This document provides an overview of routine coagulation assays including PT, aPTT, fibrinogen, D-dimer, and mixing studies. It discusses the clinical utility and interpretation of these assays for monitoring coagulation and detecting coagulation disorders or abnormalities. Key points include that PT measures the extrinsic pathway and factors VII, X, and prothrombin, while aPTT is more sensitive to deficiencies in the intrinsic pathway and contact factors. D-dimer has high negative predictive value for ruling out VTE but low positive predictive value due to non-specific elevations. Mixing studies can help distinguish between factor deficiencies and inhibitors like lupus anticoagulant.
1. 1
Routine Assays
in the Coagulation Laboratory
Larry Smith, PhD
Director, Coagulation/Hemostasis Laboratories
Assistant Attending
2. 2
Objectives
• Review of the traditional coagulation cascade
• Discuss routine assays frequently ordered
• Describe how these assay work
• Discuss interpretation and clinical utility
4. 4
Prothrombin Time (PT)
• Armand Quick 1935
• Measures the time required for fibrin clot to
form when plasma is added to [TF + Ca2+] mixture Ca2+
• TF combines with FVII to form the “extrinsic” +
tenase complex Thromboplastin
• Measures (TF)
a. FVII of the extrinsic pathway
b. FX, FV, FII, FI of the common pathway Patient Plasma
c. Measures 3 of the vitamin K-dependent
factors
• II, VII, X – does not measure IX
• PT is prolonged in
– Deficiencies of I, II, V, VII, X
– Liver Disease
– OAT
– Increased sensitivity to reduction in VKDF’s
– Increased FDP’s, antibiotics
– High dose heparin therapy and DTI’s
• PT is shortened following treatment with rVIIa
• Most common use of PT monitoring OAT
5. 5
Monitoring OAT
• Problems with the PT • INR developed by WHO using an IRP to
▫ Commercially available thromboplastins which all other thromboplastins can be
vary in their sensitivity to VKDF’s compared
▫ PT values that result from using different ▫ Recommended that a PT value be expressed
thromboplastins are not interchangeable as a ratio by normalizing it to the IRP
(ISI)
Reagent PT (sec) ISI INR INR = Patient’s PT
GM Normal PT
A 11 3.2 2.6
▫ ISI = measure of the sensitivity and
responsiveness of a particular
B 20 2.4 2.6 thromboplastin reagent to warfarin-
induced reduction of the VKDF’s
C 12 3.0 2.6 ▫ ISI of the IRP = 1.0
Advantages: INR for monitoring patients on OAT
D 18 2.2 2.6 1. Minimizes the variation in the PT assay
2. Allows comparability of PT results among
E 31 different laboratories
0.9 2.6
6. 6
Limitations of Warfarin Therapy
• PT/INR
Limitations Consequence
Slow onset of action Need to overlap with a parenteral Narrow therapeutic index
anticoagulant
Early procoagulant effect Early prothrombotic effect
Multiple drug/food Frequent monitoring required
interactions
Genetic variation in Variable dose requirement
metabolism
ADRs ~177,000/yr, ~30,000 ER visits/yr, ~3%
major bleeds, ~0.6% fatal bleeds
Narrow therapeutic index Bleeding or thrombosis
• INR should ONLY be used for patients who stabilized on OAT
• INR should NEVER be used as a substitute for the PT in patients who are NOT on OAT
1. Exponential nature of the INR may obscure interpretation of a mildly prolonged PT
WHY??? that may be suggestive of a coagulopathy
2. Sufficient studies have NOT been done to demonstrate how well the INR correlates
with diagnosis or outcome
7. 7
aPTT
• PTT – Langdell, Wagner and Brinkhous 1953
• aPTT – Proctor and Rappaport – 1961
Ca2+
• Measures
a. Time required for fibrin clot to form when a
plasma is incubated with an PL
[activator + partial thromboplastin + Ca2+ ] +
a. Activation of the contact factors activator
PK, HMWK, FXII, FXI
a. Intrinsic pathway factors Plasma
FXII, XI, IX, VIII
a. Common pathway
FX, V, II, I
• Prolonged in
1. Deficiencies of all factors except VII and XIII
2. Presence of inhibitors
Specific inhibitors – FVIII and FIX
Nonspecific inhibitors—LA, Heparin, DTI’s
• Shortened
▫ Elevated FVIII
