Tromboelastography - State of the Art

Tromboelastography

state of the art

Dionisio Colella MD

Plasma

Volume

Plasma

Volume

FFP

Advantages

•Reduction in mortality if FFP transfused aggressively; •FFP:RBC ratio varyng between 1:1 1:2

•Aggressive dosing regimen 30ml/kg .

Disadvantages

•TACO ( transfusion associated circulatory overload) 1-11% •TRALI

•Allergic reactions

J Trauma2009;66:693-7 J Trauma 2003; 54: 454-63 J Trauma 2006; 60: s91-6

Fenger-Eriksen C et al. J Thromb Haemost 2009

N=20, *significant different from baseline, ** expected

Baseline 30% Haemodilution Relative decrease % Haematocrit 0.43 ± 0.03 0.29 ± 0.02 - 32 ± 5 Platelet count (10*9L) 248 ± 65 181 ± 50* - 27± 5 P-fibrinogen (µmol/L) 9.5 ± 1.9 5.1 ± 0.8* - 44 ± 4.4** Trombin Generation (nM*min) 1471 ± 309 1532 ± 247 + 3.8 ± 11 Patient dilution

THE COAGULATION PATHWAY PROTEINS,

MINIMAL AMOUNTS NEEDED FOR SURGERY AND REPLACEMENT SOURCES

Relationship between conventional tests and post-operative bleeding

Predictive values for post operative bleeding & re-operative rates

 Activated clotting time = 30 %

 Coagulation profile = 50 %  Thrombelastograph® = 87 %

Why a “whole-blood sample”

should be the “gold standard”

Inflammation Inflammation

Inflammation

Relationships between coagulation, inflammation and endothelium:

inflammatory responds. Jerrold H. Levy, M.D.

IL-6 IL-2 TNF

bradykinin

CONTACT (XIIa) Tissue Factor

X Xa Fibrinogen THROMBIN Plasmin Plasminogen Platelet GP1b PAP complexes -2-antiplasmin Platelet BTG, PF4 Protein C APC trombomodulin nF-kB TAT tPA endotelium tPA:PAI1 IX IXa Prothrombin VIIIa, Ca++; PL Va, Ca++, PL AT III TFPI D-dimer Fibrin (M) Fibrin (Ps) Fibrin (Pi) FPA FPB FPS PTF 1.2 GPIIbIIIa Platelet

Modified from Despotis et al. 1997

VII P- selectin

ICAM PMN

The Cell-based Model of Coagulation

VIIIa IXa

Hoffman M & Munroe DM. A cell-based model of hemostasis.

Thromb Haemost 2001; 85: 958-965 + activates various factors Initiation Amplification Propagation

The Cell-based Model of Coagulation

VIIIa IXa

Hoffman M & Munroe DM. A cell-based model of hemostasis.

Thromb Haemost 2001; 85: 958-965 + activates various factors Initiation Amplification Propagation

Haemoscope Thrombelastograph® Haemostasis Analyser (TEG®)

TEG® Applications

– Liver transplants – ICU – Cardiac Surgery – Vascular Surgery – Cath lab – Trauma – Orthopedics – Obstetrics – Gyneacology

– Burn wound unit – Etc...

Time Coagulation activation Thrombin formation Fibrin formation rise of optical density (PT/aPTT) Rise of clot firmness (TEG) Fibrin polymerization Cross-linking Fibrin-platelet interaction

from Calatzis A et al.

TEG is a global assessment of hemostatic

function

Parameters of TEG analysis

• r = coagulation activation

• k = dynamics of clot formation

•



angle = kinetics of fibrin cross-linking

• MA = strenght of a clot

Parameters of TEG analysis

R

(or R-Time) = Reaction time

• time from start of measurement until the beginning of clot formation

• explores the enzimatic part of coagulation

• prolonged by anticoagulants and factor deficiencies • shortened by hypercoagulable conditions

r k

Parameters of TEG analysis

K

(or K-time) = Clot growth kinetics

• time from the beginning of clot formation until a fixed level of clot firmness (amplitude of 20 mm) is reached • explores the kinetics of clot formation

• is related to platelet function and plasma components • prolonged by anticoagulants that affect fibrinogen and platelet function

• shortened by increased levels of fibrinogen and platelet function

r k

Parameters of TEG analysis



(or



angle)

• explores the rate of clot growth • function of rate of polymerization • expression of fibrinogen level

• prolonged by anticoagulants that affect fibrinogen and platelet function

• shortened by increased levels of fibrinogen and platelet

r

MA

k

Parameters of TEG analysis

MA

= Maximum Amplitude

• maximum strenght of the developed clot • ability of the clot to form hemostasis

• depends

- on adequate fibrin formation and polymerization in a 3D network and

- on its interaction with platelet via the Gp IIb/IIIa receptor

Parameters of TEG analysis

MA

= Maximum Amplitude

• conventional coagulation tests (PT, aPTT) are based only on the time of onset of clot formation

