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Pre-analytical factors that can affect coag test results Underfilled tube High hematocrit Hemolysis Traumatic blood draw (tissue factor) Delay in testing.

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Presentation on theme: "Pre-analytical factors that can affect coag test results Underfilled tube High hematocrit Hemolysis Traumatic blood draw (tissue factor) Delay in testing."— Presentation transcript:

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2 Pre-analytical factors that can affect coag test results Underfilled tube High hematocrit Hemolysis Traumatic blood draw (tissue factor) Delay in testing Excessive agitation of blood in tube (platelet tests)

3 Thromboplastin: Tissue factor Phospholipid Calcium The Prothrombin Time Add thromboplastin (excess of tissue factor + phospholipid + calcium) to citrated plasma. Not sensitive to XI, IX, VIII levels More sensitive than aPTT to warfarin effect Usually expressed as International Normalized Ratio (INR)

4 The Prothrombin Time =

5 ISI (International Sensitivity Index) is reagent- and method-specific; higher number indicates lower sensitivity to changes in clotting factor levels INR = Patient PT Mean Normal PT ()ISI

6 INR = ( ) = 2.0 INR = ( ) = 2.0 Reagent A: ISI = 1.24, mean normal = 12.6 sec PT = 22 sec Reagent B: ISI = 2.46, mean normal = 12.2 sec PT = 16.2 sec

7 INR values with two different reagents INR values with two different reagents Patients on warfarin PATIENT #INR REAGENT E (ISI 2.98)REAGENT B (ISI 0.96)

8 INTERPRETING A LONG PT/INR If aPTT normal –Factor VII deficiency –Mild deficiency of factors in common pathway (warfarin, vit K deficiency etc) If aPTT long: –Liver disease –Vitamin K deficiency –Warfarin –DIC –High level of heparin –Inhibitor affecting common pathway (eg, direct thrombin or Xa inhibitor) –Isolated deficiency of X, V, II, fibrinogen (rare)

9 Activated partial thromboplastin time (aPTT) “Partial thromboplastin” Phospholipid + Activator (provides surface for generation of XIIa) Incubate citrated plasma with phospholipid + activator (generates XIIa→XIa→IXa). Then add calcium to allow clotting to proceed to completion. Not sensitive to VII level. More sensitive to heparin than PT

10 Activated partial thromboplastin time (aPTT) =

11 CAUSES OF A LONG aPTT WITH A NORMAL PT/INR Deficiency of VIII, IX, XI or contact factor (usually XII) Heparin Factor VIII inhibitor Lupus-type inhibitor (antiphospholipid antibody)

12 Problems with the aPTT Long aPTT may not indicate a bleeding tendency –Contact factor deficiency –Lupus anticoagulant A normal (or short) aPTT is not necessarily an indication of an intact coagulation system –High factor VIII levels (eg, from endothelial injury) mask deficiencies of other factors –Circulating VIIIa can markedly shorten aPTT in DIC and other coagulopathies –Hemolysis shortens aPTT (lab rejects hemolyzed specimens)

13 The aPTT is not a good screening test The aPTT is not a good screening test Results of 1025 consecutive tests, excluding heparin monitoring Robbins and Rose, Ann Intern Med 1979;90:796 # TESTS# PATIENTS Abnormal result14397 On anticoagulant6437 Liver disease4127 No cause found, no bleeding 1514 Normal on repeat testing99 Known hemophilia54 History of intestinal bypass54 Other malabsorption (CF)21 Technical problem with test11 Newly dx'd bleeding disorder 00 # abnormal: 143 (14%)

14 Add thrombin (human or bovine) to plasma, measure clotting time Very sensitive to heparin and other thrombin inhibitors – If there is enough heparin in the plasma to prolong the aPTT the thrombin time should be very long Also prolonged by low fibrinogen, dysfibrinogenemia, high levels of fibrin degradation products – Reptilase time (snake venom clots fibrinogen) not affected by heparin but sensitive to these other conditions The Thrombin Time PT/INR aPTT Thrombin time

15 Mixing Study Purpose: to determine whether long aPTT or PT is due to clotting factor deficiency or circulating inhibitor (eg, factor VIII inhibitor, heparin, lupus- type inhibitor) Mix patient plasma 1:1 with normal plasma, measure aPTT or PT Incubate mixture for one hour, repeat aPTT or PT –Certain inhibitors (eg, factor VIII antibody) take time to work Failure to correct prolonged clotting time by mixing with normal plasma implies presence of a circulating inhibitor

16 Clotting factor assay Serial dilutions of patient plasma in factor-deficient plasma Serial dilutions of normal plasma in factor-deficient plasma (calibration curve) Measure aPTTs of both sets Semi-log plot - % of normal factor vs aPTT

