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)

Effect of high hematocrit on coag tests Elevated citrate concentration may prolong clotting times

The Prothrombin Time Thromboplastin: Tissue factor Phospholipid Calcium Also contains a heparin- neutralizing agent 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)

Why the INR? Tissue factor in the thromboplastin may be recombinant, or derived from human or animal tissue Phospholipid composition varies among thromboplastins As a result different thromboplastins have varying sensitivity to the effect of warfarin The INR system makes PT results from different laboratories comparable to one another in patients receiving vitamin K antagonists (not in liver disease or other coagulopathies)

( ) ISI Patient PT INR = Mean Normal PT 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 1.24 ( ) 22.0 12.6 INR = = 2.0 Reagent B: ISI = 2.46, mean normal = 12.2 sec PT = 16.2 sec 2.46 ( ) 16.2 12.2 INR = = 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

Uses of the PT/INR Best single test of the integrity of the fibrin clotting system Detects most clinically significant acquired coagulopathies Does not detect the most common inherited clotting factor deficiencies (VIII, IX, XI) Routinely used to monitor warfarin therapy Insensitive to heparin at usual therapeutic concentrations

Activated partial thromboplastin time (aPTT) 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 “Partial thromboplastin” Phospholipid + Activator (provides surface for generation of XIIa)

Uses of the PTT Screen for inherited clotting factor deficiency (hemophilia, factor XI) Monitor heparin therapy Screen for acquired coagulation inhibitors Factor VIII antibody Lupus anticoagulant A long PTT does not always indicate a bleeding disorder Factor XII deficiency

The aPTT should be ordered selectively Results of 1025 consecutive tests, excluding heparin monitoring Robbins and Rose, Ann Intern Med 1979;90:796 # TESTS # PATIENTS Abnormal result 143 97 On anticoagulant 64 37 Liver disease 41 27 No cause found, no bleeding 15 14 Normal on repeat testing 9 Known hemophilia 5 4 History of intestinal bypass Other malabsorption (CF) 2 1 Technical problem with test Newly dx'd bleeding disorder # abnormal: 143 (14%)

What causes a long PT/INR and a normal PTT? Factor VII deficiency Mild deficiency of “common pathway” factors Warfarin Vitamin K deficiency Liver disease PTT PT/INR

What would cause a long PTT with a normal INR? PT/INR Deficiency of VIII, IX, XI Deficiency of a contact factor (usually XII) (does not cause bleeding) Heparin Factor VIII inhibitor Lupus-type inhibitor (antiphospholipid antibody)

What if both PT/INR and PTT are long? Liver disease Vitamin K deficiency Warfarin DIC High level of heparin Other inhibitor affecting common pathway (eg, direct thrombin inhibitor) Isolated deficiency of X, V, II, fibrinogen (rare) PTT PT/INR

Other tests aPTT Thrombin time PT/INR Thrombin time: thrombin + plasma. Very sensitive to heparin and other thrombin inhibitors. Prolonged by low fibrinogen, dysfibrinogenemia, high levels of fibrin degradation products. Urea solubility: clot immersed in concentrated urea (breaks noncovalent bonds) clot dissolves unless crosslinked by factor XIIIa). For diagnosis of severe factor XIII deficiency (v. rare)

Mixing Study Purpose: to determine whether long aPTT or PT is due to clotting factor deficiency or circulating inhibitor (eg, factor VIII inhibitor, heparin, lupus-type 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 Note: warfarin does not act as a circulating inhibitor. It inhibits synthesis of clotting factors by the liver

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%

Patient C

100/.5 = 200% Patient C

Lupus inhibitor or other non-competitive clotting inhibitor → non-parallel plot 100% ≥50% Patient 50% % test plasma 10% Normal plasma 5% 1% 20 40 60 80 aPTT (sec)

100% Patient 50% Factor VIII Inhibitor % test plasma 10% Normal plasma 5% 1% 20 40 60 80 aPTT (sec)

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

Bethesda Assay Residual factor activity dilution pt plasma 50% 1:1 1:10 1:100 1:1000 100 BU

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 Abnormal test result → test for specific defects in primary hemostasis Test not useful if platelets <100K or if patient taking ASA, etc PFA-100

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

AA agg ADP agg AA rel ADP rel Saline agg Thrombin rel Collagen agg Low High Collagen rel Risto low agg Risto high agg 2 nM ATP Ch 1 Ch 2

Risto low Risto high Pt Control Type I VWD

Pt Normal Collagen agg Low High AA agg ADP agg AA rel Collagen rel ADP rel Normal

Took Excedrin 5 days ago

Taking ASA 81 mg/d and Plavix 75 mg/d Pt AA agg ADP agg AA rel ADP rel Normal Taking ASA 81 mg/d and Plavix 75 mg/d

PFA: Coll/ADP 91 (nl 65-120) Coll/Epi 139 (nl 85-175) Low High AA agg Coll agg AA rel Coll rel Low High Coll release Low High PFA: Coll/ADP 91 (nl 65-120) Coll/Epi 139 (nl 85-175)

Assessment of the fibrinolytic system Fibrinogen (dilute thrombin time assay) D-dimer (immunoassay) α2-antiplasmin activity (chromogenic substrate assay) 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 to guide blood product use 30 min

World J Transplant 2012;2:1

Effect of Coagulation Factor Deficiency on TEG This tracing demonstrates an enzymatic pathway abnormality in a patient who is bleeding. The elongated R and normal MA values suggest factor deficiency or dysfunction. The depressed angle value demonstrates the effect of factor deficiencies on the rate of fibrin-platelet clot formation. Normal Factor deficiency

Effect of platelet abnormality on TEG Platelet abnormalities are typically identified by a low MA value. In cases where a patient is bleeding and the MA is low, reduced platelet count and/or function should be suspected as the cause. Thrombocytopenia or dysfunctional platelets Normal

Effect of hyperfibrinolysis on TEG Normal Hyperfibrinolysis

Chromogenic substrate-based assay Peptide containing target sequence of enzyme linked to chromophore Colored cleavage product (in this case nitroaniline) detectable by spectrophotometry Enzyme specificity determined by target sequence Rate of color generation proportional to enzyme activity

Examples of chromogenic assays Anti-Xa assay Patient plasma added to mixture of Xa and chromogenic substrate (± antithrombin) Residual Xa activity inversely proportional to inhibitor level Protein C activity Patient plasma + venom enzyme that selectively activates protein C; activated protein C cleaves substrate

Von Willebrand factor measurements VWF antigen: via ELISA VWF activity (must be sensitive to total VWF protein, integrity of binding sites, presence of large multimers) Ristocetin cofactor assay: patient plasma + ristocetin + formalin fixed platelets Ristocetin unfolds VWF, which then clumps platelets Alternative assay uses beads coated with monoclonal Ab against GP1b binding site in VWF rather than platelets Multimer analysis: via gel electrophoresis