Martius Scarlet Blue Research Articles

Increased activity of vascular adenosine deaminase in atherosclerosis and therapeutic potential of its inhibition.

Extracellular nucleotides and adenosine that are formed or degraded by membrane-bound ecto-enzymes could affect atherosclerosis by regulating the inflammation and thrombosis. This study aimed to evaluate a relation between ecto-enzymes that convert extracellular adenosine triphosphate to adenine dinucleotide phosphate, adenosine monophosphate, adenosine, and inosine on the surface of the vessel wall with the severity or progression of experimental and clinical atherosclerosis. Furthermore, we tested whether the inhibition of adenosine deaminase will block the development of experimental atherosclerosis. Vascular activities of ecto-nucleoside triphosphate diphosphohydrolase 1, ecto-5'-nucleotidase, and ecto-adenosine deaminase (eADA) were measured in aortas of apolipoprotein E-/- low density lipoprotein receptor (ApoE-/-LDLR-/-) and wild-type mice as well as in human aortas. Plaques were analysed in the entire aorta, aortic root, and brachiocephalic artery by Oil-Red O and Orcein Martius Scarlet Blue staining and vascular accumulation of macrophages. The cellular location of ecto-enzymes was analysed by immunofluorescence. The effect of eADA inhibition on atherosclerosis progression was studied by a 2-month deoxycoformycin treatment of ApoE-/-LDLR-/- mice. The vascular eADA activity prominently increased in ApoE-/-LDLR-/- mice when compared with wild type already at the age of 1 month and progressed along atherosclerosis development, reaching a 10-fold difference at 10 months. The activity of eADA correlated with atherosclerotic changes in human aortas. High abundance of eADA in atherosclerotic vessels originated from activated endothelial cells and macrophages. There were no changes in ecto-nucleoside triphosphate diphosphohydrolase 1 activity, whereas ecto-5'-nucleotidase was moderately decreased in ApoE-/-LDLR-/- mice. Deoxycoformycin treatment attenuated plaque development in aortic root and brachiocephalic artery of ApoE-/-LDLR-/- mice, suppressed vascular inflammation and improved endothelial function. This study highlights the importance of extracellular nucleotides and adenosine metabolism in the atherosclerotic vessel in both experimental and clinical setting. The increased eADA activity marks an early stage of atherosclerosis, contributes to its progression and could represent a novel target for therapy.

Novel methodology to replicate clot analogs with diverse composition in acute ischemic stroke.

Translational research on clot composition may be advanced by the use of clot analogs for the preclinical evaluation of mechanical thrombectomy devices. This work describes a novel set of clot analogs to represent a diverse range of fibrin and red blood cell (RBC) compositions for use in acute ischemic stroke (AIS) occlusion models. Fresh whole blood obtained from ovine species was used to create seven different clot analog types. Five replicates were formed for each clot type. Varying amounts of whole blood constituents were mixed with thrombotic factors to create clots of varying compositions. Following histological processing, five sections from each clot were stained with H&E and Martius Scarlet Blue. Fibrin, RBC and white blood cell compositions were quantified. Histological examination demonstrated that the clot types had a distinct RBC and fibrin composition. No significant difference in composition was shown between replicates (p>0.05), indicating that the method of clot formation was reproducible. Percentage fibrin composition of the clot types was 1%, 8%, 31%, 38%, 64%, 79%, and 100%. A significant difference in fibrin and RBC composition between clot types was observed (p<0.05). Seven different clot types were developed to replicate common AIS thrombi. These clot analogs may be beneficial for the preclinical evaluation of endovascular therapies, and may be applied to interventional technique training.

Platform Technologies for Directly Reconstructing 3D Living Biomaterials.

Bio-electrospraying and cell electrospinning is explored for reconstructing living biomaterials for regenerative biology and medicine. The investigations carried out in this study demonstrate these approaches as platform biotechnologies for tissue reconstruction for repair, replacement, and rejuvenation of damaged and/or ageing tissues and/or organs.

Detection of Intraplaque Hemorrhage in Mouse Atherosclerotic Lesions.

