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)
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)
The Prothrombin Time
=
Patient
PT
INR =
Mean Normal PT
(
ISI
)
ISI (International Sensitivity Index) is reagent- and method-specific;
higher number indicates lower sensitivity to changes in clotting
factor levels
Reagent A: ISI = 1.24, mean normal = 12.6 sec
PT = 22 sec
22.0
INR =
12.6
(
1.24
)
= 2.0
Reagent B: ISI = 2.46, mean normal = 12.2 sec
PT = 16.2 sec
16.2
INR =
12.2
(
2.46
)
= 2.0
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
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)
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
Activated partial thromboplastin time
(aPTT)
=
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)
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)
The aPTT is not a good screening test
Results of 1025 consecutive tests, excluding
heparin monitoring
# abnormal: 143 (14%)
Abnormal result
On anticoagulant
Liver disease
No cause found, no
bleeding
Normal on repeat testing
Known hemophilia
History of intestinal bypass
Other malabsorption (CF)
Technical problem with test
Newly dx'd bleeding
disorder
# TESTS
143
64
41
15
9
5
5
2
# PATIENTS
97
37
27
14
9
4
4
1
1
1
0
0
Robbins and Rose, Ann Intern Med 1979;90:796
The Thrombin Time
aPTT
Thrombin time
PT/INR
•
•
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
Mixing Study
• Purpose: to determine whether long aPTT or PT is
due to clotting factor deficiency or circulating
inhibitor (eg, factor VIII inhibitor, heparin, lupustype 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
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
3%
<1%
100/.5 = 200%
100%
≥50%
% test plasma
50%
10%
5%
1%
20
40
aPTT (sec)
60
80
100%
% test plasma
50%
Factor VIII
Inhibitor
10%
5%
1%
20
40
aPTT (sec)
60
80
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
50%
Residual factor activity
Bethesda Assay
100 BU
1:1
1:10 1:100 1:1000
dilution pt plasma
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
The bleeding time accurately detects
aspirin use
Rodgers and Levin, Semin Thromb Hemost 1990; 16:1
The bleeding time does not predict
surgical bleeding
Rodgers and Levin, Semin Thromb Hemost 1990; 16:1
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
Bleeding time vs PFA for detection of VWD
C-ADP
C-Epi
BT
Thromb Haemost 2003;90:483
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
PFA-100
• Abnormal test result → test for specific
defects in primary hemostasis
• Test not useful if platelets <100K or if
patient taking ASA, etc
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
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
Saline agg
AA agg
ADP agg
Thrombin rel
Collagen agg
Low
High
2 nM ATP
Ch 1
Ch 2
AA rel
ADP rel
Risto low agg
Risto high agg
High
Low
Collagen rel
Pt
Risto low
Control
Type I VWD
Risto high
Pt
Collagen agg
Low
High
AA agg
ADP agg
Normal
High
Low
Collagen rel
AA rel
ADP rel
Took Excedrin 5 days ago
Pt
AA agg
ADP agg
Normal
Taking ASA 81 mg/d and Plavix 75 mg/d
AA rel
ADP rel
Coll agg
Low
High
Coll release
Low High
AA agg
Coll agg
PFA: Coll/ADP 91 (nl 65-120)
Coll/Epi 139 (nl 85-175)
AA rel Coll rel
Assessment of the fibrinolytic system
•
•
•
•
Fibrinogen
D-dimer
α2-antiplasmin activity
Thromboelastography
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
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
Effect of Coagulation Factor
Deficiency on TEG
Normal
Factor deficiency
Effect of platelet abnormality on TEG
Normal
Thrombocytopenia or
dysfunctional platelets
Effect of hyperfibrinolysis on TEG
Normal
Hyperfibrinolysis
Effect of hypercoagulability on TEG
Normal
Hypercoagulable
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
A
B
If B < A platelet response to ADP or AA is inhibited