• Monitor Heparin Therapy
8. 8
Laboratory Monitoring of UFH
• aPTT
▫ 4-6 hours after bolus dosage and every
24 hours thereafter
A dose adjustment requires
monitoring 6 hours after the dose
adjustment
▫ Target range 1.5-2.5 x “normal”
ACCP, CAP
Correlated to 0.3-0.7 anti-Xa
U/mL using a chromogenic anti-Xa
Heparin Assay
Monitor platelet counts
• Anti-factor Xa assay (UFH)
▫ 4 hours after administration
▫ Therapeutic target—0.3 - 0.7 anti-Xa
U/mL Current therapeutic range
▫ Monitor platelet count daily 55 – 80 seconds
9. 9
Therapeutic aPTT versus Anti-Xa Ranges
Therapeutic aPTT Ranges Therapeutic Ranges with anti-Xa
Drug Range
Drug Ratio
UFH 0.3 – 0.7 U/mL
1.5 – 2.5
UFH 2x/day
55.0 – 80.0 sec 0.5 – 1.1 U/mL
LMWH 1x/day
N/A 1.1 – 2.0 u/mL
LWMH
Not sensitive to
Pentasaccharide
LMWH
Prophylaxis of VTE
Trough – 0.14 – 0.19
Argatroban 1.5 – 3.0 Fondaparinux
Peak – 0.39 – 0.50
Treatment of VTE
Trough – 0.46 – 0.62
Lepirudin Peak – 1.20 – 1.26
1.5 - 2.5
(Refludan)
10. Reagent Factor Sensitivity
• aPTT is sensitive to deficiencies of contact and intrinsic factors
• MILD deficiency may result in a normal aPTT
11. 11
Fibrinogen
• Clauss Technique
▫ Functional assay
▫ Fibrinogen concentration is inversely High conctr
proportional to the thrombin time of thrombin
diluted plasma
▫ A reference (standard) curve is prepared 1:10
dilution
using known fibrinogen concentrations plasma
versus their respective thrombin times
• Detects
▫ Quantitative
Hypofibrinogenemia
Hyperfibrinogenemia
▫ Qualitative deficiency
Dysfibrinogenemia
• Acute phase protein elevated in
▫ Inflammation
▫ Trauma
▫ Infection
▫ Increases with age
▫ Associated with CVD and thrombosis
12. 12
Fibrinogen
• Low levels suggest bleeding
▫ DIC
▫ Thrombolytic therapy
Results in increased levels of FDP’s (>190 ug/mL)
Interfere with fibrin monomer polymerization
▫ Liver disease
1. Decreased synthesis of fibrinogen
2. Abnormal fibrinogen may be seen due to abnormal/increased sialic acid content
3. May result in elevated levels of fibrinolytic activators and decreased levels of fibrinolytic
inhibitors
▫ Some patients following treatment with l-asparaginase
▫ Heparin (UFH) may lead to underestimation
• Increased levels
▫ Increasing age
▫ Pregnancy
▫ OCT
▫ Disseminated malignancy
DTI’s falsely decrease the fibrinogen level — DON’T ORDER!!!
13. 13
Thrombin Time / Reptilase Time
Thrombin Time
• Measure the conversion of fibrinogen to fibrin
• Cleaves fibrinopeptides A and B
• Screen for heparin contamination
• Prolonged
▫ Heparin therapy (UFH)
▫ Hypofibrinogenemia, Dysfibrinogenemia
▫ Paraproteins (Amyloidosis, Myeloma)
▫ Severe LD
▫ Elevated FDP’s
▫ DTI’s
▫ Bovine thrombin glues
Reptilase time
• Cleaves fibrinopeptide A only
• Unaffected by Heparin
• Unaffected by bovine thrombin glues
• Same as above
14. 14
D-Dimer
Specific degradation product of fibrin clots
that results from the action of
1. Thrombin
Converted fibrinogen into fibrin
clots
1. FXIIIa
Cross-linked fibrin monomers
clots
1. Plasmin
Cleaved the cross-linked fibrin
clot
D e D D D
15. 15
D-Dimer
• Monoclonal antibody raised
against specific epitopes on D-
dimer that react with cross-linked
fibrin
▫ Does not react with
Fibrinogen degradation
products
Non-cross-linked fibrin
degradation products
▫ Ensures high specificity for D-
dimer as a biomarker of fibrin
formation and stabilization
16. 16
Clinical Utility
• Diagnosis of VTE in combination with pretest clinical probability
▫ High negative predictive value for exclusion of DVT
▫ Poor positive predictive value for DVT
Elevated in conditions unrelated to thrombosis
Almost all patients with acute disease will have elevated D-dimer levels
• Clinical use
▫ Identification of individuals at increased risk thrombotic events (arterial and venous)
▫ Identification of individuals at increased risk of recurrent VTE
Elevated levels following discontinuation of anticoagulant therapy
▫ Establishing of optimal duration of secondary prophylaxis after a first episode of VTE
