutic
Heterozygotes have levels 35-65% of normal; first thrombotic event occurs between ages 10-50 years; 50% risk by age 45
Homozygotes with severely decreased levels present as newborns with DIC and purpura fulminans, leading to death unless anticoagulation and replacement therapy with fresh frozen plasma is started
Type I (2/3): low free and total protein S antigen, with decreased APC cofactor activity
Type II (rare): normal free and total protein S antigen, and decreased APC cofactor activity
Type III (1/3): normal to low total protein S, low free protein S antigen, and an elevated fraction of protein S bound to C4B protein
Testing recommended: individual with family history who requests testing, to confirm abnormal protein S result; must interpret with caution
Testing not recommended: during pregnancy or postpartum, during inflammatory, thrombotic or surgical event; within 30 days of taking warfarin; must delay longer periods for vitamin K antagonists (Phenprocoumon)
Clinical note: acquired protein S deficiency is often seen during pregnancy due to increased C4b, which may reduce levels to 40% or less
References: Archives 2002;126:1349
Prothrombin gene mutation (G20210A) / hyperprothrombinemia
Mutation in G to A transition at nucleotide 20210 in 3’ untranslated portion of prothrombin gene, which introduces a new Hind III site
Associated with (may not directly cause) increased prothrombin levels, 2-5x increased risk of venous thrombosis
Risk is multiplicative if taking oral contraceptives and have factor V Leiden gene
Heterozygous form occurs in 1-2% of normal individuals, 6-20% of patients with venous thrombosis
Testing via nucleic acid based assay is preferred as the lack of linearity of the clot-based Factor II assay at the high end makes this unsuitable for diagnostic use
Testing recommended: patients with recurrent venous thromboembolic, first episode before age 50, first unprovoked venous thromboemboli at any age, thromboses at unusual anatomic sites (cerebral, mesenteric, portal or hepatic veins), venous thromboemboli in patient with first degree relative with venous thromboemboli before age 50 years; venous thromboemboli related to pregnancy or estrogen use, or unexplained pregnancy loss in second or third trimesters; young individuals with myocardial infarction and no other risk factors; also test factor V Leiden and other mutations (combination most clearly impacts clinical decision making)
Testing not recommended: as general population screen, routine test during pregnancy, routine test before or during oral contraceptive use or hormone replacement therapy; as newborn initial test, as initial test in patients with arterial thrombotic events
Treatment: patients with thromboemboli and this mutation should receive similar treatment as other patients with venous thromboemboli
References: Archives 2002;126:1319
Associated with increases in thrombin generation, fibrinolytic activation, platelet activation, increased antiphospholipid antibodies, decreased levels of circulating anticoagulants and contact factors, increased circulating levels of tissue factor and endothelial cells expressing a tissue factor phenotype
Have hypercoagulable state based on thermoelastographic profiles (Archives 2005;129:760)
Coagulation laboratory tests
Coagulation laboratory tests-general
Performed in almost all hospitals in US
Necessary for diagnosis, treatment and management of bleeding and hypercoagulation disorders, to screen for coagulation disorders and to monitor anticoagulant therapy
CAP requires laboratories to notify medical staff immediately if a critical value is obtained; CLIA requires laboratory to immediately alert
individual or entity that requested the test, or if applicable, the individual responsible for using the test results
Specimens: (a) tubes with 3.2% citrate are preferred over 3.8% citrate (higher concentration prolongs PT and PTT if tube not filled to the recommended level); (b) do not draw specimens from indwelling catheters (which contain anticoagulants); (c) if multiple tubes are drawn, draw coagulation tube after the red top and before the EDTA, heparin or oxalate/fluoride tubes; (d) try to fill the sample tube completely; (e) notify laboratory if patient is on anticoagulants and specify which ones; (e) don’t delay transport of tubes to laboratory; if delay cannot be avoided, separate plasma or serum from cells as soon as possible; store plasma (or serum) on ice for up to 4 hours, or store frozen
References: Archives 2005;129:47
Important to prevent laboratory errors
CLIA regulations require patient and control specimens be tested in duplicate only for manual coagulation tests, but not for automated tests
CLIA regulations require calibration and calibration verification procures to substantiate continued accuracy throughout the laboratory’s reportable range of test results
PT normal, PTT prolonged: deficiency of intrinsic pathway factors VIII, IX, XI, XII; less commonly prekallikrein or high molecular weight kininogen (flowchart); also prolonged delay in transporting specimen to lab (affects factors V and VIII)
PT prolonged, PTT normal: deficiency of extrinsic pathway factor VII; occasionally due to deficiency of common pathway factors fibrinogen, prothrombin, factors V or X
PT and PTT prolonged: deficiency of common pathway factors fibrinogen, prothrombin, factors V or X, or multiple factor deficiencies
Whole blood clotting assay, usually performed in operating room or catheterization lab, to monitor high-dose heparin anticoagulation (cardiopulmonary bypass surgery) or to immediately measure heparin (ECMO, hemodialysis, cardiac catheterization)
Note: test is necessary for high-dose heparin monitoring because PTT is often unclottable at very high heparin levels
Whole blood is collected into tube with celite (diatomaceous earth), kaolin, glass particles or other activator of intrinsic pathway; tube may need to be shaken to disperse the activator; tube is monitored by instrument that records time until clot is formed
Do not collect blood from line containing heparin
Target reference range depends on the method: usually 70-180 seconds, 400+ seconds for cardiopulmonary bypass operations
Does not correlate well with PTT but heparin level can be measured using an anti-Xa assay
Affected by platelet count and function, lupus
anticoagulant, factor deficiencies, patient and ambient temperature,
hemodilution, aprotinin (reversible platelet inhibitor that prolongs
celite-based tests)
Activated protein C resistance assay
See Factor V Leiden assay (below)
One of the two main types of antiphospholipid antibodies (other is lupus anticoagulant)
Indications: patients with venous thromboembolism (particularly if no family history or associated with autoimmune disease); unexplained stroke (young person or autoimmune disease), cerebral venous thrombosis, recurrent or late pregnancy loss; may be considered for arterial thrombosis (particularly in young patient or no documented atherosclerosis)
Specimen: serum (red top)
Methodology: ELISA test to recognize proteins (not actually cardiolipin) bound to a microtiter plate; tests for anti-prothrombin and anti-beta2-GPI antibodies have had limited prospective studies
IgG recommended to evaluated hypercoagulability (risk with IgM or IgA antibodies is uncertain); increased titers are most closely associated with hypercoagulability
200-400x more sensitive than VDRL, but patient with syphilitic infection may have positive anticardiolipin antibody test results
High sensitivity plates have greater antigenic density on microtiter plate, may be irradiated for greater antigenic density and to facilitate bivalent bonding of plasma antibodies
