International Journal of Legal Medicine

, Volume 123, Issue 3, pp 235–240 | Cite as

Time-dependent appearance of intrathrombus neutrophils and macrophages in a stasis-induced deep vein thrombosis model and its application to thrombus age determination

  • Mizuho Nosaka
  • Yuko Ishida
  • Akihiko Kimura
  • Toshikazu KondoEmail author
Original Article


We immunohistochemically examined neutrophils and macrophages in venous thrombi, which developed in the ligation of the inferior vena cava (IVC). Myeloperoxidase (MPO)-positive neutrophils and F4/80-positive macrophages were detected in the whole course of thrombi after IVC ligation. Morphometrically, the number of neutrophils was greatest at 1 day after IVC ligation and, thereafter, gradually decreased with an increase of the post-ligation interval. In contrast, the number of macrophages peaked at 7 days after ligation. The number of intrathrombus neutrophils was significantly higher than that of intrathrombus macrophages at 1 and 3 days, and the average ratios of neutrophils to macrophages (N/M ratios) were 6.8 ± 1.1 (4.8–9.0) and 2.5 ± 0.4 (1.7–4.2) at 1 and 3 days, respectively. After more than 5 days, all samples had N/M ratios of <2.0 (0.2–1.4). These observations suggest that an N/M ratio of >2.0 indicates a thrombus age of 1–3 days. To differentiate between 1- and 3-day-old thrombi, an N/M ratio markedly exceeding 5.0 strongly indicates an age of 1 day. Furthermore, an N/M ratio of 1.0 or less probably indicates an age of more than 5 days. The present study demonstrated that the immunohistochemical detection of intrathrombus neutrophils and macrophages was suitable to determine the age of venous thrombi.


Forensic pathology Thrombus age determination Immunohistochemistry Neutrophils Macrophages 



We thank Ms. Mariko Kawaguchi for her excellent assistance in the preparation of this manuscript. This study was financially supported in part by a Grant-in-Aid for Scientific Research (A) from the Ministry of Education, Culture, Sports, Science, and Technology of the Japanese Government.


