Histochemistry 57, 97- 105 (1978)
Histochemistry 9 by Springer-Verlag 1978
lmmunoenzymehistochemical Detection of Fibrin Microthrombi During Disseminated Intravascular Coagulation in Rats H. Craane 1, J.J. Emeis 2, J. Lindeman 1, and W. Nieuwenhuizen 2 1Department of Pathology, University Medical Centre, Wassenaarseweg 62, Leiden, The Netherlands 2Gaubius Institute, Health Research Organization TNO, Herenstraat 5d, Leiden, The Netherlands
Summary. In tissue of rats with disseminated intravascular coagulation, fibrin microthrombi can be sensitively detected by immunohistochemical methods, using antisera against rat fibrinogen or fibrin monomer. An indirect immunoperoxidase procedure on paraplast-embedded sections yields best results with regard to the morphology of the thrombi and their localization in the tissuel Only fibrillar immunoreactive material, oriented lengthwise in the vessels, should be regarded as microthrombi formed in vivo.
Introduction Disseminated intravascular coagulation (DIC) is a syndrome occurring in many different diseases of diverse etiology (for review see Mtiller-Berghaus, 1977). Morphologically, DIC is characterized by the occurrence of fibrin microthrombi in various organs not affected by primary pathological processes. According to Robboy et al. (1972) the "finding of even a single fibrin thrombus is presumptive evidence" of DIC. Therefore, in order to detect DIC in either autopsy or biopsy material, a sensitive, specific, and reliable method for the detection of fibrin microthrombi is required. In a previous study (Emeis and Lindeman, 1976) those histological and histochemical procedures in common use were found insensitive, as well as revealing only a small portion of the fibrin microthrombi actually present. Since immunohistochemical procedures are both more specific and sensitive, we developed an immunoenzymehistochemical fibrin staining procedure applicable to paraplast-embedded tissue. The method was evaluated on tissue of rats with experimentally-induced DIC. Besides, criteria for the morphological identification of fibrin microthrombi formed in vivo were formulated.
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Materials and Methods Male Wistar rats, weighing 200-220 g, were anaesthetized by injecting Nembutal | i.p. (6 rag/100 g), 30 rain prior to experimentation. Rat fibrinogen was purified as described elsewhere (van Ruijven-Vermeer and Nieuwenhuizen, 1978). Rat fibrin monomer was prepared from pure rat fibrinogen as described (Haverkate and Timan, 1976), and stored in 0.02 N acetic acid at - 2 0 ~ C. Prior to injecting, this rat fibrin monomer solution was diluted with an equal volume of 6 M urea in 0.05 M Tris (pH = 10.4), and subsequently diluted to 5 mg/ml with 3 M urea in 0.05 M Tris: HC1 buffer, p H = 7 . 4 (Mtiller-Berghaus et al., i976). Bovine thrombin (Leo Pharmaceuticals, Copenhagen, Denmark) was dissolved in 0.15 M NaC1 (final concentration 100 NIH units/ml). Antisera against rat fibrinogen and rat fibrin monomer were raised in rabbits as described (van Ruijven-Vermeer and Nieuwenhuizen, 1978). The specificities of obtained antisera were assessed by both double immunodiffusion (Ouchterlony, 1968) and immunoelectrophoresis (Grabar, i964) against rat plasma, serum and purified fibrinogen. The IgG-fraction of said antisera was isolated and conjugated with fluorescein isothiocyanate (FITC) as described for FITC-protein A conjugates (Nieuwenhuizen et al., 1977). Peroxidase-conjugated goat-anti-rabbit lgG was obtained from Nordic Immunological Laboratories (Tilburg, The Netherlands). Rat IgG was obtained from Miles Laboratories (Elkhart, Ind. USA). Methods. DIC was induced by rapidly injecting either bovine thrombin (15 NIH units/100 g), or rat fibrin monomer (1 mg/100 g) into the femoral vein. Inhibition of fibrinolysis was induced by the i.v. injection ofTrasylol | (Bayer, Leverkusen, W. Germany; 1500 KIE/100 g). Rats were anticoagulated by the i.v. injection of heparin (Leo Pharmaceuticals; 25 U/100 g). Pieces of liver, lung, kidney, spleen and small intestine were obtained 10, 20, 30, 40 and 60 rain after the induction of DIC. As described above, I rain before autopsy rats were injected with Trasylol and heparin in order to prevent post-mortem clotting and fibrinolysis. Occasionally,see R e s u l t s - p r i o r to autopsy liver and kidney were perfused with phosphate-buffered saline (PBS; pH=7.4), either through the aorta (20 ml/min) or the portal vein (10 ml/min). Parts of mentioned organ specimens were rapidly fiozen in isopentane ( - 7 0 ~ C) and stored in liquid nitrogen. Other parts were fixed for 18-96 h in Bouin; 96% ethanol; 4% p-formaldehyde (freshly prepared) in PBS or in 0.I M phosphate buffer, p H = 7 . 4 ; or 2% p-formaldehyde plus 0.5 or 1.0% glutaraldehyde in 0.1 M phosphate buffer. Fixed specimens were routinely embedded in paraplast (maximum temperature 57 ~ C). For immunoenzymehistochemistry 4 gm frozen or paraplast-embedded sections were used. The latter were dried overnight in air at 37 ~ C, thereupon deparaffinized in xylene (2 • 30 rain), rehydrated in a graded alcohol series and transferred to PBS (Taylor and Burns, 1974). Endogenous peroxidatic activity was blocked by 0.3% H2Oa in methanol for 30 min (Streefkerk, 1972); then the sections were treated for 10 rain with normal goat serum diluted 1:8 in PBS. Subsequently, said sections were incubated for 30 min with 1 : 100 to i : 300 dilutions of antisera, washed in PBS (4 x 5 rain), for 30 min incubated in a 1 : 50 dilution of peroxidase-conjugated goatanti-rabbit IgG, and then washed in PBS (4 x 5 rain). Tissue-bound peroxidase was visualized, for 5 min, at room temperature in the dark, with 0.05% 3,3"-diaminobenzidine. 