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Coagulation

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Revised: 30 September 2014
Copyright: (c) 2001-2014, PathologyOutlines.com, Inc.


General

Hemostasis - general


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 9 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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

Diagrams
=========================================================================



Hemostasis Overview

Additional references
=========================================================================

Goodnight Jr, Scott & Hathaway (Eds) (2001). Disorders of Hemostasis & Thrombosis: A Clinical Guide: McGraw-Hill



General

Normal hemostasis


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 9 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Primary hemostasis: initial step of forming platelet plug to stop bleeding from damaged vessel
● Secondary hemostasis: platelet plug is reinforced by fibrin clot; then fibrin clot is stabilized by activated factor XIII, which cross-links fibrin strands
● Tertiary hemostasis: as fibrin clot is formed, plasmin is generated to break down the clot
● Fibrin clot may occur via intrinsic or extrinsic pathway or both; in vivo it occurs via a hybrid model

Coagulation factors
=========================================================================

● 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 originally called accelerin later discovered to be activated Factor V
      ● 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
      ● High Molecular Weight Kininogen: occasionally called Fitzgerald factor
      ● Prekallikrein: occasionally called Fletcher factor

Diagrams
=========================================================================



Coagulation


Intrinsic, common, and extrinsic pathways; chart credit to Kendall Crookston, M.D., PhD


The in vivo coagulation cascade; chart credit to Kendall Crookston, M.D., PhD

Additional references
=========================================================================

Chandler WI (2005). Physiology of hemostasis. In B Spiess, R Spence, A Shander (Eds), Perioperative Transfusion Medicine (pp 77-92). Lippincott, Williams, & Wilkins.



General

Intrinsic pathway


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 9 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Involves factors VIII, IX, XI, XII (Hageman factor), prekallikrein, high molecular weight kininogen (Wikipedia)
● 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)
● 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
● The activated partial thromboplastin time (aPTT) measures the intrinsic and common pathways

Diagrams
=========================================================================


       
Intrinsic pathway

Additional references
=========================================================================

● Kolde, Hans-Jurgen (2004). Haemostasis. Basel, Switzerland: Pentapharm Ltd.



General

Extrinsic pathway


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 9 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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)
● 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
● The prothrombin time (PT) measures the extrinsic and common pathways

Diagrams
=========================================================================



Coagulation extrinsic pathway – by Jeremy Parsons, M.D.

Additional references
=========================================================================

● Kolde, Hans-Jurgen (2004). Haemostasis. Basel, Switzerland: Pentapharm Ltd.



General

Common pathway


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 8 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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
● While both PT and aPTT include the common pathway, the PT is a more sensitive screening test for common pathway problems

Diagrams
=========================================================================


See normal hemostasis

Additional references
=========================================================================

M Blomback, J Antovic (Eds), Essential Guide to Blood Coagulation 2010; Wiley-Blackwell: pages 13-18



General

Protein C / Protein S anticoagulant pathway


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 9 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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)
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 and “S” because it was discovered in Seattle, WA)

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
● Protein C has the shortest half life of the vitamin K dependent proteins; when placed on warfarin the patient loses the anticoagulant properties of protein C much more quickly than the pro-coagulation effects of factor VII, II, IX and X; this leads to warfarin necrosis which can be prevented by bridging with heparin

Diagrams
=========================================================================



Protein C / Protein S anticoagulant pathway

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1337, Goodnight & Hathaway (Eds) (2000). Disorders of hemostasis & thrombosis: A clinical guide: McGraw-Hill



General

Thrombomodulin


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 7 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Intrinsic membrane glycoprotein on luminal surface of endothelial cells that binds thrombin 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
● See also discussion in Stains chapter

Diagrams
=========================================================================



Thrombomodulin / Protein C relationships and function


Thrombomodulin protein

Additional references
=========================================================================

BMC Neurology 2004;4:21



General

Antithrombin


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 8 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Formerly called antithrombin III
● Member of serine protease inhibitor (serpin) gene family on #1q23-25
● 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)

Diagrams
=========================================================================



The body makes its own natural anticoagulants. Chart credit to Kendall Crookston, M.D., PhD.

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1326, Disorders of hemostasis & thrombosis: A clinical guide (2nd ed); 2000



General

Fibrinolysis pathway


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 9 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Process of degrading the fibrin clot when it is no longer needed
● Also prevents extension of clot beyond site of injury

tPA and uPA
=========================================================================

● Fibrinolysis 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 do 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 found in 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
=========================================================================

● 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

Deficiency conditions
=========================================================================

● 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

Diagrams
=========================================================================



Coagulation cascade and fibrinolytic system


The fibrinolytic pathway counterbalances the clotting pathway by breaking down clots. Chart credit to Kendall Crookston, M.D., PhD.

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1376, Clin Lab Med 2009;29:159



General

Contact System


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 8 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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
● Contact pathway may play a large role in inflammation, as patients with factor XI deficiency often have repeated infections
● Recommended to not measure their activity in routine evaluation of patients with arterial or venous thromboembolism or acute coronary syndromes (Arch Pathol Lab Med 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

Additional references
=========================================================================

Roberts H, Escobar M (2007). Less common congenital disorders of hemostasis. In C Kitchens, B Alving, C Kessler (Eds). Consultative hemostasis and thrombosis (pp 71-73). Saunders Elsevier



Bleeding disorders

Bleeding disorders - general


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Bleeding disorders are often classified as defects of primary hemostasis (platelets, vessels, etc.) or of secondary hemostasis (coagulation cascade and its regulation)

Clinical history is important:
(a) single site (suggests structural lesion) vs. multiple sites (coagulopathy)
(b) for coagulopathies: hereditary (family history of bleeding or bleeding since childhood) vs. acquired (no previous bleeding history)
(c) time from “hemostasis challenge” to bleeding symptoms: immediate suggests platelet/primary hemostasis disorder (inability to form normal platelet plug) versus late suggests coagulopathy/secondary hemostasis (breakthrough bleeding occurs after platelet plug due to impaired fibrin formation); even further delay suggests a Factor XIII deficiency (fibrin clot forms to stop bleeding but is unstable and breaks down due to lack for crosslink stabilization)
(d) physical exam: petechiae (primary hemoastasis/platelet disorders) vs. hematoma or hemarthrosis (secondary hemostasis/coagulation defects) vs. mucous membrane bleeding or bruising (nonspecific, often primary hemostasis)

Gross images
=========================================================================



Petechiae


Hematoma



Bleeding disorders

Laboratory approach


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 12 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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)

Additional references
=========================================================================

Dzik, W. (2009). Blood components to achieve hemostasis for surgery and invasive procedures. In T. Simon, E. Snyder, C. Stowell, R. Strauss, B. Solheim, M. Petrides (Eds.). Rossi's Principles of Transfusion Medicine. Bethesda, MD: Blackwell Publishing Ltd.



Acquired bleeding disorders

Acute phase reaction


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, MD, University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 8 October 2010, last major update September 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition / General
=========================================================================

● Many serum proteins become elevated due to illness, injury, inflammation or stress; also pregnancy
● Elevated levels return to normal after condition resolves

Terminology
=========================================================================

● Also called positive acute phase proteins

Etiology
=========================================================================

● In response to injury, neutrophils and macrophages secrete IL-1, IL-6, IL-8 and TNF-a into the bloodstream; liver then produces acute-phase reactants (Wikipedia, COPE)
● Production of other proteins is reduced ("negative" acute phase reactants)

Laboratory
=========================================================================

● Associated with increase in fibrinogen, factor VIII, vWF (up to 3x normal levels) and PAI-1 (up to 50x)
● Associated with decrease in PTT, decrease in protein S (due to binding to increased C4b)



Acquired bleeding disorders

Acquired dysfibrinogenemia


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, MD, University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 9 April 2013, last major update June 2010
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

Definition
=========================================================================

● Abnormal fibrinogen molecule that causes a decrease in the rate of fibrin polymerization
● Rarely causes bleeding or thrombosis

Epidemiology
=========================================================================

● 80% prevalence in patients with liver disease
● 8% prevalence in patients with obstructive jaundice

Sites
=========================================================================

● Bleeding tends to be mucosal (menorrhagia) or into soft tissues
● Thromboses can be venous or arterial

Etiology
=========================================================================

● Usually caused by liver or biliary tract disease or acute phase reaction; also monoclonal immunoglobulin that binds to fibrinogen
● Abnormal fibrinogen has increased sialic acid residues, which increases the net negative charge of the molecule, promoting charge repulsion between fibrin monomers, leading to decreased fibrin polymerization
● In cancer-associated dysfibrinogenemia (hepatocellular carcinoma, cervical carcinoma, breast carcinoma, renal cell carcinoma), tumor cells may secrete abnormal fibrinogen
● Usually does not cause bleeding or thrombosis, but may in alcoholic liver disease

Clinical features
=========================================================================

● Patients are usually asymptomatic
● Rarely bleeding or thromboses

Laboratory
=========================================================================

● Screening tests include reptilase time and thrombin time
● Fibrinogen clotting activity/antigen ratio is confirmatory test
● Patients usually have abnormal liver function tests
● Should rule out dysfibrinogenemia in family members (i.e. rule out congenital form)
● Dysfibrinogenemia typically resolves if underlying disease improves (i.e. liver disease improves or cancer undergoes remission)



Laboratory testing algorithm

Prognostic factors
=========================================================================

● Difficult to assess as patients with liver disease often have coagulation defects that could contribute to bleeding or thrombosis

Case reports
=========================================================================

● 63 year old man with monoclonal light chain that binds fibrinogen (Haematologica 2007;92:e111)
● 72 year old man with myeloma paraprotein that interacts with fibrinogen (Acta Haematol 2008;120:75)

Treatment
=========================================================================

● Treat clinical findings (i.e. if patient is bleeding, can give cryoprecipitate; if patient has thrombosis, can give heparin followed by oral anticoagulants)

Differential diagnosis
=========================================================================

● Congenital dysfibrinogenemia
● Autoantibodies against fibrinogen

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:499, eMedicine



Acquired bleeding disorders

Acquired von Willebrand disease (AVWD)


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, MD, University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 9 April 2013, last major update June 2010
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

Definition
=========================================================================

● A rare bleeding disorder of primary hemostasis that appears either spontaneously or associated with lymphoproliferative or myeloproliferative diseases, solid tumors, autoimmune disorders, cardiovascular disorders, drugs or other miscellaneous causes (Acta Haematol 2009;121:177)
● Mimics congenital von Willebrand disease in terms of clinical presentation and laboratory findings

Terminology
=========================================================================

● Also called acquired von Willebrand syndrome (AVWS)

Epidemiology
=========================================================================

● Usually older adults, but can occur in children

Sites
=========================================================================

● Typically mucocutaneous or gastrointestinal bleeding

Etiology
=========================================================================

● Not completely understood and most likely multifactorial; underlying mechanisms include:
● Autoantibodies to vWF or Factor VIII causing inhibition or increased clearance
● Cell-mediated or drug-induced proteolysis of vWF
● Abnormal vWF binding to tumor cells causing increased clearance of Factor VIII-vWF complex
● Decreased synthesis

Clinical features
=========================================================================

● Mild to moderately severe mucocutaneous or gastrointestinal bleeding in a patient with previously normal coagulation and no family history of coagulopathy (Am J Hematol 2007;82:368)
● May be underlying cause of bleeding tendency in hypothyroid patients (Haemophilia 2008;14:423)
● Many patients present with normal or increased test results, emphasizing the importance of multimer analysis in all patients with suspected disease (J Thromb Haemost 2008;6:569)
Algorithm for clinical evaluation (Mayo Medical Laboratories)

Laboratory
=========================================================================

Patients may have any of the following:
● Prolonged bleeding time
● Platelet assay showing prolonged closure times (PFA-100)
● Reduced vWF activity
● Reduced vWF antigen
● Reduced factor VIII activity
● Prolonged aPTT
● Most patients exhibit type II pattern on multimer electrophoresis, but can see type I as well as type III
● Use of VWF multimer assay recommended only when initial VWD testing identifies an abnormal result, or clinical information suggests a high likelihood of abnormal VWF multimer analysis (Mayo Medical Laboratories)

Prognostic factors
=========================================================================

● Depends on underlying disorder and other comorbidities

Case reports
=========================================================================

● Transient neonatal acquired von Willebrand syndrome due to transplacental transfer of maternal monoclonal antibodies (Pediatr Blood Cancer 2009;53:655)
● 55 year-old man with gastrointestinal angiodysplasia (Haemophilia 2006;12:452)

Treatment
=========================================================================

● Aimed at both control of the acute bleeding episode and of the underlying disorder (i.e. hypothyroidism)
● DDAVP is usually initiated first, followed by replacement therapy with plasma derived Factor VIII-vWF concentrates
● If there is no success, then IVIG (immunoglobulin) may be tried, especially if the underlying cause is thought to be autoimmune in nature
● There is little published data on the effectiveness of plasma exchange
● Immunosuppressive agents and corticosteroids have been used, but are reported to be less effective

Differential diagnosis
=========================================================================

● Congenital von Willebrand disease
● Bernard-Soulier Syndrome (Arch Pathol Lab Med 2007;131:1834)

Additional references
=========================================================================

Consultative Hemostasis and Thrombosis: Elsevier, 2007, Wikipedia, eMedicine, ARUP Consult



Acquired bleeding disorders

Amyloidosis


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, MD, University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 9 April 2013, last major update July 2010
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Amyloidosis is a generic term for multiple disorders with extracellular tissue deposition of insoluble low molecular weight fibrils
● May be inherited or acquired
● May cause acquired Factor X deficiency due to Factor X binding to amyloid, causing a shortened half time
● Bleeding usually does not occur unless Factor X levels fall below 10% (Am J Hematol 2010;85:171)

● Some patients have normal levels of coagulation factors, but severe hemorrhage can result from amyloid deposition in small to medium sized vessel walls, leading to wall damage
● For example, cerebral amyloid angiopathy is usually asymptomatic, but can cause primary lobar intracerebral hemorrhage in the elderly

Epidemiology
=========================================================================

● Amyloidosis is rare, but is found in 15% with multiple myeloma

Sites
=========================================================================

● Amyloid deposits between the endothelium and the basement membrane of vessel walls
● Generally involves the microvasculature of any organ but can involve the great vessels of the brain
● Amyloid deposits in spleen can cause splenic rupture and severe hemorrhage (Amyloid 2009;16:47)
● Deposition in the liver can cause decreased synthesis of coagulation factors in patients with advanced liver disease

Etiology
=========================================================================

● Factor X deficiency is due to (a) binding to amyloid fibrils primarily in the liver and spleen and (b) decreased synthesis of coagulation factors in patients with advanced liver disease

AL ("amyloid light chain") amyloidosis (primary):
● AL amyloidosis is caused by deposition of immunoglobulin light chain fragments
● Usually due to a plasma cell dyscrasia; most patients have monoclonal immunoglobulin light chains detectable in the serum or urine
● 9% have Factor X levels that are <= 50% of normal (Blood 2001;97:1885)

AA ("amyloid associated") amyloidosis (secondary):
● Composed of fragments of amyloid A
● Seen with chronic diseases associated with ongoing inflammation – rheumatoid arthritis, chronic infection, inflammatory bowel disease

Beta-2 microglobulin amyloid:
● Amyloid composed of fibrils from beta-2-microglobulin
● Seen with long term hemodialysis

Transthyretin (prealbumin) amyloid:
● Amyloid composed of a mutant form of transthyretin (prealbumin)
● This type of amyloid is deposited in familial amyloid polyneuropathies and in the heart in senile systemic amyloidosis

Beta amyloid:
● Found in Alzheimer disease brain lesions

Amylin (islet amyloid polypeptide) amyloid:
● Found in pancreas of patients with type 2 diabetes

Clinical features
=========================================================================

● Factor X levels in amyloidosis typically range from 2% to 50%
● Bleeding symptoms usually do not arise unless Factor X levels fall below 10%
● Frequently presents as purpuric bleeding, typically occurs at pressure points
● Purpura may occur in periorbital regions (raccoon eyes) after minor trauma or valsalva maneuver
● Some patients with abnormal bleeding have no abnormality in any coagulation test (JAMA 1983;249:1322); amyloid infiltration of blood vessels may contribute to the bleeding diathesis
● Bleeding due to acquired von Willebrand disease has been described in AL amyloidosis (Am J Hematol 2007;82:363)
● An interaction between amyloid beta-peptide and tissue-type plasminogen activator also may contribute to the tendency to hemorrhage in cerebral amyloid angiopathy, as in vitro studies have shown that amyloid beta-peptide analogues markedly stimulate plasminogen activation by tissue-type plasminogen activator

Laboratory
=========================================================================

● Diagnosis of amyloidosis requires confirmation by tissue biopsy
● Fat pad aspiration biopsy has a low sensitivity for amyloidosis in patients with single organ involvement, but higher sensitivity in those with multiorgan involvement
● Because fat pad aspiration biopsy is less likely than liver, renal, or rectal biopsy to be complicated by serious bleeding, it is suggested for initial biopsy in patients with other than single organ involvement

Prognostic factors
=========================================================================

● Prognosis varies by etiology

Case reports
=========================================================================

● 20 year old woman with corpus luteum hemorrhage and hemoperitoneum (The Internet Journal of Anesthesiology 2007;13(2))
● 42 year old woman with easy bruising (ASH)

Treatment
=========================================================================

● Treatment is directed toward the underlying condition
● For dialysis related amyloidosis, altering the mode of dialysis or considering renal transplantation is recommended
● For the hereditary amyloidoses in which the mutant amyloid precursor protein is produced by the liver (eg: transthyretin, apolipoprotein A-I, and fibrinogen Aa), liver transplantation may prevent further deposition of amyloid and lead to regression of established deposits

Differential diagnosis
=========================================================================

● Thrombocytopenia (platelet count < 20,000/uL)
● Scurvy
● Hemophilias
● Vitamin K deficiency
● Warfarin ingestion
● Heparin
● Factitious purpura

Additional references
=========================================================================

Blood 2009;114:3147, eMedicine



Acquired bleeding disorders

Bovine coagulation factor inhibitors


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, M.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 17 August 2010, last major update August 2010
Copyright: (c) 2006-2010, PathologyOutlines.com, Inc.

