Coagulation

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 elite - based tests)

Activated protein C resistance / Factor V Leiden

Table of Contents

Definition / 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

Etiology

Factor V Leiden mutations:
- 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:
- Factor V Hong Kong (Arg306Gly)
- HR2 haplotype with mutation 4070A to G (His199Arg) in exon 13 of Factor V gene (associated with other polymorphisms)

Diagrams / tables

Images hosted on other servers:

Protein C pathway

Laboratory

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

Case reports

51 year old woman with heterozygous factor V Leiden and dural sinus thrombosis (Arch Pathol Lab Med 2003;127:1359)

Treatment

Treat venous thromboemboli similarly regardless of the presence of factor V Leiden

Additional references

Algorithm for workup of hereditary bleeding disorders

Table of Contents

Definition / 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

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

Diagrams / 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 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	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 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: 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, Clin Lab Haematol 2000;22:9

Amyloidosis

Table of Contents

Definition / 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:
- Binding to amyloid fibrils primarily in the liver and spleen
- 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))

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

Factitious purpura
Hemophilias
Heparin
Scurvy
Thrombocytopenia (platelet count Vitamin K deficiency
Warfarin ingestion

Additional references

Blood 2009;114:3147, eMedicine - Factor X Deficiency

Anticardiolipin antibodies

Antiplasmin assay

Table of Contents

Definition / 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 ( 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

Antiplatelet agents (pending)

[Pending]

Antithrombin assay

Table of Contents

Definition / general | Etiology | Laboratory | Additional references

Definition / 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 patients antithrombin
- 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)
- 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

Etiology

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

Laboratory

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)

Additional references

Arch Pathol Lab Med 2002;126:1326, Thromb Diath Haemorrh 1965;13:516

Antithrombin deficiency

Table of Contents

Definition / general | Etiology | Treatment | Molecular / cytogenetics description | Additional references

Definition / general

Hereditary deficiencies occur in 0.07 - 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

Etiology

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%)

Molecular / cytogenetics description

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

Additional references

Bleeding time

Table of Contents

Definition / 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 patients 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 - 9.5 minutes (less in newborns)
Prolonged values:
- Platelet count less than 100K, low hemoglobin, use of aspirin or other platelet inhibitors
- Also von Willebrands and other hereditary platelet disorders, uremia

Additional references

Blood 1991;77:2547

Clot retraction

Table of Contents

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 - overview

Table of Contents

Definition / 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

Laboratory

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)
Do not draw specimens from indwelling catheters (which contain anticoagulants)
If multiple tubes are drawn, draw coagulation tube after the red top and before the EDTA, heparin or oxalate / fluoride tubes
Try to fill the sample tube completely
Notify laboratory if patient is on anticoagulants and specify which ones
Do not 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

General algorithm

Bleeding tests

Clotting tests

Additional references

Arch Pathol Lab Med 2005;129:47

Cryoglobulin / cryofibrinogen assays

Table of Contents

Definition / general | Clinical images | Additional references

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
- Dont 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

D-dimer / dimerized plasmin fragment D

Table of Contents

Definition / 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 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 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+ Castlemans 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

Images hosted on other servers:

Fibrin split products / D-dimer

Additional references

Antovic, J.P. and Egberg, N: Laboratory investigations, Essential Guide to Blood Coagulation, 1st Ed., 2010

Dabigatran

Table of Contents

Definition / general | Diagrams / tables

Definition / 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;52:119S, J Med Econ 2012;15:695) 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;98:186)
INR levels are not necessary, and are substantially higher using Hemochron Jr. point-of-care device compared with laboratory values (Am J Med 2012;125:417)
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)

Diagrams / tables

Images hosted on other servers:

Structure and site of inhibition in coagulation cascade

Direct oral anticoagulants (pending)

[Pending]

Disseminated intravascular coagulation (DIC)

Table of Contents

Definition / general

First descriptions of DIC appeared in the 19th century (Semin Thromb Hemost 2014;40:874)
DIC is a systemic activation of the coagulation system, which results in microvascular thrombosis and simultaneously potentially life threatening hemorrhage attributed to consumption of platelets and coagulation factors
DIC is a complication to various underlying clinical conditions, including infection, malignancies, obstetrical complications, trauma (especially head trauma) and vascular disorders (e.g. Kasaback-Merritt syndrome, aortic aneurysms, etc.); of these conditions, infection related DIC is most common
Based on the severity and stage, DIC can be categorized as nonovert (early) and overt (decompensated); DIC can present either acutely or chronically and can be subclinical
In general, DIC patients can suffer from both bleeding and thrombosis, although thrombosis may not be readily apparent

Essential features

Essential features of DIC can be classified as clinical and laboratory features

Terminology

Most widely used definition of DIC was issued by a subcommittee of the Scientific and Standardization Committee (SSC) of the International Society on Thrombosis and Hemostasis in 2001 (Thromb Haemost 2001;86:1327)

ICD coding

ICD-10: D65 - Disseminated intravascular coagulation (defibrination syndrome)

Epidemiology

Prevalence of DIC in patients with various medical conditions:
- 1% of hospitalized patients are estimated to develop DIC
- 10 - 30% of patients in the intensive care unit (ICU)
- 20% of patients with acute respiratory distress syndrome (ARDS)
- 30 - 40% of patients with severe head trauma
- 30 - 60% of patients with severe sepsis
See Table 1 for secondary causes
Reference: Br J Haematol 2021;192:803

Pathophysiology

Increased tissue factor activity and thrombin generation
- Tissue factor is expressed on circulating activated monocytes in sepsis related DIC and on the surface of the malignant cells or circulating tumor derived microparticles in cancer related DIC
- In obstetric DIC, placental abruption and amniotic fluid embolism expose the circulating blood to tissue factor
- Through activation of coagulation factor (F) VII and FX, increased tissue factor activity leads to thrombin generation
Increased platelet activation in DIC occurs through interaction with activated endothelium and the direct action of thrombin on platelets; in sepsis related DIC, inflammatory cells, cytokines and pathogens interact directly with platelets and contribute to their activation
Levels of antithrombin, protein C and protein S decrease due to consumption, decreased synthesis in the liver and degradation by neutrophil elastase
Fibrinolysis can be either impaired (hypofibrinolysis) or enhanced (hyperfibrinolysis) according to the pathophysiology of the underlying disease
- Both tissue plasminogen activator (tPA) and plasminogen activator inhibitor 1 (PAI1) are released from the activated endothelium, such as in patients with vascular malformations (see Table 1)
- In sepsis, the net effect is impaired fibrinolysis in most cases; in contrast, cancer is often associated with increased fibrinolytic activity
- Primary hyperfibrinolysis is a common feature of acute promyelocytic leukemia; these patients often present with severe hyperfibrinolysis and bleeding due to increased plasminogen activation on the malignant cell surface
Reference: Br J Haematol 2021;192:803

Diagrams / tables

Images hosted on other servers:

Clinical diagnosis / management of patients with DIC

Key events in DIC

Table 1: secondary causes

Condition	Examples
Severe infectious diseases	Gram positive or negative organisms, malaria, hemorrhagic fevers
Malignancy	Solid tumors (e.g. adenocarcinomas), acute promyelocytic leukemia or monocytic leukemia
Trauma	Multitrauma, brain injury, burns
Obstetrical complications	Abruptio placentae, amniotic ﬂuid embolism
Vascular malformations	Kasabach-Merrit syndrome, giant hemangiomas Other vascular malformations, large aortic aneurysms
Severe immunologic reactions	Transfusion reaction
Heat stroke
Postcardiopulmonary resuscitation

Table 2: DIC scoring systems by the JAAM and the ISTH (AMIA Annu Symp Proc 2015;2015:804)

Japanese Association for Acute Medicine (JAAM)
*SIRS criteria**
≥ 3	+1
0 to 2	0
Platelet count
< 80 × 10⁹/L or > 50% decrease within 24 hours	+3
≥ 80 < 120 × 10⁹/L or > 30% decrease within 24 hours	+1
≥ 120 × 10⁹/L	0
Prothrombin time (value of patient / normal value)
≥ 1.2	+1
< 1.2	0
Fibrin / fibrinogen degradation products
≥ 25 mg/L	+3
≥ 10 < 25 mg/L	+1
< 10 mg/L	0
Diagnosis: if ≥ 4, there is positive diagnosis of DIC
*Systemic inflammatory response syndrome

International Society of Thrombosis and Hemostasis (ISTH)
Platelet count
< 50 × 10⁹/L	+2
≥ 50 < 100 × 10⁹/L	+1
≥ 100 × 10⁹/L	0
Elevated fibrin related marker
Strong increase	+3
Moderate increase	+2
No increase	0
Prolonged prothrombin time
≥ 6 seconds	+2
≥ 3 < 6 seconds	+1
< 3 seconds	0
Fibrinogen level
< 100 g/mL	+1
≥ 100 g/mL	0
Diagnosis: if > 5, there is positive diagnosis of overt DIC; if < 5, suggestive (not affirmative) of nonovert DIC

Clinical features

Without adequate treatment, DIC can eventually lead to multiorgan dysfunction / failure
Patients can present with bleeding, thrombosis or both
Septic patients are more likely to have thrombosis than bleeding
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
Causes of DIC can be acute (meningococcemia), 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
Without adequate treatment, DIC can eventually lead to multiorgan dysfunction / failure
Reference: Br J Haematol 2021;192:803

Diagnosis

Most widely used DIC diagnostic scoring systems are the JAAM and ISTH (Crit Care 2016;20:287, Thromb Haemost 2001;86:1327)
- See Table 2 for a comparison of both scoring systems

Laboratory

Prolonged prothrombin time (PT), activated partial thromboplastin time (APTT) and thrombin time (TT)
Elevated D dimers and other fibrin degradation products (but D dimer may be falsely positive in HIV+ Castleman disease due to interference from monoclonal gammopathy (Am J Clin Pathol 2004;122:178, Arch Pathol Lab Med 2004;128:328)
Fall in platelet count (usually not lower than 30,000 - 40,000 x 10⁹/L)
Consumptive deficiency of fibrinogen, antithrombin, alpha 2 antiplasmin and plasminogen
Presence of schistocytes and microspherocytes on peripheral blood smear
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

DIC is a devastating condition with a poor prognosis; the clinical course is primarily determined by the age of the patient, presence of comorbidities, identification and treatment of underlying etiologies, initial treatment response and severity of organ dysfunction, including the degree of hemostatic abnormalities
A 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)
48 year old woman, 63 year old man and 68 year old man with DIC (Blood 2018;131:845)
66 year old woman with DIC due to underlying lymphoma (Chest 2017;151:e41)

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 (ASFA Category III indication), platelet transfusions and immunoabsorption
- Because prothrombin concentrates (PCC) may increase the risk for thromboembolism, they are not recommended for patients with DIC, with an only rare exception in patients with potentially life threatening coagulation factor deficiency when no other alternative treatment is available
If platelet count is lower than 50,000 x 10⁹/L with active bleeding or lower than 10,000 x 10⁹/L, give platelet transfusion
If thrombosis predominates (chronic DIC), heparinization should be considered
See Diagram 1 for diagnosis and treatment strategies
Reference: Br J Haematol 2021;192:803

Board review style question #1

Tissue factor can be released from which of the following blood cells during infection?