4 HCI (DAB) and 0.002% H202 according to Graham and Karnovsky (1966). Sections were lightly counterstained with Mayer's haematoxylin. For purposes of immunofluorescence, 4 gm frozen sections were fixed in aceton ( - 2 0 ~ C) or 96% ethanol (4 ~ C), and incubated for 30 min with the FITC-conjugated IgG-fractions diluted 1:20 in PBS, and then washed in PBS (4 x 5 rain). For controls, non-immune rabbit serum, unrelated antisera and antisera fully absorbed with antigen were used in the first step; besides, unconjugated goat-anti-rabbit IgG serum was applied in excess prior to the conjugated antiserum in the second step. DAB without HzO z was used as a staining control; sections not treated with antisera were incubated with DAB and HaO 2 in order to detect any remaining endogenous peroxidatic activity. For comparison, 2 gm paraplast-embedded sections were stained with histological methods for the detection of fibrin (see Emeis and Lindeman 1976).
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Results
Antisera The antisera against rat fibrinogen and rat fibrin monomer generally showed a single strong precipitation line against rat plasma and fibrinogen, and no line against serum. Occasionally, antiserum against fibrinogen showed a weak second line, which could be absorbed with rat serum or rat IgG. Both antisera showed a strong cross-reactivity with human and bovine fibrinogen (see van Ruijven-Vermeer and Nieuwenhuizen, 1978)
Tissue Preparation For the evaluation of fixation and embedding effects, we used kidney, liver, and lung from Trasylol-pretreated rats with thrombin-induced DIC, as in these rats large amounts of fibrin are found (Emeis and Lindeman, 1976). Frozen sections and paraplast-embedded sections were compared regarding intensity of immunoreactivity, morphological detail of microthrombi obtainable, possible accurate localization of thrombi, and general tissue morphology. Because of a somewhat diffuser staining of the fibrin and a higher non-specific background staining, which impeded a precise localization and characterization of the thrombi, results in the frozen sections were inferior to those in paraplastembedded sections. Aldehyde fixatives reduced the immunoreactivity of the fibrin, but both the morphology of the thrombi and the general tissue morphology were superior to those in unfixed or ethanol-fixed tissue. Of note is the resistance of fibrin antigenicity to paraformaldehyde-glutaraldehyde fixation. In subsequent investigations both ethanol and 4% p-formaldehyde were used as fixatives.
Fluorescence vs Enzyme Procedures Comparison of both procedures showed the methods used to be equally sensitive (Figs. 1, 2). However, the immunoenzyme procedure gave a crisp staining of fibrin strands (Figs 3, 4) as opposed to a somewhat diffuser staining in immunofluorescence. Besides, said immunoenzyme method simplified the localization of fibrin in tissue, the more so as the tissue structure and immunoreactive material could be simultaneously observed (compare Figs l and 2). Background staining of the immunoenzyme-stained tissues was negligible, apart from some non-specific staining of connective tissue. Nuclei were unstained.
General Pattern of Immunoreactivity As the antisera used will not discriminate between fibrinogen, fibrin monomer, fibrin polymer and fibrin degradation products, the term fibrin(ogen)-related
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Figs. 1 and 2. Fibrin microthrombi in the liver of a rat with disseminated intravascular coagulation, 10 rain after injecting thrombin Fig. 1. PBS perfusion; frozen section; aceton fixation; direct immunofluorescence method. Magnification x 300 Fig. 2. PBS perfusion; formaldehyde fixation; paraplast embedding; indirect immunoperoxidase method. Magnification x 300
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Figs. 3 and 4. Fibritl microthrombi in liver (Fig. 3) and kidney (Fig. 4) of a Trasylol-pretreated rat with thrombin-induced DIC. At higher magnification the fibrillar, oriented nature of the fibrin stratxds is clearly visible. PBS perfusion; formaldehyde-glutaraldehyde fixation; paraplast embedding; indirect immunoperoxidase method. Magnifications: Fig. 3 x 600; Fig. 4 • 300
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Fig. 5. Granular (arrow) and rim-like (arrowhead) FRM in a glomerulus of a non-perfused, ethanolfixed kidney. From a rat with fibrin monomer-induced DIC. Paraplast embedding; indirect immunoperoxidase method. Magnification x 300 Fig. 6. Lung from a rat with DIC, 10 rain after injecting fibrin monomer. Formaldehyde fixation; paraplast embedding; indirect in immunoperoxidasemethod. Magnification • 200 material (FRM) will be used for any material in tissue that can be demonstrated by said antisera. We found, in rats with DIC, three morphologically distinct types of F R M : fibrillar material, often directed lengthwise in the vessels (Figs 3, 4); a finelygranular material, mainly found in larger vessels; and occasionally a thin rim or diffuse band of F R M , present on or near the endothelium of mostly liver sinusoids and glomerular capillaries (Fig. 5). Both finely-granular and rim-like F R M was more evident in monomerinduced D I C than in thrombin-induced DIC, and again more evident if preceded by ethanol fixation than following aldehyde fixation (e.g. Fig. 5). In those kidneys and livers which, prior to fixation and in order to remove blood components, were perfused with PBS, no finely-granular and rim-like F R M could be found, whereas oriented strands of F R M remained present (Figs. 3, 4). As will be discussed below, these strands are designated as fibrin microthrombi.