Definition
========================================================================

● Antibodies that develop against bovine proteins after exposure to topical bovine thrombin preparations which can potentially cross-react with corresponding human coagulation factors
● Antibodies can form against bovine thrombin, factor V/Va, and fibrinogen, and less commonly against bovine factors VII and X
● Some fibrin glues contain bovine thrombin and cryoprecipitate (containing human fibrinogen)
● After use of bovine “fibrin glue” to achieve hemostasis, 1.7% develop a clinically significant inhibitor

Epidemiology
========================================================================

● One study reported 10% of patients exposed to bovine thrombin developed bovine thrombin antibodies; the rate was 5% for those singly exposed and 39% for those with multiple exposures
● The incidence of serious bleeds in bovine thrombin exposed patients was 1.7%, compared to 17% for those with bovine thrombin antibodies (Arch Pathol Lab Med 1998;122:887)
● One review found that 40-66% of cardiac surgery patients and 20% of neurosurgery patients developed factor V antibodies after bovine thrombin exposure (Transfusion 2002;42:18)

Clinical features
========================================================================

● Elevated PT and PTT (see below) that does not normalize with vitamin K or fresh frozen plasma (FFP) administration
● Patients may not have bleeding, but mucocutaneous and surgical site bleeding, retroperitoneal hematoma, petechiae and hematuria have been reported
● Recent review articles have concluded that there is insufficient evidence for a definitive association between elevations in anti-bovine antibodies and the development of clinically significant adverse events in surgical patients (Patient Saf Surg 2008;2:5, Clin Ther 2009;31:679, Curr Med Res Opin 2008;24:2071)

Laboratory
========================================================================

● Prolonged PT and PTT that does not correct with mixing studies

Case reports
========================================================================

● Case reports of 2 children who developed anti-factor V antibodies following bovine thrombin exposure (Pediatr Blood Cancer 2007;49:1025)

Treatment
========================================================================

● Steroids, cyclophosphamide, cyclosporine A, IV immunoglobulin
● Management of acute bleeding includes plasmapheresis, platelet-transfusions and immunoabsorption

Differential diagnosis
========================================================================

Dysfibrogenemia
Disseminated invtravascular coagulation (DIC)



Acquired bleeding disorders

Disseminated intravascular coagulation (DIC)


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, M.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 17 August 2010, last major update August 2010
Copyright: (c) 2006-2010, PathologyOutlines.com, Inc.

Definition and pathophysiology
========================================================================

● A common acquired syndrome arising from various causes (see etiology below), characterized by massive, sustained and excessive activation of coagulation with the eventual inundation (overwhelming) of the anticoagulant and fibrinolytic systems
● Leads to disseminated microthrombi and tissue ischemia; consumption of platelets, coagulation factors and natural anticoagulants; and variable bleeding
● Activation of inflammatory pathways via cytokines also plays a role
● Ultimately free, circulating, unopposed thrombin and plasmin are generated (the two key agents responsible for DIC) which then leads to:
- Activation and consumption of platelets, coagulation factors, fibrinogen and fibrin
- Consumption and depletion of anticoagulant proteins (protein C, protein S and antithrombin)
- Generation of D-dimers and fibrin degradation productsn
- Formation of microthrombi leading to tissue ischemia (large thrombi can also be formed, particularly in cancer patients)
- Schistocytes (fragmented red blood cells) are formed as red blood cells are severed flowing through fibrin strands (microthrombi within vasculature)
- Variable bleeding

Diagrams
========================================================================


   

Key events in DIC

Epidemiology
========================================================================

● 1% of hospitalized patients are estimated to develop DIC
● 20% of patients with Acute Respiratory Distress Syndrome (ARDS) develop DIC and 20% of patients with DIC develop ARDS
● 20% of patients with gram-negative sepsis develop DIC

Other causes
========================================================================

● Infection/sepsis: most common cause; includes bacterial, viral, fungal, rickettsial and protozoal organisms
● Tissue damage: trauma, burns
● Malignancy: solid and hematologic
● Obstetric complications: abruptio placentae, retained dead fetus syndrome, preeclampsia/eclampsia, amniotic fluid embolism, acute fatty liver of pregnancy, septic abortion
● Miscellaneous: near drowning, fat embolism, snake bites, aortic aneurism, acute hemolytic transfusion reaction, adult respiratory distress syndrome, giant hemangioma, homozygous protein C deficiency)

Clinical features
========================================================================

● Patients can have either bleeding or thrombosis or both
● Septic patients are more likely to have thrombosis than bleeding
● If severe and prolonged, will eventually lead to multiorgan dysfunction/failure
● Causes of DIC can be acute (meningococcemia) or chronic (retained dead fetus), localized (abdominal aortic aneurysm) or systemic (acute promyelocytic leukemia)
● Chronic causes of DIC are typically malignancy, liver disease, retained dead fetus syndrome, abdominal aortic aneurysm, giant hemangioma and head trauma
● Bleeding can present as surgical site, venipuncture site or mucocutaneous bleeding (most common); gastrointestinal bleeding, CNS bleeding, hematuria or ecchymoses
● Thrombosis can present as purpura fulminans (manifestation of subdermal microthrombi with skin necrosis); cold, pulseless limb; sudden loss of vision; oliguria; mental status changes, seizures, behavioral changes or adrenal insufficiency

Laboratory
========================================================================

● Prolonged PT and PTT
● Elevated D-dimers (Am J Clin Pathol 2004;122:178) and other fibrin degradation products (but D-dimer may be falsely positive in HIV+ Castleman’s disease due to interference from monoclonal gammopathy (Arch Pathol Lab Med 2004;128:328)
● Fall in platelet count (usually not lower than 30,000-40,000 x 109/L)
● Drop in fibrinogen
● Presence of schistocytes on peripheral blood smear (not specific for DIC)
● With chronic causes, fibrinogen and platelets may actually be elevated as acute phase reactants
● All coagulation factors may be variably decreased due to factor activation and consumption
● Multiorgan dysfunction may manifest as elevated cardiac enzymes or elevated BUN/creatinine
● Baseline coagulation studies and serial follow-up are needed to follow the trends

Prognostic factors
========================================================================

● One multicenter study of critically ill patients with DIC found that the 28 day mortality was 21.9%, which was significantly higher than non-DIC patients (11.2%) (Crit Care Med 2008;36:145)
● Another study found the mortality rate was significantly higher in sepsis patients than trauma patients (Thromb Haemost 2008;100:1099)

Case reports
========================================================================

● 30 year old woman with DIC due to amniotic fluid embolism (Arch Pathol Lab Med 2002;126:869)

Treatment
========================================================================

● Treat underlying disease
● Keep fibrinogen levels above 100 mg/dL with cryoprecipitate or fresh frozen plasma
● Monitor PT, PTT, platelet count, fibrinogen and possibly antithrombin levels
● If bleeding predominates, replace coagulation factors and fibrinogen with fresh frozen plasma (FFP) and cryoprecipitate; consider plasmapheresis, platelet-transfusions and immunoabsorption
● If platelet count is lower than 50,000 x 109/L with active bleeding, or lower than 10,000 x 109/L, give platelet infusion
● If thrombosis predominates (chronic DIC), heparinization should be considered

Micro images
========================================================================


Schistocyte

Amniotic fluid embolism - uterine wall - figure 1: amniotic fluid debris; figure 2: lanugo hair

Differential diagnosis
========================================================================

● Severe hepatic cirrhosis
● Dilutional coagulopathy (but may coexist with DIC)
● HELLP syndrome (H-hemolysis; EL-elevated liver enzymes; LP-low platelets); can also degenerate to DIC
● TTP/HUS



Acquired bleeding disorders

Factor V inhibitor


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, MD, University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 17 August 2010, last major update August 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition
=========================================================================

● An acquired alloantibody that develops against factor V that promotes either its increased clearance from the circulation or interference with its coagulation function
● Most are polyclonal IgG (which may also be associated with IgM or IgA)
● May behave like factor VIII inhibitor in mixing studies, with increasing PTT or PT after 1-2 hours

Epidemiology
=========================================================================

● 20% of cases are idiopathic
● In one study, 42% of cardiac surgery patients and 20% of neurosurgery patients developed factor V inhibitors after bovine thrombin exposure (Transfusion 2002;42:18)

Sites
=========================================================================

● Mucocutaneous and surgical site bleeding are the most common
● Hematuria, gastrointestinal bleeding, intracranial bleeding and hemospermia have also been observed

Etiology
=========================================================================

● Most cases of acquired factor V inhibitors are iatrogenic and are caused by exposure to bovine protein (i.e. bovine thrombin preparations)
● Antibodies are generated as an immune response to bovine factor V which then cross-reacts with endogenous factor V
● Other risk factors associated with factor V inhibitor development include recent surgery, drug exposure (e.g. aminoglycosides, B-lactam antibiotics), infections, blood transfusions, autoimmune disorders, malignancy and pregnancy

Clinical features
=========================================================================

● Highly variable
● Bleeding is typically severe and frequently fatal, however, in some instances there may be little to no bleeding
● Inhibitors usually manifest 7-10 days postoperatively and then disappear within 8 to 10 weeks; non-iatrogenic inhibitors may persist for significantly longer periods
● The titer of the inhibitor correlates with clinical severity
● Bovine thrombin associated factor V inhibitors are often accompanied by antibodies to other coagulation proteins such as fibrinogen, prothrombin and thrombin

Laboratory
=========================================================================

● Prolongation of PT and PTT with failure to correct with mixing studies
● A nonlinear curve in a factor assay is often a clue to the presence of an inhibitor
● Thrombin times may be prolonged if thrombin inhibitor is also present
● The Bethesda assay is performed to detect and quantitate the presence of inhibitor by diluting inhibitor patient plasma with pooled normal plasma
● Each Bethesda unit indicates a decrease of factor V concentration in the assay by 50% (1 unit: reduction from 100% to 50%; 2 units: to 25%; 3 units: to 12.5%, etc.)
● The Nijmegen assay is also used (Thromb Haemost 1995;73:247)

Prognostic factors
=========================================================================

● Non-iatrogenic inhibitors are associated with more severe bleeding and fatal outcomes

Case reports
=========================================================================

● Factor V inhibitor associated with cold agglutinin disease (Ann Hematol 1998;76:49)

Treatment
=========================================================================

● Steroids, cyclophosphamide, cyclosporine A, IV immunoglobulin
● Management of acute bleeding includes plasmapheresis, platelet transfusions and immunoabsorption

Differential diagnosis
=========================================================================

● May behave like factor VIII inhibitor in mixing studies, with increasing PTT or PT after 1-2 hours
● Combined factor V-factor VIII deficiency

Additional references
=========================================================================

Clin Appl Thromb Hemost 2006;12:485



Acquired bleeding disorders

Factor VIII inhibitor


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, MD, University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 9 April 2013, last major update June 2010
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

Definition
=========================================================================

● An acquired anti-factor VIII alloantibody ("antibody produced by one individual that reacts with alloantigens of another individual of the same species") that develops following infusion of factor VIII concentrates (plasma derived and recombinant) causing either increased clearance from the circulation or interference with coagulation function (e.g. inhibition of interaction of factor VIII with phospholipid, etc.)

Terminology
=========================================================================

When challenged with factor VIII concentrate, inhibitor patients are either low or high responders:
● Low responders are patients who develop a low titer of inhibitor (<5 Bethesda Units/BU); 50% of inhibitors are low titer and transient
● High responders are patients who develop a high titer of inhibitor (>5 BU)

● "Bypassing agents" are coagulation factor treatment products that do not contain factor VIII

Epidemiology
=========================================================================

● Develops in 10-20% of patients with severe hemophilia A after infusion of factor VIII containing products, less often with mild/moderate disease
● Rarely occurs de novo in patients without hereditary hemophilia, causing acquired hemophilia A (click here for topic)

Risk factors for development of inhibitors:
● Specific factor VIII genotype (i.e major deletions or rearrangements have higher risk than those with small deletions or missense mutations)
● Increased severity of hemophilia A (most likely due to more aggressive treatment)
● Younger age
● Race (blacks and Hispanics more affected than whites)
● Family history of factor VIII inhibitors

Sites
=========================================================================

● Typically associated with intra-articular and soft tissue bleeding, similar to hemophilia B

Etiology
=========================================================================

● Multiple genetic and environmental factors (see above)

Clinical features
=========================================================================

● Occurs after at least one infusion of factor concentrate, at a median of 9-12 exposures
● Are predominantly polyclonal IgG, but isolated instances of IgM and IgA have been reported
● Titer of inhibitor often increases after treatment with factor VIII containing products - this does not happen with autoimmune factor VIII inhibitor
● The presence of an inhibitor should be suspected when a hemophilia patient shows a decreased response to replacement therapy (i.e. when a sufficient dose of factor concentrate does not control an acute bleeding episode)

Laboratory
=========================================================================

● Prolonged PTT that does not correct with mixing studies. Note: PTT may initially be normal and then increase after 1-2 hours incubation
● Normal PT
● A nonlinear curve in a factor assay is often a clue to the presence of an inhibitor
● The Bethesda assay is performed to detect and quantitate the presence of inhibitor by diluting inhibitor patient plasma with pooled normal plasma; each Bethesda unit indicates a decrease of factor VIII concentration in assay by 50% (1 unit: reduction from 100% to 50%; 2 units: to 25%; 3 units: to 12.5%, etc.)
● The Nijmegen assay is also used (Thromb Haemost 1995;73:247)

Prognostic factors
=========================================================================

● Patients with high-titer inhibitor (>5 BU) are less likely to respond to treatment
● Factor VIII genotypes that includes large gene deletions, inversions, nonsense and splice site mutations show less response to treatment (J Thromb Haemost 2009;7:1809)
● Data is inconclusive regarding relative inhibitor risk of plasma-derived vs. recombinant factor concentrates-both can lead to formation

Case reports
=========================================================================

● Acquired factor VIII inhibitor in association with myelodysplastic syndrome (Intern Med J 2009;39:e7)

Treatment
=========================================================================

● Acute bleeding episodes can be treated with high dose human factor VIII for low titer patients (to overwhelm inhibitor) or porcine factor VIII (if no cross reactivity with inhibitor)
● For high-titer inhibitors, factor VIII bypassing agents (prothrombin complex concentrates, FEIBA or recombinant factor VIIa (J Thromb Haemost 2004;2:899)
● FEIBA can also be used for minor or major surgical procedures (Haemophilia 2009;15:1300)
● Immunosuppression for autoimmune based inhibitors, with a possible role for plasmapheresis

Differential diagnosis
=========================================================================

● Occasionally a lupus-like anticoagulant can cause a false-positive inhibitor screen by prolonging the PTT and leading to a nonlinear curve in a factor assay

Additional references
=========================================================================

Am J Clin Pathol 2009;131:552, Clin Rev Allergy Immunol 2009;37:58, Blood 2009;113:11, eMedicine



Acquired bleeding disorders

Autoimmune based inhibitors in nonhemophiliac patients


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, MD, University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 9 April 2013, last major update June 2010
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

Definition
=========================================================================

● An acquired hemophilia that occurs after development of an autoantibody directed against a specific coagulation protein in patients with no prior coagulation defect

Epidemiology
=========================================================================

● Incidence of 0.2 to 1.0 per million per year
● Median age at presentation is 60-70 years and is seen equally in males and females

Etiology
=========================================================================

● Associated with autoimmune disorders, solid tumors, hematologic malignancies, dermatologic disorders, inflammatory bowel disease, respiratory diseases, diabetes, acute hepatitis (B and C), severe drug reactions, post-childbirth
● 50% occur in patients with no known medical problems

Clinical features
=========================================================================

● Most common inhibitor is anti-factor VIII (Semin Thromb Hemost 2009;35:760)
● Mortality rate of 8-22% with severe bleeding in up to 90% of affected individuals
● Patients present with soft tissue bleeding such as gastrointestinal, urinary tract or intramuscular (vs. intra-articular bleeding in hereditary hemophilia)
● An acquired hemophilia should be suspected in patients with a new onset bleeding disorder accompanied by an isolated prolonged PTT (Curr Gerontol Geriatr Res 2010;2010:927503)
● Patients should be referred to a hemophilia center with expertise in managing inhibitors (Haematologica 2009;94:566)
● Poor prognostic factors are advanced age and lack of treatment (Semin Thromb Hemost 2009;35:769)

Laboratory
=========================================================================

● Prolonged PTT that does not correct with mixing studies. Note: PTT may initially be normal and then increases after 1-2 hours incubation
● Normal PT
● A nonlinear curve in a factor assay is often a clue to the presence of an inhibitor
● The Bethesda assay is performed to detect and quantitate presence of inhibitor by diluting inhibitor patient plasma with pooled normal plasma; each Bethesda unit of inhibitor indicates a decrease of factor VIII concentration in assay by 50% (1 unit: a reduction from 100% to 50%; 2 units: to 25%; 3 units: to 12.5%, etc.)

Treatment
=========================================================================

● Prothrombin complex concentrates, recombinant factor VIIa, DDAVP, factor VIII concentrates, immunosuppressive agents (BMC Res Notes 2010;3:161), plasmapheresis (variable success)

Additional references
=========================================================================

Blood 2008;112:250, Arch Pathol Lab Med 2000;124:730



Acquired bleeding disorders

Factor IX inhibitor


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, M.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 17 August 2010, last major update August 2010
Copyright: (c) 2006-2010, PathologyOutlines.com, Inc.

Definition
========================================================================

● An acquired anti-factor IX alloantibody (“antibody produced by one individual that reacts with alloantigens of another individual of the same species”) that develops following infusion of factor IX concentrates (plasma derived and recombinant) causing either increased clearance from the circulation or interference with coagulation function (e.g. inhibition of interaction of factor IX with phospholipid, etc.)

