Effect of Hemodilution in vitro and in vivo on the Hemostatic System

The aim of the study was to determine experimentally the effect of hemodilution by the 2:1 sterofundin/gelofusine (SG) solution on hemostatic parameters in vitro and in vivo.

Material and methods. Experiments were carried out on 75 male Wistar rats weighing 270–380 g and anesthetized with intramuscular tiletamine-zolazepam (40 mg/kg) + xylazine (10 mg/kg). Animals were divided randomly into 4 groups: Group 1 — in vitro 25-percent dilution of carotid blood samples by the SG solution (n=12), Group 2 — in vitro 37.5-percent dilution of similar samples (n=11), Group 3 — in vivo 25-percent dilution (n=10), Group 4 — the controls (n=42) with no dilution. The first stage of the study compared the in vitro dilution groups with the control group and with each other; the second stage compared the in vivo dilution group with the control group. The parameters of low-frequency piezoelectric tromboelastography (LFPTEG), clotting tests and complete blood count were studied to evaluate the effect of hemodilution.

Results. At a 25-percent hemodilution with 2:1 CG solution in vitro and in vivo, the hemostatic parameters retained within the reference limits, but a trend to increased intensity of the enzymatic reactions of the coagulation cascade and a significant increase in clot polymerization in vitro due to relative anticoagulant deficiency became evident. In vitro 37.5-percent blood dilution significantly reduced the blood level of fibrinogen and platelet count, inhibited the intensity of the proteolytic stage of coagulation, reduced the clot density at the T3 gelation point, at 5 minutes after reaching it and the maximum amplitude (MA) of the LFPTEG curve, as well as significantly reduced anticoagulant activity of the blood. The observed changes in hemostatic parameters were significantly outside the reference limits, which may affect the interpretation of the experimental results and be clinically important. We found negative correlation between clot density and platelet activity at 25-percent dilution in vivo, whereas at 37.5-percent dilution in vitro an additional positive correlations between platelet count and fibrinogen levels were determined.

Conclusion. A 25-percent hemodilution with 2:1 CG solution should be considered «safe» for the in vivo hemostatic system providing minimal effect on the in vitro parameters in the exper-iment.

1. Martini W.Z. Coagulation complications following trauma. Mil. Med. Res. 2016; 3: 35. DOI: 10.1186/s40779-016-0105-2

2. Baraich A.I., Sychev A.A., Savin I.A., Polunin A.A., Oshirov A.V., Potapov A.A. Hemostasis disturbances in patients in the acute period of isolated traumatic brain injure (review). Obshchaya Reanimatologiya=General Reanimatology. 2018; 14 (5): 85–95. [In Russ.]. DOI: 10.15360/1813-9779-2018-5-85-95

3. Spahn D. R., Bouillon B., Cerny V., Duranteau J., Filipescu D., Hunt B. J., Komadina R., Maegele M., Nardi G., Riddez L., Samama C-M., Vincent J-L., Rossaint R. The European guideline on management of major bleeding and coagulopathy following trauma: fifth edition. Critical Care. 2019; 23: 98. DOI: 10.1186/s13054-019-2347-3.

4. Mullier F., Lessire S., De Schoutheete J-C., Chatelain B., Deneys V., Mathieux V., Hachimi Idrissi S., Dogne J-M., Watelet J-B., Gourdin M., Dincq A-S. Facing coagulation disorders after acute trauma. B-ENT. 2016; 26 (1): 67-85.