Eosinophils
Lymphocytes
Monocytes
Neutrophils
Red cells

Board review style answer #1

C. Monocytes

Comment Here

Reference: Disseminated intravascular coagulation (DIC)

Board review style question #2

Which of the following laboratory findings is typical in a patient with DIC?

Decreased fibrinogen
Elevated plasminogen
Elevated protein S and C
Normal clotting times (PT, APTT and TT)
Thrombocytosis

Board review style answer #2

A. Decreased fibrinogen

Comment Here

Reference: Disseminated intravascular coagulation (DIC)

Board review style question #3

Which of the following is true of disseminated intravascular coagulation?

Clotting times are usually shortened
DIC rarely happens in patients with cancer or a hematologic malignancy
Patient’s fibrinogen activity is usually elevated
Patients with promyelocytic leukemia may have markedly elevated D dimer levels
Treatment strategy should focus on stopping bleeding for virtually all cases of DIC

Board review style answer #3

D. Patients with promyelocytic leukemia have an increased risk of DIC and may show marked elevated D dimer levels

Comment Here

Reference: Disseminated intravascular coagulation (DIC)

Board review style question #4

Which of the following is the most common underlying condition or mechanism that leads to DIC?

Patient with breast cancer
Pregnant woman (30 weeks) with severe hypertension
Patient with end stage liver disease due to hepatitis C
Patient was involved in an automobile accident with fractures of his lower extremities
Leukemic patient who received chemotherapy and developed a high fever and hypotension

Board review style answer #4

E. Leukemic patient who received chemotherapy and developed a high fever and hypotension

Comment Here

Reference: Disseminated intravascular coagulation (DIC)

Dysfibrinogenemia

Table of Contents

Definition / general | Laboratory | Additional references

Definition / 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:
- defective thrombin binding to fibrin, causing increased thrombin in circulation and more thrombosis
- 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

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, Colman: Hemostasis and Thrombosis - Basic Principles and Clinical Practice (Periodicals), 4th Edition, 2000

Ecarin clotting time

Table of Contents

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 - 29.0 seconds
- Prolonged if hirudin or argatroban (direct thrombin inhibitors) present; also hypofibrinogenemia, dysfibrinogenemia
- False positive if prothrombin deficiency is present

Elevated coagulation factor levels in plasma

Table of Contents

Definition / 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:
  - factor XI assays are not generally available
  - one step clotting assay and PTT based assays may be too variable
  - 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

Factor assays

Table of Contents

Definition / 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 patients plasma
Used to determine the etiology of a prolonged PT or PTT
Factor levels are expressed as a percentage of normal plasma concentration, or units per mL of normal plasma
Reference range is often 60 - 140% (should be determined based on the laboratorys 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: Haemostasis, 1st ed., 2004

Factor I (fibrinogen) assay

Table of Contents

Definition / general

Major plasma protein that is synthesized by the liver and is converted to fibrin by thrombin
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
Levels can be increased in an acute phase reaction, malignancy, females (particularly post-menopausal), pregnancy, oral contraceptive use
Reference range 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
- Immunological 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

Factor I (fibrinogen) deficiency

Table of Contents

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 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: Fibrinogen [Accessed 18 June 2020])
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: autoimmune disease, DIC, liver disease, medications

Additional references

Semin Thromb Hemost 2009;35:356, Haemophilia 2008;14:1151, Kitchens: Consultative Hemostasis and Thrombosis, 2nd Edition, 2007

Factor II (prothrombin) deficiency

Table of Contents

Definition / general

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

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

40 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

Diagrams / tables

Images hosted on other servers:

Coagulation cascade

Clinical features

Complete deficiency is incompatible with life
Severe hemorrhage typically occurs when levels are 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 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

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

Additional references

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

Factor IX deficiency (hemophilia B)

Table of Contents

Definition / general

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:
- 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 ( 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

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

Additional references

Haemophilia 2010;16 Suppl 6:1, Haemophilia 2010;16 Suppl 6:13, Guidelines for the Management of Hemophilia: World Federation of Hemophilia, 2012, Consultative Hemostasis and Thrombosis: Elsevier, 2007

Factor V deficiency

Table of Contents

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, Owrens 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 ( Levels dont 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: Elsevier, 2007, Haemophilia 2008;14:1164, Haemophilia 2009;15:1143, Semin Thromb Hemost 2009;35:382

Factor V Leiden assay

Table of Contents

Definition / general | Case reports

Definition / 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:

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)
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)
Calculate ratio of PTT with versus without exogenous activated protein C; normal is 2.0 or more, factor V Leiden usually
Many feel that positive results should be confirmed with a genetic assay
Other assays:
- Prothrombin - based factor V assay with factor V deficient plasma (no interference from lupus anticoagulant)
- Modified Russell viper venom time test (high phospholipids neutralizes lupus anticoagulant)
- Factor Xa - based assay with factor V deficient plasma
- 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

Additional references

Arch Pathol Lab Med 1998;122:430

Factor VII deficiency

Table of Contents

Definition / general

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

Epidemiology

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

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)

Sites

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

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

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Coagulation cascade

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, 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

Factor VIII deficiency (hemophilia A)

Table of Contents

Definition / general

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

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

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

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

Diagrams / tables

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Coagulation cascade

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 (
- 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 (Factor VIII inhibitor)
- Formation of factor VIII alloantibodies is highest in individuals with intron 22 inversions, large deletions and nonsense mutations (Blood Coagul Fibrinolysis 2003;14: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

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

Additional references

Hematol Oncol Clin North Am 2010;24:181, Blood 2007;110:815, Guidelines for the Management of Hemophilia: World Federation of Hemophilia, 2012, Consultative Hemostasis and Thrombosis: Elsevier, 2007

Factor X deficiency

Table of Contents

Definition / general

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

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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 ( A small percentage of patients develop factor X inhibitors with factor replacement therapy

Laboratory

Prolonged PT and PTT that correct with mixing study
Prolonged Russells 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

Factor Xa assay

Table of Contents

Definition / general | Laboratory | Interpretation | Additional references

Definition / general

Used to determine levels of heparin, low molecular weight heparin, danaparoid, etc.
Measures ability of heparin or other drugs in patients 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 dont 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
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

Laboratory

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

Interpretation

Low levels of factor Xa are due to:
- Not collecting specimen at right time or delayed transportation to lab (see Laboratory above)
- Higher therapeutic dose needed
High levels of factor Xa are due to:
- Renal failure
- Heparin contamination (specimen drawn from indwelling line containing heparin)
- Lower therapeutic dose needed

Additional references

Thromb Haemost 2012;107:379

Factor XI deficiency

Table of Contents

Definition / general

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

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

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

Diagrams / tables

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Coagulation cascade

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 erythematous
Combined familial factor deficiency
Heparin contamination
Liver dysfunction
Other factor deficiency causing prolonged PTT: factor VIII, IX, XII, but factor XII deficiency is not associated with bleeding manifestations
von Willebrand Disease

Additional references

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

Factor XII deficiency

Table of Contents

Definition / general

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%

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)

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

Etiology

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

Diagrams / tables

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Coagulation cascade

Mechanisms of factor XII activation

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
Factor XII deficiency in association with von Willebrand disease, factor VIII or factor IX deficiency
Heparin contamination
High molecular weight kininogen deficiency
Liver disease
Lupus anticoagulants
Prekallikrein deficiency

Additional references

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

Factor XIII deficiency

Table of Contents

Definition / 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)

Diagrams / tables

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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 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 - 20 U/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

Heparin

Table of Contents

Definition / general

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

Pathophysiology

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 doesnt 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

Diagrams / tables

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Heparin 2-D structure

Function of heparin

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

Laboratory

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

Treatment

Protamine sulfate (for emergency reversal of heparin)

Heparin - low molecular weight

Table of Contents

Definition / general | Diagrams / tables | Clinical features | Laboratory | Additional references

Definition / 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

Diagrams / tables

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Function of LMWH

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
Unlike regular heparin, does not as readily inhibit thrombin or factor IXa (this also contributes to a lower number of bleeding side effects)

Laboratory

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

Additional references

Clin Lab Med 2009;29:283

Heparin cofactor II deficiency

Table of Contents

Definition / 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)

Heparin induced thrombocytopenia

Table of Contents

Definition / general | Diagrams / tables | Diagnosis

Definition / 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

Diagrams / tables

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4-T score chart

Diagnosis

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)
4-T score:
- Developed to determine pre-test probability of HIT by assigning scores of 0, 1 or 2 in 4 clinical categories:
  - Thrombocytopenia severity
  - Timing of symptoms in relation to heparin exposure
  - Presence or absence of thrombosis
  - 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)

Heparin induced thrombocytopenia (HIT)

Table of Contents

Definition / general | Diagrams / tables | Clinical features | Treatment

Definition / general

Common complication of heparin therapy, may cause life threatening venous or arterial thrombosis (Postgrad Med J 2007;83:575)
Prevent by monitoring platelet count for at least 20 days after initiation of heparin therapy
Caused by formation of antibodies against heparin and platelet factor 4 complex
Testing: ELISA assay screen followed by serotonin release assay for conformation
Use 4T score (Online 4T Score Calculator) to evaluate pre-test probability of HIT

Diagrams / tables

Images hosted on other servers:

Pathophysiology of HIT

Clinical features

Clinical note: platelet counts often fall slightly during the first 24 hours of heparin treatment, unrelated to heparin induced thrombocytopenia

Treatment

Permanently discontinue heparin, avoid platelet transfusions
Recommended to not use low molecular weight heparin (may cross react) or warfarin (may cause venous limb gangrene)
Instead use danaparoid, hirudin or argatroban

Heparinase / heparin contamination assay

Table of Contents

Definition / general | Diagnosis

Definition / 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

Diagnosis

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 - general

Table of Contents

Definition / general

Hereditary bleeding disorders are a diverse group of diseases that occur due to platelet dysfunction or absence / deficiency of specific clotting proteins, resulting in abnormalities of primary or secondary hemostasis
Most common hereditary bleeding disorders:
- Von Willebrand disease
- Hemophilia A (factor VIII deficiency)
- Hemophilia B (factor IX deficiency)
Less common hereditary bleeding disorders:
- Factor I (fibrinogen) deficiency / dysfunction
- Factor II (prothrombin) deficiency
- Factor V deficiency
- Factor VII deficiency
- Factor X deficiency
- Factor XI deficiency (also known as hemophilia C)
- Factor XIII deficiency
Rare disorders:
- α2 antiplasmin deficiency
- Combined factor deficiencies: combined factor V and VIII, combined vitamin K dependent clotting factors deficiency
- Glanzmann thrombasthenia
- Bernard-Soulier syndrome
- Gray platelet syndrome

Essential features

Hereditary bleeding disorders could occur due to platelet dysfunction (i.e., primary hemostasis disorder) or absence / deficiency of specific clotting proteins (i.e., secondary hemostasis disorder)
Primary hemostasis: formation of a platelet plug which involves the endothelium, platelets and von Willebrand factor (vWF)
Secondary hemostasis: formation of fibrin plug by activated coagulation factors
Von Willebrand disease is the most common hereditary bleeding disorder (M = F), followed by hemophilia A and B (M > F)
Patients with primary hemostasis abnormalities are more likely to manifest mucocutaneous bleeding, while those with secondary hemostasis abnormalities tend to have muscle and joint bleeds