Terminology
========================================================================

● When challenged with factor IX concentrate, inhibitor patients are either low or high responders
● Low responders are patients who develop a low titer of inhibitor (<5 Bethesda Units/BU); 50% of inhibitors are low titer and transient
● “Bypassing agents” are coagulation factor treatment products that do not contain factor IX

Epidemiology
========================================================================

● Develops in 1.5-3% of patients with severe hemophilia B after transfusion of factor IX containing products, less commonly with mild/moderate disease
● The lower inhibitor rate in hemophilia B is due to the lower proportion of severe cases of hemophilia B (~30%) compared with hemophilia A (~60%)
● Rarely arises in non-hemophilia patients with autoimmune disorders causing acquired hemophilia B

Risk factors for development of inhibitors:
● Specific factor IX genotype (i.e. major deletions or nonsense mutations have higher risk than those with small deletions or missense mutations)
● Increased severity of hemophilia B (most likely due to more aggressive treatment)
● Younger age
● Race (particularly individuals of Scandinavian descent)
● Family history of factor IX inhibitors

Sites
========================================================================

● Typically associated with intra-articular and soft tissue bleeding, similar to hemophilia A

Etiology
========================================================================

● Multiple genetic and environmental factors (see above)

Clinical features
========================================================================

● Occurs after at least one infusion of factor concentrate, at a median of 9-12 exposures
● Antibodies are predominantly polyclonal IgG
● Titer of inhibitor often increases after treatment with factor IX containing products
● The presence of an inhibitor should be suspected when a hemophilia patient shows a decreased response to replacement therapy (i.e. when a sufficient dose of factor concentrate does not control an acute bleeding episode)
● With the development of inhibitors, some patients may experience allergic or anaphylactic reactions following exposure to concentrate

Laboratory
========================================================================

● Prolonged PTT that does not correct with mixing studies
● Note: with factor VIII inhibitors, the PTT may initially be normal and then increase after 1-2 hours incubation; in contrast, factor IX inhibitors immediately inactivate factor IX activity and therefore do not require prolonged incubation
● Normal PT
● A nonlinear curve in a factor assay is often a clue to the presence of an inhibitor
● The Bethesda assay is performed to detect and quantitate the presence of inhibitor by diluting inhibitor patient plasma with pooled normal plasma
● Each Bethesda unit indicates a decrease of factor IX concentration in assay by 50% (1 unit: reduction from 100% to 50%; 2 units: to 25%; 3 units: to 12.5%, etc.)
● The Nijmegen assay is also used (Thromb Haemost 1995;73:247)

Prognostic factors
========================================================================

● Patients with a high titer of inhibitor (>5 BU) are less likely to respond to treatment
● Data is inconclusive regarding the relative inhibitor risk of plasma-derived vs. recombinant factor concentrates - both can lead to formation of inhibitors

Case reports
========================================================================

● Development of an IgA factor IX inhibitor (Am J Hematol 1984;17:321)

Treatment
========================================================================

● Treatment is primarily supportive; aggressive treatment should be aimed at correcting the underlying cause
● Acute bleeding episodes can be treated with high dose human factor IX for low titer patients (to overwhelm the inhibitor) or porcine factor IX (if no cross reactivity with inhibitor)
● For high titer inhibitors, factor IX bypassing agents (prothrombin complex concentrates, FEIBA or recombinant factor VIIa (J Thromb Haemost 2004;2:899)
● FEIBA can also be used for minor or major surgical procedures (Haemophilia 2009;15:1300)
● For autoimmune based inhibitors, use immunosuppression and possibly plasmapheresis

Differential diagnosis
========================================================================

● Occasionally a lupus-like anticoagulant can cause a false-positive inhibitor screen by prolonging the PTT and leading to a nonlinear curve in a factor assay

Additional references
========================================================================

Semin Thromb Hemost 2009;35:760



Acquired bleeding disorders

Liver dysfunction


Reviewer: Nat Pernick, M.D. (see Reviewers page)
Revised: 23 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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; 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



Coagulation laboratory tests

Lupus anticoagulant


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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 (Arch Pathol Lab Med 2002;126:1424, Kelley's Textbook of Rheumatology (9th ed, 2012))
● 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, Arch Pathol Lab Med 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) and (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 6-12 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 and other coagulopathies
● Values prolonged by bivalirudin, lepirudin, argatroban and fondaparinux (Arch Pathol Lab Med 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)



Acquired bleeding disorders

Proteinuria


Reviewer: Nat Pernick, M.D. (see Reviewers page)
Revised: 23 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Proteinuric patients are in a more prothrombotic state than healthy controls, but antiproteinuric therapy ameliorates the prothrombotic state (J Thromb Haemost 2011;9:2416)
● Patients with nephrotic syndrome may have decreased factors XI and XII



Acquired bleeding disorders

Vitamin K deficiency / warfarin use


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, MD, University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 10 March 2011, last major update March 2011
Copyright: (c) 2002-2011, PathologyOutlines.com, Inc.

Definition
=========================================================================

● Hemorrhage is a complication of both warfarin therapy and vitamin K deficiency (see etiology below)

Warfarin (Coumadin):
● The most widely prescribed therapeutic anticoagulant for the prevention or treatment of venous thrombosis, pulmonary embolism, and thromboembolic complications
● Its mechanism of action relates to its ability to impair the regeneration of the active form of vitamin K, which is required in the carboxylation of glutamic acid residues of coagulation factors II, VII, IX and X (as well as anticoagulant proteins C and S)

Vitamin K:
● A fat-soluble vitamin that participates as a cofactor in carboxylation of glutamic acid residues of factors II, VII, IX and X and proteins C and S

Terminology
=========================================================================

● The International Normalized Ratio (INR) is defined as the ratio of a patient’s PT to the control sample (normal) PT raised to the ISI value of the control sample:




Each manufacturer assigns an ISI value (International Sensitivity Index) for any tissue factor they manufacture. The ISI value indicates how the particular batch of tissue factor compares to an internationally standardized sample

The INR was developed to standardize results due to differences in tissue factor between manufacturers that can lead to differences in PT values

Epidemiology
=========================================================================

● The incidence of major bleeding events in patients prescribed warfarin is approximately 2-13%
● The incidence of vitamin K deficiency bleeding in newborns (hemorrhagic disease of the newborn) is reported between 1.6-8.6 per 100,000 live births

Sites
=========================================================================

● Skin and mucosa, soft tissue, genitourinary tract, gastrointestinal tract, CNS, respiratory tract, abdomen (see clinical features below)

Etiology
=========================================================================

Warfarin-related bleeding
● There is significant inter-individual variability in the dosing of warfarin that is a result of many variables including genetic factors [due to polymorphisms in the genes that encode hepatic cytochrome P450 enzyme (CYP2C9) and vitamin K epoxide reductase (VKORC1)], drug-drug interactions, specific disease states (e.g. hypo/hyperthyroidism) and dietary vitamin K intake that can lead to increased risk of bleeding complications

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

Clinical features
=========================================================================

● Easy bruising, soft tissue hematoma, gastrointestinal bleeding, epistaxis, intracranial bleeding, hematuria, hemoptysis, intraperitoneal and retroperitoneal bleeding
● Bleeding in vitamin K deficiency can include the above as well as bleeding after circumcision and umbilical cord stump bleeding

Laboratory
=========================================================================

● Both warfarin administration and vitamin K deficiency cause prolonged PT (severe cases of vitamin K deficiency can also result in prolonged PTT)
● Anticoagulant effect of warfarin is monitored by PT/INR

Prognostic factors
=========================================================================

● Risk factors associated with hemorrhagic complications of warfarin include age (>65 yrs), concomitant comorbidities (e.g. atrial fibrillation, gastrointestinal bleeding, renal insufficiency, cerebrovascular disease), concomitant medications (e.g. aspirin, amiodarone)

Case reports
=========================================================================

● Spontaneous supraglottic hemorrhage in a patient on warfarin therapy (Emerg Med J 2001;18:406)
● Vitamin K deficiency presenting as impending brain herniation in a 2 month-old male infant (J Pediatr Neurosci 2010;5:55)

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)

Differential diagnosis
=========================================================================

● Liver disease
● DIC

Additional references
=========================================================================

Am J Clin Pathol 2008;129:876, Discov Med 2009;8:196. Thromb Res 2008;122 Suppl 2:S13, J Thromb Thrombolysis 2008;25:151



Hereditary bleeding disorders

Hereditary bleeding disorders - general


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, MD, University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 9 April 2013, last major update September 2010
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

Definition
=========================================================================

● Hereditary bleeding disorders are a diverse group of diseases that include abnormalities of primary and secondary hemostasis

The most common congenital bleeding disorders include:
● von Willebrand disease
● Hemophilia A (factor VIII deficiency)
● Hemophilia B (factor IX deficiency)

Less common congenital bleeding disorders include:
● Factor I (fibrinogen) deficiency
● Factor II (prothrombin) deficiency
● Factor V deficiency
● Factor VII deficiency
● Factor X deficiency
● Factor XI deficiency
● Factor XIII deficiency
● Platelet disorders

Extremely rare disorders include:
● α2-antiplasmin deficiency
● α1-antitrypsin Pittsburgh (Antithrombin III Pittsburgh) deficiency (Haematologica 2009;94:881)
● Combined factor deficiencies: 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)

Epidemiology
=========================================================================


Bleeding disorder Prevalence Inheritance pattern
Factor I (fibrinogen) deficiency

● Afibrinogenemia

● Hypofibrinogenemia

● Dysfibrinogenemia
More than 200 cases reported








Autosomal recessive

Autosomal dominant or recessive

Autosomal dominant or recessive
Factor II (prothrombin) deficiency Less than 100 cases reported Autosomal recessive
Factor V deficiency Less than 1 in 1,000,000 Autosomal recessive
Factor VII deficiency 1 in 500,000 Autosomal recessive
Factor VIII deficiency 1 in 5000 male births X-linked recessive
Factor IX deficiency 1 in 30,000 male births X-linked recessive
Factor X deficiency 1 in 500,000 Autosomal recessive
Factor XI deficiency 4% in Ashkenazi Jews, otherwise rare Autosomal recessive
Factor XIII deficiency More than 200 cases reported Autosomal recessive
Combined factor deficiencies

●Factor V-Factor VIII

● Factor II, VII, IX, X


> 30 families reported

< 15 families reported


Autosomal recessive

Autosomal recessive
a2-antiplasmin deficiency > 10 families reported Autosomal recessive
a1-antitrypsin Pittsburgh deficiency Only 3 cases reported Autosomal dominant
von Willebrand Disease (VWD)

● Type I

● Type II

● Type III
1 in 100








Autosomal dominant

Autosomal dominant

Autosomal recessive
Glanzmann thrombasthenia 1 in 1,000,000 Autosomal recessive
Bernard-Soulier syndrome < 1 in 1,000,000 Autosomal recessive
Gray platelet syndrome Rare Autosomal dominant, recessive or X-linked recessive
Wiskott-Aldrich syndrome 1 in 1,000,000 X-linked recessive

Etiology
=========================================================================

● Primary hemostasis involves formation of the platelet plug which involves platelets, the blood vessel wall and von Willebrand factor; abnormalities can include problems in platelet number, adhesion or aggregation
● Secondary hemostasis involves the formation of fibrin through the humoral coagulation cascade; abnormalities include deficiencies of coagulation factors or contact factors, deficiencies or abnormalities of fibrinogen or connective tissue diseases
● Mutations can be inherited in an autosomal dominant, recessive or X-linked pattern

Disorders of Primary Hemostasis
von Willebrand disease
Glanzmann thrombasthenia
Bernard-Soulier syndrome
Platelet storage pool disease
Gray platelet syndrome
Wiskott-Aldrich syndrome

Disorders of Secondary Hemostasis
Factor I (fibrinogen) abnormalities

● Afibrinogenemia

● Hypofibrinogenemia

● Dysfibrinogenemia
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 XIII deficiency
Combined factor deficiencies
a2-antiplasmin deficiency
a1-antitrypsin deficiency
Ehlers-Danlos syndrome
Osler-Weber-Rendu syndrome
Scurvy (vitamin C deficiency)

Clinical features
=========================================================================

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, hematuria
● 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

Laboratory
=========================================================================

● Basic screening tests include CBC, PT/PTT, bleeding time or platelet function assay (e.g. PFA-100), thrombin time, peripheral blood smear review (for platelet and erythrocyte morphology), fibrinogen
● Testing for vWD includes Factor VIII activity, vWF antigen, vWF activity (often done by the "ristocetin cofactor" method)
● These results may lead to obtaining vWF multimer assays and blood type (type O patients have reduced vWF activity)
● For suspected coagulation factor abnormalities: mixing studies, factor levels, Bethesda assay (to detect coagulation factor inhibitors); can confirm hereditary deficiency by determining factor levels in relatives
● For suspected platelet disorders: platelet aggregation studies, bone marrow aspirate and biopsy, platelet-associated immunoglobulin levels
● Factor XIII assay if delayed bleeding is present (often done by "urea clot lysis" method)
● More esoteric assays include PAI-1 activity and antiplasmin

Treatment
=========================================================================

● Specific treatment recommendations are dependent on type and severity of bleeding disorder, but generally factor replacement therapy for factor deficiencies is the mainstay of treatment with the exception of factor II, factor V and factor X deficiencies, which are treated with FFP and cryoprecipitate (Haemophilia 2008;14:671)
● For vVWD, use DDAVP (desmopressin), vWF concentrates and antifibrinolytic agents
● For platelet-related bleeding disorders, use platelet transfusions and recombinant factor VIIa

Differential diagnosis
=========================================================================

● 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
● Acquired platelet defects due to anti-platelet medications (aspirin, glycoprotein IIB/IIIA inhibitors, clopidogrel, ticlopidine) are much more common than inherited platelet abnormalities

Additional references
=========================================================================

Consultative Hemostasis and Thrombosis: Elsevier, 2007



Hereditary bleeding disorders

Algorithm for workup of hereditary bleeding disorders


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, MD, University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 9 April 2013, last major update September 2010
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Hereditary bleeding disorders are a diverse group of diseases that include abnormalities of primary and secondary hemostasis (see etiology)
● Evaluation of a patient for a hereditary bleeding disorder is a multistep process that involves a complete and accurate history followed by laboratory evaluation
● Important considerations are whether the bleeding is truly congenital vs. a possible acquired coagulopathy, and whether the clinical symptoms suggest a disorder of primary vs. secondary hemostasis (see clinical features)
● Causes of acquired bleeding include liver disease, renal disease, vitamin K related factor deficiency (warfarin, prolonged antibiotic use, malabsorption syndromes, nutritional deficiency), DIC and dilutional coagulopathy in massive transfusion

A complete history should include:
● Surgical history: dental procedures, bleeding during surgery vs. postoperative bleeding
● Nature of bleeding: habitual epistaxis, menorrhagia, hemarthrosis, postpartum hemorrhage, petechiae, purpura, etc.
● For patients with menorrhagia: history of severe iron deficiency anemia, multiple red blood cell transfusions, procedures such as D&C or hysterectomy for excessive bleeding
● Family history of bleeding (but a negative family history of bleeding does not rule out a congenital bleeding disorder)
● Medication use: warfarin, heparin, aspirin or other NSAIDs, antibiotics (affecting vitamin K dependent clotting factors) and herbal medications
● Medical problems: renal failure, severe liver disease, malabsorption syndromes

Etiology / Pathophysiology
=========================================================================

● Primary hemostasis involves formation of the platelet plug, which involves platelets, the blood vessel wall and von Willebrand factor
● Abnormalities can include problems in platelet number, adhesion or aggregation
● Secondary hemostasis involves the formation of fibrin through the humoral coagulation cascade
● Abnormalities include deficiencies of coagulation factors or contact factors, deficiencies or abnormalities of fibrinogen or connective tissue diseases
● Mutations can be inherited in an autosomal dominant, recessive or X-linked pattern

Tables
=========================================================================


Bleeding disorder Prevalence Inheritance pattern
Factor I (fibrinogen) deficiency

● Afibrinogenemia

● Hypofibrinogenemia

● Dysfibrinogenemia
More than 200 cases reported








Autosomal recessive

Autosomal dominant or recessive

Autosomal dominant or recessive
Factor II (prothrombin) deficiency Less than 100 cases reported Autosomal recessive
Factor V deficiency Less than 1 in 1,000,000 Autosomal recessive
Factor VII deficiency 1 in 500,000 Autosomal recessive
Factor VIII deficiency 1 in 5000 male births X-linked recessive
Factor IX deficiency 1 in 30,000 male births X-linked recessive
Factor X deficiency 1 in 500,000 Autosomal recessive
Factor XI deficiency 4% in Ashkenazi Jews, otherwise rare Autosomal recessive
Factor XIII deficiency More than 200 cases reported Autosomal recessive
Combined factor deficiencies

●Factor V-Factor VIII

● Factor II, VII, IX, X


> 30 families reported

< 15 families reported


Autosomal recessive

Autosomal recessive
a2-antiplasmin deficiency > 10 families reported Autosomal recessive
a1-antitrypsin Pittsburgh deficiency Only 3 cases reported Autosomal dominant
von Willebrand Disease (VWD)

● Type I

● Type II

● Type III
1 in 100








Autosomal dominant

Autosomal dominant

Autosomal recessive
Glanzmann thrombasthenia 1 in 1,000,000 Autosomal recessive
Bernard-Soulier syndrome < 1 in 1,000,000 Autosomal recessive
Gray platelet syndrome Rare Autosomal dominant, recessive or X-linked recessive
Wiskott-Aldrich syndrome 1 in 1,000,000 X-linked recessive

Disorders of Primary Hemostasis
von Willebrand disease
Glanzmann thrombasthenia
Bernard-Soulier syndrome
Platelet storage pool disease
Gray platelet syndrome
Wiskott-Aldrich syndrome

