4. Preanalytical Variables
Specimen Collection
3.2% sodium citrate
9:1 volume of blood to anticoagulant
Hct 25% or 50% may affect results
5. Preanalytical Variables
Stability
PT (good up to 72 h, closed tube at RT)
APTT (good up to 4 h, closed tube at RT)
Special tests – plasma must be frozen at –80C,
if not assayed within 4 h of collection
Specimen Processing
Preparation of Platelet Poor Plasma
• Plt 10,000/ ml
• Centrifugation (10 – 20 min, 1000g)
• Assays performed on Plasma
6. Plasma vs Serum
Plasma
Anticoagulated
• Only citrate is
acceptable
Common
Abbreviations:
PPP – platelet poor plasma
NPP – normal pool plasma
PNP – pooled normal
plasma
Serum
Not anticoagulated
Consumption of
coagulation factors,
particularly fibrinogen,
F V, F VIII, F II
12. Monitoring Heparin
Clot based assays:
aPTT (sec)
aPTT reagent sensitivity varies
aPTT Ratio (1.5 – 2.5 x)
• use median value of normal range
• use patient’s baseline aPTT
13. Monitoring Heparin Therapy
Using the APTT
APTT response to heparin therapy may
be exaggerated
Numerous factors may elevate the APTT:
• Concomitant warfarin therapy
• Lupus anticoagulant
• Factor deficiency
• Liver disease
14. Monitoring Heparin Therapy
Using the APTT
APTT response to anticoagulants may be blunted
Factor VIII and fibrinogen elevate
• Can shorten the APTT in a clinically significant manner
• Increase in factor VIII from 100% to 250% can shorten APTT by
10%
Under-estimates level of anticoagulation
Cause of in vitro drug “resistance”
15. Monitoring Heparin (LMWH)
Chromogenic assays
Anti Xa assay= Heparin Assay
• Specify anticoagulant
• Need separate assay for Fondaparinux
Role of AT III
16. 200
150
100
50
0
0 0,2 0,4 0,6 0,8 1
aPTT [sec]
UFH: Correlation of APTT and
Anti-FXa poor;
LMWH: very low sensitivity
Fondaparinux: insensitive
UFH
LMWH
Monitoring Heparin
Therapy
APTT
Responsiveness varies with
reagent, instrument, drug
Standardization attempts
(INR like) have failed
18. DOACs
Dabigatran
etexilate
Rivaroxaban Apixaban
Target Thrombin F Xa F Xa
Prodrug Converted via
esterase catalyzed
hydrolysis to
dabigatran
No No
Bioavailability 6.5% 80% ~ 66%
Peak 2h 2.5 – 4 h 3 h
½ life 12-14h 7-13h 8-13h
Routine monitoring No No No
Dosing Fixed, BID Fixed, BID Fixed, BID
Elimination 80% renal 67% renal, 33%
fecal
25% renal, 75%
fecal
23. Thrombin Time
Evaluates 3rd Stage of Coagulation
Dysfibrinogenemia (follow with Reptilase Time)
Thrombin: FPA FPB
Reptilase: FPA
Need fibrinogen result for interpretation
(Hypofibrinogenemia/DIC)
better alternatives: fibrinogen, D-dimers
Heparin Contamination
24. Fibrinogen
Assay and
Thrombin Time
3rd Stage of Coagulation
Neither assay
measures
crosslinked fibrin
To measure F XIII
activity- F XIII
assay or
quantitative F XIII
antigen
25. Sensitivity of Screening Tests
PT/APTT : prolonged by single factor
deficiency 30% (variable)
PT: highly sensitive to multiple Vit K
dependent factor deficiencies
APTT: more sensitive to heparin
– Variably sensitive to LMWH
26. Short PT/APTT
In vitro sample activation
problematic venopuncture
under anticoagulation/low Hct
High F VIII (APTT)
FEIBA, r F VIIa
27. D-dimers vs FDPs
D-dimer = crosslinked
degradation product
FPD = fibrinogen or
fibrin degradation
product
primary fibrinolysis
tPA
uPA
SK
Fibrinolysis
28. Quantitative D-dimer Assays
MoAb to D-dimers on microbeads
Agglutination of beads in the presence of
D-dimers
Reference Range: 230 ng/ml
Rule out thrombotic event:
NPV 100%
Specificity 49%
29. Elevated D-dimers
Recent thrombosis
DIC
Inflammatory conditions
Cancer
30. Interpretation of Prolonged PT
and/or APTT Results
Factor Deficiency
Single vs multiple deficiencies
Circulating Anticoagulant
Lupus-like anticoagulant
Specific factor inhibitor
Paraproteins
Anticoagulants: UFH, LMWH,
Direct Oral Anticoagulants
31. Mixing Studies
Patient Results
PNP
Patient : PNP
Interpretation: What is correction?
