Week 6: Secondary Hemostasis
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Plasmatic factors
Intrinsic pathway
Extrinsic pathway
Specimen
PPP preparation
PT, INR
APTT
TT
Vitamin K
Liver disease
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Factor deficiency
Circulating inhibitors
Heparin
Coumarin
Lupus inhibitor
Factor assay
Mixing and substitution
studies
5M urea test
Lee-White clotting time
Plasmatic Factors
Intrinsic pathway activated by contact to
collagen: HMWK, prekallikrein, XII, XI,
IX, VIII
 Extrinsic pathway activated by tissue
thromboplastin: VII
 Common pathway: X, V, II, I
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RBC’s trapped in fibrin strands SEM x 6,400
Fibrin Formation
D and E domains on fibrinogen
 Thrombin cleaves fibrinopeptides
 Spontaneous polymerization (unstable)
 Disulfide cross-linkages between D
domains by the action of XIII
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Inhibitors
Anti-thrombin III with heparin: II, IX, X,
XI, XII
 Protein C and protein S: slow down VIII
and V
 Heparin: quick acting but short lived and
need AT-III
 Coumarin: vitamin K antagonist
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Vitamin K Dependent Factors
Caboxylation to chelate Ca++
 II, VII, IX, X, protein C, protein S
 Liver synthesis inhibited by coumarin
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Specimen
Clean phlebotomy required
 3.2% vs 3.8% citrate
 1:9 citrate to blood ratio
 Transport to lab quickly and separate
plasma
 Platelet poor plasma (PPP)
 Test without delay or store frozen
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Instrumentation
Electro-mechanical (e.g., Fibrometer)
 Physical detection of clot
 Cannot be automated
 Optical (e.g., MLA, ACL)
 Interference with icteria or lipemia
 Can be automated
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Basic Hemostasis Tests
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Plasmatic factors
 Thrombin time
 Prothrombin time
 Activated Partial
Thromoplastin time
 5M urea test (factor
XIII)
Platelet
 Platelet count
 Platelet function test
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Vascular integrity
 Bleeding time
 Tourniquet test
Others
 FDP, D-dimer
 Factor assays
 Anti-thrombin III
 Proteins C and S
 Factor V Leiden
Activated Partial Thromboplastin
Time (APTT)
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For intrinsic pathway factors
Lee-White clotting time
o
 Whole blood at 37 C
 Glass test tube for surface
Phospholipid platelet substitute
Activator: kaolin
0.02M CaCl2
Monitor heparin therapy
Prothrombin Time (PT)
For extrinsic pathway factors
 Tissue thromboplastin (rabbit brain) with
Ca++
 European labs use Tpl from human source,
so more sensitive
 INR = (Pt PT/normal PT)ISI
 Monitor coumarin therapy
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Thrombin Time (TT)
Fibrinogen screen
 Thrombin reagent
 Clotting time corresponds to fibrinogen
level
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Other Tests
Factor assay: reconstitute patient plasma
with known deficient plasma and determine
clotting time
 FDP and D-dimer tests for fibrinolysis
 Fibrinogen assay: modified TT
 5M urea test for factor XIII
 Plasma protamine-sulfate paracoagulation
test (3P) for fibrin monomers
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Mixing and Substitution Tests
1:1 with normal plasma: screen for
circulating inhibitor
 Aged serum has: II, VII, IX, XI, XII
 Barium sulfate adsorbed plasma has: I, V,
VIII, XI, XII
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Abnormal Coagulation Tests
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Check specimen collection
 Phlebotomy, anticoagulant
 Patient condition
 Medication
Check specimen integrity
 Storage temperature
 PPP preparation
Check reagent integrity
Check instrument QC
Abnormal APTT
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Hemophilia A (VIII) if male
Christmas disease (IX) if male
Liver disease: multi-factor deficiency
Hypofibrinogenemia
Heparin
Anti-phospholipid antibody (Lupus inhibitor): do
a 1:1 mix with normal plasma
Von Willebrand’s: variable
Abnormal PT
Coumarin therapy
 Vitamin K deficiency, especially in newborn
 More sensitive to common pathway factors
than APTT
 Heparin
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Abnormal TT
Dysfibrinogenemia
 Afibrinogenemia
 Hypofibrinogenemia
 Heparin
 FDP: forms abnormal complex
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Hyper-coagulable State
Deep vein thrombosis due to inappropriate
coagulation
 Protein C and protein S deficiency
 Anti-thrombin III deficiency
 Factor V Leiden mutation: does not
respond to protein C (activated protein C
resistance, APCR)
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