5. Baraich A.I., Sychev A.A., Savin I.A., Polunin A.A., Oshirov A.V., Potapov A.A. Coagulopathy in the acute phase of traumatic brain injury. Obshchaya Reanimatologiya=General Reanimatology. 2020; 16 (1): 14 (5): 85–95[In Russ.]. DOI: 10.15360/1813-9779-2020-1-27-34

6. Kugaevskaya E.V., Gureeva T.A., Timoshenko O.S., Solovyeva N.I. Urukinase-type plasminogenactivator system in norm and life-threatening processes (review). Obshchaya Reanimatologiya=General Reanimatology. 2018; 14 (6): 61–79 [In Russ.]. DOI: 10.15360/1813-9779-2018-6-61-79

7. Hampton D. A., Fabricant L. J., Differding J., Diggs B., Underwood S., De La Cruz D., Holcomb J. B., Brasel K. J., Cohen M. J., Fox E. E., Alarcon L. H., Rahbar M. H., Phelan H. A., Bulger E. M., Muskat P., Myers J. G., del Junco D. J., Wade C. E., Cotton B.A., Schreiber M. A. Prehospital intravenous fluid is associated with increased survival in trauma patients. J Trauma Acute Care Surg. 2013; 75 (1): 9-15. DOI: 10.1097/TA.0b013e318290cd52

8. Boyd C.J., Claus M.A., Raisis A.L., Hosgood G., Sharp C.R., Smart L. Hypocoagulability and platelet dysfunction are exacerbated by synthetic colloids in a canine hemorrhagic shock model. Front Vet Sci. 2018; 5: 279-290. DOI: 10.3389/fvets.2018.0027

9. Sevcikova S., Vymazal T., Durila M. Effect of balanced crystalloid, gelatin and hydroxyethyl starch on coagulation detected by rotational thromboelastometry in vitro. Clin Lab. 2017; 63 (10): 1691-1700. DOI: 10.7754/Clin.Lab.2017.170505

10. Kozek-Langenecker S.A. Fluids and coagulation. Curr Opin Crit Care. 2015; 21 (4); 285-291. DOI: 10.1097/mcc.0000000000000219

11. Wu R., Peng L-G., Zhao H-M. Diverse coagulopathies in a rabbit model with different abdominal injuries. World J. Emerg. Med. 2017; 8 (2): 141-147. DOI: 10.5847/wjem.j.1920-8642.2017.02.011

12. Dyer M., Haldeman S., Gutierrez A., Kohut L., Gupta A.S., Neal M.D. Uncontrolled hemorrhagic shock modeled via liver laceration in mice with real time hemodynamic monitoring. J Vis Exp. 2017; 123: 55554. DOI: 10.3791/55554

13. Wang H., Cao H., Zhang X., Ge L., Bie L. The effect of hypertonic saline and mannitol on coagulation in moderate traumatic brain injury patients. Am J Emerg Med. 2017; 35 (10): 1404-1407. DOI: 10.1016/j.ajem.2017.04.020

14. Ponschab M., Schöchl H., Keibl C., Fischer H., Redl H., Schlimp C.J. Preferential effects of low volume versus high volume replacement with crystalloid fluid in a hemorrhagic shock model in pigs. BMC Anesthesiol. 2015; 15: 133. DOI: 10.1186/s12871-015-0114-9

15. Solovyev M.A., Tyutrin I.I., Udut V.V., Klimenkova V.F. Experience in the diagnosis and monitoring of critical hemostasis disorders. Med.biol. i sots.-psikhol. probl. bezopasnosti v chrezv. situatsiyakh. 2013; 4: 55–60. [In Russ.] DOI: 10.25016/2541-7487-2013-0-4-55-60

16. Udut V.V., Tyutrin I.I., Kotlovskaya L.Yu., Solovyev M.A., Zhukov E.L., Lastovetskiy A.G., Borodulina E.V., Kotlovsky M.Yu. Technology of low-frequency piezothromboelastography in the assessment of hemostatic potential. Vestnik novikh meditsinskikh tekhnologiy. 2016; 4 [In Russ.] DOI: 10.12737/22220

17. Kinzersky A.A., Dolgikh V.T., Korzhuk M.S. Method for obtaining reference values of low-frequency piezothromboelastography in male rats of the Wistar line. Sibirsky meditsinsky zhurnal (Irkutsk). 2016; 142 (3): 25-28 [In Russ.].

