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Licensed Unlicensed Requires Authentication Published by De Gruyter September 29, 2018

Next-generation rapid serum tube technology using prothrombin activator coagulant: fast, high-quality serum from normal samples

  • Kong-Nan Zhao , Goce Dimeski , John de Jersey , Lambro A. Johnson , Michael Grant , Paul P. Masci and Martin F. Lavin EMAIL logo

Abstract

Background

Incomplete blood clotting or latent clotting in serum is a common laboratory problem, especially for patients on anticoagulant therapy or when serum tubes are centrifuged before clotting is completed. We describe a novel approach to producing high-quality serum using snake venom prothrombin activator complex (OsPA) as an additive in blood collection tubes for non-anticoagulated (normal) individuals.

Methods

Plasma clotting assays were performed using a Hyland-Clotek instrument. Blood clotting was visually observed, and thromboelastography was also performed to determine the important parameters of coagulation. Thrombin generation was assayed using the chromogenic substrate S-2238, and biochemical analytes in the serum were determined on chemistry and immunoassay analysers. Fibrinogen was determined by either ELISA or Clauss fibrinogen assay.

Results

We initially showed that OsPA had strong coagulation activity in clotting not only recalcified citrated plasma and recalcified citrated whole blood, but also fresh whole blood in a clinical setting. The use of TEG clearly showed improved speed of clotting and generation of a firmer clot. We also showed that the use of OsPA to produce serum did not interfere with the determination of commonly measured biochemical analytes. The underlying clotting mechanism involves a burst of thrombin production at the initial stages of the clotting process upon contact with prothrombin in blood.

Conclusions

These results demonstrate rapid generation of high-quality serum, contributing to faster turnaround times with standardised quality samples, for accurate analyte determinations in normal individuals.

Acknowledgments

We wish to thank the Australian Red Cross Blood Supply (ARCBS) for the provision of blood for some experiments. The project was in part funded by a development grant from the National Health and Medical Research Council of Australia and by Q-Sera Pty Ltd., Australia, Grant Number: Clotting tube product development. We wish to thank Nathan Dunstan, Venom Supplies, South Australia, for providing snake venom and Ms Connie Solano, Haematology Department, Princess Alexandra Hospital, Brisbane, for supply of blood.

  1. Author contributions: M.F.L, J.D.J., P.P.M., G.D., K.N.Z. and M.G. contributed to the conception of this project and designed the experiments. P.P.M., K.N.Z., L.A.J. and G.D. performed the experiments. K.N.Z. and G.D. organised and analysed data, and all authors reviewed and discussed. K.N.Z. and M.F.L wrote the paper with input from all authors on the drafting of the manuscript. All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

  2. Research funding: None declared.

  3. Employment or leadership: None declared.

  4. Honorarium: None declared.

  5. Competing interests: MFL, PM, JdJ, MG and GD have Employee Share Options Scheme in Q-Sera. The funding organisation(s) played no role in the study design; in the collection, analysis and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.

References

1. Vignati G, Chiecchio A, Osnaghi B, Giovanelli L, Meloncelli C. Different biological matrices (serum and plasma) utilization in consolidation processes: evaluation of seven access immunoassays. Clin Chem Lab Med 2008;46:264–70.10.1515/CCLM.2008.032Search in Google Scholar PubMed

2. Lee BS, Lee YU, Kim HS, Kim TH, Park J, Lee JG, et al. Fully integrated lab-on-a-disc for simultaneous analysis of biochemistry and immunoassay from whole blood. Lab Chip 2011;11:70–8.10.1039/C0LC00205DSearch in Google Scholar PubMed

3. Bowen RA, Sattayapiwat A, Gounden V, Remaley AT. Blood collection tube-related alterations in analyte concentrations in quality control material and serum specimens. Clin Biochem 2014;47:150–7.10.1016/j.clinbiochem.2013.11.003Search in Google Scholar PubMed PubMed Central

4. Aoki N, Sakata Y, Ichinose A. Fibrin-associated plasminogen activation in alpha 2-plasmin inhibitor deficiency. Blood 1983;62:1118–22.10.1182/blood.V62.5.1118.1118Search in Google Scholar

5. Cuhadar S, Atay A, Koseoglu M, Dirican A, Hur A. Stability studies of common biochemical analytes in serum separator tubes with or without gel barrier subjected to various storage conditions. Biochem Med (Zagreb) 2012;22:202–14.10.11613/BM.2012.023Search in Google Scholar PubMed PubMed Central

6. Dimeski G, Masci PP, Trabi M, Lavin MF, De Jersey J. Evaluation of the Becton-Dickinson rapid serum tube: does it provide a suitable alternative to lithium heparin plasma tubes? Clin Chem Lab Med 2010;48:651–7.10.1515/CCLM.2010.141Search in Google Scholar PubMed