Trasylol and Heparin Pretreatment Pretreatment of rats with Trasylol for the purpose of inhibiting fibrinolysis did not influence the morphology of microthrombi.
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Heparinisation prevented thrombin-induced fibrin formation, but had no effect on the morphology or distribution of fibrin monomer-induced microthrombi.
Distribution of Fibrin Microthrombi At 10 min after induction of thrombin- or fibrin monomer-induced DIC, both in liver and lung large amounts of fibrin were found (Figs. 1 6). In the lung, microthrombi showed no preferential localization, whereas in the liver, microthrombi were found mainly periportally on or near Kupffer cells. No fibrin was found near the central vein of the liver lobules. In the kidney, microthrombi were always found in peritubular capillaries and in the vasa recta. In glomerular capillaries, microthrombi were found mainly during monomer-induced DIC, and only very occasionally after thrombin injection. Trasylol treatment of rats prior to thrombin injection, however, resulted in the presence of microthrombi in glomerular capillaries, in number comparable to monomerinduced microthrombi (Fig. 4). In other organs only minor amounts of fibrin were detected. Thirty minutes after induction of DIC, fibrin was found but occasionally (and mainly in the lung) ; after forty minutes all microthrombi had disappeared. After 30 and 60 min the amount and tissue distribution of fibrin in rats pretreated with Trasylol were comparable to those found after 10 min.
Imrnunohistochemical versus Histological Procedures Compared to immunohistochemically-stained tissue, in tissue stained with histochemical or histological procedures much less fibrin could be demonstrated.
Discussion
For the detection of fibrin microthrombi in autopsy or biopsy material a method is required which is both sensitive and specific and will allow a precise localization of microthrombi in the tissue. The histological and histochemical staining procedures available (Beneke, 1971) proved too insensitive (Emeis and Lindeman, 1976) and of doubtful specificity (Davidson et al., 1973; MacIver, 1972; Nordstoga, 1977). Electron microscopy (e.g. Margaretten et al., 1969; Emeis and Lindeman, 1976) allows the detection and accurate localization of very small amounts of fibrin, but is very time consuming, while only small samples of tissue can be studied. The immunoenzymehistochemical method outlined above meets all requirements for the detection of fibrin microthrombi: specificity, sensitivity and accurate localization of microthrombi in tissues. It has the added advantage that large areas of tissue can be scanned rapidly. Since antisera against rat fibrinogen
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or fibrin m o n o m e r cross-react with h u m a n fibrinogen, the method proved to be applicable to paraplast-embedded human autopsy material as well (ms in prep). The use of an animal model allowed differentiation between the morphological varieties of F R M found during DIC. As, after perfusion, only fibrillar F R M was found, the granular staining found in larger vessels and the rim-like staining in capillaries and sinusoids might be due to an artefactual precipitation of circulating fibrinogen and/or fibrin m o n o m e r during fixation. As rim-like staining is most evident in monomer-induced D I C (especially after ethanol fixation) this staining might be due to the precipitation of fibrin monomer, in agreement with Bleyl's observation that ethanol makes an excellent fixative for fibrin m o n o m e r (Bleyl 1977; Bleyl et al., 1969; compare Godal and Abildgaard, 1966). Thus, only fibrillar strands of F R M are thought to represent in vivo-formed fibrin microthrombi, whereas the diffusely-granular and perivascular F R M are postfixation artefacts. The lengthwise intravascular orientation of these strands is another argument again for the in vivo-formation of these microthrombi (Boyd, 1965). The diffuse pericapillary and perisinusoidal staining as observed by us in non-perfused tissue, and described by others in normal and diseased human liver tissue (e.g. Arias and Mancilla-Jimenez, 1970; Orfila et al., 1976), should not be considered as proof of DIC, but at the most suggests increased levels of fibrin m o n o m e r in the circulation. The organ distribution and tissue localization of fibrin microthrombi as found during D I C induced by thrombin or fibrin monomers, are practically identical. Of note is t h a t - unless fibrinolysis is inhibited by Trasylol - g l o m e r u l a r microthrombi are found in monomer-induced D I C only. The reason for this discrepancy is unclear. The very rapid disappearance (within 30 40 min) of fibrin microthrombi from the circulation is remarkable. Therefore, as far as rats are concerned, one should be cautious not to rely too much on histological observations for the diagnosis of DIC. In conclusion: we demonstrated that fibrin microthrombi can be reliably, sensitively and specifically detected by immunoenzymehistochemistry on paraplast-embedded tissue sections, whereas fibrillar, length-wise oriented F R M only should be considered as fibrin microthrombi formed in vivo.