62% positivity in patients with SLE or other autoimmune results
To demonstrate persistence, positive test must be confirmed by repeat testing after 6 weeks
Transient antibodies are not strongly associated with thrombosis
References: Archives 2002;126:1424
Also called anti-alpha2-antiplasmin, plasmin inhibitor
An uncommon assay usually sent to reference laboratories
Indications: familial bleeding disorder, after ruling out more common bleeding disorders such as von Willebrand disease
Specimen: plasma in citrate tube, without epsilon-aminocaproic acid, aprotinin, heparin or other fibrinolysis inhibitors
Reference range: approximately 48-80 mg/dL, lower during first 5 days of life
Functional assays: add specific amount of excess plasmin to patient’s plasma, measure plasmin that is unbound to antiplasmin in patient’s serum by detecting color change spectrophotometrically; amount of unbound plasmin detected is inversely proportional to patient’s antiplasmin level
Antigenic (immunologic) assay: patient’s plasma in placed in the cylindrical well of an agarose gel containing antiplasmin antibody, which defuses into the well and forms an antigen-antibody complex and precipitin ring; the size of the ring is proportional to the patient’s antiplasmin
Acquired causes of decreased antiplasmin: liver disease, thrombolytic therapy, DIC
Assays detect antigenic (type I, reduced normal protein, quantitative) or functional (type II, normal amount of defective protein, qualitative) deficiencies of antithrombin (formerly called antithrombin III)
Perform functional assay first - if decreased, perform antigenic assay on fresh specimen; family studies may be helpful
Functional assays: are chromogenic, use predominantly amidolytic methods (i.e. through cleavage of an amide bond), employing a synthetic peptide that mimics the natural target of the enzyme; patient plasma is incubated with excess thrombin and heparin; antithrombin neutralizes thrombin, and remaining thrombin is then quantitated with a chromogenic substance; the amount detected is inversely proportional to the patient’s antithrombin
Functional assay limitations - false levels may be produced if high levels of heparin cofactor II are present; this is eliminated by assays that use inhibition of factor Xa rather than thrombin; newer assays have protease inhibitors to minimize nonspecific substrate cleavage and bovine thrombin; hirudin or argatroban anticoagulation may interfere with thrombin based assays
Antigenic assays: quantification is usually via radial immunodiffusion techniques, although they have coefficients of variation of 40-50%; amidolytic assays have CV of only 9-14%; also used are latex particles coated with antithrombin antibodies (e.g. LIA); light absorbance is related to the amount of antithrombin in the specimen
Antigenic assay limitations - does not detect functional deficiencies by itself
If initial antithrombin result is low, should do confirmatory test on repeat specimen; also family studies (first degree relatives); must also exclude acquired causes
Specimen: plasma in sodium citrate tube
Levels are lower in newborns; rise to adult levels (112-140 mg/liter) by age 6-12 months
Mildly decreased values (70-80%) are unlikely to be associated with thrombosis
Indications: evaluation of individuals with thrombophilia (strong family history or young patient); also analyze for factor V Leiden and prothrombin G20210A; preferable to not test during the acute phase of a thrombotic event (normal antithrombin value makes antithrombin deficiency unlikely, although cannot interpret mildly abnormal values)
Acquired causes of low antithrombin levels: clot formation, surgical procedures, liver disease, nephrotic syndrome, DIC, heparin (full dose therapy decreases levels by up to 30%), L-asparaginase therapy, possibly pregnancy or oral contraceptives
Acquired causes of high antithrombin levels: warfarin therapy
References: Archives 2002;126:1326
A relatively nonspecific and nonsensitive test of platelet function, whose use is declining
This test should usually be avoided, particularly if definitive testing, such as a von Willebrand panel is available; preoperative bleeding time does NOT predict surgical bleeding
Test is affected by use of aspirin or other NSAIDs; patients should abstain from their use for 1 week prior to testing
Test is also affected by how incision is made (very difficult to standardize)
Procedure: place blood pressure cuff on arm at 40 mm Hg; then trained technologist makes a small incision on patient’s arm, blots the blood gently every 30 seconds with filter paper, without touching the clot, to see if bleeding has stopped and records the time when it stops; then apply bandage
Duke bleeding time: uses earlobe or fingertip pierced with lancet
Ivy bleeding time: blood pressure cuff at 40 mm Hg on arm, and forearm cut by lancet
Mielke (template) bleeding time: template placed on skin with spring loaded blade that cuts through template, to standardize the size and depth of cut; more reproducible than standard bleeding time but still quite variable
Reference range: varies, sample range is 1.5 to 9.5 minutes (less in newborns)
Prolonged values: platelet count less than 100K, low hemoglobin, use of aspirin or other platelet inhibitors; also von Willebrand’s and other hereditary platelet disorders, uremia
Obsolete test
Uses whole blood in red top tube; examine clot at 1, 2, 4 and 24 hours for clot retraction; after clot forms, remaining 40-60% consists of serum and red blood cell “fall-out” from clot
Reduced clot formation: Glanzmann thrombasthenia (reduced glycoprotein IIb/IIIa causes reduced platelet aggregation and clot retraction); DIC, hypofibrinogenemia, dysfibrinogenemia (small clot with increased red blood cell “fall-out”)
Cryoglobulin / cryofibrinogen assays
Either asymptomatic or causes cutaneous symptoms at cold-exposed areas
Cryofibrinogen consists of fibrinogen and other substances that precipitate at cold temperatures (cryoglobulins are immunoglobulins that precipitate at cold temperatures)
Either primary, or associated with malignancy, infection, inflammatory conditions, diabetes, pregnancy, oral contraceptives; may exhibit leukocytoclastic vasculitis in skin biopsies
Specimen: two sodium citrate or EDTA tubes plus one red top tube for cryoglobulin; place immediately in warm water (or use warmer for heal sticks or other warming method) and transport to laboratory within 2 hours; don’t use heparin-containing specimens (heparin precipitates fibrinogen in this assay)
Indication: for patients with unexplained cutaneous ulcers or ischemia on cold-exposed areas
Procedure: centrifuge at 37C, refrigerate plasma, centrifuge at 4C; each mm of visible precipitate represents 1% of cryofibrinogen; cyrocrit is %volume of precipitate compared to total plasma
Also perform cryoglobulin test to ensure that plasma precipitate is not a cryoglobulin
D-dimer / dimerized plasmin fragment D
Marker of ongoing procoagulant activity
Fibrin degradation products (fibrin split products) that are formed only by plasmin degradation of fibrin, not by plasmin degradation of intact fibrinogen; thus indicating that fibrin has been formed
D-regions of fibrinogen are crosslinked by factor XIII after the fibrin clot is formed; plasmin cannot cleave the bond between the D-regions, so dimers are also found when a clot is broken down
Normal plasma level is probably due to physiologic clotting activity
Specimen: usually plasma with citrate anticoagulant
Values < 0.5 mg/L with quantitative ELISA assays have good negative predictive value for thromboembolic disorders; other values are not predictive (Archives 2004;128:519)