Disorders of Secondary Hemostasis
Factor I (fibrinogen) abnormalities

● Afibrinogenemia

● Hypofibrinogenemia

● Dysfibrinogenemia
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 XIII deficiency
Combined factor deficiencies
a2-antiplasmin deficiency
a1-antitrypsin deficiency
Ehlers-Danlos syndrome
Osler-Weber-Rendu syndrome
Scurvy (vitamin C deficiency)

Clinical features
=========================================================================

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, hematuria
Note: Pregnant women with mild to moderate vWD (von Willebrand disease) or Hemophilia A carriers typically are asymptomatic due to elevated vWF and Factor VIII during pregnancy

Some symptoms are more suggestive of either a primary or secondary hemostatic defect and can help narrow the differential diagnosis:
● Epistaxis, menorrhagia, melena, intraoperative or immediate postoperative bleeding and petechiae suggest a platelet disorder or vWD
● Delayed postoperative bleeding is usually due to a coagulation factor deficiency, fibrinogen abnormalities or a collagen disorder
● Spontaneous hemarthrosis and intramuscular bleeds suggest coagulation factor deficiencies
Note: hemarthrosis is common in classic hemophilias but also can occur in acquired hemophilias

Laboratory
=========================================================================

● Basic screening tests include CBC, PT/PTT, bleeding time or platelet function assay (e.g. PFA-100), thrombin time, peripheral blood smear review (for platelet and erythrocyte morphology), fibrinogen
● Testing for vWD includes Factor VIII activity, vWF antigen, vWF activity (often done by the "ristocetin cofactor" method)
● These results may lead to ordering vWF multimer assays and blood type determination (type O patients have reduced vWF activity)
● For suspected coagulation factor abnormalities: mixing studies, factor levels, Bethesda assay (to detect coagulation factor inhibitors); can confirm hereditary deficiency by determining factor levels in relatives
● For suspected platelet disorders: platelet aggregation studies, bone marrow aspirate and biopsy, platelet-associated immunoglobulin levels
● Perform Factor XIII assay if delayed bleeding is present (often done by "urea clot lysis" method)
● More esoteric assays include PAI-1 activity and antiplasmin

● Note: lupus anticoagulants can cause prolongation of PTT, but are associated with thrombosis, not bleeding
● Deficiencies of Factor XII, prekallikrein or high molecular weight kininogen do not cause bleeding but cause prolongation of PTT

Prognostic factors
=========================================================================

● Heterozygous patients have 30-60% of normal values of affected factors, usually with no or minor bleeding disorder
● 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

Case reports
=========================================================================

● Bleeding due to familial platelet cyclo-oxygenase deficiency (Thromb Res 2005;116:483)

Treatment
=========================================================================

● Specific treatment recommendations depend on the type and severity of bleeding disorder, but in general, factor replacement therapy for factor deficiencies is the mainstay of treatment, with the exception of factor II, factor V and factor deficiencies; FFP and cryoprecipitate are also common (Haemophilia 2008;14:671)
● For von Willebrand disease: DDAVP (desmopressin), vWF concentrates, antifibrinolytic agents
● For platelet-related bleeding disorders: platelet transfusion, recombinant factor VIIa

Differential diagnosis
=========================================================================

● Acquired factor deficiencies: are due to liver disease, DIC, lupus anticoagulants, heparin, warfarin or other anticoagulants, inhibitors; are more common than hereditary factor deficiencies, and should be ruled out first
● Acquired platelet defects due to anti-platelet medications (aspirin, glycoprotein IIB/IIIA inhibitors, clopidogrel, ticlopidine) are much more common than inherited platelet abnormalities

Additional references
=========================================================================

Consultative Hemostasis and Thrombosis: Elsevier, 2007, Clin Lab Haematol 2000;22 Suppl 1:91



Hereditary bleeding disorders

Factor I (fibrinogen) deficiency


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 15 October 2010, last major update September 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition / General
=========================================================================

● Factor I (fibrinogen) deficiency is an inherited bleeding disorder that is characterized by a variable bleeding diathesis or paradoxically by thrombosis
● Deficiencies are classified as quantitative (afibrinogenemia and hypofibrinogenemia) or qualitative (dysfibrinogenemia) defects of the fibrinogen molecule, or both (hypodysfibrinogenemia)
● Fibrinogen is a soluble glycoprotein that is converted to fibrin by thrombin; it is synthesized mainly in the liver as a hexamer comprised of two sets of three different chains
● Normal fibrinogen levels are 1.5–3.5 g/L, with a half-life of 2-4 days

Terminology
=========================================================================

● Afibrinogenemia is the most severe form of fibrinogen deficiency; patients have no detectable circulating fibrinogen in the plasma or platelets
● Hypofibrinogenemia is a milder form of fibrinogen deficiency; patients have fibrinogen levels < 1.5 g/L
● Dysfibrinogenemia is the synthesis of abnormal fibrinogen molecules
● Hypodysfibrinogenemia is characterized by both low levels of fibrinogen and abnormal fibrinogen molecules

Epidemiology
=========================================================================

● Estimated prevalence of afibrinogenemia is 1 in 1,000,000
● More than 400 cases of dysfibrinogenemia have been reported
● Hypofibrinogenemia and dysfibrinogenemia are more frequent than afibrinogenemia, but prevalence is difficult to establish due to the large number of asymptomatic patients

Sites
=========================================================================

● Bleeding of skin and mucosa, joint and muscle, genitourinary tract, gastrointestinal tract, CNS (see clinical features below)

Etiology
=========================================================================

● Encoded by FGA, FGB and FGG on chromosome 4 (Wikipedia)
● Afibrinogenemia is inherited in an autosomal recessive pattern
● Hypofibrinogenemia is inherited in an autosomal dominant or recessive pattern; mutations in all three genes have been described, and include deletions, frameshift, nonsense or splicing mutations; these mutations can lead to problems that affect fibrinogen synthesis, assembly, intracellular processing (with or without endoplasmic retention), domain stability and protein secretion
● Dysfibrinogenemia is inherited in an autosomal dominant or autosomal recessive pattern; most patients are heterozygous for missense mutations in one of the three fibrinogen genes, which can cause absent or delayed release of Aa and Bb, delayed or enhanced polymerization, defective cross-linking, decreased thrombin binding and defective assembly of the fibrinolytic system

Clinical features
=========================================================================

● Afibrinogenemia is typically diagnosed in the neonatal period due to umbilical cord stump bleeding or bleeding after circumcision; patients can have bleeding or thrombosis
● Hypofibrinogenemia is associated with milder bleeding episodes, usually due to trauma or surgical challenge; patients are usually asymptomatic
● Dysfibrinogenemia is usually diagnosed in adulthood; patients can be asymptomatic, have bleeding or thrombosis or both
● Bleeding symptoms can include umbilical cord stump bleeding, bleeding after circumcision, easy bruising, mucosal bleeding, GI/GU bleeding, hemarthrosis, intracranial hemorrhage, recurrent fetal loss, menorrhagia, menometrorrhagia, placental abruption or postpartum hemorrhage; rarely patients can have hemopericardium, hemoperitoneum or spontaneous splenic rupture
● Arterial and venous thrombosis may occur

Laboratory
=========================================================================

● Prolonged PT/PTT that corrects with mixing study (may not correct with mixing study in patients with dysfibrinogenemia)
● Prolonged thrombin time
● Low or absent fibrinogen; levels may be normal in dysfibrinogenemia
● Prolonged reptilase time
● Specific fibrinogen assays including clotting and immunologic assays
● Mild thrombocytopenia has been reported in 25% of patients with afibrinogenemia

Case reports
=========================================================================

● Patient with congenital afibrinogenemia with retrochorionic hematoma during pregnancy (Am J Hematol 2007;82:317)

Treatment
=========================================================================

● Use cryoprecipiate to keep level at 50-100 mg/dL in acute bleeding episodes, preoperative state or during pregnancy
● FFP or fibrinogen concentrates may also be used

Differential diagnosis
=========================================================================

● Acquired fibrinogen deficiency (DIC, liver disease, medication use, autoimmune disease)

Additional references
=========================================================================

Semin Thromb Hemost 2009; 35;356, Haemophilia 2008;14:1151, Consultative Hemostasis and Thrombosis: Elsevier, 2007



Hereditary bleeding disorders

Factor II (prothrombin) deficiency


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, M.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 23 August 2010, last major update August 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition
=========================================================================

● Congenital deficiency of prothrombin (factor II) that results in reduced prothrombin activity and normal or reduced prothrombin antigen levels
● Prothrombin (factor II) is a vitamin-K dependent cofactor which is activated by factor Xa to form thrombin, which then converts fibrinogen to fibrin


Coagulation cascade

Terminology
=========================================================================

● Hypoprothrombinemia (type I deficiency) is a decrease in the overall synthesis of prothrombin and is characterized by reduced prothrombin activity and antigen levels
● Dysprothrombinemia (type II deficiency) is the synthesis of dysfunctional prothrombin molecules and is characterized by reduced activity and normal antigen levels

Epidemiology
=========================================================================

● Rare; fewer than 100 cases have been reported
● Prevalence rate of approximately 1:2,000,000 in the general population

Sites
=========================================================================

● Mucocutaneous and soft tissue bleeding (see below)

Etiology
=========================================================================

● Forty different mutations in the prothrombin gene have been identified, the majority of which are missense mutations (~80%). However, insertion or deletion mutations (~10%) and nonsense mutations (~4%) have also been identified.
● The gene is transmitted in an autosomal recessive pattern with homozygous, heterozygous or compound heterozygous genotypes
● Homozygous individuals have functional prothrombin levels of 2-25%
● Heterozygous patients have prothrombin levels of 40-60%
● Biologic half life is 48-120 hours

Clinical features
=========================================================================

● Complete deficiency is incompatible with life
● Severe hemorrhage typically occurs when levels are < 5%
● Need levels of 10-40% for surgical hemostasis
● Patients have lifelong history or family history of bleeding; varies from asymptomatic to minor bleeding to severe bleeding (depending on the mutation)
● Also easy bruising, postoperative bleeding, epistaxis, menorrhagia, miscarriage, postpartum hemorrhage, hemarthroses and intracranial bleeding (if severe deficiency)

Laboratory
=========================================================================

● Prolonged PT and PTT that correct with mixing study (1:1 mixture of patient and normal pooled plasma)
● Type I (true deficiency) patients will have both activity and antigen levels <10%
● Type II patients will have normal antigen levels and low activity

Treatment
=========================================================================

● Recombinant factor VIIa, alternatively 10-20 ml fresh frozen plasma/kg, then 3 ml/kg every 12-24 hours as necessary
● Plasma infusion for recurrent bleeding episodes every 3-5 weeks
● Prothrombin complex concentrates may be used for serious bleeding

Differential diagnosis
=========================================================================

● Must rule out other coagulation factor deficiencies that cause prolonged PT and PTT (e.g. factor V deficiency or factor X deficiency)
● Acquired prothrombin deficiency due to severe liver disease, vitamin K deficiency or anti-prothrombin antibodies (antiphospholipid syndrome)

Additional references
=========================================================================

Semin Thromb Hemost 2009;35:367, Consultative Hemostasis and Thrombosis (2007)



Hereditary bleeding disorders

Factor V deficiency


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, M.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 20 September 2010, last major update August 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition / General
=========================================================================

● Factor V deficiency is a rare congenital bleeding disorder that is inherited as an autosomal recessive trait and is characterized by decreased or absent factor V activity

Terminology
=========================================================================

● Also called autoprothrombin I deficiency, Owren’s disease, labile factor deficiency or parahemophilia (severe deficiency)
● Deficiency classified as either:
  - Type I: both reduced antigen level and activity (quantitative defect)
  - Type II: normal or mildly reduced antigen level and reduced activity (qualitative defect)

Epidemiology
=========================================================================

● Rare, incidence of less than 1 per 1,000,000 in the homozygous form
● Over 200 cases have been described in the literature

Sites
=========================================================================

● Autosomal recessive trait with variable heterozygote expressivity
● There have been 56 mutations described including insertion/deletion, missense, splice site and nonsense mutations (in order of decreasing frequency)
● Parental consanguinity is often present, especially in countries (Muslim or southern India) where consanguineous marriages are common

Pathophysiology
=========================================================================

● Factor V is a plasma glycoprotein that is synthesized in the liver and is also present in platelet a-granules
● Platelet factor V accounts for approximately 20% of the total body pool of factor V
● Factor V is a plasma cofactor for the prothrombinase complex that converts prothrombin to thrombin
● Deficiency leads to predisposition for hemorrhage, while some mutations (most notably factor V Leiden) predispose to thrombosis

Clinical features
=========================================================================

● 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; however hemarthroses are not as common
● There are mild, moderate and severe forms
● Patients with mild to moderate deficiency (>20-30%) have a heterozygous genotype and are usually asymptomatic and may not be diagnosed until adulthood
● Patients with severe deficiency (<10%) have either homozygous or compound heterozygous genotype and typically present within the first 6 months of life
● Levels don’t always correlate with severity of symptoms
● Complications of treatment include the development of factor V alloantibodies (inhibitors)

Laboratory
=========================================================================

● Prolonged PT/PTT with correction after mixing study with normal pooled plasma
● Normal thrombin time
● Specific clot-based factor V assay for diagnosis

Prognostic factors
=========================================================================

● Prognosis is good for most patients with factor V deficiency
● Most severe cases have been in patients who present in the perinatal period with intracranial bleeding

Case reports
=========================================================================

● Severe gestational factor V deficiency presenting with intracranial hemorrhage detected by ultrasound (Haemophilia 2007;13:432)

Treatment
=========================================================================

● Since there are no factor V concentrates available, fresh frozen plasma (FFP) is the mainstay of treatment (15 to 20 mL/kg followed by smaller amounts, such as 5 mL/kg every 12 hours, adjusting the dosage on the basis of Factor V levels, PT and APTT)
● Cryoprecipitate and prothrombin complex concentrates do not contain factor V
● For refractory patients or patients with inhibitors, prothrombin complex concentrates, recombinant activated FVIIa and platelet transfusions have been successfully used
● Patients with inhibitors may need immunosuppression

Differential diagnosis
=========================================================================

● Acquired factor V deficiency (seen in liver disease or patients with DIC)
Acquired factor V inhibitors
● Combined Factor VIII/Factor V deficiency

Additional references
=========================================================================

Consultative Hemostasis and Thrombosis (2007), Haemophilia 2008;14:1164, Haemophilia 2009;15:1143, Semin Thromb Hemost 2009; 35:382



Hereditary bleeding disorders

Factor VII deficiency


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, M.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 16 October 2010, last major update September 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition
=========================================================================

● Factor VII deficiency is a rare congenital bleeding disorder that is inherited as an autosomal recessive trait, and is characterized by variable bleeding symptoms, ranging from asymptomatic to life-threatening hemorrhage

Terminology
=========================================================================

● Also known as proconvertin deficiency
● The deficiency is classified as:
     -Type I: decreased synthesis or increased clearance (quantitative defect)
     -Type II: dysfunctional molecule (qualitative defect)

Pathophysiology
=========================================================================

● Factor VII is a vitamin K-dependent coagulation factor that is synthesized in the liver (Wikipedia)
● Circulating factor VII forms a complex with exposed tissue factor (from injured vascular endothelium), and is activated by proteases to initiate the extrinsic coagulation cascade to form a fibrin clot
● Biologic half-life is 3.5 hours

Coagulation cascade

Epidemiology
=========================================================================

● Factor VII deficiency is the most common of the rare congenital coagulation disorders
● Incidence is 1 in 500,000

Sites
=========================================================================

● Bleeding into skin and mucosa, joint and muscle, genitourinary tract, gastrointestinal tract, CNS (see clinical features below)

Etiology
=========================================================================

● Autosomal recessive inheritance
● More than 130 mutations have been described, predominantly missense and splice-site mutations and less commonly small deletion and nonsense mutations
● Other environmental and genetic factors influence phenotype since patients with identical factor VII gene mutations have been shown to have discordant bleeding severity

Clinical features
=========================================================================

● Patients with a homozygous or compound heterozygous genotype develop bleeding symptoms while heterozygotes are typically asymptomatic
● There is little correlation of factor levels and bleeding symptoms
● Patients exhibit easy bruising, epistaxis, soft tissue hematoma, menorrhagia, menometrorrhagia, postpartum bleeding, postoperative bleeding, hemarthrosis, retroperitoneal bleeding, gastrointestinal bleeding, intracranial hemorrhage
● Paradoxically, thrombotic episodes have been reported (e.g. deep venous thrombosis) in 3-4%, but in most cases, thrombotic risk factors were identified (Haemophilia 2008;14:564)
● Few cases have been reported of inhibitor development after replacement therapy

Laboratory
=========================================================================

● Prolonged PT
● Normal PTT (although may be prolonged)
● Obtain specific factor VII activity assay to confirm

Prognostic factors
=========================================================================

● Patients with severe deficiency typically have life-threatening bleeds (e.g. intracranial, gastrointestinal) within the first 6 months of life

Case reports
=========================================================================

● Multiple cerebral aneurysms in congenital factor VII deficiency (AJNR Am J Neuroradiol 2004;25:784)

Treatment
=========================================================================

● For mild hemorrhage, recommended to maintain factor levels of 5%-10% of normal to stop bleeding; for surgical procedures, recommended to maintain levels of 15%-20% of normal
● Recombinant factor VIIa is the treatment of choice; single dose for mild to moderate bleeding, or every 4-6 hours for severe bleeding episodes
● Due to the short half-life of factor VII (3.5 hr), it is difficult to give fresh frozen plasma (FFP) every 4-6 hours to maintain levels without producing volume overload
● Plasma derived factor VII concentrates and prothrombin complex concentrates are associated with post-treatment thrombosis

Differential diagnosis
=========================================================================

● Acquired factor VII deficiency (due to warfarin, vitamin K deficiency, severe liver disease)
● Acquired factor VII inhibitors
● Familial combined factor deficiencies (e.g. factor VI/factor VIII or factor II, VII, IX and X)

