Week 6: Secondary Hemostasis

Plasmatic factorsPlasmatic factors Intrinsic pathwayIntrinsic pathway Extrinsic pathwayExtrinsic pathway SpecimenSpecimen PPP preparationPPP preparation PT, INRPT, INR APTTAPTT TTTT Vitamin KVitamin K Liver diseaseLiver disease

Factor deficiencyFactor deficiency Circulating inhibitorsCirculating inhibitors HeparinHeparin CoumarinCoumarin Lupus inhibitorLupus inhibitor Factor assayFactor assay Mixing and substitution Mixing and substitution

studiesstudies 5M urea test5M urea test Lee-White clotting timeLee-White clotting time

Plasmatic Factors

Intrinsic pathway activated by contact to Intrinsic pathway activated by contact to collagen: HMWK, prekallikrein, XII, XI, collagen: HMWK, prekallikrein, XII, XI, IX, VIIIIX, VIII

Extrinsic pathway activated by tissue Extrinsic pathway activated by tissue thromboplastin: VIIthromboplastin: VII

Common pathway: X, V, II, ICommon pathway: X, V, II, I

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

Fibrin Formation

D and E domains on fibrinogenD and E domains on fibrinogen Thrombin cleaves fibrinopeptidesThrombin cleaves fibrinopeptides Spontaneous polymerization (unstable)Spontaneous polymerization (unstable) Disulfide cross-linkages between D Disulfide cross-linkages between D

domains by the action of XIIIdomains by the action of XIII

Inhibitors

Anti-thrombin III with heparin: II, IX, X, Anti-thrombin III with heparin: II, IX, X, XI, XIIXI, XII

Protein C and protein S: slow down VIII Protein C and protein S: slow down VIII and Vand V

Heparin: quick acting but short lived and Heparin: quick acting but short lived and need AT-IIIneed AT-III

Coumarin: vitamin K antagonistCoumarin: vitamin K antagonist

Vitamin K Dependent Factors

Caboxylation to chelate CaCaboxylation to chelate Ca++++

II, VII, IX, X, protein C, protein SII, VII, IX, X, protein C, protein S Liver synthesis inhibited by coumarinLiver synthesis inhibited by coumarin

Specimen

Clean phlebotomy requiredClean phlebotomy required 3.2% vs 3.8% citrate3.2% vs 3.8% citrate 1:9 citrate to blood ratio1:9 citrate to blood ratio Transport to lab quickly and separate Transport to lab quickly and separate

plasmaplasma Platelet poor plasma (PPP)Platelet poor plasma (PPP) Test without delay or store frozenTest without delay or store frozen

Instrumentation

Electro-mechanical (e.g., Fibrometer)Electro-mechanical (e.g., Fibrometer) Physical detection of clotPhysical detection of clot Cannot be automatedCannot be automated

Optical (e.g., MLA, ACL)Optical (e.g., MLA, ACL) Interference with icteria or lipemiaInterference with icteria or lipemia Can be automatedCan be automated

Basic Hemostasis Tests

Plasmatic factorsPlasmatic factors Thrombin timeThrombin time Prothrombin timeProthrombin time Activated Partial Activated Partial

Thromoplastin timeThromoplastin time 5M urea test (factor 5M urea test (factor

XIII)XIII) PlateletPlatelet

Platelet countPlatelet count Platelet function testPlatelet function test

Vascular integrityVascular integrity Bleeding timeBleeding time Tourniquet testTourniquet test

OthersOthers FDP, D-dimerFDP, D-dimer Factor assaysFactor assays Anti-thrombin IIIAnti-thrombin III Proteins C and SProteins C and S Factor V LeidenFactor V Leiden

Activated Partial Thromboplastin Time (APTT) For intrinsic pathway factorsFor intrinsic pathway factors Lee-White clotting timeLee-White clotting time

Whole blood at 37Whole blood at 37ooCC Glass test tube for surfaceGlass test tube for surface

Phospholipid platelet substitutePhospholipid platelet substitute Activator: kaolinActivator: kaolin 0.02M CaCl0.02M CaCl22

Monitor heparin therapyMonitor heparin therapy

Prothrombin Time (PT)

For extrinsic pathway factorsFor extrinsic pathway factors Tissue thromboplastin (rabbit brain) with Tissue thromboplastin (rabbit brain) with

CaCa++++

European labs use Tpl from human source, European labs use Tpl from human source, so more sensitiveso more sensitive

INR = (Pt PT/normal PT)INR = (Pt PT/normal PT)ISIISI

Monitor coumarin therapyMonitor coumarin therapy

Thrombin Time (TT)

Fibrinogen screenFibrinogen screen Thrombin reagentThrombin reagent Clotting time corresponds to fibrinogen Clotting time corresponds to fibrinogen

levellevel

Other Tests

Factor assay: reconstitute patient plasma Factor assay: reconstitute patient plasma with known deficient plasma and determine with known deficient plasma and determine clotting timeclotting time

FDP and D-dimer tests for fibrinolysisFDP and D-dimer tests for fibrinolysis Fibrinogen assay: modified TTFibrinogen assay: modified TT 5M urea test for factor XIII5M urea test for factor XIII Plasma protamine-sulfate paracoagulation Plasma protamine-sulfate paracoagulation

test (3P) for fibrin monomerstest (3P) for fibrin monomers

Mixing and Substitution Tests

1:1 with normal plasma: screen for 1:1 with normal plasma: screen for circulating inhibitorcirculating inhibitor

Aged serum has: II, VII, IX, XI, XIIAged serum has: II, VII, IX, XI, XII Barium sulfate adsorbed plasma has: I, V, Barium sulfate adsorbed plasma has: I, V,

VIII, XI, XIIVIII, XI, XII

Abnormal Coagulation Tests

Check specimen collectionCheck specimen collection Phlebotomy, anticoagulantPhlebotomy, anticoagulant Patient conditionPatient condition MedicationMedication

Check specimen integrityCheck specimen integrity Storage temperatureStorage temperature PPP preparationPPP preparation

Check reagent integrityCheck reagent integrity Check instrument QCCheck instrument QC

Abnormal APTT

Hemophilia A (VIII) if maleHemophilia A (VIII) if male Christmas disease (IX) if maleChristmas disease (IX) if male Liver disease: multi-factor deficiencyLiver disease: multi-factor deficiency HypofibrinogenemiaHypofibrinogenemia HeparinHeparin Anti-phospholipid antibody (Lupus inhibitor): do Anti-phospholipid antibody (Lupus inhibitor): do

a 1:1 mix with normal plasmaa 1:1 mix with normal plasma Von Willebrand’s: variableVon Willebrand’s: variable

Abnormal PT

Coumarin therapyCoumarin therapy Vitamin K deficiency, especially in Vitamin K deficiency, especially in

newbornnewborn More sensitive to common pathway factors More sensitive to common pathway factors

than APTTthan APTT HeparinHeparin

Abnormal TT

DysfibrinogenemiaDysfibrinogenemia AfibrinogenemiaAfibrinogenemia HypofibrinogenemiaHypofibrinogenemia HeparinHeparin FDP: forms abnormal complexFDP: forms abnormal complex

Hyper-coagulable State

Deep vein thrombosis due to inappropriate Deep vein thrombosis due to inappropriate coagulationcoagulation

Protein C and protein S deficiencyProtein C and protein S deficiency Anti-thrombin III deficiencyAnti-thrombin III deficiency Factor V Leiden mutation: does not Factor V Leiden mutation: does not

respond to protein C (activated protein C respond to protein C (activated protein C resistance, APCR)resistance, APCR)