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

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)
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)=

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 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/dPt
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