Age-Dependent Differential Staining of Fibrin in Blood Clots and Thrombi

  • Rafael R. KhismatullinEmail author
  • Asia Z. Shakirova
  • John W. Weisel
  • Rustem I. Litvinov


It has been known for a long time that histologically fibrin can be visualized as a fibrous structure of variable colors, even when stained with the same histochemical technique. The reason for this phenomenon called metachromasia remains unknown. We hypothesized that metachromasia is related to fibrin structural maturation and age. To establish a link between the color of fibrin in histological preparations and the age of blood clots and thrombi. Using the Picro-Mallory staining technique, we studied changes in fibrin color in histological preparations of in vitro human plasma clots at various time points within 48 h after formation in the absence and presence of platelets. Also, we used the same stain to visualize fibrin in histological sections of ex vivo human cerebral thrombi of different maturity. In histological preparations of plasma clots formed either in the absence or in the presence of platelets, fibrin was distinctively polychromic depending on the time lapse between formation and chemical fixation of the clot. In the 30-min and 6-h clots (“young” clots), fibrin was red; after 6–12 h (“mature” clots), fibrin was purple or violet; at 24 or 48 h (“old” clots), fibrin was blue. In thrombi removed from cerebral arteries of ischemic stroke patients, fibrin also stained differentially. The colors generally corresponded to the time from the onset of stroke to the time of intravital thrombectomy, such that fibrin in the younger thrombi was red or purple, while in the older thrombi, fibrin was blue. The Picro-Mallory stain can be used to assess histologically the maturity of fibrin in blood clots, thrombi, and thrombotic emboli based on the age-dependent differential staining of fibrin. A color-temporal scale is proposed that can help pathologists to estimate the age of a blood clot or thrombus.


Fibrin Blood clotting Thrombosis Histochemistry Picro-Mallory 


Funding Information

The work was supported by NIH grants HL135254, HL116330 grant RFBR 19-015-00075 and the Program for Competitive Growth at Kazan Federal University.

Compliance with Ethical Standards

Conflict of Interest

The authors declare that they have no conflict of interest.