Overview

Hemostasis means stopping bleeding
Vasoconstriction is the initial response following vessel injury (Ann Med 2012;44:405)
Primary hemostasis involves formation of a weak platelet plug, where damaged endothelium exposes the procoagulant subendothelial matrix, leading to platelet adhesion by and von Willebrand factor (Ann Med 2012;44:405)
Normal platelet function can be summarized with the triple A mnemonic: adhesion, activation and aggregation (Hematol Oncol Clin North Am 2021;35:1069)
Secondary hemostasis involves the formation of cross linked fibrin by activated coagulation factors (Ann Med 2012;44:405)
Inheritance pattern can be autosomal dominant, recessive or X linked
In many patients, there is a family history of bleeding (Int J Lab Hematol 2018;40:6)

Epidemiology

Disorders of primary hemostasis
Bleeding disorder	Prevalence	Inheritance pattern / genetic mutation
Von Willebrand disease (vWD) Type I Type II (A, B, M and N subtypes) Type III	1 in 100 (based on abnormal laboratory results); 1 in 1,000 (symptomatic patients) (Hematol Oncol Clin North Am 2021;35:1085)	Chr 12p, VWF gene, multiple mutations identified Autosomal dominant Autosomal dominant (type 2N is autosomal recessive) Autosomal recessive
Glanzmann thrombasthenia	1 in 1,000,000	Autosomal recessive / chr 17q21, ITGA2B or ITGB3 genes (StatPearls: Glanzmann Thrombasthenia [Accessed 2 December 2021])
Bernard-Soulier syndrome	< 1 in 1,000,000	Autosomal recessive; rare cases of autosomal dominant inheritance / GPIbα, GPIbβ or GPIX genes on chromosomes 17p12, 22q11.2 and 3q21, respectively (Orphanet J Rare Dis 2006;1:46)
Gray platelet syndrome	Rare (Autosomal dominant, recessive or X linked recessive / chr 3p21.31, NBEAL2 gene (J Blood Med 2021;12:719)
Quebec platelet disorder	1 in 300,000 in Quebec, Canada	Autosomal dominant / chr 10q22, PLAU gene (Expert Rev Hematol 2011;4:137)
Wiskott-Aldrich syndrome	1 - 10 in 1,000,000	X linked recessive / Xp 11.22-23, WAS gene (StatPearls: Wiskott-Aldrich Syndrome [Accessed 2 December 2021])
Hermansky-Pudlak syndrome	1 - 9 in 1,000,000; 1 in 1,800 in Puerto Rico (Semin Respir Crit Care Med 2020;41:238)	Autosomal recessive / mutations in 1 of 10 genes (HPS1, AP3B1, HPS3, HPS4, HPS5, HPS6, DTNBP1, BLOC1S3, PLDN and AP3D1)
Chédiak-Higashi syndrome	Rare (Autosomal recessive / chr 1q42.1-q42.2, LYST gene (StatPearls: Chediak Higashi Syndrome [Accessed 2 December 2021])

Disorders of secondary hemostasis
Bleeding disorder	Prevalence	Inheritance pattern / genetic mutation
Factor I (fibrinogen) deficiency Afibrinogenemia Hypofibrinogenemia Dysfibrinogenemia Hypodysfibrinogenemia	1 in 1,000,000 True incidence is unknown; more common than afibrinogenemia > 500 reported cases Unknown	Chr 4, mutations in FGA, FGB or FGG genes (Blood Rev 2021;48:100793) Autosomal recessive Autosomal dominant or recessive Autosomal dominant Autosomal dominant
Factor II (prothrombin) deficiency	1 in 2,000,000	Autosomal recessive / chr 11p11.2, F2 gene (Hematol Oncol Clin North Am 2021;35:1181)
Factor V deficiency	1 in 1,000,000	Autosomal recessive / chr 1q24.2, F5 gene (Hematol Oncol Clin North Am 2021;35:1181)
Factor VII deficiency	1 in 500,000	Autosomal recessive / chr 13q34, F7 gene (Hematol Oncol Clin North Am 2021;35:1181)
Factor VIII deficiency (hemophilia A)	1 in 5,000 male births	X linked recessive / chr Xq28 (Haematologica 2019;104:1702)
Factor IX deficiency (hemophilia B)	1 in 30,000 male births	X linked recessive / chr Xq27 (Haematologica 2019;104:1702)
Factor X deficiency	1 in 500,000 - 1,000,000	Autosomal recessive / chr 13q34, F10 gene (Hematol Oncol Clin North Am 2021;35:1181)
Factor XI deficiency (hemophilia C)	1 in 1,000,000; 5% in Ashkenazi Jews	Autosomal recessive / chr 4q35.2, F11 gene (Expert Rev Hematol 2016;9:629)
Factor XIII deficiency	1 in 2,000,000 - 3,000,000	Autosomal recessive / FXIIA gene (chr 6p25.1) or FXIIIB gene (chr 1q31.3) (StatPearls: Factor XIII Deficiency [Accessed 2 December 2021])
Combined factor deficiencies Factors V and VIII Combined vitamin K dependent clotting factors deficiency (deficiency of factors II, VII, IX, X, protein C and protein S)	1 in 1,000,000	Autosomal recessive / mutations in LMAN1 gene (chr 18q21) or MCFD2 gene (chr 2p21) (Semin Thromb Hemost 2013;39:613) Autosomal recessive / VKORC gene (chr 16) or GGCX gene (chr 2) (Orphanet J Rare Dis 2010;5:21)
α2 antiplasmin deficiency	Unknown	Autosomal recessive / chr 17pter-p12, SERPINF2 gene (Haemophilia 2008;14:1250)
Plasminogen activator inhibitor type 1 (PAI1) deficiency	Unknown	Autosomal recessive / chr 7q21.3-22, SERPINE1 gene (Hematol Oncol Clin North Am 2021;35:1197)

Clinical features

Symptoms: bleeding associated with surgery, trauma, dental extractions, postpartum, circumcision or umbilical stumps, easy bruising, epistaxis, menorrhagia, gastrointestinal bleeding, hemarthrosis, soft tissue hematomas, hematuria, intracranial bleeding
Patients with primary hemostasis abnormalities are more likely to manifest mucocutaneous bleeding (Eur J Pediatr 2012;171:1)
Reasons to suspect hereditary platelet disorder (Int J Mol Sci 2021;22:4521):
1. Persistence of neonatal thrombocytopenia or onset of bleeding symptoms in childhood
2. Family history of thrombocytopenia or mucocutaneous bleeding
3. Association with other diseases (check for unique physical characteristics and findings on peripheral blood smear)
4. Bleeding out of proportion to the platelet count
Patients with secondary hemostasis abnormalities tend to have muscle and joint bleeds (Eur J Pediatr 2012;171:207)
Heterozygous patients have 30 - 60% of normal values of affected factors, usually with no or minor bleeding disorder (UpToDate: Rare Inherited Coagulation Disorders [Accessed 26 October 2021])
Factor I, X, XI or XIII deficient heterozygotes may have bleeding symptoms (UpToDate: Rare Inherited Coagulation Disorders [Accessed 26 October 2021])
Homozygous deficient patients have UpToDate: Rare Inherited Coagulation Disorders [Accessed 26 October 2021])
In hemophilia A and B, small differences in factor levels (i.e., 5%) may markedly affect the clinical presentation, disease course and management (UpToDate: Clinical Manifestations and Diagnosis of Hemophilia [Accessed 26 October 2021])

Laboratory

Basic screening tests include complete blood count (CBC), prothrombin time (PT) / activated partial thromboplastin time (aPTT), platelet function assay (e.g., PFA 100 / PFA 200), thrombin time, peripheral blood smear review (for platelet and erythrocyte morphology) and fibrinogen (Int J Lab Hematol 2018;40:6)
For suspected platelet disorders: platelet aggregation studies, bone marrow aspirate and biopsy, flow cytometry, electron microscopy, next generation sequencing (Hematol Oncol Clin North Am 2021;35:1069)
Testing for von Willebrand disease includes factor VIII activity, vWF antigen and vWF activity (measured by ristocetin cofactor activity or vWF GPIbM activity assay) (Pediatr Clin North Am 2013;60:1419)
- These results may lead to obtaining vWF multimer analysis and blood type (group O patients have reduced vWF antigen and activity) (Pediatr Clin North Am 2013;60:1419)
For suspected coagulation factor abnormalities: mixing studies, factor levels, Bethesda assay (to detect coagulation factor inhibitor titers) (Int J Lab Hematol 2018;40:6)
Factor XIII assay if delayed bleeding is present with normal PT and aPTT (measured by factor XIII activity assay or urea clot lysis method) (Hematol Oncol Clin North Am 2021;35:1171)

Treatment

Bleeding in patients with hereditary platelet disorders is managed with platelet transfusions, antifibrinolytic agents, DDAVP (desmopressin) or recombinant factor VIIa, depending on the defect (Hematol Oncol Clin North Am 2021;35:1069)
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 exceptions of factor V (which is treated with plasma) and factor II and factor X deficiencies (which are treated with prothrombin complex concentrates) (Haemophilia 2008;14:671)
For von Willebrand disease, use vWF concentrates, DDAVP (desmopressin; contraindicated in vWD type 2B) or antifibrinolytic agents (Hematology Am Soc Hematol Educ Program 2016;2016:683)

Differential diagnosis

Antiplatelet medications, such as aspirin, glycoprotein IIB / IIIA inhibitors, clopidogrel, ticlopidine
Anticoagulants (heparin, warfarin or other anticoagulants) (Wikipedia: Anticoagulant [Accessed 19 April 2022])
Liver disease:
Disseminated intravascular coagulation (DIC):
- Can be caused by sepsis, malignancy, trauma, obstetrical complications, among others
- Complete blood count may show increased white blood cell count and low platelet count
- Prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT), low fibrinogen, increased D-dimer
- Peripheral smear may show evidence of microangiopathic hemolytic anemia (MAHA), with schistocytes and helmet cells
Vitamin K deficiency:
- Results in deficiency in vitamin K dependent coagulation factors: factors II, VII, IX, X, protein C and S
- Certain patients are at increased risk: on warfarin, taking antibiotics, have fat malabsorption (celiac disease, cystic fibrosis, short bowel syndrome, on diet lacking in vitamin K)
Acquired von Willebrand syndrome:
- Due to any qualitative, structural or functional disorder of vWF
- Negative family history
- Lack of previous bleeding
- Decreased vWF activity to antigen ratio, normal vWF propeptide antigen, loss of high molecular weight multimers
- Often associated lymphoproliferative, myeloproliferative, cardiovascular and immunological disorders

Board review style question #1

What is the most common hereditary bleeding disorder?

Fibrinogen deficiency
Glanzmann thrombasthenia
Hemophilia A
Hemophilia B
Von Willebrand disease

Board review style answer #1

E. Von Willebrand disease

Comment Here

Reference: Hereditary bleeding disorders - general

Board review style question #2

Which of the following findings is suggestive of a primary hemostasis disorder?