▫ Pregnancy monitoring
▫ Diagnosis/monitoring of DIC
Sensitive but NOT specific marker for DIC
1. A positive D-dimer is NOT specific for VTE
2. Negative D-dimer is highly unlikely for VTE
3. The greatest utility of D-dimer is its negative predictive value for VTE!
17. 17
Fibrin(ogen) Degradation Products
• Patient plasma mixed with latex particles
coated with monoclonal anti-FDP
antibodies
• Positive FDP assay indicates
▫ Fibrin and/or fibrinogen is being
degraded by plasmin
• Elevated FDPs
▫ Dysfibrinogenemia
▫ LD, DIC, DVT, PE, MI
▫ Thrombolytic therapy
▫ Primary and secondary
fibrinogenolysis
• What would the D-dimer and FSP levels be in a person who
has a congenital FXIII deficiency?
• What would the D-dimer and FSP levels be in individuals
with FXIII deficiency due to DIC?
18. 18
Algorithm of Mixing Tests
Prolongation of the Coagulation
Time of the Screening Test
Mixing: Patient Plasma + Normal Plasma
Correction No Correction
Factor Deficiency Lupus Anticoagulant Specific Factor Inhibitor
19. 19
Mixing Studies
• Look for the presence of:
▫ Factor Deficiency versus Inhibitor
Step 1
Mix and Run
TUBE 1 TUBE 2 TUBE 3
300 uL PNP
1 mL PNP 300 uL patient 1 mL Run immediately
plasma patient
plasma
Incubate @ 37oC
Step 2
Mix and Run
TUBE 1 TUBE 2 TUBE 3
300 uL PNP Run after 1 hour incubation
PNP 300 uL Patient
patient plasma
plasma
20. 20
Interpreting Mixing Studies
If the 50/50 corrects after the immediate and remains corrected after the 60 minute
incubation
Factor Deficiency
Follow-up with specific factor assays
If the 50/50 corrects after the immediate, but prolongs after the 60 minute incubation
Time-dependent inhibitor – usually a specific factor inhibitor
~ 15% of LA’s may be time-and-temperature dependent
Follow-up with a specific factor assay and specific factor inhibitor assay
If the 50/50 prolongs after the immediate and remains prolonged after the 60 minute
incubation
Nonspecific inhibitor such as a lupus anticoagulant
Follow-up with Lupus anticoagulant assay
Mixing studies on samples minimally prolonged (<3 sec) may produce confusing results
Addition of normal plasma sometimes dilutes a weak inhibitor lead to a “false”
correction
21. 21
Mixing Study Panel
• Look for the presence of:
▫ Factor Deficiency versus Inhibitor • Thrombin Time
▫ Rule-out the presence of
aPTT Mixing Study Panel heparin
1.aPTT Patient • PTT-FS
2.50/50 mix immediate
▫ Lupus-insensitive reagent for
3.Normal Plasma
the aPTT
4.aPTT Patient Not prolonged in the
5.50/50 mix 60 minutes
presence of LA
6.Normal Plasma Sensitive to FD
7.Thrombin Time
8.PTT-FS*
23. 23
Lupus Anticoagulant/APAs
• Paradox
▫ LA is a riddle wrapped in a
mystery inside an enigma
Prolonged clotting time in vitro
Thrombosis in vivo
• Lupus Anticoagulant
▫ Auto-antibodies directed
against phospholipid-binding
proteins
▫ Targets
β2GPI—thrombosis
Prothrombin—bleeding
PC, PS, Annexin V—
thrombosis
24. 24
ISTH Criteria for Lupus Anticoagulant Testing
The ISTH has defined the minimum diagnostic criteria for lupus
anticoagulants to include
1. A prolonged clotting time in a screening assay such as the aPTT
2. Mixing studies indicating the presence of an inhibitor
3. Confirmatory studies demonstrating phospholipid dependence of the
inhibitor
a. Screen – decreased amount of phospholipids prolonged
clotting time
b. Confirm—increased amount of phospholipids shortened
clotting time
4. No evidence of other inhibitor-based coagulopathies
Specific factor assays if the confirmatory step is negative or there is
evidence of a specific factor inhibitor
25. 25
ISTH Criteria for Lupus Anticoagulant Testing
• Updated ISTH guidelines (2009) ISTH
▫ 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
▫ Choice of tests
1. Two tests based on different principles
2. dRVVT should be the first test considered
3. Seconds test should be a sensitive aPTT (low phospholipids and silica
as activator)
4. LA should be considered as positive if one of the two tests gives a
positive result
26. 26
Detection of LA
• Assays
dRVVT* Clot-based assays
SCT dPT
SCT* Why do we see so few LA’s
on the extrinsic side???