• blood clot firmness is an important parameter for in-vivo haemostasis because the clot must

Parameters of TEG analysis

G

• 5000 A / (100 - A) dyn/cm2

• elastic shear modulus

r

MA

k

 30 min

Parameters of TEG analysis

LY30, LY60

= Clot stability

• measures the percentage change in clot strenght at 30 or

60 min after MA

• refers to the potential of the clot to redissolve as a result

of circulating fibrinolytic activators which activate the plasminogen incorporated in the clot

Interpreting Test Results

MA Platelets + Fibrin

MA Fibrin

MA Platelets

Parameters of TEG analysis

CI

= Coagulation Index

• derived from r, k, MA and 

• describes global balance of coagulation • CI > + 3 = hypercoagulation

TEG® Tracing

LY30 measures the rate of amplitude reduction 30 minutes after MA. This measurement gives an indication of the stability of the clot.

LY30 (EPL)

MA, or Maximum Amplitude, is a direct function of the maximum dynamic properties of fibrin and platelet bonding and represents the ultimate strength of the fibrin clot.

MA

K time is a measure of the rapidity to reach a certain level of clot strength.

K

The  value measures the rapidity (kinetics) of fibrin build-up and cross-linking, that is, the speed of clot strengthening.



R is the time of latency from the time that the blood was placed in the TEG® analyzer until the initial fibrin formation.

MA

R time (reaction time)– Time taken to first fibrin strand formation

Reaction time

K & Angle

K &  – Reflect the kinetics of the interaction of fibrin/fibrinogen with platelets

K=Time to reach amplitude of 20mm 

MA

MA, maximum clot strength

MA- Maximum Amplitude – Reflects the ultimate strength of the clot

due to fibrin/fibrinogen interaction with the GPIIb/IIIa receptor on functioning platelets

Thrombin generation

How to detect?

PF 1.2 (ELISA)

TAT (ELISA)

TEG Thrombin Generation

(point of care)

Thrombus generation

The velocity transformation

Profile is divided into equal time intervals = ∆time (∆t)

• Within each time interval: ∆Amplitude (∆A)

Velocity = ∆A/∆t

Thrombus generation

TTG = total thrombus generation

MTG = maximum rate of thrombus generation TMG = time to maximum rate of thrombus generation

Thrombus generation

Parameters defined

• TMG = time to maximum thrombus generation • MTG = maximum thrombus generation • TTG = total thrombus generation • TML = time to maximum lysis • MRL = maximum rate of lysis • ACL = ****

Continuous Real Time measurement of thrombin generation

Thrombin generation test

(methods)

Plasma

TF / phospholipids

CaCl₂ / fluorogenic (chromogenic) substrate

0 100 200 300 400 500 600 700 0 5 10 15 Time (min) F lu o re s c e n c e (R F U ) Detector Raw data ETP Corrections First derivative

Thrombin generation curve

0 50 100 150 200 250 300 350 0 5 10 15 Time (min) T h ro m b in (n M ) Castoldi, modified

0 100 200 300 400 500 600 700 0 5 10 15 Time (min) F lu o re s c e n c e (R F U ) Fluorometer Raw data

Thrombin generation test +

APC

Plasma

TF / phospholipids + APC

CaCl₂ / fluorogenic substrate

Corrections First derivative

Thrombin generation curve

0 50 100 150 200 250 300 350 0 5 10 15 Time (min) T h ro m b in (n M ) -APC +APC -APC +APC Castoldi, modified

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TEG® monitored patients Reduction (%) Post-operative

haemorrhage 27

Fresh Frozen Plasma 62

Platelets 28

Cryoprcipitate 94

Overall (FFP,Platelets,Cryo) 49

Incidence of re-exploration 3,9 to 0,66

Relationship between conventional tests and post-operative bleeding

Elongated R

• Intrinsic contact pathway inibition (aprotinin) • Tissue factor inibition

• Warfarin therapy

• FXa inibition (LMWH) • Low prothrombin level

• Thrombin inibition (HMWH; r-irudin; etc.) • Low fibrinogen level

Fibrinolysis

Fibrinolysis

8,8-20,7 632-861 228-642

Timing for the use of heparinase and

TEG in cardiac surgery

• Pre-CPB

• Shortly before the termination of CPB (with heparinase)

• 10 minutes and 60 minutes after protamine administration (with and without heparinase)

In case of elongated R-time TEG with heparinase or HMS-plus for detection of HMWH!