Suggested guidelines for D-dimer testing to rule out pulmonary emboli in patients with low clinical suspicion (if moderate or high clinical suspicion, should do imaging studies): age < 70 years, and no unexplained hypoxemia, unilateral leg swelling, recent surgery, hemoptysis, pregnancy or prolonged duration of symptoms (Acad Emerg Med 2005;12:20)
Elevated levels are sensitive but not specific for DIC
Elevated levels after completion of oral anticoagulation are associated with venous thromboemboli
LIA assay: mix patient plasma with latex particles coated with monoclonal anti-D-dimers or fibrin degradation product antibodies; detect agglutination with coagulation analyzer and semiquantitate with dilutions
Note: although this is called a Latex ImmunoAssay, it is different from the qualitative latex agglutination assay that is NOT predictive of pulmonary emboli
ELISA method: also available
False positives: recent surgery, HIV+ Castleman’s disease due to interference from monoclonal gammopathy (Archives 2004;128:328), high rheumatoid factor, liver disease, cancer patients, pregnancy
Note: some platforms use fibrinogen equivalent units, which product results half the numerical value of d-dimer units
References: CAP Today; May 2005 (cutoff values for venous thromboemboli), CAP Today; April 2005 (new tests and uses)
Measures activity of hirudin in plasma, important since severe bleeding can occur with hirudin overdose and no antidote is known
Ecarin catalyzes prothrombin to meizothrombin, an active form that is inhibited by hirudin; test measures ability of hirudin to complex with / inhibit meizothrombin as ecarin produces it from prothrombin
Specimen: sodium citrate tube filled to top; freeze immediately until testing occurs; specimens with clots or hemolysis are unacceptable, unless hemolysis is due to cardiopulmonary bypass and is produced in vivo; do not draw from heparinized catheter
Reference range: 22.6 to 29.0 seconds
Prolonged if hirudin or argatroban (direct thrombin inhibitors) present; also hypofibrinogenemia, dysfibrinogenemia
False positive if prothrombin deficiency is present
PT (factors II, V, VII, X) or PTT (factors V, VIII, IX, XI, XII) based reactions, performed by mixing patient plasma with plasma that is deficient in the factor being measured; the rate limiting reactant is the deficient factor which must be supplied by the patient
PT or PTT is compared to standard curve, to determine amount of factor present in patient’s plasma
Used to determine the etiology of a prolonged PT or PTT
Factor levels are expressed as % of normal plasma concentration, or units per mL of normal plasma; reference range is often 60-140% (should be determined based on the laboratory’s patient population)
Perform at multiple dilutions to rule out an inhibitor - at higher dilutions, inhibitor interference should decrease due to dilution of the inhibitor (this gives a nonlinear curve)
Can also use chromogenic assays to quantitate
Levels at birth of factors other than factor VIII are 10-100% of adult levels, but reach adult levels at 6 months
Clauss method: a functional test that is essentially a dilute thrombin time (making fibrinogen the rate-limiting reagent); add diluted patient plasma to high concentration of thrombin, which converts fibrinogen to fibrin; clotting time is inversely proportionate to fibrinogen in sample; values are falsely decreased by heparin > 0.6 units/mL or fibrin degradation products
Ellis method: thrombin (less than Clauss method) is added to undiluted patient plasma and spectrophotometer records change in turbidity
PT based method: thromboplastin (tissue factor with phospholipids) is added to undiluted patient plasma and light scatter or turbidity is measured; the measured optical change (before and after fibrin clot formation) is proportional to amount of fibrinogen
Immunologic methods: use anti-fibrinogen antibodies targeted to different parts of the fibrinogen molecule; usually a send-out test
Values decrease with liver disease (usually late), DIC, thrombolytic therapy, fibrinolysis
Values falsely decreased by bivalirudin, lepirudin, argatroban, and fondaparinux (Archives 2004;128:1142)
Values increased in acute phase reactions and during pregnancy
Reference range: 150-400 mg/dL
Factor V Leiden assay
Term is used interchangeably with activated protein C resistance (APCR) since Factor V Leiden causes most (but not all) cases of APCR
Uses plasma (in citrate tube) for screening assay and whole blood for DNA based confirmation assay
Procedure:
(1) dilute patient plasma 1:5 with factor V deficient plasma (dilutes the effect of other factor deficiencies or elevations) and add polybrene (neutralizes unfractionated heparin or low molecular weight heparin)
(2) if lupus anticoagulant is present, must perform DNA based test for Factor V Leiden (or perhaps 1:40 dilution of plasma or add phospholipids to neutralize lupus anticoagulant)
(3) calculate ratio of PTT with versus without exogenous activated protein C; normal is 2.0 or more, factor V Leiden usually < 2.0 (sensitivity and specificity approach 100%, because these patient’s activated factor V resists activated protein C degradation)
Many feel that positive results should be confirmed with a genetic assay
Other assays: (a) prothrombin-based factor V assay with factor V deficient plasma (no interference from lupus anticoagulant)
(b) modified Russell viper venom time test (high phospholipids neutralizes lupus anticoagulant)
(c) factor Xa-based assay with factor V deficient plasma
(d) DNA based tests such as PCR (using whole blood, not plasma); absence of MnlI cleavage at mutation site, guanine to adenine at #1691, or arginine to glutamine at amino acid #506 indicates factor V Leiden mutation
References: Archives 1998;122:430 (advantages of step 1 above)
Usually a one stage, prothrombin based assay
Results distorted by cold activation of factor VII, by variable sensitivity of thromboplastins to activity of factor VII vs. VIIa
Can also use a chromogenic assay
Assay is usually a PTT-based clotting assay that measures factor VIII activity
Use severely deficient factor VIII plasma as substrate for one stage clotting assay, although there is tremendous interlaboratory variability
Can also use chromogenic assay, particularly when assessing B-chain deleted recombinant Factor VIII (i.e. “Refacto”)
Low levels: hemophilia, vWF disease with decreased vWF antigen levels, delay in transporting specimen to lab
Note: levels are not greatly decreased at birth or throughout childhood; levels may increase during pregnancy
Factor VIII inhibitor assay / Bethesda assay
Methodology: prepare serial dilutions of patient plasma in citrated saline from 1:1 to 1:160 (or higher); mix each dilution with an equal volume of normal plasma containing a normal amount of coagulation factors, incubate for 2 hours, then perform factor VIII assay; titer of inhibitor is dilution that inhibits 50% of factor VIII in assay
Can use porcine Factor VIII to assess cross-reactivity and assist in therapeutic decisions
A linear, dose-dependent, false decrease is caused by bivalirudin, lepirudin, argatroban, and fondaparinux (Archives 2004;128:1142)
Used to determine levels of heparin, low molecular weight heparin, danaparoid, etc.
Note: the term “anti-Xa level” should be avoided because this suggests measuring a factor X inhibitor, but the factor Xa assay is really a drug level using chromogenic Xa inhibition as the methodology; as the reference range and standard curve vary with the drug tested, the clinician should indicate the drug (heparin, danaparoid, etc.)
