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Pre-analytical factors that can affect coag test results

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Presentation on theme: "Pre-analytical factors that can affect coag test results"— Presentation transcript:


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 The Prothrombin Time Thromboplastin:
Tissue factor Phospholipid Calcium 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 Patient PT INR = Mean Normal PT
ISI (International Sensitivity Index) is reagent- and method-specific; higher number indicates lower sensitivity to changes in clotting factor levels

6 Reagent A: ISI = 1.24, mean normal = 12.6 sec
PT = 22 sec 1.24 ( ) 22.0 12.6 INR = = 2.0 Reagent B: ISI = 2.46, mean normal = 12.2 sec PT = 16.2 sec 2.46 ( ) 16.2 12.2 INR = = 2.0

7 INR values with two different reagents Patients on warfarin
REAGENT E (ISI 2.98) REAGENT B (ISI 0.96) PATIENT # INR INR 1 3.4 2.7 2 2.8 2.5 3 3.5 2.3 4 2.6 2 5 2.2 1.2 6 2.3 2.4 7 1.9 1.7 8 3 2.8 9 2.2 2.7 10 4 4

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)
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 “Partial thromboplastin” Phospholipid + Activator (provides surface for generation of XIIa)

10 Activated partial thromboplastin time (aPTT)

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 Results of 1025 consecutive tests, excluding heparin monitoring Robbins and Rose, Ann Intern Med 1979;90:796 # TESTS # PATIENTS Abnormal result 143 97 On anticoagulant 64 37 Liver disease 41 27 No cause found, no bleeding 15 14 Normal on repeat testing 9 Known hemophilia 5 4 History of intestinal bypass Other malabsorption (CF) 2 1 Technical problem with test Newly dx'd bleeding disorder # abnormal: 143 (14%)

14 The Thrombin Time PT/INR aPTT Thrombin time 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

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


18 3% <1%

19 Patient C

20 100/.5 = 200% Patient C

21 100% 50% 10% 5% 1% 20 40 60 80 aPTT (sec) % test plasma Normal plasma Patient ≥50%

22 100% Patient 50% Factor VIII Inhibitor % test plasma 10% Normal plasma 5% 1% 20 40 60 80 aPTT (sec)

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% 1:1 1:10 1:100 1:1000 100 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 The bleeding time accurately detects aspirin use
Rodgers and Levin, Semin Thromb Hemost 1990; 16:1

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

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


30 Bleeding time vs PFA for detection of VWD
C-ADP C-Epi BT 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


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

36 Risto low Risto high Pt Control Type I VWD

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

38 Took Excedrin 5 days ago

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

40 PFA: Coll/ADP 91 (nl 65-120) Coll/Epi 139 (nl 85-175) Low High
AA agg Coll agg AA rel Coll rel 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
30 min 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

44 Effect of Coagulation Factor Deficiency on TEG
This tracing demonstrates an enzymatic pathway abnormality in a patient who is bleeding. The elongated R and normal MA values suggest factor deficiency or dysfunction. The depressed angle value demonstrates the effect of factor deficiencies on the rate of fibrin-platelet clot formation. Normal Factor deficiency

45 Effect of platelet abnormality on TEG
Platelet abnormalities are typically identified by a low MA value. In cases where a patient is bleeding and the MA is low, reduced platelet count and/or function should be suspected as the cause. Thrombocytopenia or dysfunctional platelets Normal

46 Effect of hyperfibrinolysis on TEG
Normal Hyperfibrinolysis

47 Effect of hypercoagulability on TEG
Normal Hypercoagulable

48 Platelet mapping 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 B A If B < A platelet response to ADP or AA is inhibited

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