3. 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/ µl
• Centrifugation (10 – 20 min, 1000g)
• Assays performed on Plasma
3
4. Plasma vs Serum
Plasma
Anticoagulated
• Only citrate is
acceptable
Common
Abbreviations:
Serum
Not anticoagulated
Consumption of
coagulation factors,
particularly fibrinogen,
F V, F VIII, F II
PPP – platelet poor plasma
NPP – normal pool plasma
PNP – pooled normal
plasma
4
11. Monitoring Heparin
Clot based assays:
aPTT Ratio (1.5 – 2.5 x)
• use median value of normal range
• use patient’s baseline aPTT
Chromogenic assays
Anti Xa assay= Heparin Assay
Role of AT III
11
12. 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
12
13. Monitoring HeparinTherapy
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”
13
14. Responsiveness varies with
reagent, instrument, drug
Standardization attempts
(INR like) have failed
UFH
LMWH
150
aPTT [sec]
Monitoring Heparin
Therapy
APTT
200
100
50
0
0
0,2
0,4
0,6
0,8
UFH: Correlation of APTT and
Anti-FXa poor;
LMWH: very low sensitivity
Fondaparinux: insensitive
14
1
16. New direct Xa inhibitors
Rivaroxaban
Mw 436 g/mole
Rapid onset
Apixaban
MW 459.5 g/mole
Rapid onset
• ~ 3h
• 3-4 h
Half-life
Half-life
• ~ 12h
• 10-14h
Excretion
Excretion
• Renal
• Hepatic metabolism
– Substrate for CYP 3A4
• Renal
• Hepatic metabolism
– Substrate for CYP 3A4
16
17. Oral Direct Xa inhibitors: Effect
on clot based tests
Variable effects depending on reagents
Prolonged PT
• 2x control at ~138 -764nM
Prolonged APTT
• 2x control at ~897-2050nM
Thrombin time
• Insensitive ~2500nM
All clot based factor assays show a concentration
dependent effect
17
19. Thrombin Time
Heparin Contamination
Dysfibrinogenemia (follow with Reptilase
Time)
Thrombin: FPA & FPB
Reptilase: FPA
(Hypofibrinogenemia/DIC)
better alternatives: fibrinogen, D-dimers
Need fibrinogen result for interpretation!
19
20. 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
– sensitive to LMWH
20
21. Short PT/APTT
In vitro sample activation
problematic venopuncture
under anticoagulation/low Hct
High F VIII (APTT)
FEIBA, r F VIIa
21
23. 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%
23
25. 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 Xa
Inhibitors, DTI
25
26. Mixing Studies
Patient Results
PNP
Patient : PNP
Interpretation: What is correction?
Factor deficiency vs. circulating
anticoagulants
APTT Actin FS
Lupus anticoagulant insensitive reagent
26
27. 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: 19.1 sec (9.4 – 12.8 sec)
APTT: 79.6 sec (23.8-36.3 sec)
27
31. Clot Based
Specific Factor Assays
PT or APTT based
Patient plasma
Deficient plasma
Pooled normal plasma/assayed reference
plasma
Patient plasma assayed at 3 dilutions
Anticoagulant effect: rising factor level
with increasing dilution
31
32. 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
32
33. Detection of Lupus
Anticoagulant
Dilute Russel Viper Venom Time
Silica Clotting Time
• (APTT based test)
Tests based on neutralization of lupus
anticoagulant with excess phospholipid
33
35. Sensitivity of Screening APTT to Lupus Anticoagulant
A normal APTT does not rule out the
presence of a lupus anticoagulant
20% False Negative on samples with low titer LAC
depending on APTT reagent
35
36. Evaluating a Lupus Anticoagulant
(ISTH Recommendations)
Prolonged Coagulation Screening Test
(PT, APTT-LA, dRVVT)
Uncorrected Screening Test by Mixing
Study (R/O factor deficiency)
Shortening of Abnormal Clotting Time with
Addition of Excess Phospholipid
Exclusion of specific factor inhibitor
36
37. 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
37
38. 4T Scoring System for Pretest Probability
(T.E. Warkentin, 2006)
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
unclear or plt
fall after 10
days
Plt fall <5 days and
without recent
heparin
<1 day if previous
heparin within 100
days
Score <3: < 5% chance of HIT
Score 4-5: Intermediate risk
Score > 6: Very high risk of HIT
Thrombosis
New thrombosis, skin
necrosis
Progressive or
recurrent
thrombosis,
some skin
lesions e.g.
erythema
None
Other causes of
Thrombocytopenia
None
Possible
Other causes clearly
identified
38
39. ConfirmatoryHIT/T Testing
Heparin Induced Platelet Aggregation
~ 50% sensitive
Serotonin Release Assay
Gold standard
• not available locally
• 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
39