Used to monitor heparin, particularly if PTT has baseline prolongation due to lupus anticoagulant or factor XII deficiency
Note: can cautiously use PTT to monitor heparin, even if lupus anticoagulant present, if factor Xa assay demonstrates that it is not affected by the lupus anticoagulant
Also used to monitor low molecular weight heparin and danaparoid, which don’t prolong PTT; however, these products have more predictable results, and don’t need to be monitored except if renal failure, pregnancy (increased dosage needed in third trimester), newborns (increased dosage needed), over- or underweight patients, prolonged use or high risk for bleeding/thrombosis
Measures ability of heparin or other drugs in patient’s plasma to inhibit known amount of factor Xa; usually reported out based on standard curve for the drug in question
Sampling: draw specimen 4 hours after subcutaneous injection of low molecular weight heparin or 6 hours after subcutaneous injection of damaparoid to avoid falsely low values; must deliver to laboratory immediately (or separate plasma from cells within 1 hour), because platelets release platelet factor 4, which neutralizes heparin; delays may cause falsely low values
Approximate therapeutic range for treatment of existing deep venous thrombosis: heparin – 0.3 to 0.7 anti-Xa international units/mL; low molecular weight heparin – either 0.4 to 1.1 units/mL for twice a day dosing or 1 to 2 units/mL for once daily dosing; danaparoid – 0.5 to 0.8 units/mL
Chromogenic factor X assays: used to monitor warfarin in the presence of a lupus anticoagulant, hirudin or argatroban (which prolong the PT and increase the INR), because warfarin decreases factor X (also factors II, VII, IX), and the chromogenic assay has no interference from lupus anticoagulant, hirudin or argatroban; patient plasma is added to a known amount of excess factor Xa with excess antithrombin; anticoagulant binds to antithrombin and inhibits factor Xa; residual factor Xa is inversely proportional to anticoagulant in plasma, cleaves a chromogenic substrate, and colored compound is detected by spectrophotometer; results reported in antifactor Xa units/mL
Interpretation: low levels of factor Xa are due to (a) not collecting specimen at right time or delayed transportation to lab (see Sampling above), (b) higher therapeutic dose needed; high levels of factor Xa are due to (a) renal failure, (b) heparin contamination (specimen drawn from indwelling line containing heparin), (c) lower therapeutic dose needed
Determine by one step clotting activity assay, comparing dilutions of patient plasma to clotting times of dilutions of pooled plasma from normals
Use plasma from individuals with <1% activity of immunodepleted normal plasma
Can also use chromogenic substrate after add inhibitors to factor XIIa and kallikrein
Levels may decrease during pregnancy
Indications: patients with familial bleeding disorder but normal PT and PTT and normal von Willebrand panel
Factor XIII deficiency causes delayed bleeding after clot formation due to deficient crosslinking of the fibrin clot
Screening assay: evaluates clot stability in 5M urea; add calcium to patient plasma to make it clot, incubate for 30 minutes at 37C, then place clot in 5M urea for 24 hours at room temperature; normal patients have stable cots, but patients with factor XIII deficiency of 1-2% of normal have clots that dissolve in urea; screening assay does not detect heterozygotes
Quantitative assay: reference range is 70-140% of normal; detects values of 50% of normal (heterozygous deficiencies); expensive and not readily available, factor XIII is activated by thrombin, attaches glycine ethyl ester to a peptide substrate, releasing ammonia detected by photometer; high serum ammonia levels falsely decrease the result
Note: newborns may have lower levels than adults
Heparin induced thrombocytopenia
Determine if thrombosis or thrombocytopenia in a patient exposed to heparin is due to anti-heparin antibody (actually antibody to heparin bound to platelet factor 4 on platelet surface)
Heparin exposure may be minimal (heparin-coated catheter)
Note: up to 8% of heparinized patients have antibody without symptoms, 1-5% have thrombocytopenia, 1/3 of these develop arterial or venous thrombosis, 20-30% of these die and 20-30% become disabled
Affected patients usually have reduction in platelet count within 4-20 days after heparin exposure for the first time, 1-3 days after reexposure to heparin, platelet count typically decreases 50% or more to under 100K; starts to rise 2-3 days after ceasing heparin, with normal levels at 4-10 days after heparin cessation; however thrombosis may occur for several weeks after heparin is stopped
Antibody binds to heparin-platelet factor 4 complex, antibody then binds to platelet Fc receptor, which activates the platelet, causing thrombocytopenia and thrombosis
Test should be performed in acute setting, before antibody disappears
Note: initial test may be negative and need to be repeated after several days; a negative test by itself has very poor predictive value
Methodology: either ELISA (90% sensitive; heparin complexed to platelet factor 4 as antigen), platelet aggregation (add patient plasma/serum to donor platelets and heparin, check for platelet aggregation) or serotonin release assays (add patient plasma/serum and heparin to donor platelets with radiolabeled serotonin, check for release of serotonin from platelets activated by the antibody)
Heparinase / heparin contamination assay
To detect heparin contamination of specimens, which may cause a prolonged PTT
Also used to remove heparin from specimens so coagulation tests can be performed without interference
Heparinase degrades unfractionated and low molecular weight heparin at multiple sites, including the antithrombin binding site (pentasaccharide sequence), producing fragments up to 1000 daltons, which lack anticoagulant activity
Methodology: measure PTT before and after heparinase (add 1 mL of patient plasma to one vial of heparinase, keep at room temperature for 15 minutes); alternative is to add heparin-binding cellulose to specimens, which binds to heparin, then centrifuge and use supernatant plasma (free of heparin)
Notes: normal thrombin time rules out heparin prolonging the PTT; may have coagulation abnormality in addition to heparin contamination; marked reduction of PTT, but with elevated value, may indicate residual heparin
Note: the PTT may never totally correct in normal patients with large amounts of heparin contamination
High molecular weight kininogen assay
Interference occurs in these assays if patient on heparin, hirudin or argatroban, possibly danaparoid
Lower levels in newborns, increase to adult levels by age 6 months
Indications: rarely used test to determine cause of prolonged PTT, if PTT normalizes in mixing study, factors VIII, IX, XI and XII are normal, PT and fibrinogen are normal, lupus anticoagulant is negative
Methodology: mix patient plasma with high molecular weight kininogen deficient plasma, perform PTT, and compare to standard curve of high molecular weight kininogen vs. PTT
70% of homocysteine is bound to albumin, 30% is oxidized to disulfides, 2% is free
Reference range is 5-15 micromolar (reflects free, non-bound form)
Gender and local population specific reference ranges are strongly recommended, because levels are affected by dietary intake of methionine and vitamins, gender and age (lower in premenopausal women)
High levels may also be due to vitamin B12 deficiency, post-myocardial infarction or stroke
Usually recommended to measure after 10 hour fast, although this may not be necessary
Increase test specificity by measuring 3-6 hours after methionine load of 0.1 g of L-methionine/kg
Must put specimen on ice if plasma separation cannot be performed within 30 minutes, because homocysteine is produced and exported by red blood cells and levels rise after collection in EDTA-anticoagulated tubes; alternatively can use acid citrate tubes and hold for up to 6 hours
Methodology: reduce all forms of homocysteine to free homocysteine, then quantify using either (a) high performance liquid chromatography; (b) fluorescence based immunoassay (Abbott’s IMx analyzer) - reduce using dithiothreitol, then convert to S-adenosyl-L-homocysteine (SAH) via SAH hydrolase; SAH is measured with monoclonal antibody and fluorescent tracer; (c) conventional amino acid analyzer with separation column (slow, but can also detect related amino acids, such as methionine, cystathionine, cysteine)
References: Archives 2002;126:1367
Panels are useful to identify all factors predisposing to thrombosis
Laboratory must be notified if patient is receiving therapeutic anticoagulants (heparin, warfarin, danaparoid, hirudin, argatroban)
