The document discusses various assays and models used to evaluate global hemostasis and coagulation, emphasizing the importance of thrombin and clot waveform analysis in diagnosing bleeding disorders and predicting thrombotic risks. It elaborates on the advancements in thrombin generation assays and viscoelastic testing techniques like TEG and ROTEM for clinical applications, particularly in trauma and surgical settings. Recommendations for testing protocols and interpretations are provided to enhance the management of coagulation-related issues.

1. TESTS OF GLOBAL HAEMOSTASIS
Meenakshi Bhatia
28/06/2024

2. CASCADE MODEL OF COAGULATION CELL BASED MODEL OF COAGULATION
Hoffman,
Maureane
&
Monroe,
Dougald.
(2001).
A
Cell-based
Model
of
Hemostasis.
Thrombosis
and
haemostasis.
85.
958-65.
10.1055/s-0037-1615947.

3. ■ Thrombin, terminal enzyme in the ?cascade (Janus-faced
protein): adopts opposing procoagulant and anticoagulant
(thrombomodulin on endothelial cells) functions.
■ PT/APTT normal in protein C, S deficiency: not
shortened.
■ Plasma tends to clot after 5% of the thrombin has been
formed: 95% thrombin formation not accounted for.
PROCOAGULANT
DRIVERS
ANTICOAGULANT
DRIVERS

4. Elbaz C, Sholzberg M. An illustrated review of bleeding assessment tools and common coagulation tests. Research and Practice in Thrombosis and Haemostasis. 2020 Jul;4(5):761-773. DOI: 10.1002/rth2.12339. PMID: 32685885;
PMCID: PMC7354401.

5. Clot Waveform Analysis
APTT

6. APTT Clot Waveform Analysis
Measured by photo-optical coagulometer which
monitors changes in light transmittance over
entire course of clot formation being reflected
as a waveform, which is processed
mathematically to give various parameters like:
■ First Derivative: (dT/dt): Rate of change in
light Transmittance –velocity of clotting process
– Min1: the minimum value of the first
derivative of the aPTT waveform
■ Second Derivative (d2T/dt2): Magnitude of
change in light Transmittance with respect to
time – coagulation acceleration
– Min2: minimum value of the second
derivative of the aPTT waveform.
– Max2: maximum value of the second
derivative of the aPTT waveform.
Nair
SC,
Dargaud
Y,
Chitlur
M,
Srivastava
A.
Haemophilia.
2010
Jul;16
Suppl
5:85-92.
dT/dt
d2T/dt2
Max2
Min1
Min2

7. ?
• Study of 1187 consecutive admissions to
the intensive care unit.
• BPW preceded the time of DIC diagnosis
by 18 hours, on average,
• BPW better predicts for DIC than either
CRP or triglyceride alone.
• Simple, rapid test within frequently
requested parameter for assessing
coagulation in ITU setting.

8. Clot waveform of normal plasma by monitoring of absorbance in ACL-
Department
of
Transfusion
Medicine
and
Immunohematology,
CMC
Vellore

9. Wada H, Shiraki K, Matsumoto T, Shimpo H, Shimaoka M. Clot Waveform Analysis for Hemostatic Abnormalities. Ann Lab Med
2023;43:531-538.
mAbs (milliabsorbance)

11. RECOMMENDATIONS
TEST PLASMA To be prepared from fresh citrated whole
blood
REFERENCE PLASMA Pooled plasma from normal
individuals/commercial normal plasma
APTT REAGENTS (COLOR) Colourless APTT reagents without opacity
(sensitive to changes in transmittance/
absorbance)
APTT REAGENTS (TRANSMITTANCE)
{MDA-II/CS series}
Thrombocheck APTT-SLA/0.02M CaCl2
APTT REAGENTS (ABSORBANCE)
{ACL-Top series}
HemosIL SynthASiL
• Other instrument/reagent combinations need to be tested prior to use.
• APTT reagents for APLA antibodies not recommended (low sensitivity for assessing low levels of clotting
function)

12. RECOMMENDED
CLINICAL
APPLICATIONS Clotting function of various
bleeding disorders
Dose-dependent waveform changes in
plasma containing various conc. Of factor
VIII
Waveform changes in haemophilia A with
various levels of Factor VIII
CWA parameters and clinical severity of
severe haemophilia A

13. ‘CWA is a reliable test in patients with both hypercoagulable and
hypocoagulable states.’
‘Routine use of clot waveform analysis may be promoted even in
laboratories not dedicated to haemostasis and thrombosis.’

