Secondary Hemostasis

Plasmatic factors
Intrinsic pathway
Extrinsic pathway
Specimen
PPP preparation
PT, INR
APTT
TT
Vitamin K
Liver disease

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

RBCs 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

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

Vitamin K Dependent Factors

Caboxylation to chelate Ca++
II, VII, IX, X, protein C, protein S
Liver synthesis inhibited by coumarin

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

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

Basic Hemostasis Tests

Plasmatic factors
Thrombin time
Prothrombin time
Activated Partial
Thromoplastin time
5M urea test (factor XIII)
Platelet
Platelet count
Platelet function test

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)
For intrinsic pathway factors
Lee-White clotting time
Whole blood at 37oC
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

Thrombin Time (TT)

Fibrinogen screen
Thrombin reagent
Clotting time corresponds to fibrinogen level

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

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

Abnormal Coagulation Tests

Check specimen collection
Phlebotomy, anticoagulant
Patient condition
Medication
Check specimen integrity
Storage temperature
PPP preparation
Check reagent integrity
Check instrument QC

Abnormal APTT

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 Willebrands: variable

Abnormal PT
Coumarin therapy
Vitamin K deficiency, especially in newborn
More sensitive to common pathway factors
than APTT
Heparin

Abnormal TT

Dysfibrinogenemia
Afibrinogenemia
Hypofibrinogenemia
Heparin
FDP: forms abnormal complex

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)