Bleeding into joints
Mucosal bleeding
Negative family history of bleeding
Prolonged prothrombin time and activated partial thromboplastin time

Board review style answer #2

B. Mucosal bleeding

Comment Here

Reference: Hereditary bleeding disorders - general

Hereditary bleeding disorders - general

Table of Contents

Definition / general

Hereditary bleeding disorders are a diverse group of diseases that occur due to platelet dysfunction or absence / deficiency of specific clotting proteins and result in abnormalities of primary or secondary hemostasis
Most common hereditary bleeding disorders
- Von Willebrand disease
- Hemophilia A (factor VIII deficiency)
- Hemophilia B (factor IX deficiency)
Less common hereditary bleeding disorders
- Factor I (fibrinogen) deficiency / dysfunction
- Factor II (prothrombin) deficiency
- Factor V deficiency
- Factor VII deficiency
- Factor X deficiency
- Factor XI deficiency (also known as hemophilia C)
- Factor XIII deficiency
Rare disorders
- α2 antiplasmin deficiency
- Combined factor deficiencies: combined factor V and VIII, combined vitamin K dependent clotting factors deficiency
- Glanzmann thrombasthenia
- Bernard-Soulier syndrome
- Gray platelet syndrome

Essential features

Hereditary bleeding disorders could occur due to platelet dysfunction (i.e., primary hemostasis disorder) or absence / deficiency of specific clotting proteins (i.e., secondary hemostasis disorder)
Primary hemostasis: formation of a platelet plug that involves the endothelium, platelets and von Willebrand factor (vWF)
Secondary hemostasis: formation of fibrin plug by activated coagulation factors
Von Willebrand disease is the most common hereditary bleeding disorder (M = F), followed by hemophilia A and B (M > F)
Patients with abnormalities in primary hemostasis are more likely to manifest mucocutaneous bleeding, while those with abnormalities in secondary hemostasis tend to have muscle and joint bleeds

Overview

Hemostasis means stopping bleeding
Vasoconstriction is the initial response following vessel injury (Ann Med 2012;44:405)
Primary hemostasis involves the formation of a weak platelet plug, where damaged endothelium exposes the procoagulant subendothelial matrix, leading to platelet adhesion by von Willebrand factor (Ann Med 2012;44:405)
Normal platelet function can be summarized with the triple A mnemonic: adhesion, activation and aggregation (Hematol Oncol Clin North Am 2021;35:1069)
Secondary hemostasis involves the formation of cross linked fibrin by activated coagulation factors (Ann Med 2012;44:405)
Inheritance pattern can be autosomal dominant, recessive or X linked
In many patients, there is a family history of bleeding (Int J Lab Hematol 2018;40:6)

Epidemiology

Disorders of primary hemostasis
Bleeding disorder	Prevalence	Inheritance pattern / genetic mutation
Von Willebrand disease (vWD) Type I Type II (A, B, M and N subtypes) Type III	1 in 100 (based on abnormal laboratory results); 1 in 1,000 (symptomatic patients) (Hematol Oncol Clin North Am 2021;35:1085)	Chr 12p, VWF gene, multiple mutations identified Autosomal dominant Autosomal dominant (type 2N is autosomal recessive) Autosomal recessive
Glanzmann thrombasthenia	1 in 1,000,000	Autosomal recessive / chr 17q21, ITGA2B or ITGB3 genes (StatPearls: Glanzmann Thrombasthenia [Accessed 1 November 2024])
Bernard-Soulier syndrome	< 1 in 1,000,000	Autosomal recessive; rare cases of autosomal dominant inheritance / GPIbα, GPIbβ or GPIX genes on chromosomes 17p12, 22q11.2 and 3q21, respectively (Orphanet J Rare Dis 2006;1:46)
Gray platelet syndrome	Rare (Autosomal dominant, recessive or X linked recessive / chr 3p21.31, NBEAL2 gene (J Blood Med 2021;12:719)
Quebec platelet disorder	1 in 300,000 in Quebec, Canada	Autosomal dominant / chr 10q22, PLAU gene (Expert Rev Hematol 2011;4:137)
Wiskott-Aldrich syndrome	1 - 10 in 1,000,000	X linked recessive / Xp 11.22-23, WAS gene (StatPearls: Wiskott-Aldrich Syndrome [Accessed 1 November 2024])
Congenital amegakaryocytic thrombocytopenia	Rare (Autosomal recessive / chr 1p34.2, MPL gene (Haematologica 2021;106:2439)
Hermansky-Pudlak syndrome	1 - 9 in 1,000,000; 1 in 1,800 in Puerto Rico (Semin Respir Crit Care Med 2020;41:238)	Autosomal recessive / mutations in 1 of 10 genes (HPS1, AP3B1, HPS3, HPS4, HPS5, HPS6, DTNBP1, BLOC1S3, PLDN and AP3D1)
Chédiak-Higashi syndrome	Rare (Autosomal recessive / chr 1q42.1-q42.2, LYST gene (StatPearls: Chediak-Higashi Syndrome [Accessed 1 November 2024])

Disorders of secondary hemostasis
Bleeding disorder	Prevalence	Inheritance pattern / genetic mutation
Factor I (fibrinogen) deficiency Afibrinogenemia Hypofibrinogenemia Dysfibrinogenemia Hypodysfibrinogenemia	1 in 1,000,000 True incidence is unknown; more common than afibrinogenemia > 500 reported cases Unknown	Chr 4, mutations in FGA, FGB or FGG genes (Blood Rev 2021;48:100793) Autosomal recessive Autosomal dominant or recessive Autosomal dominant Autosomal dominant
Factor II (prothrombin) deficiency	1 in 2,000,000	Autosomal recessive / chr 11p11.2, F2 gene (Hematol Oncol Clin North Am 2021;35:1181)
Factor V deficiency	1 in 1,000,000	Autosomal recessive / chr 1q24.2, F5 gene (Hematol Oncol Clin North Am 2021;35:1181)
Factor VII deficiency	1 in 500,000	Autosomal recessive / chr 13q34, F7 gene (Hematol Oncol Clin North Am 2021;35:1181)
Factor VIII deficiency (hemophilia A)	1 in 5,000 male births	X linked recessive / chr Xq28 (Haematologica 2019;104:1702)
Factor IX deficiency (hemophilia B)	1 in 30,000 male births	X linked recessive / chr Xq27 (Haematologica 2019;104:1702)
Factor X deficiency	1 in 500,000 - 1,000,000	Autosomal recessive / chr 13q34, F10 gene (Hematol Oncol Clin North Am 2021;35:1181)
Factor XI deficiency (hemophilia C)	1 in 1,000,000; 5% in Ashkenazi Jews	Autosomal recessive / chr 4q35.2, F11 gene (Expert Rev Hematol 2016;9:629)
Factor XIII deficiency	1 in 2,000,000 - 3,000,000	Autosomal recessive / FXIIA gene (chr 6p25.1) or FXIIIB gene (chr 1q31.3) (StatPearls: Factor XIII Deficiency [Accessed 1 November 2024])
Combined factor deficiencies Factors V and VIII Combined vitamin K dependent clotting factors deficiency (deficiency of factors II, VII, IX, X, protein C and protein S)	1 in 1,000,000 < 15 families reported	Autosomal recessive / mutations in LMAN1 gene (chr 18q21) or MCFD2 gene (chr 2p21) (Semin Thromb Hemost 2013;39:613) Autosomal recessive / VKORC gene (chr 16) or GGCX gene (chr 2) (Orphanet J Rare Dis 2010;5:21)
α2 antiplasmin deficiency	Unknown	Autosomal recessive / chr 17pter-p12, SERPINF2 gene (Haemophilia 2008;14:1250)
Plasminogen activator inhibitor type 1 (PAI1) deficiency	Unknown	Autosomal recessive / chr 7q21.3-22, SERPINE1 gene (Hematol Oncol Clin North Am 2021;35:1197)

Clinical features

Symptoms: bleeding associated with surgery, trauma, dental extractions, postpartum, circumcision or umbilical stumps, easy bruising, epistaxis, menorrhagia, gastrointestinal bleeding, hemarthrosis, soft tissue hematomas, hematuria, intracranial bleeding
Patients with primary hemostasis abnormalities are more likely to manifest mucocutaneous bleeding (Eur J Pediatr 2012;171:1)
Reasons to suspect hereditary platelet disorder (Int J Mol Sci 2021;22:4521)
1. Persistence of neonatal thrombocytopenia or onset of bleeding symptoms in childhood
2. Family history of thrombocytopenia or mucocutaneous bleeding
3. Association with other diseases (check for unique physical characteristics and findings on peripheral blood smear)
4. Bleeding out of proportion to the platelet count
Patients with secondary hemostasis abnormalities tend to have muscle and joint bleeds (Eur J Pediatr 2012;171:207)
Heterozygous patients have 30 - 60% of normal values of affected factors, usually with no or minor bleeding disorder (UpToDate: Rare Inherited Coagulation Disorders [Accessed 1 November 2024])
Factor I, X, XI or XIII deficient heterozygotes may have bleeding symptoms (UpToDate: Rare Inherited Coagulation Disorders [Accessed 1 November 2024])
Homozygous deficient patients have UpToDate: Rare Inherited Coagulation Disorders [Accessed 1 November 2024])
In hemophilia A and B, small differences in factor levels (i.e., 5%) may markedly affect the clinical presentation, disease course and management (UpToDate: Clinical Manifestations and Diagnosis of Hemophilia A and B [Accessed 1 November 2024])

Laboratory

Basic screening tests include complete blood count (CBC), prothrombin time (PT) / activated partial thromboplastin time (aPTT), platelet function assay (e.g., PFA 100 / PFA 200), thrombin time, peripheral blood smear review (for platelet and erythrocyte morphology) and fibrinogen (Int J Lab Hematol 2018;40:6)
For suspected platelet disorders: platelet aggregation studies, bone marrow aspirate and biopsy, flow cytometry, electron microscopy, next generation sequencing (Hematol Oncol Clin North Am 2021;35:1069)
Testing for von Willebrand disease includes factor VIII activity, vWF antigen and vWF activity (measured by ristocetin cofactor activity or vWF GPIbM activity assay) (Pediatr Clin North Am 2013;60:1419)
- These results may lead to obtaining vWF multimer analysis and blood type (group O patients have reduced vWF antigen and activity) (Pediatr Clin North Am 2013;60:1419)
For suspected coagulation factor abnormalities: mixing studies, factor levels, Bethesda assay (to detect coagulation factor inhibitor titers) (Int J Lab Hematol 2018;40:6)
Factor XIII assay if delayed bleeding is present with normal PT and aPTT (measured by factor XIII activity assay or urea clot lysis method) (Hematol Oncol Clin North Am 2021;35:1171)

Treatment

Bleeding in patients with hereditary platelet disorders is managed with platelet transfusions, antifibrinolytic agents, DDAVP (desmopressin) or recombinant factor VIIa, depending on the defect (Hematol Oncol Clin North Am 2021;35:1069)
Specific treatment recommendations are dependent on the type and severity of bleeding disorder but generally factor replacement therapy for factor deficiencies is the mainstay of treatment, with the exceptions of factor V (which is treated with plasma), factor II and factor X deficiencies (which are treated with prothrombin complex concentrates) (Haemophilia 2008;14:671)
For von Willebrand disease, use vWF concentrates, DDAVP (desmopressin; contraindicated in vWD type 2B) or antifibrinolytic agents (Hematology Am Soc Hematol Educ Program 2016;2016:683)