7. Dimeski G, Solano C, Petroff MK, Hynd M. Centrifugation protocols: tests to determine optimal lithium heparin and citrate plasma sample quality. Ann Clin Biochem 2011;48:218–22.10.1258/acb.2010.010230Search in Google Scholar PubMed

8. Bowen RA, Remaley AT. Interferences from blood collection tube components on clinical chemistry assays. Biochem Med (Zagreb) 2014;24:31–44.10.11613/BM.2014.006Search in Google Scholar PubMed PubMed Central

9. Prusa R, Doupovcova J, Warunek D, Stankovic AK. Improving laboratory efficiencies through significant time reduction in the preanalytical phase. Clin Chem Lab Med 2010;48:293–6.10.1515/CCLM.2010.036Search in Google Scholar PubMed

10. Dimeski G, Johnston J, Masci PP, Zhao KN, Brown N. Evaluation of the Greiner Bio-One serum separator BCA fast clot tube. Clin Chem Lab Med 2017;55:1135–41.10.1515/cclm-2016-0806Search in Google Scholar PubMed

11. Bompiani KM, Lohrmann JL, Pitoc GA, Frederiksen JW, Mackensen GB, Sullenger BA. Probing the coagulation pathway with aptamers identifies combinations that synergistically inhibit blood clot formation. Chem Biol 2014;21:935–44.10.1016/j.chembiol.2014.05.016Search in Google Scholar

12. Brummel-Ziedins K, Undas A, Orfeo T, Gissel M, Butenas S, Zmudka K, et al. Thrombin generation in acute coronary syndrome and stable coronary artery disease: dependence on plasma factor composition. J Thromb Haemost 2008;6:104–10.10.1111/j.1538-7836.2007.02799.xSearch in Google Scholar

13. Ruggeri ZM, Ruggeri ZM. Platelet and von Willebrand factor interactions at the vessel wall. Hamostaseologie 2004;24:1–11.10.1055/s-0037-1619601Search in Google Scholar

14. Undas A, Szuldrzynski K, Brummel-Ziedins KE, Tracz W, Zmudka K, Mann KG. Systemic blood coagulation activation in acute coronary syndromes. Blood 2009;113:2070–8.10.1182/blood-2008-07-167411Search in Google Scholar

15. Stubbs MT, Bode W. Coagulation factors and their inhibitors. Curr Opin Struct Biol 1994;4:823–32.10.1016/0959-440X(94)90263-1Search in Google Scholar

16. Matafonov A, Sarilla S, Sun MF, Sheehan JP, Serebrov V, Verhamme IM, et al. Activation of factor Xi by products of prothrombin activation. Blood 2011;118:437–45.10.1182/blood-2010-10-312983Search in Google Scholar

17. Walker FJ, Owen WG, Esmon CT. Characterization of the prothrombin activator from the venom of Oxyuranus scutellatus scutellatus (Taipan Venom). Biochemistry 1980;19:1020–3.10.1021/bi00546a029Search in Google Scholar

18. Kini RM. The intriguing world of prothrombin activators from snake venom. Toxicon 2005;45:1133–45.10.1016/j.toxicon.2005.02.019Search in Google Scholar

19. Speijer H, Govers-Riemslag JW, Zwaal RF, Rosing J. Prothrombin activation by an activator from the venom of Oxyuranus scutellatus (Taipan Snake). J Biol Chem 1986;261:13258–67.10.1016/S0021-9258(18)69299-3Search in Google Scholar

20. Speijer H, Govers-Riemslag JW, Zwaal RF, Rosing J. Platelet procoagulant properties studied with snake venom prothrombin activators. Thromb Haemost 1987;57:349–55.10.1055/s-0038-1651132Search in Google Scholar

21. Bos MH, Camire RM. Procoagulant adaptation of a blood coagulation prothrombinase-like enzyme complex in Australian elapid venom. Toxins (Basel) 2010;2:1554–67.10.3390/toxins2061554Search in Google Scholar PubMed PubMed Central

22. Bos MH, Camire RM. Blood coagulation factors V and VIII: molecular mechanisms of procofactor activation. J Coagul Disord 2010;2:19–27.Search in Google Scholar

23. Austen DE, Rhymes IL. Laboratory Manual of Blood Coagulation. Oxford: Blackwell Scientific Publications, 1975:109.Search in Google Scholar

24. Swallow RA, Agarwala RA, Dawkins KD, Curzen NP. Thromboelastography: potential bedside tool to assess the effects of antiplatelet therapy? Platelets 2006;17:385–92.10.1080/09537100600757521Search in Google Scholar PubMed