TEG trace in cardiac surgey

Pre-CPB

During CPB (test without heparinase)

During CPB (test with heparinase)

During CPB (test with heparinase)

Post CPB after protamine administration

Effects of temperature on TEG

37° 30° 25° 20° 15° Colella D, MD

Heparinase cup _{Plain cup}

Protamine overdose

(in vitro)

LMWH and TEG

12 h after LMWH withdrawal

Chest drainage

0 _{1 2 3 4 5 6 7 8} 500 1000 1500 2000 2500 3000 0

ACT vs Heparin levels (Anti factor Xa)

ACT

Group 1 Group 2 Group 3 120 140 160 180 200 220 ACT value (95% CI)

0 _{1 2 3 4 5 6} 300 400 500 600 700 800 900 1000 1100 ACT (seconds) Anti Xa (IU/ml)

200 400 600 800 1000 ACT (seconds) Anti Xa (IU/ml) 0 _{1 2 3 4 5 6}

ECMO

UFH 500 U/h

UFH 200 U/h PLT 6 U

UFH 100 U/h

UFH 100 U/h + Dipiridamolo

CORRELATION OF F. 1.2 LEVELS WITH TIMES FROM START OF

SURGERY



PRESENTATION OF

PROTHROMBIN FRAGMENT 1.2 OVER THE COURSE OF CPB



 COMPUTER MODEL OF THROMBIN GENERATION DURING CPB

Thrombin generation

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8,8-20,7 632-861 228-642

8,8-20,7 632-861 228-642

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TEG

®

Decision Tree

Hypercoagulable Branch

For kaolin samples

Gray tracing represents normal TEG® tracing.

U.S. Patent 6,787,363

Assessing Risk of Thrombosis

McCrath DJ et al. Anesth Analg 2005

General surgical patients

Cardiac patients (Post PCI)

Gurbel et al. “Platelet Reactivity in Patients and Recurrent Events Post-Stenting” J Am Coll Cardiol 2005; 46: 1820-1826

15 750 440

Fattori della coagulazione contenuti _{antitrombotiche}Proteine

II VII IX X C S

Differente contenuto di fattori ed inibitori nei diversi preparati di CPC*

* quantità riportate in RCP dei singoli prodotti

* per i prodotti più recenti vengono indicati i range (UI/mL), secondo le normative AIFA

(UI/mL) (UI/mL) (UI/mL) _(UI/mL) (UI/mL)

Confidex Prothromplex TIM 3 Uman complex 20 - 48 10 - 25 20 - 31 22 - 60 15 - 45 13 - 26 30 NO 30 30 NO NO 25 NO 25 20 NO NO

Coumadin

Coumadin Base Coumadin Base

Post Confidex 25 U/Kg

Thrombin Generation Software

Post ulteriori

25 U/Kg

Dabigatran etexilate:

A novel new oral

anticoagulant

Jeffrey I Weitz, MD, FRCP(C), FACP

Professor of Medicine and Biochemistry McMaster University

Canada Research Chair in Thrombosis Heart & Stroke Foundation/ J.F. Mustard Chair

Heparin Antithrombin

Heparin Direct thrombin inhibitors

Reversible direct thrombin inhibitor Fibrin-Bound Thrombin X Fibrin-Bound Thrombin

Adapted from Di Nisio M, et al. N Engl J Med. 2005;353:1028–40

148

15 750 440

Novoseven 90 mcg/Kg

Thrombelastographic analysis of recombinant factor VIIa in cardiac surgery

Paolo Prati, Dionisio Colella et al.

CONCLUSION

rFVIIa leads to

Improvement in thrombin generation

kinetics

No change in total amount of thrombin

generated

START BIVALIRUDIN

0,15 mg/Kg/h

ACT 123 aPTT 1,45 plt 45.000

ACT 197 s Plt 50.000 aPTT 1,80 POST BIVALIRUDIN

Bivalirudin 0,15 mg/Kg/h

Bivalirudin 0,25 mg/kg/h

After 3 days of Bivalirudin: addition of Coumadin 1,25 mg

STOP Bivalirudin; Coumadin 2,25

6 days after sospension of bivalirudin

ACT 111s Plt 140.000 aPTT 1,12 INR 1,73 10 days after sospension of bivalirudin

147 127 79 63 ₅₅ 45 60 127 140 170 0 20 40 60 80 100 120 140 160 180 0 1 2 3 4 5 6 7 8 9

CVVHDF CITRATO

CVVHDF CITRATO

CVVHDF CITRATO

CVVHDF CITRATO

CVVHDF CITRATO

CVVHDF CITRATO

Dilutional coagulopathy

Dilutional coagulopathy occurs after massive haemorrage in trauma and surgery, and it adversely affects

morbidity and mortality.

The degree of coagulopathy often relates to the extent of abnormality in three

critical factors: •Hypotermia; •Acidosis

Dilutional coagulopathy

MASSIVE

BLEEDING Intravenously fluid resuscitation

diluition

DILUTIONAL COAGULOPATHY

• Loss, consumption or dilution of coagulation factors, platelets and RBC.