Acknowledgements. The authors are indebted to Mrs. C.M. van Sabben for excellent assistance. References
Arias, F., Mancilla-Jimenez, R.: Hepatic fibrinogen deposits in pre-eclampsia. N. Engl. J. Med. 295, 578 582 (1976) Beneke, G. : Demonstration of plasma proteins in microscopicsections with emphasis on the identification of fibrin. In: Thrombosis and bleeding disorders (ed. N.U. Bang, F.K. Beller, E. Deutsch, E.F. Mammen), p. 524-533. Stuttgart: Georg Thieme 1971 Bleyl, U. : Morphologic diagnosis of disseminated intravascular coagulation : histologic, histochemical and electronmicroscopicstudies. Semin.Thromb. Hemostasis 3, 247 267 (1977)
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Bleyl, U., Sebening, H., Kuhn, W.: Morphologischer Nachweis yon Fibrinomeren im histologischen Schnitt. Thromb. Diath. Haemorrh. 22, 68-86 (1969) Boyd, J.F. : Disseminated fibrin thrombo-embolism among stillbirths and neonatal deaths. J. Pathol. Bact. 90, 53-63 (1965) Davidson, A.M., Thomson, D., Macdonald, M.K., Rae, J.K., Uttley, W.S., Clarkson, A.R. : Identification of intrarenal fibrin deposition. J. clin. Pathol. 26, 102-112 (1973) Emeis, J.J., Lindeman, J.: Rat liver macrophages will not phagocytose fibrin during disseminated intravascular coagulation. Haemostasis 5, 193 210 (1976) Godal, H.C., Abildgaard, U.: Gelation of soluble fibrin in plasma by ethanol. Scand. J. Haemat. 3, 342-350 (1966) Grabar, P.: The immuno-electrophoretic method of analysis. In: Immunoelectrophoretic analysis (ed. P. Grabar, P. Burtin), p. 3-29. Amsterdam: Elsevier 1964 Graham, R.C., Karnovsky, M.J. : The early stages of absorbtion of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302 (1966) Haverkate, F., Timan, G.: Preparation of highly purified bovine fibrin plates. In: Progress in chemical fibrinolysis and thrombolysis (ed. J.F. Davidson, M.M. Samama, P.C. Desnoyers), p. 67 71. New York: Raven Press t976 MacIver, A.G.: A comparison of tinctorial and immunohistological methods for the detection of fibrinoid change and fibrin deposition in the kidney. Histochem. J. 4, 169-176 (1972) Margaretten, W., Csavossy, I , McKay, D.G.: An electron microscopic study of thrombin-induced disseminated intravascular coagulation. Blood 29, 269-275 (1967) Mueller-Berghaus, G.: Pathophysiology of generalized intravascular coagulation. Semin. Thromb. and Hemostasis 3, 209 246 (1977) Mueller-Berghaus, G., Mahn, I., K6veker, G., Maul, F.D. : In vivo behavior of homologous ureasoluble 131I-fibrin and 12sI-fibrinogen in rabbits. Brit. J. Haematol. 33, 61-79 (1976) Nieuwenhuizen0 W., Emeis, J.J., Sabben, C.M. van: Localization of lipase-like immunoreactivity in porcine adipose, aortic and myocardial tissue. Atherosclerosis 27, 97-106 (1977) Nordstoga, K. : Disseminated intravascular coagulation: a pathologist's view. Thromb. Haemostas. 37, 180 (1977) Orfila, C., Duffaut, M., Mignon-Conte, M., Rumeau, J.-L, Bugat, R., Suc J.-M: Localisation en immunofluorescence directe dans le tissu h6patique de I'AgHBs, des immunoglobulines, de la fibrine et de la fraction C3 du compl~ment. Pathol. Biol. 24, 313-318 (1976) Ouchterlony O. : Handbook of immunodiffusion and immunoelectrophoresis, p. 25. London: Ann Arbor-Humphrey Science 1968 Robboy S.J., Colman R.W., Minna J.D.: Pathology of disseminated intravascular coagulation. Human Pathol. 3, 327-343 (1972) Ruijven-Vermeer, I.A.M, van, Nieuwenhuizen, W. : Purification of rat fibrinogen and its constituent chains. Biochem. J. 169, 653-658 (1978) Streefkerk, J.G.: Inhibition of erythrocyte pseudoperoxidase activity by treatment with hydrogen peroxide following methanol. J. Histochem. Cytochem. 20, 829-831 (1972) Taylor, C.R., Burns, J. : The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase-labelled antibody. J. clin. Pathol. 27, 14 20 (1974)
Received April 22, 1978
Histochemistry 9 by Springer-Verlag 1978
lmmunoenzymehistochemical Detection of Fibrin Microthrombi During Disseminated Intravascular Coagulation in Rats H. Craane 1, J.J. Emeis 2, J. Lindeman 1, and W. Nieuwenhuizen 2 1Department of Pathology, University Medical Centre, Wassenaarseweg 62, Leiden, The Netherlands 2Gaubius Institute, Health Research Organization TNO, Herenstraat 5d, Leiden, The Netherlands
Summary. In tissue of rats with disseminated intravascular coagulation, fibrin microthrombi can be sensitively detected by immunohistochemical methods, using antisera against rat fibrinogen or fibrin monomer. An indirect immunoperoxidase procedure on paraplast-embedded sections yields best results with regard to the morphology of the thrombi and their localization in the tissuel Only fibrillar immunoreactive material, oriented lengthwise in the vessels, should be regarded as microthrombi formed in vivo.