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18 3% <1%

19 Patient C

20 100/.5 = 200%

21 100% 50% 10% 5% 1% aPTT (sec) % test plasma Normal plasma Patient ≥50%

22 100% 50% 10% 5% 1% aPTT (sec) % test plasma Normal plasma Patient Factor VIII Inhibitor

23 Bethesda Assay for Inhibitors Serial dilutions of patient plasma in normal plasma Incubate 2 hours Assay residual factor activity 1 Bethesda Unit neutralizes 50% of factor in an equivalent volume of normal plasma Example: 1:100 dilution of patient plasma + normal plasma → 50% residual factor activity, so inhibitor titer is 100 BU

24 Bethesda Assay Residual factor activity dilution pt plasma 50% 1:11:101:1001: BU

25 The decline and fall of the bleeding time Advantage: an in vivo test that theoretically measures both vascular and platelet function Disadvantages –Poor standardization –Accuracy depends on experience of operator –Poor sensitivity, very poor specificity –Does not predict bleeding risk

26 Rodgers and Levin, Semin Thromb Hemost 1990; 16:1 The bleeding time accurately detects aspirin use

27 Rodgers and Levin, Semin Thromb Hemost 1990; 16:1 The bleeding time does not predict surgical bleeding

28 Platelet function analysis Whole blood passed through capillary tube coated with collagen plus either ADP or epinephrine (high shear) Time to occlusion measured Moderate sensitivity to platelet function defects, VWD PFA-100

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30 Bleeding time vs PFA for detection of VWD BT C-Epi C-ADP Thromb Haemost 2003;90:483

31 Platelet function analysis Advantages vs bleeding time –In vitro test –Well-standardized –Somewhat better sensitivity and specificity Disadvantages –Does not assess vascular function –Does not predict bleeding risk Abnormal test result → test for specific defects in primary hemostasis Test not useful if platelets <100K or if patient taking ASA, etc PFA-100

32 Bleeding disorders that may be missed by screening tests von Willebrand disease –Use specific assays Factor XIII deficiency –Send out test for XIII activity Some platelet function disorders –Aggregometry, EM, genetic testing Fibrinolytic disorders Vascular disorders

33 Platelet aggregometry Various platelet agonists added to whole blood –Thrombin, ADP, collagen (2 concentrations), arachidonic acid, ristocetin (2 concentrations) Aggregation decreases electrical conductance Release measured by chemiluminesence Significantly more sensitive than PFA Many abnormal results nonspecific Expensive

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35 Saline agg Thrombin rel Risto low agg Risto high agg Collagen agg Low High Low Collagen rel AA agg ADP agg AA rel ADP rel 2 nM ATP Ch 1 Ch 2

36 Risto low Risto high Pt Control Type I VWD

37 Collagen agg Low High Low Collagen rel AA agg ADP agg AA rel ADP rel Pt Normal

38 Took Excedrin 5 days ago

39 AA agg ADP agg AA rel ADP rel Pt Normal Taking ASA 81 mg/d and Plavix 75 mg/d

40 AA agg Coll agg AA rel Coll rel Coll agg Low High Coll release Low High PFA: Coll/ADP 91 (nl ) Coll/Epi 139 (nl )

41 Assessment of the fibrinolytic system Fibrinogen D-dimer α2-antiplasmin activity Thromboelastography

42 Global assessment of clotting: thromboelastography Measures mechanical strength of clot vs time Sensitive to most major defects in fibrin clot formation, platelet plug formation, excessive fibrinolysis Can also detect hypercoagulability Useful “point of care” test in OR, etc

43 Clotting parameters in thromboelastography R value: time from adding activator until clot formation starts (analogous to aPTT, sensitive to clotting factor deficiency, heparin) Angle: Rate of initial clot formation (sensitive to fibrinogen concentration and platelet number/function) MA: maximal amplitude of clot strength (mainly determined by platelet number/function) LY30: degree of clot lysis at 30 min 30 min

44 Effect of Coagulation Factor Deficiency on TEG NormalFactor deficiency

45 Effect of platelet abnormality on TEG Normal Thrombocytopenia or dysfunctional platelets

46 Effect of hyperfibrinolysis on TEG NormalHyperfibrinolysis

47 Effect of hypercoagulability on TEG Normal Hypercoagulable

48 Platelet mapping If B < A platelet response to ADP or AA is inhibited B A Black line = standard TEG (fibrin + thrombin-activated platelets Green line = fibrin only (venom protease generates fibrin) Purple line = venom-generated fibrin + AA or ADP-activated platelets Arrows = contribution of platelets to clot strength under test conditions


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