•Dys-functional fibrinogen with compromised polymerization

• Reduces clot strength with reduced thrombin generation

• Increases bleeding

Fries et al. Br J Anaest 95(2):172-7 (2005) Fenger-Eriksen et al. Br J Anaest 94(3)324-29 (2005) E de Jonge et al. Crit Care Med 2001 Vol 29 1261-67

Haas T et al Anesth Analg 2008 Apr; 106(4):1078-86 Hiippala ST et al., Anesth Analg. 1995 Aug;81(2):360-5

Fenger-Eriksen C et al. J Thromb Haemost 2009

N=20, *significant different from baseline, ** expected

Baseline 30% Haemodilution Relative decrease % Haematocrit 0.43 ± 0.03 0.29 ± 0.02 - 32 ± 5 Platelet count (10*9L) 248 ± 65 181 ± 50* - 27± 5 P-fibrinogen (µmol/L) 9.5 ± 1.9 5.1 ± 0.8* - 44 ± 4.4** Trombin Generation (nM*min) 1471 ± 309 1532 ± 247 + 3.8 ± 11 Patient dilution

TEG and dilutional coagulopathy

•1 ml native blood •1ml HES

•Kaolin

Conclusion

•Increase in coagulation time (K); •Decrease in clot formation rate (α);

•Decrease in strenght of the clot (MA) for dilution of 50% •No pathological clot lysis caused by haemodilution;

•Reduction in MRTG,TMRTG and TG in 80% emodilution in accordance with litterature

Post Haemocomplettan 2 gr

TEG® Decision tree (cardiac surgery)

275

Suggested treatment (cardiac surgery)

anticoagulant of choice Prothrombotic state

LY30 < 7.5%, C.I. > 3.0

anticoagulant of choice Secondary fibrinolysis

LY30 at 7.5% or greater, C.I. > 3.0

antifibrinolytic of choice Primary fibrinolysis

LY30 at 7.5% or greater, C.I. < 3.0

.06 u/kg cryo  fibrinogen level  less than 45° x10 platelet units  platelet function MA at 40 mm or less x5 platelet units  platelet function MA between 41 -48 mm 0.3mcg/kg DDAVP  platelet function MA between 49 -54 mm x 4 FFP or 16 ml/kg  clotting factors

R greater than 14 min

x 2 FFP or 8 ml/kg  clotting factors R between 11-14 min x 1 FFP or 4 ml/kg  clotting factors R between 7 - 10 min Suggested Treatment Clinical cause TEG® value Treatment protocol

Sampling Protocol — All samples are Kaolin activated

Sample

# When Cup type

1 On induction Heparinase bonded (blue) cup and pin

2 At rewarming (approx 36°C) on CPB Heparinase bonded (blue) cup and pin

3 & 4 10 min post protamine Split sample:

Heparinase bonded (blue) cup and pin Plain (clear) cup and pin

5 & 6 Post op Split sample:

Heparinase bonded (blue) cup and pin Plain (clear) cup and pin

TEG® Results Interpretation (If HIT, treat with Hirudin)

Sample # Measures If Suggested Treatment

1 Baseline hemostasis profile

Prothrombotic state: AT III deficiency or others (To test for AT III deficiency,

see AT III protocol)

 AT III or FFP

 Antifibrinolytic drugs are contraindicated unless patient treated with Plavix, ReoPro, Aggrastat, or Integrilin, in which case Aprotinin is recommended.

2 Coagulopathy, if any, developed during bypass phase

Coagulopathy  Treat hyperfibrinolysis. See protocol below.

 Order blood product. See protocol below. 3 & 4  Post-CPB

hemostasis profile

 Heparin reversal

Heparinase R and Plain R are

within normal limits, heparin is effectively reversed

None

Heparinase R normal, Plain R

above normal limits, heparin is not completely reversed

Protamine

Coagulopathy See protocol below 5 & 6 Post-op hemostasis

profile

Normal None

Coagulopathy / heparin rebound

See protocol below

Interpretation

Conclusions I

• TEG is very sensible to the presence of HMWH • TEG is a very sensible test but does not identify

any specific alteration of clotting factors

• TEG parameter MA is both a sensible and specific test for platelet dysfunction

• TEG is both a sensible and specific test for fibrinolysis

Conclusions II

• Modified TEG - native whole-blood - celite - kaoline - heparinase

- ADP, AA and novel activator (Platelet Mapping) - tissue factor (Hemoliance)

- abciximab

- ATIII (heparin resistance detection) - urokinase

- heparinase/abciximab

- heparinase/fresh frozen plasma

Conclusions III

• Modified TEG is important as a research tools and for pilot studies

• It is important to use few common tests to achieve higher standardization and to assure quality

• Ongoing international cardiac surgery enrollement as well as large institution databases will facilitate

further standardization