Introduction Disseminated intravascular coagulation (DIC) is a syndrome occurring in many different diseases of diverse etiology (for review see Mtiller-Berghaus, 1977). Morphologically, DIC is characterized by the occurrence of fibrin microthrombi in various organs not affected by primary pathological processes. According to Robboy et al. (1972) the "finding of even a single fibrin thrombus is presumptive evidence" of DIC. Therefore, in order to detect DIC in either autopsy or biopsy material, a sensitive, specific, and reliable method for the detection of fibrin microthrombi is required. In a previous study (Emeis and Lindeman, 1976) those histological and histochemical procedures in common use were found insensitive, as well as revealing only a small portion of the fibrin microthrombi actually present. Since immunohistochemical procedures are both more specific and sensitive, we developed an immunoenzymehistochemical fibrin staining procedure applicable to paraplast-embedded tissue. The method was evaluated on tissue of rats with experimentally-induced DIC. Besides, criteria for the morphological identification of fibrin microthrombi formed in vivo were formulated.
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Materials and Methods Male Wistar rats, weighing 200-220 g, were anaesthetized by injecting Nembutal | i.p. (6 rag/100 g), 30 rain prior to experimentation. Rat fibrinogen was purified as described elsewhere (van Ruijven-Vermeer and Nieuwenhuizen, 1978). Rat fibrin monomer was prepared from pure rat fibrinogen as described (Haverkate and Timan, 1976), and stored in 0.02 N acetic acid at - 2 0 ~ C. Prior to injecting, this rat fibrin monomer solution was diluted with an equal volume of 6 M urea in 0.05 M Tris (pH = 10.4), and subsequently diluted to 5 mg/ml with 3 M urea in 0.05 M Tris: HC1 buffer, p H = 7 . 4 (Mtiller-Berghaus et al., i976). Bovine thrombin (Leo Pharmaceuticals, Copenhagen, Denmark) was dissolved in 0.15 M NaC1 (final concentration 100 NIH units/ml). Antisera against rat fibrinogen and rat fibrin monomer were raised in rabbits as described (van Ruijven-Vermeer and Nieuwenhuizen, 1978). The specificities of obtained antisera were assessed by both double immunodiffusion (Ouchterlony, 1968) and immunoelectrophoresis (Grabar, i964) against rat plasma, serum and purified fibrinogen. The IgG-fraction of said antisera was isolated and conjugated with fluorescein isothiocyanate (FITC) as described for FITC-protein A conjugates (Nieuwenhuizen et al., 1977). Peroxidase-conjugated goat-anti-rabbit lgG was obtained from Nordic Immunological Laboratories (Tilburg, The Netherlands). Rat IgG was obtained from Miles Laboratories (Elkhart, Ind. USA). Methods. DIC was induced by rapidly injecting either bovine thrombin (15 NIH units/100 g), or rat fibrin monomer (1 mg/100 g) into the femoral vein. Inhibition of fibrinolysis was induced by the i.v. injection ofTrasylol | (Bayer, Leverkusen, W. Germany; 1500 KIE/100 g). Rats were anticoagulated by the i.v. injection of heparin (Leo Pharmaceuticals; 25 U/100 g). Pieces of liver, lung, kidney, spleen and small intestine were obtained 10, 20, 30, 40 and 60 rain after the induction of DIC. As described above, I rain before autopsy rats were injected with Trasylol and heparin in order to prevent post-mortem clotting and fibrinolysis. Occasionally,see R e s u l t s - p r i o r to autopsy liver and kidney were perfused with phosphate-buffered saline (PBS; pH=7.4), either through the aorta (20 ml/min) or the portal vein (10 ml/min). Parts of mentioned organ specimens were rapidly fiozen in isopentane ( - 7 0 ~ C) and stored in liquid nitrogen. Other parts were fixed for 18-96 h in Bouin; 96% ethanol; 4% p-formaldehyde (freshly prepared) in PBS or in 0.I M phosphate buffer, p H = 7 . 4 ; or 2% p-formaldehyde plus 0.5 or 1.0% glutaraldehyde in 0.1 M phosphate buffer. Fixed specimens were routinely embedded in paraplast (maximum temperature 57 ~ C). For immunoenzymehistochemistry 4 gm frozen or paraplast-embedded sections were used. The latter were dried overnight in air at 37 ~ C, thereupon deparaffinized in xylene (2 • 30 rain), rehydrated in a graded alcohol series and transferred to PBS (Taylor and Burns, 1974). Endogenous peroxidatic activity was blocked by 0.3% H2Oa in methanol for 30 min (Streefkerk, 1972); then the sections were treated for 10 rain with normal goat serum diluted 1:8 in PBS. Subsequently, said sections were incubated for 30 min with 1 : 100 to i : 300 dilutions of antisera, washed in PBS (4 x 5 rain), for 30 min incubated in a 1 : 50 dilution of peroxidase-conjugated goatanti-rabbit IgG, and then washed in PBS (4 x 5 rain). Tissue-bound peroxidase was visualized, for 5 min, at room temperature in the dark, with 0.05% 3,3"-diaminobenzidine. 4 HCI (DAB) and 0.002% H202 according to Graham and Karnovsky (1966). Sections were lightly counterstained with Mayer's haematoxylin. For purposes of immunofluorescence, 4 gm frozen sections were fixed in aceton ( - 2 0 ~ C) or 96% ethanol (4 ~ C), and incubated for 30 min with the FITC-conjugated IgG-fractions diluted 1:20 in PBS, and then washed in PBS (4 x 5 rain). For controls, non-immune rabbit serum, unrelated antisera and antisera fully absorbed with antigen were used in the first step; besides, unconjugated goat-anti-rabbit IgG serum was applied in excess prior to the conjugated antiserum in the second step. DAB without HzO z was used as a staining control; sections not treated with antisera were incubated with DAB and HaO 2 in order to detect any remaining endogenous peroxidatic activity. For comparison, 2 gm paraplast-embedded sections were stained with histological methods for the detection of fibrin (see Emeis and Lindeman 1976).