Venous thrombosis panel typically includes assays for activated protein C resistance (factor V Leiden), protein C, protein S, antithrombin, prothrombin G20210A mutation assay, antiphospholipid antibodies, homocysteine; less common are assays for plasminogen, dysfibrinogenemia (e.g. reptilase time), heparin cofactor II or platelet hyperaggregability
Arterial thrombosis panel may include antiphospholipid antibodies, homocysteine levels, lipoprotein (a) [if arterial thrombosis occurs with coronary artery disease, myocardial infarction or stroke]
In special circumstances, arterial thrombosis may be due to thrombotic diatheses tested on the venous thrombosis panel
International normalized ratio (INR)
Used to standardize prothrombin time (PT) results for patients taking warfarin (coumadin)
Therapeutic goal is usually a value of 2 to 3
Intended to make comparisons similar between different labs by compensating for variable thromboplastins used in PT test
Defined as patient PT divided by mean normal PT, with the result raised to the power (exponent) of the ISI:
Results can be improved with a calibration curve (Archives 2004;128:308)
Results are affected by different thromboplastin reagents, not by storage at room temperature for up to 24 hours (Archives 1998;122:972)
References: J Clin Path 2003;56:48 (recommendations for reporting), J Clin Path 2002;55:845 (patient self testing)
International sensitivity index (ISI)
Measure of sensitivity of particular PT reagent - determined by manufacturer
Used to resolve interlaboratory variations in PT
Different PT reagents have different sensitivities to factor deficiencies
High ISI (3.0) means insensitive reagent vs. low ISI (1.0) means sensitive reagent
Labs should use reagents with an ISI of 1.0 to 1.5 if possible
References: Archives 2004;128:308 (ISI calibration), J Clin Path 2003;56:114 (ISI calibration for home PT monitors)
Low molecular weight heparin (LMWH)
Recommended to monitor using chromogenic antifactor Xa assay on specimens obtained up to 4 hours after subcutaneous injection of LMWH
Recommended to use different calibrations for LMWH and unfractionated heparin, and to establish calibration curves for each lot and type of LMWH
Don’t use PTT to monitor because LMWH doesn’t affect thrombin or factor IXa
Also called lupus inhibitor
One of the two main types of antiphospholipid antibodies (other is anticardiolipin antibodies)
Common in patients with systemic lupus erythematosus, but most cases occur in patients without SLE
May cause increased PTT (not time dependent), increased or normal PT
Prolongs clotting times by binding to phospholipid cofactors in coagulation cascade (note: often not true for HIV+ patients, Archives 1993;117:595)
Indications: patients with venous thromboembolism (particularly if no family history or associated with autoimmune disease); unexplained stroke (young person or autoimmune disease), cerebral venous thrombosis, recurrent or late pregnancy loss; may be considered for arterial thrombosis (particularly in young patient or no documented atherosclerosis)
Specimen: plasma (citrate tube)
Methodology: an algorithm combining several tests is necessary; all are clotting time based: (a) Russell viper venom time (sensitive to abnormalities in factors X and V, diluted for screening), (b) kaolin clotting time, (c) dilute PT (tissue thromboplastin inhibition test), (d) PTT-based assays (should have low concentration of phospholipids to enhance sensitivity), (e) less commonly Textarin (obtained from venomous Australian snake, not sensitive to abnormalities of factor X but sensitive to abnormalities of factor V), (f) less commonly Taipan venom (insensitive to abnormalities of factors X or V)
Note: all venom assays are sensitive to abnormalities in factor II, calcium and platelets
Use of commercially available, integrated test systems is recommended: Staclot procedure - (1) add diluent to tube 1 and egg phosphatidylethanolamine to tube 2; (2) add platelet poor plasma with polybrene (neutralizes heparin) to both tubes, incubate and add PTT reagent; PTT in tube 2 should be 12+ seconds shorter than tube 1 to be a positive test for lupus anticoagulant
To demonstrate persistence, positive test must be confirmed by repeat testing after 6 weeks
Screening assay has low concentration of phospholipids to enhance sensitivity; should have platelet count less than 10K
Abnormal (prolonged) PTT results may be repeated after mixing with equal amount of normal platelet-poor plasma; continued prolongation of clotting time indicates an inhibitor (not a factor deficiency); confirmed by adding excess phospholipids, which should shorten clotting time towards normal; must also rule out factor VIII inhibitors, heparin, other coagulopathies
Values prolonged by bivalirudin, lepirudin, argatroban, and fondaparinux (Archives 2004;128:1142)
Results vary based on dilutions in factor XII, XI, IX and VIII assays
May be mistaken for a factor VIII inhibitor if dilutions to abnormal factor assays are not done
Don’t test patients being treated with anticoagulants (or interpret with caution)
References: Archives 2002;126:1424, CAP Today; January 2003-warfarin monitoring
Used to determine if etiology of prolonged PT or PTT is due to a factor deficiency or an inhibitor
Laboratory should be notified of presence of therapeutic anticoagulant
Add heparinase to remove any heparin present (or perform thrombin time to check for even small amounts of heparin)
Experienced laboratories may omit mixing studies and move to more definitive testing, based on patient presentation; although understanding the theory of mixing studies is educational, interpreting the actual data is not always straightforward
Methodology: add patient plasma to equal volume of normal plasma and repeat PTT
Various incubation times are usually assessed; the most rigorous testing uses 0, 30, 60 and 120 minutes; less rigorous testing omits the 30 and 120 minute incubations, although detecting some inhibitors requires the 120 minute incubation
Prolonged PTT becomes normal after mixing study and stays normal after 2 hours: indicates factor deficiency; perform assays for factors VIII, IX, XI and XII; if PT also prolonged, consider assays for common pathway factors
Prolonged PTT remains prolonged after mixing study: indicates inhibitor; most common is lupus anticoagulant; also therapeutic anticoagulant; rarely due to inhibitors to factors IX, XI or XII
Prolonged PTT becomes normal after mixing study but prolonged after 1-2 hour incubation: indicates factor VIII inhibitor, rarely factor V inhibitor
Either functional (based on plasmin activity) or immunologic (based on concentration of plasminogen antigen)
Functional assays: determine plasmin enzyme activity with plasmin-specific chromogenic substrate; add streptokinase to patient plasma, complex cleaves a chromogen releasing a colored compound; color is measured spectrophotometrically, and is proportional to plasminogen in sample; expressed as percentage of normal plasma (reference range 75-130%)
Immunologic assays: radial immunodiffusion methods; used if dysplasminogenemia is being evaluated; ratio of functional activity to antigen is significantly decreased compared to controls
Plasminogen levels are increased by oral contraceptives (which increase cholesterol levels), pregnancy, acute phase reactions
Plasminogen levels are decreased by liver disease, thrombolytic therapy, DIC; newborns have levels that are 60% of adults, increase to adult values by age 6 months
Indications: patients with familial venous thrombosis but no evidence of other hypercoagulable states, occasionally used to monitor thrombolytic therapy or for patients with ligneous conjunctivitis
Plasminogen activator antigen-1
Uncommon test; perform if strong evidence of familial bleeding disorder but normal results for von Willebrand disease or possibly if unexplained premature myocardial infarction
Not a known risk factor for hypercoagulability (Archives 2002;126:1401), although high levels are associated with arterial thrombosis; low levels are associated with rare familial bleeding disorder
Has circadian rhythm, with highest values in morning; in one study, mean level was 23 ng/mL at 9 am vs. 10 ng/mL at 4 pm; also is acute phase reactant, so don’t measure immediately following thrombosis; also elevated during pregnancy
Collection: collect blood from steadily flowing venipuncture, discard first 3-5 mL (if this is the only test), avoid platelet contamination of plasma (platelets contain PAI1) by separating plasma from cells or storing on ice
Reject specimen if antifibrinolytic agent is present in specimen
Reference range: 4-40 ng/mL for antigen assay, 0-12 units/mL for functional assay
Functional assay: add patient plasma to known amount of urokinase/tPA, which binds to patient PAI1; residual urokinase is detected by adding plasminogen, which converts it to plasmin, which cleaves a chromogenic substrate; amount of released color is inversely proportional to patient PAI1 (note: inhibitors of antiplasmin and plasmin are present to prevent their interference)
