Fundamentals of Coagulation Testing Robert Gosselin, CLS University of California, Davis Health System Department of Clinical Pathology and Lab Medicine Sacramento, CA Robert.gosselin@ucdmc.ucdavis.edu

Goals and Objectives The more painful first one… Quick review of hemostasis Technical aspects of laboratory tests Methods Limitations Outside the lab stuff Subsequent presentations Disease states and laboratory tests for Dx Review session, case studies, lab test caveats…

Goals and Objectives Resident driven… What do you want? What do you need? Bringing questions to the table…

Hemostasis Cellular Fluidic Endothelium Platelets Red blood cells White blood cells Fluidic Procoagulant factors and regulators Fibrinolytic factors and regulators

Role of endothelium Procoagulant Collagen vWF stored in WP bodies Tissue factor expression Cytokine/chemokine

Role of endothelium Anticoagulant Heparin sulfate In presence of thrombin Prostacylcin production  PLT aggregation Nitric oxide- vasdilation  PLT aggregation Express thrombomodulin  thrombin production Release TFPI  thrombin production Release plasminogen activators  fibrinolysis and promote wound healing

Cytokines/Chemokines TNF-α IL1-β IL-6 IL-8 CD54 - transmembrane adhesion molecule to facilitate WBC to endothelium CD62E and CD62p - adhesion properties Complement C3a C5a induce IL release from endothelium C3a:C4aC5a induce elastase release TF expression

Cellular components Platelets Receptors Intracellular Adhesion Aggregation Factors Intracellular Procoagulant factors

AD Shapiro, WFH 1999;19

Role of platelets vWF-GPIb-IX TF collagen exposure P selectin exposure GPIIb-IIIa Release of ATP,ADP TF serotonin, B-TG Fbg, Thrombin P selectin exposure Monocyte adhesion CR Fbg Platelet IX X V II

Cellular components RBCs WBCs Facilitate platelets to the endothelial surface Thromboplastin source WBCs Tissue factor expression-MC Cytokine expression Elastase production-PMNs

Revised Cascade Tissue Factor VII IX X IXa Xa Thrombin VIIa-TF VIIa-TF IX X XIa IXa neutralized VIIIa VIII Xa VIIa-TF-Xa TFPI V Va Prothrombin Thrombin F1.2

Cytokine release THROMBIN Procoagulant Anticoagulant Fibrinogen cleavage: fibrin monomer generation Complex with thrombomodulin: protein C activation Activates factor VIII and V Generate TAFI: fibrinolysis inhibitor Induces platelet aggregation Endothelial release of tPA Activate factor XIII

CLOT LYSIS Thrombin XIII Fibrinogen Fibrin XIIIa FDP Stabilized Fibrin Plasminogen uPA or tPA Plasmin uPAR FDP uPAR:uPA CLOT LYSIS MMP D-dimer Extracellular degradation

D E E E D FIBRIN D D D D D tPA D-dimer Plasminogen Plasmin uPA FIBRINOGEN D D E E D D E Fragment Y Fragment X D Fragments D & E Fragment D

Traditional “Waterfall” Cascade HMWK Pre Traditional “Waterfall” Cascade XII XIIa XI XIa VII Ca+2 TF IX IXa VIIa PF4 Ca+2 VIII VIIIa X Xa PF4 Ca+2 V Va Prothrombin Thrombin F1.2 Fibrinogen Fibrin FPA FPB

Primary Coagulation Regulators HMWK Pre  Primary Coagulation Regulators XII XIIa   AT  PC  PS  C1 Inh  HCFII  TFPI  2-MG  XI XIa   VII Ca+2 TF IX IXa  VIIa  PF4 Ca+2 VIII VIIIa   X Xa   PF4 Ca+2 V Va   Prothrombin Thrombin    Fibrinogen Fibrin

CLOT LYSIS Thrombin XIII Fibrinogen Fibrin  PAI-I TAFI 2-AP 2-MG XIIIa FDP Stabilized Fibrin Plasminogen uPA or tPA  Plasmin uPAR  FDP uPAR:uPA CLOT LYSIS MMP D-dimer Extracellular degradation

Tests for endothelial function Nada… All indirect measurements Soluble factors vWF, Endothelial-1 Cytokine

Test for WBC and RBC function Indirect assessment Soluble factors Flow cytometry Aggregates Cytokine production RBC HCT

Tests for platelet function In addition to absolute numbers Platelet function testing Screening methods PFA and others Aggregation methods Whole blood versus PRP Flow cytometry All kinds of possibilities… Drug occupancy Activation