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Results
Antisera The antisera against rat fibrinogen and rat fibrin monomer generally showed a single strong precipitation line against rat plasma and fibrinogen, and no line against serum. Occasionally, antiserum against fibrinogen showed a weak second line, which could be absorbed with rat serum or rat IgG. Both antisera showed a strong cross-reactivity with human and bovine fibrinogen (see van Ruijven-Vermeer and Nieuwenhuizen, 1978)
Tissue Preparation For the evaluation of fixation and embedding effects, we used kidney, liver, and lung from Trasylol-pretreated rats with thrombin-induced DIC, as in these rats large amounts of fibrin are found (Emeis and Lindeman, 1976). Frozen sections and paraplast-embedded sections were compared regarding intensity of immunoreactivity, morphological detail of microthrombi obtainable, possible accurate localization of thrombi, and general tissue morphology. Because of a somewhat diffuser staining of the fibrin and a higher non-specific background staining, which impeded a precise localization and characterization of the thrombi, results in the frozen sections were inferior to those in paraplastembedded sections. Aldehyde fixatives reduced the immunoreactivity of the fibrin, but both the morphology of the thrombi and the general tissue morphology were superior to those in unfixed or ethanol-fixed tissue. Of note is the resistance of fibrin antigenicity to paraformaldehyde-glutaraldehyde fixation. In subsequent investigations both ethanol and 4% p-formaldehyde were used as fixatives.
Fluorescence vs Enzyme Procedures Comparison of both procedures showed the methods used to be equally sensitive (Figs. 1, 2). However, the immunoenzyme procedure gave a crisp staining of fibrin strands (Figs 3, 4) as opposed to a somewhat diffuser staining in immunofluorescence. Besides, said immunoenzyme method simplified the localization of fibrin in tissue, the more so as the tissue structure and immunoreactive material could be simultaneously observed (compare Figs l and 2). Background staining of the immunoenzyme-stained tissues was negligible, apart from some non-specific staining of connective tissue. Nuclei were unstained.
General Pattern of Immunoreactivity As the antisera used will not discriminate between fibrinogen, fibrin monomer, fibrin polymer and fibrin degradation products, the term fibrin(ogen)-related
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Figs. 1 and 2. Fibrin microthrombi in the liver of a rat with disseminated intravascular coagulation, 10 rain after injecting thrombin Fig. 1. PBS perfusion; frozen section; aceton fixation; direct immunofluorescence method. Magnification x 300 Fig. 2. PBS perfusion; formaldehyde fixation; paraplast embedding; indirect immunoperoxidase method. Magnification x 300
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Figs. 3 and 4. Fibritl microthrombi in liver (Fig. 3) and kidney (Fig. 4) of a Trasylol-pretreated rat with thrombin-induced DIC. At higher magnification the fibrillar, oriented nature of the fibrin stratxds is clearly visible. PBS perfusion; formaldehyde-glutaraldehyde fixation; paraplast embedding; indirect immunoperoxidase method. Magnifications: Fig. 3 x 600; Fig. 4 • 300
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Fig. 5. Granular (arrow) and rim-like (arrowhead) FRM in a glomerulus of a non-perfused, ethanolfixed kidney. From a rat with fibrin monomer-induced DIC. Paraplast embedding; indirect immunoperoxidase method. Magnification x 300 Fig. 6. Lung from a rat with DIC, 10 rain after injecting fibrin monomer. Formaldehyde fixation; paraplast embedding; indirect in immunoperoxidasemethod. Magnification • 200 material (FRM) will be used for any material in tissue that can be demonstrated by said antisera. We found, in rats with DIC, three morphologically distinct types of F R M : fibrillar material, often directed lengthwise in the vessels (Figs 3, 4); a finelygranular material, mainly found in larger vessels; and occasionally a thin rim or diffuse band of F R M , present on or near the endothelium of mostly liver sinusoids and glomerular capillaries (Fig. 5). Both finely-granular and rim-like F R M was more evident in monomerinduced D I C than in thrombin-induced DIC, and again more evident if preceded by ethanol fixation than following aldehyde fixation (e.g. Fig. 5). In those kidneys and livers which, prior to fixation and in order to remove blood components, were perfused with PBS, no finely-granular and rim-like F R M could be found, whereas oriented strands of F R M remained present (Figs. 3, 4). As will be discussed below, these strands are designated as fibrin microthrombi.