HEX
Kaolin CT
dPT DRVVT
27. 27
dRVVT Screen (Normal plasma)
X
dRVVT
Xa
Prothrombin
Xa
Phospholipid
Va Ca2+
(PF3)
Thrombin
Fibrinogen Fibrin
28. 28
dRVVT Screen (Lupus Anticoagulant)
X
dRVVT
Xa
Prothrombin
Xa
Va Ca2+
Low
Phospholipid Thrombin
Content
Fibrinogen Fibrin
29. 29
dRVVT Confirm (LA)
X
dRVVT
Xa
Prothrombin
Xa
Va Ca2+
High
Phospholipid Thrombin
Content
Fibrinogen Fibrin
30. 30
Assays to Detect APA’s
• All LA’s are antiphospholipid antibodies, BUT not all APA’s are LA’s
• Antiphospholipid antibodies
ELISA-based assays
Anticardiolipin antibodies (IgG, IgM, IgA)
β2-Glycoprotein I antibodies
• Systematic reviews have consistently reported
▫ LA is a stronger risk factor than aCL aβ2GPI for both arterial and venous
thrombosis and obstetric complications
▫ aCL and aβ2GPI only show some significant association with thrombosis
and obstetric complications at high titer
▫ Independent of LA and aCL neither aβ2GPI nor antiprothrombin
antibodies are associated with arterial or venous thrombosis
31. 31
Patients with LA on Warfarin
• LA can influence PT/INR can lead to INRs that do not accurately reflect the true level
of anticoagulation
a. Use of the INR (to standardize PTs) may be invalid for some patients with LA
b. To prevent supratherapeutic or subtherapeutic anticoagulation these patients
must be individually monitored with a test that is insensitive to LA
Ann Intern Med. 1997;127:177-185
• Chromogenic Factor X
• Therapeutic Range = 23-47%
• [INR 4.0 (23%) – INR 2.0 (47%)]
Probably the most commonly ordered test in the coagulation laboratory Most sensitivity to decrease in FVII Moderate sensitivity to FV and FX Insensitive to “intrinsic” factors Liver disease versus VKD FV and FVII – FV is NOT VKDF, so if decreased then probably due to LD Sensitive to fibrinogen levels less than 100 mg/dL VDK – seen in malnutrition, broad-spectrum antibiotics (destroy gut flora), newborns (have decreased levels of VKDFs) Hemorrhage is likely in VKD – PT is probably the most sensitive indicator When FVIII and FIX are decreased – PT is not affected due to increased concentration of TF in the reagent – these factors are bypassed during fibrin polymerization Heparin may prolong the PT – most reagents contain polybrene to neutralize the effect of heparin Some thromboplastins are prolonged by LA may be partially neutralized by the thromboplastin reagent How useful is the PT for screening asymptomatic surgical patient to predict intraoperative bleeding? Current data doesn’t support UNLESS the patient is a member of a
During the 1 st week of warfarin therapy physicians should rely on the PT (seconds) rather than the INR. Warfarin works by slowing down the process in the liver that uses vitamin K to make certain proteins { clotting factors } that cause clotting. Warfarin interferes with the action of vitamin K. Vitamin K is essential for the production of prothrombin and other proteins necessary for blood to clot. If there is less vitamin K action, there will be less prothrombin action, and blood clotting will be inhibited.
