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Coag testing for hema fellows mskcc 10 15 2015 dr peerschke
1. Introduction to Coagulation Testing
Ellinor I. Peerschke, Ph.D., F.A.H.A.
Vice Chair, Laboratory Medicine
Chief, Hematology & Coagulation Laboratory Services
MSKCC
2. Hemostasis
Balance between bleeding and clotting
Cellular Components
• Vascular endothelial cells
• Platelets – Primary hemostasis
Fluid phase components
• Coagulation proteins and enzymes
• Secondary Hemostasis
3. Learning Objectives
List major Clinical Laboratory Coagulation
Screening Tests
Discuss use of screening tests and their principles
in the evaluation of patients with bleeding
disorders or for monitoring patients receiving
anticoagulation therapy
Interpret test results
7. Preanalytical Considerations
Specimen Stability
PT (stable up to 72 h, closed tube at RT)
APTT (stable 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
9. Types of Assays
• Functional Assays
Clot-based assays
Good screening assays
Based on a functioning coagulation cascade
Subject to exogenous and intrinsic interferences
Chromogenic assays
Discreet measure of the activity of a specific enzyme
Not affected by most preanalytical variables
Enzyme of interest
Peptide pNA Peptide pNA
Color develops
Quantify
spectrophotometrically
Absorbance correlates
with activity
(Substrate)
+
10. Types of Assays
Immunologic assays
LIA- or ELISA-based technologies
Measure the amount of protein present rather than functional activity
• Sandwich ELISA
• Immuno capture
• Immuno detection with enzyme conjugated secondary antibody
• Substrate cleaved by conjugated enzyme
• Color development
• Spectrophotometric quantification
• Latex Agglutination
• Antibody coated latex beads
• Agglutination in presence of antigen
• Agglutination is measured optically
11. Screening Tests of Hemostasis
PT
APTT
Fibrinogen
Thrombin Time
D-Dimer
Identify underlying
coagulation defect
Monitor/assess
anticoagulation
therapy
Evaluate for DIC
Rule out DVT/PE
Clot
based
Optical
LIA Test
12. Major Uses: Hemostasis Screening
Monitoring Warfarin Anticoagulation
From: Jesty & Morrison, Stony Brook University,
Stony Brook, NY
14. Major Uses: Hemostasis Screening
Monitoring UFH heparin therapy
From: Jesty & Morrison, Stony Brook
University, Stony Brook, NY
15. APTT reagents are variably sensitive to
UFH
Laboratories establish reagent specific
therapeutic range
Reagent standardization has not been
successful
APTT: Monitoring UFH Therapy
16. APTT response to heparin may be exaggerated by
Conditions that elevate the APTT:
• Concomitant warfarin therapy
• Lupus anticoagulant
• Liver disease
APTT response to heparin may be blunted by
Conditions that shorten the APTT:
• Elevated Factor VIII
• Antithrombin deficiency
Under-estimates level of anticoagulation
• Cause of in vitro drug “resistance”
APTT: Monitoring UFH Therapy
17. Thrombin Time
Highly sensitive to UFH Contamination
Highly sensitive to Direct Thrombin Inhibitors
Evaluates 3rd
Stage of Coagulation
Dysfibrinogenemia (follow with Reptilase Time)
Thrombin: FPA & FPB
Reptilase: FPA
Need fibrinogen result for interpretation
Hypo/Dysfibrinogenemia
Compare functional fibrinogen with fibrinogen antigen
18. Fibrinogen & Thrombin Time
Fibrinogen Assay
Patient plasma + excess thrombin
Thrombin Time
Patient plasma+ limited thrombin
Reaction
Fibrinogen + thrombin
• Cleavage of FPA, FPB
Fibrin monomers
Fibrin monomer polymerization CLOT
Factor XIII + thrombin F XIIIa
F XIIIa crosslinking of fibrin into stable
clot
19. Fibrinogen Assay and Thrombin Time
• Neither assay measures crosslinked fibrin or
F XIII activity
• Reptilase Time
• Reptilase cleaves FPA
• Insensensitive to heparin
20. Limitations of Coagulation
Screening Tests
Do not detect defects in F XIII
Do not detect defects in fibrinolysis
Fibrinolysis inhibitors
• Tissue plasminogen activator
• Plasmin inhibitor
21. Sensitivity of Screening Tests
PT/APTT : prolonged by single factor
deficiency <30% (variable)
PT: highly sensitive to Vit K dependent
factor deficiencies
Variably sensitive to DOAC
APTT: sensitive to unfractionated heparin
– Variably sensitive to LMWH
– Variably sensitive to DOAC
22. Monitoring LMWH: Anti-Xa Heparin Assay
Specifically determines anticoagulant
activity of LMWH (and UFH) by
measuring ability of heparin-bound
antithrombin to inhibit F Xa
More specific than aPTT since it
measures inhibition of a single
enzyme
Major advantage is lack of biologic
interference
Limitations of Heparin Assay
Clinical data examining
outcomes is limited
Eikelboom JW. Thromb Haemost 2006;96:547-52.
Francis JL. Pharmacotherapy 2004;24:108S-19S.