Trasylol and Heparin Pretreatment Pretreatment of rats with Trasylol for the purpose of inhibiting fibrinolysis did not influence the morphology of microthrombi.
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Heparinisation prevented thrombin-induced fibrin formation, but had no effect on the morphology or distribution of fibrin monomer-induced microthrombi.
Distribution of Fibrin Microthrombi At 10 min after induction of thrombin- or fibrin monomer-induced DIC, both in liver and lung large amounts of fibrin were found (Figs. 1 6). In the lung, microthrombi showed no preferential localization, whereas in the liver, microthrombi were found mainly periportally on or near Kupffer cells. No fibrin was found near the central vein of the liver lobules. In the kidney, microthrombi were always found in peritubular capillaries and in the vasa recta. In glomerular capillaries, microthrombi were found mainly during monomer-induced DIC, and only very occasionally after thrombin injection. Trasylol treatment of rats prior to thrombin injection, however, resulted in the presence of microthrombi in glomerular capillaries, in number comparable to monomerinduced microthrombi (Fig. 4). In other organs only minor amounts of fibrin were detected. Thirty minutes after induction of DIC, fibrin was found but occasionally (and mainly in the lung) ; after forty minutes all microthrombi had disappeared. After 30 and 60 min the amount and tissue distribution of fibrin in rats pretreated with Trasylol were comparable to those found after 10 min.
Imrnunohistochemical versus Histological Procedures Compared to immunohistochemically-stained tissue, in tissue stained with histochemical or histological procedures much less fibrin could be demonstrated.
Discussion
For the detection of fibrin microthrombi in autopsy or biopsy material a method is required which is both sensitive and specific and will allow a precise localization of microthrombi in the tissue. The histological and histochemical staining procedures available (Beneke, 1971) proved too insensitive (Emeis and Lindeman, 1976) and of doubtful specificity (Davidson et al., 1973; MacIver, 1972; Nordstoga, 1977). Electron microscopy (e.g. Margaretten et al., 1969; Emeis and Lindeman, 1976) allows the detection and accurate localization of very small amounts of fibrin, but is very time consuming, while only small samples of tissue can be studied. The immunoenzymehistochemical method outlined above meets all requirements for the detection of fibrin microthrombi: specificity, sensitivity and accurate localization of microthrombi in tissues. It has the added advantage that large areas of tissue can be scanned rapidly. Since antisera against rat fibrinogen
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or fibrin m o n o m e r cross-react with h u m a n fibrinogen, the method proved to be applicable to paraplast-embedded human autopsy material as well (ms in prep). The use of an animal model allowed differentiation between the morphological varieties of F R M found during DIC. As, after perfusion, only fibrillar F R M was found, the granular staining found in larger vessels and the rim-like staining in capillaries and sinusoids might be due to an artefactual precipitation of circulating fibrinogen and/or fibrin m o n o m e r during fixation. As rim-like staining is most evident in monomer-induced D I C (especially after ethanol fixation) this staining might be due to the precipitation of fibrin monomer, in agreement with Bleyl's observation that ethanol makes an excellent fixative for fibrin m o n o m e r (Bleyl 1977; Bleyl et al., 1969; compare Godal and Abildgaard, 1966). Thus, only fibrillar strands of F R M are thought to represent in vivo-formed fibrin microthrombi, whereas the diffusely-granular and perivascular F R M are postfixation artefacts. The lengthwise intravascular orientation of these strands is another argument again for the in vivo-formation of these microthrombi (Boyd, 1965). The diffuse pericapillary and perisinusoidal staining as observed by us in non-perfused tissue, and described by others in normal and diseased human liver tissue (e.g. Arias and Mancilla-Jimenez, 1970; Orfila et al., 1976), should not be considered as proof of DIC, but at the most suggests increased levels of fibrin m o n o m e r in the circulation. The organ distribution and tissue localization of fibrin microthrombi as found during D I C induced by thrombin or fibrin monomers, are practically identical. Of note is t h a t - unless fibrinolysis is inhibited by Trasylol - g l o m e r u l a r microthrombi are found in monomer-induced D I C only. The reason for this discrepancy is unclear. The very rapid disappearance (within 30 40 min) of fibrin microthrombi from the circulation is remarkable. Therefore, as far as rats are concerned, one should be cautious not to rely too much on histological observations for the diagnosis of DIC. In conclusion: we demonstrated that fibrin microthrombi can be reliably, sensitively and specifically detected by immunoenzymehistochemistry on paraplast-embedded tissue sections, whereas fibrillar, length-wise oriented F R M only should be considered as fibrin microthrombi formed in vivo.