Hereditary thrombocytopenia

Hereditary thrombocytopenia (inherited thrombocytopenia) encompasses a group of rare genetic disorders characterized by a bleeding tendency
Most individuals with hereditary thrombocytopenia present with mild thrombocytopenia and a minimal or no risk of spontaneous bleeding
Diagnosing hereditary thrombocytopenia can be challenging, requiring the exclusion of other causes of low platelet counts, such as infections or immune disorders
Over the past decade, the advent of high throughput sequencing technologies, including whole exome and whole genome sequencing, has significantly advanced our understanding of these conditions
Currently, more than 40 genes have been implicated in various forms of hereditary thrombocytopenia
Recent discoveries have also highlighted that certain genetic mutations associated with hereditary thrombocytopenia may predispose individuals to additional congenital abnormalities, hematological malignancies or bone marrow failure
Given that the most common forms of hereditary thrombocytopenia pose little to no risk of spontaneous bleeding, most patients require only regular monitoring and in some cases, intervention during hemostatic challenges
Historically, platelet transfusion was the only treatment option for hereditary thrombocytopenia; however, thrombopoietin receptor agonists (TPO RAs) have emerged as a promising therapy, reducing the need for platelet transfusions in preparation for surgical or hemostatic challenges and offering longterm management for patients with significant spontaneous bleeding
For the most severe forms, such as Wiskott-Aldrich syndrome and congenital amegakaryocytic thrombocytopenia, hematopoietic stem cell transplantation remains the treatment of choice

Differential diagnosis

Antiplatelet medications, such as aspirin, glycoprotein IIB / IIIA inhibitors, clopidogrel, ticlopidine
Anticoagulants (heparin, warfarin or other anticoagulants) (Wikipedia: Anticoagulant [Accessed 1 November 2024])
Liver disease:
- Patients with liver disease (e.g., liver dysfunction, acute liver failure, cirrhosis) may have varying degrees of thrombocytopenia
- Mechanism can be due to impaired platelet production from decreased synthesis of thrombopoietin, platelet sequestration in the spleen in the setting of portal hypertension, bone marrow suppression from infection (e.g., hepatitis C virus), alcohol use or antibiotic and antiviral treatment
Disseminated intravascular coagulation (DIC):
- Can be caused by sepsis, malignancy, trauma, obstetrical complications, among others
- Complete blood count may show increased white blood cell count and low platelet count
- Prolonged prothrombin time (PT) and activated partial thromboplastin time (aPTT), low fibrinogen, increased D-dimer
- Peripheral smear may show evidence of microangiopathic hemolytic anemia (MAHA), with schistocytes and helmet cells
Vitamin K deficiency:
- Results in deficiency in vitamin K dependent coagulation factors: factors II, VII, IX, X, protein C and S
- Certain patients are at increased risk: on warfarin, taking antibiotics, have fat malabsorption (celiac disease, cystic fibrosis, short bowel syndrome, on a diet lacking in vitamin K)
Acquired von Willebrand syndrome:
- Due to any qualitative, structural or functional disorder of vWF
- Negative family history
- Lack of previous bleeding
- Decreased vWF activity to antigen ratio, normal vWF propeptide antigen, loss of high molecular weight multimers
- Often associated lymphoproliferative, myeloproliferative, cardiovascular and immunological disorders

Board review style question #1

What is the most common hereditary bleeding disorder?

Fibrinogen deficiency
Glanzmann thrombasthenia
Hemophilia A
Hemophilia B
Von Willebrand disease

Board review style answer #1

E. Von Willebrand disease has a prevalence of 1 in 1,000 - 1 in 100. Answer A is incorrect because fibrinogen deficiency is a rare inherited bleeding disorder with a prevalence of 1 in 1,000,000. Answer B is incorrect because Glanzmann thrombasthenia is a rare disorder with a prevalence of 1 in 1,000,000. Answer C is incorrect because hemophilia A is diagnosed in 1 of 5,000 male births. Answer D is incorrect because hemophilia B is diagnosed in 1 of 30,000 male births.

Comment Here

Reference: Hereditary bleeding disorders - general

Board review style question #2

Which of the following findings is suggestive of a primary hemostasis disorder?

Bleeding into joints
Mucosal bleeding
Negative family history of bleeding
Prolonged prothrombin time or activated partial thromboplastin time

Board review style answer #2

B. Mucosal bleeding. Patients with primary hemostasis abnormalities are more likely to manifest mucocutaneous bleeding. Answer A is incorrect because bleeding into joints manifests in patients with secondary hemostasis. Answer C is incorrect because primary hemostasis disorders are inherited and can run in families. Answer D is incorrect because prolonged prothrombin time or activated partial thromboplastin time are often seen in secondary hemostasis disorders.

Comment Here

Reference: Hereditary bleeding disorders - general

Hereditary bleeding disorders - testing

Table of Contents

Factor VII assay

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 versus VIIa
Can also use a chromogenic assay (Haemostasis 1983;13:161)

Factor VIII assay

Assay is usually a PTT based clotting assay that measures factor VIII activity
Use severely deficient factor VIII plasma as substrate for one stage clotting assay, although there is tremendous interlaboratory variability
Can also use chromogenic assay, particularly when assessing B chain deleted recombinant Factor VIII (i.e., refacto)
Low levels: hemophilia, vWF disease with decreased vWF antigen levels, delay in transporting specimen to lab (Goldman: Cecil Medicine: Expert Consult, 23rd Edition, 2007)
- Levels are not greatly decreased at birth or throughout childhood
- Levels may increase during pregnancy

Factor VIII inhibitor assay / Bethesda assay

Methodology (Am J Clin Pathol 2009;131:552):
- 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

Factor IX assay

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, Goldman: Cecil Medicine: Expert Consult, 23rd Edition, 2007)

Factor XI assay

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 (J Clin Pathol 1975;28:332)

Factor XIII assay

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 37°C, 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

Protein C assays

Definition / 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; patients 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; patients 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, sso use 3 standard deviations as cutoff
- ELISA: uses antibody to protein C immobilized to microtiter place; add plasma; add secondary antiprotein 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

Etiology

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

Diagnosis

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)
Nonindications: screening before oral contraceptives or oral anticoagulants (discontinue for 10 days or test family members)

Interpretation

Values falsely increased by bivalirudin, lepirudin, argatroban and fondaparinux, lowered by warfarin (must discontinue for 10 days prior to testing) (Arch Pathol Lab Med 2004;128:1142)
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

Protein S assays

Definition / 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

Etiology

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

Laboratory

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, lupus anticoagulants (Arch Pathol Lab Med 2004;128:1142)
Reference ranges (nmol/L)
- 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

Prothrombin gene 20210A testing

Definition / 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

Laboratory

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

Hereditary thrombophilia - general

Table of Contents

Definition / general | Diagnosis

Thrombotic disorders due in part to deficiencies in natural anticoagulant or fibrinolytic systems
Acquired risk factors, such as oral contraceptive use, may synergistically increase the risk for thrombosis
Disorders often occur at a young age; usually affects venous system
Workup includes documenting all acquired risk factors for thrombosis

High molecular weight kininogen deficiency / assay

Table of Contents

Definition / general

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

Homocysteine assay

Table of Contents

Definition / 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

Diagnosis

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)

Hypercoagulation panel

Table of Contents

Definition / general | Diagrams / tables | Additional references

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

Hyperhomocysteinemia

Table of Contents

Definition / 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 / tables

Images hosted on other servers:

Folate metabolism

Additional references

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

Inhibitors

Table of Contents

Autoimmune based inhibitors in nonhemophiliac patients | Bovine coagulation factor inhibitors | Factor V inhibitor | Factor VIII inhibitor | Factor IX inhibitor

Autoimmune based inhibitors in nonhemophiliac patients

Definition / general

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
M = F

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 antifactor 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 (versus 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 → 12.5%, etc.)

Treatment

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

Additional references

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

Bovine coagulation factor inhibitors

Definition / general

Antibodies that develop against bovine proteins after exposure to topical bovine thrombin preparations which can potentially crossreact 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

Laboratory

Prolonged PT and PTT that does not correct with mixing studies

Case reports

3 month old and 11 year old children who developed antifactor 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

Factor V inhibitor

Definition / general

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 crossreacts 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 - 10 weeks
- Noniatrogenic 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

Noniatrogenic 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

Factor VIII inhibitor

Definition / general

An acquired antifactor 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 (Autoimmune based inhibitors)
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 Epidemiology)

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 → 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 versus 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

Lupus-like anticoagulant:
- Occasionally 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: Hemophilia A Treatment [Accessed 11 October 2022]

Factor IX inhibitor

Definition / general

An acquired antifactor 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 nonhemophilia 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 Epidemiology)

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 → 25%, 3 units → 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 versus 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

Lupus-like anticoagulant:
- Occasionally 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

International sensitivity index (ISI)

Table of Contents

Measure of sensitivity of particular PT reagent - determined by manufacturer
Used to resolve interlaboratory variations in PT (Arch Pathol Lab Med 2004;128:308, J Clin Pathol 2003;56:114)
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

Liver dysfunction

Table of Contents

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

Low molecular weight heparin (LMWH)

Table of Contents

Definition / general | Diagnosis | Antiphospholipid antibodies

Recommended to monitor using chromogenic antifactor Xa assay on specimens obtained up to 4 hours after subcutaneous injection of LMWH (Chest 2001;119:64S)
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

Lupus anticoagulation & antiphospholipid antibodies

Table of Contents

Definition / general

Also called lupus inhibitor
One of the 2 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, Firestein: Kelley's Textbook of Rheumatology, 9th Edition, 2012)
May cause increased PTT (not time dependent), increased or normal PT
Prolongs clotting times by binding to phospholipid cofactors in coagulation cascade; 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)

Diagnosis

An algorithm combining several tests is necessary
All are clotting time based:
- Russell viper venom time (sensitive to abnormalities in factors X and V, diluted for screening)
- Kaolin clotting time
- Dilute PT (tissue thromboplastin inhibition test)
- PTT based assays (should have low concentration of phospholipids to enhance sensitivity)
- Less commonly Textarin (obtained from venomous Australian snake, not sensitive to abnormalities of factor X but sensitive to abnormalities of factor V
- 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: add diluent to tube 1 and egg phosphatidylethanolamine to tube 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)

Antiphospholipid antibodies

Acquired antibodies against phospholipid protein complexes
Occurs in 3 - 5% of general population; most common cause of acquired thrombophilia
Rate of thrombosis per year is 1% if no history of thrombosis, 4% in systemic lupus erythematosus (SLE) patients, 5.5% in patients with a history of thrombosis, 6% if high titer of IgG anticardiolipin
Includes lupus anticoagulant (most patients do not have SLE), anticardiolipin antibody and anti β2 glycoprotein antibodies
Antibodies are against phospholipids (usually transient, secondary to infection) or various plasma protein antigenic targets (β2-glycoprotein I, protein C, protein S, annexin V, high and low molecular weight kininogens, thrombomodulin, prothrombin, factors XI and XII, complement factor H)
First described by Wassermann in 1906 (Wasserman test was complement fixation procedure using saline liver extracts from fetuses with congenital syphilis)
Associated with an increased risk of arterial or venous thrombosis, thrombocytopenia, recurrent miscarriages; causes 1 / 3 of strokes in patients younger than age 50 years (often due to mitral or aortic valve emboli), 15% of deep venous thromboses, 5 - 15% of recurrent spontaneous abortions, eclampsia, maternal DVT's; also multi infarct dementia, chorea, migraine, livedo reticularis in skin
Catastrophic antiphospholipid syndrome resembles TTP-HUS
Antiphospholipid antibody syndrome: diagnosis requires a positive lupus anticoagulant or anticardiolipin antibody on 2 separate occasions, at least 6 - 12 weeks apart and either venous or arterial thrombosis, thrombocytopenia or recurrent fetal loss
References: Arch Pathol Lab Med 2002;126:1424, J Thromb Haemost 2006;4:295

Mixing studies

Table of Contents

Definition / general

Mixing studies are typically used to investigate abnormal clotting time results
Mixing studies help distinguish clotting time prolongation due to a coagulation factor deficiency or an inhibitor (specific or nonspecific)
Mixing study may direct further coagulation testing but it is not by itself diagnostic

Essential features

Mixing studies help distinguish clotting time prolongation due to a coagulation factor deficiency or an inhibitor, e.g. lupus anticoagulant
To perform a mixing study, mix patient plasma and normal pooled plasma and measure the clotting time that was initially prolonged
If the clotting time corrects, this suggests a factor deficiency; if the clotting time does not correct, this suggests presence of a circulating inhibitor
Mixing study may direct further coagulation testing but it is not by itself diagnostic, e.g. may aid in selecting factor assays

Terminology

Mixing test
Inhibitor screen
Inhibitor assay
Circulating anticoagulant
Prothrombin time (PT) 1:1 mix
Partial thromboplastin time (PTT) 1:1 mix
Partial thromboplastin time (PTT) correction

ICD coding

ICD-10: R79.1 - abnormal coagulation profile
ICD-10: D68.9 - coagulation defect, unspecified

Diagrams / tables

Contributed by Tori Seasor, M.D. and Karen A. Moser, M.D.