Platelet Function Assessment Chronolog Corporation Single channel impedance method Dade Behring Incorporated Shear rate induced aggregation Medtronics Modified ACT using PAF Accumetrics Optical detection of fibrinogen coated beads Hemadyne Platelet contractile force Helena Plateletworks-changes in impedance (2 tubes) Diamed Cone and Plate(let)

Results reported as “Closure time” in seconds Principle of the PFA-100® Collagen/Epinephrine (CEPI) — primary screening cartridge Collagen/ADP (CADP) — differentiates dysfunction due to aspirin Results reported as “Closure time” in seconds

Coagulation methods-functional Clot detection Screening versus specific e.g. aPTT versus factor VIII Light scatter vs mechanical Chromogenic Immunologic

Coagulation methods-antigenic ELISA Electrophoresis Immunologic

Coagulation Testing in the Clinical Laboratory Prothrombin times (PT) PP Plasma + Activator (+ CaCl2) Clot Detection 37oC Activated partial thromboplastin times (aPTT) PP Plasma + Activators CaCl2 Clot Detection Optical methods: change in turbidity Mechnical methods: change in motion

Clot based tests optical density time

CRUMMY BLOOD DRAW Increased activation Delays in testing:  PF4 release optical density time  Factor levels Factor VIII Fibrinogen Drugs Fxs DDAVP PCCs Novo7 Foods (e.g. caffeine) Exercise (Physiological) Stress

 Factor activity Drugs- UFH, DTI, Xigris Lipemia, icterus +/- Lupus anticoagulant Inhibitors optical density time Crummy blood draw: Factor consumption Delays in testing:  Fx VIII activity

AT-heparin-Xa complex + residual fXa Anti-Xa activity plasma [heparin] + exogenous antithrombin Excess fXa AT-heparin-Xa complex + residual fXa Chromogenic substrate yellow color

Instrument reading—changes in optical density secondary to aggregates HIT antibodies IgG (+) Conjugated Anti-human IgG antibody ¤ Chromogenic tag  Wash Wash Microwell containing target antigen: PF4-heparin complex ¤ + ¤ + ¤ Color +    ¤ ¤ ¤ + + + + + + Amount of color proportional to amount of antibody present Amount of color proportional to amount of antibody present Incubate Incubate Reagent beads coated with anti-vWF Patient vWF                 Instrument reading—changes in optical density secondary to aggregates   Incubate Testing well

Test Validation

aPTT PT HMWK Pre XII XIIa XI XIa VII Ca+2 TF IX IXa VIIa PF4 Ca+2 VIII VIIIa X X Xa V PF4 Ca+2 V Va II Prothrombin Thrombin Fibrinogen Fibrin aPTT PT

aPTT Screen for factor deficiencies Monitoring drug effect: Other UFH DTI Factor VIII & IX replacement Rx Other LA

PT/INR Factor deficiency Drug monitoring Effect of UFH Rx varies Oral vitamin K antagonist Effect of UFH Rx varies Most with no effect up to 1.0 U/ml Anti-Xa Variable LA effect

A quantitative fibrinogen is extrapolated using the clotting time obtained plotted against a calibration curve Fibrinogen: with concentrated thrombin reagent, no effect of UFH up to 2.0 U/ml Thrombin time: with dilute thrombin reagent, alternative test for UFH or DTI monitoring in patients with elevated baseline (pre-treatment) aPTT

Influence on coagulation testing Preanalytical In-vivo Ex-vivo Analytical In-vivo—drugs, diet, physiological stress, inflammation, OC, HRT, etc Ex-vivo—poor phlebotomy, short draw, lysis, increased tourniquet time, etc Analytical—clot, chromogenic, immunologic, instrument variability, etc

Preanalytical variables Blood sample Too little versus too much Too long Difficult phlebotomy 3.2% vs 3.8% buffered sodium citrate Venipuncture vs line draws Tube sequence?? Unintended effects: Drugs Other nonpathologic events e.g. stress, diet, oral contraceptives and other hormonal changes, etc

Analytical variables Instrumentation Reagents optical versus mechanical clot detection Affected by interferences lipemia, bilirubinemia, etc. Reagents PT source (recombinant, brain extract, etc) aPTT activator phospholipid source and concentration designed for heparin, factor VIII & IX, poss LA

Summary Most coagulation screening tests are crude in nature All coagulation results should be interpreted with caution until pre-analytical and analytical variables have been excluded to prevent false positive/negative results Good luck with validation process…