Color development is Inversely
proportional to the anticoagulant
concentration in the plasma sample
Excess
FXa
Plasma [heparin] +
(Antithrombin)
[AT-Heparin-Xa] + Residual FXa
Chromogenic
substrate
pNA
23. Specific Assays for DOAC
Rivaroxaban Level
Chromogenic Xa
Dabigatran Level
Dilute thrombin time
• Diluted Patient plasma
NYS approved at MSKCC
24. Short PT/APTT
In vitro sample activation
traumatic venopuncture
under anticoagulation/low Hct
High F VIII (APTT)
PCCs, rFVIIa
25. D-dimer
D-dimer = crosslinked fibrin degradation product
Presence indicates activation of both coagulation
and fibrinolysis (plasmin)
26. Quantitative D-dimer Assay
MoAb to D-dimers linked to microbeads
Agglutination of beads occurs in the
presence of D-dimers
Agglutination is measured optically
Cut off: <230 ng/ml
Rule out thrombotic event:
• NPV 100%
• Specificity 49%
28. Interpretation of Prolonged PT
and/or APTT Results
Factor Deficiency
Single vs multiple deficiencies
Specific Anticoagulants
Specific factor inhibitor
F VIII, F V
Global Anticoagulant
Lupus anticoagulant
Paraproteins
Therapeutic Anticoagulants: UFH, LMWH,
Direct Oral
29. Prolonged PT/APTT Work-Up:
Mixing Studies
Compare Clotting Time Results
Patient Plasma
Pooled Normal Plasma (PNP)
Patient : PNP mix (1:1 mix)
Interpretation:
Correction
• Factor deficiency
Lack of Correction
• Circulating anticoagulant
30. Mixing Studies
Immediate Mix
Incubated Mix (60 min, 37o
C)
To detect time dependent inhibitors
• F VIII Inhibitors
– Less correction of Incubated Mix than Immediate Mix
31. Mixing Study at MSKCC
APTT Actin FS
Lupus anticoagulant insensitive reagent
Normal result rules out a significant factor
deficiency
32. Clot Based
Specific Factor Assays
PT or APTT based
Constituents
Patient plasma
Factor deficient plasma
Reference Plasma
Assay Principle
Patient plasma reconstitutes factor deficient
plasma
Assayed reference plasma is used for
quantitation
33. Factor Assays
Determines factor level as
% activity relative to reference plasma
Procedure
Patient plasma or reference plasma
(PNP)
Dilute plasma 1/10, 1/20/ 1/40 with
deficient plasma (deficient in a
single factor)
Perform PT or aPTT and compare
clotting time (seconds) of patient
plasma to reference plasma
(standard curve)
Patient plasma is run in multiple
dilutions (usually 3) to check for
the presence of an inhibitory
substance
Reference
Plasma
Patient 1
Patient 2
Patient 1: Factor Deficiency
Patient 2: Inhibitor Effect
34. Lupus Anticoagulant
Heterogeneous antibodies against phospholipids and phospholipid
binding proteins
Not usually associated with bleeding
• Arterial/venous thrombosis
• Rarely patients may also have antibodies against F II
– Check PT for prolongation
Prolongs screening APTT
• Reagent dependent
– APTT Actin FS
» LA insensitive
• Most clinical APTT reagents are moderately sensitive to LA
– Normal APTT does not rule out a LA
35. ISTH Guidelines for Lupus Anticoagulant Testing
• Two tests based on different principles
• dRVVT
• sensitive aPTT (low phospholipids and silica as activator)
• LA should be considered positive if one of the two tests
gives a positive result
(Pengo V, Tripodi A, Reber G, Rand JH, Ortel TL, Galli M, de Groot PG. Update of the
guidelines for lupus anticoagulant detection. J Thromb Haemost 2009; 7: 1737–40)
• False negative rate
• ~20% for low and intermediate titer antibodies
• False positive rate ~ 10%
• Repeat testing in 12 weeks for confirmation
(Dembitzer et al, Am J Clin Pathol 2010; 134:764-773)
36. Lupus Anticoagulant Testing: dRVVT Screen
Xa
X
Va
Xa
Prothrombin
dRVVT
Thrombin
Ca2+
Fibrinogen Fibrin
Low
Phospholipid
Content
Prolonged Clotting Time
What types of assays are there for identifying patients at risk and how good are they?
Functional assays for type I deficiencies
Antigenic assays for type II deficiencies
DNA-based assays for confirmation
The general principle of a chromogenic assay, in its simplest form: A protein is converted to its active formThe cleaved peptide releases the chromophore, in this example pNA, which gives off a color.
In this example, PNA is used. PNA = para-nitroaniline, which appears as a yellow color.
What types of assays are there for identifying patients at risk and how good are they?
Functional assays for type I deficiencies
Antigenic assays for type II deficiencies
DNA-based assays for confirmation
The general principle of a chromogenic assay, in its simplest form: A protein is converted to its active form. the. The cleaved peptide releases the chromophore, in this example pNA, which gives off a color.
In this example, PNA is used. PNA = para-nitroaniline, which appears as a yellow color.