Intraplaque hemorrhage is defined as the presence of fresh or lysed erythrocytes, iron deposits in macrophages, and/or a fibrin clot in an atherosclerotic plaque. These features can be detected by hematoxylin and eosin, Martius scarlet Blue, and Perl's iron histological stainings. It is noteworthy that intraplaque hemorrhage is only present in murine atherosclerotic plaques after additional interventions or additional genetic traits affecting matrix degradation or thrombosis. In this chapter, we describe methods to detect intraplaque hemorrhage in mouse atherosclerotic lesions.

1572 HISTOLOGICAL CHANGES IN THE HUMAN PROSTATE AFTER PHOTOSELECTIVE VAPORIZATION OF THE PROSTATE COMPARED TO STANDARD TRANSURETHRAL RESECTION OF THE PROSTATE

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research1 Apr 20121572 HISTOLOGICAL CHANGES IN THE HUMAN PROSTATE AFTER PHOTOSELECTIVE VAPORIZATION OF THE PROSTATE COMPARED TO STANDARD TRANSURETHRAL RESECTION OF THE PROSTATE Amanda Chung, Venu Chalasani, Fiona Maclean, and Henry Woo Amanda ChungAmanda Chung Sydney, Australia More articles by this author , Venu ChalasaniVenu Chalasani Sydney, Australia More articles by this author , Fiona MacleanFiona Maclean Sydney, Australia More articles by this author , and Henry WooHenry Woo Sydney, Australia More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2012.02.1344AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The histological changes in the human prostate following photoselective vaporization of the prostate (PVP) have not been documented. The objective of this study is to examine and document the histological changes in the human prostate after PVP versus transurethral resection of the prostate (TURP). METHODS The prostates of 3 men who underwent 532nm PVP followed by retropubic radical prostatectomy (RRP) were compared histologically to the prostates of 5 contemporaneous men who underwent bipolar diathermy TURP followed by RRP. Time from PVP or TURP to RRP (for prostate cancer) was greater than 6 months. A single surgeon performed the PVP, TURP and RRP. A single uropathologist, who was blinded to the initial treatment modality, examined all the specimens. The prostates were fixed in formalin, examined macroscopically, and sliced in step sections. Paraffin sections were taken and stained with hematoxylin and eosin (H&E), Masson's trichrome (MT), Martius scarlet blue (MSB) and Verhoeff-Van Gieson (VVG). RESULTS The mean age of the PVP group of men and TURP group of men was 61.5 years and 58.7 years respectively. Amongst the PVP group, H&E staining of the prostatic specimens showed patchy foci of residual coagulative necrosis in the submucosa of the prostatic urethra. MT and MSB staining demonstrated submucosal fibrosis in both PVP and TURP groups, but with differing patterns of distribution. In the PVP group, fibrosis was discontinuous, as discrete foci, extending as tongues into adjacent fibromuscular stroma. In the TURP group, a continuous band of submucosal fibrosis formed a “tight cuff” around the prostatic urethra, which also demonstrated more prominent squamous metaplasia. CONCLUSIONS This histopathological study is the first to document the medium-term effects of PVP on human prostates. We discover an absence of a continuous submucosal fibrosis “cuff”, leading to the hypothesis that PVP may be associated with less risk of complication with bladder neck contracture, compared with TURP. © 2012 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 187Issue 4SApril 2012Page: e637 Advertisement Copyright & Permissions© 2012 by American Urological Association Education and Research, Inc.MetricsAuthor Information Amanda Chung Sydney, Australia More articles by this author Venu Chalasani Sydney, Australia More articles by this author Fiona Maclean Sydney, Australia More articles by this author Henry Woo Sydney, Australia More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Abstract 3750: Embolus Adhesion to Activated Endothelium After Embolization: A Parameter To Predict Outcomes Of Mechanical Thrombectomy In Acute Ischemic Stroke