  1. 1.
    Mitchell RN (2005) Hemodynamic disorders, thromboembolic disease, and shock. In: Kumar V, Abbas AK, Fausto N (eds) Robbins and Cotran pathologic basis of disease, 7th edn. Elsevier, Philadelphia, pp 119–144Google Scholar
  2. 2.
    Ro A, Kageyama N, Tanifuji T, Fukunaga T (2008) Pulmonary thromboembolism: overview and update from medicolegal aspects. Leg Med (Tokyo) 10:57–71Google Scholar
  3. 3.
    Betz P (1994) Histological and enzyme histochemical parameters for the age estimation of human skin wounds. Int J Legal Med 107:60–68PubMedCrossRefGoogle Scholar
  4. 4.
    Raekallio J (1972) Determination of the age of wounds by histochemical and biochemical methods. Forensic Sci Int 1:3–16CrossRefGoogle Scholar
  5. 5.
    Kondo T (2007) Timing of skin wounds. Leg Med 9:109–114CrossRefGoogle Scholar
  6. 6.
    Oehmichen M (2004) Vitality and time course of wounds. Forensic Sci Int 144:221–231PubMedCrossRefGoogle Scholar
  7. 7.
    Dreßler J, Bachmann L, Kasper M, Hauck JG, Müller E (1997) Time dependence of the expression of ICAM (CD-54) in human skin wounds. Int J Legal Med 110:299–304PubMedCrossRefGoogle Scholar
  8. 8.
    Dreßler J, Bachmann L, Koch R, Müller E (1998) Enhanced expression of selectins in human skin wounds. Int J Legal Med 112:39–44CrossRefGoogle Scholar
  9. 9.
    Dreßler J, Bachmann L, Koch R, Müller E (1999) Estimation of wound age and VCAM-1 in human skin. Int J Legal Med 112:159–162PubMedCrossRefGoogle Scholar
  10. 10.
    Ishida Y, Kimura A, Takayasu T, Eisenmenger W, Kondo T (2008) Expression of oxygen-regulated protein 150 (ORP150) in skin wound healing and its application for wound age determination. Int J Legal Med 122:409–414PubMedCrossRefGoogle Scholar
  11. 11.
    Takamiya M, Fujita S, Saigusa K, Aoki Y (2008) Simultaneous detection of eight cytokines in human dermal wounds with a multiplex bead-based immunoassay for wound age estimation. Int J Legal Med 122:143–148PubMedCrossRefGoogle Scholar
  12. 12.
    Hayashi T, Ishida Y, Kimura A, Takayasu T, Eisenmenger W, Kondo T (2004) Forensic application of VEGF expression to skin wound age determination. Int J Legal Med 118:320–325PubMedCrossRefGoogle Scholar
  13. 13.
    Irninger W (1963) Histologische Altersbestimmung von Thrombosen und Embolien. Virch Arch Path Anat 336:220–226CrossRefGoogle Scholar
  14. 14.
    Schäfer V (1977) Altersbestimmung von Thrombosen und Embolien unter besonderer Berücksichtigung forensischer Gesichtspunkte. Med Diss HamburgGoogle Scholar
  15. 15.
    Astrup T, Henrichsen J, Kwaan HC (1967) Protease content and fibrinolytic activity of human leukocytes. Blood 29:134–138PubMedGoogle Scholar
  16. 16.
    McGuinness CL, Humphries J, Waltham M, Burnand KG, Collins M, Smith A (2001) Recruitment of labelled monocytes by experimental venous thrombi. Thromb Haemost 85:1018–1024PubMedGoogle Scholar
  17. 17.
    Wakefield TW, Linn MJ, Henke PK, Kadell AM, Wilke CA, Wrobleski SK, Sarkar M, Burdick MD, Myers DD, Strieter RM (1999) Neovascularization during venous thrombosis organization: a preliminary study. J Vasc Surg 30:885–892PubMedCrossRefGoogle Scholar
  18. 18.
    Knighton DR, Fiegel VD (1989) Macrophage-derived growth factors in wound healing: regulation of growth factor production by the oxygen microenvironment. Am Rev Respir Dis 140:1108–1111PubMedGoogle Scholar
  19. 19.
    Knighton DR, Fiegel VD (1989) The macrophages: effector cell wound repair. Prog Clin Biol Res 299:217–226PubMedGoogle Scholar
  20. 20.
    Henke PK, Pearce CG, Moaveni DM, Moore AJ, Lynch EM, Longo C, Varma M, Dewyer NA, Deatrick KB, Upchurch GR Jr, Wakefield TW, Hogaboam C, Kunkel SL (2006) Targeted deletion of CCR2 impairs deep vein thombosis resolution in a mouse model. J Immunol 177:3388–3397PubMedGoogle Scholar
  21. 21.
    Henke PK, Varga A, De S, Deatrick CB, Eliason J, Arenberg DA, Sukheepod P, Thanaporn P, Kunkel SL, Upchurch GR Jr, Wakefield TW (2004) Deep vein thrombosis resolution is modulated by monocyte CXCR2-mediated activity in a mouse model. Arterioscler Thromb Vasc Biol 24:1130–1137PubMedCrossRefGoogle Scholar
  22. 22.
    Varma MR, Varga AJ, Knipp BS, Sukheepod P, Upchurch GR, Kunkel SL, Wakefield TW, Henke PK (2003) Neutropenia impairs venous thrombosis resolution in the rat. J Vasc Surg 38:1090–1098PubMedCrossRefGoogle Scholar
  23. 23.
    Ishida Y, Kondo T, Takayasu T, Iwakura Y, Mukaida N (2004) The essential involvement of cross-talk between IFN-γ and TGF-β in the skin wound-healing process. J Immunol 172:1848–1855PubMedGoogle Scholar
  24. 24.
    Ishida Y, Kondo T, Kimura A, Matsushima K, Mukaida N (2006) Absence of IL-1 receptor antagonist impaired wound healing along with aberrant NF-κB activation and a reciprocal suppression of TGF-β signal pathway. J Immunol 176:5598–5606PubMedGoogle Scholar
  25. 25.
    Kondo T, Ohshima T, Eisenmenger W (1999) Immunohistochemical and morphometrical study on the temporal expression of interleukin-1α (IL-1α) in human skin wounds for forensic wound age determination. Int J Legal Med 112:249–252PubMedCrossRefGoogle Scholar
