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. Effect of high hematocrit on coag tests
Elevated citrate concentration may prolong clotting times

4. The Prothrombin Time Thromboplastin:
Tissue factor
Phospholipid
Calcium
Also contains a heparin-
neutralizing agent
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)

5. Why the INR?
Tissue factor in the thromboplastin may be recombinant, or derived from human or animal tissue
Phospholipid composition varies among thromboplastins
As a result different thromboplastins have varying sensitivity to the effect of warfarin
The INR system makes PT results from different laboratories comparable to one another in patients receiving vitamin K antagonists (not in liver disease or other coagulopathies)

6. ( ) 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

7. 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

8. 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

9. Uses of the PT/INR
Best single test of the integrity of the fibrin clotting system
Detects most clinically significant acquired coagulopathies
Does not detect the most common inherited clotting factor deficiencies (VIII, IX, XI)
Routinely used to monitor warfarin therapy
Insensitive to heparin at usual therapeutic concentrations

10. 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)

11. Uses of the PTT
Screen for inherited clotting factor deficiency (hemophilia, factor XI)
Monitor heparin therapy
Screen for acquired coagulation inhibitors
Factor VIII antibody
Lupus anticoagulant
A long PTT does not always indicate a bleeding disorder
Factor XII deficiency

12. The aPTT should be ordered selectively 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%)

13. What causes a long PT/INR and a normal PTT?
Factor VII deficiency
Mild deficiency of “common pathway” factors
Warfarin
Vitamin K deficiency
Liver disease
PTT
PT/INR

14. What would cause a long PTT with a normal INR?
PT/INR
Deficiency of VIII, IX, XI
Deficiency of a contact factor (usually XII) (does not cause bleeding)
Heparin
Factor VIII inhibitor
Lupus-type inhibitor (antiphospholipid antibody)

15. What if both PT/INR and PTT are long?
Liver disease
Vitamin K deficiency
Warfarin
DIC
High level of heparin
Other inhibitor affecting common pathway (eg, direct thrombin inhibitor)
Isolated deficiency of X, V, II, fibrinogen (rare)
PTT
PT/INR

16. Other tests
aPTT
Thrombin time
PT/INR
Thrombin time: thrombin + plasma. Very sensitive to heparin and other thrombin inhibitors. Prolonged by low fibrinogen, dysfibrinogenemia, high levels of fibrin degradation products.
Urea solubility: clot immersed in concentrated urea (breaks noncovalent bonds) clot dissolves unless crosslinked by factor XIIIa). For diagnosis of severe factor XIII deficiency (v. rare)

17. 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
Note: warfarin does not act as a circulating inhibitor. It inhibits synthesis of clotting factors by the liver

18. 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

20. 3%
<1%

21. Patient C

22. 100/.5 = 200%
Patient C

23. Lupus inhibitor or other non-competitive clotting inhibitor → non-parallel plot
100%
≥50%
Patient
50%
% test plasma
10%
Normal plasma 5% 1% 20 40 60 80
aPTT (sec)

24. 100%
Patient
50%
Factor VIII
Inhibitor
% test plasma
10%
Normal plasma 5% 1% 20 40 60 80
aPTT (sec)

25. 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

26. Bethesda Assay
Residual factor activity
dilution pt plasma
50%
1:1
1:10
1:100
1:1000
100 BU

27. 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

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

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

30. 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

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

33. 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

34. 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

36. 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

37. Risto low
Risto high Pt
Control
Type I VWD

38. Pt Normal Collagen agg Low High AA agg ADP agg AA rel Collagen rel
ADP rel
Normal

39. Took Excedrin 5 days ago

40. 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

41. 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 )

42. Assessment of the fibrinolytic system
Fibrinogen (dilute thrombin time assay)
D-dimer (immunoassay)
α2-antiplasmin activity (chromogenic substrate assay)
Thromboelastography

43. 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 to guide blood product use
30 min

45. World J Transplant 2012;2:1

46. 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

47. 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

48. Effect of hyperfibrinolysis on TEG
Normal
Hyperfibrinolysis

49. Chromogenic substrate-based assay
Peptide containing target sequence of enzyme linked to chromophore
Colored cleavage product (in this case nitroaniline) detectable by spectrophotometry
Enzyme specificity determined by target sequence
Rate of color generation proportional to enzyme activity

50. Examples of chromogenic assays
Anti-Xa assay
Patient plasma added to mixture of Xa and chromogenic substrate (± antithrombin)
Residual Xa activity inversely proportional to inhibitor level
Protein C activity
Patient plasma + venom enzyme that selectively activates protein C; activated protein C cleaves substrate

51. Von Willebrand factor measurements
VWF antigen: via ELISA
VWF activity (must be sensitive to total VWF protein, integrity of binding sites, presence of large multimers)
Ristocetin cofactor assay: patient plasma + ristocetin + formalin fixed platelets
Ristocetin unfolds VWF, which then clumps platelets
Alternative assay uses beads coated with monoclonal Ab against GP1b binding site in VWF rather than platelets
Multimer analysis: via gel electrophoresis