ELISA (antigen) assay: also available
Used to assess platelet function if a familiar bleeding disorder is suspected, but the PT, PTT, platelet count and von Willebrand tests are normal (which is unusual)
May include platelet responses to adenosine diphosphate (ADP), epinephrine, collagen and arachidonic acid
Agglutination with ristocetin may also be assessed
Usually 60% or more platelets aggregate with the above agonists, but not spontaneously; aggregation is decreased in newborns
Methodology: aggregometry with platelet-rich plasma to measure optical transmission or electric impendence; whole blood aggregation with a lumiaggregometer can measure both aggregation and ATP release
Abnormalities are often due to medications (aspirin - affects arachidonate aggregation; other platelet-inhibiting agents); also uremia, monoclonal gammopathy, myeloproliferative disorders
Note: testing is labor intensive and must be scheduled in advance because a normal control must be drawn simultaneously; a platelet function assay (e.g. PFA-100) may be used to assess platelet function; although easier to perform, it is not as robust as platelet aggregation and must be interpreted with caution
Hereditary disorders: consider in patients with bleeding histories, no obvious acquired cause, but abnormal platelet aggregation study repeated at least once, same abnormality in family members; may be a platelet storage pool disorder (deficiency in alpha or dense platelet granules), Glanzmann thombasthenia (deficiency of platelet glycoprotein IIb/IIIa, reduced aggregation by all agonists except ristocetin), Bernard-Soulier disease (deficiency of platelet glycoprotein Ib, causes decreased ristocetin-induced aggregation only)
More detailed discussion in platelet chapter (pending)
Either autoimmune (idiopathic thrombocytopenic purpura), alloimmune (neonatal alloimmune thrombocytopenia, post-transfusion purpura, platelet transfusion refractoriness) or heparin-induced
These tests must be ordered and interpreted cautiously, considering the clinical presentation
ELISA: test for specific antiplatelet antibodies; antigen of interest is bound to surface of microtiter plate, then add patient plasma, antibody will bind to antigen
Antigen capture immunoassay: specific antigens are bound to solid phase, then add patient serum, patient antibodies will bind to antigens
Platelet antigen typing by antigen capture immunoassays: patient’s platelet antigens are immobilized by monoclonal antibodies onto a solid phase; then add antibodies of known specificity
Flow cytometry: may be used
Lymphocytotoxicity assay: determine percent reactive antibody (HLA antibodies in patients who are refractory to platelet transfusions)
Polymerase chain reaction: can be used to identify patient’s platelet antigens
Platelet antibody disorders
Drug-induced thrombocytopenia: detected by a difficult serotonin release assay (add patient plasma/serum plus drug and platelets with radiolabeled serotonin; drug antibodies, if present, stimulate platelets and radioactive serotonin is released); also detected with flow cytometry; offending drugs include quinine and quidinine, sulfonamides, sulfonylureas, gold salts, salicylates; mechanism is either nonimmune (marrow suppression or nonimmune destruction) or immune (platelet counts due to immune causes may drop to < 10K, return to normal within 7 days of stopping offending drug)
Idiopathic thrombocytopenic purpura (ITP): autoantibody against platelets, usually directed against GP IIb/IIIa, less commonly GP Ib/IX; diagnosis of exclusion; usually resolves in children but is chronic in adults; tests to order include peripheral blood smear, CBC, HIV, thyroid function tests, liver function tests, bone marrow biopsy
Neonatal alloimmune thrombocytopenia (NAIT): incidence of 1 per 1-5K live births; father and newborn have antigen that mother lacks, mother produces antibodies to this antigen (usually PI-A1 component of GP IIb/IIIa) which crosses the placenta and destroys fetal platelets; newborn platelet counts are <100K at birth, return to normal within 2 weeks
Platelet refractoriness: in thrombocytopenic patients with multiple platelet transfusions, due to formation of HLA-A, HLA-B or less commonly ABO antibodies that destroy transfused platelets; platelet crossmatch using immobilized platelets may be performed in referral centers
Post-transfusion purpura: patient has antibody directed against transfused platelet antigen absent on patient’s platelets; for unknown reasons, these antibodies also destroy platelet’s own antigens; antigen is most commonly PI-A1 component of GP IIb/IIIa; patients have sudden onset of severe thrombocytopenia 5-12 days after transfusion of platelet product, resolves 14 days after transfusion
Platelet hyperaggregation studies
Hyperaggregation may rarely be associated with hypercoagulability, including myocardial infarction, strokes, venous thrombosis
Tested patients should have abstained from aspirin, NSAIDs or platelet-inhibiting drugs for 7 days prior to testing
Indications: patients with unexplained hypercoagulability and normal values in hypercoagulation panel
Methodology: similar to platelet aggregation; centrifuge citrated plasma gently to draw red and white blood cells into a pellet, which leaves platelets suspended in the plasma; then add various agonists at multiple low concentrations and a control (no agonist, to measure spontaneous aggregation), and measure platelet aggregation with an aggregometer (which measures optical density); must carefully evaluate patient’s use of medications (including over the counter); must compare to normal control, and results can be subjective
Screening assays: preincubate PTT sample for 10 minutes prior to adding calcium; a prolonged PTT that shortens after the 10 minute preincubation is suspicious for prekallikrein deficiency
PTT performed with elagic acid as the intrinsic pathway activator will be normal in prekallikrein deficiency
Specific assay: preincubate patient plasma with prekallikrein-deficient plasma for 1 minute, then add calcium and perform PTT; prekallikrein level is determine from a standard curve of prekallikrein vs. PTT
Interference is due to hirudin, argatroban, danaparoid, heparin (add heparinase)
Reference range is 60-140% of normal; newborn levels are lower, but increase to near adult levels by age 6 months
Indications: prolonged PTT corrected with mixing study, factors VIII, IX, XI and XII are normal, PT and fibrinogen are normal, lupus anticoagulant assays are negative
Deficiencies are either quantitative (type I, reduced amount of normal protein) or qualitative (type II, normal amount of defective protein) Assays are either functional (measure protein activity) or antigenic (immunoassays that measure quantity, not function)
Perform functional assay first - if decreased, perform antigenic assay; must exclude acquired causes (below)
Low values should be confirmed on a new specimen
Assays should be performed with platelet poor plasma, using sodium citrate collection tubes
Functional assays are clot-based or chromogenic
Clot based functional assays: detects all known type I and II variants; patient’s protein C is activated by Southern Copperhead venom (Agkistrodon contortrix contrortrix), which degrades synthetic substrate, factor Va or factor VIIIa with clot based PTT assay; the prolongation of clotting time is proportional to the amount of factor activity
PT based assay or amidolytic assays are affected by lupus anticoagulants (raises protein C result), elevations of factor VIII > 200% (decreases the result), acute phase reactions, factor V Leiden mutation (decrease the result); cannot perform on patients taking hirudin or argatroban
Chromogenic functional assays: not affected by lupus anticoagulants, factor VIII levels, factor V Leiden or other coagulation abnormalities that interfere with clot-based functional assays; may not detect qualitative deficiencies detected by clot-based assays; patient’s protein C is activated by snake venom, which cleaves a synthetic substrate, which releases a chromogenic that is measured spectrophotometrically
Antigenic assays: either ELISA, electroimmunoassay (Laurell rocket method) or radioimmunoassay; variable levels, so use 3 standard deviations as cutoff
ELISA: uses antibody to protein C immobilized to microtiter place; add plasma; add secondary anti-protein C antibody coupled to an enzyme for colorimetric detection; use standard curve to determine plasma protein C
Laurell rocket antigenic assay: agarose gel has antibody to protein C; plasma samples are put into wells and electrophoresed; antigen-antibody complexes precipitate during electrophoresis, and height of precipitin arc is proportional to plasma protein C, which is compared to standard curve using pooled normal plasma; may be unable to detect protein C levels < 5%