Mixing study performance and interpretation

*Differential diagnosis of prolonged PT or aPTT incorporating mixing study results (correction versus noncorrection)*
Clotting time prolonged	Mixing study result	Key differential diagnoses
Prothrombin time (PT)	Corrects	Factor deficiency (VII, X, V, II) Warfarin Vitamin K deficiency Liver disease
Prothrombin time (PT)	Does not correct	Specific factor inhibitor (VII, X, V, II) Direct Xa inhibitor
Activated partial thromboplastin time (aPTT)	Corrects	Factor deficiency (VIII, IX, XI, contact factors) Rarely, von Willebrand disease (if VIII is low enough to prolong aPTT)
Activated partial thromboplastin time (aPTT)	Does not correct	Specific factor inhibitor (VIII, IX, XI, contact factors) Heparin Direct thrombin inhibitor Lupus anticoagulant
PT and aPTT	Corrects	Common pathway factor deficiency (X, V, II) Warfarin (high doses) or superwarfarin Vitamin K deficiency Disseminated intravascular coagulation Liver disease
PT and aPTT	Does not correct	Common pathway factor inhibitors (X, V, II) Direct thrombin inhibitors Direct Xa inhibitors Lupus anticoagulant (rarely) Heparin (very high doses)

Specimen

Platelet poor plasma in 3.2% sodium citrate

Principles

To perform a mixing study, mix patient plasma and normal pooled plasma (NPP) and measure the clotting time that was initially prolonged
In factor deficiency: NPP adds sufficient clotting factors to overcome the deficiency and correct the clotting time
If an inhibitor is present, it typically inhibits the clotting factors in patient plasma and NPP so the clotting time remains prolonged
Inhibitors can be specific inhibitors (antibodies directed against a specific coagulation factor), nonspecific inhibitors (e.g. lupus anticoagulant) or medications such as heparin, direct thrombin inhibitors or direct Xa inhibitors
Mixing study results aid in selection of further coagulation testing, such as assays for specific factor deficiencies or inhibitors

Laboratory

CLSI H47-A2 includes general guidance for the performance of PT and aPTT mixing studies (CLSI: H47-A2 - One-Stage Prothrombin Time (PT) Test and Activated Partial Thromboplastin Time (APTT) Test, 2nd Edition, 2008)
Mixing study is performed for the prolonged clotting time only (e.g. if the PT is prolonged, a PT mixing study will be performed)
Mixing studies vary in the ratio of patient plasma to normal pooled plasma (NPP) and in incubation time
- Patient plasma to NPP ratio
  - 1:1 mixing study (equal parts patient plasma and NPP) is most commonly used
  - 4:1 mixing study (4 parts patient plasma and 1 part NPP) is sometimes used to evaluate minimally prolonged aPTT as it may be more sensitive in this scenario (Am J Clin Pathol 2002;117:62)
- Incubation time
  - Immediate: clotting time is evaluated immediately after mixing patient plasma and NPP
  - Incubated: mixed patient plasma and NPP is incubated at 37 °C for 1 - 2 hours, depending on local laboratory protocols
    - Incubation control consists of patient plasma and NPP incubated separately at 37 °C for same amount of time as mix; at end of incubation, patient plasma and NPP are mixed and clotting time is measured
      - Incubation control accounts for loss of labile factors during incubation
    - Incubation performed if immediate mixing study does not show an inhibitor pattern
    - Incubated aPTT mixing studies are useful to identify time dependent inhibitors (such as factor VIII inhibitors and some lupus anticoagulants)
    - Incubated PT mixing studies are not typically useful
Definition of normal pooled plasma is also not standardized
- NPP can be purchased from a vendor
- Making NPP locally is not preferred but if necessary it is recommended to use platelet poor plasma from a minimum of 20 - 30 donors with coagulation factor levels of approximately 100% for NPP (Semin Thromb Hemost 2014;40:195)

Interpretation of results

If a clotting time corrects in the immediate (PT) or immediate and incubated mixing study (aPTT), it is interpreted as a factor deficiency pattern
- Principle: at least 50% activity of a given factor is sufficient to produce a normal clotting time
- Example:
  
  Patient factor activity NPP factor activity 1:1 mix factor activity Mixing study interpretation
  2% 100% 51% Correction
If a clotting time does not correct in the immediate (PT) or immediate or incubated mixing study (aPTT), it is interpreted as an inhibitor pattern
- Principle: the inhibitor binds all available factor, from both the patient and NPP and the clotting time remains prolonged
There is no standard definition of correction of aPTT or PT mixing studies
- CLSI H60-A (CLSI: H60-A - Laboratory Testing for the Lupus Anticoagulant, 1st Edition, 2014) recommends interpreting mixing studies in the setting of lupus anticoagulant testing by using either
  - 1:1 mix normalized ratio (ratio = 1:1 mix result(s) / 1:1 mix mean of reference interval(s))
  - Rosner index (see below)
Strategy for interpretation should be determined and validated locally by individual laboratories
Example strategies for determining mixing study correction (Semin Thromb Hemost 2013;39:283)
- Clotting time of mix returns to within laboratory's reference interval
- Clotting time of mix falls within a 2 - 3 standard deviation range of the mean normal clotting time
- Compare the clotting time of mix to the NPP clotting time (ratio)
- Estimated factor correction method takes into account effects of single versus multiple factor deficiencies (Blood Coagul Fibrinolysis 2016;27:90)
- Rosner index and Chang percentage are calculations created to assist in interpretation of mixing study results
  - Rosner index is used in conjunction with a 1:1 mixing study; originally described for use in lupus anticoagulant testing
    - Rosner index > 15 suggests presence of an inhibitor (Thromb Haemost 1987;57:144)
      
      Rosner index = Clotting time of 1:1 mix − clotting time of NPP × 100
      Clotting time of patient sample
  - Chang percentage is used in conjunction with a 4:1 mixing study (optimal sensitivity) (Am J Clin Pathol 2002;117:62)
    - Chang percentage result of < 50% suggests the presence of an inhibitor
      
      Chang percentage = Initial patient clotting time − clotting time 4:1 mix × 100
      Initial patient clotting time − clotting time NPP

Pitfalls and preanalytic variables

Multiple factor deficiencies may cause mixing study noncorrection, which could be incorrectly interpreted as an inhibitor (Semin Thromb Hemost 2014;40:195)
High hematocrit or underfilled collection tubes can cause prolonged aPTT and PT due to excess citrate anticoagulant in the sample
Anticoagulant effect that appears as an inhibitor should be taken into account when interpreting mixing studies
- Heparin: affects aPTT mixing studies
  - May be detected with a thrombin time or anti-Xa activity assay and corrected by a neutralization procedure
- Direct thrombin inhibitor (DTI) may affect aPTT > PT mixing studies
- Direct Xa inhibitors may affect PT > aPTT mixing studies
If a specimen is not properly centrifuged to ensure removal of platelets, platelets can interfere with clotting time results by neutralization of lupus anticoagulant effect (if present) by phospholipids present in platelet cell membranes (J Thromb Haemost 2009;7:1737)
Rare occurrence of increased prolongation of aPTT (rather than shortening) when a patient's sample containing lupus anticoagulant is mixed with NPP (lupus cofactor effect) (Semin Thromb Hemost 2012;38:385)
- Lupus cofactor effect is reagent dependent and may represent a prozone effect (Am J Clin Pathol 2016;146:262)
- Decreased factor II in patient plasma may account for lupus cofactor effect in some cases (Am J Clin Pathol 2020;153:229, TH Open 2020;4:e40)

Additional references

Bennett: Laboratory Hemostasis, 2nd Edition, 2014, Transfus Apher Sci 2019;58:39

Board review style question #1

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are performed on a 64 year old man. The patient is taking no anticoagulants or other medications. The results of a 1:1 mixing study are

Patient aPTT: 51 s (reference range 38 - 42 s)
Immediate 1:1 aPTT mixing study: 36 s

Which of the following is the next best test to order?

4:1 mixing study
Factor VIII activity
Factor X activity
von Willebrand factor activity

Board review style answer #1

B. Factor VIII activity. The mixing study indicates a corrected result which suggests a factor deficiency as a cause of the prolonged aPTT. The next step would be to investigate which factor deficiency is causing this, which can be accomplished with factor VIII, IX and XI activity testing. Factors VIII, IX and XI are factors in the intrinsic coagulation pathway that can prolong the aPTT and are associated with clinical bleeding. Lupus anticoagulant testing could also be considered, as some weak lupus anticoagulants may show apparent correction in mixing studies.

Comment Here

Reference: Mixing studies

Board review style question #2

Coagulation laboratory tests are drawn on a 35 year old woman with the recent diagnosis of an autoimmune disease. The physician ordered a 1:1 mixing study after the original results revealed a prolonged activated partial thromboplastin time (aPTT).

Patient aPTT: 51 s (reference range 38 - 42 s)
Immediate 1:1 aPTT mixing study: 50 s

Which of the following do these laboratory results suggest?

Factor V Leiden mutation
Factor VII deficiency
Lupus anticoagulant
Vitamin K deficiency

Board review style answer #2

C. Lupus anticoagulant. The aPTT was prolonged in the patient sample and the addition of NPP in the immediate mixing study did not correct the aPTT to the normal reference range. This result suggests an inhibitor that overcomes the normal coagulation factors in the patient sample as well as the NPP. Lupus anticoagulants behave as nonspecific inhibitors in aPTT mixing studies and are a common cause of unexpected aPTT prolongation. The recent history of an autoimmune disorder diagnosed in a young woman would also raise concern for a lupus anticoagulant as an explanation for her prolonged aPTT.

Comment Here

Reference: Mixing studies

Other anticoagulants & thrombolytic therapy

Table of Contents

Danaparoid (Orgaran) | Hirudin | Thrombolytic therapy | Warfarin (Coumadin)

Danaparoid (Orgaran)

Definition / 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.