Additional references
=========================================================================

Semin Thromb Hemost 2009; 35:400, Haemophilia 2008;14:1170



Hereditary bleeding disorders

Factor VIII deficiency (Hemophilia A)


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, M.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 16 October 2010, last major update October 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition
=========================================================================

● Factor VIII deficiency (hemophilia A) is the most common congenital bleeding disorder that is inherited as an X-linked recessive trait
● It is characterized by mild, moderate or severe bleeding episodes

Terminology
=========================================================================

● Factor VIII is also known as the anti-hemophilic factor

Epidemiology
=========================================================================

● Almost exclusively affects males
● 1 in every 10,000 births or 1 in 5000 male births
● Rarely affects females (see etiology)
● Female carriers are unaffected

Pathophysiology
=========================================================================

● Factor VIII is a plasma protein produced in the liver and by the reticuloendothelial system (Wikipedia)
● It circulates mainly bound to von Willebrand factor protein
● It functions as a cofactor along with activated factor IX to activate factor X, which in turn with its cofactor, factor Va, activates thrombin
● Normal plasma activity is 50%-150% (0.5-1.5 IU/mL)
● Biologic half life is 8-12 hours

Coagulation cascade

Sites
=========================================================================

● Bleeding into muscle, soft tissue or joints (hemarthrosis), GI/GU tract bleeding, easy bruising, excessive bleeding after surgery, trauma, dental procedures or circumcision; epistaxis, poor wound healing, intracranial hemorrhage, scalp hematoma, development of pseudotumors with repetitive hematoma formation, menorrhagia

Incidence of sites of bleeding:
● hemarthrosis: 70-80%
● muscle/soft tissue: 10-20%
● other major bleeds: 5-10%
● central nervous system: <5%

Incidence of bleeding into joints:
● Knee: 45%
● Elbow: 30%
● Ankle: 15%
● Shoulder: 3%
● Wrist: 3%
● Hip: 2%
● Other: 2%

Etiology
=========================================================================

● Inherited as an X-linked recessive trait, however 30% are due to spontaneous mutations
● The gene for factor VIII is a large gene located on a fragile region of the X chromosome
● The most common mutation involves inversion of intron 22; less common mutations include missense, large deletions, small point mutations and insertions/deletions
● Hemophilic females develop disease due to:
     - high degree of X-inactivation in carriers
     - hemizygosity of the X chromosome in females with Turner syndrome (XO karyotype)
     - homozygosity in female progeny of a hemophilia A carrier and an affected hemophilic male

Clinical features
=========================================================================

● Clinical severity is dependent on factor levels:
   - Mild (>5% activity; >0.05 IU/mL); occurs in 30%-40% and typically presents with bleeding after surgery or trauma
   - Moderate (1%-5% activity; 0.01-0.05 IU/mL); occurs in 10% and presents with bleeding after surgery or trauma, less commonly with spontaneous bleeding
   - Severe (<1% activity; <0.01 IU/mL); occurs in 50% and presents with spontaneous bleeding into joints, muscles and with life-threatening hemorrhage
● 30% of cases are due to spontaneous mutations, and have no family history of bleeding
● 35% of patients with severe hemophilia A will develop alloantibody inhibitors to factor VIII after replacement therapy (link)
● Formation of factor VIII alloantibodies is highest in individuals with intron 22 inversions, large deletions and nonsense mutations (Blood Coagul Fibrinolysis 2003;14 Suppl 1:S17)

Laboratory
=========================================================================

● Prolonged PTT with correction after mixing study (at 0 and 2 hr)
● Normal PT and bleeding time
● Measure both factor VIII and IX activity by functional plasma clot-based assay or chromogenic substrate-based assay
● Rule out vWD by vWF antigen and ristocetin cofactor activity
● Bethesda assay for quantitation of inhibitor
● Candidates for genetic testing include patients who have a diagnosis of hemophilia A or B, at-risk women who are related to an affected man (proband) who has a known mutation, and female carriers of hemophilia A or B seeking prenatal diagnosis
● First-line testing involves identification of the inversion of intron 22
● Obtain linkage analysis using restriction fragment length polymorphism (RFLP) if no inversion is detected or family members are available for testing
● Cord blood sampling for measurement of factor VIII activity in male fetus of known carrier

Prognostic factors
=========================================================================

● Chronic complications of hemophilia include musculoskeletal problems (e.g.chronic synovitis, arthropathy, fractures, contractures), inhibitor formation (which complicates treatment), and transfusion-related infections (e.g. HIV, HBV, HCV, etc.)

Case reports
=========================================================================

● Intraosseous hematoma in a newborn (AJNR Am J Neuroradiol 2000;21:308)
● Intracranial pseudotumor (Br J Neurosurg 2009;23:455))

Treatment
=========================================================================

● Need 80-100% of normal factor VIII levels for surgical hemostasis with major surgery or major bleeding, 30-50% postoperatively or to prevent minor bleeding
● Use plasma-derived or recombinant factor VIII concentrates (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
     - Mild/moderate bleeding - DDAVP (if patients respond to DAVP)
     - Prophylaxis in severe hemophilia A – 25-40 units/kg three times weekly
● Antifibrinolytic and topical agents (e.g. epsilon-aminocaproic acid, tranexamic acid, fibrin sealants) as adjuvant therapy
● Treatment of acute bleeding episodes in patients with inhibitors:
     - For low titer inhibitors: high dose factor VIII (to overwhelm inhibitor), porcine factor VIII (if no cross reactivity with inhibitor)
     - For high-titer inhibitors: factor VIII bypassing agents (prothrombin complex concentrates, FEIBA, recombinant factor VIIa)

Differential diagnosis
=========================================================================

von Willebrand Disease (particularly type 2N or type 3)
Factor IX deficiency (hemophilia B)
Acquired hemophilia A (autoantibody against factor VIII)
● Other factor deficiencies (XI, XII)
● Familial combined factor deficiencies (typically have an autosomal recessive pattern)

Additional references
=========================================================================

Hematol Oncol Clin North Am 2010;24:181, Blood 2007;110:815, World Federation of Hemophilia. Guidelines for the management of hemophilia, Consultative Hemostasis and Thrombosis: Elsevier, 2007



Hereditary bleeding disorders

Factor IX deficiency (Hemophilia B)


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 16 October 2010, last major update September 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition
=========================================================================

● Factor IX deficiency (hemophilia B) is the second most common congenital bleeding disorder that is inherited as an X-linked recessive trait
● Characterized by mild, moderate or severe bleeding episodes

Terminology
=========================================================================

● Also known as Christmas disease

Epidemiology
=========================================================================

● 1 in 30,000 male births
● Almost exclusively affects males
● Rarely affects females (see etiology)
● Female carriers are unaffected

Sites
=========================================================================

● Bleeding into muscle, soft tissue or joints (hemarthrosis), GI/GU tract bleeding, easy bruising, excessive bleeding after surgery, trauma, dental procedures or circumcision; epistaxis, poor wound healing, intracranial hemorrhage, scalp hematoma, development of pseudotumors with repetitive hematoma formation, menorrhagia

Incidence of sites of bleeding:
● hemarthrosis: 70-80%
● muscle/soft tissue: 10-20%
● other major bleeds: 5-10%
● central nervous system: <5%

Incidence of bleeding into joints:
● Knee: 45%
● Elbow: 30%
● Ankle: 15%
● Shoulder: 3%
● Wrist: 3%
● Hip: 2%
● Other: 2%

Pathophysiology
=========================================================================

● Factor IX is a vitamin K-dependent serine protease produced in the liver
● It circulates in the plasma in its inactive form
● It is activated by factor VIIIa, and catalyzes the conversion of factor X to Xa
● It can also be activated directly by the Tissue Factor-Factor VIIa complex in the extrinsic pathway
● Factor IX has normal plasma activity of 50%-150% (0.5-1.5 IU/mL)
● Its biologic half life is 18-24 hrs

Etiology
=========================================================================

● Factor IX deficiency is inherited as an X-linked recessive trait, but 30% of cases are due to spontaneous mutations
● The gene for factor IX is located on a fragile region of the X chromosome
● More than 300 mutations have been identified; the most common are single point mutations; numerous point and deletion mutations produce defective, nonfunctional but immunologically detectable factor IX
● Large gene deletions and nonsense mutations are most susceptible to formation of factor IX alloantibodies

Hemophilic females develop disease due to:
● High degree of X-inactivation in carriers
● Hemizygosity of the X chromosome in females with Turner syndrome (XO karyotype)
● Homozygosity in female progeny of a hemophilia B carrier and an affected hemophilic male

Clinical features
=========================================================================

Clinical severity is dependent on factor levels:
● Mild (>5% activity; >0.05 IU/mL); occurs in 30%-40%; presents with bleeding after surgery or trauma
● Moderate (1%-5% activity; 0.01-0.05 IU/mL); occurs in 10%; presents with bleeding after surgery or trauma and less commonly with spontaneous bleeding
● Severe (<1% activity; <0.01 IU/mL); occurs in 50%; presents with spontaneous bleeding into joints, muscles and with life-threatening hemorrhage
● 30% of cases are due to spontaneous mutations and have no family history of bleeding
● 1%-4% of patients with hemophilia B will develop alloantibody inhibitors after replacement therapy

Laboratory
=========================================================================

● Prolonged PTT with correction after mixing study (at 0 and 2 hr)
● Normal PT and bleeding time
● Measure both factor VIII and IX activity by functional plasma clot-based assay or chromogenic substrate-based assay
Note: diagnosis is confounded in neonates since factor IX levels are significantly reduced at birth and up to 6 months post-partum
● Rule out vWD by vWF antigen and ristocetin cofactor activity
● Bethesda assay for quantitation of inhibitor
● Candidates for genetic testing include patients who have a diagnosis of hemophilia A or B, at-risk women who are related to an affected man (proband) who has a known mutation, and female carriers of hemophilia A or B seeking prenatal diagnosis
● Genetic testing uses RFLP analysis

Prognostic factors
=========================================================================

● Chronic complications of hemophilia include musculoskeletal problems (e.g. chronic synovitis, arthropathy, fractures, contractures), inhibitor formation (which complicates treatment), and transfusion-related infections (e.g. HIV, HBV, HCV, etc.)

Case reports
=========================================================================

● Spontaneous hemopericardium in a patient with hemophilia B (J Invasive Cardiol 2008;20:E296)

Treatment
=========================================================================

● Need 50-80% of normal levels for surgical hemostasis with major surgery or major bleeding, 40% postoperatively, 30-50% to prevent minor bleeding

Plasma-derived or recombinant factor IX concentrates (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 every 12-24 hours as necessary
● Prophylaxis in severe hemophilia B - 25-40 units/kg two times weekly

Treatment of acute bleeding episodes in patients with inhibitors:
● For low titer inhibitors: high dose factor IX (to overwhelm inhibitor), porcine factor IX (if no cross reactivity with inhibitor)
● For high-titer inhibitors: factor IX bypassing agents (prothrombin complex concentrates, FEIBA, recombinant factor VIIa

Differential diagnosis
=========================================================================

von Willebrand Disease (particularly type 2N or type 3)
Factor VIII deficiency (hemophilia A)
● Acquired hemophilia B (autoantibody against factor IX)
● Other factor deficiencies (XI, XII)

Additional references
=========================================================================

Haemophilia 2010;16 Suppl 6:1, Haemophilia 2010;16 Suppl 6:13, World Federation of Hemophilia. Guidelines for the management of hemophilia, Consultative Hemostasis and Thrombosis: Elsevier, 2007



Hereditary bleeding disorders

Factor X deficiency


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, M.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 16 October 2010, last major update October 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition
=========================================================================

● Factor X deficiency is a rare congenital bleeding disorder that is inherited as an autosomal recessive trait, and is characterized by a variable bleeding tendency

Terminology
=========================================================================

● Also known as Stuart-Prower Factor Deficiency
● Deficiency classified as either:
     - Type I: decreased functional activity and antigen level (quantitative defect)
     - Type II: decreased functional activity and near normal antigen level (qualitative defect)

Epidemiology
=========================================================================

● Estimated incidence of 1 in 500,000 to 1,000,000; however, in countries where consanguinity is more common (e.g. Iran), incidence is reported to be 1 in 200,000
● Estimated carrier incidence of 1 in 500

Sites
=========================================================================

● Bleeding into skin and mucosa, joint and muscle, genitourinary tract, gastrointestinal tract, CNS (see clinical features below)

Pathophysiology
=========================================================================

● Factor X is a vitamin K-dependent serine protease produced in the liver
● It is the first enzyme in the common pathway to form a fibrin clot
● Its activated form (in complex with factor Va, Ca++ and phospholipid) cleaves prothrombin to thrombin
● It has a long half-life of 20-40 hours

Coagulation cascade

Etiology
=========================================================================

● Inherited as an autosomal recessive trait
● More than 80 mutations have been identified, which include small deletions, missense and frameshift mutations

Clinical features
=========================================================================

● May be associated with bruising, epistaxis, menorrhagia, GI/GU bleeding, umbilical stump bleeding or bleeding after surgery, trauma, dental procedures, pregnancy or circumcision, recurrent fetal loss
● Bleeding symptoms in severe deficiency are similar to that seen in patients with factor VIII (Hemophilia A) and factor IX (Hemophilia B) deficiency
● Heterozygotes are usually asymptomatic but may have mild mucocutaneous bleeds
● Patients with severe deficiency are either homozygous or compound heterozygous
● Bleeding symptoms tend to correlate with factor X activity levels: mild (>6%-10%), moderate (1%-5%) or severe (<1%)
● A small percentage of patients develop factor X inhibitors with factor replacement therapy

Laboratory
=========================================================================

● Prolonged PT and PTT that correct with mixing study
● Prolonged Russell’s viper venom time (measures the direct activation of factor X)
● Normal thrombin and bleeding time
● Factor X specific functional and immunologic assays for confirmation

Prognostic factors
=========================================================================

● Particular genotypes [Gly(-20)Arg, Gly94Arg and Gly380Arg mutations] are associated with higher rates of hemarthrosis and intracranial bleeding

Case reports
=========================================================================

● Factor X deficiency presenting as chronic bilateral subdural hematomas in a 7 month-old infant (Pediatr Neurosurg 2010;46:54)

Treatment
=========================================================================

● For minor bleeding episodes, maintain factor X levels at 10%-15% of normal using FFP (15-20 mL/kg followed by 3-6 mL/kg every 24 hours)
● For major bleeding episodes, trauma or surgical procedures, factor X rich prothrombin complex concentrates can be used to maintain factor X levels at 50% of normal

Differential diagnosis
=========================================================================

● Acquired factor X deficiency (liver disease, vitamin K deficiency): also exhibits reduced levels of other coagulation factors; isolated factor X deficiency is associated with respiratory infection, AML and other malignancies, amyloidosis
● Acquired factor X inhibitors in patients without congenital factor X deficiency are rare but have been reported in leprosy and chemical exposure
● Other factor deficiencies (e.g. factor V, prothrombin)

Additional references
=========================================================================

Haemophilia 2008;14:1176, Consultative Hemostasis and Thrombosis: Elsevier, 2007



Hereditary bleeding disorders

Factor XI deficiency


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, M.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 16 October 2010, last major update October 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition
=========================================================================

● Factor XI deficiency is a rare bleeding disorder that is inherited most commonly as an autosomal recessive trait, and is characterized by variable bleeding episodes

Terminology
=========================================================================

● Also known as plasma thromboplastin antecedent deficiency or hemophilia C

Epidemiology
=========================================================================

● Incidence of 1 in 450 in Ashkenazi Jewish populations and 1 in 1,000,000 in non-Jewish populations

Sites
=========================================================================

● Bleeding into skin and mucosa, genitourinary tract, gastrointestinal tract (see clinical features below)

Pathophysiology
=========================================================================

● Factor XI is a serine protease that is produced in the liver, and is involved in the contact (intrinsic) pathway
● It is activated by factor XIIa, thrombin or autoactivated, and activates factor IX, which in turn activates factor X to convert prothrombin to thrombin
● Its biologic half-life is 60-80 hrs

Coagulation cascade

Etiology
=========================================================================

● Most commonly inherited as an autosomal recessive trait
● An autosomal dominant pattern of inheritance is possible, as the dimeric structure of factor XI may result in a dominant negative effect through intracellular heterodimer formation (Blood 2004;104:128)
● The gene for factor XI is located on the long arm of chromosome 4
● To date, over 180 mutations have been described with three main genotypes: type I are splice site mutations, type II are nonsense mutations and type III are missense mutations
● Type II and III mutations are predominantly seen in Ashkenazi Jews
● Type II mutations are also seen in Iraqi Jews and Israeli Arabs

Clinical features
=========================================================================

● Deficiency can occur in homozygous, heterozygous or a combined heterozygous form
● Bleeding manifestations do not correlate with factor XI levels
● Most bleeding episodes in patients with severe deficiency are injury-related
● Spontaneous bleeding is rare
● May be associated with bruising, epistaxis, menorrhagia, GI/GU bleeding, umbilical stump bleeding or bleeding after surgery, trauma, dental procedures, pregnancy or circumcision
● Up to 33% of patients with severe deficiency develop inhibitors after replacement therapy

Laboratory
=========================================================================

● Prolonged PTT that corrects with mixing study
● Normal PT, thrombin time
● Specific factor XI assay

Case reports
=========================================================================

● Cerebellar hemorrhage due to factor XI deficiency (Cerebrovasc Dis 2005;19:138)

Treatment
=========================================================================

● 10-20 ml fresh frozen plasma/kg, then 5-10 ml/kg every 24 hours as necessary
● Antifibrinolytic therapy has been used in women with factor XI deficiency and menorrhagia
● Patients with inhibitors have been treated successfully with plasma, prothrombin complex concentrates, and recombinant activated factor VII
● Note: factor XI concentrates may promote thromboembolic complications

