Tests of Global Hemostasis Hematological

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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 (d 2 T/dt 2 ): 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 d 2 T/dt 2 Max2 Min1 Min2

7. ? S tudy of 1187 consecutive admissions to the intensive care unit . B PW 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-TOP (IL) 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 )

10. 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)

11. 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

12. ‘ 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.’

13. 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 M odified CWA can provide a useful tool for assessing global coagulation activity in patients with hemophilia treated with emicizumab

14. (Adjusted CWA to remove impact of fibrinogen conc.n .) Fig. AdMin1 in FVIII incubated with emicizumab

15. HYPERCOAGUABLE STATES VENOUS THROMBOEMBOLISM CWA parameters are significantly higher i n patients with VT E compared to controls. CANCER E levated peak height of CWA- sTF / FIXa may be considered as a risk factor for thrombosis in patients with cancer.

16. 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.

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

18. 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 )

19. 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

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

21. Emphasis has been placed on more reliable, more standardized and more user-friendly assays that have now reached a high level of maturity. Pre-analytical conditions have been extensively studied leading to precise yet manageable recommendations. 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. Altogether, this has paved the way for an imminent transition of TGA from research to routine use.

22. ESTABILISHING STANDARDIZED TGA A specific analyzer optimized for TGA including tightly controlled temperature and light intensity V alidated pipetting schemes for the respective reagent including optimized measurement times W ell controlled substrate including traceable thrombin and fluorescence standards A vailability of specific QC controls covering the analytical measuring range U se of standardized reagents or triggers

23. 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 parallel to the samples

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

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

26. Viscoelastic tests of coagulation TEG ROTEM Setting Cardiac surgery Trauma Post partum hemorrhage Liver transplantation

27. 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

28. 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

29. 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

30. 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,

31. 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

32. ROTEM TYPE REAGENT INTERPRETATION INTEM Reagent contains ellagic acid activator. A ssess 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

33. R otational thromboelastometry -guided bleeding management. Korean J Anesthesiol . 2019

34. Semin Thromb Hemost . 2010 Oct;36(7):757-63

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

36. CARDIAC SURGERY

37. 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

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

39. 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,

40. 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

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

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

43. 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

44. 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

45. 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 x10 9 /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 10 9 /L Thromboprophylaxis once bleeding is controlled – atleast 10 d ays POST-PARTUM HAEMORRHAGE

46. 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.

47. POST-PARTUM HAEMORRHAGE

48. 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 P atients 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

49. 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

50. 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'

51. 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.

52. Department of Transfusion Medicine and Immunohematology, CMC Vellore

53. Thank you