Background: In the MERCI trial and a prior animal study (Catch device), it was reported that when the thrombus retrieval maneuver was performed, the deployed device failed to mobilize the thrombus. Increased pulling force was applied and resulted in device rupture. These experiences lead us to hypothesize that the occlusive mass interacts with the activated endothelium, and the stability of the embolus on the vessel wall is related to the outcomes of mechanical thrombectomy. The aim of this study is to establish a methodology to quantitatively characterize the embolus-vessel wall adhesion. Methods: A rabbit CCA stenosis model was developed for creation of embolic occlusion with allogeneic clot. To evaluate the morphology of the embolus analogue (EA)-endothelium interface, two occluded vessel segments were explanted at 5-hour for Martius Scarlet Blue (MSB) staining and double immunofluorescence labeling for glycoprotein (GP) IIb/IIIa and fibrin. A modified test rig with a carbon steel wire (0.6 mm in diameter) fixture was used to detach the EA from the vessel wall (n=4). The wire moved down at the test speed of 0.1mm/s. The mean force was recorded and the energy to dislodge the EA from the vessel wall was calculated by numerical integration of the force versus distance curve below. Data were presented as mean±standard error. Results: MSB results showed that, the EA conformed to the shape of the underlying tunica intima and the undulated internal elastic lamina tended to flatten out after the occlusion. Immunofluorescent staining showed that GP IIb/IIIa (red) co-localized with fibrin(green), mainly observed at the EA-endothelium interface (yellow) ( Fig 1 A). In the detachment tests, a test distance of 6.07±1.18 mm was achieved after imposing 422.9±113.1 gram-force*cm of energy ( Fig 1 B). The mean force was 73.08±8.5 gram-force. Conclusions: Presence of GP IIb/IIIa and fibrin immunoreactivity indicates the interactions between border of the EA and the luminal surface, and this finding is supported by the quantitative adhesion force acquired during the detachment test. This project is ongoing to investigate thrombus-vessel wall interactions caused by acute thrombosis. The fibrin crosslink number and the platelet volume will be characterized by using confocal laser scanning microscope, and correlated with the adhesion results.

Histopathological analysis and in situ localisation of Australian tiger snake venom in two clinically envenomed domestic animals

Objective To assess histopathological changes in clinically envenomed tiger snake patients and identify tissue specific localisation of venom toxins using immunohistochemistry. Samples One feline and one canine patient admitted to the Murdoch Pet Emergency Centre (MPEC), Murdoch University with tiger snake ( Notechis sp.) envenoming. Both patients died as a result of envenomation. Non-envenomed tissue was also collected and used for comparison. Methodology Biopsy samples (heart, lung, kidney andskeletal muscle tissue) were retrieved 1–2 h post death and processed for histopathological examination using Haemotoxylin and Eosin, Martius Scarlet Blue and Periodic Acid Schiff staining. Tissues were examined by light microscopy and tissue sections subjected to immunohistochemical staining using in-house generated monoclonal and polyclonal antibodies against Notechis venoms. Results Venom-induced pathological changes were observed in the lungs, kidneys and muscle tissue of both patients. Evidence, not previously noted, of procoagulant venom effects were apparent, with formed thrombi in the heart, lungs (small fibrillar aggregates and larger, discrete thrombi) and kidneys. Immunohistochemical assays revealed venom present in the pulmonary tissue, in and around the glomerular capsule and surrounding tubules in renal tissue and scattered throughout the Gastrocnemius muscle tissue. Conclusion This work has shown pathological evidence of procoagulant venom activity supporting previous suggestions that an initial thrombotic state occurs in envenomed patients. We have shown that venom toxins are able to be localised to specific tissues, in this case, venom was detected in the lung, kidney and muscle tissues of clinically envenomed animals. Future work will examine specific toxin localisation using monoclonal antibodies and identify if antivenom molecules are able to reach their target tissues.

P-002 Realistic clot models with different mechanical characteristics for preclinical evaluation of endovascular recanalization devices

IntroductionEfficacy and safety of devices for the endovascular recanalization of acute ischemic stroke (AIS) are mostly validated in experimental vascular models and in vivo animal models. Efforts have been made...

Activated Protein C Resistance (APCR) and Placental Fibrin Deposition

Activated protein C resistance (APCR) results in an ineffective anticoagulant response leading to an increased risk of thrombosis, particularly during pregnancy. Adverse pregnancy outcomes including pre-eclampsia (PET), intrauterine growth restriction (IUGR), recurrent miscarriage and placental abruption have been linked with thrombotic lesions compromising the utero-placental circulation. Using histological staining including Martius Scarlet Blue (MSB) and Haematoxylin and Eosin (H&E) and microscopy, we studied placental fibrin deposition and histological abnormalities in subjects ( n = 23) with APCR (APCR group), based on a ratio of less than or equal to 2.1 s with the Coatest ® classic test and subjects ( n = 11) with an APC ratio in the normal range, greater than 2.1 s (APCN group). Fibrin deposition was significantly higher (3.3-fold) in the APCR group compared to the APCN group. An inverse correlation between APC ratio and placental fibrin deposition was determined for the study group. Histological abnormalities were more than 2-fold higher in the APCR group compared to the APCN group. Molecular screening identified common thrombophilic mutations, FVL and FII-G20210A in the APCR group but not in the APCN group.

