Week 6: Secondary Hemostasis Plasmatic factors Plasmatic factors Intrinsic pathway Intrinsic pathway Extrinsic pathway Extrinsic pathway Specimen Specimen PPP preparation PPP preparation PT, INR PT, INR APTT APTT TT TT Vitamin K Vitamin K Liver disease 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 Intrinsic pathway activated by contact to collagen: HMWK, prekallikrein, XII, XI, IX, VIII Extrinsic pathway activated by tissue thromboplastin: VII Extrinsic pathway activated by tissue thromboplastin: VII Common pathway: X, V, II, I Common pathway: X, V, II, I

RBC’s trapped in fibrin strands SEM x 6,400

Fibrin Formation D and E domains on fibrinogen D and E domains on fibrinogen Thrombin cleaves fibrinopeptides Thrombin cleaves fibrinopeptides Spontaneous polymerization (unstable) Spontaneous polymerization (unstable) Disulfide cross-linkages between D domains by the action of XIII Disulfide cross-linkages between D domains by the action of XIII

Inhibitors Anti-thrombin III with heparin: II, IX, X, XI, XII Anti-thrombin III with heparin: II, IX, X, XI, XII Protein C and protein S: slow down VIII and V Protein C and protein S: slow down VIII and V Heparin: quick acting but short lived and need AT-III Heparin: quick acting but short lived and need AT-III Coumarin: vitamin K antagonist Coumarin: vitamin K antagonist

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

Specimen Clean phlebotomy required Clean phlebotomy required 3.2% vs 3.8% citrate 3.2% vs 3.8% citrate 1:9 citrate to blood ratio 1:9 citrate to blood ratio Transport to lab quickly and separate plasma Transport to lab quickly and separate plasma Platelet poor plasma (PPP) Platelet poor plasma (PPP) Test without delay or store frozen Test without delay or store frozen

Instrumentation Electro-mechanical (e.g., Fibrometer) Electro-mechanical (e.g., Fibrometer)  Physical detection of clot  Cannot be automated Optical (e.g., MLA, ACL) Optical (e.g., MLA, ACL)  Interference with icteria or lipemia  Can be automated

Basic Hemostasis Tests Plasmatic factors Plasmatic factors  Thrombin time  Prothrombin time  Activated Partial Thromoplastin time  5M urea test (factor XIII) Platelet 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 For intrinsic pathway factors Lee-White clotting time Lee-White clotting time  Whole blood at 37 o C  Glass test tube for surface Phospholipid platelet substitute Phospholipid platelet substitute Activator: kaolin Activator: kaolin 0.02M CaCl M CaCl 2 Monitor heparin therapy Monitor heparin therapy

Prothrombin Time (PT) For extrinsic pathway factors For extrinsic pathway factors Tissue thromboplastin (rabbit brain) with Ca ++ Tissue thromboplastin (rabbit brain) with Ca ++ European labs use Tpl from human source, so more sensitive European labs use Tpl from human source, so more sensitive INR = (Pt PT/normal PT) ISI INR = (Pt PT/normal PT) ISI Monitor coumarin therapy Monitor coumarin therapy

Thrombin Time (TT) Fibrinogen screen Fibrinogen screen Thrombin reagent Thrombin reagent Clotting time corresponds to fibrinogen level Clotting time corresponds to fibrinogen level

Other Tests Factor assay: reconstitute patient plasma with known deficient plasma and determine clotting time Factor assay: reconstitute patient plasma with known deficient plasma and determine clotting time FDP and D-dimer tests for fibrinolysis FDP and D-dimer tests for fibrinolysis Fibrinogen assay: modified TT Fibrinogen assay: modified TT 5M urea test for factor XIII 5M urea test for factor XIII Plasma protamine-sulfate paracoagulation test (3P) for fibrin monomers Plasma protamine-sulfate paracoagulation test (3P) for fibrin monomers

Mixing and Substitution Tests 1:1 with normal plasma: screen for circulating inhibitor 1:1 with normal plasma: screen for circulating inhibitor Aged serum has: II, VII, IX, XI, XII Aged serum has: II, VII, IX, XI, XII Barium sulfate adsorbed plasma has: I, V, VIII, XI, XII Barium sulfate adsorbed plasma has: I, V, VIII, XI, XII

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

Abnormal APTT Hemophilia A (VIII) if male Hemophilia A (VIII) if male Christmas disease (IX) if male Christmas disease (IX) if male Liver disease: multi-factor deficiency Liver disease: multi-factor deficiency Hypofibrinogenemia Hypofibrinogenemia Heparin Heparin Anti-phospholipid antibody (Lupus inhibitor): do a 1:1 mix with normal plasma Anti-phospholipid antibody (Lupus inhibitor): do a 1:1 mix with normal plasma Von Willebrand’s: variable Von Willebrand’s: variable

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

Abnormal TT Dysfibrinogenemia Dysfibrinogenemia Afibrinogenemia Afibrinogenemia Hypofibrinogenemia Hypofibrinogenemia Heparin Heparin FDP: forms abnormal complex FDP: forms abnormal complex

Hyper-coagulable State Deep vein thrombosis due to inappropriate coagulation Deep vein thrombosis due to inappropriate coagulation Protein C and protein S deficiency Protein C and protein S deficiency Anti-thrombin III deficiency Anti-thrombin III deficiency Factor V Leiden mutation: does not respond to protein C (activated protein C resistance, APCR) Factor V Leiden mutation: does not respond to protein C (activated protein C resistance, APCR)