14. HYPOCOAGUABLE STATES
HEMOPHILIA
Highly significant correlation between CWA and
thrombin generation throughout a wide range of
concentrations (including lower levels) of both FVIII
and FIX.
Useful for obtaining information on the possible
heterogeneity of clinical phenotypes among patients
with severe or moderate hemophilia.
EMICIZUMAB
Modified CWA with a PT/aPTT mixed reagent (TF and
ellagic acid) used to create an adjusted Min1
(first derivative).
addition of emicizumab to hemophiliac plasma with/without
inhibitors led to concentration-dependent increases in adjusted
Min1
Modified CWA can provide a useful tool for assessing
global coagulation activity in patients with
hemophilia treated with emicizumab

15. (Adjusted CWA to remove impact of fibrinogen Fig. AdMin1 in FVIII incubated with emicizumab

16. HYPERCOAGUABLE STATES
VENOUS THROMBOEMBOLISM
CWA parameters are significantly higher in patients
with VTE compared to controls.
CANCER
Elevated peak height of CWA-sTF/FIXa may be
considered as a risk factor for thrombosis in patients
with cancer.

17. VTE PATIENTS vs CONTROLS
Mean APTT similar
Min1, p<0.001
Min2, p=0.001
Max2, p=0.002
Deltachange, p<0.001
Clot waveform analysis data from patients
(N = 45) with objectively proven acute VTE
who had an aPTT performed prior to
initiation of anticoagulation were compared
with controls (N = 111). The CWA
parameters measured were min1, min2,
max2 and delta change.

18. Kobayashi
M,
Wada
H,
Fukui
S,
et
al.
Clot
waveform
analysis
showing
a
hypercoagulable
state
in
patients
with
malignant
neoplasms.
J
Clin
Med.
2021

19. Thrombin generation assays for global evaluation of the hemostatic system: perspectives and limitations, Revista Brasileira de Hematologia e
Hemoterapia, Volume 39, Issue 3, 2017,
THROMBIN GENERATION TEST
THROMBINOSCOPE BV SOFTWARE (RFU > nM)

20. Peak Height (PH)
Endogenous Thrombin
Potential (ETP): Area Under
The Curve
Lag Time
Time To Peak (TTP)
Start Tail
Tripodi
A.
Thrombin
generation:
a
global
coagulation
procedure
to
investigate
hypo-
and
hyper-coagulability.
Haematologica
2020;105(9):2196-2199

21. Tripodi
A.
Thrombin
generation:
a
global
coagulation
procedure
to
investigate
hypo-
and
hyper-coagulability.
Haematologica
2020;105(9):2196-2199

22. 1. Emphasis has been placed on more reliable, more standardized and more user-friendly assays that have
now reached a high level of maturity.
2. Pre-analytical conditions have been extensively studied leading to precise yet manageable
recommendations.
3. Researchers have looked at the TGA potential in various clinical and industrial settings and demonstrated
the contributions of TGA in patient's diagnosis, prognosis evaluation and treatment monitoring.
4. Altogether, this has paved the way for an imminent transition of TGA from research to routine use.