  26. 26.
    Kondo T, Ohshima T, Mori R, Guan DW, Ohshima K, Eisenmenger W (2002) Immunohistochemical detection of chemokines in human skin wounds and its application to wound age determination. Int J Legal Med 116:87–91PubMedCrossRefGoogle Scholar
  27. 27.
    Ali T, Humphries J, Burnand K, Sawyer B, Bursill C, Channon K, Greaves D, Rollins B, Charo IF, Smith A (2006) Monocyte recruitment in venous thrombus resolution. J Vasc Surg 43:601–608PubMedCrossRefGoogle Scholar
  28. 28.
    Betz P, Nerlich A, Wilske J, Tübel J, Wiest I, Penning R, Eisenmenger W (1992) Immunohistochemical localization of fibronectin as a tool for the age determination of human skin wounds. Int J Legal Med 105:21–26PubMedCrossRefGoogle Scholar
  29. 29.
    Betz P, Nerlich A, Wilske J, Tübel J, Penning R, Eisenmenger W (1993) Analysis of the immunohistochemical localization of collagen type III and V for the time-estimation of human skin wounds. Int J Legal Med 105:329–332PubMedCrossRefGoogle Scholar
  30. 30.
    Betz P, Nerlich A, Wilske J, Tübel J, Penning R, Eisenmenger W (1993) Immunohistochemical localization collagen types I and VI in human skin wounds. Int J Legal Med 106:31–34PubMedCrossRefGoogle Scholar
  31. 31.
    Betz P, Nerlich A, Tübel J, Wiest I, Hausmann R (1997) Detection of cell death in human skin wounds of various ages by an in situ end labeling of nuclear DNA fragments. Int J Legal Med 110:240–243PubMedCrossRefGoogle Scholar
  32. 32.
    Hausmann R, Betz P (2001) Course of glial immunoreactivity for vimentin, tenascin and alpha1-antichymotrypsin after traumatic injury to human brain. Int J Legal Med 114:338–342PubMedCrossRefGoogle Scholar
  33. 33.
    Hausmann R, Betz P (2000) The time course of the vascular response to human brain injury–an immunohistochemical study. Int J Legal Med 113:288–292PubMedCrossRefGoogle Scholar
  34. 34.
    Hausmann R, Riess R, Fieguth A, Betz P (2000) Immunohistochemical investigations on the course of astroglial GFAP expression following human brain injury. Int J Legal Med 113:70–75PubMedCrossRefGoogle Scholar
  35. 35.
    Hausmann R, Kaiser A, Lang C, Bohnert M, Betz P (1999) A quantitative immunohistochemical study on the time-dependent course of acute inflammatory cellular response to human brain injury. Int J Legal Med 112:227–232PubMedCrossRefGoogle Scholar
  36. 36.
    Dreßler J, Hanisch U, Kuhlisch E, Geiger KD (2007) Neuronal and glial apoptosis in human traumatic brain injury. Int J Legal Med 121:365–375PubMedCrossRefGoogle Scholar
  37. 37.
    Hausmann R, Seidl S, Betz P (2006) Hypoxic changes in Purkinje cells of the human cerebellum. Int J Legal Med 12:175–183Google Scholar
  38. 38.
    Hausmann R, Biermann T, Wiest I, Tübel J, Betz P (2004) Neuronal apoptosis following human brain injury. Int J Legal Med 118:32–36PubMedCrossRefGoogle Scholar
  39. 39.
    Oehmichen M, Walter T, Meissner C, Friedrich HJ (2003) Time course of cortical hemorrhages after closed traumatic brain injury: statistical analysis of posttraumatic histomorphological alterations. J Neurotrauma 20:87–103PubMedCrossRefGoogle Scholar
  40. 40.
    Hayashi T, Ishida Y, Mizunuma S, Kimura A, Kondo T (2009) Differential diagnosis between freshwater drowning and saltwater drowning based on intrapulmonary aquaporin-5 expression. Int J Legal Med 123:7–13PubMedCrossRefGoogle Scholar
  41. 41.
    Tsokos M, Schalinski S, Paulsen F, Sperhake JP, Püschel K, Sobottka I (2008) Pathology of fatal traumatic and nontraumatic clostridial gas gangrene: a histopathological, immunohistochemical, and ultrastructural study of six autopsy cases. Int J Legal Med 122:35–41PubMedCrossRefGoogle Scholar
  42. 42.
    Ishikawa T, Zhu BL, Miyaishi S, Ishizu H, Maeda H (2007) Increase in clusterin-containing follicles in the adenohypophysis of drug abusers. Int J Legal Med 121:395–402PubMedCrossRefGoogle Scholar
  43. 43.
    Kitamura O, Tokunaga I, Gotohda T, Kubo S (2007) Immunohistochemical investigation of dopaminergic terminal markers and caspase-3 activation in the striatum of human methamphetamine users. Int J Legal Med 121:163–168PubMedCrossRefGoogle Scholar
  44. 44.
    Bacci S, Romagnoli P, Norelli GA, Forestieri AL, Bonelli A (2006) Early increase in TNF-alpha-containing mast cells in skin lesions. Int J Legal Med 120:138–142PubMedCrossRefGoogle Scholar
  45. 45.
    Marschall S, Rothschild MA, Bohnert M (2006) Expression of heat-shock protein 70 (Hsp70) in the respiratory tract and lungs of fire victims. Int J Legal Med 120:355–359PubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2009

Authors and Affiliations

  • Mizuho Nosaka
    • 1
  • Yuko Ishida
    • 1
  • Akihiko Kimura
    • 1
  • Toshikazu Kondo
    • 1
    Email author
  1. 1.Department of Forensic MedicineWakayama Medical UniversityWakayamaJapan

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