2 nd most commonly ordered test in the coagulation laboratory Partial thromboplastin LACKS TF DIC prolongs the PTT due to consumption of the clotting factors VKD leads to decreased levels of 2,7,9, and 10 eventually prolonging the PTT. Therefore the aPTT is NOT as sensitive to VKD or warfarin therapy as the PT. Sensitive to deficiencies of contact and intrinsic factors – however, a MILD deficiency may result in a normal aPTT – so we do factor sensitivity studies for all of the factors Clinical applications : Screen for coagulation factor deficiencies Monitor UFH therapy May detect both lupus anticoagulant and circulating anticoagulants
Anti-factor Xa measures the concentration of heparin rather than its anticoagulant effect . The heparin concentration is correlated to 1.5 – 2.5 “X” normal.
PTT is sensitive to deficiencies of contact and intrinsic factors – however, a MILD deficiency may result in a normal aPTT – so we do factor sensitivity studies for all of the factors
Procedure Uses dilutions of a reference plasma over a range of fibrinogen concentrations these dilutions are correlated to individual clotting times Determine the clotting time of a 1:10 dilution of patient plasma (considered 100%) by adding a high concentration of thrombin (100U/mL). The 1:10 dilution minimizes the effect of heparin and elevated FDP’s. Bovine or human thrombin is added to dilute plasma. Catalyzes the conversion of fibrinogen to fibrin. Time to clot is compared to a reference curve. Diluted plasma is clotted with a high concentration of thrombin—note: a high concentration of thrombin (typically 100 U/mL) ensures that the clotting times are independent of the thrombin concentration over a wide range of fibrinogen levels . A calibration curve is constructed using reference plasma over a series of dilutions (1:5-1:40). Clotting time of each dilution is established and the results are converted to mg/dL of fibrinogen. The patient plasma clotting time is read from the standard curve. Diluting the PPP minimizes the antithrombotic effects of heparin, FDPs and paraproteins. Heparin levels <0.6 U/mL and FDP’s <100 mcg/mL do NOT affect the fibrinogen assay when the fibrinogen levels if >150 mg/dL. Hypofibrinogenemia is associated with severe LD and DIC. Hyperfibrinogenemia may be produced in moderately severe LD, pregnancy, chronic inflammation. Clauss—based on the time for a fibrin clot to form PT-derived—fibrinogen derived upon the PT that is derived by optical density change for a range of plasma dilutions with known fibrinogen levels. Immunological—measures fibrinogen antigen concentration rather than functional fibrinogen. Gravimetric—method based upon the weight of the clot—clot is compressed to extrude plasma and unused reagents, washed, dried and then weighed. Fetal fibrinogen and liver disease have abnormal sialic acid content.
1. Patients exposed to bovine thrombin develop antibodies that cross-react with human thrombin which prolongs the thrombin time but does NOT affect the reptilase time. Fetal fibrinogen (in neonates) consists of increased sialic acid content leading to temporary dysfibrinogenemia. Hyperfibrinogenemia may interfere with fibrin assembly due to the excess fibrinogen. Hypofibrinogenemia may interfere with fibrin polymerization but may be more an in-vitro phenomenon.
What would the D-dimer and FSP levela be in a person who is FXIII deficient? D-dimer and FSP levels be in individuals with FXIII deficiency due to DIC?
What would the D-dimer and FSP levela be in a person who is FXIII deficient? D-dimer and FSP levels be in individuals with FXIII deficiency due to DIC?
Patient plasma is mixed with latex particles which are coated with monoclonal anti-FDP antibodies. 3 If FDP are present in the patient plasma, the latex particles agglutinate as FDP bind to the antibodies on the particles. These large agglutinated clumps are detected visually by the technologist. Various dilutions of patient plasma can be tested to provide an estimation of the FDP titer (semiquantitative result). D-dimer is a specific FDP that is formed only by plasmin degradation of fibrin, and not by plasmin degradation of intact fibrinogen. Thus, the presence D-dimer is a specific FDP that is formed only by plasmin degradation of fibrin, and not by plasmin degradation of intact fibrinogen. In contrast, a positive FDP assay indicates that fibrin and/or fibrinogen is being degraded by plasmin, because the FDP assay detects fibrin degradation products, including D-dimers, and fibrinogen degradation products.