23. ESTABILISHING STANDARDIZED TGA
■ A specific analyzer optimized for TGA including tightly controlled temperature
and light intensity
■ Validated pipetting schemes for the respective reagent including optimized
measurement times
■ Well controlled substrate including traceable thrombin and fluorescence
standards
■ Availability of specific QC controls covering the analytical measuring range
■ Use of standardized reagents or triggers

24. RECOMMENDATIONS
SUBSTRATE (FLUOROGENIC)
Z-Gly-Gly-Arg-AMC (ZGGR-AMC)
(7-Amino-4-methylcoumarin: fluorophore that generates
390 nm excitation/460 nm emission)
SUBSTRATE (CHROMOGENIC)
H-β-Ala-Gly-Arg-pNA
(absorption at 405 nm, requires defibrination, no longer
commercially available)
TRIGGER/REAGANT
• For bleeding tendencies testing on PPP
low TF concentrations 0.4 to 2 pM + phospholipid 0.4 to 4
µM
• Trigger reagents for thrombophilia testing
TF between 5 and 40 pM + phospholipid 0.4 to 4 µM
• To overcome anticoagulation
20 and 50 pM TF + 4µM phospholipids
CALIBRATION
Thrombin calibrator with known thrombin activity run

26. Summaries of most relevant TGA studies performed to assess bleeding
conditions.

27. Summaries of most relevant TGA studies for thrombotic
conditions.

28. Viscoelastic tests of coagulation
■ TEG
■ ROTEM
Setting
1. Cardiac surgery
2. Trauma
3. Post partum hemorrhage
4. Liver transplantation

30. TEG ROTEM INTERPRETATION
PHYSIOLOGICAL
CORRELATE
Reaction rate (R) (min) Clotting time (CT) (s)
Time for the trace to reach an
amplitude of 2 mm
Activation of coagulation,
thrombin generation, time to
initial clot formation, and
influence of anticoagulants
Kinetics time (K) (min)
Clot formation time (CFT)
(s)
Time for the clot amplitude to
reach from 2 mm to 20 mm
Fibrin activation and
polymerization (ie, speed of
clot propagation)
Angle (α) (degrees Angle (α) (degrees)
Angle created by drawing a
tangent line from the point of
clot initiation (R or CT) to the
slope of the developing curve
Fibrin activation and
polymerization (ie, speed of
clot propagation)
Maximum amplitude (MA)
(mm)
Maximum clot firmness
(MCF) (mm)
Peak amplitude or strength of
the clot
Fibrinogen and platelet
contribution to the strength of
the clot
Lysis 30 (LY 30) (%) Lysis index 30 (LI 30) (%) *** Fibrinolysis
Lysis 60 (LY 60) (%)
Percentage reduction in the
area under the TEG curve
(assuming MA remains
constant) that occurs 60 min
after MA is reached
Fibrinolysis
Maximum lysis (ML) (%)
Degree of fibrinolysis relative
to MCF achieved during the
measurement
Fibrinolysis
Whiting,
D.
and
DiNardo,
J.
A.
(2014),
TEG
and
ROTEM:
Technology
and
clinical
applications.
Am.
J.
Hematol.,
89:
228–232.
doi:
10.1002/ajh.23599

31. TEG v/s standard tests of coagulation
Whiting,
D.
and
DiNardo,
J.
A.
(2014),
TEG
and
ROTEM:
Technology
and
clinical
applications.
Am.
J.
Hematol.,
89:
228–232.
doi:
10.1002/ajh.23599

32. Müller,
M.C.,
Meijers,
J.C.,
Vroom,
M.B.
et
al.
Utility
of
thromboelastography
and/or
thromboelastometry
in
adults
with
sepsis:
a
systematic
review.
Crit
Care
18,
R30
(2014).
https://doi.org/10.1186/cc13721

33. Jan Hartmann, Daniela Hermelin, Jerrold H. Levy, Viscoelastic testing: an illustrated review of technology and clinical applications,
Research and Practice in Thrombosis and Haemostasis, Volume 7, Issue 1, 2023,

34. PARAMETER AB35946 NORMAL RANGE
CT 363 secs 324-565 secs
CFT 323 sec 112-224 secs
Alpha-angle 41 50-68 degrees
MCF 41mm 55-66 mm
ML 8% 0-15%
LAB PARAMETERS AB35946
PT 19.8 secs
INR 1.51
PT correction studies 16 secs
APTT 43.8 secs
APTT correction studies 31.3 secs