The Microvacular Thrombi of Colonic Tissue in Ulcerative Colitis

Mucosal microvascular thrombi in rectal biopsies were observed in some ulcerative colitis (UC). Heparin may be effective in steroid resistant UC in some studies, however, the new results of meta-analysis demonstrated a non-significant effect of heparin in controlled clinical trials, differing markedly from observational studies. The objective of this study was to identify colonic microvascular thrombi in larger cases with UC, and analyse its possible risk factors: age, gender, histologic score, extent of lesions and operation or biopsy specimens, and assess the significance of microvascular thrombosis in patients with UC. The microvascular thrombi were identified by immunohistochemical staining with anti-CD61 monoclonal antibody and Martius scarlet blue (MSB) staining in 40 colonic tissue samples of UC (31 biopsy specimens and nine operated cases) and 12 cases of normal colon tissue from operated colonic carcinoma. Logistic regression analysis was used to assess the relationship of age, gender, degree of histology, origin of the specimens, extent of lesions and microvascular thrombi examined. Microvascular thrombi were positive in 14 of 40 UC cases, and none in the controls. The presence of microvascular thrombi was related to operation specimens with odds ratio 11.667, P=0.0179, it might be also related to histologic score (OR=1.350) and extent of lesions (OR=1.619). These results suggest that microvascular thrombosis may be one of the important pathogenesis in some UC, and that the effect of anticoagulant treatment still needs to be assessed.

Thrombocytopenia in the experimental leptospirosis of guinea pig is not related to disseminated intravascular coagulation

BackgroundThrombocytopenia is commonly observed in severe leptospirosis. However, previous studies on coagulation alterations during leptospirosis resulted in inconsistent conclusions. Some findings showed that the prominent levels of thrombocytopenia observed in severe leptospirosis did not reflect the occurrence of disseminated intravascular coagulation (DIC) syndrome, while the others reached the conclusion that the hemorrhages observed in leptospirosis were due to DIC. The aim of this study is to elucidate whether DIC is an important feature of leptospirosis.MethodsThe leptospirosis model of guinea pig was established by intraperitoneal inoculation of Leptospira interrogans strain Lai. Hematoxylin and eosin (HE) staining, electron microscopy and immunohistochemistry staining were used to detect the pathologic changes. Platelet thrombus or fibrin thrombus was detected by HE, Martius Scarlet Blue (MSB) staining and electron microscopy. Hemostatic molecular markers such as 11-dehydrogenate thromboxane B2 (11-DH-TXB2), thrombomodulin (TM), thrombin-antithrombin III complex (TAT), D-Dimer and fibrin (ogen) degradation products (FDPs) in the plasma were examined by quantitative enzyme-linked immunosorbent assay (ELISA) to evaluate the hematological coagulative alterations in leptospirosis models.ResultsPulmonary hemorrhage appeared in the model guinea pig 24 hours after leptospires intraperitoneal inoculation, progressing to a peak at 96 hours after the infection. Leptospires were detected 24 hours post-inoculation in the liver, 48 hours in the lung and 72 hours in the kidney by immunohistochemistry staining. Spiral form of the bacteria was initially observed in the liver, lung and kidney suggestive of intact leptospires, granular form of leptospires was seen as the severity increased. Platelet aggregation in hepatic sinusoid as well as phagocytosis of erythrocytes and platelets by Kupffer cells were both observed. Neither platelet thrombus nor fibrin thrombus was found in the liver, lung or kidney via morphological observation. Thrombocytopenia was observed in all infected guinea pigs of our experimental leptospirosis study. Analysis of hematologic molecular markers showed that 11-DH-TXB2 and TM in the plasma were elevated significantly. TAT that reflects the thrombin activation had a trend of decline after infection. Although D-dimer and FDPs increased statistically, the increasing may not bear clinical significance.ConclusionPathologic and hematological studies for experimental leptospirosis of guinea pig indicated that the thrombocytopenia found in guinea pigs did not correlate with the occurrence of DIC. The platelet aggregation and Kupffer cells phagocytosis might be the potential causes of thrombocytopenia in severe leptospirosis.