Differential diagnosis
=========================================================================

● Acquired factor XI deficiency associated with systemic lupus erythematosus
● Other factor deficiency causing prolonged PTT (factor VIII, IX, XII, but factor XII deficiency is not associated with bleeding manifestations)
von Willebrand Disease
● Heparin contamination
● Liver dysfunction
● Combined familial factor deficiency

Additional references
=========================================================================

J Thromb Haemost 2009;7 Suppl 1:84, Semin Thromb Hemost 2009;35:416



Hereditary bleeding disorders

Factor XII deficiency


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, M.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 30 October 2010, last major update October 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition
=========================================================================

● Factor XII deficiency is a congenital disorder that is most commonly inherited as an autosomal recessive trait and is not associated with a bleeding diathesis
● It is typically discovered in individuals with an isolated prolonged PTT
● Homozygous individuals have undetectable factor XII levels
● Heterozygous individuals have factor XII levels between 20-60%

Terminology
=========================================================================

● Factor XII is also known as the Hageman factor
● Factor XII deficiency is also known as Hageman factor deficiency or Hageman trait

Pathophysiology
=========================================================================

● Factor XII is a coagulation protein which is either autoactivated by contact with a number of artificial or biologic negatively charged surfaces (contact activation) or by proteolytic activation on the surface of endothelial cells by prekallikrein/kallikrein and high molecular weight kininogen
● Activated factor XII converts prekallikrein to kallikrein (which activates more factor XII, liberates bradykinin from high molecular weight kininogen, and activates complement components C3 and C5), activates factor XI which eventually leads to thrombin generation via the intrinsic pathway, and also activates C1 esterase, thereby activating the complement system

     

Coagulation cascade


     

Mechanisms of Factor XII activation

Epidemiology
=========================================================================

● Actual prevalence difficult to determine since individuals are asymptomatic
● One study of 300 healthy blood donors reported a prevalence of 2.3% (Thromb Haemost 1994;71:68)

Sites
=========================================================================

● Rarely skin and mucosa (see clinical features below)

Etiology
=========================================================================

● Most commonly inherited in an autosomal recessive pattern
● Autosomal dominant inheritance has been described in one family (Blood 1972;40:412)

Clinical features
=========================================================================

● Not associated with bleeding episodes, even after major surgical procedures or trauma
● Most patients are detected by routine preoperative coagulation studies (isolated prolonged PTT)
● Very rarely may manifest with epistaxis or easy bruising
● There is some debate about the association of factor XII deficiency and an increased risk of arterial and venous thrombosis, myocardial infarction and pulmonary embolism

Laboratory
=========================================================================

● Prolonged PTT that corrects with mixing study
● Normal PT, thrombin time and bleeding time
● Specific factor XII assay is diagnostic

Case reports
=========================================================================

● Combined factor VIII and factor XII deficiency (Am J Hematol. 1992;39:137)
● Acquired factor XII deficiency by orthotopic liver transplant (Am J Transplant 2006;6:1743)

Differential diagnosis
=========================================================================

● Acquired factor XII deficiency: associated with nephrotic syndrome, liver transplantation, autoimmune disease)
● Heparin contamination
● Liver disease
Lupus anticoagulants
Prekallikrein deficiency
High molecular weight kininogen deficiency
● Factor XII deficiency in association with von Willebrand disease, factor VIII or factor IX deficiency

Additional references
=========================================================================

J Clin Invest 2008;118:3006, Consultative Hemostasis and Thrombosis (2007)



Hereditary bleeding disorders

Factor XIII deficiency


Reviewers: Julie Gober-Wilcox, M.D., Resident, University of New Mexico; Kendall Crookston, M.D., Ph.D., University of New Mexico (see Reviewers page)
Revised: 24 July 2011, last major update July 2011
Copyright: (c) 2002-2011, PathologyOutlines.com, Inc.

General
=========================================================================

● Factor XIII deficiency is a congenital disorder that is inherited as an autosomal recessive trait and is associated with a variable bleeding tendency
● Acquired factor XIII deficiency is associated with liver failure, inflammatory bowel disease, leukemia, disseminated intravascular coagulation, Henoch-Schonlein purpura, systemic lupus erythematosus and exposure to certain drugs (phenytoin, isoniazid, valproate)

Terminology
=========================================================================

● Factor XIII is also known as fibrin stabilizing factor

Epidemiology
=========================================================================

● Estimated at 1 in 2 to 5 million births
● More frequent in regions where consanguineous marriages are more common

Sites
=========================================================================

● Skin and mucosa, joints, soft tissue, gastrointestinal tract, genitourinary tract, CNS (see clinical features below)

Pathophysiology
=========================================================================

● Factor XIII is a transglutaminase that circulates as a zymogen comprised of 2 catalytic A subunits and 2 carrier B subunits
● The A subunit is synthesized in platelets, monocytes and macrophages while the B subunit is synthesized in the liver; the A and B dimers then assemble in the plasma to form a heterotetramer
● Factor XIII is activated by thrombin and is responsible for catalyzing the final step in the coagulation cascade by cross-linking fibrin (in the presence of calcium)
● Deficiency is due to a defect in either the A gene (type 2) or B gene (type 1)


Diagram of factor XIII activation

Etiology
=========================================================================

● Inherited as an autosomal recessive trait
● Most cases are due to mutations in A subunit gene on chromosome 6
● More than 70 mutations have been identified, most of which are missense and nonsense mutations
● Only 5 mutations in FXIII B deficient patients have been identified; gene is on chromosome 1

Clinical features
=========================================================================

● Variable bleeding tendency, from mild to severe depending on factor levels
● Umbilical cord stump bleeding, intracranial hemorrhage, soft tissue hematoma, bleeding after circumcision, gastrointestinal bleeding, gingival bleeding, epistaxis, hematuria, surgical site bleeding, menorrhagia, joint bleeding, delayed healing, spontaneous abortion, recurrent miscarriage
● Plasma half –life is 9-12 days
● Factor XIII levels above 3-5% are usually sufficient to prevent spontaneous bleeding
● Severe bleeding typically occurs in individuals with <1% circulating levels
● Compound heterozygotes are usually asymptomatic

Laboratory
=========================================================================

● Normal PT, PTT, thrombin time, fibrinogen
● Screening test for factor XIII deficiency uses the clot solubility test in which patient plasma is incubated with thrombin and calcium; deficiency will cause the clot to dissolve in the presence of urea or acid
● A standard mixing test using patient plasma and normal pooled plasma is usually performed to rule out the presence of an inhibitor
● Confirmatory testing uses a quantitative factor XIII activity assay

Prognostic factors
=========================================================================

● Although there is a life-long risk of bleeding, prognosis is excellent due to good response to treatment; subsequent risk of development of inhibitors is low

Case reports
=========================================================================

● Spontaneous splenic rupture in a patient with factor XIII deficiency (Pediatr Blood Cancer 2008;50:113)

Treatment
=========================================================================

● Factor XIII concentrate, FFP or cryoprecipitate for replacement therapy or for treatment of acute bleeding episodes:
● Factor XIII concentrate: 10-20U/kg at 4-6 week intervals
● FFP: 10 mL/kg every 4-6 weeks
● Cryoprecipitate: 1 bag per 10-20 kg every 3-4 weeks
● To prevent miscarriage, maintain factor XIII levels >10% in early gestation and >30% at time of delivery to prevent significant bleeds

Differential diagnosis
=========================================================================

● Acquired factor XIII deficiency

Additional references
=========================================================================

J Thromb Haemost 2003;1:1852, Semin Thromb Hemost 2009;35:426, Haemophilia 2008;14:1190



Hereditary bleeding disorders

High molecular weight kininogen deficiency / assay


Reviewers: Julie Gober-Wilcox, M.D., Kendall Crookston, M.D., Ph.D., Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update July 2011
Copyright: (c) 2002-2011, PathologyOutlines.com, Inc.

Definition
=========================================================================

● High molecular weight kininogen deficiency is a rare congenital disorder inherited as an autosomal recessive trait, not associated with a bleeding diathesis
● Typically discovered in individuals with an isolated prolonged PTT

Terminology
=========================================================================

● High molecular weight kininogen is also known as Fitzgerald factor, Williams factor and Flaujeac factor

Pathophysiology
=========================================================================

● High molecular weight kininogen is a protein produced by the liver (with no inherent catalytic activity) that is involved in the early steps of the intrinsic coagulation pathway; it functions as a cofactor and binds with prekallikrein and factor XI to help facilitate their activation by factor XIIa
● The kininogens (low and high molecular weight) are also involved in the kinin-kallikrein system and function to inhibit thrombin activation of platelets and stimulate liberation of nitric oxide, prostacyclin and tissue plasminogen activator

Clinical features
=========================================================================

● Not associated with bleeding

Laboratory
=========================================================================

● Isolated prolonged PTT
● Negative lupus anticoagulant
● Specific assay to test for deficiency uses high molecular weight kininogen deficient plasma mixed with patient plasma; a PTT is performed and is compared to a standard curve of high molecular weight kininogen vs. PTT
● Interference occurs in these assays if patient is on heparin, hirudin or argatroban, possibly danaparoid
● Lower levels in newborns, increase to adult levels by age 6 months

Case reports
=========================================================================

● 66 year old man evaluated for cardiac surgery (Thromb Haemost 2001;85:195)

Differential diagnosis
=========================================================================

Factor XII deficiency
Lupus anticoagulant



Hereditary bleeding disorders

Prekallekrein deficiency


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, MD, University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 6 December 2014, last major update October 2010
Copyright: (c) 2002-2014, PathologyOutlines.com, Inc.

Definition / General
=========================================================================

● Prekallikrein deficiency is a rare congenital disorder that causes an isolated prolonged PTT but is not associated with a bleeding tendency

Terminology
=========================================================================

● Also known as Fletcher factor deficiency

Epidemiology
=========================================================================

● Rare

Pathophysiology
=========================================================================

● Prekallikrein is a contact factor that complexes with high molecular weight kininogen and is cleaved by factor XII (Hageman factor) to produce kallikrein in the initial steps of the intrinsic pathway

Etiology
=========================================================================

● Autosomal recessive inheritance
● Homozygous individuals have < 1% activity
● Heterozygous individuals have 20%-60% of normal activity
● There are rare variants of abnormal prekallikrein molecules

Clinical features
=========================================================================

● No bleeding tendency
● Usually detected in asymptomatic individuals after the incidental finding of isolated prolonged PTT
● There have been anecdotal reports of prekallikrein deficiency and increased risk of arterial and venous thrombosis, but usually thrombotic risk factors were identified (Acta Haematol 2010;123:210)

Laboratory
=========================================================================

● Prolonged PTT that corrects after mixing study
● Normal PT, thrombin time and bleeding time
● Specific functional prekallikrein assay is diagnostic

Case reports
=========================================================================

● Prekallikrein deficiency resulting in mucosal hemorrhage (Am J Med Sci 2009;338:429)

Treatment
=========================================================================

● Typically not required

Differential diagnosis
=========================================================================

Factor XII deficiency
High molecular weight kininogen deficiency
● Acquired prekallikrein deficiency due to DIC or liver disease, rarely due to antibodies to prekallikrein

Additional references
=========================================================================

Consultative Hemostasis and Thrombosis (2007)



Hereditary bleeding disorders
von Willebrand's disease

Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 7 April 2013, last major update April 2013
Copyright: (c) 2003-2013, PathologyOutlines.com, Inc.

See subtypes and molecular basis below

General
=========================================================================
Diagrams
=========================================================================
Binding of von Willebrand factor
Diagnosis
=========================================================================
Treatment
=========================================================================


Subtypes
General
=========================================================================
Type 1 (70-80%): Type 2 (15-20%): Type 2A: Type 2B: Type 2C: Type 2M: Type 2N: Type 3: Platelet type or pseudo von Willebrand's disease:

Molecular basis of von Willebrand’s disease
General
=========================================================================


Therapy related coagulopathies

Warfarin (Coumadin™)


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 12 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Therapeutic anticoagulant to prevent thromboembolism by impairing regeneration of active vitamin K (warfarin is a synthetic derivation based on coumarin)
● The name incorporates the acronym for the organization which funded the key research (WARF, for Wisconsin Alumni Research Foundation) and the ending -arin, indicating its link with coumarin

Pathophysiology
=========================================================================

● Vitamin K is a cofactor in reactions that carboxylate glutamic acid residues in factors II, VII, IX, X, protein C, protein S
● These “GLA” domains enable the coagulation factors to bind to phospholipid membranes in the presence of calcium
● Therapeutic warfarin or Vitamin K deficiency cause decreased activity of these proteins, making the enzymes less able to participate in the clotting cascade, which prolongs the PT

Clinical features
=========================================================================

● Takes 4-5 days for complete therapeutic effect due to long half-life of factors II and X
● Must be supplemented with another anticoagulant such as heparin (“bridged”) until INR is in therapeutic range for 2 consecutive days to prevent Warfarin Skin Necrosis in those with low Protein C
● 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
● Warfarin should not be used alone for acute heparin-induced thrombocytopenia because it causes paradoxical thrombosis - must add a rapid acting anticoagulant (hirudin, danaparoid, argatroban) until INR is therapeutic
● Note: PTT may be normal if low warfarin levels

Treatment of bleeding/overdose:
● Vitamin K, fresh frozen plasma; also prothrombin complex concentrates (J Clin Pathol 2004;57:1132, J Thromb Haemost 2006;4:1853)

Diagrams
=========================================================================




Biochemistry of coumarins



Therapy related coagulopathies

Danaparoid (Orgaran)


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 12 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Approved to prevent deep venous thromboses
● Also an alternative to heparin for patients with heparin induced thrombocytopenia (Crit Care 2007;11:R102)
● Use is decreasing due to newer anticoagulants
● Composed of low molecular weight glycosaminoglycans (mixture of heparan sulfate, dermatan sulfate and chondroitin sulfate) that primarily inhibit factor Xa, factor IIa to a much lesser extent

Clinical features
=========================================================================

● 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, but incomplete reversal is shown with protamine sulfate
● Not marketed in the US since 2004, still available in some countries.



Therapy related coagulopathies

Heparin


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 12 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Also called unfractionated heparin
● Short acting anticoagulant with half life of approximately 90 minutes

Mechanism of action
=========================================================================

● Derived from porcine intestinal mucosa or bovine lung, which contain heparin-rich mast cells
● Markedly enhances 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
● 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

Clinical features
=========================================================================

● 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
● Complications include hemorrhage (overcoagulation) and heparin-induced thrombocytopenia (up to 3% of patients)
● Recommended that 90% of patients should achieve therapeutic anticoagulation within 24 hours

Monitoring
=========================================================================

● 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 (Clin Lab Med 2009;29:283)
● Also monitor platelet count within 72 hours, with platelet monitoring to continue periodically for 20 days; PT is usually normal
Note: often the cause of prolonged PTT is heparin in sample collected through indwelling line; identify by treating with heparinase

Diagrams
=========================================================================



Heparin 2-D structure


Function of heparin

Treatment
=========================================================================

● Protamine sulfate (for emergency reversal of heparin)



Therapy related coagulopathies

Heparin - low molecular weight


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 13 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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

Clinical features
=========================================================================

● 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

Monitoring
=========================================================================

● Typically do not monitor except for periodic platelet counts
● Indications for monitoring include pregnancy, renal failure, obesity, prolonged use, infants and children, patients at high risk for bleeding / thrombosis
● Monitor by measuring anti-factor Xa activity, drawn 4 hours after injection
● Typical therapeutic range is 0.6 to 1.0 U/ml for twice a day dosing, higher for once a day dosing, 1.0 to 2.0 U/ml for prophylactic dosing
● Effects are reversed with protamine sulfate

Diagrams
=========================================================================



Function of LMWH

Additional references
=========================================================================

Clin Lab Med 2009;29:283



Therapy related coagulopathies

Hirudin


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 21 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Anticoagulant approved by FDA to treat thrombosis in patients with heparin induced thrombocytopenia (does not itself cause this type of syndrome)
● Derivatives include lepirudin, refludan
● Recombinant protein, cloned from a leech (Wikipedia), which directly inhibits factor IIa (thrombin)
● Also has fibrinolytic properties
● 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, but more specific assays are also used to monitor, such as ecarin clotting time

● Half-life ~one hour, may be prolonged if antibodies develop, dramatically prolonged in renal failure
● No reversal agent

Additional references
=========================================================================

Thromb Haemost 2008;99:819



Therapy related coagulopathies

Thrombolytic therapy


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 13 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Used to treat myocardial infarction, pulmonary embolism, arterial or venous thrombosis, thrombotic stroke
● Thrombolytic agents include recombinant t-PA, urokinase, streptokinase (only tPA is widely available in the USA)

Laboratory
=========================================================================

● Presence of fibrin degradation products and D-dimers, decreased fibrinogen and plasminogen, prolonged thrombin time, PT and PTT

Diagrams
=========================================================================



t-PA action

Additional references
=========================================================================

Clin Lab Med 2009;29:159



Acquired thrombophilia / hypercoagulopathies
Acquired thrombophilia-general

Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 7 April 2013, last major update April 2013
Copyright: (c) 2003-2013, PathologyOutlines.com, Inc.

General
=========================================================================
Diagrams
=========================================================================
Initiation and propagation of blood coagulation
Activation of protein C by thrombin-thrombomodulin
Degradation of FVa and FVIIIa by APC
Blood coagulation and protein C anticoagulant system
Activation and degradation of normal FV and FV Leiden
20210G>A mutation in the prothrombin gene
Regulation of hemostasis
Balance of hemostasis
Genetic polymorphisms of various procoagulant proteins
Activated protein C (APC)
Transsulfuration pathway and metabolism of homocysteine
Case reports
=========================================================================
Clinical images
=========================================================================
Thrombi in major trunks of pulmonary artery and large thrombus near left iliac vein.
Micro images
=========================================================================

Fig. 1/2: old, recanalized thrombi in portal veins;
Fig. 3: recanalization of portal vein webs with intimal tags;
Fig. 4: bone marrow is hypercellular with large, dysplastic, abnormally clustered megakaryocytes
Additional references
=========================================================================


Acquired thrombophilia / hypercoagulopathies
Antiphospholipid antibodies

Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 7 April 2013, last major update April 2013
Copyright: (c) 2003-2013, PathologyOutlines.com, Inc.