35. ROTEM TYPE REAGENT INTERPRETATION
INTEM
Reagent contains ellagic acid
activator.
Assess the contact activation
pathway of coagulation
EXTEM
Reagent contains tissue factor
activator.
Assesses the tissue factor–
initiated pathway of coagulation
FIBTEM
Reagent contains tissue factor and
cytochalasin D
Qualitatively assess the
contribution of fibrinogen to clot
strength independent of platelets
APTEM
Reagent contains aprotinin
(fibrinolysis inhibitor).
Allows discrimination between
fibrinolysis and platelet-mediated
clot retraction
HEPTEM
Reagent contains ellagic acid
activator and lyophilized
heparinase for neutralizing
unfractionated heparin
Assess heparin effect
Whiting,
D.
and
DiNardo,
J.
A.
(2014),
TEG
and
ROTEM:
Technology
and
clinical
applications.
Am.
J.
Hematol.,
89:
228–232.
doi:
10.1002/ajh.23599

36. Rotational
thromboelastometry-guided
bleeding
management.
Korean
J
Anesthesiol.
2019

38. CARDIAC SURGERY
www.nice.org.uk/guidance/dg13 published 20th August 2014

39. CARDIAC
SURGERY

40. CARDIAC
SURGERY
Wikkelsø A, Wetterslev J, Møller AM, Afshari A. Thromboelastography (TEG) or thromboelastometry (ROTEM) to monitor haemostatic treatment versus usual care in adults or children with bleeding. Cochrane Database Syst
Rev. 2016

41. CARDIAC
SURGERY
Korean J Anesthesiol. 2019 Aug
Implementation of VET into an
integrated transfusion algorithm
during cardiac surgery can lead
to:
1. Reduction in major post
surgical bleeding
2. Reduced product transfusions
3. Reduced red cell transfusions

42. CARDIAC
SURGERY
Jan Hartmann, Daniela Hermelin, Jerrold H. Levy, Viscoelastic testing: an illustrated review of technology and clinical applications,
Research and Practice in Thrombosis and Haemostasis, Volume 7, Issue 1, 2023,

44. Standard
treatment:
INR >1.5 – 2 unit
FFP
Fib <150 – 10 unit
Cryo
Plt<1 lac – 1 SDP
Gonzalez E, Moore EE, Moore HB, Chapman MP, Chin TL, Ghasabyan A, Wohlauer MV, Barnett CC, Bensard DD, Biffl WL, Burlew CC, Johnson JL, Pieracci FM, Jurkovich GJ, Banerjee A, Silliman CC, Sauaia A.
Goal-directed Hemostatic Resuscitation of Trauma-induced Coagulopathy: A Pragmatic Randomized Clinical Trial Comparing a Viscoelastic Assay to Conventional Coagulation Assays. Ann Surg. 2016
Jun;263(6):1051-9.
TRAUMA

45. Cochrane Database Syst Rev. 2015 Feb 16;(2):CD010438.
TRAUMA

46. Viscoelastic haemostatic assay augmented protocols for major trauma haemorrhage (ITACTIC): a randomized, controlled trial.
Intensive Care Med. 2021
TRAUMA

47. Jan Hartmann, Daniela Hermelin, Jerrold H. Levy, Viscoelastic testing: an illustrated review of technology and clinical applications,
Research and Practice in Thrombosis and Haemostasis, Volume 7, Issue 1, 2023,
TRAUMA

48. Charbit B, Mandelbrot L, Samain E, Baron G, Haddaoui B, Keita H, Sibony O, Mahieu-Caputo D, Hurtaud-Roux
MF, Huisse MG, Denninger MH, de Prost D, for the PPH Study Group. The decrease of fibrinogen is an early
predictor of the severity of postpartum hemorrhage. J Thromb Haemost 2007; 5: 266–73.
POST-PARTUM
HAEMORRHAGE