Recruitment of Labelled Monocytes by Experimental Venous Thrombi

Infusing monocytes that have been stimulated to produce fibrinolytic activators and factors that regulate cell proliferation, migration and maturation, might enhance venous thrombus resolution. The aim of this study was to determine the time course of infused monocyte recruitment into venous thrombus in an appropriate model of this disease. Thrombus was induced in the inferior vena cava of male Wistar rats using reduced flow (80-90% stenosis). The vessel wall was examined at 1hr by scanning electron microscopy. Resolving thrombi with surrounding vena cava were obtained at 1, 7, 14 and 21 days after induction (n = 8). Sections, taken at 0.5 mm intervals (10-15 sections per thrombus), were stained using haematoxylin, Martius Scarlet Blue and antibodies against monocytes, platelets and fibrin. Sections from human venous thrombi (n = 4) were similarly stained. The area occupied by monocytes (in relative pixel units, RPU) was determined using computer aided image analysis. Peripheral rat blood monocytes were extracted, fluorescently labelled and injected intravenously into 7 rats prior to thrombus induction, Vena cava with thrombus was harvested 1 h, 2, 3, 4, 7, 14 and 25 days after induction and their fluorescence measured. The fluorescent content of the caval wall and thrombus was analysed in greater detail at 2 and 25 days after thrombus induction (n = 4 at each time interval). Experimental thrombi were structurally similar to human thrombus and resolved within 14-21 days. Scanning electron microscopy showed minimal endothelial damage at 1 h with signs of early thrombus formation (platelet, red cell leukocyte and fibrin deposition). Neutrophils were the predominant leukocyte in the thrombus at 1 day, with monocytes making up only 0.3% (0.04% sem) of the area of the thrombus. There was a steady increase in thrombus monocyte content and by 21 days the percentage area of thrombus covered by monocytes had increased by over 35 fold to 11.5% (2.3% sem) (p <0.001). Initially, monocytes appeared around the edge of the thrombus and became more evenly distributed through the thrombus as resolution progressed. Labelled monocytes could be found in the circulation up to 1 week after infusion. The fluorescent content (RPU) of the thrombus increased over 25 days (mean RPU At 2 days 0.012, sem 0.005; mean RPU at 25 days 1.062, sem 0.252, p = 0.008). The number of labelled monocytes in the vessel wall peaked at 2 days and decreased thereafter. The structure of thrombi produced by this model was comparable to that of human venous thrombi. Endogenous and injected monocytes migrated into the thrombus during natural resolution, possibly via the vein wall. Monocyte targeting could therefore be used to develop novel treatments for venous thrombosis, with the aim of reducing post-thrombotic complications.

Pathology of idiopathic megarectum and megacolon

Background—The aetiology and pathology of both idiopathic megarectum and idiopathic megacolon are unknown. In particular, it is unknown whether there are abnormalities involving enteric nerves or smooth muscle.Methods—Resected tissue was...

Microvascular obstruction in avascular necrosis. Immunohistochemistry of 14 femoral heads.

14 femoral heads with late stage avascular necrosis of different etiologies were histologically examined, with special attention to vascular structures. Decalcified slices were stained with hematoxylin-eosin, safranin-O, van Gieson stain, and Martius Scarlet Blue. Immunohistochemical techniques with antibodies against Factor VIII, and Ulex Europeus Lectin were used to visualize the endothelium of the blood vessels. 5 distinct zones of the necrotic femoral head could be identified. The necrotic zone contained areas with richly vascularized connective tissue. In the transitional zone, several areas with intravascular aggregations of newly formed and older fibrin clots were noticed, mainly on the venous side of the vascular system. Other small vessels were collapsed, with a few endothelial cells clumped together in the center of a concentric fibrous tissue. We suggest that obstruction to the venous outflow due to intravascular thrombosis as well as to perivascular fibrosis is important in the pathogenesis of non-traumatic avascular necrosis of the femoral head.

Altered collagen (fibrinoid change) at the site of post-mortem injuries.