Acknowledgements. The authors are indebted to Mrs. C.M. van Sabben for excellent assistance. References
Arias, F., Mancilla-Jimenez, R.: Hepatic fibrinogen deposits in pre-eclampsia. N. Engl. J. Med. 295, 578 582 (1976) Beneke, G. : Demonstration of plasma proteins in microscopicsections with emphasis on the identification of fibrin. In: Thrombosis and bleeding disorders (ed. N.U. Bang, F.K. Beller, E. Deutsch, E.F. Mammen), p. 524-533. Stuttgart: Georg Thieme 1971 Bleyl, U. : Morphologic diagnosis of disseminated intravascular coagulation : histologic, histochemical and electronmicroscopicstudies. Semin.Thromb. Hemostasis 3, 247 267 (1977)
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Bleyl, U., Sebening, H., Kuhn, W.: Morphologischer Nachweis yon Fibrinomeren im histologischen Schnitt. Thromb. Diath. Haemorrh. 22, 68-86 (1969) Boyd, J.F. : Disseminated fibrin thrombo-embolism among stillbirths and neonatal deaths. J. Pathol. Bact. 90, 53-63 (1965) Davidson, A.M., Thomson, D., Macdonald, M.K., Rae, J.K., Uttley, W.S., Clarkson, A.R. : Identification of intrarenal fibrin deposition. J. clin. Pathol. 26, 102-112 (1973) Emeis, J.J., Lindeman, J.: Rat liver macrophages will not phagocytose fibrin during disseminated intravascular coagulation. Haemostasis 5, 193 210 (1976) Godal, H.C., Abildgaard, U.: Gelation of soluble fibrin in plasma by ethanol. Scand. J. Haemat. 3, 342-350 (1966) Grabar, P.: The immuno-electrophoretic method of analysis. In: Immunoelectrophoretic analysis (ed. P. Grabar, P. Burtin), p. 3-29. Amsterdam: Elsevier 1964 Graham, R.C., Karnovsky, M.J. : The early stages of absorbtion of injected horseradish peroxidase in the proximal tubules of mouse kidney: ultrastructural cytochemistry by a new technique. J. Histochem. Cytochem. 14, 291-302 (1966) Haverkate, F., Timan, G.: Preparation of highly purified bovine fibrin plates. In: Progress in chemical fibrinolysis and thrombolysis (ed. J.F. Davidson, M.M. Samama, P.C. Desnoyers), p. 67 71. New York: Raven Press t976 MacIver, A.G.: A comparison of tinctorial and immunohistological methods for the detection of fibrinoid change and fibrin deposition in the kidney. Histochem. J. 4, 169-176 (1972) Margaretten, W., Csavossy, I , McKay, D.G.: An electron microscopic study of thrombin-induced disseminated intravascular coagulation. Blood 29, 269-275 (1967) Mueller-Berghaus, G.: Pathophysiology of generalized intravascular coagulation. Semin. Thromb. and Hemostasis 3, 209 246 (1977) Mueller-Berghaus, G., Mahn, I., K6veker, G., Maul, F.D. : In vivo behavior of homologous ureasoluble 131I-fibrin and 12sI-fibrinogen in rabbits. Brit. J. Haematol. 33, 61-79 (1976) Nieuwenhuizen0 W., Emeis, J.J., Sabben, C.M. van: Localization of lipase-like immunoreactivity in porcine adipose, aortic and myocardial tissue. Atherosclerosis 27, 97-106 (1977) Nordstoga, K. : Disseminated intravascular coagulation: a pathologist's view. Thromb. Haemostas. 37, 180 (1977) Orfila, C., Duffaut, M., Mignon-Conte, M., Rumeau, J.-L, Bugat, R., Suc J.-M: Localisation en immunofluorescence directe dans le tissu h6patique de I'AgHBs, des immunoglobulines, de la fibrine et de la fraction C3 du compl~ment. Pathol. Biol. 24, 313-318 (1976) Ouchterlony O. : Handbook of immunodiffusion and immunoelectrophoresis, p. 25. London: Ann Arbor-Humphrey Science 1968 Robboy S.J., Colman R.W., Minna J.D.: Pathology of disseminated intravascular coagulation. Human Pathol. 3, 327-343 (1972) Ruijven-Vermeer, I.A.M, van, Nieuwenhuizen, W. : Purification of rat fibrinogen and its constituent chains. Biochem. J. 169, 653-658 (1978) Streefkerk, J.G.: Inhibition of erythrocyte pseudoperoxidase activity by treatment with hydrogen peroxide following methanol. J. Histochem. Cytochem. 20, 829-831 (1972) Taylor, C.R., Burns, J. : The demonstration of plasma cells and other immunoglobulin-containing cells in formalin-fixed, paraffin-embedded tissues using peroxidase-labelled antibody. J. clin. Pathol. 27, 14 20 (1974)
Received April 22, 1978