25. Masci PP, Whitaker AN, De Jersey J. Purification and characterization of a prothrombin activator from the venom of the Australian brown snake, Pseudonaja textilis textilis. Biochem Int 1988;17:825–35.Search in Google Scholar

26. Moore GW, Tugnait S, Savidge GF. A new-generation dilute Russell’s viper venom time assay system for lupus anticoagulants: evaluation of detection utilising frozen reagents and controls. Br J Biomed Sci 2005;62:127–32.10.1080/09674845.2005.11732699Search in Google Scholar PubMed

27. Nowak G. The Ecarin clotting time, a universal method to quantify direct thrombin inhibitors. Pathophysiol Haemost Thromb 2003;33:173–83.10.1159/000081505Search in Google Scholar PubMed

28. Van Cott EM, Smith EY, Galanakis DK. Elevated fibrinogen in an acute phase reaction prolongs the reptilase time but typically not the thrombin time. Am J Clin Pathol 2002;118: 263–8.10.1309/WUB3-72JT-E50M-EU8JSearch in Google Scholar PubMed

29. Herrera M, Paiva OK, Pagotto AH, Segura A, Serrano SM, Vargas M, et al. Antivenomic characterization of two antivenoms against the venom of the Taipan, Oxyuranus scutellatus, from Papua New Guinea and Australia. Am J Trop Med Hyg 2014;91:887–94.10.4269/ajtmh.14-0333Search in Google Scholar PubMed PubMed Central

30. Arslan FD, Karakoyun I, Basok BI, Aksit MZ, Baysoy A, Ozturk YK, et al. The local clinical validation of a new lithium heparin tube with a barrier: Bd Vacutainer(R) Barricor Lh Plasma tube. Biochem Med (Zagreb) 2017;27:030706.10.11613/BM.2017.030706Search in Google Scholar PubMed PubMed Central

31. Carraro P, Servidio G, Plebani M. Hemolyzed specimens: a reason for rejection or a clinical challenge? Clin Chem 2000;46:306–7.10.1093/clinchem/46.2.306Search in Google Scholar

32. Lippi G, Cervellin G, Mattiuzzi C. Critical review and meta-analysis of spurious hemolysis in blood samples collected from intravenous catheters. Biochem Med (Zagreb) 2013;23:193–200.10.11613/BM.2013.022Search in Google Scholar PubMed PubMed Central

33. Monneret D, Mestari F, Atlan G, Corlouer C, Ramani Z, Jaffre J, et al. Hemolysis indexes for biochemical tests and immunoassays on roche analyzers: determination of allowable interference limits according to different calculation methods. Scand J Clin Lab Invest 2015;75:162–9.10.3109/00365513.2014.993691Search in Google Scholar PubMed

34. Rao VS, Kini RM. Pseutarin C, a prothrombin activator from Pseudonaja textilis venom: its structural and functional similarity to mammalian coagulation factor Xa-Va complex. Thromb Haemost 2002;88:611–9.10.1055/s-0037-1613264Search in Google Scholar

35. St Pierre L, Masci PP, Filippovich I, Sorokina N, Marsh N, Miller DJ, et al. Comparative analysis of prothrombin activators from the venom of Australian Elapids. Mol Biol Evol 2005;22:1853–64.10.1093/molbev/msi181Search in Google Scholar PubMed

36. Bos MH, Boltz M, St Pierre L, Masci PP, De Jersey J, Lavin MF, et al. Venom Factor V from the common brown snake escapes hemostatic regulation through procoagulant adaptations. Blood 2009;114:686–92.10.1182/blood-2009-02-202663Search in Google Scholar PubMed PubMed Central

37. Plebani M, Lippi G. Improving diagnosis and reducing diagnostic errors: the next frontier of laboratory medicine. Clin Chem Lab Med 2016;54:1117–8.10.1515/cclm-2016-0217Search in Google Scholar PubMed

38. Lippi G, Plebani M, Favaloro EJ. The model list of essential in vitro diagnostics: nuisance or opportunity? Diagnosis (Berl) 2018. pii: /j/dx.ahead-of-print/dx-2018-0035/dx-2018-0035.xml. Doi: 10.1515/dx-2018-0035. [Epub ahead of print].10.1515/dx-2018-0035Search in Google Scholar PubMed

39. Plebani M, Lippi G. Improving the post-analytical phase. Clin Chem Lab Med 2010;48:435–6.10.1515/CCLM.2010.113Search in Google Scholar PubMed


Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/cclm-2018-0397).


Received: 2018-04-17
Accepted: 2018-08-31
Published Online: 2018-09-29
Published in Print: 2019-03-26

©2019 Walter de Gruyter GmbH, Berlin/Boston

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