General
=========================================================================
Additional references
=========================================================================


Acquired thrombophilia / hypercoagulopathies
Heparin induced thrombocytopenia (HIT)

Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 7 April 2013, last major update April 2013
Copyright: (c) 2003-2013, PathologyOutlines.com, Inc.

General
=========================================================================
Diagrams
=========================================================================
Pathophysiology of HIT

Clinical features
=========================================================================
Treatment
=========================================================================


Hereditary thrombophilia / hypercoagulopathies
Hereditary thrombophilia-general

Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 7 April 2013, last major update April 2013
Copyright: (c) 2003-2013, PathologyOutlines.com, Inc.

General
=========================================================================


Hereditary thrombophilia / hypercoagulopathies

Activated protein C resistance


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 16 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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
● Approximately 64% of people with venous thrombosis have activated protein C deficiency
● Does not appear to reduce life expectancy
● Acquired forms of activated protein C deficiency can lead to elevated factor VIII levels

Diagrams
=========================================================================



Protein C pathway

Additional references
=========================================================================

J Am Board Fam Pract 2000;13:111, Semin Vasc Med 2003;3:33, Kolde, Hans-Jurgen (2004). Haemostasis. Basel, Switzerland: Pentapharm Ltd


Factor V Leiden mutations

General
=========================================================================

● 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 5-10x 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 smoking, malignancy, trauma, surgery, oral contraceptive use, estrogen replacement therapy, antiphospholipid antibody, heterozygosity for prothrombin G20210A, elevated serum homocysteine
● Other low frequency factor V mutations, which have unclear association with venous thrombosis, are Factor V Cambridge (Arg306Thr), Factor V Hong Kong (Arg306Gly), HR2 haplotype with mutation 4070A to G (His199Arg) in exon 13 of Factor V gene (associated with other polymorphisms)

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 (Arch Pathol Lab Med 2003;127:1359)

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:577, Goodnight Jr, Scott & Hathaway, William (Eds) (2001) Disorders of hemostasis & thrombosis: A clinical guide: McGraw-Hill.



Hereditary thrombophilia / hypercoagulopathies

Antithrombin deficiency


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 19 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Hereditary deficiencies occur in 0.07 to 0.17% of general population
● 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; overall 50% have thrombosis
● First thrombotic event occurs between ages 10-50 years
● Often occurs with other genetic or acquired risk factors
● Heterozygotes have levels 35-75% of normal

Genetics / types of mutations
=========================================================================

● Many mutations exist (qualitative or quantitative)
● Usually autosomal dominant
● 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 and heparin binding site
      ● IIb: mutations affect reactive site of target protease
      ● IIc: isolated decreased heparin binding

Acquired cause
=========================================================================

● Acute hemolytic transfusion reaction
● Acute thrombotic episodes
● Burns (extensive)
● Heparin therapy
● Inflammatory bowel disease
● L-asparaginase therapy
● Liver disease
● Malignancy
● Malnutrition
● Nephrotic syndrome
● Plasmapheresis
● Preeclampsia
● Protein poor diet
● Thrombosis-recent or active (including DIC)

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%)

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1326, eMedicine #1, #2
Goodnight Jr, Scott & Hathaway, William (Eds) (2001). Disorders of hemostasis & thrombosis: A clinical guide. McGraw-Hill



Hereditary thrombophilia / hypercoagulopathies

Dysfibrinogenemia


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Disorders of fibrinogen structure (over 350 described)
● 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

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1387, Arch Pathol Lab Med 2002;126:499
McDonagh, J. (2001). Dysfibrinogenemia and other disorders of fibrinogen structure or function. In R. Colman, J. Hirsh, V. Marder, A. Clowes, J. George, Hemostasis and Thrombosis (4th ed.) (pp.855–892). Philadelphia, PA: Lippincott Williams & Wilkins.



Hereditary thrombophilia / hypercoagulopathies

Elevated coagulation factor levels in plasma


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 21 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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 prevent ischemic heart disease

Factor II (prothrombin):
● High levels associated with increased risk of venous thrombosis
● See also prothrombin G20210A mutation
● 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 (Arch Pathol Lab Med 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

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1405, Thromb Haemost 2009;102:936
Goodnight Jr., Scott, & Hathaway, William (Eds.). (2001). Disorders of hemostasis & thrombosis: A clinical guide. New York, NY: McGraw-Hill.



Hereditary thrombophilia / hypercoagulopathies

Heparin cofactor II deficiency


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 13 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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 (Arch Pathol Lab Med 2002;126:1394)

Case reports
=========================================================================

● Family with homozygous deficiency, but no significant symptoms (Circulation 2004;110:1303)



Hereditary thrombophilia / hypercoagulopathies

Hyperhomocysteinemia


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 21 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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, but 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
● Mutation in the methioninesynthetase gene (MTR) also can lead to increased homocystein levels but, as with MTHFR mutations, are not thought to be associated with 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, folic acid, trimethylglycine, 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

Diagrams
=========================================================================



Folate metabolism

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1367, Hematol J 2001;2:38



Hereditary thrombophilia / hypercoagulopathies

Protein C deficiency


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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
● Homozygous protein C deficiency can be cured with liver transplant; however, this is usually too risky so replacement is preferred treatment
● 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

Clinical images
=========================================================================



Warfarin induced skin necrosis

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1337, Haemophilia 2008;14:1214



Hereditary thrombophilia / hypercoagulopathies

Protein S deficiency


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 21 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1349, Haemophilia 2008;14:1222



Hereditary thrombophilia / hypercoagulopathies

Prothrombin gene mutation (G20210A) / hyperprothrombinemia


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 21 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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
● Patients with G20210A mutation have increased levels of TAFI (Thrombin Activated Fibrinolysis I, a protein that inhibits clot lysis), which makes clots exist longer, leading to increased thrombotic episodes
● 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

Recommended patients to test
=========================================================================

● Patients with recurrent venous thromboembolic events
● 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

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1319, Blood 2004;103:2157



Hereditary thrombophilia / hypercoagulopathies

Sickle cell disease


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Associated with increases in thrombin generation, fibrinolytic activation, platelet activation, increased antiphospholipid antibodies, decreased levels of circulating anticoagulants and contact factors
● Also increased circulating levels of tissue factor and endothelial cells expressing a tissue factor phenotype
● Have hypercoagulable state based on thermoelastographic profiles (Arch Pathol Lab Med 2005;129:760)

Additional references
=========================================================================

J Clin Pathol 1980;33:622



Coagulation laboratory tests

General


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

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
● For critical values, 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
(f) 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

Additional references
=========================================================================

Arch Pathol Lab Med 2005;129:47



Coagulation laboratory tests

Quality assurance


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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 procedures to substantiate continued accuracy throughout the laboratory’s reportable range of test results



Coagulation laboratory tests

Abnormal PT and PTT - causes


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 16 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

PT normal, PTT prolonged:
● Deficiency of intrinsic pathway factors VIII, IX, XI, XII
● Less commonly prekallikrein or high molecular weight kininogen
● Also prolonged delay in transporting specimen to lab (affects factors V and VIII), presence of heparin in specimen

PT prolonged, PTT normal:
● Deficiency of extrinsic pathway factor VII
● Occasionally due to deficiency of common pathway factors fibrinogen, prothrombin, factors V or X, early Vitamin K deficiency (dietary or warfarin)

PT and PTT prolonged:
● Deficiency of common pathway factors fibrinogen, prothrombin, factors V or X, or multiple factor deficiencies



Coagulation laboratory tests

Activated clotting time


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 16 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Whole blood clotting assay, performed at point of case, including operating room or catheterization lab (Am J Clin Pathol 2011;135:741), 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)

Additional references
=========================================================================

Lab Report for Physicians 1982;4:17



Hereditary thrombophilia / hypercoagulopathies

Activated protein C resistance


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 16 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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
● Approximately 64% of people with venous thrombosis have activated protein C deficiency
● Does not appear to reduce life expectancy
● Acquired forms of activated protein C deficiency can lead to elevated factor VIII levels

Diagrams
=========================================================================



Protein C pathway

Additional references
=========================================================================

J Am Board Fam Pract 2000;13:111, Semin Vasc Med 2003;3:33, Kolde, Hans-Jurgen (2004). Haemostasis. Basel, Switzerland: Pentapharm Ltd


Factor V Leiden mutations

General
=========================================================================

● 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 5-10x 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 smoking, malignancy, trauma, surgery, oral contraceptive use, estrogen replacement therapy, antiphospholipid antibody, heterozygosity for prothrombin G20210A, elevated serum homocysteine
● Other low frequency factor V mutations, which have unclear association with venous thrombosis, are Factor V Cambridge (Arg306Thr), Factor V Hong Kong (Arg306Gly), HR2 haplotype with mutation 4070A to G (His199Arg) in exon 13 of Factor V gene (associated with other polymorphisms)

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 (Arch Pathol Lab Med 2003;127:1359)

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:577, Goodnight Jr, Scott & Hathaway, William (Eds) (2001) Disorders of hemostasis & thrombosis: A clinical guide: McGraw-Hill.



Coagulation laboratory tests

Anticardiolipin antibodies


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 19 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● One of the two main types of antiphospholipid antibodies (other is lupus anticoagulant) often seen in conjunction with beta2-glycoprotein antibodies

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
● Test 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 antibody persistence, positive test must be confirmed by repeat testing after 6 weeks
● Transient antibodies are not strongly associated with thrombosis

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1424, Lancet 2010;376:1498



Coagulation laboratory tests

Antiplasmin assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 19 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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
● True alpha2-antiplasmin deficiency is a rare condition (<20 cases)
● Most cases are caused by inhibitors (antibodies)

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

Additional references
=========================================================================

J Thromb Haemost 2007;5:812



Coagulation laboratory tests

Antithrombin assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 19 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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
● Also family studies (first degree relatives)

Antigenic assay limitations
● Does not detect functional deficiencies by itself
● If initial antithrombin result is low, should do confirmatory test on repeat specimen
● Must also exclude acquired causes

Specimen / reference ranges
=========================================================================

● 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
=========================================================================

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

Additional references
=========================================================================

Arch Pathol Lab Med 2002;126:1326, Thromb Diath Haemorrh 1965;13:516



Coagulation laboratory tests

Bleeding time


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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

Additional references
=========================================================================

Blood 1991;77:2547



Coagulation laboratory tests

Clot retraction


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Obsolete test
● Evaluates how well platelets keep the clot adhered to the sides of specimen tube
● 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
● Must have normal fibrinogen and hematocrit for test be accurate

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”)



Coagulation laboratory tests

Cryoglobulin / cryofibrinogen assays


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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 (especially hepatitis C), 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
● If the cryoglobulin test is positive, serum protein electorpheresis with immune fixation should be run to determine what type of cryoglobulin is present



Coagulation laboratory tests

D-dimer / dimerized plasmin fragment D


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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 (Arch Pathol Lab Med 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
● Although this is called a Latex ImmunoAssay, it differs 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 (Arch Pathol Lab Med 2004;128:328)
● High rheumatoid factor
● Liver disease
● Cancer patients
● Pregnancy

Note:
● Some platforms use fibrinogen equivalent units, which are 50% the numerical value of d-dimer units

Clinical images
=========================================================================



Fibrin split products/ D-dimer

Additional references
=========================================================================

Antovic, J.P. and Egberg, N. (2010). Laboratory investigations. In M. Blomback and J.P. Antovic (Eds.), Essential Guide to Blood Coagulation (pp.11-33). Oxford, UK: Wiley-Blackwell.



Coagulation laboratory tests

Dabigatran


Reviewer: Nat Pernick, M.D. (see Reviewers page)
Revised: 15 March 2012, last major update March 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Oral anti-coagulant (also rivaroxaban, apixaban) with minimal food and drug interactions that does not require coagulation monitoring
● Drug is very expensive, and is affected by humidity (Wikipedia)
● Dabigatran etexilate is prodrug of dabigatran, a direct thrombin inhibitor
● Alternative to warfarin for reducing the risk of stroke and systemic embolism in patients with nonvalvular atrial fibrillation (J Clin Pharmacol 2012 Jan 31 [Epub ahead of print], J Med Econ 2012 Mar 7 [Epub ahead of print]) or venous thromboembolism (N Engl J Med 2009;361:2342, Vasc Health Risk Manag 2012;8:45)
● Good patient compliance (Orthop Traumatol Surg Res 2012 Mar 2 [Epub ahead of print])
● INR levels are not necessary, and are substantially higher using Hemochron Jr. point-of-care device compared with laboratory values (Am J Med 2012 Feb 3 [Epub ahead of print])
● Can use HEMOCLOT direct thrombin inhibitor assay to determine plasma concentrations of dabigatran (Blood Coagul Fibrinolysis 2012;23:138)
● Contraindications: nonvalvular atrial fibrillation patients with renal insufficiency (Rinsho Shinkeigaku 2011;51:1004), INR 2.0 or greater, age 80 years or greater (N Engl J Med 2012;366:864)

Drawings
=========================================================================


   

Structure and site of inhibition in coagulation cascade



Coagulation laboratory tests

Ecarin clotting time


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Measures activity of hirudin in plasma, important since severe bleeding can occur with hirudin overdose and no antidote is known
● Also measures activity of lepirudin, a recombinant form of hirudin (J Extra Corpor Technol 2001;33:117)
● Ecarin catalyzes prothrombin to meizothrombin, an active form that is inhibited by hirudin (Pathophysiol Haemost Thromb 2004;33:173)
● Test measures ability of hirudin to complex with / inhibit meizothrombin as ecarin produces it from prothrombin
● Ecarin comes from the venon of the saw-scaled viper

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



Coagulation laboratory tests

Factor assays


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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

Additional references
=========================================================================

Kolde, Hans-Jurgen. (2004). Haemostasis. Basel, Switzerland: Pentapharm Ltd.



Coagulation laboratory tests

Factor I (fibrinogen) assay


Reviewers: Kendall Crookston, M.D., Ph.D., University of New Mexico; Lizabeth Rosenbaum, M.D., University of New Mexico; Julie Gober-Wilcox, M.D., Resident, University of New Mexico (see Reviewers page)
Revised: 22 June 2012, last major update August 2010
Copyright: (c) 2002-2010, PathologyOutlines.com, Inc.

Definition / General
=========================================================================

● Fibrinogen (factor I) is a major plasma protein that is synthesized by the liver and is converted to fibrin by thrombin
● Fibrinogen levels can be reduced in disseminated intravascular coagulation (DIC), liver disease, inherited and acquired fibrinogen disorders, thrombolytic therapy, dilutional coagulopathy following massive transfusion and in L-asparaginase therapy
● Fibrinogen levels can be increased in an acute phase reaction, malignancy, females (particularly post-menopausal), pregnancy, oral contraceptive use
● The reference range for fibrinogen is usually 1.5-4.0 g/L

Purpose of fibrinogen assay =========================================================================

Fibrinogen assays are typically used to investigate the following:
● Investigation of abnormal coagulation tests (e.g. PT/PTT)
● Investigation of unexplained bleeding
● Investigation of suspected inherited or acquired disorders of fibrinogen (afibrinogenemia, hypofibrinogenemia and dysfibrinogenemia)
● Evaluation of DIC
● Establishment and monitoring of thrombolytic therapy
● Assessment of cardiovascular risk

Types of fibrinogen assays
=========================================================================

● There are different assays that measure fibrinogen levels and are selected based upon the clinical scenario

Clauss assay: A functional assay which is based upon the time for fibrin clot formation
● Diluted patient plasma is clotted using a high concentration of thrombin and then the clotting time is measured
● The high concentration of thrombin ensures that the clotting times are independent of the amount of thrombin present
● A calibration curve is generated by clotting times of dilutions of reference plasma samples of known fibrinogen concentrations
● The clotting time is inversely proportional to the amount of fibrinogen in the sample
● Most laboratories use an automated method based on optical density in which fibrin formation decreases light transmission and increases scattered light
● Values can be affected by turbid specimens (e.g. hyperbilirubinemia, hyperlipidemia)
● Values are falsely decreased by heparin > 0.6 units/mL or fibrin degradation products
● The most widely used laboratory method
● Typically used in investigation of bleeding diathesis, congenital and acquired fibrinogen abnormalities, DIC or thrombolytic therapy

Immunologcal assays: An immunological method based on measuring fibrinogen antigen rather than functional fibrinogen
● Uses antibodies directed against fibrinogen
● Techniques include enzyme linked immunoabsorbent assays (ELISA), radial immunodiffusion or electrophoresis
● Typically used in investigation of congenital fibrinogen disorders where there is a discrepancy between functional activity and antigen level

Clottable protein assay: Method based upon clot weight
● Thrombin is added to patient plasma in the absence of calcium ions and then the clot is washed and dissolved by alkaline urea or other reagents
● Spectrophotometry is performed, and since the majority of the protein present in the clot is fibrin, the protein concentration will be equivalent to the fibrinogen concentration
● Very labor intensive and time consuming to perform
● Occasionally used in evaluation of congenital fibrinogen disorders

PT-based assay: Fibrinogen levels are indirectly measured (derived) and are calculated based on the prothrombin time (PT)
● A calibration curve is generated using prothrombin times and changes in optical density of a series of plasma dilutions of known fibrinogen concentration
● A PT is performed on the patient sample and the fibrinogen level is calculated based on the change in optical density
● Simple and inexpensive test
● Not recommended for routine laboratory use since values can vary based on type of reagents and analyzers used and therefore results are not interchangeable between laboratories or hospitals. Also, when compared to results given by the Clauss assay, the PT-based method has been shown to produce higher values in certain conditions such as liver disease, DIC, renal disease, dysfibrinogenemia, and in those receiving anticoagulants or thrombolytic therapy

Note: Assays should not be performed on samples collected within 4 hours of heparin administration or on samples collected from heparin-contaminated arterial or venous lines, as heparin may lead to falsely low fibrinogen levels

Additional references
=========================================================================

Br J Haematol 2003;121:396, Fibrinogen Assays



Coagulation laboratory tests

Factor V Leiden assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Term is used interchangeably with activated protein C resistance (APCR) since Factor V Leiden causes most (but not all) cases of APCR
● Test 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

Diagrams
=========================================================================



Testing algorithm

Additional references
=========================================================================

Arch Pathol Lab Med 1998;122:430



Coagulation laboratory tests

Factor VII assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 20 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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

Additional references
=========================================================================

Haemostasis 1983;13:161



Coagulation laboratory tests

Factor VIII assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 21 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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

Additional references
=========================================================================

Kessler, C. (2007). Hemorrhagic disorders: Coagulation factor deficiencies. In L. Goldman, D. Ausiello (Eds.), Cecil Medicine (pp.1301-1313). Philadelphia, PA: Saunders Elsevier.