Hirudin

Definition / 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, which directly inhibits factor IIa (thrombin) (Wikipedia: Hirudin [Accessed 19 April 2021])
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 ~1 hour, may be prolonged if antibodies develop, dramatically prolonged in renal failure
No reversal agent
Reference: Thromb Haemost 2008;99:819

Thrombolytic therapy

Definition / 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)
Presence of fibrin degradation products and D-dimers, decreased fibrinogen and plasminogen, prolonged thrombin time, PT and PTT
References: Clin Lab Med 2009;29:159

Diagrams / tables

Images hosted on other servers:

t-PA action

Warfarin (Coumadin)

Definition / general

Therapeutic anticoagulant to prevent thromboembolism by impairing regeneration of active vitamin K (warfarin is a synthetic derivation based on coumarin)
Name incorporates the acronym for the organization which funded the key research (WARF) and the ending -arin, indicating its link with coumarin (Wikipedia: Wisconsin Alumni Research Foundation [Accessed 19 April 2021])

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
PTT may be normal even 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 / tables

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Biochemistry of coumarins

Physiology

Table of Contents

Definition / general

Hemostasis is a highly complex process of clot formation in response to blood vessel injury (Indian J Anaesth 2014;58:515)
Disorders in the hemostatic pathway can lead to either hypercoagulability (in which excessive clot formation occurs) or bleeding disorders (in which clot formation does not adequately stop bleeding)

Essential features

Hemostasis can be categorized into 3 stages (Curr Med Chem 2004;11:2245):
- Primary hemostasis is the formation of a weak platelet plug at the site of vascular injury
- Secondary hemostasis is the formation of a fibrin plug by coagulation factors; coagulation factor activation occurs via the intrinsic and extrinsic pathways, which merge to form the common pathway
- Tertiary hemostasis describes the anticoagulation and fibrinolytic balance to promote healing and prevent excessive clotting
The 3 major constituents of the hemostatic system are platelets, coagulation proteins and endothelium (Cardiovasc Diagn Ther 2018;8:568)

Primary hemostasis

During injury to vascular endothelium, the first response to prevent blood loss is vasoconstriction which activates endothelial cells (Ann Med 2012;44:405)
Formation of the platelet plug in primary hemostasis is primarily orchestrated by interactions between the endothelium, von Willebrand factor (vWF) and platelets (Arterioscler Thromb Vasc Biol 2020;40:1441)
Activated endothelial cells secrete vWF, which recruits platelets to the site of injury and results in a sequence of platelet adhesion, activation and aggregation (Int J Biochem Cell Biol 2021;131:105900, Hematol Oncol Clin North Am 2021;35:1069)
Platelet adhesion occurs when vWF, bound to exposed subendothelial collagen, interacts with glycoprotein Ib / IX / V on platelets (Arterioscler Thromb Vasc Biol 2008;28:403)
vWF is a plasma glycoprotein, stored in alpha granules secreted by platelets and Weibel-Palade bodies in the endothelium (Blood 2015;125:2019)
Platelet activation results in changes in platelet and membrane morphology and secretion of microparticles from intracellular granules
Upon activation, platelet cytoplasm and cytoskeleton rearrange to increase surface area and enable adherence to endothelium (Thromb Haemost 2002;88:186)
Platelet aggregation is mediated by glycoprotein IIb / IIIa (GpIIb / IIIa) on the platelet surface
During platelet activation, GpIIb / IIIa undergoes a conformational change into the active form; activated GpIIb / IIIa has high affinity for ligand binding and promotes platelet aggregation into the initial platelet plug (Blood 2007;109:5087)

Secondary hemostasis

In secondary hemostasis, the fibrin plug is formed by the activated coagulation factors (Ann Med 2012;44:405)
- Fibrinogen is cleaved into fibrin by factor II (thrombin) at the end of the common pathway (Arterioscler Thromb Vasc Biol 2011;31:494)
- Insoluble fibrin is crosslinked by factor XIII, which strengthens the fibrin plug
Coagulation factors are synthesized in the liver, except factor VIII and vWF (Blood Coagul Fibrinolysis 2000;11:S69)
Coagulation proteins circulate as inactive zymogens; factors must undergo enzymatic cleavage to function (Hemodial Int 2006;10:S2)
Factors II, VII, IX and X and protein C and S are synthesized in a vitamin K dependent manner; vitamin K epoxide reductase is a key enzyme in the vitamin K cycle (Vitam Horm 2008;78:23)
- Warfarin is an inhibitor of vitamin K epoxide reductase and suppresses the synthesis of active factors II, VII, IX and X and protein C and S
- Protein C and S are anticoagulant proteins that degrade factor Va and VIIIa (Adv Clin Exp Med 2013;22:459)
The model of secondary hemostasis is comprised of 2 separate pathways, the intrinsic and extrinsic pathway, that merge to form a common pathway; in vivo, it appears that these pathways may not be so distinctly separated

Intrinsic pathway

Intrinsic pathway is comprised of factors VIII, IX, XI, XII, prekallikrein and high molecular weight kininogen (Indian J Anaesth 2014;58:515)
The pathway is initiated when factor XII (Hageman factor) is activated by exposure to negatively charged molecules such as inorganic polyphosphate released by platelets (Cell 2009;139:1143)
Factor XIIa initiates a chain of activation involving factor XI, IX, VIII and ultimately factor X, initiating the common pathway with the activation of thrombin (factor II) (Arterioscler Thromb Vasc Biol 2019;39:331)
- Factor VIIIa increases the ability of factor IXa to activate factor X (Thromb Res 2007;119:1)
- Hemophilia A, B and C are inherited deficiencies of factor VIII, IX and XI, respectively; hemophilia A and B are X linked recessive disorders (Acta Neuropathol Commun 2021;9:111)
Factor XIIa additionally converts prekallikrein to kallikrein; the functions of kallikrein include converting prorenin to renin, kininogen to bradykinin and further activating factor XII (J Clin Invest 2002;109:1007)
The function of the intrinsic and common pathways of coagulation is measured with activated partial thromboplastin time (aPTT); aPTT measures the time it takes plasma to form a clot when exposed to factor XII activator (Methods Mol Biol 2013;992:111)
- Prolonged aPTT can be indicative of a factor deficiency, such as hemophilia or an inhibitor to the intrinsic or common pathway; an aPTT mixing study is used to differentiate between these causes (Eur J Haematol 2020;104:519)

Extrinsic pathway

Extrinsic pathway is comprised of tissue factor and factor VII (Indian J Anaesth 2014;58:515)
Tissue factor (TF) is a glycosylated protein expressed on subendothelial tissues; TF is activated in response to vascular wall damage (Curr Pharm Des 2015;21:1152)
Activated TF binds to and activates factor VII
Factor VIIa converts factor X into factor Xa, initiating the common pathway
Clinically, the extrinsic and common coagulation pathways are assessed by prothrombin time (PT); PT is a measurement of the time it takes for clot formation when plasma is exposed to tissue factor (StatPearls: Prothrombin Time [Accessed 15 June 2022])
- Prolonged PT can reveal a clotting factor deficiency or inhibitor to the extrinsic or common pathway; a PT mixing study can differentiate between these causes (Mayo Clin Proc 2007;82:864)
- International normalized ratio (INR) is the ratio between a patient's PT and a standardized, control PT value (StatPearls: Prothrombin Time [Accessed 15 June 2022])

Common pathway

Both the intrinsic and extrinsic pathways of coagulation lead to the activation of factor X
Common pathway involves fibrinogen (factor I), factors II, V and X
Prothrombinase complex is formed by factor Xa, factor V and calcium; this complex converts prothrombin (factor II) to thrombin (factor IIa) (Indian J Anaesth 2014;58:515)
Thrombin converts fibrinogen to fibrin, further activates factors V and VIII and cleaves factor XIII (Blood 2005;106:2605)
Factor XIII further stabilizes the clot by covalently crosslinking fibrin (Blood 2005;106:2605)
Antithrombin is a protease inhibitor that inhibits activated coagulation factors, primarily thrombin and Xa (J Thromb Haemost 2020;18:3142)
- Heparin inhibits thrombin generation by increasing antithrombin activity (Prog Mol Biol Transl Sci 2019;163:1)

Tertiary hemostasis

Tertiary hemostasis is the process of fibrinolysis (clot dissolution), in which fibrin is cleaved to prevent excessive clotting
Tissue type plasminogen activator (tPA) is produced by endothelial cells to mediate clot breakdown; urokinase type plasminogen activator (uPA) also functions in fibrinolysis (Front Immunol 2019;10:1348)
In the presence of fibrin, tPA and uPA cleave plasminogen to plasmin (J Thromb Haemost 2007;5:804)
Plasmin is a serine protease that degrades fibrin clot to soluble fibrin and fibrinogen degradation products (Int J Mol Sci 2021;22:2758)
- D dimer is a soluble fibrin degradation product that is used as an important clinical marker for coagulation and fibrinolysis (Am J Hematol 2019;94:833)
Fibrinolysis inhibitors, such as antiplasmin (which binds and inactivates fibrin) and plasminogen activator inhibitor 1 (PAI1; inhibitor of uPA / tPA), prevent excessive fibrinolysis (FEBS J 2005;272:4852, Transfus Apher Sci 2019;58:572)

Diagrams / tables

Contributed by Zaher K. Otrock, M.D.

Summary of secondary hemostasis

Images hosted on other servers:

Coagulation

Intrinsic pathway

Thrombomodulin function

Thrombomodulin protein

Board review style question #1

What are the basic components of hemostasis?

Phospholipids, platelets and coagulation proteins
Tissue factor, platelets and phospholipids
Vascular endothelium, platelets and coagulation proteins
Vascular endothelium, platelets and fibrinogen
Vascular endothelium, tissue factor and platelets

Board review style answer #1

C. Vascular endothelium, platelets and coagulation proteins

Comment Here

Reference: Coagulation - Physiology

Board review style question #2

Which coagulation factor deficiency has normal PT and aPTT?