49. ■ Either PT/ aPTT/platelet count and Clauss fibrinogen or viscoelastic tests - local algorithm and a quality
control protocol
■ Fibrinogen level of at least 2 g/L (Fibtem A5 about 12 mm)
■ 15 mL/kg FFP- if PT/aPTT prolonged 1.5 times normal
■ After 4 units of RBC, infuse 4 units of FFP and 1 : 1 RBC : FFP transfusion maintained until test results are
known
■ Platelets transfusion if platelet count is < 75 x109/L
■ 1 gm IV tranexamic acid
■ Massive bleeding requiring 8 units RBCs + 8 units FFP - no coagulation results or platelet count available -
then give two pools of cryoprecipitate and one pool of platelets
■ Upto 2 doses of rFVIIa 60 mcg/kg can be tried if fib >2g/L and plt>50 x 109/L
■ Thromboprophylaxis once bleeding is controlled – atleast 10 days
POST-PARTUM
HAEMORRHAGE

50. ■ FIBTEM A5 <5 mm or A10 <6 mm - 5 to 15 units of cryoprecipitate - Goal - A10 of 8 mm if
bleeding controlled and 10 mm for patients with ongoing hemorrhage
■ Repeat test at the end of the cryoprecipitate transfusion and every 20 to 60 min thereafter till
bleeding stops
■ If CFT and alpha angle demonstrated hypocoagulation accompanied by a decrease in EXTEM
MCF to <45 mm - one bag (5 units) of platelet concentrate, followed by repeat testing.
■ If isolated CT prolongation was noticed in EXTEM and INTEM assays - 2 units of FFP, followed by
repeat testing.
■ Packed red blood cell transfusions were provided as needed in all the cases to maintain the
hematocrit above 21%
Snegovskikh
D,
Souza
D,
Walton
Z,
Dai
F,
Rachler
R,
Garay
A,
Snegovskikh
VV,
Braveman
FR,
Norwitz
ER.
Point-of-
care
viscoelastic
testing
improves
the
outcome
of
pregnancies
complicated
by
severe
postpartum
hemorrhage.
J
Clin
Anesth.
2018
Feb;44:50-56.

51. POST-PARTUM
HAEMORRHAGE

52. Lisman T, Hernandez-Gea V, Magnusson M, Roberts L, Stanworth S, Thachil J, Tripodi A. The
concept of rebalanced hemostasis in patients with liver disease: Communication from the
ISTH SSC working group on hemostatic management of patients with liver disease. J Thromb
Haemost. 2021 Apr
Patients with liver disease are in haemostatic balance due to the
simultaneous decline of pro‐ and anticoagulant drivers. The value
of the PT and APTT in patients with liver disease is limited and
may also provide misleading information
Prophylactic correction of abnormal laboratory tests of hemostasis
with blood products or pharmacological prohemostatic agents
aiming to prevent spontaneous or procedure‐related bleeding is
not indicated.
REBALANCE HEMOSTASIS
IN PATIENTS WITH LIVER DISEASE

53. Hepatology 2016;63:566-573.
LIVER
DISEASE
There was no increase in procedure-related bleeding complications despite a
significant reduction in component transfusion, suggesting that the ROTEM-
based transfusion strategy may safely reduce unnecessary blood component
transfusion without a concomitant increase in risk of bleed

54. Global assays such as ROTEM/TEG may be an attractive means to
reassure the proceduralist that haemostasis is ‘normal’ as these
assays are frequently normal despite prolonged coagulation times.
‘ We recommend ROTEM/TEG over measuring INR,
platelet, fibrinogen in critically ill patients with
ALF/ACLF undergoing procedures strongly'

55. Smart
L,
Mumtaz
K,
Scharpf
D,
Gray
NO,
Traetow
D,
Black
S,
Michaels
AJ,
Elkhammas
E,
Kirkpatrick
R,
Hanje
AJ.
Rotational
Thromboelastometry
or
Conventional
Coagulation
Tests
in
Liver
Transplantation:
Comparing
Blood
Loss,
Transfusions,
and
Cost.
Ann
Hepatol.
2017;16(6):916-923.

59. Department
of
Transfusion
Medicine
and
Immunohematology,
CMC
Vellore

60. THANK YOU