Radioimmunoassay: similar to ELISA, but uses single, radiolabeled antibody
Values falsely increased by bivalirudin, lepirudin, argatroban, and fondaparinux (Archives 2004;128:1142), lowered by warfarin (must discontinue for 10 days prior to testing)
Reference range: 70-140% of normal; newborns levels are 20-30% of adult values; usually rise to near adult levels by age 6 months, but may remain below adult normal levels until age 10 years
Indications: necrotic skin in newborns days 1-3 of life (purpura fulminas neonatorum, can also test parents also for heterozygosity); evaluation of cause of venous thromboembolism (recommended to use chromogenic protein C assays initially)
Non-indications: screening before oral contraceptives or oral anticoagulants (discontinue for 10 days, or test family members)
Acquired causes of low Protein C levels: more common than hereditary deficiencies - clot formation, surgery, liver disease, warfarin (should be discontinued at least 10-30 days prior to testing), DIC, vitamin K deficiency, vitamin K antagonist therapy, L-asparaginase therapy; repeat protein C test once these conditions are no longer present
Acquired causes of increased Protein C (may mask protein C deficiency): ischemic heart disease, pregnancy, postmenopausal women, hormone replacement therapy, oral contraceptives
References: Archives 2002;126:1337
Deficiencies are either quantitative (type I, reduced normal protein) or qualitative (type II, normal amount of defective protein)
Assays are either functional (measure protein activity) or antigenic (immunoassays that measure quantity, not function)
Gold standard to measure free protein S or APC cofactor activity of protein S is considered the polyclonal ELISA with or without polyethylene glycol precipitation, although this procedure has poor reproducibility
Perform functional assay first (detects all types of deficiencies)
Functional assays are clot-based, cannot be performed in patients taking hirudin or argatroban
Methodology: clot based protein S method is based on the addition of activated protein C, which in the presence of protein S, accelerates the inhibition of thrombin-activated factors VIII and V; the prolongation of clotting time is proportional to the amount of factor S activity; interference may occur with elevated factor VIII (acute phase reactions or otherwise); values falsely increased by bivalirudin, lepirudin, argatroban, and fondaparinux (Archives 2004;128:1142), lupus anticoagulants
Reference ranges are in nmol/liter (each lab should establish its own, values in acute phase plasma are higher):
Total protein S:- 65% of value in pooled normal human plasma (289-397)
Free protein S: 71-115
C4 binding protein beta+: 228-310
Total C4 binding protein: 257-423
Antigenic assays measure free protein S (functionally active form) or total (bound plus free) protein S - usually 60% of protein S is bound to C4b-binding protein
Free protein S levels in protein S deficient patients are very sensitive to timing, temperature and dilutional conditions of assays compared to normal individuals
Acquired causes of low Protein S levels: more common than hereditary deficiencies - clot formation, surgery, liver disease, warfarin (should be discontinued at least 10 days prior to testing), nephrotic syndrome, DIC, L-asparaginase therapy, any stimulus to acute phase response (increases C4b binding protein, decreases free protein S), newborns (12-60% of adult levels, rise to adult levels by 6 months), women (lower than men before menopause, while taking oral contraceptives, during pregnancy or with hormone replacement therapy), vitamin K antagonist drugs, vitamin K deficiency, elevated factor VIII levels (>200%) in PTT based functional assays, thrombosis; also nephrotic syndrome, varicella infection, HIV infection
Classification of deficiencies: all have low functional protein S; I - also low free and total protein S; II / IIb - also normal free and total protein S; III / IIa - low free but normal total protein S
Prothrombin gene 20210A testing
Mutation in G to A transition at nucleotide 20210 in 3’ untranslated portion of prothrombin gene, which introduces a new Hind III restriction site
Methodology: usually PCR amplification of 3’ untranslated region of prothrombin gene surrounding the 20210 polymorphism, then either gel electrophoresis, radioisotopic probing or restriction endonuclease digestion with Hind III to detect the nucleotide sequence
Can identify heterozygotes and homozygotes
Multiplexed arrays test for factor V Leiden, MTHFR C677T and other sequences
Specimen is whole blood
Most commonly performed laboratory coagulation test
Measures clotting time from factor VII activation through fibrin formation (i.e. extrinsic and common pathway)
Used as screening test and to monitor warfarin anticoagulation; can only detect single factor deficiencies if level is 15-45% of normal
Methodology: mix patient plasma with calcium (neutralizes citrate anticoagulant) and thromboplastin (a tissue extract, such as from brain, that contains abundant phospholipids and tissue factor), measure time to clot formation
Anticoagulant is usually 3.2% sodium citrate (recommended by Clinical and Laboratory Standards Institute; 3.8% sodium citrate causes prolonged PT if samples are <80% filed compared to 100% filled; no difference in result with 3.2% citrate between filled volumes of 70% and 100%)
Test should use a thromboplastin that is insensitive to heparin in therapeutic range
Note: Warfarin is monitored using INR (international normalized ratio), which standardizes PT results for patients on oral anticoagulants; goal is INR of 2-3; calculated as patient PT divided by mean normal PT; PT/INR should be checked daily at onset of warfarin use until dose and INR are stable (usually at least a week since half life of factors II and X are long), then decreased gradually to every 4 weeks
May be improved by instrument-specific International Sensitivity Index (ISI) values, in-house calibrators or calibration curves (Archives 2004;128:308); ISI measures sensitivity of PT reagent to factor deficiencies (1.0 is sensitive, 3.0 is insensitive, value determined by manufacturer)
Reference interval should be established using at least 120 subjects for each reference population or subclass, and verified using at least 20 subjects
Usual reference range is 10-14 seconds, up to 16 seconds at birth, decreasing to adult values at age 6 months
Limitations: lupus anticoagulants, use of hirudin or argatroban - must use alternative assays, such as chromogenic factor X assays
Prolonged PT: usually due to deficiencies of factors I (fibrinogen), II, V, VII, X, less commonly due to an inhibitor or anticoagulant (heparin, hirudin, argatroban), rarely lupus anticoagulant or specific factor inhibitor
Prolonged PT with normal PTT: warfarin or vitamin K deficiency (decreases function of factors II, VII, IX, X, protein C, protein S), liver dysfunction (decreases hepatic synthesis of all coagulation factors except factor VIII), DIC
Markedly prolonged values may be due to long acting warfarin-like rodenticide toxicity (Archives 2004;128:e181)
References: Archives 1997;121:956 (INR differs with 3.8 vs. 3.2% citrate), CAP today; March 2005-home monitoring
Algorithms for working up a prolonged PTT:
(1) add heparinase; if PT corrects to normal, prolongation is due to presence of heparin
(2) mixing study (determine if etiology if factor deficiency or factor inhibitor); mix patient plasma with equal amount of normal plasma and determine the PT of the mixture after incubation for 2 hours
(a) if PT of mixture is normal, prolonged PT is due to factor deficiency; do assays for factors I, II, V, VII, X
(b) if PT of mixture is still prolonged, suggests presence of inhibitor (rare)
(c) if PTT of mixture is initially normal but becomes prolonged after incubation for 1-2 hours, may be due to factor V inhibitor (rare)
PTT - Partial thromboplastin time
Also called activated partial thromboplastin time (aPTT)
Second most commonly performed coagulation test (after PT)
Measures clotting time from factor XII activation through fibrin formation (i.e. intrinsic and common pathway); more sensitive to intrinsic factor deficiencies
Methodology: mix patient plasma with excess calcium (to counteract the citrate anticoagulant) and phospholipid (called partial thromboplastin since tissue factor is not present) and intrinsic pathway activator such as silica, kaolin, celite or elagic acid; after drawing, invert gently to mix
Used to monitor heparin or direct thrombin inhibitors such as hirudin; target ratio is 1.5 to 3.0 (compared to nonheparinized samples)
Incorrect values may cause bleeding, thrombosis, morbidity or death
3.2% citrate tube is recommended; use of 3.8% citrate as anticoagulant causes prolonged PTT if samples are <90% filed compared to 100% filled ( no difference in result with 3.2% citrate between filled volumes of 60% and 100%)