18. Kinzersky A.A., Dolgikh V.T., Korzhuk M.S. Normal values of low-frequency piezotromboelastography of male Wistar rats obtained under xylazine + tiletamine-zolazepam anesthesia during blood sampling from the carotid artery. Certificate of state registration of the database No.2016620346. Bull. 2016. 4 [In Russ.].

19. Kinzersky A.A., Petrova Yu.A., Korzhuk M.S., Dolgikh V.T. Normal values of the general, biochemical analysis of blood and coagulogram of male rats of the Wistar line. Certificate of state registration of the database No. 2017620486. Bull. 2017; 5 [In Russ.]

20. Lipatov V.A., Severinov D.A., Kryukov A.A., Saakyan A.R. Ethical and legal aspects of experimental biomedical research in vivo. Part II. Rossiysy medico-biologichesky vestnik im. I.P. Pavlova. 2019; 27 (2): 245-257. DOI: 10.23888/PAVLOVJ2019272245-257 [In Russ.]

21. Kinzersky A.A., Dolgikh V.T., Korzhuk M.S. Temporal and structural indicators of thrombogenesis dynamics of low-frequency piezotromboelastography of male Wistar rats obtained under xylazine + tiletamine-zolazepam anesthesia during blood sampling from the carotidartery. Certificate of state registration of the database No. 201662071. Bull. 2016; 6 [In Russ.]

22. Mastitsky S.E., Shitikov V.K. Statistical analysis and visualization of data using R. Khaydelberg-London-Toliyatti. 2014: 401 [In Russ.]

23. Haas T., Mauch J., Weiss M., Schmugge M. Management of dilutional coagulopathy during pediatric major surgery. Transfus Med Hemother. 2012; 39 (2): 114–119. DOI: 10.1159/000337245

24. Dolgov V.V., Svirin P.V. Laboratory diagnostics of hemostatic disorders. M.- Tver: Triad Publishing Company LLC; 2005: 227 [In Russ.].

25. Ruttmann T.G., Lemmens H.J.M., Malott K.A., Brock-Utne J.G. The haemodilution enhanced onset of coagulation as measured by the thrombelastogram is transient. Eur J Anaesthesiol. 2006; 23 (7): 574-579. DOI: 10.1017/S0265021506000238

26. Veigas P.V., Callum J., Rizoli S., Nascimento B., Luz L.T. A systematic review on the rotational thrombelastometry (ROTEM®) values for the diagnosis of coagulopathy, prediction and guidance of blood transfusion and prediction of mortality in trauma patients. Scand. J. Trauma Resusc. Emerg. Med. 2016; 24 (1): 114. DOI: 10.1186/s13049-016-0308-2

27. Morris B.R., Laforcade A, Lee J., Palmisano J., Meola D., Rozanski E. Effects of in vitro hemodilution with crystalloids, colloids, and plasma on canine whole blood coagulation as determined by kaolin‐activated thromboelastography. J Vet Emerg Crit Care (San Antonio). 2016; 26 (1): 58-63. DOI: 10.1111/vec.12345

28. Schäfer N., Driessen A., Bauerfeind U., Fröhlich M., Ofir J., Stürmer E. K., Maegele M. In vitro effects of different sources of fibrinogen supplementation on clot initiation and stability in a model of dilutional coagulopathy. Transfus Med. 2016; 26 (5): 373–380. DOI: 10.1111/tme.12333

29. Sevcikova S., Durila M., Vymazal T. Rotational thromboelastometry assessment of ballanced crystalloid, hydroxyethyl starch and gelatin effects on coagulation: a randomized trial. Rev Bras Anestesiol. 2019; 69 (4): 383–389. DOI: 10.1016/j.bjan.2019.03.009

30. Kam P., Varanasi S., Yang K.X. The effects of haemodilution with succinylated gelatin solution on coagulation in vitro as assessed by thromboelastometry and impedance (multiple electrode) aggregometry. Anaesth Intensive Care. 2018; 46 (3): 272–277. DOI: 10.1177/0310057X1804600304