Research Involving Humans and Animals Statement


Informed Consent



  1. 1.
    Lendrum, A. C., Fraser, D. S., Slidders, W., et al. (1962). Studies on the character and staining of fibrin. Journal of Clinical Pathology, 15(5), 401–413.CrossRefGoogle Scholar
  2. 2.
    Cattaneo, C., Andreola, S., Marinelli, E., et al. (2010). The detection of microscopic markers of hemorrhaging and wound age on dry bone: a pilot study. The American Journal of Forensic Medicine and Pathology, 31(1), 22–26.CrossRefGoogle Scholar
  3. 3.
    Tutwiler, V., Wang, H., Litvinov, R. I., et al. (2017). Interplay of platelet contractility and elasticity of fibrin/erythrocytes in blood clot retraction. Biophysical Journal, 112(4), 714–723.CrossRefGoogle Scholar
  4. 4.
    Mehta, B. P., & Nogueira, R. G. (2012). Should clot composition affect choice of endovascular therapy? Neurology., 79(1), 63–67.CrossRefGoogle Scholar
  5. 5.
    Liebeskind, D. S., Sanossian, N., Yong, W. H., et al. (2011). CT and MRI early vessel signs reflect clot composition in acute stroke. Stroke., 42, 1237–1243.CrossRefGoogle Scholar
  6. 6.
    Froehler, M. T., Tateshima, S., Duckwiler, G., et al. (2013). The hyperdense vessel sign on CT predicts successful recanalization with the Merci device in acute ischemic stroke. Journal of NeuroInterventional Surgery, 5(4), 289–293.CrossRefGoogle Scholar
  7. 7.
    Vidmar, J., Serša, I., Kralj, E., et al. (2015). Unsuccessful percutaneous mechanical thrombectomy in fibrin-rich high-risk pulmonary thromboembolism. Thrombosis Journal, 13(1), 30.CrossRefGoogle Scholar
  8. 8.
    Silvain, J., Collet, J. P., Nagaswami, C., et al. (2011). Composition of coronary thrombus in acute myocardial infarction. Journal of the American College of Cardiology, 57(12), 1359–1367.CrossRefGoogle Scholar
  9. 9.
    Silvain, J., Collet, J. P., Guedeney, P., et al. (2017). Thrombus composition in sudden cardiac death from acute myocardial infarction. Resuscitation., 113, 108–114.CrossRefGoogle Scholar
  10. 10.
    Simons, N., Mitchell, P., Dowling, R., et al. (2014). Thrombus composition in acute ischemic stroke: a histopathological study of thrombus extracted by endovascular retrieval. Journal of Neuroradiology, 42(2), 86–92.CrossRefGoogle Scholar
  11. 11.
    Maegerlein, C., Friedrich, B., Berndt, M., et al. (2018). Impact of histological thrombus composition on preinterventional thrombus migration in patients with acute occlusions of the middle cerebral artery. Interventional Neuroradiology, 24(1), 70–75.CrossRefGoogle Scholar
  12. 12.
    Autar, A. S. A., Hund, H. M., Ramlal, S. A., et al. (2018). High-resolution imaging of interaction between thrombus and stent-retriever in patients with acute ischemic stroke. Journal of the American Heart Association, 7(13), e008563.CrossRefGoogle Scholar
  13. 13.
    Muszbek, L., Bereczky, Z., Bagoly, Z., et al. (2011). Factor XIII: a coagulation factor with multiple plasmatic and cellular functions. Physiological Reviews, 91(3), 931–972.CrossRefGoogle Scholar
  14. 14.
    Bowley, S. R., & Lord, S. T. (2009). Fibrinogen variant BβD432A has normal polymerization but does not bind knob “B”. Blood., 113(18), 4425–4430.CrossRefGoogle Scholar
  15. 15.
    Muszbek, L., Bereczky, Z., Bagoly, Z., et al. (2010). Factor XIII and atherothrombotic diseases. Seminars in Thrombosis and Hemostasis, 36(1), 18–33.CrossRefGoogle Scholar
  16. 16.
    Tsurupa, G., Mahid, A., Veklich, Y., et al. (2011). Structure, stability, and interaction of fibrin αC-domain polymers. Biochemistry., 50(37), 8028–8037.CrossRefGoogle Scholar
  17. 17.
    Boeckh-Behrens, T., Schubert, M., Förschler, A., et al. (2016). The impact of histological clot composition in embolic stroke. Clinical Neuroradiology, 26(2), 189–197.CrossRefGoogle Scholar
  18. 18.
    Niesten, J. M., van der Schaaf, I. C., van Dam, L., et al. (2014). Histopathologic composition of cerebral thrombi of acute stroke patients is correlated with stroke subtype and thrombus attenuation. PLoS One, 9(2), e88882.CrossRefGoogle Scholar
  19. 19.
    Singh, P., Doostkam, S., Reinhard, M., et al. (2013). Immunohistochemical analysis of thrombi retrieved during treatment of acute ischemic stroke: does stent-retriever cause intimal damage? Stroke., 44(6), 1720–1722.CrossRefGoogle Scholar
  20. 20.
    Sallustio, F., Arnò, N., Di Legge, S., et al. (2015). Histological features of intracranial thrombo-emboli predict response to endovascular therapy for acute ischemic stroke. Journal of Neurological Disorders & Stroke, 3(3), 1105.Google Scholar
  21. 21.
    Marder, V. J., Chute, D. J., Starkman, S., et al. (2006). Analysis of thrombi retrieved from cerebral arteries of patients with acute ischemic stroke. Stroke., 37(8), 2086–2093.CrossRefGoogle Scholar
  22. 22.
    Staessens, S., Denorme, F., François, O., et al. (2019). Structural analysis of ischemic stroke thrombi: histological indications for therapy resistance. Haematologica. [Epub ahead of print].

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© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of General PathologyKazan State Medical UniversityKazanRussian Federation
  2. 2.Department of Cell and Developmental BiologyUniversity of Pennsylvania School of MedicinePhiladelphiaUSA
  3. 3.Institute of Fundamental Medicine and BiologyKazan Federal UniversityKazanRussian Federation

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