Quality control: therapeutic range for heparin should be determined specific to each laboratory’s reagent and instrument system, and redetermined if method changes; determine by comparing ex vivo specimens preferably with an appropriated validated heparin assay or with a previously calibrated PTT specimen using a method to control for reagent drift; determine equivalence using ex vivo plasma samples obtained from patients treated with unfractionated heparin, not spiked in vitro heparinized plasma samples
Can assay heparinized samples up to 4 hours after phlebotomy if centrifuged within 1 hour of collection; have shorter PTT if stored uncentrifuged at room temperature (up to 50% decrease at 4 hours), due to release of PF4 from platelets, which neutralizes heparin
Markedly prolonged values may be due to long acting warfarin-like rodenticide toxicity (Archives 2004;128:e181)
Interpretation: values are normally higher in newborns (up to 55 seconds), decreases to adult levels at age 6 months; values are smaller with acute phase reactions, which elevate Factor VIII levels
References: CAP Today; October 2004 (validating heparin sensitivity of PTT); Archives 1997;121:956 (INR differs with 3.8 vs. 3.2% citrate)
Algorithms for working up a prolonged PTT:
(1) add heparinase; if PTT corrects to normal, prolongation is due to presence of heparin
(2) mixing study (determine if etiology is factor deficiency or factor inhibitor); mix patient plasma with equal amount of normal plasma and determine the PTT of the mixture after incubation for 2 hours
(a) if PTT of mixture is normal, prolonged PTT is likely due to factor deficiency; do assays for factors VIII, IX, XI and XII; if PT is also prolonged, consider common pathway factor assays also
(b) if PTT of mixture is still prolonged, suggests presence of inhibitor, usually lupus coagulant; perform lupus coagulant assay; also possible if heparin is present (should have tested for in step (1) above) or rare factor inhibitors
(c) if PTT of mixture is initially normal but becomes prolonged after incubation for 1-2 hours, may be due to factor VIII inhibitor; perform factor VIII assay - if decreased, perform assay for factor VIII inhibitor
Clotting time similar to thrombin time, but uses snake venom (Reptilase) instead of thrombin
Measure rate of fibrin clot formation after addition of reptilase to citrated plasma
Generates a fibrin clot by cleaving fibrinopeptide A from fibrinogen
Prolonged by decreased or dysfunctional fibrinogen, or high levels of fibrin degradation products; also amyloidosis (inhibits fibrinogen conversion to fibrin)
Used to diagnose dysfibrinogenemia (also thrombin time)
Not prolonged by heparin or hirudin (unlike thrombin time)
References: Mass General handbook
Measures rate of fibrin clot formation after addition of standard concentration of thrombin to citrated plasma; thrombin cleaves fibrinogen, releasing fibrinopeptides A and B, and converting fibrinogen to fibrin
Useful to diagnose dysfibrinogenemia after more common disorders are excluded
Reference range: 10-13 or 16-24 seconds, depending on reaction conditions and thrombin concentration
Prolonged if even small amounts of heparin, hirudin or argatroban anticoagulants are present
Also prolonged with dysfibrinogenemia, amyloidosis (inhibits fibrinogen conversion to fibrin), DIC, thrombolytic therapy, thrombin inhibitors in patients exposed to bovine thrombin
tPA activity / antigen and PAI-1 activity / antigen are used to evaluate fibrinolysis; often PAI-1 activity and tPA antigen are ordered together
PAI-1 rapidly forms a complex with free tPA in the specimen
Methodology: must first inhibit interaction of tPA with PAI1 (its functional inhibitor) by acidifying plasma; determine activity by measuring plasmin activity from conversion of plasminogen
Not recommended for routine clinical laboratories due to complexity and special handling requirements
Can also measure tPA plasma concentration by ELISA or other immunologic assays
Resting level is usually low, not clinically significant
Post-stimulation level (such as after venous occlusion for 10 minutes) may be more useful
von Willebrand disease testing - general
Often need to repeat tests, because von Willebrand factor and factor VIII are elevated during acute phase reactions, pregnancy, estrogen use and in newborns - can measure fibrinogen (acute phase reactant) to determine if acute phase condition exists
Tests: von Willebrand factor antigen assay, von Willebrand factor activity (ristocetin cofactor activity), factor VIII levels, fibrinogen (or other acute phase reaction marker), multimer analysis
Interpretation:
- All results normal (considering ABO blood type) - unlikely to have vWD if no acute phase reaction, pregnancy, estrogen use, newborn
- All results normal but elevated fibrinogen / factor VIII - acute phase reaction may mask abnormalities; repeat when fibrinogen and factor VIII levels are normal
- Reduced antigen, activity, factor VIII - likely type 1 vWD
- Severely reduced (<10%) or undetectable antigen, activity, factor VIII - likely type 3 vWD
- Activity reduced more than antigen and factor VIII - possibly type 2 vWD; perform multimer analysis and low dose ristocetin cofactor
. normal multimer analysis - likely type 2M vWD
. missing high molecular weight multimers - likely type 2A vWD
. missing high and intermediate molecular weight multimers - likely type 2B or platelet type vWD
. increased low dose ristocetin aggregation - likely type 2B or platelet type vWD
. normal or decreased low dose ristocetin aggregation - not type 2B or platelet type vWD
. reduced factor VIII (5-40%), normal activity and activity - possibly type 2N vWD or in males, mild hemophilia A; also possibly factor VIII degradation due to processing delay
von Willebrand factor antigen assay (vWF)
Levels can increase 2-3x with injury, infection or other acute phase reactant stimulus (30% level at baseline can increase to 90% by the time the patient is tested); can determine presence of acute phase reaction by measuring fibrinogen
Reference range is higher in children < 6 months old than adults (abnormal value in 3 month old may be normal for an adult)
Minor injuries may produce major bleeds in children, leading to false accusations of child abuse
Type O patients have significantly lower vWF antigen levels (75%) compared to type A (106%), type B (117%), type AB (123%); although bleeding symptoms may depend on vWF antigen levels regardless of ABO type
Methodology: (a) ELISA assay measures quantity of vWF, not function (quality); (b) also latex particles coated with anti-vWF antibodies, measure light absorbance; (c) rocket immunoelectrophoresis
Used to determine if patient with personal or family history of bleeding has von Willebrand disease; also to assist in determining hemophilia A carrier status in females
von Willebrand factor activity
Also called ristocetin cofactor activity
Ristocetin is an antibiotic that causes vWF to bind to and activate platelets
Test measures function of von Willebrand factor
Platelets from healthy individuals are mixed with standard concentrations of ristocetin and patient plasma is added to cause platelet agglutination (measured in aggregometer), which is proportional to the vWF concentration
Used to confirm type 2B von Willebrand’s disease (increased agglutination due to increased affinity of vWF for GPIb); similar results for platelet type von Willebrand’s disease (although the defect is in GPIb)
Note: “aggregation” of platelets implies linkage via fibrinogen and GP IIb/IIIa; ristocetin links platelets through vWF and GP Ib, and appropriate term is actually “agglutination”
Other functional test: collagen-binding ELISA assay, functional vWF binds to collagen and is detected
Low dose ristocetin platelet aggregation assay
To diagnose type 2B von Willebrand disease
Similar to von Willebrand factor activity test, but uses patient’s platelets and lower dose of ristocetin
Platelets from patient are mixed with standard concentrations of ristocetin and patient plasma is added to cause platelet agglutination (measured in aggregometer); increased aggregation in type 2B vWD in this assay due to GP Ib mutation, which increases affinity for vWF
von Willebrand factor multimer analysis
To detect type 2 von Willebrand disease
Involves separation of multimers by size using agarose gel electrophoresis of patient’s plasma
Then detect multimers using radiolabeled or enzyme linked anti-vWF antibody
Normal in von Willebrand’s disease types 1, 2N or 2M (type 1 has reduced quantity of all sizes, but difficult to identify on gel)
No/reduced high molecular weight multimers in types 2A and 2B von Willebrand’s disease
No/reduced intermediate molecular weight multiples in type 2A
No/marked reduction in all multimers in type 3
End of Coagulation chapter/outline