Secondary HemostasisPlasmatic factorsIntrinsic pathwayExtrinsic pathwaySpecimenPPP preparationPT, INRAPTTTTVitamin KLiver diseaseFactor deficiencyCirculating inhibitorsHeparinCoumarinLupus inhibitorFactor assayMixing and substitution studies5M urea testLee-White clotting time

Plasmatic FactorsIntrinsic pathway activated by contact to collagen: HMWK, prekallikrein, XII, XI, IX, VIIIExtrinsic pathway activated by tissue thromboplastin: VIICommon pathway: X, V, II, I

RBCs trapped in fibrin strands SEM x 6,400

Fibrin FormationD and E domains on fibrinogenThrombin cleaves fibrinopeptidesSpontaneous polymerization (unstable)Disulfide cross-linkages between D domains by the action of XIII

InhibitorsAnti-thrombin III with heparin: II, IX, X, XI, XIIProtein C and protein S: slow down VIII and VHeparin: quick acting but short lived and need AT-IIICoumarin: vitamin K antagonist

Vitamin K Dependent FactorsCaboxylation to chelate Ca++II, VII, IX, X, protein C, protein SLiver synthesis inhibited by coumarin

SpecimenClean phlebotomy required3.2% vs 3.8% citrate1:9 citrate to blood ratioTransport to lab quickly and separate plasmaPlatelet poor plasma (PPP)Test without delay or store frozen

InstrumentationElectro-mechanical (e.g., Fibrometer)Physical detection of clotCannot be automatedOptical (e.g., MLA, ACL)Interference with icteria or lipemiaCan be automated

Basic Hemostasis TestsPlasmatic factorsThrombin timeProthrombin timeActivated Partial Thromoplastin time5M urea test (factor XIII)PlateletPlatelet countPlatelet function testVascular integrityBleeding timeTourniquet testOthersFDP, D-dimerFactor assaysAnti-thrombin IIIProteins C and SFactor V Leiden

Activated Partial Thromboplastin Time (APTT)For intrinsic pathway factorsLee-White clotting timeWhole blood at 37oCGlass test tube for surfacePhospholipid platelet substituteActivator: kaolin0.02M CaCl2Monitor heparin therapy

Prothrombin Time (PT)For extrinsic pathway factorsTissue thromboplastin (rabbit brain) with Ca++European labs use Tpl from human source, so more sensitiveINR = (Pt PT/normal PT)ISIMonitor coumarin therapy

Thrombin Time (TT)Fibrinogen screenThrombin reagentClotting time corresponds to fibrinogen level

Other TestsFactor assay: reconstitute patient plasma with known deficient plasma and determine clotting timeFDP and D-dimer tests for fibrinolysisFibrinogen assay: modified TT5M urea test for factor XIIIPlasma protamine-sulfate paracoagulation test (3P) for fibrin monomers

Mixing and Substitution Tests1:1 with normal plasma: screen for circulating inhibitorAged serum has: II, VII, IX, XI, XIIBarium sulfate adsorbed plasma has: I, V, VIII, XI, XII

Abnormal Coagulation TestsCheck specimen collectionPhlebotomy, anticoagulantPatient conditionMedicationCheck specimen integrityStorage temperaturePPP preparationCheck reagent integrityCheck instrument QC

Abnormal APTTHemophilia A (VIII) if maleChristmas disease (IX) if maleLiver disease: multi-factor deficiencyHypofibrinogenemiaHeparinAnti-phospholipid antibody (Lupus inhibitor): do a 1:1 mix with normal plasmaVon Willebrands: variable

Abnormal PTCoumarin therapyVitamin K deficiency, especially in newbornMore sensitive to common pathway factors than APTTHeparin

Abnormal TTDysfibrinogenemiaAfibrinogenemiaHypofibrinogenemiaHeparinFDP: forms abnormal complex

Hyper-coagulable StateDeep vein thrombosis due to inappropriate coagulationProtein C and protein S deficiencyAnti-thrombin III deficiencyFactor V Leiden mutation: does not respond to protein C (activated protein C resistance, APCR)