The terms fibrinoid degeneration and fibrinoid substance were first introduced by Neumann in 1880 to describe some alterations in the staining characteristics of collagen. Collagen fibres which have undergone this change are eosinophilic, homogeneous and take some of the tinctorial properties of fibrin. Lendrum et al. (1962) developed Martius-Scarlet-Blue (MSB) stain, which is preferentially taken up by fibrin. The principle of the method described by Lendrum and his colleagues is the use of acid dyes of different molecular size (Martius yellow, Brilliant Crystal Scarlet 6R, methyl blue) in accordance with the alteration in structure of fibrin at different stages of development. The newest fibrin likely to be found in sections is presumably the fine network of postmortem fibrin. Much of this takes a yellow stain with the MSB method, as do erythrocytes. Slightly older fibrin (16 hours) takes a bright red stain with the MSB. Complete validity of the method depends on prolonged fixation in formal-sublimate. It gives brilliant staining with acid dyes and enhances metachromasia. In the Department of Forensic Medicine in Leeds it was noticed that an alteration in collagen staining with MSB stain was taking place in samples collected from different kinds of injuries, e.g. ligature marks, electrical injuries and burns. Similar changes were only occasionally seen in abrasions. It was therefore decided to inflict different kinds of injuries on the skins of rats and to fresh pieces of human skin in vitro, to observe any alterations in collagen staining with MSB and to assess the significance of these changes in relation to the timing of injuries.(ABSTRACT TRUNCATED AT 250 WORDS)

Pathological changes in levator palpebrae superioris muscle treated with botulinum toxin in a case of carotico-cavernous fistula.

We describe the case of a patient with carotico-cavernous fistula who had botulinum toxin A to induce a protective ptosis 4.5 days before death. The levator palpebrae superioris muscle from both sides and the superior rectus muscle from the injected side were obtained for examination. The preserved samples were stained with haematoxylin and eosin, Martius scarlet blue, Glees, S100, dehydrogenase, ATPase, and toluidine blue as well as being examined by electron microscopy. Inflammation and oedema were found that were probably due to the carotico-cavernous fistula. Axonal and some myelin sheath damage were also seen.

Simultaneous demonstration of connective tissue elastica and fibrin by a combined Verhoeff's elastic-Martius-scarlet-blue trichrome stain.

A method for the routine combined demonstration of elastica, connective tissue in general, and fibrin is described. Elastica, stained blue-black by Verhoeff's iron hematoxylin, is contrasted with muscle and collagen, stained red and blue or green respectively, by a modification of the Martius-scarlet-blue (MSB) trichrome for fibrin of Lendrum et al. The MSB technique selectively stains fresh, mature and old fibrin orange-yellow, red and blue respectively; the Masson trichrome does not distinguish between erythrocytes and fibrin. Nuclei are stained at the same time as the elastica. The technique takes approximately one and a half hours and is ideal for the study of connective tissue and vascular pathology, especially the necrotizing vasculitides.

Identification of intrarenal fibrin deposition.

The site and extent of intrarenal fibrin deposition has been examined by routine histological staining, immunofluorescence, and electron microscopy in 109 patients with a wide variety of renal diseases. The findings have been correlated with the amount of urinary fibrin/fibrinogen degradation product (FDP) excretion as measured by the tanned red cell haemagglutination inhibition immunoassay. The results show that routine histological stains (Martius Scarlet Blue and Picro Mallory) are unreliable, particularly where there is mesangial accumulation of material or where the amount of fibrin deposited is small and confined to a subendothelial position. Similarly the electron microscope may overassess the degree of fibrin deposition, particularly if this is associated with the deposition of immunoglobulins and complement. There is a close relationship between the site and extent of fibrin, as detected by immunofluorescence, and the maximal urinary FDP excretion.

A comparison of tinctorial and immunohistological methods for the detection of fibrinoid change and fibrin deposition in the kidney.

Renal tissue from six cases with fibrinoid change or with glomerular ‘fibrin’ deposits was examined by the standard tinctorial methods (Lendrum's Martius Scarlet Blue, phosphotungsticacid-Haematoxylin and Lendrum's picro-Mallory) for the demonstration of fibrin. It was found that all three methods should be used to detect its presence. Examination by immunofluorescence techniques indicated that little or no fibrinogen or fibrin need be present in areas giving positive tinctorial reactions.