Factor II
Factor V
Factor X
Factor XIII

Board review style answer #2

D. Factor XIII

Comment Here

Reference: Coagulation - Physiology

Plasminogen activator antigen-1

Table of Contents

Definition / general | Interpretation

Definition / 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

Laboratory

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 (inhibitors of antiplasmin and plasmin are present to prevent their interference)
ELISA (antigen) assay: also available

Plasminogen assay

Table of Contents

Definition / 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) or immunologic (based on concentration of plasminogen antigen) (Haemostasis 1988;18:47)
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

Platelet aggregation studies

Table of Contents

Definition / 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)

Laboratory

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

Platelet antibodies

Table of Contents

Definition / 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)
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
  - 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 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 patients platelets
    - For unknown reasons, these antibodies also destroy platelets 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)

Laboratory

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: patients 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 patients platelet antigens

Platelet hyperaggregation studies

Table of Contents

Definition / general | Laboratory | Additional references

Definition / 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

Laboratory

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, Jacobs and DeMott: The Laboratory Test Handbook, 5th Edition, 2001 (Page 327)

Prekallikrein assay

Table of Contents

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

Prekallikrein deficiency

Table of Contents

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 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

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

Additional references

Consultative Hemostasis and Thrombosis: Elsevier, 2007

Procoagulants (pending)

[Pending]

Protein C deficiency

Table of Contents

Definition / general | Etiology | Clinical images | Additional references

Definition / 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

Etiology

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

Images hosted on other servers:

Warfarin induced skin necrosis

Additional references

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

Protein S deficiency

Table of Contents

Definition / 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

Proteinuria

Table of Contents

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

Prothrombin gene mutation (G20210A) / hyperprothrombinemia

Table of Contents

Definition / general | Laboratory | Treatment | Additional references

Definition / 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

Laboratory

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

PT - Prothrombin time

Table of Contents

Definition / general | Laboratory | Interpretation

Definition / 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 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

Laboratory

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 (seebelow)
- 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)
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)

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)

PT / INR and aPTT

Table of Contents

Definition / general

Prothrombin time (PT) and activated partial thromboplastin time (aPTT) are common initial tests in the evaluation of patients with suspected bleeding disorders
PT evaluates extrinsic and common pathways
aPTT measures intrinsic and common pathways
International normalized ratio (INR) is a calculation allowing for the standardization and comparison of PT clotting time results between laboratories

Essential features

PT and aPTT assays measure time to fibrin clot formation
PT and aPTT results are reported in seconds
INR is calculated from PT clotting time values and allows for standardization to monitor warfarin anticoagulant therapy

Terminology

Coagulation screening tests
Clotting times

ICD coding

ICD-10:
- D68.9 - coagulation defect, unspecified
- R79.1 - abnormal coagulation profile
- Z79.01 - long term (current) use of anticoagulants
- Z86.711 - personal history of pulmonary embolism
- Z86.718 - personal history of other venous thrombosis and embolism

Laboratory

Clinical & Laboratory Standards Institute (CLSI) H47-A2 includes general guidance for the performance of PT and aPTT (CLSI: H47-A2 - One-Stage Prothrombin Time (PT) Test and Activated Partial Thromboplastin Time (APTT) Test, 2nd Edition, 2008)
PT and aPTT are clot based coagulation tests measuring time to formation of fibrin clot (Lab Med 2017;48:295)
- Coagulation analyzers use 1 of 2 strategies for clot detection
  - Electromechanical
  - Optical
Methods (Lab Med 2017;48:295, Semin Thromb Hemost 2014;40:195, Res Pract Thromb Haemost 2020;4:761):
- Whole blood sample for testing: platelet poor plasma in 3.2% sodium citrate (collection tube with light blue top)
  - Centrifuged at 1,500 x g (3,000 RPM) for 15 minutes to separate out plasma that contains < 10,000/uL of platelets
- PT and aPTT results are reported in seconds
- PT: thromboplastin (tissue factor combined with phospholipid) and calcium chloride are added to the sample to initiate clotting
  - PT reagents also contain heparin neutralizers (up to 2 U/mL) (Mayo Clin Proc 2007;82:864)
  - PT measures effects of factors in extrinsic and common pathways
- INR is a calculated value to allow for comparison of PT results from different laboratories or reagent lots (Semin Thromb Hemost 2014;40:195)
- aPTT: a contact factor activator (e.g., kaolin, silica) combined with phospholipid and calcium chloride are added to the sample to initiate clotting
  - aPTT measures effects of factors in intrinsic and common pathways
Reagents (Lab Med 2017;48:295, Clin Lab Med 2009;29:253, Mayo Clin Proc 2007;82:864):
- Reagents used for PT and aPTT testing vary between labs and show different factor sensitivities
  - PT reagents differ in phospholipid concentration and source as well as source of tissue factor
  - aPTT reagents differ in contact activator, phospholipid type and phospholipid concentration
Considerations when validating new reagent lots (Lab Med 2017;48:295, Clin Lab Med 2009;29:253)
- PT
  - Must determine mean normal prothrombin time and input this into instrument or laboratory information system (LIS) for calculation of INR when new lot put into use
  - Must input new mean normal prothrombin time into instrument or LIS for calculation of INR when new lot put into use
- APTT
  - Must determine heparin therapeutic range if aPTT will be used to monitor unfractionated heparin (UFH)
    - Heparin therapeutic range determined by measuring aPTT and UFH anti-Xa activity on a set of at least 20 plasmas from patients treated with UFH (Semin Thromb Haemost 2017;43:253)
      - Draw line of best fit
      - aPTT heparin therapeutic range represents aPTT values in seconds corresponding to UFH anti-Xa activity between 0.3 and 0.7 IU/mL
- Optional studies
  - Determination of factor sensitivity of PT and aPTT reagents is considered a best practice but is not a required study under Clinical Laboratory Improvement Amendments (CLIA)
    - Some reagent manufacturers provide lot level information about factor sensitivity in PT and aPTT package inserts
  - Saline dilution curves help describe level of combined factor deficiencies, at which a clotting time prolongs
    - Prepared by measuring clotting time of serial dilutions of normal plasma with saline
Preanalytic variables (Mayo Clin Proc 2007;82:864, Lab Med 2017;48:295)
- Variables that may affect either PT or aPTT
  - Underfilled collection tube
    - Not collecting blood to fill line
    - May occur if phlebotomist fails to account for dead space volume in a butterfly collection device
  - High hematocrit
  - Heparin contamination from a line draw
    - Tends to affect aPTT more than PT due to heparin neutralizers present in PT reagents
    - May affect PT at very high levels of heparin
  - Wrong anticoagulant in collection tube (e.g., ethylenediaminetetraacetic acid [EDTA], heparin)
- Samples must be processed (centrifuged, plasma removed from cells and testing performed or plasma frozen for later testing) within a specified time for valid results (Semin Thromb Hemost 2019;45:443)
  - PT: 24 hours
  - aPTT (not for heparin monitoring): 4 hours
  - aPTT (for heparin therapeutic monitoring): 1 hour

Clinical features

PT testing: can be used clinically for evaluation of bleeding, assessment of liver disease, screening evaluation of factors II, V, VII, X and fibrinogen (Mayo Clin Proc 2007;82:864, Lab Med 2017;48:295, Res Pract Thromb Haemost 2020;4:761)
- See Abnormal PT / aPTT values for differential diagnosis
- Therapeutic monitoring
  - INR used to monitor warfarin therapy
  - PT / INR not used to monitor heparin or direct oral anticoagulant therapy (DOAC)
- INR is a component of the model for end stage liver disease (MELD) score that assesses severity of end stage liver disease (MDCalc: MELD Score [Accessed 4 May 2021])
- PT / INR may also be measured on point of care (POC) instruments
  - POC PT / INR are only approved for monitoring of vitamin K antagonists
  - May be used in limited clinical settings
    - Anticoagulation clinics
    - Home use for carefully selected patients
  - Comparison between POC PT / INR and central laboratory values may be challenging and varies by instrument and reagent
  - Possible sources of error to consider specifically with POC PT / INR performed outside of a clinical laboratory
    - Inadequate training for users
    - Inattention to quality control procedures
    - Inappropriate patient selection
    - Failure to participate in External Quality Assessment (EQA) program
    - Failure to refer unexpected or questionable results for central laboratory PT / INR
aPTT testing: can be used clinically for monitoring heparin and certain direct thrombin inhibitor therapy, screening evaluation of factors II, V, VIII, IX, XI, XII, fibrinogen, prekallikrein and HMWK, screening for lupus anticoagulant (lupus sensitive aPTT reagents) (Mayo Clin Proc 2007;82:864, Lab Med 2017;48:295, Res Pract Thromb Haemost 2020;4:761)
- See Abnormal PT / aPTT values for differential diagnosis
- Therapeutic monitoring
  - Unfractionated heparin (if aPTT has heparin therapeutic range determined by laboratory)
  - Argatroban, an anticoagulant (direct thrombin inhibitor) for treating patients with thrombosis and heparin induced thrombocytopenia
  - aPTT not used to monitor low molecular weight heparin or dabigatran therapy

Interpretation

Reference intervals are dependent upon the instrument, anticoagulant, tube type, reagent and reagent lot
Determination of a laboratory specific reference interval that is applicable to the current reagent lot and testing methods in use is crucial (Clin Lab Med 2009;29:253, Lab Med 2017;48:295)
- PT results are reported in seconds with common values of 10 - 13 seconds (exact interval determined by individual laboratory)
- aPTT results are reported in seconds with common values 25 - 40 seconds (exact interval determined by individual laboratory)
- INR
  - INR improves comparison of prolonged PT values between laboratories for the purpose of monitoring warfarin therapy
  - INR therapeutic range for warfarin = 2.0 to 3.0 for most indications (Blood Adv 2020;4:4693)
Critical values
- Determined by individual laboratories but may be similar to the following recommended values from the International Council for Standardization in Haematology (ICSH) (Semin Thromb Hemost 2020;46:398)
  - PT / INR: INR 4.0 - 6.0
  - aPTT: should be locally determined

Board review style question #1

The laboratory supervisor tells you that a new lot of reagent is to be started next week and the international sensitivity index (ISI) needs to be updated. If this is done incorrectly, which laboratory result(s) will be affected?

aPTT
INR
PT
PT and aPTT
Thrombin time

Board review style answer #1

B. INR

The international normalized ratio (INR) is used to standardize PT across different laboratories to monitor warfarin therapy. The international sensitivity index (ISI) value is specific to a reagent lot and analyzer. The INR is then calculated by the following equation:

INR =	[	PTpatient	]
		PTnormal

^ISI

Comment Here

Reference: PT / INR and aPTT

Board review style question #2

A patient has an unexpectedly prolonged aPTT of > 150 seconds and a normal PT. The patient is not receiving any anticoagulants and has no history of bleeding or thrombosis. The laboratory investigates this unexpected value and identifies a prolonged thrombin time of > 150 seconds. What is the most likely preanalytic variable that would explain this pattern of results?

EDTA anticoagulant in tube
Heparin contamination
High hematocrit without citrate volume correction
Underfilled collection tube

Board review style answer #2

B. Heparin contamination

The marked prolongation of both the aPTT and thrombin time in this case is best explained by heparin contamination. Although all of the other preanalytic variables listed could cause falsely prolonged clotting times, they would be expected to also falsely prolong the PT (which was normal in this case) and may not show such extreme aPTT and thrombin time prolongation as can be seen with heparin.

Comment Here

Reference: PT / INR and aPTT

Board review style question #3

Your laboratory is provided a reagent lot with an international sensitivity index (ISI) of 1.31 on your instrument. The PT is performed with this reagent in 40 normal donors. With this information, the arithmetic mean is calculated to be 12.9 seconds and the geometric mean is calculated is 13.0 seconds. A patient sample PT is 18.1 seconds using this reagent lot in your laboratory. What is the international normalized ratio (INR) for this patient sample?

Board review style answer #3

B. 1.5

The calculated patient INR for this given ISI and PTnormal is 1.5, with the PTpatient (18.1 seconds) and the PTnormal being the PT of normal donors using the geometric mean (13.0 seconds) using the formula:

INR =	[	PTpatient	]
		PTnormal

^ISI

Comment Here

Reference: PT / INR and aPTT

Quality assurance

Table of Contents

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 laboratorys reportable range of test results

Reptilase time

Table of Contents

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)

Thrombin time

Table of Contents

Measures rate of fibrin clot formation after addition of standard concentration of thrombin to citrated plasma (Key, Makris, O'Shaughnessy and Lillicrap (Eds): Practical Hemostasis and Thrombosis, 2nd Edition, 2009 (Page 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

tPA assay

Table of Contents