Coagulation laboratory tests

Factor VIII inhibitor assay / Bethesda assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 21 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

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

Additional references
=========================================================================

Am J Clin Pathol 2009;131:552



Coagulation laboratory tests

Factor IX assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 11 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● To detect congenital or acquired factor IX deficiency
● Congenital factor IX deficiency (hemophilia B) is inherited as a sex-linked recessive in 1 per 30,000 males
● Acquired factor IX deficiency is seen in liver disease, vitamin K deficiency, Coumadin therapy
● A linear, dose-dependent, false decrease is caused by bivalirudin, lepirudin, argatroban, and fondaparinux (Arch Pathol Lab Med 2004;128:1142)

Additional references
=========================================================================

Goldman: Cecil Medicine (23rd ed, Chapter 180)



Coagulation laboratory tests

Factor Xa assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 21 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● Used to determine levels of heparin, low molecular weight heparin, danaparoid, etc.
● 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
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; these drugs usually do not 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

Sampling
=========================================================================

● Draw specimen 4 hours after subcutaneous injection of low molecular weight heparin or 6 hours after subcutaneous injection of danaparoid 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

Additional references
=========================================================================

Thromb Haemost 2012;107:379



Coagulation laboratory tests

Factor XI assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 13 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

● 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

Additional references
=========================================================================

J Clin Pathol 1975;28:332



Coagulation laboratory tests

Factor XIII assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 21 June 2012, last major update June 2012
Copyright: (c) 2002-2012, PathologyOutlines.com, Inc.

General
=========================================================================

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
● 2% acetic acid can also be used as a clot stability screening reagent, but will only detect patients down to 4% deficiency

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
● Newborns may have lower levels than adults

Additional references
=========================================================================

Parsons, J.C. Factor XIII Deficiency. LabQ 2012 (ASCP);CL-11:79



Coagulation laboratory tests

Heparin induced thrombocytopenia


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 9 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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

4-T score
=========================================================================

● Developed to determine pre-test probability of HIT by assigning scores of 0, 1 or 2 in 4 clinical categories: (a) thrombocytopenia severity, (b) timing of symptoms in relation to heparin exposure, (c) presence or absence of thrombosis and (d) possible alternatives to the symptoms
● Score 0-3: HIT very unlikely; 4-5: intermediate probability; 6-8: high likelihood (Postgrad Med J 2007;83:575)



4-T score chart


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)



Coagulation laboratory tests

Heparinase / heparin contamination assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 9 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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)

Note:
● 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
● The PTT may never totally correct in normal patients with large amounts of heparin contamination



Hereditary bleeding disorders

High molecular weight kininogen deficiency / assay


Reviewers: Julie Gober-Wilcox, M.D., Kendall Crookston, M.D., Ph.D., Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update July 2011
Copyright: (c) 2002-2011, PathologyOutlines.com, Inc.

Definition
=========================================================================

● High molecular weight kininogen deficiency is a rare congenital disorder inherited as an autosomal recessive trait, not associated with a bleeding diathesis
● Typically discovered in individuals with an isolated prolonged PTT

Terminology
=========================================================================

● High molecular weight kininogen is also known as Fitzgerald factor, Williams factor and Flaujeac factor

Pathophysiology
=========================================================================

● High molecular weight kininogen is a protein produced by the liver (with no inherent catalytic activity) that is involved in the early steps of the intrinsic coagulation pathway; it functions as a cofactor and binds with prekallikrein and factor XI to help facilitate their activation by factor XIIa
● The kininogens (low and high molecular weight) are also involved in the kinin-kallikrein system and function to inhibit thrombin activation of platelets and stimulate liberation of nitric oxide, prostacyclin and tissue plasminogen activator

Clinical features
=========================================================================

● Not associated with bleeding

Laboratory
=========================================================================

● Isolated prolonged PTT
● Negative lupus anticoagulant
● Specific assay to test for deficiency uses high molecular weight kininogen deficient plasma mixed with patient plasma; a PTT is performed and is compared to a standard curve of high molecular weight kininogen vs. PTT
● Interference occurs in these assays if patient is on heparin, hirudin or argatroban, possibly danaparoid
● Lower levels in newborns, increase to adult levels by age 6 months

Case reports
=========================================================================

● 66 year old man evaluated for cardiac surgery (Thromb Haemost 2001;85:195)

Differential diagnosis
=========================================================================

Factor XII deficiency
Lupus anticoagulant



Coagulation laboratory tests

Homocysteine assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Suspected risk factor for arerial or venous thrombosis, although evidence is weak (Arch Pathol Lab Med 2002;126:1367)
● 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:
● High performance liquid chromatography is standard (Am J Clin Pathol 2008;130:969)
● 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
● Conventional amino acid analyzer with separation column (slow, but can also detect related amino acids, such as methionine, cystathionine and cysteine)



Coagulation laboratory tests

Hypercoagulation panel


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 January 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Panels are useful to identify all factors predisposing to thrombosis; vary by institution
● 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 and 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
● Note: clinicians often confuse Factor V Leiden (to workup thrombosis) with Factor V assay (not the correct test); prevent with use of hypercoagulation panel



Coagulation laboratory tests

International normalized ratio (INR)


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Used to standardize prothrombin time (PT) results for patients taking warfarin (coumadin)
● Intended to make comparisons similar between different labs by compensating for variable thromboplastins used in PT test
● Often used as an arbitrary level of “bleeding likelihood” (J Clin Pathol 2003;56:48 (recommendations for reporting))
● Generally only helpful when a patient is on warfarin and one is evaluating if patient is currently in warfarin therapeutic ranges, or if a warfarin reversal is being considered for upcoming intervention
● Patient self management may be effective (J Clin Pathol 2002;55:845 (patient self testing))

Interpretation
=========================================================================

● Therapeutic goal is usually a value of 2 to 3
● 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 (Arch Pathol Lab Med 2004;128:308)
● Results are affected by different thromboplastin reagents, not by storage at room temperature for up to 24 hours (Arch Pathol Lab Med 1998;122:972)



Coagulation laboratory tests

International sensitivity index (ISI)


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Measure of sensitivity of particular PT reagent - determined by manufacturer
● Used to resolve interlaboratory variations in PT (Arch Pathol Lab Med 2004;128:308 (ISI calibration), J Clin Pathol 2003;56:114 (ISI calibration for home PT monitors)
● 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



Coagulation laboratory tests

Low molecular weight heparin (LMWH)


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Recommended to monitor using chromogenic antifactor Xa assay on specimens obtained up to 4 hours after subcutaneous injection of LMWH (Chest 2001;119(1 Suppl):64S)
● 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



Coagulation laboratory tests

Lupus anticoagulant


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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 (Arch Pathol Lab Med 2002;126:1424, Kelley's Textbook of Rheumatology (9th ed, 2012))
● 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, Arch Pathol Lab Med 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) and (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 6-12 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 and other coagulopathies
● Values prolonged by bivalirudin, lepirudin, argatroban and fondaparinux (Arch Pathol Lab Med 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)



Coagulation laboratory tests

Mixing studies


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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 (Thromb Res 2007;119:369); 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



Coagulation laboratory tests

Plasminogen assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

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
● Either functional (based on plasmin activity, Haemostasis 1988;18 Suppl 1:47) 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

Interpretation
=========================================================================

● Plasminogen levels are increased by oral contraceptives (which increase cholesterol levels), pregnancy and acute phase reactants
● Plasminogen levels are decreased by liver disease, thrombolytic therapy and DIC; newborns have levels that are 60% of adults, increase to adult values by age 6 months



Coagulation laboratory tests

Plasminogen activator antigen-1


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Uncommon test; perform if strong evidence of familial bleeding disorder, but normal results for von Willebrand disease or possibly if unexplained premature myocardial infarction
● May predict severe hepatic veno-occlusive disease after allogeneic bone marrow transplantation (Br J Haematol 2002;118:1087)
● Not a known risk factor for hypercoagulability (Arch Pathol Lab Med 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

Methodology
=========================================================================

Collection: collect blood from steadily flowing venipuncture, discard first 3-5 mL (if this is the only test) and 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



Coagulation laboratory tests

Platelet aggregation studies


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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 (Br J Haematol 1988;68:53)
● 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) or Bernard-Soulier disease (deficiency of platelet glycoprotein Ib, causes decreased ristocetin-induced aggregation only)

Methodology
=========================================================================

● Aggregometry with platelet-rich plasma to measure optical transmission or electric impendence (J Thromb Haemost 2009;7:1029)
● 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 and myeloproliferative disorders



Coagulation laboratory tests

Platelet antibodies


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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 (Blood 1997;89:1112)

Types of tests
=========================================================================

ELISA: test for specific antiplatelet antibodies; antigen of interest is bound to surface of microtiter plate, then add patient plasma and antibody will bind to antigen
Antigen capture immunoassay: specific antigens are bound to solid phase, then add patient serum and 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 (Am J Clin Pathol 1990;93:552)
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 non-immune (marrow suppression or non-immune 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 and bone marrow biopsy
● Although not technically contraindicated, platelet transfusions tend to be futile until the offending antibody is removed via some kind of immunosuppression

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
● Newborn can sometimes develop intracranial hemorrhage due to extremely low platelet count
● If possible, give platelets negative for the antigen the mother lacks; otherwise, treatment of choice is washed maternal platelets

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
● Typically antigen is PI-A1 component of GP IIb/IIIa or an HLA antigen the patient lacks
● Patients have sudden onset of severe thrombocytopenia 5-12 days after transfusion of platelet product, resolves 14 days after transfusion
● In thrombocytopenic phase, common anti-platelet antibodies (GPIIb-IIIa, GPIb-IX and GPIa-IIa) are present, in addition to antibodies to the specific antigen the donor platelets express that precipitated the event
● After resolution of the PTP episode, the general platelet antibodies will disappear, while the donor specific platelet antibodies will persist (Vox Sang 1999;76:120)



Coagulation laboratory tests

Platelet hyperaggregation studies


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Hyperaggregation may rarely be associated with hypercoagulability, including myocardial infarction, strokes and 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

Additional references
=========================================================================

Semin Thromb Hemost 1999;25:361, The Laboratory Test Handbook 2001:327



Coagulation laboratory tests

Prekallikrein assay


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 Feburary 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Prekallikrein is also known as Fletcher factor
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 determined from a standard curve of prekallikrein vs. PTT (Thromb Res 2002;105:463)
● Interference is due to hirudin, argatroban, danaparoid and 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



Coagulation laboratory tests

Protein C assays


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

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)

General
=========================================================================

● Deficiencies are either quantitative (type I - reduced amount of normal protein) or qualitative (type II - normal amount of defective protein, Thromb Haemost 1984;51:1)
● 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 (Arch Pathol Lab Med 2002;126:1337)
● 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
=========================================================================

● Detect 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 or 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

Other assays
=========================================================================

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

Interpretation
=========================================================================

● Values falsely increased by bivalirudin, lepirudin, argatroban and fondaparinux (Arch Pathol Lab Med 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

Acquired causes of altered levels
=========================================================================

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 and 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 and oral contraceptives



Coagulation laboratory tests

Protein S assays


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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
● 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

Reference ranges (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

Acquired causes
=========================================================================

● 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 or thrombosis; also nephrotic syndrome, varicella infection and 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

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, Thromb Res 1995;77:375)
● Values falsely increased by bivalirudin, lepirudin, argatroban and fondaparinux (Arch Pathol Lab Med 2004;128:1142), lupus anticoagulants



Coagulation laboratory tests

Prothrombin gene 20210A testing


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 10 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Mutation in G to A transition at nucleotide 20210 in 3’ untranslated portion of prothrombin gene, which introduces a new Hind III restriction site
● Can identify heterozygotes and homozygotes
● G20210A mutation in heterozygotes is associated with increased risk of first venous thromboembolic episode (Br J Haematol 2001;113:630)
● Multiplexed arrays test for factor V Leiden, MTHFR C677T and other sequences
● Specimen is whole blood

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



Coagulation laboratory tests

PT - Prothrombin time


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 11 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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
● 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%, Arch Pathol Lab Med 1997;121:956)
● Test should use a thromboplastin that is insensitive to heparin in therapeutic range
● PT is more sensitive to deficiencies in common pathway than aPTT

Warfarin monitoring
=========================================================================

● 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 INR = (patient PT/mean normal PT)ISI, where ISI is the International Sensitivity Index which is used to calibrate a particular batch of thromboplastin reagent to a universal standard (see below)
● 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 need to check decreases gradually to every 4 weeks
● May be improved by instrument-specific International Sensitivity Index (ISI) values, in-house calibrators or calibration curves (Arch Pathol Lab Med 2004;128:308); ISI measures sensitivity of PT reagent to factor deficiencies (1.0 is sensitive, 3.0 is insensitive, value determined by manufacturer)

Interpretation
=========================================================================

● 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 and 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) and 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) and DIC
● Markedly prolonged values may be due to long acting warfarin-like rodenticide toxicity (Arch Pathol Lab Med 2004;128:e181)

Algorithm for working up a prolonged PT
=========================================================================

● (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)



Coagulation laboratory tests

aPTT - Activated partial thromboplastin time


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 11 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Also called partial thromboplastin time (PTT); originally described in 1953 (J Lab Clin Med 1953;41:637)
● 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
● Used to monitor heparin or direct thrombin inhibitors such as hirudin

Methodology
=========================================================================

● 3.2% citrate tube is recommended; use of 3.8% citrate as anticoagulant causes prolonged PTT if samples are < 90% filled compared to 100% filled (no difference in result with 3.2% citrate between filled volumes of 60% and 100%)
● 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
● Mix patient plasma with excess calcium (to counteract the citrate anticoagulant), 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

Interpretation
=========================================================================

● Target ratio is 1.5 to 3.0 (compared to nonheparinized samples); abnormal values may cause bleeding, thrombosis, morbidity or death
● Markedly prolonged values may be due to long acting warfarin-like rodenticide toxicity (Arch Pathol Lab Med 2004;128:e181)
● Values are normally higher in newborns (up to 55 seconds), decrease to adult levels at age 6 months; values are smaller with acute phase reactions, which elevate Factor VIII levels
● INR differs with 3.8 vs. 3.2% citrate (Arch Pathol Lab Med 1997;121:956)

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

Algorithms for working up a prolonged PTT:
=========================================================================

● Add heparinase; if PTT corrects to normal, prolongation is due to presence of heparin
● 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
     ● 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
     ● 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 first step above) or rare factor inhibitors
     ● 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



Coagulation laboratory tests

Reptilase time


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 11 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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); also prolonged by increased fibrinogen associated with acute phase reaction (Am J Clin Pathol 2002;118:263)
● Used to diagnose dysfibrinogenemia (also thrombin time)
● Not prolonged by heparin or hirudin (unlike thrombin time)



Coagulation laboratory tests

Thrombin time


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 11 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● Measures rate of fibrin clot formation after addition of standard concentration of thrombin to citrated plasma (Practical Hemostasis and Thrombosis 2009:53)
● Thrombin cleaves fibrinogen, releasing fibrinopeptides A and B, 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 or thrombin inhibitors in patients exposed to bovine thrombin



Coagulation laboratory tests

tPA


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 11 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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
● 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
● Higher levels in patients with acute paraquat intoxication (J Korean Med Sci 2011;26:474)

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



Coagulation laboratory tests

von Willebrand disease testing - general


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 11 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

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) or multimer analysis

Interpretation (Thromb Haemost 1994;71:520):
● All results normal (considering ABO blood type) - unlikely to have vWD if no acute phase reaction, pregnancy, estrogen use or 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 and factor VIII - likely type 1 vWD
● Severely reduced (< 10%) or undetectable antigen, activity and 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



Coagulation laboratory tests

von Willebrand factor antigen assay (vWF)


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 11 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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%) and type AB (123%); although bleeding symptoms may depend on vWF antigen levels, regardless of ABO type
● 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

Methodology
=========================================================================

● ELISA assay measures quantity of vWF, not function (Can J Vet Res 2008;72:420)
● Can also use latex particles coated with anti-vWF antibodies, measure light absorbance; rocket immunoelectrophoresis



Coagulation laboratory tests

von Willebrand factor activity


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 11 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● 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; appropriate term is actually “agglutination”
Other functional tests: collagen-binding ELISA assay, functional vWF binds to collagen and is detected (Br J Haematol 2002;116:187)

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



Coagulation laboratory tests

von Willebrand factor multimer analysis


Reviewer: Jeremy Parsons, M.D. (see Reviewers page)
Revised: 11 February 2013, last major update November 2012
Copyright: (c) 2002-2013, PathologyOutlines.com, Inc.

General
=========================================================================

● To detect type 2 von Willebrand disease
● Involves separation of multimers by size using agarose gel electrophoresis of patient’s plasma (Thromb Res 2010;126:543)
● 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 > Superpage


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