Fundamentals of Fundamentals of Coagulation TestingCoagulation Testing

Robert Gosselin, CLSRobert Gosselin, CLSUniversity of California, Davis Health SystemUniversity of California, Davis Health System

Department of Clinical Pathology and Lab MedicineDepartment of Clinical Pathology and Lab MedicineSacramento, CASacramento, CA

Robert.gosselin@ucdmc.ucdavis.eduRobert.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…

HemostasisHemostasis

• Cellular– 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

• Adhesion • Aggregation• Factors

– Intracellular• Adhesion• Procoagulant factors

AD Shapiro, WFH 1999;19

Role of platelets

collagen exposure

vWF-GPIb-IX

GPIIb-IIIa

Release of ATP,ADP TF serotonin,

B-TG Fbg,

ThrombinPlatelet

P selectin exposureMonocyte adhesion

IXX

VII

TF

CR Fbg

Cellular components

• RBCs– Facilitate platelets to the endothelial

surface– Thromboplastin source

• WBCs– Tissue factor expression-MC– Cytokine expression– Elastase production-PMNs

Tissue Factor

VIIa-TF

XaVIIa-TF-Xa

VIIa-TF

VII

V

IXa

IX X

Prothrombin

Va

TFPI

neutralized

Thrombin

VIIIVIIIa

F1.2

XIa

Revised Cascade

THROMBIN

AnticoagulantProcoagulant

Fibrinogen cleavage: fibrin monomer generation

Activates factor VIII and V

Induces platelet aggregation

Activate factor XIII

Generate TAFI: fibrinolysis inhibitor

Complex with thrombomodulin: protein C activation

Endothelial release of tPA

Cytokine release

Thrombin

Stabilized Fibrin

XIIIa

Plasminogen

Plasmin

Fibrinogen Fibrin

XIII

uPA or tPA

CLOT LYSIS

uPAR

uPAR:uPA

Extracellular degradation

MMP

D-dimer

FDP

FDP

DE

D D

ED

Plasminogen PlasmintPA

uPA

FIBRIN

D

E

D

D

D

E

E

D

DD

D

E

D-dimer

Fragment X

Fragment D

Fragment Y

Fragments D & E

FIBRINOGEN

XII

VII

XIIa

XIaXI

IXaIX

VIIIaVIII

VIIa

Prothrombin

VaV

XaX

Thrombin

FibrinFibrinogen

HMWK Pre

TF

PF4 Ca+2

PF4 Ca+2

Ca+2

Traditional “Waterfall” Cascade

F1.2

FPA FPB

XII

VII

XIIa

XIa XI

IXa IX

VIIIa VIII

VIIa

Prothrombin

Va V

Xa X

Thrombin

FibrinFibrinogen

HMWK Pre

TF

PF4 Ca+2

PF4 Ca+2

Ca+2

AT

PC

PS

C1 Inh

HCFII

TFPI

2-MG

Primary Coagulation Regulators

Thrombin

Stabilized Fibrin

XIIIa

Plasminogen

Plasmin

Fibrinogen Fibrin

XIII

uPA or tPA

CLOT LYSIS

uPAR

uPAR:uPA

Extracellular degradation

MMP

D-dimer

FDP

FDP

PAI-ITAFI2-AP2-MG

Tests for endothelial function

• Nada…

– All indirect measurements• Soluble factors

– vWF, Endothelial-1– Cytokine

Test for WBC and RBC function

• WBC– 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

• HemadynePlatelet contractile force

• Helena – Plateletworks-changes in impedance (2 tubes)

• Diamed– Cone and Plate(let)

• Collagen/Epinephrine (CEPI) — primary screening cartridge

• Collagen/ADP (CADP) — differentiates dysfunction due to aspirin

• Collagen/Epinephrine (CEPI) — primary screening cartridge

• Collagen/ADP (CADP) — differentiates dysfunction due to aspirin

Principle of the PFA-100®

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

37oC

Optical methods: change in turbidity

Mechnical methods: change in motion

op

tica

l d

ensi

ty

time

Clot based testsClot based tests

op

tica

l d

ensi

ty

time

CRUMMY BLOOD DRAWCRUMMY BLOOD DRAW

Increased activation Increased activation

Delays in testing: Delays in testing:

PF4 releasePF4 release

Factor levels Factor levels

Factor VIIIFactor VIII

FibrinogenFibrinogen

DrugsDrugs

FxsFxs

DDAVPDDAVP

PCCsPCCs

Novo7Novo7 Foods (e.g. caffeine)Foods (e.g. caffeine) ExerciseExercise (Physiological) Stress(Physiological) Stress

op

tica

l d

ensi

ty

time

Crummy blood draw:Crummy blood draw:

Factor consumptionFactor consumption

Delays in testing:Delays in testing:

Fx VIII activityFx VIII activity

Factor activityFactor activity

Drugs- UFH, DTI, XigrisDrugs- UFH, DTI, Xigris

Lipemia, icterusLipemia, icterus

+/- Lupus +/- Lupus anticoagulantanticoagulant

InhibitorsInhibitors

Anti-Xa activity

Excess fXa

Chromogenic substrate

plasma [heparin] + exogenous antithrombin

AT-heparin-Xa complex + residual fXa

yellow color

Microwell containing

target antigen:

PF4-heparin complex

HIT antibodies IgG (+)

Conjugated Anti-human IgG antibody ¤

Amount of color proportional to amount of antibody present

++ +

+++

Incubate

¤¤ ¤

Wash

+++ ¤¤¤

Incubate

Wash Chromogenic tag

Color

Amount of color proportional to amount of antibody present

Patient vWF

Testing well

Reagent beads coated with anti-vWF

Instrument reading—changes in optical density secondary to aggregates

Incubate

Test Validation

Accuracy

Comparison of new method to existing method or reference method.

Precision

Determining the precision of running a single sample multiple times concurrently (within-run precision) as well as the same sample (usually control material) over a period of days (day-to-day precision) to determine the coefficient of variation (CV).

Reportable range (Linearity)

Assesses the reportable range (high and low), as well a reproducibility of diluted samples

Verify manufacturer’s

reference interval (normal range)

Appropriate for laboratory’s patient population. Pediatric reference ranges may be cited from acceptable references

Notes

If modifications to an FDA-approved test is used, or if in-house test is used, then additional performance characteristics must be evaluated and documented to include aforementioned accuracy, precision, reportable range, reference intervals, but additionally:

Analytical sensitivity Analytical specificity, including interfering

substances Other performance characteristics required

for testing

XII

VII

XIIa

XIaXI

IXaIX

VIIIaVIII

VIIa

Prothrombin

VaV

XaX

Thrombin

FibrinFibrinogen

HMWK

Pre

TF

PF4 Ca+2

PF4 Ca+2

Ca+2

aPTT PT

XII

XI

IX

VIII

VII

X

V

II

aPTT

• Screen for factor deficiencies

• Monitoring drug effect:– UFH– DTI– Factor VIII & IX replacement Rx

• Other– LA

PT/INR

• Factor deficiency

• Drug monitoring– Oral vitamin K antagonist

• Effect of UFH Rx varies– Most with no effect up to 1.0 U/ml Anti-Xa

• Variable LA effect

Fibrinogen: with concentrated thrombin reagent, no effect of UFH up to 2.0 U/mlThrombin time: with dilute thrombin reagent, alternative test for UFH or DTI monitoring in patients with elevated baseline (pre-treatment) aPTT

A quantitative fibrinogen is extrapolated using the clotting time obtained plotted against a calibration curve

Influence on coagulation testing

• Preanalytical– In-vivo– Ex-vivo

• Analytical

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

– 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

Possible causes of INR and/or aPTT Possible causes of INR and/or aPTT Preanalytical Short draw – excess sodium citrate in plasma Elevated HCT (>55%) - common in neonates – excess citrate in plasma Clotted sample Sample >4 hours old: ↑ aPTT Sample >24 hours old: ↑ INR Hemodilution-drawn above IV site; drawn via arterial line without proper

clearance

Preanalytical Low HCT (<25%) Elevated Calcium levels

Poorly collected sample ↓ INR and/or aPTT due to activated sample

Samples placed on ice ↓ INR Sample > 2 hours for patients on UFH: ↓ aPTT due to platelet degranulation and PF4 release, which neutralizes heparin.

Factor deficiencies PT factors VII, X, V, II and to a lesser degree, fibrinogen aPTT factors XII, XI, IX, VIII, X, V, II, and to a lesser degree, fibrinogen Physiological decrease: Hereditary deficiencies: Factor VIII and IX most common, with

higher incidence of factor XI deficiencies in Azhkenazi Jewish population

Immature liver: premature infants and neonates Liver disease ↑ INR with normal or slight ↑ aPTT Consumptive coagulopathy Hemodilution RBC transfusion without FFP Blood volume expanders Deficiencies can also be associated with antibody directed against

factors

Elevated factor levels Inflammatory response ↓ aPTT due to elevated factor VIII and/or fibrinogen Cryoprecipitate - contains high levels of factor VIII and

fibrinogen Drugs INR: Activated factor VII (Novseven) Prothrombin complexes (Proplex or Autoplex) aPTT: Prothrombin complexes (Proplex or Autoplex) Direct factor therapy (Humate, Kogenate, Alphanine)

Drugs PT: Oral vitamin K antagonists Daptomycin Direct thrombin inhibitors Argatroban > bivalirudin > lepirudin Activated protein C (Xigris) Systemic fibrinolytic activators (e.g. urokinase) aPTT: Unfractionated heparin Direct thrombin inhibitors Activated protein C (Xigris) Systemic fibrinolytic activators (e.g. urokinase) Hydroxy-ethyl starch, hematin, sumatin, taularidine

Antiphospholipid antibodies – varies with reagent, may ↑ INR in addition to aPTT

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…