Factor deficiency vs. circulating anticoagulants
APTT Actin FS
Lupus anticoagulant insensitive reagent
Normal result rules out a significant factor
deficiency
32. Case Study
23 Y Female, newly diagnosed Hodgkins
Lymphoma with widespread metastatic
disease to bones, liver, spleen, and
lymphadenopathy
Scheduled for possible right pleural biopsy
and mediastinal lymph node biopsy
PT: 22.1 sec (9.4 – 12.8 sec)
APTT: 79.6 sec (23.8-36.3 sec)
36. Clot Based
Specific Factor Assays
PT or APTT based
Constituents
Patient plasma
Factor deficient plasma
Assay Principle
Patient plasma reconstitutes factor deficient
plasma
Pooled normal plasma/assayed reference
plasma is used for quantitation
37. Effect of anticoagulants on factor assays
Heparin
Direct thrombin or Xa inhibitors –
all clot based assays are invalid
falsely DECREASED Factors I - XII
falsely INCREASED anticoagulation factors
38. Detection of Lupus
Anticoagulant
No single screening test
Requires a panel of screening and confirmatory
assays demonstrating neutralization of lupus
anticoagulant with excess phospholipid
Dilute Russel Viper Venom Time
APTT based tests
• Silica Clotting Time (IL)
• StaClot LA (Stago)
Results affected by anticoagulation therapy
39. Sensitivity of Screening APTT to Lupus Anticoagulant
A normal APTT does not rule out the
presence of a lupus anticoagulant
Most commercial APTT reagents are moderately
sensitive to LA
Proficiency Testing Survey data
~20% False Negative on samples with low titer LAC
40. HIT Testing
Screening ELISA
Antibodies to heparin-PF4 complexes
• IgG titers
– IgG titer (OD) more specific
– Involved in platelet activation
• Specificity: heparin spike
• High Negative Predictive Value
• Poor specificity (false positives)
– Improves with consideration of pre-test probability
41. 4T Scoring System for Pretest Probability
2 1 0
Thrombocytopenia 50% fall in plt or plt
nadir of 20K-100K
30-50% fall in
plt or plt nadir
10K-19K
30% fall in plt or
plt nadir of 10K
Timing 5-10 d post heparin
1 day if previous
heparin within 100
days
unclear or plt
fall after 10
days
Plt fall 5 days and
without recent
heparin
Thrombosis New thrombosis, skin
necrosis
Progressive or
recurrent
thrombosis,
some skin
lesions e.g.
erythema
None
Other causes of
Thrombocytopenia
Score 3: 5% chance of HIT
Score 4-5: Intermediate risk
Score 6: Very high risk of HIT
None Possible Other causes clearly
identified
(Lo GK, Juhl D, Warkentin TE, Sigouin CS, Eichler P, Greinacher A. Evaluation of pretest clinical
score (4 T’s) for the diagnosis of heparin-induced thrombocytopenia in two clinical settings. J Thromb
Haemost 2006; 4: 759–65.)
42. ConfirmatoryHIT/T Testing
Serotonin Release Assay
Gold standard
• Send-out test
• Use to clarify ELISA test result
– intermediate positives (O.D. 0.8 and 1.0), or
– high positives (O.D. 1.0) not responsive to heparin spike
